Escherichia coli

J Immunol Methods, 1984 Jul 6, 71(2), 203 - 10
Development of a sensitive solid-phase radioimmunoassay technique for quantification of class-specific Escherichia coli antibodies; Stephens S; A specific and sensitive, quantitative solid-phase radioimmunoassay (RIA) has been developed for the detection of Escherichia coli antibodies in serum and secretions . Preparation of affinity-purified anti-E . coli standards allows accurate quantification of G, M and A classes of antibody down to 10 ng/ml using only 20 microliters of sample . This technique has considerable advantages over indirect haemagglutination in sensitivity and accuracy of immunoglobulin class detection . RIA also compares favourably with ELISA in sensitivity and sample size required . Affinity-purified standards may also be used to quantify the ELISA test.

J Mol Biol, 1984 Jul 5, 176(3), 431 - 42
Roles of the 5' leader region of the ompA mRNA; Green PJ et al.; OmpA protein, a major outer membrane protein of Escherichia coli, is synthesized from a messenger RNA containing a 134-nucleotide 5' leader region . The role of this leader region in efficient ompA expression was investigated using a series of ompA-lacZ fusion plasmids . These plasmids differ in the amount of DNA encoding the ompA leader region which is fused to the lacZ structural gene . The fusion containing all but six nucleotides of the ompA leader produced the highest beta-galactosidase activity, while the fusion containing the shortest leader synthesized only 4% as much beta-galactosidase . Fusions with leaders intermediate in length produced between 6% and 24% of the activity found in the most efficient fusion . Quantitation of lacZ mRNA synthesis by DNA-RNA hybridization revealed differences in lacZ mRNA production reflecting the observed differences in beta-galactosidase activity . The primary effect of the ompA leader in maintaining high levels of mRNA is discussed in terms of the roles of mRNA secondary structure.

Biochemistry, 1984 Jul 3, 23(14), 3330 - 5
Probing the conformation of 26S rRNA in yeast 60S ribosomal subunits with kethoxal; Hogan JJ et al.; The conformation and accessibility of 26S rRNA in yeast 60S ribosomal subunits were probed with kethoxal . Oligonucleotides originating from reactive sites were isolated by diagonal electrophoresis and sequenced . From over 70 oligonucleotide sequences, 26 kethoxal-reactive sites could be placed in the 26S rRNA sequence . These are in close agreement with a proposed secondary structure model for the RNA that is based on comparative sequence analysis . At least seven kethoxal-reactive sites in yeast 26S rRNA are in positions that are exactly homologous to reactive positions in E . coli 23S rRNA; each of these sites has previously been implicated in some aspect of ribosomal function.

Biochemistry, 1984 Jul 3, 23(14), 3136 - 43
Elementary steps in the reaction mechanism of the alpha-ketoglutarate dehydrogenase multienzyme complex from Escherichia coli: kinetics of succinylation and desuccinylation; Waskiewicz DE et al.; The kinetics of the succinylation and the desuccinylation of the alpha-ketoglutarate dehydrogenase complex from Escherichia coli have been studied at 4 degrees C in 2 mM thiamin pyrophosphate, 2 mM MgCl2, and 20 mM potassium phosphate (pH 7.0) by steady-state and quenched-flow techniques . The initial steady-state velocity for the reaction of the complex is inhibited by high concentrations of alpha-ketoglutarate . The data are consistent either with cooperative interactions between two catalytic sites or with the existence of an alpha-ketoglutarate regulatory site . The time course of the succinylation by alpha-ketoglutarate of the unmodified complex or the complex in which a fraction of the alpha-ketoglutarate decarboxylase subunits (E1) has been inhibited with N-ethylmaleimide reveals a complex kinetic process . A mechanism consistent with the kinetic data is proposed in which some E1 subunits succinylate one lipoic acid per E1 and other E1 subunits succinylate two lipoic acids per E1 . Furthermore, each succinylation reaction occurs via a two-step process with rate constants of 49 and 89 s-1 at saturating concentrations of alpha-ketoglutarate for the first and second steps, respectively . At long times, 13-16 mol of succinate binds per mol of unmodified complex . The stoichiometry of binding obtained with N-ethylmaleimide-treated complex is initially lower but approaches the same values as for the unmodified complex over the course of minutes . Coenzyme A removes the succinyl groups on the unmodified enzyme with a rate constant greater than or equal to 200 s-1 . The results obtained suggest a limited accessibility between sites on the complex.

Biochemistry, 1984 Jul 3, 23(14), 3111 - 4
Exploring the conformational roles of signal sequences: synthesis and conformational analysis of lambda receptor protein wild-type and mutant signal peptides; Briggs MS et al.; Secretion of the Escherichia coli lambda receptor protein (LamB protein) appears from genetic evidence to be correlated with the predicted tendency of its signal sequence to adopt an alpha-helical conformation {Emr, S . D., & Silhavy, T . J . (1983) Proc . Natl . Acad . Sci . U . S . A . 80, 4599} . We have tested this hypothesis by synthesizing major portions of signal sequences from the wild-type and mutant LamB proteins and analyzing their conformations by circular dichroism . The wild-type signal sequence contains a seven-residue hydrophobic region flanked by a proline and a glycine . Chou-Fasman rules predict that this segment will adopt an alpha-helical conformation . An export-deficient mutant is missing four residues from this region; the helix-breaking glycine and proline are thus separated by only three residues, and an alpha helix is not predicted to form . In each of the export-restored revertants, either the glycine or the proline is replaced with a residue which promotes helix formation . The helix content of the synthetic signal sequence fragments on the basis of CD measurements supports the secondary structure hypothesis described above . The relative helicity in aqueous sodium dodecyl sulfate, lysolecithin, or trifluoroethanol is as follows: wild type greater than R2 (Pro----Leu) greater than R1 (Gly----Cys) much greater than deletion mutant.

Biochemistry, 1984 Jul 3, 23(14), 3322 - 30
Probing the conformation of 18S rRNA in yeast 40S ribosomal subunits with kethoxal; Hogan JJ et al.; Yeast 40S ribosomal subunits have been reacted with kethoxal to probe the conformation of 18S rRNA . Over 130 oligonucleotides were isolated by diagonal electrophoresis and sequenced, allowing identification of 48 kethoxal-reactive sites in the 18S rRNA chain . These results generally support a secondary structure model for 18S rRNA derived from comparative sequence analysis . Significant reactivity at positions 1436 and 1439, in a region shown to be base paired by comparative analysis, lends support to the earlier suggestion {Chapman, N.M., & Noller, H.F . (1977) J . Mol . Biol 109, 131-149} that part of the 3'-major domain of 16S-like rRNAs may undergo a biologically significant conformational rearrangement . Modification of positions in 18S rRNA analogous to those previously found for Escherichia coli 16S rRNA argues for extensive structural homology between 30S and 40S ribosomal subunits, particularly in regions thought to be directly involved in translation.

Eur J Biochem, 1984 Jul 2, 142(1), 161 - 4
Two different aldolase A mRNA species in rat tissues; Tsutsumi R et al.; Double-stranded DNA was synthesized with reverse transcriptase from size-fractionated poly(A)-containing RNA from rat ascites hepatoma cells . The cDNA was introduced into Escherichia coli HB101 using pBR322 DNA as a cloning vector . Several plasmids containing aldolase A cDNA were identified by colony hybridization with 32P-labeled cDNA prepared from immunologically purified aldolase A mRNA . The partial amino acid sequence of the cDNA sequence was determined, and found to coincide with that of rabbit aldolase A . Using aldolase A cDNA as a hybridization probe, the aldolase A mRNA concentrations in various rat tissues were analysed, and two aldolase A mRNA species differing in nucleotide length were found; the smaller mRNA (about 1550 nucleotides) in muscle, and the larger one (about 1650 nucleotides) in brain and hepatoma cells.

Cell, 1984 Jul, 37(3), 1001 - 7
Heterogeneous mitochondrial DNA D-loop sequences in bovine tissue; Hauswirth WW et al.; Mitochondrial DNA from bovine tissue contains heterogeneous sequences located within an evolutionary conserved cytosine homopolymer sequence near the 5' end of the D-loop region . This part of the mammalian mitochondrial genome is known to contain the origin of heavy strand DNA synthesis and the major transcriptional promoter for each strand . Nucleotide sequence analysis of cloned DNA and electrophoretic analysis of appropriate small fragments from animal tissue reveal a population of length polymorphs containing from nine to 19 cytosine residues . No individual length species represents more than 40% of the population . These data imply a state of significant intraanimal mtDNA sequence heterogeneity, which most likely occurs intracellularly as well . The localization of variability to a homopolymer run suggests that replication slip-page generated the sequence population . We also report that when recombinant clones containing this region are repeatedly passaged in E . coli, they begin to regenerate length variation similar to that seen in animal mtDNA.

Br J Surg, 1984 Jul, 71(7), 537 - 9
The effect of tetracycline lavage and trauma on visceral and parietal peritoneal ultrastructure and adhesion formation; Phillips RK et al.; The clinical efficacy of tetracycline lavage (1 mg/ml) in the management of abdominal sepsis has led to advocacy of its use in potentially contaminated cases . Yet at higher concentrations (6 mg/ml), tetracycline is a pleural sclerosant . The possibility of early ultrastructural peritoneal damage and later adhesion formation has been examined in syngeneic female Wag rats . At high concentration (10 mg/ml), tetracycline caused adhesions in the absence of peritoneal trauma and there was an associated loss of serosal microvilli . Lavage with low concentration tetracycline (1 mg/ml) or saline after clean abdominal surgery led to more adhesions than if no lavage was employed . There was an unexplained paradoxically low incidence of adhesions if prior mild contamination of the peritoneal cavity with 1 ml 10(5) E . coli had been performed.

Cancer Res, 1984 Jul, 44(7), 2855 - 7
O6-alkylguanine-DNA alkyltransferase activity in normal human tissues and cells; Grafstrom RC et al.; Normal adult human tissues and cultured bronchial epithelial cells and fibroblasts exhibit O6-alkylguanine-DNA alkyltransferase activity in vitro by catalyzing the repair of the promutagenic alkylation lesion O6-methylguanine from DNA . The amount repaired by extracts of liver, peripheral lung, and colon extracts was proportional to the amount of extract protein . Repair of O6-methylguanine led to stoichiometric regeneration of guanine in the DNA and stoichiometric formation of S-methylcysteine in protein . Alkyltransferase activity varies in the different human tissues tested in the decreasing order of liver greater than colon greater than esophagus greater than peripheral lung greater than brain . Extracts of lung tissues, cultured human bronchial epithelial cells, and fibroblasts had similar alkyltransferase activities . Various human tissues exhibit 2- to 10-fold higher alkyltransferase activity than corresponding rat tissues . Whereas the interindividual variation of the activity was 4- to 5-fold in ten or more human lung and colon specimens, the interindividual variation in the inbred rat was less than 20% . The present results show that different human tissues and cells have a several-fold higher capacity to repair O6-methylguanine in DNA than do rat tissues and that the repair process occurs via a mechanism similar to that shown previously in other mammalian cells and Escherichia coli.

Biokhimiia, 1984 Jul, 49(7), 1059 - 65
{Availability of DNP-modified 5'- and 3'-ends of the poly(U) fragment bound to the 30S ribosomal subunit for interaction with antibodies}; Evstaf'eva AG et al.; Modification of the 5'- or 3'-terminal nucleotide residues of poly(U) by dinitrophenyl haptens was carried out . The accessibility of the modified ends of short poly(U) (20-70 nucleotide residues) bound to the 30S subunit of E . coli ribosomes for the interaction with antibodies that are specific against the dinitrophenyl group, was investigated . Some peculiarities of the structural organization of the "entry" (3'-end) and "exit" (5'-end) sites of the template polynucleotide on the 30S subunit and the possibility of the template polynucleotide folding on the ribosome are discussed.

Mol Pharmacol, 1984 Jul, 26(1), 135 - 40
Lethality associated with incorporation of 5-fluorouracil into preribosomal RNA; Herrick D et al.; We have previously demonstrated a highly significant relationship between incorporation of 5-fluorouracil (FUra) into total cellular RNA and loss of clonogenic survival . The present study extends these findings by demonstrating a similar relationship between incorporation of FUra into preribosomal nuclear RNA (45 S and 32 S) and lethal cellular events . The results also demonstrate that the extent of FUra incorporation into preribosomal RNA (prRNA) correlates significantly with inhibition of maturation to cytoplasmic 28 S and 18 S rRNA . These findings suggest that the incorporation of FUra into prRNA alters recognition sites involved in the processing of 45 S and 32 S RNA . These data are further supported by our finding of an enhanced degradation of (FUra)prRNA by RNase III, an enzyme implicated in the maturation of Escherichia coli prRNA and T7 mRNA . These observations suggest that the incorporation of FUra into prRNA is responsible for altered processing to cytoplasmic rRNA and cell lethality.

J Clin Microbiol, 1984 Jul, 20(1), 59 - 64
Simple and reliable enzyme-linked immunosorbent assay with monoclonal antibodies for detection of Escherichia coli heat-stable enterotoxins; Thompson MR et al.; We have developed a sensitive and specific competitive enzyme-linked immunosorbent assay for Escherichia coli heat-stable enterotoxins consisting of methanol-soluble, suckling mouse active peptides with similar core sequences (STa) by using monoclonal antibodies prepared against STa purified from a human isolate . The assay can detect 3 to 20 pg of purified STa, depending on the monoclonal antibody used in the assay . The assay is rapid, requiring ca . 1 h to complete . With this competitive enzyme-linked immunosorbent assay, we measured STa production by enterotoxigenic E . coli directly in Casamino Acid-yeast extract culture supernatants . The assay was suitable for measuring STa in culture supernatants from human, bovine, and porcine E . coli isolates . No cross-reactivity was observed with heat-labile enterotoxin, cholera toxin, or heat-stable enterotoxin STb, which is a methanol-insoluble peptide(s) active in the ligated pig jejunal loop test . A 100% correlation of toxin production was found by comparing the enzyme-linked immunosorbent assay with the previously established radioimmunoassay for STa and with suckling mouse activity.

Vet Microbiol, 1984 Jul, 9(3), 287 - 99
The effect of oral immunization on the population of lymphocytes migrating to the mammary gland of the sow; Kortbeek-Jacobs JM et al.; Sows were immunized orally with live Escherichia coli according to various immunization schedules . Six pregnant gilts were used; 4 immunized at various intervals during the last month of gestation, 1 control immunized after parturition following suppression of lactation by weaning and 1 non-immunized control . The effect of oral vaccination on cell populations from lymphoid organs was studied . The in vitro proliferative responses of the cell populations to K88 antigen, anti-Ig sera and mitogens were used to demonstrate the distribution of sensitized lymphocytes over different lymphoid organs . The capacity of these cells to produce antigen-specific Ig was determined by in ovo translation of their mRNA . Oral administration of antigen resulted in the appearance of K88-positive cells in lymphoid organs . In lactating sows, sensitized cells preferentially occurred in the mammary lymph nodes, whereas after suppression of lactation such a distribution was not seen . A possible route of migration of sensitized lymphocytes is discussed in relation to the local immune response . The antibody isotype produced by sensitized lymphocytes seemed to depend on the immunization schedule . The most effective schedule was one starting early in gestation and comprising frequent administration of antigen . This caused an optimal distribution of sensitized lymphocytes capable of IgA production.

Rev Infect Dis, 1984 Jul-Aug, 6(4), 528 - 31
Lipid A-dependent lymphocyte proliferation in "endotoxin-nonresponder" mice; Vukajlovich SW et al.; Lipopolysaccharides (LPS) with a subunit composition reduced in heterogeneity were prepared by gel-filtration chromatography of detergent-dissociated LPS from the smooth strain of Escherichia coli 055:B5 . The splenocyte mitogenic activity of reassociated column-fraction pools was markedly dependent on the ratio of O-antigen polysaccharide to lipid A, where the activity of the fraction richest in lipid A (F5 LPS) was approximately three orders of magnitude greater than the least active fraction (F1 LPS) . The F5 LPS, which contains only a trace of O-antigen polysaccharide, induces a spleen-cell proliferative response in C3H/HeJ "LPS nonresponder" spleen cells . This mitogenic activity is not present in either unfractionated LPS or the other column fractions . These latter findings indicate that LPS macromolecular aggregates of the appropriate physico-chemical structure and/or compositions have the capacity to elicit C3H/HeJ spleen-cell proliferative responses.

Rev Infect Dis, 1984 Jul-Aug, 6(4), 488 - 92
Physical interaction between lipid A and phospholipids: a study with spin-labeled phospholipids; Takeuchi Y et al.; When mixed bilayers containing spin-labeled phosphatidylethanolamine (or phosphatidylglycerol) and Escherichia coli B lipopolysaccharide were prepared, electron spin resonance signals indicated that the patches that were initially present and contained only phospholipids or only lipopolysaccharides were unusually stable and that little lateral diffusion of phospholipids into lipopolysaccharide domains, or vice versa, took place . These results explain how the outer layer of the outer membrane, which essentially contains only lipopolysaccharides in addition to proteins, can be generated and maintained stably in bacterial cells . Furthermore, the stability of pure lipopolysaccharide domains may have important implications in the mode of action of endotoxins in the body of the host . For example, lipopolysaccharide molecules may tend to form stable domains or patches spontaneously in the animal cell membrane, and special mechanisms (such as the binding to a special receptor) may be needed to disperse the lipopolysaccharide molecules within the host cell membranes.

Rev Infect Dis, 1984 Jul-Aug, 6(4), 478 - 82
Recent developments in the organic synthesis of lipid A in relation to biologic activities; Shiba T et al.; For elucidation of a relationship between chemical structure and biologic activity of lipid A, syntheses of 13 derivatives of glucosamine beta (1-6) disaccharide, which were acylated at 3, 4, and 6'-hydroxyl groups and/or phosphorylated at positions 1 and 4', were carried out . The results showed the importance of the presence of the phosphate group at either position 1 or 4' and of the beta-hydroxyl group in acyl function, particularly in N-linked fatty acids, for the exhibition of biologic activities characteristic of lipid A . New evidence on the positions of fatty acids in natural lipid A of Escherichia coli species obtained by means of 2D-H nuclear magnetic resonance was also demonstrated; the findings indicate that the 3'-hydroxyl group is acylated and the 6'-hydroxyl group in the molecule must be free.

Gene, 1984 Jul-Aug, 29(1-2), 27 - 31
One-step purification of hybrid proteins which have beta-galactosidase activity; Ullmann A; A one-step purification method of hybrid proteins exhibiting beta-galactosidase activity, based on affinity chromatography in the presence of high salt concentration, is described . Starting from crude bacterial extracts, several milligrams of near-homogeneous proteins can be obtained in a few hours with an overall yield of 85 to 95% . The purified hybrid proteins can be used to obtain antibodies against the foreign portion of the protein fusion.

Mutat Res, 1984 Jul-Aug, 132(1-2), 15 - 20
Expression of the SOS response following simultaneous treatment with methyl-nitrosoguanidine and mitomycin C in Escherichia coli; Vericat JA et al.; Simultaneous treatment of Escherichia coli cultures with methyl-nitrosoguanidine and mitomycin C induces recA-dependent inhibition of respiration but not inhibition of cell division . This pattern of SOS functions expression is the same as that is found following treatment with methyl-nitrosoguanidine alone and contrary to the pattern induced after mitomycin C addition . The same result is obtained when a culture of E . coli RecA441 (formerly tif) is shifted to 42 degrees C and treated simultaneously with methyl-nitrosoguanidine . The suppressor effect of this compound over the pattern of SOS functions expression induced by mitomycin C or high temperature in recA441 mutants is directly related to the increase in its dose . Moreover, the division temperature-sensitive mutant ftsA treated with methyl-nitrosoguanidine and high temperature does not show any decrease in its normal filamentous growth when cultured at 42 degrees C . This indicates that the effect of methyl-nitrosoguanidine on the recA-independent inhibition of cell division is not due to any indiscriminate effect of this compound over the division process . These results suggest that the specific kind of lesion caused in DNA is very important in determining which SOS function is induced.

Carbohydr Res, 1984 Jul 1, 129, 243 - 55
Chemical modification and serological properties of the 3-deoxy-alpha-D-manno-2-octulosonic acid-containing polysaccharide from Escherichia coli LP1092; Jennings HJ et al.; The alpha-D configuration of the 3-deoxy-D-manno-2-octulosylonic acid residues in the E . coli LP1092 polysaccharide was definitively assigned by a comparison of its c.d . spectrum with those of methyl 3-deoxy-alpha- and -beta-D-manno-2-octulopyranosidonic acid . The c.d . spectrum of the polysaccharide showed a negative n----pi transition at 211 nm, associated with carboxyl groups, which correlated well with the negative band at 218 nm exhibited in the c.d . spectrum of the methyl alpha-D-glycoside . In contrast, the methyl beta-D-glycoside gave a positive band in the same region of its c.d . spectrum, at 221 nm . Treatment of the E . coli LP1092 polysaccharide with 3-(3-dimethylaminopropyl)-1-ethylcarbodiimide hydrochloride yielded a modified polysaccharide containing O-acylisourea groups and intramolecular lactones, in addition to some unesterified carboxyl groups . Both forms of ester could be reduced to hydroxymethyl groups by sodium borohydride . Although immuno-precipitation of an antiserum specific for the E . coli LP1092 polysaccharide is not sensitive to the introduction of O-acylisourea groups into the polysaccharide, precipitation was completely eliminated when approximately half of the carboxyl groups of the polysaccharide were converted either into lactone or hydroxymethyl groups . Failure to precipitate the antiserum can be attributed to significant conformational changes in the polysaccharide brought about by the introduction of the latter two groups.

EMBO J, 1984 Jul, 3(7), 1567 - 72
Antibodies against a fused 'lacZ-yeast mitochondrial intron' gene product allow identification of the mRNA maturase encoded by the fourth intron of the yeast cob-box gene; Jacq C et al.; Several missense or nonsense mutations have been localized in the fourth intron open reading frame (ORF) of the yeast mitochondrial cytochrome b gene . These results and the phenotypes of mutants strongly suggested that a mRNA maturase, controlling the expression of both cytochrome b and cytochrome oxidase subunit I (COXI) genes, is encoded in this ORF . To investigate more directly the biosynthesis of mRNA maturase we raised antibodies against a part of the putative ORF translation product . For that purpose we inserted a fragment of the ORF sequence, in phase, into the C-terminal EcoRI site of lacZ gene . The hybrid gene was then expressed in Escherichia coli under the control of either the wild-type lac promoter or the thermoregulated lambda system PR/cI857 . The hybrid protein was partially purified and antibodies were raised against it . These antibodies recognized a mitochondrially coded protein, p27, in intron mutants, whereas no such protein was detected in the wild-type cell . These results demonstrate that the p27 protein, previously shown to be associated with the mRNA maturase activity, is actually translated from the intron ORF . The autoregulated mRNA maturase synthesis model is discussed in relation to these results.

Biochem Pharmacol, 1984 Jul 1, 33(13), 2041 - 6
N-glucuronide formation of carcinogenic aromatic amines in rat and human liver microsomes; Lilienblum W et al.; (1) Sensitive fluorimetric assays were developed for the determination of microsomal UDP-glucuronosyltransferase activities towards 1- and 2-naphthylamine and 4-aminobiphenyl . (2) In rat liver microsomes, enzyme activity towards 1-naphthylamine was orders of magnitude higher than the activities towards 2-naphthylamine, 4-aminobiphenyl or aniline . The differences were less marked with human liver microsomes . (3) Glucuronidation of aniline and 4-aminobiphenyl was not appreciably altered in rat liver microsomes from 3-methylcholanthrene- or phenobarbital-treated rats . UDP-glucuronosyltransferase activities towards 1- and 2-naphthylamine were selectively increased (about 2-fold) by 3-methylcholanthrene-treatment . However the increases were less marked than those observed with representative substrates of the 3-methylcholanthrene-inducible enzyme form . The results suggest that the arylamines investigated are predominantly conjugated by constitutive enzyme forms in rat liver . (4) Arylamine N-glucuronides were found to be susceptible to hydrolysis by E . coli beta-glucuronidase suggesting the release of carcinogenic arylamines in the gut and their enterohepatic circulation.

J Urol, 1984 Jul, 132(1), 179 - 83
The ultrastructure of acute pyelonephritis in the monkey; Fussell EN et al.; P-fimbriated E . coli, which have specific receptors on urothelial cells for their fimbriae, were used to produce pyelonephritis . Using both scanning and electron microscopy, it was shown that the bacteria attached to ureteral epithelium by 12 hours after inoculation . They developed into a biolayer of bacteria covering the epithelium by 48 hours, but did not invade within that time . In the kidney, adherence was seen by 6 hours in collecting proximal and distal tubules . While many bacteria were seen, tubular epithelium remained normal through 24 hours . The inflammatory response was first seen at 48 hours . Phagocytosis was accompanied by mortal damage to both phagocytes and surrounding tubules . This was followed by bacterial invasion of the interstitium.

Mol Biol Evol, 1984 Jul, 1(4), 317 - 34
Immunological similarities between specific chloroplast ribosomal proteins from Chlamydomonas reinhardtii and ribosomal proteins from Escherichia coli; Schmidt RJ et al.; Polyclonal antibodies were elicited against seven of the 33 different proteins of the large subunit of the chloroplast ribosome from Chlamydomonas reinhardtii . Three of these proteins are synthesized in the chloroplast and four are made in the cytoplasm and imported . In western blots, six of the seven antisera are monospecific for their respective large subunit ribosomal proteins, and none of these antisera cross-reacted with any chloroplast small subunit proteins from C . reinhardtii . Antisera to the three chloroplast-synthesized ribosomal proteins cross-reacted with specific Escherichia coli large subunit proteins of comparable charge and molecular weight . Only one of the four antisera to the chloroplast ribosomal proteins synthesized in the cytoplasm cross-reacted with an E . coli large subunit protein . None of the antisera cross-reacted with any E . coli small subunit proteins . On the assumption of a procaryotic, endosymbiotic origin for the chloroplast, those chloroplast ribosomal proteins still synthesized within the organelle appear to have retained more antigenic sites in common with E . coli ribosomal proteins than have those which are now the products of cytoplasmic protein synthesis . Antisera to this cytoplasmically synthesized group of chloroplast ribosomal proteins did not recognize any antigenic sites among C . reinhardtii cytoplasmic ribosomal proteins, suggesting that the genes for the cytoplasmically synthesized chloroplast ribosomal proteins either are not derived from the cytoplasmic ribosomal protein genes or have evolved to a point where no antigenic similarities remain.

Vopr Med Khim, 1984 Jul-Aug, 30(4), 127 - 32
{Method of determining low-molecular weight oligoamines in various biological materials}; Chudinov AA et al.; A modified procedure is described for thin-layer chromatography of dansyl-spermine, -spermidine, -cadaverine and -putrescine . Distinct separation of the dansyl derivatives studied from dansyl-ammonium, which usually causes difficulties, was achieved . This procedure enabled to estimate the pyckomolar quantities of biogenic oligoamines . The method does not require radioactive compounds, enables to carry out the measurements without the use of spectrofluorimeter or fluorescent densitometer . The densitometry of the chromatogram photo-prints was successfully used for quantitative estimation of the oligoamines.

Mol Biol (Mosk), 1984 Jul-Aug, 18(4), 1042 - 52
{Study of the regulation of colicin E1 and K synthesis}; Rekesh AN et al.; Synthesis of colicins E1 and K and other plasmids was studied using DNA templates of corresponding plasmids in a cell-free system of coupled transcription and translation . Up to 15 plasmid-specific polypeptides differing by molecular weight were detected electrophoretically in conditions of complete denaturation . Samples of polypeptides related to plasmids ColE1 and ColK were similar in size . A complete or partial substitution of plasmid-free cell extracts by plasmid-containing cell extracts in a cell-free system leads to repression of the synthesis of colicins and some other proteins which is indicated by diminishing or complete disappearance of electrophoretic bands of corresponding polypeptides . The repression is relieved by addition of the recA gene protein into a cell-free system . Based on the obtained results, a conclusion is made that the repressors of colicin genes are not strictly specific and are, probably, coded by plasmids.

Stain Technol, 1984 Jul, 59(4), 221 - 4
A method to identify microinjected nephrons of rat; Shimamura T et al.; This paper describes a technique for identifying individual nephrons that have been subjected to micropuncture . The general location of the nephron is marked on the surface of the kidney by implanting two micropipette tips on opposite sides of it two or three tubule diameters away . The tubule itself is marked by the injection into the lumen of a tracer material, for purposes of this account, a suspension of E . coli . After perfusion fixation the kidneys are removed and a block of tissue containing the extrapapillary portion of the nephron is excised . This block is cut into thin slices parallel to the surface of the kidney; these are embedded in plastic for subsequent sectioning . On sectioning, the marker material makes the nephron in question readily discernible under the microscope . A major advantage of this technique is that it allows the tubule of interest to be located as much as 48 hours later.

Plasmid, 1984 Jul, 12(1), 37 - 40
A mutational hot spot in the incompatibility gene incC of mini-F plasmid; Seelke RW et al.; The incC incompatibility locus of the mini-F plasmid is also a replication control locus . We have selected and sequenced six independent incC mutations . Each mutation causes a partial loss of incompatibility . All mutations are GC----AT transitions . Five of six mutations occur in the same base pair located at coordinate 46.070 kb; the sixth mutation occurs in an adjacent base pair at 46.069 kb . Both mutational sites are in a hexanucleotide sequence common to several origins of replication.

Plasmid, 1984 Jul, 12(1), 19 - 36
Factors affecting the isolation of CCC DNA from Streptomyces lividans and Escherichia coli; Kieser T; Based on the results of a systematic study of factors affecting plasmid yield and purity, a procedure suitable for the rapid screening for and isolation of covalently closed circular DNA from Streptomyces lividans and Escherichia coli was developed . The method consists of lysis of lysozyme-treated bacteria combined with alkaline denaturation of DNA at high temperature . Renaturation of CCC DNA and precipitation of single-stranded DNA together with protein is achieved by the addition of a minimal amount of phenol/chloroform . The screening procedure uses only a single tube and the samples can be analyzed by agarose gel electrophoresis about 30 min after lysis . Removal of phenol and further purification of the plasmid preparation is achieved by consecutive precipitations with isopropanol and spermine, followed by extraction with ethanol, producing samples suitable for restriction endonuclease digestion, ligation, and transformation of S . lividans protoplasts or competent E . coli cells in about 2 h . All steps of the procedure are explained in detail with information about the effects of changing parameters . This should help the experimenter to obtain reproducible results and may be useful if the method has to be adapted to new strains or plasmids.

Infection, 1984 Jul-Aug, 12(4), 309 - 12
Studies on immunity against Escherichia coli K13 with monoclonal anti-K13 and anti-anti-K13; Soderstrom T et al.; The structural basis for the cross-reactivity between the Escherichia coli K13, K20 and K23 capsular polysaccharides is the ----)-beta-ribofuranosyl-(1----7)-beta-2-keto-3-deoxyoctonate polymer . Monoclonal antibodies against E . coli K13 which require O-acetyl-2-keto-3-deoxyoctonate for binding were further investigated . Such antibodies, of both the IgG and the IgM isotype, opsonized E . coli K13 in vitro and protected against intraperitoneal infection in mice as well as ascending pyelonephritis in rats . A monoclonal IgG1 anti-idiotype, specific for the K13 polysaccharide combining site of a protective IgM idiotype, primed for protection against intraperitoneal infection with live E . coli K13 following K13 injections at four as well as 12 weeks of age . the K13 polysaccharide alone did not immunize and protect . The monoclonal anti-K13 idiotype only primed for protection at four weeks of age . These findings suggest a strong effect of a single idiotype on the outcome of a bacterial infection.

Gene, 1984 Jul-Aug, 29(1-2), 125 - 34
Overproduction of a gastrointestinal hormone, secretin, in Escherichia coli cells and its chemical characterization; Sumi S et al.; A cloned synthetic DNA fragment coding for 27-desamidosecretin (DAS), which differs from natural porcine secretin by the lack of the amide group at the carboxyl-terminal valine residue, was fused to the beta-galactosidase gene (lacZ) at the EcoRI site near the 3'-terminus of the gene . The fusion gene was efficiently expressed in Escherichia coli, yielding 1.15 X 10(6) fused-protein molecules per cell . After the treatment of the fused protein with cyanogen bromide, DAS was purified by a combination of ion-exchange column chromatography and reverse-phase high-performance liquid chromatography (HPLC) . The structure of bacterial DAS was confirmed by tryptic mapping and amino acid composition analyses . The bacterial DAS was not amidated at its carboxyl-terminal valine residue . Nevertheless, it stimulated pancreatic secretion in rats as does authentic porcine secretin, indicating that the carboxyl-terminal amide is not essential to secretin activity.

Vet Q, 1984 Jul, 6(3), 134 - 40
Some pharmacokinetic aspects of four sulphonamides and trimethoprim, and their therapeutic efficacy in experimental Escherichia coli infection in poultry; Goren E et al.; Pharmacokinetic studies in broilers and layers of different sulphonamides indicate a good absorption and a long elimination half-life (of sulphaquinoxaline, sulphadimidine and to a lesser degree sulphadiazine) resulting in high plasma concentrations during drinking water medication in the recommended therapeutic doses . In contrast drinking water medication with high concentrations of trimethoprim (up to 1,320 mg/liter) resulted in a maximal mean plasma concentration of 1.2 micrograms/ml . Very good therapeutic effects were demonstrated in broilers experimentally infected with a sulphonamide-susceptible E . coli strain when treated with sulphaquinoxaline (200 mg/liter), sulphadimidine sodium (2 gram/liter), sulphachloropyridazine 30 per cent (1 gram/liter) and to a lesser degree sulphadiazine sodium (250 mg/liter) . Synergism was demonstrated between trimethoprim and sulphadiazine (1:5) . The combination of trimethoprim with sulphaquinoxaline (1:3) did not induce better therapeutic effects than sulphaquinoxaline in proportional doses . However, significant synergism was demonstrated between trimethoprim and both sulphonamides in treatment of experimental infection with sulphonamide-resistant E . coli . No signs resembling sulphonamide intoxication were observed during these studies.

Kardiologiia, 1984 Jul, 24(7), 14 - 9
{Septic endocarditis in cardiosurgical clinical practice}; Solov'ev GM et al.; The authors analyze the experience of the treatment of eight patients with primary acute septic endocarditis (SE), 35 patients with rheumatic heart disease complicated by secondary subacute SE, 68 patients with early postsurgery SE including 25 who had had closed mitral comissurotomy, and 43 with prosthetic SE . Surgical treatment consisting in the removal of the infection source (the impaired valve or prosthesis) and the implantation of a new prosthesis should be carried out before the development of irreversible alterations of the internal organs.

Biosci Rep, 1984 Jul, 4(7), 565 - 72
The immunity genes of colicins E2 and E8 are closely related; Lau PC et al.; We have determined the nucleotide sequence of the newly characterized colicin E8 imm gene which exists in tandem with the colicin E3 imm gene in the ColE3-CA38 plasmid . Comparison of these immunity structures reveals considerable sequence divergence, but the ColE8 imm gene is markedly homologous to the colicin E2 imm gene from the ColE2-P9 plasmid.

Biochem Int, 1984 Jul, 9(1), 105 - 13
Purification and characterisation of a 12 K dalton peptide with an affinity for oriC containing DNA fragments from Escherichia coli K12 membrane fractions; Chen YQ et al.; A DNA binding protein capable of interacting specifically with oriC containing DNA was purified to near homogeneity from E . coli membrane fractions . It has a molecular weight of 12 KD in denaturing conditions . The specific binding to oriC DNA is more resistant to the presence of salt than the non specific binding . Neither the DNA binding activity nor the specificity for oriC DNA was destroyed by heating at 100 degrees C for 2 min.

Arch Microbiol, 1984 Jul, 138(3), 191 - 4
Expression of surface hydrophobicity encoded by R-plasmids in Escherichia coli laboratory strains; Ferreiros CM et al.; The inclusion of drug-resistance plasmids (R-plasmids) in Escherichia coli strains has been shown to determine the formation of specific surface structures which could modify bacterial surface characteristics relevant for pathogenic processes . Thirtyone R-plasmids (from different incompatibility groups) have been transferred to three E . coli laboratory strains, and surface hydrophobicity modifications have been measured by three methods: "salting-out", adsorption to hexadecane and adsorption to xylene . The results obtained show that the presence of R-plasmids produced variations which are dependent on the receptor strains and measuring method employed . Also, it has been found that the plasmids behave differently depending on the strain in which they are included . The results obtained by the "salting-out" method are not correlative with those obtained by adsorption to hydrocarbons, probably due to the implication of different hydrophobic molecules in the interaction with salt or hydrocarbons . Concluding, the choice of receptor strain and measuring method are of great importance for the investigation of surface hydrophobicity (and probably other characteristics) encoded by R-plasmids.

Antimicrob Agents Chemother, 1984 Jul, 26(1), 10 - 2
Identification of aminoglycoside-acetylating enzymes by high-pressure liquid chromatographic determination of their reaction products; Lovering AM et al.; A method to identify the aminoglycoside-acetyltransferase (AAC) enzymes AAC(3), AAC(2') and AAC(6') by high-pressure liquid chromatographic characterization of their products of reaction with tobramycin or sisomicin is described . Conditions are given for the chromatography of kanamycin A, netilmicin, neomycin, and apramycin, and their products of reaction, if any, with the three AAC enzymes are listed.

Tsitol Genet, 1984 Jul-Aug, 18(4), 268 - 71
{Cytogenetic characteristics of 5 experimental mammalian pathology models}; Zolotareva GN et al.; A cytogenetic study of bone marrow cells was carried out first under conditions of toxic hepatopathy, experimental toxic nephropathy, alloxan diabetes in rats and under conditions of colibacillar and pyocyanic sepsis in mice in different periods of pathology . These experimental models may be used in research on medicinal mutagenesis.

Rev Infect Dis, 1984 Jul-Aug, 6(4), 463 - 71
The enzymatic synthesis of lipid A: molecular structure and biologic function of monosaccharide precursors; Raetz CR; Certain Escherichia coli mutants, deficient in phosphatidylglycerol, accumulate novel, glucosamine-derived phospholipids that appear to be very early precursors of lipid A . The simplest of these, lipid X, is a derivative of glucosamine-1-phosphate substituted with beta-hydroxymyristoyl moieties at positions 2 and 3 . The discovery of lipid X makes it possible to predict a biosynthetic pathway and a unified structure for lipid A . In addition, the minimal molecular requirements for the mitogenic and endotoxic properties of lipopolysaccharides can be elucidated with compounds of this kind.

J Gen Microbiol, 1984 Jul, 130 ( Pt 7), 1839 - 44
The effect of plasmid R391 and other IncJ plasmids on the survival of Escherichia coli after UV irradiation; Pembroke JT et al.; The presence of the IncJ plasmids R391, R997, R705, R706, R748 and R749 was shown to sensitize Escherichia coli AB1157 and both its uvrA and lexA derivatives to UV irradiation . No alteration in post-irradiation survival was observed in a recA mutant containing these plasmids, compared with the non-plasmid-containing recA strain . Analysis of recombination frequency in Hfr crosses to recA+ cells containing plasmid R391 indicated a reduction in recombination frequency compared with that obtained in similar crosses to a non-plasmid-containing strain . This effect was not due to plasmid-encoded restriction or entry exclusion systems and therefore must be considered as a real block in recombination . When cells containing plasmid R391 were irradiated and allowed to photoreactivate, an increase in survival was observed which was comparable to that observed in the non-plasmid-containing derivative . This indicated that post-irradiation processing of UV-induced damage, or lack of such processing, by mechanisms other than photoreactivation was responsible for the UV sensitivity associated with plasmid R391.

J Gen Microbiol, 1984 Jul, 130 ( Pt 7), 1651 - 63
Molecular gentic analysis of the trfB and korB region of broad host range plasmid RK2; Smith CA et al.; A 3.2 kb region of the broad host range IncP plasmid RK2 (indistinguishable from RP1, RP4, R68 and R18) anticlockwise from the EcoRI site may be separated phenotypically into three loci . The trfB/korA/korD locus both complements a temperature-sensitive maintenance mutation and suppresses the deleterious effects of the loci kilA and kilD; the incC locus expresses incompatibility towards complete RK2-like replicons, and the korB locus suppresses the host lethal effect the kilB locus . Transcriptional fusions of the galK gene to various segments of this region revealed that all three loci are transcribed anticlockwise from a common promoter . A weak secondary promoter may also contribute to the expression of korB . Analysis of the sizes of the polypeptides produced from these segments led to the identification of two cistrons, the first encoding a polypeptide of 38 kDal associated with incC function and the second a polypeptide of 49 kDal associated with korB activity . The trfB/korA/korD activities are associated with a polypeptide of 14kDal which may be an N-terminal fragment of the incC-associated polypeptide.

J Gen Microbiol, 1984 Jul, 130 ( Pt 7), 1641 - 50
Genetic evidence for the direction of transcription of the trfA gene of broad host range plasmid RK2; Thomas CM; For replication of broad host range plasmid RK2 in Escherichia coli two regions of the plasmid genome are essential, oriV RK2 between 12.0 and 12.7 kb on the genome (defined clockwise from the unique EcoRI site) and trfA, located between 16.0 and 17.4 kb, which provides a trans-acting product necessary for oriV RK2 function . The properties of an insertion mutant of a mini-RK2/ColE1 hybrid plasmid suggest that the trfA promoter lies clockwise from the 17.4 kb RK2 coordinate . Fusion of the trfA gene lacking its normal promoter to the E . coli trpE gene in a hybrid plasmid confirmed that trfA is transcribed anticlockwise, towards the Tcr gene of RK2 . Repression of trpE promoter activity in these fusions showed that replication of an RK2 replicon can be regulated by varying the level of trfA gene expression . The results also indicate the presence of a transcription unit running anticlockwise through the trfB region located between 54.0 and 56.0 kb on the RK2 genome.

Biull Eksp Biol Med, 1984 Jul, 98(7), 117 - 9
{Determination of the nucleotide and isoplith composition of DNA by scanning thin-layer chromatograms in ultraviolet light}; Vasil'ev VK; The author studied the UV spectra of the bases and pyrimidine sequences of DNA after separation in thin layers of cellulose, and calculated the coefficients permitting the determination of nucleotide and isoplith composition of DNA from UV light reflection during thin-layer chromatogram scanning.

Proc Natl Acad Sci U S A, 1984 Jul, 81(14), 4290 - 3
Ribonuclease T: new exoribonuclease possibly involved in end-turnover of tRNA; Deutscher MP et al.; Examination of double mutants lacking one of the exoribonucleases, RNase II, RNase D, RNase BN, or RNase R, and also devoid of tRNA nucleotidyltransferase has suggested that none of these RNases participates in the end-turnover of tRNA . This prompted a search for and identification of a new exoribonuclease, termed RNase T . RNase T could be detected in mutant Escherichia coli strains lacking as many as three of the known exoribonucleases, and it could be separated from each of the four previously described RNases . RNase T is optimally active at pH 8-9 and requires a divalent cation for activity . The enzyme is sensitive to ionic strengths greater than 50 mM and is rapidly inactivated by heating at 45 degrees C . Its preferred substrate is tRNA-C-C-{14C}A, with much less activity shown against tRNA-C-C . RNase T is an exoribonuclease that initiates attack at the 3' hydroxyl terminus of tRNA and releases AMP in a random mode of hydrolysis . The possible involvement of RNase T in end-turnover of tRNA and in RNA metabolism in general are discussed.

Mol Immunol, 1984 Jul, 21(7), 609 - 20
Interaction of human complement proteins with serum-sensitive and serum-resistant strains of Escherichia coli; Taylor PW et al.; Exposure of serum-susceptible Escherichia coli strains to lethal concns of lysozyme-free human serum resulted in stable binding of complement components to the outer membrane (OM), but not to the cytoplasmic membrane (CM) . The short prekilling phase of the reaction was accompanied by binding of C3b; loss of viability was immediately preceeded by stable deposition onto the OM of the component proteins of the membrane attack complex . During the early stages of the active killing phase, bound monomeric C9 could be resolved into two distinct bands on SDS-polyacrylamide gels . Serum exposure lead to a progressive loss of CM recoverability, which appeared to result from partial degradation of CM phospholipids . In contrast, exposure of a resistant E, coli strain to human serum resulted in little change in the membrane profile and very little stable deposition of terminal complement components onto the OM.

JPEN J Parenter Enteral Nutr, 1984 Jul-Aug, 8(4), 440 - 2
Clearance rate of intravenously administered lipid emulsion in canine endotoxemia; Iriyama K et al.; The effect of endotoxemia on the initial catabolism of intravenously given lipid emulsion was investigated in dogs . Two types of endotoxemia were prepared . One was produced by peritonitis which was established by ligation of the artery and vein of an isolated intestine (group 1, n = 6) . The other was made by an intravenous injection of Escherichia coli lipopolysaccharide in a dose of 1.5 mg/kg of body weight (group 2, n = 6) . Group 1 showed evident peritonitis with a positive limulus test 48 hr after the procedure, but no significant changes of blood sugar level and lactate/pyruvate ratio, while group 2 demonstrated profound hypoglycemia, significant elevation of lactate/pyruvate ratio, and low arterial pressure 3 to 5 hr after the injection of lipopolysaccharide . The clearance rate of intravenously administered lipid emulsion (K value) of group 1 before the peritonitis was 0.0105 +/- 0.0017 and after the peritonitis it was 0.0105 +/- 0.0019 . The difference was not significant, while the K value of group 2 which was 0.0133 +/- 0.0056 before the injection of lipopolysaccharide decreased significantly to 0.0069 +/- 0.0024 after the injection of lipopolysaccharide . These results suggest that, in case of endotoxemia with normally maintained oxidation-reduction potential, the initial catabolism of intravenously given lipid emulsion is kept in a normal level, while oxidation-reduction potential is impaired, it is inhibited.

EMBO J, 1984 Jul, 3(7), 1609 - 12
RNA secondary structure and translation inhibition: analysis of mutants in the rplJ leader; Christensen T et al.; We have carried out measurements of the stable binding of the ribosomal protein (r-protein) complex L10-L7/L12 to mutant forms of the mRNA leader of the rplJ operon of Escherichia coli . One of the point mutations, base 1548, which lies within the L10-L7/L12-protected region, almost completely abolishes in vitro formation of a stable complex of L10-L7/L12 with rplJ mRNA leader, and a second point mutation, base 1634, strongly reduces it . These observations constitute strong support for the proposition that L10-L7/L12 binds to the rplJ leader in bringing about translational feedback . To account for the action of these and other mutations, and to explain the mechanism of translation feedback inhibition, we suggest a secondary structure model involving alternate forms of the rplJ mRNA leader.

EMBO J, 1984 Jul, 3(7), 1513 - 9
The korB gene of broad host range plasmid RK2 is a major copy number control element which may act together with trfB by limiting trfA expression; Thomas CM et al.; For replication, plasmid RK2 encodes a vegetative replication origin, oriV RK2, and a gene, trfA, whose polypeptide product(s) is essential for oriV RK2 activity . The trfA gene is transcribed as part of a polycistronic operon which also includes kilD . Transcription of this operon is negatively regulated by the products of the trfB/korD/korA and korB loci . Mini replicons previously studied in detail lack the korB locus and have copy numbers significantly higher than RK2 itself . Here we report that korB in trans expresses incompatibility towards RK2 replicons either when the korB gene dosage is high or when it is expressed from a strong foreign promoter . This incompatibility can be largely overcome if a trfA gene which is expressed from a foreign promoter, and is therefore not regulated by korB, is supplied in trans . When korB is introduced in cis to mini RK2 replicons the copy number is reduced to within the range estimated for parental RK2 . Deletions in the oriV RK2 region which otherwise cause quite large increases in plasmid copy number have only a small effect when korB is present in cis . These results suggest that korB in combination with trfB may be the overriding copy number control element in RK2 reducing trfA expression to levels limiting for replication.

Am J Surg, 1984 Jul, 148(1), 117 - 24
Oriental cholangitis; Carmona RH et al.; Oriental cholangitis is a poorly understood syndrome consisting of intrahepatic pigment stone formation with chronically recurrent exacerbations and remissions . Endemic to Asia, it is being encountered more frequently in the United States due to increased immigration of asians . Twenty-one patients with oriental cholangitis (9 men and 12 women), 19 to 84 years of age, all of whom immigrated from asian countries, were treated between 1970 and 1983 . All had histories of episodic abdominal pain, most with jaundice, chills, and fever . Laboratory results were nonspecific but frequently included leukocytosis and hyperbilirubinemia . All patients were operated on with 15 having cholecystectomy, common duct exploration, and a bilioenteric anastomosis . E . coli was cultured from specimens obtained from the biliary tracts of all patients, and 13 patients had more than one organism . Four patients had a previous history of parasitic infection, and four different patients had parasites identified in the biliary tract intraoperatively . Early recognition and appropriate operation will decrease morbidity and mortality.

Proc Natl Acad Sci U S A, 1984 Jul, 81(13), 4139 - 43
Multiple regulatory signals in the control region of the Escherichia coli carAB operon; Bouvier J et al.; The first reaction in pyrimidine and arginine biosynthesis in Escherichia coli is catalyzed by a single enzyme, carbamoyl-phosphate synthetase (EC 6.3.5.5), the product of the carAB operon . Expression of this operon is cumulatively repressed by arginine and pyrimidines . The nucleotide sequence of the carAB control region was determined and transcriptional starts were localized . Two adjacent promoters, 70 base pairs apart, appear to be used in vivo, the downstream one overlapping a typical arginine operator . The absence of any attenuation-like sequence excludes such a mechanism for pyrimidine-mediated repression . Various fragments of the carA promoter-proximal region were fused in vitro with the lacZ gene . Results obtained with these fusions indicate that (i) translation of the carA gene can be initiated in vivo without an AUG codon but very likely with an UUG or an AUU codon; (ii) the carAB downstream promoter is repressed by arginine; and (iii) the carAB upstream promoter is repressed by pyrimidines and subject to stringent control . When carried by a multicopy plasmid the carAB control region escapes repression by arginine and pyrimidines . The existence of a pyrimidine repressor, present in limiting amounts in the cell, is therefore postulated.

Proc Natl Acad Sci U S A, 1984 Jul, 81(13), 4120 - 4
Regulation of the araC gene of Escherichia coli: catabolite repression, autoregulation, and effect on araBAD expression; Miyada CG et al.; The araC gene encodes a positive regulatory protein required for L-arabinose utilization in Escherichia coli . Transcription from the araC promoter has been shown to be under positive control by cAMP receptor protein and under negative control by its protein product (autoregulation) . This work describes the identification of the region of the araC promoter that interacts with the cAMP receptor protein to mediate catabolite repression . A 3-base-pair deletion centered 60 base pairs from the transcriptional initiation site results in a mutant araC promoter that, in the absence of araC protein, reduces transcriptional activity when compared with the wild-type promoter and is unresponsive to various concentrations of intracellular cAMP in vivo . The same deletion results in a lowered affinity of the araC promoter for cAMP receptor protein in vitro . However, this lowered affinity for the mutant araC promoter does not result in substantial reduction of intracellular araC protein because autoregulation of the araC gene dominates catabolite repression . The 3-base-pair deletion in the cAMP receptor protein binding site of the araC promoter does not affect catabolite repression of the adjacent araBAD operon . The implications of these results on current models for expression of the araBAD operon and the araC gene are discussed.

Proc Natl Acad Sci U S A, 1984 Jul, 81(13), 4037 - 40
Structure of unligated aspartate carbamoyltransferase of Escherichia coli at 2.6-A resolution; Ke HM et al.; The three-dimensional structure of the allosteric enzyme aspartate carbamoyltransferase (EC 2.1.3.2) has been refined to a crystallographic R-factor of 0.24 at 2.6-A resolution in the space group P321, where a and b are 122.1 A and c is 142.2 A . This structure is isomorphous to the form of the enzyme complexed to the allosteric inhibitor cytidine triphosphate . All sources of sequence information have been evaluated against the electron density . The corrected amino acid sequences of the catalytic and regulatory proteins have been incorporated in the model, and three regions in the active site are described: (i) near arginine-105, histidine-134, and arginine-167, (ii) near lysine-232 and arginine-229, and (iii) near lysine-83 and lysine-84.

Proc Natl Acad Sci U S A, 1984 Jul, 81(13), 3973 - 7
Model of specific complex between catabolite gene activator protein and B-DNA suggested by electrostatic complementarity; Weber IT et al.; Calculation of the electrostatic potential energy surfaces of Escherichia coli catabolite gene activator protein (CAP) dimer suggests a model for the complex between CAP and a specific DNA sequence . The positive electrostatic charge density of CAP lies on the two COOH-terminal domains and about 20-30 A from the molecular 2-fold axis . Assuming that the 2-fold axes of the CAP dimer and the DNA to which it binds are coincident, the positions of the positive electrostatic potential surfaces strongly suggest the rotational orientation of the DNA relative to the protein . A specific complex between CAP and its DNA binding site in the lac operon has been built with the DNA in this orientation . The amino ends of the two protruding F alpha-helices interact in successive major grooves of the DNA . Four side chains emanating from each F helix can form hydrogen bonds with the exposed edges of four bases in the major groove . Electrostatic considerations as well as the necessity to make interactions between CAP and a DNA site as much as 20 base pairs long require us to bend or kink the DNA . In our model of CAP complexed with B-DNA, as with those proposed for Cro and lambda cI repressors, the protruding second helices of the two-helix motif from both subunits interact in successive major grooves of B-DNA . However, unlike Cro and similar to lambda cI, the protruding alpha-helices are nearly parallel to the bases rather than the groove.

Proc Natl Acad Sci U S A, 1984 Jul, 81(13), 3969 - 72
Cross-linking of tRNA at two different sites of the elongation factor Tu; Van Noort JM et al.; Recently, we reported on the induction by kirromycin of two tRNA binding sites on elongation factor Tu . To obtain independent information on the existence of these two sites and to characterize them further, 3' oxidized tRNA was cross-linked to elongation factor Tu by {3H}borohydride reduction . Specific cross-linking occurred exclusively in the presence of kirromycin . In the case of elongation factor Tu X GDP X kirromycin, cross-linking was found at lysine-208; in elongation factor Tu X GTP X kirromycin, cross-linking was at lysine-208 and lysine-237 . In both elongation factor Tu complexes, kirromycin itself was found cross-linked to lysine-357 . The tRNA cross-linking sites are in agreement with the idea of two different binding sites of tRNA on elongation factor Tu.

J Bacteriol, 1984 Jul, 159(1), 421 - 3
Kinetics of methylation in Escherichia coli K-12; Lyons SM et al.; Newly synthesized DNA is undermethylated in E . coli K-12 . The amount of N6-methyl deoxyadenylic acid in labeled DNA varied from 0.3 mol% of total adenine for a 2-min pulse to 1.7 mol% for DNA that was labeled for more than two generations.

J Bacteriol, 1984 Jul, 159(1), 368 - 74
Chemosensory and thermosensory excitation in adaptation-deficient mutants of Escherichia coli; Imae Y et al.; Methyl-accepting chemotaxis protein-methyltransferase-deficient mutants, cheR mutants, of Escherichia coli showed a tumble response to repellents only at low temperatures, and the resultant tumbling lasted unless the condition was changed . The swimming pattern of the repellent-treated cells was different at different temperatures, indicating that the absolute temperature is a determinant of the tumbling frequency of those cells . The tumbling of those cells was also suppressed by the addition of attractants . Under a suitable repellent concentration, the tumbling frequency of the cells was found to be simply determined by the ligand occupancy of chemoreceptors for many attractants . In a methyl-accepting chemotaxis protein-methylesterase-deficient mutant, a cheB deletion mutant, the tumbling frequency was also determined by receptor occupancy of some attractants . These results indicate that in the adaptation-deficient mutants, sensory signals are produced in proportion to the amount of ligand-bound or of thermally altered receptors and transmitted to the flagellar motors without any modification . Thus, it is concluded that the adaptation system, namely, the methylation-demethylation system of methyl-accepting chemotaxis proteins, is not concerned with the step of chemosensory or thermosensory excitation . A simple model is proposed to explain how the swimming pattern of the adaptation-deficient mutants is determined.

J Bacteriol, 1984 Jul, 159(1), 360 - 7
Conditional inversion of the thermoresponse in Escherichia coli; Mizuno T et al.; Mutants in Escherichia coli having defects in one of the methyl-accepting chemotaxis proteins, Tsr protein, which is the chemoreceptor and transducer for L-serine, showed a reduced but similar type of thermoresponse compared with wild-type strains; the cells showed smooth swimming upon temperature increase and tumbling upon temperature decrease . However, when the mutant cells were adapted to attractants such as L-aspartate and maltose, which are specific to another methyl-accepting chemotaxis protein, Tar protein, the direction of the thermoresponse was found to be inverted; a temperature increase induced tumbling and a temperature decrease induced smooth swimming . Consistent with this, the mutant cells showed inverted changes in the methylation level of Tar protein upon temperature changes . Wild-type strains but not Tar protein-deficient mutants exhibited the inverted thermoresponse when the cells were simultaneously adapted to L-aspartate and L-serine, indicating that Tar protein has a key role in the inversion of the thermoresponse . Thus, besides Tsr protein, Tar protein has a certain role in thermoreception . A simple model for thermoreception and inversion of the thermoresponse is also discussed.

J Bacteriol, 1984 Jul, 159(1), 329 - 34
Acquisition of Escherichia coli outer membrane proteins by Bdellovibrio sp . strain 109D; Diedrich DL et al.; The ability of Bdellovibrio sp . to acquire the OmpF major outer membrane protein from its Escherichia coli prey was examined to determine if there were other outer membrane proteins which could or could not be acquired . Growth of bdellovibrios on mutant prey which were defective in the expression of outer membrane proteins revealed that Bdellovibrio sp . could acquire the OmpC protein in the absence of the OmpF protein . However, the OmpA, LamB, and protein 2 proteins could not be found in the Bdellovibrio Triton-insoluble outer membrane . The disappearance of the OmpF and OmpC proteins from the bdelloplast surface was measured, and it was determined that Bdellovibrio sp . exhibited a kinetic and temporal preference for the OmpF protein . Bdellovibrios could be grown on porin-deficient prey, and the progeny bdellovibrios possessed outer membranes with a protein mass deficiency.

Infect Immun, 1984 Jul, 45(1), 278 - 80
Species differences in Kupffer cells and endotoxin sensitivity; McCuskey RS et al.; The relative species sensitivity to Escherichia coli O111:B4 endotoxin was found to be guinea pig greater than hamster greater than mouse greater than rat . The 50% lethal dose of this endotoxin correlated with both the rate at which single latex particles were phagocytosed by individual Kupffer cells and the number of Kupffer cells in hepatic lobules that phagocytosed latex . The results suggests that the intrahepatic density and the level of activation of Kupffer cells participate in determining endotoxin sensitivity.

Infect Immun, 1984 Jul, 45(1), 242 - 7
Molecular homogeneity of heat-stable enterotoxins produced by bovine enterotoxigenic Escherichia coli; Saeed AM et al.; Heat-stable enterotoxins (STs) from four strains of bovine enterotoxigenic Escherichia coli representing four serogroups were purified to homogeneity by utilizing previously published purification schemata . Biochemical characterization of the purified STs showed that they met the basic criteria for the heat-stable enterotoxins of E . coli . Amino acid analysis of the purified STs revealed that they were peptides of identical amino acid composition . This composition consisted of 18 residues of 10 different amino acids, 6 of which were cysteine . The amino acid composition of the four ST peptides was identical to that reported for the STs of human and porcine E . coli . In addition, complete sequence analysis of two of the ST peptides and partial sequencing of several others revealed strong homology to the sequences of STs from human and porcine E . coli and to the sequence predicted from the last 18 codons of the transposon Tn1681 . There was also substantial homology to the sequence predicted from the ST-coding genetic element of human E . coli, which may indicate the existence of identical bioactive configuration among ST peptides of E . coli strains of various host origins . These data support the hypothesis that STs produced by human, bovine, and porcine E . coli are coded by a closely related genetic element which may have originated from a single, widely disseminated transposon.

Proc Soc Exp Biol Med, 1984 Jul, 176(3), 292 - 6
Changes in protein degradation in chickens due to an inflammatory challenge; Klasing KC et al.; Tissue-specific changes in protein catabolism were examined in chicks 16 hr following an inflammatory challenge . It was determined that tyrosine was not catabolized or converted to phenylalanine in muscle, thymus, bursa, or spleen . Therefore, rates of tyrosine release from protein were used to estimate rates of protein catabolism in these tissues . Arginine was not catabolized to urea by chick liver; consequently, arginine release from liver protein was used to measure protein catabolism in this tissue . An injection of sheep red blood cells (SRBC) or Escherichia coli did not change rates of protein catabolism in liver or bursa as compared to saline-injected controls . SRBC significantly increased protein catabolism in muscle and spleen by 29 and 15%, respectively . E . coli resulted in significant increases in muscle, spleen, and thymus of 43, 30, and 34%, respectively . These changes in protein catabolism, together with known changes in protein synthesis, suggest that an inflammatory response to SRBC and E . coli result in increased protein accretion in the bursa and liver, and net protein loss from muscle.

Proc Soc Exp Biol Med, 1984 Jul, 176(3), 285 - 91
Changes in protein synthesis due to an inflammatory challenge; Klasing KC et al.; Rates of protein synthesis in various chick tissues were examined 16 hr after an inflammatory challenge . Protein synthetic rates were calculated from the rate at which {14C}leucine was incorporated into protein and the specific activity of {14C}leucine in the precursor pool . An injection of either Escherichia coli or sheep red blood cells (SRBC) decreased the rate of protein synthesis in the gastrocnemius muscle, and increased the rate in liver, bursa, spleen, and thymus . E . coli, but not SRBC, decreased protein synthesis in the pectoralis muscle . E . coli significantly decreased the aggregation of pectoralis muscle polysomes and increased the aggregation of polysomes in the thymus, bursa, and spleen . E . coli increased the aggregation of free, but not bound, polysomes in liver, suggesting an increase in synthesis of export proteins . SRBC significantly increased polysomal aggregation in bursa and spleen only . A crude preparation of leukocyte endogenous mediator, isolated from peritoneal macrophages, decreased muscle-polysomal aggregation . These studies indicate that tissue-specific changes in protein synthesis occur after a noninfectious inflammatory challenge . These changes may be part of a homeostatic mechanism which supports the immune response.

Proc Natl Acad Sci U S A, 1984 Jul, 81(13), 4149 - 53
Construction of mutants of Moloney murine leukemia virus by suppressor-linker insertional mutagenesis: positions of viable insertion mutations; Lobel LI et al.; A highly efficient method for the generation of insertion mutations is described . The procedure involves the use of a 220-base-pair (bp) EcoRI fragment bearing the SuIII+ suppressor tRNA gene as an insertional mutagen . The plasmid DNA to be mutagenized is linearized by a variety of means, and the suppressor fragment is ligated into the site of cleavage . Successful insertion mutants can be readily detected in Escherichia coli carrying lac- amber mutations on MacConkey lactose plates; virtually 100% of the red colonies contain insertions of the fragment . Subsequent removal of the SuIII+ gene and recyclization leaves a 12-bp insertion if the original cleavage was blunt-ended and a 9-bp insertion if the original cleavage generated 3-bp cohesive termini . This technique, as well as conventional linker mutagenesis with decamer and dodecamer linkers, was used to generate a large library of insertion mutations in cloned DNA copies of the genome of Moloney murine leukemia virus . A number of viable mutants were isolated bearing 9-, 10-, and 12-bp insertions in various domains of the genome . The map positions of the viable mutations suggest that the viral long terminal repeats and portions of the gag and env genes are quite insensitive to alteration . Although most of the mutations were stable for many passages, some of the mutants lost the inserted DNA; we presume that the insertion was somewhat deleterious in these mutants and that continued passage of the virus selected for overgrowth by a revertant.

Proc Natl Acad Sci U S A, 1984 Jul, 81(13), 4134 - 8
DNA sequence of the carA gene and the control region of carAB: tandem promoters, respectively controlled by arginine and the pyrimidines, regulate the synthesis of carbamoyl-phosphate synthetase in Escherichia coli K-12; Piette J et al.; The carAB operon of Escherichia coli K-12, which encodes the two subunits of carbamoyl-phosphate synthetase (glutamine hydrolyzing) {carbon-dioxide: L-glutamine amido-ligase (ADP-forming, carbamate-phosphorylating); EC 6.3.5.5}, is cumulatively repressed by arginine and the pyrimidines . We describe the structure of the control region of carAB and the sequence of the carA gene . Nuclease S1 mapping experiments show that two adjacent tandem promoters within the carAB control region serve as initiation sites . The upstream promoter P1 is controlled by pyrimidines; the downstream promoter P2 is regulated by arginine . Attenuation control does not appear to be involved in the expression of carAB . A possible mechanism by which control at these promoters concurs to produce a cumulative pattern of repression is discussed . The translational start of carA is atypical; it consists of a UUG or AUU codon.

Proc Natl Acad Sci U S A, 1984 Jul, 81(13), 4110 - 4
Induction of specific Escherichia coli genes by sublethal treatments with alkylating agents; Volkert MR et al.; Fusions of the lac operon to genes induced by treatment with sublethal levels of alkylating agents have been selected from random insertions of the Mu-dl(ApRlac) phage by screening for induction of beta-galactosidase activity in the presence of methyl methanesulfonate . Genetic analysis reveals that these fusions resulted from insertion of Mu-dl(ApRlac) into two regions of the chromosome . One region (aidA) is near his and, based on phenotypic effects, appears to represent insertion into the alkA gene . The other region (aidB) is in the 92.3- to 98-min region, which harbors no previously identified genes involved in repair of alkylation damage . The aidB fusions caused increased resistance to alkylating agents and caused little or no change in the biological effects of adaptation to alkylating agents . Unlike the aidA fusions, aidB fusions showed increased beta-galactosidase activity in untreated cells in a growth phase-dependent fashion . The ada-5 mutation, which blocks expression of the adaptive response, decreased induction of beta-galactosidase activity in both aidA and aidB fusions after alkylation treatments . Thus, both aidA and aidB share with adaptive response a common regulatory mechanism involving the ada gene . The growth phase-dependent control of the aidB fusions, however, is unaffected by ada, suggesting that a second regulatory mechanism exists that controls only aidB.

Proc Natl Acad Sci U S A, 1984 Jul, 81(13), 4051 - 4
Accurate initiation of human epsilon-globin RNA synthesis by Escherichia coli RNA polymerase in isolated nuclei of K562 erythroleukemia cells; Gilmour RS et al.; The human epsilon-globin gene was transcribed in vitro in isolated K562 cell nuclei by using exogenous Escherichia coli RNA polymerase (EC 2.7.7.6) . Newly formed RNA transcripts were distinguished from nuclear RNA molecules by (i) incorporating mercurated UTP into RNA under conditions in which the endogenous polymerase II is inactive and (ii) subsequently isolating the mercurated RNA by affinity chromatography on thiolated Sepharose . A specific 5'-end-labeled probe spanning the epsilon-globin gene cap site was used in nuclease S1 mapping studies to examine the in vitro initiation site of the isolated transcripts . It was found that transcription occurred from the coding strand only and originated almost entirely from a point that was identical to that of the major cap site for epsilon-globin mRNA in vivo.

Proc Natl Acad Sci U S A, 1984 Jul, 81(13), 3983 - 7
Intracellular targeting and import of an F1-ATPase beta-subunit-beta-galactosidase hybrid protein into yeast mitochondria; Douglas MG et al.; The gene coding for the yeast mitochondrial F1-ATPase beta subunit (ATP2) has been fused to the Escherichia coli lacZ gene . The chimeric ATP2-lacZ gene codes for a hybrid protein consisting of some 350 amino acids of the F1-ATPase beta subunit at its amino terminus and a large enzymatically active portion of the lacZ gene product, beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23), at its carboxyl terminus . The beta-subunit-beta-galactosidase hybrid protein is expressed in both E . coli and yeast . In yeast, this hybrid molecule is targeted to the mitochondrion and is protected in isolated mitochondria from added protease under conditions in which an outer membrane enzymatic marker is digested . Yeast cells carrying the ATP2-lacZ gene fusion on plasmid p beta Z1 are unable to grow on a nonfermentable carbon source . Upon loss of the p beta Z1 plasmid, growth of the cured host strain on the nonfermentable substrate is restored . In the presence of the beta-subunit-beta-galactosidase hybrid protein, the energy-transducing capacity of the mitochondrial membrane as measured by the 32Pi-ATP exchange reaction is only 9% of that measured in the absence of the gene fusion product . The results indicate that it is the presence of the beta-subunit-beta-galactosidase hybrid protein within mitochondria that interferes with function(s) essential for respiratory growth . These observations open up the prospect of genetic characterization of the signals and cellular machinery responsible for mitochondrial protein delivery.

J Invest Dermatol, 1984 Jul, 83(1 Suppl), 82s - 87s
Transforming functions associated with Epstein-Barr virus; Sugden B et al.; Epstein-Barr virus (EBV) transforms the human B-lymphocytes it infects into lymphoblasts that are competent to proliferate indefinitely in tissue culture {1} . The dividing transformed lymphoblasts carry multiple and complete copies of EBV DNA as plasmids {1} . No viral functions that are necessary for EBV-induced transformation have been identified and characterized to date . We have developed an assay that aids in the identification of viral functions needed to maintain the viral plasmid in transformed cells . The assay employs a plasmid vector that encodes resistance to ampicillin and can replicate in E . coli . It also encodes resistance to the neomycin derivative G418, so that its presence can be selected in mammalian cells {2} . Fragments of viral DNA that span the genome have been molecularly cloned into this vector . These recombinant molecules have been transfected into four cell types, and survivors to G418 have been scored . The DNA from one region of EBV gives a 10- to 100-fold higher rate of survival per microgram of added DNA than does the DNA from all other recombinants, as tested in cells that already contain EBV genomes . This recombinant fragment of EBV is maintained in an apparently unrearranged state as a plasmid and therefore carries at least those cis-acting viral elements necessary for plasmid replication.

J Bacteriol, 1984 Jul, 159(1), 93 - 9
Transposon Tn10-dependent expression of the lamB gene in Escherichia coli; Brass JM et al.; Among Tn10 insertions isolated in or near the malB region of Escherichia coli, one (zjb-729::Tn10) mapped between malK and lamB or late in malK and allowed MalT-independent expression of lamB . Tn10-dependent expression of a lamB-lacZ protein fusion was 25% of the expression of the fusion from the malK-lamB operon promoter in malTc constitutive strains . The maltoporin content of a strain carrying this Tn10 was about 20% that of a malTc malB+ strain . Transport of maltose at concentrations of below 10(-6) M was reduced about threefold . When maltoporin was present at about 50% of the level of malTc malB+ strains, maltose transport was largely restored . We conclude that maltoporin is not rate limiting for maltose transport in wild-type cells but becomes rate limiting when the ratio of maltoporin to other maltose transport components is reduced more than twofold.

J Bacteriol, 1984 Jul, 159(1), 57 - 62
Nucleotide sequence of Escherichia coli pabB indicates a common evolutionary origin of p-aminobenzoate synthetase and anthranilate synthetase; Goncharoff P et al.; Biochemical and immunological experiments have suggested that the Escherichia coli enzyme p-aminobenzoate synthetase and anthranilate synthetase are structurally related . Both enzymes are composed of two nonidentical subunits . Anthranilate synthetase is composed of proteins encoded by the genes trp(G)D and trpE, whereas p-aminobenzoate synthetase is composed of proteins encoded by pabA and pabB . These two enzymes catalyze similar reactions and produce similar products . The nucleotide sequences of pabA and trp(G)D have been determined and indicate a common evolutionary origin of these two genes . Here we present the nucleotide sequence of pabB and compare it with that of trpE . Similarities are 26% at the amino acid level and 40% at the nucleotide level . We propose that pabB and trpE arose from a common ancestor and hence that there is a common ancestry of genes encoding p-aminobenzoate synthetase and anthranilate synthetase.

J Bacteriol, 1984 Jul, 159(1), 424 - 6
Physical mapping of hemolysin plasmid pCW2, which codes for virulence of a nephropathogenic Escherichia coli strain; Waalwijk C et al.; The hemolysin plasmid pCW2, which enhances the virulence of Escherichia coli, has been mapped . Comparison of the region coding for hemolysin with other hemolysin determinants reveals differences that could explain differences in the ability to confer virulence.

J Bacteriol, 1984 Jul, 159(1), 418 - 20
Cloning of the iron superoxide dismutase gene (sodB) in Escherichia coli K-12; Sakamoto H et al.; A clone overproducing iron superoxide dismutase has been isolated from an Escherichia coli cosmid bank . Subcloning located the gene responsible for iron superoxide dismutase overproduction on a 6.6-kilobase PstI restriction endonuclease fragment . Maxicell analysis, followed by immunological identification of iron superoxide dismutase protein, demonstrated that the structural gene, sodB, of iron superoxide dismutase has been cloned.

J Bacteriol, 1984 Jul, 159(1), 404 - 6
Isolation and analysis of inhibitors of transposon Tn3 site-specific recombination; Fennewald MA et al.; We have constructed a genetic bioassay for inhibitors of site-specific recombination by transposon Tn3 resolvase . Of 6,000 compounds tested, 26 inhibited in vivo, and 5 of these 26 inhibited in vitro . At least two inhibitors also inhibit the topoisomerase of resolvase . We have also identified analogs of A1062 which inhibit.

J Bacteriol, 1984 Jul, 159(1), 283 - 7
lon gene product of Escherichia coli is a heat-shock protein; Phillips TA et al.; The product of the pleiotropic gene lon is a protein with protease activity and has been tentatively identified as protein H94.0 on the reference two-dimensional gel of Escherichia coli proteins . Purified Lon protease migrated with the prominent cellular protein H94.0 in E . coli K-12 strains . Peptide map patterns of Lon protease and H94.0 were identical . A mutant form of the protease had altered mobility during gel electrophoresis . An E . coli B/r strain that is known to be defective in Lon function contained no detectable H94.0 protein under normal growth conditions . Upon a shift to 42 degrees C, however, the Lon protease was induced to high levels in K-12 strains and a small amount of protein became detectable at the H94.0 location in strain B/r . Heat induction of Lon protease was dependent on the normal allele of the regulatory gene, htpR, establishing lon as a member of the high-temperature-production regulon of E . coli.

J Bacteriol, 1984 Jul, 159(1), 265 - 70
Purine metabolism in Acholeplasma laidlawii B: novel PPi-dependent nucleoside kinase activity; Tryon VV et al.; Acholeplasma laidlawii B-PG9 was examined for 16 cytoplasmic enzymes with activity for purine salvage and interconversion . Phosphoribosyltransferase activities for adenine, guanine, xanthine, and hypoxanthine were shown . Adenine, guanine, xanthine, and hypoxanthine were ribosylated to their nucleoside . Adenosine, inosine, xanthosine, and guanosine were converted to their base . No ATP-dependent phosphorylation of nucleosides to mononucleotides was found . However, PPi-dependent phosphorylation of adenosine, inosine, and guanosine to AMP, inosine monophosphate, and GMP, respectively, was detected . Nucleotidase activity for AMP, inosine monophosphate, xanthosine monophosphate, and GMP was also found . Interconversion of GMP to AMP was detected . Enzyme activities for the interconversion of AMP to GMP were not detected . Therefore, A . laidlawii B-PG9 cannot synthesize guanylates from adenylates or inosinates . De novo synthesis of purines was not detected . This study demonstrates that A . laidlawii B-PG9 has the enzyme activities for the salvage and limited interconversion of purines and, except for purine nucleoside kinase activity, is similar to Mycoplasma mycoides subsp . mycoides . This is the first report of a PPi-dependent nucleoside kinase activity in any organism.

J Bacteriol, 1984 Jul, 159(1), 260 - 4
Evidence for antitermination in Escherichia coli RRNA transcription; Aksoy S et al.; The stable RNA operons of Escherichia coli do not exhibit polarity, even though they make an RNA product that is not translated . By contrast, most E . coli operons that specify proteins exhibit polarity if their translation is interrupted . The transcriptional component of this polarity depends on the action of Rho protein on the exposed mRNA, which results in premature transcription termination . Here we examine how a stable RNA operon (rrnG) transcript is protected from the Rho protein-mediated polarity response . We compared transcription from the ara and the rrnG promoters through a 16S DNA segment . In each case, the promoter-16S sequences were joined to a trp-lac fusion, and lacZ mRNA was examined in rho+ and rho-115 strains . We found significant Rho protein-dependent termination of transcripts from the ara promoter but little or no Rho protein effect on transcription from the rrnG promoter . We concluded that the transcript of the 16S ribosomal DNA segment does contain Rho protein-dependent transcription terminators, but there is an antitermination system in the rrnG control region that allows it to transcribe through those terminators.

J Bacteriol, 1984 Jul, 159(1), 19 - 25
Identification of the phoM gene product and its regulation in Escherichia coli K-12; Ludtke D et al.; Plasmids containing the chromosome region of Escherichia coli encoding phoM, whose product is a positive regulator of alkaline phosphatase expression, were isolated from the Clarke and Carbon plasmid bank . A 9.9-kilobase EcoRI fragment of plasmid pLC17-39 (subcloned into pBR322) was able to complement both phoM and thrB mutations . Restriction endonuclease analysis and in vitro mutagenesis of the hybird plasmids enabled the localization of the phoM gene locus to 3 kilobases of the cloned chromosomal fragment . The phoM gene product was identified, with maxicell techniques, as a protein with an approximate molecular weight of 55,000 . A phoM-lacZ protein fusion was constructed by using a plasmid carrying the phoM gene and a derivative of phage lambda, lambda plac Mu2 . Restriction endonuclease analysis of the plasmid carrying the fusion indicated that phoM is transcribed in a clockwise direction on the circular E . coli chromosome . Analysis of strains bearing the fusion on a multiple-copy plasmid or integrated at the lambda attachment site of the chromosome indicated that the synthesis of the phoM gene product was unaffected by phosphate limitation of growth . The expression of the phoM gene was studied in strains with mutations in genes encoding effectors of the pho regulon . A threefold increase in phoM expression was seen in a phoU strain in comparison with the wild-type strain.

J Bacteriol, 1984 Jul, 159(1), 184 - 90
Regulatory changes in the formation of chromosomal dihydrofolate reductase causing resistance to trimethoprim; Flensburg J et al.; High resistance to trimethoprim mediated by the several hundredfold overproduction of the drug target enzyme, dihyrofolate reductase, in a clinically isolated Escherichia coli strain, 1810, was cloned onto several vector plasmids and seemed to be comprised of a single dihydrofolate reductase gene, which by DNA-DNA hybridization and restriction enzyme digestion mapping was very similar to the corresponding gene of E . coli K-12 . Determination of mRNA formation in the originally isolated resistant strain and strains with cloned trimethoprim resistance determinant demonstrated an about 15-fold increase in production of dihydrofolate reductase mRNA compared with that in E . coli K-12 . This was explained by the occurrence of a promoter up mutation in the resistant isolate accompanied by changes in the restriction enzyme digestion pattern found by comparison with the corresponding pattern from E . coli K-12.

J Bacteriol, 1984 Jul, 159(1), 159 - 66
Construction in vitro of a cloned nar operon from Escherichia coli; Rondeau SS et al.; To clone the nar operon of Escherichia coli without an effective selection procedure for the nar+ phenotype, a strategy utilizing nar::Tn5 mutants was employed . Partial segments of the nar operon containing Tn5 insertions were cloned into plasmid pBR322 by using the transposon resistance character for selection . A hybrid plasmid was constructed in vitro from two of these plasmids and isolated by a procedure that involved screening a population of transformed nar(Ts) mutant TS9A for expression of thermal stable nitrate reductase activity . A detailed restriction site map of the resulting plasmid, pSR95, corresponded closely to the composite restriction endonuclease map deduced for the nar region from maps of the cloned nar::Tn5 fragments . When transformed with pSR95, wild-type strain PK27 overproduced the alpha, beta, and gamma subunits of nitrate reductase, although nitrate reductase activity was only slightly increased . The alpha and beta subunits were overproduced about 5- to 10-fold and accumulated mostly as an inactive aggregate in the cytoplasm; the gamma subunit overproduction was detected as a threefold increase in the specific content of cytochrome b555 in the membrane fraction . Functional nitrate reductase and the cytochrome spectrum associated with functional nitrate reductase were restored in the nar::Tn5 mutant EE1 after transformation with pSR95 . Although the specific activity of nitrate reductase in this case was less than that of the wild type, both the alpha and beta subunits appeared to be overproduced in an inactive form . In both strains PK27(pSR95) and EE1(pSR95), the formation of nitrate reductase activity and the accumulation of inactive subunits were repressed during aerobic growth . From these observations and the accumulation of inactive subunits were repressed during aerobic growth . From these observations and the demonstration that pSR95 contains a functional nor operon that encodes the alpha, beta, gamma subunits of nitrate reductase.

Biochem J, 1984 Jul 1, 221(1), 43 - 51
An additional acidic residue in the membrane portion of the b-subunit of the energy-transducing adenosine triphosphatase of Escherichia coli affects both assembly and function; Jans DA et al.; Glycine at position 9 is replaced by aspartic acid in the mutant b-subunit of Escherichia coli F1F0-ATPase coded for by the uncF476 allele . The mutant b-subunit is not assembled into the membrane in haploid strains carrying the uncF476 allele, but, if the mutant allele is incorporated into a multicopy plasmid, then some assembly of the mutant b-subunit occurs . Two revertant strains were characterized, one of which (AN2030) was a full revertant, the other (AN1953) a partial revertant . DNA sequencing indicated that in strain AN2030 the uncF476 mutation had reverted to give the sequence found in the normal uncF gene . The partial-revertant strain AN1953, however, retained the DNA sequence of the uncF476 allele, and complementation analysis indicated that the second mutation may be in the uncA gene . Membranes prepared from the partial-revertant strain carried out oxidative phosphorylation, although the membranes appeared to be impermeable to protons, and the ATPase activity was sensitive to the inhibitor dicyclohexylcarbodi-imide.

Mol Biol (Mosk), 1984 Jul-Aug, 18(4), 907 - 18
{Affinity modification of Escherichia coli ribosomes with photoactivated analogs of mRNA}; Gimautdinova OI et al.; Oligoribonucleotide derivatives containing the photoactivated arylazidogroup at 5'-end of the oligonucleotide fragment {2-(N-2,4-dinitro-5-azidophenyl) aminoethyl} phosphamides of the oligoribonucleotides, azido-NH (CH2)2NHpN (pN) n-1, were prepared . It was demonstrated that azido-NH(CH2)2NHpA(pA)4 and azido-NH (CH2)2NHpU (pU)3 stimulate the binding of the codonspecific aminoacyl-tRNA with ribosome . After irradiation of the ternary complex ribosome-azido-NH (CH2)2NHpU (pU) n-1 X tRNA with UV-light (lambda greater than 350 nm) covalent binding of the reagent to ribosome occurs . Up to 10% of the reagent, bound in the ternary complex with ribosome, is cross-linked with the ribosomal proteins of 30S and 50S subunits . The ribosomal RNA are not modified by azido-NH (CH2)2NHpU (pU) n-1 . The proteins of 30S and 50S subunits, modified with azido-NH (CH2)2NHpU (pU) n-1 with n = 4,7 and 8, were identified . It is shown that proteins of 30S subunits S3, S4, S9, S11, S12, S14, S17, S19, S20 undergo modification . The proteins of 50S subunits L2, L13, L16, L27, L32, L33 are modified . The set of the modified proteins essentially depends on the length of the oligonucleotide part of the reagent and on occupancy of ribosome A-site by a molecule of tRNA.

Ann Clin Biochem, 1984 Jul, 21 ( Pt 4), 310 - 7
Evaluation of beta-D-galactosidase from Escherichia coli and horseradish peroxidase as labels by sandwich enzyme immunoassay technique; Imagawa M et al.; beta-D-galactosidase from Escherichia coli and horseradish peroxidase were evaluated as labels of Fab' in dose-response curves for human alpha-fetoprotein and human chorionic gonadotropin by sandwich enzyme immunoassay technique using fluorogenic substrates for enzyme assay . The non-specific binding of Fab'-peroxidase conjugates to IgG-coated polystyrene balls was less than that of Fab'-beta-D-galactosidase conjugates, and the affinity-purified Fab'-peroxidase conjugates gave more sensitive dose-response curves for these antigens than the corresponding beta-D-galactosidase conjugates . However, a large quantity of Fab'-peroxidase conjugates was required and a longer incubation was necessary for the peroxidase assay, since the peroxidase assay was much less sensitive than the beta-D-galactosidase assay . Other advantages and disadvantages of the two enzymes are discussed.

Acta Paediatr Scand, 1984 Jul, 73(4), 426 - 32
In-vivo immune responses of breast- and bottle-fed infants to tetanus toxoid antigen and to normal gut flora; Stephens S et al.; The effects of breast- and bottle-feeding on serum immunoglobulin levels and specific antibody responses have been examined in 30 infants on five occasions from 6 days until 9 months of age . No significant differences were found on any sample occasion between the two feeding groups in total immunoglobulin levels of G, M and A classes or in class-specific antibody responses to tetanus toxoid vaccine . This suggests that the capacity of the two groups to make serum antibodies develops similarly . Concentrations of antibodies to commensal Escherichia coli 'O' lipopolysaccharide antigens, however, were significantly greater in the bottle-fed group, and it is suggested that this difference is due to an increase in the exposure of the systemic immune system to these gut antigens in the bottle-fed infants . There are several possible explanations for this increased exposure and the resulting effects on the infants' immune system . These experiments also illustrate a possible role of breast milk in stimulating the immune system.

Proc Natl Acad Sci U S A, 1984 Jul, 81(14), 4465 - 9
cis-acting mutations that affect rop protein control of plasmid copy number; Moser DR et al.; A number of pMB1 derivatives provide a trans-acting function that can suppress lethal runaway replication of a temperature-sensitive copy-number mutant of NTP1 . Deletion analysis indicates that the region of the pMB1 genome that contains the rop gene is required for this suppression . Mutant derivatives of the temperature-sensitive copy-number mutant plasmid whose conditional lethal phenotype is not suppressed in trans by the region encoding the rop gene have been isolated . These rop-insensitive derivatives contain single nucleotide changes within the RNA I coding region.

Mol Immunol, 1984 Jul, 21(7), 673 - 7
Some monoclonal antibodies raised with a native protein bind preferentially to the denatured antigen; Friguet B et al.; Recent studies with monoclonal antibodies directed against different epitopes of the beta 2-subunit of Escherichia coli tryptophan synthase have shown that some of these antibodies bind rapidly in solution to the native protein; others bind very slowly to native beta 2 in solution while they recognize quite rapidly this antigen adsorbed on a microtitration plate . In the present work, an enzyme-linked immunosorbent assay competition test with either the native or a denatured form of the antigen has been developed . It allowed us to show that the rapidly binding antibodies recognize epitopes present on the native protein while those which react very slowly in solution bind preferentially to the denatured form of the protein . These results prompted us to emphasize how important it is, in the characterization of antibodies, to ascertain that the antigen they recognize remains native in the specificity test.

Proc Natl Acad Sci U S A, 1984 Jul, 81(13), 3959 - 63
Tandem promoters preceding the gene for the M1 RNA component of Escherichia coli ribonuclease P; Motamedi H et al.; The nucleotide sequence of a cloned gene for the RNA component of Escherichia coli ribonuclease P, M1 RNA, is presented . The sequence determined extends 320 nucleotides upstream of the 377-base-pair (bp) structural gene and includes three sequences homologous to the consensus E . coli promoter sequence . Two nucleotides found in the M1 RNA structural gene sequence were not found in a previously determined gene sequence of another M1 RNA clone {Reed, R . E., Baer, M . F., Guerrier-Takeda, C., Donis-Keller, H . & Altman, S . (1982) Cell 30, 627-636} . In vitro transcription of supercoiled plasmid DNA containing the M1 RNA gene resulted in a major transcript arising from the strong promoter nearest to the mature M1 RNA . RNAs encoded by the M1 RNA clone in vivo were examined by S1 nuclease mapping . The results indicated that in vivo transcripts originate from all three promoters preceding the M1 RNA gene . These transcripts are apparently processed in a multistep pathway to generate the 5' end of mature M1 RNA.

J Cell Physiol, 1984 Jul, 120(1), 75 - 82
Induction of ornithine decarboxylase, RNA, and protein synthesis in macrophage cell lines stimulated by immunoadjuvants; Prosser FH et al.; Early biochemical changes associated with adjuvant stimulation of macrophage protein synthesis were studied using two murine macrophage cell lines, PU5-1.8 and J774.1 . An induction of ornithine decarboxylase (ODC) was detected 2 hours after exposure of PU5-1.8 and J774.1 cells to two crude immunoadjuvants, BCG cell walls (BCGcw) and lipopolysaccharides from Escherichia coli (LPS) . The chemically defined immunoadjuvant glycopeptide, N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDPL) also promoted an increase in ODC activity at 2 hours that was maximal after 4 hours, while little or no effect was observed with the D-alanyl analog (MDPD) that is devoid of adjuvant activity . The increase in ODC activity promoted by BCGcw in PU5-1.8 and J774.1 cells returned toward control levels by 6 to 8 hours . BCGcw also stimulated RNA and protein synthesis which remained elevated for at least 24 hours and was associated with a decrease in DNA synthesis and cell proliferation . ODC induction by BCGcw and MDPL was enhanced by the addition of PGE2 in both cell lines . Indomethacin slightly depressed the magnitude of ODC stimulation by BCGcw in J774.1 cells but failed to alter the response of PU5-1.8 cells . Additional observations indicated that the induction of ODC by BCGcw in both cell lines was preceded by an activation of cyclic AMP-dependent protein kinase . These observations suggest that a cyclic AMP-mediated induction of ODC may be an early biochemical marker of adjuvant stimulation in macrophages.

J Bacteriol, 1984 Jul, 159(1), 63 - 70
Energy-requiring translocation of the OmpA protein and alkaline phosphatase of Escherichia coli into inner membrane vesicles; Rhoads DB et al.; In developing a reliable in vitro system for translocating bacterial proteins, we found that the least dense subfraction of the membrane of Escherichia coli was superior to the total inner membrane, both for a secreted protein (alkaline phosphatase) and for an outer membrane protein (OmpA) . Compounds that eliminated the proton motive force inhibited translocation, as already observed in cells; since protein synthesis continued, the energy for translocation appears to be derived from the energized membrane and not simply from ATP . Treatment of the vesicles with protease, under conditions that did not interfere with subsequent protein synthesis, also inactivated them for subsequent translocation . We conclude that export of some proteins requires protein-containing machinery in the cytoplasmic membrane that derives energy from the proton motive force.

Infect Immun, 1984 Jul, 45(1), 203 - 9
Escherichia coli O18ac antigen: structure of the O-specific polysaccharide moiety; Gupta DS et al.; The O-specific polysaccharide moiety (O18ac polysaccharide) of the O18ac antigen (lipopolysaccharide) from Escherichia coli 2980 (O18ac:K5:Fim+:H-) was isolated in pure form by degradation of the lipopolysaccharide and chromatography on Sephadex G-50 . The primary structure of the O18ac polysaccharide was elucidated by composition, fragmentation procedures, methylation analysis, and nuclear magnetic resonance spectroscopy . The polysaccharide consists of repeating units of the pentasaccharide: (formula; see text) which are joined in the polymer by alpha-1,2 linkages.

J Pharm Pharmacol, 1984 Jul, 36(7), 467 - 70
Rifampicin curing of plasmids in Escherichia coli K12-rifampicin resistant host; Obaseiki-Ebor EE; The in-vitro rifampicin curing of R-plasmids JR225, BN102, BN106 and F'-plasmid F'lac+/R386 in Escherichia coli K12 rifampicin-resistant host J62-2 is described . The minimum rifampicin curing concentration and the frequency of curing of plasmids from the rifampicin-resistant host cell J62-2 and the isogenic rifampicin-sensitive J62-1 host cell were similar . However, rifampicin did not cure some test R-plasmids such as R222 and RP4 from rifampicin-resistant and rifampicin-sensitive host cells, indicating that it is the R-plasmid itself and not the host strain that determines rifampicin curing.

Infect Immun, 1984 Jul, 45(1), 299 - 301
Pilus-mediated adherence of Escherichia coli K1 to human oral epithelial cells; Clegg H et al.; We examined the effect of host age and health status on the adherence of mannose-sensitive piliated Escherichia coli K1 to human oral epithelial cells . Mannose-sensitive piliated bacteria adhered in comparable numbers to newborn, older infant, and adult cells (125 +/- 61, 198 +/- 54, and 139 +/- 69 bacteria per cell, respectively) . Prematurity and serious illness did not alter adherence in newborns . The increased susceptibility of premature newborns to E . coli K1 cannot be explained by enhanced epithelial cell adherence.

Mol Cell Biol, 1984 Jul, 4(7), 1411 - 5
Detection of deletion mutations in pSV2gpt-transformed cells; Tindall KR et al.; We have developed a system to study mutations that affect xanthine-guanine phosphoribosyltransferase gene (gpt) expression in hypoxanthine-guanine phosphoribosyltransferase-deficient CHO cells that have been transformed by the plasmid vector pSV2gpt . One isolated transformant, designated AS52, carries a single copy of the Escherichia coli gpt gene stably integrated into the high-molecular-weight DNA and expresses the bacterial gene for the enzyme xanthine-guanine phosphoribosyltransferase . Mutants deficient in this enzyme can be induced in the AS52 cell line by a variety of mutagens, and spontaneous or induced mutants can be selected for resistance to 6-thioguanine (Tgr) . Two Tgr clones derived from the AS52 line were analyzed by Southern blot hybridization and were found to contain deletions involving at least a portion of the gpt gene . Because of the small size and stability of the integrated pSV2gpt plasmid, and the well-defined selection protocol for mutant isolation, the AS52 line offers promise as a system suitable for the study of mutation at the molecular level in CHO cells.

Mol Biol (Mosk), 1984 Jul-Aug, 18(4), 1081 - 9
{Directed modification of the Tcr gene region of the plasmid pBR322 using complementary single-stranded DNA fragments carrying alkylating groups}; Mazin AV et al.; A method is suggested for chemical modification of preselected regions of plasmid DNA by complementary single-stranded restriction fragments of DNA (srf DNA), carrying alkylating reagents . The gene coding for tetracycline resistance of plasmid pBR322 was used as a target . Srf DNA was prepared by a partial digestion of a double-stranded EcoRI-BamHI restriction fragment (377 base pairs) from Tcr by E . coli exonuclease III . The residues of an alkylating reagent N,N,N'-tri(beta-chlorethyl)-N'-(p-formylphenyl) propylenediamine 1,3 (TFP) were attached covalently to 4-5% of sfr DNA bases . The alkylating derivative of the sfr DNA was hybridized with supercoiled pBR322 plasmid DNA . The hybridization conditions (37 degrees C, 40% formamide, 0,2 M NaCl, 0,1 M Tris-HCl pH 7,5, 0,001 M EDTA) under which the bases carrying TFP residues are not eliminated from the sfr DNA, and transforming activity of pBR322 DNA does not decrease were established . It was shown that about 20% of plasmid pBR322 molecules form D-loops with alkylating sfr DNA under these conditions . It was shown that sfr DNA, carrying TFP can alkylate the complementary region of plasmid DNA, forming cross-linked D-loops . A method for the site-directed mutagenesis of switching off the preselected genes or non-transcribed DNA functional regions (promotors, introns etc) integrated into plasmids of other vectors is suggested.

Gene, 1984 Jul-Aug, 29(1-2), 41 - 9
Expression of cloned calf prochymosin cDNA under control of the tryptophan promoter; Nishimori K et al.; To increase yields of calf prochymosin (PC) produced in Escherichia coli, PC cDNA was cloned in a plasmid vector under control of the trp promoter . The hybrid plasmid pCR501 constructed for this purpose contains cDNA coding for PC (from the 5th Arg to the C-terminal Ile) fused to the N-terminal fragment of the trpE gene preceded by the trp promoter and attenuator region . E . coli C600 harboring this plasmid produces approx . 300 000 molecules of PC per cell . This is about a tenfold increase above the amount obtained using lacUV5 promoter {Nishimori et al., Gene 19 (1982) 337-344} . A similar plasmid, pCR601, which contains the same coding sequence fused to the trp promoter and N-terminal fragment of the trpL gene, directs the production of PC at the same rate as pCR501 . In pCR601 the trp attenuator is deleted . Another plasmid, pCR701, in which construction of a sequence coding for fMet-PC cDNA that was aided by chemical synthesis, was placed under direct control of the trp promoter, produced PC at a much lower rate . Extracts prepared from all these bacterial transformants in the presence of urea showed distinct milk-clotting activity after renaturation and processing.

Gene, 1984 Jul-Aug, 29(1-2), 251 - 4
Synthesis of human insulin gene . VIII . Construction of expression vectors for fused proinsulin production in Escherichia coli; Guo LH et al.; We have constructed two families of plasmids suitable for the cloning of genes and for directing the synthesis of large amounts of fused proteins in Escherichia coli . The plasmids include the E . coli lac promoter and a portion of the coding sequence for beta-galactosidase, which can code for approx . 590 or 450 amino acids . The truncated beta-galactosidase gene ends with a poly-linker region at the 3' end, which can be cleaved by any one of the eight common restriction enzymes and joined to the gene coding for any desired protein . Each family includes three plasmids that enable fusion to be made in all three of the translational reading frames . We have cloned a synthetic human proinsulin gene into these plasmids, and 30% of the total E . coli protein was represented by the 590 amino acid-long truncated beta-galactosidase fused to proinsulin . The yield of proinsulin in this system is more than twice the amount produced by using a 1007 amino acid-long beta-galactosidase gene for fusion.

Gene, 1984 Jul-Aug, 29(1-2), 243 - 6
Molecular cloning of the hamster papovavirus genome in Escherichia coli plasmid vector pBR322; Zimmermann W et al.; The complete genome of the hamster papovavirus (HaPV) which was isolated from virions found in multiple skin tumors of Syrian hamsters was cloned in Escherichia coli using the plasmid vector pBR322 . The cloned viral DNAs were identified by digestion of the recombinant DNAs with various restriction enzymes followed by comparison of their electrophoretic mobilities in agarose gels with that of similarly digested uncloned DNAs . The cloned HaPV DNAs showed the same migration pattern as the corresponding fragments from the restricted uncloned DNAs, indicating that no major insertions or deletions occurred during cloning and plasmid propagation . The electrophoretic data were confirmed by Southern blot hybridization.

Gene, 1984 Jul-Aug, 29(1-2), 231 - 41
A technique for integrating any DNA fragment into the chromosome of Escherichia coli; Raibaud O et al.; We describe a technique that allows the insertion of any DNA fragment into the EcoRI-site-containing malPpa, the promoter of malPQ, one of the three maltose operons of Escherichia coli . DNA fragments were cloned into the unique EcoRI site of the pBR322-derived plasmid pOM40, which carries malPpa . In the next step these fragments were transposed into the chromosome by homologous recombination events occurring on both sides of malPp . Cells in which such insertion of the entire recombinant plasmid have occurred can be conveniently selected . Excision and curing of the vector plasmid could then occur spontaneously at a high frequency, leaving behind the inserted fragment that can be manipulated as any chromosomal marker . When the inserted fragment contains a properly positioned promoter, its promoting activity can be estimated by assaying amylomaltase, the product of malQ . When required, the inserted fragment can be easily transferred back onto pOM40 . As examples of application we have transferred two different fragments into the chromosome of E . coli: one contained the ceaC-ceiC operon, which encodes colicin E3 and its immunity protein, and the other contained the lac promoter of E . coli.

Gene, 1984 Jul-Aug, 29(1-2), 211 - 9
The replication origin of pSC101: the nucleotide sequence and replication functions of the ori region; Yamaguchi K et al.; The nucleotide sequence of a 770-bp ori region of plasmid pSC101 is presented . The sequence shows homologies to some parts of Escherichia coli oriC and phage G4 ori . Several other features are an 80-bp A + T-rich region overlapping a part of the region homologous to oriC, three direct repeats of an 18-bp sequence adjacent to the A + T-rich region, a typical promoter sequence just upstream of the longest open reading frame (ORF) and a long inverted repeat sequence overlapping the putative promoter region . Analysis of successive deletions by BAL31 exonuclease demonstrated that one of the regions homologous to oriC along with the A + T-rich region are essential for autonomous replication of the plasmid . The three 18-bp repeats are responsible for incompatibility phenotype . The region containing the promoter-like sequence is required for expression of a trans-acting function.

Gene, 1984 Jul-Aug, 29(1-2), 175 - 84
Characterization and nucleotide sequence of a colicin-release gene in the hic region of plasmid ColE3-CA38; Watson RJ et al.; Downstream from its colicin and immunity genes (col . imm), Escherichia coli plasmid ColE3-CA38 contains a 0.81-kb DNA segment, the hic region, which is required for high colicin production . Characterization of derived plasmids, carrying the col-imm operon but varying in the hic region, showed that the latter functions in lacuna production, colicin release, cell death, and lysis . The hic gene expression after induction was shown to be dependent on the col gene promoter . The nucleotide sequence of the 0.81-kb region was determined and the hic gene localized to its imm-distal portion following an open reading frame (ORF) with no known function . There are two overlapping ORFs in that portion of the sequence, one of which was identified as the hic gene by its partial homology to lysis gene H of CloDF13 . The 3' half of the hic gene is non-essential and contains a terminator-like DNA sequence . Preceding the gene, there are also inverted repeats which may attenuate its transcription.

Gene, 1984 Jul-Aug, 29(1-2), 145 - 55
Characterisation of the ColE8 plasmid, a new member of the group E colicin plasmids; Mark G et al.; We have determined the restriction map of the ColE8-J plasmid after cloning it into the pBR322 vector . By subcloning and transposon mutagenesis we have localized the colicin immunity gene, the colicin structural gene, and lys, the region that determines MC sensitivity . In contrast to the ColE3-CA38 plasmid, the genes coding for colicin E8 production and immunity cannot be cloned on a single EcoRI fragment . Insertion of Tn5 transposons into the colicin structural gene region of the recombinant plasmid inactivated colicin production and MC sensitivity . Insertion of transposons into the lys region reduced colicin E8 production and MC induced lysis, the extent of which was dependent upon the precise site of insertion . We propose that the colicin E8 structural gene and lys must be transcribed from a common promoter situated proximal to the structural gene, whilst the colicin E8 immunity gene is transcribed from a second promoter . The lys region is responsible both for cell lysis after MC induction and positive regulation of colicin E8 synthesis.

Gene, 1984 Jul-Aug, 29(1-2), 113 - 24
Structure and function of the yeast URA3 gene: expression in Escherichia coli; Rose M et al.; The expression of the cloned Saccharomyces cerevisiae URA3 gene in Escherichia coli on both plasmid and phage vectors was studied . Isolates of the gene from two different laboratory strains of yeast differ in their ability to be expressed in E . coli in the absence of external adjacent promoters of transcription . The DNA sequence of the two genes was determined and revealed several differences in the DNA flanking the structural gene . One base change alters the "Pribnow-box" of an E . coli promoter present in the yeast sequences . Three amber alleles of the yeast gene were also cloned from yeast . Two of the alleles could be suppressed in E . coli by a tRNA suppressor mutation . One of the amber alleles was determined to be a mutation in the seventh codon of the structural gene, thereby establishing the reading frame and extent of the coding sequence . The initiator codon of the reading frame encoding the URA3 structural gene is preceded by two other ATG codons in a different reading frame 61 and 79 bp away . The nearer ATG begins an open reading frame that overlaps the structural gene sequences by 17 bp . With the DNA sequence of the URA3 gene many of the common yeast vector plasmids are now completely known at the level of DNA sequence.

J Gen Microbiol, 1984 Jul, 130 ( Pt 7), 1597 - 601
The genomes of Desulfovibrio gigas and D . vulgaris; Postgate JR et al.; Two-dimensional electrophoresis of sequential double-restriction digests showed that the genome of Desulfovibrio gigas compromised 1.63 x 10(6) bp (1.09 x 10(9) Dal) of DNA; an ammonia-limited chemostat population possessed an average of nine genomes per cell and a multiplying batch culture possessed approximately 17 genomes per cell . The genome size of D . vulgaris (Hildenborough) was 1.72 x 10(6) bp (1.14 x 10(9) Dal); a population from an ammonia-limited batch culture contained four genomes per cell . Control digestions and analyses with Escherichia coli GM4 agreed reasonably with published values: a genome size of 3.95 x 10(6) bp and approximately two genomes per cell from a stationary batch culture in glucose minimal medium . Desulfovibrio gigas carried two plasmids of approximately 70 MDal (1.05 x 10(5) bp) and approximately 40 MDal (6 x 10(4) bp); D . vulgaris (Hildenborough) contained one of approximately 130 MDal (1.95 x 10(5) bp) . Single plasmids were also detected in a second strain of D . vulgaris and in strain Berre sol of D . desulfuricans but not in 10 other desulfovibrios including representatives of D . desulfuricans, D . vulgaris, D . salexigens and D . africanus.

Proc Natl Acad Sci U S A, 1984 Jul, 81(14), 4490 - 4
Cell-division control in Escherichia coli: specific induction of the SOS function SfiA protein is sufficient to block septation; Huisman O et al.; Blocks in DNA replication cause a rapid arrest of cell division in Escherichia coli . We have previously established that the function SfiA (SulA), induced under these conditions as part of the SOS response, is involved in this inhibition of division . To separate the effects of SfiA from those of other SOS functions, we have constructed a plac-sfiA operon fusion, permitting specific induction of SfiA protein by addition of the lac operon inducer isopropyl beta-D-thiogalactopyranoside (IPTG) . In lon mutants, in which the unstable SfiA protein has a longer half-life, IPTG caused a rapid arrest of cell division . Under these conditions, there is no concomitant induction of the SOS response . IPTG also caused a rapid arrest of cell division in lon+ strains . These results demonstrate that induction of the SfiA protein is sufficient to cause inhibition of division . Mutations in the sfiB gene can suppress IPTG-induced SfiA-mediated inhibition of division . At higher SfiA concentrations, however, even sfiB mutants cease division; an additional mutation genetically inseparable from sfiB restores normal division . These observations reinforce the hypothesis that the SfiB protein, probably required for cell septation, is the target of action of the SfiA division inhibitor.

Proc Natl Acad Sci U S A, 1984 Jul, 81(14), 4294 - 7
Primary structure of the Escherichia coli ribonucleoside diphosphate reductase operon; Carlson J et al.; The nucleotide sequence of the Escherichia coli K-12 DNA comprising the operon for the structural genes of the subunits of ribonucleotide diphosphate reductase has been determined . The DNA sequenced maps at 48.5 minutes on the E . coli chromosome and includes a total length of 8557 nucleotides . An open reading frame between nucleotides 3506 and 5834, encoding a 776-amino acid polypeptide chain with a molecular weight of 87,532, has been identified as the nrdA gene . An open reading frame between nucleotides 6012 and 7139, encoding a 375-amino acid polypeptide with a molecular weight of 43,466, has been identified as the nrdB gene . The sequences reveal not only the primary structures for both subunits, but also some interesting aspects of potential regulatory sites.

Science, 1984 Jun 29, 224(4656), 1431 - 3
Site-specific mutagenesis of the human interleukin-2 gene: structure-function analysis of the cysteine residues; Wang A et al.; The gene encoding human interleukin-2 (IL-2) has been cloned from human spleen cells, peripheral blood lymphocytes, and the Jurkat cell line . Nucleotide sequence analysis of the gene revealed that the encoded IL-2 protein has three cysteines located at amino acid residues 58, 105, and 125 of the mature protein . Site-specific mutagenesis procedures were used to modify the IL-2 gene by changing each of the cysteine codons individually to serine codons . Substitution of serine for cysteine residues at either position 58 or 105 of the IL-2 protein substantially reduced biological activity, indicating that the cysteines at these positions are necessary for maintenance of the biologically active conformation and may therefore be linked by a disulfide bridge . The modified IL-2 protein containing a substitution at position 125 retained full biological activity, suggesting that the cysteine at this position is not involved in a disulfide bond and that a free sulfhydryl group at that position is not necessary for receptor binding.

Biochim Biophys Acta, 1984 Jun 29, 799(3), 282 - 90
Increased activity of rat liver N2-guanine tRNA methyltransferase II in response to liver damage; Wainfan E et al.; Alterations in rat liver transfer RNA (tRNA) methyltransferase activities have been observed after liver damage by various chemicals or by partial hepatectomy . The qualitative and quantitative nature of these activity changes and the time course for their induction have been studied . Since homologous tRNAs are essentially fully modified in vivo, E . coli tRNAs were used as in vitro substrates for the rat liver enzymes in these studies . Each of the liver-damaging agents tested rapidly caused increases in activities of the enzyme(s) catalyzing methyl group transfer to tRNAs that have an unmodified guanine at position 26 from the 5' end of the molecule . This group of tRNAs includes E . coli tRNANfmet, tRNAAla1, tRNALeu1, or Leu2, and tRNASer3 (Group 1) . In each case N2-methylguanine and N2,N2-dimethylguanine represented 90% or more of the products of these in vitro methylations . The product and substrate specificity observed are characteristic of N2-guanine methyltransferase II (S-adenosyl-L-methionine : tRNA (guanine-2)-methyltransferase, EC 2.1.1.32) . In crude and partially purified preparations derived from livers of both control and treated animals this enzyme activity was not diminished significantly by exposure to 50 degrees C for min . The same liver-damaging agents induced little or no change in the activities of enzymes that catalyze methyl group transfer to various other E . coli tRNAs that do not have guanine at position 26 (Group 2) . The results of mixing experiments appear to rule out the likelihood that the observed enzyme activity changes are due to stimulatory or inhibitory materials present in the enzyme preparations from control or treated animals . Thus, our experiments indicate that liver damage by each of several different methods, including surgery or administration of chemicals that are strong carcinogens, hepatotoxins, or cancer-promoting substances, all produce changes in liver tRNA methyltransferase activity that represent a selective increase in activity of N2-guanine tRNA methyltransferase II . It is proposed that the specificity of this change is not fortuitous, but is the manifestation of an as yet unidentified regulatory process.

J Biol Chem, 1984 Jun 25, 259(12), 7495 - 503
Separation of the presynaptic and synaptic phases of homologous pairing promoted by recA protein; Kahn R et al.; Homologous pairing of single strands with duplex DNA promoted by recA protein occurred without a lag only when the protein was preincubated with ATP and single-stranded DNA . The rate-limiting presynaptic interaction of recA protein and single strands showed a high temperature coefficient: it proceeded 30 times more slowly at 30 degrees C than at 37 degrees C, whereas synapsis showed a normal temperature coefficient . Thus, the presynaptic phase could be separated experimentally from the rest of the reaction by preincubation of single strands with recA protein and ATP at 37 degrees C, followed by a shift to 30 degrees C before double-stranded DNA was added . The presynaptic phase was an order of magnitude more sensitive to inhibition by ADP than was subsequent strand exchange . Presynaptic complexes that were formed at 37 degrees C decayed only slowly at 30 degrees C, but Escherichia coli single strand binding protein caused complexes to form rapidly at 30 degrees C which indicates that single strand binding protein accelerated the rate of formation of complexes . Preincubation synchronized the initial pairing reaction, and further revealed the rapid formation of nascent heteroduplex DNA 250-300 base pairs in length.

Nucleic Acids Res, 1984 Jun 25, 12(12), 5109 - 22
Heterogeneity of mammalian DNA ligase detected on activity and DNA sequencing gels; Mezzina M et al.; A new method to detect DNA ligase activity in situ after NaDodSO4 polyacrylamide gel electrophoresis has been developed . After renaturation of active polypeptides the ligase reaction occurs in situ by incubating the intact gel in the presence of Mg++ and ATP . Further treatment with alkaline phosphatase removes the unligated 5'-32P-end of oligo (dT) used as a substrate and active polypeptides having ligase activity are identified by autoradiography . Analysis on DNA sequencing gels of the oligo (dT) reaction products present in the activity bands ensures that the radioactive material detected in activity gels or in standard in vitro ligase assays corresponds unambiguously to a ligase activity . Using these methods, we have analysed the purified phage T4 DNA ligase, and the activities present in crude extracts and in purified fractions from monkey kidney (CV1-P) cells . The purified T4 enzyme yields one or two active peptides with Mr values of 60,000 and 70,000 . Crude extracts from CV1-P cells contain several polypeptides having DNA ligase activity . Partial purification of these extracts shows that DNA ligase I isolated from hydroxylapatite column is enriched in polypeptides with Mr 200,000, 150,000 and 120,000, while DNA ligase II is enriched in those with Mr 60,000 and 70,000.

Nucleic Acids Res, 1984 Jun 25, 12(12), 4997 - 5003
Structure and function of tryptophan tRNA from wheat germ; Ghosh K et al.; The coding properties of tRNATrp from yeast and wheat germ were studied . Unlike E . coli tRNATrp or mitochondrial tRNATrp, eukaryotic tRNATrp did not recognize the UGA codon in vitro . The sequence of wheat germ tRNATrp as determined by {32P} post-labelling techniques is: {sequence in text} The interesting features are: (i) Presence of a C11:G24 base pair in contrast to the U11:G24 in E . coli Su- tRNATrp . (ii) The anticodon sequence is -CmCA- compared to -CCA- in E . coli tRNATrp . (iii) Lack of a hypermodified base i6A adjacent to the 3'-end of the anticodon . (iv) Presence of -T psi CG- sequence instead of -psi psi CG- sequence present in mammalian tRNATrp.

J Biol Chem, 1984 Jun 25, 259(12), 7998 - 8003
Characterization of the cytochrome d terminal oxidase complex of Escherichia coli using polyclonal and monoclonal antibodies; Kranz RG et al.; The cytochrome d terminal oxidase complex is one of two terminal oxidases in the aerobic respiratory chain of Escherichia coli . Previous work has shown by dodecyl sulfate-polyacrylamide gel electrophoresis that this enzyme contains two subunits (I and II) and three cytochrome components, b558 , a1, and d . Reconstitution studies have demonstrated that the enzyme functions as a ubiquinol-8 oxidase and catalyzes an electrogenic reaction, i.e . turnover is accompanied by a charge separation across the membrane bilayer . In this paper, monoclonal and polyclonal antibodies were used to obtain structural information about the cytochrome d complex . It is shown that antibodies directed against subunit I effectively inhibit ubiquinol-1 oxidation by the purified enzyme in detergent, whereas antibodies which bind to subunit II have no effect on quinol oxidation . The oxidation rate of N,N,N',N'-tetramethyl-p-phenylenediamine, in contrast, is unaffected by antisubunit I antibodies, but is inhibited by antibodies against subunit II . It is concluded that the quinol oxidation site is on subunit I, previously shown to be the cytochrome b558 component of the complex, and that N,N,N',N'-tetramethyl-p-phenylenediamine oxidation occurs at a secondary site on subunit II . The antibodies were also used to analyze the results of a protein cross-linking experiment . Dimethyl suberimidate was used to cross-link the subunits of purified, solubilized oxidase . Immunoblot analysis of the products of this cross-linking clearly indicate that subunit II probably exists as a dimer within the complex . Finally, it is shown that the purified enzyme contains tightly bound lipopolysaccharide . This was revealed after discovering that one of the monoclonal antibodies raised against the purified complex is actually directed against lipopolysaccharide . The significance of this finding is not known.

J Biol Chem, 1984 Jun 25, 259(12), 7994 - 7
Identification of subunit I as the cytochrome b558 component of the cytochrome d terminal oxidase complex of Escherichia coli; Green GN et al.; The cytochrome d terminal oxidase complex was recently purified from Escherichia coli membranes (Miller, M . J., and Gennis , R . B . (1983) J . Biol . Chem . 258, 9159-1965) . The complex contains two polypeptides, subunits I and II, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and three spectroscopically defined cytochromes, b558 , a1, and d . A mutant that failed to oxidize N,N,N',N'-tetramethyl-p-phenylenediamine was obtained which was lacking this terminal oxidase complex and was shown to map at a locus called cyd on the E . coli genome . In this paper, localized mutagenesis was used to generate a series of mutants in the cytochrome d terminal oxidase . These mutants were isolated by a newly developed selection procedure based on their sensitivity to azide . Two classes of mutants which map to the cyd locus were obtained, cydA and cydB . The cydA phenotype included the lack of all three spectroscopically detectable cytochromes as well as the absence of both polypeptides, determined by immunological criteria . Strains manifesting the cydB phenotype lacked cytochromes a1 and d, but had a normal amount of cytochrome b558 . Immunological analysis showed that subunit I (57,000 daltons) was present in the membranes, but that subunit II (43,000 daltons) was missing . These data justify the conclusion that subunit I of this two-subunit complex can be identified as the cytochrome b558 component of the cytochrome d terminal oxidase complex.

J Biol Chem, 1984 Jun 25, 259(12), 7990 - 3
Nucleoside triphosphate binding to DNA polymerase III holoenzyme of Escherichia coli . A direct photoaffinity labeling study; Biswas SB et al.; The physical basis of ATP binding and activation of DNA polymerase III holoenzyme was studied by an ultraviolet irradiation cross-linking technique . ATP and dATP were photocrosslinked to the alpha, tau, gamma, and delta subunits of holoenzyme; photocrosslinking of dATP was competitively inhibited by ATP . No photocrosslinking was observed with GTP or CTP, nor did GTP, CTP, or UTP inhibit cross-linking of ATP . ADP and adenosine 5'-O-(3-thio)-triphosphate, both potent inhibitors of ATP activation of holoenzyme, inhibited cross-linking of ATP to tau, gamma, and delta subunits, but not to the alpha subunit, suggesting that one or more of these subunits are ATP (or dATP)-binding sites . Photocrosslinking of dTTP to the ATP-activated holoenzyme was exclusively to the epsilon subunit, the dnaQ ( mutD ) gene product; dCTP and dGTP were not photocrosslinked to any subunit . Binding of dTTP was enhanced by ATP, but by no other nucleotide (or deoxynucleotide) . This binding of dTTP to epsilon, a subunit likely responsible for regulation of proofreading by the holoenzyme, may function in the control of the fidelity of replication.

J Biol Chem, 1984 Jun 25, 259(12), 7802 - 6
Mechanism of proline transport in Escherichia coli K12 . III . Inhibition of membrane potential-driven proline transport by syn-coupled ions and evidence for symmetrical transition states of the 2H+/proline symport carrier; Mogi T et al.; Specific inhibition of 2H+/proline symport by syn-coupled ions (Na+, Li+, and H+) was investigated using cytoplasmic membrane vesicles prepared from the proline carrier-overproducing strain MinS/ pLC4 -45 of Escherichia coli K12 . The 2H+/proline symport driven by the membrane potential generated via respiration with 20 mM ascorbate/Tris, 0.1 mM phenazine methosulfate was specifically inhibited by Na+ . The inhibition by Na+ was described by a fully noncompetitive mechanism, and the apparent Ki for Na+ was 15 mM . A linear correlation between the apparent Vmax and the apparent Kd was observed . Li+ stimulated the transport activity 2-fold at 10 mM and inhibited it at concentrations above 50 mM . H+ caused fully noncompetitive inhibition of 2H+/proline symport, and its apparent Ki was 0.6 microM . These results indicate that the concentrations of Na+ and H+ strictly and independently regulate the amount of the active C state carrier responsible for 2H+/proline symport driven by the membrane potential by inhibiting the transition from the C* state carrier which exhibits Na+- and H+-dependent binding of proline and is predominant in nonenergized conditions.

J Biol Chem, 1984 Jun 25, 259(12), 7797 - 801
Mechanism of proline transport in Escherichia coli K12 . II . Effect of alkaline cations on binding of proline to a H+/proline symport carrier in cytoplasmic membrane vesicles; Mogi T et al.; The substrate binding reaction of the proline carrier was investigated in nonenergized conditions using cytoplasmic membrane vesicles prepared from the proline carrier-overproducing strain MinS/ pLC4 -45 of Escherichia coli K12 . The binding activity specifically required both alkaline cations (X+), Na+ and Li+, and protons . The Na+-dependent binding activity was dependent on the proline carrier, which is the product of the putP gene, and was not affected by ionophores and energy transduction inhibitors . The parameters of proline binding were determined by double reciprocal plots in reaction media with various combinations of Na+ and H+ concentrations . The apparent dissociation constant was greatly affected by the Na+ and H+ concentrations of the medium and could be expressed as a combination of the reciprocals of the Na+ and H+ concentrations, while the maximum number of binding sites remained constant . The characteristics of proline binding to the carrier can be explained by a mechanism in which the unloaded carrier forms a carrier/H+/X+ (CH+X+) complex by a random equilibrium and only the CH+X+ complex binds substrate in nonenergized conditions, as proposed for the Na+/H+/glutamate symport carrier of E . coli B ( Fujimura , T., Yamato , I., and Anraku , Y . (1983) Biochemistry 22, 1954-1959).

J Biol Chem, 1984 Jun 25, 259(12), 7791 - 6
Mechanism of proline transport in Escherichia coli K12 . I . Effect of a membrane potential on the kinetics of 2H+/proline symport in cytoplasmic membrane vesicles; Mogi T et al.; The stoichiometric coupling mechanism of the membrane potential (delta psi) in the reaction of H+/proline symport was investigated kinetically, using cytoplasmic membrane vesicles of the proline carrier-overproducing strain of Escherichia coli MinS/ pLC4 -45 . When a delta psi was imposed across the cytoplasmic membrane by respiration, the Michaelis constant of transport (Kt) was lowered to about 1 microM, which was 2 orders of magnitude smaller than that of passive influx and efflux, and the maximum velocity (Vmax) was concomitantly enhanced as an exponential function of delta psi . Thermodynamically, the carrier translocated proline with a stoichiometry of 2 mol of protons versus 1 mol of substrate when driven by a delta psi at pH 8.0 . Data on the delta psi dependence of Vmax of proline transport could be explained quantitatively by the Geck-Heinz hypothesis (Geck, P., and Heinz, E . (1976) Biochim, Biophys . Acta 443, 49-63) . A symmetrical model of the 2H+/proline symport via formation of a carrier/H+/substrate (CH+H+S) intermediate is proposed . In this model, the effect of delta psi on the Kt was resolved as stimulation of formation of a transport intermediate, whereas the effect of delta psi on the Vmax was explained by enhancement of translocation of loaded carriers between the two sides of the membrane.

J Biol Chem, 1984 Jun 25, 259(12), 7532 - 9
DNA polymerase I and DNA primase complex in yeast; Plevani P et al.; Chromatographic analysis of poly(dT) replication activity in fresh yeast extracts showed that the activities required co-fractionate with the yeast DNA polymerase I . Since poly(dT) replication requires both a primase and a DNA polymerase, the results of the fractionation studies suggest that these two enzymes might exist as a complex in the yeast extract . Sucrose gradient analysis of concentrated purified yeast DNA polymerase I preparations demonstrates that the yeast DNA polymerase I does sediment as a complex with DNA primase activity . Two DNA polymerase I peptides estimated at 78,000 and 140,000 Da were found in the complex that were absent from the primase-free DNA polymerase fraction . Rabbit anti-yeast DNA polymerase I antibody inhibits DNA polymerase I but not DNA primase although rabbit antibodies are shown to remove DNA primase activity from solution by binding to the complex . Mouse monoclonal antibody to yeast DNA polymerase I binds to free yeast DNA polymerase I as well as the complex, but not to the free DNA primase activity . These results suggest that these two activities exist as a complex and reside on the different polypeptides . Replication of poly(dT) and single-stranded circular phage DNA by yeast DNA polymerase I and primase requires ATP and dNTPs . The size of the primer produced is 8 to 9 nucleotides in the presence of dNTPs and somewhat larger in the absence of dNTPs . Aphidicolin, an inhibitor of yeast DNA polymerase I, is not inhibitory to the yeast DNA primase activity . The primase activity is inhibited by adenosine 5'-(3-thio)tri-phosphate but not by alpha-amanitin . The association of yeast DNA polymerase I and yeast DNA primase can be demonstrated directly by isolation of the complex on a column containing yeast DNA polymerase I mouse monoclonal antibody covalently linked to Protein A-Sepharose . Both DNA polymerase I and DNA primase activities are retained by the column and can be eluted with 3.5 M MgCl2 . Part of the primase activity can be dissociated from DNA polymerase on the column with 1 M MgCl2 and this free primase activity can be detected as poly(dT) replication activity in the presence of Escherichia coli polymerase I.

Nucleic Acids Res, 1984 Jun 25, 12(12), 5123 - 41
Production of infectious poliovirus from cloned cDNA is dramatically increased by SV40 transcription and replication signals; Semler BL et al.; Sub-genomic cDNA clones representing the entire genomic RNA of poliovirus Type 1 (Mahoney) have been isolated in E . coli . Construction of a complete cDNA copy of the poliovirus genome in the EcoRI site of plasmid vector pBR325 from these clones is described . Introduction of plasmid DNA containing the complete cDNA copy of polio RNA into cultured primate cells by transfection produces infectious poliovirus . The virus produced by such a transfection appears to be identical to wild type poliovirus . Isolation of a polio recombinant plasmid containing SV40 transcription and replication signals is also described . Transfection of COS-1 cells with this plasmid yields greater than 1,600 plaque-forming units (PFU) per microgram of input DNA.

Nucleic Acids Res, 1984 Jun 25, 12(12), 4893 - 906
Genes and pseudogenes in a reiterated rat tRNA gene cluster; Rosen A et al.; A 13.4 kb rat genomic DNA fragment containing two related tRNA gene clusters was isolated from a rat lambda recombinant and analyzed for gene arrangement and nucleotide sequence . One cluster was found to contain a tRNALeuCUG gene while the second contained a tRNALeuCUA pseudogene with multiple base substitutions . The tRNALeu gene was found to possess an intact coding region and a functional transcription termination signal at the 3' end as demonstrated by in vitro transcription and processing of precursors to mature size tRNA . The first tRNA gene cluster was found to contain in addition to tRNALeu, three other transcribable genes coding for tRNAAspGAC(U), tRNAGlyGGA(G) and tRNAGluGAG; the second cluster contained in addition to tRNALeu pseudogene, the tRNAAsp tRNAGly and tRNAGlu genes . Examination of flanking sequences of the corresponding tRNA genes in the two clusters shows no homology at the 5' ends and partial conservation of sequences at the 3'-end region . Genomic rat DNA blot hybridizations show that the tRNALeu gene is distributed together with the tRNAAsp, tRNAGly and tRNAGlu on a 10 fold repeat of 3.2 kb EcoRI fragment.

Nucleic Acids Res, 1984 Jun 25, 12(12), 4849 - 63
Nucleotide sequence of the repressor gene of the TN10 tetracycline resistance determinant; Postle K et al.; The Tn10 tetR gene encodes the repressor that regulates transcription of the Tn10 tetracycline resistance determinant . We have determined the DNA sequence of the tetR gene and a 905 base pair region immediately 3' to tetR . The tetR gene is located on a 701 base pair HincII restriction fragment . Deletions at either end of this region eliminate synthesis of the wild-type TetR protein in E . coli minicells, and eliminate TetR activity as measured by repression of beta-galactosidase synthesis in tetA-lacZ operon fusion strains . Taken together, the DNA sequence and the genetic data indicate that tetR encodes a 207 amino acid protein with a calculated molecular weight of 23,328 . This value is in good agreement with estimates of 23,000-25,000 based on electrophoretic mobility in SDS-polyacrylamide gels . There is 47% amino acid sequence homology between the deduced sequences of the Tn10 and RP1/Tn1721 TetR proteins . There is, in addition, significant amino acid sequence homology between an NH2-terminal region of the Tn10 TetR repressor and the DNA recognition regions of other DNA-binding proteins.

J Biol Chem, 1984 Jun 25, 259(12), 8015 - 26
The organization and complete nucleotide sequence of the PstI restriction-modification system; Walder RY et al.; We have determined the nucleotide sequence of a 4.0-kilobase DNA fragment containing the genes of the PstI restriction-modification system . Two large open reading frames were identified within the sequence and were ascribed to the restriction enzyme and methylase by the analysis of a series of deletion mutants . The two genes are encoded on opposite DNA strands, and hence must be transcribed from separate promoters rather than as a polycistronic message . The sequence of the first 10 amino acids of the restriction endonuclease was determined by sequential Edman degradation of the purified protein, permitting the alignment of the polypeptide with the DNA sequence . The NH2 terminus of the modification enzyme was established by sequential Edman degradation of the protein synthesized in bacterial minicells with different radiolabeled amino acids . The initiation codons of the two genes are separated by 130 base pairs . The deduced amino acid sequences indicate that the restriction endonuclease contains 326 amino acids with a calculated Mr = 37,370; the modification enzyme is composed of 507 amino acids with a calculated Mr = 56,830 . There is no significant homology between the two proteins at the level of the primary structure . Antibody raised against the purified restriction endonuclease did not immunoprecipitate the modification enzyme . The transcription initiation sites were mapped using mung bean nuclease . Both of the transcripts begin with adenosine . The initiation sites are separated by only 70 base pairs . This close proximity suggests that the promoters for the two divergent genes overlap . DNase I protection experiments show that Escherichia coli RNA polymerase has a higher affinity for the methylase promoter than for the restriction enzyme promoter.

J Biol Chem, 1984 Jun 25, 259(12), 7602 - 6
Suppression of lymphocyte proliferation by copper-albumin chelates; Anderson WL et al.; The copper-albumin chelate (Cu2+-Alb), at concentrations less than 100 micrograms/ml, has potent noncytolytic antiproliferative activity for murine splenocytes stimulated by phytohemagglutinin-M, lipopolysaccharide (Escherichia coli 055:B5), or allogeneic cells and for phytohemagglutinin-M-stimulated human leukocytes . Inhibitory effects on the incorporation of {3H}leucine into trichloroacetic acid-precipitable protein is observed only at concentrations of Cu2+-Alb above 1 mg/ml . Only albumins with a histidine residue at position number 3 (rabbit, human, bovine) which bind one copper molecule at a high affinity site are capable of eliciting Cu2+-dependent suppression . Canine albumin, which has a tyrosine residue at position 3 and does not bind Cu2+, is nonsuppressive . Copper-albumin is suppressive in both the G1 and S phases of the cell cycle, thus clearly differentiating its suppressive activity from that of normal human plasma . It is not clear, however, if the Cu2+-Alb chelate is the active suppressive species or whether albumin is more efficient than other Cu2+ chelates in donating Cu2+ to another suppressive molecule . The biological significance of Cu2+-Alb-induced suppression is unknown . Although several possibilities are discussed, the potential to generate "artifactual" suppression by the formation of Cu2+-Alb chelates as a result of protein isolation procedures using Cu2+-contaminated reagents is considered to be an important potential problem.

J Biol Chem, 1984 Jun 25, 259(12), 7570 - 6
Topology of phage lambda receptor protein . Mapping targets of proteolytic cleavage in relation to binding sites for phage or monoclonal antibodies; Schenkman S et al.; Phage lambda receptor protein of Escherichia coli (LamB protein or maltoporin ) was purified in a mild detergent and subjected to prolonged proteolysis by either trypsin or subtilisin . Cleavage occurred at a limited number of sites without affecting the trimeric structure of the protein . Fragments could be dissociated only by heating in sodium dodecyl sulfate to 100 degrees C . The positions of purified fragments were determined with respect to the uncleaved 421-residue polypeptide by chemical analyses . The regions containing target sites were mapped around residues 159, 203, 245, and 370 . Based on kinetics of appearance of the different peptides, early cleavage events occurred at sites near residues 159, 203, and 245 and could be distinguished from late events around residue 370 . Information regarding the topological orientation of the cleavage sites could be obtained from the effect of in vitro proteolysis on the ability of the protein to bind phage lambda or monoclonal antibodies . Loss of phage lambda neutralizing activity coincided with early cleavage events, whereas loss of antigenic determinants, known to be exposed at the cell surface, appeared late . Cleavage regions are thus likely to be exposed at the cell surface, a conclusion compatible with the location of mutations affecting the interaction of LamB protein with phage in vivo.

Nucleic Acids Res, 1984 Jun 25, 12(12), 5025 - 36
Chemical synthesis of 5-azacytidine nucleotides and preparation of tRNAs containing 5-azacytidine in its 3'-terminus; Zielinski WS et al.; 5-azacytidine-5'-triphosphate prepared from 5-azacytidine by chemical phosphorylation is a substrate for AMP (CMP) tRNA nucleotidyl transferase from yeast . tRNAsPhe from yeast containing 5-azacytidine in their 3'-termini were prepared enzymatically . tRNAPhe-Cpn5CpA and tRNAPhe-n5Cpn5CpA can be aminoacylated by phenylalanyl-tRNA synthetase from yeast and they are active in the poly(U)-dependent synthesis of poly(Phe) on E . coli ribosomes . The decomposition of 5-azacytidine via hydrolysis of the triazine ring is significantly accelerated by a phosphate group on the 5'-position of the nucleotide . After the incorporation of 5-azacytidine-5'-phosphate into a polynucleotide chain the rate of hydrolysis of the triazine ring decreases considerably.

Br Med J (Clin Res Ed), 1984 Jun 23, 288(6434), 1864 - 6
Evaluation of test immunisation in the assessment of antibody deficiency syndromes; Webster AD et al.; Antibody responses after immunisation with pneumococcal polysaccharide did not correlate with the severity and frequency of infections in 22 patients with severe hypogammaglobulinaemia, when these were measured by a Farr radioimmunoassay . Five "healthy" patients with severe hypogammaglobulinaemia not only failed to make antipneumococcal polysaccharide antibody, when measured by radioimmunoassay, but also had very low or unrecordable antibody responses to Escherichia coli and failed to produce antibody when immunised with tetanus toxoid . Some of these subjects, however, did make small amounts of IgM antipneumococcal polysaccharide antibody when this was measured by an enzyme linked immunosorbent assay, while others retained some ability to produce IgM or IgA or both in their saliva . These findings show that the measurement of serum antibody responses after immunisation, with the possible exception of IgM antibodies to polysaccharides, is unlikely to be helpful in assessing the requirement for gammaglobulin replacement therapy in patients with hypogammaglobulinaemia.

Science, 1984 Jun 22, 224(4655), 1343 - 6
Adenovirus E1a gene product expressed at high levels in Escherichia coli is functional; Ferguson B et al.; The human type C adenovirus E1a 13S messenger RNA encodes a gene product, that positively regulates the transcription of viral genes and certain cellular genes and is involved in the transformation of primary mammalian cells . The E1a gene product was expressed at high levels in Escherichia coli . In a Xenopus oocyte microinjection assay, the purified Escherichia coli-produced protein activated the E1a-responsive adenovirus E3 promoter and functioned as efficiently as the E1a gene itself.

Nature, 1984 Jun 21-27, 309(5970), 682 - 7
Use of light for footprinting DNA in vivo; Becker MM et al.; A new ' photofootprinting ' technique, which uses light to detect protein-DNA contacts as well as changes in the structure of DNA at the base pair level, has been developed and used to detect contacts between lac repressor and the lac operator in Escherichia coli cells.

Biochemistry, 1984 Jun 19, 23(13), 2952 - 7
7S RNA, containing 5S ribosomal RNA and the termination stem, is a specific substrate for the two RNA processing enzymes RNase III and RNase E; Szeberenyi J et al.; The 7S RNA, a precursor of 5S rRNA that contains 5S rRNA and the termination stem and loop, is a substrate for RNase E and is also a substrate for RNase III . The cleavage by RNase III is in the stem, 11 nucleotides downstream from the 3' end of the mature 5S rRNA and 8 nucleotides downstream from the RNase E cleavage site . Near the cleaved nucleotides there are three base pairs that appear in the same relative positions in most known RNase III cleavage sites . The large product of the RNase III cleavage reaction, which is a 5S rRNA that contains 11 extra nucleotides at the 3' end, is a substrate for RNase E . This suggests that the information for the 3'-end cleavage by RNase E resides mainly in the 5S rRNA itself . Using rnc rne strains, carrying the plasmid that leads to the accumulation of 7S RNA, we showed that the 7S RNA does not result from an RNase III cleavage but is apparently a proper transcription termination product.

Biochemistry, 1984 Jun 19, 23(13), 2933 - 9
Photochemical cross-linking of lac repressor to nonoperator 5-bromouracil-substituted DNA; Barbier B et al.; Ultraviolet irradiation of lac repressor bound to 5-bromouracil-substituted nonoperator DNA leads to the formation of cross-links between the protein and the nucleic acid . The cross-links are formed between the DNA and the 1-51 N-terminal peptide of the repressor, the "headpiece" . The tetrameric core (4 X 60-360 amino acids) was never found to be cross-linked to the DNA . With isolated headpieces, which are able to bind DNA, no cross-link was detected . These results are discussed considering the fundamental role of the core in keeping the headpieces in an adequate geometry for the DNA-repressor interaction . It has been possible to cross-link two DNA molecules to one repressor molecule, thus showing the existence of at least two binding sites for nonoperator DNA on the repressor . The attached peptides were analyzed after extensive proteolytic cleavage, and the most abundant peptide found was peptide 23-33 . Histidine-29 seems to be the photo-cross-linked amino acid . Analysis of the results required a computation method discussed in the Appendix.

Biochemistry, 1984 Jun 19, 23(13), 2914 - 22
Stimulation of Escherichia coli DNA photoreactivating enzyme activity by adenosine 5'-triphosphate; Koka P; A purification procedure consisting of Biorex-70, single-stranded DNA-agarose, and ultraviolet (UV) light irradiated DNA-cellulose chromatography has been adopted for the Escherichia coli photoreactivating enzyme . The purpose of this purification was to obtain enzyme preparations that are free of extraneous nucleic acid or nucleotides . The purification yields high specific activities (75 000 pmol h-1 mg-1) with a 50% recovery . Enzyme preparations have also been obtained from UV-irradiated DNA-cellulose by exposure to visible light . These enzyme preparations contain oligoribonucleotides, the largest found to be 26 nucleotides in length in relation to DNA size markers . However, the oligoribonucleotides associated with the enzyme are not essential for enzymatic activity . When the enzyme is preincubated with exogenous ATP for 4-10 h at 3 degrees C, a 10-fold stimulation in the enzyme activity has been observed . It has been determined by polyacrylamide gel electrophoresis and high-voltage diethylaminoethyl paper electrophoresis that the light-released enzyme samples from a preincubated and washed mixture of the enzyme, {gamma-32P}ATP, and UV-irradiated DNA-cellulose contained exogenous {gamma-32P}ATP . {gamma-32P}ATP eluted with the enzyme-containing fractions when subjected to Bio-Gel P-30 chromatography . GTP caused a slight enhancement of the enzyme activity while ADP strongly inhibited photoreactivation, at the same concentration and conditions as those for ATP . Higher (X5) concentrations of ADP and adenosine 5'-(beta, gamma-methylenetriphosphate) totally inhibited the enzyme activity . Dialysis of a photoreactivating enzyme preparation against a buffer solution containing 1 mM ATP caused a 9-fold stimulation of the enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1984 Jun 19, 23(13), 2900 - 5
Amino acid sequence of Escherichia coli citrate synthase; Bhayana V et al.; Detailed evidence for the amino acid sequence of allosteric citrate synthase from Escherichia coli is presented . The evidence confirms all but 11 of the residues inferred from the sequence of the gene as reported previously {Ner, S . S., Bhayana, V., Bell, A . W., Giles, I . G., Duckworth, H . W., & Bloxham, D . P . (1983) Biochemistry 22, 5243}; no information has been obtained about 10 of these (residues 101-108 and 217-218), and we find aspartic acid rather than asparagine at position 10 . Substantial regions of sequence homology are noted between the E . coli enzyme and citrate synthase from pig heart, especially near residues thought to be involved in the active site . Deletions or insertions must be assumed in a number of places in order to maximize homology . Either of two lysines, at positions 355 and 356, could be formally homologous to the trimethyllysine of pig heart enzyme, but neither of these is methylated . It appears that E . coli and pig heart citrate synthases are formed of basically similar subunits but that considerable differences exist, which must explain why the E . coli enzyme is hexameric and allosterically inhibited by NADH, while the pig heart enzyme is dimeric and insensitive to that nucleotide.

Biochemistry, 1984 Jun 19, 23(13), 2843 - 8
Photoaffinity labeling of DNA-dependent RNA polymerase from Escherichia coli with 8-azidoadenosine 5'-triphosphate; Woody AY et al.; A photoaffinity analogue of adenosine 5'-triphosphate (ATP), 8-azidoadenosine 5'-triphosphate (8-N3ATP), has been used to elucidate the role of the various subunits involved in forming the active site of Escherichia coli DNA-dependent RNA polymerase . 8-N3ATP was found to be a competitive inhibitor of the enzyme with respect to the incorporation of ATP with Ki = 42 microM, while uridine 5'-triphosphate (UTP) incorporation was not affected . UV irradiation of the reaction mixture containing RNA polymerase and {gamma-32P}-8-N3ATP induced covalent incorporation of radioactive label into the enzyme . Analysis by gel filtration and nitrocellulose filter binding indicated specific binding . Subunit analysis by sodium dodecyl sulfate and sodium tetradecyl sulfate gel electrophoresis and autoradiography of the labeled enzyme showed that the major incorporation of radioactive label was in beta' and sigma, with minor incorporation in beta and alpha . The same pattern was observed in both the presence and absence of poly{d(A-T)} and poly{d(A-T)} plus ApU . Incorporation of radioactive label in all bands was significantly reduced by 100-150 microM ATP, while 100-200 microM UTP did not show a noticeable effect . Our results indicate major involvement of the beta' and sigma subunits in the active site of RNA polymerase . The observation of a small extent of labeling of the beta and alpha subunits, which was prevented by saturating levels of ATP, suggests that these subunits are in close proximity to the catalytic site.

J Mol Biol, 1984 Jun 15, 176(1), 155 - 9
In vitro replication of a dam methylated and non-methylated ori-C plasmid; Hughes P et al.; We have examined the replication of a dam methylated and non-methylated ori-C plasmid in an in vitro ori-C dependent replication system . The results show that the non-methylated plasmid is 50% to 80% less efficient in the initiation of DNA synthesis; that the methylation state of the plasmid does not change the site of initiation at ori-C, and that in both cases initiation at this region requires the presence of exogenously furnished dnaA protein.

Biochim Biophys Acta, 1984 Jun 14, 787(2), 165 - 73
Purification, molecular and kinetic properties of glucosamine-6-phosphate isomerase (deaminase) from Escherichia coli; Calcagno M et al.; Glucosamine-6-phosphate isomerase (deaminase), (2-amino-2-deoxy-D-glucose-6-phosphate ketol isomerase (deaminating), EC 5.3.1.10) has been purified to homogeneity from Escherichia coli B as judged by several criteria of purity . The procedure included ammonium sulfate fractionation, anion-exchange chromatography and a biospecific affinity chromatography step with N-epsilon-amino-n- caproyl -D-glucosamine 6-phosphate bound to agarose as the ligand, the elution being performed with GlcNAc6 P . The enzyme appears to be an hexamer of about 178 kDa, composed of six subunits of 29 700 +/- 300 Da; the isoelectric point was 6.0-6.1 and the sedimentation constant 9.0 S . The amino-acid composition of the enzyme was determined and a value for E1%275 of 4.55 was calculated . The molecular activity was 1800 s-1 for the deamination reaction and 455 s-1 for the reaction of GlcN6 P formation . A positive homotropic cooperativity was found for both sugar substrates; it was stronger for GlcN6 P in the deamination reaction (Hill number 2.7 at pH 7.7) . Ammonia behaved as a Michaelian substrate . Cooperativity was abolished by 0.1 mM GlcNAc6 P; this allosteric modulator activated the reaction in both directions, with a positive K-effect upon both sugar phosphates, but had no effect on Km for ammonia . The initial velocity patterns for the amination reaction were obtained under conditions of hyperbolic kinetics produced by GlcNAc6 P; the Km values for the allosteric substrates were determined under the same conditions, and their dependence upon pH was studied.

FEBS Lett, 1984 Jun 11, 171(2), 207 - 10
Cooperative and salt-resistant binding of lexA protein to non-operator DNA; Schnarr M et al.; The interaction of the lexA repressor of E . coli with poly{d(A-T)} has been studied by circular dichroism . The binding induces an about 2-fold increase of the circular dichroism intensity at 263 nm, pointing out a conformational change of the nucleic acid . The observed spectral changes are very similar to those observed for the binding of the lac repressor to poly{d(A-T)} and natural DNA . At elevated ionic strength the binding isotherms do show a pronounced sigmoidal shape indicating a cooperative mode of binding.

Nucleic Acids Res, 1984 Jun 11, 12(11), 4757 - 68
A ribosome binding site sequence is necessary for efficient expression of the distal gene of a translationally-coupled gene pair; Das A et al.; Expression of trpB and trpA of the Escherichia coli tryptophan operon is shown to be "translationally coupled", i.e., efficient translation of the trpA coding region is dependent on prior translation of the trpB coding region and termination of translation at the trpB stop codon . To examine the dependence of trpA expression on the ribosome binding site sequence in the distal segment of trpB, deletions were produced that replaced this trpB sequence . Analysis of trpA expression in these deletion mutants established that the ribosome binding site sequence is required for efficient translation of the trpA segment of trp mRNA . A modest effect of translation over the trpA ribosome binding site on independent initiation at that site was also observed.

FEBS Lett, 1984 Jun 11, 171(2), 245 - 8
The gene coding for lipoprotein signal peptidase (lspA) and that for isoleucyl-tRNA synthetase (ileS) constitute a cotranscriptional unit in Escherichia coli; Yamada H et al.; The lspA gene coding for lipoprotein signal peptidase is located very close to the ileS gene coding for isoleucyl-tRNA synthetase on the Escherichia coli chromosome . Deletions were generated in vitro from both ends of the 4.3 kb fragment that carries the lspA gene and the ileS gene, and the expression of the two genes was examined before and after insertion of the trp promoter-operator at one end . The results indicate that the lspA and ileS genes constitute a cotranscriptional unit in the order of promoter- ileS - lspA . The gene order of dnaJ - rpsT - ileS - lspA - dapB around 0.5 min on the E . coli chromosome map was also determined.

Nucleic Acids Res, 1984 Jun 11, 12(11), 4731 - 45
Primary structure of rabbit 18S ribosomal RNA determined by direct RNA sequence analysis; Connaughton JF et al.; The primary structure of rabbit 18S ribosomal RNA was determined by nucleotide sequence analysis of the RNA directly . The rabbit rRNA was specifically cleaved with T1 ribonuclease, as well as with E . coli RNase H using a Pst 1 DNA linker to generate a specific set of overlapping fragments spanning the entire length of the molecule . Both intact and fragmented 18S rRNA were end-labeled with {32P}, base-specifically cleaved enzymatically and chemically and nucleotide sequences determined from long polyacrylamide sequencing gels run in formamide . This approach permitted the detection of both cistron heterogeneities and modified bases . Specific nucleotide sequences within E . coli 16S rRNA previously implicated in polyribosome function, tRNA binding, and subunit association are also conserved within the rabbit 18S rRNA . This conservation suggests the likelihood that these regions have similar functions within the eukaryotic 40S subunit.

J Biol Chem, 1984 Jun 10, 259(11), 7317 - 24
The NH2-terminal domain of Escherichia coli ribosomal protein L11 . Its three-dimensional location and its role in the binding of release factors 1 and 2; Tate WP et al.; Ribosomes from three previously described mutants of Escherichia coli lacking L11 ( AM68 , AM76 , and AM77 ) supported in vitro termination with release factor 1 very poorly, but with release factor 2 had a severalfold elevation in activity for this function compared with ribosomes from a control strain or from a mutant containing unmethylated L11 . L11 exerts its effect on the binding of the factors into a functional ribosomal complex with the termination codon . Reconstitution of L11 back into the L11-lacking ribosomes restored them to the control phenotype . The NH2-terminal part of L11 (amino acids 1-64) seems critical in modulating release factor binding . This part of L11 has been localized with the use of fragment-specific antibodies on the three-dimensional model of the 50 S subunit in the region from where the L7/L12 stalk originates . IgG antibodies from an antiserum specific for this fragment but not a middle fragment of L11 (amino acids 65-102) strongly inhibited in vitro termination . The activities of the two factors were inhibited differentially by several anti-L11 preparations recognizing antigenic determinants in the NH2-terminal part of L11 . In all but one case, release factor 1 was more sensitive . These studies indicate that there are significant differences in the binding domains for the two release factors which are affected by the NH2-terminal part of L11.

J Biol Chem, 1984 Jun 10, 259(11), 6798 - 805
Promoter helical structure variation at the Escherichia coli polymerase interaction sites; Nussinov R; There is evidence that the Escherichia coli polymerase recognizes and binds to three sites on the promoter DNA: the -10, -35, and -16 regions . Sequence homology was noted among the -10 sites (Pribnow box) and among the -35s with consensus sequences, TATAAT and TTGACA , respectively . Weak nucleotide sequence homology was detected at -16 . Since the polymerase recognizes these sites in a multitude of promoters, one expects similarities in the three-dimensional structures . To date, no data directly bearing on such structures exist . Recently, Calladine ( Calladine , C.R . (1982) J . Mol . Biol . 161, 343-352) and, subsequently, Dickerson ( Dickerson , R.E . (1983) J . Mol . Biol . 166, 419-441) suggested "rules" for doublestranded DNA structures which were tested against data from several known crystals . Using these rules, I compare the deviations from "ideal" B-DNA of the twist angles, base pair roll, sideways shift, and propeller suppression in 56 promoters at the three sites . I also appended to these the twist angle computations on additional 77 promoters from the recently published compilation of promoter sequences . For the latter, additional nucleotides from the spacer regions were added . The results display similarities at the -10 site . Equally strong similarities were obtained for the -35 and the -16 contact regions . The existence of structural differences for some sites is likely to account for the different degrees of efficiency of the polymerase recognition and transcriptional regulation.

J Biol Chem, 1984 Jun 10, 259(11), 6826 - 32
Xylose isomerase from Escherichia coli . Characterization of the protein and the structural gene; Schellenberg GD et al.; The gene that codes for xylose isomerase in Escherichia coli has been cloned by complementation of a xylose isomerase-negative E . coli mutant . The structural gene is 1320 nucleotides in length and codes for a protein of 440 amino acids . An additional 209 nucleotides 5' and 82 nucleotides 3' to the structural gene were also sequenced . To verify that the cloned gene encodes E . coli xylose isomerase, the enzyme was purified to homogeneity and the sequence of the first 25 amino acid residues was determined by a semimicromanual Edman procedure . These results establish that the NH2-terminal methionine of xylose isomerase is specified by an ATG which is 7 nucleotides downstream from a Shine-Dalgarno sequence.

J Biol Chem, 1984 Jun 10, 259(11), 7206 - 11
Molecular cloning of DNA sequences complementary to mouse thymidylate synthase messenger RNA; Geyer PK et al.; We describe the isolation of recombinant cDNA clones containing sequences corresponding to mouse thymidylate synthase mRNA . Double-stranded cDNA was made from poly(A+) RNA isolated from the 5-fluoro-2'-deoxyuridine-resistant mouse cell line LU3 -7 that overproduces thymidylate synthase 50- to 100-fold as compared to the parental mouse 3T6 fibroblasts . The cDNA was inserted into pBR322 using the poly(dC)-poly(dG) tailing procedure and transformed into Escherichia coli HB101 . A library of 30,000 colonies was screened for thymidylate synthase cDNA sequences by differential colony hybridization . Plasmid DNA was purified from a colony that gave a positive signal . RNA corresponding to this plasmid was isolated by hybridization and translated in vitro to yield a protein that coelectrophoresed with authentic thymidylate synthase . The identity of the translation product was confirmed by immunoprecipitation with thymidylate synthase antiserum and by peptide analysis . A restriction fragment from this plasmid was used to rescreen the library to give a collection of thymidylate synthase cDNA plasmids with overlapping restriction maps . Several plasmids contained cDNA inserts greater than 1 kilobase pair in length . The size and content of thymidylate synthase mRNA in the overproducing and parental cell lines was determined by RNA blot analysis . Multiple thymidylate synthase mRNA species were identified . The predominant species was 1.3 kilobase pairs in length . Thymidylate synthase mRNA was 50 times more abundant in LU3 -7 than in 3T6 cells.

Science, 1984 Jun 8, 224(4653), 1068 - 74
Structure and expression of a complementary DNA for the nuclear coded precursor of human mitochondrial ornithine transcarbamylase; Horwich AL et al.; Most mitochondrial proteins are encoded in the nucleus and are translated on free cytoplasmic ribosomes as larger precursors containing amino-terminal "leader" sequences, which are removed after the precursors are taken up by mitochondria . We have deduced the complete primary structure of the precursor of a human mitochondrial matrix enzyme, ornithine transcarbamylase (OTC), from the nucleotide sequence of cloned complementary DNA . The amino-terminal leader peptide of OTC is 32 amino acids in length and contains four arginines but no acidic residues . Cleavage of the leader peptide from the "mature" protein occurs between glutamine and asparagine residues . The sequence of mature human OTC resembles that of the subunits of both OTC and aspartate transcarbamylase from Escherichia coli . The biological activity of the cloned OTC complementary DNA was tested by joining it with SV40 (an animal virus) regulatory elements and transfecting cultured HeLa cells, which do not normally express OTC . Both the precursor and mature forms of the OTC subunit were identified; in stable transformants, enzymatic activity was also detected.

Brain Res, 1984 Jun 8, 302(2), 277 - 80
Anterior hypothalamic lesions prevent the endotoxin-induced reduction of beta-adrenoceptor number in guinea pig lung; Van Oosterhout AJ et al.; Bacterial endotoxin (E . coli O111:B4) induces, 4 days after intraperitoneal injection, a 30% reduction of guinea pig lung beta-adrenoceptor number (Bmax) . No change in affinity (Kd) for the receptors occurred . Bilateral electrolytic lesions centered in the anterior hypothalamic nucleus prevent this reduction in Bmax and even reverse the reduction into a small increase in beta-adrenoceptor number . Since it is known from the literature data that anterior hypothalamic lesions as well as beta-adrenoceptor stimulants have an inhibitory influence on the immune system, the mechanism by which these lesions inhibit the reduction of beta-adrenoceptor sites after bacterial endotoxin and influence immune functions, may be related.

J Immunol Methods, 1984 Jun 8, 71(1), 97 - 105
The separation of human serum IgG into subclass fractions by immunoaffinity chromatography and assessment of specific antibody activity; Bird P et al.; Murine monoclonal antibodies ( McAbs ) with specificity for subclass-specific or subclass-restricted determinants on human IgG have been coupled to Sepharose to generate affinity columns . The judicial use of positive and negative chromatography and the exploitation of the special properties of individual McAb affinity columns has allowed the preparation of individual IgG subclasses from polyclonal IgG containing less than 1% contamination by any other IgG subclass . The specificity of the antibodies present in each polyclonal IgG subclass preparation has been assayed against a bacterial toxoid (tetanus), 2 bacterial cell wall antigens (E . coli and pneumococcal) and coat antigen(s) of a DNA virus (CMV) . Antibodies were predominantly IgG1 to tetanus toxoid, IgG2 to pneumovax and E . coli cell walls, and IgG1, 2 and 3 to CMV coat antigens.

Biochemistry, 1984 Jun 5, 23(12), 2753 - 8
Catalytic mechanism of the dihydrofolate reductase reaction as determined by pH studies; Stone SR et al.; The variation with pH of the kinetic parameters of the reaction catalyzed by dihydrofolate reductase from Escherichia coli has been determined with the aim of elucidating the chemical mechanism of the reaction . The (V/K)DHF and V profiles indicated that protonation enhances the observed rate of interaction of dihydrofolate (DHF) with the enzyme-NADPH complex as well as the maximum velocity of the reaction . The pKa value of 8.09 observed in the (V/K)DHF profile is similar to that of 7.9 observed in the Ki profile for 2,4-diamino-6,7-dimethylpteridine while the pKa value of the V profile is displaced to 8.4 . From the magnitude of the pH-independent value for (V/K)DHF, it is concluded that unprotonated dihydrofolate must react, at neutral pH, with the protonated form of the enzyme . The D(V/K)DHF value is independent of pH and equal to unity whereas the DV value varies as a wave function of pH with limiting values of 1.5 and 1.0 at low and high pH, respectively . It is proposed that dihydrofolate reacts with the unprotonated enzyme-NADPH complex to form a dead-end complex and with the protonated form of the same complex to form a productive complex . Further, it is considered that the protonated carboxyl of Asp-27 at the active site of the enzyme is responsible for the protonation of the N-5 nitrogen of dihydrofolate and that this protonation precedes and facilitates hydride transfer.

Biochemistry, 1984 Jun 5, 23(12), 2679 - 83
Preferential stimulation of the in vivo synthesis of a protein by polyamines in Escherichia coli: purification and properties of the specific protein; Mitsui K et al.; The possibility that polyamines can stimulate the synthesis of special kinds of proteins has been examined by using a polyamine-requiring mutant of Escherichia coli . It was found that the synthesis of some proteins, particularly one with a molecular weight (Mr) of 62K, was significantly stimulated following polyamine supplementation of polyamine-starved cells . The preferential stimulation of the synthesis of this polyamine-induced protein of Mr 62K (PI protein) was followed by the stimulation of overall protein synthesis by polyamines . PI protein was purified to homogeneity and some of its properties were examined . From studies on the effect of PI protein on MS2 RNA directed protein synthesis, it was shown that this protein stimulated the synthesis of RNA replicase by 2.2-fold in the presence of 1 mM spermidine.

J Mol Biol, 1984 Jun 5, 175(4), 511 - 28
Assembly pathway of newly synthesized LamB protein an outer membrane protein of Escherichia coli K-12; Vos-Scheperkeuter GH et al.; The assembly of newly induced LamB protein (phage lambda receptor) was investigated in an operon fusion strain of Escherichia coli, in which the lamB gene is expressed under lac promoter control . The induction kinetics both for total cellular and for cell surface-exposed LamB protein were studied by immunochemical detection methods, using two distinct antisera directed against detergent-solubilized LamB trimers and completely denatured LamB monomers, respectively . Anti-trimer antibodies recognized both monomers and trimers, whereas anti-monomer antibodies only reacted with monomers . Provided appropriate solubilization conditions were used, both antisera were able to immunoprecipitate intracellular mature LamB protein quantitatively . Following induction, the first LamB antigenic determinants were detected after 60 to 80 seconds; detection of the newly synthesized protein by anti-monomer antibodies slightly preceded that by anti-trimer antibodies, a finding that could be partly explained by the observation that anti-monomer antibodies recognized a larger fraction of nascent LamB than did anti-trimer antibodies . Exposure of antigenic determinants at the cell surface was delayed for 30 to 50 seconds with respect to their synthesis . Therefore, either translocation or conformational changes must be rate-limiting in the series of processes that eventually convert the newly synthesized protein into its mature outer membrane state . LamB protein was found to occur in at least three clearly distinguishable states . State I is the LamB monomer, state II corresponds to a metastable trimer that dissociates in sodium dodecyl sulphate above 60 degrees C, and state III is the state LamB trimer that dissociates in sodium dodecyl sulphate only at temperatures above 90 degrees C . The chase kinetics of these states showed that conversion of newly synthesized LamB monomers to stable LamB trimers occurred in two stages: state I monomers were chased into metastable state II trimers rapidly (t 1/2 = 20 s), whereas stabilization of state II trimers to state III trimers was a relatively slow (t 1/2 = 5.7 min) process . Based on our results, a timing sequence in the assembly of outer membrane LamB protein is proposed.

Biochemistry, 1984 Jun 5, 23(12), 2673 - 9
Identification of a neutral flavin radical and characterization of a second chromophore in Escherichia coli DNA photolyase; Jorns MS et al.; DNA photolyase from Escherichia coli is a blue protein exhibiting absorption maxima at 580, 475, and 384 nm . One of the two chromophores present in this enzyme has been identified as the blue neutral flavin adenine dinucleotide (FAD) radical on the basis, in part, of visible absorption and electron spin resonance (ESR) data . The enzyme-bound radical (epsilon 580 = 3.6 X 10(3) M-1 cm-1) is stable toward O2 or K3Fe(CN)6, is reversibly reduced by dithionite, and is converted to oxidized FAD upon aerobic denaturation . Disproportionation of the radical is observed upon anaerobic denaturation, consistent with an N-5 unsubstituted radical . The absorbance of the enzyme at lambda greater than 500 nm is due only to the FAD radical whereas the band at 384 nm reflects contributions from both the radical and a second chromophore . The latter is labile when protein free a neutral pH (lambda max = 360 nm, k = 5.5 X 10(-2) min -1 +/- O2), a reaction that is readily monitored by the loss of an intense absorption band at 360 nm following enzyme denaturation under conditions where radical oxidation is immediate . This decomposition is pH dependent and the chromophore is stable at acid pH . Native photolyase is fluorescent (emission lambda max = 470 nm, excitation lambda max = 398 nm) . An unlikely fluorescent flavin radical can be excluded by the position of the emission maximum . The enzyme fluorescence is attributed to the second chromophore.

Nature, 1984 Jun 28-Jul 4, 309(5971), 810 - 2
Generation of beta-globin by sequence-specific proteolysis of a hybrid protein produced in Escherichia coli; Nagai K et al.; High-level expression of many eukaryotic genes has proved difficult to achieve even when a strong promoter and the ribosome binding sequence from highly expressed Escherichia coli genes have been placed in front of the coding sequences . To overcome this problem, many eukaryotic proteins have been efficiently produced as hybrids after fusion of their genes with a coding sequence of E . coli genes . However, such hybrid proteins are not suitable for functional studies or clinical use unless the authentic protein sequence can be released by specific cleavage . Here, we have inserted the sequence Ile-Glu-Gly-Arg between the 31 amino-terminal residues of lambda cII protein and Val 1 of human beta-globin, and produced this hybrid in high yield in E . coli . We then cleaved the hybrid specifically at the single arginine, using blood coagulation factor Xa and thus liberated the authentic beta-globin chain . As factor Xa is specific for the tetrapeptide Ile-Glu-Gly-Arg, which is rare in protein sequences, our expression/cleavage system is applicable to the efficient production of many eukaryotic proteins.

DNA, 1984 Jun, 3(3), 269 - 77
Biotin-labeled oligonucleotides: enzymatic synthesis and use as hybridization probes; Murasugi A et al.; An oligonucleotide probe (23-mer) containing a single biotinylated deoxyuridine residue at the 3' terminus was prepared by a primer extention reaction using Escherichia coli DNA polymerase I (Klenow fragment) . For efficient synthesis of the probe, it was necessary to add about 16-fold molar excess of the template oligonucleotide (pentadecanucleotide) to the primer oligonucleotide (nonadecanucleotide) in the reaction mixture and to continue the reaction for 2.5 hr at 4 degrees C . The probe was purified by polyacrylamide gel electrophoresis under denaturing conditions . The probe could be specifically and tightly bound with Avidin D (Vector Laboratories) in 1 M NaCl . It could be hybridized to a plasmid DNA containing a perfectly matched complementary sequence, but not to a DNA containing five consecutive noncomplementary bases . The hybridized biotinylated probe could be detected by Avidin D and biotinylated alkaline phosphatase, even when 1.8 ng of the plasmid DNA (0.5 fmoles) was used . A general approach to the enzymatic synthesis of oligonucleotides containing a single biotinylated deoxyuridine at the 3' end is described.

J Surg Res, 1984 Jun, 36(6), 553 - 62
A physiologic explanation for cardiac deterioration in septic shock; Demeules JE; Utilizing analysis of multiple mechanical and electrophysiologic parameters, a technique for describing the myocardial depressant effects of septic shock was employed . Rabbit papillary muscle was exposed to septic shock serum obtained from dogs made hypotensive by infusing Escherichia coli 1-1.9 X 10(9) colony forming units per cubic centimeter (7 cc/kg) . The exposure produced depression in action potential amplitude (47%), duration (32%), resting membrane potential (37%), velocities of phases 0 (31%), 2 (100%); phase 3 was preserved . Also, peak tension (62%) and velocity of contraction (51%) and relaxation (56%) were decreased . If the muscle was first exposed to 30 mM KCL solution, these depressions were eliminated . These data suggest the myocardial defects are mediated through abnormalities in fast channel activity.

Eur J Immunol, 1984 Jun, 14(6), 566 - 77
Human T cell antigens involved in cytotoxicity against allogeneic or autologous chemically modified targets . Association of the Leu 2a/T8 antigen with effector-target cell binding and of the T3/Leu 4 antigen with triggering; Platsoucas CD; Monoclonal antibodies (mAb) recognizing human T cell differentiation antigens were employed to analyze the role of these antigens on T cell-mediated cytotoxicity against autologous 2,4,6-trinitrophenyl (TNP)-modified targets . The OKT3/anti-Leu 4 and anti-Leu 2a/OKT8 mAb inhibited T cell-mediated cytotoxicity against autologous or unrelated TNP-modified targets, in the absence of complement and at the effector cell level . These cytotoxic effector cells were T3+, T8+, T11+, T4- . To analyze the role of the T3/Leu 4 and Leu 2a/T8 T cell differentiation antigens in the cytolytic process, we investigated the stages of this process that were inhibited by the OKT3/anti-Leu 4 and anti-Leu 2a/OKT8 mAb . Using: (a) visual adhesions, and (b) a single cell cytotoxicity assay in agarose, we observed that the OKT3 and anti-Leu 4 mAb did not inhibit binding of effector cells to allogeneic targets or to autologous E rosette-negative TNP-modified targets, although they significantly inhibited both the proportions of target cells in conjugates that were lysed, and the 51Cr release . In contrast, the anti-Leu 2a and OKT8 mAb blocked cytotoxicity by inhibiting binding of effector cells to the allogeneic or to autologous chemically modified targets . To further analyze the stages of the cytolytic process (adhesion; programming for lysis or lethal hit; and cytotoxic cell-independent lysis) that were inhibited by these mAb, we employed the detachment and dispersion method . This method is based on the differential temperature requirements of adhesion (which occurred both at 37 degrees C or 20 degrees C) and of programming for lysis (which occurred at 37 degrees C but not at 20 degrees C) . Operational adhesions were determined by the 51Cr-release assay after dispersion of effector and target cells in a 10% solution of dextran (mol . wt . 500 000) . Programming for lysis was determined by the 51Cr-release assay after detachment of effector-target cell conjugates with 10 mM EDTA and dispersion in 10% dextran solution . Using this method we determined that mAb recognizing the Leu 2a/T8 antigen blocked cytotoxicity by inhibiting adhesion and binding of effector cells to target cells . These antibodies do not affect post-adhesion stages of the cytolytic process . In contrast, the OKT3 and anti-Leu 4 mAb inhibit a post-adhesion step of the cytolytic process, that occurs before irreversible events of the programming for lysis stage have taken place.(ABSTRACT TRUNCATED AT 400 WORDS)

Microcirc Endothelium Lymphatics, 1984 Jun, 1(3), 307 - 27
Pulmonary vascular and hemodynamic effects of prostaglandin E1 in unanesthetized sheep; Ogletree ML et al.; We prepared fully instrumented sheep with chronic lung lymph fistulas and measured lung lymph and hemodynamic responses to intravenous infusions of prostaglandin (PG) E1 . PGE1 decreased lung lymph flow from 8.0 +/- 0.8 S.E.M . ml/h during baseline to 6.5 +/- 0.7 ml/h (p less than 0.05) and 5.4 +/- 0.8 ml/h (p less than 0.05) at 50 and 250 micrograms/min, respectively . Lymph to plasma protein concentration ratio (L/P) did not change during PGE1 infusion and increased after PGE1 was stopped . Systemic and pulmonary vascular pressures decreased without producing reflex tachycardia . PGE1 consistently caused hypoxemia and hemoconcentration . Multiple indicator dilution studies showed consistent decreases in the 14C-urea permeability surface area product (PS urea) during PGE1 infusion . To distinguish effects of PGE1 on perfused lung vascular surface area from effects on vascular permeability, we mechanically increased lung vascular pressures, achieved a steady state lung lymph response, and infused PGE1 (50 micrograms/min) while maintaining constant left atrial pressure . PGE1 decreased lung lymph flow from 14.1 +/- 2.0 ml/h to 10.0 +/- 1.8 ml/h (p less than 0.05) . Systemic and pulmonary arterial pressures fell . Cardiac output did not change and heart rate decreased . L/P increased significantly, indicating that the decrease in lymph flow could be attributed to decreased lung microvascular pressure without obligate change in microvascular permeability . During increased lung vascular permeability caused by E . coli endotoxin, PGE1 (250 micrograms/min) decreased lung lymph flow, and systemic and pulmonary vascular pressures fell . Although PGE1 decreased lymph protein clearance, L/P did not change . Decreased lymph flow with PGE1 could be attributed to decreased lung vascular pressure, decreased exchanging vessel surface area, or both.

Biokhimiia, 1984 Jun, 49(6), 1046 - 9
{Localization of the redox-center of cytochrome b562 on the internal (M-) side of the mitochondrial membrane}; Konstantinov AA et al.; In the inside-out submitochondrial particles, cytochrome b-562 is readily reduced by a hydrophilic redox mediator Ru(NH3)6(2)+; this reaction is not inhibited by antimycin and myxothiazol . In mitochondria, cytochromes b do not virtually interact with Ru(NH3)6(2)+ . The accessibility of cytochrome b-562 to Ru(NH3)6(2)+ in submitochondrial particles and its inaccessibility in mitochondria suggest the localization of the hemoprotein redox center on the inner surface of the mitochondrial membrane.

Acta Pathol Microbiol Immunol Scand {B}, 1984 Jun, 92(3), 177 - 9
The Limulus Amoebocyte Lysate (LAL) assay for the detection of endotoxin in fat emulsions for total parenteral nutrition (TPN); Paulssen J et al.; Fat emulsions spiked with Escherichia coli endotoxin were tested with Limulus Amoebocyte Lysate (LAL) reagent, using both the micro method and the tube method . The effect of vortexing, ultrasonication and addition of Pyrosperse on the dispersion of endotoxin in these emulsions was compared . The advantage of using the tube method, rather than the micro method, is shown . Ultrasonication may substitute vortex mixing . Pyrosperse had no convincing effect.

Anal Biochem, 1984 Jun, 139(2), 459 - 62
Purification of component A of the soluble methane monooxygenase of Methylococcus capsulatus (Bath) by high-pressure gel permeation chromatography; Woodland MP et al.; A major improvement in the purification of the oxygenase protein (component A) of the methane monooxygenase has been effected . By employing high-pressure gel permeation chromatography several purification steps may be omitted from the previously published scheme . Furthermore the yield of the protein is enhanced and more importantly the recovered protein displays an increased specific activity, unlike that purified by other techniques.

J Rheumatol, 1984 Jun, 11(3), 315 - 7
A comparison of serologic reactivity among SLE patients with or without anti-Ro (SS-A) antibodies; Bell DA et al.; The serum from 112 patients with systemic lupus erythematosus (SLE) was examined to compare serologic reactivity among anti-Ro positive and anti-Ro negative patients . While hypergammaglobulinemia, rheumatoid factor (RF) and elevated Clq binding were significantly more frequent among the anti-Ro positive group there was no increase in the frequency of anti-dsDNA antibody measured by the Farr assay or antibodies to ssDNA, dsDNA, poly dG . poly dC, poly (dA-dT) and cardiolipin measured by ELISA . Patients with the highest levels of anti-DNA antibody by the Farr assay did not have any increased frequency of anti-Ro antibodies . We concluded that anti-Ro and anti-DNA antibodies are independently regulated . The frequent occurrence of RF in anti-Ro positive SLE patients may provide a useful screening assay for this autoantibody among pregnant SLE patients.

Eur J Biochem, 1984 Jun 1, 141(2), 331 - 7
Prolipoprotein modification and processing in Escherichia coli . A unique secondary structure in prolipoprotein signal sequence for the recognition by glyceryl transferase; Giam CZ et al.; An Escherichia coli mutant (lpp-14-1), with an alteration of glycine to aspartic acid at the 14th amino acid residue of the prolipoprotein signal sequence, has previously been shown to contain unmodified and unprocessed prolipoprotein in its cell envelope . Both the wild-type and the lpp-14-1 alleles of the lpp gene have been cloned onto a phage lambda vector . Two pseudorevertant alleles of lpp-14-1 (14R21 and 6a) have been isolated, cloned and sequenced . Amino acid sequences, deduced from the DNA sequences of the two revertant lipoprotein alleles, and biochemical characterization of the revertant lipoproteins, show that a conversion of the aspartic acid (residue 14) to asparagine completely restores the modification and processing of the 14R21 revertant prolipoprotein, while a change of the threonine-16 to isoleucine-16 partially enhances the modification and processing of the 6a prolipoprotein, which retains the aspartate-14 substitution . Secondary structure analysis of the revertant prolipoprotein signal sequences according to the Chou and Fasman rules revealed that the specific coil region in residues 14 and 15, and the beta-sheet structure in residues 16-18 of signal sequence may be important for prolipoprotein modification . These results suggest essential roles of both a unique secondary structure and hydrophobicity in residues 14-18 of prolipoprotein signal sequence for the proper recognition by the glyceryl transferase.

J S Afr Vet Assoc, 1984 Jun, 55(2), 57 - 60
The number and location of air sacs in broiler chickens and the implication in Escherichia coli infection; Mitchell JR; Broiler chicken carcasses were injected with latex to determine the number and location of the air sacs and the presence of diverticula . The adverse affect of Escherichia coli air sacculitis spreading into the diverticula is discussed.

Acta Pathol Microbiol Immunol Scand {B}, 1984 Jun, 92(3), 171 - 3
Neutrophil phagocytosis of treponema denticola as indicated by extracellular release of lactoferrin; Hurlen B et al.; Lysosomal enzyme release from viable human neutrophils occurs during phagocytic activity in vivo . Phagocytosis of a strain of T . denticola, an oral spirochete, was indicated by the finding of lactoferrin in the extracellular medium of neutrophils challenged with this organism . The extracellular release of lactoferrin was dependent on duration of bacterial challenge, but peak concentration appeared at a later stage than seen in phagocytosis experiments with Escherichia coli, which served as a control . Neutrophil phagocytosis of T . denticola may be of importance as a defence factor in periodontal disease.

J Pharmacobiodyn, 1984 Jun, 7(6), 407 - 13
A comparative study on 1-nitropyrene and nitrofurazone reductases in Escherichia coli; Narai N et al.; 1-Nitropyrene and nitrofurazone reductases in Escherichia coli B/r were studied comparatively . Nitrofurazone reductase activity was oxygen-insensitive, whereas 1-nitropyrene reductase activity was markedly inhibited by oxygen in both intact cells and cell-free preparations . The former activity depended upon reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate, whereas the latter activity upon flavin-adenine dinucleotide (FAD) as well as the reduced pyridine nucleotide . E . coli B/r acquired resistance to nitrofurazone in two mutational steps, associated with stepwise loss of the oxygen-insensitive nitrofuran reductase activity . However, 1-nitropyrene reductases were not affected at all by the mutation . These facts indicated that the major enzymes responsible for the reduction of 1-nitropyrene and nitrofurazone in E . coli B/r were different from each other . 1-Nitropyrene reductases were resolved by diethylaminoethyl-cellulose column chromatography into four enzymes all of which seem to reduce FAD, too . Among them, three enzymes appear to be able also to catalyze the reduction of nitrofurazone under anaerobic conditions.

J Gen Microbiol, 1984 Jun, 130 ( Pt 6), 1535 - 42
Tryptophanase synthesis in Escherichia coli: the role of indole replacement in supplying tryptophan and the nature of the constitutive mutation tnaR3; Edwards RM et al.; The properties of the tryptophanase constitutive mutation tnaR3 have been investigated . It has previously been reported that mutants carrying tnaR3 grow poorly on medium that selects for constitutive expression of tryptophanase . Our results now show that this poor growth can be explained by the inability of tryptophanase to catalyse the synthesis of L-tryptophan from indole, pyruvate and ammonia at a rate sufficient to allow normal growth . Improved media for the characterization of tryptophanase constitutive mutants are described . The mutation tnaR3 rendered tryptophanase synthesis constitutive (at a different rate that at 37 degrees C is 30% of the fully induced wild-type) and not further inducible . Diploid studies showed that tnaR3 is cis-dominant, but no evidence was found for any effect in trans . In addition to rendering tryptophanase synthesis constitutive, tnaR3 affects the response of tryptophanase synthesis to different growth temperatures.

J Gen Microbiol, 1984 Jun, 130 ( Pt 6), 1409 - 18
Electrophoretic analysis of proteins from Mycoplasma capricolum and related serotypes using extracts from intact cells and from minicells containing cloned mycoplasma DNA; Andersen H et al.; The acidic proteins of six different mycoplasma serotypes causing bovine or caprine pleuropneumonia were compared by two-dimensional gel electrophoresis of extracts of 35S-labelled cells . The organisms investigated were Mycoplasma mycoides subsp . mycoides (PG1), M . mycoides subsp . mycoides (Y-goat), M . mycoides subsp . capri (PG3), M . capricolum (California kid), the unclassified bovine serogroup 7 of Leach (PG50) and the F38-like group (F38) . The results suggested a close relationship between M . capricolum and F38 and a similarly close relationship between the different M . mycoides subspecies, whereas the two M . mycoides subspecies appeared to be quite distant from M . capricolum and F38 . The representative strain of the bovine serogroup 7 of Leach was equally distant from F38, M . capricolum and the three strains of M . mycoides . Strikingly, all six mycoplasma strains apparently shared six proteins in the two-dimensional gels . In Escherichia coli minicells, DNA from strain PG50 cloned in the vector pBR325 gave rise to incorporation of radioactive label into proteins which were identified as mycoplasma proteins by two-dimensional electrophoresis and immunoprecipitation.

J Gen Microbiol, 1984 Jun, 130 ( Pt 6), 1391 - 8
Origin and fate of the lysophosphatidylethanolamine in a chain-forming mutant (envC) of Escherichia coli; Michel GP et al.; The role of phospholipid metabolism in the functioning of the bacterial envelope was investigated in the chain-forming Escherichia coli envC . Lysophosphatidylethanolamine (LPE) which accumulated in this strain during growth was identified as the product of phosphatidylethanolamine (PE) hydrolysis by a phospholipase A1, i.e . 2-acylLPE . Isotopically labelled LPE transferred into intact mutant and parent cells by liposome/bacteria interaction was rapidly reacylated to PE . However, in envC the final PE/LPE ratio was lower than that in the parent, thus showing that the fate of LPE is modified . Crude cell extracts degraded LPE to a lesser extent in envC than in the parent but were unable to promote reacylation activity under our experimental conditions . In both strains, the lysophospholipase activity was neither calcium-dependent nor inhibited by the SH-group inhibitors pHMB or pCMPS, and hydrolysed 1-acylLPE as well as 2-acylLPE . These results indicate the existence of a deacylation-reacylation cycle in E . coli and show that this cycle is perturbed in envC cells, especially at the lysophospholipase step.

J Gen Microbiol, 1984 Jun, 130 ( Pt 6), 1311 - 4
Use of homocysteic acid for selecting mutants at the gltS locus of Escherichia coli K12; Essenberg RC; L-Homocysteic acid is toxic to Escherichia coli K12 . Sensitivity to this compound is higher in cells which can utilize glutamate as sole carbon source via the Na+-dependent glutamate transport system . Such cells become resistant by mutation at the gltS locus . Sensitivity of both wild-type and glutamate-utilizing strains is greater if cells are growing on acetate as compared with glucose as major carbon source.

Can J Biochem Cell Biol, 1984 Jun, 62(6), 507 - 15
A highly thermostable proline:tRNA ligase from Thermus aquaticus . Purification and enzyme-tRNA recognition at elevated temperatures; Rivera JA et al.; Proline:tRNA ligase from Thermus aquaticus was purified to homogeneity and characterized . Its molecular weight was found to be 127 000, consisting of two identical subunits . It catalysed the prolylation of tRNAPro from Escherichia coli with a bell-shaped pH dependence peaking at about pH 7 and exhibited extreme thermostability . The Vm/Km ratios of steady-state kinetics for proline and ATP as well as tRNAPro were not extensively diminished even at 85 degrees C, but prolylation became insignificant at 90 degrees C . Since the melting of tRNAPro was in progress, yet incomplete, at 85 degrees C, these findings suggest that some threshold level of conformational integrity of tRNAPro, rather than the entire unmelted conformation of the molecule, is essential to effective recognition by the proline:tRNA ligase.

Biochem Genet, 1984 Jun, 22(5-6), 467 - 82
Isolation of unselected mutants of alkaline phosphatase in Escherichia coli through nitrosoguanidine comutation and comparison with natural variants; del Castillo F et al.; We have devised a general procedure to isolate enzymatic variants without selecting or screening for related phenotypic peculiarities of the organism . A high mutation rate at phoA, the structural gene for alkaline phosphatase, is found among N-methyl-N'-nitro-N-nitrosoguanidine-induced proC revertants of Escherichia coli . About 1.6% of such revertants lack alkaline phosphatase, and many others exhibit altered enzyme parameters . Three mutants studied in detail had full enzyme activity but differed from the wild type in electrophoretic mobility, thermostability, and, in one case, optimum pH for enzyme activity . Four other phosphatase variants were discovered in a survey of 50 natural E . coli isolates; their electrophoretic mobility and thermostability were different from those of the wild type . Natural and induced enzyme variants are similar enough to suggest the absence of strong selective pressures in natural populations.

J Dairy Sci, 1984 Jun, 67(6), 1306 - 15
Reduced set of phages for typing Escherichia coli; Gershman M et al.; A phage typing set composed of 32 phages is described for differentiating Escherichia coli . Eight hundred sixty-six isolates from cases of bovine mastitis were used in this effort . Of these cultures 829, or 96%, were characterized successfully, and 178 phage types were observed . Thirty-seven isolates were not typable.

Cancer Lett, 1984 Jun, 23(2), 159 - 65
Endotoxin-induced antitumor activity in the mouse is highly potentiated by muramyl dipeptide; Bloksma N et al.; The ability of aqueous solutions of various endotoxin preparations, muramyl dipeptide (MDP) and combinations of endotoxin and MDP, to induce necrosis and regression of subcutaneous Meth A transplants in mice and their toxicity were studied . While intravenously injected toxic endotoxins, in contrast to a detoxified preparation and to MDP, induced considerable necrosis and regression of their own, addition of MDP potentiated the antitumor potential of both toxic and detoxified endotoxins to the same high degree . Detoxified endotoxin combined with MDP, however, was far less toxic than toxic preparations alone or combined with MDP . This indicates that toxicity is not directly related to therapeutic potential.

Can J Physiol Pharmacol, 1984 Jun, 62(6), 673 - 7
Subacute endotoxemia in dogs with experimental cirrhosis and ascites: effects on kidney function; Levy M et al.; To study the effect of low-grade continuous endotoxemia in normal and cirrhotic dogs, osmotic minipumps were filled with Escherichia coli endotoxin, implanted subcutaneously and arranged so that the endotoxin could be infused intravenously over a 7-day period in doses ranging from 2.5 to 100 micrograms/h . Observations were made at 3 and 7 days postinfusion . In normal dogs (N = 9), there was no effect on cardiac output or arterial pressure when doses as high as 50 micrograms/h were delivered into the circulation . Neither was there an effect on inulin or p-aminohippurate (PAH) clearances . At doses of 100 micrograms/h, dogs suffered a marked decrement in cardiac output, blood pressure, and renal perfusion and became lethargic at 3-7 days . In cirrhotic dogs, doses of 25 micrograms/h which had no effect in the control dogs, caused a significant decline in the glomerular filtration rate (59-21.5 mL/min) and CPAH (147-66 mL/min) at a time when cardiac output and blood pressure remained normal . At doses of 50 micrograms/h, cardiac output and blood pressure declined markedly and the dogs deteriorated quickly following 3-5 days of endotoxin . When endotoxin (25 micrograms/h) was given to dogs with acute biliary obstruction (serum bilirubin = 9.8 +/- 0.1 mg/dL) or to dogs with chronic thoracic caval constriction (which produced portal hypertension and ascites), no effect was observed on either central hemodynamics or renal perfusion . The selective renal vasoconstrictor effect observed in cirrhotic dogs could not be abolished by intravenous phentolamine or propranolol, inhibitors of alpha- and beta-adrenergic activity, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Biull Eksp Biol Med, 1984 Jun, 97(6), 704 - 6
{Mobilization for transfer of the nonconjugative plasmid pAP/57Hly by different conjugative plasmids}; Gigani OB et al.; The ability for mobilization of E . coli pAP57Hly nonconjugative plasmid which codes for beta-hemolysis production was investigated . It was shown that mobilization of pAP57Hly nonconjugative plasmid does not depend on the incompatibility groups, pili nature, host range of transmissible activity of conjugative plasmids . The data obtained exclude the mechanism of mobilization in which the cointegrative structures are formed.

J Hyg (Lond), 1984 Jun, 92(3), 377 - 84
Distribution of multilocus genotypes of Escherichia coli within and between host families; Caugant DA et al.; Isolates from the intestinal Escherichia coli flora of 28 members of five families (including parents, children, and household pets) in Amherst, Massachusetts, and Rochester, New York, were characterized by the electrophoretic mobilities of 12 enzymes to estimate the extent of sharing of strains among associated and unassociated hosts . Among the 655 isolates examined, 60 different combinations of electromorphs (electrophoretic types or ETs), each representing a distinctive multilocus genotype, were identified, of which 85% were recovered from only a single individual . On average, 11% of the ETs isolated from the same family were shared by two or more members; 4.9% of ETs were shared among members of unassociated families living in the same city; and only 2% were shared by families in different cities . All three ETs that were recovered from multiple hosts in the present study are widespread clones that have been isolated from many other host individuals in North America and Sweden.

Eur J Biochem, 1984 Jun 1, 141(2), 361 - 74
Nucleotide sequence of the sucB gene encoding the dihydrolipoamide succinyltransferase of Escherichia coli K12 and homology with the corresponding acetyltransferase; Spencer ME et al.; The nucleotide sequence of the sucB gene, which encodes the dihydrolipoamide succinyltransferase component (E2o) of the 2-oxoglutarate dehydrogenase complex of Escherichia coli K12, has been determined by the dideoxy chain-termination method . The results extend by 1440 base pairs the previously reported sequence of 3180 base pairs, containing the sucA gene . The sucB structural gene comprises 1209 base pairs (403 codons excluding the initiating AUG), and it is preceded by a 14-base-pair intercistronic region containing a good ribosomal binding site . The absence of a typical terminator sequence and the presence of an IS-like sequence downstream of sucB suggest that there may be further gene(s) in the suc operon . The IS-like sequence is homologous with other intercistronic sequences including that between the sdhB and sucA genes, the overall gene organisation being: sdhB-IS-sucAsucB-IS- . The patterns of codon usage indicate that sucB may be more strongly expressed than sucA, consistent with the disproportionate contents of their products in the oxoglutarate dehydrogenase complex . The predicted amino acid composition and Mr (43 607) of the succinyltransferase component agree with previous studies on the purified protein . Comparison with the corresponding acetyltransferase component of the pyruvate dehydrogenase complex (E2p, aceF gene product) indicates that each contains two analogous domains, an amino-terminal lipoyl domain linked to a carboxy-terminal catalytic and subunit binding domain . The lipoyl domain of the acetyltransferase (E2p) comprises three tandemly repeated approximately 100-residue lipoyl binding regions containing two short (approximately 19 residues) internal repeats, whereas the lipoyl domain of the succinyltransferase (E2o) contains just one approximately 100-residue lipoyl binding region, with approximately 27% homology to each of the three comparable regions in E2p, and no detectable internal repeats . The catalytic and subunit binding domains, each approximately 300 residues, have an overall homology of 34% and, consistent with their combination of analogous and specific functions, some regions are more homologous than others . Both sequences feature segments rich in proline and alanine . In E2p these occur at the carboxy-terminal ends of each of the three lipoyl binding regions, there being a particularly extended sequence at the end of the third repeat, whereas in E2o the main proline-alanine segment is found approximately 50 residues into the subunit binding domain.(ABSTRACT TRUNCATED AT 400 WORDS)

Eur J Biochem, 1984 Jun 1, 141(2), 351 - 9
Nucleotide sequence of the sucA gene encoding the 2-oxoglutarate dehydrogenase of Escherichia coli K12; Darlison MG et al.; The nucleotide sequence of a 3180-base-pair segment of DNA, containing the sucA gene encoding the 2-oxoglutarate dehydrogenase component (E1o) of the 2-oxoglutarate dehydrogenase complex of Escherichia coli, has been determined by the dideoxy chain-termination method . The sucA structural gene contains 2796 base pairs (932 codons, excluding the initiation codon AUG) and encodes a polypeptide having a glutamine residue at the amino terminus, a glutamate residue at the carboxy-terminus and a calculated Mr = 104905 . The predicted amino acid composition is in good agreement with published information obtained by hydrolysis of the purified enzyme . There is a striking lack of sequence homology between the 2-oxoglutarate dehydrogenase (E1o) and the corresponding pyruvate dehydrogenase (E1p), which suggests that the two components are not closely related in evolutionary terms . The location and polarity of the sucA gene, relative to the restriction map of the corresponding segment of DNA, are consistent with it being the proximal gene of the suc operon, as defined in previous genetic and post-infection labelling studies, but it could also form part of a more complex regulatory unit . The sucA gene is preceded by a segment of DNA that contains many substantial regions of hyphenated dyad symmetry including an IS-like sequence of the type that is thought to function as an intercistronic regulatory element . This segment also contains three putative RNA polymerase binding sites and a good ribosome binding site.

Circ Res, 1984 Jun, 54(6), 623 - 35
Interactions of granulocytes with the lungs; Brigham KL et al.; Under normal conditions, there is a sizeable pool of marginated granulocytes in the lung circulation which is in dynamic equilibrium with the circulating granulocyte pool . The number of granulocytes in the lungs' microcirculation may depend on pulmonary blood flow or biochemical interactions between granulocytes and pulmonary vascular endothelium, or both . There is some evidence that normal lung function may be affected by granulocytes . Several acute and chronic diseases may result, at least in part, from interactions of granulocytes with the lungs . Acute diffuse lung injury (adult respiratory distress syndrome) is characterized by diffuse pulmonary inflammation, and, in animal models, some of the lung dysfunction depends on the presence of granulocytes . Bronchoconstriction and airway hyperreactivity, characteristic of asthma, may be influenced by granulocyte-generated products of arachidonic acid . Granulocyte-derived proteases and oxidants may contribute to the pathogenesis of pulmonary emphysema and may affect connective tissue synthesis in interstitial pulmonary fibrosis . There is some evidence suggesting a connection between granulocytes and chronic pulmonary hypertension . The fact that some interventions which cause pulmonary leukostasis do not cause severe, persistent lung injury indicates that as yet unknown factors may determine whether interactions of granulocytes with the lungs are benign or pathological . Such factors could include the generation of humoral substances, and metabolites of arachidonic acid are particularly interesting in this regard . Research related to interactions of granulocytes with the lungs suggests strongly that such interactions are integral to the pathogenesis of several lung diseases . Understanding those diseases will require further basic studies of granulocyte behavior and the modes of communication between cells intrinsic to the lung and granulocytes.

Biochem Pharmacol, 1984 Jun 1, 33(11), 1741 - 6
Stereochemistry of the inactivation of 4-aminobutyrate: 2-oxoglutarate aminotransferase and L-glutamate 1-carboxylase by 4-aminohex-5-ynoic acid enantiomers; Danzin C et al.; Incubation of rat brain or bacterial 4-aminobutyrate aminotransferase (EC 2.6.1.19) with both (S)-(+)- and (R)-(-)-enantiomers of 4- aminohex -5- ynoic acid results in a time-dependent irreversible loss of enzymatic activity . Rat brain glutamate decarboxylase (EC 4.1.1.15) is inactivated by the (S)-(+)-enantiomer while the bacterial glutamate decarboxylase is inactivated by the (R)-(-)-enantiomer . In addition, we demonstrate that (R)-(-)-4- aminohex -5- ynoic acid is a selective and effective inhibitor of rat brain 4-aminobutyrate aminotransferase in vivo.

Gan To Kagaku Ryoho, 1984 Jun, 11(6), 1284 - 9
{Mechanisms of production of tumor necrosis factor (TNF)--reconstitution experiment white nude mice}; Watanabe N et al.; In order to investigate the role of T cells in the production of tumor necrosis factor (TNF), a reconstitution experiment was performed with nude mice (Balb/c, nu/nu) . The results obtained were as follows: The cytotoxic activity of tumor necrosis serum (TNS) from Balb/c, nu/nu mice treated with P . acnes-LPS was 1/22 against that from Balb/c, nu/+ mice . TNF activity increased 14 times in reconstituted nude mice against Balb/c, nu/nu mice . Investigation of the production of the cytotoxic activity per cell was carried out using T cell and macrophage fractions separated from the spleens of both Balb/c, nu/nu and Balb/c, nu/+ mice treated with P . acnes as a priming agent . Elicitation employing LPS was done in vitro . Cytotoxic activity released into culture medium was observed in the macrophage fraction, but not in the T cell fraction . However, no significant difference was shown in species . With P . acnes treatment, the population of macrophages in the spleens from Balb/c, nu/+ mice increased 25.5 times, whereas that from Balb/c, nu/nu mice only increased 6.8 times . The above results suggest that the mechanism of the incremental effect of T cells on TNF production was due to the promotion of macrophage proliferation during the priming period after injection of P . acnes.

Proc Natl Acad Sci U S A, 1984 Jun, 81(12), 3830 - 4
Intragenic regions required for LamB export; Benson SA et al.; Escherichia coli strains containing a series of lamB-lacZ fusions have been isolated and characterized . Each of these fusions specifies a hybrid protein with LamB sequences at the NH2 terminus and a large functional COOH-terminal fragment of beta-galactosidase . The amount of LamB present in the various hybrid proteins ranges from as few as 4 amino acids to a complete signal sequence (25 amino acids) plus 49 amino acids of the mature protein . With respect to hybrid protein export these fusions fall into three classes . Hybrid proteins with an incomplete LamB signal sequence or those that have a complete signal sequence plus 27 or fewer amino acids of the mature LamB protein are not exported and remain in the cytoplasm . In contrast, fusion strains attempt to export hybrid proteins that contain a complete signal sequence plus 39 or 43 amino acids of mature LamB . However, these proteins are not localized to the outer membrane . Finally, a hybrid protein that is slightly larger, containing 49 amino acids of mature LamB, is found in the outer membrane in appreciable amounts . These fusions, together with previously described lamB-lacZ fusions, have enabled us to define more precisely the minimal amount of lamB required to initiate the process of protein export . Moreover, they genetically locate a signal that appears to guide LamB to the outer membrane . This signal is within a region of amino acid homology shared by other major outer membrane proteins { Nikaido , H . & Wu, H . C . P . (1984) Proc . Natl . Acad . Sci . USA 81, 1048-1052}.

Proc Natl Acad Sci U S A, 1984 Jun, 81(12), 3821 - 4
Removal of UV light-induced pyrimidine-pyrimidone(6-4) products from Escherichia coli DNA requires the uvrA, uvrB, and urvC gene products; Franklin WA et al.; Ultraviolet light induces the formation of cyclobutane pyrimidine dimers and pyrimidine- pyrimidone (6-4) photoproducts in cellular DNA . In Escherichia coli, the uvrA, uvrB, and uvrC genes are necessary for excision of cyclobutane dimers . To determine whether the uvrABC gene products are required for (6-4) product removal from DNA, a sensitive HPLC assay was developed that allows the separation and quantitation of both types of photoproducts . Both the T T cyclobutane dimer and the T-C(6-4) product were completely removed from the DNA after 2 hr of repair in a wild-type strain . Both products were also removed in the wild-type strain in the presence of chloramphenicol, an inhibitor of protein synthesis . No decrease in the amount of either T T cyclobutane dimer or of T-C(6-4) products was observed in strains that were deficient in any one of the three uvr gene products under similar conditions . We conclude the uvrABC enzyme complex is required for excision of (6-4) photoproducts from E . coli DNA.

Proc Natl Acad Sci U S A, 1984 Jun, 81(12), 3708 - 12
Nucleotide sequence of the Escherichia coli prolipoprotein signal peptidase (lsp) gene; Innis MA et al.; The nucleotide sequence of the prolipoprotein signal peptidase (lsp) gene has been determined . The lsp gene was found to be adjacent to the isoleucyl-tRNA synthetase ( ileS ) gene, such that the termination codon of the ileS gene overlaps with the initiation codon of lsp . These two genes are transcribed in the same direction and the major promotor for the lsp gene appears to be upstream of ileS . Identification of the lsp gene was established by amplification of prolipoprotein signal peptidase activity in strains carrying a subcloned 1.1-kilobase Stu I-Acc I fragment and was further confirmed by introducing mutational alterations in the COOH terminus of the protein that caused a decrease in prolipoprotein signal peptidase activity . The deduced amino acid sequence indicates that prolipoprotein signal peptidase contains 164 residues . Unlike most exported proteins, there is no apparent signal peptide sequence for the lsp protein . Computer-assisted secondary structure analysis of the deduced amino acid sequence identified four hydrophobic regions that share features common to transmembrane segments in integral membrane proteins.

Proc Natl Acad Sci U S A, 1984 Jun, 81(12), 3690 - 4
Identification and expression in Escherichia coli of merozoite stage-specific genes of the human malarial parasite Plasmodium falciparum; McGarvey MJ et al.; The key steps in the development of a malaria vaccine through gene cloning are the identification of the proteins involved in host protective immunity and the cloning, identification, and expression of the genes coding for these proteins . Recent data have indicated that certain proteins synthesized at the late schizont-merozoite stage of Plasmodium falciparum play a major role in malaria immunity . This paper reports the identification, in a cDNA library, of recombinant clones corresponding to genes expressed specifically during the late schizont-merozoite stage of P . falciparum development . The 132 cDNA clones thus identified out of 10,000 were found to correspond to only 12 different genes, probably representing most of the major schizont-merozoite specific genes . The stage-specific cDNAs can be efficiently expressed in Escherichia coli cells . The protein products of some of these clones are recognized by monoclonal antibodies specific for late schizont-merozoite proteins . We conclude that only a small set of genes is specifically induced in the schizont-merozoite stage and that the stage-specific cDNA clones we have isolated are very likely to include the genes coding for the immunologically relevant proteins of P . falciparum.

Proc Natl Acad Sci U S A, 1984 Jun, 81(11), 3332 - 6
Copper metallothionein of yeast, structure of the gene, and regulation of expression; Butt TR et al.; Addition of copper to yeast cells leads to the induction of a low molecular weight, cysteine-rich protein that binds copper . This protein, termed copper chelatin or thionein, is related to the metallothionein family of proteins that are induced in response to cadmium and zinc in vertebrate cells . We have determined the structure of the yeast copper-binding protein by DNA sequence analysis of the gene . Although the 6573-dalton yeast protein is substantially divergent from vertebrate metallothioneins, the arrangement of 12 cysteine residues, which is a hallmark of metal-binding proteins, is partially conserved . We analyzed the regulatory DNA sequence of the gene by fusing it with the Escherichia coli galactokinase gene and assaying the levels of enzyme activity in yeast in response to copper . The transcriptional activation has a specific requirement for copper . Zinc, cadmium, and gold were unable to regulate the galactokinase activity . The yeast copper metallothionein regulatory sequences represent a previously unreported class of yeast promoter that is regulated by copper.

Proc Natl Acad Sci U S A, 1984 Jun, 81(11), 3287 - 91
Structure of the Trg protein: Homologies with and differences from other sensory transducers of Escherichia coli; Bollinger J et al.; Transducer proteins are central to chemotaxis in Escherichia coli . Three transducer genes comprise a homologous gene family, while a fourth gene, trg, is more distantly related . We have determined the nucleotide sequence of trg . The deduced sequence of the Trg protein has features in common with other transducers as well as regions of significant divergence . The protein sequence suggests the same transmembrane structure postulated for other transducers: an extra cytoplasmic NH2-terminal domain connected by a membrane-spanning region to an intracellular COOH-terminal domain . The COOH-terminal domain of Trg exhibits substantial sequence identity with the corresponding domains of the other transducers, particularly near the sites of covalent modification . Trg appears to have the same five methyl-accepting sites identified in the Tsr protein . Two of those sites are glutamines that are deamidated to yield methyl-accepting glutamates, while the remainder are synthesized as glutamates . Conservation in number but not in position of modified glutamines in Trg compared to the other transducers is consistent with the notion that uncharged glutamines at a specific number of modification sites serve to balance the signaling state of newly synthesized transducers . The NH2-terminal domain of Trg exhibits no significant homology with other transducers, implying that trg may be a fusion of the common COOH-terminal transducer sequence with an unrelated NH2-terminal sequence . The location of specific mutations within trg provides support for the suggestion that ligand-binding sites are in the NH2-terminal domains.

Proc Natl Acad Sci U S A, 1984 Jun, 81(11), 3273 - 7
Generation of antibody activity from immunoglobulin polypeptide chains produced in Escherichia coli; Cabilly S et al.; Plasmids have been constructed that direct the synthesis in Escherichia coli of heavy chains and/or light chains of an anti-carcinoembryonic antigen (CEA) antibody . Another plasmid was constructed for expression of a truncated form of heavy chain (Fd' fragment) in E . coli . Functional CEA-binding activity was obtained by in vitro reconstitution in E . coli extracts of heavy chain or Fd' fragment mixed with extracts containing light chain.

J Surg Res, 1984 Jun, 36(6), 625 - 30
The pulmonary effects of opiate blockade in septic shock; Mamazza J et al.; Sepsis remains the most common associated factor in acute respiratory failure (ARF) . Endogenous opiates are known to have both respiratory and cardiovascular depressant effects . Because there is a high level of circulating endogenous opiates in sepsis, the possible role of opioids in the ARF syndrome seen in sepsis was studied . Sixteen piglets were infused with an LD100 dose (7.5 X 10(10) organisms/kg) of live Escherichia coli (Type 09-41) . The pigs were hemodynamically monitored . Serial blood samples were taken for arterial blood gases and lactate . Serial lung biopsies were taken for determining wet/dry lung weight ratios and for histology . Group I (n = 8): septic shock controls without naloxone; group II (n = 8): naloxone treated, given as 2 mg/kg/hr intravenous boluses, starting within 1 min of E . coli infusion . All animals died of septic shock . Survivors at 150 min in group II had a higher blood pressure than group I (67.7 +/- 5.33 SEM vs 39.0 +/- 9.39) and cardiac output was also greater (1.07 +/- 0.23 vs 0.25 +/- 0.25) . By 210 min, group I had no survivors (0/8) while 3/8 in group II survived . Pulmonary vascular resistance in group II at 90 and 120 min (870.8 +/- 274.1 and 942.5 +/- 12.9, respectively) was lower than in group I (2868.3 +/- 843.6 and 4156 +/- 1067) . The PO2 was markedly better in group II and at 90 min; controls had a PO2 70.7 +/- 13.0, while group II had a PO2 111.4 +/- 8.4 (P less than 0.05) . PCO2 levels showed a progressive rise in group I from 39.25 +/- 1.4 to 49.4 +/- 8.57.(ABSTRACT TRUNCATED AT 250 WORDS)

J Clin Pathol, 1984 Jun, 37(6), 645 - 50
Localisation of enteropathogens in paraffin embedded tissue by immunoperoxidase; Parsons KR et al.; An indirect immunoperoxidase technique has been used to identify enteropathogens in formol-sublimate fixed paraffin embedded sections of calf intestine . Infections with bovine rotavirus, bovine coronavirus, Newbury agent SRV -1, and K99+ Escherichia coli have been detected in the intestines from experimentally infected and conventially reared diarrhoeic or normal calves . The ability to visualize enteropathogenic agents in histological sections resulted in the demonstration of virus infected cells at sites not previously shown to be infected using the immunofluorescence technique.

J Bacteriol, 1984 Jun, 158(3), 769 - 76
Quantities of individual aminoacyl-tRNA families and their turnover in Escherichia coli; Jakubowski H et al.; The cellular content of all 20 aminoacyl-tRNA species was determined in small cultures of Escherichia coli by labeling cells with 3H-amino acids and extraction of 3H-amino acid-labeled nucleic acid by standard procedures . Of 3H-amino acid-labeled material, 25 to 90% was identified as 3H-aminoacyl-tRNA by the following criteria: sensitivity to base hydrolysis with expected kinetics; association of 3H counts released by base treatment of the 3H-amino acid-labeled nucleic acid with amino acid standards upon paper chromatography of the hydrolysate; and changes in the amount of 3H-amino acid-labeled nucleic acid recovered from cells as a function of time . Individual aminoacyl-tRNA content was determined with as few as 8 X 10(7) to 4 X 10(8) E . coli cells . Although the total number of aminoacyl-tRNA molecules per cell varied only by 10 to 20% among various strains of E . coli, some individual aminoacyl-tRNA families varied two- to threefold among strains . For a given amino acid, the number of aminoacyl-tRNA molecules per cell in E . coli strain K38 growing with a doubling time of 60 min varied from 730 (glutamyl-tRNA) to 7,910 (valyl-tRNA) with a mean value of 3,200 . The total number of aminoacyl-tRNA molecules per cell (6.4 X 10(4)) in E . coli K38 was 5.5-fold higher than the number of ribosomes and was equal to 84% of the amount of elongation factor Tu molecules per cell . The ratio of aminoacyl-tRNA to synthetase for 10 amino acids varied from about 1 to 15 with a mean value of 4.7 . The turnover of individual aminoacyl-tRNA families in E . coli cells was estimated to be in the range of 1.7 to 8.1 s-1 with a mean value of 3.7 s-1 . An estimate of minimum in vivo molecular activity of aminoacyl-tRNA synthetases gives values of 2 to 48 s-1 for individual enzymes.

J Bacteriol, 1984 Jun, 158(3), 1191 - 4
Immunological analysis of the heme proteins present in aerobically grown Escherichia coli; Kranz RG et al.; Immunological methods were used to obtain information about Escherichia coli heme proteins . There is a membrane-bound catalase which consists of a single subunit (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis) which is also present in the soluble fraction . Antibodies raised against purified, soluble cytochrome b562 showed that this cytochrome is not related to any of the membrane-bound cytochromes, including the b562 component of the cytochrome o complex . Cytochrome b556 is immunologically unrelated to the cytochrome b556 NR associated with the nitrate reductase system . Cytochrome b556 and cytochrome o are not present in a constant ratio in the membrane.

Infect Immun, 1984 Jun, 44(3), 599 - 608
Ultrastructural study of adherence to and penetration of cultured cells by two invasive Escherichia coli strains isolated from infants with enteritis; Knutton S et al.; The adherence of invasive Escherichia coli strains 444-3 and 469-3 to human erythrocytes and to cultured HeLa and HEp-2 cells has been examined by electron microscopy . Bacteria elaborating type 1 fimbriae, glycocalyces , and nonfimbrial mannose-resistant hemagglutinins specific for human erythrocytes were identified in cultures of both strains, and each of these different bacterial surface components appeared to be involved in attachment of 444-3 and 469-3 to cultured epithelial cells or human erythrocytes (or to both) . Both strains, which were isolated from infants with dysentery-like illness, penetrated cultured epithelial cells and existed within membrane-bounded intracellular vesicles . Mutants of 444-3 and 469-3 selected for deficiency in mannose-resistant hemagglutination did not adhere to or penetrate cultured cells . These ultrastructural studies demonstrate the complexity of the bacterial surface and show that E . coli strains 444-3 and 469-3 can elaborate several different adhesions , each of which could function to promote attachment to host intestinal epithelial cells . Mucosal invasion may also be an important virulence property of these strains.

Infect Immun, 1984 Jun, 44(3), 592 - 8
Characterization of nonfimbrial mannose-resistant protein hemagglutinins of two Escherichia coli strains isolated from infants with enteritis; Williams PH et al.; Escherichia coli strains 444-3 and 469-3, isolated from patients with severe infantile enteritis, are able to adhere to and penetrate human epithelial cells in culture . In addition to type 1 fimbriae and glycocalyces , both strains elaborate mannose-resistant nonfimbrial protein hemagglutinins specific for human erythrocytes . Purified agglutinins are aggregates (greater than 4 X 10(6) daltons) of a single protein subunit of apparent Mr 14,000 (469-3) to 14,500 (444-3) . The optimal temperature for expression of the agglutinins is 37 degrees C . Bacteria grown at 22 degrees C, which show 1% or less of maximal activity, and mutants deficient in the ability to agglutinate human erythrocytes do not synthesize detectable levels of these surface proteins and, moreover, do not adhere to cultured epithelial cells . Coupled with the observation that purified agglutinins competitively inhibit bacterial adherence to cultured cells, these data indicate that the nonfimbrial surface proteins expressed by strains 444-3 and 469-3 are essential for adherence both to erythrocytes and to cultured epithelial cells.

Cell, 1984 Jun, 37(2), 675 - 82
Specificity of mutagenesis resulting from the induction of the SOS system in the absence of mutagenic treatment; Miller JH et al.; Strains in which the E . coli SOS system is continuously induced, in the absence of mutagenic treatment, have been used to generate nonsense mutations in the lacl gene . The examination of over 600 independently occurring amber and ochre mutations reveals that inducing the SOS system stimulates specifically G:C----T:A and, to a lesser extent, A:T----T:A transversions . This specificity is similar to that seen for a number of carcinogens that form bulky adducts to DNA, such as benzo(a)pyrene diolepoxide (BPDE) and aflatoxin B1 (AFB1), and which are dependent on the SOS system to mutagenize bacteria . However, G:C----T:A transversions resulting from SOS induction alone display a unique site specificity . One possibility is that the SOS-induced mutations result from cryptic, spontaneous lesions, such as apurinic sites, which cannot induce the SOS system themselves but which can target mutations once the SOS system is induced.

Am J Physiol, 1984 Jun, 246(6 Pt 2), R915 - 21
Common origin of linguistic and movement abilities; Bellman KL et al.; We start with the view that the development of systems of symbols is rooted in the regulation of cellular processes and the behavior of unicellular animals . Animals would thereafter start to externalize these internal symbol systems, to coordinate movements with each other . We propose that the brains of multicellular animals can be understood as a continuing elaboration of the early chemical symbol systems of unicellular animals: the labile symbols of the unicellular animal are replaced by hormones, more stable chemical compounds, and nerves that are seen as more stable and more specific routes of activation; and brains developed layers of symbols such that the domain of a symbol is not a set of bodily processes but rather a set of brain processes . Human language is very much in the "style" of the rule-governed symbol manipulation required by all behaving animals, although unique in its complexity . We suggest that the essential question is not how humans have evolved symbolic and linguistic abilities from a primitive sensorimotor brain but rather how do symbols come to exist in biological systems and what is useful and necessary about a system of symbols for the coordination of action within animals and among animals.

Mol Cell Biol, 1984 Jun, 4(6), 1169 - 71
Enhanced transformation of human cells by UV-irradiated pSV2 plasmids; Spivak G et al.; Irradiating the plasmid pSV2-gpt with UV (254 nm) doses up to 200 J m-2 caused a dose-dependent increase in the yield of Gpt+ transformants when the plasmid was introduced into human cells by calcium phosphate coprecipitation . UV doses greater than 1 kJ m-2 were required to reduce the efficiency of transformation below that obtained with unirradiated DNA.

Mol Cell Biol, 1984 Jun, 4(6), 1020 - 34
Model for homologous recombination during transfer of DNA into mouse L cells: role for DNA ends in the recombination process; Lin FL et al.; We have constructed phage lambda and plasmid DNA substrates (lambda tk2 and ptk2) that contain two defective herpesvirus thymidine kinase (tk) genes that can be used to detect homologous recombination during the transfer of DNA into mouse L cells deficient in thymidine kinase activity . The recombination event reconstructs a wild-type tk gene and is scored because it converts Tk- cells to Tk+ . Using this system, we have shown that (i) both intramolecular and intermolecular homologous recombination can be detected after gene transfer; (ii) the degree of recombination decreases with decreasing tk gene homology; and (iii) the efficiency of recombination can be stimulated 10- to 100-fold by cutting the tk2 DNA with restriction enzymes at appropriate sites relative to the recombining sequences . Based on the substrate requirements for these recombination events, we propose a model to explain how recombination might occur in mammalian cells . The essential features of the model are that the cut restriction site ends are substrates for cellular exonucleases that degrade DNA strands . This process exposes complementary strands of the two defective tk genes, which then pair . Removal of unpaired DNA at the junction between the paired and unpaired regions permits a gap repair process to reconstruct an intact gene.

Eur J Biochem, 1984 Jun 1, 141(2), 409 - 14
The regulatory properties of isocitrate dehydrogenase kinase and isocitrate dehydrogenase phosphatase from Escherichia coli ML308 and the roles of these activities in the control of isocitrate dehydrogenase; Nimmo GA et al.; Isocitrate dehydrogenase kinase can use ATP but not other nucleoside triphosphates as a phosphate donor . It responds hyperbolically to both ATP and isocitrate dehydrogenase . The kinase is inhibited sigmoidally by low concentrations of DL-isocitrate and hyperbolically by ADP, AMP, NADPH, phosphoenolpyruvate and several other effectors . Isocitrate dehydrogenase phosphatase requires a nucleotide for activity; ADP and ATP are the best activators . The phosphatase responds hyperbolically to ADP or ATP, to Mg2+ ions and to phosphorylated isocitrate dehydrogenase . The phosphatase is activated twofold to threefold by AMP, oxaloacetate, pyruvate, phosphoenolpyruvate, 2-oxoglutarate and DL-isocitrate . It is inhibited hyperbolically by NADPH . The pH optima and the Km values for substrates of the kinase and the phosphatase are reported . We propose that the role of the phosphorylation of isocitrate dehydrogenase during growth of Escherichia coli on acetate is to render this enzyme rate-limiting in the citric acid cycle; this should cause an increase in the level of isocitrate and divert the flux of carbon through the glyoxylate bypass . We suggest that the phosphorylation state of isocitrate dehydrogenase in intact cells is controlled by the levels of isocitrate, phosphoenolpyruvate, NADPH and the adenine nucleotides . This theory can explain many recent observations on the control of the activity of isocitrate dehydrogenase.

Eur J Biochem, 1984 Jun 1, 141(2), 401 - 8
Partial purification and properties of isocitrate dehydrogenase kinase/phosphatase from Escherichia coli ML308; Nimmo GA et al.; Isocitrate dehydrogenase kinase and isocitrate dehydrogenase phosphatase were purified over 1000-fold from Escherichia coli ML308 by procedure involving fractionation with (NH4)2SO4 and chromatography on DEAE-cellulose, blue-dextran-Sepharose and Sephadex G150 . The kinase and phosphatase activities copurified, in agreement with the observation {Laporte, D.C . and Koshland, D.E . (1982) Nature (Lond.) 300, 458-460} that a single protein bears both activities . Isocitrate dehydrogenase kinase catalysed the phosphorylation of homogeneous active isocitrate dehydrogenase with a stoichiometry of just under one phosphate group incorporated per subunit . This almost completely inactivated the dehydrogenase . There was a good correlation between phosphorylation and inactivation . Analysis of a partial acid hydrolysate of phosphorylated isocitrate dehydrogenase showed that the only phosphoamino acid present was phosphoserine . Isocitrate dehydrogenase phosphatase catalysed the release of 32P from 32P-phosphorylated isocitrate dehydrogenase; it required either ADP or ATP for activity . In the presence of ADP, or ATP plus an inhibitor of the kinase, the phosphatase catalysed full reactivation of isocitrate dehydrogenase and there was a good correlation between reactivation and the release of phosphate . In the presence of ATP alone the phosphatase catalysed the release of 32P from phosphorylated isocitrate dehydrogenase but the activity of the dehydrogenase remained low, indicating that the kinase and phosphatase were active simultaneously in these conditions . The active and inactive forms of isocitrate dehydrogenase can be resolved by non-denaturing gel electrophoresis; the two forms of the enzyme were interconverted by phosphorylation and dephosphorylation in vitro . The extent of the interconversion correlated well with the changes in isocitrate dehydrogenase activity.

Proc Natl Acad Sci U S A, 1984 Jun, 81(11), 3351 - 5
Biologically active protease of foot and mouth disease virus is expressed from cloned viral cDNA in Escherichia coli; Klump W et al.; Foot and mouth disease virus O1K cDNA had been cloned in Escherichia coli . Here we report on in vitro recombination of cDNA fragments according to the cDNA restriction map and on expression of viral proteins in E . coli . Use was made of the expression vector pPLVP1 , which is known to express the virus capsid protein VP1 . Recombined cDNAs of various sizes were inserted downstream from the VP1 gene . The constructed plasmids differ from each other in the number of virus genes coding for nonstructural proteins . The effects caused by their expression in E . coli are compared . It is shown that the virus protease is expressed in E . coli as an active enzyme that recognizes the simultaneously produced virus-specific polyprotein as substrate . The virus protease gene was mapped 5'-adjacent to the virus replicase gene.

J Virol, 1984 Jun, 50(3), 822 - 31
Expression in COS-1 cells of Epstein-Barr virus nuclear antigen from a complete gene and a deleted gene; Robert MF et al.; In a previous study the BamHI-K fragment of Epstein-Barr virus DNA was shown to induce a nuclear antigen, Epstein-Barr virus nuclear antigen (EBNA), when cotransfected with the herpes simplex virus thymidine kinase gene into mouse LTK- cells . We have now inserted the BamHI-K fragment and a BamHI/HindIII subfragment, I1f , into shuttle vectors containing the origin of replication of simian virus 40 . These plasmids have been introduced into COS-1, which are monkey kidney cells transformed by an origin-defective simian virus 40 genome . This expression system permitted rapid characterization of antigens, mRNAs, and proteins related to EBNA . The same-sized EBNA protein (approximately 78,000) was made after transfection with BamHI-K (5.2 kilobase pairs {kbp}) or the I1f subfragment (2.9 kbp) . A deletion of about 600 bp occurred when the I1f fragment was propagated on the pSV2 plasmid in Escherichia coli . The deleted fragment gave rise to a smaller protein (approximately 52,000) . These data provide evidence that EBNA is encoded by the 2.9-kbp I1f and is not an induced cellular protein . Nuclear antigen and polypeptide expression occurred equally well when the Epstein-Barr virus DNA was cloned on PSV2 -gpt or pSVOd . The latter plasmid lacks sequences allowing for efficient early gene transcription as well as splicing and polyadenylation signals which are present in pSV2 . Preliminary mapping of the EBNA gene transcripts demonstrated that two mRNAs (2.9 and 2.4 kilobases {kb}) are homologous to the I1f fragment . Taken together, the data suggest that the 2.9-kbp I1f fragment contains the structural gene for EBNA synthesis . COS-1 cells will thus provide a valuable system in which to analyze functional domains of the EBNA gene.

J Bacteriol, 1984 Jun, 158(3), 934 - 42
Structure of an Escherichia coli tRNA operon containing linked genes for arginine, histidine, leucine, and proline tRNAs; Hsu LM et al.; A plasmid containing a gene for the most abundant Escherichia coli leucine isoacceptor tRNA, tRNALeu1 (anticodon CAG) was isolated from the Clarke-Carbon bank of cloned E . coli DNA . The clone contains a 12.3-kilobase DNA insert which was mapped by F' DNA hybridization analysis to the region 82 to 89 min on the chromosome . The cloned tDNALeu corresponds to the minor of two chromosomal regions containing different amounts of DNA complementary to tRNALeuCAG . Sequencing of the tDNA region revealed it to contain a multimeric transcription unit consisting of four different tRNA genes . The genes are in the arrangement 5'-leader- tRNAArgCCG -57 base pairs- tRNAHisGUG -20 base pairs- tRNALeuCAG -42 base pairs- tRNAProUGG -3' . Coordinate expression of the component tRNAs in vivo and the absence of intercistronic promoters indicated that all four tDNAs reside in the same operon . The tDNA sequence is bounded by a promoter element showing good agreement with the procaryotic consensus sequence and a GC-rich stem-loop element that corresponds to a rho-independent terminator . The promoter region contains a GC-rich sequence that agrees with a suggested consensus stringency control element and two domains possessing dyad symmetry which flank the Pribnow box and include the putative stringency control region.

J Bacteriol, 1984 Jun, 158(3), 910 - 9
Construction of a single-copy promoter vector and its use in analysis of regulation of the transposon Tn10 tetracycline resistance determinant; Bertrand KP et al.; The construction and characterization of a promoter expression vector, lambda RS205 , is described . lambda RS205 can be used for the in vitro construction of transcriptional (operon) fusions to the lacZ gene of Escherichia coli K-12 . The level of beta-galactosidase activity in lysogens of lambda RS205 fusion phages provides a quantitative measure of promoter function under single-copy conditions . The regulation of the Tn10 tetracycline resistance gene ( tetA ) and the Tn10 tet repressor gene (tetR) was examined by inserting DNA fragments that span the tetR- tetA promoter-operator region into lambda RS205 . Levels of beta-galactosidase in tetA -lacZ and tetR-lacZ fusion strains indicate that the tetA and tetR promoters are strong promoters; the tetA promoter is fourfold more active than the tetR promoter . Introduction of tetR+ plasmids into tetA -lacZ and tetR-lacZ fusion strains represses beta-galactosidase synthesis 15- to 60-fold and 6- to 15-fold, respectively . The concentration of tetracycline required to induce half-maximal beta-galactosidase synthesis in these tetR+ tet-lac strains depends on both the tetracycline resistance phenotype and the level of tetR repressor in the fusion strain . However, the induction of beta-galactosidase in isogenic tetA -lacZ and tetR-lacZ strains is coordinate . The data presented here support the current model of Tn10 tet gene organization and regulation and provide quantitative information about the regulation of tetA and tetR in vivo.

J Bacteriol, 1984 Jun, 158(3), 866 - 71
Detection and characterization of Tn2501, a transposon included within the lactose transposon Tn951; Michiels T et al.; The DNA sequence spanning coordinates 9.9 to 16.4 kilobases of the lactose transposon Tn951 ( Cornelis et al., Mol . Gen . Genet . 160:215-224, 1978) constitutes a transposable element by itself . Unlike Tn951 ( Cornelis et al., Mol . Gen . Genet . 184:241-248, 1981), this element, called Tn2501 , transposes in the absence of any other transposon . Transposition of Tn2501 proceeds through transient cointegration and duplicates 5 base pairs of host DNA . Tn2501 is flanked by nearly perfect inverted repeats (44 of 48), related to the inverted repeats of Tn21 ( Zheng et al., Nucleic Acids Res . 9:6265-6278, 1982) . Unlike Tn21 , Tn2501 does not confer mercury resistance.

J Bacteriol, 1984 Jun, 158(3), 849 - 59
Defined set of cloned termination suppressors: in vivo activity of isogenetic UAG, UAA, and UGA suppressor tRNAs; Raftery LA et al.; We have cloned an isogenetic set of UAG, UAA, and UGA suppressors . These include the Su7 -UAG, Su7 -UAA, and Su7 -UGA suppressors derived from base substitutions in the anticodon of Escherichia coli tRNATrp and also Su9 , a UGA suppressor derived from a base substitution in the D-arm of the same tRNA . These genes are cloned on high-copy-number plasmids under lac promoter control . The construction of the Su7 -UAG plasmid and the wild-type trpT plasmid have been previously described ( Yarus , et al., Proc . Natl . Acad . Sci . U.S.A . 77:5092-5097, 1980) . Su7 -UAA ( trpT177 ) is a weak suppressor which recognizes both UAA and UAG nonsense codons and probably inserts glutamine . Su7 -UGA ( trpT176 ) is a strong UGA suppressor which may insert tryptophan . Su9 ( trpT178 ) is a moderately strong UGA suppressor which also recognizes UGG (Trp) codons, and it inserts tryptophan . The construction of these plasmids is detailed within . Data on the DNA sequences of these trpT alleles and on amino acid specificity of the suppressors are presented . The efficiency of the cloned suppressors at certain nonsense mutations has been measured and is discussed with respect to the context of these codons.

J Bacteriol, 1984 Jun, 158(3), 820 - 5
Construction and characterization of an Escherichia coli strain with a uncI mutation; Gay NJ; A strain of Escherichia coli with a mutation in the promoter proximal gene ( uncI ) of the unc operon has been constructed by using a new gene replacement method . The mutation is a deletion of a defined sequence of 196 base pairs . It was constructed by homologous integration and segregation of a ColE1-derived recombinant plasmid containing the mutation, in a temperature-sensitive polA strain . The mutant strain is phenotypically unc+ but has a reduced growth yield compared to a normal sibling strain.

J Bacteriol, 1984 Jun, 158(3), 1202 - 3
Genetic basis of minicell formation in Escherichia coli K-12; Davie E et al.; Hfr- and P1-mediated genetic transfer experiments failed to confirm the presence of a " minA " gene in Escherichia coli K-12, leading to the conclusion that mutation at a single locus, the minB locus, is sufficient to cause minicell production in this species.

J Bacteriol, 1984 Jun, 158(3), 1198 - 201
Overlapping functional units in a cell division gene cluster in Escherichia coli; Sullivan NF et al.; The ftsZ ( sulB ) coding sequence is preceded by two promoters, at least one of which lies within the coding sequence of the neighboring gene, ftsA . This region of the ftsA gene is required for full biological activity of ftsZ .

J Bacteriol, 1984 Jun, 158(3), 1078 - 83
Mutations altering aspartyl-61 of the omega subunit (uncE protein) of Escherichia coli H+ -ATPase differ in effect on coupled ATP hydrolysis; Fillingame RH et al.; Mutations in the H+-translocating ATPase complex (F1F0) of Escherichia coli have been described in which aspartyl-61 of the omega subunit ( uncE protein) is substituted by either glycine ( uncE105 ) or asparagine ( uncE107 ) . Either substitution blocks the H+-translocation activity of the F0 sector of the complex . Here we report a difference in the effects of the two substitutions on the coupled ATPase activity of F1 bound to F0 . Wild-type F1 was bound to the F0 of either mutant with affinities comparable to wild-type . The ATPase activity of F1 bound to uncE107 F0 was inhibited by 50%, whereas that bound to uncE105 F0 was not inhibited . Complementation studies with a pBR322-derived plasmid that carried the E gene of the unc operon only indicated that a single mutation in the host strain was responsible for the respective phenotypes . In mutants complemented by the uncE + plasmid, restoration of wild-type biochemical properties was only partial and may be attributed to a mixing of wild-type and mutant omega subunits in a hybrid F0 complex . The activity of membrane-bound F1 was less inhibited in the uncE +/ uncE107 hybrid . Paradoxically, complementation of uncE105 by the uncE + plasmid resulted in substantial inhibition of the activity of membrane-bound F1 . The results indicate that a glycine-versus-asparagine substitution for aspartyl-61 must lead to altered conformations of omega and that these differences in conformation are important in the coupling between the F0 and F1 sectors of the complex.

J Bacteriol, 1984 Jun, 158(3), 1048 - 53
Fructose bisphosphatase of Escherichia coli: cloning of the structural gene (fbp) and preparation of a chromosomal deletion; Sedivy JM et al.; The fbp locus at 96 min on the Escherichia coli chromosome governs fructose bisphosphatase (fructose-1,6-P2 1-phosphatase) . We have cloned and subcloned fbp on vector pBR322 to obtain strains with high levels of the enzyme . In vivo mutagenesis of the clone was used to show that fbp is the structural gene . The gene was deleted on the plasmid in vitro, and the chromosomal wild-type locus was replaced with this deletion by a method involving stabilization of a heterozygous intermediate resulting from plasmid integration, followed by segregation of the wild-type gene.

J Bacteriol, 1984 Jun, 158(3), 1041 - 7
Promoter exchange between ompF and ompC, genes for osmoregulated major outer membrane proteins of Escherichia coli K-12; Matsuyama S et al.; Expression of the ompF and ompC genes coding for major outer membrane proteins OmpF and OmpC is regulated in opposite directions by medium osmolarity . Chimera genes were constructed by a reciprocal exchange of the promoter-signal sequence region between the two genes . The chimera gene construction was designed so that the proteins synthesized by these genes were essentially the same as the OmpC and OmpF proteins . Studies with the chimera genes demonstrated that the osmoregulation of the OmpF-OmpC synthesis was promoter dependent . They also showed that cells grew normally even when the osmoregulation took place in opposite directions . The effects of the ompR2 and envZ mutations, which suppress ompC and ompF expression, respectively, also became reversed . The reduced expression was still subject to the promoter-controlled osmoregulation . Based on these observations, the mechanism of regulation of the ompF-ompC gene expression and its physiological importance are discussed.

Cell, 1984 Jun, 37(2), 683 - 91
Duplex-duplex interactions catalyzed by RecA protein allow strand exchanges to pass double-strand breaks in DNA; West SC et al.; Using gapped circular DNA and homologous duplex DNA cut with restriction nucleases, we show that E . coli RecA protein promotes strand exchanges past double-strand breaks . The products of strand exchange are heteroduplex DNA molecules that contain nicks, which can be sealed by DNA ligase, thereby effecting the repair of double-strand breaks in vitro . These results show that RecA protein can promote pairing interactions between homologous DNA molecules at regions where both are duplex . Moreover, pairing leads to strand exchanges and the formation of heteroduplex DNA . In contrast, strand exchanges are unable to pass a double-strand break in the gapped substrate . This apparent paradox is discussed in terms of a model for RecA-DNA interactions in which we propose that each RecA monomer contains two nonequivalent DNA-binding sites.

Cell, 1984 Jun, 37(2), 661 - 7
Site-specific excision of integrated polyoma DNA; Sylla BS et al.; Cyp cells are permissive murine cells carrying a thermosensitive polyoma virus genome that remains integrated at 39 degrees C, but is effectively excised and replicated after transfer to 33 degrees C . In rare subclones of the Cyp line, temperature shift-down yields predominantly homogeneous populations of chimeric molecules that appear to reflect the circularization of defined segments of DNA spanning one of the junctions between the integrated viral genome and the adjacent cellular DNA . Such accurate and frequent excision requires a specific recombination mechanism . We examined both the cellular and the viral sequences that cross-over to generate one of these chimeric molecules, Rm I . The homology at the cross-over site is one of 1 or 2 base pairs at most; patches of homology, amounting in total to 19 or 20 base pairs, are found in perfect register on both sides of this site; and the two stretches of DNA that are joined to form RM I contain similar 12-14 base pair sequences (5'- CTCCTTTACAGAGG -3' and 5'- CTCCTTTCAAGG -3') in opposite orientations.

Gene, 1984 Jun, 28(3), 337 - 42
Nucleotide sequence of gene pfkB encoding the minor phosphofructokinase of Escherichia coli K-12; Daldal F; The nucleotide sequence of a 1.3-kb DNA fragment containing the entire pfkB gene which codes for Pfk-2 of Escherichia coli, a minor phosphofructokinase (Pfk) enzyme, is reported . The Pfk-2 protein subunit is encoded by 924 bp, has 308 amino acids and an Mr of 33 000 . Like other weakly expressed E . coli genes the codon usage in the pfkB gene is random; there is no strong bias for the usage of major tRNA isoaccepting species, and the codon preference rules of Grosjean and Fiers {Gene, 18 (1982) 199-209} are followed . This is the first report of the complete gene sequence of a phosphofructokinase.

Transplantation, 1984 Jun, 37(6), 556 - 61
Cellular interactions in marrow-grafted patients . II . Normal monocyte antigen-presenting and defective T-cell-proliferative functions early after grafting and during chronic graft-versus-host disease; Tsoi MS et al.; The proliferation of T cells of marrow donor origin in response to Escherichia coli, an ubiquitous antigen, presented by circulating monocytes of marrow donor origin was investigated in 30 human allogeneic marrow transplant recipients . Compared with cells from healthy marrow donors, T cell proliferation was found to be deficient in all recipients studied 36-71 days after grafting, regardless of the presence or absence of acute graft-versus-host disease and in most patients with chronic graft-versus-host disease studied 118-1804 days postgrafting . In contrast, lymphocytes from most long-term patients without chronic graft-versus-host disease studied 363-2673 days had immune reactivity comparable to that of lymphocytes from their marrow donors . Results of cell-mixing experiments showed that (1) monocytes from most marrow recipients were capable of presenting antigens to normal T cells of marrow donors, and (2) T cells from short-term patients and from long-term patients with active chronic graft-versus-host disease were not induced to proliferate by E-coli-pulsed monocytes from the marrow donors . This inability of T cells to proliferate was likely the result of ineffective interactions among T cell subsets.

Proc Natl Acad Sci U S A, 1984 Jun, 81(12), 3612 - 6
Formation of termination-resistant transcription complex at phage lambda nut locus: effects of altered translation and a ribosomal mutation; Warren F et al.; Transcription antitermination by lambda N gene product is affected in a mutant Escherichia coli with altered ribosomal protein S10, caused by the nusE71 mutation . To study the role of translation in antitermination, we have fused the phage nutR locus, the site of action of N, with the lac regulatory region . We have monitored N action by measuring galactokinase, whose synthesis depends on suppression of terminators located between nutR and the galK cistron . We show that a deletion removing potential ribosome binding signals and AUG codons from the upstream region of nut site does not affect N action . Moreover, the lack of translation upstream of nutR does not overcome the antitermination defect caused by nusE mutation . When the upstream region is translated, however, N action is impaired if translation terminates 19 base pairs upstream of nutR . Termination of translation at further upstream sites, such as 23 or 97 base pairs upstream, does not interfere with N action . Our results suggest that the S10 ribosomal protein is required for N action without involving translation . These results also suggest that the nut site RNA itself plays an important role in the formation of a termination-resistant transcription complex.

Proc Natl Acad Sci U S A, 1984 Jun, 81(11), 3447 - 51
The rnh gene is essential for growth of Escherichia coli; Kanaya S et al.; We have determined that a functional gene coding for ribonuclease H seems to be essential for cell growth in Escherichia coli . A strain was made with two copies of the rnh gene by lysogenizing an E . coli strain with a lambda phage bearing a copy of the rnh gene . Inactivation of one of the two copies of the rnh gene was accomplished by transformation with a linear DNA molecule that had the gene for chloramphenicol acetyltransferase inserted near the middle of the rnh gene . In recombinants that had an inactive gene replacing the normal chromosomal rnh gene, the lambda rnh prophage supplies an intact functional copy of the rnh gene . Curing the cells of the lambda rnh prophage left the cell with an inactive rnh gene and resulted in cell death . An intact functional rnh gene provided on a plasmid permits normal curing, and cured survivors were readily obtained . The technique described is probably generally applicable for assessing the requirement for other E . coli genes.

Proc Natl Acad Sci U S A, 1984 Jun, 81(11), 3278 - 82
DNA methylation pattern is determined by the intracellular level of the methylase; Szyf M et al.; Extrachromosomal plasmid DNA is transiently undermethylated in Escherichia coli during amplification in the presence of chloramphenicol . In addition, undermethylation of phage lambda DNA was observed after thermal induction of a lambda c1857 lysogen while the integrated lambda phage DNA was found to be fully methylated . These methylation pattern changes occur under conditions (extensive replication) in which the intracellular methylase level becomes limiting . In an E . coli strain that harbors a plasmid that carries the dam methylase gene and therefore overproduces dam methylase, there is no undermethylation of dam sites in either of the extrachromosomal DNAs . The sites that are methylated by the mec methylase in both plasmid and lambda phage DNAs were undermethylated in the dam overproducer as well . These results indicate that the intracellular level of the E . coli methylase determines the DNA methylation pattern.

J Bacteriol, 1984 Jun, 158(3), 878 - 83
Mutation prlF1 relieves the lethality associated with export of beta-galactosidase hybrid proteins in Escherichia coli; Kiino DR et al.; The 42-1 lamB-lacZ gene fusion confers a conditionally lethal, export-dependent phenotype known as maltose sensitivity . A maltose-resistant mutant showing decreased beta-galactosidase activity of the hybrid protein, designated prlF1 (protein localization), was unlinked to the lamB-lacZ fusion . This mutation mapped at 70 min on the Escherichia coli linkage map and conferred maltose resistance, a 30-fold reduction in beta-galactosidase activity, and a 30% decrease in cellular growth rate at 30 degrees C that was independent of the presence of a gene fusion . prlF1 also decreased the beta-galactosidase activity and relieved the maltose sensitivity conferred by fusions of lacZ to the gene specifying the periplasmic maltose-binding protein, malE . The decrease in beta-galactosidase activity, however, was specific for exported hybrid proteins . When export of the hybrid protein was blocked by a signal sequence mutation, prlF1 decreased the beta-galactosidase activity only 2.5-fold . Similarly, prlF1 did not affect the beta-galactosidase activity of fusions of lacZ to a gene specifying a nonexported protein, malK.

Cell Biophys, 1984 Jun, 6(2), 117 - 29
Raman spectroscopy and order in biological systems; Del Giudice E et al.; The Raman spectra in the low 5-200 cm-1 frequency region of metabolically active E . coli cells have been analyzed to determine whether they are indicators of a possible in vivo underlying order by applying standard concepts derived from the Raman spectroscopy of crystalline systems with varying degrees of order . The analysis suggests that in-vivo space-time ordered structures involving amino acids associated with DNA exist since the low frequency lines of metabolically active cells can be assigned to lines seen in the spectra of crystals of given amino acids known to associate with DNA early in the lifetime of a cell.

EMBO J, 1984 Jun, 3(6), 1365 - 9
Control of pMB1 replication: inhibition of primer formation by Rop requires RNA1; Cesareni G et al.; The rop gene participates in the control of plasmid copy number by interfering with transcripts originating from the primer promoter . Here we show that this inhibition mechanism requires RNA1 in trans . Mutations in the RNA1 coding sequence that result in plasmids with altered incompatibility properties do not affect the ability of the molecule to participate in the Rop inhibition mechanism . Furthermore we show that the target of the Rop-RNA1 inhibitory mechanism is located, at least in part, after the 52nd nucleotide of the sequence encoding the primer transcript.

EMBO J, 1984 Jun, 3(6), 1295 - 300
Amino acid sequence requirements in the epitope recognized by the alpha-tubulin-specific rat monoclonal antibody YL 1/2; Wehland J et al.; We have characterized the epitope of the rat monoclonal antibody YL 1/2 in detail using synthetic peptides and several alpha-tubulin derivatives . The epitope seems to be provided by the linear sequence spanning the carboxy-terminal residues of tyrosinated alpha-tubulin . By competitive ELISA, dipeptides covering the carboxyl end could be antigenically recognized . Three sites were deduced at the dipeptide level: a negatively charged side chain in the penultimate position followed by an aromatic residue which must carry the free carboxylate group . Experiments with longer peptides point to a further negative charge provided by a carboxylate group on the third residue from the end . Thus the tripeptide Glu-Glu-Tyr was only 5-fold less active than the octapeptide spanning the carboxy-terminal alpha-tubulin sequence . The octapeptide itself showed only a 40-fold lower activity than tyrosinated alpha-tubulin . In line with the emerging epitope requirements of YL 1/2, the Escherichia coli rec A protein, the catalytic subunit of the cyclic AMP-dependent muscle protein kinase as well as performic acid-oxidized actin were recognized by YL 1/2 in immunoblots . These results thus define the sequence requirements within a probably linear epitope and give rise to some general questions concerning experiments where monoclonal antibodies are microinjected into cells in order to assess the contribution of a known antigen to cellular physiology.

J Med Microbiol, 1984 Jun, 17(3), 311 - 5
Rapid detection of bacteraemia by early subculture; Ganguli LA et al.; Inspection and blind subculture was carried out on 7031 consecutive blood cultures at 10 p.m . on the day they were received . Analysis of the results after a 2-year period showed that 119 out of 237 (50%) bacteraemic patients were detected at this stage . Preliminary sensitivity tests were done at this time and their results were available within 24 h of the blood cultures being received . Thus earlier specific therapy was possible in 50% of the cases of bacteraemia.

J Gen Microbiol, 1984 Jun, 130 ( Pt 6), 1489 - 502
Characteristics and function of thick and thin conjugative pili determined by transfer-derepressed plasmids of incompatibility groups I1, I2, I5, B, K and Z; Bradley DE; Eleven transfer-derepressed plasmids from incompatibility groups I1, I5, B, K and Z were constructed using the dnaG3 mutant Escherichia coli strain BW86 . All were found to determine thin flexible and thick rigid pili constitutively . Immune electron microscopy was used to relate thick and thin pilus serotypes with incompatibility grouping . Mutant plasmids that determined only thick pili constitutively transferred efficiently on an agar surface but not in a liquid, whereas plasmids with both kinds of pili transferred equally well in both environments . A mutant of the IncI2 plasmid R721 determined thin pili constitutively, and thick pili at a repressed level, as indicated by electron microscopy . Experiments with this indicated that thin pili were apparently not involved directly in conjugation but were only used to stabilize mating aggregates.

Bioorg Khim, 1984 Jun, 10(6), 824 - 43
{Phage T7 RNA-polymerase: gene cloning and its structure}; Grachev MA et al.; Gene 1.0 to T7 phage coding for a phage-specific RNA polymerase has been cloned in a plasmid vector . One of the clones obtained, viz . E . coli K12 HB101 (pSK-T7-1.0a), produced an active T7 RNA polymerase as revealed by the fact that its extract stimulated RNA synthesis on the T7 DNA template in the presence of rifampicin . Analysis of gene 1.0 fragments isolated from pSK-T7-1.0a plasmid made it possible to refine the sequence of this gene which has been recently published by the authors of the present paper and, independently, by other workers . Another plasmid, pSK-T7-1.0b, did not differ from pSK-T7-1.0a in the restriction map . However, it failed to induce production of a rifampicin-insensitive RNA polymerase . Sequencing revealed a deletion of one T residue in gene 1.0 present in this plasmid.

Arch Microbiol, 1984 Jun, 138(2), 106 - 12
Two-dimensional electrophoretic analysis of the regulation of SOS proteins in three ssb mutants; Johnson BF; A previously undescribed mutation in the ssb gene, which codes for a major single strand DNA binding protein essential for DNA replication, was mapped on the Escherichia coli chromosome . Three ssb mutants were analyzed under parallel physiological conditions for the induction of SOS proteins (products of recA, uvrA, and an unknown gene), the production of mutants, the induction of lambda prophage, and sensitivity to DNA damaging agents . Two-dimensional electrophoretic techniques were used to quantitate changes in the rate of synthesis of proteins . The previously unpublished position of the uvrA gene-product in the two-dimensional matrix of E . coli proteins was described . These ssb strains exhibited varying sensitivities to ultraviolet irradiation and methylmethane sulfonate that correlated with the rate of constitutive synthesis of SOS proteins, spontaneous commitment to virulent growth of lambda lysogens, and elevation of endogenous mutation rates.

J Biochem (Tokyo), 1984 Jun, 95(6), 1645 - 53
Temperature dependence of action of polymyxin B on Escherichia coli; Katsu T et al.; The temperature dependence of the action of polymyxin B on Escherichia coli was studied by using K+, Ca2+, and tetraphenylphosphonium (TPP+) ion-selective electrodes . At room temperature (27 degrees C), Ca2+ was released immediately after addition of polymyxin, while the efflux of K+ occurred after 30 s . The rapid release of Ca2+ was not affected by incubation temperature, while the efflux of K+ was significantly lowered at temperatures below about 25-30 degrees C . The uptake of TPP+ also increased after polymyxin addition . The release of Ca2+ and the uptake of TPP+ supported the disruption of the outer membrane structure reported previously . In experiments with isolated membrane vesicles (the cytoplasmic membrane being exposed), the efflux of K+ was not delayed, but was lowered at temperatures below about 15-20 degrees C . This temperature range differed significantly from that of whole cells, and was interpreted as representing a difference in membrane fluidity between the outer and cytoplasmic membranes . The phase transition temperature of the outer membrane is known to be higher than that of the cytoplasmic membrane; and the temperature dependence of efflux of K+ from membrane vesicles was compatible with the phase transition temperature of liposomes prepared with phospholipids (not containing lipopolysaccharides) extracted from E . coli . Thus, it was speculated that, with whole cells, polymyxin molecules passed through the outer membrane at temperatures above the phase transition and reached the cytoplasmic membrane, increasing its K+ permeability . The mechanism of the permeability change is discussed in terms of deformation of the cytoplasmic membrane structure induced by polymyxin molecules.

Zh Mikrobiol Epidemiol Immunobiol, 1984 Jun, (6), 50 - 5
{Growth and bioenergetics characteristics of a producer of EcoK restriction endonuclease under batch cultivation}; Gruber IM et al.; The conditions necessary for the controlled single- and multicycle process of the batch cultivation of E . coli CK, capable of producing E . coli CK specific endonuclease, have been established . This process can be regulated with respect to a number of parameters (temperature, pH, pO2, eH) . The possibility of using thermodynamic characteristics, calculated on the basis of the redox potential and disclosing the energetics of growth, for evaluating the effectiveness of controlled batch cultivation . The optimum results have been obtained during the isolation of E . coli CK restriction endonuclease, active and containing no admixture of other endonucleases, at the period from the maximum specific growth rate to the end of the exponential growth phase, i.e . to the beginning of the stationary phase.

Gene, 1984 Jun, 28(3), 311 - 8
Vectors with restriction-site banks . I . pJRD158, a 3903-bp plasmid containing 28 unique cloning sites; Davison J et al.; A DNA fragment has been constructed that contains many unique cloning sites not present in currently used Escherichia coli plasmid cloning vehicles . Insertion of this fragment into a modified version of pBR322 results in an AmpRTetR vector (pJRD158) of 3903 bp containing 28 unique cloning sites, four "almost unique" cloning sites, and eight unassigned unique 6-bp palindromes . The plasmid has the additional advantages of very high copy number and altered incompatibility . The latter permits it to be stably maintained in the same host as pBR322.

EMBO J, 1984 Jun, 3(6), 1377 - 82
Transcription of an artificial ribosomal RNA gene in yeast; Kempers-Veenstra AE et al.; We constructed an artificial yeast rRNA gene and studied its transcription after introduction into a recipient yeast strain . The artificial gene comprised a fragment containing the sequence from position -207 to +128 relative to the site of initiation of Saccharomyces carlsbergensis 37S pre-rRNA, followed by a marker fragment from Spirodela oligorhiza chloroplast DNA and finally a fragment containing the sequence from position -36 to +101 relative to the 3' end of the 26S rRNA gene . The resulting construct was cloned into the yeast-Escherichia coli shuttle vector pJDB207 . Both Northern blot hybridization and R-loop analysis of RNA from transformed Saccharomyces cerevisiae cells revealed a discrete transcript of the expected length . S1 nuclease mapping as well as primer extension analysis showed that the major proportion of the transcripts was initiated at exactly the same site as 37S pre-rRNA . These results show that the respective rDNA fragments contain the information for correct initiation of transcription and formation of the 3' end . A minor proportion of the transcripts was initiated at a number of sites between positions -1 and -100 upstream of the predominant start . The proportion and the pattern of these upstream starts is affected by the vector context of the artificial rRNA gene.

DNA, 1984 Jun, 3(3), 259 - 68
Restriction site bank vectors . II . DNA sequence analysis of plasmid pJRD158; Heusterspreute M et al.; pJRD158 is a small plasmid vector (3903 bp) derived from pBR327 and specifying resistance to ampicillin and tetracycline . It contains 28 unique restriction sites (and 4 nonunique restriction sites) that can be used for cloning . The DNA sequence and computer-assisted restriction site analysis of pJRD158 are reported . Evidence is also presented that suggests a 2-bp revision of the DNA sequence of pBR322 in the RNA primer region.

Mol Biol Med, 1984 Jun, 2(3), 207 - 21
The ring-infected erythrocyte surface antigen (RESA) polypeptide of Plasmodium falciparum contains two separate blocks of tandem repeats encoding antigenic epitopes that are naturally immunogenic in man; Cowman AF et al.; We showed previously that the ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum contains a repetitive amino acid sequence . We have investigated here the sequence and antigenic relationships between RESA from FC27, a Papua New Guinea isolate, and from NF7, a Ghanaian isolate . The complete nucleotide sequences of eight different cDNA clones demonstrate that RESA from the two strains are closely homologous over the region that can be compared . A series of related eight, four and three amino acid repeats is located at the 3' end, forming the C terminus of RESA in FC27 . RESA contains a second block of repeats based on an 11 amino acid sequence and separated from the 3' block by 381 amino acids in NF7 . Antibodies from Papua New Guineans react with RESA from the African isolate, and vice-versa . Antigenic determinants that are naturally immunogenic in man are present in the 5' repeats of RESA, as well as in the 3' repeats, and antibodies that cross-react with both blocks of repeats were detected by reacting affinity-purified human antibodies with cloned subfragments of the cDNA clones, expressed in Escherichia coli.

Biochem Biophys Res Commun, 1984 May 31, 121(1), 55 - 61
The mitogenic principle of Escherichia coli lipoprotein: B-lymphocyte mitogenicity of the synthetic analogue palmitoyl-tetrapeptide (Pam-Ser-Ser-Asn-Ala); Bessler WG et al.; N-Palmitoyl-(S)-seryl-(S)-seryl-(S)-asparaginyl-(S)-alanine (Palmitoyl-tetrapeptide) is an analogue of the N-terminal part of the lipoprotein from the outer membrane of Escherichia coli . It was prepared by chemical synthesis and tested for biological activity in in vitro lymphocyte culture systems . In spleen cells of the inbred mouse strains C3H/HeJ, C3H/He/Bom/ nunu , and Balb/c, the compound exhibited stimulatory activity towards B-lymphocytes comparable to the effect of native lipoprotein, as measured by the incorporation of 3H-thymidine and 3H-uridine, and by a hemolytic plaque assay . The B-lymphocyte tumor cell line BCL1 was also activated by the compound . The results demonstrate, that the N-terminal tetrapeptide moiety of lipoprotein, linked to a lipophilic molecule, constitutes by itself a novel B-lymphocyte mitogen.

Biochem Biophys Res Commun, 1984 May 31, 121(1), 34 - 40
Affinity engineering of maltoporin: variants with enhanced affinity for particular ligands; Clune A et al.; Affinity-chromatographic selection on immobilized starch was used to selectively enhance the affinity of the maltodextrin-specific pore protein ( maltoporin , LamB protein, or lambda receptor protein) in the outer membrane of E . coli . Selection strategies were established for rare bacteria in large populations producing maltoporin variants with enhanced affinities for both starch and maltose, for starch but not maltose and for maltose but not starch . Three classes of lamB mutants with up to eight-fold increase in affinity for particular ligands were isolated . These mutants provide a unique range of modifications in the specificity of a transport protein.

Biochem Biophys Res Commun, 1984 May 31, 121(1), 41 - 6
Nucleotide sequence of the promoter region of the melibiose operon of Escherichia coli; Shimamoto T et al.; The nucleotide sequence of the promoter region of the melibiose operon of E . coli was determined . Consensus sequences for the -35 region, the Pribnow box and the binding site for cyclic AMP receptor protein were found in this region . The possible secondary structure of this DNA region was very similar to that of the promoter region of the lactose operon . A possible initiation ATG preceded by a Shine-Dalgarno sequence with proper spacing was present just downstream of the promoter region . The possible sequence of 52 amino acid residues in the NH2 terminus of the alpha-galactosidase were determined.

Biochem Biophys Res Commun, 1984 May 31, 121(1), 331 - 9
Structural asymmetry of the F1 of Escherichia coli as indicated by reaction with dicyclohexylcarbodiimide; Lotscher HR et al.; Dicyclohexylcarbodiimide (DCCD) inhibits the ATPase activity of F1 from Escherichia coli by covalent modification of a single glutamic acid in the beta subunit . 95% inhibition was obtained after incorporation of around 1 mole of DCCD per mole F1, i.e . 1 mole of reagent per 3 beta subunits; and up to 2 moles of DCCD per mole F1 were readily incorporated into the protein . One of the 3 beta subunits per F1 can be crosslinked to the epsilon subunit by 1-ethyl-3-{3(dimethylamino)propyl}carbodiimide (EDC) . This beta subunit (beta 1) is here shown to be shielded from reaction with DCCD, presumably by its association with epsilon and also possibly the gamma subunit . Thus the three beta subunits are not equivalent in the enzyme complex.

J Mol Biol, 1984 May 25, 175(3), 403 - 8
Identification of the Escherichia coli cya gene product as authentic adenylate cyclase; Danchin A et al.; The Escherichia coli cya gene has been fused in the same register with the lacZ gene . The corresponding hybrid cya-lacZ gene is expressed as a bifunctional protein that exhibits both adenylate cyclase and beta-galactosidase activities, thus proving that cya is the structural gene for adenylate cyclase . The hybrid protein was purified to homogeneity and has been used to raise antibodies that recognize wild-type adenylate cyclase . Finally, the protein has been submitted to amino acid sequence analysis . It has been found that the first ten amino acids fit the predicted sequence obtained from DNA sequence analysis, thus substantiating the prediction that the cya translation initiation codon is UUG .

J Biol Chem, 1984 May 25, 259(10), 6334 - 9
Analysis of fluorescence quenching of ribosome-bound virginiamycin S; Di Giambattista M et al.; The two virginiamycin components VM and VS interact synergistically with bacterial ribosomes in vitro and in vivo . Ribosome affinity for virginiamycin S increases about 10-fold upon incubation with virginiamycin M . This effect has been previously traced by spectrofluorimetric measurement based on the enhancement of virginiamycin S fluorescence upon its binding to the 50 S ribosomal subunit . In the present work the action of two virginiamycin S fluorescence quenchers, acrylamide and iodide, has been explored to gather information about the accessibility of ribosome-bound virginiamycin S and the variation of the accessibility level in the presence of virginiamycin M . Both acrylamide (non-ionized quencher) and iodide (ionized quencher) proved powerful quenchers of free virginiamycin S solutions . Since a comparable effect was obtained on 3- hydroxypicolinamide , the latter was indicated as the part of the molecule involved in the fluorescence effect . Fluorescence quenching by either agent was of the dynamic, i.e . collisional, type . Such an inference was based on the fact that these quenchers merely modified the emission spectrum (not the absorption spectrum), the bimolecular rate constant for the quenching process decreased linearly with the viscosity of the medium (static-type quenching is viscosity-independent), and that linear Stern-Volmer plots were obtained . The quenching ability of both agents underwent a sharp decrease in the presence of ribosomes; however, the Stern-Volmer equation was followed only in the case of acrylamide, whereas Lehrer 's relationship had to be applied in the case of iodide . When ribosomes were incubated with virginiamycin M, the fluorescence quenching ability of acrylamide and iodide was significantly reduced . Conclusions are as follows: a) the 3- hydroxypicolinyl residue of virginiamycin S is buried within an open well on the ribosome surface and is likely to be involved in the interaction with the binding site; b) the accessibility to the well is partly controlled by electrostatic forces; c) interaction of ribosomes with virginiamycin M entails a conformational change whereby the access to the well is reduced . These findings provide a molecular explanation for the previously observed increase of the association constant of virginiamycin S to ribosomes incubated with virginiamycin M which was found to be due to the decrease of the dissociation rate constant (the association rate constant remains practically the same).

J Biol Chem, 1984 May 25, 259(10), 6195 - 200
Effects of replacing serine and threonine residues within the signal peptide on the secretion of the major outer membrane lipoprotein of Escherichia coli; Vlasuk GP et al.; We have investigated the importance of serine and threonine residues within the signal peptide in the secretion and processing of the major outer membrane lipoprotein precursor prolipoprotein in Escherichia coli . This was accomplished by systematically replacing these residues with alanine utilizing oligodeoxyribonucleotide-directed mutagenesis . The results demonstrated that the replacement of serine 15 but not threonine 16 alone caused an initial accumulation of membrane-bound unmodified prolipoprotein . In addition, replacement of both serine 15 and threonine 16 resulted in a greater accumulation of this membrane-bound precursor . The accumulated prolipoprotein could be matured to lipoprotein in a quantitative manner, and this process was inhibited by globomycin and carbonyl cyanide m-chlorophenylhydrazone . These results will be discussed in terms of the contribution that serine and threonine have in determining the overall secondary structure of the signal peptide and its importance in secretion and/or processing.

J Mol Biol, 1984 May 25, 175(3), 395 - 401
Further sequence analysis of the phage lambda receptor site . Possible implications for the organization of the lamB protein in Escherichia coli K12; Charbit A et al.; We present the DNA sequence alterations due to seven lamB missense mutations yielding resistance to phages lambda and K10 . They reveal five different amino acid positions in the LamB protein . Three positions (245, 247 and 249) define a new region required for phage adsorption . The two other positions (148 and 152) belong to a region where mutations to phage resistance has already been detected . These two regions are hydrophilic and could belong to turns of the protein located at the surface of the cell . All the missense mutational alterations to phage resistance sequenced in the LamB protein correspond to 10 sites located in four different segments of the polypeptide chain . We discuss their location in terms of the notion of phage receptor site and of a working model for the organization of this protein in the outer membrane of Escherichia coli.

J Mol Biol, 1984 May 25, 175(3), 299 - 312
Genetic analysis of the tryptophan operon regulatory region using site-directed mutagenesis; Kolter R et al.; The regulatory region of the tryptophan operon of Escherichia coli was subjected to in vitro site-directed mutagenesis using sodium bisulfite as the mutagen . The mutagenized DNA was used to transform cells to drug resistance, and plasmid DNAs from individual transformants were isolated and sequenced . Overall, 22% of the plasmids sequenced contained alterations within the region of interest . Many of the mutants obtained had characteristics that bear on aspects of the alternative secondary structure model of attenuation . Expression analyses with several of the mutants provided evidence suggesting that ribosome dissociation at the leader peptide stop codon may be rapid and that this dissociation is responsible for setting the steady-state level of expression observed in cultures growing in the presence of excess tryptophan . One mutation altered the amino acid composition of the leader peptide without affecting a transcript secondary structure . The behavior of this mutant supports the prediction that the leader peptide per se plays no role in attenuation, rather it is the act of synthesis of the peptide that has regulatory significance . Several of the mutations provide information on the structure of the RNA antiterminator . Additional mutations support the conclusion that the last stem and loop structure in the terminated transcript, structure 3:4, is sufficient to cause transcription termination.

J Biol Chem, 1984 May 25, 259(10), 6593 - 600
Cruciform transitions in DNA; Sinden RR et al.; The rates of transition between the cruciform and linear conformations of a perfectly inverted repeated lac operator DNA sequence have been measured using a trimethylpsoralen intrastrand cross-linking assay . The rate and extent of the linear to cruciform transition were dependent on temperature and on the superhelical density of the DNA . Apparent half-lives for this transition were between 4-9 min at 37 degrees C for supercoiled DNAs as isolated from cells . The half-life for the cruciform to linear transition in relaxed DNA was about 30 s at 37 degrees C . Mg2+ stabilized both conformations but stabilized the linear form to a greater degree than the cruciform . The rates of transition were temperature dependent suggesting enthalpies of activation of 26.3 kcal mol-1 for the cruciform to linear transition and 33.4 kcal mol-1 for the linear to cruciform transition . The rate of the linear to cruciform transition was slower at 50 than 37 degrees C . Heating above 70 degrees C resulted in the loss of the cruciform structure.

J Biol Chem, 1984 May 25, 259(10), 6559 - 63
The binding site for ribosomal protein complex L8 within 23 s ribosomal RNA of Escherichia coli; Beauclerk AA et al.; The ribosomal protein complex L8 of Escherichia coli consists of two dimers of protein L7/L12 and one monomer of protein L10 . This pentameric complex and ribosomal protein L11 bind in mutually cooperative fashion to 23 S rRNA and protect specific fragments of the latter from digestion with ribonuclease T1 . Oligonucleotides protected either by the L8 complex alone or by the complex plus protein L11 were isolated from such digests and shown to rebind specifically to these proteins . They were also subjected to nucleotide sequence analysis . The longest oligonucleotide, protected by the L8 complex alone, consisted of residues 1028-1124 of 23 S rRNA and included all the other RNA fragments produced in this study . Previously, protein L11 had been shown to protect residues 1052-1112 of 23 S rRNA . It is concluded that the binding sites for the L8 protein complex and for protein L11 are immediately adjacent within 23 S rRNA of E . coli.

J Biol Chem, 1984 May 25, 259(10), 6188 - 94
Turnover of fatty acids in the 1-position of phosphatidylethanolamine in Escherichia coli; Rock CO; Phosphatidylethanolamine is the major membrane phospholipid of Escherichia coli, and two experimental approaches were used to investigate the metabolic activity of the fatty acids occupying the 1-position of this phospholipid . {3H}Acetate pulse-chase experiments with logarithmically growing cells indicated that 3-5% of the acyl groups were removed from the phosphatidylethanolamine pool/generation . The reacylation aspect of the turnover cycle was demonstrated by the incorporation of fatty acids into the 1-position of pre-existing phosphatidylethanolamine when de novo phospholipid biosynthesis was inhibited using the plsB acyltransferase mutant . 2- Acylglycerophosphoethanolamine would be the intermediate in a 1-position turnover cycle, and this lysophospholipid was identified as a membrane component that could re-esterified by a membrane-bound acyltransferase . The acyltransferase either utilized acyl-acyl carrier protein directly as an acyl donor or activated fatty acids for acyl transfer in the presence of ATP and Mg2+ . Acyl-acyl carrier protein was also indicated as an intermediate in the latter reacylation reaction by the complete inhibition of phosphatidylethanolamine formation from fatty acids by acyl carrier protein-specific antibodies and by the observation that the inhibition of the acyltransferase by LiCl was reversed by the addition of acyl carrier protein . Coenzyme A thioesters were not substrates for this acyltransferase . These results suggest the existence of a metabolic cycle for the utilization of 1-position acyl moieties of phosphatidylethanolamine followed by the resynthesis of this membrane phospholipid from 2- acylglycerophosphoethanolamine by an acyl carrier protein-dependent 1-position acyltransferase.

J Biol Chem, 1984 May 25, 259(10), 6142 - 6
Calcium efflux from Escherichia coli . Evidence for two systems; Ambudkar SV et al.; Calcium transport into everted membrane vesicles of Escherichia coli was found to have two components, one phosphate-dependent and the other phosphate-independent . In vesicles prepared in a glycerol buffer, calcium/proton exchange was phosphate independent but net uptake of 45Ca2+ required phosphate . In vesicles prepared in a sucrose buffer both phosphate-independent and phosphate-dependent accumulation of 45Ca2+ occurred . Both calcium/proton exchange and phosphate-independent uptake of 45Ca2+ were inactivated by treatment with trypsin, chymotrypsin, or N,N'-dicyclohexylcarbodiimide but not by N-ethylmaleimide . Phosphate-dependent uptake of 45Ca2+ was inhibited by N-ethylmaleimide but not by trypsin, chymotrypsin, or N,N'-dicyclohexylcarbodiimide . Under conditions permitting only phosphate-dependent uptake of 45Ca2+, concomitant uptake of 32Pi was also observed . Both uptake and efflux of the two ions occurred with a 1:1 ratio . These results imply the existence of two calcium transport systems in everted membrane vesicles, one of which catalyzes exchange of calcium ions and protons, the other of which catalyzes cotransport of calcium and phosphate.

J Biol Chem, 1984 May 25, 259(10), 6098 - 104
Studies on the modification and processing of prolipoprotein in Escherichia coli . Effects of structural alterations in prolipoprotein on its maturation in wild type and lpp mutants; Tokunaga H et al.; We have previously shown that an Escherichia coli mutant ( mlpA allele) containing a structurally altered murein prolipoprotein due to substitution of Gly14 by Asp14 , is globomycin resistant . In addition, the mutant prolipoprotein is not modified with glyceride and consequently remains uncleaved . Spontaneous revertants possessing a mature lipoprotein of apparent normal structure can be isolated by EDTA selection . Three revertants were chosen in the present study which included the analysis of kinetics of lipoprotein maturation and the determination of globomycin sensitivity . These pseudorevertants in the lpp gene which could be recognized by the anomalous prolipoprotein mobility in sodium dodecyl sulfate gels, exhibited altered globomycin sensitivity in vivo . Our results indicate that alterations in prolipoprotein structure affect the kinetics of prolipoprotein modification and processing reactions, both in vivo and in vitro . Pulse-chase experiments revealed the transient existence of unmodified prolipoprotein and modified prolipoprotein as biosynthetic intermediates of mature lipoprotein . The rate of prolipoprotein modification appeared to be slightly faster than that of processing in the wild type cell . In contrast, modification of prolipoprotein was rate limiting in a pseudorevertant strain 14R21 , and the processing of 14R21 modified prolipoprotein appeared to proceed more rapidly than that of wild type prolipoprotein, both in vitro and in vivo.

Nucleic Acids Res, 1984 May 25, 12(10), 4191 - 206
Functional analysis of the steroid hormone control region of mouse mammary tumor virus; Lee F et al.; Gene fusions between the mouse mammary tumor virus long terminal repeat and the E . coli lacZ gene have been shown to exhibit hormone dependent expression of beta-galactosidase activity . These constructions were used in transient expression experiments to assess the effects of specific modifications introduced into the region upstream of the transcription initiation site . 5' deletions demonstrate that sequences sufficient for wild-type promoter function are contained downstream of residue -64 relative to the initiation site . Other deletions define a region of approximately 80 base pairs between -220 and -140 which contains sequences essential for hormonal control . Between this control region and the promoter lie sequences dispensable for both functions.

J Mol Biol, 1984 May 25, 175(3), 331 - 48
A 37 X 10(3) molecular weight plasmid-encoded protein is required for replication and copy number control in the plasmid pSC101 and its temperature-sensitive derivative pHS1; Armstrong KA et al.; Nucleotide sequences were determined for a region essential for autonomous replication and partitioning of pSC101, a plasmid whose replication is dependent on the Escherichia coli dnaA gene product . The essential replication region contains one long coding sequence, rep101 , for a protein composed of 316 amino acids, and a polypeptide approximately 37 X 10(3) Mr in size was identified as the rep101 gene product . rep101 is preceded by two inverted repeat sequences, three directly repeated sequences and a region of high A + T content containing a sequence similar to the E . coli oriC consensus sequence . Because the lesions in seven replication-deficient insertion mutants, four mutants with increased copy number and one temperature-sensitive replication mutant occur within rep101 , the rep101 gene product must control pSC101 replication and copy number . par, a region adjacent to the replication region, which functions in stable plasmid inheritance, contains several inverted repeat sequences.

J Mol Biol, 1984 May 25, 175(3), 263 - 84
Role of the F factor oriV1 region in recA-independent illegitimate recombination . Stable replicon fusions of the F derivative pOX38 and pBR322-related plasmids; O'Connor MB et al.; We have used a mating protocol to isolate recA-independent recombinants of pOX38 , an F factor derivative, and the non-conjugative plasmid pMBO311 . Plasmid pMBO311 is a derivative of pBR322 carrying a DNA insertion that contains IS121 and shows no extensive sequence homology to pOX38 . Twenty-seven cointegrate molecules formed during independent mobilizations of pMBO311 by pOX38 were examined by restriction and Southern hybridization analysis . In general, there were two classes of recombinants . A minority class appears to have been mediated by IS121 , resulting in the formation of cointegrate molecules containing IS121 at the junctions between the two plasmids . The majority class (23/27) apparently involved reciprocal recombination between sites on pOX38 and pMBO311 . IS121 does not seem to be responsible for the formation of this type of cointegrate molecule, since similar structures were generated at approximately the same frequency during mobilization of control plasmids that do not contain IS121 . We have localized the regions involved in this second class of recombination events and find that most (17/23) occur at or near oriV1 , the primary replication initiation site of pOX38 . Twelve of the cointegrate molecules showed identical restriction and Southern hybridization patterns demonstrating a preferred region on pMBO311 as well . This site was localized just distal to the tet genes, within a 640-base AvaI-PvuII segment in the pBR322 portion of the molecule.

J Biol Chem, 1984 May 25, 259(10), 6659 - 66
Overproduction in Escherichia coli K-12 and purification of the TraJ protein encoded by the conjugative plasmid F; Cuozzo M et al.; The TraJ protein encoded by the conjugative plasmid F has been designated an Escherichia coli K-12 envelope protein that participates in a mechanism of gene regulation . We have purified the TraJ protein, using an Flac::lambda traJ lysogen that overproduces the protein after heat induction of the prophage . Sufficient TraJ protein was synthesized within 40 min after induction to follow the purification by sodium dodecyl sulfate/polyacrylamide gel electrophoresis . Our purification exploited the observation that the TraJ protein remains insoluble after repeated Triton X-100/EDTA extractions of crude envelope fractions . The protein was then solubilized in sodium dodecyl sulfate at 60 degrees C and fractionated further by gel filtration and hydroxylapatite chromatography, both in the presence of sodium dodecyl sulfate . After hydroxylapatite chromatography, the protein was greater than 95% pure . The identity of the purified protein was confirmed by analysis of its NH2-terminal amino acid sequence, which was the same as that predicted from the partial nucleotide sequence of the traJ gene (Thompson, R., and Taylor, L . (1982) Mol . Gen . Genet . 188, 513-518) . This analysis also indicated that the TraJ protein is localized in the cell without proteolytic modification of its NH2-terminus . We discuss the possible significance of these observations with respect to the cellular functions of the TraJ protein.

J Biol Chem, 1984 May 25, 259(10), 6311 - 7
Chemical synthesis of a biologically active gene for human immune interferon-gamma . Prospect for site-specific mutagenesis and structure-function studies; Jay E et al.; A 453-base pair DNA duplex consisting of a gene coding for human interferon-gamma and initiation and termination signals plus appropriate restriction enzyme sites for plasmid insertion has been totally synthesized . The synthesis involved preparation of 66 oligodeoxynucleotides by a modified, solid phase phosphite procedure and enzymatic ligation of the oligonucleotides . The gene, when inserted into a previously constructed expression vector, was expressed in Escherichia coli, demonstrating functional activity for the synthetic gene . Several strategically located restriction cleavage sites have been introduced into the sequence . This provides a convenient system for site-specific mutagenesis for structure-function studies.

J Biol Chem, 1984 May 25, 259(10), 6261 - 6
Nucleotide sequence reveals overlap between T4 phage genes encoding dihydrofolate reductase and thymidylate synthase; Purohit S et al.; We have determined the nucleotide sequence of a 1075-base-pair HindIII fragment of the T4 phage genome . This fragment contains the structural gene (frd) for dihydrofolate reductase and part of the gene (td) encoding thymidylate synthase . The fragment contains a 579-base-pair open reading frame, encoding a 193-residue polypeptide with a calculated mass of 21,603 Da, in agreement with our reported subunit molecular mass of 23,000 . The deduced amino acid sequence shows partial homology with other dihydrofolate reductases, with most of the identities lying in regions known to be involved in substrate binding and catalysis . The 3' end of the coding strand overlaps the coding region for thymidylate synthase; the sequence - ATGA -includes an opal terminator for the frd gene and an initiating triplet for the td gene . The deduced amino acid sequence from this initiating ATG is identical, for the first 20 residues, with the NH2-terminal 20 residues reported for the td protein (M . Belfort , A . Moelleken , G . F . Maley , and F . Maley (1983) J . Biol . Chem . 258, 2045-2051) . The sequenced HindIII fragment was transferred into a high expression plasmid vector for large scale production of homogeneous T4 dihydrofolate reductase . The experimentally determined sequence of 20 residues at the NH2-terminus of this protein is identical with that deduced from the nucleotide sequence for T4 dihydrofolate reductase.

J Biol Chem, 1984 May 25, 259(10), 6147 - 52
Kinetic mechanism of phosphofructokinase-2 from Escherichia coli . A mutant enzyme with a different mechanism; Campos G et al.; The kinetic mechanisms of Escherichia coli phosphofructokinase-2 (Pfk-2) and of the mutant enzyme Pfk-2 were investigated . Initial velocity studies showed that both enzymes have a sequential kinetic mechanism, indicating that both substrates must bind to the enzyme before any products are released . For Pfk-2, the product inhibition kinetics was as follows: fructose-1,6-P2 was a competitive inhibitor versus fructose-6-P at two ATP concentrations (0.1 and 0.4 mM), and noncompetitive versus ATP . The other product inhibition patterns, ADP versus either ATP or fructose-6-P were noncompetitive . Dead-end inhibition studies with an ATP analogue, adenylyl imidodiphosphate, showed uncompetitive inhibition when fructose-6-P was the varied substrate . For Pfk-2, the product inhibition studies revealed that ADP was a competitive inhibitor versus ATP at two fructose-6-P concentrations (0.05 and 0.5 mM), and noncompetitive versus fructose-6-P . The other product, fructose-1, 6-P2, showed noncompetitive inhibition versus both substrates, ATP and fructose-6-P . Sorbitol-6-P, a dead-end inhibitor, exhibited competitive inhibition versus fructose-6-P and uncompetitive versus ATP . These results are in accordance with an Ordered Bi Bi reaction mechanism for both enzymes . In the case of Pfk-2, fructose-6-P would be the first substrate to bind to the enzyme, and fructose-1,6-P2 the last product to be released . For Pfk-2, ATP would be the first substrate to bind to the enzyme, and APD the last product to be released.

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