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 AT