Escherichia coli

J Biol Chem, 1985 Jun 25, 260(12), 7281 - 8
Purification and structure of the intact Ada regulatory protein of Escherichia coli K12, O6-methylguanine-DNA methyltransferase; Nakabeppu Y et al.; The ada gene of Escherichia coli K12, the regulatory gene for the adaptive response of bacteria to alkylating agents, was cloned and placed under the control of the lac regulatory region on a multicopy runaway plasmid, thereby yielding a hybrid plasmid pYN3059 . Ada protein with a molecular weight of about 38,000 was overproduced when cells harboring pYN3059 were incubated at 42 degrees C in the presence of a lac inducer, isopropyl-beta-D-thiogalactoside . Taking advantage of overproduction of Ada protein, we purified the protein to apparent physical homogeneity . The purified 38,000-dalton Ada protein transferred the methyl group from the O6-methylguanine residue of alkylated DNA to the Ada protein, per se . Although the Ada protein was degraded to smaller polypeptides when crude extracts or partially purified preparations were incubated in a high ionic-strength buffer at neutral pH, the purified Ada protein remained stable under the same conditions, indicating that the Ada protein may not undergo autodegradation . An amino-terminal sequence and total amino acid composition of the purified Ada protein were in accord with nucleotide sequence of the ada gene, determined by the dideoxy method using M13 phage . It was deduced that Ada protein comprises 354 amino acids and its molecular weight is 39,385 . The promoter for the ada gene was determined by S1 nuclease mapping.

J Biol Chem, 1985 Jun 25, 260(12), 7206 - 13
Characterization of the lep operon of Escherichia coli . Identification of the promoter and the gene upstream of the signal peptidase I gene; March PE et al.; The DNA sequence flanking the gene (lep) encoding signal peptidase I of Escherichia coli has been determined . The upstream flanking sequence contains a gene (lepA) that encodes a polypeptide of 598 amino acid residues and terminates 18 base pairs from the initiation codon of the lep gene . The position of the lep promoter was determined by both gene fusion with the lacZ gene as well as by S1 nuclease mapping of the lep mRNA to be 73 base pairs upstream from the initiation codon of the lepA gene . The lepA gene was cloned into a high expression vector (pIN-III), and its gene product was identified to be a protein of apparent molecular weight of 76,000 . This gene product was preferentially localized in the cytoplasmic membrane and periplasmic fractions upon subcellular fractionation . The DNA sequence immediately downstream of the lep gene contains features consistent with a rho factor-independent transcriptional termination site, indicating that the lep operon encodes only two proteins (lepA and lep).

J Biol Chem, 1985 Jun 25, 260(12), 7214 - 8
A monoclonal antibody that recognizes the functional domain of Escherichia coli single-stranded DNA binding protein that includes the ssb-113 mutation; Chase JW et al.; We have isolated a monoclonal antibody against Escherichia coli single-stranded DNA binding protein (SSB) that recognizes the functional domain specified by the ssb-113 temperature-sensitive mutation, a domain which is distinct from the DNA-binding site . Although the ssb-113 and ssb-1 mutations result in many similar phenotypic defects, they differ significantly in others, indicating that they affect different functional domains of the protein . Whereas the SSB-1 mutant protein is clearly defective in tetramer formation and is also unable to bind single-stranded DNA at nonpermissive temperatures, no similar in vitro defects have yet been found in the SSB-113 mutant protein . In fact, the only reported in vitro effect of the ssb-113 mutation on the protein is a slight increase in its helix destabilizing ability . Competition radioimmunoassays using a monoclonal antibody demonstrated that SSB-113 mutant protein, containing a single amino acid substitution at position 176 (the penultimate residue), did not compete with SSB while SSB-1 protein (with a single change at position 55) did compete with SSB . This analysis was refined by studies with a proteolysis fragment and with peptides derived from both SSB and SSB-113 . The results indicate that the antibody recognizes a determinant near the COOH-terminal end of the protein and that the SSB-113 mutation lies within or very close to this determinant.

J Mol Biol, 1985 Jun 25, 183(4), 529 - 41
Identification and characterization of mutants affecting transcription termination at the threonine operon attenuator; Lynn SP et al.; Mutations that map in or delete the attenuator of the threonine (thr) operon of Escherichia coli were isolated and characterized . These mutations disrupt or delete the transcription termination structure encoded by the attenuator leading to increased transcriptional readthrough into the thr operon structural genes . Most of the base substitutions and single base-pair insertions and deletions map in the G + C-rich region of dyad symmetry in the attenuator and decrease the calculated stabilities of the attenuator RNA secondary structures to similar extents (from -30.8 kcal/mol to approximately -21 kcal/mol) . Most of the mutants showed a three- to fourfold increase in homoserine dehydrogenase (thrA gene product) synthesis relative to the wild-type parent strain . The mutation in one mutant (thrL153 + G) lowered the calculated stability of the RNA secondary structure only slightly (from -30.8 to 27.8 kcal/mol) but the mutant still exhibited high levels of homoserine dehydrogenase synthesis . In addition, three base substitution mutants (thrL135U, thrL139A and thrL156U) showed only slightly (1.5 to 2-fold) elevated levels of homoserine dehydrogenase activity, even though the calculated stabilities of the attenuator RNA secondary structures were reduced as much as most of the other mutants . Two of the mutations (thrL135U and thrL156U) mapped in the G + C-rich-A + T-rich junction of the attenuator . The third mutation (thrL139A) creates an A X C pair in the center of the G + C-rich region of the attenuator stem . The results obtained for these mutants show that the stability of the RNA secondary structure does not always correlate with the efficiency of transcription termination . Finally, analysis of the base changes in the substitution mutations showed that the mutational changes do not appear to be random.

Biochim Biophys Acta, 1985 Jun 24, 825(2), 207 - 13
Stabilisation of human interferon beta synthesis in Escherichia coli by zinc ions; Gross G et al.; Human interferon beta synthesized in Escherichia coli is unstable and toxic for the bacterial cell . Zinc ions are able to stabilise interferon beta in E . coli probably by inhibiting the action of cell internal proteinase(s) which affect the half-life of this foreign protein . As a result up to one order of magnitude more active IFN-beta can be detected in Zn2+-treated E . coli cells.

Biochim Biophys Acta, 1985 Jun 24, 825(2), 161 - 8
Covalent cross-linking of tRNAGly1 to the ribosomal P site via the dihydrouridine loop; Chen JK et al.; The dihydrouracil residue at position 20 of Escherichia coli tRNAGly1 has been replaced by the photoaffinity reagent, N-(4-azido-2-nitrophenyl)glycyl hydrazide (AGH) . The location of the substituent was confirmed by the susceptibility of the modified tRNA to cleavage with aniline . When N-acetylglycyl-tRNAGly1 derivatized with AGH was bound noncovalently to the P site of E . coli 70 S ribosomes, 5-6% on average was photochemically cross-linked to the ribosomal particles in a reaction requiring poly(G,U), irradiation and the presence of the AGH label in the tRNA . Approximately two-thirds of the covalently attached tRNA was associated with 16 S RNA in the 30 S subunit . This material was judged to be in the P site by the criterion of puromycin reactivity . As partial RNAase digestion of the tRNA-16 S RNA complex produced labeled fragments from both 5' and 3' segments of the rRNA, there appeared to be more than one site of cross-linking in the 30 S subunit . The small amount of N-acetylglycyl-tRNAGly1 associated with the 50 S subunit was also linked mainly to rRNA, but it was not puromycin-reactive.

Biochim Biophys Acta, 1985 Jun 24, 825(2), 255 - 60
Function and structure of microvirid phage alpha 3 genome . DNA sequence of H gene and properties of missense H mutant; Kodaira K et al.; The nucleotide sequence of wild-type alpha 3 H gene and its surrounding region was determined and compared with those of phi X174 and G4 . The corresponding DNA regions in double mutants amJH22, amJH69 and amJH76 were also sequenced and their missense mutation sites located . A phage strain missH22 having a single missense mutation in gene H was constructed by replacing the J region of amJH22 in vitro with the wild-type DNA . Like amJH22, the missense mutant coded for H protein with aberrant electrophoretic mobility, but formed normal plaques on suppressor-deficient Escherichia coli . Heat stability, plating efficiency on certain hosts and rate of eclipse were higher in strain missH22 than in wild-type phage.

Nature, 1985 Jun 20-26, 315(6021), 641 - 7
Cloning, sequence and expression of two distinct human interleukin-1 complementary DNAs; March CJ et al.; Two distinct but distantly related complementary DNAs encoding proteins sharing human interleukin-1 (IL-1) activity (termed IL-1 alpha and IL-1 beta), were isolated from a macrophage cDNA library . The primary translation products of the genes are 271 and 269 amino acids long, although expression in Escherichia coli of the carboxy-terminal 159 and 153 amino acids produces IL-1 biological activity.

J Chromatogr, 1985 Jun 19, 326, 157 - 61
Purification of a uridine-specific acid nuclease from chicken liver by fast protein liquid chromatography; Ellis B et al.; A rapid purification procedure for a novel uridine-specific nuclease from chicken liver based on the Pharmacia fast protein liquid chromatography (FPLC) system is presented . The purification was achieved by applying crude extract to a Mono S cation-exchange column equilibrated with 10 mM potassium phosphate buffer (pH 6.0) . The enzyme was eluted in 20 min with a potassium chloride gradient at a flow-rate of 2 ml/min . The enzyme was then chromatographed on a Superose 12 size-exclusion column in less than 1 h at a flow-rate of 0.5 ml/min (Kav = 0.77) . The enzyme was re-chromatographed on a second Mono S column to concentrate the protein . The uridine-specific nuclease hydrolyzed poly(U) and Escherichia coli 5S RNA . Poly(A) was hydrolyzed by the nuclease less efficiently (about 10% of the poly(U) activity) . No hydrolysis was detected with poly(C), poly(G), poly(dT) or poly(dA) as substrate . The speed with which each purification step could be carried out facilitated the determination of optimal chromatographic conditions . We found that the resolution of the Mono S and Superose 12 columns was superior to that of conventional ion exchangers and size-exclusion columns respectively.

J Chromatogr, 1985 Jun 19, 326, 357 - 61
Characterization of recombinant human interleukin-2 with micromethods; Lahm HW et al.; Highly purified recombinant human interleukin-2, expressed in Escherichia coli, was analyzed by micromethods . N-Terminal sequence analysis showed that methionine at position 0 was found in 90% of the molecules and not completely removed in post-ribosomal processing . A complete peptide map of the reduced and S-carboxymethylated protein was obtained by high-performance liquid chromatography after tryptic digestion, and the fragments were identified by amino acid analysis and automated Edman sequence analysis . Using a double-label S-carboxymethylation procedure, it was determined that there is a disulfide linkage between the cysteine residues at positions 58 and 105 . The third cysteine residue at position 125 was found to be present as the free sulfhydryl.

J Chromatogr, 1985 Jun 19, 326, 251 - 61
High-performance liquid chromatography of DNA restriction fragments; Hecker R et al.; High-performance liquid chromatography on Nucleogen-DEAE 4000-10 has been applied to several problems of the isolation of DNA restriction fragments . Large amounts of DNA fragments of high purity are necessary for biophysical studies and for molecular hybridization in basic research, as well as in medical diagnosis . The influence of various parameters, such as buffer, pH, eluting salt, gradient slope, flow-rate and the addition of urea on the resolution of fragments by high-performance liquid chromatography were studied on an analytical scale, and the optimal conditions were then used for the large-scale preparation of milligram amounts . The best resolution of fragments between 25 and 1500 base pairs was obtained with a linear gradient from 500 mM to 1200 mM sodium chloride in 6 M urea -30 mM sodium phosphate (pH 6.0) . Quantitative data are given for the purity and recovery of the sample, and the capacity and lifetime of the column . The following applications of high-performance liquid chromatography of restriction fragments are described: preparation of 2 mg of fragments, separation of 1 mg of DNA insert from 7 mg of its plasmid vector, and analysis of DNA-RNA hybrids.

Biochemistry, 1985 Jun 18, 24(13), 3240 - 5
Characterization of effects of anti-beta and anti-beta' monoclonal antibodies on the activity of the RNA polymerase from Escherichia coli; Rockwell P et al.; Monoclonal antibodies directed against antigenic determinants on the beta and beta' subunits of the Escherichia coli RNA polymerase were characterized by using d(A-T)n-directed transcription assays . Antibodies were prepared by using purified subunits as immunogens, and seven anti-beta and five anti-beta' monoclonal antibodies were generated . Inhibitory anti-beta monoclonal antibodies were found to affect RNA polymerase during synthesis of r(A-U)n, abortive initiation of pApU and UpApU, and elongation by preformed ternary complexes . A comparative enzyme study of r(A-U)n synthesis showed the core polymerase to be more sensitive to inhibition by the anti-beta monoclonal antibody than was the holoenzyme . In contrast, the inhibition effected by the anti-beta' monoclonal antibody was found to be 90% or greater for each of the d(A-T)n-directed assays used . The different inhibitory patterns exhibited by the anti-beta and anti-beta' monoclonal antibodies suggest that the beta and beta' subunits engage in different roles during transcription . Kinetic analysis of the abortive initiation reaction in the presence and absence of the inhibitory antibodies resulted in distinctive but complex modes of inhibition . Inhibition by the anti-beta monoclonal antibody 210E8 was noncompetitive with regard to UTP and competitive for UpA incorporation; at increased UpA concentration, the inhibition was completely reversed . Inhibition of the abortive synthesis of UpApU by the anti-beta' monoclonal antibody 311G2 was noncompetitive with regard to both UpA and UTP incorporation . When the preformed ternary elongation complex was used, inhibition by the anti-beta monoclonal antibody was mixed with regard to the ribonucleoside triphosphate substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1985 Jun 18, 24(13), 3233 - 9
Conservation of RNA sequence and cross-linking ability in ribosomes from a higher eukaryote: photochemical cross-linking of the anticodon of P site bound tRNA to the penultimate cytidine of the UACACACG sequence in Artemia salina 18S rRNA; Ciesiolka J et al.; The complex of Artemia salina ribosomes and Escherichia coli acetylvalyl-tRNA could be cross-linked by irradiation with near-UV light . Cross-linking required the presence of the codon GUU, GUA being ineffective . The acetylvalyl group could be released from the cross-linked tRNA by treatment with puromycin, demonstrating that cross-linking had occurred at the P site . This was true both for pGUU- and also for poly(U2,G)-dependent cross-linking . All of the cross-linking was to the 18S rRNA of the small ribosomal subunit . Photolysis of the cross-link at 254 nm occurred with the same kinetics as that for the known cyclobutane dimer between this tRNA and Escherichia coli 16S rRNA . T1 RNase digestion of the cross-linked tRNA yielded an oligonucleotide larger in molecular weight than any from un-cross-linked rRNA or tRNA or from a prephotolyzed complex . Extended electrophoresis showed this material to consist of two oligomers of similar mobility, a faster one-third component and a slower two-thirds component . Each oligomer yielded two components on 254-nm photolysis . The slower band from each was the tRNA T1 oligomer CACCUCCCUVACAAGp, which includes the anticodon . The faster band was the rRNA 9-mer UACACACCGp and its derivative UACACACUG . Unexpectedly, the dephosphorylated and slower moving 9-mer was derived from the faster moving dimer . Deamination of the penultimate C to U is probably due to cyclobutane dimer formation and was evidence for that nucleotide being the site of cross-linking . Direct confirmation of the cross-linking site was obtained by "Z"-gel analysis {Ehresmann, C., & Ofengand, J . (1984) Biochemistry 23, 438-445}.(ABSTRACT TRUNCATED AT 250 WORDS)

Eur J Biochem, 1985 Jun 18, 149(3), 517 - 23
Purification and properties of reconstitutively active nicotinamide nucleotide transhydrogenase of Escherichia coli; Clarke DM et al.; The nicotinamide nucleotide transhydrogenase of Escherichia coli has been purified from cytoplasmic membranes by pre-extraction of the membranes with sodium cholate and Triton X-100, solubilization of the enzyme with sodium deoxycholate in the presence of 1 M potassium chloride, and centrifugation through a 1.1 M sucrose solution . The purified enzyme consists of two subunits, alpha and beta, of apparent Mr 50000 and 47000 . During transhydrogenation between NADPH and 3-acetylpyridine adenine dinucleotide by both the purified enzyme reconstituted into liposomes and the membrane-bound enzyme, a pH gradient is established across the membrane as indicated by the quenching of the fluorescence of 9-aminoacridine . Treatment of transhydrogenase with N,N'-dicyclohexylcarbodiimide results in an inhibition of proton pump activity and transhydrogenation, suggesting that proton translocation and catalytic activities are obligatory linked . NADH protected the enzyme against inhibition by N,N'-dicyclohexylcarbodiimide, while NADP, and to a lesser extent NADPH, appeared to increase the rate of inhibition . {14C}Dicyclohexylcarbodiimide preferentially labelled the 50000-Mr subunit of the transhydrogenase enzyme . The presence of an allosteric binding site which reacts with NADH, but not with reduced 3-acetylpyridine adenine dinucleotide, has been demonstrated.

Eur J Biochem, 1985 Jun 18, 149(3), 609 - 16
Molecular cloning and analysis of cDNA sequences for two ribosomal proteins from Artemia . The coordinate expression of genes for ribosomal proteins and elongation factor 1 during embryogenesis of Artemia; Maassen JA et al.; The large subunit of eukaryotic ribosomes contains acidic phosphoproteins which are related to L7/L12 from Escherichia coli . In the brine shrimp Artemia these proteins are designated eL12 and eL12' . We have isolated cDNA clones for these proteins from a cDNA bank that was constructed by the use of size-fractionated poly(A)-rich RNA (8-10S fraction) from Artemia and a synthetic oligonucleotide as primer . Clones containing DNA sequences coding for eL12 and eL12 were characterized by hybrid-selected translation and DNA sequencing . The proteins eL12 and eL12' share an identical peptide of 22 amino acids at their carboxy termini whereas the remaining part of the protein shows little sequence homology . The nucleotide sequences show a different codon use for the amino acids in the common carboxy terminus, thereby excluding a common exon coding for this part of both proteins . Despite the differences in amino acid sequence in the major part of eL12 and eL12' the proteins have a considerable degree of homology on the basis of the distribution of hydrophobic and hydrophilic amino acids over the polypeptide chains, in agreement with a related folding and function of both proteins . Relative levels of mRNA coding for eL12, eL12' and elongation factor 1 alpha were determined during the development of Artemia from a dormant cyst to a nauplius . The data show a coordinate expression of the genes for EF-1 alpha and both ribosomal proteins, excluding a differential expression of the genes for these related ribosomal proteins during embryogenesis . Analysis of the gene copy number for eL12 and eL12' indicates the presence of a few genes for each protein.

Biochem Pharmacol, 1985 Jun 15, 34(12), 2069 - 72
Inhibition of a soman- and diisopropyl phosphorofluoridate (DFP)-detoxifying enzyme by Mipafox; Hoskin FC; Mipafox, N,N'-diisopropylphosphordiamidofluoridate, has been found to be a reversible competitive inhibitor of a diisopropyl phosphorofluoridate hydrolyzing enzyme (DFPase) isolated from hog kidney and Escherichia coli . Heretofore, this DFPase was characterized by its more rapid hydrolysis of Soman (1,2,2-trimethylpropyl methylphosphonofluoridate), its stimulation by Mn2+, and its wide distribution . In sharp contrast, Mipafox did not inhibit the DFPase found only in cephalopod nerve, hepatopancreas, and saliva, and further characterized by its more rapid hydrolysis of DFP than of Soman, and its indifference to Mn2+ . Neither of these two DFPases hydrolyzed Mipafox.

Carbohydr Res, 1985 Jun 15, 139, 13 - 22
Empirical 13C-n.m.r.-correlations between the Escherichia coli K 13 and LP 1092 capsular polysaccharides and model oligosaccharides containing D-ribose and 3-deoxy-D-manno-2-octulosonic acid; Neszmelyi A et al.; The proton-decoupled, Fourier-transform, 13C-n.m.r . spectra of the two anomeric sodium (methyl 3-deoxy-7-O-beta-D-ribofuranosyl-alpha- and beta-D-manno-2-octulopyranosid)onates, of the two anomeric sodium {methyl 3-deoxy-7-O-(2-O-beta-D-ribofuranosyl-beta-D-ribofuranosyl)-alpha- or -beta-D-manno-2-octulopyranosid}onates, and of methyl 2-O-beta-D-ribofuranosyl-beta-D-ribofuranoside have been recorded . The constitutions of these compounds correspond to repeating units and partial structures of the capsular polysaccharides from Escherichia coli K 13, K 20, K 23, and LP 1092 strains . The 13C-n.m.r.-line patterns of these oligosaccharide derivatives and the corresponding polysaccharides show striking differences dependent upon the anomeric configurations of the KDO residues . These differences may be used for the identification, by visual or computer-assisted pattern analysis, of the anomeric configurations of KDO-residues in oligo- or poly-saccharides . Thus, it was confirmed that the KDO residues in the K 13, K 20, and K 23 polysaccharides have the beta anomeric configuration, whereas those in the LP 1092 polysaccharide have the alpha anomeric configuration.

Carbohydr Res, 1985 Jun 15, 139, 261 - 71
Structure of the amino acid-containing capsular polysaccharide (K54 antigen) from Escherichia coli O6:K54:H10; Hofmann P et al.; The structure of the K54-antigenic polysaccharide (K54 antigen) of Escherichia coli O6:K54:H10 was elucidated by determination of the composition, 1H- and 13C-n.m.r . spectroscopy, periodate oxidation, and a study of the oligosaccharides obtained by partial hydrolysis with acid . The K54 polysaccharide consists of----3)-beta-D-glucosyluronic acid-(1----3)-alpha-L-rhamnosyl-(1----repeating-units . Of the glucuronic acid residues, approximately 85% are substituted in the ratio 9:1 with L-threonine and L-serine amidically linked to the carboxyl group . The K54 polysaccharide has a molecular weight of approximately 160,000, corresponding to approximately 380 repeating-units.

Experientia, 1985 Jun 15, 41(6), 764 - 7
Differential sensitivity to tributyltin of cytochrome-containing and cytochrome-deficient cells of Escherichia coli SASX76; Singh AP et al.; The effect of tributyltin (TBT) chloride on the growth of cytochrome-deficient and cytochrome-containing cells of Escherichia coli SASX76 was examined . The former cells were found to be at least 20 times more sensitive to TBT . It is proposed that the differential sensitivity of these two cell types to the biocide, TBT, may be due to a different mode of energy generation by cytochrome-deficient and cytochrome-sufficient cells . In addition to the energy state, the pH change caused by the presence and absence of cytochromes which occurred during growth also resulted in a differential sensitivity of these cells.

Biochem Biophys Res Commun, 1985 Jun 14, 129(2), 479 - 84
Immunochemical comparison of lipoamide dehydrogenases from various sources and reactivity of various lipoamide dehydrogenases with rat heart pyruvate dehydrogenase-subcomplex; Matuda S et al.; Lipoamide dehydrogenases from various sources were purified and their immunochemical properties were compared . Antibody against rat lipoamide dehydrogenase reacted with rat, human, pig, pigeon and frog enzymes, but not with enzymes from E . coli, yeast and Ascaris . Anti-Ascaris enzyme and anti-E . coli enzyme antibodies reacted with Ascaris and E . coli enzymes, respectively . The pyruvate dehydrogenase subcomplex, which consists of pyruvate dehydrogenase and lipoate acetyltransferase, was prepared by releasing the lipoamide dehydrogenase from rat heart pyruvate dehydrogenase complex by anti-lipoamide dehydrogenase antibody . Lipoamide dehydrogenases from various sources were added to rat pyruvate dehydrogenase subcomplex and the complex overall activity was measured . Each lipoamide dehydrogenase effectively recovered the overall activity of rat pyruvate dehydrogenase subcomplex to 80% of the original activity.

Biochem Biophys Res Commun, 1985 Jun 14, 129(2), 321 - 7
Specific and non specific Escherichia coli ribonucleic acid polymerase DNA complexes are not hydrodynamically equivalent in analytical band sedimentation; Schmitt B et al.; We have measured the sedimentation coefficients (s) of different DNA molecules of a few thousand base bairs in the presence of increasing amounts of E . coli RNA polymerase under conditions where tight binding complexes are formed . The measured s does not increase linearly with n(n=RNA Polymerase/DNA molar ratio); the s vs n plot can be decomposed into two parts; first the increase in s is small until n reaches a value n0 approximately equal to the number of strong promoters of the DNA molecule under consideration, then when n greater than n0 the slope of s(n) is much higher . The observations are in agreement with a model which postulates that strong specific polymerase binding leads to an increase in frictional coefficient of the RNA Polymerase-DNA complex, while non specific(or less specific)RNAP binding leads to a contraction of the RNA Polymerase-DNA complexes.

J Immunol Methods, 1985 Jun 12, 80(1), 55 - 66
Monoclonal antibodies to human interferon-gamma . I . Antibodies to a synthetic carboxyl-terminal peptide; Ichimori Y et al.; Two types of hybridomas secreting monoclonal antibodies (MAB) against human interferon-gamma (HuIFN-gamma) were obtained by somatic cell hybridization between mouse myeloma P3U1 cells and spleen cells from BALB/c mice immunized with a conjugate of a synthetic carboxyl-terminal peptide (residues 131-146) of HuIFN-gamma and bovine thyroglobulin . One of the antibodies bound to recombinant HuIFN-gamma produced in E . coli as well as to natural HuIFN-gamma, while the others bound only to recombinant HuIFN-gamma . These 2 types of MAB did not neutralize the anti-viral activity of HuIFN-gamma . They were useful for effectively purifying recombinant HuIFN-gamma and quantitatively determining it by an enzyme-linked immunosorbent assay.

J Immunol Methods, 1985 Jun 12, 80(1), 7 - 13
Passive immunization: a method of enhancing the immune response against antigen mixtures; Thalhamer J et al.; Antigenic competition is known to be a widespread phenomenon when using crude extracts of antigens (e.g., Escherichia coli cytoplasmic proteins) for immunization . This non-specific form of immune suppression can be partially overcome by passive immunization with antibodies against dominant antigens (which are the suppressive molecules) before injection of the antigenic mixture . Blocking these immunodominant antigens or antigenic determinants by a passively administered antibody permits antibody responses against hitherto weakly or non-immunogenic molecules.

Nucleic Acids Res, 1985 Jun 11, 13(11), 3931 - 40
Salt induced transitions between multiple conformations of poly (rG-m5dC).poly (rG-m5dC); Wu HY et al.; Salt induced transitions between four conformations of the methylated ribo-deoxyribo co-polymer poly (rG-m5dC).poly (rG-m5dC) have been studied using phosphorous-NMR, Raman spectroscopy, and circular dichroism . A high salt A-Z transition is observed for the polymer . However, the methylated polymer does not enter the high salt Z form more readily than the analogous unmethylated polymer, unlike the effect of methylation on the fully deoxy polymer poly (dG-dC).poly (dG-dC) . The methylated polymer fails to undergo a low salt A-Z transition in 5 mM Tris buffer, unlike the unmethylated poly (rG-dC).poly (rG-dC) . However, if the counterion is changed to triethanolamine buffer, an A-Z transition does take place . In 5 mM Tris buffer the phosphorous-NMR spectrum of poly (rG-m5dC).poly (rG-m5dC) shows one resonance in the absence of NaCl that splits into two closely spaced resonances as the NaCl level is increased to 30 mM . The Raman spectrum of poly (rG-m5dC).poly (rG-m5dC) shows that it is in the A conformation at intermediate salt concentrations . From this we conclude that poly (rG-m5dC).poly (rG-m5dC) is in a regular A conformation in Tris buffer at low Na+ levels, shifting to an alternating A conformation with a dinucleotide repeat at intermediate salt concentrations.

Biochim Biophys Acta, 1985 Jun 11, 816(1), 77 - 82
Biotin uptake: influx, efflux and countertransport in Escherichia coli K12; Piffeteau A et al.; Biotin uptake by Escherichia coli K12 has been reinvestigated . The vitamin uptake is an active process depending on energy and inhibited by uncouplers . The kinetic parameters (Km = 0.27 microM, Vmax = 6.8 pmol/min per mg dry cells) are close to those previously determined for a biotin-dependent strain E . coli C162 (Piffeteau, A., Zamboni, M . and Gaudry, M . (1982) Biochim . Biophys . Acta 688, 29-36) . By use of biotin p-nitrophenyl ester, an affinity label of the biotin transport system, it was shown, under conditions of steady state, that the efflux of biotin is not energy dependent and is mainly mediated by a diffusion mechanism . Reexamination of the regulation of the biotin transport by biotin, revealed that only 50% of the biotin uptake system is under control by the vitamin.

Nucleic Acids Res, 1985 Jun 11, 13(11), 4113 - 23
Codon-defined ribosomal pausing in Escherichia coli detected by using the pyrE attenuator to probe the coupling between transcription and translation; Bonekamp F et al.; This communication describes an assay for the relative translation efficiency of individual codons which makes use of the pyrE attenuator to probe the coupling between transcription and translation at the end of an artificial leader peptide . By cloning of short synthetic DNA fragments the codons to be tested were placed in the middle of the leader peptide and the downstream transcription of a pyrE"lacZ gene was monitored by measuring beta-galactosidase activity . The substitution, one by one, of three AGG codons for arginine with three CGT codons for the same amino acid residue was found to cause a two fold increase per codon of transcription over the pyrE attenuator, such that an eight fold higher frequency of pyrE expression was seen when all three AGG codons were replaced by CGT codons . No such effect of codon composition was observed, when the cells were grown with a low UTP pool which causes a reduction of the mRNA chain growth rate.

Nucleic Acids Res, 1985 Jun 11, 13(11), 4097 - 112
Variants within the yeast Ty sequence family encode a class of structurally conserved proteins; Fulton AM et al.; The Ty transposable elements of Saccharomyces cerevisiae form a heterogeneous family within which two broad structural classes (I and II) exist . The two classes differ by two large substitutions and many restriction sites . We show that, like class I elements a class II element, Tyl-17, also appears to contain at least two major protein coding regions, designated TYA and TYB, and the organisational relationship of these regions has been conserved . The TYA genes of both classes encode proteins, designated p1 proteins, with an approximate molecular weight of 50 Kd and, despite considerable variation between the TYA regions at the DNA level, the structures of these proteins are remarkably similar . These observations strongly suggest that the p1 proteins of Ty elements are functionally significant and that they have been subject to selection.

Nucleic Acids Res, 1985 Jun 11, 13(11), 4011 - 27
Nucleotide sequence of the yeast ILV2 gene which encodes acetolactate synthase; Falco SC et al.; We have determined the nucleotide sequence of the yeast ILV2 gene which codes for the amino acid biosynthetic enzyme acetolactate synthase (ALS) . ALS has recently been shown to be the target in bacteria, yeast and plants, of the potent new herbicide sulfometuron methyl . The coding sequence for the ILV2 polypeptide contains 2061 base pairs . Comparison of deduced amino acid sequences indicates considerable conservation between the yeast protein and the large subunits of the E . coli ALS II and ALS III isozymes . A major distinction between the three proteins is the presence of an additional 90 amino acids at the amino terminal of the yeast protein . The amino acid sequence in this region shows similarities to yeast mitochondrial transit sequences and may function as such, since yeast ALS is localized in the mitochondria . Consensus sequences for initiation and termination of transcription that are consistent with the ends of the ILV2 mRNA, as well as general amino acid control regulatory sequences have been identified.

Nucleic Acids Res, 1985 Jun 11, 13(11), 3995 - 4010
The nucleotide sequence of the ilvBN operon of Escherichia coli: sequence homologies of the acetohydroxy acid synthase isozymes; Wek RC et al.; Three acetohydroxy acid synthase isozymes, AHAS I (ilvBN), AHAS II (ilvGM) and AHAS III (ilvIH) catalyze the first step of the parallel isoleucine-valine biosynthetic pathway in Escherichia coli . Previous DNA sequence and protein purification data have shown that AHAS II and AHAS III are composed of large and small subunits encoded in the ilvGMEDA and ilvIH operons, respectively . Recent protein purification and characterization data have demonstrated that the AHAS I isozyme is also composed of large and small subunits (L . Eoyang, L . and P . M . Silverman {1984} J . Bacteriol . 157:184-189) . Now the complete DNA sequence of the operon encoding the AHAS I isozyme has been determined . These data show that both AHAS I subunits (Mr 60,400 and Mr 11,100) are encoded in this operon . The coordinant regulation of both genes of the ilvBN operon has also been demonstrated . Comparisons of the DNA sequences of the genes encoding all three AHAS isozymes have been performed . Conserved homologies were observed between both the large and small subunits of all three isozymes . The closest homology was seen between the AHAS I and AHAS II isozymes . On the basis of these comparisons a rationale for the evolution of the AHAS isozymes in E . coli has been proposed.

Nucleic Acids Res, 1985 Jun 11, 13(11), 3979 - 93
The ilvB locus of Escherichia coli K-12 is an operon encoding both subunits of acetohydroxyacid synthase I; Friden P et al.; The ilvB locus of Escherichia coli K-12 encloses two open reading frames defining polypeptides of 60,000 and 11,200 molecular weight . The entire locus, about 2.3 kb, is co-transcribed as an operon . The molecular weights and amino acid compositions of the presumptive operon polypeptides agree with those of the large and small subunit polypeptides of acetohydroxyacid synthase (AHAS) I, for which ilvB is the structural locus . We reserve the designation ilvB for the promoter proximal (longer) cistron and designate the promoter distal cistron ilvN . The molecular weight and amino acid sequence of the ilvB polypeptide are strikingly similar to those of the I1vI (larger subunit of AHAS III) and I1vG (larger subunit of AHAS II) polypeptides . There is less size uniformity among the I1vN, I1vH (smaller subunit of AHAS III), and I1vM (smaller subunit of AHAS II) polypeptides . Nevertheless, there is significant amino acid sequence homology among the three small subunit polypeptides . Thus, all three AHAS isozymes of E . coli K-12 probably have a common evolutionary origin.

Nucleic Acids Res, 1985 Jun 11, 13(11), 3891 - 903
Nucleotide sequence of the alpha ribosomal protein operon of Escherichia coli; Bedwell D et al.; In Escherichia coli some 19 transcription units encoding the 52 ribosomal proteins are scattered throughout the genome . One of the units, the alpha operon, encodes genes for the ribosomal proteins S13, S11, S4 and L17 as well as the alpha subunit of RNA polymerase . We report here the complete 3.0 kb nucleotide sequence of the alpha operon . In addition, we have determined by S1 nuclease mapping the site of transcription termination in this operon.

Nucleic Acids Res, 1985 Jun 11, 13(11), 3823 - 39
Overproduction of the EcoR V endonuclease and methylase; Bougueleret L et al.; Strains overproducing the EcoR V endonuclease and methylase have been obtained by inserting each of the two genes in expression vectors containing the lambda PL promoter . The methylase is overproduced to a level reaching 5-10% of the total cellular proteins, which represents a 50-100 fold increase . A 30 fold overproduction of endonuclease was achieved by randomly positioning the EndRV gene downstream of the lambda PL promoter . The situation in the endonuclease overproducing clone resembles that encountered in maxi-cells . The strains described here allowed a quick purification of both enzymes in sufficient amounts for crystallisation attempts.

Nucleic Acids Res, 1985 Jun 11, 13(11), 4171 - 90
Further characterization of the extremely small mitochondrial ribosomal RNAs from trypanosomes: a detailed comparison of the 9S and 12S RNAs from Crithidia fasciculata and Trypanosoma brucei with rRNAs from other organisms; Sloof P et al.; We have determined the nucleotide sequence of a maxi-circle segment from the insect trypanosome Crithidia fasciculata mitochondrial DNA, on which the genes for the major maxicircle transcripts of 9S and 12S are localized . The 5'-terminal sequences of these RNAs were determined by wandering spot analysis . The map coordinates of the 9S and 12S RNAs from Trypanosoma brucei were adjusted with respect to a previous report with the aid of primer extension analysis with reverse transcriptase . This approach allowed us to align the corresponding genes from both organisms which show an overall sequence homology of 77% . The 9S and 12S RNA genes from the two trypanosome species contain sequences, closely related to some of the regions that are universally conserved among ribosomal RNAs from members of the three primary kingdoms and their organelles, even though the overall level of sequence homology is extremely low . These universal sequences occur at positions in the 9S and 12S RNAs that are analogous to those occupied by their counterparts in authentic ribosomal RNAs . The characteristic secondary structure elements flanking these universal sequences in genuine ribosomal RNAs can also be formed in the trypanosomal 9S and 12S RNAs . These results provide unequivocal evidence for a ribosomal function of the 9S and 12S RNAs of trypanosomal mitochondria, notwithstanding their extremely small size (estimated to be 612 and 1141 nucleotides in C . fasciculata, 611 and 1150 nucleotides in T . brucei) and their unusual base composition (83% A+U).

Nucleic Acids Res, 1985 Jun 11, 13(11), 3941 - 52
Structure of the human alpha 1-acid glycoprotein gene: sequence homology with other human acute phase protein genes; Dente L et al.; We have determined the sequence coding for human alpha 1-acid glycoprotein from two independently isolated cDNA clones and a genomic clone . The aminoacid sequences deduced from the three clones, deriving from three different individuals, are identical . Southern blot analysis on human DNA indicates that there are at least two genes coding for alpha 1-AGP . We propose that alpha 1-AGP found in plasma is a mixture of the products of these two different genes . This is the simpler explanation for the heterogeneity in the aminoacid composition in purified alpha 1-AGP observed by Schmid et al . (1) . DNA sequence comparison with cDNA clones coding for human alpha 1-antitrypsin and haptoglobin shows a conserved sequence within the 5' untranslated region which may play a role in the acute phase response.

Nucleic Acids Res, 1985 Jun 11, 13(11), 3917 - 30
Cloning of cDNA sequences for an Artemia salina hnRNP protein: evidence for conservation through evolution; Cruz-Alvarez M et al.; A cDNA clone was isolated for Artemia salina protein HD40, a component of heterogenous nuclear ribonucleoproteins . Enriched Artemia 15S poly(A)+ RNA was used as a template and double-stranded cDNA sequences were inserted into the Pst I restriction endonuclease site of E . coli plasmid pBR322 . Recombinant colonies were analyzed by positive hybrid selection of poly(A)+ RNA that directs the synthesis of protein HD40 in an in vitro assay . In vitro translation of the mRNA selected by recombinant clone 87HD yields a protein that is immunoprecipitated by anti-HD40 antibodies and that comigrates with authentic HD40 on gel electrophoresis . Partial proteolysis of protein HD40 and the in vitro translated product selected by clone 87HD produces the same peptide patterns . The size of the cloned insert is about 820 bp . The length of HD40 mRNA as determined by Northern blot analysis, is about 1500 nucleotides . Southern blot analysis performed with DNA of different species (plant, avian, mammal) shows cross-hybridizing bands when probed with clone 87HD DNA suggesting that the HD40 gene is evolutionarily conserved.

J Biol Chem, 1985 Jun 10, 260(11), 7067 - 71
Purification and characterization of Escherichia coli RNase T; Deutscher MP et al.; RNase T, a nuclease thought to be involved in end-turnover of tRNA, has been purified about 4,000-fold from extracts of Escherichia coli . At this stage of purification, the enzyme was judged to be at least 95% pure based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The native molecular weight of RNase T determined from gel filtration and sedimentation analyses is about 50,000, whereas the monomer molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 25,000, suggesting that the protein is an alpha 2 dimer . Purified RNase T is extremely sensitive to inactivation by oxidation, sulfhydryl group reagents, and temperature . The ribonuclease activity against tRNA-C-C-{14C}A is optimal at pH 8-9 in the presence of 2-5 mM MgCl2 and ionic strengths of less than 50mM . Although RNase T is highly specific for intact tRNA-C-C-A as a substrate and can hydrolyze all species in a mixed population of tRNA, it is inhibited by other RNAs, such as poly(A), rRNA, 5 S RNA, and tRNA-C-C . RNase T is an exoribonuclease which initiates attack at a free 3' terminus of tRNA and releases AMP; aminoacyl-tRNA is not a substrate . The role of RNase T in the end-turnover of tRNA and its possible involvement in other aspects of RNA metabolism are discussed.

J Biol Chem, 1985 Jun 10, 260(11), 7122 - 5
Transcriptional organization of the convergent overlapping dnaQ-rnh genes of Escherichia coli; Nomura T et al.; The transcriptional organization was determined for the DNA polymerase III epsilon subunit (dnaQ) and the ribonuclease H (rnh) genes, which are closely spaced and organized in a face-to-face system (Maki, H., Horiuchi, T., and Sekiguchi, M . (1983) Proc . Natl . Acad . Sci . U . S . A . 80, 7137-7141) . Nuclease S1 mapping revealed that transcription starts from a single promoter for rnh and two promoters for dnaQ and proceeds in opposite directions . The 5' terminus of the rnh RNA overlaps about 100 and 20 nucleotide sequences with the 5' terminus of the dnaQ-1 and dnaQ-2 RNA, respectively . The levels of in vivo transcription for the three RNA species increased altogether by more than 10-fold in cells carrying a multicopy plasmid with intact dnaQ-rnh genes, keeping the relative level for the convergent transcription units nearly constant.

J Biol Chem, 1985 Jun 10, 260(11), 6755 - 60
Resonance Raman study of cytochrome b562-o complex, a terminal oxidase of Escherichia coli in its ferric, ferrous, and CO-ligated states; Uno T et al.; Cytochrome b562-o complex, a terminal oxidase in the respiratory chain of aerobically grown Escherichia coli, has been studied by resonance Raman spectroscopy in its air-oxidized, dithionite-reduced, and reduced and CO-ligated states . In the reduced state, with a 406.7-nm excitation, there appeared 1494 and 1473 cm-1 lines, indicating that low spin and high spin components are included in the cytochrome b562-o complex . For the air-oxidized protein, resonance Raman lines were observed at 1372, 1503, and 1580 cm-1 with a 413.1-nm excitation, indicating that there is a ferric low spin heme . In addition, a weak but appreciable Raman line was observed at 1480 cm-1 assignable to a ferric high spin heme . Accordingly, it was concluded that low spin and high spin components are included in the cytochrome b562-o complex in the reduced and the air-oxidized states . In the CO-ligated state, with a defocused laser beam of 413.1 nm, two Raman bands assignable to the Fe-CO stretching mode have been observed at 489 and 523 cm-1, as a major and a minor component, respectively . When the laser beam was focused upon the sample to cause a photodissociation of CO from the heme moiety, the intensity of the major band at 489 cm-1 was reduced as expected . On the other hand, the minor band at 523 cm-1 remained still obvious . It was suggested that the cytochrome b562-o complex may have an additional anomalous site for CO that is resistant to photodissociation.

J Biol Chem, 1985 Jun 10, 260(11), 7015 - 22
Cloned mouse ribonucleotide reductase subunit M1 cDNA reveals amino acid sequence homology with Escherichia coli and herpesvirus ribonucleotide reductases; Caras IW et al.; We have isolated and sequenced overlapping cDNA clones containing the entire coding region of mouse ribonucleotide reductase subunit M1 . The coding region comprises 2.4 kilobases and predicts a polypeptide of 792 amino acids (Mr 90,234) which shows striking homology with ribonucleotide reductases from Escherichia coli and the herpesviruses, Epstein-Barr virus and herpes simplex virus . The homologies reveal three domains: an N-terminal domain common to the mammalian and bacterial enzymes, a C-terminal domain common to the mammalian and viral ribonucleotide reductases, and a central domain common to all three . We speculate on the functional basis of this conservation.

J Biol Chem, 1985 Jun 10, 260(11), 6522 - 7
Preparation and characterization of two monoclonal antibodies against different epitopes in Escherichia coli ribosomal protein L7/L12; Sommer A et al.; Two monoclonal antibodies with specificities for Escherichia coli 50 S ribosomal subunit protein L7/L12 were isolated . The antibodies and Fab fragments thereof were purified by affinity chromatography using solid-phase coupled L7/L12 protein as the immunoadsorbent . The two antibodies were shown to recognize different epitopes; one in the N-terminal and the other in the C-terminal domain of protein L7/L12 . Both intact antibodies strongly inhibited polyuridylic acid-directed polyphenylalanine synthesis, ribosome-dependent GTPase activity, and the binding of elongation factor EF-G to the ribosome . Ratios of antibody to ribosome of 4:1 or less were effective in inhibiting these activities . Neither antibody prevented the association of ribosomal subunits to form 70 S ribosomes . The Fab fragments showed similar effects.

Nature, 1985 Jun 6-12, 315(6019), 515 - 6
Human transforming growth factor-alpha causes precocious eyelid opening in newborn mice; Smith JM et al.; Both murine and human epidermal growth factors (EGFs) are known to cause precocious opening of the eyelids in newborn mice . Another set of peptides that are structurally and functionally homologous to murine and human EGFs are the murine and human type-alpha transforming growth factors (TGF-alpha s), TGF-alpha s have been found in many cancer cells and it has been suggested that their autocrine action may play an important part in malignant transformation . In several in vitro systems murine and human TGF-alpha s are functionally interchangeable with murine and human EGFs . However, the in vivo activity of the TGF-alpha s has not been characterized, as only small amounts of these peptides were available until recently . The cloning of the gene for human TGF-alpha and its expression in Escherichia coli now allow us to demonstrate that human TGF-alpha is as active as murine EGF in promoting eyelid opening in newborn mice . Furthermore, we show in a dose-dependent eyelid opening assay that human EGF is as potent as its murine homologue with respect to this biological property.

J Mol Biol, 1985 Jun 5, 183(3), 461 - 77
A molecular dynamics study of the C-terminal fragment of the L7/L12 ribosomal protein . Secondary structure motion in a 150 picosecond trajectory; Aqvist J et al.; A 150 picosecond molecular dynamics computer simulation of the C-terminal fragment of the L7/L12 ribosomal protein from Escherichia coli is reported . The molecular dynamics results are compared with the available high-resolution X-ray data in terms of atomic positions, distances and positional fluctuations . Good agreement is found between the molecular dynamics results and the X-ray data . The form and parameters of the interaction potential energy function and the procedures for deriving it are discussed . Some current misunderstandings concerning the ways of evaluating the efficiency of molecular dynamics algorithms and of application of bond-length constraints in protein simulations are cleared up . The 150 picosecond trajectory has been scanned in a search for correlated motions within and between secondary structure elements . The beta-strands have diffusional stretching modes, and uncorrelated transversal displacements . The dynamic analysis of alpha-helices shows a variety of features . The atomic fluctuations differ between the helix ends; this effect reflects long time-scale motions . Two alpha-helices, alpha A and alpha C, show diffusive longitudinal stretching modes . The third helix, alpha B, has a correlated asymmetric longitudinal stretching; the N-terminal part dominates this behaviour . Furthermore, alpha B presents a librational motion with respect to the other parts of the molecule with a frequency of approximately 5 cm-1 . This motion is coupled to helix stretching . Interestingly, the regions of highly conserved residues contain the most mobile parts of the molecule.

J Mol Biol, 1985 Jun 5, 183(3), 341 - 51
DNA binding and mutation spectra of the carcinogen N-2-aminofluorene in Escherichia coli . A correlation between the conformation of the premutagenic lesion and the mutation specificity; Bichara M et al.; When the chemical carcinogen N-2-acetylaminofluorene binds to DNA in vivo, two major adducts are formed, both at position C-8 of the guanine residue . One of these (the acetylaminofluorene adduct) retains the acetyl group, while the other (the aminofluorene adduct) is the corresponding deacetylated form . Unlike -AAF adducts, which trigger important structural changes of the DNA secondary structure (either the insertion-denaturation model or the induction of a Z-DNA structure, depending upon the local nucleotide sequence), -AF adducts bind to the C-8 of guanine residues without causing any major conformational change of the B-DNA structure . Well-defined adducts (either -AF or -AAF) can be formed in vitro by reacting DNA with either N-hydroxy-N-2-aminofluorene or N-acetoxy-N-2-acetylaminofluorene . Specific cleavage of the phosphodiester backbone at -AF adducts can be achieved by treating -AF-modified DNA in 1 M-piperidine at 90 degrees C . This observation led us to construct the spectrum for -AF binding to a defined DNA restriction fragment . It is found that only guanine residues react to form alkali-labile lesions and that the reactivity among the different guanines is similar . In a forward mutation assay, namely the inactivation of the tetracycline resistance gene, we found previously that more than 90% of mutations induced by -AAF adducts are frameshift mutations . Using the same assay, we show here that -AF adducts induce primarily base substitution mutations (85%), mainly of the G to T transversion type . There is therefore a strong correlation between the nature of the carcinogen-induced conformational change of the DNA structure and the corresponding mutation specificity . The -AF-induced base substitution mutations depend upon the umuC gene function(s) . The data obtained in our forward mutation assay are compared to the data previously obtained in the histidine reversion assay (Ames test).

Biochemistry, 1985 Jun 4, 24(12), 3043 - 9
Lactose repressor protein modified with dansyl chloride: activity effects and fluorescence properties; Hsieh WT et al.; Chemical modification using 5-(dimethylamino)naphthalene-1-sulfonyl chloride (dansyl chloride) has been used to explore the importance of lysine residues involved in the binding activities of the lactose repressor and to introduce a fluorescent probe into the protein . Dansyl chloride modification of lac repressor resulted in loss of operator DNA binding at low molar ratios of reagent/monomer . Loss of nonspecific DNA binding was observed only at higher molar ratios, while isopropyl beta-D-thiogalactoside binding was not affected at any of the reagent levels studied . Lysine residues were the only modified amino acids detected . Protection of lysines-33 and -37 from modification by the presence of nonspecific DNA correlated with maintenance of operator DNA binding activity, and reaction of lysine-37 paralleled operator binding activity loss . Energy transfer between dansyl incorporated in the core region of the repressor protein and tryptophan-201 was observed, with an approximate distance of 23 A calculated between these two moieties.

Biochemistry, 1985 Jun 4, 24(12), 3006 - 11
Monoclonal antibodies against the membrane-bound, flavin-linked D-lactate dehydrogenase of Escherichia coli: preparation, characterization, and use in immunoaffinity chromatography; Santos E et al.; Three mouse hybridoma cell lines are described that produce monoclonal antibodies directed against the membrane-bound, flavin adenine dinucleotide linked D-lactate dehydrogenase of Escherichia coli . In contrast to polyclonal antibodies produced in rabbits, none of the monoclonal antibodies inhibits enzyme activity . Immunoblots of D-lactate dehydrogenase proteolytic fragments indicate that each antibody is directed against a different region of the molecule . One monoclonal antibody, 1B2a, reacts with native, undigested D-lactate dehydrogenase only and is used to purify the enzyme in a single step . The protocol involves chromatography of a Triton X-100 extract of membrane vesicles containing D-lactate dehydrogenase on a column made with the monoclonal antibody coupled to a solid support . After the column is washed free of unadsorbed protein, elution at high pH in the presence of guanidine hydrochloride yields a fraction containing highly purified, catalytically active D-lactate dehydrogenase.

Biochemistry, 1985 Jun 4, 24(12), 3049 - 54
Measurement of DNA-protein equilibria using gel chromatography: application to the HinfI restriction endonuclease; Frankel AD et al.; A method is described for measuring equilibrium constants of DNA-protein interactions using gel chromatography . This technique has been used to study the sequence-specific interaction of the HinfI restriction endonuclease with DNA . HinfI has a monomeric molecular weight of 31000 and exists as a dimer in its active form . The protein binds to supercoiled DNA molecules containing its recognition site with an apparent free energy of -13.9 kcal/mol of sites . This interaction is highly salt sensitive and causes a release of 3.4 ion pairs . The affinity of the nuclease for its recognition site is largely independent of both pH (6.5-8.5) and temperature (7-35 degrees C) and was not affected by variations in the degenerate middle position of the site . Linear DNA fragments containing the HinfI recognition site were bound as tightly as supercoiled molecules . Binding to nonspecific DNA sites or to methylated DNA sites was approximately 6 orders of magnitude weaker . In general, enzyme activity and binding affinity paralleled each other.

FEBS Lett, 1985 Jun 3, 185(1), 83 - 8
Properties of a mutant lactose carrier of Escherichia coli with a Cys148----Ser148 substitution; Neuhaus JM et al.; The cysteine residue at position 148 in the lactose carrier protein of Escherichia coli has been replaced by serine using oligonucleotide-directed, site-specific mutagenesis of the lac Y gene . The mutant carrier is incorporated into the cytoplasmic membrane to the same extent as the wild-type carrier, confers a lactose-positive phenotype on cells, and actively transports lactose and other galactosides . However, the maximum rate of transport for several substrates is reduced by a factor of 6-10 while the apparent affinity is reduced by a factor of 2-4 . Carrier activity in the mutant is much less sensitive to sulfhydryl reagents (HgCl2, p-(chloromercuri)benzenesulfonate and N-ethylmaleimide) than in the wild type, and beta-D-galactosyl 1-thio-beta-D-galactoside does not protect the mutant carrier against slow inactivation by N-ethylmaleimide . It is concluded that the Cys148 residue is not essential for carrier-catalyzed galactoside: proton symport and that its alkylation presumbly prohibits access of the substrate to the binding site by steric hindrance . A serine residue at position 148 in the amino acid sequence appears to alter the protein structure in such a way that one or more sulfhydryl groups elsewhere in the protein become accessible to alkylating agents thereby inhibiting transport . Recently, Trumble et al . {(1984) Biochem . Biophys . Res . Commun . 119, 860-867} arrived at similar conclusions by investigating a mutant carrier with a Cys148----Gly148 replacement.

FEBS Lett, 1985 Jun 3, 185(1), 51 - 6
Mutant species of EF-Tu, altered at position 375, exhibit a reduced affinity for aminoacylated transfer-RNAs; Sam T et al.; The interaction between EF-Tu X GTP and aminoacyl-tRNA is shown to be influenced by mutations at site 375 of this three-domain protein . Site 375 is located in domain II near the interface with domain I {(1984) EMBO J . 3, 113-120} . Replacement of the alanine at this site by a threonine or valine residue results in lower binding constants with Phe-tRNA and Tyr-tRNA, as was evaluated by the hydrolysis protection technique . The data are discussed in the light of what is known about the three-dimensional structure of the protein and its interaction sites with aminoacyl-tRNA.

FEBS Lett, 1985 Jun 3, 185(1), 57 - 62
Topography of RNA in the ribosome: location of the 5 S RNA residues A39 and U40 on the central protuberance of the 50 S subunit; Evstafieva AG et al.; The internal site of 5 S RNA comprising residues A39 and U40 has been localized on the E . coli 50 S ribosomal subunit by immune electron microscopy . It has been found to be located on the interface side of the central protuberance at the position distinctly apart but very close to the position of the 5 S RNA 3'-end providing evidence for a quite compact folded conformation of the 5 S RNA in situ.

FEBS Lett, 1985 Jun 3, 185(1), 162 - 4
Isolation of gram quantities of isoleucyl-tRNA synthetase from an overproducing strain of Escherichia coli and its use for purification of cognate tRNA; Kawakami M et al.; The ileS gene coding for isoleucyl-tRNA synthetase was cloned on a runaway-replication plasmid . From the cells harboring the plasmid, gram quantities of the synthetase were isolated using two column procedures . The synthetase was used for the purification of cognate tRNA . Isoleucine tRNAGAU of greater than 90% purity was easily isolated by taking advantage of a specific complex formation of the synthetase with cognate tRNA.

FEBS Lett, 1985 Jun 3, 185(1), 121 - 4
Affinity of protein HU for different nucleic acids; Holck A et al.; The binding of protein HU from Escherichia coli to nucleic acids was investigated by affinity chromatography under various conditions, by a nitrocellulose retention assay and by isopycnic centrifugations in metrizamide gradients . The results indicate that HU has a preference for binding to RNA and single-stranded DNA over double-stranded DNA . The affinity of HU for supercoiled DNA was also less than that of the corresponding relaxed DNA.

Acta Pathol Microbiol Immunol Scand {C}, 1985 Jun, 93(3), 131 - 7
Cytotoxic factor(s) released from stimulated mouse peritoneal macrophages; Morland B; Mechanisms of macrophage-mediated cytotoxicity against a tumor-cell line (L-929 cells) were analyzed . Culture supernatants were harvested from mouse peritoneal macrophages cultivated for 3 days in the absence or presence of the stimulating agents Escherichia coli endotoxin or zymosan . The supernatants from stimulated cultures were cytotoxic for the tumor cells, evaluated by measuring release of radio-activity during subsequent 4 days' culture of 14C-thymidine-labelled tumor cells in the supernatants . Cytotoxicity was verified by counting cells per culture . Corresponding results were obtained from co cultures of stimulated macrophages and tumor cells, in accordance with a previous study . Selective release of af lysosomal enzyme (beta-glucuronidase) was shown in the supernatants from endotoxin- or zymosan-stimulated cultures, while reduced levels of glucose were seen in all supernatants from macrophage cultures . Dialysis of supernatants against fresh medium reduced the toxic activity somewhat . Dialysis restored the glucose content to optimal levels, while the enzyme activity was unchanged . Heating of supernatants to 56 degrees C for 30 min reduced the cytotoxicity along with a reduction in enzyme activity; 70 degrees C for 30 min removed both cytotoxic activity completely . Heating had no effect on the glucose content of the supernatants . The present data indicate that macrophage-mediated tumor cytotoxicity may be performed through release of heat-labile soluble factor(s) which co-variate with the secretion of a lysosomal enzyme from stimulated macrophages.

EMBO J, 1985 Jun, 4(6), 1425 - 30
Sequencing of the chicken non-erythroid spectrin cDNA reveals an internal repetitive structure homologous to the human erythrocyte spectrin; Wasenius VM et al.; Immunological screening of a chicken gizzard cDNA expression library was used to isolate two clones encoding a part of the non-erythroid spectrin-like protein . Clones were identified by immunoblotting of the polypeptides synthesized in Escherichia coli cells transformed with cDNA cloned in the pUC8 plasmid vector using polyclonal rabbit antibodies raised against bovine non-erythroid spectrin . The sequence of an approximately 1.5-kb cDNA insert of one clone was determined . Analysis of the predicted amino acid sequence reveals that, despite differences in immunological cross-reactivity and peptide maps, the chicken non-erythroid and the human erythrocyte spectrins are highly homologous proteins . Like the human erythrocyte spectrin, the chicken smooth muscle spectrin appears also to be constructed from repeated, homologous structures of 106 amino acid residues . This is probably a universal structure motif of spectrins.

Anal Biochem, 1985 Jun, 147(2), 369 - 73
Labeling of cysteine-containing peptides with 2-nitro-5-thiobenzoic acid; Sliwkowski MX et al.; A method for specific labeling of cysteine-containing peptides has been developed using Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) . Prior to cleavage with proteases or chemical reagents, proteins are reacted with DTNB, resulting in the formation of a mixed disulfide between the protein sulfhydryl group and 2-nitro-5-thiobenzoic acid (TNB) . The formation of the mixed disulfide introduces a chromophore, with an absorbance peak at 328 nm . By monitoring peptide maps generated by HPLC at 210 and 328 nm, peptides containing cysteine residues are readily identified . The stability of the derivative was tested using glutathione-TNB as a model compound . Glutathione-TNB is stable to conditions used for CNBr cleavage, as well as those for tryptic cleavage . The TNB label may also increase the hydrophobicity of small peptides, which otherwise might not bind to reverse-phase matrices . This was the case for an oxidatively modified tetrapeptide isolated from Escherichia coli glutamine synthetase.

Am J Physiol, 1985 Jun, 248(6 Pt 2), R674 - 8
Effect of intracerebroventricular vasopressin on body temperature and endotoxin fever of macaque monkey; Lee TF et al.; In the adult monkey (Macaca fasicularis) acclimated to a primate restraint chair, intracerebroventricular (ICV) cannulas were stereotaxically implanted bilaterally in the lateral cerebral ventricle . During an experiment, colonic and skin temperatures were continuously recorded, and in selected cases heart and respiratory rates as well as O2 consumption were similarly monitored . Arginine vasopressin (AVP) was infused ICV in a volume of 500 microliter in one of six doses ranging from 16 to 4,240 ng . The results showed that AVP in all doses infused failed to alter the resting core and skin temperatures of the monkey or other recorded physiological responses . After a fever was produced in the monkey by 1/10-1/100 dilution of Escherichia coli endotoxin infused ICV, AVP at doses of 16-260 ng did not augment the febrile response . Further, AVP infused in the same range of doses caused only a negligible and inconsistent antipyretic action; i.e., the 65-ng dose of AVP lowered the mean core temperature of the febrile monkey by only 0.5 degrees C . This was in marked contrast to the antipyretic effect of dipyrone administered systemically . In addition, no reproducible changes occurred in respiratory, heart, or metabolic rates after ICV infusion of AVP . These results indicate that AVP does not affect the normal thermoregulatory function of this species of monkey and, in contrast to other species, a febrile response is neither enhanced nor antagonized in the monkey by a central action of AVP . Although AVP thus does not appear to be an endogenous antipyretic in the monkey, anatomic investigations will be required to substantiate this conclusion in the subhuman primate.

Am J Physiol, 1985 Jun, 248(6 Pt 2), H818 - 26
Development of myocardial dysfunction in endotoxin shock; Parker JL et al.; Isolated heart muscle preparations were used to investigate the onset and development of myocardial inotropic dysfunction during endotoxin shock in guinea pigs . Left atrial muscles were removed from separate groups of animals at increasing time intervals after administration of either 4 mg/kg purified Escherichia coli endotoxin (shock groups) or an equivalent volume of isotonic saline (control groups) . Peak developed contractile tension (CT) and maximal rate of tension development (+dT/dtmax) were significantly depressed in shock tissues as early as 2 h postendotoxin (P less than 0.01), with the magnitude of the contractile deficit progressively increasing during 4, 6, and 12 h postendotoxin . Contractility remained significantly depressed (P less than 0.001) at 16 and 24 h postendotoxin but progressively recovered toward control levels during 16, 24, 48, and 72 h postendotoxin . Shock-induced myocardial dysfunction was characterized by altered contractile responsiveness to low-Ca2+ medium (0.5 mM), gentamicin (4 mM), and hypoxia; altered inotropic reactivity to these interventions followed similar temporal development as the postendotoxin changes in basal contractile parameters . Left ventricular papillary muscles obtained at 16 h postendotoxin corroborated the shock-induced contractile depression observed in atria . These studies provide evidence for early and progressive intrinsic myocardial dysfunction in endotoxin shock and demonstrate that this dysfunction can be unmasked through the study of in vitro atrial and ventricular heart muscle preparations isolated from in vivo shocked animals.

Mutat Res, 1985 Jun-Jul, 150(1-2), 77 - 84
Methylation-induced blocks to in vitro DNA replication; Larson K et al.; Single-stranded primed M13mp2 templates and double-stranded templates were treated with either dimethyl sulfate (DMS) or N-methyl-N'-nitro-N-nitrosoguanidine and used for DNA synthesis in vitro . Methylation inhibits the ability of the molecules to serve as templates . When either E . coli DNA polymerase I or AMV reverse transcriptase were used as polymerases, DNA synthesis terminated one nucleotide 3' to the site of adenine residues in the template . Heating of the templates resulted in the appearance of additional termination bands one nucleotide before the site of G's in the template . We assume that methylated A's but not methylated G's are blocks to in vitro DNA synthesis and that heating converts a portion of the sites of methylated G to AP sites which are blocks to synthesis.

Cell, 1985 Jun, 41(2), 597 - 605
N4 virion RNA polymerase sites of transcription initiation; Haynes LL et al.; Coliphage N4 virion encapsulated RNA polymerase shows a marked preference for denatured N4 DNA as a template . We show that initiation on denatured N4 virion DNA occurs with in vivo specificity . The location of the in vivo and in vitro initiation sites and the corresponding DNA sequences were determined . The N4 virion RNA polymerase promoters contain extensive sequence homology from position -18 to position 1, with a conserved GC-rich heptamer centered at -12, and two sets of short inverted repeats . We suggest that the N4 virion RNA polymerase recognizes the promoter only in a novel single-stranded form, and that the formation of the initiation complex is facilitated in vivo by supercoiling and E . coli single-stranded DNA binding protein.

Cancer Res, 1985 Jun, 45(6), 2440 - 4
Lack of miscoding properties of 7-(2-oxoethyl)guanine, the major vinyl chloride-DNA adduct; Barbin A et al.; Chloroethylene oxide, an ultimate carcinogenic metabolite of vinyl chloride, was reacted with poly(deoxyguanylate-deoxycytidylate); the nucleic acid base adducts, 7-(2-oxoethyl)guanine and 3,N4-ethenocytosine, were analyzed by reverse-phase high-performance liquid chromatography . Chloroethylene oxide-modified poly(deoxyguanylate-deoxycytidylate) was assayed as template in a replication fidelity assay with Escherichia coli DNA polymerase I, and the newly synthesized product was subjected to nearest-neighbor analysis . Misincorporation rates of deoxyadenosine monophosphate and thymidine monophosphate were found to increase with the level of template modification . About 80% of the mispairing events were located opposite minor cytosine lesions . 7-(2-Oxoethyl)guanine, the major adduct identified (greater than 98% of the adducts), did not miscode for either thymine or adenine, failing to support an earlier hypothesis that the cyclic hemiacetal form, O6,7-(1'-hydroxyethano)guanine, could, by analogy with O6-methyl- and O6-ethylguanine, simulate adenine . Our results indicate that direct miscoding of 7-(2-oxoethyl)-guanine may contribute only slightly to the induction of mutations by chloroethylene oxide or vinyl chloride.

Pathol Biol (Paris), 1985 Jun, 33(5 Pt 2), 628 - 30
{In vitro release of lipopolysaccharide in Escherichia coli by addition of antiseptic and disinfectant products}; Agbalika F et al.; Release of lipopolysaccharide (LPS) by E . coli following addition of antiseptics and disinfectants was investigated using the Limulus test . The five following compounds were studied: glutaraldehyde (cidex 2%), chlorhexidine 0.05%, povidone iodine 5%, aqueous formol 37%, and cetavlon 20% . These compounds were used in concentrations 2 to 4 times greater than the previously determined minimal inhibitory concentrations . LPS concentration falls sharply following addition of glutaraldehyde (cidex) to a suspension of E . coli (approximately 10(6)/ml) . Released LPS is completely inhibited by formol and cetavlon . In contrast, high levels of LPS persist in the presence of chlorhexidine and povidone iodine . These results illustrate the need for thoroughly and generously washing all non-autoclavable equipment exposed to these chemical agents.

J Biol Response Mod, 1985 Jun, 4(3), 264 - 72
Recombinant interferon-gamma (immuneron): results of a phase I trial in patients with cancer; van der Burg M et al.; Recombinant DNA-produced interferon-gamma (rIFN-gamma) was administered intravenously to patients with solid tumors in a Phase I study . The rIFN gamma was prepared from Escherichia coli and purified to greater than 95% with a specific activity of greater than or equal to 30 X 10(6) units/mg protein . Twenty patients received intravenous bolus injections once weekly for 4 consecutive weeks . They were assigned to one of six dose groups ranging from 1 to 81 X 10(6) units/m2 body surface area; intrapatient dose escalation was not allowed . Patients were monitored intensively for toxicity, but no dose-limiting toxicity was demonstrated . Fever was the predominant side effect, occurring in all patients treated, and usually reached 38-40 degrees C . Short-term somnolence and fatigue were also observed, but no chronic fatigue was seen . Decreases in white blood cell and platelet counts, generally within the normal range, were observed; however, the counts rose again after intervals of 2-5 days . There was no firm evidence of a relationship between adverse effects and dose . No life-threatening side effects were noted and no antibodies developed to either rIFN gamma or E . coli proteins . The pharmacokinetics of rIFN gamma did not appear to alter from week 1 to week 4 . Calculated half-lives were from 0.8 to 3.5 h . Doses greater than 9 X 10(6) units/m2 gave measurable serum levels for at least 12 h . A partial response of 8 weeks' duration was observed in a patient with hepatoma.

Gut, 1985 Jun, 26(6), 570 - 8
Enteropathogenic Escherichia coli enteritis: evaluation of the gnotobiotic piglet as a model of human infection; Tzipori S et al.; The pathogenicity of classical enteropathogenic Escherichia coli strains of human origin was investigated in gnotobiotic piglets . One to two day old piglets in groups of four were infected perorally with approximately 10(8) colony forming units of one of eight enteropathogenic E coli strains or a non-pathogenic control strain . Animals were necropsied 24 or 48 hours after infection and their intestines were subjected to histological examination, quantitative bacterial culture and estimation of lactase activity . Four enteropathogenic E coli strains caused mild to moderate diarrhoea in nine of the 16 piglets inoculated with them . Piglets given two of these strains later became moribund . One enteropathogenic E coli strain caused a severe illness unaccompanied by diarrhoea . Inflammation of the intestinal mucosa occurred with all eight enteropathogenic E coli strains, but not with the control strain . Pathological changes were most pronounced in the distal ileum and colon and adherent bacteria were seen on the surface of the inflamed mucosa . The extent of the inflammatory response in infected piglets for the most part paralleled the severity of the clinical signs, the degree of bacterial colonisation and the reduction in lactase activity . Electron microscopic examination of tissue from piglets infected with three different strains showed that bacterial adherence to the apical plasma membrane of epithelial cells was accompanied by distinctive ultrastructural changes . These included degeneration of the microvillous brush border, together with cupping and pedestal formation of the plasma membrane at sites of bacterial attachment . The same changes have been seen in naturally occurring enteropathogenic E coli diarrhoea in humans and rabbits . The combined clinical and pathological findings indicate that the neonatal gnotobiotic piglet is a suitable model of infection with enteropathogenic E coli.

J Bacteriol, 1985 Jun, 162(3), 1339 - 41
Identification of facultatively heterotrophic, N2-fixing cyanobacteria able to receive plasmid vectors from Escherichia coli by conjugation; Flores E et al.; Plasmid vectors transferable by conjugation from Escherichia coli to obligately photoautotrophic strains of Anabaena spp . are also transferred to and maintained in heterotrophic, filamentous cyanobacteria of the genus Nostoc . These organisms can be used for the genetic analysis of oxygenic photosynthesis, chromatic adaptation, nitrogen fixation, and heterocyst development.

J Bacteriol, 1985 Jun, 162(3), 1227 - 37
Genetic characterization and partial sequence determination of a Treponema pallidum operon expressing two immunogenic membrane proteins in Escherichia coli; Hansen EB et al.; A detailed physical and genetic map of a previously cloned 5.5-kilobase segment of Treponema pallidum DNA is described . This segment expressed two proteins that are cell membrane associated in Escherichia coli . The structural genes of these treponemal membrane proteins, tmpA and tmpB, are coordinately expressed, and transcription in E . coli can start from at least two different treponemal promoters . The tmpA and tmpB proteins are the products of in vivo proteolytic cleavage from precursor proteins which are 2 and 4 kilodaltons larger, respectively, than the mature proteins . Because the sizes of the corresponding proteins produced in T . pallidum were identical to those of the mature membrane proteins in E . coli, we concluded that a similar proteolytic processing takes place in both E . coli and T . pallidum . Although tmpA and tmpB were controlled by the same transcription signals, tmpB was expressed to a higher extent than tmpA, and only the tmpB product could be overproduced by placing the left lambda promoter in front of the structural genes . The nucleotide sequence of the T . pallidum tmpA gene was established . This is the first T . pallidum gene sequenced . Codon usage and the nature of transcriptional and translational signals are discussed . The deduced amino acid sequence indicated the presence of a sequence that was characteristic for a signal peptide . This sequence information allowed the construction of hybrid genes coding for proteins having beta-galactosidase enzyme activity as well as TmpA epitopes . The enzyme-linked antigen was expressed at a high level in E . coli when transcriptional and translational signals from coliphage lambda were used . In this case the protein produced was a sandwich protein consisting of 21 amino acids of the lambda cro protein, 204 amino acids of the T . pallidum TmpA protein, and 1,020 amino acids of the E . coli lambda-galactosidase . The potential use of this enzyme-linked antigen for the serodiagnosis of syphilis is discussed.

J Biomol Struct Dyn, 1985 Jun, 2(6), 1221 - 34
A structural transition in d(AT)n.d(AT)n inserts within superhelical DNA; Panyutin I et al.; We have constructed plasmids carrying d(AT)n.d(AT)n inserts of different lengths . Two-dimensional gel electrophoresis patterns show that an increase in the negative superhelicity of these DNAs brings about a structural transition within the inserts, resulting in a reduction of the superhelical stress . However, this reduction corresponds to the expected values neither for cruciform nor the Z form . Those DNA topoisomers in which the structural transition had occurred proved to be specifically recognizable by single-strand-specific endonuclease S1, with the cleavage site situated at the centre of the insert . These data, as well as kinetic studies, suggest that the cloned d(AT)n.d(AT)n sequences adopt a cruciform rather than the Z-form structure . We discuss plausible reasons of the discrepancy between the observed superhelical stress release and that expected for the transition of the insert to the cruciform state.

J Gen Microbiol, 1985 Jun, 131 ( Pt 6), 1305 - 11
The IncI plasmids R144, R64 and ColIb belong to one exclusion group; Hartskeerl R et al.; The exclusion relationship between the IncI plasmids R144, R64 and ColIb was studied in such a way that incompatibility interference was avoided . Genetic crosses with an R144-derived Hfr donor, crosses with recipient strains carrying R144-derived exclusion genes on a recombinant plasmid compatible with R144, and measurement of transmission frequencies of a recombinant plasmid compatible with IncI plasmids after mobilization by R144 revealed that R144, R64 and ColIb belong to one exclusion group.

Acta Pathol Microbiol Immunol Scand {B}, 1985 Jun, 93(3), 253 - 4
Fatal outcome of splenectomy in rats with experimental biliary infection; Tanaka N et al.; The effect of splenectomy in normal rats receiving retrograde intrabiliary (RI) or intravenous (IV) injection of 10(5) Escherichia coli was studied . IV injection of the bacteria did not cause any deaths, independent of whether simultaneous ligation of the common bile duct was performed or not . In contrast, RI injection immediately after splenectomy resulted in the death of 9/12 animals although only 1/12 RI-injected rats with intact spleens died (p less than 0.005) . The findings might have implications for the performance of splenectomy in patients with combined hepato-biliary diseases.

Vet Immunol Immunopathol, 1985 Jun, 9(2), 143 - 60
In vivo proteolytic activity of the mammary gland . Contribution to the origin of secretory component, beta 2-microglobulin and bovine-associated mucoprotein (BAMP) in cows milk; Pringnitz DJ et al.; Milk samples were collected from Holstein-Friesian cows at various times after milking (10-30 min; 30 min-10 hr) and treated with a protease inhibitor or control solution . Samples were then fractionated into whole, skimmed and cell-free skimmed milk aliquots . Some animals were treated with E . coli endotoxin prior to sample collection . The concentrations of three membrane-associated proteins (MAP), beta 2-microglobulin (beta 2M), secretory component (SC) and bovine-associated mucoprotein (BAMP) as well as albumin were measured in each aliquot to determine if in vivo proteolysis of milk elements could explain the origin of these MAP in milk . All three MAP could be localized on milk fat globules (MFG) and alveolar epithelial cells of the gland . Data revealed that all BAMP in milk can be accounted for by in vivo proteolytic degradation of MFG while most beta 2M is derived by similar degradation, from cellular elements in milk, presumably monocytes . Experiments with endotoxin which elevate PMN levels, failed to influence the release of any MAP while elevating albumin levels by greater than 10-fold . Based on these studies, SC release into milk cannot be ascribed to a protease-dependent mechanism.

Genetika, 1985 Jun, 21(6), 1068 - 9
{Generation of deletions in the region of the crp gene on the Escherichia coli chromosome}; Kulakauskas ST et al.; Deletions in the argD, crp, cysG genes (73-74 min of the Escherichia coli genetic map) were obtained by heat induction of the phage lambda c1857 b221 rex::Tn5 integrated previously into the cysG gene by homologous recombination in the cysG::Tn5 mutant . Properties of the deletions obtained suggest the gene order: argD-crp-cysG.

Bangladesh Med Res Counc Bull, 1985 Jun, 11(1), 20 - 7
Diarrhoeagenic Escherichia coli in hospitalized children in Dhaka; Moyenuddin M et al.; One hundred and four cases of acute diarrhoea upto the age of eight years and seventy four age and sex matched controls were studied to isolate enteropathogenic (EPEC), enterotoxigenic (ETEC), and enteroinvasive (EIEC) Escherichia coli . About 23% EPEC and 9% ETEC were isolated from diarrhoeal patients in contrast to 8% and 1% from control respectively . Among the twelve different serotypes of EPEC isolated, O2Oa O2Oc: K61 (33.3%) was the most predominant . No EIEC was isolated by sereny test . In 8 diarrhoeal cases, no other enteropathogen except EPEC was isolated . The study has highlighted the important role that enteropathogenic Esch . coli could play in causing endemic diarrhoea in Bangladesh.

Am J Vet Res, 1985 Jun, 46(6), 1287 - 93
Alterations in coagulation and hemograms of horses given endotoxins for 24 hours via hepatic portal infusions; Duncan SG et al.; This experiment was designed to establish a model for the study of gastrointestinal disturbances as a result of prolonged endotoxin uptake in the horse . The hepatic portal vein of 7 horses was catheterized (through flank incisions) to give chronic hepatic portal infusions of lipopolysaccharide (LPS, endotoxin) . Lipopolysaccharide was infused at a rate of 1 microgram/kg of body weight/hr for 24 hours . Two of the horses were infused with saline solution for 12 hours before LPS infusions were given . Lipopolysaccharide was shown to affect behavior and hematologic and coagulation values . The 1st hour was critical for the LPS-infused horses; yet by 4 hours, the horses had apparently become refractory to continued infusion of LPS . During the 1st hour, all horses collapsed without an accompanying hypotension . A decrease in polymorphonuclear leukocytes (neutrophils) was seen during this time and was accompanied by a shortening of the recalcification tests, 1-stage prothrombin time, and activated partial thromboplastin time . There was an increased concentration of circulating fibrinogen/fibrin degradatory products . All of the LPS-infused horses showed signs of hoof discomfort and either stood with the 4 feet together beneath the body or continually shifted their weight from one front foot to the other . Hoof temperature decreased approximately 3 degrees (C) during this time and remained decreased for the duration of the experiment.

Sci Sin {B}, 1985 Jun, 28(6), 618 - 25
Expression of surface antigen gene of human hepatitis B virus serotype adr in Escherichia coli; Ao SZ et al.; The construction of an expression plasmid of hepatitis B virus surface antigen (HBsAg) gene from the cloned hepatitis B virus (HBV) genome subtype adr is reported . The expression products of this plasmid in E . coli were detected by means of radioimmunoassay in competitive suppression and polyacrylamide-SDS gel electrophoresis . The presence of a fusion protein containing HBsAg was confirmed.

Med Radiol (Mosk), 1985 Jun, 30(6), 37 - 40
{Relative biological effectiveness of highly active mixed gamma-neutron 252Cf radiation}; Aleksandrov ID et al.; Comprehensive radiobiological studies of the relative biological and genetic efficacy (RBE and RGE) of powerful 252Cf radiation (the ANET-B unit) were conducted using research tools of various radiosensitivity (bacteria, Drosophila, Chinese hamster cells, murine thymocytes, human and murine bone marrow stem cells, human peripheral blood lymphocytes, Lewis lung carcinoma cells) . It was shown in the tests of reproductive or interphase death and chromosome aberrations that the RBE and the RGE values of a 252Cf new source varied within the same limits from 1.3 to 3.0 whereas in the tests of gene mutations the RGE of the source did not exceed the efficacy of 60Co gamma-radiation and in some cases it was much lower . Thus the RBE of the new source in induced lethal and chromosome damages was 2-4 times lower than the efficacy of a low-activity 252Cf source used now in radiotherapy.

J Mol Cell Cardiol, 1985 Jun, 17(6), 575 - 85
Reduction of intrinsic contractile reserves of the left ventricle by Escherichia coli endotoxin shock in guinea-pigs; Adams HR et al.; To test the hypothesis that cardiodynamic responses during endotoxemia are limited by intrinsic myocardial dysfunction, we studied contractile properties of isovolumic left ventricular (LV) preparations isolated from E . coli endotoxin-shocked guinea pigs . Compared to control hearts, shock hearts developed significantly lower LV systolic pressures (54 +/- 7 v . 84 +/- 2 mmHg; P less than 0.001) and maximal rates of LV pressure rise (+dP/dtmax; 886 +/- 106 v . 1246 +/- 39 mmHg/s; P less than 0.006) and fall (-dP/dtmax; 702 +/- 98 v . 1103 +/- 26 mmHg/s; P less than 0.001) . The LV mechanical disadvantage of shock hearts was not correlated with changes in beating frequency, active state duration, or tissue water content; neither was it surmounted by pyruvate nor by maximally effective increases in coronary flow, diastolic stretch, or extracellular Ca2+ concentration . These findings suggest that endotoxin pathogenesis encompasses a decrease in intrinsic contractile reserves of the left ventricle, and that the resulting changes in myocardial contractile mechanisms may underlie cardiac involvement in endotoxin shock syndromes.

J Pediatr Gastroenterol Nutr, 1985 Jun, 4(3), 498 - 501
Chronic granulomatous disease mimicking Crohn's disease; Isaacs D et al.; A 34-month-old boy with intermittent diarrhoea and abdominal distension from 2 months of age, a chronic microabscess of the cheek, gastric antral narrowing, and perianal abscesses containing granulomata was found at colonscopy to have extensive, noncaseating, submucosal ileal and colonic granulomata . He was initially thought to have Crohn's disease, but then developed a cervical abscess, and a diagnosis of chronic granulomatous disease was established . This is an important, although rare, differential diagnosis of chronic inflammatory bowel disease in childhood.

Biull Eksp Biol Med, 1985 Jun, 99(6), 714 - 6
{Characteristics of the reaction of immunologically tolerant mice to the polyclonal stimulant salmozan}; Kondrat'eva TK et al.; The nonspecific stimulant of the immune response salmozan (Sal) increases the number of PFC against SRBC in intact mice, B mice (thymectomized, irradiated and reconstituted with embryonic liver cells), and in mice pretreated with cyclophosphamide (CY) . This effect was decreased in mice pretreated with SRBC and CY . The subsequent injections of SRBC and Sal into tolerant mice did not increase the response under study . It is concluded that the effect observed is due to partial alteration of antigen-specific B cells of tolerant mice and this alteration cannot be explained by the lack of the regeneration of membrane immunoglobulins.

Biull Eksp Biol Med, 1985 Jun, 99(6), 661 - 3
{Effect of litonit and teturam on the course of biochemical processes in infectious inflammatory lesions of the kidneys in alcoholized animals}; Kostev FI; The time-course of changes in the activity of sorbitol dehydrogenase, catalase and the intensity of lipid peroxidation in the blood and kidneys of 250 albino rats was studied in the course of the development of alcoholic dependence when suffering from secondary infection . Litonit, a new antialcoholic drug, was found to be effective for the treatment of infectious inflammatory renal lesions in alcoholism . Unlike teturam, litonit promoted the decrease of sorbitol dehydrogenase and catalase activity in the kidneys and abated the intensity of lipid peroxidation . The possibility of using litonit as one of the remedies of pathogenetic therapy for infectious inflammatory renal lesions in alcoholism is under discussion.

Biophys J, 1985 Jun, 47(6), 799 - 807
Microwave-field-driven acoustic modes in DNA; Edwards GS et al.; The direct coupling of a microwave field to selected DNA molecules is demonstrated using standard dielectrometry . The absorption is resonant with a typical lifetime of 300 ps . Such a long lifetime is unexpected for DNA in aqueous solution at room temperature . Resonant absorption at fundamental and harmonic frequencies for both supercoiled circular and linear DNA agrees with an acoustic mode model . Our associated acoustic velocities for linear DNA are very close to the acoustic velocity of the longitudinal acoustic mode independently observed on DNA fibers using Brillouin spectroscopy . The difference in acoustic velocities for supercoiled circular and linear DNA is discussed in terms of solvent shielding of the nonbonded potentials in DNA.

Anal Biochem, 1985 Jun, 147(2), 497 - 502
One-step fluorometric microassay of DNA in procaryotes; Legros M et al.; A fluorometric DNA microassay in procaryotes is proposed . The fluorescent dye employed is 4',6-diamidino-2-phenylindole X 2HCl, which exhibits very high specificity for DNA . The assay can be directly made on whole bacteria without DNA purification . Bacteria are treated with toluene and the fluorescent dye is added . The range of linearity has been explored . The stability, reproducibility, and accuracy of fluorescence measurements permit one to determine the equivalent number of genomes per cell and to distinguish between two different but very similar modes of increase in DNA quantity: bilinear or exponential synthesis.

Anal Biochem, 1985 Jun, 147(2), 458 - 61
Ionic-exchange high-performance liquid chromatography of Escherichia coli ribosomal small-subunit proteins; Flamion PJ et al.; Ion-exchange high-performance liquid chromatography was applied to the separation of proteins from the 30S ribosomal subunit . The proteins present in each peak have been identified by polyacrylamide gel electrophoresis analysis . The purification has been made using either unmodified proteins or proteins specifically labeled at their SH group . The results clearly show that the method can be used to purify and identify ribosomal proteins.

Anal Biochem, 1985 Jun, 147(2), 336 - 41
Purification of aspartase and aspartokinase-homoserine dehydrogenase I from Escherichia coli by dye-ligand chromatography; Karsten WE et al.; Improved purification schemes are reported for the enzymes L-aspartase and aspartokinase-homoserine dehydrogenase I from Escherichia coli . Dye-ligand chromatography on commercially available dye matrices are incorporated as key steps in these purifications . Red A-agarose has a high affinity for L-aspartase, which is then eluted as a homogeneous protein fraction with 1 mM L-aspartic acid . Green A-agarose shows a high binding affinity for the bifunctional enzyme aspartokinase-homoserine dehydrogenase I . Purification is accomplished by elution with NADP+, followed by formation of a ternary complex with NADP and cysteine, a good competitive inhibitor of the homoserine dehydrogenase activity, and rechromatography on Green A-agarose . The final specific activity of each purified enzyme equaled or exceeded previously reported values, the overall yield of enzymes obtained was significantly higher, and these improved purification schemes were found to be more amenable to being scaled up for the production of large quantities of purified enzyme.

J Gen Virol, 1985 Jun, 66 ( Pt 6), 1215 - 9
Expression of T4 early genes 62, 44, 45 and 46 in the lambda-T4 recombinant phage lambda 806-17; Yu CY et al.; A lambda-T4 recombinant phage, lambda 806-17, which carries the T4 early genes 62, 44, 45 and 46, was studied inside a homoimmune lysogen . Under such conditions, gene expression from the lambda promoters is represented . Results showed extensive expression of gene 46, and significant expression of genes 62 and 45 . The expression of these early T4 genes is presumed to depend on T4 promoters included in the cloned fragment . A new promoter proximal to gene 46 is implicated . The results also indicate that the extent of gene expression, in terms of complementation, increases with the time allowed for expression.

J Appl Physiol, 1985 Jun, 58(6), 2033 - 40
Respiratory muscle fatigue: a cause of ventilatory failure in septic shock; Hussain SN et al.; The effect of endotoxic shock on the respiratory muscle performance was studied in spontaneously breathing dogs given Escherichia coli endotoxin (Difco Laboratories, 10 mg/kg) . Diaphragmatic (Edi) and parasternal intercostal (Eic) electromyograms were recorded using fishhook electrodes . The recorded signals were then rectified and electrically integrated . Pleural, abdominal, and transdiaphragmatic (Pdi) pressures were recorded by a balloon-catheter system . After a short control period, the endotoxin was administered slowly intravenously (within 5 min) . Death was secondary to respiratory arrest in all animals . All animals died within 150-270 min after the onset of endotoxic shock . Within 45-80 min of the endotoxin administration, mean blood pressure and cardiac output dropped to 42.1 +/- 4.1 and 40.1 +/- 6.0% (mean +/- SE) of control values, respectively, with little change afterward . Mean inspiratory flow rate and Pdi increased from control values of 0.27 +/- 0.03 l X s-1 and 5.75 +/- 0.7 cmH2O to mean values of 0.44 +/- 0.3 l X s-1 and 8.70 +/- 1.05 cmH2O and then decreased to 0.17 +/- 0.03 l X s-1 and 3.90 +/- 0.30 cmH2O before the death of the animals . There were no major changes in the mechanics of the respiratory system . Edi and Eic increased progressively to mean values of 360 +/- 21 and 263 +/- 22% of control, respectively, before the death of the animals . None of the dogs were hypoxic . Arterial PCO2 decreased from a control value of 42.9 +/- 1.7 Torr to a mean value of 29.9 +/- 2.8 Torr and then increased to 51 +/- 4.3 Torr before the death of the animals.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA, 1985 Jun, 4(3), 221 - 32
Efficient expression in Escherichia coli of two species of human interferon-alpha and their hybrid molecules; Mizoguchi J et al.; A new type of interferon (IFN)-alpha cDNA (IFN-alpha I') was identified in a cDNA library constructed from Namalva cells infected with Sendai virus . The nucleotide sequence of this cDNA showed homology, with the exception of two nucleotides in the coding region, with the previously identified IFN-alpha I gene (Lawn et al., 1981) . An expression plasmid which directs the synthesis of the mature IFN-alpha I' peptide was constructed using vectors carrying the lpp/lac promoter and "runaway" replicon . Furthermore, hybrid genes were constructed by in vitro recombination of IFN-alpha I' and IFN-alpha A at a common restriction endonuclease site located at amino acid positions 121-122 . While the specific antiviral and anticellular activities of IFN alpha A and IFN-alpha I' on human cells were comparable, the antiviral activity of IFN-alpha I' on mouse cells was 125-fold higher than that of IFN-alpha A . The specific antiviral activities of the hybrid IFNs on human and bovine cells were similar to that of the amino-terminal parental IFN peptide, while the anticellular activities on human cells of the alpha A/alpha I' hybrid were higher and that of the alpha I'/alpha A hybrid were lower than the parental IFN-alpha A and IFN-alpha I'.

Proc Natl Acad Sci U S A, 1985 Jun, 82(12), 4193 - 7
Mutagenic repair in Escherichia coli: products of the recA gene and of the umuD and umuC genes act at different steps in UV-induced mutagenesis; Bridges BA et al.; When excision-deficient Escherichia coli carrying umuC or umuD alleles were exposed to visible light several hours after ultraviolet irradiation, base-pair-substitution mutations were induced in these normally non-UV-mutable bacteria . It is argued that delayed photoreversal of pyrimidine dimers removes blocks to DNA replication and allows the "survival" and expression of misincorporated bases . A model for UV mutagenesis is proposed with two steps: (i) misincorporation opposite a photoproduct, which can be mediated directly by RecA protein, and (ii) bypass, only the latter process requiring umuD+ and umuC+ alleles . Basal levels of gene products are sufficient for at least some misincorporation events, although induced levels of umuD and umuC gene products are necessary for the bypass step . umuC bacteria containing the recA441 allele showed a greater yield of mutants, and those containing recA430 a reduced yield, following delayed photoreversal . The lexA51 allele (which results in constitutive derepression of RecA protein production) did not significantly alter the yield of mutants but caused them to appear marginally sooner in a recA441 umuC strain . These results emphasize that the nature of the RecA protein and not its concentration is paramount in determining the level of misincorporation . Experiments with recA441 umuC bacteria at 43 degrees C and 30 degrees C suggest that the misincorporation effect is unlikely to be attributable to cleavage of a DNA binding protein such as a repressor or a component of the polymerase complex . Moreover, misincorporation seems to occur without the need for induced synthesis of any other protein under recA control.

Proc Natl Acad Sci U S A, 1985 Jun, 82(12), 3959 - 63
Isolation and nucleotide sequencing of lactose carrier mutants that transport maltose; Brooker RJ et al.; The wild-type lactose carrier of Escherichia coli has a poor ability to transport the disaccharide maltose . However, it is possible to select lactose carrier mutants that have an enhanced ability to transport maltose by growing E . coli cells on maltose minimal plates in the presence of isopropyl thiogalactoside (an inducer of the lac operon) . We have utilized this approach to isolate 18 independent lactose permease mutants that transport maltose . The relevant DNA sequences have been determined, and all of the mutations were found to be single base pair changes either at triplet 177 or at triplet 236 . The nucleotide changes replace alanine-177 with valine or threonine, or tyrosine-236 with phenylalanine, asparagine, serine, or histidine . Transport experiments indicate that all of the mutants have faster maltose transport compared with the wild-type strain . Position 177 mutants retain the ability to transport galactosides, such as lactose and melibiose, at rates similar to the rate of the wild-type strain . In contrast, the position 236 mutants are markedly defective in the ability to transport galactosides . With regard to secondary structure, alanine-177 and tyrosine-236 are located on adjacent hydrophobic segments of the lactose carrier that are predicted to span the membrane . Thus, the results of this study indicate that the substrate recognition site of the lactose carrier is located within the plane of the lipid bilayer . In addition, a tertiary structure model is proposed that suggests how certain transmembrane segments might be localized relative to one another.

Proc Natl Acad Sci U S A, 1985 Jun, 82(11), 3766 - 70
Deletion of the terminus region (340 kilobase pairs of DNA) from the chromosome of Escherichia coli; Henson JM et al.; A strain of Escherichia coli with a 7-minute (340 kilobase pairs of DNA) deletion of the terminus region of the chromosome was isolated . This deletion was probably an IS10-promoted event and its extent was characterized by both genetic and DNA hybridization analyses . The most dramatic property of strains harboring this deletion was the absence of the sites that inhibit clockwise- and counterclockwise-traveling replication forks . These strains also grew slowly, produced many nonviable cells, were filamentous, and appeared to have an induced SOS system.

Proc Natl Acad Sci U S A, 1985 Jun, 82(11), 3577 - 81
Characterization of rate-controlling steps in vivo by use of an adjustable expression vector; Walsh K et al.; Citrate synthase (EC 4.1.3.7) was varied from 10% to 5000% the level found in wild-type Escherichia coli by means of recombinant DNA techniques . When acetate was the sole carbon source, cell growth and carbon flow through the Krebs cycle were greatly affected by the under-production of citrate synthase . In contrast, when glucose was the main nutrient, the same underproduction of citrate synthase had little effect on either growth or carbon flux . When the enzyme was overproduced 50-fold, cultures would grow on glucose but cell division could be abruptly stopped by adding acetate to the medium . These results indicate that the regulatory properties of citrate synthase are highly dependent on the carbon-source composition of the medium . Furthermore, recombinant DNA technology can be used to alter rate-controlling steps in biological pathways and elucidate the regulatory properties of metabolic systems.

Proc Natl Acad Sci U S A, 1985 Jun, 82(11), 3543 - 7
Chemical synthesis of a gene encoding the human complement fragment C5a and its expression in Escherichia coli; Mandecki W et al.; A gene coding for the C5a fragment of the fifth component of human complement has been chemically synthesized, cloned, and expressed in Escherichia coli . The 253-base-pair gene fragment was built through a two-step enzymic assembly of 16 oligonucleotides, the average length of each being 32 residues . The oligonucleotides were synthesized by using the phosphoramidite method . The gene was cloned in a pBR322-derivative plasmid downstream from the lac up-promoter mutant, UV5-D . The expression of C5a was detected and measured by immunoassay and a radioligand binding assay . C5a from E . coli was comparable to C5a purified from human serum in inhibiting binding of human 125I-labeled C5a to its putative receptor on polymorphonuclear leukocytes . Studies of smooth muscle contraction in isolated guinea pig ileum showed that the recombinant C5a was biologically active and produced cross-tachyphylaxis with human serum-derived C5a . The results demonstrate the feasibility of expressing C5a anaphylatoxin in bacteria and provide a system for mutagenesis of the C5a protein.

J Bacteriol, 1985 Jun, 162(3), 859 - 64
Mechanism of mutation by thymine starvation in Escherichia coli: clues from mutagenic specificity; Kunz BA et al.; To probe the mechanisms of mutagenesis induced by thymine starvation, we examined the mutational specificity of this treatment in strains of Escherichia coli that are wild type (Ung+) or deficient in uracil-DNA-glycosylase (Ung-) . An analysis of Ung+ his-4 (ochre) revertants revealed that the majority of induced DNA base substitution events were A:T----G:C transitions . However, characterization of lacI nonsense mutations induced by thymine starvation demonstrated that G:C----A:T transitions and all four possible transversions also occurred . In addition, thymineless episodes led to reversion of the trpE9777 frameshift allele . Although the defect in uracil-DNA-glycosylase did not appear to affect the frequency of total mutations induced in lacI by thymine deprivation, the frequency of nonsense mutations was reduced by 30%, and the spectrum of nonsense mutations was altered . Furthermore, the reversion of trpE9777 was decreased by 90% in the Ung- strain . These findings demonstrate that in E . coli, thymine starvation can induce frameshift mutations and all types of base substitutions . The analysis of mutational specificity indicates that more than a single mechanism is involved in the induction of mutation by thymine depletion . We suggest that deoxyribonucleoside triphosphate pool imbalances, the removal of uracil incorporated into DNA during thymine starvation, and the induction of recA-dependent DNA repair functions all may play a role in thymineless mutagenesis.

J Bacteriol, 1985 Jun, 162(3), 1314 - 6
Starvation for ilvB operon leader amino acids other than leucine or valine does not increase acetohydroxy acid synthase activity in Escherichia coli; Tsui P et al.; Eleven different amino acids are encoded in the ilvB leader mRNA . Starvation for leucine or valine, but not for any of the other nine amino acids, resulted in high levels of acetohydroxy acid synthase I . These results are discussed in terms of a report (C.A . Hauser and G.W . Hatfield, Proc . Natl . Acad . Sci . U.S.A . 81:76-79, 1984) which suggests that threonine and alanine, in addition to leucine and valine, are involved in the regulation of the ilvB operon.

J Bacteriol, 1985 Jun, 162(3), 1307 - 10
Enhanced sensitivity of Escherichia coli umuC to photodynamic inactivation by angelicin (isopsoralen); Miller SS et al.; Escherichia coli umuC cells were inactivated four times more rapidly than umuC+ cells by angelicin (a monofunctional psoralen) plus near-UV irradiation . With other DNA-damaging treatments, either no or much smaller differences in sensitivity were observed . These results show that functions associated with the UmuC+ phenotype contribute to the repair (or tolerance) of some categories of DNA damage more efficiently than others.

J Bacteriol, 1985 Jun, 162(3), 1270 - 5
In vitro and in vivo activation of L-serine deaminase in Escherichia coli K-12; Newman EB et al.; Escherichia coli L-serine deaminase (L-SD) in crude extracts made in glycylglycine could be activated by incubation with iron sulfate and dithiothreitol . This activation could also be demonstrated in vitro in two mutants which were physiologically deficient in L-SD activity in vivo . This suggests that these mutants were deficient not in L-SD but in an enzyme(s) activating L-SD . The suggestion is made that production of a functional L-SD in vivo requires activation of the structural gene product by an enzyme or enzymes that reduce the protein to an active form.

J Bacteriol, 1985 Jun, 162(3), 1196 - 202
Construction and characterization of a deletion mutant lacking micF, a proposed regulatory gene for OmpF synthesis in Escherichia coli; Matsuyama S et al.; A method is presented for the construction of a deletion mutant lacking the micF gene, which has been proposed to negatively regulate expression of the ompF gene . The method includes (i) construction of a temperature-sensitive plasmid containing a chromosomal fragment that carries both flanking regions of the micF gene but does not carry micF itself and (ii) replacement of the corresponding chromosomal domain with the fragment . The method is applicable to construction of a deletion mutant for any Escherichia coli chromosomal gene provided that it is dispensable . The micF deletion was confirmed by genetic and biochemical tests, including nucleotide sequence analysis . ompF expression in the micF deletion mutant thus constructed was normally regulated and was not enhanced . When micF was cloned into a high-copy-number plasmid it repressed ompF gene expression, whereas when cloned into a low-copy-number plasmid it did not . From these results, it is concluded that a single copy of the micF gene on the E . coli chromosome does not play a critical role in ompF gene expression.

J Bacteriol, 1985 Jun, 162(3), 1162 - 5
Regulation of the SOS response analyzed by RecA protein amplification; Calsou P et al.; A split UV light dose procedure was used in Escherichia coli to induce an SOS function, RecA protein amplification, which was measured by an immunoradiometric assay . The SOS system was partially induced after the first UV irradiation, and the inducing effects of subsequent identical UV doses were quantified . Variations in the inducing effects of successive UV doses were related to modulations of the SOS signal level during SOS induction . A reduction in the level of SOS signal was found after 20 min in the wild-type strain, hypothesized to result from negative control of repair functions . A few DNA repair mutants were tested by the same procedure; the uvrA, recF, and umuC genes were involved in SOS induction control, but we found differences in their respective kinetics of expression . On the contrary, in a recB mutant, only a slight effect was obtained on this control.

J Bacteriol, 1985 Jun, 162(3), 1156 - 61
Pantothenate transport in Escherichia coli; Vallari DS et al.; The transport system for pantothenic acid uptake in Escherichia coli was characterized . This transport system was specific for pantothenate, had a Kt of 0.4 microM, and had a maximum velocity of 1.6 pmol/min per 10(8) cells (45 pmol/min per mg {dry weight}) . Pantothenate uptake was not reduced in osmotically shocked cells or by ATP depletion with arsenate, but was reduced greater than 90% by the dissipation of the membrane electrochemical gradient with 2,4-dinitrophenol . Sodium ions stimulated pantothenate uptake (Kt, 0.8 mM) by reducing the Kt for pantothenate by an order of magnitude . Intracellular pantothenate was rapidly phosphorylated, but phosphorylation of pantothenate was not required for uptake since pantothenate was the only labeled intracellular compound concentrated by ATP-depleted, glucose-energized cells . The data were consistent with the presence of a high-affinity pantothenate permease that concentrates the vitamin by sodium cotransport.

J Bacteriol, 1985 Jun, 162(3), 1005 - 7
Quantitation with monoclonal antibodies of Escherichia coli H protein suggests histone function; Lutz H et al.; The abundance of the histonelike H protein of Escherichia coli (U . Hubscher, H . Lutz, and A . Kornberg, Proc . Natl . Acad . Sci . U.S.A . 77:5097-5101, 1980) was determined by using monoclonal antibodies against H protein, immunoblotting, and homogeneous H protein as a standard . H protein was found to be present at approximately 120,000 monomeric molecules per fast-growing E . coli cell . This amount of H protein corresponds to a ratio of one H protein molecule to approximately 200 base pairs of the bacterial chromosome . Together with previous results, these findings suggest that H protein has histonelike function similar to that of histone protein H2A, its counterpart in the eucaryotic cell.

J Bacteriol, 1985 Jun, 162(3), 1000 - 4
UDP-N-acetylmuramylpentapeptide as acceptor in murein biosynthesis in Escherichia coli membranes and ether-permeabilized cells; Kraus W et al.; Two widely used in vitro systems of Escherichia coli capable of synthesizing murein were evaluated by using high-pressure liquid chromatography for murein analysis . Comparison of the composition of murein synthesized by either a membrane preparation or ether-treated cells with native murein revealed that both in vitro systems failed to synthesize murein that was identical to murein formed in vivo . Furthermore, neither system attached the lipoprotein to the murein . Ether-treated cells, however, were superior to the membrane preparation in catalyzing the formation of the remarkable A2pm-A2pm cross-linkage . In both systems an atypical transpeptidation reaction was found to take place in which exogenously supplied UDP-N-acetylmuramylpentapeptide was directly linked to the murein without participation of the bactoprenol lipid carrier . The direct transpeptidation yields preferentially trimeric peptide bridges with the UDP-linked muramylpentapeptide serving as the acceptor.

Infect Immun, 1985 Jun, 48(3), 818 - 23
Elicitation of enteroluminal neutrophils by enterotoxigenic and nonenterotoxigenic strains of Escherichia coli in swine; Rose R et al.; In intact neonatal piglets, two strains of enterotoxigenic Escherichia coli (ETEC), which could adhere to epithelial cells and thus colonize the small intestine, attracted greater numbers of neutrophils into the lumen and wall of the intestine than did a nonenteropathogenic strain of E . coli . Ligated loops of small intestine in 8-week-old pigs were used in attempts to identify the attributes of ETEC involved in stimulating an increased enteroluminal migration of neutrophils . A nonenteropathogenic strain of E . coli did not attract neutrophils into the intestinal lumen in this model . However, three of the five ETEC strains tested did so . The three positive strains all produced heat-stable enterotoxin type b (STb) . Neither of the negative ETEC strains produced STb . An STb-containing culture supernatant prepared from a strain of E . coli which contained an STb plasmid did not attract significantly more neutrophils than did a control supernatant prepared from the same strain of E . coli without the plasmid . The ETEC strains which attracted neutrophils in loops did not associate intimately with loop villi more consistently, nor did they grow to higher numbers in loops than strains which did not . It was concluded that there are increased numbers of neutrophils in the intestinal lumen during ETEC infection of newborn pigs . However, attempts to identify the attribute(s) of ETEC responsible for eliciting enteroluminal neutrophils were not successful.

Cell, 1985 Jun, 41(2), 607 - 14
An artificial anchor domain: hydrophobicity suffices to stop transfer; Davis NG et al.; A hydrophobic sequence of 23 contiguous, uncharged residues anchors the coliphage f1 gene III protein (pIII) to the Escherichia coli cytoplasmic membrane; mutations removing this domain allow secretion of the protein to the periplasm . Multiple copies of an oligonucleotide encoding the hydrophobic repeat, Leu-Ala-Leu-Val, were introduced into genes for secreted forms of pIII . Artificial domains of 16 or more hydrophobic residues function to anchor the protein . Pronase protection experiments demonstrate that the new sequences act to halt transfer of the protein across the membrane, thus specifying a transmembrane topology . Relocating the hydrophobic domain within the polypeptide chain predictably alters the resultant protein/membrane topology . Repeats of a polar sequence were inserted with no effect on secretion . Furthermore, an unrelated hydrophobic sequence, uncovered by a gene III frameshift mutation, acts to anchor the protein . We conclude that function simply reflects hydrophobicity and not some more subtle feature of structure or sequence.

Proc Natl Acad Sci U S A, 1985 Jun, 82(12), 4100 - 4
Identification of an IgE-binding protein by molecular cloning; Liu FT et al.; The synthesis and function of IgE are dependent on IgE-binding proteins, which include cell surface IgE receptors and IgE-binding lymphokines . To further our understanding of the IgE system, we have engaged in the molecular cloning of genes for some of these proteins . In studying the in vitro translation products of mRNA from rat basophilic leukemia (RBL) cells, we have identified a Mr 31,000 polypeptide that binds IgE and is also reactive with antibodies to proteins affinity-purified from RBL cells with IgE immunoadsorbent . For the molecular cloning, double-stranded cDNA was synthesized from sucrose gradient-fractionated RBL mRNA, inserted into plasmid pBR322, and used to transform Escherichia coli . By screening transformants with a hybridization-selection/in vitro translation procedure, we identified one clone containing cDNA that hybridized to mRNA coding for a Mr 31,000 IgE-binding protein . The DNA sequence of this cloned cDNA (571 base pairs) was determined and the amino acid sequence corresponding to part of the protein was deduced . In RNA blot analysis, the cDNA hybridized with a mRNA of 1100 nucleotides found in RBL cells but absent in cells not expressing IgE receptors . This cloned cDNA most likely codes for the Mr 31,000 IgE-binding protein identified in RBL cells, which appears to be related to the IgE-binding phenotype of the cells and which may have a significant role in the IgE-mediated activation of basophils and mast cells.

Proc Natl Acad Sci U S A, 1985 Jun, 82(12), 3983 - 7
In vitro expression of chloroplast genes in lysates of higher plant chloroplasts; Bard J et al.; A DNA-dependent in vitro-coupled transcription-translation system has been prepared from lysates of isolated chloroplasts . These lysates are comparable to those of Escherichia coli in transcriptional and translational fidelity and efficiency in response to a given template DNA . When Nicotiana tabacum chloroplast DNA is used as template with chloroplast lysates (N . tabacum or spinach) or E . coli lysates, NaDodSO4 gel analysis reveals similar polypeptide patterns that are distinct from the patterns obtained with E . coli DNA . Genes in recombinant plasmids containing chloroplast DNA are also expressed in these in vitro systems . DNA . RNA hybridization experiments show that transcripts are synthesized from most of the chloroplast genome . Newly synthesized large subunit of ribulosebisphosphate carboxylase/oxygenate and a transcript of the large subunit gene (rbcL) are observed in chloroplast lysates using as template chloroplast DNA or cloned fragments of tobacco chloroplast DNA that contain the large subunit gene . Results suggest that differential expression of chloroplast genes occurs in vitro . By using cloned chloroplast DNA templates in this homologous system, it is possible to identify and map structural genes for chloroplast proteins.

J Surg Res, 1985 Jun, 38(6), 582 - 91
Low-dose dopamine preserves renal blood flow in endotoxin shocked dogs treated with ibuprofen; Fink MP et al.; Drugs that inhibit prostaglandin (PG) biosynthesis improve hemodynamics and survival in experimental endotoxic and septic shock . The therapeutic utility of these agents in the management of septic patients may be limited, however, by their tendency to decrease renal blood flow (RBF) in animals and humans stressed by experimental manipulations or disease states that promote renal vasoconstriction . In the present study, we addressed this question: can low-dose intravenous (iv) dopamine (4 micrograms/kg/min), a known renal vasodilator, improve renal perfusion in endotoxin-shocked dogs treated with the PG synthesis inhibitor, ibuprofen . RBF was measured in pentobarbital anesthetized dogs using an electromagnetic flow meter . After obtaining baseline hemodynamics, Escherichia coli endotoxin (1.5 mg/kg) was given iv . The dogs were randomized 30 min later into three groups: Group I received saline; Group II received ibuprofen (12.5 mg/kg, iv); Group III received ibuprofen plus dopamine . Comparison of Groups I and II revealed that ibuprofen increased mean arterial pressure (MAP) and systemic vascular resistance (SVR) (P less than 0.0001 and P = 0.002, respectively) and decreased RBF (P = 0.019) . Adding low-dose dopamine (Group II vs Group III) did not significantly affect MAP or SVR, but did augment RBF (P less than 0.001) . We conclude that low-dose dopamine improves renal hemodynamics in ibuprofen-treated endotoxemic dogs.

Mol Cell Biol, 1985 Jun, 5(6), 1449 - 55
Biochemical properties of a highly purified v-rasH p21 protein overproduced in Escherichia coli and inhibition of its activities by a monoclonal antibody; Hattori S et al.; The v-rasH oncogene of Harvey murine sarcoma virus encodes a 21,000-dalton p21 protein which has been expressed at a high level as a fusion protein in Escherichia coli . We have purified the p21 to over 90% in purity without the use of any detergent or protein denaturant . The purified p21 possesses full biochemical activities of GTP/GDP binding, autokinase, and GTPase . Scatchard analysis indicates a single class of binding sites with Kd values of 0.83 X 10(-8)M for GTP and 1.0 X 10(-8)M for GDP . The binding site can be specifically labeled with a {3H}GTP photoaffinity analog, P3-(4-azidoanilido)-5' GTP . To probe for the active center of p21, we used a battery of six monoclonal antibodies to p21 to examine their effects on p21 activities . We found that only one monoclonal antibody, Y13-259, was capable of inhibiting both GTP/GDP binding and autokinase enzymatic activities, suggesting that these p21 activities are related activities conferred by a single active center within the p21 molecule . These observations together with the recent finding that microinjection of the same monoclonal antibody into NIH 3T3 cells specifically blocks p21 in vivo function (Mulcahy et al., Nature {London} 313:241, 1985) strongly suggest that p21 in vitro activities are responsible for its cellular function.

Chemioterapia, 1985 Jun, 4(3), 199 - 201
Inhibition of conjugal transfer by new quinolinic compounds; Oliva B et al.; We found that nalidixic acid is a good inhibitor of conjugal transfer of R-plasmids and that related compounds show the same properties . We investigated recently synthetized quinolinic molecules . The inhibition was not due to bacterial activity of the compounds against donors, recipients or R transconjugants; in fact, the drug concentrations were twice or four times lower than the M.I.C . values . The new molecules showed a marked inhibitory effect in various R-mating experiments.

J Gen Virol, 1985 Jun, 66 ( Pt 6), 1209 - 13
Influence of C-terminal modifications of phi X174 lysis gene E on its lysis-inducing properties; Blasi U et al.; The phi X174 gene E product (gpE) causes lysis of Escherichia coli by inducing the host autolytic system . Experiments were carried out to ascertain which part of the 91 amino acid polypeptide carries the functional site for this process . For this purpose fusion genes were created comprising the first 51 codons of gene E and unrelated sequences coding for 102 or 33 amino acids respectively . The chimeric protein of 153 amino acids consisting of the N-terminal part of gpE and a fragment of beta-galactosidase, was neither able to lyse E . coli nor to restore beta-galactosidase activity by alpha-complementation . Expression of the 84 amino acid polypeptide, however, was able to induce lysis of E . coli . It is therefore concluded that the functional lysis-inducing site of gpE is located within the cloned N-terminal part of gene E . In the shorter chimeric protein the sequence following the functional site was tolerated or necessary for stabilization, but in the longer chimeric protein, the C-terminal sequence disturbed the lysis-inducing conformation.

J Bacteriol, 1985 Jun, 162(3), 1166 - 72
Mutation-dependent suppression of recB21 recC22 by a region cloned from the Rac prophage of Escherichia coli K-12; Willis DK et al.; Using pBR322 as a vector, we cloned a 5.95-kilobase fragment of the Rac prophage together with 1.70 kilobases of a flanking Escherichia coli chromosome sequence . The resulting plasmid (pRAC1) was unable to suppress the mitomycin and UV sensitivity and recombination deficiency of a recB21 recC22 strain . Five spontaneous mitomycin-resistant derivatives contained deletion mutant plasmids . These plasmids also suppressed the UV sensitivity and recombination deficiency of their recB21 recC22 hosts . All five deletions were contained within a 2.45-kilobase EcoRI-to-HindIII segment of the plasmid . By substituting the corresponding 2.45-kilobase EcoRI-toHindIII fragments of Rac prophage isolated from sbcA+, sbcA6, and sbcA23 strains for the shortened segment of one of the deletion mutant plasmids, we were able to show that sbcA mutations map in this region . Also in this region is the site (or closely linked sites) at which previous studies had shown that insertion of Tn5 and IS50 leads to suppression of recB21 recC22 . The sequence in this region that must be altered or circumvented to allow suppression is discussed . Also presented are data correlating the expression of nuclease activity with the degree of suppression.

J Immunol, 1985 Jun, 134(6), 4062 - 8
Osmotic stress and the freeze-thaw cycle cause shedding of Fc and C3b receptors by human polymorphonuclear leukocytes; Takahashi T et al.; A major problem in the cryopreservation of human polymorphonuclear leukocytes (PMN) is the loss of phagocytic function in cryopreserved cells . This is not a problem with cryopreserved monocytes . To study the reasons for this difference in detail, PMN and monocytes were either osmotically stressed in hypertonic media or were frozen to various temperatures . Cells were then returned to conditions of physiologic osmolarity and temperature . All cells remained viable . However, the ability of PMN to phagocytize bacteria and to bind sheep erythrocytes (E) opsonized with IgG, C3b, or C3bi decreased sharply after exposure to media of 600 mOsM or greater and after freezing to -1.5 degrees C . In contrast, monocytes were unaffected until a concentration of 1500 mOsM or a freezing temperature of -5 degrees C was exceeded . To determine whether the functional losses of surface receptor activity in PMN resulted from a loss of receptors from the membranes or from inactivation or internalization of receptors, opsonized E were incubated in the supernatants from stressed PMN . On subsequent incubation with healthy PMN, these E made fewer rosettes than control opsonized E . The inhibitory effect of the supernatants on rosetting of IgG-sensitized E could be removed by preincubation with IgG bound to Sepharose 4B . Immunoprecipitation of C3b and C3bi receptors from surface-iodinated, osmotically stressed, and control PMN suggested that about 50% of cell surface complement receptors were lost from the cell surface during osmotic stress . These experiments suggest that receptors for IgG and C3 are extruded from PMN cell membranes as a result of hyperosmotic stress, which is associated with the freeze-thaw cycle . This may be an early event in the functional damage done to PMN during attempts at cryopreservation.

Virology, 1985 Jun, 143(2), 435 - 41
Complementary DNA cloning and expression of the papaya ringspot potyvirus sequences encoding capsid protein and a nuclear inclusion-like protein in Escherichia coli; Nagel J et al.; Three cDNA clones that express viral gene products in Escherichia coli JM83 were derived from a watermelon mosaic virus-1 strain of papaya ringspot virus (PRSV-W) . DNAs complementary to portions of the viral RNA were inserted into the pUC8 and pUC9 plasmids, and the expressed polypeptides were fusion products with the amino terminus of beta-galactosidase . Clones W1-77 and W2-1 expressed fusion products with apparent molecular weights of 40,000 (40K) and 14K, respectively, which were serologically related to PRSV capsid protein . A 52K product serologically related to a 54K nuclear inclusion protein of tobacco etch virus was produced by clone W1-18 . The sequences encoding the capsid and 57K nuclear inclusion-like proteins of PRSV were physically mapped to adjacent positions through Southern blot analyses of clones W1-77 and W1-18.

Biochimie, 1985 Jun, 67(6), 643 - 9
Overproduction and purification of initiation factor IF2 and pNUSA proteins from a recombinant plasmid bearing strain; Dondon J et al.; The genes for translational initiation factor, IF2 and pNusA have been cloned into a plasmid vector where they are placed under the control of the inducible lambdapL promoter and the c1857 thermosensitive repressor . When a strain carrying this plasmid is heat induced, IF2 alpha, IF2 beta and pNusA are overproduced 15 to 20 fold . This has allowed us to purify the IF2 and NusA proteins in large amounts.

Biochimie, 1985 Jun, 67(6), 583 - 8
Sequence analysis of a Dictyostelium discoideum gene coding for an active dihydroorotate dehydrogenase in yeast; Jacquet M et al.; A Dictyostelium discoideum DNA fragment isolated on the basis of its ability to complement the ural mutation of yeast, codes for a dihydroorotate dehydrogenase activity . The complete nucleotide sequence of this 1898 bp fragment has been determined and reveals an open reading frame capable of coding for a 369 amino acid polypeptide of molecular mass 47.000 . The gene shows preferential use of codons with weak pairing forces . Eleven codons, mainly those with a G in the third position, are absent . The flanking sequences are unusually rich in A + T (80%) . Several direct and inverted repeats exist in the 5' flanking sequence.

J Gen Microbiol, 1985 Jun, 131 ( Pt 6), 1523 - 30
Molecular homology and incompatibility relationships between F and IncH1 plasmids; Taylor DE et al.; IncH1 plasmids and the F plasmid of Escherichia coli display one-way compatibility . An entering IncH1 plasmid is incompatible with a resident F plasmid, but is compatible when it is the resident plasmid . There is little molecular homology between IncH1 plasmids and the F plasmid . A single 5 MDal EcoRI restriction enzyme fragment from digests of several IncH1 plasmids hybridizes with probes constructed from the primary replication region of F . Homology can be demonstrated only with the gene for the essential replication protein of F (gene E), but the expression of incompatibility behaviour appears to be associated with the presence of the secondary replicon of the F plasmid . Thus R27 and F are compatible under growth conditions allowing replication and maintenance of F by the secondary replicon . However, a mutant F plasmid which lacks the secondary replicon of F is incompatible with R27 in both directions, irrespective of the growth conditions used.

Mol Cell Biol, 1985 Jun, 5(6), 1247 - 59
Recombination between poly{d(GT).d(CA)} sequences in simian virus 40-infected cultured cells; Stringer JR; CVI cells were transfected with oversized simian virus 40 (SV40) genomes that could be reduced to packageable size by alternative homologous recombination pathways involving either two polydeoxyguanylic-thymidylic acid X polydeoxycytidylic-adenylic acid (poly{d(GT).d(CA)}; abbreviated hereafter as poly(GT)} tracts or two tracts of homologous SV40 sequence . Plaque-forming viruses rescued by this procedure were found to contain genomes formed by homologous and nonhomologous recombination events . Half of the viable viral DNA molecules recovered were the result of recombination between two tracts of poly(GT) . Approximately 20% of the rescued viral genomes were produced by homologous recombination between tracts of SV40 DNA . Nonhomologous recombination involving SV40 sequences was also a major pathway of deletion, producing ca . 30% of the viral plaques . Tracts of poly(GT) generated by recombination were variable in length, suggesting that recombination between poly(GT) tracts was usually unequal . On a per-nucleotide basis, poly(GT) recombination occurred eight times more frequently than did recombination between homologous SV40 DNA . This eightfold difference is the maximum recombinatory enhancement attributable to poly(GT) sequences . Although DNA sequence analysis showed that tracts of poly(GT) generated by recombination retained the alternating G-T repeat motif throughout their length, the contribution of the nonhomologous pathway to poly(GT) recombination cannot be ruled out, and the relative proclivity of a given length of d(GT).d(CA) sequence to undergo homologous recombination is probably less than eight times greater than that of an SV40 sequence of the same length.

EMBO J, 1985 Jun, 4(6), 1593 - 7
Folding patterns of porin and bacteriorhodopsin; Paul C et al.; Porin spans the outer membrane of Escherichia coli with most of the protein embedded within the membrane . It lacks pronounced hydrophobic domains and consists predominantly of beta-pleated sheet . These observations require the accommodation of polar and ionizable residues in an environment that has a low dielectric constant . Owing to a currently limited understanding of the constraints governing membrane protein structure, a minimal approach to structure prediction is proposed that identifies segments causing polypeptides to reverse their direction (turn identification) . The application of this procedure avoids hydrophobicity parameters and yields a model of porin which is in good agreement with all experimental data available . The presence of polar and ionizable residues within membrane boundaries implies a dense (saturating) network of hydrogen bond donor and acceptor groups . Application to a paradigm of hydrophobic membrane proteins, bacteriorhodopsin, reveals a pattern consistent with its alpha-helical folding . The postulated structure includes significantly more polar residues in the membrane domain than have been assumed previously, suggesting that there are also hydrogen bonding networks in bacteriorhodopsin . Extensive networks permeating protein interior and surfaces would explain the extraordinary stability and the tight interactions between functional units in the formation of crystalline arrays of both proteins.

Arch Biochem Biophys, 1985 Jun, 239(2), 467 - 74
Cloning and expression of the metE gene in Escherichia coli; Chu J et al.; A lambda-transducing phage was isolated that contains the metE gene . This gene codes for N5-methyl-H4-folate:homocysteine methyltransferase (EC 2.1.1.14), an enzyme that catalyzes the terminal reaction in methionine biosynthesis . A 9.1-kb EcoR1 fragment of this phage, containing the metE gene, was then cloned into pBR325 . This plasmid, pJ19, was used to transform Escherichia coli strain 2276, a metE mutant, and restore the MetE+ phenotype . Although the transformed cells produced large amounts of the metE protein in vivo, in vitro studies using pJ19 as template showed low synthesis of the metE protein.

Proc Natl Acad Sci U S A, 1985 Jun, 82(11), 3562 - 6
Initiation of enzymatic replication at the origin of the Escherichia coli chromosome: contributions of RNA polymerase and primase; Ogawa T et al.; Replication of plasmids that depend on the 245-base-pair origin of the Escherichia coli chromosome (oriC) requires many purified proteins that (i) direct initiation to oriC (e.g., dnaA protein), (ii) influence initiations elsewhere (e.g., auxiliary proteins), and (iii) prime and extend DNA chains (e.g., priming and synthesis proteins) . For the RNA priming and initiation of new DNA chains, the requirements for both primase and RNA polymerase (RNA pol) {Kaguni, J . M . & Kornberg, A . (1984) Cell 38, 183-190} have been further analyzed . Depending on the levels of auxiliary proteins (topoisomerase I and protein HU), three priming systems can operate: primase alone, RNA pol alone, or both combined . At low levels of auxiliary proteins, primase alone sustains an effective priming system . At higher levels, primase action is blocked, but RNA pol alone can initiate replication, albeit feebly; at these high levels of auxiliary proteins, primase and RNA pol act synergistically . When RNA pol is stalled by an inhibitor or lack of a ribonucleoside triphosphate, primase action is also inhibited . Based on these and other data {van der Ende, A., Baker, T . A., Ogawa, T . & Kornberg, A . (1985) Proc . Natl . Acad . Sci . USA 82, in press}, RNA pol can counteract inhibition by auxiliary proteins and thus activate the origin for the priming by primase of the leading strand of the replication fork.

Mutat Res, 1985 Jun-Jul, 150(1-2), 147 - 58
Complementation of a pKM101 derivative that decreases resistance to UV killing but increases susceptibility to mutagenesis; Langer PJ et al.; The drug resistance plasmid pKM101 makes Escherichia coli resistant to the lethal effects of ultraviolet (UV) irradiation and more susceptible to mutagenesis by a variety of agents . The plasmid operon responsible for increasing mutagenesis has been termed mucAB (Mutagenesis, UV and chemical) . We have isolated a derivative of pKM101 called pGW1975 which makes cells more sensitive to killing by UV but which retains the ability of pKM101 to increase susceptibility to methyl methanesulfonate (MMS) mutagenesis . pGW1975 increases UV mutagenesis less than pKM101 in a uvrA+ strain but more than pKM101 in a uvrA- strain . muc- point and insertion mutants of pKM101 and pGW1975 complement to restore the plasmid-mediated: (i) ability to reactivate UV-irradiated phage, (ii) resistance to killing by UV, and (iii) level of susceptibility to UV mutagenesis . We have identified a 2.0 kb region of pKM101 which is responsible for the complementation and which maps counterclockwise of mucAB.

J Virol, 1985 Jun, 54(3), 817 - 24
The alpha sequence of the cytomegalovirus genome functions as a cleavage/packaging signal for herpes simplex virus defective genomes; Spaete RR et al.; Although herpes simplex virus (HSV) 1 and human cytomegalovirus (CMV) differ remarkably in their biological characteristics and do not share nucleotide sequence homology, they have in common a genome structure that undergoes sequence isomerization of the long (L) and short (S) components . We have demonstrated that the similarity in their genome structures extends to the existence of an alpha sequence in the CMV genome as previously defined for the HSV genome . As such, the alpha sequence is predicted to participate as a cis-replication signal in four viral functions: (i) inversion, (ii) circularization, (iii) amplification, and (iv) cleavage and packaging of progeny viral DNA . We have constructed a chimeric HSV-CMV amplicon (herpesvirus cis replication functions carried on an Escherichia coli plasmid vector) substituting CMV DNA sequences for the HSV cleavage/packaging signal in a test of the ability of this CMV L-S junction sequence to provide the cis signal for cleavage/packaging in HSV 1-infected cells . We demonstrate that the alpha sequence of CMV DNA functions as a cleavage/packaging signal for HSV defective genomes . We show the structure of this sequence and provide a functional demonstration of cross complementation in replication signals which have been preserved over evolutionary time in these two widely divergent human herpesviruses.

J Virol, 1985 Jun, 54(3), 665 - 74
Two major outer envelope glycoproteins of Epstein-Barr virus are encoded by the same gene; Beisel C et al.; Two major outer envelope glycoproteins of Epstein-Barr virus, gp350 and gp220, are known to be encoded by 3.2- and 2.5-kilobase RNAs which map to the same DNA fragment (M . Hummel, D . Thorley-Lawson, and E . Kieff, J . Virol . 49:413-417) . These RNAs have the same 5' and 3' ends . The larger RNA is encoded by a 2,777-base DNA segment which is preceded by TATTAAA, has AATAAA near its 3' end, and contains a 2,721-base open reading frame . The smaller RNA has one internal splice which maintains the same open reading frame . Translation of the 3.2- and 2.5-kilobase RNAs yielded proteins of 135 and 100 kilodaltons (Hummel et al., J . Virol . 49:413-417) . The discrepancy between the 907 codons of the open reading frame and the 135-kilodalton size of the gp350 precursor is due to anomalous behavior of the protein in gel electrophoresis, since a protein translated from most of the Epstein-Barr virus open reading frame in Escherichia coli had similar properties . Antisera raised in rabbits to the protein expressed in E . coli specifically immunoprecipitated gp350 and gp220, confirming the mapping and sequencing results and the translational reading frame . The rabbit antisera also reacted with the plasma membranes of cells that were replicating virus and neutralized virus, particularly after the addition of complement . This is the first demonstration that the primary amino acid sequence of gp350 and gp220 has epitopes which can induce neutralizing antibody . We propose a model for the gp350 protein based on the theoretical analysis of its primary sequence.

J Cell Biol, 1985 Jun, 100(6), 1968 - 76
The small subunit of ribonucleotide reductase is encoded by one of the most abundant translationally regulated maternal RNAs in clam and sea urchin eggs; Standart NM et al.; In both clam oocytes and sea urchin eggs, fertilization triggers the synthesis of a set of proteins specified by stored maternal mRNAs . One of the most abundant of these (p41) has a molecular weight of 41,000 . This paper describes the identification of p41 as the small subunit of ribonucleotide reductase, the enzyme that provides the precursors necessary for DNA synthesis . This identification is based mainly on the amino acid sequence deduced from cDNA clones corresponding to p41, which shows homology with a gene in Herpes Simplex virus that is thought to encode the small subunit of viral ribonucleotide reductase . Comparison with the B2 (small) subunit of Escherichia coli ribonucleotide reductase also shows striking homology in certain conserved regions of the molecule . However, our attention was originally drawn to protein p41 because it was specifically retained by an affinity column bearing the monoclonal antibody YL 1/2, which reacts with alpha-tubulin (Kilmartin, J . V., B . Wright, and C . Milstein, 1982, J . Cell Biol., 93:576-582) . The finding that this antibody inhibits the activity of sea urchin embryo ribonucleotide reductase confirmed the identity of p41 as the small subunit . The unexpected binding of the small subunit of ribonucleotide reductase can be accounted for by its carboxy-terminal sequence, which matches the specificity requirements of YL 1/2 as determined by Wehland et al . (Wehland, J., H . C . Schroeder, and K . Weber, 1984, EMBO {Eur . Mol . Biol . Organ.} J., 3:1295-1300) . Unlike the small subunit, there is no sign of synthesis of a corresponding large subunit of ribonucleotide reductase after fertilization . Since most enzymes of this type require two subunits for activity, we suspect that the unfertilized oocytes contain a stockpile of large subunits ready for combination with newly made small subunits . Thus, synthesis of the small subunit of ribonucleotide reductase represents a very clear example of the developmental regulation of enzyme activity by control of gene expression at the level of translation.

J Bacteriol, 1985 Jun, 162(3), 933 - 7
Alteration in cation specificity of the melibiose transport carrier of Escherichia coli due to replacement of proline 122 with serine; Yazyu H et al.; The structural genes (melB) for the melibiose carrier of five mutants of Escherichia coli showing altered cation specificity for melibiose transport were cloned . The mutations were mapped in a 248-base-pair DNA fragment by a recombinational assay by using the mutants transformed with hybrid plasmids carrying various portions of the wild-type melB gene . The nucleotide sequences of the corresponding DNA fragments derived from mutated melB genes were determined, and the amino acid sequences of the carrier were deduced . Proline 122 was replaced with serine in the melibiose carrier of all five mutants (which were isolated independently) . We conclude that this amino acid replacement caused the alteration in cation specificity (loss of coupling to H+) of the melibiose carrier.

J Bacteriol, 1985 Jun, 162(3), 1221 - 6
Mapping of transfer regions within incompatibility group HI plasmid R27; Taylor DE et al.; Plasmids of incompatibility group HI are large (greater than 150 kilobases {kb}) and possess an unusual thermosensitive mode of conjugative transfer . R27, the prototype IncHi1 plasmid, encodes resistance to tetracycline via a determinant which is related to transposon Tn10 . A restriction endonuclease map of R27 (size, 182 kb) was recently constructed with ApaI, PstI, and XbaI . Transfer genes within R27 were mapped by insertion of Tn5 and Tn7 . At least two different regions of the plasmid were concerned with transfer functions . Insertions into either region completely abolished transfer . None of the insertions had any effect on entry exclusion (Eex) of other IncH plasmids . However, a deletion mutant which lacked the Eex function was obtained, allowing us to map the probable site of the gene encoding Eex to one of the two transfer regions . The tetracycline resistance determinant in R27 was located within an 8-kb region between the two main transfer regions . The transfer genes, therefore, are not located together in R27 but are situated in at least two major widely separated transfer regions.

J Bacteriol, 1985 Jun, 162(3), 1173 - 9
Genetic analysis of mutations that compensate for loss of Escherichia coli DNA topoisomerase I; Raji A et al.; A transposon Tn10 insertion in topA, the structural gene of Escherichia coli DNA topoisomerase I, behaves as an excluded marker in genetic crosses with many strains of E . coli . However, derivative strains that accept this mutant topA allele are readily selected . We show that many of these topA mutant strains contain additional mutations that compensate for the loss of DNA topoisomerase I . Genetic methods for mapping and manipulating such compensatory mutations are described . These methods include a plate-mating test for the ability of strains to accept a topA::Tn10 allele and a powerful indirect selection for transferring compensatory mutations from male strains into non-compensatory female strains . One collection of spontaneous compensatory mutants is analyzed in detail and is shown to include compensatory mutations at three distinct loci: gyrA and gyrB, the genes that encode the subunits of DNA gyrase, and a previously unidentified locus near tolC . Mutations at this third locus, referred to as toc (topoisomerase one compensatory) mutations, do not behave as point mutations in transductional crosses and do not result in lowered DNA gyrase activity . These results show that wild-type strains of E . coli require DNA topoisomerase I, and at least one class of compensatory mutations can relieve this requirement by a mechanism other than reduction of DNA gyrase activity.

J Biochem (Tokyo), 1985 Jun, 97(6), 1807 - 10
Value of heptyl-beta-D-thioglucoside, a new nonionic detergent, in studies on membrane proteins; Shimamoto T et al.; A new nonionic detergent, heptylthioglucoside, was synthesized and found to be more soluble than octylthioglucoside in water at low temperatures . Use of this detergent for solubilization and reconstitution of membrane proteins of Escherichia coli was examined . Heptylthioglucoside was as effective as octylthioglucoside and octylglucoside in solubilizing membrane proteins, and by the heptylthioglucoside-dilution procedure the H+-translocating ATPase (F1F0) and melibiose carrier could easily be reconstituted into liposomes . It is concluded that heptylthioglucoside is very useful in studies on membrane proteins.

J Clin Microbiol, 1985 Jun, 21(6), 951 - 4
Indications that the erythrocyte receptor involved in enterotoxigenic Escherichia coli attachment is a sialoglycoconjugate; Bartus H et al.; A reverse hemagglutination assay was used to study adherence to human erythrocytes by Escherichia coli H10407, which possesses colonization factor antigen I . Pretreatment of erythrocytes with trypsin, chymotrypsin, papain, protease, and neuraminidase completely abolished attachment reactivity . In addition, the hemagglutination reaction was prevented by the presence of urea and guanidine . In contrast, the lipases, nucleotide hydrolases, exoglycosidases, and reagents affecting disulfide or sulfhydryl moieties did not alter receptor reactivity . Glycoconjugates containing sialic acid inhibited the hemagglutination reaction . Furthermore, a sialoglycoprotein isolated from the erythrocyte membrane inhibited the hemagglutination reaction . Collectively, these data indicate that the erythrocyte receptor responsible for attachment by E . coli possessing colonization factor antigen I is a sialoglycoconjugate.

J Urol, 1985 Jun, 133(6), 1068 - 75
Experimental pyelonephritis in the monkey . VII . Ascending pyelonephritis in the absence of vesicoureteral reflux; Roberts JA et al.; Six adult male nonrefluxing monkeys were experimentally infected by inoculation of P-fimbriated E . coli into the bladder . Eight control monkeys were inoculated with a non-P-fimbriated E . coli strain . Inoculation with the P-fimbriated E . coli resulted in marked leukocytosis, prolonged bacteriuria and loss of renal function with a 66 per cent incidence of pyelonephritis . Death secondary to bilateral pyelonephritis was seen in 2 monkeys inoculated with P-fimbriated E . coli . Pyelonephritis was not seen in any of the monkeys inoculated with non-P-fimbriated E . coli . The study shows that ascending pyelonephritis can occur in monkeys in the absence of vesicoureteral reflux.

J Bacteriol, 1985 Jun, 162(3), 1293 - 301
Globoside-specific adhesins of uropathogenic Escherichia coli are encoded by similar trans-complementable gene clusters; Lund B et al.; Uropathogenic Escherichia coli frequently express globoside-specific adhesins, shown to mediate binding to uroepithelial cells . For one gene cluster pap, it recently has been demonstrated that globoside binding is not dependent on expression of the pilus subunit gene papA . Instead, two other pap genes papF and papG are specifically required for globoside binding (F . P . Lindberg et al., EMBO J . 3:1167-1173, 1984) . By restriction enzyme mapping, DNA hybridization, DNA sequencing, and protein expression in minicells, we show that three gene clusters encoding globoside binding have a very similar structure and gene organization, although they were cloned from different E . coli isolates . Major differences between the adhesin clones were restricted to the central part of the pilin gene (papA) and to one of the two adhesin gene (papG) . The three functional units required for biogenesis of globoside-binding pili, i.e., pilin synthesis, pilin export, and pilin assembly, as well as expression of adhesion function, were all trans complementable among the gene clusters.

J Bacteriol, 1985 Jun, 162(3), 1285 - 92
Genetic organization of the afimbrial adhesin operon and nucleotide sequence from a uropathogenic Escherichia coli gene encoding an afimbrial adhesin; Labigne-Roussel A et al.; The uropathogenic Escherichia coli KS52 strain expresses a mannose-resistant hemagglutinin AFA-I, which recognizes a human erythrocyte site distinct from the alpha-digalactoside glycosphingolipid receptor common to uropathogenic E . coli strains specifying a P adhesin . A 6.7-kilobase chromosomal DNA fragment was cloned from KS52 into pBR322 and was shown to be necessary for host cell mannose-resistant hemagglutination expression and uroepithelial cell adherence (Labigne-Roussel et al., Infect . Immun . 46:251-259, 1984) . The genetic organization of the 6.7-kilobase DNA fragment was investigated by generating derivative plasmids, and the polypeptides encoded by those plasmids in isolated minicells were analyzed on polyacrylamide gel . The 6.7-kilobase insert expresses five polypeptides of molecular mass 13,000, 16,000, 18,500, 30,000, and 100,000 daltons encoded, respectively, by the afaA, afaE, afaD, afaB, and afaC genes . The five genes were localized and were shown to belong to the same transcriptional unit . The afaB, afaC, and afaE gene products are required for mannose-resistant hemagglutination expression, whereas mutations in or deletions of either afaA or afaD do not modify host mannose-resistant hemagglutination expression . The afaE gene was identified as the structural gene encoding hemagglutinin . The gene has been sequenced; it encodes a 152-residue protein containing a typical 21-residue procaryote signal sequence and a 131-residue mature polypeptide, the AFA-I adhesin.

J Bacteriol, 1985 Jun, 162(3), 1075 - 8
Expression of Actinomyces viscosus antigens in Escherichia coli: cloning of a structural gene (fimA) for type 2 fimbriae; Donkersloot JA et al.; A cosmid gene library of Actinomyces viscosus T14V was prepared in Escherichia coli to examine the expression of A . viscosus antigens and to gain insight into the structure of A . viscosus type 1 and type 2 fimbriae . Out of this library of 550 clones, 28 reacted in a colony immunoassay with antibodies against A . viscosus cells . The proteins responsible for these reactions were identified in three clones . Clones AV1209 and AV2009 displayed nonfimbrial antigens with subunits of 40 and 58 kilodaltons, respectively . Clone AV1402 showed a 59-kilodalton protein that reacted with monospecific antibody against type 2 fimbriae and that comigrated with a subunit of type 2 fimbriae during sodium dodecyl sulfate-polyacrylamide gel electrophoresis . This indicates that AV1402 expresses a gene (fimA) for a subunit of A . viscosus type 2 fimbriae.

Infect Immun, 1985 Jun, 48(3), 824 - 31
Adhesion of enterotoxigenic Escherichia coli to human small intestinal enterocytes; Knutton S et al.; An improved enterocyte adhesion assay has been used to examine a collection of 44 strains of enterotoxigenic Escherichia coli (ETEC) for their ability to adhere to the brush border of isolated human duodenal enterocytes . Fourteen strains showed good adhesion; in each case the ability to adhere correlated with the production of colonization factor antigen I or II (CFA/I or CFA/II) fimbriae . CFA/II-positive producing coli surface antigens 1 and 3 (CS1 and CS3), coli surface antigens 2 and 3 (CS2 and CS3), and only coli surface antigen 3 (CS3) each showed good adhesion . CS3-mediated brush border attachment of CFA/II-positive ETEC was demonstrated by electron microscopy with monospecific antibody and an immunogold labeling technique . One CFA/I-positive ETEC strain was nonadherent in the assay, as were ETEC producing type 1 somatic fimbriae . Five animal ETEC strains producing K88, K99, F41, and 987P fimbriae were slightly more adhesive than control strains, but adhesion was significantly less than that of CFA-positive ETEC . Twenty five human ETEC strains that lacked CFA/I and CFA/II were nonadherent, suggesting either that the surface antigens responsible for adhesion to human intestinal mucosa in these strains were not being produced or that mucosal receptors for these strains are present in regions of the small intestine other than the duodenum.

Proc Natl Acad Sci U S A, 1985 Jun, 82(11), 3799 - 803
Cross-reactive idiotypes and common antigen binding specificities expressed by a series of murine B-cell lymphomas: etiological implications; Pennell CA et al.; A series of 27 B-cell lymphomas (designated the CH series), induced in B10.H-2aH-4b p/Wts mice by intense adoptive immunization with sheep erythrocytes, was found to represent a subset of the total B-cell repertoire . This subset was characterized by expression of a limited number of Ig heavy chain variable regions, as evidenced by the presence of cross-reactive idiotypes and common antigen binding specificities . Twenty-one of the 27 CH lymphomas studied were classified into five groups, defined by a particular cross-reactive idiotype; four of these groups were linked in a single network . Seven of 16 idiotypes defined by absorption analysis were present on lymphomas bearing either kappa or lambda light chains and so were localized to the heavy chain variable region . The surface Ig on 14 CH lymphomas was found to be specific for epitopes on certain erythrocytes (bromelain-treated autologous erythrocytes, sheep, and chicken erythrocytes) or E . coli . We propose that the CH lymphomas represent the malignant counterparts of a subset of idiotypically related, normal B cells in B10.H-2aH-4b p/Wts mice . Perturbation of this idiotype network, by hyperimmunization with an antigen for which some of the members are specific (sheep erythrocytes), increases the risk for neoplasia . Possible mechanisms for this are discussed.

J Bacteriol, 1985 Jun, 162(3), 1039 - 46
Promoter mapping and transcriptional regulation of the iron assimilation system of plasmid ColV-K30 in Escherichia coli K-12; Bindereif A et al.; The promoter of the high-affinity iron assimilation system coded in an approximately 8-kilobase-pair segment of the large Escherichia coli plasmid ColV-K30 was localized to a 0.7-kilobase HindIII-SalI fragment by in vitro runoff transcription . By an S1 nuclease protection assay, with in vitro-transcribed RNA and total in vivo-synthesized RNA, the major start site for transcription was mapped within this fragment and found to be identical in vitro and in vivo . A minor initiation site was located about 50 base pairs upstream from the major site . DNA sequencing of the HindIII-SalI fragment revealed the presence of two promoter-like structures within an extremely AT-rich region with transcriptional initiation sites at 30 and about 80 base pairs upstream from the initiation codon for the first structural gene . Numerous potential secondary structures were found in the DNA sequence around the major promoter . The major transcriptional start site was determined precisely by sequencing the 5' end of in vitro-transcribed RNA . The effect of iron on both the level of specific RNA, as determined by a quantitative S1 nuclease mapping assay, and on beta-galactosidase activity in a iucA'-'lacZ protein fusion, showed that the aerobactin operon is regulated at the transcriptional level . The iron-regulatory sequences are contained within a 152-base-pair Sau3A fragment of the promoter region.

Int J Radiat Biol Relat Stud Phys Chem Med, 1985 Jun, 47(6), 663 - 71
Inhibition of excision repair of DNA in u.v.-irradiated Escherichia coli by phenethyl alcohol; Tachibana A et al.; Membrane-specific drugs such as procaine and chlorpromazine have been shown to inhibit excision repair of DNA in u.v.-irradiated E . coli . One possible mechanism is that, if association of DNA with the cell membrane is essential for excision repair, this process may be susceptible to drugs affecting the structure of cell membranes . We examined the effect of phenethyl alcohol, which is a membrane-specific drug and known to dissociate the DNA-membrane complex, on excision repair of DNA in u.v.-irradiated E . coli cells . The cells were irradiated with u.v . light and then held at 30 degrees C in buffer (liquid-holding) in the presence or absence of phenethyl alcohol . It was found that phenethyl alcohol inhibits the liquid-holding recovery in both wild-type and recA strains, corresponding to its dissociating action on the DNA-membrane complex . Thus, the association of DNA with cell membrane is an important factor for excision repair in E . coli . Procaine did not show the dissociating effect, suggesting that at least two different mechanisms are responsible for the involvement of cell membrane in excision repair of DNA in E . coli.

Infect Immun, 1985 Jun, 48(3), 735 - 40
A completely synthetic toxoid vaccine containing Escherichia coli heat-stable toxin and antigenic determinants of the heat-labile toxin B subunit; Houghten RA et al.; The immunodeterminant regions of the Escherichia coli heat-labile toxin B subunit were identified by determining the antigenicity, by using enzyme-linked immunosorbent assays, of synthetically produced peptides corresponding to various segments of its 124-amino-acid sequence . The addition of the 18-amino-acid sequence of heat-stable toxin (ST) to some of these peptides enhanced their B subunit antigenicity . Peptide residues containing the 26 amino acids of B subunit sequence 58 to 83 joined to the 18-amino-acid sequence of ST yielded a 44-amino-acid peptide whose antigenicity was 50% that of both native B subunit and ST . This peptide was completely nontoxic when tested in Chinese hamster ovary tissue culture, suckling mouse, and rat ligated ileal loop assays . Peroral immunization of rats with the polymeric form of this peptide yielded a dose-dependent response of intestinal immunoglobulin A antitoxin titers to both the ST and B subunit components and provided strong protection against challenge with viable ST- and heat-labile toxin-producing E . coli strains . The immunogenicity of the synthetic peptide in rats was the same as that of ST and about 50% that of native B subunit . The completely synthetic peptide vaccine has the following advantages over previously described toxoid vaccines that consist of synthetic ST chemically cross-linked to native B subunit derived from bacterial cultures: it is produced by a single synthetic process, it is completely nontoxic, and it is immunogenic for both ST and B subunit.

Genetika, 1985 Jun, 21(6), 902 - 13
{Characteristics of the phenotypic manifestation of hpt and gpt mutations blocking 6-oxypurine utilization and their effect on the expression of catabolite-sensitive genes in the cells of an Escherichia coli K-12 purine auxotroph}; Brikun IA et al.; The Escherichia coli purine auxotrophs with the complete block of utilization of hypoxanthine, guanine and xanthine by means of phosphoribosyltransferases, as a result of the hpt and gpt mutations, have the Rel phenotype . In the purD hpt gpt bacteria, under conditions of amino acid starvation synthesis of RNA continues and accumulation of ppGpp is not found . Upon a study of expression of the deo-operon, uridine phosphorylase and beta-galactosidase genes, data were obtained showing that activity of catabolite sensitive promoters of these genes is inhibited in the purD hpt gpt cells . Inhibition of activity of the catabolite sensitive cytP promoter in the deo-operon seems to be accompanied by an increase in activity of the preceding deoP promoter.

Biochem J, 1985 Jun 1, 228(2), 505 - 12
Respiration-dependent uptake of dihydrostreptomycin by Escherichia coli . Its irreversible nature and lack of evidence for a uniport process; Nichols WW et al.; The transport of {3H}dihydrostreptomycin into the cytoplasm of Escherichia coli was distinguished, by its respiration-dependent nature, from binding within the cell envelope . 1 . Of the radiolabel in the cytoplasm, 70-90% was dissolved in, or quickly equilibrated with, the cytoplasmic aqueous phase because this proportion rapidly left cells treated with toluene or with butan-1-ol . 2 . After a period of respiration-dependent uptake of {3H}dihydrostreptomycin, cells were washed repeatedly by centrifugation and resuspension . Radiolabel did not leave the cells at any appreciable rate . 3 . Uptake of dihydrostreptomycin (at an exogenous concentration of 1 mg of base/ml) was monitored for 2h to an apparent equilibrium . Then the specific radioactivity of exogenous dihydrostreptomycin was raised without significantly altering its chemical concentration . There was no exchange of radiolabel between the exogenous pool and the cytoplasmic pool . 4 . Dihydrostreptomycin was not taken up by respiring, cytoplasm-free membrane vesicles which accumulated L-proline in control experiments . These data support the view that respiration-dependent uptake of dihydrostreptomycin by E . coli is not simply a secondary translocation process such as uniport.

Anal Biochem, 1985 Jun, 147(2), 296 - 300
Analysis of RNA structure by ultraviolet crosslinking and denaturation gel electrophoresis; Quarless SA et al.; Electrophoresis in polyacrylamide gels containing both formamide and urea is a high-resolution technique for the analysis of crosslinked RNA species . Combined with a specific crosslinking agent like uv irradiation, it allows a rapid fingerprint of structural differences between RNA forms . The technique reveals significant differences in the pattern of uv crosslinking of free Escherichia coli 16 S ribosomal RNA compared with the RNA in active or inactive 30 S subunits . Ultraviolet photocrosslinks seen only in the 30 S particle are likely to be tertiary structure contacts.

Arch Biochem Biophys, 1985 Jun, 239(2), 427 - 35
Ribosomal activity of the 16 S.23 S RNA complex; Burma DP et al.; It has been demonstrated in this laboratory that 16 S and 23 S RNAs form a binary complex like 30 S and 50 S ribosomes under certain specific conditions, and 5 S RNA can be incorporated into the complex in stoichiometric amounts in presence of three ribosomal proteins, L5, L18, and L15/25 . These studies raised the basic question of whether such complex will have biological activity . Therefore, the following steps in protein synthesis were examined with the complex in place of the ribosomes: (i) poly-U-dependent binding of phenylalanyl tRNA; (ii) EF-G-dependent GTPase activity; (iii) initiation complex formation; (iv) peptidyl transferase activity; and (v) poly-U-dependent polyphenylalanine synthesis . All the steps could be unequivocally demonstrated by the addition of a limited number of proteins although the complex had comparatively much less activity than 70 S ribosomes . It appears that rRNAs are directly involved in various steps of protein synthesis . Furthermore, the 16 S.23 S RNA complex might have acted as a primitive ribosome, as suggested by Crick and Orgel.

Proc Natl Acad Sci U S A, 1985 Jun, 82(12), 3954 - 8
Initiation of enzymatic replication at the origin of the Escherichia coli chromosome: primase as the sole priming enzyme; van der Ende A et al.; The enzymatic replication of plasmids containing the unique (245 base pair) origin of the Escherichia coli chromosome (oriC) can be initiated with any of three enzyme priming systems: primase alone, RNA polymerase alone, or both combined (Ogawa, T., Baker, T . A., van der Ende, A . & Kornberg, A . (1985) Proc . Natl . Acad . Sci . USA 82, 3562-3566) . At certain levels of auxiliary proteins (topoisomerase I, protein HU, and RNase H), the solo primase system is efficient and responsible for priming synthesis of all DNA strands . Replication of oriC plasmids is here separated into four stages: (i) formation of an isolable, prepriming complex requiring oriC, dnaA protein, dnaB protein, dnaC protein, gyrase, single-strand binding protein, and ATP; (ii) formation of a primed template by primase; (iii) rapid, semiconservative replication by DNA polymerase III holoenzyme; and (iv) conversion of nearly completed daughter molecules to larger DNA forms . Optimal initiation of the leading strand of DNA synthesis, over a range of levels of auxiliary proteins, appears to depend on transcriptional activation of the oriC region by RNA polymerase prior to priming by primase.

Mech Ageing Dev, 1985 May 31, 30(3), 285 - 97
Programmed aging or error catastrophe? An examination by two-dimensional polyacrylamide gel electrophoresis; Johnson TE et al.; We have examined newly synthesized proteins in the young adult and in older populations of the nematode Caenorhabditis elegans using two-dimensional polyacrylamide gel electrophoresis (2D PAGE) . A temperature-sensitive mutant strain, DH26, with a mean life span of about 15 days, under our conditions, was used to block progeny development . Nematodes of several different ages were pulse-labeled for 5 h, in vivo, with 35S-labeled E . coli, A subsequent 30-min chase with unlabeled E . coli served to rid the worms of endogenous labeled E . coli proteins . We resolve 700 or more proteins by 2D PAGE polyacrylamide gel electrophoresis of extracts of young nematodes . The patterns of these proteins are highly reproducible in comparisons of independent repeats of identical experiments . No new major proteins are synthesized at any time during the adult phase (4-22 days) nor are any of the most abundant proteins not made during this period . At our level of detectability (estimated as a satellite spot containing 4% of the amount of label in a major spot) we see no misincorporation of radioactive amino acids into newly synthesized proteins . These data are inconsistent with predictions by any one of several, so called, "error catastrophe" models of senescence and also show that modulation of the highest abundancy classes of proteins are also not involved in senescence.

Science, 1985 May 31, 228(4703), 1096 - 9
In vivo function and membrane binding properties are correlated for Escherichia coli lamB signal peptides; Briggs MS et al.; Wild-type and pseudorevertant signal peptides of the lamB gene product of Escherichia coli interact with lipid systems whereas a nonfunctional deletion mutant signal peptide does not . This conclusion is based on interaction of synthetic signal peptides with a lipid monolayer-water surface, conformational changes induced by presence of lipid vesicles in an aqueous solution of signal peptide, and capacities of the peptides to promote vesicle aggregation . Analysis of the signal sequences and previous conformational studies suggest that these lipid interaction properties may be attributable to the tendency of the functional signal peptides to adopt alpha-helical conformations . Although the possibility of direct interaction between the signal peptide and membrane lipids during protein secretion is controversial, the results suggest that conformationally related amphiphilicity and consequent membrane affinity of signal sequences are important for function in vivo.

Biochim Biophys Acta, 1985 May 31, 807(3), 238 - 44
Inhibition of Escherichia coli H+-ATPase by venturicidin, oligomycin and ossamycin; Perlin DS et al.; The antibiotics venturicidin, oligomycin and ossamycin were investigated as potential inhibitors of the Escherichia coli H+-ATPase . It was found that venturicidin strongly inhibited ATP-driven proton transport and ATP hydrolysis, while oligomycin weakly inhibited these functions . Inhibition of the H+-ATPase by venturicidin and oligomycin was correlated with inhibition of F0-mediate proton transport . Both inhibitors were found to interfere with the covalent reaction between dicyclohexyl{14C}carbodiimide and the F0 subunit c (uncE protein) . Ossamycin had no direct inhibitory effect on E . coli F0 or F1; rather, it was found to uncouple ATP hydrolysis from proton transport.

Biochim Biophys Acta, 1985 May 29, 840(1), 29 - 36
Further purification, characterization and salt activation of acyl-CoA synthetase from Escherichia coli; Kameda K et al.; Acyl-CoA synthetase was further purified from Escherichia coli in good yield and fold purification by affinity chromatography on CoA-Sepharose 4B . The molecular weight of the active form of the purified enzyme was estimated as 45 000 by Sephadex G-100 and 47 000 by Sephadex G-200 . Sedimentation equilibrium ultracentrifugation analysis revealed a molecular weight of 50 000 . The sedimentation coefficient was calculated as 4.4 S . An absorption maximum at 276 nm was observed in the ultraviolet light absorption spectrum . The molar extinction coefficient was 9.2 X 10(4) . Kinetic constants were determined for trans fatty acids . All ions tested, including chaotropic and lyotropic ions, stimulated or inhibited acyl-CoA synthetase activity depending on their concentrations in the assay system . In a series of chaotropes, the lower concentration required to maximally activate acyl-CoA synthetase in increasing order of potency of chaotropic ions . The inhibitory effect of chaotrope on the enzyme activity was reversible . These data suggest that salts have a common mode of action and influence acyl-CoA synthetase activity primarily through their effect on the solution structure.

J Biol Chem, 1985 May 25, 260(10), 5891 - 4
Inhibition of prolipoprotein signal peptidase by globomycin; Dev IK et al.; Globomycin inhibits the prolipoprotein-specific signal peptidase activity by binding to the enzyme in a noncompetitive manner (Ki = 36 nM) . The Km of prolipoprotein signal peptidase for the prolipoprotein substrate is 6 (+/- 1) microM.

J Biol Chem, 1985 May 25, 260(10), 6329 - 33
Nucleotide sequence of cloned cDNA specific for rat ribosomal protein S11; Tanaka T et al.; A cDNA clone specific for rat ribosomal protein S11 was isolated by hybrid-selected translation from the cDNA library made for 8-9 S poly(A) RNA from regenerating rat liver . Since this cDNA had not enough length, another clone was selected by colony hybridization using a fragment of isolated cDNA as a probe . The nucleotide sequence of the cDNA was determined . The sequence contains 2 base pairs from the 5' noncoding region, the entire coding region of 477 base pairs, and the 3' noncoding region of 55 base pairs besides the poly(A) tail . The primary structure of the protein S11 was deduced from the nucleotide sequence . It consists of 157 amino acids . Its molecular weight is 18,299 . The calculated amino acid composition is consistent with the reported composition of S11 determined on the protein hydrolysate . The amino acid sequence showed a marked homology with that of S16 of Halobacterium cutirubrum and an appreciable homology with that of S17 of Escherichia coli.

J Mol Biol, 1985 May 25, 183(2), 239 - 46
Essential interaction between lambdoid phage 21 terminase and the Escherichia coli integrative host factor; Feiss M et al.; Lambdoid phage 21 requires the Escherichia coli integrative host factor (IHF) for growth . lambda-21 hybrids that have 21 DNA packaging specificity also require IHF . IHF-independent (her) mutants have been isolated . her mutations map in the amino-terminal half of the 21 1 gene . The 1 gene encodes the small subunit of the 21 terminase, and the amino-terminal half of the 1 polypeptide is a functional domain for specifically binding 21 DNA . Hence changes in the DNA-binding domain of terminase, her mutations, render 21 terminase able to function in the absence of IHF . Three of four her mutations studied are trans-dominant . An in vitro system was used to show that packaging of 21 DNA is IHF-dependent . IHF is directly required during the early, terminase-dependent steps of assembly . It is concluded that IHF is a host factor required for function of the 21 terminase . It is proposed, in analogy to the role of IHF in lambda integration, that IHF facilitates proper binding of 21 terminase to phage DNA . Consistent with this proposal, possible IHF-binding sites are present in the 21 cohesive end site.

J Mol Biol, 1985 May 25, 183(2), 165 - 77
Interaction of RNA polymerase with lacUV5 promoter DNA during mRNA initiation and elongation . Footprinting, methylation, and rifampicin-sensitivity changes accompanying transcription initiation; Carpousis AJ et al.; We have used enzymatic and chemical probes to follow the movement of Escherichia coli RNA polymerase along lacUV5 promoter DNA during transcription initiation . The RNA polymerase does not escape from the promoter but remains tightly bound during the synthesis of the initial bases of the transcript . This initial phase of RNA synthesis involves the reiterative synthesis and release of RNA chains up to ten bases long via the RNA polymerase cycling reaction and the enzyme remains sensitive to rifampicin inhibition . When longer chains are made, promoter-specific binding is disrupted and the enzyme forms a rifampicin-resistant elongation complex with downstream DNA sequences . This elongation complex covers less than half as much DNA and lacks the DNase I-hypersensitive sites and the base-specific contacts that characterize promoter-bound RNA polymerase . These results lead us to suggest that lacUV5 mRNA synthesis is primed by a promoter-bound enzyme complex that synthesizes the initial nine or ten bases in the mRNA chain . Subsequently, when a chain of ten bases, or slightly longer, is made, contacts with promoter DNA are irreversibly disrupted, sigma subunit is lost, and a "true" elongation complex is formed.

Nucleic Acids Res, 1985 May 24, 13(10), 3581 - 97
rRNA processing: removal of only nineteen bases at the gap between 28S alpha and 28S beta rRNAs in Sciara coprophila; Ware VC et al.; We have determined the sequence of the rDNA region between the 28S alpha and 28S beta rRNA coding segments (termed a "gap") in the insect Sciara coprophila, and have used S1 nuclease mapping and cDNA primer extension to define the 5' and 3' boundaries of the gap . Only 19 bases found in rDNA at the gap region are absent from mature 28S rRNA . Eukaryotic rRNAs contain stretches of nucleotides ("expansion segments") which are absent in E . coli rRNA . The gap region in Sciara is located within expansion segment V . Therefore, the excision of 19 bases in the Sciara gap suggests that a large portion of expansion segment V plays no function in mature ribosomes . Specific sequences conserved in Sciara and Drosophila are considered as candidates for recognition signals for the excision of the gap transcript.

Biochim Biophys Acta, 1985 May 24, 825(1), 12 - 20
Specific incorporation of 5-fluorocytidine into Escherichia coli RNA; Kaiser II et al.; RNAs isolated from Escherichia coli B grown in the presence of 5-fluorouracil have high levels of the analog replacing uridine and uridine-derived modified nucleosides . Cytidine has also been shown to be replaced in these RNAs by 5-fluorocytidine, a metabolic product of 5-fluorouracil, but to a considerably lesser extent . When 5-fluorocytidine is added to cultured of E . coli B little 5-fluorocytidine (0.20 mol%) is incorporated into cellular RNAs because of the active cytosine/cytidine deaminase activities . Addition of the cytidine deaminase inhibitor tetrahydrouridine (70 micrograms/ml) increases 5-fluorocytidine incorporation to about 3 mol% in tRNAs, but does not eliminate 5-fluorouridine incorporation . E . coli mutants lacking cytosine/cytidine deaminase activities are able to more than double the extent of 5-fluorocytidine incorporation into their transfer and ribosomal RNAs, replacing cytidine with no detectable 5-fluorouridine incorporation . Levels of 5-methyluridine, pseudouridine and dihydrouridine in tRNAs are not affected . These fluorocytidine-containing tRNAs show amino acid-accepting activities similar to control tRNAs . Fluorocytidine was found to be quite susceptible to deamination under alkaline conditions . Its conversion to primarily 5-fluorouridine follows pseudo-first-order reaction kinetics with a half-life of 10 h in 0.3 M KOH at 37 degrees C . This instability in alkali probably explains why 5-fluorocytidine was not found earlier in RNAs isolated from cells treated with 5-fluorouridine, since most early RNA hydrolyses were carried out in alkali . It may also explain the mild mutagenic properties observed in some systems following 5-fluorouridine treatment . Initial 19F-NMR measurements in fluorocytidine-containing tRNAs indicate that this modified tRNA may be useful in future structural studies of tRNAs and in probing tRNA-protein complexes.

Biochim Biophys Acta, 1985 May 24, 825(1), 1 - 11
Expression of srnB gene of F plasmid by altered RNA polymerase in Escherichia coli; Ito R et al.; Degradation of otherwise stable rRNA and tRNA takes place in the presence of rifampin, dependent on the F plasmid srnB gene . We have reported that a protein newly synthesized in the presence of rifampin might be a product of the srnB gene required for stable RNA degradation (Ito, R . and Ohnishi, Y . (1983) Biochim . Biophys . Acta 739, 27-34) . Here we have further studied the mechanism of srnB expression . Among eighteen mutants with altered RNA polymerase, two (TJ2470 (rpoC4) and TJ302 (rpoC56)) showed RNA degradation at high temperature (42 degrees C) when the srnB gene was present . Labeling proteins at 42 degrees C in strain TJ2470 indicated that a protein of molecular weight 12 000 was a product of the srnB gene, and that expression of the srnB gene provoked RNA degradation . Using plasmid pTK4, in which the srnB gene is inserted downstream of the promoter of lacZ, lac promoter-dependent expression of the srnB gene, with production of the putative protein product, also induced RNA degradation at 42 degrees C, with no requirement for added rifampin or altered RNA polymerase . RNA degradation in these conditions was quite similar to that in the case of the addition of rifampin; e.g., it showed some responses to Mg2+, temperature and RNAase I content of the cells . Expression of the srnB gene dependent on lac promoter was also observed in minicells . Thus, it is inferred that the srnB gene is probably repressed under normal conditions with its own promoter; its expression initiates RNA turnover.

J Immunol Methods, 1985 May 23, 79(2), 263 - 75
The influence of murine macrophage-conditioned medium on cloning efficiency, antibody synthesis, and growth rate of hybridomas; Sugasawara RJ et al.; Murine B-cell hybridomas made with the P3X63-AG8.653 myeloma showed increases in cloning efficiency and efficiency of growth in hypoxanthine-aminopterin-thymidine (HAT) medium of 50-100-fold in the presence of medium conditioned by primary mouse peritoneal macrophages (MCM) . Similar effects were elicited by MCM from 3 continuous macrophage lines . The J774A.1 line conditioned the medium as efficiently as primary macrophages without induction . Conditioning by the P388D1 line was several-fold less efficient, but could be increased by treating the cells with Escherichia coli lipopolysaccharide . By contrast, the BJ-1 macrophage line required treatment with the lipopolysaccharide to induce expression of the hybridoma growth factor(s) . Four commercially available serum supplements could not substitute for MCM, but addition of MCM and the supplements together stimulated the growth rate of hybridomas in media with 4% or less fetal bovine serum . The rate of antibody synthesis paralleled the growth rate, and the amount of antibody synthesized per cell was approximately the same for hybridomas grown in medium supplemented with MCM or adapted to growth in the absence of MCM . The results indicate that MCM has advantages as an alternative to 'feeder cells' and serum supplements in hybridoma cultures, and suggest that MCM may be useful for hybridoma culture at reduced serum concentrations . The nature of the soluble factor(s) in MCM which promote these effects remains unknown.

Biochemistry, 1985 May 21, 24(11), 2812 - 8
Large-scale purification, oligomerization equilibria, and specific interaction of the LexA repressor of Escherichia coli; Schnarr M et al.; A rapid large-scale procedure for the purification of the LexA repressor of Escherichia coli is described . This procedure allows one to get more than 100 mg of purified protein from 100 g of bacterial paste with a purity of at least 97% . This method is comparable to earlier, far more complicated purification procedures giving clearly smaller yields . It is shown that the LexA protein may be identified spectroscopically by a large A235/A280 ratio and very pronounced ripples in the absorption spectrum arising from a high amount of phenylalanine residues with respect to that of the other aromatic amino acids . Polyacrylamide gel electrophoresis has been used to study the specific interaction of LexA with a recA operator fragment . The quaternary structure of LexA has been studied by equilibrium ultracentrifugation and sedimentation velocity measurements . The sedimentation coefficient increases with increasing LexA concentration, indicating that LexA is involved in self-association . This finding has been confirmed by equilibrium ultracentrifugation . The results are best described by a monomer-dimer and a subsequent dimer-tetramer equilibrium, with an association constant of 2.1 X 10(4) M-1 for the dimer and 7.7 X 10(4) M-1 for the tetramer formation . These relatively small association constants determined under near-physiological pH and salt conditions suggest that in vivo LexA should be essentially in the monomeric state . The degree to which LexA decreases the electrophoretic mobility of a 175 base pair fragment harboring the recA operator suggests that the recA operator interacts nevertheless with a LexA dimer . However, our results may be also explained by the binding of a LexA monomer with a simultaneous bending of the DNA fragment.

Biochemistry, 1985 May 21, 24(11), 2723 - 31
Changes in the DNA structure of the lac UV5 promoter during formation of an open complex with Escherichia coli RNA polymerase; Spassky A et al.; By chemical and enzymatic methods, two stable complexes between Escherichia coli RNA polymerase and a linear DNA fragment carrying the lac UV5 promoter have been identified . In these binary complexes, DNA can adopt two alternate conformations as a function of temperature . Contacts between RNA polymerase and the DNA phosphate backbone are indistinguishable in these two forms, as revealed by probing with pancreatic DNase I . Protection of enhancement of the reactivity of the bases toward (CH3)2SO4 occurs, however, only in the form that predominates above 22 degrees C, RPo . The form stable at low temperature, RPi, is a "closed" complex since no single-stranded region is detectable in the DNA . The strong temperature dependence of the equilibrium constant, the midpoint value of the transition, and the rate of conversion between these two forms are in close agreement with a series of measurements performed by using a transcriptional assay and reported in the preceding paper {Buc, H., & McClure, W . R . (1985) Biochemistry (preceding paper in this issue)} . These data further support the postulated mechanism of open complex formation involving three sequential steps: R + P in equilibrium RPc in equilibrium RPi in equilibrium RPo . The binary complex RPc, which accumulates transiently at 37 degrees C before the isomerization leading to open complex formation, is not significantly protected against enzymatic cleavage or chemical modification and is therefore distinct from RPi and RPo.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1985 May 21, 24(11), 2712 - 23
Kinetics of open complex formation between Escherichia coli RNA polymerase and the lac UV5 promoter . Evidence for a sequential mechanism involving three steps; Buc H et al.; The forward and reverse kinetics of open complex formation between Escherichia coli RNA polymerase and the lac UV5 promoter have been studied in the temperature range of 15-42 degrees C . The standard two-step model, involving the formation of a closed intermediate, RPc, followed by an isomerization that leads to the active complex RPo, could not account for the present data . The promoter-enzyme lifetime measurements showed an inverse temperature dependence (apparent activation energy, -35 kcal/mol) . A third step, which is very temperature dependent and which is very rapid at 37 degrees C, was postulated to involve the unstacking of DNA base pairs that immediately precedes open complex formation . Evidence for incorporating a new binary complex, RPi, in the pathway was provided by experiments that distinguished between stably bound species and active promoter after temperature-jump perturbations . These experiments allowed measurement of the rate of reequilibration between the stably bound species and determination of the corresponding equilibrium constant . They indicated that the third step became rate limiting below 20 degrees C; this prediction was checked by an analysis of the forward kinetics . A quantitative evaluation of the parameters involved in this three-step model is provided . Similar experiments were performed on a negatively supercoiled template: in this case the third equilibrium was driven toward formation of the open complex even at low temperature, and the corresponding step was not rate limiting.

Biochim Biophys Acta, 1985 May 20, 829(1), 83 - 96
A spectroscopic investigation of the structure and redox properties of Escherichia coli cytochrome b-562; Moore GR et al.; The six-coordinate monohaem ferricytochrome b-562 from Escherichia coli exhibits two haem-linked pH-dependent transitions detected by NMR and optical spectroscopy . Only one of these transitions, that of the Fe(III)-coordinated His-102, is detected by EPR and MCD; the ionisation of a haem propionate is not . Both ionisations are redox-state-dependent and the midpoint redox potential of the protein is markedly pH-dependent . Over the pH range 5.0 to 8.5 the potential drops from 260 mV to 110 mV and at least five single proton ionisations are responsible for this . In addition to the two spectroscopically identified ferricytochrome ionisations, there are at least three unidentified ionisations, two of which occur in the ferrous protein . From a consideration of the X-ray structure, together with NMR data, it seems probable that at least one of these ionisations involves an amino acid carboxylate . The X-ray structure also suggests that the relatively low pKa of His-102 is a result of its proximity to Arg-98 . However, an appreciable interaction between these groups requires that the solution conformation differs slightly from the X-ray structure . The fast rate of electron self-exchange, over 4 X 10(6) M-1 X s-1 at 315 K and pH* 7, may be a reflection of the fact that, as shown by the X-ray structure, a large amount of the haem and axial histidine ligand are exposed at the molecular surface with an asymmetric distribution of charged groups surrounding them.

Experientia, 1985 May 15, 41(5), 676 - 7
Mutagenicity and toxicity of chloroethylene oxide and chloroacetaldehyde; Perrard MH; Exposure of several trp-auxotrophic Escherichia coli strains, carrying base-pair substitutions, to chloroethylene oxide or chloroacetaldehyde (two metabolites of vinyl chloride) increased the mutation frequency to tryptophan prototrophy . Strong cytotoxic and mutagenic effects were observed with 2.5 mM chloroethylene oxide, while a higher concentration of chloroacetaldehyde (100 mM) exhibited a mutagenic effect which was 400 times lower.

Experientia, 1985 May 15, 41(5), 643 - 4
Effect of phosphonic analogues of glutamic acid on glutamate decarboxylase; Lacoste AM et al.; Among the phosphonic analogues of glutamic acid, only 4-amino-4-phosphono butyric acid, the compound which shows the highest affinity for pyridoxal phosphate, inhibits competitively both Escherichia coli and rat brain glutamate decarboxylases . Phosphinothricin, 2-amino-4-(methylphosphino)butyric acid, is a strong inhibitor of the mammalian enzyme.

Eur J Biochem, 1985 May 15, 149(1), 129 - 33
Functional role of cysteinyl residues in tryptophanase; Nihira T et al.; Holotryptophanase inactivated by oxidation of cysteinyl residues showed a different absorption spectrum from the native enzyme . At pH 8.0, the native enzyme preferentially existed as a 337-nm species (active form), whereas in the inactive enzyme a 420-nm species (inactive form) was dominant . During the reactivation of the enzyme by reduction with dithiothreitol, an increase at 337 nm and a decrease at 420 nm were observed with concomitant increase in enzymatic activity, which was accompanied by the appearance of two cysteinyl residues per monomer . Specific S-cyanylation of cysteinyl residues by nitrothiocyanobenzoic-acid-inactivated apotryptophanase with the modification of one cysteinyl residue per monomer, whereas holotryptophanase was highly resistant to inactivation with nitrothiocyanobenzoic acid . The essential role of the active-site-bound pyridoxal 5'-phosphate in protection against inactivation was confirmed by the agreement of the K1/2 (protection) of 5.0 microM for pyridoxal 5'-phosphate with Km of 2.0 microM in enzyme catalysis . The inactivation by nitrothiocyanobenzoic acid caused a similar shift in the equilibrium between the 337-nm species and 420-nm species, i.e . decrease of the 337-nm species and increase of the 420-nm species . From the pH dependence of the equilibrium between these two species, pKa of 7.9 and 7.4 was obtained for the inactive and the dithiothreitol-activated enzyme, respectively, indicating that cysteinyl residue(s) participated in lowering the pKa of the interconversion between the 337-nm species (active form) and 420-nm species (inactive form) . The possible role of cysteinyl residues in the function of tryptophanase is discussed.

Eur J Biochem, 1985 May 15, 149(1), 95 - 9
The primary structure of the iron-sulfur subunit of ubiquinol-cytochrome c reductase from Neurospora, determined by cDNA and gene sequencing; Harnisch U et al.; The primary structure of the iron-sulfur subunit of ubiquinol-cytochrome c reductase from Neurospora mitochondria was determined by cDNA and genomic DNA sequencing . A first cDNA was identified from a cDNA bank cloned in Escherichia coli by hybridization selection of mRNA, cell-free protein synthesis and immunoadsorption . Further cDNA and geonomic DNA were identified by colony filter hybridization . The N-terminal sequence of the mature protein was determined by automated Edman degradation . From the sequence a molecular mass of 24749 Da results for the precursor protein and of 21556 Da for the mature protein . The presequence consists of 32 amino acids with four arginines as the only charged residues . The mature protein consists of 199 amino acids . It is characterized by a small N-terminal hydrophilic part of 29 residues, a hydrophobic stretch of 25 residues and a large C-terminal hydrophilic domain of 145 residues . The only four cysteines of the protein, which are assumed to bind the 2 Fe-2S cluster, are located in a moderate hydrophobic region of this large domain . Cysteines 3 and 4 are unusually arranged in that they are separated by only one proline . From sequence data the arrangement of the subunit in the membrane is deduced.

Eur J Biochem, 1985 May 15, 149(1), 113 - 8
Maturation of the 3' end of 5-S ribosomal RNA from Escherichia coli; Szeberenyi J et al.; The 3' ends of 5-S rRNA isolated from Escherichia coli cells were analyzed and identified after different durations of labeling with 32Pi, with and without blocking of protein synthesis . These experiments suggest that the 5-S rRNA starts as a species containing 126 nucleotides, three at each end, and that the extra nucleotides are removed from the 5' and 3' ends in parallel at comparable but different rates . Inhibition of protein synthesis with chloramphenicol blocks, in addition to the 5'-end maturation, the trimming of the extra nucleotides from the 3' end . The trimming of extra nucleotides from both ends of the 5-S rRNA is also affected by the structure of the molecular stalk of 5-S rRNA . A number of observations suggest that the trimmings from both ends are independent processes, which are carried out probably by different enzymes.

Vet Rec, 1985 May 11, 116(19), 519 - 21
Clinical and experimental modifications of plasma iron and zinc concentrations in cattle; Depelchin BO et al.; Plasma iron and zinc concentrations were studied in pyrexic cattle or in cattle experimentally infected with infectious bovine rhinotracheitis virus or Escherichia coli endotoxin . Plasma iron and zinc levels tended to decline in the animals given endotoxin and in the pyrexic cattle, but the plasma iron level was only modified after experimental infectious bovine rhinotracheitis infection . These changes were not always related to pyrexia . Plasma iron and zinc concentrations taken together may be used as an indicator of infection.

Nucleic Acids Res, 1985 May 10, 13(9), 3213 - 20
Structure and transcription of the tRNAPro1 gene from Escherichia coli; Kuchino Y et al.; A 5 kbp DNA fragment containing the tRNAPro1 gene from Escherichia coli was cloned into Charon 21A phage and sequenced by the M13 DNA sequencing technique . When the cloned DNA fragment was used as a template for in vitro transcription with E . coli RNA polymerase, a tRNAPro1 precursor of 120 nucleotide residues was obtained . The tRNAPro1 gene transcribed as a single transcription unit was followed by two unusual repeating sequences, both of 108 bp . These two repeating sequences were separated by a 60 bp spacer sequence . The 5'-portion of each repeating sequence overlapped 19 bp of the 3'-terminal region of the tRNAPro1 gene just like the repeating sequences in the E . coli tRNATyr1 gene . A rho-independent termination was present in the first repeating unit.

J Biol Chem, 1985 May 10, 260(9), 5683 - 90
Amplified expression of streptomyces endo-beta-N-acetylglucosaminidase H in Escherichia coli and characterization of the enzyme product; Trumbly RJ et al.; The endo-beta-N-acetylglucosaminidase H (Endo H) gene from Streptomyces plicatus has been cloned into the Escherichia coli plasmid pKC30 (Shimatake, H., and Rosenberg, M . (1981) Nature 272, 128-132), thus placing expression of this gene under control of the strong lambda promoter pL . The construction, pKCE3, which includes a properly positioned E . coli ribosome binding site from the lac operon (Robbins, P.W., Trimble, R . B., Wirth, D.F., Hering, C., Maley, F . Maley, G . F., Das, R., Gibson, B.W., and Biemann, K . (1984) J . Biol . Chem . 259, 7577-7583), was used to transform an E . coli strain lysogenic for a lambda prophage containing a temperature-sensitive repressor . By shifting cultures of pKCE3 lysogens to 42 degrees C, the production of Endo H commenced and was linear for about 1 h . Enzyme yields were amplified 150-fold above those obtained from comparable cultures of S . plicatus and represented 3 to 4% of total cellular protein, which enabled purification of Endo H to homogeneity by a rapid fourstep procedure . Although most of the cloned Endo H was secreted into the periplasmic space by E . coli, its 4 kDa leader sequence peptide (Robbins et al . (1984} was only partially removed during processing . As a result the purified pKCE3 Endo H was a heterogeneous population of molecules with an average molecular mass of 31 kDa compared to the 28.9 kDa fully processed product normally secreted by S . plicatus . Despite the residual approximately 2 kDa of leader sequence on the cloned pKCE3 product, there were no detectable differences in either the substrate specificity or the stability characteristics of the enzyme purified from E . coli or from S . plicatus . Of particular value for studies on glycoproteins was the finding that the genetically engineered Endo H was completely free of proteolytic contaminants.

Nucleic Acids Res, 1985 May 10, 13(9), 3305 - 16
Oligonucleotide-directed mutagenesis by microscale 'shot-gun' gene synthesis; Grundstrom T et al.; We describe a rapid and efficient microscale method for in vitro site-directed mutagenesis by gene synthesis . Mutants are constructed by "shot-gun ligation" of overlapping synthetic oligonucleotides yielding double stranded synthetic DNA of more than 120 nucleotides in length . The terminal oligonucleotides of the DNA segment to be synthesized are designed to create sticky ends complementary to unique restriction sites of a polylinker present in an M13 vector . The oligonucleotides are hybridized and ligated to the M13 vector without any purification of the synthetic DNA segment . After cloning, about half of the progeny from such shot-gun ligations contained the predicted sequence demonstrating the efficacy of this method for gene synthesis and its potential for the extensive mutational analysis of genes.

J Biol Chem, 1985 May 10, 260(9), 5498 - 504
Bovine interferon alpha genes . Structure and expression; Velan B et al.; The bovine genome contains a gene family of interferon-alpha s (bIFN-alpha) that consists of at least five distinct members . Four of the bIFN-alpha genes isolated show a high degree of homology (97% in the nucleotide sequence and 93% in amino acid sequence) . The overall homology in amino acid sequence of bIFN-alpha to human, murine, and rat IFNs-alpha is approximately 60% . Yet there are amino acid clusters (positions 28-41 and 118-146) which are highly conserved throughout the mammalian evolution and in which the overall homology can be as high as 86% . Within the C terminus conserved cluster there is a sequence containing 9 amino acids completely conserved in 16 mammalian IFNs-alpha and of these, 7 are also shared with a similar domain in some bacterial toxins, implying a common functional role for these domains . One of the genes, IFN-alpha C, was expressed in Escherichia coli . The purified bacterial IFN (specific activity, 2 X 10(8) units/mg) exhibited antiviral activity on bovine cells but no detectable activity was demonstrated on human and simian cells.

J Biol Chem, 1985 May 10, 260(9), 5480 - 5
Evidence for an essential arginine residue in the active site of Escherichia coli 2-keto-4-hydroxyglutarate aldolase . Modification with 1,2-cyclohexanedione; Vlahos CJ et al.; Treatment of homogeneous preparations of Escherichia coli 2-keto-4-hydroxyglutarate aldolase with 1,2-cyclohexanedione, 2,3-butanedione, phenylglyoxal, or 2,4-pentanedione results in a time- and concentration-dependent loss of enzymatic activity; the kinetics of inactivation are pseudo-first order . Cyclohexanedione is the most effective modifier; a plot of log (1000/t 1/2) versus log {cyclohexanedione} gives a straight line with slope = 1.1, indicating that one molecule of modifier reacts with each active unit of enzyme . The kinetics of inactivation are first order with respect to cyclohexanedione, suggesting that the loss of activity is due to modification of 1 arginine residue/subunit . Controls establish that this inactivation is not due to modifier-induced dissociation or photoinduced structural alteration of the aldolase . The same Km but decreased Vmax values are obtained when partially inactivated enzyme is compared with native . Amino acid analyses of 95% inactivated aldolase show the loss of 1 arginine/subunit with no significant change in other amino acid residues . Considerable protection against inactivation is provided by the substrates 2-keto-4-hydroxyglutarate and pyruvate (75 and 50%, respectively) and to a lesser extent (40 and 35%, respectively) by analogs like 2-keto-4-hydroxybutyrate and 2-keto-3-deoxyarabonate . In contrast, formaldehyde or glycolaldehyde (analogs of glyoxylate) under similar conditions show no protective effect . These results indicate that an arginine residue is required for E . coli 2-keto-4-hydroxyglutarate aldolase activity; it most likely participates in the active site of the enzyme by interacting with the carboxylate anion of the pyruvate-forming moiety of 2-keto-4-hydroxyglutarate.

J Biol Chem, 1985 May 10, 260(9), 5366 - 9
Substrate-induced hysteresis in the activity of Escherichia coli dihydrofolate reductase; Penner MH et al.; Full time course studies of the kinetic activity of Escherichia coli dihydrofolate reductase show that there is an increase in activity with time . The half-time for this hysteretic behavior is about 9 s . Preincubation of the enzyme with either of the substrates abolishes the lag and results in initial velocities which are 2-2.3-fold faster than those observed for the non-preincubated enzyme . The kinetic properties of the activated and nonactivated forms of the enzyme appear to be similar as measured by the full time course of the reaction . The results are consistent with observations for NADPH binding studies that the enzyme exists in two interconvertible forms, one of which is incapable of binding NADPH (Cayley, P . J., Dunn, S . M . J., and King, R . W . (1981) Biochemistry 20, 874-879).

J Biol Chem, 1985 May 10, 260(9), 5621 - 4
Molecular cloning of DNA sequences complementary to rat liver glucose-6-phosphate dehydrogenase mRNA . Nutritional regulation of mRNA levels; Kletzien RF et al.; The nutritional regulation of rat liver glucose-6-phosphate dehydrogenase was studied using a cloned DNA complementary to glucose-6-phosphate dehydrogenase mRNA . The recombinant cDNA clones were isolated from a double-stranded cDNA library constructed from poly(A+) RNA immunoenriched for glucose-6-phosphate dehydrogenase mRNA . Immunoenrichment was accomplished by adsorption of polysomes with antibodies directed against glucose-6-phosphate dehydrogenase in conjunction with protein A-Sepharose and oligo(dT)-cellulose chromatography . Poly(A+) RNA encoding glucose-6-phosphate dehydrogenase was enriched approximately 20,000-fold using these procedures . Double-stranded cDNA was synthesized from the immunoenriched poly(A+) RNA and inserted into pBR322 using poly(dC)-poly(dG) tailing . Escherichia coli MC1061 was transformed, and colonies were screened for glucose-6-phosphate dehydrogenase cDNA sequences by differential colony hybridization . Plasmid DNA was purified from clones which gave positive signals, and the identity of the glucose-6-phosphate dehydrogenase clones was verified by hybrid-selected translation . A collection of glucose-6-phosphate dehydrogenase cDNA plasmids with overlapping restriction maps was obtained . Northern blot analysis of rat liver poly(A+) RNA using nick-translated, 32P-labeled cDNA inserts revealed that the glucose-6-phosphate dehydrogenase mRNA is 2.3 kilobases in length . RNA blot analysis showed that refeeding fasted rats a high carbohydrate diet results in a 13-fold increase in the amount of hybridizable hepatic glucose-6-phosphate dehydrogenase mRNA which parallels the increase in enzyme activity . These results suggest that the nutritional regulation of hepatic glucose-6-phosphate dehydrogenase occurs at a pretranslational level.

J Biol Chem, 1985 May 10, 260(9), 5820 - 5
Rat liver glutathione S-transferases . Construction of a cDNA clone complementary to a Yc mRNA and prediction of the complete amino acid sequence of a Yc subunit; Telakowski-Hopkins CA et al.; Using polysomal immunoselected rat liver glutathione S-transferase mRNAs, we have constructed cDNA clones using DNA polymerase I, RNase H, and Escherichia coli ligase (NAD+)-mediated second strand cDNA synthesis as described by Gubler and Hoffman (Gubler, U., and Hoffman, B . S . (1983) Gene 25, 263-269) . Recombinant clone, pGTB42, contained a cDNA insert of 900 base pairs whose 3' end showed specificity for the Yc mRNA in hybrid-select translation experiments . The nucleotide sequence of pGTB42 has been determined, and the complete amino acid sequence of a Yc subunit has been deduced . The cDNA clone contains an open reading frame of 663 nucleotides encoding a polypeptide comprising 221 amino acids with a molecular weight of 25,322 . The NH2-terminal sequence deduced from pGTB42 is in agreement with the first 39 amino acids determined for a Ya-Yc heterodimer by conventional protein-sequencing techniques . A comparison of the nucleotide sequence of pGTB42 with the sequence of a Ya clone, pGTB38, described previously by our laboratory (Pickett, C . B., Telakowski-Hopkins, C . A., Ding, G . J.-F., Argenbright, L., and Lu, A.Y.H . (1984) J . Biol . Chem . 259, 5182-5188) reveals a sequence homology of 66% over the same regions of both clones; however, the 5'- and 3'-untranslated regions of the Ya and Yc mRNAs are totally divergent in their sequences . The overall amino acid sequence homology between the Ya and Yc subunits is 68%, however, the NH2-terminal domain is more highly conserved than the middle or carboxyl-terminal domains . Our data suggest that the Ya and Yc subunits of the rat liver glutathione S-transferases are products of two different mRNAs which are derived from two related yet different genes.

J Biol Chem, 1985 May 10, 260(9), 5729 - 38
The DNA restriction endonuclease of Escherichia coli B . II . Further studies of the structure of DNA intermediates and products; Endlich B et al.; The DNA intermediates and final products formed by the Type I restriction endonuclease, EcoB, were further characterized . DNA cleaved on only one strand (hemi-restricted DNA) contains gaps of approximately 70-100 nucleotides, while the fully restricted products contain 3'-single-stranded tails averaging approximately 70-100 nucleotides for each strand cleaved . The gaps and tails are formed with the release of an equal number of nucleotides as small oligonucleotides that are soluble in acid . After purification, neither the hemi-restricted nor the fully restricted DNAs are cleaved again by EcoB . There is no apparent specificity for which strand of a duplex is initially cleaved by EcoB, nor is there specificity with respect to the composition of the 3'-terminal nucleotide formed on the DNA or the 3'- or 5'-terminal nucleotides of the acid-soluble oligonucleotides released during DNA cleavage . The structure formed at the 5' terminus of the DNA product which blocks phosphorylation by T4 polynucleotide kinase remains unknown, but its removal with phage lambda exonuclease allows at least some reutilization of recognition sites by EcoB as well as phosphorylation of the newly formed 5' termini . To explain the complex mechanism of this enzyme, it is suggested that the unidentified 5'-tails prevent wasteful rerestriction from occurring, whereas the 3'-single-stranded tails create DNA which, when nonhomologous to chromosomal DNA, cannot be rescued because such tails are not substrate for DNA polymerases . However, when homologous chromosomal DNA exists, the randomly cleaved large fragments with these tails can easily be assimilated by recA-mediated genetic recombination, thus stimulating DNA exchange between related organisms.

J Biol Chem, 1985 May 10, 260(9), 5720 - 8
The DNA restriction endonuclease of Escherichia coli B . I . Studies of the DNA translocation and the ATPase activities; Endlich B et al.; Electron microscopic examination of DNA intermediates formed by the restriction endonuclease of Escherichia coli B revealed supercoiled loops that are presumably formed during an ATP-dependent DNA translocation process in which the enzyme remains bound to the recognition site while tracking along the DNA helix to a cleavage site . The rate of DNA translocation during this process is at least 5000 base pairs/min at 37 degrees C . Even after all cleavages have been completed, complexes are seen that contain terminal loops or loop plus tail structures . During this later phase of the reaction, ATP is hydrolyzed at a rate which is dependent upon the size of the largest possible loop (or loop plus tail); this ATP hydrolysis can be terminated by one double-strand cleavage within the loop region between the recognition site and the terminus . To explain these results, it is hypothesized that after cleavage the enzyme cycles between a tracking (and possibly back-tracking) mode which is fueled by ATP hydrolysis and a relatively long static period in which ATP hydrolysis does not occur . While tracking, the enzyme would be bound both to the recognition site and to a distal site but, while static, the enzyme would be bound only at the recognition site of nonlooped molecules . This post-nuclease phase of the reaction is hypothesized to reflect a reaction whereby the enzyme initially scans DNA molecules before making a strand cleavage.

J Biol Chem, 1985 May 10, 260(9), 5625 - 30
Folylpoly-gamma-glutamate synthetase-dihydrofolate synthetase . Cloning and high expression of the Escherichia coli folC gene and purification and properties of the gene product; Bognar AL et al.; The Escherichia coli gene for folylpolyglutamate synthetase-dihydrofolate synthetase was localized to plasmids pLC22-45, 24-31, and 28-44 of the Clarke-Carbon E . coli colony bank (Clarke, L., and Carbon, J . (1976) Cell 9, 91-99) by screening the bank by replica mating with an E . coli folC mutant . The folC gene was subcloned from pLC22-45 and inserted into a high copy number plasmid containing the lambda replication control region under the control of the temperature-sensitive cI857 repressor and into a high expression plasmid containing the lambda PL promoter and the cI857 repressor . The folC structural gene was located on a 1.52-kilobase PvuI fragment, sufficient to code for a protein of maximum Mr 55,000 . E . coli transformants containing the recombinant plasmids, when induced by culturing at 42 degrees C, had folylpolyglutamate synthetase and dihydrofolate synthetase levels that were 100- to 400-fold higher than in wild type strains and which represented up to 4% of the soluble cell protein . The E . coli folylpolyglutamate synthetase-dihydrofolate synthetase has been purified to homogeneity from the transformants . Both activities are catalyzed by a single protein of Mr 47,000 . Some kinetic properties of the enzymes and a new spectrophotometric method for assaying dihydrofolate synthetase activity are described.

J Biol Chem, 1985 May 10, 260(9), 5616 - 20
Characterization of the ileS-lsp operon in Escherichia coli . Identification of an open reading frame upstream of the ileS gene and potential promoter(s) for the ileS-lsp operon; Kamio Y et al.; The preceding paper presents evidence for the co-transcriptional expression of the ileS and lsp genes in Escherichia coli . To identify the promoter for the ileS-lsp operon, we have determined the nucleotide sequence of an 1.8-kilobase DNA fragment between the rpsT and IleS genes . The sequence data have revealed an open reading frame, designated gene X, which encodes a polypeptide with 312 amino acid residues . Both in vivo and in vitro expressions of the x gene result in the synthesis of a soluble protein with an apparent Mr of 35,000 . The x gene is transcribed in the same direction as that of the ileS-lsp operon and opposite to that of the upstream adjacent rpsT gene . No transcription termination sequence can be discerned in the intercistronic region between the x and ileS genes . DNase I footprinting experiment revealed a RNA polymerase binding site at 170-151 base pairs upstream of the x gene.

J Biol Chem, 1985 May 10, 260(9), 5588 - 95
Expression in vivo of a mutant human initiator tRNA gene in mammalian cells using a simian virus 40 vector; Drabkin HJ et al.; We have cloned both the wild type (A54) and mutant (T54) human initiator genes described in the preceding paper (Drabkin, H . J., and RajBhandary, U . L . (1985) J . Biol . Chem . 260, 5580-5587) as 141-base pair fragments into the SV40-pBR322 vector pSV1GT3 . These vectors were subsequently used to transfect monkey kidney CV-1 cells to obtain recombinant virus stocks carrying each of the initiator tRNA genes . Following infection of CV-1 cells by the recombinant virus stocks, both the wild type and mutant tRNAs are produced in large quantities during a 48-h period . Fingerprint analysis of 32P-labeled tRNAs was used to characterize the tRNAs made in vivo and to show that the sequence AUCG in loop IV of the wild type tRNA is replaced by T psi CG in the mutant tRNA . Modified nucleotide composition analysis of the {32P}tRNAs overproduced in vivo shows that they contain all the modified nucleotides found in human placenta initiator tRNA . Both wild type and mutant initiator tRNAs can be aminoacylated by either mammalian or Escherichia coli methionyl-tRNA synthetases . Furthermore, the mutant tRNA can be easily separated from the endogenous monkey initiator tRNA by RPC-5 column chromatography.

J Biol Chem, 1985 May 10, 260(9), 5456 - 63
The iron-sulfur cluster composition of Escherichia coli nitrate reductase; Johnson MK et al.; Nitrate reductase from Escherichia coli has been investigated by low-temperature magnetic circular dichroism and electron paramagnetic resonance (EPR) spectroscopies, as well as by Fe-S core extrusion, to determine the Fe-S cluster composition . The results indicate approximately one 3Fe and three or four {4Fe-4S}2+,1+ centers/molecule of isolated enzyme . The magnetic circular dichroism spectra and magnetization characteristics show the oxidized and reduced 3Fe and {4Fe-4S} centers to be electronically analogous to those in bacterial ferredoxins . The form and spin quantitation of the EPR spectra from {4Fe-4S}1+ centers in the reduced enzyme were found to vary with the conditions of reduction . For the fully reduced enzyme, the EPR spectrum accounted for between 2.9 and 3.5 spins/molecule, and comparison with partially reduced spectra indicates weak intercluster magnetic interactions between reduced paramagnetic centers . In common with other Fe-S proteins, the 3Fe center was not extruded intact under standard conditions . The results suggest that nitrate reductase is the first example of a metalloenzyme where enzymatic activity is associated with a form that contains an oxidized 3Fe center . However, experiments to determine whether or not the 3Fe center is present in vivo were inconclusive.

J Biol Chem, 1985 May 10, 260(9), 5610 - 5
Identification of prolipoprotein signal peptidase and genomic organization of the lsp gene in Escherichia coli; Tokunaga M et al.; The product of the lsp gene of Escherichia coli, i.e . the prolipoprotein signal peptidase, was identified by both in vivo pulse labeling experiments using a high expression lambda PL promoter vector and by an in vitro transcription/translation coupled system . The molecular weight of prolipoprotein signal peptidase was estimated to be approximately 18,000 by its mobility on polyacrylamide gel electrophoresis and was found to be the same as that of SPase II purified from the wild-type cells . Analysis of SPase II activities in strains containing various subclones, deletion derivatives generated from plasmid pMT521, and analysis of protein products in a strain harboring an ileS-lsp-fused gene indicated that ileS and lsp genes are transcribed on the same mRNA . This was further supported by the observation that Tn5 insertions in the ileS gene resulted in a reduced expression of the lsp gene . In addition to an upstream promoter shared by the ileS and lsp genes, these analyses also revealed the presence of an internal promoter for the lsp gene within the coding region of the ileS gene.

Nature, 1985 May 9-15, 315(6015), 151 - 4
An HTLV-III peptide produced by recombinant DNA is immunoreactive with sera from patients with AIDS; Chang NT et al.; Human T-cell lymphotropic retrovirus type III (HTLV-III), also called lymphadenopathy-associated virus (LAV), has been identified as the aetiological agent of acquired immune deficiency syndrome (AIDS) . The sera of most patients with AIDS or AIDS-related complexes, and of asymptomatic individuals infected with HTLV-III, contain antibodies against antigens of HTLV-III . The characterization of these antibodies and their corresponding viral antigens is important not only for understanding immunity against HTLV-III and the pathology of AIDS, but also for the development of diagnostic methods and preventive vaccine for AIDS . Following the successful establishment of a long-term T-cell line permissive for HTLV-III replication, large quantities of virus have been produced, facilitating the purification of viral proteins and the development of mouse monoclonal antibodies against several viral antigens . More recently, the structure of HTLV-III proviral DNA has been elucidated . We now report the production, by genetic engineering methods, of a peptide encoded by a gene segment of HTLV-III . A 1.1-kilobase (kb) EcoRI DNA segment from an isolate of HTLV-III was inserted into a lpp and lac promoter-coupled expression vector, pIN-III-ompA . Escherichia coli transformants of this plasmid produced a peptide of relative molecular mass (Mr) 15,000 (15K) which was strongly immunoreactive with anti-HTLV-III antibodies present in sera from AIDS patients . Lysates of the clones expressing this 15K peptide inhibited the reactivity of the p31 virion protein with AIDS sera, suggesting that it is a fragment of the viral p31 protein . The peptide reacted with sera from all 20 AIDS patients but none of the 8 normal controls tested . These results suggest that the peptide may be useful for detecting anti-HTLV-III antibodies in blood samples.

Biochim Biophys Acta, 1985 May 8, 839(3), 233 - 9
Purification and characterization of Escherichia coli xanthine-guanine phosphoribosyltransferase produced by plasmid pSV2gpt; Deo SS et al.; The enzyme xanthine-guanine phosphoribosyltransferase from Escherichia coli cells harboring the plasmid pSV2gpt has been purified 30-fold to near homogeneity by single-step GMP-agarose affinity chromatography . It has a Km value of 2.5, 42 and 182 microM for the substrates guanine, xanthine and hypoxanthine, respectively, with guanine being the most preferred substrate . The enzyme exhibits a Km value of 38.5 microM for PRib-PP with guanine as second substrate and of 100 microM when xanthine is used as the second substrate . It is markedly inhibited by 6-thioguanine, GMP and to a lesser extent by some other purine analogues . Thioguanine has been found to be the most potent inhibitor . The subunit molecular weight of xanthine-guanine phosphoribosyltransferase was determined to be 19 000 . The in situ activity assay on a nondenaturing polyacrylamide gel electrophoresis gel has indicated that a second E . coli phosphoribosyltransferase preferentially uses hypoxanthine as opposed to guanine as a substrate, and it does not use xanthine.

Biochemistry, 1985 May 7, 24(10), 2425 - 31
Escherichia coli pyruvate dehydrogenase complex: particle masses of the complex and component enzymes measured by scanning transmission electron microscopy; CaJacob CA et al.; Particle masses of the Escherichia coli pyruvate dehydrogenase (PDH) complex and its component enzymes have been measured by scanning transmission electron microscopy (STEM) . The particle mass of PDH complex measured by STEM is 5.28 X 10(6) with a standard deviation of 0.40 X 10(6) . The masses of the component enzymes together with their standard deviations are (2.06 +/- 0.26) X 10(5) for the dimeric pyruvate dehydrogenase (E1), (1.15 +/- 0.17) X 10(5) for dimeric dihydrolipoyl dehydrogenase (E3), and (2.20 +/- 0.17) X 10(6) for dihydrolipoyl transacetylase (E2), the 24-subunit core enzyme . The latter value corresponds to a subunit molecular weight of (9.17 +/- 0.71) X 10(4) for E2 . The subunit molecular weight measured by polyacrylamide gel electrophoresis in sodium dodecyl sulfate is 8.6 X 10(4) . STEM measurements on PDH complex incubated with excess E3 or E1 failed to detect any additional binding of E3 but showed that the complex would bind additional E1 under forcing conditions (high concentrations with glutaraldehyde) . The additional E1 subunits were bound too weakly to represent binding sites in an isolated or isolable complex . The mass measurements by STEM are consistent with the subunit composition 24:24:12 when interpreted in the light of the flavin content of the complex and assuming 24 subunits in the core enzyme (E2).

J Mol Biol, 1985 May 5, 183(1), 43 - 51
A mutant lactose repressor with altered inducer and operator binding parameters; Chakerian AE et al.; The lactose repressor protein from the mutant Escherichia coli BG185 contains valine at position 81 instead of alanine . Spectroscopic, chemical and direct binding measurements demonstrate that the BG185 protein exhibits properties similar to the wild-type repressor-inducer complex . Kinetic measurements of inducer binding to BG185 repressor yielded rate constants that were more than two orders of magnitude smaller than those observed for wild-type repressor; these results suggest that the structural transitions required for inducer binding are markedly impaired by the mutation . The fluorescence spectral shift in response to inducer binding was identical for mutant and wild-type proteins . This identity indicates direct effects of inducer binding on the tryptophan(s) near the sugar binding site rather than environmental changes consequent to conformational shifts . Analogy to the bacterial sugar binding proteins suggest that the Ala to Val change at position 81 in BG185 repressor yields a molecule that is fixed in a closed, sugar-binding conformation.

Eur J Biochem, 1985 May 2, 148(3), 509 - 14
Cloning of the gene encoding the hydrogenase from Desulfovibrio vulgaris (Hildenborough) and determination of the NH2-terminal sequence; Voordouw G et al.; The gene encoding the hydrogenase from Desulfovibrio vulgaris (Hildenborough) has been cloned in Escherichia coli . D . vulgaris DNA was digested with the restriction endonucleases EcoRI and SalI and ligated into the vector pUC9 {Vieira, J . & Messing, J . (1982) Gene 19, 259-268}, which had been cut with these same enzymes . Approximately 9000 recombinant clones were obtained by transformation of E . coli JM 101 followed by growth on rich plates with ampicillin for selection and isopropyl-beta-D-thiogalactoside and 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside present for detection of recombinants . The recombinant clones were then screened for production of immunoreactive proteins with rabbit antisera against purified hydrogenase and 125I-labelled protein A . 28 positive clones were found in this initial screening . These were further tested in an immunocompetition experiment, which showed that the protein product from one clone behaved identically to purified hydrogenase . The plasmid pHV15 isolated from this clone has a 4.7 X 10(3)-base-pair SalI/EcoRI insert . Cells of E . coli JM 101 transformed with pHV 15 produce a hydrogenase polypeptide of molecular mass 46 kDa as detected by Western blotting . The mass, as well as the Cleveland mapping pattern of the polypeptide produced by E . coli, are identical with those of the hydrogenase isolated from D . vulgaris (Hildenborough) . Southern blotting of restriction-enzyme-digested D . vulgaris DNA, using the nick-translated 4.7 X 10(3)-base-pair SalI/EcoRI fragment as a probe, indicates the presence of a single gene with an internal PstI site . The NH2-terminal sequence of the hydrogenase was determined to be: (sequence in text) . This information should allow an unambiguous identification of the hydrogenase gene.

Eur J Biochem, 1985 May 2, 148(3), 493 - 7
Comparison of two hydrolytic murein transglycosylases of Escherichia coli; Keck W et al.; Escherichia coli has two murein transglycosylases, which are found in the soluble and the particulate fraction, respectively . The enzymes have been purified and have been shown to differ in some of their molecular properties {Mett, H., Keck, W., Funk, A . & Schwarz, U . (1980) J . Bacteriol . 144, 45-52} . We improved and simplified the purification procedure for the membrane-derived transglycosylase and characterized the two enzymes in more detail by peptide mapping and by immunological procedures . The peptide pattern obtained after tryptic digestion of the purified enzymes differed for the two enzymes . Antisera to the transglycosylases reacted only with their own antigen as shown by specific inhibition of the enzymatic activity, double immunodiffusion and by immunochemical staining of protein blots on nitrocellulose filters . Thus we conclude that the transglycosylases are two distinct proteins and that the one is not a precursor of the other.

Rev Infect Dis, 1985 May-Jun, 7 Suppl 2, S300 - 4
Prospects for improved laboratory diagnoses of treponemal infections and species differentiation; Hardy PH; The serologic diagnosis of treponemal infections has depended in the past on a variety of tests in which specificity was defined on an epidemiologic rather than on an immunologic basis . The lipoidal antigen tests possess no immunologic specificity . Tests based on whole treponemal antigens, although they do have some immunologic specificity, react with antibodies other then those generated in the course of syphilis and yaws infections . Recent developments in biotechnology now permit the identification of immunologically specific antigens in Treponema pallidum, and cloning of appropriate genetic information in Escherichia coli has led to the production of pure specific reagents . These developments will finally place the serologic diagnosis of treponemal infections on a sound immunologic basis.

Appl Environ Microbiol, 1985 May, 49(5), 1295 - 303
Application of DNA-DNA colony hybridization to the detection of catabolic genotypes in environmental samples; Sayler GS et al.; The application of preexisting DNA hybridization techniques was investigated for potential in determining populations of specific gene sequences in environmental samples . Cross-hybridizations among two degradative plasmids, TOL and NAH, and two cloning vehicles, pLAFR1 and RSF1010, were determined . The detection limits for the TOL plasmid against a nonhomologous plasmid-bearing bacterial background was ascertained . The colony hybridization technique allowed detection of one colony containing TOL plasmid among 10(6) Escherichia coli colonies of nonhomologous DNA . Comparisons between population estimates derived from growth on selective substrates and from hybridizations were examined . Findings indicated that standard sole carbon source enumeration procedures for degradative populations lead to overestimations due to nonspecific growth of other bacteria on the microcontaminant carbon sources present in the media . Population estimates based on the selective growth of a microcosm population on two aromatic substrates (toluene and naphthalene) and estimates derived from DNA-DNA colony hybridizations, using the TOL or NAH plasmid as a probe, corresponded with estimates of substrate mineralization rates and past exposure to environmental contaminants . The applications of such techniques are hoped to eventually allow enumeration of any specific gene sequences in the environment, including both anabolic and catabolic genes . In addition, this procedure should prove useful in monitoring recombinant DNA clones released into environmental situations.

Biochem Pharmacol, 1985 May 1, 34(9), 1571 - 5
Inhibition of platelet and neutrophil phospholipase A2 by hydroxyeicosatetraenoic acids (HETES) . A novel pharmacological mechanism for regulating free fatty acid release; Chang J et al.; The present study demonstrated that acid-extracted platelet phospholipase A2 (PLA2) exhibited marked hydrolytic activity against both {1-14C}oleic acid- and {1-14C}arachidonic acid-labeled Escherichia coli . The rate of hydrolysis was linear up to 30 min and was directly proportional to the amount of enzyme added to the reaction mixture . The data further indicated that 5-hydroxy-6,8,11,15-eicosatetraenoic acid (5-HETE) inhibited platelet PLA2 in a dose-dependent manner (IC50 = 42 microM), whereas 5-lactone HETE had no inhibitory effect up to 100 microM . The degree of inhibition of PLA2 activity was unaffected by Ca2+ concentrations but was reduced in the presence of increasing amounts of E . coli substrate . Both 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) and 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) also inhibited platelet PLA2 activity (IC50 = 26 and 72 microM respectively) . Furthermore, the inhibitory effects of these monoHETEs were confirmed with a PLA2 preparation derived from rat neutrophils . Thus, these data suggest a novel pharmacological action of HETEs on PLA2 which may have potential ramifications in the regulation of arachidonic acid metabolism.

J Bacteriol, 1985 May, 162(2), 746 - 51
Lesions in citrate synthase that affect aerobic nitrogen fixation by Azotobacter chroococcum; Ramos JL et al.; A class of Azotobacter chroococcum mutants induced by Tn1 that were defective in normal aerobic nitrogen fixation when grown on sugars (Fos-) were corrected by provision of alpha-ketoglutarate or glutamate . In a representative mutant, Fos252, rates of evolution of 14CO2 from {14C}acetate or {14C}glucose were 5% of the parental values, although uptake and incorporation were normal for both substrates . The results suggest that a lesion affects the entry of substrates into the tricarboxylic acid cycle . The activity of citrate synthase in Fos252 in vitro was 5% that of the parents . The citrate synthase (gltA) gene from Escherichia coli was cloned into broad-host-range vectors and mobilized into Fos252 . The plasmids restored parental citrate synthase activities to Fos252 and complemented the inability to fix N2 in air . The data indicate that a mutation causing an intrinsic limitation in respiratory capacity abolishes normal aerobic N2 fixation, which is consistent with the hypothesis of respiratory protection for nitrogenase in Azotobacter species.

Jpn J Antibiot, 1985 May, 38(5), 1254 - 9
{Results of the clinical use of cefminox in the field of obstetrics and gynecology}; Tateno M et al.; Cefminox (CMNX, MT-141) was used in the treatment of obstetrical and gynecological infections with following results . In the treatment of a total of 8 cases including 4 cases of intrauterine infections, 2 cases of pyosalpinx and 2 cases of pelvioperitonitis, clinical results obtained were rated as excellent in 4 cases (50%) and good in 2 cases (25%) . When good and excellent results were combined as representing effectiveness, the effective rate was 75% (6/8 cases) . The 2 cases, which gave poor results, were under treatment for ovarian carcinoma . CMNX was found effective for intrauterine infections (postpartal) and pyosalpinx, especially those caused by E . coli . None of the cases experienced side effects.

J Biochem (Tokyo), 1985 May, 97(5), 1429 - 36
Secretion of human interferon-alpha induced by using secretion vectors containing a promoter and signal sequence of alkaline phosphatase gene of Escherichia coli; Miyake T et al.; We constructed a new vector containing the promoter and the signal sequence of E . coli phoA gene, the structural gene for the periplasmic alkaline phosphatase . One of the most useful characteristics of this vector is the unique HindIII restriction site located just at the end of the phoA signal sequence . This restriction site was generated by oligonucleotide-directed site-specific mutagenesis without changing the amino acid sequence of the signal peptide . Any kind of foreign structural gene can be easily inserted into the HindIII site by using synthetic oligonucleotides to construct a hybrid gene which has neither an extra sequence nor a deletion between the phoA signal sequence and the foreign structural gene . Human alpha-interferon gene was inserted into this HindIII site . When this hybrid gene was expressed under the control of the phoA promoter region, a low but significant activity was recovered in the cold water wash of the cells after an osmotic shock procedure.

Proc Natl Acad Sci U S A, 1985 May, 82(10), 3096 - 100
Sequence context effects in DNA replication blocks induced by aflatoxin B1; Refolo LM et al.; The genotoxic effects of the potent mutagenic carcinogen aflatoxin B1 (AFB1) are believed to be mediated by its reaction with the N-7 atom of guanine residues in DNA . We have analyzed the effect of AFB1-induced chemical modification on the template function of single-stranded DNA in vitro . The experimental strategy involves the elongation of a primer on a modified template by Escherichia coli DNA polymerase I (large fragment) and analysis of the products by high-resolution gel electrophoresis . Our data show that (i) AFB1 induces specific replication blocks one nucleotide 3' to the sites of occurrence of guanine residues on template DNA; (ii) AFB1-induced replication blocks occur predominantly at sequences capable of participation in intrastrand base pairing; (iii) within the intrastrand base-paired regions there are strong sequence context effects, in accordance with the previously described {Muench, K . F., Misra, R . P . & Humayun, M . Z . (1983) Proc . Natl . Acad . Sci . USA 80, 6-10} specificity "rules" that apply to the reaction of AFB1 with guanine residues in double-stranded DNA; (iv) there is evidence that the (7-guanyl)-AFB1 adducts as well as secondary derivatives such as the formamidopyrimidine-AFB1 act as replication blocks . In summary, these data suggest that previously observed inhibition of DNA replication and transcription by AFB1 is directly attributable to (7-guanyl)-AFB1 adducts or their secondary reaction products.

Mol Cell Biol, 1985 May, 5(5), 1015 - 24
Purification and characterization of human H-ras proteins expressed in Escherichia coli; Gross M et al.; The full-length normal and T24 mutant human H-ras proteins and two truncated derivatives of the T24 mutant were expressed efficiently in Escherichia coli . The proteins accumulated to 1 to 5% of total cellular protein, and each was specifically recognized by anti-ras monoclonal antibodies . The two full-length proteins as well as a carboxyl-terminal truncated derivative (deleted for 23 amino acid residues) were soluble upon cell lysis and were purified to 90% homogeneity without the use of denaturants . In contrast, an amino-terminal truncated ras derivative (deleted for 22 amino acid residues) required treatment with urea for its solubilization . The guanine nucleotide binding activity of these four proteins was assessed by a combination of ligand binding on proteins blots, immunoprecipitation, and standard filter binding procedures . The full-length proteins showed similar binding kinetics and a stoichiometry approaching 1 mol of GTP bound per mol of protein . The showed similar binding kinetics and a stoichiometry approaching 1 mol of GTP bound per mol of protein . The carboxyl-terminal truncated protein also bound GTP, but to a reduced extent, whereas the amino-terminal truncated protein did not have binding activity . Apparently, the carboxyl-terminal domain of ras, although important for transforming function, does not play a critical role in GTP binding.

Mol Gen Mikrobiol Virusol, 1985 May, (5), 36 - 41
{Increased transformation ability of Escherichia coli strains carrying the plasmid pLD4}; Rudchenko ON; The effect of resident plasmid pLD4, a derivative of plasmid Hly241, on transformability of the host bacteria cells has been studied . Plasmid pLD4 was transferred into the different strains of E . coli subsequently transformed by the DNA of plasmids pBR322, pBR325, pAL-R2, pMB9 . The majority of strains harbouring pLD4 obtain the increased ability to be transformed as compared with the ability of isogenic plasmidless strains . The similar but less expressed effect was conferred by the plasmid Hly241 . Another hemolytic plasmid Hly195 and its derivatives, carrying the different transposons, as well as plasmid F' tet Hly did not increase the transformability of host bacteria . The optimal parameters for transformation of the strains harbouring pLD4 and plasmidless strains coincide, but the number of competent cells is considerably higher for plasmid containing strains, due to preliminary results.

Mol Gen Mikrobiol Virusol, 1985 May, (5), 31 - 6
{Recombinant plasmids carrying multiple markers: isolation during yeast co-transformation}; Kozhina TN et al.; Cotransformants of yeast cells by two partially homologous plasmids, one of which is incapable of autonomous replication, has been used to construct multiply marked recombinant plasmids . Only simultaneous elimination of three yeast markers was registered when episomal plasmid, carrying Ade2 gene, and integrative plasmid, carrying yeast genes LEU2 and URA3, were cotransformed . Transformants, in which yeast genes LEU2, URA3 and HIS3 are linked, have been isolated by analogous technique . The genetic analysis has confirmed existence of plasmid cointegrates in the transformant cells, which carry three yeast genes, bacterial DNA fragment and 2 micrometers DNA fragment, coding for replicative functions . Recombination in the region of bacterial plasmid pBR322 might have resulted in formation of such plasmids . Plasmid recombination in cotransformants has been used to construct multiply marked circular chromosomes, having included yeast genes LEU2, URA3 and TRP1, centromere of the IV yeast chromosome and the sequence coding for their replication in yeast as well as in E . coli cells.

Radiat Res, 1985 May, 102(2), 232 - 40
Promotion of septation in irradiated Escherichia coli by a cytoplasmic membrane preparation; Gill JS et al.; Septation can be promoted in an X-irradiated lon mutant of Escherichia coli K-12 by the addition of an E . coli B/r cytoplasmic membrane preparation to the postirradiation plating medium . The promotion of septation was not associated with an inhibition of growth rate . Two distinct cytoplasmic membrane-associated properties were necessary to promote septation . One of these, the cytochrome-based electron transport system, produced anaerobic conditions by the reduction of oxygen dissolved in the medium . The second system, functioning independently from the first, altered substances found in the peptone and yeast extract components of the postirradiation plating medium . When both systems were operative, significant repair of the cell division mechanism occurred.

Prikl Biokhim Mikrobiol, 1985 May-Jun, 21(3), 372 - 7
{Isolation and study of the properties of the hemagglutinins produced by Streptomyces badius actinomycetes}; Beliaev OA et al.; Hemagglutinin inducing polyagglutination of erythrocytes in the presence of the group blood sera was isolated from the supernatant of the culture fluid of Streptomyces badius 0626 by precipitation with ammonium sulfate and subsequent chromatography on Sephadex . Hemagglutinin proved to be a protein containing no lipid or carbohydrate components and possessing no bacterioagglutinating ability . Examination of the culture fluid supernatants of 150 cultures did not reveal any strains definitely exhibiting the transformation capability for human blood groups.

Prikl Biokhim Mikrobiol, 1985 May-Jun, 21(3), 334 - 41
{Comparison of the aspartase activity and its stability in Escherichia coli cells immobilized on polyacrylamide gel and carrageenan}; Zueva NN et al.; The conditions for immobilization of Escherichia coli cells (Soviet strain 85) on the natural polysaccharide carrier carrageenan (Soviet-made) were investigated and kinetic regularities of the aspartase reaction catalysed by immobilized in carrageenan cells of E . coli 85 were established . The conditions for retaining a high aspartase activity and stability of biocatalysts based on the E . coli 85 cells immobilized in PAAG and carrageenan were determined using full-loaded tanks for continuous synthesis of L-aspartic acid . The time-stable aspartase activity of the biocatalyst can be increased by treating the beads of the catalyst with bifunctional reagents (hexamethylenediamine, glutaraldehyde), the most active catalyst for the biotechnological synthesis of L-aspartic acid being obtained when carrageenan is used.

Mol Biol (Mosk), 1985 May-Jun, 19(3), 800 - 4
{Ribosomal proteins interacting with Phe-tRNAPhe during enzymatic binding with translating ribosome before and after the release of the elongation factor EF-Tu}; Abdurashidova GG et al.; Proteins, directly interacting with tRNA in R- and A-sites of E . coli ribosome were determined by means of ultraviolet-induced RNA-protein cross-links . It is shown, that tRNAPhe in the R-site (upon enzymatic binding of the ternary complex Phe-tRNAPhe . X Tu X GMPPCP to ribosome) directly interact with factor Tu and ribosomal proteins S4, S5, S8 and L6, while in the A-site (upon binding of Phe-tRNAPhe X Tu X GTP, GTP hydrolysis, Tu release and transpeptidation)--with proteins S5, S10, L6, L16 and S13/S14/L27.

Mol Biol (Mosk), 1985 May-Jun, 19(3), 617 - 22
{Stimulation of peptidyltransferase activity of 50S subunits by alcohols}; Maimets TO et al.; We have demonstrated that in certain conditions 50S subunits can transfer peptide moiety from peptidyl-tRNA to puromycin in the absence of alcohol . Monovalent cations NH4+ and K+ support this reaction, while Na+ and Li+ are ineffective . Optimal concentration for NH4+ is 1.8 M . Mg2+ ion concentrations above 12 mM are needed as well as an elevated temperature (30 degrees C) . Using the alcohol-free puromycin reaction of 50S subunits we show that alcohol activates the peptidyl transferase center, but does not participate in the puromycin reaction . Neither does it change the protein composition of subunits.

Biochimie, 1985 May, 67(5), 475 - 83
The origins of the strategy of codon use; Henaut A et al.; We analyzed the DNA sequences taking as an elementary pattern segments of increasing length from the codon to the gene . We have thus been able to identify part of the constraints from which originates the use of the code degeneracy in each gene . Our results show that the strategy of codon use is not solely related to the translation apparatus characteristics.

Mol Cell Biochem, 1985 May, 67(1), 65 - 76
Redox interconversion of glutathione reductase from Escherichia coli . A study with pure enzyme and cell-free extracts; Mata AM et al.; The glutathione reductase from E . coli was rapidly inactivated following aerobic incubation of the pure and cell-free extract enzymes with NADPH, NADH and other reductants . The inactivation of the pure enzyme depended on the time and temperature of incubation (t 1/2 = 2 min at 37 degrees C), and was proportional to the {NADPH}/{enzyme} ratio, reaching 50% in the presence of 0.3 microM NADPH and 45 microM NADH respectively, at a subunit concentration of 20 nM . Higher pyridine nucleotide concentrations were required to inactivate the enzyme from cell-free extracts . Two apparent pKa, corresponding to pH 5.8 and 7.3, were determined for the redox inactivation . The enzyme remained inactive even after eliminating the excess NADPH by gel chromatography . E . coli glutathione reductase was protected by oxidized and reduced glutathione against redox inactivation with both pure and cell-free extract enzymes . Ferricyanide and dithiothreitol protected only the pure enzyme, while NADP+ exclusively protected the cell-free extract enzyme . The inactive glutathione reductase was reactivated by treatment with oxidized and reduced glutathione, ferricyanide, and dithiothreitol in a time-and temperature-dependent process . The oxidized form of glutathione was more efficient and specific than the reduced form in the protection and reactivation of the pure enzyme . The molecular weight of the redox-inactivated E . coli glutathione reductase was similar to that of the dimeric native enzyme, ruling out aggregation as a possible cause of inactivation . A tentative model is discussed for the redox inactivation, involving the formation of an 'erroneous' disulfide bridge at the glutathione-binding site.

Isr J Med Sci, 1985 May, 21(5), 410 - 4
Escherichia coli adherence to the intestine of mice; Golderman L et al.; Bacterial attachment to the intestine is the first step in the initiation of many intestinal infections . The effect of mucin and its major constituents on the adherence of a mannose-positive Escherichia coli strain to various intestinal segments of mouse intestine was examined . Removal of intestinal mucus led to increased adherence of E . coli to the ileal and colonic mucosal layers . Ileal mucin significantly decreased E . coli attachment to the ileum . Galactose and galactosamine were the major constituents of ileal mucin that reduced E . coli adherence to the ileum . We conclude that ileal mucin protects the epithelial cell from adherence by this mannose-positive E . coli strain . This protection is dependent on the presence of other sugar moieties that may be of importance in the adherence process.

Genetika, 1985 May, 21(5), 756 - 62
{Enhancement of expression of Escherichia coli uridine phosphorylase gene as a result of duplication}; Alkhimova RA et al.; The thymine-requiring (thy) Escherichia coli strains normally convert thymine to thymidine through the action of thymidine phosphorylase, an enzyme, whose synthesis is directed by tpp gene of the deo operon . Selection for efficient thymine utilization in the thy mutants carrying a deletion in the tpp gene allows to obtain clones with constitutive synthesis of an alternative enzyme, uridine phosphorylase coded by the udp gene . Such clones are usually formed, due to "promoter-up" constitutive udpP mutants . Further selection for increased expression of the udp gene was performed on the background of udpP1 and udpP18 "promoter-up" constitutive mutants isolated previously . Several lines of evidence obtained by using transposon insertions suggest that the increased synthesis of uridine phosphorylase in some newly isolated mutants is due to duplications of the udp gene . In two mutants the adjacent metE gene is involved in duplications simultaneously with the udp gene.

Genetika, 1985 May, 21(5), 693 - 706
{Organization of the Escherichia coli genome}; Sukhodolets VV; A review of literature data reveals that for the last years, the molecular biology techniques have been of an increasing use in the study of the Escherichia coli genome, having supplemented the standard genetic mapping . For the proper understanding of the Escherichia coli genome organization, recombinational events occurring in the course of evolution should be considered . The bacterial genome seems to carry traces of both "long-term" evolution, possibly responsible for appearance of the bacterial cell itself, and "current" evolution, consisting mainly of periodic genome entering by new plasmid-originated genes . It is supposed that in the process of stabilization within a genome, every new gene undergoes a stage of the "transgene", that is the gene situated in a transposon on the chromosome . In parallel with integration of new genes into the genome, some genes deleting should also take place . The formation of deletions could occur by unequal crossing over in segments of direct homologous repeats which seem to be ordinarily revealed in the experimental study of the tandem gene duplications.

Eur J Pediatr, 1985 May, 144(1), 49 - 52
Plasma fibronectin concentrations in healthy and septic infants; Domula M et al.; The concentration of plasma fibronectin was determined by Laurell's electroimmunoassay in 75 preterm or term newborns within the first 2 days of life, in 97 healthy infants aged from 3 days to 12 months, in 40 septic infants and in 38 healthy adult subjects . The mean fibronectin concentration in citrated plasma of normal adults was 318 +/- 84 ml/l . Healthy eutrophic term newborns 1-2 days old had approximately one-third of the fibronectin concentration of adults . There was no significant difference in the values between healthy term and eutrophic preterm newborns or between eutrophic and hypotrophic newborns . The plasma fibronectin increased strongly over the 1st month of life . No significant difference was observed between fibronectin levels in infant boys and girls . The values in septic newborns and septic older infants were significantly lower when compared with those of age-matched healthy controls . It is speculated that this deficiency, because of linkage to fibrin in disseminated intravascular coagulation or due to increased utilisation as a nonspecific opsonin and sequestration at sites of tissue injury, may contribute to organ failure in septicaemia.

Biophys J, 1985 May, 47(5), 629 - 39
Projected structure of the pore-forming OmpC protein from Escherichia coli outer membrane; Chang CF et al.; A single-projection structure analysis of a bacterial outer membrane protein, OmpC, has been carried out by electron microscopy of frozen hydrated specimens . Two distinct crystal polymorphs have been observed in the frozen-hydrated samples, and projection structures of both forms have been obtained to a resolution of 13.5 A . Preliminary examination of negatively stained samples revealed the expected, trimeric appearance of pores in the OmpC specimens . Electron microscopy of unstained, frozen-hydrated OmpC reveals the trimeric pore structure with equal clarity . In addition, the overall molecular envelope of the protein is readily discerned, and a major lipid-containing domain can also be seen . Because of the small coherent patch size, mosaic disorder, and unpredictable polymorphism of the presently available specimens, three-dimensional reconstruction of frozen-hydrated OmpC has not been carried out.

Tijdschr Diergeneeskd, 1985 May 1, 110(9), 345 - 55
{Quality of the colostrum in relation to neonatal diarrhea due to enteropathogenic Escherichia coli in calves}; Geene JJ; Of 107 calves studied, forty-one animals (38 per cent) showed diarrhoea during the first week of life of life; only one of the forty-one calves showing diarrhoea died . E . coli K99+ was isolated from twelve calves . Serological studies showed that the concentrations of IgG1 and IgG2 on the first and second days of life were almost equally high in calves with and those without diarrhoea . The differences were more marked as regards the concentrations of IgA and IgM . The Ig fractions of the colostrum immediately after parturition (t = 0), which was supplied to the calves (later, with and without diarrhoea) did not differ; the concentrations of IgG1 and IgM in the colostrum within 24 hours after parturition were significantly lower in those dams, the calves of which subsequently showed diarrhoea . The above results are discussed in greater detail in the comment.

Res Vet Sci, 1985 May, 38(3), 255 - 8
Protection of lambs against enteric colibacillosis by vaccination of ewes; Pugh CA et al.; Pregnant ewes were vaccinated twice, seven weeks and three weeks before lambing, with a multivalent formalin-killed Escherichia coli vaccine containing an added K99, F41 antigen preparation . Lambs born to vaccinated and unvaccinated ewes were exposed to oral infection with E coli B44 (09:K30, K99, F41) . All 10 lambs from vaccinated ewes were protected whereas all 10 control lambs developed severe diarrhoea and five died or were killed in extremis . In the following year, previously immunised ewes were given a single dose of the vaccine two weeks before lambing . Eleven of their 12 lambs were protected against a similar challenge, which caused the death of six of eight control lambs and severe diarrhoea in the two survivors . Higher levels of antibody to the K99, F41 preparation were detected by enzyme-linked immunosorbent assay in the serum and colostrum from vaccinated ewes and in the serum of their lambs when compared with similar samples from control ewes and lambs.

J Appl Bacteriol, 1985 May, 58(5), 471 - 7
A comparison of the ecology of Escherichia coli in the intestine of healthy unweaned pigs and pigs after weaning; Hinton M et al.; A total of 51 clinically healthy pigs (14 unweaned and 37 weaned) from five litters, and aged 21 to 35 d, were studied . Escherichia coli isolates from the duodenum, jejunum, ileum, caecum and colon were differentiated on the basis of O-serogroup, biotype and resistance pattern . The complexity of the flora was influenced considerably by the presence or absence of the enterotoxigenic serotype 0149: K91, K88a,c (Abbotstown strain) . When it was absent the E . coli flora of both weaned and unweaned pigs was complex with up to 25 strains being identified . The majority of these E . coli strains identified in each pig were isolated from only one of the five intestinal sites sampled . On the other hand, when the enterotoxigenic strain was present (14 pigs) it tended to dominate the E . coli flora at all levels of the intestine and this dominance was reflected in a corresponding fall in the total number of E . coli strains isolated per pig.

Exp Eye Res, 1985 May, 40(5), 711 - 9
Endotoxin-induced ocular inflammation increases prostaglandin E2 synthesis by rabbit lens; Fleisher LN et al.; Twenty-four hours after the intravitreal injection of 0.1-100 ng of Escherichia coli endotoxin into one eye of the New Zealand white rabbit, lenses from the inflamed eyes synthesized significantly more prostaglandin E2 (PGE2) than their contralateral, control lenses at all doses of endotoxin greater than 0.1 ng . PGE2 elevations were also seen in the aqueous and vitreous humors from inflamed eyes . Lenses did not synthesize 6-keto-PGF1alpha (a stable metabolite of PGI2) . Incubation of untreated lenses with 1 microgram ml-1 of endotoxin for 24 h did not increase PGE2 production . These results indicate that rabbit lens can synthesize PGE2, that this synthesis is significantly increased 24 hr after the intravitreal infusion of E . coli endotoxin, and that this increased PGE2 synthesis is most likely not due to a direct action of endotoxin on the lens.

EMBO J, 1985 May, 4(5), 1319 - 26
Importance of state of methylation of oriC GATC sites in initiation of DNA replication in Escherichia coli; Smith DW et al.; In vivo and in vitro evidence is presented implicating a function of GATC methylation in the Escherichia coli replication origin, oriC, during initiation of DNA synthesis . Transformation frequencies of oriC plasmids into E . coli dam mutants, deficient in the GATC-specific DNA methylase, are greatly reduced compared with parental dam+ cells, particularly for plasmids that must use oriC for initiation . Mutations that suppress the mismatch repair deficiency of dam mutants do not increase these low transformation frequencies, implicating a new function for the Dam methylase . oriC DNA isolated from dam- cells functions 2- to 4-fold less well in the oriC-specific in vitro initiation system when compared with oriC DNA from dam+ cells . This decreased template activity is restored 2- to 3-fold if the DNA from dam- cells is first methylated with purified Dam methylase . Bacterial origin plasmids or M13-oriC chimeric phage DNA, isolated from either base substitution or insertion dam mutants of E . coli, exhibit some sensitivity to digestion by DpnI, a restriction endonuclease specific for methylated GATC sites, showing that these dam mutants retain some Dam methylation activity . Sites of preferred cleavage are found within the oriC region, as well as in the ColE1-type origin.

EMBO J, 1985 May, 4(5), 1307 - 11
Cloning an Aspergillus nidulans developmental gene by transformation; Johnstone IL et al.; We have developed a transformation system for Aspergillus nidulans giving a frequency of transformation high enough to screen a gene bank from which we were able to isolate and clone the A . nidulans developmental gene brlA by visual selection . The vector contains the selective marker argB+, and with it a frequency of transformation of 500 stable transformants/micrograms plasmid DNA can regularly be achieved . The evidence suggests that transformation is by integration but spontaneous excision of integrated plasmids is apparently frequent enough to allow the recovery of transforming plasmids in Escherichia coli.

Arthritis Rheum, 1985 May, 28(5), 522 - 8
Induction of chronic polyarthritis in rabbits by hyperimmunization with Escherichia coli . I . Pathologic and serologic features in two breeds of rabbits; Aoki S et al.; Hyperimmunization of 147 rabbits (outbred Japanese white rabbits and New Zealand white {NZW} rabbits bred in a closed colony) with heat-killed Escherichia coli 0:14 in Freund's incomplete adjuvant resulted in the animals developing a chronic polyarthritis resembling rheumatoid arthritis (RA) . While both Japanese white and NZW rabbits showed a high incidence of the induced arthritis, a higher proportion of NZW rabbits developed the disease, suggesting that genetic influence is important in the development of RA-like illness . This experimental model may be useful for the study of RA.

Appl Environ Microbiol, 1985 May, 49(5), 1323 - 5
Mobilization of Thiobacillus ferrooxidans plasmids among Escherichia coli strains; Rawlings DE et al.; Nonconjugative Thiobacillus ferrooxidans plasmids were mobilized at high frequencies among Escherichia coli strains by the IncP plasmid RP4 and at low frequencies by the IncN plasmid R46, but not by the IncW plasmid pSa . The mobilization region of a nonconjugative T . ferrooxidans plasmid was located on a 5.3-kilobase T . ferrooxidans DNA fragment.

Am J Vet Res, 1985 May, 46(5), 1117 - 20
Effect of endotoxin administration on body fluid compartments in the horse; Spurlock GH et al.; Plasma volume, extracellular fluid volume (ECFV), and total body water (TBW) were measured before and after endotoxin (Escherichia coli) administration in 6 conscious adult horses . Evan's blue dye, sodium thiocyanate, and antipyrine were the test substances used to estimate plasma volume, ECFV, and TBW, respectively . Pharmacokinetic analysis of plasma concentration vs time was used to determine changes in body fluid compartments . The pathophysiologic effects of endotoxin were monitored by clinical evaluation, blood chemical changes, and blood gas determinations . All horses became dyspneic within 15 minutes of endotoxin administration and clinical signs of colic were evident 30 to 45 minutes after endotoxin administration . After endotoxin administration, serum glucose and creatinine concentrations were significantly (P less than 0.05) elevated, and all horses became hypoxic and developed marked metabolic acidosis, and plasma volume decreased approximately 15% (P less than 0.05) . A significant change in ECFV or TBW during the 300-minute experimental period was not observed.

Plasmid, 1985 May, 13(3), 193 - 9
A selection system for unequal crossing-over: plasmid construction and initial studies in Escherichia coli; Clevenger W et al.; Unequal crossing-over is involved in genetic duplication and deletion in such diverse genetic systems as Drosophila, bacteria, and animal viruses . It is proposed to be involved in the form of unequal sister chromatid exchange in gene amplification in cultured animal cells and during carcinogenesis . Studies of the process of unequal crossing-over have been hampered by the lack of genetic systems allowing specific selection for cells that have undergone such unequal crossing-over . We report here on the construction of plasmids designed to provide specific selection of unequal crossing-over . One such plasmid was studied in Escherichia coli . We show that kanamycin resistance is generated, as predicted, by the expected unequal crossover event.

Proc Natl Acad Sci U S A, 1985 May, 82(10), 3078 - 81
Rapid "footprinting" on supercoiled DNA; Gralla JD; A DNase protection technique is described and applied to the interaction of three lac control proteins with supercoiled lac DNA . The technique uses end-labeled oligonucleotide primers to probe specific DNA regions as an alternative to protocols requiring restriction endonuclease cleavage or blotting . Thus DNA may be probed with high resolution in its native state . It is demonstrated that the introduction of supercoiling into DNA accelerates the rate of lac ps promoter binding by RNA polymerase but does not alter the positions at which polymerase, c-AMP-binding protein, or lac repressor bind to lac DNA.

Mutat Res, 1985 May-Jun, 143(1-2), 29 - 34
Chloramphenicol-promoted increase in resistance to UV damage in Escherichia coli B/r WP2 trpE65: development of the capacity for successful repair of otherwise mutagenic or lethal lesions in DNA; Doudney CO et al.; The ultraviolet radiation survival curve of exponentially growing cultures of Escherichia coli B/r WP2 trpE65 was modified by a short period (20 min) of chloramphenicol treatment before UV exposure, which produced an extended exponential section of intermediate slope between the shoulder and the final exponential slope . More prolonged incubation with chloramphenicol (up to 90 min) resulted in little further extension of the intermediate exponential slope, but caused a progressive expansion of the shoulder region . With each period of chloramphenicol pretreatment, a major surge of mutation to tryptophan independence always occurred after that UV fluence promoting the transition from the shoulder to the intermediate exponential slope of the survival curve, and another major surge occurred after that fluence promoting the transition from the intermediate exponential slope to the final exponential slope . A minor surge of mutation occurred after low fluences . The 3 surges in mutation and the increased slopes of the survival curve are ascribed to UV-inactivation of 3 qualitatively different DNA-repair systems, each with differentially increased resistances to UV caused by pretreatment by chloramphenicol.

J Appl Physiol, 1985 May, 58(5), 1463 - 8
Effect of endotoxemia on hypoxic pulmonary vasoconstriction in unanesthetized sheep; Hutchison AA et al.; This study examined the effect of acute endotoxemia on hypoxic pulmonary vasoconstriction (HPV) in awake sheep . Thirteen sheep were chronically instrumented with Silastic catheters in the pulmonary artery, left atrium, jugular vein, and carotid artery; with a Swan-Ganz catheter in the main pulmonary artery; with a chronic lung lymph fistula; and with a tracheostomy . Base-line HPV was determined by measuring the change in pulmonary vascular resistance (PVR) while sheep breathed 12% O2 for 7 min . Concentrations of immunoreactive 6-keto-PGF1 alpha and thromboxane B2 (TXB2) were measured in lung lymph during the hypoxic challenge . Escherichia coli endotoxin (0.2-0.5 micrograms/kg) was infused intravenously . Four hours after endotoxemia, HPV was measured . In five sheep, meclofenamate was infused at 4.5 h after endotoxemia and HPV measured again . During the base-line hypoxic challenge, PVR increased by 36 +/- 9% (mean +/- SE) . There was no significant change in lung lymph 6-keto-PGF1 alpha or TXB2 levels with hypoxia . Twelve of the 13 sheep showed a decrease in HPV 4 h after endotoxemia; the mean change in PVR with hypoxia was -8 +/- 5%, which was significantly (P less than 0.05) reduced compared with base-line HPV . The infusion of meclofenamate at 4.5 h after endotoxin did not restore HPV.

Dis Colon Rectum, 1985 May, 28(5), 353 - 7
Colonic malacoplakia and abdominal tuberculosis in a child . Report of a case with review of the literature; Satti MB et al.; A 4 1/2-year-old girl had colonic malacoplakia of two years' duration, the presenting symptom being rectal bleeding . Abdominal tuberculosis and Escherichia coli lumbar abscess were diagnosed at the age of 3 1/2 years . Despite antituberculous treatment, there was no improvement and she died from protein-losing enteropathy . The patient is discussed and the literature reviewed, with special emphasis on the incidence of malacoplakia in children, the aggressive nature of colonic malacoplakia, and the lack of response to treatment . A brief review of the pathogenesis of malacoplakia is considered.

Dis Colon Rectum, 1985 May, 28(5), 291 - 3
Hemorrhoidal banding . A warning; Russell TR et al.; Band ligation of symptomatic internal hemorrhoids is a well-established and accepted outpatient procedure . The purpose of this paper is to alert the medical profession to potential complications and death following this procedure . Each of the four patients described in this report experienced pain and inability to urinate following banding . This report does not condemn banding but, rather, focuses on problems associated with a procedure perceived by many to be risk free.

Cytometry, 1985 May, 6(3), 186 - 90
Detection of Escherichia coli in blood using flow cytometry; Mansour JD et al.; A rapid method for the detection of Escherichia coli in blood has been developed . The method employs blood cell lysis, staining of bacteria with ethidium bromide, and detection of stained bacteria using flow cytometry . The detection protocol requires less than 2 h sample handling time and is not dependent on bacterial growth . This method has been applied to human donor blood specimens seeded with various E . coli concentrations and to two rabbit model systems . Bacterial detection is evident from the in vitro human blood studies at levels of 10 E . coli/ml and from in vivo rabbit model studies at less than 100 E . coli/ml.

Cell, 1985 May, 41(1), 153 - 63
RecBC enzyme nicking at Chi sites during DNA unwinding: location and orientation-dependence of the cutting; Taylor AF et al.; Homologous recombination by the E . coli RecBC pathway occurs at elevated frequency near Chi sites . We reported previously that Chi induces RecBC enzyme to cleave one DNA strand--that containing the Chi sequence 5'G-C-T-G-G-T-G-G3' . We report here that the Chi-dependent cleavage occurs four, five, or six nucleotides to the 3' side of the Chi octamer and produces nicks with 3'-OH and 5'-PO4 groups . Chi-dependent cleavage occurs if RecBC enzyme approaches the Chi sequence from the right, but not if it approaches only from the left, during unwinding of the duplex DNA substrate . A single RecBC enzyme molecule appears to cleave the DNA and to release part of it as a single-stranded fragment . These and previous results indicate that Chi-dependent cleavage is concomitant with DNA unwinding by RecBC enzyme and provide an enzymatic basis for the orientation-dependence of Chi recombinational hotspot activity . These observations demonstrate a key step of a proposed model of recombination in which RecBC enzyme produces a potentially invasive single-stranded DNA tail extending from Chi to its left . We discuss the relation between the action of Chi sites and that of special sites enhancing eukaryotic recombination.

Cell, 1985 May, 41(1), 145 - 51
Chi-dependent DNA strand cleavage by RecBC enzyme; Ponticelli AS et al.; Chi sites enhance in their vicinity homologous recombination by the E . coli RecBC pathway . We report here that RecBC enzyme catalyzes Chi-dependent cleavage of one DNA strand, that containing the Chi sequence 5'G-C-T-G-G-T-G-G3' . Chi-specific cleavage is greatly reduced by single base pair changes within the Chi sequence and by mutations within the E . coli recC gene, coding for a RecBC enzyme subunit . Although cleavage occurs preferentially with double-stranded DNA, the product of the reaction is single-stranded DNA . These results demonstrate the direct interaction of RecBC enzyme with Chi sites that was inferred from the genetic properties of Chi and recBC, and they support models of recombination in which Chi acts before the initiation of strand exchange.

Proc Soc Exp Biol Med, 1985 May, 179(1), 128 - 31
Inhibition of ascorbic acid uptake by endotoxin: evidence of mediation by serum factor(s); Aleo JJ et al.; The effect of bacterial endotoxin on the ascorbic acid uptake by 3T6 fibroblasts was studied . Endotoxin inhibited ascorbic acid uptake by fibroblasts in a dose dependent manner . The inhibition by endotoxin takes place only in the presence of unheated serum; decomplementing serum by heat inactivation at 56 degrees C for 30 minutes eliminates endotoxin's inhibitory effect on ascorbic acid uptake . The effect of endotoxin appears to be instantaneous since the inhibition seen in the cells without any preexposure was similar to the cells preexposed to endotoxin for up to 6 hours . Polymyxin B sulfate which is known to bind the lipid A portion of endotoxin did not reverse the inhibition of ascorbic acid uptake caused by endotoxin.

Proc Natl Acad Sci U S A, 1985 May, 82(9), 2688 - 92
Active site and complete sequence of the suicidal methyltransferase that counters alkylation mutagenesis; Demple B et al.; The inducible resistance to alkylation mutagenesis and killing in Escherichia coli (the adaptive response) is controlled by the ada gene . The Ada protein acts both as a positive regulator of the response and as a DNA repair enzyme, correcting premutagenic O6-alkylguanine in DNA by suicidal transfer of the alkyl group to one of its own cysteine residues . We have determined the DNA sequence of the cloned ada+ gene and its regulatory region . The data reveal potential sites of ada autoregulation . Amino acid sequence determinations show that the active center for the O6-methylguanine-DNA methyltransferase is located close to the polypeptide COOH terminus and has the unusual sequence -Pro-Cys-His-, preceded by a very hydrophobic region . These same structural features are present at the active site of thymidylate synthase, suggesting a common chemical mechanism for activation of the cysteine.

Proc Natl Acad Sci U S A, 1985 May, 82(9), 2679 - 83
Consensus sequence for Escherichia coli heat shock gene promoters; Cowing DW et al.; We have identified promoters for the Escherichia coli heat shock operons dnaK and groE and the gene encoding heat shock protein C62.5 . Transcription from each promoter is heat-inducible in vivo, and each is recognized in vitro by RNA polymerase containing sigma 32, the sigma factor encoded by rpoH (htpR) but not by RNA polymerase containing sigma 70 . We compared the sequences of the heat shock promoters and propose a consensus promoter sequence, having T-N-t-C-N-C-c-C-T-T-G-A-A in the -35 region and C-C-C-C-A-T-t-T-a in the -10 region . These sequences differ from the consensus sequence recognized by holoenzyme containing sigma 70, the major sigma in E . coli . We suggest that the accumulated consensus sequences of promoters recognized by alternate forms of holoenzyme are compatible with a model in which sigma recognizes only the -10 region of the promoter.

Proc Natl Acad Sci U S A, 1985 May, 82(9), 2593 - 7
A chemically cleavable biotinylated nucleotide: usefulness in the recovery of protein-DNA complexes from avidin affinity columns; Shimkus M et al.; A biotinylated nucleotide analog containing a disulfide bond in the 12-atom linker joining biotin to the C-5 of the pyrimidine ring has been synthesized . This analog, Bio-SS-dUTP, is an efficient substrate for Escherichia coli DNA polymerase I . Bio-SS-dUTP supported DNA synthesis in a standard nick-translation reaction at 35%-40% the rate of an equal concentration of the normal nucleotide, TTP . DNA containing this analog was bound to an avidin-agarose affinity column and subsequently eluted after reduction of the disulfide bond by dithiothreitol . The ability to recover biotinylated DNA from an avidin affinity column under nondenaturing conditions should prove useful in the isolation of specific protein-DNA complexes . As a demonstration of this approach, Bio-SS-DNA was reconstituted with histones to form 11S monomer nucleosomes . Bio-SS-nucleosomes were shown to selectively bind to avidin-agarose . Ninety percent of the bound Bio-SS-nucleosomes were recovered from the affinity column by elution with buffer containing 50-500 mM dithiothreitol . The recovered nucleosomes were shown to be intact 11S particles as judged by velocity sedimentation in a sucrose gradient . This approach may prove to be generally useful in the isolation of protein-DNA complexes in a form suitable for further analysis of their native unperturbed structure.

J Surg Res, 1985 May, 38(5), 501 - 8
Prostanoid production by lipopolysaccharide-stimulated Kupffer cells; Bowers GJ et al.; Although some data suggest that macrophages in the reticuloendothelial system (RES) are important sources of thromboxane A2 (TxA2) and prostacyclin (PGI2) during endotoxic shock, we are unaware of data documenting the ability of hepatic macrophages (Kupffer cells) to release either TxA2 or PGI2 when exposed to lipopolysaccharide (endotoxin, LPS) . In this study, Kupffer cells were examined for their ability to release prostaglandin E2 (PGE2), TxA2, and PGI2 following stimulation with 0, 1.0, 50.0, and 100.0 micrograms/ml of Escherichia coli LPS . Kupffer cells were obtained from rat livers by enzymatic digestion with 0.05% collagenase followed by enrichment of the macrophage population on the basis of differences in density and adherence among the various cell populations isolated . Based on several criteria (phagocytosis of opsonized sheep erythrocytes, positive staining for esterase and peroxidase, failure to replicate), 95% of adherent cells were Kupffer cells . After 4 days of incubation, cells were stimulated with various doses of LPS for 4 and 8 hr . Prostanoid concentrations in culture supernatants were determined by radioimmunoassay . Increasing doses of LPS significantly (P less than 0.001) increased the concentration of immunoreactive PGE2 (iPGE2) and iTxB2 (the stable metabolite of TxA2) . The concentration of i6-keto-PFG1 alpha (stable metabolite of PGI2) increased following stimulation with 1.0 microgram/ml of LPS, but declined as the dose of LPS was increased . The results provide evidence that endotoxin-activated Kupffer cells, like other macrophage populations, release several metabolites of arachidonic acid . Kupffer cell-derived prostanoids, particularly TxA2, may be important mediators of some of the pathophysiologic manifestations of acute endotoxemia.

J Bacteriol, 1985 May, 162(2), 837 - 9
Isolation and nucleotide sequence analysis of tRNAAlaGGC from Escherichia coli K-12; Mims BH et al.; An alanine tRNA with the anticodon 5'-GGC-3' has been identified in Escherichia coli K-12 . It is the first sequenced alanine tRNA with G in the 5' position of the anticodon . tRNAAlaGGC has A in the "semi-invariant" position 32 . At the "invariant" position 8 we observed both U and another, unknown, nucleoside.

J Bacteriol, 1985 May, 162(2), 830 - 2
Novel dnaG mutation in a dnaP mutant of Escherichia coli; Murakami Y et al.; Reexamination of the dnaP18 mutant strain of Escherichia coli revealed that the mutation responsible for the arrest of DNA replication and cell growth at high temperatures resides in the dnaG gene rather than in the dnaP locus as previously thought; this mutation has been designated dnaG2903.

J Bacteriol, 1985 May, 162(2), 822 - 5
Separate regulation of purA and purB loci of Escherichia coli K-12; Wolfe SA et al.; We isolated a strain of Escherichia coli K-12 in which the lac structural genes are fused to the purB control region and used this strain to study the regulation of the purA and purB loci . The purA locus was derepressed in response to either limiting adenine or guanine growth conditions in the presence of excess guanine or adenine, respectively . The presence of hypoxanthine in the culture medium did not have any effect on the expression of the purA locus . The purB locus responded to limiting adenine growth conditions in the presence of either excess hypoxanthine or guanine alone but not when both hypoxanthine and guanine were present.

J Bacteriol, 1985 May, 162(2), 661 - 7
Genetic mapping of katG, a locus that affects synthesis of the bifunctional catalase-peroxidase hydroperoxidase I in Escherichia coli; Loewen PC et al.; A locus unlinked to either katE or katF that affected catalase levels in Escherichia coli was identified and localized between metB and ppc at 89.2 min on the genome . The locus was named katG . Mutations in katG which prevented the formation of both isoenzyme forms of the bifunctional catalase-peroxidase HPI were created both by nitrosoguanidine and by transposon Tn10 insertions . All katG+ recombinants and transductants contained both HPI isoenzymes . Despite the common feature of little or no catalase activity in four of the catalase-deficient strains, subtle differences in the phenotypes of each strain resulted from the different katG mutations . All three mutants caused by nitrosoguanidine produced a protein with little or no catalase activity but with the same subunit molecular weight and with similar antigenic properties to HPI, implying the presence of missense mutations rather than nonsense mutations in each strain . Indeed one mutant produced an HPI-like protein that retained peroxidase activity, whereas the HPI-like protein in a second mutant exhibited no catalase or peroxidase activity . The third mutant responded to ascorbate induction with the synthesis of near normal catalase levels, suggesting a regulatory defect . The Tn10 insertion mutant produced no catalase and no protein that was antigenically similar to HPI.

J Bacteriol, 1985 May, 162(2), 607 - 10
Genetic mapping of nth, a gene affecting endonuclease III (thymine glycol-DNA glycosylase) in Escherichia coli K-12; Weiss B et al.; The nth gene of Escherichia coli affects the production of endonuclease III, a glycosylase-endonuclease that attacks DNA damaged by oxidizing agents or by ionizing radiation . An nth insertion mutant and a deletion mutant were studied . nth is located between add and tyrS on the linkage map of E . coli K-12 and was 97% linked to tyrS in a transduction with phage P1.

Clin Sci (Lond), 1985 May, 68(5), 589 - 96
Cholesterol gallstone pathogenesis: a study of potential nucleating agents for cholesterol crystal formation in bile; Whiting MJ et al.; Cholesterol monohydrate crystal formation was measured quantitatively in model bile solutions, which were supersaturated with cholesterol, by a radiochemical method and qualitatively in human gallbladder bile by polarizing microscopy . Various agents, which have been postulated to act as nucleating factors for cholesterol crystal and gallstone formation, were added to bile and their effect on the appearance of cholesterol crystals was determined . These agents included calcium salts found in gallstones (calcite, aragonite, apatite, bilirubinate), Escherichia coli bacteria, pigment residues from cholesterol gallstones, bilirubin and several mucin preparations . Human gallbladder bile, which was collected from patients with and without cholesterol gallstones, was also mixed with model bile to examine whether nucleating or anti-nucleating factors were present . None of the agents tested markedly and consistently promoted cholesterol monohydrate crystal formation in model or human bile, except seed crystals of cholesterol monohydrate which were used as a control . Human gallbladder bile from obese patients without gallstones delayed the appearance of cholesterol crystals in model bile solutions, whereas gallbladder bile from gallstone patients did not . These results do not provide experimental support for the hypothesis that calcium salts and pigment material found in gallstones, or gallbladder mucin at concentrations less than 10 mg/ml, act as nucleating agents for cholesterol crystal and stone formation . The difference between gallbladder biles from patients with and without gallstones in their propensity to form cholesterol crystals may be due to the presence of an anti-nucleating factor in normal bile.

Eur J Cell Biol, 1985 May, 37, 196 - 202
Multimeric complexes of differentiation-inducing protein bound to DNA; Weisinger G et al.; Myeloid hematopoietic precursor cells are induced to differentiate by the macrophage and granulocyte differentiation-inducing protein MGI-2 (DF) . This differentiation-inducing protein bound to double-stranded but not to single-stranded mammalian DNA . The bound MGI-2 was not eluted by high salt, but was eluted by sodium dodecyl sulfate (SDS) . MGI-2 also bound to double-stranded E . coli DNA, but with this DNA the bound MGI-2 was eluted by high salt . This indicated a difference in the binding affinities of MGI-2 to mammalian and E . coli DNA . MGI-2 bound to DNA was examined by electron microscopy . The results indicate that MGI-2 formed a multimeric complex with double-stranded DNA and that the size of the complex was correlated with the strength of protein binding to the DNA . The multimeric complex bound to DNA was disrupted by deoxyribonuclease . The data indicated that binding of this differentiation-inducing protein to DNA involves the formation of a multimeric complex in which the monomers are held together by DNA . It is suggested that the formation of such multimeric complexes of MGI-2 and DNA may allow activation of the multiple pathways of gene expression that is required for differentiation.

Proc Natl Acad Sci U S A, 1985 May, 82(10), 3129 - 33
A dimer of AraC protein contacts three adjacent major groove regions of the araI DNA site; Hendrickson W et al.; Contact sites of AraC protein to the regulatory site araI of the Escherichia coli araBAD operon have been determined by the chemical-interference technique . DNA fragments were chemically modified an average of once per molecule, and fragments that no longer bound AraC were separated by gel electrophoresis from the DNA fragments still able to bind the protein . The contact sites were then determined by comparing the positions of modifications in the two DNA samples . Strong contacts were found with guanines in three consecutive major groove regions and the adjacent phosphates along one side of the DNA . The conserved bases of the AraC-binding DNA consensus sequence are also found in the same positions . The gel electrophoresis assay was used to determine the stoichiometry of binding, and AraC protein was found to bind the araI and araO1 regulatory sites as a dimer . Therefore, AraC appears to bind DNA differently from the other well-characterized regulatory proteins such as phage lambda repressor.

Proc Natl Acad Sci U S A, 1985 May, 82(9), 2574 - 8
Replication initiator protein of plasmid R6K autoregulates its own synthesis at the transcriptional step; Kelley W et al.; The replication initiator protein of plasmid R6K preferentially repressed transcription initiated in vitro from the promoter of the initiator protein cistron . DNase I protection experiments revealed that the sequences in the region of the promoter recognized by the initiator protein partially overlapped the sequences of the same promoter recognized by RNA polymerase of Escherichia coli . Competitive DNase I protection experiments revealed that the initiator not only prevented the RNA polymerase from binding to the promoter sequence but also displaced RNA polymerase from preformed enzyme-promoter binary complexes . Thus, the initiator protein acts as a transcriptional repressor of its own cistron by either preventing RNA polymerase from binding to the promoter or by displacing RNA polymerase from promoter-enzyme complexes.

Genetika, 1985 May, 21(5), 707 - 15
{Mechanism of non-tandem integration of prophage phi 80 into the chromosome of the wild-type Escherichia coli}; Kholodii GIa et al.; We studied the ability of lambda, phi 80 and their hybrid lambda att80 to lysogenize homoimmune monolysogens and examined the prophage locations on the chromosome of the resulting polylysogens . We observed an effective integration of phi 80 and lambda att80, in contrast to lambda, into the host chromosome, exclusively, at the attachment sites that were not occupied by the resident prophage (nontandem) . Besides, the lambda att80 (int+) prophage was observed to ensure effective nontandem integration of a homoimmune int mutant DNA . Hence, we inferred that the expression of the int gene in the phi 80 prophage is constitutive, cI-independent and results in nontandem integration of the homoimmune prophage . The validity of this inference has been supported experimentally: (i) the only lysogen that was found to contain a phi 80 tandem was highly unstable (spontaneous segregation of monolysogens occurred 6-7 times more frequently than with the lambda tandem); (ii) an int inactivating mutation stabilized the phi 80 tandem; as a result, the int mutant has the frequency of tandem integration as high as that of lambda, while no nontandem integration was observed . A hypothesis is proposed which accounts for the instability of the phi 80 tandems and explains the relation between this phenomenon and the prophage ability to integrate into secondary attachment sites in the presence of the primary (normal) one.

Arch Microbiol, 1985 May, 141(4), 359 - 63
The seleno-polypeptide of formic dehydrogenase (formate hydrogen-lyase linked) from Escherichia coli: genetic analysis; Pecher A et al.; The site of integration of phage M mu d (Ap lac) in mutant M9s which leads to deficiency of formic dehydrogenase (benzylviologen-linked) activity was determined . It was shown that the phage had inserted into the gene for the seleno-polypeptide of the enzyme (80 kd) leading to the formation of a truncated peptide (60 kd) still able to incorporate Se . Synthesis of the truncated polypeptide is subject to the same regulatory signals as that of the wild-type enzyme . The formation of the 110 kd seleno-polypeptide, which is a constituent component of the formic dehydrogenase from the formate-nitrate respiratory pathway, is unimpaired in mutant M9s . The location of the gene for the 80 kd seleno-polypeptide was mapped at 92.4 min of the Escherichia coli chromosome.

Chem Biol Interact, 1985 May, 53(3), 351 - 64
Filamentation of Escherichia coli K12 elicited by some monomeric, dimeric and trimeric complexes of ruthenium in various oxidation states; Gibson JF et al.; A number of ruthenium complexes were tested for their ability to induce filamentation in Escherichia coli . These included monomeric and dimeric complexes with ruthenium in the II or III oxidation states, as well as mixed-valence complexes with ruthenium in the (II,III) oxidation states . In general, dimeric mixed-valence Ru(II,III) complexes were the most active class of compound, although some complexes of this type were relatively inactive . These were pyrazine- or bipyridyl-bridged complexes which are known to involve strong metal-ligand interaction, which stabilizes the Ru(II) oxidation state . Some Ru(III) complexes were also significantly active in induction of filamentous growth in E . coli . One of these was {Ru(NH3)5Cl}Cl2, which did not inhibit electron transport, Mg2+-ATPase activity or DNA synthesis in E . coli, but like {Ru2(NH3)6Br3}Br2 X H2O was a potent inhibitor of respiration-driven calcium transport in the organism . Filament-inducing activity of the complex was reduced in the presence of NaCl, but not in the presence of added Ca2+, ethanol, calcium pantothenate, or E . coli 'division promoting extract' . This behaviour is also similar to that of {Ru2(NH3)6Br3}Br2 X H2O . It is suggested that both complexes may induce filamentation in E . coli by a common mechanism, which may involve interference with calcium metabolism, or a wall or membrane target, rather than interaction with DNA.

Proc Natl Acad Sci U S A, 1985 May, 82(10), 3325 - 9
Dual role for Escherichia coli RecA protein in SOS mutagenesis; Ennis DG et al.; Induction of the Escherichia coli SOS system increases the ability of the cells to perform DNA repair and mutagenesis . Previous work has shown that this increased mutagenesis is the result of derepression of specific genes through a complex regulatory mechanism controlled by LexA and RecA proteins . One role of RecA protein in this process is to facilitate proteolytic cleavage of LexA protein (the repressor) in response to an inducing signal that reversibly activates RecA protein to perform this function . We show that activated RecA protein plays a second role in SOS mutagenesis, as revealed by analyzing repair of UV-damaged phage lambda in host mutants with alterations in the SOS regulatory system . First, phage mutagenesis was not expressed constitutively in a mutant that is derepressed through lack of functional LexA protein; activated RecA protein was still required . Second, phage mutagenesis was constitutively expressed in the presence of recA mutations that alter RecA protein so that it is activated in normally growing cells . There was also RecA-dependent constitutive expression of SOS mutagenesis in host mutants that lack functional LexA protein and carry plasmids . We discuss several possible biochemical mechanisms for this second role of activated RecA protein in SOS mutagenesis.

J Bacteriol, 1985 May, 162(2), 840 - 4
Isolation and characterization of delta ompB strains of Escherichia coli by a general method based on gene fusions; Garrett S et al.; We isolated a series of delta ompR delta envZ mutants by inducing a strain carrying a lambda prophage in the closely linked gene malP and screening the bacterial survivors for loss of the major outer membrane porins OmpF and OmpC . Characterization of these deletion strains showed that both OmpR and EnvZ were necessary for transcription of ompF and ompC and that neither gene was essential for cell viability . Moreover, the deletion strains did not exhibit the pleiotropic membrane protein deficiency observed with certain envZ mutants . The method described should allow the simple isolation of deletions in any region of the chromosome.

Mutat Res, 1985 May, 145(3), 137 - 44
Weigle reactivation of phage lambda in a recA mutant of Escherichia coli: dependence on the excess amounts of photoreactivating enzyme in the dark; Yamamoto K et al.; The plating efficiency of ultraviolet-light-irradiated phage lambda in the dark is increased when an Escherichia coli recA host, which is transformed with a multicopy plasmid, pKY1, carrying the phr gene of E . coli, is irradiated with 254 nm UV prior to infection (Weigle (W) reactivation) . Such W reactivation in lexA3 and umuC strains, with or without pKY1, is almost undetectable . Addition of umu mutations to recA56/pKY1 cells blocks this process, but addition of the lexA3 mutation instead gives high levels of W reactivation . Fusion of the lacZ gene with recA or umuC promoters permitted measurement of the effects of UV radiation on transcription of these SOS operons for various mutated cells, with and without the phr+ plasmid . The presence of the recA56 allele totally abolishes UV induction of both recA and umuC gene expression in the cells, both with and without the plasmid . Thus, the observed W reactivation in the recA strain carrying the phr+ plasmid requires only the constitutive amount of the Umu proteins, and does not require the lexA+ recA+-dependent inducible process . While we observed an increase in the W reactivation of UV-irradiated lambda phage in a recA56/pKY1 cell, there is no corresponding mutagenesis of the UV-irradiated phage in UV-irradiated cells . We propose that the E . coli phr gene product facilitates error-free pathways of DNA repair in the absence of photoreactivation . The fact that a recA/pKY1 strain permits almost normal levels of W reactivation, but completely blocks W mutagenesis, leads us further to suggest that the recA gene product, in general, functions in a regulatory manner in W reactivation and in both regulatory and mechanistic ways in W mutagenesis.

Mol Gen Mikrobiol Virusol, 1985 May, (5), 7 - 13
{Isolation of Escherichia coli K12 mutants with deficient in precise excision of transposons}; Markov AP et al.; Plasmid pNM1, the derivative of R100.1, has been constructed by insertion of transposon Tn5 into structural tet genet (Tn10) of the parental plasmid . The frequency of precise excision of Tn5 from plasmidic genome is 10(-5) . The high frequency of precise excision obtained in this system permits one, to use it for isolation of mutants having low frequencies of precise excision . Two mutants were isolated in which the frequencies of precise excision of Tn5 were decreased for two orders . The pex1 and pex2 mutations responsible for the effect decrease the precise excision of Tn5 from R100.1 as well as from RP4 genomes.

Virology, 1985 May, 143(1), 280 - 9
Phage P22 lysis genes: nucleotide sequences and functional relationships with T4 and lambda genes; Rennell D et al.; Wild-type and amber mutant alleles of the lysis genes of P22 were cloned and sequenced . Gene 13 encodes an 11,520-Da basic hydrophobic protein that has 89% amino acid homology to lambda S protein . Gene 19 encodes a protein that has a small degree of amino acid homology with T4 lysozyme, but we could detect no homology to lambda R or RZ proteins . The protein product of gene 19 was purified; its amino terminal amino acid sequence is as predicted by the DNA sequence . It starts with a single amino terminal methionine residue and is a basic protein with a molecular weight of 15,968 . Plasmids expressing P22 gene 19, lambda genes R and RZ, and T4 gene e were constructed . All of these plasmids were able to complement both lambda R- and P22 19-.

Virology, 1985 May, 143(1), 175 - 84
Correlation of the genetic and physical maps of phage T1; Liebeschuetz J et al.; A series of BglII- and Sau3A-derived fragments of phage T1 DNA were cloned into the plasmid vector pLV59 and the clones were tested for their ability to complement and recombine with infecting genomes of am mutants representing all known T1 essential genes . The resulting data were used to correlate the T1 genetic and physical maps . This analysis led to the following conclusions: the large map distances observed at the left (early) end of the genetic map arise partly from increased genetic recombination at the chromosomal left end and partly from increased physical distances between identified genetic markers in this region, the right chromosomal end is also recombinogenic, genes 3.5 to 18, which occupy the right two-thirds of the DNA molecule, are tightly compacted with little space for additional protein-specifying sequences in this region.

Bioorg Khim, 1985 May, 11(5), 621 - 7
{Effective method of oligonucleotide-controlled mutagenesis of DNA fragments}; Efimov VA et al.; A procedure has been designed for changing specific nucleotides in a DNA sequence with efficiency . The method involves the use of the specially constructed cloning vectors pBRS1, pHS1, and pHS2 . These plasmids are derivatives of pBR322 in which the EcoRI-HindIII region has been replaced by synthetic duplexes carrying SmaI, HpaI and XhoI sites, in addition to EcoRI and HindIII sites . The DNA fragment to be mutagenized is cloned in pHS1 (or pBRS1, or pHS2) using restriction sites close to the SmaI and HpaI sites . The recombinant plasmid obtained is digested with one of these enzymes to produce double-stranded DNA with blunt ends . This linear DNA is a substrate for exonuclease III (or T4 DNA polymerase) . The digestion under controlled conditions produces duplex with protruding single-stranded 5'-regions which include the site of the desired mutation . The synthesis of DNA by DNA-polymerase I (Klenow's fragment), primed in part by the synthetic oligonucleotide containing the desired mutation, leads to the linear heteroduplex . The closed circular heteroduplex is formed by ligation . After transformation into E . coli, DNA replication generates homoduplexes, some of which contain the mutation . Colony hybridization with the same 32P-labeled oligonucleotide is used to select mutants . The yield of the mutants is 10-20% . This technique can be extended to replicative form of M13 vectors . It can be also applied to any DNA sequence which has a unique site of restriction endonuclease generating blunt ends.

Mol Biol (Mosk), 1985 May-Jun, 19(3), 818 - 32
{The region of phage T4 W-29 genes: cloning and expression}; Zograf IuN et al.; The EcoRI fragment of T4 DNA containing the W-29 genes and its subfragments were cloned in the pBR322 and the singlestranded M13 phage . Hybridization with a cloned DNA showed that in T4 infected cells the transcription of the late genes 25-29 depends on the phage-induced RNA polymerase changes and on replication of phage DNA . At a late infection stage one also observes an enhanced transcription of the (early) genes uvsW and uvsY, which depends on viral DNA replication . Both early and late genes within recombinant plasmids are also expressed in uninfected cells carrying a plasmid regardless of the inserted fragment orientation and independently of the vector promoters . Hybrid plasmids demonstrated a high frequency of recombination with phage DNA in the infected cell . An RNA polymerase from uninfected cells binds itself to the late cloned genes to form "open" complexes . A purified RNA polymerase transcribes both early and late genes within recombinant plasmids . The relative transcription of the late cloned genes is enhanced if one uses an RNA polymerase from T4-infected cells . The super-helicity of template DNA is essential for transcription of early and late genes.

Eur J Cell Biol, 1985 May, 37, 1 - 6
Immunoelectron microscopic localization of the restriction endonuclease EcoRI in Escherichia coli BS 5; Kohring GW et al.; Purified restriction endonuclease EcoRI isolated from Escherichia coli BS 5 was used for the production of enzyme-specific IgG antibodies in rabbits . For enzyme localization experiments, paraformaldehyde-glutaraldehyde-fixed cells were embedded and polymerized by a low-temperature procedure using Lowicryl K4M . The immuno electron microscopic protein A-gold technique and an immuno-gold method revealed that 70% of the enzyme-specific labeling were located in the cell envelope whereas 30% were found in the cytoplasm . In metal-shadowed preparations, no indications for the presence of the enzyme could be found on the cell surface; however, on the surface of cell protoplasts enzyme specific labeling could be detected . The results indicate the presence of the major amount of EcoRI in the periplasmic space of the cell where it might be loosely bound or even freely diffusible.

Anal Biochem, 1985 May 1, 146(2), 434 - 6
Assay of aminoglycoside phosphotransferase in situ; Kiselev VI et al.; A method for the evaluation of the aminoglycoside phosphotransferase activity in bacterial colonies directly is described . The method is based on the ability of the enzyme to modify the substrate immobilized on carboxymethylcellulose paper . The sensitivity and accuracy of the method were tested by comparing the results of the present assay to those obtained with conventional procedures . The method seems to be particularly useful for the detection within a bacterial population producing aminoglycoside phosphotransferase of those cells which do not make the enzyme and for rapid determination of the relative levels of enzyme produced by different clones.

Anal Biochem, 1985 May 1, 146(2), 423 - 8
Propidium iodide and S1 nuclease: tools for studying DNA reassociation kinetics; Davies W et al.; A method for the determination of the amount of double-stranded DNA in a reassociation mixture is described . Reassociated DNA resistant to S1 nuclease digestion is measured fluorometrically using propidium iodide . A direct comparison is made between this method and an established method in which radiolabeled Escherichia coli DNA resistant to S1 digestion is measured by scintillation counting after separation of nucleotides by Sephadex G-100 chromatography . Reassociation curves determined for calf thymus and E . coli DNA are presented.

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