|
|
|
|
|
| Electrophoresis, 1996 May, 17(5), 932 - 7 Investigation on minor degraded derivatives of the recombinant hirudin variant HM2 from Hirudinaria manillensis isolated by isoelectric focusing in multicompartment electrolyzers; Bossi A et al.; On isoelectric focusing in immobilized pH gradients (IPG) a preparation of recombinant hirudin from Hirudinaria manillensis, purified to homogeneity, was found to still contain a total of 5% minor components: three with higher pI values (pIs 4.10, 4.25 and 4.31), one with a lower pI value (pI 3.98) as compared with the main form (pI 4.03) . Multicompartment electrolyzers with isoelectric membranes and micropreparative IPG gel slabs allowed the recovery of pure fractions of such minor components, which were further characterized by electrospray mass spectra, limited proteolysis, and sequence analysis . All four minor isoforms were found to be cleavage products of the parent, full-length hirudin molecule (molecular mass 6797 Da), as follows: the pI 4.31 (5032 Da) had lost sixteen amino acids from the N-terminus, the pI 4.25 (6212 Da) lacked five amino acids from the C-terminus, the pI 4.10 (2980 Da) was a cleavage product at residue Cys37, and the pI 3.98 (6610 Da) lacked the dipeptide Val-Ser at the N-terminus . Combining the extreme resolving power of IPGs with the high accuracy of mass spectra was found to be an attractive strategy in decoding post-synthetic modifications often encountered in r-DNA proteins. Environ Health Perspect, 1996 May, 104 Suppl 3, 683 - 6 Distinction of mutagenic carcinogens from a mutagenic noncarcinogen in the big blue transgenic mouse; Cunningham ML et al.; The aromatic amines 2,4-diaminotoluene (2,4-DAT) and 2,6-diaminotoluene (2,6-DAT) are structural isomers that have been extensively studied for their mutagenic and carcinogenic characteristics . Both compounds are rapidly absorbed after oral administration and are equally mutagenic in the Ames test; however, 2,4-DAT is a potent hepatocarcinogen, whereas 2,6-DAT does not produce an increased incidence of tumors in rats or mice at similar doses . The Big Blue transgenic B6C3F1 mouse carries multiple copies of the lacl mutational target gene . Our studies were designed to determine whether the Big Blue system could be used to detect differences in the vivo mutagenic activity between the carcinogen-noncarcinogen pair 2,4-DAT and 2,6-DAT and to determine whether the in vivo mutagenesis assay results correspond to the rodent carcinogen bioassay results . Male B6C3F1 transgenic mice were exposed to 2,4-DAT or 2,6-DAT at 0 or 1,000 ppm in the diet for 30 and 90 days or to dimethylnitrosamine as a positive control . Mutant frequencies were nearly identical for all three groups at 30 days, while at 90 days the mutant frequency for the hepatocarcinogen 2,4-DAT (12.1 +/- 1.4 x 10(-5)) was significantly higher (p < 0.01) as compared to both age-matched (spontaneous) controls (5.7 +/- 2.9 x 10(-5)) and the 2,6-DAT-exposed group (5.7 +/- 2.4 x 10(-5)) . Results from this study demonstrate that the Big Blue transgenic mutation assay can distinguish differences in vivo between the mutagenic responses of hepatic carcinogens ad a noncarcinogen; is sensitive to mutagens through subchronic dietary exposure; and yields a differential response depending upon the length of time mice are exposed to a mutagen. Environ Health Perspect, 1996 May, 104 Suppl 3, 557 - 62 Involvement of cytochrome P450, glutathione S-transferase, and epoxide hydrolase in the metabolism of aflatoxin B1 and relevance to risk of human liver cancer; Guengerich FP et al.; In recent years there has been considerable interest in the effect of variations in activities of xenobiotic-metabolizing enzymes on cancer incidence . This interest has accelerated with the development of methods for analyzing genetic polymorphisms . However, progress in epidemiology has been slow and the contributions of polymorphisms to risks from individual chemicals and mixtures are often controversial . A series of studies is presented to show the complexities encountered with a single chemical, aflatoxin B1 (AFB1) . AFB1 is oxidized by human cytochrome P450 enzymes to several products . Only one of these, the 8,9-exo-epoxide, appears to be mutagenic and the others are detoxication products . P450 3A4, which can both activate and detoxicate AFB1, is found in the liver and the small intestine . In the small intestine, the first contact after oral exposure, epoxidation would not lead to liver cancer . The (nonenzymatic) half-life of the epoxide has been determined to be approximately 1 sec at 23 degrees C and neutral pH . Although the half-life is short, AFB1-8,9-exo-epoxide does react with DNA and glutathione S-transferase . Levels of these conjugates have been measured and combined with the rate of hydrolysis in a kinetic model to predict constants for binding of the epoxide with DNA and glutathione S-transferase . A role for epoxide hydrolase in alteration of AFB1 hepatocarcinogenesis has been proposed, although experimental evidence is lacking . Some inhibition of microsome-generated genotoxicity was observed with rat epoxide hydrolase; further information on the extent of contribution of this enzyme to AFB1 metabolism is not yet available. J Neurochem, 1996 May, 66(5), 1819 - 25 A novel rat tyrosine hydroxylase mRNA species generated by alternative splicing; Laniece P et al.; Tyrosine hydroxylase (TH) catalyzes the first and rate-limiting step in the biosynthesis of catecholamines . Among the various mechanisms implicated in the regulation of TH activity, alternative splicing of TH primary transcript has been described as a characteristic of higher primates and Drosophila . We investigated whether there is such a regulatory mechanism in the rat . Reverse transcriptase-PCR experiments were performed with RNA from PC12 cells . A new TH mRNA species was evidenced, resulting from the use of an alternative donor site in exon 2 . RNase protection assays and in situ hybridization experiments detected this mRNA species in the adrenal medulla but not in the main catecholaminergic nuclei of the CNS . The corresponding putative protein lacks 33 amino acids in the N-terminal regulatory domain . A recombinant protein was produced in E . coli . Its in vitro specific activity was similar to that of the previously identified TH protein. Res Commun Mol Pathol Pharmacol, 1996 May, 92(2), 225 - 32 Copper replacement of cadmium-binding alpha-fragment of metallothionein expressed in Escherichia coli; Kurasaki M et al.; Titration studies of Cd-binding alpha-fragment, the carboxyl-terminal half of human metallothionein-2, which was independently expressed in Escherichia coli as a Cd-binding form, with Cu were performed . The Cu saturated alpha-fragment was not obtained when the alpha-fragment incubated with Cu (I), although completely saturated MT with Cu (I) was successfully obtained in the same conditions . The stoichiometry of the alpha-fragment replaced with Cu (I) was Cu 4 and Cd 1 . It is considered that the Cu-binding alpha-fragment required the presence of Cd (II) to form a stable structure . Our results could suggest that each fragment was independent in the metal binding processes, e.g . alpha-fragment for Cd-binding. J Hepatol, 1996 May, 24(5), 532 - 8 An ELISA for the detection of antibody to the core antigen of hepatitis C virus: use in diagnosis and analysis of indeterminate samples; Trowbridge R et al.; AIMS: In order to examine more carefully the natural history of hepatitis C virus infection and to determine a role for anti-core in the discrimination of indeterminate samples, a solid phase ELISA to detect antibody of the immunoglobulin G class to the hepatitis C virus core antigen was developed using purified protein expressed in Escherichia coli from a major portion of the core antigen coding region . METHODS/RESULTS: In a study which examined 651 samples submitted for routine testing by a commercial ELISA (Ortho), only 11 samples showed discrepant results; of these, 10 were Ortho ELISA positive, anti-core negative and one was Ortho ELISA negative anticore positive . Supplemental tests showed that 5/10 of these samples were anti-HCV negative by RIBA and the reciprocal 5 were negative for anti-C22 but positive for anti-C100 and anti-C33 . The Ortho ELISA negative, anticore positive sample was weakly positive for anti-C22 . The anti-core ELISA was then used to examine 67 indeterminate samples from the blood bank; 11/11 samples which were HCV-RNA positive were anti-core positive and 7/56 samples which were HCV-RNA negative were anti-core positive . The anti-core titre was then examined in two groups of indeterminate samples; group 1, polymerase chain reaction-positive, anti-core positive and group 2, polymerase chain reaction-negative, anti-core positive . The geometric mean anti-core titres in these groups were 1 x 10(-3.6) and 1 x 10(-2.3), respectively . Thus in this group of indeterminate samples, all samples (except one) with an anti-core titre > or = 1/200 were polymerase chain reaction-positive, confirming a close correlation between anti-core levels and hepatitis C viraemia . Anti-core was detected with equal efficiency in patients infected with genotypes which differed to that used to express the recombinant core antigen. Arch Pediatr, 1996 May, 3(5), 470 - 2 {Cystic adenomatoid malformation of the lung revealed in a newborn infant by an image of a lung abscess}; Kieffer F et al.; BACKGROUND: Cystic adenomatoid malformation, a rare pulmonary malformation, usually appears as a cystic mass, radiologically . It may be infected and confusion has also arisen in distinguishing it from pneumonia with pneumatoceles . CASE REPORTS: A full-term boy suffered from severe neonatal respiratory distress . Pregnancy had been uneventful despite the fact that his mother had insulin-dependent diabetes . Prenatal ultrasonographies did not reveal any abnormality . On day 2, X-rays showed a right pulmonary mass that appeared solid . The patient was treated for E Coli sepsis . Subsequently, the pulmonary mass became lacent, cystic, fluid-filled, resembling an abscess; the CT scan confirmed these features . As the lesion increased in volume, a limited resection was performed . Histologic examination showed adenomatoid proliferation of bronchiolar elements with formation of cysts and necrosis . CONCLUSION: Infection of cystic adenomatoid malformation may supervene the first days of life resulting in a lung abscess appearance. Plant Mol Biol, 1996 May, 31(2), 659 - 65 The cyanobacterial genome contains a single copy of the ffh gene encoding a homologue of the 54 kDa subunit of signal recognition particle; Packer JC et al.; Cyanobacteria possess thylakoid membranes that differ in their protein composition from the cytoplasmic membrane . To study possible pathways of protein targeting to these membranes, we have investigated whether or not cyanobacteria have a homologue or homologues of the signal recognition particlelike chaperone Ffh . We have amplified a fragment of ffh by polymerase chain reaction and established that ffh is present as a single copy in the genomes of three cyanobacterial species . We have cloned and sequenced ffh from Synechococcus sp . PCC7942 and predict that Ffh functions as a ribonucleoprotein in cyanobacteria and chloroplasts. Plant Mol Biol, 1996 May, 31(2), 405 - 12 Characterization of eight new members of the calmodulin-like domain protein kinase gene family from Arabidopsis thaliana; Hrabak EM et al.; A family of calcium-responsive protein kinases is abundant in plant cell extracts but has not been identified in animals and fungi . These enzymes have a unique structure consisting of a protein kinase catalytic domain fused to carboxy-terminal autoregulatory and calmodulin-like domains . In this report, we present the amino acid sequences for eight new Arabidopsis cDNA clones encoding isoforms of this enzyme . Three isoforms were expressed as fusion proteins in Escherichia coli and exhibited calcium-stimulated protein kinase activity . We propose CPK as the gene designation for this family of enzymes and describe a phylogenetic analysis for all known isoforms. Biokhimiia, 1996 May, 61(5), 786 - 99 {Cytochrome bd: structure and properties}; Borisov VB; Literary evidence concerning the arrangement and functioning of the cytochrome bd complex is reviewed with particular emphasis on ligand-binding properties of the enzyme . Some novel data on cytochrome bd interaction with carbon monoxide, cyanide and hydrogen peroxide are presented. Genome Res, 1996 May, 6(5), 414 - 30 A P1-based physical map of the Drosophila euchromatic genome; Kimmerly W et al.; A PCR-based sequence-tagged site (STS) content mapping strategy has been used to generate a physical map with 90% coverage of the 120-Mb euchromatic portion of the Drosophila genome . To facilitate map completion, the bulk of the STS markers was chosen in a nonrandom fashion . To ensure that all contigs were localized in relation to each other and the genome, these contig-building procedures were performed in conjunction with a large-scale in situ hybridization analysis of randomly selected clones from a Drosophila genomic library that had been generated in a P1 cloning vector . To date, the map consists of 649 contigs with an STS localized on average every 50 kb . This is the first whole genome that has been mapped based on a library constructed with large inserts in a vector that is maintained in Escherichia coli as a single-copy plasmid. J Cell Sci, 1996 May, 109 ( Pt 5), 1071 - 9 Activation of the Xenopus cyclin degradation machinery by full-length cyclin A; Jones C et al.; The entry into mitosis is dependent on the activation of mitotic forms of cdc2 kinase . In many cell types, cyclin A-associated kinase activity peaks just prior to that of cyclin B, although the precise role of cyclin A-associated kinase in the entry into mitosis is still unclear . Previous work has suggested that while cyclin B is capable of triggering cyclin destruction in Xenopus cell-free systems, cyclin A-associated kinase is not able to support this function . Here we have expressed a full-length human cyclin A in Escherichia coli and purified the protein to homogeneity by virtue of an N-terminal histidine tag . We have found that when added to Xenopus cell-free extracts free of cyclin B and incapable of protein synthesis, the temporal pattern of cyclin A-associated cdc2 kinase activity showed distinct differences that were dependent on the concentration of cyclin A added . When cyclin A was added to a concentration that generated levels of cdc2 kinase activity capable of inducing nuclear envelope breakdown, the histone H1 kinase activity profile was bi-phasic, consisting of an activation phase followed by an inactivation phase . Inactivation was found to be due to cyclin destruction, which was prevented by mos protein . Cyclin destruction was followed by nuclear reassembly and an additional round of DNA replication, indicating that there is no protein synthesis requirement for DNA replication in this embryonic system . It has been suggested that the evolutionary recruitment of cyclin A into an S phase function may have necessitated the loss of an original mitotic ability to activate the cyclin destruction pathway . The results presented here indicate that cyclin A has not lost the ability to activate its own destruction and that cyclin A-mediated activation of the cyclin destruction pathway permitted destruction of cyclin B1 as well as cyclin A, indicating that there are not distinct cyclin A and cyclin B destruction pathways . Thus the ordered progression of the cell cycle requires the careful titration of cyclin . A concentration in order to avoid activation of the cyclin destruction pathway before sufficient active cyclin B/cdc2 kinase has accumulated. Eur J Cell Biol, 1996 May, 70(1), 42 - 53 Immunodetection of alpha 1-3 fucosyltransferase (FucT-V); Borsig L et al.; The fucosyltransferases constitute a family of glycosyltransferases incorporating fucose residues into glycoprotein or glycolipid glycans . They afford one of the possible termination steps of glycoconjugate biosynthesis creating the sialyl Lewisx or sialyl Lewisa determinant, which play an important role in cell-cell interaction . While cDNA, chromosomal localization and kinetic properties of a number of fucosyltransferases are known, immunocytochemical localization and trafficking studies have been delayed because of the lack of specific antibodies due to the pronounced homology of alpha 1, 3 fucolsyltransferases III, V and VI . Here we report development and characterization of monospecific polyclonal antibodies to alpha 1-3 fucosyltransferase V (FucT-V) and their application for immunodetection in transfected cells . Antisera against FucT-V were raised in two different ways: first by producing a fusion protein beta-galactosidase-FucT-V in Escherichia coli, and by synthesizing a peptide stretch specific for FucT-V . Polyclonal antisera were raised against each of both antigens and characterized by enzyme-linked immunosorbent assay, neutralization of activity, immunoblotting, immunofluorescence and immunoprecipitation of metabolically labeled COS cells, transiently transfected with cDNA encoding FucT-V . Both antibodies recognized only FucT-V . No cross-reactivity to FucT-III or FucT-VI was observed . FucT-V was localized mainly to the Golgi apparatus by colocalization with beta 1, 4-galactosyltransferase, and to the cell surface of COS, CHO and HeLa cells . Expression of FucT-V in COS cells revealed three enzyme forms of 58, 53 and 50 kDa, respectively . These size differences arose by post-translational modifications, as shown by pulse-chase experiments . Our results indicate that alpha 1-3 fucosyltransferase is a Golgi-associated enzyme and suggest its possible occurrence on the cell surface. Inflamm Res, 1996 May, 45(5), 254 - 8 Nitric oxide enhances cyclooxygenase activity in articular cartilage; Manfield L et al.; Nitric oxide (NO) is a small messenger molecule synthesized by a family of enzymes, the nitric oxide synthases . Cyclooxygenases are a group of proinflammatory enzymes that release prostaglandins including prostaglandin E2 (PGE2) . Both nitric oxide synthase and cyclooxygenase are involved in the inflammatory cascade of arthritis . However, the relationship between these two enzymes and their products has not been explored in articular cartilage . Here we show that in cultured bovine chondrocytes and explants of human osteoarthritic cartilage both nitric oxide synthase and cyclooxygenase activities were induced by the inflammatory mediators, lipopolysaccharide, and interleukin-1 beta or tumor necrosis factor-alpha . When nitric oxide synthase activity was inhibited, PGE2, synthesis was inhibited . NO donors also induced PGE2 synthesis and NO scavengers inhibited cyclooxygenase activity . Taken together, these results support the concept that PGE2 synthesis is directly related to NO formation and that NO may modulate cyclooxygenase activity in articular cartilage. Inflamm Res, 1996 May, 45(5), 246 - 53 Human whole blood assays for inhibition of prostaglandin G/H synthases-1 and -2 using A23187 and lipopolysaccharide stimulation of thromboxane B2 production; Young JM et al.; When freshly drawn, heparinized human whole blood is incubated with 50 microM calcium ionophore A23187, platelets are stimulated to produce thromboxane B2 (TxB2) by activation of prostaglandin G/H synthase-l (PGHS-1) . TxB2 concentration, as measured by immunoassay, is maximal at 20-30 min and declines thereafter . Addition of acetylsalicylic acid (IC50 = 2.8 microM) or other nonsteroidal antiinflammatory drugs (NSAIDs) 15 min or 4.5h prior to 30 min stimulation with ionophore results in concentration dependent inhibition of TxB2 production . When blood is incubated with 0.01-10 micrograms/ml E . colilipopolysaccharide (LPS), PGHS-2 is induced and TxB2 levels become detectable at 3h and continue to increase through 24h . Using a 5h incubation with 10 micrograms/ml LPS, aspirin (10 microM added at 0 h), which is rapidly metabolized to salicylic acid, had no effect on 10 micrograms/ml LPS-induced TxB2, but inhibited TxB2 production by ionophore A23187 added at 4.5h through acetylation of pre-existing PGHS-1 . In a 5h assay, NSAIDs added at 0 h were compared for inhibition of TxB2 production stimulated by addition of ionophore A23187 at 4.5h (PGHS-1), or by addition of LPS at 0 h (PGHS-2) . Most NSAIDs were more potent against PGHS-1 than PGHS-2 . Diclofenac, naproxen and flufenamic acid were equipotent or slightly selective for PGHS-2 . Diflunisal and nimesulide were > 4-fold selective for PGHS-2, and NS-398 was > 30-fold selective for PGHS-2. Mol Microbiol, 1996 May, 20(3), 645 - 55 Identification and characterization of FrzZ, a novel response regulator necessary for swarming and fruiting-body formation in Myxococcus xanthus; Trudeau KG et al.; The frz genes of Myxococcus xanthus constitute a signal-transduction pathway that processes chemotactic information in a manner analogous to that found in enteric bacteria . Ultimately, these genes regulate the frequency of individual cell reversal . We report here the identification of a novel component of this signal-transduction pathway, designated frzZ, which was discovered as an open reading frame located 5' to the frz operon but transcribed in the opposite orientation . The translational start site of frzZ is 170 base pairs from that of frzA.frzZ utilizes a promoter similar to the sigma 70 promoters of Escherichia coli, and encodes a 290-amino-acid soluble protein, FrzZ (M(r) 30,500) . FrzZ contains two domains, both of which show strong homology to CheY and other members of the response-regulator family . Linking these domains is a 39-amino-acid region that is very rich in alanine and proline (38% Ala and 33% Pro) . A frzZ null mutant showed abnormally low reversal rates when compared to the wild-type control and was unable to form fruiting bodies on starvation medium, but it did form 'frizzy' aggregates . In addition, the frzZ mutant was defective in swarming, particularly on soft agar (0.3% w/v) . However, unlike most frz mutants, the frzZ mutant was able to respond to attractants and repellents in the spatial chemotaxis assay . The discovery of FrzZ demonstrates that the M . xanthus frz signal-transduction pathway utilizes multiple response-regulator (CheY-like) proteins. Mol Microbiol, 1996 May, 20(3), 621 - 32 Analysis of nitrate regulatory protein NarL-binding sites in the fdnG and narG operon control regions of Escherichia coli K-12; Darwin AJ et al.; During anaerobic growth, expression of the fdnGHI and narGHJI operons of Escherichia coli is induced by the NarL protein in response to nitrate . The fdnG operon control region contains four NarL-binding sites (termed NarL heptamers) between positions -70 and -130 . The two central NarL heptamers of fdnG are arranged as an inverted repeat and are essential for regulation by NarL . We used mutational analysis of these central heptamers to investigate the precise sequence requirements for NarL-dependent induction . Mutations were examined for their effects on NarL-dependent expression in vivo . Substitutions at position 1 of either heptamer had the strongest effect whereas substitutions at position 7 had the weakest effect . For some positions, alterations in both heptamers had a stronger effect than either of the single changes . The 2 bp spacing between these NarL heptamers was also important for normal nitrate induction . The narG operon control region has at least eight NarL heptamers arranged in two groups . Previous work has shown that nucleotide substitutions in two of these heptamers, centred at positions -195 and -89, severely reduce nitrate induction of narG operon expression in vivo and significantly interfere with NarL-DNA interactions in vitro . Substitutions in heptamers -185 and -101 affected narG operon induction only when the concentration of phospho-NarL was low (during growth in the presence of nitrite) . Changes in each of the other four NarL heptamers studied had little or no effect on nitrate or nitrite induction of narG operon expression or on NarL-DNA interactions in vitro. Mol Microbiol, 1996 May, 20(3), 613 - 20 Accessory proteins impose site selectivity during ColE1 dimer resolution; Guhathakurta A et al.; The cer-Xer dimer resolution system of plasmid ColE1 is highly selective, acting only at sites on the same molecule and in direct repeat . Recombination requires the XerCD recombinase and accessory proteins ArgR and PepA . The Escherichia coli chromosome dimer resolution site dif and the type II hybrid site use the same recombinase but are independent of ArgR and PepA and show no site selectivity . This has led to the proposal that ArgR and PepA are responsible for the imposition of constraint . We describe here the characterization of a novel class of "conditionally constrained' multimer resolution sites whose properties support this hypothesis . In the presence of ArgR and PepA, plasmids containing conditionally constrained sites are monomeric, but in their absence, extensive multimerisation is seen . A mutant ArgR derivative (ArgR110), which is defective in cer-mediated dimer resolution, remains able to prevent plasmid multimerisation by a conditionally constrained site . This implies that the accessory factors block recombination in trans rather than ensuring rapid multimer resolution . When the distance between the ArgR and XerCD binding sites in a conditionally constrained site was altered by a non-integral number of helical turns, the site became unconstrained . Constraint was restored by the insertion of a full helical turn. Mol Microbiol, 1996 May, 20(3), 549 - 58 Regulation of the expression of the traM gene of the F sex factor of Escherichia coli; Penfold SS et al.; Conjugative F-plasmid transfer is mediated by the transfer (tra) region which encodes nearly 40 genes, 25 of which are essential for this process in Escherichia coli . TraM is required for conjugation and is encoded on a separate operon between the origin of transfer and the traJ gene . The traJ gene product is the positive regulator of transcription of the 30 kb tra operon, the first gene of which is traY . Using primer-extension assays and immunoblots on the F plasmid itself and its derivatives, we demonstrate that F TraM regulates its own expression from two promoters and that it requires TraY as well as expression of the tra operon for maximal traM transcription . traY is the first gene in the tra operon under the control of the TraJ regulator, which is in turn negatively regulated by the antisense RNA, FinP, and the FinO protein . Thus, a control circuit has been established whereby traM is negatively regulated by the FinOP fertility inhibition system through its repression of TraJ expression, which adversely affects transcription of the traY gene. Mol Microbiol, 1996 May, 20(3), 489 - 96 Phosphorylation-dependent binding of BvgA to the upstream region of the cyaA gene of Bordetella pertussis; Karimova G et al.; In Bordetella pertussis, transcription of virulence-associated genes is regulated by the BvgS and BvgA proteins, members of the bacterial two-component signal-transduction family . BvgS is the transmembrane sensor and BvgA, in its phosphorylated form, is believed to be the key transcriptional activator in B . pertussis . However, the BvgA recognition sites in most virulence promoters have not yet been identified . To investigate the interaction of BvgA with the upstream region of cyaA, the gene encoding adenylate cyclase haemolysin, we have produced large amounts of BvgA in Escherichia coli . The protein was purified from inclusion bodies and then phosphorylated by acetyl phosphate . Using electrophoretic mobility-shift and footprinting assays, we provide evidence that BvgA cannot bind to the cyaA promoter unless it is phosphorylated . The phosphorylated form of BvgA (BvgA-P) is able to bind specifically to the upstream region of cyaA . Analysis of this region revealed that an unexpectedly large sequence, from -137 to -51, appears to be the target for BvgA-P binding, and probably contains multiple binding sites. Mol Microbiol, 1996 May, 20(3), 467 - 73 Very short patch repair: reducing the cost of cytosine methylation; Lieb M et al.; In Escherichia coli and related bacteria, the product of gene dcm methylates the second cytosine of 5'-CCWGG sequences (where W is A or T) . Deamination of 5-methylcytosine (5meC) results in C to T mutations . The mutagenic potential of 5meC is reduced by a system called very short patch (VSP) repair, which replaces T with C . T:G and U:G mispairs in the methylatable sequence and in related sequences are recognized by the product of vsr, a gene adjacent to dcm . Vsr creates a nick just 5' of the mispaired pyrimidine to initiate the repair . Additional products known to be required for VSP repair are DNA polymerase I and DNA ligase . MutS and MutL have a stimulatory role but are not required . The ability of Vsr to recognize T:G mispairs in sequences related to CCWGG is probably responsible for over- and under-representation of certain tetranucleotides in the E . coli genome . Although VSP repair reduces spontaneous mutations at 5meCs in replicating bacteria, mutation hot-spots persist at these sites . Under conditions that more accurately mimic the natural environment of E . coli, VSP repair appears to be effective in preventing mutation at 5meC. Br J Pharmacol, 1996 May, 118(1), 198 - 204 Effects of the endothelin receptor antagonist, SB 209670, on circulatory failure and organ injury in endotoxic shock in the anaesthetized rat; Ruetten H et al.; 1 . This study investigates the effects of the non-selective ETA/ETB receptor antagonist, SB 209670, on systemic haemodynamics, renal function, liver function, acid-base balance and survival in a rat model of endotoxic shock . 2 . Injection of E . coli lipopolysaccharide (LPS, 10 mg kg-1, i.v.) resulted in increases in the serum levels of tumour necrosis factor-alpha (TNF-alpha, maximum 60 min after LPS), endothelin-1, (ET-1; maximum 120 min after LPS), and interferon-gamma (IFN-gamma, maximum 180 min after LPS) . 3 . Injection of LPS also resulted in a fall in blood pressure from 113 +/- 3 mmHg (time = 0) to 84 +/- 4 mmHg at 360 min (n = 15) as well as a hyporeactivity to the vasoconstrictor responses elicited by noradrenaline (NA, 1 microgram kg-1, i.v.) . Pretreatment of rats with a continuous infusion of SB 209670 (3 mg kg-1, i.v . bolus + 100 micrograms kg-1, i.v . infusion commencing 15 min prior to LPS) significantly augmented the hypotension as well as the vascular hyporeactivity to NA caused by endotoxaemia . 4 . Pretreatment of LPS-rats with SB 209670 (3 mg kg-1, i.v . bolus given 15 min prior to LPS) or infusion of SB 209670 (bolus dose and infusion as above) resulted in a reduction in 6 h-survival from 71% (control) to 30% and 13%, respectively . 5 . Endotoxaemia for 4 h resulted in rises in the serum levels of urea and creatinine (indicators of renal failure), but not in the serum levels of bilirubin, GPT and GOT (indicators of liver dysfunction and/or hepatocellular injury) . Pretreatment of LPS-rats with SB 209670 (3 mg kg-1, i.v . bolus 15 min prior to LPS) significantly augmented the serum levels of creatinine, bilirubin, GPT and GOT caused by endotoxin . In addition, endotoxaemia caused, within 15 min, an acute metabolic acidosis (falls in pH, HCO3- and base excess) which was compensated by hyperventilation (fall in PaCO2) . Pretreatment of LPS-rats with SB 209670 (3 mg kg-1, i.v . bolus) significantly augmented the metabolic acidosis caused by LPS . 6 . Thus, the non-selective ETA/ETB receptor antagonist, SB 209670, augments the degree of (i) hypotension, (ii) vascular hyporeactivity to noradrenaline, (iii) renal dysfunction and (iv) metabolic acidosis caused by endotoxin in the anaesthetized rat . In contrast to rats treated with LPS alone, LPS-rats treated with SB 209670 exhibited liver dysfunction and hepatocellular injury . We propose that the release of endogenous ET-1 serves to maintain blood pressure and subsequently organ perfusion in septic shock. Br J Pharmacol, 1996 May, 118(1), 179 - 85 Nitric oxide synthase-cyclo-oxygenase pathways in organum vasculosum laminae terminalis: possible role in pyrogenic fever in rabbits; Lin JH et al.; 1 . Fever was induced in rabbits by administration of Escherichia coli endotoxin (lipopolysaccharide; LPS; 0.001-10 micrograms) into the organum vasculosum laminae terminalis (OVLT) . Deep body temperature was evaluated over a period of 7 h . 2 . The LPS-induced febrile response was mimicked by intra-OVLT injection of the nitric oxide (NO) donors, S-nitroso-acetylpenicillamine (SNAP, 1-10 micrograms), sodium nitroprusside (SNP, 50 micrograms), or hydroxylamine (10 micrograms), the cyclic GMP analogue 8-bromo-cyclic GMP (8-Br-cyclic GMP, 10-100 micrograms), or prostaglandin E2 (PGE2, 0.2 micrograms) . 3 . Dexamethasone (Dex, a potent inhibitor of the transcription of inducible NO synthase, iNOS, 10 micrograms), anisomycin (a protein synthesis inhibitor, 100 micrograms), L-N5-(1-iminoethyl)ornithine (L-NIO; an irreversible NOS inhibitor, 10-200 micrograms), aminoguanidine (a specific iNOS inhibitor, 1000 micrograms), or NG-methyl-L-arginine acetate (L-NMMA, a NOS inhibitor, 100 micrograms) inhibited fever induced by LPS when injected into the OVLT 1 h before LPS injection . An intra-OVLT dose of 1000 micrograms of NG-nitro-L-arginine methyl ester (L-NAME, a potent inhibitor of constitutive NOS) did not exhibit antipyretic effects . 4 . Methylene blue (an inhibitor of NOS and soluble guanylate cyclase, 1-10 micrograms), 6-(phenylamino)-5,8-quinolinedione (LY-83583; an inhibitor of soluble guanylate cyclase and NO release, 20 micrograms), or indomethacin (an inhibitor of cyclo-oxygenase, COX, 400 micrograms) inhibited fever induced by LPS when injected into the OVLT 1 h before LPS injection . Pretreatment with methylene blue or haemoglobin (a NO scavenger, 100 micrograms) attenuated the fever induced by intra-OVLT injection of SNAP . 5 . The PGE2-induced fever was potentiated, rather then attenuated, by pretreatment with an intra-OVLT dose of animoguanidine (1000 micrograms), L-NMMA (100 micrograms) or L-NIO (200 micrograms) . 6 . These results suggest that iNOS-COX pathways in the OVLT represent an important mechanism for modulation of pyrogenic fever in rabbits. Protein Sci, 1996 May, 5(5), 914 - 22 A novel family of phospholipase D homologues that includes phospholipid synthases and putative endonucleases: identification of duplicated repeats and potential active site residues; Ponting CP et al.; Phosphatidylcholine-specific phospholipase D (PLD) enzymes catalyze hydrolysis of phospholipid phosphodiester bonds, and also transphosphatidylation of phospholipids to acceptor alcohols . Bacterial and plant PLD enzymes have not been shown previously to be homologues or to be homologous to any other protein . Here we show, using sequence analysis methods, that bacterial and plant PLDs show significant sequence similarities both to each other, and to two other classes of phospholipid-specific enzymes, bacterial cardiolipin synthases, and eukaryotic and bacterial phosphatidylserine synthases, indicating that these enzymes form an homologous family . This family is suggested also to include two Poxviridae proteins of unknown function (p37K and protein K4), a bacterial endonuclease (nuc), an Escherichia coli putative protein (o338) containing an N-terminal domain showing similarities with helicase motifs V and VI, and a Synechocystis sp . putative protein with a C-terminal domain likely to possess a DNA-binding function . Surprisingly, four regions of sequence similarity that occur once in nuc and o338, appear twice in all other homologues, indicating that the latter molecules are bi-lobed, having evolved from an ancestor or ancestors that underwent a gene duplication and fusion event . It is suggested that, for each of these enzymes, conserved histidine, lysine, aspartic acid, and/or asparagine residues may be involved in a two-step ping pong mechanism involving an enzyme-substrate intermediate. Protein Sci, 1996 May, 5(5), 904 - 13 Enzymatic and fluorescence studies of four single-tryptophan mutants of rat testis fructose 6-phosphate,2-kinase:fructose 2,6-bisphosphatase; Watanabe F et al.; In order to determine environments around four tryptophan residues, located in the N-terminus, in the kinase and in the phosphatase domains of rat testis Fru 6-P,2-kinase:Fru 2,6-bisphosphatase, mutant enzymes containing a single tryptophan were constructed by site-directed mutagenesis . The kinetic constants of these mutant enzymes were similar to those of the wild-type enzyme . The sum of the fluorescence intensities of the enzymes was 1.5 x that of the wild-type enzyme, and Trp 299, Trp 64, Trp 15, and Trp 320 contributed 38%, 28%, 17%, and 17%, respectively . The fluorescence polarization of the wild-type enzyme was significantly lower than any of the mutant enzymes, suggesting proximity of two tryptophan residues in the wild-type enzyme . The polarization in the presence of Fru 6-P affected only Trp 15, which suggested that it is located near the Fru 6-P binding site, but Trp 64 is not . Inactivation of both enzyme activities and unfolding of these enzymes in guanidine were monitored by activity assays and fluorescence intensities and maxima . Both Fru 6-P,2-kinase and Fru 2,6-bisphosphatase activities of all these enzymes were inactivated between 0.7 and 1 M guanidine . Enzymes containing Trp 64 or Trp 15 showed biphasic fractional unfolding curves, but those of Trp 299 or Trp 320 showed gradual steady changes . Fluorescence quenching by iodide indicated that Trp 64 was not accessible and that other Trp residues were only slightly accessible to solvent . These results suggest that all the Trp residues are in heterogeneous environments and that none are exposed on the protein surface. Protein Sci, 1996 May, 5(5), 814 - 24 Sequence replacements in the central beta-turn of plastocyanin; Ybe JA et al.; The role of beta-turns in dictating the structure of a beta-barrel protein is assessed by probing the tolerance of the central beta-turn of poplar plastocyanin to substitution by arbitrary sequences . Native plastocyanin binds copper and is colored bright blue . However, when the wild-type Pro47-Ser48-Gly49-Val50 turn sequence is replaced by arbitrary tetrapeptides, the vast majority (92/98 = 94%) of mutant proteins cannot fold into the native blue structure . Characterization of the colorless mutant proteins demonstrates that the majority of substitutions in this type II beta-turn disrupt the native structure severely . Gross structural changes are indicated by major differences in the CD spectra of the mutants relative to the wild-type protein, and by the much larger apparent size of mutant proteins in gel filtration experiments . These mutant proteins do not bind copper . Furthermore, Cys84 forms a disulfide bond readily in the colorless mutant proteins, indicating that it has moved away from the buried position it occupies in the native copper binding site and has become exposed . These results indicate that the central beta-turn in plastocyanin is not merely a default structure arising in response to the surrounding context; rather, sequence information in this turn plays an active role in dictating the location of a chain reversal in the beta-barrel structure . These findings are discussed in terms of their implications for the folding of natural proteins, as well as the design of de novo proteins. J Inorg Biochem, 1996 May 1, 62(2), 89 - 102 Reactivity of the N-terminal cysteine residues in active and inactive forms of FNR, and O2-responsive, Fe containing transcriptional regulator of Escherichia coli; Six S et al.; FNR, the O2-responsive gene regulator of anaerobic respiratory genes in Escherichia coli, contains an N-terminal cluster of four cysteine residues (Cys16-X3-Cys20-X2-Cys23-X5-Cys29), three of which are thought to be involved in the binding of an iron cofactor . The accessibility of the cysteine residues for iodoacetate is known to increase upon switch from the active (anaerobic) to the inactive (aerobic or metal depleted) state . It was analyzed which residues become accessible under either condition . Up to four modified forms, FNR-I, FNR-II, FNR-III, and FNR-IV, containing approximately 1, 2, 3.5, and 5 carboxymethyl groups, were obtained either by reaction in vivo and in vitro under conditions of aerobiosis, anaerobiosis, or iron limitation . By N-terminal sequencing, the carboxymethylated cysteine residues were identified . The amount of label in each of the four cysteine residues increased rather uniformly and gradually from FNR-I to FNR-IV irrespective of the condition of labeling; only Cys16 was preferentially labeled to some extent . It is concluded that the four essential cysteine residues change their accessibility in a similar way in the switch from active to inactive (aerobic or metal depleted) FNR, without specific differences in their reaction or function . Potential modes of Fe-binding by the cysteine residues are discussed . In addition, a different type of interaction of Fe(II) with FNR is described . The interaction occurred also in FNR carboxymethylated at approximately three cysteine residues. Proteins, 1996 May, 25(1), 139 - 41 Crystallization and preliminary X-ray analysis of Escherichia coli methionyl-tRNA(fMet) formyltransferase; Schmitt E et al.; Methionyl-tRNA(fMet) formyltransferase from Escherichia coli, a monomer of 34kDa, was overexpressed from its cloned gene fmt (Guillon, J.M., Mechulam, Y., Schmitter, J.M., Blanquet, S., and Fayat, G., J . Bacteriol . 174:4294-4301, 1992) and crystallized using ammonium sulphate as precipitant . The crystals are trigonal and have unit cell parameters a = b = 151.0 A, c = 81.8 A . They belong to space group P3(2)21 and diffract to 2.0 A resolution . The structure is being solved by multiple isomorphous replacement. Proteins, 1996 May, 25(1), 137 - 8 Crystallization and preliminary crystallographic analysis of RepA1, a replication control protein of the RepFIC replicon of enterotoxin plasmid EntP307; Song H et al.; RepA1 protein is essential for replication of the RepFIC replicon of enterotoxin plasmid EntP307 and is thought to interact directly with the origin of replication . We have purified RepA1 from an over-producing expression system and have prepared single crystals using a macroseeding technique . The crystals belong to space group P2(1)2(1)2(1) or P2(1)2(1)2, with cell dimensions a = 61 A, b = 67 A, and c = 243 A . They diffract X-rays to 3.3 A resolution and probably contain two 40,000 molecular weight RepA1 molecules per asymmetric unit. Proteins, 1996 May, 25(1), 112 - 9 Purification, stabilization, and crystallization of a modular protein: Grb2; Guilloteau JP et al.; We report here the purification and the crystallization of the modular protein Grb2 . The protein was expressed as a fusion with glutathione-S-transferase and purified by affinity chromatography on glutathione agarose . It was apparent from reverse phase chromatography that the purified protein was conformationally unstable . Instability was overcome by the addition of 100 mM arginine to the buffers . Because Grb2 appeared to be extremely sensitive to oxidation, crystallization experiments were performed with a dialysis button technique involving daily addition of fresh DTT to the reservoirs . The presence of 8 to 14% glycerol was necessary to obtain monocrystals . These results are discussed in relation with the modular nature of Grb2. Proteins, 1996 May, 25(1), 79 - 88 Study of global motions in proteins by weighted masses molecular dynamics: adenylate kinase as a test case; Elamrani S et al.; The weighted masses molecular dynamics (WMMD) technique is applied to the protein adenylate kinase . A novel set of restraints has been developed to allow the use of this technique with proteins . The WMMD simulation is successful in predicting the flexibility of the two mobile domains of the protein . The end product of the simulation is similar to the known open and AMP bound forms of the enzyme . The biological relevance of the restraints used and potential methods of improving the technique are discussed. Proteins, 1996 May, 25(1), 48 - 78 The three-dimensional structure of Escherichia coli porphobilinogen deaminase at 1.76-A resolution; Louie GV et al.; Porphobilinogen deaminase (PBGD) catalyses the polymerization of four molecules of porphobilinogen to form the 1-hydroxymethylbilane, preuroporphyrinogen, a key intermediate in the biosynthesis of tetrapyrroles . The three-dimensional structure of wild-type PBGD from Escherichia coli has been determined by multiple isomorphous replacement and refined to a crystallographic R-factor of 0.188 at 1.76 A resolution . the polypeptide chain of PBGD is folded into three alpha/beta domains . Domains 1 and 2 have a similar overall topology, based on a five-stranded, mixed beta-sheet . These two domains, which are linked by two hinge segments but otherwise make few direct interactions, form an extensive active site cleft at their interface . Domain 3, an open-faced, anti-parallel sheet of three strands, interacts approximately equally with the other two domains . The dipyrromethane cofactor is covalently attached to a cysteine side-chain borne on a flexible loop of domain 3 . The cofactor serves as a primer for the assembly of the tetrapyrrole product and is held within the active site cleft by hydrogen-bonds and salt-bridges that are formed between its acetate and propionate side-groups and the polypeptide chain . The structure of a variant of PBGD, in which the methionines have been replaced with selenomethionines, has also been determined . The cofactor, in the native and functional form of the enzyme, adopts a conformation in which the second pyrrole ring (C2) occupies an internal position in the active site cleft . On oxidation, however, this C2 ring of the cofactor adopts a more external position that may correspond approximately to the site of substrate binding and polypyrrole chain elongation . The side-chain of Asp84 hydrogen-bonds the hydrogen atoms of both cofactor pyrrole nitrogens and also potentially the hydrogen atom of the pyrrole nitrogen of the porphobilinogen molecule bound to the proposed substrate binding site . This group has a key catalytic role, possibly in stabilizing the positive charges that develop on the pyrrole nitrogens during the ring-coupling reactions . Possible mechanisms for the processive elongation of the polypyrrole chain involve: accommodation of the elongating chain within the active site cleft, coupled with shifts in the relative positions of domains 1 and 2 to carry the terminal ring into the appropriate position at the catalytic site; or sequential translocation of the elongating polypyrrole chain, attached to the cofactor on domain 3, through the active site cleft by the progressive movement of domain 3 with respect to domains 1 and 2 . Other mechanisms are considered although the amino acid sequence comparisons between PBGDs from all species suggest they share the same three-dimensional structure and mechanism of activity. Cancer Gene Ther, 1996 May-Jun, 3(3), 163 - 7 Recombinant vaccinia expressing interleukin-2 for cancer gene therapy; Qin H et al.; Use of a recombinant vaccinia virus expressing human interleukin-2 (IL-2) was evaluated for preparation of tumor vaccines . A/J mice were immunized against neuroblastoma (C1300) cells using a preparation of C1300 cells infected/transfected with the recombinant virus, vCF13, expressing IL-2 . A second recombinant vaccinia, vSC8, expressing Escherichia coli beta-galactosidase, was used as a control . After three weekly immunizations with virus-transfected cells, the mice were challenged with 1 x 10(6) unmodified C1300 cells and tumor development was monitored . Tumor development in the mice was inhibited by immunization with vCF13-transfected cells, compared to those vaccinated with vSC8-transfected cells (P < .008) . A group of mice (7/15) immunized with vCF13-transfected cells followed by tumor challenge survived more than 60 days, at which time all mice immunized with the control vaccine were dead (p < .006) . Five of the mice treated with the vCF13 vaccine were alive for more than 75 days (P < .05), after which they were rechallenged with another dose of 1 x 10(6) unmodified tumor cells . Tumor development was not apparent in these mice for more than 45 days following the second challenge, suggesting that these mice were completely protected by this immunization . These results demonstrate that recombinant vaccinia virus expressing IL-2 may be useful for cancer gene therapy. Cancer Gene Ther, 1996 May-Jun, 3(3), 155 - 62 Escherichia coli gpt gene sensitizes rat glioma cells to killing by 6-thioxanthine or 6-thioguanine; Tamiya T et al.; Genes that encode enzymes that convert inactive "prodrugs" into anticancer metabolites may be therapeutically useful against brain tumors . Unlike other genes tested to date in brain tumor models, the Escherichia coli gpt gene is unique in that it not only sensitizes cells to the prodrug 6-thioxanthine (6TX) but also encodes resistance to a different regimen (mycophenolic acid, xanthine, and hypoxanthine), thus providing a means to select for gpt-positive cells . In the present study, rat C6 glioma cells were infected with a retrovirus vector that transduces this gene . A clonal line (C6GPT-7) was derived that exhibited significant 6TX susceptibility in vitro with an ID50 of 2.5 mumol/L, whereas 50% growth inhibition of parental C6 cells was not achieved at concentrations tested (up to 50 mumol/L) . This line also exhibited significant sensitivity to 6-thioguanine (6TG), with an ID50 of 0.05 mumol/L, whereas 50% growth inhibition of parental C6 cells was achieved at 0.5 mumol/L . In a "bystander" assay, C6GPT-7 tumor cells efficiently transferred 6TX sensitivity to C6 cells at ratios as low as 1:9 (C6GPT-7:C6) . This in vitro bystander effect was abrogated when C6GPT-7 and C6 cells were separated by a microporous membrane, suggesting that it was not mediated by highly diffusible metabolites . In vivo both 6TX and 6TG significantly inhibited the growth of subcutaneously transplanted C6GPT-7 cells but not that of C6 cells in athymic mice . In an intracerebral model, both 6TX and 6TG exhibited significant antiproliferative effects against tumors formed by C6GPT-7 cells . These findings provide a basis for exploring further gene therapy strategies based on in vivo transfer of the E coli gpt gene to provide chemosensitivity against 6TX and 6TG. Biotechniques, 1996 May, 20(5), 862 - 4, 866-8 Sequencing homopolymer tracts and repetitive elements; Robbins CM et al.; We investigated the use of Taq dye primer and Taq terminator sequencing chemistry to optimize the quality of sequence data obtained from templates containing homopolymer tracts and repetitive elements . In direct side-by-side comparisons using the Applied Biosystems Model 373A Fluorescent Sequencer, the Taq terminator sequencing chemistry gave much cleaner and more consistent results on long homopolymer tracts and dinucleotide repeats . We also investigated various thermal cycling conditions and determined that higher annealing temperatures and longer denaturation times improved the ability to sequence through these problem templates. Am J Vet Res, 1996 May, 57(5), 659 - 63 Attempt to pharmacologically modulate procoagulant activity of lipopolysaccharide-stimulated adherent bovine alveolar macrophages; Olchowy TW et al.; OBJECTIVE--To investigate the effects of anti-inflammatory drugs on lipopolysaccharide-induced procoagulant activity of bovine alveolar macrophages . DESIGN--Procoagulant activity was induced in bovine alveolar macrophages from 4 healthy Holstein calves aged 6 to 16 weeks by incubation with lipopolysaccharide . 3 anti-inflammatory drugs were used at 4 concentrations and 3 times to pretreat the alveolar macrophages . Results were analyzed to determine whether drug, concentration, or exposure period had a significant (P > 0.05) effect . PROCEDURE--Bovine alveolar macrophages, harvested by volume-controlled bronchoalveolar lavage, were pretreated for 30, 60, or 120 minutes with an anti-inflammatory compound (dexamethasone, flunixin meglumine, or phenylbutazone) at several concentrations ( 0, 1, 10, and 100 microM) . Bovine alveolar macrophages were exposed to lipopolysaccharide (Escherichia coli O55:B5) in the presence and absence of fetal bovine serum for 4 hours . Procoagulant activity was measured, using a chromogenic assay . RESULTS--None of the drugs was associated with a modification of procoagulant activity expression . CONCLUSION--Use of these 3 anti-inflammatory drugs is unlikely to modify the extent of the fibrinous reaction commonly observed in cases of acute bovine respiratory tract disease complex . CLINICAL RELEVANCE--The alveolar macrophage has a key role in fibrin production . Assuming in vivo events mimic the in vitro model, is appears unlikely that administration of anti-inflammatory drugs will reduce the procoagulant activity of the bovine alveolar macrophages and the directly associated pulmonary fibrosis. J Neurosci Res, 1996 May 1, 44(3), 235 - 42 Lipopolysaccharide stimulates differential expression of myristoylated protein kinase C substrates in murine microglia; Rose SD et al.; Microglia rapidly respond to lipoplysaccharide (LPS) by transformation from resting to active states and secretion of several neuro- and immuno-regulators including tumour necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta), and interleukin 6 (IL-6) . With longer LPS treatment, microglia are converted to reactive or phagocytic states with characteristics similar to macrophages in inflammation and injury processes . We have investigated LPS-mediated changes in two myristoylated substrates of protein kinase C (PKC): MARCKS (myristoylated alaninerich C kinase substrate) and MRP (MARCKS-related protein) . Within 6 hours of addition, LPS induced a twofold increase in {3H}myristoylated and immunoreactive MARCKS protein and a sevenfold increase in MRP . The differential effect of LPS on expression of MRP vs . MARCKS was even more dramatic at the level of transcription: S1 nuclease protection assays revealed a 40-fold increase in MRP mRNA levels (maximum at 4-6 hours), whereas a threefold increase was observed for MARCKS . TNF alpha and colony-stimulating factor 1 (CSF-1), two cytokines which are induced by LPS, did not reproduce the observed effect of LPS on MARCKS and MRP gene transcription . CSF-1 also induced differential transcription of MRP, but of lower magnitude (threefold) and more sustained than by LPS . Accordingly, these two substrates for PKC are differentially up-regulated by LPS, apparently independent of TNF alpha or CSF-1. Genetics, 1996 May, 143(1), 27 - 36 Orientation dependence in homologous recombination; Yamamoto K et al.; Homologous recombination was investigated in Escherichia coli with two plasmids, each carrying the homologous region (two defective neo genes, one with an amino-end deletion and the other with a carboxyl-end deletion) in either direct or inverted orientation . Recombination efficiency was measured in recBC sbcBC and recBC sbcA strains in three ways . First, we measured the frequency of cells carrying neo+ recombinant plasmids in stationary phase . Recombination between direct repeats was much more frequent than between inverted repeats in the recBC sbcBC strain but was equally frequent in the two substrates in the recBC sbcA strain . Second, the fluctuation test was used to exclude bias by a rate difference between the recombinant and parental plasmids and led to the same conclusion . Third, direct selection for recombinants just after transformation with or without substrate double-strand breaks yielded essentially the same results . Double-strand breaks elevated recombination in both the strains and in both substrates . These results are consistent with our previous findings that the major route of recombination in recBC sbcBC strains generates only one recombinant DNA from two DNAs and in recBC sbcA strains generates two recombinant DNAs from two DNAs. Genetics, 1996 May, 143(1), 15 - 26 Long-term experimental evolution in Escherichia coli . IV . Targets of selection and the specificity of adaptation; Travisano M et al.; This study investigates the physiological manifestation of adaptive evolutionary change in 12 replicate populations of Escherichia coli that were propagated for 2000 generations in a glucose-limited environment . Representative genotypes from each population were assayed for fitness relative to their common ancestor in the experimental glucose environment and in 11 novel single-nutrient environments . After 2000 generations, the 12 derived genotypes had diverged into at least six distinct phenotypic classes . The nutrients were classified into four groups based upon their uptake physiology . All 12 derived genotypes improved in fitness by similar amounts in the glucose environment, and this pattern of parallel fitness gains was also seen in those novel environments where the limiting nutrient shared uptake mechanisms with glucose . Fitness showed little or no consistent improvement, but much greater genetic variation, in novel environments where the limiting nutrient differed from glucose in its uptake mechanisms . This pattern of fitness variation in the novel nutrient environments suggests that the independently derived genotypes adapted to the glucose environment by similar, but not identical, changes in the physiological mechanisms for moving glucose across both the inner and outer membranes. Genetics, 1996 May, 143(1), 5 - 13 Differential suppression of priA2::kan phenotypes in Escherichia coli K-12 by mutations in priA, lexA, and dnaC; Sandler SJ et al.; First identified as an essential component of the phi X174 in vitro DNA replication system, PriA has ATPase, helicase, translocase, and primosome-assembly activities . priA1::kan strains of Escherichia coli are sensitive to UV irradiation, deficient in homologous recombination following transduction, and filamentous . priA2::kan strains have eightfold higher levels of uninduced SOS expression than wild type . We show that (1) priA1::kan strains have eightfold higher levels of uninduced SOS expression, (2) priA2::kan strains are UVS and Rec-, (3) lexA3 suppresses the high basal levels of SOS expression of a priA2::kan strain, and (4) plasmid-encoded priA300 (K230R), a mutant allele retaining only the primosome-assembly activity of priA+, restores both UVR and Rec+ phenotypes to a priA2::kan strain . Finally, we have isolated 17 independent UVR Rec+ revertants of priA2::kan strains that carry extragenic suppressors . All 17 map in the C-terminal half of the dnaC gene . DnaC loads the DnaB helicase onto DNA as a prelude for primosome assembly and DNA replication . We conclude that priA's primosome-assembly activity is essential for DNA repair and recombination and that the dnaC suppressor mutations allow these processes to occur in the absence of priA. Gut, 1996 May, 38(5), 747 - 52 Lipopolysaccharide induced apoptosis of rat pancreatic acinar cells; Laine VJ et al.; BACKGROUND--Bacterial lipopolysaccharide (LPS) has been proposed to participate in the pathogenesis of pancreatic inflammatory disease . AIMS--This study investigated the role of endotoxaemia in the pathogenesis of pancreatic acinar cell injury . METHODS--Sixty eight male Spraque-Dawley rats were used in the study . Escherichia coli LPS (5 mg/kg) was injected into the peritoneal cavity of the rats . The concentration of pancreatic phospholipase A2 (PLA2) in plasma was measured and pancreatic tissue examined by histology, in situ detection of free DNA 3'-ends, and electrophoretic DNA analysis . RESULTS--The concentration of pancreatic PLA2 increased in plasma and the catalytic activity of PLA2 increased in pancreatic tissue after an LPS injection . Apoptosis in pancreatic acinar cells and fragmentation of DNA typical of apoptosis in pancreatic tissue was seen 24 hours after an LPS injection . Pancreatic acinar atrophy was seen 72 hours after the LPS injection . CONCLUSIONS--These data show that LPS causes release of pancreatic PLA2 into blood plasma, activation of PLA2 in pancreatic tissue, and apoptosis of acinar cells. Crit Care Med, 1996 May, 24(5), 807 - 14 Effect of human hemoglobin on systemic and regional hemodynamics in a porcine model of endotoxemic shock; Aranow JS et al.; OBJECTIVE: Excessive release of nitric oxide has been implicated as being an important factor contributing to systemic arterial hypotension in septic shock . Hemoglobin is an effective nitric oxide scavenger . The purpose of this study was to test the hypothesis that treatment with cross-linked human hemoglobin can ameliorate systemic arterial hypotension and improve organ perfusion in a porcine model of normodynamic endotoxemic shock . DESIGN: Prospective, randomized, controlled trial . SETTING: Laboratory at a university medical center . SUBJECTS: Fourteen, male, random-bred swine . INTERVENTIONS: All animals were challenged with Escherichia coli lipopolysaccharide (400 microg/kg) infused from t = 0 to 90 mins . Pigs in group 1 (n = 7) were infused with cross-linked human hemoglobin (150 mg/kg) at t = 30 mins . Pigs in group 2 (n = 7) were infused at t = 30 mins with 150 mg/kg of dextran (average molecular weight 70,000 daltons) as a 5% (weight per volume) solution . MEASUREMENTS AND MAIN RESULTS: After infusion of endotoxin, mean arterial pressure decreased significantly (p < .05) but baseline cardiac index was maintained in both groups . In hemoglobin-treated pigs (group 1), mean arterial pressure was higher than in controls (group 2) from t = 60 to 120 mins (p < .05) . There were no significant differences between the two groups in systemic vascular resistance index, renal blood flow, mesenteric blood flow, systemic oxygen delivery, or systemic oxygen extraction . Ileal mucosal blood flow was lower (p < .07) in group 1 than in group 2 . Mean pulmonary arterial pressure increased relative to baseline in both groups, but was significantly greater in group 1 as compared with group 2 . Compared with controls, infusion of hemoglobin significantly exacerbated endotoxin-induced arterial hypoxemia (p < .05) . CONCLUSIONS: Treatment with hemoglobin improved mean arterial pressure in endotoxemic swine without significantly impairing blood flow to the renal or mesenteric vascular beds . Infusion of hemoglobin, however, significantly exacerbated endotoxin-induced pulmonary hypertension and arterial hypoxemia . Additional pharmacologic strategies may be necessary to ameliorate the potential adverse pulmonary effects of administering hemoglobin solutions to patients with sepsis. Clin Diagn Lab Immunol, 1996 May, 3(3), 355 - 7 Recombinant Toxoplasma gondii surface antigen 1 (P30) expressed in Escherichia coli is recognized by human Toxoplasma-specific immunoglobulin M (IgM) and IgG antibodies; Harning D et al.; The immunodominant surface antigen of Toxoplasma gondii, surface antigen 1 (SAG1), was expressed in Escherichia coli as a fusion protein containing a majority of the SAG1 protein supplied with six histidyl residues in the N-terminal end . The recombinant protein was purified on a Ni-chelate column and then on a fast-performance liquid chromatography column and was in a nonreduced condition . It was recognized by T . gondii-specific human immunoglobulin G (IgG) and IgM antibodies as well as by a mouse monoclonal antibody (S13) recognizing only nonreduced native SAG1 . Antibodies induced in mice by the recombinant SAG1 recognized native SAG1 from the T . gondii RH isolate in culture . Recombinant SAG1 is suitable for use in diagnostic systems for detecting anti-SAG1-specific IgG and IgM antibodies. Microbiology, 1996 May, 142 ( Pt 5), 1141 - 8 Reactions of the Escherichia coli flavohaemoglobin (Hmp) with NADH and near-micromolar oxygen: oxygen affinity of NADH oxidase activity; Poole RK et al.; The soluble flavohaemoglobin (Hmp) of Escherichia coli, product of the hmp gene, contains haem B and FAD in a single polypeptide of molecular mass 44 kDa . The function of this protein (and of the similar proteins identified in several bacteria and yeast) is unknown, but the observation that the binding of oxygen to haem modulates the reduction level of FAD has suggested that Hmp could act as an oxygen sensor . Here, stopped-flow, rapid-scan spectroscopy has shown that the oxidized protein reacts rapidly with NADH to form an oxygenated species, even when efforts are made to reduce oxygen concentrations to sub-micromolar levels, suggesting a high affinity for this ligand . As is the case at high oxygen concentrations (130 microM), oxygenated species formation was kinetically and spectrally heterogeneous . Between 12 ms and 1 s after mixing, following transient formation of the deoxy form and its reaction with dioxygen, a steady-state level of the oxygenated species was attained . During the oxygenated steady state, the flavin remained largely oxidized, as observed previously at 130 microM oxygen . Hmp is an NADH oxidase; on exhaustion of oxygen by reduction (in < 10 s under these conditions), the oxygenated species disappeared to generate the deoxy Fe(II) haem, whereupon the flavin was reduced . The affinity for oxygen during NADH oxidation was measured by continuous dual-wavelength monitoring of the deoxygenation of oxymyoglobin . The Km for oxygen was 2.6 microM, much higher than the Km values determined, using the same method, for the membrane-bound terminal oxidases cytochromes bo' and bd . These results show that the oxidase activity of Hmp, but not necessarily oxygen binding, would be minimal at oxygen concentrations that limit terminal oxidase function. Acta Crystallogr A, 1996 May 1, 52 ( Pt 3), 480 - 9 Direct phasing in protein electron crystallography--phase extension and the prospects for ab initio determinations; Dorset DL; Zonal diffraction amplitudes and crystallographic phases, derived from an averaged electron micrograph of two-dimensionally crystalline E . coli Omp F outer membrane porin (plane group p31m, a = 72 A), embedded in glucose, were used as a model data set to test the feasibility of direct phase extension and ab initio direct phase determination . If 17 phase terms derived from e.g . a 10 A (diffraction) resolution image are expanded to 6 A by the Sayre-Hughes equation, the unknown phases are found with reasonable accuracy (mean error 43 degrees for 25 reflections) . This, however, is not the most optimal starting point . As a function of initial image resolution, the accuracy of the phase extension to 6 A is approximately a parabolic function . That is, an optimal basis resolution, found at 11 A (i.e . 14 defined reflections), produces a least mean error of 18 degrees for 28 new reflections . In addition, ab initio phase determination is possible via a multisolution technique, using a test for density flatness as a figure of merit . The success of the determination, again is sensitive to the size of the starting basis set generated from the permuted unknown reflections . If an annealing step is used to improve the basis set, the test for flatness will identify which reflections should be changed in phase . However, this figure of merit is not absolutely reliable for finding the exact value of the unknown phases. Phytochemistry, 1996 May, 42(2), 321 - 4 Effect of cucurbitacins on mRNA coding for laccase in Botrytis cinerea; Gonen L et al.; The effect of cucurbitacin and of Ecballium extract on the formation of mRNA coding for laccase was examined in cultures of Botrytis cinerea grown with inducers of laccase formation, in the presence or absence of the inhibitory compounds . RNA was isolated from the cultures and probed with specific DNA probes for laccase . As an internal control, the RNA was probed for Botrytis beta-tubulin mRNA . From an analysis of the results it is clear that cucurbitacin I and Ecballium extract specifically repress the amount of mRNA coding for laccase . This could account for the previously observed repression of laccase formation by cucurbitacins. Plant Physiol, 1996 May, 111(1), 61 - 71 A putative Mg chelatase subunit from Arabidopsis thaliana cv C24 . Sequence and transcript analysis of the gene, import of the protein into chloroplasts, and in situ localization of the transcript and protein; Gibson LC et al.; We have isolated and sequenced a cDNA from Arabidopsis thaliana cv C24 that encodes a putative Mg chelatase subunit . The deduced amino acid sequence shows a very high level of identity to a gene previously characterized from Antirrhinum majus (olive and also high similarity to bchH, a bacterial gene involved in the Mg chelatase reaction of bacteriochlorophyll biosynthesis . We suggest that this gene be called CHL H . Northern blot analyses were used to investigate the expression of CHL H, another putative Mg chelatase gene, ch-42, and ferrochelatase . The CHL H transcript was observed to undergo a dramatic diurnal variation, rising almost to its maximum level by the end of the dark period, then increasing slightly at the onset of the light and declining steadily to a minimum by the end of the light period; in contrast, transcripts for ch-42 and ferrochelatase remained constant . A model is proposed in which the CHL H protein plays a role in regulating the levels of chlorophyll during this cycle . In situ hybridization revealed that the transcripts are located over the surface of the chloroplasts, a feature in common with transcripts for the ch-42 gene . The CHL H protein was imported into the stromal compartment of the chloroplast and processed in an in vitro assay . Immunoblotting showed that the distribution of CHL H protein between the stroma and chloroplast membranes varies depending on the concentration of Mg+ . In situ immunofluorescence was used to establish that the CHL H and CH-42 proteins are localized within the chloroplast in vivo. Leuk Res, 1996 May, 20(5), 369 - 74 Trisomy 12 is a rare cytogenetic finding in typical chronic lymphocytic leukemia; Woessner S et al.; We have studied 61 cases of B-chronic lymphocytic leukemia (CLL), combining cytological features, conventional cytogenetics and in situ hybridization (ISH) . The comparison of these results constitutes the main subject of this study . The patients were cytologically classified according to the FAB criteria as: chronic lymphocytic leukemia (CLL) typical type (48 cases) and CLL atypical types (13 cases) . Chromosome analysis was carried out on lymphoid cells from peripheral blood . The following mitogens were used: phytohemagglutinin (PHA) 5%, pokeweed (PWM) and lipopolysaccharide from E . coli . The ISH was performed with a biotin-labeled, chromosome 12-specific alpha satellite DNA probe, pSP12-1 . Trisomy 12 was not found in any of the 48 patients with the typical type of CLL and in contradistinction it was present in some patients with atypical types . This study emphasizes the great importance of a closer link between hematological morphology and the cytogenetic approach. Transgenic Res, 1996 May, 5(3), 171 - 7 A model for the mechanism of precise integration of a microinjected transgene; McFarlane M et al.; A unique transgenic mouse line has undergone transgene integration in a very precise fashion . The phenotype displayed by mice of the line followed the predicted inheritance patterns for X-linked transgene insertion which has been confirmed . In order to investigate the mechanism of integration the DNA sequence of the transgene and cellular junctions have been determined . A comparison between wild type and transgenic mutant sequences at the site of insertion revealed that there was no loss or rearrangement of cellular DNA upon integration of the transgene . The cellular sequences at the transgene 5' and 3' joins are contiguous in the wild type . The integrant exists as a head to tail tandem dimer with minimal loss of sequence compared with the injected monomer . Analysis of the site of insertion has revealed a 5 bp homology between the 5' end of the transgene and the cellular sequences . In addition, adjacent to the site of insertion within the cellular sequences, there are several sequence motifs implicated in recombination events including a clustering of strong consensus sites of DNA topoisomerase type I and a region of homology to the human minisatellite consensus core sequence, the Escherichia coli Chi site and the meiotic recombination hotspot within the E beta gene of the murine major histocompatibility complex . This clustering of features is likely to have been factorial in the integrity of the insertion event . A model depicting the mechanism of this precise integration is proposed. Int Immunol, 1996 May, 8(5), 731 - 6 Cross-linking of cell surface ganglioside GM1 induces the selective apoptosis of mature CD8+ T lymphocytes; Nashar TO et al.; Gangliosides are glycosphingolipids found ubiquitously on the surface of mammalian cells . They contain a ceramide tail that is inserted into the membrane and exposed carbohydrate and sialic acid moieties . The non-toxic B subunit oligomer (EtxB) of Escherichia coli heat-labile enterotoxin (Etx) is a potent immunogen in vivo and has profound modulatory effects on EtxB-primed lymphocytes in vitro, properties which are dependent on its ability to bind to GM1 ganglioside receptors . Here, it is shown that cross-linking GM1 by EtxB causes a differential effect on mature CD4(+) and CD8(+) T cells from lymph node cultures proliferating in response to an unrelated antigen, ovalbumin . Addition of EtxB to such cultures led to the complete depletion of CD8(+) T cells compared with enhanced activation of CD4(+) cells {as measured by expression of CD25 (IL-2Ralpha)} . By contrast, addition of a mutant EtxB, EtxB(G33D), which does not bind to GM1, failed to trigger CD8(+) T cell depletion . When EtxB was added to isolated non-immune CD8(+) lymphocytes rapid (12-18 h) alterations in nuclear morphology and the appearance of sub-G0/G1 levels of DNA were induced; properties which are characteristic of cells undergoing apoptosis . EtxB(G33D) failed to trigger apoptosis, indicating that the induction of the apoptotic signal was dependent on the binding of GM1 . These findings provide an insight into the potent immunogenicity and immunomodulatory properties of E . coli enterotoxins as well as heralding a novel method for the selective induction of apoptosis in mature CD8(+) T lymphocytes. J Natl Med Assoc, 1996 May, 88(5), 306 - 9 Deceptive prothrombin and activated partial thromboplastin times in alcoholic cirrhosis; Sirikonda PR et al.; It is believed that perioperative hemorrhage, in the hepatoportal area, results from a coagulopathy . This study determined if this could be quantitated by a modified recalcification time (MRT) test developed in our laboratory . Unlike prothrombin (PT) and activated partial thromboplastin times (APTT), the MRT is performed with whole blood to ensure the role of blood cells and chemicals (particularly tissue factor, a potent procoagulant) in the coagulation process . Candidates for liver transplantation (n = 11) were studied . Samples (5 mL) of citrated venous blood were obtained from the patients . Aliquots (1 mL) from these samples were divided into groups of vials labeled C, S, and E . Groups C and S received 20 microL saline and group E, 20 microL of saline containing 10 micrograms of Escherichia coli endotoxin (055: B5W) . Vial C was incubated for 10 minutes and vials S and E for 120 minutes, all at 37 degrees C . Then, the MRT was determined on 300 microL of blood from each vial after adding 40 microL of 0.1M calcium chloride . Mean MRT values (minutes +/- standard deviation) for C (MRTC), for S (MRTS), and for E (MRTE) were compared with like values from healthy controls (n = 29) . Despite prolonged PT and APTT values, MRT values were shortened in patients with cirrhosis . This hypercoagulability detected by the MRT exonerates a hemorrhagic coagulopathy and possibly implicates widened and thinned gaps in the walls of the portal venous tributaries as the cause of perioperative hemorrhage. RNA, 1996 May, 2(5), 463 - 72 Phylogenetic comparative mutational analysis of the base-pairing between RNase P RNA and its substrate; Svard SG et al.; We have studied the base-pairing between the 3'-terminal CCA motif of a tRNA precursor and RNase P RNA by a phylogenetic mutational comparative approach . Thus, various derivatives of the Escherichia coli tRNA(Ser)Su1 precursor harboring all possible substitutions at either the first or the second C of the 3'-terminal CCA motif were generated . Cleavage site selection on these precursors was studied using mutant variants of M1 RNA, the catalytic subunit of E . coli RNase P, carrying changes at positions 292 or 293, which are involved in the interaction with the 3'-terminal CCA motif . From our data we conclude that these two C's in the substrate interact with the well-conserved G292 and G293 through canonical Watson-Crick base-pairing . Cleavage performed using reconstituted holoenzyme complexes suggests that this interaction also occurs in the presence of the C5 protein . Furthermore, we studied the interaction using various derivatives of RNase P RNAs from Mycoplasma hyopneumoniae and Mycobacterium tuberculosis . Our results suggest that the base-pairing between the 3'-terminal CCA motif and RNase P is present also in other bacterial RNase P-substrate complexes and is not limited to a particular bacterial species. Curr Genet, 1996 May, 29(6), 582 - 6 A small insertion in the SSU rDNA of the lichen fungus Arthonia lapidicola is a degenerate group-I intron; Grube M et al.; Insertions of less than 100 nt occurring in highly conserved regions of the small subunit ribosomal DNA (SSU rDNA) may represent degenerate forms of the group-I introns observed at the same positions in other organisms . A 63-nt insertion at SSU rDNA position 1512 (relative to the Escherichia coli SSU rDNA) of the lichen-forming fungus Arthonia lapidicola can be folded into a secondary structure with two stem loops and a pairing of the insertion and flanking sequences . The two stem loops may correspond to the P1 and P2, and the insertion-flanking pairing to the P10, of a group-I intron . Considering these small insertions as degenerate introns provides important clues to the evolution and catalytic function of group-I introns.Keywords Ribosomal DNA middle dot Small subunit middle dot 18s middle dot Degenerate introns middle dot Ascomycetes J Mol Evol, 1996 May, 42(5), 525 - 36 Codon usage and base composition in Rickettsia prowazekii; Andersson SG et al.; Codon usage and base composition in sequences from the A + T-rich genome of Rickettsia prowazekii, a member of the alpha Proteobacteria, have been investigated . Synonymous codon usage patterns are roughly similar among genes, even though the data set includes genes expected to be expressed at very different levels, indicating that translational selection has been ineffective in this species . However, multivariate statistical analysis differentiates genes according to their G + C contents at the first two codon positions . To study this variation, we have compared the amino acid composition patterns of 21 R . prowazekii proteins with that of a homologous set of proteins from Escherichia coli . The analysis shows that individual genes have been affected by biased mutation rates to very different extents: genes encoding proteins highly conserved among other species being the least affected . Overall, protein coding and intergenic spacer regions have G + C content values of 32.5% and 21.4%, respectively . Extrapolation from these values suggests that R . prowazekii has around 800 genes and that 60-70% of the genome may be coding. Arch Microbiol, 1996 May, 165(5), 333 - 41 Analysis of the hydA locus of Escherichia coli: two genes (hydN and hypF) involved in formate and hydrogen metabolism; Maier T et al.; The hydA locus of Escherichia coli is known to encode some function necessary for formation of hydrogenase activity . The locus contains two open reading frames, hydN and hypF . In this communication, an analysis of the regulation of these two genes and of the phenotype of respective mutants is presented, Both genes were expressed in a T7 promoter/polymerase system, yielding a 19-kDa (HydN) and a 81-kDa (HypF) protein . In-frame deletions were constructed for each gene and transferred to the chromosome by homologous recombination . The mutation in hydN led to a decrease of the activity of formate dehydrogenase H (FDH-H) in crude extracts, but the activity and maturation of hydrogenases were nearly unaffected . In contrast, a deletion in hypF resulted in the loss of hydrogenase activity and in the synthesis of the large subunits of the hydrogenase isoenzymes 1, 2 and 3 in the inactive precursor form . For hydrogenase 3, it was shown that this is due to a lack of incorporation of nickel into the large subunit . hydN and hypF are organised in an operon that is a member of the formate regulon . Transcription was shown to be dependent on sigma 54 and Fh1A, and an Fh1A-binding site upstream of hydN was identified . The sigma 54-dependent promoter shows a rare deviation from the consensus at positions -24/-12, namely GG/GA instead of GG/GC . In conclusion, the product of hydN appears to have some role in electron flow from or to FDH-H, and the product of hypF is connected with maturation of all three hydrogenases of E . coli. Mamm Genome, 1996 May, 7(5), 349 - 52 Mouse uroporphyrinogen decarboxylase: cDNA cloning, expression, and mapping; Wu C et al.; Uroporphyrinogen decarboxylase (URO-decarboxylase; EC 4.1.1.37), the heme biosynthetic enzyme responsible for the conversion of uroporphyrinogen III to coproporphyrinogen III, is the enzymatic defect in porphyria cutanea tarda, the most common porphyria . The mouse URO-decarboxylase cDNA was isolated from a mouse adult liver cDNA library . The longest clone of 1.5 kb, designated pmUROD-1, had 5' and 3' untranslated sequences of 281 and 97 bp, respectively, and an open reading frame of 1104 bp encoding a 367-amino acid polypeptide with a predicted molecular mass of 40,595 Da . The mouse and human coding sequences had 87.8% and 90.0% nucleotide and amino acid identity, respectively . The authenticity of the mouse cDNA was established by expression of the active enzyme in Escherichia coli . In addition, the analysis of two sets of multilocus genetic crosses localized the mouse gene, Urod, on Chromosome (Chr) 4, consistent with the map location of the human gene to a position of conserved synteny on Chr 1 . The availability of the mouse URO-decarboxylase should facilitate studies of the structure and organization of the mouse genomic sequence and the development of a mouse model of this inherited porphyria. J Membr Biol, 1996 May, 151(2), 175 - 87 Multiple mechanosensitive ion channels from Escherichia coli, activated at different thresholds of applied pressure; Berrier C et al.; Mechanosensitive ion channels from Escherichia coli were studied in giant proteoliposomes reconstituted from an inner membrane fraction, or in giant round cells in which the outer membrane and the cell wall had been disrupted by a lysozyme-EDTA treatment and a mild osmotic shock . Patch-clamp experiments revealed the presence in these two preparations of an array of different conductances (100 to 2,300 pS in 0.1 M KCl) activated by stretch . The electrical activity induced by stretch in the native membrane was complex, due to the activation of several different conductances . In contrast, patches of proteoliposomes generally contained clusters of identical conductances, which differed from patch to patch . These experiments are consistent with the notion that these different conductances correspond to different proteins in the plasma membrane of E . coli, which segregate into clusters of identical channels on dilution involved in reconstitution in proteoliposomes . These conductances could be grouped into three subfamilies of poorly selective channels . In both preparations, the higher the conductance, the higher was the negative pressure needed for activation . We discuss the putative role of these channels as parts of a multicomponent osmoregulatory system. Hum Genet, 1996 May, 97(5), 557 - 60 A novel point mutation in congenital erythropoietic porphyria in two members of Japanese family; Tanigawa K et al.; The molecular basis of the uroporphyrinogen III synthase (UROIIIS) deficiency was investigated in two members of a Japanese family . This defect in heme biosynthesis is responsible for a rare autosomal recessive disease: congenital erythropoietic porphyria (CEP) or Gnther's disease . The first patient was homoallelic for a novel missense mutation: a T to C transition of nucleotide 634 that predicted a serine to proline substitution at residue 212 (S212P) . The second patient appeared heteroallelic, carrying the same missense mutation and a nonsense mutation: a C to T change at nucleotide 745, resulting in a premature stop at codon 249, instead of a glutamine (Q249X) . The corresponding mutated proteins were expressed in Escherichia coli and no residual activity was observed . A family study was also performed to determine the carrier status. Genes Dev, 1996 May 1, 10(9), 1162 - 71 Use of a transposon (Tndif) to obtain suppressing and nonsuppressing insertions of the dif resolvase site of Escherichia coli; Kuempel P et al.; The dif locus is a RecA-independent recombination site, located in the terminus region of the chromosome of Escherichia coli . This site functions to reduce circular dimer chromosomes to monomers before cell division . Strains lacking this site exhibit the Dif phenotype, in which a fraction of the cells form extended filaments with abnormal nucleoids, and the SOS system is induced . We have used a transposon (Tndif), as well as linear transformation, to position dif in 19 locations around the chromosome . All of the suppressing insertions that we obtained were within 10 kb of the normal site, even in strains in which the normal symmetry, between the origin of replication and dif had been altered by 200 kb . We also observed that the nonsuppressing insertions in the terminus region became suppressing if a deletion occurred that extended from the ectopic site up to or past the normal location of dif . We propose that dif is normally located at the center of converging polarities in the terminus region and that deletions that restore suppression do so by placing ectopic sites once again at the center of this polarity . Similar results and conclusions are described in this issue. Genes Dev, 1996 May 1, 10(9), 1152 - 61 Restriction of the activity of the recombination site dif to a small zone of the Escherichia coli chromosome; Cornet F et al.; The recombination site dif is the target on the Escherichia coli chromosome of the site-specific recombinases XerC and XerD . The dif/XerC-D system plays a role during the cell cycle, probably by favoring sister chromosome monomerization or separation . A phenomenon of regional control over dif activity, also analyzed in this issue, is demonstrated here by translocation of dif to a series of loci close to the normal locus . We found that the site is physiologically active only within a narrow zone around its natural position . Competence for dif activity does not depend on the sequence of the normal dif activity zone (DAZ), because delta(dif) deletions larger than the DAZ result in Dif+ bacteria when dif is reinserted at the junction point . Although dif maps where replication normally terminates, termination of replication is not the elicitor . A strain with a large inversion that places dif and its surrounding region close to oriC remains Dif+, even when a Tus- mutation allows replication to terminate far away from it . Preliminary data suggest the possibility that specialized sequences separate the competent zone from the rest of the chromosome . We suspect that these sequences are members of a set of sequences involved in a polarized process of postreplicative reconstruction of the nucleoid structure . We propose that this reconstruction forces catenation links between sister chromosomes to accumulate within the DAZ, where they eventually favor recombination at dif. Genes Dev, 1996 May 1, 10(9), 1143 - 51 The RNA-binding protein HF-I, known as a host factor for phage Qbeta RNA replication, is essential for rpoS translation in Escherichia coli; Muffler A et al.; The rpoS-encoded sigma(S) subunit of RNA polymerase in Escherichia coli is a global regulatory factor involved in several stress responses . Mainly because of increased rpoS translation and stabilization of sigma(S), which in nonstressed cells is a highly unstable protein, the cellular sigma(S) content increases during entry into stationary phase and in response to hyperosmolarity . Here, we identify the hfq-encoded RNA-binding protein HF-I, which has been known previously only as a host factor for the replication of phage Qbeta RNA, as an essential factor for rpoS translation . An hfq null mutant exhibits strongly reduced sigma(S) levels under all conditions tested and is deficient for growth phase-related and osmotic induction of sigma(S) . Using a combination of gene fusion analysis and pulse-chase experiments, we demonstrate that the hfq mutant is specifically impaired in rpoS translation . We also present evidence that the H-NS protein, which has been shown to affect rpoS translation, acts in the same regulatory pathway as HF-I at a position upstream of HF-I or in conjunction with HF-I . In addition, we show that expression and heat induction of the heat shock sigma factor sigma(32) (encoded by rpoH) is not dependent on HF-I, although rpoH and rpoS are both subject to translational regulation probably mediated by changes in mRNA secondary structure . HF-I is the first factor known to be specifically involved in rpoS translation, and this role is the first cellular function to be identified for this abundant ribosome-associated RNA-binding protein in E . coli. Genes Dev, 1996 May 1, 10(9), 1120 - 30 Posterior patterning by the Caenorhabditis elegans even-skipped homolog vab-7; Ahringer J; Patterning of the posterior end in animals is not well understood . Homologs of Drosophila even-skipped (eve) have a similar posterior expression pattern in many animals, and in vertebrates they are linked physically to the "posterior" ends of homeotic clusters (HOM-C), suggesting a conserved role in posterior development . However, the function of this posterior expression is not known . Here I show that the Caenorhabditis elegans gene vab-7 encodes an eve homolog that is required for posterior development and expressed in a pattern strikingly similar to that of vertebrate eve genes . Using a four-dimensional recording system, I found that posterior body muscles and the posterior epidermis are patterned abnormally in vab-7 mutants, but commitment to muscle and epidermal fates is normal . Furthermore, vab-7 activity is required for the complete expression of the most posterior HOM-C gene egl-5 in muscle cells, supporting the idea that eve homologs may act with the HOM-C to determine posterior cell fates . The conservation of sequence and expression pattern between vab-7 and eve homologs in other animals argues that most eve genes have posterior mesodermal and ectodermal patterning functions. Genes Dev, 1996 May 1, 10(9), 1073 - 83 The Caenorhabditis elegans cell-death protein CED-3 is a cysteine protease with substrate specificities similar to those of the human CPP32 protease; Xue D et al.; The Caenorhabditis elegans cell-death gene ced-3 encodes a protein similar to mammalian interleukin-1beta-converting enzyme (ICE), a cysteine protease implicated in mammalian apoptosis . We show that the full-length CED-3 protein undergoes proteolytic activation to generate a CED-3 cysteine protease and that CED-3 protease activity is required for killing cells by programmed cell death in C . elegans . We developed an easy and general method for the purification of CED-3/ICE-like proteases and used this method to facilitate a comparison of the substrate specificities of four different purified cysteine proteases . We found that in its substrate preferences CED-3 was more similar to the mammalian CPP32 protease than to mammalian ICE or NEDD2/ICH-1 protease . Our results suggest that different mammalian CED-3/ICE-like proteases may have distinct roles in mammalian apoptosis and that CPP32 is a candidate for being a mammalian functional equivalent of CED-3. Plant J, 1996 May, 9(5), 671 - 81 Reduction of the cytosolic fructose-1,6-bisphosphatase in transgenic potato plants limits photosynthetic sucrose biosynthesis with no impact on plant growth and tuber yield; Zrenner R et al.; Sucrose produced in source leaves is the predominant carbon source for developing sink tissues in most higher plants . Consequently the rate of sucrose synthesis is likely to be important for sink development and final crop yield . Two sucrose biosynthetic enzymes are believed to possess regulatory properties with respect to the rate of sucrose synthesis: (i) cytosolic FBPase and (ii) sucrose phosphate synthase . To study the impact of reduced photosynthetic sucrose biosynthesis on plant growth and crop yield a cDNA clone encoding cytosolic FBPase was isolated from a potato leaf cDNA library and used for antisense experiments in transgenic potato plants . The cDNA clone cy-F1, containing an open reading frame of 1020 bp highly homologous (85%) to other known sequences of plant cytosolic FBPases, was cloned in reversed orientation between the 35S CaMV promoter and the octopine synthase polyadenylation signal . Out of 75 independent transformants five transgenic lines having 9 to 55% of the wild-type FBPase activity were chosen for further analysis . A 45% reduction of the cytosolic FBPase activity did not cause any measurable change in metabolite concentrations, growth behaviour or photosynthetic parameters of the transgenic plants . Inhibition of cytosolic FBPase activity below 20% of the wild-type activity led to an accumulation of 3-PGA, triose-phosphates and fructose-1,6-biphosphate in source leaves . This resulted in a reduced light-saturated rate of assimilation measured via gas exchange and a decreased photosynthetic rate under conditions of the leaf disc electrode with saturating light and CO2 . Measuring photosynthetic carbon fluxes by labelling leaf discs with 14CO2 revealed a 53-65% reduction of sucrose synthesis whereas starch synthesis decreased only by 18-24% . The flux into the anionic and cationic fraction was not altered . Despite these changes steady-state sucrose concentrations were not effected in source leaves from transgenic plants . Starch accumulated by more than a factor of 3 compared with wild-type leaves and was degraded during the night . This provides strong evidence for the hypothesis that hexoses and/or hexosephosphates are exported out of the chloroplasts, thereby circumventing the limitation of sucrose biosynthesis caused by the inhibition of cytosolic FBPase in the dark . Accordingly, plant growth and potato tuber yield remained unaltered . From these data it can be concluded that a reduced photosynthetic sucrose biosynthetic capacity can be efficiently compensated without any reduction in crop yield under greenhouse or growth chamber conditions by changing carbon export strategy . Whether the same holds true for field conditions remains to be elucidated. Nucleic Acids Res, 1996 May 1, 24(9), 1753 - 7 Accumulation of a mRNA decay intermediate by ribosomal pausing at a stop codon; Bjornsson A et al.; A RNA fragment which is protected from degradation by ribosome pausing at a stop codon has been identified in growing Escherichia coli . The fragment is 261 nt long and corresponds to the 3'-end of the mRNA expressed from a semi-synthetic model gene . The 5'-end of the RNA fragment, denoted rpRNA (ribosomal pause RNA), is located 13 bases upstream of the stop codon . In vivo decay of the complete mRNA and accumulation of rpRNA are dependent on the nature of the stop codon and its codon context . The data indicate that the rpRNA fragment arises from interrupted decay of the S3A'mRNA in the 5'-->m3'direction, in connection with a ribosomal pause at the stop codon . RF-2 decoding of UGA is less efficient than RF-1 decoding of UAG in identical codon contexts, as judged from rpRNA steady-state levels . The half-life of UGA-containing rpRNAs is at least 5 min, indicating that ribosomal pausing can be a major factor in stabilising downstream regions of messenger RNAs. Nucleic Acids Res, 1996 May 1, 24(9), 1747 - 52 Identification of promoter and stringent regulation of transcription of the fabH, fabD and fabG genes encoding fatty acid biosynthetic enzymes of Escherichia coli; Podkovyrov SM et al.; In Escherichia coli, amino acid starvation results in the coordinate inhibition of a variety of metabolic activities, including fatty acid and phospholipid biosynthesis . By using primer extension analysis we identified the fabH promoter responsible for transcription of the fabH, fabD and fabG genes encoding fatty acid biosynthetic enzymes . The response of the fabH promoter to amino acid starvation was determined in vivo . Transcripts originating from the fabH promoter were quantified by employing a ribonuclease protection assay . The fabH promoter was subject to relA-dependent stringent control and was repressed approximately 4-fold upon amino acid starvation . The results suggest that inhibition of transcription initiation of lipid biosynthetic genes in starved cells contributes to the stringent control of lipid biosynthesis. Nucleic Acids Res, 1996 May 1, 24(9), 1742 - 6 Reduction of the toxicity and mutagenicity of aziridine in mammalian cells harboring the Escherichia coli fpg gene; Cussac C et al.; Aziridine (ethyleneimine) reacts with DNA in vitro, mainly at the N7 position of guanine and N3 of adenine, then imidazole ring opening of the modified guanine results in formation of formamidopyrimidine (FaPy) residues . The Escherichia coli fpg gene encodes a DNA glycosylase that removes FaPy residues from DNA . To determine whether aziridine produces FaPy lesions in mammalian cells we have expressed the E.coli fpg gene in CHO cells . The transfected cells, expressing high levels of the bacterial protein, are more resistant to the toxic and mutagenic effects of aziridine than the control population . Less DNA damage was measured by quantitative PCR analysis in transfected than in control cells treated with equimolar concentrations of aziridine . The results suggest that aziridine produces in vivo FaPy residues that could account for the deleterious effects of this compound. Nucleic Acids Res, 1996 May 1, 24(9), 1710 - 8 Differences in mutagenesis during minus strand, plus strand and strand transfer (recombination) synthesis of the HIV-1 gene in vitro; Wu W et al.; We have developed an HIV nef-Escherichia coli lacZ fusion system in vitro that allows the detection of low frequency mutations, including frameshifts, deletions and insertions . A portion of the nef gene that encompasses a hypervariable region was fused in-frame with a downstream lacZalpha peptide coding region . The resulting lacZalpha peptide fusion protein remained functional . Any frameshift mutations in the nef insert would put the downstream lacZ alpha peptide gene out of frame, eliminating alpha complementation . With this system we compared the error rates of frameshift mutations that arise during DNA-directed and RNA-directed DNA synthesis . Results showed that DNA-directed and RNA-directed DNA synthesis did not contribute equally to the generation of mutations . DNA-directed DNA synthesis generated frameshift mutations at a frequency approximately 10-fold higher than those arising from RNA-directed DNA synthesis . RNA-directed DNA synthesis in the presence of acceptor templates showed an increase in mutation rate and differences in the mutation spectrum . The enhancement of mutation rate was caused by the appearance of mutations at three new locations that correlated with likely recombination sites . Results indicate that recombination is another source of mutations during viral replication. Nucleic Acids Res, 1996 May 1, 24(9), 1608 - 15 The polypyrimidine tract binding (PTB) protein interacts with single-stranded DNA in a sequence-specific manner; Brunel F et al.; Polypyrimidine tract binding (PTB) protein is a cellular factor whose function is unknown . Various RNA or single-stranded DNA sequences have been shown to interact with PTB . In this paper, using laser UV crosslinking and electrophoretic mobility shift assays to probe DNA-protein interactions, we demonstrate that PTB binding at a single-stranded DNA target is highly sequence-specific . We provide data showing that PTB interacts with the top strand of the adenovirus major late promoter transcriptional initiator, a sequence rich in pyrimidine residues . We also demonstrate that PTB is organised into at least two different binding domains. Eur J Biochem, 1996 May 1, 237(3), 870 - 5 Cys5 and Cys214 of NAD(P)H:flavin oxidoreductase from Escherichia coli are located in the active site; Fieschi F et al.; The NAD(P)H:flavin oxidoreductase (NADPH:riboflavin oxidoreductase) from Escherichia coli, Fre, is a monomer of 26.1 kDa, which catalyzes the reduction of free flavins by NADPH or NADH . A sequential ordered mechanism, with NADPH binding first, operates . Fre is the prototype of a class of flavin reductases able to transfer electrons with no prosthetic group . It has been previously reported that several members of this family, including Fre, were inactivated by thiol reagents such as N-ethylmaleimide (MalNEt) . Amino acid sequence similarities among these enzymes reveal that one of the three cysteines residues of Fre is highly conserved . Altogether this suggested a crucial role of cysteine residues for catalysis . The three cysteine residues were mutated to serine residues . Single-mutant and double-mutant enzymes were as active as the wild-type and Km values for both substrates remained the same . Cysteine residues are thus not important for activity . Nevertheless, we showed that cysteines 5 and 214, but not cysteine 149, were responsible for MalNEt inactivation . In addition, it has been found that riboflavin, but not NADPH, can protect Fre from MalNEt inactivation . This strongly suggested that Cys5 and Cys214 are located at the flavin-binding site of Fre and that flavin can bind to the enzyme in the absence of NADPH. Eur J Biochem, 1996 May 1, 237(3), 827 - 32 Expression in Escherichia coli of Sin a 1, the major allergen from mustard; Gonzalez De La Pena MA et al.; Sin a 1, the major yellow mustard allergen, is a seed storage protein that belongs to the 2S albumin family . It is composed of two disulfide-bonded polypeptide chains . The cloning of this allergen has been carried out by means of the polymerase chain reaction using non-degenerate oligonucleotides encoding the N-terminal and C-terminal regions of the mature protein as primers . Five genomic nucleotide sequences have been analyzed, encoding both mature polypeptide chains linked by the internal processed fragment . The sequence data show the existence of microheterogeneities at ten positions, demonstrating the polymorphism exhibited by the natural protein . One of the genomic clones was expressed in Escherichia coli by fusion to glutathione S-transferase from Schistosoma japonicum . The resulting chimeric protein was purified by affinity chromatography on a glutathione-Sepharose 4B matrix, and digested with thrombin to release the recombinant allergen . The recombinant Sin a 1 is recognized by rabbit polyclonal and mouse monoclonal antisera raised against natural Sin a 1, as well as by the IgE of mustard-sensitive human sera . In addition, recombinant Sin a 1 possesses a high resistance to trypsin digestion, like the native mustard allergen. Eur J Biochem, 1996 May 1, 237(3), 800 - 8 Analysis of the specificity of the AMP-activated protein kinase by site-directed mutagenesis of bacterially expressed 3-hydroxy 3-methylglutaryl-CoA reductase, using a single primer variant of the unique-site-elimination method; Ching YP et al.; The specificity of protein kinases is usually examined using synthetic peptide substrates, either designed variants, or, more recently random peptide libraries . However not all protein kinases utilize synthetic peptides efficiently as substrates . Even among those that do, these approaches neglect effects caused by three-dimensional protein conformation, or the existence of determinants remote from the phosphorylation site . To follow up our previous peptide studies on the specificity of the AMP-activated protein kinase (AMPK) {Dale, S., Wilson, W . A., Edelman, A.M., & Hardie, D . G . (1995) FEBS Lett . 361, 191-195}, we have expressed the C-terminal, catalytic domain of Chinese hamster hydroxymethylglutaryl-CoA reductase in Escherichia coli . The domain was expressed with an N-terminal His6 tag which allowed rapid purification on Nj(2+)-agarose . The purified protein retained full enzymic activity, and as with the native enzyme, was totally inactivated by phosphorylation by AMPK at a single site corresponding to Ser871 . Using a novel modification of the unique-site elimination method (which allowed direct mutagenesis of the double-stranded expression vector using a single oligonucleotide primer) we expressed 18 mutations involving residues around Ser871 . The results broadly confirmed the recognition motif previously proposed on the basis of peptide studies . Three of the mutants were better substrates for AMPK than the wild type, and one of these (K872A) had hydroxymethylglutaryl-CoA reductase kinetic parameters virtually indistinguishable from the wild type . This suggests that hydroxymethylglutaryl-CoA reductase may have been selected to be a sub-optimal substrate for AMPK. Eur J Biochem, 1996 May 1, 237(3), 743 - 51 Systematic mutational analysis of the receptor-binding region of the human urokinase-type plasminogen activator; Magdolen V et al.; The amino-terminal fragment of human uPA (ATF; amino acids 1-135), which contains the binding site for the uPA receptor (uPAR, CD87) was expressed in the yeast Saccharomyces cerevisiae . Recombinant yeast ATF, modified and extended by an amino-terminal in-frame insertion of a His6 tract, was purified from total protein extracts by nickel chelate affinity chromatography and shown to be functionally active since it efficiently competes with uPA for binding to cell-surface-associated uPAR . The ATF expression plasmid served as a template for the construction of a series of site-directed mutants in order to define those amino acids that are important for binding to uPAR . All mutant ATF proteins but one (deletion of Ser26) were expressed in a stable form (about 20-30 ng/mg total protein) and the binding capacity of each mutant was tested by a uPA-ligand binding assay employing recombinant uPAR immobilized to a microtiter plate . Each of the 11 amino acids of loop B of the binding region of uPA (amino acids 20-30) were individually substituted with alanine . Lys23, Tyr24, Phe25, IIe28, and Trp30 were important determinants for uPAR binding . A systematic alanine scan was also performed with chemically synthesized linear peptides spanning amino acids 14-32 of ATF . Comparable results to those with the yeast ATF mutants were obtained . In a different set of experiments, those amino acids of the uPAR-binding region of uPA that are only conserved between man and baboon but not in other species were altered: whereas substitution of Thr18 by alanine or Asn32 by serine had hardly any effect, replacement of Asn22 by tyrosine and Trp30 by arginine (both positions are strictly conserved in other mammals) led to ATF variants incapable of interacting with human uPAR . Deletion of either Val20, Ser21, Lys23, His29 or Val20 plus Ser21, respectively, also generated non-reactive ATF mutants . Finally, Lys23 in ATF was substituted with certain amino acids: whereas the replacement of Lys23 by alanine, histidine or glutamine generated ATF variants with moderate uPAR-binding activity, the introduction of a negatively charged amino acid (exchange of Lys23 by glutamic acid) completely abolished uPAR-binding activity . The results presented for the ATF mutants and uPA-derived peptides may provide clues necessary to establish the nature of the physical interaction of uPA with its receptor and may help to develop uPA-derived peptide analogues as potential therapeutic agents to block tumor cell-associated uPA/uPAR interaction. Eur J Biochem, 1996 May 1, 237(3), 592 - 600 Flavin motion in p-hydroxybenzoate hydroxylase . Substrate and effector specificity of the Tyr22-->Ala mutant; van der Bolt FJ et al.; The side chain of Tyr222 in p-hydroxybenzoate hydroxylase interacts with the carboxy moiety of the substrate . Studies on the Tyr222-->Phe mutant, {F222}p-hydroxybenzoate hydroxylase, have shown that disruption of this interaction hampers the hydroxylation of 4-hydroxybenzoate . Tyr222 is possibly involved in flavin motion, which may facilitate the exchange of substrate and product during catalysis . To elucidate the function of Tyr222 in more detail, in the present study the substrate and effector specificity of the Tyr222-->Ala mutant, {A222}p-hydroxybenzoate hydroxylase, was investigated . Replacement of Tyr222 by Ala impairs the binding of the physiological substrate 4-hydroxybenzoate and the substrate analog 4-aminobenzoate . With these compounds, {A222}p-hydroxybenzoate hydroxylase mainly acts as a NADPH oxidase . {A222}p-hydroxybenzoate hydroxylase tightly interacts with 2,4-dihydroxybenzoate and 2-hydroxy-4-aminobenzoate . Crystallographic data {Schreuder, H.A., Mattevi, A., Oblomova, G., Kalk, K.H., Hol, W.G.J., van der Bolt, F.J.T . & van Berkel, W.J.H . (1994) Biochemistry 33, 10161-10170} suggest that this is due to motion of the flavin ring out of the active site, allowing hydrogen-bond interaction between the 2-hydroxy group of the substrate analogs and N3 of the flavin . {A222}p-Hydroxybenzoate hydroxylase produces about 0.6 mol 2,3,4-trihydroxybenzoate from 2,4-dihydroxybenzoate/mol NADPH oxidized . This indicates that reduction of the Tyr222-->Ala mutant shifts the equilibrium of flavin conformers towards the productive "in' position . {A222}p-Hydroxybenzoate hydroxylase converts 2-fluoro-4-hydroxybenzoate to 2-fluoro-3,4-dihydroxybenzoate . The regioselectivity of hydroxylation suggests that {A222}p-hydroxybenzoate hydroxylase binds the fluorinated substrate in the same orientation as wild-type . Spectral studies suggest that wild-type and {A222}p-hydroxybenzoate hydroxylase bind 2-fluoro-4-hydroxybenzoate in the phenolate form with the flavin ring preferring the "out' conformation . Despite activation of the fluorinated substrate and in contrast to the wild-type enzyme, {A222}p-hydroxybenzoate hydroxylase largely produces hydrogen peroxide . The effector specificity of p-hydroxybenzoate hydroxylase is not changed by the Tyr222-->Ala replacement . This supports the idea that the effector specificity is mainly dictated by the protein-substrate interactions at the re-side of the flavin ring. Int Arch Allergy Immunol, 1996 May, 110(1), 41 - 5 Cloning Aspergillus fumigatus allergens by the pJuFo filamentous phage display system; Crameri R et al.; A cDNA library from Aspergillus fumigatus has been displayed on the surface of filamentous phage M13 and screened for gene products binding to human serum IgE . The physical linkage of cDNA gene products to the genetic information required for their production, achieved by exploiting the high-affinity interaction of the Jun and Fos leucine zippers, allows rapid and easier screening of large libraries in semifluid systems . The pJuFo cloning vector is designed to display proteins on the surface of phage and allows selective isolation of genes by gene product-ligand interaction . Thus the system is applicable to clone cDNA that encodes proteins for which a ligand is available . Herein we show that phage expressing IgE binding proteins from A . fumigatus can be enriched and separated from nonspecific phage by using serum IgE from A . fumigatus-allergic individuals coated to plastic dishes . Subsequently, the proteins can be produced in high amounts in Escherichia coli and purified for usage in allergy testing. Biochem J, 1996 May 1, 315 ( Pt 3), 989 - 94 Bovine inositol monophosphatase: enzyme-metal-ion interactions studied by pre-equilibrium fluorescence spectroscopy; Thorne MR et al.; Stopped-flow fluorescence spectroscopy has been used to determine the on-rate (kass) and the off-rate (kdiss) for the equilibrium between inositol monophosphatase and Mg2+ ions . The dissociation constant (Kd) for the equilibrium calculated from these constants suggests that the ions interact at site 1 on the enzyme with a Kd typically around 450 microM, close to values determined by equilibrium studies (270-300 microM) . The affinity of this site on the wild-type enzyme for Mg2+ ions increases as the pH is increased . This is mediated almost entirely by change in the rate kdiss . A slow increase occurs in the fluorescence intensity of the pyrene-labelled enzyme after the initial, fast, increase in fluorescence caused by the binding of the Mg2+ ion . The rate of this change is independent of the concentration of the metal ion, implying that it may be a structural change in the enzyme-Mg2+ complex . Neither the fast nor the slow change in fluorescence intensity occurs when enzyme subjected to limited proteolysis by trypsin, which removes the N-terminal 36 residues, is mixed with Mg2+ ions . The data suggest that interaction with Mg2+ ions at a high-affinity site leads to a structural change in inositol monophosphatase . The data further confirm the importance of the presence of two metal ions in the structure/function of this enzyme, and show that the binding of the metal ions is not competitive with that of H+ ions and that the variation in Kd with pH is mediated almost totally by changes in kdiss. Biochem J, 1996 May 1, 315 ( Pt 3), 931 - 8 Purification, cloning and expression of dehydroascorbic acid-reducing activity from human neutrophils: identification as glutaredoxin; Park JB et al.; Dehydroascorbic acid-reducing activity in normal human neutrophil lysates was characterized and identified by activity-based purification and measurement of newly synthesized ascorbate by HPLC . The initial reducing activity was non-dialysable and could not be accounted for by the activity of glutathione as a reducing agent . The reducing activity was purified to homogeneity as an 11 kDa protein . The protein had a specific activity of 3 mumol/min per mg of protein and was glutathione dependent . Kinetic experiments showed that the protein had a K(m) for glutathione of 2.0 mM and a K(m) for dehydroascorbic acid of 250 microM . Dehydroascorbic acid reduction by the purified protein was pH dependent and was maximal at pH 7.5 . Peptide fragments from the purified protein were analysed for amino acid sequence and the protein was identified as glutaredoxin . By using degenerate oligonucleotides based on the amino acid sequence, glutaredoxin was cloned from a human neutrophil library . Expressed purified glutaredoxin displayed reducing activity and kinetics that were indistinguishable from those of native purified enzyme . Several approaches indicated that glutaredoxin was responsible for the most of the protein-mediated dehydroascorbic acid reduction in lysates . From protein purification data, glutaredoxin was responsible for at least 47% of the initial reducing activity . Dehydroascorbic acid reduction was at least 5-fold greater in neutrophil lysates than in myeloid tumour cell lysates, and glutaredoxin was detected in normal neutrophil lysates but not in myeloid tumour cell lysates by Western blotting . Glutaredoxin inhibitors inhibited dehydroascorbic acid reduction in neutrophil lysates as much as 80% . These findings indicate that glutaredoxin plays a major role in dehydroascorbic acid reduction in normal human neutrophil lysates, and represent the first identification of dehydroascorbic acid reductase in human tissue by activity-based purification. Biochem J, 1996 May 1, 315 ( Pt 3), 761 - 6 Site-directed mutagenesis of rat liver S-adenosylmethionine synthetase . Identification of a cysteine residue critical for the oligomeric state; Mingorance J et al.; We have examined the functional importance of the cysteine residues of rat liver S-adenosylmethionine synthetase . For this purpose the ten cysteine residues of the molecule were changed to serines by site-directed mutagenesis . Ten recombinant enzyme mutants were obtained by using a bacterial expression system . The same level of expression was obtained for the wild type and mutants, but the ratio of S-adenosylmethionine synthetase between soluble and insoluble fractions differed for some of the mutant forms . The immunoreactivity against an anti-(rat liver S-adenosylmethionine synthetase) antibody was equivalent in all the cases . Effects on S-adenosylmethionine synthetase activities were also measured . Mutants C57S, C69S, C105S and C121S showed decreased relative specific activity of 68, 85, 63 and 29%, respectively, compared with wild-type, whereas C312S resulted in an increase of 1.6-fold . Separation of tetramer and dimer forms for wild type and mutants was carried out by using phenyl-Sepharose columns . The dimer/tetramer ratio was calculated based on the activity and on the protein level estimated by immunoblotting . No monomeric forms of the enzyme were detected in any case . Comparison of dimer/tetramer ratios indicates the importance of cysteine-69 (dimer/tetramer protein ratio of 88 versus 10.2 in the wild type) in maintaining the oligomeric state of rat liver S-adenosylmethionine synthetase . Moreover, all the mutations carried out of cysteine residues between cysteine-35 and cysteine-105 altered the ratio between oligomeric forms. Biochem J, 1996 May 1, 315 ( Pt 3), 733 - 8 Processing of N3, a mammalian proteasome beta-type subunit; Thomson S et al.; Proteasome subunits are encoded by members of the same gene family and can be divided into two groups based on their similarity to the alpha and beta subunits of the simpler proteasome isolated from Thermoplasma acidophilum . RN3 is the beta-type subunit, N3, of rat proteasomes which has been implicated in the peptidylglutamyl-peptide hydrolase activity of the proteinase complex . We have expressed recombinant RN3 protein in Escherichia coli in order to raise subunit-specific polyclonal antibodies . Identification of the position of RN3 on two-dimensional PAGE gels of purified rat liver proteasomes showed a single protein spot of molecular mass 24 kDa and of pI value of about 5 . This protein has a free N-terminus, having undergone post-translational processing . After immunoprecipitation from {35S}methionine-labelled human embryo lung L-132 cells using anti-RN3 antibodies, two radiolabelled spots were observed on two-dimensional PAGE gels, one corresponding to the mature N3, the other of molecular mass 28.5 kDa and pI value around 5, which was probably the unprocessed form of N3 . However, the latter protein had a higher molecular mass (31 kDa) than was predicted from the sequence of previously cloned cDNA . Therefore rapid amplification of cDNA ends ("RACE') was carried out to determine the full sequence . The lack of detectable RN3 precursor in purified rat liver proteasomes suggests that the processing probably accompanies assembly of the complex . The half-life of the processing was determined to be 31 min in growing L-132 cells . The unprocessed form of N3 was not observed after immunoprecipitation of 35S-labelled complexes with anti-proteasome antibodies . There was no evidence to suggest that unprocessed N3 is found in precursor complexes which have been implicated in the assembly of some other unprocessed beta-type subunits . Interestingly also, the site of cleavage of N3 (ITR decreases TQN) differs significantly from those of other processed animal beta-type proteasome subunits {(H/T)G decreases TT(T/L)}, many of which resemble more closely the cleavage site of the Thermoplasma acidophilum beta subunit. Biochem J, 1996 May 1, 315 ( Pt 3), 727 - 32 Purification and characterization of a recombinant human Theta-class glutathione transferase (GSTT2-2); Tan KL et al.; A cDNA encoding the human Theta-class glutathione transferase GSTT2-2 was expressed in Escherichia coli as a ubiquitin fusion protein . The co-translational removal of the ubiquitin by a cloned ubiquitin-specific protease, Ubp1, generates enzymically active GSTT2-2 without any additional N-terminal residues . The recombinant isoenzyme was purified to apparent homogeneity by DEAE anion-exchange, gel filtration, dye ligand chromatography and high resolution anion-exchange chromatography on Mono Q FPLC . The recombinant enzyme had significant activity with a range of substrates, including cumene hydroperoxide and 1-menapthyl sulphate . The activity of GSTT2-2 with a range of secondary lipid peroxidation products such as the trans,trans-alka-2,4-dienals and trans-alk-2-enals, as well as its glutathione peroxidase activity with organic hydroperoxides, suggest that it may play a significant role in protection against the products of lipid peroxidation. Lab Invest, 1996 May, 74(5), 871 - 81 In vivo cell lineage analysis during chemical hepatocarcinogenesis using retroviral-mediated gene transfer; Bralet MP et al.; We have studied the proliferation of cells in two models of chemical hepatocarcinogenesis . The cells were genetically labeled in vivo using retrovirally mediated transfer of the Escherichia coli beta-galactosidase marker gene coupled to a nuclear localization signal (nls-lacZ gene) . In the first carcinogenic model, rats were fed a choline-deficient diet containing 2-acetylaminofluorene, and their livers were perfused with recombinant retrovirus at the onset of oval cell proliferation . The second model was based on the administration of diethylnitrosamine coupled with a partial hepatectomy and is thought to induce cancer with no involvement of oval cells . Analysis of beta-galactosidase expression in the liver at various times after gene transfer revealed the presence of large clusters of positive cells in both models . Moreover, the beta-galactosidase-positive cells displayed morphologic, antigenic, and enzymatic profiles consistent with a hepatocyte phenotype . Our results, therefore, provide evidence for a strikingly similar clonal proliferation of apparently normal hepatocytes during the course of 2-acetylaminofluorene- as well as diethylnitrosamine-induced liver carcinogenesis. EMBO J, 1996 May 1, 15(9), 2306 - 12 A Drosophila ribosomal protein contains 8-oxoguanine and abasic site DNA repair activities; Yacoub A et al.; Ionizing radiation and normal cellular respiration form reactive oxygen species that damage DNA and contribute to a variety of human disorders including tumor promotion and carcinogenesis . A major product of free radical DNA damage is the formation of 8-oxoguanine, which is a highly mutagenic base modification produced by oxidative stress . Here, Drosophila ribosomal protein S3 is shown to cleave DNA containing 8-oxoguanine residues efficiently, The ribosomal protein also contains an associated apurinic/apyrimidinic (AP) lyase activity, cleaving phosphodiester bonds via a beta,delta elimination reaction . The significance of this DNA repair activity acting on 8-oxoguanine is shown by the ability of S3 to rescue the H2O2 sensitivity of an Escherichia coli mutM strain (defective for the repair of 8-oxoguanine) and to abolish completely the mutator phenotype of mutM caused by 8-oxoguanine-mediated G-->T transversions . The ribosomal protein is also able to rescue the alkylation sensitivity of an E.coli mutant deficient for the AP endonuclease activities associated with exonuclease III (xth) and endonuclease IV (nfo), indicating for the first time that an AP lyase can represent a significant source of DNA repair activity for the repair of AP sites . These results raise the possibility that DNA repair may be associated with protein translation. EMBO J, 1996 May 1, 15(9), 2150 - 9 Sac1, a putative regulator that is critical for survival of Chlamydomonas reinhardtii during sulfur deprivation; Davies JP et al.; The sac1 mutant of Chlamydomonas reinhardtii is aberrant in most of the normal responses to sulfur limitation; it cannot synthesize arylsulfatase, does not take up sulfate as rapidly as wild-type cells, and does not synthesize periplasmic proteins that normally accumulate during sulfur-limited growth . Here, we show that the sac1 mutant dies much more rapidly than wild-type cells during sulfur deprivation; this emphasizes the vital role of the acclimation process . The loss of viability of the sac1 mutant during sulfur deprivation is only observed in the light and is mostly inhibited by DCMU . During sulfur-stress, wild-type cells, but not the sac1 mutant, downregulate photosynthesis . Thus, death of the sac1 mutant during sulfur deprivation is probably a consequence of its inability to downregulate photosynthesis . Furthermore, since SAC1 is necessary for the downregulation of photosynthesis, the process must be highly controlled and not simply the result of a general decrease in protein synthesis due to sulfur limitation . Genomic and cDNA copies of the SAC1 gene have been cloned . The deduced amino acid sequence of Sac1 is similar to an Escherichia coli gene that may involved in the response of E.coli to nutrient deprivation. Carcinogenesis, 1996 May, 17(5), 1183 - 5 Involvement of Escherichia coli exonuclease III and endonuclease IV in the repair of singlet oxygen-induced DNA damage; Agnez LF et al.; Singlet molecular oxygen (1O2) has been implicated in several biological processes that may lead to genetic damage . The relevance of various repair pathways in plasmid inactivation mediated by 1O2 was investigated . Plasmid treated with 1O2, chemically generated, was transfected into Escherichia coli strains deficient in genes implicated in the DNA repair of oxidative damage . The ability to transform bacteria is significantly reduced in the double mutant xth,nfo, deficient in both exonuclease III and endonuclease IV, although it was similar to wild-type cells in single mutants . The products of these two genes are able to cleave DNA damaged by 1O2 and to remove DNA polymerization blocks from 3'-termini generated either directly by 1O2 treatment or after the action of the formamidopyrimidine-DNA-N-glycosylase (Fpg protein) . The results indicate that the exonuclease III and endonuclease IV participate in the excision of lethal lesions induced in DNA by 1O2. Can J Microbiol, 1996 May, 42(5), 519 - 23 Cyanide degradation by an Escherichia coli strain; Figueira MM et al.; Chemical formation of a glucose-cyanide complex was necessary for metabolic degradation of cyanide at concentrations up to 50.0 mg/L by a strain of Escherichia coli isolated from gold extraction circuit liquids . Ammonia accumulating during the culture log phase as the sole nitrogen by-product was further utilized for bacterial growth . Washed (intact) cells, harvested at different periods of bacterial growth on cyanide, consumed oxygen in presence of cyanide . These findings suggest that metabolism of cyanide involved a dioxygenase enzyme that converted cyanide directly to ammonia, without the formation of cyanate. FEMS Microbiol Rev, 1996 May, 18(2-3), 93 - 104 General vectors for archaeal hyperthermophiles: strategies based on a mobile intron and a plasmid; Aagaard C et al.; Although there are currently no cloning and expression vectors available for archaeal hyperthermophiles, small cryptic plasmids have been characterized for these organisms as well as viruses and introns capable of spreading between cells . Below, we review the recent progress in adapting these genetic elements as vectors for Pyrococcus furiosus and Sulfolobus acidocaldarius . An efficient and reliable transformation procedure is described for both organisms . The potential of the mobile intron from Desulfurococcus mobilis, inserted into the bacterial vector pUC18 to generate a new type of vector, was investigated in S . acidocaldarius . A polylinker was inserted upstream from the open reading frame encoding the homing enzyme I-DmoI . Both the polylinker and a 276 bp fragment of the tetracycline gene from pBR322 could be inserted into the intron-plasmid construct and spreading still occurred in the culture of S . acidocaldarius . Experiments are in progress to test the co-mobility of the alcohol dehydrogenase and beta-galactosidase genes from Sulfolobus species with the intron . A shuttle vector pCSV1 was also produced by fusing the pGT5 plasmid from Pyrococcus abyssi and the bacterial vector pUC19 which, on transformation, is stable in both organisms without selection . Growth inhibition studies indicate that both P . furiosus and S . acidocaldarius are sensitive to the antibiotics carbomycin, celesticetin, chloramphenicol and thiostrepton as well as butanol and butylic alcohol . Spontaneous mutants resistant to these drugs have been isolated carrying single site mutations in their 23S rRNA gene; they include mutants of S . acidocaldarius resistant to chloramphenicol, carbomycin and celesticetin with the mutation C2452U and thiostrepton-resistant mutants of P . furiosus carrying the mutation A1067G (both numbers corresponding to Escherichia coli 23S rRNA) . These mutated genes are being developed as selective markers . Moreover, two beta-galactosidase genes from P . furiosus have been cloned as possible phenotypic markers; one of these exhibits maximum activity at 95 degrees C with O-nitrophenyl beta-D-galactopyranoside as substrate. Mutat Res, 1996 May, 368(1), 49 - 57 Detection of genotoxic activity in native hospital waste water by the umuC test; Giuliani F et al.; The genotoxic potential of the waste water of a hospital was evaluated by the umuC test . Within 2 years over 800 native waste water samples were analysed . Genotoxic activity was found in 13% of the samples . The highest genotoxic activity occurred in the morning hours, but genotoxic samples were detected also during the day and at night . 96% of the genotoxic waste water samples revealed a genotoxic potential without growth inhibition of test bacteria monitored as OD600, in the same way as antineoplastic drugs like mitomycin C or cisplatin . 4% of the genotoxic waste water samples showed combined cytotoxic and genotoxic activities as seen in control experiments using glutaraldehyde containing disinfectants and certain antibiotics. J Med Microbiol, 1996 May, 44(5), 362 - 71 Adhesion of enteroaggregative Escherichia coli to formalin-fixed intestinal and ureteric epithelia from children; Hicks S et al.; The adhesion characteristics of enteroaggregative Escherichia coli (EAggEC) to the mucosal surfaces of formalin-fixed paediatric intestinal and ureteral tissue were studied . The technique offers a means of overcoming the problem of limited tissue access in childhood and a way of examining the initial steps of bacterial adhesion . Five EAggEC strains isolated from children with diarrhoea in the UK and a well characterised, prototype EAggEC strain (221) were examined . Five of the six EAggEC strains showed preferential adhesion to jejunal mucosa with limited adhesion to ileum and colon . Five of the six also adhered to ureteric tissue . EAggEC can adhere to proximal, as well as distal, regions of the gastrointestinal tract in children, a previously unrecognised characteristic. Aust N Z J Surg, 1996 May, 66(5), 291 - 3 Genitoperineal gangrene: experience in Singapore; Ong HS et al.; BACKGROUND: The experience with genitoperineal gangrene at the Department of Colorectal Surgery, Singapore General Hospital is documented . METHODS: The results of 12 patients treated during the 5 year period between 1 January 1990 and 31 January 1995 were studied . There were 10 men and two women . The mean age of the patients was 55.2 (range 37-83) years . RESULTS: Perianal pain and swelling were the commonest presentation . However, three patients were admitted in a toxic state with mental confusion . The gangrene progressed from anorectal sepsis in six patients . Five of these patients had diabetes mellitus . Escherichia coli was the commonest organism cultured . Early diagnosis, immediate resuscitation, antibiotics and early aggressive surgery was our management policy . Applying these principles, all except two patients survived (mortality 17%) . One had advanced malignancy and the other had extensive burn injuries . CONCLUSIONS: A high index of suspicion should be maintained in patients with diabetes and with anorectal sepsis . Should genitoperineal gangrene develop, aggressive surgery is often successful. Scand J Immunol, 1996 May, 43(5), 490 - 9 Molecular characterization of MPT83: a seroreactive antigen of Mycobacterium tuberculosis with homology to MPT70; Hewinson RG et al.; The Mycobacterium bovis antigens MPB70 and MPB83 are homologous cross-reactive proteins . It has been reported previously that MPB83 is glycosylated and exists in two forms with apparent molecular masses of 23kDa and 25kDa, whereas the apparent molecular mass of MPB70 is 22kDa . Using a monoclonal antibody, SB10, which recognizes an epitope common to both MPB70 and MPB83, we compared the expression of these proteins in M . bovis BCG, virulent M . bovis and virulent Mycobacterium tuberculosis by Western blotting of bacterial lysates . The previously described pattern of high and low producing substrains of BCG for MPB70 was also applicable for MPB83 . Virulent M . bovis was found to express high levels of MPB70 and MPB83 . Immunoblotting experiments using sera from Balb/c mice infected with live M . tuberculosis H37Rv revealed that although the MPB83 homologue of M . tuberculosis, MPT83, is expressed at low levels in M . tuberculosis when grown in vitro, the protein is highly immunogenic during infection with live bacteria . A clone from a mycobacterial shuttle cosmid library of M . tuberculosis H37Rv was isolated which expressed both MPT70 and MPT83 . Genetic analysis of this cosmid revealed that MPT70 and MPT83 were encoded by separate genes with the gene encoding MPT83 situated 2.4kb upstream of mpt70 . Both genes are transcribed in the same direction . The gene encoding MPT83 was cloned and DNA sequencing revealed an open reading frame of 660bp encoding a protein with a predicted molecular mass of 22kDa . Recombinant MPT83 was expressed in Escherichia coli from the native AUG initiation codon by translational coupling . In E . coli MPT83 was expressed as a 23kDa antigen whereas in the rapid growing mycobacterium Mycobacterium smegmatis the protein was expressed as a 25kDa protein indicating post-translational modification of the protein by M . smegmatis . In recombinant M . smegmatis MPT83 was predominantly cell associated whereas MPT70 was secreted into the culture medium . Amino acid sequence comparison between MPT83 and MPT70 revealed a 61% identity between the proteins, although little homology was apparent at the amino terminus . In MPT83 this region contained a typical lipoprotein signal peptide cleavage motif and a putative signal motif for O glycosylation . Both these motifs were absent from the amino acid sequence of MPT70. J Surg Res, 1996 May, 62(2), 197 - 200 Thymopentin modulates Th1 and Th2 cytokine response and host survival in experimental injury; Braga M et al.; OBJECTIVE: To investigate the impact of thymopentin (Thy) on mortality and in vivo cytokine release in an animal model of gut-derived sepsis which includes different combinations of allogeneic blood transfusion (T) and burn injury plus bacterial gavage (BG) . DESIGN: Randomly controlled experiments . MATERIAL: Two hundred sixteen Balb/c (H-2d) and 50 C3H/HeJ (H-2k) mice . INTERVENTIONS: In the first study 60 Balb/c mice were given Thy (1 mg/kg) . The same day of therapy onset, 40 mice were transfused with allogeneic blood (from C3H/HeJ mice) . The remaining 20 mice received aliquots of saline . Five days post-T, 20 of the 40 transfused mice were subjected to a 20% TBSA thermal injury and simultaneous gavage with 1 x 10(9) Escherichia coli and the other 20 mice underwent a sham burn . The 20 nontransfused mice also received a 20% burn plus bacterial gavage . In all animals Thy was administered for 15 days . Three control groups (n = 20 each) entered the same protocol design, but they did not receive Thy . In the second study 96 animals were randomized to six groups (n = 16 each) according to the above experimental design . Animals were sacrificed by exsanguination after burn or 5 days post-T in nonburned mice to measure TNF-alpha, IL-2, and IL-4 plasma levels . RESULTS: The highest mortality (70%) occurred when T was combined with BG . Thy significantly reduced mortality in both groups that underwent BG, regardless of the association with T . TNF-alpha was detectable in 30% of the tested samples, IL-2 in 50%, and IL-4 in 70% . Thy significantly reduced the levels of IL-4 and increased the production of IL-2 . CONCLUSIONS: The protective effect of Thy in this experimental model may be mediated by modulation of cytokine release. J Bacteriol, 1996 May, 178(10), 2986 - 8 Site-specific proteolysis of the Escherichia coli SecA protein in vivo; Mondigler M et al.; A seven-amino-acid cleavage site specific for tobacco etch virus (TEV) protease was introduced into SecA at two separate positions after amino acids 195 and 252 . Chromosomal wild-type secA was replaced by these secA constructs . Simultaneous expression of TEV protease led to cleavage of both SecA derivatives . In the functional SecA dimer, proteolysis directly indicated surface exposure of the TEV protease cleavage sites . Cleavage of SecA near residue 195 generated an unstable proteolysis product and a secretion defect, suggesting that this approach could be used to inactivate essential proteins in vivo. J Bacteriol, 1996 May, 178(10), 2941 - 7 maoB, a gene that encodes a positive regulator of the monoamine oxidase gene (maoA) in Escherichia coli; Yamashita M et al.; The structural gene for copper- and topa quinone-containing monoamine oxidase (maoA) and an unknown amine oxidase gene have been located at 30.9 min on the Escherichia coli chromosome . Deletion analysis showed that the unknown gene was located within a 1.1-kb cloned fragment adjacent to the maoA gene . The nucleotide sequence of this fragment was determined, and a single open reading frame (maoB) consisting of 903 bp was found . The gene encoded a polypeptide with a predicted molecular mass of 34,619 Da which was correlated with the migration on a sodium dodecyl sulfate-polyacrylamide gel . The predicted amino acid sequence of the MaoB protein was identical to the NH2-terminal amino acid sequence derived by Edman degradation of the protein synthesized under the self-promoter . No homology of the nucleotide sequence of maoB to the sequences of any reported genes was found . However, the amino acid sequence of MaoB showed a high level of homology with respect to the helix-turn-helix motif of the AraC family in its C terminus . The homology search and disruption of maoA on the chromosome led to the conclusion that MaoB is a transcriptional activator of maoA but not an amine oxidase . The consensus sequence of the cyclic AMP-cyclic AMP receptor protein complex binding domain was adjacent to the putative promoter for the maoB gene . By use of lac gene fusions with the maoA and maoB genes, we showed that the maoA gene is regulated by tyramine and MaoB and that the expression of the maoB gene is subject to catabolite repression . Thus, it seems likely that tyramine and the MaoB protein activate the transcription of maoA by binding to the regulatory region of the maoA gene. J Bacteriol, 1996 May, 178(10), 2926 - 33 Overexpression of an mRNA dependent on rare codons inhibits protein synthesis and cell growth; Zahn K; lambda's int gene contains an unusually high frequency of the rare arginine codons AGA and AGG, as well as dual rare Arg codons at three positions . Related work has demonstrated that Int protein expression depends on the rare AGA tRNA . Strong transcription of the int mRNA with a highly efficient ribosome-binding site leads to inhibition of Int protein synthesis, alteration of the overall pattern of cellular protein synthesis, and cell death . Synthesis or stability of int and ampicillin resistance mRNAs is not affected, although a portion of the untranslated int mRNA appears to be modified in a site-specific fashion . These phenotypes are not due to a toxic effect of the int gene product and can be largely reversed by supplementation of the AGA tRNA in cells which bear plasmids expressing the T4 AGA tRNA gene . This indicates that depletion of the rare Arg tRNA due to ribosome stalling at multiple AGA and AGG codons on the overexpressed int mRNA underlies all of these phenomena . It is hypothesized that int mRNA's effects on protein synthesis and cell viability relate to phenomena involved in lambda phage induction and excision. J Bacteriol, 1996 May, 178(10), 2883 - 9 Novel mechanisms of Escherichia coli succinyl-coenzyme A synthetase regulation; Birney M et al.; Low concentrations of ADP are shown to increase the rate of phosphoenzyme formation of E . coli succinyl-coenzyme A (CoA) synthetase (SCS) without altering the fraction of phosphorylated enzyme . This is true when either ATP or succinyl-CoA and Pi are used to phosphorylate the enzyme . The stimulatory effect of ADP is not altered by sample dilution, is retained upon partial purification of the enzyme, and reflects the binding of ADP to a site other than the catalytic site . GDP also alters the phosphorylation of the E . coli SCS but does so primarily by enhancing the level of the phosphoenzyme and only when ATP is used as the phosphate donor . GDP appears to function by neutralizing the action of a specific inhibitory protein . This inhibitor of SCS allows for interconversion of succinate and succinyl-CoA in a manner dissociated from changes in ATP-ADP metabolism . These previously unidentified and varied mechanisms by which SCS is regulated focus attention on this enzyme as an important control point in determining the cell's potential to meet its metabolic demands. J Bacteriol, 1996 May, 178(10), 2846 - 52 A single amino acid change in Escherichia coli glycerol kinase abolishes glucose control of glycerol utilization in vivo; Pettigrew DW et al.; Escherichia coli glycerol kinase (EC 2.7.1.30; ATP:glycerol 3-phosphotransferase) is a key element in glucose control of glycerol metabolism . Its catalytic activity is inhibited allosterically by the glycolytic intermediate, fructose 1,6-biphosphate, and by the phosphotransferase system phosphocarrier protein, IIIGlc (also known as IIAGlc) . These inhibitors provide mechanisms by which glucose blocks glycerol utilization in vivo . We report here the cloning and sequencing of the glpK22 gene isolated from E . C . C . Lin strain 43, a strain that shows the loss of glucose control of glycerol utilization . DNA sequencing shows a single missense mutation that translates to the amino acid change Gly-304 to Ser (G-304-S) in glycerol kinase . The effects of this substitution on the functional and physical properties of the purified mutant enzyme were determined . Neither of the allosteric ligands inhibits it under conditions that produce strong inhibition of the wild-type enzyme, which is sufficient to explain the phenotype of strain 43 . However, IIIGlc activates the mutant enzyme, which could not be predicted from the phenotype . In the wild-type enzyme, G-304 is located 1.3 nm from the active site and 2.5 nm from the IIIGlc binding site (M . Feese, D . W . Pettigrew, N . D . Meadow, S . Roseman, and S . J . Remington, Proc . Natl . Acad . Sci . USA 91:3544-3548, 1994) . It is located in the same region as amino acid substitutions in the related protein DnaK which alter its catalytic and regulatory properties and which are postulated to interfere with a domain closure motion (A . S . Kamath-Loeb, C . Z . Lu, W.-C . Suh, M . A . Lonetto, and C . A . Gross, J . Biol . Chem . 270:30051-30059, 1995) . The global effect of the G-304-S substitution on the conformation and catalytic and regulatory properties of glycerol kinase is consistent with a role for the domain closure motion in the molecular mechanism for glucose control of glycerol utilization. J Bacteriol, 1996 May, 178(10), 2836 - 45 Energy-coupled transport across the outer membrane of Escherichia coli: ExbB binds ExbD and TonB in vitro, and leucine 132 in the periplasmic region and aspartate 25 in the transmembrane region are important for ExbD activity; Braun V et al.; Ferric siderophores, vitamin B12, and group B colicins are taken up through the outer membranes of Escherichia coli cells by an energy-coupled process . Energy from the cytoplasmic membrane is transferred to the outer membrane with the aid of the Ton system, consisting of the proteins TonB, ExbB, and ExbD . In this paper we describe two point mutations which inactivate ExbD . One mutation close to the N-terminal end of ExbD is located in the cytoplasmic membrane, and the other mutation close to the C-terminal end is located in the periplasm . E . coli CHO3, carrying a chromosomal exbD mutation in which leucine at position 132 was replaced by glutamine, was devoid of all Ton-related activities . A plasmid-encoded ExbD derivative, in which aspartate at position 25, the only changed amino acid in the predicted membrane-spanning region of ExbD, was replaced by asparagine, failed to restore the Ton activities of strain CHO3 and negatively complemented ExbD+ strains, indicating an interaction of this mutated ExbD with wild-type ExbD or with another component . This component was shown to be ExbB . ExbB that was labeled with 6 histidine residues at its C-terminal end and that bound to a nickel-nitrilotriacetic acid agarose column retained ExbD and TonB specifically; both were eluted with the ExbB labeled with 6 histidine residues, demonstrating interaction of ExbB with ExbD and TonB . These data further support the concept that TonB, ExbB, and ExbD form a complex in which the energized conformation of TonB opens the channels in the outer membrane receptor proteins. J Bacteriol, 1996 May, 178(10), 2785 - 93 Sequences in the -35 region of Escherichia coli rpoS-dependent genes promote transcription by E sigma S; Wise A et al.; sigma S is an alternate sigma factor which functions with RNA polymerase to activate transcription of genes that are involved in a number of stress responses, including stationary-phase survival and osmoprotection . The similarity of the sigma S protein to sigma D (Escherichia coli's major sigma factor) in the regions thought to recognize and bind promoter sequences suggests that sigma S- and sigma D-associated RNA polymerases recognize promoter DNA in a similar manner . However, no promoter recognition sequence for sigma S holoenzyme (E sigma S) has been identified . An apparent conservation of cytosine nucleotides was noted in the -35 region of several sigma S-dependent promoters . Site-directed mutagenesis and reporter gene fusions were used to investigate the importance of the -35 cytosine nucleotides for sigma S-dependent transcription . Substitution of cytosine nucleotides for thymidine at the -35 site of the sigma D-dependent proU promoter effectively abolished transcription by E sigma D but allowed E sigma S to direct transcription from the mutant promoter . Inclusion of the sigma D consensus -10 hexamer strengthened transcription by E sigma S, demonstrating that both E sigma D and E sigma S can recognize the same -10 sequences . Conversely, replacement of -35 site cytosine nucleotides with thymidine in the sigma S-dependent osmY promoter reduced transcription by E sigma S and increased transcription by E sigma D . Our data suggest that DNA sequences in the -35 region function as part of a discriminator mechanism to shift transcription between E sigma D and E sigma S. Mol Cell Biol, 1996 May, 16(5), 2537 - 44 Strand specificity of mutagenic bypass replication of DNA containing psoralen monoadducts in a human cell extract; Thomas DC et al.; Psoralens are mutagenic compounds of vegetable origin that are used as photosensitizing agents in the treatment of various skin diseases, blood cell cancer, and autoimmune disorders . To study the mechanism of mutagenicity of psoralens in humans, we examined the efficiency and fidelity of simian virus 40 origin-dependent replication in a human cell extract of M13mp2 DNA randomly treated with the psoralen derivative 4'-hydroxymethyl-4,5',8-trimethyl psoralen plus UVA irradiation . Replication of DNA treated with variable amounts of 4'-hydroxymethyl-4,5',8-trimethyl psoralen and a fixed UVA fluence was inhibited in a concentration-dependent manner . However, covalently closed monomer-length circular replication products were observed . Product analysis by renaturing agarose gel electrophoresis after cross-linking with 250- to 280-nm UV light indicated that approximately 1 of 9 psoralen monoadducts was bypassed during in vitro replication . Introduction of product DNA into Escherichia coli to score replication errors in the lacZalpha reporter gene demonstrated that replication of the damaged DNA was more mutagenic than was replication of undamaged DNA . Sequence analysis of lacZ mutants revealed that damage-dependent replication errors were predominantly T.A-->C.G transitions, transversions at C.G base pairs, and deletions of single A.T base pairs, the last occurring most frequently in homopolymeric runs . A comparison of error specificities with two substrates having the replication origin asymmetrically placed on opposite sides of the mutational target suggests that the lagging-strand replication apparatus is less accurate than the leading-strand replication apparatus for psoralen monoadduct-dependent deletion errors . A model is proposed based on the preferential loopout of the monoadducted base from the strand that templates retrograde discontinuous synthesis. Mol Cell Biol, 1996 May, 16(5), 2496 - 503 Identification of neurofibromin mutants that exhibit allele specificity or increased Ras affinity resulting in suppression of activated ras alleles; Morcos P et al.; Neurofibromin plays a critical role in the downregulation of Ras proteins in neurons and Schwann cells . Thus, the ability of neurofibromin to interact with Ras is crucial for its function, as mutations in NF1 that abolish this interaction fail to maintain function . To investigate the neurofibromin-Ras interaction in a systematic manner, we have carried out a yeast two-hybrid screen using a mutant of H-ras, H-rasD92K, defective for interaction with the GTPase-activated protein-related domain (GRD) of NF1 . Two screens of a randomly mutagenized NF1-GRD library led to the identification of seven novel NF1 mutants . Characterization of the NF1-GRD mutants revealed that one class of mutants are allele specific for H-raSD92K . These mutants exhibit increased affinity for H-raSD92K and significantly reduced affinity for wild-type H-ras protein . Furthermore, they do not interact with another H-ras mutant defective for interaction with GTPase-activating proteins . Another class of mutants are high-affinity mutants which exhibit dramatically increased affinity for both wild-type and mutant forms of Ras . They also exhibit a striking ability to suppress the heat shock sensitive traits of activated RAS2G19v in yeast cells . Five mutations cluster within a region encompassing residues 1391 to 1436 (region II) . Three NF1 patient mutations have previously been identified in this region . Two mutations that we identified occur in a region encompassing residues 1262 to 1276 (region I) . Combining high-affinity mutations from both regions results in even greater affinity for Ras . These results demonstrate that two distinct regions of NF1-GRD are involved in the Ras interaction and that single amino acid changes can affect NF1's affinity for Ras. Mol Cell Biol, 1996 May, 16(5), 2283 - 94 Role of EGR1 in regulation of stimulus-dependent CD44 transcription in B lymphocytes; Maltzman JS et al.; The immediate-early gene egr-1 encodes a transcription factor (EGR1) that links B-cell antigen receptor (BCR) signals to downstream activation events through the regulation of previously unidentified target genes . Here we identify the gene encoding the lymphocyte homing and migration protein CD44 as a target of EGR1 regulation in B cells . BCR-induced increases in CD44 mRNA expression and transcription levels are shown to occur in EGR1-expressing but not in nonexpressing subclones of the B-cell line WEHI-231 . Kinetics of egr-1 transcription and the appearance of nuclear EGR1 protein precede CD44 induction and occur within 30 min after stimulation in the EGR1-expressing subclone . A single EGR1 binding motif is demonstrated at bp -301 of the human CD44 promoter . Cotransfection of a CD44 promoter-chloramphenicol acetyltransferase reporter construct with an egr-1 expression vector resulted in a 6.5- to 8.5-fold induction of transcriptional activity relative to an empty expression vector . The EGR1 binding motif was shown to be necessary for stimulus-induced expression of a CD44 promoter-chloramphenicol acetyltransferase reporter construct in nontransformed B lymphocytes and was required for transactivation by an EGR1 expression vector in a B-cell line . These studies identify EGR1 as an intermediary linking BCR-derived signals to the induction of CD44 . The relevance of these molecular events to BCR signal transduction and antigen-stimulated B-cell-mediated immune responses is discussed. Mol Cell Biol, 1996 May, 16(5), 2065 - 73 Characterization of the cooperative function of inhibitory sequences in Ets-1; Jonsen MD et al.; DNA binding by the eukaryotic transcription factor Ets-1 is negatively regulated by an intramolecular mechanism . Quantitative binding assays compared the DNA-binding activities of native Ets-1, three deletion mutants, and three tryptic fragments . Ets-1 and activated Ets-1 polypeptides differed in DNA-binding affinity as much as 23-fold . Inhibition was mediated by two regions flanking the minimal DNA-binding domain . Both regions regulated affinity by enhancing dissociation of the protein-DNA complex . Three lines of evidence indicated that inhibition requires cooperative interaction between the two regions: first, the two inhibitory regions acted through a common mechanism; second, neither region functioned independently of the other; finally, mutation of the C-terminal inhibitory region altered the conformation of the N-terminal inhibitory region . In addition, partial proteolysis detected an identical altered conformation in the N-terminal inhibitory region of Ets-1 bound to DNA . This finding suggested that repression is transiently disrupted during DNA binding . These results provide evidence that the two inhibitory regions of Ets-1 are structurally, as well as functionally, coupled . In addition, conformational change is shown to be a critical component of the inhibition mechanism . A cooperative, allosteric model of autoinhibition is described . Autoinhibition of Ets-1 could be relieved by either protein partner(s) or posttranslational modifications. J Virol, 1996 May, 70(5), 3313 - 8 Analysis of Rous sarcoma virus Gag protein by mass spectrometry indicates trimming by host exopeptidase; Pepinsky RB et al.; We have used electrospray ionization-mass spectrometry to investigate Gag protein structure and processing in Rous sarcoma virus, the prototype of the avian sarcoma and leukemia viruses . Molecular masses determined for the mature virion proteins MA, CA, NC, and PR agree closely with those predicted by currently accepted models for their structures . However, the data for p10 imply that only about 10% of the product has the predicted mass while the remainder is missing the C-terminal methionine residue . Molecular masses also were obtained for products generated by PR cleavage in vitro of a Gag precursor polyprotein expressed in Escherichia coli . The data confirm the predicted Gag cleavage sites for PR . Thus, carboxypeptidase activity appears to be responsible for generating the des-Met form of p10 . The same activity may account for the small amount of the mature des-Met CA, as previously reported . Analysis of cleavage products generated in vitro also serves to define the PR processing site separating the p2a and p2b peptides, Asn-164-Cys-165 . In conjunction with published characterizations of these two peptides processed from the segment of Gag between MA and p10, these data suggest trimming of p2b by an aminopeptidase . Finally, the molecular masses determined for the MA-related species p19f, p23, and p35 now accurately define the structures of these proteins. J Virol, 1996 May, 70(5), 3302 - 6 Human endogenous retrovirus K10 encodes a functional integrase; Kitamura Y et al.; We cloned a human endogenous retrovirus K1O DNA fragment encoding integrase and expressed it as a fusion protein with Escherichia coli maltose-binding protein . Integrase activities were measured in vitro by using a double-stranded oligonucleotide as a substrate mimicking viral long terminal repeats (LTR) . The fusion protein was highly active for both terminal cleavage and strand transfer in the presence of Mn2+ on the K1O LTR substrate . It was also active on both Rous sarcoma virus and human immunodeficiency virus type 1 LTR substrates, whereas Rous sarcoma virus and human immunodeficiency virus type 1 integrases were active only on their corresponding LTR substrates . The results strongly suggest that K1O encodes a functional integrase with relaxed substrate specificity. J Virol, 1996 May, 70(5), 3018 - 25 Human cytomegalovirus uracil DNA glycosylase is required for the normal temporal regulation of both DNA synthesis and viral replication; Prichard MN et al.; Human cytomegalovirus (CMV) encodes a gene, UL114, whose product is homologous to the uracil DNA glycosylase and is highly conserved in all herpesviruses . This DNA repair enzyme excises uracil residues in DNA that result from the misincorporation of dUTP or spontaneous deamination of cytosine . We constructed a recombinant virus, RC2620, that contains a large deletion in the UL114 open reading frame and carries a 1.2-kb insert containing the Escherichia coli gpt gene . RC2620 retains the capacity to replicate in primary human fibroblasts and reaches titers that are similar to those produced by the parent virus but exhibits a significantly longer replication cycle . Although the rate of expression of alpha and beta gene products appears to be unaffected by the mutation, DNA synthesis fails to proceed normally . Once initiated, DNA synthesis in mutant virus-infected cells proceeds at the same rate as with wild-type virus, but initiation is delayed by 48 h . The mutant virus also exhibits two predicted phenotypes: (i) hypersensitivity to the nucleoside analog 5-bromodeoxyuridine and (ii) retention of more uracil residues in genomic DNA than the parental virus . Together, these data suggest UL114 is required for the proper excision of uracil residues from viral DNA but in addition plays some role in establishing the correct temporal progression of DNA synthesis and viral replication . Although such involvement has not been previously observed in herpesviruses, a requirement for uracil DNA glycosylase in DNA replication has been observed in poxviruses. J Virol, 1996 May, 70(5), 2982 - 91 Biophysical characterization of recombinant proteins expressing the leucine zipper-like domain of the human immunodeficiency virus type 1 transmembrane protein gp41; Shugars DC et al.; Envelope oligomerization is thought to serve several crucial functions during the life cycle of human immunodeficiency virus type 1 (HIV-1) . We recently reported that virus entry requires coiled-coil formation of the leucine zipper-like domain of the HIV-1 transmembrane envelope glycoprotein gp41 (C . Wild, T . Oas, C . McDanal, D . Bolognesi, and T . Matthews, Proc . Natl . Acad . Sci . USA 89:10537-10541, 1992; C . Wild, J . W . Dubay, T . Greenwell, T . Baird, Jr., T . G . Oas, C . McDanal, E . Hunter, and T . Matthews, Proc . Natl . Acad . Sci . USA 91:12676-12680, 1994) . To determine the oligomeric state mediated by this region of the envelope, we have expressed the zipper motif as a fusion partner with the monomeric maltose-binding protein of Escherichia coli . The biophysical properties of this protein were characterized by velocity and equilibrium sedimentation, size exclusion chromatography, light scattering, and chemical cross-linking analyses . Results indicate that the leucine zipper sequence from HIV-1 is capable of multimerizing much larger and otherwise monomeric proteins into extremely stable tetramers . Recombinant proteins containing an alanine or a serine substitution at a critical isoleucine residue within the zipper region were also generated and similarly analyzed . The alanine- and serine-substituted proteins behaved as tetrameric and monomeric species, respectively, consistent with the influence of these same substitutions on the helical coiled-coil structure of synthetic peptide models . On the basis of these findings, we propose that the fusogenic gp4l structure involves tetramerization of the leucine zipper domain which is situated approximately 30 residues from the N-terminal fusion peptide sequence. J Virol, 1996 May, 70(5), 2950 - 6 Locations of herpes simplex virus type 2 glycoprotein B epitopes recognized by human serum immunoglobulin G antibodies; Goade DE et al.; Herpes simplex virus type 2 (HSV-2) glycoprotein B (gB-2) gene segments were expressed as recombinant proteins in Escherichia coli . gB-2 recombinant proteins were reacted with human serum immunoglobulin G (IgG) antibodies in Western immunoblot assays . Initially, samples were tested for the presence of HSV-1-specific antibodies and HSV-2-specific antibodies by using HSV-infected cell lysates as antigen targets in Western blot assays . Serum samples that contained HSV-2-specific IgG (n = 58), HSV-1-specific IgG (n = 33), or no detectable HSV antibodies (n = 31) were tested for reactivities with the gB-2 recombinant proteins . In 58 of 58 samples that contained HSV-2-specific IgG, antibodies were present that reacted strongly with a gB-2 amino-proximal segment between amino acids (aa) 18 and 75 . Three of 33 serum samples that contained HSV-1- and not HSV-2-specific IgG (as defined by the HSV lysate Western blot assay) reacted with this segment . Both HSV-2 antibodies and HSV-1 antibodies reacted strongly with a carboxy-terminal gB-2 segment between aa 819 and 904; a second minor cross-reactive region was mapped to a gB-2 segment between aa 564 and 626 . The gB-2 segment from aa 18 to 75 may constitute a useful reagent for the virus type-specific serodiagnosis of HSV-2 infections . Further studies will be required to determine the relative sensitivities and specificities of the assay for gB-2 aa 18 to 75, HSV gG assays, and HSV lysate Western blot assays for detecting virus type-specific antibody responses in acute and chronic HSV-2 infections. J Virol, 1996 May, 70(5), 2697 - 705 Identification and characterization of a filament-associated protein encoded by Amsacta moorei entomopoxvirus; Alaoui-Ismaili MH et al.; A novel protein which is expressed at high levels in insect cells infected with Amsacta moorei entomopoxvirus was identified by our laboratory . This viral gene product migrates as a 25/27-kDa doublet when subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels . It is expressed at late times of infection and is present in infected cells but is absent in purified extracellular virions and occlusion bodies . The gene encoding this polypeptide was mapped on the viral genome, and cDNA clones were generated and sequenced . The predicted protein was shown to be phosphorylated and contained an unusual 10-unit proline-glutamic acid repeat element . A polyclonal antiserum was produced against a recombinant form of the protein expressed in Escherichia coli, and a monoclonal antibody which reacted with the proline-glutamic acid motif was also identified . Immunofluorescence and immunoelectron microscopy techniques revealed that this protein is associated with large cytoplasmic fibrils which accumulate in the cytoplasm between 96 and 120 h postinfection . We subsequently called this viral polypeptide filament-associated late protein of entomopoxvirus . The fibrils containing this polypeptide are closely associated with occlusion bodies and may play a role in their morphogenesis and maturation. J Bacteriol, 1996 May, 178(9), 2718 - 20 Synonymous codon selection controls in vivo turnover and amount of mRNA in Escherichia coli bla and ompA genes; Deana A et al.; A number of silent codon changes were made in two Escherichia coli genes . For the ompA gene, the replacement of seven consecutive frequently used codons with synonymous infrequently used codons reduced the ompA mRNA level and its half-life . For the bla gene, the exchange of 24 codons for the most frequently used synonymous codons extended the bla mRNA half-life . A modification of ribosome traffic could account for these observations. J Bacteriol, 1996 May, 178(9), 2715 - 7 Integration host factor amplifies the induction of the aceBAK operon of Escherichia coli by relieving IclR repression; Resnik E et al.; A binding site for integration host factor (IHF) was identified upstream of the aceBAK promoter . Under inducing conditions, IHF activates aceB::lacZ expression by opposing IclR repression . In contrast, IHF has little effect on aceB::lacZ expression under repressing conditions . The ability of IHF to relieve repression under inducing but not repressing conditions allows this protein to amplify the induction of aceBAK. J Bacteriol, 1996 May, 178(9), 2712 - 4 Intracellular inducer Hg2+ concentration is rate determining for the expression of the mercury-resistance operon in cells; Yu H et al.; Experiments involving mercury resistance mer operon-lacZ fusions, point mutations in the mercuric ion reductase merA gene, and transcomplementation have revealed that in Hg2+-resistant cells, the inducer Hg2+ concentration is rate determining for activation of transcription . mer operon expression is activated by the presence of nanomolar concentrations of Hg2+ in liquid media only when the mercuric ion reductase function is artificially inactivated in cells, whereas cells with active mercuric ion reductase require micromolar concentrations of Hg2+ for effective induction of the operon. J Bacteriol, 1996 May, 178(9), 2662 - 7 The apparent coupling between synthesis and posttranslational modification of Escherichia coli acyl carrier protein is due to inhibition of amino acid biosynthesis; Keating DH et al.; Acyl carrier protein (ACP) is modified on serine 36 by the covalent posttranslational attachment of 4'-phosphopantetheine from coenzyme A (CoA), and this modification is required for lipid biosynthesis . Jackowski and Rock (J . Biol . Chem 258:15186-15191, 1983) reported that upon depletion of the CoA pool by starvation for a CoA precursor, no accumulation of the unmodified form of ACP (apo-ACP) was detected . We report that this lack of apo-ACP accumulation results from decreased translation of the acpP mRNAs because of the limitation of the synthesis of glutamate and other amino acids made directly from tricarboxylic acid cycle intermediates. J Bacteriol, 1996 May, 178(9), 2629 - 36 How to achieve constitutive expression of a gene within an inducible operon: the example of the nagC gene of Escherichia coli; Plumbridge J; The nagC gene, encoding the NagC repressor/activator of the nag regulon, is part of the nagBACD operon . When the promoter-proximal nagB and nagA genes are induced 20- to 40-fold, the nagC gene is induced only two- to threefold . In addition to being transcribed as part of the polycistronic nagBACD mRNA, nagC is also expressed from two promoters located within the upstream nagA gene . These promoters are comparable in strength to the induced nagB promoter, resulting in a high basal level of the nagC mRNA . This means that when the nagBA genes are induced, there is a much smaller effect on the amount of nagC mRNA . The nagC gene is subject to low-level translation so that the amount of NagC protein is kept low despite the relatively high transcription levels. J Bacteriol, 1996 May, 178(9), 2613 - 28 Enteropathogenic Escherichia coli: identification of a gene cluster coding for bundle-forming pilus morphogenesis; Sohel I et al.; Sequence flanking the bfpA locus on the enteroadherent factor plasmid of the enteropathogenic Escherichia coli (EPEC) strain B171-8 (O111:NM) was obtained to identify genes that might be required for bundle-forming pilus (BFP) biosynthesis . Deletion experiments led to the identification of a contiguous cluster of at least 12 open reading frames, including bfpA, that could direct the synthesis of a morphologically normal BFP filament . Within the bfp gene cluster, we identified open reading frames that share homology with other type IV pilus accessory genes and with genes required for transformation competence and protein secretion . Immediately upstream of the bfp gene cluster, we identified a potential replication origin including genes that are predicted to encode proteins homologous with replicase and resolvase . Restriction fragment length polymorphism analysis of DNA from six additional EPEC serotypes showed that the organization of the bfp gene cluster and its juxtaposition with a potential plasmid origin of replication are highly conserved features of the EPEC biotype. J Bacteriol, 1996 May, 178(9), 2580 - 5 Specific in vivo protein-protein interactions between Escherichia coli SOS mutagenesis proteins; Jonczyk P et al.; One of the components of the RecA-LexA-controlled SOS response in Escherichia coli cells is an inducible error-prone DNA replication pathway that results in a substantial increase in the mutation rate . It is believed that error-prone DNA synthesis is performed by a multiprotein complex that is formed by UmuC, UmuD', RecA, and probably DNA polymerase III holoenzyme . It is postulated that the formation of such a complex requires specific interactions between these proteins . We have analyzed the specific protein-protein interactions between UmuC, UmuD, and UmuD' fusion proteins, using a Saccharomyces cerevisiae two-hybrid system . In agreement with previous in vitro data, we have shown that UmuD and UmuD' are able to form both homodimers (UmuD-UmuD and UmuD'-UmuD') and a heterodimer (UmuD-UmuD') . Our data show that UmuC fusion protein is capable of interacting exclusively with UmuD' and not with UmuD . Thus, posttranslational processing of UmuD into UmuD' is a critical step in SOS mutagenesis, enabling only the latter protein to interact with UmuC . Our data seem to indicate that the integrity of the entire UmuC sequence is essential for UmuC-UmuD' heterotypic interaction . Finally, in our studies, we used three different UmuC mutant proteins: UmuC25, UmuC36, and UmuC104 . We have found that UmuC25 and UmuC36 are not capable of associating with UmuD' . In contrast, UmuC104 protein interacts with UmuD' protein with an efficiency identical to that of the wild-type protein . We postulate that UmuC104 protein might be defective in interaction with another, unknown protein essential for the SOS mutagenesis pathway. J Bacteriol, 1996 May, 178(9), 2559 - 63 Substitution of mucAB or rumAB for umuDC alters the relative frequencies of the two classes of mutations induced by a site-specific T-T cyclobutane dimer and the efficiency of translesion DNA synthesis; Szekeres ES Jr et al.; We have examined the effect of replacing umuDC with mucAB or rumAB on the mutagenic properties of a T-T cyclobutane dimer in an attempt to determine the molecular basis for the differences in UV-induced mutagenesis that are associated with these structurally and functionally related genes . A single-stranded vector carrying a site-specific T-T cis-syn cyclobutane dimer was transfected into a set of isogenic Escherichia coli delta umuDC strains harboring low-copy-number plasmids expressing UmuDC, MucAB, RumAB, or their genetically engineered and mutagenically active counterparts UmuD'C, MucA'B, and RumA'B, respectively . Although the overall mutation frequency was similar for all strains, the relative frequencies of the two classes of mutation induced by the T-T dimer varied according to the mutagenesis operon expressed . In umuDC strains, 3' T-->A mutations outnumbered 3' T-->C mutations, but the reverse was true for the mucAB and rumAB strains . We also found that the T-T dimer was bypassed with differing efficiencies in unirradiated cells expressing wild-type UmuDC, MucAB, and RumAB proteins . These differences can probably be attributed to the relative efficiency of the normal cellular posttranslational activation of UmuD, MucA, and RumA, respectively, since recombinant constructs expressing the mutagenically active UmuD'C, MucA'B, and RumA'B proteins all promoted similarly high levels of bypass in UV-irradiated cells . These results suggest that the UmuD'/UmuC complex and its homologs may differ in their relative abilities to promote elongation from T - T and T - G mismatched termini . Alternatively, they may differentially influence the efficiency with which these mismatches are edited or influence nucleotide insertion by the catalytic subunit of the DNA polymerase III. J Bacteriol, 1996 May, 178(9), 2539 - 50 Characterization of the regulatory region of a cell interaction-dependent gene in Myxococcus xanthus; Fisseha M et al.; omega 4403 is the site of a Tn5 lac insertion in the Myxococcus xanthus genome that fuses lacZ expression to a developmentally regulated promoter . Cell-cell interactions that occur during development, including C-signaling, are required for expression of Tn5 lac omega 4403 . We have cloned DNA upstream of the omega 4403 insertion site, localized the promoter, and identified a potential open reading frame . From the deduced amino acid sequence, the gene disrupted by Tn5 lac omega 4403 appears to encode a serine protease that is dispensable for development . The gene begins to be expressed between 6 and 12 h after starvation initiates development, as determined by measuring mRNA or beta-galactosidase accumulation in cells containing Tn5 lac omega 4403 . The putative transcriptional start site was mapped, and sequences centered near -10 and -35 bp relative to this site show some similarity to the corresponding regions of promoters transcribed by Escherichia coli sigma70 RNA polymerase . However, deletions showed that an essential promoter element lies between -80 and -72 bp, suggesting the possible involvement of an upstream activator protein . DNA downstream of -80 is sufficient for C-signal-dependent activation of this promoter . The promoter is not fully expressed when fusions are integrated at the Mx8 phage attachment site in the chromosome . Titration of a limiting factor by two copies of the regulatory region (one at the attachment site and one at the native site) can, in part, explain the reduced expression . We speculate that the remaining difference may be due to an effect of chromosomal position . These results provide a basis for studies aimed at identifying regulators of C-signal-dependent gene expression. Dev Biol, 1996 May 1, 175(2), 314 - 24 Regulation of two pair-rule stripes by a single enhancer in the Drosophila embryo; Small S et al.; Previous studies on the regulation of the segmentation gene even-skipped (eve) have centered on the transcription of stripe 2 . Here, we characterize another enhancer module contained within the complex eve promoter that directs expression of stripes 3 and 7 . This enhancer is approximately 500 bp in length and maps approximately 3.3 kb upstream of the transcription start site . The stripe 3 + 7 enhancer appears to be regulated by one or more ubiquitously distributed activators, including components of a JAK-Stat pathway . The two-stripe pattern results via multiple tiers of repressors which delimit this ubiquitous activation . The zinc finger repressor hunchback appears to be responsible for establishing the anterior border of stripe 3 and the posterior border of stripe 7 . knirps, a member of the nuclear receptor family of transcription factors, appears to establish the posterior border of stripe 3 and the anterior border of stripe 7 . Activator and repressor proteins bind in vitro to several sites within the enhancer . These findings suggest a general model for the regulation of segmentation stripes, whereby enhancers integrate positional information provided by broadly distributed activators and spatially restricted repressors. Dig Dis Sci, 1996 May, 41(5), 1030 - 7 Role of platelet-activating factor in pathogenesis of galactosamine-lipopolysaccharide-induced liver injury; Komatsu Y et al.; In an attempt to clarify the role of platelet-activating factor (PAF) in the pathogenesis of hepatic injury induced by galactosamine (GalN) plus lipopolysaccharide (LPS), effects of WEB 2086 (PAF receptor antagonist) on hepatic injury in vivo as well as on neutrophil adherence to hepatic endothelial cells in vitro have been investigated, as we have recently clarified the role of neutrophils in this experimental model of hepatic injury . Although an enhanced serum TNF-alpha level after GalN-LPS administration was not reduced by WEB 2086, hepatic injury and hepatic neutrophil accumulation in the liver after GalN-LPS administration were attenuated by WEB 2086 . An in vitro study revealed that an enhanced neutrophil adhesion to hepatic endothelial cells by stimulation with the sera that were collected from the GalN-LPS-treated rats, was reduced in the presence of WEB 2086 in a dose-dependent manner . In addition, LPS, TNF-alpha, and PAF were found to enhance the neutrophil adherence to hepatic endothelial cells, which was reduced in the presence of WEB 2086 . These results suggest that PAF play an important role in the GalN-LPS induced hepatic injury and that PAF receptor antagonist reduces the neutrophil adherence to hepatic endothelial cells in the liver. Clin Exp Immunol, 1996 May, 104(2), 351 - 8 Production of neutralizing granulocyte-macrophage colony-stimulating factor (GM-CSF) antibodies in carcinoma patients following GM-CSF combination therapy; Wadhwa M et al.; In this study, the development of neutralizing and non-neutralizing GM-CSF antibodies and the clinical consequences related to the induction of these antibodies were analysed in 20 patients with metastatic colorectal carcinoma receiving a combination therapy of Escherichia coli-derived GM-CSF and a colon carcinoma-reactive MoAb in the absence of any concomitant chemotherapy . The recombinant human GM-CSF was administered subcutaneously for 10 days every month for 4 months . Following the first cycle of treatment, no GM-CSF antibodies were detected, but during subsequent therapy, 19 of the 20 patients studied developed GM-CSF binding antibodies . However, only a proportion (40%) of the 19 antibody-positive patients developed antibodies that neutralized the biological activity of GM-CSF in an in vitro bioassay . The presence of GM-CSF neutralizing antibodies was associated with a significant reduction in GM-CSF-induced expansion of leucocytes, neutrophils and eosinophils . Such clinical effects were not apparent in patients with non-neutralizing antibodies . Further characterization of sera from patients with neutralizing antibodies showed that, in most cases, the antibodies neutralized the biological activity of GM-CSF preparations derived using different expression systems (Chinese hamster ovary cells and yeast), suggesting that these antibodies may have the potential to cross-react with endogenously produced GM-CSF . These effects should be considered before therapeutic use of cytokines, particularly in patients who are not immunosuppressed, and therefore capable of mounting an effective immune response . Our results indicate that assessment of production of neutralizing antibodies induced during cytokine therapy can be used to predict diminished clinical response to further therapy. Virology, 1996 May 1, 219(1), 77 - 86 Catalytic activities of the human T-cell leukemia virus type II integrase; Balakrishnan M et al.; Despite the widespread nature of HTLV-II in New World populations and intravenous drug users, the enzymatic activities of the pol genes have not been reported . To ascertain the activity of the HTLV-II(G12) integrase (IN), the coding region was isolated and the encoded protein was purified, using nickel-affinity chromatography, to greater than 90% homogeneity . HTLV-II(G12) IN proved active on HTLV-II(G12) and HIV-1 integration and disintegration substrates . Distinct differences in requirements for enzyme concentration for 3'-processing, strand-transfer, and disintegration reactions were observed . Catalysis of integration reactions occurred in the presence of either Mn2+ or Mg2+, although strand-transfer activity preferred Mn2+ . In comparison, HTLV-II(G12) IN catalyzed disintegration reactions with almost 10-fold less protein, was not selective for Mn2+ or Mg2+, and tolerated higher NaCl concentrations than integration . HTLV-II(G12) IN was unable to catalyze the "splicing" reaction, which suggests that this may not be an activity ubiquitous to all retroviral integrases. Virology, 1996 May 1, 219(1), 115 - 24 The Escherichia coli retrons Ec67 and Ec86 replace DNA between the cos site and a transcription terminator of a 186-related prophage; Dodd IB et al.; Retrons are unusual, reverse transcriptase-encoding elements found in bacteria . Although there are a number of indications that retrons are mobile elements, their transposition has not been observed . The Escherichia coli retrons Ec67 and Ec86 are different retrons inserted at the same site and we have further characterized this site in search of clues to the mechanism of retron transposition . We confirm, by extending previous sequence analysis, that Ec67 and Ec86 are inserted into prophages related to coliphage 186 . Comparison with the recently published sequence of the 186 96-2% region indicates that the retrons have replaced approximately 180 bp of DNA between the phage cohesive end site (cos) and the transcription terminator of a phage DNA-packaging gene . These features--DNA replacement at the insertion site and the location of retron junctions near transcription terminators or DNA cleavage sites--are shared with other retrons and suggest ways in which retron transposition might have occurred. Virology, 1996 May 1, 219(1), 105 - 14 Defining the SOS operon of coliphage 186; Brumby AM et al.; We have sequenced the LexA-controlled operon of coliphage 186 that carries the tum gene, whose product is necessary for UV induction of the 186 prophage . The operon consists of orf95 and orf97, and we have identified orf95 as the tum gene . The major translation products from orf95 result from internal initiations and modulate Tum activity . Tum is the product of the full-length Orf95 protein . The second gene of the operon, orf97, is of unknown function but, while it has little effect on prophage induction, its presence in the cell totally blocks infection of that cell by 186. J Lab Clin Med, 1996 May, 127(5), 448 - 55 Lipopolysaccharide increases fibronectin production and release from cultured lung fibroblasts partially through proteolytic activity; Adachi Y et al.; Fibronectin is a major product of fibroblasts and can mediate diverse functions including wound healing . Chronic bacterial infections are generally associated with a marked decreased in the ability to repair . We therefore hypothesized that bacterial endotoxin, lipopolysaccharide (LPS), might alter fibroblast fibronectin production . LPS augmented fibronectin production by fibroblasts and also stimulated the release of fibronectin from cell layers . An increase in new protein synthesis appeared to account for part of the increased fibronectin, because the inhibitor of protein synthesis, cycloheximide, inhibited the increase in total production of fibronectin . Cycloheximide did not attenuate the increased release of fibronectin into the culture medium . This increased release appeared to be caused, at least in part, by fragmentation of fibronectin by proteases contained in LPS preparations . In this regard all preparations of LPS tested were found to cleave fibronectin . Finally, zymograms indicated that LPS could also cleave gelatin with at least two bands of proteolytic activity but that it did not cleave bovine serum albumin or ovalbumin . These results indicate that the ability of bacterial products to alter fibronectin production and to degrade this macromolecule may account for altered wound repair that occurs with chronic bacterial infection. Arch Biochem Biophys, 1996 May 1, 329(1), 73 - 81 The ubiquitously expressed human CYP51 encodes lanosterol 14 alpha-demethylase, a cytochrome P450 whose expression is regulated by oxysterols; Stromstedt M et al.; Sterol biosynthesis requires the removal of the 14 alpha-methyl group from lanosterol in animals and fungi and from obtusifoliol in plants . This reaction is catalyzed by a microsomal cytochrome P450, the sterol 14 alpha-demethylase (P450(14DM), which is the only P450 described so far to be expressed in different phyla . A cDNA encoding human P450(14DM) was isolated from a liver cDNA library using a partial rat lanosterol 14 alpha-demethylase cDNA probe . The deduced amino acid sequence is 93% and 38--42% identical to rat and fungal P450(14DM), respectively . Expression of the human CYP51 cDNA in Escherichia coli showed that the cDNA encodes an enzyme having lanosterol 14 alpha-demethylase activity . Northern blot analysis showed that CYP51 mRNA is ubiquitously expressed with highest levels in testis, ovary, adrenal, prostate, liver, kidney, and lung . Many genes involved in cholesterol homeostasis are regulated by cholesterol or its metabolites . In the case of CYP51, cholesterol deprivation led to a 2.6- to 3.8-fold induction of mRNA levels in human adrenocortical H295R cells and this effect was suppressed by the addition of 25-hydroxycholesterol . In human hepatoma HepG2 cells, no effect of cholesterol deprivation was observed; however, the levels of CYP51 mRNA were reduced 4- to 6-fold by the addition of 25-hydroxycholesterol . Thus, like several other genes in the cholesterol biosynthetic pathway, including the 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase, HMG CoA reductase, squalene synthase, and farnesyl diphosphate synthase, the expression of the human CYP51 is suppressed by oxysterols. J Immunol, 1996 May 1, 156(9), 3389 - 401 Chemotactic factors stimulate CD18-dependent canine neutrophil adherence and motility on lung fibroblasts; Burns AR et al.; The mechanisms by which neutrophils migrate through the alveolar interstitium during acute lung inflammation are unknown . We wished to determine whether platelet-activating factor (PAF) and IL-8, two important mediators in neutrophil transendothelial migration, stimulated neutrophil adherence and motility on lung fibroblasts . Canine fibroblasts grown from lung explants were characterized by light and electron microscopy, and flow cytometry . Unstimulated neutrophils adhered poorly (less than 2%) to cultured fibroblasts . However, neutrophils stimulated with PAF (20-200 nM) showed a dose-dependent increase in adherence that was largely (70%) mediated by the beta 2 (CD11/CD18) integrins; adherence was less dependent (50%) on fibroblast intercellular adhesion molecule-1 . Conversely, neutrophils stimulated with canine rIL-8 did not increase their adherence to fibroblasts . PAF-stimulated neutrophils were nonmotile on the surface of the fibroblast, but subsequent addition of rIL-8 (10(-8) M) induced motility that was entirely CD1 8 dependent . Fibroblasts stimulated with human rTNF-alpha or Escherichia coli endotoxin (LPS) were a significant source of IL-8 mRNA . In response to rTNF-alpha (50 U/ml), IL-8 mRNA was detected at 2 h by northern blot analysis; it peaked at 6 h and returned to baseline by 24 h . Fibroblasts stimulated with rTNF-alpha secreted IL-8 protein into the culture medium; secreted IL-8 was chemotactic for neutrophils . These data suggest that fibroblasts can function not only as an adhesive substrate, but also as a source of stimulation for neutrophil migration through the inflamed alveolar interstitium. Nat Med, 1996 May, 2(5), 581 - 4 Inducible nitric oxide synthase gene expression in the brain during systemic inflammation; Wong ML et al.; Inducible nitric oxide synthase (iNOS) is a transcriptionally regulated enzyme that synthesizes nitric oxide from L-arginine that has a key role in the pathophysiology of systemic inflammation and sepsis . Transgenic animals with a null mutation for the iNOS gene are resistant to hypotension and death caused by Escherichia coli lipopolysaccharide (LPS) . The regulation of peripheral iNOS has been well studied in sepsis, but little is known about iNOS regulation in the brain during systemic inflammation or sepsis . We know that at baseline there is no detectable iNOS gene expression in the brain, but a detailed neuroanatomical study reveals that early in the course of systemic inflammation there is a profound induction of iNOS messenger RNA in vascular, glial and neuronal structures of the rat brain, accompanied by the production of nitric oxide (NO) metabolites in brain parenchyma and cerebrospinal fluid (CSF) . We propose that the spillover of nitrite into the CSF has the potential to be a diagnostic marker for systemic inflammation and sepsis . Pharmacological interventions aimed at regulating iNOS function in the brain might represent a new treatment strategy in sepsis . Brain iNOS may be relevant to the pathophysiology, diagnosis and treatment of systemic inflammation and sepsis. J Trauma, 1996 May, 40(5), 702 - 9 Immunosuppression after endotoxin shock: the result of multiple anti-inflammatory factors; Junger WG et al.; OBJECTIVES: Endotoxin induced suppression of cellular immune function is thought to contribute to septic complications in trauma patients . A rabbit model of endotoxemia was used to determine the relative roles of the anti-inflammatory factors interleukin-4 (IL-4), interleukin-10 (IL-10), transforming growth factor beta1 (TGFbeta1), and prostaglandin E2 (PGE2) in addition to other factors, in inducing immunosuppression . DESIGN: T-cell suppressive factors (TSF) in serum ultrafiltrates were separated and tested for the presence of the known suppressive factors PGE2, IL-4, IL-10, and TGFbeta1 . MATERIAL AND METHODS: New Zealand rabbits were injected with 50 microg/kg of purified Escherichia coli lipopolysaccharide . Animals were exsanguinated after 48 hours and serum was separated by ultrafiltration (cutoff 50 kd), TSK HW-40 size exclusion chromatography, and Q-Sepharose anion exchange chromatography . TSF activities of chromatographic fractions and serum samples were measured with a mitogen induced in vitro T-cell proliferation assay . Levels of PGE2, IL-4, IL-10, and TGFbeta1 were measured with enzyme immunoassays . MEASUREMENTS AND MAIN RESULTS: Serum TSF activity, and levels of PGE2, IL-4, IL-10, and TGFbeta1 were increased after endotoxemia . Size exclusion chromatography revealed three major fractions (TSF1-3) with up to 600 times more TSF activity compared with controls . IL-4 and IL-10 were found in TSF1 and TSF3 . Further separation of TSF1 by anion exchange chromatography revealed a total of eight different T-cell suppressive factors . TGFbeta1 probably remained in the retentate after ultrafiltration, while PGE2 eluted at a higher retention time . The known anti-inflammatory factors TGFbeta1, IL-10, IL-4, and PGE2 only accounted for 13% of the total serum TSF activity of 614 U/mL . CONCLUSIONS: Lipopolysaccharide shock results in the release of multiple T-cell suppressive factors in addition to known immunosuppressive factors, all of which contribute to the anti-inflammatory response. J Trauma, 1996 May, 40(5), 688 - 92; discussion 692-3 Starvation and endotoxin act independently and synergistically to coordinate hepatic glutamine transport; Fischer CP et al.; OBJECTIVE: Because hepatic glutamine transport is markedly enhanced during critical illness, we tested the hypothesis that nutrient starvation and endotoxemia act coordinately to augment transport activity . DESIGN: Fed or starved (48 hours) rats received Escherichia coli endotoxin (LPS, 10 mg/kg of body weight, intraperitoneally) or saline before hepatocyte isolation for measurement of glutamine transport . MATERIALS AND METHODS: Hepatocytes were isolated from fed or fasted rats 4 hours after LPS treatment . {3H}glutamine uptake was measured and normalized to cellular protein . Data (mean +/- standard deviation, three separate determinations) were analyzed by Student's t test and analysis of variance . MAIN RESULTS: Starvation induced a 1.6-fold increase in glutamine transport, while LPS treatment of fed rats increased transport activity 2.6-fold . Treatment of fasted animals with LPS induced a sixfold increase in glutamine transport . Kinetically, this effect in endotoxemic starved rats was mediated by both an increase in System N Vmax and the induction of a high affinity System A amino acid carrier which transports glutamine . CONCLUSIONS: Starvation and endotoxemia regulate hepatocyte glutamine transport independently and synergistically . This hepatic response provides glutamine and other amino acids to support key metabolic pathways in the liver during critical illness. Infect Immun, 1996 May, 64(5), 1794 - 9 Leucine auxotrophy restricts growth of Mycobacterium bovis BCG in macrophages; Bange FC et al.; The ability of slow-growing mycobacteria to replicate within host mononuclear phagocytes is thought to be central to the pathogenesis of mycobacterial infection . However, because of the lack of a mycobacterial mutant defective for intracellular replication, it has not been possible to test this hypothesis directly . Previously, we showed that a BCG leucine auxotroph with a transposon disruption of the leuD gene is unable to grow in mice . Here we demonstrate that this mutant is also incapable of replicating within cultured macrophages in vitro . Complementation of the leuD mutation with the leuCD genes of Escherichia coli restored wild-type levels of growth in macrophages, establishing that the defect for intracellular replication was due to leucine auxotrophy per se and not to a polar effect of the transposon insertion on an adjacent gene . These results suggest that the inability of the leucine auxotroph to grow in mice was due to its sequestration, after phagocytosis, in an intracellular compartment from which it could not obtain leucine. Infect Immun, 1996 May, 64(5), 1736 - 43 Coordinate synthesis and turnover of heat shock proteins in Borrelia burgdorferi: degradation of DnaK during recovery from heat shock; Cluss RG et al.; The synthesis and turnover of heat shock proteins (Hsps) by Borrelia burgdorferi, the Lyme disease spirochete, was investigated by radiolabeling of whole spirochetes and spheroplasts, comparison of one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and use of immunochemistry . The approximately 72-kDa DnaK homolog and three additional Hsps of 39, 27, and 21 kDa increased in amount by 3- to 15-fold between 2 and 6 h following temperature upshift from 28 to 39 degrees C . Temperature downshift experiments following the transfer of spirochetes from 40 to 28 degrees C showed that within 15 to 30 min, synthesis of most of the major Hsps returned to levels seen in spirochetes statically maintained at the lower temperature . Spheroplasts of B . burgdorferi produced by treatment with EDTA and lysozyme were radiolabeled, and specific Hsps were localized to either the cytoplasm or membrane fraction . Further analysis by two-dimensional electrophoresis demonstrated three constitutively expressed DnaK isoforms with pIs near 5.5 . A pattern suggestive of DnaK degradation was observed following recovery from heat shock but not in spirochetes maintained entirely at a low temperature . Some of these putative degradation products were recognized by monoclonal antibodies directed against the B . burgdorferi DnaK protein . These data suggest that following a period of peak synthesis, DnaK is actively degraded as the spirochete reestablishes its metabolic thermometer . These findings provide a new interpretation of previous work suggesting that 10 to 15 B . burgdorferi polypeptides, including DnaK have a common epitope. Infect Immun, 1996 May, 64(5), 1714 - 9 Interaction of verotoxin 2e with pig intestine; Waddell TE et al.; In pigs with edema disease, verotoxin 2e (VT2e) is produced in the intestine and transported to tissues, but neither the mechanism by which toxin passes through the intestine nor its failure to induce an enterotoxic reaction is understood . Binding of VT2e to pig intestine was examined by enzyme-linked immunosorbent assay involving microvillus membranes (MVM) and crude mucus; thin-layer chromatographic overlay immunoassay with total lipids extracted from MVM; and indirect immunofluorescence of toxin bound to thin sections of jejunum, ileum, and colon . VT2e bound significantly to MVM from pig jejunum and ileum but not to crude mucus . Verotoxin 2e-binding glycolipids, globotetraosylceramide and globotriaosylceramide, were detected by thin-layer chromatographic overlay immunoassay in extracts of MVM from jejunum and ileum . Indirect immunofluorescence showed that VT2e bound to vessels within the submucosa and muscularis mucosa of the jejunum, ileum, and colon and to enterocytes at the lower portion but not at the tips of villi in the jejunum and ileum . Receptors for VT2e are therefore present in the intestine of the pig, but their role in absorption of VT2e is unclear since intraintestinal inoculation of pigs with large quantities of VT2e does not result in edema disease . Previously reported lack of enterotoxicity of verotoxins in pig intestine may be explained by the absence of toxin receptors in the villus absorptive enterocytes. Infect Immun, 1996 May, 64(5), 1694 - 705 Mitotic block and delayed lethality in HeLa epithelial cells exposed to Escherichia coli BM2-1 producing cytotoxic necrotizing factor type 1; De Rycke J et al.; The cytopathic effect (CPE) of Escherichia coli producing cytotoxic necrotizing factor type 1 (CNF1) was investigated by using a human epithelial cell (HeLa) model of infection with CNF1-producing E . coli BM2-1 . This strain was shown to bind loosely, but massively, to HeLa cells . A 4-h interaction between bacteria and eukaryotic cells triggered the delayed appearance of a progressive dose-dependent CPE characterized by (i) intense swelling of cells accompanied by the formation of a dense network of actin stress fibers, (ii) inhibition of cell division due to a complete block in the G2 phase of the cell cycle, and (iii) nucleus swelling and chromatin fragmentation . These alterations resulted in cell death starting about 5 days after interaction . The absence of multinucleation clearly distinguished the CPE from the effect produced by cell-free culture supernatants of infected cells nor prevented by a CNF1-neutralizing antiserum . Pathogenicity was completely abolished after Tn5::phoA insertion mutagenesis in the cnf-1 structural gene but not restored by trans complementation with a recombinant plasmid containing intact cnf-1 and its promoter . These results suggest that a gene downstream of cnf-1, essential to the induction of the CPE, was affected by the mutation . On the other hand, transformation of the wild-type strain BM2-1 with the same recombinant plasmid leads to a significant increase in both CNF1 activity and CPE, demonstrating the direct contribution of CNF1 to the CPE . In conclusion, the pathogenicity of E . coli BM2-1 for HeLa cells results from a complex interaction involving cnf-1 and associated genes and possibly requiring a preliminary step of binding of bacterial organisms to target cells. Fertil Steril, 1996 May, 65(5), 1065 - 6 Ruptured tubo-ovarian abscess complicating transcervical cryopreserved embryo transfer; Friedler S et al.; OBJECTIVE: To present a rare infectious complication related to transcervical ET, without prior transvaginal puncture . DESIGN: Case report . SETTING: Hadassah University Hospital, IVF-ET unit . PATIENT: One patient undergoing cryopreserved-thawed ET . INTERVENTIONS: Artificial preparation of the endometrium with E2 and P, followed by transcervical intrauterine cryopreserved-thawed embryo transfer . RESULTS: After ET, severe pelvic inflammatory disease (PID) with ruptured tubo-ovarian abscess was diagnosed and treated . CONCLUSIONS: Severe PID including tubo-ovarian abscess formation should be considered a potential complication after ET, even without transvaginal oocyte aspiration. J Gen Virol, 1996 May, 77 ( Pt 5), 947 - 51 DNA sequence variation as a clue to the phylogenesis of orthopoxviruses; Douglas NJ et al.; We have sequenced DNA equivalent to the E5R ORF of Copenhagen vaccinia virus from an additional strain of vaccinia and from cowpox (three strains), camelpox (two strains), taterapox and ectromelia viruses . None of these showed the disruptions previously reported in the equivalent region of monkeypox virus . We also constructed a viable recombinant of vaccinia virus strain Dairen in which the E5R sequence was disrupted by a 436 bp deletion and substitution of the E . coli gpt gene . Quantitative analysis of the sequences, including available sequences from monkeypox, variola and vaccinia viruses revealed four main groupings, namely cowpox, ectromelia, monkeypox and a cluster which includes variola, camelpox, taterapox and vaccinia viruses . It was noted that, at over 75 % of the positions which differentiated species . all species but one had a common nucleotide . Although the analysis covers one single gene only, the results accord with what is known of the biology of the viruses. J Gen Virol, 1996 May, 77 ( Pt 5), 889 - 97 Beet necrotic yellow vein virus 42 kDa triple gene block protein binds nucleic acid in vitro; Bleykasten C et al.; The triple gene block (TGB) of beet necrotic yellow vein virus RNA 2 is required for cell-to-cell movement of the virus RNA . The protein P42 encoded by the 5'-proximal gene of the TGB has consensus sequence motifs characteristic of an ATP/GTP-dependent helicase . P42 was over-expressed in Escherichia coli and shown to bind both single- and double-stranded RNA and DNA by Northwestern blotting . Site-directed mutagenesis located the nucleic acid-binding domain to the N-terminal 24 amino acids of the protein and a point mutation or deletions in the region of P42 containing the helicase consensus sequences did not affect nucleic acid-binding activity of the immobilized protein . Electrophoretic mobility-shift assays revealed that P42 also binds nucleic acids in solution and that deletion of the N-terminal region inhibits this binding . Mutations in both the N-terminal nucleic acid-binding domain and the helicase domain blocked infection of leaves, indicating that both regions of P42 are important for its activity in vivo. J Gen Virol, 1996 May, 77 ( Pt 5), 879 - 88 RNA-binding activities of barley stripe mosaic virus gamma b fusion proteins; Donald RG et al.; The barley stripe mosaic virus (BSMV) gamma-b gene encodes a 17 kDa cysteine-rich protein known to affect virulence and to have a role in regulating viral gene expression . We have constructed recombinant gamma-b-glutathione S-transferase fusion proteins in Escherichia coli and have determined the ability of the purified fusion proteins and various mutant derivatives to bind nucleic acids in vitro . Gel-shift analyses revealed that the wild-type gamma-b-fusion protein is able to bind RNA cooperatively . The binding affinity is highly selective for single-stranded RNA because double-stranded RNA, single-stranded and double-stranded DNA, and transfer RNA were unable to compete for binding with the labelled RNA probes . However, BSMV-specific sequence binding was not observed since a chloroplast RNA competed for binding with 32P-labelled transcripts derived from the BSMV genome . The first 44 amino acids of the 152 amino acid gamma-b fusion protein encompassing one of two cysteine-rich 'zinc finger-like' motifs, and a basic region separating the finger-like motifs are required for RNA binding . Site- specific amino acid substitutions within two groups of lysine and arginine residues located in the basic motif reduced the binding affinity of the fusion protein greatly, but cysteine and histidine substitutions designed to disrupt the finger-like motifs failed to have appreciable effects on binding . These findings indicate that the regulatory properties of gamma-b may be mediated in part by RNA binding activities. J Gen Virol, 1996 May, 77 ( Pt 5), 869 - 77 Nucleic acid-binding properties of a bacterially expressed potato virus Y helper component-proteinase; Maia IG et al.; The potyvirus helper component-proteinase (HC-Pro) is a multifunctional protein previously reported to have affinity for polyribonucleotides . To investigate further the ability of HC-Pro to bind nucleic acids, the potato virus Y (PVY) LYE84 isolate HC-Pro gene was amplified, cloned in an Escherichia coli expression vector and sequenced . HC-Pro was expressed as a fusion with the maltose-binding protein and purified by affinity chromatography . Electrophoretic mobility-shift assays demonstrated that HC-Pro acts as a sequence non-specific RNA-binding protein and suggest that more than one molecule of protein was bound per molecule of RNA . The HC-Pro RNA-binding activity was stable in 400 mm-NaCl and temperature sensitive . The recombinant protein preferentially bound ssRNA over DNA or dsRNA and showed little, if any, affinity for poly(A) . The possible implications of the RNA-binding activity of HC-Pro in potyvirus replication and movement are discussed. DNA Res, 1996 Apr 30, 3(2), 93 - 4 The effect of the crp genotypes on the transformation efficiency in Escherichia coli; Umemoto A et al.; We have found that a significant difference exists in transformation efficiency between the crp+/crp- isogenic pair of strains of Escherichia coli, with the efficiency being much higher in crp- than in crp+ . The ratio of transformation efficiency between crp+ and crp- strains depends very little on the plasmid size . This observation suggests that the difference of the transformation efficiency is due to mechanisms other than a crp-regulated endonuclease . The crp gene is one of the first specific genes that have been shown to affect transformation efficiency. Proc Natl Acad Sci U S A, 1996 Apr 30, 93(9), 4437 - 41 Escherichia coli trigger factor is a prolyl isomerase that associates with nascent polypeptide chains; Hesterkamp T et al.; Correct folding of newly synthesized proteins is proposed to be assisted by molecular chaperones and folding catalysts . To identify cellular factors involved in the initial stages of this process we searched for proteins associated with nascent polypeptide chains . In an Escherichia coli transcription/translation system synthesizing beta-galactosidase we identified a 58-kDa protein which associated with translating ribosomes but dissociated from these ribosomes upon release of nascent beta-galactosidase . N-terminal sequencing identified it as trigger factor, previously implicated in protein secretion . Direct evidence for association of trigger factor with nascent polypeptide chains was obtained by crosslinking . In a wheat germ translation system complemented with E . coli lysates, epsilon-4-(3-trifluoromethyldiazirino)benzoic acid-lysine residues were incorporated into nascent secretory preprolactin and a nonsecretory preprolactin mutant . Trigger factor crosslinked to both types of nascent chains, provided they were ribosome bound . Trigger factor contains key residues of the substrate-binding pocket of FK506-binding protein-type peptidyl-prolyl-cis/trans-isomerases and has prolyl isomerase activity in vitro . We propose that trigger factor is a folding catalyst acting cotranslationally. Proc Natl Acad Sci U S A, 1996 Apr 30, 93(9), 4380 - 5 Mutator tRNAs are encoded by the Escherichia coli mutator genes mutA and mutC: a novel pathway for mutagenesis; Slupska MM et al.; We have previously described the mutator alleles mutA and mutC, which map at 95 minutes and 42 minutes, respectively, on the Escherichia coli genetic map and which stimulate transversions; the A.T-->T.A and G.C-->T.A substitutions are the most prominent . In this study we show that both mutA and mutC result from changes in the anticodon in one of four copies of the same glycine tRNA, at either the glyV or the glyW locus . This change results in a tRNA that inserts glycine at aspartic acid codons . In view of previous studies of missense suppressor tRNAs, the mistranslation of aspartic acid codons is assumed to occur at approximately 1-2% . We postulate that the mutator tRNA effect is exerted by generating a mutator polymerase and suggest that the epsilon subunit of DNA polymerase, which provides a proofreading function, is the most likely target . The implications of these findings for the contribution of mistranslation to observed spontaneous mutation rates in wild-type strains, as well as other cellular phenomena such as aging, are discussed. Proc Natl Acad Sci U S A, 1996 Apr 30, 93(9), 4374 - 9 Mutation detection with MutH, MutL, and MutS mismatch repair proteins; Smith J et al.; Escherichia coli methyl-directed mismatch repair is initiated by MutS-, MutL-, and ATP-dependent activation of MutH endonuclease, which cleaves at d(GATC) sites in the vicinity of a mismatch . This reaction provides an efficient method for detection of mismatches in heteroduplexes produced by hybridization of genetically distinct sequences after PCR amplification . Multiple examples of transition and transversion mutations, as well as one, two, and three nucleotide insertion/deletion mutants, have been detected in PCR heteroduplexes ranging in size from 400 bp to 2.5 kb . Background cleavage of homoduplexes is largely due to polymerase errors that occur during amplification, and the MutHLS reaction provides an estimate of the incidence of mutant sequences that arise during PCR. Proc Natl Acad Sci U S A, 1996 Apr 30, 93(9), 4311 - 5 Modulation of promoter occupancy by cooperative DNA binding and activation-domain function is a major determinant of transcriptional regulation by activators in vivo; Tanaka M; Binding of transcriptional activators to a promoter is a prerequisite process in transcriptional activation . It is well established that the efficiency of activator binding to a promoter is determined by the affinity of direct interactions between the DNA-binding domain of an activator and its specific target sequences . However, I describe here that activator binding to a promoter is augmented in vivo by the effects of two other determinants that have not been generally appreciated: (i) the number of activator binding sites present in a promoter and (ii) the potency of activation domains of activators . Multiple sites within a promoter can cooperatively recruit cognate factors regardless of whether they contain an effective activation domain . This cooperativity can result in the synergistic activation of transcription . The second effect is the enhancement of activator binding to a promoter by the presence of activation domains . In this case, activation domains are not simply tethered to the promoter by the DNA-binding domain but instead assist the DNA-binding domain being tethered onto the promoter . This effect of activation domains on DNA binding is instrumental in determining how potent activators can induce steep transcriptional increases at low concentrations. Proc Natl Acad Sci U S A, 1996 Apr 30, 93(9), 4273 - 7 Modulation of the transcriptional activity of thyroid hormone receptors by the tumor suppressor p53; Yap N et al.; Thyroid hormone nuclear receptors (TRs) are ligand-dependent transcriptional factors that regulate growth, differentiation, and development . The molecular mechanisms by which TRs mediate these effects are unclear . One prevailing hypothesis suggests that TRs may cooperate with other transcriptional factors to mediate their biological effects . In this study, we tested this hypothesis by examining whether the activity of TRs is modulated by the tumor suppressor p53 . p53 is a nuclear protein that regulates gene expression via sequence-specific DNA binding and/or direct protein-protein interaction . We found that the human TR subtype beta 1 (h-TR beta 1) physically interacted with p53 via its DNA binding domain . As a result of this physical interaction, binding of h-TR beta 1 to its hormone response elements either as homodimer or as a heterodimer with the retinoic X receptor was inhibited by p53 in a concentration-dependent manner . In transfected cells, wild-type p53 repressed the hormone-dependent transcriptional activation of h-TR beta 1 . In contrast, mutant p53 either had no effect or activated the transcriptional activity of h-TR beta 1 depending on the type of hormone response elements . These results indicate the gene regulating activity of TRs was modulated by p53, suggesting that the cross talk between these two transcriptional factors may play an important role in the biology of normal and cancer cells. Proc Natl Acad Sci U S A, 1996 Apr 30, 93(9), 4202 - 6 Truncated elongation factor G lacking the G domain promotes translocation of the 3' end but not of the anticodon domain of peptidyl-tRNA; Borowski C et al.; The mechanism by which elongation factor G (EF-G) catalyzes the translocation of tRNAs and mRNA on the ribosome is not known . The reaction requires GTP, which is hydrolyzed to GDP . Here we show that EF-G from Escherichia coli lacking the G domain still catalyzed partial translocation in that it promoted the transfer of the 3' end of peptidyl-tRNA to the P site on the 50S ribosomal subunit into a puromycin-reactive state in a slow-turnover reaction . In contrast, it did not bring about translocation on the 30S subunit, since (i) deacylated tRNA was not released from the P site and (ii) the A site remained blocked for aminoacyl-tRNA binding during and after partial translocation . The reaction probably represents the first EF-G-dependent step of translocation that follows the spontaneous formation of the A/P state that is not puromycin-reactive {Moazed, D . & Noller, H . F . (1989) Nature (London) 342, 142-148} . In the complete system--i.e., with intact EF-G and GTP--the 50S phase of translocation is rapidly followed by the 30S phase during which the tRNAs together with the mRNA are shifted on the small ribosomal subunit, and GTP is hydrolyzed . As to the mechanism of EF-G function, the results show that the G domain has an important role, presumably exerted through interactions with other domains of EF-G, in the promotion of translocation on the small ribosomal subunit . The G domain's intramolecular interactions are likely to be modulated by GTP binding and hydrolysis. Proc Natl Acad Sci U S A, 1996 Apr 30, 93(9), 3865 - 9 Proteins associated with RNase E in a multicomponent ribonucleolytic complex; Miczak A et al.; The Escherichia coli endoribonuclease RNase E is essential for RNA processing and degradation . Earlier work provided evidence that RNase E exists intracellularly as part of a multicomponent complex and that one of the components of this complex is a 3'-to-5' exoribonuclease, polynucleotide phosphorylase (EC 2.7.7.8) . To isolate and identify other components of the RNase E complex, FLAG-epitope-tagged RNase E (FLAG-Rne) fusion protein was purified on a monoclonal antibody-conjugated agarose column . The FLAG-Rne fusion protein, eluted by competition with the synthetic FLAG peptide, was found to be associated with other proteins . N-terminal sequencing of these proteins revealed the presence in the RNase E complex not only of polynucleotide phosphorylase but also of DnaK, RNA helicase, and enolase (EC 4.2.1.11) . Another protein associated only with epitope-tagged temperature-sensitive (Rne-3071) mutant RNase E but not with the wild-type enzyme is GroEL . The FLAG-Rne complex has RNase E activity in vivo and in vitro . The relative amount of proteins associated with wild-type and Rne-3071 expressed at an elevated temperature differed. Biochemistry, 1996 Apr 30, 35(17), 5571 - 6 Effect of the tyrosyl radical on the reduction and structure of the Escherichia coli ribonucleotide reductase protein R2 diferric site as probed by EPR on the mixed-valent state; Davydov R et al.; It was recently shown by EPR that high yields of a sterically constrained mixed-valent species may be formed in radical free protein metR2 of Escherichia coli ribonucleotide reductase by gamma-irradiation at 77 K {Davydov, R., Kuprin, S., Graslund, A., & Ehrenberg, A . (1994) J . Am . Chem . Soc . 116, 11120} . This species, with S = 1/2, essentially retains the ligand geometry of the original diferric center and should be a sensitive probe for structural changes in the diferric centers . Here we apply this probe and demonstrate that there is a structural difference between the diferric iron center of the complete site of protein R2, with a tyrosyl radical, and that of metR2, without radical . The EPR spectrum of the mixed-valent species of metR2 shows pure axial symmetry, while complete sites show rhombic distortion and a shifted high-field turning point . Differences also remain in the EPR of the first S = 9/2 species obtained by annealing at 165 K, but disappear after relaxation at 200 K . In addition, the diferric center of a complete site is not reduced radiolytically until the associated tyrosyl radical has been reduced, indicating that an electron first reaching the iron center may be transferred to the radical . This route of electron transfer and the influence of the radical on the structure of the iron center are likely to have functional roles for the formation of the proposed substrate radical and regulation of redox processes within the enzyme . The sensitivity of the structure of the iron site to the structure of the Tyr-122 site is also demonstrated by the strong influence the mutation Y122F has on the EPR spectra of the corresponding mixed-valent species. Biochemistry, 1996 Apr 30, 35(17), 5528 - 37 High-level expression and characterization of a purified 142-residue polypeptide of the prion protein; Mehlhorn I et al.; The major, and possible only, component of the infectious prion is the scrapie prion protein (PrPSc); the protease resistant core of PrPSc is PrP 27-30, a protein of approximately 142 amino acids . PrPSc is derived from the cellular PrP isoform (PrPC) by a post-transliatonal process in which a profound conformational change occurs . Syrian hamster (SHa) PrP genes of varying length ranging from the N- and C- terminally truncated 90-228 up to the full-length mature protein 23-231 were inserted into various secretion and intracellular expression vectors that were transformed into Escherichia coli deficient for proteases . Maximum expression was obtained for a truncated SHaPrP containing residues 90-231, which correspond to the sequence of PrP 27-30; disruption of the bacteria using a microfluidizer produced the highest yields of this protein designated rPrP . After solubilization of rPrP in 8 M GdnHC1, it was purified by size exclusion chromatography and reversed phase chromatography . During purification the recovery was approximately 50%, and from each liter of E . coli culture, approximately 50 mg of purified rPrP was obtained . Expression of the longer species containing the basic N-terminal region was less successful and was not pursued further . The primary structure of rPrP was verified by Edman sequencing and mass spectrometry, and secondary structure determined by circular dichroism and Fourier transform infrared spectroscopy . When rPrP was purified under reducing conditions, it had a high beta-sheet content and relatively low solubility similar to PrPSc, particularly at pH values > 7 . Refolding of rPrP by oxidation to form a disulfide bond between the two Cys residues of this polypeptide produced a soluble protein with a high alpha-helical content similar to PrPC . These multiple conformations of rPrP are reminiscent of the structural plurality that characterizes the naturally occurring PrP isoforms . The high levels of purified rPrP which can now be obtained should facilitate determination of the multiple tertiary structures that Prp can adopt. Biochemistry, 1996 Apr 30, 35(17), 5509 - 17 Thermodynamic analysis of small ligand binding to the Escherichia coli repressor of biotin biosynthesis; Xu Y et al.; BirA is the transcriptional repressor of biotin biosynthesis and a biotin holoenzyme synthetase . It catalyzes synthesis of biotinyl-5'-AMP from the substrates biotin and ATP . The adenylate is the activated intermediate in the biotin transfer reaction as well as the positive allosteric effector for site-specific DNA binding . The affinity of BirA for the adenylate is considerably greater than its affinity for biotin, and both binding reactions are coupled to changes in the conformation of the protein . The temperature dependencies of the two binding interactions have been determined using kinetic techniques . Van't Hoff analysis of the equilibrium dissociation constants derived from the kinetic data indicate that while the two binding processes are characterized by large negative enthalpies, the entropic contributions are small for both . Binding enthalpies have also been determined by isothermal titration calorimetry . Consistent with the results of the van't Hoff analyses, the calorimetric enthalpies are large and negative . The greater precision of the calorimetric measurements allowed more accurate estimation of the entropic contributions to the binding processes, which are of opposite sign for the two ligands . In addition, the heat capacity changes associated with the two binding reactions are small . The measured thermodynamic parameters for binding of biotin and bio-5'-AMP to BirA have been utilized to dissect out structural contributions to the binding energetics . Results of these calculations indicate equivalent contributions of burial of polar and apolar surface area to both binding processes . The total loss of solvent accessible surface area is, however, greater for biotin binding . The analysis indicates furthermore that although both binding reactions are coupled to losses in configurational entropy, the magnitude of the conformational change is significantly larger for biotin binding. Biochemistry, 1996 Apr 30, 35(17), 5359 - 65 On the dynamics and conformation of the HA2 domain of the influenza virus hemagglutinin; Kim CH et al.; To investigate the dynamics and conformation of the membrane-interacting HA2 domain of the hemagglutinin protein of influenza virus, the peripheral part of the HA2 domain (aa 1-127) was expressed in Escherichia coli . Four consecutive single-cysteine mutants, F63C, H64C, Q65C, and I66C, were generated using site-directed mutagenesis . This region is proposed to undergo a conformational change from a loop to a helical coiled-coil when going from the native to the fusion-active state {Bullough et al . (1994) Nature (London) 371, 37-43} . In the trimeric coiled-coil geometry positions 63 and 66 belong to the core so that cysteines from individual monomers are spatially close . On the other hand, positions 64 and 65 face the aqueous phase so that cyteines from monomers are spatially remote . The mutants were studied with cysteine-cysteine cross-linking and the spin-labeling electron paramagnetic resonance (EPR) in both the membrane-bound state and in the detergent-solubilized state . Extensive intramolecular cysteine-cysteine cross-linking was observed not only for F63C and I66C but also for H64C . Rates of cross-linking were comparable for these three mutants at physiological temperatures . These results are inconsistent with what is expected for a well-defined coiled-coil and suggest that the region containing the mutation sites is flexible . However, a characteristic cross-linking pattern consistent with a well-defined coiled-coil developed at very low temperatures . Line shapes of EPR spectra also indicate that this region is dynamic at ambient temperatures . Such flexibility perhaps arises from an equilibrium between a coiled-coil and a random coil conformation . No significant changes of the EPR spectra were observed upon lowering the pH to fusogenic conditions, suggesting that this flexible structure is the stable conformation at both neutral and low pH . The dynamic flexibility of this region may have important implications for the mechanism of HA-induced membrane fusion; for example it may be required for the apposition of the viral and endosomal membranes. Philos Trans R Soc Lond B Biol Sci, 1996 Apr 29, 351(1339), 543 - 50 Protein-protein interactions during transcription activation: the case of the Escherichia coli cyclic AMP receptor protein; Savery N et al.; The Escherichia coli cyclic AMP receptor protein (CRP) is a homodimeric transcription activator triggered by cyclic AMP . Escherichia coli contains more than 100 different promoters that can be activated by CRP: in most cases the CRP acts by making direct contact with RNA polymerase . Remarkably, there is considerable variation in the location of the DNA site for CRP from one CRP-dependent promoter to another . Genetic methods have been used to locate the activating regions of CRP that make contact with RNA polymerase at promoters of different architectures . At promoters where the DNA site for CRP is centred near to positions -61, -71 or -81 (i.e . 61, 71 or 81 base pairs upstream of the transcript start-point, respectively), a single surface-exposed loop (Activating Region 1) in the downstream subunit of the CRP dimer makes contact with RNA polymerase . The contact site in RNA polymerase is located in one of the C-terminal domains of two RNA polymerase alpha subunits . At promoters where the DNA site for CRP is centred near to position-41, both subunits of the CRP dimer make contact with RNA polymerase via three separate surface exposed regions (Activating Regions 1, 2 and 3) . At these promoters, where bound CRP overlaps with RNA polymerase-binding elements, the C-terminal domains of the polymerase alpha subunits are displaced and bind upstream of CRP . Activation at a number of E . coli promoters is dependent on binding of two CRP dimers, with one dimer bound near to position-41 and the other dimer bound further upstream . In these cases, both bound CRP dimers contact RNA polymerase . The CRP dimer bound around position-41 contacts RNA polymerase via Activating Regions 1, 2 and 3, whereas the upstream bound CRP dimer contacts one of the displaced alpha C-terminal domains via Activating Region 1 in the downstream CRP subunit . Thus in these cases, codependence on two activators is due to simultaneous contacts between separate activators and RNA polymerase . This mechanism allows great flexibility, as any activator that can contact the C-terminal domain of the RNA polymerase alpha subunits can act cooperatively with CRP. Philos Trans R Soc Lond B Biol Sci, 1996 Apr 29, 351(1339), 527 - 35 Structure and function of Escherichia coli met repressor: similarities and contrasts with trp repressor; Phillips SE et al.; Transcription of genes encoding enzymes for the biosynthesis of methionine and trytophan in Escherichia coli is regulated by the ligand-activated met and trp repressors . X-ray crystallographic studies show how these two small proteins, although similar in size and function, have totally different three-dimensional structures and specifically recognize their respective DNA operator sequences in different ways . A common feature is that both repressors bind as cooperative arrays to tandem repeats of 8 base-pair 'Met' or 'Trp boxes' respectively, and the consensus sequences share the rare tetranucleotide CTAG . A series of structural and functional studies have shown how the two repressors discriminate between their operators, using a combination of direct contacts between side chains and bases, and indirect sensing of conformational properties of the DNA. Philos Trans R Soc Lond B Biol Sci, 1996 Apr 29, 351(1339), 475 - 82 A structure/function analysis of Escherichia coli RNA polymerase; Gross CA et al.; Control of RNA polymerase is a common means of regulating gene expression . A detailed picture of both the structure and how the structural details of RNA polymerase encode function is a key to understanding the molecular strategies used to regulate RNA polymerase . We review here data which ascribes functions to some regions of the primary sequence of the subunits (alpha, beta beta' sigma) which make up E . coli RNA polymerase . We review both genetic and biochemical data which place regions of the primary sequence that are distant from one another in close proximity in the tertiary structure . Finally we discuss the implications of these findings on the quaternary structure of RNA polymerase. FEBS Lett, 1996 Apr 29, 385(1-2), 91 - 5 The C-terminal domain of peptide deformylase is disordered and dispensable for activity; Meinnel T et al.; Upon trypsinolysis, the 18 C-terminal residues of Escherichia coli peptide deformylase were removed but the resulting form exhibited full activity . Moreover, a mutant fms gene encoding the first 145 out of the 168 residues of the enzyme was able to complement a fms(Ts) strain and exhibited full activity . Upon progressive truncation up to residue 139, both activity and stability decreased up to complete inactivation . Mutagenesis of residues of the 138-145 region highlights the importance of Leu-141 and Phe-142 . N-Terminal deletions were also carried out . Beyond two residues off, the enzyme showed a dramatic instability . Finally, NMR and thermostability studies of the full-length enzyme and comparison to the 1-147 form strongly suggest that the dispensable residues are disordered in solution. FEBS Lett, 1996 Apr 29, 385(1-2), 67 - 71 Identification of the prolyl isomerase domain of Escherichia coli trigger factor; Hesterkamp T et al.; E . coli trigger factor is a protein of 48 kDa which was recently identified as a ribosome-bound peptidyl-prolyl-cis/transisomerase (PPIase) capable of catalysing protein folding in vitro . We found trigger factor in association with nascent polypeptide chains, suggesting a function in the co-translational folding of proteins . Sequence comparisons revealed a homology of a segment of trigger factor with PPIases of the FK506 binding protein (FKBP) family . Here, we report on the purification of trigger factor and a domain assignment of its polypeptide chain by microsequencing and mass spectroscopy of proteolytic fragments . Two proteases generated fragments of 12-13 kDa molecular weight that encompass the predicted FKBP domain and possess PPIase activity in vitro . Sequence alignment of the known trigger factor proteins demonstrates a high degree of conservation within this central functional domain of the protein. FEBS Lett, 1996 Apr 29, 385(1-2), 105 - 8 Calculation of Cys 30 delta pKa's and oxidising power for DsbA mutants; Warwicker J et al.; DsbA possesses a redox active disulphide, with the equilibrium strongly shifted towards the reduced form as compared to its structural homologue, thioredoxin . It is widely believed that the two amino acids that separate the active site cysteines play a crucial role in determining oxidising power within the thioredoxin family . Data concerning redox and pKa properties for DsbA mutants in this region are available . Electrostatics calculations show reasonable agreement with the experimental data, and support the suggestion that amino acids outside of the CXXC active site sequence are as important in determining oxidising power within the thioredoxin family as are those within it. J Chromatogr B Biomed Appl, 1996 Apr 26, 679(1-2), 61 - 7 One-step purification of rat heart-type fatty acid-binding protein expressed in Escherichia coli; Schaap FG et al.; Heart-type fatty acid-binding protein (H-FABP) is a member of a family of 14-15 kDa lipid binding proteins which are believed to enhance intracellular transport of lipids by facilitating their cytoplasmic diffusion . To obtain sufficient amounts of protein for in vitro studies, we expressed rat H-FABP in Escherichia coli and compared its biochemical properties with the protein isolated from rat heart . An effective method was developed to purify recombinant rat H-FABP from cell lysates in a single step using anion-exchange chromatography . This method also proved to be applicable for purifying heterologously expressed human H-FABP . Recombinant rat H-FABP, which made up approximately 25% of the soluble proteins in E . coli, was obtained in a yield of 30-40 mg/l culture . Characterization showed that recombinant rat H-FABP was indistinguishable from the protein isolated from rat heart regarding molecular mass and oleic acid binding . Some heterogeneity upon isoelectric focusing was observed, presumably due to differences in N-terminal processing of the proteins . In conclusion, a method is presented for efficient high-yield production of recombinant rat H-FABP. J Biol Chem, 1996 Apr 26, 271(17), 10419 - 24 Post-translational modification of human brain type I inositol-1,4,5-trisphosphate 5-phosphatase by farnesylation; De Smedt F et al.; In brain, type I inositol-1,4,5-trisphosphate 5-phosphatase (InsP3 5-phosphatase) is the major isoenzyme hydrolyzing the calcium-mobilizing second messenger InsP3 . Activity of this enzyme could be measured in both soluble and particulate fractions of tissue homogenates . The protein sequence showed a putative C-terminal isoprenylation site (CVVQ) . In this study, two mutants have been generated . The first mutant (C409S) has a serine replacing a cysteine at position 409 of the wild-type enzyme . The second mutant (K407D1) is a deletion mutant that lacks the last five C-terminal amino acids . These constructs were individually expressed by transfection in COS-7 cells . Western blot analysis of wild-type transfected cells indicated that both soluble and particulate fractions had a 43-kDa immunoreactive band, with a higher proportion of the original homogenate associated with the particulate part . On the contrary, when the two mutated constructs were transfected in COS-7 cells, the phosphatase was predominantly soluble . Confocal immunofluorescence studies showed the wild-type enzyme to be present on the cell surface of transfected COS-7 cells and in subcellular compartments around the nucleus . This was not observed for the two mutants, where uniform immunofluorescence labeling was observed throughout the cytosol . Recombinant type I InsP3 5-phosphatase expressed in Escherichia coli was a substrate of purified farnesyltransferase . Altogether, the data therefore suggest a direct participation of Cys-409 in a C-terminally anchored InsP3 5-phosphatase by farnesylation. J Biol Chem, 1996 Apr 26, 271(17), 10413 - 8 Functionally active recombinant alpha and beta chain-peptide complexes of human major histocompatibility class II molecules; Nag B et al.; Major histocompatibility (MHC) class II molecules are cell surface heterodimeric (alphabeta) glycoproteins that display processed antigens to T cell receptors (TCRs) of CD4-positive T cells . The present study describes that individual recombinant alpha and beta chains of human MHC class II molecules lacking the transmembrane region (alpha-Tm and beta-Tm) are capable of binding antigenic peptide and that these complexes of chain-peptide are recognized by TCRs to induce antigen-specific apoptosis in restricted T cells . The alpha-Tm and the beta-Tm of human HLA-DR2 (DRB5*0101) were cloned, expressed in Escherichia coli, and purified in large scale by conventional chromatographic methods . The in vitro binding of an immunodominant epitope from the myelin basic protein (MBP-(83-102)Y83) to purified DR2 alpha-Tm and DR2 beta-Tm was demonstrated with biotinylated and fluoresceinated MBP-(83-102)Y83 peptide . The specificity of the MBP-(83-102)Y83 peptide binding to both DR2 alpha-Tm and DR2 beta-Tm was demonstrated in a competitive peptide binding assay . When exposed to a transformed T cell clone (SS8T) restricted to DR2(DRB5*0101) and MBP-(84-102) peptide, complexes of DR2 alpha-Tm and DR2 beta-Tm with MBP-(83-102)Y83 peptide were able to specifically recognize TCRs as measured by the increase in gamma-interferon (gamma-IFN) cytokine . Such recognition of TCRs by soluble alpha-MBP-(83-102)Y83 and beta-MBP-(83-102)Y83 complexes led to the induction of antigen-specific apoptosis in SS8T cells as measured by double fluorescence flow cytometry and electron microscopy . These results provide the first evidence that soluble complexes of antigenic peptide and individual chains of human MHC class II molecules lacking the transmembrane region can recognize TCRs and induce antigen-specific apoptosis in T cells . Since activated CD4-positive T cells are involved in pathogenesis of various autoimmune diseases, the apoptosis triggered by individual soluble chain-peptide complexes has significant potential for eliminating autoreactive T cells. J Biol Chem, 1996 Apr 26, 271(17), 10291 - 8 In vivo assembly of the tau-complex of the DNA polymerase III holoenzyme expressed from a five-gene artificial operon . Cleavage of the tau-complex to form a mixed gamma-tau-complex by the OmpT protease; Pritchard AE et al.; A plasmid was constructed that encodes all five subunits of the Escherichia coli tau-complex on a single artificially constructed operon under the control of an inducible promoter . The proteins tau, delta, delta , chi, and psi overproduced from this artificial operon assemble efficiently in vivo, providing an efficient source of homogeneous tau-complex . The gamma subunit is a truncated form of tau that is produced by a translational frameshift . When protein expression was induced in bacterial strains containing the outer membrane protein T (OmpT) protease, tau was proteolyzed after lysis to a gamma-like protein, gammaP, and a peptide, C-tau, corresponding to the C terminus of tau . N-terminal sequencing of C-tau revealed a cleavage site between two lysines at positions 429 and 430 of tau . The deduced sequence of gammaP is, therefore, only two amino acids shorter than natural gamma . The proteolysis by OmpT was also shown directly by using purified OmpT and tau-complex in an in vitro reaction . A gammaP-complex and a mixed tau-gammaP-complex were purified from ompT+ cells . When the tau-complex proteins were overexpressed in ompT- bacteria, intact tau-complex lacking gammaP could be purified. J Biol Chem, 1996 Apr 26, 271(17), 10121 - 9 Phosphorylation of Vif and its role in HIV-1 replication; Yang X et al.; Vif is a 23-kDa protein encoded by human immunodeficiency virus, type 1 (HIV-1) which is important for virion infectivity . Here, we describe the phosphorylation of HIV-1 Vif and its role in HIV-1 replication . In vivo studies demonstrated that Vif is highly phosphorylated on serine and threonine residues . To identify phosphorylation sites and characterize the Vif kinase(s), Vif was expressed in Escherichia coli and purified for use as a substrate in in vitro kinase assays . The purified Vif protein was phosphorylated in vitro on serine and threonine residues by a kinase(s) present in both cytosol and membrane fractions . Phosphorylation of Vif was stimulated by phorbol 12-myristate 13-acetate and inhibited by staurosporine and hypericin, a drug with potent anti-HIV activity . The Vif kinase(s) was resistant to inhibitors of protein kinase C, cAMP-dependent kinase, and cGMP-dependent kinase, suggesting that it is distinct from these enzymes . To identify the phosphorylation sites, 32P-labeled Vif was digested by V8 protease and the peptides were resolved by reverse-phase high performance liquid chromatography . Radioactive peptide sequencing identified three phosphorylation sites within the C terminus, Ser144, Thr155, and Thr188 . Two-dimensional tryptic phosphopeptide mapping indicated that these sites are also phosphorylated in vivo . Both Ser144 and Thr188 are contained in the recognition motifs (R/KXXS*/T* and R/KXXXS*/T*) used by serine/threonine protein kinases such as cGMP-dependent kinase and PKC . Ser144 is present in the motif SLQXLA, which is the most highly conserved sequence among all lentivirus Vif proteins . Mutation of Ser144 to alanine resulted in loss of Vif activity and >90% inhibition of HIV-1 replication . These studies suggest that phosphorylation of Vif by a serine/threonine protein kinase(s) plays an important role in regulating HIV-1 replication and infectivity. J Biol Chem, 1996 Apr 26, 271(17), 10109 - 15 Mutation of Tyr235 in the NAD(H)-binding subunit of the proton-translocating nicotinamide nucleotide transhydrogenase of Rhodospirillum rubrum affects the conformational dynamics of a mobile loop and lowers the catalytic activity of the enzyme; Diggle C et al.; The Tyr residue in the mobile loop region of the soluble, domain I polypeptide (called Ths) of the proton-translocating transhydrogenase from Rhodospirillum rubrum has been substituted by Asn and by Phe . The recombinant proteins were expressed at high levels in Escherichia coli and purified to homogeneity . The two well defined resonances at 6.82 and 7.12ppm, observed in the one-dimensional proton NMR spectrum of wild-type protein, and previously attributed to the Tyr residue, were absent in both mutants . In the Tyr235 --> Phe mutant Ths, they were replaced by two new resonances at 7.26 and 7.33 ppm, characteristic of a Phe residue . In both mutants, narrow resonances attributable to Met residues (and in the Tyr235 --> Phe mutant, resonances attributable to Ala residues) were shifted relative to the wild type, but other features in the NMR spectra were unaffected . The conformational dynamics of the mobile loop closure in response to nucleotide binding by the protein were altered in the two mutants . The fluorescence emission from Trp72 was unaffected by both Tyr substitutions, and the fluorescence was still quenched by NADH . The mutant Ths proteins bound to chromatophore membranes depleted of their native Ths with undiminished affinity . In these reconstituted systems, the Km values for thio-NADP+ and NADH, during light-driven transhydrogenation, were similar to those of wild-type, but the kcat values were decreased about 2-fold . In reverse transhydrogenation, the Kmvalues for NADPH were slightly decreased in the mutants relative to wild-type, but those for acetyl pyridine adenine dinucleotide were increased about 10- and 13-fold, respectively, and the kcat values were decreased about 2- and 5-fold, respectively, in the Tyr235 --> Phe and Tyr235 --> Asn mutants . It is concluded that Tyr235 may contribute to the process of nucleotide binding and that substitution of this residue prevents proper functioning of the mobile loop in catalysis. J Biol Chem, 1996 Apr 26, 271(17), 10103 - 8 Interaction of nucleotides with the NAD(H)-binding domain of the proton-translocating transhydrogenase of Rhodospirillum rubrum; Bizouarn T et al.; Transhydrogenase catalyzes the reduction of NADP+ by NADH coupled to the translocation of protons across a membrane . The polypeptide composition of the enzyme in Rhodospirillum rubrum is unique in that the NAD(H)-binding domain (called Ths) exists as a separate polypeptide . Ths was expressed in Escherichia coli and purified . The binding of nucleotide substrates and analogues to Ths was examined by one-dimensional proton nuclear magnetic resonance (NMR) spectroscopy and by measuring the quenching of fluorescence of its lone Trp residue . NADH and reduced acetylpyridine adenine dinucleotide bound tightly to Ths, whereas NAD+, oxidized acetylpyridine adenine dinucleotide, deamino-NADH, 5'-AMP and adenosine bound less tightly . Reduced nicotinamide mononucleotide, NADPH and 2'-AMP bound only very weakly to Ths . The difference in the binding affinity between NADH and NAD+ indicates that there may be an energy requirement for the transfer of reducing equivalents into this site in the complete enzyme under physiological conditions . Earlier results had revealed a mobile loop at the surface of Ths (Diggle, C., Cotton, N . P . J., Grimley, R . L., Quirk, P . G., Thomas, C . M., and Jackson, J . B . (1995) Eur . J . Biochem . 232, 315-326); the loop loses mobility when Ths binds nucleotide; the reaction involves two steps . This was more clearly evident, even for tight-binding nucleotides, when experiments were carried out at higher temperatures (37 degrees C), where the resonances of the mobile loop were substantially narrower . The binding of adenosine was sufficient to initiate loop closure; the presence of a reduced nicotinamide moiety in the dinucleotide apparently serves to tighten the binding . Two-dimensional 1H NMR spectroscopy of the Ths-5'-AMP complex revealed nuclear Overhauser effect interactions between protons of amino acid residues in the mobile loop (including those in a Tyr residue) and the nucleotide . This suggests that, in the complex, the loop has closed down to within 0.5 nm of the nucleotide. J Biol Chem, 1996 Apr 26, 271(17), 10035 - 41 Assessment of the ATP binding properties of Hsp90; Jakob U et al.; Hsp90, one of the most prominent proteins in eucaryotic cells under physiological and stress conditions, chaperones protein folding reactions in an ATP-independent way . Surprisingly, ATP binding and ATPase activity of Hsp90 has been reported by several groups . To clarify this important issue, we have reinvestigated the potential ATP binding properties and ATPase activity of highly purified Hsp90 using a number of different techniques . Hsp90 was compared to the well characterized ATP-binding chaperone Hsc70 and to two control proteins, immunoglobulin G and bovine serum albumin, that are known to not bind ATP . Hsp90 behaved very similarly to the non-ATP-binding proteins and very differently from the ATP-binding protein Hsc70 . Like bovine serum albumin and immunoglobulin G, Hsp90 (i) did not bind to immobilized ATP, (ii) could not be specifically photocross-linked with azido-ATP, (iii) failed to exhibit significant changes in intrinsic protein fluorescence upon ATP addition, and (iv) did not bind to three fluorescent ADP analogues . In contrast, Hsc70 strongly bound ATP and ADP, specifically cross-linked with azido-ATP, and exhibited major shifts in fluorescence upon addition of ATP . Finally, reexamination of the amino acid sequence of Hsp90 failed to reveal any significant homologies to known ATP-binding motifs . Taken together, we conclude that highly purified Hsp90 does not bind ATP . Weak ATPase activities associated with Hsp90 preparations may be due to minor impurities or kinases copurifying with Hsp90. J Biol Chem, 1996 Apr 26, 271(17), 10004 - 9 Characterization of the interaction between RhoGDI and Cdc42Hs using fluorescence spectroscopy; Nomanbhoy TK et al.; The GDP-dissociation-inhibitor (GDI) for Rho-like GTP-binding proteins is capable of three different biochemical activities . These are the inhibition of GDP dissociation, the inhibition of GTP hydrolysis, and the stimulation of the release of GTP-binding proteins from membranes . In order to better understand how GDI interactions with Rho-like proteins mediate these different effects, we have set out to develop a direct fluorescence spectroscopic assay for the binding of the GDI to the Rho-like protein, Cdc42Hs . We show here that when the GDI interacts with Cdc42Hs that contains bound N-methylanthraniloyl GDP (Mant-GDP), there is an approximately 20% quenching of the Mant fluorescence . The GDI-induced quenching is only observed when Mant-GDP is bound to Spodoptera frugiperda-expressed Cdc42Hs and is not detected when the Mant nucleotide is bound to Escherichia coli-expressed Cdc42Hs and thus shows the same requirement for isoprenylated GTP-binding protein as that observed when assaying GDI activity . A truncated Cdc42Hs mutant that lacks 8 amino acids from the carboxyl terminus and is insensitive to GDI regulation also does not show changes in the fluorescence of its bound Mant-GDP upon GDI addition . Thus, the GDI-induced quenching of Mant-GDP provides a direct read-out for the binding of the GDI to Cdc42Hs . Titration profiles of the GDI-induced quenching of the Mant-GDP fluorescence are saturable and are well fit to a simple 1:1 binding model for Cdc42Hs-GDI interactions with an apparent Kd value of 30 nM . A very similar Kd value (28 nM) is measured when titrating the GDI-induced quenching of the fluorescence of Mant-guanylyl imidotriphosphate, bound to Cdc42Hs . These results suggest that the GDI can bind to the GDP-bound and GTP-bound forms of Cdc42Hs equally well . We also have used the fluorescence assay for GDI interactions to demonstrate that the differences in functional potency observed between the GDI molecule and a related human leukemic protein, designated LD4, are due to differences in their binding affinities for Cdc42Hs . This, together with the results from studies using GDI/LD4 chimeras, allow us to conclude that a limit region within the carboxyl-terminal domain of the GDI molecule is responsible for its ability to bind with higher affinity (compared with LD4) to Cdc42Hs. Science, 1996 Apr 26, 272(5261), 557 - 60 Transcription-coupled repair deficiency and mutations in human mismatch repair genes; Mellon I et al.; Deficiencies in mismatch repair have been linked to a common cancer predisposition syndrome in humans, hereditary nonpolyposis colorectal cancer (HNPCC), and a subset of sporadic cancers . Here, several mismatch repair-deficient tumor cell lines and HNPCC-derived lymphoblastoid cell lines were found to be deficient in an additional DNA repair process termed transcription-coupled repair (TCR) . The TCR defect was corrected in a mutant cell line whose mismatch repair deficiency had been corrected by chromosome transfer . Thus, the connection between excision repair and mismatch repair previously described in Escherichia coli extends to humans . These results imply that deficiencies in TCR and exposure to carcinogens present in the environment may contribute to the etiology of tumors associated with genetic defects in mismatch repair. J Mol Biol, 1996 Apr 26, 258(1), 74 - 87 Dynamics of the GroEL-protein complex: effects of nucleotides and folding mutants; Sparrer H et al.; Chaperonins are a ubiquitous class of ring-shaped oligomeric protein complexes that are of crucial importance for protein folding in vivo . Analysis of the underlying functional principles had relied mainly on model proteins the (un)folding of which is dominated by irreversible side-reactions . We used maltose-binding protein (MBP) as a substrate protein for GroEL, since the refolding of this protein is completely reversible and thus allows a detailed analysis of the molecular parameters that determine the interaction of GroEL with non-native protein . We show that MBP folding intermediates are effectively trapped by GroEL in a diffusion-controlled reaction . This complex is stabilized via unspecific hydrophobic interactions . Stabilization energies for wild-type MBP increasing linearly with ionic strength from 50 kJ/mol to 60 kJ/mol . Depending on the intrinsic folding rate and the hydrophobicity of the substrate protein, the interaction of GroEL with MBP folding intermediates leads to a dramatically decreased apparent refolding rate of MBP (wild-type) or a complete suppression of folding (MBP folding mutant Y283D) . On the basis of our data, a quantitative kinetic model of the GroEL-mediated folding cycle is proposed, which allows simulation of the partial reactions of the binding and release cycles under all conditions tested . In the presence of ATP and non-hydrolysable analogues, MBP is effectively released from GroEL, since the overall dissociation constant is reduced by three orders of magnitude . Interestingly, binding of nucleotide does not change the off rate by more than a factor of 3 . However the on-rate is decreased by at least two orders of magnitude . Therefore, the rebinding reaction is prevented and folding occurs in solution. J Mol Biol, 1996 Apr 26, 258(1), 14 - 24 Transcription activation parameters at ara pBAD; Zhang X et al.; We studied the formation of open complexes of RNA polymerase and promoter DNA as activated by the AraC protein at the Escherichia coli araBAD promoter pBAD and by the cyclic AMP receptor protein at the galKTE promoter P1 . The DNA migration retardation assay was demonstrated to be suitable for the detection and quantitation of open complexes by the correspondence in the properties of open complexes in solution and retarded complexes observed in gels . These included, on the ara promoter, heparin resistance, lifetime, DNAseI footprinting, exonuclease III footprinting, permanganate footprinting and disappearance upon transcription, and on the gal promoter, the correspondence between the kinetic parameters Kd and k2 obtained with established techniques and those obtained with the migration retardation assay . On the pBAD promoter we obtained kinetic parameters of Kd = 0.3 nM and K2 = 1 minute(-1) . The unusually tight binding of polymerase in the presence of AraC suggests that AraC binds polymerase tightly. Nature, 1996 Apr 25, 380(6576), 730 - 4 Context-dependent secondary structure formation of a designed protein sequence; Minor DL Jr et al.; Protein secondary structures have been viewed as fundamental building blocks for protein folding, structure and design . Previous studies indicate that the propensities of individual amino acids to form particular secondary structures are the result of a combination of local conformational preferences and non-local factors . To examine the extent to which non-local factors influence the formation of secondary structural elements, we have designed an 11-amino-acid sequence (dubbed the 'chameleon' sequence) that folds as an alpha-helix when in one position but as a beta-sheet when in another position of the primary sequence of the IgG-binding domain of protein G (GB1) . Both proteins, chameleon-alpha and chameleon-beta, are folded into structures similar to native GB1, as judged by several biophysical criteria . Our results demonstrate that non-local interactions can determine the secondary structure of peptide sequences of substantial length . They also support views of protein folding that favour tertiary interactions as dominant determinants of structure. Nature, 1996 Apr 25, 380(6576), 727 - 30 Structure of the UmuD' protein and its regulation in response to DNA damage; Peat TS et al.; For life to be sustained, mistakes in DNA repair must be tolerated when damage obscures the genetic information . In bacteria such as Escherichia coli, DNA damage elicits the well regulated 'SOS response' . For the extreme case of damage that cannot be repaired by conventional enzymes, there are proteins that allow the replication of DNA through such lesions, but with a reduction in the fidelity of replication . Essential proteins in this mutagenic process are RecA, DNA polymerase III, UmuD, UmuD' and UmuC (umu: UV mutagenesis) . Regulation of this response involves a RecA-mediated self-cleavage of UmuD to produce UmuD' . To understand this system in more detail, we have determined the crystal structure of the E . coli UmuD' mutagenesis protein at 2.5 A resolution . Globular heads folded in an unusual Beta-structure associate to form molecular dimers, and extended amino-terminal tails associate to produce crystallized filaments . The structure provides insight into the mechanism of the self-cleavage reaction that UmuD-like proteins undergo as part of the global SOS response. Mol Gen Genet, 1996 Apr 24, 251(1), 1 - 12 Cell cycle-dependent expression of the mouse Rad51 gene in proliferating cells; Yamamoto A et al.; The mouse Rad51 gene is a mammalian homologue of the Escherichia coli recA and yeast RAD51 genes, both of which are involved in homologous recombination and DNA repair in mitosis and meiosis . The expression of mouse Rad51 mRNA was examined in synchronized mouse m5S cells . The Rad51 transcript was observed from late G1 phase through to M phase . During the period of late G1-S-G2, the RAD51 proteins were observed exclusively in nuclei . Activation by mitogens of T cell and B cell proliferation in spleen induced the expression of Rad51 mRNA . By immunohistochemical analyses, in mouse RAD51 protein was detected in proliferating cells: spermatogonia in testis, immature T cells in thymus, germinal center cells of the secondary lymphatic nodules of spleen and intestine, follicle cells in ovary and epithelial cells in uterus and intestine . It was also expressed in spermatocytes during early and mid-prophase of meiosis and in resting oocytes before maturation . Thus, mouse Rad51 expression is closely related to the state of cell proliferation and is presumably involved in DNA repair coupled with DNA replication, as well as in meiotic DNA recombination in spermatocytes. Biochemistry, 1996 Apr 23, 35(16), 5333 - 8 Cysteine-scanning mutagenesis of helix VI and the flanking hydrophilic domains on the lactose permease of Escherichia coli; Frillingos S et al.; Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in putative transmembrane helix VI and the flanking hydrophilic loops (residues 164- 211) was replaced individually with Cys . Of the 48 mutants, 43 accumulate lactose at highly significant rates to > 80% of the steady state observed with C-less permease . Three mutants (Phe185--> Cys, Ala187--> Cys, and Phe208--> Cys) exhibit lower but significant levels of accumulation (30-60% of C-less) . Cys replacement for Ala177 or Leu184 results in low transport activity (ca . 20%) in the C-less background but much higher activity (60-70%) in the wild type . Immunoblot analysis reveals that all of the mutants are inserted into the membrane at concentrations comparable to that of C-less perrmease . The transport activity of the great majority of the mutants is unaffected by treatment with N-ethylmaleimide (NEM) . Relatively modest but significant inactivation (ca . 50%) is observed with mutants Phe170--> Cys, Gly173--> Cys, and Ala187--> Cys, and these positions cluster on the same face of the helix VI . Moreover, the two positions where single Cys replacements result in low activity (Ala177 and Lcu184) are on the same face of helix VI . The results demonstrate the following . (i) Permease function is not disrupted by replacement of most residues with Cys, but function is disrupted when some of the residues are further altered by addition of the NeM moiety . (ii) The latter residues lie on a stripe down one face of an alpha-helix, and within the same stripe are residues where Cys substitution itself leads ti inhibition of function. Biochemistry, 1996 Apr 23, 35(16), 5318 - 26 Inhibitors of Escherichia coli RNA polymerase specific for the single-stranded DNA of transcription intermediates . Tetrahedral cuprous chelates of 1,10-phenanthrolines; Perrin DM et al.; Single-stranded DNA of the lacUV-5 promoter formed at the active site of Escherichia coli RNA polymerase during transcription is specifically cleaved by the redox active tetrahedral cuprous chelates of 1,10-phenanthroline and its derivatives . The cleavage sites are observed in the open, initiating, and elongating complexes . Redox-inert, tetrahedral cuprous chelates of neocuproine (2,9-dimethyl-1,10- phenanthroline) and its 5-phenyl and 4-phenyl derivatives protect the template strand of DNA from scission within these steady state intermediates and inhibit transcription . Although these cuprous chelates of neocuproine bind at multiple sites within three distinct enzyme intermediates, the highest affinity site is within the elongation complex . The I50 of 5 microM for the 2:1 5-phenylneocuproine cuprous complex ((5 phi NC)2Cu+) in runoff transcription therefore primarily reflects its intermediate . The neocuproine cuprous chelates are novel transcription inhibitors because they bind to single-stranded DNA sites generated during the course of catalysis by RNA polymerase. Biochemistry, 1996 Apr 23, 35(16), 5300 - 7 Dimerization of the human cytomegalovirus protease: kinetic and biochemical characterization of the catalytic homodimer; Margosiak SA et al.; The single-chain 28 kDa human cytomegalovirus (HCMV) protease catalytic domain containing the A143Q mutation has been kinetically and conformationally characterized . The specific activity of the HCMV A143Q protease (HCMVp) increases as the protease concentration increases, suggesting that this protease oligomerizes at high protein concentration to form a more active species . Both cross-linking and light-scattering studies of HCMVp show the existence of a homodimer with an apparent molecular mass of 56 kDa under low ionic strength and high protein concentration . The cosolvent and solute effects of glycerol, trisodium citrate, and NaCl as well as the temperature effects on the HCMVp activity and quaternary structure were investigated . The effects induced by cosolvents and temperature can largely be explained by their influences in the dimerization or oligomerization state of HCMVp . The dissociation constant (Kd) for the HCMVp homodimer was determined to be 8 +/- 1 microM with all activity attributed to the dimeric form . Monomeric HCMVp is inactive . This report demonstrates that in vitro, HCMV A143Q protease exists as an obligate catalytic homodimer . This protease dimerization may have regulatory significance during viral replication. Biochemistry, 1996 Apr 23, 35(16), 5280 - 91 The reaction catalyzed by Escherichia coli aspartate aminotransferase has multiple partially rate-determining steps, while that catalyzed by the Y225F mutant is dominated by ketimine hydrolysis; Goldberg JM et al.; The mechanism of transamination catalyzed by Escherichia coli wild-type aspartate aminotransferase (AATase) and the mutant AAtase in which Tyr-225 is converted to Phe (Y225F) was investigated . The absorbance spectrum of wild-type AATase in the presence of excess L-Asp and oxalacetate is dominated by species absorbing near 330 nm . The primary C alpha 2H-Asp kinetic isotope effects (KIEs) on reactions catalyzed by wild-type AAtase at pH 8.9 and 7.5 on kcat/KMAsp are approximately 2, and the KIEs on kcat are 1.9 (pH 8.9) and 1.4 (pH 7.5) . The C alpha 2H-Asp KIEs on reactions catalyzed by Y225F are near unity at both pH values . The solvent deuterium KIEs (SKIEs) on kcat for reactions with L-Asp catalyzed by wild-type AATase and Y225F at their pH/pD maxima approximately 2, and the SKIE on kcat/kMAsp is increased from 1.3 to 2.3 by the mutation . The C4' (S)-2H-pyridoxamine 5'-phosphate KIE values on reactions of alpha-ketoacids with both enzymes are near unity . The viscosity effects on kcat/KMAsp and kcat for wild-type AAtase at pH 9 are 0.10 and 0.31, respectively, indicating that the reaction is partially diffusion limited . The viscosity effects on kcat/KMAsp and kcat for Y225F are reduced to -0.02 and 0.06, respectively, indicating that the mutant catalyzed reaction is almost fully chemistry-limited . A free-energy profile for the L-Asp-to-oxalacetate half-reaction was constructed for wild-type AAtase . C alpha H abstraction, ketimine hydrolysis, and oxalacetate dissociation are partially rate-determining . Ketimine hydrolysis is the sole rate-determining step for the corresponding Y225F- catalyzed reaction. Biochemistry, 1996 Apr 23, 35(16), 5272 - 9 A highly salt-dependent enthalpy change for Escherichia coli SSB protein-nucleic acid binding due to ion-protein interactions; Lohman TM et al.; We have examined the linkage between salt concentration and temperature for the equilibrium binding of the tetrameric Escherichia coli single-stranded binding (SSB) protein to three single-stranded nucleic acids, poly(U), dA(pA)69, and dT(pT)69, by van't Hoff analysis and isothermal titration calorimetry (ITC) . For SSB binding to poly(U) in its (SSB)65 mode, the equilibrium association constant, Kobs, decreases with increasing salt concentration at all temperatures examined, and binding is enthalpy-drive; however, the value of {symbol see text} log Kobs/ {symbol see text} log {NaCl} is highly temperature- dependent, varying from -9.3 +/- 0.3 at 10 degrees C to -5.1 +/- 0.4 at 37 degrees C . This indicates that delta Hobs for SSB-poly(U) binding is strongly dependent on {NaCl}; based on van't Hoff analyses, delta Hobs varies from -57 +/- 3 kcal/mol at 0.18 M NaCl to -34 +/- 3 kcal/mol at 042 M NaCl ({symbol see text} delta Hobs/{symbol see text} log {NaCl} = 60 +/- 5 kcal/mol) . However, {symbol see text} delta Hobs/{symbol see text} log {NaF} is independent of temperature (25-37 degrees C), indicating that the effect of {NaCl} on delta Hobs is due primarily to Cl- . Similar effects were also observed for SSB binding to dA(pA)69 . We also measured delta Hobs and its dependence on {NaCl} for SSB binding dT(pT)69 by ITC and find delta Hobs = -144 +/- 4 kcal/mol (0.175 M NaCl, pH 8.1, 25 degrees C) and {symbol see text} delta Hobs/ {symbol see text} log {NaCl} = 46 +/- 2 kcal/ mol (0.175-2.0 M NaCl) . These large effects of {NaCl} on delta Hobs appear to result, at least partly, from the release of preferentially bound Cl- from SSB protein upon binding nucleic acid, with the release of Cl- being linked to a process with delta H > > 0 . Effects of salt concentration on delta Hobs are not observed for processes in which only monovalent cations are released from the nucleic acid, presumably since Na+ of K+ are bound to linear nucleic acids as delocalized, fully hydrated cations . Such salt effects on delta Hobs may serve as a signature for differential ion-protein binding . These results underscore the need to examine the linkage of {salt} to delta Hobs, as well as delta Hobs degrees and delta S(obs) degrees, in order to understand the bases for stability and specificity of protein-nucleic acid interactions. Biochemistry, 1996 Apr 23, 35(16), 5220 - 8 Multiple structural elements define the specificity of recombinant human inhibitor-1 as a protein phosphatase-1 inhibitor; Endo S et al.; The cDNA encoding human brain protein phosphatase inhibitor-1 (I-1) was expressed in Escherichia coli . Following PKA phosphorylation at a threonine, recombinant human I-1 was indistinguishable from rabbit skeletal muscle I-1 as a potent and specific inhibitor of the type-1 protein serine/threonine phosphatase (PP1) . N-Terminal phosphopeptides of I-1 that retained the selectivity of intact human I-1 highlighted a functional domain that mediates PP1 inhibition . Substituting alanine in place of threonine-36 eliminated I-1 phosphorylation by PKA and its phosphatase inhibitor activity . An acidic residue was substituted in place of the phosphoacceptor to produce I-1(T35D), a constitutive phosphate inhibitor . I-1(T35D) was an equally effective inhibitor of PP1 and the type-2 phosphatase, PP2A . However, CNbr digestion of I-1(T35D) yielded an N-terminal peptide that showed 100-fold increased specificity as a PP1 inhibitor . This provided new insight into a unique conformation of the phosphorylated I-1 that accounts for selective inhibition of PP1 activity . Truncation of an active I-1 phosphopeptide identified an N-terminal sequence that was reduced in addition to threonine-35 phosphorylation to inhibit PP1 activity . Biosensor studies demonstrated that PP1 bound to both Phosphorylated and dephosphorylated I-1 and suggested that distinct elements of I-1 structure accounted for PP1 binding and inhibition . Our data point to multiple interactions between the I-1 functional domain . and the PP1 catalytic subunit that define this phosphoprotein as a physiological regulator of the type-1 protein phosphatase. Biochemistry, 1996 Apr 23, 35(16), 5213 - 9 Purification and functional characterization of the C-terminal half of the lactose permease of Escherichia coli; Wu J et al.; The lactose permease has been expressed in contiguous, non-overlapping polypeptide fragments containing the N-terminal (N6) and C-terminal (C6) transmembrane domains of the protein {Bibi, E., & Kaback, H . R . (1990) Proc . Natl . Acad . Sci . U.S.A . 87, 4325; Zen, K., et al . (1994) Biochemistry 33, 8198} . When expressed individually, N6 and C6 are unstable and do not catalyze active transport . However, when expressed simultaneously, the polypeptides stabilize each other and form a complex that catalyzes active lactose transport . Moreover, a deletion construct containing the first transmembrane domain and the six C-terminal transmembrane domains mediates downhill lactose translocation {Bibi et al . (1991) proc . Natl . Acad . Sci . U.S.A . 88, 7271} . Here we report that C6 can be expressed independently in a relatively stable form that binds monoclonal antibodies 4B1 and 4B11, which interact with conformationally dependent epitopes on the periplasmic and cytoplasmic surfaces of the membrane, respectively . In addition, C6 retains the ability to catalyze lactose translocation down a concentration gradient in a specific manner . Finally, as observed with full-length Val331Cys permease, beta-D-galactopyranosyl 10thio-beta-D-galactopyranoside quenches the fluorescence of 2-(4'-maleimidylanilino)naphthalene- 6-sulfonic acid (MIANS)- labeled C6 with a single-Cys residue in place of Val331, exhibiting as apparent Kd of 0.2 mM . Unlike full-length Val331Cys permease, however, ligand does not induce a chance in the position of the emission maximum of MIANS-labeled C6(Val331Cys) permease not in the reactivity of C6 (Val331Cys) permease with MINAS . the results indicate that C6 retains a conformation similar to that on the native permease and that most of the structure required of high-affinity binding and substrate translocation is located in the C-terminal half of the molecule. Biochemistry, 1996 Apr 23, 35(16), 5199 - 206 The cytoplasmic fragment of the aspartate receptor displays globally dynamic behavior; Seeley SK et al.; A number of cloned soluble fragments if the bacterial chemotaxis transmembrane receptors retain partial function . Prior studies of a fragment corresponding to the cytoplasmic domain (c-fragment) of the Escherichia coli aspartate receptor have correlated the signaling state of mutant receptors with the oligomerization state of the c-fragments: equilibria of smooth-swimming mutants are shifted toward oligomeric states; tumble mutants are shifted toward monomeric states {Long, D . G., & Weis, R . M . (1992) Biochemistry 31, 9904-9911} . We have applied several experimental probes of local and global structural flexibility to two signaling states, the wild-type (monomeric) and S461L smooth mutant (predominantly dimeric) c-fragments . Featureless near-UV CD spectra are observed, which indicate that the single Trp residue is in a symmetric environment (most likely averaged by fluctuations) and suggest that the C-termini of both proteins are highly mobile . Both proteins undergo extremely rapid proteolysis and enhance ANS fluorescence, which indicates that many sites are accessible to trypsin cleavage and hydrophobic sites are accessible to ANS binding . The global nature of the flexibility is demonstrated by 1H NMR studies . Lack of chemical shift dispersion suggests that fluctuations average the environments of side chains and backbone protons . Rapid exchange of 99% of the observable amide protons suggests that these fluctuations give high solvent accessibility to nearly the entire backbone . This evidence indicates that both monomeric and dimeric c-fragments are globally flexible proteins, with properties similar to "molten-globule" states . The significance of this flexibility depends on whether it is retained in functioning receptors: the c-fragment structure may lack important tertiary contacts, protein-protein interactions, or topological constraints needed to stabilize a nondynamic native structure, or the cytoplasmic domain of the native receptor may retain flexibility which may be modulated in the mechanism of transmembrane signaling. Biochemistry, 1996 Apr 23, 35(16), 5166 - 74 Characterization of two glycolipid: alpha 2-3sialyltransferases, SAT-3 (CMP-NeuAc:nLcOse4Cer alpha 2-3sialyltransferase) and SAT-4 (CMP-NeuAc:GgOse4Cer alpha 2-3sialyltransferase), from human colon carcinoma (Colo 205) cell line; Basu SS et al.; Sialyltransferase activities, SAT-3 (CMP-NeuAc:nLcOse4Cer alpha 2-3sialyltransferase) and SAT-4 (CMP-NeuAc:GgOse4Cer alpha 2-3sialyltransferase), in Colo 205 cells catalyze the transfer of sialic acid to the terminal galactose of GlcNc-- and GalNAc-containing glycolipid substrates, respectively . Competition kinetic studies with nLcOse4Cer and GM1 as substrates in a sialyltransferase assay show that these two activities are catalyzed by two different catalytic entities . The two enzymes were co-solubilized with taurochlorate and resolved by DEAE--Cibacron Blue--Sepharose column chromatography into two elution peaks . The column eluent with SAT-3 activity failed to transfer sialic acid to asialo alpha(1)-acid glycoprotein, indicating that this enzyme is different from the sialyltransferase (ST3N) that synthesizes NeuAc alpha 2-3Gal linkage in asparagine-linked oligosaccharides of glycoprotein . However, SAT-3 activity can be immunoprecipitated with a polyclonal antibody produced against a protein expressed in Escherichia coli as GST-fusion protein from an ECB cDNA homolog of an alpha 2-3 sialyltransferase SAT-3 or STZ) the has been cloned from human melanoma cell and human placenta . Thus a concentration-dependent decrease in the residual SAT-3 activity relative to SAT-4 activity was observed in the supernatant after precipitation of the immune complex . Expression of SAT-3 (STZ) cDNA was also detected in Colo 205 cell by RT-PCR, followed by sequence analysis of the RT-PCR product . Characterization of the catalytic reaction products of SAT-3 and SAT-4 with thin-layer chromatography, sialidase treatment, and binding to specific antibodies indicates that both SAT-3 and SAT-4 catalyze the formation of alpha 2-3 linkage between sialic acid and terminal galactose of glycolipid substrates. FEBS Lett, 1996 Apr 22, 384(3), 289 - 93 cDNAs encoding spinach stromal and thylakoid-bound ascorbate peroxidase, differing in the presence or absence of their 3'-coding regions; Ishikawa T et al.; Two cDNA clones encoding stromal (SAP28) and thylakoid-bound (SAP22) ascorbate peroxidase were isolated from a spinach cDNA library constructed by greening cotyledons . The SAP22 and SAP28 contained an open reading frame encoding mature protein of 295 and 345 amino acids with calculated molecular mass of 32239 Da and 37710 Da, respectively, preceded by the common transit peptides of 70 amino acid residues . Interestingly, the N-terminal 364 amino acids of SAP22 were 100% identical with SAP28 except for one C-terminal amino acid residue (Asp-365), and the C-terminal of SAP22, which is the putative transmembrane segment, was 50 amino acids longer than that of SAP28. FEBS Lett, 1996 Apr 22, 384(3), 265 - 8 Vaccinia virus DNA topoisomerase I preferentially removes positive supercoils from DNA; Fernandez-Beros ME et al.; Type I DNA topoisomerases homologous to Escherichia coli topoisomerase I normally only remove negative supercoils from DNA . Topoisomerases I from various eukaryotes share sequence homology and remove both positive and negative supercoils from DNA . Here we report that vaccinia virus topoisomerase I has significant difference in substrate preference from the other homologous type I topoisomerases . Vaccinia virus topoisomerase I shows a definite preference for removal of positive supercoils . In contrast, topoisomerase I from human, wheat germ and Saccharomyces cerevisiae has little preference between positive and negative supercoils . The vaccinia enzyme may have evolved for functions required for optimal viral growth . topoisomerases. J Mol Biol, 1996 Apr 19, 257(5), 992 - 1007 Role of the N terminus in RNase A homologues: differences in catalytic activity, ribonuclease inhibitor interaction and cytotoxicity; Boix E et al.; A number of biochemical properties differ dramatically among homologues within the pancreatic ribonuclease superfamily . Human pancreatic ribonuclease (hRNase) has high enzyme activity, extreme sensitivity to ribonuclease inhibitor (RI) and is non-toxic, whereas a homologous RNase from frog eggs, called onconase, has much lower enzyme activity, is not sensitive to RI and is cytotoxic to cancer cell lines and animals . To explore the structural basis of these differences among members in the RNAse family we synthesized genes for onconase, hRNase, a mutant onconase (K9Q) and onconase-hRNase N-terminal hybrids and expressed the proteins in Escherichia coli with final yields of 10 to 50 mg per liter of culture after purification . A recombinant version of onconase with an N-terminal methionine instead of the native pyroglutamyl residue had decreased cytotoxicity and enzyme activity . Cleavage of the recombinant onconase Met-1 residue, and cyclization of the Gln1 residue to reform the pyroglutamyl N terminus, reconstituted cytotoxicity and enzyme activity . Thus a unique role of the pyroglutamyl residue in the active site of amphibian RNases is indicated . Replacement of one to nine residues of onconase with the homologous residues of hRNase increased the enzymatic activity against most of the substrates tested with a simultaneous shift in the enzyme specificity from high preference for poly(U) to slight preference for poly(C) . Cytotoxicity of the chimera decreased, dissociating cytotoxicity from enzymatic activity . The molecular basis for the low binding affinity of onconase for RI has been examined experimentally with the recombinant RNases and by fitting onconase and RNase A structures to the coordinates from the recently published RNase A-RI complex. J Mol Biol, 1996 Apr 19, 257(5), 977 - 91 The type I restriction endonuclease R.EcoR124I: over-production and biochemical properties; Janscak P et al.; In this paper we describe a two-plasmid system which allows over-production of the R.EcoR124I restriction endonuclease . The endonuclease has been purified to homogeneity in milligram amounts and has been shown to be fully active for both restriction and modification . Unexpectedly, the enzyme was found to require only ATP and Mg2+ for ATPase activity and DNA cleavage; S-adenosyl methionine (SAM), which has been described as a cofactor of type I restriction enzymes, is not required by R.EcoR124I . However, SAM was found to stimulate the rate of ATPase activity and DNA cleavage . This may occur through an increase in specific DNA binding by R.EcoR124I in the presence of SAM, as indicated by our surface plasmon resonance experiments . These functional differences from the well described R.EcoKI restriction endonuclease are reflected in a possible structural difference between the two enzymes, namely that the stoichiometry of R.EcoR124I appears to be R1M2S1 while that of R.EcoKI is R2M2S1 . Supercoiled DNA with one or two SR124I recognition sites is cleaved by the same mechanism inferring co-operation between specifically bound and excess enzymes . Nicked-circle DNA is an intermediate of cleavage reaction . Cleavage of DNA was inhibited by an increased degree of negative supercoiling, which may reflect an increased difficulty for the enzyme to translocate the DNA . Hemi-methylated DNA was the preferred substrate for methylation. J Mol Biol, 1996 Apr 19, 257(5), 970 - 6 Binding and incision activities of UvrABC excinuclease on slipped DNA intermediates that generate frameshift mutations; Delagoutte E et al.; Previous in vivo studies involving sequence 5'-CCCG1G2G3-3' (SmaI site) have demonstrated that adducts of N-2-acetylaminofluorene (AAF) to any of the three guanine residues of the SmaI sequence induce, with different efficiencies, two classes of -1 frameshift events, namely -G and -C mutations, referred to as targeted and semitargeted mutations, respectively . It has been proposed that both events occur during replication as a consequence of slippage events involving slipped mutagenic intermediates (SMIs) . In order to evaluate the potential role of the UvrABC excinuclease in frameshift mutagenesis, we have studied the interaction of this enzyme with DNA molecules mimicking SMIs in vitro . In all of our constructions, when present, the AAF adduct was located on the third guanine residue of the SmaI site (5'-CCCG1G2G3-3') . This strand was referred to as the top strand, the complementary strand being the bottom strand . Double-stranded heteroduplexes mimicking the targeted and semitargeted SMIs contained a deletion of a C and a G within the SmaI sequence in the bottom strand and were designated deltaC/3 and deltaG/3 when modified with the AAF on the third guanine residue in the top strand or deltaC/O and deltaG/O when unmodified . The modified homoduplex was designated SmaI/3 . deltaC/O and deltaG/O were weakly recognized by UvrA2B, but not incised . All three AAF-modified substrates were recognized with similar efficiency and much more efficiently than unmodified heteroduplexes . With AAF-monomodified substrates, dissociation of UvrA2 from the UvrA2B-DNA complex occurred more readily in heteroduplexes than in the homoduplex . SmaI/3 and deltaC/3 were incised with equal efficiency, while deltaG/3 was less incised . The position of the AAF lesion dictated the position of the incised phosphodiester bonds, suggesting that the presence of a bulge can modulate the yield but not the incision pattern of AAF-modified substrates . The finding that UvrABC excinuclease acts on substrates that mimic SMIs suggests that the nucleotide excision repair pathway may help in fixing frameshift mutations before the following round of replication. J Mol Biol, 1996 Apr 19, 257(5), 960 - 9 ATPase activity of the type IC restriction-modification system EcoR124II; Dreier J et al.; We have investigated the ATPase activity of the type IC restriction-modification (R-M) system EcoR124II . As with all type I R-M systems EcoR 124II requires ATP hydrolysis to cut DNA . We determined the KM for ATP to be 10(-5) to 10(-4) M . By measuring ATP hydrolysis under different conditions and by simultaneously monitoring DNA restriction, methylation and ATP hydrolysis we propose that the order of events during restriction is: (1) binding of EcoR124II to a non-methylated recognition sequence, (2) start of DNA-dependent ATP hydrolysis which continues even after restriction is complete, (3) restriction of DNA, (4) methylation of the product . Non-cleavable DNA substrates, such as recognition site containing oligonucleotides, also support ATP hydrolysis . Methylation can also occur prior to ATP hydrolysis and prevent DNA degradation. J Mol Biol, 1996 Apr 19, 257(5), 909 - 18 Residues in the RNP1-like sequence motif of Rho protein are involved in RNA-binding affinity and discrimination; Martinez A et al.; The termination of transcription in Escherichia coli by action of Rho factor is dependent on the ability of this homohexameric protein to make productive interactions with the nascent RNA molecule to be terminated . The roles of two residues in a phylogenetically conserved sequence motif in the RNA-binding domain of Rho, Asp60 and Phe62, were analyzed by studies of the biochemical properties of pure mutant proteins . F62S Rho had greatly reduced affinity for lambda cro RNA, very poor ability to terminate transcription in vitro by itself and only partial termination activity (at a level consistent with its in vivo defect) in the presence of NusG . D60G Rho had a high affinity for lambda cro RNA but a much lower ability to discriminate against RNA molecules lacking cis-acting Rho-utilization sequences, and a reduced efficiency of termination that was not improved by NusG . These results indicate a major role for Phe62 in stabilizing the binding of Rho to RNA through hydrophobic interactions, while Asp60 provides an electrostatic repulsive force that allows a rapid dissociation of non-productive complexes with RNA. J Mol Biol, 1996 Apr 19, 257(5), 895 - 908 Mutational analysis and secondary structure model of the RNP1-like sequence motif of transcription termination factor Rho; Martinez A et al.; The function of transcription termination factor Rho from Escherichia coli is dependent upon its ability to bind to specific sites on nascent RNA molecules . The roles of 19 individual amino acid residues (Ile49 to Ser67) in and near a phylogenetically conserved sequence segment of Rho that is similar to the RNP1 motif found in many RNA-binding proteins were examined by testing the phenotypic consequences of mutational changes that were introduced into rho by a random-sequence cassette mutagenesis procedure . The tests of each mutant included the ability of the cells to survive at 42 degrees C in the absence of wild-type rho, the efficiency of termination at a Rho-dependent terminator (lambdatR1) in vivo, the relative level of expression of the mutant protein, and the ability of some of the mutant proteins to bind RNA . The results revealed that residues in the RNP1-like sequence of DGFGFLR (residues 60 to 66) were more important than residues 49 to 59 for termination function and RNA binding, and identified three residues that were particularly sensitive to mutation: Asp60, Phe62 and Arg66 . The properties of the mutants are consistent with a secondary structure model, derived from phylogenetic analysis, that has the RNP1-like sequence on one of the three beta-strands of an antiparallel beta-sheet with Asp60 and Gly61 in a turn and the side-chains of Phe62, Phe64 and Arg66 accessible on the same face of the beta-structure for interaction with RNA. J Mol Biol, 1996 Apr 19, 257(5), 1070 - 87 Analysis of the low frequency normal modes of the T-state of aspartate transcarbamylase; Thomas A et al.; Aspartate transcarbamylase (ATCase) is an important control enzyme in the pyrimidine biosynthetic pathway in Escherichia coli . It is a classic example of an allosteric protein and has been extensively studied biochemically, kinetically and structurally . As yet, however, a detailed model for the cooperative transition between the tensed (T) and relaxed (R) forms of the protein does not exist . In this work we have calculated the low frequency normal modes of the CTP-ligated T-state of ATCase with the aim of identifying some of the motions that could be important in initiating the transition . The calculated modes, of frequencies lower than 5 per cm, produce root-mean-square coordinate deviations for the atoms which are a substantial fraction of those derived from the crystallographic B-factors . Some of the modes result in displacements which change the quaternary structure of the protein (in particular the elongation of the protein and the relative rotation of the subunits) in such a way that the R-state structure is approached . The implication of these mode motions for the overall T-->R transition process is discussed. J Biol Chem, 1996 Apr 19, 271(16), 9730 - 8 Human T-cell leukemia virus type I tax masks c-Myc function through a cAMP-dependent pathway; Semmes OJ et al.; Human T-cell leukemia virus type I Tax is a pleiotropic gene regulator that functions through CREB/ATF- and NF-kappaB-mediated pathways . In most contexts, Tax is a potent gene activator . Here, we describe an unexpected finding of Myc repression by Tax . In cells that overexpress human T-cell leukemia virus type I Tax, the detection of c-Myc protein in the nucleus by a monoclonal antibody was masked . Tax prevented immunological visualization of a Myc epitope contained within amino acids 45-104, resulting in interference with Myc function in transcription and in anchorage-independent cell growth . Tax did not affect steady-state protein levels since detection of c-Myc with other antibodies was unperturbed . Four observations suggest that this Tax-Myc interaction is mediated through CREB/ATF signal transduction . 1) Tax point mutants, selectively defective for activation of CREB/ATF but not NF-kappaB, failed to mask c-Myc; 2) masking of Myc was abolished when Tax-expressing cells were treated with protein kinase inhibitor H-9; 3) Tax-specific shielding of Myc is absent in cells (B1R) that are genetically defective for cAMP signaling; and 4) forskolin treatment of cells mimicked Tax in masking the Myc epitope . Considered collectively, these findings suggest a regulation of Myc function at the level of localized protein conformation. J Biol Chem, 1996 Apr 19, 271(16), 9723 - 9 Mutations in the B subunit of Escherichia coli DNA gyrase that affect ATP-dependent reactions; O'Dea MH et al.; We have previously reported specific labeling of Escherichia coli DNA gyrase by the ATP affinity analog pyridoxal 5'-diphospho-5'adenosine (PLP-AMP), which resulted in inhibition of ATP-dependent reactions . The analog was found to be covalently bound at Lys103 and Lys110 on the gyrase B subunit (Tamura, J . K., and Gellert, M . (1990) J . Biol . Chem . 265, 21342-21349) . In this study, the importance of these two lysine residues is examined by site-directed mutagenesis . Substitutions of Lys 103 result in the loss of ATP-dependent functions . These mutants are unable to supercoil DNA, to hydrolyze ATP, or to bind a nonhydrolysable ATP analog, 5'-adenylyl-beta,gamma-imidodiphosphate (ADPNP) . The ATP-independent functions of gyrase, such as relaxation of negatively supercoiled DNA and oxolinic acid-induced cleavage of double-stranded DNA, are unaffected by these mutations, suggesting that the mutant B subunits are assembling correctly with the A subunits . Gyrase with substitutions of Lys110 retains all activities . However, the affinity of ATP is decreased . The DNA supercoiling activity of gyrase A2B2, tetramers reconstituted with varying ratios of inactive mutant and wild-type gyrase B subunits is consistent with a mechanism of DNA supercoiling that requires the interdependent activity of both B subunits in ATP binding and hydrolysis. J Biol Chem, 1996 Apr 19, 271(16), 9716 - 22 Structural analysis of the predicted coiled-coil rod domain of the cytoplasmic bullous pemphigoid antigen (BPAG1) . Empirical localization of the N-terminal globular domain-rod boundary; Tang HY et al.; The bullous pemphigoid antigen BPAG1 is required for keratin filament linkage to the hemidesmosome, an adhesion complex in epithelial basal cells . BPAG1 structural organization is similar to the intermediate filament-associated proteins desmoplakin I (DPI) and plectin . All three proteins have predicted dumbbell-like structure with central alpha-helical coiled-coil rod and regions of N- and C-terminal homology . To characterize the size of the N-terminal globular domain in BPAG1, two polypeptides spanning possible boundaries with the coiled-coil rod domain of BPAG1 were expressed in Escherichia coli . BP-1 (Mr = 111,000), containing amino acids 663-1581 of BPAG1 (Sawamura, D., Li, K., Chu, M.-L., and Uitto, J . (1991) J . Biol . Chem . 266, 17784-17790), and BP-1A, with a 186 amino acid N-terminal deletion, were purified . BP-1 and BP-1A behave as highly asymmetric dimers in aqueous solution according to velocity sedimentation and gel filtration . Both have globular heads with rod-like tails of roughly equal length, 55-60 nm, upon rotary shadowing . BP-1A content of alpha-helix, determined by circular dichroism, is approximately 90%, consistent with alpha-helical coiled-coil formation in the rod-like tails . The estimated rod length, 383 +/- 57 amino acids (0.15 nm/amino acid), implies that globular folding in the BPAG1 N-terminal extends to the end of N-terminal homology with DPI and plectin . These findings support the existence of a common domain structure in the N-terminal regions of the BPAG1/DPI/plectin family. J Biol Chem, 1996 Apr 19, 271(16), 9648 - 59 Preparation of figure 8 and cruciform DNAs and their use in studies of the kinetics of branch migration; Mulrooney SB et al.; We have re-examined the kinetics of the branch migration of double-stranded DNA that is mediated by the stepwise movement of the Holliday junction . This work revises and extends our previous treatment (Thompson, B . J., Camien, M . N., and Warner, R . C . (1976) Proc . Natl . Acad . Sci . U.S.A . 73, 2299-2303) . New methodology and new highly purified substrates have been used . The latter include figure 8s prepared from phage G4 DNA by annealing single-stranded components and two sizes of a novel cruciform . We treat the process as a one-dimensional diffusion based on the random walk, the mathematical basis of which is discussed in detail . The step rate is shown to be 3 orders of magnitude slower than we reported previously . The most important contribution to the erroneously high rate was a result of the presence of EDTA in the spreading solution used for electron microscopy at that time . A second contribution of about 4-fold resulted from catalysis by EcoRI and other proteins . The rates reported here are for the uncatalyzed reaction. J Biol Chem, 1996 Apr 19, 271(16), 9519 - 25 Crystal structure of PotD, the primary receptor of the polyamine transport system in Escherichia coli; Sugiyama S et al.; PotD protein is a periplasmic binding protein and the primary receptor of the polyamine transport system, which regulates the polyamine content in Escherichia coli . The crystal structure of PotD in complex with spermidine has been solved at 2.5-A resolution . The PotD protein consists of two domains with an alternating beta-alpha-beta topology . The polyamine binding site is in a central cleft lying in the interface between the domains . In the cleft, four acidic residues recognize the three positively charged nitrogen atoms of spermidine, while five aromatic side chains anchor the methylene backbone by van der Waals interactions . The overall fold of PotD is similar to that of other periplasmic binding proteins, and in particular to the maltodextrin-binding protein from E . coli, despite the fact that sequence identity is as low as 20% . The comparison of the PotD structure with the two maltodextrin-binding protein structures, determined in the presence and absence of the substrate, suggests that spermidine binding rearranges the relative orientation of the PotD domains to create a more compact structure. J Biol Chem, 1996 Apr 19, 271(16), 9473 - 82 Equilibrium studies of kinesin-nucleotide intermediates; Rosenfeld SS et al.; We have examined the energetics of the interactions of two kinesin constructs with nucleotide and microtubules to develop a structural model of kinesin-dependent motility . Dimerization of the constructs was found to reduce the maximum rate of the microtubule-activated kinesin ATPase 5-fold . Beryllium fluoride and aluminum fluoride also reduce this rate, and they increase the affinity of kinesin for microtubules . By contrast, inorganic phosphate reduces the affinity of a dimeric kinesin construct for microtubules . These findings are consistent with a model in which the kinesin head can assume one of two conformations, "strong" or "weak" binding, determined by the nature of the nucleotide that occupies the active site . Data for dimeric kinesin are consistent with a model in which kinesin.ATP binds to the microtubule in a strong state with positive cooperativity; hydrolysis of ATP to ADP+P(i) leads to dissociation of one of the attached heads and converts the second, attached head to a weak state; and dissociation of phosphate allows the second head to reattach . These results also argue that a large free energy change is associated with formation of kinesin.ADP.P(i) and that this step is the major pathway for dissociation of kinesin from the microtubule. J Biol Chem, 1996 Apr 19, 271(16), 9429 - 36 Pro-OmpA derivatives with a His6 tag in their N-terminal "translocation initiation domains" are arrested by Ni2+ at an early post-targeting stage of translocation; Yoshihisa T et al.; We examined in vitro translocation of pro-OmpA derivatives with a His6 tag at various positions in their mature proteins and with a c-Myc tag at their C termini across inverted membrane vesicles of Escherichia coli . Those with a His6 tag in the N-terminal region of the mature domain, which corresponds to the "translocation initiation domain" proposed previously (Andersson, H., and von Heijne, G . (1991) Proc . Natl . Acad . Sci . U . S . A . 88, 9751-9754), could not be translocated in the presence of 100 micron Ni2+, while OmpA derivatives with a His6 tag in the middle of or at the C terminus did not show such Ni2+ sensitivity . The inhibitory action of Ni2+ on pro-3His-OmpA' (with a His6 tag after the third amino acid of the mature OmpA-c-Myc region) translocation was exerted only during early events, after which it became ineffective . The inhibition point of Ni2+ was suggested to lie between membrane targeting and exposure of the signal cleavage site to the periplasm since the unprocessed and membrane-bound form of pro-3His-OmpA' was accumulated by the addition of Ni2+ . The Ni(2+)-"trapped" precursor was released from its translocation block by 30 mM histidine, which should compete with the His6 tag on the precursor protein for formation of a Ni2+ chelating complex . We propose that Ni2+ confers a reversible positive charge effect on the His6-tagged initiation domain of the pro-OmpA derivatives and inhibits an early event(s) of protein translocation, such as presentation of the precursor to the membranous part of the translocase . This system will be useful in dissecting early events of the protein translocation pathway. J Biol Chem, 1996 Apr 19, 271(16), 9410 - 6 The anaerobic Escherichia coli ribonucleotide reductase . Subunit structure and iron sulfur center; Ollagnier S et al.; During anaerobic growth Escherichia coli uses a specific ribonucleoside triphosphate reductase for the production of deoxyribonucleoside triphosphates . The active species of this enzyme was previously found to be a large homodimer of 160 kDa (alpha 2) with a stable, oxygen-sensitive radical located at Gly-681 of the 80-kDa polypeptide chain . The radical is formed in an enzymatic reaction involving S-adenosylmethionine, NADPH, a reducing flavodoxin system and an additional 17.5-kDa polypeptide, previously called activase . Here, we demonstrate by EPR spectroscopy that this small protein contains a 4Fe-4S cluster that joins two peptides in a 35-kDa small homodimer (beta 2) . A degraded form of this cluster may have been responsible for an EPR signal observed earlier in preparations of the large 160-kDa subunit that suggested the presence of a 3Fe-4S cluster in the reductase . These preparations were contaminated with a small amount of the small protein . The large and the small proteins form a tight complex . From sucrose gradient centrifugation, we determined a 1:1 stoichiometry of the two proteins in the complex . The anaerobic reductase thus has an alpha 2 beta 2 structure . We speculate that the small protein interacts with S-adenosylmethionine and forms a transient radical involved in the generation of the stable glycyl radical in the large protein that participates in the catalytic process. J Biol Chem, 1996 Apr 19, 271(16), 9347 - 54 A conserved HPD sequence of the J-domain is necessary for YDJ1 stimulation of Hsp70 ATPase activity at a site distinct from substrate binding; Tsai J et al.; The 46-kDa protein YDJ1 is one of several known yeast homologues of the Escherichia coli DnaJ protein . Like all J homologues, it shares homology with the highly conserved NH2-terminal "J-domain" of DnaJ . A component of the DnaK (Hsp70) chaperone machinery that mediates protein folding, DnaJ is necessary for survival at elevated temperatures . It stimulates ATP hydrolysis by DnaK and effects the release of DnaK-bound polypeptides . Previous genetic and biochemical studies indicate that the J-domain is necessary for these functions . Using peptides corresponding to J-domain sequence, we show that a peptide containing the highly conserved His-Pro-Asp sequence at positions 34-36 in the J-domain competes off YDJ1 stimulation of Hsp70 ATPase activity . Inhibitory concentrations of peptide do not prevent binding of folding substrates, therefore YDJ1 must interact with Hsp70 at a site distinct from that for substrate binding . This interaction is critical for Hsp70 activity, since a mutant YDJ1 protein harboring a H34Q change (ydj1Q34) stimulates neither Hsp70 ATPase nor substrate release . The importance of the proper function of this region of the protein is supported by the poor growth and temperature-sensitive phenotype of yeast expressing ydj1Q34. J Biol Chem, 1996 Apr 19, 271(16), 9291 - 7 The role of arsenic-thiol interactions in metalloregulation of the ars operon; Shi W et al.; The ars operon of the Escherichia coli plasmid R773 that confers arsenical and antimonial resistance is negatively regulated by the ArsR repressor . ArsR residues Cys-32 and Cys-34 were previously identified as involved in induction by arsenite and antimonite, suggesting coordination between As(III) and the two cysteine thiolates . However, in small molecule thiolate-As(III) complexes, arsenic is frequently three-coordinate . A site-directed mutagenic approach was employed in a search for a third arsenic ligand . ArsR proteins with C32G, C34G, and C32G/C34G substitutions were active repressors, but were not inducible in vivo . In vitro, the altered repressor-ars DNA complexes could not be dissociated by inducers . Alteration of Cys-37 and Ser-43, residues located in or near the putative helix-turn-helix DNA-binding region of the protein, had no effect on the inducibility of the operon . While these results indicated that neither the thiolate of Cys-37 nor the hydroxyl oxygen of Ser-43 is required for induction, they did not eliminate either atom as a potential arsenic ligand . Another approach involved reaction with an alternative inducer, phenylarsine oxide, which can form only two coordinations . Phenylarsine oxide was shown to be as effective as or more effective than arsenite or antimonite in induction in vivo . In vitro, the organic arsenical was more effective than either arsenite or antimonite in dissociating the repressor-promoter complex . Thus, two ArsR-arsenic bonds are sufficient for induction . The interaction of ArsR proteins with As(III) was examined using a phenylarsine oxide affinity resin . ArsR proteins containing any two of the three cysteine residues Cys-32, Cys-34, and Cys-37 bound to the resin . Alteration of any two of the three resulted in loss of binding . Arsenic X-ray absorption spectroscopy of ArsR treated stoichiometrically with arsenite confirmed the average arsenic coordination as AsS3 These results suggest that all three cysteine thiolates are arsenic ligands, but binding to only two, the Cys-32 and Cys-34 thiolates, is required to produce the conformational change that results in release of the repressor from the DNA and induction. J Biol Chem, 1996 Apr 19, 271(16), 9254 - 8 EPR study of NO complex of bd-type ubiquinol oxidase from Escherichia coli; Hori H et al.; The heme axial ligands of bd-type ubiquinol oxidase of Escherichia coli were studied by EPR and optical spectroscopies using nitric oxide (NO) as a monitoring probe . We found that NO bound to ferrous heme d of the air-oxidized and fully reduced enzymes with very high affinity and to ferrous heme b595 of the fully reduced enzyme with low affinity . EPR spectrum of the 14NO complex of the reduced enzyme exhibited an axially symmetric signal with g-values at g = 2.041 and g = 1.993 and a clear triplet of triplet (or a triplet of doublet for the 15NO complex) superhyperfine structure originating from a nitrogenous proximal ligand trans to NO was observed . This EPR species was assigned to the ferrous heme d-NO complex . This suggests that the proximal axial ligand of heme d is a histidine residue in an anomalous condition or other nitrogenous amino acid residue . Furthermore, the EPR line shape of the ferrous heme d-NO was slightly influenced by the oxidation state of the heme b595 . This indicates that heme d exists in close proximity to heme b595 forming a binuclear center . Another axially symmetric EPR signal with g-values at g(parallel) = 2.108 and g(perpendicular) = 2.020 appeared after prolonged incubation of the reduced enzyme with NO and was attributed to the ferrous heme b595-NO complex. J Biol Chem, 1996 Apr 19, 271(16), 9201 - 4 A cDNA clone for taxadiene synthase, the diterpene cyclase that catalyzes the committed step of taxol biosynthesis; Wildung MR et al.; The committed step of taxol (paclitaxel) biosynthesis is catalyzed by taxa-4(5),11(12)-diene synthase, a diterpene cyclase responsible for transforming the ubiquitous isoprenoid intermediate geranylgeranyl diphosphate to the parent olefin with a taxane skeleton . To obtain the corresponding cDNA clone, a set of degenerate primers was constructed based on consensus sequences of related monoterpene, sesquiterpene, and diterpene cyclases . Two of these primers amplified a 83-base pair fragment that was cyclase-like in sequence and that was employed as a hybridization probe to screen a cDNA library constructed from poly(A)+ RNA extracted from Pacific yew (Taxus brevifolia) stems . Twelve independent clones with insert size in excess of 2 kilobase pairs were isolated and partially sequenced . One of these cDNA isolates was functionally expressed in Escherichia coli, yielding a protein that was catalytically active in converting geranylgeranyl diphosphate to a diterpene olefin that was confirmed to be taxa-4(5),11(12)-diene by combined capillary gas chromatography-mass spectrometry . The sequence specifies an open reading frame of 2586 nucleotides, and the complete deduced polypeptide, including a long presumptive plastidial targeting peptide, contains 862 amino acid residues and has a molecular weight of 98,303, compared with about 79,000 previously determined for the mature native enzyme . Sequence comparisons with monoterpene, sesquiterpene, and diterpene cyclases of plant origin indicate a significant degree of similarity between these enzymes; the taxadiene synthase most closely resembles (46% identity, 67% similarity) abietadiene synthase, a diterpene cyclase from grand fir. J Biol Chem, 1996 Apr 19, 271(16), 9173 - 6 Essential amino acids for substrate binding and catalysis of human flap endonuclease 1; Shen B et al.; Human flap endonuclease 1 (FEN-1) is a member of the structure-specific endonuclease family and is involved in DNA repair . Eight restrictively conserved amino acids in FEN-1 have been converted individually to an alanine to elucidate their roles in specific DNA substrate binding and catalysis . Flap endonuclease activity of the wild type and mutant enzymes was measured by kinetic flow cytometry . Mutants D34A, D86A, and D181A lost their cleavage activity completely but retained substrate binding ability, as measured by their ability to inhibit the wild type enzyme in a competition assay . This indicates that these amino acids contribute to integrity of the enzyme active site . Loss of both binding and cleavage competency for the flap substrate by mutants E156A, G231A, and D233A suggests that these amino acids are involved in substrate binding . Mutants R103A and D179A retained wild type-like enzyme activity. Science, 1996 Apr 19, 272(5260), 401 - 4 Switching from cut-and-paste to replicative Tn7 transposition; May EW et al.; The bacterial transposon Tn7 usually moves through a cut-and-paste mechanism whereby the transposon is excised from a donor site and joined to a target site to form a simple insertion . The transposon was converted to a replicative element that generated plasmid fusions in vitro and cointegrate products in vivo . This switch was a consequence of the separation of 5'- and 3'-end processing reactions of Tn7 transposition as demonstrated by the consequences of a single amino acid alteration in an element-encoded protein essential for normal cut-and-paste transposition . The mutation specifically blocked cleavage of the 5' strand at each transposon end without disturbing the breakage and joining on the 3' strand, producing a fusion (the Shapiro Intermediate) that resulted in replicative transposition . The ability of Tn7 recombination products to serve as substrates for both the limited gap repair required to complete cut-and-paste transposition and the extensive DNA replication involved in cointegrate formation suggests a remarkable plasticity in Tn7's recruitment of host repair and replication functions. Oncogene, 1996 Apr 18, 12(8), 1609 - 16 A Herpes saimiri oncogene causing peripheral T-cell lymphoma in transgenic mice; Kretschmer C et al.; Herpesvirus saimiri is an oncogenic virus causing rapid T-cell lymphomas in New World primates and rabbits . Deletion analysis of one strain of H saimiri has indicated an open reading frame, StpA, necessary for oncongenicity in monkeys . We have investigated the function of StpA in tumor induction by the generation of transgenic mice . Expression of two different constructs caused the development of peripheral lymphomas . The infiltrating cells were of T-cell origin, expressing mainly the CD4 phenotype and restricted sets of V beta chains . Thus, StpA is not only necessary for the oncogenicity of Herpesvirus saimiri, but is also sufficient for the induction of peripheral pleomorphic T-cell lymphomas. Gene, 1996 Apr 17, 170(1), 73 - 6 Characterisation of the mcpA and mcpB genes capable of encoding methyl-accepting type chemoreceptors in Rhodobacter capsulatus; Michotey V et al.; Two contiguous mcp genes, mcpA and mcpB, transcribed from the same DNA strand and capable of encoding methyl-accepting chemotaxis proteins (Mcp) have been isolated from Rhodobacter capsulatus (Rc), sequences and overexpressed in Escherichia coli (Ec) . The deduced proteins (McpA, 69 171 Da; McpB, 81 629 Da) show a structure similar to that of Ec Mcp . The products of mcpA and mcpB, overproduced in Ec, were recognized by anti-Ec Mcp (Trg) antibodies. Gene, 1996 Apr 17, 170(1), 51 - 5 Increased efficiency of alkaline phosphatase production levels in Escherichia coli using a degenerate PelB signal sequence; Le Calvez H et al.; To obtain an expression vector that will optimize secretion of proteins with disulfide bridges in Escherichia coli, we fused the phoA gene, encoding the bacterial alkaline phosphatase (PhoA), to the sequence encoding the pectate lyase B signal sequence (PelBSS) . We used an extensively degenerate pelBSS with silent mutations to study their effects on the production level and activity of PhoA . 11 representative clones differed by a factor of five between the lowest and the highest level of activity, and by a factor greater than seven for the production levels . The efficiency of translocation seems to be the result of an equilibrium between production and secretion levels that favours the secretion of active PhoA according to the competence of the fusion protein being translocated . Free energy calculations and the predicted mRNA secondary structures of the translation initiation regions showed that the high stability of the secondary structure decreased production and secretion levels of PhoA and vice versa . A stem-loop encompassing the degenerate positions downstream from the AUG start codon appears to be responsible for the differences in the production levels. Gene, 1996 Apr 17, 170(1), 45 - 50 In vivo intermolecular recombination in Escherichia coli: application to plasmid constructions; Degryse E; Repair of a double-strand break (DSB) was investigated by intermolecular recombination in Escherichia coli (Ec) recBC sbcBC cells with restriction enzyme-cleaved model plasmids . Circular plasmids were generated when a linearized plasmid (vector) containing an origin of replication was co-transformed with a DNA fragment (template) containing a homologous sequence . The influence of the position of the DSB in the vector was analyzed using templates which contain various genetic markers, non-homologous sequences and/or deletions relative to the vector . In all cases, when a DSB occurs within a marker, this marker is lost in the resulting plasmid, whereas markers flanked by homologous regions located in the vicinity of a DSB are transmitted . Insertions (deletions), substitutions and shuffling of genetic markers are possible by in vivo recombination using Ec and can be applied to plasmid constructions . It is shown that recombination can occur from both template ends or from one vector and one template end . A D-loop nuclease is suggested to participate in the resolution of the recombination intermediates. Gene, 1996 Apr 17, 170(1), 147 - 8 Construction of an alkaline phosphatase fusion-generating transposon, mTn10phoA; McClain MS et al.; We constructed a derivative of the mini-transposon mTn10 that generates translational fusions to the phoA gene from Escherichia coli and carries the KmR determinant from Tn5 . This new transposon, mTn10phoA, is carried on a mobilizable plasmid with both selectable and counterselectable markers . The plasmid carrying mTn10phoA was introduced into Legionella pneumophila . Southern hybridization analysis indicated that the mTn10phoA insertions were randomly distributed. Gene, 1996 Apr 17, 170(1), 145 - 6 Introducing StuI sites improves vectors for the expression of fusion proteins with factor Xa cleavage sites; Gorlach J et al.; The Arg-encoding triplet (AGG) in the recognition sequence Ile-Glu-Gly-Arg for factor Xa can be used to generate a StuI restriction site (AGGCCT) which greatly facilitates the construction of DNA fragments encoding fusion proteins . Following proteolytic cleavage with factor Xa, a protein with the desired N terminus can be obtained. Gene, 1996 Apr 17, 170(1), 141 - 2 Construction of mini-F plasmid vectors for plasmid shuffling in Escherichia coli; Kato J et al.; We have constructed two Escherichia coli mini-F plasmid vectors, pJK286 and pJK289, which have unique EcoRI, BamHI and HindIII sites downstream from the lac promoter . The mini-F vectors are useful for plasmid shuffling, with which we can efficiently carry out localized mutagenesis. Biophys Chem, 1996 Apr 16, 59(3), 289 - 97 A bifunctional fusion protein containing the maltose-binding polypeptide and the catalytic chain of aspartate transcarbamoylase: assembly, oligomers, and domains; Yang YR et al.; The in vivo synthesis of many target proteins or polypeptides has been enhanced dramatically and their purification facilitated through the use of gene fusion techniques which lead to the expression of fusion proteins . This approach was used to characterize the product formed in Escherichia coli encoded by a DNA construct comprising malE, the gene encoding maltose binding protein, linked to a small 30 nucleotide region which, in turn, was linked to pyrB, the gene encoding the catalytic (c) chains of aspartate transcarbamoylase (ATCase) . The resulting fusion protein, MBP-C, was produced in excellent yield and readily purified in two steps because of its binding to an amylose column and displacement by maltose . The complex was studied by both sedimentation velocity and sedimentation equilibrium and shown to be a trimer of c chains with one MBP linked covalently to each chain . Treatment of the fusion protein with factor Xa cleaved each chain at the tetrapeptide encoded by the linker region yielding purified MBP with a minor modification at the C-terminus and the catalytic (C) trimer of ATCase . The MBP-C complex was fully active as an enzyme and could be reversibly denatured in 6 M urea . Scanning calorimetry studies on the fusion protein demonstrated that the MBP domain melted at the same temperature as did the purified protein . Similarly, the Tm for the C trimer in the complex was identical to the value for C trimer isolated from ATCase . Moreover, the thermal stability of the C trimer in the MBP-C complex was greatly enhanced by the addition of the bisubstrate ligand, N-(phosphonacetyl)-L-aspartate (PALA), just as observed with purified C trimer . Analogous denaturation experiments with varying concentrations of guanidine-HCl indicated that the fusion protein was denatured at much lower concentration of denaturant than observed for C trimer . These experiments demonstrate that the linker between the two structural genes encodes a polypeptide of sufficient length to permit independent folding and assembly of each protein and permit the subsequent specific cleavage at the factor Xa recognition site, thereby yielding both active proteins. Biophys Chem, 1996 Apr 16, 59(3), 231 - 46 Specificity mechanisms in the control of transcription; von Hippel PH et al.; In this overview we analyze and illustrate the principles underlying some of the specificity mechanisms that control the initiation, elongation, and termination phases of transcription . Thermodynamic mechanisms dominate in the first steps of initiation, where promoters at various levels of activation can be considered to be in competition for a limiting supply of core RNA polymerase . In the later stages of initiation, as well as in elongation and termination, the regulatory mechanisms that control specificity are largely kinetic, involving rate competition between branching reaction pathways where the outcome depends on the rates (and equilibria) of reaction and interconversion of different forms of the transcription complex . Elongation complexes are very stable at most positions along the DNA template, meaning that only RNA chain elongation (and editing) can occur at these positions . However, the stability of transcription complexes decreases abruptly when termination sequences are encountered, and here the outcome can be easily switched between elongation and termination (RNA release) by minor changes in the relative rates of these competing processes . Cis effectors, defined as sites at which regulatory proteins bind to upstream activation loci on either the DNA or the nascent RNA, play important roles in the control of both initiation and of the elongation-termination decision . Examples, drawn from studies of phage lambda N-dependent antitermination and E . coli rho-dependent termination processes, illustrate the flexibility and additivity of regulatory components within control mechanisms in transcription that involve multiple determinants . The generality of such regulatory principles are stressed. Biochemistry, 1996 Apr 16, 35(15), 4858 - 66 Altering substrate specificity at the heme edge of cytochrome c peroxidase; Wilcox SK et al.; Two mutants of cytochrome c peroxidase (CCP) are reported which exhibit unique specificities toward oxidation of small substrates . Ala-147 in CCP is located near the delta-meso edge of the heme and along the solvent access channel through which H2O2 is thought to approach the active site . This residue was replaced with Met and Tyr to investigate the hypothesis that small molecule substrates are oxidized at the exposed delta-meso edge of the heme . X-ray crystallographic analyses confirm that the side chains of A147M and A147Y are positioned over the delta-meso heme position and might therefore modify small molecule access to the oxidized heme cofactor . Steady-state kinetic measurements show that cytochrome c oxidation is enhanced 3-fold for A147Y relative to wild type, while small molecule oxidation is altered to varying degrees depending on the substrate and mutant . For example, oxidation of phenols by A147Y is reduced to less than 20% relative to the wild-type enzyme, while Vmax/e for oxidation of other small molecules is less affected by either mutation . However, the "specificity inverted question mark of aniline oxidation by A147M, i.e., (Vmax/e)/Km, is 43-fold higher than in wild-type enzyme, suggesting that a specific interaction for aniline has been introduced by the mutation . Stopped-flow kinetic data show that the restricted heme access in A147Y or A147M slows the reaction between the enzyme and H202, but not to an extent that it becomes rate limiting for the oxidation of the substrates examined . The rate constant for compound ES formation with A147Y is 2.5 times slower than wild-type CCP . These observations strongly support the suggestion that small molecule oxidations occur at sites on the enzyme distinct from those utilized by cytochrome c and that the specificity of small molecule oxidation can be significantly modulated by manipulating access to the heme edge . The results help to define the role of alternative electron transfer pathways in cytochrome c peroxidase and may have useful applications in improving the specificity of peroxidase with engineered function. Biochemistry, 1996 Apr 16, 35(15), 4837 - 45 Probing the cytochrome c peroxidase-cytochrome c electron transfer reaction using site specific cross-linking; Pappa HS et al.; Engineered cysteine residues in yeast cytochrome c peroxidase (CCP) and yeast iso-1-cytochrome c have been used to generate site specifically cross-linked peroxidase-cytochrome c complexes for the purpose of probing interaction domains and the intramolecular electron transfer reaction . Complex 2 was designed earlier {Pappa, H.S., & Poulos, T.L . (1995) Biochemistry 34, 6573-6580} to mimic the known crystal structure of the peroxidase-cytochrome c noncovalent complex {Pelletier, H., & Kraut, J . (1992) Science 258, 1748-1755} . Complex 3 was designed such that cytochrome c is tethered to a region of the peroxidase near Asp148 which has been suggested to be a second site of interaction between the peroxidase and cytochrome c . Using stopped flow methods, the rate at which the ferrocytochrome c covalently attached to the peroxidase transfers an electron to peroxidase compound I is estimated to be approximately 0.5-1 s-1 in complex 3 and approximately 800 s-1 in complex 2 . In both complexes the Trp191 radical and not the Fe4+=O oxyferryl center of compound I is reduced . Conversion of Trp191 to Phe slows electron transfer about 10(3) in complex 2 . Steady state kinetic measurements show that complex 3 behaves like the wild type enzyme when either horse heart or yeast ferrocytochrome c is used as an exogenous substrate, indicating that the region blocked in complex 3 is not a functionally important interaction site . In contrast, complex 2 is inactive toward horse heart ferrocytochrome c at all ionic strengths tested and yeast ferrocytochrome c at high ionic strengths . Only at low ionic strengths and low concentrations of yeast ferrocytochrome c does complex 2 give wild type enzyme activity . This observation indicates that in complex 2 the primary site of interaction of CCP with horse heart and yeast ferrocytochrome c at high ionic strengths is blocked . The relevance of these results to the pathway versus distance models of electron transfer and to the interaction domains between peroxidase and cytochrome c is discussed. Biochemistry, 1996 Apr 16, 35(15), 4828 - 36 Complete coordination of the four Fe-S centers of the beta subunit from Escherichia coli nitrate reductase . Physiological, biochemical, and EPR characterization of site-directed mutants lacking the highest or lowest potential {4Fe-4S} clusters; Guigliarelli B et al.; The beta subunit of the nitrate reductase A from Escherichia coli contains four groups of cysteine residues (I-IV) which are thought to bind the four iron-sulfur centers (1-4) of the enzyme . The fourth Cys residue of each group was replaced by Ala by site-directed mutagenesis, which led to the C26A, C196A, C227A, and C263A mutants . Physiological and biochemical effects of the mutations were investigated on both the membrane-bound and the soluble forms of the enzyme . In addition, detailed redox titrations of the mutants were monitored by EPR spectroscopy . The C196A and C227A mutations resulted in the full loss of the four Fe-S clusters and of the Mo-cofactor, leading to inactive enzymes . In contrast, the C26A and C263A mutants retained significant nitrate reductase activities . The EPR analysis showed that the highest redox potential {4Fe-4S} cluster (center 1) was selectively removed by the C263A mutation and that the C26A replacement likely eliminated the lowest potential {4Fe-4S} cluster (center 4) . In both mutants, the three remaining Fe-S clusters kept the same spectral and redox properties as in the wild type enzyme . These results enabled the determination of the Cys ligands of center 1 to be completed and led to a proposed model for the coordination of the four Fe-S centers by the four Cys groups of the beta subunit . In this model, the four clusters are organized in two pairs, (center 1, center 4) and (center 2, center 3), which is in good agreement with the magnitude of intercenter magnetic interactions observed by EPR and with the stability of the different mutants . The possible implications on the intramolecular electron transfer pathway are discussed. Biochemistry, 1996 Apr 16, 35(15), 4704 - 12 Enzyme-monitored turnover of Escherichia coli thioredoxin reductase: insights for catalysis; Lennon BW et al.; Thioredoxin reductase from Escherichia coli is a member of the pyridine nucleotide-disulfide oxidoreductase family, and contains one FAD and one redox-active disulfide per subunit . It is known that two other well-studied members of this family, lipoamide dehydrogenase and glutathione reductase, cycle between the two electron-reduced and fully oxidized forms in catalysis . Enzyme-monitored turnover shows that the spectrum of thioredoxin reductase during turnover represents fully reduced flavin with NADP(H) bound . Whether the pyridine nucleotide bound is NADPH or NADP+ is dependent on the concentration of each species, i.e., how far turnover has progressed . It is also shown that the midpoint potentials of this enzyme are increased through the differential binding of NADP+ to the oxidized and reduced form of the enzyme . When combined with other kinetic and oxidation/reduction studies of this enzyme, these results indicate that thioredoxin reductase cycles between the four-electron-reduced and two-electron-reduced forms in catalysis, and that it does so with pyridine nucleotide bound . These results clarify the mechanism of thioredoxin reductase in relation to the known structure the enzyme, and provide support for earlier work in which we proposed that this enzyme utilizes a ternary complex mechanism in catalysis. Biochemistry, 1996 Apr 16, 35(15), 4697 - 703 Enzyme-substrate complexes of adenosine and cytidine deaminases: absence of accumulation of water adducts; Shih P et al.; Adenosine deaminase has been reported to bind the product inosine (the substrate for the reverse reaction) as inosine 1,6-hydrate considered similar in structure to the transition state for adenosine deamination (Wilson & Quiocho, 1994) Accumulation on the enzyme of inosine 1,6-hydrate would be surprising, because this compound is an actual intermediate, probably approaching the transition state, in oxygen exchange between water and the C==O group of inosine, a reaction previously shown to be catalyzed by adenosine deaminase (Wolfenden & Kirsch, 1968) . The equilibrium constant for conversion of ES to ES*, in the oxygen exchange reaction, is less than 10-12 . To investigate the structure of enzyme-bound inosine in a different way, we labeled deoxyinosine with 13C, excepting an upfield shift of 70-110 ppm if significant rehybridization to sp3 had occurred at the carbonyl group . Instead, the results show a very small shift (1.3 ppm), indicating that C-6 of 2'-deoxyinosine retains its sp2 hybridization after binding by calf intestinal adenosine deaminase . In a separate series of experiments, {4,5-13C}-2'-deoxyuridine was synthesized and found to retain its sp2 hybridization at C-4, after binding by Escherichia coli cytidine deaminase, an enzyme that catalyzes 18O exchange from water into uridine . These findings are consistent with the general expectation, based on the unfavorable equilibrium of activation of enzyme-bound substrates, that enzymes should not accumulate appreciable concentrations of intermediates whose free energies approach that of the transition state in substrate transformation. Biochemistry, 1996 Apr 16, 35(15), 4670 - 7 Crystallographic identification of metal-binding sites in Escherichia coli inorganic pyrophosphatase; Kankare J et al.; We report refined crystal structures of the hexameric soluble inorganic pyrophosphatase from Escherichia coli (E-PPase) to R-factors of 18.3% and 17.1% at 2.2 and 2.3 angstroms, respectively . Both structures contain two independent monomers in the asymmetric unit of an R32 cell . The difference between the structures is that the latter contains 1.5 Mg2+ ions per monomer . One metal ion binds to the "tight" metal-binding site identified by equilibrium dialysis studies, and is coordinated to Asp65, Asp70, and Asp102 . The other metal ion, shared between two monomers at a hitherto unidentified metal-binding site in the dyad interface between trimers, is coordinated through water molecules to Asp26s and Asn24s from two monomers . The hexamers with metal bound to them are more tightly associated than the ones without metal bound to them . Combined with our other mechanistic and structural data, the results suggest that, at high metal concentrations, E-PPase may bind at least 4.5 metals per monomer: two in the active site before binding substrate, two with substrate, and 0.5 in the dyad interface . Glu20 interacts via a water molecule with Asp70 and appears in the related yeast PPase structure (Heikinheimo, manuscript in preparation) to be involved in binding the second metal ion . Magnesium ion therefore stabilizes the hexamer form through both direct and indirect effects . The direct effect is by tighter association at the subunit interface; the indirect effect occurs because magnesium stabilizes the correct conformation of the loop between Glu20 and Ile32, a loop involved a trimer-trimmer interactions . Our results thus provide a structural explanation for the solution studies that show that the E20D variant is partially hexameric and that the hexamer form can be stabilized by binding magnesium ion. Biochemistry, 1996 Apr 16, 35(15), 4662 - 9 Effect of E20D substitution in the active site of Escherichia coli inorganic pyrophosphatase on its quaternary structure and catalytic properties; Volk SE et al.; Glutamic acid 20 is an evolutionarily conserved residue found within the active site of the inorganic pyrophosphatase of Escherichia coli (E-PPase) . Here we determine the effect of E20D substitution on the quaternary structure and catalytic properties of E-PPase . In contrast to wild-type enzyme, which is hexameric under a variety of conditions, E20D-PPase can be dissociated by dilution into nearly inactive trimers, as shown by electrophoresis of cross-linked enzyme, analytical ultracentrifugation, and measurement of catalytic activity as a function of enzyme concentration . Hexamer stability is increased in the presence of both substrate and Mg2+, is maximal at pH 6.5, and falls off sharply as the pH is lowered or raised from this value . Measured at saturating substrate, 20 mM Mg2+ and pH 7.2, E20D substitution (a) decreases activity towards inorganic pyrophosphate (PPi) hydrolysis and oxygen exchange between water and inorganic phosphate (P1), (b) increases the rate of net PPi synthesis, and (c) decreases the amount of enzyme-bound PPi in equilibrium with Pi in solution . Measurements of PPi hydrolysis rate as a function of both Mg2+ concentration and pH for the E20D variant show that its decreased activity is largely accounted for on the basis of an increased pKa of the catalytically essential base at the active site, and the need for a Mg2+ stoichiometry of 5 in the enzyme-substrate complex, similar to what is seen for the D97E variant . By contrast, wild-type PPase catalysis over a wide range of Mg2+ concentration and pH is dominated by an enzyme-substrate complex having a total of four Mg2+ ions . These results are consistent with a supporting role for Glu20 in PPase catalysis and demostrate that even conservative mutation at the active site can perturb the quaternary structure of the enzyme. Biochemistry, 1996 Apr 16, 35(15), 4655 - 61 Catalysis by Escherichia coli inorganic pyrophosphatase: pH and Mg2+ dependence; Baykov AA et al.; Steady-state rates of PPi hydrolysis by Escherichia coli inorganic pyrophosphatase (E-PPase) were measured as a function of magnesium pyrophosphatase (substrate) and free Mg2+ ion (activator) in the pH range 6.0-10.0 . Computer fitting of hydrolysis data in combination with direct measures of Mg2+ binding to enzyme has resulted in a model that quantitatively accounts for our results . The major features of this model are the following: (a) E-PPase catalysis proceeds both with three and with four (and possibly with five) Mg2+ ions per active site; (b) catalysis requires both an essential base and an essential acid, and the pKas of these groups are modulated by the stoichiometry of bound Mg2+; and (c) the four-metal route predominates for concentrations of free Mg2+>0.2mM . The model straightforwardly accounts for the apparent linkage between increased pKa of an essential base and activity requirements for higher Mg2+ concentration observed for several active site variants . Microscopic rate constants for overall catalysis of PPi-Pi equilibration were determined at pH 6.5-9.3 by combined analysis of enzyme-bound PPi formation and rates of PPi hydrolysis, PPi synthesis, and Pi-H2O oxygen exchange . The catalytic activity of E-PPase at saturating substrate increases toward PPi hydrolysis and decreases toward PPi synthesis and Pi-H2O oxygen exchange with increasing pH . These changes are mainly due to an increased rate of dissociation of the second released Pi and a decreased rate of enzyme-bound PPi synthesis from enzyme-bound Pi, respectively, as the pH is raised . Proc Natl Acad Sci U S A, 1996 Apr 16, 93(8), 3653 - 7 Human TOP3: a single-copy gene encoding DNA topoisomerase III; Hanai R et al.; A human cDNA encoding a protein homologous to the Escherichia coli DNA topoisomerase I subfamily of enzymes has been identified through cloning and sequencing . Expressing the cloned human cDNA in yeast (delta)top1 cells lacking endogenous DNA topoisomerase I yielded an activity in cell extracts that specifically reduces the number of supercoils in a highly negatively supercoiled DNA . On the basis of these results, the human gene containing the cDNA sequence has been denoted TOP3, and the protein it encodes has been denoted DNA topoisomerase III . Screening of a panel of human-rodent somatic hybrids and fluorescence in situ hybridization of cloned TOP3 genomic DNA to metaphase chromosomes indicate that human TOP3 is a single-copy gene located at chromosome 17p11.2-12. Proc Natl Acad Sci U S A, 1996 Apr 16, 93(8), 3525 - 9 Creation of drug-specific herpes simplex virus type 1 thymidine kinase mutants for gene therapy; Black ME et al.; Herpes simplex virus type 1 (HSV-1) thymidine kinase is currently used as a suicide agent in the gene therapy of cancer . This therapy is based on the preferential phosphorylation of nucleoside analogs by tumor cells expressing HSV-1 thymidine kinase . However, the use of HSV-1 thymidine kinase is limited in part by the toxicity of the nucleoside analogs . We have used random sequence mutagenesis to create new HSV-1 thymidine kinases that, compared with wild-type thymidine kinase, render cells much more sensitive to specific nucleoside analogs . A segment of the HSV-1 thymidine kinase gene at the putative nucleoside binding site was substituted with random nucleotide sequences . Mutant enzymes that demonstrate preferential phosphorylation of the nucleoside analogs, ganciclovir or acyclovir, were selected from more than one million Escherichia coli transformants . Among the 426 active mutants we have isolated, 26 demonstrated enhanced sensitivity to ganciclovir, and 54 were more sensitive to acyclovir . Only 6 mutant enzymes displayed sensitivity to both ganciclovir and acyclovir when expressed in E . coli . Analysis of 3 drug-sensitive enzymes demonstrated that 1 produced stable mammalian cell transfectants that are 43-fold more sensitive to ganciclovir and 20-fold more sensitive to acyclovir. Proc Natl Acad Sci U S A, 1996 Apr 16, 93(8), 3383 - 7 Transcription termination factor La is also an initiation factor for RNA polymerase III; Maraia RJ; La RNA-binding protein is a transcription termination factor that facilitates recycling of template and RNA polymerase (pol) 111 . Transcription complexes preassembled on immobilized templates were depleted of pol III after a single round of RNA synthesis in the presence of heparin and sarkosyl . The isolated complexes could then be complemented with highly purified pol III and/or recombinant La to test if La is required for transcription reinitiation . VA1, 7SL, and B1 transcription complexes cannot be transcribed by supplemental pol III in single or multiple-round transcription assays unless La is also provided . La mediates concentration-dependent activation of pol III initiation and thereby controls the use of preassembled stable transcription complexes . The initiation factor activity of La augments its termination factor activity to produce a novel mechanism of activated reinitiation . A model in which La serves pol III upon transcription initiation and again at termination is discussed. Proc Natl Acad Sci U S A, 1996 Apr 16, 93(8), 3313 - 8 Molecular characterization of a positively photoregulated nuclear gene for a chloroplast RNA polymerase sigma factor in Cyanidium caldarium; Liu B et al.; We have cloned the gene for a putative chloroplast RNA polymerase sigma factor from the unicellular rhodophyte Cyanidium caldarium . This gene contains an open reading frame encoding a protein of 609 amino acids with domains highly homologous to all four conserved regions found in bacterial and cyanobacterial sigma 70-type subunits . When Southern blots of genomic DNA were hybridized to the "rpoD box" oligonucleotide probe, up to six hybridizing hands were observed . Transcripts of the sigma factor gene were undetectable in RNA from dark-grown cells but were abundant in the poly(A)+ fraction of RNA from illuminated cells . The sigma factor gene was expressed in Escherichia coli, and antibodies against the expressed sigma factor fusion protein cross-reacted with a 55-kDa protein in partially purified chloroplast RNA polymerase . Antibodies directed against a cyanobacterial RNA polymerase sigma factor also cross-reacted with a 55-kDa protein in the same enzyme preparation . Immunoprecipitation experiments showed that this enzyme preparation contains proteins with the same molecular weights as the alpha, beta, beta', and beta" subunits of chloroplast RNA polymerase in higher plants . This study identifies a gene for a plastid RNA polymerase sigma factor and indicates that there may be a family of nuclear-encoded sigma factors that recognize promoters in subsets of plastid genes and regulate differential gene expression at the transcriptional level. Proc Natl Acad Sci U S A, 1996 Apr 16, 93(8), 3243 - 7 The C-terminal region of human angiogenin has a dual role in enzymatic activity; Russo N et al.; The ribonucleolytic activity of angiogenin (Ang) is essential to Ang's capacity to induce blood vessel formation . Previous x-ray diffraction and mutagenesis results have shown that the active site of the human protein is obstructed by Gln-117 and imply that the C-terminal region of Ang must undergo a conformational rearrangement to allow substrate binding and catalysis . As a first step toward structural characterization of this conformational change, additional site-directed mutagenesis and kinetic analysis have been used to examine the intramolecular interactions that stabilize the inactive conformation of the protein . Two residues of this region, Ile-119 and Phe-120, are found to make hydrophobic interactions with the remainder of the protein and thereby help to keep Gln-117 in its obstructive position . Furthermore, the suppression of activity by the intramolecular interactions of Ile-119 and Phe-120 is counterbalanced by an effect of the adjacent residues, Arg-121, Arg-122, and Pro-123 which do not appear to form contacts with the rest of the protein structure . They contribute to enzymatic activity, probably by constituting a peripheral subsite for binding polymeric substrates . The results reveal the nature of the conformational change in human Ang and assign a key role to the C-terminal region both in this process and, presumably, in the regulation of human Ang function. Proc Natl Acad Sci U S A, 1996 Apr 16, 93(8), 3221 - 6 Protein-RNA interactions in the active center of transcription elongation complex; Markovtsov V et al.; By using a crosslinkable probe incorporated into the 3' terminus of nascent transcript, three sites were mapped in Escherichia coli RNA polymerase that are contacted by the RNA in the productive elongation complex . Two of these sites are in the beta subunit and one is in the beta' subunit . During elongation, the transcription complex occasionally undergoes an arrest whereby it can neither extend nor release the RNA transcript . It is demonstrated that in an arrested complex, the three contacts of RNA 3' terminus are lost, while a new beta' subunit contact becomes prominent . Thus, elongation arrest appears to involve the disengagement of the bulk of the active center from the 3' terminus of RNA and the transfer of the terminus into a new protein environment. Biochim Biophys Acta, 1996 Apr 16, 1293(2), 243 - 53 Conformational analysis of pentapeptide sequences matching a proposed recognition motif for lysosomal degradation; Gorinsky B et al.; A selective pathway for the degradation of specific long-lived cytosolic proteins is activated in response to starvation in vivo or to serum withdrawal from cultured cells . It involves recognition of a targeting motif by a member of the hsp70 family . A 5-residue targeting motif has been proposed on the basis of sequence comparisons . We investigate whether there is any structural basis for this motif being the true recognition signal . We examine the conformations of four motif peptides in proteins that are either known to be serum regulated or are from related vertebrate species, and two equivalent peptides in bacterial proteins that closely resemble other regulated proteins . Our studies show that all the motif sequences are located near the ends of surface helices with one or more of the residues buried in the structure, yet it is known that members of the hsp70 family tend to interact with extended peptide chains . Furthermore, recognition by these proteins generally requires a specific ordering of key residues, yet the motif implies a largely order-independent sequence characterized by residue type only . We conclude that the proposed motif is unlikely to be the true targeting signal for lysosomal degradation unless additional factors apply. Biochim Biophys Acta, 1996 Apr 16, 1293(2), 238 - 42 Inhibition of Escherichia coli beta-galactosidase by 2-nitro-1-(4,5-dimethoxy-2-nitrophenyl) ethyl, a photoreversible thiol label; Golan R et al.; 1-Nitro-2-phenylethene (beta-nitrostyrene, 1) which is a thiol-protecting reagent (Jung, G., Fouad, H . and Heusel, G . (1975) Angew . Chem . Int . Ed . Engl . 14, 817-818), was demonstrated in this work to be an irreversible inhibitor of beta-galactosidase (EC 3.2.1.23), an enzyme known to be inhibited by some thiol reagents or through modifying a methionine residue at the active site . No reversal of the inhibition was observed upon subsequent incubation with mercaptoethanol or irradiation (350 nm) . 1-(4,5-dimethoxy-2-nitrophenyl)-2-Nitroethene 2) was also shown to be an irreversible inhibitor (94% inhibition, pH 8.3) of the enzyme . Kcat values of beta-galactosidase at pH 8.3 with o-nitrophenyl beta-D-galactopyranoside (ONPG) as the substrate and at the highest inhibitor concentrations employed for compound 1 (4.06 x 10(-4) M) ranged from 1.67 x 10(4) S-1 after 30 min of preincubation to <0.07 x 10(4) S-1 after 180 min preincubation . For compound 2 (9.5 x 10(-5) M) Kcat values ranged from 2.70 x 10(4) S-1 following 30 min preincubation to 1.15 x 10(4) S-1 after 180 min of preincubation; the changes in Km(app), however, were small . The activity was not recovered following incubation with mercaptoethanol . Since compound 2 and the inhibited enzyme are 2-nitrobenzyl derivatives, they are expected to be photosensitive and indeed, irradiation of the inhibited enzyme in the presence of mercaptoethanol resulted in recovery (89%, pH 8.3) of the enzyme activity. Biochem Biophys Res Commun, 1996 Apr 16, 221(2), 328 - 32 cDNA sequence analysis and expression of the a chain of beta-bungarotoxin from Bungarus multicinctus (Taiwan banded krait); Chang LS et al.; The cDNA encoding the A chain of beta-bungarotoxin (beta-Bgt) was constructed from the cellular RNA isolated from the venom glands of Bungarus multicinctus (Taiwan banded krait) . The deduced amino acid sequence encoding the A chain revealed that the determined one was different from the known A chains (A1, A2, A3 and A4) . Nevertheless, the amino acid sequence and cDNA sequence of the new A chain (A5) was highly homologous with those of other A chains . The A5 chain was subcloned into the expression vector pT7-7 and transformed into BL21(DE3) E . coli strain . The expressed protein was isolated from the inclusion bodies of E . coli, and the refolded A chain was purified by reversed phase high performance liquid chromatography . The purified recombinant A chain exhibited an about 16% phospholipase activity of beta-Bgt . These results strongly suggest that the A chain is an active subunit responsible for the phospholipase activity of beta-Bgt. Eur J Biochem, 1996 Apr 15, 237(2), 399 - 405 Identification of residues of Rhodobacter capsulatus ferredoxin I important for its interaction with nitrogenase; Naud I et al.; In Rhodobacter capsulatus, ferredoxin I (FdI) serves as natural electron donor to nitrogenase . In order to probe amino acid residues possibly involved in the interaction with dinitrogenase reductase, FdI was subjected to site-specific mutagenesis . A three-dimensional structure of FdI was designed by computer modelling and used for selecting target residues . Mutant ferredoxins bearing substitutions of surface residues, as well as a variant having a Met2 --> Tyr replacement in the vicinity of one cluster, have been constructed . All FdI variants were expressed to similar levels both in Escherichia coli and in a FdI-deleted mutant of the natural host . Once purified, the mutant ferredoxins exhibited molecular and spectroscopic properties almost identical to wild-type FdI . Determination of the reduction potential of FdI by cyclic voltammetry gave an E'o of -510 mV (pH 7.6) for both clusters, which is one of the lowest values reported for a 2{4Fe-4S} ferredoxin . Only the {Tyr2}FdI variant showed a significant difference in redox potential (delta E'o = -15 mV) . Based on in vitro assays, a {Glu27, Glu28}FdI double mutant exhibited a twofold decrease in the electron transfer rate to dinitrogenase reductase while the affinity of this mutant for the enzyme was barely affected . On the other hand, an Asp36 --> His substitution resulted in a sevenfold increase of the apparent Km for dinitrogenase reductase . Unlike FdI and the other mutant ferredoxins, the {His36}FdI variant also failed to form a cross-linked complex with dinitrogenase reductase upon incubation with a carbodiimide . It is concluded that Asp36 in FdI probably participates in the interaction between the two protein partners . Nevertheless, all the FdI mutants proved competent in restoring a wild-type phenotype when expressed in a FdI-deleted mutant background, indicating that none of the studied residues was absolutely critical for electron transfer to nitrogenase. Eur J Biochem, 1996 Apr 15, 237(2), 383 - 92 Sequence-specific resonance assignments of the 1H-NMR spectra and structural characterization in solution of the HIV-1 transframe protein p6; Beissinger M et al.; The frameshift protein p6* encoded directly upstream of the protease in the human immunodeficiency virus type 1 (HIV-1) pol reading frame is thought to be a natural inhibitor of protease activation and to play a role in the polyprotein processing of Gag and Gag-Pol precursors . To allow structural characterization of the p6* transframe protein, the p6* coding region was cloned into the vector pGEX-KG and expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST) under the control of the tac promoter . Thrombin cleavage of the construct resulted in a 70-amino-acid polypeptide which is extended by two additional residues at the N-terminus compared to the natural p6* sequence . The native purification procedure including an affinity and a size-exclusion chromatography step yielded sufficient amounts of highly pure protein suitable for NMR spectroscopy . Fluorescence, circular dichroism and 1H-NMR spectroscopy were applied to characterize the structure of protein . Two-dimensional NMR spectra provided essentially complete sequence-specific resonance assignments at pH 5.9 . Although there is evidence for a helix-forming tendency in the N-terminus of the protein, the experiments indicate that p6* has no overall stable secondary or tertiary structure with the single tryptophan exposed in aqueous solution . However, the results reported herein open the way to characterize further the interaction of p6* with the HIV-1 protease in structural and functional in vitro studies. Eur J Biochem, 1996 Apr 15, 237(2), 367 - 72 cDNA cloning, expression, and chromosomal localization of Caenorhabditis elegans DNA topoisomerase I; Kim J et al.; By screening Caenorhabditis elegans cDNA libraries, overlapping cDNA clones encoding DNA topoisomerase I were obtained . An open reading frame of 751 amino acids was found in 3.2-kb cDNA sequence . The open reading frame has 54% and 50% identities to the amino acid sequences of human and Drosophila melanogaster DNA topoisomerases I, respectively . Northern blot analysis showed the presence of an mRNA of 3.4 kb which suggests that the cDNA sequences is close to full length . The 72-kDa C-terminal polypeptide expressed in Escherichia coli cells showed catalytic DNA topoisomerase I activity . The DNA topoisomerase I gene was mapped to position 18 of chromosome I by screening polytene YAC plasmid DNAs. Arch Biochem Biophys, 1996 Apr 15, 328(2), 317 - 23 Kinetic characterization of human immunodeficiency virus type 1 protease: determination of inhibitor rate constants during dynamic monomer-dimer interconversion; Morelock MM et al.; A numerical method was applied to a system of differential rate equations describing the monomer-dimer-inhibitor (M-D-I) interaction involving human immunodeficiency virus type 1 protease and a peptidomimetic, competitive inhibitor . Two pairs of progress curves were obtained, one involving the M-D interaction and the other the M-D-I interaction . Each pair of reactions was designed to begin with extreme conditions and end at the identical equilibrium position . The results were compared with analytical (exact mathematical) methods reported previously . Good agreement between the two methods was observed at high- and low-salt conditions for the rates of monomer association and dimer dissociation . Not surprisingly, however, the major difference was observed in the analyses involving the M-D-I interaction, since analytical methods cannot account for dimer dissociation in the presence of inhibitor . While the estimates for the inhibitor off rate were comparable for high-salt conditions (where dimer dissociation is minimized), the analytical method underestimated this parameter for low-salt conditions by an order of magnitude, the consequence of mistaking inactive M for inactive DI. Arch Biochem Biophys, 1996 Apr 15, 328(2), 295 - 301 Further characterization of Escherichia coli alanyl-tRNA synthetase; Sood SM et al.; Selected physical and thermodynamic parameters for Escherichia coli alanyl-tRNA synthetase (AlaRS) have been determined primarily to assess the quaternary structure of this enzyme . The extinction coefficient (epsilon) at 280 nm was determined experimentally to be 0.71 ml mg-1 cm-1, and the partial specific volume (nu) was calculated from the amino acid composition to be 0.73 ml g-1 . From viscosity experiments the intrinsic viscosity (eta) of AlaRS was extrapolated to be 3.4 ml g-1 and the degree of hydration (delta 1) estimated to be 0.67 gH2O g(-1)(AlaRS) . Laser light-scattering studies indicated some heterogeneity; a radius of 6.3 nm was calculated for the major fraction with a diffusion coefficient (D20,W) of 3.89 x 10(-7) cm2 s-1 . In 50 mM Hepes, pH 7.5, 20 mM KCl, 2 mM 2-mercaptoethanol and at a protein concentration of 4.2 mg ml-1 the sedimentation coefficient (S20,W) was 6.36 S; this value increased slightly when the protein concentration was decreased . The combination of S20,W and D20,W under these conditions yielded a molecular weight of approximately 186,000 Da, corresponding to a dimer . The S20,W was virtually independent of temperature in the range of 10-37 degrees C, while an Arrhenius plot of aminoacylation activity was biphasic . The isoelectric point was determined experimentally to be 4.9 . Sedimentation equilibrium data were best fit to a decamer association complex in which dimeric AlaRS is the predominant species at 25 degrees C. Nucleic Acids Res, 1996 Apr 15, 24(8), 1561 - 5 Mutation spectra of TA*, the major photoproduct of thymidylyl-(3'5')-deoxyadenosine, in Escherichia coli under SOS conditions; Zhao X et al.; The biological activity of TA*, the major photoproduct of thymidylyl-(3',5')-deoxyadenosine, has remained speculative since it was identified a decade ago . To determine the mutagenicity of TA* in Escherichia coli, we constructed the replicative form of an M13mp18-derived phage containing TA* in the (-)-strand by polymerase-catalyzed elongation of a TA*-containing 49mer opposite a uracil-containing (+)-strand of the phage . The in vitro synthesis mixture was transfected into an ung+, phr- E.coli host and the progeny were screened with a hybridization probe unique for the (-)-strand . TA* was found to block DNA replication substantially in the absence of SOS, but under SOS, TA* was bypassed more efficiently and was highly mutagenic . Among 56 analyzed (-)-strand progeny from two transfections, 46 (82%) were mutants, including six (11%) tandem mutants . The most abundant mutation was a 3'A-->T substitution (31/46, 56%) . The possible biological consequences of TA* formation in the highly conserved TATA box consensus sequence on gene expression are discussed in light of the mutagenicity of TA*. Nucleic Acids Res, 1996 Apr 15, 24(8), 1497 - 503 The catalytic core of RNase P; Green CJ et al.; A deletion mutant of the catalytic RNA component of Escherichia coli RNase P missing residues 87-241 retains the ability to interact with the protein component to form a functional catalyst . The deletion of this phylogenetically conserved region significantly increases the Km, indicating that the deleted structures may be important for binding to the precursor tRNA substrate but not for the cleavage reaction . Under some reaction conditions, this RNase P deletion mutant can become a relatively non-specific nuclease, indicating that this RNA's catalytic center may be more exposed . The catalytic core of the RNase P is formed by less than one third of the 377 residues of the RNase P RNA. Nucleic Acids Res, 1996 Apr 15, 24(8), 1404 - 11 Regulation of Cre recombinase activity by the synthetic steroid RU 486; Kellendonk C et al.; To create a strategy for inducible gene targeting we developed a Cre-lox recombination system which responds to the synthetic steroid RU 486 . Several fusions between Cre recombinase and the hormone binding domain (HBD) of a mutated human progesterone receptor, which binds RU 486 but not progesterone, were constructed . When tested in transient expression assays recombination activities of all fusion proteins were responsive to RU 486, but not to the endogenous steroid progesterone . However, the observed induction of recombination activity by the synthetic steroid varied between the different fusion proteins . The fusion with the highest activity in the presence of RU 486 combined with low background activity in the absence of the steroid was tested after stable expression in fibroblast and embryonal stem (ES) cells . We could demonstrate that its recombination activity was highly dependent on RU 486 . Since the RU 486 doses required to activate recombination were considerably lower than doses displaying anti-progesterone effects in mice, this system could be used as a valuable tool for inducible gene targeting. EMBO J, 1996 Apr 15, 15(8), 2003 - 9 Restriction by EcoKI is enhanced by co-operative interactions between target sequences and is dependent on DEAD box motifs; Webb JL et al.; One subunit of both type I and type III restriction and modification enzymes contains motifs characteristic of DEAD box proteins, which implies that these enzymes may be DNA helicases . This subunit is essential for restriction, but not modification . The current model for restriction by both types of enzyme postulates that DNA cutting is stimulated when two enzyme complexes bound to neighbouring target sequences meet as the consequence of ATP-dependent DNA translocation . For type I enzymes, this model is supported by in vitro experiments, but the predicted co-operative interactions between targets have not been detected by assays that monitor restriction in vivo . The experiments reported here clearly establish the required synergistic effect but, in contrast to earlier experiments, they use Escherichia coli K-12 strains deficient in the restriction alleviation function associated with the Rac prophage . In bacteria with elevated levels of EcoKI the co-operative interactions are obscured, consistent with co-operation between free enzyme and that bound at target sites . We have made changes in three of the motifs characteristic of DEAD box proteins, including motif III, which in RecG is implicated in the migration of Holliday junctions . Conservative changes in each of the three motifs impair restriction. EMBO J, 1996 Apr 15, 15(8), 1983 - 91 Transfer RNA-dependent cognate amino acid recognition by an aminoacyl-tRNA synthetase; Hong KW et al.; An investigation of the role of tRNA in the catalysis of aminoacylation of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) has revealed that the accuracy of specific interactions between GlnRS and tRNAGln determines amino acid affinity . Mutations in GlnRS at D235, which makes contacts with nucleotides in the acceptor stem of tRNAGln, and at R260 in the enzyme's active site were found to be independent during tRNA binding but interactive for aminoacylation . Characterization of mutants of GlnRS at position 235, showed amino acid recognition to be tRNA mediated . Aminoacylation of tRNA(CUA)Tyr {tyrT (UAG)} by GlnRS-D235H resulted in a 4-fold increase in the Km for the Gln, which was reduced to a 2-fold increase when A73 was replaced with G73 . These and previous results suggest that specific interactions between GlnRS and tRNAGln ensure the accurate positioning of the 3' terminus . Disruption of these interactions can change the Km for Gln over a 30-fold range, indicating that the accuracy of aminoacylation is regulated by tRNA at the level of both substrate recognition and catalysis . The observed role of RNA as a cofactor in optimizing amino acid activation suggests that the tRNAGln-GlnRS complex may be partly analogous to ribonucleoprotein enzymes where protein-RNA interactions facilitate catalysis. EMBO J, 1996 Apr 15, 15(8), 1933 - 40 Yeast SUB1 is a suppressor of TFIIB mutations and has homology to the human co-activator PC4; Knaus R et al.; Activation of transcription in eukaryotes depends upon the interplay between transcriptional activators and general transcription factors . While direct contacts between activators and general factors have been demonstrated in vitro, an additional class of proteins, termed co-activators, is also required of transcriptional activation . Here we describe a yeast protein, SUB1, that was isolated as a suppressor of the cold-sensitive TFIIB R78H mutant . The N-terminal third of SUB1 is highly similar to the mammalian co-activator PC4 . We show that increased expression of SUB1 suppresses two alleles of TFIIB (E62G, R78H) specifically and that the deletion of SUB1 is lethal in combination with these same two alleles . We show that SUB1 binds to TFIIB in vitro and that it specifically inhibits the formation of TBP-TFIIB-promoter complexes . Furthermore we show that increasing the copy number of SUB1 stimulates transcriptional activation in vivo . Based on our results and recent observations of others, we propose that SUB1 plays a role in the release of TFIIB from the transcription complex during transcription initiation. EMBO J, 1996 Apr 15, 15(8), 1818 - 25 The conserved amino-terminal domain of hSRP1 alpha is essential for nuclear protein import; Weis K et al.; Nuclear proteins are targeted through the nuclear pore complex (NPC) in an energy-dependent reaction . The import reaction is mediated by nuclear localization sequences (NLS) in the substrate which are recognized by heterodimeric cytoplasmic receptors . hSRP1 alpha is an NLS-binding subunit of the human NLS receptor complex and is complexed in vivo with a second subunit of 97 kDa (p97) . We show here that a short amino-terminal domain in hSRP1 alpha is necessary and sufficient for its interaction with p97 . This domain is conserved in other SRP1-like proteins and its fusion to a cytoplasmic reporter protein is sufficient to promote complete nuclear import, circumventing the usual requirement for an NLS receptor interaction . The same amino-terminal domain inhibits import of NLS-containing proteins when added to an in vitro nuclear transport assay . While full-length hSRP alpha is able to leave the nucleus, the amino-terminal domain alone is not sufficient to promote exit . We conclude that hSRP1 alpha functions as an adaptor to tether NLS-containing substrates to the protein import machinery. Biochem J, 1996 Apr 15, 315 ( Pt 2), 443 - 6 Expression of soluble cloned porcine pepsinogen A in Escherichia coli; Tanaka T et al.; A system for the production of soluble porcine pepsinogen A (EC 3.4.23.1) was developed by fusing the pepsinogen and thioredoxin genes and then expressing the fused product (Trx-PG) in Escherichia coli . The expressed fusion protein was purified using a combination of ion-exchange and hydrophobic chromatography . Trypsin digestion of the fusion protein yielded pepsinogen which was one residue longer than the intrinsic length . Acidification of either the fusion protein or pepsinogen (tryptic digestion of Trx-PG) yielded recombinant pepsin A (r-pepsin) . When compared with commercial porcine pepsin A, r-pepsin had similar milk-clotting and proteolytic activities, kinetic parameters and pH dependency . The above results indicate that an expression system was developed which yielded fully active soluble pepsin(ogen) from Escherichia coli. Biochem J, 1996 Apr 15, 315 ( Pt 2), 363 - 7 A network of specific minor-groove contacts is a common characteristic of paired-domain-DNA interactions; Pellizzari L et al.; Pax proteins are a family of transcription factors conserved during evolution and able to bind specific DNA sequences through a domain called a "paired domain' . The DNA-binding specificity of the Pax-8 paired domain was investigated . Site-selection experiments indicate that Pax-8 binds to a consensus sequence similar to those bound by Pax-2 and Pax-5 . When consensus sequences of various paired domains are observed in light of recent structural studies describing paired-domain-DNA interaction {Xu, Rould, Jun, Desplan and Pabo (1995) Cell 80, 639-650}, it appears that base-pairs contacted in the minor groove are conserved, while most of the base-pairs contacted in the major groove are not . Therefore a network of specific minor groove contacts is a common characteristic of paired-domain-DNA interactions . The functional importance of such a network was successfully tested by analysing the effect of consensus-based mutations on the Pax-8 binding site of the thyroglobulin promoter. FEBS Lett, 1996 Apr 15, 384(2), 193 - 7 Construction of a fusion protein between protein A and green fluorescent protein and its application to western blotting; Aoki T et al.; Aequorea green fluorescent protein (GFP) and protein A were fused and expressed in Escherichia coli . The fluorescent native fusion protein (PA-GFP) migrated at 47 kDa in SDS-PAGE . However, the non-fluorescent denatured PA-GFP migrated at 57 kDa which corresponds to the theoretical molecular mass . Although the reason(s) for this mobility shift between fluorescent and non-fluorescent molecules remains unclear, the small ring structure within the native molecules may affect their mobility . The cell extract, prepared from an E . coli strain producing PA-GFP, was used in Western and dot blots . The sensitivity and specificity of the PA-GFP detection were sufficient for rapid and easy screening. Virology, 1996 Apr 15, 218(2), 335 - 42 Identification of a set of proteins (C' and C) encoded by the bicistronic P gene of the Indiana serotype of vesicular stomatitis virus and analysis of their effect on transcription by the viral RNA polymerase; Peluso RW et al.; Previous work (C.F . Spiropoulou and S.T . Nichol, 1993, J . Virol . 67, 3103-3110) has demonstrated the existence in cells infected with the New Jersey serotype of vesicular stomatitis virus (VSV) of two small carboxy-coterminal proteins encoded by the P mRNA . These proteins have been named C' and C . We are interested in studying the function of these proteins in the virus life cycle . Toward this end, we have cloned the ORF encoding the potential C' protein of the Indiana serotype as a histidine-tagged fusion protein, purified the expressed protein from Escherichia coli, and used the fusion protein as an immunogen to raise antiserum in a rabbit . We have used this anti-C' protein serum to demonstrate that both of the predicted C' and C proteins are synthesized in cells infected with the Indiana serotype of VSV . In addition we have localized a portion of these proteins to nucleocapsids isolated from infected cells, suggesting that they may play a role in RNA synthesis . Reconstitution of the viral polymerase activity by expressing the L and P protein subunits with or without the C proteins failed to demonstrate any effect of the presence of these latter proteins on reconstituted transcription using purified nucleocapsids as templates . However, we have been able to show a dramatic stimulation of the polymerase activity in purified virions by the addition of purified C' protein to in vitro transcription reactions . Both the level and the fidelity of mRNA synthesis are stimulated by this protein . Evidence for the specificity of this effect comes from the fact that stimulation appears to be serotype specific; C' protein of the Indiana serotype stimulates transcription by purified Indiana serotype virions but has a minimal effect on transcription by purified virions of the New Jersey serotype . We are continuing our studies to determine the mechanism of this stimulation. J Med Chem, 1996 Apr 12, 39(8), 1720 - 8 6-Substituted and 5,6-disubstituted derivatives of uridine: stereoselective synthesis, interaction with uridine phosphorylase, and in vitro antitumor activity; Felczak K et al.; Stereoselective procedures are described for the synthesis of 6-alkyluridines by Lewis acid-catalyzed condensation of (a) trimethylsilylated 6-alkyl-4-alkylthiouracils with 1-O-acetyl-2,3,5-tri-O-benzoyl-beta-D-ribofuranose (ABR) and (b) trimethylsilylated 6-alkyl-3-benzyluracils with ABR . The 4-methylthio group was subsequently removed with the use of 1 N trifluoroacetic acid and the 3-benzyl group by a new modified procedure with the use of the complex BBr3-THF . Furthermore, 6-(hydroxymethyl)uridine (39) and 5-fluoro-6-(hydroxymethyl)uridine (40) were obtained by sequential oxidation with SeO2 and reduction with tetrabutylammonium borohydride of the 6-methyl group of 6-methyluridine (5) and 5-fluoro-6-methyluridine (35), and their corresponding 6-fluoromethyl congeners 41 and 42 were obtained by DAST treatment of 39 and 40, respectively . For all the foregoing nucleosides in the fixed syn conformation about the glycosyl bond, 1H NMR spectroscopy further demonstrated that the pentose rings exist predominantly in the conformation N (3'-endo) . Most of the nucleosides were weak substrates of Escherichia coli pyrimidine nucleoside phosphorylase . Enhanced susceptibility to phosphorolysis was exhibited by two of them, 39 and 41, with 6-CH2OH and 6-CH2F substituents capable of formation of an additional hydrogen bond with the enzyme . The 5-fluoro-6-substituted uridines were the poorest substrates . Cytotoxicities of the nucleosides were examined vs the human tumor cell lines MOLT-3, U-937, K-562, and IM-9, as well as PHA-stimulated human lymphocytes . Two of the analogues, 5-fluoro-6-(fluoromethyl)uridine (42) and 5-fluoro-6-(hydroxymethyl)uridine (40), exhibited cytotoxicities comparable to that of 5-fluorouracil. J Mol Biol, 1996 Apr 12, 257(4), 790 - 803 Posttranscriptional regulation of EcoP1I and EcoP15I restriction activity; Redaschi N et al.; Efficient establishment of a DNA restriction-modification (R-M) system in a non-modified cell requires a tight control of the potentially lethal activity of the restriction enzyme . The type III R-M systems EcoP1I and EcoP15I can be transferred to non-modified Escherichia coli cells by transfection, conjugation or transformation and become established without difficulty . Modification activity is expressed immediately after the R-M genes enter the cell, whereas the expression of restriction activity is delayed until complete protection of the cellular DNA is achieved by methylation . We have shown by Western blot analysis that the expression of the modification polypeptide subunit positively regulates the amount of restriction subunit present in the cell . The finding that ribosomal alterations affected the expression of restriction activity pointed to additional control at the translational level . The analysis of EcoP1I expression in E . coli strains mutated in either of the ribosomal proteins S12 (rpsL) or S4 (rpsD) suggests that the level of in vivo restriction activity can be modulated both by a decrease in the efficiency of translation and by varying ribosomal accuracy conditions . In addition, we have preliminary evidence from in vivo gene fusion studies that the res gene may code for more than one gene product. J Biol Chem, 1996 Apr 12, 271(15), 9039 - 45 The role of the carboxyl-terminal amino acid residues in Escherichia coli DNA topoisomerase III-mediated catalysis; Zhang HL et al.; The role that the carboxyl-terminal amino acids of Escherichia coli DNA topoisomerase I (Topo I) and III (Topo III) play in catalysis was examined by comparing the properties of Topo III with those of a truncated enzyme lacking the generalized DNA binding domain of Topo III, Topo I, and a hybrid topoisomerase polypeptide containing the amino-terminal 605 amino acids of Topo III and the putative generalized DNA binding domain of Topo I . The deletion of the carboxyl-terminal 49 amino acids of Topo III decreases the affinity of the enzyme for its substrate, single-stranded DNA, by approximately 2 orders of magnitude and reduces Topo III-catalyzed relaxation of supercoiled DNA and Topo III-catalyzed resolution of DNA replication intermediates to a similar extent . Fusion of the carboxyl-terminal 312 amino acid residues of Topo I onto the truncated molecule stimulates topoisomerase-catalyzed relaxation 15-20-fold, to a level comparable with that of full-length Topo III . However, topoisomerase-catalyzed resolution of DNA replication intermediates was only stimulated 2-3-fold . Therefore, the carboxyl-terminal amino acids of these topoisomerases constitute a distinct and separable domain, and this domain is intimately involved in determining the catalytic properties of these polypeptides. J Biol Chem, 1996 Apr 12, 271(15), 8996 - 9001 Reconstitution of barley photosystem I with modified PSI-C allows identification of domains interacting with PSI-D and PSI-A/B; Naver H et al.; The PSI-C subunit of photosystem I shows similarity to soluble 2{4Fe-4S} ferredoxins . Alignment analysis clearly shows that PSI-C contains an 8-residue internal loop and a 15-residue C-terminal extension that are absent in the ferredoxins . The remaining residues in PSI-C are likely to be folded in a way similar to the soluble 2{4Fe-4S} ferredoxins . Two modified PSI-C subunits lacking either the 8-residue loop or 10 residues of the C terminus were expressed in Escherichia coli and used to reconstitute a barley P700-FX core prepared to specifically lack PSI-C, PSI-D, and PSI-E . As shown by EPR spectroscopy, the modified proteins carry two {4Fe-4S} clusters with characteristics similar to those of native PSI-C . Western blot analysis of the reconstituted photosystem I complexes showed that the modified PSI-C proteins bind to the P700-FX core . Flash photolysis revealed that in photosystem I complexes reconstituted in the presence of PSI-D with the C-terminally deleted PSI-C, the FA/FB back-reaction was less efficiently restored than with wild-type PSI-C . The loop-deleted PSI-C was even less efficient . We attribute these differences to altered binding properties of the modified proteins . Comparison of reconstitutions performed in the presence and absence of PSI-D shows that the loop-deleted PSI-C is unable to bind without PSI-D, whereas the C-terminally deleted PSI-C binds only weakly with PSI-D . These results imply that the internal loop of PSI-C interacts with the PSI-A/B heterodimer and that the C terminus of PSI-C interacts with PSI-D. J Biol Chem, 1996 Apr 12, 271(15), 8863 - 8 Structure of the heme d of Penicillium vitale and Escherichia coli catalases; Murshudov GN et al.; A heme d prosthetic group with the configuration of a cis-hydroxychlorin gamma-spirolactone has been found in the crystal structures of Penicillium vitale catalase and Escherichia coli catalase hydroperoxidase II (HPII) . The absolute stereochemistry of the two heme d chiral carbon atoms has been shown to be identical . For both catalases the heme d is rotated 180 degrees about the axis defined by the alpha-gamma-meso carbon atoms, with respect to the orientation found for heme b in beef liver catalase . Only six residues in the heme pocket, preserved in P . vitale and HPII, differ from those found in the bovine catalase . In the crystal structure of the inactive N201H variant of HPII catalase the prosthetic group remains as heme b, although its orientation is the same as in the wild type enzyme . These structural results confirm the observation that heme d is formed from protoheme in the interior of the catalase molecule through a self-catalyzed reaction.
|
© 2005
Transgalactic Ltd (manufacturer of Bioscreen C software) |
Privacy Statement | P.O. Box
1393, 00101 Helsinki, Finland,
Last modified: May 25, 2005
| ||||||
