Infect Immun, 1984 Feb, 43(2), 678 - 83 Effect of the estrous cycle on uterine infection induced by Escherichia coli; Nishikawa Y et al.; Escherichia coli was inoculated into the uterine lumen of rats and rabbits at different estrous stages; one uterine horn of each animal was ligated at the cervical end . In rats, a large number of E . coli were retained in the ligated horns regardless of the estrous stage . E . coli inoculated at diestrus or pseudopregnancy induced purulent endometritis, but when inoculated at proestrus-estrus the organism caused asymptomatic infection . In nonligated horns, few E . coli were recovered, and marked histopathological changes were not observed . Large numbers of E . coli were retained in the nonligated horn at proestrus as a result of physiological constriction of the cervix . E . coli inoculated at proestrus never caused purulent endometritis in either the ligated horn or the nonligated horn . In rabbits, E . coli infused into ligated horns brought about purulent inflammation irrespective of ovarian states . The number of recoverable E . coli was reduced rapidly at the follicular phase as compared with the luteal phase . These results suggest that the stage of the estrous cycle when animals are inoculated with E . coli influences the course of the uterine infection. Infect Immun, 1984 Feb, 43(2), 491 - 6 Role of granulocytes and monocytes in experimental Escherichia coli endocarditis; Meddens MJ et al.; The role of granulocytes and monocytes during the induction and course of Escherichia coli endocarditis was investigated in rabbits by selectively depleting monocytes from the circulation with the drug VP16-213 and granulocytes and monocytes with nitrogen mustard . For induction, the number of E . coli needed to infect the vegetations in 50% of the rabbits was significantly lower in rabbits with combined granulocytopenia and monocytopenia than in those with selective monocytopenia or in control rabbits, whereas the number of E . coli needed to infect 50% of the rabbits did not differ between the latter two . During the course of the endocarditis in endocardial vegetations, the numbers of CFU per gram of vegetation were significantly higher in the rabbits with combined granulocytopenia and monocytopenia than in the monocytopenic and control rabbits but did not differ between the latter two . The numbers of granulocytes in the circulation and the numbers of CFU per gram of vegetation showed a significant negative correlation that was not measurably influenced by the duration of the disease but was dependent on the number of E . coli injected for the induction of endocarditis . Granulocytes were found to be most effective at the lowest numbers of bacteria injected . In the circulation, too, the numbers of CFU per milliliter were significantly higher in rabbits with combined granulocytopenia and monocytopenia than in those with selective monocytopenia and control rabbits, and there was a significant negative correlation between the numbers of granulocytes and CFU per milliliter of blood . From these findings we conclude that granulocytes play a protective role during the induction and course of E . coli endocarditis in rabbits, whereas no role is demonstrable for monocytes. Eur J Biochem, 1984 Feb 1, 138(3), 543 - 9 Studies on reconstituted partially purified glycerophosphate acyltransferase from Escherichia coli; Kessels JM et al.; Solubilized glycerophosphate acyltransferase from Escherichia coli was reconstituted in small unilamellar vesicles consisting of phosphatidylcholine/phosphatidylglycerol in a molar ratio of 4:1 . Glycerol 3-phosphate, trapped inside these vesicles, cannot be acylated by the enzyme upon addition of extra-vesicular palmitoyl-CoA . Thus, substrate-binding sites and active sites are asymmetrically oriented in the model membrane . When up to 10 mol/100 mol lysophosphatidic acid was incorporated in the vesicles a decrease in glycerophosphate acyltransferase activity is observed at amounts exceeding 1 mol% lysophosphatidate . Similar experiments, using lysophosphatidylcholine and phosphatidic acid, suggest the decrease to result from an increase in negative surface charge . Reconstituted glycerophosphate acyltransferase exhibits a preference for palmitoyl-CoA over oleoyl-CoA . This preference increases considerably at elevated temperatures . The glycerophosphate acyltransferase could, therefore, participate in the temperature-dependent changes in the fatty acid composition of the phospholipids in E . coli. Eur J Biochem, 1984 Feb 1, 138(3), 497 - 508 Purification of the lactose:H+ carrier of Escherichia coli and characterization of galactoside binding and transport; Wright JK et al.; The lactose carrier, a galactoside:H+ symporter in Escherichia coli, has been purified from cytoplasmic membranes by pre-extraction of the membranes with 5-sulfosalicylate, solubilization in dodecyl-O-beta-D-maltoside, Ecteola-column chromatography, and removal of residual impurities by anti-impurity antibodies . Subsequently, the purified carrier was reincorporated into E . coli phospholipid vesicles . Purification was monitored by tracer N-{3H}ethylmaleimide-labeled carrier and by binding of the substrate p-nitrophenyl-alpha-D-galactopyranoside . All purified carrier molecules were active in substrate binding and the purified protein was at least 95% pure by several criteria . Substrate binding to the purified carrier in detergent micelles and in reconstituted proteoliposomes yielded a stoichiometry close to one molecule substrate bound per polypeptide chain . Large unilamellar proteoliposomes (1-5-micron diameter) were prepared from initially small reconstituted vesicles by freeze-thaw cycles and low-speed centrifugation . These proteoliposomes catalyzed facilitated diffusion and active transport in response to artificially imposed electrochemical proton gradients (delta mu H+) or one of its components (delta psi or delta pH) . Comparison of the steady-state level of galactoside accumulation and the nominal value of the driving gradients yielded cotransport stoichiometries up to 0.7 proton/galactoside, suggesting that the carrier protein is the only component required for active galactoside transport . The half-saturation constants for active uptake of lactose (KT = 200 microM) or beta-D-galactosyl-1-thio-beta-D-galactoside (KT = 50-80 microM) by the purified carrier were found to be similar to be similar to those measured in cells or cytoplasmic membrane vesicles . The maximum rate for active transport expressed as a turnover number was similar in proteoliposomes and cytoplasmic membrane vesicles (kcat = 3-4 s-1 for lactose) but considerably smaller than in cells (kcat = 40-60 s-1) . Possible reasons for this discrepancy are discussed. Biochimie, 1984 Feb, 66(2), 151 - 8 Yeast cytochrome b2 gene: isolation with antibody probes; Guiard B et al.; An efficient technique was used to clone the gene for yeast cytochrome b2, (a nuclear encoded mitochondrial protein) using the expression vector, lambda gt11 (lac 5 nin 5 c1857 S100) . This enables the insertion of yeast DNA into the beta-galactosidase structural gene (lacZ) and promotes synthesis of hybrid proteins . Screening of antigen producing clones in the lambda gt11 recombinant genomic library was achieved using antiserum against cytochrome b2 according to Young and Davis (1983) Two recombinants containing part of the gene coding for cytochrome b2 were isolated and characterized as follows: by their expression in Escherichia coli cells, examined by immuno-blotting with antibodies to pure cytochrome b2 . by DNA sequence analysis . One recombinant carries a 3 Kb yeast DNA insert which contains the whole nucleotide sequence encoding cytochrome b2 and a few amino acids of the amino terminal presequence. J Gen Microbiol, 1984 Feb, 130 ( Pt 2), 401 - 10 Molecular comparisons of plasmids isolated from colicinogenic strains of Escherichia coli; Lehrbach PR et al.; The plasmid content of 14 colicinogenic strains of Escherichia coli has been examined . Four strains contained miniplasmids (1.2-2.0 kb) . Small plasmids (4-7 kb) were detected in all the strains specifying group A colicins (colicins A, E1, E2, E3 and K; group I plasmids) . Larger plasmids (55-130 kb) were detected in seven out of nine strains specifying group B colicins (colicins B, H, Ia, Ib, M, V and S1; group II plasmids) . DNA-DNA hybridization with group II plasmids showed a wide variation in the degree of DNA sequence homology among its members . In contrast little (if any) DNA sequence homology was detected with the group I plasmids when the same group II plasmid DNAs were used as hybridization probes . The results of DNA-DNA hybridization studies with the two small group II plasmids (pcolG-CA46 and pcolD-CA23) suggested that these plasmids are equivalent to deleted forms of larger group II plasmids . Our hybridization data thus support the division of colicin plasmids into the two groups (I and II) that have been previously defined from genetic and physiological studies. J Biochem (Tokyo), 1984 Feb, 95(2), 521 - 7 Turn-over of phospholipids in Selenomonas ruminantium; Watanabe T et al.; The particulate enzyme prepared from Selenomonas ruminantium subsp . lactilytica catalyzed the formation of phosphatidylserine (PS) from CDP-diglyceride and serine, and phosphatidylethanolamine (PE) from PS . This indicates that PS and PE in this organism are synthesized through a similar pathway to that in Escherichia coli . In turn-over experiments with {32P}orthophosphate and {14C}caproate, a rapid turn-over of PE was observed, while ethanolamine plasmalogen was relatively stable . The decrease of 14C-radioactivity in PE side-chains was accompanied by an increase of 14C-radioactivity in side-chains of ethanolamine plasmalogen . In pulse label-chase experiments with {32P}orthophosphate, significant amounts of 32P-radioactivities were incorporated into plasmalogens at the beginning of the chase and a precursor product relationship was observed between serine plasmalogen and ethanolamine plasmalogen . On the contrary, in pulse-label-chase experiments using {14C}caproate and {3H}glycerol, no significant radioactivity was incorporated into plasmalogens at the beginning of the chase and radioactivities in plasmalogens slowly increased during the chase . These results indicate that 1-O-alk-1'-enyl-2-acyl-glycerol moieties of plasmalogens are derived from a large precursor pool, but the phosphorous moiety is not . This concept was supported by the fact that synthesis of plasmalogens occurred in the absence of fatty acid synthesis . We wish to propose the possibility that 1-O-alk-1'-enyl-2-acyl-glycerol moieties of plasmalogens are derived from the diglyceride moieties of diacyl phospholipids. EMBO J, 1984 Feb, 3(2), 365 - 71 Plasmidial maintenance in rodent fibroblasts of a BPV1-pBR322 shuttle vector without immediately apparent oncogenic transformation of the recipient cells; Meneguzzi G et al.; A recombinant plasmid was constructed (pV69) which comprises a subgenomic fragment of bovine papilloma virus type 1 (BPV1) DNA, part of plasmid pBR322 DNA and a drug resistance gene expressed in both mammalian fibroblasts and Escherichia coli . This gene (vv2) is a modified form of the bacterial neomycin resistance gene (neo) linked to the herpes simplex virus thymidine kinase (tk) promoter (plasmid pAG60), to which the original bacterial neo promoter from transposon Tn5 was added back, upstream of the eukaryotic promoter . It induced kanamycin resistance in E . coli, as well as resistance to the drug G418 in rat and mouse fibroblasts . Its expression in FR3T3 rat cells was enhanced as compared with the original tk-neo construction . After transfer of plasmid pV69 into C127 mouse cells or FR3T3 rat cells, the number of resistant colonies selected in medium containing G418 was one to two orders of magnitude higher than that of transformed foci in normal medium . In eight independent cell lines selected by drug resistance, pV69 DNA was found to be maintained in a plasmidial state, without any detectable rearrangement or deletion and could be transferred back in E . coli . In contrast, cell lines selected by focus formation in normal medium maintained deleted forms of the original plasmid DNA, and only part of them were resistant to G418 . Most of the drug-resistant clones had kept the morphology and growth control of the normal fibroblasts . However, with further passages in culture, these cells spontaneously produced transformed foci with increasing frequencies. EMBO J, 1984 Feb, 3(2), 315 - 20 Gene expression in a temperature-sensitive gyrB mutant of Escherichia coli; Wahle E et al.; The effect of gyrase inactivation on gene expression was studied by examining the activities of different promoters in a temperature-sensitive gyrB mutant of Escherichia coli . The relative activities of promoters affected by cAMP-binding protein (CAP), e.g., the lac promoter, are not reduced by gyrase inactivation but can, on the contrary, be enhanced . This stimulation depends on the promoter location or its structure . The tnaA promoter is activated when located near the origin of replication, suggesting a differential effect of gyrase inactivation on various chromosomal domains . Only silent or mutant promoters such as the non-functional wild-type bgl or the lacIq can be activated . No differential effect of gyrase inactivation on the lambda pL and the trp promoters carried by the phage can be detected. Proc Natl Acad Sci U S A, 1984 Feb, 81(4), 1040 - 4 RNase H confers specificity in the dnaA-dependent initiation of replication at the unique origin of the Escherichia coli chromosome in vivo and in vitro; Ogawa T et al.; Escherichia coli rnh mutants defective in RNase H activity display the features of previously described sdrA (stable DNA replication) and dasF (dnaA suppressor) mutants: (i) sustained DNA replication in the absence of protein synthesis, (ii) lack of requirement for dnaA protein and the origin of replication (oriC), and (iii) sensitivity of growth to a rich medium . Both the sdrA mutants (selected for continued DNA replication in the absence of protein synthesis) and the dasF mutants (selected as dnaA suppressors) are defective in RNase H activity, measured in vitro . Furthermore, a 760-base-pair fragment containing the rnh+ structural gene complements the phenotype of each of the rnh, sdrA, and dasF mutants, indicative of a single gene . One function of RNase H in vivo is in the initiation of a cycle of DNA replication at oriC dependent on dnaA+ . In keeping with these results, RNase H contributes to the specificity of dnaA protein-dependent replication initiated at oriC in a partially purified enzyme system. Proc Natl Acad Sci U S A, 1984 Feb, 81(3), 824 - 8 Unusual alleles of recB and recC stimulate excision of inverted repeat transposons Tn10 and Tn5; Lundblad V et al.; Precise and nearly precise excision of transposon Tn10 occur by host-mediated processes unrelated to transposition . Both types of excision involve interactions between short (9 or 24 base-pair) direct repeat sequences at or near the termini of the transposon and are stimulated by the large (1,329-base-pair) inverted repeats that form the ends of Tn10 . We describe here three mutations of Escherichia coli K-12, designated texA, that enhance excision of Tn10 and of the structurally analogous transposon Tn5 . Genetic mapping and complementation analysis show that these mutations are unusual alleles of the recB and recC genes that alter but do not abolish RecBC function . As Tn10 excision normally does not depend on RecA or RecBC functions, texA mutations appear to provide another pathway for excision that depends on altered RecBC function; for one texA allele, excision has become dependent on RecA function as well . The available evidence suggests that texA mutations alter the stimulatory interaction between the inverted repeats of Tn10. Proc Natl Acad Sci U S A, 1984 Feb, 81(3), 654 - 8 Replication of the broad host range plasmid RSF1010: requirement for three plasmid-encoded proteins; Scherzinger E et al.; Cloning of specific regions of plasmid RSF1010, in conjunction with in vitro replication studies, has revealed three novel genes: repA, repB, and repC . They are clustered in one region of the plasmid, separated from the origin of replication by regions that are not essential for plasmid viability in an Escherichia coli host . In vivo, a 2.1-kilobase segment of the plasmid, bearing the replication origin, can establish itself as an autonomous replicon if the DNA region carrying the three rep genes is present in the same cell on an independent plasmid . In vitro, RSF1010 DNA is efficiently replicated by an ammonium sulfate fraction from the E . coli extract, provided the extracts are prepared from cells that can supply the required rep gene products . Using cells containing the cloned rep gene region as a source of elevated levels of the rep proteins, we have partially purified these proteins in functional form . When added to an enzyme fraction derived from plasmid-free cells, they specifically promote the replication of plasmid DNA bearing the RSF1010 origin. Arch Surg, 1984 Feb, 119(2), 145 - 50 Mechanisms of adrenocortical depression during Escherichia coli shock; Catalano RD et al.; The response of the adrenal cortex to corticotropin during sepsis is variable . We have previously demonstrated a significant decrease of corticosterone production by rat adrenocortical cells in response to corticotropin stimulation after incubation with septic shock plasma (SP) as compared with control plasma (CP) . We have studied the mechanisms of this depression . The following defects were demonstrated . (1) Cells bound less radioiodinated corticotropin analog after SP treatment (2.9 +/- 0.4 femtomoles/50 micrograms DNA) than after CP treatment (6.4 +/- 0.3 fmole/50 micrograms DNA) . (2) Cyclic adenosine monophosphate (cAMP) production was less after SP treatment (59.3 +/- 4 pmole per 10(5) cells per two hours) compared with CP treatment (110.3 +/- 11.3 pmole per 10(5) cells per two hours) . (3) Exogenously added dibutyryl cAMP was unable to correct the defect in corticosterone production after SP treatment (4.96 +/- 0.7 micrograms/24 hr) as compared with CP treatment (6.99 +/- 0.5 micrograms/24 hr) . Our studies suggest this defect is located in the synthesis of pregnenolone from cholesterol . These mechanisms may be responsible for the low cortisol levels previously observed in humans during septic shock. J Virol, 1984 Feb, 49(2), 588 - 90 Evidence that Escherichia coli virus T1 induces a DNA methyltransferase; Auer B et al.; DNA of Escherichia coli virus T1 is resistant to MboI cleavage and appears to be heavily methylated . Analysis of methylation by the isoschizomeric restriction enzymes Sau3AI and DpnI revealed that recognition sites for E . coli DNA adenine methylase (dam methylase) are methylated . The same methylation pattern was found for virus T1 DNA grown on an E . coli dam host, indicating a T1-specific DNA methyltransferase. J Virol, 1984 Feb, 49(2), 490 - 6 Synergistic antiviral and antiproliferative activities of Escherichia coli-derived human alpha, beta, and gamma interferons; Czarniecki CW et al.; The antiviral and antiproliferative effects of highly purified Escherichia coli-derived human interferons (IFNs) were examined in human melanoma cells (Hs294T) . Antiproliferative activity was monitored by measuring inhibition of cell multiplication, and antiviral activity was determined by inhibition of herpes simplex virus type 1 replication . Treatment of cells with IFN-gamma in combination with IFN-alpha A or IFN-beta 1 resulted in potentiation of both antiproliferative and antiviral activities . In contrast, combination treatments composed of IFN-alpha A and IFN beta 1 yielded inconsistent results . Some combinations reflected additive responses, whereas others were antagonistic . To examine correlations between IFN-induced biological activities and interactions of the different IFNs with cell surface receptors, in vivo {35S}methionine-labeled IFN-alpha A was prepared . Binding studies indicated the presence of 2,980 +/- 170 receptors per cell, each with an apparent Kd of (8.4 +/- 1.3) X 10(-11) M . Results from competitive binding studies suggested that Hs294T cells possess at least two types of IFN receptors: one which binds IFN-alpha A and IFN-beta 1 and another to which IFN-gamma binds. J Virol, 1984 Feb, 49(2), 426 - 36 Characterization of guinea pig cytomegalovirus DNA; Isom HC et al.; The genome of guinea pig cytomegalovirus (GPCMV) was analyzed and compared with that of human cytomegalovirus (HCMV) . GPCMV and HCMV DNAs were isolated from virions and further purified by CsCl centrifugation . Purified GPCMV DNA sedimented as a single peak in a neutral sucrose gradient and was infectious when transfected into guinea pig embryo fibroblast cells . The cytopathology was characteristic of that seen after infection with GPCMV . Virus DNA purified from virions isolated from infected GPEF or 104C1 cells had a CsCl buoyant density of 1.713 g/cm3, which corresponds to a guanine plus cytosine content of 54.1% . The CsCl buoyant density of GPCMV DNA was slightly less than that of HCMV DNA (1.716 g/cm3), but sufficiently different so that the two virus DNA peaks did not coincide . GPCMV DNA cosedimented with T4 DNA in a neutral sucrose gradient . Restriction endonuclease cleavage of GPCMV or HCMV DNAs with HindIII, XbaI, or EcoRI yielded fragments easily separable by agarose gel electrophoresis and ranging from 1.0 X 10(6) to 25.8 X 10(6) daltons . The number, size, and molarity of GPCMV DNA fragments generated by restriction enzymes were determined . Hybridization of restriction endonuclease-cleaved GPCMV DNA with radioactively labeled HCMV DNA and, conversely, hybridization of restriction endonuclease-cleaved HCMV DNA with radioactively labeled GPCMV DNA indicated sequence homology between the two virus DNAs. J Virol, 1984 Feb, 49(2), 343 - 8 Construction of replication-competent Herpesvirus saimiri deletion mutants; Desrosiers RC et al.; DNA fragments derived from the left end of Herpesvirus saimiri 11 L-DNA were cloned in Escherichia coli by using vector pBR322 . Deletions were introduced within a cloned 7.4-kilobase-pair sequence by using restriction endonucleases that cut once or twice within this sequence . Permissive owl monkey kidney-cultured cells were transfected with parental strain 11 viral DNA plus cloned DNA with specific sequences deleted . By screening the progeny of these transfections with a limiting-dilution spot hybridization assay, we isolated recombinant viruses containing deletions in this region . A contiguous 4.5-kilobase-pair sequence representing 4.1% of the coding capacity of the virus was found to be unnecessary for virus replication in cultured cells . These deletion mutants will allow us to test whether sequences in this region are required for the lymphoma-inducing capacity of H . saimiri . These same procedures should also allow us to introduce foreign DNA sequences into this region for studying their expression. J Bacteriol, 1984 Feb, 157(2), 622 - 6 Isolation of catalase-deficient Escherichia coli mutants and genetic mapping of katE, a locus that affects catalase activity; Loewen PC; A number of catalase-deficient mutants of Escherichia coli which exhibit no assayable catalase activity were isolated . The only physiological difference between the catalase mutants and their parents was a 50- to 60-fold greater sensitivity to killing by hydrogen peroxide . For comparison, mutations in the xthA and recA genes of the same strains increased the sensitivity of the mutants to hydrogen peroxide by seven- and fivefold, respectively, showing that catalase was the primary defense against hydrogen peroxide . One class of mutants named katE was localized between pfkB and xthA at 37.8 min on the E . coli genome . A second class of catalase mutants was found which did not map in this region. J Bacteriol, 1984 Feb, 157(2), 582 - 90 Plasmid ColE3 specifies a lysis protein; Jakes KS et al.; Tn5 insertion mutations in plasmid ColE3 were isolated and characterized . Several of the mutants synthesized normal amounts of active colicin E3 but, unlike wild-type colicinogenic cells, did not release measurable amounts of colicin into the culture medium . Cells bearing the mutant plasmids were immune to exogenous colicin E3 at about the same level as wild-type colicinogenic cells . All of these lysis mutants mapped near, but outside of, the structural genes for colicin E3 and immunity protein . Cells carrying the insertion mutations which did not release colicin E3 into the medium were not killed by UV exposure at levels that killed cells bearing wild-type plasmids . The protein specified by the lysis gene was identified in minicells and in mitomycin C-induced cells . A small protein, with a molecular weight between 6,000 and 7,000, was found in cells which released colicin into the medium, but not in mutant cells that did not release colicin . Two mutants with insertions within the structural gene for colicin E3 were also characterized . They produced no colicin activity, but both synthesized a peptide consistent with their map position near the middle of the colicin gene . These two insertion mutants were also phenotypically lysis mutants--they were not killed by UV doses lethal to wild-type colicinogenic cells and they did not synthesize the small putative lysis protein . Therefore, the lysis gene is probably in the same operon as the structural gene for colicin E3. J Bacteriol, 1984 Feb, 157(2), 568 - 75 Genetic and molecular analyses of Escherichia coli K1 antigen genes; Silver RP et al.; The plasmid pSR23, composed of a 34-kilobase E . coli chromosomal fragment inserted into the BamHI site of the pHC79 cosmid cloning vector, contains genes encoding biosynthesis of the K1 capsular polysaccharide . Deletions, subclones, and Tn5 insertion mutants were used to localize the K1 genes on pSR23 . The only deletion derivative of pSR23 that retained the K1 phenotype lacked a 2.7-kilobase EcoRI fragment . Subclones containing HindIII and EcoRI fragments of pSR23 did not produce K1 . Cells harboring pSR27, a subclone containing a 23-kilobase BamHI fragment, synthesized K1 that was not detectable extracellularly . Six acapsular Tn5 insertion mutants of three phenotypic classes were observed . Class I mutants synthesized K1 only when N-acetylneuraminic acid (NANA) was provided in the medium . Reduced amounts of K1 were detectable in cell extracts of class II mutants . Class III mutants did not produce detectable K1 in either extracts or when cells were provided exogenous NANA . All mutants had sialyltransferase activity . Analysis in the E . coli minicell system of proteins expressed by derivatives of pSR23 identified a minimum of 12 polypeptides, ranging in size from 18,000 to 80,000 daltons, involved in K1 biosynthesis . The 16-kilobase coding capacity required for the proteins was located in three gene clusters designated A, B, and C . We propose that the A cluster contains a NANA operon of two genes that code for proteins with apparent molecular weights of 45,000 and 50,000 . The A region also includes a 2-kilobase segment involved in regulation of K1 synthesis . The B region encoding five protein species appears responsible for the translocation of the polymer from its site of synthesis on the cytoplasmic membrane to the cell surface . The C region encodes four protein species . Since the three gene clusters appear to be coordinately regulated . we propose that they constitute a kps regulon. J Bacteriol, 1984 Feb, 157(2), 552 - 9 Antagonistic transcriptional regulation of the putrescine biosynthetic enzyme agmatine ureohydrolase by cyclic AMP and agmatine in Escherichia coli; Satishchandran C et al.; The putrescine biosynthetic enzyme agmatine ureohydrolase (AUH) (agmatinase; EC 3.5.3.11) catalyzes the conversion of agmatine to putrescine in Escherichia coli . The specific activity of AUH was determined in crude extracts prepared from wild-type strains and from strains with mutations in the adenylate cyclase gene (cya) or the cAMP receptor protein gene (crp) or both . In glucose minimal medium, a delta cya strain exhibited 70 to 90% higher AUH activity than a cya+ strain . Addition of 1 to 10 mM cAMP to cya+ and delta cya strains cultured in glucose repressed AUH activity in a dose-dependent manner . Addition of 1 to 10 mM cAMP to a delta crp strain failed to repress AUH activity . Addition of agmatine resulted in a three- to fourfold induction of AUH in delta cya and delta crp strains . This induction could be blocked by the addition of chloramphenicol . Simultaneous additions of various proportions of cAMP and agmatine resulted in reduced levels of induction and repression of AUH activity . This antagonistic regulation was shown to be exerted by independent mechanisms since AUH activity could be induced by agmatine in a delta crp strain supplemented with cAMP . These results suggest that both agmatine and cAMP antagonistically regulate AUH activity at the level of transcription . In minimal medium supplemented with 1 mM putrescine, the strains did not exhibit repression of AUH activity . In contrast, in minimal medium supplemented with 1 mM ornithine or arginine, cya+ or delta cya strains exhibited induced AUH activity as a result of conversion of these substrates to agmatine . Further experiments in vitro demonstrated that the effects observed with cAMP, agmatine, and arginine were not post-translationally mediated. J Bacteriol, 1984 Feb, 157(2), 545 - 51 Purification and characteristics of a gamma-glutamyl kinase involved in Escherichia coli proline biosynthesis; Smith CJ et al.; gamma-Glutamyl kinase, the first enzyme of the proline biosynthetic pathway, was purified to homogeneity from an Escherichia coli strain resistant to the proline analog 3,4-dehydroproline . The enzyme had a native molecular weight of 236,000 and was apparently comprised of six identical 40,000-dalton subunits . Enzymatic activity of the protein was detectable only in assays containing highly purified gamma-glutamyl phosphate reductase, the second enzyme of the proline pathway . Plots of gamma-glutamyl kinase activity as a function of glutamate concentration were sigmoidal, with a half-saturation value for glutamate of 33 mM, whereas plots of enzyme activity as a function of ATP concentration displayed typical Michaelis-Menten kinetics with a Km for ATP of 4 X 10(-4) M . Enzyme activity was insensitive to the glutamate analog L-methionine-DL-sulfoximine, but ADP was a potent competitive inhibitor . Characteristics of the enzyme were compared with those of a gamma-glutamyl kinase partially purified from a 3,4-dehydroproline-sensitive E . coli . These results indicated that the only major difference was that the enzyme from the 3,4-dehydroproline-resistant strain was 100-fold less sensitive to feedback inhibition by proline. J Bacteriol, 1984 Feb, 157(2), 526 - 32 Characterization of the cloned fip gene and its product; Russel M et al.; A DNA fragment encoding the fip (filamentous phage production) gene from Escherichia coli, when cloned in a filamentous phage vector, restored to the phage ability to assemble progeny in fip mutant hosts . The fip gene was located just upstream of and transcribed in the same direction as the rho gene . Minicells containing fip+ phage or plasmids synthesized a 12,500-dalton protein that was missing or truncated when the Fip+ phenotype was inactivated by insertion of Tn5 . The fip protein was cytoplasmic and was partially purified. J Bacteriol, 1984 Feb, 157(2), 490 - 7 Mutations in the DNA gyrB gene that are temperature sensitive for lambda site-specific recombination, Mu growth, and plasmid maintenance; Friedman DI et al.; We report the isolation of two mutations in the gyrB gene of Escherichia coli K12 obtained from an initial selection for resistance to coumermycin A1 and a subsequent screening for bacteria that fail to support site-specific recombination of phage lambda, i.e., Him- . These two mutations have a temperature-sensitive Him- phenotype, supporting site-specific recombination efficiently at low temperature, but inefficiently at high temperatures . Like other Him mutants, the gyrB-him mutants fail to plate phage Mu; again this defect is observed only at high temperatures . Additional thermally sensitive characteristics have also been observed; growth of lambda as well as maintenance of the plasmids pBR322 and F' gal are reduced at high temperature . Restriction of foreign DNA imposed by a P1 prophage is also reduced in these mutants . The temperature-sensitive phenotypic characteristics imposed by both the gyrB-him-230(Ts) and gyrB-him-231(Ts) mutations correlate with in vitro studies that show decreased gyrase activity, especially at higher temperatures, and in vivo studies showing reduced supercoiling of lambda DNA in the mutants at high temperature. J Bacteriol, 1984 Feb, 157(2), 440 - 4 Isolation and characterization of an Escherichia coli mutant deficient in dTMP kinase activity; Daws TD et al.; Escherichia coli LD0181 is sensitive to 15 micrograms of 2',3'-dideoxythymidine per ml . A derivative that was resistant to 40 micrograms of the same chemical per ml at 30 degrees C and that had lost the ability to grow on enriched medium at 42 degrees C was isolated after nitroso-guanidine mutagenesis . This mutant, TD105, produced a dTMP kinase with 25-fold lower specific activity and a 5-fold higher Km for dTMP than the parental strain . The dTMP pool in TD105 was 4.4-fold higher than in the parent . In addition to temperature sensitivity and resistance to 2',3'-dideoxythymidine, the mutant exhibited a hypersensitivity to 5-bromo-2'-deoxyuridine . All three of these phenotypes are cotransducible . The tmk gene was mapped by cotransduction to approximately 30 min on the E . coli map. J Bacteriol, 1984 Feb, 157(2), 413 - 9 Physical organization of the metJB component of the Escherichia coli K-12 metJBLF gene cluster; Liljestrand-Golden CA et al.; The structures of a series of plaque-forming metJB transducing phage were studied by restriction endonuclease mapping and enzyme activity assay . One of these phage, lambda pmet100, was inactivated by heat shock in the presence of EDTA, and deletion mutants were selected from the survivors . Two of these mutants, lambda pmet100 delta 1 and lambda pmet100 delta 2, were used to confirm the gene order metJ metB when moving clockwise on the linkage map of Escherichia coli K-12 . Additional results indicate that the metB gene can be expressed independently of any other component of the met regulon and that the metJ gene also forms a separate transcription unit. J Bacteriol, 1984 Feb, 157(2), 363 - 7 Translational coupling of the trpB and trpA genes in the Escherichia coli tryptophan operon; Aksoy S et al.; We investigated whether there is translational coupling between the tryptophan operon trpB and trpA genes in Escherichia coli . A trp-lac fusion system was used in which part of the trpA gene is fused to the lacZ gene . This fusion protein has the translation initiation site of trpA but retains beta-galactosidase activity . We introduced a frameshift mutation early in trpB and measured its effect on transcription and translation of the trp-lac fusion . The mutation resulted in a 10-fold drop in beta-galactosidase activity but only a 2-fold drop in lacZ mRNA or galactoside transacetylase levels . An rho mutation restored the lacZ mRNA and transacetylase levels to those of the control but only increased the beta-galactosidase level to 20% that of the control . We conclude from these results that if the trpB gene is not translated, efficient translation of the trpA'-lac'Z mRNA does not occur and, thus, that these genes are translationally coupled . The implication of this finding for other studies with gene fusions is discussed. Eur J Biochem, 1984 Feb 1, 138(3), 617 - 22 Chemical modification of the F0 part of the ATP synthase (F1F0) from Escherichia coli . Effects on proton conduction and F1 binding; Steffens K et al.; The purified F0 part of the ATP synthase complex from Escherichia coli was incorporated into liposomes and chemically modified by various reagents . The modified F0-liposomes were assayed for H+ uptake and, after reconstitution with F1, for total and dicyclohexylcarbodiimide-sensitive ATPase activity . The water-soluble carbodiimide, 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide methiodide, (1.2 mM), inhibited H+ uptake to a great extent . Binding of F1 was almost unaffected, but the hydrolysis of ATP was uncoupled from H+ transport . This is reflected by the inhibition of dicyclohexylcarbodiimide-sensitive ATPase activity . Woodward's reagent K, N-ethyl-5-phenylisoxazolium-3'-sulfonate, inhibited both H+ uptake and total ATPase activity . Modification of arginine residues by phenylglyoxal (20 mM) was followed by inhibition of the F1 binding activity by 80% of the control . H+ translocation was reduced to 70% . Diethylpyrocarbonate (3 mM) exhibited a strong inhibiting effect on H+ uptake but not on F1 binding . Modification of tyrosine (by tetranitromethane) as well as lysine residues (by succinic anhydride) did not affect F0 functions . From the data presented we conclude that carboxyl-groups, different from the dicyclohexylcarbodiimide-binding site, are involved in H+ translocation through F0 and, in part, in the functional binding of F1 . Furthermore, for the latter function, also arginine residues seem to be important . The role of histidine residues remains unclear at present. Cell, 1984 Feb, 36(2), 391 - 401 Characterization of the bovine papilloma virus plasmid maintenance sequences; Lusky M et al.; Bovine Papilloma Virus (BPV-1) establishes itself as a multicopy nuclear plasmid in somatic mammalian cells in culture . We report here that two discontinuous regions within the viral genome can independently support extrachromosomal replication of the Tn5 neomycinr gene in cells that provide viral factors in trans . The viral plasmid maintenance sequences (PMS) act in cis and will integrate along with the marker gene in cell lines that do not provide BPV-1 gene products . PMS-1 is localized within a 521 bp region upstream of the BPV-1 early transcription unit; PMS-2 has been localized to a 140 bp region within the putative reading frame for the E1 protein of the viral genome . Recombinant plasmids carrying either of the PMS elements are unrearranged and stably maintained at a constant copy number supernumerary to the resident BPV-1 genomes even in the absence of selective pressure . Specific deletion mutants within the viral genome show that BPV-1 gene products required for morphological transformation are dispensable for plasmid maintenance . In mouse cells cotransformed with such deletion derivatives and an unlinked marker gene (neomycinr or Tk), the marker genes integrate into the host genome while the BPV molecules are nonselectively carried as nuclear plasmids . This result implies that the BPV-1 genome must have signals that specifically preclude integration in the presence of transacting factors. Cancer Genet Cytogenet, 1984 Feb, 11(2), 161 - 8 Chromosomes and causation of human cancer and leukemia . LII . Chromosome findings in treated patients with B-cell chronic lymphocytic leukemia; Sadamori N et al.; The chromosomes of stimulated lymphocytes in 40 treated cases of B-cell chronic lymphocytic leukemia (B-CLL) were examined . The polyclonal B-cell activators (PBA) used were: pokeweed mitogen (PWM), Epstein-Barr virus (EBV), and lipopolysaccharide W from Escherichia coli 055:B5 (LPS) . Thirty-three (83%) of the 40 cases contained an adequate number of metaphases that were suitable for banding . Of 15 cases with abnormal clones, 7 cases had trisomy #12 . Occasionally, trisomy #1, 6q-, i(7q), 14q+, trisomy #16, and trisomy #18 were seen . In 5 cases, marker chromosomes of unknown origin existed . The findings indicate that trisomy #12 may be a unique and nonrandom karyotypic change in B-CLL. EMBO J, 1984 Feb, 3(2), 361 - 4 Isolation of the human insulin-like growth factor I gene using a single synthetic DNA probe; Ullrich A et al.; A single synthetic oligonucleotide was employed as hybridization probe to detect and enable isolation of the human insulin-like growth factor I (IGF-I) gene from a human genomic DNA library . The synthetic oligonucleotide probe coded for the B-chain of IGF-I and was designed for expression in Escherichia coli . Despite numerous interspersed mismatches, the synthetic probe hybridized specifically with seven recombinant lambda phage containing almost the entire B-chain region of the human IGF-I gene . The usefulness of this approach was further demonstrated by the detection of lambda phage containing human preproinsulin, using A and B chain synthetic oligonucleotides, 90 and 63 nucleotides in length, as hybridization probes . The nucleotide sequence of the human IGF-I exon suggests that IGF-I is synthesized as a larger precursor molecule. Proc Natl Acad Sci U S A, 1984 Feb, 81(4), 1021 - 5 Functional molecular weight of the lac carrier protein from Escherichia coli as studied by radiation inactivation analysis; Goldkorn T et al.; Cytoplasmic membrane vesicles prepared from Escherichia coli containing multiple copies of the lac y gene were frozen in liquid nitrogen before or after generation of a proton electrochemical gradient (interior negative and alkaline) and irradiated with a high-energy electron beam at -135 degrees C . Subsequently, the lac carrier protein was extracted into octyl beta-D-glucopyranoside, reconstituted into proteoliposomes, and assayed for transport activity . Under all conditions tested, activity decreased as a single exponential function of radiation dosage, allowing straightforward application of target theory for determination of functional molecular mass . When lac carrier activity solubilized from nonenergized vesicles was assayed, the results obtained were consistent with a functional molecular size of 45-50 kDa, a value similar to the size of the protein as determined by other means . Similar values were obtained when the octyl beta-D-glucopyranoside extract was irradiated, and the target size observed for D-lactate dehydrogenase was in good agreement with the molecular size of this enzyme . Strikingly, when the same procedures were carried out with vesicles that were energized with appropriate electron donors prior to freezing and irradiation, a functional molecular size of 85-100 kDa was obtained for the lac carrier with no change in the target size of D-lactate dehydrogenase . In contrast, when the vesicles were energized under conditions in which the proton electrochemical gradient was collapsed, the target mass of the lac carrier returned to 45-50 kDa . The results indicate that the functional mass of the lac carrier protein is no greater than a dimer and suggest that the proton electrochemical gradient may cause an alteration in subunit interactions. J Bacteriol, 1984 Feb, 157(2), 484 - 9 Synergistic effect of himA and gyrB mutations: evidence that him functions control expression of ilv and xyl genes; Friedman DI et al.; We have constructed Escherichia coli strains containing mutations at two different loci, both originally selected for failure to support lambda site-specific recombination: himA and gyrB-him(Ts) . Although the gyrB-him(Ts) mutations by themselves reduce supercoiling at high temperature, the double mutants show a far greater effect on supercoiling . Our studies show that growth of phage lambda is severely inhibited and that maintenance of plasmid pBR322 is extremely unstable in the double mutants . Physiological studies also reveal that the double mutants are isoleucine auxotrophs at 42 degrees C . The fact that himA mutants are isoleucine auxotrophs at 42 degrees C in the presence of leucine suggests that a significant component of the isoleucine auxotrophy of the double mutants is a result of the himA mutation . The himA gene encodes the alpha subunit of a protein called the integration host factor . Since mutations in the hip or himD gene encoding beta, the other subunit of the integration host factor, also result in isoleucine auxotrophy in the presence of leucine, we suggest that the integration host factor regulates the synthesis of at least one of the enzymes in the ilv pathway, acetohydroxyacid synthase I, which is encoded by the ilvB gene . Studies of the utilization of various sugars as the sole carbon source suggest that the integration host factor controls expression of some gene(s) involved in the utilization of xylose. Neurochem Res, 1984 Feb, 9(2), 249 - 62 Evidence that multiple species of aminoacylated transfer RNA are present in regenerating optic axons of goldfish; Zanakis MF et al.; This study reports that 4S RNA present in regenerating optic axons of goldfish is likely to be transfer RNA . Evidence is also presented which indicates that this transfer RNA is similar to transfer RNA found in tectal cells and that its aminocylation is likely to occur both in retinal ganglion cells prior to axonal transport as well as in the axon itself . Fish with regenerating optic nerves received intraocular injections of {3H}uridine followed 4 days later by intracranial injections of {14C}uridine . Radioactive tectal 4S RNA was isolated 6 days after {3H}uridine injections and chromatographed by BD cellulose chromatography . Optical density as well as radioactivity profiles for both {14C}4S RNA (from tectal cells) and {3H}4S RNA (90% of which originated from regenerating optic axons) were found to be similar to E . coli transfer RNA optical density profiles, indicating that the intra-axonal 4S RNA is likely to be transfer RNA . Moreover, comparisons of 3H/14C suggest that intra-axonal and cellular 4S RNAs are composed of similar species of transfer RNA . Results of other experiments indicate that aminoacylation of axonally transported tRNA occurs both in the retina and in optic axons subsequent to axonal transport. Gene, 1984 Feb, 27(2), 161 - 72 Toxicity of an overproduced foreign gene product in Escherichia coli and its use in plasmid vectors for the selection of transcription terminators; Brosius J; A rat insulin gene which was fused to Escherichia coli signals for the initiation of translation could not be retained when expressed from the strong rrnB ribosomal RNA promoters or an induced trp/lac (= tac) hybrid promoter . When the latter promoter was repressed by transformation into a lac-repressor-overproducing strain, the insulin gene fragment could be retained . Upon induction of the promoter with isopropyl-beta-D-galactosidase the growth rate of the cells was reduced, and in most cases the cells subsequently lysed . Deletion of the translational initiation signals, changing the reading frame, or insertion of an efficient transcription terminator between the promoter and the rat insulin gene each permitted retention of the fragment . The first two observations indicate that overproduction of the specific polypeptide, and not of the RNA, is detrimental to the cell . The third finding has been exploited for the testing and selection of transcription terminators . The rpoC terminator, which is located distal to the rplJL /rpoBC operon, has been shown to terminate transcripts from the rrnB promoters . It was also shown that the putative rrnB terminators, T1 and T2, each function separately in vivo. J Infect Dis, 1984 Feb, 149(2), 184 - 93 The importance of the K1 capsule in invasive infections caused by Escherichia coli; Cross AS et al.; We examined 534 clinical isolates of Escherichia coli for sensitivity to rough lipopolysaccharide-specific and K1-specific phages . Twenty-eight percent of bacteremic isolates were sensitive to rough-specific phages . Forty-two percent of these strains, against only 20% of bacteremic isolates insensitive to rough-specific phages, had K1 capsule (P less than 0.001) . K1-positive strains were usually resistant to phagocytic killing, whereas strains lacking the K1 capsule were more likely to be killed regardless of capsular type . Eighty-two percent of strains were typable with O-specific, 57% with K-specific, and 74% with H-specific antisera . Sixty percent of E coli were agglutinated by only 10 O-specific antisera . K1 was the most common capsular type, followed by K5, K2, and K12, whereas four H antigens accounted for nearly half of the H-typable strains . We conclude that (1) the combination of rough-specific and K1-specific phage sensitivity defines functionally similar groups of bacteria and (2) a polyvalent vaccine against invasive E coli is possible given the relatively limited number of invasive O:K:H serotypes. Chem Biol Interact, 1984 Feb, 48(2), 145 - 52 Genetic activity of bleomycin in Escherichia coli; Yamamoto K et al.; The induction of umuC gene expression, cell lethality, induction of W-reactivation of UV-irradiated lambda-phage and the induction of mutagenesis caused by bleomycin (Blm) were studied in Escherichia coli K-12 strains with special references to the effects of SOS repair deficiencies . (1) The umuC gene is inducible by Blm and the induction is regulated by the lexA and recA genes . (2) The lexA and recA mutants are slightly more sensitive to Blm-killing than wild-type strain . (3) The plating efficiency of UV-irradiated lambda-phage increased by Blm treatment of the host cell . This increase was not observed in the umuC mutant . The plating efficiency of UV-irradiated lambda-phage was drastically reduced in the lexA and recA strains treated with Blm . (4) No significant increase of the reversion of nonsense mutation (his-4 to His+) in AB1157 by the treatment of Blm was observed . Possible implications of these results are discussed. J Bacteriol, 1984 Feb, 157(2), 694 - 6 Participation of Escherichia coli K-12 groE gene products in the synthesis of cellular DNA and RNA; Wada M et al.; Cells having the temperature-sensitive mutation groES131(Ts) were isolated from Escherichia coli K-12 strain C600T by thymineless death selection at 44 degrees C . This conditionally expressed mutation affected both cellular DNA and RNA syntheses at nonpermissive temperature, in addition to rendering cells unable to propagate phage lambda at permissive temperature. Cell, 1984 Feb, 36(2), 513 - 22 Effects of point mutations on formation and structure of the RNA primer for ColE1 DNA replication; Masukata H et al.; We report mutations affecting several steps of CoIE1 primer formation . Primer precursor forms the hybrid with the template DNA and is cleaved by RNAase H to form the primer . Point mutations at positions -264, -265, -268, and -308 (base pairs upstream of the replication origin) reduce efficiency of hybrid formation . A suppressor mutation at -18 increases the efficiency for a mutant transcript and reduces that for a wild-type transcript . Therefore, hybrid formation probably starts after transcription passes this position . A mutation at -10 affects the site of cleavage by RNAase H . The site appears to be determined by the distance from a stem-loop structure immediately upstream . A double mutation at -186 and -188 results in formation of an RNA secondary structure that prevents use of an RNAase H-cleaved transcript as primer. Infect Immun, 1984 Feb, 43(2), 508 - 14 Organization and expression of genes involved in the biosynthesis of K99 fimbriae; de Graaf FK et al.; Escherichia coli K-12 minicells were used to study the expression of plasmid pFK99 encoding for the production of K99 fimbriae . Plasmid pFK99 is composed of a 6.7-kilobase pair DNA fragment derived from the wild-type K99 plasmid and the vector pBR322 . The cloned K99 DNA expressed seven polypeptides with apparent masses of 18.2, 19.0, 21.0, 21.5, 26.5, 33.5, and 76.0 kilodaltons (kd) . The 18.2-kd polypeptide was identified as the K99 fimbrial subunit by reaction with specific anti-K99 antibodies . The fimbrial subunit and the 19.0-, 26.5-, 33.5-, and 76.0-kd polypeptides appeared to be synthesized in a precursor form which was ca . 2 kd larger than the mature polypeptide . The location of the structural genes encoding the seven polypeptides on the physical map of pFK99 was established by analyzing a set of deletion derivatives of pFK99 . The gene encoding the fimbrial subunit was located at the promoter proximal end of the K99 operon . Only mutants with a deletion in the gene encoding the 33.5- or the 19.0-kd polypeptide or both showed a weak expression of the K99 antigen and a comparably weak agglutination of horse or sheep erythrocytes . None of the deletion mutants was able to adhere to calf intestinal epithelial cells. Biochemistry, 1984 Jan 31, 23(3), 558 - 62 Determination of the metabolic origins of the sulfur and 3'-nitrogen atoms in biotin of Escherichia coli by mass spectrometry; DeMoll E et al.; Two steps in the biosynthesis of biotin in Escherichia coli, incorporation of the nitrogen atom of methionine into 7-keto-8-aminopelargonic acid and of the sulfur atom into dethiobiotin, were examined . Sulfur and nitrogen metabolism were monitored by gas chromatography-mass spectrometry of volatile derivatives of internal (protein-bound) amino acids and excreted biotin . We were able to show that internal cysteine and excreted biotin were labeled to the same extent with 34S from either of two exogenous sulfur sources, 34SO4(2)-or L-{sulfane-34S}thiocystine . Internal methionine was eliminated from consideration, while cysteine, or possibly a closely related intermediate, was implicated as providing the sulfur atom for biotin biosynthesis . Also, in experiments designed to follow the metabolism of the nitrogen atom of methionine, it was found that biotin excreted into the culture medium by this organism grown with 95 atom % {15N}methionine contained greater than 70 atom % excess 15N in one of the nitrogens over that obtained from cultures grown with methionine of natural abundance 15N . These results provide evidence for the direct transfer of the methionine nitrogen as the role of S-adenosylmethionine in the conversion of 7-keto-8-aminopelargonic acid to 7,8-diaminopelargonic acid. Biochemistry, 1984 Jan 31, 23(3), 445 - 53 Reconstitution of the membrane-bound, ubiquinone-dependent pyruvate oxidase respiratory chain of Escherichia coli with the cytochrome d terminal oxidase; Koland JG et al.; Pyruvate oxidase is a flavoprotein dehydrogenase located on the inner surface of the Escherichia coli cytoplasmic membrane and coupled to the E . coli aerobic respiratory chain . In this paper, the role of quinones in the pyruvate oxidase system is investigated, and a minimal respiratory chain is described consisting of only two pure proteins plus ubiquinone 8 incorporated in phospholipid vesicles . The enzymes used in this reconstitution are the flavoprotein and the recently purified E . coli cytochrome d terminal oxidase . The catalytic velocity of the reconstituted liposome system is about 30% of that observed when the flavoprotein is reconstituted with E . coli membranes . It is also shown that electron transport from pyruvate to oxygen in the liposome system generates a transmembrane potential of at least 180 mV (negative inside), which is sensitive to the uncouplers carbonyl cyanide p-(tri-chloromethoxy)phenylhydrazone and valinomycin . A trans-membrane potential is also generated by the oxidation of ubiquinol 1 by the terminal oxidase in the absence of the flavoprotein . It is concluded that (1) the flavoprotein can directly reduce ubiquinone 8 within the phospholipid bilayer, (2) menaquinone 8 will not effectively substitute for ubiquinone 8 in this electron-transfer chain, and (3) the cytochrome d terminal oxidase functions as a ubiquinol 8 oxidase and serves as a "coupling site" in the E . coli aerobic respiratory chain . These investigations suggest a relatively simple organization for the E . coli respiratory chain. Biochemistry, 1984 Jan 31, 23(3), 470 - 8 Structure of the divalent metal ion activator binding site of S-adenosylmethionine synthetase studied by vanadyl(IV) electron paramagnetic resonance; Markham GD; The structure of the divalent metal ion binding site of S-adenosylmethionine synthetase from Escherichia coli has been studied by using the vanadyl(IV) ion (VO2+) as probe . VO2+ binds at a single site per subunit in the presence or absence of substrates . Single turnover experiments measuring S-adenosylmethionine (AdoMet) formation from methionine and the ATP analogue 5'-adenylyl imidodiphosphate show that complexes containing VO2+ and either Mg2+ or Ca2+ as a second metal ion are catalytically active, while a complex containing VO2+ alone is inactive . Electron paramagnetic resonance spectra of the enzyme-VO2+ complex, as well as complexes also containing AdoMet or methionine, indicate the coordination of two water molecules and at least two protein ligands to the VO2+ . In complexes with polyphosphate substrates or products (e.g., enzyme-VO2+-ATP-methionine, enzyme-VO2+-PPi-Mg2+), EPR spectral changes reveal ligand substitutions on the VO2+, and 8.5-G isotropic superhyperfine coupling to two 31P nuclei can be resolved . 17O superhyperfine coupling from {17O}pyrophosphate indicates coordination of two oxygen atoms of PPi to the VO2+ ion . Thus the polyphosphate compounds are bidentate ligands to the VO2+, demonstrating that the VO2+ binds at the active site and suggesting a catalytic role for the protein-bound metal ion. Biochem Biophys Res Commun, 1984 Jan 30, 118(2), 508 - 13 Role of actin binding protein phosphorylation in platelet cytoskeleton assembly; Zhuang QQ et al.; Actin binding protein from human blood platelets is shown to exist in the resting platelet as a phosphorylated protein and contains two residues of phosphate per 260,000 kd . Removal of one-half of these residues with E . coli alkaline phosphatase results in the loss of its ability to crosslink F-actin into a low speed sedimentable complex (its cytoskeleton) and to bind to an F-actin affinity column . Thus, phosphorylation-dephosphorylation of ABP may be an important regulatory mechanism by which the platelet regulates its shape via its cytoskeletal structure. J Biol Chem, 1984 Jan 25, 259(2), 1201 - 5 Cloning of cDNA sequences for murine ATP-citrate lyase . Construction of recombinant plasmids using an immunopurified mRNA template and evidence for the nutritional regulation of ATP-citrate lyase mRNA content in mouse liver; Sul HS et al.; Mouse liver mRNA that was enriched in sequences coding for ATP-citrate lyase by polysome immunoadsorption was used as a template for cDNA synthesis . Double-stranded cDNA sequences were inserted into the plasmid pBR322 and cloned in Escherichia coli RR1 . Twenty-seven plasmids containing putative cDNA sequences for ATP-citrate lyase were identified by differential hybridization with single-stranded 32P-cDNAs synthesized from immunopurified mRNA, sucrose gradient-purified ATP-citrate lyase mRNA, and mRNA isolated from the livers of mice that were nutritionally induced or de-induced for ATP-citrate lyase biosynthesis . A subgroup of five recombinant plasmids was characterized further in hybridization-selection experiments . Each of these plasmids positively selected ATP-citrate lyase mRNA as determined by in vitro translation and specific immunoprecipitation . The length of ATP-citrate lyase mRNA was estimated to be 4900 bases in a Northern blot analysis . A 32P-cDNA probe derived from a 1500-base pair insert was used to investigate the basis for the 20-30-fold induction of ATP-citrate lyase that occurs when starved animals are fed a high carbohydrate/low fat diet . Dot-blot hybridization analysis disclosed that the relative content of liver ATP-citrate lyase mRNA increased 25-fold after 15 h of refeeding, indicating that the synthesis of the lipogenic enzyme is controlled at a pretranslational level in the nutritional paradigm. J Biol Chem, 1984 Jan 25, 259(2), 805 - 14 Characterization of the Escherichia coli SSB-113 mutant single-stranded DNA-binding protein . Cloning of the gene, DNA and protein sequence analysis, high pressure liquid chromatography peptide mapping, and DNA-binding studies; Chase JW et al.; The ssb-113 (formerly lexC113) gene encoding a mutant single-stranded DNA binding protein (SSB) has been cloned into plasmid pSC101 resulting in 5- to 10-fold more mutant protein than strains carrying only one (chromosomal) copy of the gene . Analysis of tryptic and chymotryptic peptides of the mutant protein by high pressure liquid chromatography and solid phase protein sequencing has shown that the ssb-113 mutation results in the substitution of serine for proline at residue 176 of SSB . This change could only occur in one step by a C leads to T transition in the DNA sequence . Physicochemical studies of the homogeneous mutant protein have shown that it binds as well as wild type SSB to single-stranded DNA and that it is a slightly better helix-destabilizing protein than wild type SSB as measured by its ability to lower the thermal melting transition of poly{d(A-T)} . In vivo studies of ssb-113 strains carrying the cloned ssb-113 gene in pSC101 have shown that overproduction of the mutant protein does not complement the temperature-sensitive conditional lethality caused by the ssb-113 mutation when present in single gene copy in contrast to effects recently observed in ssb-1 strains overproducing the ssb-1 encoded protein (Chase, J . W., Murphy, J . B., Whittier, R . F., Lorensen, E., and Sninsky, J . J . (1983) J . Mol . Biol . 164, 193-211) . Also noted in this report are two corrections to the DNA sequence of wild type SSB, one of which places glycine (codon GGC) at residue 133 rather than serine as previously reported (Sancar, A., Williams, K . R., Chase, J . W., and Rupp, W . D . (1981) Proc . Natl . Acad . Sci . U.S.A . 78, 4274-4278) . The second correction to the DNA sequence is in the serine 39 codon, previously reported to be TCA and now correctly shown to be TCC. J Biol Chem, 1984 Jan 25, 259(2), 757 - 60 Apolipoprotein, an intermediate in the processing of the major lipoprotein of the Escherichia coli outer membrane; Inukai M et al.; A new intermediate (apolipoprotein) in the synthesis of the major lipoprotein of the Escherichia coli outer membrane has been identified . The accumulation of this new form of the lipoprotein was observed when excessive production of lipoprotein was induced or when the membrane fraction containing the prolipoprotein accumulated in the presence of globomycin was incubated at 60 degrees C . The new form of the lipoprotein could be chased into the mature lipoprotein . In addition, from sequential analysis of this new protein by Edman degradation, the NH2 terminus was found to be cysteine, containing a free unmodified amino group and a glyceride-modified sulfhydryl group . These results indicate that this protein is an intermediate in the conversion of glyceride-modified prolipoprotein to the mature lipoprotein . It is believed that the lipoprotein signal peptidase directly cleaves the lipoprotein signal peptide at the peptide bond between the glycine residue at position 20 and the cysteine residue at position 21 of the prolipoprotein . The resulting intermediate, designated here as apolipoprotein, is subsequently acylated at its free amino group to yield the final mature lipoprotein. J Biol Chem, 1984 Jan 25, 259(2), 729 - 33 Trypsin modification of Escherichia coli alkaline phosphatase; Roberts CH et al.; A trypsin-modified form of Escherichia coli alkaline phosphatase has been isolated, purified, and characterized . The native enzyme, previously thought to be resistant to proteases, shows a loss of 20% of its activity after a 30-min exposure to 10% trypsin . No further loss is seen after 3 h; this is in contrast to the apoenzyme which loses essentially all restorable activity (addition of saturating Zn(II) and Mg(II) restores activity to the apoenzyme) when exposed to trypsin . Under these conditions, a single major peptide is produced, cleaved at the Arg-10 Ala-11 bond, which is purified using a chromatographic technique that separates proteins according to their pI (chromatofocusing) . This modified alkaline phosphatase has a Vmax of 2000 mumol/h/mg (1 M Tris, pH 8.0, 20 degrees C, 1 mM p-nitrophenolphosphate) which is 22% less than the Vmax for the native enzyme . The Km for p-nitrophenolphosphate is lower for trypsin-modified alkaline phosphatase than for the native enzyme, 1.9 X 10(-5) and 4 X 10(-5) M, respectively . The KI for Pi for the native enzyme is 1.5 X 10(-5) M and for trypsin-modified alkaline phosphatase is 1 X 10(-5) M, suggesting that the reduction in Vmax is due to a reduction in the rate constant for Pi dissociation . Differential scanning calorimetry results indicate differences in the stabilities of the two species . The trypsin-modified alkaline phosphatase has a Tm of 90 degrees C which is lower than that for the reconstituted apoenzyme (93.5 degrees C) or for the native enzyme (98.5 degrees C) . This modified form of alkaline phosphatase may prove to be valuable in studies concerning subunit interactions in this system as the deleted decapeptide occurs at the subunit interface region in the native structure. J Biol Chem, 1984 Jan 25, 259(2), 703 - 6 Immunological cross-reactivity between carbamyl phosphate synthetases I, II, and III; Devaney MA et al.; Four types of carbamyl phosphate synthetase have been previously distinguished on the basis of catalytic properties and metabolic role . Immunoblot assay has now demonstrated cross-reactivity between rat liver carbamyl phosphate synthetase I and the following other three types of synthetases: carbamyl phosphate synthetase II from SV40-transformed baby hamster kidney cells, carbamyl phosphate synthetase III from spiny dogfish liver and from largemouth bass liver, and Escherichia coli carbamyl phosphate synthetase . The strongest cross-reactivity was observed between carbamyl phosphate synthetases I and III . These findings indicate at least partial structural homology among the various synthetases and constitute the first demonstration of such a relationship among the enzymes. J Biol Chem, 1984 Jan 25, 259(2), 1105 - 9 Close range interactions between nucleotide bases and tryptophan residues in an Escherichia coli single-stranded DNA binding protein-mercurated poly(uridylic acid) complex . A study by optically detected magnetic resonance spectroscopy; Cha TA et al.; Optically detected triplet state magnetic resonance spectra are reported for the complex formed between mercurated poly(Urd) and Escherichia coli single-stranded DNA binding protein . Upon forming a complex, the triplet state properties of Trp residue(s) in the protein are perturbed by the heavy mercury atom and are characterized by a shortened triplet state lifetime and the appearance of a strong D + E slow passage optically detected magnetic resonance signal . These features, which signal an external heavy atom effect, provide direct evidence for a close range interaction between mercurated nucleotide bases and Trp residues owing to the requirement of a van der Waals contact between the perturbed molecule and the heavy atom perturber . The amplitude-modulated phosphorescence microwave double resonance technique selectively displays the phosphorescence spectrum of the heavy atom-perturbed Trp triplet states . A van der Waals contact manifested through a stacked structure of the mercurated uridine base and the indole moiety of Trp is strongly suggested as the most plausible mode of interaction from steric considerations, since other approaches of the mercury atom are blocked by the covalent attachment of 2-mercaptoethanol to mercury . The magnitude of the heavy atom perturbation also is consistent with Hg approach to the pi-system from above or below the indole aromatic plane, and is at least an order of magnitude larger than effects expected from an edge-on approach. Biochim Biophys Acta, 1984 Jan 25, 769(2), 348 - 56 Properties of chemically modified porin from Escherichia coli in lipid bilayer membranes; Benz R et al.; Purified porin OmpF from Escherichia coli outer membrane was chemically modified by acetylation and succinylation of amino groups and by amidation of the carboxyl groups . Native and chemically modified porins were incorporated into lipid bilayer membranes and the permeability properties of the pores were studied . Acetylation and succinylation of the porin trimers had almost no influence on the single channel conductance in the presence of small cations and anions and the cation selectivity remained essentially unchanged as compared with the native porin . Amidation had also only little influence on the single channel conductance and changed the pore conductance at maximum by less than 50%, whereas the cation selectivity of the porin is completely lost after amidation . The results suggest that the structure of the porin pore remains essentially unchanged after chemical modification of the pores and that their cation selectivity is caused by an excess of negatively charged groups inside the pore and/or on the surface of the protein . Furthermore, it seems very unlikely that the pore contains any positively charged group at neutral pH. Nucleic Acids Res, 1984 Jan 25, 12(2), 887 - 99 The nucleotide recognized by the Escherichia coli A restriction and modification enzyme; Kroger M et al.; The nucleotide recognition sequence for the restriction-modification enzyme of Escherichia coli A (EcoA) has been determined to be GAG-7N-GTCA . This sequence is fairly similar, but distinctly different from the two other type I restriction enzyme recognition sites known for E . coli B and E . coli K12, respectively . N6-adenosine methylation has been observed at nucleotide positions 2 and 12 within that sequence after modification by EcoA . As a reference point for mapping the single EcoA site in lambda, the position of lambda point mutation Oam29 has been determined also. Nucleic Acids Res, 1984 Jan 25, 12(2), 1287 - 99 A potential stem-loop structure and the sequence CAAUCAA in the transcript are insufficient to signal rho-dependent transcription termination at lambda tR1; Lau LF et al.; It has been suggested that a sequence in the RNA transcript that can form a stem and loop structure, followed by the sequence CAAUCAA, is the signal for rho-dependent transcription termination . We tested this hypothesis by synthesizing a DNA duplex whose sequence corresponds to a region of the lambda tR1 terminator that contains these structural features . We cloned this synthetic DNA fragment under the control of the lacUV5 promoter, and showed that it does not cause rho-dependent termination in vitro . RNA polymerase pauses during in vitro transcription across the synthetic sequence, although less efficiently than at the corresponding sequence on the lambda template . No rho-mediated termination was detected even under conditions that prolonged transcriptional pausing at the synthetic site, indicating that the synthetic sequence is defective as a transcript release site . We suggest that unlike rho-independent terminators, rho-dependent terminators require sequences in addition to those immediately before the sites of termination. Nucleic Acids Res, 1984 Jan 25, 12(2), 1277 - 85 Transcription of multimeric tRNA genes; Ciliberto G et al.; We have constructed a set of plasmids carrying a tRNAPro gene from C . elegans in a head to tail dimeric and trimeric arrangement with virtually no spacer sequence . We show that in two different transcriptional systems, each coding region functions as an internal promoter directing the synthesis of independent transcriptional products . This is in contrast with the property of natural head to tail dimeric arrangements found in yeast, where only one coding region functions as promoter (Mao et al., 1980 Cell 20, 589; Schmidt et al., 1980 Nature 287, 750) . The evolutionary significance of our finding is discussed. Nucleic Acids Res, 1984 Jan 25, 12(2), 1227 - 42 Structure and expression in Escherichia coli of a cloned rat interferon-alpha gene; Dijkema R et al.; DNA synthesized by in vitro transcription on rat interferon (IFN) mRNA has been cloned and amplified as recombinant DNA . The nucleotide sequence of these rat IFN cDNA clones revealed i . the partial presence of the coding region of the gene and ii all cDNA clones were derived from the same subtype of rat IFN-alpha mRNA . Purified inserted fragments were used as a hybridisation probe against chromosomal "Southern blots" to show that at least twelve rat IFN-alpha-related sequences are present in the genome . A lambda-linked rat gene library was screened with the cDNA probes, resulting in an equivalent number of rat IFN-alpha-related hybrid phages . By use of a 3'-noncoding region as a probe, the chromosomal counterpart of the cDNA clones could be detected and the nucleotide sequence of its coding region has been determined . Expression of the coding region in E . coli yielded biologically active IFN, when tested for in vitro or in vivo antiviral activity. Nucleic Acids Res, 1984 Jan 25, 12(2), 1219 - 26 Effect of ligand binding on the buoyant density of DNA in Nycodenz gradients; Ford TC et al.; The effect of ligand binding upon the buoyant density of DNA in Nycodenz gradients has been studied using DNAs of differing base compositions . The effect of both intercalating ligands (ethidium bromide and proflavin) and non-intercalating ligands (distamycin A, DAPI and netropsin) has been studied . The binding of intercalating ligands to DNA has essentially no effect on the buoyant density of DNA in Nycodenz gradients . The non-intercalating ligands were found to increase the buoyant density of DNA in a base specific manner . The increase in buoyant density can be interpreted in terms of disruption of the hydration shell of the DNA molecule caused by the binding of the ligand along the minor groove of the DNA helix. Nucleic Acids Res, 1984 Jan 25, 12(2), 1203 - 17 Transcription analysis of the lexA gene of Escherichia coli: attenuation and cotranscription with the neighboring region; Miki T et al.; The lexA gene of Escherichia coli encodes a repressor of the genes whose expression is induced by the agents that result in DNA damage . In vivo transcripts of the lexA gene consisted of two species; mRNA-1 of 673 bases and mRNA-2 of approximately 3,000 bases . The transcription in vivo started at a site which was two-base pairs downstream from the in vitro initiation site reported previously . The majority of the transcription stopped at a series of T residues preceeded by a dyad symmetry located immediately after the lexA gene . A small fraction of the transcription passed through the termination site to form the mRNA of downstream gene(s) which would be related to the "SOS functions". J Mol Biol, 1984 Jan 25, 172(3), 263 - 82 Kinetics and mechanism in the reaction of gene regulatory proteins with DNA; Fried MG et al.; We have measured the kinetic properties of the Escherichia coli cAMP receptor protein (CAP) and lac repressor interacting with lac promoter restriction fragments . Under our reaction conditions (10 mM-Tris X HCl (pH 8.0 at 21 degrees C), 1 mM-EDTA, 10 microM-cAMP, 50 micrograms bovine serum albumin/ml, 5% glycerol), the association of CAP is at least a two-step process, with an initial, unstable complex formed with rate constant kappa a = 5(+/- 2.5) X 10(7) M-1 s-1 . Subsequent formation of a stable complex occurs with an apparent bimolecular rate constant kappa a = 6.7 X 10(6) M-1 s-1 . At low total DNA concentration, the dissociation rate constant for the specific CAP-DNA complex is 1.2 X 10(-4) s-1 . The ratio of formation and dissociation rate constants yields an estimate of the equilibrium constant, Keq = 5 X 10(10) M-1, in good agreement with static results . We observed that the dissociation rate constant of both CAP-DNA and repressor-DNA complexes is increased by adding non-specific "catalytic" DNA to the reaction mixture . CAP dissociation by the concentration-dependent pathway is second-order in added non-specific DNA, consistent with either the simultaneous or the sequential participation of two DNA molecules in the reaction mechanism . The results imply a role for distal DNA in assembly-disassembly of specific CAP-DNA complexes, and are consistent with a model in which the subunits in the CAP dimer separate in the assembly-disassembly process . The dissociation of lac repressor-operator complexes was found to be DNA concentration-dependent as well, although in contrast to CAP, the reaction is first-order in catalytic DNA . Added excess operator-rich DNA gave more rapid dissociation than equivalent concentrations of non-specific DNA, indicating that the sequence content of the competing DNA influences the rate of repressor dissociation . The simplest interpretation of these observations is that lac repressor can be transferred directly from one DNA molecule to another . A comparison of the translocation rates calculated for direct transfer with those predicted by the one-dimensional sliding model indicates that direct transfer may play a role in the binding site search of lac repressor. J Mol Biol, 1984 Jan 25, 172(3), 241 - 62 Equilibrium studies of the cyclic AMP receptor protein-DNA interaction; Fried MG et al.; The binding of the Escherichia coli cyclic AMP receptor protein (CAP) to restriction fragments containing the lac promoter-operator region has been investigated as a function of cAMP concentration, using a sensitive gel electrophoresis assay . Under standard conditions (13 mM ionic strength), the equilibrium constant for CAP binding to its primary site on a 203 base-pair lac promoter fragment is 6.3 X 10(8) M-1 at 0.2 microM-cAMP, and increases to 8.4 X 10(10) M-1 at 5.0 microM-cAMP . The latter is about 10(5) times larger than the equilibrium constant for binding to an isolated, non-specific site . The L8 mutation, which renders the lac promoter unresponsive to CAP in vivo, lowers this binding affinity by five- to tenfold . Analysis of the cAMP dependency of binding over the concentration range of 0.2 microM to 10 microM reveals that uptake of a single equivalent of cAMP is required for site-specific binding . Similarly, the transfer of CAP from a non-specific DNA site to a specific site requires the net uptake of a single molecule of cAMP . In contrast, co-operative non-specific binding to DNA was found to be independent of cAMP concentration with an equilibrium binding constant of 6 X 10(6) M-1 . We conclude that the cAMP affinity of the two CAP subunits in the specific promoter complex is not equal, and that the complex structure therefore deviates significantly from twofold symmetry . A model for the regulation of the lac promoter by the intracellular cAMP concentration is proposed on the basis of the equilibrium binding results. J Mol Biol, 1984 Jan 25, 172(3), 355 - 62 Molecular cloning and sequence analysis of trp-lac fusion deletions; Yu XM et al.; DNA fragments containing deletions that fuse the trp operon to the lac operon were cloned and the end-points of the fusions were determined . The results from DNA sequence analysis correlated well with those from genetic, biochemical and physiological studies previously reported . The sequence data from this study, in combination with the known properties of these fusion strains, provided information on: (1) the precise lac operon distal boundary of the lac operator; (2) the nature of the trp operon terminator; and (3) the messenger RNA sequences that result in inhibition of lacZ translation initiation in trp-lac fused mRNAs. J Mol Biol, 1984 Jan 25, 172(3), 283 - 300 Effects of the mutant sigma allele rpoD800 on the synthesis of specific macromolecular components of the Escherichia coli K12 cell; Gross CA et al.; Escherichia coli K12 strains containing the mutant sigma allele rpoD800 are temperature-sensitive for growth . We have compared gene expression in isogenic rpoD+ and rpoD800 cells during steady-state growth and after temperature shift, in order to define the role of the sigma subunit in vivo . We have shown that sigma synthesis is regulated . After temperature shift-up, sigma behaves like other heat-shock proteins . The stimulation of sigma synthesis by heat shock is greater in mutant than in wild-type cells, possibly because the cell is responding to sigma limitation at high temperature by over-producing sigma . Mutant cells continue protein synthesis for a short time after shift-up and then shut off the synthesis of all proteins in response to the decreasing intracellular concentration of sigma . During the initial period of high protein synthesis, the relative expression of many proteins is changed in mutant cells . We argue that these changes are predominantly an indirect, rather than a direct effect of mutant sigma and are due to a change in the physiological state of mutant cells . Finally, we have shown that degradation of mutant sigma results in a decrease in synthesis of all major messenger RNA and stable RNA species . If other sigma factors are present in exponentially growing cells, they do not appear to be involved in a significant fraction of RNA synthesis. FEBS Lett, 1984 Jan 23, 166(1), 194 - 8 A new reaction useful for chemical cross-linking between nucleic acids and proteins; Nitta N et al.; Cytosine in nucleic acids can be converted into N4-aminocytosine by treatment with a mixture of hydrazine and bisulfite . The hydrazino group thus formed at position 4 of the pyrimidine ring can be linked to a sulhydryl group in proteins by the use of bromopyruvate as a linker . Successful use of this scheme of chemical cross-linking between nucleic acid and protein was demonstrated in the linking of poly(C) with glutathione, and of RNA with protein in the E . coli 30 S ribosomal subunit. FEBS Lett, 1984 Jan 23, 166(1), 53 - 6 Ribosomal protein L16 binds to the 3'-end of transfer RNA; Maimets T et al.; Escherichia coli 50 S ribosomal subunits were reconstituted with and without protein L16 present . The latter particles, although active in puromycin reaction, were unable to use CACCA-Phe as an acceptor substrate . We also found that L16 interacts directly with this oligonucleotide and, in the complex with tRNA, protects its 3'-end from pancreatic ribonuclease digestion . We suggest that the role of L16 is in the fixation of the aminoacyl stem of tRNA to the ribosome at its A-site. FEBS Lett, 1984 Jan 23, 166(1), 179 - 82 The major outer membrane lipoprotein and new lipoproteins share a common signal peptidase that exists in the cytoplasmic membrane of Escherichia coli; Yamada H et al.; The cell envelope of Escherichia coli possesses several lipoproteins including the major outer membrane lipoprotein . These lipoproteins are synthesized as a signal peptide-carrying precursor that is subsequently modified with glyceride . In this work, lipoprotein signal peptidase that processes the precursor of the major lipoprotein was partially purified from cells harboring a plasmid that carries the gene for this enzyme (1spA) . The enzyme was also active against the glyceride-containing precursors of the peptidoglycan-associated lipoprotein and many additional membrane lipoproteins . The unmodified precursor of the major lipoprotein was not attacked by the enzyme . The enzyme was exclusively localized in the cytoplasmic membrane. FEBS Lett, 1984 Jan 23, 166(1), 67 - 70 Expression of human alpha 1-antitrypsin in Escherichia coli; Bollen A et al.; Complementary DNA coding for human alpha 1-antitrypsin has been placed under the control of the lambda PR promotor carrier by the expression vector pCQV2 {1} . In conditions which allow transcription from this promotor (thermoinactivation of the repressor), Escherichia coli cells harbouring the recombinant plasmid pULB1114 express human alpha 1-antitrypsin (+/- 9000 molecules/cell) . The product has a Mr of 44000, corresponding to mature unglycosylated alpha 1-antitrypsin. FEBS Lett, 1984 Jan 23, 166(1), 120 - 4 Use of the pH sensitive fluorescence probe pyranine to monitor internal pH changes in Escherichia coli membrane vesicles; Damiano E et al.; Measurements of the fluorescent properties of 8-hydroxy-1,3,6-pyrenetrisulfonate (pyranine) enclosed within the internal space of Escherichia coli membrane vesicles enable recordings and quantitative analysis of: (i) changes in intravesicular pH taking place during oxidation of electron donors by the membrane respiratory chain; (ii) transient alkalization of the internal aqueous space resulting from the creation of outwardly directed acetate diffusion gradients across the vesicular membrane . Quantitation of the fluorescence variations recorded during the creation of transmembrane acetate gradients shows a close correspondence between the measured shifts in internal pH value and those expected from the amplitude of the imposed acetate gradients. FEBS Lett, 1984 Jan 23, 166(1), 19 - 22 Amino acid sequence of the oligomycin sensitivity-conferring protein (OSCP) of beef-heart mitochondria and its homology with the delta-subunit of the F1-ATPase of Escherichia coli; Ovchinnikov YA et al.; The complete amino acid sequence of the oligomycin sensitivity-conferring protein (OSCP) of beef-heart mitochondria is reported . The protein contains 190 amino acids and has a molecular mass of 20 967 . Its structure is characterized by a concentration of charged amino acids in the two terminal segments (N 1-77 and C 128-190) of the protein, whereas its central region is more hydrophobic . The earlier reported homology of the protein with the delta-subunit of E . coli F1, based on the terminal amino acid sequences of OSCP, is further substantiated. J Theor Biol, 1984 Jan 21, 106(2), 89 - 102 Cellular control models with linked positive and negative feedback and delays . I . The models; Mahaffy JM; Basic techniques from biochemical kinetics are used to develop models for a cellular control system with linked positive and negative feedback . The models are represented by a system of nonlinear differential equations with delays . The lac operon provides an example of a control system where the transcription of the operon is controlled by induction or positive feedback control and catabolite repression or negative feedback control . These processes are linked through the metabolism of lactose. J Theor Biol, 1984 Jan 21, 106(2), 103 - 18 Cellular control models with linked positive and negative feedback and delays . II . Linear analysis and local stability; Mahaffy JM; An analysis of local behavior is made of two nonlinear models which incorporate both an induction or positive feedback control mechanism and a repression or negative feedback control mechanism . The systems of differential equations with delays are linearized about their equilibria . The related characteristic equations which are exponential polynomials are studied to determine the local stability of the models . Computer studies are included to show the range of stability for different parameter values, and the biological significance is discussed briefly. Science, 1984 Jan 20, 223(4633), 285 - 6 Catalytic activity of an RNA molecule prepared by transcription in vitro; Guerrier-Takada C et al.; Ribonuclease P is a ribonucleoprotein that cleaves precursors to transfer RNA (tRNA) molecules to yield the correct 5' terminal sequences of the mature tRNA's . The RNA moiety M1 RNA of ribonuclease P from Escherichia coli and the unprocessed transcript prepared in vitro of the gene for M1 RNA can both perform the cleavage reactions of the canonical enzyme in the absence of the protein moiety . When the transcript of the M1 RNA gene is combined with the protein moiety not only is a tRNA precursor cleaved but also the precursor to 4.5S RNA from Escherichia coli. Biochemistry, 1984 Jan 17, 23(2), 288 - 98 Nuclear magnetic resonance studies of amino acids and proteins . Deuterium nuclear magnetic resonance relaxation of deuteriomethyl-labeled amino acids in crystals and in Halobacterium halobium and Escherichia coli cell membranes; Keniry MA et al.; We have obtained deuterium (2H) Fourier transform nuclear magnetic resonance (NMR) spectra of zwitterionic L-{beta-2H3}alanine, DL-{gamma-2H6}valine, DL-{beta, gamma-2H4}threonine, L-{delta-2H3}leucine, and L-{alpha, beta, gamma, gamma', delta-2H10}isoleucine in the crystalline solid state and have determined the deuteriomethyl group spin-lattice relaxation rates as a function of temperature . The results yield the Arrhenius activation energies (delta E) for methyl rotation, and through use of a suitable mathematical model, rotational correlation times, tau c . For alanine, valine, threonine, leucine, and isoleucine at 37 degrees C, tau c and delta E values are 780, 100, 40, 38, and 18 ps and 22, 14.0, 17.6, 15.5, and 8.6 kJ, respectively . For L-{beta-2H3}alanine in the zwitterionic lattice, a spin-lattice relaxation time (T1) minimum of 2.1 +/- 0.3 ms is observed (at 0 degree C), in excellent agreement with the 1.92-ms prediction of the mathematical model . Similar tau c and delta E measurements are reported for bacteriorhodopsin in the purple membrane of Halobacterium halobium R1 and for Escherichia coli cell membranes . Overall, our results demonstrate a great similarity between the dynamics in amino acid crystals and in membrane proteins . However, threonine exhibits a nonlinear Arrhenius behavior in bacteriorhodopsin, and in the valine-, leucine-, and isoleucine-labeled membrane samples at higher temperatures (approximately greater than 37 degrees C), there is evidence of an additional slow side-chain motion . The lipid phase state in E . coli does not appear to influence, on the average, the dynamics of the valine side chains . These results indicate that the sensitivity of the deuterium NMR technique is now adequate to study in moderate detail the dynamics of most types of amino acids in a membrane protein and that adequate sensitivity, in some instances, should be available for the study of individual amino acids in suitably labeled membrane proteins. Biochemistry, 1984 Jan 17, 23(2), 211 - 5 Vicinal dithiol-disulfide distribution in the Escherichia coli mannitol specific carrier enzyme IImtl; Roossien FF et al.; Escherichia coli mannitol specific EII in membrane vesicles can be inhibited by the action of the oxidizable substrate-reduced phenazine methosulfate (PMS) in a manner similar to E . coli enzyme IIGlc {Robillard, G . T., & Konings, W . (1981) Biochemistry 20, 5025-5032} . The fact that reduced PMS and various oxidizing agents protect the enzyme from inactivation by the sulfhydryl reagents N-ethylmaleimide and bromopyruvate suggests that the active form possesses a dithiol which can be protected by conversion to a disulfide . The sulfhydryl-disulfide distribution has been examined in purified EIImtl by labeling studies with N-{1-14C}ethylmaleimide ( {14C}NEM) . EIImtl can be alkylated at three positions per peptide chain . When alkylation takes place in 8 M urea, only two positions are labeled . The third position becomes labeled in urea only after treatment with DTT, suggesting that the native enzyme is composed of two subunits linked by a disulfide bridge . The remaining two sulfhydryl groups per peptide chain appear to undergo changes in oxidation state as indicated by the following results . (1) Treatment of the active enzyme with NEM leads to complete inactivation and incorporation of 1 mol of {14C}NEM per peptide chain . Oxidizing agents protect the activity and prevent labeling presumably by forming a disulfide . (2) Phosphorylating the enzyme (one phosphoryl group per peptide chain) fully protects the activity, but 1 mol of NEM per peptide chain is still incorporated . Subsequent dephosphorylation by adding mannitol causes a second mole of {14C}NEM to be incorporated and results in complete inactivation.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1984 Jan 17, 23(2), 182 - 90 Phenylalanyl-tRNA synthetase of Escherichia coli K10 . Effects of zinc(II) on partial reactions of diadenosine 5',5"'-P1,P4-tetraphosphate synthesis, conformation, and protein aggregation; Goerlich O et al.; The synthesis of diadenosine 5',5"'-P1-,P4-tetraphosphate (Ap4A) catalyzed by phenylalanyl-tRNA synthetase in the presence of Zn2+ involves the same partial reactions (synthesis of phenylalanyladenylate and transfer of the adenylate moiety to ATP) as occur in the absence of this metal ion . However, transfer is strongly stimulated while adenylate synthesis is depressed . Also inhibited are pyrophosphorolysis of phenylalanyladenylate and transfer of phenylalanine from the adenylate to cognate tRNA, because overall tRNA phenylalanylation is depressed {Mayaux, J.-F., & Blanquet, S . (1981) Biochemistry 20, 4647-4654}, whereas binding of tRNA to the synthetase is not . At moderate concentrations of Zn2+, and in the presence of 5 microM phenylalanine and 0.5 mM ATP, transfer of AMP is rate limiting, while at higher concentrations of Zn2+ synthesis of adenylate is rate determining . The Zn2+ concentration optimum for stimulation depends on the concentration of phenylalanine and ATP . The effects of Zn2+ are mediated through two classes of binding site(s) on the synthetase, the half-saturations of which are 1-4 and 20-30 microM Zn2+, respectively . Binding of Zn2+ to the second class of site(s) causes inhibition of the synthetase, whereas binding to the first class is responsible for activation and inhibition, which may be caused by a conformational change . Evidence for the latter is the observed decrease in protein intrinsic fluorescence intensity and the decrease in fluorescence intensity of 6-(p-toluidinyl)naphthalene-2-sulfonate, which is used as a reporter group . The kinetics of the binding reaction show a saturation dependence on Zn2+, also suggesting that a conformational change occurs.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1984 Jan 17, 23(2), 197 - 203 Amplification and isolation of Escherichia coli nusA protein and studies of its effects on in vitro RNA chain elongation; Schmidt MC et al.; The Escherichia coli nusA gene product is an RNA polymerase binding protein which has been implicated in a variety of cellular and viral termination and antitermination processes . To facilitate large-scale preparation and biochemical studies of the nusA protein, we have cloned the nusA gene into a lambda PL-derived overexpression vector . E . coli strains bearing the resulting plasmid (pMS7) produce large amounts of nusA protein when induced, and the protein is easily purified to homogeneity . Biochemical studies of nusA protein reveal that it inhibits in vitro RNA chain elongation by E . coli RNA polymerase with a variety of templates . Two modes of inhibition are found . Inhibition of elongation with poly{d(A-T} ) template is completely competitive with nucleoside triphosphates and shows an inhibitory constant (Ki) of 3 X 10(-7) M . In contrast, inhibition of elongation with T7 DNA as template is mixed . One component of the inhibition is competitive with nucleoside triphosphate substrates and is reversed at elevated substrate concentrations . A second inhibitory component remains even at saturating substrate concentrations; this sequence-dependent mode of inhibition shows a much lower Ki of 2 X 10(-8) M . The existence of two different modes of inhibition might be explained if two molecules of nusA protein can bind to each RNA polymerase complex . The interaction of nusA protein with elongating RNA polymerase molecules is not processive but appears to be characterized by rapid association and dissociation . Under proper conditions, a sigma-nusA cycle {Greenblatt, J., & Li, J . (1981) Cell (Cambridge, Mass.) 24, 421-428} can be demonstrated in vitro in which each polymerase goes through multiple rounds of transcription involving successive interactions with sigma and the nusA protein. J Mol Biol, 1984 Jan 15, 172(2), 223 - 7 Escherichia coli DNA photolyase is a flavoprotein; Sancar A et al.; Escherichia coli DNA photolyase (photoreactivating enzyme) was purified to homogeneity from a strain that greatly overproduces the protein . The purified enzyme has absorption peaks at 280 and 380 nm, a fluorescence emission peak at 480 nm and, upon denaturation, releases a chromophore that has the spectroscopic properties of flavin adenine dinucleotide (FAD), indicating that FAD is an intrinsic chromophore of the enzyme. J Mol Biol, 1984 Jan 15, 172(2), 177 - 84 Nucleotide substitution in the amino acid acceptor stem of lysine transfer RNA causes missense suppression; Prather NE et al.; Previous results from this laboratory indicated that, in Escherichia coli K12, a new class of missense suppressors, which read the lysine codons AAA and AAG, may be misacylated lysine transfer RNAs . We therefore isolated and determined the nucleotide sequence of the lysine tRNA from two of the suppressor strains . In each case, we found both wild-type and mutant species of lysine tRNA, a result consistent with evidence that there are two genes for lysine tRNA in the E coli genome . The wild-type sequence was essentially identical to that reported for lysine tRNA from E . coli B . The mutant species isolated from each suppressor strain had a U for C70 nucleotide substitution, demonstrating that the AAG suppressor is a mutant lysine tRNA . The nucleotide substitution in the amino acid acceptor stem is consistent with the in vivo evidence that the suppressor corrects AAA and AAG missense mutations by inserting an amino acid other than lysine during polypeptide synthesis . This report represents the first verification of missense suppression caused by misacylation of a mutant tRNA. J Mol Biol, 1984 Jan 15, 172(2), 185 - 201 Control of expression of the Tn10-encoded tetracycline resistance operon . II . Interaction of RNA polymerase and TET repressor with the tet operon regulatory region; Hillen W et al.; The promoter and operator sequences of the Tn10-encoded tetracycline resistance operon are determined in vitro by transcription studies of purified DNA restriction fragments, protection of guanosine from methylation by dimethylsulphate, and DNase I footprinting employing the purified TET repressor protein . In vitro transcription reveals three promoters with overlapping consensus sequences . Two of them, designated PR1 and PR2, are directed towards the tet repressor gene and the third, called PA, initiates transcription of the tet resistance gene . All three promoters are regulated simultaneously by the TET repressor protein, as demonstrated by in vitro transcription . Tetracycline functions as an inducer in these experiments . Two palindromic operator sequences in the tet operon control region, called O1 and O2, are occupied simultaneously by the TET repressor . Four guanosine residues in symmetric positions close to the centre of the palindromic operator sequences are protected from methylation in the repressor-operator complex . However, only one guanosine residue exhibits an enhanced reaction with dimethylsulphate under these conditions . Footprinting experiments reveal protection of phosphodiester bonds against DNase I slightly further than the palindromic sequence arrangement . Several phosphodiester bonds between the two operators are accessible for cleavage by DNase I in the repressor-operator complex . Two phosphodiester bonds within each operator sequence are cleaved by DNase I . This feature shows a clear assymmetry with the two inside cleavage positions of O1 and O2 being much less accessible for DNase I as compared to the two outside positions . A molecular mechanism of regulation of the Tn10-encoded tetracycline resistance operon is presented based on these and previous results. Lancet, 1984 Jan 14, 1(8368), 63 - 6 Identification by DNA hybridisation of enterotoxigenic Escherichia coli in homes of children with diarrhoea; Echeverria P et al.; The DNA hybridisation technique to detect genes coding for Escherichia coli enterotoxin was used to identify enterotoxigenic E coli (ETEC) in homes of children with diarrhoea in Thailand . ETEC was found in 30 (14%) of 221 children with diarrhoea and in 9% (8/88) of their household contacts, 8% (8/101) of their neighbours, and 2% (32/1379) of inhabitants of 382 homes not associated with ETEC infections . ETEC was found significantly more often in water and food and on mothers' hands in homes of children with ETEC-associated diarrhoea and of their neighbours than in homes of children without ETEC infections (8/360 vs 3/2290; p less than 0.001) . ETEC was identified in 80% (71/89) of specimens that hybridised with the enterotoxin gene probes by testing E coli isolated from the same specimen in the Y-1 adrenal and suckling-mouse assays . The DNA hybridisation assay to detect genes coding for E coli enterotoxin is an effective method of identifying ETEC in a large number of human and environmental specimens and will be a valuable tool to define further the epidemiology of this enteric pathogen. Biochem Biophys Res Commun, 1984 Jan 13, 118(1), 270 - 7 Reversion of the effects of a threonine deaminase regulatory mutant by a mutation in ilvH in Escherichia coli K-12; Singer PA et al.; In a strain carrying an ilvA538 mutation, the ilvGEDA operon expression is decreased (hyperattenuated) and the activity and/or expression of isoleucyl- and valyl- tRNA synthetases is decreased . We have isolated two revertants of ilvA538 owing to mutations in the ilvH gene, whose product is acetohydroxy acid synthase III . The regulatory properties of these revertants are consistent with a dual role for threonine deaminase as an effector of the ilvGEDA operon and the isoleucyl- and valyl- tRNA synthetase structural genes. Nucleic Acids Res, 1984 Jan 11, 12(1 Pt 2), 789 - 800 Escherichia coli promoter sequences predict in vitro RNA polymerase selectivity; Mulligan ME et al.; We describe a simple algorithm for computing a homology score for Escherichia coli promoters based on DNA sequence alone . The homology score was related to 31 values, measured in vitro, of RNA polymerase selectivity, which we define as the product KBk2, the apparent second order rate constant for open complex formation . We found that promoter strength could be predicted to within a factor of +/-4.1 in KBk2 over a range of 10(4) in the same parameter . The quantitative evaluation was linked to an automated (Apple II) procedure for searching and evaluating possible promoters in DNA sequence files. Nucleic Acids Res, 1984 Jan 11, 12(1 Pt 2), 551 - 67 Measurements of the effects that coding for a protein has on a DNA sequence and their use for finding genes; Staden R; Protein genes can be found either by searching the DNA sequence for signals such as ribosome binding sites or by looking for the effects that coding for a protein has on the coding sequence . This paper describes how these coding effects can be measured and used to detect protein coding regions. Nucleic Acids Res, 1984 Jan 11, 12(1 Pt 2), 521 - 38 Graphic methods to determine the function of nucleic acid sequences; Staden R; We describe an interactive computer program (ANALYSEQ) that is used from a simple graphics terminal . The main purpose of the program is to determine the function of nucleic acid sequences but it also offers the simpler listing, searching and counting options . It contains methods to locate genes by looking for the effects that coding for a protein has on the coding sequence, to locate tRNA genes by looking for secondary structure and conserved bases, and methods to locate signals such as promoters . Techniques to identify unusual regions of sequence and to search for potential Z DNA-forming regions are also included . Most of the routines produce graphical output which gives ease of interpretation and allows superposition of several independent forms of analysis. Nucleic Acids Res, 1984 Jan 11, 12(1 Pt 2), 505 - 19 Computer methods to locate signals in nucleic acid sequences; Staden R; This paper describes computer methods for locating signals in nucleic acid sequences . The signals include ribosome binding sites, promoter sequences and splice junctions . The methods are of use both to those trying to interpret the function of newly determined sequences and to those studying the molecular mechanisms involved in the recognition of these special signal sequences. Nucleic Acids Res, 1984 Jan 11, 12(1 Pt 2), 657 - 64 JINN, an integrated software package for molecular geneticists; Johnsen M; I describe JINN, a microcomputer-based system designed to maintain and search a strain collection, to enter, modify and analyze sequences, and to use the EMBL Sequence Data Base . The major objective during development of this program has been integration of individual program modules to ensure a consistent and helpful user interface . The system is running under the CP/M operating system and requires little in the way of particular hardware configuration. J Biol Chem, 1984 Jan 10, 259(1), 487 - 90 The primary structure of rat liver ribosomal protein L39; Lin A et al.; The covalent structure of the rat liver 60 S ribosomal subunit protein L39 was determined . Fourteen tryptic peptides were purified, and the sequence of each was established by a micromanual procedure; they accounted for all 50 residues of L39 . The sequence of the NH2-terminal 32 residues of L39, obtained by automated Edman degradation of the intact protein, provided the alignment of the first seven tryptic peptides . Two peptides, CNI (28 residues) and CNII (22 residues), were produced by cleavage of protein L39 with cyanogen bromide and the sequence of CNII was determined by automated Edman degradation . This sequence established the order of tryptic peptides T8 through T14 . The carboxyl-terminal amino acids were identified after carboxypeptidase A treatment . Protein L39 contains 50 amino acids and has a molecular weight of 7308 . There are indications that a portion of rat L39 is related to a fragment of Escherichia coli ribosomal protein S1. J Biol Chem, 1984 Jan 10, 259(1), 526 - 33 Protein phosphorylation in Escherichia coli and purification of a protein kinase; Enami M et al.; More than 40 protein species including RNA polymerase were found to be phosphorylated in Escherichia coli on analyses of 32P-labeled cell lysates by single and two-dimensional gel electrophoresis and autoradiography . The protein species and the level of phosphorylation varied depending on the cell growth phase . With {gamma-32P}ATP as a substrate, cell lysates phosphorylated endogenous proteins in vitro which were predominantly phosphorylated in vivo . Both serine and threonine were the major phosphate acceptors in whole cell lysates . Starting from a partially purified RNA polymerase preparation with the protein phosphorylation activity and using an E . coli protein with an apparent Mr = 90K (K represents X 1000) as the substrate, we purified a protein kinase with a native Mr approximately 120K to apparent homogeneity . The protein kinase is either a heterodimer of 61K and 66K polypeptides or a homodimer of one of these polypeptides . We also isolated a 100K protein with self-phosphorylation activity. J Biol Chem, 1984 Jan 10, 259(1), 378 - 82 Cloning and expression in Escherichia coli of a yeast mannosyltransferase from the asparagine-linked glycosylation pathway; Couto JR et al.; The yeast Saccharomyces cerevisiae temperature-sensitive lethal mutant alg1-1, has been previously shown to lack the activity necessary for the addition of the first mannose residue in the synthesis of lipid-linked precursor oligosaccharide . The gene ALG1 has been cloned by complementation of the temperature-sensitive mutation alg1-1 with a total genomic DNA library . The original DNA fragment isolated was 11,300 base pairs and has been subcloned to a 1,500-base pair fragment which is still capable of complementing alg1-1 . The gene ALG1 has been mapped on chromosome II at a distance of 2.1 map units from LYS2 . The ALG1 gene product has been shown to catalyze the transfer of a mannosyl residue from GDP-mannose to the lipid-linked acceptor GlcNAc2, yielding Man beta 1-4GlcNAc2-lipid, in lysates from Escherichia coli transformants . This result proves that ALG1 is the structural gene for the first mannosyltransferase in lipid-linked oligosaccharide assembly. J Biol Chem, 1984 Jan 10, 259(1), 284 - 9 Nuclear magnetic resonance studies on the role of intrinsic metals in Escherichia coli RNA polymerase . Effect of DNA template on the nucleotide-enzyme interaction; Chatterji D et al.; Nuclear magnetic resonance studies were performed to investigate the effect of DNA template on the interaction of initiating nucleotide ATP with Escherichia coli RNA polymerase (RPase) in which one of the two intrinsic Zn ions was substituted with a Co(II) (Co-Zn RPase) or Mn(II) (Mn-Zn RPase) ion . This intrinsic metal ion is located at the initiation site in the beta subunit of RPase . The paramagnetic effects of Co-Zn and Mn-Zn RPases on the relaxation rates of 1H- and 31P-nuclei of ATP were used to determine the distances from the intrinsic metal to various atoms of ATP bound at the initiation sites in the presence of DNA . The distances from the metal to H2, H8, H1', alpha-P, beta-P, and gamma-P atoms were estimated to be 6.7 +/- 0.9, 4.1 +/- 0.6, 6.0 +/- 1.2, 7.5 +/- 0.8, 9.4 +/- 1.0, and 9.8 +/- 1.0 A, respectively . These distances were compared with those measured in the absence of DNA (Chatterji, D., and Wu, F . Y.-H . (1982) Biochemistry 21, 4657) . In both the presence and absence of DNA, the close proximity between the intrinsic metal and the H8 atom strongly indicates that the metal is coordinated directly to the base moiety of ATP . Such a coordination may provide a structural basis for the selection of a purine nucleotide during the initiation process . The presence of DNA causes the H2 atom to move away (greater than 2 A) from the intrinsic metal, whereas all three phosphorus atoms shift closer (greater than 3 A) toward the metal . The possible mechanistic implications of the conformational alteration of ATP at the initiation site induced by the DNA template is discussed. J Biol Chem, 1984 Jan 10, 259(1), 190 - 6 Purification of a small receptor-binding peptide from the central region of the colicin E1 molecule; Brunden KR et al.; An Mr = 16,000 receptor-binding fragment of colicin E1 has been obtained by cyanogen bromide digestion of colicin E1 . The purified 16-kDa fragment shows binding properties similar to those of an Mr = 38,000 colicin E1 receptor-binding fragment generated by thermolysin treatment . Treatment of the 38-kDa fragment with cyanogen bromide also yields the 16-kDa fragment . By comparing the NH2-terminal amino acid sequence of the 16-kDa fragment with the known colicin E1 sequence, the receptor-binding fragment can be shown to occupy the central region of the colicin molecule, extending from residue 231 to 370 . It is inferred that the 16-kDa fragment binds efficiently to the colicin receptor because it is able to protect sensitive cells against the lethal effects of colicins E1 and E2 and, when pre-adsorbed to the cell, to physically displace colicin E1 . Unlike the 38-kDa receptor-binding fragment, the 16-kDa fragment was found to be devoid of channel-forming ability previously shown to be associated with the COOH-terminal region of the colicin E1 polypeptide. J Biol Chem, 1984 Jan 10, 259(1), 97 - 101 Nucleotide sequence of dnaB and the primary structure of the dnaB protein from Escherichia coli; Nakayama N et al.; We have determined the nucleotide sequence of the dnaB gene and the primary structure of the dnaB protein of Escherichia coli (Arai, K., Yasuda, S., and Kornberg, A . (1981) J . Biol . Chem . 256, 5247-5252) . The coding region for the dnaB protein is 1413 base pairs followed by double stop codons and preceded by a possible promoter sequence .