Escherichia coli

Tokushima J Exp Med, 1993 Jun, 40(1-2), 13 - 7
Production of dihydrofolate reductase by an improved continuous flow cell-free translation system using wheat germ extract; Endo Y et al.; We have examined the characteristics of protein synthesis in an improved continuous flow cell-free translation system prepared from wheat germ extract with dihydrofolate reductase mRNA as the translated message . Continuous buffer flow and separation of the product from the reaction mixture were accomplished by the use of a modified Amicon ultrafiltration chamber as the reaction vessel . The system worked for 19 hours and produced 1.52 nmol (27.4 micrograms) of enzymatically active dihydrofolate reductase.

Plant Physiol, 1993 Jun, 102(2), 529 - 36
Identification of the uridine-binding domain of sucrose-phosphate synthase . Expression of a region of the protein that photoaffinity labels with 5-azidouridine diphosphate-glucose; Salvucci ME et al.; The uridine diphosphate-glucose (UDP-Glc) binding domain of sucrose-phosphate synthase (SPS) was identified by overexpressing part of the gene from spinach (Spinacia oleracea) . Degenerate oligonucleotide primers corresponding to two tryptic peptides common to both the full-length 120-kD SPS subunit and an 82-kD form that photoaffinity labeled with 5-azidouridine diphosphate-glucose (5-N3UDP-Glc) were used in a polymerase chain reaction to isolate a partial cDNA clone . Comparison of the deduced amino acid sequence of spinach SPS with the sequences of potato sucrose synthase showed that the partial cDNA included one region that was highly conserved between the proteins . Expression of the partial cDNA clone of SPS in Escherichia coli produced a 26-kD fusion protein that photoaffinity labeled with 5-N3UDP-Glc . Photoaffinity labeling of the 26-kD fusion protein was specific, indicating that this portion of the SPS protein harbors the UDP-Glc-binding domain . Isolation of a modified peptide from the photolabeled protein provided tentative identification of amino acid residues that make up the uridine-binding domain of SPS.

Plant Physiol, 1993 Jun, 102(2), 387 - 99
Isolating the Arabidopsis thaliana genes for de novo purine synthesis by suppression of Escherichia coli mutants . I . 5'-Phosphoribosyl-5-aminoimidazole synthetase; Senecoff JF et al.; We have initiated an investigation of the de novo purine nucleotide biosynthetic pathway in the plant Arabidopsis thaliana . Functional suppression of Escherichia coli auxotrophs allowed the direct isolation of expressed Arabidopsis leaf cDNAs . Using this approach we have successfully suppressed mutants in 4 of the 12 genes in this pathway . One of these cDNA clones, encoding 5'-phosphoribosyl-5-aminoimidazole (AIR) synthetase (PUR5) has been characterized in detail . Analysis of genomic DNA suggests that the Arabidopsis genome contains a single AIR synthetase gene . Analysis of the cDNA sequence and mRNA size suggests that this enzyme activity is encoded by a monofunctional polypeptide, similar to that of bacteria and unlike other eukaryotes . The Arabidopsis AIR synthetase contains a basic hydrophobic transit peptide consistent with transport into chloroplasts . Comparison of both the predicted amino acid and nucleotide sequence from Arabidopsis to those of eight other distant organisms suggests that the plant sequence is more similar to the bacterial sequences than to other eukaryotic sequences . This study provides the groundwork for future investigations into the regulation of de novo purine biosynthesis in plants . Additionally, we have demonstrated that functional suppression of bacterial mutants may provide a useful method for cloning a variety of plant genes.

Bioorg Khim, 1993 Jun, 19(6), 612 - 22
{Synthesis of somatostatin in Escherichia coli cells: isolation and characteristics}; Karpova SK et al.; A synthetic gene coding for somatostatin-14 (SST) was cloned in plasmid expression vectors in frame with the chloramphenicol acetyl transferase (CAT) gene, both genes being divided by a Met residue . The hybrid gene was expressed under the control of the CAT gene promoter (Pcat) or the tryptophan operon promoter (Ptrp) . Them fused genes gave insoluble polypeptide products amounting from 5% of the total cellular protein under constitutive biosynthesis conditions (Pcat) to 30% upon induction (Ptrp) . SST was liberated from the fused polypeptide by treatment with cyanogen bromide, purified to homogeneity by gel-filtration and reverse phase HPLC, and finally refolded by dilution and air oxidation . The renaturated recombinant SST showed the specific biological and immunological activities of the native peptide.

Genome, 1993 Jun, 36(3), 459 - 66
Molecular cloning and expression of a cDNA encoding the proliferating cell nuclear antigen from Brassica napus (oilseed rape); Markley NA et al.; A cDNA clone encoding the proliferating cell nuclear antigen (PCNA) has been isolated from a Brassica napus apical meristem cDNA library . The putative full-length cDNA contains an open reading frame of 1004 nucleotides, which predicts a protein of 263 amino acids (M(r) = 29,231) . Sequence analysis has revealed that the plant PCNA exhibits 81.6% amino acid similarity with the human PCNA . Genomic Southern blot analysis indicates the presence of at least two copies of PCNA per genome . The B . napus PCNA mRNA (1.0 kb) was expressed in rapidly dividing tissues such as flower buds, apical meristems, and young leaves, while mature stem and fully expanded leaves showed significantly lower levels of PCNA transcript . The B . napus PCNA cDNA was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase (GST) in the bacterial expression vector pGEX-2T . A broad specificity monoclonal antibody raised against rabbit PCNA cross-reacted with the GST-PCNA fusion peptide but not with the GST moiety alone . This antibody also recognized the human PCNA (36 kDa) polypeptide, confirming the structural similarities between the human and plant PCNA . The high degree of structural conservation of PCNA from such diverse organisms as humans and higher plants suggests that the plant PCNA may function in a manner analogous to that found in mammals with respect to plant cell DNA replication . Such conservation suggests that PCNA is also a critical component of the plant cell DNA replication complex.

J Crit Care, 1993 Jun, 8(2), 117 - 27
Organ blood flow and distribution of cardiac output in dopexamine- or dobutamine-treated endotoxemic rats; van Lambalgen AA et al.; Endotoxemia causes a decrease of blood flow to most organs . If this could be prevented, chances of survival might improve . In endotoxemic rats, we studied the effect of a therapeutic infusion of dopexamine (dopaminergic, beta 2-adrenergic) on blood flow and percentage of the cardiac output distributed to heart, brain, hepatic artery, stomach, intestines, spleen, pancreas, kidneys, adrenals, diaphragm, skeletal muscle, and skin . Dopexamine action was compared with that of dobutamine (beta 1-adrenergic) . Endotoxin shock was induced in 28 rats with infusion of 8 mg/kg Escherichia coli O127:B8 endotoxin from 0 to 60 minutes; the rats were then divided into 3 groups, which received from 60 to 135 minutes of an infusion of saline (ES; n = 10), dopexamine hydrochloride (DX, 3 x 10(-8) mol/kg.min; n = 10) or dobutamine (DB, 10(-7) mol/kg.min; n = 8) . A fourth group served as time-matched controls (C, saline from 0 to 135 minutes; n = 8) . In the untreated endotexemic rats, cardiac output decreased and organ blood flow decreased except in the diaphragm, heart, and brain; the percentage of the cardiac output to those organs increased . Dopexamine and dobutamine similarly improved cardiac output in endotoxemic rats . All organs benefitted to the same extent from the increased cardiac output . Therapeutic infusion of dopexamine during endotoxemia did not favor flow to any particular organ; redistribution of cardiac output changed little after administration of dopexamine, and its effects were not significantly different from those of dobutamine.

Antimicrob Agents Chemother, 1993 Jun, 37(6), 1278 - 84
Functional studies of truncated soluble intercellular adhesion molecule 1 expressed in Escherichia coli; Martin S et al.; We have expressed in Escherichia coli the two N-terminal immunoglobulin (Ig)-like domains of the intercellular adhesion molecule 1 (ICAM-1) . The first 188 residues of ICAM-1 were expressed with an N-terminal methionine (MP188) or as a maltose-binding fusion protein which was cleaved with factor Xa (XP188) . After refolding, both MP188 and XP188 were active in binding to the leukocyte integrin lymphocyte function-associated antigen 1, which has previously been shown to bind to the N-terminal Ig domain of ICAM-1 . The major group of rhinoviruses and malaria-infected erythrocytes bind to distinct sites within the first Ig-like domain of ICAM-1 . Both MP188 and XP188 bound to malaria-infected erythrocytes; however, only XP188 inhibited human rhinovirus plaque formation . A product (MdQ1P188) with the initiation methionine fused to residue 2, i.e., with glutamine 1 deleted, inhibited plaque formation . MdQ1P188 was able to induce a conformational change of the virus capsid as shown by conversion of 149S particles to 85S particles, whereas MP188 had no effect . These results show that functionally active fragments of ICAM-1 can be produced in E . coli, that glycosylation is not required for ligand binding, and that the N-terminal residue of ICAM-1 is proximal to or part of the human rhinovirus-binding site.

J Bacteriol, 1993 Jun, 175(11), 3679 - 84
Close linkage of genes encoding glutamine synthetases I and II in Frankia alni CpI1; Hosted TJ et al.; Frankia alni CpI1 has two glutamine synthetases (GSs), GSI and GSII . The GSI gene (glnA) was isolated from a cosmid library of F . alni CpI1 DNA by heterologous probing with glnA from Streptomyces coelicolor . The glnA gene was shown to be located upstream of the GSII gene (glnII) by DNA-DNA hybridization . The nucleotide sequences of the 1,422-bp CpI1 glnA gene and of the 449-bp intervening region between glnA and glnII were determined, and the glnA amino acid sequence was deduced . In common with GSIs from other organisms, CpI1 GSI contains five conserved regions near the active site and a conserved tyrosine at the adenylylation site . F . alni CpI1 glnA complemented the glutamine growth requirement of the Escherichia coli glnA deletion strain YMC11 but only when expressed from an E . coli lac promoter . While the functional significance of maintaining two GSs adjacent to one another remains unclear, this arrangement in F . alni provides support for the recently proposed origin of GSI and GSII as resulting from a gene duplication early in the evolution of life.

Infect Immun, 1993 Jun, 61(6), 2526 - 31
Piglet ileal mucus contains protein and glycolipid (galactosylceramide) receptors specific for Escherichia coli K88 fimbriae; Blomberg L et al.; The aim of this study was to characterize the Escherichia coli K88-specific receptors in mucus from the small intestines of 35-day-old piglets with the isogenic strains E . coli K-12(pMK005) (K88+) and E . coli K-12(pMK002) (K88-) . These strains differed only in that the latter one cannot produce intact K88 fimbriae because of a deletion in the gene coding for the major fimbrial subunit . Adhesion was studied by incubating 3H-labeled bacteria with crude mucus, pronase-treated whole mucus, mucus fractionated by gel filtration, delipidated mucus, or extracted lipids immobilized in microtiter wells . In addition, E . coli strains were tested for adhesion to glycolipids extracted from mucus by overlaying glycolipid chromatograms with 125I-labeled bacteria . The recently reported finding that K88 fimbriae bind to glycoproteins in mucus from the piglet small intestine was confirmed in two ways . Pronase treatment of immobilized mucus reduced adhesion by 82%, and adhesion to delipidated mucus was 14 times greater for the K88+ than for the K88- strain . E . coli K88+ adhered to several of the fractions collected after gel filtration of crude mucus, including the void volume (M(r), > 250,000) . Receptor activity specific for the K88 fimbriae was demonstrated in the lipids extracted from mucus, as the neutral lipids contained six times as much receptor activity as the acidic lipid fraction . Specificity was confirmed by demonstrating that adhesion to the total lipids could be inhibited by pretreatment of the immobilized lipids with K88 fimbriae . Relative to K-12 (K88-), the K-12 (K88+) bacterial cells bound more avidly to galactosylceramide when the neutral lipids were separated on thin-layer chromatography plates . No adhesion to lipids in the acidic fraction separated on thin-layer plates was detected . Relative to adhesion of K-12 (K88-), adhesion of K-12 (K88+) to commercially available galactosylceramide immobilized in microtiter wells confirmed the results with the thin-layer plates . It can be concluded that 35-day-old piglet mucus contains both protein and glycolipid receptors specific for K88 fimbriae, the latter being galactosylceramide.

Infect Immun, 1993 Jun, 61(6), 2453 - 61
Structure and copy number analyses of pap-, sfa-, and afa-related gene clusters in F165-positive bovine and porcine Escherichia coli isolates; Maiti SN et al.; Pathogenic F165-positive Escherichia coli isolates of porcine and bovine origin possess gene clusters related to extraintestinal E . coli fimbrial operons pap, sfa, and afaI . Probes from different segments of the pap, sfa, and afaI operons were used in Southern hybridization to analyze 18 F165-positive, mannose-resistant hemagglutinating E . coli isolates possessing pap- and sfa-, pap- and afa-, or pap-related sequences . Only single copies of the pap-, sfa-, or afa-related sequences were found among the isolates, and the position of each sequence with respect to those of adjacent sequences was variable . Expression of the P and F adhesins individually or concurrently was associated with the presence of a single copy of pap-related sequences . Gene clusters related to pap were structurally similar to the pap operon in certain isolates or the prs operon in others . sfa-related sequences showed few internal structural polymorphisms and were structurally similar to the foc rather than the sfa operon . afa-related sequences showed many internal structural polymorphisms compared with the afa-related sequences from prototype strain KS52 . These results demonstrate that the pap- and sfa-related sequences in F165-positive isolates are closely related to the prototype pap operon and foc operon of the P family and Sfa family, respectively . afa-related sequences, on the other hand, display heterogeneity and differ from the prototype afaI operon.

J Virol, 1993 Jun, 67(6), 3534 - 43
Mutational analysis of the central domain of adenovirus virus-associated RNA mandates a revision of the proposed secondary structure; Pe'ery T et al.; Protein synthesis in adenovirus-infected cells is regulated during the late phase of infection . The rate of initiation is maintained by a small viral RNA, virus-associated (VA) RNAI, which prevents the phosphorylation of eukaryotic initiation factor eIF-2 by a double-stranded RNA-activated protein kinase, DAI . On the basis of nuclease sensitivity analysis, a secondary-structure model was proposed for VA RNA . The model predicts a complex stem-loop structure in the central part of the molecule, the central domain, joining two duplexed stems . The central domain is required for the inhibition of DAI activation and participates in the binding of VA RNA to DAI . To assess the significance of the postulated stem-loop structure in the central domain, we generated compensating, deletion, and substitution mutations . A substitution mutation which disrupts the structure in the central domain abolishes VA RNA function in vitro and in vivo . Base-compensating mutations failed to restore the function or structure of the mutant, implying that the stem-loop structure may not exist . To confirm this observation, we tested mutants with alterations in the hypothetical loop and short stem that constitute the main features of the wild-type model structure . The upper part of the hypothetical loop could be deleted without abolishing the ability of the RNA to block DAI activation in vitro, whereas other loop mutations were deleterious for function and caused major rearrangements in the molecule . Base-compensating mutations in the stem did not restore the expected base pairing, even though the mutant RNAs were still functional in vitro . Surprisingly, a mutant with a noncompensating substitution mutation in the stem was more effective than wild-type VA RNAI in DAI inhibition assays but was ineffective in vivo . The structural and functional consequences of these mutations do not support the proposed model structure for the central domain, and we therefore suggest an alternative structure in which tertiary interactions may play a significant role in shaping the specificity of VA RNA function in the infected cell . Discrepancies between the functionality of mutant forms of VA RNA in vivo and in vitro are consistent with the existence of additional roles for VA RNA in the cell.

J Neurochem, 1993 Jun, 60(6), 2181 - 91
Development of polyclonal anti-D2 dopamine receptor antibodies to fusion proteins: inhibition of D2 receptor-G protein interaction; Boundy VA et al.; Portions of the cDNA encoding the third intracellular loop (i3 loop) of the long and short isoforms of the rat D2 dopamine receptor were subcloned into the vector pNMHUBpoly and expressed in Escherichia coli as fusion proteins . The fusion proteins were gel-purified and used to immunize rabbits for the production of polyclonal anti-receptor antisera . The anti-fusion protein antisera recognized synthetic peptides corresponding to segments of the i3 loops of D2 dopamine receptors in a solid-phase radioimmunoassay . Antisera were tested in an immunoprecipitation assay using the reversible D2 antagonist {125I}NCQ 298 and digitonin-solubilized extracts of canine and rat caudate . {125I}-NCQ 298 bound reversibly and with high affinity (KD = 0.14 nM) to receptors in solubilized extracts enriched by chromatography on heparin-agarose . The anti-UBI-D2i3L and anti-UBI-D2i3s antisera were able to immunoprecipitate quantitatively D2 dopamine receptors labeled with {125I}NCQ 298 from solubilized rat caudate . The antibodies were tested for their ability to affect the coupling of D2 dopamine receptors to GTP-binding proteins in digitonin-solubilized rat caudate . Both anti-UBI-D2i3L and anti-UBI-Di3s antisera were able to inhibit the high-affinity binding of the agonist N-propylnorapomorphine to digitonin-solubilized rat caudate . These findings indicate that the i3 loop of the D2 dopamine receptor is an important determinant for coupling of the G protein.

C R Acad Sci III, 1993 Jun, 316(6), 593 - 7
Identification of a Kin nuclear protein immunologically related to RecA protein in the rat CNS; Araneda S et al.; A polypeptide, called Kin, has been identified in cells of the central nervous system (CNS) by using antibodies raised against RecA protein of E . coli and by in situ hybridization with identified cDNA . RecA protein is a recombination enzyme associated with DNA repair . The RecA cross-reacting polypeptide was immunocytochemically demonstrated in the nuclei of various cells of adult rats . Following Western blot analysis using anti-RecA antibodies, the Kin protein showed a band with an apparent molecular weight of 41 kDa . Double labelling experiments, using in situ hybridization of Kin-17 mRNA with serotonin immunocytochemistry, demonstrated a cytoplasmic distribution of radiolabelling indicating the translation of this messenger RNA in serotonergic neurons . These data indicate the presence of a Kin nuclear protein in the CNS and suggest that neurons may possess some DNA-repair pathways analogous to those described in bacteria.

Development, 1993 Jun, 118(2), 553 - 62
The chicken CdxA homeobox gene and axial positioning during gastrulation; Frumkin A et al.; The chicken homebox containing gene, CdxA (formerly CHox-cad), was previously shown to be expressed during gastrulation . Localization of CdxA transcripts by in situ hybridization to tissue sections revealed that, during gastrulation, expression of this gene exhibits a posterior localization along the primitive streak . The transcripts are localized to epiblast cells in the vicinity of the primitive streak, to cells of the primitive streak itself and in the definitive endoderm as it replaces the hypoblast . In order to study in greater detail the pattern of expression of the CdxA gene during gastrulation, we expressed the full-length CdxA protein as a fusion protein in E . coli and generated monoclonal antibodies against it . Chicken embryos at different stages of gastrulation were processed for whole-mount immunohistochemical localization of the protein using anti-CdxA antibodies . Once the pattern of expression in the whole embryo was determined, the same embryos were sectioned to determine the identity of the cells expressing the CdxA protein . Detailed analysis of the CdxA protein in embryos, from the onset of primitive streak formation to the beginning of the tail bud stage (stages 2 to 10), has shown different patterns of expression during primitive streak elongation and regression . The CdxA protein is initially detected at the posterior marginal zone and the expression moves rostrally into the primitive streak during mid-streak stages . As the primitive streak elongates, the CdxA stripe of expression moves anteriorly . By definitive streak stages, the CdxA stripe of expression delineates a position along the anterior-posterior axis in the primitive streak . CdxA, like its Drosophila homologue cad, is expressed during gastrulation in a stripe localized to the posterior region of the embryo . These observations suggest that CdxA as a homebox gene may be part of a regulatory network coupled to axial determination during gastrulation in the early chick embryo.

Afr J Med Med Sci, 1993 Jun, 22(2), 39 - 41
Cloning and characterization of the tetracycline resistance determinant from enteropathogenic Escherichia coli p1479; Daini OA et al.; As part of an epidemiological study, R plasmids coding for tetracycline resistance were isolated from enteropathogenic Escherichia coli . A 1.4kb Pst 1 fragment of one of them p1479 (size 5.7kb) has been cloned into plasmid pGL101 . This recombinant plasmid containing the tetracycline gene was then transformed into E . coli DHI where the tetracycline resistant gene was fully expressed . Attempts to develop this clone to a diagnostic probe is now in progress.

J Biotechnol, 1993 Jun, 29(3), 299 - 306
Enhanced production of pL-controlled recombinant proteins and plasmid stability in Escherichia coli RecA+ strains; Benito A et al.; Overexpression of pL-controlled foot-and-mouth disease virus recombinant proteins was studied in Escherichia coli RecA+ strains and in a recA mutant . Higher protein yield and extractable plasmid DNA amounts were found in wild type cells, in absence of detectable RecA proteolytic activity . Minor but still significant differences in pBR322 DNA amounts were also detected between RecA+ and its recA13 and lexA1 derivatives . These data should be seriously considered to select expression systems and to design production processes for recombinant proteins, specially if they are expected to be toxic for Escherichia coli cells.

Appl Microbiol Biotechnol, 1993 Jun, 39(3), 324 - 8
Optimization of the synthesis of porcine somatotropin in Escherichia coli; Wang H et al.; We report on the influence of choice of promoter and RNA polymerase, 5'-untranslated regions and ribosome binding sites, codon usage, leader peptide coding sequences and poly A tail in the 3'-untranslated region on the synthesis of porcine somatotropin (PST) in Escherichia coli . A total of 12 different constructs were tested in this study for the production of porcine somatotropin (PST) in E . coli . Several factors have significant effects on PST synthesis . In the presence of a strong promoter and a strong ribosome binding site, the next most important factor seems to be the combination of sequences at the 5'-end of the mRNA including both the 5'-untranslated region and the start of the coding sequence . Codon usage in the 5'-coding sequence per se is not important in determining the level of PST synthesis where high level expression is achieved from a strong ribosome binding site . However, where low level synthesis of recombinant PST (rPST) is achieved, codon usage in the 5'-coding sequence is important in determining the level of PST synthesis . Leader sequences dramatically reduce the level of PST synthesis . The presence of a poly A tail in the 3'-untranslated region has no significant effect on PST synthesis.

Appl Microbiol Biotechnol, 1993 Jun, 39(3), 309 - 17
Cloning and characterization of the Methylobacterium extorquens polyhydroxyalkanoic-acid-synthase structural gene; Valentin HE et al.; A cosmid gene bank of partially EcoRI-digested genomic DNA from Methylobacterium extorquens IBT no . 6 was screened for DNA fragments restoring polyhydroxyalkanoic-acid (PHA) accumulation in the PHA-negative mutant Alkaligenes eutrophus H16 PHB-4 . The M . extorquens PHA-synthase structural gene phaCMex was mapped on a 23-kbp EcoRI fragment by complementation studies, by hybridization experiments with heterologous DNA probes from A . eutrophus H16 encoding for phaA, phaB and phaC and by nucleic acid sequence analysis . Evidence for the presence of genes for a beta-ketothiolase or an acetoacetyl-coenzyme A reductase on this fragment was not obtained . The nucleotide sequence of a 3.7-kbp region was obtained . It contained the entire 1.815-kbp phaCMex plus approximately each 900-bp upstream and downstream of phaCMex.PhaCMex encoded a protein of 605 amino acids with a relative molecular mass (M(r)) of 66742, which exhibited 38.1% amino acid identity with the A . eutrophus PHA synthase . Determination of the N-terminal amino acid sequence of an M(r) 65,000 protein, which was enriched concomitantly with the purification of PHA granules in sucrose gradients, revealed a sequence that was identical with the amino acid sequence deduced from the most probable translation start codon except for a valine, which was obviously removed post-translationally . Enzyme analysis, which was done with the native gene and a phaCMex'-'lacZ fusion gene, gave no evidence for expression of phaCMex in Escherichia coli.

J Membr Biol, 1993 Jun, 134(2), 85 - 92
A carboxy-terminal fragment of colicin Ia forms ion channels; Ghosh P et al.; A carboxy-terminal, 18 kD fragment of colicin Ia, a bacterial toxin, forms ion channels in artificial phospholipid bilayers . This fragment, which comprises a quarter of the intact 70 kD molecule, is resistant to extensive protease digestion and probably constitutes a structural domain of the protein . The ion channels formed by the 18 kD fragment are functionally heterogeneous, having conductances that range from 15 to 30 pS at positive voltages and from 70 to 250 pS at negative voltages, and open lifetimes that range from at least 25 msec to 5 sec . In contrast, ion channels formed by whole colicin Ia open only at negative voltages, at which their conductances range from 6 to 30 pS, and their open lifetimes range from 1 sec to 3 min . Additionally, the open state of the 18 kD fragment channel is characterized by noisy fluctuations in current, while the open state of the whole molecule ion channel is often marked by numerous, stable subconductance states . Since the properties of the fragment channel differ substantially from those of the whole molecule channel, we suggest that portions of the molecule outside of the 18 kD fragment are involved in forming the whole molecule ion channel.

J Electron Microsc (Tokyo), 1993 Jun, 42(3), 202 - 4
Visualization of lipopolysaccharide aggregates by freeze-fracture and negative staining; Risco C et al.; The morphology of Escherichia coli 0111:B4 lipopolysaccharide (LPS) in aqueous medium was studied by freeze-fracture and negative staining . Samples processed by freeze-fracture showed LPS aggregates that were mainly spherical or elliptical and of rather homogeneous size . Negative staining, however, showed a more heterogeneous population, although globular structures revealed by both procedures had a similar size . Considering the mechanisms involved in the processing of the samples, we suggest that the genuine shape of LPS aggregates is more likely to be globular, thus being artefactual those forms visualized by negative staining, traditionally associated with the organization of LPS in aqueous suspension.

Biometrics, 1993 Jun, 49(2), 543 - 55
Phylogenetic inference: linear invariants and maximum likelihood; Navidi WC et al.; We develop a new statistical method for inferring phylogenies, based on a likelihood ratio test . This method does not require parameter constraints but does require identical evolutionary processes in the sites considered . Another method of phylogenetic inference is the method of linear invariants, described by Cavender (1989, Molecular Biology and Evolution 6, 301-316), based on a notion of Lake (1987, Molecular Biology and Evolution 4, 167-191) . We describe a sound mathematical basis for the use of linear invariants . We show that the validity of the method requires parameter constraints, but does not require that the evolutionary processes in differing sites be identical . We show that the method of linear invariants is asymptotically equivalent to a less powerful version of our likelihood ratio test, and is thus essentially a maximum likelihood technique.

J Cardiovasc Pharmacol, 1993 Jun, 21(6), 926 - 30
Effects of methylene blue on blood pressure and reactivity to norepinephrine in endotoxemic rats; Paya D et al.; The effects of methylene blue, an inhibitor of the activation of the soluble guanylyl cyclase by nitric oxide (NO), were studied on blood pressure (BP) and on hyporesponsiveness to norepinephrine (NE) induced by Escherichia coli lipopolysaccharide (LPS) in pentobarbital-anesthetized rats . Methylene blue intravenous (i.v.) injection (3 mg/kg) produced a transient increase in BP which, in LPS-treated rats, was followed by a more sustained increase in BP . Methylene blue restored the reactivity to NE in LPS-treated rats but did not change either BP or reactivity to NE in saline-infused control rats . Cyclic GMP level was significantly increased in small femoral resistance arteries removed from LPS-treated rats as compared with controls (125.2 +/- 19.5 and 83.5 +/- 18.8 fmol/mg DNA, respectively, n = 8) . In rats receiving methylene blue, there was no significant difference in cyclic GMP content of the arteries of LPS-treated rats as compared with controls (59.4 +/- 8.1 and 78.5 +/- 6.1 fmol/mg DNA, respectively, n = 8) . These results support the involvement of increased stimulation of arterial guanylyl cyclase in hyporeactivity to NE elicited by LPS . They show that in vivo administration of methylene blue is able to restore both vascular cyclic GMP level and pressor responses to NE to control levels in LPS-treated rats.

Antimicrob Agents Chemother, 1993 Jun, 37(6), 1227 - 31
Inhibition of Pneumocystis carinii dihydropteroate synthetase by para-acetamidobenzoic acid: possible mechanism of action of isoprinosine in human immunodeficiency virus infection; Kovacs JA et al.; Isoprinosine has been reported to decrease progression to AIDS, primarily by preventing Pneumocystis carinii pneumonia (PCP), in human immunodeficiency virus-infected patients, but the mechanism of action is unknown . para-Acetamidobenzoic acid (PAcBA), one component of isoprinosine, is structurally related to para-aminobenzoic acid (PABA), a precursor of de novo folate synthesis . This pathway is known to be important for P . carinii because sulfonamides, which are effective anti-P . carinii agents, inhibit incorporation of PABA into folate precursors by the enzyme dihydropteroate synthetase (DHPS) . Inhibition of P . carinii DHPS by PAcBA was investigated by using two assays . In short-term cultures of P . carinii from rats, {3H}PABA incorporation into reduced folates was inhibited by both isoprinosine (mean +/- standard error 50% inhibitory concentration {IC50}, 20 +/- 8.4 microM) and PAcBA free acid (IC50, 240 +/- 100 microM); a soluble PAcBA salt was more potent than PAcBA free acid alone (IC50, 29 +/- 48 microM) . The activity of PAcBA free acid was confirmed in a cell-free DHPS inhibition assay (IC50, 120 +/- 120 microM) . Inosine and dimethylaminopropanol, two other components of isoprinosine, were poor inhibitors of PABA incorporation (IC50, > 1,000 microM) . PAcBA free acid also showed activity in inhibiting the DHPS of Toxoplasma gondii, but was a poor inhibitor of the DHPSs of Escherichia coli and Saccharomyces cerevisiae . In a rat model of PCP, the PAcBA salt administered intraperitoneally demonstrated no activity against established PCP either alone or when used in combination with trimethoprim; the lack of efficacy in this model may be due to the rapid metabolism of the drug . Prevention of PCP by PaCBA through inhibition of P . carinii DHPS may explain the activity of isoprinosine in decreasing the progression to AIDS in human immunodeficiency virus-infected patients.

Genomics, 1993 Jun, 16(3), 551 - 61
DNA sequence and analysis of 136 kilobases of the Escherichia coli genome: organizational symmetry around the origin of replication; Burland V et al.; The DNA sequence of a 136-kb segment (81.5 to 84.5 min) from the Escherichia coli origin of replication region has been determined and analyzed . Of the 122 protein coding regions that were found, we could assign no gene name or function to half of them, even in this well-studied part of the genome . The newly sequenced region also includes five RNA genes . The arrangement of open reading frames and potential promoters suggests 63 transcription units . The sequence was also analyzed for bend sites and two types of repeated sequence elements . Together with our sequence of the 84.5 to 86.5 min region, this new determination forms a 227-kb contiguous region centered on oriC . A global analysis of this region reveals a remarkable symmetry: most genes are transcribed divergently from the replication origin, and Chi octanucleotide (5'GCTGGTGG3') recombinational hot spots are also strikingly oriented with respect to the directions of replication and translation.

Mol Gen Genet, 1993 Jun, 239(3), 400 - 8
Nitrogen regulation in an Escherichia coli strain with a temperature sensitive glutamyl-tRNA synthetase; Osorio AV et al.; Escherichia coli cells carrying the gltX351 allele are unable to grow at 42 degrees C (Ts phenotype) due to an altered glutamyl-tRNA synthetase . We found that gltX351 cells display a new phenotype termed Gsd-, i.e . an inability to raise glutamine synthetase activity above low constitutive levels in minimal medium with 6.8 mM glutamine as sole nitrogen source . When 0.5 mM NH4+ or 12 mM glutamate replaced glutamine, the glutamine synthetase activities of gltX351 cells were raised to wild-type levels . Northern experiments showed that the Gsd- phenotype is the result of an impairment in transcription initiation from the Ntr-regulated promoter, glnAp2 . Intragenic and extragenic secondary mutations appeared frequently in gltX351 cells, which suppressed their Gsd- but not their Ts phenotype . Moreover, in heterozygous gltX+/gltX351 partial diploids, gltX351 was dominant for the Gsd- phenotype and recessive for the Tr phenotype . A slight increase in the glutamine pool and in the intracellular glutamine: 2-oxoglutarate ratio was also observed but this could not account for the Gsd- phenotype of gltX351 cells . In cells carrying gltX351 and a suppressor of the Gsd- phenotype, sup-1, tightly linked to gltX351, the glutamine pool and glutamine: 2-oxoglutarate intracellular ratio were even higher than in the gltX351 single mutant . These results indicate that the gltX351 mutant polypeptide may be the direct cause of the Gsd- phenotype . The possibility that it interacts with one or more components that trigger the Ntr response is discussed.

Protein Expr Purif, 1993 Jun, 4(3), 187 - 99
Expression of polypeptides of human immunodeficiency virus-1 reverse transcriptase in Escherichia coli; Becerra SP et al.; We have prepared a plasmid, pRC-RT, for expression of HXB2 HIV-1 reverse transcriptase (RT) in Escherichia coli (Becerra et al., Biochemistry 30, 11707-11719, 1991) . Here we describe the optimization of RT overexpression and its purification . In pRC-RT, the precise RT coding region of HXB2 proviral DNA is flanked by start and stop codons, and expression is driven by the phage lambda pL promoter in a temperature-inducible system . The 64,484-Da RT polypeptide (termed p66) is expressed as approximately 10% of total cell protein after 2 h of induction, and the RT is readily solubilized and purified free of DNA Pol I and to near homogeneity as a homodimer of p66 or as a heterodimer of p66 and p51, resembling the natural enzyme . After achieving appropriate expression of the full-length p66 RT, we next created vectors to express multiple individual segments of the p66 polypeptide . These segments are: a 51,000-Da peptide, representing C-terminal truncation of p66, and several peptides representing consecutive N-terminal, central, and C-terminal segments of p66 . The latter peptide, corresponding to the RNase H domain of RT, has been purified in large quantities and is currently under study for solution of its structure by NMR . This peptide is devoid of enzyme activity and of substrate-binding capacity, but exists in solution as a folded globular protein with structure resembling that of E . coli ribonuclease H and that of a similar HIV-1 RT RNase H domain peptide examined by X-ray crystallography (Becerra et al., FEBS Lett . 270, 67-80, 1990) . Various other RT peptides described here should prove to be similarly useful for structural studies, as well as other approaches.

Lab Invest, 1993 Jun, 68(6), 629 - 36
New Ki-67-equivalent murine monoclonal antibodies (MIB 1-3) generated against bacterially expressed parts of the Ki-67 cDNA containing three 62 base pair repetitive elements encoding for the Ki-67 epitope; Key G et al.; BACKGROUND: The monoclonal antibody Ki-67 reacts with a human nuclear cell proliferation-associated antigen that is expressed in all cells that are not in G0 . Recently, we could demonstrate that Ki-67 detects a double band in Western blots of proliferating cells with apparent molecular weights of 345 kilodaltons and 395 kilodaltons, respectively . Furthermore, initial molecular biologic data favored the view that the epitope detected by Ki-67 might be encoded by a repetitive 66 bp element . EXPERIMENTAL DESIGN: In order to verify this assumption, parts of the Ki-67 cDNA were bacterially expressed, and the fusion proteins obtained were used to elicit new monoclonal antibodies . The specificities of the new reagents were tested by immunohistochemistry, Western blot, and enzyme-linked immunosorbent assay techniques . RESULTS: The somatic cell fusions revealed a number of antibodies with immunoreactivities comparable to Ki-67 . Three antibodies, designated MIB 1-3, were further characterized . Besides the fact that their immunostaining reactivity is identical with that of Ki-67, all new antibodies react in Western blots with native Ki-67 antigen . Furthermore, Western blot and competitive binding assays by enzyme-linked immunosorbent assay clearly demonstrate that MIB 1 and MIB 3, like the original Ki-67 antibody, react with an epitope that is encoded by the 66 bp repetitive element mentioned above . MIB 2, however, reacts with an epitope distinct from this latter structure . In addition, after antigen unmasking by microwave treatment, MIB 1 and MIB 3 detect the Ki-67 antigen in paraffin sections . CONCLUSIONS: Our results demonstrate that it is possible to use bacterially expressed parts of the Ki-67 antigen as immunogen to elicit antibodies that react with the native antigen . While MIB 1 and MIB 3 detect the same or a very similar epitope as the original antibody Ki-67, MIB 2 clearly differs in its fine specificity . Our results provide a circle of evidence that the cDNA sequence thus far determined encodes for the Ki-67 antigen . Furthermore, the new antibodies may become powerful tools for routine histopathology and for further functional characterization of the Ki-67 antigen.

Exp Parasitol, 1993 Jun, 76(4), 358 - 70
Schistosoma mansoni: comparison of cloned tropomyosin antigens shared between adult parasites and Biomphalaria glabrata; Weston DS et al.; Rabbit antisera, raised against whole, homogenized hepatopancreas from uninfected Biomphalaria glabrata, were used to screen a cDNA library prepared from adult Schistosoma mansoni . Of 1.8 x 10(5) recombinants screened, 34 clones were specifically immunoreactive with the antisera . Twenty of the clones were subcloned for further analysis, and 13 of the 20 were demonstrated to hybridize to a cDNA probe encoding schistosome tropomyosin I (pSMTM) at high stringency (Xu et al . Experimental Parasitology 69, 373-392, 1989) . One partial cDNA clone (pSM4), which failed to hybridize to pSMTM, was used to rescreen the adult worm cDNA library and a 1381-bp cDNA clone, SmTMII, was identified and characterized . SmTMII contained a single 284-amino acid open reading frame and was shown to be 65.8% homologous to SMTM . Computer-assisted analysis of the predicted protein structure depicted a hydrophilic molecule with the extensive alpha helical secondary structure characteristic of tropomyosins . SmTMII was expressed in Escherichia coli and the resulting 45-kDa fusion protein was recognized by both a polyclonal antiserum specific for SMTM and the antiserum of mice chronically infected with S . mansoni . Northern analysis showed the SmTMII mRNA in the adult stage to be about 1.6 kb in size . Analysis of the SmTMII gene by Southern blot revealed a complex hybridization pattern suggesting the presence of introns or multiple gene copies . The 650-bp EcoRI fragment of SmTMII was used to screen a cDNA library prepared from uninfected Biomphalaria glabrata and two clones, each encoding a novel snail tropomyosin, BgTMII, were isolated and characterized . The two clones, consisting of 2816 and 2314 bp, respectively, differed from each other only in the polyadenylation signal used and each contained a single and identical 284-amino acid open reading frame having 94.7% homology with the previously reported snail tropomyosin, BG39 (Dissous et al . Molecular and Biochemical Parasitology 43, 245-256, 1990) . BgTMII was shown to have a higher amino acid homology with SMTM (68.3%) than with BG39 (65.1%) and a higher amino acid homology with SMTM than the two schistosome tropomyosins (SMTM and SmTMII) have with each other (65.8%) . Implications of these observations in relation to the concept of host-parasite antigen mimicry are discussed.

J Bacteriol, 1993 Jun, 175(12), 3767 - 75
Methanol oxidation genes in the marine methanotroph Methylomonas sp . strain A4; Waechter-Brulla D et al.; Methanol dehydrogenase has been purified from the type I marine methanotroph Methylomonas sp . strain A4 and found to be similar to other methanol dehydrogenase enzymes in subunit composition, molecular mass, and N-terminal sequence of the two subunits . A heterologous gene probe and a homologous oligonucleotide have been used to identify a DNA fragment from Methylomonas sp . strain A4 which contains moxF, the gene encoding the large subunit of methanol dehydrogenase . Protein expression experiments with Escherichia coli, immunoblotting of expression extracts, and partial DNA sequence determination have confirmed the presence of moxF on this DNA fragment . In addition, expression and immunoblot experiments have shown the presence of the genes for the small subunit of methanol dehydrogenase (moxI) and for the methanol dehydrogenase-specific cytochrome c (moxG) . The moxG gene product has been shown to be cytochrome c552 . The expression experiments have also shown that two other genes are present on this DNA fragment, and our evidence suggests that these are the homologs of moxJ and moxR, whose functions are unknown . Our data suggest that the order of these genes in Methylomonas sp . strain A4 is moxFJGIR, the same as in the facultative methylotrophs . The transcriptional start site for moxF was mapped . The sequence 5' to the transcriptional start does not resemble other promoter sequences, including the putative moxF promoter sequence of facultative methylotrophs . These results suggest that although the order of these genes and the N-terminal amino acid sequence of MoxF and MoxI are conserved between distantly related methylotrophs, the promoters for this gene cluster differ substantially.

EMBO J, 1993 Jun, 12(6), 2483 - 94
The Escherichia coli FIS protein is not required for the activation of tyrT transcription on entry into exponential growth; Lazarus LR et al.; The Escherichia coli DNA bending protein factor for inversion stimulation (FIS), is neither necessary nor responsible for the stimulation of transcription from the wild type promoter for the tyrT operon (encoding a species of tyrosine tRNA) that occurs upon resumption of exponential growth . This conclusion is unexpected given that the regulatory element required for optimal transcription of tyrT contains three binding sites for FIS protein . In addition, it is in apparent conflict with reports from other laboratories which have described FIS-dependent activation of the stable RNA promoters rrnB P1 and thrU(tufB) in vivo . However, tyrT transcription is stimulated in a FIS-dependent manner both in vivo and in vitro when promoter function is impaired by mutation of the promoter itself or by the addition of the polymerase effector guanosine 5'-diphosphate 3'-diphosphate . These conditions, which expose a requirement for activation of stable RNA synthesis by FIS, suggest that FIS serves an adaptive role permitting high levels of stable RNA transcription on nutritional shift-up when RNA polymerase levels are depleted . In principle such a mechanism could confer a significant selective advantage thus accounting for the conservation of FIS binding sites in the regulatory regions of stable RNA promoters.

Proc Natl Acad Sci U S A, 1993 Jun 1, 90(11), 4902 - 6
Kinetics of p56lck and p60src Src homology 2 domain binding to tyrosine-phosphorylated peptides determined by a competition assay or surface plasmon resonance; Payne G et al.; Src homology 2 (SH2) domains are phosphotyrosine-binding modules found within various signal-transducing proteins . We have determined by 125I competition assay and surface plasmon resonance that the SH2 domains of Src and Lck bind to a variety of phosphopeptides with similar affinity and specificity . Both bound with highest affinity {Kd(app) approximately 3.7 nM; ka = 2.4 x 10(5) M-1 x s-1; kd = 1.2 x 10(-3) s-1} a phosphopeptide having a Tyr(P)-Glu-Glu-Ile motif found in the hamster polyomavirus middle-sized tumor antigen . Intermediate affinity (5- to 40-fold lower) was observed with phosphopeptides corresponding to the regulatory domains of Src and Lck, containing Tyr527 and Tyr505, respectively . Lowest affinity (80- to 300-fold lower) was observed with phosphopeptides corresponding to phosphorylated tyrosines of GTPase-activating protein, insulin receptor substrate 1, and SH2 domain-containing protein-tyrosine-phosphatase 1.

Mutat Res, 1993 Jun, 291(3), 181 - 92
A plasmid system to monitor gene conversion and reciprocal recombination in vitro; Oppliger T et al.; A plasmid system allowing for the detection of recombinagenic activities in cell-free extracts is described . Two truncated alleles of the bacterial neomycin resistance gene (neo), differing from each other at a polymorphic restriction site, were constructed . Recombinations involving both alleles mediated by Drosophila embryo nuclear protein extracts or Drosophila larva whole cell protein extracts were selected by their ability to confer kanamycin resistance to E . coli . Restriction analysis of plasmids recovered from E . coli transformants allowed the monitoring of the two molecular mechanisms which can lead to functional neo genes, gene conversion and reciprocal recombination . A dose dependent increase in the recombination frequency with increasing amounts of cell extract was observed . Recombination was further increased by linearizing one of the two substrate plasmids . The Drosophila cell extracts catalyzed recombination in vitro since after incubation a recombination product could be identified by polymerase chain reaction (PCR) technology . The recombination was absolutely dependent on the presence of an active cell extract, since no diagnostic PCR product was detected in a reaction where extract was omitted . Analysis of a representative number of recombinant plasmids by restriction analysis revealed that in the absence of an exogenous recombinational system less than 2% of kanamycin resistant recombinant plasmids occurred by gene conversion upon transformation into E . coli . In contrast, recombinants exhibiting restriction patterns diagnostic for gene conversion were observed at frequencies between 5.1% and 9.8% after incubation with Drosophila larva cell extracts . These results strongly argued that gene conversion is a prominent mechanism of recombination in Drosophila mitotic cells.

Biochemistry, 1993 Jun 1, 32(21), 5508 - 17
A large deletion in the connection subdomain of murine leukemia virus reverse transcriptase or replacement of the RNase H domain with Escherichia coli RNase H results in altered polymerase and RNase H activities; Post K et al.; The functional relationship between the polymerase and RNase H domains of reverse transcriptase (RT) was investigated by studying the activities of AKR murine leukemia virus (MuLV) enzymes . In addition to the wild type, an RNase H-minus RT missing the entire RNase H domain and two other mutants having abnormal polymerase:RNase H ratios were expressed . These mutants include (i) a chimeric protein in which the MuLV RNase H domain was replaced by the entire Escherichia coli RNase H sequence and (ii) an RT with a 126 amino acid deletion in a region analogous to the "connection" subdomain in the p66 subunit of human immunodeficiency virus type 1 RT (Kohlstaedt, L . A., Wang, J., Friedman, J . M., Rice, P . A., & Steitz, T . A . (1992) Science 256, 1783-1790) . With the wild-type RT, the major RNase H cleavage reaction was coordinated with DNA synthesis and occurred at a position corresponding to 15 nucleotides from the 3'-terminus of the DNA primer . Additional cleavages closer to the 5'-end of the RNA were explained in terms of a model relating binding of the RNA.DNA hybrid substrate and enzyme structure . The chimeric RT behaved like E . coli RNase H, exhibited 300-fold higher RNase H activity than wild-type RT, and was limited in its ability to synthesize DNA . Qualitative and quantitative changes in the polymerase and RNase H activities of the deletion mutant were also observed . The RNase H domain appeared to function independently of the polymerase domain, supporting the idea that the proper spatial relationship between the two active centers was disrupted by the mutation . Taken together, our results indicate that alteration of the normal polymerase:RNase H ratio can have profound effects on both polymerase and RNase H cleavage activities, as expected for an enzyme with two interdependent domains.

Oncogene, 1993 Jun, 8(6), 1477 - 85
Identification of a human guanine nucleotide-releasing factor (H-GRF55) specific for Ras proteins; Schweighoffer F et al.; A critical step in the activation of cellular Ras is the release of bound GDP . Oligonucleotide primers derived from a mouse cDNA sequence homologous to the Saccharomyces cerevisiae CDC25 gene product were used to screen a human brain cDNA library . The cloning led to the isolation of a 2.8-kb cDNA predicted to encode a protein of 488 amino acids . This protein was produced in Escherichia coli as a glutathione S-transferase fusion protein and functioned in vitro as a specific guanine nucleotide-releasing factor . Polyclonal antibodies raised against the last 281 amino acids of the protein allowed a protein in the molecular weight range of 55 kDa to be identified in human cortex homogenates . Analysis by Northern blotting led to the identification of a 5.5-kb mRNA in brain poly(A)+ RNA . The functionality of the encoded protein was evaluated after expression in different cells: (i) in Saccharomyces cerevisiae the effects of the cdc25.5 and RAS2 Ala-22 mutations were reversed; (ii) in chinese hamster ovary cells, a RAS-responsive element was transactivated as demonstrated by the expression of a CAT reporter gene under the control of the polyomavirus enhancer . Finally, in situ hybridization on of human chromosomes revealed a localization on band 15q2.4.

J Virol, 1993 Jun, 67(6), 3696 - 701
Hybrid hepatitis B virus nucleocapsid bearing an immunodominant region from hepatitis B virus surface antigen; Borisova G et al.; A hepatitis B core antigen (HBcAg) gene bearing the 39-amino-acid-long domain A of hepatitis B surface antigen (HBsAg) within the HBcAg immunodominant loop has been constructed and expressed in Escherichia coli . Chimeric capsids demonstrated HBs but not HBc antigenicity and elicited in mice B-cell and T-cell responses against native HBcAg and HBsAg.

J Virol, 1993 Jun, 67(6), 3489 - 96
Mutational analysis of the envelope gene of Moloney murine leukemia virus; Gray KD et al.; The env gene products of Moloney murine leukemia virus are required for binding and entry of the virus into the target cell . Thirty-three linker insertion mutations were constructed throughout the env gene of Moloney murine leukemia virus . Twenty of the mutations were located in the surface protein (SU), and the remaining thirteen were located in the transmembrane protein (TM) . The viability of the viruses containing these env gene mutations was determined by performing transient transfections and screening for the release of reverse transcriptase . Eleven viable mutants were isolated, nine in SU and two in TM . Three of the viable mutants were temperature sensitive . Four of the viable mutants were clustered in the carboxy terminus of SU . The env gene products of transfected cell lines which produced viable virus were analyzed . Our results indicated two regions of SU important for the stability of the SU/TM heteropolymer and one region important for the interaction of the env gene products with the viral core.

Mutat Res, 1993 Jun, 294(1), 43 - 50
Enzymatic recognition and biological effects of DNA damage induced by 3-carbethoxypsoralen plus UVA; Boiteux S et al.; The specific recognition of DNA modifications by repair endonucleases was used to characterize damage induced by 3-carbethoxypsoralen (3-CPs) plus UvA in M13mp8 replicative form I (RF-I) DNA . Under the conditions used, 3-CPs plus UVA generates DNA base modifications which are recognized by the UvrABC complex and the Fpg protein of E . coli . The rate of formation of UvrABC sensitive sites is 3-4-fold higher than that of Fpg sensitive sites . In addition a small number of sites of base loss (sensitive to Nfo protein) were observed . M13mp8 RF-I DNA treated with 3-CPs plus UVA was tested for transfection efficiency in E . coli mutants defective in either Fpg protein and/or UvrABC complex . The survival of 3-CPs plus UVA damaged M13mp8 RF-I DNA was significantly reduced when transfected into uvrA mutants compared to that in the wild-type strain . On the other hand, the survival of 3-CPs plus UVA damaged RF-I DNA was not altered in fpg-1 mutants . These results show that nucleotide excision repair mediated by the UvrABC complex is the major repair pathway involved in the elimination of lethal lesions induced in DNA by 3-CPs plus UVA . Our data suggest that in vitro exposure of M13mp8 RF-I DNA to 3-CPs plus UVA produces predominantly thymine photoaddition and to a lesser extent guanine photooxidation partially due to singlet oxygen generated during photoreaction . The photoaddition products are primarly responsible for the observed lethal effect.

Mutat Res, 1993 Jun, 294(1), 1 - 8
The level of GC-->AT transitions induced by MMS is not affected by the adaptive response in Escherichia coli K12; Sledziewska-Gojska E; I have examined the effect of the adaptive response on the frequency of MMS-induced umuC-dependent AT-->TA transversions and umuC-independent, GC-->AT transitions . It was found that the induction of the adaptive response causes a moderate decrease (50-60%) in the frequency of AT-->TA transversions and surprisingly has no effect on the level of GC-->AT transitions . In contrast, a dramatic decrease in MNNG-induced mutations has been observed in adapted cultures . However, this effect was completely abolished by MMS pretreatment of adapted cells before MNNG challenge . A mechanism for the MMS interference with the repair of MNNG-induced mutations in adapted cells is proposed.

Eur J Neurosci, 1993 Jun 1, 5(6), 624 - 32
The proximal region of the MBP gene promoter is sufficient to induce oligodendroglial-specific expression in transgenic mice; Goujet-Zalc C et al.; To characterize regulatory DNA sequences involved in oligodendroglial expression of myelin basic protein (MBP), transgenic mice carrying a 256 bp fragment of the mouse MBP promoter fused to an Escherichia coli lacZ gene were generated . Of four transgenic families, two (lines 2 and 4) expressed beta-galactosidase activity in the nervous system but not in most other tissues . Histochemical and immunohistochemical analysis of adult brain from these two lines showed oligodendroglial-specific expression of the transgene . In line 2, only a small proportion of oligodendrocytes expressed the transgene, and in labelled cells the product of the enzymatic reaction with beta-galactosidase was confined to a small round vesicle in the vicinity of the nucleus . In contrast, in tissue sections from line 4 adult brain and spinal cord beta-galactosidase activity was much more intense and at least 80-90% of oligodendrocytes expressed the transgene . Detection of the MBP-lacZ transcript by in situ hybridization showed that the transgene mRNA was confined to the oligodendrocyte cell body . These results suggest that cis-acting regulatory elements, specifying oligodendrocytes identity, are located within 256 bp upstream from the MBP gene.

Braz J Med Biol Res, 1993 Jun, 26(6), 591 - 603
Expression of the VP3-VP1 sequence of foot-and-mouth disease virus in Escherichia coli; Rossetti ML et al.; 1 . cDNA recombinants containing the VP3 and VP1 sequences of foot-and-mouth disease virus were isolated and the VP3-VP1 sequence was reconstructed . 2 . The reconstructed VP3-VP1 sequence was subcloned into expression vector pEX31b and a fusion protein of about 62,000 Da was expressed . 3 . When injected into mice, the fusion protein was able to elicit the production of antibodies that recognized viral VP1 and VP3 . 4 . Antibodies present in sera from mice immunized with VP3-VP1 protein did not neutralize the foot-and-mouth disease virus in vitro.

Gene, 1993 May 30, 127(2), 215 - 9
Cloning and expression of the gene encoding Acacia confusa trypsin inhibitor that is active without post-translational proteolysis; Hung CH et al.; A recombinant plasmid containing the coding regions for Acacia confusa trypsin inhibitor (ACTI) has been constructed and expressed in Escherichia coli cells, as a fusion protein between ACTI and glutathione S-transferase (GST) . The GST-fusion was produced as a soluble protein which did not require denaturing agents such as urea to solubilize it . The recombinant ACTI (reACTI) was obtained by treating the GST-fusion protein with thrombin . Both the reACTI and fusion protein have a strong inhibitory effect on trypsin activity without post-translational proteolysis.

Gene, 1993 May 30, 127(2), 193 - 7
Functional starch-binding domain of Aspergillus glucoamylase I in Escherichia coli; Kusnadi AR et al.; We have fused three starch-binding domains (SBD) encoding gene fragments (residues 511-616, 495-616 and 481-616) of glucoamylase I (GAI) to the 3' end of the Escherichia coli malE gene encoding maltose-binding protein (MBP) . The fusion proteins were produced in E . coli and were purified by chromatography on cross-linked amylose . Factor Xa digestion of the fusion proteins resulted in the release of functional SBD fragments which were separated from MBP on the basis of their differential binding to cross-linked amylose . The amino acid (aa) composition of the purified SBD fragments agreed with the respective amino acid compositions of GAI . The sizes of the SBD fragments were 11.9, 13.8 and 15.6 kDa, respectively.

Gene, 1993 May 30, 127(2), 221 - 5
Nucleotide sequence and expression in Escherichia coli of cDNAs encoding papaya proteinase omega from Carica papaya; Revell DF et al.; We have cloned and sequenced two similar, but distinct, cDNAs from both fruit and leaf tissues of Carica papaya . The C-terminal portion of the predicted amino acid (aa) sequence of one of the clones has complete identity with the mature enzyme sequence of the cysteine proteinase papaya proteinase omega (Pp omega) . The second clone contains ten individual bp changes compared with the first and encodes a protein with three single-aa substitutions, only one of which is located in the mature sequence, but most noticeably carries an additional 19-aa C-terminal extension . The clones encode pre-pro precursor isoforms of Pp omega . The former of these clones has been expressed in Escherichia coli using a T7 polymerase expression system to produce insoluble pro-enzyme which has been solubilized and refolded to yield auto-activable pro-Pp omega.

Biochem Biophys Res Commun, 1993 May 28, 193(1), 453 - 9
Binding activity of the human transcription factor TFIID; Icard-Liepkalns C; In order to investigate the conformational state of the human TFIID, we studied the structure of the TATA-box binding protein (TBP) which is the DNA-binding subunit of the transcription factor TFIID required for transcriptional initiation by RNA polymerase II . We showed that TBP was able to form dimers and tetramers by chemical crosslinking, subunit exchange, ultracentrifugation and gel shift experiment . These findings indicate that the TBP homodimers could be the inactive binding form of TFIID and therefore could explain the lack of Gal4-activated transcriptional activity of the E . coli-expressed human TBP.

Biochem Biophys Res Commun, 1993 May 28, 193(1), 228 - 34
Sec-Y protein is localized in both the cytoplasmic and thylakoid membranes in the cyanobacterium Synechococcus PCC7942; Nakai M et al.; Members of the SecY protein family mediate protein export in bacterial cells . Southern analyses showed that secY is likely a single copy gene in the cyanobacterium Synechococcus PCC7942 . Then the subcellular location of the cyanobacterial SecY protein was determined; i) antiserum raised against a fusion protein between the SecY fragment and maltose binding protein were used for immunoblotting of the membrane fractions, and ii) a modified SecY protein carrying the c-Myc peptide tag was expressed in the cyanobacterial cells, and the subcellular distribution of the SecY-c-Myc fusion protein was analyzed with the anti-c-Myc antibodies . The obtained results suggest that the SecY protein is localized in the thylakoid membrane as well as the cytoplasmic membrane; the SecY protein probably mediates protein translocation across both the cytoplasmic and thylakoid membranes in Synechococcus PCC7942.

Biochem Biophys Res Commun, 1993 May 28, 193(1), 155 - 60
Expression of bovine seminal ribonuclease in Escherichia coli; de Nigris M et al.; Bovine seminal RNAase (BS-RNAase), an unusually dimeric member of the pancreatic-like ribonuclease superfamily, is also a multifunctional biological effector, with antitumor, immunosuppressive, and antispermatogenic activities . We report here the cloning of a semi-synthetic cDNA coding for the protein subunit chain, its expression with a T7 expression system in Escherichia coli inclusion bodies, the dimerization of correctly reoxidized monomeric protein, followed by the purification in high yields of the recombinant enzyme, and by its conversion to a protein undistinguishable from BS-RNAase as isolated from seminal vesicles, both in its catalytic activity and in the micro-heterogeneity of its quaternary structure.

Brain Res, 1993 May 28, 612(1-2), 165 - 71
Interleukin-6 and tumor necrosis factor in cerebrospinal fluid: changes during pyrogen fever; Coceani F et al.; Several peptides (cytokines), viz., interleukin-1 (IL-1), interferon-alpha (IFN-alpha), interleukin-6 (IL-6), tumor necrosis factor (TNF), are formed in response to conditions causing tissue inflammation or damage and are implicated in reactive changes of the host, including fever, while IL-1 has been considered an important mediator of fever, the other cytokines, specifically IL-6 and TNF, have recently acquired prominence . The present study extends earlier research on IL-1 and addresses the question of the role of IL-6 and TNF in the genesis of fever . Experiments were conducted in the conscious cat, and IL-6 and TNF were assayed concomitantly in cerebrospinal fluid (CSF) from the third ventricle using specific bioassays . In the absence of fever, IL-6 was usually below the threshold of the assay (4-32 pg/ml), while TNF appeared measurable (424 +/- 57 pg/ml) in most experiments . A single intravenous injection of endotoxin (bolus) or continuous infusion of IL-1 at doses eliciting a sustained fever increased CSF levels of IL-6, but had no effect on concentrations of TNF . Intracerebroventricular injection of a pyrogenic dose of endotoxin led to an elevation of TNF and IL-6 and, in either case, the effect was manifest during the latent period before the fever . In addition, by the same route, IL-1 caused a rise in IL-6 . We conclude that brain is intrinsically capable of producing both IL-6 and TNF depending on the site of challenge . However, since IL-6 CSF levels are elevated regardless of the site of pyrogen injection, IL-6 lends itself better to a role in the pathogenesis of fever.

Nucleic Acids Res, 1993 May 25, 21(10), 2459 - 64
The DNA binding affinity of HhaI methylase is increased by a single amino acid substitution in the catalytic center; Mi S et al.; The HhaI methyltransferase recognizes the sequence GCGC and transfers a methyl group to C5 of the first cytosine residue . All m5C-methyltransferases contain a highly conserved sequence motif called the P-C motif . The cysteine residue of this motif is involved in catalysis by forming a covalent bond with the 6-position of cytosine prior to methyl group transfer . For the EcoRII methyltransferase, it has been shown that substitution of this catalytic cysteine by glycine is cytotoxic to E.coli cells expressing the mutant methyltransferase (Wyszynski et al . Nucl . Acids Res . 20: 319, 1992) . We now show that this observation can be extended to the HhaI system and suggest that the cytotoxicity is due to abnormally tight DNA binding by the mutant methyltransferase, which probably interferes with replication or transcription.

Nucleic Acids Res, 1993 May 25, 21(10), 2423 - 7
African swine fever virus encodes two genes which share significant homology with the two largest subunits of DNA-dependent RNA polymerases; Yanez RJ et al.; A random sequencing strategy applied to two large SalI restriction fragments (SB and SD) of the African swine fever virus (ASFV) genome revealed that they might encode proteins similar to the two largest RNA polymerase subunits of eukaryotes, poxviruses and Escherichia coli . After further mapping by dot-blot hybridization, two large open reading frames (ORFs) were completely sequenced . The first ORF (NP1450L) encodes a protein of 1450 amino acids with extensive similarity to the largest subunit of RNA polymerases . The second one (EP1242L) codes for a protein of 1242 amino acids similar to the second largest RNA polymerase subunit . Proteins NP1450L and EP1242L are more similar to the corresponding subunits of eukaryotic RNA polymerase II than to those of vaccinia virus, the prototype poxvirus, which shares many functional characteristics with ASFV . ORFs NP1450L and EP1242L are mainly expressed late in ASFV infection, after the onset of DNA replication.

Nucleic Acids Res, 1993 May 25, 21(10), 2363 - 7
DNA sequence determinants of LexA-induced DNA bending; Lloubes R et al.; The LexA repressor from Escherichia coli induces DNA bending upon interaction with the two overlapping operators which regulate the transcription of the colicin A encoding gene caa . Both caa operators harbor T-tracts adjacent to their recognition motifs . These tracts have been suggested to be especially favorable for the promotion of LexA-induced DNA bending . Here we show that this is indeed the case, since disruption of the TTTT-tract adjacent to operator O1 by the replacement of the two central thymine bases by AA, GA or CG markedly reduces LexA-induced DNA bending . Simple A.T-richness in this position is thus not sufficient to promote full LexA-induced bending, albeit a TAAT sequence is always more efficient to promote bending than those sequences containing one or two C/G base pairs.

Nucleic Acids Res, 1993 May 25, 21(10), 2309 - 13
Gene structure and expression of the MboI restriction--modification system; Ueno T et al.; The genes from Moraxella bovis encoding the MboI restriction--modification system were cloned and expressed in Escherichia coli . Three open reading frames were found in the sequence containing the genes . These genes, which we named mboA, mboB, and mboC, had the same orientation in the genome . Genes mboA and mboC encoded MboI methyltransferases (named M.MboA and M.MboC) with 294 and 273 amino acid residues, respectively . The mboB gene coded for MboI restriction endonuclease (R.MboI) with 280 amino acid residues . Recombinant E.coli-MBOI, which contained the whole MboI system, overproduced R.MboI . R.MboI activity from E.coli-MBOI was 480-fold that of M.bovis . The amino acid sequences deduced from these genes were compared with those of other restriction--modification systems . The protein sequences of the MboI system had 38-49% homology with those of the DpnII system.

Biochemistry, 1993 May 25, 32(20), 5455 - 65
Reversible electrochemistry of fumarate reductase immobilized on an electrode surface . Direct voltammetric observations of redox centers and their participation in rapid catalytic electron transport; Sucheta A et al.; Fumarate reductase (Escherichia coli) can be immobilized in an extremely electroactive state at an electrode, with retention of native catalytic properties . The membrane-extrinsic FrdAB component adsorbs to monolayer coverage at edge-oriented pyrolytic graphite and catalyzes reduction of fumarate or oxidation of succinate, depending upon the electrode potential . In the absence of substrates, reversible redox transformations of centers in the enzyme are observed by cyclic voltammetry . The major component of the voltammograms is a pair of narrow reduction and oxidation signals corresponding to a pH-sensitive couple with formal reduction potential E degree' = -48 mV vs SHE at pH 7.0 (25 degrees C) . This is assigned to two-electron reduction and oxidation of the active-site FAD . A redox couple with E degree' = -311 mV at pH 7 is assigned to center 2 ({4Fe-4S}2+/1+) . Voltammograms for fumarate reduction at 25 degrees C, measured with a rotating-disk electrode, show high catalytic activity without the low-potential switch-off that is observed for the related enzyme succinate dehydrogenase . The catalytic electrochemistry is interpreted in terms of a basic model incorporating mass transport of substrate, interfacial electron transfer, and intrinsic kinetic properties of the enzyme, each of these becoming a rate-determining factor under certain conditions . Electrochemical reversibility is approached under conditions of low turnover rate, for example, as the supply of substrate to the active site is limited . In this situation, electrocatalytic half-wave potentials, E1/2, are similar for oxidation of bulk succinate and reduction of bulk fumarate and coincide closely with the E degree' value assigned to the FAD . At 25 degrees C and pH 7, the apparent KM for fumarate reduction is 0.16 mM, and kcat is 840 s-1 . Accordingly the second-order rate constant, kcat/KM, is 5.3 x 10(6) M-1 s-1 . Under the same conditions, oxidation of succinate is much slower . As the supply of fumarate to the enzyme is raised to increase turnover, the electrochemical reaction eventually becomes limited by the rate of electron transfer from the electrode . Under these conditions a second catalytic wave becomes evident, the E1/2 value of which corresponds to the reduction potential of the redox couple suggested to be center 2 . This small boost to the catalytic current indicates that the low-potential {4Fe-4S} cluster can function as a second center for relaying electrons to the FAD.

Biochemistry, 1993 May 25, 32(20), 5442 - 7
Flow-flash study of the reaction between cytochrome bo and oxygen; Svensson M et al.; The reaction between reduced cytochrome bo from Escherichia coli and oxygen has been studied using flash photolysis of the CO complex of the reduced protein after rapid mixing with oxygen . Absorbance changes were monitored in the alpha and Soret spectral regions . Two kinetic phases taking place at catalytically competent rates could be detected . The apparent rate constant obtained for both the first and second phase showed a hyperbolic dependence on the oxygen concentration . For the first phase, we obtained limiting first- and second-order rate constants at saturating and low oxygen concentrations of 4.5 x 10(4) s-1 and 1.6 x 10(8) M-1 s-1, respectively . The corresponding values for the second phase were 5 x 10(3) s-1 and 1.7 x 10(7) M-1 s-1 . The first phase accounted for 30% of the total absorbance change in the Soret band (430 nm) and 15% of the total absorbance change in the alpha band (555 nm) . These reactions are followed by a very slow phase with a lifetime of about 1 s . We have also studied the interaction between the fully oxidized enzyme and hydrogen peroxide, and we have found that peroxide binding induces an absorbance increase in the alpha band and a red shift of the Soret band . A consideration of the magnitude of the absorbance changes taking place during the first phase suggests that this reaction includes at least partial oxidation of the low-spin cytochrome b.

Biochemistry, 1993 May 25, 32(20), 5419 - 24
Exchange, efflux, and substrate binding by cysteine mutants of the lactose permease of Escherichia coli; van Iwaarden PR et al.; In this study, wild-type lac permease and lac permease mutated at each of the eight cysteinyl residues in the molecule were solubilized from the membrane, purified, and reconstituted into proteoliposomes . Lactose equilibrium exchange and efflux activities of mutants with Ser in place of Cys117, Cys176, Cys234, Cys333, Cys353, or Cys355 are essentially the same as wild-type permease . In contrast, mutants in Cys148 and Cys154 exhibit diminished exchange and efflux activities . These mutants in Cys148 and Cys154, except for the C148S mutant, have previously been shown to slow down active transport as well {Van Iwaarden, P.R., Driessen, A . J . M., Menick, D . R., Kaback, H.R., & Konings, W . N . (1991) J . Biol . Chem . 266, 15688-15692} . C148S permease shows monophasic kinetics with a high apparent KM with respect to external lactose in the exchange reaction under nonequilibrium conditions, whereas wild-type permease exhibits biphasic kinetics with both a high and low KM component . Moreover, the absence of the low Km pathway in the C148S permease is correlated with the absence of a high-affinity binding site for p-nitrophenyl alpha-D-galactopyranoside (NPG) . Interestingly, the affinity of the permease for NPG appears to increase with the hydrophobicity of the side chain at position 154 (Ser < Cys < Gly < Val) . Finally, the presence of a high-affinity binding site for NPG in C154V is consistent with the biphasic exchange kinetics exhibited by this mutant . The results are discussed in the context of a model in which lac permease has two substrate binding sites, a catalytic site and a regulatory site.

Biochemistry, 1993 May 25, 32(20), 5282 - 90
Structure and stability of an early folding intermediate of Escherichia coli trp aporepressor measured by far-UV stopped-flow circular dichroism and 8-anilino-1-naphthalene sulfonate binding; Mann CJ et al.; The refolding kinetics of Escherichia coli trp aporepressor were monitored using stopped-flow far-ultraviolet circular dichroism and 8-anilino-1-naphthalene sulfonate fluorescence spectroscopy . Significant gains in secondary structure and the development of hydrophobic surface, respectively, were observed within the dead time of mixing (4-5 ms) . These initial increases, or burst phase amplitudes, plotted as a function of final urea concentration, exhibited sigmoidal, coincident unfolding transition curves . The transition curves were fit to a two-state model, and the resulting free energies of folding in the absence of denaturant were found to be similar (approximately 3.3 kcal/mol) . Three subsequent slow refolding phases exhibited relaxation times and amplitudes similar to those previously observed for tryptophan fluorescence {Gittelman, M . S., & Matthews, C . R . (1990) Biochemistry 29, 7011-7021} . These results support the proposals that a stable, monomeric intermediate is rapidly formed during the folding of trp aporepressor and that this species contains a significant amount of secondary structure and hydrophobic surface . This early intermediate is then processed through folding and association reactions that result in the formation of the remaining secondary, tertiary, and quaternary structure.

Biochemistry, 1993 May 25, 32(20), 5273 - 81
Multiple magnesium ions in the ribonuclease P reaction mechanism; Smith D et al.; The ribozyme ribonuclease (RNase) P cleaves precursor transcripts to produce the mature 5'-end of tRNAs . This hydrolysis reaction has a divalent cation requirement that is primarily catalytic, rather than structural; RNase P can be considered a metalloenzyme . Kinetic analysis shows that the RNase P catalytic mechanism has a cooperative dependence upon Mg2+ concentration . At least three Mg2+ ions are required for optimal activity, suggesting a multiple metal ion mechanism . The 2'-OH at the site of substrate cleavage may act as a ligand for a catalytically important Mg2+: deoxyribose substitution reduces the apparent number of Mg2+ bound from three to two and increases the apparent dissociation constant for Mg2+ from the micromolar to the millimolar range . In addition to these cation effects, the deoxyribose substitution reduces the rate of catalysis by 3400-fold; substitution with 2'-O-methyl at the cleavage site reduces the catalytic rate 10(6)-fold . If we presume no significant conformational effects of the substitutions, these results suggest that the 2'-OH serves as hydrogen-bond donor . The kinetic analysis of the catalytic mechanism is based upon the characterization of the pH dependence of the reaction . There is a hyperbolic (saturable) dependence on hydroxide concentration, with the half-maximal rate achieved at pH 8.0-8.5 . The rate of the cleavage step is about 200 min-1 at pH 8.0, which is 500-fold faster than the steady-state parameter kcat.

J Biol Chem, 1993 May 25, 268(15), 11143 - 51
Preparation and characterization of a set of deoxyoligonucleotide 49-mers containing site-specific cis-syn, trans-syn-I, (6-4), and Dewar photoproducts of thymidylyl(3'-->5')-thymidine; Smith CA et al.; Deoxyoligonucleotide 49-mers containing a central cis-syn, trans-syn-I, (6-4), or Dewar photoproduct of TpT were constructed for use in repair and replication studies by ligation of shorter photoproduct-containing oligonucleotides . A (6-4) product-containing 6-mer was prepared in 3.4% yield by 254 nm irradiation of d(AATTAA) and converted in nearly quantitative yield to the Dewar isomer by irradiation with Pyrex- and Mylar-filtered medium-pressure mercury arc light . d(CGAATTAAGC) containing a site-specific cis-syn or trans-syn-I dimer was prepared via automated solid-phase DNA synthesis utilizing photoproduct building blocks . The photoproduct-containing 49-mers were characterized by their susceptibility to base cleavage and a number of enzymes routinely used to map the sites of DNA photoproduct formation . 1 M piperidine at 90 degrees C cleaved the Dewar product faster than the (6-4) product, but did not cleave the cyclobutane dimers . The 3'-->5' exonuclease activity of T4 DNA polymerase was completely blocked by all the lesions except the (6-4) product, which it slowly bypassed . T4 endonuclease V did not cleave the (6-4) or Dewar photoproduct, but unexpectedly cleaved the trans-syn-I dimer at most 1% the rate of the cis-syn dimer in double-stranded DNA . The trans-syn-I dimer was cleaved at a 50-fold higher rate in double- than in single-stranded DNA . Escherichia coli photolyase was found to be specific for the cis-syn dimer at low concentrations . Implications of this work to methodology for mapping and quantifying DNA photoproducts are also discussed.

J Biol Chem, 1993 May 25, 268(15), 11028 - 40
Mutational analysis of the human DNA polymerase alpha . The most conserved region in alpha-like DNA polymerases is involved in metal-specific catalysis; Copeland WC et al.; Five site-directed mutations were introduced at the most conserved amino acids in region I (YGDTDS) of the human DNA polymerase alpha catalytic subunit . Mutant proteins were expressed in the baculovirus system by an improved method and purified by a rapid one-step purification in high yield and high specific activity . The Asp1004 to Asn mutation produced a protein with no detectable polymerase activity while other mutations gave activities from 1 to 20% of the wild type polymerase activity . Steady state kinetic analysis of the active mutants indicates that none of the mutations caused a change in Km(dNTP) or KD(DNA), but all active mutants showed a decrease in kcat . Thus, the effect of these conserved mutations is manifest in altered rates of catalysis . Two mutations, Asp1002 to Asn and Thr1003 to Ser, caused the enzyme to utilize Mn2+ more effectively in catalysis than Mg2+, suggesting that these amino acids are involved in metal binding . Rates of catalysis by the D1002N and T1003S mutants, as well as Y1000F mutant were improved 80-, 30-, and 70-fold, respectively, on homopolymer templates when Mn2+ replaced Mg2+ as the activator metal . The results from these mutational studies suggest that this highly conserved region binds the metal which is essential for catalysis . The Asp1002 may participate directly in chelating the metal . Results from the T1003S mutant suggest that the beta-methyl group of the threonine side chain might be locked in a hydrophobic pocket preventing free rotation around the C alpha-C beta bond, thus positioning the Thr1003 hydroxyl group to form a crucial bond with the metal ion . In addition, D1002N and T1003S displayed a 20-fold resistance to aphidicolin compared to the wild type polymerase alpha, and all of the active mutants displayed altered sensitivity to butylphenyl-dGTP . Models of the involvement of region I in catalysis and aphidicolin interaction are proposed . The mutational studies presented in this report will serve as a prototype for the functional role of region I in catalysis for all alpha-like DNA polymerases.

J Biol Chem, 1993 May 25, 268(15), 10914 - 9
Interaction of GTPase activating proteins (GAPs) with p21ras measured by a novel fluorescence anisotropy method . Essential role of Arg-903 of GAP in activation of GTP hydrolysis on p21ras; Brownbridge GG et al.; Ras GTPase activating proteins (GAPs) contain an invariant motif, -FLR-, within the most conserved region of their catalytic domains . Certain mutations in this motif have greatly reduced activity (Skinner, R . H., Bradley, S., Brown, A . L., Johnson, N . J., Rhodes, S., Stammers, D . K., and Lowe, P . N . (1991) J . Biol . Chem . 266, 14163-14166), but it was not determined whether the reduced activity was due to loss of binding or impaired catalysis . In order to address this question, we have developed a simple physical method to study formation of GAP.p21ras complexes . This utilizes the increase of fluorescence anisotropy upon binding of GAP to p21ras complexed with 2'(3')-O-(N-methylanthraniloyl) (mant) derivatives of guanine nucleotides . Dissociation constants obtained for the catalytic domains of either p120-GAP (GAP-344) or neurofibromin (NF1-GRD) with normal and Leu-61 p21ras proteins are comparable with those obtained by kinetic methods . In the course of these studies, we found, in contrast to previous observations, that both GAP and NF1-GRD can weakly activate the GTPase of Leu-61 mutant p21, showing that Gln-61 is not absolutely required for the stimulation of GTPase activity by GAPs . The fluorescence anisotropy method allowed us to show that mutation of Arg-903, within the FLR motif of GAP, can result in protein defective in catalysis but not in binding to p21ras . These data suggest a direct role for this residue in catalyzing GTP hydrolysis on p21ras, possibly by contributing a catalytic group to the p21 active site . This method is independent of the catalytic activity of the proteins, and so it could be extended generally to the measurement of binding of effector molecules, exchange factors, or other macromolecules to guanine nucleotide-binding proteins.

J Biol Chem, 1993 May 25, 268(15), 10826 - 35
Differential usage of the carboxyl-terminal region among aldolase isozymes; Berthiaume L et al.; Sequence homology among nonconserved residues 357-362 of the COOH-terminal region in fructose-1,6-bisphosphate aldolases correlates with isozyme classification of aldolases . Recombinant chimers of human liver and maize aldolases were constructed by exchanging residues 357-362 with those from muscle, maize, and liver isozyme and by insertion in the maize sequence at position 349 rabbit muscle and liver residues 346-349 . Activity variation among the chimers relative to native controls ranged from less than 10% to greater than 300% of Vm . Exchange of residues 357-362 significantly affected both Vm and Km without modifying catalytic efficiency kcat/Km, whereas insertion of residues 346-349 modified Vm and Km and increased catalytic efficiency . Steady state carbanion oxidation rates varied inversely with activity and were differentially affected with respect to equilibrium oxidation rates . Sequence exchange of residues 357-362 appears to modulate carbanion proton exchange, whereas sequence insertion of residues 346-349 modifies substrate and aldehyde interaction with C6 phosphate binding locus . Low intrinsic susceptibility to carboxypeptidase A degradation of the COOH terminus in liver aldolase is consistent with tight association of this COOH terminus in a conformation unfavorable for promoting high catalytic activity . Efficient carbanion protonation promoted by specific sequences 357-362 represents a mechanistic feature which distinguishes catalytically active maize and muscle isozymes from less active liver isozyme . Conservation of active site residues among aldolases suggests that isozyme diversity among aldolases arose from divergent evolution of the COOH-terminal sequence.

J Biol Chem, 1993 May 25, 268(15), 10760 - 5
Refolding of luciferase subunits from urea and assembly of the active heterodimer . Evidence for folding intermediates that precede and follow the dimerization step on the pathway to the active form of the enzyme; Ziegler MM et al.; Conditions have been established that allow reversible refolding of luciferase from 5 M urea . The kinetics of formation of the active enzyme showed a concentration-independent lag, suggesting the existence of intermediate structures on the pathway of refolding . The rate of approach to the final level of activity was strongly concentration-dependent at protein concentrations below 10 micrograms/ml, but at concentrations above about 20 micrograms/ml, the rate of approach to the final activity value did not change with concentration . The concentration dependence presumably reflects the second-order step yielding the heterodimeric structure . The finding that at concentrations above 20 micrograms/ml, the rate becomes insensitive to concentration suggests that under these conditions, some step subsequent to dimerization become rate-limiting . When the refolding reaction was initiated by dilution out of 5 M urea at 50 micrograms/ml followed at various times by a secondary dilution to a final concentration of 5 micrograms/ml, it was found that the increase in activity continued at the rate characteristic of the higher protein concentration for a period of about 1-2 min following the dilution before slowing to the rate expected for the lower protein concentration . These observations indicate that there are inactive heterodimeric species that form from assembly of the individual subunits and that these species must undergo further folding to yield the active heterodimeric species . At protein concentrations of 5-50 micrograms/ml, the final yield of active enzyme was about 65-85%, decreasing at higher and lower concentrations . At higher concentrations, aggregation probably accounts for the limit in recovery, whereas at lower concentrations, it appears that the reduced yield of activity is due to the competing process of the folding of one or both individual subunits into some form incompetent to interact with each other . These experiments demonstrate the existence of slow steps in the refolding of luciferase subunits from urea and the formation of the active heterodimeric structure, both preceding and following the dimerization . Furthermore, the failure of protein at low concentrations to efficiently reassemble into the active heterodimer is consistent with the prior finding that luciferase subunits produced independently in Escherichia coli fold into conformations that cannot interact to form the active heterodimer upin mixing (Waddle, J . J., Johnston, T . C., and Baldwin, T . O . (1987) Biochemistry 26, 4917-4921).

Biochemistry, 1993 May 25, 32(20), 5431 - 5
Redesign of the interior hydrophilic region of mitochondrial cytochrome c by site-directed mutagenesis; Davies AM et al.; Heme propionate-7 in cytochrome c is an ionizable group located in a region of the protein that is inaccessible to bulk solvent . Electrostatic stabilization of this functional group appears to be achieved through interaction of heme propionate-7 with several amino acid residues that occur within hydrogen-bonding distance of it . To investigate the functional and spectroscopic roles of the amino acid residues that contribute to the immediate environment of heme propionate-7, the following variant forms of yeast (Saccharomyces cerevisiae) cytochrome c have been prepared and characterized by electrochemical and spectrochemical analyses: Arg38Ala, Tyr48Phe, Ala38Phe, Tyr48Phe/Trp59Phe, and Arg38Ala/Tyr48Phe/Trp59Phe . For each protein, the dependence of midpoint reduction potential and NMR spectrum on pH was determined, and the UV (250-450 nm) circular dichroic (CD) spectrum was measured . All of the variant proteins exhibited decreased reduction potentials with the greatest difference (-65 to -70 mV) exhibited by the multiply mutated proteins . The electrostatic properties of the variant proteins as reflected by the oxidation-state dependence of the His-39 pKa value were similar to those of the wild-type protein . Previous indirect assignments of minima in the CD spectrum of cytochrome c at 282 and 289 nm to Trp-59 are confirmed by spectra of the variant cytochromes in which this residue is replaced by Phe . The present results establish that the electrochemical effects of eliminating hydrogen-bonding interactions with heme propionate-7 are not additive and that the functional modulation of cytochrome c through regulation of the heme propionate-7 dielectric environment involves a complex combination of solvation effects and electrostatic or hydrogen-bonding interactions.

J Biol Chem, 1993 May 25, 268(15), 11435 - 9
Cloning and characterization of two distinct human extracellular signal-regulated kinase activator kinases, MEK1 and MEK2; Zheng CF et al.; Mitogen-induced signal transduction is mediated by a cascade of protein phosphorylation and dephosphorylation . One of the immediate responses of mitogen stimulation is the activation of a family of protein kinases known as mitogen-activated protein kinase or extracellular signal-regulated kinase (ERK) . MEK (MAP kinase or ERK kinase) is the immediate upstream activator kinase of ERK . Two cDNAs, MEK1 and MEK2, were cloned and sequenced . MEK1 and MEK2 encode 393 and 400 amino acid residues, respectively . The human MEK1 shares 99% amino acid sequence identity with the murine MEK1 and 80% with human MEK2 . Both MEK1 and MEK2 were expressed in Escherichia coli and shown to be able to activate recombinant human ERK1 in vitro . The purified MEK2 protein stimulated threonine and tyrosine phosphorylation on ERK1 and concomitantly activated ERK1 kinase activity more than 100-fold . The recombinant MEK2 showed lower activity as an ERK activator as compared with MEK purified from tissue . However, the recombinant MEK2 can be activated by serum-stimulated cell extract in vitro . MEKs, in a manner similar to ERKs, are likely to consist of a family of related proteins playing critical roles in signal transduction.

J Biol Chem, 1993 May 25, 268(15), 10754 - 9
Phospholipase C-gamma 1 associates with viral and cellular src kinases; Nakanishi O et al.; A tyrosine kinase inhibitor, genistein, caused the subcellular translocation of phosphoinositide-specific phospholipase C (PLC) activity from membrane fractions to cytosolic fractions in rat 3Y1 fibroblasts and their transformants by Rous sarcoma virus, SR-3Y1 . The ratio of PLC activities associated with the membrane fractions to those of the homogenate fractions was greater in SR-3Y1 (32.6%) than in 3Y1 (20.8%) whereas membrane-associated PLC activities were strikingly reduced to the same levels in both cells by treatment with genistein . Moreover, it was found by immunoblotting analyses of membrane fractions that the amounts of PLC-gamma 1 isozyme were reduced to 20.4% of initial level in SR-3Y1 and to 30.2% of that in 3Y1 cells . While the levels of PLC-delta, another detectable PLC isozyme, were not altered by genistein suggesting that tyrosine kinase plays an important role in the association of PLC-gamma 1 with membranes . PLC-gamma 1 molecules were detected in anti-p60arc antibody immunoprecipitates of both 3Y1 and SR-3Y1 cells . The amounts of PLC-gamma 1 co-immunoprecipitating with src kinases were higher in SR-3Y1 than 3Y1 cells and were reduced in both cell types by treatment with genistein . In addition, it was confirmed that PLC-gamma 1 purified from rat liver was phosphorylated at a tyrosine residue and associated with viral src kinase and that src kinases associated with the recombinant SH2 region of PLC-gamma 1, expressed in Escherichia coli, depending upon phosphorylation of tyrosine residues . These findings suggest that both viral and cellular src kinases associate with PLC-gamma 1 and may mediate cellular signaling in normal and transformant cell growth.

J Biol Chem, 1993 May 25, 268(15), 10946 - 52
Acetyl-CoA carboxylase from yeast is an essential enzyme and is regulated by factors that control phospholipid metabolism; Hasslacher M et al.; We have isolated a 1.2-kilobase pair cDNA fragment in a screening for yeast genes regulated at the level of transcription by soluble lipid precursors, inositol and choline . Sequence analysis and comparison of the deduced amino acid sequence to protein databases unveiled 68% similarity of a 374-amino acid peptide fragment to published C termini of chicken and rat acetyl-CoA carboxylase and almost 100% identity to the product of the FAS3 gene from yeast . Several lines of evidence confirm that the cloned gene represents the yeast structural gene ACC1 encoding acetyl-CoA carboxylase . Overexpression of the ACC1 gene from a high copy number plasmid resulted in overexpression of a 250-kDa biotin-enzyme and enzymatic activity of acetyl-CoA carboxylase . Disruption of one ACC1 allele in a diploid wild-type strain resulted in 50% reduction of ACC1-specific mRNA and acetyl-CoA carboxylase specific activity and a marked decrease of biotin associated with a 250-kDa protein, compared to wild-type . After sporulation of diploid disruptants, spores containing the disrupted acc1 allele failed to enter vegetative growth, despite fatty acid supplementation, suggesting that acetyl-CoA carboxylase activity is essential for a process other than de novo fatty acid synthesis and that only a single functional copy of the ACC1 gene exists . ACC1 transcription was repressed 3-fold by lipid precursors, inositol and choline, and was also controlled by regulatory factors Ino2p, Ino4p, and Opi1p, providing evidence that the key step of fatty acid synthesis is regulated in conjunction with phospholipid synthesis at the level of gene expression . The 5'-untranslated region of the ACC1 gene contains a sequence reminiscent of an inositol/choline-responsive element identified in genes encoding phospholipid biosynthetic enzymes.

Nucleic Acids Res, 1993 May 25, 21(10), 2429 - 35
Domain I of 23S rRNA competes with a paused transcription complex for ribosomal protein L4 of Escherichia coli; Zengel JM et al.; Ribosomal protein L4 of Escherichia coli regulates expression of its own eleven gene S10 operon both by inhibiting translation and by stimulating premature termination of transcription . Both regulatory processes presumably involve L4 recognition of the S10 leader RNA . To help define L4's regulatory target, we have investigated the protein's cognate target on 23S rRNA . Binding of L4 to various fragments of the 23S rRNA was monitored by determining their ability to sequester L4 in an in vitro transcription system and thereby eliminate the protein's effect on transcription . Using this approach we identified a region of about 110 bases within domain I of 23S rRNA which binds L4 . A two base deletion within this region, close to the base to which L4 has been cross-linked in intact 50S subunits, eliminates L4 binding . These results also confirm the prediction of the autogenous control model, that L4 bound to its target on rRNA is not active in regulating transcription of the S10 operon.

J Biol Chem, 1993 May 25, 268(15), 11449 - 55
Aldehyde dehydrogenase-derived omega-crystallins of squid and octopus . Specialization for lens expression; Zinovieva RD et al.; omega-Crystallin of the octopus lens is related to aldehyde dehydrogenases (ALDH) of vertebrates (Tomarev, S . I., Zinovieva, R . D., and Piatigorsky, J . (1991) J . Biol . Chem . 266, 24226-24231) and ALDH1/eta-crystallin of elephant shrews (Wistow, G., and Kim, H . (1991) J . Mol . Evol . 32, 262-269) . Only very low amounts of omega-crystallin are present in the squid lens . Here, we have cloned omega-crystallin cDNAs of the octopus (Octopus dofleini) and squid (Ommastrephes sloani pacificus) lenses . The deduced amino acid sequences of omega-crystallin from these species are 78% identical to each other, 56-58% identical to cytoplasmic ALDH1 and mitochondrial ALDH2 of vertebrates (which are 66-68% identical to each other), and 40% identical to Escherichia coli and spinach ALDHs . These data are consistent with the idea that the ALDH1/ALDH2 gene duplication in vertebrates occurred after divergence of cephalopods from the line giving rise to vertebrates, but before the separation of squid and octopus . Southern blot hybridization indicated that omega-crystallin is encoded by few genes (possibly just one) in octopus and squid . Northern blot hybridization revealed two bands (2.7 and 9.0 kilobases) of omega-crystallin RNA in the octopus lens and one band (4.2 kilobases) in the squid lens; omega-crystallin RNAs were undetectable in numerous non-lens tissues of octopus and squid, suggesting lens-specific expression of this gene(s) . Finally, extracts of the octopus lens had no detectable ALDH activity using different substrates, consistent with omega-crystallin having no enzymatic activity . Taken together, our results suggest that omega-crystallin evolved by duplication of an ancestral gene encoding ALDH and subsequently specialized for refraction in the transparent lens while losing ALDH activity and expression in other tissues.

J Biol Chem, 1993 May 25, 268(15), 11409 - 16
Translational repression of endogenous thymidine kinase mRNA in differentiating and arresting mouse cells; Knofler M et al.; We observed that decline of thymidine kinase (TK) enzyme activity was severalfold faster than the decay of full length TK mRNA during growth arrest of 3T6 mouse fibroblasts or during differentiation of myoblasts (C2Cl12) or F9 embryonal carcinoma cells . In order to study the molecular mechanism of this disparate behavior, a polyclonal antiserum against mouse TK was raised in rabbit . High level expression of mouse TK polypeptide in Escherichia coli was achieved with a T7 RNA polymerase-directed expression system . Using the antiserum in immunoblotting, no indication for a pool of inactive enzyme was found during differentiation of F9 or growth arrest of 3T6 cells . Pulse labeling of these cells in vivo with {35S}methionine showed a more than 6-fold decrease in the rate of TK-protein synthesis of in F9 cells after 3 days of treatment with retinoic acid as well as in 3T6 cells after 16 h under low serum . This was not due to increased turnover of the protein as measured in pulse chase experiments . In addition, full length TK mRNA stayed associated with polysomes under these conditions in F9 as well as 3T6 cells . Taken together the results suggest that endogenous TK mRNA becomes translationally repressed under a variety of conditions when mouse cells cease to grow.

J Biol Chem, 1993 May 25, 268(15), 11335 - 9
FK-506-binding protein: three-dimensional structure of the complex with the antagonist L-685,818; Becker JW et al.; L-685,818 differs only slightly in structure from the immunosuppressive drug FK-506, and both compounds bind with comparable affinity to the 12-kDa FK-506-binding protein (FKBP12), the major intracellular receptor for the drug . Despite these similarities, L-685,818 is a potent antagonist of both the immunosuppressive and toxic effects of the drug . Here, we present a structural analysis of this problem . Although FK-506 and L-685,818 differ greatly in pharmacology, we have found that the three-dimensional structures of their complexes with FKBP12 are essentially identical . Approximately half of each ligand is in contact with the receptor protein, and half is exposed to solvent; the exposed region includes the two sites where the compounds differ . These results indicate that the profound differences in the pharmacology of these two compounds are not caused by any difference in their interaction with FKBP12 . Rather, these effects arise because relatively minor changes in the exposed part of a bound ligand have a strong effect on how FKBP12-ligand complexes interact with calcineurin, their putative intracellular target . In addition, FK-506 complexes with FKBP12 proteins from several species all inhibit mammalian calcineurin . Analysis of the three-dimensional structure of the complex with respect to residues conserved among these proteins suggests a small number of surface residues near the bound ligands that may play a critical role in interactions between the protein-drug complex and calcineurin.

J Biol Chem, 1993 May 25, 268(15), 10851 - 62
Characterization of RNA and DNA synthesis in Escherichia coli strains devoid of ppGpp; Hernandez VJ et al.; The synthesis rates of DNA, rRNA, bulk mRNA, protein, and RNA polymerase beta- and beta'-subunits were determined as functions of growth rate in a wild-type Escherichia coli strain, which produces guanosine tetraphosphate (ppGpp), and in a delta relA delta spoT mutant which does not produce ppGpp . The rate of stable RNA synthesis per amount of protein depends on three factors: RNA polymerase concentration, RNA polymerase activity, and the distribution of active RNA polymerase between stable and mRNA genes, measured as the stable RNA synthesis rate/total RNA synthesis rate, rs/rt . In the wild-type strain, all three factors increase with growth rate . In the ppGpp-deficient strains, only RNA polymerase synthesis and activity, but not rs/rt, increased with growth rate . Thus, adjustments of rs/rt require ppGpp . In the absence of ppGpp, the synthesis of rRNA and bulk mRNA both varied in direct proportion to the concentration of active RNA polymerase, in contrast to the wild-type strain, in which only rRNA synthesis increased with growth rate, while mRNA synthesis remained constant . Thus, a control specific for rRNA is absent in strains lacking ppGpp . In rich media, the ppGpp-deficient strain synthesized up to 4-fold more mRNA than wild-type bacteria, which was associated with a similarly increased RNA polymerase activity . We propose that RNA polymerase is rendered inactive in wild-type bacteria due to ppGpp-dependent transcriptional pausing during the synthesis of mRNA . Finally, the control of replication initiation was altered in ppGpp-less bacteria, apparently reflecting indirect changes in the cell physiology, rather than a direct effect of ppGpp on replication initiation.

J Biol Chem, 1993 May 25, 268(15), 10802 - 7
Functional size analysis of F-ATPase from Escherichia coli by radiation inactivation; Ma JT et al.; A radiation inactivation technique was employed to determine the functional size of adenosine triphosphatase from Escherichia coli (EF0EF1-ATPase) . Functional units of the membrane-bound and the soluble ATPases were estimated to be 300 +/- 39 and 295 +/- 32 kDa, respectively . The presence of the free radical scavenger dithiothreitol was crucial in measuring the radiation inactivation size of ATPase . When gramicidin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone were added, an increase in the functional mass of membrane-bound ATPase was observed . In contrast, valinomycin and KCl had hardly any effect on the functional size of ATPase . We also determined a functional unit of 355 +/- 33 kDa for proton translocation by a fluorescence quenching technique . A reconstitution study using irradiated coupling factor 1 (EF1)-depleted membrane revealed that the functional mass of the proton channel was 96 +/- 11 kDa . A similar functional size for ATP-Pi exchange and ATP hydrolysis implies that both reactions might utilize identical machinery . Furthermore, functional units of soluble EF1 for unisite (nonsteady state) and multisite (steady state) ATP hydrolysis were calculated as 200 +/- 32 and 298 +/- 32 kDa, respectively . A working hypothesis was proposed from radiation inactivation analysis to elucidate the structure and mechanism of F1-ATPase.

J Biol Chem, 1993 May 25, 268(15), 10705 - 8
Evaluation of mutagenesis for epitope mapping . Structure of an antibody-protein antigen complex; Prasad L et al.; The location and description of epitopes on proteins describe the basis of immunological specificity . The 2.8-A structure of the phosphocarrier protein, HPr from Escherichia coli, complexed to the Fab fragment of the monoclonal antibody, Jel42, has been determined . This allows the first comparison of epitope predictions from extensive site-directed mutagenesis experiments, coupled with biological activity studies (Sharma, S., Georges, F., Klevit, R . E., Delbaere, L . T . J., Lee, J . S., and Waygood, E . B . (1991) Proc . Natl . Acad . Sci . U.S.A . 88, 4877-4881), with those from x-ray analysis . There are 14 amino acid residues of E . coli HPr that interact with the Jel42 antigen-binding site . Nine of these were correctly assigned by the mutagenesis studies . Of the 5 remaining residues, Met-1 could not be altered; two others appear to have critical roles in determining protein conformation; the other 2 residues have a minimal effect on antibody binding since they are located on the periphery of the epitope with one face of their side chains in van der Waals contact with the antibody and the other face in contact with solvent . Four residues were incorrectly assigned to the epitope . These residues were located adjacent to epitope residues that were likely perturbed by these mutations . This study demonstrates that mutations which caused greater than 10-fold changes in antibody binding affinity were correctly assigned to the epitope by the mutagenesis experiments . Guidelines are also presented in order to minimize incorrect assignments.

J Chromatogr, 1993 May 21, 638(1), 9 - 19
Reversed-phase capillary high-performance liquid chromatography with on-line UV, fluorescence and electrospray ionization mass spectrometric detection in the analysis of peptides and proteins; Heath TG et al.; Analysis of peptide mixtures by reversed-phase capillary HPLC with gradient elution using three detectors in series: UV (214 nm), fluorescence (lambda exc . = 280 nm, lambda emiss . = 356 nm), and electrospray ionization mass spectrometry (ES-MS) is reported . The chromatographic integrity of the system and the detection limits were evaluated . The effect of the mass spectrometer's acquisition rate on the total ion current (TIC) profile was also examined . The utility of fluorescence monitoring with UV and ES-MS detection was demonstrated in the analysis of proteolytic digests of proteins . The native fluorescence character of tryptophan-containing peptides provides selectivity in peptide mapping, while monitoring UV absorption at 214 nm affords detection of the peptide bond . Three tryptophan-containing tryptic peptides of bovine serum albumin were immediately located by fluorescence among many UV peaks and ES-MS provided molecular masses allowing the peptides to be identified.

J Theor Biol, 1993 May 21, 162(2), 153 - 86
Variation in concentrations of RNAs and proteins involved in gene expression of Escherichia coli; Mahaffy JM; A growing cell is not a steady-state situation . At some point in the cell cycle each gene doubles its concentration, which may result in a doubling of the production rate of its mRNA . The variations in concentration of mRNAs and proteins in an individual exponentially growing cell are studied for a gene which is constitutive, autorepressed, or autoactivated . Analysis of the mathematical model for a constitutive gene shows a fixed variation in concentration of a stable RNA between its minimum and maximum concentration of approximately 6%, independent of cell cycle time or gene location . The variation in the concentrations of the mRNA and protein for a constitutive gene are studied as gene position, molecular stability, and cell cycle time are varied . One result shows that doubling the growth rate can more than double the percentage of product from a gene near the origin of replication compared to one near the terminus . Results from these theoretical studies compare favorably to experimental results for rRNAs and the fully induced lac gene . Additional studies were performed to determine the effects of autorepression and autoactivation on the variation of concentration of mRNAs and proteins . The studies show that RNA and protein products of an autorepressed gene have almost the same variation in concentration as products of a constitutively expressed gene though the absolute concentrations are decreased . It is shown that the stability of an mRNA or protein affects variation in its concentration throughout the cell cycle, much more than the type of genetic control . The strength of repression has no effect on the variation in concentration of RNA and protein gene products through a cell cycle . Studies of an autoactivated gene show that it is significantly more responsive to a shift up or down, such as those caused by nutritional changes . An example is provided where the autoactivated gene is not expressed at one growth rate, but turns on at a higher growth rate . Furthermore, it is shown that gene position may determine whether or not the autoactivated gene is expressed.

J Mol Biol, 1993 May 20, 231(2), 274 - 92
Solution structure of selenocysteine-inserting tRNA(Sec) from Escherichia coli . Comparison with canonical tRNA(Ser); Baron C et al.; Selenocysteine-inserting tRNAs (or tRNA(Sec)) are structurally untypical tRNAs that are charged by seryl-tRNA synthetase before being recognized by the selenocysteine synthase that converts serine into selenocysteine . tRNA(Sec) from Escherichia coli contains 95 nucleotides and is the longest tRNA known to date, in contrast to canonical tRNA(Ser), 88 nucleotides-long . We have studied its solution conformation by chemical and enzymatic probing . Global structural features were obtained by cobra venom and S1 nuclease mapping, as well as by probing with Pb2+ . Accessibilities of phosphate groups were measured by ethylnitrosourea probing . Information about positions in bases involved in Watson-Crick pairing, in stacking or in tertiary interactions were obtained by chemical probing with dimethylsulfate, diethylpyrocarbonate, kethoxal and carbodiimide . On the basis of these chemical data, a three-dimensional model was constructed by computer modeling and compared to that of canonical tRNA(Ser) . tRNA(Sec) resembles tRNA(Ser) at the level of its T-arm and anticodon-arm conformations, as well as at the joining of the D- and T-loops by a tertiary Watson-Crick G19-C56 interaction . Its extra-long variable arm is a double-stranded structure closed by a four nucleotide loop that is linked to the body of the tRNA in a way different from that found in tRNA(Ser) . As anticipated from the peculiar features of the sequence in the D-loop and at the junction of amino acid and D-arms, tRNA(Sec) possesses a novel but restricted set of tertiary interactions in the core of its three-dimensional structure: a G8-A21-U14 triple pair and a novel interaction between C16 of the D-loop and C59 of the T-loop . A third triple interaction involving C15-G20a-G48 is suggested but some experimental evidence for it is still lacking . It is furthermore concluded that the D-arm has six base-pairs instead of three, as in canonical class II tRNA(Ser), with the D-loop containing only four nucleotides . Finally, the amino acid accepting arm forms a stack of eight Watson-Crick base-pairs (instead of 7 in other tRNAs) . The biological relevance of this model with regard to interaction with seryl-tRNA synthetase and enzymes from the selenocysteine metabolism is discussed.

J Mol Biol, 1993 May 20, 231(2), 219 - 29
Recognition nucleotides of Escherichia coli tRNA(Leu) and its elements facilitating discrimination from tRNASer and tRNA(Tyr); Asahara H et al.; In order to study how Escherichia coli leucyl-tRNA synthetase recognizes tRNA(Leu) and discriminates it from the other two class II tRNAs, tRNA(Ser) and tRNA(Tyr), various mutations were introduced into class II tRNA transcripts . The discriminator base A73, but not the anticodon sequence, was found to serve as a critical recognition element of tRNA(Leu) . A base substitution at the invariant nucleotide A14, but not at any of the other nucleotides characteristic of the E . coli tRNA(Leu) isoacceptors among the three class II tRNAs, caused significantly damaged aminoacylation with leucine . A two base-pair deletion in the long variable arm also resulted in no significant decrease of activity . Transplanting the three tertiary elements characteristic of E . coli tRNA(Leu) (i.e . the location of the G18G19 sequence in the D-loop, the A15 U48 base-pair and the stem pairing pattern of the long variable arm) besides the discriminator base change introduced the leucine charging activity in terms of Vmax/Km, up to 0.1 of that for the normal sequence of tRNA(Leu) into both tRNA(Ser) and tRNA(Tyr) . These results indicate that A73 and A14 (or its vicinity) are involved in recognition by leucyl-tRNA synthetase, and that several tertiary elements play a significant role in the discrimination of tRNA(Leu) from the other two class II tRNAs.

J Mol Biol, 1993 May 20, 231(2), 205 - 18
AraC protein can activate transcription from only one position and when pointed in only one direction; Reeder T et al.; At the araBAD promoter, the RNA polymerase-proximal half-site for AraC binding partially overlaps the -35 region . Random and explicit spacing experiments show that both this partial overlapping and AraC binding to the polymerase-proximal half-site are necessary and sufficient for strong transcriptional activation . Normally, this occupancy is generated by the presence of arabinose, which shifts AraC from a DNA looping interaction involving the polymerase-distal half-site and the araO2 site 210 base-pairs away, to an interaction with the two half-sites adjacent to RNA polymerase . Changing the polymerase-proximal half-site to a higher affinity AraC binding site gives activation in the absence of arabinose . Thus, arabinose is not required to transform AraC into an activating conformation . Because the two half-sites of araI are direct repeats, the RNA polymerase proximal and distal surfaces of AraC are not identical . When the araI site was turned around, no spacings were found from which AraC could activate transcription . In light of the strict spacing and orientation requirements for AraC activation, the interactions between AraC and RNA polymerase are likely to be specific and inflexible.

J Mol Biol, 1993 May 20, 231(2), 191 - 5
Structural differences between wild-type NADP-dependent glutathione reductase from Escherichia coli and a redesigned NAD-dependent mutant; Mittl PR et al.; NAD and NADP are ubiquitous coenzymes in biological redox reactions . They have distinct metabolic functions, yet they differ only by an additional phosphate group esterified at the 2'-hydroxyl group of the AMP moiety of NADP . The natural specificity of Escherichia coli glutathione reductase for NADP has previously been converted into a marked preference for NAD by introducing seven point mutations into the beta alpha beta-fold of the NADP-binding domain of the protein based on the known structure of the human enzyme . Among them was the replacement of Ala179 by glycine (A179G) in the alpha-helix of the fold, a change suggested by a difference in a sequence fingerprint previously found in the dinucleotide-binding domains of a number of dehydrogenases . Although this position is at a distance of 10 A from the bound 2'-phosphate group of NADP in glutathione reductase, the A179G mutation was found to be synergistic and beneficial . We have now carried out X-ray crystallographic analyses of the NAD-dependent mutant without and with bound NADH . A comparison of the structures of the mutant and wild-type enzymes reveals a flip of the peptide bond between Gly174 and Ala175 such that the side-chain of another introduced amino acid, Glu197, is fixed and can participate in binding the adenine ribose of NAD, thereby contributing to the ability of the mutated enzyme to exert its selectivity for the "wrong" coenzyme.

J Mol Biol, 1993 May 20, 231(2), 513 - 5
Purification, circular dichroism analysis, crystallization and preliminary X-ray diffraction analysis of the F plasmid CcdB killer protein; Steyaert J et al.; Large crystals of the Escherichia coli F plasmid CcdB killer protein were grown from solutions containing 32% ammonium sulphate . The crystals belong to space group P4(2)2(1)2 with a = b = 104.52 A and c = 88.45 A or P2(1)2(1)2(1) with a = 77.62 A, b = 93.28 A and c = 141.44 A . Both crystal forms diffract to 2.6 A resolution . Structure determination by multiple isomorphous replacement is under way.

J Mol Biol, 1993 May 20, 231(2), 261 - 73
Mutations that affect separate functions of OmpR the phosphorylated regulator of porin transcription in Escherichia coli; Russo FD et al.; OmpR is a member of a family of bacterial transcriptional regulators whose activity is controlled by phosphorylation . It regulates the transcription of two genes, serving as an activator of ompC, and as both an activator and a repressor of ompF . A previously isolated collection of ompR mutations was analyzed for the effect of each on the expression of both genes simultaneously . The results of this analysis indicate that the activation, repression, and DNA binding functions of OmpR can be disrupted independently, and that mutations interfering with each of these functions cluster within the sequence of the OmpR protein . The nature of these mutations is discussed in terms of the mechanisms by which OmpR regulates transcription, and potentially similar mechanisms operating within closely related response regulators.

J Mol Biol, 1993 May 20, 231(2), 489 - 97
tRNA-guanine transglycosylase from Escherichia coli . Overexpression, purification and quaternary structure; Garcia GA et al.; tRNA-guanine transglycosylase (TGT) is the enzyme responsible for the posttranscriptional modification of specific tRNAs (Asn, Asp, His and Tyr) with queuine . In E . coli this modification occurs via a two-step reaction: (1) TGT-catalyzed base exchange of guanosine-34 with preQ1 (7-aminomethyl-7-deazaguanine) and (2) addition of a cyclopentenediol moiety to the preQ1-34 tRNA . E . coli TGT is normally expressed at very low levels (approximately 1 mg from 500 g cells) . The sequence of the queuine operon of E . coli has recently been reported by Reuter et al . (1991) . We have cloned the tgt gene into an overexpressing vector in order to provide a more efficient preparation of TGT . A simple, four-step purification scheme yields 78 mg of homogeneous TGT per liter of cell culture (A600 = 5 to 6) . Amino-terminal protein sequencing confirms the identity of the recombinant protein and indicates that the initiator methionine is retained in the mature form . Native-PAGE of TGT and SDS-PAGE of cross-linked TGT are most consistent with a hexameric quaternary structure for the enzyme . The cross-linking data also suggests that the enzyme exists as a dimer of trimers of identical 42.5 kDa subunits (total M(r) = 255 kDa . The enzyme is inactivated by cross-linking with the bisimidoester, dimethylsuberimidate . Substrate (tRNA) protects the enzyme against cross-linking and inactivation by dimethylsuberimidate and against inactivation by modification with ethylacetimidate, a monofunctional, imidoester . This indicates that the enzymic residues (presumably lysines) that are involved in cross-linking and the inactivation are in the active site of the enzyme.

Biochemistry, 1993 May 18, 32(19), 5267 - 72
Dissection of a class II tRNA synthetase: determinants for minihelix recognition are tightly associated with domain for amino acid activation; Buechter DD et al.; The ten class II aminoacyl-tRNA synthetases are large homo- and hetero-oligomeric proteins that share three conserved sequence motifs . Within this class, Escherichia coli alanyl-tRNA synthetase is the only homotetramer and is comprised of subunits of 875 amino acids . The enzyme aminoacylates sequence-specific RNA oligonucleotides that recreate as few as four base pairs of the acceptor stem of tRNA(Ala) . A monomeric 461 amino acid N-terminal fragment (461N) was previously shown to have full adenylate synthesis activity . However, fragment 461N has significant, but reduced, efficiency of charging of tRNA(Ala), when compared to native enzyme {Ho, C., Jasin, M., & Schimmel, P . (1985) Science 229, 389-393} . We show here that, in contrast, the fragment and the native enzyme aminoacylate a 12 base pair acceptor-T psi C stem minihelix and a four base pair RNA tetraloop with the same efficiency . We also show that fragment 461N makes footprint contacts both on and outside the acceptor helix of bound tRNA(Ala) . With one possible exception, the contacts observed with fragment 461N are identical to those seen with the native enzyme . In spite of contacts outside the acceptor helix, fragment 461N charges the native tRNA, minihelix, and tetraloop with similar efficiency . Thus, all minihelix contacts required for activation for charging are tightly associated with the adenylate synthesis domain and, at least in the fragment, are not influenced by additional RNA-protein contacts outside the minihelix domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 May 18, 32(19), 5239 - 46
tRNA-guanine transglycosylase from Escherichia coli: gross tRNA structural requirements for recognition; Curnow AW et al.; tRNA-guanine transglycosylase (TGT) is the enzyme responsible for the post-transcriptional modification of specific tRNAs (those for Asn, Asp, His, and Tyr) with the hypermodified base, queuine . In Escherichia coli this enzyme catalyzes the exchange of guanine-34 in the anticodon with preQ1, which is subsequently further modified to queuine . There is evidence that such hypermodified tRNA molecules may play a role in the control of cell proliferation and differentiation . In order to perform detailed, in vitro mechanistic studies and to probe the tRNA-enzyme interaction, we have generated unmodified E . coli tRNA(Tyr) and truncated analogues using an in vitro RNA synthesis system suggested by Milligan and Uhlenbeck {Milligan, J . F., & Uhlenbeck, O . C . (1989) Methods Enzymol . 180, 51-62} . From this system we have generated three tRNA analogues totally devoid of any post-transcriptional modifications . In order to compare the unmodified tRNA with the true physiological substrate for TGT, that is, tRNA that contains all modified bases except queuine, we have isolated E . coli tRNA(Tyr) from an overexpressing clone in a TGT-deficient strain of E . coli . We report here that unmodified, full-length tRNA(Tyr) serves as a substrate for TGT with kinetic parameters that are, within experimental error, the same as those for in vivo isolated tRNA(Tyr) . This indicates that other post-transcriptional modifications have negligible effects upon TGT recognition of tRNA . A 17-base oligoribonucleotide, corresponding to the anticodon loop and stem, is also a substrate for TGT with only a 20-fold loss in Vmax/KM, versus the full-length tRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 May 18, 32(19), 5214 - 21
Role of native disulfide bonds in the structure and activity of insulin-like growth factor 1: genetic models of protein-folding intermediates; Narhi LO et al.; Insulin and insulin-related proteins contain three motif-specific disulfide bonds . Here we examine the role of these disulfide bonds in the folding and function of one family member, human insulin-like growth factor 1 (IGF-1) . Analogues containing pariwise Cys-->Ser or Cys-->Ala substitutions were expressed in Escherichia coli, purified, and analyzed with respect to receptor-binding, solution structure, and thermodynamic stability . An analogue lacking all three disulfide bonds (designated des-Cys-IGF-1) is inactive and unfolded . Introduction of the {18-61} disulfide bond, previously shown to occur in an early intermediate in oxidative refolding {Miller, J . A., Owers-Narhi, L., Hua, Q . X., Rosenfeld, R., Arakawa, T., Rohde, M., Prestrelski, S., Lauren, S., S . Stoney, K . S., Tsai, L., & Weiss, M . A . (1993) Biochemistry (preceding paper in this issue)}, results in a compact partially folded state with low but significant biological activity . Additional but incomplete structural organization and biological activity are observed following introduction of either the {6-48} or the {47-52} disulfide bonds . Native function, structure, and stability require the presence of all three disulfide bonds . These analogues provide genetic models of IGF-1 protein-folding intermediates . Their characterization suggests that bifurcation of the IGF-1 folding pathway reflects alternative late steps in the folding of a molten-globule intermediate.

Biochemistry, 1993 May 18, 32(19), 5177 - 86
Imidazole glycerol phosphate synthase: the glutamine amidotransferase in histidine biosynthesis; Klem TJ et al.; Two proteins essential for the biosynthesis of the amino acid histidine in Escherichia coli have been overexpressed and purified to apparent homogeneity . The protein encoded by the hisF gene has an ammonia-dependent activity that results in the conversion of the biosynthetic intermediate N1-{(5'-phosphoribulosyl)formimino}-5-aminoimidazole-4- carboxamide ribonucleotide (PRFAR) to imidazole glycerol phosphate (IGP) and 5-aminoimidazole-4-carboxamido-1-beta-D- ribofuranosyl 5'-monophosphate (AICAR) . The second protein encoded by the hisH gene exhibits no detectable catalytic properties with biosynthetic intermediate PRFAR, glutamine, or ammonia . In combination, the proteins are capable of a stoichiometric conversion of glutamine and PRFAR to form AICAR, IGP, and glutamate . Neither protein alone is capable of mediating a conversion of the nucleotide substrate to a free metabolic intermediate . The HisH and HisF proteins form a stable 1:1 dimeric complex that constitutes the IGP synthase holoenzyme . Steady-state kinetic parameters for the holoenzyme indicate that glutamine is a more efficient substrate relative to ammonium ion by a factor of 10(3) . The HisF subunit will support an ammonia-dependent reaction with a turnover number similar to that of the holoenzyme with glutamine . The glutaminase activity for the holoenzyme is 0.8% of that in the presence of the nucleotide substrate PRFAR . There are critical subunit interactions that mediate the catalytic properties for glutamine hydrolysis . The catalytic turnover of glutamine can be increased up to 37-fold by the addition of either the product IGP or the biosynthetic precursor N1-{(5'-phosphoribosyl)formimino}-5-aminoimidazole-4-carboxamide ribonucleotide (5'-ProFAR) . The mechanistic significance of this glutaminase activity compared to other trpG type glutamine amidotransferases is discussed.

Biochemistry, 1993 May 18, 32(19), 5167 - 76
Characterization of recombinant human farnesyl-protein transferase: cloning, expression, farnesyl diphosphate binding, and functional homology with yeast prenyl-protein transferases; Omer CA et al.; We have isolated cDNAs encoding the alpha and beta subunits of human farnesyl-protein transferase (FPTase) . The proteins encoded by these two cDNAs are 93-95% identical to the corresponding subunits of bovine and rat FPTase and show regions of homology with proteins encoded by Saccharomyces cerevisiae prenyl-protein transferase genes . Human FPTase expressed in Escherichia coli from a translationally coupled operon had kinetic properties similar to those of FPTase isolated from bovine brain . Examination of farnesyl diphosphate binding indicated that while neither individual subunit was capable of isoprenoid binding, a radiolabeled farnesyl diphosphate analog could be specifically photo-cross-linked to the beta subunit of FPTase holoenzyme . To further analyze subunit structure-function and to detect functional similarities with yeast prenyl-protein transferases (FPTase and two geranylgeranyl-protein transferases), amino acid changes homologous to those found in mutant yeast prenyl-protein transferase subunits were made in the subunits of human FPTase . Substitutions in either the alpha or beta subunits that decrease the activity of yeast prenyl-protein transferases were also observed to impair human FPTase . Kinetic analyses showed that these mutant human FPTases have Km and kcat values that are altered with respect to wild-type human FPTase.

Biochemistry, 1993 May 18, 32(19), 5132 - 8
Serine92 (F7) contributes to the control of heme reactivity and stability in myoglobin; Smerdon SJ et al.; The effects of mutation of the conserved serine92 residue to alanine, valine, and leucine in pig myoglobin have been determined . In myoglobin crystal structures, the hydroxyl group of serine92 is within hydrogen-bonding distance of the N delta-H of histidine93, whose N epsilon coordinates the iron atom of the heme prosthetic group . The association equilibrium constants of the ferrous forms of the mutant myoglobins for O2, CO, and methyl and ethyl isocyanide are increased 1.3-13-fold relative to the wild-type protein . The rates of azide association with the mutant ferric proteins at neutral pH are decreased by factors of 2-5 consistent with an increased affinity for the iron-bound water molecule which must be displaced . The dissociation rates for azide appear to be decreased 4-10-fold, suggesting that the affinity of the mutant proteins for this ligand is also higher . Thus, the overall affinities are increased regardless of the chemical nature of the liganded species, indicating that the reactivity of the heme iron itself has been raised . Time courses for association of methyl and ethyl isocyanide at high concentrations show fast and slow phases in which the absorbance at 445 nm drops and then rises, respectively . Comparison of these traces with spectra following the reaction of isocyanide ligands with chelated proton heme in soap micelles indicates that the slow phase is associated with the breaking of the iron-proximal histidine bond and the binding of a second isocyanide species in the proximal heme pocket.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 May 18, 32(19), 5083 - 92
The reactive and destabilizing disulfide bond of DsbA, a protein required for protein disulfide bond formation in vivo; Zapun A et al.; The protein DsbA facilitates disulfide bond formation in the periplasm of Escherichia coli . It has only two cysteine residues that are separated in the sequence by two other residues and are shown to form a disulfide bond reversibly . Chemical modification studies demonstrate that only one of the cysteine residues has an accessible thiol group in the reduced protein . Equilibrium and kinetic characterization of thiol-disulfide exchange between DsbA and glutathione showed that the DsbA disulfide bond was 10(3)-fold more reactive than a normal protein disulfide . Similarly, the mixed disulfide between the accessible cysteine residue and glutathione was 10(4)-fold more reactive than normal . The overall equilibrium constant for DsbA disulfide bond formation from GSSG was only 8 x 10(-5) M . These properties indicate that disulfide-bonded DsbA is a potent oxidant and ideally suited for generating protein disulfide bonds . Disulfide bonds generally increase the stabilities of folded proteins, when the folded conformation reciprocally stabilizes the disulfide bonds . In contrast, the disulfide bond of DsbA was so unstable in the folded state that its stability increased by 4.5 +/- 0.1 kcal.mol-1 when the protein unfolded . This implies that the disulfide bond destabilizes the folded conformation of DsbA . This was confirmed by demonstrating that the reduced protein was 3.6 +/- 1.4 kcal.mol-1 more stable than that with the disulfide bond.

Biochemistry, 1993 May 18, 32(19), 5099 - 108
Removal of the high-potential {4Fe-4S} center of the beta-subunit from Escherichia coli nitrate reductase . Physiological, biochemical, and EPR characterization of site-directed mutated enzymes; Augier V et al.; The beta-subunit of the nitrate reductase of Escherichia coli contains four groups of Cys residues (I-IV) which are thought to bind the single {3Fe-4S} center and the three {4Fe-4S} centers . The first or second Cys residue of group I was substituted by site-directed mutagenesis with Ala or Ser . Physiological, biochemical, and EPR studies were performed on the mutated enzymes . With small variations, the properties of these mutant enzymes do not differ from one another . They were found to be as abundant and as stably bound to the membrane as the native enzyme, provided the gamma-subunit was present . Although physiological activity was reduced, it was sufficient to allow growth on nitrate . The study of variations in EPR intensity as a function of the redox potential indicated that these enzymes only contained three iron-sulfur centers instead of the usual four in the native enzyme . Spectral EPR analysis showed that the {4Fe-4S} center of high redox potential (center 1, +80 mV) was missing . The loss of this center did not affect the stable integration of the other three centers . The data presented here are in total contrast to those we have reported for each of the other three centers (centers 2-4), the loss of which was detrimental to the integration of all centers and to the integration of the molybdenum cofactor (Augier et al., in press) . Taken together, our results demonstrated that the first and second Cys residues of group I are the ligands of the {4Fe-4S} center (center 1, +80 mV) and that this center participates in electron transfer, but is dispensable . On the basis of these results, it is proposed that the {3Fe-4S} center (center 2, +60 mV) also plays a biological role and that in the native enzyme both high-potential centers, centers 1 and 2, contribute independently and in parallel to the electron transfer to the molybdenum cofactor.

Biochemistry, 1993 May 18, 32(19), 5093 - 8
Crystal structure analysis of a mutant Escherichia coli thioredoxin in which lysine 36 is replaced by glutamic acid; Nikkola M et al.; The structure of a mutant Escherichia coli thioredoxin with a glutamic acid substituted for a conserved lysine at position 36 adjacent to the active site has been solved using molecular replacement and refined at 2.0-A resolution to a crystallographic residual of 19.9% . The mutant was crystallized in an orthorhombic space group with one molecule in the asymmetric unit . The structure of the mutant thioredoxin shows overall good agreement with the wild-type E . coli thioredoxin . The root-mean-square deviations for all C alpha s are 0.45 and 0.79 A between the mutant structure and the two molecules in the asymmetric unit of the wild-type crystals . Structural changes are seen in several residues in the active-site region preceding the disulfide . A reverse turn of residues 29-32 changes the conformation from a type I to a type II turn . This change may be related to the loss of a hydrogen bond from Lys-36 to the main-chain carbonyl of residue 30 due to the mutation . The C alpha atom of Trp-31 has moved 1.9 A and the indole ring no longer makes hydrogen bonds to the carboxyl group of Asp-61 but instead participates in a crystal contact . The structural differences seen in the mutant thioredoxin may be influenced by the crystal packing . The substituted Glu-36 makes extensive crystal contacts . The static fluorescence of this mutant thioredoxin has a different pH dependence than the wild type.

FEBS Lett, 1993 May 17, 322(3), 277 - 9
Autostimulation of the DnaK (HSP 70) ATPase of Escherichia coli; Richarme G et al.; The ATPase activity of DnaK, the 70-kDa chaperone of Escherichia coli, is stimulated by an unfolded protein . However, the stimulation of the DnaK ATPase by unfolded bovine pancreatic trypsin inhibitor can only be observed at low DnaK protein concentrations . At higher DnaK concentrations, the ATPase activity of DnaK cannot be stimulated by the addition of unfolded bovine pancreatic trypsin inhibitor . This is a consequence of the autostimulation of the DnaK ATPase at higher DnaK concentrations . The autostimulation of DnaK is reflected by a non-linear dependence of ATP hydrolysis on DnaK concentration . Furthermore, DnaK exists as a mixture of monomers and dimers in equilibrium, and the dimers dissociate into monomers in the presence of ATP.

Eur J Biochem, 1993 May 15, 214(1), 59 - 65
Site directed mutagenesis of DNA polymerase I (Klenow) from Escherichia coli . The significance of Arg682 in catalysis; Pandey VN et al.; We have reported that a domain containing Arg682 in the Klenow fragment of Escherichia coli DNA polymerase I (pol I) is important for the template-dependent dNTP-binding function {Pandey, V.N., Kaushik, N . A., Pradhan, D . S . & Modak, M . J . (1990) J . Biol . Chem . 265, 3679-3884} . In order to further define the role of Arg682 in the catalytic process, we have performed site-directed mutagenesis of this residue . For this purpose the Klenow-coding region of the DNA-pol-I gene was selectively amplified from the genomic DNA of E . coli and was cloned in an expression vector, pET-3a . This clone under appropriate conditions overproduces the Klenow fragment in E . coli . Using this clone (pET-3a-K) as the template, two mutant polymerase clones were constructed in which arginine has been replaced with either alanine, {R682A} pol I, or lysine {R682K} pol I . Both mutant enzymes showed significantly lower specific activity as compared to the wild-type enzyme . The kinetic analyses of the mutant enzymes indicated a 3-4-fold increase in the Km for the substrate dNTP, a 20-25-fold decrease in the Vmax and an overall decrease in the processive nature of DNA synthesis in both the mutant enzymes . The reverse mutation of Ala682 to the wild-type form Arg682 fully restored the processive nature and the polymerase activity of the enzyme . These observations suggest that the positively charged guanidino group in the side chain of Arg682 is catalytically important but not absolutely essential for synthesis of DNA . Furthermore it appears to maintain high processivity of the DNA synthesis catalyzed by the enzyme.

Eur J Biochem, 1993 May 15, 214(1), 287 - 93
Efficient secretion of attacin from insect fat-body cells requires proper processing of the prosequence; Gunne H et al.; The attacins constitute a group of immune proteins induced after bacterial infection in the moth Hyalophora cecropia . They are synthesized as preproproteins and undergo post-translational modification during transport to the hemolymph . The processing and transport rates of attacin were studied in its natural host as a response to infection . Monensin totally inhibited the processing from proattacin to attacin and the radiolabeled proattacin remained intracellular . This observation indicated that the prosequence is removed at or after the trans-Golgi compartment . It is also suggested that the processing of the prosequence does not occur in acidic vesicles, as the process was not inhibited by the weak base chloroquine . To study prosequence function, the attacin gene and genes with mutations in the prosequence were cloned into the baculovirus Autographa californica nuclear polyhedrosis virus . The processing of proattacin and the transport of attacin were studied by pulse-chase experiments with fat body isolated from Trichoplusia ni larvae . The rate of secretion from fat body was lowest for proattacin, which could not be processed to attacin, intermediate for attacins lacking the prosequence and highest for natural attacin . We could not detect any biological activity for proattacin.

Eur J Biochem, 1993 May 15, 214(1), 17 - 25
Molecular cloning and nucleotide sequence of cDNA encoding rat kidney long-chain L-2-hydroxy acid oxidase . Expression of the catalytically active recombinant protein as a chimaera; Belmouden A et al.; Long-chain L-alpha-hydroxy acid oxidase from rat kidney is a member of the family of FMN-dependent alpha-hydroxy-acid-oxidizing enzymes . With the knowledge of the recently determined amino acid sequence, the cDNA encoding the enzyme has now been cloned using the polymerase chain reaction . The 1648-bp cDNA contains an open reading frame coding for the 352 residues of the previously determined sequence, preceded by a methionine codon . In addition, several clones were found to present a nine-base insertion, predicting the existence of an isoform with a tripeptide VRK inserted between residues 188 and 189 of the mature protein . The presence of about 10% of this isoform in the oxidase purified from rat kidney was indeed identified by amino acid sequencing . A recombinant active enzyme was obtained as a protein fused to glutathione S-transferase using the bacterial expression plasmid pGEX-3X . Physico-chemical characterization indicated, for the fused enzyme, properties similar to those of the rat kidney protein . When the chimaera was submitted to factor Xa, proteolysis at the engineered cleavage point was poor . Separation of hydroxy acid oxidase from glutathione S-transferase could not be achieved with trypsin either . With both proteases, the initial cleavage point appeared to be in a peptide loop internal to the hydroxy acid oxidase sequence, close to or in the tripeptide insertion locus and not at the engineered factor-Xa-cleavage point . Comparative tryptic proteolysis of the rat kidney enzyme yielded a form cleaved in the same loop.

Proc Natl Acad Sci U S A, 1993 May 15, 90(10), 4626 - 30
Detection and characterization of mammalian DNA polymerase beta mutants by functional complementation in Escherichia coli; Sweasy JB et al.; We have designed and utilized a bacterial complementation system to identify and characterize mammalian DNA polymerase beta mutants . In this complementation system, wild-type rat DNA polymerase beta replaces both the replicative and repair functions of DNA polymerase I in the Escherichia coli recA718 polA12 double mutant; our 263 DNA polymerase beta mutants replace E . coli polymerase I less efficiently or not at all . Of the 10 mutants that have been shown to contain DNA sequence alterations, 2 exhibit a split phenotype with respect to complementation of the growth defect and methylmethanesulfonate sensitivity of the double mutant; one is a null mutant . The mutants possessing a split phenotype contain amino acid residue alterations within a putative nucleotide binding site of DNA polymerase beta . This approach for the isolation and evaluation of mutants of a mammalian DNA polymerase in E . coli may ultimately lead to a better understanding of the mechanism of action of this enzyme and to precisely defining its role in vertebrate cells.

Proc Natl Acad Sci U S A, 1993 May 15, 90(10), 4389 - 93
Evidence for lack of DNA photoreactivating enzyme in humans; Li YF et al.; Photoreactivating enzyme (DNA photolyase; deoxyribocyclobutadipyrimidine pyrimidine-lyase, EC 4.1.99.3) repairs UV damage to DNA by utilizing the energy of near-UV/visible light to split pyrimidine dimers into monomers . The enzyme is widespread in nature but is absent in certain species in a seemingly unpredictable manner . Its presence in humans has been a source of considerable controversy . To help resolve the issue we used a very specific and sensitive assay to compare photoreactivation activity in human, rattlesnake, yeast, and Escherichia coli cells . Photolyase was easily detectable in E . coli, yeast, and rattlesnake cell-free extracts but none was detected in cell-free extracts from HeLa cells or human white blood cells with an assay capable of detecting 10 molecules per cell . We conclude that humans most likely do not have DNA photolyase.

Biochem J, 1993 May 15, 292 ( Pt 1), 189 - 95
Cloning and characterization of the major hepatic glutathione S-transferase from a marine teleost flatfish, the plaice (Pleuronectes platessa), with structural similarities to plant, insect and mammalian Theta class isoenzymes; Leaver MJ et al.; A cDNA clone (PLGSTA) of 896 bp, containing an open reading frame encoding a 225-amino-acid polypeptide of M(r) 25,723, was isolated from a cDNA library constructed in lambda gt11 from the liver of a marine teleost flatfish, the plaice (Pleuronectes platessa) . The identification of this cDNA as that coding for the subunit of the major cytosolic glutathione S-transferase of plaice liver, GST-A, was supported by its heterologous expression in and purification of its protein product from Escherichia coli . The recombinant-derived protein exhibited identical M(r) and immunoreactivity and a similar substrate specificity to GST-A previously isolated from plaice liver . Comparison of the deduced amino acid sequence of the plaice GST-A polypeptide with the primary structures of GSTs from other Phyla revealed that it showed the greatest similarity to plant, insect and mammalian Theta class GSTs . Southern blot analysis of plaice DNA hybridized to the PLGSTA cDNA showed a banding pattern indicative of the presence of a single gene . Northern blot analysis of a variety of plaice tissues showed hybridizing bands of approx . 1100 nucleotides in all tissues tested, with the highest relative amounts in liver and intestinal mucosa . A marked increase in hybridization intensity was observed in hepatic RNA samples from plaice treated with trans-stilbene oxide, suggesting that GST-A is induced by epoxides in this species.

J Inorg Biochem, 1993 May 15, 50(3), 193 - 210
Binding of the ferric uptake regulation repressor protein (Fur) to Mn(II), Fe(II), Co(II), and Cu(II) ions as co-repressors: electronic absorption, equilibrium, and 57Fe Mössbauer studies; Hamed MY; The binding of the repressor protein (Fur) to Fe(II) as co-repressor was studied . Other transition metal ions such as Mn(II), Co(II), and Cu(II) were also studied as models . From the equilibrium studies Kd values of 55, 85, 36, and 10 microM were obtained for the Fur complex with Fe(II), Mn(II), Co(II), and Cu(II), respectively . The ratio of metal to Fur monomer was 1:1 in both the Fe(II) and Mn(II) complexes . Fur mutants were also studied . Electronic absorption spectra of the Co(II) Fur complex gave evidence of a distorted tetrahedral Co(II) site bound to sulfur . Frozen solution 57Fe Mossbauer spectra of the Fe(II) Fur indicated the presence of Fe(II) in a high spin distorted octahedral environment . The role of the metal ion as co-repressor in the binding of Fur to DNA is discussed in view of the above results.

J Biol Chem, 1993 May 15, 268(14), 10694 - 700
Effect of sequence, adduct type, and opposing lesions on the binding and repair of ultraviolet photodamage by DNA photolyase and (A)BC excinuclease; Svoboda DL et al.; The cis,syn and (6-4) products of dipyrimidine sites are the major mutagenic and lethal UV photoproducts in DNA . To investigate their relative susceptibilities to repair and other factors such as sequence context and lesions in the complementary strand that might influence repair efficiencies, we constructed 49-mer duplexes containing site-specific photoproducts of thymidylyl(3',5')thymidine sites at central locations . Using these substrates, we measured the binding of Escherichia coli DNA photolyase to cis,syn dimers in four sequence contexts and to two cis,syn dimers in close proximity and on opposing strands . We found that the sequence within a 10-base pair region had little effect on binding and that two enzyme molecules bound to substrate containing two dimers in the 5'-staggered orientation, but not in the 3'-staggered one . Similarly, the excision of a cis,syn dimer by (A)BC excinuclease was not influenced by the sequence in the immediate vicinity of the dimer, and the enzyme was active on 5'-staggered cis,syn dimers, but not on 3'-staggered ones . Of special significance, we found that (A)BC excinuclease removed the cis,syn, trans,syn-I, (6-4), and Dewar photoproducts at vastly differing relative rates of 1:6:9:9, respectively.

J Biol Chem, 1993 May 15, 268(14), 10517 - 23
Rough morphological variants of Mycobacterium avium . Characterization of genomic deletions resulting in the loss of glycopeptidolipid expression; Belisle JT et al.; Previously, a gene cluster, termed ser2, which encodes for the synthesis of the specific oligosaccharide of the glycopeptidolipid antigen of Mycobacterium avium serovar 2 strain TMC 724, was defined . DNA probes from this cloned ser2 gene cluster have now been used to clone and characterize the ser2 region from a strain of M . avium which produces rough and smooth colony forms and to identify the genetic differences between these morphotypes . Interstrain differences were seen to exist between the ser2 gene cluster of M . avium strains TMC 724 and 2151 . In addition, two distinct rough (Rg) genotypes of strain 2151 were defined by this analysis . The first of these, present in the M . avium Rg-0 and Rg-1 variants, was attributed to a deletion of approximately 28 kilobases from smooth variants, including the entire ser2 gene cluster . This particular deletion is thought to be mediated by recombination between repetitive sequences that flank both sides of the 28-kilobase excised region . The second genotype, seen in M . avium Rg-3 and Rg-4 variants, results from the deletion of an undefined amount of DNA from the right of the ser2 gene cluster . Reported separately (Belisle, J . T., McNeil, M . R., Chaterjee, D., Inamine, J . M., and Brennan, P . J . (1993) J . Biol . Chem . 268, 10510-10516) are the results of biochemical analyses of the glycopeptidolipid/lipopeptide population of the Rg genotypes which revealed that Rg-0 and Rg-1 possess lipopeptides devoid of all of the sugars of the glycopeptidolipids and are obviously biosynthetic precursors of the glycopeptidolipids . These studies help formulate a definition of the physiological effects of glycolipid expression, the biosynthetic and genetic mechanisms involved in their formation, and toward an understanding of the role of M . avium as a serious opportunistic pathogen.

J Biol Chem, 1993 May 15, 268(14), 10238 - 45
Molecular cloning of the cDNA for the catalytic subunit of human DNA polymerase epsilon; Kesti T et al.; The cDNA encoding the catalytic polypeptide of human DNA polymerase epsilon was cloned . The deduced amino acid sequence reveals that the catalytic polypeptide is 2257 amino acids in length and its calculated molecular mass is 258 kDa . A single RNA message of 7.5 kilobases was recognized by isolated cDNA clones . The identity of the cDNA was verified by direct amino acid sequencing of tryptic fragments derived from the catalytic polypeptide of the HeLa DNA polymerase epsilon . The primary structure comparison with multiple DNA polymerases indicates that human DNA polymerase epsilon catalytic polypeptide is a homolog of the yeast Saccharomyces cerevisiae DNA polymerase II catalytic polypeptide . The proteins are 39% identical . In the region containing known DNA polymerase consensus motifs, the identity is 63% . The expression of the mRNA encoding DNA polymerase epsilon is strongly dependent on cell proliferation.

Gene, 1993 May 15, 127(1), 99 - 103
A new cloning vector and expression strategy for genes encoding proteins toxic to Escherichia coli; Brown WC et al.; Here, we describe a modification of a plasmid, pT7-7 {Tabor and Richardson, Proc . Natl . Acad . Sci . USA 262 (1985) 1074-1078}, that allows expression of inserted genes from the phage T7 RNA polymerase promoter . The modification is designed to suppress readthrough transcription from cryptic promoters and start points on the plasmid, in order to reduce expression in the absence of T7 RNA polymerase and thus improve the vector for use in the expression of highly toxic gene products . This vector (pT7SC) was used to stably clone the POL3 gene (encoding DNA polymerase delta) of Saccharomyces cerevisiae, which destabilizes all other cloning and expression vectors tested . Previously described expression strategies proved ineffective in overexpressing the POL3 gene . A new strategy was developed which relies on induction by infection with mutant T7 phage . This system efficiently overproduced the POL3 gene product.

Gene, 1993 May 15, 127(1), 47 - 52
Cloning and sequencing the recA+ genes of Acetobacter polyoxogenes and Acetobacter aceti: construction of recA- mutants of by transformation-mediated gene replacement; Tayama K et al.; The recA+ gene of Acetobacter polyoxogenes was cloned as a gene that conferred methyl methanesulfonate resistance (MMSR) on the RecA- Escherichia coli HB101 . The cloned recA+ gene also conferred (i) resistance to UV irradiation, (ii) enhanced intrachromosomal recombination, and (iii) permitted prophage phi 80 induction in E . coli recA- lysogens . Nucleotide sequence determination revealed that the recA product consists of 348 amino acids (aa) corresponding to 38 kDa, and shows significant similarity to RecA proteins from other Gram- bacteria . Next, a portion of recA from Acetobacter aceti was cloned by using polymerase chain reaction with oligodeoxyribonucleotide primers design based on the A . polyoxogenes recA sequence . Due to availability of efficient host-vector and transformation systems in A . aceti, recA mutants of A . aceti were obtained by transformation-mediated gene replacement with the cloned A . aceti recA gene which was inactivated by insertion of the kanamycin-resistance-encoding gene from pACYC177 . The recA mutants obtained in this way showed similar phenotypes to those of E . coli recA strains, such as increased sensitivity to MMS and to UV irradiation, and decreased homologous recombination.

Arch Biochem Biophys, 1993 May 15, 303(1), 107 - 13
Purification and characterization of two phytases from Escherichia coli; Greiner R et al.; Two periplasmatic phytases, called P1 and P2, were purified about 16,500-fold to an apparent homogeneity with a recovery of 7 and 18%, respectively . The enzymes behave as monomeric proteins with molecular masses of about 42 kDa . Because of the limited amounts recovered, the amino terminal sequence of only one of the phytases was determined . Both enzymes are very specific for phytate and have little or no activity on other phosphate esters tested . The kinetic parameters for the hydrolysis of Na-phytate and p-nitrophenyl phosphate are kcat/KM 478 x 10(5) s-1 M-1 and 0.6 x 10(5) s-1 M-1 at pH 4.5 . The hydrolysis pathway for phytate was elucidated for P2; consequently, this enzyme is a 6-phytase . The chemical and kinetic properties of the purified phytase P2 points to an identity with an enzyme described by Dassa et al . (1982, J . Biol . Chem . 257, 6669-6676) as a pH 2.5 acid phosphatase . Because of the kinetic parameters it would be better to denote this enzyme as a phytase.

J Biol Chem, 1993 May 15, 268(14), 10659 - 67
Post-transcriptional regulation of tissue-specific isoforms . A bovine cytosolic RNA-binding protein, COLBP, associates with messenger RNA encoding the liver-form isopeptides of cytochrome c oxidase; Preiss T et al.; Regulation of the liver isopeptides of bovine cytochrome c oxidase is reported to be post-transcriptional . Extensive interspecies sequence homologies exist in the 3'-untranslated regions for transcripts encoding these liver isoforms, suggesting that these regions may be involved in mediating regulation of mRNA expression . To explore this possibility, several bovine tissue homogenates were assayed for any trans-acting factors that recognized the transcript encoding the liver isoform of subunit VIII (BCOL8) . Such a protein factor (COLBP: cytochrome c oxidase L-form transcript-binding protein) was identified in liver, kidney, and lung tissue and was shown to require free sulfhydryl groups for activity . No binding activity, however, was found in muscle-type homogenates . Furthermore, this binding protein also recognized the subunit VIIa-liver transcript but was unable to associate with the mRNA encoding the heart isoform of subunit VIII . Intriguingly, the tissue-specific distribution of COLBP activity parallels the presence of the liver isopeptides in the mature oxidase complex . It is therefore suggested that COLBP may mediate the tissue-specific regulation of cytochrome c oxidase liver isoform mRNA expression.

J Biol Chem, 1993 May 15, 268(14), 10490 - 4
A chimeric type II/type I interleukin-1 receptor can mediate interleukin-1 induction of gene expression in T cells; Heguy A et al.; The type I interleukin-1 receptor (IL-1R) is capable of transducing a signal resulting in promoter activation in T cells . This signal transduction is dependent on the cytoplasmic domain, which consists of 213 amino acids . In contrast to the type I receptor, the type II IL-1R has a small cytoplasmic tail, and it is not clear whether this receptor is a signal-transducing or a regulatory molecule . Here we report that the type II IL-1R does not mediate gene activation in Jurkat cells . However, a hybrid receptor composed of the extracellular and transmembrane regions of the human type II interleukin-1 fused to the cytoplasmic domain of the human type I IL-1R was capable of transducing a signal across the membrane resulting in a pattern of gene activation identical to that mediated by the type I IL-1R . Our results indicated that the extracellular domain of the type II IL-1R was capable of functionally interacting with interleukin-1 and transmitting the resulting signal to a heterologous cytoplasmic domain.

J Biol Chem, 1993 May 15, 268(14), 10168 - 75
Overexpression, purification, and characterization of recombinant human 5-phosphoribosyl-1-pyrophosphate synthetase isozymes I and II; Nosal JM et al.; Human 5-phosphoribosyl-1-pyrophosphate synthetase isozymes I and II (PRSI and PRSII) have been isolated independently and characterized in pure form . cDNAs for PRSI and PRSII were overexpressed in an Escherichia coli strain which lacks the bacterial 5-phosphoribosyl-1-pyrophosphate synthetase . The recombinant isoforms were purified to virtual homogeneity with specific activities of 25.0 and 35.7 units/mg, respectively, values which are 5-10-fold higher than any previously reported for this enzyme from human sources . Despite 95% amino acid sequence identity, the isoforms differed significantly in several physical and kinetic properties . PRSII was more sensitive to heat inactivation at 55 degrees C and more susceptible to disaggregation to inactive forms in the absence of Mg2+ and ATP than was PRSI . The isoforms were separable on the basis of isoelectric point . PRSI and PRSII also differed significantly in Km values for MgATP and ribose 5-phosphate, pH optima, and Mg2+ and Pi activation curves . PRSII was less sensitive to feedback inhibition by purine nucleotides and more sensitive to inhibition by 2,3-diphosphoglycerate than PRSI . Differences in kinetic properties between PRSI and PRSII are consistent with the suggestion that PRSII predominates in rapidly proliferating cells.

FEMS Microbiol Lett, 1993 May 15, 109(2-3), 317 - 22
Actinobacillus pleuropneumoniae haemolysin II is secreted from Escherichia coli by A . pleuropneumoniae pleurotoxin secretion gene products; Macdonald J et al.; Actinobacillus pleuropneumoniae serotype 2 secretes type II haemolysin and pleurotoxin activities . Here, the genes for type II haemolysin were cloned in Escherichia coli, but type II haemolysin antigen and haemolysin activity were only detected intracellularly and not exported to culture supernatant . It has been reported that the genes for type II haemolysin are not linked to functional secretion genes, while those for pleurotoxin are . In this report the means of secretion of type II haemolysin was examined by constructing a hybrid plasmid carrying the genes required for type II haemolysin expression, together with determinants which allow secretion of pleurotoxin and are linked to the pleurotoxin toxin genes . These genes facilitated the export of type II haemolysin from E . coli, and may perform this function in A . pleuropneumoniae.

FEMS Microbiol Lett, 1993 May 15, 109(2-3), 311 - 5
Production of heat-stable enterotoxin II by chicken clinical isolates of Escherichia coli; Akashi N et al.; Enterotoxigenic Escherichia coli isolated from diarrhea stools of chickens were examined for production of heat-stable enterotoxin II which is considered to be implicated only in diarrhea of pigs . Seven out of 38 strains examined were found to contain heat-stable enterotoxin II gene, determined by colony hybridization and the polymerase chain reaction . The culture supernatants of these strains caused fluid accumulation in the mouse intestinal loop test . This fluid accumulation activity was not lost by heating at 100 degrees C and was neutralized by anti-heat-stable enterotoxin II antiserum . Purified heat-stable enterotoxin II caused fluid accumulation in the chicken intestinal loop assay . These results indicate that STII-producing E . coli is implicated in chicken diarrhea.

FEMS Microbiol Lett, 1993 May 15, 109(2-3), 269 - 72
Characterization of an endo-1,3(4)-beta-D-glucanase gene from Cellvibrio mixtus; Sakellaris H et al.; An endo-1,3(4)-beta-D-glucanase gene (cwd2) of Cellvibrio mixtus encoding laminarinase activity was cloned on a 3.9-kb PstI fragment . The Cwd2 enzyme, extracted from recombinant Escherichia coli, degraded both beta-1,3 glucans and beta-1,3-1,4 mixed-linkage glucans, was endohydrolytic and so conformed to the enzyme class 3.2.1.6 . The pH and temperature optima of the enzyme were approximately 7 and 40 degrees C respectively . The M(r) of specifically labelled Cwd2 was approximately 34,000 . This gene was quite distinct from two other C . mixtus beta-1,3 glucanases previously described.

FEMS Microbiol Lett, 1993 May 15, 109(2-3), 219 - 23
Tyr26 and Phe73 are essential for full biological activity of the Fd gene 5 protein; O'Donohue MJ et al.; Using site-saturation mutagenesis, we have established all possible amino acid substitutions at Tyr26 and Phe73 that are compatible with biological activity of the gene 5 protein in vivo . No substitutions were found at either site that gave rise to a fully functional gene 5 protein, indicating that these two amino acid residues are crucial . However, partial activity was found if either residue was replaced by another aromatic amino acid (Y26F, Y26W, F73Y, F73W) . The results suggest that both Tyr26 and Phe73 are important for base stacking in the nucleoprotein complex . The functional consequences of the removal of the hydroxyl group from Tyr26 argue that this residue may, in addition, be involved in hydrogen bond formation to confer greater stability on the complex . In contrast, the addition of such a group to Phe73 reduces activity.

J Biol Chem, 1993 May 15, 268(14), 9957 - 9
The N terminus of the molecular chaperonin GroEL is a crucial structural element for its assembly; Horovitz A et al.; The Escherichia coli heat-shock protein GroEL is a member of the highly conserved family of tetradecameric chaperonins 60, which assist in the folding and assembly of other proteins . Using site-directed mutagenesis, it is shown that replacement of the absolutely conserved amino acid residue Lys-3 by arginine or isoleucine destabilizes the GroEL particle and that the replacement Lys-3-->Glu completely blocks its formation . The rank order of effects of these mutations on the stability of the GroEL particle correlates with the associated changes in net charge at that position . Our results show that the N terminus of GroEL is a crucial structural element for its assembly.

J Biol Chem, 1993 May 15, 268(14), 10356 - 63
Adhesion of S-fimbriated Escherichia coli to brain glycolipids mediated by sfaA gene-encoded protein of S-fimbriae; Prasadarao NV et al.; In an attempt to further assess the role of S-fimbriae in the pathogenesis of Escherichia coli meningitis, the adherence of E . coli strains with or without S-fimbriae were examined for this study to purified glycolipids using thin layer chromatography overlay assays . Only S-fimbriated E . coli strains bound to sulfatide, seminolipid, galactosyl ceramide, and lactosyl ceramide but not to gangliosides including sialyl neolacto-series and other neutral glycolipids . The binding of S-fimbriated E . coli to sulfatide was temperature dependent (i.e . maximal at 37 degrees C) and inhibited by S-fimbriae, anti-S-fimbriae, and anti-S-adhesin antibodies as well as by sulfatide, galactosyl ceramide, and lactosyl ceramide . E . coli transformants which lack the sfaA gene from the Sfa gene cluster showed no binding to the glycolipids, while other transformants lacking the adhesin gene sfaS or sfaG or H and mutants obtained by site-directed mutagenesis in the sfaS gene exhibited a similar binding to the glycolipids compared to the parent S-fimbriated strain . A large amount of sulfated glycolipids was demonstrated on brain endothelial cells and the binding of S-fimbriated E . coli to brain endothelial cells was inhibited by these glycolipids . These findings suggest that the binding of S-fimbriated E . coli to brain endothelial cells occurs in part via glycolipids containing terminal Gal(3SO4)beta-1 residues and in part by S-fimbriae protein SfaA . S-adhesin was not involved in the binding of S-fimbriae to these glycolipids.

J Biol Chem, 1993 May 15, 268(14), 10345 - 50
Oxidation, cross-linking, and insolubilization of recombinant tropoelastin by purified lysyl oxidase; Bedell-Hogan D et al.; The use of recombinant human tropoelastin (rTE) and selected variants thereof as substrates for the assay of lysyl oxidase activity in vitro was explored . The possibility was also assessed that an insoluble elastin-like product could be generated from this elastin precursor in the absence of other macromolecules found associated with elastin in vivo . rTE was more efficiently oxidized by lysyl oxidase than the insoluble chick aorta elastin substrate conventionally used . Anionic amphiphilic elastin ligands strongly inhibited rTE oxidation consistent with the importance of electrostatic enzyme-substrate interactions previously noted with the insoluble elastin substrate . An rTE variant, rTE delta 26A, lacking the hydrophilic sequence coded by exon 26A, was a less effective substrate than rTE, largely due to an increase in Km, while the kinetic parameters for the oxidation of rTE delta 36, lacking the C-terminal polybasic sequence coded by exon 36, were quite similar to those for rTE . Incubation of rTE delta 26A with lysyl oxidase not only resulted in the generation of peptidyl alpha-aminoadipic-delta-semialdehyde and lysine-derived cross-linkages, but also yielded a product insoluble in hot 0.1 N NaOH, consistent with the properties of insoluble elastin . Thus, oxidation, cross-linking and insolubilization of elastin substrates by lysyl oxidase can occur in the absence of other macromolecules implicated as being involved in this process in vivo, although such macromolecules may be essential to obtain the proper alignment between tropoelastin units for specifically placed cross-linkages and optimally functional elastic fibers.

J Biol Chem, 1993 May 15, 268(14), 10246 - 51
The folding and stability of rhodanese are influenced by the replacement of glutamic acid 17 in the NH2-terminal helix by proline but not by glutamine; Luo GX et al.; Two site-directed mutants of the enzyme rhodanese which replace glutamic acid 17 with either glutamine (E17Q) or with proline (E17P) were produced and purified . Both mutants displayed specific activities similar to the wild type enzyme . E17Q was equivalent to the wild type enzyme in all assayed characteristics, except that the mutant had slightly more solvent exposure of hydrophobic surfaces . Results with E17Q suggest that the charge on Glu17 is not required for helix stabilization, nor is its titration required for the low pH structural transitions seen previously . In contrast, E17P was significantly different from the wild type enzyme . For example, E17P had (a) higher exposure of hydrophobic surfaces in the unperturbed state; (b) considerably lower stability to perturbation by urea; (c) easier exposure of organized hydrophobic surfaces on initial unfolding, even though denaturation to the final disorganized state was the same as for the wild type; (d) the ability to refold without assistants but with lower yields and somewhat slower folding; and (e) similar susceptibility to trypsin and evidence of a new clip site closer to the NH2 terminus . However, E17P and the wild type enzyme had very similar recoveries with chaperonin-assisted refolding, and the chaperonin protein groEL had a very similar ability to suppress unassisted refolding . These results indicate that changes in the NH2-terminal sequence can have dramatic effects on the stability of rhodanese and on its ability to be refolded in the absence of assistants . They further suggest that interactions with chaperonins do not rely exclusively on the detailed conformation at the NH2 terminus . A model that incorporates observations here includes step(s) in which the NH2-terminal sequence folds onto the NH2-terminal domain late in the folding process after the protein had adopted a near native conformation.

FEMS Microbiol Lett, 1993 May 15, 109(2-3), 335 - 42
Recognition of the colicin A N-terminal epitope 1C11 in vitro and in vivo in Escherichia coli by its cognate monoclonal antibody; Geli V et al.; We demonstrate that the 1C10 monoclonal antibody (mAb) directed against the N-terminal domain of the colicin A recognizes a 13 residue-region (13Thr-Gly-Trp-Ser-Ser-Glu-Arg-Gly-Ser-Gly-Pro- Asp-Pro25) . When this peptide is inserted into a protein in the amino-terminal or an internal position, the tagged protein is efficiently detected by the 1C11 mAb either by immunoblotting or immunoprecipitation . In vitro, the minimal structure required for detection using the pepscan system is 19Arg-Gly-Ser-Gly-Pro-Glu-Pro25, indicating that in vivo the proper exposure of the epitope requires additional residues . The construction of a versatile vector allowing overproduction of tagged proteins is described . Various applications of the 1C11 epitope are mentioned . This epitope did not alter the function of any of the proteins so far tested.

Eur J Biochem, 1993 May 15, 214(1), 295 - 304
Novel hirudin variants from the leech Hirudinaria manillensis . Amino acid sequence, cDNA cloning and genomic organization; Scacheri E et al.; Novel hirudin variants isolated from the leech Hirudinaria manillensis, a leech more specialized for mammalian parasitism, are described . Isolation of antithrombin polypeptides was performed by ion-exchange chromatographies followed by an affinity chromatography step on immobilized thrombin . The major active component, antithrombin polypeptide peak 2 (HM2) and a second polypeptide, named HM1, were purified to homogeneity and their complete amino acid sequences were determined . The protein structure of the two hirudin variants include 64 amino acids with 6 cysteine residues at highly conserved positions . Comparison of the amino acid sequences of HM1 and HM2 with other known hirudins shows differences mainly in the central part and in the C-terminal region of the polypeptides . Particularly significant is the lack of a sulfated tyrosine residue in the C-terminal portion of the molecule which is replaced by aspartic acid . Polymerase chain reaction cloning techniques were used to isolate and characterize the cDNAs and determine the genomic structures of these hirudin-like polypeptides . The cDNA clones coding for the two variants indicate the expression of pre-hirudins of 84 amino acids where the first 20 residues constitute the signal peptide required for extracellular secretion . The leader sequence appears to be highly conserved for both isoforms and shares a complete similarity with the partial hirudin variant 2 (HV2) signal peptide sequence previously reported . The HM1 and HM2 gene fragments show the presence of four exons: the first one corresponding to a 20-amino-acid signal peptide while the other three exons share the full primary structure of the antithrombin polypeptides . HM2 was also efficiently produced in recombinant Escherichia coli by expressing a periplasmic construction containing the synthetic gene.

Eur J Biochem, 1993 May 15, 214(1), 259 - 65
Structural analysis of O4-reactive polysaccharides from recombinant Escherichia coli . Changes in the O-specific polysaccharide induced by cloning of the rfb genes; Kogan G et al.; In previous studies it had been shown that lipopolysaccharide from O4-specific recombinant Escherichia coli, had serological reactivities and a chemical composition that differed from wildtype O4 LPS {Haraguchi, G.E., Zahringer, U., Jann, B., Jann, K., Hull, R.A . & Hull, S.I . (1991) Microb . Pathog . 10, 351-361} . Here we present the structural elucidation of the O-specific moieties from lipopolysaccharides of some of the recombinant strains obtained in previous studies . Compositional analysis, methylation, chemical reactions and NMR spectroscopy showed that, during genetic manipulations (recombination, cosmid cloning, plasmid subcloning), a gradual structural change in the O-specific polysaccharides was observed in the recombinant strains . These changes comprised of an alteration in the position of glucose (side chain) substitution, a change in the anomeric configuration of the main-chain N-acetylglucosamine and an exchange of alpha-L-rhamnopyranose for beta-D-galactofuranose . The relevance of these results for lipopolysaccharide cloning and lipopolysaccharide biosynthesis are discussed.

Proc Natl Acad Sci U S A, 1993 May 15, 90(10), 4399 - 403
Allosteric mechanism for translational repression in the Escherichia coli alpha operon; Spedding G et al.; The ribosomal protein S4 is a translational repressor that binds to a complex mRNA pseudoknot structure containing the ribosome binding site for the first gene of the alpha operon . Either 30S subunits or S4 protein bound to the mRNA causes Moloney murine leukemia virus reverse transcriptase to pause near the 3' terminus of the pseudoknot . There is no competition between subunits and S4 for mRNA binding . The kinetics of forming S4-30S-mRNA complexes are biphasic, and the fraction of mRNA molecules reacting more rapidly decreases as the temperature is increased from 30 degrees C to 40 degrees C . The complex cannot be detected with mRNA mutants that cannot be repressed . We have previously shown similar kinetic behavior for the formation of tRNA(fMet) initiation complexes with tRNA(fMet), 30S subunits, and mRNA, except that the fraction reacting rapidly increases when the temperature is increased over the same 30-40 degrees C range . Thus the two sets of experiments show that there are two forms of 30S-mRNA complexes that differ in their abilities to bind S4 and tRNA(fMet) . The results support an allosteric model for translational repression in which S4 traps the mRNA in a conformation able to bind 30S subunits but unable to form an initiation complex with tRNA(fMet).

Proc Natl Acad Sci U S A, 1993 May 15, 90(10), 4394 - 8
Ribosomal protein S15 from Escherichia coli modulates its own translation by trapping the ribosome on the mRNA initiation loading site; Philippe C et al.; From genetic and biochemical evidence, we previously proposed that S15 inhibits its own translation by binding to its mRNA in a region overlapping the ribosome loading site . This binding was postulated to stabilize a pseudoknot structure that exists in equilibrium with two stem-loops . Here, we use "toeprint" experiments with Moloney murine leukemia virus reverse transcriptase to analyze the effect of S15 on the formation of the ternary mRNA-30S-tRNA(fMet) complex . We show that the binding of the 30S subunit on the mRNA stops reverse transcriptase near position +10, corresponding to the 3' terminus of the pseudoknot, most likely by stabilizing the pseudoknot conformation . Furthermore, S15 is found to stabilize the binary 30S-mRNA complex . When the ternary 30S-mRNA-tRNA(fMet) complex is formed, a toeprint is observed at position +17 . This toeprint progressively disappears when the ternary complex is formed in the presence of increasing concentrations of S15, while a shift from position +17 to position +10 is observed . Beside, RNase T1 footprinting experiments reveal the simultaneous binding of S15 and 30S subunit on the mRNA . Otherwise, we show by filter binding assays that initiator tRNA remains bound to the 30S subunit even in the presence of S15 . Our results indicate that S15 prevents the formation of a functional ternary 30S-mRNA-tRNA(fMet) complex, the ribosome being trapped in a preternary 30S-mRNA-tRNA(fMet) complex.

Proc Natl Acad Sci U S A, 1993 May 15, 90(10), 4350 - 3
Substitution of the 3' terminal adenosine residue of transfer RNA in vivo; Reuven NB et al.; We have altered by site-directed mutagenesis the 3' terminal adenosine residue of a tRNA(Tyrsu3+) gene encoded on a single-copy plasmid and examined the consequences of these substitutions on suppressor activity in vivo . Our data show that mutant su3 genes containing 3'-CCC, -CCG, or -CCU termini instead of -CCA can be efficiently transcribed and processed in Escherichia coli to generate functional suppressor tRNAs . However, in contrast to normal tRNA genes, both tRNA nucleotidyltransferase and exoribonuclease activities are required to obtain suppression by the mutant tRNAs, indicating that removal of the incorrect 3' terminal residue and resynthesis of the normal -CCA terminus are occurring in this situation . In addition, a low level of suppressor activity and tRNA repair was found in cells devoid of tRNA nucleotidyltransferase, suggesting that an additional activity able to partially repair the 3' end of tRNA is present in E . coli . The use of mutant strains lacking one or several exoribonucleases revealed that the various RNAses have very different specificities for removal of incorrect 3' residues and that these differ greatly from their action on CCA-ending tRNA . These data show that the 3' terminal adenosine residue is necessary for tRNA function in vivo and that cells can compensate for its alteration by changes in the normal pathway of tRNA metabolism.

J Biol Chem, 1993 May 15, 268(14), 10471 - 81
Identification of sites for feedback regulation of glutamine 5-phosphoribosylpyrophosphate amidotransferase by nucleotides and relationship to residues important for catalysis; Zhou G et al.; Glutamine phosphoribosylpyrophosphate amidotransferase, the key regulatory enzyme for de novo purine nucleotide synthesis, is subject to feedback regulation by adenine and guanine nucleotides . Affinity labeling with 5'-p-fluorosulfonylbenzoyladenosine (FSBA) and 8-azidoadenosine 5'-monophosphate (N3-AMP) was used to identify purine nucleotide sites for feedback control of the Escherichia coli amidotransferase . FSBA inactivated the amidotransferase with saturation kinetics . Specificity for inactivation was shown by the covalent attachment of 2.0-2.4 eq of {3H} sulfobenzoyladenosine (SBA) per subunit and protection by GMP and AMP against inactivation and incorporation of {3H}SBA . Six chymotryptic peptides modified with {3H}SBA were isolated and identified by differential labeling followed by high performance liquid chromatography and radioactivity . Mass spectrometry and Edman degradation analysis were used to identify 5 residues that were covalently modified by {3H}SBA: Tyr74, Tyr258, Lys326, Tyr329, and Tyr465 . Tyr258 was also modified by N3-AMP . Mutant enzymes K326Q and Y329A had activity similar to that of the wild type enzyme . However, both mutants exhibited decreased sensitivity to inhibition by GMP and decreased binding of GMP but were inhibited by AMP . Mutant enzymes Y74A and Y258F were normally feedback-inhibited but were defective in glutamine amide transfer and synthase functions, respectively . Therefore Tyr74 and Tyr258 are important for activity and modification by FSBA and N3-AMP accounts for enzyme inactivation . These results localize residues important for catalysis in close proximity to a site for nucleotide binding . Two additional mutant enzymes, G331I and N351A, were constructed which were refractory to inhibition by GMP with little change in inhibition by AMP . A replacement of Tyr465 indicates that this residue is not essential for catalysis or feedback inhibition . Overall, these results are interpreted in terms of a two-nucleotide site model with Lys326, Tyr329, Gly331, and Asn351 defining a site required for inhibition by GMP . A second nucleotide site not affinity labeled by analogs is very close to or overlaps with the catalytic site.

J Biol Chem, 1993 May 15, 268(14), 10380 - 5
Tissue-specific expression and chromosomal localization of the alpha subunit of mouse meprin A; Jiang W et al.; Meprins, membrane-bound oligomeric metalloendopeptidases, contain alpha and/or beta subunits . Their activities have been found in the mouse and rat kidney . The cloned cDNA for the mouse alpha subunit of meprin A (EC cloned cDNA for the mouse alpha subunit of meprin A (EC 3.4.24.18) was used here to survey mRNA expression in kidney of different mouse strains and in various tissues of mice and rats . A single message of 3.6 kilobases was found in kidney of random bred (ICR) and inbred mice (C57BL/6, DBA/2) that contain high meprin A activity and in Sprague-Dawley rat kidney . The alpha subunit message was undetectable in the kidney of C3H/He and CBA mice, inbred strains that do not express meprin A activity . Therefore, meprin A activity in the kidney of mouse strains correlates with the amount of alpha subunit mRNA present . The 3.6-kilobase mRNA meprin alpha subunit message was also detected in the small intestine of the rat but not in mice . No message was detected in brain, heart, skeletal muscle, liver, lung, or spleen of mice or rats . Polymerase chain reaction amplification or Southern blot analysis of genomic DNA revealed that the gene for the alpha subunit is present in all mouse strains as well as in human, monkey, rat, mouse, dog, cow, rabbit, and chicken, but it was not detected in yeast . There is one gene copy present in the mouse genome . The gene was localized to mouse chromosome 17 centromeric to the major histocompatibility complex (H-2) by the interspecific backcrossing method . The localization of this allele to Mep-1, the gene previously found to regulate the expression of meprin A activity in mice, supports the proposal that Mep-1 is the structural gene for the alpha subunit.

J Biol Chem, 1993 May 15, 268(14), 10324 - 34
Error-prone polymerization by HIV-1 reverse transcriptase . Contribution of template-primer misalignment, miscoding, and termination probability to mutational hot spots; Bebenek K et al.; We have observed previously that DNA template-directed polymerization by the type 1 human immunodeficiency virus reverse transcriptase is error-prone for single-nucleotide substitution, addition and deletion errors at homopolymeric sequences . We have also noted strong termination of processive synthesis at these positions (Bebenek, K., Abbotts, J., Roberts, J . D., Wilson, S . H., and Kunkel, T . A . (1989) J . Biol . Chem . 264, 16948-16956) . Here we have tested three models to explain errors at these hot spots: template-primer misalignment for deletion errors, and dislocation and direct miscoding for substitution errors . The approach involves introducing single-nucleotide changes within or flanking the homopolymeric hot spots and examining the effects that these changes have on human immunodeficiency virus type 1 (HIV-1) reverse transcriptase error rate, error specificity, and termination probability . The results obtained suggest that single-nucleotide deletion errors in homopolymeric runs result from template-primer misalignment and that both direct miscoding and template-primer dislocation contribute to the base substitution hot spots . The data also suggest that base substitution errors at one position can be templated by the preceding nucleotide or either of the next two nucleotides . Frameshift error rates at homopolymeric sites were affected by changes in the sequences flanking the runs, including single-nucleotide differences in the single-stranded template strand and in the double-stranded primer region as many as six nucleotides distant from the hot spot . Both increases and decreases in frameshift fidelity were observed, and most of these correlated with concomitant increases or decreases in the probability that HIV-1 reverse transcriptase terminated processive synthesis within the run . These data provide further support for a relationship between the frameshift fidelity and the processivity of DNA-dependent DNA synthesis by HIV-1 reverse transcriptase.

J Biol Chem, 1993 May 15, 268(14), 10312 - 23
Mechanism of HIV-1 reverse transcriptase . Termination of processive synthesis on a natural DNA template is influenced by the sequence of the template-primer stem; Abbotts J et al.; During processive DNA synthesis in vitro, the human immunoefficiency virus, type 1 (HIV-1) reverse transcriptase encounters template nucleotide positions at which continued synthesis is difficult . At these positions, the enzyme has a relatively high probability of dissociating from the template, and product molecules of corresponding length accumulate as the incubation proceeds . These positions, which are known as termination sites, could be associated with template secondary structures in some cases, but many termination sites appear to be template sequence-related rather than secondary structure-related . Mechanisms producing these blocks in processive DNA synthesis are not well understood . In this study, to examine further the effects of template sequence on termination, we engineered selected single-base changes in the M13mp2 template, and we found that such changes can influence termination . Several general trends emerged from the study . First, strong termination sites rarely correspond to dATP as the "incoming" substrate opposite template T . Second, the sequence of the template-primer stem is more important for termination than the sequence of the single-stranded template ahead of the primer . Thus, we note the phenomenon of action at a distance: changing sequence at one nucleotide position in the template-primer stem alters termination at other positions, a few nucleotides distant at the primer 3' end . A and C as template bases in the template-primer stem have opposite effects . A is the strongest terminator residue, and C is the weakest terminator residue, followed by G . Since termination sites are produced by reverse transcriptase dissociation from the template-primer, the results suggest that the HIV-1 reverse transcriptase has properties reminiscent of a sequence-specific double-stranded DNA-binding protein in that its binding mechanism can distinguish both base residues and positions in the double-stranded DNA template-primer stem.

J Biol Chem, 1993 May 15, 268(14), 10176 - 84
Intramembrane helix-helix interactions as the basis of inhibition of the colicin E1 ion channel by its immunity protein; Zhang YL et al.; It had previously been hypothesized that the ability of a small number of immunity protein molecules in the cytoplasmic membrane to confer protection against the lethal effects of a channel-forming colicin involves a complex stabilized by electrostatic or polar interactions between immunity protein, the colicin channel, and specific sites on the cytoplasmic membrane surface defined by the presence of the tol gene translocation proteins . The hypothesis was tested (a) by constructing a hybrid colicin molecule, IaE1, containing the E1 channel domain, and the translocation and receptor domains of Ia, and (b) by altering charged residues in all peripheral regions of the immunity protein to neutral residues . It was concluded that the specificity of immunity protein requires neither specific translocation proteins, nor a specific arrangement of charged residues in the immunity protein . (c) In addition, by making 65 site-directed mutations, "immunity by-pass" mutants were found at five different loci, Ala474, Ser477, His440, Phe443, and Gly444, on two proposed membrane-spanning helices of the open colicin channel, one hydrophobic (A471-A488) and one amphiphilic (V441-W460) . The mutants in the hydrophobic helix showed a larger bypass effect . The "bypass" phenotype could be assayed by (i) cytotoxicity and (ii) K+ efflux in imm+ cells caused by a bypass mutant but not wild-type colicin . It is concluded that the immunity protein exerts its specific effect through rapid lateral diffusion in the cytoplasmic membrane and helix-helix recognition and interaction with at least one hydrophobic and one amphiphilic trans-membrane helix of the colicin channel . Interaction with the amphiphilic helix implies that the immunity protein can react with the channel in the open state.

Biochem Biophys Res Commun, 1993 May 14, 192(3), 1230 - 7
Expression and activity of pre-dianthin 30 and dianthin 30; Legname G et al.; Dianthin 30 is a ribosome inactivating protein (RIP 1) found in different tissues of the carnation (Dianthus caryophyllus) . Recently we have isolated and sequenced a cDNA clone from a lambda gt11 expression library {Legname et al . (1991) Biochim . Biophys . Acta 1090, 119-122} . Here we describe specific PCR amplifications of either the full length pre-dianthin 30 or dianthin 30, the mature polypeptide lacking the 23 amino acid signal peptide . In vitro expression of both proteins in reticulocyte lysate generated products of the expected molecular weight . Moreover, the activity of both proteins has been evaluated confirming the characteristics of the natural product . A first attempt to produce recombinant dianthin 30 in Escherichia coli is described.

Biochem Biophys Res Commun, 1993 May 14, 192(3), 1023 - 9
Rat 12-lipoxygenase: mutations of amino acids implicated in the positional specificity of 15- and 12-lipoxygenases; Watanabe T et al.; Site directed mutagenesis, based on the polymerase chain reaction, was carried out on a rat 12-lipoxygenase cDNA within a region that encodes several amino acids believed to be of importance for the positional specificity of 15- and 12-lipoxygenases . The following mutants were constructed to increase the 15-lipoxygenase activity of rat 12-lipoxygenase; {Leu397}12-lipoxygenase, {Ile389, Leu397}12-lipoxygenase, {Gln416}12-lipoxygenase, {Ile417}12-lipoxygenase, and {Gln416, Ile417}12-lipoxygenase . Each mutated cDNA was expressed in Escherichia coli and 12- and 15-lipoxygenase activity was assayed in the 10,000 x g supernatants of homogenized cells . When compared to wild type enzyme, none of the mutants exhibited significantly increased 15-lipoxygenase activity . Thus, considering the primary structure of wild type enzyme and our results of mutagenetic replacements, we have not found evidence indicating that amino acids in positions 416, 417, or 418 are critical for the positional specificity of rat 12-lipoxygenase.

Biochem Biophys Res Commun, 1993 May 14, 192(3), 1409 - 14
Effect of topoisomerase inhibitors on the in vitro HIV DNA integration reaction; Carteau S et al.; Retroviral growth requires as an obligatory step the integration of a DNA copy of the viral RNA into the genomic DNA of the host . Recombinant human immunodeficiency virus type I (HIV-1) integrase (IN) expressed in Escherichia coli efficiently catalyzes the overall in vitro integration reaction, namely, the processing of the LTR ends and the strand transfer reaction . Using the 3' end of synthetic oligonucleotides which match the termini of the HIV-I U5 LTR as substrate and supercoiled pSP65 DNA as target, we have measured the effect of various topoisomerase inhibitors on the functional activity of the IN protein . Among the various drugs tested, the antitumor drug 2N-Methyl, 9-hydroxyellipticinium (NMHE) displays a marked inhibitory effect on the IN-catalyzed U5 insertion . This effect is related to the DNA binding properties of the drug rather than to a selective effect on the IN protein or the DNA-IN protein complex.

Biochem Biophys Res Commun, 1993 May 14, 192(3), 1403 - 8
Sulfite reductase of Escherichia coli is a ferrisiderophore reductase; Coves J et al.; A soluble ferrisiderophore reductase activity of Escherichia coli was purified to homogeneity and identified as the sulfite reductase . The pure enzyme catalyzes the reduction of ferric citrate, ferriaerobactin, ferrioxamin, ferricrocin, ferrichrome and ferrifusarinin by NADPH . Free flavins, riboflavin, FMN, FAD were absolutely required, suggesting that this activity resides in the flavin reductase activity of sulfite reductase.

Nucleic Acids Res, 1993 May 11, 21(9), 2109 - 15
Two regions of the Escherichia coli 16S ribosomal RNA are important for decoding stop signals in polypeptide chain termination; Brown CM et al.; Two regions of the 16S rRNA, helix 34, and the aminoacyl site component of the decoding site at the base of helix 44, have been implicated in decoding of translational stop signals during the termination of protein synthesis . Antibiotics specific for these regions have been tested to see how they discriminate the decoding of UAA, UAG, and UGA by the two polypeptide chain release factors (RF-1 and RF-2) . Spectinomycin, which interacts with helix 34, stimulated RF-1 dependent binding to the ribosome and termination . It also stimulated UGA dependent RF-2 termination at micromolar concentrations but inhibited UGA dependent RF-2 binding at higher concentrations . Alterations at position C1192 of helix 34, known to confer spectinomycin resistance, reduced the binding of f{3H}Met-tRNA to the peptidyl-tRNA site . They also impaired termination in vitro, with both factors and all three stop codons, although the effect was greater with RF-2 mediated reactions . These alterations had previously been shown to inhibit EF-G mediated translocation . As perturbations in helix 34 effect both termination and elongation reactions, these results indicate that helix 34 is close to the decoding site on the bacterial ribosome . Several antibiotics, hygromycin, neomycin and tetracycline, specific for the aminoacyl site, were shown to inhibit the binding and function of both RFs in termination with all three stop codons in vitro . These studies indicate that decoding of all stop signals is likely to occur at a similar site on the ribosome to the decoding of sense codons, the aminoacyl site, and are consistent with a location for helix 34 near this site.

Nucleic Acids Res, 1993 May 11, 21(9), 2095 - 101
Consensus sequences for good and poor removal of uracil from double stranded DNA by uracil-DNA glycosylase; Eftedal I et al.; We have purified uracil DNA-glycosylase (UDG) from calf thymus 32,000-fold and studied its biochemical properties, including sequence specificity . The enzyme is apparently closely related to human UDG, since it was recognised by a polyclonal antibody directed towards human UDG . SDS-PAGE and western analysis indicate an apparent M(r) = 27,500 . Bovine UDG has a 1.7-fold preference for single stranded over double stranded DNA as a substrate . Sequence specificity for uracil removal from dsDNA was examined for bovine and Escherichia coli UDG, using DNA containing less than one dUMP residue per 100 nucleotides and synthetic oligonucleotides containing one dUMP residue . Comparative studies involving about 40 uracil sites indicated similar specificities for both UDGs . We found more than a 10-fold difference in rates of uracil removal between different sequences . 5'-G/CUT-3' and 5'-G/CUG/C-3' were consensus sequences for poor repair whereas 5'-A/TUAA/T-3' was a consensus for good repair . Sequence specificity was verified in double stranded oligonucleotides, but not in single stranded ones, suggesting that the structure of the double stranded DNA helix has influence on sequence specificity . Rate of uracil removal appeared to be slightly faster from U:A base pairs as compared to U:G mis-matches . The results indicate that sequence specific repair may be a determinant to be considered in mutagenesis.

Nucleic Acids Res, 1993 May 11, 21(9), 2045 - 9
Excision of 3-methylguanine from alkylated DNA by 3-methyladenine DNA glycosylase I of Escherichia coli; Bjelland S et al.; Escherichia coli has two DNA glycosylases for repair of DNA damage caused by simple alkylating agents . The inducible AlkA DNA glycosylase (3-methyladenine {m3A} DNA glycosylase II) removes several different alkylated bases including m3A and 3-methylguanine (m3G) from DNA, whereas the constitutively expressed Tag enzyme (m3A DNA glycosylase I) has appeared to be specific for excision of m3A . In this communication we have reexamined the substrate specificity of Tag by using synthetic DNA rich in GC base pairs to facilitate detection of any possible methyl-G removal . In such DNA alkylated with {3H}dimethyl sulphate, we found that m3G was excised from double-stranded DNA by both glycosylases, although more efficiently by AlkA than by Tag . This was further confirmed using both N-{3H}methyl-N-nitrosourea- and {3H}dimethyl sulphate-treated native DNA, from which Tag excised m3G with an efficiency that was about 70 times lower than for AlkA . These results can explain the previous observation that high levels of Tag expression will suppress the alkylation sensitivity of alkA mutant cells, further implying that m3G is formed in quantity sufficient to represent an important cytotoxic lesion if left unrepaired in cells exposed to alkylating agents.

Biochemistry, 1993 May 11, 32(18), 4761 - 8
Mutagenesis of ribosomal protein S8 from Escherichia coli: expression, stability, and RNA-binding properties of S8 mutants; Wu H et al.; Protein S8, a 129 amino acid component of the Escherichia coli ribosome, plays an essential role in the assembly of the 30S ribosomal subunit and in the translational regulation of the spc operon by virtue of its capacity to bind specifically to rRNA and mRNA . To study structure-function relationships within the protein, we have constructed a vector for its high-level expression in vivo and developed efficient methods for its purification . Under our conditions, S8 accumulates to a level of 35% of the cellular protein and can be prepared at a purity of over 98% using either HPLC or a combination of ion-exchange and gel-filtration chromatography . The unique cysteine residue at position 126 was replaced by alanine or serine by oligonucleotide-directed mutagenesis, and the two mutant proteins, CA126 and CS126, were expressed and isolated . The effects of the mutations on the RNA-binding ability, secondary structure, and stability of S8 were assessed . CD spectra indicated that wild-type S8 and the two mutant proteins have very similar secondary structures at 25 degrees C . In addition, both mutants are metabolically stable in vivo as inferred from pulse-chase labeling and immunoprecipitation experiments . However, while CA126 exhibits the same affinity for RNA and the same susceptibility to urea and thermal denaturation as wild-type S8, CS126 is severely impaired in its ability to interact with RNA and displays a dramatic reduction in conformational stability . Our results suggest that Cys126 is unlikely to play a specific role in RNA recognition but that it is an integral part of the RNA-binding domain of protein S8.

Biochemistry, 1993 May 11, 32(18), 4881 - 94
Conformational behavior of Escherichia coli OmpA signal peptides in membrane mimetic environments; Rizo J et al.; Nuclear magnetic resonance and circular dichroism (CD) studies of isolated peptides corresponding to WT and mutant OmpA signal sequences are reported; all of the peptides adopt substantial amounts of alpha-helical structure both in 1:1 (v/v) trifluoroethanol (TFE)/water and in sodium dodecyl sulfate (SDS) micelles . In TFE/water, the helix begins after the positively charged N-terminal residues and is most stable in the hydrophobic core, which correlates with results obtained previously for other signal sequences . The helix is weaker between the hydrophobic core and the C-terminus; such a break in the helix appears to be common to other signal peptides studied previously and could be of functional importance . No clear correlation could be established between the helicity of the peptides in TFE/water and their in vivo activities . All the peptides have a higher alpha-helix content in SDS than in TFE/water, and there is a good correlation between helix content in SDS and in vivo activity . Helicity in SDS for the functional peptides increases both at the N-terminus and in the hydrophobic core, and is driven by a strong association of the core with the hydrophobic chains of the detergent . The extension of the helix toward the N-terminus may be a result of neutralization of the N-terminal positive charges by the headgroups of the micelles, which removes unfavorable electrostatic interactions with the helix dipole . All these comparisons were facilitated by the use of upfield shifts of H alpha protons in helical regions relative to random coil chemical shifts, which also yielded estimates of helical content that correlated well with the CD results.

Biochemistry, 1993 May 11, 32(18), 4873 - 80
Kinetics and processivity of ATP hydrolysis and DNA unwinding by the RecBC enzyme from Escherichia coli; Korangy F et al.; The RecB and RecC subunits of the RecBCD enzyme from Escherichia coli were purified from cells containing plasmids overproducing these proteins {Boehmer, P.E., & Emmerson, P.T . (1991) Gene 102, 1-6} . RecB hydrolyzes ATP in the presence of either single- or double-stranded DNA . RecC stimulates ATP hydrolysis by RecB, particularly with double-stranded DNA . The steady-state kinetic parameters for ATP hydrolysis by RecBC with double-stranded DNA are kcat = 1600 min-1, Km = 8.1 microM, and kcat/Km(ATP) = 1.97 x 10(8) M-1 min-1 . The RecBC enzyme acts processively, as measured by the effect of heparin on ATP hydrolysis stimulated by double-stranded DNA . About 2400 ATP molecules are hydrolyzed per enzyme bound to the end of a DNA molecule, using DNA substrates of 6250 or 21,400 base pairs . The enzyme is capable of unwinding a 6250 base pair double-stranded DNA molecule, in the presence of the single-stranded DNA binding protein of Escherichia coli . The steady-state kinetic parameters and the processivity are close to those found previously for the RecBCD-K177Q enzyme, with a lysine-to-glutamine mutation in the consensus ATP binding sequence in the RecD subunit, and are reduced compared to the RecBCD holoenzyme {Korangy, F., & Julin, D . A . (1992) J . Biol . Chem . 267, 1733-1740} . The most salient difference between RecBC and RecBCD-K177Q is the nuclease activity . RecBCD-K177Q produces a significant amount of acid-soluble DNA fragments from double-stranded DNA, while RecBC does not, even though the DNA does become unwound.

Nucleic Acids Res, 1993 May 11, 21(9), 2173 - 8
Prediction of alternative RNA secondary structures based on fluctuating thermodynamic parameters; Le SY et al.; In this paper we present a new method for predicting a set of RNA secondary structures that are thermodynamically favored in RNA folding simulations . This method uses a large number of 'simulated energy rules' (SER) generated by perturbing the free energy parameters derived experimentally within the range of the experimental errors . The structure with the lowest free energy is computed for each SER . Structural comparisons are used to avoid multiple generation of similar structures . Computed structures are evaluated using the energy distribution of the lowest free energy structures derived in the simulation . Predicted be graphically displayed with their occurring frequencies in the simulation by dot-plot representations . On average, about 90% of phylogenetic helixes in the known models of tRNA, Group I self-splicing intron, and Escherichia coli 16 S rRNA, were predicted using the method.

FEBS Lett, 1993 May 10, 322(2), 201 - 4
Effects of sulfhydryl reagents on the Cys65 mutant of the transposon Tn10-encoded metal-tetracycline/H+ antiporter of Escherichia coli; Yamaguchi A et al.; The Cys65 mutant of the Tn10-encoded metal-tetracycline/H+ antiporter is the only one which is inactivated by sulfhydryl reagents among the Cys mutants of the putative loop2-3 region {Yamaguchi, A . et al . (1992) J . Biol . Chem . 267, 19155-19162} . The tetracycline transport activity of the Cys65 mutant was completely abolished by N-ethylmaleimide; however, methyl methanethiosulfonate only abolished 45% of the activity, even in the presence of an excess of the reagent . Since N-ethylmaleimide did not further inactivate the methyl methanethiosulfonate-treated antiporter, it is clear that the modified antiporter molecule with a small substituent, a thiomethyl group, had significant but lower activity than the unmodified antiporter . The binding of {14C}N-ethylmaleimide to the Cys65 mutant was inhibited in the presence of tetracycline . These findings indicate that position 65 is close to the site of the interaction with the substrate and the modification of the side chain at this position caused steric hindrance as to substrate translocation.

J Theor Biol, 1993 May 7, 162(1), 75 - 80
A simple method to forecast good promoters of Escherichia coli by means of doublet frequencies; Recknagel RD et al.; A training sample (taken offhand) of 14 promoter sequences, 70 or 69 base pairs in length each, with their (relative) promoter strengths given by Deuschle et al . (1986), is used to find a relation between some sequence characteristics and the promoter strength . The analysis is based on the doublet frequencies of purines and pyrimidines in the promoter sequence under study.

Science, 1993 May 7, 260(5109), 805 - 7
Protein-induced bending as a transcriptional switch; Perez-Martin J et al.; The question of whether protein-induced DNA bending can act as a switch factor when placed upstream of an array of promoters located in tandem was investigated in vivo . The catabolite activating protein binding site of the fur operon was replaced by the binding site of the RepA repressor protein, which is able to bend DNA immediately after binding . Appropriately phased induced bending could act as a transcriptional switch factor in vivo.

Nature, 1993 May 6, 363(6424), 85 - 8
Guanine-nucleotide-releasing factor hSos1 binds to Grb2 and links receptor tyrosine kinases to Ras signalling; Li N et al.; Many of the actions of receptor tyrosine kinases are mediated by the protein Ras, including the activation of various downstream serine/threonine kinases and the stimulation of growth and differentiation . The human protein Grb2 binds to ligand-activated growth factor receptors and downstream effector proteins through its Src-homology (SH) domains SH2 and SH3, respectively, and like its homologue from Caenorhabditis elegans, Sem-5, apparently forms part of a highly conserved pathway by which these receptors can control Ras activity . Here we show that the SH3 domains of Grb2 bind to the carboxy-terminal part of hSos1, the human homologue of the Drosophila guanine-nucleotide-releasing factor for Ras, which is essential for control of Ras activity by epidermal growth factor receptor and sevenless . Moreover, a synthetic 10-amino-acid peptide containing the sequence PPVPPR specifically blocks the interaction . These results indicate that the Grb2/hSos1 complex couples activated EGF receptor to Ras signalling.

Nature, 1993 May 6, 363(6424), 38 - 45
Recognition by Max of its cognate DNA through a dimeric b/HLH/Z domain; Ferre-D'Amare AR et al.; The three-dimensional structure of the basic/helix-loop-helix/leucine zipper domain of the transcription factor Max complexed with DNA has been determined by X-ray crystallography at 2.9 A resolution . Max binds as a dimer to its recognition sequence CACGTG by direct contacts between the alpha-helical basic region and the major groove . This symmetric homodimer, a new protein fold, is a parallel, left-handed, four-helix bundle, with each monomer containing two alpha-helical segments separated by a loop . The two alpha-helical segments are composed of the basic region plus helix 1 and helix 2 plus the leucine repeat, respectively . As in GCN4, the leucine repeat forms a parallel coiled coil.

J Mol Biol, 1993 May 5, 231(1), 29 - 40
The LexA repressor binds within the deep helical groove of the activated RecA filament; Yu X et al.; The RecA protein of Escherichia coli, as a result of DNA damage, catalyzes the cleavage of its own repressor, the LexA protein, and thereby initiates the SOS response . Using a non-cleavable LexA mutant, we have obtained a co-complex of both the RecA and LexA proteins on DNA . Mass analysis using scanning transmission electron microscopy suggests that the site size of the LexA repressor on RecA is two, which would be consistent with a nearest-neighbor exclusion model for binding . Three-dimensional reconstruction of electron micrographs of these filaments shows that the LexA protein is bound in the deep groove of the RecA filament, with two strong regions of contact that span adjacent RecA protomers within the filament . One contact is consistent with a proposed LexA binding site in the RecA crystal structure . The other contact maps onto a region that has been postulated to be a second DNA-binding site within RecA, which can explain the inhibition of RecA cleavage of LexA by excess DNA.

J Mol Biol, 1993 May 5, 231(1), 145 - 7
Crystallization and preliminary X-ray analysis of the periplasmic dipeptide binding protein from Escherichia coli; Dunten PW et al.; The periplasmic dipeptide-binding protein from Escherichia coli has been purified, freed of bound endogenous ligands, and crystallized . Crystals of the protein in complex with added dipeptides have been subjected to X-ray analysis . The crystals grow as hexagonal bipyramids or eye-shaped disks which have the symmetry of space group P6(1) . The unit cell dimensions are a = b = 183 A, c = 212 A, and the diffraction pattern extends to 3.2 A resolution with a conventional X-ray source.

J Mol Biol, 1993 May 5, 231(1), 141 - 4
Purification and crystallization of fumarase C from Escherichia coli; Weaver TM et al.; Fumarase C purified from Escherichia coli has been crystallized in the presence of polyethylene glycol in both a citrate buffer at pH 5.3 and a 3-(4-morpholino)-propanesulfonic acid buffer at pH 7.5 yielding two crystal forms . An orthorhombic C222(1) form was obtained in citrate at pH 5.3 and an orthorhombic I222 form was obtained in 3-(4-morpholino)-propanesulfonic acid (pH 7.5) . Complete native data sets have been collected on both crystal forms: the C222(1) form is complete to 2.10 A and the I222 form is complete to 2.20 A.

J Mol Biol, 1993 May 5, 231(1), 1 - 5
Genetic evidence for the interaction between cluster I and cluster III rifampicin resistant mutations; Singer M et al.; Rifampicin-resistant (Rifr) mutations of Escherichia coli map to the central portion of the rpoB gene, which encodes the beta subunit of RNA polymerase . These mutations are located in three distinct clusters, designated I, II and III . Three intragenic suppressors of the cluster III Rifr mutation, rpoB3406(RH687), restore the ability of the mutant strain to grow at low and high temperatures and map to a single locus in cluster I . These suppressors are identical to two previously characterized Rifr alleles, rpoB3401(RC529) and rpoB3402(RS529) . None of the other 14 previously identified Rifr mutations that we have characterized confers this phenotype . We suggest that this allele-specific suppression results from interaction between Cluster I and Cluster III of the beta subunit.

J Biol Chem, 1993 May 5, 268(13), 9681 - 9
Cloning, sequencing, distribution, and expression in Escherichia coli of flavin-containing monooxygenase 1C1 . Evidence for a third gene subfamily in rabbits; Atta-Asafo-Adjei E et al.; Two full-length cDNA clones (2.2 kilobases) encoding a newly recognized form of mammalian flavin-containing monooxygenase (FMO) have been isolated from independent libraries constructed with mRNA from different rabbits . The cDNAs encode a polypeptide of 533 amino acids which contains two putative pyrophosphate binding domains and a hydrophobic carboxyl terminus characteristic of FMOs . This sequence is 52 and 57% identical to sequences of the rabbit "hepatic" and "pulmonary" FMOs, respectively, and 55% identical to the sequence of "liver form 2" published recently by Ozols (Ozols, J . (1991) Arch . Biochem . Biophys . 290, 103-115) . cDNA for the new FMO (FMO 1C1) hybridizes with two species of mRNA, one of 2.6 kilobases and one of about 5.4 kilobases, from liver or kidney, but not lung . Guinea pig, hamster, rat, and mouse all express this form of FMO in liver, kidney, and lung . FMO 1C1 has been tentatively characterized following expression in Escherichia coli . It is inactive with methimazole as substrate but highly active with n-octylamine . The temperature lability, responses to ions and detergent, and pH optimum of FMO 1C1 are similar to values reported for hepatic FMO . Sequence comparisons and analysis of rabbit and human genomic DNA indicate that FMO 1C1, as well as the pulmonary and hepatic FMOs, comprise a single gene family made up of distinct gene subfamilies (A, B,C,D, .. . N), each appearing to contain a single gene . A nomenclature, based on these interrelationships and following the same designations used for classifying cytochromes P-450, is proposed.

J Biol Chem, 1993 May 5, 268(13), 9629 - 35
Wild type GAL4 binds cooperatively to the GAL1-10 UASG in vitro; Kang T et al.; Transcription of the genes required for utilization of galactose in Saccharomyces cerevisiae is controlled primarily by the transcriptional activator protein GAL4 . The upstream activating sequences for galactose (UASG) of most GAL genes have multiple sites to which GAL4 can bind . In this report we compare the binding properties of wild type GAL4 and derivatives of GAL4 bearing the N-terminal DNA-binding domain to multiple DNA-binding sites in vitro . To produce wild type GAL4, we constructed a recombinant baculovirus for expression in insect cells . Recombinant wild type GAL4 was found to bind efficiently to an oligonucleotide containing a near-consensus 17-mer GAL4 DNA-binding site in electrophoretic mobility shift assays . Footprinting experiments revealed that wild type GAL4 binds cooperatively to the four GAL4 DNA-binding sites of the GAL1-10 UASG; however, in contrast an N-terminal fragment of GAL4 containing only the DNA-binding/dimerization domains binds to each of these sites with slightly different affinity . With increasing concentrations of GAL4(1-147), the four sites become filled in the following order: site II, site IV, site I, and site III . In experiments with wild type GAL4, these four sites become fully occupied at approximately the same concentration of protein . In footprints of wild type GAL4 on the USAG, enhancements and protections of DNase I-sensitive cleavages are detectable between sites III and IV, indicative of formation of a loop between these distantly spaced sites . Binding of wild type GAL4 to a strong near-consensus binding site assists binding to an adjacent mutant site in both electrophoretic mobility shift and footprinting assays . GAL4(1-147) and GAL4(1-147) fused to portions of GAL4's activating region II were incapable of cooperative DNA binding in our assays . We conclude from these observations that wild type GAL4 has a cooperative DNA-binding function that is distinct from the DNA binding and dimerization or transcriptional activation functions, and likely plays and important role in precise regulation of GAL gene transcription.

J Biol Chem, 1993 May 5, 268(13), 9442 - 7
Charged residues render pro-OmpA potential dependent for initiation of membrane translocation; Geller B et al.; We have examined the effects of positively and negatively charged residues on the translocation of outer membrane protein A precursor (pro-OmpA) across the bacterial inner membrane . Pro-OmpA does not translocate across the membrane when 2 positively charged residues are inserted immediately after the leader peptide, whereas it does insert when 2 neutral or negatively charged residues are introduced . Using a cell-free translocation system, we show that the membrane potential stimulated the rate of initial insertion of pro-OmpA with negatively charged residues, inhibited pro-OmpA with positively charged residues, and had no effect on neutral pro-OmpA . Thus, acidic residues render pro-OmpA potential-dependent for loop formation, which then initiates the translocation process.

J Biol Chem, 1993 May 5, 268(13), 9191 - 3
Presteady state kinetics of an S-adenosylmethionine-dependent enzyme . Evidence for a unique binding orientation requirement for EcoRI DNA methyltransferase; Reich NO et al.; We present the first presteady state kinetic analysis of an S-adenosylmethionine-dependent enzyme . The target enzyme is the bacterial EcoRI DNA methyl-transferase, which transfers the methyl group to the second adenine in the DNA sequence GAATTC . The rate constant for conversion of the central complex (enzyme-DNA-S-adenosylmethionine) to products (enzyme-methylated DNA-S-adenosylhomocysteine) (41 +/- 7 s-1) is over 300-fold faster than kcat, consistent with our demonstration that steps after methyl transfer are rate-limiting (Reich, N . O., and Mashhoon, N . (1991) Biochemistry 30, 2933-2939) . Methyl transfer at the N6 amino moiety of adenine on each strand requires a single binding orientation.

J Biol Chem, 1993 May 5, 268(13), 9466 - 72
Isolation of a human liver fructose-1,6-bisphosphatase cDNA and expression of the protein in Escherichia coli . Role of ASP-118 and ASP-121 in catalysis; el-Maghrabi MR et al.; A cDNA encoding human liver fructose-1,6-bisphosphatase was isolated from a lambda gt11 library by screening with a rat liver fructose-1,6-bisphosphatase cDNA . The cDNA (1421 base pairs) contains an open reading frame encoding 337 amino acids, corresponding to a protein with an estimated molecular weight of 36,697 . Its primary sequence is highly homologous to that of the pig kidney and rat liver enzymes . The human liver cDNA was used to construct a T7 RNA polymerase-transcribed expression vector, and the enzyme was expressed in Escherichia coli BL21 (DE3) . Approximately 50% of the expressed human fructose-1,6-bisphosphatase was soluble and enzymatically active, and the enzyme was purified to homogeneity by heat treatment, ammonium sulfate fractionation, and substrate/AMP elution from carboxymethyl-Sephadex . Expressed human liver fructose-1,6-bisphosphatase had a specific activity (9.8 mumol/min/mg of protein) that was half that of the rat liver enzyme, but had an identical Km for substrate . However, the human enzyme was more sensitive to inhibition by fructose-2,6-bisphosphate (Ki = 0.3 microM) and AMP (Ki = 12 microM) than the rat liver form (fructose 2,6-P2, Ki = 4 microM; AMP, Ki = 40 microM) . Crystallographic analyses have suggested that Asp-118 and Asp-121 are catalytic residues located in a negatively charged pocket that binds divalent metal cations . These residues were mutated to alanine, and the E . coli-expressed mutant enzymes were purified to homogeneity . The Asp-118-->Ala and Asp-121-->Ala mutants had 1/5000 and 1/20,000 lower Kcat values than the wild-type enzyme, respectively, consistent with their critical role in fructose-1,6-bisphosphatase catalysis.

J Mol Biol, 1993 May 5, 231(1), 58 - 64
Mutation Ala2-->Ser destabilizes intersubunit interactions in the molecular chaperone GroEL; Horovitz A et al.; The mutation Ala2-->Ser in the molecular chaperone GroEL increases positive co-operativity in ATP hydrolysis, as reflected by a change in the Hill coefficient from 2.36(+/- 0.23) for wild-type to 3.19(+/- 0.17) for the mutant . This amino acid replacement destabilizes the oligomeric structure of GroEL . It is shown that adenine nucleotides also have a specific destabilizing effect which is more pronounced in the case of the Ala2-->Ser mutant . Addition of GroES or the non-folded protein ligand rhodanese blocks the destabilizing effect of adenine nucleotides for both wild-type and mutant . The results are interpreted using the Monod-Wyman-Changeux (MWC) model for co-operativity.

J Biol Chem, 1993 May 5, 268(13), 9238 - 45
Escherichia coli thioesterase I, molecular cloning and sequencing of the structural gene and identification as a periplasmic enzyme; Cho H et al.; The structural gene for Escherichia coli thioesterase I (called tesA) has been cloned by use of sequence data obtained from the purified protein . The tesA gene was mapped at 530 kilobase pair of the E . coli physical map (minute 11.6 of E . coli genetic map) . The DNA sequence of the tesA gene was obtained and the deduced protein sequence showed that thioesterase I consists of 182 amino acids and has a molecular mass of 20.5 kDa . Comparison of the DNA and protein sequence data suggested that a leader sequence of 26 amino acid residues is cleaved from the primary translation product, and this processing was confirmed by NH2-terminal sequencing of the primary translation product synthesized in vitro . These data predicted that thioesterase I (long believed to be a cytoplasmic protein) is exported to the cell periplasm, a prediction supported by release of the enzyme from cells upon osmotic shock . The TesA protein sequence does not exhibit any significant overall sequence similarity with other known proteins, although the sequence does contain two small sequence elements found in several other thioesterases . One of these elements is a sequence similar to the serine esterase active sites found in serine proteases and four other thioesterases . A serine residue within this TesA element was shown to be covalently labeled with {3H} diisopropyl fluorophosphate, a potent inhibitor of TesA activity . The second sequence element is a histidine-containing sequence found close to the carboxyl terminus that is also found in the carboxyl termini of the four known active serine thioesterases . The physiological role of thioesterase I is unclear . A strain carrying a null mutation of the tesA gene was constructed and found to have no growth phenotype . Moreover, a strain carrying a plasmid that gave massive overproduction of TesA (approximately 100-fold higher than that of the wild type) also grew normally . In addition a strain containing double null mutations in both tesA and tesB (the structural gene for E . coli thioesterase II) also failed to display any growth phenotype . Analysis of the fatty acid compositions of phospholipid, lipid A, and lipoprotein of the above strains showed no significant changes from a wild type strain.

J Biol Chem, 1993 May 5, 268(13), 9793 - 802
Template-directed pausing of DNA synthesis by HIV-1 reverse transcriptase during polymerization of HIV-1 sequences in vitro; Klarmann GJ et al.; Replication of human immunodeficiency virus type 1 (HIV-1) requires reverse transcriptase (RT) to synthesize double-stranded proviral DNA (9.7 kilobases) through a complex mechanism utilizing both RNA and DNA templates . We have examined DNA synthesis by HIV-1 RT on RNA and DNA templates derived from the HIV-1 genome using a primer extension assay in vitro . Analysis of polymerization products on sequencing gels revealed strong pauses in synthesis, on both RNA and DNA templates, in homopolymeric nucleotide runs, and at regions of predicted secondary structure . Polymerization pauses occurred in runs of template rGs (> or = 4 bases) and rCs (> or = 3 bases) during minus-strand synthesis on RNA templates, and in most runs (> or = 4 bases) of template dTs and dAs during plus-strand synthesis on DNA templates . Pausing also occurred on both templates within the first few nucleotides of the predicted hairpin structures of the Rev response element . The locations of pauses were dependent on template sequence and were unaffected by primer positioning, RT concentration, and ionic strength . Recombinant and virion-derived HIV-1 RTs showed similar pausing patterns . DNA products that accumulated at HIV-1 RT pause sites on RNA templates were extended by continued incubation with excess RT from Moloney murine leukemia virus, showing that the RNA templates were not broken or otherwise unable to support polymerization . Polymerizations conducted in the presence of a poly(rA) oligo(dT) trap showed that pausing results from two mechanisms: 1) RT remaining bound to the primer-template and polymerizing at a greatly reduced rate, or 2) RT dissociating from the primer-template . These results demonstrate that specific HIV-1 RNA and DNA template sequences are capable of interrupting processive DNA synthesis by HIV-1 RT in vitro . Pausing may serve specific functions in HIV-1 replication and mutagenesis . Moreover, these data suggest that one or more accessory factors are required to complete proviral DNA synthesis in vivo and that efficient HIV-1 DNA synthesis may require multiple origins.

J Biol Chem, 1993 May 5, 268(13), 9329 - 36
Analysis of gas vesicle gene expression in Haloferax mediterranei reveals that GvpA and GvpC are both gas vesicle structural proteins; Englert C et al.; Gas vesicle synthesis in Haloferax mediterranei involves several gene products encoded by a 9.4-kilobase pair DNA region (mc-vac region) that contains 13 genes in addition to gvpA encoding the major structural gas vesicle protein . The expression of part of this region, encompassing the genes gvpA, gvpC, gvpN, and gvpO was investigated . These genes are transcribed from a common promoter located upstream of gvpA . Transcripts of 0.34 (gvpA only), 1.8 (gvpA/C), 2.4 (gvpA/C/N) and 3 kilobases (gvpA/C/N/O) were observed, with the gvpA transcript being the predominant mRNA species . The majority of the mRNA formed terminates 64 base pairs downstream of gvpA at the cytosine of the sequence 5' TTTTTC 3' . The synthesis of the GvpA and GvpC proteins was investigated by Western analyses . An antiserum raised against isolated gas vesicles of Hf . mediterranei detects, in addition to gas vesicle fragments, the GvpA protein of the M(r) of approximately 8,000 in lysates derived from different halobacteria or from Escherichia coli expressing gvpA . In samples containing isolated gas vesicles, mainly partially disaggregated gas vesicle fragments hybridize, but a minor amount of monomeric GvpA is also seen . For the detection of the GvpC protein, two versions of the gvpC gene (full length and gvp delta C lacking the 3' part encoding the acidic C terminus) were expressed in E . coli, and the resulting proteins were purified . The two antisera raised against these GvpC versions indicate the expression of gvpC in different halobacteria . By Western analysis, GvpC is also detectable in samples containing isolated gas vesicles demonstrating that GvpC is a second, but minor, gas vesicle structural protein.

J Biol Chem, 1993 May 5, 268(13), 9323 - 8
The interaction of illimaquinone, a selective inhibitor of the RNase H activity, with the reverse transcriptases of human immunodeficiency and murine leukemia retroviruses; Loya S et al.; Illimaquinone, a natural marine product, was shown by us to inhibit preferentially the ribonuclease H (RNase H) activity of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) . We have also shown that illimaquinone inhibits the RNase H activity of HIV-2 RT in addition to that of HIV-1 RT, murine leukemia virus RT, and Escherichia coli RNase H . Chemical modifications of HIV-1 RT by sulfhydryl-specific reagents, such as N-ethylmaleimide (NEM) have been demonstrated to specifically inhibit the RNase H activity of the enzyme . Since our previous studies have suggested that cysteine 280 in HIV-1 RT interacts with the sulfhydryl reagents, we have examined the possibility that illimaquinone interacts with the RT molecules via amino acid residues located in the vicinity of cysteine 280 in both HIV-1 and HIV-2 RTs . In the combined effect studies of illimaquinone and NEM, the two structurally unrelated compounds were shown to be mutually exclusive, exhibiting an antagonistic interaction with both HIV-1 and murine leukemia virus-associated RNase H activities . This implicates cysteine 280, in both HIV-1 and HIV-2 RTs, to be in close proximity to the putative binding site of the enzyme to illimaquinone . The above conclusion is further supported by the fact that the RNase H activity of an enzymatically active mutant of HIV-1 RT, in which cysteine 280 was replaced by serine, was substantially more resistant to illimaquinone than the corresponding activity of the wild-type enzyme . The fact that NEM failed to inhibit E . coli RNase H as opposed to illimaquinone highlights a major difference between the retroviral and bacterial RNase H.

Biochemistry, 1993 May 4, 32(17), 4677 - 84
Expression, purification, and characterization of the Drosophila kinesin motor domain produced in Escherichia coli; Gilbert SP et al.; The Drosophila kinesin heavy-chain gene was truncated to obtain the N-terminal 401 amino acid motor domain (designated K401) containing both the microtubule and ATP binding sites . The plasmid construct with the truncated kinesin gene was used to transform Escherichia coli . After induction, K401 was expressed as soluble kinesin protein at high levels and purified to homogeneity in milligram quantities . The purified protein was active and behaved as native kinesin with respect to its steady-state kinetic properties: K401 demonstrated a very low ATPase activity (kcat = 0.01 s-1) which was stimulated approximately 1000-fold by the addition of microtubules (kcat = 10 s-1; K0.5,MT = 0.9 microM tubulin; Km,ATP = 31 microM) . Like native kinesin, K401 when purified contained ADP tightly bound at its active site, and the release of ADP from the active site occurred at a rate equal to the steady-state ATPase kcat . Active-site measurements using {alpha-32P}ATP demonstrated a stoichiometry of one ATPase site per K401 molecule . Like native kinesin, K401 can also hydrolyze MgGTP, and in the presence of microtubules, the rate of hydrolysis was increased dramatically from 0.03 to 16 s-1 (K0.5,MT = 2 microM tubulin; Km,GTP = 3.5 mM) . These results establish that an active kinesin motor domain can be bacterially expressed and that this domain, the N-terminal 401 amino acids of the Drosophila kinesin heavy chain without light chains or additional eukaryotic factors, has full catalytic activity with microtubules.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 May 4, 32(17), 4560 - 3
Inter-tryptophan distances in rat cellular retinol binding protein II by solid-state NMR; McDowell LM et al.; Structural constraints for the tryptophans in rat cellular retinol binding protein II (CRBP II) have been obtained by rotational-echo double-resonance (REDOR) solid-state NMR . CRBP II was labeled with L-{6-19F}tryptophan and L-{2-13C}tryptophan . The 13C-19F dipolar coupling was determined for various possible tryptophan geometries . The allowed distance between the closest two of the four tryptophans in CRBP II was obtained for each geometry . The minimum possible distance between these two tryptophans in CRBP II is 7 A, and the maximum possible distance is 11 A.

FEBS Lett, 1993 May 3, 322(1), 6 - 9
Chaperonins dependent increase of Cu,Zn superoxide dismutase production in Escherichia coli; Battistoni A et al.; Over-expression of the chaperonins GroEL and GroES significantly suppressed the temperature-dependent pattern of expression of Cu,Zn superoxide dismutases in Escherichia coli and increased the yield of active enzyme . The results obtained indicate that chaperonins prevent degradation of metal-deficient enzyme molecules . GroEL was shown to form a complex with unfolded Cu,Zn superoxide dismutase in vitro, confirming that GroEL can interact with beta-stranded proteins.

Klin Lab Diagn, 1993 May-Jun, (3), 44 - 7
{Causes of false-positive results in serological studies of HIV infection}; Orlova VM et al.; Analysis of the results of testing 150880 serum samples with the Antigen and Recombinant-HIV types test systems, manufactured in Russia, has demonstrated the relationship between the false-positive results and the type of the test system employed and the category of the examined subjects . Depletion of the sera with NaCl and CO2 reduced the incidence of false-positive results in analyses of the pregnant women's sera . Examinations of the sera in the Combi-HIV test system have revealed E . coli antigens in 8.8% of the sera.

Izv Akad Nauk Ser Biol, 1993 May-Jun, (3), 468 - 71
{The ultrastructural and cytointerferometric changes in blood cells induced by endotoxin action}; Kharlanova NG et al.; The results of ultrastructural and cytointerferometric changes of different blood cells in the main target-organ microcirculation are present in the dynamics of endotoxin shock . It is established that the development of endotoxin shock is accompanied by a decrease in dry mass and content of dense substance in red blood cells and leucocytes . Ultrastructural alterations of blood cells have a stable enough character and may be the action of humoral mediator systems or due to direct effects of lipopolysaccharide . Ultrastructural and cytointerferometric changes are discussed in the context of possible receptor-ligand interrelations.

Appl Environ Microbiol, 1993 May, 59(5), 1391 - 7
Identification of procaryotic repetitive DNA suitable for use as fingerprinting probes; Waterhouse RN et al.; We have developed a method which enables the cloning and identification of procaryotic repetitive DNA suitable for use as DNA fingerprinting probes . The method involves shotgun cloning of restricted genomic DNA with subsequent selection of clones containing repetitive DNA by reverse-probed genomic hybridizations, in which the plasmid DNA clones are probed with labelled genomic DNA . Confirmation that the clones contained repeated sequences was by Southern hybridization, gene copy equivalence, and DNA sequencing . The sequences were used for highly specific and sensitive detection of bacteria and as target sequences for the mediation of chromosomal integration of reporter gene constructs.

J Virol Methods, 1993 May, 42(2-3), 309 - 22
Primer extension analysis provides a sensitive tool for the identification of PCR-amplified DNA from HIV-1; Boni J et al.; Primer extension analysis was evaluated as a means to identify PCR-amplified DNA from HIV-1 . Solution hybridization with radioactive labeled oligonucleotides followed by an extension reaction with Klenow fragment of Escherichia coli DNA polymerase I and subsequent separation on denaturing polyacrylamide gels reveals single stranded DNA products of the predicted size . The specificity of the reaction is further demonstrated by specific endonuclease digestion . The analysis is more sensitive than Southern blotting and about equally sensitive as Slot blot analysis when double-stranded DNA probes are used for hybridization . With end-labeled oligonucleotide probes, primer extension analysis proved an order of magnitude more sensitive than membrane hybridization . The analysis also allows quantitation of amplified DNA from 1 pg to about 1 ng of DNA product . Under the conditions described for amplification, primer extension analysis is capable of detecting a single HIV-1 plasmid DNA molecule in the presence of 1 microgram of total DNA . 3'-end mismatching of the oligonucleotide probe does not result in a significantly altered detection limit . Primer extension analysis can also be carried out with at least three different PCR-amplified DNAs in the same reaction tube.

Exp Hematol, 1993 May, 21(5), 660 - 4
Production of a functional single-chain Fv fragment from the pan-leukocyte antibody WM65 using splicing by asymmetric PCR; Ward RL et al.; A method of assembly of a single-chain Fv fragment is described, whereby asymmetric polymerase chain reactions (APCR) and primer extension were used to join immunoglobulin heavy and light chain variable region genes via a linker sequence . In this procedure heavy and light chain genes, together with a linker gene containing complementary sequences, were amplified by APCR to generate single-stranded products . The single stranded heavy or light chain genes were hybridized to the relevant single-stranded link product, and extended to produce double-stranded heavy-link and link-light genes . These genes then underwent another round of APCR, resulting in two single-stranded genes (heavy-link and link-light) containing extensive overlapping sequences . Hybridization and extension of these two single-stranded products allowed the formation of the complete heavy chain-link-light chain double-stranded product . Using this method, a functional single-chain Fv fragment based on the pan-leukocyte antibody WM65 was expressed and purified from E . coli . This method of immunoglobulin gene assembly by polymerase chain reaction (PCR) offers an alternative to the current methods of the genetic engineering of antibody fragments.

Biotechniques, 1993 May, 14(5), 818 - 23
Recombinant retroviruses containing novel reporter genes; Schreiber JH et al.; Two histochemical marker genes, Drosophila alcohol dehydrogenase (ADH) and human placental alkaline phosphatase (ALP), were cloned into the recombinant retroviral vectors pLJ and pgag beta-actin . The resulting vectors were transfected into retroviral producer cell lines, psi CRE and psi CRIP, and stable recombinant retrovirus producers were isolated . Recombinant virus was harvested and used to transduce genes into several cell lines, singly or in conjunction with lacZ (Escherichia coli beta-galactosidase)-containing retrovirus . Cell lines were then stained using standard histochemical methods for recombinant gene expression . We found that multiple gene products could be identified in the same cell populations and in the case of ALP and beta-galactosidase, in the same cells . The resulting reagents should be useful for a variety of cell-marking studies including those involving multiple clonal analysis and developmental studies for gene therapy.

Anal Biochem, 1993 May 1, 210(2), 344 - 50
A radioimmunoassay-based method for measuring the true affinity of a monoclonal antibody with trace amounts of radioactive antigen: illustration with the products of a cell-free protein synthesis system; Friguet B et al.; This communication describes a novel highly sensitive method for measuring the affinity of a monoclonal antibody for its antigen . It is based on a radioimmunoassay in which the antigen is labeled with radioactivity . It is therefore particularly well adapted to the study of trace amounts of radiolabeled polypeptide chains produced either in vivo, or in vitro by a cell free protein synthesis system or by chemical radiolabeling . It offers several advantages over previously described methods . Though making use of insolubilized antibody, it does measure the true affinity constant of the monoclonal antibody in solution for the antigen . It can be used even when the antigen is present at concentrations far below the dissociation constant of the antibody/antigen complex . It does not require the antigen or the antibody to be purified . In most cases, it requires no sophisticated equipment . This method could be easily adapted to the determination of the equilibrium constant of any type of protein/ligand system.

Rev Clin Esp, 1993 May, 192(8), 383 - 5
{The association of pleural empyema and perinephritic infection: apropos 4 cases}; Vargas Puerto A et al.; Empyema of renal origin is very rare (3% in our series) . We discuss 4 cases of empyemas associated to perirenal infection . Two of them were diabetic and all of them have renal lithiasis . They made their debut through pleural effusions, isolating Escherichia coli in all of them . Treatment was antibiotics and drainage in both foci, three of them evolved to healing and one of them died being the abdominal foci without drainage . This association must be suspected when no clear etiology of the empyema is present in patients with history of renal lithiasis and diabetes.

Mol Gen Genet, 1993 May, 239(1-2), 72 - 6
Mutations in the mutY gene of Escherichia coli enhance the frequency of targeted G:C-->T:a transversions induced by a single 8-oxoguanine residue in single-stranded DNA; Moriya M et al.; Oxidative damage to guanine in DNA results in the formation of 8-oxoguanine, which has been shown to induce G-->T transversions targeted to this site . The mutagenicity of this lesion was studied in several mutator strains of Escherichia coli, using single-stranded DNA containing a single 8-oxoguanine residue . The frequencies of targeted G-->T transversions increased markedly in mutY strains, while this mutagenic event was not affected in mutM or mutS strains . Introduction of a mutM mutation into a mutY strain caused a somewhat higher frequency of G-->T transversions than that in the mutY strain and the effect of a mutS mutation was marginal . We conclude that the mutY gene plays a crucial role in preventing targeted G-->T mutations derived from misreplication of the 8-oxoguanine-containing template DNA.

Mol Gen Genet, 1993 May, 239(1-2), 137 - 44
Induction of only one SOS operon, umuDC, is required for SOS mutagenesis in Escherichia coli; Sommer S et al.; The actions of UmuDC and RecA proteins, respectively in SOS mutagenesis are studied here with the following experimental strategy . We used lexAl (Ind-) bacteria to maintain all SOS proteins at their basal concentrations and then selectively increased the concentration of either UmuDC or RecA protein . For this purpose, we isolated operator-constitutive mutations oc in the umuDC and umuD'C operons and also used the oc98-recA mutation . The oc1-umuDC mutation prevents LexA repressor from binding to the operator and improves the Pribnow box consensus sequence . As a result, 5000 UmuD and 500 UmuC molecules per cell were produced in lexAl bacteria . This concentration is sufficient to restore SOS mutagenesis . The level of RecA protein present in the repressed state promoted full UmuD cleavage . Overproduction of RecA alone did not promote SOS mutagenesis . Increasing the level of RecA in the presence of high concentrations of UmuDC proteins has no further effect on SOS mutagenesis . We conclude that, after DNA damage, umuDC is the only SOS operon that must be induced in Escherichia coli to promote SOS mutagenesis.

Plant Mol Biol, 1993 May, 22(2), 347 - 60
Structure and expression of a barley acidic beta-glucanase gene; Malehorn DE et al.; A barley acidic beta-1,3-glucanase gene was recovered from a barley genomic library by homology with a partial cDNA of barley basic beta-1,3-glucanase isoenzyme GII . The gene, Abg2, is homologous to the PR2 family of pathogenesis-related beta-1,3-glucanase genes . The ABG2 protein has 81% amino acid similarity to barley basic beta-1,3-glucanase GII . The ABG2 protein is encoded as a preprotein of 336 amino acids including a 28 amino acid signal peptide . A 299 bp intron occurs within codon 25 . The mature ABG2 protein has a predicted mass of 32,642 Da and a calculated isoelectric point of 4.9 . The second exon of the Abg2 gene shows a strong preference for G + C in the third position of degenerate codons . The Abg2 gene was functionally expressed in Escherichia coli . Abg2 mRNA is constitutively expressed in barley root; leaf expression of Abg2 mRNA is induced by mercuric chloride and infection by Erysiphe graminis f . sp . hordei . Southern blot analysis indicates that Abg2 is a member of a small gene family.

Plant Mol Biol, 1993 May, 22(2), 301 - 12
Identification and expression of a cDNA from Daucus carota encoding a bifunctional aspartokinase-homoserine dehydrogenase; Weisemann JM et al.; Aspartokinase (EC 2.7.2.4) and homoserine dehydrogenase (EC 1.1.1.3) catalyze steps in the pathway for the synthesis of lysine, threonine, and methionine from aspartate . Homoserine dehydrogenase was purified from carrot (Daucus carota L.) cell cultures and portions of it were subjected to amino acid sequencing . Oligonucleotides deduced from the amino acid sequences were used as primers in a polymerase chain reaction to amplify a DNA fragment using DNA derived from carrot cell culture mRNA as template . The amplification product was radiolabelled and used as a probe to identify cDNA clones from libraries derived from carrot cell culture and root RNA . Two overlapping clones were isolated . Together the cDNA clones delineate a 3089 bp long sequence encompassing an open reading frame encoding 921 amino acids, including the mature protein and a long chloroplast transit peptide . The deduced amino acid sequence has high homology with the Escherichia coli proteins aspartokinase I-homoserine dehydrogenase I and aspartokinase II-homoserine dehydrogenase II . Like the E . coli genes the isolated carrot cDNA appears to encode a bifunctional aspartokinase-homoserine dehydrogenase enzyme.

Plant Mol Biol, 1993 May, 22(2), 227 - 37
Isolation and characterization of wheat triticin cDNA revealing a unique lysine-rich repetitive domain; Singh NK et al.; Polyclonal antibodies were raised against a purified 22 kDa triticin polypeptide (delta) and were used to screen a wheat seed cDNA library in the Escherichia coli expression vector lambda gt11 . The isolated cDNA clones were grouped into three families based on their cross-hybridization reactions in DNA dot-blot studies . Southern blots of genomic DNAs extracted from ditelocentric and nullisomic-tetrasomic lines of Chinese Spring wheat, probes with the excised cDNA inserts, indicated that one of the three families (9 clones) had triticin clones . This was finally confirmed by comparing the predicted amino acid sequences of two of these clones (lambda Tri-12, lambda Tri-25) with the published tryptic peptide sequences of triticin . The Southern blots also showed that there is at least one triticin gene located on the short arm of each of the homoeologous group 1 chromosomes (1A, 1B, 1D), although till now no triticin protein product has been identified for the chromosome 1B . The nucleotide sequence of the largest triticin cDNA clone lambda Tri-25 (1567 bp) is presented here, and its predicted amino acid sequence shows strong homology with the legumin-like proteins of oats (12S globulin), rice (glutelin) and legume seeds . A unique feature of the triticin sequence is that it contains a lysine-rich repetitive domain, inserted in the hypervariable region of the typical legumin-like genes . Northern blotting of total RNA extracted from different stages of the developing wheat seed revealed that the triticin gene expression is switched on 5-10 days after anthesis (DAA) . There was a steady increase in the level of triticin mRNA until 20 DAA, after which it started decreasing . The maximum mRNA accumulation occurred between 17 and 20 DAA . These observations conform closely with the published data on triticin protein accumulation during grain development.

JPEN J Parenter Enteral Nutr, 1993 May-Jun, 17(3), 226 - 30
Addition of glucagon to lipid-free total parenteral nutrition reduces production of prostaglandin E2 by stimulated splenic macrophages; Zamir O et al.; Sepsis is a major complication of total parenteral nutrition (TPN) . Impaired immunity has been suggested as being responsible for TPN-related sepsis, but it is unknown how the immune system is affected by TPN . We recently found that administration of lipid-free TPN resulted in an increase in prostaglandin E2 (PGE2) release by stimulated splenic macrophages . This observation suggested that TPN may impair immunity through the prominent immunosuppressive effects of PGE2 . In the present study, we tested the hypothesis that addition of glucagon to TPN solution may protect against the immunosuppressive effect of TPN by modifying PGE2 secretion . Adult, male Sprague-Dawley rats (n = 18) underwent jugular vein cannulation: group 1 (n = 7) received intravenous saline and chow ad libitum; group 2 (n = 6) received TPN (80 mL/24 h); and group 3 (n = 5) received TPN (80 mL/24 h) plus glucagon (100 micrograms/24 h) . After 10 days, spleens were removed and splenic macrophages were isolated and cultured for 24 h in plain M199 medium (nonstimulated) or in medium containing Escherichia coli lipopolysaccharide (5 micrograms/mL) (stimulated) . PGE2 release was determined by enzyme-linked immunosorbent assay . There were no differences in PGE2 release between the groups of nonstimulated cells, but when stimulated with lipopolysaccharide, the macrophages from the TPN rats (group 2) released more PGE2 (81.68 +/- 25.99 ng/2.5 x 10(6) cells) than the control group (16.04 +/- 3.26 ng/2.5 x 10(6) cells) . The release of PGE2 was normalized in the TPN animals treated with glucagon (15.71 +/- 3.33 ng/2.5 x 10(6) cells) . This difference was significant, with p < .05 by Tukey's test after analysis of variance.(ABSTRACT TRUNCATED AT 250 WORDS)

Eur J Biochem, 1993 May 1, 213(3), 995 - 1002
FAD and substrate analogs as probes for lysine N6-hydroxylase from Escherichia coli EN 222; Macheroux P et al.; Lysine N6-hydroxylase catalyzes the hydroxylation of the N-terminal amino function of L-lysine at the expense of NADPH and molecular oxygen . The enzyme also requires FAD for its catalytic activity . Unlike other flavoprotein monooxygenases, binding of FAD is rather weak with a Kd of 30 microM at 4 degrees C . The spectral properties of FAD bound to lysine N6-hydroxylase are very similar to free oxidized FAD . In the absence of substrate, the enzyme has an NADPH oxidase activity which results in the generation of hydrogen peroxide . With increasing concentration of L-lysine, the NADPH oxidase activity is enhanced up to 10-fold and the generation of hydrogen peroxide decreases . At the same time, the substrate is hydroxylated . Km values for L-lysine and FAD were determined as 105 microM and 0.7 microM, respectively . Utilizing FAD analogs, we could demonstrate that L-lysine exerts its effector role mostly on the reductive half reaction of the overall catalytic cycle . Prolonged incubation of the enzyme with either 8-chloro- or 8-fluoro-FAD gave rise to a covalently attached flavin which is formed as a result of the nucleophilic attack of a thiolate on the 8-position of the flavin . Several lines of evidence indicate that the reaction takes place in the FAD binding site of the protein . The substrate specificity was investigated using amino acids with various lengths of side chain . L-Lysine and derivatives with similar side chain length are hydroxylated by lysine N6-hydroxylase . Ornithine, the lower homolog of lysine, was not hydroxylated and did not affect the NADPH oxidase activity of the enzyme . On the other hand, homolysine accelerated the rate of NADPH oxidation but was not hydroxylated . Additional requirements for efficient hydroxylation were also investigated using a variety of substrate analogs . From these studies a schematic structure of the active site of the enzyme was deduced . Sequence comparison of the FAD binding site of various flavoproteins revealed possible factors for weak binding of the cofactor in the case of lysine N6-hydroxylase.

Carcinogenesis, 1993 May, 14(5), 789 - 94
Mutational specificities of environmental carcinogens in the lacI gene of Escherichia coli . VI: Analysis of methylene chloride-induced mutational distribution in Uvr+ and UvrB- strains; Zielenska M et al.; To better understand the mechanisms of mutagenesis by the carcinogen, methylene chloride (DCM), we have determined the nature and distribution of forward mutations induced by DCM in the N-terminal region of the lacI gene of Escherichia coli . A total of 116 lacI-d mutations (50 from Uvr+, 66 from UvrB- strain) were characterized by DNA sequencing . Both similarities and differences were observed . Although in both strains base substitutions predominated (74-88%) the distribution among the classes differed . In the case of the Uvr+ strain, DCM substantially increased the frequency of G:C-->C:G transversion and duplication events . Direct repeats were not observed at the endpoints of the duplications, however, all endpoints were in an A:T-rich region . In contrast, in the UvrB- strain, DCM induced A:T-->G:C, A:T-->C:G, G:C-->C:G events as well as deletions . The mutational spectra presented here represent a first step in the elucidation of the mechanism(s) of DCM-induced mutation.

Anal Chem, 1993 May 1, 65(9), 1147 - 51
Enzyme-linked immunosorbent assay for an octapeptide based on a genetically engineered fusion protein; Witkowski A et al.; Traditional chemical means of preparing enzyme-ligand conjugates for use in enzyme-linked immunosorbent assays (ELISAs) lead to the production of multisubstituted enzyme-ligand conjugates with a high degree of variability in the site of ligand attachment . A genetically engineered fusion protein was prepared in order to investigate the feasibility of controlled production of conjugates for use in ELISAs . Specifically, a synthetic octapeptide was fused with bacterial alkaline phosphatase . The resulting enzyme-peptide conjugate is monosubstituted (one peptide per subunit), has a single site of attachment, and results in assays with good response characteristics . The use of such fusion proteins, which combine small analyte peptides with enzyme labels, can lead to a new approach to improved assays for numerous biomolecules, including peptide pharmaceuticals, neurotransmitters, hormones, cell surface antigens, etc.

Am J Physiol, 1993 May, 264(5 Pt 2), F774 - 80
LPS-induced MCP-1, IL-1 beta, and TNF-alpha mRNA expression in isolated erythrocyte-perfused rat kidney; Xia Y et al.; The capacity of the lipopolysaccharide (LPS)-stimulated isolated erythrocyte-perfused rat kidney (IEPK) to produce monocyte chemoattractant protein-1 (MCP-1), interleukin-1 beta (IL-1 beta), and tumor necrosis factor-alpha (TNF-alpha) mRNA was investigated . The IEPK was chosen to exclude the influence of circulating neutrophils and monocytes that can produce both these mediators when exposed to LPS . The control minimal LPS group (LPS < 10 pg/ml) showed a small increase in mRNA expression for MCP-1, IL-1 beta, and TNF-alpha in the cortex and medulla after 80 min of perfusion when compared with the unperfused left kidney in which no IL-1 beta or TNF-alpha mRNA and only minimal amounts of MCP-1 mRNA were detected . LPS stimulation (1 microgram/ml for 40 or 80 min) increased MCP-1, IL-1 beta, and TNF-alpha mRNA expression, which was found predominately in peritubular capillary endothelial cells by in situ hybridization . The changes were not due to a marked perturbation of LPS on renal hemodynamics . The renal vascular resistance (RVR) remained constant (40 min LPS exposure) or increased only slightly during the last 5-10 min (80 min LPS exposure) compared with a progressive increase in RVR of the minimal LPS group . The hemodynamic effects of LPS on the IEPK appear to counteract the gradual increase in RVR seen in the minimal LPS group.

Am J Physiol, 1993 May, 264(5 Pt 1), G828 - 34
Attenuation of endotoxin-induced intestinal microcirculatory damage by eicosapentanoic acid; Miura S et al.; The major objective of this study is to investigate whether oral administration of eicosapentanoic acid (EPA) has any preventive effect on endotoxin-induced microcirculatory damage of rat small intestine . EPA in a daily dose of 300 mg/kg was orally given to male Wistar rats for 3 wk . Submucosal microvessels of the ileum were observed by intravital microscopy equipped with a high-speed video camera system after the intra-arterial infusion of endotoxin at a dose of 2 mg.kg-1.h-1 . The number of sticking leukocytes was significantly increased at 30 min after the treatment of endotoxin especially along the smaller branch of intestinal venules . It reached the maximal plateau at 45 min after treatment . The pretreatment of EPA significantly attenuated the increase in sticking leukocytes induced by endotoxin . A platelet-activating factor (PAF) antagonist 2-{N-acetyl-N-(2-methoxy-3-octadecylcarbamoyloxy propoxycarbonyl) aminomethyl}-1-ethylpyridinium chloride (CV-6209) significantly prevented the increased leukocyte sticking to the same extent as EPA treatment . Thirty minutes after endotoxin infusion, red blood cell (RBC) velocity was significantly decreased in both arterioles and venules . RBC velocity appeared to be continuously decreased thereafter and reached its minimum value at approximately 60 min . EPA treatment was revealed to prevent the decrease in RBC velocity of microvessels induced by endotoxin . CV-6209 also significantly attenuated the decreased RBC velocity . The remarkable elevation of PAF content in the ileal mucosa as observed by endotoxin infusion was also significantly attenuated by administration of EPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Proteins, 1993 May, 16(1), 57 - 63
Recombinant antineuraminidase single chain antibody: expression, characterization, and crystallization in complex with antigen; Malby RL et al.; The variable heavy (VH) and variable light (VL) genes of NC10, a monoclonal antibody with specificity toward N9 neuraminidase (NA), were cloned and sequenced . A single chain Fv (scFv) fragment of NC10, consisting of VH and VL domains joined by a peptide linker, was designed, constructed and expressed in the E . coli expression vector pPOW . The N-terminal secretion signal PelB directed the synthesized protein into the periplasm where it was associated with the insoluble membrane fraction . An octapeptide (FLAG) tail was fused to the C-terminus of the single chain Fv to aid in its detection and remained intact throughout the protein purification process . NC10 scFv was purified by solubilization of the E . coli membrane fraction with guanidinium hydrochloride followed by column chromatography . The purified NC10 scFv showed binding affinity for its antigen, NA, 2-fold lower than that of the parent Fab . The complex between NA and the scFv has been crystallized by the vapor diffusion method . The crystals are tetragonal, space group P42(1)2, with unit cell dimensions a = b = 141 A, c = 218 A.

Protein Sci, 1993 May, 2(5), 717 - 26
Redox properties of protein disulfide isomerase (DsbA) from Escherichia coli; Wunderlich M et al.; The redox properties of periplasmic protein disulfide isomerase (DsbA) from Escherichia coli were analyzed by measuring the equilibrium constant of the oxidation of reduced DsbA by oxidized glutathione . The experiments are based on the finding that the intrinsic tryptophan fluorescence of DsbA increases about threefold upon reduction of the enzyme, which can be explained by the catalytic disulfide bridge quenching the fluorescence of a neighboring tryptophan residue . From the specific fluorescence of DsbA equilibrated in the presence of different ratios of reduced and oxidized glutathione at pH 7, an equilibrium constant of 1.2 x 10(-4) M was determined, corresponding to a standard redox potential (E'0) of DsbA of -0.089 V . Thus, DsbA is a significantly stronger oxidant than cytoplasmic thioredoxins and its redox properties are similar to those of eukaryotic protein disulfide isomerase . The equilibrium constants for the DsbA/glutathione equilibrium were found to be strongly dependent on pH and varied from 2.5 x 10(-3) M to 3.9 x 10(-5) M between pH 4 and 8.5 . The redox state-dependent fluorescence properties of DsbA should allow detailed physicochemical studies of the enzyme as well as the quantitative determination of the oxidized protein by fluorescence titration with dithiothreitol and open the possibility to observe bacterial protein disulfide isomerase "at work" during catalysis of oxidative protein folding.

Am J Pathol, 1993 May, 142(5), 1373 - 82
Regulation of the differentiation of diploid and some aneuploid rat liver epithelial (stemlike) cells by the hepatic microenvironment; Coleman WB et al.; Following intrahepatic transplantation in adult syngeneic Fischer 344 rats, diploid cultured rat liver epithelial cells (WB-F344), modified to carry the Escherichia coli beta-galactosidase reporter gene and/or the fluorescent membrane dye PKH26-GL, integrate into hepatic plates and acquire the size and nuclear structure of mature hepatocytes . Additionally, of two aneuploid, neoplastically transformed derivatives of WB-F344 cells, both of which produce aggressively growing tumors when transplanted subcutaneously, cells of one line (GN6TF) do not produce tumors in the liver but integrate into hepatic plates and morphologically differentiate . The other transformed line (GP7TB) retains tumorigenicity in the liver, but cells in the intrahepatic tumors are more differentiated morphologically than are tumors at subcutaneous sites . These results suggest that WB-F344 cells are stemlike cells for hepatocytes and that the hepatic microenvironment induces them to incorporate into hepatic plates and differentiate . Our results also suggest that the hepatic microenvironment regulates the differentiation of some neoplastically transformed hepatic stemlike cells, thereby eliminating or reducing their tumorigenic potential.

J Gen Virol, 1993 May, 74 ( Pt 5), 893 - 6
Site-directed mutagenesis of a potyvirus coat protein and its assembly in Escherichia coli; Jagadish MN et al.; Multiple copies of the Johnsongrass mosaic virus coat protein synthesized in Escherichia coli can readily assemble to form potyvirus-like particles . This E . coli expression system has been used to identify some of the key amino acid residues, within the core region of the coat protein, required for assembly . The two charged residues R194 and D238 previously proposed theoretically to be involved as a pair in the construction of a salt bridge crucial for the assembly process were targeted for site-directed mutagenesis . The results from our experiments suggest that the two residues are required for the assembly process but are not necessarily involved as a pair in a common salt bridge.

J Bacteriol, 1993 May, 175(10), 3204 - 7
Frameshifting in the expression of the Escherichia coli trpR gene is modulated by translation initiation; Benhar I et al.; The Escherichia coli trpR gene encodes the 108-amino-acid-long Trp repressor . We have shown previously that a +1 frameshifting event occurs during the expression of trpR, resulting in the synthesis of an additional (+1 frame) polypeptide . Using trpR-lac'Z fusions, we have recently found that the transition from the 0 to the +1 frame occurs via the bypassing of a 55-nucleotide-long segment of the trpR+1-lac'Z mRNA (I . Benhar, and H . Engelberg-Kulka, Cell 72:121-130, 1993) . Here we show that the frequency of trpR frameshifting (or bypassing) can be regulated both in vivo and in vitro . This frequency is inversely proportional to the rate of initiation of translation of the trpR gene . Hence, modulating the level of translation initiation affects the frequency of frameshifting.

J Bacteriol, 1993 May, 175(10), 3195 - 203
Component A2 of methylcoenzyme M reductase system from Methanobacterium thermoautotrophicum delta H: nucleotide sequence and functional expression by Escherichia coli; Kuhner CH et al.; The gene for component A2 of the methylcoenzyme M reductase system from Methanobacterium thermoautotrophicum delta H was cloned, and its nucleotide sequence was determined . The gene for A2, designated atwA, encodes an acidic protein of 59,335 Da . Amino acid sequence analysis revealed partial homology of A2 to a number of eucaryotic and bacterial proteins in the ATP-binding cassette (ABC) family of transport systems . Component A2 possesses two ATP-binding domains . A 2.2-kb XmaI-BamHI fragment containing atwA and the surrounding open reading frames was cloned into pGEM-7Zf(+) . A cell extract from this strain replaced purified A2 from M . thermoautotrophicum delta H in an in vitro methylreductase assay.

J Bacteriol, 1993 May, 175(10), 3161 - 73
Suppression of replication-deficient mutants of IncFII plasmid NR1 can occur by two different mechanisms that increase expression of the repA1 gene; Wu R et al.; Replication-proficient (Rep+) revertants were isolated from mutants of IncFII plasmid NR1 that were replication defective (Rep-) . The parental Rep- plasmids contained a mutation that inactivated promoter PE for transcription of RNA-E, a trans-acting repressor of translation of the essential RepA1 replication initiation protein of NR1 . The PE mutation also introduced a nonsense codon into a leader peptide gene that precedes and slightly overlaps the repA1 translation initiation site in the mRNA . This reduced the rate of synthesis of RepA1 by uncoupling its translation from that of the leader peptide . The reduced rate of RepA1 synthesis was responsible for the Rep- phenotype . All Rep+ revertants retained the PE mutation and contained second-site mutations responsible for suppression of the Rep- phenotype . One Rep+ revertant contained a second mutation adjacent to the Shine-Dalgarno sequence of repA1 . Another Rep+ revertant contained a mutation in the repA2 gene, which encodes the trans-acting repressor of transcription of repA1 . By using translational lacZ gene fusions, it was found that both kinds of suppressor mutation increased the expression of repA1 to a level sufficient to support replication . In both cases, the synthesis of RepA1 remained uncoupled from that of the leader peptide . The Shine-Dalgarno mutation increased the rate of leader peptide-independent translation of repA1 mRNA and also reduced the sensitivity of repA1 mRNA to inhibition by RNA-E . The repA2 mutation inactivated the RepA2 repressor and increased the rate of transcription of repA1 mRNA . The translational lacZ gene fusions were used to assess the range of regulation of expression of repA1 provided by each of the RNA-E and RepA2 regulatory circuits . By constructing miniplasmids that contained various combinations of the mutations, the contributions of the RNA-E and RepA2 regulatory circuits were assessed with respect to control of plasmid copy number and stable inheritance . Plasmids that lacked either circuit were less stable than wild-type plasmids.

J Bacteriol, 1993 May, 175(10), 3020 - 5
Energetic efficiency of Escherichia coli: effects of mutations in components of the aerobic respiratory chain; Calhoun MW et al.; The aerobic respiratory chain of Escherichia coli can function with either of two different membrane-bound NADH dehydrogenases (NDH-1 and NDH-2) and with either of two ubiquinol oxidases (bd-type and bo-type) . The amounts of each of these enzymes present in the E . coli membrane depend on growth conditions in general and particularly on the dissolved oxygen concentration . Previous in vitro studies have established that NDH-1 and NDH-2 differ in the extent to which they are coupled to the generation of an energy-conserving proton motive force . The same is true for the two ubiquinol oxidases . Hence, the bioenergetic efficiency of the aerobic respiratory chain must depend on the electron flux through each of the specific enzyme components which are being utilized . In this work, the specific rates of oxygen consumption for cells growing under glucose-limited conditions are reported for a series of isogenic strains in which one or more respiratory components are genetically eliminated . The results are compatible with the proton translocation values of the various components reported from in vitro measurements . The data show that (i) the bd-type oxidase is less efficient than is the bo-type oxidase, but the former is still a coupling site in the respiratory chain; and (ii) under the conditions employed, the wild-type strain uses both the NDH-1 and NDH-2 NADH dehydrogenases to a significant degree, but most of the electron flux is directed through the bo-type oxidase.

Genes Dev, 1993 May, 7(5), 870 - 82
Oxytricha telomere-binding protein: separable DNA-binding and dimerization domains of the alpha-subunit; Fang G et al.; A telomere-binding protein heterodimer of 56-kD (alpha) and 41-kD (beta) subunits binds to the single-stranded (T4G4)2 terminus of each Oxytricha nova macronuclear DNA molecule . The alpha-subunit by itself binds to telomeric DNA . The beta-subunit alone does not bind to DNA specifically but interacts with the alpha-subunit to form a very stable ternary complex . We show that the formation of alpha-beta-DNA ternary complex is extremely cooperative . Furthermore, the binary complex (alpha-DNA) has a dissociation half-life of much less than 1 min; addition of the beta-subunit increases the half-life to approximately 100 hrs . Libraries of plasmids with random deletions of the open reading frame for the alpha-subunit were introduced into Escherichia coli, and extracts were subsequently checked for both protein expression and DNA-binding activity with or without added beta-subunit . The alpha-subunit was found to contain two structurally separable domains with distinct functions . The amino-terminal two-thirds is necessary and sufficient for sequence-specific DNA binding . The carboxy-terminal one-third is responsible for alpha/beta-subunit interactions . When expressed separately in E . coli, purified, and mixed together, these two domains reconstitute the activity of the wild-type alpha-subunit (trans-complementation in vitro) . The amino-terminal two-thirds of the beta-subunit is necessary and sufficient both for alpha/beta-subunit interactions and for ternary complex formation . We conclude that the alpha-subunit of the telomere-binding protein, like many transcription factors, has separable DNA-binding and protein-protein interaction domains.

EMBO J, 1993 May, 12(5), 2109 - 17
Overproduction of the poly(ADP-ribose) polymerase DNA-binding domain blocks alkylation-induced DNA repair synthesis in mammalian cells; Molinete M et al.; The zinc-finger DNA-binding domain (DBD) of poly (ADP-ribose) polymerase (PARP, EC 2.4.2.30) specifically recognizes DNA strand breaks induced by various DNA-damaging agents in eukaryotes . This, in turn, triggers the synthesis of polymers of ADP-ribose linked to nuclear proteins during DNA repair . The 46 kDa DBD of human PARP, and several derivatives thereof mutated in its first or second zinc-finger, were overproduced in Escherichia coli, in CV-1 monkey cells or in human fibroblasts to study their DNA-binding properties, the trans-dominant inhibition of resident PARP activity, and the consequences on DNA repair, respectively . A positive correlation was found between the in vitro DNA-binding capacity of the recombinant DBD polypeptides and their inhibitory effect on PARP activity stimulated by the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) . Furthermore, overproduced wild-type DBD blocked unscheduled DNA synthesis induced in living cells by MNNG treatment, but not that induced by UV irradiation . These results define a critical role for the second zinc-finger of PARP for DNA single-stranded break binding and furthermore underscore the importance for PARP to act as a critical regulatory component in the repair of DNA damage induced by alkylating agents.

EMBO J, 1993 May, 12(5), 1897 - 905
Strategies for differential sensory responses mediated through the same transmembrane receptor; Yaghmai R et al.; Trg mediates chemotaxis of Escherichia coli to galactose and ribose by recognition of respective, sugar-occupied binding proteins . Although both attractants act through one transmembrane receptor, maximal response is approximately 50% greater to ribose . This phenomenon was investigated by mutational analysis of a 20-residue segment of Trg implicated in ligand interaction and signalling . Among 17 defective receptors, responses to the two chemoattractants were reduced equivalently for seven and differentially for 10, in some cases reversing the preference order . Mutational substitutions with equivalent effects occurred throughout the segment, but those with a greater effect on galactose or ribose response were segregated to the amino-terminal two-thirds or the carboxy-terminal one-third, respectively, a segregation corresponding in large part to a functional division based on signalling phenotypes . A model for binding protein-mediated recognition revealed two strategies for differential responses . The wild-type preference for ribose probably reflects a balance of receptor affinities and a limiting supply of binding proteins . Mutants with reversed preference probably have differentially reduced receptor affinities and those with an accentuated ribose preference probably have altered signalling abilities . Two-step recognition of ligand allows functional separation of detection and response.

Biochem J, 1993 May 1, 291 ( Pt 3), 687 - 92
Purification and reactivation of recombinant Synechococcus phytoene desaturase from an overexpressing strain of Escherichia coli; Fraser PD et al.; The Synechococcus phytoene desaturase has been isolated from an overexpressing strain of Escherichia coli . The plasma pPDSde135 mediated the overexpression of the full-length polypeptide directly . The recombinant protein comprised 5% of the total cellular protein and was found predominantly in the inclusion body fraction . Urea was used to solubilize the recombinant protein from the inclusion fraction and the protein was subsequently purified to homogeneity on a DEAE-cellulose column . The purification scheme yielded 4.0 mg of homogeneous desaturase protein after a 20-fold purification, recovering 40% of the original protein from a 100 ml suspension culture of E . coli . The recombinant desaturase had an apparent molecular mass of 53 kDa on SDS/PAGE and crossreacted with an antiserum raised against the expressed protein . Desaturase activity was restored upon the removal of urea . The enzyme catalysed the conversion of phytoene to zeta-carotene via phytofluene . These products of the desaturase reaction existed predominantly in a cis configuration . Lipid replenishment enhanced activity . NAD+ and NADP+ were observed to be involved, whilst FAD was an ineffective electron acceptor.

Arch Biochem Biophys, 1993 May, 302(2), 402 - 8
Domains of rat heme oxygenase-2: the amino terminus and histidine 151 are required for heme oxidation; McCoubrey WK Jr et al.; Cleavage of heme b (Fe-protoporphyrin IX) at the alpha-meso carbon bridge is catalyzed by heme oxygenase isozymes, HO-1 and HO-2, to form biliverdin IX alpha . Currently, we have examined the requirement for the amino terminus and the hydrophobic carboxy terminus of rat HO-2 expressed in Escherichia coli for heme degradation activity and have assessed the importance of His 151 for this activity . His 151 is in the longest span of amino acids (24 residues) which are present, with only a single conservative substitution, in seven cloned heme oxygenases including the apparent single isozyme in chicken . We show His 151 is essential for cleavage of heme, as substitution of alanine for this residue by site-directed mutagenesis resulted in expression of an inactive protein with immunoreactivity toward antibody to rat HO-2 . A cDNA construct in which nucleotides encoding the 33 N-terminal amino acid residues were deleted, when expressed, produced a protein of predicted size and immunoreactivity with antibody to HO-2 but also devoid of heme degrading activity . The presence of additional residues at this terminus, for the most part, accounts for the larger size of HO-2 compared to HO-1 . Conversely, the hydrophobic region at the carboxy terminus did not appear to be essential for heme degradation . A construct in which the sequence encoding the primarily hydrophobic amino acids of the carboxy terminus was replaced by a sequence encoding predominantly hydrophilic residues expressed a protein which retained full capability to convert heme to biliverdin . Further, the construct with a hydrophilic carboxy terminus was not appreciably associated with bacterial membranes, suggesting that the carboxy terminus in the wild-type protein serves as a membrane anchor for this enzyme.

Pharmacology, 1993 May, 46(5), 254 - 67
Fluid resuscitation improves survival of endotoxemic or septicemic rats: possible contribution of tumor necrosis factor; Smith EF 3rd et al.; The present study was designed to investigate the effects of fluid administration on survival in endotoxemic or septicemic male Sprague-Dawley rats . Endotoxemia was induced by intravenous injection of Escherichia coli lipopolysaccharide (LPS), and septicemia produced by cecal ligation and puncture (CLP) . In endotoxemic animals deprived of fluid resuscitation, 7-day survival following injection of LPS at doses of 1, 3, or 10 mg/kg LPS were 70% (n = 10), 30% (n = 10), and 0% (n = 10), respectively . In rats resuscitated with 3.3 ml/kg/h of 0.9% NaCl, the dose-response curve for survival was shifted 5-fold rightward in a parallel manner (p < 0.001, between the fluid-resuscitated and nonfluid resuscitated LPS groups), indicating a reduced sensitivity to the effects of LPS following fluid resuscitation . LPS increased serum tumor necrosis factor (TNF alpha) concentrations in fluid-resuscitated endotoxemic animals from a baseline value of 20 U/ml to 2,350 U/ml at 1 h, which returned to 200 U/ml at 2 h . In endotoxemic animals not receiving fluid resuscitation, serum TNF alpha levels at 1 and 2 h were 5-fold and 27-fold higher, respectively, than in fluid-resuscitated animals . There were no differences in arterial blood pressure or heart rate between the two groups of endotoxemic animals; total peripheral resistance was significantly lower at 1 h, and cardiac index was significantly greater at 3 h in the fluid-resuscitated LPS group; otherwise there were no further differences in hemodynamic parameters between the two groups . The survival rate at 4 days following CLP without fluid resuscitation was 14%, whereas CLP with fluid resuscitation improved survival to 74% (p < 0.01) . TNF alpha was undetectable (i.e., < 20 U/ml) in the serum of animals subjected to CLP . The improvement in survival with fluid infusion in the LPS and CLP models cannot be attributed to catheter implantation, or to improved hemodynamic parameters in the LPS model . The improvement in survival in the LPS model with fluid infusion was associated with attenuated increases in TNF alpha levels . Furthermore, these studies illustrate that fluid-resuscitated and nonfluid-resuscitated experimental animal models should not be considered equivalent.

J Clin Invest, 1993 May, 91(5), 2275 - 80
Alternative splicing: a mechanism for phenotypic rescue of a common inherited defect; Morisaki H et al.; Approximately 2% of Caucasians and African-Americans are homozygous for a nonsense mutation in exon 2 of the AMPD1 (AMP deaminase) gene . These individuals have a high grade deficiency of AMPD activity in their skeletal muscle . More than 100 patients with AMPD1 deficiency have been reported to have symptoms of a metabolic myopathy, but it is apparent many individuals with this inherited defect are asymptomatic given the prevalence of this mutant . Results of the present study provide a potential molecular explanation for "correction" of this genetic defect . Alternative splicing eliminates exon 2 in 0.6-2% of AMPD1 mRNA transcripts in adult skeletal muscle . Expression studies document that AMPD1 mRNA, which has exon 2 deleted, encodes a functional AMPD peptide . A much higher percentage of alternatively spliced transcripts are found during differentiation of human myocytes in vitro . Transfection studies with human minigene constructs demonstrate that alternative splicing of the primary transcript of human AMPD1 is controlled by tissue-specific and stage-specific signals . Alternative splicing of exon 2 in individuals who have inherited this defect provides a mechanism for phenotypic rescue and variations in splicing patterns may contribute to the variability in clinical symptoms.

Scand J Immunol, 1993 May, 37(5), 581 - 6
Cyclosporin A does not inhibit IL-1 alpha-induced epithelial cell IL-6 secretion; Hedges S et al.; Trauma and infection activated a murine mucosal IL-6 response in different ways: the IL-6 response to bacteria was sensitive to Cyclosporin A (CsA); the IL-6 response to trauma was not . The aim of the present study was to identify possible activators of the CsA-insensitive IL-6 secretion at the epithelial cell level . Two human epithelial cell lines from the kidney (A498) and bladder (J82) were exposed to Escherichia coli Hu734, interleukin-1 alpha (IL-1 alpha) and tumour necrosis factor alpha (TNF-alpha) . The E . coli strain had been used for the in vivo experiments which led to this study, and IL-1 alpha and TNF-alpha were likely to be released during infections and trauma . The secretion of IL-6 into the supernatants was compared between cells stimulated in the presence or absence of CsA . E . coli Hu734, IL-1 alpha and TNF-alpha stimulated an IL-6 response in the two epithelial cell lines . The IL-1 alpha-induced IL-6 response was rapid, and the secreted IL-6 levels were significantly higher than those induced by E . coli Hu734 or TNF-alpha . The IL-6 response to IL-1 alpha was insensitive to CsA . By contrast, the IL-6 response to E . coli Hu734 and TNF-alpha was inhibited by CsA . These results demonstrated that the inhibitory effect of CsA depends on the stimulus triggering the IL-6 response . IL-1 alpha may play a role in the induction of trauma-associated CsA-insensitive IL-6 secretion.

Proc Natl Acad Sci U S A, 1993 May 1, 90(9), 4181 - 5
Interaction of translation factor SELB with the formate dehydrogenase H selenopolypeptide mRNA; Baron C et al.; The SELB protein from Escherichia coli is a specialized elongation factor required for the UGA-directed insertion of the amino acid selenocysteine into selenopolypeptides . Discrimination of the UGA codon requires the presence of a recognition element within the mRNA, which is located at the 3' side of the UGA codon; a hairpin structure can be formed within this mRNA region . By gel shift assays, a specific interaction between SELB and the mRNA recognition element could be demonstrated . Footprinting experiments, using nucleases or iodine as cleaving agents, showed that SELB binds to the loop region of the hairpin structure . In the presence of selenocysteinyl-tRNA, SELB formed a complex with the charged tRNA and the mRNA . The results indicate that targeted insertion of selenocysteine is accomplished by the binding of the SELB protein to this mRNA recognition element, resulting in the formation of a selenocysteinyl-tRNA.SELB complex at the mRNA in the immediate neighborhood of the UGA codon.

Proc Natl Acad Sci U S A, 1993 May 1, 90(9), 4161 - 5
Translation of mRNAs with degenerate initiation triplet AUU displays high initiation factor 2 dependence and is subject to initiation factor 3 repression; La Teana A et al.; The influence of the rare initiation triplet AUU on mRNA translation was investigated by comparing the activity of two pairs of model mRNAs that differ in the length of Shine-Dalgarno and spacer sequences . Irrespective of the initiation triplet (AUG or AUU), all mRNAs had similar template activity in vitro, but translation of AUU mRNAs depended more on initiation factor (IF) 2 and less on IF3 than that of AUG mRNAs . Increasing the IF3/ribosome ratio from 2 to 10 progressively inhibited the AUU mRNAs and abolished their capacity to compete for translating ribosomes with other mRNAs but did not affect activity of the AUG mRNAs . The effects induced by IF3 are from its different influence on on- and off-rates of the transition 30S preinitiation complex<==>30S initiation complex; depending on the nature of the initiation triplet (AUG or AUU) of the mRNA, IF3 shifts the position of equilibrium toward binding or dissociation of fMet-tRNA, respectively . Stimulation of fMet-tRNA binding and dissociation yields superimposable IF3 titration curves that saturate at an IF3/30S ratio of approximately 1, indicating that the data are from the interaction of one molecule of IF3 with the same 30S binding site . Both effects are either lost or strongly reduced with 30S mutants defective in IF3 binding . Translational repression of AUU mRNAs by IF3 is from the factor-dependent dissociation of fMet-tRNA from 30S subunits, which becomes relevant when excess IF3 interferes with the formation of 70S initiation complex, presumably by interacting with 50S subunit.

Proc Natl Acad Sci U S A, 1993 May 1, 90(9), 3983 - 7
Evidence from in vitro replication that O6-methylguanine can adopt multiple conformations; Dosanjh MK et al.; The effect of O6-methylguanine (m6G) on replication, in a partially double-stranded defined 25-base oligonucleotide, has been studied under nonlimiting conditions of unmodified dNTPs and over an extended time period, using the Klenow fragment of Escherichia coli DNA polymerase I . The sequence surrounding m6G has flanking cytosines (C-m6G-C), and the initial steady-state kinetics have been reported . When the primer was annealed so that the first base to be replicated was m6G, replication was virtually complete in approximately 5 min, although the reaction appears biphasic . When annealed with a primer where thymine or cytosine is paired opposite template m6G, about half the molecules were replicated in the first 15 sec, and no significant further replication was seen over a 1-hr period . When m6G was dealkylated by DNA-O6-methylguanine-methyltransferase, replication was rapid with no blockage . These data suggest that there can be two (or more) conformations of m6G . In these studies the term syn refers to conformers interfering with base-pairing, whereas anti refers to those allowing such base-pairing . Previous physical studies by others indicate that syn- and anti-conformers of the methyl group relative to the N1 of guanine are possible . Here molecular modeling/computational studies are described, suggesting that syn- and anti-m6G can be of similar energy in DNA, and, therefore, these two conformers may explain the two types of species observed during in vitro replication . An alternative explanation could be the possibility that the different species may manifest differential interactions of m6G with Klenow fragment . These results may provide a rationale for why m6G lesions in vivo have been reported to be lethal as well as mutagenic.

Proc Natl Acad Sci U S A, 1993 May 1, 90(9), 3904 - 7
Tandem double CC-->TT mutations are produced by reactive oxygen species; Reid TM et al.; Oxidative damage to DNA is mutagenic and thus may play a role in carcinogenesis . Because of the large number of different DNA lesions formed by oxidative species, no genetic alteration so far identified is exclusively associated with oxygen damage . Tandem double CC-->TT mutations are known to occur via UV damage to DNA and are thought to be a specific indicator of UV exposure . Using a sensitive reversion assay that can detect both single and double mutations within the same codon of the M13-encoded lacZ alpha gene, we show that treatments that produce reactive oxygen species can also produce tandem double CC-->TT mutations . The frequency at which these mutations occur is less than that for single base mutations by a factor of approximately 30 . The induction of these mutations is inhibited by treatment that scavenges hydroxyl radicals . This unique mutation provides a marker of oxygen free radical-induced mutagenesis in cells that are not exposed to UV-irradiation and an indicator for assessing the involvement of oxidative damage to DNA in aging and tumor progression.

Proc Natl Acad Sci U S A, 1993 May 1, 90(9), 3875 - 9
Biochemical interaction of the Escherichia coli RecF, RecO, and RecR proteins with RecA protein and single-stranded DNA binding protein; Umezu K et al.; The Escherichia coli RecF, RecO, and RecR proteins were analyzed for their effect on RecA-mediated pairing of single-stranded circular DNA and homologous linear duplex DNA substrates . As shown by other workers, joint molecule formation by RecA was inhibited by E . coli single-stranded DNA binding protein (SSB) when it was added to single-stranded DNA before RecA . This inhibitory effect was overcome by the addition of RecO and RecR or RecF, RecO, and RecR . Both the rate and extent of joint molecule formation were restored to the maximal level observed when SSB was added after RecA . RecF, RecO, and RecR proteins had no effect on the conversion of joint molecules to final products and only appeared to stimulate an early step in the pairing reaction . The stimulatory effect of RecF, RecO, and RecR was not seen without SSB or when SSB was added after RecA . RecF protein by itself inhibited reactions in mixtures containing RecA and SSB, and this inhibition was overcome by the addition of RecO and RecR . These data suggest that RecO and RecR, and possibly RecF, help RecA overcome inhibition by SSB and utilize SSB-single-stranded-DNA complexes as substrates.

Am J Clin Nutr, 1993 May, 57(5), 643 - 9
Rapid incorporation of fish or olive oil fatty acids into rat hepatic sinusoidal cell phospholipids after continuous enteral feeding during endotoxemia; Palombo JD et al.; Therapeutic modalities that downregulate macrophage and endothelial production of eicosanoid mediators by displacing membrane arachidonic acid (20:4 omega 6) may benefit patients at increased risk of septic complications . The objective of this study in rats was to assess the incorporation of fish or olive oil fatty acids into hepatic Kupffer and endothelial (K&E) cell phospholipids after 4 d of continuous enteral feeding during endotoxemia . Either endotoxin (ETX) (0.5-1 mg-1.day-1) or vehicle was infused intravenously during the last 72 h . Dietary fish and olive oil fatty acids were rapidly incorporated into both K&E and plasma phospholipids irrespective of ETX cotreatment . Rats infused with the fish oil-enriched diet had a significantly lower relative percent of both K&E linoleic acid (18:2 omega 6) and 20:4 omega 6, whereas rats infused with the olive oil-enriched diet only had a lower relative percent of 18:2 omega 6 compared with control rats receiving corn oil . Provision of specific dietary lipids by continuous enteral infusion may prove efficacious for the rapid modulation of hepatic sinusoidal cell membrane fatty acids under either normal or endotoxemic conditions.

Virology, 1993 May, 194(1), 332 - 7
RNA binding of recombinant nucleocapsid proteins of hantaviruses; Gott P et al.; Genes encoding the nucleocapsid (N) proteins of two hantaviruses, Hantaan virus strain 76-118 (HTN) and Puumala virus strain CG 18-20 (PUU), were expressed in Escherichia coli as histidine-tagged proteins . They were purified by metal-chelate affinity chromatography under native or denaturing conditions to near homogeneity . The soluble form of HTN N protein was associated with RNA of E . coli . Renatured N proteins were shown to bind in vitro transcribed RNA representing the hantaviral small genomic (S) RNA segment . RNA binding was shown by affinity to filter-immobilized N proteins and by gel mobility shift assays . Competition experiments using tRNA, poly(U) and poly(A)+ U indicated that binding of RNA by the N protein is nonspecific . However, direct binding of ds-RNA resulted in efficient formation of large complexes suggesting that double-stranded nucleic acids are bound preferentially . Carboxyterminal fragments of HTN and PUU N proteins containing about 100 amino acids of the carboxy termini retained full binding capacity indicating that RNA binding occurs via a carboxyterminal domain.

Virology, 1993 May, 194(1), 128 - 36
A multicomponent cis-activator of transcription of the E1b gene of adenovirus type 5; Spector DJ et al.; We report that transcription of the adenovirus type 5 E1b gene is activated substantially in cis by sequences located between positions -362 and -49 with respect to the RNA start site . DNA fragments consisting of the -362 to -49 sequences, or subsets thereof, were inserted into a reporter plasmid containing a minimal E1b promoter (positions -48 to +14) joined to the Escherichia coli cat gene . In the presence of cotransfected E1a and E1b genes in trans, CAT enzyme synthesis in transfected KB cells was stimulated about 20-fold by sequences from -362 to -49 (XY) in cis and to a lesser extent by sequences from either -362 to -128 (X) or -127 to -49 (Y) . Adenoviruses were isolated lacking the X, Y, or XY sequences and KB cells were infected with one of the mutants, as well as wild-type virus to provide E1a and E1b in trans . Deletion of both X and Y resulted in a 20-fold reduction in early E1b RNA and a 12-fold reduction in late RNA . Deletion of X or Y alone produced up to 5-fold reductions in early or late E1b RNA accumulation . In vitro DNA-protein interactions in the Y sequence were revealed by modification of the procedure used for previous detection of X region footprints . These data indicate that X and Y sequences, which include E1a protein coding and 3' untranslated DNA, also participate in DNA-protein interactions necessary for high levels of E1b promoter activity . The presence of such overlapping genetic elements raises the interesting possibility that functional E1a and E1b mRNAs must be synthesized from separate templates.

Oncogene, 1993 May, 8(5), 1183 - 93
Wild-type but not mutant p53 can repress transcription initiation in vitro by interfering with the binding of basal transcription factors to the TATA motif; Ragimov N et al.; It has previously been shown that excess wild type (wt) p53 can repress the transcriptional activity of a variety of promoters in intact cells . To determine whether this transcriptional repression represented a direct effect of p53, wt and mutant p53 were prepared from E . coli-produced p53 and from insect cells infected with a recombinant baculovirus . When added into an in vitro transcription system, wt p53, but not mutant p53 reduced markedly transcription from the c-myc promoter, as well as from an array of other promoters, with the exception of an MHC class I gene promoter . The presence of wt p53 seemed to affect specifically the formation of the transcription preinitiation complex because preformed initiation complexes were completely refractory to wt p53, as was also the process of transcript elongation . Wild-type but not mutant p53 interfered with the stable binding of TBP and TFIIA to the TATA motif, although both wt and mutant p53 could associate in vitro with purified TBP . We propose that upon binding to TBP, wt but not mutant p53 specifically blocks the ability of TBP to engage in interactions required for efficient transcriptional initiation . This may account, at least in part, for the ability of excess wt p53 to inhibit cell proliferation and to interfere with neoplastic processes.

J Bacteriol, 1993 May, 175(9), 2788 - 91
Cell division and transcription of ftsZ; Smith RW et al.; For normal cell division, the ftsZ gene must be transcribed from a number of promoters that are located within the proximal upstream genes (ddlB, ftsQ, and ftsA) . We show that the main promoters have identical responses to changes in growth rate, i.e., under all conditions, the frequency of transcription per septum formed is approximately constant and independent of cell size or growth rate per se . We also show that transcription from these promoters is independent of stationary-phase transcription factor sigma s.

J Bacteriol, 1993 May, 175(9), 2613 - 24
The Escherichia coli heat shock gene htpY: mutational analysis, cloning, sequencing, and transcriptional regulation; Missiakas D et al.; We have identified a new heat shock gene, designated htpY, located 700 bp upstream of the dnaK dnaJ operon . We cloned it and showed that it is transcribed clockwise vis-a-vis the Escherichia coli genetic map, in the same direction as the dnaK dnaJ operon . The htpY gene encodes a 21,193-Da polypeptide . Promoter mapping experiments and Northern (RNA) analysis showed that the htpY gene belongs to the classical heat shock gene family, because the transcription from its major promoter is under the positive control of the rpoH gene product (sigma 32) and resembles canonical E sigma 32-transcribed consensus promoter sequences . This conclusion has been strengthened by the construction and analysis of a phtpY-lacZ promoter fusion . Despite the fact that htpY null bacteria are viable, the expression of various E sigma 32 heat shock promoters is significantly decreased, suggesting that HtpY plays an important role in the regulation of the heat shock response . Consistent with this interpretation, overproduction of the HtpY protein results in a generalized increase of the heat shock response in E . coli.

J Bacteriol, 1993 May, 175(9), 2564 - 7
Escherichia coli shows two types of behavioral responses to osmotic upshift; Li C et al.; Behavioral responses to osmotic upshift were characterized by temporal assays of free-swimming cells of Escherichia coli . Small osmotic upshifts (200 to 300 mosM) elicited tumble responses which were chemotaxis dependent, while large osmotic upshifts (400 to 500 mosM) elicited stopping followed by pseudotumbling which was chemotaxis independent.

J Bacteriol, 1993 May, 175(9), 2552 - 63
Deletion analysis of the avermectin biosynthetic genes of Streptomyces avermitilis by gene cluster displacement; MacNeil T et al.; Streptomyces avermitilis produces a group of glycosylated, methylated macrocyclic lactones, the avermectins, which have potent anthelmintic activity . A homologous recombination strategy termed gene cluster displacement was used to construct Neor deletion strains with defined endpoints and to clone the corresponding complementary DNA encoding functions for avermectin biosynthesis (avr) . Thirty-five unique deletions of 0.5 to > 100 kb over a continuous 150-kb region were introduced into S . avermitilis . Analysis of the avermectin phenotypes of the deletion-containing strains defined the extent and ends of the 95-kb avr gene cluster, identified a regulatory region, and mapped several avr functions . A 60-kb region in the central portion determines the synthesis of the macrolide ring . A 13-kb region at one end of the cluster is responsible for synthesis and attachment of oleandrose disaccharide . A 10-kb region at the other end has functions for positive regulation and C-5 O methylation . Physical analysis of the deletions and of in vivo-cloned fragments refined a 130-kb physical map of the avr gene cluster region.

Infect Immun, 1993 May, 61(5), 2203 - 6
Identification of eae sequences in enteropathogenic Escherichia coli strains from rabbits; Pohl PH et al.; DNA sequences coding for attachment and for verotoxin production were investigated in a collection of enteropathogenic Escherichia coli strains from rabbits . All of the strains produced diarrhea after experimental infection, attached to the brush borders of the intestinal lining, and possessed homology to the eae probe, whereas strains isolated from healthy rabbits did not . Sequences homologous to the AF/R1 fimbriae of strain RDEC-1 were not found . One strain reacted with the probe for the Shiga-like toxin type I gene.

Infect Immun, 1993 May, 61(5), 1917 - 25
Similar mechanisms of action of defined polysaccharides and lipopolysaccharides: characterization of binding and tumor necrosis factor alpha induction; Otterlei M et al.; Little has been reported about the effects of different polysaccharides on cytokine production from human monocytes . In this study, we show that several well-defined polysaccharides, including polymers with different sizes of beta 1-4-linked D-mannuronic acid (poly-M, high-M alginate, and M-blocks) and cellulose oxidized in the C-6 position, induced human monocytes to produce tumor necrosis factor alpha (TNF-alpha) . Poly-M was the most efficient polysaccharide tested and, on a weight basis, was approximately as efficient as lipopolysaccharide (LPS) from Escherichia coli . TNF-alpha production was shown to depend strongly on the molecular weights of poly-M and high-M alginate, with maximal TNF-alpha production occurring at molecular weights above 50,000 and 200,000, respectively . G-blocks, alpha 1-4-linked L-guluronic acid polymers that did not induce cytokine production from monocytes, reduced the cytokine production induced by the beta 1-4-linked polyuronic acids and LPS . Furthermore, both G-blocks and LPS were found to inhibit the binding of poly-M to monocytes, as measured by flow cytometry . In addition, we found that the binding of LPS to monocytes was inhibited by G-blocks, M-blocks, and poly-M . Our results indicate that beta 1-4-linked polyuronic acids and LPS may stimulate monocytes to produce TNF-alpha by similar mechanisms and may bind to a common receptor.

Infect Immun, 1993 May, 61(5), 1799 - 809
Cloning and expression of a high-molecular-mass major antigen of Helicobacter pylori: evidence of linkage to cytotoxin production; Tummuru MK et al.; A high-molecular-mass (120- to 128-kDa) Helicobacter pylori antigen has been associated with peptic ulcer disease . We created a bank of 40,000 random chromosomal fragments of H . pylori 84-183 by using lambda ZapII . Screening of this bank in Escherichia coli XL1-Blue with absorbed serum from an H . pylori-infected person permitted the isolation and purification of a clone with a 3.5-kb insert . Subcloning of this insert (pMC3) permitted the expression of a recombinant H . pylori protein that had a mass of approximately 96 kDa and that was recognized by the human serum . Sera that were obtained from H . pylori-infected persons and that recognized the native 120- to 128-kDa H . pylori antigen recognized the recombinant 96-kDa pMC3 protein to a significantly greater extent than did sera that did not recognize the native H . pylori antigen . All 19 H . pylori isolates producing the 120- to 128-kDa antigen hybridized with pMC3; none of 13 nonproducers did so (P < 0.001) . Because all 15 isolates producing the vacuolating cytotoxin hybridized with pMC3, we called the gene cagA (cytotoxin-associated gene) . Sequence analysis of pMC3 identified an open reading frame of 859 amino acids, without a termination codon . Parallel screening of a lambda gt11 library with human serum revealed positive plaques with identical 0.6-kb inserts and sequences matching the sequence of the downstream region of pMC3 . To clone the full-length gene, we used the 0.6-kb fragment as a probe and isolated a clone with a 2.7-kb insert from the lambda ZapII genomic library . Nucleotide sequencing of this insert (pYB 2) revealed a 785-bp sequence that overlapped the downstream region of pMC3 . Translation of the complete nucleotide sequence of cagA revealed an open reading frame of 1,181 amino acids yielding a protein of 131,517 daltons . There was no significant homology with any previously reported protein sequence . These findings indicate the cloning and characterization of a high-molecular-mass H . pylori antigen potentially associated with virulence and with cytotoxin production.

Infect Immun, 1993 May, 61(5), 1722 - 9
Lipopolysaccharide from Brucella abortus behaves as a T-cell-independent type 1 carrier in murine antigen-specific antibody responses; Betts M et al.; In order to determine the carrier nature of lipopolysaccharide from Brucella abortus (LPS-BA) in evoking humoral responses, normal and immunodeficient mice were immunized with trinitrophenyl (TNP)-conjugated LPS-BA (TNP-LPS-BA) and the responses were compared with those to known T-dependent and T-independent antigens . TNP-LPS-BA, like T-independent type 1 (TI-1) antigens such as TNP-BA and TNP-LPS from Escherichia coli (TNP-LPS-EC), generated anti-TNP responses in BALB/c, athymic BALB/c nu/nu, and CBA/N mice . In contrast, N-2,4-dinitrophenyl-beta-alanylglycylglycyl-substituted keyhole limpet hemocyanin, a typical T-dependent antigen, was not immunogenic in athymic mice, and TNP-Ficoll (T-independent type 2) was ineffective in eliciting humoral responses in CBA/N mice . These results indicate that LPS from B . abortus acts as a TI-1 carrier in generating antibody responses . In C3H/HeJ mice, TNP-LPS-BA generated higher-titer immunoglobulin G1 (IgG1), IgG2a, and IgG2b anti-TNP antibodies than TNP-LPS-EC . Compared with those from BALB/c mice, pure resting B cells isolated from C3H/HeJ mice exhibited a 30-fold lower proliferative response to LPS-EC, whereas the LPS-BA response was reduced to a lesser extent (5-fold) . This suggests that the disparity observed in antibody titers was due to different abilities of LPS from B . abortus and E . coli to stimulate C3H/HeJ B cells . The ability of LPS from B . abortus to act as a carrier in generating humoral immune responses indicates that LPS-BA can be substituted for whole B . abortus organisms in vaccine development.

Endocrinology, 1993 May, 132(5), 2246 - 53
Tumor necrosis factor mediates the effects of endotoxin on cholesterol and triglyceride metabolism in mice; Memon RA et al.; The host response to infection is frequently accompanied by changes in cholesterol and triglyceride (TG) metabolism . To determine the role of cytokines in mediating these changes, we studied the effects of endotoxin (LPS), tumor necrosis factor-alpha (TNF) and interleukin-1 beta (IL-1) on cholesterol and TG metabolism in C57Bl/6 (LPS-sensitive) mice and in C3H/HeJ (LPS-resistant) mice whose macrophages do not produce TNF and IL-1 in response to LPS . Sixteen hours after administration, LPS (1 micrograms/mouse) produced a 41% increase in serum cholesterol and a 62% increase in serum TG levels in C57Bl/6 mice whereas a 100-fold higher dose of LPS did not have a significant effect in C3H/HeJ mice . LPS (1 microgram/mouse) also produced a 8.6-fold increase in hepatic cholesterol synthesis and a 2.7-fold increase in hepatic fatty acid synthesis in C57Bl/6 mice but had no effect in C3H/HeJ mice . This suggests that macrophage produced cytokines such as TNF and IL-1 may be involved in mediating these effects of LPS . Additionally, LPS also increased the activity of hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in cholesterol synthesis . As seen with LPS, TNF and IL-1 also increased serum cholesterol and TG levels in C57Bl/6 mice . Moreover, TNF and IL-1 produced a 2.3- and 2.1-fold increase in hepatic HMG-CoA reductase activity, respectively . Finally, pretreatment of mice with anti-TNF antibodies, but not with an IL-1 receptor antagonist, blocked the effect of LPS on serum cholesterol and TG levels, hepatic cholesterol and fatty acid synthesis, and hepatic HMG-CoA reductase activity . These results suggest that whereas both TNF and IL-1 mimic the effects of LPS on cholesterol and TG metabolism, TNF may be the in vivo mediator of these late effects of LPS in mice.

Circ Res, 1993 May, 72(5), 1132 - 8
In vivo gene transfer and expression in normal uninjured blood vessels using replication-deficient recombinant adenovirus vectors; Lemarchand P et al.; Replication-deficient recombinant adenovirus vectors do not require target cell replication for transfer and expression of exogenous genes and thus may be useful for in vivo gene therapy in the endothelium . To evaluate the feasibility of adenovirus-mediated gene transfer in vivo in normal intact blood vessels, adenovirus vectors containing the Escherichia coli lacZ gene or a human alpha 1-antitrypsin (alpha 1AT) cDNA were injected in vivo into the lumen of an occluded vessel segment of sheep jugular vein and/or carotid artery . After 15 minutes of incubation, circulation was restored; the vessels were harvested 1-28 days later and evaluated for gene transfer and expression . Three days after in vivo exposure to the lacZ adenovirus vector, the endothelium of jugular veins and carotid arteries expressed beta-galactosidase . Exposure of jugular veins and carotid arteries in vivo to the alpha 1AT adenovirus vector resulted in the expression of alpha 1AT mRNA transcripts detected by Northern analysis and in the synthesis and secretion of alpha 1AT detected by ex vivo {35S}methionine labeling . Expression with the adenovirus vectors was efficient and easily detectable 1-14 days after injection, with maximum expression at 7 days . Expression was no longer evident at 28 days . Thus, adenovirus vectors are capable of transferring exogenous genes to the endothelium of normal arteries and veins with expression for at least 2 weeks, suggesting that these vectors have the potential for a variety of cardiovascular experimental and clinical applications.

Proc Soc Exp Biol Med, 1993 May, 203(1), 26 - 9
Legionella pneumophila induced tumor necrosis factor production in permissive versus nonpermissive macrophages; Arata S et al.; The ability of an opportunistic intracellular bacterial pathogen, Legionella pneumophila, to induce tumor necrosis factor (TNF) in macrophages from susceptible A/J or resistant BDF1 and BALB/c mice was determined . Cultures of peritoneal elicited macrophages from these mouse strains produced TNF in response to the Legionella . The TNF levels produced by the macrophages stimulated with either heat-killed Legionella vaccine or lipopolysaccharide were similar and dose dependent, although the amount of TNF produced by macrophages from permissive A/J mice was 2- to 4-fold higher than that produced by macrophages from the nonpermissive mice . Similar differences in TNF levels occurred when macrophages from either permissive or non-permissive mice were infected with viable Legionella . The TNF levels produced by the A/J mouse macrophages increased as a function of time after infection, with a peak of activity on Day 1 or 2, depending upon the initial concentration of the bacteria . Infection of the A/J mouse macrophages with avirulent Legionella resulted in induced levels comparable to those induced by a virulent strain . Although it is widely believed that TNF production by mouse macrophages is related to resistance to infections, the results of this study did not show a relationship between TNF production by macrophages in vitro and resistance versus susceptibility of the macrophage donor mouse strain to Legionella infection.

Mol Cell Biol, 1993 May, 13(5), 3113 - 21
The orphan receptor Rev-ErbA alpha activates transcription via a novel response element; Harding HP et al.; Rev-ErbA alpha (Rev-Erb) is a nuclear hormone receptor-related protein encoded on the opposite strand of the alpha-thyroid hormone receptor (TR) gene . This unusual genomic arrangement may have a regulatory role, but the conservation of human and rodent Rev-Erb amino acid sequences suggests that the protein itself has an important function, potentially as a sequence-specific transcriptional regulator . However, despite its relationship to the TR, Rev-Erb bound poorly to TR binding sites . To determine its DNA-binding specificity in an unbiased manner, Rev-Erb was synthesized in Escherichia coli, purified, and used to select specific binding-sites from libraries of random double-stranded DNA sequences . We found that Rev-Erb binds to a unique site consisting of a specific 5-bp A/T-rich sequence adjacent to a TR half-site . Rev-Erb contacts this entire asymmetric 11-bp sequence, which is the longest nonrepetitive element specifically recognized by a member of the thyroid/steroid hormone receptor superfamily, and mutations in either the A/T-rich or TR half-site regions abolished specific binding . The binding specificity of wild-type Rev-Erb was nearly identical to that of C- and N-terminally truncated forms . This binding was not enhanced by retinoid X receptor, TR, or other nuclear proteins, none of which formed heterodimers with Rev-Erb . Rev-Erb also appeared to bind to the selected site as a monomer . Furthermore, Rev-Erb activates transcription through this binding site even in the absence of exogenous ligand . Thus, Rev-Erb is a transcriptional activator whose properties differ dramatically from those of classical nuclear hormone receptors, including the TR encoded on the opposite strand of the same genomic locus.

Mol Cell Biol, 1993 May, 13(5), 2870 - 81
Casein kinase I-like protein kinases encoded by YCK1 and YCK2 are required for yeast morphogenesis; Robinson LC et al.; Casein kinase I is an acidotropic protein kinase class that is widely distributed among eukaryotic cell types . In the yeast Saccharomyces cerevisiae, the casein kinase I isoform encoded by the gene pair YCK1 and YCK2 is a 60- to 62-kDa membrane-associated form . The Yck proteins perform functions essential for growth and division; either alone supports growth, but loss of function of both is lethal . We report here that casein kinase I-like activity is associated with a soluble Yck2-beta-galactosidase fusion protein in vitro and that thermolabile protein kinase activity is exhibited by a protein encoded by fusion of a temperature-sensitive yck2 allele with lacZ . Cells carrying the yck2-2ts allele arrest at restrictive temperature with multiple, elongated buds containing multiple nuclei . This phenotype suggests that the essential functions of the Yck proteins include roles in bud morphogenesis, possibly in control of cell growth polarity, and in cytokinesis or cell separation . Further, a genetic relationship between the yck2ts allele and deletion of CDC55 indicates that the function of Yck phosphorylation may be related to that of protein phosphatase 2A activity.

Mol Cell Biol, 1993 May, 13(5), 2802 - 14
(CT)n (GA)n repeats and heat shock elements have distinct roles in chromatin structure and transcriptional activation of the Drosophila hsp26 gene; Lu Q et al.; Previous analysis of the hsp26 gene of Drosophila melanogaster has shown that in addition to the TATA box and the proximal and distal heat shock elements (HSEs) (centered at -59 and -340, relative to the start site of transcription), a segment of (CT)n repeats at -135 to -85 is required for full heat shock inducibility (R.L . Glaser, G.H . Thomas, E.S . Siegfried, S.C.R . Elgin, and J.T . Lis, J . Mol . Biol . 211:751-761, 1990) . This (CT)n element appears to contribute to formation of the wild-type chromatin structure of hsp26, an organized nucleosome array that leaves the HSEs in nucleosome-free, DNase I-hypersensitive (DH) sites (Q . Lu, L.L . Wallrath, B.D . Allan, R.L . Glaser, J.T . Lis, and S.C.R . Elgin, J . Mol . Biol . 225:985-998, 1992) . Inspection of the sequences upstream of hsp26 has revealed an additional (CT)n element at -347 to -341, adjacent to the distal HSE . We have analyzed the contribution of this distal (CT)n element (-347 to -341), the proximal (CT)n element (-135 to -85), and the two HSEs both to the formation of the chromatin structure and to heat shock inducibility . hsp26 constructs containing site-directed mutations, deletions, substitutions, or rearrangements of these sequence elements have been fused in frame to the Escherichia coli lacZ gene and reintroduced into the D . melanogaster genome by P-element-mediated germ line transformation . Chromatin structure of the transgenes was analyzed (prior to gene activation) by DNase I or restriction enzyme treatment of isolated nuclei, and heat-inducible expression was monitored by measuring beta-galactosidase activity . The results indicate that mutations, deletions, or substitutions of either the distal or the proximal (CT)n element affect the chromatin structure and heat-inducible expression of the transgenes . These (CT)n repeats are associated with a nonhistone protein(s) in vivo and are bound by a purified Drosophila protein, the GAGA factor, in vitro . In contrast, the HSEs are required for heat-inducible expression but play only a minor role in establishing the chromatin structure of the transgenes . Previous analysis indicates that prior to heat shock, these HSEs appear to be free of protein . Our results suggest that GAGA factor, an abundant protein factor required for normal expression of many Drosophila genes, and heat shock factor, a specific transcription factor activated upon heat shock, play distinct roles in gene regulation: the GAGA factor establishes and/or maintains the DH sites prior to heat shock induction, while the activated heat shock factor recognizes and binds HSEs located within the DH sites to trigger transcription.

J Virol, 1993 May, 67(5), 2628 - 36
Comparison of DNA binding and integration half-site selection by avian myeloblastosis virus integrase; Grandgenett DP et al.; Insertion of the linear retrovirus DNA genome into the host DNA by the virus-encoded integrase (IN) is essential for efficient replication . We devised an efficient virus-like DNA plasmid integration assay which mimics the standard oligonucleotide assay for integration . It permitted us to study, by electron microscopy and sequence analysis, insertion of a single long terminal repeat terminus (LTR half-site) of one plasmid into another linearized plasmid . The reaction was catalyzed by purified avian myeloblastosis virus IN in the presence of Mg2+ . The recombinant molecules were easily visualized and quantitated by agarose gel electrophoresis . Agarose gel-purified recombinants could be genetically selected by transformation of ligated recombinants into Escherichia coli HB101 cells . Electron microscopy also permitted the identification and localization of IN-DNA complexes on the virus-like substrate in the absence of the joining reaction . Intramolecular and intermolecular DNA looping by IN was visualized . Although IN preferentially bound to AT-rich regions in the absence of the joining reaction, there was a bias towards GC-rich regions for the joining reaction . Alignment of 70 target site sequences 5' of the LTR half-site insertions with 68 target sites previously identified for the concerted insertion of both LTR termini (LTR full-site reaction) indicated similar GC inflection patterns with both insertional events . Comparison of the data suggested that IN recognized only half of the target sequences necessary for integration with the LTR half-site reaction.

J Virol, 1993 May, 67(5), 2583 - 91
Effects of baculovirus infection on IE1-mediated foreign gene expression in stably transformed insect cells; Jarvis DL; Previously, we produced transformed insect cell lines that can express a selected foreign protein constitutively, in the absence of baculovirus infection (D . L . Jarvis, J . G . W . Fleming, G . R . Kovacs, M . D . Summers, and L . A . Guarino, Bio/Technology 8:950-955, 1990) . These cells contain stably integrated copies of chimeric genes consisting of the promoter from an immediate-early baculovirus gene, IE1, and the sequences encoding either human tissue plasminogen activator or Escherichia coli beta-galactosidase . Transcription of the integrated genes in these cells is specifically controlled by the IE1 promoter . The purpose of this study was to determine how baculovirus infection influences IE1-mediated foreign protein production by these stably transformed insect cell lines . The results showed that viral infection transiently stimulated and then strongly inhibited the production of both tissue plasminogen activator, a secreted protein, and beta-galactosidase, an intracellular protein . These effects reflected virus-induced changes in the steady-state levels of RNA produced by the integrated genes . Transient assays showed that expression of the viral IEN gene alone could account for the increased levels of RNA observed early in infection . The precise mechanism accounting for the decreased levels of RNA observed later in infection was not determined . However, we obtained evidence that the native IE1 promoter remains active throughout infection, which suggested indirectly that the integrated IE1 promoter is transcriptionally inactivated at late times of baculovirus infection . Thus, the same promoter behaved quite differently late in infection, depending on its local environment . Neither methylation nor degradation appeared to be responsible for inactivating IE1-mediated expression of the integrated genes . The significance of these results with respect to the baculovirus-host interaction and the practical applications of stably transformed insect cell lines are discussed.

Can J Physiol Pharmacol, 1993 May-Jun, 71(5-6), 414 - 24
Anatomical distribution of brainstem sites where PGE1 induces hyperthermia in macaque species; Simpson CW et al.; The neuroanatomical distribution of sites in the diencephalon and mesencephalon within which a prostaglandin (PG) of the E series elicits hyperthermia was characterized in Macaca mulatta and Macaca nemestrina . In 420 experiments undertaken in 13 animals, 225 loci were examined for their reactivity to PGE1 microinjected in a dose of 30 or 100 ng given in a volume of 1.0-1.5 microL . The regions of the brainstem for injection extended rostrally from the thermosensitive cells of the anterior hypothalamic, preoptic area (AH/POA) to the caudal border of the mesencephalon . Colonic and skin temperatures of the monkeys were measured continuously by thermistor probes . A hyperthermic response of > or = 0.5 degrees C and a latency of < or = 45 min was evoked by PGE1 within sites located primarily in the AH/POA . When PGE1 was microinjected at loci located caudal to the AH/POA, the elevation in body temperature (Tb) not only was less intense but rose at a slower rate . A higher concentration of PGE1 in these caudal regions was required to induce hyperthermia comparable with that elicited at loci within the AH/POA . In a second series of experiments either 1.0-5.0 micrograms 5-hydroxytryptamine (serotonin) or a concentration of 10(8) organisms/mL of Escherichia coli was microinjected at PGE1-reactive sites . A close anatomical concordance within the AH/POA of the animal was found in terms of the temporal characteristics and magnitude of the hyperthermia evoked by the indoleamine or lipopolysaccharide . The present results coincide with the reported neuroanatomical distribution of sites in the diencephalon and mesencephalon of other species in which PGE1 causes hyperthermia . Furthermore, these findings support the concept that the local neuronal mechanism of action of a pyrogen in the brainstem of the primate may involve phasic changes in the endogenous activity of both the serotonergic pathway and cyclo-oxygenase system in the AH/POA . In turn, their commonality of action suggests a functional similarity in their effect of shifting the set point for Tb.

Mol Microbiol, 1993 May, 8(5), 797 - 802
Transcription activation at Class I CAP-dependent promoters; Ebright RH; Catabolite gene activator protein (CAP)-dependent promoters can be grouped into three classes, based on the requirement for transcription activation and the position of the DNA site for CAP . Class I CAP-dependent promoters require only CAP for transcription activation and have the DNA site for CAP located upstream of the DNA site for RNA polymerase . Amino acids 156 to 162 of the promoter-proximal subunit of CAP are essential for transcription activation at Class I CAP-dependent promoters, but are not essential for DNA binding, and are not essential for DNA bending . In the structure of the CAP-DNA complex, these amino acids are located in a surface loop and form a cluster on the surface of the CAP-DNA complex . Amino acids 261, 265, and 270 of the alpha subunit of RNA polymerase are essential for response to transcription activation by CAP at Class I CAP-dependent promoters . Several lines of evidence indicate that transcription activation at Class I CAP-dependent promoters requires a direct protein-protein contact between amino acids 156 to 162 of the promoter-proximal subunit of CAP and a molecule of RNA polymerase bound adjacent to CAP on the same face of the DNA helix . It is a strong possibility that this direct protein-protein contact involves amino acids 261 and 265 of the alpha subunit of RNA polymerase.

J Photochem Photobiol B, 1993 May, 18(2-3), 211 - 4
An assay for DNA photolyases using non-radioactive DNA and restriction enzymes; Dutta K et al.; Restriction enzymes, such as Eco RI, Hind III, etc., which have a potential pyrimidine dimer site in their recognition sequence, fail to cleave DNA if their recognition site is modified by the formation of pyrimidine dimers as a result of UV irradiation of DNA (J . E . Cleaver, J . Mol . Biol., 170 (1983) 305-317) . We have made use of this functional property of restriction enzymes to develop a rapid and sensitive assay for DNA photolyases . UV-irradiated plasmid pBR322 DNA is only partially digested when incubated with a single site enzyme Hind III even at high concentration (20 units (micrograms DNA)-1) . The amount of DNA not cleaved by Hind III is determined by agarose gel electrophoresis . The effect of UV irradiation is reversed by the photoreactivation of DNA . The decrease in the amount of Hind III-resistant DNA on treatment with photolyase gives a measure of the enzymatic activity of the photolyase preparation . The advantage of using non-radioactive DNA and the high speed and simplicity of this assay make it especially suitable for use in the purification of photolyases.

J Biochem (Tokyo), 1993 May, 113(5), 620 - 4
Heat-induced DNA relaxation in vitro by mouse DNA topoisomerase I in the presence of ethidium bromide; Haq S et al.; In general, covalently-closed circular DNAs that are relaxed by DNA topoisomerase I at higher temperatures are more negatively (or less positively) supercoiled when analyzed at the same temperature . We found that in the presence of ethidium bromide the reaction products were less negatively supercoiled at higher temperatures . Consequently, the linking number of substrate DNA changed reversibly with temperature-shift: DNA relaxation occurred on temperature shift-up, and re-supercoiling on temperature shift-down . Analyses of the migration of covalently closed circular DNA on agarose gel electrophoresis and of the fluorescence intensity of ethidium bromide bound to DNA indicated decrease in binding of the drug to DNA at higher temperatures . Thus, the thermal effects on the topology of the reaction products of mouse DNA topoisomerase I in the presence of ethidium bromide can be explained by the temperature-sensitive interaction of the drug with DNA.

J Antimicrob Chemother, 1993 May, 31(5), 637 - 44
Development of a specific probe for the aminoglycoside-(3)-N-acetyltransferase resistance gene; Ho BS et al.; Aminoglycoside-(3)-N-acetyltransferase isoenzyme V (AAC(3)V) is produced by > 90% of aminoglycoside-resistant Escherichia coli isolates from Hong Kong . A gentamicin resistance determinant from a conjugative plasmid carried by one of these E . coli strains producing AAC(3)V was cloned as a 6-kb fragment in the PstI site of the Bluescript II KS-phagemid vector . A 0.8-kb SphI-SalI fragment from the cloned insert was used as a probe in an epidemiological study . The specificity of this probe was evaluated by colony hybridization with 17 control strains producing different known aminoglycoside resistance enzymes . Hybridization was observed only with strains producing AAC(3)V . The probe demonstrated the presence of the AAC(3)V gene in 95.5% to 133 gentamicin-resistant E . coli isolates . This result agreed with the earlier data on the distribution of this enzyme that were based on the susceptibility profile.

Mol Microbiol, 1993 May, 8(3), 521 - 4
Transformation with katG restores isoniazid-sensitivity in Mycobacterium tuberculosis isolates resistant to a range of drug concentrations; Zhang Y et al.; Isoniazid-resistant isolates of Mycobacterium tuberculosis were transformed with a plasmid vector carrying the functional catalase-peroxidase (katG) gene . Expression of katG restored full drug susceptibility in isolates initially resistant to concentrations ranging from 3.2 to > 50 micrograms ml-1 . Transformation with the corresponding katG gene from Escherichia coli resulted in low-level expression of catalase and peroxidase activities and conferred partial isoniazid sensitivity.

Mol Microbiol, 1993 May, 8(3), 495 - 506
Expression and analysis of two gyrB genes from the novobiocin producer, Streptomyces sphaeroides; Thiara AS et al.; A novobiocin producer, Streptomyces sphaeroides, contains two genes, designated gyrBS and gyrBR, that encode novobiocin-sensitive and -resistant DNA gyrase B proteins, respectively . The cloning of gyrBR was reported earlier; here, we describe the cloning of gyrBS . Both genes have been sequenced (the deduced products of gyrBS and gyrBR have M(r) values of 87.6K and 86.5K, respectively) and their transcripts have been mapped . Downstream of gyrBS, and co-transcribed with it, is the sole gyrA gene (encoding DNA gyrase A protein) . By constructing hybrid gyrB genes, using fragments of gyrBS and gyrBR, a specific portion of the N-terminal domain of the gyrase B protein (corresponding to amino acid residues 134-256 of Escherichia coli gyrase B) has been implicated in the binding of novobiocin.

Circ Shock, 1993 May, 40(1), 1 - 8
Effects of endotoxemia on the redox level of brain cytochrome a,a3 in rats; Schaefer CF et al.; We conducted the present study to determine the effect of endotoxin challenge on brain oxidative metabolism, after finding evidence in previous studies suggesting early uncoupling of mitochondrial oxidative phosphorylation in the rat small intestine during endotoxemia . Twenty male Sprague-Dawley rats were divided into two groups (N = 10 each) which received E . coli endotoxin (20 mg/kg BW) or an equal volume of 0.9% saline (1 ml/kg) by i.v . bolus . Catheter implantation and the subsequent data collection were conducted using isoflurane anesthesia with controlled ventilation . Hemodynamic and metabolic data were recorded for 30 min before and 60 min after endotoxin or saline injection . Tissue oxidative metabolism was monitored in vivo using differential multiwavelength near-infrared spectrophotometry . Optrodes were positioned on either side of the rat's head (transillumination mode) to monitor the redox state of mitochondrial cytochrome a,a3 (AA3) as well as the supply of oxygen to the brain as reflected by tissue oxyhemoglobin (HbO2) . In contrast to our previous results for the small intestine, where the decrease in AA3 oxidation level was disproportionately greater than the concomitant HbO2 decrease, we found that the endotoxin-induced impairment in blood flow to the head was associated with a decrease in brain AA3 redox level, which was proportional to the decrease in tissue HbO2 . This finding of an apparent oxygen-dependent AA3 redox shift in the brain during endotoxemia is similar to previous findings of others in hemorrhagic hypotension and hypoxic hypoxia . Possible mechanisms for the different mitochondrial AA3 redox responses to endotoxin in the brain and small intestine are discussed.

FEMS Microbiol Lett, 1993 May 1, 109(1), 107 - 12
Calf small intestine receptors for K99 fimbriated enterotoxigenic Escherichia coli; Teneberg S et al.; Non-acid and acid glycolipids were isolated from the small intestine of a newborn calf and tested for the ability to bind Escherichia coli carrying K99 fimbriae . The bacteria did not bind to any of the non-acid glycolipids, whereas in the acid glycolipid fraction several gangliosides were detected which bind to K99 fimbriae . Gangliosides capable of binding K99 fimbriated E . coli were characterized as NeuGc-GM3, NeuGc-GM2, NeuGc-GD1a NeuAc-SPG and NeuAc-SPG . No binding was detected to NeuAc-GM3 and NeuGc-GM1.

Appl Environ Microbiol, 1993 May, 59(5), 1668 - 70
Inactivation of Escherichia coli by titanium dioxide photocatalytic oxidation; Ireland JC et al.; Titanium dioxide in the anatase crystalline form was used as a photocatalyst to generate hydroxyl radicals in a flowthrough water reactor . Experiments were performed on pure cultures of Escherichia coli in dechlorinated tap water and a surface water sample to evaluate the disinfection capabilities of the reactor . In water devoid of significant amounts of inorganic-radical scavengers, rapid cell death was observed with both pure cultures and members of the indigenous flora in a natural water sample.

Mol Gen Genet, 1993 May, 239(1-2), 90 - 6
Analysis of secondary integration sites for IS117 in Streptomyces lividans and their role in the generation of chromosomal deletions; Smokvina T et al.; IS117, the 2.6 kb mini-circle of Streptomyces coelicolor A3(2), is a transposable element previously shown to be integrated into two distant sites in the chromosome . When introduced into S . lividans, IS117 integrates into one preferred chromosomal site, but when this site was artificially deleted, IS117 integrated into many secondary sites . Nucleotide sequence analysis of several secondary integration sites revealed varying degrees of similarity with the preferred site, but no consensus sequence . Nevertheless, sites more similar to the preferred site tended to be occupied more often than those that are less similar . Insertion of IS117 into secondary sites in the chromosome of S . lividans sometimes mediated chromosomal rearrangements . It was shown that some strains containing IS117 integrated into secondary sites had suffered deletions of chromosomal DNA . Deletions were adjacent to the inserted element and were at least several kilobases long . The proposed model implicates homologous recombination between IS117 copies integrated into two different secondary sites in the same chromosome as a cause of the deletions.

Mol Gen Genet, 1993 May, 239(1-2), 177 - 87
Formation of the tandem repeat (IS30)2 and its role in IS30-mediated transpositional DNA rearrangements; Olasz F et al.; Plasmids carrying two IS30 elements in the same orientation, as in the composite transposon Tn2706, are structurally unstable in Escherichia coli . A primary segregation product is formed by site-specific deletion of the sequences carried between the two IS30 elements . The resulting covalently closed replicon carries the two IS30 elements as tandem repeats separated by only 2 bp . This (IS30)2 structure is extremely unstable, but it can nevertheless be isolated on its vector plasmid and, after purification, can be reintroduced into host cells by transformation . Among the descendants of transformants of recA- bacteria, replicated copies of the introduced (IS30)2 structure are still present, together with various kinds of segregation products which provide evidence for the efficient generation of DNA rearrangements . Most abundant is the product of another site-specific recombination between two identical ends of the IS30 elements involved, which results in the presence of just one intact IS30 on the plasmid . Apart from this, and depending on the presence of appropriate targets for IS30 transposition, various transposition products of (IS30)2 are also seen . Intramolecular reactions lead to DNA inversions and deletions with breakpoints other than IS30 ends . In intermolecular reactions inverse transposition occurs at high frequency and one also obtains simple transposition and cointegration . A mutational study revealed the requirement in cis of one intact IS30 transposase gene and of both proximal ends of the two IS30 elements concerned not only for the formation of (IS30)2, but also for its further rearrangement reactions, including the efficient formation of site-specific deletions . A model is proposed, which postulates that (IS30)2 intermediates play a key role in IS30 transposition pathways in which the formation of (IS30)2 may be rate-limiting . Once this structure is formed, it gives rise to a burst of transpositional rearrangements in the subclone carrying (IS30)2 . Evolutionary implications of these findings are discussed.

Eur J Biochem, 1993 May 1, 213(3), 1055 - 66
Inhibitor-2 functions like a chaperone to fold three expressed isoforms of mammalian protein phosphatase-1 into a conformation with the specificity and regulatory properties of the native enzyme; Alessi DR et al.; Three isoforms of mammalian protein phosphatase-1 (PP1 alpha, PP1 beta and PP1 gamma) were expressed in Escherichia coli and purified to near homogeneity . The activities of all isoforms towards phosphorylase, phosphorylase kinase and myosin and their sensitivities to inhibitor-2 were similar to the native PP1 catalytic subunit (PP1C) isolated from vertebrate tissues . Like PP1C, they each formed a complex with the glycogen-targetting(G) subunit which directs PP1C to glycogen particles in skeletal muscle . However, other properties differed strikingly from native PP1C . The expressed isoforms were 100-600-fold less sensitive to inhibitor-1, 3-5-fold less sensitive to okadaic acid, 5-100-fold less sensitive to microcystin-LR and approximately 20-fold more active in dephosphorylating histone H1 than native PP1C . Although PP1 gamma (like PP1C) was active in the absence of Mn2+, expressed PP1 alpha and PP1 beta were completely dependent on Mn2+ for activity . PP1 beta, like PP1C, interacted with the myofibrillar-targetting(M) complexes from skeletal-muscle and smooth-muscle producing species with enhanced myosin-phosphatase activity, whereas expressed PP1 alpha and PP1 gamma did not . The expressed isoforms of PP1 combined with inhibitor-2 to form an inactive complex (PP1I) that could be reactivated by the glycogen-synthase-kinase-3(GSK3)-catalysed phosphorylation of inhibitor-2 . This procedure transformed the properties of all three expressed isoforms to those of native PP1C . Their sensitivities to inhibitor-1, okadaic acid and microcystin-LR were increased greatly, their histone-phosphatase activities decreased and the activities of PP1 alpha and PP1 beta became independent of Mn2+ . Furthermore PP1 alpha and PP1 gamma now interacted with the M complexes in a similar manner to PP1 beta and PP1C . Conversely, incubation of native PP1C with 50 mM NaF caused conversion to a Mn(2+)-dependent form with properties similar to those of the expressed isozymes . The G subunit from skeletal muscle or the M complex from smooth muscle could displace PP1C from activated PP1I, but not inactive PP1I, to form G-subunit/PP1C and M-complex/PP1C heterodimeric complexes . Inhibitor-2 was also found to be essential for the reactivation of PP1C from 6 M guanidinium chloride in the absence of Mn2+ . Taken together, the results suggest that inhibitor-2 is critical for the correct folding of nascent PP1C polypeptides, that its function is similar to that of a molecular chaperone and that it acts as a cytosolic reservoir of PP1C molecules which can be directed to the required subcellular locations following the synthesis of specific targetting subunits.

Am J Physiol, 1993 May, 264(5 Pt 1), C1210 - 8
Phosphatidic acid modulates Cl- secretion in T84 cells: varying effects depending on mode of stimulation; Vajanaphanich M et al.; Cl- secretion in T84 cells evoked by a stimulus that activates protein kinase C, carbachol, was associated with elevated levels of 32P-labeled phosphatidic acid (PA) . PA's role in the regulation of Cl- secretion was explored by examining the effect of exogenous PA (10(-4) M) on Cl- secretion and intracellular Ca2+ levels ({Ca2+}i) in monolayers . PA potentiated the effect of carbachol on {Ca2+}i and Cl- secretion, although it did not stimulate Cl- secretion by itself . PA had divergent effects on cyclic nucleotide-dependent Cl- secretion . It delayed Cl- secretion induced by vasoactive intestinal polypeptide {VIP, adenosine 3',5'-cyclic monophosphate (cAMP) dependent} but potentiated that induced by the heat-stable enterotoxin of Escherichia coli (STa; guanosine 3',5'-cyclic monophosphate dependent) . PA did not alter AMP or GMP levels, suggesting that PA acts at a site distal to the generation of these second messengers . PA caused a slight increase in phosphorylation of protein kinase C substrates but not of cAMP-dependent protein kinase substrates . However, PA is probably not acting through a classical protein kinase C pathway, because we have previously shown that phorbol esters inhibit carbachol's actions, and the protein kinase C inhibitor staurosporine failed to block the effect of PA on VIP- or STa-stimulated Cl- secretion . Thus PA differentially regulates stimulated Cl- secretion in T84 cells, depending on the nature of the agonist.

Protein Sci, 1993 May, 2(5), 826 - 37
Electrostatic stabilization in four-helix bundle proteins; Robinson CR et al.; Charge substitutions generated by site-directed mutagenesis at the termini of adjacent anti-parallel alpha-helices in a four-helix bundle protein were used to determine a precise value for the contribution of indirect charge-charge interactions to overall protein stability, and to simulate the electrostatic effects of alpha-helix macrodipoles . Thermodynamic double mutant cycles were constructed to measure the interaction energy between such charges on adjacent anti-parallel helices in the four-helix bundle cytochrome b562 from Escherichia coli . Previously, theoretical calculations of helix macrodipole interactions using modeled four-helix bundle proteins have predicted values ranging over an order of magnitude from 0.2 to 2.5 kcal/mol . Our system represents the first experimental evidence for electrostatic interactions such as those between partial charges due to helix macrodipole charges . At the positions mutated, we have measured a favorable interaction energy of 0.6 kcal/mol between opposite charges simulating an anti-parallel helix pair . Pairs of negative or positive charges simulating a parallel orientation of helices produce an unfavorable interaction of similar magnitude . The interaction energies show a strong dependence upon ionic strength, consistent with an electrostatic effect . Indirect electrostatic contacts do appear to confer a limited stabilization upon the association of anti-parallel packing of helices, favoring this orientation by as much as 1 kcal/mol at 20 mM K phosphate.

J Bacteriol, 1993 May, 175(10), 3146 - 50
The proton motive force drives the outer membrane transport of cobalamin in Escherichia coli; Bradbeer C; Cells of Escherichia coli pump cobalamin (vitamin B12) across their outer membranes into the periplasmic space, and it was concluded previously that this process is potentiated by the proton motive force of the inner membrane . The novelty of such an energy coupling mechanism and its relevance to other outer membrane transport processes have required confirmation of this conclusion by studies with cells in which cobalamin transport is limited to the outer membrane . Accordingly, I have examined the effects of cyanide and of 2,4-dinitrophenol on cobalamin uptake in btuC and atp mutants, which lack inner membrane cobalamin transport and the membrane-bound ATP synthase, respectively . Dinitrophenol eliminated cobalamin transport in all strains, but cyanide inhibited this process only in atp and btuC atp mutant cells, providing conclusive evidence that cobalamin transport across the outer membrane requires specifically the proton motive force of the inner membrane . The coupling of metabolic energy to outer membrane cobalamin transport requires the TonB protein and is stimulated by the ExbB protein . I show here that the tolQ gene product can partly replace the function of the ExbB protein . Cells with mutations in both exbB and tolQ had no measurable cobalamin transport and thus had a phenotype that was essentially the same as TonB- . I conclude that the ExbB protein is a normal component of the energy coupling system for the transport of cobalamin across the outer membrane.

J Bacteriol, 1993 May, 175(10), 3013 - 9
Demonstration of separate genetic loci encoding distinct membrane-bound respiratory NADH dehydrogenases in Escherichia coli; Calhoun MW et al.; The nature of the Escherichia coli membrane-bound NADH dehydrogenases and their role in the generation of the proton motive force has been controversial . One E . coli NADH:ubiquinone oxidoreductase has previously been purified to homogeneity, and its corresponding gene (ndh) has been isolated . However, two biochemically distinct E . coli NADH:ubiquinone oxidoreductase activities have been identified by others (K . Matsushita, T . Ohnishi, and H . R . Kaback, Biochemistry 26:7732-7737, 1987) . An insertional mutation in the ndh gene has been introduced into the E . coli chromosome, and the resulting strain maintains membrane-bound NADH dehydrogenase activity, demonstrating that a second genetically distinct NADH dehydrogenase must be present . By standard genetic mapping techniques, the map position of a second locus (nuo) involved in the oxidation of NADH has been determined . The enzyme encoded by this locus probably translocates protons across the inner membrane, contributing to the proton motive force.

J Bacteriol, 1993 May, 175(10), 2799 - 808
Rhs elements of Escherichia coli K-12: complex composites of shared and unique components that have different evolutionary histories; Zhao S et al.; The complete sequences of the RhsB and RhsC elements of Escherichia coli K-12 have been determined . These sequence data reveal a new repeated sequence, called H-rpt (Hinc repeat), which is distinct from the Rhs core repetition that is found in all five Rhs elements . H-rpt is found in RhsB, RhsC, and RhsE . Characterization of H-rpt supports the view that the Rhs elements are composite structures assembled from components with very different evolutionary histories and that their incorporation into the E . coli genome is relatively recent . In each case, H-rpt is found downstream from the Rhs core and is separated from the core by a segment of DNA that is unique to the individual element . The H-rpt's of RhsB and RhsE are very similar, diverging by only 2.1% . They are 1,291 bp in length, and each contains an 1,134-bp open reading frame (ORF) . RhsC has three tandem copies of H-rpt, all of which appear defective in that they are large deletions and/or have the reading frame interrupted . Features of H-rpt are analogous to features typical of insertion sequences; however, no associated transposition activity has been detected . A 291-bp fragment of H-rpt is found near min 5 of the E . coli K-12 map and is not associated with any Rhs core homology . The complete core sequences of RhsB and RhsC have been compared with that of RhsA . As anticipated, the three core sequences are closely related, all having identical lengths of 3,714 bp each . Like RhsA, the RhsB and RhsC cores constitute single ORFs that begin with the first core base . In each case, the core ORF extends beyond the core into the unique sequence . Of the three cores, RhsB and RhsA are the most similar, showing only 0.9% sequence divergence, while RhsB and RhsC are the least similar, diverging by 2.9% . All three cores conserve the 28 repetitions of a peptide motif noted originally for RhsA . A secondary structure is proposed for this motif, and the possibility of its having an extracellular binding function is discussed . RhsB contains one additional unique ORF, and RhsC contains two additional unique ORFs . One of these ORFs includes a signal peptide that is functional when fused to TnphoA.

EMBO J, 1993 May, 12(5), 1979 - 86
The c-mos proto-oncogene protein kinase turns on and maintains the activity of MAP kinase, but not MPF, in cell-free extracts of Xenopus oocytes and eggs; Nebreda AR et al.; During studies of the activation and inactivation of the cyclin B-p34cdc2 protein kinase (MPF) in cell-free extracts of Xenopus oocytes and eggs, we found that a bacterially expressed fusion protein between the Escherichia coli maltose-binding protein and the Xenopus c-mos protein kinase (malE-mos) activated a 42 kDa MAP kinase . The activation of MAP kinase on addition of malE-mos was consistent, whereas the activation of MPF was variable and failed to occur in some oocyte extracts in which cyclin A or okadaic acid activated both MPF and MAP kinase . In cases when MPF activation was transient, MAP kinase activity declined after MPF activity was lost, and MAP kinase, but not MPF, could be maintained at a high level by the presence of malE-mos . When intact oocytes were treated with progesterone, however, the activation of MPF and MAP kinase occurred simultaneously, in contrast to the behaviour of extracts . These observations suggest that one role of c-mos may be to maintain high MAP kinase activity in meiosis . They also imply that the activation of MPF and MAP kinase in vivo are synchronous events that normally rely on an agent that has still to be identified.

Biochem J, 1993 May 1, 291 ( Pt 3), 811 - 6
Lys-197 and Asp-414 are critical residues for binding of ATP/Mg2+ by rat brain inositol 1,4,5-trisphosphate 3-kinase; Communi D et al.; Rat brain inositol 1,4,5-trisphosphate (InsP3) 3-kinase A was expressed in Escherichia coli in order to identify the amino acid residues involved in substrate ATP/Mg2+ binding . Two amino acid regions that are conserved in the catalytic domain of InsP3 3-kinase isoenzymes A and B had characteristics consistent with two ATP/Mg(2+)-binding motives . Site-directed mutagenesis was performed on residues Lys-197, Lys-207 and Asp-414 to generate three mutant enzymes, referred to as C5 K197I, C5 K207I and C5 D414N . Comparison of the wild-type and mutant proteins with regard to enzymic activity revealed that C5 K197I exhibited 10% of control enzyme activity, C5 D414N was totally inactive and C5 K207I was fully active . The reduced levels of enzyme activity for C5 K197I and C5 D414N were correlated with an altered ability of the mutant enzymes to bind ATP/Mg2+, as determined by ATP-agarose affinity chromatography . Neither Ca2+/calmodulin binding nor InsP3 binding appeared to be affected . Mutant C5 K207I showed the same characteristics as the wild-type enzyme . Taken together, these results strongly indicated (i) that amino acid residues Lys-197 and Asp-414 are necessary for InsP3 3-kinase activity and form part of the ATP/Mg(2+)-binding domain, and (ii) that amino acid residues Lys-197, Lys-207 and Asp-414 are not involved in either InsP3 binding or enzyme stimulation by Ca2+/calmodulin.

Arch Biochem Biophys, 1993 May, 302(2), 372 - 9
Characterization of cis-acting regulatory mutations causing anaerobic expression of the sodA gene in Escherichia coli; Bowen SW et al.; The biosynthesis of Mn-containing superoxide dismutase is regulated in response to stimuli that affect the redox potential of the cell . To further investigate the mode of regulation of the gene (sodA) encoding this enzyme, cis-acting regulatory mutations in a strain containing a sodA::lacZ gene fusion were studied . The mutant strains expressed beta-galactosidase under anaerobic conditions, whereas the wild-type did not . Furthermore, the mutants were not induced in response to the presence of iron chelator, 2,2'-dipyridyl, or to the redox cycling compound, paraquat . The wild-type, however, did respond to these effectors . In vivo cloning was used to isolate the cis-acting regulatory elements from the mutants (NC4 and NC5) . Replacement of the wild-type 5'-regulatory region with either of the mutants' cis-acting regulatory element resulted in the anaerobic expression of active Mn-superoxide dismutase . Sequence and restriction analysis revealed the presence of an IS2 insertion element in the promoter region of one of the mutants (NC5) . This insertion caused the displacement of the 5'-regulatory region of sodA and the formation of a functional hybrid promoter consisting of the resident-10 region from sodA and -35 from IS2 . The second mutation (from NC4) was similarly analyzed, and an IS5 element was identified . The insertion site of IS5 (in NC4) was 6 bp (5'-TTAATT-3') upstream from the IS2 site (in NC5) . Anaerobic expression of sodA in NC4 was lower than in NC5 . This difference was almost eliminated in an arc- background, suggesting that the sequence 5'-TTAATT-3' might be essential for negative regulation by ArcA.

Proc Natl Acad Sci U S A, 1993 May 1, 90(9), 4012 - 6
Thymidine kinase mutants obtained by random sequence selection; Munir KM et al.; Knowledge of the catalytic properties and structural information regarding the amino acid residues that comprise the active site of an enzyme allows one, in principle, to use site-specific mutagenesis to construct genes that encode enzymes with altered functions . However, such information about most enzymes is not known and the effects of specific amino acid substitutions are not generally predictable . An alternative approach is to substitute random nucleotides for key codons in a gene and to use genetic selection to identify new and interesting enzyme variants . We describe here the construction, selection, and characterization of herpes simplex virus type 1 thymidine kinase mutants either with different catalytic properties or with enhanced thermostability . From a library containing 2 x 10(6) plasmid-encoded herpes thymidine kinase genes, each with a different nucleotide sequence at the putative nucleoside binding site, we obtained 1540 active mutants . Using this library and one previously constructed, we identified by secondary selection Escherichia coli harboring thymidine kinase mutant clones that were unable to grow in the presence of concentrations of 3'-azido-3'-deoxythymidine (AZT) that permits colony formation by E . coli harboring the wild-type plasmid . Two of the mutant enzymes exhibited a reduced Km for AZT, one of which displayed a higher catalytic efficiency for AZT over thymidine relative to that of the wild type . We also identified one mutant with enhanced thermostability . These mutants may have clinical potential as the promise of gene therapy is increasingly becoming a reality.

Proc Natl Acad Sci U S A, 1993 May 1, 90(9), 3830 - 4
A bacterially expressed single-chain Fv construct from the 2B4 T-cell receptor; Kurucz I et al.; A single-chain Fv construct of the 2B4 T-cell receptor has been made and expressed in Escherichia coli as bacterial inclusion bodies . After solubilization in 6 M guanidine hydrochloride and formation of mixed disulfides with glutathione, the protein was refolded by diluting out the denaturant and allowing intramolecular disulfide bridges to form by disulfide exchange . Approximately 65-100 mg of refolded protein was obtained from 1 liter of bacterial culture, an appreciable fraction of which was monomeric in nondenaturing solvents . This protein bound to three monoclonal antibodies specific for allotypic or idiotypic determinants on the native 2B4 variable region but did not bind several other anti-T-cell-receptor monoclonal antibodies that lacked such specificity . These experiments show that T-cell-receptor variable regions, like the V regions of antibodies, can form a well-behaved single-chain Fv molecule and provide large amounts of recombinant single-chain Fv T-cell receptor that can be used to study T-cell function.

Virology, 1993 May, 194(1), 314 - 31
Molecular cloning and characterization of PEBP2 beta, the heterodimeric partner of a novel Drosophila runt-related DNA binding protein PEBP2 alpha; Ogawa E et al.; Polyomavirus enhancer binding protein, PEBP2 (PEA2), is a heterodimer of two distinct subunits, alpha and beta, of which the former directly binds to DNA and the latter acts auxiliary to enhance the DNA binding . Recent cloning studies has revealed that the alpha subunit is homologous to the products of the Drosophila segmentation gene runt and the human AML1 gene, and that it functions as a major regulator for the T cell-specific gene expression . We have currently cloned cDNAs for the beta subunit . The isolated cDNAs contain three isoforms that are presumed to arise from alternative RNA splicing and encode polypeptides consisting of 187, 182, and 155 amino acids, respectively . These polypeptides neither show any significant homology with known other proteins including the alpha subunit nor have any known DNA-binding and dimerization domains . Thus, PEBP2, as the complex of these subunits, is thought to constitute an entirely novel category of heteromeric transcriptional regulator together with the Runt and AML1 proteins . Gel retardation assays of the cDNA-encoded proteins produced in an in vitro translation system or in Escherichia coli demonstrated that the larger two beta isoforms, but not the smallest one, can dimerize with the alpha subunit . Furthermore, this heterodimerization was shown to cause a marked increase in the intrinsic DNA binding affinity of the alpha subunit.

Oncogene, 1993 May, 8(5), 1311 - 6
Activation of fibroblast growth factor (FGF) receptors by recombinant human FGF-5; Clements DA et al.; We have purified biologically active recombinant human fibroblast growth factor 5 (FGF-5) from Escherichia coli . In the presence of heparin, recombinant FGF-5 is as active as native growth factor, demonstrating that glycosylation does not significantly potentiate FGF-5 activity . FGF-5 can bind and induce autophosphorylation of human FGF receptors (FGFR) 1 and 2 . Competition binding studies show that the KD for FGF-5-FGFR-1 and FGF-5-FGFR-2 interactions are both between 0.5 and 1.5 x 10(-9) M.

J Bacteriol, 1993 May, 175(9), 2645 - 51
Isolation and characterization of Escherichia coli strains containing new gene fusions (soi::lacZ) inducible by superoxide radicals; Mito S et al.; Gene fusions in Escherichia coli that showed increased beta-galactosidase expression in response to treatment with a superoxide radical (O2-) generator, methyl viologen (MV), were obtained . These fusions were constructed by using a Mud(Ap lac) phage to insert the lactose structural genes randomly into the E . coli chromosome . Ampicillin-resistant colonies were screened for increased expression of beta-galactosidase on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) plates containing MV at 1.25 micrograms/ml . Other O2- generators, menadione and plumbagin, also induced beta-galactosidase activity in these fusion strains . The induction by these drugs occurred only under aerobic conditions . Hyperoxygenation also elicited an induction of the fusions . On the other hand, no significant induction was observed with hydrogen peroxide and cumene hydroperoxide . The induction of these fusions by MV was not dependent on the peroxide stress control mediated by the oxyR gene or on the recA-dependent SOS system . These fusions were named soi (superoxide inducible)::lacZ . The induction of beta-galactosidase was significantly reduced by introducing a soxS::Tn10 locus into the fusion strains, indicating that the soi genes are members of the soxRS regulon . Five of the fusions were located in 6 to 26 min of the E . coli genetic map, while three fusions were located in 26 to 36 min, indicating that these fusions are not related to genes already known to be inducible by O2- under the control of soxRS . At least five mutants containing the soi::lacZ fusion were more sensitive to MV and menadione than the wild-type strain, suggesting that the products of these soi genes play an important role in protection against oxidative stress.

J Bacteriol, 1993 May, 175(9), 2599 - 606
Characterization of the reaction product of the oriT nicking reaction catalyzed by Escherichia coli DNA helicase I; Matson SW et al.; DNA helicase I, encoded on the Escherichia coli F plasmid, catalyzes a site- and strand-specific nicking reaction within the F plasmid origin of transfer (oriT) to initiate conjugative DNA strand transfer . The product of the nicking reaction contains a single phosphodiester bond interruption as determined by single-nucleotide resolution mapping of both sides of the nick site . This analysis has demonstrated that the nick is located at precisely the same site previously shown to be nicked in vivo (T . L . Thompson, M . B . Centola, and R . C . Deonier, J . Mol . Biol . 207:505-512, 1989) . In addition, studies with two oriT point mutants have confirmed the specificity of the in vitro reaction . Characterization of the nicked DNA product has revealed a modified 5' end and a 3' OH available for extension by E . coli DNA polymerase I . Precipitation of nicked DNA with cold KCl in the presence of sodium dodecyl sulfate suggests the existence of protein covalently attached to the nicked DNA molecule . The covalent nature of this interaction has been directly demonstrated by transfer of radiolabeled phosphate from DNA to protein . On the basis of these results, we propose that helicase I becomes covalently bound to the 5' end of the nicked DNA strand as part of the reaction mechanism for phosphodiester bond cleavage . A model is presented to suggest how helicase I could nick the F plasmid at oriT and subsequently unwind the duplex DNA to provide single-stranded DNA for strand transfer during bacterial conjugation.

J Virol, 1993 May, 67(5), 2592 - 600
Characterization of equine infectious anemia virus dUTPase: growth properties of a dUTPase-deficient mutant; Threadgill DS et al.; The putative dUTPase domain was deleted from the polymerase (pol) gene of equine infectious anemia virus (EIAV) to produce a recombinant delta DUpol Escherichia coli expression cassette and a delta DU proviral clone . Expression of the recombinant delta DUpol polyprotein yielded a properly processed and enzymatically active reverse transcriptase, as determined by immunoblot analysis and DNA polymerase activity gels . Transfection of delta DU provirus into feline (FEA) cells resulted in production of virus that replicated to wild-type levels in both FEA cells and fetal equine kidney cells . In contrast, the delta DU virus replicated poorly (less than 1% of wild-type levels) in primary equine macrophage cultures, as measured by reverse transcriptase assays . Preparations of delta DU virus contained negligible dUTPase activity, which confirms that virion-associated dUTPase is encoded in the pol gene region between the RNase H domain and integrase, as has been demonstrated previously for feline immunodeficiency virus (J . H . Elder, D . L . Lerner, C . S . Hasselkus-Light, D . J . Fontenot, E . Hunter, P . A . Luciw, R . C . Montelaro, and T . R . Phillips, J . Virol . 66:1791-1794, 1992) . Our results suggest that virus-encoded dUTPase is dispensable for virus replication in dividing cells in vitro but may be required for efficient replication of EIAV in nondividing equine macrophages, the natural host cells for this virus.

J Virol, 1993 May, 67(5), 2486 - 95
Mutations in the putative calcium-binding domain of polyomavirus VP1 affect capsid assembly; Haynes JI 2nd et al.; Calcium ions appear to play a major role in maintaining the structural integrity of the polyomavirus and are likely involved in the processes of viral uncoating and assembly . Previous studies demonstrated that a VP1 fragment extending from Pro-232 to Asp-364 has calcium-binding capabilities . This fragment contains an amino acid stretch from Asp-266 to Glu-277 which is quite similar in sequence to the amino acids that make up the calcium-binding EF hand structures found in many proteins . To assess the contribution of this domain to polyomavirus structural integrity, the effects of mutations in this region were examined by transfecting mutated viral DNA into susceptible cells . Immunofluorescence studies indicated that although viral protein synthesis occurred normally, infective viral progeny were not produced in cells transfected with polyomavirus genomes encoding either a VP1 molecule lacking amino acids Thr-262 through Gly-276 or a VP1 molecule containing a mutation of Asp-266 to Ala . VP1 molecules containing the deletion mutation were unable to bind 45Ca in an in vitro assay . Upon expression in Escherichia coli and purification by immunoaffinity chromatography, wild-type VP1 was isolated as pentameric, capsomere-like structures which could be induced to form capsid-like structures upon addition of CaCl2, consistent with previous studies . However, although VP1 containing the point mutation was isolated as pentamers which were indistinguishable from wild-type VP1 pentamers, addition of CaCl2 did not result in their assembly into capsid-like structures . Immunogold labeling and electron microscopy studies of transfected mammalian cells provided in vivo evidence that a mutation in this region affects the process of viral assembly.

Sci China B, 1993 May, 36(5), 557 - 67
A stable vector for high-level expression and secretion of human interferon alpha A in yeast; Huo KK et al.; Yeast high stable plasmid vector pHC11 was constructed by introducing pEM-BL Yi27 cleaved with SmaI into the SnaBI site of intact 2 microns plasmid . The result of plasmid stability assay revealed that 82% of the host cells still harbored the vector after 50-generations growth in non-selective medium, which confirmed the existence of a non-functional region in 2 microns plasmid . The human interferon alpha A (IFN alpha A) gene expression-secretion cassette was inserted into pHC11, and the yeast transformant was cultured in complex medium . The data showed that the expressed product was 36.8% of the total protein amount in the culture supernatant and the IFN alpha A biological activity was 2.6 x 10(10) units per liter, demonstrating that high-level expression and secretion of IFN alpha A were achieved in yeast by using the stable vector pHC11.

Microb Pathog, 1993 May, 14(5), 399 - 409
Clonal relationships and variation in virulence among Escherichia coli strains of avian origin; White DG et al.; Average genetic relatedness among 44 Escherichia coli strains of serotypes O1, O2, and O78 isolated mainly from birds with colibacillosis or swollen-head syndrome from France or Saudi Arabia was estimated based on allelic variation detected by multilocus enzyme electrophoresis . For 20 enzyme-encoding loci, we resolved 2.8 alleles per locus and distinguished 17 electrophoretic types (ETs) that were used to mark naturally occurring cell lineages or clones . On average, ETs differed at 37% of their loci . Forty-eight percent of the isolates represent three ETs, two of which belong to previously defined complexes of clones identified in avian disease in North America and Europe . Virulence of strains, assessed in experimental infections of day-old chicks, showed little variation among isolates of a clone, but was significantly variable among isolates of different clone complexes . These findings add support to the evidence that a majority of avian isolates that cause colibacillosis belong to a few cosmopolitan pathogenic clones and indicate a substantial between-clone component of pathogenicity.

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