Escherichia coli

Mutat Res, 1987 Mar, 183(2), 103 - 8
Induction of SOS responses in Escherichia coli by 5-fluorouracil; Oda Y; The inducibility of SOS responses by 5-fluorouracil (5-FU), which has been used as an antitumor drug, was studied in Escherichia coli cells which have different DNA repair capacities for UV lesions . Expression of the umuC gene was apparently induced by 5-FU in the wild-type and uvrA strains, but not in lexA and recA strains . The inducibility of the umuC gene by 5-FU, the metabolite of which inhibits thymidylate synthetase, was abolished in cultures containing deoxythymidine monophosphate which is converted from deoxyuridine monophosphate by thymidylate synthetase . These results suggest that 5-FU may exert its SOS inducibility by inhibiting thymidylate synthetase and then disturbing DNA metabolism but not by incorporating 5-FU residues into RNA . Further, 5-FU weakly induced mutations in E . coli.

J Immunol, 1987 Mar 1, 138(5), 1542 - 5
Diacylglycerol mass measurements in stimulated HL-60 phagocytes; Preiss JE et al.; The mass of sn-1,2-diacylglycerol in crude lipid extracts from differentiated HL-60 phagocytes was measured by quantitative conversion of the diacylglycerol to {32P}-labeled phosphatidic acid catalyzed by E . coli diacylglycerol kinase . The chemotactic peptide N-formyl-Met-Leu-Phe caused a time- and concentration-dependent increase in diacylglycerol that was maximal at 4 min . Diacylglycerol returned toward basal levels by 15 min . The basal level of diacylglycerol was 290 +/- 25 pmol/10(7) cells (n = 36) . Maximally effective concentrations of N-formyl-Met-Leu-Phe and N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys increased diacylglycerol to 176% +/- 16 of basal (n = 8) and 198% +/- 15 of basal (n = 4), respectively . t-Boc-Phe-Leu-Phe-Leu-Phe, a competitive antagonist of formyl peptide receptor function, competitively inhibited the N-formyl-Met-Leu-Phe-induced diacylglycerol increase . Pretreatment of the cells with pertussis toxin abolished the stimulated rise in diacylglycerol, whereas depletion of extracellular Ca2+ markedly inhibited the increase . The Ca2+ ionophore A23187 stimulated a large (450% of basal) and persistent (greater than 30 min) increase in diacylglycerol . These data suggest that agents which raise intracellular Ca2+ levels in differentiated HL-60 cells produce a prolonged increase in cellular diacylglycerol which may activate protein kinase C.

J Korean Med Sci, 1987 Mar, 2(1), 65 - 70
Purification of heat-labile enterotoxin from an enterotoxin from an enterotoxigenic Escherichia coli of human origin by monoclonal immunoaffinity chromatography; Cho MJ et al.; Heat-labile enterotoxin (LT) was purified from an enterotoxigenic Escherichia coli 015H11 of human origin . The purification steps included French pressure cell disruption of the bacteria, salting-out, DEAE-Sephacel on chromatography . Application of this procedure resulted in a 95.1-fold purification of LT with a yield of 19.9% as determined by rabbit ileal loop assay . The final LT preparation showed only one protein-staining band on polyacrylamide gel electrophoresis, indicating that the purified LT was homogeneous.

Plasmid, 1987 Mar, 17(2), 117 - 36
Characterization of the maintenance functions of IncFIV plasmid R124; Campbell IG et al.; The genetic arrangement of the regions involved in R124 replication and incompatibility have been located and their homology to the IncFI basic replicons has been assessed . We show that R124 has homology with all three basic replicons, RepFIA, RepFIB, and RepFIC, and that these regions, FIVA, RepFIVB, and RepFIVC, are widely separated on the R124 genome . Cloning of autonomously replicating fragments has shown that RepFIVB and RepFIVC are functional in R124 and express incompatibility . The FIVA region was unable to form a functional replicon and when cloned into pUC8 lacked incompatibility activity . A fourth region of R124 was identified, which although not essential for replication stabilized mini-R124 plasmid replication and exhibited incompatibility with R124 . This region, designated IncIV, showed no homology to RepFIA, RepFIB, or RepFIC . Incompatibility expression of IncIV required only the EcoRI fragment E13 but the strength of the reaction was modified in the presence of other fragments . The replication and incompatibility properties of an R124 deletion derivative indicated that R124 can switch its replication to either RepFIVB or RepFIVC when in the presence of an incompatible plasmid . The ambiguous incompatibility reactions reported for R124 is a result of the expression of the two functional replicons, RepFIVB and RepFIVC, and that expressed by IncIV.

Bioorg Khim, 1987 Mar, 13(3), 350 - 8
{Expressing plasmid vectors on the basis of Escherichia coli beta-galactosidase gene fragments}; Chakhmakhcheva OG et al.; With the use of synthetic DNA fragments, a set of new plasmid vectors has been obtained . The vectors provided high level expression of peptides and small proteins in E . coli as fusions with fragments of beta-galactosidase of various length . These vectors were used to achieve expression of a synthetic gene for a functionally active fragment of bacteriorhodopsin . The yields of hybrid proteins consisting of beta-galactosidase and bacteriorhodopsin fragments were in the range of 5-30% from the total amount of cellular protein.

Biochem J, 1987 Mar 1, 242(2), 539 - 50
Galactoside-proton symport in a lacYUN mutant of Escherichia coli investigated by analysis of transport progress curves; Page MG; The kinetics of galactoside-proton symport catalysed by a wild-type strain and one carrying a mutation, previously reported to cause uncoupling of the symport reaction, have been examined . The mutation does not affect the stoichiometry during the initial period of uptake, when the internal concentration of galactoside is low, but it does result in much greater competition from the galactoside as it is accumulated . Simple methods for the analysis of the uptake progress curves have been developed and used to estimate the initial rate of uptake and affinity for internal galactoside . The maximum rate of uptake is decreased by a factor of 2 at most whereas the affinity for internal galactoside is increased up to 50-fold by the mutation . The pH-dependence of the galactoside efflux reaction is changed in a manner which suggests that the defect is in the interaction between proton-binding and galactoside-binding sites rather than in the structure of either site.

Mol Gen Genet, 1987 Mar, 206(3), 519 - 21
Replication of mini-F plasmid in vitro promoted by purified E protein; Muraiso K et al.; An in vitro system for replication of mini-F plasmid DNA was constructed . This system consists of an ammonium sulfate fraction II (Fuller et al . 1981) from Escherichia coli extract, exogenously added purified E protein encoded by mini-F plasmid, and mini-F DNA in a closed circular form . Experiments with this system showed that the 217 bp DNA region which contains the A + T rich cluster and the four 19 bp direct repeats responsible for incB incompatibility is essential for mini-F DNA replication.

Mol Gen Genet, 1987 Mar, 206(3), 505 - 9
Isolation and characterization of the regulatory HEX2 gene necessary for glucose repression in yeast; Niederacher D et al.; The HEX2 gene which is necessary for glucose repression and is involved in the regulation of hexokinase PII synthesis and maltose uptake, has been cloned by complementation of a hex2 mutant, and selection for restored growth on maltose . Glucose repression in the transformants was like that in the wild type . The HEX2 gene was localized within a 2.15 kb fragment . The restriction map was confirmed by Southern hybridization of genomic DNA . Based on 30 tetrads, the linkage between HEX2 and TRP1 was determined as 10 cM . Plasmid integration directed to the genomic site of the cloned gene also gave a similar linkage distance between the amino acid auxotroph plasmid marker and genomic TRP1 . Gene disruption of HEX2 yielded nonrepressible transformants with elevated hexokinase PII activity showing inhibition by maltose; this provides clear evidence that the HEX2 gene has been isolated.

Mol Gen Genet, 1987 Mar, 206(3), 452 - 9
Cloning and characterization of the immunity region of phage phi 80; Coste G et al.; The immunity region of phage phi 80 has been localized . It codes for at least three proteins: a protein of 34 kDa which has the biological properties of the phage repressor, and two other proteins of 9 kDa and 18 kDa which are the first proteins on the rightward operon . These two proteins are negatively regulated by the 34 kDa protein at a divergent promoter site . By position analogy with phage lambda, but not by its biological activity, the 9 kDa protein could be the cro product . The 18 kDa protein is able to block totally UV induction of phage phi 80.

Mol Gen Genet, 1987 Mar, 206(3), 419 - 27
Comparison of the organisation of the genomes of phenotypically diverse plasmids of incompatibility group P: members of the IncP beta sub-group are closely related; Smith CA et al.; Comparison of physical maps of the broad host range plasmids R751, R906 and R772, belonging to the IncP beta sub-group of the Escherichia coli incompatibility group P, reveals two large regions of similarity, separated by dissimilar regions which contain the majority of the cleavage sites for restriction endonucleases with hexanucleotide recognition sites . Mapping of the regions of these plasmids which show homology to probes specific for genetically characterised segments of the distantly related IncP alpha plasmid RK2, involved in plasmid maintenance or conjugal transfer, reveals that all four plasmids share a similar genetic organisation . In each case the homologous plasmid backbone is interrupted by heterologous segments both between the essential replication loci oriV and trfA, and between the conjugal transfer regions tra1 and tra2, although in the case of R772 the segment of the backbone carrying the trfA and tra2 regions is inverted relative to that of the other plasmids . However, in the case of pJP4, shown to be a fourth member of the IncP beta sub-group, the backbone is interrupted only by a single large segment adjacent to the trfA region . Mapping of the regions of the four IncP beta plasmids which show homology to Tn501 and nucleotide sequence determination at the ends of the homologous regions reveals that R906, R772 and pJP4 share a common mercury resistance region . This region, which appears to have been inactivated in R772, was probably inserted into a common ancestor of these plasmids by the transposition of an element related to an ancestor of Tn501 . R751 shows no trace of the mercury resistance region, but contains a short relict of Tn501, derived from an independent insertion event.

Mol Gen Genet, 1987 Mar, 206(3), 368 - 76
Regulation of the ban gene containing operon of prophage P1; Heisig A et al.; A physical map of the ban gene of P1 and sites relevant to its regulation has been deduced from cloning of the appropriate regions of P1 wild-type and of P1 ban regulatory mutants . The cloning required the presence of P1 repressor in the cell confirming the existence of a repressible ban operon (Austin et al . 1978) . Evidence for additional member(s) of that operon is presented . Of particular interest for understanding the regulation of ban are the relative positions of a binding site for the P1 repressor and of the regulatory mutations bac and crr that render ban expression constitutive . The results reveal a repressible operon-like structure of about 4 kb within the P1 EcoRI-3 fragment that comprises a c1 repressor binding site/bac - additional gene(s) - crr/ban in the clockwise direction of the circular map of P1.

EMBO J, 1987 Mar, 6(3), 737 - 42
Yeast DNA polymerase--DNA primase complex; cloning of PRI 1, a single essential gene related to DNA primase activity; Lucchini G et al.; The immunopurified yeast DNA polymerase--DNA primase complex is constituted by DNA polymerase I polypeptides and by three other protein species, called p74, p58 and p48, which we show to be immunologically unrelated . The gene encoding the p48 polypeptide has been identified by immunological screening of a lambda gt11 yeast genomic DNA library . Antiserum specific for p48 inhibits DNA primase, and immunoreactive, inhibitory antibodies are affinity-purified by the clone-encoded protein, thus relating the p48 polypeptide to DNA primase activity . The entire gene has been cloned, and the 1.45-kb p48 mRNA is overproduced in cells containing the gene in high copy number . Gene disruption and Southern hybridization experiments demonstrate that the p48 protein is encoded by a single gene and it performs an essential function.

EMBO J, 1987 Mar, 6(3), 729 - 36
The product of the mei3+ gene, expressed under control of the mating-type locus, induces meiosis and sporulation in fission yeast; McLeod M et al.; In fission yeast the ability to undergo meiosis and sporulation is conferred by the matP+ and matM+ genes of the mating-type locus . Inactivation of ran1+, a negative regulator of meiosis, is thought to be an essential step in meiotic initiation . We have isolated a further meiotic control gene mei3+, and have shown the following: a null allele of mei3 totally inhibits meiosis; the mei3+ RNA transcript and its translational product are expressed only in matP+/matM+ diploids entering meiosis; forced expression of mei3+ in vegetative cells provokes haploid meiosis and sporulation . We suggest that the product of mei3+ gene, a protein of 21 kd, initiates meiosis by inactivating ran1+.

EMBO J, 1987 Mar, 6(3), 713 - 21
Three suppressor mutations which cure a mitochondrial RNA maturase deficiency occur at the same codon in the open reading frame of the nuclear NAM2 gene; Labouesse M et al.; Dominant mutations of the nuclear NAM2 gene are able to compensate for a deficiency of the maturase encoded by the fourth intron of the mitochondrial cytochrome b gene . We have determined the complete nucleotide sequence of the NAM2-1 suppressor allele . The results of S1 nuclease protection experiments show that two overlapping poly(A)+ RNAs are transcribed from the gene using different promoters . The longer transcript contains two open reading frames (ORFs), a long ORF which could encode a protein of 894 amino acids, mol . wt 102,000 daltons, and a short ORF of 51 codons which is omitted from the shorter transcript . The wild-type nam2+ and two other suppressor alleles, NAM2-6 and NAM2-7, have been cloned . A comparison of the sequence of the wild-type and the three suppressor alleles shows that on three separate occasions the same codon specifying glycine was mutated (once to serine and twice to cysteine) . Finally sequence comparisons identified two regions in the long ORF, distinct from the position of the suppressor mutations, that could correspond to binding domains for a nucleotide and a nucleic acid.

Anal Biochem, 1987 Mar, 161(2), 272 - 9
Direct mapping of rare messenger RNAs by means of oligomer-directed ribonuclease H cleavage; Berger SL; A method has been developed for characterizing rare messenger RNAs in the bulk population by using oligodeoxyribonucleotide: RNA hybrids as substrates for Escherichia coli ribonuclease H . Two 1.3-kb mRNAs in lymphocyte cytoplasm, interferon-gamma (0.002% of polyadenylated mRNA), and prothymosin-alpha, have been studied . Interferon-gamma mRNA was cut virtually completely into two fragments, each about 0.6 kb in length, by using an interferon-specific 24-mer to direct cleavage . Prothymosin-alpha mRNA in the same bulk population was unaffected by this treatment . When the 24-mer was replaced by a 12-mer, whose sequence was based on an incomplete cDNA clone for prothymosin-alpha, the products included two fragments of prothymosin-alpha mRNA . The sum of the fragment lengths equaled the length of the mRNA . Although the reaction directed by the smaller oligomer did not go to completion, the 12-mer, and hence the cDNA clone from which it was derived, could nevertheless be oriented with respect to prothymosin-alpha mRNA . With this technique, sequences in mRNA can be mapped without first isolating full-length cDNA clones.

Mol Gen Mikrobiol Virusol, 1987 Mar, (3), 3 - 6
{Detailed mapping of the Hly-region of the plasmid pHly241 . Homology of the hemolysis determinants of plasmids pHly241 and pHly195}; Berezkina NE et al.; Data on the hemolysin synthesis determined by the plasmid pHly241, belonging to the incompatibility group I2, and its derivatives are presented in this paper . The restriction analysis of the pHly241 plasmid has resulted in the detailed mapping of the hly determinant region in the plasmid . The substantial homology has been demonstrated between the regions of the plasmids pHly241 and pHly195 coding for hemolysin synthesis and excretion from the bacterial cell.

Genetika, 1987 Mar, 23(3), 397 - 404
{Plasmid vectors of "insertion inactivation" of the trimethoprim resistance marker}; Gorovits RL et al.; We demonstrate the possibility of using the T4 phage frd gene as an insertion inactivation marker within pBR322, in plasmids with changing copy number and expression of foreign genes under control . The structural part of the frd genes contains unique recognition sites for EcoRI and SalI endonucleases . Transformants with recombinant plasmids carrying the frd gene grow on media with up to 500 mkg/ml trimethoprim, whatever the gene dosage.

Proc Natl Acad Sci U S A, 1987 Mar, 84(5), 1215 - 8
The E2 "gene" of bovine papillomavirus encodes an enhancer-binding protein; Moskaluk C et al.; The E2 early open reading frame (presumably gene) of bovine papillomavirus-1 was fused in frame with the collagen-beta-galactosidase-encoding region of the vector pJG200 and was expressed in and partially purified from Escherichia coli . The hybrid protein specifically bound to the enhancer region of bovine papillomavirus at several sites . DNase I-cleavage protection analysis of one such site revealed the protected sequence . A comparison of the protected sequence with the remainder of the DNA sequences that also have affinity for the protein revealed a consensus sequence having the motif AATTGGCGGNNCG, in which N is any nucleotide . The protected region also includes a sequence with 2-fold rotational symmetry--ATCGGTG/CACCGAT.

Proc Natl Acad Sci U S A, 1987 Mar, 84(5), 1210 - 4
Regulation of c-myc and c-fos mRNA levels by polyomavirus: distinct roles for the capsid protein VP1 and the viral early proteins; Zullo J et al.; The levels of c-myc, c-fos, and JE mRNAs accumulate in a biphasic pattern following infection of quiescent BALB/c 3T3 mouse cells with polyomavirus . Maximal levels of c-myc and c-fos mRNAs were seen within 1 hr and were nearly undetectable at 6 hr after infection . At 12 hr after infection mRNA levels were again maximal and remained elevated thereafter . Empty virions (capsids) and recombinant VP1 protein, purified from Escherichia coli, induced the early but not the late phase of mRNA accumulation . Virions, capsids, and recombinant VP1 protein stimulated {3H}thymidine nuclear labeling and c-myc mRNA accumulation in a dose-responsive manner paralleling their affinity for the cell receptor for polyoma . The second phase of mRNA accumulation is regulated by the viral early gene products, as shown by polyomavirus early gene mutants and by a transfected cell line (336a) expressing middle tumor antigen upon glucocorticoid addition . These results suggest that polyomavirus interacts with the cell membrane at the onset of infection to increase the levels of mRNA for cellular genes associated with cell competence for DNA replication, and subsequently these levels are maintained by the action of the early viral proteins.

Mutat Res, 1987 Mar, 177(1), 17 - 26
Induction of the SOS system in a dam-3 mutant: a diagnostic strain for chemicals causing DNA mismatches; Quillardet P et al.; We have developed a strain of E . coli in which expression of the SOS function sfiA, monitored by means of a sfiA::lacZ operon fusion, is efficiently triggered by the two base analogues 2-aminopurine and 5-bromo-2'-deoxyuridine . This strain resulted from introduction of a dam-3 mutation into a Uvr+, Rfa+ derivative of strain PQ37 used in the SOS chromotest, a bacterial colorimetric assay for genotoxins (Quillardet et al., 1982) . The dam-3 mutation affects the mismatch correction system in E . coli . We show that the SOS-inducing capacity of a weak SOS inducer such as the alkylating agent ethyl methanesulfonate was also increased in the dam-3 strain . We provide evidence that the increase in SOS inducibility due to the dam-3 mutation is specific for compounds causing DNA mismatches and propose the use of the dam-3 derivative of PQ37 as a diagnostic strain for such agents . This diagnostic strain can be a useful addition to the SOS chromotest.

J Gen Virol, 1987 Mar, 68 ( Pt 3), 633 - 42
Synthesis in Escherichia coli and immunological characterization of a polypeptide containing the cleavage sites associated with trypsin enhancement of rotavirus SA11 infectivity; Arias CF et al.; About 45% of the rotavirus SA11 VP3 gene was inserted into a thermoinducible expression plasmid under the control of phage lambda PL promoter . The primary translation product predicted on the basis of the plasmid construction was a hybrid protein in which the 98 amino-terminal amino acids of phage MS2 polymerase were followed by amino acids 42 to 387 of the VP3 protein, which included the region containing the cleavage sites associated with trypsin enhancement of infectivity . On induction, a polypeptide that had the expected mol . wt . and contained VP3-related amino acid sequences as judged by immunological criteria, was synthesized to a level representing about 15% of the total bacterial protein . When a bacterial lysate enriched for the fusion polypeptide was injected into mice, it induced antibodies which inhibited haemagglutination and neutralized SA11 rotavirus infectivity.

J Bacteriol, 1987 Mar, 169(3), 981 - 9
Capsule synthesis in Escherichia coli K-12 is regulated by proteolysis; Torres-Cabassa AS et al.; lon mutants of Escherichia coli K-12 are defective in an ATP-dependent protease, are UV sensitive, and overproduce the capsular polysaccharide colanic acid . Six structural genes needed for capsular polysaccharide synthesis (cps) are transcriptionally regulated by lon as well as by three other regulatory genes, rcsA, -B, and -C (S . Gottesman, P . Trisler, and A . S . Torres-Cabassa, J . Bacteriol . 162:1111-1119, 1985) . We have cloned rcsA, the gene for a positive regulator of capsule synthesis, onto multicopy plasmids and defined the gene by both insertions and deletions . The product of rcsA has been identified as an unstable protein of 27 kilodaltons . RcsA has a half-life of 5 min in lon+ cells and one of 20 min in lon cells . The availability of RcsA is the limiting factor for capsule synthesis; doubling the gene dosage of rcsA+ significantly increases expression of cps genes . Our results are consistent with a model in which the presence of a lon mutation increases the synthesis of capsular polysaccharide via stabilization of RcsA.

J Bacteriol, 1987 Mar, 169(3), 1286 - 90
secD, a new gene involved in protein export in Escherichia coli; Gardel C et al.; New mutants of Escherichia coli altered in protein export were identified in phoA-lacZ and lamB-lacZ gene fusion strains by searching for mutants that showed an altered lactose phenotype . Several mutations mapped in a new gene, secD . These mutants were, in general, cold sensitive for growth, and the mutations led to an accumulation of precursor of exported proteins . The secD gene is closely linked to tsx on the E . coli chromosome, but separable from another gene proposed to be involved in export, ssaD, which maps nearby . A plasmid carrying secD+ was identified and used to show that the mutations are recessive . The secD gene may code for a component of the cellular export machinery.

J Bacteriol, 1987 Mar, 169(3), 1272 - 8
Fusions of the Escherichia coli gyrA and gyrB control regions to the galactokinase gene are inducible by coumermycin treatment; Menzel R et al.; We have previously shown that the genes encoding the two subunits of Escherichia coli DNA gyrase are regulated in a manner which is dependent on DNA conformation . When the DNA encoding the gyrA and gyrB genes is relaxed, both genes are expressed at a high level; in negatively supercoiled DNA they are expressed at a low level . In this paper we describe fusions of both the gyrA and gyrB 5' sequences to the E . coli galactokinase gene . In such fusions we found that galactokinase can be induced by treating the cells with coumermycin A1, an inhibitor of DNA gyrase . Our results suggest that the regulation occurs at the transcriptional level and that only a small region of DNA is necessary for coumermycin-induced gene expression.

J Bacteriol, 1987 Mar, 169(3), 1254 - 9
Influence of GATC sequences on Escherichia coli DNA mismatch repair in vitro; Lu AL; The effect of the number and position of DNA adenine methylation (dam) sites, i.e., d(GATC) sequences, on mismatch repair in Escherichia coli was investigated . The efficiency of repair was measured in an in vitro assay which used an f1 heteroduplex containing a G/T mismatch within the single EcoRI site . Both an increase in the number of dam sites and a shortened distance between dam site and mismatched site increased the efficiency of mismatch repair . The sequences adjacent to d(GATC) also affected the efficiency of methylation-directed mismatch repair . Furthermore, heteroduplexes with one extra dam site located close to either the 5' or 3' end of the excised base increased the repair efficiency to about the same extent . The findings suggest that the mismatch repair pathway has no preferred polarity.

J Bacteriol, 1987 Mar, 169(3), 1246 - 53
Chemotaxis in Escherichia coli: construction and properties of lambda tsr transducing phage; Callahan AM et al.; The tsr gene of Escherichia coli, located at approximately 99 min on the chromosomal map, encodes a methyl-accepting protein that serves as the chemoreceptor and signal transducer for chemotactic responses to serine and several repellents . To determine whether any other chemotaxis or motility genes were located in the tsr region, we constructed and characterized two lambda tsr transducing phages that each contain about 12 kilobases of chromosomal material adjacent to tsr . lambda tsr70 carries sequences from the promoter-proximal side of tsr; lambda tsr72 carries sequences from the promoter-distal side of tsr . Restriction maps of the bacterial inserts in these phages and Southern hybridization analyses of the bacterial chromosome indicated that the tsr gene is transcribed in the counterclockwise direction on the genetic map . Insert deletions were isolated in lambda tsr70 and transferred into the host chromosome to examine the null phenotype of tsr . All such strains exhibited wild-type swimming patterns and chemotactic responses to a variety of stimuli, but were specifically defective in serine taxis and other Tsr-mediated responses . In addition, UV programming experiments demonstrated that Tsr and several of its presumptive degradation products were the only bacterial proteins encoded by lambda tsr70 and lambda tsr72 that required host FlbB/FlaI function for expression . These findings indicate that there are probably no other chemotaxis-related genes in the tsr region . A series of tsr point mutations were isolated by propagating lambda tsr70 on a mutD host and used to construct a fine-structure map of the tsr locus . These mutations should prove valuable in exploring structure-function relationships in the Tsr transducer.

J Bacteriol, 1987 Mar, 169(3), 1174 - 81
Multiple SecA protein isoforms in Escherichia coli; Liebke HH; To define the anti-SecA-LacZ antiserum, immunoprecipitates produced with either whole anti-SecA-LacZ rabbit antiserum or affinity-purified antibodies were used to analyze nondenatured lysates of Escherichia coli . The antiserum contains antibodies that recognize different proteins . Antibody purified by preadsorption to the SecA-LacZ hybrid protein precipitated only the SecA protein from extracts . In contrast, antibody purified from the intact SecA protein precipitated several additional proteins with SecA protein . Ribosomal protein L7L12 is one of the polypeptides coprecipitated with SecA protein by antibody purified by immunoadsorption to the intact SecA protein as well as by unfractionated anti-SecA-LacZ antiserum . Two-dimensional gel electrophoresis of the SecA protein immunoprecipitated by either antiserum or purified antibody indicated that the SecA protein exists in at least two, and probably four, isoforms . Only one of the SecA isoforms is present in a ribosomal preparation.

J Bacteriol, 1987 Mar, 169(3), 1080 - 8
The 65-kilodalton antigen of Mycobacterium tuberculosis; Shinnick TM; The immune response of the host to the antigens of Mycobacterium tuberculosis plays the key role in determining immunity from infection with as well as the pathogenicity of this organism . A 65-kilodalton (kDa) protein has been identified as one of the medically important antigens of M . tuberculosis . The gene encoding this antigen was isolated from a lambda gt11-M . tuberculosis recombinant DNA library using monoclonal antibodies directed against the 65-kDa antigen as the specific probes . The nucleotide sequence of this gene was determined, and a 540-amino-acid sequence was deduced . This sequence was shown to correspond to that of the 65-kDa antigen by constructing a plasmid in which this open reading frame was fused to the lacZ gene . The resulting fusion protein reacted specifically with the anti-65-kDa protein antibodies . A second long open reading frame was found downstream of the 65-kDa antigen gene which could encode a protein of 517 amino acids . This putative protein contained 29 tandemly arranged partial or complete matches to a pentapeptide sequence.

J Bacteriol, 1987 Mar, 169(3), 1061 - 70
Structural analysis of the Escherichia coli K-12 hisT operon by using a kanamycin resistance cassette; Arps PJ et al.; We constructed a series of recombinant plasmids containing a kanamycin resistance (Kmr) cassette upstream from, within, and downstream from hisT, which encodes the tRNA modification enzyme pseudouridine synthase I . These Kmr insertions were then crossed directly into the bacterial chromosome . We determined growth characteristics, assayed in vivo hisT expression, and mapped in vivo hisT operon transcripts for the Kmr insertion mutants . We also analyzed polypeptides synthesized in minicells from plasmids containing Kmr cassettes . The combined results from these experiments demonstrate new features concerning the structure and expression of the complex operon that contains hisT . We show that the minimum size of the operon is approximately 3,500 base pairs and that it contains at least four genes, which are arranged in the order usg-2 (pdxB), usg-1, hisT, and dsg-1 and encode polypeptides with apparent molecular masses of 42,000, 45,000, 31,000, and 17,000 daltons, respectively . Of these genes, only the functions of usg-2 (pdxB) and hisT are known, and genetic evidence suggests that these two genes do not require usg-1 or dsg-1 for function, usg-2 (pdxB) is required for growth of bacteria on minimal medium at 37 degrees C . In contrast, the three genes at the end of the hisT operon are dispensable and form a transcription unit that is expressed from a relatively strong internal promoter . The phenotypes of the Kmr insertion mutants and results from gene expression experiments further confirm the position of the internal promoter and locate additional genetic signals in the DNA sequence around hisT . The experiments reported here also indicate several interesting properties of the Kmr cassette as a tool for probing complex operons.

J Bacteriol, 1987 Mar, 169(3), 1056 - 60
In vivo D-serine deaminase transcription start sites in wild-type Escherichia coli and in dsdA promoter mutants; Bornstein-Forst SM et al.; The D-serine deaminase structural (dsdA) and regulatory (dsdC) genes are transcribed with opposite polarity from an intergenic region comprising more than 600 base pairs . The order of genes in the dsd region is supN-dsdA-dsdC-aroC---his . The DNA sequence of the intergenic region has been slightly revised from a previously published version (E . McFall and L . Runkel, J . Bacteriol . 154:1508-1512, 1983) . The dsdA gene is preceded by a long open reading frame . The dsdA in vivo transcription start sites for the wild type (base pair +1) and for three phenotypically distinct promoter constitutive mutants were determined by the S1 nuclease method . They are identical and are located about 81 base pairs upstream of the translation start site . D-Serine deaminase regulation is normal in rho mutants . Possible mechanisms for dsdA activation are discussed.

J Bacteriol, 1987 Mar, 169(3), 1029 - 36
Molecular cloning and characterization of the Streptomyces hygroscopicus alpha-amylase gene; Hoshiko S et al.; We have isolated and sequenced a gene (amy) coding for alpha-amylase (EC 3.2.1.1.) from the Streptomyces hygroscopicus genome (H . Hidaka, Y . Koaze, K . Yoshida, T . Niwa, T . Shomura, and T . Niida, Die Starke 26:413-416, 1974) . Amylase was purified to obtain amino acid sequence information which was used to synthesize oligonucleotide probes . amy-containing Escherichia coli cosmids identified by hybridization did not express amylase activity . Subcloning experiments indicated that amy could be expressed from the lac promoter in E . coli or from its own promoter in S . lividans . The amy nucleotide sequence indicated that it coded for a protein of 52 kilodaltons (478 amino acids) . Secreted alpha-amylase contained amino- and carboxy-terminal as well as internal amino acid sequences which were consistent with the nucleotide sequence . The 30-residue leader sequence showed similarities to those found in other procaryotes . The DNA sequence 5' to the amy structural gene contained a sequence complementary to the 3'-terminal sequence of 16S rRNA of S . lividans (M . J . Bibb and S . N . Cohen, Mol . Gen . Genet . 187:265-277, 1982) . The transcriptional start points of amy were determined by mung bean nuclease mapping, but the promoter of amy was not similar to the consensus sequence found in other procaryotes.

J Virol, 1987 Mar, 61(3), 866 - 75
Posttranslational processing of an Epstein-Barr virus-encoded membrane protein expressed in cells transformed by Epstein-Barr virus; Baichwal VR et al.; The BamHI Nhet fragment of the B958 strain of Epstein-Barr virus (EBV) encodes a membrane protein (BNLF-1) that is present in cells transformed by EBV . We made a hybrid protein in which a polypeptide sequence from the carboxyl-terminal part of BNLF-1 is fused to Escherichia coli beta-galactosidase . This hybrid protein was used to immunize rabbits, and the resulting antiserum was purified by immunoaffinity chromatography . The antiserum was able to immunoprecipitate BNLF-1 from cell lysates . We found that BNLF-1 is phosphorylated at serines in EBV genome-positive B-cell lines . Pulse-chase analyses with {35S}methionine indicated that BNLF-1 is turned over in lymphoblasts with a half-life of approximately 5 h . Protein immunoblots of EBV genome-positive B-cell lines revealed both a 62,000-molecular-mass band corresponding to BNLF-1 and a myriad of lower-molecular-mass bands . We postulate that these lower-molecular-mass bands are degradation products resulting from the turnover of BNLF-1 in cells . The BNLF-1 gene was expressed in COS cells, and the protein was both phosphorylated and turned over in these cells.

Gene Anal Tech, 1987 Mar-Apr, 4(2), 23 - 6
Storage of spheroplasts at -70 degrees C for transfection with phi X174 RF DNA; Malling HV et al.; A method was developed for preparation of spheroplasts used for transfection with phi X174 RF DNA . This method had a high-level competence and retained the competence for up to one year of storage in 7% DMSO at -70 degrees C.

Proc Natl Acad Sci U S A, 1987 Mar, 84(6), 1624 - 8
Large palindromes in the lambda phage genome are preserved in a rec+ host by inhibiting lambda DNA replication; Shurvinton CE et al.; A large palindrome carried by phage lambda has been shown to prevent growth of the phage on a rec+ strain of Escherichia coli . The phage do form plaques on recBC sbcB strains, but the palindrome is not stable--deletions that either destroy the palindrome or diminish its size overgrow the original engineered palindrome-containing phage . We have prepared stocks of lambda carrying a palindrome that is 2 X 4200 base pairs long . These phage stocks are produced by induction of a lysogen in which the two halves of the palindrome are stored at opposite ends of the prophage and are of sufficient titer (10(9) phage per ml) to enable one-step growth experiments with replication-blocked phage . We find that the large palindrome as well as a lesser palindrome of 2 X 265 base pairs are recovered intact among particles carrying unreplicated chromosomes following such an infection of a rec+ host . We propose that DNA replication drives the extrusion of palindromic sequences in vivo, forming secondary structures that are substrates for the recBC and sbcB gene products.

Microb Pathog, 1987 Mar, 2(3), 195 - 209
Molecular cloning and characterization of the genetic determinant encoding CS3 fimbriae of enterotoxigenic Escherichia coli; Boylan M et al.; The genetic determinant encoding the synthesis and surface expression of CS3 fimbriae of colonization factor antigen II-(CFA/II-) positive enterotoxigenic Escherichia coli was cloned on a 5.1 kb HindIII DNA fragment in pBR322 from the wild-type plasmid pCS001 to yield the CS3+ plasmid pCS100 . Subcloning of EcoRI fragments of 1.8 kb and 2.5 kb into vector plasmid pACYC184 and the isolation of a series of pCS100::Tn5 insertion mutants revealed that more than one cistron was involved in the synthesis and expression of CS3 fimbriae . Polypeptides of 94, 26, 24, 17 and 15 kDa were detected in E . coli minicells harbouring pCS100 . In Western immunoblotting the 17 kDa and 15 kDa polypeptides reacted with specific anti-CS3 fimbriae serum . The 15 kDa polypeptide comigrated with the structural subunit of CS3 fimbriae . Inhibition of protein processing in minicells by ethanol confirmed that the 17 kDa polypeptide was the precursor form of the 15 kDa structural subunit . A physical map of the cloned DNA was constructed showing the location and direction of transcription of the genes for the 17 and 94 kDa polypeptides . Using the 5.1 kb HindIII fragment of pCS100 as a genetic probe for the CS3 determinant, Southern hybridization analysis of plasmid and total cellular DNA was performed in wild-type enterotoxigenic E . coli strains.

Biochem Int, 1987 Mar, 14(3), 517 - 24
Antipili antibody affords protection against ascending pyelonephritis in rat: evaluated by renal brush border membrane enzymes; Garg UC et al.; Kinetic parameters (Km and Vmax) of renal brush border membrane (BBM) enzymes alkaline phosphatase, maltase, leucine aminopeptidase and gamma-glutamyltranspeptidase were worked out in control, infected and immunized-infected rats . There was no significant change in the Km of all the enzymes studied in three groups . The Vmax of all the enzymes studied decreased significantly (p less than 0.05) 3 or 4 days postinfection and onwards in the left obstructed kidney of infected and immunised-infected animals . However, in the right unobstructed kidney the Vmax of alkaline phosphatase and leucine aminopeptidase increased significantly (p less than 0.05) in the early stages and decreased (p less than 0.05) in later stages in both the experimental groups . The significant difference (p less than 0.05) in the Vmax of infected and immunized-infected groups at various stages of infection revealed the partial protective role of antipili antibody against ascending pyelonephritis.

Tsitologiia, 1987 Mar, 29(3), 338 - 44
{Genetic transformation of somatic cells . XII . Mutations in human satellite DNA introduced into Chinese hamster cells}; Tomilin NV et al.; Data are presented about detailed restriction mapping, hybridization and sequence analysis of plasmid pRT301 rescued into E . coli 25-30 cell divisions after transfection of Chinese hamster cells . This plasmid contains an insert of human satellite III fragment (HS3), which was replicated many times in mammalian cells, and we found multiple point mutations in this non-selectable fragment, mainly single inserts of GC base pair . Specificity of mutagenesis of HS3 induced by transfection and replication in Chinese hamster cells differs from mutagenesis specificity found by other authors for selectable genes.

EMBO J, 1987 Mar, 6(3), 749 - 59
Characterization and localization of the even-skipped protein of Drosophila; Frasch M et al.; On the basis of homeo box cross-homology we have isolated the pair-rule gene even-skipped (eve) of Drosophila . The eve transcription unit appears to be less than 1.5 kb in length, and encodes a single mRNA of approximately 1.4 kb . The nucleotide sequence of genomic and cDNA clones indicates that the eve protein is composed of 376 amino acid residues, and that its homeo domain shares only approximately 50% amino acid identity with the homeo domains of previously characterized genes . Using antibodies raised against a beta-galactosidase fusion protein we show that the eve protein is distributed in a series of seven transverse stripes at the cellular blastoderm stage, and is localized primarily within the nuclear regions of those embryonic cells that express the gene . After gastrulation, seven weakly stained stripes of eve expression appear, resulting in a transient pattern that consists of a total of 14 evenly spaced stripes . Both the original and new stripes gradually disappear during germ band elongation . A second expression pattern emerges during neurogenesis, whereby eve protein is detected in discrete subsets of neurons in each of the ventral ganglia.

EMBO J, 1987 Mar, 6(3), 705 - 11
Independent mutations at the amino terminus of a protein act as surrogate signals for mitochondrial import; Vassarotti A et al.; Intracellular delivery of the mitochondrial F1-ATPase beta-subunit precursor from the cytoplasm into the matrix of mitochondria is prevented by deletion of its mitochondrial import signal, a basic amphipathic alpha-helix at its amino terminus . Using a complementation assay, we have selected spontaneous mutations which restore the correct in vivo localization of the protein containing the import signal deletion . Analysis of these mutations revealed that different functional surrogate mitochondrial targeting signals formed within a narrow region of the extreme amino terminus of the import signal deleted beta-subunit . These modifications specifically replace different acidic residues with neutral or basic residues to generate a less acidic amphipathic helix within a region of the protein which is accessible for interaction with the membrane surface . The observations of this study confirm the requirement for amphipathicity as part of the mitochondrial import signal and suggest how mitochondrial targeting signals may have evolved within the extreme amino terminus of mitochondrial proteins.

J Hosp Infect, 1987 Mar, 9(2), 175 - 81
Role of R-plasmids in adherence and hydrophobicity in Escherichia coli; Sainz V et al.; Twenty-five R-plasmids from different incompatibility groups were conjugatively transferred to the laboratory strain Escherichia coli JE2571 and the variations in surface hydrophobicity and adherence to human buccal epithelial cells were investigated . It was found that some R-plasmids produce significant variations in adherence and/or hydrophobicity but that these variations show no quantitative or qualitative correlation.

Proc Natl Acad Sci U S A, 1987 Mar, 84(6), 1575 - 9
Cloning and sequence of the human nuclear protein cyclin: homology with DNA-binding proteins; Almendral JM et al.; A full-length cDNA clone for the human nuclear protein cyclin has been isolated by using polyclonal antibodies and sequenced . The sequence predicts a protein of 261 amino acids (Mr 29,261) with a high content of acidic (41, aspartic and glutamic acids) versus basic (24, lysine and arginine) amino acids . The identity of the cDNA clone was confirmed by in vitro hybrid-arrested translation of cyclin mRNA . Blot-hybridization analysis of mouse 3T3 and human MOLT-4 cell RNA revealed a mRNA species of approximately the same size as the cDNA insert . Expression of cyclin mRNA was undetectable or very low in quiescent cells, increasing after 8-10 hr of serum stimulation . Inhibition of DNA synthesis by hydroxyurea in serum-stimulated cells did not affect the increase in cyclin mRNA but inhibited 90% the expression of H3 mRNA . These results suggest that expression of cyclin and histone mRNAs are controlled by different mechanisms . A region of the cyclin sequence shows a significant homology with the putative DNA binding site of several proteins, specially with the transcriptional-regulator cAMP-binding protein of Escherichia coli, suggesting that cyclin could play a similar role in eukaryotic cells.

Infect Immun, 1987 Mar, 55(3), 555 - 8
F41 pili as protective antigens of enterotoxigenic Escherichia coli that produce F41, K99, or both pilus antigens; Runnels PL et al.; Pigs suckling dams that have been vaccinated with pilus antigen are protected against challenge with enterotoxigenic Escherichia coli (ETEC) strains that express the same pilus antigen . However, some ETEC strains express more than one pilus antigen . Pregnant swine were vaccinated either with E . coli HB101 that harbored a recombinant plasmid coding for F41 expression (F41+) or with the HB101 parent strain that carries the pHC79 vector (F41-) . Suckling pigs born to vaccinated dams were challenged with ETEC that expressed either K99, F41, or both pilus antigens . Production of F41 in vivo was demonstrated by immunofluorescence assay of sections of ileum and by seroconversion against F41 antigen by pigs challenged with F41+ and K99+ F41+ ETEC strains . The F41+ vaccine protected against challenge with an F41+ ETEC strain . In contrast, F41+ vaccination did not protect against challenge with K99+ or K99+ F41+ ETEC strains . The F41- vaccine did not protect against challenge with any strain used . The results indicate that K99+ F41+ ETEC strains produce F41 antigen in the small intestine during disease and that F41+ vaccination can be a protective antigen if the challenge strain expresses only F41 antigen, but that F41+ vaccination may not protect against strains that produce both K99 and F41 antigens.

Infect Immun, 1987 Mar, 55(3), 534 - 40
Stimulation of human polymorphonuclear leukocyte oxidative metabolism by type 1 pili from Escherichia coli; Goetz MB et al.; We compared the degree to which Escherichia coli phase variants which do (T1P+ E . coli) or do not (T1P- E . coli) express type 1 pili (T1P) stimulate human polymorphonuclear leukocyte (PMN) oxidative activity . Unopsonized T1P+ E . coli stimulated the release of 0.20 to 0.24 nmol of H2O2 per 10(6) PMN per min and the consumption of 1.4 to 4.0 nmol of O2 per 10(6) PMN per min; no measurable PMN oxidative activity was stimulated by unopsonized T1P- E . coli . In the presence of serum opsonins, T1P+ E . coli stimulated the release of 1.12 to 1.16 nmol of H2O2 per 10(6) PMN per min and the consumption of 5.0 to 6.0 nmol of O2 per 10(6) PMN per min, whereas T1P- E . coli stimulated the release of 0.42 to 0.43 nmol of H2O2 per 10(6) PMN per min and the consumption of 0.6 to 2.0 nmol of O2 per 10(6) PMN per min . Although unaggregated T1P did not stimulate PMN, latex beads coated with T1P (T1P-latex) stimulated alpha-methylmannoside-inhibitable, opsonin-independent PMN oxidative activity . The activity stimulated by either T1P+ E . coli or T1P-latex was susceptible to inhibition by cytochalasin B . Latex particles coated with bovine serum albumin or mannose-resistant pili did not stimulate PMN . These data indicate that T1P+ E . coli stimulate PMN oxidative metabolism more effectively than do T1P- E . coli and that a similar PMN oxidative response follows cellular stimulation by either unopsonized T1P+ or opsonized T1P- E . coli . Furthermore, T1P-latex faithfully mimics the ability of T1P+ E . coli to stimulate PMN oxidative metabolism . Such particles may be useful in further analyses of cellular responses to T1P+ E . coli.

J Immunol, 1987 Mar 1, 138(5), 1397 - 402
The localization of the antibody response in milk or bile depends on the nature of the antigen; Dahlgren UI et al.; Immunization in the Peyer's patches of rats with horse spleen ferritin or Escherichia coli 06 carrying type 1 pili resulted in an IgA antibody response detected in milk and bile and an IgG and IgM antibody response in serum, milk, and bile . The IgA antibody response to type 1 pili was as a mean 5.0-fold higher in milk than in bile . In contrast IgA antibody activity to 06 LPS was as a mean 6.3-fold higher in bile than in milk . The IgA antibodies to ferritin were randomly distributed between milk and bile . The IgG and IgM antibody activity to all three antigens studied were higher in the milk than in the bile . The secretory antibody response could be transferred from immunized rats to unimmunized rats with mesenteric lymph node cells (MLN) taken from donor rats 4 days after immunization in the Peyer's patches . IgA antibodies to pili and ferritin appeared solely in the milk of the recipients, whereas IgA antibodies to the 06 LPS only appeared in the bile . The ratios serum:milk and serum:bile for the IgG and IgM antibodies indicated an antigen-specific direction of homing with local production of these two isotypes primarily in the mammary gland . Antibody-forming cells of the IgA class could not be detected in the MLN on the day the cells were transferred . It is concluded that the difference seen in antibody distribution between milk and bile is not due to dissemination of antigen, but instead a result of different homing or expansion at the mucosal-glandular site dependent on the antigen specificity of the migrating cells.

J Gen Microbiol, 1987 Mar, 133 ( Pt 3), 597 - 607
Investigation of Escherichia coli fumarate reductase subunit function using transposon Tn5; Latour DJ et al.; Seventy two Tn5 transposon insertions were isolated in the frd operon carried on the multicopy plasmid pFRD79 . The polar nature of these mutations permitted examination of the expression and localization of the frd polypeptides in novel subunit combinations . The minimal catalytic unit is the FRDA plus B dimer . A transposon within frdB (frdB::Tn5) produces inactive, soluble FRDA polypeptide which has covalently attached 8 alpha(N3-histidyl)flavin adenine dinucleotide cofactor . A transposon mutation within frdC (frdC::Tn5) produces soluble, catalytically active dimer . An insertion in frdD (frdD::Tn5) produces both a soluble trimer composed of FRDABC, and a tetramer of FRDABC and truncated FRDD bound to the inner membrane . Eighty percent of the activity is in the soluble form . Using this mutant, the requirement for FRDD both for optimal activity of the catalytic domain and for proper anchorage in the cytoplasmic membrane was demonstrated.

Mol Biol (Mosk), 1987 Mar-Apr, 21(2), 506 - 14
{Comparison of the conformation of RNA from phage MS2 and 16S rRNA . Accessibility to nucleases S1 and SV specific to secondary structure and thermal stability}; Grechko VV et al.; The hydrolysis of E . coli 16S rRNA by nucleases specific to the secondary structure elements (S1 and SV), the melting of the RNA after partial hydrolysis by nuclease S1 and the electrophoretic mobility of hydrolysis products after denaturation-renaturation of RNA were studied . It was shown that the sensitivity of 16S rRNA to nuclease S1 depends on Zn or Mg ions concentration . The melting curves after partial hydrolysis by nuclease S1 were characterized by a decrease of the hyperchromic effect (by approximately 15%) and by a increase of Tm (by 3 degrees) . After RNA denaturation followed by slow or fast renaturation the electrophoretic patterns of the hydrolysis products were not changed, as in the case of phage MS2 RNA . It was supposed, that the rRNA molecule has a stable "nucleus" (or "nuclei"), which is organized as an intramolecular association of parallelly oriented double-stranded fragments of this RNA . Previously, such a mode of the spatial organization was proposed by us for phage MS2 RNA.

Biochem J, 1987 Mar 1, 242(2), 565 - 72
Escherichia coli endonuclease III is not an endonuclease but a beta-elimination catalyst; Bailly V et al.; The oligonucleotide {5'-32P}pdT8d(-)dTn, containing an apurinic/apyrimidinic (AP) site {d(-)}, yields three radioactive products when incubated at alkaline pH: two of them, forming a doublet approximately at the level of pdT8dA when analysed by polyacrylamide-gel electrophoresis, are the result of the beta-elimination reaction, whereas the third is pdT8p resulting from beta delta-elimination . The incubation of {5'-32P}pdT8d(-)dTn, hybridized with poly(dA), with E . coli endonuclease III yields two radioactive products which have the same electrophoretic behaviour as the doublet obtained by alkaline beta-elimination . The oligonucleotide pdT8d(-) is degraded by the 3'-5' exonuclease activity of T4 DNA polymerase as well as pdT8dA, showing that a base-free deoxyribose at the 3' end is not an obstacle for this activity . The radioactive products from {5'-32P}pdT8d(-)dTn cleaved by alkaline beta-elimination or by E . coli endonuclease III are not degraded by the 3'-5' exonuclease activity of T4 DNA polymerase . When DNA containing AP sites labelled with 32P 5' to the base-free deoxyribose labelled with 3H in the 1' and 2' positions is degraded by E . coli endonuclease VI (exonuclease III) and snake venom phosphodiesterase, the two radionuclides are found exclusively in deoxyribose 5-phosphate and the 3H/32P ratio in this sugar phosphate is the same as in the substrate DNA . When DNA containing these doubly-labelled AP sites is degraded by alkaline treatment or with Lys-Trp-Lys, followed by E . coli endonuclease VI (exonuclease III), some 3H is found in a volatile compound (probably 3H2O) whereas the 3H/32P ratio is decreased in the resulting sugar phosphate which has a chromatographic behaviour different from that of deoxyribose 5-phosphate . Treatment of the DNA containing doubly-labelled AP sites with E . coli endonuclease III, then with E . coli endonuclease VI (exonuclease III), also results in the loss of 3H and the formation of a sugar phosphate with a lower 3H/32P ratio that behaves chromatographically as the beta-elimination product digested with E . coli endonuclease VI (exonuclease III) . From these data, we conclude that E . coli endonuclease III cleaves the phosphodiester bond 3' to the AP site, but that the cleavage is not a hydrolysis leaving a base-free deoxyribose at the 3' end as it has been so far assumed . The cleavage might be the result of a beta-elimination analogous to the one produced by an alkaline pH or Lys-Trp-Lys . Thus it would seem that E . coli 'endonuclease III' is, after all, not an endonuclease.

Biokhimiia, 1987 Mar, 52(3), 484 - 92
{Effect of cAMP modifiers on the cytotoxic activity of macrophages}; Neskoromnyi AG et al.; The effects of various cAMP modifiers on the changes in the intracellular cAMP level and on the coupling of the cAMP system with realization of macrophage cytotoxicity depending on their functional activity were studied . Nonactivated and activated by E . coli polysaccharides peritoneal macrophages of BALB/c mice and macrophage-like cells of J744 mice were incubated in the presence of cAMP modifiers and further assayed for cytolytic and cytostatic activities . Cells of tetraploid strain of Ehrlich carcinoma G10 were used as target cells . Among other modifiers only dibutyryl-cAMP caused a steady increase of the intracellular nucleotide content, whereas methylisobutylxanthine and isoproterenol in combination with methylisobutylxanthine caused only a temporary increase of the cAMP level . Isoproterenol did not induce any appreciable changes in the intracellular cAMP level . All modifiers under study suppressed the cytotoxic activity of macrophages irrespective of the nature of changes in the intracellular cAMP content . It was assumed that cAMP accomplishes a triggering function in the regulation of the cytotoxic activity of macrophages and that the cAMP system is universal in the regulation of cytotoxicity at various functional states of macrophages.

Biokhimiia, 1987 Mar, 52(3), 452 - 8
{Interpretation of the phenomenon of K+ transport activation in Escherichia coli during transition to anaerobiosis}; Puchkov EO et al.; Earlier it was demonstrated that the transition of E . coli K-12 cells to anaerobiosis is accompanied by the activation of K+ uptake . K+ that are additionally accumulated during the transition to anaerobiosis are released from the cells after the turning on of the respiratory chain . The K+ accumulation by the cells is potential-dependent both under aerobic and anaerobic conditions . A correlation was found between the degree of acidification of the cytoplasm and the rate of K+ uptake during the transition to anaerobiosis . It was assumed that under aerobic conditions the functioning of the electrogenic system of K+ uptake is concomitant with the operation of the K+ release system, K+/H+ antiporter, which is inactivated at the beginning of anaerobiosis, presumably as a result of cytoplasm acidification . This effect manifests itself as the activation of K+ uptake . The trigger function of the K+/H+ antiporter in E . coli cells was suggested to provide for the control of the intracellular pH as well as the switching from the aerobic to the anaerobic pathway of energy metabolism.

Prikl Biokhim Mikrobiol, 1987 Mar-Apr, 23(2), 179 - 84
{Distribution of enzymes of nucleic acid biosynthesis and their precursors during fractionation of Escherichia coli MRE-600 extract}; Khomov VV et al.; Distribution of the enzymes of template-dependent and template-independent polynucleotide syntheses (DNA-polymerase I, RNA-polymerase, polynucleotide phosphorylase) as well as those of the biosynthesis of nucleic acids precursors (nucleotide kinases, acetokinase and nucleoside deoxyribosyltransferases) during fractionation of Escherichia coli MRE-600 cell extract was studied . On the basis of the results obtained a technological scheme was developed that enabled to combine routine procedures of purification of the above mentioned enzymes.

Proc Natl Acad Sci U S A, 1987 Mar, 84(5), 1399 - 403
Synthetic gene construct expressing a repeated and highly immunogenic epitope of the Plasmodium falciparum antigen Pf155; Aslund L et al.; The Plasmodium falciparum-derived antigen Pf155 contains two blocks of tandemly repeated amino acid sequences . A pair of complementary oligonucleotides, encoding the C-terminally located repeat Val-Glu-His-Asp-Ala-Glu-Glu-Asn, were synthesized . The oligonucleotides were polymerized by ligation, and the resulting multimers were cloned into an expression vector . One construct that contained four copies of the repeat was expressed in Escherichia coli . The product, a fusion protein, was soluble and produced in high amounts . It reacted in immunoblotting with a monoclonal antibody to a synthetic octapeptide (Glu-Glu-Asn-Val-Glu-His-Asp-Ala) . Rabbits immunized with partially purified fusion protein, either with or without adjuvant, formed antibodies against this octapeptide . These antibodies reacted with Pf155 both in parasite extracts and when deposited in the membrane of infected erythrocytes . Furthermore, these antibodies inhibited merozoite reinvasion in vitro as efficiently as human antibodies to the octapeptide sequence in Pf155, induced by natural infection . The results suggest that products of synthetic gene constructs may be a suitable basis for an anti-merozoite vaccine.

Proc Natl Acad Sci U S A, 1987 Mar, 84(5), 1345 - 9
Isolation of temperature-sensitive tyrosine kinase mutants of v-abl oncogene by screening with antibodies for phosphotyrosine; Kipreos ET et al.; Temperature-sensitive protein-tyrosine kinase (EC 2.7.1.112) mutants of the oncogene v-abl have been obtained by a direct screening of kinase mutants in bacteria . The v-abl oncogene was expressed in Escherichia coli as a trpE/v-abl fusion protein from the trp promoter . The expression plasmid was mutagenized in vitro and then transfected into E . coli . Bacteria that produced defective tyrosine kinases were distinguished from those producing wild-type v-abl kinases by hybridization with antibodies specific for phosphotyrosine . Two independent mutations that generated temperature-sensitive tyrosine kinases were found to be located in a 12-amino acid region in the tyrosine kinase domain of the v-abl-encoded protein . These mutant v-abl oncogenes displayed temperature-sensitive transforming activity when expressed in NIH 3T3 cells . Cells transformed by these temperature-sensitive tyrosine kinase mutants could be shifted between the transformed and untransformed states by changing their growth temperature . These results confirmed the crucial role of tyrosine kinase activity in the v-abl-mediated oncogenesis.

Proc Natl Acad Sci U S A, 1987 Mar, 84(5), 1152 - 6
Site-directed mutagenesis of the COOH-terminal region of colicin A: effect on secretion and voltage-dependent channel activity; Baty D et al.; A large number of mutants introducing point mutations and deletions into the COOH-terminal domain of colicin A have been constructed by using site-directed mutagenesis . The COOH-terminal domain carries the channel activity . The effects of the alterations in the polypeptide chain on the secretion of colicin A by colicinogenic cells have been investigated . All deletions and some mutations were found to lead to protein aggregation in the cytoplasm, thereby preventing release into the medium . The mutated colicin A proteins have been purified, and their activity in vivo (on sensitive cells) and in vitro (in planar lipid bilayers) has been assayed . Deletions in the region containing putative helices 4, 5, and 6 (predicted to be involved in pore formation) and the transitions (Ala----Asp-492, Phe----Pro-493) in helix 4 abolished the activity . No correlation was observed between mutations leading to protein aggregation and those leading to loss of channel activity . Some mutations were found to alter characteristic properties of the single channels, such as stability, current-relaxation kinetics, voltage dependence, and pore conductance . Site-directed mutagenesis provides a powerful tool for studying structure-function relationships of voltage-sensitive ionic channels.

Proc Natl Acad Sci U S A, 1987 Mar, 84(5), 1147 - 51
Partly native epitopes are already present on early intermediates in the folding of tryptophan synthase; Blond S et al.; Two monoclonal antibodies directed against the native beta 2 subunit of Escherichia coli tryptophan synthase {L-serine hydro-lyase (adding indoleglycerol-phosphate), EC 4.2.1.20}, one recognizing the C-terminal F1 domain and the other the N-terminal F2 domain, were used to detect immunoreactive intermediates in the folding of the protein . For that purpose, the association of the monoclonal antibodies with either the beta 2 subunit or its isolated domains was studied by using fluorescence energy transfer between tryptophan residues of the antibodies and a dansyl group covalently linked to the antigen . It is shown that the association of both monoclonal antibodies with the antigen occurs within a few seconds after initiation of the renaturation, whereas complete refolding of the beta 2 subunit requires several minutes under the same experimental conditions . Thus, immunoreactive intermediates appear to be formed at an early stage of the folding process . While the isolated F1 domain alone is able to rapidly refold into a conformational intermediate already well recognized by the anti-native-beta 2 antibody, it cannot, in the absence of the F2 domain, reach its native conformation . However, its association with the anti-native-beta 2 antibody induces a structural change of F1 that brings it closer to the conformation it normally adopts when interacting with F2 inside the native beta 2 subunit.

J Bacteriol, 1987 Mar, 169(3), 1217 - 22
Activities of the RNAI and RNAII promoters of plasmid pBR322; Lin-Chao S et al.; The synthesis rates of the replication control RNAs of plasmid pBR322, RNAI, an inhibitor of replication, and RNAII, the preprimer, have been determined by hybridizing in vivo pulse-labeled RNA to specific, single-stranded DNA probes for RNAI and RNAII . In Escherichia coli growing in glycerol minimal medium, RNAI transcripts were made at a rate of one molecule per 30 s per plasmid; RNAII was transcribed fivefold less, at a rate of one molecule per 3 min per plasmid . It is estimated that only 1 in 20 prepriming events leads to replication.

Biochim Biophys Acta, 1987 Feb 27, 908(2), 131 - 42
Absolute values of ras p21 defined by direct binding liquid competition radioimmunoassays; Horan Hand P et al.; Several distinct and high-conserved genes comprise the ras gene family of rodents and humans, i.e., rodent Harvey and Kirsten, and human Harvey, Kirsten and neuroblastoma . Transformation, either by a point-mutation resulting in a change in one amino acid of the 21 kDa ras gene product (p21), or by increased expression of ras p21, has been demonstrated to be mediated by members of this gene family . We report here the development of direct binding liquid competition radioimmunoassays for the detection and quantitation of the ras oncogene and proto-oncogene products . Using these radioimmunoassays and ras p21 purified from Escherichia coli containing the full-length T24 mutant human Harvey ras gene protein product as a standard, we have defined the actual amount of ras p21 per micrograms of total cellular protein, or per cell, in various ras transformed and 'normal' mammalian cell lines . One of the radioimmunoassays developed is group-specific, since the antigenic determinant recognized is shared by both the point-mutated and proto-forms of Harvey, Kirsten and neuroblastoma members of the ras gene family, while the second may be termed type-selective, since it recognizes an antigenic determinant localized primarily on the Harvey ras p21 . Both radioimmunoassays are interspecies, since they detect a ras p21 antigenic determinant common to cells of human and rodent origin . These studies thus describe the first means for defining absolute values of any oncogene or proto-oncogene protein product . The assays described, when used in combination with specific c-DNA probes to define specific ras proto-oncogenes or point-mutated oncogenes being expressed, will now permit truly quantitative analyses of ras p21 expression in experimental cell culture systems, animal models and human biopsy material.

Biochem Biophys Res Commun, 1987 Feb 27, 143(1), 323 - 8
A sensitive colorimetric detection of virus DNA and oncogene; Inoue H et al.; Advantage of cloning probe DNA fragment in phage M13 DNA was taken to provide a larger single stranded DNA as a hybridization probe . High level of direct enzyme labels was introduced via the M13 DNA moiety as well as probe DNA . A highly sensitive colorimetric detection of virus DNA and oncogene was developed.

Biochem Biophys Res Commun, 1987 Feb 27, 143(1), 104 - 9
Proofreading of a mutagenic nucleotide, N4-aminodeoxycytidylic acid, by Escherichia coli DNA polymerase I; Takahashi M et al.; N4-Aminodeoxycytidine triphosphate, a putative metabolite of N4-aminocytidine which is a potent mutagen, is incorporated, in vitro, into polynucleotides in place of dCTP and at a much lesser extent, but significantly, in place of dTTP by E . coli DNA polymerase I large fragment . The activity of the polymerase to proofread this unnatural nucleotide has now been investigated . The results indicate that the 3'-5' exonuclease in the polymerase recognizes N4-aminocytosine as an incorrect base when N4-aminocytosine is incorporated opposite adenine but the enzyme cannot distinguish N4-aminocytosine from cytosine when it is incorporated opposite guanine.

Biochim Biophys Acta, 1987 Feb 27, 908(2), 113 - 22
Organization of genes for ribosomal proteins S7 and S12, elongation factors EF-Tu and EF-G in the cyanobacterium Spirulina platensis; Tiboni O et al.; The gene encoding ribosomal proteins S12 and probably S7 as well as protein synthesis elongation factors Tu (EF-Tu) and G (EF-G) of Spirulina platensis have been identified and cloned . Gene expression was determined for ribosomal protein S12 by genetic complementation of the appropriate Escherichia coli mutant, whereas for the EF-Tu gene it was determined by production of the protein in E . coli minicells . On the basis of these experiments we suggest the following gene order in the S . platensis chromosome: S12, S7, EF-G, EF-Tu.

Biochem Biophys Res Commun, 1987 Feb 27, 143(1), 300 - 8
Repetitive region of calpastatin is a functional unit of the proteinase inhibitor; Maki M et al.; A cDNA portion coding for one of the repetitive regions of pig heart calpastatin (107 kDa) was subcloned into E . coli plasmid pUC119 to express the portion of the proteinase inhibitor gene in bacteria . The expressed protein was a chimaeric protein whose calpastatin segment (130 amino acid residues) was fused with an amino-terminus portion (7 amino acid residues) of beta-galactosidase . The chimaeric protein could inhibit proteolytic activity of calpain (Ca2+-dependent cysteine proteinase), and maintained properties of the authentic calpastatin concerning inhibition specificity and heat stability . These findings led us to conclude that the repetitive region is a functional unit of the proteinase inhibitor.

Cell, 1987 Feb 27, 48(4), 555 - 66
The inducible lac operator-repressor system is functional in mammalian cells; Hu MC et al.; We have investigated the use of the Escherichia coli lac operator-repressor system to regulate expression of transfected genes in mammalian cells . We show that lac repressor produced in mouse L cells by transfection of a lacl expression vector blocks transcription of an MSV-CAT fusion gene when the lac operator is inserted at any one of the following sites within the promoter region: between the initiation codon (ATG) and the transcription start site; between the transcription start and TATA box regions; or upstream of the TATA box region . This last result suggests that the repressor may prevent protein-protein interactions involved in transcription activation . The inducer IPTG causes a marked derepression of CAT expression . The lac repressor-operator complex may be useful as an on/off "switch" in the regulation of gene expression for gene transfer experiments.

J Immunol Methods, 1987 Feb 26, 97(1), 19 - 27
A sensitive micro-immunoassay using beta-galactosidase/anti-beta-galactosidase complexes; Durbin H et al.; This paper describes a new sensitive microELISA based on enzyme/anti-enzyme complexes following an unlabelled antibody bridging step . beta-Galactosidase/anti-beta-galactosidase complexes were made using a monoclonal antibody raised against bacterial (E . coli) beta-galactosidase and enzyme activity was quantified with a fluorogenic substrate . Because of its high sensitivity the assay is particularly suitable for the detection of limited amounts of antigen . One application illustrated is the analysis of Class I and Class II histocompatibility antigens on peripheral blood lymphocytes using 5000 cells/well in 60-well Terasaki or 96-well microtitre plates.

Nucleic Acids Res, 1987 Feb 25, 15(4), 1763 - 77
Substrate specificity of DNA polymerases . I . Enzyme-catalysed incorporation of 5-'1-alkenyl)-2'-deoxyuridines into DNA; Otvos L et al.; A series of (E)-5-(1-alkenyl)-dUTPs as well as 5-vinyl-and (Z)-5-(1-propenyl)-dUTP have been synthesized to study steric requirements in DNA polymerase reactions . Experiments were carried out in E . coli DNA polymerase I Klenow fragment enzyme system . Substrates were characterized by KM and Vmax-values, initial incorporation rates as well as by total extent of incorporation of the analogues into poly(dA-dT) as a template-primer . Incorporation of the analogues could be best correlated with Vmax-values as well as the very similar initial incorporation rate values . Reactivity (Vmax/KM) showed no correlation with the extent of incorporation . 5-Vinyl-dUTP proved to be as good a substrate of the enzyme as dTTP, whereas (E)-5-(1-heptenyl)-and (E)-5-(1-octenyl)-dUTPs were very poor substrates, their incorporation was strongly limited and they also proved to be very efficient inhibitors of DNA replication, as shown by Ki-values . Substrate specificity of the Klenow enzyme can be explained by the steric hindrance of C-5 substituent, by the "orientational steric substituent effect" concept.

J Biol Chem, 1987 Feb 25, 262(6), 2590 - 6
The kinetics of bovine growth hormone folding are consistent with a framework model; Brems DN et al.; The framework model of protein folding requires the hydrogen-bonded secondary structure to be formed early in folding (i.e . the formation of secondary structure precedes the tertiary structure) (Kim, P . S., and Baldwin, R . L . (1982) Annu . Rev . Biochem . 51, 459-489) . To test the framework model directly the kinetics of bovine growth hormone (bGH) folding were compared utilizing two methods of detection, one that measures the secondary structure (far ultraviolet circular dichroism) and another that measures the tertiary structure (near ultraviolet absorbance) . The results demonstrate that, under identical folding conditions, the kinetics observed by far ultraviolet circular dichroism are faster than those observed by ultraviolet absorption . The faster kinetics observed by circular dichroism indicate the existence of a helix-containing intermediate which is consistent with the framework model . The effect of protein concentration and denaturant concentration on the kinetics of refolding were studied . The rate of refolding measured by absorbance and circular dichroism was dependent on protein concentration . The protein concentration dependence on refolding is due to the transient formation of an associated intermediate . The concentration dependence of folding is taken as evidence that folding is a sequential process with partially folded monomers responsible for the observed association effect . At dilute protein concentrations the refolding can be studied independent of the association phenomena . The growth hormones utilized in this study were derived from Escherichia coli through recombinant DNA technology and from bovine pituitaries . The pituitary-derived bGH has been shown to be heterogeneous at the NH2 terminus (Lorenson, M . F., and Ellis, S . (1975) Endocrinology 96, 833-838), whereas the recombinant DNA-derived bGH contains a single NH2 terminus . No differences in the folding kinetics between the recombinant DNA and pituitary derived-bGH were observed . It is concluded that the heterogeneity of the NH2 terminus of growth hormone obtained from bovine pituitaries does not affect the observed in vitro folding kinetics.

Nucleic Acids Res, 1987 Feb 25, 15(4), 1521 - 42
Effect of the deletion of upstream DNA sequences on expression from the ilvGp2 promoter of the ilvGMEDA operon of Escherichia coli K-12; Ortuno MJ et al.; Transcription in vitro of the regulatory region of the ilvGMEDA operon yields two attenuated RNAs initiated from the tandem promoters ilvGp1 and ilvGp2 . Both S1 nuclease analysis and the fusion of ilvGp1 to galK indicate that transcription is not initiated in vivo from ilvGp1 . However deletion of DNA sequences 150 to 100 bp upstream of ilvGp2 drastically reduces expression in vivo from ilvGp2 . Both the distance separating ilvGp2 from the upstream DNA sequences and their relative orientation to each other on the DNA helix affect expression from ilvGp2 . Deletion of DNA sequences approximately 400 bp upstream of ilvGp2 increases expression in vivo from this promoter . Analysis of products of transcription in vitro indicates that the effects observed in vivo are probably not due to DNA conformation or interactions of RNA polymerase.

J Biol Chem, 1987 Feb 25, 262(6), 2852 - 3
Crystallization and preliminary X-ray investigation of uridine phosphorylase from Escherichia coli; Cook WJ et al.; Crystals of uridine phosphorylase from Escherichia coli K12 have been grown from solutions of polyethylene glycol 4000 . The crystals are trigonal, space group R3; the hexagonal axes are a = 154.4 A and c = 49.4 A . The crystals are quite stable to x-rays and diffract beyond 2.6 A resolution . It appears that the molecule is a hexamer with a subunit molecular weight of 27,500 and utilizes the 3-fold symmetry of the space group, resulting in two subunits/asymmetric unit.

J Biol Chem, 1987 Feb 25, 262(6), 2478 - 84
Physical characteristics of ribosomal protein S4 from Escherichia coli; Dodd J et al.; A hydrodynamic study of protein S4 from Escherichia coli 30 S ribosomal subunits indicates that this protein is moderately asymmetric . A sedimentation coefficient of 1.69 S and a diffusion coefficient of 7.58 X 10(-7) cm2/s suggest that S4 has an axial ratio of about 5:1 using a prolate ellipsoidal model . This structure should give a radius of gyration of about 29-30 A from small-angle neutron or small-angle x-ray scattering studies . This study has utilized quasi-elastic light scattering as an analytical tool to obtain a diffusion coefficient as well as a method to monitor sample quality . Using quasi-elastic light scattering in this manner allows an assessment of problems associated with protein purity which may be responsible for the many disparate results reported for ribosomal proteins and especially protein S4.

Nucleic Acids Res, 1987 Feb 25, 15(4), 1615 - 25
Characterization of the 5'-flanking region for the human fibrinogen beta gene; Huber P et al.; To identify the possible regulatory sequences in the genetic expression of fibrinogen, a human genomic DNA library raised in lambda EMBL 4 phage was screened using cDNA probes coding for the A alpha, B beta and gamma chains of human fibrinogen . The entire fibrinogen locus was characterized and its organization analysed by means of hybridization and restriction mapping . Among the clones identified, a single recombinant lambda phage contained the beta gene and its 5'- and 3'-flanking regions . A 1.5 kb fragment of the immediate 5'-flanking region was sequenced and S1 mapping experiments revealed three transcription start points . Comparison of this sequence with that previously reported for the same region upstream from the human gamma gene revealed no significant homology which suggests that the potential promoting sequences of these genes are different . In contrast, comparison of the 5'-flanking regions of human and rat beta genes revealed a 142 bp sequence of 80% homology situated 16 bp upstream from the human beta gene . This highly conserved region may well represents a potential candidate for a regulatory sequence of the human beta gene.

J Biol Chem, 1987 Feb 25, 262(6), 2502 - 6
Circular dichroism investigation of Escherichia coli adenylate kinase; Monnot M et al.; We examined by circular dichroism (CD) spectroscopy in far- and near-ultraviolet three different molecular forms of Escherichia coli adenylate kinase: the wild type protein, the enzyme carboxymethylated at a single cysteine residue (Cys-77), and the thermosensitive adenylate kinase . The thermosensitive enzyme differs from the wild type protein in that a serine is substituted for a proline residue at position 87 (Gilles, A.-M., Saint Girons, I., Monnot, M., Fermandjian, S., Michelson, S., and Barzu, O . (1986) Proc . Natl . Acad . Sci . U . S . A., 83, 5798-5802) . We also examined the CD spectra of isolated peptides resulting from chemical cleavage of adenylate kinase at Cys-77 (C1, residues 1-76; C2, residues 77-214) . The secondary structure composition of wild type bacterial adenylate kinase (50% alpha-helix and 15% beta-sheet) was close to that derived from x-ray analysis of pig muscle enzyme (Schulz, G.E., Elzinga, M., Marx, F., and Schirmer, R . H . (1974) Nature 250, 120-123) . Carboxymethylation of wild type protein did not greatly affect the CD spectrum . The secondary structure of the thermosensitive adenylate kinase was observed to be significantly different from that of the wild type enzyme (reduction in alpha-helix content to 39%) . Changes in ellipticities at 222 nm as a function of temperature indicated that the melting temperature for thermosensitive adenylate kinase was 38 degrees C and that for the wild type enzyme was 54 degrees C . Isolated C1 and C2 peptides had a large proportion of unordered structures . When mixed, C1 and C2 fragments reassociated into structures resembling native, uncleaved adenylate kinase . The recovery of ordered structures, indicated by CD spectroscopy, paralleled the recovery of catalytic activity.

J Biol Chem, 1987 Feb 25, 262(6), 2468 - 71
Regulation of pantothenate kinase by coenzyme A and its thioesters; Vallari DS et al.; Pantothenate kinase catalyzes the rate-controlling step in the coenzyme A (CoA) biosynthetic pathway, and its activity is modulated by the size of the CoA pool . The effect of nonesterified CoA (CoASH) and CoA thioesters on the activity of pantothenate kinase was examined to determine which component of the CoA pool is the most effective regulator of the enzyme from Escherichia coli . CoASH was five times more potent than acetyl-CoA or other CoA thioesters as an inhibitor of pantothenate kinase activity in vitro . Inhibition by CoA thioesters was not due to their hydrolysis to CoASH . CoASH inhibition was competitive with respect to ATP, thus providing a mechanism to coordinate CoA production with the energy state of the cell . There were considerable differences in the size and composition of the CoA pool in cells grown on different carbon sources, and a carbon source shift experiment was used to test the inhibitory effect of the different CoA species in vivo . A shift from glucose to acetate as the carbon source resulted in an increase in the CoASH:acetyl-CoA ratio from 0.7 to 4.3 . The alteration in the CoA pool composition was associated with the selective inhibition of pantothenate phosphorylation, consistent with CoASH being a more potent regulator of pantothenate kinase activity in vivo . These results demonstrate that CoA biosynthesis is regulated through feedback inhibition of pantothenate kinase primarily by the concentration of CoASH and secondarily by the size of the CoA thioester pool.

Biochemistry, 1987 Feb 24, 26(4), 1132 - 6
Role of cysteine residues in the lac permease of Escherichia coli; Menick DR et al.; Oligonucleotide-directed, site-specific mutagenesis has been utilized to replace cysteine residues 117, 333, or 353 and 355 with serine in the lac permease of Escherichia coli . Replacement of Cys-117 or Cys-333 has no significant effect on permease activity, while permease with serine residues in place of Cys-353 and Cys-355 has about 50% of wild-type permease activity . The results provide a clear demonstration that cysteine residues at positions 117, 333, 353, and 355 are not obligatory for lactose/H+ symport . When considered in conjunction with previous findings, the results indicate that, of the eight cysteine residues in the lac permease, only Cys-154 is important for lactose transport . As discussed, the conclusion has important implications for the hypothesis that sulfhydryl-disulfide interconversion plays an important role in the symport mechanism.

FEBS Lett, 1987 Feb 23, 212(2), 271 - 5
Simplified in vitro synthesis of mutated RNA molecules . An oligonucleotide promoter determines the initiation site of T7RNA polymerase on ss M13 phage DNA; Krupp G et al.; We describe a simplified method for the in vitro synthesis of mutated RNA molecules . The method makes use of an oligodeoxyribonucleotide (T7-oligo) which contains the T7RNA polymerase promoter sequence . In combination with a second oligonucleotide, a series of transcripts initiating and terminating at any chosen position on a cloned ss DNA (e.g . M13 phage DNA) can be generated . The phage DNA represents the non-coding DNA strand for the desired transcript; the T7-oligo determines the transcription start site, whereas the second oligonucleotide permits the choice of the transcription termination site . The synthesis of the required template DNA is achieved by hybridizing the two oligonucleotides to the phage DNA and subsequently synthesizing the coding DNA strand by a fill-in reaction with Klenow enzyme . The reaction product is used directly as a template for T7RNA polymerase; cloning of mutants is not required.

J Mol Biol, 1987 Feb 20, 193(4), 723 - 50
Selection of DNA binding sites by regulatory proteins . Statistical-mechanical theory and application to operators and promoters; Berg OG et al.; We present a statistical-mechanical selection theory for the sequence analysis of a set of specific DNA regulatory sites that makes it possible to predict the relationship between individual base-pair choices in the site and specific activity (affinity) . The theory is based on the assumption that specific DNA sequences have been selected to conform to some requirement for protein binding (or activity), and that all sequences that can fulfil this requirement are equally likely to occur . In most cases, the number of specific DNA sequences that are known for a certain DNA-binding protein is very small, and we discuss in detail the small-sample uncertainties that this leads to . When applied to the binding sites for cro repressor in phage lambda, the theory can predict, from the sequence statistics alone, their rank order binding affinities in reasonable agreement with measured values . However, the statistical uncertainty generated by such a small sample (only 6 sites known) limits the result to order-of-magnitude comparisons . When applied to the much larger sample of Escherichia coli promoter sequences, the theory predicts the correlation between in vitro activity (k2KB values) and homology score (closeness to the consensus sequence) observed by Mulligan et al . (1984) . The analysis of base-pair frequencies in the promoter sample is consistent with the assumption that base-pairs at different positions in the sites contribute independently to the specific activity, except in a few marginal cases that are discussed . When the promoter sites are ordered according to predicted activities, they seem to conform to the Gaussian distribution that results from a requirement for maximal sequence variability within the constraint of providing a certain average activity . The theory allows us to compare the number of specific sites with a certain activity to the number that would be expected from random occurrence in the genome . While strong promoters are "overspecified", in the sense that their probability of random occurrence is very low, random sequences with weak promoter-like properties are expected to occur in very large numbers . This leads to the conclusion that functional specificity is based on other properties in addition to primary sequence recognition; some possibilities are discussed . Finally, we show that the sequence information, as defined by Schneider et al . (1986), can be used directly (at least in the case of equilibrium binding sites) to estimate the number of protein molecules that are specifically bound at random "pseudosites" in the genome.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochim Biophys Acta, 1987 Feb 20, 923(2), 275 - 85
Studies on the catalytic rate constant of ribosomal peptidyltransferase; Synetos D et al.; A detailed kinetic analysis of a model reaction for the ribosomal peptidyltransferase is described, using fMet-tRNA or Ac-Phe-tRNA as the peptidyl donor and puromycin as the acceptor . The initiation complex (fMet-tRNA X AUG X 70 S ribosome) or (Ac-Phe-tRNA X poly(U) X 70 S ribosome) (complex C) is isolated and then reacted with excess puromycin (S) to give fMet-puromycin or Ac-Phe-puromycin . This reaction (puromycin reaction) is first order at all concentrations of S tested . An important asset of this kinetic analysis is the fact that the relationship between the first order rate constant kobs and {S} shows hyperbolic saturation and that the value of kobs at saturating {S} is a measure of the catalytic rate constant (k cat) of peptidyltransferase in the puromycin reaction . With fMet-tRNA as the donor, this kcat of peptidyltransferase is 8.3 min-1 when the 0.5 M NH4Cl ribosomal wash is present, compared to 3.8 min-1 in its absence . The kcat of peptidyltransferase is 2.0 min-1 when Ac-Phe-tRNA replaces fMet-tRNA in the presence of the ribosomal wash and decreases to 0.8 min-1 in its absence . This kinetic procedure is the best method available for evaluating changes in the activity of peptidyltransferase in vitro . The results suggest that peptidyltransferase is subjected to activation by the binding of fMet-tRNA to the 70 S initiation complex.

J Mol Biol, 1987 Feb 20, 193(4), 651 - 9
Specific strand loss in N-2-acetylaminofluorene-modified DNA; Koffel-Schwartz N et al.; N-2-Acetylaminofluorene (AAF), a well-known chemical carcinogen, when covalently linked to guanine residues constitutes a premutagenic lesion that is converted in vivo into frameshift mutations . In Escherichia coli, it is thought that -AAF adducts block the replication fork and that the mutagenic processing of the -AAF adducts is mediated by the SOS response . The construction in vitro of plasmids containing -AAF adducts in one strand only of a double-stranded DNA molecule enabled us to investigate the segregation of the strands and the mutagenicity of the lesions in vivo . The two DNA strands were "genetically labelled" by means of a single base-pair mismatch in the tetracycline-resistance gene, one strand carrying the wild-type allele and the other strand a mutant tetracycline-sensitive allele . The two strands contained either no -AAF adducts, -AAF adducts in one strand or -AAF adducts in both strands . When such constructions are used to transform bacterial cells the following are found . When no -AAF adducts are present on either strand of the DNA, a mixture of plasmids having information from both parent strands is found in 80% of the transformed bacterial clones . With -AAF adducts present in one strand only, in 90% of the transformants there is a consistent loss of the parent strand information that contained the -AAF adducts . In the constructions having -AAF adducts in both strands, the transformed bacteria carry either one or the other allele in a pure form . Our results suggest that when blocking lesions (-AAF adducts) are present in one strand only, they trigger the specific loss of that strand . The forward mutation frequency (i.e . the tetracycline-resistance gene inactivation frequency) was found to be more than ten times lower when the -AAF adducts are bound to one strand only compared with the situation where both strands carry the premutagenic lesions . Moreover, when the isolated mutants were sequenced, the mutations were found to consist of a mixture of true -AAF-induced mutations (i.e . -1 or -2 frameshift mutation at previously determined mutation hot spots) and of mutations that are not targeted at -AAF adducts . We suggest that these "background" mutants arose from the mutagenic processing of cryptic lesions present in our DNA . The low mutagenic efficiency of -AAF adducts, when present in one strand only of a duplex DNA, most probably results from the above-described loss of the damaged strand.(ABSTRACT TRUNCATED AT 400 WORDS)

J Mol Biol, 1987 Feb 20, 193(4), 637 - 41
Ultraviolet light-induced mutagenesis in the Escherichia coli chromosome . Sequences of mutants in the cI gene of a lambda lysogen; Wood RD et al.; DNA sequences were determined for 56 mutations induced by ultraviolet light in the lambda cI gene of an Escherichia coli uvr+ lysogen, which should reflect those occurring in the E . coli chromosome . The spectrum of mutagenesis was similar to that found in the cI gene of irradiated phase assayed in uvr- host cells, except that the fraction of transversions is about 35% in prophage and about 15% in phage . The cause of this difference is not known . Of 17 frameshifts in phage and prophage, six have an accompanying base substitution . These double mutational events are consistent with a model in which a photoproduct in a template can cause a DNA polymerase to insert a wrong base and destabilize the next few bases added, thus leading to a frameshift by a slippage mechanism.

J Mol Biol, 1987 Feb 20, 193(4), 609 - 36
Mechanisms of frameshift mutagenesis by aflatoxin B1-2,3-dichloride; Refolo LM et al.; In order to characterize frameshift mutagenesis by aflatoxin B1-2,3-dichloride (AFB1Cl2), we have introduced a +1 (BK8) or a -1 (HS8) frameshift within the lacZ alpha gene segment contained in the phage M13mp8 to obtain lacZ alpha- derivatives . BK8 or HS8 replicative form DNA was modified with AFB1Cl2 in vitro, transfected into appropriate Escherichia coli hosts and lacZ alpha+ revertants scored and defined by DNA sequencing . The -1 frameshift (BK8) results suggest the following . (1) The E . coli recA gene is not absolutely required for AFB1Cl2-induced frameshift mutagenesis; however, in recA+ cells, ultraviolet light (SOS) induction enhances AFB1Cl2 mutagenesis, but such ultraviolet induction is not required . The plasmid pGW270 (mucAB+) significantly enhances the AFB1Cl2-induced frameshift mutagenesis . The uvrABC+ excision system plays a major role in the repair of AFB1Cl2-induced damage . (2) Sequence analysis reveals that AFB1Cl2 induces two classes of -1 frameshift mutations: the simple class in which the frameshift is due to the loss of one base-pair, and the complex class in which the loss of a base-pair is coupled to a vicinal base substitution . Both types of mutations occur predominantly at G.C runs, which are hotspots for AFB1Cl2 damage . The complex mutations appear to be concerted events targeted by a single AFB1Cl2 adduct . The frequency of these complex mutations is significantly enhanced by mucAB activity . In this system, recA activity is required for generation of significant levels of complex mutations . An analysis of the +1 frameshifts (HS8) reveals that AFB1Cl2 induces +1 frameshifts with an efficiency comparable to that for -1 frameshifts . Most +1 frameshifts occur by the addition of a base, and a third of the additions are complex mutations because they are accompanied by at least one base substitution . All simple additions occur at G.C runs; however, in a striking contrast to spontaneous insertions, a majority of the induced events introduce an A.T pair at these sites . Our data suggest a model for the generation of base substitution as well as simple and complex frameshift mutations induced by AFB1Cl2 . To the extent determined, the frameshift specificity of aflatoxin B1 activated by metabolic enzymes is similar to that of AFB1Cl2.

J Mol Biol, 1987 Feb 20, 193(4), 661 - 71
Sites of action of two ribosomal RNA methylases responsible for resistance to aminoglycosides; Beauclerk AA et al.; Methylation of either of two residues (G-1405 or A-1408) within bacterial 16 S ribosomal RNA results in high level resistance to specific combinations of aminoglycoside antibiotics . The product of a gene that originated in Micromonospora purpurea (an actinomycete that produces gentamicin) gives resistance to kanamycin plus gentamicin by converting residue G-1405 to 7-methylguanosine . Resistance to kanamycin plus apramycin results from conversion of residue A-1408 to 1-methyladenosine catalysed by the product of a gene from Streptomyces tenjimariensis.

Nature, 1987 Feb 19-25, 325(6106), 720 - 3
T gamma protein is expressed on murine fetal thymocytes as a disulphide-linked heterodimer; Nakanishi N et al.; During the search for genes coding for the mouse alpha and beta subunits of the antigen-specific receptor of mouse T cells we encountered a third gene, subsequently designated gamma . This gene has many properties in common with the alpha and beta genes, somatic assembly from gene segments that resemble the gene segments for immunoglobulin variable (V), joining (J) and constant (C) regions; rearrangement and expression in T cells and not in B cells; low but distinct sequence homology to immunoglobulin V, J and C regions; other sequences that are reminiscent of the transmembrane and intracytoplasmic regions of integral membrane proteins; and a cysteine residue at the position expected for a disulphide bond linking two subunits of a dimeric membrane protein . Despite these similarities the gamma gene also shows some interesting unique features . These include a relatively limited repertoire of the germ-line gene segments, more pronounced expression at the RNA level in immature T cells such as fetal thymocytes and an apparent absence of in-frame RNA in some functional, alpha beta heterodimer-bearing T cells or cultured T clones and hybridomas . To understand the function of the putative gamma protein it is essential to define the cell population that expresses this protein . To this end we produced a fusion protein composed of Escherichia coli beta-galactosidase and the gamma-chain (hereafter referred to a beta-gal-gamma) using the phage expression vector lambda gt11 and raised rabbit antisera against the gamma determinants . Using the purified anti-gamma antibody we detected a polypeptide chain of relative molecular mass 35,000 (Mr 35K) on the surface of 16-day old fetal thymocytes . The gamma-chain is linked by a disulphide bridge to another component of 45K . No such heterodimer was detected on the surface of a cytotoxic T lymphocyte (CTL) clone 2C from which an in-phase gamma cDNA clone was originally isolated.

Eur J Biochem, 1987 Feb 16, 163(1), 67 - 71
Expression of bovine pancreatic ribonuclease A in Escherichia coli; Nambiar KP et al.; A synthetic gene for bovine pancreatic ribonuclease A (RNase A) has been expressed in Escherichia coli as a fusion protein with beta-galactosidase linked by the tetrapeptide Ile-Glu-Gly-Arg . RNase A was cleaved from the fusion using factor Xa, and the resulting product purified and reconstituted . The isolated RNase A was chromatographically, catalytically, and immunologically identical with authentic RNase A . This work argues that the method suggested by Nagai and Thogersen {Nagai, K . & Thogersen, H . C . (1984) Nature (Lond.) 309, 810-812} for releasing fusion proteins is quite general, even when applied to particularly complicated expression problem . The procedure here makes RNase A available for the first time as a model for studying structure-function relationships in proteins using site-directed mutagenesis.

Eur J Biochem, 1987 Feb 16, 163(1), 113 - 21
Studies on the functional topography of Escherichia coli RNA polymerase . Highly selective affinity labelling by analogues of initiating substrates; Grachev MA et al.; RNA polymerase was treated in the presence of promoter-containing templates with 16 affinity reagents, derivatives on NMPs, NDPs and NTPs with reactive substituents at the terminal phosphate . This treatment was followed by addition of a pyrimidine {alpha-32P}NTP . Due to 'catalytic competence' of some of the residues of the affinity reagents bound covalently near the active center at the first stage, active-center-catalyzed synthesis of a phosphodiester bond occurred, and radioactive residues with the general formula -pNpN (where p = radioactive phosphate) appeared covalently attached to the enzyme . Such affinity labelling was super-selective because affinity reagent residues bound outside the active center were not elongated and thus remained non-radioactive . Labelling took place only when the combination of the reagent and {alpha-32P}NTP corresponded to the sequence of nucleotides of the promoter . With reagents having short 'arms', only the beta subunit was labelled; the targets were His and/or Lys residues . With reagents having longer 'arms', the sigma subunit was also labelled.

Eur J Biochem, 1987 Feb 16, 163(1), 141 - 6
Modulation of human neutrophil function by C-reactive protein; Buchta R et al.; Investigation of the effect of C-reactive protein (CRP), an acute-phase reactant, on the functional capacities of human neutrophils was carried out as the basis for elucidating the biological function of C-reactive protein . An initial stimulation at low concentrations, followed by inhibition of superoxide production, and secretion of vitamin-B12-binding protein in the presence of two stimulants, phorbol myristate acetate and concanavalin A, and of neutrophil chemotaxis with increasing concentration of CRP was observed . Correlation between modulation of cell function, at least at relatively high CRP concentrations (greater than 50 micrograms/ml) and an increase in the intracellular level of cAMP is suggested . CRP was also found to enhance neutrophil phagocytosis of particles not containing phosphorylcholine, the native CRP ligand . The proposed role of CRP in neutrophil function is one of regulation and as a negative feedback for potential cytotoxic neutrophil functions.

Biochem J, 1987 Feb 15, 242(1), 261 - 6
Characterization of argininosuccinate lyase (EC 4.3.2.1) from Chlamydomonas reinhardtii; Farrell K et al.; We have isolated and characterized argininosuccinate lyase (ASL; EC 4.3.2.1) from the photosynthetic green alga, Chlamydomonas reinhardtii . The general properties of Chlamydomonas ASL are very similar to those described previously for ASLs from phylogenetically diverse organisms . The algal ASL has a native Mr, determined by gel-filtration chromatography, of 218,000 +/- 25,000, and a pI of 5.4-5.6 . The Km for argininosuccinate at 37 degrees C and pH 7.5 is 0.26 mM . The subunit Mr of Chlamydomonas ASL is approx . 50,000, determined by SDS/polyacrylamide-gel electrophoresis, in contrast with a previously reported value of 39,000 . Rabbit antisera prepared against the Mr-50,000 protein completely abolished ASL activity in vitro . In contrast, serum prepared against the Mr-39,000 protein was ineffective in inhibiting ASL activity . Despite the general similarity of the physical properties of Chlamydomonas ASL and those of other ASLs, antiserum raised against the algal ASL did not cross-react with ASL preparations from Escherichia coli, Saccharomyces cerevisiae or bovine liver.

J Biol Chem, 1987 Feb 15, 262(5), 2121 - 30
Properties of initiation complexes formed between Escherichia coli DNA polymerase III holoenzyme and primed DNA in the absence of ATP; Kwon-Shin O et al.; In the presence of ATP, the beta subunit of the Escherichia coli DNA polymerase III holoenzyme can induce a stable initiation complex with the other holoenzyme subunits and primed DNA that is capable of highly processive synthesis . We have recently demonstrated that the ATP requirement for processive synthesis can be bypassed by an excess of the beta subunit (Crute, J., LaDuca, R., Johanson, K., McHenry, C., and Bambara, R . (1983) J . Biol . Chem . 258, 11344-11349) . To examine the complex formed with excess beta subunit, and the lengths of the products of processive synthesis, we have designed a uniquely primed DNA template . Poly(dA)4000 was tailed with dCTP by terminal deoxynucleotidyl transferase and the resulting template annealed to oligo(dG)12-18 . In the presence of excess beta, the lengths of processively extended primers nearly equaled the full-length of the DNA template . Similar length synthesis occurred in the presence or absence of spermidine or single-stranded DNA-binding protein . When the beta subunit was present at normal holoenzyme stoichiometry it could induce highly processive synthesis without ATP, although inefficiently . Both ATP and excess beta increased the amount of initiation complex formation, but complexes produced with excess beta did so without the time delay observed with ATP, suggesting different mechanisms for formation . Almost 50% of initiation complexes formed without ATP survived a 30-min incubation with anti-beta IgG, reflecting a stability similar to those formed with ATP . The ability to form initiation complexes in the absence of ATP permitted the demonstration that cycling of the holoenzyme to a new primer, after chain termination with a dideoxynucleotide, is not affected by the presence of ATP.

Experientia, 1987 Feb 15, 43(2), 174 - 6
Ornithine decarboxylase, S-adenosyl-L-methionine decarboxylase and arginine decarboxylase from Mycobacterium bovis (BCG); Paulin L et al.; Ornithine decarboxylase (ODC), S-adenosyl-L-methionine decarboxylase (AMDC) and arginine decarboxylase (ADC) activities were detected for the first time in extracts of Mycobacterium bovis (BCG) . All the decarboxylases differed from corresponding known bacterial decarboxylases in that: a) ODC did not require GTP for activity; b) ODC was not inhibited by any known inhibitor of bacterial ODCs; c) AMDC and ADC did not require Mg2+-ion for activity and were not markedly inhibited by any known inhibitor of the decarboxylases of other bacteria.

Arch Biochem Biophys, 1987 Feb 15, 253(1), 241 - 8
Purification and properties of the manganese superoxide dismutase from the liver of bullfrog, Rana catesbeiana; Abe Y et al.; A manganese superoxide dismutase (Mn-SOD) from the liver of bullfrog, Rana catesbeiana, was purified to electrophoretic homogeneity . The enzyme has a molecular weight of about 84,000 and is composed of four identical subunits, each containing one manganese atom . The amino acid composition of the enzyme is similar to that of Mn-SODs isolated from human and chicken livers, but differs considerably from that of the Escherichia coli enzyme (D . Barra et al . (1984) J . Biol . Chem . 259, 12595-12601; R . A . Weisiger and I . Fridovich (1973) J . Biol . Chem . 248, 3582-3592; H . M . Steinman (1978) J . Biol . Chem . 253, 8708-8720) . The N-terminal amino acid is lysine . The sequence of 23 amino acid residues in the N-terminal region was determined . It shows excellent homologies with those of the human and chicken enzymes (H . M . Steinmam and R . L . Hill (1973) Proc . Natl . Acad . Sci . USA 70, 3725-3729; C . Ditlow et al . (1982) Carlsberg Res . Commun . 47, 81-91) . The frog liver enzyme is also located exclusively in the mitochondrial matrix . Immunologically the same enzyme is also found in the tadpole liver, in an amount of about one-half of that in the adult bullfrog.

J Biol Chem, 1987 Feb 15, 262(5), 2358 - 62
In vitro translocation of protein across Escherichia coli membrane vesicles requires both the proton motive force and ATP; Yamane K et al.; The energy requirement for protein translocation across membrane was studied with inverted membrane vesicles from an Escherichia coli strain that lacks all components of F1F0-ATPase . An ompF-lpp chimeric protein was used as a model secretory protein . Translocation of the chimeric protein into membrane vesicles was totally inhibited in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP) or valinomycin and nigericin and partially inhibited when either valinomycin or nigericin alone was added . Depletion of ATP with glucose and hexokinase resulted in the complete inhibition of the translocation process, and the inhibition was suppressed by the addition of ATP-generating systems such as phosphoenolpyruvate-pyruvate kinase or creatine phosphate-creatine kinase . These results indicate that both the proton motive force and ATP are required for the translocation process . The results further suggest that both the membrane potential and the chemical gradient of protons (delta pH), of which the proton motive force is composed, participate in the translocation process.

J Biol Chem, 1987 Feb 15, 262(5), 2304 - 9
Formation of rolling-circle molecules during phi X174 complementary strand DNA replication; Mok M et al.; The primosome is a mobile multiprotein priming apparatus that requires seven Escherichia coli proteins for assembly (the products of the dnaB, dnaC and dnaG genes; replication factor Y (protein n'); and proteins i, n, and n") . While the primosome is analagous to the phage T7 gene 4 protein and phage T4 gene 41/61 proteins in its DNA G-catalyzed priming function, its ability to act similarly also as a DNA helicase has remained equivocal . The role of the primosome in unwinding duplex DNA strands was investigated in the coliphage phi X174 SS(c)----replicative form DNA replication reaction in vitro, which requires the E . coli single-stranded DNA binding protein, the primosomal proteins, and the DNA polymerase III holoenzyme . Multigenome-length, linear, double-stranded DNA molecules were generated in this reaction, presumably via a rolling circle-type mechanism . Synthesis of these products required the presence of a helicase-catalyzed strand-displacement activity to permit multiple cycles of continuous complementary (-) strand synthesis . The participation of the primosome in this helicase activity was supported by demonstrating that other SS(c) DNA templates (G4 and alpha-3), which lack primosome assembly sites, failed to support significant linear multimer production and that replication of phi X174 with the general priming system (the DNA B and DNA G proteins and DNA polymerase III holoenzyme) resulted in a 13-fold lower rate of linear multimer synthesis.

J Immunol, 1987 Feb 15, 138(4), 1109 - 14
Expression in Escherichia coli of fully active recombinant human IL 1 beta: comparison with native human IL 1 beta; Tocci MJ et al.; Although complementary DNA (cDNA) encoding human interleukin 1 beta (IL 1 beta) have been cloned in several laboratories, there are as yet no data demonstrating that recombinant IL 1 beta (rIL 1 beta) molecules expressed from such cDNA are faithful, fully active replicas of the native protein secreted by human monocytes . To this purpose, cDNA sequences corresponding to the exact NH2-terminus and amino acid sequence of mature, monocyte-derived human IL 1 beta were placed under control of the inducible trp-lac (TAC) fusion promoter and were expressed in E . coli strain JM105 . rIL 1 beta was purified to homogeneity by high pressure liquid chromatography (HPLC) . Yields of 10 to 20 mg of rIL 1 beta/liter/10(11) cells were obtained . Purified rIL 1 beta was then compared in a number of biochemical and biologic assays to purified native IL 1 beta . Native and rIL 1 beta co-migrated on SDS-polyacrylamide gels as 17.5 kd polypeptides and reacted with specific polyclonal antisera raised to three synthetic peptides of human IL 1 beta in immunoblot experiments . Amino acid sequence analysis of rIL 1 beta showed that the native amino terminus, an ALA residue, was faithfully maintained . Purified native and rIL 1 beta co-chromatographed on reverse-phase HPLC . Specific biologic activities of rIL 1 beta were indistinguishable from those of the native protein in murine thymocyte and human dermal fibroblast proliferation assays, with half-maximal stimulation occurring at concentrations of 25 pM in the murine thymocyte assay and 2 pM in the human dermal fibroblast assay . Similarly, native and rIL 1 beta competed equally well for high affinity IL 1 receptor binding sites, each exhibiting a Ki of 20 pM on MRC-5 human embryonic lung fibroblasts . These observations indicate that E . coli efficiently expresses large quantities of rIL 1 beta, which emulates exactly the properties of the native protein.

J Biol Chem, 1987 Feb 15, 262(5), 2180 - 6
Catalytic properties of the F1-adenosine triphosphatase from Escherichia coli K-12 and its genetic variants as revealed by 18O exchanges; Wood JM et al.; We have examined intermediate Pi-water oxygen exchange during {gamma-18O}ATP hydrolysis by the F1 adenosine triphosphatase from Escherichia coli K-12 . Water oxygen incorporation into each Pi released was increased as ATP concentration was lowered as observed previously for the same reaction catalyzed by the enzyme from eukaryotic sources . Heterogeneous distributions of 18O in product Pi were produced by coexisting epsilon subunit-replete and epsilon subunit-depleted enzyme molecules . The epsilon-replete enzyme showed a much higher probability for oxygen exchange . These data imply that the epsilon subunit inhibits net ATP hydrolysis by imposing conformational constraints which reduce the cooperative conformational interactions that promote ADP and Pi release . Four enzyme variants altered in alpha or beta subunit structure with reduced net hydrolytic activity showed sharply increased oxygen exchange during ATP hydrolysis . Heterogeneity was apparent in the 18O distribution of the product Pi, however . That behavior could reflect hindered conformational interactions and/or increased affinity of the alpha 3 beta 3 gamma delta complex for the epsilon subunit . In contrast, enzyme from mutant uncA401 showed very little oxygen exchange accompanying hydrolysis of 20 microM ATP . This is the only enzyme so far reported with this unusual property . Its rate limitation appears to be in the hydrolytic rather than the product release step of the catalytic sequence.

J Biol Chem, 1987 Feb 15, 262(5), 2101 - 10
Purification of a protease from Escherichia coli with specificity for oxidized glutamine synthetase; Roseman JE et al.; A soluble Escherichia coli protease has been identified and purified to homogeneity . The protease cleaves glutamine synthetase which has been modified by mixed function oxidation; native glutamine synthetase is not a substrate . Using {14C}glutamine synthetase as a substrate (prepared by growing E . coli on 14C-labeled amino acids), protease activity was assayed by determining the release of trichloroacetic acid-soluble material . The pure protease cleaves glutamine synthetase near the carboxyl terminus yielding 4,500 and 47,000 Mr products . The characteristics of this enzyme distinguish it from proteases previously purified from E . coli . These characteristics include a molecular weight of 75,000, alkaline pH optimum, lack of inhibition by serine protease inhibitors, and the ability to degrade insulin and casein . Oxidation of glutamine synthetase and other enzymes can be catalyzed by a variety of mixed function oxidase systems from bacterial and mammalian sources . Mixed function oxidation may be a "signal" or "marker" which consigns a protein for proteolytic degradation . Susceptibility to oxidation is subject to metabolic regulation, thereby providing control of proteolytic turnover . Isolation of a protease specific for modified glutamine synthetase provides the enzymatic basis for the specificity of this scheme.

Arch Biochem Biophys, 1987 Feb 15, 253(1), 62 - 72
Inactivation of enzymes and oxidative modification of proteins by stimulated neutrophils; Oliver CN; Differentiated, stimulated HL-60 cells and freshly isolated, stimulated neutrophils inactivate glutamine synthetase (L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2) either inside or outside of Escherichia coli . Stimulated neutrophils also inactivate at least four endogenous enzymes which are inactivated by mixed-function oxidation (MFO) systems in vitro (L . Fucci, C.N . Oliver, M.J . Coon, and E.R . Stadtman (1983) Proc . Natl . Acad . Sci . USA 80, 1521-1525) . The inactivation of glutamine synthetase by stimulated neutrophils exhibits characteristics similar to those previously described using both enzymic and nonenzymic MFO systems (R.L . Levine, C.N . Oliver, R.M . Fulks, and E.R . Stadtman (1981) Proc . Natl . Acad . Sci . USA 78, 2120-2124) . Although the reaction occurs in the absence of Fe(III), it is stimulated by added Fe (III) . Inactivation required molecular oxygen and is partially inhibited by Mn(II), catalase, superoxide dismutase, and metal chelators, ethylenediaminetetraacetic acid and o-phenanthroline . Both the kinetics and the extent of glutamine synthetase inactivation differ when neutrophils are stimulated with phorbol esters compared with formylated peptides . Glutamine synthetase inactivation catalyzed by MFO systems is accompanied by the formation of protein carbonyl derivatives which form stable hydrazones when treated with 2,4-dinitrophenylhydrazine . Multiple carbonyl derivatives are formed in the soluble protein fraction of stimulated neutrophils and these derivatives collectively exhibit an absorbance spectrum similar to that of glutamine synthetase inactivated by liver microsomal cytochrome P-450 MFO system (K . Nakamura, C.N . Oliver, and E.R . Stadtman (1985) Arch . Biochem . Biophys . 240, 319-329).

Arch Biochem Biophys, 1987 Feb 15, 253(1), 73 - 80
Activation and inhibition of the Escherichia coli F1-ATPase by monoclonal antibodies which recognize the epsilon subunit; Dunn SD et al.; The properties of two monoclonal antibodies which recognize the epsilon subunit of Escherichia coli F1-ATPase were studied in detail . The epsilon subunit is a tightly bound but dissociable inhibitor of the ATPase activity of soluble F1-ATPase . Antibody epsilon-1 binds free epsilon with a dissociation constant of 2.4 nM but cannot bind epsilon when it is associated with F1-ATPase . Likewise epsilon cannot associate with F1-ATPase in the presence of high concentrations of epsilon-1 . Thus epsilon-1 activates F1-ATPase which contains the epsilon subunit, and prevents added epsilon from inhibiting the enzyme . Epsilon-1 cannot bind to membrane-bound F1-ATPase . The epsilon-4 antibody binds free epsilon with a dissociation constant of 26 nM . Epsilon-4 can bind to the F1-ATPase complex, but, like epsilon-1, it reverses the inhibition of F1-ATPase by the epsilon subunit . The epsilon subunit remains crosslinkable to both the beta and gamma subunits in the presence of epsilon-4, indicating that it is not grossly displaced from its normal position by the antibody . Presumably the activation arises from more subtle conformational effects . Antibodies epsilon-4 and delta-2, which recognizes the delta subunit, both bind to F1F0 in E . coli membrane vesicles, indicating that these subunits are substantially exposed in the membrane-bound complex . Epsilon-4 inhibits the ATPase activity of the membrane-bound enzyme by about 50%, and Fab prepared from epsilon-4 inhibits by about 40% . This inhibition is not associated with any substantial change in the major apparent Km for ATP . These results suggest that inhibition of membrane-bound F1-ATPase arises from steric effects of the antibody.

Biochem Biophys Res Commun, 1987 Feb 13, 142(3), 893 - 7
The catalytic mechanism of Escherichia coli aspartate carbamoyltransferase: a molecular modelling study; Gouaux JE et al.; Based on molecular modelling study, we propose that the reaction between L-aspartate an carbamoylphosphate, catalyzed by E . coli aspartate carbamoyltransferase, may proceed via a tetrahedral intermediate and that the breakdown of the intermediate is facilitated by an intramolecular proton transfer between the amino group of L-aspartate and a terminal phosphate oxygen of carbamoylphosphate.

Science, 1987 Feb 13, 235(4790), 783 - 7
Leader peptidase of Escherichia coli: critical role of a small domain in membrane assembly; Dalbey RE et al.; Leader peptidase spans the Escherichia coli plasma membrane with its amino-terminal domain facing the cytoplasm and its carboxyl terminus facing the periplasm . It is made without a cleavable leader sequence . The three apolar domains near the amino terminus of the peptidase are candidates for internal "signal sequences" and they anchor the protein to the lipid bilayer . Oligonucleotide-directed deletion was used to show that only the second domain has an essential function in membrane assembly . While this second apolar domain is crucial for membrane assembly, its continued function when disrupted by arginine suggests that its apolar character per se is not its only important feature.

Biochem Biophys Res Commun, 1987 Feb 13, 142(3), 879 - 84
lon-sulA regulatory function affects the efficiency of transposition of Tn5 from lambda b221 cI857 Pam Oam to the chromosome; Sasakawa C et al.; In transposition of Tn5 from lambda b221 cI857 Pam Oam rex::Tn5 to the chromosome, lon mutants of Escherichia coli K12 are less efficient than lon+ parents . Reduced frequency of transposition of Tn5 is recovered to the wild type level by adding DL-pantoyl lactone, an agent which suppresses Lon- phenotypes . A suppressor mutation, sulA (or sfiA), suppresses the deficiency of transposition as well as other known lon-associated phenotypes, whereas a lon sulB (or sfiB) double mutant is phenotypically Lon+ but still transposition-deficient . An additive effect of the sulA genes on teh chromosome and on a high copy number plasmid is found in inhibiting transposition of Tn5 . A hypothesis that the SulA product someway regulates transposition of Tn5 negatively is proposed.

Biochem Biophys Res Commun, 1987 Feb 13, 142(3), 1084 - 8
Identification of the Rts 1 DNA fragment responsible for temperature sensitive growth of host cells harboring a drug resistant factor Rts 1; Okawa N et al.; We have placed a kanamycin resistance SalI fragment (3.65 Kb) from the drug resistance factor Rts1 into pUC19 and pBR322 . These chimeric plasmids containing the kanamycin resistance fragment from Rts1 cause temperature sensitive growth in E coli . The orientation of the kanamycin resistance fragment in the vector plasmids does not influence this phenotype.

Biochim Biophys Acta, 1987 Feb 12, 897(1), 159 - 68
Lipid and temperature dependence of the kinetic and thermodynamic parameters for active amino acid transport in Escherichia coli K1060; Eze MO et al.; The influence of membrane physical state on the kinetic and thermodynamic parameters for the active transport systems for two amino acids has been investigated in Escherichia coli K1060, an unsaturated fatty acid auxotrophic mutant . The apparent Michaelis constant (Km) for the uptake of L-{14C}glutamine (0.05 to 0.08 microM) or L-{14C}proline (1 microM approx.) is invariant with temperature for this mutant grown on elaidate (18:1t), palmitelaidate (16:1t), oleate (18:1c), palmitoleate (16:1c) and linoleate (18:2c,c) . Arrhenius plots of the maximum velocities (Vmax) for L-glutamine transport in cells grown on 16:1t, 18:1c and 16:1c are biphasic within a limited temperature range peculiar to each UFA supplementation . Above an upper temperature limit also displayed by 18:1t and 18:2c,c-cells, Vmax decreases with temperature . A characteristic temperature (Tb) marks the point of intersection of the biphasic slope of the Arrhenius plots, and activation energy (Ea) is lower above than below Tb . Differential thermal analysis considered with membrane lipid fatty acyl profiles indicates that the upper temperature limit is governed by both membrane lipid acyl chain fluidity and heterogeneity, while Tb is governed by fluidity alone . Data on L-proline transport Vmax are similar, but the upper temperature limit and Tb are each shifted to lower temperatures relative to L-glutamine . We suggest that membrane defects related to energy-coupling and caused by abnormal fluidity and physical state are responsible for the peculiar temperature dependences of Vmax for these active transport processes.

Biochim Biophys Acta, 1987 Feb 12, 897(1), 112 - 26
The role of protons in the mechanism of galactoside transport via the lactose permease of Escherichia coli; Page MG; The kinetic mechanism of lactose transport across the cytoplasmic membrane has been investigated and the results related to standard models for the lactose-H+ symport reaction using computer simulation . It is shown that the biphasic kinetics reported for lactose uptake (Kaczorowski, G.J . and Kaback, H.R . (1979) Biochemistry 18, 3691-3697) are consistent with random binding of lactose and protons and rapid subsequent translocation of the ternary lactose-H+-permease complex . Such a model is also shown to explain the observed dependence of the kinetic parameters on the magnitude of the protonmotive force . Both sugar and protons are shown to cause product inhibition of lactose flux and the ability of standard models to account for the pattern of inhibition is discussed . Three apparent dissociation constants have been determined for the protonation reactions in the external medium: two (pKa 6.3 and 9.6) control the activity of the permease, whilst the third (pKa 8.3) controls the affinity of the permease for galactosides . A similar set of dissociation constants has been determined for the internal reactions . Again two (pKa 6 and 9.8) control activity and a third (pKa 8.8) controls the affinity for galactosides . The dissociation reactions characterised by pKa 8.3, 8.8, 9.6 and 9.8 are attributed to the dissociation of the substrate (symported) proton from the binary proton-permease complexes (pKa 8.3 and 8.8) and the ternary proton-galactoside-permease complexes (pKa 9.6 and 9.8) . The third pair (pKa 6.3 and 6.0) must be interpreted as describing a separate protonation reaction which may have a regulatory or auxiliary role in transport.

Nucleic Acids Res, 1987 Feb 11, 15(3), 1281 - 95
The codon Adaptation Index--a measure of directional synonymous codon usage bias, and its potential applications; Sharp PM et al.; A simple, effective measure of synonymous codon usage bias, the Codon Adaptation Index, is detailed . The index uses a reference set of highly expressed genes from a species to assess the relative merits of each codon, and a score for a gene is calculated from the frequency of use of all codons in that gene . The index assesses the extent to which selection has been effective in moulding the pattern of codon usage . In that respect it is useful for predicting the level of expression of a gene, for assessing the adaptation of viral genes to their hosts, and for making comparisons of codon usage in different organisms . The index may also give an approximate indication of the likely success of heterologous gene expression.

J Immunol Methods, 1987 Feb 11, 96(2), 247 - 53
Production of IgG-producing hybridomas by in vitro stimulation of murine spleen cells; Takahashi M et al.; An in vitro stimulation protocol has been established which allows production of IgG-secreting murine hybridomas . This procedure has been examined using jack bean urease and human luteinizing hormone as antigens . Parameters which have been optimized include selection of media and serum supplements, thymocyte-conditioned media, antigen dosage, length of stimulation and the effect of medium changes during stimulation and additions of polyclonal mitogen . Murine spleen cells (1 X 10(8) in 10 ml) were incubated with varying doses of jack bean urease and human luteinizing hormone in a six-well plate in supplemented DMEM with 5% normal rabbit serum and 10% thymocyte-conditioned media . Following 5 and/or 8 days stimulation, the spleen cells were fused with SP2/0 cells and plated in 96-well plates . Stable hybridomas were obtained for both antigens from over 25% of the wells identified in initial screening for specific antibody production . All monoclonal antibodies obtained in the LH stimulation experiments, with one exception, were of the IgM isotype . A large number of IgG-producing hybridomas were isolated following prolonged (8 day) stimulation with high concentrations of urease, during which time the medium remained unchanged . Addition of polyclonal mitogen (E . coli lipopolysaccharide) at 10 micrograms/ml markedly increased the production of hybridomas secreting anti-urease, but most were of IgM class.

Nucleic Acids Res, 1987 Feb 11, 15(3), 905 - 19
The alpha protein ICP0 does not appear to play a major role in the regulation of herpes simplex virus gene expression during infection in tissue culture; Sandri-Goldin RM et al.; The herpes simplex virus type 1 (HSV-1) alpha protein ICP0 trans-activates HSV-1 early genes in transient expression assays . To investigate the function of ICP0 during HSV-1 infection, we have lowered the level of ICP0 by use of a recombinant plasmid that has been engineered to express the antisense message . Cell lines were constructed which stably carry the antisense plasmid . Total protein profiles from infected antisense cell lines showed that the level of ICP0 was reduced to less than 10% of the wild type level in two of the cell lines . However, reducing the level of ICP0 did not have a significant effect on the expression of HSV-1 early or late genes . The polypeptide patterns for the remaining infected cell polypeptides were similar in that no bands were absent although there were some quantitative differences . The level of two early proteins, glycoprotein B and glycoprotein D was reduced in one of the cell lines, however, levels were nearly equivalent to the control infection for two other cell lines tested . Virus yields were the same for the antisense cell lines and for parent cells . Decreased ICP0 levels did not lead to more restrictive phenotypes for an alpha 4 or alpha 27 mutant as protein patterns were similar for these mutants in antisense and parent cells . Therefore, while ICP0 has been demonstrated to be a strong inducer of gene expression in transient expression assays, it does not appear to have a major role as an activator during the productive infection of tissue culture cells.

Nucleic Acids Res, 1987 Feb 11, 15(3), 1185 - 98
On the fidelity of DNA replication: herpes DNA polymerase and its associated exonuclease; Abbotts J et al.; Procaryotic DNA polymerases contain an associated 3'----5' exonuclease activity which provides a proofreading function and contributes substantially to replication fidelity . DNA polymerases of the eucaryotic herpes-type viruses contain similar associated exonuclease activities . We have investigated the fidelity of polymerases purified from wild type herpes simplex virus, as well as from mutator and antimutator strains . On synthetic templates, the herpes enzymes show greater relative exonuclease activities, and greater ability to excise a terminal mismatched base, than procaryotic DNA polymerases which proofread . On a phi X174 natural DNA template, the herpes enzymes are more accurate than purified eucaryotic DNA polymerases; the error rate is similar to E . coli polymerase I . However, conditions which abnegate proofreading by E . coli polymerase I have little effect on the herpes enzymes . We conclude that either these viral polymerases are accurate in the absence of proofreading, or the conditions examined have little effect on proofreading by the herpes DNA polymerases.

Nucleic Acids Res, 1987 Feb 11, 15(3), 1165 - 72
Shufflon: multi-inversion of four contiguous DNA segments of plasmid R64 creates seven different open reading frames; Komano T et al.; The IncI alpha plasmid R64 was found to bear a highly mobile DNA segment which was designated as a clustered inversion region (J . Bacteriol . 165, 94-100, 1986) . The clustered inversion region consists of four DNA segments designated respectively as A, B, C and D which differ in molecular size and restriction sites . The four DNA segments invert independently or in groups resulting in a complex DNA rearrangement . We now show the nucleotide sequence of the clustered inversion region of R64 . The present results suggest that the clustered inversion region is a biological switch to select one of seven open reading frames whose primary structures at the region proximal to N-termini are constant while those at the C-terminal region are variable . A name, "Shufflon" was proposed to call this kind of the clustered inversion region.

Nucleic Acids Res, 1987 Feb 11, 15(3), 1153 - 63
Structure of the gene for the stringent starvation protein of Escherichia coli; Serizawa H et al.; The nucleotide sequence of the gene for the stringent starvation protein (SSP) of E . coli was determined . The deduced amino acid sequences shows that the SSP is composed of 212 amino acid residues, rich in both positively and negatively charged amino acids and has a molecular weight of 24,305 . Primer extension experiments and nuclease S1 mapping analysis showed a site on the chromosome DNA corresponding to the 5' end of the transcript of the SSP gene . However, the consensus promoter sequences were not found at the upstream region . In the 3' flanking region a long coding frame was found immediately following the SSP gene, suggesting that the SSP gene is a member of a multicistronic operon.

Nucleic Acids Res, 1987 Feb 11, 15(3), 1005 - 17
Cloning and characterization of the gene for Escherichia coli seryl-tRNA synthetase; Hartlein M et al.; Seryl-tRNA synthetase is the gene product of the serS locus in Escherichia coli . Its gene has been cloned by complementation of a serS temperature sensitive mutant K28 with an E . coli gene bank DNA . The resulting clones overexpress seryl-tRNA synthetase by a factor greater than 50 and more than 6% of the total cellular protein corresponds to the enzyme . The DNA sequence of the complete coding region and the 5'- and 3' untranslated regions was determined . Protein sequence comparison of SerRS with all available aminoacyl-tRNA synthetase sequences revealed some regions of significant homology particularly with the isoleucyl- and phenylalanyl-tRNA synthetases from E . coli.

Biochim Biophys Acta, 1987 Feb 11, 890(2), 195 - 204
Amino acid substitutions in the epsilon-subunit of the F1F0-ATPase of Escherichia coli; Cox GB et al.; A mutant strain of Escherichia coli was isolated in which Gly-48 of the mature epsilon-subunit of the energy-transducing adenosine triphosphatase was replaced by Asp . This amino acid substitution caused inhibition of ATPase activity (about 70%), loss of ATP-dependent proton translocation and lowered oxidative phosphorylation, but did not affect proton translocation through the F0 . Purified F1-ATPase from the mutant strain bound to stripped membranes with the same affinity as the normal F1-ATPase . Partial revertant strains were isolated in which Pro-47 of the epsilon-subunit was replaced by Ser or Thr . Pro-47 and Gly-48 are predicted to be residues 2 and 3 in a Type II beta-turn and the Gly-48 to Asp substitution is predicted to cause a change from a Type II to a Type I or III beta-turn . Space-filling models of the beta-turn (residues 46-49) in the normal, mutant and partial revertant epsilon-subunits indicate that the peptide oxygen between Pro-47 and Gly-48 is in a different position to the peptide oxygen between Pro-47 and Asp-48 and that the substitution of Pro-47 by either Ser or Thr restores an oxygen close to the original position . It is suggested that the peptide oxygen between Pro-47 and Gly-48 of the epsilon-subunit is involved either structurally in inter-subunit H-bonding or directly in proton movements through the F1-ATPase.

Nucleic Acids Res, 1987 Feb 11, 15(3), 1047 - 61
A monoclonal antibody to triplex DNA binds to eucaryotic chromosomes; Lee JS et al.; A monoclonal antibody (Jel 318) was produced by immunizing mice with poly{d(TmC)}.poly{d(GA)}.poly{d(mCT) which forms a stable triplex at neutral pH . Jel 318 did not bind to calf thymus DNA or other non pyrimidine.purine DNAs such as poly{d(TG)}.poly{d(CA)} . In addition the antibody did not recognize pyrimidine.purine DNAs containing mA (e.g . poly{d(TC)}.poly{d(GmA)}) which cannot form a triplex since the methyl group blocks Hoogsteen base-pairing . The binding of Jel 318 to chromosomes was assessed by immunofluorescent microscopy of mouse myeloma cells which had been fixed in methanol/acetic acid . An antibody specific for duplex DNA (Jel 239) served as a control . The fluorescence due to Jel 318 was much weaker than that of Jel 239 but binding to metaphase chromosomes and interphase nuclei was observed . The staining by Jel 318 was unaffected by addition of E . coli DNA but it was obliterated in the presence of triplex . Since an acid pH favours triplex formation, nuclei were also prepared from mouse melanoma cells by fixation in cold acetone . Again Jel 318 showed weak but consistent staining of the nuclei . Therefore it seems likely that triplexes are an inherent feature of the structure of eucaryotic DNA.

Biochemistry, 1987 Feb 10, 26(3), 964 - 9
Synthesis of dihydrothymidine and thymidine glycol 5'-triphosphates and their ability to serve as substrates for Escherichia coli DNA polymerase I; Ide H et al.; 5,6-Dihydrothymidine 5'-triphosphate (DHdTTP) was synthesized by catalytic hydrogenation of thymidine 5'-triphosphate (dTTP) . Thymidine glycol 5'-triphosphate (dTTP-GLY) was prepared by bromination of dTTP followed by treatment with Ag2O . The modified nucleotides were extensively purified by anion-exchange high-performance liquid chromatography (HPLC) . Alkaline phosphatase digestion of DHdTTP and dTTP-GLY gave the expected products (5,6-dihydrothymidine and cis-thymidine glycol), the identities of which were confirmed by reverse-phase HPLC using authentic markers . HPLC analysis of the alkaline phosphatase digested DHdTTP revealed that DHdTTP was a mixture of C5 diastereoisomers {(5S)- and (5R)-DHdTTP} . Despite the significant distortion of the pyrimidine ring in DHdTTP, it was incorporated in place of dTTP during primer elongation catalyzed by Escherichia coli DNA polymerase I Klenow fragment . The rate of incorporation of DHdTTP was about 10-25-fold lower than that of dTTP . On the other hand, dTTP-GLY, which also has a distorted pyrimidine ring, did not replace dTTP, and no elongation of the primer was observed . In order to study the preference of incorporation of the diastereoisomers of DHdTTP into DNA, salmon testes DNA, activated by exonuclease III, was used as a template for DNA polymerase I Klenow fragment in the presence of {3H}DHdTTP (S and R mixture) and normal nucleotides . After enzymatic digestion of the DNA to nucleosides, the products were analyzed by HPLC . The ratio of the isomers incorporated into DNA (S:R = 73.27) was virtually the same as that of the {3H}DHdTTP substrates (S:R = 79.21).(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1987 Feb 10, 26(3), 943 - 50
Kinetic investigation of the DNA platination reaction: evidence for a transient adduct between deoxyribonucleic acid and cis-platinum(II); Schaller W et al.; Kinetics of the synthesis of adducts between salmon testis DNA and platinum(II) compounds were measured by their effects on DNA synthesis, circular dichroism, and ethidium bromide dependent fluorescence . Transient incorporation of {14C}cyanide into DNA adducts of of cis-diammineaquochloroplatinum(II) and respectively cis-diamminediaquoplatinum(II) compounds but not of trans-diammineaquochlorplatinum(II) was observed . A minimal kinetic scheme is derived, in which a transient monodentate DNA-platinum(II) adduct is formed in a bimolecular reaction between DNA and aquated platinum(II) compounds . Second-order rate constants are 2000-3000 M-1 min-1 for cis-diamminediaquoplatinum(II) and 280-400 M-1 min-1 for cis- and trans-diammineaquochloroplatinum(II), respectively . The dependence of pseudo-first-order rate constants is not linear for high concentrations of DNA, suggesting competitive formation of more than one primary adduct . The monodentate adducts inhibit DNA polymerase catalyzed DNA synthesis . The biomolecular reaction is followed by a rearrangement (rate constant 0.22 min-1) that gives rise to most of the decrease in the fluorescence intensity and that depends on the state of aquation of the DNA-bound platinum(II) complex . By exchange of coordinated water with a second nucleotide, the monodentate adduct can form cross-links in a reaction joining the rearrangement . Adducts containing a chloro group liberate it by hydrolysis prior to cross-linking . In the case of the trans-platinum(II) adduct, the hydrolysis is aided by the trans effect of the bound first nucleotide.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1987 Feb 10, 26(3), 863 - 71
Spectroscopic characterization of thioredoxin covalently modified with monofunctional organoarsenical reagents; Brown SB et al.; Thioredoxin upon reduction with mercaptoethylamine was subjected to covalent modification by the monofunctional organoarsenical reagents H2NPhAsO and HO(CH2)4AsCl2 . The degree of modification was monitored by the percentage loss in free thiol content as measured by the reaction with the thiol reagent 5,5'-dithiobis(2-nitrobenzoic acid) . The modification results in the formation of a stable 15-membered cyclic dithioarsenite ring that readily extrudes the arsenic moiety upon the addition of 2,3-dimercaptopropanol . The conformational effects of this modification were monitored by steady-state fluorescence and circular dichroism . On the basis of circular dichroic spectra, it appeared that the protein experiences no significant backbone conformational change from this modification . The degree of conformational change was found to be within the range observed upon reduction of the oxidized thioredoxin . Steady-state fluorescence revealed that the arsenicals caused strong quenching of the tryptophan fluorescence . Stern-Volmer titrations revealed that the quenching was a function of both the nature of the organic group and its covalent attachment to the "spatially close" thiols . The analysis of the spectroscopic results obtained with the arsenical reagents provides further insight into the nature of the conformational change that has been observed upon reduction of thioredoxin.

Biochemistry, 1987 Feb 10, 26(3), 854 - 62
Dynamics of fd coat protein in lipid bilayers; Leo GC et al.; The dynamics of backbone and side-chain sites of the membrane-bound form of fd coat protein are described with solid-state 2H and 15N NMR experiments . The samples were isotopically labeled coat protein in phospholipid bilayers in excess water . The protein itself is immobile and does not undergo rapid rotation within the bilayer . Like the structural form of the protein, the membrane-bound form has four mobile residues at the N-terminus . The membrane-bound form differs from the structural form in having several mobile residues at the C-terminus . Many of the side chains of residues with immobile backbone sites undergo large amplitude jump motions . The dynamics are generally similar in both the structural and membrane-bound forms of the protein.

Biochemistry, 1987 Feb 10, 26(3), 950 - 6
Specific activation of transcription initiation by the sequence-specific DNA-binding agents distamycin A and netropsin; Bruzik JP et al.; A series of promoters with nine base-pair substitutions in the spacer DNA separating the -10 and -35 regions was used to demonstrate that Escherichia coli RNA polymerase is sensitive to events affecting the spacer DNA--a region not directly contacted by the enzyme . The drugs distamycin A and netropsin specifically enhanced the rate of functional complex formation at a promoter bearing a substitution of nonalternating A-T base pairs . The effect is exerted at an early step in the RNA polymerase-promoter interaction . We hypothesize that a drug-induced structural alteration in the spacer DNA occurs, similar to that normally resulting from RNA polymerase binding . These findings are relevant to an understanding of potential mechanisms of transcription activation.

Biochemistry, 1987 Feb 10, 26(3), 897 - 904
Dimerization of the operator binding domain of phage lambda repressor; Weiss MA et al.; Dimerization of lambda repressor is required for its binding to operator DNA . As part of a continuing study of the structural basis of the coupling between dimer formation and operator binding, we have undertaken 1H NMR and gel filtration studies of the dimerization of the N-terminal domain of lambda repressor . Five protein fragments have been studied: three are wild-type fragments of different length (1-102, 1-92, and 1-90), and two are fragments bearing single amino acid substitutions in residues involved in the dimer interface (1-102, Tyr-88----Cys; 1-92, Ile-84----Ser) . The tertiary structure of each species is essentially the same, as monitored by the 1H NMR resonances of internal aromatic groups . However, significant differences are observed in their dimerization properties . 1H NMR resonances of aromatic residues that are involved in the dimer contact allow the monomer-dimer equilibrium to be monitored in solution . The structure of the wild-type dimer contact appears to be similar to that deduced from X-ray crystallography and involves the hydrophobic packing of symmetry-related helices (helix 5) from each monomer . Removal of two contact residues, Val-91 and Ser-92, by limited proteolysis disrupts this interaction and also prevents crystallization . The Ile-84----Ser substitution also disrupts this interaction, which accounts for the severely reduced operator affinity of this mutant protein.

Biochemistry, 1987 Feb 10, 26(3), 890 - 7
1H NMR aromatic spectrum of the operator binding domain of the lambda repressor: resonance assignment with application to structure and dynamics; Weiss MA et al.; The aromatic 1H NMR resonances of the operator binding domain of lambda repressor are completely assigned . Since the resonances of this 23-kilodalton domain are too broad for the application of two-dimensional strategies for sequence-specific assignment, an alternative approach has been used . Assignments are obtained by a combination of one- and two-dimensional NMR methods, by the study of genetically altered domains, and by the biosynthetic incorporation of deuterium labels . The resulting assignments provide sensitive markers for tertiary and quaternary structure . Nuclear Overhauser enhancements demonstrate that the major features of the crystal structure, including the dimer contacts, are retained in solution . The rates of aromatic ring rotation indicate that the globular domain is not rigid; significant barriers to ring rotation are observed only in the dimer contact.

FEBS Lett, 1987 Feb 9, 212(1), 21 - 7
RNA polymerase molecules initiating transcription at tandem promoters can collide and cause premature transcription termination; Ponnambalam S et al.; Using purified E . coli RNA polymerase we have studied the transcription in vitro of a series of DNA fragments carrying two tandemly arranged promoters, where the corresponding transcription start points were separated by 263, 138, 83 and 78 base pairs . In the case where the transcription start points are 83 base pairs apart, there is an interaction between RNA polymerase molecules transcribing from the two promoters . This interaction results in premature termination of the upstream transcript at a precise site . We propose that this is the result of RNA polymerase transcribing from the upstream promoter bumping into polymerase at the downstream promoter . The interaction between the two polymerase molecules is crucially dependent on the distance between the two promoters.

J Biol Chem, 1987 Feb 5, 262(4), 1855 - 9
Promoter selectivity of Escherichia coli RNA polymerase . Purification and properties of holoenzyme containing the heat-shock sigma subunit; Fujita N et al.; An RNA polymerase holoenzyme containing sigma 32, the heat-shock sigma subunit, has been purified from heat-shocked Escherichia coli cells, and its functional properties including the promoter selectivity have been analyzed using the in vitro mixed transcription system . The holoenzyme correctly recognized a heat-shock promoter for the groE gene and efficiently initiated transcription at the same site as that found in vivo . The enzyme was, however, unable to recognize the promoters which are usually transcribed by the regular holoenzyme (E sigma 70), such as tufB, nusA, supP, lacUV5, araS, rpsA, recA, rplJ, dnaQ, rnh, and trp promoters . On the other hand, the regular holoenzyme did not recognize the groE promoter . These observations altogether indicate that strict difference exists in the promoter selectivity between two molecular species of the RNA polymerase holoenzyme . The groE transcription in vitro was not affected significantly within the temperature range from 32 to 42 degrees C . The two sigma subunits could be replaced in vitro on the same core enzyme, supporting the view that the spectrum of gene expression in E . coli is under a dynamic control by intracellular levels of individual sigma subunits.

J Biol Chem, 1987 Feb 5, 262(4), 1725 - 33
Stacking interactions of tryptophan residues and nucleotide bases in complexes formed between Escherichia coli single-stranded DNA binding protein and heavy atom-modified poly(uridylic) acid . A study by optically detected magnetic resonance spectroscopy; Khamis MI et al.; The complexes formed between Escherichia coli single-stranded DNA binding protein (SSBP) and the heavy atom-modified single-stranded polynucleotides poly(5-BrU) and poly(5-HgU) are investigated using optically detected magnetic resonance (ODMR) methods . In these complexes the triplet state properties of the tryptophan residues are subjected to the external heavy atom effect generated by bromine and mercury atoms and are characterized by a shortened triplet state lifetime and the appearance of the otherwise dark {D} + {E} slow passage ODMR signal . These features provide direct evidence for close range interactions between tryptophan residue(s) and the nucleotide bases in the complexes . The extent of the triplet state lifetime reduction in the case of the SSBP-poly(5-HgU) complex together with steric considerations of the complex structure is consistent only with a van der Waals contact between the perturbed molecule and the heavy atom perturber by means of a stacking interaction . Fast passage ODMR measurements show a lifetime for a sublevel of the perturbed tryptophan chromophore(s) in this complex on the order of 1 ms . The amplitude-modulated phosphorescence microwave double resonance technique captures selectively the broadened and red-shifted phosphorescence spectrum of the heavy atom-perturbed tryptophan residue(s) . This work supports a model for the binding of SSBP to single-stranded polynucleotides in which the bases are inserted into hydrophobic regions of the protein, where they are likely to undergo stacking interactions with the indole moiety of buried tryptophan residues.

J Biol Chem, 1987 Feb 5, 262(4), 1720 - 4
The beta subunit dissociates readily from the Escherichia coli DNA polymerase III holoenzyme; Lasken RS et al.; Purified DNA polymerase III holoenzyme (holoenzyme) was separated by glycerol gradient sedimentation into the beta subunit and the subassembly that lacks it (pol III) . In the presence of ATP, beta subunit dimer dissociated from holoenzyme with a KD of 1 nM; in the absence of ATP, the KD was greater than 5 nM . The beta subunit was known to remain tightly associated in the holoenzyme upon formation of an initiation complex with a primed template and during the course of replication . With separation from the template, holoenzyme dissociated into beta and pol III . Cycling to a new template depended on the reformation of holoenzyme . Holoenzyme was in equilibrium with pol III and the beta subunit in crude enzyme fractions as well as in pure preparations.

J Biol Chem, 1987 Feb 5, 262(4), 1674 - 9
RNA polymerase . Limit cognate primer for initiation and stable ternary complex formation; Ruetsch N et al.; Various lengths of oligoribonucleotides corresponding to regions flanking the initiation site of the A1 promoter of the T7 delta D111 template were examined in order to determine their ability to function as primers of transcription for the DNA-dependent RNA polymerase from Escherichia coli . The oligoribonucleotides which functioned as primers were also examined as precursors for the formation of a stable ternary complex (enzyme X DNA template X oligoribonucleotide product) by systematically extending each primer with one or more specific cognate substrate nucleotide triphosphates . A stable ternary complex (resistant to a salt jump challenge) was formed whenever the primer oligoribonucleotide and augmenting nucleotide triphosphate(s) allowed the formation of the normal third phosphodiester bond of the transcript to occur on the enzyme surface . An oligoribonucleotide (a cognate having the correct base-pairing substituents) containing the preformed third phosphodiester bond does not function as a primer . For example the cognate oligoribonucleotide corresponding to the region flanking the A1 promoter of the T7 delta D111 is: formula; see text The limit primer is the oligoribonucleotide trimer AUC (+1...+3) . Other acceptable primers are constructed by adding to this limit primer, cognate bases in the negative registry . The oligonucleotide containing one base added to this limit primer in the positive registry (e.g . AUCG) is completely inactive as a primer . We have also demonstrated these phenomena for the A2 and the A3 promoter of the T7 template.

J Biol Chem, 1987 Feb 5, 262(4), 1427 - 9
Protein translocation into Escherichia coli membrane vesicles is inhibited by functional synthetic signal peptides; Chen L et al.; A synthetic peptide corresponding to the signal sequence of wild type Escherichia coli lambda-receptor protein (LamB) inhibits in vitro translocation of precursors of both alkaline phosphatase and outer membrane protein A into E . coli membrane vesicles (half-maximal inhibition at 1-2 microM) . By contrast, the inhibitory effect was nearly absent in a synthetic peptide corresponding to the signal sequence from a mutant strain that harbors a deletion mutation in the LamB signal region and displays an export-defective phenotype for this protein in vivo . Two peptides derived from pseudorevertant strains that arose from the deletion mutant and exported LamB in vivo were found to inhibit in vitro translocation with effectiveness that correlated with their in vivo export ability . Controls indicated that these synthetic signal peptides did not disrupt the E . coli membrane vesicles . These results can be interpreted to indicate that the presequences of exported proteins interact specifically with a receptor either in the E . coli inner membrane or in the cytoplasmic fraction . However, biophysical data for the family of signal peptides studied here reveal that they will spontaneously insert into a lipid membrane at concentrations comparable to those that cause inhibition . Hence, an indirect effect mediated by the lipid bilayer of the membrane must be considered.

J Mol Biol, 1987 Feb 5, 193(3), 579 - 84
Sequence comparison of single-stranded DNA binding proteins and its structural implications; Prasad BV et al.; The primary sequences were compared among several proteins: gene product 5 protein (GP5) from phage M13; PIKE from phage Ike; gene product 32 protein (GP32) from phage T4; RecA, SSB and SSF from Escherichia coli . These proteins bind strongly and cooperatively to single-stranded DNA with no sequence specificity . GP5 is the smallest in this group and its three-dimensional structure is well-characterized . Using the entire sequence of GP5 as a template we searched for the regions in other single-stranded DNA binding proteins yielding the best alignment of aromatic and basic residues . The identified domains show alignment of five aromatic and four charged residues in these proteins . The domains in PIKE, GP32 and RecA exhibit statistically significant sequence homology with GP5 . These observations strongly favor the hypothesis that the protein-single-stranded DNA complex in this class of proteins is stabilized by the stacking interaction of the aromatic residues with the bases of the DNA, and by the electrostatic interaction of the basic residues with the phosphate groups of the DNA . We also find that the DNA binding domains of these proteins have similar secondary structural preferences, mainly beta structures . The triple-stranded beta-sheet may be a common motif in the DNA binding domains of these proteins.

J Mol Biol, 1987 Feb 5, 193(3), 447 - 64
Searching for potential Z-DNA in genomic Escherichia coli DNA; Hoheisel JD et al.; The Clarke-Carbon library with Escherichia coli DNA cloned into plasmid ColE1 was partially screened for Z-DNA with the monoclonal antibody Z-D11 using the retardation of the covalently closed circular DNA-protein complex by nitrocellulose filters . About 85% of the plasmids tested at "natural" supercoil density bound to the filter . Together with binding studies of the iodinated antibody, one Z-DNA segment per about 18,000 base-pairs of E . coli DNA is observed . One clone containing the region around the lactose operon, pLC20-30, was studied in detail . Subcloning a partial Sau3A digest and selection with antibodies gave three different Z-forming sites . They were mapped to within about +/- 20 base-pairs by preparing unidirectional deletion clones, selection of protein binding plasmids on nitrocellulose filters and subsequent sizing on agarose gels . The size of the Z-DNA-forming segments was estimated from two-dimensional gels of topoisomer mixtures . Together with results from sequencing of the plasmid DNA using exonuclease III to create single-stranded templates, stretches of alternating purine-pyrimidine tracts of 12 to 15 base-pairs were found to be responsible for Z-DNA formation . One of the sites was found in the middle of the lacZ gene, where it might be an obstacle for RNA polymerase . The methods used here should also be helpful for studying other DNA-protein sites, especially if they exist only in supercoiled DNA.

J Biol Chem, 1987 Feb 5, 262(4), 1698 - 705
Inhibition of phospholipase A2 by "lipocortins" and calpactins . An effect of binding to substrate phospholipids; Davidson FF et al.; The "lipocortins" are a group of proteins that have been reported to inhibit phospholipase A2 by direct interaction with enzyme . Two proteins which have been identified as lipocortin on the basis of inhibition of phospholipase A2 activity have recently been cloned and sequenced . These have been shown to be identical to the calpactins, which are membrane cytoskeletal proteins serving as major substrates of the tyrosine protein kinases . We have now found that two forms of calpactin (I and II) inhibit porcine pancreatic phospholipase A2 in an assay using Escherichia coli cells or extracted phospholipid vesicles as substrate, but only when the substrate concentration is very low . Both calpactins, as well as another, 73-kDa inhibitory protein, were found to bind purified phospholipids and E . coli cell membranes directly . Kinetic studies show that the inhibition of phospholipase A2 by calpactin can be overcome by high phospholipid substrate concentrations, whether E . coli cells or isolated phospholipid vesicles are used . For example, in the presence of 5 X 10(-10) M phospholipase A2 and 1 X 10(-7) M calpactin, the inhibition decreases from 100 to 0% as phospholipid in vesicles is raised from 2 to 8 microM . The evidence reported here strongly suggests that in vitro inhibition of phospholipase A2 by lipocortin is due to sequestering of the phospholipid substrate by the inhibitor protein, rather than a direct interaction with the phospholipase . These results raise questions about the physiological significance of the inhibition of phospholipases by calpactins.

Invest Ophthalmol Vis Sci, 1987 Feb, 28(2), 320 - 9
Intercellular junctions of the ciliary epithelium in anterior uveitis; Freddo TF; The intercellular junctions of the anterior ciliary and iridial epithelia of the inflamed rabbit eye were examined by use of an ultrastructural tracer, conventional electron microscopy, and the freeze-fracture technique . In normal control eyes, intravascularly injected horseradish peroxidase was prevented from entering the posterior chamber by the zonulae occludentes of the nonpigmented ciliary epithelium . In freeze-fracture studies these junctions appeared as a series of 5-12 branching and anastomosing strands on the P-fracture face, which were complemented by a network of shallow grooves with discontinuous rows of particles at their bases on the E-fracture face . Gap junctions were abundant, particularly between the apical surfaces of the pigmented and nonpigmented layers where they were accompanied by discontinuous tight junctional strands . In eyes inflamed by intravitreal injection of E . coli 055:B55 endotoxin, peroxidase leaked into the posterior chamber primarily from the crests of anterior ciliary and iridial processes . Freeze-fracture electron microscopy of these same areas demonstrated primarily a simplification in junctional complexity and reduction in the number of occluding strands . Severe junctional disorganization and complete junctional fragmentation were rarely seen . A profound reduction in the complement of gap junctions was observed particularly between the apical surfaces of the pigmented and nonpigmented layers . The possible functional significance of the observed changes is discussed.

Am J Clin Pathol, 1987 Feb, 87(2), 267 - 70
A new solid phase passive hemagglutination test for IgM antibody to hepatitis B core antigen; Petchclai B et al.; There is a need for a simple, sensitive, specific, and inexpensive test for immunoglobulin M antibody to hepatitis B core antigen (anti-HBc IgM) . A solid phase passive hemagglutination test (SP-PHA) was developed for this purpose and compared with the enzyme-linked immunosorbent assay (ELISA) test . Hepatitis B core antigen (HBcAg) used in PHA and SP-PHA was synthesized in Escherichia coli . Human IgM was captured to a microtiter plate coated with anti-human IgM, and the presence of anti-HBc IgM was demonstrated by the adherence of HBcAg-sensitized erythrocytes to the bottom of a U-shaped microtiter plate . ELISA and SP-PHA were made at 1:100 and 1:1,000 serum dilution, respectively . Both were positive in 100% of 36 cases of acute hepatitis B, 68.18% of 22 cases of chronic hepatitis B, and 20% of 75 healthy carriers of hepatitis B surface antigen (HBsAg) but none in 65 anti-HBc-positive blood donors that had negative results for HBsAg . Results of both tests were identical but were false positive because rheumatoid factor was found only in ELISA . End-point titration by SP-PHA and PHA was also found useful for the differentiation of acute hepatitis B from chronic hepatitis B and HBsAg carriers.

Virology, 1987 Feb, 156(2), 396 - 403
Assembly of influenza ribonucleoprotein in vitro using recombinant nucleoprotein; Kingsbury DW et al.; The influenza A virus nucleoprotein previously expressed in Escherichia coli after fusion to 32 heterologous amino acids has now been purified and tested for its ability to form complexes with RNA in vitro . By using a simple filter binding assay, we show that ribonucleoprotein (RNP) complexes form readily with single-stranded RNA of viral or nonviral origin but not with double-stranded RNA . The RNP complexes formed were similar to authentic influenza virus RNPs in appearance under the electron microscope, in buoyant density in gradients of cesium chloride, and in sensitivities to pancreatic ribonuclease, to chaotropic reagents, and to high salt . We conclude that nucleoprotein synthesized in E . coli has all the properties required for correct assembly into ribonucleoprotein.

Virology, 1987 Feb, 156(2), 197 - 203
The c4 gene of phage P1; Baumstark BR et al.; The c4 gene of phage P1 has been localized to 335 bp of the P1EcoRI-9 fragment, within 50 bp of the EcoRI-9/14 junction . DNA sequence analysis of this fragment reveals a single open reading frame of 66 amino acids . The location of two c4 mutations, both of which produce changes in the predicted amino acid sequence in this reading frame, suggests that the reading frame codes for the c4 repressor . A region with high homology to the E . coli promoter consensus sequence is located approximately 50 bp upstream from the reading frame . Deletion of this potential promoter region abolishes expression of c4, as indicated by the loss of complementation of c4 mutants for lysogeny . Complementation is restored by the introduction of a heterologous promoter (the T7 phi 10 promoter), indicating that c4 expression is absolutely dependent on transcription of the 66-amino acid reading frame.

Environ Res, 1987 Feb, 42(1), 149 - 54
Amoebae isolated from the atmosphere of Mexico City and environs; Rivera F et al.; A protozoological analysis was performed from June to August, 1982 to isolate small free-living amoebae from the atmosphere by using an air vacuum sampler and several filters . Monoxenic medium (nonnutritive agar plus Escherichia coli) and axenic medium (De Jonckheere, 1977) were used to culture the isolates . The species isolated included Naegleria sp . Alexeieff emend . Calkins, Acanthamoeba polyphaga Puschkarew, Vahlkampfia jugosa Page, Acanthamoeba astronyxis Ray and Hayes, Acanthamoeba castellanii Douglas, Acanthamoeba culbertsoni Singh y Das, Vahlkampfia ustiana Page, Saccamoeba stagnicola Page, Hyalodiscus sp . Hertwig and Lesser, Platyamoeba placida Page, Rugipes sp . Schaeffer, Vannella platypodia Glaser, Vahlkampfia russelli Singh, Vahlkampfia ovis Schmidt, and Leptomyxa flabellata Goodey . Among the species isolated Naegleria sp., A . polyphaga, A . culbertsoni, and A . castellanii include strains, known to be pathogenic in humans.

J Hepatol, 1987 Feb, 4(1), 22 - 8
Detection of antibodies against pre-S1 proteins in sera of patients with hepatitis B virus (HBV) infection; Theilmann L et al.; Pre-S1 (large S) proteins are components of the envelope of HBV . The presence of pre-S1 proteins is correlated with viral replication . To test sera of patients with HBV infection for the presence of antibodies against pre-S1 proteins (anti-pre-S1), an E . coli extract containing a pre-S fusion protein covering the greater part of the pre-S1 region was subjected to Western blotting and probed with sera of patients . Anti-pre-S1 was present in the sera of all 4 patients with acute self-limited HBV infection and in the sera of 3 patients with a fulminant course . The antibodies could not be detected in the sera of 6 patients with acute HBV infection entering chronicity, in the sera from 10 HBsAg carriers or in the sera of 25 patients with chronic liver disease, positive for HBsAg and antibodies to hepatitis Delta virus . In an additional study, anti-pre-S1 could not be found in 11 sera from 12 patients with previous HBV infection, positive for anti-HBs and anti-HBc . Antibodies to pre-S1 proteins appear at the early stage of acute resolving HBV infection and seem to play a role in the elimination of the virus . The antibodies are absent in the sera of patients with acute HBV infection entering a chronic course and in the sera of chronic HBsAg-carriers.

Eur J Cell Biol, 1987 Feb, 43(1), 128 - 33
Flow-cytometric characterization of stimulation, free radical formation, peroxidase activity and phagocytosis of human granulocytes with 2,7-dichlorofluorescein (DCF); Burow S et al.; A standardized four-step assay for the flow cytometric determination of the oxidative activity of human polymorphonuclear leukocytes (PMNL) from normal human individuals and from septic patients was developed, using 2,7-dichlorofluorescin-diacetate (DCFH-DA) as indicator for the intracellular formation of H2O2 and free radicals . Spontaneous H2O2 and free radical formation was measured by preincubation of buffy coat PMNLs from fresh peripheral venous blood at 37 degrees C and pH 7.4 with 10 microM DCFH-DA . Intracellular peroxidase activity was determined by addition of 1 mM external H2O2 to this assay . A maximum of granulocyte oxidative burst activity was elicited by the addition of 150 nM phorbol-myristate-acetate (PMA) . A physiological burst was generated by incubating buffy coat PMNLs together with E . coli bacteria . The DNA of dead cells was in all instances simultaneously counterstained with propidium iodide (PI) . Quiescent or H2O2 or bacteria treated granulocytes moved as a single cell cluster to higher fluorescences . Stimulation with PMA, in contrast, generated always a bimodal distribution of granulocyte fluorescence with the high activity cell cluster being approximately sevenfold more active than the low activity cell cluster . Roughly half of the granulocytes in normal individuals had high fluorescence . An increase of the high activity granulocytes was observed in septic patients . Model experiments with the nonfluorescent DCFH-DA cleavage product DCFH (2,7-dichlorofluorescin) showed that DCFH was quickly photo-oxidized to fluorescent DCF (2,7-dichlorofluorescein) by UV-light and to a lower degree by daylight . DCFH even slowly autooxidized in the dark.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem Med Metab Biol, 1987 Feb, 37(1), 73 - 80
Liver glycogen metabolism in endotoxin shock . II . Endotoxin administration increases glycogen phosphorylase activities in dog livers; Liu MS et al.; The effects of E . coli endotoxin administration on hepatic glycogen phosphorylase activities in dogs were investigated . Hepatic glycogen phosphorylase activities in both control and endotoxic dogs were inactivated spontaneously by preincubation of enzyme preparations at 25 degrees C . Total glycogen phosphorylase activity was not significantly altered during preincubation . The activity of glycogen phosphorylase a was increased by 83 and 80% at 1 and 2 hr postendotoxin, respectively, without preincubation; and by 203 and 133% at 1 and 2 hr postendotoxin, respectively, after 30 min preincubation . Without preincubation, the glycogen phosphorylase percentage a activity was increased from the control value of 37 to 58% at 1 hr postendotoxin and to 53% at 2 hr postendotoxin . After 30 min preincubation, the glycogen phosphorylase percentage a activity was increased from the control value of 10 to 28% at 1 hr postendotoxin and to 20% at 2 hr postendotoxin . The time required for half maximum inactivation of percentage a activity was 16.5, 33, and 24 min for control, 1 and 2 hr postendotoxin, respectively . Although the Vmax and Km for glucose-1-P for total glycogen phosphorylase were not affected by endotoxin administration, the Vmax for glucose-1-P for glycogen phosphorylase a was increased by 57.3 and 42.7% at 1 and 2 hr postendotoxin, respectively, with no change in the Km values . Glucose inhibited glycogen phosphorylase a activity both in control and endotoxin-injected dogs, but the I50 value was increased by 35% in endotoxin-injected (2 hr) dogs . AMP activated glycogen phosphorylase b activity both in control and endotoxin-injected dogs with no change in A0.5 values between the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Biotechnol Appl Biochem, 1987 Feb, 9(1), 39 - 52
S-adenosylmethionine: studies on chemical and enzymatic synthesis; Matos JR et al.; Several methods for the chemical and enzymatic synthesis of (-)-S-adenosylmethionine (AdoMet) are described and compared . Studies on the effects of solvents, pH, methylating reagents, and KI on the coupling of sodium homocysteine thiolate and 5'-chloro-5'-deoxyadenosine led to an improved procedure for the synthesis of (+/-)-AdoMet . The use of trimethylsulfonium iodide as a methylating agent under acidic conditions results in a higher content of the desired (-)-epimer than does the use of CH3I . The enzymatic synthesis of (-)-AdoMet using AdoMet synthetase from an over-producing strain of Escherichia coli is demonstrated and the effect of product inhibition on preparative-scale synthesis is illustrated . A new HPLC technique for separation of the epimeric mixture of AdoMet, which allows small-scale preparation of optically pure AdoMet from the enzyme product, has been developed . With this HPLC technique, evidence that (-)-AdoMet is the sole epimer formed by the enzyme has been shown.

Vet Immunol Immunopathol, 1987 Feb, 14(2), 157 - 71
Influence of the Hal locus and standardized stress on antibody response and in vitro reactivity of peripheral blood lymphocytes in pigs; Edfors-Lilja I et al.; The antibody response to two Escherichia coli antigens (O149 and K88) and the in vitro reactivity of peripheral blood lymphocytes (PBLs) to pokeweed mitogen was studied in growing pigs after exposure to varying degrees of stress . Three experimental groups were used; transportation by lorry, transportation by lorry after a previous injection of a tranquillizing drug (amperozide), no transportation but an amperozide injection . Another group was used as a control group . This group was not transported and received no amperozide injection . The animals were the offspring of boars and sows heterozygous (Nn) for the Hal gene, and all 3 Hal genotypes (NN, Nn, nn) were identified and could thus be compared within litters . Immediately after the experimental treatment, the highest PBL reactivity was found for the amperozide-treated animals and for the non-transported animals, with no differences in reactivity between Hal genotypes . Two weeks later, the treatments caused different effects on pigs of the 3 Hal genotypes, both with regard to the PBL reactivity and the IgG response to O149 . The NN pigs had a higher PBL reactivity than the nn pigs for all treatments except the 'drug + transport' class where the reverse rank order was found . The NN pigs also had a higher IgG response to O149 than the nn pigs in the 'drug + no transport' class . The amperozide treatment was followed by a higher PBL reactivity in non-transported NN pigs and in transported nn pigs . The amperozide-treated non-transported NN pigs also had a higher IgG response to O149 . The highest PBL reactivity and IgG response to O149 were found mainly in pigs with the lowest cortisol levels . Pronounced differences between litters were found for both the antibody response and the PBL reactivity.

Vet Microbiol, 1987 Feb, 13(2), 167 - 78
Heat-stable (STa) enterotoxin of enterotoxigenic Escherichia coli: binding of the enterotoxin to coagulated milk and casein; Sriranganathan N et al.; Oral or intragastric inoculation of the STa enterotoxin of Escherichia coli has been the standard laboratory test for that toxin . We demonstrated that the severity of the secretory response of 2-4-day-old mice to a single dose of the toxin was influenced by weaning of these mice . After oral application of 50-250 fmol of purified, radioiodinated STa toxin (12-13 Ci mmol-1) approximately 55-65% of the administered toxin bound to the stomach contents . Binding of the toxin did not destroy its biological activity . The binding was pH dependent; stomach contents bound 75% of the toxin at pH 2.3 and only 7.4% at pH 10.1 . The principle in the stomach contents which bound the toxin was also demonstrated in murine and bovine milk . Further studies with renin-coagulated milk and commercial casein indicated that milk casein bound the toxin . Based on these findings, we speculate that in cases of food-poisoning involving casein-containing foods STa toxin may be present in the absence of cultivable enterotoxigenic E . coli.

EMBO J, 1987 Feb, 6(2), 515 - 22
Control of replication of plasmid R1: translation of the 7k reading frame in the RepA mRNA leader region counteracts the interaction between CopA RNA and CopT RNA; Wagner EG et al.; Replication of IncFII plasmids is regulated through the expression of a gene, repA . The RepA protein is rate-limiting for initiation of replication . The main negative control is exerted by a countertranscript, CopA RNA, that binds to the complementary region of the RepA mRNA, thereby inhibiting the formation of the RepA protein . The target region for CopA RNA, CopT, is located upstream of the RepA coding region . An open reading frame for a putative 7k protein overlaps the CopT sequence . Here we show by using lacZ fusions that the 7k gene is expressed . We constructed a translation start mutation in order to abolish formation of the 7k protein . This resulted in a 10-fold decrease in repA expression . The 7k protein produced in trans did not reverse this effect, so the 7k protein per se does not control expression of repA . However, translation of the 7k coding sequence must influence CopA/CopT RNA recognition, since ribosomes will transiently disrupt the target hairpin . We propose here a novel mechanism that affects the level of gene expression: the 7k region of the RepA mRNA is a leader sequence that is involved in expression of the downstream gene; translation of the 7k region competes with a negative control system involving RNA-RNA interaction.

EMBO J, 1987 Feb, 6(2), 477 - 83
Enzymatic conversion of guanosine 3' adjacent to the anticodon of yeast tRNAPhe to N1-methylguanosine and the wye nucleoside: dependence on the anticodon sequence; Droogmans L et al.; N1-Methylguanosine (m1G) or wye nucleoside (Y) are found 3' adjacent to the anticodon (position 37) of eukaryotic tRNAPhe . The biosynthesis of these two modified nucleosides has been investigated . The importance of the type of nucleosides in the anticodon of yeast tRNAPhe on the potentiality of this tRNA to be a substrate for the corresponding maturation enzyme has also been studied . This involved microinjection into Xenopus laevis oocytes and incubation in a yeast extract of restructured yeast tRNAPhe in which the anticodon GmAA and the 3' adjacent Y nucleoside were substituted by various tetranucleotides ending with a guanosine . The results obtained by oocyte microinjection indicate: that all the restructured yeast tRNAsPhe are efficient substrates for the tRNA (guanosine-37 N1)methyltransferase . This means that the anticodon sequence is not critical for the tRNA recognition by this enzyme; in contrast, for Y nucleoside biosynthesis, the anticodon sequence GAA is an absolute requirement; the conversion of G-37 into Y-37 nucleoside is a multienzymatic process in which m1G-37 is the first obligatory intermediate; all the corresponding enzymes are cytoplasmic . In a crude yeast extract, restructured yeast tRNAPhe with G-37 is efficiently modified only into m1G-37; the corresponding enzyme is a S-adenosyl-L-methionine-dependent tRNA methyltransferase . The pure Escherichia coli tRNA (guanosine-37 N1) methyltransferase is unable to modify the guanosine-37 of yeast tRNAPhe.

Bioorg Khim, 1987 Feb, 13(2), 259 - 62
{Antiviral activity of mutant interferons . A new approach to the study of structural-functional organization of interferons}; Petrenko VA et al.; The developed approach to investing the structure-functional organization of interferon has been developed consisting in: 1) fusing genes of interferon and alpha-peptide of beta-galactosidase, the resultant protein having the interferon properties and being determined by the beta-galactosidase alpha-complementation test; 2) constructing mutant genes of interferon by the localized mutagenesis; 3) determining the mutant interferon activity; 4) deducing the amino acid sequence of mutant interferon by sequencing mutant genes; 5) analyzing structure-functional organization of interferon . In accordance with this approach, ten mutant interferons with up to 15 changes of amino acid substitutions are obtained and their antiviral activity is determined . The role of some amino acid residues in antiviral activity of interferon alpha 2 is revealed.

Mol Biochem Parasitol, 1987 Feb, 23(1), 91 - 102
Putative glycophorin-binding protein is secreted from schizonts of Plasmodium falciparum; Bianco AE et al.; A cDNA clone expressing an antigen of Plasmodium falciparum, selected by screening an expression library cloned in Escherichia coli, encodes a portion of the protein identified as a glycophorin-binding protein {Kochan et al . (1986) Cell 44, 689-696} . Human antibodies affinity-purified on extracts from this clone were used to characterize the antigen by immunoblotting . This protein was present in all isolates tested, restricted to mature trophozoites and schizonts . It was abundant in culture supernatants at the time of merozoite release but present in minor amounts if at all in merozoites . The pattern of antigen distribution over schizont-infected cells observed by immunoelectron microscopy differed from that of the precursor of the major merozoite surface antigens in that most of the antigen appeared to be located over the erythrocyte cytoplasm without any obvious association with organelles . It thus appears unlikely that this antigen is present on the merozoite surface prior to schizont rupture.

Mol Gen Mikrobiol Virusol, 1987 Feb, (2), 43 - 7
{Analysis of mutations affecting the expression of catabolite-sensitive operons in Escherichia coli K12 mutants defective in the HPr-component of the carbohydrate transport system}; Erlagaeva RS et al.; Expression of catabolite-sensitive operons in mutants devoid of HPr (a component of the glucose transport system) is severely repressed . ptsH mutants do not utilize substrates of the phosphoenolpyruvate: carbohydrate system (PTS) and many other sugars . Analysis of mutations suppressing the effect of the delta ptsH mutation revealed a new class of reversions which restore the growth of bacteria on different substrates . This mutation (named ptsS) intensifies the growth rate of ptsH mutants and increases the differential rate of beta-galactosidase production . ptsS mutation was mapped in the region of ptsF gene (coding for the fructose specific enzyme II of the PTS) on the 46th min . of the E . coli chromosome map . The effect of the ptsS mutation on the expression of catabolite-sensitive operons manifests only in the presence of the intact enzyme I of the PTS.

J Antibiot (Tokyo), 1987 Feb, 40(2), 209 - 16
A single assay for simultaneously testing effectors of alanine racemase and/or D-alanine: D-alanine ligase; Vicario PP et al.; The biosynthesis from L-alanine of D-alanyl-D-alanine, required for the peptidoglycan layer of the cell wall of many bacterial species, is catalyzed by two enzymes in series, alanine racemase and D-alanine: D-alanine ligase . A simple in vitro method, called the combined assay, for simultaneously testing for effectors of either or both enzymes in a single assay by coupling these enzymes to each other is described here . The experiments used to derive the optimum conditions for the assay are also described . Each enzyme is included in the assay in rate-limiting amounts, wherein the product of the initial racemase reaction, D-alanine, becomes the substrate for the subsequent ligase . The product of the overall reaction, {14C}-D-alanyl-D-alanine, is separated chromatographically from the L-{1-14C}alanine substrate, and from any D-{1-14C}alanine intermediate, at the end of the incubation, is counted and the percent conversion of substrate to product calculated . The inhibitory effects of 3-fluoro-D-alanine-2d, a known inhibitor of the racemase, and D-cycloserine and DL-1-aminoethylphosphonic acid, inhibitors of both enzymes, were readily detectable . The sensitivity of the combined assay to these inhibitors appears similar to that of earlier assays . This assay has the advantage over previous ones of being able to detect inhibitors of either enzyme in a single assay, thereby avoiding the need to screen each compound in a separate assay of each enzyme.

J Biol Response Mod, 1987 Feb, 6(1), 69 - 87
Augmentation of human natural killer cell activity by influenza virus antigens produced in Escherichia coli; Rees RC et al.; A series of Escherichia coli cloned influenza viral gene products were assayed for their ability to augment human natural cytotoxicity in overnight cultures (18 h) at 37 degrees C . Nylon wool nonadherent peripheral blood mononuclear cells (PBMC) proved responsive to stimulation by a number of cloned viral proteins, the most effective being the nonstructural (NS1) protein (but not NS2 protein) and haemagglutinin and matrix antigen components fused to the N-terminal 81 amino acid sequence of NS1 . Furthermore, interferon (IFN) was generated in cultures in which enhanced cytotoxicity was detected and was identified as mostly IFN alpha (greater than 90%) with less than 10% IFN gamma contamination . The cell type responding to antigen stimulation was present in Percoll fractions enriched for large granular lymphocytes (LGLs); furthermore PBMC activated by NS1 protein fractionated in the low density Percoll fractions (LGL enriched) . Using specific anti-IFN antisera, it was shown that IFN alpha but not IFN gamma was responsible for the enhancement of cytotoxicity . Interferon induction and activation of cytotoxicity could not be ascribed to the presence of contaminating bacterial products . These results suggest that a particular NS1 protein configuration is capable of activating human natural killer cells via the induction of IFN alpha.

J Appl Physiol, 1987 Feb, 62(2), 706 - 10
Effect of endotoxin on diaphragm lymph contamination in unanesthetized sheep; Drake RE et al.; The preparation for collecting lung lymph from sheep caudal mediastinal lymph node (CMN) efferent vessels is widely used to study the effects of endotoxin on lung microvascular permeability . However, there are nonpulmonary lymph vessels that drain into the CMN along with the afferent lymph vessels from the lung . Thus CMN lymph is a mixture of lymph from the lung and diaphragm lymph vessels as well as from other nonpulmonary lymph vessels . We studied the effect of 0.5-1.0 microgram/kg Escherichia coli endotoxin on the flow rates in diaphragm and CMN efferent lymph vessels (Qdi and QCMN, respectively) in unanesthetized sheep . For the time period between 2 and 5.5 h after endotoxin QCMN was increased from its base line of 7.2 +/- 4.4 (SD) to 17.3 +/- 10.6 ml/h and the lymph-to-plasma protein concentration ratio (L/PCMN) had increased from 0.68 +/- 0.11 to 0.81 +/- 0.06 . During the same time period, Qdi was 4.5 +/- 3.1 ml/h compared with 1.0 +/- 0.8 ml/h at base line and the diaphragm lymph-to-plasma protein concentration ratio (L/Pdi) was 0.92 +/- 0.07 (base line = 0.74 +/- 0.15) . The increases in flow rate and protein concentration were significant for each type of vessel (P less than 0.05) . We conclude that the period of increased QCMN and L/PCMN after endotoxin is associated with an increase in Qdi and L/Pdi . Thus, it is difficult to determine how much of the CMN lymph response comes from the lungs and how much comes from diaphragm lymph vessels.

J Clin Microbiol, 1987 Feb, 25(2), 338 - 43
Detection of enterotoxigenic Escherichia coli by dot blot hybridization with biotinylated DNA probes; Bialkowska-Hobrzanska H; A dot blot hybridization test was developed for the detection of enterotoxigenic E . coli without the use of radioisotopes . Three biotin-labeled DNA (Bio-DNA) probes corresponding to structural genes specifying heat-labile and heat-stable enterotoxins of porcine and human origin were prepared by random priming; label incorporation was significantly higher than that obtained from the use of nick translation . Bio-DNA probes were highly specific when reacted with protein- and RNA-free DNA preparations in a dot blot hybridization assay . The Bio-DNA probe, in which 40% of available thymidines were replaced by a biotin-labeled deoxyuridine, readily detected 160 pg of target DNA mixed with 6 micrograms of carrier DNA . The minimum amount of total DNA required for reliable identification of a single-copy enterotoxin gene of porcine origin within a 5,000-kilobase chromosome was found to be approximately 5 micrograms . Complete agreement among the results of Bio-DNA probe hybridization, {32P}DNA hybridization, and biological assay was demonstrated for 15 (100%) of the clinical isolates . This procedure provides a more suitable approach for the diagnosis of enterotoxigenic E . coli infections in clinical settings than the hybridization assay based on 32P-labeled DNA probes.

Am J Physiol, 1987 Feb, 252(2 Pt 2), H291 - 300
Effect of vasopressors on organ blood flow during endotoxin shock in pigs; Breslow MJ et al.; A volume-resuscitated porcine endotoxin shock model was used to evaluate the effect on organ blood flow of increasing systemic arterial blood pressure with vasopressors . Administration of 0.05-0.2 mg/kg of Escherichia coli endotoxin (E) reduced mean arterial blood pressure (MAP) to 50 mmHg, decreased systemic vascular resistance to 50% of control, and did not change cardiac output or heart rate . Blood flow to brain, kidney, spleen, and skeletal muscle was reduced during endotoxin shock, but blood flow to left ventricle, small and large intestine, and stomach remained at pre-endotoxin levels throughout the study period . Four groups of animals were used to evaluate the effect of vasopressor therapy . A control group received E and no vasopressor, whereas the other three groups received either norepinephrine, dopamine, or phenylephrine . Vasopressors were administered starting 60 min after E exposure, and the dose of each was titrated to increase MAP to 75 mmHg . Despite the increase in MAP, brain blood flow did not increase in any group . Norepinephrine alone increased blood flow to the left ventricle . Kidney, splanchnic, and skeletal muscle blood flow did not change with vasopressor administration . The dose of norepinephrine required to increase MAP by 20-25 mmHg during E shock was 30 times the dose required for a similar increase in MAP in animals not receiving E . We conclude that hypotension in the fluid resuscitated porcine E shock model is primarily the result of peripheral vasodilatation, that the vascular response to vasoconstrictors in this model is markedly attenuated following E administration, that blood pressure elevation with norepinephrine, dopamine, and phenylephrine neither decreases blood flow to any organ nor increases blood flow to organs with reduced flow, and that norepinephrine, dopamine, and phenylephrine affect regional blood flow similarly in this model.

Radiat Res, 1987 Feb, 109(2), 256 - 74
Radiation protection of Escherichia coli B/r by hydroxyl radical scavengers; Ewing D et al.; We have used Escherichia coli B/r to test the proposal that hydroxyl radicals (.OH) are major contributors to lethal damage when bacteria in equilibrium with air or 100% nitrogen are exposed to ionizing radiation . In addition, we have tested the hypothesis that oxygen sensitizes bacterial cells to radiation by reacting at radical sites previously formed by reactions of .OH . Our results with B/r indicate that the involvement of OH radicals in damage may have been overestimated . We believe that simple .OH removal provides B/r with only a relatively small amount of protection in N2 and air . Although some .OH scavengers can have large protective effects in air, evidence supports the tentative conclusion that these effects are not based on simple .OH removal . If this conclusion is correct, then radiation sensitization by oxygen--at least of this bacterial strain--would be unrelated to reactions of .OH.

Proc Natl Acad Sci U S A, 1987 Feb, 84(3), 690 - 4
3' homologous free ends are required for stable joint molecule formation by the RecA and single-stranded binding proteins of Escherichia coli; Konforti BB et al.; The RecA protein of Escherichia coli is important for genetic recombination in vivo and can promote synapsis and strand exchange in vitro . The DNA pairing and strand exchange reactions have been well characterized in reactions with circular single strands and linear duplexes, but little is known about these two processes using substrates more characteristic of those likely to exist in the cell . Single-stranded linear DNAs were prepared by separating strands of duplex molecules or by cleaving single-stranded circles at a unique restriction site created by annealing a short defined oligonucleotide to the circle . Analysis by gel electrophoresis and electron microscopy revealed that, in the presence of RecA and single-stranded binding proteins, a free 3' homologous end is essential for stable joint molecule formation between linear single-stranded and circular duplex DNA.

Proc Natl Acad Sci U S A, 1987 Feb, 84(3), 634 - 7
Vitamin K-dependent carboxylase: possible role of the substrate "propeptide" as an intracellular recognition site; Suttie JW et al.; The liver microsomal vitamin K-dependent carboxylase catalyzes the posttranslational conversion of specific glutamate residues to gamma-carboxyglutamate residues in a limited number of proteins . A number of these proteins have been shown to contain a homologous basic amino acid-rich "propeptide" between the leader sequence and the amino terminus of the mature protein . Plasmids encoding protein C, a vitamin K-dependent protein, containing or lacking a propeptide region were constructed and the protein was expressed in Escherichia coli . The protein products were assayed as substrates in an in vitro vitamin K-dependent carboxylase system . Only proteins containing a propeptide region were substrates for the enzyme . These data support the hypothesis that this sequence of the primary gene product is an important recognition site for this processing enzyme.

Lab Invest, 1987 Feb, 56(2), 198 - 210
Rat pulmonary artery restructuring and pulmonary hypertension induced by continuous Escherichia coli endotoxin infusion; Kirton OC et al.; We have studied the effect of continuous endotoxin infusion on rat pulmonary structure and function (69.4 ng/100 gm body weight/min for 24 hours) . After 6 days of endotoxin infusion, lack of filling of pre- and intraacinar arteries was evident on pulmonary arteriograms . Microscopy demonstrated lumen narrowing in preacinar arteries and occlusion of intraacinar arteries . Morphometry of patent intraacinar arteries established dilation and increased wall muscle . Widespread alveolar wall injury was evident . After 24 hours of infusion, pulmonary artery pressure was raised (delta 9 mmHg; p less than or equal to 0.001); it then fell but was again increased by day 6 (delta 6 mmHg; p less than or equal to 0.05) . Pulmonary vascular resistance was markedly increased at 24 hours (day 0 = 0.1 +/- 0.011 dyne/sec/cm-5; 24 hours endotoxin = 0.572 +/- 0.102 dyne/sec/cm-5; p less than or equal to 0.02) . It remained elevated during the infusion period but was not significant . At day 6 the alveolar-arterial oxygen diffusion gradient (A-aDO2) was increased (day 0 = 19.6 +/- 1.39 mmHg, day 6 endotoxin = 33.8 +/- 0.1 mmHg; p less than or equal to 0.001) . The arterial oxygen tension (PaO2) was decreased (day 0 = 86.5 +/- 1.8 mmHg, day 6 endotoxin = 74 +/- 2.52 mmHg; p less than or equal to 0.05), as was the arterial carbon dioxide tension (PaCO2) (day 0 = 36.0 +/- 0.73 mmHg, day 6 endotoxin = 30 +/- 1.9 mmHg; p less than or equal to 0.05) . Thrombocytopenia occurred during the first 72 hours of infusion (day 0 = 7.41 +/- 0.41 X 10(5)/mm3, day 1 endotoxin = 2.43 +/- 0.30 X 10(5)/mm3, day 3 endotoxin = 2.32 +/- 0.31 X 10(5)/mm3; p less than or equal to 0.001) but by day 6 the platelet count had returned to basal levels (9.9 +/- 0.65 X 10(5)/mm3) . Endotoxin increased the number of leukocytes in peripheral blood (day 0 = 12.8 +/- 1.2 X 10(3)/mm3, day 3 endotoxin = 17.0 +/- 1.86 X 10(3)/mm3, day 6 endotoxin = 22.5 +/- 1.8 X 10(3)/mm3; p less than or equal to 0.01 for day 6) . Plasma concentrations of 6-keto-prostaglandin F1 alpha decreased during the first 24 hours of infusion (day 0 = 0.56 +/- 0.076 ng/ml, 24 hours endotoxin = 0.27 +/- 0.026 ng/ml; p less than or equal to 0.05) and thromboxane (TX) B2 in the first 15 hours (day 0 = 0.23 +/- 0.058 ng/ml, 15 hours endotoxin = 0.09 +/- 0.14 ng/ml; p less than or equal to 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)

J Bacteriol, 1987 Feb, 169(2), 885 - 7
An alkaline shift induces the heat shock response in Escherichia coli; Taglicht D et al.; Activation of heat shock response was observed after an alkaline shift of extracellular pH: it peaked at 5 to 10 min, as was previously reported for the heat-induced response, and was dependent on a functional rpoH gene, which is the positive regulator of the heat shock response . An induction of over sixfold was observed for dnaK and groE . The response was induced by the alkalization of extracellular pH but not by the alkalization of intracellular pH . An acidic shift of extracellular pH failed to activate the heat shock response, showing that the response is specific to the alkaline shift.

J Bacteriol, 1987 Feb, 169(2), 811 - 22
Repression and catabolite gene activation in the araBAD operon; Lichenstein HS et al.; Catabolite gene activation of the araBAD operon was examined by using catabolite gene activator protein (CAP) site deletion mutants . A high-affinity CAP-binding site between the divergently orientated araBAD and araC operons has been previously identified by DNase I footprinting techniques . Subsequent experiments disagreed as to whether this site is directly involved in stimulating araBAD expression . In this paper, we present data showing that deletions generated by in vitro mutagenesis of the CAP site led to a five- to sixfold reduction in single-copy araBAD promoter activity in vivo . We concluded that catabolite gene activation of araBAD involves this CAP site . The hypothesis that CAP stimulates the araBAD promoter primarily by relieving repression was then tested . The upstream operator araO2 was required for repression, but we observed that the magnitude of CAP stimulation was unaffected by the presence or absence of araO2 . We concluded that CAP plays no role in relieving repression . Other experiments showed that when CAP binds it induces a bend in the ara DNA; similar bending has been reported upon CAP binding to lac DNA . This conformational change in the DNA may be essential to the mechanism of CAP activation.

J Bacteriol, 1987 Feb, 169(2), 785 - 9
Loss of aldehyde dehydrogenase in an Escherichia coli mutant selected for growth on the rare sugar L-galactose; Zhu Y et al.; Escherichia coli K-12 converts L-fucose to dihydroxyacetone phosphate (C-1 to C-3) and L-lactaldehyde (C-4 to C-6) by a pathway specified by the fuc regulon . Aerobically, L-lactaldehyde serves as a carbon and energy source by the action of an aldehyde dehydrogenase of broad specificity; the product, L-lactate, is then converted to pyruvate . Anaerobically, L-lactaldehyde serves as an electron acceptor to regenerate NAD from NADH by the action of an oxidoreductase; the reduced product, L-12-propanediol, is excreted . A strain selected for growth on L-galactose (a structural analog of L-fucose) acquired a broadened inducer specificity because of an altered fucR gene encoding the activator protein for the fuc regulon (Y . Zhu and E . C . C . Lin, J . Mol . Evol . 23:259-266, 1986) . In this study, a second mutation that abolished aldehyde dehydrogenase activity was discovered . The L-fucose pathway converts L-galactose to dihydroxyacetone phosphate and L-glyceraldehyde . Aldehyde dehydrogenase then converts L-glyceraldehyde to L-glycerate, which is toxic . Loss of the dehydrogenase averts the toxicity during growth on L-galactose, but reduces by one-half the aerobic growth yield on L-fucose . When mutant cells induced in the L-fucose system were incubated with radioactive L-fucose, accumulation of radioactivity occurred if the substrate was labeled at C-1 but not if it was labeled C-6 . Complete aerobic utilization of carbons 4 through 6 of L-fucose depends not only on an adequate activity of aldehyde dehydrogenase to trap L-lactaldehyde as its anionic acid but also on the lack of L-1,2-propanediol oxidoreductase activity, which converts L-lactaldehyde to a readily excreted alcohol.

J Bacteriol, 1987 Feb, 169(2), 728 - 34
Constitutive expression of the SOS response in recA718 mutants of Escherichia coli requires amplification of RecA718 protein; McCall JO et al.; In recA718 lexA+ strains of Escherichia coli, induction of the SOS response requires DNA damage . This implies that RecA718 protein, like RecA+ protein, must be converted, by a process initiated by the damage, to an activated form (RecA) to promote cleavage of LexA, the cellular repressor of SOS genes . However, when LexA repressor activity was abolished by a lexA-defective mutation {lexA(Def)}, strains carrying the recA718 gene (but not recA+) showed strong SOS mutator activity and were able to undergo stable DNA replication in the absence of DNA damage (two SOS functions known to require RecA activity even when cleavage of LexA is not necessary) . lambda lysogens of recA718 lexA(Def) strains exhibited mass induction of prophage, indicative of constitutive ability to cleave lambda repressor . When the cloned recA718 allele was present in a lexA+ strain on a plasmid, SOS mutator activity and beta-galactosidase synthesis under LexA control were expressed in proportion to the plasmid copy number . We conclude that RecA718 is capable of becoming activated without DNA damage for cleavage of LexA and lambda repressor, but only if it is amplified above its base-line level in lexA+ strains . At amplified levels, RecA718 was also constitutively activated for its roles in SOS mutagenesis and stable DNA replication . The nucleotide sequence of recA718 reveals two base substitutions relative to the recA+ sequence . We propose that the first allows the protein to become activated constitutively, whereas the second partially suppresses this capability.

J Bacteriol, 1987 Feb, 169(2), 694 - 8
Verification of the protein in the outer membrane of Bdellovibrio bacteriovorus as the OmpF protein of its Escherichia coli prey; Talley BG et al.; Two research groups showed that several Bdellovibrio strains incorporated into their outer membranes intact OmpF porin proteins derived from their Escherichia coli prey . These results could not be reproduced by another group using Bdellovibrio bacteriovorus 109J . They showed that a major protein appearing in the Bdellovibrio Triton X-100-insoluble outer membrane was coded for by the bdellovibrios . We reconciled these results by examining the strain used by this group and by reviving a freeze-dried culture of strain 109J which had been stored for almost 9 years . B . bacteriovorus 109J failed to acquire substantial amounts of the OmpF protein from E . coli ML35, and a protein coded for by the bdellovibrios was expressed in its place . However, B . bacteriovorus 109J incorporated the OmpF protein from rough K-12 strains of E . coli, and the revived 9-year-old culture of B . bacteriovorus 109J incorporated more of the OmpF protein from the smooth E . coli ML35 than did its contemporary counterpart . The protein isolated from the outer membrane of the bdellovibrios was identified as the OmpF protein of E . coli by its protease peptide profile on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by Western blot analysis . This confirmed that bdellovibrios relocalize outer membrane proteins from their prey, but relocalization may be an unstable trait which can be influenced by the prey.

J Bacteriol, 1987 Feb, 169(2), 682 - 6
Transfer of phosphoethanolamine residues from phosphatidylethanolamine to the membrane-derived oligosaccharides of Escherichia coli; Miller KJ et al.; The membrane-derived oligosaccharides (MDO) of Escherichia coli are periplasmic constituents composed of glucose residues linked by beta-1,2 and beta-1,6 glycosidic bonds . MDO are substituted with phosphoglycerol, phosphoethanolamine, and succinic acid moieties . The phosphoglycerol residues present on MDO are derived from phosphatidylglycerol (B . J . Jackson and E . P . Kennedy, J . Biol . Chem . 258:2394-2398, 1983), but evidence as to the source of the phosphoethanolamine residues has been lacking . We now report that phosphatidylethanolamine, exogenously added to intact cells of E . coli, provides a source of phosphoethanolamine residues that are transferred to MDO . The biosynthesis of phosphoethanolamine-labeled MDO is osmotically regulated, with maximum synthesis occurring during growth in medium of low osmolarity.

J Bacteriol, 1987 Feb, 169(2), 670 - 4
Operator-constitutive mutations of the Escherichia coli metF gene; Belfaiza J et al.; The Escherichia coli metF gene codes for 5,10-methylene-tetrahydrofolate reductase, the enzyme that leads to the formation of N-methyltetrahydrofolate, supplying the methyl group of methionine . Transcription of metF, as well as most of the methionine genes, is repressed by the metJ gene product complexed with S-adenosylmethionine . A metF'-'lacZ gene fusion was used to isolate mutants that have altered expression from the metF promoter . The nucleotide sequences of the metF regulatory region from five such mutants were determined . The mutations were located in the region previously defined as the potential target of the methionine repressor by its similarity to other binding sites . The mutationally defined metF operator thus consists of a 40-base-pair-long region, with five 8-base-pair imperfect palindromes spanning the metF transcription start . The altered operators do not recognize the purified repressor in an in vitro transcription-translation system, although the repressor binds efficiently to the metF wild-type operator.

J Bacteriol, 1987 Feb, 169(2), 558 - 64
Roles of the divergent branches of the meta-cleavage pathway in the degradation of benzoate and substituted benzoates; Harayama S et al.; The TOL plasmid-specified meta-cleavage pathway for the oxidative catabolism of benzoate and toluates branches at the ring cleavage products of catechols and reconverges later at 2-oxopent-4-enoate or its corresponding substituted derivatives . The hydrolytic branch of the pathway involves the direct formation of 2-oxopent-4-enoate or its derivatives, whereas the oxalocrotonate branch involves three enzymatic steps effected by a dehydrogenase, an isomerase, and a decarboxylase, which produce the same compounds . Evidence is presented which shows that benzoate and p-toluate can, under certain circumstances, be catabolized by the hydrolytic branch . However, in a fully functional pathway, only m-toluate is dissimilated via this branch, and benzoate and p-toluate are catabolized almost exclusively by the oxalocrotonate branch . The biochemical basis of this selectivity was found to reside in the high affinity of the dehydrogenase for ring fission products derived from benzoate and p-toluate and its inability to attack the ring fission product derived from m-toluate . Although isomerization of 4-oxalocrotonate occurs spontaneously in vitro, enzymatic isomerization was found to be essential for effective functioning of this branch of the pathway in vivo.

J Bacteriol, 1987 Feb, 169(2), 553 - 7
Feedback inhibition of ammonium (methylammonium) ion transport in Escherichia coli by glutamine and glutamine analogs; Jayakumar A et al.; When cultured with glutamate or glutamine as the nitrogen source, Escherichia coli expresses a specific ammonium (methylammonium) transport system . Over 95% of the methylammonium transport activity in washed cells was blocked by incubation with 100 microM L-glutamine in the presence of chloramphenicol (100 micrograms/ml) . The time course for the onset of this glutamine inhibition followed a first-order rate expression with a t1/2 of 2.8 min . The inhibition of transport by L-glutamine was noncompetitive (Ki = 18 microM) with respect to the {14C}methylammonium substrate . D-Glutamine had no significant effect . The glutamine analogs gamma-L-glutamyl hydroxamate (Ki = 360 microM) and gamma-L-glutamyl hydrazide (Ki = 800 microM) were also noncompetitive inhibitors of methylammonium transport, suggesting that glutamine metabolism is not required . The role of the intracellular glutamine pool in the regulation of ammonium transport was investigated by using mutants carrying defects in the operon of glnP, the gene for the glutamine transporter . The glnP mutants had normal rates of methylammonium transport but were refractory to glutamine inhibition . Glycylglycine, a noncompetitive inhibitor of methylammonium uptake in wild-type cells (Ki = 43 microM), was equipotent in blocking transport in glnP mutants . Although ammonium transport is also subject to repression by growth of E . coli in the presence of ammonia, this phenomenon is unrelated to glutamine inhibition . A GlnL RegC mutant which constitutively expressed ammonium transport activity exhibited a sensitivity to glutamine inhibition similar to that of wild-type cells . These findings indicate that ammonium transport in E . coli is regulated by the internal glutamine pool via feedback inhibition.

J Bacteriol, 1987 Feb, 169(2), 540 - 5
Repair of N-methyl-N'-nitro-N-nitrosoguanidine-induced DNA damage by ABC excinuclease; Van Houten B et al.; Escherichia coli has several overlapping DNA repair pathways which act in concert to eliminate the DNA damage caused by a diverse array of physical and chemical agents . The ABC excinuclease which is encoded by the uvrA, uvrB, and uvrC genes mediates both the incision and excision steps of nucleotide excision repair . Traditionally, this repair pathway has been assumed to be active against DNA adducts that cause major helical distortions . To determine the level of helical deformity required for recognition and repair by ABC excinuclease, we have evaluated the substrate specificity of this enzyme by using DNA damaged by N-methyl-N'-nitro-N-nitrosoguanidine . ABC excinuclease incised methylated DNA in vitro in a dose-dependent manner in a reaction that was ATP dependent and specific for the fully reconstituted enzyme . In vivo studies with various alkylation repair-deficient mutants indicated that the excinuclease participated in the repair of DNA damage induced by N-methyl-N'-nitro-N-nitrosoguanidine.

Infect Immun, 1987 Feb, 55(2), 329 - 34
Association and dissociation of Escherichia coli heat-stable enterotoxin from rat brush border membrane receptors; Cohen MB et al.; Escherichia coli heat-stable enterotoxin (ST) binds to receptors on rat intestinal cells and brush border membranes (BBM) . We devised experiments to examine the reversibility of ST binding . We found that both 125I-labeled ST and native ST were spontaneously dissociable from the BBM receptor . Radiolabeled ST bound to BBM was also dissociated by the addition of avid goat anti-ST antiserum . Furthermore, using a computer program for analysis of ligand binding, we calculated an apparent Ka of 10(8) liters/mol from competitive inhibition and saturation-binding data . This is significantly lower than the value previously reported by others . Our findings, of a lower Ka and a reversible ST-binding process, suggest that a therapeutic strategy of removing bound ST from its receptor or competing with the enterocyte receptor for unbound ST might be successful in terminating ST-induced secretion.

Radiology, 1987 Feb, 162(2), 573 - 4
Unilateral hypertrophic osteoarthropathy in a patient with an infected axillary-axillary bypass graft; Ho A et al.; Hypertrophic osteoarthropathy (HO) is a specific clinicoradiologic entity, the most common cause of which is the presence of pulmonary lesions . Eight cases of aortic graft infection and aortoenteric fistulae in association with HO have been recognized . A case of an infected axillary-axillary graft presenting as unilateral HO of the upper limb is reported, and its unusual features are used to postulate a mechanism underlying HO.

Clin Immunol Immunopathol, 1987 Feb, 42(2), 218 - 28
The pathogenesis of virus-associated encephalopathies: a prospective study of immunological mechanisms; Kennedy CR et al.; Thirty-two patients, including 29 children, presenting with acute unexplained encephalopathies were studied prospectively for evidence of virus infection, immunodeficiency, and immunologic involvement in the pathogenesis of their illnesses . Twenty-five of these patients had a clinical diagnosis of encephalitis . Twenty-two of these 25 had laboratory evidence of active virus infection, the majority with viruses usually associated with self-limiting illness outside the central nervous system . In patients with encephalitis, immune competence, as reflected by T-cell numbers and subsets in peripheral blood, in vitro interferon production, natural killer activity, and specific antiviral antibody production, was normal . Transudation of albumin into the cerebrospinal fluid (CSF), a measure of blood-brain barrier breakdown, was seen in 40% of patients . Intrathecal antibody synthesis was suggested by an elevated IgG index in 9/20 CSF/serum pairs but was confirmed by an elevated specific IgG ratio in only 3 . The serum IgG1 and IgG3 subclass levels were significantly elevated at the time of the illness and remained elevated 8 months later; IgG2 and IgG4 levels were normal . IgE levels were elevated in 50% of patients . Serum levels of IgM antibodies against Escherichia coli measured 8 months after the neurological illness were also significantly higher in encephalitis patients than in age-matched healthy controls . Human myelin basic protein did not induce proliferation in peripheral blood lymphocytes in any patient . We conclude that most encephalopathies associated with viral infections are not due to an underlying generalized immunodeficiency, and probably result from an inappropriately vigorous immune response.

Microb Pathog, 1987 Feb, 2(2), 91 - 9
Cloning and expression of a common Legionella outer membrane antigen in Escherichia coli; Hindahl MS et al.; A genomic library of Legionella pneumophila was constructed by inserting L . pneumophila knoxville-1 strain (LPK-1) chromosome fragments into cosmid vector pHC79 . Screening of the library with antibodies directed against a major outer membrane protein/lipopolysaccharide complex from LPK-1 resulted in the identification of six clones that reacted with the antiserum . Western blot analysis indicated that a 19,000 dalton (19 kDa) component was the reactive antigen in all of the clones . Western blot analysis of outer membranes from L . pneumophila serogroups and other Legionella species revealed that the cloned 19 kDa antigen was common to all serogroups and all but one of the five other Legionella species examined . One of the 19 kDa expressing clones was used as an immunoabsorbent to recover antibody to the 19 kDa antigen thus confirming the surface localization of this L . pneumophila antigen in E . coli.

J Gen Microbiol, 1987 Feb, 133 ( Pt 2), 347 - 51
2-Phenylethylamine catabolism by Escherichia coli K12; Parrott S et al.; Escherichia coli K12 grows on 2-phenylethylamine as sole carbon and energy source by converting it, via phenylacetaldehyde, to phenylacetic acid . Phenylacetaldehyde was formed by the action of an inducible amine oxidase and catalase activity was increased sixfold, presumably to ensure removal of the H2O2 that was expected to be a product of the amine oxidation . The phenylacetaldehyde was oxidized to phenylacetic acid by an inducible NAD+-dependent dehydrogenase . Mutants defective in phenylacetaldehyde dehydrogenase cannot grow on 2-phenylethylamine as carbon and energy source but can still use it as a nitrogen source.

J Gen Microbiol, 1987 Feb, 133 ( Pt 2), 341 - 6
Location and function of fruC, a gene involved in the regulation of fructose utilization by Escherichia coli; Kornberg HL et al.; Procedures are described for the selection of Escherichia coli mutants that constitutively take up and phosphorylate fructose, and convert it to fructose 1,6-bisphosphate . The phenotype of such mutants is described . The altered regulatory gene, fruC, is highly co-transducible with leu and other markers located at min 2 on the genome . In merozygotes, fruC+ is dominant to fruC . Mutants can be readily isolated that are fruC at 42 degrees C but fruC+ at 30 degrees C; moreover, the integration of a Tn10 transposon in the genome at min 2 converts fruC+ strains to fruC . It is therefore likely that the fruC+ regulatory gene specifies a repressor protein.

J Gen Microbiol, 1987 Feb, 133 ( Pt 2), 317 - 22
Cloning, characterization and expression in Escherichia coli of a leucine biosynthetic gene from Streptomyces rochei; Hercomb J et al.; Leucine and histidine biosynthetic genes from Streptomyces rochei HP1 that complemented auxotrophic mutations in S . lividans TK54 were cloned in pIJ61 . DNA from one leucine recombinant plasmid was subcloned into pBR322 . From the latter, a recombinant plasmid was obtained that complemented the leuA mutation in Escherichia coli CV512 but not other leucine markers in E . coli . Analysis of this and several subclones, including mutant plasmids constructed in vitro, established that the cloned S . rochei gene was expressed in E . coli from the tetracycline promoter of pBR322 to produce a polypeptide of 67 kDa; the corresponding coding region was shown to be within a 1.7 kbp DNA fragment . Blot hybridization revealed corresponding homologous genes in several other streptomycetes.

Biochem J, 1987 Feb 1, 241(3), 877 - 81
A novel approach to study of the structural basis of enzyme polymorphism . Analysis of carboxylesterase B of Escherichia coli as model; Picard B et al.; In order to understand the structural basis of charge differences among enzyme variants without undertaking purification and sequencing of the protein, an original approach was developed . The approach is applicable to any enzyme or protein provided that there is a specific staining procedure . This consists, as a first step, in the projection of electrophoretically obtained mobility values versus pI of all variants into a two-dimensional profile . In a second step, starting from the most common variant, various theoretical possibilities of substitutions are envisaged, taking into consideration the pH of the electrophoretic conditions, pI of the variants and range of variations of the pK values of several amino acid side chains . In a third step, verification of the theoretical data is obtained through comparative protein titration curves by combined isoelectrofocusing-electrophoresis of several pairs of relevant variants . The validity of this approach is tested on the highly polymorphic carboxylesterase B enzyme of Escherichia coli and is found to provide valuable information.

Biochem J, 1987 Feb 1, 241(3), 699 - 704
An investigation of transient intermediates in the reaction of 2-methylglutamate with glutamate decarboxylase from Escherichia coli; Grant PL et al.; Several intermediates in the reaction of 2-methylglutamate with glutamate decarboxylase from Escherichia coli were detected by stopped-flow spectrophotometry and by rapid-scanning spectrophotometry after conventional mixing . Structures were assigned to intermediates on the basis of kinetic and spectral evidence . In the early stages of the reaction an intermediate with the properties expected of a geminal diamine accumulated significantly . Changes consistent with the conversion of this species into the external aldimine were also observed . The course of product formation was determined and linked with spectral changes taking place in the bound coenzyme . The effect of the minor decarboxylation-dependent transamination that accompanies the major reaction was analysed.

Poult Sci, 1987 Feb, 66(2), 270 - 2
Effects of fasting, water deprivation, and adrenal-blocking chemicals on resistance to Escherichia coli challenge; Gross WB et al.; Fasting for 2 days plus 1 day of feeding resulted in increased resistance to Escherichia coli challenge . If feed during the postfasting period contained metyrapone (a blocker of adrenal synthesis of corticosterone), resistance to E . coli challenge was reduced . Fasting for 1.75 days or water deprivation for 2 days resulted in decreased resistance to E . coli challenge . Deprivation of water for two days of chickens with blocked adrenals resulted in an E . coli challenge response similar to that of controls.

Mol Gen Genet, 1987 Feb, 206(2), 356 - 7
A newly discovered tRNA(1Asp) gene (aspV) of Escherichia coli K12; Horiuchi T et al.; We report a new tRNA(1Asp) gene near the dnaQ gene, which is located at 5 min on the Escherichia coli linkage map . We named it aspV . The sequence corresponding to the mature tRNA is identical with that of the two previously identified tRNA(1Asp) genes (aspT and aspU), but there is no homology in the sequences of their 3'- and 5'-flanking regions.

Mol Gen Genet, 1987 Feb, 206(2), 246 - 51
Cloning and expression of the exbB gene of Escherichia coli K-12; Eick-Helmerich K et al.; The exbB locus of Escherichia coli is involved in the uptake of certain iron(III) siderophore compounds, of vitamin B12 and of certain colicins . Outer membrane receptor proteins are essential constituents of the corresponding uptake systems . The DNA carrying the exbB locus was cloned into pACYC184 and subcloned into pUC18 . With the use of insertion mutagenesis employing transposon Tn1000 and by deletion analysis, the exbB locus was confined to a 1.9 kb DNA fragment . An in vitro transcription/translation system and minicells programmed by exbB+ plasmids expressed a protein with an apparent molecular weight of 26,000 . One plasmid, designated pKE7, expressed this protein to an extent that it became a prominent band in the membrane fraction of transformants . In contrast, chromosomally encoded ExbB protein could not be detected . The plasmid-encoded ExbB protein was mainly localized in the cytoplasmic membrane . Ferrichrome transport in exbB mutants was restored by exbB+ plasmids . Moderate overexpression of ExbB resulted in an enhanced ferrichrome transport, strong overexpression reduced the transport rate compared to a wild-type strain . The ExbB function shares some properties with the TonB function.

Mol Gen Genet, 1987 Feb, 206(2), 238 - 45
Mutations affecting activity and transport of haemolysin in Escherichia coli; Ludwig A et al.; Temperature-sensitive mutants that exhibit an altered haemolytic phenotype were isolated from Escherichia coli harbouring the plasmid pHly152 . Complementation with recombinant plasmids carrying one of the four hly genes (C, A, B or D) allowed localization of the hly(ts) mutations . A ts mutation in hlyC leads to a pro----leu exchange in amino acid position 53 of HlyC . Two ts mutations in HlyA were found in positions 312 (ser----pro) and 315 (thr----ile) . Both amino acid exchanges are located in the same hydrophobic domain of HlyA which extends from amino acids 299 to 327 . Two different mutations were introduced by site-specific mutagenesis in this hlyA domain: one by an exchange of ala, val to asp, glu (positions 313, 314) altering the hydrophobicity of this region and another which removes most of this hydrophobic portion . Both mutants have entirely lost the haemolytic activity but the mutant haemolysins are still efficiently transported across both membranes when hlyB and hlyD are provided . Functional HlyC is not required for the transport of the mutant haemolysins . Two site-specific mutations at the N-terminal end of hlyA (one at amino acid position 2 leading to a thr----pro exchange and another deleting ile and thr at positions 4 and 5) also do not affect the transport of the altered haemolysins . The thr----pro exchange enhances the haemolytic activity of the corresponding mutant, whereas the ile, thr deletion exhibits little or no effect on the haemolytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Gen Genet, 1987 Feb, 206(2), 226 - 37
Cytoplasmic ribosomal proteins from Chlamydomonas reinhardtii: characterization and immunological comparisons; Fleming GH et al.; Experiments were undertaken to characterize the cytoplasmic ribosomal proteins (r-proteins) in Chlamydomonas reinhardtii and to compare immunologically several cytoplasmic r-proteins with those of chloroplast ribosomes of this alga, Escherichia coli, and yeast . The large and small subunits of the C . reinhardtii cytoplasmic ribosomes were shown to contain, respectively, 48 and 45 r-proteins, with apparent molecular weights of 12,000-59,000 . No cross-reactivity was seen between antisera made against cytoplasmic r-proteins of Chlamydomonas and chloroplast r-proteins, except in one case where an antiserum made against a large subunit r-protein cross-reacted with an r-protein of the small subunit of the chloroplast ribosome . Antisera made against one out of five small subunit r-proteins and three large subunit r-proteins recognized r-proteins from the yeast large subunit . Each of the yeast r-proteins has been previously identified as an rRNA binding protein . The antiserum to one large subunit r-protein cross-reacted with specific large subunit r-proteins from yeast and E . coli.

Mol Gen Genet, 1987 Feb, 206(2), 220 - 5
N-methyl-N'-nitro-N-nitrosoguanidine-induced resistance to ionizing radiation; Morse ML et al.; N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) pretreatments increase the resistance of Escherichia coli to gamma-radiation . The increased resistance is dependent on functional polA, recA, recB, recC, and lexA genes and is partly dependent on recN . The MNNG-induced resistance is additive to resistance induced by pretreatment with gamma-radiation but not by increases induced by hydrogen peroxide . The MNNG-induced resistance occurs in adaptive response mutants and at pretreatment levels of MNNG that do not activate cells to reactivate UV-inactivated lambda phage . The MNNG-induced resistance appears to be distinct from other inductions to gamma-radiation resistance.

J Biochem (Tokyo), 1987 Feb, 101(2), 525 - 34
New approaches for the high-level expression of human interleukin-2 cDNA in Escherichia coli; Sato T et al.; We constructed several expression plasmids of human IL-2 gene, some of which directed high-level synthesis of mature IL-2 protein in E . coli . In all the plasmids reported here, we installed the E . coli trp promoter and SD sequence upstream of the IL-2 cDNA . When DNA sequences containing the rho-independent transcription terminator such as those involved in the trpA and lpp gene were inserted downstream of the IL-2 cDNA sequence, the expression level of the IL-2 gene increased up to 5-fold . Moreover, the deletion of either the whole region including A-T and G-C tails or a part of the 3' non-coding sequence resulted in further increase of the expression of the IL-2 gene up to 500-fold . The mature IL-2 produced in E . coli exhibited biological and immunological activities indistinguishable from those of purified IL-2 from a human T cell line, Jurkat-111 . The manipulations described here may be useful for the high-level expression of eukaryotic genes in E . coli.

J Biochem (Tokyo), 1987 Feb, 101(2), 387 - 96
Isolation and characterization of deletion mutants of ompR and envZ, regulatory genes for expression of the outer membrane proteins OmpC and OmpF in Escherichia coli; Mizuno T et al.; Expression of the ompC and ompF genes coding for the major outer membrane proteins, OmpC and OmpF, respectively, is known to be controlled by at least two regulatory genes, ompR and envZ, which together comprise a single ompB operon . We constructed chromosomal mutants with either ompR-envZ deletion or envZ deletion . Characterization of these deletion strains showed that the OmpR protein is necessary for transcription of the ompC and ompF genes, and the EnvZ protein is essential for normal regulation of the ompC and ompF expression, which is affected by the medium osmolarity . We also constructed several plasmids carrying different portions of the ompB operon . Characterization of these plasmids allowed us to identify the OmpR protein with an apparent molecular weight of 29 kilodaltons (kDa) and the EnvZ protein with an apparent molecular weight of 50 kDa . The initiation codon for EnvZ translation appeared to overlap with the termination codon for OmpR translation . It was also found that a truncated EnvZ polypeptide (44 kDa) which lacks the N-terminal 55 amino acid residues can complement the envZ deletion mutant . Based on these results, the structure and function of the ompB operon are discussed in relation to the regulation of ompC and ompF expression.

Bioorg Khim, 1987 Feb, 13(2), 263 - 5
{Determination of beta-galactosidase activity on nitrocellulose plates using 5-bromo-3-indolyl-beta-D-galactopyranoside and tetrazolium salts}; Markarian AN et al.; A simple and convenient technique has been developed for detection of beta-galactosidase from E . coli on nitrocellulose sheets using a mixture of 5-bromoindol-3-yl-beta-D-galactopyranoside and nitro blue tetrazolium, which enables rapid detection of fmole (10(-15) mole) quantities of the enzyme at pH 9.5 . The technique has the following advantages: the substrates are stable for a long period; reaction products give non-fading intense blue colour, resolution is extremely good with essentially no diffusion.

Biochimie, 1987 Feb, 69(2), 157 - 61
One step purification of Escherichia coli beta-glucuronidase; Blanco C et al.; beta-glucuronidase was purified by affinity chromatography on thiophenyl-glucuronide coupled to Sepharose . The enzyme was more than 95% pure . This enzyme is a tetramer composed of identical 74 kDa monomers . The amino-terminal sequence determined was: NH2-Met-Leu-Arg-Pro-Val.

Biochem Med Metab Biol, 1987 Feb, 37(1), 61 - 72
Liver glycogen metabolism in endotoxin shock . I . Endotoxin administration decreases glycogen synthase activities in dog livers; Liu MS et al.; The effects of E . coli endotoxin administration on hepatic glycogen content and glycogen synthase activities in dogs were studied . Liver glycogen content was decreased by 80% 2 hr after endotoxin injection . When enzyme preparations were preincubated at 25 degrees C for 3 hr prior to their assays, 75% of total glycogen synthase was in I form in control dogs . Under such conditions, endotoxin administration decreased the percentage I activity from 75 to 37%; decreased the Vmax and Km for UDP-glucose for total glycogen synthase by 62.2 and 35.3%, respectively; decreased the Vmax and Km for UDP-glucose for glycogen synthase I by 75.6 and 15.6%, respectively; increased the A0.5 for glucose-6-P for the activation of glycogen synthase D by 126% at high (10 mM) and by 18-fold at low (1 mM) UDP-glucose concentration; increased the percentage D activity from 24 to 72%; decreased the I50 for ATP for the inhibition of total glycogen synthase by 49.7%; decreased the I50 for ATP for the inhibition of glycogen synthase I by 26.4%; and decreased the percentage I activity from 78 to 33% at ATP concentrations below 6 mM . When enzyme preparations were not preincubated prior to their assays, 90% of total glycogen synthase was in D form in control dogs . Under such conditions, endotoxin administration decreased the Vmax and Km for UDP-glucose for total glycogen synthase by 47.1 and 33.3%, respectively, and increased the A0.5 for glucose-6-P for the activation of glycogen synthase D by 24.2% at high (10 mM) and by 106% at low (1 mM) UDP-glucose concentration . From these results, it is clear that endotoxin administration greatly impaired hepatic glycogenesis by decreasing the activity of glycogen synthase; this impairment is at least in part responsible for the depletion of liver glycogen content in endotoxin shock . Kinetic analyses revealed that the decrease in the activity of glycogen synthase in endotoxic shock is a result of a decrease in the interconversion of this enzyme from inactive to active form and an increase in the interconversion from active to inactive form.

Am J Gastroenterol, 1987 Feb, 82(2), 171 - 2
Escherichia coli peritonitis after left-sided colonoscopy in a patient on continuous ambulatory peritoneal dialysis; Petersen JH et al.; A 69-yr-old man on chronic ambulatory peritoneal dialysis presented with Escherichia coli peritonitis 36 h after left-sided colonoscopy . No evidence for colonic perforation was found and the infection cleared with intravenous and intraperitoneal antibiotics . This case supports the need for further studies evaluating the use of prophylactic antibiotics during colonoscopy of chronic ambulatory peritoneal dialysis patients with diverticula.

J Lab Clin Med, 1987 Feb, 109(2), 211 - 6
Filterability of L-forms; Darwish RZ et al.; We investigated the recovery of L-forms through micropore filtration . The findings indicate that L-form recovery and viability are a function of filter size and preparation technique . Current methods in use for L-form isolation appear to give erroneously low results, underestimating the potential role of L-forms in human disease.

Mol Gen Genet, 1987 Feb, 206(2), 352 - 5
Cloning of seven differently complementing DNA fragments with chl functions from Escherichia coli K12; Reiss J et al.; Seven genomic libraries of chromosomal Escherichia coli K12 wild-type DNA were constructed in plasmid vectors . These were used to transform chl insertion mutants . Selection for growth on nitrate under anaerobic conditions yielded four plasmids which complemented mutants of the chlA, B, E and G types . The chromosomal fragments were mapped with restriction enzymes and subcloned . Three complementation groups were observed among the chlA mutants and two among the chlE mutants . The established complementation groups plus mutants of the chlD type represent eight distinct functions, which are all believed to be required for the molybdenum cofactor activity in the reduction of nitrate to nitrite by E . coli.

Mol Gen Genet, 1987 Feb, 206(2), 185 - 91
Multivalent regulation of the nusA operon of Escherichia coli; Ishihama A et al.; The rate of synthesis and intracellular content of the NusA protein, a transcription termination factor, were determined for wild-type and nusA and/or nusB mutants of Escherichia coli . Both the rate and content of NusA in wild-type strains were similar to that of the RNA polymerase sigma subunit, a transcription initiation factor, on a molar basis, and about 30%-40% the levels of RNA polymerase beta beta' subunits . At the stationary phase of cell growth, the values increased in parallel for both transcription factors up to approximately the level of the beta beta' subunits . In nus mutants, the rate of synthesis and the content of the sigma subunit were significantly increased . These observations together suggest that the two transcription factors are coordinately regulated.

EMBO J, 1987 Feb, 6(2), 507 - 13
Unusual properties of promoter-up mutations in the Escherichia coli galactose operon and evidence suggesting RNA polymerase-induced DNA bending; Kuhnke G et al.; Two mutations are described, each of which renders the Pribnow box sequence of one of the two overlapping promoters of the Escherichia coli galactose operon identical to the consensus sequence TATAAT . Both double exchanges were specifically introduced into the original context by oligonucleotide-directed mutation construction . Each of the mutant promoters exhibits a greatly enhanced capacity to form stable complexes with RNA polymerase, as judged by nuclease protection experiments and by assaying shifts of electrophoretic mobility . On the other hand, the effect of the same mutations on the rates of transcription from the two gal promoters is strikingly different . Unexpectedly, when complexed with RNA polymerase, DNA fragments carrying one of the two double exchanges were found to differ from each other as well as from the corresponding wild-type fragment with respect to their electrophoretic mobilities . These observations are indicative of different three-dimensional structures of these complexes which may reflect different forms of DNA bending induced in these otherwise identical fragments by complex formation with RNA polymerase.

EMBO J, 1987 Feb, 6(2), 501 - 5
Recombinant forms of M13 procoat with an OmpA leader sequence or a large carboxy-terminal extension retain their independence of secY function; Kuhn A et al.; The assembly of phage M13 procoat protein into the plasma membrane of Escherichia coli is independent of the secY protein . To test whether this is caused by the unusually small size of procoat, we fused DNA encoding 103 amino acids to the carboxy-terminal end of the procoat gene . The resulting fusion protein, which attains the same membrane-spanning conformation as mature coat protein, still does not require the secY function for membrane assembly . To determine whether the leader sequence governs interaction with the secY protein, we genetically exchanged the leader peptides between procoat and pro-OmpA, a protein which does require secY for its membrane assembly . Each of the resulting hybrid proteins assembles across the plasma membrane, though at a reduced rate . Membrane assembly of the fusion of procoat leader and OmpA required secY function, whereas assembly of the pro-OmpA leader/coat protein fusion was independent of secY . Properties of the entire procoat molecule, rather than its small size or a specific property of its leader peptide determines its mode of membrane assembly.

EMBO J, 1987 Feb, 6(2), 355 - 61
Expression and rescuing of a cloned human tumour necrosis factor gene using an EBV-based shuttle cosmid vector; Kioussis D et al.; A cosmid vector carrying the Epstein-Barr virus origin of replication, the EBNA-1 gene, the hygromycin phosphotransferase (hph) gene and pBR322 sequences has been constructed . This cosmid can replicate autonomously in the nucleus of human tissue culture cells, even when it carries a 35-kb long insert . The cosmid can be rescued from the transfected cells by cloning it directly into ampicillin-sensitive Escherichia coli . A gene for human tumour necrosis factor (TNF) cloned into this cosmid vector was introduced in tissue culture cells, where it was transcribed into mature mRNA.

Bioorg Khim, 1987 Feb, 13(2), 213 - 7
{The role of structural elements of ColE1-related plasmids in replication control}; Gurevich AI et al.; Effect of deletions downstream from the replication origin on copy number of ColE1 related (pBR322 derived) plasmids has been studied . Along with main control elements (RNAI of defined secondary structure, the repressor protein, effectiveness of promoter upstream of gene of RNA primer) replication is influenced by transcription level propagated into the replicon region from promoters even in distal parts of plasmid . Structure of the region adjacent to the ori is important for startpoint recognition specificity, so that deletion of this region reduces the effectiveness of replication.

Biochem Int, 1987 Feb, 14(2), 227 - 34
Transport of fatty acid is obligatory coupled with H+ entry in spheroplasts of Escherichia coli K12; Kameda K et al.; Transport of palmitate by spheroplasts of Escherichia coli K12 was studied . {14C}Palmitate was accumulated in spheroplasts approximately 1700-fold over the extracellular concentration of unbound {14C}palmitate . Uptake of {14C}palmitate was inhibited to 13% by addition of H+ uncoupler carbonyl cyanide-m-chlorophenylhydrazone (CCCP) . Spheroplasts exhibited the uptake of 9-aminoacridine depending on the addition of palmitate to the incubation mixture . The rate of {14C}palmitate uptake by the spheroplasts pre-equilibrated in a buffer at pH 7.5 or 8.0 significantly increased in comparison with the spheroplasts pre-equilibrated in a buffer at pH 7.0 when the spheroplasts were incubated at an external pH of 7.0.

Mol Gen Mikrobiol Virusol, 1987 Feb, (2), 17 - 20
{Protein Z is not an endonuclease of the recF recombination pathway in Escherichia coli K12}; Kil' IuV et al.; A 74 kD protein was extracted from Escherichia coli cells and purified under the physiological conditions . The protein is able to catalyze the reactions of endonucleolytic degradation of plasmid DNA . The genetic determinant coding for the 74 KD protein synthesis has been localized between 17 and 27 min on Escherichia coli chromosomal map . The endonuclease previously described as a recF gene dependent "protein Z" (Krivonogov S . V., Novitskaja V . A . Mol . Gen . Genet., 1982, v, 187, p . 302) is shown to be independent of the integrity of Escherichia coli recF gene.

Genetika, 1987 Feb, 23(2), 197 - 201
{Effect of promoter duplication in the synthetic interferon gene on the level of its expression}; Il'ichev AA et al.; The expression of the artificial gene for the human leukocyte interferon (IFN) synthesized by the chemo-enzymic method has been studied in Escherichia coli cells . The genetic constructions in which the IFN gene transcription is carried out from A2 and B promoters of T7 phage or from E . coli lacZ UV5 gene promoter have been obtained, based on pEMB101 plasmid . In addition, the level of interferon production, depending on the promoter used, has been studied . It has been shown that the tandemly located promoters increase efficiency of the IFN gene expression.

Am J Pathol, 1987 Feb, 126(2), 350 - 7
Mechanism of Escherichia coli alpha-hemolysin-induced injury to isolated renal tubular cells; Keane WF et al.; Alpha-hemolysin (AH) is a 110,000-dalton protein secreted extracellularly by certain Escherichia coli . This protein is an acknowledged virulence factor for E coli and recently has been implicated as an important determinant in the pathogenesis of E coli pyelonephritis . Recombinant engineered strains of E coli were used that varied only in their ability to secrete AH extracellularly . The effect of AH on vital dye exclusion, oxygen consumption rate (QO2) adenosine triphosphate (ATP) levels, superoxide (O2-) and hydrogen peroxide (H2O2) production in preparations of isolated rat cortical renal tubular cells (RTCs) was assessed . Approximately 5-10 pg of AH dramatically stimulated QO2 by nearly 150% . This was associated with a marked increase in production of O2- and H2O2, to 13.9 +/- 1.7 and 13.2 +/- 2.1 nM/mg cell protein, respectively (P less than 0.05), as well as a 38% decrease in cellular ATP . These biochemical effects were all seen after a 30-minute exposure to AH and by 120 minutes were associated with 15.7% +/- 1.1% of RTCs that were unable to exclude vital dye . The effect of AH on QO2 and O2- formation was prevented by pretreatment of RTCs with ouabain, which indicates that the effect of AH on oxygen metabolism is linked to Na-K ATPase activity . However, when ouabain-treated RTCs were exposed to AH, ATP remained depressed despite the inhibition of QO2 and O2- production . In contrast, in ouabain-pretreated RTCs, cell membrane integrity was dramatically protected, because only 2.4% +/- 0.4% of RTCs were not unable to exclude vital dye . Thus, the data demonstrate that E coli AH provokes at least two biochemical events that may be injurious to RTC: increased oxygen intermediates (O2- and H2O2 and ATP depletion . These findings with ouabain suggest that the first mechanism of injury may be a more proximate cause of cell death . Moreover, the data suggest that endogenous production of reactive oxygen molecules may be critical modulators of RTC membrane injury.

Mutat Res, 1987 Feb, 190(2), 77 - 81
Inducible DNA polymerase I synthesis in a UV hyper-resistant mutant of Escherichia coli; Ahmad SI et al.; A mutant of Escherichia coli which is more resistant to shortwave UV light than its wild-type parent strain and which can synthesise DNA polymerase I constitutively has been further analysed . It carries two mutational alleles which are located about 1.5 min apart and cotransducible by P1 with the argH locus . The two mutational alleles have been segregated and their analysis shows that one of them is responsible for UV hyper-resistance whereas the other mutation confers UV sensitivity . Recombinant plasmids carrying various sections of the polA regulatory region, linked to a galK gene, were introduced into the mutant strains . Analysis of galactokinase shows that the enzyme activity in the UV hyper-resistant mutant is increased . The results suggest that the synthesis of DNA polymerase I in E . coli is inducible.

J Virol, 1987 Feb, 61(2), 621 - 4
A nonstructural protein of feline panleukopenia virus: expression in Escherichia coli and detection of multiple forms in infected cells; Carlson JO et al.; Sequences coding for the nonstructural protein NS1 of the autonomous parvovirus feline panleukopenia virus were expressed in Escherichia coli as fusion proteins . The fusion proteins were specifically bound by antisera from canine parvovirus-infected dogs . Antisera against one of the fusion proteins bound to several proteins found only in feline panleukopenia virus-infected feline cells.

J Virol, 1987 Feb, 61(2), 446 - 53
Primary structure and transcription of the genes coding for the two virion phosphoproteins pp65 and pp71 of human cytomegalovirus; Ruger B et al.; Human cytomegalovirus contains a phosphorylated matrix protein of 65,000 apparent molecular weight (65K phosphoprotein; pp65) and a related phosphoprotein of 71,000 molecular weight (pp71) . The 65K phosphoprotein is usually by far the most abundant structural component found in culture-grown purified virus particles . This study describes the precise mapping of the genes for both polypeptides, giving the entire nucleotide sequences and the exact positions of the respective transcripts . The 65K phosphoprotein is coded for by the 5'-terminal part of an abundant 4-kilobase (kb) mRNA . The 71K phosphoprotein corresponds to the single translational reading frame of a rare nonspliced 1.9-kb mRNA that is coterminal with the 4-kb transcript . The promoter for 4-kb mRNA appears to be unusual in structure; it does not contain a characteristic TATA sequence . The expression of antigenic epitopes from pp65 may allow improved serodiagnosis of human cytomegalovirus infections.

J Virol, 1987 Feb, 61(2), 302 - 7
Expression of the Kirsten ras viral and human proteins in Escherichia coli; Nakano ET et al.; The expression vectors pINIII-A and pINIII (lpp p5) were used to construct plasmids which direct the synthesis in Escherichia coli of the Kirsten ras viral (v-Ki-ras) and human cellular (c-Ki-ras) oncogene products as fusion proteins containing 9 and 10 extra amino acids, respectively, at their N termini . Authenticity of the bacterially produced proteins was determined by immunoprecipitation and immunoblot analyses with ras-specific monoclonal antibodies . After induction with isopropyl-beta-D-thiogalactopyranoside, the viral protein represented approximately 20% of the total cellular protein . The majority of the protein was found in the postsonication low-speed centrifugation pellet . The synthesized viral protein was active in GTP binding, as judged by autophosphorylation and photoaffinity labeling assays.

J Bacteriol, 1987 Feb, 169(2), 888 - 90
Reduced transposition in rho mutants of Escherichia coli K-12; Datta AR et al.; Substantially reduced frequencies of transposition for the transposons Tn5 and Tn9 and the insertion sequences IS1 and IS5 were observed in several rho mutants of Escherichia coli K-12 compared with those observed in their isogenic wild-type counterparts . The lower transposition frequencies could be due to decreased supercoiling of DNA, to altered expression of required genes, or to aberrant transcription of transposon or target DNA resulting from the lack of transcription termination at Rho-sensitive sites in rho mutants.

J Bacteriol, 1987 Feb, 169(2), 751 - 7
Processing of the initiation methionine from proteins: properties of the Escherichia coli methionine aminopeptidase and its gene structure; Ben-Bassat A et al.; Methionine aminopeptidase (MAP) catalyzes the removal of amino-terminal methionine from proteins . The Escherichia coli map gene encoding this enzyme was cloned; it consists of 264 codons and encodes a monomeric enzyme of 29,333 daltons . In vitro analyses with purified enzyme indicated that MAP is a metallo-oligopeptidase with absolute specificity for the amino-terminal methionine . The methionine residues from the amino-terminal end of the recombinant proteins interleukin-2 (Met-Ala-Pro-IL-2) and ricin A (Met-Ile-Phe-ricin A) could be removed either in vitro with purified MAP enzyme or in vivo in MAP-hyperproducing strains of E . coli . In vitro analyses of the substrate preference of the E . coli MAP indicated that the residues adjacent to the initiation methionine could significantly influence the methionine cleavage process . This conclusion is consistent, in general, with the deduced specificity of the enzyme based on the analysis of known amino-terminal sequences of intracellular proteins (S . Tsunasawa, J . W . Stewart, and F . Sherman, J . Biol . Chem . 260:5382-5391, 1985).

J Bacteriol, 1987 Feb, 169(2), 624 - 31
Tn2501, a component of the lactose transposon Tn951, is an example of a new category of class II transposable elements; Michiels T et al.; Tn2501 is a cryptic class II transposon found as part of the lactose transposon Tn951 . Insertional inactivation and nucleotide sequence analysis of Tn2501 allowed us (i) to localize the transposase (tnpA) and the resolvase (tnpR) genes as well as the resolution site (res) of Tn2501 and (ii) to compare Tn2501 with other well-known elements of the two subgroups of class II transposons (Tn3, gamma delta, Tn951, IS101; and Tn21, Tn501, Tn1721) . The genetic organization of Tn2501 is similar to that of Tn3 with divergent transcription of the tnpA and tnpR genes away from the intervening res site . The tnpR gene of Tn2501 shows weak homology with that of Tn3 and even less with those of Tn21 and Tn501 . However, the tnpA gene and the inverted repeat sequences of Tn2501 present more homology with those of Tn21 and Tn501 than with those of Tn3 . Complementation studies showed that TnpA- mutants of Tn2501 can be complemented, at a low frequency, by the Tn21 transposase . None of the tested transposons complemented TnpR- mutants of Tn2501.

J Bacteriol, 1987 Feb, 169(2), 619 - 23
Repressor gene finO in plasmids R100 and F: constitutive transfer of plasmid F is caused by insertion of IS3 into F finO; Yoshioka Y et al.; Fertility factor F confers bacterial conjugation, a process which involves at least 20 tra genes . Resistance plasmids such as R100, R6-5, and R1 have homology with F in the tra region . Conjugal transfer of these plasmids is, however, repressed, while transfer of F is constitutive . Repression of R transfer is due to the existence of the two genes, called finO and finP; constitutive transfer of F is believed to be due to a lack of finO in F . In this paper, we report the identification and DNA sequence of the finO gene of R100, encoding a protein of 21,265 daltons . We show that F does actually encode finO, but the gene has been inactivated by insertion of IS3 . Lederberg and Tatum (Nature {London} 158:558, 1946), who discovered sexuality in bacteria, may have had an Escherichia coli K-12 strain harboring such an finO F factor, which facilitated the generation of recombinant progeny useful for genetic analysis of bacteria and established the foundation for molecular genetics.

J Bacteriol, 1987 Feb, 169(2), 600 - 4
Effects of deletion and insertion mutations in the ilvM gene of Escherichia coli; Lu MF et al.; A plasmid was constructed that carried the ilvG and ilvM genes and the associated promoter and leader regions derived from the K-12 strain of Escherichia coli . The ilvG gene contained a + 1 frameshift mutation that enabled the plasmid to specify acetohydroxyacid synthase II . The plasmid was modified by deletions in the terminus of and within the ilvM gene and by insertions into the ilvM gene . The effects of these modifications on the phenotypes of the plasmids were examined in a host strain that lacked all three isozymes of acetohydroxyacid synthase . Most of the ilvM mutant plasmids so obtained permitted growth of the host strain in the absence of isoleucine but not in the absence of valine . Growth in the presence of valine, however, was very slow . No significant acetohydroxyacid synthase activity could be detected even when the cells were grown in a valine-supplemented minimal medium . It thus appears that, at most, only a very low level of acetohydroxyacid synthase activity occurred with ilvG in the absence of ilvM and that low activity was more effective for acetohydroxy butyrate formation than for acetolactate formation . The ilvM gene product could be formed under the control of the lac promoter in the presence of a plasmid that carried an in-frame gene fusion between lacZ and the downstream portion of ilvG . Extracts from the host strain that contained such an IlvG(-)-IlvM+ plasmid could be combined with extracts from cells that contained one of the IlvG+-IlvM- plasmids to yield acetohydroxyacid synthase activity . Thus, the ilvM and ilvG genes could be expressed independently of each other.

J Bacteriol, 1987 Feb, 169(2), 593 - 9
Indirect role of adenylate cyclase and cyclic AMP in chemotaxis to phosphotransferase system carbohydrates in Escherichia coli K-12; Vogler AP et al.; Most strains of Escherichia coli K-12 lacking the enzyme adenylate cyclase showed normal chemotaxis toward carbohydrates taken up and phosphorylated by the phosphoenolpyruvate-dependent carbohydrate: phosphotransferase system . The normal reaction was observed even in the absence of externally added cyclic adenosine 3',5'-phosphate, provided that the enzyme II chemoreceptors and the flagella were synthesized . In the CA8306 series of strains, however, the cya-854 deletion abolished chemotaxis toward phosphotransferase system carbohydrates even though growth on and transport of these carbohydrates were not affected . This abnormal phenotype was due to the presence of a specific mutation in strain CA8306 which mapped in or close to the crp locus and apparently prevented expression of a hitherto unidentified molecule involved in enzyme II-mediated signal transduction . This molecule is neither a pts protein nor a cyclic adenosine 3',5'-phosphate-binding protein.

J Bacteriol, 1987 Feb, 169(2), 546 - 52
Expression of a tRNA gene in the context of the lacZ mRNA; Murakawa GJ et al.; Fusions of the gene for tyrosine suppressor tRNA, tyrT(Sup3), and the lacZ gene of Escherichia coli were constructed such that the tRNA gene could be expressed from either its own promoter or that of the lac operon . These chimeras, carried on phage M13 vectors, were tested for the expression of the tRNA in E . coli . The tRNA gene was expressed on the order of 10-fold more weakly from the lac promoter than from its own promoter . To examine whether pausing or premature termination of transcription played a role in determining the relative strength, the fusions were tested in a variety of genetic backgrounds and under different physiological conditions that uncouple transcription and translation . The expression of the tRNA was not enhanced in backgrounds in which polarity was weakened or under the other conditions tested, although a dependence on nusB function was observed when the tRNA was transcribed from the lac promoter . These results indicate that pausing or premature termination of transcription did not play a role in the weak expression of the gene fusions . The results further suggest that the transcription of the tyrT gene does not normally require relief from polarity as imposed by any of the known transcriptional termination systems, in contrast to the antitermination system thought to be involved in the expression of the rRNAs.

J Bacteriol, 1987 Feb, 169(2), 526 - 32
Divergent transcription of the sn-glycerol-3-phosphate active transport (glpT) and anaerobic sn-glycerol-3-phosphate dehydrogenase (glpA glpC glpB) genes of Escherichia coli K-12; Ehrmann M et al.; The glpTQ operon and the glpA and glpB genes are located adjacent to one another near min 49 of the linkage map of Escherichia coli K-12 . The positions and directions of transcription of the glpA and glpB genes with respect to the glpTQ operon were determined in the present work . Strains harboring Mu d1(Ap lac) fusions in either glpA or glpB were converted to the respective lambda p1(209) lysogens . Induction of these lysogens with mitomycin C resulted in production of Lac+ phage progeny which carried adjacent chromosomal DNA . Genetic crosses with a collection of glpT mutant strains were performed with several such phage lines . A fine-structure deletion map of the glpT gene was thus constructed . All phages used for this mapping carried DNA starting with the promoter-proximal end of glpT . This indicated that the glpTQ operon and the glpA and glpB genes are transcribed divergently . Additional evidence supporting this conclusion was obtained by physical mapping of restriction endonuclease cleavage sites in plasmids carrying these genes and in plasmids carrying glpA-lacZ or glpB-lacZ fusions . A new designation (glpC) for the gene encoding the 41,000-Mr subunit of the anaerobic sn-glycerol-3-phosphate dehydrogenase was proposed to distinguish it from the glpA gene, which encodes the 62,000-Mr subunit of the dehydrogenase, and the glpB gene, which encodes a membrane anchor subunit of the dehydrogenase . These three genes were present in an operon transcribed in the order glpA glpC glpB in the clockwise direction on the linkage map of E . coli.

J Bacteriol, 1987 Feb, 169(2), 507 - 13
Cloning and characterization of the aerobic sn-glycerol-3-phosphate dehydrogenase structural gene glpD of Escherichia coli K-12; Schweizer H et al.; The glpD gene encoding aerobic sn-glycerol-3-phosphate dehydrogenase of Escherichia coli K-12 was cloned into pACYC177 from a lambda glpD transducing phage . The recombinant plasmid, designated pSH55, carried a 7.4-kilobase-pair HindIII fragment containing the glpD and glpR genes . The glpD gene was subcloned into pACYC177 on a 4.4-kilobase-pair BamHI-HindIII fragment . Expression of the cloned glpD gene was regulated in the manner previously described for the chromosomal glpD gene . The position of glpD on this plasmid was determined by Tn1000 insertional inactivation experiments . The glpD gene product, a polypeptide of Mr 55,000, was detected in a maxicell system . Truncated polypeptides replaced the 55,000-molecular-weight polypeptide when plasmid derivatives harboring Tn1000 insertions that inactivate glpD were used as templates . The sizes of these polypeptides confirmed the previously determined direction of transcription and allowed estimation of the translation start site . Determination of the apparent Mr of a hybrid protein encoded by a glpD'-'lacZ fusion provided additional evidence for the position of the glpD control region . The amino-terminal 30 to 60 amino acids of this hybrid protein (provided by glpD) were sufficient for efficient membrane localization of glpD'-'lacZ-encoded beta-galactosidase activity . The glpD3 mutation was mapped within the glpD gene, providing additional evidence that glpD is the structural gene for aerobic sn-glycerol-3-phosphate dehydrogenase.

J Histochem Cytochem, 1987 Feb, 35(2), 229 - 31
Polymyxin B binding sites in Escherichia coli as revealed by polymyxin B-gold labeling; Morioka H et al.; A complex of polymyxin B, bovine serum albumin, and colloidal gold was prepared and used for the ultrastructural localization of polymyxin B binding sites on thin sections of Epon-embedded Escherichia coli cells . Gold particles were found on the outer membrane of E . coli, which is consistent with reported biochemical findings . We concluded that gold labeling with polymyxin B is useful in localizing the binding sites of polymyxin.

Scand J Dent Res, 1987 Feb, 95(1), 59 - 64
In vitro studies of monocyte function in two siblings with Papillon-Lefèvre syndrome; Preus HR et al.; Blood monocytes were isolated from two siblings with Papillon-Lefevre syndrome (PLs) and compared to corresponding cells from their healthy cousin . The number of monocytes isolated were within normal limits in all three test participants . Aggregating tendency was increased when PLs monocytes were cultured in the presence of autologous sera . The monocyte ability of specific immune phagocytosis was decreased in PLs patients . The monocyte morphology, non-specific phagocytosis, lysosomal enzyme activities, and response to E . coli endotoxin were similar in patients and control.

Microb Pathog, 1987 Feb, 2(2), 113 - 21
Monoclonal antibodies raised against Pap fimbriae recognize minor component(s) involved in receptor binding; de Ree JM et al.; Pap fimbriae were purified from a recombinant strain and used for the production of monoclonal antibodies (MAbs) . These MAbs were screened in a fimbriae ELISA with eight different P fimbriae as well as 1A and 1C fimbriae . Five MAbs were specific for Pap fimbriae whereas one MAb did react with Pap, F7(2) and F11 fimbriae . Previously, we described two F11 MAbs which also reacted with Pap, F7(2) and F11 fimbriae . In a whole bacteria ELISA it was shown that the MAbs, which recognized Pap, F7(2) and F11 fimbriae, reacted with recombinant strains which did not express Pap or F11 fimbriae, but still expressed the globoside binding properties . Not one of the five MAbs which are specific for Pap fimbriae reacted with these globoside binding recombinant strains . In a haemagglutination and adherence assay it was shown that only the MAbs which recognized the Pap, F7(2) and F11 fimbriae inhibited the adhesive properties of the globoside binding recombinant strain . Therefore it is concluded that in the present study MAbs are presented which recognize the minor components responsible for adhesion.

J Gen Microbiol, 1987 Feb, 133 ( Pt 2), 323 - 30
Isolation of a repeated DNA sequence from Bordetella pertussis; McPheat WL et al.; A repeated DNA sequence in the genome of Bordetella pertussis has been demonstrated . At least 20 copies of this sequence could be observed in either BamHI or EcoRI restriction enzyme digests of chromosomal DNA; fragments carrying the repeated DNA sequence ranged in size from about 1.5 to 20 kbp . The repeated DNA sequence was cloned from two separate regions of the B . pertussis genome, as shown by restriction enzyme site maps of the two clones and by hybridization studies . A small number of differences in the pattern of hybridization of the repeated DNA sequence to chromosomal DNA from several strains of B . pertussis was observed . No repeated DNA sequences were observed in one strain each of B . parapertussis and B . bronchiseptica, and there was no hybridization of B . pertussis DNA to Escherichia coli chromosomal DNA . The repeated DNA sequence was subcloned on a 2.54 kbp BamHI fragment from one of the two original clones . Restriction enzyme digests and hybridization studies showed that the repeated DNA sequence was about 1 kbp in size and had a single, internal ClaI site.

J Biochem (Tokyo), 1987 Feb, 101(2), 511 - 7
Molecular cloning and sequencing of cDNA encoding goat pre alpha-lactalbumin; Kumagai I et al.; In poly(A)+RNA extracted from a lactating goat mammary gland, mRNA of about 750 nucleotides was shown to encode pre alpha-lactalbumin by using in vitro translation and immunoprecipitation . From the total poly(A)+RNA, the cDNA library was constructed using the Escherichia coli plasmid pUC18; it was screened with the oligodeoxyribonucleotide probe corresponding to the amino acid sequence of Trp60-Gln65 of goat alpha-lactalbumin . A plasmid containing almost full-length cDNA of goat pre alpha-lactalbumin, pGLA-1, was identified . The cDNA insert of pGLA-1 comprises 727 base pairs and contains the signal peptide and mature protein sequence.

Arch Microbiol, 1987 Feb, 147(1), 1 - 7
Osmoregulation in Escherichia coli by accumulation of organic osmolytes: betaines, glutamic acid, and trehalose; Larsen PI et al.; It has been shown previously that externally added glycine betaine is accumulated in Escherichia coli in response to the external osmotic strength . Here we have shown, by using nuclear magnetic resonance spectroscopy and radiochemical methods, that E . coli growing in a glucose-mineral medium of elevated osmotic strength generated with NaCl, had the same capacity to accumulate proline betaine and glycine betaine . Its capacity to accumulate gamma-butyrobetaine was, however, 40 to 50% lower . Accordingly, externally added proline betaine and glycine betaine stimulated aerobic growth of osmotically stressed cells equally well, and they were more osmoprotective than gamma-butyrobetaine . In cells grown at an osmotic strength of 0.64, 1.01, or 1.47 osmolal, respectively, the molal cytoplasmic concentration of the two former betaines corresponded to 29, 38, or 58% of the external osmotic strength . Nuclear magnetic resonance spectroscopy revealed that trehalose and glutamic acid were the only species of organic osmolytes accumulated in significant amounts in cells grown under osmotic stress in glucose-mineral medium without betaines . Their combined molal concentration in the cytoplasm of cells grown at 1.01 osmolal corresponded to 27% of the external osmotic strength.

J Bacteriol, 1987 Feb, 169(2), 735 - 41
Inhibition of adhesive activity of K88 fibrillae by peptides derived from the K88 adhesin; Jacobs AA et al.; A cyanogen bromide fragment derived from the K88ab adhesin inhibited the hemagglutinating activity of K88 fibrillae . Smaller fragments which inhibited the adherence of K88 fibrillae to erythrocytes or to intestinal epithelial cells were obtained by digestion of K88ab fibrillae with alpha-chymotrypsin . Active peptides were isolated from the digestion mixture and identified as Ser-Leu-Phe and Ala-Ile-Phe . Both tripeptides correspond to the peptide stretches Ser-148-Leu-Phe-150 and Ala-156-Ile-Phe-158, respectively, which are part of conserved regions in the primary structure of the K88 variants ab, ac, and ad . The isolated tripeptides inhibited the hemagglutinating activity of purified K88 fibrillae in the 1 to 5 microM range, while adherence of the fibrillae to intestinal epithelial cell brush borders was inhibited in the 10 to 50 microM range . Furthermore, the tripeptides were capable of eluting attached bacteria from agglutinated erythrocytes . The inhibitory activity of the isolated peptides was confirmed by testing various synthetic peptides for their ability to inhibit the interaction of the different K88 variants with various species of erythrocytes . The significance of these findings for the localization of the receptor-binding domain is discussed.

J Bacteriol, 1987 Feb, 169(2), 640 - 5
Identification and characterization of genes determining receptor binding and pilus length of Escherichia coli type 1 pili; Maurer L et al.; We describe the identification and characterization of two genes and their gene products responsible for determining receptor binding and pilus length in type 1-piliated Escherichia coli . One gene, pilE, conferred the ability of piliated cells to agglutinate guinea pig erythrocytes . The other gene, pilF, determined pilus length, in that mutants having lesions in pilF had very long pili . The two genes were detected after Tn5 mutagenesis of a cloned segment of DNA that normally complemented a pilE lesion in the chromosome . Thus, lesions in pilE or pilF on the cloned segment resulted in mutants having the PilE- phenotype (piliated but unable to agglutinate erythrocytes) . Introduction of the plasmid-encoded mutant alleles of pilE and pilF into the chromosome followed by electron microscopic examination of the mutants showed that only lesions in pilF conferred the striking increase in pilus length . Mutations in pilF could be complemented in trans by the original cloned segment to produce cells with parental-length pili . Minicell transcription and translation of the cloned pilE and pilF genes having representative Tn5 insertion mutations showed that the pilE gene product was a protein of ca . 31 kilodaltons and that the pilF gene product was a protein of ca . 18 kilodaltons . We believe that the pilF gene product may act as a competitive inhibitor of pilus polymerization . Thus, pilus length may be controlled by the ratio of pilin to pilF gene product present within the cell.

Infect Immun, 1987 Feb, 55(2), 393 - 402
Adherence to human colonocytes of an Escherichia coli strain isolated from severe infantile enteritis: molecular and ultrastructural studies of a fibrillar adhesin; Hinson G et al.; Escherichia coli 469-3 (O21:H-) was isolated from a child with severe enteritis . Ultrastructural analysis of the surface of the strain indicated the presence of very fine fimbriae which mediated mannose-resistant hemagglutination of human blood and caused the bacteria to adhere to human epithelial cell lines and to brush borders of isolated human colonic, but not duodenal, enterocytes . A cosmid library of total DNA of the strain, expressed in laboratory strains of E . coli, was screened by a rapid hemadsorption method, and a number of positive clones were identified . Restriction endonuclease fragments specifying mannose-resistant adherence were subcloned from the cosmid DNA of a strongly hemagglutinating clone in a plasmid vector . The identity of the adhesin was confirmed by biochemical, electron-microscopic, and immunological comparisons with the adhesin synthesized by the clinical isolate . It comprised a high-molecular-weight aggregate of a 14,000-dalton subunit protein which bound antiserum raised against the mannose-resistant adhesin of strain 469-3 . The adhesin was synthesized by both the clone and the parental strain at growth temperatures above 18 degrees C but by only a fraction of the cells in a pure culture, although all the bacteria which adhered to human cells expressed the protein.

J Med Microbiol, 1987 Feb, 23(1), 19 - 28
A model of acute infectious neonatal diarrhoea; Newsome PM et al.; Oral inoculation of neonatal MFI mice with enterotoxigenic strains of Escherichia coli that possessed the K99 or F41 antigen or both resulted in severe diarrhoea with high mortality . The diarrhoea was associated with increased fluid in the gut, greatly increased numbers of E . coli in gut homogenates and reduced weight gain compared to control animals . Further studies with strain B44 demonstrated greatly increased numbers of E . coli on the surface of the intestinal mucosa and haemo-concentration . The infection was transmissible between litter-mates . There was no evidence of invasion of the intestinal tissue of infected animals . Gnotobiotic Balb C mice and endotoxin-resistant mice were susceptible to oral inoculation with bovine enterotoxigenic E . coli strains, but neonatal rats were not susceptible to infection with enterotoxigenic E . coli strains B44 or 431 . Porcine strains of E . coli that possessed K88 or 987P antigen did not infect neonatal MFI mice but an "atypical" porcine strain (431) which possessed both K99 and F41 antigens caused diarrhoea and a high mortality . The disease in neonatal mice resembled acute diarrhoea caused by these bacteria in other species, particularly the calf, and the model should be of value in assessing the efficacy of therapeutic agents.

EMBO J, 1987 Feb, 6(2), 485 - 91
A malaria protein exported into a new compartment within the host erythrocyte; Simmons D et al.; A Plasmodium falciparum protein which is exported into a new compartment in the host erythrocyte has been located . This protein, exp-1, has a variable region recognized by a monoclonal antibody . Naturally occurring mutants of this region have been characterized . All mutants studied so far have the same A----G transition abolishing the target for the antibody . The exp-1 gene has a complex structure containing two introns . It is highly conserved in five independent, genetically defined parasite lines, suggesting that exp-1 has an important function . We discuss the possible role of exp-1 in P . falciparum infections.

Anal Biochem, 1987 Feb 1, 160(2), 332 - 6
A procedure for large-scale isolation of RNA-free plasmid and phage DNA without the use of RNase; Lev Z; A preparative procedure for the large-scale isolation of plasmid DNA without the use of RNAse is described . Crude plasmid DNA is prepared using a standard boiling method . High-molecular-weight RNA is removed by precipitation with LiCl, and low-molecular-weight RNA is removed by sedimentation through high-salt solution . The procedure is inexpensive, rapid, simple, and particularly suitable for processing several large-scale preparations simultaneously . A similar procedure has been developed for preparation of lambda-phage DNA.

Can J Physiol Pharmacol, 1987 Feb, 65(2), 185 - 90
Effects on the murine mononuclear phagocyte system of chronic administration of liposomes containing cytotoxic drug or lipid A compared with empty liposomes; Allen TM et al.; In experiments designed to examine the adverse effects of chronic liposome administration in vivo on the mononuclear phagocyte system (reticuloendothelial system), the presence of drug entrapped in the liposomes may increase the level of reticuloendothelial impairment . We have compared the effects on the mononuclear phagocyte system in mice of chronic administration of empty liposomes with the effects of liposomes containing the anti-leishmanial drug meglumine antimoniate . We have also examined the effect on the mononuclear phagocyte system of continued injections of liposomes containing lipid A, a component of bacterial lipopolysaccharide, which is responsible for macrophage activation . Ten intravenous injections of multilamellar liposomes composed of dipalmitoylphosphatidylcholine and cholesterol (1:0.75 M ratio) were given to ICR mice over a 25-day period . Two individual groups of mice received endotoxin-free liposomes in which meglumine antimoniate was either present or absent . One addition group received liposomes containing lipid A derived from Escherichia coli lipopolysaccharide . A control group received sterile saline injections . In each group, a depression of the phagocytic index, as measured by reduction of uptake of particulate carbon, was observed among some of the individual animals 24 h after the first injection . In many mice a marked splenomegaly was observed . A depressed phagocytic index and splenomegaly were most marked for mice receiving lipid A liposomes . However, there was a large individual variability among mice receiving these preparations and some mice in each group had normal spleen size and a nearly normal phagocytic index . Tissue distribution of liposomes containing {14C}dipalmitoylphosphatidylcholine as a phospholipid marker was examined in all groups in mice 24 h after the last injection.(ABSTRACT TRUNCATED AT 250 WORDS)

J Gen Virol, 1987 Feb, 68 ( Pt 2), 315 - 23
A neutralizing epitope on human rhinovirus type 2 includes amino acid residues between 153 and 164 of virus capsid protein VP2; Skern T et al.; Use has been made of a monoclonal antibody (designated 8F5) to map a neutralizing epitope on the viral capsid protein VP2 of human rhinovirus 2 (HRV2) . This antibody which was raised against the native virus, neutralizes HRV2 and is also capable of recognizing denatured VP2 on Western blots . To examine the binding site of 8F5, VP2 of HRV2 was expressed in Escherichia coli . Deletions starting at the 3' end were then introduced into the gene for VP2 using Bal-31 nuclease . Polypeptides shortened at the carboxy terminus of VP2 were obtained from the deletions and were blotted onto nitrocellulose . The samples were then probed with monoclonal antibody 8F5 . Recognition by 8F5 was maintained as long as the expressed polypeptide contained the VP2 sequence up to amino acid 164 or beyond . However, when the VP2 sequence was truncated to amino acid 153 or less 8F5 was no longer able to bind . The neutralization epitope (or part of it) recognized by 8F5 on VP2 is therefore located between amino acids 153 and 164.

J Virol, 1987 Feb, 61(2), 534 - 42
Proteolytic processing of avian sarcoma and leukosis viruses pol-endo recombinant proteins reveals another pol gene domain; Alexander F et al.; Three pol gene products have been identified in avian retroviral particles: the full-length 95-kilodalton (kDa) beta chain of reverse transcriptase and two proteolytic cleavage products of beta, a 63-kDa reverse transcriptase alpha chain derived from the amino terminus of beta and a 32-kDa (pp32) endonuclease from its carboxy terminus . By using molecularly cloned retroviral DNA and synthetic oligonucleotides to introduce initiator ATGs and codons corresponding to the authentic N termini, we constructed two bacterial-expression clones; one clone contains the entire pol gene, and the other contains the region encoding the pp32 domain . A 99-kDa protein was synthesized in Escherichia coli by the full-length clone, and a 36-kDa protein was synthesized by the endonuclease domain clone . The recombinant proteins exceeded the size of both the mature viral beta chain and the pp32, respectively, by approximately 4 kDa . These larger sizes, however, are consistent with predictions from the DNA sequence of the pol gene . Processing of the recombinant pol proteins was examined by using p15 protease purified from virus particles and antisera directed against synthetic peptides corresponding to three domains in pol . Proteolytic digestion of the 99-kDa product with p15 produced a 63-kDa protein that comigrated on polyacrylamide gels with the alpha chain of reverse transciptase and a 36-kDa fragment that comigrated with the endonuclease domain product . Further digestion of the 36-kDa protein yielded a 32-kDa protein that comigrated with viral pp32 endonuclease . Thus, we concluded that two p15-sensitive sites exist in pol . Cleavage at the previously identified site produces alpha, and cleavage at the newly discovered site removes approximately 4 kDa from the C terminus of the primary protein product . Since the 36-kDa protein was also detected in protein isolated from virus particles, it seems probable that processing at the C-terminal site is a normal step in the production of mature beta and pp32 endonuclease products.

Biochim Biophys Acta, 1987 Jan 30, 911(2), 180 - 90
Nonspecific inhibition of Escherichia coli ornithine decarboxylase by various ribosomal proteins: detection of a new ribosomal protein possessing strong antizyme activity; Kashiwagi K et al.; Escherichia coli ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) was found to be inhibited by several basic proteins . When ribosomal proteins were tested, major ribosomal proteins, with the exceptions of S1, S5, S6, S8, S10, L3, L5, L6, L7/L12, L8, L9 and L10 proteins, showed antizyme activity in addition to the recognized antizymes (S20/L26 and L34 proteins) . Furthermore, it was found that L20 protein and a new ribosomal protein, tentatively named X1 protein and bound to 50 S ribosomal subunits, showed stronger antizyme activity than S20/L26 and L34 proteins . The antizyme activity of S20/L26 and L34 proteins was at most 10% of the total antizyme activity of ribosomal proteins . Several basic polypeptides also showed antizyme activity in the order polyarginine greater than protamine greater than histone greater than polylysine . Ribosomal proteins and basic polypeptides inhibited ornithine decarboxylase activity competitively . Ribosome-bound antizymes were inactive as antizymes, and antizyme inhibition of ornithine decarboxylase was eliminated by ribosomes . When E . coli extracts were separated into ribosomes and 100,000 X g supernatant fraction, no significant antizyme activity was observed in the supernatant fraction . Results of these in vitro experiments infer that basic antizymes may not function as inhibitors of ornithine decarboxylase in vivo.

J Chromatogr, 1987 Jan 30, 387, 139 - 54
Effect of zinc ions on tRNA structure . I . Reversed-phase chromatography; Flanagan JM et al.; The effect of zinc on the chromatographic behavior of four tRNAs was examined on RPC-5 and Aminex A-28 columns . RPC-5 contains dichlorodifluoroethylene beads coated with a quaternary ammonium compound where the substituents are: R1 = methyl, and R2-4 = C8-10 hydrocarbons . Aminex A-28 contains quaternary ammonium covalently attached to styrene-divinylbenzene copolymer lattice and R1-3 are methyl groups . The retentions of tRNAVal, tRNAIle, and tRNALys of E . coli and yeast tRNAPhe on RPC-5 were all markedly increased by Zn2+ ions . In contrast, no increased retention due to Zn2+ was observed when tRNAPhe was chromatographed on Aminex A-28 . A model for chromatography on RPC-5 is developed which treats the elution behavior of tRNAs from this matrix as the sum of ion-exchange and hydrophobic interactions . The chromatography of tRNA in the presence and absence of Zn2+ is interpreted in terms of this model and the effects of sodium chloride concentration, temperature, and pH were explored as the experimental variables . These experiments suggest that in the absence of Zn2+ tRNA does not interact appreciably with the hydrophobic surface of the column . The addition of Zn2+ has three effects on chromatography: a decrease in the number of anionic sites on the tRNA which interact with the positively charged ammonium ion, an increase in affinity of the tRNA for these ionic sites, and an increase in affinity of tRNA for hydrophobic sites on the column . All three effects were fully reversed by the addition of Cd2+ (10 mM) or Mg2+ (35 mM), but only partially reversed at lower concentrations of these competing ions . These results show that chromatography on RCP-5 can be a sensitive physical chemical technique for examination of the structure of tRNA, and probably for other nucleic acids as well.

Biochim Biophys Acta, 1987 Jan 28, 908(1), 97 - 102
Reactivity of essential histidine residues in EF-Tu.GDP and EF-Tu.GTP from Escherichia coli; Jonak J et al.; The microenvironment of histidine residues located in the binding site of elongation factor EF-Tu from Escherichia coli for the 3' terminus of aminoacyl-tRNA is altered during transition of EF-Tu.GDP to EF-Tu.GTP.

Biochemistry, 1987 Jan 27, 26(2), 597 - 603
Stereochemistry and mechanism of a new single-turnover, half-transamination reaction catalyzed by the tryptophan synthase alpha 2 beta 2 complex; Miles EW; Tryptophan synthase is a versatile enzyme that catalyzes a wide variety of pyridoxal phosphate dependent reactions that are also catalyzed in model systems . These include beta-replacement, beta-elimination, racemization, and transamination reactions . We now show that the apo-alpha 2 beta 2 complex of tryptophan synthase will bind two unnatural substrates, pyridoxamine phosphate and indole-3-pyruvic acid, and will convert them by a single-turnover, half-transamination reaction to pyridoxal phosphate and L-tryptophan, the natural coenzyme and a natural product, respectively . This enzyme-catalyzed reaction is more rapid and more stereospecific than an analogous model reaction . The pro-S 4'-methylene proton of pyridoxamine phosphate is removed during the reaction, and the product is primarily L-tryptophan . We conclude that pyridoxal phosphate enzymes may be able to catalyze some unnatural reactions involving bound reactants and bound coenzyme since the coenzyme itself has the intrinsic ability to promote a variety of reactions.

Biochemistry, 1987 Jan 27, 26(2), 549 - 56
Nuclear magnetic resonance and molecular genetic studies of the membrane-bound D-lactate dehydrogenase of Escherichia coli; Rule GS et al.; In this study we demonstrate the potential of combining fluorine-19 nuclear magnetic resonance (NMR) spectroscopy with molecular genetics . We are using the membrane-bound enzyme D-lactate dehydrogenase of Escherichia coli as a model system to characterize interactions between proteins and lipids . We have labeled D-lactate dehydrogenase with 4-, 5-, and 6-fluorotryptophans and obtained high-resolution fluorine-19 NMR spectra showing five resonances, in agreement with the five tryptophan residues expected from the DNA sequence . The five 19F resonances in the spectra have been assigned to the specific tryptophan residues in the primary sequence of D-lactate dehydrogenase by site-directed oligonucleotide mutagenesis of the cloned gene . We observe large differences in the relative fluorine-19 chemical shifts of each tryptophan residue when labeled by different isomers of fluorotryptophan . We have determined by NMR methods that two tryptophans are exposed to the solvent and that none of the tryptophan residues are within 10 A of the lipid phase . On the basis of 19F NMR spectroscopy of the labeled tryptophan residues, the conformation of D-lactate dehydrogenase is similar in aqueous solution and in the presence of a variety of lipids and detergents . This result indicates that the presence of lipids or detergents is not required to maintain the tertiary structure of this membrane-bound enzyme . In contrast, Triton X-100 induces a change to an abnormal conformation of the enzyme as judged from both NMR spectroscopy and the effect of temperature on the maximal velocity of the enzyme in the presence of this detergent.

Biochemistry, 1987 Jan 27, 26(2), 351 - 60
Yeast cytochrome c peroxidase: mutagenesis and expression in Escherichia coli show tryptophan-51 is not the radical site in compound I; Fishel LA et al.; Using oligonucleotide-directed site-specific mutagenesis, we have constructed a system for the mutation and expression of yeast cytochrome c peroxidase (CCP, EC 1.11.1.5) in Escherichia coli and applied it to test the hypothesis that Trp-51 is the locus of the free radical observed in compound I of CCP {Poulos, T . L., & Kraut, J . (1980) J . Biol . Chem . 255, 8199-8205} . The system was created by substituting a CCP gene modified by site-directed mutagenesis, CCP(MI), for the fol gene in a vector previously used for mutagenesis and overexpression of dihydrofolate reductase . E . coli transformed with the resulting plasmid produced the CCP(MI) enzyme in large quantities, more than 15 mg/L of cell culture, of which 10% is holo- and 90% is apo-CCP(MI) . The apoenzyme was easily converted to holoenzyme by the addition of bovine hemin . Purified CCP(MI) has the same catalytic activity and spectra as bakers' yeast CCP . A mutation has been made in CCP(MI), Trp-51 to Phe . The Phe-51 mutant protein CCP(MI,F51) is fully active, and the electron paramagnetic resonance (EPR) spectrum, at 89 K, of its oxidized intermediate, compound I, displays a strong sharp resonance at g = 2.004, which is very similar to the signal observed for compound I of both bakers' yeast CCP and CCP(MI) . However, UV-visible and EPR spectroscopy revealed that the half-life of CCP(MI,F51) compound I at 23 degrees C is only 1.4% of that observed for the compound I forms of CCP(MI) or bakers' yeast CCP . Thus, Trp-51 is not necessary for the formation of the free radical observed in compound I but appears to exert a significant influence on its stability.

Biochemistry, 1987 Jan 27, 26(2), 343 - 51
Predicted structures of cAMP binding domains of type I and II regulatory subunits of cAMP-dependent protein kinase; Weber IT et al.; The mammalian cAMP-dependent protein kinases have regulatory (R) subunits that show substantial homology in amino acid sequence with the catabolite gene activator protein (CAP), a cAMP-dependent gene regulatory protein from Escherichia coli . Each R subunit has two in-tandem cAMP binding domains, and the structure of each of these domains has been modeled by analogy with the crystal structure of CAP . Both the type I and II regulatory subunits have been considered, so that four cAMP binding domains have been modeled . The binding of cAMP in general is analogous in all the structures and has been correlated with previous results based on photolabeling and binding of cAMP analogues . The model predicts that the first cAMP binding domain correlates with the previously defined fast dissociation site, which preferentially binds N6-substituted analogues of cAMP . The second domain corresponds to the slow dissociation site, which has a preference for C8-substituted analogues . The model also is consistent with cAMP binding in the syn conformation in both sites . Finally, this model has targeted specific regions that are likely to be involved in interdomain contacts . This includes contacts between the two cAMP binding domains as well as contacts with the amino-terminal region of the R subunit and with the catalytic subunit.

Biochemistry, 1987 Jan 27, 26(2), 461 - 5
Monoclonal antibodies to epitopes in both C-terminal and N-terminal domains of Escherichia coli ribosomal protein L7/L12 inhibit elongation factor binding but not peptidyl transferase activity; Nag B et al.; Two monoclonal antibodies against different epitopes in Escherichia coli ribosomal protein L7/L12, one within residues 74-120 and the other within residues 1-73, shown before to inhibit the binding of EF-G, have been tested for their effects on the binding to E . coli ribosomes of EF-Tu-aminoacyl-tRNA-GTP ternary complex and on peptidyl transferase activity . Both antibodies inhibit the binding of ternary complex and EF-Tu-dependent GTPase but have no inhibitory effect on peptidyl transferase activity . The inhibition of binding of both elongation factors is indicative of overlapping binding sites for EF-G and EF-Tu . The inhibition by both antibodies implies the contribution of both domains of L7/L12 to this binding site . This implies the location of one or more of the C-terminal domains of L7/L12 on the body of the 50S subunit . The absence of any inhibition of peptidyl transferase activity shows distinct separation of this site from the factor binding site.

Nucleic Acids Res, 1987 Jan 26, 15(2), 717 - 29
Nucleotide sequence and analysis of the coliphage T3 S-adenosylmethionine hydrolase gene and its surrounding ribonuclease III processing sites; Hughes JA et al.; To understand better the characteristics of the coliphage T3 S-adenosyl-L-methionine (AdoMet) hydrolase (AdoMetase, E.C . 3.3.1.2) and its expression in phage-infected Escherichia coli, we determined the DNA sequence of the cloned gene and its surrounding ribonuclease (RNase) III mRNA transcript processing sites . The AdoMetase gene contains two in-frame protein translation initiation sites specifying peptides 17105 and 13978 daltons in size . Both proteins terminate at the same ochre codon making the shorter peptide identical to the carboxy terminal 82% of the 17 kd protein . Our data explain the existence of two AdoMetase-related peptides in preparations of the purified enzyme as well as identify sequences that might serve to regulate the enzyme's expression . Comparisons between this T3 sequence and the homologous 0.3 gene region of the closely related coliphage T7 show both the nucleotide and amino acid sequences to be unrelated . The RNase III mRNA processing sites that bracket these genes in T3 and T7 are highly conserved in both their primary and secondary structures.

Biochim Biophys Acta, 1987 Jan 26, 896(2), 319 - 22
The influence of maltoporin affinity on the transport of maltose and maltohexaose into Escherichia coli; Ferenci T et al.; The kinetics of maltose transport and its inhibition by maltohexaose were investigated using Escherichia coli strains with engineered modifications of maltoporin . The permeation of lactose through maltoporin was also measured, as well as its inhibition by maltohexaose . Based on these results, the role of the maltoporin binding site in transport was evaluated.

Nucleic Acids Res, 1987 Jan 26, 15(2), 709 - 16
Cleavage of single stranded oligonucleotides by EcoRI restriction endonuclease; Bischofberger N et al.; The 31mer 5'-TCA ACG CTA GAA TTC GGA TCC ATC GCT TGG T, the complementary 33mer 5'-CCA AGC GAT GGA TCC GAA TTC TAG CGT TGA GAT, the 40mer 5'-GGC CAG GAT GGT GAA GAA TTC GAT CCG GTA CGT AGC TAA G, and the complementary 42mer 5'-TAC TTA GCT ACG TAC CGG ATC GAA TTC TTC ACC ATC CTG GCC were synthesized and their reactivity towards EcoRI was studied . It was found that the 31mer and the 40mer were cleaved at a comparable rate to the 31mer-33mer hybrid and the 40mer-42mer hybrid, respectively . The rate of cleavage of the 33mer and the 42mer was an order of magnitude lower . To rule out possible intermolecular duplex formation, the 33mer was immobilized on cellulose by ligation and labeled with alpha 32P-dCTP using Klenow fragment of E . coli DNA polymerase . EcoRI cleaved this immobilized oligomer into specific fragments.

Nucleic Acids Res, 1987 Jan 26, 15(2), 477 - 90
A DNA binding protein showing sequence specificity for a region containing the replication origin of Xenopus laevis mitochondrial DNA; Cordonnier AM et al.; In Xenopus laevis mitochondria up to 14 different polypeptides with affinity for the DNA, have been identified by the protein blotting technique . Under stringent binding conditions only one polypeptide displayed specific affinity for a restriction fragment containing the H strand origin of replication of the Xenopus laevis mt chromosome . The proteins were fractionated by double stranded DNA cellulose chromatography . Under conditions which favor high affinity interactions between proteins and DNA, a protein of the 2M NaCl step shows specific binding to the DNA fragments containing the D-loop region . Some physical properties of the protein have been studied . It has a MW of 21.5 Kd and a globular shape as can be inferred from the relationship between MW and sedimentation coefficient (2.7 S) . It binds non cooperatively to DNA and forms relatively stable complexes as demonstrated by DNA competition experiments.

Nucleic Acids Res, 1987 Jan 26, 15(2), 465 - 75
Escherichia coli rep gene: sequence of the gene, the encoded helicase, and its homology with uvrD; Gilchrist CA et al.; The sequence of a 2.67-kilobase section of the Escherichia coli chromosome that contains the rep gene has been determined . This gene codes for a protein of predicted Mr 72,800, a DNA helicase, which is also a single-stranded DNA-dependent ATPase . The sequenced region contains an open reading frame of the correct length and orientation to encode the Rep protein . A secondary structure for the protein can be formulated from the amino acid sequence . We have compared both the primary and the secondary structures of Rep with other proteins and find the greatest homology between Rep and E . coli helicase II, the product of the uvrD gene.

J Biol Chem, 1987 Jan 25, 262(3), 1337 - 43
recA protein binding to the heteroduplex product of DNA strand exchange; Pugh BF et al.; Following DNA strand exchange, recA protein remains associated with the heteroduplex DNA product of the reaction . This association exists without significant changes (as measured by nuclease protection, topological state of the heteroduplex DNA, and rates of ATP hydrolysis) for at least 30 min after strand exchange is complete . The heteroduplex DNA is unwound as a result of this association to an extent at least as great as the unwinding observed when recA protein is bound to duplex DNA at pH 6.35 . The extensive unwinding, in combination with the rates of ATP hydrolysis reported elsewhere, provide evidence that recA protein binding is contiguous throughout the heteroduplex DNA . This binding is disrupted upon challenge with heterologous single-stranded DNA, with rapid migration of recA protein to the challenging DNA . These results are discussed in relation to the mechanism of recA protein-promoted branch migration.

J Biol Chem, 1987 Jan 25, 262(3), 1326 - 36
Stable binding of recA protein to duplex DNA . Unraveling a paradox; Pugh BF et al.; recA protein binding to duplex DNA is a complicated, multistep process . The final product of this process is a stably bound complex of recA protein and extensively unwound double-stranded DNA . recA monomers within the complex hydrolyze ATP with an apparent kcat of approximately 19-22 min-1 . Once the final binding state is achieved, binding and ATP hydrolysis by this complex becomes pH independent . The weak binding of recA protein to duplex DNA reported in previous studies does not, therefore, reflect an intrinsically unfavorable binding equilibrium . Instead, this apparent weak binding reflects a slow step in the association pathway . The rate-limiting step in this process involves the initiation rather than the propagation of DNA binding and unwinding . This step exhibits no dependence on recA protein concentration at pH 7.5 . Extension or propagation of the recA filament is fast relative to the overall process . Initiation of binding is pH dependent and represents a prominent kinetic barrier at pH 7.5 . ATP hydrolysis occurs only after the duplex DNA is unwound . The binding density of recA protein on double-stranded DNA is approximately one monomer/4 base pairs . A model for this process is presented . These results provide an explanation for several paradoxical observations about recA protein-promoted DNA strand exchange . In particular, they demonstrate that there is no thermodynamic requirement for dissociation of recA protein from the heteroduplex DNA product of strand exchange.

J Biol Chem, 1987 Jan 25, 262(3), 1111 - 5
The primary structure of rat ribosomal protein L19 . A determination from the sequence of nucleotides in a cDNA and from the sequence of amino acids in the protein; Chan YL et al.; The covalent structure of rat ribosomal protein L19 was inferred from the sequence of nucleotides in a recombinant cDNA and confirmed from the sequence of amino acids in a portion of the protein . Ribosomal protein L19 contains 196 amino acids and has a molecular weight of 26,971 . There are indications that a segment of 23 residues in rat L19 is related to sequences of the same length in Escherichia coli ribosomal proteins L30, L18, and S2.

J Biol Chem, 1987 Jan 25, 262(3), 1420 - 4
Amino-terminal deletions in the presequence of an imported mitochondrial protein block the targeting function and proteolytic cleavage of the presequence at the carboxy terminus; Hurt EC et al.; Subunit IV of yeast cytochrome oxidase is made in the cytosol with a 25-residue presequence . This presequence targets subunit IV into mitochondria and is removed by a protease in the matrix space . Here we show that removal of as few as 4 amino-terminal residues from the subunit IV presequence (which had been attached to the cytosolic protein dihydrofolate reductase) blocks import of the protein into mitochondria and proteolytic removal of the presequence by the soluble matrix protease . Thus, this protease requires not only an appropriate cleavage site at the carboxy-terminal end of the presequence, but also information at the extreme amino terminus of the presequence.

J Biol Chem, 1987 Jan 25, 262(3), 1412 - 9
Purification and characterization of an outer membrane-bound protein involved in long-chain fatty acid transport in Escherichia coli; Black PN et al.; We report the purification and localization of the fadL gene product (FLP), an essential component of the long-chain fatty acid transport machinery in Escherichia coli . FLP was extracted from total membranes by differential extraction with the nonionic detergents Tween 20 and Triton X-100 . This protein was further purified from a Tween 20-insoluble-Triton X-100-soluble extract by salt fractionation, gel filtration chromatography, and hydrophobic interaction chromatography . This regime results in a 95-fold purification of FLP from total membranes . The purified protein preparation was homogeneous based on silver staining and gave the characteristic behavior established for the fadL gene product in the presence of sodium dodecyl sulfate at different temperatures prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr of 33,000 when heated at 25 degrees C and Mr of 43,000 when heated at 100 degrees C) and on two-dimensional polyacrylamide gels (pI of 4.6 and a Mr of 33,000) . Purified FLP was rich in hydrophobic residues accounting for approximately 45% of the total amino acid composition . To localize FLP, antisera were raised against the purified protein and were used to probe differentially fractionated membranes by Western immunoblotting . This procedure demonstrated the presence of this protein only in the outer membrane fraction of fadL+ strains . We confirmed the outer membrane localization of FLP by measuring long-chain fatty acid transport in fadL+ and fadL strains treated with EDTA to alter outer membrane permeability and in spheroplasts generated from fadL+ and fadL strains . Both EDTA-treated cells and spheroplasts transported long-chain fatty acids at essentially the same rate regardless of whether they contained a wild-type or mutant fadL gene . These data imply that FLP is a protein in the outer membrane which is specifically involved in long-chain fatty acid transport.

Nature, 1987 Jan 22-28, 325(6102), 355 - 7
DNA ligase I deficiency in Bloom's syndrome; Willis AE et al.; Certain rare human diseases with autosomal recessive mode of inheritance are associated with a greatly increased cancer frequency which may reflect specific defects in DNA repair or replication . These disorders include xeroderma pigmentosum, ataxia-telangiectasia, Fanconi's anaemia and Bloom's syndrome . Cells from individuals with Bloom's syndrome usually grow slowly in culture and exhibit increased chromosomal breakage and rearrangement, an elevated frequency of sister chromatid exchanges, retarded rates of progression of DNA replication forks, delayed conversion of replication intermediates to high-molecular-weight DNA, and slightly increased sensitivity to DNA-damaging agents . Several of these features are also characteristic of Escherichia coli and yeast mutants with a defective DNA ligase . In this investigation we show that one of the two DNA ligases of human cells, ligase I, is defective in a representative lymphoid cell line of Bloom's syndrome origin.

J Theor Biol, 1987 Jan 21, 124(2), 127 - 35
How the slot machine led biologists astray; Opadia-Kadima GZ; An extensive search, in the literature, for experiments in which a new enzyme did evolve, produced only two: the first by Campbell et al . in 1973; the second by Hall & Hartl in 1974 . Since the experiments provide the only means of gaining a first hand view of how new enzymes evolve, they were scrutinized, minutely, with the objective of ascertaining whether the mutations involved were random or non-random . We report here that they were non-random . Further, with the benefit of hindsight, we highlight the weakness in the insights and the reasoning which produced the belief that new enzymes evolve purely by chance.

J Mol Biol, 1987 Jan 20, 193(2), 423 - 5
Crystals of seryl-tRNA synthetase from Escherichia coli . Preliminary crystallographic data; Leberman R et al.; Crystals of seryl-tRNA synthetase from Escherichia coli can be grown from ammonium sulphate/octyl glucoside solutions in two days . The crystals appear to be very suitable for X-ray analysis, diffracting to at least 2.8 A resolution and being resistant to radiation damage . The crystals are monoclinic (space group C2) with cell parameters a = 148.2 A, b = 90.6 A, c = 69.5 A and beta = 119.0 degrees . Depending on whether the asymmetric unit is the enzyme monomer (Mr 48,414) or dimer the Vm value would be either 4.12 or 2.10 A3/dalton . Although the former would indicate a rather high solvent content, other proteins crystallized in the presence of octyl glucoside have Vm values similar to this.

J Mol Biol, 1987 Jan 20, 193(2), 293 - 302
Promoter properties and negative regulation of the uvrA gene by the LexA repressor and its amino-terminal DNA binding domain; Bertrand-Burggraf E et al.; A comparative study of the interaction of the LexA repressor of Escherichia coli and of its amino-terminal DNA binding domain to the uvrA operator has been undertaken . Most of the binding constants are determined from competition experiments with RNA polymerase by measuring the time-course of the abortive initiation transcriptional activity . The presence of repressor increases the lag time, tau, without affecting the final maximum activity . The inhibition of transcription by LexA, at least in the case of the uvrA gene, is thus a transient, time-dependent phenomenon, because once the RNA polymerase is engaged in a stable "open" complex, it is quasi-irreversibly trapped in this state . A study of the binding constants as a function of ionic strength suggests the formation of 5.5(+/- 1) salt bridges between the uvrA operator and a LexA dimer . Surprisingly, the binding affinity of the amino-terminal domain was only about one order of magnitude smaller than that of the entire LexA repressor . The determination of the binding constant of the RNA polymerase to the "closed" uvrA promoter (KB approximately 1 X 10(7) to 2 X 10(7) M-1) allowed us to determine theoretical repression curves for the two repressor species . These calculations show that the binding constant found for LexA is sufficiently high to account for substantial or complete repression, and that of the amino-terminal domain is sufficiently low to account for partial or nearly full induction . Under solvent conditions used by others for the determination of binding constants to other SOS operators by DNAase I footprinting, the uvrA operator turns out to be a rather weak one (K approximately 3 X 10(7) M-1), being comparable with that of the uvrB gene . The uvrA promoter is "association-limited" with a KB X k2 product fitting very nicely the homology score for the promoter of 55.

Biochim Biophys Acta, 1987 Jan 20, 923(1), 74 - 82
Nucleoside-triphosphatase and hydrolysis of thiamin triphosphate in Escherichia coli; Nishimune T et al.; A membrane-bound nonspecific triphosphatase of E . coli was solubilized and purified to a homogeneous SDS-acrylamide gel electrophoresis band . It was found to be a single polypeptide of 16 kDa requiring no Mg2+, with an optimal pH at 6.5 . The substrate specificity was broad and a nonspecific Mg2+-independent ribonucleoside-triphosphatase (NTPase) activity was expressed together with thiamin-triphosphatase activity . The molecular size and characteristics were clearly different from the known NTPase (EC 3.6.1.15) . Using the purified thiamin-triphosphatase II, ATP:thiamin-diphosphate phosphoryl transferase (EC 2.7.4.15) activity was demonstrated with an optimal pH of approx . 5.3 . Considering its kinetic parameters and other characteristics, however, the thiamin triphosphate synthesizing activity was not thought to take part in cellular thiamin triphosphate synthesis . The possibility that thiamin-triphosphatase II plays a part in the hydrolysis of thiamin triphosphate to control its cellular level is suggested.

J Mol Biol, 1987 Jan 20, 193(2), 279 - 92
Comparison of the open complexes formed by RNA polymerase at the Escherichia coli lac UV5 promoter; Straney DC et al.; In transcription initiation at the lac UV5 promoter, Escherichia coli RNA polymerase forms two open complexes, called Ou and O1, which can be separated by electrophoresis on native polyacrylamide gels . We have compared the properties of these two open complexes, with the objective of rationalizing the functional difference previously reported between the two forms: the complex which is dominant at high temperature (Ou) is better able to escape abortive transcriptional cycling into productive mRNA elongation . Methylation protection and binding domain probing with exonuclease III were used to investigate differences in polymerase binding strength to particular DNA domains . Also, we examined the difference in the extent and temperature dependence of promoter unwinding in the two complexes, as probed by methylation of unpaired cytosines and cleavage by phage T7 endonuclease . We find that O1 has stronger promoter interactions in the DNA domain whose upstream edge is defined by an exonuclease III stop at -24 . These -24 domain interactions, which presumably aid in promoter binding and nucleation of DNA unwinding, are inferred to be strong enough to hinder escape of the polymerase from the open complex contacts that are maintained during abortive initiation . The Ou complex has weaker binding to the -24 domain, partially compensated by better upstream interactions and a better ability to accommodate extensive DNA unwinding . It thus escapes abortive initiation more readily because of weaker critical open complex contacts that must be lost when stable initiation occurs from the corresponding stressed intermediates.

J Mol Biol, 1987 Jan 20, 193(2), 267 - 78
A stressed intermediate in the formation of stably initiated RNA chains at the Escherichia coli lac UV5 promoter; Straney DC et al.; We report experiments designed to elucidate the mechanism by which RNA polymerase advances from the open complex to synthesis of a stably bound RNA chain during transcription initiation . Techniques used include deoxyribonuclease I footprinting, methylation protection, and exonuclease III digestion through upstream domains, each applied to the open, abortive and productive transcription complexes of Escherichia coli RNA polymerase with the lac promoter . The results show a slight loss of upstream open complex contacts during abortive transcription of a 6-mer and 8-mer, but a large loss of these contacts upon escape from abortive cycling into productive transcription at the 11-mer . We propose a model for early initiation in which competition between open complex polymerase-DNA contacts on one hand and initiated complex polymerase-DNA-RNA interactions on the other produces a "stressed intermediate" during formation of a short RNA-DNA duplex . The strain energy is relieved either by ejecting the short RNA, resulting in aborted initiation, or by eliminating the sigma subunit and breaking the open complex contacts, thereby escaping abortive cycling into productive transcription . Further evidence for this model is based on the observation that destabilization of interactions specific for either open complex or initiated complex has the predicted effect on the amount of abortive cycling . The model predicts a complicated relationship between overall promoter strength and DNA sequence changes that alter polymerase-DNA interactions.

Biochim Biophys Acta, 1987 Jan 20, 923(1), 66 - 73
Organothallium(III) reagents for modification of biomacromolecules: interaction of thallium derivatives with transfer RNA; Baindur SR et al.; As an extension of work on the inhibition of enzymes by arylthallium(III) reagents, the thallium analogues of the organomercurials, we have studied the interactions of these molecules with transfer RNA . In contrast to thallous acetate, thallium(III) derivatives (thallic trifluoroacetate, p-methylphenylthallium(III) bis-trifluoroacetate (MPT) and o-carboxyphenylthallium(III) bis-trifluoroacetate) bound to Escherichia coli tRNA . The interaction was fully reversible upon Sephadex G-25 gel filtration, and binding constants and stoichiometries were evaluated by a number of procedures . The likely site of interaction was shown to be the thiouridine residue (s4U8) based on changes induced by MPT on the absorbance around 330 nm . No changes in stacking interactions could be detected from the absorption or circular dichroic spectra . The detailed structure of the groups on thallium(III) affected the interaction with tRNA . Thalliation at s4U8 affects the absorbance at 335 nm and the amino-acid uptake capacity of E . coli tRNAPhe in parallel, the latter being progressively inhibited by increasing amounts of MPT . In a model nucleoside system, uridine disulphide is probably formed from reduced thiouridine by the oxidative action of the Tl(III) reagents . No evidence of cross-linking of E . coli tRNA molecules under gel electrophoretic conditions was obtained in contrast to the model nucleoside . The easily reversible interaction of MPT with sulphur sites in E . coli tRNA contrasts with the stable (to gel filtration) bonds formed between MPT and (thiol) sites in enzymes.

FEBS Lett, 1987 Jan 19, 211(1), 78 - 82
Anomalous behavior of human leukocyte interferon subtypes on polyacrylamide gel electrophoresis in the presence of dodecyl sulfate; Ohara O et al.; 35S-labeled human leukocyte interferon (IFN) subtypes produced in a cell-free system derived from Escherichia coli were analyzed by polyacrylamide gel electrophoresis in the presence of SDS (SDS-PAGE) . Some IFN subtypes anomalously showed lower electrophoretic mobilities than those expected from their formula molecular masses . The results with hybrid IFNs and esterification suggest that this anomaly of IFN subtypes on SDS-PAGE is due to the introduction of one or two negative charges in the middle of the molecule.

FEBS Lett, 1987 Jan 19, 211(1), 23 - 6
c-Ha-ras gene products are potent inhibitors of cathepsins B and L; Hiwasa T et al.; c-Ha-ras proteins produced by Escherichia coli inhibited the activities of cathepsins B and L which had been partially purified from rat kidney . Furthermore, amino acid sequence homology between c-Ha-ras proteins and thiol proteinase inhibitors has been found.

FEBS Lett, 1987 Jan 19, 211(1), 1 - 4
Voltage gating in porin channels; Lakey JH; Data from experiments in which porin channels are reconstituted into planar bilayer membranes are reviewed for their relevance to porin channel gating in vivo . Contradictory evidence concerning voltage gating indicates that the different results may stem from the variety of purification techniques employed . The likelihood of voltage gating as a property of E . coli porins in vivo is discussed in relation to the possible magnitude of the membrane potential across the outer membrane.

Biochim Biophys Acta, 1987 Jan 16, 890(1), 97 - 105
Escherichia coli F1 ATPase is reversibly inhibited by intra- and intersubunit crosslinking: an approach to assess rotational catalysis; Kandpal RP et al.; Reaction of the multisubunit F1 ATPase from Escherichia coli (EF1) with a bifunctional cleavable crosslinker, 3,3'-dithiobis(succinimidylpropionate) (DSP), has been used to explore the possibility that during catalysis a rotational movement of catalytic subunits relative to noncatalytic subunits occurs . The premise is that such rotational catalysis is tenable if intersubunit crosslinking of a major subunit with one of the minor subunits inhibits the enzyme activity and if upon cleavage of the crosslinks, the enzyme regains activity . The results presented in this paper show that crosslinking of about 5-6 reactive groups on EF1 with DSP is accompanied by a loss of 2/3 of the enzyme activity . Both intra- and intersubunit crosslinks are formed . The most prominent intersubunit crosslinks are those of gamma and delta subunits with the alpha subunit . Nearly complete recovery of activity can be attained by cleaving the disulfide bond in the crosslinker with dithiothreitol . Because the chemical modification of enzyme groups remains after the crosslinker is cleaved, the loss in activity before cleavage can be ascribed to conformational restraints . The results show that catalysis by the EF1 ATPase is highly sensitive to the restrictions of crosslinking, and are consistent with the view that catalysis is accompanied by appreciable movements of the major subunits with respect to the minor subunits, as suggested for rotational catalysis.

Biochem Pharmacol, 1987 Jan 15, 36(2), 265 - 8
Effects of lead acetate on DNA and RNA synthesis by intact HeLa cells, isolated nuclei and purified polymerases; Frenkel GD et al.; The effects of lead acetate on DNA and RNA synthesis have been investigated with intact HeLa cells, isolated nuclei, and purified DNA and RNA polymerases . No inhibition of DNA or RNA synthesis in intact cells was found even after exposure to 0.5 mM lead acetate for 18 hr . In contrast, both DNA and RNA synthesis in isolated nuclei were inhibited by lead (with 50% inhibition at approximately 150 and 80 microM respectively) . Similarly, both HeLa DNA polymerase alpha and RNA polymerase II were inhibited, with 50% inhibition obtained at approximately 150 and 20 microM lead acetate respectively . The inhibition of nucleic acid synthesis in isolated nuclei can thus be accounted for by inhibition of the polymerases . The sensitivity of Escherichia coli DNA polymerase I to lead acetate was found to be significantly greater than the HeLa DNA polymerase alpha (50% inhibition at only 10 microM), but the sensitivity of the E . coli RNA polymerase was the same as that of the HeLa enzyme.

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