J Biochem (Tokyo), 1988 Sep, 104(3), 317 - 8 Three-dimensional structure of aspartate aminotransferase from Escherichia coli at 2.8 A resolution; Kamitori S et al.; The crystal structure of aspartate aminotransferase of Escherichia coli was determined by X-ray structure analysis at 2.8 A resolution . The structure was solved by the molecular replacement method and refined to an R-factor of 0.27, and it was found that the overall structure of AspAT of E . coli is similar to that of those of higher animals. Protein Eng, 1988 Sep, 2(3), 233 - 7 Apomyoglobin as a molecular recognition surface: expression, reconstitution and crystallization of recombinant porcine myoglobin in Escherichia coli; Dodson G et al.; Recombinant porcine myoglobin has been produced in Escherichia coli using the lambda cII fusion expression system of Nagai and Thogersen {Nature, 309, 810-812 (1984)} . After processing and reconstitution with haem, the protein is gel-electrophoretically and spectrophotometrically indistinguishable from native pig myoglobin . Large crystals of both native and recombinant porcine myoglobin were grown from 50 mM sodium phosphate, pH 7.1, 80% ammonium sulphate . The crystals belong to space group C2 (a = 156.9 A, b = 42.0 A, c = 92.2 A, beta = 127.9 degrees) and diffract to a nominal 2.5 A resolution . We plan to explore apomyoglobin as a binding surface in studies combining site-directed mutagenesis and X-ray analysis . These experiments will be extended by studying the binding of haem analogues to the mutant apoproteins. Protein Eng, 1988 Sep, 2(3), 227 - 31 Conversion of the guanine nucleotide binding sites of ras protein resulting in the reduction of base specificity; Miura K et al.; A gene coding for the novel ras protein, p21X, in which the domains of guanine binding and phosphate binding were exchanged, was constructed and expressed in Escherichia coli . The gene product, p21X, showed GTP binding activity, but no GPTase activity . In addition, p21X revealed binding activity toward ATP and CTP . In a competitive binding assay, {3H}GTP binding to p21X was inhibited in the presence of ATP, CTP and UTP, ITP as well as GDP, GTP and dGTP. Biol Chem Hoppe Seyler, 1988 Sep, 369(9), 1045 - 54 Expression of normal and mutagenized apolipoprotein CII in procaryotic cells . Structure-function relationship; Holtfreter C et al.; A full-length human apo CII cDNA clone has been constructed by completing the 5' end of an incomplete cDNA with a 44 bp long synthetic oligonucleotide . This apo CII cDNA insert was cloned into the pSP19 expression vector and transcribed and translated in vitro . Its N-terminal signal sequence (23 amino-acid residues) was accurately cleaved during cotranslational translocation through endoplasmic reticulum membranes to yield the mature apo CII . Mature apo CII was expressed on a preparative scale as fusion protein apo CII-beta-galactosidase with the full-length apo CII cDNA integrated into the pUR291 vector . Furthermore it was expressed in E . coli transformed with the pKK233-2 apo CII clone . The preform was accurately processed by the host cell . C-Terminal apo CII deletion mutants generated by partial Bal31 digestion of the pKK233-2 apo CII vector yielded well-defined truncated apo CII polypeptides on a preparative scale which allowed the determination of the polypeptide domain responsible for the activation of the serum lipoprotein lipase. Mol Gen Genet, 1988 Sep, 214(1), 80 - 4 Multicopy plasmid stability in Escherichia coli requires host-encoded functions that lead to plasmid site-specific recombination; Stirling CJ et al.; The heritable stability of the multicopy plasmid ColE1 and its natural relatives, requires the presence in the plasmid of a site (cer in ColE1) that acts as a substrate for site-specific recombination, thereby maintaining plasmids in the monomeric state . Multimerization, promoted by homologous recombination, leads to plasmid loss . Here we show that the Escherichia coli chromosome encodes at least two unlinked functions that act on cer and its analogous sites, to promote stabilizing site-specific recombination . One of these functions is encoded by a gene residing on a cosmid that also contains the argI and pyrB genes, mapping it to the 96-97 min region of the E . coli map. Arzneimittelforschung, 1988 Sep, 38(9), 1381 - 6 Prophylactic and curative effects of recombinant human superoxide dismutase in lethal rat endotoxemia; Schneider J; The continuous infusion of E . coli lipopolysaccharide (100 mg/kg in 4 h) caused a 100%-mortality in pentobarbital-anesthetized rats within 6 h . Recombinant human superoxide dismutase (r-HSOD) infused concomitantly with the E . coli endotoxin dose-dependently (0.1-1.0 mg/kg per min) increased survival rate up to 90% . Significant improvement of survival rate was also obtained when r-HSOD-infusion (0.464 mg/kg per min) was only started up to 3 h after beginning of the endotoxin application . Also, two single bolus injections of r-HSOD (20 mg/kg each) during endotoxemia significantly increased survival rate . Decrease of heart rate was prevented and decline of arterial blood pressure was diminished by r-HSOD (0.464 mg/kg per min) as compared to vehicle-treated endotoxemic rats . Lactic acidosis occurred with no significant statistical difference between r-HSOD- and vehicle-treated groups . Increase of hematocrit in endotoxemic control rats was balanced by fluid uptake . In contrast, in the groups treated with endotoxin plus r-HSOD or saline alone hematocrit decreased identically . Decrease of whole blood leukocytes (to 30.2 +/- 9.5% of baseline in endotoxemic controls) was less pronounced in the r-HSOD group (fall to 49.2 +/- 6.5% of baseline), but this difference did not reach statistical significance . Marked thrombocytopenia (to 12.9 +/- 3.2% of baseline) and consumption of plasma fibrinogen (to 39.5 +/- 10.3% of baseline) were significantly attenuated in r-HSOD-treated rats, where thrombocytes only decreased to 28.1 +/- 3.6% and plasma fibrinogen to 76.6 +/- 5.0% of baseline values at the end of endotoxin infusion.(ABSTRACT TRUNCATED AT 250 WORDS) Am J Vet Res, 1988 Sep, 49(9), 1641 - 3 Effect of acupuncture on young pigs with induced enteropathogenic Escherichia coli diarrhea; Hwang YC et al.; Thirty-four preweaning pigs with induced enteropathogenic Escherichia coli diarrhea were treated with electroacupuncture, traditional acupuncture, or neomycin . In the group treated with electroacupuncture, points GV-1, bilateral ST-36, and Bai-hui were stimulated electrically . In the group treated with traditional acupuncture, points GV-1, bilateral ST-36, BL-20, bulb points, bilateral ear tip, and Shan-gen were used . Acupuncture points CV-12 and bilateral ST-25 also were treated with moxibustion (applying heat generated by a burning herb, Artemisia argyi) . Hemoacupuncture also was applied to Shan-gen, bilateral ear tip, and bulb points . Pigs in the third group were given neomycin orally . Five pigs were inoculated with E coli, but were not treated and served as nontreated controls . At postinoculation day 5, 60% of control pigs and greater than 80% of pigs in treated groups recovered from diarrhea . However, at postinoculation day 3, recovery rates for pigs in the control and group treated with electroacupuncture were only 20 and 27.3%, respectively; whereas pigs treated with acupuncture or neomycin attained 81.8 and 71.4% of recovery rates, respectively . Seemingly, traditional acupuncture, but not electroacupuncture, was effective in controlling induced E coli diarrhea in pigs at its early stage. Mol Biol (Mosk), 1988 Sep-Oct, 22(5), 1301 - 6 {Effect of a fragment of pBR322 plasmid in the region of tetracycline resistance gene on the stability of this plasmid}; Kolot MN et al.; A fragment destabilizing the pBR322 plasmid has been localized in the region of pBR322 tet gene promoter . On the basis of stability analysis of pBR322 derivatives comprising the modified region of tet gene, we deduced that it is the DNA sequence localized at the beginning of tet gene in the region of HindIII splitting site that ensures the plasmid destabilization, and not tet gene product or the active transcription of this region . The destabilizing effect of this fragment of the plasmid is manifested both in cis and in trans . Possible molecular mechanisms of the phenomenon are discussed. Mol Biol (Mosk), 1988 Sep-Oct, 22(5), 1198 - 203 {Accumulation of N-terminal fragment of recA protein in the htpR- mutant impairs the SOS-function of Escherichia coli cells}; Kiselev VI et al.; It has been established that the plasmid encoding the N-terminal domain of RecA protein (50 amino acids residues) disturbs SOS functions of htpR- mutants of E . coli . This is expressed in increased UV-sensitivity of strains and in the poor transcription of the aminoglycosidephosphoransferase gene in the hybrid operon recA APT . The discovered effect is due to an increase in the stability of the peptide whose accumulation seems to impede the production of RecA protease . Mutation of gene lon does not lead to the accumulation of N-terminal fragment, therefore an increased stability of the peptide in htpR mutants is probably related to the absence of an unidentified peptidase. Genetics, 1988 Sep, 120(1), 37 - 45 The genetic dependence of recombination in recD mutants of Escherichia coli; Lovett ST et al.; RecBCD enzyme has multiple activities including helicase, exonuclease and endonuclease activities . Mutations in the genes recB or recC, encoding two subunits of the enzyme, reduce the frequency of many types of recombinational events . Mutations in recD, encoding the third subunit, do not reduce recombination even though most of the activities of the RecBCD enzyme are severely reduced . In this study, the genetic dependence of different types of recombination in recD mutants has been investigated . The effects of mutations in genes in the RecBCD pathway (recA and recC) as well as the genes specific for the RecF pathway (recF, recJ, recN, recO, recQ, ruv and lexA) were tested on conjugational, transductional and plasmid recombination, and on UV survival . recD mutants were hyper-recombinogenic for all the monitored recombination events, especially those involving plasmids, and all recombination events in recD strains required recA and recC . In addition, unlike recD+ strains, chromosomal recombination events and the repair of UV damage to DNA in recD strains were dependent on one RecF pathway gene, recJ . Only a subset of the tested recombination events were affected by ruv, recN, recQ, recO and lexA mutations. Hinyokika Kiyo, 1988 Sep, 34(9), 1665 - 7 {A case of pyogenic orchitis with abscess formation}; Numa H et al.; A 34-year-old man visited our clinic because of right testicular swelling, pain and scrotal erythema . At first he was diagnosed as having acute epididymitis and received medical treatment . Nevertheless, his course was poor and was hospitalized after 1 month . Incision to the scrotum was performed and about 30 ml of dark yellow pus was drained . Culture of the pus yielded E . coli which was susceptible to all kinds of antibiotics . The local symptoms subsided, but the right testicular pain still persisted . Right epididymectomy was attempted on the 22nd hospital day . At the operation an abscess formation in testis was found but no marked changes in the epididymis were found . Right orchiectomy was performed . Histology of testis showed extensive necrosis and severe infiltration of many inflammatory cells . We report this rare case of pyogenic orchitis with abscess formation. Mol Gen Mikrobiol Virusol, 1988 Sep, (9), 8 - 14 {Molecular cloning of plasmid ColIb-P9 genes responsible for its mutator function}; Kolot MN et al.; The localization of plasmid ColIb-P9 muc genes mediating the plasmids protective and mutagenesis-increasing activity has been determined . The increase of muc genes dose by cloning them within the multicopy vector has been shown to repress the mutator function of the plasmid . No essential homology has been revealed between ColIb-P9 muc gene nucleotide sequences, pKM101 muc genes with a similar function, and umuDC chromosome genes . It has been shown that the synthesis of 38 KD protein is essential for the manifestation of the mutator function of the plasmid. Biokhimiia, 1988 Sep, 53(9), 1474 - 8 {Purification of restriction endonuclease EcoRII using monoclonal antibodies}; Kosykh VG et al.; Monoclonal antibodies against EcoRII endonuclease were obtained after immunization of two BALB/c mice with a homogeneous enzyme prepared by conventional methods . IgG from ascitic fluid was purified and coupled to CNBr-activated Sepharose 4B to give a specific column used to isolate EcoRII endonuclease . The isolated EcoRII endonuclease produced a single band during SDS gel electrophoresis. Scanning Microsc, 1988 Sep, 2(3), 1567 - 86 Ultrastructural and functional effects of lipopolysaccharide and interleukin-2 on human NK cells; Kang YH et al.; Bacterial endotoxin (lipopolysaccharide, LPS) and interleukin-2 (IL-2) are known to stimulate NK cell mediated cytotoxicity against tumor cells . In the present report we sought to correlate the stimulatory effect of LPS and IL-2 on NK cell activity with ultrastructural changes which occurred as a result of such stimulation . Peripheral blood mononuclear cells (PBMC) were purified from healthy donors by a Ficoll-Hypaque density gradient technique . Leu-11a+ NK cells were isolated by flow microfluorometry using a monoclonal FITC conjugated anti-Leu-11a antibody and a FACS II cell sorter . The PBMC were incubated, respectively, with E . coli LPS or recombinant IL-2 (IL-2) for various time periods . Sorted Leu-11a+ NK cells were incubated with LPS for 24 hours . The NK cytotoxicity in the PBMC and sorted Leu-11a+ cells was assessed by a 51Cr release technique using K562 tumor cells as targets . Leu-11a+ NK cells were identified by immunoelectron microscopy using anti-Leu-11a antibody and labeling with horseradish peroxidase or colloidal gold . Results showed that both LPS and IL-2 significantly enhanced the cytotoxic activity of PBMC . The cytotoxicity of sorted Leu-11a+ cells was augmented by LPS . Recombinant IL-2 induced a significant increase in the number of dense granules, hypertrophy of Golgi apparatus and rough endoplasmic reticulum, and mitosis of Leu-7+ cells and Leu-11a+ cells 4 or 7 days after stimulation . These data indicate that: (1) the effect of LPS on the enhancement of NK cytotoxicity in PBMC may be a direct and/or indirect process involving production of lymphokines; (2) LPS has a direct effect on sorted Leu-11a+ cells; (3) IL-2 stimulates mitosis of Leu-7+ cells and Leu-11a+ cells; and (4) the LPS or IL-2 induced ultrastructural changes in Leu-11a+ cells are consistent with the enhanced NK cytotoxicity. Immunol Lett, 1988 Sep, 19(1), 65 - 9 The use of a 'universal' yeast expression vector to produce an antigenic protein of Mycobacterium leprae; Booth RJ et al.; This report describes the use of a recombinant yeast expression vector to synthesize and secrete the Mycobacterium leprae 18 kDa antigenic protein . The protein is secreted with a short hydrophilic 'flag' octapeptide fused to its amino-terminus . The fusion protein can be purified directly from yeast culture supernatant through an anti-flag antibody affinity column and the flag octapeptide removed using enterokinase . The method provides a simple and rapid means of obtaining recombinant 18 kDa antigen in quantities suitable for immunological studies. Anal Biochem, 1988 Sep, 173(2), 376 - 82 A direct nonchromatographic assay for 1-acyl-sn-glycerol-3-phosphate acyltransferase; Rajasekharan R et al.; 1-Acyl-sn-glycerol-3-phosphate acyltransferase (also called lysophosphatidic acid acyltransferase) which catalyzes the acylation of 1-acyl-sn-glycerol-3-phosphate to phosphatidic acid is generally assayed by the use of a radioactive substrate followed by a time-consuming chromatographic separation of substrate and product . We report a direct and highly sensitive nonchromatographic assay for this enzyme based on the ability of Escherichia coli alkaline phosphatase to dephosphorylate 1-acyl-sn-glycerol-3-phosphate but not phosphatidic acid . This selective hydrolysis coupled with the use of 32P-labeled 1-acyl-sn-glycerol-3-phosphate as substrate permits measurement of the product, 32P-labeled phosphatidic acid by solvent extraction or precipitation . We also report a series of enzymatic reactions for the efficient conversion of 32Pi to 32P-labeled 1-acyl-sn-glycerol-3-phosphate. Anal Biochem, 1988 Sep, 173(2), 241 - 5 Identification of Escherichia coli ribosomal proteins by an alternative two-dimensional electrophoresis system; Datta DB et al.; We have developed a two-dimensional gel electrophoretic system for the identification of Escherichia coli ribosomal proteins that involves the use of acid-urea in the first dimension and sodium dodecyl sulfate in the second dimension . This system has high sensitivity, resolution, and speed, and it is more convenient than others previously described . We have identified individual E . coli ribosomal proteins by this system. Mol Microbiol, 1988 Sep, 2(5), 553 - 62 Alterations in the carboxy-terminal half of cloacin destabilize the protein and prevent its export by Escherichia coli; van Putten AJ et al.; Several overlapping carboxy-terminal and internal deletions were constructed in the cloacin structural gene . The expression, the binding of the cloacin DF13 immunity protein and the release into the culture medium of the mutant cloacin polypeptides were studied by immunoblotting and ELISAs . Minor alterations at the carboxy-terminal end of the cloacin did not affect protein expression, stability or release to a large extent, but larger carboxy-terminal deletions strongly destabilized the protein and no release was observed . The removal of a particular region within the carboxy-terminal portion of cloacin strongly destabilized the polypeptide and made it a target for proteolytic degradation . Binding of immunity protein did not affect stability and release of the mutant polypeptides . By using immunoelectron microscopy, the polypeptides that were not exported were located in the cytoplasm of producing cells . Large aggregates of these mutant polypeptides were not observed in the cytoplasm: the polypeptides were present in a soluble form. J Clin Microbiol, 1988 Sep, 26(9), 1626 - 9 Mannose-resistant hemagglutination of human erythrocytes by enterotoxigenic Escherichia coli with colonization factor antigen II; Evans DJ Jr et al.; The human erythrocyte receptor which mediates mannose-resistant hemagglutination by enterotoxigenic Escherichia coli possessing colonization factor antigen II is not universally distributed among donors . Mannose-resistant hemagglutination-positive erythrocytes are more common among black donors than nonblack donors; tests with erythrocytes of known antigenic makeup confirm this correlation . Colonization factor antigen II receptor activity of mannose-resistant hemagglutination-positive erythrocytes is unstable when whole blood is stored at 4 degrees C . Also, screening of donors is best performed with enterotoxigenic E . coli possessing colonization factor antigen II composed of the coli surface antigen 1 (CS1) plus CS3, since these consistently produce stronger hemagglutination reactions than strains with colonization factor antigen II composed of either CS2 plus CS3 or CS3 only. Biochem J, 1988 Sep 1, 254(2), 427 - 35 Purification, crystallization and properties of porphobilinogen deaminase from a recombinant strain of Escherichia coli K12; Jordan PM et al.; Porphobilinogen deaminase has been purified and crystallized from an overproducing recombinant strain of Escherichia coli harbouring a hemC-containing plasmid which has permitted the purification of milligram quantities of the enzyme . Determination of the Mr of the enzyme by SDS/polyacrylamide-gel electrophoresis (35,000) and gel filtration (32,000) agrees with the gene-derived Mr of 33,857 . The enzyme has a Km of 19 +/- 7 microM, an isoelectric point of 4.5 and an N-terminal sequence NH2-MLDNVLRIAT . The substrate, porphobilinogen, binds to the active-site dipyrromethane cofactor to form three intermediate complexes: ES, ES2 and ES3 . The gene-derived primary structure of the E . coli deaminase is compared with that derived from the cDNA of the human enzyme. Anticancer Res, 1988 Sep-Oct, 8(5A), 995 - 1004 Oncodevelopmental expression and structure of alkaline phosphatase genes; Millan JL; Alkaline phosphatases (APs) are members of a multigene family, that in humans include four different genes . Their wide distribution in nature, ranging from bacteria to man, indicates that APs are involved in fundamental biochemical processes . Information on the primary structure of eukaryotic APs is accumulating very rapidly . There is a high degree of similarity between the eukaryotic APs and Escherichia coli AP . Structural comparisons with the E . coli enzyme have helped identify those residues that may participate in the active site pocket, as well as predict functional-structural features unique to eukaryotic APs . The general structure of the AP genes has now been revealed through the cloning of the germ cell AP gene in humans . The entire nucleotide sequence of the gene reveals the existence of 11 exons interrupted by 10 small introns . Elucidation of the mechanism of regulation and tissue-specific expression of AP genes will be highly relevant to understanding the re-expression of these enzymes in testicular and ovarian tumors . Two vitally important developmental processes, i.e., germ cell differentiation and early embryogenesis, provide experimentally accessible models to attempt to unravel the elusive function of APs. Radiat Res, 1988 Sep, 115(3), 617 - 23 Absence of pyrimidine salvage and prevention of thymineless radiosensitization in Escherichia coli thyA cells fed dihydrothymine or thymine glycol; Claycamp HG et al.; Little information is available concerning the metabolic fate of radiation-induced thymine base damage products once they have been excised from DNA . The present study was an attempt to determine whether or not thymine-requiring mutants of Escherichia coli could grow on dihydrothymine (DHT) and thymine glycol (TG) by "salvaging" the altered thymines . A second test of thymine product utilization was prevention of thymineless radiosensitization . Results showed that very low growth of Thy- cells on DHT or TG could be explained by the presence of less than or equal to 1% contaminating thymine in the mixtures . Radiation dose-modification factors (DMFs) for thyA cells fed DHT or TG for 3 h were 1.38 +/- 0.28 and 1.26 +/- 0.24, respectively, whereas the DMF for 3 h thymine-starved cells was 1.63 +/- 0.05 . The small (approximately 25%) amelioration of thymineless radiosensitization observed in DHT- or TG-fed cells could probably be explained by contaminating thymine in the medium . Although DHT is a normal metabolite in some cells, neither DHT nor TG could be used efficiently by thymine-requiring cells in the protocol presented. J Med Microbiol, 1988 Sep, 27(1), 71 - 4 The role of diarrhoeagenic Escherichia coli in acute diarrhoeal diseases in Bandar-Abbas, Iran; Katouli M et al.; The prevalance of different types of diarrhoea-producing Escherichia coli was measured in 273 patients attending 12 out-patient clinics in Bandar-Abbas, State of Hormozgan, Iran, during March 1984 . Enteropathogenic E . coli (EPEC) belonging to 12 different serogroups, of which O128 and O126 were the most common, were found in almost 31% of the patients . Enterotoxigenic strains of E . coli (ETEC) were the next most frequent group (21.9%); among these, 36 (60%) strains produced heat-stable enterotoxin (ST), 14 (23.3%) strains produced both heat-labile enterotoxin (LT) and ST, and 10 (16.7%) strains produced LT only . The same pattern of toxigenicity was observed among the EPEC isolates . Ten of the 12 serogroups encountered in this study contained toxin producers, amongst which strains producing ST were dominant . Enteroinvasive E . coli (EIEC) strains were not isolated . These findings suggest that enterotoxin-producing E . coli may be an important cause of diarrhoea in this part of Iran. Mutat Res, 1988 Sep, 201(1), 219 - 28 N-methyl-N'-nitro-N-nitrosoguanidine-induced mutation in a RecA strain of Escherichia coli; Gordon AJ et al.; 274 N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced forward mutations in the lacI gene of an Escherichia coli RecA- strain were cloned and sequenced . Base substitutions accounted for 264 mutations and consisted of 261 G:C----A:T transitions (including one double mutant with two G:C----A:T transitions separated by 25 base pairs), two A:T----G:C transitions and one A:T----T:A transversion . Therefore, 263 of the 274 mutations (all the transitions) can be explained as a result of the direct mispairing of O6-methylguanine, and O4-methylthymine residues during DNA synthesis . The source of the transversion is not known . The remaining mutations, one 16-base pair deletion, two -1 frameshifts and 7 frameshifts at the lacI frameshift hotspot, are located in runs of identical bases or flanked by directly repeated DNA sequences and can therefore be explained by template slippage events during DNA synthesis . The observed distribution of mutations recovered is identical to that found in a RecA+ background indicating little involvement of RecA function in MNNG-induced mutation . Analysis of neighbouring base sequence revealed that the G:C----A:T transition was 6 times more likely to be recovered if the mutated guanine residue was preceded by a purine rather than a pyrimidine . A most striking aspect of this distribution concerns particular residues in the core domain of the lac repressor protein . Within this domain the great majority of mutations generate nonsense codons or alter Gly codons. Mutat Res, 1988 Sep, 201(1), 107 - 12 Post-replication repair and recombination in uvrA umuC strains of Escherichia coli are enhanced by vanillin, an antimutagenic compound; Ohta T et al.; Effects of vanillin on UV killing of umuC mutant strains of E . coli were investigated in order to analyze the antimutagenic role of vanillin in mutagenesis . UV-irradiated uvrA umuC cells showed higher survival when plated on medium containing vanillin rather than medium without vanillin . This increased survival associated with exposure to vanillin was observed more clearly in uvrA umuC lexA(Ind-) and uvrA umuC recF strains . However, the effect was inhibited by additional recB recC mutations and completely blocked by an additional recA mutation . As far as tested the increased survival of UV-treated cells by vanillin was dependent on a capacity for genetic recombination . The effect of vanillin on recombination frequency between 2 plasmid DNA, pATH4 (Cmr Tcs) and pBMX7 (Apr Tcs), in a uvrA umuC background was investigated . A significantly higher frequency of plasmid recombination was observed when vanillin was present in the culture medium . These findings suggest that the antimutagenic effect of vanillin is the result of enhancement of a recA-dependent, error-free, pathway of post-replication repair. Clin Sci (Lond), 1988 Sep, 75(3), 251 - 5 Interleukin-1 and tumour necrosis factor cause hypotension in the conscious rabbit; Weinberg JR et al.; 1 . The cardiovascular effects of intravenous injections of interleukin-1 (IL-1) and tumour necrosis factor (TNF) have been investigated in the conscious rabbit . They have been compared with the effects of bacterial lipopolysaccharide (LPS) because both IL-1 and TNF are released from macrophages by LPS . 2 . IL-1, TNF and Escherichia coli J5-LPS all caused hypotension when given intravenously in a dose with low mortality . The time course of the hypotension caused by IL-1 and LPS was similar, although the maximal fall in mean blood pressure occurred earlier after IL-1 . TNF produced a more sustained fall in blood pressure . Hypotension was not accompanied by a compensatory tachycardia after any of the test substances . Hypotension was associated with a fever after TNF, hypothermia after LPS and no significant change in temperature after IL-1 . 3 . The packed cell volume did not change during hypotension in any of the study groups, implying that the hypotension was not due to fluid loss resulting from increased capillary permeability . 4 . IL-1 and TNF are candidates for the role of effectors of LPS-induced hypotension. Proc Natl Acad Sci U S A, 1988 Sep, 85(18), 6731 - 2 Tryptophan repressor of Escherichia coli shows unusual thermal stability; Bae SJ et al.; Differential scanning calorimetry demonstrates that the tryptophan repressor of Escherichia coli is unusually resistant to thermal denaturation . The dimeric protein undergoes reversible dissociative unfolding at pH 7.5 centered at about 90 degrees C . The thermal stability may be due in part to the unusual structure of the protein, which is composed of two identical intertwined polypeptide chains. Proc Natl Acad Sci U S A, 1988 Sep, 85(18), 6627 - 31 Discrimination between glutaminyl-tRNA synthetase and seryl-tRNA synthetase involves nucleotides in the acceptor helix of tRNA; Rogers MJ et al.; Analysis of the in vivo amber suppressor activity of mutants derived from two Escherichia coli serine tRNAs shows that substitution of 2 base pairs in the acceptor helix changes a serine suppressor tRNA to an efficient glutamine acceptor . Determination of the amino acid inserted in vivo into protein by this tRNA shows that these changes reduce the tRNA recognition by seryl-tRNA synthetase while increasing that of glutaminyl-tRNA synthetase . This implies that misaminoacylation in vivo is dependent on the competition by different synthetases for the tRNA . In addition, the "translational efficiency" of tRNA is an integral part in observing misaminoacylation in vivo. Proc Natl Acad Sci U S A, 1988 Sep, 85(17), 6498 - 502 Immunoregulatory activities of human immunodeficiency virus (HIV) proteins: effect of HIV recombinant and synthetic peptides on immunoglobulin synthesis and proliferative responses by normal lymphocytes; Nair MP et al.; Recombinant and synthetic peptides corresponding to envelope proteins of the human immunodeficiency virus (HIV) were examined for their effects on the activities of lymphocytes from normal donors in vitro . Although lymphocytes cultured with env-gag peptides produced significant amounts of IgG, addition of env-gag peptides to a pokeweed mitogen-induced B-cell activation system resulted in suppression of immunoglobulin synthesis by normal lymphocytes . Recombinant antigens, env-gag and env-80 dihydrofolate reductase (DHFR), produced a substantial proliferative response by peripheral blood mononuclear cells (PBMC) as determined by {3H}thymidine incorporation . PBMC precultured with HIV synthetic peptide env 578-608 also manifested significant proliferative responses as compared to control cultures . CD3+ lymphocytes precultured with recombinant HIV antigens, env-gag and env-80 DHFR, and synthetic HIV peptide, env 487-511, showed moderate but significant proliferative responses . Both recombinant antigens and synthetic peptides also produced a dose-dependent stimulatory effect on proliferation by CD3- lymphocytes . Stimulation of CD3+ and CD3- lymphocyte subpopulations induced by env-gag peptides was specifically inhibited by goat anti-env-gag polyclonal antibodies, demonstrating the specificity of the reaction . These studies demonstrate that recombinant and synthetic peptides of the HIV genome express immunoregulatory T- and B-cell epitopes . Identification of unique HIV epitopes with immunogenic and immunoregulatory activities is necessary for the development of an effective vaccine against HIV infection. Proc Natl Acad Sci U S A, 1988 Sep, 85(17), 6427 - 31 Selection of the mRNA translation initiation region by Escherichia coli ribosomes; Calogero RA et al.; Two genes specifying model mRNAs of minimal size and coding capacity, with or without the Shine-Dalgarno (SD) sequence, were assembled, cloned, and transcribed in high yields . These mRNAs, as well as synthetic polynucleotides, phage MS2 RNA, and a deoxyoctanucleotide complementary to the 3' end of 16S rRNA were used to study the mechanism of translation initiation in vitro . Escherichia coli 30S ribosomal subunits interact with all these nucleic acids, albeit with different affinities; the affinity for the mRNA with the SD sequence (Ka approximately 2 x 10(7) M-1) is more than an order of magnitude higher than that for the mRNA lacking this sequence . The initiation factors are equally required, regardless of the presence of the SD sequence, for 30S and 70S initiation complex formation and for mRNA translation, but the initiation factors do not affect the SD interaction or the binding of the mRNAs to the ribosomes . The SD interaction is also mechanistically irrelevant for 30S initiation complex formation and is not essential for translation in vitro or for the selection of the mRNA reading frame . It is suggested that the function of the SD interaction is to ensure a high concentration of the initiation triplet near the ribosomal peptidyl-tRNA binding site, whereas the selection of the translational start is achieved kinetically, under the influence of the initiation factors, during decoding of the initiator tRNA. Proc Natl Acad Sci U S A, 1988 Sep, 85(17), 6262 - 6 Integration host factor interacts with the DNA replication enhancer of filamentous phage f1; Greenstein D et al.; We present data which show that the Escherichia coli integration host factor (IHF) is an activator of phage f1 DNA replication . Phage f1 poorly infects bacterial strains lacking IHF because IHF is required for efficient expression of F-pili, the receptor for f1 phage . However, when F- strains are transfected with f1 DNA the phage replicates in IHF mutants (himA, himD, or himA himD) at a rate of only 3% of that in wild-type bacteria . A plasmid dependent on the f1 replicon fails to transform IHF mutants . By gel retardation analysis, we show that IHF specifically binds to the origin of replication . DNase I "footprinting" experiments demonstrate that IHF binds to multiple sites within the replication enhancer sequence, a cis-acting, A + T-rich sequence that potentiates f1 DNA replication . Moreover, the effect of IHF mutation on f1 growth is suppressed by initiator protein (f1 gene II) mutations that restore efficient replication from origins that lack a functional replication enhancer sequence . This genetic evidence supports the conclusion that the replication enhancer sequence is the site of action of IHF. Proc Natl Acad Sci U S A, 1988 Sep, 85(17), 6237 - 41 Biosynthesis of a protein containing a nonprotein amino acid by Escherichia coli: L-2-aminohexanoic acid at position 21 in human epidermal growth factor; Koide H et al.; Endeavoring to develop a method to biosynthesize proteins substituted with nonprotein amino acids, we attempted the incorporation of L-2-aminohexanoic acid (Ahx) into human epidermal growth factor (hEGF) . Escherichia coli YK537 strain harboring plasmid pTA1522, which has the phoA promoter-phoA signal peptide-hEGF gene, was used . Cells were cultured first in high-phosphate medium and then, for induction of the hEGF-encoding gene, transferred to low-phosphate medium containing Ahx (0.25 mg/ml) . hEGF and Ahx-substituted hEGF, {Ahx21}hEGF, secreted into the periplasm were recovered . After treatment with H2O2, {Ahx21}-hEGF was clearly separated from methionine-oxidized hEGF by one-step reverse-phase HPLC . Substitution of the methionine residue of hEGF with Ahx was confirmed by the amino acid analysis of {Ahx21}hEGF . The three biological activities of {Ahx21}hEGF were the same as those of hEGF . From the successful production of {Ahx21}hEGF, a basic strategy was established for preparing proteins substituted with nonprotein amino acid (alloprotein) . Induction of the phoA promoter of pho regulon and secretion of the product to the periplasm may depress heat shock-like responses and subsequent hydrolysis of the product by cytoplasmic protease. Mutat Res, 1988 Sep, 194(2), 109 - 20 SOS independent survival against mitomycin C induced lethality in a rifampicin-nalidixic acid-resistant mutant of Escherichia coli; Kumaresan KR et al.; A combination of specific rifampicin-resistant (rpoB87) and nalidixic acid-resistant (gyrA87) mutations results in a marked increase in the survival of Escherichia coli against mitomycin C-induced lethality in mutants defective for SOS induction and excision repair . Although the response does not seem to be obligatorily dependent upon the RecA protein, the efficiency is markedly increased in its presence, even in a conventionally inactive form . This response is not elicited against lethality due to ultraviolet radiation or N-methyl-N' -nitro-N-nitrosoguanidine exposure . The combination of rpoB87 and gyrA87 mutations also greatly alleviates post-mitomycin C degradation of DNA under SOS non-inducible conditions . It is proposed that the rpoB subunit of RNA polymerase and gyrA subunit of DNA gyrase could participate in the repair of certain types of DNA damage, such as cross-links, in a mode independent of SOS-regulated excision repair and post-replication repair. J Med Chem, 1988 Sep, 31(9), 1768 - 72 Synthesis and evaluation of multisubstrate inhibitors of an oncogene-encoded tyrosine-specific protein kinase . 2; Kruse CH et al.; Tyrosine-specific protein kinases that transfer the terminal phosphate from ATP to protein acceptors are associated with certain transforming viruses and cell surface growth factor receptors . Here we describe the synthesis and testing of potential multisubstrate inhibitors of this class of enzymes . The inhibitors were prepared by covalent attachment of the terminal phosphate of ATP or its tetraphosphate analogue to tyrosine mimics . Testing against p60v-abl, the tyrosine kinase from the Abelson murine leukemia virus, showed that the series of inhibitors was moderately potent (IC50 values as low as 13 microM) . However, structural modification of the tyrosine mimic, including replacement with a serine-like moiety, had little effect on potency . It is therefore concluded that the ATP moiety is largely responsible for binding and that the enzyme requires additional structural features for recognition of the tyrosine-containing substrate. J Bacteriol, 1988 Sep, 170(9), 4415 - 9 Nucleotide sequence of katG, encoding catalase HPI of Escherichia coli; Triggs-Raine BL et al.; The gene katG, encoding catalase HPI of Escherichia coli, was sequenced, predicting a 726-amino-acid protein . The sequence was confirmed by identification of potential regulatory elements and amino acid sequencing of peptides . HPI shows no homology to other catalases . The distances between katG, metF, and ppc were defined. J Bacteriol, 1988 Sep, 170(9), 4411 - 4 Purification of Rts1 RepA protein and binding of the protein to mini-Rts1 DNA; Kamio Y et al.; RepA protein, essential for the replication of plasmid Rts1, was purified, and its binding to mini-Rts1 subregions was examined by a DNase I protection assay . RepA protected the incI and incII iterons, a region immediately upstream of the repA promoter, and a 10-base-pair region located between the most external incII iteron and a GATC box . The protection was less efficient when preheated RepA was used. J Bacteriol, 1988 Sep, 170(9), 4395 - 8 In vitro insertion of leader peptidase into Escherichia coli membrane vesicles; Moore KE et al.; Leader peptidase is an integral protein of the Escherichia coli cytoplasmic membrane whose topology is known . We have taken advantage of this knowledge and available mutants of this enzyme to develop a genetic test for a cell-free protein translocation reaction . We report that leader peptidase inserted into inverted plasma membrane vesicles in its correct transmembrane orientation . We have examined the in vitro membrane assembly characteristics of a variety of leader peptidase mutants and found that domains required for insertion in vivo are also necessary for insertion in vitro . These data demonstrate the physiological validity of the in vitro insertion reaction and strengthen the use of this in vitro protein translocation reaction for the dissection of this complex sorting pathway. J Bacteriol, 1988 Sep, 170(9), 4392 - 4 A gene encoding an SOS inhibitor is present in different conjugative plasmids; Golub E et al.; In 9 of 20 conjugative plasmids of different incompatibility groups, including F and R100 (or R6-5), coexist two sequences which are homologous, respectively, to the gene psiB, which encodes an inhibitor of SOS induction, and to the gene ssb, which encodes a single-stranded-DNA-binding protein. J Bacteriol, 1988 Sep, 170(9), 4385 - 7 Cloning and nucleotide sequence of the oriT region of the IncI1 plasmid R64; Komano T et al.; The nucleotide sequence at the oriT region of the IncI1 plasmid R64 was determined . A recombinant plasmid carrying a 141-base-pair R64 sequence was mobilized with a normal frequency, while a plasmid carrying only 44 base pairs of this R64 sequence was mobilized with a frequency 1/10 that of the original plasmid . The oriT region of the R64 plasmid contains two inverted-repeat sequences. J Bacteriol, 1988 Sep, 170(9), 4266 - 71 Regulation of DNA superhelicity by rpoB mutations that suppress defective Rho-mediated transcription termination in Escherichia coli; Arnold GF et al.; The highly defective rho-15 mutant of Escherichia coli produces plasmid DNA that is 22% less negatively supercoiled than DNA from an isogenic wild-type strain (J . S . Fassler, G . F . Arnold, and I . Tessman, Mol . Gen . Genet . 204:424-429, 1986) . We extended our measurements of plasmid superhelicity to additional rho mutants and to strains containing mutations that suppress rho transcription termination defects; the suppressor mutations were in the rpoB and the rho genes . The superhelicity of plasmid DNA was reduced by 11 and 10%, respectively, in the rho-702 and rho-201 mutants, both of which are less defective in Rho-mediated transcription termination than rho-15 . Plasmid superhelicity was restored in all the suppressed rho mutants; in one rpoB mutant, plasmid DNA was even more negatively supercoiled than in rpoB+ cells, whether in a rho+ or rho mutant background . Suppression of rho mutants enabled them to maintain plasmids that could not be maintained in the mutants in the absence of the suppressor mutations . The results indicate that in addition to DNA gyrase, topoisomerase I, and Rho, RNA polymerase is also a determinant of DNA superhelicity, and its effect is modified by the Rho protein . We propose that Rho may increase the degree of DNA unwinding by the transcription complex, possibly at transcription termination sites. J Bacteriol, 1988 Sep, 170(9), 4209 - 15 Structures of the promoter and operator of the glpD gene encoding aerobic sn-glycerol-3-phosphate dehydrogenase of Escherichia coli K-12; Ye SZ et al.; The nucleotide sequence of a 690-base-pair DNA segment containing the control region for the glpD gene encoding aerobic sn-glycerol-3-phosphate dehydrogenase was determined . An ATG translation initiation codon with an adjacent ribosome-binding site was found which preceded an open reading frame continuing 61 codons to the end of the DNA that was sequenced . The start site for transcription, identified by using primer extension analysis, was located 42 base pairs upstream from the proposed Met start codon . The transcription start site was preceded by a region containing typical -10 and -35 sequences found in bacterial promoters . A binding site for the cyclic AMP-cyclic AMP receptor protein complex (identified by comparison with the consensus-binding sequence and verified by using DNase I footprinting) was located just upstream from the -35 sequence, centered at position -63 . The interaction site for the glp repressor was identified by using DNase I footprinting . It consisted of a 49-base-pair region which started at the -10 sequence and continued to position +38 . This region contained two directly repeated sequences, each possessing hyphenated dyad symmetry, which suggests that the operator is tandemly repeated . The presence of two adjacent operators may explain why expression of the glpD gene is the most sensitive to repressor when compared with expression of the other operons that are members of the glp regulon. J Bacteriol, 1988 Sep, 170(9), 4153 - 60 Effect of a mutation preventing lipid modification on localization of the pCloDF13-encoded bacteriocin release protein and on release of cloacin DF13; Luirink J et al.; The pCloDF13-encoded bacteriocin release protein (BRP; Mr 2,871) is essential for the translocation of cloacin DF13 across the cell envelope of producing Escherichia coli cells . Overproduction of this BRP provokes lysis (quasilysis) of cells . Construction and analysis of a hybrid BRP-beta-lactamase protein (BRP-Bla) demonstrated that the BRP contains a lipid modified cysteine residue at its amino terminus and is mainly located in the outer membrane . The significance of lipid modification for the localization and functioning of the BRP was investigated . Site-directed mutagenesis was used to substitute the cysteine residue for a glycine residue in the lipobox of the BRP and the BRP-Bla protein . The mutated BRP was unable to bring about the release of cloacin DF13 and could not provide the lysis (quasilysis) of host cells . However, the mutated BRP strongly inhibited the colony-forming ability of the cells, indicating that induction of the mutated protein still affected cell viability . In contrast to the wild-type BRP-Bla protein, the mutated BRP-Bla protein was mainly located in the cytoplasmic membrane, indicating that the mutation prevented the proper localization of the protein . The results indicated that lipid modification of the BRP is required for its localization and release of cloacin DF13, but not for its lethality to host cells. J Bacteriol, 1988 Sep, 170(9), 4097 - 102 Cloning and sequencing of the Escherichia coli chlEN operon involved in molybdopterin biosynthesis; Nohno T et al.; The nucleotide sequence of a HinPI-HpaII restriction nuclease fragment which complemented a delta chlE strain of Escherichia coli was determined . Two open reading frames were deduced to be the structural genes for ChlE and ChlN proteins, which have molecular weights of 44,067 and 26,719, respectively . Both proteins were required for complementing a chromosomal deletion of the chlE locus . The chlE and chlN genes were transcribed from a common promoter, chlEp, located upstream of chlE . Transcriptional and translational signal sequences were recognized in this region. J Bacteriol, 1988 Sep, 170(9), 3937 - 45 Common ancestry of Escherichia coli pyruvate oxidase and the acetohydroxy acid synthases of the branched-chain amino acid biosynthetic pathway; Chang YY et al.; A number of enzymes require flavin for their catalytic activity, although the reaction catalyzed involves no redox reaction . The best studied of these enigmatic nonredox flavoproteins are the acetohydroxy acid synthases (AHAS), which catalyze early steps in the synthesis of branched-chain amino acids in bacteria, yeasts, and plants . Previously, work from our laboratory showed strong amino acid sequence homology between these enzymes and Escherichia coli pyruvate oxidase, a classical flavoprotein dehydrogenase that catalyzes the decarboxylation of pyruvate to acetate . We have now shown this homology (i) to also be present in the DNA sequences and (ii) to represent functional homology in that pyruvate oxidase has AHAS activity and a protein consisting of the amino-terminal half of pyruvate oxidase and the carboxy-terminal half of E . coli AHAS I allows native E . coli AHAS I to function without added flavin . The hybrid protein contains tightly bound flavin, which is essential for the flavin substitution activity . These data, together with the sequence homologies and identical cofactors and substrates, led us to propose that the AHAS enzymes are descended from pyruvate oxidase (or a similar protein) and, thus, that the flavin requirement of the AHAS enzymes is a vestigial remnant, which may have been conserved to play a structural rather than a chemical function. J Bacteriol, 1988 Sep, 170(9), 3910 - 4 Starvation-induced cross protection against heat or H2O2 challenge in Escherichia coli; Jenkins DE et al.; Glucose- or nitrogen-starved cultures of Escherichia coli exhibited enhanced resistance to heat (57 degrees C) or H2O2 (15 mM) challenge, compared with their exponentially growing counterparts . The degree of resistance increased with the time for which the cells were starved prior to the challenge, with 4 h of starvation providing the maximal protection . Protein synthesis during starvation was essential for these cross protections, since chloramphenicol addition at the onset of starvation prevented the development of thermal or oxidative resistance . Starved cultures also demonstrated stronger thermal and oxidative resistance than did growing cultures adapted to heat, H2O2, or ethanol prior to the heat or H2O2 challenge . Two-dimensional gel electrophoresis of 35S-pulse-labeled proteins showed that subsets of the 30 glucose starvation proteins were also synthesized during heat or H2O2 adaptation; three proteins were common to all three stresses . Most of the common proteins were among the previously identified Pex proteins (J.E . Schultz, G . I . Latter, and A . Matin, J . Bacteriol . 170:3903-3909, 1988), which are independent of cyclic AMP positive control for their induction during starvation . Induction of starvation proteins dependent on cyclic AMP was not important in these cross protections, since a delta cya strain of E . coli K-12 exhibited the same degree of resistance to heat or H2O2 as the wild-type parent did during both growth and starvation. Dig Dis Sci, 1988 Sep, 33(9), 1116 - 20 Unconjugated bilirubin in hepatic bile with brown pigment gallstones and cholangitis; Nakano T et al.; To investigate the role of cholangitis in hydrolysis of bilirubin in bile with brown pigment gallstones, bilirubin composition and bacterial growth in hepatic bile with and without cholangitis were studied . The study included 38 brown pigment gallstone cases (28 without cholangitis and 10 with cholangitis) . The proportion of unconjugated bilirubin in hepatic bile with cholangitis (16.9 +/- 8.5%, mean +/- SD) was significantly higher than that without cholangitis (3.7 +/- 1.8%, P less than 0.001) . A positive correlation was found between bacterial population with beta-glucuronidase activity and the proportion of unconjugated bilirubin in bile in cases of brown pigment stones with cholangitis (P less than 0.05) but not in those without cholangitis despite the fact that bacterial species and population are similar regardless of the presence of cholangitis . In cholangitis, pH of bile becomes lower toward the optimal pH of bacterial beta-glucuronidase . Together the lower concentration of bile acid and the lower pH in bile result in lower solubility of unconjugated bilirubin, promoting its precipitation . Thus occasional bouts of cholangitis may result in periodic deposition of bilirubinate on brown pigment stones with layered structures by inducing cyclic changes of bile composition in situ. Carcinogenesis, 1988 Sep, 9(9), 1607 - 10 Mutational specificity of N-methyl-N-nitrosourea in the lacI gene of Escherichia coli; Burns PA et al.; The mutational specificity of the carcinogenic alkylating agent N-methyl-N-nitrosourea (MNU) was investigated through the DNA sequence characterisation of 104 lacI-d mutations induced by this agent in the lacI gene of Escherichia coli . The vast majority (95%) of MNU-induced mutations were G:C----A:T transitions . An analysis of neighbouring base sequence revealed that this transition was almost 10 times more likely to occur if the mutated guanine residue was preceded (5') by a purine (R) rather than a pyrimidine (Y); apart from this 5'-R-G-3' influence no other site specificity of mutation was observed . This 5'-flanking base influence on mutability has also been observed in this system with other mechanistically similar alkylating agents. Carcinogenesis, 1988 Sep, 9(9), 1529 - 32 Mutation site specificity of N-nitroso-N-methyl-N-alpha-acetoxybenzylamine: a model derivative of an esophageal carcinogen; Horsfall MJ et al.; A total of 171 mutations induced by N-nitroso-N-methyl-N-alpha-acetoxybenzylamine within the first 180 base pairs of the lacI gene of Escherichia coli were characterized at the DNA sequence level . Consistent with the methylating ability of this compound and the predicted mutagenic specificity of the O6-methylguanine lesion, all but two of these mutations were G:C----A:T transitions . An analysis of neighboring sequences revealed the same 5' flanking sequence influence on mutability reported for other SN1-type direct-acting alkylating agents . G:C----A:T transitions were found to be six times more likely to occur at G:C base pairs at which the guanine residues were flanked (5') by a purine than at those preceded by a pyrimidine . This mutagenic and site specificity appeared to be independent of the dose and likely reflects the behaviour of the activated parent carcinogen, N-nitroso-N-methyl-N-benzylamine in the esophagus. Cancer Res, 1988 Sep 1, 48(17), 4823 - 6 Gene expression caused by alkylating agents and cis-diamminedichloroplatinum(II) in Escherichia coli; Fram RJ et al.; Previous work has demonstrated heterogeneous effects of methylating agents on induction of DNA damage inducible genes in Escherichia coli . These studies employed E . coli mutants that have fusions of the lac operon to genes induced by treatment with sublethal levels of alkylating agents . These mutants were selected from random insertions of the Mu-dl (Apr lac) phage by screening for induction of beta-galactosidase activity in the presence of methylmethanesulfonate or N-methyl-N'-nitro-N-nitrosoguanidine . The current report extends these findings by analyzing gene expression caused by mechlorethamine, chloroethylnitrosoureas and cis-diamminedichloroplatinum(II) (cis-DDP) . The results demonstrate heterogeneous effects by these agents on gene expression . While 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea induces alkA, other nitrosoureas, mechlorethamine, and cis-DDP do not cause expression of this gene . Further, while all nitrosoureas caused expression of aidC, mechlorethamine and cis-DDP did not . Lastly, cis-DDP caused marked expression of a sulA fusion mutant while not inducing any of the other E . coli fusion mutants. Obstet Gynecol, 1988 Sep, 72(3 Pt 2), 496 - 7 Fetal bradycardia associated with maternal hypothermia; Jadhon ME et al.; A case of fetal bradycardia associated with severe maternal hypothermia (92.9F) is reported . Until maternal temperature was corrected, the baseline fetal heart rate (FHR) remained between 90-110 beats per minute without other evidence of fetal distress and despite normal maternal blood pressure and pulse . With rewarming, the FHR gradually returned to normal . Upon return of maternal hypothermia, fetal bradycardia recurred, again responding only to rewarming . This evidence suggests that low maternal temperature alone may lead to alterations of FHR. Genetics, 1988 Sep, 120(1), 7 - 21 Chain-bias of Escherichia coli Rec-mediated lambda patch recombinants is independent of the orientation of lambda cos; Rosenberg SM; Chi is a hotspot for homologous recombination mediated by the RecBCD (Rec) pathway of Escherichia coli . For Rec-mediated recombination of phage lambda, the orientation of lambda cos in the lambda chromosome dictates the direction of travel of RecBCD enzyme through DNA and dictates which orientation of Chi or Chi-like sequences will be active in stimulating recombination . I previously found that Rec-mediated lambda patch heteroduplexes, stimulated by Chi or not, are chain-biased; at the lambda P locus, recombinant information resides on the lambda r chain . This bias exists in the presence or absence of Chi sites . Reported herein is the finding that r-chain-bias at the P locus is independent of the orientation of lambda cos and thus also independent of the orientation of active Chi's or Chi-like sequences and of the direction of travel of RecBCD enzyme . These results disprove previously elaborated models in which a chain-specific nick at Chi initiates recombination, and imply that some other chain-distinguishing process is involved with recombination . Replication and transcription are candidates for such a process. Genetics, 1988 Sep, 120(1), 173 - 80 Analysis of the promoter of the Rh2 opsin gene in Drosophila melanogaster; Mismer D et al.; We have analyzed the cis-acting regulatory sequences of the Drosophila melanogaster Rh2 gene that encodes the protein component of a rhodopsin which is expressed in ocellar photoreceptor cells . DNA fragments containing the start point of transcription of the Rh2 gene were fused to either the Escherichia coli chloramphenicol acetyltransferase (CAT) or lacZ (beta-galactosidase) genes and introduced into the Drosophila germline by P-element-mediated transformation . Expression of the E . coli genes was then used to assay the ability of various sequences from the Rh2 gene to confer upon the indicator genes the Rh2 pattern of expression . Fragments containing between 4.3 kb and 183 bp upstream of the start of transcription plus the first 32 bp of the 5'-untranslated leader were found to result in nearly identical levels of head-specific CAT expression . Deletion of Rh2 sequences distal to position -112 bp resulted in loss of detectable CAT expression from these Rh2/CAT fusion constructs . We have, therefore, defined a region essential for head-specific expression of the Rh2 gene to a region extending from -183 to -112 . We have determined the DNA sequence of the Rh2 promoter from -448 to +32 and have found an 11-bp sequence which is also present in the upstream flanking sequences of two other photoreceptor-specific genes (ninaE and ninaC) . By histochemical staining of beta-galactosidase expressed under the control of the Rh2 promoter and by analyzing the effect of the ocelliless mutation on the expression of an Rh2/CAT fusion gene, we have been able to demonstrate that this promoter is active in ocelli. EMBO J, 1988 Sep, 7(9), 2919 - 23 PL of coliphage lambda: an alternative solution for an efficient promoter; Knaus R et al.; Promoter PL of coliphage lambda is highly active in vivo although it is recognized 15-30 times less efficiently by RNA polymerase when compared with promoters of similar strength . Moreover, it differs significantly from the consensus sequence for Escherichia coli promoters . Sequence variants of PL which are more homologous to consensus promoters bind RNA polymerase with increased efficiency . They are nevertheless significantly reduced in their in vivo strength . High activity can be restored by a downstream sequence of a typical consensus-like promoter . Evidently, such elements are required for the efficient release of a stably bound RNA polymerase into a transcriptional elongation complex . We propose that the functional programme encoded in a promoter sequence can be optimized in alternative ways. Proc Natl Acad Sci U S A, 1988 Sep, 85(17), 6483 - 7 Isolation and characterization of cDNAs coding for the beta subunit of the high-affinity receptor for immunoglobulin E; Kinet JP et al.; Among receptors that bind the Fc region of immunoglobulins ("Fc receptors"), only the one with high affinity for immunoglobulin E (IgE) is known to consist of more than a single polypeptide . In addition to the IgE-binding alpha chain, the receptor contains a single beta chain and two, disulfide-linked, gamma chains . From a cDNA library of a rat mucosal mast cell tumor, from which we recently cloned cDNAs coding for the alpha chain, we have now isolated cDNAs coding for the beta subunit . In vitro transcription-translation of the cDNA directed the synthesis of a polypeptide reactive with two distinctive anti-beta monoclonal antibodies and whose molecular weight was identical to that of authentic beta chains . Polyclonal antibodies to beta peptides expressed in Escherichia coli reacted with intact receptors and isolated beta chains . The gene encodes a protein of 243 residues with no leader sequence . A hydropathicity plot suggests that the polypeptide crosses the plasma membrane four times . The epitope recognized by one of the monoclonal antibodies was localized to the NH2 terminus; that by the other was localized to the COOH terminus . Since those antibodies react with membranes and not with intact cells, we suggest that both ends of the beta subunit are cytoplasmic . RNA transfer blots at high stringency failed to reveal mRNA for beta chains in a variety of cells (in particular, monocytes) that do not contain the high-affinity receptor for IgE. J Med Chem, 1988 Sep, 31(9), 1762 - 7 Synthesis and evaluation of multisubstrate inhibitors of an oncogene-encoded tyrosine-specific protein kinase . 1; Kruse CH et al.; The synthesis and testing of potential multisubstrate inhibitors of tyrosine-specific protein kinases are described . One of the substrates, ATP, was mimicked by the known kinase inhibitor 5'-{4-(fluorosulfonyl)benzoyl}adenosine, which was covalently linked via the sulfonyl moiety to tyrosine mimics . The resulting multisubstrate inhibitors were tested for their ability to inhibit the transfer of phosphate from ATP to a protein acceptor by p60v-abl, the tyrosine kinase encoded by the transforming gene (v-abl) of the Abelson murine leukemia virus (A-MuLV) . Although the series of inhibitors displayed moderately potent activity (IC50 values as low as 19 microM), the absence of large effects produced by modification of the tyrosine mimic suggests that they do not behave as multisubstrate inhibitors but bind primarily through the adenosine moiety common to all the inhibitors . This interpretation is strengthened by the finding that the inhibitors lack specificity, inhibiting a serine kinase at comparable concentrations. J Bacteriol, 1988 Sep, 170(9), 4174 - 80 Novel activation of araC expression and a DNA site required for araC autoregulation in Escherichia coli B/r; Cass LG et al.; Mutations in the araC gene have been isolated which alter both the activator and autoregulatory functions of AraC protein (L.G . Cass and G . Wilcox, J . Bacteriol . 166:892-900, 1986) . In this study, the effect of each araC mutation on autoregulation was characterized in vivo and in vitro in the presence of L-arabinose . The effect of L-arabinose in some of these araC mutants revealed a novel activation of araC expression which was not observed in the araC+ cell . Experiments were therefore focused on understanding the mechanism of this novel activation . We describe a systematic analysis of the effect of mutations within the known regulatory binding sites for araBAD and araC transcription on araC expression . Our results suggest that the novel activation of araC expression requires the AraC activator-binding site, araI, and the cyclic AMP receptor protein-cyclic AMP complex-binding site . We also found that in the absence of L-arabinose, the araI site was required for maximal autoregulation by the wild-type AraC protein. Mol Cell Endocrinol, 1988 Sep, 59(1-2), 155 - 9 The beta-subunit of the bovine mitochondrial F1 ATPase specifically binds the amino terminal domain of parathyroid hormone; Laethem R et al.; A protein which specifically binds the amino terminal domain of parathyroid hormone (PTH) on nitrocellulose blots of polyacrylamide gels was fragmented with cyanogen bromide (CNBr), and two fragments were sequenced through 20 residues . The sequence obtained was 100% homologous with the beta-subunit of bovine F1 mitochondrial ATPase . Purified F1 ATPase from bovine heart and Escherichia coli were obtained and the binding of PTH examined on the blots . The beta-subunit of the bovine enzyme bound PTH specifically through its amino terminal domain . However, both the alpha- and beta-subunit of the E . coli enzyme were found to bind the hormone . This binding was also specific for the amino terminal domain of the hormone . The subcellular distribution of the PTH-binding protein from bovine kidney was also examined further . While the mitochondria and plasma membrane appear to possess similar PTH-binding capability, submitochondrial particles enriched in F1 ATPase were also enriched in PTH-binding activity. Proc Natl Acad Sci U S A, 1988 Sep, 85(18), 6711 - 5 Acetyladenylate plays a role in controlling the direction of flagellar rotation; Wolfe AJ et al.; Cells of Escherichia coli deleted for genes that code for the transducers and all the known cytoplasmic Che proteins except CheY responded reversibly to the addition of acetate by spinning their flagellar motors clockwise . By varying growth conditions and using metabolic inhibitors and mutants deficient in acetate metabolism, this effect was shown to require acetate-CoA synthetase {acetate:CoA ligase (AMP-forming); EC 6.2.1.1}, an enzyme that catalyzes the formation of acetyl-CoA from acetate by an acetyladenylate intermediate . A mutant deficient in this enzyme but retaining the chemotaxis genes was deficient for chemotaxis . Thus, acetyladenylate appears to play a role in generating clockwise rotation at the level of CheY or the motor. J Bacteriol, 1988 Sep, 170(9), 3983 - 90 Gene clusters for S fimbrial adhesin (sfa) and F1C fimbriae (foc) of Escherichia coli: comparative aspects of structure and function; Ott M et al.; Fimbrial adhesins enable bacteria to attach to eucaryotic cells . The genetic determinants for S fimbrial adhesins (sfa) and for F1C ("pseudotype I") fimbriae (foc) were compared . Sfa and F1C represent functionally distinct adhesins in their receptor specificities . Nevertheless, a high degree of homology between both determinants was found on the basis of DNA-DNA hybridizations . Characteristic differences in the restriction maps of the corresponding gene clusters, however, were visible in regions coding for the fimbrial subunits and for the S-specific adhesin . While a plasmid carrying the genetic determinant for F1C fimbriae was able to complement transposon-induced sfa mutants, a plasmid carrying the genetic determinant for a third adhesin type, termed P fimbriae, was unable to do so . Proximal sfa-specific sequences carrying the S fimbrial structural gene were fused to sequences representing the distal part of the foc gene cluster to form a hybrid cluster, and the foc proximal region coding for the structural protein was ligated to sfa distal sequences to form a second hybrid . Both hybrid clones produced intact fimbriae . Anti-F1C monoclonal antibodies (MAbs) only recognized clones which produced F1C fimbriae, and an anti-S adhesin MAb marked clones which expressed the S adhesin . However, one of four other anti-S fimbriae-specific MAbs reacted with both fimbrial structures, S and F1C, indicating a common epitope on both antigens . The results presented here support the view that sfa and foc determinants code for fimbriae that are similar in several aspects, while the P fimbriae are members of a more distantly related group. J Bacteriol, 1988 Sep, 170(9), 3810 - 6 Formation of the chlorophyll precursor delta-aminolevulinic acid in cyanobacteria requires aminoacylation of a tRNAGlu species; O'Neill GP et al.; In the chloroplasts of higher plants and algae, the biosynthesis of the chlorophyll precursor delta-aminolevulinic acid (ALA) involves at least three enzymes and a tRNA species . Here we demonstrate that in cell extracts of the unicellular cyanobacterium Synechocystis sp . strain PCC 6803 ALA was formed from glutamate in a series of reactions in which activation of glutamate by glutamyl-tRNAGlu formation was the first step . The activated glutamate was reduced by a dehydrogenase which displayed tRNA sequence specificity . Fractionation of strain 6803 tRNA by reverse-phase chromatography and polyacrylamide gel electrophoresis yielded two pure tRNAGlu species which stimulated ALA synthesis in vitro . These tRNAs had identical primary sequences but differed in the nucleotide modification of their anticodon . The 6803 tRNAGlu was similar to the sequences of tRNAGlu species or tRNAGlu genes from Escherichia coli and from chloroplasts of Euglena gracilis and higher plants . Southern blot analysis revealed at least two tRNAGlu gene copies in the 6803 chromosome . A glutamate-1-semialdehyde aminotransferase, the terminal enzyme in the conversion of glutamate to ALA in chloroplasts, was detected in 6803 cell extracts by the conversion of glutamate-1-semialdehyde to ALA and by the inhibition of this reaction by gabaculin. J Bacteriol, 1988 Sep, 170(9), 3803 - 9 Cloning and nucleotide sequence of a gene for Actinomyces naeslundii WVU45 type 2 fimbriae; Yeung MK et al.; A genomic library of Actinomyces naeslundii WVU45 DNA in Escherichia coli was screened for antigen expression with rabbit antibody against A . naeslundii fimbriae . Western blotting (immunoblotting) of one recombinant clone carrying a 13.8-kilobase-pair insert revealed a 59-kilodalton (kDa) immunoreactive protein . A protein of similar electrophoretic mobility was detected from the isolated fimbrial antigen . Expression of the 59-kDa cloned protein in E . coli was directed by a promoter from the insert . The DNA sequence of the subunit gene was determined, and an open reading frame of 1,605 nucleotides was identified which was preceded by a putative ribosome-binding site and followed by two inverted repeats of 14 and 17 nucleotides, respectively . The reading frame encoded a protein of 534 amino acids (calculated molecular weight, 57,074), and the N-terminal sequence resembled that of a signal peptide . The presence of a 32-amino-acid signal peptide was indicated by amino-terminal sequencing of the fimbriae from A . naeslundii . The sequence, as determined by Edman degradation, was identical to that deduced from the DNA sequence beginning at predicted residue 33 of the latter sequence . Moreover, the amino acid composition of the predicted mature protein was similar to that of the isolated fimbriae from A . naeslundii . Thus, the cloned gene encodes a subunit of A . naeslundii fimbriae. Infect Immun, 1988 Sep, 56(9), 2330 - 5 Detection of pilus subunits (pilins) and filaments by using anti-P pilin antisera; Salit IE et al.; P pilus filaments are important in binding to globoside through an adhesin located at the tip of the pilus . There is considerable antigenic variation among P pili, and the immunologic response is usually serotype specific . We purified denatured pilin subunits and used them as immunogens to prepare more broadly cross-reactive antisera . Although antifilament antisera (AFA) detected predominantly the homologous strain, antisubunit antisera (ASA) prepared from two different strains detected P pili in 16 of 16 and 14 of 16 P-piliated strains by Western blotting (immunoblotting) . The binding of ASA to the homologous pilus filament was inhibited by only 3 of 17 strains . ASA agglutinated only two of nine heterologous strains and immunoprecipitated pili from one of three heterologous strains . By immunoelectron microscopy ASA was seen to bind to pilus filaments but not as strongly as AFA . Antiserum raised to the denatured pilin subunit was not substantially more reactive with pilus filaments derived from heterologous strains than was AFA . ASA was, however, a very useful probe for detecting most P pilins. Infect Immun, 1988 Sep, 56(9), 2317 - 23 Expression of K99 adhesion antigen controlled by the Escherichia coli tryptophan operon promoter; Baecker PA et al.; The genetic determinant for the K99 adhesin of enterotoxigenic Escherichia coli B41 {O101:K99} has been cloned as a 7.0-kilobase BamHI-generated DNA fragment into the vector pBR322 by us and others (J . D . A . van Embden, F . K . de Graaf, L . M . Schouls, and J . S . Teppma, Infect . Immun . 29:1125-1133, 1980) . Cells harboring one such construction, known as pK99-64, are capable of expressing K99 antigen on the cell surface . We replaced the natural promoter sequence for the gene encoding the K99 pilus subunit with a strong, inducible exogenous promoter, the E . coli tryptophan (trp) operon promoter, to construct the plasmid pBR-TrpK99 . E . coli cells harboring pBR-TrpK99 or a similar construction in the plasmid pDR540, known as pKO-TrpK99, upon induction with 3-beta-indoleacrylic acid, produced about fourfold more K99 antigen than did cells bearing pK99-64 with the natural promoter . Expression of the pilus antigen was found to be under control of the tryptophan promoter . Plasmid instability was encountered, however, in cells bearing pKO-TrpK99 when the trp promoter was derepressed . Introduction of the aminoglycoside 3'-phosphotransferase gene of transposable element Tn5 into pKO-TrpK99 to generate pKON-TrpK99 effectively stabilized the plasmid in cells grown under identical conditions in medium containing kanamycin. J Gen Microbiol, 1988 Sep, 134 ( Pt 9), 2513 - 24 Localization of a pyrroloquinoline quinone biosynthesis gene near the methanol dehydrogenase structural gene in Methylobacterium organophilum DSM 760; Mazodier P et al.; A partial Sau3AI genomic bank of Methylobacterium organophilum DSM 760 was constructed in the cosmid pSUP106 and moxF, the structural gene for methanol dehydrogenase, was isolated . In M . organophilum, pSUP106 behaves as a suicide plasmid . This property was used to insert Tn5 into the bacterial chromosome, in the vicinity of moxF, by marker exchange . Mobilization of the Tn5-labelled chromosomal region by a broad-host-range plasmid, pJB3J1 (an R68-45 derivative), allowed the selection of several large R' hybrid plasmids in Escherichia coli HB101 . Most of them were able to complement both mutants of the moxF region and mutant MTM1, the first mutant of the pyrroloquinoline quinone (PQQ) biosynthesis pathway in M . organophilum . The gene involved, pqqA, was subcloned and localized. Plasmid, 1988 Sep, 20(2), 143 - 7 Spontaneous insertion of an IS2 element into the promoter region of the lac operon; De Togni P et al.; A spontaneous mutation in pUC18 has revealed the insertion of a chromosomal insertion sequence (IS)2 element into the promoter region of the lac operon . The IS2 insertion site, at the pentanucleotide sequence TCGAG, is unlike previously described junctional sequences. Plasmid, 1988 Sep, 20(2), 106 - 12 Restriction pattern and polypeptide homology among plasmid-borne mercury resistance determinants; Jobling MG et al.; The structural and functional properties of mercury resistance determinants cloned from a series of independently isolated conjugative plasmids were compared with those of the prototype HgR determinants from Tn501 and plasmid R100 (containing Tn21) . Restriction endonuclease mapping classified the HgR determinants into at least three different but related structural groups which are distantly related to those from Tn501 and R100 . These relationships were confirmed by the functional analysis of sub-clones and gamma delta insertion mutations and from the polypeptides specified by the cloned HgR determinants . Each mercury resistance clone synthesized polypeptides equivalent in size to the merA, merT, and merP gene products . However, those for merA and merT showed considerable size variation . No polypeptide equivalent to merD or merC of R100 was detected. Mol Gen Genet, 1988 Sep, 214(1), 62 - 7 Inosine induced mutations; Nordmann PL et al.; Two complementary 24 base single stranded oligonucleotides containing randomly located inosine residues were synthesized in vitro . Once annealed, the two oligonucleotides were cloned into derivatives of ColE1 and transformed into Escherichia coli . Sequence analysis of 157 clones yielded 305 mutations . The pattern of the mutations revealed the following: (1) The frequency of inosine induced mutations was significantly less than predicted from its content in the oligonucleotides; (2) Inosine incorporation resulted almost exclusively in base changes to guanine; (3) The mutation distribution is biased towards A/T to G/C substitutions; (4) There were reproducible position biases; and (5) There was a reproducible strand bias which was independent of the cassette orientation with respect to the plasmid origin of replication. Mol Gen Genet, 1988 Sep, 214(1), 1 - 5 Formation of deletion in Escherichia coli between direct repeats located in the long inverted repeats of a cellular slime mold plasmid: participation of DNA gyrase; Saing KM et al.; We constructed a recombinant plasmid containing the 2.1 kb HindIII fragment of plasmid pDG1, isolated from the cellular slime mold (Dictyostelium sp . strain GA11), and using pAG60 as cloning vector . We found that deletions of the recombinant plasmid took place frequently in Escherichia coli wild-type cells . However, the deletion was not observed when the plasmid was introduced into a strain that was an isogenic temperature-sensitive mutant of the gyrA gene . These results suggest that E . coli DNA gyrase is involved in the mechanisms of the deletion formation . It was shown that the 1.0 kb deletant derived from the 2.1 kb HindIII insert was produced by elimination of a 1.1 kb region . Sequence analysis of the deletants showed that cutting and rejoining took place between two out of the six nearly perfect direct repeats {21 bp palindromic sequences; AAAAAA(T/C)GGC(G/C)GCC(A/G)TTTTTT}, located near the distal ends of the inverted repeats, preserving one copy of the repeats . These sequences consist of local short inverted repeats, where cutting and rejoining occur at one of the two regions. Mol Cell Biol, 1988 Sep, 8(9), 3703 - 9 Isolation and characterization of an autonomously replicating sequence from Ustilago maydis; Tsukuda T et al.; DNA fragments that function as autonomously replicating sequences (ARSs) have been isolated from Ustilago maydis . When inserted into an integrative transforming vector, the fragments increased the frequency of U . maydis transformation several-thousandfold . ARS-containing plasmids were transmitted in U . maydis as extrachromosomal elements through replication . They were maintained at a level of about 25 copies per cell but were mitotically unstable . One ARS characterized in detail, which we called UARS1, was localized to a 1.7-kilobase fragment . UARS1 contained a cluster of active sequences . This element could be reduced further into three separate subfragments, each of which retained ARS activity . The smallest one was 383 base pairs (bp) long . Although not active itself in yeast, this small fragment contained seven 8-bp direct repeats, two contiguous 30-bp direct repeats, and five 11-bp units in both orientations with sequences similar but not identical to the consensus sequence found to be crucial for ARS activity in Saccharomyces cerevisiae. Mol Gen Mikrobiol Virusol, 1988 Sep, (9), 14 - 7 {The effect of mutations in recB and recC genes on the precise excision of Tn5 from pNM1 plasmid genome}; Davenis SV et al.; The affect of mutations in chromosomal genes determining the realization of RecBC and RecF pathways of recombination in E . coli K12 on the frequency of transposon Tn5 precise excision from the genome of the conjugative plasmid pNM1 has been demonstrated . The pNM1 plasmid is a derivative of R100.1 and differs from the latter in the presence of Tn5 inactivating the tet gene of transposon Tn10. Mol Microbiol, 1988 Sep, 2(5), 581 - 8 Mapping and characterization of mutants of the Escherichia coli cell division gene, ftsA; Robinson AC et al.; Eight independent temperature-sensitive mutants of the cell division protein FtsA have been studied . They fall into two classes in terms of their behaviour at 42 degrees C and recovery at 30 degrees C . The first class shows salt-dependent temperature-sensitivity and reversible inactivation of FtsA protein at 42 degrees C . The second shows irreversible inactivation which is not prevented by salt . Recovery of the ability to divide at 30 degrees C is rapid in mutants of the first group, but is delayed for approximately a generation time in the second group . This suggests that irreversible inactivation of FtsA causes extensive damage to the division machinery . The amino acid substitutions show clustering to a limited domain of the protein, and one particular substitution is found in three of the mutants. Biull Eksp Biol Med, 1988 Sep, 106(9), 347 - 9 {Genetic properties of transposons Tn6-1, Tn6-2 and Tn19-1}; Gigani OB et al.; The level and range transposition of the transposons Tn6-1, Tn6-2, Tn19-1, and their ability to influence plasmid transfer has been studied . The widest range of transposition was shown for transposon Tn6-2 . Insertions of each of the studied transposons into different conjugative plasmids genomes resulted in change of frequencies of plasmids transfer and change of plasmids mobilization activity. Arch Biochem Biophys, 1988 Sep, 265(2), 227 - 33 Redistribution of phosphate pools and the regulation of Escherichia coli adenylate cyclase activity; Peterkofsky A; The enzyme adenylate cyclase plays a key role in mediating the phenomenon of catabolite repression in Escherichia coli . The mechanism by which one sugar prevents the expression of the gene for another catabolite depends on the capacity of the cell to take up the sugar . Sugars that are most effective in the repression mechanism are those that are transported by the phosphoenolpyruvate-energized phosphotransferase system . The hypothesis presented here is that one or more of the proteins associated with this sugar transport system interact with adenylate cyclase and, when they are in their phosphorylated form, activate the enzyme, provided other factors that permit this activation are present . Another essential activator of adenylate cyclase is inorganic orthophosphate . When E . coli are starved for sugars, the pool of total phosphate is accounted for primarily as inorganic orthophosphate, ATP, phosphoenolpyruvate, and transport proteins in their phospho-forms, a condition that promotes activation of adenylate cyclase . When cells are exposed to sugars, the phosphate pool becomes drastically redistributed, such that the level of inorganic orthophosphate and transport phosphoproteins decreases markedly while the pool of sugar phosphate increases . This translation of the extracellular availability of carbon sources into an intracellular phosphate redistribution is the immediate event that is responsible for catabolite repression. Virology, 1988 Sep, 166(1), 229 - 39 Tumorigenic poxviruses: fine analysis of the recombination junctions in malignant rabbit fibroma virus, a recombinant between Shope fibroma virus and myxoma virus; Upton C et al.; Malignant rabbit fibroma virus (MRV) has been shown to be a lethal tumorigenic poxvirus of rabbits derived from a recombination event between Shope fibroma virus (SFV), which induces benign fibromas in rabbits, and myxoma virus, the agent of myxomatosis . We have cloned and sequenced all of the MRV recombination junctions, which are located near the left and right terminal inverted repeat (TIR) regions, and present a composite map of the MRV genome with respect to the relevant gene products . The two junctions closet to the MRV termini, at identical positions at the left and right ends, are at nucleotide 5272 and result in an in-frame fusion protein (ORF T-5) in which the N-terminal 232 aa are derived from an SFV sequence linked to a C-terminus derived from myxoma . At the left MRV TIR the recombination junction distal from the terminus maps to nucleotide 9946 but leaves the adjacent gene virtually unchanged from its SFV homolog . At the right terminus, the relevant junction sequences from MRV and myxoma could not be cloned in wild-type Escherichia coli but were maintained stably in a recA recBC sbcB host . The SFV/myxoma junction at this location maps 5' to a growth factor gene (SFGF) which is related to those encoding epidermal growth factor and transforming growth factor-alpha . As a result, the myxoma growth factor gene has been deleted in MRV and replaced in toto by the SFV gene . The recombination junction upstream from the SFGF gene creates an in-frame fusion in ORF T11-R in which the N-terminal amino acids are derived from myxoma and the remainder from SFV . In summary, MRV has received the following ORFs from SFV: at the left terminus T5 (fusion), T6, T7, and T8; at the right terminus, T5 (fusion), T6, T7, T8, T9-R, SFGF, and T11-R (fusion). Proc Natl Acad Sci U S A, 1988 Sep, 85(18), 6987 - 91 Lysogeny and transformation in mycobacteria: stable expression of foreign genes; Snapper SB et al.; Requisite to a detailed understanding of the molecular basis of bacterial pathogenesis is a genetic system that allows for the transfer, mutation, and expression of specific genes . Because of the continuing importance of tuberculosis and leprosy worldwide, we initiated studies to develop a genetic system in mycobacteria and here report the use of two complementary strategies to introduce and express selectable genetic markers . First, an Escherichia coli cosmid was inserted into the temperate mycobacteriophage L1, generating shuttle phasmids replicating as plasmids in E . coli and phage capable of lysogenizing the mycobacterial host . These temperate shuttle phasmids form turbid plaques on Mycobacterium smegmatis and, upon lysogenization, confer resistance to superinfection and integrate within the mycobacterial chromosome . When an L1 shuttle phasmid containing a cloned gene conferring kanamycin resistance in E . coli was introduced into M . smegmatis, stable kanamycin-resistant colonies--i.e., lysogens--were obtained . Second, to develop a plasmid transformation system in mycobacteria, M . fortuitum/E . coli hybrid plasmids containing mycobacterial and E . coli replicons and a kanamycin-resistance gene were constructed . When introduced into M . smegmatis or BCG (Mycobacterium tuberculosis typus bovinus var . Bacille-Calmette-Guerin) by electroporation, these shuttle plasmids conferred stable kanamycin resistance upon transformants . These systems should facilitate genetic analyses of mycobacterial pathogenesis and the development of recombinant mycobacterial vaccines. Proc Natl Acad Sci U S A, 1988 Sep, 85(18), 6592 - 6 Properties of a mutant recA-encoded protein reveal a possible role for Escherichia coli recF-encoded protein in genetic recombination; Madiraju MV et al.; A mutation partially suppressing the UV sensitivity caused by recF143 in a uvrA6 background was located at codon 37 of recA where GTG (valine) became ATG (methionine) . This mutation, originally named srf-803, was renamed recA803 . Little if any suppression of the recF143 defect in UV induction of a lexA regulon promoter was detected . This led to the hypothesis that a defect in recombination repair of UV damage was suppressed by recA803 . The mutant RecA protein (RecA803) was purified and compared with wild-type protein (RecA+) as a catalyst of formation of joint molecules . Under suboptimal conditions, RecA803 produces both a higher rate of formation and a higher yield of joint molecules . The suboptimal conditions tested included addition of single-stranded DNA binding protein to single-stranded DNA prior to addition of RecA . We hypothesize that the ability of RecA803 to overcome interference by single-stranded DNA binding protein is the property that allows recA803 to suppress partially the deficiency in repair caused by recF mutations in the uvrA6 background . Implications of this hypothesis for the function of RecF protein in recombination are discussed. Proc Natl Acad Sci U S A, 1988 Sep, 85(17), 6468 - 72 Tn5tac1, a derivative of transposon Tn5 that generates conditional mutations; Chow WY et al.; Conditional lethal mutations are valuable for analyzing essential genes . We describe here a derivative of the bacterial transposon Tn5 called Tn5tac1 and its use in an innovative strategy for making mutations with conditional phenotypes . The 4.6-kilobase Tn5tac1 element contains a strong, regulatable, outward-facing promoter (Ptac) near one end and is polar on the expression of distal genes when the inducer of Ptac {isopropyl beta-D-thiogalactoside (IPTG)} is absent . Our results show that two unusual conditional mutant phenotypes can result from Tn5tac1 insertion in Escherichia coli: one is corrected by IPTG while the other is induced by IPTG . The broad host range of Tn5 and the conditional nature of these mutant phenotypes makes Tn5tac1 well suited for identifying essential genes in diverse bacterial species. J Immunol, 1988 Sep 1, 141(5), 1596 - 601 A rabbit Ig lambda L chain C region gene encoding C21 allotopes; Duvoisin RM et al.; Southern blot analyses of germ-line DNA obtained from rabbits expressing lambda chains of C7 and/or C21 allotypes were performed with a rabbit C lambda region-specific probe; a 12-kbp EcoRI- and a 2-kbp BamHI-hybridizing fragment were detected only in the DNA from rabbits expressing the C21 allotype . The 12-kbp EcoRI fragment was cloned and shown to contain two C lambda region-encoding genes in the same orientation . Each is preceded by a J lambda gene segment . Nonamer-12-bp spacer-heptamer recombination signal sequences were found 5' of each J lambda segment, and splicing signals were identified at the 3' ends of the J lambda segments and the 5' ends of the corresponding C lambda genes . The C lambda 5 gene, which exhibits a sequence identical with that found in several cDNA clones, is carried by the 2-kbp BamHI fragment missing from the genomic DNA of rabbits which do not express the C21 allotype . The second C lambda gene, C lambda 6, lies 3' of C lambda 5, in a 1.6-kbp BamHI fragment which is present in genomic DNAs of all tested rabbits, irrespective of their phenotype . Its sequence is identical with that found in one cDNA clone and differs from that of C lambda 5 in 17 base positions resulting in four amino acid substitutions . A fragment of a cDNA, with a J-C region sequence identical with that encoded by the J lambda 5-C lambda 5 gene pair, was subcloned into a plasmid expression vector . The resulting polypeptide product could be specifically immunoprecipitated by anti-C21 but not anti-C7 alloantisera, showing that some, if not all, C21 allotopes are encoded by the C lambda 5 gene . In contrast, the C lambda 6 gene product was not precipitable, either by anti-C7 or by anti-C21 alloantisera, although it was readily immunoprecipitated by a goat anti-rabbit lambda chain antiserum. J Bacteriol, 1988 Sep, 170(9), 4338 - 42 Cell division control in Escherichia coli K-12: some properties of the ftsZ84 mutation and suppression of this mutation by the product of a newly identified gene; Phoenix P et al.; The Fts proteins play an important role in the control of cell division in Escherichia coli . These proteins, which possibly form a functional complex, are encoded by genes that form an operon . In this study, we examined the properties of the temperature-sensitive mutation ftsZ84 harbored by low- or high-copy-number plasmids . Cells of strain AB1157, which had the ftsZ84 mutation, did not form colonies on salt-free L agar at 30 degrees C . When a low-copy-number plasmid containing the ftsZ84 mutation was present in these mutant cells, colony formation was restored on this medium at 30 degrees C, suggesting that FtsZ84 is probably less active than the wild-type protein and is therefore limiting in its capacity to trigger cell divisions . On the other hand, when the ftsZ84 mutation was harbored by the high-copy-number plasmid pBR325, colony formation was prevented on salt-free L agar plates whether the recipients were ftsZ84 mutant or parental cells, suggesting that, at high levels, FtsZ84 acts as a division inhibitor . The fact that colony formation was also prevented at 42 degrees C indicates that the FtsZ84 protein is not inactivated at the nonpermissive temperature . The possibility that FtsZ84 is a more efficient division inhibitor than the wild-type FtsZ is discussed . Evidence is also presented showing that a gene adjacent to mutT codes for a product that, under certain conditions, suppresses the ftsZ84 mutation. J Bacteriol, 1988 Sep, 170(9), 4322 - 9 Structure and regulation of the Escherichia coli ruv operon involved in DNA repair and recombination; Shinagawa H et al.; The ruv gene of Escherichia coli, which is involved in DNA repair and recombination, was cloned on a plasmid vector . The DNA of the ruv region was sequenced; it had two open reading frames in tandem that could code for 22- and 37-kilodalton proteins . The proteins encoded by these open reading frames were identified by the maxicell method . The two genes were aligned in the same orientation and regulated by the SOS system, so the two genes probably constitute an operon . The distal one complemented the ruv mutations . Transcription of the operon was studied both in vivo and in vitro . Two transcription initiation sites were identified upstream of the coding frames, and the transcription from both sites was repressed by the LexA repressor . A DNA sequence that is homologous to the SOS box and bound by LexA protein was found in the regulatory region of the operon . The amino acid sequence of Ruv protein deduced from the DNA sequence shows a high degree of homology to the consensus sequence shared by ATP-binding proteins. J Bacteriol, 1988 Sep, 170(9), 4293 - 8 Location of sites that inhibit progression of replication forks in the terminus region of Escherichia coli; Pelletier AJ et al.; We used a Southern hybridization assay to locate precisely the sites at which DNA replication is arrested in the terminus region of the Escherichia coli chromosome . The assay was based on the properties of restriction fragments that contain stalled replication forks . Replication forks that entered the terminus from the clockwise direction with respect to the genetic map were inhibited near manA at a site called T2, which we located at kilobase 442 on the physical map of Bouche (J . P . Bouche, J . Mol . Biol . 154:1-20, 1982) . Those that entered the terminus region traveling in the counterclockwise direction were inhibited near pyrF at a site called T1, which we located at kilobase 90 . In each case we found only a single, precise site of arrest . Inhibition at T1 was not detectable in our assay in strains lacking the trans-acting locus tus, which is located near T2 and is required for T1 to function . We demonstrated that the sites of inhibition are also used during termination of replication in exponentially growing, wild-type cells . In all previous studies on the terminus of E . coli, inhibition has only been detected in strains that were modified so that the origin used was placed near the terminus to force the use of the sites of inhibition. J Bacteriol, 1988 Sep, 170(9), 4125 - 35 Characteristics of a ugp-encoded and phoB-dependent glycerophosphoryl diester phosphodiesterase which is physically dependent on the ugp transport system of Escherichia coli; Brzoska P et al.; The ugp-encoded transport system of Escherichia coli accumulates sn-glycerol-3-phosphate with high affinity; it is binding protein mediated and part of the pho regulon . Here, we report that glycerophosphoryl diesters (deacylated phospholipids) are also high-affinity substrates for the ugp-encoded system . The diesters are not taken up in an unaltered form but are hydrolyzed during transport to sn-glycerol-3-phosphate plus the corresponding alcohols . The enzyme responsible for this reaction is not essential for the translocation of sn-glycerol-3-phosphate or for the glycerophosphoryl diesters but can only hydrolyze diesters that are in the process of being transported . Diesters in the periplasm or in the cytoplasm were not recognized, and no enzymatic activity could be detected in cellular extracts . The enzyme is encoded by the last gene in the ugp operon, termed ugpQ . The product of the ugpQ gene, expressed in minicells, has an apparent molecular weight of 17,500 . We present evidence that only one major phoB-dependent promoter controls all ugp genes. J Bacteriol, 1988 Sep, 170(9), 4065 - 71 Regulation of ribulose bisphosphate carboxylase expression in Rhodospirillum rubrum: characteristics of mRNA synthesized in vivo and in vitro; Leustek T et al.; The synthesis of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase) in Rhodospirillum rubrum was regulated by the CO2 concentration in the culture medium . The specific activity of RuBPCase in cells grown photolithotrophically in low concentrations of CO2 (1.5%) was five to ten times higher than that in cultures grown at high concentrations of CO2 (10%) . Increased enzyme activity was reflected by an increase in both RuBPCase mRNA and RuBPCase protein . RuBPCase expression was also studied in vitro with a plasmid-borne genomic clone (pRR117) as the template in a partially defined Escherichia coli system containing either E . coli or R . rubrum RNA polymerase . With both enzymes there was excellent synthesis of RuBPCase mRNA, but no significant synthesis of RuBPCase was detected . The promoter region of the RuBPCase gene was sequenced, and mRNA start sites were mapped . A single major in vivo transcriptional start site was detected in RuBPCase mRNA extracted from R . rubrum . However, transcripts synthesized from pRR117 in vitro or from E . coli transformed with pRR117 started at upstream sites that were different from the in vivo transcription site . Two major features of the RuBPCase promoter region are three 6-base-pair direct repeats and a 31-base-pair region of dyad symmetry. J Bacteriol, 1988 Sep, 170(9), 4001 - 7 Synthesis and export of the outer membrane lipoprotein in Escherichia coli mutants defective in generalized protein export; Watanabe T et al.; Export of the outer membrane lipoprotein in Escherichia coli was examined in conditionally lethal mutants that were defective in protein export in general, including secA, secB, secC, and secD . Lipoprotein export was affected in a secA(Ts) mutant of E . coli at the nonpermissive temperature; it was also affected in a secA(Am) mutant of E . coli at the permissive temperature, but not at the nonpermissive temperature . The export of lipoprotein occurred normally in E . coli carrying a null secB::Tn5 mutation; on the other hand, the export of an OmpF::Lpp hybrid protein, consisting of the signal sequence plus 11 amino acid residues of mature OmpF and mature lipoprotein, was affected by the secB mutation . The synthesis of lipoprotein was reduced in the secC mutant at the nonpermissive temperature, as was the case for synthesis of the maltose-binding protein, while the synthesis of OmpA was not affected . Lipoprotein export was found to be slightly affected in secD(Cs) mutants at the nonpermissive temperature . These results taken together indicate that the export of lipoprotein shares the common requirements for functional SecA and SecD proteins with other exported proteins, but does not require a functional SecB protein . SecC protein (ribosomal protein S15) is required for the optimal synthesis of lipoprotein. J Bacteriol, 1988 Sep, 170(9), 3967 - 77 Gene organization in the region containing a new gene involved in chromosome partition in Escherichia coli; Kato J et al.; A new mutation, parC, causing abnormal chromosome segregation was identified in two thermosensitive mutants of Escherichia coli . The thermosensitive growth of the mutants was corrected by pLC4-14 in the Clarke-Carbon collection . This plasmid carries a putative gene which can suppress the cell division defect due to ftsI (pbpB) and has hence been termed sufI (sui) . The nearness of parC to metC was confirmed, and cotransduction frequency of parC was 59% with metC and 20% with glc . The parC-sufI region was analyzed by subcloning the chromosome region of pLC4-14 . The parC and the sufI gene products were electrophoretically identified as proteins of 75 and 55 kilodaltons (kDa), respectively . The allelism of parC+ on pLC4-14 to parC1215 was confirmed by cloning parC1215 . The sufI gene appeared to be dispensable for cell viability, and overproduction of its product caused suppression of ftsI . An essential gene coding for a 25-kDa protein was found between the parC and the sufI gene . These three genes were transcribed in the same direction and may be organized into an operon, with parC to the proximal side and with internal promoters at least for the distal genes . The localization of the gene products was examined in maxicells . The sufI protein was synthesized as a precursor which could be chased into a mature form . The major part of the mature form was found in the soluble fraction . The 25-kDa protein was found almost exclusively in the membrane fraction . The parC protein was associated with the membrane fraction in the presence of Mg2+ but found in the soluble fraction when Mg2+ was sequestered with EDTA. J Bacteriol, 1988 Sep, 170(9), 3961 - 6 Biosynthesis and degradation both contribute to the regulation of coenzyme A content in Escherichia coli; Vallari DS et al.; Escherichia coli mutants {coaA16(Fr); Fr indicates feedback resistance} were isolated which possessed a pantothenate kinase activity that was refractory to feedback inhibition by coenzyme A (CoA) . Strains harboring this mutation had CoA levels that were significantly elevated compared with strains containing the wild-type kinase and also overproduced both intra- and extracellular 4'-phosphopantetheine . The origin of 4'-phosphopantetheine was investigated by using strain SJ135 {panD delta(aroP-aceEF)}, in which synthesis of acetyl-CoA was dependent on the addition of an acetate growth supplement . Rapid degradation of CoA to 4'-phosphopantetheine was triggered by the conversion of acetyl-CoA to CoA following the removal of acetate from the media . CoA hydrolysis under these conditions appeared not to involve acyl carrier protein prosthetic group turnover since {acyl carrier protein} phosphodiesterase was inhibited equally well by acetyl-CoA or CoA . These data support the view that the total cellular CoA content is controlled by modulation of biosynthesis at the pantothenate kinase step and by degradation of CoA to 4'-phosphopantetheine. J Bacteriol, 1988 Sep, 170(9), 3864 - 9 Cloning of the fic-1 gene involved in cell filamentation induced by cyclic AMP and construction of a delta fic Escherichia coli strain; Kawamukai M et al.; PA3092 is an Escherichia coli mutant that forms filaments at 43 degrees C in the presence of cyclic AMP (cAMP) . The mutation responsible for this phenotype is called fic-1 . We cloned fic-1 from PA3092 by selection for the neighboring argD gene . The fic-1 gene product had a relative molecular mass of 21 kilodaltons by the maxicell method . A strain with the fic gene completely deleted was constructed by replacing fic with a kanamycin resistance gene . In one of the fic-deleted strains derived from PA3092, cAMP did not induce cell filamentation at 43 degrees C, but it did in the same strain harboring a plasmid containing the fic-1 gene . These results indicate that the fic-1 gene product is necessary for the induction of cell filamentation by cAMP but is dispensable to the cell . We also found that high levels of NaCl suppressed the cell filamentation induced by cAMP. J Bacteriol, 1988 Sep, 170(9), 3847 - 54 Cloning and expression of the Mycobacterium bovis BCG gene for extracellular alpha antigen; Matsuo K et al.; The gene for the extracellular alpha antigen of Mycobacterium bovis BCG was cloned by using a single probe restricted to G or C in the third position . This technique should have great potential for the isolation of mycobacterial antigen genes . The gene analysis revealed that the alpha antigen gene encoded 323 amino acid residues, including 40 amino acids for signal peptide followed by 283 amino acids for mature protein . This is the first report on the structure of the mycobacterial signal peptide . The promoter-like sequence and ribosome-binding site were observed upstream of the open reading frame . In the coding region, the third position of the codon showed high G + C content (86%) . The gene was expressed as an unfused protein in Escherichia coli by using an E . coli expression vector . This protein, which reacted with polyclonal antibody raised against alpha antigen from Mycobacterium tuberculosis, would be applicable to the immunodiagnosis of tuberculosis. J Virol, 1988 Sep, 62(9), 3463 - 73 Identification of sequence requirements and trans-acting functions necessary for regulated expression of a human cytomegalovirus early gene; Staprans SI et al.; We analyzed the regulation of expression of a human cytomegalovirus (HCMV) early transcription unit encoded by EcoRI fragments R and d (map units, 0.682 to 0.713), located within the long unique segment of the genome . This region specified a 2.2-kilobase class of spliced transcripts which encode several related proteins . To define important upstream regulatory elements of this gene, we generated hybrid plasmids in which 5'-promoter sequences were fused to the Escherichia coli chloramphenicol acetyltransferase (CAT) gene and tested these hybrid genes in transient expression assays in human fibroblast cells . The stimulation of CAT activity in HCMV-infected cells was found to reflect an induction of correctly initiated hybrid mRNA, which was dependent on the de novo synthesis of some virally induced factor(s) . A time course experiment showed the correct early kinetics of CAT expression . Analysis of a series of 5'-promoter deletion plasmids, ending between -323 and -7 base pairs relative to the transcription start site, showed a stepwise reduction in inducible CAT activity, suggesting that this HCMV early promoter consists of multiple elements . One of these elements resembles the binding site of a previously identified cellular "transcription" factor . We also examined the role of specific virus-encoded factors in the transactivation of this promoter . Cotransfection of human fibroblasts with the 2.2-kilobase RNA promoter-CAT construct and plasmids containing different immediate-early genes showed that expression of CAT from this promoter was stimulated by the region of the HCMV genome encoding the immediate-early 1 and 2 gene products. J Virol, 1988 Sep, 62(9), 3319 - 27 Comparison of the bovine herpesvirus 1 gI gene and the herpes simplex virus type 1 gB gene; Whitbeck JC et al.; In a previous report, we localized the gene for a 130-kilodalton envelope glycoprotein (gI) of bovine herpesvirus 1 (BHV-1) to a 3.6-kilobase HpaI-KpnI restriction endonuclease fragment from the long unique region of the BHV-1 genome (map position 0.405 to 0.432) and showed that a herpes simplex virus 1 (HSV-1) glycoprotein B (gB) probe uniquely hybridized to this BHV-1 restriction fragment . Here we present the complete nucleotide sequence of the BHV-1 gI gene and the predicted 932-amino-acid sequence of the gI primary translation product . Comparison with the published nucleotide sequence of the HSV-1 (KOS) gB gene (D . J . Bzik, B . A . Fox, N . A . DeLuca, and S . Person, Virology 133:301-314, 1984) reveals a similarity of 56.3% at the nucleotide level and 45.9% at the amino acid level . Upstream of the proposed gI coding region are potential mRNA transcriptional promoter elements including a TATA box and multiple Sp1 binding sites (GC boxes) . Downstream of the gI coding region are two sequence elements associated with mRNA cleavage and polyadenylation (AATAAA and a GT-rich region roughly 30 nucleotides further downstream) . Like HSV-1 gB, the predicted gI amino acid sequence exhibits two broad hydrophobic regions likely to represent a transient amino-terminal signal sequence and a transmembrane anchor domain (near the carboxyl terminus) . Additional features shared with gB include 6 potential N-linked glycosylation sites and 10 highly conserved cysteine residues in the gI extracellular domain . Two regions of nonsimilarity between gI and gB are a centrally located 22-amino-acid region of gI for which there is essentially no gB counterpart and the transient amino-terminal leaders which differ in both size and sequence . The hydrophobic signal sequence of the gI leader, unlike that of gB, is preceded by an unusually large region of predominantly hydrophilic amino acids . The unusual length of the gI leader may result from an overlap between that portion of the gI coding region and a potential upstream coding region. J Virol, 1988 Sep, 62(9), 3309 - 18 Induction of complement-dependent and -independent neutralizing antibodies by recombinant-derived human cytomegalovirus gp55-116 (gB); Britt WJ et al.; The human cytomegalovirus (HCMV) envelope glycoprotein complex gp55-116 was expressed in both Escherichia coli and cells infected with a recombinant vaccinia virus . E . coli produced a single protein of Mr 100,000 which approximated the size of the nonglycosylated gp55-116 precursor found in HCMV-infected cells . Cells infected with the recombinant vaccinia virus contained three intracellular forms of Mr 160,000, 150,000, and 55,000 which were detected by a monoclonal antibody reactive with gp55 . Comparison of the immunological properties of these recombinant proteins indicated that several of the HCMV gp55-116 monoclonal antibodies and sera from patients infected with HCMV reacted with the vaccinia virus-derived proteins whereas a more restricted group of monoclonal antibodies recognized the E . coli-produced protein . Immunization of mice with either E . coli or vaccinia virus recombinant HCMV gp55-116 resulted in production of virus-neutralizing antibodies . In contrast to the almost exclusive production of complement-dependent neutralizing antibodies following immunization with recombinant vaccinia virus, the E . coli-derived protein induced complement-independent neutralizing antibodies. J Virol, 1988 Sep, 62(9), 3295 - 300 Alterations in the regulatory region of the human papillomavirus type 6 genome are generated during propagation in Escherichia coli; Kasher MS et al.; We analyzed the long control regions (LCRs) of seven human papillomavirus type 6b (HPV-6b) clones, which contained prototype HPV-6b sequences recloned into various plasmid vectors and propagated in different strains of Escherichia coli . Southern blot analysis and DNA sequencing demonstrated three different sequences, each distinct from the published prototype HPV-6b sequence . Two of the plasmids contained insertions of 24 and 94 base pairs (bp) and a 1-bp deletion . Four plasmids contained insertions of 24 and 58 bp and a deletion of 49 bp . One plasmid contained a single insertion of 77 bp . The 94-, and 58-bp insertions occurred at the same site and had 100% positional identity across their shared lengths . All changes were located in the purine-thymidine-rich region of the LCR (nucleotides 7292 to 7400) . Two additional LCR sequences were detected by restriction analysis of two other HPV-6b clones . We conclude that the purine-thymidine-rich region of the LCR is a hot spot for recombination in E . coli and that the alterations are the result of recA-independent events . These results emphasize the need to rigorously prove that a cloned isolate is an authentic copy of the genomic DNA present in the original lesion . In addition, the data indicate that the HPV-6b LCR sequences employed in different laboratories may be different, even if their parental DNAs were identical . Finally, we discuss the need for caution in assigning biological significance to alterations in this region, in view of the limited data available on the true identity of the HPV-6b LCR. Mol Biol (Mosk), 1988 Sep-Oct, 22(5), 1405 - 10 {Selective inhibition by 3'-azido-2',3-dideoxythymidine-5'- triphosphate of reverse transcription in retrovirus A particles from the rat liver}; Pyrinova GB et al.; The data presented demonstrate that 3'-azido-2',3'-dideoxythymidine 5'-triphosphate {dTTP (3N3)} specifically inhibits reverse transcription of viral