Escherichia coli

Biokhimiia, 1994 Nov, 59(11), 1621 - 37
{Nucleotide sequence of the Escherichia coli B38 flagellin gene}; Zheleznaia LA et al.; The fliC gene of the E . coli B38 flagellin has been cloned and its nucleotide sequence determined using the terminator method . According to the sequencing data, the flagellin contains 565 amino acid residues which exceeds by 65 residues the number of amino acid residues in the earlier decoded E . coli K12 flagellin . Strong homology was observed in the two flagellins among the 160 initial and 89 tail-ended residues, whereas the central, variable parts showed no homology . Similar to the K12 flagellin, the B38 flagellin has no serines, cysteines or tryptophans . The variable part of the fliC E . coli B38 gene contains a Chi-site which initiates the genetic recombination in E . coli and related species.

Bioconjug Chem, 1994 Nov-Dec, 5(6), 660 - 5
Preparation and characterization of a bifunctional fusion enzyme composed of UDP-galactose 4-epimerase and galactose-1-P uridylyltransferase; Tamada Y et al.; A fusion enzyme consisting of UDP-galactose 4-epimerase and galactose-1-P uridylyltransferase with an intervening Ala3 linker was constructed by in-frame fusion of E . coli gene galT to the 3'-terminus of the E . coli gene galE that had been extended with the coding sequence for three alanine residues, all contained within a high-expression plasmid . The fusion enzyme was expressed in E . coli and purified 24-fold to about 98% homogeneity by chromatography on hydroxylapatite and Q-Sepharose . On the basis of the comparison of the elution profile for enzyme activities upon gel permeation chromatography (Sephacryl S-400) with the molecular weight of 80,000 determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, the fusion enzyme appears to exist in monomeric, dimeric, and tetrameric forms, all of which exhibit both enzymatic activities . The Km values of the fusion enzyme for substrates were similar to those for the corresponding native enzymes, except for UDP-glucose, but the kcat values were smaller than those for the native enzymes . The fusion enzyme shows kinetic advantages in that the initial velocity to produce glucose-1-P from UDP-galactose and galactose-1-P is about 20% faster than that for a mixture of equal activities of the separate enzymes.

Antimicrob Agents Chemother, 1994 Nov, 38(11), 2623 - 7
Comparison of inhibition of Escherichia coli topoisomerase IV by quinolones with DNA gyrase inhibition; Hoshino K et al.; In order to examine the inhibitory activities of quinolones against topoisomerase IV, both subunits of this enzyme, ParC and ParE, were purified from Escherichia coli . The specific activity of topoisomerase IV decatenation was found to be more than five times greater than that of topoisomerase IV relaxation . Thus, the decatenation activity of topoisomerase IV seems the most relevant activity for use in studies of drug inhibition of this enzyme . Although topoisomerase IV was less sensitive to quinolones than DNA gyrase, the 50% inhibitory concentrations for decatenation were significantly lower than those for type I topoisomerases . Moreover, there was a positive correlation between the inhibitory activity against topoisomerase IV decatenation and that for DNA gyrase supercoiling . These results imply that topoisomerase IV could be a target for the quinolones in intact bacteria and that quinolones could inhibit not only supercoiling of DNA gyrase but also decatenation of topoisomerase IV when high concentrations of drug exist in bacterial cells.

Mol Biol Cell, 1994 Nov, 5(11), 1253 - 63
Nuclear mRNA accumulation causes nucleolar fragmentation in yeast mtr2 mutant; Kadowaki T et al.; We have identified a set of genes that affect mRNA transport (mtr) from the nucleus to the cytoplasm of Saccharomyces cerevisiae . One of these genes, MTR2, has been cloned and shown to encode a novel 21-kDa nuclear protein that is essential for vegetative growth . MTR2 shows limited homology to a protein implicated in plasmid DNA transfer in Escherichia coli . PolyA+RNA accumulates within the nucleus of mtr2-1 in two to three foci at 37 degrees C . mRNA, tRNA, and rRNA synthesis continue as do pre-mRNA splicing, tRNA processing, and rRNA export at 37 degrees C . Under these conditions the polyA tail length increases, and protein synthesis is progressively inhibited . Nucleolar antigens also redistribute to two to three nuclear foci at 37 degrees C, and this redistribution depends on ongoing transcription by RNA polymerase II . Surprisingly, these foci coincide with the sites of polyA+RNA accumulation . Comparable colocalization and dependance on RNA polymerase II transcription is seen for the mtr1-1 mutant . The disorganization of the nucleolus thus depends on mRNA accumulation in these mutants . We discuss the possible functions of MTR2 and the yeast nucleolus in mRNA export.

Anal Biochem, 1994 Nov 1, 222(2), 479 - 82
Preparation of high-molecular-weight DNA: application to mycobacterial cells; Imai T et al.; A method for isolating high-molecular-weight DNA from bacteria is described . A special feature of the method is the treatment of whole bacterial cells with an organic solvent (chloroform-methanol (2:1, v/v) or ethanol-ether (1:1, v/v)) prior to DNA extraction from the cells . The DNA preparations obtained from organic solvent-pretreated bacterial cells such as Mycobacterium smegmatis, M . phlei, and Escherichia coli contained highly polymerized DNA, as revealed by pulse-field gel electrophoresis . The size and yield of the DNA obtained from E . coli pretreated with the organic solvent were quite similar to that of the DNA obtained from protoplasts . The results strongly suggest that the organic solvent pretreatment is effective for extracting very large DNA from bacterial cells and especially from bacteria whose protoplasts cannot be easily formed.

Anal Biochem, 1994 Nov 1, 222(2), 456 - 60
Differential suppression of background mammalian lysosomal beta-galactosidase increases the detection sensitivity of LacZ-marked leukemic cells; Hendrikx PJ et al.; A method is described for the detection of Escherichia coli beta-galactosidase-expressing leukemic cells in ex vivo bone marrow samples . 4-Methylumbelliferyl-beta-D-galactopyranoside is used as a substrate in a kinetic assay . D-Galactose is used to suppress endogenous lysosomal beta-galactosidase activity, yielding a sixfold increase in sensitivity . With this assay, the detection limit is one leukemic cell per 10(4) normal bone marrow cells.

Thromb Res, 1994 Nov 1, 76(3), 253 - 67
Fluorescence studies on plasminogen activator inhibitor 1: reactive centre cysteine mutants remain active after fluorophore attachment; Strandberg L et al.; To investigate structural-functional aspects of plasminogen activator inhibitor 1 (PAI-1) we have taken advantage of the lack of cysteines in the PAI-1 molecule and replaced Ser344 (P3) and Asn329 (P18) with cysteine residues, thereby creating unique attachment sites for extrinsic fluorescent probes . After expression in E . coli and purification to homogeneity, both of the mutant proteins were found to have similar biochemical characteristics as wild type PAI-1 (wtPAI-1) . Following labelling with 4-chloro-7-nitrobenzofurazan (NBD) and 2-(4'-iodoacetamido-anilino)naphtalene-6-sulfonic acid (IAANS) the mutant inhibitors showed similar inhibitory activities and heat stability as wtPAI-1 . The purified complex between uPA and NBD-labelled P3cys mutant was found to be extremely stable, suggesting that no slow cleavage or reversible reaction occurs in complexes that have been properly formed . The rate of labelling of both mutants was decreased when the mutants were in the latent form indicating that these cysteine residues may be less accessible in the latent configuration . The PAI-1 mutants labelled with both NBD and IAANS could convert from the active to the latent form, but P3cys labelled with the larger IAANS chromophore showed a two fold decrease in the rate of conversion to latency, suggesting that a large chromophore in the P3 position may interfere with the active to latent conversion . The fluorescence spectra of the two NBD labelled mutants were similar, but the intensity was three times higher for the P3cys mutant than for P18cys . No significant spectral changes could be seen when the P3cys mutant was transferred to latency . In contrast, the P18cys mutant showed a major change in the excitation spectra characteristic of migration of the NBD chromophore from a thiol to an amine . Complex formation with uPA had no effect on the fluorescence spectrum of P18cys-NBD while the spectrum of P3cys-NBD revealed changes consistent with a restriction of the mobility of NBD probe in the uPA-PAI-1 complex.

Hum Exp Toxicol, 1994 Nov, 13(11), 764 - 75
Evaluation of the genetic toxicity of the peroxisome proliferator and carcinogen methyl clofenapate, including assays using Muta Mouse and Big Blue transgenic mice; Lefevre PA et al.; The rodent liver carcinogen and hepatic peroxisome proliferator methylclofenapate (MCP) has been evaluated for genetic toxicity in a range of in vitro and rodent genotoxicity assays . It gave a negative response in each of the following assays: mutagenicity to S . typhimurium and E . coli (+/- S9 mix, plate and pre-incubation assays), clastogenicity to cultured human lymphocytes and CHO cells (+/- S9 mix), a mouse bone marrow micronucleus assay (24h and 48h sampling), a rat liver assay for UDS in vivo (12h sampling), assays for lac I (Big Blue) and lac Z (Muta Mouse) mutations in the liver of transgenic mice, and an assay of the ability of MCP to modify the mutagenicity to the liver of dimethylnitrosamine in both transgenic mutation assays . The micronucleus and UDS assays were conducted using a single administration of MCP at its maximum tolerated dose, while the transgenic assays were conducted using nine daily administrations of MCP at its cancer bioassay dose level . These nine daily administrations were shown to double the weight of the liver of non-transgenic, Big Blue and Muta Mice, as well as leading to a dramatic proliferation of peroxisomes (electron microscopy) in the livers of each strain . These changed parameters had returned to control levels when the mutation analyses were conducted (10 days after the final dose of MCP) . Despite the liver enlargement observed following MCP administration, no evidence of mitotic activity was observed in treated livers, although an increased number of cells were undergoing replicative DNA synthesis during the final 3 days of the 9 days of administration (BUdR assessment of S-phase) . Liver biochemistry parameters (ALT, AST, AP, CK, GGT and albumin) were unaffected by the chronic (9 day) administration of MCP indicating an absence of hepatic toxicity . These combined observations favour a non-genotoxic mechanism of action for the hepatic carcinogenicity of MCP . The clastogenicity in vitro of the perixisome proliferator Wyeth 14,643 has been confirmed in CHO cells, but it is noted that this chemical is more soluble than is MCP . In particular, at the highest dose level at which MCP could be tested, Wy 14,643 was also non-clastogenic.

Hum Exp Toxicol, 1994 Nov, 13(11), 759 - 63
Selective inhibition of gastrointestinal beta-glucuronidase by poly(vinylbenzyl D-glucaro(1,4)lactonate) . Part 2 . Poly(vinylbenzyl D-glucaro(1,4) lactonate) in vitro inhibition studies; Sacco C et al.; In vitro inhibition studies with beta-glucuronidase from purified E . coli and mouse intestinal contents indicated that the polymer, poly(vinylbenzyl D-glucaro(1,4)lactonate, is an effective beta-glucuronidase inhibitor . Purified E . coli beta-glucuronidase was inhibited by 99.6% with 177 mM D-glucaro(1,4)lactone using the polymer-inhibitor . Similarly, 95% inhibition of beta-glucuronidase activity of mouse intestinal contents was obtained with 177 mM and 50% inhibition was obtained with 31.5 mM D-glucaro(1,4)lactone based on the modified polymer . The structural requirements of an effective beta-glucuronidase inhibitor based on the structure of the polymer-inhibitor are also discussed.

Virus Res, 1994 Nov, 34(2), 178 - 86
High level expression of the envelope glycoprotein (gp53) of bovine viral diarrhoea virus (Singer) and its potential use as diagnostic reagent; Yu M et al.; A 1.74-kb cDNA fragment containing the gp53 coding region has been cloned from bovine viral diarrhoea virus (BVDV) strain Singer by reverse transcription polymerase chain reaction (RT-PCR) . Sequence analysis indicated that gp53 of BVDV strains Singer, NADL and SD-1 shared extensive sequence homology at both the RNA (85-94%) and protein (82-91%) levels . Nineteen cysteine residues and five potential N-linked glycosylation sites were identified within the sequenced region, all of which were conserved . These observations suggest that although the homology at the nucleotide sequence level may vary, there was strong structural conservation among bovine viral diarrhoea virus envelope proteins . Full-length gp53 was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase (GST) . The N-terminal half of gp53 was also synthesised in E . coli as a 28-KDa recombinant protein using the T7 RNA polymerase-directed expression system . Both recombinant proteins were expressed at high levels (approximately 30-50 mg/l) . The recombinant proteins were recognised in ELISA and Western blot analyses by polyclonal serum raised against a mixture of BVDV and classical swine fever virus (CSFV) . Rabbit antiserum raised against the 28-kDa recombinant protein reacted with different BVDV strains in ELISA and immunofluorescent antibody test, but not with CSFV in the same tests . These results demonstrated that the bacterial recombinant proteins have similar immunological properties to that of the native viral protein and, in conjunction with its homologous antisera, can be useful as diagnostic reagents.

Vet Immunol Immunopathol, 1994 Nov, 43(4), 401 - 11
Caprine arthritis encephalitis virus infection changes caprine blood monocyte responsiveness to lipopolysaccharide stimulation in vitro; Werling D et al.; The effects of caprine arthritis encephalitis virus (CAEV) infection on cytokine activity of caprine monocytes stimulated with Escherichia coli lipopolysaccharide (LPS) were examined . Compared with supernatants from LPS-stimulated monocytes of CAEV-negative goats, supernatants from CAEV-positive goats stimulated less proliferation of murine thymocytes in the MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide) assay, showed about 50% less IL-1 activity on the IL-1-dependent cell line LBRM-33 1 A-5, and showed about 200% more tumor necrosis factor (TNF) activity on the TNF-sensitive murine fibroblast cell line L-929 . These results indicate that CAEV infection changes caprine monocyte cytokine responsivity.

Pediatr Pathol, 1994 Nov-Dec, 14(6), 1017 - 28
Role of asphyxia and feeding in a neonatal rat model of necrotizing enterocolitis; Caplan MS et al.; Necrotizing enterocolitis (NEC) is a common gastrointestinal disorder affecting premature infants . To investigate critically the importance of the purported risk factors of NEC (formula feeding, asphyxia, bacteria, and prematurity), we developed a neonatal rat model that closely mimics the human disease . Full-term and premature newborn rats were stressed with formula feeding, asphyxia, and/or exogenous bacterial colonization and subsequently evaluated grossly and histologically for the development of intestinal injury . We found that most animals treated with asphyxia, formula feeding, and bacteria developed NEC (77%) and died (86%) by 96 h . All maternally fed animals treated with asphyxia and bacterial colonization survived and had normal intestinal histology . Furthermore, asphyxia was a critical instigating factor, because formula and bacterial exposure without asphyxia resulted in normal intestine and minimal mortality (12%) . Enteral bacterial colonization was not a significant determinant of NEC in this model . We conclude that the neonatal rat model is an excellent test system for the study of NEC . As in the human disease, asphyxia and formula feeding play an important role in the pathophysiology of experimental NEC.

Mutagenesis, 1994 Nov, 9(6), 527 - 35
Resolution and conservation of mismatches in DNA end joining; Pfeiffer P et al.; DNA end joining is a major pathway for the elimination of double-strand breaks from chromosomal DNA of higher eucaryotic cells . Extracts of Xenopus laevis eggs rejoin such breaks even when their short single-stranded termini are expected to form imperfectly matched overlaps . However, end-joined products cloned in Escherichia coli, necessarily give rise to perfectly matched products . Therefore it has not been possible to determine whether the end joining process creates mismatched products, perfectly matched (resolved) products or both . To investigate whether mismatch resolution was the result of the X . laevis end joining process or of activities of the bacterial host we used denaturing gradient gel electrophoresis to analyse joined products . We found that the end joining process does include mismatch resolution, the degree of which varies with regard to the nature of the original overlap structure . Mismatches 3' to a gap are completely resolved, mismatches 3' to a nick and 5' to a nick or gap are resolved to some extent but are generally conserved . Mismatches between base matches are always conserved . These findings suggest competing processes of ligation, DNA fill-in synthesis or exonucleolytic excision of mismatched bases next to a gap or nick . At mismatches 3' to a nick the probability of ligation is greater than that of excision while at mismatches 3' to a gap the probability of excision is greater than elongation of a given mismatch . At mismatches 5' to nicks or gaps it appears that ligation or elongation and ligation, respectively, are the most probable pathways but products resulting from mismatch excision, elongation and ligation are also detected.

Gen Comp Endocrinol, 1994 Nov, 96(2), 179 - 88
Cloning, expression, and characterization of a recombinant gilthead seabream growth hormone; Martinez-Barbera JP et al.; cDNA clones coding for the gilthead seabream (Sparus aurata) growth hormone (sbGH) were isolated from a pituitary expression library using a flounder cDNA probe . The nucleotide sequence of a GH cDNA clone containing an insert of 896 nucleotides was determined . The cDNA encoded a polypeptide of 204 amino acids including a signal peptide of 17 amino acids and contained a 5' and a 3' untranslated region of 48 and 233 nucleotides, respectively . The mRNA determined by Northern blot was approximately 1 kb . Amino acid sequence homologies of 97.1% with red seabream GH, 88.9% with the tuna GH, and 67% with the coho salmon GH was found . Transient expression of a sbGH cDNA was done in HeLa cells by induction with a vaccinia virus system, and the expressed GH was detected by immunofluorescence and immunoprecipitation with a specific antibody to the native sbGH . The sbGH cDNA was expressed in Escherichia coli by using the pGEX-3X and the pET-3a expression systems . The recombinant sbGH expressed in the pET-3a system was similar, if not identical, to the native hormone when analyzed by homologous radioimmunoassay and receptor binding assay.

Cell Mol Biol (Noisy-le-grand), 1994 Nov, 40(7), 981 - 8
Expression of branching enzyme II of maize endosperm in Escherichia coli; Guan HP et al.; A cDNA clone encoding maize branching enzyme II (BEII) has been independently isolated from a maize endosperm cDNA library . The deduced protein sequence of maize BEII was compared with that of BE from diverse sources . The gene encoding mature BEII of maize endosperm has been expressed in E . coli using the T7 promoter . The expressed BEII was purified to near homogeneity so that amylolytic activity and bacterial BE could be completely eliminated from the BE preparation . The expressed enzyme showed very similar properties to those of BEII purified from developing maize endosperm . This result confirmed our earlier report that BEII had a lower rate of branching amylose and the rate of branching amylopectin was twice that of branching amylose . This study also showed a greater advantage of purifying BEII from the bacterial expression system than from developing maize endosperm . Most importantly, this study has established a useful tool to study the structure-function relationships of the maize BE using site-directed mutagenesis.

Cell Mol Biol (Noisy-le-grand), 1994 Nov, 40(7), 899 - 905
A role for polyamines in the control of ppGpp levels in Escherichia coli; Goldemberg SH; As an approach to understand the involvement of polyamines in the variation of intracellular guanosine 5'-diphosphate 3'-diphosphate (ppGpp) levels, kinetic studies with several polyamine-requiring relA and spoT Escherichia coli mutants have been carried out . The accumulation and turnover of the nucleotide have been followed under conditions of aminoacid depletion or energy source starvation . The results obtained strongly suggest an important role of the polycations mainly in the degradation of ppGpp, and also in its synthesis mediated by ppGpp synthetase I (PSI).

Cell Mol Biol (Noisy-le-grand), 1994 Nov, 40(7), 891 - 7
Protoporphyrinogen accumulation in cultured hepatocytes treated with the diphenyl ether herbicide, acifluorfen; Sinclair PR et al.; Diphenyl ether (DPE) herbicides such as acifluorfen inhibit both the plant and mammalian forms of protoporphyrinogen oxidase, a heme biosynthetic enzyme . Only small amounts of protoporphyrin accumulated in primary cultures of chick embryo and rat hepatocytes treated with acifluorfen and the porphyrin precursor, 5-aminolevulinic acid . However, there was a large accumulation of the porphyrin precursor, protoporphyrinogen, which was detected after oxidation to protoporphyrin by an E . coli membrane enzyme . In contrast, conventional methods of porphyrin analysis which depend on quantitative autoxidation of protoporphyrinogen failed to detect this accumulation of protoporphyrinogen . This is the first demonstration that protoporphyrinogen can accumulate to high levels and remain stable in liver cells . In addition, we found that the effect of a protoporphyrinogen oxidase inhibitor such as acifluorfen on the regulation of heme synthesis in hepatocyte cultures differed from that of an iron chelator.

Biotechniques, 1994 Nov, 17(5), 974 - 80
Automated determination of beta-galactosidase specific activity; Bianco PR et al.; We describe a modification of an automated kinetic assay for beta-galactosidase (beta-gal) activity . This modification includes an assay to quantitate the amount of protein added to each assay . The determination of specific activity includes the amount of protein in the calculation which produces a specific activity with units of pmol product produced/minute/mg protein . In addition to this modification, we present a series of macros written in Microsoft Excel for either the Macintosh or Windows on the PC . These macros decrease the amount of time required to analyze the data from beta-gal assays.

Biotechniques, 1994 Nov, 17(5), 922 - 6
Quantifying radiolabeled macromolecules and small molecules on a single gel; Morrison TB et al.; A protein phosphorylation cascade involved in chemotactic signaling in Escherichia coli was investigated with purified components in vitro . CheA, an auto-phosphorylating histidine kinase, was mixed with {gamma-32P}ATP, and the labeled protein was purified for use as a reagent in the assays . CheY, a response regulator protein, can acquire phosphate groups from CheA but then undergoes rapid hydrolysis, which releases inorganic phosphate . To follow the kinetics of the CheA-CheY phospho-transfer reaction and the subsequent dephosphorylation of phospho-CheY, we separated the reaction components by polyacrylamide gel electrophoresis and measured the amount of 32P label in the CheA . CheY and inorganic phosphate bands with phosphor storage screens . By reducing the time needed to separate and quantify the reaction products, we minimized diffusive spreading of the low molecular weight inorganic phosphate, which enabled us to measure it accurately on the same gel with the much larger proteins . In principle, any radiolabeled molecules that can be separated by relatively rapid means, such as acrylamide gel electrophoresis, and that are detectable with a phosphor storage screen, should be amenable to this technique.

Bioessays, 1994 Nov, 16(11), 833 - 9
DNA damage tolerance, mismatch repair and genome instability; Karran P et al.; DNA mismatch repair is an important pathway of mutation avoidance . It also contributes to the cytotoxic effects of some kinds of DNA damage, and cells defective in mismatch repair are resistant, or tolerant, to the presence of some normally cytotoxic base analogues in their DNA . The absence of a particular mismatch binding function from some mammalian cells confers resistance to the base analogues O6-methylguanine and 6-thioguanine in DNA . Cells also acquire a spontaneous mutator phenotype as a consequence of this defect . Impaired mismatch binding can cause an instability in DNA microsatellite regions that comprise repeated dinucleotides . Microsatellite DNA instability is common in familial and sporadic colon carcinomas as well as in a number of other tumours . Several independent lines of investigation have identified defects in mismatch repair proteins that are causally related to these cancers.

Neurosurgery, 1994 Nov, 35(5), 910 - 5; discussion 915-6
Stereotactic delivery of a recombinant adenovirus into a C6 glioma cell line in a rat brain tumor model; Badie B et al.; The dismal results of conventional therapy for primary malignant brain tumors has justified exploring gene therapy approaches for this disease . Transduction of animal brain tumor models in vivo has been reported previously with retroviruses and herpes viruses . Because adenoviruses have the advantage of transducing quiescent and actively dividing tumor cells, they may prove to be more effective in such therapy . We used a replication-deficient recombinant adenovirus bearing the Escherichia coli beta-galactosidase gene in a rat C6 glioma tumor model . Transduced cells were detected by X-5-bromo-4-chloro-3-indolyl beta-D-galactoside staining to reveal beta-galactosidase activity . Initial experiments in vitro showed 50% and 90% transduction at vector titers of approximately 10(7) and 10(8) plaque-forming units/ml, respectively . Although no cytopathic effects were seen at 10(7) plaque-forming units/ml, more than 50% reduction in tumor cell growth was noted at 10(8) plaque-forming units/ml both in vitro and in vivo . Stereotactic delivery of the recombinant adenovirus into the frontal lobe of normal rat brains resulted in intense staining of all cell types, that is, neurons, astrocytes, and ependymal cells . Stereotactic injection into C6 glioma brain tumors in rats stained 25 to 30% of the tumor cells . We conclude that adenovirus vectors can be used to transfer genes to central nervous system tumors in vivo . Using stereotactic delivery, adenovirus vectors can transfer genes into the central nervous system intended for tumor therapy.

Free Radic Biol Med, 1994 Nov, 17(5), 379 - 88
Modulation of streptonigrin cytotoxicity by nitroxide SOD mimics; Krishna MC et al.; Nitroxides are cell-permeable, stable radicals that react readily with paramagnetic species such as transition metals or short-lived free radicals, though not generally with diamagnetic molecules . Nitroxides can undergo one-electron selective redox reactions and thereby potentially modify the activity of cytotoxic drugs . Streptonigrin (SN) toxicity requires bioreduction to yield the semiquinone radical, and the toxicity is reportedly mediated by transition metals and oxygen-derived reactive species via redox-cycling of the semiquinone intermediate . The present study shows that (1) nitroxides protected isolated DNA and also aerated or hypoxic bacterial cells from SN toxicity; (2) H2O2 potentiated the hypoxic cytotoxicity of the drug but inhibited the damage to aerated cells; (3) pretreatment of cells with H2O2 conferred some protection, but not when the drug alone was preexposed to H2O2; and (4) desferrioxamine and 2,2-dipyridyl, though neither diethylenetriamino pentaacetate, exogenous catalase, or superoxide dismutase, decreased SN-induced cell killing . The mechanisms by which nitroxides protect from SN toxicity involve both a selective radical-radical reaction with SN semiquinone and the reoxidation of reduced cellular transition metal ions . On the other hand, H2O2 appears to exert two opposing effects: (1) facilitation of cell killing by the Fenton reaction and (2) lowering the cellular level of reducing equivalents, thus inhibiting the bioreductive activation of SN.

Surg Endosc, 1994 Nov, 8(11), 1332 - 4
Delayed gallstone abscess following laparoscopic cholecystectomy; Mellinger JD et al.; Delayed infectious complications following elective laparoscopic cholecystectomy have not been well delineated in the medical literature . Irretrievable spillage of gallbladder contents at the time of laparoscopic cholecystectomy is not rare, and has generally been felt to be of little consequence, particularly in the nonacute setting . The case presented documents an instance of delayed gallstone abscess formation after elective laparoscopic cholecystectomy . While rare, such cases highlight the need for refined techniques to prevent gallbladder, perforation during this procedure and to allow laparoscopic recovery of small gallstones spilled at the time of cholecystectomy.

Kansenshogaku Zasshi, 1994 Nov, 68(11), 1381 - 9
{Studies on local immune response in mouse model of experimental Escherichia coli intrauterine infections}; Satoh T et al.; Studies were conducted to elucidate the immune cell response at infection sites by performing immunostaining of immune cells with a monoclonal antibody in an experimental Escherichia coli (E . coli) mouse uterine infection model . 1 . The incidence of uterine infection by E . coli decreased with the passage of time: 4/4 on Day 1, 4/6 on Day 3, 2/6 on Day 7, and 1/6 on each of Days 14 and 21 . It was surmised that clearance of the bacteria from the infection sites was being carried out by immune cells . 2 . Beginning from infection Day 1, the infected uterine tissue was observed to undergo a moderate degree of invasion by neutrophils, macrophages, CD4+ T cells, CD8+ T cells and IgA+ B cells . Then, beginning from infection Day 3, there was a mild degree of invasion of the infected uterine tissue by IgM+ B cells and IgG+ B cells . The number of neutrophils in the tissue decreased beginning from infection Day 14, but the degree of invasion of the infected tissue by the other kinds of immune cells remained almost constant through infection Day 21 . 3 . A comparison was made of the immune responses to local infection by E . coli, and Chlamydia trachomatis (C . trachomatis), an intracellular parasite . It was found that the invasion of the infection site by immune cells occurred earlier in the case of E . coli infection than C . trachomatis infection . In addition, the C . trachomatis infection site was observed to contain greater numbers of macrophages and CD8+ T cells play important roles in the immune defense at sites of infection by C . trachomatis.

Kansenshogaku Zasshi, 1994 Nov, 68(11), 1330 - 7
{Molecular cloning of alpha antigen like protein gene of Mycobacterium leprae and its over production in Escherichia coli}; Yin Y; I have constructed the genomic library of M . leprae Thai 53 strain, and cloned the alpha antigen like protein gene by plaque hybridization method by using M . leprae alpha antigen DNA fragment as probe which was characterized in the previous study, I have termed it as alpha 2 antigen gene . The alpha 2 antigen gene has been characterized by sequencing . By comparing the deduced amino acid sequence of alpha and alpha 2 antigen with 85 complex antigen of other mycobacteria . I have found the higher homology between alpha 2 antigen and 85A antigen and between alpha antigen and 85B antigen . We have constructed the over expression system of M . leprae alpha and alpha 2 antigen gene in E . coli using vector pMALc-RI . Recombinant alpha and alpha 2 antigen has been purified by amylose column chromatography at the purity of more than 95% . More than 6 mg and more than 10 mg of recombinant alpha and alpha 2 antigen has been obtained from 200 ml of liquid culture, respectively . ELISA tests have been performed with the sera of leprosy patient and healthy control against the recombinant alpha and alpha 2 antigens . The antibody titers in sera of leprosy patient against the two kinds of antigens were all much higher than healthy controls . The antibody titer against the alpha 2 antigen was higher than that against alpha antigen . Recombinant alpha and alpha 2 antigens in this study could be used as a new specific antigen for serodiagnosis of leprosy.

Gut, 1994 Nov, 35(11), 1613 - 6
Effect of Escherichia coli enterotoxins on macromolecular absorption; Verma M et al.; Macromolecular absorption of gliadin, a wheat protein and alpha lactalbumin, a milk protein was evaluated in control and Escherichia coli enterotoxin (heat-stable, heat-labile, and both heat-stable and heat-labile enterotoxin) treated mice . The peak concentration of gliadin and lactalbumin was two hours and three hours after their ingestion, respectively . There was also a significant increase (p < 0.01) in the absorption of both the proteins in all the three toxin treated groups compared with the control group . These results suggest that intestinal permeability and macromolecular absorption changes after E coli infection.

J Exp Biol, 1994 Nov, 196, 443 - 56
Molecular physiology of the Na+/H+ antiporter in Escherichia coli; Padan E et al.; All living cells maintain an inwardly directed Na+ gradient and a constant intracellular pH . Na+/H+ antiporters have been assigned an essential role in these homeostatic mechanisms in all cells . In Escherichia coli, two Na+/H+ antiporter genes, nhaA and nhaB, have been cloned . Deletion of either one or both showed that NhaA is essential for adaptation to high salinity, for growth at alkaline pH in the presence of Na+ and for challenging Li+ toxicity . NhaB confers tolerance to low levels of Na+ and becomes essential when the activity of NhaA limits growth . The adaptive response to Na+ is mediated by the positive regulator nhaR, which transduces the signal (intracellular Na+) to expression of the nhaA gene . We have identified Glu-134 of NhaR as part of the 'Na+ sensor' of NhaA . In agreement with the role of NhaA in pH homeostasis, its Na(+)-dependent expression is enhanced at alkaline pH . Reconstitution of pure NhaA and NhaB in proteoliposomes demonstrates that, whereas both are electrogenic (the H+/Na+ stoichiometry of NhaA is 2), only NhaA is pH-dependent, increasing its activity 1000-fold between pH 7 and 8.5 . Mutating all the histidines of NhaA shows that His-226 is part of the 'pH sensor' of NhaA.

J Exp Biol, 1994 Nov, 196, 213 - 28
Porters and neurotransmitter transporters; Nelson N et al.; Uptake of neurotransmitters involves multiple transporters acting in different brain locations under different physiological conditions . The vesicular transporters are driven by a proton-motive force generated by a V-ATPase and their substrates are taken up via proton/substrate exchange . The plasma membrane transporters are driven by an electrochemical gradient of sodium generated by a Na+/K(+)-ATPase . Two distinct families of transporters were identified in this group . One cotransports sodium with glutamate and other amino acids and requires additionally an outwardly directed potassium gradient . The second cotransports sodium, chloride and a variety of neurotransmitters, including gamma-aminobutyric acid (GABA), glycine and monoamines . Genes and cDNA encoding several members of the latter family have been cloned and studied in detail . The structure and function as well as the evolutionary relationships among these neurotransmitter transporters are discussed.

J Exp Biol, 1994 Nov, 196, 183 - 95
The lactose permease meets Frankenstein; Kaback HR et al.; The lactose permease (lac) of Escherichia coli is a paradigm for membrane transport proteins . Encoded by the lacY gene, the permease has been solubilized, purified to homogeneity, reconstituted into phospholipid vesicles and shown to catalyse the coupled translocation of beta-galactosides and H+ with a stoichiometry of unity . Circular dichroism and other spectroscopic approaches demonstrate that the purified permease is about 80% helical . Based on hydropathy analysis of the primary amino-acid sequence, a secondary structure has been proposed in which the protein has 12 hydrophobic domains in alpha-helical conformation that traverse the membrane in zigzag fashion connected by hydrophilic loops . A variety of other approaches are consistent with the model and demonstrate that both the N and C termini are on the inner surface of the membrane, and studies on an extensive series of lac permease/alkaline phosphatase fusion proteins provide exclusive support for the topological predictions of the 12-helix motif . This presentation concentrates on the use of site-directed fluorescence spectroscopy to study structure-function relationships in the permease.

Arch Dis Child Fetal Neonatal Ed, 1994 Nov, 71(3), F192 - 7
IgA antibodies in human milk: epidemiological markers of previous infections?
Nathavitharana KA, Catty D, McNeish AS.
The concept of an enteromammary link in secretory IgA (SIgA) antibody production was tested by hypothesising that specific SIgA antibody profiles in human milk might be an epidemiological marker for enteropathogens in a community . Milk from three subject groups was studied: 64 Sri Lankan women living in poor suburbs of Colombo, 20 Asian immigrant women domiciled in Birmingham, for a median period of five years (range 14 days-16 years), and 75 white women living in Birmingham . An enzyme linked immunosorbent assay (ELISA) was developed for the detection and measurement of SIgA antibodies to a panel of 14 crude O and 10 pure lipopolysaccharide antigens of diarrhoeagenic Escherichia coli strains well known to be endemic in the Indian subcontinent . The number of Sri Lankan and Asian immigrant women with SIgA antibodies to all 14 diarrhoeagenic E coli antigens (except O127 in Asian women) was significantly higher than in the white controls . The amount of E coli O antigen specific SIgA antibody activity as a percentage of total SIgA also gave significantly higher median values in Sri Lankan (6%) and in Asian immigrant (4%) women than in white controls (0.7%) . SIgA antibodies were highly O serogroup specific and showed excellent concordance between crude O and the corresponding purified lipopolysaccharide antigens . These results suggest that milk antibody profiles represent an epidemiological marker of exposure to enteral pathogens . The continuing specific milk antibody response in Asian women who have been domiciled in the United Kingdom for many years may indicate 'memory' in the human secretory immune system.

Br J Haematol, 1994 Nov, 88(3), 520 - 6
Impaired TCR signal transduction, but normal antigen presentation, in a patient with common variable immunodeficiency; Fischer MB et al.; We describe a 27-year-old white man with common variable immunodeficiency (CVID) who has two healthy histoidentical brothers and one IgA-deficient sister who shares one HLA haplotype with the patient . T cells from the patient with CVID showed an impaired response to recall antigens (tetanus toxoid, E . coli), whereas his IgA-deficient sister and his two healthy histoidentical brothers responded normally . Cross-mixing experiments using isolated monocytes and T cells from the CVID patient and one histoidentical brother revealed that the patient's monocytes were fully functional in processing and presenting antigen to resting T cells of his brother, and provided normal accessory cell function for superantigen-induced activation of his brother's resting T cells . In contrast, the patient's T cells were unable to respond to antigen presented by the brother's monocytes and failed to respond with an increase in intracellular free Ca++ to stimulation with superantigen, which is known to bind to the TCR V beta-chain outside the antigen-binding groove . However, stimulation with a combination of PMA and IM, directly activating protein kinase C and increasing intracellular free Ca++ by bypassing membrane receptors, induced normal Ca++ flux . These data indicate that the patient with CVID has a defect in TCR-mediated signalling at the level of the T cells which is not present in his histoidentical healthy brothers or in his haploidentical IgA-deficient sister.

Mol Gen Genet, 1994 Nov 1, 245(3), 390 - 6
Gene density and organization in a small region of the Arabidopsis thaliana genome; Le Guen L et al.; We have characterized a 6.4 kb genomic fragment from Arabidopsis thaliana ecotype Columbia overlapping the 5' end of the AKin10 gene which encodes a protein Ser/Thr kinase . Using, as probes, various restriction fragments located upstream of AKin10, two cDNA clones have been isolated from a cDNA library prepared from young shoot tissue . A comparison between the cDNA and the above genomic sequences allowed us to locate two novel genes, Atcys1 and Athyp1 (for Arabidopsis thaliana cystathionine gamma-synthase 1 and hypothetical protein 1) . The coding sequences of both genes are interrupted by introns and the exons match the sequences of the corresponding cDNAs . Further analysis of the genomic fragment revealed the presence of an open reading frame (ORF) of 609 nucleotides situated between the two genes . Atcys1, Athyp1, AKin10 and the ORF are very close to each other and organized in the same polarity; hence, the intergenic regions probably contain, within less than 0.5 kb, all the regulatory elements necessary to govern initiation and termination of transcription . The deduced protein sequence of Atcys1 shows a high degree of similarity with the cystathionine gamma-synthase from Escherichia coli . The putative product of the Athyp1 gene contains seven hydrophobic regions flanked by hydrophilic domains, reminiscent of membrane-spanning proteins . Southern blot hybridization experiments suggest the presence of one copy of Atcys1, Athyp1 and AKin10 per haploid genome, and Northern blot analysis demonstrates that the three genes are differentially expressed in roots, shoots and leaves.

Mol Gen Genet, 1994 Nov 1, 245(3), 294 - 300
Recombination between repeats in Escherichia coli by a recA-independent, proximity-sensitive mechanism; Lovett ST et al.; We have examined the influence of proximity on the efficiency of recombination between repeated DNA sequences in Escherichia coli . Our experiments have employed a plasmid-based assay to detect deletions between direct repeats of 100 bp . The rate of deletion of the juxtaposed direct repeats was reasonably high at 6 x 10(-5) per cell . A comparison of recA+ and recA mutant strains showed that these deletion events are primarily the result of recA-independent recombination at these homologous sequences . Random restriction fragments of yeast or E . coli genomic DNA were used to separate the two repeats . Deletion rates decreased over two orders of magnitude with increasing separation of up to 7 kb . There was a surprisingly strong effect of even short sequence separations, with insertions of a few hundred base pairs exhibiting 10-fold reductions of deletion rates . No effect of recA on the efficiency of deletion was observed at any distance between repeats.

Mol Gen Genet, 1994 Nov 1, 245(3), 279 - 85
MucAB but not UmuDC proteins enhance -2 frameshift mutagenesis induced by N-2-acetylaminofluorene at alternating GC sequences; Janel-Bintz R et al.; N-2-acetylaminofluorene has been shown efficiently to induce both -1 and -2 frameshift mutations in Escherichia coli as well as in mammalian cells . In E . coli, the genetic characteristics of -1 and -2 frameshift mutations were found to be distinct . The -1 frameshift mutation pathway occurs at monotonous runs of G residues (i.e . GGG-->GG) . This pathway exhibits the same genetic requirements as UV light-induced base substitution mutagenesis . Indeed, optimal mutagenesis requires the expression of both UmuDC and the activated form of RecA . The -2 frameshift mutation pathway operates at short alternating GpC sequences, such as the NarI sequence (i.e . GGCGCC-->GGCC) . In contrast to the -1 frameshift mutation pathway, optimal induction does not require the UmuDC and RecA proteins . This pathway involves a LexA-repressed function tentatively called Npf (for NarI processing factor) . In this paper, we show that MucAB efficiently stimulates the -2 frameshift mutation pathway . However, unlike the Npf pathway, MucAB-mediated stimulation requires expression of the RecA protein.

Microbiology, 1994 Nov, 140 ( Pt 11), 2981 - 90
Regulation of transfer genes of promiscuous IncP alpha plasmid RK2: repression of Tra1 region transcription both by relaxosome proteins and by the Tra2 regulator TrbA; Zatyka M et al.; The Tra1 region of broad host range IncP alpha plasmid RK2 encodes proteins essential for its promiscuous conjugative transfer and includes oriT, the site at which nicking occurs to initiate transfer replication . Unregulated expression of the Tra1 region genes would be likely to place a major burden on the host . To investigate the control of these genes the three transcriptional promoters from this region were cloned by PCR and inserted into xylE promoter probe vectors . The strength of traJp and traKp was estimated to be six to eightfold less than the strong trfA promoter which is required for expression of genes for vegetative replication of RK2 . The traG promoter was about one-tenth the strength of the other two . These promoters are not repressed by products of the central control operon of RK2 . However, traJp and traKp, which are arranged as back to back divergent promoters in the oriT region, are repressed by TraK which constitutes part of the relaxosome necessary for nicking at oriT . A second relaxosome protein, TraJ, represses traJp . traGp is not repressed by any relaxosome proteins . All three promoters are repressed by TrbA, which is encoded at the start of the trb operon containing the rest of the transfer genes (the Tra2 region) . These circuits provide: (i) an autoregulatory way of ensuring production of enough relaxosome proteins without overburdening the host; and (ii) a means of coordinating expression of both blocks of transfer genes.

Anal Chem, 1994 Nov 1, 66(21), 3840 - 7
Engineering the maltose binding protein for reagentless fluorescence sensing; Gilardi G et al.; This paper describes a mutant of the maltose binding protein (MBP) in which the serine residue at position 337 is replaced by a cysteine residue using site-directed mutagenesis . The mutant MBP has an approximately 2-fold lower affinity for maltose, and the cysteine residue can be modified with 4-{N-(2-(iodoacetoxy)ethyl)-N-methylamino}-7-nitrobenz-2-oxa-1,3-diazole (IANBD) and 6-acryloyl-2-(dimethylamino)-naphthalene (acrylodan) . This combined genetic and chemical modification places the fluorophores close to the maltose binding site such that when the ligand is added the fluorescence intensity of the labels increases by 60-180% over that of the ligand-free form . This change is consistent with the fluorophores being buried when the conformation of the protein changes with maltose binding . Titration of the labeled mutant proteins yields dissociation constants for maltose of 62 +/- 0.2 and 0.8 +/- 0.01 microM respectively for the IANBD and acrylodan modifications . The application of this strategy of combined genetic and chemical modification to the development of reagentless fluorescence sensing is discussed.

Parasitology, 1994 Nov, 109 ( Pt 4), 525 - 30
Excretion of host immunoglobulin in tick saliva and detection of IgG-binding proteins in tick haemolymph and salivary glands; Wang H et al.; Host immunoglobulin G (IgG) crossed the gut wall into the haemocoel of adult Rhipicephalus appendiculatus female ticks when they fed on guinea-pigs . Guinea-pig IgG was also found in saliva of the feeding ticks . The concentration and antibody activity of IgG in haemolymph, salivary gland extract (SGE) and saliva at different stages of tick feeding were detected by enzyme-linked immunoassay . Specific activity of the IgG in tick samples was determined by feeding ticks on guinea-pigs which were immunized with killed Escherichia coli: 35-42% of the antibody activity in guinea-pig immune serum remained in the tick samples . The high relative concentration of IgG in tick saliva at later stages of feeding suggests that the tick may have a mechanism for getting rid of foreign proteins via the salivary gland . Such a mechanism could involve IgG binding proteins (IGBPs) which were found in both haemolymph and SGE of female ticks at day 6 of feeding using a guinea-pig IgG-agarose affinity column . In female ticks, the M(r) of IGBPs in SGE (23 and 57 kDa) were less than those in haemolymph (78 and > 100 kDa) . The existence of IGBPs in both the tick salivary gland and haemolymph indicate that haemolymph and salivary gland cooperate to remove foreign proteins, e.g . host immunoglobulin, from the body during feeding . This mechanism may be a part of the tick self-defence system.

Appl Microbiol Biotechnol, 1994 Nov, 42(2-3), 313 - 8
Overproduction and purification of Lon protease from Escherichia coli using a maltose-binding protein fusion system; Sonezaki S et al.; Lon protease, which plays a major role in degradation of abnormal proteins in Escherichia coli, was overproduced and efficiently purified using the maltose-binding protein (MBP) fusion vector . The MBP-Lon fusion protein was expressed in a soluble form in E . coli and purified to homogeneity by amylose resin in a single step . Lon protease was split from MBP by cleaving a fusion point between MBP and Lon with factor Xa and purified by amylose resin and subsequent gel filtration . In this simple method, Lon protease was purified to homogeneity . Purified MBP-Lon fusion protein and Lon protease showed similar breakdown activities with a peptide (succinyl-L-phenylalanyl-L-leucyl-phenylalanyl-beta-D-methoxynaphthyl amide) and protein (alpha-casein) in the presence of ATP . Therefore, the gene-fusion approach described in this study is useful for the production of functional Lon protease . MBP-Lon fusion protein, which both binds to the amylose resin and has ATP-dependent protease activity, should be especially valuable for its application in the degradation of abnormal proteins by immobilized enzymes.

Appl Microbiol Biotechnol, 1994 Nov, 42(2-3), 304 - 8
Sequence of the region downstream of the Vitreoscilla hemoglobin gene: vgb is not part of a multigene operon; Liu SC et al.; The 1668 base pairs (bp) downstream of the Vitreoscilla hemoglobin gene were sequenced in the hope of finding related genes that might be part of an operon . Instead, a sequence was found that constituted an open reading frame (ORF) of 569 amino acids (apparently the carboxy-terminal part of a larger ORF), in the direction opposite to the hemoglobin gene . This sequence was found to have 64% similarity with the 1685 bp at the 3' end of the Escherichia coli uvrA gene . The inferred amino acid sequence of the Vitreoscilla DNA has 69% similarity with the corresponding sequence of the E . coli uvrA protein, with similarities of 90, 100, and 85% in the helix-turn-helix, C-terminal ATP binding, and C-terminal zinc finger domains, respectively . The distance between the 3' ends of the Vitreoscilla hemoglobin and uvrA genes is 63 bp.

Biotechnol Prog, 1994 Nov-Dec, 10(6), 648 - 51
Ammonium-mediated reduction of plasmid copy number and recombinant gene expression in Escherichia coli; Vila P et al.; The effect of ammonium as a medium supplement on plasmid-encoded recombinant beta-galactosidase synthesis was explored in Escherichia coli cells during aerobic growth in complex medium . After induction, only doses of ammonium chloride below 1 g/L are able to transiently enhance the yield . However, the presence of nontoxic ammonium chloride concentrations of up to 10 g/L results in lower values of beta-galactosidase in a concentration-dependent fashion . A significant reduction in plasmid DNA content explains the decrease in the yield by a gene-dosage-involving mechanism.

Trends Biotechnol, 1994 Nov, 12(11), 456 - 63
Recent developments in heterologous protein production in Escherichia coli; Hockney RC; During the past three to four years, remarkable progress has been made in our understanding of protein folding, protein translocation across biological membranes, and the role of molecular chaperones in these processes . In conjunction with recent developments in Escherichia coli expression systems, this understanding has led to an improved capability to accumulate proteins in a soluble form, secrete proteins from the cell cytoplasm, accumulate proteins in the cytoplasmic membrane, and direct proteins to the outer membrane of the cell for surface display . These advances suggest that E . coli should now be considered seriously for applications that, only a few years ago, would have been thought beyond the scope of this organism.

Lett Appl Microbiol, 1994 Nov, 19(5), 312 - 6
Loss of salt-tolerance and transformation efficiency in Escherichia coli associated with sub-lethal injury by centrifugation; Wyber JA et al.; Sub-lethal injury of Escherichia coli has been detected following centrifugation at g-forces between 5 and 30 kg . The extent of injury was measured either as a reduction in colony forming ability when plated onto NaCl-containing plates (2% w/v), or as a reduction in transformation efficiency associated with plasmid pBR322 encoding ampicillin resistance . In both cases, the extent of sub-lethal injury was found to increase with increasing centrifugal force and probably reflects structural damage to the cell envelope.

Bioorg Med Chem, 1994 Nov, 2(11), 1119 - 32
Synthetic carbohydrate vaccines: synthesis and immunogenicity of Tn antigen conjugates; Toyokuni T et al.; A tumor-associated carbohydrate antigen, Tn antigen (GalNAc alpha 1-->O-Ser), was synthesized with a spacer arm, and assembled to dimeric and trimeric structures using N-tert-butyloxycarbonyl-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-alpha- D-galactopyranosyl)-L-serine as a key building block . The synthetic antigens were conjugated with OSA and their immunogenicity examined in mice . Mice immunized with dimeric or trimeric Tn antigen showed a stronger antibody (IgM) response to a Tn-glycoprotein (asialo-ovine submaxillary mucin) than mice immunized with monomeric Tn antigen . The dimeric and trimeric Tn antigens also induced measurable IgG responses . The dimeric Tn antigen was further coupled to a Starburst dendrimer (5th generation) and to tripalmitoyl-S-glycerylcysteinyl-serine, a synthetic lipopeptide of the active moiety of a major lipoprotein of Escherichia coli . Unexpectedly, the Starburst dendrimer conjugate did not stimulate any immune response specific to Tn antigen . On the other hand, immunization of mice with the lipopeptide conjugate produced not only a high IgM response but also significant IgG anti-Tn response without any carrier molecules or additional adjuvants . The production of IgG antibody is quite significant since carbohydrate antigens are in general known to produce only IgM antibody response . Being a totally synthetic, low-molecular weight, and carrier-free immunogen, the lipopeptide conjugate could be a prototype of synthetic carbohydrate vaccines.

Res Microbiol, 1994 Nov-Dec, 145(9), 699 - 709
The role of the adsorption complex in the termination of filamentous phage assembly; Gailus V et al.; The adsorption complex of filamentous phage fd consists of two minor coat proteins, g3p and g6p, and is considered to be not only a structural entity, but also a functional unit to terminate phage assembly . Cells were infected with phage M13am8H1, which cannot assemble because it lacks the major coat protein g8p, although producing all of the other minor coat proteins . The membranes of infected cells were solubilized and analysed by non-denaturing PAGE and gel filtration . The data suggest the presence of the adsorption complex in these membranes . Furthermore, the non-polar gene 3 amber-mutant phage R171 was shown to lack g6p in the phage coat as well . The termination of assembly of this phage is disturbed, resulting in synthesis of polyphages . Electron micrographs and transient electrical birefringence show that these polyphages are eight times longer as compared to unit length phage . From these results, we conclude that the formation of the g3p-g6p complex is essential for correct termination of filamentous phage assembly.

Shock, 1994 Nov, 2(5), 344 - 50
Pulmonary capillary wedge pressure estimates of left ventricular preload are inaccurate in endotoxin shock: contribution of Starling resistor forces to septic pulmonary hypertension; Krahmer RL et al.; We tested the hypothesis that Starling resistor forces play a significant role in the increase in pulmonary vascular resistance during endotoxin shock . Anesthetized pigs (n = 9) were given Escherichia coli endotoxin (ETX; .5 mg/kg intravenously over 30 min) . Mean pulmonary arterial pressure (MPAP) and pulmonary capillary wedge pressure (PCWP) were recorded through a Swan-Ganz catheter . Pulmonary capillary pressure (Pc) was obtained from the analysis of the transient pulmonary artery pressure decay curve upon balloon inflation . Both proximal (Ra) and distal (Rv) pulmonary vascular resistance were calculated from cardiac output (CO), MPAP, Pc, and PCWP . Left atrial pressure (LAP) was measured directly via a left atrial catheter . Left ventricular end-diastolic wall thickness (LV-EDWT) was monitored by sonomicrometry, and used as an index of left ventricular preload . The results at baseline (t = 0) and t = 60 (30 min after the cessation of endotoxin infusion) were compared with saline control animals (n = 6) . Data were analyzed with a two-way ANOVA followed by contrast of residuals (p < or = .05) . After endotoxin, arterial blood pressure and CO fell significantly, an effect not seen in control pigs . In the control group neither LAP nor PCWP changed significantly over time, and remained equivalent to each other . In the septic shock group there was no difference between LAP and PCWP at t = 0 . However, by t = 60 LAP dropped and PCWP rose significantly . This fall in LAP and increase in PCWP were significantly different from the time-matched control values, and from each other.(ABSTRACT TRUNCATED AT 250 WORDS)

Shock, 1994 Nov, 2(5), 336 - 43
The impact of infection on gluconeogenesis in the conscious dog; McGuinness OP; The effect of infection on gluconeogenesis was assessed in the chronically catheterized conscious dog . Dogs were studied 42 h after implantation of a sterile (n = 7) or an Escherichia coli containing (n = 7) fibrinogen clot into the peritoneum (54 h fasted) . Infection increased arterial plasma glucagon and cortisol (4.2- and 2.1-fold, respectively), but it did not alter arterial plasma insulin, catecholamines, or glucose concentrations . Infection increased tracer ({3-3H}glucose) determined glucose production and utilization and net hepatic glucose output by 35% . Net hepatic alanine and lactate uptake were also increased by 34 and 54%, respectively, without an alteration in their net hepatic fractional extraction . The intrahepatic efficiency of conversion of {14C}alanine to {14C}glucose was not decreased in the septic dog (.77 +/- .08) vs . .95 +/- .10 in noninfected and infected, respectively) . Intestinal glucose uptake and lactate release were increased approximately twofold . The increase in intestinal lactate release accounted for 35% of the increase in net hepatic lactate delivery seen in response to infection . In conclusion, a good model of hypermetabolic infection was developed in which the characteristic increases in hepatic glucose production and gluconeogenesis were observed in the fasted state . The increase in gluconeogenesis was due to an increase in hepatic gluconeogenic precursor uptake with no impairment in the net fractional hepatic extraction of gluconeogenic precursors or the efficiency of gluconeogenesis . In addition, the intestine is a significant contributor to the increase in gluconeogenic precursor supply seen in response to infection.

Shock, 1994 Nov, 2(5), 332 - 5
Generation of a CD11b/c upregulating and chemotactic factor by hepatocytes of endotoxic rats--nonidentity with interleukin-8; Zhang P et al.; To further clarify the mechanism of polymorphonuclear leukocyte (PMN) recruitment into the liver associated with short term endotoxin infusion (1), we investigated the effect of a noval factor generated by hepatocytes of such endotoxic rats on the expression of PMN adhesion molecules CD11b/c and chemotactic activity . Conditioned medium of hepatocytes from endotoxin-infused rats shows a fast induction and dose-dependent activity for upregulating CD11b/c expression in and chemotactic activity for blood PMN of naive rats . Supernatants of naive control rats cultured in the presence of endotoxin and Kupffer cells and liver PMNs of endotoxic rats also produce activation, but to a much lesser extent . The upregulating activity can be reduced significantly by heat inactivation at 100 degrees C for 10 min and by pronase hydrolysis at 37 degrees C for 60 min . Generation of the activity does not depend on cyclooxygenase products or phospholipase A2 activity, and it does not seem to be associated with the complement pathway . The activity is associated with molecular masses of 9-12 and 27-32 kDa and cannot be reduced by antiserum to rat interleukin-8 in serial dilutions ranging from 1:50 to 1:25,600 . The results show that hepatocytes from acutely endotoxin infused rats generate a small molecular weight protein factor (or factors) that is capable of upregulating PMN 11b/c expression and chemotactic activity and is seemingly different from rat interleukin-8 . Thus, hepatocytes in endotoxemia may play an important role in modulating neutrophil function and contributing to the mechanism of neutrophil sequestration into the liver.

Agents Actions, 1994 Nov, 43(1-2), 13 - 6
In vitro effect of cetirizine on PGE2 release by rat peritoneal macrophages and human monocytes; Roch-Arveiller M et al.; Cetirizine was first described as a specific anti-H1 molecule displaying potent antiallergic activity . It was later found that its pharmacological properties extended to cellular actions as on eosinophil recruitment at inflammatory sites in allergic patients . Monocytes and macrophages participate in allergic mechanisms, particularly through high affinity H1 and H2 membrane receptors and generation of pro- and anti-inflammatory agents; among them histamine-induced factors, IL-1 and prostanoids are of importance . The aim of this work was to investigate the effect exerted by various concentrations of cetirizine (0.1-10 micrograms/ml) applied in vitro to human monocytes and peritoneal rat macrophages cultured for 24 h . Peritoneal macrophages were collected either from normal or experimentally inflamed rats . Human monocytes, isolated from peripheral blood, were studied either in a resting state or after stimulation by LPS from Escherichia coli (1 and 10 micrograms/ml) . Cetirizine (10 micrograms/ml) significantly enhanced IL-1 release by human monocytes stimulated by a weak LPS concentration (1 microgram/ml) but could not modify the maximal increase of IL-1 release induced by 10 micrograms/ml of LPS . It did not exert any effect on resting cells . Cetirizine (0.1-10 micrograms/ml) enhanced PGE2 release by resting human monocytes . Concentrations of 1 and 10 micrograms/ml enhanced PGE2 release by LPS-stimulated monocytes, and by healthy and inflamed rat macrophages . This effect was concentration-dependent . Our findings point to an anti-inflammatory action of cetirizine via PGE2 release and histamine H2 interactions . Cetirizine did not directly modify IL-1 generation by resting monocytes but the IL-1 production observed after LPS stimulation could promote the mechanisms by which PGE2 is released.

Mol Gen Mikrobiol Virusol, 1994 Nov-Dec, (6), 8 - 12
{In vitro and in vivo study of the activity of secreted alkaline phosphatase mRNA ribozyme gene}; Zakharchuk AN et al.; A ribozyme was constructed of the catalytic domain of tobacco ringspot virus satellite RNA and flanking sequences complementary to the target, secreted alkaline phosphatase (SEAP) mRNA . The ribozyme specifically cleaved the substrate in vitro; compared with that constant temperature . The relationship between specific endoribonuclease activity of the ribozyme and Mg2+ concentration was shown . The ribozyme was active in 293 cell line which was cotransfected with plasmids carrying the SEAP gene under control of RSV LTR promoter and the ribozyme gene under early HCMV promoter: SEAP activity reduced by half.

Eur Cytokine Netw, 1994 Nov-Dec, 5(6), 533 - 8
Molecular studies on interleukin-1 alpha; Furutani Y; When rabbit alveolar macrophages were treated with phorbol-12-myristate-13-acetate (PMA) and lipopolysaccharide (LPS), the synthesis of interleukin-1 (IL-1), as well as tumor necrosis factor (TNF), was greatly increased . These inducible cytokines were subjected to cloning by the differential colony hybridization method and the subsequent mRNA hybridization-translation assay . Cloned rabbit IL-1 cDNA was disclosed to encode the sequence of the counterpart of the mouse IL-1 alpha . This cDNA was used as a hybridization probe to screen a human cDNA library which was constructed from induced HL-60 cells, a human promyelocytic leukemia cell line . Isolated human IL-1 alpha cDNA was shown to direct the synthesis of a polypeptide with IL-1 activity in E . coli expression system . The chromosomal gene for human IL-1 alpha was isolated and characterized to elucidate the structural organization of this gene . To identify the region that is essential for regulating IL-1 alpha gene expression, various CAT (chloramphenicol acetyltransferase) fusion plasmids were constructed and analysed for their ability to direct CAT synthesis in a transient expression system . The unpublished results obtained in the early stages of these experiments are also presented and discussed in this review.

Microb Pathog, 1994 Nov, 17(5), 313 - 22
Immune response against the L-lactate dehydrogenase of Mycoplasma hyopneumoniae in enzootic pneumonia of swine; Frey J et al.; The L-lactate dehydrogenase (LDH) of Mycoplasma hyopneumoniae, formerly named protein P36, belongs to the predominant immunogenic proteins in pigs which were naturally or experimentally infected with M . hyopneumoniae . The antigenic reaction against M . hyopneumoniae LDH has been shown to be species specific . Recombinant M . hyopneumoniae LDH, which was genetically engineered to contain six histidine residues at its C-terminal end, was expressed in E . coli and purified to a high degree using Ni-chelate affinity chromatography . The genetically engineered LDH still showed the same biochemical activity and immunological specificity as the wild-type LDH and was used as an antigen for a M . hyopneumoniae LDH ELISA . Using this assay, we showed that pigs experimentally infected with M . hyopneumoniae raised antibodies against LDH in two steps . An early, relatively weak anti-LDH response was detected between 5 to 10 weeks post-infection when clinical signs and lung lesions occur . This first minor raise of anti-LDH antibodies occurred simultaneously with the strong appearance of antibodies against an antigen consisting of membrane proteins of M . hyopneumoniae prepared with Tween 20 extraction . A second, strong raise in anti-LDH antibodies was observed from the twelfth week after infection, at a time when the disease signs and the infectious agent disappeared . The high anti-LDH titer persisted until 21 weeks post-infection, in contrast to the antibody titer against the membrane proteins which started to decrease after its peak at 12 weeks post-infection . A LDH-ELISA may also be useful for detecting past infections.

Development, 1994 Nov, 120(11), 3173 - 83
The mouse tissue plasminogen activator gene 5' flanking region directs appropriate expression in development and a seizure-enhanced response in the CNS; Carroll PM et al.; Tissue plasminogen activator (t-PA) is a secreted serine protease implicated in multiple aspects of development . In the adult rat brain, transcription of t-PA is an immediate-early response in the hippocampus following treatments that induce neuronal plasticity . To study the sequence elements that govern transcription of this gene, in situ analysis was used to define t-PA's temporal and spatial expression pattern in midgestation embryos . Transgenic mice were then generated carrying t-PA 5' flanking sequences linked to the E . coli lacZ gene . Constructs containing 4 kb of the flanking sequences (4.0TAMGAL) confer beta-galactosidase activity mostly to the same tissues that exhibit high levels of t-PA mRNA by in situ analysis . In 4.0TAMGAL embryos from embryonic day 8.5 (E8.5) to 13.5 (E13.5), the majority of expression observed is localized to neural ectoderm-derived tissues . beta-galactosidase activity is first detected in restricted neuromeres in the midbrain and diencephalon, at E8.5 and E9.5 respectively . At E10.5, transgene expression is observed in neural crest-derived cranial nerves and dorsal root ganglia, but not placode-derived cranial nerves . From E10.5 to E13.5, beta-galactosidase activity is observed in postmitotic neurons of the midbrain, spinal cord, neural retina and the developing olfactory system . beta-galactosidase activity is also detected in areas undergoing tissue remodeling such as the pinna of the ear, whisker follicles and the limbs . In adult mice, lacZ is expressed in the hippocampus and this expression was found to be enhanced upon seizure in the giant pyramidal neurons of CA3 . These results reinforce the concept that t-PA plays a role in neurogenesis and morphogenesis, and identifies the promoter region that directs its transcriptional regulation both in development and in the CNS.

Biochem Mol Biol Int, 1994 Nov, 34(5), 955 - 61
Specific interaction of the Escherichia coli chaperone GroEL (60-KDA heat shock protein) with the liganded form of the galactose binding protein; Richarme G et al.; The Escherichia coli chaperone GroEL interacts more strongly with the liganded form of the galactose binding protein (the galactose binding protein-galactose complex), than with its unliganded form . This specific interaction is reflected by the stimulation of the ATPase activity of GroEL by the liganded galactose binding protein . Interactions between native proteins and chaperones could be more frequent than generally suspected, and may help to detect protein conformational changes.

Protein Sci, 1994 Nov, 3(11), 2055 - 63
Protein structural similarities predicted by a sequence-structure compatibility method; Matsuo Y et al.; A method for protein structure prediction has been developed, which evaluates the compatibility of an amino acid sequence with known 3-dimensional structures and identifies the most likely structure . The method was applied to a large number of sequences in a database, and the structures of the following proteins were predicted: (1) shikimate kinase (SKase), (2) the hydrophilic subunit of mannose permease (IIABMan), (3) rat tyrosine aminotransferase (Tyr AT), and (4) threonine dehydratase (TDH) . The functional and evolutionary implications of the predictions are discussed . (1) The structural similarity between SKase and adenylate kinase was predicted . Alignment of their sequences reveals that the ATP-binding type A sequence motif and 2 ATP-binding arginine residues are conserved . The prediction suggests a similarity in their functional mechanisms as well as an evolutionary relationship . (2) The structural similarity between IIABMan and galactose/glucose-binding protein (GGBP) was predicted . The IIA and IIB domains are aligned with the N- and C-terminal domains of GGBP, respectively . The 2 phosphorylated residues, His 10 and His 175, of IIABMan are threaded onto loops located in the substrate-binding cleft of GGBP . The prediction accounts for the phosphoryl transfer from His 10 to His 175, and to the sugar substrate . (3) The structural similarity between rat Tyr AT and Escherichia coli aspartate AT was predicted, as well as (4) the structural similarity between TDH and the tryptophan synthase beta subunit . Predictions (3) and (4) support the previous predictions based on observations of the functional similarities between the proteins.

Protein Sci, 1994 Nov, 3(11), 2005 - 14
Kinetics and crystal structure of a mutant Escherichia coli alkaline phosphatase (Asp-369-->Asn): a mechanism involving one zinc per active site; Tibbitts TT et al.; Using site-directed mutagenesis, an aspartate side chain involved in binding metal ions in the active site of Escherichia coli alkaline phosphatase (Asp-369) was replaced, alternately, by asparagine (D369N) and by alanine (D369A) . The purified mutant enzymes showed reduced turnover rates (kcat) and increased Michaelis constants (Km) . The kcat for the D369A enzyme was 5,000-fold lower than the value for the wild-type enzyme . The D369N enzyme required Zn2+ in millimolar concentrations to become fully active; even under these conditions the kcat measured for hydrolysis of p-nitrophenol phosphate was 2 orders of magnitude lower than for the wild-type enzyme . Thus the kcat/Km ratios showed that catalysis is 50 times less efficient when the carboxylate side chain of Asp-369 is replaced by the corresponding amide; and activity is reduced to near nonenzymic levels when the carboxylate is replaced by a methyl group . The crystal structure of D369N, solved to 2.5 A resolution with an R-factor of 0.189, showed vacancies at 2 of the 3 metal binding sites . On the basis of the kinetic results and the refined X-ray coordinates, a reaction mechanism is proposed for phosphate ester hydrolysis by the D369N enzyme involving only 1 metal with the possible assistance of a histidine side chain.

Protein Sci, 1994 Nov, 3(11), 1998 - 2004
Glu-50 in the catalytic chain of Escherichia coli aspartate transcarbamoylase plays a crucial role in the stability of the R quaternary structure; Tauc P et al.; Glu-50 of aspartate transcarbamoylase from Escherichia coli forms a set of interdomain bridging interactions between the 2 domains of the catalytic chain; these interactions are critical for stabilization of the high-activity high-affinity form of the enzyme . The mutant enzyme with an alanine substituted for Glu-50 (Glu-50-->Ala) exhibits significantly reduced activity, little cooperativity, and altered regulatory behavior (Newton CJ, Kantrowitz ER, 1990, Biochemistry 29:1444-1451) . A study of the structural consequences of replacing Glu-50 by alanine using solution X-ray scattering is reported here . Correspondingly, in the absence of substrates, the mutant enzyme is in the same, so-called T quaternary conformation as is the wild-type enzyme . In the presence of a saturating concentration of the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA), the mutant enzyme is in the same, so-called R quaternary conformation as the wild-type enzyme . However, the Glu-50-->Ala enzyme differs from the wild-type enzyme, in that its scattering pattern is hardly altered by a combination of carbamoyl phosphate and succinate . Addition of ATP under these conditions does result in a slight shift toward the R structure . Steady-state kinetic studies indicate that, in contrast to the wild-type enzyme, the Glu-50-->Ala enzyme is activated by PALA at saturating concentrations of carbamoyl phosphate and aspartate, and that PALA increases the affinity of the mutant enzyme for aspartate . These data suggest that the enzyme does not undergo the normal T to R transition upon binding of the physiological substrates and verifies the previous suggestion that the interdomain bridging interactions involving Glu-50 are critical for the creation of the high-activity, high-affinity R state of the enzyme.

Protein Eng, 1994 Nov, 7(11), 1401 - 6
The effects of induction conditions on production of a soluble anti-tumor sFv in Escherichia coli; Sawyer JR et al.; CC49 is a second generation monoclonal antibody (mAb) with high affinity to a pancarcinoma antigen, TAG-72 . A single-chain Fv (sFv) of CC49 may have a role in managing human carcinomas . Most reported sFvs have been expressed as insoluble products that must be solubilized and renatured . Soluble sFv expression is advantageous as activity can be assayed directly from the periplasmic fraction . Also, gene-level immunoconjugates may not be amenable to refolding protocols . Using a vector that carries the tac promoter and omp A signal, we have examined the effects of four variables on the expression and accumulation of soluble CC49 sFv: (i) linker sequence joining VL and VH, (ii) isopropylthio-beta-D-galactoside concentration for induction, (iii) temperature, and (iv) the addition of non-metabolizable sugars to the medium . We have been able to demonstrate, using rapidly prepared periplasmic extracts, that the yield of soluble sFv improves by the addition of 0.4 M sucrose to the medium and by inducing expression with a very low concentration of IPTG (0.02-0.03 mM) . Under these induction conditions periplasmic extracts demonstrate increased expression of the sFv, as shown by the larger amount of a 27 kDa band on SDS-polyacrylamide gel, and an increased ability to inhibit binding of the mAb CC49 to immobilized tumor extracts.

Protein Eng, 1994 Nov, 7(11), 1395 - 9
The role of the sequence extensions in beta-crystallin assembly; Kroone RC et al.; The modular construction of the eye lens beta gamma-crystallins makes them good candidates for protein engineering to ascertain the rules of assembly of oligomers . X-ray studies have shown that although the polypeptide chains of beta B2-crystallin and gamma-crystallins fold to form similar N- and C-terminal domains, the conformation of the connecting peptides are such that the gamma-crystallins are monomers and the beta-crystallin is a dimer . Unlike gamma-crystallins, the numerous beta-crystallins have extensions of variable sequence from the globular domains . We have tested the effect of removing the N- and C-terminal extensions from rat beta B2-crystallin using a bacterial expression system . Abundant proteins were produced in Escherichia coli using the pET or pQE vectors . Full-length and truncated proteins were purified and checked for refolding using circular dichroism . Sizing of the truncated proteins using gel filtration chromatography showed that the absence of either the N- or C-terminal extension does not affect dimerization of beta B2-crystallin.

Protein Eng, 1994 Nov, 7(11), 1387 - 93
Human aldolase B: liver-specific properties of the isozyme depend on type B isozyme group-specific sequences; Kusakabe T et al.; A series of chimeric enzymes between two human aldolases A, B or C were constructed to identify the molecular regions responsible for isozyme-specific functions . Chimeras constructed between aldolases A and B were AB34 (an AB chimera connected at position 34), ABA34-306 and ABA212-306 (the ABA chimeras) . Those between aldolases B and C are BC243, BC263 and BC306 (the BC chimeras connected at positions as indicated), as well as CB55, CB243, CB263 and CB306 (the CB chimeras connected at positions as indicated), CBC55-263 (a CBC chimera), and BCB55-193, BCB55-306, BCB79-193 and BCB79-306 (the BCB chimeric enzymes) . Through the analysis of the properties of these chimeras, it was found that for aldolase B, isozyme B group-specific sequences (IGSs)-1 and -4 were required for exerting type B-specific functions, while the IGSs-2 and -3 enhanced, in collaboration with the IGS-1, the catalytic activity of aldolase B . In addition, the alpha/beta-barrel and the restricted stretches, which were not specified but occupied two distinct regions spanning the amino acid positions 108-137 (designated connector 1) and 243-306 (designated connector 2), were found to be indispensable for showing full catalytic activity of aldolase B.

Chem Res Toxicol, 1994 Nov-Dec, 7(6), 823 - 8
Reductive metabolism of 1-nitropyrene accompanies deamination of cytosine; Malia SA et al.; 1-Nitropyrene (1-NP), a common environmental pollutant, is a mutagen and tumorigen . Nitroreduction is a major pathway by which 1-NP is metabolized . In order to study the mutational specificity of reductively activated 1-NP, single-stranded M13mp18 DNA was treated with tritium-labeled 1-nitrosopyrene in the presence of ascorbic acid to generate N-hydroxy-1-aminopyrene in situ . HPLC analysis of the treated DNA, following enzymatic digestion, showed that > 95% of tritium was located in one major adduct, N-(deoxyguanosin-8-yl)-1-aminopyrene . Transfection of these adducted M13 DNA in Escherichia coli indicated a dose-dependent reduction in viability with concomitant enhancement in mutagenesis in the lacZ gene fragment . Without SOS functions, the major type of mutation was C-->T transition (48%) . Further studies have shown that cytosine deamination occurred during ascorbic acid-induced nitroreduction, which was likely responsible for the C-->T transitions . Deamination of cytosine alco occurred at a significant frequency when nitroreduction of either 1-NP or 1-nitrosopyrene was catalyzed by xanthine oxidase, a mammalian nitroreductase.

Arch Oral Biol, 1994 Nov, 39(11), 995 - 1000
Increased prostaglandin I2 and thromboxane A2 production by rat dental pulp after intravenous administration of endotoxin; Hirafuji M et al.; The effect of systemic endotoxin (lipopolysaccharide from Escherichia coli 0111:B4) on prostaglandin I2 (PGI2) and thromboxane A2 (TXA2) production by rat dental pulp was investigated . Intravenous injection of endotoxin increased ex vivo production of both PGI2 and TXA2 by the pulp tissue, when determined by radioimmunoassay . A significant effect on PGI2 and TXA2 production was observed with endotoxin doses of greater than 2 and 0.4 mg/kg, respectively . A significant increase was also observed at 30 min after injection of 10 mg/kg endotoxin, reaching a maximum after 60 min for both PG and TX production . Endotoxin (10 mg/kg for 60 min) also increased TXA2 but not PGI2 production in lung tissue, but had no effect in jejunal tissue . Indomethacin (10 microM) completely inhibited PGI2 and TXA2 production by the pulp of physiological saline- and endotoxin-treated rats . Further, arachidonic acids (10 microM) significantly increased PG and TX production by the pulp of saline- but not of endotoxin-treated rats . Endotoxin (100 micrograms/ml) had no in vitro effect on PG or TX production when incubated with isolated pulp, lung and jejunal tissues, suggesting that the endotoxin-induced increases in PG and TX production are an indirect effect . The endotoxin-induced increase in TXA2 production, but not in PGI2 production, by the pulp tissue was significantly suppressed by WEB 2170, a platelet-activating factor (PAF) antagonist . These results indicate that arachidonate metabolism in pulp tissue is susceptible to endotoxaemia in comparison with the lung and jejunum, and further suggest that the endotoxin-induced increase in, at least, TXA2 production by the pulp is mediated by PAF.

J Endod, 1994 Nov, 20(11), 558 - 9
Hazards of laser smoke during endodontic therapy; McKinley IB Jr et al.; The purpose of this study was to evaluate the potential for spreading bacterial contamination from the root canal to the patient and the dental team via the smoke produced by the laser . Five extracted teeth were deliberately inoculated with a specific strain of Escherichia coli . The canals were subjected to an agron laser . The smoke plume was captured and cultured . All of the cultures were positive for growth of the E . coli used . It was concluded that the laser smoke does present a hazard of bacterial dissemination and that precautions must be taken to protect against spreading infections when using lasers in the root canal.

Nat Struct Biol, 1994 Nov, 1(11), 765 - 70
Critical elements in proteasome assembly; Zwickl P et al.; Coexpression of both subunits of the Thermoplasma proteasome in Escherichia coli yields fully assembled and proteolytically active proteasomes . Post-translational processing of the beta-subunit occurs in E . coli as it does in Thermoplasma . Coexpression of the alpha-subunit and the beta delta pro-subunit, a mutant beta-subunit lacking the propeptide, also yields fully assembled and active proteasomes . This indicates that the beta-propeptide is not essential for the folding and assembly of Thermoplasma proteasomes . Separately expressed alpha-subunits assemble into heptameric rings indistinguishable from the terminal rings of a proteasome . Mutational analysis shows that the amino terminus, which is highly conserved in all proteasomal alpha-type proteins, is essential for assembly . In the absence of alpha-subunits the beta-subunits are monomeric and post-translational processing of the beta-propeptide does not occur.

Circ Shock, 1994 Nov, 44(3), 154 - 9
Effect of estradiol on endotoxin-induced changes in steroid hormone levels and lethality in male rats; Christeff N et al.; We examined the effect of exogenous estradiol on the changes in serum steroid hormone levels induced by a nonlethal dose of Escherichia coli endotoxin in male rats and the deaths due to nonlethal and lethal doses of endotoxin . Injection of estradiol 5 min before a nonlethal dose of endotoxin changed the serum sex steroid hormone response of male rats to endotoxin . The serum estrogen concentrations of estradiol + endotoxin-treated rats decreased by 50% (P < 0.001), while those of the endotoxin-treated rats increased (2- to 5-fold) . The serum androgen concentrations of estradiol + endotoxin-treated rats did not change significantly, while those of endotoxin-treated rats dropped to 30-40%, P < 0.001 . Exogenous estradiol also appeared to influence the percentage of endotoxin-induced deaths in a dose-dependent manner . It reduced the number of deaths induced by nonlethal (2 mg/kg) dose of endotoxin but increased the number of deaths induced by a highly lethal dose (8 mg/kg) . These results, together with the known relationships between estrogen and the immune response, suggest that estrogens affect the course of septic shock in a complex fashion and may have either protective or deleterious effect.

Circ Shock, 1994 Nov, 44(3), 148 - 53
Modulation of lung injury by platelet-activating factor antagonism in nonhypotensive porcine endotoxemia; Abu-Zidan FM et al.; Endotoxemia was induced by intravenous infusion of Escherichia coli endotoxin in 18 anesthetized pigs in a dose of 36 micrograms/kg/hr . Nine pigs were pretreated with BB-882, a novel platelet-activating factor (PAF) antagonist, 33 mg/kg/hr, starting 30 min before endotoxin, and nine pigs received a similar volume of vehicle . Normotension was maintained with intravenous crystalloid resuscitation . Six pigs received only BB-882 and served as controls . Endotoxemia induced an acute transient 300% increase in pulmonary vascular resistance, identical in both groups . The initial increase was followed by a second, more gradual, rise in resistance, which was significantly attenuated by BB-882 (P < 0.01, repeated measurements ANOVA) . Endotoxin-induced arterial deoxygenation and fall in lung/thorax compliance was not significantly altered by BB-882 . Hematocrit was less in endotoxic pigs receiving BB-882 (P < 0.02) . There were no significant changes compared to baseline in the control group . The results indicate that PAF is a minor determinant of early pulmonary dysfunction in nonhypotensive porcine endotoxemia.

Braz J Med Biol Res, 1994 Nov, 27(11), 2551 - 5
Lethal interaction between hydrogen peroxide and o-phenanthroline in Escherichia coli; Asad NR et al.; The iron chelator o-phenanthroline enhances the lethal effect of H2O2 about four hundred times in Escherichia coli when both substances are added simultaneously to the culture medium . If o-phenanthroline is added for increasing periods of time prior to the addition of H2O2, there is a shift from this lethal interaction to protection by the chelator about seven hundred times . It is known that the Fe(2+)-o-phenanthroline(I) and Fe(2+)-o-phenanthroline(II) complexes are formed quickly whereas the final and more stable Fe(2+)-o-phenanthroline(III) complex is formed slowly . Moreover, the mono and bis complexes react with H2O2 to produce OH., whereas the tris complex is stable towards H2O2 . Therefore, the lethal effect could be explained by the kinetics of reaction of o-phenanthroline with intracellular Fe2+, i.e., the mono and bis complexes are more reactive than intracellular Fe2+.

Can J Physiol Pharmacol, 1994 Nov, 72(11), 1308 - 12
Nitric oxide synthase induction and cytoprotection of rat gastric mucosa from injury by ethanol; Tepperman BL et al.; The present study determined the effects of nitric oxide (NO) synthase induction on ethanol-mediated damage to rat gastric mucosa . NO synthase activity was determined by {14C}arginine conversion to radiolabeled citrulline . Ca(2+)-independent NO synthase activity was determined by citrulline formation in the presence of EGTA (1 mM) in the incubation mixture . Intraluminal ethanol administration (2 mL; 40% w/v) to control rats resulted in an increase in mucosal damage characterized as vasocongestion and hemorrhagic necrosis and a reduction in Ca(2+)-dependent NO synthase activity . Administration of Escherichia coli lipopolysaccharide (LPS; 3 mg/kg i.v.) augmented Ca(2+)-independent NO synthase activity (determined 4 h later) and reduced damage in response to intraluminal ethanol instillation . Ethanol treatment did not significantly affect induction of NO synthase activity . Dexamethasone pretreatment (1 mg/kg i.v . 2 h before LPS administration) reduced both Ca(2+)-independent NO synthase activity and the gastroprotective effect of LPS against ethanol-mediated mucosal injury . Likewise, concurrent administration of the NO synthase inhibitor NG-nitro-L-arginine methyl ester (10 mg/kg s.c.) inhibited the gastroprotection associated with LPS treatment, an effect abolished by pretreatment with the NO substrate L-arginine (300 mg/kg s.c.) . Indomethacin (5 mg/kg i.v.) was ineffective in suppressing LPS-mediated gastroprotection . These results suggest that while Ca(2+)-dependent NO formation is inhibited by ethanol treatment, the inducible Ca(2+)-independent NO synthase plays a role in LPS-mediated gastroprotection against ethanol-mediated damage to the gastric mucosa.

Zentralbl Bakteriol, 1994 Nov, 281(4), 433 - 41
rRNA gene restriction patterns as possible taxonomic tools for the genus actinomyces; Barsotti O et al.; Species delineation in the genus Actinomyces remains unclear, particularly regarding the two taxa, A . naeslundii and A . viscosus . The ribotyping patterns of 64 strains of Actinomyces, representing 8 species and comprising different serotypes, were studied as possible taxonomic tools, using an acetyl-aminofluorene (AAF)-labelled E . coli 16S + 23S rRNA probe . Similarities between patterns were assessed using Jaccard's coefficient and clustering achieved using the unweighted pair-group method with average linkage (UPGMA) on a Macintosh II (Apple, Cupertino, USA) computer . The dendrogram obtained from the ribotypes gave results which were in reasonable agreement with many previous reports: A . bovis, A . gerensceriae, A . israelii, A . meyerii, A . odontolyticus and A . pyogenes were found to be distinct species but the two taxa A . naeslundii and A . viscosus remained unclear . Further investigations, using a larger number of A . naeslundii and A . viscosus strains and other endonucleases, need to be carried out to provide more information concerning the relatedness of these two taxa . Nevertheless, these preliminary results suggested that the Actinomyces chromosome contains multiple rRNA operons which may be used as an epidemiological and taxonomical tool.

Biochem Mol Biol Int, 1994 Nov, 34(5), 1035 - 48
Amino acid functional groups involved in the binding of Escherichia coli ribosomal protein S1 to ribosomes and nucleic acids; Ravi K et al.; Histidine, arginine, tyrosine, lysine and cysteine residues of protein S1 were modified with diethyl pyrocarbonate & rose bengal, 2,3-butanedione (diacetyl), tetranitromethane, pyridoxal 5-phosphate, and N-ethyl-maleimide, respectively . Modification of the residues and the number of modified residues were determined by either fluorescence or UV spectroscopy . The effect of chemical modification on the function of protein S1 was studied with respect to nucleic acid (poly U and M13 ssDNA) binding and ribosome binding properties of the protein . We tested S1 binding to these two types of polynucleotides because of their reported (Draper et al . (1977) PNAS 74, 4786-4790) binding to two different sites on S1 . The results indicate that histidine and lysine residues of S1 play an important role in the binding of S1 to both types of nucleic acids and that histidine and to some extent tyrosine residues are involved in the binding of S1 to ribosomes . The Data indicate the need for a re-evaluation of the two nucleic acid binding-site model.

Mol Microbiol, 1994 Nov, 14(4), 731 - 41
mRNA degradation in Escherichia coli: a novel factor which impedes the exoribonucleolytic activity of PNPase at stem-loop structures; Causton H et al.; Stem-loop structures can protect upstream mRNA from degradation by impeding the processive activities of 3'-5' exoribonucleases . The ability of such structures to impede exonuclease activity in vitro is insufficient to account for the stability they can confer on mRNA in vivo . In this study we identify a factor from Escherichia coli which specifically impedes the processive activity of the 3'-5' exonuclease PNPase at stem-loop structures in vitro . This factor can, potentially, reconcile the apparent discrepancy between the ability of 3' stem-loop structures to stabilize upstream mRNA in vitro and in vivo . Its mechanism of action, and possible role in regulating mRNA degradation, is discussed.

Mol Microbiol, 1994 Nov, 14(3), 427 - 36
The FinO protein of IncF plasmids binds FinP antisense RNA and its target, traJ mRNA, and promotes duplex formation; van Biesen T et al.; Most of the genes required for the conjugative transfer of DNA are encoded by the 33 kb transfer (tra) operon of F-like conjugative plasmids . Transcription of the tra operon is positively regulated by the TraJ transcriptional activator which, in turn, is negatively regulated by the FinOP fertility inhibition system . The FinOP system consists of an antisense RNA, FinP, and a 21.2 kDa protein, FinO, which together inhibit TraJ expression . Previously, it has been demonstrated that FinO increases the in vivo stability of the FinP RNA in the absence of the traJ mRNA target . Using electrophoretic mobility shift assays, we have shown that FinO is an RNA-binding protein that binds to one of the two stem-loops in FinP and to its complementary structure in traJ mRNA . This interaction presumably protects FinP RNA from degradation in vivo and increases the rate of formation of the FinP-traJ mRNA duplex fivefold . Thus, TraJ expression appears to be influenced by a unique RNA-protein interaction that precedes duplex formation between the FinP antisense RNA and its target traJ mRNA.

FEMS Immunol Med Microbiol, 1994 Nov, 10(1), 19 - 24
Determination of specificities of rabbit antisera against the O-antigenic polysaccharide from Escherichia coli O126; Bhattacharyya T et al.; Rabbit polyclonal antibodies against the lipopolysaccharide of Escherichia coli O126 were serologically characterized by ELISA . The antibody specificities were determined by studying the inhibitory effects of the methyl glycosides of both anomeric configurations of the constituent monosaccharides and the oligosaccharides derived from the O-antigenic polysaccharide of E . coli O126 . It was found that, amongst the monosaccharides, beta-D-N-acetyl glucosamine was the most effective inhibitory sugar in the O126 polysaccharide and the major specificity of the polyclonal antibodies was found to be directed against the trisaccharide having the structure alpha-D-Galp (1-->3)-beta-D-GlcpNAc(1-->2)-D-Manp.

Mamm Genome, 1994 Nov, 5(11), 717 - 22
Genomic structure of the mouse apurinic/apyrimidinic endonuclease gene; Takiguchi Y et al.; A mammalian apurinic/apyrimidinic endonuclease (AP endonuclease) is known to have two distinct functional domains . One domain is responsible for regulating the activity of Fos/Jun proto-oncogene products to bind to DNA at specific recognition sites . The other domain, which is highly conserved from bacteria to mammals, is responsible for repairing DNA damage caused by ionizing radiation, oxidative damage, and alkylating agents . This study reports on the isolation and characterization of the genomic structure of the mouse AP endonuclease gene (Apex) . The genomic sequence of the Apex gene was 2.14 kb in length and contained four exons . Exon 1 contained a 0.24-kb untranslated 5' region upstream of the initiation codon . Consensus sequences for two CAAT boxes and a GC box were found upstream of the end of exon 1 . A polymorphism was noted in the untranslated region of exon 1 in a comparison of a number of mouse strains . These data indicate that the 5' end of the mouse gene (Apex) differs from the previously isolated human gene (Ape), which has five exons and an untranslated region between exons 1 and 2 . Data are also presented that suggest the presence of two pseudogenes in the mouse.

Differentiation, 1994 Nov, 58(1), 53 - 64
Sequences 5' of the bovine keratin 5 gene direct tissue- and cell-type-specific expression of a lacZ gene in the adult and during development; Ramirez A et al.; Expression of keratin K5 (and K14) in multilayered epithelia occurs predominantly in the basal layer of proliferating keratinocytes . When a keratinocyte becomes committed to terminal differentiation, it moves out of the basal layer towards the epithelial surface . As part of this program of terminal differentiation, the expression of K5 (and K14) is downregulated in suprabasal cells, and new pairs of differentiation-specific keratins are expressed . To define the cis-acting DNA sequences required for K5 cell-type- and differentiation-specific expression, chimeric gene fusions between portions of the bovine keratin K5 locus and the Escherichia coli lacZ gene were used to generate transgenic mice . In the genomic fragment consisting of 5.3 kb of 5' flanking sequences, 6.1 kb corresponding to the body of the gene and 4.5 kb of 3' flanking sequences, the subfragment extending from -5300 bp to +138 bp was the smaller region that directed lacZ expression to stratified epithelia in a manner analogous to the endogenous keratin K5 . Proximal sequences from -1300 bp to +138 bp were inactive . We also determined the expression pattern of keratin K5 during mouse development using an antiserum specific for mouse keratin K5 . Expression was first detected in ectodermal cells of 11.5 days postcoitum embryos, and from day 13.5 postcoitum onwards K5 was detected in the precursors of most epithelia and organs which express K5 at adult stages . This pattern was reproduced, with few differences, by the construct with sequences from -5300 bp to +138 bp fused to the lacZ gene . These findings identify sequences between -5.3 kb and -1.3 kb of the bovine K5 gene as being important for cell-type- and differentiation-specific gene expression both during mouse development and in the adult.

Eur J Pharmacol, 1994 Nov 1, 292(1), 111 - 4
Lipopolysaccharide induces Ca(2+)-independent nitric oxide synthase activity in rat gastric mucosal cells; Brown JF et al.; Ca(2+)-independent nitric oxide synthase was detected in gastric mucosal cells isolated from rats injected 4 h previously with Escherichia coli lipopolysaccharide (3 mg/kg i.v.) . Induced nitric oxide synthase was located in an elutriated cell fraction of intermediate size which contained epithelial cells, but was absent from the parietal cell fraction . Administration of dexamethasone (2 mg/kg i.p.) 1 h before lipopolysaccharide inhibited the appearance of Ca(2+)-independent nitric oxide synthase, and prevented the observed reduction in cell viability (trypan blue exclusion) . Ca(2+)-independent nitric oxide synthase activity can thus be induced in certain cells of the gastric mucosa, and may contribute to gastric pathologies where there is activation of the immune system.

Cell Mol Biol (Noisy-le-grand), 1994 Nov, 40(7), 933 - 44
Interactions between polyamine analogs with antiproliferative effects and tRNA: a 15N NMR analysis; Fernandez CO et al.; N-Bisalkylpolyamine analogs have been shown to exert antiproliferative effects in many tumor models, with the bisethyl derivatives exerting the greatest activities . 15N NMR spectroscopy was used to explore the interactions between these analogs and tRNA . When tRNA was added to solutions of 15N-enriched homospermine (4-4-4), bisethylhomospermine (BE-4-4-4), bismethylhomospermine (BM-4-4-4), bisethylspermine (BE-3-4-3) and 1,19-bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4), the spin-lattice relaxation times T1 of the nitrogens were strongly reduced . From the temperature dependence of these T1's we calculated the rotational activation energies (Ea) of the correlation times of the amino groups in the presence and absence of tRNA . These data indicate that: i) the N-bisethyl derivatives bind strongly to tRNA through their-NH2(+)-groups (most likely, through hydrogen bonding); ii) the binding is weakest in the N-bismethyl derivative and iii) homospermine binds very weakly and mainly through its -NH3(+)-group (most likely, through electrostatic binding) . The binding of the polyamine analogs to tRNA was also estimated by the increase of the half-line widths (D1/2) of the -NH2(+)-groups, derived from the effects that tRNA has on the spin-spin relaxation time T2 . The decrease of the V1/2 values of the -NH2(+)-groups in the (15N-polyamine)-tRNA complexes when the analogs were chased away by an excess of spermine confirmed the stronger binding of the bisethyl- with respect to the bismethyl derivatives, as well as the weak binding of homospermine to tRNA . A correlation was also found between the binding strengths of the analyzed polyamine analogs and their antiproliferative activities.

Mol Gen Genet, 1994 Nov 1, 245(3), 355 - 62
Role of guanosine tetraphosphate in gene expression and the survival of glucose or seryl-tRNA starved cells of Escherichia coli K12; Nystom T; The concentration of guanosine 3',5'-bispyrophosphate (ppGpp) increases in bacteria in response to amino acid or carbon/energy source starvation . An Escherichia coli K12 delta relA delta spoT mutant lacking the ability to synthesize ppGpp lost viability at an increased rate during both glucose and seryl-tRNA starvation . Also, the deleterious effect of chloramphenicol on starved wild-type cells could be overcome by inducing expression of RelA from a plasmid carrying the relA gene transcribed from a tac promoter, prior to starvation and chloramphenicol treatment . As demonstrated by two dimensional gel electrophoresis, this induction of the RelA protein resulted in global alterations in gene expression including increased synthesis of some rpoS-dependent proteins . The delta relA delta spoT mutant maintained high expression of several ribosomal proteins during starvation and appeared to exhibit significantly decreased translational fidelity, as demonstrated by an unusual heterogeneity in the isoelectric point of several proteins and the failure to express higher molecular weight proteins during starvation . Moreover, both rpoS-dependent and independent genes failed to exhibit increased expression in the mutant . It is suggested that the deleterious effects on the cells of the relA, spoT deletions are not due solely to the inability of these cells to induce the sigma factor sigma s, but also to deficiencies in translational fidelity and failure to exert classical stringent regulation.

Mol Gen Genet, 1994 Nov 1, 245(3), 265 - 71
Expression of the linear DNA plasmid pRS64 in the plant pathogenic fungus Rhizoctonia solani; Hongo M et al.; The plant pathogenic isolate RI-64 of anastomosis group 4 of Rhizoctonia solani possesses three linear DNA plasmids (pRS64-1, -2, and -3) . Unique poly(A)- RNA, 0.5 kb in length and hybridizable with the pRS64 DNAs was found in mycelial cells of the isolate RI-64 . The overall homology at the nucleotide level between pRS64-1, -2, and -3, and the cDNA prepared from the poly(A)- RNA was 100%, 73%, and 84%, respectively . The open reading frames found in pRS64-1, -2, and -3 (ORF1-1, ORF2-1, and ORF3-1) are 68 amino acids long . The amino acids sequence showed no significant homology with known proteins . Extracts from Escherichia coli cells expressing ORF1-1 contain a specific protein of 7 kDa . Antisera raised against the ORF1-1 product obtained from E . coli cells cross-reacted with the specific proteins found in the mycelia . The results indicate that the DNA plasmids found in R . solani contain a sequence that encodes a specific protein which may be involved in determination of plant pathogenicity.

J Chem Technol Biotechnol, 1994 Nov, 61(3), 219 - 24
Liquid-liquid extraction of a recombinant protein with reverse micelles; Pires MJ et al.; Recombinant cytochrome b5 was extracted into the reversed micelle phase of an anionic surfactant (AOT) in octane and back-extracted to a final aqueous phase . The extraction of the protein was controlled by an electrostatic mechanism, since it was dependent on the global charge of the protein . This was directly demonstrated by experiments with native and mutant cytochromes obtained by site directed mutagenesis . The back-extraction of cytochrome b5 to a fresh aqueous phase was decreased by factors that reduced the size of the water pool of the organic phase, such as high salt concentrations (1-2 mol dm-3 NaCl) and low temperatures (4 degrees C), probably because of an increase in a favourable interaction of this protein with the surfactant at closer distances.

Mutat Res, 1994 Nov, 315(3), 229 - 37
The XPA protein is a zinc metalloprotein with an ability to recognize various kinds of DNA damage; Asahina H et al.; The XPA (xeroderma pigmentosum group A) gene encodes a protein of 273 amino acids with a zinc finger motif . The human XPA cDNA was placed in an Escherichia coli expression vector for the synthesis of the recombinant XPA protein . The molecular weight of the wild-type protein was about 40 kDa in SDS-PAGE . Microinjection of the wild-type protein specifically restored the defect of UV-induced unscheduled DNA synthesis in XP-A cells . Thus, the bacterially expressed XPA protein retains biochemical properties identical to those of natural sources . The wild-type protein binds preferentially to UV-, cis-diamminedichloroplatinum(II) (cisplatin)- or osmium tetroxide (OsO4)-damaged DNA as assayed by retention on nitrocellulose filters . In addition, the data from atomic absorption and UV-CD spectra revealed that the wild-type protein is a zinc metalloprotein with secondary structure . Furthermore, the mutant protein, of which the cysteine-103 residue in the zinc finger motif was replaced with serine, has a vastly different protein conformation resulting in a loss of XP-A correcting and DNA-binding activities . These findings indicate that the XPA protein is a zinc-binding protein with affinity for various DNA damages, and a cysteine residue in the C4-type zinc finger motif is indispensable for normal protein conformation.

Mutat Res, 1994 Nov 1, 311(1), 57 - 67
Spontaneous mutations in lacI-containing lambda lysogens derived from transgenic mice: the observed patterns differ in liver and spleen; Knoll A et al.; The pattern of somatic mutation observed in tumor suppressor genes, such as the p53 gene, vary dramatically with tumor type . Some of the observed differences are due to tissue specific effects of mutagens, but it is also possible that some differences may reflect the tissue/cell type specificity of spontaneous mutation . Transgenic mouse models with recombinant shuttle vectors containing the lacI or lacZ target genes may shed light on the extent to which spontaneous mutation displays tissue specificity . Herein we utilize a recently described selectable system to obtain spontaneous mutants for analysis of the molecular lesions . Spontaneous mutations were isolated in the lacI gene recovered from five transgenic mice carrying a lambda shuttle vector . Seventy-three and 67 independent mutations derived from liver and spleen DNA, respectively, were defined in the amino terminal region of lacI . Although technical barriers preclude a direct assessment of the E . coli derived pattern of mutation in this system, five pieces of circumstantial evidence suggest that many of the mutations arose in mouse rather than in E . coli . In DNA from both liver and spleen, mutations at CpG dinucleotides predominate (58% and 51%, respectively) . In spleen, most of the mutations at CpG are transitions, while in liver most are transversions . In addition, liver has a higher frequency of GC-->TA transversions at non-CpG dinucleotides while spleen had a higher frequency of deletions and insertions . The data provide evidence that the spontaneous pattern of mutation is tissue specific . In addition, the high frequency of transversions at CpG suggests the need to reevaluate the mechanisms by which mutations occur at this methylated dinucleotide.

Mutat Res, 1994 Nov 1, 311(1), 103 - 9
Ruv and recG genes and the induced precise excision of Tn10 in Escherichia coli; Nagel R et al.; Induction of precise excision of Tn10 by UV or mitomycin C (MMC) is dependent on the expression of the SOS system . Ruv mutants of Escherichia coli, which are defective in DNA repair and recombination, showed diminished frequencies of both spontaneous and UV- or MMC-induced excision of Tn10 inserted in gal . RecG mutants, which are also defective in DNA repair and recombination, showed decreased induction of Tn10 excision with MMC, but not after UV treatment . A recG ruv double mutant showed a greater decrease in induction of excision with MMC than either single mutant . One can speculate that the Ruv proteins, which are known to be involved in the resolution of Holliday junctions, might also be involved in the resolution of putative intermediates generated during the precise excision of Tn10 . RecG protein, whose function partially overlaps those of Ruv proteins, might also have some role in this process.

J Gen Virol, 1994 Nov, 75 ( Pt 11), 3177 - 84
Localization of functional regions of the cucumber mosaic virus RNA replicase using monoclonal and polyclonal antibodies; Hayes RJ et al.; Monoclonal antibodies were produced using a purified cucumber mosaic virus (CMV) replicase complex, and Escherichia coli-expressed CMV 1a and 2a proteins, as immunogens . Five out of eight monoclonal antibodies, which bound to the 1a and 2a proteins in immunoblots, inhibited the RNA-dependent RNA polymerase (RdRp) activity of the purified replicase complex in vitro . Epitope mapping showed that two of the inhibitory antibodies interacted with regions of the 1a protein containing putative helicase and methyltransferase domains respectively . Two other inhibitory antibodies mapped to a region of the 2a protein containing the GDD motif which is highly conserved in RdRps . Prior interaction of the latter antibodies with a peptide containing the GDD motif prevented the antibody-mediated inhibition of the replicase . Polyclonal antibodies which inhibited the RdRp activity of the replicase complex were also produced using peptides corresponding to conserved helicase and polymerase motifs in the 1a and 2a proteins . The greatest inhibition was shown by antibodies to a peptide containing the GDD motif . These results demonstrate the functional importance of the identified sequence motifs in CMV RNA replication and indicate that the motifs are located in the replicase complex at positions accessible to antibodies, consistent with roles in interacting with the RNA template, RNA primer and enzyme substrates.

J Exp Med, 1994 Nov 1, 180(5), 1949 - 54
Apolipoprotein E (ApoE), a novel heparin-binding protein inhibits the development of Kaposi's sarcoma-like lesions in BALB/c nu/nu mice; Browning PJ et al.; Recombinant apolipoprotein E-3 (ApoE-3), expressed in Escherichia coli, was purified and used in an in vitro and an in vivo model system for acquired immunodeficiency syndrome-associated Kaposi's sarcoma (AIDS-KS) . This protein blocked cell proliferation and chemotaxis of AIDS-KS cells in response to activated lymphocyte conditioned medium (AL-CM) and oncostatin M (OSM) . ApoE-3 also inhibited the formation of neoangiogenic lesions induced in BALB/c nu/nu mice by AIDS-KS cells . These findings represent a novel and potentially less toxic therapeutic approach for the treatment of AIDS-KS.

J Cell Biol, 1994 Nov, 127(4), 1041 - 8
Characterization of the KLP68D kinesin-like protein in Drosophila: possible roles in axonal transport; Pesavento PA et al.; This paper describes the molecular and biochemical properties of KLP68D, a new kinesin-like motor protein in Drosophila melanogaster . Sequence analysis of a full-length cDNA encoding KLP68D demonstrates that this protein has a domain that shares significant sequence identity with the entire 340-amin acid kinesin heavy chain motor domain . Sequences extending beyond the motor domain predict a region of alpha-helical coiled-coil followed by a globular "tail" region; there is significant sequence similarity between the alpha-helical coiled-coil region of the KLP68D protein and similar regions of the KIF3 protein of mouse and the KRP85 protein of sea urchin . This finding suggests that all three proteins may be members of the same family, and that they all perform related functions . KLP68D protein produced in Escherichia coli is, like kinesin itself, a plus-end directed microtubule motor . In situ hybridization analysis of KLP68D RNA in Drosophila embryos indicates that the KLP68D gene is expressed primarily in the central nervous system and in a subset of the peripheral nervous system during embryogenesis . Thus, KLP68D may be used for anterograde axonal transport and could conceivably move cargoes in fly neurons different than those moved by kinesin heavy chain or other plus-end directed motors.

J Cell Biol, 1994 Nov, 127(3), 609 - 22
The amino-terminal region of the retinoblastoma gene product binds a novel nuclear matrix protein that co-localizes to centers for RNA processing; Durfee T et al.; The tumor suppressing capacity of the retinoblastoma protein (p110RB) is dependent on interactions made with cellular proteins through its carboxy-terminal domains . How the p110RB amino-terminal region contributes to this activity is unclear, though evidence now indicates it is important for both growth suppression and regulation of the full-length protein . We have used the yeast two-hybrid system to screen for cellular proteins which bind to the first 300 amino acids of p110RB . The only gene isolated from this screen encodes a novel 84-kD nuclear matrix protein that localizes to subnuclear regions associated with RNA processing . This protein, p84, requires a structurally defined domain in the amino terminus of p110RB for binding . Furthermore, both in vivo and in vitro experiments demonstrate that p84 binds preferentially to the functionally active, hypophosphorylated form of p110RB . Thus, the amino terminus of p110RB may function in part to facilitate the binding of growth promoting factors at subnuclear regions actively involved in RNA metabolism.

J Bacteriol, 1994 Nov, 176(22), 7079 - 84
Role of the rfe gene in the biosynthesis of the Escherichia coli O7-specific lipopolysaccharide and other O-specific polysaccharides containing N-acetylglucosamine; Alexander DC et al.; We report that rfe mutants of wild-type strains of Escherichia coli O7, O18, O75, and O111 did not express O-specific polysaccharide unless the rfe mutation was complemented by a cloned rfe gene supplied in a plasmid . The O polysaccharides in these strains are known to have N-acetylglucosamine (GlcNAc) in their O repeats . In addition, in vitro transferase assays with bacterial membranes from either the O7 wild-type strain or its isogenic rfe mutant showed that GlcNAc is the first carbohydrate added onto the lipid acceptor in the assembly of the O7 repeat and that this function is inhibited by tunicamycin . Our results indicate that the rfe gene product is a general requirement for the synthesis of O polysaccharides containing GlcNAc.

Eur J Biochem, 1994 Nov 1, 225(3), 1211 - 20
Structure of the Escherichia coli O24 and O56 O-specific sialic-acid-containing polysaccharides and linkage of these structures to the core region in lipopolysaccharides; Gamian A et al.; The lipopolysaccharides from Escherichia coli O24 and O56 could be separated into higher-molecular-mass and lower-molecular-mass fractions . Mild acid hydrolysis of lipopolysaccharides of both serotypes released an O-specific polysaccharide and a tetrasaccharide repeating unit . Oligomers of the repeating unit, the core and the oligosaccharide that contains a fragment of the repeating unit linked to the core region were also obtained according to hydrolysis conditions . On the basis of sugar and methylation analyses, Smith degradation, fast-atom-bombardment mass spectrometry and NMR spectroscopy of the hydrolysis products, the biological repeating units of the O-specific polysaccharides were shown to be the following tetrasaccharides: {formula: see text} The structures differ from the structures proposed previously by Kogan et al . {Kogan, G., Shashkov, A . S., Jann, B . & Jann, K . (1993) Carbohydr . Res . 238, 261-270; Kogan, G., Jann, B . & Jann, K . (1993) Carbohydr . Res . 238, 335-338} . The O-specific repeating unit in E . coli O24 lipopolysaccharide is linked to O6 of the terminal D-galactose in the core region, whereas in O56 LPS the repeating unit is linked to O4 of a subterminal D-glucose residue in an R2 type core.

EMBO J, 1994 Nov 1, 13(21), 5070 - 4
Decay-accelerating factor CD55 is identified as the receptor for echovirus 7 using CELICS, a rapid immuno-focal cloning method; Ward T et al.; Using an anti-receptor mAb that blocks the attachment of echovirus 7 and related viruses (echoviruses 13, 21, 29 and 33), we have isolated a complementary DNA clone that encodes the human decay-accelerating factor (CD55) . Mouse cells transfected with the CD55 clone bind echovirus 7, and this binding is blocked by the anti-receptor mAb . The method used (CELICS) allows rapid and direct cloning of genes encoding cell surface receptors . It is based on episomal replication and high efficiency expression of complementary DNA clones in the vector pCDM8 in COS or WOP cells, in conjunction with a sensitive immuno-focal screen that uses antibody probes linked to beta-galactosidase . Receptor positive cells were identified by a colour change and isolated individually using a micromanipulator . DNA extracted from a small number of cells was then cloned directly in Escherichia coli.

Mutat Res, 1994 Nov, 325(2-3), 75 - 9
RecN SOS gene and induced precise excision of Tn10 in Escherichia coli; Chan A et al.; A mutant defective in the induced excision of Tn10 was isolated by decreased papillation on MacConkey-galactose plates with mitomycin C . The mutation involved was characterized as recN by genetic mapping and complementation . This mutant, as well as a previously characterized recN mutant (recN262), showed a markedly decreased frequency of excision of Tn10 after treatment with UV or mitomycin C . These observations indicate that recN is involved in the induced excision of Tn10.

J Virol, 1994 Nov, 68(11), 7067 - 74
Artificial mosaic protein containing antigenic epitopes of hepatitis E virus; Khudyakov YE et al.; A synthetic gene encoding an artificial polypeptide composed of antigenic epitopes of the hepatitis E virus (HEV) proteins was constructed from short oligodeoxyribonucleotides by using PCR . The polypeptide comprises a mosaic of three antigenically active dominant regions from the protein encoded by open reading frame 2 (ORF2), one antigenically active region from the protein encoded by ORF3 of the Burmese HEV strain, and one antigenically active region from the protein encoded by ORF3 of the Mexican HEV strain . The mosaic protein was expressed in Escherichia coli as a chimera with glutathione S-transferase or beta-galactosidase . Guinea pig sera containing antibodies to the corresponding HEV synthetic peptides were used to demonstrate by Western immunoblot analysis and enzyme immunoassay the presence and accessibility of all HEV-specific antigenic epitopes introduced into the mosaic protein . Both the glutathione S-transferase and beta-galactosidase hybrid proteins were analyzed by using a panel of human anti-HEV-positive and -negative sera . The data obtained strongly indicate a diagnostic potential for the mosaic protein.

FEBS Lett, 1994 Oct 31, 354(1), 37 - 40
A cryoelectron microscopy study of the interaction of the Escherichia coli F1-ATPase with subunit b dimer; Wilkens S et al.; A complex between the Escherichia coli F1-ATPase and a truncated form of the ECF0-b subunit was formed and examined by cryoelectron microscopy in amorphous ice . Image analysis of single particles in the hexagonal projection revealed that the polar domain of the b subunit interacts with a beta subunit different from the one which interacts with the epsilon subunit . The cavity in the enzyme, visible in the hexagonal projection, is not filled by the b polypeptide, therefore leaving enough room for extensive conformational changes of the gamma and epsilon subunits within the native F1F0 complex.

Biochem Biophys Res Commun, 1994 Oct 28, 204(2), 828 - 34
Secretion of immunoreactive corticotropin releasing factor and adrenocorticotropic hormone by T- and B-lymphocytes in response to cellular stress factors; Kravchenco IV et al.; The ability of human lymphocytes and mouse splenocytes to secrete corticotropin-releasing factor (CRF) in response to hyperthermia, hyperosmolarity and hypoxia has been shown . Both human T- and B-lymphocytes appear to have this ability . E . coli lipopolysaccharide and concanavalin A can stimulate CRF secretion by B- and T-lymphocytes, respectively, whereas hydrocortisone inhibits the CRF secretion induced by any agent tested . Adrenocorticotropic hormone (ACTH) secretion in response to hyperthermia, hyperosmolarity and hypoxia was demonstrated also, and this secretion could be inhibited by anti-CRF antibodies and by hydrocortisone . We demonstrate that CRF could provide a necessary link between external stimuli such as cellular stress factors and ACTH secretion by immunocytes leading to corticosteroid production and, subsequently, to non-specific defence reaction of the organism . We also describe a model designed to explain the results obtained.

Biochem Biophys Res Commun, 1994 Oct 28, 204(2), 701 - 9
Identification of functional cysteine residues in human galactosyltransferase; Wang Y et al.; The functions of the five cysteine residues in human galactosyltransferase were investigated using site-directed mutagenesis to determine the location of the disulfide bond as well as the role of the sulfhydryl groups . The enzyme remains active when three of its cysteine residues at positions 171, 264 and 340 are mutated to serine separately . However, enzymatic activity is lost when either cysteine-129 or cysteine-245 is replaced with serine . The loss of GT activity suggests that these two cysteine residues form a disulfide bond . The three active mutated enzymes were studied kinetically . The kinetic constants of the enzymes with cysteine-171 or cysteine-264 replaced with serine are not significantly different from those of GT that does not have these substitutions . When cysteine-340 was mutated, however, the kinetic constant for UDP-galactose increased about 30 fold, while that for N-acetylglucosamine and Mn2+ remained unchanged . In addition, sulfhydryl inhibition studies reveal that cysteine-340 is the only cysteine residue that reacts with the sulfhydryl reagents . These results indicate that cysteine-340 may be involved in the binding of UDP-galactose.

Biochem Biophys Res Commun, 1994 Oct 28, 204(2), 646 - 52
Cloning and expression of recombinant human growth/differentiation factor 5; Hotten G et al.; The complete amino acid sequence of human Growth/differentiation factor 5 (huGdf5), a new member of the TGF-beta superfamily, has been determined through initial degenerate PCR and subsequent cloning and nucleotide sequencing of genomic DNA and cDNA encoding the precursor and flanking regions . The huGdf5 gene consists of only two coding exons . The protein is highly homologous to its murine equivalent, particularly the mature part which differs only by one amino acid . Expression in HuTK- cells using recombinant vaccinia virus revealed the expected processed dimeric mature protein . Antibodies against huGdf5 were raised in chicken.

Biochem Biophys Res Commun, 1994 Oct 28, 204(2), 525 - 36
Detection of retinoid X receptors using specific monoclonal and polyclonal antibodies; Rochette-Egly C et al.; Because of the growing importance of the Retinoid X Receptors (RXR alpha, beta and gamma) in the retinoid acid signalling pathway, we have prepared polyclonal and monoclonal antibodies directed against these proteins . For this purpose, either the whole mouse RXR alpha protein expressed in E.Coli, or synthetic peptides corresponding to amino acid sequences common to all RXRs or unique to RXR alpha, beta or gamma, were used as antigens . Antibodies recognizing either all three RXR types (alpha, beta and gamma) or specific for each RXR type were obtained . The antibodies were characterized by immunocytochemistry, immunoblotting, immunoprecipitation and electromobility shift assay (EMSA) . These antibodies allowed us to detect the presence of RXR alpha proteins in mouse embryos and in mouse embryonal carcinoma cells (F9 and P19 cell lines) by immunoblotting, immunoprecipitation and EMSA whereas RXR beta could be detected only by EMSA and RXR gamma could not be detected by any of these techniques.

J Mol Biol, 1994 Oct 28, 243(3), 402 - 12
Mutations in helix 34 of Escherichia coli 16 S ribosomal RNA have multiple effects on ribosome function and synthesis; Moine H et al.; Helix 34 of E . coli 16 S rRNA (1046 to 1067 and 1189 to 1211) has been proposed to participate directly in the termination of translation at UGA stop codons . We have constructed mutations in this helix in plasmid-encoded rDNA to explore the specific functional roles of the sequence UCAUCA (1199 to 1204) and a secondary structure also involving positions 1054 and 1057-1058 . The rRNA mutations were analyzed for their effects on in vivo translational accuracy (stop codon readthrough and frameshifting) as well as growth rate, ribosome synthesis and incorporation into polysomes . Mutations at positions 1054, 1057, 1058, 1199 and 1200 had significant effects on translational accuracy, causing non-specific readthrough of all three stop codons as well as enhanced +1 and -1 frameshifting . Mutations at 1202 and 1203, however, had no effect . The incorporation of deleterious mutant subunits into 70 S ribosomes and polysomes was severely reduced and was associated with a slower growth rate and increased synthesis of host-encoded ribosomes . These data support the proposal that helix 34 is an essential component of the decoding center of the 30 S ribosomal subunit and is not restricted in function to UGA-codon specific termination.

J Mol Biol, 1994 Oct 28, 243(3), 397 - 401
Two lines of allosteric communication in the oligomeric chaperonin GroEL are revealed by the single mutation Arg196-->Ala; Yifrach O et al.; Sequence homology between GroEL and Escherichia coli DNA polymerase I, together with the fact that both proteins bind adenine nucleotides, suggested to us that they may have a similar nucleotide binding site . Arg196 in GroEL corresponds to Arg425 in DNA polymerase I, which is near its nucleotide binding site . Here, we report the striking effects of the mutation Arg196-->Ala in GroEL on its kinetic and allosteric properties with respect to ATP . The mutation reduces positive co-operativity in ATP hydrolysis found in wild-type GroEL . It also gives rise to strong substrate (ATP) inhibition, which is not apparent in the wild-type protein . The dual effect of the mutation reflects the presence of two lines of allosteric communication between ATP binding sites in GroEL and suggests the existence of nested co-operativity.

J Biol Chem, 1994 Oct 28, 269(43), 27136 - 42
Human cathepsin O . Molecular cloning from a breast carcinoma, production of the active enzyme in Escherichia coli, and expression analysis in human tissues; Velasco G et al.; A cDNA encoding a novel member of the cysteine proteinase family of proteins has been cloned from a human breast carcinoma cDNA library, by using a polymerase chain reaction-based cloning strategy . The isolated cDNA contains an open reading frame coding for a polypeptide of 321 amino acids that has been tentatively called cathepsin O . This protein presents all the structural features characteristic of the different cysteine proteinases identified to date, including the active site cysteine residue that is involved in covalent intermediate formation during peptide hydrolysis . The cathepsin O cDNA was expressed in Escherichia coli, and after purification and refolding, the recombinant protein was able to degrade the synthetic peptides benzyloxycarbonyl-Phe-Arg-7-amido-4- methylcoumarin and benzyloxycarbonyl-Arg-Arg-7-amido-4-methylcoumarin widely used as substrates for cysteine proteinases . Cathepsin O proteolytic activity was abolished by trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64), an inhibitor of this subclass of proteolytic enzymes, thus providing additional evidence that the isolated cDNA codes for an authentic cysteine proteinase . Northern blot analysis of poly(A)+ RNAs isolated from a variety of human tissues demonstrated that cathepsin O is expressed in all examined tissues, which is consistent with a putative role of this protein as a proteolytic enzyme involved in normal cellular protein degradation and turnover.

J Biol Chem, 1994 Oct 28, 269(43), 27051 - 8
Kinetics and regulation of pantothenate kinase from Escherichia coli; Song WJ et al.; Pantothenate kinase catalyzes the rate-controlling step in coenzyme A (CoA) biosynthesis and is regulated by feedback inhibition by CoA . Pantothenate kinase was purified to homogeneity from Escherichia coli and was shown to exist as a homodimer . Kinetic analysis indicated the presence of two ATP binding sites that exhibited positive cooperativity with a Hill coefficient of 1.46 . Site-directed mutagenesis of lysine 101 to methionine (K101M) resulted in the inactivation of the enzyme, although dimer formation was not altered . The K101M mutant was unable to bind either adenosine 5'-O-(3-thiotriphosphate) or CoA, supporting the conclusion from kinetic analysis that both the substrate and inhibitor bind to the same site on the enzyme . CoA binding was not cooperative . Coexpression of the K101M mutant gene on a high copy number plasmid in the presence of a chromosomal copy of the wild-type gene resulted in the production of heterodimers between active and inactive subunits . Kinetic analysis of the chimeric heterodimers showed the absence of cooperative ATP interactions and indicated a sequential kinetic mechanism for pantothenate kinase with ATP binding first and pantothenate second . Thus, pantothenate kinase regulation involves the competitive binding of CoA to the ATP site, which blocks ATP binding at one site and prevents positive cooperative ATP binding to the second site on the dimer.

J Biol Chem, 1994 Oct 28, 269(43), 26982 - 7
Multiple sites of the propeptide region of human stromelysin-1 are required for maintaining a latent form of the enzyme; Freimark BD et al.; Latency of matrix metalloproteinase 3 (MMP-3) is regulated by the interaction of a free cysteine residue (Cys-75) in the conserved amino acid sequence Pro-Arg-Cys-Gly-Val-Pro-Asp located in the COOH-terminal portion of the propeptide with a chelated zinc atom in the active site of the catalytic domain . Proteolytic activation of full-length human pro-MMP-3 involves the removal of approximately 35 amino acids from the NH2-terminal portion of the propeptide, forming a 53-kDa unstable intermediate that undergoes intermolecular autocatalysis to form the 45-kDa mature active enzyme . In this study, we have evaluated the contribution of the NH2-terminal 35 amino acids to the maintenance of latency . Full-length human pro-MMP-3 was expressed in Escherichia coli and refolded to form latent pro-MMP-3 capable of activation by chymotrypsin or aminophenylmercuric acetate . Renaturation of pro-MMP-3 expressed in bacteria with 20 or more amino acids removed from the NH2-terminal region of the propeptide yielded only an active enzyme . COS-7 cells transiently transfected with pro-MMP-3 expression vectors containing the single amino acid substitutions Y20A, L21A, and C75S also secreted active forms of the enzyme . These data suggest that simultaneous interactions of the NH2- and COOH-terminal regions of the propeptide are required for maintenance of the latent form of the enzyme.

J Biol Chem, 1994 Oct 28, 269(43), 26976 - 81
High conservation of subunit composition of RNA polymerase I(A) between yeast and mouse and the molecular cloning of mouse RNA polymerase I 40-kDa subunit RPA40; Song CZ et al.; Mouse RNA polymerase I (or A) was purified from an ascites cell line MH134 to virtual homogeneity using a novel purification procedure and examined for subunit composition . In marked contrast to older purifications that reported 5-8 subunits, polymerase I was found to have 11 subunits with remarkable correspondence to those of yeasts . The cDNA encoding a 40-kDa subunit of this enzyme, designated RPA40, was isolated . It predicts a polypeptide of 355 amino acids (M(r) = 40,065) and is encoded by a single copy gene . Protein sequence analysis reveals that RPA40 is the homolog of yeast RPC40, having homology to alpha subunit of Escherichia coli RNA polymerase, yeast RPB3, and human RPB33 RNA polymerase II subunits . The high conservation of this subunit among distant eukaryotes and different RNA polymerases suggests functional importance of this protein as a core subunit.

J Biol Chem, 1994 Oct 28, 269(43), 26959 - 68
Tus prevents overreplication of oriC plasmid DNA; Hiasa H et al.; Minichromosome plasmid DNA templates containing oriC and two Ter sites, oriented as they are on the Escherichia coli chromosome, have been used to study the role of Tus in termination of bidirectional replication . In replication reactions reconstituted with purified proteins where it could be demonstrated that each active template was replicating bidirectionally, Tus was required to prevent extensive overreplication . In the presence of Tus, both replication forks terminated DNA synthesis at one or the other Ter site in an apparent stepwise manner . First, the progress of one replication fork was arrested by a properly oriented Tus-Ter complex . Then, either because of steric hindrance resulting from the stalled replication machinery of the first fork or because of the formation of a branched DNA structure, the progression of the second opposing fork was halted at the same site on the DNA template . In the absence of Tus, overreplication required DNA ligase and arose via a template strand-switching mechanism . Thus, the role of Tus in E . coli is more likely to prevent overreplication rather than to ensure accurate termination.

J Biol Chem, 1994 Oct 28, 269(43), 26953 - 8
Biosynthesis of heparin/heparan sulfate . Purification of the D-glucuronyl C-5 epimerase from bovine liver; Campbell P et al.; The D-glucuronyl C-5 epimerase involved in the biosynthesis of heparin/heparan sulfate was purified from the high speed supernatant fraction of a homogenate of bovine liver by chromatography on immobilized O-desulfated heparin, red Sepharose, phenyl Sepharose, and concanavalin A-Sepharose . After close to 1 million-fold purification, in 10-15% yield, the product gave a single band on SDS-polyacrylamide gel electrophoresis with silver staining and had a mobility corresponding to an M(r) of approximately 52,000 . Since the epimerase assay used in the course of purification was based on release of tritium, as {3H}H2O, from a {5-3H}uronyl-labeled substrate, it was important to establish that the purified enzyme did indeed catalyze the actual conversion of D-glucuronyl to L-iduronyl residues . Upon incubation of the purified enzyme with 3H-labeled heparosan N-sulfate, prepared by metabolic labeling (with D-{1-3H}glucose) of a capsular polysaccharide from Escherichia coli K5 and subsequent chemical partial N-deacetylation and N-sulfation, approximately 30% of the D-glucuronyl residues located between two N-sulfated glucosamine units were converted to L-iduronyl units.

J Biol Chem, 1994 Oct 28, 269(43), 26904 - 11
A novel strategy for stabilization of Escherichia coli ribonuclease HI involving a screen for an intragenic suppressor of carboxyl-terminal deletions; Haruki M et al.; A strategy to genetically select Escherichia coli ribonuclease HI mutants with enhanced thermostability is described . E . coli strain MIC3001, which shows an RNase H-dependent, temperature-sensitive growth phenotype, was used for this purpose . Introduction of the rnhA gene permits the growth of this temperature-sensitive strain, whereas the gene for the truncated protein, 142-RNase HI, which lacks the carboxyl-terminal 13 residues, cannot . Analyses of the production levels and the stability of a series of mutant proteins with COOH-terminal truncations suggested that 142-RNase HI is nonfunctional in vivo because of a dramatic decrease in the protein stability . Polymerase chain reaction-mediated random mutagenesis of the rnhA142 gene, encoding 142-RNase HI, followed by selection of revertants, allowed us to isolate 11 single amino acid substitutions that render 142-RNase HI functional in vivo . Of them, eight substitutions were shown to enhance the thermal stability of the wild-type RNase HI protein, and of these, six were novel . The genetic selection strategy employed in this experiment was thus shown to be effective for identifying amino acid substitutions that enhance the thermal stability of E . coli RNase HI . Such a strategy would be versatile if a protein of interest could be destabilized by a deletion or a truncation and a conditional-lethal strain were available.

J Biol Chem, 1994 Oct 28, 269(43), 26898 - 903
Synergistic insertion of two hydrophobic regions drives Sec-independent membrane protein assembly; Cao G et al.; We have studied the membrane insertion of two proteins from the inner membrane of Escherichia coli, both with two transmembrane segments connected by a short periplasmic loop: the M13 procoat protein and a mutant "inverted" leader peptidase . Neither molecule depends on the Sec machinery for insertion . We show that the introduction of a charged residue in the second transmembrane segment completely blocks insertion of both proteins . In contrast, a Sec-dependent procoat mutant, where the periplasmic region has been lengthened, inserts into the membrane even in the presence of a charged residue in the second hydrophobic domain . In addition, a large deletion within the second transmembrane domain of the leader peptidase mutant allows membrane translocation, but only under conditions where the SecA protein is functional . Furthermore, we show that the first hydrophobic domain is required for insertion of the short periplasmic loop of the "inverted" leader peptidase . These results suggest that Sec-independent insertion occurs by a synergistic entry of the two neighboring hydrophobic domains into the lipid bilayer.

J Biol Chem, 1994 Oct 28, 269(43), 26858 - 64
Cloning, expression, and purification of a functional nonacetylated mammalian mitochondrial chaperonin 10; Dickson R et al.; An intact mouse mitochondrial chaperonin 10 has been cloned, sequenced, and overexpressed in Escherichia coli as a fusion protein harboring an oligohistidine tail at its COOH terminus . The latter was added to simplify protein purification . The purified protein is free of contaminating groES from the bacterial host cells . Edman degradation reveals that the initiator Met residue of the recombinant protein is removed in vivo, similar to the authentic chaperonin 10 purified from rat liver mitochondria . However, in contrast to the latter, the amino-terminal Ala residue of the recombinant protein is not acetylated; the molecular mass determined by electrospray ionization mass spectrometry is 12,350.9 +/- 2.6 daltons, in agreement with that predicted for the nonacetylated protein (12,351.2 daltons) . Facilitated protein folding experiments with ribulose-biphosphate carboxylase, under "nonpermissive" in vitro conditions, demonstrate that the recombinant protein is fully functional with groEL . Thus, both the initial rates of protein folding and final yields observed with this heterologous combination are virtually identical to those obtained with groEL and groES . More important, like the authentic protein purified from mitochondria, the recombinant mitochondrial chaperonin 10, but not groES, is functionally compatible with the heptameric chaperonin 60 of mammalian mitochondria.

J Biol Chem, 1994 Oct 28, 269(43), 26830 - 5
Structure-function studies of human arylamine N-acetyltransferases NAT1 and NAT2 . Functional analysis of recombinant NAT1/NAT2 chimeras expressed in Escherichia coli; Dupret JM et al.; The human arylamine N-acetyltransferases NAT1 and NAT2 catalyze the biotransformation of primary aromatic amine or hydrazine drugs and xenobiotics . These enzymes share 81% amino acid sequence identity, yet differ markedly with respect to their acceptor substrate selectivities and intrinsic in vitro stabilities . To define the contribution of large regions of NAT1 and NAT2 polypeptide structure to enzyme integrity and catalytic specificity, we used selected restriction endonuclease digestions and fragment religation into the tac promoter-based phagemid pKEN2 to construct a panel of 18 NAT1/NAT2 hybrid gene vectors for heterologous expression in Escherichia coli . Induction of hybrid gene expression in recombinant transformants of E . coli strain XA90 led to the production of soluble, catalytically active acetylating enzymes in all cases . Chimeric proteins produced in this fashion were then compared to wild-type NAT1 and NAT2 with respect to their enzyme kinetic constants (apparent Km, Vmax, and Vmax/Km) for the NAT1-selective and NAT2-selective substrates p-aminosalicylic acid and sulfamethazine, respectively, and for their in vitro stabilities at 37 degrees C . The ratio of the Vmax/Km for sulfamethazine to that for p-aminosalicylic acid allowed for the unambiguous classification of each enzyme as either NAT1 or NAT2 type, except for one novel chimera possessing a low Michaelis constant and a high maximal velocity for the acetylation of both substrates . A central region (amino acids 112-210) within the 290-residue polypeptide appeared to play a role in determining NAT1- or NAT2-type behavior . On the other hand, the region (residues 47-111) encompassing the putative active site cysteine (Cys68) was important in contributing to a low apparent Km for p-aminosalicylic acid but not for sulfamethazine, while amino acids 211-250 affected Km for sulfamethazine and 251-290 influenced Km for both substrates . Maximal velocities were highest for both substrates when the central 112-210 amino acid region was derived from NAT1 . Finally, the region from amino acids 211-250 in NAT2 was important in determining its greater intrinsic enzyme stability than that exhibited by NAT1.

J Biol Chem, 1994 Oct 28, 269(43), 26789 - 95
Arginine 30 and asparagine 74 have functional roles in the glutamine dependent activities of Escherichia coli asparagine synthetase B; Boehlein SK et al.; Although Arg-30, Asn-74, and Asn-79 appear totally conserved throughout the purF glutamine-dependent amidotransferases, their potential roles in catalysis and binding remain unexplored for any member of the enzyme family . Here we report the overexpression, purification, and kinetic characterization of a series of AS-B mutants which allow an examination of the functional roles of these 3 residues in glutamine-dependent nitrogen transfer . While Asn-79 appears to possess no catalytic function in AS-B, site-directed mutagenesis of Asn-74 has implicated this residue as playing a role in catalysis of nitrogen transfer from glutamine . The kinetic properties of the Asn-74 AS-B mutant enzymes appear consistent with both ammonia-mediated nitrogen transfer and two apparently novel mechanistic suggestions for this reaction involving either an oxyanion or imide intermediate (Richards, N . G . J., and Schuster, S . M . (1992) FEBS Lett . 313, 98-102) . We also demonstrate that replacement of Arg-30 by an alanine residue yields an AS-B mutant for which the apparent Km for glutamine is increased in the glutamine-dependent synthesis of asparagine . In addition, ATP-dependent stimulation of the glutaminase activity of AS-B is modified or completely eliminated when Arg-30 is replaced by other amino acids . The latter observation may indicate the existence of a molecular switch involving Arg-30 which coordinates the two half-reactions catalyzed by the glutamine-dependent amidotransferases and synthetase domains of cellular AS-B.

J Biol Chem, 1994 Oct 28, 269(43), 26759 - 66
The 50-kDa primase subunit of Drosophila melanogaster DNA polymerase alpha . Molecular characterization of the gene and functional analysis of the overexpressed protein; Bakkenist CJ et al.; The gene encoding the 50-kDa subunit of Drosophila melanogaster DNA polymerase alpha has been cloned . A comparison of the predicted polypeptide sequence of the Drosophila protein with the equivalent subunits from mouse and yeast suggests that they are closely related and defines three conserved regions which are likely to be important for enzyme activity . The expression patterns of both the 50-kDa protein and its transcript (a single RNA message of 1.6 kilobases) throughout development are consistent with a role of the protein in DNA replication . When overexpressed and purified the 50-kDa subunit displays DNA primase activity . The products of the reaction, mainly oligoribonucleotides 12-14 nucleotides in length, plus dimers and some trimers, are similar to those synthesized by either the intact DNA polymerase alpha, or the biochemically isolated primase heterodimer . The isolated primase also shows similar sensitivity to antibodies, magnesium and monovalent cations, and the same nucleotide requirements as complexed forms of the primase . The isolated subunit, however, is more thermally labile, suggesting a role for the additional subunits in DNA polymerase alpha in stabilizing the primase activity of the 50-kDa primase subunit.

J Biol Chem, 1994 Oct 28, 269(43), 26739 - 45
Expression and characterization of recombinant human eosinophil-derived neurotoxin and eosinophil-derived neurotoxin-anti-transferrin receptor sFv; Newton DL et al.; The gene for the human recombinant eosinophil-derived neurotoxin (rEDN) was synthesized and fused to the gene encoding a single chain antibody (sFv) to the human transferrin receptor (EDNsFv) . Both rEDN and EDNsFv were expressed as insoluble proteins in inclusion bodies in Escherichia coli BL21(DE3) . Following denaturation and renaturation, EDN and EDNsFv were partially purified by chromatography on heparin-Sepharose . Final purification of EDN was achieved by Sephadex G-100, whereas EDNsFv which contained a 6-histidyl residue carboxyl terminus was highly purified using the metal chelate resin, Ni(2+)-nitriloacetic acid . Whereas the recombinant EDN had ribonuclease activity that was similar to the native protein, the fusion protein had enzymatic activity that was 6-13% that of native EDN . The fusion protein was able to bind to the human transferrin receptor . In contrast to rEDN that had no inherent cytotoxicity to human tumor cells, the EDNsFv fusion protein was cytotoxic to human leukemia cells that express the human transferrin receptor with an IC50, 0.2-1 nM . At 1.3 nM EDNsFv, no cytotoxicity was observed on cells that lack the human transferrin receptor . Free antibody to the human transferrin receptor, E6, inhibited the cytotoxicity of the EDNsFv . Human enzymes may be engineered to acquire cytotoxic properties by fusing them to antibodies . Thus, they may be candidates for the construction of immunofusion proteins that may be less immunogenic than immunotoxins containing bacterial- or plant-derived toxin moieties.

J Biol Chem, 1994 Oct 28, 269(43), 26705 - 10
Introduction of a disulfide bond into ricin A chain decreases the cytotoxicity of the ricin holotoxin; Argent RH et al.; Wild type ricin A chain (RTA) contains two cysteine residues (Cys171 and Cys259) . Cys259 forms the interchain disulfide bond of ricin holotoxin with Cys4 of ricin B chain (RTB) . We have used site-directed mutagenesis of RTA cDNA to convert Cys171 to Ser and to introduce a disulfide bond into RTA by converting Ser215 and Met255 to Cys residues . Mutant RTA was expressed in Escherichia coli and directed to the oxidizing environment of the periplasmic space where the Cys215-Cys255 disulfide bond was formed . The disulfide-containing RTA mutant had an in vitro catalytic activity similar to that of an identical form of recombinant RTA that lacked the S215C and M255C mutations . In the presence of glutathione and protein disulfide isomerase, this RTA variant reassociated with RTB to form ricin holotoxin . Incubation of this holotoxin with increasing concentrations of dithiothreitol showed that the interchain disulfide bond joining RTA and RTB was more readily reduced than the intrachain disulfide bond in RTA . Ricin in which the RTA moiety contained the disulfide bond was 15-18-fold less cytotoxic to HeLa or Vero cells than ricin in which the RTA did not contain the stabilizing disulfide cross-link . Since these ricin molecules had identical RTB cell binding and RTA catalytic activities, we suggest that the observed reduction in cytotoxicity caused by the introduced disulfide bond resulted from a constraint on the unfolding of RTA, indicating that such unfolding is necessary for the membrane translocation of RTA during its entry into the cytosol.

J Biol Chem, 1994 Oct 28, 269(43), 26684 - 90
Differential and tissue-specific expression of a gene family for tyrosine/dopa decarboxylase in opium poppy; Facchini PJ et al.; Two early and potential rate-limiting steps in the biosynthesis of isoquinoline alkaloids, such as morphine and codeine, in opium poppy (Papaver somniferum) involve decarboxylation of L-tyrosine and L-dihydroxyphenylalanine (L-dopa) to yield tyramine and dopamine, respectively . A DNA fragment was amplified by polymerase chain reaction (PCR) using degenerate primers designed to two highly conserved domains found in other aromatic amino acid decarboxylases . A poppy seedling cDNA library was screened with this PCR product and a cDNA (cTYDC1) for tyrosine/dopa decarboxylase (TYDC/DODC) was isolated . Two other independent cDNAs (cTYDC2 and cTYDC3) encoding TYDC/DODC were isolated by heterologous screening with a plant tryptophan decarboxylase (TDC) cDNA as probe . A poppy genomic library was screened with cTYDC1 and two intronless genomic clones (gTYDC1 and gTYDC4) were isolated . The deduced amino acid sequences of all poppy clones share extensive identity with other reported pyridoxal phosphate-dependent decarboxylases from both plants and animals . Based on sequence homology, members of the gene family were divided into two subsets (cTYDC1 and gTYDC4; cTYDC2 and cTYDC3) of proteins with predicted M(r) = 56,983 and 59,323, respectively . Within each subset the clones exhibit greater than 90% identity, whereas clones between subsets share less than 75% identity . Expression of gTYDC1 and cTYDC2 as beta-galactosidase fusion proteins in Escherichia coli resulted in catalytically active enzymes immunodetectable with TDC-specific polyclonal antibodies . Each enzyme showed marginally higher substrate specificity for L-dopa over L-tyrosine, but did not accept L-tryptophan and L-phenylalanine as substrates . Genomic DNA blot-hybridization analysis revealed 6 to 8 genes homologous to cTYDC1 and 4 to 6 genes homologous to cTYDC2 in the tetraploid poppy genome . A premature translation stop codon was found in the gTYDC4 clone suggesting that it may not encode a functional protein . RNA blot-hybridization with probes specific to the gTYDC1- or cTYDC2-like subsets showed that members of the TYDC gene family are differentially expressed in various plant tissues.

Mol Gen Genet, 1994 Oct 28, 245(2), 255 - 9
The nucleoid-associated DNA-binding protein H-NS is required for the efficient adaptation of Escherichia coli K-12 to a cold environment; Dersch P et al.; The hns gene is a member of the cold-shock regulon, indicating that the nucleoid-associated, DNA-binding protein H-NS plays an important role in the adaptation of Escherichia coli to low temperatures . We show here that the ability to cope efficiently with a cold environment (12 degrees C and 25 degrees C) is strongly impaired in E . coli strains carrying hns mutations . Growth inhibition is much more pronounced in strains carrying the hns-206 allele (an ampicillin resistance cassette inserted after codon 37) than in those carrying the hns-205 mutation (a Tn10 insertion located in codon 93) . A protein fragment (H-NS*) is synthesized in strains carrying the hns-205::Tn10 mutation, suggesting that this truncated polypeptide is partially functional in the cold adaptation process . Analysis of the growth properties of strains harbouring four different low-copy-number plasmid-encoded hns' genes that result in the production of C-terminally truncated H-NS proteins supports this proposal . H-NS* proteins composed of 133, 117 or 94 amino-terminal amino acids partially complemented the severe cold-sensitive growth phenotype of the hns-206 mutant . In contrast, synthesis of a truncated H-NS protein with only 75 amino-terminal amino acids was insufficient to restore growth at low temperature.

Mol Gen Genet, 1994 Oct 28, 245(2), 218 - 23
Effect of mutations in the -10 region of the phoE promoter in Escherichia coli on regulation of gene expression; Scholten M et al.; The phoE promoter region in Escherichia coli contains a -10 region, typical of sigma 70-dependent promoters and, instead of a normal -35 region, a so-called pho box, to which the transcriptional activator phospho-PhoB binds under low phosphate conditions . A second pho box is present upstream of the first one and is required for full expression of phoE during phosphate starvation . To determine whether the lack of expression under high phosphate conditions is due solely to the absence of a genuine -35 box, the -10 region was further optimized towards the consensus -10 sequence and promoter activity was measured using alkaline phosphatase as a reporter . The mutations resulted in a drastic increment in the basal level of expression under high phosphate conditions, indicating that the deviations from consensus in the -10 region also play a role in determining the poor expression of the wild-type promoter under these conditions . The expression under high phosphate conditions was partly dependent on the presence of the phoB gene, showing that a small amount of active PhoB must be present under these circumstances . During phosphate starvation, the activity of the mutant promoters was further induced . The upstream pho box was not required for full expression from the mutant promoters under these conditions . Apparently, the wild-type phoE promoter is carefully balanced by deviations from the optimal Pribnow box sequence that reduce expression under high phosphate conditions and by the presence of several copies of the pho box, which enhance expression under phosphate starvation.

Mol Gen Genet, 1994 Oct 28, 245(2), 203 - 11
Identification and characterization of two functional domains of the hemolysin translocator protein HlyD; Schulein R et al.; Secretion of Escherichia coli hemolysin is mediated by a sec-independent pathway which requires the products of at least three genes, hlyB, hlyD and tolC . Two regions of HlyD were studied . The first region (region A), consisting of the 33-amino acid, C-terminal part of the HlyD protein, is predicted to form a potential helix-loop-helix structure . This sequence is conserved among HlyD analogues of similar transport systems of other bacterial species . Using site-directed mutagenesis, we showed that the amino acids Leu475, Glu477 and Arg478 of this region are essential for HlyD function . The last amino acid of HlyD, Arg478, is possibly involved in the release of the HlyA protein, since cells bearing a hlyD gene mutant at this position produce similar amounts of HlyA to the wild-type strain, but most of the protein remains cell-associated . Competition experiments between wild-type and mutant HlyD proteins indicate that region A interacts directly with a component of the secretion apparatus . The second region of HlyD (region B), located between amino acids Leu127 and Leu170, is highly homologous to the otherwise unrelated outer membrane protein TolC . Deletion of this region abolishes secretion of hemolysin . This sequence of HlyD also seems to interact with a component of the hemolysin secretion machinery since a hybrid HlyD protein carrying the corresponding TolC sequence, although inactive in the transport of HlyA, is able to displace wild-type HlyD from the secretion apparatus.

Mol Gen Genet, 1994 Oct 28, 245(2), 195 - 202
Isolation of an osmotic stress- and abscisic acid-induced gene encoding an acidic endochitinase from Lycopersicon chilense; Chen RD et al.; We have identified one osmotic stress- and abscisic acid-responsive member of the endochitinase (EC 3.2.1.14) gene family from leaves of drought-stressed Lycopersicon chilense plants, a natural inhabitant of extremely arid regions in South America . The 966-bp full-length cDNA (designated pcht28) encodes an acidic chitinase precursor with an amino-terminal signal peptide . The mature protein is predicted to have 229 amino acid residues with a relative molecular mass of 24,943 and pI value of 6.2 . Sequence analysis revealed that pcht28 has a high degree of homology with class II chitinases (EC 3.2.1.14) from tomato and tobacco . Expression of the pcht28 protein in Escherichia coli verified that it is indeed a chitinase . Northern blot analysis indicated that this gene has evolved a different pattern of expression from that of other family members reported thus far . It is highly induced by both osmotic stress and the plant hormone abscisic acid . Southern blot analysis of genomic DNA suggested that the pcht28-related genes may form a small multigene family in this species . The efficiency of induction of the gene by drought stress, in leaves and stems, is significantly higher in L . chilense than in the cultivated tomato . It is speculated that, besides its general defensive function, the pcht28-encoded chitinase may play a particular role in plant development or in protecting plants from pathogen attack during water stress.

J Med Chem, 1994 Oct 28, 37(22), 3739 - 48
Novel acyclic nucleotides and nucleoside 5'-triphosphates imitating 2',3'-dideoxy-2',3'-didehydronucleotides: synthesis and biological properties; Shirokova EA et al.; A series of pyrophosphoryl (Z)-(phosphonomethoxy)but-2-enyl derivatives of pyrimidines and purines 9a-d and the corresponding phosphonates 10a-d were synthesized . The prepared compounds contain the phosphonate group as an alpha-phosphate mimic as well as an acyclic residue emulating the sugar moiety in 2',3'-dideoxy-2',3'-didehydronucleoside 5'-triphosphates known as highly potent chain terminators of DNA polymerases . Phosphonates 10a-d were obtained by alternative alkylations of the nucleic bases followed by condensation with ethyl {{(p-tolylsulfonyl)oxy}methyl}phosphonate . Pyrophosphorylation of 10a-d afforded phosphonate diphosphates 9a-d . Their substrate properties were evaluated in cell-free systems containing various DNA polymerases including viral reverse transcriptases . Compounds 9a-d manifested good terminating substrate properties toward HIV-1 and AMV reverse transcriptases . They exhibited high selectivity and were not recognized by human DNA polymerases alpha and epsilon, DNA polymerase beta from rat liver, Escherichia coli DNA polymerase I, and HSV-1 and CMV DNA polymerases . Phosphonates 10b-d displayed no activity in HIV-1-infected MT-4 cells cultures; 10a was moderately effective (ED50 = 9 microM).

Nature, 1994 Oct 27, 371(6500), 796 - 9
Krox-20 controls myelination in the peripheral nervous system; Topilko P et al.; The molecular mechanisms controlling the process of myelination by Schwann cells remain elusive, despite recent progress in the identification and characterization of genes encoding myelin components (reviewed in ref . 1) . We have created a null allele in the mouse Krox-20 gene, which encodes a zinc-finger transcription factor, by in-frame insertion of the Escherichia coli lacZ gene, and have shown that hindbrain segmentation is affected in Krox-20-/- embryos . We demonstrate here that Krox-20 is also activated in Schwann cells before the onset of myelination and that its disruption blocks Schwann cells at an early stage in their differentiation, thus preventing myelination in the peripheral nervous system . In Krox-20-/- mice, Schwann cells wrap their cytoplasmic processes only one and a half turns around the axon, and although they express the early myelin marker, myelin-associated glycoprotein, late myelin gene products are absent, including those for protein zero and myelin basic protein . Therefore Krox-20 is likely to control a set of genes required for completion of myelination in the peripheral nervous system.

Nature, 1994 Oct 27, 371(6500), 762 - 7
Insulin-dependent stimulation of protein synthesis by phosphorylation of a regulator of 5'-cap function; Pause A et al.; The cloning is described of two related human complementary DNAs encoding polypeptides that interact specifically with the translation initiation factor eIF-4E, which binds to the messenger RNA 5'-cap structure . Interaction of these proteins with eIF-4E inhibits translation but treatment of cells with insulin causes one of them to become hyperphosphorylated and dissociate from eIF-4E, thereby relieving the translational inhibition . The action of this new regulator of protein synthesis is therefore modulated by insulin, which acts to stimulate the overall rate of translation and promote cell growth.

Nucleic Acids Res, 1994 Oct 25, 22(21), 4520 - 6
The cooperative binding of chromosomal protein HMG-14 to nucleosome cores is reduced by single point mutations in the nucleosomal binding domain; Postnikov YV et al.; Mutants of human chromosomal protein HMG-14 were generated by site directed mutagenesis and used to study functional domains in this protein . A replacement of serine by cysteine at position 7 did not affect the binding of the protein to nucleosome cores . The sulfhydryl group in the nucleosome-bound protein is accessible to modifying agents suggesting that position 7 in the protein is not in close contact with either the DNA or the histones in the core particles . Under cooperative binding conditions, replacements of alanine by proline at position 21, or of lysine by cysteine at position 26, decreased the affinity of the protein for nucleosome cores 6.7- and 3-fold respectively . In contrast, the non-cooperative mode of binding was only minimally affected . A replacement of glutamic acid by glutamine at position 76 caused only minor changes in the binding of the protein to the cores . The results indicate that single point mutations, which change either the conformation or change in the nucleosomal binding domain of the protein, significantly reduce the ability of the HMG-14 protein to bind to nucleosome cores . We suggest that in chromatin the protein binds to nucleosomes in a cooperative manner and that upon binding to nucleosomes the protein acquires a distinct conformation.

Nucleic Acids Res, 1994 Oct 25, 22(21), 4482 - 8
DsaV methyltransferase and its isoschizomers contain a conserved segment that is similar to the segment in Hhai methyltransferase that is in contact with DNA bases; Gopal J et al.; The methyltransferase (MTase) in the DsaV restriction--modification system methylates within 5'-CCNGG sequences . We have cloned the gene for this MTase and determined its sequence . The predicted sequence of the MTase protein contains sequence motifs conserved among all cytosine-5 MTases and is most similar to other MTases that methylate CCNGG sequences, namely M.ScrFI and M.SsoII . All three MTases methylate the internal cytosine within their recognition sequence . The 'variable' region within the three enzymes that methylate CCNGG can be aligned with the sequences of two enzymes that methylate CCWGG sequences . Remarkably, two segments within this region contain significant similarity with the region of M.HhaI that is known to contact DNA bases . These alignments suggest that many cytosine-5 MTases are likely to interact with DNA using a similar structural framework.

Nucleic Acids Res, 1994 Oct 25, 22(21), 4454 - 61
Efficient concerted integration of retrovirus-like DNA in vitro by avian myeloblastosis virus integrase; Vora AC et al.; We report the efficient concerted integration of a linear virus-like DNA donor into a 2.8 kbp circular DNA target by integrase (IN) purified from avian myeloblastosis virus . The donor was 528 bp, contained recessed 3' OH ends, was 5' end labeled, and had a unique restriction site not found in the target . Analysis of concerted (full-site) and half-site integration events was accomplished by restriction enzyme analysis and agarose gel electrophoresis . The donor also contained the SupF gene that was used for genetic selection of individual full-site recombinants to determine the host duplication size . Two different pathways, involving either one donor or two donor molecules, were used to produce full-site recombinants . About 90% of the full-site recombinants were the result of using two donor molecules per target . These results imply that juxtapositioning an end from each of two donors by IN was more efficient than the juxtapositioning of two ends of a single donor for the full-site reaction . The formation of preintegration complexes containing integrase and donor on ice prior to the addition of target enhanced the full-site reaction . After a 30 min reaction at 37 degrees C, approximately 20-25% of all donor/target recombinants were the result of concerted integration events . The efficient production of full-site recombinants required Mg2+; Mn2+ was only efficient for the production of half-site recombinants . We suggest that these preintegration complexes can be used to investigate the relationships between the 3' OH trimming and strand transfer reactions.

Nucleic Acids Res, 1994 Oct 25, 22(21), 4395 - 404
Replacement of invariant bZip residues within the basic region of the yeast transcriptional activator GCN4 can change its DNA binding specificity; Suckow M et al.; Two residues are invariant in all bZip basic regions: asparagine -18 and arginine -10 (we define the first leucine of the leucine zipper of GCN4 as +1) . X-ray structures of two specific GCN4-DNA complexes (Ellenberger et al., Cell, 71, 1223-1237, 1992; Konig & Richmond, J . Mol . Biol., 233, 139-154, 1993) demonstrate the involvement of both residues in specific base pair recognition . We replaced either asparagine -18 or arginine -10 with all other amino acids and tested the DNA binding properties of the resulting mutant peptides by gel mobility shift assays . Peptides with histidine -18 or tyrosine -10 bind with changed specificities to variants of the ATF/CREB site 5'A4T3G2A1C0*G0'T1'C2'A3'T4'3' with symmetric exchanges in positions 2/2' or 0/0', respectively . The double mutant with histidine -18 and tyrosine -10 combines the features of the parental single mutants and binds specifically to the respective double exchange target . Furthermore, the tyrosine -10 mutant clearly prefers the palindrome 5'ATGATATCAT3' over the corresponding pseudo-palindrome 5'ATGATTCA-T3', whereas the lysine -10 mutant binds better to the pseudo-palindromic AP1 site 5'ATGACTCAT3' than to the palindromic ATF/CREB site . Thus, although invariant within natural bZip proteins, asparagine -18 or arginine -10 can be functionally replaced by other amino acids, and their replacement can lead to new DNA binding specificities.

Nucleic Acids Res, 1994 Oct 25, 22(21), 4375 - 80
Interactions between the cyclic AMP receptor protein and the alpha subunit of RNA polymerase at the Escherichia coli galactose operon P1 promoter; Attey A et al.; DNAase I footprinting has been used to study open complexes between Escherichia coli RNA polymerase and the galactose operon P1 promoter, both in the absence and the presence of CRP (the cyclic AMP receptor protein, a transcription activator) . From the effects of deletion of the C-terminal part of the RNA polymerase alpha subunit, we deduce that alpha binds at the upstream end of both the binary RNA polymerase-galP1 and ternary RNA polymerase-CRP-galP1 complexes . Disruption of the alpha-upstream contact suppresses open complex formation at galP1 at lower temperatures . In ternary RNA polymerase-CRP-galP1 complexes, alpha appears to make direct contact with Activating Region 1 in CRP . DNAase I footprinting has been used to detect and quantify interactions between purified alpha and CRP bound at galP1.

Nucleic Acids Res, 1994 Oct 25, 22(21), 4364 - 74
Structural features of adenovirus 2 virus-associated RNA required for binding to the protein kinase DAI; Clarke PA et al.; The double-stranded RNA activated protein kinase DAI contains an RNA binding domain consisting of two copies of a double-stranded RNA binding motif . We have investigated the role of RNA structure in the interaction between DAI and the structured single-stranded RNA, adenovirus VA RNAI, which inhibits DAI activation . Mutations in the apical stem, terminal stem, and central domain of the RNA were tested to assess the contribution of these elements to DAI binding in vitro . The data demonstrate that over half a turn of intact apical stem is required for the interaction and that there is a correlation between the binding of apical stem mutants and their ability to function both in vivo and in vitro . There was also evidence of preference for GC-rich sequence in the proximal region of the apical stem . In the central domain the correlation between binding and function of mutant RNAs was poor, suggesting that at least some of this region plays no direct role in binding to DAI, despite its functional importance . Exceptionally, central domain mutations that encroached on the phylogenetically conserved stem 4 of VA RNA disrupted binding, and complementary mutations in this sequence partially restored binding . Measurement of the binding of wild-type VA RNAI to DAI and p20, a truncated form of the protein containing the RNA binding domains alone, under various ionic conditions imply that the major interactions are electrostatic and occur via the protein's RNA binding domain . However, differences between full-length DAI and p20 in their binding to mutants in the conserved stem suggest that regions outside the RNA binding domain also participate in the binding . The additional interactions are likely to be non-ionic, and may be important for preventing DAI activation during virus infection.

Nucleic Acids Res, 1994 Oct 25, 22(21), 4361 - 3
Two-base DNA hairpin-loop structures in vivo; Davison A et al.; In vitro studies have revealed that DNA hairpin-loops usually contain four unpaired bases . However, a small subset of sequences can form two-base loops . We have previously described an in vivo assay that is sensitive to tight loop formation and have set out to test whether DNA sequences known to form two-base loops in vitro also form tight loops in vivo . It is shown that the sequences 5'dCNNG and 5'dTNNA behave as predicted if they favour two-base loop formation in vivo, a result that is consistent with previously described in vitro studies . The ability of specific DNA sequences to form tight loops in vivo has implications for their potential to form transient structures involved in gene regulation, recombination and mutagenesis.

Proc Natl Acad Sci U S A, 1994 Oct 25, 91(22), 10747 - 51
DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution; Stemmer WP; Computer simulations of the evolution of linear sequences have demonstrated the importance of recombination of blocks of sequence rather than point mutagenesis alone . Repeated cycles of point mutagenesis, recombination, and selection should allow in vitro molecular evolution of complex sequences, such as proteins . A method for the reassembly of genes from their random DNA fragments, resulting in in vitro recombination is reported . A 1-kb gene, after DNase I digestion and purification of 10- to 50-bp random fragments, was reassembled to its original size and function . Similarly, a 2.7-kb plasmid could be efficiently reassembled . Complete recombination was obtained between two markers separated by 75 bp; each marker was located on a separate gene . Oligonucleotides with 3' and 5' ends that are homologous to the gene can be added to the fragment mixture and incorporated into the reassembled gene . Thus, mixtures of synthetic oligonucleotides and PCR fragments can be mixed into a gene at defined positions based on homology . As an example, a library of chimeras of the human and murine genes for interleukin 1 beta has been prepared . Shuffling can also be used for the in vitro equivalent of some standard genetic manipulations, such as a backcross with parental DNA . The advantages of recombination over existing mutagenesis methods are likely to increase with the numbers of cycles of molecular evolution.

Proc Natl Acad Sci U S A, 1994 Oct 25, 91(22), 10571 - 5
KorA protein of promiscuous plasmid RK2 controls a transcriptional switch between divergent operons for plasmid replication and conjugative transfer; Jagura-Burdzy G et al.; The trfA and trb operons, encoding genes essential for replication and conjugative transfer of broad host range plasmid RK2, are transcribed divergently . Deletion analysis presented here indicates that trfAp and trbAp are arranged as face to face promoters . The presence of the korA gene, whose product is known to repress seven operons on RK2, including the trfA operon, is shown here to stimulate trbAp . The effect of korA on trbAp is mimicked by the trfAp-1 promoter down mutation, suggesting that a reduction in the activity of trfAp is required for derepression of trbAp activity . The trfAp-1 mutation reduces RNA polymerase binding and open complex formation at trfAp but does not stimulate melting at trbAp in vitro . Therefore, the inhibition of trbAp is most probably due to forward transcription initiated at trfAp . The simultaneous inhibition/stimulation by KorA is seen even in the presence of the other repressors KorB and TrbA, which act at this region, thus providing a dominant mode of coordinating plasmid replication and transfer . This may be one of the keys to understanding how the maintenance and spread of promiscuous plasmids are balanced in different environments.

Proc Natl Acad Sci U S A, 1994 Oct 25, 91(22), 10521 - 5
Topological analysis of the human beta 2-adrenergic receptor expressed in Escherichia coli; Lacatena RM et al.; We have investigated the topology of the human beta 2-adrenergic receptor expressed in Escherichia coli, using the genetic method described by Beckwith and coworkers . We found that fusions with alkaline phosphatase beyond a certain point on the human beta 2-adrenergic receptor sequence were assembled into the bacterial membrane with the same topology as the human beta 2-adrenergic receptor in the mammalian membrane . The pattern that might have been expected on the basis of the topology of the human beta 2-adrenergic receptor in mammalian membranes was not reflected in the levels of alkaline phosphatase activity of the fusions occurring between the N-terminal region and positions close to the second external domain . Our data suggest that the correct positioning of the N terminus of the receptor depends on the presence of its C-terminal portions.

Proc Natl Acad Sci U S A, 1994 Oct 25, 91(22), 10389 - 93
Functional transfer RNAs with modifications in the 3'-CCA end: differential effects on aminoacylation and polypeptide synthesis; Liu M et al.; The trinucleotide CCA sequence is present at the 3' terminus of all mature tRNAs . Despite this high degree of conservation, we have been able to prepare in vitro transcripts of Escherichia coli tRNA(Val) with altered 3' termini that are readily aminoacylated and can function in polypeptide synthesis . Replacement of the 3'-terminal adenosine with either cytidine or uridine yields a tRNA(Val) variant that retains almost full aminoacylation activity, having specificity constants (Vmax/Km) 40-50% that of wild-type tRNA(Val) . The tRNA(Val) variant with a 3'-terminal guanosine remains fully chargeable but is a poor substrate for valyl-tRNA synthetase, largely as the result of a decrease in the catalytic constant . End-group analysis revealed the absence of adenosine at the 3' end of the tRNA(Val) mutants and identified the nucleotide expected from the sequence of the DNA template as the predominant 3'-terminal residue; Val-cytidine was isolated from the aminoacylated C76 mutant . Val-tRNA(Val) with 3'-CCG is active in poly(U,G)-directed (Val, Phe) copolypeptide synthesis, whereas the tRNA(Val) mutants terminating in 3'-CCC and 3'-CCU, which are readily aminoacylated, are inactive . The differential effects of nucleotide substitution on aminoacylation and polypeptide synthesis suggest that the universally conserved 3'-CCA end of tRNAs is monitored at two or more steps in protein synthesis that have different nucleotide recognition specificities.

Proc Natl Acad Sci U S A, 1994 Oct 25, 91(22), 10345 - 9
The ATP hydrolysis-dependent reaction cycle of the Escherichia coli Hsp70 system DnaK, DnaJ, and GrpE; Szabo A et al.; Molecular chaperones of the Hsp70 class bind unfolded polypeptide chains and are thought to be involved in the cellular folding pathway of many proteins . DnaK, the Hsp70 protein of Escherichia coli, is regulated by the chaperone protein DnaJ and the cofactor GrpE . To gain a biologically relevant understanding of the mechanism of Hsp70 action, we have analyzed a model reaction in which DnaK, DnaJ, and GrpE mediate the folding of denatured firefly luciferase . The binding and release of substrate protein for folding involves the following ATP hydrolysis-dependent cycle: (i) unfolded luciferase binds initially to DnaJ; (ii) upon interaction with luciferase-DnaJ, DnaK hydrolyzes its bound ATP, resulting in the formation of a stable luciferase-DnaK-DnaJ complex; (iii) GrpE releases ADP from DnaK; and (iv) ATP binding to DnaK triggers the release of substrate protein, thus completing the reaction cycle . A single cycle of binding and release leads to folding of only a fraction of luciferase molecules . Several rounds of ATP-dependent interaction with DnaK and DnaJ are required for fully efficient folding.

Proc Natl Acad Sci U S A, 1994 Oct 25, 91(22), 10280 - 4
A distinct segment of the sigma 32 polypeptide is involved in DnaK-mediated negative control of the heat shock response in Escherichia coli; Nagai H et al.; Induction of heat shock proteins in Escherichia coli is caused by a transient increase in the cellular level of sigma 32 (the rpoH gene product), a protein required for transcription of heat shock genes . Both increased synthesis and stabilization of sigma 32 contribute to the increase in sigma 32 . We previously showed that heat-induced translation of sigma 32-beta-galactosidase fusion protein encoded by an rpoH-lacZ gene fusion was mediated by an mRNA secondary structure formed between two 5'-proximal segments (A and B) of rpoH coding sequence spanning some 200 nt . We now report that a portion of the sigma 32 polypeptide that corresponds to further downstream (designated region C) is involved in the DnaK-mediated negative control resulting in the shutoff of heat-induced synthesis and degradation of fusion protein . Gene fusions carrying the 5' half (433 nt) or more of the rpoH coding sequence exhibited normal shutoff of synthesis, and the fusion proteins produced were very unstable, like authentic sigma 32; both the shutoff of synthesis and the instability of protein were markedly affected by the dnaK and dnaJ mutations . In contrast, gene fusions carrying < or = 364 nt (lacking region C) and a fusion carrying most of the rpoH sequence but with a frameshift mutation specifically affecting region C exhibited little or no shutoff and produced stable proteins . These results indicate that a distinct segment of sigma 32 plays a critical role in the negative feedback control of sigma 32 . The control may be exerted during or after completion of sigma 32 synthesis mediated by interaction between nascent or mature sigma 32 and DnaK/DnaJ proteins.

Biochemistry, 1994 Oct 25, 33(42), 12656 - 64
Affinity probing of flavin binding sites . 2 . Identification of a reactive cysteine in the flavin domain of Escherichia coli DNA photolyase; Raibekas AA et al.; 8-(Methylsulfonyl)FAD reacts with a single cysteine residue (Cys293) in the flavin domain of Escherichia coli DNA photolyase to form an 8-(cysteinyl)FAD derivative covalently bound to the protein . About 80% protection against covalent attachment with 8-(methylsulfonyl)FAD was observed in the presence of an equimolar amount of FAD . Flavinylated photolyase retains the ability to repair pyrimidine dimers (15% of native activity) and to bind its antenna chromophore, 5,10-methenyltetrahydrofolate . Comparison of the properties of flavinylated enzyme with photolyase containing noncovalently bound 8-(methylthio)-FAD indicate that a perturbation is necessary to accommodate covalent bond formation . 8-(Methylthio)-FAD-reconstituted enzyme exhibits 95% of native activity . The aerobic stability of fully reduced and radical forms of 8-(methylthio)FAD enzyme is similar to that of native enzyme, whereas a radical form is not detected with flavinylated enzyme and the fully reduced enzyme is more easily oxidized by oxygen . The flavin in 8-(methylthio)FAD enzyme or flavinylated photolyase is shielded from solvent . However, the flavin environment in flavinylated enzyme is less hydrophobic as judged by spectral comparison with model 8-(alkylthio)flavins in various solvents . Enzyme containing noncovalently bound 8-(methylsulfonyl)-FAD was prepared by reconstitution with the fully reduced flavin which does not undergo covalent attachment . Covalent attachment was observed after reoxidation but probably involved dissociation and rebinding of oxidized 8-(methylsulfonyl)FAD . The results show that 8-(cysteinyl)FAD in flavinylated photolyase is at or near the normal flavin binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Proc Natl Acad Sci U S A, 1994 Oct 25, 91(22), 10675 - 9
Structural and functional alterations of a colicin-resistant mutant of OmpF porin from Escherichia coli; Jeanteur D et al.; A strain of Escherichia coli, selected on the basis of its resistance to colicin N, reveals distinct structural and functional alterations in unspecific OmpF porin . A single mutation {Gly-119-->Asp (G119D)} was identified in the internal loop L3 that contributes critically to the formation of the construction inside the lumen of the pore . X-ray structure analysis to a resolution of 3.0 A reveals a locally altered peptide backbone, with the side chain of residue Asp-119 protruding into the channel, causing the area of the constriction (7 x 11 A in the wild type) to be subdivided into two intercommunicating subcompartments of 3-4 A in diameter . The functional consequences of this structural modification consist of a reduction of the channel conductance by about one-third, of altered ion selectivity and voltage gating, and of a decrease of permeation rates of various sugars by factors of 2-12 . The structural modification of the mutant protein affects neither the beta-barrel structure nor those regions of the molecule that are exposed at the cell surface . Considering the colicin resistance of the mutant, it is inferred that in vivo, colicin N traverses the outer membrane through the porin channel or that the dynamics of the exposed loops are affected in the mutant such that these may impede the binding of the toxin.

Proc Natl Acad Sci U S A, 1994 Oct 25, 91(22), 10660 - 4
A transcribing RNA polymerase molecule survives DNA replication without aborting its growing RNA chain; Liu B et al.; We have demonstrated elsewhere that a precisely placed, stalled Escherichia coli RNA polymerase ternary transcription complex (polymerase-RNA-DNA) stays on the DNA template after passage of a DNA replication fork . Moreover, the bypassed complex remains competent to resume elongation of its bound RNA chain . But the simplicity of our experimental system left several important questions unresolved: in particular, might the observation be relevant only to the particular ternary complex that we studied, and can the finding be generalized to a transcribing instead of a stalled RNA polymerase? To address these issues, we have created three additional ternary transcription complexes and examined their fates after passage of a replication fork . In addition, we have examined the fate of moving RNA polymerase molecules during DNA replication . The results suggest that our previous finding applies to all transcription intermediates of the E . coli RNA polymerase.

Proc Natl Acad Sci U S A, 1994 Oct 25, 91(22), 10591 - 5
ard-1: a human gene that reverses the effects of temperature-sensitive and deletion mutations in the Escherichia coli rne gene and encodes an activity producing RNase E-like cleavages; Wang M et al.; The Escherichia coli rne gene affects a variety of bacterial functions, including the activity of RNase E . We report the existence of a human gene (ard-1, for activator of RNA decay) that complements temperature-sensitive and deletion mutations of rne in E . coli, allowing growth of rne-defective cells, correcting abnormal cell shape, activating chemical decay of bulk mRNA, and producing site-specific cleavages characteristic of RNase E in vivo and in vitro . ard-1 encodes a highly basic 13.3-kDa proline-rich peptide that has features in common with Rne and also with eukaryotic proteins implicated in RNA binding and macromolecular transport.

Biochemistry, 1994 Oct 25, 33(42), 12708 - 14
Escherichia coli proline tRNA synthetase is sensitive to changes in the core region of tRNA(Pro); Liu H et al.; To investigate the relationship between tRNA conformation and specific recognition by aminoacyl-tRNA synthetases, a full-length tRNA molecule was assembled by annealing together two oligonucleotides representing fragments of Escherichia coli tRNA(Pro) . A shorter chemically synthesized 5'-fragment (7-18 nucleotides) was combined with an in vitro transcribed 3'-fragment (59 nucleotides) . Despite a break in the phosphodiester backbone between nucleotides U17a and G18, this tRNA molecule was an efficient substrate for class II Escherichia coli proline tRNA synthetase . While the deletion of three D-loop nucleotides (U17a, U17, and C16) was tolerated, removal of G15 and A14 significantly reduced aminoacylation efficiency . Hybrid DNA-RNA "annealed" substrates were also prepared and assayed for aminoacylation . Native gel electrophoresis was used to compare the global folding of the various substrates tested . The results of these studies suggest that proline tRNA synthetase is sensitive to changes in the core region of tRNA(Pro) through which information required for efficient aminoacylation may be transmitted . In particular, nucleotides in the D-loop and backbone functional groups in the D-stem appear to be critical for maintaining a tRNA structure that is optimal for recognition by proline tRNA synthetase in vitro.

FEBS Lett, 1994 Oct 24, 353(3), 312 - 4
Inactivation of Acacia confusa trypsin inhibitor by site-specific mutagenesis; Hung CH et al.; Native Acacia confusa trypsin inhibitor (ACTI) contains two disulphide bonds; one is an intrachain disulphide bond (Cys40-Cys86), located in the A-chain, while the other is an interchain disulphide bond (Cys133-Cys141) connecting the A- and B-chain; the inhibitor consists of 175 amino acid residues, 136 residues in the A-chain and 39 residues in the B-chain . The putative reactive site of ACTI is located at Lys64, while for all other Kunitz family trypsin inhibitors it is at Arg64 . When the Lys64 residue of ACTI was converted into Ile or Arg by site-specific mutagenesis, the K64I mutant completely lost its inhibitory activity but the K64R mutant retained most of its inhibitory activity . The C133G mutant lost its inhibitory activity while the C40G mutant did not . This suggests that the interchain disulphide bond (Cys133-Cys141) linking two beta-strands of the six-strand beta-barrel is essential for ACTI inhibitory activity, while the intrachain disulphide bond (Cys40-Cys86) connecting the two loops is non-essential.

FEBS Lett, 1994 Oct 24, 353(3), 305 - 8
cDNA cloning of a cytosolic protein tyrosine phosphatase (RKPTP) from rat kidney; Moriyama T et al.; A rat cDNA encoding a non-receptor type phosphotyrosine phosphatase (PTPase; EC 3.1.3.48) was identified . The 1608 bp cDNA contains a single open reading frame that predicts a 382 amino acid protein with M(r) 44,438 . The predicted protein has no apparent signal or transmembrane sequences, suggesting that it is a cytosolic protein . The C-terminal region has a PTPase catalytic domain that has 40-50% nucleic acid homology to other known PTPases . The N-terminal region has little amino acid sequence homology to any other known sequences . The recombinant protein of the cloned cDNA expressed in Escherichia coli was shown to possess PTPase activity using myelin basic protein, tyrosine phosphorylated by p43v-abl tyrosine kinase, as a substrate.

FEBS Lett, 1994 Oct 24, 353(3), 269 - 72
Expression of GATA-binding transcription factors in rat hepatocytes; Matsuda K et al.; Recently, we demonstrated that a DNA-binding protein(s) is involved in transcriptional repression of the rat serine dehydratase gene in fetal liver . Here, we report that a GAT(A/T) motif is the target sequence for the DNA-binding protein . By screening a fetal liver cDNA library, we isolated a rat homolog of GATA-1 . Rat GATA-1 expressed as a GST-fusion protein in E . coli bound to the GAT(A/T) motif in the serine dehydratase gene . Northern analysis show that GATA-1 and GATA-4 mRNAs are expressed in fetal hepatocytes.

J Theor Biol, 1994 Oct 21, 170(4), 415 - 21
Computer aided identification of a potential 5'-3' exonuclease gene encoded by Escherichia coli; Sayers JR; The predicted amino acid sequence of a previously overlooked open reading frame (ORF) from E . coli has been subjected to sequence analysis . The ORF, sequenced independently by two research groups, appears to encode a protein of 251 amino acids residues (25 kDa) . The protein sequence shares a high degree of similarity with the amino acid termini of E . coli DNA polymerase I and other bacterial DNA polymerases (and phage-encoded enzymes) known to possess 5'-3' exonuclease activity . The newly identified E . coli gene, designated exo, is positioned downstream of signals characteristic of an efficient translation initiation sequence . Codon bias analysis indicates that the ORF has good protein coding potential . These observations indicate that the gene is likely to be expressed . The gene is 60% identical with the 5'-region of the E . coli DNA PolI gene (polA) over a 260 base-pair region . These two genes may have arisen by duplication of an ancestral gene or polA may have arisen by a recombination event involving exo . A comparison of the predicted secondary structures of 5'-3' exonucleases revealed the presence of several conserved regions of secondary structure, including a potential helix-turn-helix motif.

Gene, 1994 Oct 21, 148(2), 321 - 5
Gene cloning, overproduction and purification of a functionally active cytoplasmic fatty acid-binding protein (Sj-FABPC) from the human blood fluke Schistosoma japonicum; Becker MM et al.; We report the gene cloning, molecular characterisation and purification of a 14.7-kDa functionally active recombinant (re) cytoplasmic fatty acid-binding protein (Sj-FABPC) from the Chinese strain of the human bloodfluke Schistosoma japonicum (Sj) . As schistosomes are unable to synthesise long chain fatty acids and sterols de novo and must, therefore, take up these lipids from the host, Sj-FABPC is an attractive vaccine and/or drug target . Clone 39 (C39), which contains the entire Sj-FABPC gene, was isolated from a Sj lambda ZAPII cDNA expression library immunoscreened with hyperimmune rabbit serum (HRS) raised against soluble adult Sj proteins . The complete ORF (open reading frame) of Sj-FABPC encodes a protein of 132 amino acids (aa) of 14.7 kDa . The aa sequence of Sj-FABPC exhibits 91% identity to a FABP of S . mansoni (Sm14) and 45% identity to a FABP of Fasciola hepatica (Fh15), putative vaccine candidates for schistosomiasis . Sj-FABPC was subcloned into the QIAexpress vector, pQE-10, and subsequently expressed in Escherichia coli . The re-Sj-FABPC, purified under non-denaturing conditions, was recognized by sera from patients with acute and chronic schistosomiasis japonica . The purified re-Sj-FABPC was also shown to bind to palmitic acid with high affinity . The functional expression of Sj-FABPC will facilitate studies on re-Sj-FABPC to assess its potential as a drug and/or vaccine candidate.

Gene, 1994 Oct 21, 148(2), 299 - 304
Cloning by genomic PCR and production of peanut agglutinin in Escherichia coli; Sharma V et al.; Using the polymerase chain reaction, the coding sequence for peanut agglutinin (PNA) was cloned and expressed in Escherichia coli . Amplified PNA is identical to previously reported cDNA, suggesting the absence of any introns in PNA gene . Recombinant (re-) PNA forms inclusion bodies in E . coli . Production of PNA was confirmed by probing Western blots with polyclonal anti-PNA immunoglobulin G . Inclusion bodies were solubilized with 6 M guanidine-HCl and renatured by rapid dilution in the presence of metal ions . The renatured lectin was then purified by affinity chromatography . The re-lectin shows carbohydrate-binding properties similar to the natural PNA . This expression system provides a model for future mutagenesis studies of the carbohydrate-binding site and thus facilitates ongoing efforts to explore the molecular basis for the specificity of lectin-carbohydrate interaction.

Gene, 1994 Oct 21, 148(2), 277 - 84
Isolation of human DNA-unwinding elements as sites of DNA polymerase alpha/primase entry; Pack RA et al.; Human DNA libraries were screened for DNA synthesis activity in vitro using purified DNA polymerase alpha/primase and a viral DNA helicase (simian virus 40 large tumor antigen) . Three clones exhibited a high activity distinguishable from the rest . The DNA synthesis was dependent on negative supertwisting and initiated at a unique region in the human DNA insert . Functional subclone DNA fragments which could be shortened to less than 1 kb are located in the initiation region . Binding with a single-stranded DNA-binding protein and digestion with nuclease P1 demonstrated that these DNAs have a highly single-stranded nature at a certain site in a closed circular plasmid . The minimal functional sequences coincide with the single-stranded region and contain a characteristic dinucleotide repeat sequence . These repeats have an extremely low free energy for DNA strand separation and are defined as DNA-unwinding elements, which are frequently observed at regions flanking replication origins in Escherichia coli and Saccharomyces cerevisiae chromosomes . We suggest that such a repeating sequence would have an important role during initiation of DNA replication and function as a site to recruit replication proteins.

J Mol Biol, 1994 Oct 21, 243(2), 364 - 6
Revision in sequence of CAD aspartate transcarbamylase domain of Drosophila; Davidson JN et al.; The Drosophila CAD gene, also known as rudimentary, encodes a protein that carries out the first three enzymatic steps of de novo pyrimidine biosynthesis . The sequence for this gene, as previously published, appears to contain several errors . The correction of six bases in a 250 bp stretch encoding the aspartate transcarbamylase domain leads to changes of frame in two areas of the predicted amino acid sequence, consisting of lengths of 30 and 15 amino acid residues, respectively . The revised sequence shows significantly improved positional identity with both Syrian hamster and Escherichia coli aspartate transcarbamylases.

J Mol Biol, 1994 Oct 21, 243(2), 227 - 44
Caulobacter flagellar function, but not assembly, requires FliL, a non-polarly localized membrane protein present in all cell types; Jenal U et al.; Caulobacter crescentus has a single polar flagellum, which is assembled in the predivisional cell . Known flagellar genes encode structural and regulatory components that are required for flagellar assembly and function . These genes are organized in several classes which form a transcriptional regulatory hierarchy . A member of the Class II genes, the fliLM operon, encodes homologs of the Escherichia coli flagellar switch protein, FliM, and a protein with a hitherto unknown function, FliL . We report here that flagellar rotation requires the FliL protein . In-frame deletions in the chromosomal copy of the fliL gene result in cells that form a flagellum but are non-motile . The FliL protein was found to be associated with the inner membrane and to be present in all cell types . This is the first report of a Caulobacter crescentus protein that is essential for motility but is not spatially restricted to the region of the flagellar basal body . Although FliL is required for flagellar function, it is not part of the transcriptional hierarchy, supporting the hypothesis that, as is the case for the enterics, the regulatory hierarchy responds to assembly cues rather than directly to the expression of flagellar proteins.

J Mol Biol, 1994 Oct 21, 243(2), 208 - 15
Hexameric rings of Escherichia coli RuvB protein . Cooperative assembly, processivity and ATPase activity; Mitchell AH et al.; The Escherichia coli RuvA and RuvB proteins mediate ATP-dependent branch migration of Holliday junctions during homologous genetic recombination . RuvA is a DNA-binding protein with high affinity for Holliday junctions, to which it directs RuvB (a DNA-dependent ATPase) . Electron microscopic studies have shown that RuvB forms double hexameric rings on duplex DNA . To determine whether the rings are biologically active, the conditions required for their formation and activity have been analysed . The quaternary structure of RuvB appears to be dependent upon the binding of ATP, magnesium ions, and the presence of RuvA . In the presence of Mg2+ and ATP, RuvB forms hexamers; however, in the presence of Mg2+ alone, dodecamers were observed . Both forms of the protein are stable and have been isolated by gel filtration . Performed dodecamers and, to a lesser extent, hexamers assembled in the absence of DNA lack ATPase activity . Maximal ATPase activity was observed when RuvB assembled directly on DNA in the presence of Mg2+ and ATP . Moreover, under these conditions, a direct interaction between RuvB hexamers and tetramers of RuvA was observed.

J Mol Biol, 1994 Oct 21, 243(2), 190 - 8
Topological scanning of the P1 plasmid partition site; Hayes F et al.; The parS site of the P1 plasmid promotes active partition of P1 to daughter cells when the P1 ParA and ParB proteins are provided . The structure of parS was modified by substituting portions of the sequence with synthetic oligonucleotides and testing partition activity of the resulting mutants in an in vivo assay . The boundaries of the site were defined . They enclose a 74 bp region with a central integration host factor (IHF) binding region flanked by two arms containing heptamer and hexamer ParB binding motifs . The IHF binding region was shown to be important for partition activity but could be replaced by sequences containing A tracts that induce static bends in the DNA . The properties of sites with spacer sequences of different lengths inserted at one of five different locations led to the following conclusions . (1) The spacing between the heptamer and hexamer ParB binding motifs in both arms is critical for function . (2) Optimum partition activity requires that the parS site arms are bent toward each other with specific faces of the two helices facing each other . (3) Both arms show torsional rigidity in the active complex . (4) The left arm is laterally inflexible and activity is lost when it is extended unless the right arm is similarly extended . (5) The right arm is laterally flexible so that, when it is extended by an integral number of turns of the helix, it can still align properly with a left arm of wild-type length . The results suggest that right-arm flexibility is promoted by an A + T-rich region that is essential for IHF binding and lies adjacent to the IHF binding consensus motif . Inherent flexibility of this A + T-rich region also appears to account for the residual activity of parS sites in which the IHF binding consensus has been destroyed by multiple point mutations . The results are consistent with a proposed structure in which specific alignment of the parS site arms by an IHF-promoted bend allows them to be linked by bifunctional ParB protein binding . We suggest that such a structure might be involved in the specific pairing and unpairing of daughter plasmids during partition by an isomerization reaction.

J Biol Chem, 1994 Oct 21, 269(42), 26584 - 90
Guanosine tetraphosphate inhibition of fatty acid and phospholipid synthesis in Escherichia coli is relieved by overexpression of glycerol-3-phosphate acyltransferase (plsB); Heath RJ et al.; The accumulation of the alarmone guanosine-3',5'-bispyrophosphate (ppGpp) in response to amino acid starvation or energy source depletion mediates the coordinate inhibition of macromolecular and membrane phospholipid biosynthesis in Escherichia coli . Accumulation of ppGpp triggered by the induced expression of either the relA gene or an unregulated, truncated relA gene that encodes a constitutively active ppGpp synthetase I, inhibited both de novo fatty acid and phospholipid biosynthesis and the incorporation of exogenous fatty acids into phospholipid . ppGpp inhibition of fatty acid and phospholipid synthesis was associated with an accumulation of long-chain acyl-ACP, the end products of fatty acid biosynthesis, and substrates for the sn-glycerol-3-phosphate acyltransferase (the plsB gene product) . Overexpression of the plsB gene product relieved the inhibition of fatty acid and phospholipid synthesis, prevented the accumulation of long-chain acyl-ACPs, and allowed an increase in cell size following elevation of intracellular ppGpp . However, stable RNA accumulation and cell division were still blocked by ppGpp accumulation . These data show that the sn-glycerol-3-phosphate acyltransferase mediates the ppGpp-dependent regulation of fatty acid and phospholipid biosynthesis in E . coli.

J Biol Chem, 1994 Oct 21, 269(42), 26552 - 8
Targeted versus non-targeted DNA helicase activity of the RuvA and RuvB proteins of Escherichia coli; Tsaneva IR et al.; The RuvA and RuvB proteins of Escherichia coli promote the branch migration of Holliday junctions in vitro . To understand the relationship between branch migration and the intrinsic 5'-->3' DNA helicase activity of RuvAB, the requirements and substrate specificity of the helicase reaction have been studied in more detail . We find that RuvAB-mediated DNA unwinding and branch migration reactions show similar requirements for Mg2+ and ATP and are inhibited to a similar extent by ADP and ATP gamma S (adenosine 5'-O-(3-thiotriphosphate)) . The helicase activity, measured by the dissociation of a short fragment from circular single-stranded DNA, requires both RuvA and RuvB and is stimulated by subsaturating concentrations of single-strand binding protein (SSB) . In contrast, saturating concentrations of SSB are inhibitory . Using substrates that contain a DNA junction, which permits the specific binding of RuvA, we find that the RuvA and RuvB proteins promote two types of helicase reactions: nonspecific reactions, which are sensitive to inhibition by stoichiometric amounts of SSB, and junction-targeted reactions, which are not inhibited by SSB . Using three-armed structures, we observe that junction-targeted reactions display a polarity and result in asymmetric product formation . Junction-specific binding and the subsequent initiation of DNA unwinding are likely to represent early steps in the process of branch migration.

J Biol Chem, 1994 Oct 21, 269(42), 26390 - 5
Interaction of Fe-protoporphyrin IX and heme analogues with purified recombinant heme oxygenase-2, the constitutive isozyme of the brain and testes; Rublevskaya I et al.; Heme oxygenase-2 (HO-2) is the predominant form of heme oxygenase in the brain and testes . The enzyme is not readily amenable to isolation from mammalian tissues and has not been characterized for its kinetic properties and interaction with metalloporphyrins . Presently a rat HO-2 cDNA (Rotenberg, M.O., and Maines, M . D . (1990) J . Biol . Chem . 265, 7501-7506) was used to generate a construct with a neutral hydrophobicity profile at its COOH terminus for expression of nearly full-length HO-2 protein in Escherichia coli . The procedures used for HO-1 were of no utility in purification of HO-2 . A multistep protocol developed for isolation of HO-2 resulted in a homogeneous protein with a specific activity up to 6,500 nmol of bilirubin/mg/h . Based on SDS-polyacrylamide gel electrophoresis and Western blot analyses, the protein had an apparent molecular mass of approximately 34 kDa . HO-2 binds Fe-protoporphyrin (heme) at near molar unity to give a complex with the absorption maximum at 403 nm . The Soret band has a blue shift to 430 nm when heme iron is reduced, with distinct alpha and beta bands at 485 and 550 nm, respectively . The Soret band of the CO complex of ferrous heme.HO-2 is at 420 nm, and alpha and beta bands are at 540 and 572 nm, respectively . The apparent Km for Fe-protoporphyrin is 0.33 microM, with a Vmax of 0.45 nmol of bilirubin/mg/h . Zn-protoporphyrin is a strong mixed inhibitor of enzyme activity, whereas Co-protoporphyrin is a poor competitive inhibitor of activity . When HO-2 was preincubated (10 min at 4 degrees C) with Fe-protoporphyrin, the cobalt complex did not inhibit enzyme activity, whereas the Zn-protoporphyrin effectively inhibited activity . Calorimetric measurements suggest that HO-2/heme interaction involves one type of association producing a single heat absorption peak upon melting of the complex and that the unfolding is not reversible . The association increases the enthalpy of HO-2 (130 kcal/mol versus 184 kcal/mol) and increases the stability to heat denaturation by 9 degrees C . Heat duration of zinc complex involves at least two stages of unfolding.

J Biol Chem, 1994 Oct 21, 269(42), 26274 - 9
Kinetic properties of NhaB, a Na+/H+ antiporter from Escherichia coli; Pinner E et al.; NhaB, a Na+/H+ antiporter from Escherichia coli, was overproduced, purified, and reconstituted in a functional state, demonstrating that a single polypeptide, the product of the nhaB gene, can catalyze full activity . NhaB is a minor protein that accounts for less than 0.1% of the total membrane protein . The use of proteoliposomes made possible the determination of important kinetic and pharmacological properties in the absence of passive and mediated leaks . The activity of NhaB was found to have some pH dependence; the apparent Km for Na+ changes by 10-fold from 1.55 mM at pH 8.5 to 16.66 mM at pH 7.2, while the Vmax remains constant . It was demonstrated that NhaB is electrogenic and translocates more H+ than Na+ per cycle; the rate of sodium efflux from proteoliposomes was accelerated by a membrane potential, negative inside, and NhaB activity generated a membrane potential as monitored by two techniques . The stoichiometry of NhaB was estimated by a thermodynamic method in which the magnitude of delta psi generated by NhaB was measured at various Na+ gradients . A kinetic method, in which the electrophoretic movement of 86Rb+ (in the presence of valinomycin) was monitored in parallel with measurements of NhaB-mediated 22Na+ uptake, allowed us to determine a stoichiometry of 3H+/2Na+ . The significance of the existence of two antiporters with different stoichiometries, NhaA and NhaB, active in the same cell, is discussed.

J Biol Chem, 1994 Oct 21, 269(42), 26220 - 6
Escherichia coli DNA helicase I catalyzes a sequence-specific cleavage/ligation reaction at the F plasmid origin of transfer; Sherman JA et al.; Recent studies have shown that the Escherichia coli F plasmid-encoded traI gene product (TraIp), also known as DNA helicase I, catalyzes the formation of the site- and strand-specific nick that initiates F plasmid DNA transfer . Scission of the phosphodiester bond at the nic site within the origin of transfer (oriT) is accompanied by the covalent attachment of TraIp to the 5'-phosphate of the nicked DNA strand . This mechanism suggests that TraIp may also be capable of catalyzing a DNA ligation reaction using the energy stored in the protein-DNA intermediate . To test this possibility, an in vitro assay was designed that utilized short single-stranded DNA oligonucleotides of different lengths derived from the region within oriT that spanned the nic site . Purified TraIp was capable of efficiently cleaving single-stranded DNA that contained a nic site, and upon cleavage, the protein became covalently linked to the 5'-end of the nic site . When TraIp was incubated with two oligonucleotides of different length that contained the nic site, there was formation of novel recombinant products resulting from a TraIp-catalyzed cleavage/ligation reaction . Furthermore, the cleavage and ligation reactions were both sequence-specific . These data suggest that TraIp plays an important role in the initiation and termination of conjugative DNA transfer.

J Biol Chem, 1994 Oct 21, 269(42), 26201 - 7
Mutational analysis of residues in and around the active site of human fibroblast-type collagenase; Windsor LJ et al.; Mutants in and around the catalytic zinc-binding site of human fibroblast-type collagenase have been expressed in Escherichia coli . Replacement of each of the three zinc ligands, His-199, His-203, and His-209, in the active site sequence: VAAHEXGHXXGXXH, not only destroyed catalytic activity but also led to improper folding of the polypeptide, suggesting that this sequence also serves as a structural zinc-binding site . By comparison, mutation of His-194 immediately preceding this sequence had no measurable effect on catalytic activity or on folding . Replacement of Glu-200 in the active site yielded enzymes that either were completely inactive (E200Q) or had greatly diminished (E200D) catalytic activity . Both Glu-200 mutants, however, were fully capable of forming complexes with tissue inhibitor of metalloproteinases-1 (TIMP-1) after reaction with organomercurials . Formation of complexes with TIMP-1 appear to require a properly folded, but not necessarily catalytically competent, active site . By contrast, complexes with alpha 2-macroglobulin form only with mutants with a catalytically competent active site . Two mutants identified in this study (E200Q and D212E) appeared to be properly folded but unable to generate any catalytic activity when exposed to either p-aminophenylmercuric acetate, trypsin, or SDS.

J Biol Chem, 1994 Oct 21, 269(42), 26052 - 7
Allosteric control of the substrate specificity of the anaerobic ribonucleotide reductase from Escherichia coli; Eliasson R et al.; The reduction of ribonucleotides is catalyzed by different enzymes in aerobic and anaerobic Escherichia coli, each with a different primary and quaternary structure . Here, we describe the allosteric regulation of the substrate specificity of the anaerobic ribonucleoside triphosphate reductase . The enzyme reduced ribonucleotides at a low basal rate . Reduction was stimulated up to 10-fold by an appropriate modulator (dGTP for ATP reduction, ATP for CTP and UTP reduction, and dTTP for GTP reduction) . dGTP and dTTP inhibited the reduction of the "incorrect" substrate; dATP inhibited reduction of all four . From kinetic, effector binding, and competition experiments we conclude that the enzyme has two classes of sites, one that binds ATP and dATP and regulates pyrimidine ribonucleotide reduction ("pyrimidine site"), the other that binds dATP, dGTP, and dTTP and regulates purine ribonucleotide reduction ("purine site") . This model differs slightly from the model for the aerobic reductase, but the physiological consequences remain the same and explain how a single enzyme can provide a balanced supply of the four dNTPs . The similarity of a highly sophisticated control mechanism for the aerobic and anaerobic enzymes suggests that both arose by divergent evolution from a common ancestor, in spite of their different structures.

J Mol Biol, 1994 Oct 21, 243(2), 152 - 6
Isolated P2 rRNA promoters of Escherichia coli are strong promoters that are subject to stringent control; Gafny R et al.; Ribosomal RNA synthesis in Escherichia coli is under stringent control . The seven rRNA operons are highly conserved and are each transcribed from two tandem promoters, P1 and P2, which are located about 120 base-pairs apart . In exponentially growing cells the majority of the transcripts are initiated at the P1 promoters . The P1 promoters are highly regulated, and are under stringent as well as growth rate controls . Here we demonstrate that transcription from the rrnA P1 promoter diminishes P2 expression . In the absence of P1, the P2 promoter acts as a rather strong promoter . Insertion of a transcription terminator between P1 and P2 eliminates the inhibition of P2 by P1, suggesting that the physical movement of RNA polymerase originating at P1 and progressing along the P2 promoter is necessary for the interference process to take place . Similarly to P1, the solitary P2 promoter is subject to stringent control.

J Biol Chem, 1994 Oct 21, 269(42), 26424 - 30
Identity of a peptide domain of human C9 that is bound by the cell-surface complement inhibitor, CD59; Chang CP et al.; The CD59 antigen is a plasma membrane glycoprotein that serves as an inhibitor of the C5b-9 complex of complement . This inhibitory activity appears related to the capacity of CD59 to bind with high affinity to sites that are nascently exposed in the alpha-chain subunit of human C8, as well as within the C9b domain (amino acid residues 245-538) of human C9, during assembly of the C5b-9 complex on the target membrane (Ninomiya, H., and Sims, P . J . (1992) J . Biol . Chem . 267, 13675-13680) . The CD59 binding site in C9 was first investigated by N-terminal sequencing of CD59-binding peptides generated by limited digest of the isolated C9b domain . These experiments revealed a 17-kDa fragment (starting at C9 residue Thr-320) that retained affinity for CD59, suggesting the possibility for localizing the CD59 binding site by mapping with small C9-derived peptides . Peptides spanning the entire C9b sequence were expressed in Escherichia coli and then probed with CD59 . CD59 bound specifically to all peptides starting N-terminal to C9 residue 359 with C termini extending beyond residue 411 . Little to no CD59 binding was observed for various C9-derived peptides that started C-terminal to residue 359 or that were truncated N-terminal to residue 411 . Affinity-purified antibody against C9 residues 320-411 inhibited CD59 binding to C9 by > 50% and completely inhibited its binding to the isolated C9b domain . Little to no specific binding of CD59 was detected for peptides restricted to the putative hinge domain within C9b (residues 245-271) . These results indicate that a CD59 binding site is located between residues 320 and 411 of the C9 polypeptide and suggest that the affinity of this site is principally determined by residues 359-411.

Nature, 1994 Oct 20, 371(6499), 707 - 11
Cloning of an avermectin-sensitive glutamate-gated chloride channel from Caenorhabditis elegans; Cully DF et al.; The avermectins are a family of macrocyclic lactones used in the control of nematode and arthropod parasites . Ivermectin (22,23-dihydroavermectin B1a) is widely used as an anthelmintic in veterinary medicine and is used to treat onchocerciasis or river blindness in humans . Abamectin (avermectin B1a) is a miticide and insecticide used in crop protection . Avermectins interact with vertebrate and invertebrate GABA receptors and invertebrate glutamate-gated chloride channels . The soil nematode Caenorhabditis elegans has served as a useful model to study the mechanism of action of avermectins . A C . elegans messenger RNA expressed in Xenopus oocytes encodes an avermectin-sensitive glutamate-gated chloride channel . To elucidate the structure and properties of this channel, we used Xenopus oocytes for expression cloning of two functional complementary DNAs encoding an avermectin-sensitive glutamate-gated chloride channel . We find that the electrophysiological and structural properties of these proteins indicate that they are new members of the ligand-gated ion channel superfamily.

Biochim Biophys Acta, 1994 Oct 19, 1208(2), 332 - 7
Expression of rat alpha-fetoprotein cDNA and its mutants in cultured mouse fibroblasts and identification of glycosylation sites related to electrophoretic variants; Koyama Y et al.; Rat alpha-fetoprotein (RAFP) shows two discrete electrophoretic variants, slow and fast, with molecular weights of 72,500 and 69,500, respectively . To elucidate the structural basis of this heterogeneity, we developed the expression system of RAFP cDNA and its mutants with the use of cultured mouse fibroblasts and a retrovirus vector . This produced recombinant wild-type and three mutant proteins and has several advantages over the use of Escherichia coli or Saccharomyces cerevisiae . The mutants were designed to lack either one or both of the two possible glycosylation sites on RAFP . Electrophoretic analyses relating to the glycosylation states of the recombinant proteins as well as of natural RAFP produced by rat cells before and after digestion with glycopeptidase F indicated that the slow variant has carbohydrate units located at Asn-93 and Asn-229 and the fast one has one at Asn-229.

Biochim Biophys Acta, 1994 Oct 18, 1219(2), 521 - 8
Cloning and characterization of a major allergen of the house dust mite, Dermatophagoides pteronyssinus, homologous with glutathione S-transferase; O'Neill GM et al.; A major allergen of the house dust mite, Dermatophagoides pteronyssinus, has been identified and characterized from a lambda gt11 cDNA library of the mite . IgE antibodies from the sera of allergic patients that recognise the cloned polypeptide bind to an approximately 26 kDa polypeptide on a Western blot of reduced mite polypeptides . Nucleotides sequencing of the clone revealed a 219 amino acid open reading frame encoding a protein with a derived molecular mass of 25,589 Da and a pI of 6.3 . Comparison of the deduced amino acid sequence with amino acid sequence databanks revealed a strong homology with glutathione S-transferases . The nucleotide sequence of the clone displayed a strong homology with the active glutathione binding site of glutathione transferases and contained all but one of the 19 positionally conserved amino acid residues found in glutathione transferases . The cloned polypeptide was expressed in Escherichia coli and affinity-purified on glutathione agarose.

Biochim Biophys Acta, 1994 Oct 18, 1219(2), 449 - 56
Cloning, sequencing and expression of dinoflagellate luciferase DNA from a marine alga, Gonyaulax polyedra; Bae YM et al.; The marine dinoflagellate, Gonyaulax polyedra emits light in a reaction involving the enzymatic oxidation of its tetrapyrrole luciferin by molecular oxygen; its luciferase (LCF) single chain has an estimated molecular mass of 130 kDa, and exhibits a circadian rhythm in its activity . A cDNA expression library in the lambda ZAPII vector was constructed from the polyadenylated RNA isolated from the Gonyaulax cells during the early night phase, the time at which LCF synthesis is believed to be greatest . Of the approx . 1.2 . 10(5) phages from the library screened with antibody against Gonyaulax LCF, 13 positive plaques were obtained . The nucleotide sequences of two of the larger inserts (2.4 kb and 1.6 kb in length), both carrying the poly(A) tail, were determined and found to be identical in the overlapping region . When expressed in Escherichia coli, both cDNA clones produced active luciferase . A Northern hybridization using the cDNA as a probe showed that the length of the lcf mRNA is approx . 4.1 kb, sufficiently long to encode the 130 kDa LCF . Analyses of polymerase chain reaction products, prepared using both the cloned cDNA and Gonyaulax chromosomal DNA as templates, indicated that the cloned region of the luciferase gene does not carry any introns . This represents the first dinoflagellate luciferase to be cloned and sequenced; its deduced amino acid sequence bears no significant homologies with that of any other luciferase, or any other sequence in the data base.

Biochim Biophys Acta, 1994 Oct 18, 1219(2), 441 - 8
Transfer of a functional arabinose operator-repressor system to Drosophila melanogaster Schneider line-2 cells; Raman V et al.; Regulatory elements from the arabinose operon of Escherichia coli were transfected into Drosophila melanogaster Schneider line 2 cells to test their ability to function in animal cells . A construct containing an araC fusion gene (encoding AraC protein) under the control of an act5C (encoding actin) promoter and a construct containing a lacZ fusion gene (reporter gene) also under the control of an act5C promoter were used to build a regulatory circuit in the D . melanogaster cells . We have demonstrated that a AraC fusion protein can be synthesised in Schneider cells where it is able to repress the transcription of the reporter gene containing AraC-binding sites inserted between the transcription start point and the initiation codon . The reporter gene activity can be further modulated by the addition of arabinose to the medium.

Biochim Biophys Acta, 1994 Oct 18, 1219(2), 425 - 34
Functional interactions between translation, transcription and ppGpp in growing Escherichia coli; Faxen M et al.; Strains with a relA mutation together with three different alleles of spoT were used to study the effects of different levels of ppGpp on production time for beta-galactosidase, transcriptional polarity and readthrough of a stop codon by near-cognate tRNA or a suppressor tRNA . The influences of an rpsL(S12) allele and a miaA mutation, together giving decreased efficiency of translation, as well as an rpoB mutation, coding for an altered RNA polymerase, were also investigated . The spoT alleles which give total deficiency for ppGpp, or a level which is increased several-fold (Sarubbi et al . (1988) Mol . Gen . Genet . 213, 214-222), had at the most a marginal effect on the production time for a beta-galactosidase molecule or translational misreading of a nonsense mutation . The efficiency of an amber tRNA suppressor is not affected by ppGpp in strains with an otherwise wildtype translational machinery . These data suggest that ppGpp does not influence directly the translational process in vivo . Instead, ppGpp is found to interfere with transcriptional readthrough in a manner which is dependent on the rpsL224, miaA, as well as the rpoB mutations . Similarly, bacterial growth is affected by ppGpp in a manner which is dependent on properties of both the transcriptional and translational apparatus together . It is suggested that the primary effect of ppGpp is on transcriptional readthrough, but this effect is modified by translational/transcriptional coupling.

Biochim Biophys Acta, 1994 Oct 18, 1219(2), 398 - 404
The expression of Escherichia coli diaminopimelate decarboxylase in mouse 3T3 cells; Saqib KM et al.; We have subcloned the coding sequence for the Escherichia coli lysA gene coding for diaminopimelic acid decarboxylase (DAP decarboxylase) into a eukaryotic expression vector based on the SV40 early promoter . The activities of a series of constructs with different lengths of non-coding DNA at the 5' and 3' ends of the coding region have been compared by measuring the synthesis of lysine from diaminopimelic acid (DAP) in mouse 3T3 cells . A short non-coding sequence at the 3' end reduced the expression of enzyme activity . Stable lines of 3T3 cells have been produced by co-transfection of the chimeric gene with a plasmid coding for G-418 resistance . Cells were grown in medium containing G-418 and resistant clones were screened for an ability to synthesise lysine from DAP . {3H}Lysine produced from {3H}DAP was incorporated into cell proteins . An enzyme extract from a cell line which had incorporated two copies of the gene synthesised 0.082 nmol of lysine/min per mg protein . In the intact cell the rate of lysine synthesis is limited by the uptake of DAP which is taken up at only 5% of the rate of lysine . lysA has a potential as a reporter gene in studies of gene expression in mammalian cells.

Biochim Biophys Acta, 1994 Oct 18, 1219(2), 260 - 6
Mutagenesis of a highly conserved lysine 340 of the PRD1 DNA polymerase; Zhu W et al.; All known family B DNA polymerases contain a conserved region of amino acids, KX6-7YG, which appears to be correspond to the 'finger' alpha helix O of the Klenow fragment of E . coli DNA polymerase I, a family A DNA polymerase . Toward the goal of establishing the evolutionary relationship between the family A and B DNA polymerases, we have employed site-directed mutagenesis to access the functional role of the invariant amino acid lysine-340 of the PRD1 DNA polymerase . We have replaced the lysine-340 with three amino acids: histidine, asparagine and glutamic acid, respectively . Mutant DNA polymerases were overexpressed and purified to near homogeneity . Our results showed that the modification of the lysine-340 of the PRD1 DNA polymerase abolishes the polymerase activity without affecting the 3' to 5' exonuclease activity . These results support the proposal that the KX6-7YG motif of the family B DNA polymerases may be analogous to the KX7YG motif of the family A DNA polymerases, suggesting that two family DNA polymerases share a common ancestor.

Biochemistry, 1994 Oct 18, 33(41), 12347 - 55
Sequential assignments and identification of secondary structure elements of the colicin E9 immunity protein in solution by homonuclear and heteronuclear NMR; Osborne MJ et al.; 1H-1H, 1H-15N, and 1H-1H-15N multidimensional NMR spectroscopic studies of the 86 amino acid protein that provides immunity against the DNase action of colicin E9 are reported . Through a combination of 2D NOESY and TOCSY and 3D TOCSY-HMQC, NOESY-HMQC, and HMQC-NOESY-HMQC experiments, almost complete 1H NMR and backbone 15N NMR assignments have been obtained, and the secondary structure of the protein has been partially elucidated . Approximately 50% of the protein forms three helices . The specificity determining region of the DNase immunity protein, identified from previously reported biochemical studies to include residues 32-40, is helical, indicating that the protein-protein interaction involves residues from at least one helix.

EMBO J, 1994 Oct 17, 13(20), 4991 - 5001
Human DNA helicase II: a novel DNA unwinding enzyme identified as the Ku autoantigen; Tuteja N et al.; Human DNA helicase II (HDH II) is a novel ATP-dependent DNA unwinding enzyme, purified to apparent homogeneity from HeLa cells, which (i) unwinds exclusively DNA duplexes, (ii) prefers partially unwound substrates and (iii) proceeds in the 3' to 5' direction on the bound strand . HDH II is a heterodimer of 72 and 87 kDa polypeptides . It shows single-stranded DNA-dependent ATPase activity, as well as double-stranded DNA binding capacity . All these activities comigrate in gel filtration and glycerol gradients, giving a sedimentation coefficient of 7.4S and a Stokes radius of approximately 46 A, corresponding to a native molecular weight of 158 kDa . The antibodies raised in rabbit against either polypeptide can remove from the solution all the activities of HDH II . Photoaffinity labelling with {alpha-32P}ATP labelled both polypeptides . Microsequencing of the separate polypeptides of HDH II and cross-reaction with specific antibodies showed that this enzyme is identical to Ku, an autoantigen recognized by the sera of scleroderma and lupus erythematosus patients, which binds specifically to duplex DNA ends and is regulator of a DNA-dependent protein kinase . Recombinant HDH II/Ku protein expressed in and purified from Escherichia coli cells showed DNA binding and helicase activities indistinguishable from those of the isolated protein . The exclusively nuclear location of HDH II/Ku antigen, its highly specific affinity for double-stranded DNA, its abundance and its newly demonstrated ability to unwind exclusively DNA duplexes, point to an additional, if still unclear, role for this molecule in DNA metabolism.

EMBO J, 1994 Oct 17, 13(20), 4919 - 25
Correlation between the structure and biochemical activities of FtsA, an essential cell division protein of the actin family; Sanchez M et al.; Cell division protein FtsA, predicted to belong to the actin family, is present in different cell compartments depending on its phosphorylation state . The FtsA fraction isolated from the cytoplasm is phosphorylated and capable of binding ATP, while the membrane-bound form is unphosphorylated and does not bind ATP . A variant of the protein FtsA102, in which the nucleotide binding site was destroyed by mutagenesis of a highly conserved residue predicted to be needed for the binding, does not bind ATP . Another variant, FtsA104, cannot be phosphorylated because the predicted phosphorylatable residue has been replaced by a non-phosphorylatable one . This protein although unable to bind ATP in vitro, is able to rescue the reversible ftsA2, the irreversible ftsA3 and, almost with the same efficiency, the ftsA16 amber alleles . Consequently, phosphorylation and ATP binding may not be essential for the function of FtsA . Alternatively they may have a regulatory role on the action of FtsA in the septator.

FEBS Lett, 1994 Oct 17, 353(2), 197 - 200
Yta10p, a member of a novel ATPase family in yeast, is essential for mitochondrial function; Tauer R et al.; The yeast gene, YTA10, encodes a member of a novel family of putative ATPases . Yta10p, as deduced from the nucleotide sequence, is 761 amino acids in length (predicted molecular mass 84.5 kDa) . The amino acid sequence of Yta10p exhibits high similarity to two other yeast proteins, Yta11 and Yta12, and to E . coli FtsH . Several features of Yta10p are compatible with its localization in mitochondria . We report here that Yta10p is a yeast mitochondrial protein and that import is dependent on a membrane potential and accompanied by processing to a protein of approximately 73 kDa . Disruption of YTA10 leads to a nuclear petite phenotype and to a loss of respiratory competence, as shown by spectrophotometric measurement of the activities of respiratory complexes I-III and IV, respectively . These findings together with the high similarity of Yta10p to several ATP-dependent proteases suggest that Yta10p is a mitochondrial component involved, directly or indirectly, in the correct assembly and/or maintenance of active respiratory complexes.

FEBS Lett, 1994 Oct 17, 353(2), 194 - 6
Visualisation by electron microscopy of the unique part of the cytoplasmic domain of a desmoglein, a cadherin-like protein of the desmosome type of cell junction; Rutman AJ et al.; Part of the cytoplasmic domain of a human desmoglein, Dsg1, a cadherin-like protein found in desmosomes of epithelial cells, has been visualised by electron microscopy . The cloned fragment contains five repeats of a 29 +/- 4 residue sequence unique to desmogleins, followed by a glycine-rich region . In rotary shadowed preparations the molecule consists of a globular head attached to a thin tail, the latter perhaps corresponding to the glycine-rich region . This portion of the molecule is thought to span the width of the inner dense plaque . The structure and dimensions concur well to the configuration deduced from the protein sequence.

FEBS Lett, 1994 Oct 17, 353(2), 173 - 6
The role of the CCA sequence of tRNA in the peptidyl transfer reaction; Tamura K; Peptidyl transfer is a key step in the process of protein biosynthesis . To examine the role of the universal CCA terminal sequence of tRNA in the process of peptidyl transfer, various mutant transcripts of Escherichia coli valine tRNA were constructed . Peptidyl transferase activity, monitored by the 'fragment reaction' with a slight modification, was decreased by mutation at any one base of CCA . The effect of mutation was moderate in the UCA, CUA and CCG mutants . Replacement of A76 by a pyrimidine nucleotide, or replacement of either C74 or C75 by a purine nucleotide caused a marked decrease in the activity . These findings suggested that the universal CCA terminus of tRNA makes a functional interaction with ribosomal RNA by base-pairing.

FEBS Lett, 1994 Oct 17, 353(2), 143 - 6
Effects of substitutions of amino acids on the thermal stability of the Fv fragments of antibodies; Yasui H et al.; The thermal stability of Fv fragments was examined by circular dichroism (CD) spectrometry and high-performance liquid chromatography . We analyzed three Fv fragments: that of a monoclonal antibody D1.3 and two derivatives of it . After separation of wild-type VH and VL fragments, thermal denaturation of each fragment was monitored by CD spectrometry . The results indicated that the dissociation of Fv into VH and VL fragments seemed to be coupled with the denaturation of each fragment and that the thermal denaturation of VH and VL fragments was prevented when they were associated with one another . The analysis of the three Fv fragments also indicated that, in some cases, differences in amino acids even within the CDRs could have significant effects on the thermal stability of the complex between VH and VL fragments.

Mol Gen Genet, 1994 Oct 17, 245(1), 25 - 31
Glutathione transferase gene family from the housefly Musca domestica; Syvanen M et al.; Three new glutathione transferase (GST) genes from the housefly Musca domestica are described . These genes, identified as MdGST-2, -3, and -4, were from cDNA clones obtained from a cDNA bank in phage lambda . The bank was prepared using poly(A)+ RNA from a housefly that is highly resistant to organophosphate insecticides because of enhanced expression of multiple members of the glutathione transferase gene family . The DNA sequence of each is reported and has a complete open reading frame that specified an amino acid sequence similar to other dipteran glutathione transferases . Based on phylogenetic analysis, we can conclude that the insect glutathione transferase gene family falls into two groups, each of which evolves at a different rate, presumably due to differences in functional constraints . We show that MdGST-1 (and their homologues from Drosophila and Lucilia) evolve at a significantly slower rate than the other members of the gene family . Each housefly GST cDNA was inserted into a bacterial plasmid expression system and a glutathione transferase activity was expressed in Escherichia coli . The transcription pattern of each of these glutathione transferases was examined in a variety of different housefly strains that are known to differ in their resistance to organophosphate insecticides due to different patterns of glutathione transferase expression . We found that the level of transcription for two of our clones was positively correlated with the level of organophosphate resistance.

Mol Gen Genet, 1994 Oct 17, 245(1), 53 - 60
Selection for transport competence of C-terminal polypeptides derived from Escherichia coli hemolysin: the shortest peptide capable of autonomous HlyB/HlyD-dependent secretion comprises the C-terminal 62 amino acids of HlyA; Jarchau T et al.; Escherichia coli hemolysin (HlyA) is secreted by a specific export machinery which recognizes a topogenic secretion signal located at the C-terminal end of HlyA . This signal sequence has been variously defined as comprising from 27 to about 300 amino acids at the C-terminus of HlyA . We have used here a combined genetic and immunological approach to select for C-terminal HlyA peptides that are still secretion-component . A deletion library of HlyA mutant proteins was generated in vitro by successive degradation of hylA from the 5' end with exonuclease III . Secretion competence was tested by immunoblotting of the supernatant of each clone with an antiserum raised against a C-terminal portion of hemolysin . It was found that the hemolysin secretion system has no apparent size limitation for HlyA proteins over a range from 1024 to 62 amino acids . The smallest autonomously secretable peptide isolated in this selection procedure consists of the C-terminal 62 amino acids of HlyA . This sequence is shared by all secretion-competent, truncated HlyA proteins, which suggests that secretion of the E . coli hemolysin is strictly post-translational . The capacity of the hemolysin secretion machinery was found to be unsaturated by the steady-state level of its natural HlyA substrate and large amounts of truncated HlyA derivatives could still be secreted in addition to full-length HlyA.

Carbohydr Res, 1994 Oct 17, 263(2), 209 - 15
Structural studies of the Escherichia coli O90 O-antigen polysaccharide; Ratnayake S et al.; The O-specific side-chain of the lipopolysaccharide from Escherichia coli O90 has been investigated using methylation analysis, partial hydrolysis, and NMR spectroscopy as the principal methods . It is concluded that the polysaccharide is composed of tetrasaccharide repeating-units having the following structure . {formula: see text} The polysaccharide contains approximately one mole of O-acetyl groups per repeating unit, located on the fucose residue.

EMBO J, 1994 Oct 17, 13(20), 4870 - 6
Base pairing between Escherichia coli RNase P RNA and its substrate; Kirsebom LA et al.; Base pairing between the substrate and the ribozyme has previously been shown to be essential for catalytic activity of most ribozymes, but not for RNase P RNA . By using compensatory mutations we have demonstrated the importance of Watson-Crick complementarity between two well-conserved residues in Escherichia coli RNase P RNA (M1 RNA), G292 and G293, and two residues in the substrate, +74C and +75C (the first and second C residues in CCA) . We suggest that these nucleotides base pair (G292/+75C and G293/+74C) in the ribozyme-substrate complex and as a consequence the amino acid acceptor stem of the precursor is partly unfolded . Thus, a function of M1 RNA is to anchor the substrate through this base pairing, thereby exposing the cleavage site such that cleavage is accomplished at the correct position . Our data also suggest possible base pairing between U294 in M1 RNA and the discriminator base at position +73 of the precursor . Our findings are also discussed in terms of evolution.

FEBS Lett, 1994 Oct 17, 353(2), 185 - 8
Isolation and characterization of mutants of human mitogen-activated protein kinase (ERK2); Krishnan IS et al.; Site directed mutagenesis/charged-to-alanine scanning mutagenesis of the amino terminal portion of human ERK2 (from amino acids 1 to 150) purified as a glutathione-S-transferase fusion protein (GST-ERK2) from E . coli has been done to determine regions/amino acids important for activation by rabbit skeletal muscle MAP kinase kinase (rMEK) and kinase activity towards myelin basic protein (MBP) . Five classes of mutants have been isolated . The first class of mutants comprises of G30A/G32A, A50D and R65A/R68A/E69A, that can be phosphorylated by rMEK and have no kinase activity towards MBP, the second class includes mutants D122A/H123A and N142A which have lower kinase activities but no change in their activation by rMEK; third class being Y34A, E58A/H59A, which have neutral effect towards either activity, the fourth class that includes completely inactive mutants D42A/K46A/R48A, the deletion mutant in the same region (-9aa{40-48}) and D104A/E107A/D109A and finally the fifth class that include K53A, E94A/K97A/D99A, K112A/K115A and R133A/K136A that are phosphorylated 140-240% but with kinase activity toward MBP ranging from 50-100% of the wild type.

FEMS Microbiol Lett, 1994 Oct 15, 123(1-2), 75 - 82
Cloning and DNA sequence analysis of bmpC, a gene encoding a potential membrane lipoprotein of Borrelia burgdorferi; Aron L et al.; Immunoscreening of a lambda gt11 genomic library of Borrelia burgdorferi expressed in Escherichia coli permitted detection of a clone containing a partial sequence of a B . burgdorferi gene encoding a protein with significant homology to TmpC of Treponema pallidum . Subsequent cloning and DNA sequence analysis revealed an open reading frame encoding a protein with 353 amino acid residues . The open reading frame is preceded by putative promoter sequences and a ribosome binding site, and is initiated with a TTG . The putative protein shares 26% identity with TmpC, contains a signal peptidase II sequence, and is also homologous to the gene products of the recently described bmpA and bmpB of B . burgdorferi . This gene has been designated bmpC . Additional sequencing and restriction analysis indicate that it is located at approximately 400 kbp on the chromosomal map of B . burgdorferi, immediately upstream of bmpA and bmpB.

FEMS Microbiol Lett, 1994 Oct 15, 123(1-2), 63 - 7
Genetic evidence that hepA gene is involved in the normal deposition of the envelope of both heterocysts and akinetes in Anabaena variabilis ATCC 29413; Leganes F; The hepA gene in Anabaena sp . strain PCC 7120 is required for normal formation of the polysaccharide layer of the heterocyst envelope . A plasmid bearing hepA, interrupted by a neomycin-resistance cassette, was transferred by conjugation to wild type Anabaena variabilis ATCC 29413, so that the interrupted hepA gene replaced a homologous sequence . In the recombinant exconjugants, the envelopes of akinetes as well as of heterocysts were altered.

FEMS Microbiol Lett, 1994 Oct 15, 123(1-2), 43 - 8
Sulfonate-sulfur utilization involves a portion of the assimilatory sulfate reduction pathway in Escherichia coli; Uria-Nickelsen MR et al.; Strains of Escherichia coli lacking serine transacetylase or a positive regulator (Cys B protein) of the assimilatory sulfate reduction (ASR) pathway were unable to assimilate sulfonate-S, while single mutants in O-acetyl-L-serine sulfhydrylase (either 'A' or 'B') were able to do so . Mutants unable to reduce sulfate to sulfite were nonetheless able to form and accumulate sulfide and then cysteine from sulfonates, while strains lacking sulfite reductase were not . Thus terminal portions of the ASR pathway are involved in reduction of sulfonate-S to that of cysteine . E . coli K-12 formed cysteine more slowly, and accumulated lesser amounts of it with sulfonate-sulfur than it did from either sulfate or sulfite . These observations are consistent with our earlier report that sulfate is the preferred sulfur source when present simultaneously with a sulfonate.

FEMS Microbiol Lett, 1994 Oct 15, 123(1-2), 27 - 31
Two mutant alleles of mukB, a gene essential for chromosome partition in Escherichia coli; Yamanaka K et al.; The MukB protein is essential for chromosome partitioning in Escherichia coli and consists of 1484 amino acid residues (170 kDa) . We have determined the base changes at the mutated sites of the mukB106 mutant and a newly isolated mutant, mukB33 . These mutant mukB genes were each found to carry a single base-pair transition which leads to an amino acid substitution; a serine residue at position 33 was changed to phenylalanine in the case of mukB106, and an aspartic acid residue at position 1201 was changed to asparagine in the case of mukB33.

FEMS Microbiol Lett, 1994 Oct 15, 123(1-2), 219 - 24
Characterisation of IS1126 from Porphyromonas gingivalis W83: a new member of the IS4 family of insertion sequence elements; Maley J et al.; The nucleotide sequence of IS1126, the only insertion sequence so far isolated from the oral pathogen Porphyromonas gingivalis, has been determined . It had a nucleotide sequence of 1338 base pair (bp) flanked by 12 bp perfect inverted repeats and generated a 5 bp target site duplication . The single major open reading frame encoded a predicted protein of 361 amino acids and molecular mass of 41 kDa . The gene encoding the transposase was subcloned into pUC18 and the transposase expressed in Escherichia coli minicells . The predicted amino acid sequence of the transposase had homology to putative transposases of IS1106 and IS1186 both of which belong to the IS5 group within the IS4 super-family of insertion elements . On the basis of this homology we propose that IS1126 should also be included in the IS5 group . Southern-blot analysis of a number of P . gingivalis strains using IS1126 as a probe revealed a unique pattern of hybridisation for each strain and the absence of IS1126 from other closely related Porphyromonas species . This should allow IS1126 to be used as a rapid epidemiological tool in studying oral infections by P . gingivalis.

FEMS Microbiol Lett, 1994 Oct 15, 123(1-2), 201 - 6
Identification by Tn10 transposon mutagenesis of host factors involved in the biosynthesis of K99 fimbriae of Escherichia coli: effect of LPS core mutations; Pilipcinec E et al.; Tn10 transposon mutagenesis of Escherichia coli producing K99 fimbriae was carried out to identify host factors involved in regulation of biosynthesis of fimbriae . Two chromosomal mutants were obtained that showed a strongly reduced cell surface expression of K99 fimbriae upon colony blotting and ELISA . Analysis by inversed PCR and nucleotide sequencing showed that one mutant (EP14) contained the Tn10 transposon in rfaQ, affecting the expression of the rfaQGP gene cluster, whereas the other mutant (EP35) was affected in a, to date, unknown region of the genome . Immunoblotting analysis confirmed a Rd1 type of LPS of mutant strain EP14 . These findings for the first time indicated an effect of LPS core biosynthesis on the biogenesis of fimbriae at the cell surface . Preliminary experiments indicated that K99 major subunits, in contrast to K88 subunits, strongly bind LPS molecules.

FEMS Microbiol Lett, 1994 Oct 15, 123(1-2), 153 - 9
Peptidase D of Escherichia coli K-12, a metallopeptidase of low substrate specificity; Schroeder U et al.; Peptidase D of Escherichia coli was overproduced from a multicopy plasmid and purified to electrophoretic homogeneity . The pure enzyme was stable at 4 degrees C or -20 degrees C and had a pH optimum at pH 9, and a pI of 4.7; the temperature optimum was at 37 degrees C . As the enzyme was activated by Co2+ and Zn2+, and deactivated by metal chelators, it appears to be a metallopeptidase . By activity staining of native gels, 11 dipeptides which are preferentially cleaved by peptidase D were identified . Peptidase D activity required dipeptide substrates with an unblocked amino terminus and the amino group in the alpha or beta position . Non-protein amino acids and proline were not accepted in the C-terminal position, whereas some dipeptide amides and formyl amino acids were hydrolyzed . Km values of 2 to 5 mM indicate a relatively poor interaction of the enzyme with its substrates.

FEMS Microbiol Lett, 1994 Oct 15, 123(1-2), 107 - 12
Insertion of a 27 amino acid viral peptide in different zones of Escherichia coli beta-galactosidase: effects on the enzyme activity; Benito A et al.; Seven internal, putatively exposed regions of Escherichia coli beta-galactosidase have been explored regarding their tolerance to insertions of large foreign peptides . Small sequence modifications, including amino acid substitutions and small deletions, were introduced into the lacZ gene to generate unique BamHI restriction sites . By using these mutant genes, a 27 amino acid stretch reproducing the hypervariable loop of foot-and-mouth disease virus VP1 protein (site A) was further inserted in predefined regions of the enzyme . Among the 13 resulting engineered proteins only three, carrying sequence modifications within a short region, are active, with only moderate reduction of their specific activities . The identified permissive region, which involves amino acids 275 to 279, seems to be a flexible area that could be appropriate to incorporate and study biological properties of heterologous peptides in correctly folded beta-galactosidase chimeric proteins.

Eur J Biochem, 1994 Oct 15, 225(2), 737 - 45
Evidence for a pyrimidine-nucleotide-specific initiation site (the i site) on Escherichia coli RNA polymerase . Proximity relationship with the inhibitor binding domain; Reddy PS et al.; Escherichia coli RNA polymerase has two sites, the i and i + 1, for the binding of the first two substrates . The i site is template- and Mg(2+)-independent and purine-nucleotide-specific, whereas the i + 1 site is template- and Mg(2+)-dependent and shows no nucleotide preference . The specificity of the i site for purine nucleotides is well in accord with the fact that most promoters initiate with a purine nucleotide . But there are a few promoters that initiate with a pyrimidine nucleotide . Dinucleotide synthesis at these promoters is completely inhibited by rifampicin . Earlier studies have failed to identify an i site for pyrimidine nucleotides . In this paper, using a fluorescent analog of UTP, namely uridine 5'-{gamma-(5-sulfonic acid)naphthylamidate}-triphosphate, abbreviated as UTP{AmNS}, we are able to show its binding to RNA polymerase, with a Kd of 0.8 microM, in the absence of Mg2+ and template . This suggests the presence of an i pyrimidine nucleotide site . The fact that UTP-{AmNS} is capable of initiating RNA synthesis from the i site is further evidenced by the abortive transcription analyses at the lac promoter . Fluorescence titration studies performed in the presence and absence of purine initiator molecules indicate that this site is different from the i purine site . Scatchard analysis of the above data indicates the presence of a single binding site for UTP{AmNS} in the absence of Mg2+ . Moreover UTP{AmNS} binds to the core enzyme with a Kd of 3.0 microM implying that, unlike the i purine nucleotide site, the sigma protein confers a tighter binding of UTP-{AmNS} to the low-Kd site . Forster's energy transfer measurements using UTP{AmNS} as the donor and rifampicin as the acceptor have been used for estimation of the distance of the i pyrimidine nucleotide site from the rifampicin site . From these measurements, we infer that there is no direct interference of rifampicin with the first phosphodiester bond between two pyrimidine nucleotides.

Eur J Biochem, 1994 Oct 15, 225(2), 601 - 8
The interactions of Escherichia coli trp repressor with tryptophan and with an operator oligonucleotide . NMR studies using selectively 15N-labelled protein; Ramesh V et al.; The effects of the binding of the corepressor L-tryptophan and an operator oligonucleotide to Escherichia coli trp repressor have been studied, using selective 15N labelling to permit observation of the backbone amide resonances of 50 of the 107 residues of the protein monomer . Repressor molecules selectively labelled in turn with {15N}alanine, {15N}glutamate, {15N}isoleucine, {15N}leucine and {15N}methionine were prepared by isolating them from prototrophic E . coli cells grown in media containing a mixture of unlabelled and the appropriate 15N-enriched amino acids . Analysis of the heteronuclear correlation spectra of the labelled repressors shows the value of selective labelling in resolving the crosspeaks of, for example, the 19 leucine and 12 glutamate residues . All 50 residues studied show measurable changes in amide 1H and/or 15N chemical shift on the binding of tryptophan and/or the operator oligonucleotide, showing clearly that ligand binding has effects which are transmitted throughout almost the whole protein . Large chemical shift changes on ligand binding are seen in residues in the tryptophan binding site and in the 'helix-turn-helix' DNA-binding domain, but also in residues in helices C and F remote from the ligand binding sites . On operator binding there is selective broadening of the signals of residues in the N-terminal region of the protein and in the DNA-binding domain, perhaps reflecting a conformational equilibrium.

Lancet, 1994 Oct 15, 344(8929), 1049 - 52
The role of tyrosinase in autoimmune vitiligo; Song YH et al.; Vitiligo is a common depigmenting skin disease, associated with certain autoimmune endocrinopathies, and autoantibodies to several antigens can be found in melanoma cells . We set out to identify the antigens . We examined 26 patients with vitiligo and associated endocrine disease . Of these, 18 patients (77%) and 8 immediate family members had autoantibodies specific for a 69 kDa protein in HTB-70 human melanoma cells that was not seen in control cells . The autoantibody-positive patient sera reacted with recombinant human tyrosinase expressed in Escherichia coli seen by western blots, as did antibodies raised in rabbits against hamster tyrosinase, but not to recombinant tyrosinase-related protein . Not one of 31 normal controls or 8 patients with alopecia or systemic lupus erythematosus had tyrosinase autoantibodies but a small proportion (12%) of 42 patients with autoimmune endocrine disease without a history of vitiligo had them . The results show that tyrosinase, an enzyme important in melanin formation, is a principal autoantigen of autoimmune vitiligo.

J Immunol, 1994 Oct 15, 153(8), 3655 - 63
Evidence for glycosphingolipid modification of the type 1 IFN receptor; Ghislain J et al.; P1-determinant glycolipids that include two membrane glycosphingolipids, globotriaosylceramide (Gal alpha 1-4Gal beta 1-4Glc-ceramide, Gb3) and galabiosylceramide (Gal alpha 1-4Gal-ceramide, Gb2) are receptors for an Escherichia coli-derived subunit toxin, verotoxin (VT-1) . Studies with Daudi cells and glycosphingolipid-deficient Daudi mutants and U937 cells identified that the presence of Gb2/Gb3 correlates with IFN-alpha sensitivity . Comparison of amino acid sequences between VT-1 and the extracellular region of the 63-kDa IFN-alpha beta receptor (IFNAR) peptide reveals regions of identity, specifically in those domains in the VT-1 B subunit, that have been implicated as Gb2/Gb3 binding sites . In direct ligand binding studies, we show that membrane Gb2/Gb3 content affects the binding capacity of cells for IFN-alpha, although IFNAR cell surface expression is unaffected . Binding of IFN-alpha to the receptor leads to kinase-associated phosphorylation of the latent transcription factor, ISGF3, which activates transcription by binding to IFN-stimulated regulatory elements in IFN-sensitive genes . Electrophoretic mobility band shift assays indicated that U-937 and Daudi mutant cells, deficient in Gb2 and Gb3, exhibited reduced nuclear factor binding to the human 2-5A synthetase IFN-stimulated regulatory element when compared with wild-type Daudi cells, after exposure to IFN-alpha . Moreover, when Daudi cells were treated with a ceramide analogue, 1-phenyl-2-(decanoylamino)-3-morpholino-1-propanol, Gb2 and Gb3 synthesis was inhibited and a concomitant reduction in IFN-induced ISGF3 activation was noted . IFNAR cell surface expression was unaffected by 1-phenyl-2-(decanoylamino)-3-morpholino-1-propanol treatment . By using a fusion protein of the extracellular domain of IFNAR linked at the carboxyl terminus to the Fc portion of IgG1, we demonstrate that IFNAR is able to bind preferentially to Gb2 . These results suggest that an association of IFNAR with membrane Gal alpha 1-4Gal containing glycolipids facilitates receptor-mediated signaling.

Cancer Res, 1994 Oct 15, 54(20), 5438 - 44
Structural analysis of the Ras GTPase activating protein catalytic domain by semirandom mutagenesis: implications for a mechanism of interaction with Ras-GTP; Hettich L et al.; The bovine complementary DNA encoding the catalytic domain of Ras GTPase activating protein was mutagenized semirandomly using a variation of the polymerase chain reaction . Sixty-four mutated codons were identified with seventeen of the mutations deleterious to Ras GTPase activating function . All of the inactivating single mutations affected the structure of the catalytic fragment as assessed by large decreases in soluble protein when expressed in Escherichia coli . Upon examination of the Ras binding properties of 10 of the mutants, only 1 was measurably impaired for Ras binding and 4 appeared to have increased affinity for Ras . These results demonstrate that Ras binding and GTPase activation are two distinct properties of GTPase activating protein . Additionally, the catalytic mechanism of GTPase activating protein is much more sensitive to structural perturbation than is Ras binding.

Cancer Res, 1994 Oct 15, 54(20), 5401 - 7
Human cytidine deaminase: purification of enzyme, cloning, and expression of its complementary DNA; Laliberte J et al.; Cytidine (CR) deaminase was purified 47,000-fold to homogeneity from human placenta . The molecular mass of CR deaminase was estimated to be 48.7 kDa by gel filtration and 16.1 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that it contains three or four identical subunits . We determined the amino acid sequence of several peptide fragments and designed 5'-primers to amplify, by the polymerase chain reaction, a specific 364-base pair DNA fragment using human liver complementary DNA (cDNA) as the template . This DNA fragment, which contains the codons of one peptide, was used as a probe to screen a cDNA library from human liver . We isolated and sequenced a cDNA clone of 910 base pairs that contained a 5' nontranslated region, a 438-base pair coding region, and a 3' nontranslated region with a polyadenylated tail . The translated region of the clone contained a deduced sequence of 146 amino acids, with a predicted molecular mass of 16.2 kDa and the sequences of our peptides . The cDNA was ligated in pGEX vector and expressed in Escherichia coli . The expressed protein had a high CR deaminase activity and molecular mass of 16.3 kDa . These data demonstrate clearly that the open reading frame of our cDNA clone codes for a functional human CR deaminase . Polymerase chain reaction amplifications of gene-specific DNA fragments from human/rodent hybrid cells indicate the localization of CR deaminase gene to human chromosome 1 . The cDNA for CR deaminase will be a useful molecular probe to investigate the importance of this enzyme in chemotherapy.

Cancer Res, 1994 Oct 15, 54(20), 5318 - 23
A role for metallothionein and zinc in spontaneous mutagenesis; Goncharova EI et al.; G12, a transgenic Chinese hamster V79 cell derivative which contains a single copy of the Escherichia coli gpt gene as a target for mutagenesis, has little constitutive metallothionein (MT) expression . It was transfected with a vector containing the mouse MT-I gene, and MT-I-overproducing lines were isolated . MT-I transfectants had lower spontaneous mutation frequencies compared with the G12 parental cell line . Mutagenesis by alkylating agents was unchanged . MT expression in G12 and MT transfectants could be modulated by exposure to Zn(II) or Cd(II) . The spontaneous mutation frequencies in Zn(II)- and Cd(II)-treated cells was inversely related to MT expression . In G12 cells grown in concentrations of Zn(II) up to 12 microM, a significant dose-dependent increase in spontaneous mutagenesis was observed . At higher (but subtoxic) concentrations in which endogenous MT was induced, a dramatic decrease in spontaneous mutagenesis was observed . In contrast, MT-I transfectants exhibited much lower spontaneous mutagenesis after growth in all concentrations of Zn(II) . These data demonstrate a possible role for MT in modulating spontaneous mutagenesis and point to a role for Zn(II) in contributing to spontaneous mutagenesis . Because there is variability in human MT expression, low MT expression might be a risk factor for cancer.

Structure, 1994 Oct 15, 2(10), 915 - 24
The structure of Pneumocystis carinii dihydrofolate reductase to 1.9 A resolution; Champness JN et al.; BACKGROUND: The fungal pathogen Pneumocystis carinii causes a pneumonia which is an opportunistic infection of AIDS patients . Current therapy includes the dihydrofolate reductase (DHFR) inhibitor trimethoprim which is selective but only a relatively weak inhibitor of the enzyme for P . carinii . Determination of the three-dimensional structure of the enzyme should form the basis for design of more potent and selective therapeutic agents for treatment of the disease . RESULTS: The structure of P . carinii DHFR in complex with reduced nicotinamide adenine dinucleotide phosphate and trimethoprim has accordingly been solved by X-ray crystallography . The structure of the ternary complex has been refined at 1.86 A resolution (R = 0.181) . A similar ternary complex with piritrexim (which is a tighter binding, but less selective inhibitor) has also been solved, as has the binary complex holoenzyme, both at 2.5 A resolution . CONCLUSIONS: These structures show how two drugs interact with a fungal DHFR . A comparison of the three-dimensional structure of this relatively large DHFR with bacterial or mammalian enzyme-inhibitor complexes determined previously highlights some additional secondary structure elements in this particular enzyme species . These comparisons provide further insight into the principles governing DHFR-inhibitor interaction, in which the volume of the active site appears to determine the strength of inhibitor binding.

Eur J Biochem, 1994 Oct 15, 225(2), 511 - 9
Proteasome-associated RNAs are non-specific; Pamnani V et al.; The RNA isolated from RNase-treated proteasome preparations from human erythrocytes, HeLa cells, the archaeon Thermoplasma acidophilum and also from recombinant proteasomes of T . acidophilum expressed in Escherichia coli was characterized . The RNA associated with structurally similar protein particles, namely with the two molecular chaperones, groEL from E . coli and with the thermosome from T . acidophilum, served as controls . Electrophoretic analysis on polyacrylamide gels of the radioactively end-labelled RNA revealed a very similar size distribution pattern, irrespectively of the protein particles from which they had been isolated . The predominant RNA species were in the size ranges 80 nucleotides and 120 nucleotides, respectively . Partial sequencing of their terminal regions by mobility-shift analysis revealed that, of the proteasomes from human erythrocytes, the approximately 80-nucleotide-long RNA consists of a heterogenous population of mostly tRNA species because they carried the tRNA-specific 3'-terminal sequence motif 5'-CCA-3' . The RNA in the size range 120 nucleotides isolated from the proteasomes of human erythrocytes and of T . acidophilum was also heterogeneous and displayed, in the terminal regions, a remarkable sequence similarity to the corresponding regions of the 5S rRNA from the same and different organisms . The total content of RNA of all the protein particles was quantified and found to be consistently sub-stoichiometric . All these findings strongly suggest that RNA associated with the proteasomes and with the molecular chaperones originate from the abundant cellular pool of the tRNAs and 5S rRNAs which bind non-specifically to these large protein particles.

Biochem Biophys Res Commun, 1994 Oct 14, 204(1), 105 - 11
Nitric oxide inhibits the expression of protein kinase C delta gene in the murine peritoneal macrophages; Jun CD et al.; Since there is increasing evidence that protein kinase C (PKC) has a crucial role in the production of nitric oxide (NO) from activated macrophages, this study was undertaken to address whether NO could regulate the expression of the gene of this enzyme . Stimulation of the cells with lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA) after treatment with recombinant interferon-gamma (rIFN-gamma) resulted in the increased production of NO in the medium . rIFN-gamma in combination with either LPS or PMA showed marked inhibition of the expression of PKC delta gene, whereas rIFN-gamma alone showed modest inhibition . The inhibition of gene expression was correlated with the amount of NO produced by activated macrophages . The inhibitory effect of NO on the expression of PKC delta gene is mimicked by the treatment of NO generating agent, sodium nitroprusside (SNP) . On the other hand, a specific inhibitor for NO synthase, NG-monomethyl-L-arginine (NGMMA), blocked the inhibition of the expression of PKC delta gene by blocking the NO production in the rIFN-gamma and LPS-stimulated cells . However, production of NO did not affect the expression of both TNF-alpha and TGF-beta gene which were induced by the stimulation of macrophages, as well as beta-actin gene, which was constitutively expressed in the macrophages . In conclusion, these findings show that NO has a regulatory role for the expression of the gene of PKC delta which is crucially involved in the process of NO synthesis.

J Mol Biol, 1994 Oct 14, 243(1), 6 - 9
DNA rearrangement of the shufflon determines recipient specificity in liquid mating of IncI1 plasmid R64; Komano T et al.; The shufflon is a unique DNA rearrangement found in plasmid R64 . R64 shufflon consists of four DNA segments, which are flanked and separated by seven 19-bp repeat sequences . Site-specific recombination between any inverted repeats results in a complex DNA rearrangement where four DNA segments invert independently or in groups . The shufflon is a biological switch to select one of the seven C-terminal segments of the pilV gene . To examine the biological significance of the shufflon, R64 derivatives, where the pilV gene was fixed in seven C-terminal segments, were constructed and used as donor cells for liquid mating . The transfer frequencies depended markedly on the combinations of recipient bacterial strains and C-terminal segments of the pilV gene in donor cells, indicating that the shufflon determines the recipient specificity in liquid mating of R64 . The products of the pilV genes were found to be a component of thin pili produced by R64.

J Mol Biol, 1994 Oct 14, 243(1), 128 - 30
Crystallization and preliminary X-ray diffraction studies of recombinant human ornithine aminotransferase; Shen BW et al.; Human liver ornithine aminotransferase was expressed in Escherichia coli and purified by ammonium sulfate fractionation and anion exchange column chromatography . The purified recombinant enzyme is fully active and crystallized readily over a wide range of polyethylene glycol concentrations . The crystals belong to the trigonal space group P3(1)21 (or its enantiomorph P3(2)21) with unit cell parameters a = b = 116.3 A, and c = 190.0 A, alpha = beta = 90 degrees, gamma = 120 degrees . There are three monomers per asymmetric unit . Self-rotation function studies revealed both 2-fold and 3-fold non-crystallographic symmetry, with the local 3-fold axis being tilted 15 degrees from the c axis and perpendicular to a crystallographic dyad . A complete native data set to 2.3 A resolution was collected using synchrotron radiation.

J Mol Biol, 1994 Oct 14, 243(1), 126 - 7
Crystallization and preliminary X-ray diffraction studies of the enoyl-ACP reductase from Escherichia coli; Wagner UG et al.; A crystal of the FabI protein from Escherichia coli has been obtained from polyethylene glycol (M(r) = 400) solution with sodium citrate at pH 8.5, by the hanging-drop technique at 4 degrees C . The crystal belongs to the hexagonal space group P6(1)22 (or P6(5)22) with cell dimensions of a = b = 81.1 A and c = 331.5 A . There are two molecules in the asymmetric unit and the crystal diffracts to 2.5 A resolution.

J Mol Biol, 1994 Oct 14, 243(1), 123 - 5
Crystallization and preliminary diffraction studies of Escherichia coli lysyl-tRNA synthetase (LysU); Onesti S et al.; Crystals of Escherichia coli lysyl-tRNA synthetase (lysU gene product) have been obtained by vapour diffusion techniques . Three different crystal forms could be grown under similar conditions . The crystals that have been chosen for the structure determination belong to space group C222(1) with cell dimensions a = 144.3 A, b = 257.8 A, c = 182.1 A and contain three monomers in the asymmetric unit . They diffract to at least 2.1 A resolution, but are very sensitive to radiation damage.

J Biol Chem, 1994 Oct 14, 269(41), 25936 - 41
Two distinct class A helix-loop-helix transcription factors, E2A and BETA1, form separate DNA binding complexes on the insulin gene E box; Peyton M et al.; Mutations in the RIPE3a element have shown it to be crucial for efficient tissue-specific expression of the insulin gene . In order to isolate factors binding to this element, we used a labeled RIPE3 probe to screen an expression library derived from a hamster insulinoma cell line . We isolated a clone encoding beta-cell E-box transcriptional activator1 (BETA 1) . This clone is a member of the class A subfamily of the helix-loop-helix superfamily of transcriptional activators, as determined both by sequence analysis and by functional association with a class B member (myogenin) . This clone is related to, but distinct from, other clones isolated from the same library which are also capable of binding RIPE3a . Analysis showed these additional clones to be the hamster homologs of E12 and E47 (German, M . S., Blaner, M . A., Nelson, C., Moss, L . G., and Rutter, W . J . (1991) Mol . Endocrinol . 5, 292-299) . Antibodies were raised against BETA 1 and against a common epitope of E12 and E47 to determine which proteins were contained in the native RIPE3a binding complex . Using these antibodies, we were able to separate the complex into major and minor fractions which contained either E12/47 or BETA 1, respectively . Thus, these two gene products are found in separate fractions of the tissue-specific binding activity and are therefore both likely to be important in insulin gene regulation.

J Biol Chem, 1994 Oct 14, 269(41), 25916 - 21
Structural features of the eIF-5A precursor required for posttranslational synthesis of deoxyhypusine; Joe YA et al.; Eukaryotic translation initiation factor 5A (eIF-5A, older nomenclature, eIF-4D) is a highly conserved protein that contains the unusual amino acid hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine) . The biosynthesis of hypusine occurs posttranslationally in only this protein by modification of a single lysine residue (Lys50 in the human eIF-5A precursor) . The basis for the specificity of this modification with respect to the substrate protein was investigated using fragments of eIF-5A precursor protein, each containing this lysine residue, as substrates for deoxyhypusine synthase, the first enzyme in hypusine synthesis . Proteolytic fragments (5-6 kDa) of ec-eIF-5A (the precursor form of eIF-5A produced in Escherichia coli by expression of the human eIF-5A cDNA) generated by specific cleavage by endoproteinases Arg-C, Asp-N, or Glu-C, did not act as substrates for deoxyhypusine synthesis . A series of truncated forms of the eIF-5A precursor protein generated by expression in E . coli of recombinant deletion constructs from the human eIF-5A cDNA were tested . Truncation of up to 9 amino acid residues (Met1-Thr9) from the NH2 terminus or 64 amino acid residues (Leu91-Lys154) from the COOH terminus did not significantly decrease the substrate reactivity, but removal of an additional 10 amino acids from either side did . Deletion of 34 amino acid residues (Met1-Lys34) from the NH2 terminus or of 84 amino acid residues (Asp71-Lys154) from the carboxyl terminus caused complete loss of substrate property . The results obtained thus far define the minimum domain of the eIF-5A precursor protein required for enzymatic deoxyhypusine synthesis as Phe30-Asp80, which corresponds to a region of high amino acid conservation in this protein throughout the eukaryotic kingdom.

J Biol Chem, 1994 Oct 14, 269(41), 25911 - 5
Activity of two-chain recombinant human cytomegalovirus protease; Holwerda BC et al.; The human cytomegalovirus UL80 protease was expressed in Escherichia coli and purified by metal-chelate chromatography using a histidine tag engineered at the amino terminus . Cleavage of the 30-kDa protease at an internal site, VEA/A144, resulted in the recovery of 16- plus 14-kDa two-chain protease . The amino-terminal 16-kDa chain and the carboxyl-terminal 14-kDa chain remained associated as an active enzyme that was modified specifically at Ser132 on the 16-kDa chain by {3H}diisopropyl fluorophosphate . Disruption of the cleavage site by mutation from VEA/A to AEA/A facilitated the recovery of active 30-kDa one-chain enzyme that could be similarly modified at Ser132 by {3H}diisopropyl fluorophosphate . Both one- and two-chain enzymes cleaved recombinant assembly protein at the maturation site, VNA/S, and a peptide, GVVNASARL, mimicking this site . Internal processing does not inactivate the protease but forms a two-chain enzyme that retains activity.

J Biol Chem, 1994 Oct 14, 269(41), 25770 - 6
Pure and functionally homogeneous recombinant retinoid X receptor; Chen ZP et al.; Mouse retinoid X receptor alpha (RXR alpha) lacking the amino-terminal region A/B (RXR alpha delta AB) has been purified to more than 98% purity and functional homogeneity from bacterial and baculovirus-based recombinant expression systems with yields of 2-8 mg/liter of culture . The purified protein is soluble, and fluorescence quenching analysis demonstrated that it binds its cognate ligand 9-cis-retinoic acid (9-cis-RA) stoichiometrically, and with high affinity . Compared with RXR delta AB expressed in COS-1 cells, bacterially and baculovirus-expressed proteins bind approximately 10 and 5 times less efficiently to direct repeat 1 (DR1) DNA elements, respectively, suggesting that animal cell-specific modification of RXR or interaction with other animal cell-specific factors may modulate DNA binding . 9-cis-RA did not stimulate DR1 binding of functional RXR delta AB expressed in Escherichia coli, Sf9 or COS-1 cells . The previously reported ligand effect that can be observed with in vitro made receptor may therefore be a consequence of a conformational stabilization of improperly folded in vitro synthesized protein.

J Biol Chem, 1994 Oct 14, 269(41), 25576 - 80
delta 9 Acyl-lipid desaturases of cyanobacteria . Molecular cloning and substrate specificities in terms of fatty acids, sn-positions, and polar head groups; Sakamoto T et al.; In cyanobacteria, the biosynthesis of unsaturated fatty acids is initiated by delta 9 acyl-lipid desaturase which introduces the first double bond at the delta 9 position of a saturated fatty acid that has been esterified to a glycerolipid . We have cloned genes, designated desC, for delta 9 acyl-lipid desaturases from two cyanobacteria, namely Anabaena variabilis and Synechocystis sp . PCC 6803 . These desaturases, when expressed in Escherichia coli, desaturated stearic acid to yield oleic acid at the C-1 positions of phosphatidylethanolamine and phosphatidylglycerol, but did not desaturate palmitic acid, palmitoleic acid, and cis-vaccenic acid . These results indicate that the delta 9 acyl-lipid desaturases are specific to stearic acid esterified at the C-1 position of a glycerolipid and are nonspecific with respect to the polar head group of the glycerolipid . The deduced amino acid sequences of the delta 9 acyl-lipid desaturases are similar in part to those of stearoyl-CoA desaturases of the rat, the mouse, and Saccharomyces cerevisiae, but not to those of acyl-(acyl-carrier-protein) desaturases of higher plants.

J Biol Chem, 1994 Oct 14, 269(41), 25442 - 6
ATP-dependent arsenite transport in everted membrane vesicles of Escherichia coli; Dey S et al.; Resistance to toxic oxyanions of arsenic and antimony in Escherichia coli results from active efflux of these anions out of the cell . Extrusion is an active process mediated by an ATP-dependent pump composed of two types of subunits, the integral membrane ArsB protein and the catalytic ArsA subunit . An in vitro assay for transport in everted membrane vesicles of E . coli was developed . Uptake of 73AsO2- by everted vesicles was time- and temperature-dependent and required both pump subunits . Transport required ATP; no other nucleotide, including GTP, CTP, UTP, or the nonhydrolyzable analog adenosine 5'-O-(thiotriphosphate), could substitute for ATP . Protonophores, ionophores, or inhibitors of other types of ion-motive ATPases did not inhibit arsenite uptake . The sulfhydryl reagent N-ethylmaleimide was a potent inhibitor of ATP-dependent arsenite accumulation in vesicles . The apparent Km values for ATP and arsenite were approximately 2 and 0.1 mM, respectively . Antimonite, the most potent activator of the ArsA ATPase, inhibited arsenite uptake with an apparent Ki of 10 microM.

J Biol Chem, 1994 Oct 14, 269(41), 25359 - 64
Drosophila ribosomal protein S3 contains an activity that cleaves DNA at apurinic/apyrimidinic sites; Wilson DM 3rd et al.; A rat cDNA-encoding ribosomal protein S3 was used to clone the S3 homolog in Drosophila melanogaster . The Drosophila gene was in turn used to construct fusions between S3 and glutathione S-transferase that were overexpressed in Escherichia coli, purified on affinity columns, and subsequently used for antibody production and biochemical analysis . Antibody specific for S3 was originally tested to determine the subcellular location of S3 by Western (immunoblot) analysis . As expected, the S3 antigen was found associated with purified preparations of ribosomes, but notably the protein was also observed in the nucleus where it was found to be tightly associated with the nuclear matrix . This result, combined with the fact that S3 contains a nuclear localization signal and that the protein shares some homology to a yeast nuclease gene, suggested that S3 might possibly have a role in DNA metabolism . Tests were initially performed to see if S3 contained DNase activity, where it was subsequently determined that the protein specifically cleaved DNA containing an apurinic/apyrimidinic site via a beta-elimination reaction . The DNase activity was inactivated by antibody to S3, indicating that the apurinic/apyrimidinic lyase activity was associated with the Drosophila S3 protein . Taken together, these results suggest that S3 is among a growing class of multifunctional proteins with roles in transcription/translation and DNA repair.

J Biol Chem, 1994 Oct 14, 269(41), 25310 - 4
Escherichia coli expresses a copper- and zinc-containing superoxide dismutase; Benov LT et al.; A mutant of Escherichia coli, unable to produce manganese- or iron-containing superoxide dismutase (SOD), was found to contain modest levels of an SOD that was judged to be a copper- and zinc-containing SOD on the basis of inhibition by cyanide and inactivation by either H2O2 or diethyldithiocarbamate . Moreover, the diethyldithiocarbamate-inactivated enzyme could be reactivated with Cu(II), and this reconstituted enzyme, like the native enzyme, was unaffected by EDTA and was inhibited by cyanide . This enzyme was, furthermore, selectively released by osmotic shock, in keeping with a periplasmic localization, and it was strongly induced during aerobic growth . This enzyme was also present in the SOD-competent parental strain . Failure to detect it previously can be attributed to its periplasmic localization, thermal lability, sensitivity to pH, and to its relative paucity . It will now be interesting to explore the phenotypic consequences imposed by the absence of this SOD.

J Biol Chem, 1994 Oct 14, 269(41), 25283 - 8
Transsulfuration depends on heme in addition to pyridoxal 5'-phosphate . Cystathionine beta-synthase is a heme protein; Kery V et al.; The first committed step of transsulfuration is catalyzed by cystathionine beta-synthase (CBS), a known pyridoxal 5'-phosphate (PLP) enzyme . The inferred amino acid sequences of rat liver CBS and rat liver hemoprotein H-450 are identical . We now confirm the presence of heme b in rat and human liver CBS . Heme almost entirely accounts for the visible spectrum of CBS rather than PLP . Human CBS, expressed in Escherichia coli, acquires heme b from the host bacteria . delta-Aminolevulinate supplementation during bacterial growth increases both the heme saturation and the specific activity of the homogeneous enzyme more than 3-fold . 1 mol of the 63-kDa CBS subunit binds 1 mol of each (heme and PLP) . The presence of heme is required for PLP binding, and the amount of PLP bound is limited by the heme content . Removal of PLP, but not heme, from CBS is reversible . These findings suggest that heme is functionally incorporated into CBS only during protein folding . This report describes the first instance of an enzyme that depends upon both heme and PLP for its function.

J Biol Chem, 1994 Oct 14, 269(41), 25251 - 4
Novel isoprenylated proteins identified by an expression library screen; Biermann BJ et al.; Isoprenylated proteins are involved in eukaryotic cell growth and signal transduction . The protein determinant for prenylation is a short carboxyl-terminal motif containing a cysteine, to which the isoprenoid is covalently attached via thioether linkage . To date, isoprenylated proteins have almost all been identified by demonstrating the attachment of an isoprenoid to previously known proteins . Thus, many isoprenylated proteins probably remain undiscovered . To identify novel isoprenylated proteins for subsequent biochemical study, colony blots of a Glycine max cDNA expression library were {3H}farnesyl-labeled in vitro . Proteins identified by this screen contained several different carboxyl termini that conform to consensus farnesylation motifs . These proteins included known farnesylated proteins (DnaJ homologs) and several novel proteins, two of which contained six or more tandem repeats of a hexapeptide having the consensus sequence (E/G)(G/P)EK(P/K)K . Thus, plants contain a diverse array of genes encoding farnesylated proteins, and our results indicate that fundamental differences in the identities of farnesylated proteins may exist between plants and other eukaryotes . Expression library screening by direct labeling can be adapted to identify isoprenylated proteins from other organisms, as well as proteins with other post-translational modifications.

J Biol Chem, 1994 Oct 14, 269(41), 25922 - 7
Structure-specific cleavage of the RNA primer from Okazaki fragments by calf thymus RNase HI; Huang L et al.; Cleavage specificity of RNase HI was examined on model Okazaki fragments, to determine the likely role of this nuclease in lagging strand DNA replication . Each substrate was prepared by annealing a short RNA primer, made by transcription in vitro, to a single-stranded synthetic DNA template, and subsequently extending the primer by DNA polymerization . The calf thymus RNase HI makes a structure-specific endonucleolytic cleavage in the RNA primer, releasing it intact, and leaving a mono-ribonucleotide at the 5' terminus of the RNA-DNA junction . This specific cleavage, one nucleotide upstream of the RNA-DNA junction, is RNA primer sequence- and length-independent . Cleavage specificity is lost if the RNA primer is not extended with DNA, or if the substrate has a nick at the RNA-DNA junction . In addition, the cleavage at a single site requires Mg2+ . Cleavage in the presence of Mn2+ is less specific . Neither human immunodeficiency virus reverse transcriptase nor Escherichia coli RNases H perform such a structure-specific cleavage before an RNA-DNA junction . Our work indicates that calf RNase HI is designed to recognize Okazaki fragments . It has the specificity to remove their initiator RNA segments, except for one ribonucleotide, by a single endonucleolytic cleavage in vivo.

J Biol Chem, 1994 Oct 14, 269(41), 25255 - 8
Resistance of HIV-1 reverse transcriptase against {2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro-5''-(4''-amino-1'',2''- oxathiole-2'',2''-dioxide)} (TSAO) derivatives is determined by the mutation Glu138-->Lys on the p51 subunit; Jonckheere H et al.; Determination of the three-dimensional structure of the human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) has indicated a totally different folding for the 51-kDa subunit (p51) than for the 66-kDa subunit (p66) . The polymerase catalytic site is located on the p66 subunit . Moreover, the HIV-1-specific RT inhibitors, also designated as the non-nucleoside RT inhibitors (NNRTIs), select for amino acid mutations that afford resistance to these compounds and are clustered in the palm domain of the HIV-1 RT p66 subunit . This pocket is located in the vicinity of, but clearly distinct from, the polymerase active site . However, for the NNRTIs that belong to the class of the {2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro-5''-(4''-amino-1'',2''- oxathiole- 2'',2''-dioxide)} (TSAO) derivatives, the resistance mutation is located at position Glu138 . On the p66 subunit, this amino acid is distant from the binding site of the HIV-1-specific RT inhibitors . When the TSAO-specific resistance mutation Glu138-->Lys was introduced solely in the p51 subunit of the RT p66/p51 heterodimer, the enzyme proved completely resistant to TSAO-m3T but retained full sensitivity to TIBO R82150 and ddGTP . On the other hand, when the mutation was introduced only in the p66 subunit the enzyme remained equally sensitive to the inhibitory effects of TSAO-m3T, TIBO R82150, and ddGTP . Our data provide compelling evidence for a structural and functional role of the p51 subunit in the sensitivity and/or resistance of the enzyme to the NNRTIs.

Nature, 1994 Oct 13, 371(6498), 614 - 9
Residues in chaperonin GroEL required for polypeptide binding and release; Fenton WA et al.; Chaperonins are ring-shaped protein complexes that are essential in the cell, mediating ATP-dependent polypeptide folding in a variety of compartments . Recent studies suggest that they function through multiple rounds of binding and release of non-native proteins: with each round of ATP-driven release into the bulk solution, a substrate protein kinetically partitions between folding to the native state or rebinding to another chaperonin molecule . To gain further insight into the mechanism of polypeptide binding and release by the chaperonin GroEL from Escherichia coli, we have undertaken a mutational analysis that relates the functional properties of GroEL to its crystal structure . Our functional tests identify a putative polypeptide-binding site on the inside surface of the apical domain, facing the central channel, consisting of hydrophobic residues . These same residues are essential for binding of the co-chaperonin GroES, which is required for productive polypeptide release . A highly conserved residue, Asp 87, positioned within a putative nucleotide-binding pocket in the top of the equatorial domain, is essential for ATP hydrolysis and polypeptide release.

Proc Natl Acad Sci U S A, 1994 Oct 11, 91(21), 9980 - 4
Left-handed Z-DNA and in vivo supercoil density in the Escherichia coli chromosome; Lukomski S et al.; A system for studying Z-DNA formation in the Escherichia coli chromosome was developed . Prior investigations in recombinant plasmids showed that alternating (Pur-Pyr) sequences can adopt a left-handed Z-DNA conformation both in vitro and in vivo . We constructed mobile, transposon-based cassettes carrying cloned (Pur-Pyr) sequences containing an EcoRI site in the center . These cassettes were subsequently inserted into different locations in the E . coli chromosome in a random fashion . A number of stable insertions were characterized by Southern analysis and pulsed-field gel electrophoresis mapping . A cloned temperature-sensitive MEcoRI methylase was expressed in trans as the probe to study Z-DNA formation in vivo . In this system, the control EcoRI sites were quickly methylated when cells were placed at the permissive temperature . Strong inhibition of the methylation was observed, however, only for the EcoRI sites embedded in a 56-bp run of (C-G) . In contrast, the shorter sequence of 32 bp did not show this behavior . Prior in vitro determinations revealed that the longer tract required less energy to stabilize the Z-helix than the shorter block . We conclude that the observed inhibition of methylation is due to Z-DNA formation in the E . coli chromosome . In vitro, these sequences undergo the B- to Z-DNA transition at a supercoil density of -0.026 for the 56-bp insert and -0.032 for the 32-bp block . Since only the longer (C-G) tract but not the shorter run adopted the left-handed conformation in the chromosome, we propose that these densities establish the boundaries in the different chromosomal loci investigated; these boundaries are in good agreement with the extremes found in plasmids.

Proc Natl Acad Sci U S A, 1994 Oct 11, 91(21), 9901 - 5
Dissociation of RecA filaments from duplex DNA by the RuvA and RuvB DNA repair proteins; Adams DE et al.; The RuvA and RuvB proteins of Escherichia coli act late in recombination and DNA repair to catalyze the branch migration of Holliday junctions made by RecA . In this paper, we show that addition of RuvAB to supercoiled DNA that is bound by RecA leads to the rapid dissociation of the RecA nucleoprotein filament, as determined by a topological assay that measures DNA underwinding and a restriction endonuclease protection assay . Disruption of the RecA filament requires RuvA, RuvB, and hydrolysis of ATP . These findings suggest several important roles for the RuvAB helicase during genetic recombination and DNA repair: (i) displacement of RecA filaments from double-stranded DNA, (ii) interruption of RecA-mediated strand exchange, (iii) RuvAB-catalyzed branch migration, and (iv) recycling of RecA protein.

Proc Natl Acad Sci U S A, 1994 Oct 11, 91(21), 9828 - 31
Mechanism of GTP hydrolysis by G-protein alpha subunits; Kleuss C et al.; Hydrolysis of GTP by a variety of guanine nucleotide-binding proteins is a crucial step for regulation of these biological switches . Mutations that impair the GTPase activity of certain heterotrimeric signal-transducing G proteins or of p21ras cause tumors in man . A conserved glutamic residue in the alpha subunit of G proteins has been hypothesized to serve as a general base, thereby activating a water molecule for nucleophilic attack on GTP . The results of mutagenesis of this residue (Glu-207) in Gi alpha 1 refute this hypothesis . Based on the structure of the complex of Gi alpha 1 with GDP, Mg2+, and AlF-4, which appears to resemble the transition state for GTP hydrolysis, we believe that Gln-204 of Gi alpha 1, rather than Glu-207, supports catalysis of GTP hydrolysis by stabilization of the transition state.

Proc Natl Acad Sci U S A, 1994 Oct 11, 91(21), 9813 - 7
Two additional glutaredoxins exist in Escherichia coli: glutaredoxin 3 is a hydrogen donor for ribonucleotide reductase in a thioredoxin/glutaredoxin 1 double mutant; Aslund F et al.; Thioredoxin (Trx) and glutaredoxin (Grx1) are hydrogen donors for ribonucleotide reductase, the key enzyme for deoxyribonucleotide biosynthesis . The viability of a double mutant lacking both Trx and Grx1 implies the presence of a third, unknown hydrogen donor . This paper reports the purification and characterization of two proteins with glutaredoxin activity (using hydroxyethyl disulfide as a substrate) from an Escherichia coli mutant lacking Trx and Grx1 (delta trxA, grx::kan) . Affinity chromatography was used to bind glutaredoxin on a glutathione-containing thiol-Sepharose column . The molecular weight of Grx2, 27,000, was atypical for glutaredoxins, whereas Grx3 had a molecular weight of 10,000 . Amino acid sequence analysis revealed novel structures with putative active sites typical of glutaredoxins: Cys-Pro-Tyr-Cys . The proteins are therefore referred to as Grx2 and Grx3 . The low hydrogen donor activity for ribonucleotide reductase in the crude extract was recovered in the purification of Grx3, whereas Grx2 was inactive . As a hydrogen donor for E . coli ribonucleotide reductase, Grx3 showed approximately the same Km value (0.35 microM) as Grx1, whereas its Vmax value was only 5% that of Grx1 . The combination of the Grx3 hydrogen donor activity and a 25-fold induction of ribonucleotide reductase activity in a delta trxA, grx double mutant provides an explanation for its viability and deoxyribonucleotide biosynthesis . The physiological functions of Grx2 and Grx3 remain to be determined.

Proc Natl Acad Sci U S A, 1994 Oct 11, 91(21), 9730 - 4
The Ada protein acts as both a positive and a negative modulator of Escherichia coli's response to methylating agents; Saget BM et al.; The adaptive response of Escherichia coli protects the cells against the toxic and mutagenic effects of certain alkylating agents . The major effector molecule regulating this response is the 39-kDa Ada protein, which functions as both a DNA repair protein and a transcriptional activator . Ada removes methyl groups from phosphotriester and O6-methylguanine lesions in DNA, irreversibly transferring them to cysteine residues at positions 69 and 321, respectively . When methylated at Cys-69, Ada is converted into a potent activator of ada and alkA transcription and binds to a sequence (Ada box) present in both promoters . We have found that physiologically relevant higher concentrations of unmethylated Ada are able to inhibit the activation of ada transcription by methylated Ada, both in vitro and in vivo . In contrast, the same concentrations of unmethylated Ada do not inhibit the activation of alkA transcription by methylated Ada, either in vitro or in vivo . Deletion of the carboxyl-terminal 67 amino acids of Ada abolished the ability of the unmethylated form of the protein to inhibit activation of ada transcription but not the ability of the methylated form to activate ada or alkA transcription . Our results suggest that the Ada protein plays a pivotal role in the negative modulation of its own synthesis and therefore in the down-regulation of the adaptive response . Elements present in the carboxyl terminus of Ada appear to be necessary for this negative regulatory function.

Proc Natl Acad Sci U S A, 1994 Oct 11, 91(21), 9685 - 9
High-level expression of human superoxide dismutase in the cyanobacterium Anacystis nidulans 6301; Takeshima Y et al.; A chemically synthesized gene encoding human CuZn superoxide dismutase (hSOD) was cloned into the shuttle vector pBAX18R and expressed in Anacystis nidulans 6301 (Synechococcus sp . strain PCC 6301) under the control of a ribulose-1,5-bisphosphate carboxylase/oxygenase gene (rbc) promoter derived from A . nidulans 6301 . The sequences immediately upstream from the hSOD coding region and the distances between the ribosomal binding site and ATG initiation codon strongly affected the expression of the hSOD gene in A . nidulans cells . Optimal expression of hSOD was obtained with the expression vector pBAXSOD8-I, which contained a GGAGAG sequence . In defined conditions, irradiation with light increased hSOD enzyme activity in the transformants > 18-fold and the level of the hSOD protein reached a value of about 3% of the total soluble protein . The transformants that expressed hSOD acquired the ability to extenuate photooxidative damage induced by methyl viologen.

Proc Natl Acad Sci U S A, 1994 Oct 11, 91(21), 10059 - 63
Arabidopsis thaliana gamma-glutamylcysteine synthetase is structurally unrelated to mammalian, yeast, and Escherichia coli homologs; May MJ et al.; A mutant of Escherichia coli, JTG10, deficient in gamma-glutamylcysteine synthetase (gamma-ECS; EC 6.3.2.2) is unable to synthesize glutathione (GSH) and is sensitive to 8-hydroxyquinoline . This phenotype was exploited for the isolation of Arabidopsis thaliana gamma-ECS cDNAs by expression cloning, and clones were selected through functional complementation by growth on 8-hydroxyquinoline . High levels of gamma-ECS activity were detectable in extracts derived from cultures of JTG10 expressing the Arabidopsis gamma-ECS open reading frame, although these complemented mutants accumulated GSH to only 10% of the wild-type level . The derived amino acid sequence constitutes a polypeptide of 59.9 kDa and shows only 44-48% similarity with previously published sequences of rat kidney, human liver, yeast, and E . coli gamma-ECS . When the gamma-ECS cDNA was used as a probe, Southern blot analysis of Arabidopsis genomic DNA revealed that it is present as a low copy number gene . Furthermore, the Arabidopsis gamma-ECS cDNA probe failed to hybridize to maize and tobacco genomic DNA at low stringency, suggesting that heterogeneity in gamma-ECS structure exists between plant species . The activity of recombinant Arabidopsis gamma-ECS was inhibited by buthionine sulfoximine and GSH, indicating that, while differences in the primary and secondary structure of gamma-ECS from different sources exist, the enzymes may have similar active site structures.

Proc Natl Acad Sci U S A, 1994 Oct 11, 91(21), 10039 - 43
Site-specific integration by adeno-associated virus is directed by a cellular DNA sequence; Giraud C et al.; Different regions of an 8.2-kb cloned DNA segment containing the target for adeno-associated virus (AAV) integration in human chromosome 19q13-3-qter (AAVS1 locus) were subcloned in an Epstein-Barr virus-based shuttle vector and propagated as episomes in a derivative of the 293 human embryonic kidney cell line . Preferential recombination with an infecting AAV genome was assessed by measuring the frequency of recombinants among the shuttle vectors recovered in Escherichia coli . The signals which direct recombination with the AAV genome were localized to a 510-nt region at the 5' end of the 8.2-kb AAVS1 DNA . Hence, the results indicate that site-specific integration of AAV is directed by a specific DNA sequence on human chromosome 19 . An unusual degree of DNA heterogeneity in the recovered vector was also associated with the 510 nt at the 5' end of AAVS1 DNA, suggesting that the AAV chromosomal integration locus may be involved in genomic instability.

Proc Natl Acad Sci U S A, 1994 Oct 11, 91(21), 10014 - 8
Liver mitochondrial cytochrome P450 CYP27 and recombinant-expressed human CYP27 catalyze 1 alpha-hydroxylation of 25-hydroxyvitamin D3; Axen E et al.; A cytochrome P450 catalyzing 1 alpha-hydroxylation of 25-hydroxyvitamin D3 was purified from pig liver mitochondria . It also catalyzed 27-hydroxylation of 25-hydroxyvitamin D3 and 25-hydroxylation of vitamin D3 . The ratio between the 1 alpha-, 27-, and 25-hydroxylase activities remained essentially constant during the purification . Substrates for sterol 27-hydroxylase CYP27 inhibited and a monoclonal antibody raised against CYP27 immunoprecipitated the 1 alpha-, 27-, and 25-hydroxylase activities . Apparently homogeneous preparations of CYP27 from pig and rabbit liver mitochondria catalyzed 1 alpha-hydroxylation . Human liver mitochondrial CYP27 was expressed from its cDNA in Escherichia coli . The nucleotide sequence encoding the N terminus of CYP27 was modified in the first eight codons to achieve expression in E . coli . The purified recombinant-expressed CYP27 reconstituted with the electron-transferring system of adrenal mitochondria catalyzed 1 alpha-hydroxylation of 25-hydroxyvitamin D3 . Expression of unmodified CYP27 cDNA in simian COS cells confirmed the 1 alpha-hydroxylase activity toward 25-hydroxyvitamin D3.

Nucleic Acids Res, 1994 Oct 11, 22(20), 4167 - 75
Genetic Bit Analysis: a solid phase method for typing single nucleotide polymorphisms; Nikiforov TT et al.; A new method for typing single nucleotide polymorphisms in DNA is described . In this method, specific fragments of genomic DNA containing the polymorphic site(s) are first amplified by the polymerase chain reaction (PCR) using one regular and one phosphorothioate-modified primer . The double-stranded PCR product is rendered single-stranded by treatment with the enzyme T7 gene 6 exonuclease, and captured onto individual wells of a 96 well polystyrene plate by hybridization to an immobilized oligonucleotide primer . This primer is designed to hybridize to the single-stranded target DNA immediately adjacent from the polymorphic site of interest . Using the Klenow fragment of E . coli DNA polymerase I or the modified T7 DNA polymerase (Sequenase), the 3' end of the capture oligonucleotide is extended by one base using a mixture of one biotin-labeled, one fluorescein-labeled, and two unlabeled dideoxynucleoside triphosphates . Antibody conjugates of alkaline phosphatase and horseradish peroxidase are then used to determine the nature of the extended base in an ELISA format . This paper describes biochemical features of this method in detail . A semi-automated version of the method, which we call Genetic Bit Analysis (GBA), is being used on a large scale for the parentage verification of thoroughbred horses using a predetermined set of 26 diallelic polymorphisms in the equine genome.

Nucleic Acids Res, 1994 Oct 11, 22(20), 4119 - 24
Cloning of a marsupial DNA photolyase gene and the lack of related nucleotide sequences in placental mammals; Kato T Jr et al.; Photoreactivating enzyme, DNA photolyase, reduces lethal, mutagenic and carcinogenic effects of ultraviolet light (UV) by catalyzing near UV or visible light-dependent repair of cyclobutane pyrimidine dimers (CPDs) in DNA . The enzyme activity has been detected in a wide variety of organisms ranging from bacteria to nonplacental mammals . However, the evidence for photoreactivation in placental mammals, including humans, is controversial . As a first step to identify the presence and activity of the gene in mammalian species, we isolated a cDNA clone of this gene from a marsupial, the South American opossum Monodelphis domestica . Photolyase activity was expressed in Escherichia coli from the cDNA which is predicted to encode a polypeptide of 470 amino acid residues . The deduced amino acid sequence of this protein is strikingly similar to those of photolyases from two metazoans; the opossum photolyase shares 59% and 63% sequence identity with the Drosophila melanogaster and goldfish Carassius auratus enzymes, respectively . However, no closely related nucleotide sequence was detected in higher mammals and a homologous transcript was undetectable in a number of human tissues . These results strongly suggest that humans, as well as other placental mammals, lack the photolyase gene.

Gene, 1994 Oct 11, 148(1), 71 - 4
Positive-selection vectors using the F plasmid ccdB killer gene; Bernard P et al.; Plasmids pKIL18/19 are positive-selection cloning vectors containing an active cytotoxic ccdB gene under the control of the lacP promoter . They are derivatives of high-copy-number pUC18/19 plasmids in which the ccdB killer gene has been fused in phase downstream from the lacP MCS18 and MCS19 multiple cloning sites . When an Escherichia coli wild-type gyrA+ strain is transformed by such vectors, the ccdB gene product blocks bacterial growth . However, if ccdB is inactivated by insertion of a foreign DNA fragment, this recombinant plasmid no longer interferes with host viability . The positive selection of recombinant clones is highly efficient and bench manipulations are simplified to the utmost: E . coli transformants are plated on rich medium and only cells containing recombinant plasmids give rise to colonies . The CcdB protein is a potent poison of gyrase and the gyrA462 mutation confers total resistance to CcdB {Bernard and Couturier, J . Mol . Biol . 226 (1992) 735-745} . Therefore, pKIL18/19 vectors can be amplified and prepared in large quantities in a gyrA462 host . Like pUC vectors, pKIL vectors are designed for general cloning/sequencing procedures.

Gene, 1994 Oct 11, 148(1), 169 - 70
A new DNA cloning/sequencing vector with a built-in mechanism for generation of nested deletions using transposon Tn3; Sugino Y et al.; We have constructed a new cloning/sequencing vector suitable for sequencing of DNA fragments too long to be sequenced in a single step . This vector plasmid contains inverted repeats (IR) of transposon Tn3, the kil and cI857 genes of phage lambda, and multiple cloning sites (MCS) . Escherichia coli cells harboring plasmids containing nested deletions from one of the Tn3 IR ends, across kil, to variable end points, can be positively selected by plating at 42 degrees C . The deletion products, fractionated according to their size by agarose-gel electrophoresis, can be sequenced by using a synthetic primer whose 3'-end is located within the Tn3 IR, and the total sequence of the insert can be constructed from the partial sequences.

Gene, 1994 Oct 11, 148(1), 15 - 21
The increase in gene expression induced by introduction of rare codons into the C terminus of the template; Gursky YG et al.; Short oligodeoxynucleotides (oligos) possessing two tandem Arg codons followed by TGA stop codon were inserted near the 3' end of a modified cat gene . It was found that while being decoded in vivo, the AGGAGGTGA oligo increased the yield of gene product and, in addition, caused -1 frameshifting . The 3-10-fold increase of the yield of the polypeptide was accompanied by increased accumulation of corresponding mRNA, indicating a protection from messenger decay . Transformation of the cells by a plasmid overproducing tRNA(4Arg) gene compensates for all the anomalies.

Gene, 1994 Oct 11, 148(1), 113 - 8
Functional analysis of transcription of the Mycobacterium tuberculosis 16S rDNA-encoding gene; Verma A et al.; A functional analysis of Mycobacterium tuberculosis 16S ribosomal RNA (rRNA) transcription and processing was undertaken in this study . RNA:DNA hybridizations indicated that the maximum transcriptional activity of rRNA-encoding genes (rDNA) corresponded to the earliest period of exponential growth . Transcription start points (tsp) were mapped by primer extension analysis of RNA from M . tuberculosis H37Rv and M . tuberculosis H37Ra . An identical pattern of rRNA transcription and processing was exhibited in laboratory-grown cultures of M . tuberculosis H37Rv and H37Ra . One promoter represents the structural equivalent of the Escherichia coli rrn P2 promoter . The precursor transcripts are processed into mature 16S rRNA through a pathway that includes recognition of RNA secondary structure by ribonuclease III (RNase III) in the stem structure surrounding the 16S rRNA indicating that at least this RNA processing step is conserved in mycobacteria and E . coli . The 16S rDNA promoter region from H37Rv was cloned upstream from the promoterless chloramphenicol (Cm) acetyltransferase (CAT)-encoding gene (cat) in a shuttle plasmid vector, pSD7 . The promoter-fusion construct, pSD7.16S, was characterized by CAT assays, measurement of percent survival in Cm-containing medium and in vivo transcription analysis in M . smegmatis . The M . smegmatis transformant exhibited a CAT activity of 16,669 nmol/min per mg protein, suggesting that the 16S promoter was of exceptionally high strength . Two tsp utilized in M . tuberculosis were also employed in M . smegmatis . The cat mRNA synthesized under the direction of the ribosomal promoter was less stable, as compared to genome-derived rRNA.

Biochemistry, 1994 Oct 11, 33(40), 12323 - 8
Investigation of the GTP-binding/GTPase cycle of Cdc42Hs using fluorescence spectroscopy; Leonard DA et al.; We have developed several high-resolution assays for the nucleotide state of a rho-subfamily low molecular weight GTP-binding protein, Cdc42Hs . The first involves the use of the fluorescent N-methylanthraniloyl derivative of GDP (mant-GDP) . As has been shown for the ras protein, mant-dGDP fluorescence is significantly enhanced (approximately 20%) upon binding to Cdc42Hs . It was further found that the binding of mant-nucleotides results in an efficient energy transfer between the single tryptophan residue of Cdc42Hs and the mant moiety . The exchange of mant-dGDP for GDP bound to Cdc42Hs, as read-out either by the enhancement of the mant fluorescence or by energy transfer, is inhibited by physiological (mM) Mg2+ concentrations and correlates exactly to the rate of {3H}GDP exchange observed in filter-binding assays . Moreover, changes in the fluorescence of mant-dGDP are also sensitive to nucleotide dissociation induced by the dbl-oncogene product, a known nucleotide exchange factor for Cdc42Hs . A second fluorescence read-out for the nucleotide-bound state of Cdc42Hs involves the measurements of intrinsic fluorescence of a single tryptophan residue (W97) which is highly sensitive to whether GDP or GTP is bound in the nucleotide pocket . The hydrolysis of GTP to GDP by Cdc42Hs results in an approximately 30% enhancement of the protein fluorescence . The rate of this fluorescence change corresponds well to the rate of conversion of {gamma-32P}GTP to GDP plus {32P}Pi as measured by filter-binding assays.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1994 Oct 11, 33(40), 12267 - 75
Transient conformational states of aminoacyl-tRNA during ribosome binding catalyzed by elongation factor Tu; Rodnina MV et al.; Conformational transitions of Phe-tRNA(Phe) that take place during elongation factor Tu (EF-Tu)-dependent binding to the A site of Escherichia coli ribosomes were followed by transient fluorescence measurements . The fluorescence signal of proflavin replacing dihydrouracil at position 16 or 17 in yeast tRNA(Phe) was utilized to monitor changes of the conformation of the D loop . The ternary complex EF-Tu.GTP.Phe-TRNA(Phe)(Pf16/17) was purified by gel filtration . Upon binding of the complex to the A site of poly(U)-programmed, P-site-blocked ribosomes, the fluorescence changes in several steps . First, the rapid formation of an initial complex gives rise to a small fluorescence increase . Subsequent codon-anticodon recognition leads to a conformational rearrangement of the D loop of the tRNA that is reflected in a major fluorescence increase . Fluorescence-quenching data indicate an unfolding of the D loop in this state . The latter conformational state is short-lived, and the aminoacyl-tRNA refolds during the following rearrangement that occurs after GTP hydrolysis and accompanies the release of the aminoacyl-tRNA from EF-Tu.GDP and/or its accommodation in the A site . Further experiments show that the status of the P site influences the binding to the A site in that the two rearrangement steps are slowed down when the P site is unoccupied and even more so when it is occupied with the near-cognate tRNA(Leu2) . In contrast, the occupancy of the E site has no influence on A-site binding, and vice versa, thus excluding any coupling between the two sites.

Biochemistry, 1994 Oct 11, 33(40), 12260 - 6
A cysteine in the C-terminal region of alanyl-tRNA synthetase is important for aminoacylation activity; Wu MX et al.; Alanyl-tRNA synthetase (AlaRS) from Escherichia coli is a multimeric enzyme that catalyzes the esterification of alanine to tRNA(Ala) in the ATP-dependent aminoacylation reaction . The functional binding of all three substrates follows Michaelis-Menten kinetics . The role of cysteines in this enzyme has been evaluated via modification of these residues with p-(hydroxymercuri)phenylsulfonic acid, monobromobimane, and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) . The former two reagents induce nearly complete inactivation of AlaRS aminoacylation activity and the release of all tightly bound zinc . In the case of mild DTNB treatment, only two of the six cysteines in AlaRS are modified, with release of all zinc and partial loss of aminoacylation activity . These experiments indicate the importance of one or more cysteines, other than those thought to be coordinated with zinc, in the aminoacylation reaction . Substitution of each of the cysteine residues outside the zinc-binding motif with serine does not disrupt zinc binding . However, the cysteine most removed in primary sequence from the active site (Cys665) is identified as important in the aminoacylation step . Mutation of Cys665 to serine induces a 120-fold decrease in the catalytic efficiency of this enzyme, primarily through a kcat effect, and introduces sigmoidal kinetics (nH = 1.8) with respect to the RNA substrate . The results demonstrate that a simple manipulation in the C-terminal region can introduce positive cooperativity in this otherwise noncooperative enzyme.

Biochemistry, 1994 Oct 11, 33(40), 12166 - 71
Cysteine 148 in the lactose permease of Escherichia coli is a component of a substrate binding site . 2 . Site-directed fluorescence studies; Wu J et al.; By using site-directed fluorescence spectroscopy, we have carried out structure/function studies on lactose permease purified from Escherichia coli in dodecyl beta, D-maltoside . Initially, permease containing a single native Cys at position 148 (helix V) was studied, since this residue is protected against alkylation by substrates of the permease . In the absence of ligand, Cys 148 permease reacts rapidly with 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS), a fluorophore whose quantum yield increases dramatically upon reaction with a thiol, indicating that this residue is readily accessible to the probe . Various ligands of the permease block the reaction, and the concentration dependence is commensurate with the affinity of each ligand for the permease (i.e., beta, D-galactopyranosyl 1-thio-beta, D-galactopyranoside << lactose < galactose), but neither sucrose nor glucose has any effect whatsoever . Thus, the permease retains the ability to bind ligand specifically when the molecule is in dodecyl beta, D-maltoside . Permease containing single Cys substitutions in the vicinity of Cys 148 was also studied . Interestingly, labeling of Cys 145 which is presumed to be one helical turn removed from Cys 148 exhibits properties similar to those observed with Cys 148 permease, but the effects of ligand are far less dramatic . On the other hand, permease with a single Cys residue at position 146 or 147 behaves in a completely different manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1994 Oct 11, 33(40), 12160 - 5
Cysteine 148 in the lactose permease of Escherichia coli is a component of a substrate binding site . 1 . Site-directed mutagenesis studies; Jung H et al.; Cys 148 in the lactose permease of Escherichia coli has been replaced with hydrophobic (Ala, Val, Ile, Phe), hydrophilic (Ser, Thr), or charged (Asp, Lys) residues, and the properties of the replacement mutants have been analyzed . Although Cys 148 is not essential for transport, the size and polarity of the side chain at this position modifies transport activity and substrate specificity . Thus, small hydrophobic side chains (Ala, Val) generally increase the apparent affinity of the permease for substrate, while hydrophilic side chains (Ser, Thr, Asp) decrease apparent affinity and bulky or positively charged side chains (Phe, Lys) virtually abolish activity . In addition, hydrophilic substitutions (Ser, Thr, Asp) alter the specificity of the permease toward monosaccharides relative to disaccharides . On the basis of these and other observations, it is concluded that Cys 148 is located in a sugar binding site of lac permease and probably interacts hydrophobically with the galactosyl moiety . The postulate receives more direct support from site-directed fluorescence labeling studies presented in the following paper in this issue {Wu, J., & Kaback, H . R . (1994) Biochemistry (following paper in this issue)}.

Biochemistry, 1994 Oct 11, 33(40), 12109 - 18
Ribosomal components neighboring the conserved 518-533 loop of 16S rRNA in 30S subunits; Alexander RW et al.; We report the synthesis of a radioactive, photolabile oligodeoxyribonucleotide probe complementary to 16S rRNA nucleotides 518-526 and its exploitation in identifying 30S ribosomal subunit components neighboring its target site in 16S rRNA . Nucleotides 518-526 lie within an almost universally conserved single-stranded loop that has been linked to the decoding region of Escherichia coli ribosomes . On photolysis in the presence of activated 30S ribosomes, the probe site-specifically incorporates into proteins S3, S4, S7, and S12 (identified by SDS-PAGE, RP-HPLC, and immunological analysis); nucleotides C525, C526, and G527 adjacent to its target binding site; and the 3'-terminus of 16S rRNA . When the probe is photoincorporated into 30S subunits subjected to brief cold inactivation (SI subunits), S7 labeling is increased compared to activated subunit incorporation, while S3, S4, and S12 labeling is decreased, as is labeling to nucleotides C525, C526, and G527; labeling at the 16S rRNA 3'-terminus appears unchanged . Longer cold inactivation of the 30S subunits (LI subunits) leads to decreases in the labeling of all components . These results provide clear evidence that C526 lies within 24 A (the distance between C526 and the photogenerated nitrene) of proteins S3, S4, S7, and S12 and the 3'-terminus of 16S rRNA . The identity of the tryptic digestion patterns of S7 labeled with the probe complementary to 16S rRNA nucleotides 518-526 and with a probe complementary to nucleotides 1397-1405 {Muralikrishna, P., & Cooperman, B . S . (1994) Biochemistry 33, 1392-1398} also provides evidence for proximity between C526 and G1405 . Our results support the conclusion of Dontsova et al . {Dontsova, O., et al . (1992) EMBO J . 11, 3105-3116} in placing the 530 loop in close proximity to the decoding center of the 30S subunit but are apparently inconsistent with some protein-protein distances determined by neutron diffraction {Capel, M . S., et al . (1988) J . Mol . Biol . 200, 65-87} . This inconsistency suggests that a multistate model of subunit conformation may be required to account for the totality of results pertaining to the internal structure of the 30S subunit.

Biochemistry, 1994 Oct 11, 33(40), 12092 - 9
Use of aryl azide cross-linkers to investigate protein-protein interactions: an optimization of important conditions as applied to Escherichia coli RNA polymerase and localization of a sigma 70-alpha cross-link to the C-terminal region of alpha; McMahan SA et al.; In an effort to better understand protein-protein photoaffinity cross-linking using aryl azides, we have tested a number of factors influencing the cross-linking of the sigma 70 subunit of Escherichia coli RNA polymerase to core RNA polymerase . These factors include the effect of the incubations necessary for the derivatization of the protein on enzyme activity, the effect of overhead lighting on azide stability, the effect of reducing agents on azide stability, aggregation of the derivatized protein, and a comparison of two types of aryl azide cross-linkers, N-{(5-azido-2-nitrobenzoyl)oxy}succinimide (ANB-NOS) and (N-hydroxysuccinimidyl)-4-azidosalicylic acid (NHS-ASA) . We found that derivatization proceeds effectively in a buffer similar to the buffer used during protein purification, that overderivatization can cause protein aggregation, that room lighting does not appreciably destroy aryl azides, and that 0.1 mM DTT is a better choice of reducing agent than 5 mM 2-mercaptoethanol . The cross-link products were separated by SDS gel electrophoresis and identified on Western blots by cross-reactivity with monoclonal antibodies to the individual subunits of RNA polymerase . In agreement with previous work (Coggins et al., 1977; Hillel & Wu, 1977), it was possible to cross-link sigma 70 to all three of the subunits of RNA polymerase . With a combination of gel analysis, chemical cleavage, and immunodetection, it is possible to demonstrate that sigma 70 cross-links to the alpha subunit between residues 209 and 329.

Biochemistry, 1994 Oct 11, 33(40), 12012 - 21
Kinetic mechanism for the model reaction of NADPH-cytochrome P450 oxidoreductase with cytochrome c; Sem DS et al.; The kinetic mechanism of NADPH-cytochrome P450 oxidoreductase (P450R) has been determined for the model reaction with cytochrome c3+ . Although initial velocity studies show parallel patterns, consistent with a classical (one-site) ping-pong mechanism that precludes the formation of a ternary NADPH-P450R-cytochrome c3+ complex, product and dead-end inhibition results suggest a nonclassical (two-site) ping-pong mechanism {Northrop, D . B . (1969) J . Biol . Chem . 244, 5808-5819} . This mechanism is a hybrid of the random sequential (ternary complex) and ping-pong mechanisms, since ternary complexes can form as well as intermediate, modified forms of the enzyme that can be present in the absence of any bound substrate . The complete rate equation is derived for this mechanism, and values for Vmax, (V/K)NADPH, (V/K)cytc, and the corresponding Michaelis constants are presented in terms of microscopic rate constants along with the expected product inhibition patterns (Appendix) . Inhibition by NADP+ is competitive versus NADPH and uncompetitive versus cytochrome c3+, while inhibition by cytochrome c2+ is competitive versus cytochrome c3+ and noncompetitive versus NADPH . These inhibition patterns are consistent with the proposed two-site mechanism . This mechanism would give the same initial velocity patterns as the classical one-site ping-pong mechanism, but it allows for the formation of a ternary complex, with NADPH and cytochrome c3+ reacting independently at two separate sites on P450R . The D(V/K)NADPH isotope effect is not affected by cytochrome c3+ concentration, consistent with our assumption (in deriving the rate equation) that binding at the two sites is independent . At the high ionic strength used in this study (850 mM), the mechanism is two-site ping-pong, with the electron acceptor site itself reacting with cytochrome c3+ in a tetra uni ping-pong manner.

Gene, 1994 Oct 11, 148(1), 23 - 32
Permissible peptide insertions surrounding the signal peptide-mature protein junction of the ClpG prepilin: CS31A fimbriae of Escherichia coli as carriers of foreign sequences; Der Vartanian M et al.; The clpG gene, expressing the Escherichia coli major CS31A fimbrial subunit ClpG, was subjected to random mutagenesis by insertion of an EcoRI linker and a kanamycin-resistance (KmR) cassette into the multiple newly generated EcoRI sites . The KmR gene was then excised by PstI, which left a 48-bp linker representing the heterologous sequence . The same procedure was followed to introduce a synthetic oligodeoxyribonucleotide (oligo) corresponding to epitope C from the spike protein S from the porcine transmissible gastroenteritis coronavirus (TGEV) . Nine insertion/deletion mutants (indels) that contained long foreign peptides variously located around the ClpG signal peptide (SP) processing site were characterized . A striking feature of this study is the variety of amino acid (aa) insertions in the ClpG prepilin that have little or no effect on CS31A fimbria biogenesis . These 'permissive' sites tolerate inserts of 18 or 19 aa and accept sequences of different natures in view of their aa composition, charge and hydrophobicity . The results obtained here are also interesting in light of the high level of aa sequence conservation seen in the SP and N-terminal domains of the ClpG-related subunits . The structure-function relationship of the ClpG SP is discussed . The TGEV-C epitope fused to the N-terminal end of the mature ClpG protein was cell-surface exposed, as observed on immuno-electron microscopy . Therefore, the CS31A fimbria seems to be a potent tool for the presentation of foreign antigenic determinants or the production of heterologous polypeptides in E . coli.

Gene, 1994 Oct 11, 148(1), 119 - 24
Characterization and sequence analysis of a Streptomyces rochei A2 endoglucanase-encoding gene; Perito B et al.; A 7-kb fragment of Streptomyces rochei A2 chromosomal DNA was cloned into pAT153 and shown to confer endoglucanase (EglS) activity on Escherichia coli cells . In E . coli clones, the EglS was secreted into the periplasm . Deletion analysis revealed that an 827-bp fragment was enough for the enzymatic activity . Sequence analysis showed that the 827-bp fragment codes for the catalytic domain of the enzyme . The complete sequence of the gene (eglS) is 1149-bp long . A signal peptide, a catalytic domain and a cellulose-binding domain were identified from the nucleotide sequence, and the EglS found to belong to the family H of cellulase catalytic domains . These conclusions were substantiated by determination of the N-terminal sequence of the purified protein and zymogram analysis, which revealed protein species with a molecular mass equal to that deduced from the nt sequence analysis.

Biochem Pharmacol, 1994 Oct 7, 48(7), 1371 - 7
Potential antitumour mitosenes: relationship between in vitro DNA interstrand cross-link formation and DNA damage in Escherichia coli K-12 strains; Maliepaard M et al.; This investigation was aimed at determining the possible relationship between DNA interstrand cross-linking and the cytotoxic activity of potential antitumour mitosene compounds . Mitosenes, possessing two good leaving groups at C-1 and C-10, were found to be able to cross-link calf thymus DNA under hypoxic conditions following sodium dithionite (Na2S2O4) reduction at pH 7.0 and pH 5.5 . DNA interstrand cross-linking was pH dependent for most of the mitosenes used, with a higher amount of cross-links formed at pH 5.5 compared to pH 7.0 . Without reduction or under aerobic conditions no cross-link formation was detected . The importance of DNA damage for the toxic effect of these mitosenes was assayed by comparing the survival in a DNA repair deficient and a DNA repair proficient E . coli K-12 strain . A correlation between the number of cross-links formed in calf thymus DNA in vitro and the IC50 values in the DNA repair deficient E . coli strain was found . The effect of hypoxia on toxicity of mitosenes was studied in Chinese hamster V79 cells . In these cells, mitosenes appeared to be very active . Under severe hypoxic conditions toxicity of these mitosenes increased, most likely due to the increased lifetime of the activated mitosene species as compared to aerobic conditions . The results suggest that DNA cross-linking following reductive activation is important for the eventual activity of mitosenes in a bacterial system . Increased activity of mitosenes under hypoxic conditions in the V79 cells indicates that these mitosenes may be more active in hypoxic parts of tumours.

J Mol Biol, 1994 Oct 7, 242(5), 712 - 4
Crystallization and preliminary X-ray diffraction studies on a recombinant isopenicillin N synthase from Cephalosporium acremonium; Fujishima Y et al.; Recombinant isopenicillin N synthase from Cephalosporium acremonium was expressed in Escherichia coli and the protein was purified . After nearly 5000 crystallization trials, the apo enzyme was crystallized by the hanging drop vapour diffusion technique, using polyethylene glycol and lithium sulphate as precipitants . Two crystal forms have been obtained with either octahedral or elongated prismatic habits . The larger octahedral crystals (0.1 mm over-all dimensions) belong to space group I4 with unit cell dimensions of a = b = 124.7 A, c = 156.9 A, and diffract X-rays to about 3.5 A resolution at synchrotrons . The crystallographic asymmetric unit contains a dimer.

J Mol Biol, 1994 Oct 7, 242(5), 703 - 5
Crystallization and preliminary X-ray analysis of the two domains of glucosamine-6-phosphate synthase from Escherichia coli; Obmolova G et al.; The glutamine amidohydrolase and fructose 6-phosphate binding domains of glucosamine-6-phosphate synthase from Escherichia coli have been overexpressed, purified and crystallized for X-ray diffraction analysis . The crystals of the glutamine amidohydrolase domain belong to the orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 70.4 A, b = 82.5 A, c = 86.1 A, with two molecules in the asymmetric unit, and diffract to 1.9 A resolution . The native Patterson indicated pseudo c-face centering of the unit cell . The fructose 6-phosphate binding domain was crystallized in the hexagonal space group P6(1) or P6(5) with cell dimensions a = b = 63.5 A, c = 334.3 A and with two molecules in the asymmetric unit . Diffraction data to 2.6 A resolution have been collected.

J Mol Biol, 1994 Oct 7, 242(5), 655 - 69
Correctly folded T-cell receptor fragments in the periplasm of Escherichia coli . Influence of folding catalysts; Wulfing C et al.; The T-cell receptor is the central recognition molecule in cellular immunity . Its extracellular domains are homologous with and thought to be structurally similar to an antibody Fab fragment . Despite the biological importance of the TCR and the ease of bacterial expression of antibody fragments, there are only few reports of TCR-fragment expression in E . coli . In order to understand the difficulties of expressing correctly folded TCR fragments in E . coli, we have characterized the expression behavior of single-chain Fv analogs of three different TCRs (scTCR) . All of them can be folded into the correct conformation in the periplasm of E . coli, yet the extent of correct folding varies greatly . In order to overcome the folding problems of some of the scTCRs, we have developed a system with enhanced in vivo folding capability based on the simultaneous induction of the heat-shock response and over-expression of the E . coli disulfide isomerase DsbA at low temperature . We present a model describing the folding of the scTCRs in the periplasm of E . coli and possible points of folding assistance . The role of the periplasm as an independent folding compartment is emphasized and the existence of a general periplasmic chaperone is postulated . We have also shown that a bivalent scTCR, dimerized in vivo with helix-turn-helix modules, can be expressed in a correctly folded form.

J Mol Biol, 1994 Oct 7, 242(5), 614 - 8
Genetic implication for an interaction between release factor one and ribosomal protein L7/L12 in vivo; Zhang S et al.; Strains with mutant alleles of the genes encoding release factor one (RF1), prfA, and ribosomal protein L7/L12, rplL, have been analyzed . The prfA1 allele has previously been shown to be associated with temperature sensitivity for growth at 43 degrees C and increased misreading of the nonsense codons UAG and UAA . Here we show that the prfA1 mutation is a nucleotide substitution that results in an arginine to proline change at amino acid position 137 in RF1 . This alters the factor in a domain which may be involved in ribosome-binding . The rplL564 allele codes for a mutant form of protein L7/L12 that has a change in its conserved, translation-factor binding, domain . The rplL564 mutation suppresses the temperature sensitive phenotype and the increased translational misreading associated with the prfA1 allele, while other studied rplL mutant alleles do not . These data are consistent with an interaction between L7/L12 and RF1 in vivo.

J Mol Biol, 1994 Oct 7, 242(5), 607 - 13
Gene activation by the Escherichia coli positive regulator, OmpR . Phosphorylation-independent mechanism of activation by an OmpR mutant; Tsuzuki M et al.; In Escherichia coli, expression of the major outer membrane proteins, OmpC and OmpF, is regulated through the functions of OmpR and EnvZ at the transcriptional level in response to the medium osmolarity . OmpR is the crucial activator that helps RNA polymerase to efficiently trigger ompC and ompF transcription . This OmpR function is modulated by phosphorylation mediated by the cognate sensory kinase, EnvZ . Phosphorylation at the N-terminal domain of OmpR results in substantial enhancement of the DNA-binding ability of the C-terminal domain, thereby allowing the activation of ompC and ompF transcription by OmpR . Here we isolated an OmpR mutant which lacks the N-terminal half, but can enhance transcription in vivo . This novel type of OmpR mutant was revealed to have a single amino acid replacement of Gly227 to Cys . The newly-introduced-Cys residue allows OmpR molecules to form a stable dimer in vitro without the help of the N-terminal half . This altered C-terminal half is able to bind efficiently and specifically to the cognate DNA in vitro . It can function as an activator for ompC transcription in vitro in a phosphorylation-independent manner . These results suggest that the putative activator domain of OmpR, together with the DNA-binding domain, is most likely located in the C-terminal half . They also suggested that the phosphorylation of OmpR may not be essential for gene activation per se.

J Biol Chem, 1994 Oct 7, 269(40), 25106 - 19
Characterization of a novel tumor-derived cytokine . Endothelial-monocyte activating polypeptide II; Kao J et al.; Endothelial-monocyte activating polypeptide II (EMAP II) was initially identified in the supernatant of murine methylcholanthrene A-induced fibrosarcomas (Meth A) by its capacity to activate host effector cells (Kao, J., Ryan, J., Brett, J., Chen, J., Shen, H., Fan, Y-G., Godman, G., Familletti, P., Wang, F., Pan, Y-C., Stern, D., and Clauss, M . (1992) J . Biol . Chem . 267, 20239-20247) . Based on the NH2-terminal protein sequence, a full-length cDNA has been cloned which indicates that the precursor of EMAP II is a unique, leaderless, single polypeptide chain with predicted molecular mass approximately 34 kDa and that the mature form released by Meth A cells corresponds to approximately 20 kDa . Purified recombinant mature EMAP II (EMAP II, approximately 20 kDa form) activated endothelial cells with resulting elevation of cytosolic free calcium concentration, release of von Willebrand factor, induction of tissue factor, and expression of the adhesion molecules E-selectin and P-selectin . Neutrophils exposed to EMAP II demonstrated elevated cytosolic free calcium concentration, peroxidase generation, and chemotaxis . EMAP II also activated mononuclear phagocytes elevating cytosolic free calcium concentration, inducing tumor necrosis factor-alpha (TNF) and tissue factor, and stimulating chemotaxis . Systemic infusion of EMAP II into C3H/HeJ or Balb/c mice was associated with systemic toxicity, pulmonary congestion, and the appearance of TNF, interleukin-1 and -6 in the plasma . A single intra-tumor injection of EMAP II into Meth A sarcomas induced acute thrombohemorrhage and partial tumor regression . Local injection of EMAP II into a tumor resistant to the effects of TNF, murine mammary carcinoma, rendered it sensitive to subsequently administered TNF, which resulted in acute thrombohemorrhage and partial regression . These data suggest that recombinant EMAP II, a tumor-derived cytokine, has properties of a proinflammatory mediator with the capacity to prime the tumor vasculature for a locally destructive process.

J Biol Chem, 1994 Oct 7, 269(40), 25091 - 4
Fine tuning the specificity of the periplasmic phosphate transport receptor . Site-directed mutagenesis, ligand binding, and crystallographic studies; Wang Z et al.; Phosphorous, primarily in the form of phosphate, is a critical nutrient for the life of a cell . We have previously determined the 1.7-A resolution structure of the phosphate-binding protein, an initial receptor for the high-affinity phosphate active transport system or permease in Escherichia coli (Luecke, H., and Quiocho, F.A . (1990) Nature 347, 402-406) . This structure is the first to reveal the key role of hydrogen bonding interactions in conferring the high specificity of the permease, a specificity also shared by other phosphate transport systems . Both monobasic and dibasic phosphates are recognized by the phosphate-binding protein with Asp56 playing a key role . Here we report site-directed mutagenesis, ligand binding, and crystallographic studies of the binding protein which show that introduction of one additional Asp by mutagenesis of the Thr141 in the ligand-binding site restricts binding to only the monobasic phosphate.

J Biol Chem, 1994 Oct 7, 269(40), 24883 - 9
Mutation of polar and charged residues in the hydrophobic NH2-terminal domains of the melibiose permease of Escherichia coli; Zani ML et al.; The suggestion that acidic residues in the hydrophobic NH2-terminal domains of Mel permease (Asp-31 in helix I, Asp-51 and Asp-55 in helix II, Asp-120 in helix IV) may be essential components of a coordination network involved in cation recognition (Pourcher, T., Zani, M.L., and Leblanc, G . (1993) J . Biol . Chem . 268, 3209-3215) is further analyzed using site-directed mutagenesis . To study whether nearby polar residues also contribute to the cation recognition process, Tyr-24, Tyr-27 and Tyr-28 (aligned with Asp-31) and Tyr-109 and Tyr-116 (aligned with Asp-120) were individually converted into a phenylalanine . The effect of replacing Arg-48 (aligned with Asp-51 and Asp-55) or Asn-83 (in the middle of helix III) by an alanine was also studied . The importance of the position of the carboxylate of the residue at position 31, 51, 55, or 120 was next examined by replacing each Asp by a Glu residue . Sugar binding and/or transport activity measurements indicate that all polar-->apolar or Asp-->Glu mutants use Na+ or Li+ for active sugar transport . Moreover, two groups of mutants could be distinguished . One group, composed of Y27F, Y28F, D31E, and Y109F mutants, retains wild type permease properties . A second group (Y24F, N83A, and Y116F and also D51E, D55E, and D120E) exhibits concomitant reduction of affinity for sodium and sugars and altered sugar specificity but conserves wild type cation selectivity profile . The data reinforce the notion that Asp-51, Asp-55, and Asp-120 residues and the position of their carboxyl side chains are of primary importance for cation recognition . Finally, since Mel permease properties are predominantly modified by mutagenizing residues located in the cytoplasmic half of the permease, we propose that Mel permease has a well-like shape opened toward the periplasmic space and is closed at its cytoplasmic extremity by a gate.

J Biol Chem, 1994 Oct 7, 269(40), 24820 - 5
Unfolding of colicin A during its translocation through the Escherichia coli envelope as demonstrated by disulfide bond engineering; Duche D et al.; Three double cysteine mutants, each possessing a disulfide bond in its pore-forming domain, were used to study the translocation of colicin A through the Escherichia coli envelope . These mutated colicins were able to exert their in vivo channel activity only after their disulfide bonds had been reduced by dithiothreitol . In solution, the reduction of the disulfide bonds by dithiothreitol was a slow process whose kinetics depended on the position of the disulfide bond (t1/2 varying between 35 and 100 s) . This t1/2 was strongly decreased (t1/2 = 8-9 s) upon predenaturation of the mutated colicins with urea . The t1/2 values of reduction of the mutants bound to E . coli-sensitive cells were similar to those of predenatured colicins . This suggested that the interaction of the oxidized double cysteine mutants with the E . coli envelope triggered their unfolding . The disulfide bonds did not prevent but delayed the translocation of the colicins . The amplitude of the delay and the time at which it occurred during translocation depended on the position of the disulfide bond . We could discriminate between the delays accumulated during binding to the receptor and those during the translocation via OmpF and the Tol proteins.

J Biol Chem, 1994 Oct 7, 269(40), 24756 - 61
The short amino acid sequence Pro-His-Ser-Arg-Asn in human fibronectin enhances cell-adhesive function; Aota S et al.; Synergistic sites in the central cell-adhesive domain of fibronectin (FN) substantially enhance cell adhesion mediated by the alpha 5 beta 1 integrin receptor for fibronectin . We characterized a critical minimal sequence needed for synergistic activity using site-directed mutagenesis and homology scanning using intramolecular chimeras . The minimal cell-binding domain of FN consisting of the 9th and 10th type III FN repeat was expressed in an Escherichia coli expression system . This protein retained high biological activity when assayed using a competitive inhibition assay for FN-mediated adhesion of baby hamster kidney or HT-1080 cells . In contrast, a construct consisting of the 8th and 10th repeat displayed very low biological activity . By replacing various portions of the 8th repeat with homologous 9th repeat segments, we mapped the synergistic region to the center of the 9th repeat . When a very short peptide sequence, Pro-His-Ser-Arg-Asn (PHSRN), from the 9th repeat was substituted for the homologous pentapeptide site in the 8th repeat sequence, the recombinant protein showed markedly enhanced activity . Further mutagenesis analysis suggested that the arginine residue of this pentapeptide sequence is important for function . We also identified a weaker adjacent synergy region other than the PHSRN region . Epitope mapping of an anti-FN monoclonal antibody that inhibits FN-mediated adhesion identified the same critical regions . A synthetic peptide containing the PHSRN sequence showed neither competitive inhibitory activity in solution nor synergy with a soluble RGD-containing peptide . However, when the same synthetic peptide was positioned via a covalent bond at the corresponding site of the normally inactive 8th repeat, it mediated an enhancement of adhesive activity . These results identify a pentapeptide site that synergistically enhances the cell-adhesive activity of the FN RGD sequence.

J Biol Chem, 1994 Oct 7, 269(40), 24742 - 6
Catalytic properties of mouse carbonic anhydrase V; Heck RW et al.; A cDNA encoding the mouse carbonic anhydrase V gene was isolated by reverse transcription and polymerase chain reaction from BALB/c mouse liver mRNA . Vectors containing the full coding sequence as well as two different NH2-terminal truncated genes expressed enzymatically active protein in Escherichia coli . The carbonic anhydrase V produced by a vector containing the full coding sequence, which includes a possible NH2-terminal mitochondrial targeting signal, was proteolytically processed by E . coli and contained several amino-terminal ends . The two NH2-terminal truncated vectors deleted, respectively, 1) the 29-amino acid putative targeting sequence and 2) 51 amino acids, yielding a protein equivalent to a carbonic anhydrase (CA) V isolated from mouse liver mitochondria; and both vectors produced homogeneous protein fractions . These latter two forms of CA V had identical steady-state constants for the hydration of CO2, with maximal values of kcat/Km at 3 x 10(7) M-1 s-1 and kcat at 3 x 10(5) s-1 with an apparent pKa for catalysis of 7.4 determined from kcat/Km . In catalytic properties, mouse CA V is closest to CA I; however, in inhibition by acetazolamide, ethoxzolamide, and cyanate, CA V is very similar to CA II . Mouse CA V has a tyrosine at position 64, where the highly active isozyme II has histidine serving as a proton shuttle in the catalytic pathway . Investigation of a site-specific mutant of CA V containing the replacement Tyr64-->His showed that the unique kinetic properties of CA V are not due to the presence of tyrosine at position 64.

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