Genetika, 1991 Feb, 27(2), 210 - 6 {Effectiveness of chemical mutagenesis in multiple damage to one or both strands of plasmid DNA}; Karamysheva TV et al.; Methods for site-directed multiple modification of DNA have been developed and used for modification of either one or two strands of plasmid DNA . Plasmid DNAs modified in the region of the tet gene were transformed into Escherichia coli cells and Tet colonies were screened . It was shown that multiple lesions in one DNA strand performed using either N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or sodium bisulfite were effectively repaired in the cell by error-free mechanism . In contrast, modification of two DNA strands led to induction of mutations . The efficiency of mutagenesis in the case of modification of a local region of one DNA strand with sodium bisulfite and modification of the other strand with MNNG was 1.1-7.9% . Mutations were analysed by restriction mapping and sequencing . All of them were G----A transitions. Immunol Cell Biol, 1991 Feb, 69 ( Pt 1), 51 - 5 Cloning and sequencing of the cDNA for ovine granulocyte-macrophage colony-stimulating factor (GM-CSF); O'Brien PM et al.; Colony-stimulating factors (CSFs) are not only regulators of haemopoiesis but can also enhance the function of mature myeloid cells, and are therefore potential immune adjuvants . By use of the polymerase chain reaction (PCR) with primers based on the bovine granulocyte-macrophage CSF (GM-CSF) sequence, we have amplified the cDNA for ovine GM-CSF, produced from crude mRNA extracted from alveolar macrophages . The PCR product was cloned into pUC119, and electroporated into Escherichia coli . The complete nucleotide sequence of two clones, and the partial sequence of eight others, was determined . At the nucleotide and amino acid levels, the ovine and bovine GM-CSF sequences are 91% and 81% homologous, respectively. J Biochem (Tokyo), 1991 Feb, 109(2), 294 - 8 Papain-inhibitory activity of oryzacystatin, a rice seed cysteine proteinase inhibitor, depends on the central Gln-Val-Val-Ala-Gly region conserved among cystatin superfamily members; Arai S et al.; Oryzacystatin, a cysteine proteinase inhibitor occurring in rice seeds, contains a particular glycine residue (Gly5) near the NH2-terminal position, and the sequence Gln53-Val54-Val55-Ala56-Gly57 in a central part of the molecule . Both are conserved among most members of the cystatin superfamily . We have found from Escherichia coli expression studies that the NH2-terminal 21 residues of oryzacystatin are not essential for its papain-inhibitory activity, and that the conserved pentapeptide region may be indispensable {Abe, K., Emori, Y., Kondo, H., Arai, S., & Suzuki, K . (1988) J . Biol . Chem . 263, 7655-7659} . Here we present more detailed data based on quantitative analyses of the inhibitory activities of NH2- and COOH-terminally truncated oryzacystatin and site-directed mutants at the Gln-Val-Val-Ala-Gly region . The data indicate the following results . (1) The truncated mutants lacking the NH2-terminal 21 residues or the COOH-terminal 11 residues exhibit potent papain-inhibitory activity equivalent to the activity of wild oryzacystatin . (2) However, neither the mutant lacking the NH2-terminal 38 residues nor that lacking the COOH-terminal 35 residues is completely able to inhibit papain . (3) Site-directed mutants at the Gln residue of the Gln-Val-Val-Ala-Gly region have drastically reduced papain-inhibitory activities: the Gln----Pro mutant is completely inactive and the Gln----Leu mutant has an approximately 150 times higher Ki value than wild-type oryzacystatin.(ABSTRACT TRUNCATED AT 250 WORDS) J Biochem (Tokyo), 1991 Feb, 109(2), 288 - 93 Lysophospholipase L1 from Escherichia coli K-12 overproducer; Karasawa K et al.; After screening 900 E . coli strains of the Clarke and Carbon collection for by lysophospholipase L1 activities, we isolated a clone bearing the plasmid pLC6-34, which showed an increased level of lysophospholipase L1 activity . Strains bearing the plasmid pC124, a subclone of pLC6-34 in plasmid vector pUC8, showed approximately 11.4 times higher lysophospholipase L1 activity than that of the parental strain . Starting from those overproducing strains, the lysophospholipase L1 was purified to near homogeneity by sequential use of ammonium sulfate fractionation, Sephacryl S-300, DEAE-cellulose, hydroxyapatite and Sephacryl S-200 column chromatographies . The apparent molecular weight of the purified lysophospholipase L1 was estimated to be 20,500-22,000 both by SDS-polyacrylamide gel electrophoresis and by gel permeation chromatography . The specific activity of the homogeneous lysophospholipase L1 was 10,400 nmol/min/mg protein when 1-acyl-sn-glycero-3-phosphoethanolamine was used as the substrate . The amino acid sequence of the amino-terminal portion of purified lysophospholipase L1 was determined and was different from that of lysophospholipase L2, which had previously been purified from the envelope fraction of E . coli strains bearing its cloned structural gene, pldB {Karasawa, K., Kudo, I., Kobayashi, T., Sa-eki, T., Inoue, K., & Nojima, S . (1985) J . Biochem, 98, 1117-1125} . The gene responsible for overproduction of lysophospholipase L1 activity was designated as pldC (phospholipid degradation C) . Its restriction enzyme map was also different from that of cloned pldB . These results further confirmed that, in E . coli, there are two lysophospholipases with distinct characteristics. J Biochem (Tokyo), 1991 Feb, 109(2), 262 - 6 Purification and characterization of the Escherichia coli OxyR protein, the positive regulator for a hydrogen peroxide-inducible regulon; Tao K et al.; The Escherichia coli oxyR gene is required for the induction of a regulon that is inducible by hydrogen peroxide and confers resistance to oxidative stresses . We constructed a plasmid system that greatly overproduced OxyR protein and purified the protein . OxyR protein specifically bound to the upstream regulatory regions of the oxyR and katG genes as demonstrated by the gel-retardation assay and the DNase I footprinting experiment, and activated the transcription initiation of the katG gene in vitro . Using a plasmid carrying an oxyR'-'lacZ fusion gene, we studied the regulation of oxyR expression in vivo . The expression of oxyR was not induced by the treatment with a low concentration of hydrogen peroxide which induces the genes in the oxyR regulon . The expression of the oxyR'-'lacZ gene was higher in an oxyR-deletion strain than in the oxyR+ strain, and was repressed by overexpressing the OxyR protein . These results suggest that OxyR protein functions as a repressor for oxyR, in addition to its known function as a transcriptional activator for the genes in the oxyR regulon. Gut, 1991 Feb, 32(2), 154 - 8 Enteropathogenic Escherichia coli and life threatening chronic diarrhoea; Hill SM et al.; Enteropathogenic Escherichia coli (EPEC) infection is not generally thought to cause severe diarrhoea after the neonatal period . Patients admitted to Queen Elizabeth Hospital for Children over the three years (1984-7) with diarrhoea and EPEC infection were reviewed . Clinical details, features of small intestinal mucosa, and treatment were recorded in those who developed chronic diarrhoea with failure to thrive . Twenty six children with EPEC required hospital admission for diarrhoea and six of these (23%) developed chronic diarrhoea . In contrast only two (5%) of 42 with other serogroups of E coli (p less than 0.01) and 28 (4%) of 764 children without EPEC admitted with acute diarrhoea developed chronic symptoms (p less than 0.01) . EPEC serogroups detected in the stool of the six children with chronic diarrhoea were 0128 in three, 0114 in two, and 0119 in one . The patients' clinical characteristics were: previous good health, no significant immunodeficiency, age 4-10 months, foreign travel (three of six), severe life threatening secretory diarrhoea from 0.5 to 1.5 1 per day (four of six), small intestinal enteropathy (five of six) three of whom showed mucosal adherent, non-invasive E coli of the same serotype as that in the stool, in association with microvillous loss and pedestal formation . All were treated with hypoallergenic feeds, two with parenteral nutrition, and three with parenteral antibiotics . All eventually recovered . EPEC infection is a common treatable cause of life threatening chronic diarrhoea in infancy. Protein Eng, 1991 Feb, 4(3), 351 - 7 Temperature-sensitive production of rabbit muscle glycogen phosphorylase in Escherichia coli; Browner MF et al.; In order to understand how allosteric switches regulate both the catalytic activity and molecular interactions of glycogen phosphorylase, it is necessary to design and analyze variant proteins that test hypotheses about the structural details of the allosteric mechanism . Essential to such an investigation is the ability to obtain large amounts of variant proteins . We developed a system for obtaining milligram amounts (greater than 20 mg/l) of rabbit muscle phosphorylase from bacteria . Phosphorylase aggregates as inactive protein when a strong bacterial promoter is used under full inducing conditions and normal growth conditions . However, when the growth temperature of bacteria expressing phosphorylase is reduced to 22 degrees C we obtain active muscle phosphorylase . The degree to which the induced expression of phosphorylase protein is temperature sensitive depends on the strain of bacteria used . New assay and purification methods were developed to allow rapid purification of engineered phosphorylase proteins from bacterial cultures . The rabbit muscle phosphorylase obtained from the bacterial expression system is enzymatically identical to the enzyme purified from rabbit muscle . The expressed protein crystallizes in the same conditions used for growing crystals of protein from rabbit muscle and the crystal form is isomorphous . Rabbit muscle phosphorylase is one of the largest oligomeric mammalian enzymes successfully expressed in Escherichia coli . Our results indicate that optimization of a combination of growth and induction conditions will be important in the expression of other heterologous proteins in bacteria. Biull Eksp Biol Med, 1991 Feb, 111(2), 175 - 7 {Changes in the profile of cytotoxic mediator monocytes in patients with cancer and precancerous conditions of the stomach}; Shepetkin IA et al.; Peripheral blood monocytes from healthy subjects, patients with gastric precancer disease (chronic gastric ulcer, stomach polyps and chronic atrophic gastritis) and different stages of gastric cancer were used . Spontaneous and lipopolysaccharide (LPS)-stimulated TNF-like factors production by monocytes was significantly higher in the precancer gastric disease patients than in the healthy subjects . At the same time the spontaneous capacity of monocytes to produce NTF-like factors was 2.5 lower in the gastric cancer patients compared to the healthy subjects . Moreover, in 5/13 of the gastric cancer patients in TNF-like factors production by the LPS-stimulated and non-stimulated monocytes was 1 unit/ml less . Spontaneous and reactive CL indexes were higher in the cancer patients monocytes than in the healthy subjects . The obtained results suggest that reactive oxygen species production can be an alternative mechanism by which a cytotoxic action of monocytes is regulated. Anal Biochem, 1991 Feb 1, 192(2), 262 - 7 Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase; Guan KL et al.; Several systems have been developed to allow for rapid and efficient purification of recombinant proteins expressed in bacteria . The expression of polypeptides in frame with glutathione S-transferase (GST) allows for purification of the fusion proteins from crude bacterial extracts under nondenaturing conditions by affinity chromatography on glutathione agarose (D . B . Smith and K . S . Johnson, 1988, Gene 67, 31-40) . This vector expression system has also incorporated specific protease cleavage sites to facilitate proteolysis of the bacterial fusion proteins . In our hands, the cleavage of these fusion proteins at a thrombin cleavage site proceeded slowly . To facilitate the cleavage of fusion proteins, we have introduced a glycine-rich linker (glycine kinker) containing the sequence P.G.I.S.G.G.G.G.G located immediately following the thrombin cleavage site . This glycine kinker greatly increases the thrombin cleavage efficiency of several fusion proteins . The introduction of the glycine kinker into fusion proteins allows for the cleavage of the fusion proteins while they are attached to the affinity resin resulting in a single step purification of the recombinant protein . More than 2 mg of the highly purified protein was obtained from the equivalent of 100 ml of bacterial culture within a few hours when a protein tyrosine phosphatase was employed as a test protein . The vector, pGEX-KG, has also been modified to facilitate cloning of a variety of cDNAs in all reading frames and has been successfully used to express several eukaryotic proteins. Mol Gen Mikrobiol Virusol, 1991 Feb, (2), 19 - 23 {A comparative study of the functions of recBCD-nuclease from Escherichia coli and "recombinase", encoded by plasmid R1drd-19}; Terent'ev MA et al.; The RecBCD nuclease of Escherichia coli and "recombinase" determined by R1drd-19 plasmid (the latter is able to replace at least partially the indicated cellular enzyme) were shown to differ from each other in some essential features . The product encoded by the plasmid as distinct from RecBCD nuclease practically is not sensitive to inhibition by GamS protein of the lambda phage . Earlier, it was found that the presence of R1drd-19 plasmid in the recBC cells restores the level of the total ATP-dependent exonuclease activity because of appearance in such cells of a new exonuclease activity also ATP-dependent . The exonuclease activity determined by R1drd-19 plasmid was found to differ from the corresponding activity of the RecBCD enzyme . The plasmid enzyme was able to prevent reproduction of T4g2- mutant on recBC cells . The ability of the plasmid "recombinase" to some stimulation of intrachromosomal recombination in recA mutant witness to incomplete RecA-dependence of its function . No significant homology was registered between Escherichia coli DNA fragment containing the recB, recC, recD genes and the EcoRI-C-fragment of R1drd-19 carrying the sequences responsible for recombination and repair functions of the plasmid. Mol Gen Mikrobiol Virusol, 1991 Feb, (2), 12 - 5 {Design of a gene coding encoding a polypeptide, mixing the enzyme activity of Escherichia coli homoserine kinase and threonine synthetase}; Manucharian KG et al.; The functioning of Escherichia coli threonine operon isolated genes thrB and thrC was studied by using the genetic complementation and enzymatic activity determination techniques . A new gene thrBC was obtained by the genes merging . The genes thrB and thrC were shown to function in Escherichia coli cells independent of the operon and the polipeptide encoded by the thrBC gene combined the functions to express the products of both genes in bacterial cell . At the same time the enzyme coded for by the merged genes demonstrates the level of activity compared with the ones of the isolated genes. Antimicrob Agents Chemother, 1991 Feb, 35(2), 387 - 9 4-Quinolone resistance mutations in the DNA gyrase of Escherichia coli clinical isolates identified by using the polymerase chain reaction; Oram M et al.; Seven nalidixic acid-resistant clinical isolates of Escherichia coli were shown to carry resistance mutations in their gyrase A proteins . Six had serine-83 to leucine or tryptophan changes; the seventh had an aspartate-87 to valine substitution . The frequent occurrence of a mutation at serine-83 implies a key role for this residue in quinolone action. J Chemother, 1991 Feb, 3(1), 16 - 22 Modulation of the intrinsic antiviral activity by Escherichia coli endotoxin in macrophages from patients with neoplasia; Merendino RA et al.; Macrophages from patients with breast cancer showed an impairment of their antiviral activity . The capability to hinder herpes simplex virus type 2 replication of macrophages from healthy donors and from patients with breast cancer was compared to the in-vitro treatment with Escherichia coli lipopolysaccharide (LPS) . The LPS showed a dose-dependent effect on the different macrophage populations studied . Nevertheless, macrophages from healthy donors appeared to be more sensitive to LPS in comparison with macrophages from the patients under our observation . On these cells LPS treatment was not able to modify the antiviral property, when these macrophages were differentiated in autologous serum. Gene, 1991 Feb 1, 98(1), 77 - 82 Effects of mcr restriction of methylated CpG islands of the L1 transposons during packaging and plating stages of mammalian genomic library construction; Doherty JP et al.; The use of optimally methylation-tolerant mcrA- mcrB- strains has been shown to produce an over tenfold increase in the plating efficiencies of mammalian genomic libraries, compared to a superior conventional phage host strain LE392 which is mcrB+ . However, there is an even more significant effect of mcr restriction . Amongst the recombinants recovered with an mcrB+ host, we have found that there is an additional 30-fold reduction in the frequencies of clones containing the heavily methylated 5'-CpG island sequences of both the human and rat L1 repetitive elements . The mcrA product was also found to restrict clones of these methylated genomic segments, but not as strongly as mcrB . However, the use of packaging extracts made from mcrA+ lysogens did not result in convincing reductions in the recoveries of these dispersed methylated elements . The magnitude of mcr restriction during plating due to methylated dispersed elements is sufficient to make a significant proportion of mammalian genomes unclonable from genomic libraries constructed previously using conventional mcr+ hosts. Gene, 1991 Feb 1, 98(1), 45 - 52 Gene expression in Deinococcus radiodurans; Smith MD et al.; We previously reported that the Escherichia coli drug-resistance determinants aphA (kanamycin-resistance) and cat (chloramphenicol-resistance) could be introduced to Deinococcus radiodurans by transformation methods that produce duplication insertion . However, both determinants appeared to require dramatic chromosomal amplification for expression of resistance . Additional studies described here, confirming this requirement for extensive amplification, led us to the use of promoter-probe plasmids in which the E . coli promoter has been deleted, leaving only coding sequences for the marker gene . We find that the insertion of D . radiodurans sequences immediately upstream from the promoterless drug-resistance determinant produces drug-resistant transformants without significant chromosomal amplification . Furthermore, a series of stable E . coli-D . radiodurans shuttle plasmids was devised by inserting fragments of D . radiodurans plasmid pUE10 in an E . coli plasmid directly upstream from a promoterless cat gene . These constructions replicated in D . radiodurans by virtue of the pUE10 replicon and expressed the cat determinant because of D . radiodurans promoter sequences in the pUE10 fragment . Of three such constructions, none expressed the cat gene in E . coli . Similar results were obtained using a promoterless tet gene . Translational fusions were made between D . radiodurans genes and E . coli 5'-truncated lacZ . Three fusions that produced high levels of beta Gal in D . radiodurans were introduced into E . coli, but beta Gal was produced in only one . The results demonstrate that the E . coli genes cat, tet and lacZ can be efficiently expressed in D . radiodurans if a D . radiodurans promoter is provided, and that D . radiodurans promoters often do not function as promoters in E . coli. J Bacteriol, 1991 Feb, 173(4), 1492 - 501 Genetic interaction between the beta' subunit of RNA polymerase and the arginine-rich domain of Escherichia coli nusA protein; Ito K et al.; The nusA11 mutation causes reduced transcription termination and temperature-sensitive growth of Escherichia coli . Suppressor mutations that restored growth of nusA11 mutant cells were isolated and named sna mutations . The intergenic suppressor mutation sna-10 was located in the rpoC gene at 90 min, which encodes the beta' subunit of RNA polymerase . sna-10 complemented the defect in tR1 termination caused by nusA11 and by itself stimulated termination of transcription at the lambda tR1 terminator . sna-10 is specific to the nusA11 allele and unable to suppress cold-sensitive growth of the nusA10 mutant . nusA10 carried two base substitutions at positions 311 and 634, causing two amino acid changes from the wild-type sequence . During these studies, we found three -1 frameshift errors in the wild-type nusA sequence; the correct sequence was confirmed by the peptide sequence and gene fusion analyses . The revised sequence revealed that nusA1 and nusA11 are located in an arginine-rich peptide region and substitute arginine and aspartate for leucine 183 and glycine 181, respectively . The intragenic suppressor study indicated that the nusA11 mutation can be suppressed by changing the mutated aspartate 181 to alanine or changing aspartate 84 to tyrosine. J Gen Virol, 1991 Feb, 72 ( Pt 2), 399 - 404 Immunological studies on the Epstein-Barr virus encoded alkaline deoxyribonuclease found in virus-producing lymphoblastoid cells; Baylis SA et al.; Antisera were raised against a purified recombinant form of the Epstein-Barr virus (EBV) alkaline deoxyribonuclease (DNase) expressed in Escherichia coli . These sera were shown to be reactive with lymphoblastoid cells expressing EBV antigens (B95-8, P3HR-1 and Raji) . Immunostaining studies of cells expressing EBV antigens revealed that the DNase was a component of the restricted early antigen complex of EBV . Western blot analysis of these chemically induced cells revealed that the polypeptide associated with the EBV DNase has an Mr of approximately 55,000, slightly greater than that of the recombinant form, suggesting that the protein undergoes some form of posttranslational modification during virus replication . The DNase enzymic activities observed in B95-8, P3HR-1 and Raji cells following chemical induction were neutralized using the specific antiserum . A detailed examination of protein extracts from the nude mouse-passaged nasopharyngeal carcinoma cell line C-15 failed to detect any antigenic or biochemical evidence for the presence of the DNase . Immunostaining of biopsies of oral 'hairy' leukoplakia with the antisera against EBV DNase revealed high level expression in the more differentiated spinous layers of the epithelium, a pattern of reactivity identical to that observed for other lytic cycle antigens. J Cell Biol, 1991 Feb, 112(4), 665 - 76 Domain structure in actin-binding proteins: expression and functional characterization of truncated severin; Eichinger L et al.; Severin from Dictyostelium discoideum is a Ca2(+)-activated actin-binding protein that severs actin filaments, nucleates actin assembly, and caps the fast growing ends of actin filaments . Sequence comparison with functionally related proteins, such as gelsolin, villin, or fragmin revealed highly conserved domains which are thought to be of functional significance . To attribute the different activities of the severin molecule to defined regions, progressively truncated severin polypeptides were constructed . The complete cDNA coding for 362 (DS362) amino acids and five 3' deletions coding for 277 (DS277), 177 (DS177), 151 (DS151), 117 (DS117), or 111 (DS111) amino acids were expressed in Escherichia coli . The proteins were purified to homogeneity and then characterized with respect to their effects on the polymerization or depolymerization kinetics of G- or F-actin solutions and their binding to G-actin . Furthermore, the Ca2+ binding of these proteins was investigated with a 45Ca-overlay assay and by monitoring Ca2(+)-dependent changes in tryptophan fluorescence . Bacterially expressed DS362 showed the same Ca2(+)-dependent activities as native severin . DS277, missing the 85 COOH-terminal amino acids of severin, had lost its strict Ca2+ regulation and displayed a Ca2(+)-independent capping activity, but was still Ca2+ dependent in its severing and nucleating activities . DS151 which corresponded to the first domain of gelsolin or villin had completely lost severing and nucleating properties . However, a residual severing activity of approximately 2% was detectable if 26 amino acids more were present at the COOH-terminal end (DS177) . This locates similar to gelsolin the second actin-binding site to the border region between the first and second domain . Measuring the fluorescence enhancement of pyrene-labeled G-actin in the presence of DS111 showed that the first actin-binding site was present in the NH2-terminal 111 amino acids . Extension by six or more amino acids stabilized this actin-binding site in such a way that DS117 and even more pronounced DS151 became Ca2(+)-independent capping proteins . In comparison to many reports on gelsolin we draw the following conclusions . Among the three active actin-binding sites in gelsolin the closely neighboured sites one and two share the F-actin fragmenting function, whereas the actin-binding sites two and three, which are located in far distant domains, collaborate for nucleation . In contrast, severin contains two active actin-binding sites which are next to each other and are responsible for the severing as well as the nucleating function . The single actin-binding site near the NH2-terminus is sufficient for capping of actin filaments. J Bacteriol, 1991 Feb, 173(3), 1111 - 9 Transcriptional analysis, translational analysis, and sequence of the kilA-tellurite resistance region of plasmid RK2Ter; Walter EG et al.; The tellurite resistance (Ter) determinant of the IncP alpha plasmid RK2Ter, a variant of RK2 (also called RP4), is located between the kilA and korA genes involved in plasmid replication control . Transcriptional and translational fusions were constructed between the gene for beta-galactosidase and the kilA and Ter genes by using the transpositional phage mini-Mu . These fusions indicated that the Ter genes are transcribed in the same direction as kilA and that transcription and translation of the cloned kilA gene are occurring and may not be lethal to the bacterial cell even in the absence of korA . The nucleotide sequence of this region was determined, and three open reading frames (ORFs) were identified . The first ORF codes for KilA, a 28-kDa hydrophilic protein . The second ORF, telA, codes for a hydrophilic protein of 42 kDa . The third ORF, telB, codes for a hydrophobic protein of 32 kDa . This protein appears to be located in the inner membrane of the bacterial cell, since fusions of TelB to alkaline phosphatase were obtained by using TnphoA . All three proteins were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after overproduction using the T7 RNA polymerase/promoter system . The same three proteins were produced when Tes and Ter derivatives of RP4 were expressed in an in vitro transcription-translation system . A single Ser-to-Cys missense mutation in telB was found to be responsible for mutation of RK2 to Ter. J Bacteriol, 1991 Feb, 173(3), 1027 - 34 traY and traI are required for oriT-dependent enhanced recombination between lac-containing plasmids and lambda plac5; Carter JR et al.; Recombination between F42lac and lambda plac5 is typically 20- to 50-fold more efficient than recombination between chromosomal lac and lambda plac5 . This enhancement of recombination requires trans-acting factors located in the promoter-distal and promoter-proximal regions of the main traY-to-traI (traZ) operon . By testing the ability of deletion mutants of tra to support enhanced recombination, we have identified traY as the only product has been ruled out . We also report that traI is the only gene from the promoter-distal end of the traY to traI operon that is required for recombination enhancement . Of the two proposed domains of traI, we conclude that the oriT-nicking activity is essential, whereas the helicase activity is largely dispensable . The possibility of a third traI activity is also discussed. J Bacteriol, 1991 Feb, 173(3), 1021 - 6 The Escherichia coli mraY gene encoding UDP-N-acetylmuramoyl-pentapeptide: undecaprenyl-phosphate phospho-N-acetylmuramoyl-pentapeptide transferase; Ikeda M et al.; Amplification of the mraY gene, previously called open reading frame Y (ORF-Y, 1,080 bp), at 2 min in the chromosome map of Escherichia coli enhanced the activity of UDP-N-acetylmuramoyl-pentapeptide: undecaprenyl-phosphate phospho-N-acetylmuramoyl-pentapeptide transferase (EC 2.7.8.13) . This enzyme catalyzes the formation of undecaprenyl-pyrophosphoryl-N-acetylmuramoyl-pentapeptide from UDP-N-acetylmuramoyl-pentapeptide and undecaprenyl-phosphate, the first step in the lipid cycle reactions in biosynthesis of bacterial cell wall peptidoglycans . The enhanced enzyme activity was sensitive to tunicamycin, and the amino tunicamycin-sensitive N-acetylglucosamine-1-phosphate transferase of Saccharomyces cerevisiae . Very probably mraY is the structural gene for the above enzyme. J Bacteriol, 1991 Feb, 173(3), 1004 - 11 Genetic analysis of the recG locus of Escherichia coli K-12 and of its role in recombination and DNA repair; Lloyd RG et al.; We describe a transposon insertion that reduces the efficiency of homologous recombination and DNA repair in Escherichia coli . The insertion, rec-258, was located between pyrE and dgo at min 82.1 on the current linkage map . On the basis of linkage to pyrE and complementation studies with the cloned rec+ gene, rec-258 was identified as an allele of the recG locus first reported by Storm et al . (P . K . Storm, W . P . M . Hoekstra, P . G . De Haan, and C . Verhoef, Mutat . Res . 13:9-17, 1971) . The recG258 mutation confers sensitivity to mitomycin C and UV light and a 3- to 10-fold deficiency in conjugational recombination in wild-type, recB recC sbcA, and recB recC sbcB sbcC genetic backgrounds . It does not appear to affect plasmid recombination in the wild-type . A recG258 single mutant is also sensitive to ionizing radiation . The SOS response is induced normally, although the basal level of expression is elevated two- to threefold . Further genetic studies revealed that recB recG and recG recJ double mutants are much more sensitive to UV light than the respective single mutants in each case . However, no synergistic interactions were discovered between recG258 and mutations in recF, recN, or recQ . It is concluded that recG does not fall within any of the accepted groups of genes that affect recombination and DNA repair. Am Rev Respir Dis, 1991 Feb, 143(2), 289 - 93 Early post-treatment with pentoxifylline or dibutyryl cAMP attenuates Escherichia coli-induced acute lung injury in guinea pigs; Hoffmann H et al.; We examined effects of early post-treatment with the methylxanthine pentoxifylline (PTXF), or the cell-permeable adenosine 3', 5'-cyclic monophosphate (cAMP) analog dibutyryl cAMP (db-cAMP) on Escherichia-coli-induced acute lung injury in guinea pigs . Acute lung injury was assessed by measurements of lung water (lung wet/dry weight ratio; W/D ratio), the concentration ratio of 125I-albumin in bronchoalveolar lavage (BAL) fluid and lung tissue compared with plasma (albumin index; BAL-AI or tissue-AI), and total differential leukocyte count in BAL fluid . Mean arterial pressure (Pa) and peripheral WBC counts were monitored continuously over the 8-h experiment . Septicemia was induced by a bolus injection of 2 x 10(9)/kg live E . coli . Thirty minutes later the animals received a bolus injection followed by continuous infusion of PTXF (20 mg/kg + 20 mg/kg/h; n = 8) or db-cAMP (2 mg/kg + 2 mg/kg/h; n = 8) or saline (septic control; n = 8) . Nonseptic control groups were also studied . The lung W/D ratio, BAL-AI, lung tissue-AI, and BAL leukocyte count increased significantly in the septic control group . The PTXF-septic and db-cAMP-septic groups showed no significant increase in lung W/D ratio, BAL-AI, and lung tissue-AI . However, there was no difference in BAL total and differential leukocyte count as compared with the septic control group . PTXF and db-cAMP had no effect on E . coli-induced changes in peripheral WBC count and Pa . Comparison in vitro experiments demonstrated that PTXF and db-cAMP inhibited the endotoxin-induced (E . coli) chemiluminescent response of isolated guinea-pig polymorphonuclear leukocytes (PMN).(ABSTRACT TRUNCATED AT 250 WORDS) Mol Cell Biol, 1991 Feb, 11(2), 1114 - 24 Membrane insertion of uracil permease, a polytopic yeast plasma membrane protein; Silve S et al.; Uracil permease is a multispanning protein of the Saccharomyces cerevisiae plasma membrane which is encoded by the FUR4 gene and produced in limited amounts . It has a long N-terminal hydrophilic segment, which is followed by 10 to 12 putative transmembrane segments, and a hydrophilic C terminus . The protein carries seven potential N-linked glycosylation sites, three of which are in its N-terminal segment . Overexpression of this permease and specific antibodies were used to show that uracil permease undergoes neither N-linked glycosylation nor proteolytic processing . Uracil permease N-terminal segments of increasing lengths were fused to a reporter glycoprotein, acid phosphatase . The in vitro and in vivo fates of the resulting hybrid proteins were analyzed to identify the first signal anchor sequence of the permease and demonstrate the cytosolic orientation of its N-terminal hydrophilic sequence . In vivo insertion of the hybrid protein bearing the first signal anchor sequence of uracil permease into the endoplasmic reticulum membrane was severely blocked in sec61 and sec62 translocation mutants. Virology, 1991 Feb, 180(2), 648 - 58 Use of type 1/type 2 chimeric polioviruses to study determinants of poliovirus type 1 neurovirulence in a mouse model; Martin A et al.; We previously described the characteristics of a type 1/type 2 (PV-1/PV-2) chimeric poliovirus, v510, which contains the six amino acids specific for PV-2 in the B-C loop of VP1 . This virus was found to be mouse-adapted, as PV-2 and in contrast with PV-1 . Determinants of host range were studied in detail and are reported here . PV-1/PV-2 chimeras containing partial PV-1----PV-2 substitutions in the B-C loop of VP1 were obtained by making use of a mutagenesis cartridge on PV-1 cDNA . Analysis of mouse neurovirulence of these chimeras, when correlated with the three-dimensional structure of the v510 capsid, revealed that PV-2 residues important for mouse tropism are those which determine the particular conformation of the B-C loop of VP1 in v510 . The mutation of the adenine residue at position 480 of the 5' noncoding region into a guanine residue has been shown to be an important determinant of PV-1 attenuation in monkeys . We show that introduction of this mutation in the v510 genome results in a virus which is partially attenuated for mice . This suggests that analysis of genomic determinants important for PV-1 neurovirulence could be carried out in a mouse model by making use of a mouse-adapted PV-1/PV-2 chimera. J Virol, 1991 Feb, 65(2), 972 - 5 Upstream promoter elements of the herpes simplex virus type 1 glycoprotein H gene; Steffy KR et al.; To investigate the cis-acting sequence elements that are involved in the regulation of herpes simplex virus type 1 late-gene expression, recombinant viruses were constructed that express the Escherichia coli lacZ gene from the promoter of the glycoprotein H (gH) gene . Deletion experiments established an upstream boundary for the gH promoter of no more than 83 bp from the start of gH transcription and showed that the promoter sequences did not overlap with coding sequences of the upstream thymidine kinase (tk) gene . Sequences of the tk gene previously shown to be required for efficient processing of the tk transcript were essential for expression form the gH promoter and included a TATA-like element . In addition, the gH TATA element was specifically mutagenized to substitute the TATA elements of immediate-early, early, and other late viral promoters for the gH TATA element . The results indicated that the TATA element was an interchangeable component of herpes simplex virus type 1 promoters and did not regulate temporal expression. Biochimie, 1991 Feb-Mar, 73(2-3), 305 - 12 Genetic control of recombination exchange frequency in Escherichia coli K-12; Lanzov V et al.; The frequency of recombination exchanges per unit length of DNA (Freuld) can be estimated by measuring the scale of the genetic map that is the mean statistical distance between two neighboring crossovers . The scales appear to be equal for the alternative pathways of recombination, RecBCD (wild-type cells) or RecF (recBC- sbcB- sbcC- genotypes) . The absolute value of the scale depends on specific experimental conditions . recR, recQ, ruv, recJ and recN genes of the RecF pathway of recombination (recBC- sbcBC- cell genotypes) do not appear to be silent in wild-type cells where the RecBCD pathway predominates . On the contrary, these genes are responsible for the Freuld . The list recF504::Kmr greater than recQ61::Tn3 greater than ruv-54 greater than recJ284::Tn10 shows decreasing efficiency in inhibiting recombination exchanges by these mutations . The recN264 mutation gives a small, but opposite effect of increasing the frequency of recombination exchanges . The effect of the recF and recQ mutations appears to be additive, but that is not the case in combinations of ruv-54 with recF504::Kmr or recQ61::Tn3. Eur J Cell Biol, 1991 Feb, 54(1), 95 - 101 Localization of the plasma membrane and mitochondrial H(+)-ATPases in Leishmania donovani promastigotes; Liveanu V et al.; Immunochemical methods were used to characterize the proton-translocating ATPases (H(+)-ATPases) of the plasma membrane and mitochrondrion of Leishmania donovani promastigotes . Antisera directed against the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae reacted with a 66 kDa membrane protein of L . donovani promastigotes . By immunocytochemistry, the antiserum was shown to label the cell and flagellar surface of promastigotes as well as the Golgi apparatus and the membrane of intracellular organelles . The target antigen was shown to possess ATPase activity resembling the leishmanial H(+)-ATPase activity . Antisera raised against the beta-subunit of the F0F1-ATPase of Escherichia coli reacted with a 56 kDa protein in L . donovani promastigotes . Ultrastructurally, the anti-beta-subunit antibody was exclusively associated with the mitochondrion in these cells . This antiserum immunoprecipitates ATP hydrolytic activity typical of the F1 beta-subunit activity of the mitochondria of higher eukaryotes. Neuron, 1991 Feb, 6(2), 201 - 10 Tissue-specific and developmental regulation of rod opsin chimeric genes in transgenic mice; Lem J et al.; Chimeric gene fusions between 4.4 kb of rod opsin 5' flanking sequence fused to a diphtheria toxin gene and 4.4 kb or 500 bp of rod opsin 5' flanking sequence fused to the E . coli IacZ gene were used to generate transgenic mice for analysis of cell type-specific expression and temporal and spatial distribution of reporter gene product during retinal development . Opsin-diphtheria toxin transgene expression evoked photoreceptor-specific cell death . The 4.4 kb opsin-IacZ transgene followed temporal and spatial gradients of expression that approximate opsin expression . The 500 bp opsin fragment targeted expression to photoreceptors, but expression was weaker and nonuniform, suggesting that elements located upstream may be required for enhanced and uniform spatial expression. Virology, 1991 Feb, 180(2), 668 - 77 Influenza A virus in vitro transcription: roles of NS1 and NP proteins in regulating RNA synthesis; Skorko R et al.; To study the mechanisms by which the influenza A virus RNA-dependent RNA polymerase switches from transcription to replication we have devised a riboprobe protection technique with which we analyzed the 3' end sequence of (+)-strand RNA products of an in vitro transcription reaction containing purified virion-RNP complexes in the presence and the absence of the putative regulatory proteins NP and NS1 . We found that the addition of these proteins did not result in the synthesis of full-length (+)-strand RNA products resulting from read-through of the polyadenylation signal or replication . Because NS1 and NP are both phosphoproteins we searched for protein kinase activity that might play a role in regulating RNA synthesis . We showed that virion RNP complexes phosphorylated NS1 but possessed no autophosphorylating activity . Soluble NP protein derived from RNP complexes did not phosphorylate NS1, but did phosphorylate casein . When NP protein was dephosphorylated, however, it no longer phosphorylated casein . We also showed that NS1 was an ssRNA-binding protein which binds nonspecifically to all ssRNA, and that this activity is not dependent on its state of phosphorylation. Protein Expr Purif, 1991 Feb, 2(1), 66 - 8 Affinity chromatography of Escherichia coli (m5U54)-methyltransferase on tRNA-agarose; Gu X et al.; tRNA-agarose was prepared by condensing periodate-oxidized tRNA to an agarose matrix containing hydrazide functional groups . The tRNA-agarose was used to take partially purified tRNA (m5U54)-methyltransferase to homogeneity . The method is simple and reproducible and gives high yields. Protein Expr Purif, 1991 Feb, 2(1), 59 - 62 Rapid desalting of ammonium sulfate solutions by hydrophobic interaction chromatography; O'Hern PA et al.; Open-air hydrophobic interaction chromatography with alkyl carbon functional groups coupled to agarose beads has been used to desalt large volumes of ammonium sulfate-fractionated bacterial cell lysates . The protein of interest can be simultaneously desalted and concentrated in less than 4 h, and the yield is significantly better than that obtained by the standard technique of precipitation, centrifugation, and dialysis. Protein Expr Purif, 1991 Feb, 2(1), 15 - 23 High-yield purification of HIV-1 proteinase expressed by a synthetic gene in Escherichia coli; Goobar L et al.; A rapid and simple purification procedure for human immunodeficiency virus type 1 (HIV-1) proteinase from a synthetic gene expressed in Escherichia coli has been developed . The synthetic gene was constructed from oligonucleotides containing several restriction enzyme sites in order to allow simple construction of homologous genes . The protein was translated as a precursor which was autocatalytically processed into the mature protein as shown by N-terminal sequence analysis of the purified protein . Immunoblot analysis was used to verify the nature of the expression product and it was found that 2 of 10 anti-peptide antibodies, covering the whole proteinase sequence, were able to react with the enzyme in crude bacterial lysates . These two anti-peptide antibodies represent a continuous sequence partially overlapping the active site . The purification involves two initial precipitation steps followed by cation-exchange and size-exclusion chromatography . A high yield and a high specific activity were achieved. J Vasc Interv Radiol, 1991 Feb, 2(1), 156 - 8 Percutaneous management of localized emphysematous pyelonephritis; Zagoria RJ et al.; The authors report a case of emphysematous pyelonephritis that was successfully treated with radiologically guided percutaneous drainage . This case illustrates that in certain patients with focal abnormalities, functioning renal tissue can be salvaged and emphysematous pyelonephritis can be eradicated with a combination of antibiotics and radiologically guided percutaneous drainage. Biochimie, 1991 Feb-Mar, 73(2-3), 251 - 6 Identification of a mouse cDNA fragment whose expressed polypeptide reacts with anti-recA antibodies; Angulo JF et al.; We have previously reported the in vivo detection of a mouse nuclear protein that cross-reacts with antibodies raised against E coli recA protein . Here, we characterize monospecific anti-recA antibodies, their use for the immunological screening of a cDNA expression library and the isolation of a mouse cDNA fragment which codes for a polypeptide recognized by anti-recA antibodies . The cDNA fragment is 601 nucleotide long and was called KIN17(601) . It contains an open reading frame coding for a 200 amino acid polypeptide . In kin17(200) polypeptide, there are amino acids identical to those that form one of the major antigenic determinants of recA protein . Kin17(200) polypeptide also displays a significant similarity with the helix 1 motif of several homeoproteins. FEMS Microbiol Immunol, 1991 Feb, 3(1), 39 - 45 Identification of a novel B-cell epitope of restricted specificity on the hsp 65-kDa protein of Mycobacterium tuberculosis; Rambukkana A et al.; A B-cell epitope on the carboxy-terminal region of the mycobacterial 65-kDa heat shock protein that distinguishes Mycobacterium tuberculosis/Mycobacterium bovis BCG from Mycobacterium leprae was identified by two novel monoclonal antibodies (mAbs), Ne5 and Nd4 . These mAbs also showed a limited cross reactivity with mycobacterial species belonging to M . tuberculosis complex and Mycobacterium avium complex with the exception of Mycobacterium vaccae . Characterization of the epitope recognized by these mAbs was done with M . bovis BCG 65-kDa fusion proteins expressed in Escherichia coli encoding various segments of the 65-kDa protein . Our results together with those reported in literature indicated that this epitope resides in the highly divergent region of amino acid residues 525 to 540 . This B-cell epitope on the 65-kDa protein of M . tuberculosis/M . bovis BCG has not been recognized by previously reported mAbs, although the analogous epitope sequence of M . leprae 65-kDa has been identified by a known mAb (IIIC8) reported in the literature . Therefore Ne5/Nd4 epitope could be considered important in studying the differential immune response of the host against infections with M . tuberculosis complex/M . avium complex and M . leprae. Mol Biochem Parasitol, 1991 Feb, 44(2), 213 - 24 Modification of GP63 genes from diverse species of Leishmania for expression of recombinant protein at high levels in Escherichia coli; Button LL et al.; Toward the future development of a defined subunit vaccine against leishmaniasis is, high levels of recombinant GP63 for diverse species of Leishmania were produced in Escherichia coli . Several features of Leishmania GP63 genes were simultaneously modified with the polymerase chain reaction (PCR) using either cloned genes or total genomic DNA from Leishmania as template DNA for the PCR amplification reactions . The PCR products included only the coding region for the predicted mature form of GP63 that occurs on the surface of Leishmania, flanked by the appropriate translation signals and cloning sites for the production of recombinant GP63 as nonfusion protein in E . coli . When the codon usage in the GP63 gene was modified to reduce the guanine and cytosine content for the codons adjacent to the ATG initiation codon, rGP63 represented about 50% of total protein in E . coli . Mouse monoclonal antibodies raised against purified Leishmania major rGP63 had equivalent immunoblotting characteristics for native GP63 and recombinant GP63 with respect to linear determinants on GP63 expressed in diverse species of Leishmania . Human T cell lines and clones were derived from a patient infected with Leishmania braziliensis panamensis using rGP63 purified from an L . major GP63 expression clone as antigen. Mutat Res, 1991 Feb, 262(2), 79 - 84 In vivo benzo{a}pyrene diol epoxide-induced alkali-labile sites are not apurinic sites; Moran MF et al.; We have used endonuclease IV from Escherichia coli as a probe for apurinic sites in the DNA of HeLa cells following treatment with an activated diol epoxide derivative of benzo{a}pyrene . DNA strand breaks and alkali-labile sites were observed that were repaired following exposure to the carcinogenic alkylating agent . The alkali-labile sites were not substrates for the apurinic site-specific endonuclease IV . We conclude that the alkali-labile sites formed in vivo by benzo{a}pyrene derivatives are not apurinic sites and probably arise as a consequence of rearrangement of the abundant N2-guanine adducts . This finding questions the involvement of apurinic sites in the mutagenic activity of benzo{a}pyrene. Lab Invest, 1991 Feb, 64(2), 295 - 9 Recruitment of neutrophils in the local endotoxin response: association with de novo endothelial expression of endothelial leukocyte adhesion molecule-1; Munro JM et al.; Endothelium is central to the cellular infiltration that develops during inflammation, and a prominent feature of its involvement is the expression of adhesion molecules for circulating leukocytes . In the present study, we assessed the kinetics of endothelial adhesion molecule expression during the cutaneous endotoxin response in baboons . Immunostained cryostat sections and hematoxylin and eosin-stained paraffin sections of skin biopsies were examined using set scoring systems to provide semiquantitative data on the changes in endothelial phenotype and induced polymorphonuclear leucocyte (PMN) accumulation . Endothelium in control skin did not express endothelial leukocyte adhesion molecule (ELAM)-1 but did show a relatively weak expression of intercellular adhesion molecule (ICAM)-1 . By 2 hours after injection of endotoxin (500 mcg of Escherichia coli-derived lipopolysaccharide), a marked expression of ELAM-1 developed that was associated with concurrent extensive adhesion and extravasation of PMN . The ELAM-1 expression subsequently decreased and was virtually absent by 9 hours . Mean scores for endothelial expression of ICAM-1 changed comparatively little over this time course, and mononuclear cell accumulation was minimal . The response to endotoxin differs from that to tumor necrosis factor injection; the latter causes sustained ELAM-1 expression, and delayed but pronounced increases in ICAM-1, with accompanying mononuclear cell extravasation . Thus, local endotoxin administration provides a model of acute inflammation in which PMN accumulation is associated with striking endothelial expression of ELAM-1 . In this model, appreciable elevations in ICAM-1 expression are unnecessary for PMN infiltration. Biochem J, 1991 Feb 1, 273 ( Pt 3), 777 - 82 The use of thioglycollate to demonstrate DNA AP (apurinic/apyrimidinic-site) lyase activities . Biological consequences of thiol addition to the 5' product of a beta-elimination reaction at an AP site in DNA; Bricteux-Gregoire S et al.; Thioglycollate reacts with the 5' product of AP lyase activity on apurinic/apyrimidinic (AP) sites in DNA . The 3'-terminal thioglycollate-unsaturated sugar 5-phosphate adduct can be released by the use of Escherichia coli endonuclease IV or endonuclease VI, and identified by DEAE-Sephadex chromatography . In contrast, the mammalian AP endonuclease is unable to excise a 3'-terminal thiol-unsaturated sugar adduct; this lesion, which must sometimes occur in vivo, might be irreparable and have pathological consequences. J Bacteriol, 1991 Feb, 173(4), 1460 - 70 Suppressors that permit A-signal-independent developmental gene expression in Myxococcus xanthus; Kaplan HB et al.; Progression through the early stages of Myxococcus xanthus fruiting body development requires the cell-to-cell transmission of soluble material called A signal . During these early stages, expression from the gene identified by Tn5 lac insertion omega 4521 increases . A DNA probe of the omega 4521 gene was constructed . Use of this probe showed that accumulation of mRNA corresponding to the omega 4521 gene depends upon A signal . A-signal-deficient (asg) mutants fail to accumulate this RNA, and the external addition of A signal restores accumulation . To identify links between A signal and its responsive gene, omega 4521, suppressors of an asg mutation were generated . All of the suppressor alleles restored lacZ expression from omega 4521 in the absence of A signal, and they were demonstrated to be neither reversions of the asgB mutation nor mutations in the promoter of omega 4521 . Fifteen suppressor mutations map to two loci, sasA and sasB (for suppressor of asg) . sasA and sasB mutants differ phenotypically during growth and development . Mid-logarithmic-phase sasA asgB double mutants, like sas+ asg+ strains, express low levels of lacZ, whereas sasB asgB double mutants express high levels . sasA asg+ mutants form abnormal colonies, are less cohesive than wild type, and are defective in fruiting body formation and sporulation . In contrast, sasB asg+ mutants form normal colonies, are as cohesive as wild type, and appear to develop normally . The characteristics of sasA suppressors implicate the sasA+ product as a negative regulator in the A-signal-dependent regulation of omega 4521. J Bacteriol, 1991 Feb, 173(4), 1452 - 9 Analysis of the Borrelia burgdorferi GeHo fla gene and antigenic characterization of its gene product; Gassmann GS et al.; The fla gene of Borrelia burgdorferi GeHo was analyzed and expressed in Escherichia coli . The structural gene encodes a flagellar protein of 336 amino acids . Comparative sequence analysis of the amino acid sequence revealed a high degree of sequence conservation with flagellins from both phylogenetically related and unrelated bacteria . The antigenic properties of the B . burgdorferi Fla protein were studied by synthesizing overlapping octapeptides, which were screened by using a battery of different monoclonal and polyclonal antibodies from various species directed against native and denatured flagellar proteins . No single species-independent immunodominant epitope could be located . However, immunoreactive oligopeptides clustered within the variable middle region (N-180 to I-260) . This region could constitute a candidate antigen for more specific and sensitive serodiagnosis of Lyme borreliosis. Proc Natl Acad Sci U S A, 1991 Feb 1, 88(3), 1014 - 8 Continuous microspectrophotometric measurement of DNA polymerase activity: application to the Klenow fragment of Escherichia coli DNA polymerase I and human immunodeficiency virus type 1 reverse transcriptase; Baillon JG et al.; Progress of DNA- and/or RNA-directed DNA polymerization reactions can be measured continuously using circular dichroism (CD) or ultraviolet (UV) spectroscopy . In the presence of the Klenow fragment of Escherichia coli DNA polymerase I, a CD change of -0.27 +/- 0.06 millidegree at 248 nm and a UV change of -2.7 +/- 0.3 milliabsorbance units at 275 nm occur upon incorporation of 120 pmol of dTMP in a reaction volume of 120 microliters (1 microM dTMP incorporation) into a synthetic template-primer, p(dA)40-60.p(dT)20 . The transcription of poly(A).p(dT)12-18 by reverse transcriptases can also be monitored using these methods . Kinetic parameters for the polymerization reaction catalyzed by the Klenow fragment were determined from initial velocity measurements using CD or UV assays and were in close agreement with those measured by the standard single point radiochemical filtration assay . The generality of optical techniques for the measurement of DNA polymerase activity was shown by the use of a partially self-complementary hairpin-shaped oligonucleotide substrate for the Klenow fragment . Addition of a single nucleotide residue under steady-state conditions to this 35-mer at a concentration of 1.5-3 microM gave an easily measurable absorbance decrease at 275 nm, and the absorbance changes upon sequential addition of nucleotide units were additive. J Bacteriol, 1991 Feb, 173(3), 1168 - 74 An unusual correlation between ppGpp pool size and rate of ribosome synthesis during partial pyrimidine starvation of Escherichia coli; Vogel U et al.; Escherichia coli was exposed to partial pyrimidine starvation by feeding a pyrBI strain orotate as the only pyrimidine source . Subsequently, differential rates of synthesis of rRNA and of a few ribosome-associated proteins as well as the pool sizes of nucleoside triphosphates and ppGpp were measured . As the orotate concentration in the medium was reduced, the growth rate decreased and the pools of pyrimidine nucleotides, particularly UTP, declined . We did not observe the normal inverse relation between concentration of ppGpp and growth rate; rather, we observed that the ppGpp pool was low at slow growth rates . Upshifts in growth rate were made by adding uracil to a culture growing slowly on orotate . Downshifts could be provoked by adding aspartate plus glutamate to a culture growing at a high concentration of orotate . Following the upshift, both the rates of synthesis of the ribosomal components and the pool of ppGpp increased rapidly, while they all decreased after the downshift . These results are discussed in relation to the role of ppGpp in the growth rate control and the stringent response. J Virol, 1991 Feb, 65(2), 904 - 12 Inactivation of the Moloney murine leukemia virus long terminal repeat in murine fibroblast cell lines is associated with methylation and dependent on its chromosomal position; Hoeben RC et al.; The expression of a retroviral vector with the Moloney murine leukemia virus (Mo-MuLV) long terminal repeat (LTR) promoter after integration into the genome of murine fibroblast cell lines was monitored with the Escherichia coli-derived beta-galactosidase (beta-gal) gene as the reporter . Monoclonal cell lines derived after retroviral infection exhibited a marked heterogeneity in their expression of the reporter gene . We studied two monoclonal cell lines with a single unrearranged copy of the vector provirus integrated into their genome . The first, BB10, expressed the marker enzyme in only 8% of its cell population, whereas in the second, BB16, beta-gal expression could be detected in over 98% of the cells . Treatment of BB10 with the DNA-demethylating agent 5-azacytidine raised the number of beta-gal-positive cells to over 60% . Transfection experiments showed that the Mo-MuLV LTR promoter-enhancer is potentially fully functional in both the BB10 and BB16 cell lines . The inactivated provirus from BB10 cells was cloned and subsequently used to generate retrovirus stocks . The promoter-enhancer activity of its LTR after infection with these BB10-derived viruses showed a variation similar to that of the original virus stocks . Our data showed that (1) inactivation of the Mo-MuLV LTR is a frequent event in murine fibroblast cell lines, (2) inactivation is associated with de novo methylation of cytidine residues, (3) the frequency of inactivation of the provirus must be determined by its chromosomal position, (4) the process of methylation of sequences within the LTR is not necessarily the same as the transcription-repression mechanism that is operating in undifferentiated embryonal carcinoma cells. Infect Immun, 1991 Feb, 59(2), 514 - 20 Immunoreactive epitopes on an expressed recombinant flagellar protein of Borrelia burgdorferi; Collins C et al.; A recombinant Borrelia burgdorferi flagellin protein expressed in Escherichia coli is bound by a murine monoclonal antiflagellin antibody (H9724) and by antibodies in the sera of patients with Lyme disease . Immunoreactive epitopes on the flagellar protein were identified by immunoblot analysis of antibody binding to expressed truncated flagellar proteins . The epitope recognized by the murine monoclonal antibody is within the central heterologous region of the flagellar protein (amino acids 90 to 266) . However, antiflagellin antibodies in the sera of patients with Lyme arthritis bound an epitope entirely within, or whose conformation was partly formed by, the 90 NH2-terminal amino acids of the flagellar protein . The binding of antibodies in the sera of patients with Lyme arthritis to the NH2-terminal region of the flagellar protein, a region with sequence homology to the flagellar proteins of other bacterial species, suggests the possibility that antigenic mimicry contributes to the immunopathogenesis of Lyme disease . The fact that human antibodies bind to a highly conserved and hence shared portion of the flagellin reduces the specificity of serological assays for the diagnosis of Lyme disease which use the flagellar protein as antigen. Biochem J, 1991 Feb 1, 273 ( Pt 3), 787 - 90 Expression in Escherichia coli, purification and characterization of two mammalian thioesterases involved in fatty acid synthesis; Naggert J et al.; Thioesterase I, a constituent domain of the multifunctional fatty acid synthase, and thioesterase II, an independent monofunctional protein, catalyse the chain-terminating reaction in fatty acid synthesis de novo at long and medium chain lengths respectively . The enzymes have been cloned and expressed in Escherichia coli under the control of the temperature-sensitive lambda repressor . The recombinant proteins are full-length catalytically competent thioesterases with specificities indistinguishable from those of the natural enzymes. Genes Dev, 1991 Feb, 5(2), 265 - 77 Autoregulation of a segmentation gene in Drosophila: combinatorial interaction of the even-skipped homeo box protein with a distal enhancer element; Jiang J et al.; Autoregulation has been implicated in the expression of many patterning genes in Drosophila, but the molecular details of this process are largely unknown . In the case of the segmentation gene even-skipped (eve), autoregulation is important for the specification of sharp stripes of gene expression at the onset of gastrulation . Here, we use a combination of DNA binding and P-transformation assays to characterize the cis- and trans-acting factors responsible for autoregulation . We show that eve autoregulation is mediated, at least in part, by a 100-bp minimal autoregulatory sequence (MAS) located approximately 5 kb upstream from the eve transcription start site . Multimerization of a 200-bp DNA fragment that encompasses the MAS drives optimal autoregulatory activity, comparable to that obtained with the native distal enhancer element located between -5.9 and -5.2 kb . The MAS contains two eve protein-binding sites, as well as binding sites for two nuclear factors present in early embryos . Directed mutagenesis of these binding sites suggests that both the eve protein and nuclear factors are essential for autoregulation . These results provide evidence that the eve protein acts combinatorially with other transcription factors to enhance its own expression. J Bacteriol, 1991 Feb, 173(3), 1230 - 40 Genetic analysis of 987P adhesion and fimbriation of Escherichia coli: the fas genes link both phenotypes; Schifferli DM et al.; The 987P fimbrial gene cluster has recently been shown to contain eight genes (fasA to fasH) clustered on large plasmids of enterotoxigenic Escherichia coli and adjacent to a Tn1681-like transposon encoding the heat-stable enterotoxin STIa . Different genetic approaches were used to study the relationship between 987P fimbriation and adhesion . TnphoA mutagenesis, complementation assays, and T7 RNA polymerase-promoted gene expression indicated that all of the fas genes were involved in fimbrial expression and adhesion . In contrast to other fimbrial systems, the lack of expression of any single fas gene never resulted in the dissociation of fimbriation and adhesion, indicating that the adhesin is required for fimbrial expression and suggesting that FasA, the fimbrial structural subunit itself, is the adhesin . In addition, fimbrial length was shown to be modulated by the levels of expression of different fas genes. EMBO J, 1991 Feb, 10(2), 239 - 45 Heat-shock proteins can substitute for SecB function during protein export in Escherichia coli; Altman E et al.; In this study we have shown that (i) induction of the heat-shock response can substitute for SecB function in Escherichia coli, (ii) SecB itself is not a heat-shock protein and (iii) a basal level of heat-shock proteins is required for cells to grow in the absence of a functional SecB protein . Overproduction of DnaK, or GroEL/ES, which were candidates for the heat-shock proteins that could substitute for SecB function, did not rescue the export defect caused when SecB was limiting or absent . In an attempt to identify the heat-shock protein(s) which could substitute for SecB function, unlinked suppressors of secB were isolated and characterized . Interestingly, most of the suppressors mapped to the rpoH locus . Since rpoH encodes sigma 32, the heat-shock transcription factor, it is likely that these suppressors affect the synthesis levels of heat-shock proteins that can substitute for SecB function . The remaining suppressors did not map to any known heat-shock or export genes . Collectively, our data suggest that these suppressors may represent unidentified heat-shock proteins or export factors that act in a manner similar to SecB in facilitating the export process in E . coli. Res Microbiol, 1991 Feb-Apr, 142(2-3), 289 - 94 Complex phenotypes of null mutations in the htr genes, whose products are essential for Escherichia coli growth at elevated temperatures; Karow M et al.; Transposon insertion, followed by screening, has allowed the identification of a set of genes, called htr, whose products are required for Escherichia coli growth at elevated temperatures . The htrB gene has been shown to map at 23.5 min on the E . coli genetic map . It codes for a very basic, hydrophobic, 35,000-Mr polypeptide, possessing a putative membrane-spanning domain . At the non-permissive temperature, htrB mutant bacteria stop dividing, followed by the formation of bulges and eventual lysis . The htrC gene maps at 90 min, is under sigma 32 regulation and codes for a 21, 130-Mr polypeptide . At 43 degrees C, htrC mutant bacteria gradually lyse, whereas at intermediate temperatures they filament extensively . Finally, the htrM gene maps at 81 min, is under sigma 32 regulation and codes for a 35,000-Mr polypeptide . The HtrM null phenotype included inability to grow above 42 degrees C, extreme mucoidness and sensitivity to bile salts, even at the permissive temperatures . The htrM gene is identical to the rfaD gene, whose product is required for the biosynthesis of the lipopolysaccharide precursor ADP-L-glycero-D-mannoheptose (Pegues et al., J . Bact., 1990, 172, 4652-4660). Res Microbiol, 1991 Feb-Apr, 142(2-3), 269 - 77 Functional analysis of the fic gene involved in regulation of cell division; Komano T et al.; We have shown that cAMP may be a regulation factor in cell division of Escherichia coli (Utsumi et al., 1982, 1989) . The fic (filamentation induced by cAMP) gene of this system found previously (Utsumi, et al., 1982) was analysed in this study . The open reading frame of the fic gene coded for 200 amino acids . The pabA (p-aminobenzoate synthase) gene was found downstream from the fic gene . The distance between the end of fic and the start of pabA was 31 base pairs . To deduce the function of Fic protein, the fic gene was destroyed by the kanamycin-resistant (Kmr) gene and the fic gene was shown to be essential for growth of E . coli . Such mutants required PAB (p-aminobenzoate) or folate for growth . These data suggested that the Fic protein is involved in the synthesis of PAB or folate and the fic gene could be part of a pab operon . In cells starved of them, cell division was inhibited . Addition of folate also repressed the filamentation induced by cAMP at 43 degrees C in the fic-1 mutant . These results would indicate that Fic protein and cAMP are involved in a new regulatory mechanism of cell division via folate metabolism . Furthermore, it is also shown that cell division could be controlled by coordination of cAMP, Fic and Fts proteins. Res Microbiol, 1991 Feb-Apr, 142(2-3), 223 - 7 DNA replication in Escherichia coli gyrB(Ts) mutants analysed by flow cytometry; von Freiesleben U et al.; We have investigated the initiation of DNA replication in Escherichia coli gyrB (Ts) mutants and find that initiations in single cells, even those that occur at non-permissive temperature, are synchronous . Furthermore, our results indicate that the gradual arrest of DNA replication at non-permissive temperature reflects a general decrease in transcription activity and not an initiation-specific function of the DNA gyrase . At an intermediate temperature, the only effect observed was a lack of segregation (decatenation) of replicated chromosomes in a sizeable fraction of the cell population. J Toxicol Sci, 1991 Feb, 16 Suppl 1, 95 - 105 8-Hydroxyguanine, a DNA adduct formed by oxygen radicals: its implication on oxygen radical-involved mutagenesis/carcinogenesis; Kasai H et al.; Oxygen radicals have been suggested to be involved in mutation/carcinogenesis . The C-8 position of guanine residues in DNA is hydroxylated to produce 8-hydroxyguanine (8-OH-Gua) in DNA in vitro by various oxygen radical producing agents . The formation of 8-OH-Gua was also observed in cellular DNA in vivo by radiation or oxygen radical forming carcinogens . The 8-OH-Gua residue in DNA is often misread in the position of 8-OH-Gua residue itself but also at neighboring residues next to 8-OH-Gua . When second guanine in codon 12 was specifically replaced with 8-OH-Gua and transferred to NIH3T3, the recipient cells were transformed to malignant cell type . E . coli was found to contain an endonuclease which specifically recognizes 8-OH-Gua residue and cleave DNA strand before and after the modified base . The data obtained imply that 8-OH-Gua formed in DNA in vivo is recognized as an abnormal modified base which, if not repaired, play a role in the mediation of oxygen radical-involved mutation/carcinogenesis. Biochimie, 1991 Feb-Mar, 73(2-3), 187 - 90 Homologous pairing between nucleosome cores on a linear duplex DNA and nucleoprotein filaments of RecA protein-single stranded DNA; Muniyappa K et al.; The ability of E coli recA protein to promote homologous pairing with linear duplex DNA bound to HU protein (Nucleosome cores) was found to be differentially affected . The formation of paranemic joint molecules was not affected whereas the formation of plectomic joint molecules was inhibited from the start of the reaction . The formation of paranemic joint molecules between nucleoprotein filaments of recA protein-circular single stranded DNA and closed circular duplex DNA is believed to generate positive supercoiling in the duplex DNA . We found that the positively superhelical duplex DNA was inert in the formation of joint molecules but could be converted into an active substrate, in situ, by the action of wheat germ topoisomerase I . These observations initiate an understanding of the structural features of E coli chromosome such as DNA supercoiling and nucleosome-like structures in homologous recombination. J Virol Methods, 1991 Feb-Mar, 31(2-3), 229 - 38 Influence of the helper virus on expression of beta-galactosidase from a defective HSV-1 vector, pHSVlac; Geller AI; Defective herpes simplex virus type 1 (HSV-1) vectors can deliver genes into both mitotic and postmitotic cells, including neurons and these vectors, therefore, have great potential use . pHSVlac, the prototype HSV-1 vector, expresses the E . coli Lac Z gene from the HSV-1 immediate early 4/5 promoter . pHSVlac can stably express beta-galactosidase in a range of mammalian cell lines and in neurons from throughout the nervous system, both in culture and in the adult rat brain . Thus, HSV-1 vectors may be useful for studying HSV-1 latency, neuronal physiology, and performing gene therapy for neurological conditions . A virus stock of pHSVlac consists of identical HSV-1 particles containing either pHSVlac DNA or the HSV-1 helper virus DNA . Thus, a cell can be infected with the pHSVlac virus, the helper virus, or both; consequently, it is important to determine if the helper virus influences the behavior of pHSVlac . The effect of the helper virus on expression of beta-galactosidase from pHSVlac was investigated: it was demonstrated first, that pHSVlac can be efficiently packaged into HSV-1 virus particles using each of five HSV-1 temperature sensitive (ts) mutants as helper virus . Second, pHSVlac grown with each of the five HSV-1 ts mutants expressed high levels of beta-galactosidase . Third, pHSVlac grown with HSV-1 strain 17 ts K as helper virus expresses the same amount of beta-galactosidase in the absence or presence of ts K . Thus, pHSVlac can efficiently express a gene independent of the HSV-1 helper virus. Protein Eng, 1991 Feb, 4(3), 245 - 50 Structure of alpha-alpha-hairpins with short connections; Efimov AV; This paper presents the results of detailed stereochemical analysis of structures and sequences of alpha-alpha-hairpins with short connections . It is shown that alpha-alpha-hairpins of each given type have very similar patterns of hydrophobic, hydrophilic and glycine residues in their amino acid sequences . These results can be used in the prediction of alpha-alpha-hairpin conformation as well as in protein design and engineering. Vaccine, 1991 Feb, 9(2), 89 - 96 Genetically-engineered subunit vaccine against feline leukaemia virus: protective immune response in cats; Marciani DJ et al.; A recombinant retroviral subunit vaccine has been developed that successfully protects cats from infectious feline leukaemia virus (FeLV) challenge . The antigen used is a non-glycosylated protein derived from the envelope glycoprotein of FeLV subgroup A, expressed in Escherichia coli . This recombinant protein, rgp70D, includes the entire exterior envelope protein gp70, plus the first 34 amino acids from the transmembrane protein p15E . The vaccine consists of purified rgp70D absorbed on to aluminium hydroxide and used in conjunction with a novel saponin adjuvant . Cats immunized with this formulation developed a strong humoral immune response, including neutralizing and feline oncornavirus-associated cell membrane antigen antibodies . Vaccinated animals showed an anamnestic response upon intraperitoneal challenge with FeLV-A, and were protected from viral infection . In contrast, the control animals developed viraemia shortly after the challenge, which in most cases became chronic . Formulation of the same antigen with other widely used adjuvants elicited poor protective immune responses in cats. Mol Microbiol, 1991 Feb, 5(2), 433 - 7 Design of cAMP-CRP-activated promoters in Escherichia coli; Valentin-Hansen P et al.; We have studied the deoP2 promoter of Escherichia coli to define features that are required for optimal activation by the complex of adenosine 3',5' monophosphate (cAMP) and the cAMP receptor protein (CRP) . Systematic mutagenesis of deoP2 shows that the distance between the CRP site and the -10 hexamer is the crucial factor in determining whether the promoter is activated by cAMP-CRP . Based on these observations, we propose that cAMP-CRP-activated promoters can be created by correctly aligning a CRP target and a -10 hexamer . This idea has been successfully tested by converting both a CRP-independent promoter and a sequence resembling the consensus -10 hexamer to strongly cAMP-CRP-activated promoters. Biotechnology (N Y), 1991 Feb, 9(2), 170 - 2 A genetic system to elicit and monitor antipeptide antibodies without peptide synthesis; Martineau P et al.; We present a simple and flexible procedure to elicit and assay anti-peptide antibodies without peptide synthesis . It consists of expressing the peptide of interest in the form of a genetic insert within two different "recipient" bacterial proteins . One hybrid protein is used as immunogen for the induction of antibodies against the inserted peptide and the other as antigen for monitoring the anti-peptide antibodies raised . The two "recipient" proteins used are the MalE and the LamB proteins from E . coli . The MalE hybrid proteins can be affinity purified on an amylose column using mild nondenaturing conditions and can be crystalized for structural studies; LamB hybrid proteins express the inserted peptide on the cell surface so that intact bacteria can be used as a reagent . We chose, as a model peptide, a B-cell epitope from the pre-S(2) region of Hepatitis B virus . With both MalE and LamB hybrid proteins, high titres of anti-preS antibodies, able to react with native HBsAg particles, were induced in mice . The anti-peptide antibody titres recorded by ELISA were comparable to those obtained when either a synthetic peptide, or the hybrid proteins, were used as immobilized antigen. Biotechnology (N Y), 1991 Feb, 9(2), 157 - 62 Renaturation, purification and characterization of recombinant Fab-fragments produced in Escherichia coli; Buchner J et al.; Cytoplasmatic expression of murine antibody chains in Escherichia coli results in the formation of insoluble and inactive protein aggregates (inclusion bodies) . By systematic variation of the parameters influencing the folding, formation of disulfide bonds and association of the constituent polypeptide chains, we have designed a renaturation procedure allowing the production of microbially expressed Fab-fragments at yields up to 40 percent of the total amount of recombinant protein . The strategy of optimization is generally applicable for disulfide containing proteins produced as inclusion bodies in bacteria . The purified recombinant antibody fragments obtained are identical with the native murine Fab in all functional and physicochemical parameters tested. Appl Microbiol Biotechnol, 1991 Feb, 34(5), 632 - 6 Heterologous gene expression in continuous cultures of budding yeast; Porro D et al.; In a previous paper we have studied the expression of beta-galactosidase from Escherichia coli, driven from the inducible GAL1-10/CYC1 hybrid promoter, in batch cultures of budding Saccharomyces cerevisiae and have described operating conditions for maximal productivity . In this paper we show that the plasmid instability in continuous cultures can be overcome by utilizing appropriate selection markers and a high copy number vector . The maximal level of expression is influenced by the dilution rate . Moreover, enzyme accumulation appears to depend also upon the degree of oxygenation . A possible explanation of these modulations is discussed, taking into account the interactions of the UAS-GAL and TATA-CYC1 elements. Appl Microbiol Biotechnol, 1991 Feb, 34(5), 623 - 7 Application of the tryptophanase promoter to high expression of the tryptophan synthase gene in Escherichia coli; Terasawa M et al.; The application of an inducible regulation system using the tryptophanase operon promoter (TPase promoter; Ptna) was examined for its high expression of the tryptophan synthase (TS) gene in Escherichia coli . The main problem in the application of Ptna for industrial purposes is catabolite repression by glucose, since glucose is the most abundant carbon source . However, this problem could be avoided by changing glucose to an organic acid, such as succinate, fumarate, malate and acetate, in the course of cultivation after glucose initially added was completely consumed . Under these conditions, L-tryptophan was also used to induce tryptophan synthase . Thus, the specific activity of TS in E . coli strain no . 168 harbouring pBR322F-Ptna TS was increased 500-fold compared to that of the cultured host strain . About 1 mol L-tryptophan/l reaction mixture was formed from indole and L-serine at 37 degrees C for 3.5 h. Enzyme Microb Technol, 1991 Feb, 13(2), 127 - 33 Design of two immobilized cell catalysts by entrapment on gelatin: internal diffusion aspects; Castillo E et al.; Experimental results obtained during the design of two immobilized cell catalysts by entrapment on gelatin are presented . Strong diffusional limitations are found and explained with the usual parameters and models, introducing an empirical correlation between substrate concentration and effectiveness factor . The effect of particle size, enzyme load, and specific activity in the system is discussed in terms of cooperation between bioengineers and geneticists. Lett Appl Microbiol, 1991 Feb, 12(2), 39 - 42 Electroporation-induced transformation of Escherichia coli: evaluation of a square waveform pulse; Elvin S et al.; Four strains of Escherichia coli were tested for electroporation-induced plasmid transformation using a high voltage pulse in a square waveform . Conditions for optimal transformation were determined for each strain and transformation frequencies found to be comparable to those reported previously for E . coli with a sinusoidal waveform of exponential decaying pulse. J Biotechnol, 1991 Feb, 17(2), 177 - 88 Cloning of three endoglucanase genes from Thermomonospora curvata into Escherichia coli; Presutti DG et al.; A BamHI genomic library from Thermomonospora curvata was constructed in E . coli using cosmid vector pHC79 . Four clones able to hydrolyze CMC were isolated . Restriction digests and Southern gel analysis revealed the presence of three different endoglucanase genes . DNA fragments contained in all of the endoglucanase cosmids hybridized to T . curvata chromosomal DNA . The cellulase genes were expressed in E . coli, but at rather low levels. Biochem Biophys Res Commun, 1991 Jan 31, 174(2), 898 - 902 A model for histone H5-DNA interaction: simultaneous minor and major groove binding; Segers A et al.; Using the tertiary structure of the globular domain of H5 (GH5) and based on an alternative sequence homology between GH5 and DNA-binding proteins containing the helix-turn-helix motif, a model for H5-DNA interaction is proposed . From molecular graphics it follows that helix II recognizes the major groove of the DNA, as does the second helix of the helix-turn-helix motif, while helix III makes minor groove contacts, in agreement with the hypothesis of Turnell et al . (FEBS letters 232, 263-268) . In the resulting model GH5 makes contact with a full turn of DNA. Biochem Biophys Res Commun, 1991 Jan 31, 174(2), 529 - 35 Molecular cloning and expression of a human brain inositol 1,4,5-trisphosphate 3-kinase; Takazawa K et al.; A human hippocampus cDNA library was screened by hybridization with a rat brain inositol 1,4,5-trisphosphate (InsP3) 3-kinase cDNA . Sequencing of three overlapping clones identified a 1383 bp open reading frame encoding a 461 amino acid protein with a calculated molecular weight of 50988 . The coding amino acid sequence showed an overall 93% similarity with the sequence of rat brain InsP3 3-kinase . The cDNA insert of one isolated partial clone (i.e . hh 26) was in frame with the beta galactosidase fragment fused to it as a Bluescript plasmid; it displayed InsP3 3-kinase activity when expressed in Escherichia coli (E . coli) . Biochemical characterization of human brain InsP3 3-kinase by SDS polyacrylamide gel electrophoresis and regeneration of enzyme activity reveals three active fractions with apparent Mr of 58,000-64,000, 45,000-50,000 and 37,000-39,000. Biochem Biophys Res Commun, 1991 Jan 31, 174(2), 785 - 9 Crosslinking of substrates occurs exclusively to the p66 subunit of heterodimeric HIV-1 reverse transcriptase; Cheng N et al.; Photoaffinity labeling of the hetero- and homodimeric forms of HIV-1 reverse transcriptase has been carried out using {32P}rA12-18.dT10 as a representative template-primer and {alpha-32P}dTTP as a representative 2'-deoxynucleoside-5'-triphosphate . UV irradiation produces stable, covalent crosslinks between each of the reactants and both the hetero-(p66/p51) and homodimeric (p66/p66, p51/p51) forms of the enzyme . In the case of the p66/p51 heterodimer, the form of the enzyme believed to be involved in viral replication, crosslinking occurs exclusively to the p66 subunit . These results suggest that the polymerase activity of the heterodimer residues on p66. Biochim Biophys Acta, 1991 Jan 30, 1061(2), 171 - 4 Reconstitution of the L-leucine-H+ cotransporter of the plasma membrane from Chang liver cells into proteoliposomes; Mitsumoto Y et al.; L-Leucine is cotransported with H+ in the plasma membrane of Chang liver cells (Mitsumoto, Y . et al . (1986) J . Biol . Chem . 261, 4549) . The leucine transport system was solubilized from the plasma membrane of the cells with ocytl glucoside and reconstituted in proteoliposomes prepared by a rapid dilution of a mixture of the solubilized proteins, octyl glucoside and liposomes . The proteoliposomes exhibited H(+)-gradient and electrical potential-stimulated leucine uptake . The H(+)-gradient-stimulated leucine uptake could be completely inhibited by carbonyl cyanide p-trifluoro-methoxyphenylhydrazone (FCCP) and 2-aminobicyclo{2,2,1}heptane-2-carboxylic acid (BCH) . The stimulatory effect of H+ gradient on leucine uptake was shown to be mainly due to decrease of the Km, but not to change of the Vmax, of the transport kinetics . These results suggest that the leucine-H+ cotransporter is solubilized and reconstituted into proteoliposomes. Biochim Biophys Acta, 1991 Jan 29, 1076(2), 289 - 97 Possible regulation of the in vitro assembly of bovine brain tubulin by the bovine thioredoxin system; Khan IA et al.; Microtubule assembly in vitro and in vivo is highly sensitive to a variety of sulfhydryl-reactive reagents, raising the question of the possible existence of a physiological sulfhydryl-mediated system for regulating microtubule assembly . However, the specific reagents which have previously been used to inhibit microtubule assembly in vitro are either nonphysiological or, if physiological, effective only at concentrations much higher than their physiological ones . Because of reports of association in vivo between microtubules and the sulfhydryl-reactive proteins thioredoxin and thioredoxin reductase, we decided to examine the interaction in vitro between microtubules and the thioredoxin system, comprising thioredoxin, thioredoxin reductase and NADPH . At pH 6.8, both the mammalian and the Escherichia coli thioredoxin systems inhibited microtubule assembly by 4-35% (19 +/- 9%) by reducing one intra-subunit disulfide bond in the tubulin dimer . The thioredoxin-reducible disulfide of the tubulin dimer remains protected from thioredoxin in the assembled microtubules . Thioredoxin or thioredoxin reductase alone, or together in the absence of NADPH, were incapable of either reducing tubulin or inhibiting microtubule assembly . Microtubules formed from reduced tubulin were found to be stable and morphologically identical to those obtained from native tubulin dimers . Since the components of the thioredoxin system were used at concentrations similar to their physiological ones, our results suggest a potential role of the thioredoxin system in regulation of microtubule assembly in vivo. Biochim Biophys Acta, 1991 Jan 29, 1076(2), 266 - 72 Secondary structure of Escherichia coli glucosamine-6-phosphate deaminase from amino acid sequence and circular dichroism spectroscopy; Altamirano MM et al.; The secondary structure of the purified glucosamine-6-phosphate deaminase from Escherichia coli K12 was investigated by both circular dichroism (CD) spectroscopy and empirical prediction methods . The enzyme was obtained by allosteric-site affinity chromatography from an overproducing strain bearing a pUC18 plasmid carrying the structural gene for the enzyme . From CD analysis, 34% of alpha-helix, 9% of parallel beta-sheet, 11% of antiparallel beta-sheet, 15% turns and 35% of non-repetitive structures, were estimated . A joint prediction scheme, combining six prediction methods with defined rules using several physicochemical indices, gave the following values: alpha-helix, 37%; beta-sheet, 22%; turns, 18% and coil, 23% . The structure predicted showed also a considerable degree of alternacy of alpha and beta structures; 64% of helices are amphipathic and 90% of beta-sheets are hydrophobic . Overall, the data suggest that deaminase has as dominant motif, an alpha/beta structure. Biochemistry, 1991 Jan 29, 30(4), 1141 - 8 An alkaline phosphatase protection assay to investigate trp repressor/operator interactions; Marmorstein RQ et al.; We have used an alkaline phosphatase protection assay to investigate the interaction of the trp repressor with its operator sequence . The assay is based on the principle that the trp repressor will protect a terminally 5'-32P-labeled operator DNA fragment from attack by alkaline phosphatase . The optimal oligonucleotide for investigating the trp repressor/operator interaction extends two base pairs from each end of the genetically defined target sequence predicted by in vivo studies {Bass et al . (1987) Genes Dev . 1, 565-572} . The assay works well over a 10,000-fold range of protein/DNA affinity and is used to show that the corepressor, L-tryptophan, causes the liganded repressor to bind a 20 base pair trp operator duplex 6400 times more strongly than the unliganded aporepressor . The affinity of the trp repressor for operators containing symmetrical mutations was interpreted in terms of the trp repressor/operator crystal structure as follows: (1) Direct hydrogen bonds with the functional groups of G-9 of the trp operator and the side chain of Arg 69 of the trp repressor contribute to DNA-binding specificity . (2) G-6 of the trp operator is critical for DNA-binding specificity probably because of the two water-mediated hydrogen bonds between its functional groups and the N-terminus of the trp repressor's E-helix . (3) Sequence-dependent aspects of the trp operator's conformation help stabilize the trp repressor/operator complex.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1991 Jan 29, 30(4), 1091 - 7 32P-postlabeling detection of radiation-induced DNA damage: identification and estimation of thymine glycols and phosphoglycolate termini; Weinfeld M et al.; A 32-P-postlabeling assay has been developed that permits detection of several radiogenic base and sugar lesions of DNA at the femtomole level . The technique is based on the inability of DNase I and snake venom phosphodiesterase to cleave the internucleotide phosphodiester bond immediately 5' to the site of damage so that complete digestion of irradiated DNA with these nucleases and alkaline phosphatase yields lesion-bearing "dinucleoside" monophosphates . Because these fragments contain an unmodified nucleoside at the 5'-end of each molecule, they can be readily phosphorylated by T4 polynucleotide kinase and {gamma-32P}ATP and analyzed by polyacrylamide gel electrophoresis and reverse-phase HPLC . We observed a linear induction of total damage in DNA irradiated with 5-50 Gy . Virtually no damage was detected when the DNA was irradiated in solution containing 1 M DMSO, implicating hydroxyl radicals in the formation of these lesions . Evidence for the presence of thymine glycols and phosphoglycolate groups came from (i) a comparison of the radiation-induced products with those produced by OsO4 and KMnO4 and (ii) incubation of irradiated DNA with Escherichia coli endonuclease III and exonuclease III before analysis by the postlabeling procedure . This was confirmed by comigration of the radiogenic products with chemically synthesized markers . G values of 0.0022 and 0.0105 mumol J-1 were obtained for thymine glycol and phosphoglycolate production, respectively . The identity of the 5'-nucleotide of each isolated compound was obtained by nuclease P1 digestion . This analysis of nearest-neighbor bases to thymine glycols and phosphoglycolates indicated a nonrandom interaction between radiation-induced hydroxyl radicals and DNA. Biochim Biophys Acta, 1991 Jan 29, 1076(2), 225 - 32 Sequence similarities within the family of dihydrolipoamide acyltransferases and discovery of a previously unidentified fungal enzyme; Russell GC et al.; A composite protein sequence database was searched for amino acid sequences similar to the C-terminal domain of the dihydrolipoamide acetyltransferase subunit (E2p) of the pyruvate dehydrogenase complex of Escherichia coli . Nine sequences with extensive similarity were found, of which eight were E2 subunits . The other was for a putative mitochondrial ribosomal protein, MRP3, from Neurospora crassa . Alignment of the MRP3 and E2 sequences showed that the similarity extends through the entire MRP3 sequence and that MRP3 is most closely related to the E2p subunit of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae, with 54% identical residues and a further 36% that are conservatively substituted . Other features of the MRP3 gene and protein are also consistent with it being the acyltransferase subunit of a 2-oxo acid dehydrogenase complex . A multiple alignment of 13 E2 sequences indicated that 120 (34%) of 353 equivalenced residues are identical or show some degree of conservation . It also identified residues that are potentially important for the structure, catalytic activity and substrate-specificity of the acyltransferases. Biochemistry, 1991 Jan 29, 30(4), 1097 - 118 A thermodynamic analysis of RNA transcript elongation and termination in Escherichia coli; Yager TD et al.; In the first part of this paper we present a thermodynamic analysis of the elongation phase of transcription in Escherichia coli . The stability of the elongation complex is described by a "free energy of formation" function (delta G zero f) that is a sum of terms for forming (i) a locally denatured 17-base-pair DNA "bubble"; (ii) a constant-length hybrid between the 3'-terminal 12-nucleotide residues of the RNA transcript and the corresponding region of the DNA template strand; and (iii) a set of binding interactions between the polymerase and certain DNA and RNA residues within and near the "transcription bubble" . The transcriptional elongation complex is very stable at most positions along a natural DNA template and moves in a highly processive fashion . At these positions, the delta G zero f function provides a quantitative measure of the stability of the elongation complex . Besides allowing for the polymerization of the RNA transcript, the elongation complex also serves to define the context within which transcript termination occurs . In the second part of the paper the thermodynamic analysis is extended to discriminate between template positions at which the elongation complex is stable and positions at which it is rendered relatively unstable by the presence of a string of rU residues at the 3'-terminus of the RNA together with the formation of a specific RNA hairpin just upstream of this point . Most factor-independent (intrinsic) termination events are thermodynamically disallowed at the former positions and are thermodynamically allowed at the latter positions . The extended form of the analysis closely predicts the exact sites of termination at a number of intrinsic terminators (and attenuators) in the E . coli genome . It also correctly predicts bidirectional function for a number of bidirectional terminators . In some cases it may identify terminators that are similar to the intrinsic type but that require additional protein factors, unusual polymerase-nucleic acid interactions, or rate-limiting conformational changes in order to function . Finally, it successfully locates intrinsic terminators within a number of E . coli operons and discriminates between these terminators and the surrounding DNA sequence. FEBS Lett, 1991 Jan 28, 278(2), 225 - 8 Examination of elongation factor Tu for aluminum fluoride binding sites using fluorescence and 19F-NMR methodologies; Hazlett TL et al.; This article reports on a comparison of the interaction of Al3+ and F- with two GTP-binding proteins, elongation factor Tu (EF-Tu) and the hormone sensitive regulatory protein (G protein) G0 alpha . The methodologies chosen to elucidate possible interactions between protein and aluminum fluoride were fluorescence spectroscopy and nuclear magnetic resonance (19F-NMR) . Both proteins have tryptophan residues near their nucleotide binding sites, the purported site of aluminum fluoride interaction . It has been assumed for G proteins (including G0 alpha) that aluminum fluoride, in the presence of Mg2+ mimics the magnesium coordinated gamma-phosphate group for the GDP-form of the protein and shifts the protein's conformation toward the active GTP-form . Indeed, changes in intrinsic fluorescence of G0 alpha effected by aluminum fluoride are observed . The presence of aluminum fluoride did not affect the intrinsic fluorescence, spectra or lifetimes, of EF-Tu.GDP 19F-NMR was then used to directly test for bound F- . Fluoride alone or in the presence of either protein gave a single 19F-NMR peak at -10 ppm, characteristic of free F- . With the addition of aluminum to the protein and F- samples a second peak, shifted upfield from the first to -29 ppm, was observed for G0 alpha.GDP . This second peak, which has been assigned to protein-bound F-, was not observed for EF-Tu.GDP . These observations show that the interaction of Al3+ and F-, in the presence of Mg2+, may be quite different between the hormone-sensitive G proteins, which bind aluminum fluoride, and the GTP-binding proteins as a whole, which include EF-Tu . Care must therefore be exercised when structural data on the elongation factor, specifically on the nucleotide site, are used to interpret data or compose models intended to describe the hormone-sensitive regulatory G proteins. Nucleic Acids Res, 1991 Jan 25, 19(2), 313 - 8 Training back-propagation neural networks to define and detect DNA-binding sites; O'Neill MC; A three layered back-propagation neural network was trained to recognize E . coli promoters of the 17 base spacing class . To this end, the network was presented with 39 promoter sequences and derivatives of those sequences as positive inputs; 60% A + T random sequences and sequences containing 2 promoter-down point mutations were used as negative inputs . The entire promoter sequence of 58 bases, approximately -50 to +8, was entered as input . The network was asked to associate an output of 1.0 with promoter sequence input and 0.0 with non-promoter input . Generally, after 100,000 input cycles, the network was virtually perfect in classifying the training set . A trained network was about 80% effective in recognizing 'new' promoters which were not in the training set, with a false positive rate below 0.1% . Network searches on pBR322 and on the lambda genome were also performed . Overall the results were somewhat better than the best rule-based procedures . The trained network can be analyzed both for its choice of base and relative weighting, positive and negative, at each position of the sequence . This method, which requires only appropriate input/output training pairs, can be used to define and search for any DNA regulatory sequence for which there are sufficient exemplars. Nucleic Acids Res, 1991 Jan 25, 19(2), 307 - 11 Molecular cloning and characterization of the gene encoding the adenine methyltransferase M.CviRI from Chlorella virus XZ-6E; Stefan C et al.; The gene encoding the DNA methyltransferase M.CviRI from Chlorella virus XZ-6E was cloned and expressed in Escherichia coli . M.CviRI methylates adenine in TGCA sequences . DNA containing the M.CviRI gene was sequenced and a single open reading frame of 1137 bp was identified which could code for a polypeptide of 379 amino acids with a predicted molecular weight of 42,814 . Comparison of the M.CviRI predicted amino acid sequence with another Chlorella virus and 14 bacterial adenine methyltransferases revealed extensive similarity to the other Chlorella virus enzyme. J Biol Chem, 1991 Jan 25, 266(3), 1728 - 32 Modified enterotoxin signal sequences increase secretion level of the recombinant human epidermal growth factor in Escherichia coli; Morioka-Fujimoto K et al.; Amino acid substitutions were made in the heat-labile enterotoxin signal sequence of Escherichia coli by recombinant DNA techniques, and their influence on the secretion of recombinant human epidermal growth factor by E . coli was examined . The heat-labile enterotoxin signal sequence is an amino-terminal extension of the octadecapeptide chain and is comprised of three distinct regions: a positively charged amino-terminal region, a central hydrophobic region, and a carboxyl-terminal region with the cleavage site recognized by the signal peptidase . Some alterations in the signal sequence caused a 1.5-3.5-fold increase in the secretion of recombinant human epidermal growth factor . These were the introduction of: (i) polar and small residues into the carboxyl-terminal region (replacement of Pro-1 Leu-3 with Asn-Ala or Ser-Ala), which may give a favorable structure for the recognition and cleavage by the signal peptidase; and (ii) a polar residue into the central hydrophobic region (replacement of Ile-9 with Ser), which may cause an increase of the affinity to the cytoplasmic membrane . In the latter case, a large amount of the unprocessed "precursor" was accumulated . The combination of these modifications, however, did not work additively . An increase in the amino-terminal positive charge (insertion of Lys) had no effect on secretion . These results prove that the level of protein secretion is greatly dependent on the polarity of the carboxyl-terminal region and the hydrophobicity and/or the amphiphilicity of the central region . Moreover, the overall balance of the physicochemical properties of respective regions is important. J Biol Chem, 1991 Jan 25, 266(3), 1692 - 6 Yeast thioredoxin genes; Gan ZR; Based on the conserved protein sequence of thioredoxins from yeast and other organisms, two primers were synthesized for polymerase chain reaction of yeast genomic DNA . A 34-base pair (bp) sequence around the active site of yeast thioredoxin was obtained from the polymerase chain reaction product . This specific sequence was used as a probe in Southern blot analysis of total yeast genomic DNA digested with various restriction enzymes . Under conditions of high stringency, more than one DNA species hybridized with the probe, suggesting that more than one gene encodes yeast genomic library . Two Sau3A1 fragments, 825 and 2045 bp, respectively, from two different clones were cloned into pUC13 . Sequence analysis of these fragments gave two different open reading frames without introns . The 825-bp Sau3A1 fragment encodes a 103-amino acid residue protein named thioredoxin I . The 2045-bp Sau3A1 fragment contains a sequence encoding thioredoxin II which has 102 amino acid residues . This is the first report of the cloning and sequencing of eukaryotic thioredoxin genes from any source . Both yeast thioredoxins contain a dithiol active site sequence, Cys-Gly-Pro-Cys . Thioredoxins I and II show 78% amino acid sequence identity . They display more amino acid sequence similarity with mammalian thioredoxin than with Escherichia coli and plant chloroplast thioredoxins. J Biol Chem, 1991 Jan 25, 266(3), 1616 - 26 Monomer-tetramer equilibrium of the Escherichia coli ssb-1 mutant single strand binding protein; Bujalowski W et al.; The Escherichia coli single strand binding (SSB) protein is an essential protein required for DNA replication and involved in recombination and a number of repair processes . It is a stable homotetramer in solution; however the ssb-1 mutation (His-55 to Tyr) destabilizes the tetramer with respect to monomers and this defect seems to explain the observed phenotype (Williams, K . R., Murphy, J . B., and Chase, J . W . (1984) J . Biol . Chem . 259, 11804-11811) . We report a quantitative study of the SSB-1 monomer-tetramer equilibrium in vitro as a function of temperature, pH, NaCl, MgCl2, urea, and guanidine hydrochloride concentrations . The self-assembly equilibrium was monitored by the increase in intrinsic protein fluorescence anisotropy accompanying the formation of the tetramer . The experimental isotherms indicate that SSB-1 dimers are not highly populated at equilibrium, hence the formation of the tetramer is well-described as a one-step association of four monomers . At 25 degrees C, pH 8.1, the monomer concentration for 50% tetramer dissociation is (MT)1/2 = 0.87 microM, corresponding to a monomer-tetramer equilibrium constant, KT = 3 +/- 1 x 10(18) M-3 . The tetramerization constant, KT, is highly dependent upon temperature and pH, with delta H0 = -51 +/- 7 kcal/mol (pH 8.1) and delta H0 = -37 +/- 5 kcal/mol (pH 6.9) . There is no effect of NaCl on the monomer-tetramer association in the range from 0.20 to 1.0 M; however, MgCl2 decreases the stability of the SSB-1 tetramer . In the presence of high concentrations of the single-stranded oligonucleotide, dT(pT)15, the tetramerization constant is slightly increased indicating that binding of the oligonucleotide to the SSB-1 monomer promotes the assembly process, although not dramatically . The large negative delta H0 that is associated with formation of the tetramer provides a likely explanation for the temperature sensitivity of the ssb-1 mutation. J Biol Chem, 1991 Jan 25, 266(3), 1543 - 50 5-Formyltetrahydrofolate polyglutamates are slow tight binding inhibitors of serine hydroxymethyltransferase; Stover P et al.; The interaction of the mono- and triglutamate forms of 5-methyltetrahydrofolate and 5-formyltetrahydrofolate with serine hydroxymethyltransferase were determined by several methods . These methods included: determining dissociation constants by observing the absorbance at 502 nm of a ternary complex of the enzyme, glycine, and the folate compounds; determining inhibition constants from steady-state reactions; and determining the rate of formation and breakdown of the enzyme inhibitor complex by rapid reaction kinetics . Studies of the dissociation and inhibitor constants showed that both 5-methyltetrahydrofolate and 5-formyltetrahydrofolate have essentially the same affinity for the enzyme-glycine binary complex . However, rapid reaction and steady-state kinetic studies showed that the triglutamate form of 5-formyltetrahydrofolate both binds and is released much more slowly from the enzyme-glycine binary complex, compared with the triglutamate form of 5-methyltetrahydrofolate . The results also showed that only one rotamer of 5-formyltetrahydrofolate binds at the active site of serine hydroxymethyltransferase . The results are discussed in terms of the possible role of 5-formyltetrahydrofolate polyglutamates in regulation of one-carbon metabolism. J Biol Chem, 1991 Jan 25, 266(3), 1469 - 77 Processing of DNA base damage by DNA polymerases . Dihydrothymine and beta-ureidoisobutyric acid as models for instructive and noninstructive lesions; Ide H et al.; The processing of unrepaired DNA lesions is a key to understanding and predicting the biological end points of particular DNA damages . In this study, we prepared single-stranded f1 phage (f1-K12) DNA containing dihydrothymine or beta-ureidoisobutyric acid as models for instructive or noninstructive base lesions and assessed the potential biological consequences of these lesions in vitro and in vivo . To determine the effect of the two lesions on in vitro DNA synthesis, the extent of DNA synthesis was measured by 3H-labeled nucleotide incorporation, and the newly synthesized DNA was analyzed by DNA sequencing gels . The results showed that dihydrothymine in the template was at most a weak block to in vitro DNA synthesis catalyzed by Escherichia coli DNA polymerase I Klenow fragment (Pol I) and T4 DNA polymerase . In contrast, beta-ureidoisobutyric acid constituted a very strong (probably absolute) replicative block in vitro . With Pol I, termination bands were observed either opposite or one base prior to (3' to) the putative beta-ureidoisobutyric acid depending on its position in the template . However, when DNA synthesis was catalyzed by Pol I lacking a 3'----5' exonuclease activity, termination bands were only observed opposite beta-ureidoisobutyric acid, with purine nucleotides being incorporated preferentially opposite the lesion . With T4 DNA polymerase that contains a very active 3'----5' exonuclease activity, DNA synthesis was arrested almost exclusively one base prior to (3' to) the putative beta-ureidoisobutyric acid site in the template . We also measured survival of transfecting DNA containing dihydrothymine or beta-ureidoisobutyric acid in an attempt to correlate the in vitro data with in vivo processing . In keeping with the results obtained in vitro, dihydrothymine present in transfecting f1-K12 DNA did not constitute an inactivating lesion . On the other hand, it took 0.9 beta-ureidoisobutyric acid residues per molecule to inactivate transfecting f1-K12 DNA, indicating that the lesion was an absolute replicative block in vivo . When host cells were ultraviolet-irradiated to induce the SOS response, a slight increase (about 2-fold) in survival of transfecting f1-K12 DNA containing beta-ureidoisobutyric acid was observed . The potential effects of the structures of base lesions on lesion-polymerase interactions are discussed. Nucleic Acids Res, 1991 Jan 25, 19(2), 365 - 70 Sequence effect on incision by (A)BC excinuclease of 4NQO adducts and UV photoproducts; Thomas DC et al.; Nucleotide excision repair in Escherichia coli is initiated by (A)BC excinuclease, an enzyme which incises DNA on both sides of bulky adducts and removes the damaged nucleotide as a 12-13 base long oligomer . The incision pattern of the enzyme was examined using DNA modified by 4-nitroquinoline 1-oxide (4NQO) and UV light . Similar to the cleavage pattern of UV photoproducts and other bulky adducts, the enzyme incises the 8th phosphodiester bond 5' and 5th phosphodiester bond 3' to the 4NQO-modifed base, primarily guanine . The extent of DNA damage by these agents was determined using techniques which quantitatively cleave the DNA or stop at the site of the adduct . By comparison of the intensity of gel bands created by (A)BC excinuclease and the specific cleavage at the damaged site, the efficiency of (A)BC excinuclease incision at 13 different 4NQO-induced adducts and 13 different photoproducts was determined by densitometric scanning . In general, incisions made at 4NQO-induced adducts are proportional to the extent of damage, though the efficiency of cutting throughout the sequence tested varies from 25 to 75% . Incisions made at pyrimidine dimers are less efficient than at 4NQO-adducts, ranging from 13 to 65% incision relative to modification, though most are around 50% . The two (6-4) photoproducts within the region tested are incised more efficiently than any pyrimidine dimer. J Biol Chem, 1991 Jan 25, 266(3), 1672 - 8 Characterization of recombinant human Kirsten-ras (4B) p21 produced at high levels in Escherichia coli and insect baculovirus expression systems; Lowe PN et al.; Kirsten-ras is the oncogene most frequently activated in human tumors . Studies of its biological function have been limited by the nonavailability of significant amounts of the major protein product, Kirsten-ras (4B) p21 . When expressed in Escherichia coli K12, the recombinant protein was rapidly cleaved upon cell lysis in the lysine-rich C terminus region, probably by the ompT protease . However, soluble full-length protein was obtained when the Kirsten-ras gene was expressed in an E . coli strain lacking the ompT gene, and also in a baculovirus/insect cell expression system . Additionally, the baculovirus/insect cell system produced about half of the Kirsten-ras protein in a membrane-associated form, which was post-translationally modified by polyisoprenylation and carboxyl-methylation . A C-terminally truncated form (residues 1-166) was also expressed at high levels in E . coli for x-ray crystallographic studies . The kinetics of GDP release and of GTP hydrolysis of the purified proteins are similar to those of the corresponding Harvey-ras proteins, though there are small differences in the relative affinities for GDP and GTP . Biological activity of full-length Kirsten Val-12 p21 was demonstrated by microinjection into Swiss 3T3 cells, resulting in morphological transformation, with a lower potency than that of Harvey Val-12 protein. Nucleic Acids Res, 1991 Jan 25, 19(2), 371 - 7 Replication of a geminivirus derived shuttle vector in maize endosperm cells; Ugaki M et a