Escherichia coli

J Bacteriol, 1983 Nov, 156(2), 727 - 36
Restriction nuclease and enzymatic analysis of transposon-induced mutations of the Rac prophage which affect expression and function of recE in Escherichia coli K-12; Willis DK et al.; Fourteen Tn5-generated mutations of the Rac prophage, called sbc because they suppress recB21 recC22, were found to fall into two distinct types: type I mutations, which were insertions of Tn5, and type II mutations, which were insertions of IS50 . Both orientations of Tn5 and IS50 were represented among the mutants and were arbitrarily labeled A and B . All 14 of the Tn5 and IS50 insertions occurred in the same location (+/- 100 base pairs) approximately 5.6 kilobases from one of the hybrid attachment sites . Eleven of the mutants contained essentially the same amount of exonuclease VIII, the product of recE . The possibility that a promoter for recE was created by the insertion of Tn5 and IS50 was considered . Two IS50 mutants in which such a promoter could not have been created showed three to four times as much exonuclease VIII, and another showed one-half as much as the majority . The possibility was considered that a promoter internal to IS50 is responsible for this heterogeneity . Restriction alleviation was measured in all 14 mutants . An insertion of the transposon Tn10 which reduces expression of exonuclease VIII (recE101::Tn10) was located within the Rac prophage at a position 2.35 kilobases from the left hybrid attachment site . Location and orientation of the Rac prophage on the Escherichia coli genetic map are discussed in light of these results.

J Bacteriol, 1983 Nov, 156(2), 718 - 26
Genetic analysis of transposon-induced mutations of the Rac prophage in Escherichia coli K-12 which affect expression and function of recE; Fouts KE et al.; Fourteen mitomycin-resistant revertants of a recB21 recC22 strain were isolated after Tn5 mutagenesis . Eight of the mutations (type I) were essentially inseparable from aphA+ (Kanr) of Tn5; six (type II) were not . We hypothesize that the former are Tn5 and that the latter are IS50 insertions . Because of their phenotypic similarity to sbcA and sbcB mutations, which also suppress recB21 recC22, we have called them sbc mutations . sbc-lll::Tn5 was cotransducible with nirR and has thereby been located at position 29.8 on the Escherichia coli map in the vicinity of the Rac prophage and sbcA mutations . A recB21 recC22 sbc-lll::Tn5 strain was subjected to Tn10 mutagenesis, and a mitomycin- and UV-sensitive mutant was isolated . tet+ of Tn10 was 85% cotransducible with aphA+ of Tn5, locating these two transposons 0.1 map unit apart . A three-point cross located the Tn10 mutation at position 29.7 . We hypothesize that the Tn10 insertion is located in recE and that the Tn5 and IS50 insertions activate expression of this gene . sbc-lll::Tn5 was found to be cis acting and dominant to its wild-type allele as were two sbcA mutations (sbcA1 and sbcA6) . Five other type I and type II insertion mutations were dominant to their wild-type alleles . We hypothesize that the sbc insertion and sbcA mutations affect transcription regulation of recE and discuss the possibility that they do so differently.

J Bacteriol, 1983 Nov, 156(2), 710 - 7
Analysis of regulation of phoB expression using a phoB-cat fusion; Guan CD et al.; The phoB gene, which encodes a positive control factor for a number of phosphate-regulated genes in Escherichia coli, was cloned into multicopy plasmid pBR322 . A phoB-cat fusion that expressed chloramphenicol transacetylase from the phoB promoter was constructed . Studies of the expression of the phoB-cat fusion showed that the pattern of regulation of the phoB gene was similar to that of the phoA gene, the structural gene for alkaline phosphatase . The phoB gene was derepressed under conditions of phosphate starvation, was constitutively expressed in a phoR background, and required the phoM gene product for expression in a phoR strain . Finally, a functional phoB product was required for its own synthesis . Our results indicate either that phoA gene expression responds directly to the concentration of the phoB gene product in cells or that the phoA and phoB controlling elements are quite similar.

J Bacteriol, 1983 Nov, 156(2), 584 - 91
Essential genes of plasmid RK2 in Escherichia coli: trfB region controls a kil gene near trfA; Pohlman RF et al.; Plasmid RK2 encodes several kil determinants whose lethal action on Escherichia coli host cells is prevented by RK2 kor genes . Here we show that the mini-RK2 plasmid, pRK248, specifies a kilB component (kilB1) in the region of the replication gene trfA . kilB1 is different from trfA and is completely encoded within the pRK248 HaeII A fragment . Transformation of E . coli cells with hybrid plasmids containing the cloned kilB1 determinant is very inefficient and results in the selection of variant kil- plasmids, many of which show genetic and physical evidence of deletions . If another pRK248 gene (korB1) is present in the cells, kilB1+ plasmids can be established at high efficiency and without any detectable changes . KorB1 is encoded by the trfB region of pRK248 because recombinant plasmids with this region are able to control kilB1 in trans . These results substantiate our earlier explanation for the structure of pRK248 and for the perplexing requirement of the trfB region in this plasmid.

J Virol, 1983 Nov, 48(2), 384 - 95
Class I defective herpes simplex virus DNA as a molecular cloning vehicle in eucaryotic cells; Barnett JW et al.; Defective herpes simplex virus type 1 genomes are composed of head-to-tail tandem repeats of small regions of the nondefective genome . Monomeric repeat units of class I defective herpes simplex virus genomes were cloned into bacterial plasmids . The repeat units functioned as replicons since both viral and convalently linked bacterial plasmid DNA replicated (with the help of DNA from nondefective virus) when transfected into rabbit skin cells . Recombinant plasmids were packaged into virions and were propagated from culture to culture by infection with progeny virus . Replication was evidently by a rolling circle mechanism since plasmid DNA was present in a high-molecular-weight form in transfected cells . Circular recombinant plasmid DNA replicated with a high degree of fidelity . In contrast, linear plasmid DNA underwent extensive deletions of both viral and bacterial sequences when transfected into rabbit skin cells . Derivative plasmids, a fraction of the size of the parental plasmid, were rescued by transforming Escherichia coli with DNA from the transfected rabbit skin cells . These plasmids functioned as shuttle vectors since they replicated faithfully in both eucaryotic and procaryotic cells.

J Virol, 1983 Nov, 48(2), 340 - 51
Human papillomaviruses associated with epidermodysplasia verruciformis . II . Molecular cloning and biochemical characterization of human papillomavirus 3a, 8, 10, and 12 genomes; Kremsdorf D et al.; The DNAs of four human papillomaviruses (HPVs) that were found in the benign lesions of three patients suffering from epidermodysplasia verruciformis have been characterized . The flat wart-like lesions and the macular lesions of patient 1 contained two viruses, HPV-3a and HPV-8, respectively, whose genomes had previously been only partially characterized . The flat wart-like lesions of patient 2 and the macular lesions of patient 3 each contained a virus previously considered as belonging to types 3 and 5, respectively . These viruses are shown in the present study to be different from all of the HPV types so far characterized; they have tentatively been named HPV-10 and HPV-12 . The HPV-3a, HPV-8, and HPV-12 DNAs and the two SalI fragments of HPV-10 DNA (94.1 and 5.9% of the genome length) were cloned in Escherichia coli after having been inserted in plasmid pBR322 . The cloned HPV genomes have similar sizes (about 7,700 base pairs), but their guanine-plus-cytosine contents differ from 41.8% for HPV-12 DNA to 45.5% for HPV-3a DNA . The study of the sensitivity of the four HPV DNAs to 14 restriction endonucleases permitted the construction of cleavage maps . Evidence for conserved restriction sites was found only for the HPV-3a and HPV-10 genomes since 5 of the 21 restriction sites localized in the HPV-3a DNA seem to be present also in the HPV-10 DNA . Hybridization experiments, performed in liquid phase at saturation, showed a 35% sequence homology between HPV-3a and HPV-10 DNAs, 17 to 29% sequence homology among HPV-5, HPV-8, and HPV-12 DNAs, almost no sequence homology between the HPV-3a or HPV-10 DNA and the other HPV DNAs, and a weak homology between HPV-9 DNA and HPV-8 or HPV-12 DNA . Blot hybridization experiments showed no sequence homology between the HPV-3a, HPV-8, and HPV-12 DNAs and the DNAs of the HPVs associated with skin warts (HPV-1a, HPV-2, HPV-4, and HPV-7) or with mucocutaneous and mucous membrane lesions (HPV-6b and HPV-11a, respectively) . One exception was a weak sequence homology between the HPV-2 prototype and HPV-3a or HPV-10 DNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Cancer Res, 1983 Nov, 43(11), 5064 - 7
New method for detecting Epstein-Barr virus association in nonproducer lymphoblastoid cell lines; Giunta S et al.; Epstein-Barr virus association in nonproducer human lymphoblastoid cell lines can be demonstrated by the presence of the virus genome (nucleic acid hybridization studies) or by the detection of the virus-coded complement-fixing antigen (complement fixation and/or anti-complement immunofluorescent test) . This paper describes an enzyme immunoassay for the detection of Epstein-Barr virus complement-fixing antigen and its application to the demonstration of Epstein-Barr virus association in nonproducer lymphoblastoid cell lines . The assay is based on competition for complement between Epstein-Barr complement-fixing antigen and its specific antibody and a probe complex composed of Escherichia coli beta-galactosidase and specific anti-beta-galactosidase antibody . This competitive enzyme immunoassay is a specific and sensitive procedure for detecting Epstein-Barr virus association in nonproducer cell lines, allowing also quantitative estimation of the amount of antigen produced.

Gene, 1983 Nov, 25(2-3), 271 - 80
Clone bank of Nicotiana tabacum chloroplast DNA: mapping of the alpha, beta and epsilon subunits of the ATPase coupling factor, the large subunit of ribulosebisphosphate carboxylase, and the 32-kDal membrane protein; Fluhr R et al.; All of the PstI restriction fragments of the chloroplast DNA of Nicotiana tabacum have been cloned in the plasmid vector pBR322 . The cloned fragment sizes range from 0.8 to 26 kb, are stable, and can be amplified by chloramphenicol with varying efficiencies . Using these clones we have detailed a PstI physical map of the tobacco chloroplast genome . Selected clones of SalI, BamHI and PstI fragments were used to localize the map positions of the alpha, beta, and epsilon subunits of the chloroplast ATPase coupling factor, the large subunit of ribulosediphosphate carboxylase and the 32-kDal membrane protein . The gene products of these clones were characterized by RNA transcript sizing, immunoprecipitation of maxicell-directed protein synthesis, and hybrid-arrested translation.

Radiobiologiia, 1983 Nov-Dec, 23(6), 730 - 3
{Effect of MEA on DNA degradation in permeable irradiated Bac . stearothermophilus cells}; Kuznetsova EA et al.; It was shown that DNA-degrading activity of permeable, intact and gamma-irradiated cells of Bac . stearothermophilus decreased under the effect of beta-mercaptoethylamine (MEA) . MEA decreased also a DNAase activity, in a crude acellular extract of Bac . stearothermophilus, and activities of S1-nuclease and DNAase I . The data obtained prompt an assumption that MEA has an inhibitory action on the activity of endonucleases irrespective of their substrate specificity.

Genetika, 1983 Nov, 19(11), 1778 - 85
{para-Aminobenzoic acid inhibits the manifestation of inducible SOS functions in Escherichia coli K-12}; Vasil'eva SV et al.; In experiments with chemical mutagens (alkylating agents MNU, ENU, MMS and EMS), para-aminobenzoic acid (PABA) sharply inhibited the inducible processes in Escherichia coli, namely, mutagenesis, induction of lambda prophage and W-reactivation of UV-irradiated phage lambda . Based on experimental studies of E . coli strains deficient in different steps of DNA repair, the conclusion was made that PABA participates in regulation of the branch of DNA repair that is controlled by recA+ recF+ alleles.

Biochem J, 1983 Nov 1, 215(2), 343 - 50
Properties of F1-ATPase from the uncD412 mutant of Escherichia coli; Wise JG et al.; Properties of purified F1-ATPase from Escherichia coli mutant strain AN484 (uncD412) have been studied in an attempt to understand why the amino acid substitution in the beta-subunit of this enzyme causes a tenfold reduction from normal MgATP hydrolysis rate . In most properties that were studied, uncD412 F1-ATPase resembled normal E . coli F1-ATPase . Both enzymes were found to contain a total of six adenine-nucleotide-binding sites, of which three were found to be non-exchangeable and three were exchangeable (catalytic) sites . Binding of the non-hydrolysable substrate analogue adenosine 5'-{beta gamma-imido}triphosphate (p{NH}ppA) to the three exchangeable sites showed apparent negative co-operativity . The binding affinities for p{NH}ppA, and also ADP, at the exchangeable sites were similar in the two enzymes . Both enzymes were inhibited by efrapeptin, aurovertin and p{NH}ppA, and were inactivated by dicyclohexylcarbodi-imide, 4-chloro-7-nitrobenzofurazan and p-fluorosulphonyl-benzoyl-5'-adenosine . Km values for CaATP and MgATP were similar in the two enzymes . uncD412 F1-ATPase was abnormally unstable at high pH, and dissociated into subunits readily with consequent loss of activity . The reason for the impairment of catalysis in uncD412 F1-ATPase cannot be stated with certainty from these studies . However we discuss the possibility that the mutation interrupts subunit interaction, thereby causing a partial impairment in the site-site co-operativity which is required for 'promotion' of catalysis in this enzyme.

Genetics, 1983 Nov, 105(3), 469 - 88
Turn-on of inactive genes by promoter recruitment in Escherichia coli: inverted repeats resulting in artificial divergent operons; Charlier D et al.; We have characterized two rearrangements consisting of inverted repeats of the argE gene . The promoters (p) of argE and of argCBH face each other over an internal operator . The rearrangements were obtained as reactivations of argE in a strain harboring an argEp deletion on a lambda darg prophage . In both cases the repeat included argE and argCBHp on either side of a unique sequence; the result is a divergent operon in which each copy of argCBHp reads into the adjacent argE repeat . In one case, the pair of repeats adjoins the silent parental gene, forming a triplication (comes from leads to comes from) . The other rearrangement consists of a single argE palindrome, but the whole prophage is rearranged into an inverted repeat, analogous to certain lambda dv's . Both structures could be explained by breakage of a replication fork passing argE and by inaccurate rejoining of strands . The lambda dv-like rearrangement would result from breakage at both replication forks of a phage or prophage replicating during transient release of immunity . The triplication would imply breaking of a chromosomal replication fork, formation of a cyclic intermediate by recombination between the daughter duplex molecules and reinsertion into the parental argE gene . Formation of a triplication by replication errors involving appropriate strand switchings and branch migrations can not be excluded however.

Carcinogenesis, 1983 Nov, 4(11), 1445 - 50
Site specific cleavage of phi X-174 replicative form DNA after modification by N-acetoxy-N-2-acetylaminofluorene; Bases R et al.; Three kinds of structural disturbances were found in an 88 base pair (bp) fragment of phi X-174 DNA after exposure to N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF) . (i) Frequent strand scissions at two specific guanine sites on the 5' 32P-end-labeled fragment were identified by base sequence analysis . Scissions at these two sites were induced at neutral pH and they were not increased by treatment with apurinic endonuclease . They are an immediate consequence of N-Aco-AAF action and are not primarily apurinic sites . (ii) Alkali treatment with 1 M piperidine at 90 degrees C induced strand scissions at every guanine, demonstrating adduct slices, depurination and strand scissions . (iii) Adducted DNA was sensitive to single-strand specific nuclease digestion, suggesting unwound DNA . These studies indicate the prediliction of N-Aco-AAF for certain DNA sites and they suggest three kinds of DNA modifications which can be expected after adduction by this carcinogen . Some of the sites may be premutational carcinogen-induced DNA structural modifications.

J Bacteriol, 1983 Nov, 156(2), 970 - 4
Mu dX, a derivative of Mu d1 (lac Apr) which makes stable lacZ fusions at high temperature; Baker TA et al.; We describe defective Mu phage Mu dX (Mu d1 Bx::Tn9 {lac Apr Cmr}) which is useful for insertion mutagenesis and for construction of lac operon fusions in vivo . Mu dX retains the insertion properties of Mu d1 but produces temperature-resistant lysogens and transposes at a reduced frequency . A method is described to convert existing Mu d1 insertions to Mu dX.

Arch Int Physiol Biochim, 1983 Nov, 91(4), 261 - 7
{An animal model of hyperdynamic septic shock: hemodynamic effects of injection of low doses of endotoxin in the dog}; D'Orio V et al.; In anaesthetized dogs, low dose endotoxin perfusion (250 ng kg-1 min-1 during 20 min) resulted, after a delay of 15-40 min, in a decrease of mean arterial pressure (from 127 to 106 mm Hg), a transient increase in cardiac output (from 2.2 to 2.9 L/min), a fall of systemic vascular resistance (from 60 to 41 mm Hg L-1 min-1) and an increase in heart rate (from 109 to 156/min) . This haemodynamic pattern is similar to the so called "hyperdynamic" initial phase of clinical septic shock.

J Gen Microbiol, 1983 Nov, 129 ( Pt 11), 3505 - 13
Transport of nucleic acid bases into Escherichia coli; Burton K; The uptake and retention of purine bases and of uracil requires both a protonmotive force and intracellular conversion to nucleotides . Inhibition of uptake by arsenate does not imply that ATP is required for the transport process because arsenate caused a rapid fall in the level of 5-phosphoribosyl 1-pyrophosphate . A mutation defective in high-affinity adenine transport has been identified and is designated purP . This mutation has been found to lie in the neighbourhood of mtl . Competition experiments indicate that at least two other systems are used to transport guanine, xanthine and hypoxanthine.

Gene, 1983 Nov, 25(2-3), 263 - 9
A simple and very efficient method for generating cDNA libraries; Gubler U et al.; A simple method for generating cDNA libraries from submicrogram quantities of mRNA is described . It combines classical first-strand synthesis with the novel RNase H-DNA polymerase I-mediated second-strand synthesis {Okayama, H., and Berg, P., Mol . Cell . Biol . 2 (1982) 161-170} . Neither the elaborate vector-primer system nor the classical hairpin loop cleavage by S1 nuclease are used . cDNA thus made can be tailed and cloned without further purification or sizing . Cloning efficiencies can be as high as 10(6) recombinants generated per microgram mRNA, a considerable improvement over earlier methods . Using the fully sequenced 1300 nucleotide-long bovine preproenkephalin mRNA, we have established by sequencing that the method yields faithful full-length transcripts . This procedure considerably simplifies the establishment of cDNA libraries and thus the cloning of low-abundance mRNAs.

Gene, 1983 Nov, 25(2-3), 249 - 62
The expression in yeast of the Escherichia coli galK gene on CYC1::galK fusion plasmids; Rymond BC et al.; A set of gene fusions have been constructed between the transcriptional and translational initiation signals of the yeast CYC1 gene, encoding iso-1-cytochrome c, and the coding sequence of the Escherichia coli galK gene encoding galactokinase . These fusions are contained on plasmids which have both yeast and E . coli replication origins and selectable markers and, therefore, can be used to transform either yeast or E . coli cells . When galactokinase-deficient (gall-) yeasts were transformed with these plasmids the resulting Gal+ transformants were heterogeneous with respect to their galactokinase levels . The galactokinase levels in all were subject to glucose repression, characteristic of the transcriptional regulation of the CYC1 gene . The fusion points for representative plasmids were determined by DNA sequence analysis, and from these data, the differential expression of the galK gene could be explained . One fusion plasmid, YRpR1, which gave the highest level of galK expression, was characterized further . As an additional demonstration that galactokinase expression from the fusion was under CYC1 transcriptional control, a cis-dominant, CYC1-linked mutation known to drastically reduce CYC1 gene transcription was introduced into YRpR1 and shown to similarly effect galK expression . The galK mRNA produced from the fused gene of YCpR1, a centromere-containing derivative of YRpR1, consisted of the mRNA leader sequence plus the first four codons of the CYC1 gene, the galK coding sequence, then the remainder of the CYC1 coding sequence and the 175 nucleotide non-translated 3' sequence . As a demonstration of the usefulness of these plasmids for the selection of regulatory mutants, two mutants capable of greatly enhanced levels of galactokinase expression were isolated . Preliminary characterization of these mutations indicates that they likewise affect the expression of the chromosomal CYC1 gene.

Virology, 1983 Nov, 131(1), 116 - 27
Identification of the binding sites to monoclonal antibodies on A/USSR/90/77 (H1N1) hemagglutinin and their involvement in antigenic drift in H1N1 influenza viruses; Nakajima S et al.; We have determined nucleotide sequences of the HA1 portion of the hemagglutinin (HA) gene of the parental A/USSR/90/70 (H1N1) virus and its eight variants selected in vitro with six monoclonal antibodies to study antigenic determinants . The HA1 gene of one of the variants (B-1-23) was cloned in bacteria and its nucleotide sequence was determined by the Maxam-Gilbert method . The nucleotide sequence of the variant was confirmed by the dideoxy chain termination method . The gene sequences of the other viruses were determined by the latter method . Three variants with reduced reactivity in HI test only with the selecting antibodies possessed one amino acid substitution . On the other hand, most other variants which had the changed reactivity to multiple antibodies in HI test possessed more than one substitution . Comparison of the amino acid sequences of the HA1 molecule, deduced from the nucleotide sequences, suggested that the monoclonal antibodies W18 and 264 reacted with epitopes located on the area involving amino acid residues 125C and 189-190, respectively, whereas, the antibodies 22 and 70 reacted with epitopes involving amino acid residues 129, 132, and 157 . The epitope recognized by antibody 110 overlapped with that of W18, and the epitope recognized by antibody 385 was located on the area involving at least amino acid residues 129, 159, and 189, which overlapped with some of the above epitopes . The sequence analysis with B-1-23 variant selected with antibody 264 clearly showed that in A/USSR/77 viruses, a single substitution at amino acid residue 190 effectively changes the epitope and caused a significant antigenic variation detectable by postinfection ferret sera.

Exp Cell Res, 1983 Nov, 149(1), 177 - 87
Liposome-mediated insertion of intact DNA into isolated nuclei . Potential for a new in vitro transcription system; Marchetti D et al.; DNA molecules sequestered within negatively charged liposomes became nuclei-associated following interaction between isolated mouse plasmacytoma nuclei and liposomes . Few if any liposomes appeared to adhere to washed nuclei following their interaction with liposomes, suggesting that DNA was internalized . Liposome-delivered, radioactive pBR322 DNA re-extracted from nuclei appeared intact, whereas DNA from nuclei incubated with naked DNA was degraded . Up to 2 X 10(10) D of DNA were inserted into each nucleus . DNA delivered into nuclei via liposome was transcribed as shown by the fact that about 1% of the RNA synthesized in nuclei injected with pBR322 or E . coli DNA hybridized with moderate excess of homologous DNA . pBR322-specific RNA synthesized in isolated nuclei consisted of large MW transcripts . Experiments in which SV40 DNA and pBR322 DNA were delivered simultaneously in equimolar amounts into nuclei indicated that SV40 DNA was transcribed as efficiently as pBR322 DNA.

J Exp Med, 1983 Nov 1, 158(5), 1713 - 19
Mannose-sensitive and Gal-Gal binding Escherichia coli pili from recombinant strains . Chemical, functional, and serological properties; O'Hanley P et al.; Chromosomal genes encoding the MS and Gal-Gal binding properties have been cloned into separate recombinants and their respective pili characterized . Hapten inhibition of hemagglutination with synthetic carbohydrate receptor analogues and carbohydrate-adsorbed latex agglutination studies indicate that Gal-Gal and MS pili collectively exhibit the binding properties of the parent strain . MS pili migrated in SDS-PAGE with an Mr of 19 kdaltons and 17 kdaltons; the Mr of Gal-Gal pili was 17.5 kdaltons . The pili are chemically similar by amino acid composition and when the N-terminal cysteines are aligned, 8 of the 13 residues between positions 9 and 22 are homologous . Further, carboxy-terminal sequence homology was inferred from the carboxypeptidase digestion of a MS pili and the sequence of a carboxy-terminal tryptic peptide from Gal-Gal pili.

J Bacteriol, 1983 Nov, 156(2), 529 - 36
A gene affecting accumulation of the RNA moiety of the processing enzyme RNase P; Dallmann G et al.; The level of 10Sb (M1) RNA, the RNA of RNase P, is very low in growing cultures of rnpB mutants . Northern transfer experiments suggested that these strains accumulate no more than 10% of the wild-type level of 10Sb RNA . However, there is no indication that there is a limiting amount of RNase P activity in these mutants in vivo . A plasmid that directs the synthesis of 10Sb RNA does not complement the rnpB mutants, even though there is only a single gene for 10Sb RNA in the Escherichia coli genome . The 10Sb RNA synthesized from this plasmid is equivalent to wild-type 10Sb RNA since it can replace it in the reconstitution of RNase P . The 10Sb RNA, which is a rather stable molecule, is unstable in the presence of the rnpB mutation . This could explain why rnpB mutants do not accumulate 10Sb RNA . An F' plasmid that contains DNA from the rnpB region of the chromosome complements an rnpB mutant in vivo and in vitro, and it also contains the 10Sb RNA gene . A number of possible explanations for these phenomena are discussed.

J Bacteriol, 1983 Nov, 156(2), 752 - 7
Role of glutamine synthetase in the uptake and metabolism of methylammonium by Azotobacter vinelandii; Barnes EM Jr et al.; Methylammonium is a substrate for the ammonium transport system of Azotobacter vinelandii . During cellular uptake methylammonium is rapidly converted to a less polar metabolite (E . M . Barnes, Jr., and P . Zimniak, J . Bacteriol . 146:512-516, 1981) . This metabolite has been isolated from A . vinelandii and identified as gamma-glutamylmethylamide by mass spectroscopy, 1H nuclear magnetic resonance spectroscopy, and cochromatography with the authentic compound . Escherichia coli also accumulated gamma-glutamylmethylamide during methylammonium uptake . The biosynthesis of gamma-glutamylmethylamide in vitro required methylammonium, ATP, L-glutamate, and a soluble cell extract from A . vinelandii . The enzyme responsible for gamma-glutamylmethylamide synthesis was glutamine synthetase . In a crude extract, L-methionine-DL-sulfoximine was equipotent in inhibiting the activities for gamma-glutamyltransferase and for the synthesis of glutamine and gamma-glutamylmethylamide . Likewise, an antiserum against the glutamine synthetase of E . coli precipitated the transferase and both synthetic activities at similar titers . During repression by growth of cells on ammonium medium, the synthesis of glutamine and gamma-glutamylmethylamide in vitro was also inhibited coordinately . A partially purified preparation of glutamine synthetase from A . vinelandii utilized methylammonium as substrate (Km = 78 mM, Vmax = 0.30 mumol/min per mg), although less efficiently than ammonium (Km = 0.089 mM, Vmax = 1.1 mumol/min per mg) . The kinetic properties of glutamine synthetase with methylammonium as substrate as well as the insensitivity of this activity to inhibition by T1+ were strikingly different from methylammonium translocation . Thus, methylammonium (ammonium) translocation and intracellular trapping as glutamylamides are experimentally distinguishable processes.

FEBS Lett, 1983 Oct 31, 163(1), 94 - 8
Immunoelectron microscopy of ribosomes carrying a fluorescence label in a defined position . Location of proteins S17 and L6 in the ribosome of Escherichia coli; Stoffler-Meilicke M et al.; By coupling fluorescein to a defined amino acid of a single ribosomal protein and incorporating this protein into the ribosome, we have obtained ribosomes labelled at a single, defined position . A fluorescein-specific antibody preparation was used to locate the fluorescein residues bound to the two cysteines at positions 58 and 63 of protein S17 and to the cysteine at position 86 of protein L6 . This study demonstrates the advantages which accrue from the combination of electron microscopy and fluorimetry.

Biochim Biophys Acta, 1983 Oct 31, 725(1), 168 - 77
Cytochromes of the trimethylamine N-oxide anaerobic respiratory pathway of Escherichia coli; Bragg PD et al.; Escherichia coli grown anaerobically with trimethylamine N-oxide (TMAO) as a terminal electron acceptor develops a new cytochrome pathway in addition to the aerobic respiratory pathways which are still formed . Formate, NADH, and possibly other substrates derived from glucose, supply electrons to this pathway . Cytochromes with alpha-absorption peaks at about 548, 552, 554 and 557 nm are rapidly reoxidized by TMAO in a reaction which is not inhibited by 2-n-heptyl -4-hydroxyquinone N-oxide . CuSO4 inhibits the reoxidation by TMAO of the first two of these cytochromes . This suggests that the pathway of electron transfer leading to the reduction of TMAO is: substrates leads to cytochromes 548,552 leads to cytochromes 554,557 leads to trimethylamine-N-oxide reductase leads to TMAO . These cytochromes, but not those of the aerobic respiratory pathways, are reoxidized by the membrane-impermeant oxidant ammonium persulfate in intact cells . This suggests that the cytochromes of the TMAO reduction pathway and/or trimethylamine-N-oxide reductase are situated at the periplasmic surface of the cytoplasmic membrane of E . coli.

Virology, 1983 Oct 30, 130(2), 474 - 84
Mechanism of interferon action: human leukocyte and immune interferons regulate the expression of different genes and induce different antiviral states in human amnion U cells; Samuel CE et al.; The inhibition of virus replication and the induction of protein phosphorylation were examined in human amnion U and human fibroblast GM2767A cells treated with highly purified cloned human leukocyte and immune interferons synthesized in Escherichia coli . Both leukocyte interferon (IFN-alpha A) and immune interferon (IFN-gamma) possessed antiviral activity as measured by the single cycle yield reduction of vesicular stomatitis virus (VSV) in the human U and GM2767A cell lines . By contrast, only IFN-gamma and not IFN-alpha A inhibited the single cycle replication of reovirus in U and GM2767A cells . IFN-alpha A, but not IFN-gamma, efficiently induced the double-stranded RNA-dependent phosphorylation of the ribosome-associated protein P1 and the alpha subunit of protein synthesis initiation factor eIF-2 in U cells . However, neither IFN-alpha A nor IFN-gamma induced the phosphorylation of P1 and eIF-2 alpha in GM2767A cells . The antiviral activities of IFN-alpha A and IFN-gamma were synergistic for the inhibition of VSV but not for the inhibition of reovirus or the induction of protein phosphorylation . These results suggest that human leukocyte and immune interferons differentially regulate the expression of certain genes and induce mechanistically distinct antiviral states in human cells.

Virology, 1983 Oct 30, 130(2), 539 - 45
Evolution of the influenza virus neuraminidase gene during drift of the N2 subtype; Martinez C et al.; The complete genetic information for the neuraminidase (NA) gene of influenza virus A/Bangkok/1/79 has been cloned by in vitro synthesis of dsDNA, insertion into pBR322 plasmid, and transformation of Escherichia coli . The nucleotide sequence of the NA gene has been determined by the Maxam and Gilbert method . It is 1466 nucleotides long and contains a single open reading frame with a coding capacity for 469 amino acids . When compared to the NA genes of the N2 strains A/Victoria/3/75, A/Udorn/72, A/NT/60/68, and A/RI/5-/57, 90% of the nucleotide positions and 87% of the amino acid positions remained invariant . Forty-two nucleotide changes and 14 amino acid changes accumulated in the period 1975-1979, but the general structure of the protein appeared to remain constant.

Carbohydr Res, 1983 Oct 28, 122(2), 249 - 56
Structural studies of the Escherichia coli O-antigen 25; Kenne L et al.; The structure of the Escherichia coli O-antigen 25 has been investigated using n.m.r . spectroscopy, methylation analysis, and various specific degradations . It is concluded that the O-antigen is composed of pentasaccharide repeating-units having the following structure.

Biochim Biophys Acta, 1983 Oct 26, 735(1), 131 - 6
Hydration of Escherichia coli lipids . Deuterium T1 relaxation time studies of phosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine; Borle F et al.; The hydration properties of Escherichia coli lipids (phosphatidylglycerol, phosphatidylethanolamine) and synthetic 1,2-dioleoyl-sn-glycero-3-phosphocholine in H2O/2H2O mixtures (9:1, v/v) were investigated with 2H-NMR . Comparison of the 2H2O spin lattice relaxation time (T1) as a function of the water content revealed a remarkable quantitative similarity of all three lipid-H2O systems . Two distinct hydration regions could be discerned in the T1 relaxation time profile . (1) A minimum of 11-16 water molecules was needed to form a primary hydration shell, characterized by an average relaxation time of T1 approximately equal to 90 ms . (2) Additional water was found to be in exchange with the primary hydration shell . The exchange process could be described in terms of a two-site exchange model, assuming rapid exchange between bulk water with T1 = 500 ms and hydration water with T1 = 80-120 ms . Analysis of the linewidth and the residual quadrupole splitting (at low water content) confirmed the size of the primary hydration layer . However, each lipid-water system exhibited a somewhat different linewidth behavior, and a detailed molecular interpretation appeared to be preposterous.

Biochemistry, 1983 Oct 25, 22(22), 5177 - 88
Site-specific pausing of deoxyribonucleic acid synthesis catalyzed by four forms of Escherichia coli DNA polymerase III; LaDuca RJ et al.; Sites on an fd DNA template which terminate synthesis catalyzed by each of four forms of Escherichia coli DNA polymerase III have been identified at single nucleotide resolution . Results were obtained by comparing the products made by forms of DNA polymerase III with products generated from the same 3'-terminus by using the dideoxynucleotide sequencing method, on high-resolution polyacrylamide gel electrophoresis . Each form of DNA polymerase III generates products of distinct lengths ending at a limited number of preferred sites of synthesis termination . The addition of auxiliary subunits to the DNA polymerase III core form of the enzyme has a distinct functional effect on primer elongation and specificity of polymerase pausing . Most sites (65%) can be correlated to positions of potential secondary structure in the template arising via local hydrogen-bonding interactions . The proximity of polymerase pausing to sites adjacent to hairpin stems was related to the size of the enzyme since the smaller core form of DNA polymerase III generally paused at sites which were closer to the base of these structures than the larger holoenzyme . The occurrence of termination sites is markedly affected by the inclusion of spermidine or Escherichia coli single-stranded DNA binding protein in the reaction mixtures . Additionally, a nucleotide composition specificity of pause sites has been observed.

J Mol Biol, 1983 Oct 25, 170(2), 575 - 81
Crystallographic observations of the metal ion triple in the active site region of alkaline phosphatase; Sowadski JM et al.; Diffraction analysis reveals three metal ion binding sites, M1, M2 and M3, in each of two symmetric active centers 32 A apart in alkaline phosphatase from Escherichia coli with intermediate distances within the center of 4, 5 and 7 A for M1-M2, M2-M3 and M1-M3, respectively . A fourth site, M4, has been reported 25 A away . Arsenate, a product analog, binds adjacent to M1 and M2 . The active serine residue, 102, which is phosphorylated during normal enzymatic turnover, is also adjacent to M1 and M2 and arginine 166 is adjacent to the arsenate . The implication with respect to the mechanism is that M1, M2 and Arg 166 neutralize and redistribute charges within the phosphate group, activate the serine hydroxyl, and stabilize transition states during bond formation and breakage . Three sites, A, B and C, have been deduced from solution studies and defined specifically on the basis of nuclear magnetic resonance data, binding studies and activity data . The evidence suggests correspondence of A to M1, B to M2, and C to M3 . Strong antagonism between binding at M1 and M2 is evidenced crystallographically by a pseudo-saturation, which is relieved by phosphate binding . Local destabilization of the protein, particularly residues 323 through 333, is produced by removal of metals from the crystal.

J Mol Biol, 1983 Oct 25, 170(2), 381 - 402
X-ray solution-scattering studies of active and inactive Escherichia coli ribosomal subunits; Kearney KR et al.; That ribosomal subunits can exist in active and inactive functional states, and that subunits in the two states are interconvertible, has been known for some time (see Zamir et al., 1974) . The magnitude of the conformational perturbation accompanying this functional transformation, however, was not known . In the present study 30 S and 50 S subunits in the two functional states have been compared by small-angle X-ray scattering . The results indicate that the structural differences between active and inactive subunits are small, at the limit of resolution of this technique . Model studies show that the data imply conformational differences at or below the limit of resolution of other physical methods now in use to examine the detailed structure of these particles.

J Biol Chem, 1983 Oct 25, 258(20), 12641 - 3
Functional inferences from crystals of Escherichia coli trp repressor; Joachimiak A et al.; We have reproducibly grown crystals of L-tryptophan . trp aporepressor and indole-3-propionate . trp aporepressor complexes from Escherichia coli which are suitable for x-ray diffraction analysis . The active repressor, L-tryptophan . aporepressor, crystallizes in both trigonal (P3(1)21 or P3(2)21) and tetragonal (P4(1)22 or P4(3)22) forms which diffract to at least 2.0 and 2.5 A, respectively . The trigonal form has one-half of the functional dimer/asymmetric unit; therefore, the trp repressor molecule has an axis of 2-fold rotational symmetry corresponding to the lattice dyad . The inactive complex, indole-3-propionate . aporepressor, or "pseudorepressor," forms tetragonal crystals that also diffract to at least 2.5 A and are isomorphous to those of the active repressor . Slight differences between their diffraction patterns indicate modest structural differences between active and inactive complexes that are presumably mediated by the alpha-amino group of L-tryptophan and account for operator-specific binding.

J Biol Chem, 1983 Oct 25, 258(20), 12543 - 7
Theoretical study of hinge bending in L-arabinose-binding protein . Internal energy and free energy changes; Mao B et al.; The L-arabinose-binding protein of Escherichia coli is a periplasmic component of the L-arabinose transport system . Its three-dimensional structure has been determined by x-ray diffraction and shown to have two globular domains and a connecting hinge . These structural features enclose a cleft in which the L-arabinose-binding site is located . The flexibility of the protein hinge that allows hinge-bending motion is investigated here by theoretical analysis of the changes in conformational energy and molecular structure that accompany the opening and closing of the cleft . The hinge of the molecule is found to be quite permissive in that only moderate increases in the internal energy occur upon opening the cleft . Solvation changes of charged groups on the cleft-facing surfaces of the lobes are estimated to make important contributions to the overall energetics of the system . The results indicate that an open conformation for the unliganded protein is stabilized by the exposure and solvation of charged groups in the cleft, and that the cleft is induced to close upon ligand binding . This picture is consistent with experimental data on the structure and the binding kinetics of L-arabinose-binding protein, and provides a physical framework for interpreting such data.

Biochemistry, 1983 Oct 25, 22(22), 5169 - 76
Direct evidence for the preferential binding of Escherichia coli RNA polymerase holoenzyme to the ends of deoxyribonucleic acid restriction fragments; Melancon P et al.; Escherichia coli RNA polymerase holoenzyme has been observed to form a variety of nonpromoter complexes with DNA restriction fragments in experiments performed with the nitrocellulose filter assay {Melancon, P., Burgess, R . R., & Record, M . T., Jr . (1982) Biochemistry 21, 4318-4331} . Here we report the use of this assay to investigate aspects of the weak (heparin-sensitive) interactions of RNA polymerase core and holoenzyme with a 1600 base pair (bp) fragment of T7 DNA which contains no promoters or TB (tight binding; heparin-resistant) sites . Under the ionic conditions investigated {50 mM NaCl/10 mM MgCl2/10 mM sodium N-(2-hydroxyethyl)piperazine-N'-ethanesulfonic acid (pH 7.7)}, both core and holoenzyme bind to the linear DNA fragment and cause comparable levels of filter retention . When the DNA fragment is self-ligated into a circular molecule (nonsupercoiled), the extent of binding of holoenzyme (but not that of core) is dramatically reduced . This directly proves our previous hypotheses that holoenzyme recognizes and preferentially binds to the ends of DNA fragments and that this mode of binding is responsible for most of the heparin-sensitive filter retention of nonpromoter fragments . The residual mode of binding of holoenzyme detected with the circular DNAs was considered in determining the amount of protein bound at ends only . To calculate end-binding constants (KE), the amount of protein bound nonspecifically (which does not appear to cause efficient filter retention) was also taken into consideration . At 0 degrees C, we obtain a value for KE of (2.1 +/- 0.5) X 10(8) M-1, in good agreement with that determined earlier.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleic Acids Res, 1983 Oct 25, 11(20), 7145 - 56
Primary structure and genetic organization of phage T4 DNA ligase; Armstrong J et al.; The primary structure of phage T4 DNA ligase has been determined by DNA sequencing of a cloned restriction fragment containing its gene, and partial amino acid sequence analysis of the protein . The molecule has a Mr of 55,230, and contains 487 amino acids . The DNA sequence may also encode all of one and parts of two other, hitherto unidentified, T4 proteins . The four genes are closely packed, with overlaps between terminator and initiator codons of adjacent genes . Potential terminator and promoter sites for transcription are located within the coding sequence of one of the genes.

Nucleic Acids Res, 1983 Oct 25, 11(20), 7069 - 86
Structure and in vitro transcription of a human H4 histone gene; Sierra F et al.; A human H4 histone gene was isolated and the nucleotide sequences of the mRNA coding as well as the 5' and 3' flanking regions were determined . No intervening sequences were found in this gene . A series of sequences which have been assigned putative regulatory roles in histone genes and/or in other genes were identified both upstream and downstream from the H4 histone protein coding region . Deletion mutants were constructed by BAL-31 nuclease digestion of sequences in the 5' flanking region of this H4 histone gene and were assayed in an in vitro transcription system . No regions upstream from the TATA box were required for site specific initiation in vitro . Data are presented which suggest that sequences located downstream from the 3' end of the coding region may influence the in vitro transcription of this human H4 histone gene.

Nucleic Acids Res, 1983 Oct 25, 11(20), 7011 - 30
Localization of the hsp83 transcript within a 3292 nucleotide sequence from the 63B heat shock locus of D . melanogaster; Hackett RW et al.; We have determined the complete nucleotide sequence of a 3292 bp cloned segment derived from the 63B heat shock cytogenetic locus of D . melanogaster . Within this segment we have positioned the start of transcription and RNA splice sites of the unique gene that encodes the 83,000 d heat shock polypeptide (hsp83 gene) by S1 mapping and synthesis of cDNA from restriction fragment primed mRNA . The sequence begins at a point 879 bp upstream from the transcription start and includes the 149 bp nontranslated first exon, the 1139 bp intron and extends 1125 bp into the protein coding region . These data identify a single open translation reading frame for the first 375 amino acids of the 83,000 d polypeptide, beginning with the first ATG codon located at the 3' intron-exon junction . We discuss and demonstrate the use of E . coli exonuclease III generated single-strand DNA probes as an alternative to strand separation for S1 mapping of mRNA . We also use homology search criteria based upon known protein-DNA binding sites to compare our hsp83 sequence with other sequenced Drosophila heat shock genes . These comparisons indicate that a large region of approximately 80 bp centered around the transcription initiation point of the hsp83 gene shares only a 31% homology with the corresponding region of the hsp70 gene, whereas the hsp22, 23, 26, and 27 genes share a 54% homology with hsp70 in this region . The lower homology of the hsp83 gene is consistent with the deviant nature of this heat shock gene.

J Mol Biol, 1983 Oct 25, 170(2), 305 - 17
Restriction enzyme cleavage of ultraviolet-damaged simian virus 40 and pBR322 DNA; Cleaver JE; Cleavage of specific DNA sequences by the restriction enzymes EcoRI, HindIII and TaqI was prevented when the DNA was irradiated with ultraviolet light . Most of the effects were attributed to cyclobutane pyrimidine dimers in the recognition sequences; the effectiveness of irradiation was directly proportional to the number of potential dimer sites in the DNA . Combining EcoRI with dimer-specific endonuclease digestion revealed that pyrimidine dimers blocked cleavage within one base-pair on the strand opposite to the dimer but did not block cleavage three to four base-pairs away on the same strand . These are the probable limits for the range of influence of pyrimidine dimers along the DNA, at least for this enzyme . The effect of irradiation on cleavage by TaqI seemed far greater than expected for the cyclobutane dimer yield, possibly because of effects from photoproducts flanking the tetranucleotide recognition sequence and the effect of non-cyclobutane (6-4)pyrimidine photoproducts involving adjacent T and C bases.

J Mol Biol, 1983 Oct 25, 170(2), 271 - 85
Complete nucleotide sequence of the structural gene for colicin A, a gene translated at non-uniform rate; Morlon J et al.; The complete nucleotide sequence of the structural gene for colicin A has been established . This sequence consists of 1776 base-pairs . According to the predicted amino acid sequence, the colicin A polypeptide chain comprises 592 amino acids and has a molecular weight of 62,989 . The amino-terminal part is rich in proline and glycine and accordingly secondary structure prediction indicates that this region (1 to 185) is beta-structured . The rest of the molecule (residues 186 to 592) is very rich in alpha-helix . An uncharged amino acid sequence of 48 residues is located in the C-terminal part of the molecule, which is involved in the membrane depolarization caused by colicin A . A similar region has been found in colicin E1, which has the same mode of action as colicin A . Three peptides of these bacteriocins were found to be homologous, but a comparison of the bacteriocin genes did not reveal any significant homology out of the corresponding regions . The codon usage of both genes, however, exhibits some similarity and is quite different from that of genes coding for highly or weakly expressed proteins of Escherichia coli.

J Biol Chem, 1983 Oct 25, 258(20), 12624 - 31
The structure of recA protein-DNA filaments . 2 recA protein monomers unwind 17 base pairs of DNA by 11.5 degrees/base pair in the presence of adenosine 5'-O-(3-thiotriphosphate); Chrysogelos S et al.; recA protein binds to duplex DNA in the presence of Mg2+ and adenosine 5'-O-(3-thiotriphosphate) forming a stiff nucleoprotein filament with a distinct axial repeat which contains 17 +/- 1 base pairs and spans 8-9 nm along the fiber (Di Capua, E., Engel, A., Stasiak, A., and Koller, Th . (1982) J . Mol . Biol . 157, 87-103; Dunn, K., Chrysogelos, S., and Griffith, J . (1982) Cell 28, 757-765) . Measurement of the protein:DNA ratio in these filaments utilizing double label analysis and isopycnic density banding shows that there are 2 recA monomers for every 17 base pairs . The DNA is also partially unwound in this filament . Utilizing the recA-induced relaxation of naturally supertwisted SV40 DNA, we show that the DNA is unwound by 11.5 +/- 1.5 degrees/base pair which corresponds to 180-200 degrees for each repeat unit along the filament length.

J Biol Chem, 1983 Oct 25, 258(20), 12394 - 404
ATP-dependent unwinding of the double helix and extensive supercoiling by Escherichia coli recA protein in the presence of topoisomerase; Iwabuchi M et al.; recA protein, which is essential for genetic recombination in Escherichia coli, causes extensive unwinding of the double helix by an ATP-dependent reaction and accumulation of positive supercoiling in closed circular double-stranded DNA . Initiation of the extensive unwinding was largely dependent on homologous single-stranded DNA . Therefore, it is likely that the extensive unwinding is initiated mainly at the site of D-loops . "Nascent D-loops" in which the two DNA molecules did not interwind were also good initiation sites of extensive unwinding . When the concentration of Mg2+ was decreased from the standard conditions for D-loop formation (13 mM MgCl2; the higher Mg2+ condition) to the lower Mg2+ condition (1 to 2 mM MgCl2), extensive unwinding by recA protein was initiated very quickly in the absence of single-stranded DNA . Results showed that this single-stranded DNA-independent initiation of extensive unwinding (i) requires negative superhelicity of the double-stranded DNA and (ii) is a first order reaction with respect to the DNA . These observations suggest that, under the lower Mg2+ condition, the extensive unwinding starts at a transiently denatured site in the negative superhelical DNA . Once initiated, the unwinding by recA protein is propagated extensively, even under conditions that do not allow its initiation . Therefore, the propagation of unwinding is a processive reaction ("processive unwinding") . Previous studies indicated that recA protein promotes "distributive unwinding" of double helix which depends on single-stranded DNA . Therefore, recA protein promotes unwinding of the double helix by either of two distinct pathways . Stress caused by the processive unwinding could explain the dissociation of D-loops and reversible inactivation of the double-stranded DNA in a D-loop cycle.

J Biol Chem, 1983 Oct 25, 258(20), 12102 - 5
Isolation and characterization of an Escherichia coli clone overproducing prolipoprotein signal peptidase; Tokunaga M et al.; Based on the rationale that Escherichia coli cells containing increased levels of prolipoprotein signal peptidase would be highly resistant to globomycin, a specific inhibitor of the prolipoprotein signal peptidase, we have isolated a clone from the Carbon-Clarke collection, plasmid pLC3-13, which is globomycin-resistant and contains an increased level of prolipoprotein signal peptidase activity . The plasmid pMT521, a subclone of pLC3-13 in pBR322, conferred on its host cells approximately 20 times overproduction of prolipoprotein signal peptidase and an extremely high level of resistance against globomycin . The overproduced prolipoprotein signal peptidase was completely inhibited by the presence of globomycin in the in vitro assay, and the overproduced activity was found in the cell envelope fraction . Several lines of biochemical and genetic evidence suggest that the gene contained in pLC3-13 and its derivative clones is most likely the structure gene (lsp) for prolipoprotein signal peptidase.

J Biol Chem, 1983 Oct 25, 258(20), 12712 - 7
Molecular cloning of a cDNA sequence for rat malic enzyme . Direct evidence for induction in vivo of rat liver malic enzyme mRNA by thyroid hormone; Magnuson MA et al.; Malic enzyme (EC 1.1.1.40) mRNA was partially purified to 12% of total mRNA activity (greater than 150-fold enrichment) from 3-5-3'-triiodo-L-thyronine-carbohydrate-stimulated rat liver by polysome immunopurification followed by oligo(dT)-cellulose chromatography . This preparation was used as the template for synthesis of cDNA on a pBR322-SV40 hybrid plasmid DNA primer which was then circularized with linker DNA and used to transform Escherichia coli . The resulting transformants were screened by in situ differential hybridization using 32P-labeled poly(A+)RNA prepared from uninduced and 3-5-3'-triiodo-L-thyronine-carbohydrate-induced rat livers . Of 750 transformants screened, 6 were found to hybridize differentially; 1 of these, prME, contained an insert of about 1250 base pairs and hybrid-selected an mRNA which directed synthesis of malic enzyme in a cell-free translation system . Using this cDNA as a probe, we demonstrated that the level of malic enzyme mRNA after thyroid hormone treatment was markedly increased and the size of the major malic enzyme mRNA was shown by Northern analysis to be about 2700 nucleotides in length.

Eur J Biochem, 1983 Oct 17, 136(1), 83 - 7
Affinity labeling of succinyl-CoA synthetase from Escherichia coli by the 2',3'-dialdehyde derivative of adenosine 5'-diphosphate; Nishimura JS et al.; The 2'3'-dialdehyde of adenosine 5'-diphosphate, oADP, exhibited the properties of an affinity label with Escherichia coli succinyl-CoA synthetase . Inactivation of this synthetase by oADP followed pseudo-first-order kinetics and was competitively blocked by ADP . The stoichiometry of labeling of the synthetase was 1 mol/mol alpha beta or, extrapolated, 2 mol/mol inactive alpha 2 beta 2 molecule . oADP also exhibited the properties of a substrate, bringing about rapid dephosphorylation of the enzyme . Further specificity of oADP was demonstrated in partially inactivated succinyl-CoA synthetase by selective inhibition of the succinate in equilibrium succinyl-CoA exchange reaction, in comparison to the CoA in equilibrium succinyl-CoA exchange reaction . Modification of the synthetase by oADP resulted in cross-linking of the enzyme, casting uncertainty over the subunit binding site for ADP . Modification of the synthetase by ADP-2'-semialdehyde occurred at a faster rate than that by oADP but exhibited biphasic inhibitor concentration dependence and did not exhibit saturability.

Biochim Biophys Acta, 1983 Oct 17, 748(1), 99 - 108
Associative properties of the Escherichia coli galactose-binding protein and maltose-binding protein; Richarme G; The ligand-binding characteristics of periplasmic galactose-binding protein and maltose-binding protein of Escherichia coli are analyzed . The saturation function was decreased upon increasing protein concentration and the monomer-dimer equilibrium was shifted towards the monomeric protein form upon an increase of the ligand concentration . An association constant K0 = 6 X 10(8) M-1 was found for the galactose-binding protein . These data fit a monomer-dimer system in equilibrium, in which the monomer has a higher affinity for the ligand.

Biochim Biophys Acta, 1983 Oct 17, 748(1), 34 - 9
Inhibition of acetohydroxy acid synthase by leucine; Gollop N et al.; The enzymatic reaction of acetohydroxy acid synthase in crude extracts of Escherichia coli K-12 is inhibited by leucine . Inhibition is most pronounced at low pH values and is low at pH values higher than 8.0 . Both isoenzymes of acetohydroxy acid synthase present in E . coli K-12 (isoenzyme I and isoenzyme III) are inhibited by leucine . Isoenzyme I, which is responsible for the majority of acetohydroxy acid synthase activity in E . coli K-12 at physiological pH, is inhibited almost completely by 30 mM leucine at pH 6.25-7.0 and is not affected at all at pH values higher than 8.4 . Inhibition of isoenzyme I by leucine is a mixed noncompetitive process . Leucine inhibition of isoenzyme III is pH-independent and reaches only 40% at 30 mM leucine . The inhibition of acetohydroxy acid synthase by leucine at physiological pH, observed in vitro in this study, correlates with the idea that acetohydroxy acid synthase is a target for the toxicity of the abnormally high concentrations of leucine in E . coli K-12.

Eur J Biochem, 1983 Oct 17, 136(1), 147 - 50
Preparation and properties of monomercurated tRNA; Abdurashidova GG et al.; Monomercurated tRNA has been prepared by exhaustive treatment of mercaptoethanol on polymercurated tRNA under non-denaturing conditions . The remained mercury atom is stable toward ultraviolet (254 nm) irradiation up to absorption of 100 quanta/nucleotide, but could be easily removed by mercaptoethanol under denaturing conditions . Monomercurated tRNA quantitatively binds to thiopropyl-Sepharose and can be completely desorbed by mercaptoethanol-containing solutions . Monomercurated tRNA is similar to the starting tRNA preparation in efficiency and specificity of aminoacylation and in factor-dependent polypeptide synthesis in vitro.

Int J Cancer, 1983 Oct 15, 32(4), 431 - 5
Chromosomal aberrations in a case of T-cell CLL with concomitant IgA myeloma; Zech L et al.; Four consistent markers, involving chromosomes 8, 11, 14, 18, 21 and 22, were found in the majority of metaphases obtained after stimulation of peripheral blood and bone marrow from a patient with a T-cell chronic lymphocytic leukemia (CLL) and a concomitant IgA/lambda myeloma with phytohemagglutinin (PHA), lipopolysaccharide of E . coli (LPS) and S . aureus strain Cowan I bacteria (Cowan) . A second malignant clone with three additional markers was observed after stimulation with Concanavalin A (Con A) . A chromosome 14 aberration, inv(14) (q11-q32), may be of significance in the T-cell malignancy and may suggest a common clonal origin of the two tumors.

Anal Biochem, 1983 Oct 15, 134(2), 489 - 94
An ultraviolet spectrophotometric assay for aspartate transcarbamylase; Foote J; A procedure for determining the activity of aspartate transcarbamylase, based upon the greater ultraviolet absorbancy of the products of the reaction catalyzed compared to the reactants, was devised . Extinction coefficients were determined at 205, 210, and 215 nm for the compounds carbamoyl aspartate, acetyl aspartate, and aspartate . These values formed the quantitative basis for a spectrophotometric assay in which an enzymatic reaction is monitored at one of these wavelengths . Use of this procedure was illustrated in four kinetic experiments with the allosteric aspartate transcarbamylase from Escherichia coli, and the nonallosteric catalytic subunit of this enzyme: aspartate saturation curve, arsenate saturation curve (reverse reaction), allosteric activation by a transition-state analog employing acetyl phosphate as substrate, and carbamoyl phosphate progress curve (substrate depletion in the presence of excess cosubstrate) . Owing to changes in absorbance on the order of 1000 liter mol-1 cm-1 concomitant with the reaction, the sensitivity of the method is comparable to that of many procedures already in the literature.

Arch Biochem Biophys, 1983 Oct 15, 226(2), 687 - 92
Uridine phosphorylase from Escherichia coli B.: kinetic studies on the mechanism of catalysis; Vita A et al.; Using a highly purified enzyme preparation of uridine phosphorylase from Escherichia coli B, we have performed detailed kinetic studies which include initial-velocity and product-inhibition experiments in the forward and reverse directions of the reaction . These studies indicate a rapid-equilibrium random mechanism for this enzyme with the formation of an enzyme . uracil phosphate abortive complex . Lack of formation of the enzyme . uridine . ribose-1-phosphate abortive complex suggests that the ribosyl moiety of the two ligands compete for the same binding site . The random mechanism is different from the ordered addition of substrates found for uridine phosphorylase from other sources . All the kinetic constants in the forward and reverse directions and the Keq of reaction for E . coli uridine phosphorylase are reported herein.

Arch Biochem Biophys, 1983 Oct 15, 226(2), 657 - 65
Inhibition of sugar uptake by ascorbic acid in Escherichia coli; Loewen PC et al.; The uptake of glucose by the glucose phosphotransferase system in Escherichia coli was inhibited greater than 90% by ascorbate . The uptake of the nonmetabolizable analog of glucose, methyl-alpha-glucoside, was also inhibited to the same extent, confirming that it was the transport process that was sensitive to ascorbate . Similarly, it was the transport function of mannose phosphotransferase for which mannose and nonmetabolizable 2-deoxyglucose were substrates that was partially inhibited by ascorbate . Other phosphotransferase systems, including those for the uptake of sorbitol, fructose and N-acetylglucosamine, but not mannitol, were also inhibited to varying degrees by ascorbate . The inhibitory effect on the phosphotransferase systems was reversible, required the active oxidation of ascorbate, was sensitive to the presence of free-radical scavengers, and was insensitive to uncouplers . Because ascorbate was not taken up by E . coli, it was concluded that the active inhibitory species was the ascorbate free radical and that it was interacting reversibly with a membrane component, possibly the different enzyme IIB components of the phosphotransferase systems . Ascorbate also inhibited other transport systems causing a slight reduction in the passive diffusion of glycerol, a 50% inhibition of the shock-sensitive uptake of maltose, and a complete inhibition of the proton-symport uptake of lactose . Radical scavengers had little or no effect on the inhibition of these systems.

Biochem J, 1983 Oct 15, 216(1), 143 - 50
Oxidative phosphorylation by mutant Escherichia coli membranes with impaired proton permeability; Cox GB et al.; The effect on the function of the Escherichia coli F1F0-ATPase of the substitution of leucine-31 by phenylalanine in the c-subunit of the enzyme was examined . The assembly of the mutant c-subunit requires an increased gene dosage {Jans, Fimmel, Langman, James, Downie, Senior, Ash, Gibson & Cox (1983) Biochem . J . 211, 717-726}, and this was achieved by incorporation of the uncE408 or uncE463 alleles on to F-plasmids or multicopy plasmids . Membranes from strains carrying either the uncE463 or uncE408 alleles on F-plasmids or multicopy plasmids were capable of carrying out oxidative phosphorylation . In particular, membranes from strain AN1928 (pAN162, uncE463) gave phosphorylation rates and P/O ratios equal to or greater than those obtained for the control strain AN1460 (pAN45, unc+) . However, the mutant membranes, on removal of the F1-ATPase, appeared to be proton-impermeable . The ATPase activity of the mutant membranes was also resistant to the inhibitor dicyclohexylcarbodi-imide.

Virology, 1983 Oct 15, 130(1), 195 - 203
Human interferon alpha enters cells by receptor-mediated endocytosis; Zoon KC et al.; A small number of Escherichia coli-derived human interferon alpha A molecules (up to approximately 800/cell) bind specifically to high-affinity cell surface receptors on bovine kidney cells at 4 degrees . When the cells are subsequently warmed to 37 degrees, the amount of surface-bound interferon alpha (IFN-alpha) as recognized by a radiolabeled monoclonal antibody rapidly decreases . Using 125I-IFN-alpha and treatment of cells with acetic acid/sodium chloride to remove surface-bound IFN, a similar decrease of surface-bound IFN and a corresponding increase in intracellular radiolabeled material is observed following the temperature shift . An analysis of the trichloroacetic acid precipitability of the radiolabeled material in the medium, removed by acid treatment or internalized, shows that IFN is degraded intracellularly and that its degradation products are then released into the medium . The process of uptake, degradation, and release of degraded material can be inhibited by the lysosomotropic agent chloroquine . A stable conjugate of IFN with 5-nm colloidal gold was prepared without a detectable loss of antiviral activity . As shown by transmission electron microscopy, the conjugate, bound to cells at 4 degrees, was found in clathrin-coated pits and later in receptosomes following a temperature shift to 37 degrees . Morphometric quantitation showed that an excess of native IFN added during binding of the conjugate in the cold reduced the appearance of conjugate in receptosomes by 80% . These studies demonstrate that at least a portion of receptor-bound IFN enters cells by receptor-mediated endocytosis.

J Mol Biol, 1983 Oct 15, 170(1), 61 - 91
Cointegrate formation mediated by Tn9 . II . Activity of IS1 is modulated by external DNA sequences; Chandler M et al.; We have studied the effect on the transposition activity of IS1 of the position and orientation of the element in the donor replicon . Previous studies have shown that the IS1s in the compound transposon Tn9 have different activities in forming plasmid cointegrates . In this study, designed to investigate the sources of this differential activity, we have cloned Tn9 (together with 132 base-pairs of flanking DNA) into several sites in pBR322 and created various derivatives of these plasmids to be used as donor molecules in plasmid fusion experiments . These pBR322-Tn9 plasmids were allowed to form cointegrates with a conjugative, F-derived plasmid, and a large collection of independently formed cointegrates was isolated . The relative activities of the two IS1 elements of Tn9 were estimated by examining the structures of the cointegrates for the frequency of usage of each of the IS1s in cointegrate formation . The results suggest that IS1 activity is modulated by the transcription activity of adjacent DNA sequences in the donor plasmids . Transcription directed into an IS1 inhibits its activity . Confirmatory evidence for this hypothesis is provided by the observation that deletion of adjacent promoters of transcription relieves the inhibitory effect on IS1 activity.

Biochem Biophys Res Commun, 1983 Oct 14, 116(1), 222 - 9
Correlation between structure of polyoxotungstates and their inhibitory activity on polymerases; Herve M et al.; The 21-tungsto-9-antimonate (TA, HPA 23), a polyoxotungstate, has shown a significant antiviral activity in vivo and in vitro . It inhibits viral and bacterial DNA polymerases . In this paper, several compounds of two polyoxotungstic families, tungstoantimonates and tungstoarsenates, have been used to specify the mechanism of polymerase inhibition . It has been demonstrated that the inhibitory activity of polyoxotungstates is not related to the occupation of their coordinates sites by cations, nor to the nature of these bound cations . Kinetic studies and binding assays have shown that polyoxotungstates bind to the polymerases in competition with the nucleic acid template . This result seems to be related to their polyanionic nature . Furthermore, the size and charge of these compounds may play a prominent part in their affinity for the polymerases.

Biochim Biophys Acta, 1983 Oct 13, 741(1), 70 - 6
Characterization of the fluorodihydrouracil substituent in 5-fluorouracil-containing Escherichia coli transfer RNA; Horowitz J et al.; The fluorodihydrouridine derivative previously detected in one of two isoaccepting forms of FUra-substituted Escherichia coli tRNAMetf has been further characterized . This substituent is responsible for the 19F resonance observed 15 ppm upfield from free FUra (= 0 ppm) in the high resolution 19F-NMR spectra of FUra-substituted tRNA purified by chromatography on DEAE-cellulose, at pH 8.9, to remove normal tRNA . Similar highfield 19F signals have now been observed in the spectra of two other purified fluorinated E . coli tRNAs, tRNAMetm and tRNAVal1, as well as in unfractionated tRNA, indicating the widespread occurrence of the constituent . Comparison with 19F spectrum of the model compound 5'-deoxy-5-fluoro-5,6-dihydrouridine (dH56FUrd) (delta FUra = -31.4 ppm; JHF = 48 Hz) indicates that the substituent does not contain an intact fluorodihydrouridine ring . dH56FUrd is considerably more alkali labile than 5,6-dihydrouridine (H56Urd) . At pH 8.9, where H56Urd is stable, dH56FUrd is degraded to a derivative, presumably a fluoroureidopropionic acid, with a 19F resonance at - 15.7 ppm that nearly coincides with the upfield peak in the spectrum of pH 8.9-treated tRNA . The 19F-NMR spectrum of fluorinated tRNA, not exposed to pH 8.9, exhibits two peaks 31 and 32 ppm upfield of FUra, in place of the 19F signal at - 15 ppm . Hydrolysis of this tRNA with RNAase T2 produces a sharp doublet 33 ppm upfield (JHF = 45 Hz) . Similarities of the 19F chemical shift and coupling constant to those of dH56FUrd, allows assignment of the peak at -33 ppm to an intact fluorodihydrouridine residue in the tRNA . Our results demonstrate that FUra residues incorporated into E . coli tRNA at sites normally occupied by dihydrouridine can be recognized by tRNA-modifying enzymes and reduced to fluorodihydrouridine . This substituent is labile at moderately alkaline pH values and undergoes ring-opening during purification of the tRNA.

Biochim Biophys Acta, 1983 Oct 13, 741(1), 23 - 9
The effect of ionic and temperature shifts used for in vitro ribosome subunit reconstitution upon the large molecular weight ribosomal ribonucleic acids; Sykes J et al.; The behaviour of purified, intact preparations of 16 S and 23 S rRNAs has been studied in their respective ribosome subunit reconstitution systems by means of sedimentation and electrophoretic analysis . Both species undergo profound conformation changes to more compact species at the temperatures and ionic conditions commonly agreed for their reconstitution into ribosome subunits . The 16 S rRNA undergoes a complete conformation change over the input range of concentration used, whereas the change for 23 S rRNA is incomplete for inputs above 1.5 mg X ml-1 . Intact 23 S rRNA is also required and some preparations recommended for r-protein binding studies do not meet this requirement for reconstitution . These observations are discussed in relation to the overall effectiveness of ribosome subunit reconstitution systems.

Biochemistry, 1983 Oct 11, 22(21), 4905 - 15
Influence of local nucleotide sequence on substitution of 2-aminopurine for adenine during deoxyribonucleic acid synthesis in vitro; Pless RC et al.; Three highly purified DNA polymerases, Escherichia coli polymerase I (enzyme A) and the polymerases induced by wild-type T4 phage and by T4 phage mutant L141 (antimutator phenotype), have been examined with respect to their tendency to incorporate the deoxyribonucleotide of 2-aminopurine {(AP)} for deoxyadenylate at specific sites in deoxyribonucleic acid (DNA) . Using phi X174 phage DNA as a template and selected phi X174 restriction fragments as specific primers, we synthesized short sequences of phi X174 DNA in vitro by the polymerase of interest, with the 5'-triphosphate of 2-aminopurine deoxyriboside and dATP at equimolar concentration . The relative incorporation of (AP) at the various adenine sites was determined by providing the newly synthesized DNA fragment with a specific terminal radioactive label, subjecting the DNA fragment to thermal depurination as a DNA cleavage reaction highly selective for (AP), and analyzing the resulting radioactive fragments by denaturing gel electrophoresis, autoradiography, and microdensitometry . The L141 polymerase shows very pronounced site-dependent variations in (AP) incorporation . For the wild-type T4 polymerase, the pattern of (AP) incorporation follows the biases seen for the L141 enzyme, although in a less pronounced form . Sequence preferences for (AP) incorporation are least marked for E . coli polymerase I (enzyme A); in several instances, they run counter to the sequence biases observed with the T4 enzymes . For the enzyme showing the most pronounced sequence effects, L141 polymerase, the extent of (AP) incorporation was determined at 57 different sites . No simple principle governing the sequence dependence of (AP) incorporation could be deduced from these results.

Nucleic Acids Res, 1983 Oct 11, 11(19), 6787 - 802
Nucleotides in 16S rRNA that are required in unmodified form for features recognized by ribosomal protein S8; Thurlow DL et al.; Nucleotides in 16S rRNA which are required in unmodified form for specific recognition of ribosomal protein S8 from Escherichia coli were identified using a damage-selection experimental approach . Prior to complex formation with S8, 16S rRNA was treated under fully denaturing conditions with either diethyl pyrocarbonate or 25% hydrazine . Following separation of bound from unbound fragments of RNA, those associated with S8 were analyzed for their content of modified bases by treatment with aniline . Nucleotides found to be consistently unmodified in such fragments were located near the base of a stable helix (encompassing bases 581-656) or near the apex of the helix on the 3' proximal side . A minor S8 ribonucleoprotein particle was found to contain fragments which extended in the 3' direction to position 671.

Nucleic Acids Res, 1983 Oct 11, 11(19), 6821 - 35
Construction of infectious potato spindle tuber viroid cDNA clones; Cress DE et al.; Contiguous restriction fragments from two cloned partial-length potato spindle tuber viroid (PSTV) cDNAs were used to construct recombinant DNAs containing full-length monomeric and dimeric PSTV cDNA . When five different PSTV cDNA plasmids and RNA isolated from E . coli cells harboring these plasmids were tested for infectivity on tomato, plasmid DNAs containing PSTV cDNA dimers were infectious . RNA transcripts containing the sequence of PSTV from these plasmids were also infectious . The sequences of the viroid progeny and the cloned DNA were identical . In vitro mutagenesis of infectious PSTV cDNAs will allow systematic investigation of the role of specific sequences in viroid replication and pathogenesis.

Nucleic Acids Res, 1983 Oct 11, 11(19), 6647 - 66
DNA sequence elements required for regulated expression of the HSV-1 glycoprotein D gene lie within 83 bp of the RNA capsites; Everett RD; The genes of Herpes simplex virus type 1 (HSV-1) are classified into three temporally regulated groups . The Immediate-Early (IE) genes are transcribed first by the pre-existing transcription apparatus of the cell . The Early genes are transcribed only after IE-gene expression, and finally the Late genes are activated . The control of transcription of the HSV-1 glycoprotein D (gD) gene (an Early function) was studied by quantitative S1 mapping of RNA produced in HSV-1 infected HeLa cells after short-term transfection experiments using plasmids containing the gD promoter linked to the rabbit beta-globin gene . The viral promoter in the plasmid was activated in the same way as that in the virus itself; the RNA showed a similar time-course of appearance, dependence on prior IE-gene expression and pattern of RNA cap-sites . Deletion analysis showed that the DNA sequences necessary for Early promoter activation lie within 83 bp of the RNA cap-sites in this instance . Surprisingly, a plasmid-borne beta-globin promoter was also activated by HSV-1 infection . The mechanism of this activation, and DNA sequence similarities between the promoters of HSV-1 Early and rabbit beta-globin genes are discussed.

J Biol Chem, 1983 Oct 10, 258(19), 11745 - 50
Loss of positional specificity in the aminoacylation of Escherichia coli tRNAGly; Ehrenfeld GM et al.; The positional specificity in the aminoacylation of Escherichia coli tRNAGly by its cognate aminoacyl-tRNA synthetase has been studied using tRNAGlys terminating in 2'- or 3'-deoxyadenosine under conditions believed to alter tRNA conformation . Although E . coli tRNAGly terminating in 3'-deoxyadenosine has been reported not to be a good substrate for activation by the homologous glycyl-tRNA synthetase, by systematic variation of the conditions employed for aminoacylation it was possible to activate this tRNA to essentially the same extent as unmodified tRNAGly . Activation of tRNAGly terminating in 3'-deoxyadenosine was carried out optimally at 45 degrees C in an incubation mixture containing 0.3-0.4 M NaCl; 10% methanol, ethanol, and dimethyl sulfoxide were found to facilitate activation of the modified tRNA . Interestingly, the conditions employed to enhance activation of this modified tRNAGly had no effect on the activation of unmodified tRNAGly or tRNAGly terminating in 2'-deoxyadenosine . These experiments afford insight into the activation of tRNAGly by glycyl-tRNA synthetase and provide facile access to positionally defined, isomeric glycl-tRNAGlys.

J Biol Chem, 1983 Oct 10, 258(19), 12034 - 42
S1 nuclease mapping analysis of ribosomal RNA processing in wild type and processing deficient Escherichia coli; King TC et al.; S1 nuclease mapping was used to assess rRNA processing in Escherichia coli . Single-stranded DNA probes complementary to the sequences bordering each terminus of 16 S and 23 S rRNA were end-labeled, hybridized to total E . coli RNA, and treated with S1 nuclease . The resultant DNA fragments were then displayed on denaturing polyacrylamide gels . Measurements of steady state levels of precursor rRNA species and measurements of the rates of decay of precursors after transcription arrest by rifampicin gave consistent results . 1) The rRNA precursor species identified in wild type cells corresponded to those previously identified by other means . 2) In RNase III-deficient strains, mature 16 S rRNA termini form at the same rate as in wild type cells; but the normal mature termini of 23 S rRNA are never generated . 3) RNase III cleavage at the 5' end of 23 S rRNA can occur before the 3' end of the same molecule is synthesized . 4) The cleavages that generate the mature termini of 16 S rRNA are interdependent; in the BUMMER strain, slow processing at the 5' end is accompanied by slow processing at the 3' end . Thus, the kinetically observed order of processing reactions is obligate for some cleavages but not for others, and the assumption that complete rRNA processing is required for function fails for 23 S rRNA.

J Biol Chem, 1983 Oct 10, 258(19), 11879 - 82
Activation by thiol of the latent NAD glycohydrolase and ADP-ribosyltransferase activities of Bordetella pertussis toxin (islet-activating protein); Moss J et al.; Pertussis toxin (islet-activating protein) activates adenylate cyclase in susceptible cells by ADP-ribosylating an inhibitory component of the cyclase system . This toxin, assayed in a cell-free system in the presence of high concentrations of thiol, catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide . This NAD glycohydrolase activity co-chromatographed on Sephacryl G-200 in 6.5 M urea, pH 3.2, 0.1 M glycine with the ADP-ribosyltransferase activity of the toxin, as monitored by the transfer of {32P}ADP-ribose from {32P}NAD to a 41,000-Da protein in NG108-15 neuroblastoma X glioma hybrid cells . In the absence of thiol, the native holotoxin was enzymatically inactive . Following addition of 250 mM dithiothreitol to the assay, maximal enzymatic activity was evident after a delay of approximately 1 h; with 20 mM thiol, the delay was longer . The Km for NAD with the fully activated enzyme was 25 microM; the Km did not appear to vary with the extent of activation . Thiol was necessary in a cell-free system to demonstrate NAD glycohydrolase activity . When extensively washed membranes were used as a source of 41,000-Da substrate, thiol was necessary to observe ADP-ribosylation in some cases (human erythrocytes) and significantly stimulated activity in others (NG108-15 cells) . In contrast to the bacterial toxins choleragen and Escherichia coli heat-labile enterotoxin that ADP-ribosylate stimulatory components of the cyclase system, pertussis toxin did not transfer ADP-ribose to low molecular weight guanidino compounds, such as arginine or agmatine.

J Biol Chem, 1983 Oct 10, 258(19), 11571 - 5
Purification and characterization of herpes simplex virus (type 1) thymidine kinase produced in Escherichia coli by a high efficiency expression plasmid utilizing a lambda PL promoter and cI857 temperature-sensitive repressor; Waldman AS et al.; The structural gene for herpes simplex virus (type 1) thymidine kinase was cloned downstream from the lambda phage high efficiency leftward promotor in a plasmid (pHETK2) also containing the gene for the lambda cI857 temperature-sensitive repressor . Thymidine kinase is synthesized as a run-on product containing the NH2 terminus of the lambda N protein . Heat inactivation of the lambda repressor by growth at 42 degrees C results in the accumulation of thymidine kinase as approximately 4% of the total soluble cellular protein . Thymidine kinase has been purified to greater than 95% homogeneity by high speed centrifugation, ammonium sulfate fractionation, and Sephadex G-100 and hydroxylapatite column chromatography . Thymidine kinase has a subunit Mr = 42,000 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and behaves as a dimer during Sephadex G-100 chromatography and glycerol gradient centrifugation . Thymidine kinase is enzymatically active from pH 6 to 10 with maximum activity at pH 8.5 . The enzyme is protected from heat inactivation by thymidine and has a half-life at 40 degrees C of 30 min in the presence of thymidine and 3 min in its absence . Thymidine kinase displays Michaelis-Menten kinetics with apparent Michaelis constants of 0.6 and 118 microM for thymidine and ATP, respectively . Iododeoxycytidine is a competitive inhibitor of thymidine with an apparent Ki of 14 microM . The anti-herpes drug acyclovir (9-{(2-hydroxyethoxy)methyl}guanine) also appears to be a competitive inhibitor of thymidine (Ki of approximately 300 microM) but requires 3,000-fold higher concentrations than thymidine to give 50% inhibition . Other nucleoside triphosphates can substitute for ATP in the kinase reaction with the exception of dTTP which appears to inhibit thymidine kinase activity by about 50% when present in concentrations equal to that of thymidine.

J Biol Chem, 1983 Oct 10, 258(19), 11465 - 70
Nuclear genes coding the yeast mitochondrial adenosine triphosphatase complex . Isolation of ATP2 coding the F1-ATPase beta subunit; Saltzgaber-Muller J et al.; A yeast nuclear pet mutant of Saccharomyces cerevisiae lacking any detectable mitochondrial F1-ATPase activity was genetically complemented upon transformation with a pool of wild type genomic DNA fragments carried in the yeast Escherchia coli shuttle vector YEp 13 . Plasmid-dependent complementation restored both growth of the pet mutant on a nonfermentable carbon source as well as functional mitochondrial ATPase activity . Characterization of the complementing plasmid by plasmid deletion analysis indicated that the complementing gene was contained on adjoining BamH1 fragments with a combined length of 3.05 kilobases . Gel analysis of the product of this DNA by in vitro translation in a rabbit reticulocyte lysate programmed with yeast mRNA hybrid selected by the plasmid revealed a product which could be immunoprecipitated by antisera against the beta subunit of the yeast mitochondrial ATPase complex . A comparison of the protein sequence derived from partial DNA sequence analysis indicated that the beta subunit of the yeast mitochondrial ATPase complex exhibits greater than 70% conservation of protein sequence when compared to the same subunit from the ATPase of E . coli, beef heart, and chloroplast . The gene coding the beta subunit (subunit 2) of yeast mitochondrial adenosine triphosphatase is designated ATP2 . The utilization of cloned nuclear structural genes of mitochondrial proteins for the analysis of the post-translational targeting and import events in organelle assembly is discussed.

J Biol Chem, 1983 Oct 10, 258(19), 11828 - 33
Oxidative modification of glutamine synthetase . II . Characterization of the ascorbate model system; Levine RL; The first step in the proteolytic degradation of bacterial glutamine synthetase is a mixed function oxidation of one of the 16 histidine residues in the glutamine synthetase subunit (Levine, R.L . (1983) J . Biol . Chem . 258, 11823-11827) . A model system, consisting of oxygen, a metal ion, and ascorbic acid, mimics the bacterial system in mediating the oxidative modification of glutamine synthetase . This model system was studied to gain an understanding of the mechanism of oxidation and of factors which control the susceptibility of the enzyme to oxidation . Availability of substrates and the extent of covalent modification of the enzyme (adenylylation) interact to modulate susceptibility of the enzyme to oxidation . This interaction provides the biochemical basis for physiologic regulation of intracellular proteolysis of glutamine synthetase . The oxidative modification requires hydrogen peroxide . While the reaction may involve Fenton chemistry, the participation of free radicals, superoxide anion, and singlet oxygen could not be demonstrated.

J Theor Biol, 1983 Oct 7, 104(3), 401 - 16
Codon-level analysis of histone primary sequence: evidence of a repeat tetrapeptide origin and later inclusion of transcribed sequence; Brown AP; This work is directed to the question of protein sequence conservation . By reference to the genetic code the aminoacyl sequence of histones H2A, H4, H3, H2B and H1 (fragment) were rewritten as the codon sequences . The N-terminal regions were set aside on the grounds of different composition and sequence . The remainder of the molecule could be referred to simple repeat-tetrapeptide proteins by codon composition (high Gxy, low xGy content) and by sequence . Random segments of three to six residues occur characterized by composition and sequence as originating from the complimentary DNA strand, i.e . as codon "transcript" . Ancestral features are probably best seen in H3, point mutations appear to be more extensive in H2B and H1 . Segments in reverse order in H2A and in "transcript" in H4 distinguish these two from the other three histones . There is a tenuous possibility the N-terminals also originated as repeat-tetrapeptide now intensively modified . At codon-level the 50S ribosomal protein (L7/L12) of E . coli has features in common with histones (including a palindrome-containing N-terminal) . It has the composition and sequence of a well-conserved tetrapeptide-repeat strand (statistical support) . If interpretations made here are substantially correct, the 50S r-protein illustrates a significant stage in evolution of histone codon strands.

Science, 1983 Oct 7, 222(4619), 62 - 5
In vivo determination of the pyridine nucleotide reduction charge by carbon-13 nuclear magnetic resonance spectroscopy; Unkefer CJ et al.; An intracellular coenzyme has been observed by carbon-13 nuclear magnetic resonance spectroscopy . The pyridine nucleotides in Escherichia coli were specifically labeled with carbon-13 from the biosynthetic precursor, nicotinic acid . The intracellular redox status and metabolic transformations of the pyridine nucleotides were examined under a variety of conditions . A highly reduced nicotinamide adenine dinucleotide pool was observed under anaerobic conditions only in cells that were cultured aerobically on glycerol.

Nature, 1983 Oct 6-12, 305(5934), 554 - 6
Uridine-33 in yeast tRNA not essential for amber suppression; Bare L et al.; The nucleotide at position 33 on the 5' side of the anticodon of almost all tRNAs is a uridine . Crystallographic studies of different tRNAs reveal that although the precise orientation of uridine-33 is not always the same, it connects the anticodon stacked along the 3' side of the loop with the pyrimidine-32 stacked on the 5' side of the loop . The remarkably conserved nature of uridine-33 and its unique position in the anticodon loop structure has led to suggestions that this nucleotide has an essential role in the translational mechanism . We have developed a biochemical procedure to replace nucleotides 33-35 in yeast tRNATyr with any desired sequence and used it to construct amber suppressor tRNAs having different nucleotides at position 33 . As all of these synthetic amber suppressor tRNAs functioned well in eukaryotic in vitro suppression assays, we conclude that uridine-33 does not have an obligatory role in the translation mechanism.

Biochim Biophys Acta, 1983 Oct 4, 760(1), 154 - 62
Purification and properties of the antizymes of Escherichia coli to ornithine decarboxylase; Heller JS et al.; The purification of the antizymes to ornithine decarboxylase of Escherichia coli to homogeneity is detailed . An acidic component, pI 3.8, and two basic histone-like proteins, pI above 9.5, are described . The two latter proteins constitute approximately 90% of the total antizyme activity.

FEBS Lett, 1983 Oct 3, 162(1), 39 - 42
Template-independent synthesis of guanosine tetra- and pentaphosphates on ribosomes; Belitsina NV et al.; It is shown that Escherichia coli ribosomes carrying poly(Lys)-tRNA can form (p)ppGpp in the presence of stringent factor in the absence of the poly(A) template . Template-independent synthesis of (p)ppGpp is suppressed by tetracycline and partially decreases if deacylated tRNA is omitted.

FEBS Lett, 1983 Oct 3, 162(1), 11 - 5
Does the lactose carrier of Escherichia coli function as a monomer?
Wright JK, Weigel U, Lustig A, Bocklage H, Mieschendahl M, Muller-Hill B, Overath P.
The purified lactose carrier of Escherichia coli (product of the lac Y gene) is shown to be a monomer in detergent micelles of dodecyl-O-beta-D-maltoside . The negative-dominant phenotype of mutant carriers (lacY-d mutants) could not be verified by measurements of the rate of galactoside transport in lacY+/Y-d diploid strains . It is proposed that the membrane-embedded carrier functions as a monomer in galactoside-H+ symport.

Eur J Biochem, 1983 Oct 3, 135(3), 519 - 27
Nucleotide sequence of the lipoamide dehydrogenase gene of Escherichia coli K12; Stephens PE et al.; The nucleotide sequence of a 1980-base-pair segment of DNA, containing the lpd gene encoding the lipoamide dehydrogenase component (E3) of the pyruvate dehydrogenase complex of Escherichia coli K12, has been determined by the dideoxy chain-termination method . The lpd structural gene comprises 1419 base pairs (473 codons, excluding the initiating AUG codon) . It is preceded by a good promoter and an excellent ribosome binding site and it ends with a typical rho-independent terminator sequence . The results confirm that the lpd gene is an independent gene linked to, but not part of, the ace operon that encodes the E1 and E2 components of the pyruvate dehydrogenase complex . The location and transcriptional polarity of the lpd gene relative to the restriction map of the corresponding region of DNA, are completely consistent with previous genetic and post-infection labelling studies . The composition, Mr (50554 or 51274 if the FAD cofactor is included), amino-terminal sequence and carboxy-terminal sequence predicted from the nucleotide sequence are in excellent agreement with previous studies on the purified enzyme . The enzyme also exhibits a remarkable degree of sequence homology with peptides of the pig heart enzyme and with other pyridine nucleotide disulphide oxidoreductases whose sequences have been defined: human erythrocyte glutathione reductase and plasmid-encoded mercuric reductase.

Eur J Biochem, 1983 Oct 3, 135(3), 465 - 70
Cooperative effects in the peptidyltransferase center of Escherichia coli ribosomes; Bourd SB et al.; We have measured the binding isotherms of C--A--C--C--A(3'NH)-{14C}Phe to the 70S ribosomes and 50S subunits of Escherichia coli and proposed a theoretical model for adsorption when cooperative interaction occurs between ligands that are adsorbed on ribosomes . Analysis of the experimental binding isotherms leads to the following conclusions . A ribosome (or subunit) binds two C--A--C--C--A(3'NH)-Phe molecules . The binding of C--A--C--C--A(3'NH)-Phe to a ribosome (or subunit) is a cooperative process, characterized by a cooperativity coefficient tau = 40 +/- 5 or more . The binding of C--A--C--C--A(3'NH)-AcPhe at the donor site of the peptidyltransferase center (association binding constant 1.5 X 10(6) M-1) and the binding of puromycin at the acceptor site also occur cooperatively with a coefficient of 10-25, the association binding constant of puromycin at the acceptor site being (1-2) X 10(4) M-1 . The puromycin association binding constant at the donor site multiplied by the cooperativity coefficient of two interacting puromycin molecules absorbed on a ribosome equals 100-200 M-1.

FEBS Lett, 1983 Oct 3, 162(1), 5 - 10
Lack of ability of trypsin-treated mitochondrial F1-ATPase to bind the oligomycin-sensitivity conferring protein (OSCP); Hundal T et al.; Soluble beef-heart mitochondrial F1-ATPase modified in its alpha-subunit by mild trypsin treatment (alpha'-F1) can no longer bind oligomycin-sensitivity conferring protein (OSCP) but is still capable of binding to F1-depleted submitochondrial particles, giving rise to a maximally oligomycin-sensitive ATPase, provided the particles contain their native complement of OSCP . When OSCP is removed from the particles, alpha'-F1 can still bind to the particles, but added OSCP induces only a low degree of oligomycin sensitivity . The possible role of OSCP in the functional coupling of the catalytic (F1) and H+-translocating (Fo) moieties of mitochondrial ATPase is discussed . The results suggest a functional similarity between the OSCP component of mitochondrial ATPase and the delta-subunit of E . coli ATPase, which is in accordance with the structural homology recently found to exist between the two polypeptides.

Can J Physiol Pharmacol, 1983 Oct, 61(10), 1138 - 48
Effect of theophylline and heat-stable enterotoxin of Escherichia coli on transcellular and paracellular ion movement across isolated porcine colon; Argenzio RA et al.; The effect of theophylline and a heat-stable enterotoxin of Escherichia coli (ST) on ion transport was examined using an in vitro, short-circuited preparation of the porcine colon . Theophylline abolished net Na absorption and elicited net Cl secretion, which quantitatively accounted for the increase in short-circuit current (Isc) observed . In contrast, a maximal dose of ST elicited an Isc response about one-half that of theophylline and only partially reduced the net absorption of Na and Cl . A significant residual ion flux, consistent with HCO3 secretion, was elicited by ST and was sustained after theophylline addition . Ion replacement experiments showed that the Isc and net ion transport response to ST was abolished when either Cl or HCO3 were removed from the bathing solutions . Voltage clamp experiments to evaluate the contribution of the paracellular and transcellular transepithelial pathways from serosa to mucosa showed that approximately one-half of the total serosa-to-mucosa flux (Jsm) of both Na and Cl was through the cells . Theophylline and ST both significantly reduced transcellular JNasm, but did not affect JClsm . Theophylline, but not ST, caused an increase in paracellular conductance of both ions . These results demonstrate significant differences in the effects of ST or theophylline on both transcellular and paracellular ion movement, and suggest that ST induces a Cl-dependent HCO3 secretion which is unobserved under control or theophylline-stimulated conditions . In addition, results are consistent with the operation of a neutral NaCl secretory process which is normally masked by the greater net rates of the neutral Na and Cl absorptive mechanisms . Thus, both ST and theophylline appear to reduce or abolished the neutral processes and convert the neutral secretory process into an electrogenic one . This latter effect could be explained simply by an increase in the anion conductance of the mucosal membranes.

J Toxicol Environ Health, 1983 Oct-Dec, 12(4-6), 737 - 48
Different effects of vanadium ions on some DNA-metabolizing enzymes; Sabbioni E et al.; The effects of vanadium on some enzymes involved in DNA metabolism were investigated in vitro . Vanadate (V) ions competitively inhibit calf thymus terminal deoxynucleotidyl transferase with Ki = 2.5 microM . A binding of vanadium to the enzyme with no change of the amount of the Zn constituent of the protein was found at concentrations of vanadate causing inhibition . The catalytic activity of mammalian DNA polymerase alpha was also inhibited by vanadate ions at an I50 of 60 microM, while the bacterial (E . coli) DNA polymerase I was affected to the same extent only when the concentration of vanadate was raised to about 0.5 mM . In contrast to the inhibitory effects caused by vanadium on the nucleotidyl transferases, concentrations of pentavalent vanadium ions of the order of 10 microM increase 2.4-fold the hydrolytic activity of deoxyribonuclease I from bovine pancreas . These findings suggest that vanadium can interact with enzymes involved in nucleic acid metabolism.

Proc Natl Acad Sci U S A, 1983 Oct, 80(19), 5822 - 6
Isolation and characterization of monoclonal antibodies directed against the DNA repair enzyme uracil DNA glycosylase from human placenta; Arenaz P et al.; A series of monoclonal antibodies has been prepared against the base excision repair enzyme uracil DNA glycosylase isolated from human placenta . Spleen cells from BALB/c mice immunized with purified human placental uracil DNA glycosylase were fused with either P3X63 Ag8.653 or SP2/0 myeloma cells . Hybridomas producing antibodies directed against the placental glycosylase were identified in an enzyme-linked immunosorbent assay . Each positive hybridoma was cloned twice by limit dilution and tested for anti-glycosylase activity in an enzyme immunoprecipitation assay . Each of the four clones examined in detail precipitated enzyme activity in an immunoprecipitation reaction only in the presence of rabbit anti-mouse IgG as a second antibody . No anti-uracil DNA glycosylase activity was observed in a spontaneous hybridoma used as a control . Each monoclonal antibody immunoprecipitated uracil DNA glycosylases isolated from several human tissues . Partial crossreactivity was observed with rat liver glycosylase and with a hamster enzyme . In contrast, no crossreactivity was observed with yeast or Escherichia coli glycosylase . Glycerol gradient sedimentation analysis demonstrated that one of the antibodies bound to the glycosylase at a site that did not diminish its catalytic activity . A second monoclonal antibody bound at a determinant that affected catalytic activity . Analysis of antibody-glycosylase interactions suggests that human cells contain antigenically distinct glycosylase species that may be encoded by individual uracil DNA glycosylase genes . The potential use of these monoclonal antibodies in studies examining the regulation of glycosylase isoenzymes during cell proliferation in normal human cells and in cells from cancer-prone individuals is considered.

Biochimie, 1983 Oct, 65(10), 531 - 41
{Biosynthesis and transport of envelope proteins of Escherichia coli}; Pages JM; Bacterial protein synthesis takes place in the cytoplasm, thus periplasmic and outer membrane proteins pass through the cytoplasmic membrane during their dispatch to the cell envelope . The exported proteins are synthesized as precursor that contains an extra amino-terminal sequence of amino-acids . This sequence, termed "signal sequence", is essential for transport of the envelope proteins through the inner membrane and is cleaved during the exportation process . Various hypotheses for the mechanism have been presented, and it is likely that no signal model will be suitable to the export of all cell envelope proteins . This review is focused on the relationship between the cytoplasmic membrane and the precursor form . The physiological state of the membrane - fluidity, membrane potential for instance - is the strategic requirement of exportation process . Precursors can be accumulated in whole cells with various treatments which alter the cytoplasmic membrane . This inhibition of processing is obtained by modification of unsaturated to saturated fatty acids ratio or with phenylethyl alcohol which perturbs the membrane fluidity, with uncoupler agents such as carbonyl cyanide m-chlorophenyl hydrazone which dissipate the proton motive force, or with hybrid proteins which get jamming in the membrane . However, little is known about the early steps of translocation process across the cytoplasmic membrane ; for instance, it is not clear yet whether energy is required for either or both of the first interaction membrane-precursor and the crossing through the membrane . Several studies have recently shown the presence of exportation sites and of proteins which might play a prominent role in the export process, but the mechanism of discrimination between outer membrane proteins and periplasmic proteins is unknown . Considerable work has been done by genetic or biochemical methods and we have now the first lights of the expert mechanism.

Gene, 1983 Oct, 24(2-3), 219 - 25
The complete nucleotide sequence of a 23S rRNA gene from a blue-green alga, Anacystis nidulans; Kumano M et al.; The complete nucleotide sequence of a 23S rRNA gene from a blue-green alga, Anacystis nidulans, has been determined . This nucleotide sequence has 78% and 68% homologies with those of the tobacco chloroplast and Escherichia coli 23S rRNA genes, respectively . The 3'-terminal region of the A . nidulans 23S rRNA gene has strong homology with the chloroplast 4.5S rRNA.

J Bacteriol, 1983 Oct, 156(1), 95 - 100
Chemoattractants elicit methylation of specific polypeptides in Spirochaeta aurantia; Kathariou S et al.; On the basis of this investigation, chemotaxis in Spirochaeta aurantia correlates with methylation of specific polypeptides which are presumed to be analogous to the methyl-accepting chemotaxis proteins (MCPs) in bacteria such as Escherichia coli . The polypeptides exhibited apparent molecular weights in the range of 55,000 to 65,000 . Generally, two major presumptive MCP bands and three minor bands were observed on sodium dodecyl sulfate-polyacrylamide gels . Upon addition of D-glucose to S . aurantia cells, methylation of the presumptive MCPs increased for 10 to 12 min to a level greater than 4 times the level of methylation in the absence of D-glucose . Removal of D-glucose resulted in a decrease in methylation of the presumptive MCPs to a level similar to that in unstimulated cells . All attractants tested, including a non-metabolizable attractant (alpha-methyl-D-glucoside) stimulated methylation of the presumptive MCPs (from 1.7 to 4.3 times the level of methylation in unstimulated cells) . D-Mannitol, a metabolizable sugar which is not an attractant for S . aurantia, did not stimulate methylation . Stimulation of methylation by D-galactose occurred in cells induced for D-galactose taxis but not in uninduced cells . These data are indicative of an evolutionary relationship between the chemotaxis systems of spirochetes and of flagellated bacteria.

J Bacteriol, 1983 Oct, 156(1), 338 - 47
Identification of the pifC gene and its role in negative control of F factor pif gene expression; Miller JF et al.; The pif region of the F factor includes two genes, pifA and pifB, that lead to abortive T7 infection . We have identified a new gene in this region, pifC, by constructing an in vitro fusion of pif DNA at 41.6 kilobases on the F factor physical map to the lacZ gene . A PifC-LacZ fusion protein of 149,000 daltons has been identified by immunoprecipitation and polyacrylamide gel electrophoresis . This allows us to assign the N terminus of pifC to 42.5 kilobases on the F map . Using fusions of pifC, pifA, and pifB to lacZ, we have studied the regulation of pif gene expression and have shown that the product of pifC negatively controls its own expression and that of pifA and pifB.

J Biomol Struct Dyn, 1983 Oct, 1(2), 509 - 21
Possible molecular detent in the DNA structure at regulatory sequences; Lu P et al.; A common feature that appears in a number of DNA sites where proteins interact is the sequence GTG/CAC . In the lac operator this sequence leads to a region with a higher imino proton exchange rate well below the optical melting temperature . It is suggested that this reflects a structural feature recognized by proteins that bind specific sites on the DNA molecule.

Biochem Int, 1983 Oct, 7(4), 529 - 34
The Shine and Dalgarno hypothesis for termination: the 3' terminus of the 16S rRNA of the Escherichia coli ribosome can be modified or base-paired with a complementary oligonucleotide without affecting termination in vitro; Tate WP et al.; The occurrence of the nucleotides "...CCUUAOH" at the 3' terminus of the 16S rRNA of the small subunit of the Escherichia coli ribosome led to the suggestion that they may have a direct base pairing with the termination codon in the termination event of protein biosynthesis (Shine and Dalgarno 1974) . We have examined this concept with two approaches, firstly using a 30S subunit whose 16S rRNA has been modified with a fluorescein moiety on the terminal adenosine together with the antibody against the moiety, and secondly with an oligonucleotide, UAAGG, complementary to the terminal pentanucleotide sequence of the rRNA . Collectively the data suggest that the nucleotides at the 3' terminus of 16S rRNA are not critically involved in base pairing during termination codon recognition.

Nord Vet Med, 1983 Oct, 35(10), 346 - 52
Occurrence of enterotoxigenic Escherichia coli in calves with acute neonatal diarrhoea; Krogh HV; Enterotoxigenic Escherichia coli (ETEC) were isolated from 16.4% of diarrheic calves of up to 30 days old . Of 1-2-day-old calves nearly one half (45.0%) harboured ETEC, while this was the case with only 4.9% of 8-day-old calves . Among 206 9-30-day-old calves just three were found to harbour ETEC.

Anal Biochem, 1983 Oct 1, 134(1), 126 - 32
Polyacrylamide gels which contain a novel mixed disulfide compound can be used to detect enzymes that catalyze thiol-producing reactions; Harris RB et al.; The synthesis of N-{5-(hydroxyethyl)dithio-2-nitrobenzoylaminoethyl} acrylamide (I) is described . If the disulfide bond in this compound is reduced with thiol reagents, an intense yellow color develops (epsilon 412 V 13,600 at pH 7.4) due to essentially the same chromophore as 5-thio-2-nitrobenzoic acid, the reduced form of 5,5'-dithiobis(2-nitrobenzoic acid)(Ellman's reagent) . Polyacrylamide gels were prepared that were crosslinked with N,N'-methylenebisacrylamide and which contained I as an integral part of the polymerized acrylamide chain . Acetylcholinesterase (from electric eel and human brain tissue slices) and alkaline phosphatase (from Escherichia coli and calf intestine) were subjected to electrophoresis and then the gels were immersed in an appropriate thiol-substrate buffer (acetylthiocholine and cysteamine-S-phosphate, respectively) . A yellow band developed rapidly in the acrylamide gel at the site of enzyme activity . Electrophoresis on the mixed disulfide-polyacrylamide gel proved to be a rapid and sensitive technique to detect very small amounts of enzyme (approximately 0.02 fmol acetylcholinesterase) and should have wide application for detecting other enzymes that hydrolyze thiol substrates.

Acta Pathol Microbiol Immunol Scand {B}, 1983 Oct, 91(5), 333 - 7
Demonstration of the in vitro phagocytosis of Treponema denticola by human polymorphonuclear neutrophils; Lingaas E et al.; A method has been developed for the study of phagocytosis of the oral treponeme T . denticola by human polymorphonuclear neutrophils . T . denticola was labelled with 32P-orthophosphate, and phagocytosis, expressed as counts per mg cell protein, was measured after 15, 60 and 120 min of interaction between neutrophils and treponemes . Four different cell cultures were used as controls, and phagocytosis of E . coli was used as reference . The uptake of radiolabelled T . denticola was significantly larger with the PMNs than with any of the control cells . An increase of uptake was observed from 15 to 120 min . The addition of autologous human serum or rabbit T . denticola antiserum enhanced the phagocytosis 2-3 fold, and phagocytosis was decreased under an anaerobic atmosphere . Scanning electron microscopic studies also indicated that phagocytosis had taken place.

J Biochem (Tokyo), 1983 Oct, 94(4), 1289 - 99
Magnesium ion induced proton release as a probe for the polyelectrolytic structure of ribosomal RNAs and subunits; Horie K et al.; E coli ribosomes and rRNA's released 20 to 50 protons upon jump of magnesium ion concentration from 1 mM to 20 mM . The Mg2+-induced proton release was measured separately for 16S rRNA, 23S rRNA, 30S subunit, and 50S subunit by a new spectrophotometric method that had a much better sensitivity than the pH-stat method . The proton release from the subunits and rRNA's were similar in the number of protons, the pH dependence that had a minimum at neutral pH, and the upward concaveness of the Scatchard plot . From these results, the main source of protons in ribosomal subunits was assigned to nucleotide bases of rRNA's that showed a downward pKa shift upon Mg2+-ion binding . The subunits and rRNA's, however, differed in the proton release . 16S rRNA released protons somewhat more effectively than 23S rRNA, while 30S subunit released protons 2 to 5 times more effectively than 50S subunit . The marked difference between the two subunits suggest that ionizable bases in 16S and 23S rRNA's are covered and their pKa values are shifted by ribosomal proteins to different extents . The association of 30S and 50S subunits induced little proton release, showing that few ionizable groups with pKa near neutral pH are involved in the association . E . coli tRNA and poly U also showed Mg2+-induced proton release . The amounts of protons released from rRNA's, tRNA, and poly U were roughly proportional to the amount of bases not hydrogen bonded . The Mg2+-induced proton release from the natural and synthetic RNA's can be explained by the electrostatic field effect of polyphosphate backbones on bases not hydrogen bonded, as proposed in a previous paper . It also reflects the conformational structure of each RNA molecule.

Genetika, 1983 Oct, 19(10), 1573 - 81
{Instability of recombinant molecules}; Vel'kov VV; The regions and mechanisms of recombinant DNAs instability are reviewed, in particular, the mechanisms of the replication instability expressed as elimination of recombinant DNA from cells and the mechanisms of structural instability revealed as spontaneous alteration of the chemical structure of these DNAs . The replication instability is subdivided into that induced by ineffective replication systems, and that induced by disturbances in the process of correct partitioning of the plasmids between dividing cells . The structural instability is subdivided into topological one occurring due to formation in the hybrid plasmids of the anomalous elements of the secondary structure-"loops", the regulatory instability caused by nonbalanced transcription streams and metabolic instability induced because of the protein superproduction which is energy capacious and not necessary for normal cell growth.

Gastroenterol Jpn, 1983 Oct, 18(5), 436 - 9
Anti-tumor activity of endotoxin depends on activation of serum complement fragments; Yamaguchi N et al.; The anti-tumor activity of endotoxin (LPS), derived from E . coli O127, on the subcutaneous tumor MH134 inoculated into C3H/He mice in vivo was examined . On repeated examination, LPS administration of 50-100 micrograms/head induced a 50% complete cure rate . However, pretreatment with K76COONa inhibited the anti-tumor activity of LPS, and no case of cure could be found . These results showed that the main anti-tumor activity of LPS administration consisted of complementary activation, mainly from the C5 step . The fact that the intraperitoneal administration of human complement activated serum with agar apparently inhibited the tumor growth in vivo certified these phenomena.

Br J Pharmacol, 1983 Oct, 80(2), 295 - 301
The effect of a selective 5-HT2 antagonist, ketanserin, on the pulmonary responses to Escherichia coli endotoxin; Ball HA et al.; 5-Hydroxytryptamine (5-HT, 5-160 microgram kg-1) injected intravenously in pentobarbitone-anaesthetized, open-chest cats caused dose-dependent increases in pulmonary arterial and intratracheal pressures . There was also a marked systemic hypotension and bradycardia . The pulmonary effects were completely prevented by ketanserin (0.2 mg kg-1), a selective 5-HT2 blocking drug . Ketanserin (0.2 mg kg-1) itself lowered arterial pressure (by 30-40 mmHg) but this systemic hypotension was relatively transient . Endotoxin (E . coli) administration resulted in pulmonary hypertension, increases in intratracheal pressure and airways resistance and reductions in lung compliance and in arterial PO2 . Only the airways resistance response was modified by ketanserin (0.2 mg kg-1), suggesting a relatively unimportant role for 5-HT in mediating the acute, pulmonary effects of endotoxin in this species . The reductions in arterial (mixed venous) pH and in PO2 that resulted from endotoxin administration were not affected by pretreatment with ketanserin.

Avian Dis, 1983 Oct-Dec, 27(4), 972 - 9
Evaluation of the heterophil/lymphocyte ratio as a measure of stress in chickens; Gross WB et al.; The number of lymphocytes in chicken blood samples decreased and the number of heterophils increased in response to stressors and to increasing levels of corticosterone in the chicken feed . The ratio of heterophils to lymphocytes was less variable than the number of heterophil or lymphocyte cells, and the range of values for this ratio was greater than the range of values for heterophils and lymphocytes among control and experimental groups . The heterophil/lymphocyte ratio appears to be a more reliable indicator of levels of corticosterone in the feed and to social stress than were the plasma corticosteroid levels.

Paraplegia, 1983 Oct, 21(5), 318 - 21
Peculiar septic responses in traumatic tetraplegic patients; Ohry A et al.; Patients with a high level tetraplegia from a spinal injury have only been able to survive the critical initial period since the development of modern resuscitation techniques including artificial respiration . However, their lives are still threatened by many complications, such as decubitis ulcers, infections and respiratory failure . We describe four young tetraplegic patients who developed an unusual sepsis pattern several years after the injury . The sepsis was accompanied by hypothermia, leukopenia and mental deterioration . This peculiar 'silent' sepsis may also occur in elderly people who are not paralysed . The question arises, therefore, if the chronic spinal cord injured patient may become 'prematurely aged'.

J Biol Stand, 1983 Oct, 11(4), 251 - 60
The processing and collaborative assay of a reference endotoxin; Hochstein HD et al.; A preparation of Escherichia coli bacterial endotoxin, the latest of successive lots drawn from bulk material which has been studied in laboratory tests and in animals and humans for suitability as a reference endotoxin, has been filled and lyophilized in a large number of vials . Details of its characterization, including stability studies, are given . A collaborative assay was conducted by 14 laboratories using gelation end-points with Limulus amebocyte lysates . Approximate continuity of the unit of potency with the existing national unit was achieved . The lot was made from the single final bulk but had to be freeze-dried in five sublimators . An assessment was therefore made for possible heterogeneity . The results indicate that the lot can be used as a large homogeneous quantity . The advantages of using it widely as a standard for endotoxins are discussed.

J Antimicrob Chemother, 1983 Oct, 12 Suppl C, 105 - 16
Effect of clindamycin on growth and haemolysin production by Escherichia coli; Boe NM et al.; The effect of clindamycin on growth and haemolysin production by four strains of Escherichia coli was tested . Clindamycin MICs were greater than 256 mg/l for all strains . Clindamycin concentrations of 16-256 mg/l significantly inhibited growth, while concentrations from 2-32 mg/l significantly inhibited haemolysin production . Administration of clindamycin to rats with peritonitis due to haemolytic E . coli reduced mortality . Subinhibitory concentrations of clindamycin can inhibit growth and haemolysin production by E . coli and reduce mortality in an animal model of haemolytic E . coli peritonitis.

Gene, 1983 Oct, 24(2-3), 335 - 9
Cloning the Escherichia coli K-12 argD gene specifying acetylornithine delta-transaminase; Riley M et al.; The argD gene of Escherichia coli was shown to be present in plasmids pLC2-28 and pLC3-11 of the collection of Clarke and Carbon {Cell 9 (1976) 91-99} . The gene was cloned into pBR322 as a 6.3-kb BamHI fragment . Enzyme determination showed that the cloned DNA contains the structural gene for acetylornithine delta-transaminase . The argD DNA was used as a probe in hybridization experiments which indicated that the argM gene resides in a duplicated portion of E . coli DNA that is highly similar to the argD region.

Gene, 1983 Oct, 24(2-3), 317 - 26
Viability of palindromic DNA is restored by deletions occurring at low but variable frequency in plasmids of Escherichia coli; Hagan CE et al.; Palindromic arrangements of 2 X 400 to 2 X 1517 bp of nucleotide sequences were created by in vitro manipulation of Escherichia coli plasmids . As a consequence of its method of formation, each palindrome possessed at its center the recognition site for a particular restriction endonuclease . Eight out of eight palindromes, having at their centers the recognition sites for BamHI, BglI, BglII, HindIII, PstI, SalI, XhoI, and Xma III, were shown to be inviable when transformed into E . coli . The smallest of these palindromes had a half-length of approx . 400 bp . The lethality of palindromic sequences for their carrier plasmids was circumvented at low frequencies by spontaneous in vivo deletion events which removed the centers of symmetry of the palindromes . The frequencies of such deletions were less than 1% in most cases, but varied significantly both with the palindromic sequence in question and with the surrounding nonpalindromic sequences of the carrier plasmid . We confirmed the viability of a plasmid with a 147-bp palindrome {Bergsma et al., Gene 20 (1982) 157-167} and found that this palindrome (derived from SV40) does not confer viability on the plasmids with long palindromes.

Gene, 1983 Oct, 24(2-3), 309 - 15
Nucleotide sequence of the partition locus of Escherichia coli plasmid pSC101; Miller CA et al.; We report here the sequence of a 375-bp EcoRI-AvaI DNA fragment that previously has been shown to contain a locus (termed partition or par) responsible for stable maintenance of the pSC101 plasmid in growing cell populations . The DNA sequence of the par region encodes no obvious proteins and contains no segments having the structural characteristics of transcriptional or translational start signals . However, segments of the par locus appear capable of forming regions of intra-strand secondary structure, one of which resembles a rho-independent transcription terminator . Computer analysis shows regions within par that have homology with sequences found near the origin of replication of the Escherichia coli chromosome and of the pBR322 and ColE1 plasmids . The par sequence homology in the pBR322 and ColE1 plasmids maps in the vicinity of sites that interact with the E . coli replication factor Y and accomplishes initiation of DNA synthesis on single-strand templates.

Gene, 1983 Oct, 24(2-3), 265 - 79
The replication origin region of Escherichia coli: nucleotide sequence and functional units; Buhk HJ et al.; The minichromosome pCM959 contains the DNA segment from bp -677 (left) to bp + 3335 (right) of the Escherichia coli replication origin, oriC . The nucleotide sequence of this plasmid was determined . The coding regions for proteins were identified, and the possible function of those proteins is discussed . Within oriC two extended systems of dyad symmetry were found, and their possible significance is considered.

Gann, 1983 Oct, 74(5), 743 - 50
Immune-enhancing effect of recombinant human interferon-beta produced in Escherichia coli on human peripheral blood mononuclear cells in vitro; Okabe M et al.; Highly purified recombinant fibroblast interferon produced by Escherichia coli (ReIFN-beta) was tested for its ability to stimulate natural killer (NK) cell activity, antibody-dependent cell-mediated cytotoxicity (ADCC) and monocyte-macrophage phagocytic activity in human peripheral blood mononuclear cells (PBM) in comparison with natural human fibroblast interferon (IFN-beta) . The NK cell activity against target cells (K-562, Molt-3, CCRF-HSB-2, CCRF-CEM, Daudi, and HeLa S3) was enhanced by ReIFN-beta and IFN-beta . Augmentation of cytotoxicity of NK cells was observed at a concentration of ReIFN-beta as low as 1 U/ml and increased in a dose-dependent manner . The enhancing effect of ReIFN-beta was expressed sufficiently during a 1-hr incubation with effector cells, and IFN-beta showed kinetics and potency similar to those of ReIFN-beta . ADCC was measured with chicken red blood cells (CRBC) or CCRF-CEM coated with each antibody, as target cells . Pretreatment of PBM with ReIFN-beta caused a significant increase in ADCC activity against both targets, and the enhancing activity of ReIFN-beta was similar to that of IFN-beta . Phagocytic activity of adherent cells in PBM against CRBC was enhanced by ReIFN-beta and IFN-beta as determined by microscopical cytologic examination and by the 51Cr-labeled CRBC-uptake method . From these results, it can be concluded that recombinant human IFN-beta modulates NK cell activity, ADCC and phagocytic activity of human PBM as effectively as natural human IFN-beta.

Eur J Cancer Clin Oncol, 1983 Oct, 19(10), 1371 - 9
Massive BACT chemotherapy with autologous bone marrow transplantation in 17 cases of non-Hodgkin's malignant lymphoma with a very bad prognosis; Philip T et al.; A group of 12 children and 5 adults, all with diffuse non-Hodgkin's malignant lymphoma (NHML), received massive chemotherapy regimens . The stages of the disease were as follows: 7 patients were in second complete remission; 6 in a progressive phase of the disease; and 4 in first complete remission which occurred late in the course of the disease . All patients received BACT (BCNU+aracytine+cyclophosphamide+thioguanine) or TACC (idem with CCNU) at different dose levels: 6/17 received 10 Gy total-body irradiation (TBI) after BACT treatment; 16/17 received autologous bone marrow transplantation (ABMT) previously stored in liquid nitrogen to combat the medullary effects of chemotherapy . Direct therapy-related deaths occurred in 4/17 patients (1 Aspergillus endocarditis; 1 Moskowitz syndrome; 1 veno-occlusive disease of the liver; and 1 Escherichia coli pneumopathy) and 6/17 patients relapsed between days 25 and 70 of treatment . Seven out of these 17 patients are still alive NED 102-900 days (mean, 475 days) after the beginning of therapy without receiving maintenance treatment . Massive chemotherapy could thus be the best treatment for NHML in relapse, but the high percentage of early therapy-related deaths is a strong limiting factor for patients before relapse.

Biokhimiia, 1983 Oct, 48(10), 1705 - 8
{Some structural properties of 80S acid protein from pea ribosomes}; Iurina NP et al.; The 80S acid protein from pea ribosomes similar to the L7/L12 protein from E . coli was studied . This protein was found to be rich in alanine (18 mol.%) and to contain an acid amino acids excess over basic ones, the ratio of basic amino acids to acid ones was 0.42 . As in the case of other eukaryotic L7/L12 homologs studied, the N-terminal amino acid of the protein is methionine . Using the double immunodiffusion technique, no crossreaction of E . coli anti-L7/L12 with 80S acid protein from pea ribosomes was observed . It was assumed that the protein molecule contains conservative sites responsible for the specific functioning of eukaryotic L7/L12 homologs.

Neuroendocrinology, 1983 Oct, 37(4), 258 - 61
Hormonally active arginine-vasopressin suppresses endotoxin-induced fever in rats: lack of effect of oxytocin and a behaviorally active vasopressin fragment; Kovacs GL et al.; Vasopressin and oxytocin modulate memory processes which effects are dissociated from the typical peripheral endocrine effects of these neuropeptides . Recently, vasopressin has been implicated in the regulation of body temperature . In view of this, experiments were designed to determine whether the antipyretic effect of vasopressin was related to the action of the neuropeptide on memory processes . Fever was induced in rats by intracerebroventricular (i.c.v.) injection of 10 ng bacterial endotoxin (ET), which resulted in a rapid rise in colonic temperature . A second i.c.v . injection 15 min after ET administration of graded amounts (0.01, 0,1, 1 and 100 ng) or arginine-vasopressin (AVP) suppresses ET-induced fever in a dose-dependent manner, 1 ng being the minimally effective amount . Equivalent amounts of des-9-glycinamide-arginine-vasopressin (DG-AVP) or oxytocin (OXT) were ineffective . Large amounts (1,000 ng) of the latter two peptides, however, transiently mimicked the effect of AVP . On one-trial learning passive avoidance behavior, the neurohypophyseal peptides exerted a completely different pattern of effects . AVP and DG-AVP induced a dose-dependent facilitation, while OXT resulted in a dose-dependent attenuation of passive avoidance behavior . These findings suggest that AVP-induced antipyresis is related to the hormonally active AVP and dissociated from the effects of neurohypophyseal hormones and hormone fragments on other CNS processes-like learning and memory.

Mutat Res, 1983 Oct, 112(5), 275 - 86
Repair resynthesis in Escherichia coli mutants deficient in single-stranded DNA-binding protein; Whittier RF et al.; A series of Escherichia coli strains deficient in single-stranded DNA-binding protein (SSB) and DNA polymerase I was constructed in order to analyze the effects of these mutations on DNA repair resynthesis after UV-irradiation . Since SSB has been suggested to play a role in protecting single-stranded regions which may transiently exist during excision repair and since long single-stranded regions are believed to occur frequently as repair intermediates in strains deficient in DNA polymerase I, studies of repair resynthesis and strand rejoining were performed on strains containing both the ssb-1 and polA1 mutations . Repair resynthesis appears to be slightly decreased in the ssb-1 strain at 42 degrees C relative to the wild-type; however, this effect is not enhanced in a polA1 derivative of this strain . After UV-irradiation, the single-strand molecular weight of the DNA of an ssb-1 strain decreases and fails to recover to normal size . These results are discussed in the context of long patch repair as an inducible component of repair resynthesis and of the protection of intermediates in the excision repair process by SSB . A direct role for SSB in repair resynthesis involving modulation of the proteins involved in this mode of DNA synthesis (particularly stimulation of DNA polymerase II) is not supported by our findings.

J Infect Dis, 1983 Oct, 148(4), 699 - 709
Recombinant DNA risk assessment studies in humans: efficacy of poorly mobilizable plasmids in biologic containment; Levine MM et al.; Recombinant DNA risk assessment studies quantitated the mobilizability of "safe" plasmid pBR325, in comparison with readily mobilizable plasmid pJBK5 (chloramphenicol and tetracycline resistant) . Of 15 volunteers who became colonized after ingestion of 5 X 10(10) Escherichia coli HS-4, a normal human flora strain containing pJBK5 and daily oral tetracycline, nine manifested transfer of pJBK5 to normal flora by means of triparental mating . In contrast, none of 12 other volunteers cocolonized with HS-4 bearing "safe" pBR325 and normal flora showed transfer (P = 0.001), despite ingestion of tetracycline . To accomplish transfer directly, E coli HS-4 containing both pBR325 and a derepressed, conjugative plasmid (F-amp) was fed to two groups of volunteers . Transfer of pBR325 to normal flora occurred in 13 of 18 volunteers taking daily tetracycline but in none of eight who did not (P less than 0.002) . Nor were transconjugants detected, despite tetracycline ingestion, in five volunteers who ingested and excreted E coli K12 (pBR325 plus F-amp).

J Clin Microbiol, 1983 Oct, 18(4), 935 - 7
Changes in serotypes of enterotoxigenic Escherichia coli in Dhaka over time: usefulness of polyvalent antisera; Stoll BJ et al.; The finding that enterotoxigenic (ET) Escherichia coli strains from many geographic areas belong to a limited number of serogroups led investigators to hope that polyvalent antisera could be used to screen for ET E . coli in areas of the world where more complicated tests of toxigenicity are not available . We compared the serotypes of 207 ET E . coli strains obtained from patients attending the Dhaka Hospital of the International Centre for Diarrhoeal Disease Research, Bangladesh, in 1980 with results obtained from similar surveys conducted in 1976 and 1978 . During that time the distribution of serogroups changed significantly so that only 46% of our strains had O serogroups included in the polyvalent antisera capable of detecting 66% of ET E . coli strains in 1978 . O167, a serogroup which was not included previously, was the third most common serogroup in our study . These findings suggest that polyvalent antisera used to screen for ET E . coli need to be reassessed over time as well as in different geographic areas and may not be as useful for field diagnosis as was originally hoped.

J Clin Microbiol, 1983 Oct, 18(4), 798 - 802
Comparison of methods to detect Escherichia coli heat-labile enterotoxin in stool and cell-free culture supernatants; Morgan DR et al.; We compared the standard Y-1 adrenal cell (YAC) assay for heat-labile enterotoxin (LT) with newer rapid and economical immunological methods of detection . Stool samples were collected from 164 acutely ill American students in Guadalajara, Mexico . Supernatants were prepared from each stool . Stools were cultured for Escherichia coli by standard techniques . Individual E . coli-like colonies were examined for LT production by the Biken assay . Culture supernatants and stool supernatants were assayed for the presence of LT by the YAC assay, counterimmunoelectrophoresis, and enzyme-linked immunosorbent assay (ELISA) . Standard YAC assays of culture supernatants revealed that 40 of the 164 specimens (24%) were LT positive . Counterimmunoelectrophoresis detected 60% and ELISA detected 65% of the 40 known positives when culture supernatants were used . The Biken assay detected 35% of the 40 known positives . With stool supernatants, the YAC assay detected only 18% of the known positives, counterimmunoelectrophoresis detected 60%, and ELISA detected 90% . In addition, ELISA detected 13 LT-positive stool supernatants not detected by the YAC assay of culture supernatants . The ELISA in which stool supernatants are used may be a useful method to detect LT.

Cell, 1983 Oct, 34(3), 979 - 88
Localization of the target site for translational regulation of the L11 operon and direct evidence for translational coupling in Escherichia coli; Baughman G et al.; The L11 ribosomal protein operon in Escherichia coli consists of the structural genes for proteins L11 and L1 . Hybrid deletion plasmids were constructed carrying these two genes with decreasing amounts of the leader mRNA under lac transcriptional control . Measurements of mRNA and protein synthesis directed by these plasmids in vitro and in vivo demonstrate that the regulation of this operon is posttranscriptional and identifies a region of the mRNA, preceding the proximal L11 gene, important for successful feedback inhibition of L11 and L1 synthesis by L1 . Additionally, deletions extending to the ribosome binding site of the L11 gene fail to synthesize both L11 and the downstream L1 protein although synthesis of the corresponding mRNA remains unchanged . These results directly demonstrate the presence of translational coupling in this bicistronic operon.

Cell, 1983 Oct, 34(3), 1007 - 14
Maize mitochondrial DNA contains a sequence homologous to the ribulose-1,5-bisphosphate carboxylase large subunit gene of chloroplast DNA; Lonsdale DM et al.; The mitochondrial genome of maize contains a DNA sequence homologous to the chloroplast gene coding for the large subunit of ribulose-1,5-bisphosphate carboxylase (LS gene) . The presence in mitochondrial DNA of both coding and flanking sequences of this gene has been demonstrated first, by cross hybridization between the purified organelle DNAs and between cloned mitochondrial and chloroplast DNA sequences and second, by in vitro transcription-translation of cloned mitochondrial DNA in an E . coli cell free system where a 21,000 dalton polypeptide is synthesized that can be precipitated with antibodies to wheat ribulose-1,5-bisphosphate carboxylase . In contrast to the 12 kb chloroplast homologous sequence found in the mitochondrial genome (Stern and Lonsdale, 1982), the sequence homologous to the LS gene is unaltered in mitochondrial DNA isolated from the male sterile cytoplasms of maize . The LS gene homologous sequence in the mitochondrial genome is located some 65 kb from the 18S mitochondrial rRNA gene and approximately 20 kb from the mitochondrial DNA sequence having homology to the chloroplast 16S rRNA gene.

Proc Natl Acad Sci U S A, 1983 Oct, 80(20), 6219 - 23
Perturbation of lac operator DNA modification by tryptic core protein from lac repressor; Manly SP et al.; Operator DNA fragments were modified in the presence of lac repressor protein or its trypsin-resistant core . Operator DNA was alkylated or cleaved enzymatically with these related proteins present to compare the influences of their binding on the reactivities or enzymatic susceptibilities of individual bases in the sequence . These two protein species have pronounced and distinguishable effects on the reactivity of the bases of the operator fragment toward methylation by dimethyl sulfate . Perturbation of base alkylation by the trypsin-resistant core repressor is most pronounced in the inner, asymmetric region of the operator DNA, while repressor effects extend further on either end of the operator sequence . Digestion of the two protein-operator complexes by DNase I yields fragment patterns that differ primarily in extent of protection . These data extend the experimental base supporting the involvement of the core region of the lac repressor in addition to its NH2 termini in the operator-specific binding activity of this protein.

Proc Natl Acad Sci U S A, 1983 Oct, 80(20), 6152 - 6
Structural and transcriptional evidence for related thrS and infC expression; Mayaux JF et al.; The nucleotide sequence of thrS, the gene encoding dimeric Escherichia coli threonyl-tRNA synthetase {L-threonine:tRNAThr ligase (AMP forming), 6.1.1.3}, has been determined . The structural part of the gene is found upstream of and on the same DNA coding strand as infC, the gene for translational initiation factor IF3 . thrS is composed of 1,926 base pairs and accounts for a protein of molecular weight 73,906 . In addition, a 336-base-pair sequence 5' to the thrS structural gene has been determined . There are only three nucleotides between the stop codon of thrS and the initiator codon of infC . The only potent transcriptional terminator structure is 55 base pairs downstream of the infC coding sequence . This implies that thrS and infC can be expressed from a polycistronic mRNA originating from a promoter upstream to thrS . Although sequence data indicate that thrS and infC are cotranscribed, in vitro transcription and RNA sequence analyses reveal the existence of a promoter within thrS . This promoter can account for the independent expression of infC as reported {Springer, M., Plumbridge, J . A., Trudel, M., Graffe, M . & Grunberg-Manago, M . (1982) Mol . Gen . Genet . 186, 247-252} . A second promoter has been located within infC and could link the expression of infC and that of the next downstream gene, pdzA . Whether these promoters function normally in vivo is an open question.

J Clin Pathol, 1983 Oct, 36(10), 1145 - 9
An improved chromogenic substrate endotoxin assay for clinical use; Harris RI et al.; An improved quantitative assay for endotoxin in plasma was developed after evaluating three different chromogenic substrates and seven methods for removal of plasma inhibitors . Optimal storage conditions for plasma samples prior to assay were also determined . Using chromogenic substrate S2423 with plasma diluted 1/10 in water and heated to 75 degrees C for 5 min to remove inhibitors, a within-batch coefficient of variation of 4% was obtained at levels of endotoxin likely to be encountered clinically . The limit of assay sensitivity was less than 10 pg/ml . This assay provides a sensitive quantitative test for single episodes of endotoxaemia in individual patients but variable activation of the Limulus proenzyme by endotoxin from different bacterial strains limits quantitative comparisons between patients.

J Bacteriol, 1983 Oct, 156(1), 6 - 12
Repair and mutagenesis in Escherichia coli K-12 after exposure to various alkyl-nitrosoguanidines; Todd ML et al.; The mutagenic and toxic effects of a series of N-alkyl-N'-nitro-N-nitrosoguanidines were examined in Escherichia coli K-12 . The role of nucleotide excision repair, the SOS response, and the adaptive response in both the reduction and the production of the biological effects of these chemicals was tested . The effects of ethyl-nitrosoguanidine are similar in nucleotide excision repair-proficient and -deficient strains, but both the mutagenicity and the toxicity of alkyl groups larger than two carbons are significantly reduced by the presence of this repair system . Similarly, when alkyl groups are larger than two carbons, the umuC gene product is essential for the production of a fraction of the mutations that these lesions produce . The induction of the adaptive response had a significant effect on the toxicity of all of the chemicals tested, but its effect on mutagenicity was less uniform, having a larger effect on ethylating and propylating agents than on butylating and amylating agents.

J Bacteriol, 1983 Oct, 156(1), 441 - 3
Plasmid association and nucleotide sequence relationships of two genes encoding heat-stable enterotoxin production in Escherichia coli H-10407; Moseley SL et al.; Plasmid DNA from enterotoxigenic Escherichia coli strains H-10407 and H-10407-P was examined for nucleotide sequence homology to two E . coli genes encoding infant mouse-active heat-stable enterotoxins (ST) . A 62-megadalton plasmid of strain H-10407 contained sequences homologous to the gene encoding a toxin designated STIb, previously isolated from a human isolate of E . coli . A 42-megadalton plasmid of strains H-10407 and H-10407-P contained sequences homologous to the gene encoding a toxin designated STIa, previously isolated from bovine and porcine isolates of E . coli.

J Bacteriol, 1983 Oct, 156(1), 414 - 8
Membrane-bound fractions of R6K plasmid DNA in Escherichia coli; Archibold ER et al.; The intracellular location of plasmid DNA has been of interest in an effort to understand the maintenance of these molecules . We have employed a simple procedure which enables us to isolate from exponentially grown cells on sucrose gradients membrane-complexed forms of R6K plasmid DNA . Electron micrographs identified the complexing of membrane fractions to circular forms of R6K DNA . Biochemical studies of the complexed R6K molecules showed the presence of membrane-specific proteins and suggested that complexing of R6K DNA was primarily with inner membrane fractions of Escherichia coli.

J Bacteriol, 1983 Oct, 156(1), 386 - 92
Evidence for multisite growth of Escherichia coli murein involving concomitant endopeptidase and transpeptidase activities; Burman LG et al.; During diaminopimelic acid starvation of Escherichia coli W7, a large fraction of the preexisting murein cross-links are opened by murein endopeptidase and the resulting uncross-linked material is degraded . This is reflected morphologically in a general loss of rigidity of the murein sacculus long before lysis occurs . In growing cells, a dynamic situation is demonstrable . When cells whose murein sacculi are uniformly labeled with {14C}diaminopimelic acid were chased with unlabeled DAP, a significant, rapid shift of {14C}diaminopimelic acid from the donor to the acceptor half of dimers was observed . The shift can be explained by the presence of about 100 separate sites where new murein strands were being inserted between old radioactive strands of murein . Thus, the gradual loss of rigidity of the murein sacculus as endopeptidase continues to function during starvation of E . coli W7 suggests an even distribution of the active endopeptidases . This is consistent with the kinetic data which suggest that endopeptidase, along with murein synthetase and transpeptidase, acts at about 100 distinct sites to elongate the murein sacculus.

J Bacteriol, 1983 Oct, 156(1), 243 - 50
Novel mechanism of cell division inhibition associated with the SOS response in Escherichia coli; D'Ari R et al.; Certain Escherichia coli strains were shown to possess a novel system of cell division inhibition, called the SfiC+ phenotype . SfiC+ filamentation had a number of properties similar to those of sfiA-dependent division inhibition previously described: (i) both are associated with the SOS response induced by expression of the recA(Tif) mutation, (ii) both are associated with cell death, (iii) both are amplified in mutants lacking the Lon protease, and (iv) both are suppressed by sfiB mutations . SfiC+ filamentation and sfiA-dependent division inhibition differed in (i) the physiological conditions under which loss of viability is observed, (ii) the extent of amplification in lon mutants, (iii) their genetic regulation (SfiC+ filamentation is not under direct negative control of the LexA repressor), and (iv) their genetic determinants (SfiC+ filamentation depends on a locus, sfiC+, near 28 min on the E . coli map and distinct from sfiA).

J Bacteriol, 1983 Oct, 156(1), 205 - 11
Evidence for positive regulation of plasmid prophage P1 replication: integrative suppression by copy mutants; Froehlich BJ et al.; Like low-copy-number plasmids including P1 wild type, multicopy P1 mutants (P1 cop, maintained at five to eight copies per chromosome) can suppress the thermosensitive phenotype of an Escherichia coli dnaA host by forming a cointegrate . At 40 degrees C in a dnaA host suppressed by P1 cop, the only copy of P1 is the one in the host chromosome . Trivial explanations of the lack of extrachromosomal copies of P1 cop have been eliminated: (i) during integrative suppression, the P1 cop plasmid does not revert to cop+; (ii) the dnaA+ function of the host is not required to maintain P1 cop at a high copy number; and (iii) integrative recombination does not occur within the region of the plasmid involved in regulation of copy number . Since there are no more copies of the chromosomal origin (now located within the integrated P1 plasmid) than in a P1 cop+-suppressed strain, the extra initiation potential of the P1 cop is not used to provide multiple initiations of the chromosome . When a P1 cop-suppressed dnaA strain was grown at 30 degrees C so that replication could initiate from the chromosomal origin as well as from the P1 origin, multicopy supercoiled P1 DNA was found in the cells . This plasmid DNA was lost again when the temperature was shifted back to 40 degrees C.

J Bacteriol, 1983 Oct, 156(1), 19 - 29
Complementation of argG and hisA mutations of Escherichia coli by DNA cloned from the archaebacterium Methanococcus voltae; Wood AG et al.; DNA derived from the methanogenic archaebacterium Methanococcus voltae was digested with PstI restriction endonuclease and cloned into the PstI site of pBR322 . The recombinant plasmids generated were used to transform a multiply auxotrophic strain of Escherichia coli with selection for tetracycline resistance . Plasmids complementing the argG(pAW1) or hisA(pAW2) mutations were isolated and characterized . Nick-translated pAW1 and pAW2 hybridized to the predicted M . voltae PstI fragments but not to digested E . coli DNA . A novel 55,000-dalton protein was synthesized in UV-irradiated cells by pAW1, whereas pAW2 synthesized a novel 26,000-dalton protein . Derivatives of pAW1 carrying insertion elements no longer complemented the argG mutation and failed to produce the 55,000-dalton protein . When an AccI fragment was deleted from pAW2, complementation of hisA did not occur and no 26,000-dalton protein was synthesized . The effect of orientation of the cloned DNA within the vector on complementation and polypeptide synthesis was examined.

J Bacteriol, 1983 Oct, 156(1), 130 - 5
Absence of oligomeric murein intermediates in Escherichia coli; Goodell EW et al.; The intermediates in the biosynthetic pathway of murein were examined in two strains of Escherichia coli to determine whether they synthesized oligomeric precursors in vivo . No oligomeric precursors could be detected; the only intermediates found were the previously described UDP-N-acetylmuramyl peptides, and the two lipid-linked compounds, N-acetylglucosamyl-N-acetylmuramyl-(pentapeptide)-pyrophosphoryl-undecaprenol and N-acetylmuramyl-(pentapeptide)-pyrophosphoryl-undecaprenol . It was concluded that lipid-linked monomers are directly incorporated into the murein sacculus in vivo and do not pass through an oligomeric stage.

J Bacteriol, 1983 Oct, 156(1), 109 - 14
Expression of a gene in a 400-base-pair fragment of colicin plasmid ColE2-P9 is sufficient to cause host cell lysis; Pugsley AP et al.; The colicin E2 immunity (ceiB) and lysis (celB) genes of colicin plasmid ColE2-P9 were cloned as a 900-base-pair insert under the control of the lac promoter in high-copy-number plasmid pUR222 . Hosts carrying this plasmid were immune to colicin E2, produced increased amounts of immunity protein (molecular weight, 9,000) and two smaller proteins (molecular weights, 5,000 and 3,000), and lysed when incubated in medium containing isopropyl-beta-D-thiogalactopyranoside (IPTG) . A 400-base-pair lacp-distal fragment derived from the insert in this plasmid was recloned in the same orientation into pUR222 . Although hosts carrying this plasmid also lysed when grown in the presence of IPTG, they were sensitive to colicin E2 and produced increased amounts of the 5,000- and 3,000-molecular-weight proteins (but not the full-length immunity protein) when treated with IPTG . The results were consistent with the idea that expression of celB (production of the 5,000- and 3,000-molecular-weight proteins) is sufficient to cause host cell lysis in the absence of colicin production and derepression of the host cell SOS system.

Infect Immun, 1983 Oct, 42(1), 362 - 7
In vivo microscopic observations of the responses of Kupffer cells and the hepatic microcirculation to Mycobacterium bovis BCG alone and in combination with endotoxin; McCuskey RS et al.; Kupffer cell function and hepatic microvascular hemodynamics were studied by in vivo microscopy in Mycobacterium bovis BCG-infected NMRI mice before and after treatment with minute (0.01 mu mg) tolerance-producing doses and doses causing 70% lethality (0.5 micrograms) of Escherichia coli 0111:B5 endotoxin alone and in combination . BCG-induced granulomas distorted the hepatic microvasculature and impeded blood flow in many sinusoids; flow also was altered further by leukocytes adhering to the sinusoidal walls and by enlarged Kupffer cells that bulged into the lumen . Nevertheless, in BCG-infected mice, the ratio of Kupffer cells which phagocytosed latex to sinusoids containing blood flow and capable of delivering these particulates to Kupffer cells was significantly greater than that in uninfected mice . The phagocytosis of single latex particles by individual Kupffer cells also was more rapid . This indicated an expansion of the numbers and activation of Kupffer cells . In this hyperreactive state, the tolerance-inducing dose of endotoxin produced no change in the rate of phagocytosis after 2 h . In contrast, the 70% lethal dose reduced the rate by 123%, unless tolerance was induced, in which case there was no reduction in the rate of phagocytosis . Twenty-four hours after injection of the tolerance-inducing dose, however, the rate of phagocytosis was accelerated slightly (17%) . This suggested that the Kupffer cells had been activated and perhaps were more effective in clearing subsequent endotoxin from the blood but without sufficient release of toxic substances to be lethal . That some mediators were released, however, was suggested by the microvascular alterations that accompanied the above phagocytic responses . These results further support the concept of a central role for Kupffer cells in endotoxin-mediated, nonspecific host defense mechanisms.

Infect Immun, 1983 Oct, 42(1), 269 - 75
Nucleotide sequence of the gene for heat-stable enterotoxin II of Escherichia coli; Picken RN et al.; Previously, the gene for heat-stable enterotoxin II (STII) has been mapped by transposon Tn5 insertion mutagenesis in the chimeric R-Ent plasmid pCG86 (Mazaitis, A . J., R . Maas, and W . K . Maas, J . Bacteriol . 145:97-105, 1981) . DNA segments containing this gene were cloned from the wild-type and STII-insertion-mutant plasmid . The position of the Tn5 insertion was determined, and a 530-base-pair-long segment of the wild-type plasmid corresponding to the Tn5 insertion site was sequenced . An open reading frame for the STII gene was identified and is characterized by typical promoter and ribosome binding site sequences . The deduced STII structural gene codes for a protein 71 amino acids long, including a typical signal peptide of 23 amino acids and a mature protein of 48 amino acids . The size and overall structure of STII are similar to those of STI, but the amino acid compositions of the two heat-stable enterotoxins are completely different.

Infect Immun, 1983 Oct, 42(1), 264 - 8
Characterization of the gene encoding heat-stable toxin II and preliminary molecular epidemiological studies of enterotoxigenic Escherichia coli heat-stable toxin II producers; Lee CH et al.; The gene encoding heat-stable toxin II (STII) was cloned into an Escherichia coli K-12 strain, and its nucleotide sequence was determined . The deduced amino acid sequence indicates that STII is synthesized within the cell as a 71-amino-acid protein and that neither the DNA nor amino acid sequence bears any similarity to that of heat-stable toxin I . A DNA fragment containing the STII gene was used to probe enterotoxigenic E . coli clinical isolates with various toxin phenotypes and was shown to be useful in detecting all STII and only STII producers.

Infect Immun, 1983 Oct, 42(1), 245 - 9
In vivo function of hemolysin in the nephropathogenicity of Escherichia coli; Waalwijk C et al.; The role of hemolysin in the nephropathogenicity of Escherichia coli was studied in a hematogenous pyelonephritis model in mice . The nephropathogenicity of a nonhemolytic, avirulent E . coli strain was increased by simultaneous injection with its hemolytic, nephropathogenic parent . This helper mechanism could be attributed to hemolysin, since the simultaneous injection of partially purified hemolysin gave a similar enhancement of nephropathogenicity . Intraperitoneal injection of hemoglobin or iron sulfate before intravenous challenge with this avirulent strain also led to increased virulence . The nephropathogenicity-enhancing effect of hemolysin is therefore supposed to depend on increasing the level of available iron in the host . Under conditions of plentiful iron, hemolysin production was repressed, as shown by in vitro growth experiments in the presence of exogenous iron . These results suggest that the production of hemolysin is regulated by feedback inhibition.

Infect Immun, 1983 Oct, 42(1), 152 - 5
Identification of enterotoxigenic Escherichia coli in patients with diarrhea in Asia with three enterotoxin gene probes; Seriwatana J et al.; A total of 984 enterotoxigenic Escherichia coli (ETEC) and 733 non-ETEC isolated from patients with diarrhea in Asia (one isolate per patient) were examined for homology with radiolabeled fragments of DNA encoding heat-labile toxin (LT) or heat-stable toxin of porcine (ST-P) or human (ST-H) origin . A total of 246 ETEC that produced LT and ST as determined by the Y-1 adrenal and suckling mouse assays were homologous with the LT probe . Of these 246 LTST ETEC, 156 (63%) were homologous with the ST-H, 46 (19%) were homologous with the ST-P, and 44 (18%) were homologous with both probes . A total of 401 LT ETEC were homologous with the LT probe . Of 337 ST ETEC identified by the suckling mouse assay, 244 (72%) were homologous with the ST-H, 84 (25%) were homologous with the ST-P, and 9 (3%) were homologous with both probes . None of the 733 isolates that were non-enterotoxigenic as determined by the Y-1 adrenal and suckling mouse assays was homologous with genes coding for enterotoxin . Four isolates (not included among the 984 ETEC examined) that were initially considered to produce LT because sterile culture supernatants produced rounding of Y-1 adrenal cells were not homologous with the LT probe . The sterile culture supernatants of these four isolates caused rounding after 8 h and subsequent destruction after 24 h of Y-1 adrenal tissue cultures . This effect was not inhibited by convalescent human cholera antiserum, Swiss Serum Institute cholera antitoxin, or antiserum to purified LT . These isolates were also negative in the Biken test previously used to identify LT-producing E . coli . The DNA hybridization technique with three enterotoxin gene probes was developed as a specific technique to identify ETEC in large numbers of specimens in Asia.

Proc Natl Acad Sci U S A, 1983 Oct, 80(19), 5895 - 7
Multiple quantum two-dimensional 1H--15N nuclear magnetic resonance spectroscopy: chemical shift correlation maps for exchangeable imino protons of Escherichia coli tRNAMetf in water; Griffey RH et al.; A procedure based on multiple quantum two-dimensional nuclear magnetic resonance spectroscopy is described for generation of 1H--15N chemical shift correlation maps . The method is used to obtain 15N chemical shifts for the exchangeable imino protons in 1H--15N units of site-specifically labeled Escherichia coli tRNAMetf in water . The high sensitivity and excellent chemical shift dispersion of the multiple quantum two-dimensional technique make it ideally suited for studying protonated nitrogens by NMR.

Proc Natl Acad Sci U S A, 1983 Oct, 80(19), 5852 - 6
Macromolecular crowding allows blunt-end ligation by DNA ligases from rat liver or Escherichia coli; Zimmerman SB et al.; In the presence of high concentrations of any of several types of macromolecules, DNA ligase preparations from rat liver nuclei or from Escherichia coli actively catalyze the blunt-end ligation of DNA . This is in contrast to the lack of activity on such substrates by these enzymes under conventional assay conditions . In addition, the previously established activity of T4 DNA ligase on blunt-ended molecules is greatly increased in the presence of high concentrations of macromolecules . Because such crowded solutions may well be a more adequate model for intracellular conditions than assays in dilute solutions, we suggest that blunt-end ligation may be a widely occurring reaction in vivo.

Proc Natl Acad Sci U S A, 1983 Oct, 80(19), 5837 - 41
Secondary structure of the lac repressor DNA-binding domain by two-dimensional 1H nuclear magnetic resonance in solution; Zuiderweg ER et al.; A recently proposed approach for spatial structure determination in noncrystalline proteins by nuclear magnetic resonance was applied to the lac repressor DNA-binding domain . On the basis of sequence-specific 1H NMR assignments, the location of alpha-helices in the amino acid sequence was determined from nuclear Overhauser enhancement data and from amide proton exchange studies . These investigations provide detailed experimental data on the structure of a noncrystalline DNA-binding protein . The results support the hypothesis advanced by others that sequence-specific interactions between lac repressor and DNA are mediated by a particular spatial arrangement of two alpha-helices common to various different DNA-binding proteins.

J Gen Microbiol, 1983 Oct, 129 (Pt 10), 3215 - 25
Sub-cloning of the wild-type proAB region of the Escherichia coli genome; Hayzer DJ; The genes proA and proB encoding the first two enzymes of the proline biosynthetic sequence in Escherichia coli were subcloned from a ColE1 hybrid plasmid containing 23.3 kilobases of genomic DNA . proA and proB are contiguous and constitute a single operon transcribed in the direction proB-proA . The pro operon is contiguous with the gene phoE . Hybridization experiments showed no homology between proAB of E . coli and the other regions of the E . coli genome or with the DNA of several other bacterial species.

Genetika, 1983 Oct, 19(10), 1611 - 5
{Transposition of the phage D3112 genome in Escherichia coli cells}; Plotnikova TG et al.; It has been demonstrated that the genome of phage D3112 of Preudomonas aeruginosa can be transposed into Escherichia coli chromosome as a component of the hybrid plasmid RP4 TcrKms::D3112 . Also, transposition of D3112 from E . coli (D3112) chromosome into RP4 plasmid occurs . The phage stimulates the chromosome mobilizing activity of RP4 plasmid, similar to other transposons . E . coli (RP4::D3112) cells were previously shown to form no colonies at 30 degrees C . Auxotrophic mutants and mutants incapable of utilizing different carbohydrates were found among E . coli clones survived after a long incubation at 30 degrees C (at frequencies approximately 10(-3) - 10(-4) . These mutants inherited stably the capability to produce D3112 phage . E . coli auxotrophic mutants have arisen indeed as a consequence of phage integration into the E . coli chromosome, since prototrophic transductants derived from these mutants after their treatment with generalized transducing P1 phage have lost the ability to produce D3112 phage . Clones with mutations in Km or Tc genes of RP4 plasmid, occurring at high frequencies (about 3%) were found after introduction of RP4 into E . coli (D3112) . These mutant RP4 plasmids carry insertions of D3112 genomes . Clones of E . coli which lost mutant plasmids still produce D3112 and retain their initial auxotrophic mutations.

Genetika, 1983 Oct, 19(10), 1582 - 92
{Integration of plasmid RP1 with the chromosome of Escherichia coli K-12 recA . 2 classes of Hfr strains}; Danilevich VN et al.; Integration of broad host range RP1 plasmid into the chromosome of Escherichia coli K-12 recA- cells has been studied . Using temperature-sensitive for replication plasmids pVD1 and pVD3, the derivatives of RP1, it has been shown that integration of RP1 into the bacterial chromosome results in formation of two classes of Hfr strains . Properties of these Hfrs have been examined . From the data obtained, it has been concluded that the plasmid integration and formation of one of the Hfrs classes appear to be mediated by transposon Tn1 residing on RP1 . The other class of Hfr strains is formed due to a stable integration of RP1 . In the course of analysis of R+ transconjugants arising at low frequency in crosses between stable Hfrs and E . coli rec+ recipients, it has been found that the significant part of them contain plasmid-chromosome hybrids (R-prim plasmids) . On the basis of the latter results, a new simple method for R' plasmids selection has been proposed . Using restriction endonuclease analysis, the structure of plasmids that were excised from chromosomes of the stable Hfr strains and were comparable in their size to RP1, has been investigated . Probable mechanisms of the stable Hfr strains formation are discussed.

Mol Cell Biol, 1983 Oct, 3(10), 1746 - 58
Rous sarcoma virus contains sequences which permit expression of the gag gene in Escherichia coli; Mermer B et al.; Several aspects of Rous sarcoma virus gene expression, including transcription, translation, and protein processing, can occur within Escherichia coli containing cloned viral DNA . The viral long terminal repeat contains a bacterial promoter, and viral sequences at or near the authentic viral initiation codon permit the initiation of translation . These signals can direct the synthesis in E . coli of the viral gag gene precursor Pr76 or, when fused to a portion of the lacZ gene, a gag-beta-galactosidase fusion protein . Pr76 is processed into gag structural proteins in E . coli in a process which is dependent upon the gag product p15 . These observations suggest that E . coli can be used for the introduction and analysis of mutations in sequences relevant to viral gene expression.

Gene, 1983 Oct, 24(2-3), 245 - 53
A vehicle for DNA transfer and for recovery of transferred genes: lambda Charon phage-pBR322 hybrid; Hamada Y et al.; Recombinant Charon 4A phages accommodating the Herpes simplex virus (HSV-1) thymidine kinase (tk) gene, the ampicillin-resistance (ApR) gene, and the replication origin of pBR322 were constructed . The phage DNA was introduced into mouse Ltk- cells by a free DNA transfer method or phage-mediated DNA transfer method {Ishiura et al., Mol . Cell . Biol . 2 (1982) 607} . Analyses of the physical state of the transferred DNA in the recipient cell genome showed that a DNA fragment as long as 12.7 kb was integrated intact into 67% and less than 40% of the Ltk- transformant cells by phage-mediated DNA transfer and by free DNA transfer, respectively . We also developed a new rapid method for recovery of the transferred gene from the Ltk+ cell into Escherichia coli; the method depends on the fact that the recombinant lambda phage carrying the ApR gene and replication origin of pBR322 transduces lambda-lysogenic bacteria to ApR and is maintained as a plasmid . Using this method the HSV-1 tk gene from one Ltk+ transformant was rapidly and successfully recovered without any rearrangement of the target sequence.

Gene, 1983 Oct, 24(2-3), 191 - 8
Mapping restriction sites on large DNAs by electron microscopy; Moore C et al.; We have developed a novel technique to map restriction sites on large duplex DNAs by electron microscopy . In this method, the sample DNA is first cut with a restriction enzyme . The resulting fragments are briefly digested with Escherichia coli exonuclease III, and treated with wheat germ RNA polymerase II to fill-in with RNA the resulting gaps . These small RNAs, complementary to sequences immediately adjacent to either side of the restriction site, are isolated from the DNA template and R-looped to the full-length DNA . When this material is prepared by the formamide-cytochrome spreading technique, small bubbles are visible wherever there is a restriction site on the DNA . Improved methods of mapping are outlined.

Mutat Res, 1983 Oct, 112(5), 253 - 60
Effects of recB, recF and uvrA mutations on Weigle reactivation of lambda phages in Escherichia coli K12 treated with 8-methoxypsoralen or angelicin and 365-nm light; Lichtenberg B et al.; The extent of Weigle reactivation (W-R) of lambda c+ phages treated with bifunctional 8-methoxypsoralen and 365-nm light (8-MOP + UVA) and with monofunctional angelicin and 365-nm light (ANG + UVA) were compared in Escherichia coli strains with different excision repair and recombinational capacities . In uvrA6 host cells, irradiation of the cells with 254-nm radiation only decreased the survival of phages treated with 8-MOP + UVA . The extent of W-R of ANG + UVA-treated phages in uvrA6 cells, however, was much larger than that obtained in other host cells . These results indicate that monoadducts produced by ANG + UVA treatment can be repaired effectively by the repair induced without uvrA gene products, but lesions produced by 8-MOP + UVA treatment cannot be repaired by the repair . The small W-R of ANG + UVA-treated phages in wild-type cells may be due to the high repairability of the lesions by constitutive excision repair in the cells . recB21 reduced only a little of the W-R of 8-MOP + UVA- or ANG + UVA-treated phages obtained in wild-type cells . Although recF143 cells have an almost comparable host-cell repair capacity of 8-MOP + UVA- or ANG + UVA-treated phages to that of the wild-type cells, recF143 mutation reduced not only the extent of W-R of 8-MOP + UVA-treated phages to 17% of that obtained in wild-type cells but also the extent of W-R of ANG + UVA-treated phages to 12% of that obtained in uvrA6 cells.

Cell, 1983 Oct, 34(3), 815 - 22
Structure of the plasmodium knowlesi gene coding for the circumsporozoite protein; Ozaki LS et al.; The gene that codes for the surface antigen of Plasmodium knowlesi sporozoites (CS protein) is unsplit and present in the genome in only one copy . The CS protein, as deduced from DNA sequence analysis of the structural gene, has an unusual structure with the central 40% of the polypeptide chain present as 12 tandemly repeated amino acid peptide units flanked by regions of highly charged amino acids . The protein has an amino-terminal hydrophobic amino acid signal sequence and a hydrophobic carboxy-terminal anchor sequence . The coding sequence of the gene has an AT content of 53%, compared with 70% AT in the 5' and 3' flanking sequences, and is contained entirely within an 11 kb Eco RI genomic DNA fragment . This genomic fragment expresses the CS protein in E . coli, indicating that the parasite promoter and ribosome binding site signals can be recognized in E . coli.

Proc Natl Acad Sci U S A, 1983 Oct, 80(20), 6157 - 61
Enzymatic properties of purified Escherichia coli uvrABC proteins; Yeung AT et al.; The cloned uvrA and uvrB genes of Escherichia coli K-12 were amplified by linkage to the PL promoter of plasmid pKC30 . The uvrC gene was amplified in the high-copy-number plasmid pRLM 24 . The three gene products (purified in each case to greater than 95% purity) and ATP are required to effectively incise UV-damaged DNAs . The uvrABC proteins bind tightly to damaged sites in DNA, requiring the initial attachment of the uvrA protein in the presence of ATP before productive binding of the uvrB and uvrC proteins . Using a cloned tandem double insert of the lac p-o region as a damaged DNA substrate for the uvrABC complex and analyzing the incision both 5' and 3' to each pyrimidine dimer, we found that one break occurs 7 nucleotides 5' to a pyrimidine dimer and a second break is made 3-4 nucleotides 3' from the same pair of pyrimidines in the dimer . No such breaks are found in the strand complementary to the dimer . The size of the incised fragment in the DNA suggests that incision may be coordinated with excision reactions in repair processes.

J Gen Virol, 1983 Oct, 64 (Pt 10), 2261 - 70
Physical mapping of temperature-sensitive mutations of herpes simplex virus type 1 using cloned restriction endonuclease fragments; Matz B et al.; Sequences from the whole of the HSV-1 strain 17 genome were cloned into bacterial plasmid vectors, with the exception of part of BamHI v which was deleted in all cloned DNAs spanning this region of the virus DNA . The cloned DNAs were used in intratypic marker rescue experiments to map temperature-sensitive (ts) mutations on to the virus genome . Since the sequences of these DNAs overlapped, any mutation could be rapidly assigned a physical map position . This approach is particularly useful for mapping spontaneous mutations and lesions induced by mutagenesis of whole virus DNA . In this study, we mapped ten ts mutations comprising eight different complementation groups . Five lesions, representing three different cistrons, were located within BglII k (map units 0.098 to 0.166), and three mapped within EcoRIf (map units 0.321 to 0.414), two of which were in previously unidentified cistrons of HSV-1 strain 17 . One mutation analysed had a defect within the short repeat region and another had a mutation within EcoRI i (map units 0.632 to 0.720).

J Immunol, 1983 Oct, 131(4), 1784 - 8
Evidence for a nonoxidative mechanism of human natural killer (NK) cell cytotoxicity by using mononuclear effector cells from healthy donors and from patients with chronic granulomatous disease; Kay HD et al.; In vitro natural killer (NK) activity expressed by blood mononuclear cells from patients with chronic granulomatous disease of childhood (CGD) was equivalent to that expressed by cells from normal, healthy volunteers . Because neutrophils and monocytes from these same donors exhibited extremely depressed oxidative functions, our data could be interpreted to show that a) NK cells derived from a unique and separate cellular lineage unaffected by the disease-related oxidative defect, or b) the in vitro cytolytic mechanism(s) of NK cells were not dependent on oxygen metabolites . These hypotheses were examined by using as NK effector cells large granular lymphocytes (LGL) from healthy donors whose monocytes and neutrophils had normal oxidative functions . Such functions were measured in the nitroblue tetrazolium dye reduction assay, which is a qualitative measurement of superoxide anion production; by reduction of ferric cytochrome c, a more specific and quantitative measurement of superoxide anion production; and in the luminol-enhanced chemiluminescence assay, an extremely sensitive measure of several reactive oxygen radicals, including superoxide anion, hydroxyl radical, and singlet oxygen . Whereas monocytes and neutrophils from healthy donors were readily stimulated with zymosan or phorbol myristate acetate (PMA) in each of these assays . LGL produced no detectable amounts of oxygen metabolites when co-incubated either with K562 erythroleukemia cells, PMA, E . coli endotoxin, or the calcium ionophore A23187 . Thus, because NK cell activity is normal in CGD patients with major oxidative defects, and because no reactive oxygen metabolites could be detected in LGL that simultaneously exhibited potent NK activity, we conclude that in vitro NK activity by human mononuclear cells involves a lytic mechanism(s) independent of oxygen metabolites.

J Bacteriol, 1983 Oct, 156(1), 81 - 8
2-Oxoacid dehydrogenase complexes of Escherichia coli: cellular amounts and patterns of synthesis; Smith MW et al.; The oxidative decarboxylations of pyruvate and 2-oxoglutarate in Escherichia coli are carried out by two large, multienzyme complexes: pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase . The enzyme complexes each contain three subunits: two are unique to the individual complexes, the third is shared between them . Resolution of the polypeptide subunits on two-dimensional gels allowed quantitative analysis of their cellular levels and patterns of synthesis in growing cells . Cells growing in glucose-salts medium were found to contain roughly 85 to 136 pyruvate dehydrogenase complexes and 73 2-oxoglutarate complexes . Lipoamide dehydrogenase, the subunit shared by the two complexes, was found to be in significant excess of its stoichiometric demand in the two enzyme complexes under most growth conditions . The subunits unique to each of the complexes were coordinately regulated over a wide variety of growth conditions and a broad range of expression . The two complexes responded to different, but partially overlapping, regulatory signals . Most importantly, the shared subunit was actively regulated to accommodate its demand in both enzymes . These results are discussed with regard to possible mechanisms of regulation of the enzyme complexes in general and of the shared subunit specifically.

J Bacteriol, 1983 Oct, 156(1), 62 - 9
Isolation and characterization of chain-terminating nonsense mutations in a porin regulator gene, envZ; Garrett S et al.; We isolated four independent amber mutations in gene envZ, whose product is involved in the regulation of porin protein genes ompF and ompC . The envZ amber strains exhibit an OmpF-/+ OmpC- porin phenotype and express other envelope protein genes at wild-type levels . This phenotype is clearly different from that of the previously isolated class of envZ mutants that exhibit an OmpF- OmpC+ phenotype and a pleiotropic decrease in the expression of several exported protein genes, including lamB and phoA . The addition of the local anesthetic procaine to wild-type strains also causes a pleiotropic decrease in the expression of genes ompF, lamB, and phoA . However, procaine has no effect on the synthesis of LamB or PhoA protein in the envZ amber strains . Thus, although EnvZ protein is required for the full expression of ompF and ompC, it apparently is not normally involved in the expression of other envelope protein genes . One interpretation of these results is that the EnvZ protein can be altered either by mutation or by procaine to a form that interferes with the expression of several envelope protein genes other than ompF and ompC . Finally, complementation analysis with ompR insertion mutations supports the physical data of Mizuno et al . (J . Biol . Chem 257:13692-13698, 1982) that suggest that envZ is cotranscribed with ompR from a single promoter in the order ompR envZ.

J Bacteriol, 1983 Oct, 156(1), 458 - 9
Convenient construction of recA deletion derivatives of Escherichia coli; Lorence MC et al.; Two Escherichia coli K-12 Hfr strains have been constructed which transfer a recA deletion, which is highly linked to a Tn10 insertion conferring tetracycline resistance, early during conjugation . These strains transfer the recA deletion in opposite directions with different origins of transfer, allowing for preservation of desirable recipient strain markers either clockwise or counterclockwise of recA.

J Bacteriol, 1983 Oct, 156(1), 402 - 8
Outer membrane porins are important in maintenance of the surface structure of Escherichia coli cells; Nogami T et al.; Escherichia coli cells lacking the OmpF and OmpC proteins, porin proteins of the outer membrane, are often unstable and easily revert to strains which either have regained one or both of these proteins or contain a new outer membrane protein . The structural importance of porin proteins in the cell surface was studied in the present work . Tris-hydrochloride buffer at a concentration of 120 mM caused deformation of the cell surface of a strain lacking these porins; the undulated appearance of the negatively stained cell surface changed to a smooth and expanded form . The Tris-induced deformation was seldom observed with either the wild-type strain or a pseudorevertant that possessed the OmpF protein . The role of the OmpF protein in stabilizing the cell surface against Tris treatment could be slightly taken over by the LamB protein, which shares a number of unique properties with the former proteins . The deformation of the cell surface by Tris-hydrochloride buffer was accompanied by a loss of viability, the lethal damage being especially significant when the cells lacked porins . Upon induction with maltose, cells with the undulated appearance could absorb lambda phages, whereas the deformed cells could not . These results suggest that the instability of cells lacking porins is primarily due to a structural defect of the outer membrane.

J Bacteriol, 1983 Oct, 156(1), 115 - 21
Immunological characterization of an Escherichia coli strain which is lacking cytochrome d; Kranz RG et al.; The isolated membranes from an Escherichia coli mutant strain which lacks spectroscopically detectable levels of cytochromes d, a1, and b558 also have abnormally low levels of N,N,N',N'-tetramethyl-p-phenylenediamine oxidase activity . In this paper, it is shown that the material previously identified as the N,N,N',N'-tetramethyl-p-phenylenediamine oxidase is, in fact, the two-subunit cytochrome d complex . Antisera directed against the native cytochrome d complex as well as against each of two subunits apparent on sodium dodecyl sulfate-polyacrylamide gels were used to show that the mutant strain lacks both subunits of the cytochrome d complex . Introduction of F-prime F152 into the mutant strain restored the two subunits along with the spectroscopic and enzymatic activity associated with the cytochrome d complex.

Infect Immun, 1983 Oct, 42(1), 187 - 96
Molecular cloning and expression of Treponema pallidum DNA in Escherichia coli K-12; van Embden JD et al.; A gene bank of Treponema pallidum DNA in Escherichia coli K-12 was constructed by cloning SauI-cleaved T . pallidum DNA into the cosmid pHC79 . Sixteen of 800 clones investigated produced one or more antigens that reacted with antibodies from syphilitic patients . According to the separation pattern of the antigens produced on sodium dodecyl sulfate-polyacrylamide gels, six different phenotypes were distinguished among these 16 clones . These antigens reacted also with anti-T . pallidum rabbit serum . No antibodies against the cloned antigens were found in normal rabbit serum and in nonsyphilitic human serum . The antigens produced by the E . coli K-12 recombinant DNA clones comigrated in sodium dodecyl sulfate-polyacrylamide gels with antigens extracted from T . pallidum bacteria, suggesting that the treponemal DNA is well expressed in E . coli K-12 . Several of the cosmid recombinant plasmids have been subcloned, resulting in smaller T . pallidum recombinant plasmids which are more stably maintained in the cell and produce more treponemal antigen . Monoclonal antibodies were raised against T . pallidum, and one hybridoma produced antibodies that reacted not only with an antigen from T . pallidum but also with the antigen produced by one of the E . coli clones.

Proc Natl Acad Sci U S A, 1983 Oct, 80(19), 6105 - 9
Efficient expression of influenza virus NS1 nonstructural proteins in Escherichia coli; Young JF et al.; RNA segment 8 of the influenza A virus genome codes for two nonstructural proteins, NS1 and NS2, for which the functions are unknown . Cloned cDNA copies of this gene from three different influenza A virus strains were inserted into an Escherichia coli plasmid expression vector, pAS1, carrying the strong regulatable lambda phage promoter, PL . After induction, the NS1 proteins were overproduced to levels of 20-25% of total cellular protein . This was surprising in that the codon composition for these eukaryotic genes is similar to that for weakly expressed proteins in E . coli . Thus, under the appropriate conditions, it appears that high level expression of genes containing a relatively large proportion of minor codons can be obtained . The NS1 protein produced in bacteria from a cloned cDNA copy of the A/PR/8/34 virus NS gene was purified to apparent homogeneity and used to generate a high-titer monospecific rabbit antiserum . Immunoprecipitation studies showed this antibody to be crossreactive against the NS1 proteins produced by several different influenza A virus strains . Immunofluorescence experiments in Madin-Darby canine kidney cells showed the NS1 proteins to be located in the nucleoplasm early in infection for all strains examined . With some of the strains, NS1-specific immunofluorescence was observed predominantly in the nucleoli later in infection . This technology can be used to obtain other viral proteins in pure form for structural, functional, and immunological studies.

Proc Natl Acad Sci U S A, 1983 Oct, 80(19), 5970 - 4
Molecular evolution of the human adult alpha-globin-like gene region: insertion and deletion of Alu family repeats and non-Alu DNA sequences; Hess JF et al.; Previous heteroduplex studies have revealed extensive sequence homology between the two human adult alpha-globin-like genes (alpha 2 and alpha 1) and their flanking regions . These homologous regions, which are interrupted by two blocks of nonhomology, each span approximately 4 kilobases {Lauer, J., Shen, C.-K . J . & Maniatis, T . (1980) Cell 20, 119-130} . We have determined 3 kilobases of DNA sequences within and flanking the nonhomologous blocks of these two tandem duplication units . A total of three Alu family repeats has been identified . Two of them are approximately 300 base pairs long and define the 3' ends of the first homology blocks . The third Alu family member is a 600-base-pair-long sequence consisting of two monomeric Alu members arranged in a head-to-tail fashion . It is located in the 3' portion of the first block of nonhomology in alpha 2-gene-containing unit . We present direct evidence that this dimeric Alu sequence was inserted at a staggered break . The second nonhomology block is the result of insertion or deletion of a 224-base-pair sequence . From these data and the calculation of sequence divergence, we propose a history for the evolution of the human adult alpha-globin-like gene region . We also suggest that DNA insertion elements may disrupt gene correction processes in the two duplication units containing alpha 2- and alpha 1-globin genes.

Proc Natl Acad Sci U S A, 1983 Oct, 80(19), 5842 - 6
Cloning and expression of murine immune interferon cDNA; Gray PW et al.; The murine immune interferon (IFN-gamma) gene was cloned and expressed under control of the simian virus 40 early promoter in the monkey COS-1 cell line . A protein is secreted from these cells having the biological, antigenic, and biochemical characteristics of natural murine IFN-gamma . Cloned murine IFN-gamma cDNAs were obtained by using RNA from both mitogen-induced murine spleens and the transfected COS cells, and both code for identical proteins . The mature murine IFN-gamma encoded is 136 amino acids long, 10 amino acids shorter than human IFN-gamma . The nucleotide homology between the murine and human IFN-gamma genes is 60-65%, whereas the encoded proteins are only 40% homologous . Murine IFN-gamma cDNA was expressed in Escherichia coli under trp promoter control.

Proc Natl Acad Sci U S A, 1983 Oct, 80(19), 5817 - 21
Purified dnaA protein in initiation of replication at the Escherichia coli chromosomal origin of replication; Fuller RS et al.; Soluble protein fractions from Escherichia coli dnaA+ cells but not dnaA temperature-sensitive cells replicate plasmids containing the E . coli chromosomal origin of replication (oriC) . Complementation of these mutant fractions provided an assay for dnaA protein activity in initiation of replication at oriC . From a strain (constructed in vitro) that overproduces the dnaA protein more than 200-fold, the 52,000-dalton polypeptide was purified to near homogeneity . Although the protein tends to aggregate, monomer-sized protein purified by high-performance liquid chromatography is fully active for replication . It binds specifically and tightly to oriC in a supercoiled plasmid as judged by a Millipore filter-binding assay and by protection of the unique HindIII site within the oriC sequence . In the oriC replication reaction, dnaA protein acts at an early step preceding DNA synthesis.

Anal Biochem, 1983 Oct 1, 134(1), 82 - 5
A colony filter-hybridization procedure for the filamentous fungus Neurospora crassa; Stohl LL et al.; A colony filter-hybridization procedure for the filamentous fungus Neurospora crassa has been developed . The procedure is sensitive enough to detect Escherichia coli plasmid pBR322 DNA integrated into chromosomal DNA in a Neurospora transformant . Thus, it should facilitate the isolation of nuclear genes by plasmid-rescue procedures.

Mutat Res, 1983 Oct, 111(2), 119 - 33
Induction of SOS functions in Escherichia coli by lesions resulting from incorporation of 5-bromouracil into DNA; Pietrzykowska I et al.; Lesions induced by 5-bromouracil (BU), after its incorporation into DNA, led to effective induction of prophage lambda and W reactivation (or BU reactivation) . Prophage induction due to incorporated BU occurred only with the wild-type prophage, and not for the lambda c1857 mutant with a thermosensitive repressor . Antipain, a protease inhibitor, inhibited wild-type prophage induction 70-90% . This indicates that BU-induced lesions may induce the SOS repair system . The finding that such lesions provoke BU reactivation permits the inference that BU-induced mutagenesis also proceeds via involvement of the error-prone repair system, and not directly as a result of base-pairing errors . Genetic evidence suggests that induction of the SOS repair system as a result of incorporation of BU into DNA is linked to the subsequent appearance of uracil residues and apyrimidinic sites, resulting from dehalogenation of incorporated BU . Apyrimidinic sites appear to be more effective than uracil residues in induction of the SOS system.

Genetics, 1983 Oct, 105(2), 247 - 57
Specialized transduction with lambda plac5: involvement of the RecE and RecF recombination pathways; Porter RD; Several aspects of the recombination resulting from lambda plac5 transduction were investigated in strains of Escherichia coli K-12 that use the RecE or RecF recombination pathways . In a RecBC pathway strain, F42lac recombination with lambda plac5 is 20- to 50-fold higher than chromosomal lac times lambda plac5 recombination, and this recombination enhancement is largely dependent on constitutive expression of F42lac fertility functions . Here, it was observed that F42lac fertility functions do not effect the ability of F42lac to recombine with lambda plac5 in a RecE or RecF pathway strain . Therefore, the enhancement observed in a Rec+ (or RecBC pathway) strain is directly dependent on the recBC gene product . The end product of recombination between lambda plac5 and either F42lac or chromosomal lac in RecE and RecF pathway strains was monitored by scoring for addition and substitution transductants . It was observed that the percentage of addition transductants was lower in all cases for RecE and RecF pathway strains as compared with RecBC pathway or a recB strain . It is concluded that the introduction of sbcA or sbcB into a recB strain produces a change in recombination mechanism that is reflected in the nature of the end product of recombination.

Cell, 1983 Oct, 34(3), 941 - 9
Differential effects of mutations on discrete steps in transcription initiation at the lambda PRE promoter; Shih MC et al.; The effects of cy mutations on transcription initiation at the lambda PRE promoter were determined using abortive initiation analysis (McClure, 1980) . In the presence of lambda cll protein, which activates PRE, three mutations in the -10 region dramatically reduce k2, the forward rate constant for the isomerization of closed to open complexes, but only slightly affect KB, the equilibrium constant for the initial recognition by RNA polymerase to form closed complexes . In contrast, five -35 region mutations caused decreases of 30 to 150 times in KB with much smaller effects on k2 . In the absence of cll protein, the effects of mutations in the -10 region are qualitatively similar to those observed in the presence of cll protein, although the reductions in k2 are much less dramatic . In contrast, none of the mutants with defects in the -35 region is distinguishable from wildtype PRE in the absence of cll protein . Thus RNA polymerase may recognize different sequences in the -35 region in the absence of cll protein than in its presence.

Bioorg Khim, 1983 Oct, 9(10), 1388 - 94
{Synthesis and expression of the DNA fragment coding the antigenic determinant of the surface antigen protein of hepatitis B virus}; Smirnov VD et al.; With the use of chemical-enzymatic synthesis, a polynucleotide (I) has been obtained which codes the amino acid sequence 93-109 of the hepatitis B surface antigen (HBsAg) . The plasmid pHS12, constructed by insertion of (I) into pBR325 between EcoRI and BamHI sites, transformed E . coli HB101 cells . The plasmid expression in cell-free system produced a chimeric protein, its molecular weight corresponding to that a polypeptide containing the heptadecapeptide from HBsAg . An approach is discussed for creating a new type of vaccines on the basis of chimeric proteins containing the antigenic determinants of virus proteins.

Proc Natl Acad Sci U S A, 1983 Oct, 80(20), 6187 - 91
Processing of transcription products of the gene encoding the RNA component of RNase P; Sakamoto H et al.; The gene coding for the RNA component of RNase P from Escherichia coli was transcribed in vitro by using a restriction fragment carrying the gene as template . The terminal sequences of the transcription products were determined and mapped on the DNA sequence of the gene . The signals for transcription initiation and termination of the gene were thus identified . Transcription termination occurs within a region of two bases at positions 413 and 414 from the transcription start site . The transcripts carry extra stretches of 36 and 37 nucleotides at the 3' end of the RNA molecule isolated from the enzyme . The extra sequences were removed in vitro when the transcripts were incubated with a crude cell extract.

J Exp Med, 1983 Oct 1, 158(4), 1114 - 28
Antiadhesive properties of a quaternary structure-specific hybridoma antibody against type 1 fimbriae of Escherichia coli; Abraham SN et al.; The relationship between the structure and biological function of type 1 fimbriae of Escherichia coli was investigated using a set of monoclonal antibodies directed against conformation-specific antigenic determinants . Of three monoclonal antibodies tested, only one (clone CD3) prevented adhesion of the vaccine strain to epithelial cells or guinea pig erythrocytes . The antibody produced by CD3, but not that produced by the other two hybridoma clones (AA8 and GG1), precipitated isolated fimbriae by double diffusion in agar gel and was shown to bind in a highly discrete, periodic manner along the length of each of the fimbriae by immunoelectron microscopy . Immunoelectroblots of type 1 fimbrial subunits and polymers electrophoresed in SDS-gels indicated that the antibodies in AA8 and GG1 reacted only with fimbrial monomers (mol wt 17,000), whereas the antibody in CD3 reacted only with polymers of mol wt 102,000 (hexamers) or higher . ELISA inhibition assays demonstrated that dissociated fimbrial subunits lost their reactivity with antibody CD3 but gained reactivity with antibodies AA8 and GG1 . Conversely, when allowed to reassemble in vitro in the presence of 5 mM MgCl2, the reassembled fimbriae lost their reactivity with antibodies AA8 and GG1 but regained reactivity with antibody CD3 . These results demonstrated that certain antigenic epitopes are dependent on quaternary structural determinants, whereas others are independent of quaternary fimbrial structure and also are inaccessible for antibody binding in fimbriae once they have been assembled . These monoclonal antibodies should prove useful in studies of the structural determinants of the biological function of type 1 fimbriae as well as in studies of fimbrial synthesis, transport, and assembly.

Infect Immun, 1983 Oct, 42(1), 333 - 40
Isolation and characterization of a monoclonal antibody directed against type 1 fimbriae organelles from Escherichia coli; Eisenstein BI et al.; We have isolated a mouse immunoglobulin M (IgM) monoclonal antibody directed against type 1 fimbriae from the Escherichia coli K-12-derived strain CSH50 . Antibody specificity was demonstrated by (i) the ability of fimbriate but not nonfimbriate bacteria to compete with solid-phase purified fimbriae for antibody binding in an enzyme-linked immunosorbent assay, (ii) the visualized binding of antibody to fimbriae alone by electron microscopy, and (iii) the appearance in a radioimmunoprecipitation assay of a single electrophoretic band comigrating with pure type 1 fimbriae . The monoclonal antibody was further characterized by immunoblot analysis and compared with previously prepared rabbit anti-fimbrial antisera . Whereas the monospecific but polyvalent antisera recognized both fimbrial monomeric subunits and non-disaggregated fimbriae organelles, the monoclonal antibody recognized only the intact organelles even when the samples were prepared under nondenaturing conditions . The monoclonal antibody, therefore, might be directed against an epitope spanning two (or more) adjacent fimbrial subunits.

J Bacteriol, 1983 Oct, 156(1), 463 - 5
Mutation of the N-acetylmuramyl-L-alanine amidase gene of Escherichia coli K-12; Tomioka S et al.; Mutants of Escherichia coli with very low N-acetylmuramyl-L-alanine amidase activity were isolated . The gene amiA responsible for most of this enzyme activity was mapped at 51 min on the E . coli chromosome, with the most plausible gene order assumed to be amiA pts(H or I) purC . The mutant phenotype was recessive and physiologically undiscernible.

J Bacteriol, 1983 Oct, 156(1), 230 - 5
Characterization of conjugative plasmid EDP208; Finlay BB et al.; EDP208 is a conjugative plasmid belonging to incompatibility group IncF0 lac, A restriction endonuclease map of this plasmid was constructed using five restriction enzymes: BamHI, HindIII, PvuI, SstI, and XhoI . On the basis of these mapping studies, the plasmid was found to be 90 kilobases in length . Clones were constructed from four large HindIII fragments of plasmid EDP208 . One fragment, HindIII-20.5, was found to contain the lac genes and the origin of vegetative replication (oriV) . Another fragment, HindIII-27.5, was found to contain all of the genes necessary for sex pilus formation, but it was nontransmissible . However, when used to complement a plasmid carrying an adjacent fragment, HindIII-23, the transfer of the latter occurred, suggesting that HindIII-23 contains the origin of transfer (oriT) . The further localization of genes concerned with pilus biosynthesis was achieved by transposon mutagenesis . Six EDP208::Tn1 and thirty-seven EDP208::Tn5 mutants were isolated on the basis of their resistance to f1, a filamentous phage which adheres to intact pilus tips . The positions of the inserted transposons were determined on the restriction map and a 16.5-kilobase region was found to be required for pilus synthesis.

Gastroenterology, 1983 Oct, 85(4), 837 - 45
Species-specific binding of purified pili (AF/R1) from the Escherichia coli RDEC-1 to rabbit intestinal mucosa; Berendson R et al.; Species-specific colonization of rabbit intestine by RDEC-1 Escherichia coli is an accepted animal model for bacterial mucosal adherence . To determine whether RDEC-1 pili are functional as adherence factors for this organism, we grew the organism under conditions that promoted pilus expression; we isolated the pili, documented their purity, and compared their mucosal adherence properties with those of whole organisms using an indirect immunofluorescence technique . Frozen sections of rabbit, rat, guinea pig, and human small intestine were incubated with either piliated or nonpiliated RDEC-1 organisms or purified RDEC-1 pili and observed for the distribution and intensity (0-4+) of fluorescence . Piliated RDEC-1 organisms fluoresced brightly (4+) and were distributed along the entire mucosal surface of the rabbit ileum . Only a few nonpiliated RDEC-1 attached to rabbit ileum, and they were randomly scattered across the entire section of tissue . Rabbit ileum overlain with pure RDEC-1 pili showed a specific, D-mannose resistant (2-3+) fluorescence on the mucosal surface from the crypts to the villus tips . No fluorescence was seen on the guinea pig, rat, or human mucosal surface overlain with RDEC-1 pili . Purified RDEC-1 pili adhere to the rabbit intestinal mucosa in a species-specific manner and with the same distribution as whole piliated organisms . The data suggest that RDEC-1 produce pili (distinct from type 1 pili) that determine the specificity of the mucosal adherence of RDEC-1 to rabbit ileum.

J Biomol Struct Dyn, 1983 Oct, 1(1), 83 - 96
Raman spectroscopy study of the B-Z transition in (dG-dC)n.(dG-dC)n and a DNA restriction fragment; Wartell RM et al.; The B to Z conformational transition of (dG-dC)n.(dG-dC)n and a 157 bp DNA restriction fragment were followed using Raman spectroscopy . The 157 bp DNA has a 95 bp segment from the E . coli lactose operon sandwiched between 26 and 32 bp of (dC-dG) sequences . Raman spectra of the DNAs were obtained at varying sodium chloride concentrations through the region of the transition . A data analysis procedure was developed to subtract the background curves and quantify Raman vibrational bands . Profiles of relative intensity vs . sodium chloride concentration are shown for bands at 626, 682, 831-833 and 1093 cm-1 . Both (dG-dC)n.(dG-dC)n and the 157 bp DNA show changes in the guanine vibration at 682 cm-1 and backbone band at 831-3 cm-1 preceding a highly cooperative change in the 1093 cm-1 PO2- vibration . This result indicates that there are at least two conformational steps in the B to Z conformational pathway . We review the effect of the (dC-dG) portion of the 157 bp DNA on the 95 bp segment . Comparison of Raman spectra of the 157 bp DNA, the 95 bp fragment and (dG-dC)n.(dG-dC)n indicate that in 4.5 M NaCl the (dC-dG) segments are in a Z-conformation . Base stacking in the 95 bp portion of the 157 bp DNA appears to maintain a B-type conformation . However, a substantial portion of this region no longer has a B-type backbone vibration.

J Hypertens Suppl, 1983 Oct, 1(1), 25 - 30
Regulation of angiotensin converting enzyme; Fyhrquist F et al.; Angiotensin converting enzyme (ACE;EC 3.4.15.1), or kininase II, was studied in serum, cultured endothelial cells from cord artery, in macrophages of humans, and in serum and purified plasma membranes of rats following treatment with inducers of ACE biosynthesis . ACE activity was measured in biological fluids with an enzyme kinetic method employing synthetic 1-hipp-1-his-l-leu tripeptide as a substrate, and with a new method using 125I-labelled specific inhibitor of ACE as a sensitive probe for ACE binding sites . The latter technique also proved suitable for the quantification of ACE in cells . Anti-human ACE antibody was employed for immunofluorescence studies in human cells . Dexamethasone treatment caused an increase in ACE in cultured human endothelial cells, macrophages and in rat pulmonary plasma membranes, but failed to increase serum ACE activity in rats . Captopril and enalapril treatment of hypertensive patients increased total serum ACE, the increase being evident after removal of the active drug from the serum by prolonged storage or chloramine T treatment (captopril) or by dialysis (enalapril) . Captopril increased the ACE content of endothelial cells and macrophages . Macrophages appeared sensitive to captopril induction of ACE biosynthesis after pre-stimulation with Escherichia coli lipopolysaccharide . Dexamethasone treatment potentiated the known induction of ACE in rat pulmonary tissue . Thus ACE biosynthesis may be enhanced by three categories of treatment: (1) glucocorticoid; (2) macrophage activation; (3) ACE inhibitors . The precise mechanism of ACE induction and its possible biological relevance await further clarification.

J Biomol Struct Dyn, 1983 Oct, 1(1), 225 - 9
Synthesis of two polypeptide subunits of an aminoacyl tRNA synthetase as a single polypeptide chain; Keng T et al.; E . coli aminoacyl tRNA synthetases are typically comprised of a single type of polypeptide chain . Glycine tRNA synthetase is an exception, and is comprised of two different subunits . Previous work showed that glyS encodes both subunits in a tandem arrangement of coding regions which are in the same reading frame . Nine nucleotides separate the TAA stop of the first coding segment (alpha-subunit) from the ATG start of the second one (beta-subunit) . A plasmid containing glyS was put into four different ochre suppressor strains . In three of them, significant quantities of an alpha-beta fusion protein were synthesized in maxicells, in genetic backgrounds which retained cellular proteases . This shows that the fusion protein is stable in vivo and suggests that Gly-tRNA synthetase is operationally a single polypeptide which is the ancestor of the two subunits.

Biochem Biophys Res Commun, 1983 Sep 30, 115(3), 888 - 93
Tetrahydro-sepiapterin is an intermediate in tetrahydrobiopterin biosynthesis; Milstien S et al.; 7,8-Dihydrobiopterin is not an intermediate in the de novo biosynthesis of tetrahydrobiopterin, the cofactor required for aromatic amino acid hydroxylations . However, N-acetyl-serotonin inhibition of sepiapterin reductase, an enzyme whose previously only known function was the reduction of sepiapterin to 7,8-dihydrobiopterin, completely inhibited biosynthesis of tetrahydrobiopterin by bovine adrenal medulla extracts . We have now shown that sepiapterin reductase catalyzes the reduction of tetrahydro-sepiapterin to tetrahydrobiopterin and that this reaction is N-acetyl-serotonin-sensitive . A new pathway for tetrahydrobiopterin biosynthesis is proposed which takes these observations into account and which involves tetrahydro intermediates.

Biochim Biophys Acta, 1983 Sep 28, 747(3), 200 - 8
Purification and properties of ribonuclease E, an RNA-processing enzyme from Escherichia coli; Roy MK et al.; The Escherichia coli RNA-processing enzyme RNAase E was purified through a number of steps including isoelectrofocusing . The final fraction contained mainly a single polypeptide of 66 kDa . However, while all the steps in the purification yielded the same qualitative activity, the specific activity of fractions was decreased in the last steps of the purification . By combining the most-purified enzyme with earlier fractions from the purification, we could show that the cells could contain a factor that enhances RNAase E activity . The purified enzyme showed the same characteristics with respect to temperature optimum, pH and ionic requirements as less-purified preparations . Testing specific inhibitors we concluded that the enzyme requires SH groups, free amino groups, and either of the amino acids tryptophan, tyrosine, histidine or methionine for its activity.

Biochim Biophys Acta, 1983 Sep 28, 747(3), 291 - 7
Determination of molecular weight of membrane proteins by the use of low-angle laser light scattering combined with high-performance gel chromatography in the presence of a non-ionic surfactant; Maezawa S et al.; An assessment study was carried out to evaluate the performance of the low-angle laser light scattering technique combined with high-performance gel chromatography in the presence of a nonionic surfactant, octaethyleneglycol n-dodecyl ether, precision differential refractometry and ultraviolet photometry . It was found that the combined technique is highly promising as a method for the determination of the molecular weight of a membrane protein solubilized by the surfactant . For trial, molecular weights of the following membrane proteins of Escherichia coli, both solubilized in oligomeric forms, were measured; porin that forms the transmembrane diffusion pore in the outer membrane, and lambda-receptor protein that facilitates the diffusion of maltose-maltodextrins across the outer membrane . The result obtained indicates that both porin and lambda-receptor protein exist as trimers in the surfactant solution.

Biochemistry, 1983 Sep 27, 22(20), 4831 - 42
Silver and mercury probing of deoxyribonucleic acid structures in the filamentous viruses fd, If1, IKe, Xf, Pf1, and Pf3; Casadevall A et al.; Ag+ binding and Hg2+ binding to both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) have been examined in some detail, and the results have been applied to study the structures of circular ssDNA in several filamentous viruses . It has been known for some time that Ag+ and Hg2+ bind to the bases of DNA producing characteristic large changes in absorbance and circular dichroism (CD) spectra, as well as changes in sedimentation rates . In the case of Ag+, it is known that there are three modes of binding to isolated dsDNA, referred to as types I, II, and III . Type III binding, by definition, occurs when Ag+ binds to Ag-dsDNA complexes having sites for binding types I and II extensively occupied, if not saturated . It produces CD spectra, assigned in this study, and absorbance spectra that are isosbestic with those of the Ag-dsDNA complexes present prior to its onset . In phosphate buffers binding is restricted to types I and II, whereas in borate buffers weaker type III binding can occur . Characteristics of types I, II, and III were observed for the DNAs in fd, If1, IKe, and Xf, but not for those in Pf1 and Pf3 . Similarly, many of the spectral changes seen when Hg2+ binds to isolated double-stranded DNA are mimicked by Hg2+ binding to the DNAs within fd, IKe, If1, and Xf, but not for those in Pf1 and Pf3 . The Ag+ and Hg2+ results indicate the presence of right-handed DNA helices in fd, If1, IKe, and Xf, with the two antiparallel strands of the covalently closed single-stranded DNAs having the bases directed toward the virion axes . For Pf1 and Pf3, Ag+ and Hg2+ binding cause large absorbance changes but only small CD changes . The very different results for Pf1 and Pf3 are consistent with the presence of inverted DNA structures (I-DNA) with the bases directed away from the structure axes, but the two structures differ from one another . Sedimentation velocity changes with Ag+ and Hg2+ binding strongly suggest structural linkages between the DNA and the surrounding protein sheath in each of the viruses.

Biochemistry, 1983 Sep 27, 22(20), 4737 - 45
Cooperative, excluded-site binding and its dynamics for the interaction of gene 5 protein with polynucleotides; Porschke D et al.; The binding of gene 5 protein to various single-stranded polynucleotides is investigated by fluorescence titrations and stopped-flow measurements . The association state of gene 5 protein itself is analyzed by equilibrium sedimentation: the monomer-dimer equilibrium found in the micromolar concentration range is described by a stability constant of 8 X 10(5) M-1 . The fluorescence quenching upon binding to polynucleotides, studied over a broad concentration range and analyzed in terms of a cooperative excluded-site binding model, provides binding constants for "isolated" and for "cooperative" sites . The cooperativity for various ribo- and deoxyribopolymers is between 400 and 800 and is virtually independent of the ionic strength . The binding to isolated sites is strongly dependent upon the ionic strength; analysis in terms of polyelectrolyte theory indicates the compensation of 4 +/- 0.5 charges upon complex formation . The number of nucleotide residues covered by one protein molecule is also found to be 4 +/- 0.5 units . The affinity of gene 5 protein for polynucleotides increases in the series poly(C) less than poly(dA) less than poly(A) less than poly(U) much less than poly(dT); the binding constant for poly(dT) is roughly a factor of 1000 higher than that for the other polymers . Model studies with Lys-Tyr-Lys and Lys-Trp-Lys suggest that the preferential interaction with poly(dT) is not simply due to enhanced stacking interactions between the aromatic amino acids and the thymine residues . Stopped-flow reaction curves obtained by mixing of gene 5 protein with poly(dT) in the micromolar concentration range show three relaxation processes with time constants between 1 ms and 1 s.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1983 Sep 27, 22(20), 4657 - 63
Purification and properties of citrate lyase from Escherichia coli; Nilekani S et al.; Citrate lyase (EC 4.1.3.6) has been purified from Escherichia coli and the homogeneity of the preparation established from the three-component subunits obtained on sodium dodecyl sulfate/polyacrylamide gel electrophoresis . The purified enzyme has a specific activity of 120 mumol min-1 mg-1 and requires optimally 10 mM Mg2+ and a pH of 8.0 for the cleavage reaction . The native enzyme is polydispersed in the ultracentrifuge and in polyacrylamide gel electrophoresis . The enzyme complex is composed of three different polypeptide chains of 85 000, 54 000, 32 000 daltons . An estimate of subunit stoichiometry indicates that 1 mol of the largest polypeptide chain is associated with 6 mol each of the smaller ones . The polypeptide subunits have been isolated in pure state and their biological functions characterize . The 54 000-dalton subunit functions as the acyltransferase alpha subunit catalyzing the formation of citryl coenzyme A from citrate in the presence of acetyl coenzyme A and ethylenediaminetetraacetic acid . The 32 000-dalton subunit functions as the acyllyase beta subunit catalyzing the cleavage of (3S)-citryl coenzyme A to oxal-acetate and acetyl coenzyme A . The 85 000-dalton subunit, which carries exclusively the prosthetic group components, functions as the acyl-carrier protein gamma subunit in the cleavage of citrate in the presence of mg2+ and the alpha and beta subunits . The presence of a large ACP subunit and the unusual stoichiometry of the different subunits distinguish the complex from other citrate lyases . A ligase which acetylates the deacetyl{citrate lyase} in the presence of acetate and ATP has ben shown to be present in the organism.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1983 Sep 25, 258(18), 11344 - 9
Excess beta subunit can bypass the ATP requirement for highly processive synthesis by the Escherichia coli DNA polymerase III holoenzyme; Crute JJ et al.; We have previously shown that the processive synthesis of long DNA products on a poly(dA) X oligo(dT)10 primer-template is facilitated by formation of an isolable initiation complex between the Escherichia coli DNA polymerase III holoenzyme and DNA in the presence of ATP (Fay, P . J., Johanson, K . O., McHenry, C . S., and Bambara, R . A . (1982) J . Biol . Chem . 257, 5692-5699) . Here we have demonstrated that the ATP requirement for processive synthesis can be obviated by a large excess of the beta subunit of the DNA polymerase III holoenzyme . The reaction which occurs in the presence of excess beta can be distinguished from the ATP-mediated reaction by its salt sensitivity and the lack of stabile initiation complex formation between polymerase and primed DNA . A model is presented which suggest that one of the functions of ATP in the DNA polymerase III holoenzyme reaction is to lock beta into the replicative complex such that it does not readily equilibrate with solution.

J Biol Chem, 1983 Sep 25, 258(18), 11057 - 62
Leucine, isoleucine, valine-binding protein from Escherichia coli . Structure at 3.0-A resolution and location of the binding site; Saper MA et al.; The structure of the leucine, isoleucine, valine-binding protein, an integral part of the high-affinity, branched-chain aliphatic amino acid transport system in Escherichia coli, has been solved at 3.0-A resolution by x-ray crystallography . Five isomorphous heavy atom derivatives, including anomalous differences from a samarium derivative, were used . A model of the polypeptide chain backbone reveals two distinct, globular domains connected by three strands . Each domain consists of a beta-sheet core flanked by at least two helices on either side . Difference Fourier analyses of crystals soaked in L-leucine, L-isoleucine, or L-valine have located a single amino acid-binding site in the cleft formed by the two domains . Despite the lack of significant sequence homology, the bilobate and secondary structure observed were similar to that found in the structures of L-arabinose- and D-galactose-binding proteins previously determined in our laboratory.

J Biol Chem, 1983 Sep 25, 258(18), 10967 - 72
Dimerization by colicin E3 in the absence of immunity protein; Levinson BL et al.; Using small angle x-ray scattering from solutions of colicin E3-immunity protein complex and the separated proteins, we have measured the radii of gyration of each species . We find that the complex is an elongated molecule with a radius of gyration of 35.5 +/- 0.7 A . Immunity protein is more compact with a radius of gyration of 13.4 +/- 0.1 A . Upon removal of immunity protein from the complex, colicin E3 (form minus immunity protein) undergoes further elongation and dimerizes, such that its radius of gyration increases to 75.6 +/- 3.1 A . Dimerization is not reversed by simple addition of a stoichiometric amount of immunity protein to the E3 solution, even though this is sufficient to block in vitro activity . We suggest that the elongation, and perhaps dimer formation, occurs in vivo, concomitant with membrane translocation.

J Biol Chem, 1983 Sep 25, 258(18), 10956 - 9
R plasmid dihydrofolate reductase with a dimeric subunit structure; Novak P et al.; Dihydrofolate reductase specified by plasmid R483 from a trimethoprim-resistant strain of Escherichia coli has been purified 2,000-fold to homogeneity using dye-ligand chromatography, gel filtration, and polyacrylamide gel electrophoresis . The protein migrated as a single band on nondenaturing polyacrylamide gel electrophoresis and had a specific activity of 250 mumol/mg min(-1) . The molecular weight was estimated to be 32,000 by gel filtration and 39,000 by Ferguson analysis of polyacrylamide gel electrophoresis . When subjected to electrophoresis in the presence of sodium dodecyl sulfate, the protein migrated as a single 19,000-molecular weight species, a fact that suggests that the native enzyme is a dimer of similar or identical subunits . Antibody specific for R483-encoded dihydrofolate reductase did not cross-react with dihydrofolate reductase encoded by plasmid R67, T4 phage, E . coli RT500, or mouse L1210 leukemia cells . The amino acid sequence of the first 34 NH2-terminal residues suggests that the R483 plasmid dihydrofolate reductase is more closely related to the chromosomal dihydrofolate reductase than is the enzyme coded by plasmid R67.

J Biol Chem, 1983 Sep 25, 258(18), 10862 - 6
Partial NH2- and COOH-terminal sequence and cyanogen bromide peptide analysis of Escherichia coli sn-glycerol-3-phosphate acyltransferase; Green PR et al.; The sn-glycerol-3-phosphate acyltransferase from Escherichia coli, an integral membrane protein whose activity is dependent on phospholipids, was purified to near homogeneity (Green, P . R., Merrill, A . H., Jr., and Bell, R . M., (1981) J . Biol . Chem . 256, 11151-11159) . Determination of a partial NH2-terminal sequence and the COOH terminus permitted alignment of the polypeptide on the sequenced sn-glycerol-3-phosphate acyltransferase structural gene (Lightner, V . A., Bell, R . M., and Modrich, P . (1983) J . Biol . Chem . 258, 10856-10861) . Processing of the sn-glycerol-3-phosphate acyltransferase is apparently limited to the removal of the NH2-terminal formylmethionine . Thirteen of 27 possible cyanogen bromide peptides predicted from the DNA sequence were purified, characterized, and assigned to their location in the primary structure . Three peptides located at positions throughout the sequence were partially sequenced by automated Edman degradation . The partial sequence analysis of the homogeneous sn-glycerol-3-phosphate acyltransferase is fully in accord with the primary structure inferred from the DNA sequence.

J Biol Chem, 1983 Sep 25, 258(18), 10817 - 20
Peptide-specific antibody locates the COOH terminus of the lactose carrier of Escherichia coli on the cytoplasmic side of the plasma membrane; Seckler R et al.; The carboxyl-terminal decapeptide NH2-Leu-Leu-Arg-Arg-Gln-Val-Asn-Glu-Val-Ala-OH of the lactose carrier protein, the product of the lac Y gene of Escherichia coli, was synthesized, and specific anti-peptide antibodies were raised in rabbits . These antibodies bind to membrane-bound lactose carrier showing that the carboxyl terminus is accessible from the aqueous phase . The antibodies bind only to the surface of inverted cytoplasmic membrane vesicles (but not to closed, right-side-out membrane vesicles), demonstrating that the carboxyl terminus of the carrier protein is directed towards the cytoplasmic side of the plasma membrane in cells . The carboxyl terminus is a potent immunogenic epitope on the purified, detergent-solubilized carrier . Binding of peptide-specific antibodies to the carrier protein inhibits neither substrate binding nor translocation.

J Biol Chem, 1983 Sep 25, 258(18), 11296 - 304
Lac repressor mRNA transcription terminates in vivo in the lac control region; Cone KC et al.; We have isolated the in vivo messenger RNA encoding the lactose repressor protein of Escherichia coli by hybridization to lacI gene DNA, and have verified its identity by characterizing specific fragments derived from its 5' and 3' ends . The 3' end points of the RNA, as shown by oligonucleotide analysis and S1 nuclease-mapping data, are clustered in the lac control region, within 70 nucleotides beyond the end of the repressor protein coding sequence . DNA fragments from this region, when inserted between the E . coli gal operon promoter and galactokinase (galK) gene, effect an 80% reduction in downstream galK expression . These results are indicative that sequences at the gene end function in vivo to terminate lacI gene transcription.

J Biol Chem, 1983 Sep 25, 258(18), 11157 - 64
Mössbauer studies of Escherichia coli sulfite reductase complexes with carbon monoxide and cyanide . Exchange coupling and intrinsic properties of the {4Fe-4S} cluster; Christner JA et al.; Mossbauer studies of the hemoprotein subunit (SiR) of E . coli sulfite reductase have shown that the siroheme and the {4Fe-4S} cluster are exchange-coupled . Here we report Mossbauer studies of SiR complexed with either CO or CN- and of SiR in the presence of the chaotropic agent dimethyl sulfoxide (Me2SO) . The spectra of one-electron-reduced SiR X CN show that all five iron atoms reside in a diamagnetic environment; the ferroheme X CN complex is low spin and the {4Fe-4S} cluster is in the 2+ oxidation state . Titration with ferricyanide affords a CN- complex of oxidized SiR in which the siroheme iron is low spin ferric, with the cluster remaining in the 2+ state . At low temperatures, paramagnetic hyperfine interactions are observed for the iron sites of the cluster, suggesting that it is exchange-coupled to the heme iron . Reduction of one-electron-reduced SiR X CN and SiR X CO yields complexes with "g = 1.94"-type EPR signals showing that the second electron is accommodated by the iron-sulfur cluster . The fully reduced complexes yield well resolved Mossbauer spectra which were analyzed in the spin Hamiltonian formalism . The analysis shows that the cluster subsites are equivalent in pairs, one pair having properties reminiscent of ferric sites whereas the other pair has features more typical of ferrous sites . The Mossbauer spectra of oxidized SiR kept in 60% (v/v) Me2SO are virtually identical with those observed for SiR in standard buffer, implying that the coupling is maintained in the presence of the chaotrope . Fully reduced SiR displays an EPR signal with g values of g = 2.53, 2.29, and 2.07 . In 60% Me2SO, this signal vanishes and a g = 1.94 signal develops; this transition is accompanied by a change in the spin state of the heme iron from S = 1 (or 2) to S = O.

J Biol Chem, 1983 Sep 25, 258(18), 11147 - 56
Mössbauer evidence for exchange-coupled siroheme and {4Fe-4S} prosthetic groups in Escherichia coli sulfite reductase . Studies of the reduced states and of a nitrite turnover complex; Christner JA et al.; We have studied the hemoprotein subunit (SiR) of Escherichia coli NADPH-sulfite reductase with Mossbauer spectroscopy in a variety of complexes and oxidation states . We demonstrated earlier for oxidized SiR (Christner, J . A., Munck, E., Janick, P . A., and Siegel, L . M . (1981) J . Biol . Chem . 256, 2098-2101) that the two metal centers, a siroheme and a {4Fe-4S} cluster, are covalently linked as witnessed by the observation of strong exchange-coupling . The studies reported here show that the centers remain coupled in the one- and two-electron-reduced states of SiR . Exchange-coupling is also observed for the turnover complex, SiR X NO; this species can be prepared by reacting fully reduced SiR, which can function as a nitrite reductase, with nitrite . The coupling is also maintained in the presence of certain chaotropic agents . In SiR X NO, the exchange interactions between the S = 1/2 Fe(II) X siroheme X NO and the iron-sulfur cluster induce paramagnetism at the iron sites of the {4Fe-4S} cluster which is formally in the (diamagnetic) 2+ oxidation state . Analysis of the Mossbauer spectra shows that the iron sites of the cluster are equivalent in pairs, which are distinguishable by positive and negative magnetic hyperfine coupling constants . This study has revealed that the Mossbauer parameters of the siroheme, an Fe X isobacteriochlorin, are very similar to those observed for other hemes . The data obtained for the reduced forms of uncomplexed enzyme show that the siroheme iron is Fe(II), that it is paramagnetic, and possibly in the S = 1 intermediate spin state.

J Biol Chem, 1983 Sep 25, 258(18), 10856 - 61
The DNA sequences encoding plsB and dgk loci of Escherichia coli; Lightner VA et al.; We have determined the sequence of a 3865-base pair DNA segment from Escherichia coli containing plsB, the structural gene for the sn-glycerol-3-phosphate acyltransferase, and the dgk locus, believed to encode diglyceride kinase . The 806-amino acid sequence encoded within the longest open reading frame is in agreement with NH2-terminal sequences of the sn-glycerol-3-phosphate acyltransferase (Green, P., Vanaman, T . C., Modrich, P., and Bell, R . M . (1983) J . Biol . Chem . 258, 10862-10866), indicating that this is the structural gene for this protein . Furthermore, an open reading frame encoding a 122-residue polypeptide consistent with the size of diglyceride kinase has been identified and coincides with the position of dgk determined by deletion analysis.

J Biol Chem, 1983 Sep 25, 258(18), 10813 - 6
A characterization of the low temperature structural transition of Escherichia coli 5 S RNA by partial enzymatic digestion; Rabin D et al.; The low temperature structural transition (low leads to high) of 5 S RNA from Escherichia coli is investigated by partial digestion with ribonuclease T1 . In addition to a general masking of guanines from the nuclease, differential changes of accessibility are observed when Mg2+ and salt concentrations are increased to bring about the low leads to high transition . Residue G13 becomes more exposed in the high form while residues G54, G56, G61, G72, and G83-86 become less exposed . The observed cutting rate at other sites is unchanged . A possible conformational change is discussed which could explain the observed changes in RNase T1 digestion patterns as well as the physical chemical observations.

Nucleic Acids Res, 1983 Sep 24, 11(18), 6487 - 95
Primary structure of the tolC gene that codes for an outer membrane protein of Escherichia coli K12; Hackett J et al.; We present the nucleotide sequence of the tolC gene of Escherichia coli K12, and the amino acid sequence of the TolC protein (an outer membrane protein) as deduced from it . The mature TolC protein comprises 467 amino acid residues, and, as previously reported (1), a signal sequence of 22 amino acid residues is attached to the N-terminus . The C-terminus of the gene is followed by a stem-loop structure (8 base pair stem, 4 base loop) which may be a rho-independent termination signal . The codon usage of the gene is nonrandom; the major isoaccepting species of tRNA are preferentially utilised, or, among synonomous codons recognized by the same tRNA, those codons are used which can interact better with the anticodon (2,3) . In contrast to the codon usage for other outer membrane proteins of E . coli (4) the rare arginine codons AGA and AGG are used once and twice respectively.

Nucleic Acids Res, 1983 Sep 24, 11(18), 6419 - 35
Chemical synthesis of a human interferon-alpha 2 gene and its expression in Escherichia coli; Edge MD et al.; A 511-base pair DNA fragment encoding human interferon-alpha 2 has been chemically synthesised and expressed from a lac UV5 and a synthetic trp promoter in Escherichia coli . The synthesis involved preparation of 68 oligodeoxyribonucleotides and their enzymic ligation . The product expressed from the trp promoter system had high antiviral activity and displayed biological effects similar to those of Namalwa interferon on natural killer cell activity and in a Daudi cell growth inhibition assay . E.coli minicells containing plasmid DNA with the synthetic IFN-alpha 2 gene under trp promoter control produce a protein with the same electrophoretic mobility as a sample of authentic IFN-alpha 2 . The protein from E.coli cross-reacts with the monoclonal antibody NK-2 and was readily purified, close to homogeneity, by immunoadsorption chromatography on NK-2 sepharose.

Nucleic Acids Res, 1983 Sep 24, 11(18), 6289 - 300
DNA sequence of the tandem ribosomal RNA promoter for B . subtilis operon rrnB; Stewart GC et al.; A new ribosomal RNA operon designated rrnB has been identified by screening a Charon 4a library of cloned B . subtilis sequences . Clones containing the promoter region of this operon are unstable in E . coli unless a special vector possessing a transcriptional terminator is used . DNA sequence data suggests that this operon contains two tandem putative promotor regions not unlike those found in E . coli . There are 92 base pairs separating the two "-10 regions" of the promotors . The second is 180 bp upstream from the start site for mature 16S RNA . A potential 29 base pair stem structure necessary for processing of the mature 16S RNA sequence can also be predicted from this analysis.

Nucleic Acids Res, 1983 Sep 24, 11(18), 6167 - 84
Biotin and fluorescent labeling of RNA using T4 RNA ligase; Richardson RW et al.; Biotin, fluorescein, and tetramethylrhodamine derivatives of P1-(6-aminohex-1-yl)-P2-(5'-adenosine) pyrophosphate were synthesized and used as substrates with T4 RNA ligase . In the absence of ATP, the non-adenylyl portion of these substrates is transferred to the 3'-hydroxyl of an RNA acceptor to form a phosphodiester bond and the AMP portion is released . E . coli and D . melanogaster 5S RNA, yeast tRNAPhe, (Ap)3C, and (Ap)3A serve as acceptors with yields of products varying from 50 to 100% . Biotin-labeled oligonucleotides are bound selectively and quantitatively to avidin-agarose and may be eluted with 6 M guanidine hydrochloride, pH 2.5 . Fluorescein and tetramethylrhodamine-labeled oligonucleotides are highly fluorescent and show no quenching due to attachment to the acceptor . The diverse structures of the appended groups and of the chain lengths and compositions of the acceptor RNAs show that T4 RNA ligase will be a useful modification reagent for the addition of various functional groups to the 3'-terminus of RNA molecules.

Biochim Biophys Acta, 1983 Sep 20, 753(2), 227 - 35
Facilitated utilization of endogenously synthesized lysophosphatidic acid by 1-acylglycerophosphate acyltransferase from Escherichia coli; Kessels JM et al.; Complete separation of glycerophosphate acyltransferase and 1-acylglycerophosphate acyltransferase from Escherichia coli was obtained by sequential extraction with Triton X-100 . Solubilized glycerophosphate acyltransferase was reconstituted by the cholate dispersion and gel filtration method in small unilamellar vesicles . 1-Acylglycerophosphate acyltransferase could not be solubilized from the membranes and was used in endogenous membrane fragments after detergent removal . Mixing of the two preparations and subsequent incubation in the presence of glycerol 3-phosphate, palmitoyl-CoA and oleoyl-CoA resulted in the efficient synthesis of phosphatidic acid . Inclusion of exogenous lysophosphatitic acid in the assay medium resulted in a dilution of the newly synthesized lysophosphatidate . By contrast, the synthesis of phosphatidic acid from glycerol 3-phosphate by the acyltransferases present in native membrane vesicles was barely influenced by the presence of exogenous lysophosphatidic acid . When comparing the utilization of membrane-associated 14C-labeled and newly generated 3H-labeled lysophosphatidic acid, the latter appeared to be the preferred substrate . These results indicate that lysophosphatidic acid, synthesized by glycerophosphate acyltransferase, is utilized by 1-acylglycerophosphate acyltransferase without prior mixing with the total membrane-associated pool of lysophosphatidic acid, and suggest a close proximity of the two enzymes in native E . coli membranes . This property of the acyltransferases is lost upon separation and reconstitution of enzyme activities.

FEBS Lett, 1983 Sep 19, 161(2), 213 - 6
Three sequence-specific endonucleases from Escherichia coli RFL47; Janulaitis A et al.; The characterization of the new restriction enzyme Eco47III recognizing a hexanucleotide palindromic sequence 5'AGC decreases GCT and cleaving, as indicated by the arrow, is reported . It was isolated from Escherichia coli strain RFL47 . Another two specific endonuclease Eco47I (isoschizomer of AvaII) and Eco47II (isoschizomer of AsuI) were also found in this strain . There are two Eco47III recognition sites on lambda DNA at 20997 and 37060 basepairs . The central Eco47III fragment can be replaced by a cloned fragment in lambda vector mutant in tR2 gene; i.e., lambda gt.

Experientia, 1983 Sep 15, 39(9), 988 - 91
Potential toxins of acute liver failure and their effects on blood-brain barrier permeability; Zaki AE et al.; The effects of potentials toxins of hepatic coma on the blood-brain barrier (BBB) permeability of the rat have been examined using the Oldendorf technique . Classical toxins of hepatic failure such as ammonia, methyl octanoate, mercaptans, and phenol caused significant increases in BBB permeability . A slight increase in permeability occurred following infusion of peroxidized linoleic acid and unconjugated bilirubin but no increase after infusion of bile acids . E . coli endotoxin infused into rats following partial hepatectomy also increased the BBB permeability.

Biochem Biophys Res Commun, 1983 Sep 15, 115(2), 577 - 82
Biochemical studies on the potentiation of antitumor activity of cisplatin by adriamycin; Chuang RY et al.; The RNA synthesis in vitro by E . coli RNA polymerase was found to be highly sensitive to cis-diamminedichloroplatinum (cisplatin) inhibition . The degree of inhibition was in proportion to the length of time of template preincubation with cisplatin, suggesting that cisplatin-template binding was involved in the inhibition of RNA polymerase . The effect of adriamycin on this inhibition was studied and it was found that adriamycin significantly enhanced the inhibitory effect of cisplatin and that the total effect was greater than the sum of the effects of each drug used individually . This synergistic effect was not observed when the effect of the combination of adriamycin and cisplatin on in vitro DNA synthesis was studied.

J Mol Biol, 1983 Sep 15, 169(2), 507 - 19
A genetic approach to defining the sites of interaction of a membrane protein with different external agents; Manoil C; In addition to a role in maintaining outer membrane structural integrity, the OmpA protein of Escherichia coli serves as a receptor for different phages, is required in the recipient cell for efficient conjugation, and functions in the internalization of receptor-bound colicins . To determine whether the same or different sites of OmpA protein are needed for these different functions, this paper presents the properties of a collection of ompA mutants selected as being defective in phage receptor function, but containing normal amounts of OmpA protein . Of 44 mutants examined, most were affected in more than one OmpA protein function, showing that there is considerable overlap in sites needed for different functions . The pattern of this overlap can be represented in a phenotypic map of OmpA protein functions.

Eur J Biochem, 1983 Sep 15, 135(2), 351 - 7
Effect of reduced membrane lipid fluidity on the biosynthesis of lipopolysaccharide of Escherichia coli; Dirienzo JM et al.; A low molecular weight precursor of lipopolysaccharide was accumulated under conditions in which the membrane lipids of a fatty acid auxotroph of Escherichia coli were reduced to a non-fluid state . The lipopolysaccharide precursor was detected, by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiography, in membranes isolated from cells which were pulse-labeled with N-acetyl-{1-14C}glucosamine . The precursor could be chased into mature lipopolysaccharide by returning the membrane lipids to a normal fluid state . Conversion of the precursor to lipopolysaccharide was inhibited by the presence of potassium cyanide or sodium arsenate . The processing of several outer membrane protein precursors, including the promatrix proteins, was also inhibited under these conditions . Preliminary characterization of the lipopolysaccharide precursor was undertaken.

Eur J Biochem, 1983 Sep 15, 135(2), 263 - 9
The quaternary structure of Escherichia coli RNA polymerase studied with (scanning) transmission (immuno)electron microscopy; Tichelaar W et al.; A model for the quaternary structure of Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) is presented . It is based on results from classification of profiles of enzyme molecules, and from application of immuno electron microscopy . Classification of molecules, prepared with the single carbon layer technique, was first achieved for images recorded in dark field with the scanning transmission electron microscope and later on for images recorded in bright-field transmission electron microscopy . It results in five approximately equally sized groups, containing about 80% of the core enzyme profiles . Holoenzyme profiles can be grouped into the same classes, and have approximately the same dimensions (9 nm X 16 nm) . Based on the shapes and sizes of the classified profiles, a tentative model for core enzyme has been constructed . Correlation of shadow projections of this model, with the distributions of attachment sites of antibodies against alpha, beta, beta' and sigma over the profiles, has led to models for core and holoenzyme in which the subunits are localized . The model is compared with literature data on the quaternary structure of RNA polymerase.

Eur J Biochem, 1983 Sep 15, 135(2), 223 - 9
Nucleotide sequence of the Escherichia coli pyrE gene and of the DNA in front of the protein-coding region; Poulsen P et al.; Orotate phosphoribosyltransferase (EC 2.4.2.10) was purified to electrophoretic homogeneity from a strain of Escherichia coli containing the pyrE gene cloned on a multicopy plasmid . The relative molecular masses (Mr) of the native enzyme and its subunit were estimated by means of gel filtration and electrophoresis in the presence of dodecyl sulfate . The amino acid sequences at the N and C termini, as well as the amino acid composition, were determined . The nucleotide sequence of the structural pyrE gene, including 394 nucleotide residues preceding the beginning of the coding frame, was also established . From the results the following conclusions may be drawn . Orotate phosphoribosyltransferase is a dimeric protein with subunits of Mr 23 326 consisting of 211 amino acid residues . The pyrE gene is transcribed in a counter-clock wise direction from the E . coli chromosome as an mRNA with a considerable leader segment in front of the protein-coding region . This leader contains a structure with features characteristic for a (translated?) rho-independent transcriptional terminator, which is preceded by a cluster of uridylate residues . This indicates that the frequency of pyrE transcription is regulated by an RNA polymerase (UTP) modulated attenuation.

J Mol Biol, 1983 Sep 15, 169(2), 353 - 72
Partition of unit-copy miniplasmids to daughter cells . I . P1 and F miniplasmids contain discrete, interchangeable sequences sufficient to promote equipartition; Austin S et al.; Hybrids formed by insertion of the plasmid maintenance regions of P1 or F into a lambda delta att vector form stable unit-copy plasmids in their Escherichia coli host . They must therefore both be substrates for an accurate cellular partition apparatus that ensures that all daughter cells inherit a plasmid copy . Analysis of deletion mutants of both types of hybrid showed that, although the P1 and F plasmid maintenance regions differ in sequence and specificity, they are similar in general organization . Each contains an approximately 3 X 10(3) base-pair region that is essential for replication (rep) and an adjacent but separable 3 X 10(3) base-pair region that is essential for the stability of plasmid maintenance (par) . Each par region is thought to specify the recognition of the plasmid as a substrate for equipartition . The deletion mutants provide sources of isolated rep and par sequences from both P1 and F DNA . These elements were then used to construct composite plasmids with novel combinations and arrangements of rep and par sequences . Heterologous constructions containing P1 rep and F par or F rep and P1 par sequences were maintained faithfully . We conclude that par regions are both necessary and sufficient to promote equipartition of replicating plasmid DNA . This activity is exerted only in cis but otherwise seems to be independent of the position or orientation of the par sequences within the DNA . Both P1 and F par regions include DNA sequences (incB of P1, incD of F) that we propose are analogues of the centromeres of eukaryotic chromosomes . The remaining portions of the par regions are known to encode protein products that, we believe, act at the inc sites . Extra copies of these inc sites appear to exert incompatibility by competition for the cellular partition apparatus.

Nature, 1983 Sep 15-21, 305(5931), 243 - 5
A common regulatory region shared by divergently transcribed genes of the Escherichia coli SOS system; Brandsma JA et al.; The Escherichia coli single-stranded DNA binding protein (SSB) is implicated in DNA replication, recombination and repair . On the chromosome, the ssb gene is located adjacent to the excision repair gene uvrA, but the two genes are transcribed in opposite directions . uvrA has been shown to be part of the E . coli SOS system by introducing Mud(Ap, lac) insertions distal to the regulatory region of the gene in the chromosome . Recent investigations suggest that SSB is also involved in the SOS response . However, because the SSB protein is essential to the cell, the inducibility of the ssb gene cannot be investigated by the insertion method . Therefore, we used plasmids harbouring the regulatory region of ssb fused to the galK structural gene, while leaving an intact ssb gene in the chromosome . We show here that expression of the ssb gene is dependent on two promoters of which one is damage inducible . Evidence is presented that the divergently transcribed ssb and uvrA genes are controlled by a common LexA binding site.

Biochemistry, 1983 Sep 13, 22(19), 4507 - 12
Properties of a human lymphoblast AP-endonuclease associated with activity for DNA damaged by ultraviolet light, gamma-rays, or osmium tetroxide; Brent TP; An endonuclease activity for UV-irradiated DNA, gamma-irradiated DNA, and OsO4-treated DNA that was partially purified from human lymphoblasts was found to have associated with it an endonuclease activity for partially depurinated DNA . When this apurinic endonuclease (Endo A) was characterized and compared with the cells' major apurinic endonuclease (Endo B), several notable differences were observed . (1) Endo A bound to oxidized DNA-Sepharose under conditions where Endo B did not . (2) Endo A did not require Mg2+, retaining full activity in 10 mM ethylenediamine-tetraacetic acid, while Endo B showed an absolute requirement for Mg2+ . (3) Whereas the nicks made in depurinated DNA by Endo B were efficient priming sites for Escherichia coli polymerase I, those made by Endo A were not . Further characterization of the nicks indicated that Endo A incises depurinated DNA 3' to apurinic sites, leaving 3'-terminal deoxyribose, a poor priming site for DNA synthesis . Endo A action on UV-irradiated DNA produced nicks that resembled those it made in depurinated DNA, suggesting that the UV endonuclease activity acts through an apurinic/apyrimidinic site intermediate.

Biochemistry, 1983 Sep 13, 22(19), 4546 - 50
Assignment of resonances in the phosphorus-31 nuclear magnetic resonance spectrum of poly{d(A-T)} from phosphorothioate substitution; Eckstein F et al.; Two phosphorothioate analogues of poly{d(A$-T)} have been synthesized enzymatically . In one, poly{d(A$-T)}, dTMP is replaced by thymidine 5'-O-phosphorothioate; in the other, poly{d(T$-A)}, dAMP is replaced by 2'-deoxyadenosine 5'-O-phosphorothioate . The 31P NMR spectrum of poly{d-(A-T)} in solutions at low salt concentration shows two resonances at 51.80 and -4.25 ppm relative to trimethyl phosphate . The corresponding values for poly{d(T$-A)} are 51.51 and -4.43 ppm . These data allow the assignment of the downfield resonance at -4.23 ppm in poly{d(A-T)} to the phosphate group of d(TpA) and the resonance at -4.41 ppm to that of d(ApT) . Thus, strong evidence is provided for a repeating dinucleotide structure . A comparison of the 31P NMR spectra of the various polymers in solutions of 2 M CsF reveals that both resonances are shifted upfield by approximately 0.9 ppm in the case of the phosphorothioates and by 0.2 or 0.4 ppm in the case of the phosphates . An upfield shift of about 0.18 ppm can also be observed for the two corresponding dinucleoside monophosphates . Thus, the upfield shift induced by high concentrations of CsF is not specific for the polymer backbone.

Biochemistry, 1983 Sep 13, 22(19), 4518 - 26
Insertion of nucleotides opposite apurinic/apyrimidinic sites in deoxyribonucleic acid during in vitro synthesis: uniqueness of adenine nucleotides; Sagher D et al.; M13 DNA containing 20-30 apurinic/apyrimidinic (AP) sites per intact circular molecule was prepared by growing phage on an ung- dut- Escherichia coli mutant and treating the DNA with uracil N-glycosylase . AP sites obstruct in vitro DNA synthesis catalyzed by E . coli pol I . The position at which termination of synthesis occurs was determined for four enzymes . T4 DNA polymerase terminates one nucleotide before putative AP sites . DNA pol I, AMV reverse transcriptase, and DNA polymerase alpha terminate synthesis either before or at the site of an AP lesion depending on the particular sequence . We determined the identity of the nucleotide inserted opposite an AP site by synthesizing up to the lesion in a first-stage reaction using T4 DNA polymerase and then determining elongation in a second stage . Purines are inserted opposite AP sites more readily than pyrimidines, and dATP is more efficient than dGTP in promoting such elongation . The DNA-dependent conversion of dNTP to dNMP was determined in mixtures of all four dNTP's by using AP DNA . The production of dAMP from dATP occurs most readily . We conclude that there is an inherent specificity for the incorporation of adenine nucleotides opposite AP sites in this in vitro system . Insofar as the model system reflects in vivo mutational events, our data suggest that depurination should produce transversions and depyrimidination should produce transitions.

Biochemistry, 1983 Sep 13, 22(19), 4494 - 501
Secondary tritium isotope effects as probes of the enzymic and nonenzymic conversion of chorismate to prephenate; Addadi L et al.; To obtain information about the degree of concert of both the nonenzymic and the enzyme-catalyzed rearrangement of chorismate to prephenate, we have determined the secondary tritium isotope effects at the bond-making position (C-9) and the bond-breaking position (C-5) of chorismate . The isotope effects were determined by the competitive method, using either {5-3H,7-14C )chorismate or {9-3H,7-14C}chorismate as the substrate . In the nonenzymic reaction (pH 7.5, 60 degrees C), KH/kT is 1.149 +/- 0.012 for bond breaking (C-9) and 0.992 +/- 0.012 for bond making (C-5) . This indicates an asymmetric transition state in which the new bond is hardly, if at all, formed, while the bond between C-5 and oxygen is substantially broken . In the enzymic reaction (pH 7.5, 30 degrees C), the values of kH/kT in both positions are unity within experimental error . It is most likely that the isotope effects are suppressed in the enzymic process and that the rate-limiting transition state occurs before the rearrangement itself . The kinetically significant transition state presumably involves either the binding step of the small equilibrium proportion of the axial conformer of the substrate or an isomerization of enzyme-bound chorismate from the more stable conformer in which the carboxyvinyloxy group is equatorial to that in which this group is axial . Rearrangement would then proceed relatively rapidly from the higher energy axial conformer.

Biochemistry, 1983 Sep 13, 22(19), 4485 - 93
Acyl carrier protein from Escherichia coli I . Aspects of the solution structure as evidenced by proton nuclear Overhauser experiments at 500 MHz; Mayo KH et al.; The downfield aromatic (6-8 ppm) and upfield ring current shifted methyl regions (1-0 ppm) in the proton nuclear magnetic resonance spectrum of acyl carrier protein (ACP) from Escherichia coli have been examined at 500 MHz by using nuclear Overhauser methods . The data are analyzed in terms of the secondary structural model of Rock & Cronan (1979) {Rock, C . O., & Cronan, J . E., Jr . (1979) J . Biol . Chem . 254, 9778-9785}, which suggests the existence of four alpha-helical segments joined by three beta-turns, and a short coil at the C terminus of the protein . Nuclear Overhauser effects among Tyr-71, Ile-69, Ile-72, and His-75 allow refinement of the secondary structure of the C terminus . Nuclear Overhauser effects among Tyr-71, Phe-28, and three Ile's also place stringent limitations on the folding of the four alpha-helices . These data allow the proposal of a tertiary structural model for ACP.

Biochemistry, 1983 Sep 13, 22(19), 4501 - 7
Enzymic detection of uracil in a cloned and sequenced deoxyribonucleic acid segment; Weiss RB et al.; Uracil can occur in DNA either as a result of utilization of dUTP by DNA polymerases or from in situ deamination of cytosine . The latter results in transition mutations following the next round of replication . We describe a technique for the detection of uracil in DNA by a modification of the Maxam-Gilbert sequencing procedure . Reaction of end-labeled DNA with uracil-DNA glycosylase followed by 1 M piperidine results in scission products corresponding to locations of uracils . These are detected by autoradiography following electrophoresis through a sequencing gel . Comparison of these scission products with the DNA sequences elucidates the mechanism of origin of the DNA uracils . The technique was tested with a cloned human DNA sequence grown in a dut-,ung-strain of Escherichia coli, which incorporates uracil in place of thymine in its DNA, and by chemical deamination of cytosines in that sequence . The technique was expanded by use of alkaline and enzymic probes to investigate possible accumulation of uracil, base losses, and other modifications in human liver and brain DNA . No damaged DNA moieties were detected . This method is applicable to the study of any recoverable reiterated sequence by any enzyme preparation that can recognize modifications in DNA.

Biochemistry, 1983 Sep 13, 22(19), 4362 - 5
Imino proton assignments in the proton nuclear magnetic resonance spectrum of the lambda phage OR3 deoxyribonucleic acid fragment; Ulrich EL et al.; The 17 base pair duplex d(TATCACCGCAAGGGATAp) . d(TATCCCTTGCGGTGATAp) corresponding to the OR3 operator site of lambda phage has been synthesized and studied by 1H nuclear magnetic resonance spectroscopy at 470 MHz . The 13 imino proton resonances observed at 20 degrees C have been assigned to specific base pairs at positions 3-15 on the basis of nuclear Overhauser effect measurements and studies of the temperature dependence of peak intensities . Resonances from the A-T base pairs at positions 1, 2, 16, and 17 are assumed to be absent from the spectrum because of terminal fraying . Resonance from many of the base pairs suggested by Ohlendorf et al . {Ohlendorf, D . H., Anderson, W . F., Fisher, R . G., Takeda, Y., & Matthews, B . W . (1982) Nature (London) 298, 718-723} to be involved in specific binding of the lambda phage cro repressor are well resolved.

Biochemistry, 1983 Sep 13, 22(19), 4400 - 5
Transfer ribonucleic acid deprived of the C-C-A 3'-extremity can interact with elongation factor Tu; Picone D et al.; In this work we describe that uncharged tRNAs or modified tRNAs lacking all or part of the C-C-A end (i.e., tRNA minus pCpCpA, tRNA minus pA, and tRNA minus A) can still influence the GTPase activity of the elongation factor Tu (EF-Tu), thus showing that, besides the aminoacylated 3'-end, other regions of the aa-tRNA interact with EF-Tu . The existence of an interaction between EF-Tu and truncated tRNAs was also confirmed by examining the dissociation of the EF-Tu-GTP complex: the rate of this reaction is decreased upon addition of tRNAVal1 minus pCpCpA . The effect on the EF-Tu GTPase activity of tRNAs deprived of the C-C-A 3'-end is still evident in the presence of C-C-A-aa . The stimulatory pattern obtained with C-C-A-Val at 5 mM MgCl2 is decreased upon addition of tRNAVal1 minus pCpCpA, tRNAVal1 minus pA, or tRNAVal1 minus A . This shows that the effect of the aminoacylated C-C-A 3'-end can be influenced via EF-Tu by the remaining regions of the tRNA, after cleavage of a bond in the 3'-extremity . However, also with an excess of tRNAVal1 minus pCpCpA over C-C-A-Val, no "aa-tRNA-like" effect, i.e., no inhibition of the EF-Tu GTPase, was obtained, suggesting that, upon binding with EF-Tu, a specific conformational change in the aa-tRNA molecule also takes place, regulating the expression of the GTPase activity . Our results unequivocally show that different regions of the aa-tRNA are needed for a coordinated interaction with EF-Tu.

Life Sci, 1983 Sep 12, 33(11), 1033 - 7
Abdominal vagotomy does not modify endotoxic shock in rats; Pitterman AB et al.; Bilateral subdiaphragmatic vagotomy does not improve the clinical course nor the survival of Sprague-Dawley rats injected intravenously with E . coli lipopolysaccharide . These results show that whatever peripheral signals are elicited by endotoxin to generate the centrally mediated hypotensive response, they are not conveyed to the central nervous system by abdominal vagal afferent fibers.

J Biol Chem, 1983 Sep 10, 258(17), 10204 - 7
The active site regions of lacZ and ebg beta-galactosidases are homologous; Fowler AV et al.; The active site-directed inhibitor 4-nitrophenyl-beta-D-galactopyranosylmethyltriazene, previously shown (Fowler, A . V., Zabin, I., Sinnott, M . L., and Smith, P . J . (1978) J . Biol . Chem . 253, 5283-5285) to alkylate methionine 502 in lacZ beta-galactosidase, was used to label the second naturally occurring beta-galactosidase of Escherichia coli (ebgo) . The reagent was also used to label two mutant forms of the enzyme (ebga and ebgb) selected for enhanced lactase activity . In the case of ebgo and ebga, 75 and 85% of the label, respectively, was incorporated into a tryptic peptide which is homologous (38% identity) to residues 483-503 of the lacZ beta-galactosidase sequence . In the ebgo and ebga enzymes, a serine probably is alkylated . In the case of the ebgb enzyme, 61% of the label is found on a tryptic peptide homologous (69% identity) with residues 457-468 of the lacZ beta-galactosidase . In this peptide, a glutamic acid and a tyrosine residue are both alkylated.

J Biol Chem, 1983 Sep 10, 258(17), 10614 - 21
Immunological characterization of the cytochrome o terminal oxidase from Escherichia coli; Kranz RG et al.; The cytochrome o terminal oxidase from Escherichia coli was immunochemically purified and monospecific antiserum toward cytochrome o was obtained . This antiserum is able to precipitate 100% of the ubiquinol-1 oxidase activity in Triton X-100 extracts of membranes from an E . coli strain in which cytochrome o is the only terminal oxidase . Cytochrome o was analyzed and quantitated using crossed immunoelectrophoresis, rocket immunoelectrophoresis, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that cytochrome o is composed of four subunits of approximate equimolar stoichiometry with molecular weights of 51,000, 28,500, 18,000, and 12,700 . The low temperature (77 K) reduced - oxidized spectrum of the immunoprecipitate shows two peaks at 555 and 562 nm, indicating b-type cytochromes . With the anti-cytochrome o and antiserum toward the cytochrome d terminal oxidase complex which was previously obtained, it is possible to immunochemically assay for all the cytochromes in the cytoplasmic membrane of aerobically grown E . coli . Preliminary results indicate that the biosynthesis of cytochrome o is repressed when cytochrome d is induced by lowering the dissolved oxygen concentration during cell growth.

J Biol Chem, 1983 Sep 10, 258(17), 10411 - 7
Exonuclease VIII of Escherichia coli . I . Purification and physical properties; Joseph JW et al.; Exonuclease VIII is an enzyme whose synthesis is induced as a result of sbcA mutations . The enzyme has been purified to near homogeneity from an Escherichia coli strain containing an sbcA mutation and mutations in the structural genes for exonuclease III, exonuclease V, and endonuclease I . The enzyme specifically degrades linear duplex DNA in a reaction which requires magnesium ions and is susceptible to inhibition by other divalent cations and by sulfhydryl-blocking reagents . Enzyme activity occurs over a broad pH range with peak activity at pH 8.5 in Tris buffer . The protein has a subunit Mr = 140,000, a sedimentation coefficient of 8.4 +/- 0.6, and a Stokes radius of 142 +/- 6 A, which is consistent with its active form being a multimer . Exonuclease VIII has a frictional coefficient of 2.6 which indicates that it has an asymmetric structure.

J Biol Chem, 1983 Sep 10, 258(17), 10360 - 5
Recognition of termination codon by release factor in the presence of a tRNA-occupied A site . Evidence for flexibility in accommodation of the release factor on the ribosome; Tate WP et al.; Recognition of the termination codon by release factor was studied with 70 S ribosomes containing initiator tRNA at the P site and an amino acid-specifying codon as well as the corresponding cognate tRNA at the A site . In vitro termination was not excluded when either the A site was occupied with an amino acid-specifying codon or the termination codon was displaced from the A site codon position by spacer nucleotides between UAA and the P site bound AUG . The release factor may have flexibility in its codon recognizing domain to be able to adjust to this enforced change in the position of the termination codon on the ribosome without loss of functional specificity . Deacylated tRNA bound at the A site of 70 S ribosomes did not interfere with stoichiometric UAA-directed release factor binding to these particles . In contrast, a ternary complex (aminoacyl-tRNA elongation factor-Tu . GTP) abolished the binding of the release factor while aminoacyl-tRNA inhibited it only weakly . This suggests that release factor and elongation factor-Tu have overlapping binding sites but the A site binding domains of the release factor and tRNA are exclusive.

Nucleic Acids Res, 1983 Sep 10, 11(17), 6089 - 105
The tetracycline resistance determinants of RP1 and Tn1721: nucleotide sequence analysis; Waters SH et al.; Nucleotide sequences of the homologous tetracycline resistance (tet) determinants of plasmid RP1 and transposon Tn1721 have been determined . Two open reading frames of divergent polarity have been assigned to a regulatory gene (tetR) and a gene encoding a resistance protein (tetA) . The intercistronic region contains appropriate regulatory and transcription signals . The tetR gene can code for a protein of 216 amino acids (deduced mol.wt . 23,288) and the tetA gene for a protein of 399 amino acids (deduced mol . wt . 42,205) . Based on the deduced amino acid sequence, the tetA proteins of RP1/Tn1721 are 78% homologous with that of pBR322 and 45% homologous with that of Tn10 . We conclude that a single tetA gene mediates resistance in each of these tet determinants.

Nucleic Acids Res, 1983 Sep 10, 11(17), 6021 - 39
Construction and characterization of type II collagen complementary deoxyribonucleic acid clones; Lukens LN et al.; The mRNA for type II collagen was purified from embryonic chick sternum or from purified sternal chondrocytes with guanidine thiocyanate as the extractant . Double-stranded cDNAs to procollagen mRNAs from sternum were synthesized and dC-tailed . After annealing with PstI-cleaved, dG-tailed pBR322, this DNA was used to transform Escherichia coli X1776 . Transformed colonies were screened by colony hybridization to type I and II collagen cDNAs . Clones that preferentially hybridized to type II cDNA were characterized further . Four such cDNA clones, pCgII-2, 3, 10 and 12, with inserts of 400, 320, 260 and 750 bp, have been identified as type II collagen cDNA clones by several criteria, including their preference for hybridizing with type II rather than type I collagen mRNAs in hybrid-selected translation experiments.

Nucleic Acids Res, 1983 Sep 10, 11(17), 5877 - 92
Nucleotide sequence of a Euglena gracilis chloroplast genome region coding for the elongation factor Tu; evidence for a spliced mRNA; Montandon PE et al.; We characterize a 1.95 kb transcription product of the Euglena gracilis chloroplast DNA fragment Eco-N + Q by S1 nuclease analysis and DNA sequencing and show that it is the product of three splicing events . Exon 1 (0.45 kb), exon 2 (0.74 kb) and 175 nucleotides of exon 3 (0.53 kb) code for the chloroplast elongation factor protein (EF-Tu) . The remaining part of exon 3 and exon 4 (0.23 kb) have unidentified open reading frames . The chloroplast EF-Tu protein has 408 aminoacids and is to 70% homologous with the E . coli EF-Tu protein . The active site for aminoacyl-tRNA binding is highly conserved, while the active site for GTP/GDP binding lacks the cysteine present in the E . coli EF-Tu protein . The two introns separating exons 1, 2 and 3 are, respectively, 103 and 110 nucleotides long . The size of the third intron is not yet determined . The splicing rules for eukaryote mRNA are not followed.

Nucleic Acids Res, 1983 Sep 10, 11(17), 5837 - 54
Increased expression of a cloned gene by local mutagenesis of its promoter and ribosome binding site; Warburton N et al.; A strategy for local mutagenesis of DNA has been developed . The lac promoter in phage M13mp9 was replaced with the E . coli trp promoter . A restriction fragment bearing only the trp promoter region was mutagenized with nitrous acid, religated to the unmutagenized vector and transfected into E.coli . Several clones which give darker blue plaques on indicator media, suggesting increased beta-galactosidase synthesis, were selected for DNA sequencing . One clone has a G leads to A transition on the 3' side of the 'Pribnow box' which results in a constitutive promoter . Two clones have different point mutations (C leads to T and T leads to C) between the Shine-Dalgarno sequence and initiation codon which raise expression of beta-galactosidase two-fold . A secondary structure model suggests that the latter two mutations could exert their effect by destabilizing base-pairing of the lac Z coding region with the ribosome binding site (RBS), thereby allowing easier access to ribosomes . Support for the model comes from the finding that neither of the RBS mutations increase expression of a different downstream gene which forms no obvious secondary structure with the RBS region, whether or not the mutations are present . These results strengthen the hypothesis that secondary structure masking is a major determinant of RBS strength.

Nucleic Acids Res, 1983 Sep 10, 11(17), 5811 - 9
Sequence of human haptoglobin cDNA: evidence that the alpha and beta subunits are coded by the same mRNA; Raugei G et al.; We have isolated and sequenced a cDNA clone coding for human haptoglobin . Our sequence shows that haptoglobin is very likely synthesized as a single polypeptide chain which is then cleaved at an Arg residue to generate its two characteristic alpha and beta subunit . Southern blot analysis suggests that there are at least two copies of the haptoglobin gene per haploid genome.

Nucleic Acids Res, 1983 Sep 10, 11(17), 5795 - 810
In vivo regulation of the uvrA gene: role of the "-10" and "-35" promoter regions; Backendorf C et al.; The effect of increasing deletions in the uvrA promoter region on the transcriptional efficiency was quantitatively analysed by fusion to the galK structural gene . A physical analysis of uvrA messenger RNA synthesis from the different deletion plasmids was performed using the S1 mapping technique . Both methods indicate that the uvrA "-10" promoter sequence is sufficient to trigger uvrA transcription . Although not essential, the "-35" region, which is overlapping with the LexA binding site, is shown to have an enhancing function, as the exposure of this region after SOS induction results in a 3- to 4-fold increase in uvrA transcription . A model is presented which accounts both for the observed basal and induced expression of the uvrA gene on a molecular level.

J Biol Chem, 1983 Sep 10, 258(17), 10637 - 41
Primary structures of both subunits of Escherichia coli glycyl-tRNA synthetase; Webster TA et al.; Escherichia coli glycyl-tRNA synthetase is one of two aminoacyl-tRNA synthetases which is comprised of two different subunits (in an alpha 2 beta 2 structure) . The two coding regions occur in tandem in the order alpha + beta and are synthesized from a single mRNA (Keng, T., Webster, T . A., Sauer, R . T., and Schimmel, P . R . (1982) J . Biol . Chem . 257, 12503-12508) . Primary structures of both proteins were determined by DNA sequencing of each coding region and by analysis of tryptic fragments of the enzyme . The alpha-subunit is 303 codons and terminates with TAA; the beta-subunit is 689 codons followed by tandem TAA stops . S1 nuclease mapping of the 3'-end of the two-cistron glyS mRNA showed that it predominantly ends 33/34 bases beyond the tandem stops with an RNA polymerse terminator sequence . Altogether, 43% of the translated polypeptide sequences were confirmed by mass spectrometric analysis of peptide fragments including confirmation of the COOH-terminal end of the beta-chain . This involved determinations, by fast atom bombardment mass spectrometry, of the masses of numerous whole tryptic fragments (with an accuracy of better than 1 Da) and of fragments truncated by one to three cycles of Edman degradations . The primary structures of the two subunits show no homologies with each other and have no internal sequence repeats of significance . While there are no extensive homologies with five other sequenced, or partially sequenced, synthetases, the alpha-subunit has a short sequence which can be aligned with sequences found in functionally important areas of two other synthetases and in uncharacterized parts of a third and fourth synthetase.

J Biol Chem, 1983 Sep 10, 258(17), 10418 - 24
Exonuclease VIII of Escherichia coli . II . Mechanism of action; Joseph JW et al.; Exonuclease VIII from Escherichia coli was shown to preferentially degrade linear duplex DNA, although a limited amount of activity with single-stranded linear DNA substrates was detected . No nucleolytic activity was observed with double-stranded circular substrates containing single strand breaks or gaps . Exonuclease VIII was shown to degrade linear duplex DNA from the 5' termini of the molecules and proceed in the 5' to 3' direction via a processive reaction mechanism . Initiation could occur from either a 5'-hydroxyl or 5'-phosphate residue at equal rates . The products of degradation of linear duplex DNA were an equivalent amount of 5'-mononucleotides and single-stranded DNA . Possible roles for exonuclease VIII in genetic recombination are discussed.

J Biol Chem, 1983 Sep 10, 258(17), 10294 - 5
Preliminary X-ray crystallographic studies and molecular symmetry of the PII regulatory protein from Escherichia coli; Suh SW et al.; The PII regulatory protein (tetramer, Mr = 44,000) plays an important role in the regulation of glutamine synthetase in enteric bacteria . The unmodified form (PIIA) of the PII protein from Escherichia coli has been crystallized in the cubic space group I23 . The unit cell dimension is a = 88.8 (+/-0.1) A, with six molecules in the unit cell (i.e . 1 subunit/asymmetric unit) . The molecule has symmetry 222.

Biochim Biophys Acta, 1983 Sep 9, 740(4), 410 - 6
Reaction of apurinic/apyrimidinic sites with {14C}methoxyamine . A method for the quantitative assay of AP sites in DNA; Talpaert-Borle M et al.; A simple and rapid method is described for the determination of AP (apurinic/apyrimidinic) sites in DNA . The method involves the reaction of {14C}methoxyamine with the aldehyde group present in the deoxyribose moiety after a base loss . Studies with alkylated-depurinated DNA and with uracil-containing polydeoxyribonucleotides depyrimidinated by uracil-DNA glycosylase show that methoxyamine reacts with both apurinic and apyrimidinic sites in a rapid and exhaustive way . Under standard conditions (30-min incubation with 5 mM methoxyamine at 37 degrees C, pH 7.2) untreated DNA is almost unreactive and the {14C}methoxyamine incorporation in DNA is proportional to the number of AP sites . Since the methoxyamine reaction is free from any degradative effect on DNA, AP sites may be estimated from a simple determination of the acid-insoluble radioactivity.

Science, 1983 Sep 9, 221(4615), 1020 - 6
DNA-binding proteins; Takeda Y et al.; The structures of three proteins that regulate gene expression have been determined recently and suggest how these proteins may bind to their specific recognition sites on the DNA . One protein (Cro) is a repressor of gene expression, the second (CAP) usually stimulates gene expression, and the third (lambda repressor) can act as either a repressor or an activator . The three proteins contain a substructure consisting of two consecutive alpha helices that is virtually identical in each case . Structural and amino acid sequence comparisons suggest that this bihelical fold occurs in a number of proteins that regulate gene expression, and is an intrinsic part of the DNA-protein recognition event . The modes of repression and activation by Cro and lambda repressor are understood reasonably well, but the mode of action of CAP is still unclear.

Biochim Biophys Acta, 1983 Sep 9, 740(4), 362 - 8
Differential stimulation by polyamines of phage DNA-directed in vitro synthesis of proteins; Watanabe Y et al.; The effect of polyamines on T7- and lambda rifd18 DNA-directed synthesis of proteins in an Escherichia coli cell-free system has been studied . When T7 DNA was used as a template, the degree of stimulation by spermidine of protein synthesis was larger with T7 RNA polymerase than with Mr 42 K protein, while the synthesis of Mr 13.5 K protein was not stimulated significantly by spermidine . The synthesis of T7 RNA polymerase was stimulated approx . 10-fold by 1 mM spermidine . When lambda rifd18 DNA was used as a template, the synthesis of beta beta' subunits of RNA polymerase was stimulated greatly by spermidine, while the synthesis of elongation factor Tu and ribosomal proteins was not stimulated significantly by spermidine . Spermidine stimulation of T7 DNA-directed synthesis of T7 RNA polymerase was at the level of both translation and transcription . The degree of stimulation by spermidine was greater at the level of translation . Putrescine stimulated the synthesis of T7 RNA polymerase and Mr 42 K protein to a small degree at the level of translation.

J Mol Biol, 1983 Sep 5, 169(1), 345 - 50
Computer averaging of 50 S ribosomal subunit electron micrographs; Kiseley NA et al.; Micrographs of one of the views of the large Escherichia coli subunit negatively stained with uranyl acetate were computer averaged using two techniques: the maximum-likelihood method and the correlation method . In both cases, extremely similar images were obtained . The same fine structure of the subunit has also been observed using optical averaging.

Nature, 1983 Sep 29-Oct 5, 305(5933), 448 - 51
Viability of lambda phages carrying a perfect palindrome in the absence of recombination nucleases; Leach DR et al.; In Escherichia coli in vitro constructions of perfect palindromes larger than 30 base pairs (bp) long have in general been unstable . A perfect palindrome has the unique possibility of forming a cruciform structure, and it is this feature which probably results in its instability . Negative supercoiling favours the formation of the cruciform conformation, which in turn causes the molecule to relax . This relaxation may render replicons containing large perfect palindromes inviable . An alternative hypothesis for inviability has been that the cruciform interferes with replication by favouring strand switching by polymerase I . Here we show that the simultaneous absence of two recombination nucleases, the recBC product, exonuclease V, and the sbcB product, exonuclease I, confers viability on a derivative of phage lambda carrying a perfect palindrome of inverted repeat length 1,600 bases . This observation suggests a third hypothesis--that nucleolytic cleavage of the cruciform is responsible for the inviability of the phage . Such an activity has been shown in vitro for T4 exonuclease VII.

J Mol Biol, 1983 Sep 5, 169(1), 197 - 215
Characterization of the DNA binding protein encoded by the N-specific filamentous Escherichia coli phage IKe . Binding properties of the protein and nucleotide sequence of the gene; Peeters BP et al.; A DNA binding protein encoded by the filamentous single-stranded DNA phage IKe has been isolated from IKe-infected Escherichia coli cells . Fluorescence and in vitro binding studies have shown that the protein binds co-operatively and with a high specificity to single-stranded but not to double-stranded DNA . From titration of the protein to poly(dA) it has been calculated that approximately four bases of the DNA are covered by one monomer of protein . These binding characteristics closely resemble those of gene V protein encoded by the F-specific filamentous phages M13 and fd . The nucleotide sequence of the gene specifying the IKe DNA binding protein has been established . When compared to the nucleotide sequence of gene V of phage M13 it shows an homology of 58%, indicating that these two phages are evolutionarily related . The IKe DNA binding protein is 88 amino acids long which is one amino acid residue larger than the gene V protein sequence . When the IKe DNA binding protein sequence is compared with that of gene V protein it was found that 39 amino acid residues have identical positions in both proteins . The positions of all five tyrosine residues, a number of which are known to be involved in DNA binding, are conserved . Secondary structure predictions indicate that the two proteins contain similar structural domains . It is proposed that the tyrosine residues which are involved in DNA binding are the ones in or next to a beta-turn, at positions 26, 41 and 56 in gene V protein and at positions 27, 42 and 57 in the IKe DNA binding protein.

J Mol Biol, 1983 Sep 5, 169(1), 53 - 81
Specific in vitro transcription of the insertion sequence IS2; Hinton DM et al.; The insertion sequence IS2 is a small transposable element of Escherichia coli that lacks any known genetic markers . Insertion of this element in one orientation (I) within bacterial operons blocks expression of downstream genes . In the other orientation (II), IS2 has been associated with the constitutive expression of genes distal to its insertion, suggesting that IS2 might contain promoters directing transcription of IS2(II) into other genes . To test the transcription potential of IS2, we have transcribed in vitro DNA templates from gal3, a Gal- allele in which an IS2(I) is inserted between the gal promoter and the gal genes . We have detected two IS2-specific RNAs which initiate from promoters within IS2 and are transcribed in orientation II (away from the galETK genes) . Though the presence and orientation of these promoters suggests that they could be responsible for the constitutive expression of genes adjacent to an IS2(II) element, an alternative role could be for transcription of IS2-encoded genes . Although IS2(I) insertions normally block expression of adjacent genes, certain altered (e.g . mutant) IS2(I) sequences lead to the constitutive expression of downstream genes . We have transcribed DNA templates from galwc5 and galc331, which are Galc alleles that contain altered IS2(I) insertions within the gal operon . For each allele, we have detected two gal-directed transcripts initiating within the IS2 sequence . These RNAs are not detected upon transcription of the unaltered IS2(I) DNA and the promoters arise as a direct consequence of the IS2(I) alterations . This result suggests that these promoters detected in vitro are responsible for the Galc phenotype of these alleles.

Vet Rec, 1983 Sep 3, 113(10), 208 - 12
Prevalence of enterotoxigenic Escherichia coli in calves in Scotland and northern England; Sherwood D et al.; Eighty-eight of 1529 (5.7 per cent) Escherichia coli isolates from diarrhoeic and clinically normal calves in Scotland and northern England were found to possess the K99 pilus antigen (K99+) . There was complete correlation between possession of K99 antigen, heat stable enterotoxin production and ability to dilate intestinal loops . The diagnosis of calf enterotoxigenic E coli infections may therefore be based on the detection of K99 antigen alone . Enterotoxigenic E coli was isolated from 23 of 306 (7.5 per cent) diarrhoeic calves from eight of 70 (11.4 per cent) farms and was not isolated from clinically normal calves . Infected calves were between one and three days old . A survey by an enzyme-linked immunosorbent assay found 3.0 per cent and 3.9 per cent of sera from calves and cows respectively to contain antibodies to K99 antigen . The prevalence of other enteropathogenic organisms in calf faeces is also discussed.

J Clin Invest, 1983 Sep, 72(3), 911 - 8
Thromboxane A2 mediates lung vasoconstriction but not permeability after endotoxin; Winn R et al.; The effect of dazoxiben, a selective thromboxane (Tx) synthetase inhibitor, on systemic and pulmonary hemodynamics, eicosanoids, and lung permeability was assessed in awake goats with lung lymph fistulae following infusion of Escherichia coli endotoxin (1 microgram/kg) . Animals received endotoxin either with no treatment or pretreatment with a bolus (25 mg/kg) followed by a maintenance infusion (10 mg/kg per h) of dazoxiben . In untreated animals, the peak rise of 26.8 cm H2O in pulmonary artery (Ppa) and of 13.5 cm H2O in wedge (Pw) pressures occurred at the same time as the peak elevations in plasma thromboxane B2 (T X B2) . Maximum reduction in cardiac output (Qt) also occurred at the same time . Lung lymph flow (QL) increased during this period and remained elevated for at least 6 h after endotoxin . T X B2 levels had returned from a peak of 13.1 to 0.7 ng/ml by 2 h . In dazoxiben-treated animals, plasma concentrations of T X B2 were never significantly elevated . Increases in Ppa and Pw were markedly reduced and decreased Qt was transient . QL in treated animals began to increase by 30 min after endotoxin and reached a peak by 2 h . Increased QL in treated animals was not as great as in the untreated animals . Moreover, lymph-plasma protein ratios increased significantly in treated animals . Plasma prostaglandin (PG)F2 alpha and 6-keto-PGF1 alpha concentrations were elevated in both groups after endotoxin with values significantly greater in treated animals . We conclude that selective inhibition of Tx ameliorates many adverse hemodynamic consequences of endotoxemia but does not prevent lung permeability changes.

J Bacteriol, 1983 Sep, 155(3), 1192 - 9
Molecular cloning of regulatory gene xylR and operator-promoter regions of the xylABC and xylDEGF operons of the TOL plasmid; Inouye S et al.; The regulatory gene xylR of the TOL plasmid, which functions positively on both xylABC and xylDEGF operons in the presence of m-xylene or m-methylbenzyl alcohol, was cloned onto an Escherichia coli vector, pACYC177 . A fused operon consisting of the operator-promoter region of the xylABC operon and the xylE gene was cloned onto pBR322 . The xylE product, catechol 2,3-dioxygenase, was induced by m-xylene or m-methylbenzyl alcohol in the cells containing the fused operon when a 2.8-kilobase segment of the TOL plasmid was provided in trans . Therefore, the segment appeared to contain the regulatory gene xylR . The xylR gene was mapped very close to the other regulatory gene, xylS, determined previously . The xylR gene was not effective on activation of the xylDEGF operon unless an additional region containing xylS was provided together with the inducer . These results indicate that both xylR and xylS are essential to the m-methylbenzyl alcohol-dependent induction of the xylDEGF operon . The map positions of xylR and xylS were precisely determined by subcloning or insertion inactivation . In addition, the operator-promoter regions of the xylABC and xylDEGF operons were mapped to the 0.6- and 0.4-kilobase regions of the TOL plasmid, respectively.

J Virol, 1983 Sep, 47(3), 642 - 8
Complete nucleotide sequence of the nucleoprotein gene of influenza B virus; Londo DR et al.; A DNA copy of influenza B/Singapore/222/79 viral RNA segment 5, containing the gene coding for the nucleoprotein (NP), has been cloned in Escherichia coli plasmid pBR322, and its nucleotide sequence has been determined . The influenza B NP gene contains 1,839 nucleotides and codes for a protein of 560 amino acids with a molecular weight of 61,593 . Comparison of the influenza B NP amino acid sequence with that of influenza A NP (A/PR/8/34) reveals 37% direct homology in the aligned regions, indicating a common ancestor . However, influenza B NP has an additional 50 amino acids at its N-terminal end . As is the case with influenza A NP, influenza B NP is a basic protein, with its charged residues relatively evenly distributed rather than clustered . The structural homology suggests functional similarity between the NP of influenza A and B viruses.

Eur J Cell Biol, 1983 Sep, 31(2), 334 - 40
Specific visualization of ribosomal RNA in the intact ribosome by electron spectroscopic imaging; Korn AP et al.; The recently developed electron microscopic technique of electron spectroscopic imaging has been used to map the distribution of phosphorus, and therefore of RNA, in situ in the ribosomal subunits of E . coli . The results indicate that the RNA moiety of both subunits is concentrated toward the centre of the particle somewhat more than is the total mass, but reaches the outer surface at several places . The micrographs also reveal certain distinctive features in the shape of the RNA component that may be related to the overall shape of the ribosome . The method yielded a reasonably accurate estimate of the phosphorus content of the 30 S ribosome.

Appl Environ Microbiol, 1983 Sep, 46(3), 611 - 8
Predicting coliform concentrations in upland impoundments: design and calibration of a multivariate model; Kay D et al.; This paper reports on the calibration and use of a multiple regression model designed to predict concentrations of Escherichia coli and total coliforms in two upland British impoundments . The multivariate approach has improved predictive capability over previous univariate linear models because it includes predictor variables for the timing and magnitude of hydrological input to the reservoirs and physiochemical parameters of water quality . The significance of these results for catchment management research is considered.

Arch Biochem Biophys, 1983 Sep, 225(2), 722 - 30
Inhibition of aminoacyl-tRNA synthetases by p-chloroamphetamine and its role in protein synthesis inhibition; Nowak TS Jr et al.; p-Chloroamphetamine inhibited to some degree all amino acid-dependent pyrophosphate-exchange activities which could be detected in a rabbit reticulocyte extract . A detailed kinetic analysis of the reaction catalyzed by reticulocyte leucyl-tRNA synthetase demonstrated that the inhibitor affected only amino acid binding . Less rigorous studies of other synthetases from both rabbit reticulocyte and Escherichia coli could be similarly interpreted, suggesting that this compound interacts in a common manner with these several enzymes . The contribution of such effects to the inhibition of protein synthesis by the drug was investigated using cell-free translation systems in which rates of amino acid incorporation were limited to varying degrees by the synthesis and availability of aminoacyl-tRNA . In a wheat germ system programmed with globin mRNA, in which levels of amino acids and aminoacyl-tRNAs were shown to limit the rate of protein synthesis, the inhibition produced by p-chloroamphetamine could be partially reversed by increasing the concentration of the limiting amino acid . In a reticulocyte lysate, in which amino acid concentrations were not limiting, inhibition failed to show an amino acid-reversible component . Thus, while the inhibition of aminoacyl-tRNA synthetases by amphetamines can be shown in some cases to play a role in the effects of these compounds on in vitro protein synthesis, other sites of interference with initiation and/or elongation reactions may predominate, depending on the construction of the system under study.

J Trauma, 1983 Sep, 23(9), 769 - 74
Changes in red blood cell transmembrane potential, electrolytes, and energy content in septic shock; Shires GT 3rd et al.; Septic shock was induced in adult baboons by the infusion of live Escherichia coli . A progressive derangement in skeletal muscle cell function was documented by the direct measurement of declining transmembrane potential difference (PD) . A concurrent depolarization of the red blood cell (RBC) was characterized by cellular uptake of chloride, sodium, and water, and loss of potassium . The decrease in RBC PD was significantly greater than the change predicted to occur from acidosis alone . These findings are compatible with changes in membrane permeability and decreased active transport . The continuous accumulation of RBC adenosine triphosphate during shock suggests decreased energy utilization rather than decreased energy production as a factor leading to diminished active ion transport.

Biochem Pharmacol, 1983 Sep 1, 32(17), 2523 - 8
Influence of endotoxin on arachidonate metabolism in isolated rabbit peritoneum; Bult H et al.; The serous membranes of the rabbit peritoneal cavity are tissues in which cyclo-oxygenase and lipoxygenase pathways of arachidonate metabolism can be studied simultaneously . After elaboration of the optimum conditions, the metabolism of two concentrations of arachidonic acid (AA) was studied in the presence and absence of endotoxin lipopolysaccharide (LPS) from E . coli O127:B8 (0.1 and 1.0 mg/ml) . LPS suppressed the formation of radiolabelled cyclo-oxygenase products (predominantly prostacyclin) at the lower concentration of exogenous AA (0.8 microM), but not at the higher substrate concentration (34.5 microM) . The biosynthesis of lipoxygenase metabolites, i.e . monohydroxyeicosatetra-enoic acids (HETEs), was not influenced by LPS . These findings can be explained by an enhanced release of endogenous AA in the prostacyclin forming mesothelial cells in the presence of LPS . Measurements of the endogenous biosynthesis of prostacyclin supported this assumption.

J Bacteriol, 1983 Sep, 155(3), 1001 - 8
Identification of the type I trimethoprim-resistant dihydrofolate reductase specified by the Escherichia coli R-plasmid R483: comparison with procaryotic and eucaryotic dihydrofolate reductases; Simonsen CC et al.; We have isolated and determined the nucleotide sequence of a 1,626-base-pair fragment from R-plasmid R483 which encodes a trimethoprim-resistant dihydrofolate reductase . Analysis of the nucleotide sequence of this fragment revealed the presence of two open reading frames, each sufficient to encode polypeptides of approximately 17,000 daltons . Both open regions are preceded by sequences conforming closely to the canonical description of procaryotic promoters . A 490-base-pair HpaI fragment spanning one of the potential coding regions was inserted into a plasmid vector under the transcriptional control of the trp promoter . Cells transformed with this plasmid were trimethoprim resistant and produced dihydrofolate reductase activity which in vitro was resistant to moderate levels of trimethoprim . Analysis of the predicted amino acid sequence of this protein indicated that the R483-encoded trimethoprim-resistant enzyme was distantly related to the trimethoprim-sensitive bacterial homologs . The conserved amino acids were localized primarily to the region of the enzyme previously shown to comprise the hydrophobic substrate binding pocket.

Yale J Biol Med, 1983 Sep-Dec, 56(5-6), 777 - 81
Molecular biology of spiroplasma plasmids; Barber CE et al.; With one exception, all spiroplasma strains examined contained extrachromosomal DNA, most of which was in the form of covalently closed circular plasmids . One plasmid, pIJ2000, carried by Spiroplasma citri strain ASP-1, was purified and characterized and used to probe for related plasmids in other strains . Unsuccessful attempts were made to clone pIJ2000 into Escherichia coli using the vectors pAT153 and pBR322 . However, spiroplasma chromosomal DNA fragments could be cloned without difficulty.

Rev Esp Fisiol, 1983 Sep, 39(3), 321 - 5
Heat damage and repair in the Escherichia coli nucleoid: kinetics based on sedimentation analysis; Pellon JR; Changes in the structure of the Escherichia coli nucleoid during heat damage and repair were followed by sedimentation analysis in neutral sucrose gradients . Heating at 50 degrees C results first in a decrease in the sedimentation coefficient of the isolated nucleoid . Increasing the heating time, a subsequent increase in sedimentation coefficient is observed . After a heat shock (i.e . 4 min at 50 degrees C), a short incubation at 25 degrees C (i.e . 5 min) allows the nucleoid to repair and return to the sedimentation coefficient of control unheated nucleoids . The nucleoids heated at 50 degrees C for longer periods and incubated afterwards at 25 degrees C demonstrate a different pattern of structural repair . They associate with protein in the first stage of the repair period.

Tsitol Genet, 1983 Sep-Oct, 17(5), 54 - 6
{Localization of the genes coding for GTP cyclohydrolase II and riboflavin synthase on the chromosome of Escherichia coli K-12}; Tesliar GE et al.; A conjugation analysis of riboflavin-dependent mutants of Escherichia coli K-12 has been made by means of various F'-factors . It is shown that the GTP cyclohydrolase II gene is localized in the chromosome map site between 27 and 30 min; the riboflavin synthase gene--in the site between 56 and 59 min; and the gene in which mutation causes accumulation of 2,6-dioxy-5-amino-4-ribitylamino-pyrimidine--in the site limited by 61-65 min . So it may be concluded that riboflavin biosynthesis genes in the E . coli chromosome are not clustered.

Invest Radiol, 1983 Sep-Oct, 18(5), 425 - 35
Platelet kinetics and biodistribution in canine endotoxemia; Sostman HD et al.; Kinetics and magnitudes of changes in Indium-labeled platelet biodistribution were studied in dogs given E . coli endotoxin . Marked, reversible, dose-dependent shifts of platelets from blood to lung and apparently irreversible shifts to liver were demonstrated . These were contemporaneous with alterations in blood gases and in pulmonary and systemic hemodynamics . Morphologic studies revealed atelectasis, sequestration of leukocytes and platelets in the lungs, and mild interstitial pulmonary edema . This study provides in vivo quantification of labeled platelet response to a specific stimulus, and illustrates a method that could be applied to more extensive study of blood element participation in acute lung injury.

Genetika, 1983 Sep, 19(9), 1433 - 8
{Expression of the amino acid operons in Escherichia coli strains with an altered transcription and translation apparatus . IV . The effect of mutations disturbing the coupling of the transcription and translation processes on ilv-operon expression in cells carrying the mutation in the spoT gene}; Gordeev VK et al.; The rate of adaptation of Escherichia coli K-12 NF930 spoT1 cells with elevated intracellular level of ppGpp to various minimal media was studied . It has been found that the rate of adaptation of spoT cells, like that of parent and rel strains, depends mainly on the rate of derepression of the ilv operon . The maximal rate of the ilv operon derepression was observed when an optimal concentration of ppGpp was maintained in cells . Derepression of the ilv operon is sharply delayed when the level of ppGpp is elevated or reduced . Mutations altering the translation system do not change the rate of adaptation of spoT cells . Rifampicin resistance mutations which altered the structure of RNA polymerase change the rate of adaptation of spoT cells to minimal media, especially to those containing serine at high concentrations . The possible role of serine in the regulation of ppGpp degradation system is discussed.

Eur J Cell Biol, 1983 Sep, 31(2), 325 - 33
A survey of 50 S ribosomal subunits by dark field electron microscopy; Korn AP et al.; A survey of dark field electron micrographs of the 50 S ribosomal subunit of E . coli has been performed and supplemented, for comparative purposes, by examination of negatively stained or metal shadowed specimens in the bright field mode . Attention was directed to the so-called "crown" and "kidney" views . The elongated appendage seen in negatively stained crown profiles was not observed in unstained or positively stained samples examined in dark field; these showed only symmetrical crown profiles regardless of changes in buffer type and drying method and of the presence or absence of uranyl acetate treatment and glutaraldehyde fixation . The crown view occasionally displayed a bifurcation in one of the lateral lobes, while the kidney profile showed a groove near the base of the convex edge . Uranyl acetate treatment produced delicate stripes which may give an indication of the surface RNA distribution.

Appl Environ Microbiol, 1983 Sep, 46(3), 756 - 7
Colloidal clay inhibits conjugal transfer of R-plasmid R1drd-19 in Escherichia coli; Singleton P; Colloidal clay (montmorillonite) strongly inhibited the conjugal transfer of R-plasmid R1drd-19 in Escherichia coli . This finding is discussed in the context of the feasibility of plasmid transfer in environmental waters.

Appl Environ Microbiol, 1983 Sep, 46(3), 749 - 52
Binding of metallic ions to the outer membrane of Escherichia coli; Hoyle B et al.; The binding of metal ions by the outer membrane of Escherichia coli K-12 strain AB264 was investigated by using outer membrane obtained after Triton X-100 extraction of purified cell envelopes . Binding studies, conducted under saturating conditions, indicated a selective trapping of certain metallic ions . Low-dose electron microscopy of metal-loaded samples revealed an aggregative deposition of lead on one surface of the membrane which suggests that at least one distinctive binding site is asymmetrically arranged in these outer membrane vesicles.

Anal Biochem, 1983 Sep, 133(2), 292 - 5
A continuous spectrophotometric assay for Escherichia coli alanyl-transfer RNA synthetase; Roy S; A new continuous spectrophotometric assay is demonstrated for Escherichia coli alanyl-tRNA synthetase . It involves beta-gamma adenylyl imidophosphate as a substitute for ATP in the pyrophosphate exchange reaction . The net conversion of beta-gamma adenylyl imidophosphate to ATP can be linked to NADP reduction by hexokinase and glucose-6-P dehydrogenase catalyzed reactions, which can be monitored at 340 nm . This assay can be extended to other aminoacyl-tRNA synthetases which can use beta-gamma nonhydrolyzable analogs of ATP as an ATP substitute.

Res Vet Sci, 1983 Sep, 35(2), 242 - 4
Intestinal K99+ escherichia coli adhesion and absorption of colostral IgG1 in the newborn lamb: effect of fetal infusion of thyroid hormones; Cabello G et al.; Continuous infusion of thyroid hormones during the last 12 days of gestation in ovine fetuses was associated with: a reduction of the rate of appearance of blood IgG1 after colostrum feeding in the newborn lambs; an important reduction of the number of jejunal cells filled with IgG1, 12 hours post partum, suggesting a premature closure of the intestinal permeability to IgG1; and a significant reduction of the K99+ Escherichia coli adhesion on the intestinal villi . These results could partly explain relationships observed previously between perinatal thyroid function abnormalities and the occurrence of neonatal diseases.

Res Vet Sci, 1983 Sep, 35(2), 222 - 6
Increase in specific opsonic activity in bovine milk following experimental Escherichia coli mastitis; Hill AW et al.; An infection in a single bovine mammary quarter with a capsulated Escherichia coli stimulated a long lasting opsonic activity for the phagocytosis and killing of the homologous strain by polymorphonuclear leucocytes . The induced activity was associated with increases in the IgM fraction and an appearance of activity in the IgG2 fraction . The capsular polysaccharide was the major inducing antigen, with the immune whey showing opsonic activity for other strains of E coli with antigenically similar capsules.

Plasmid, 1983 Sep, 10(2), 148 - 55
The repA2 gene of the plasmid R100.1 encodes a repressor of plasmid replication; Liu CP et al.; We have constructed two miniplasmids, derived from the resistance plasmid R100.1 . In one of these plasmids 400 bp of R100.1 DNA have been replaced by DNA from the transposon Tn1000 (gamma-delta) . This substitution removes the amino-terminal end of the repA2 coding sequence of R100.1 and results in an increased copy number of the plasmid carrying the substitution . The copy number of the substituted plasmid is reduced to normal levels in the presence of R100.1 . The repA2 gene thus encodes a trans-acting repressor function involved in the control of plasmid replication.

Plasmid, 1983 Sep, 10(2), 101 - 10
Cloning and deletion mapping of the recF dnaN region of the Escherichia coli chromosome; Ream LW et al.; By cloning a 3.6-kb EcoRI fragment of the Escherichia coli chromosome with pBR322 we located more precisely recF relative to dnaN . By deletion mapping we localized functional recF to a 1.65-kb region of the cloned fragment and allowed rough mapping of the C terminus of dnaN . Cloned recF+, separated from functional flanking genes dnaN and gyrB, complemented chromosomal recF mutations presumably by coding for a cytodiffusible product . The protein encoded by dnaN was observed as a band on a polyacrylamide gel from minicells . Identification of a recF protein was not made.

Mol Biol (Mosk), 1983 Sep-Oct, 17(5), 965 - 71
{Intermediates of replication of NR1 plasmid DNA in Escherichia coli mini-cells}; Perebitiuk AN et al.; The pulse label of NRL plasmid-containing mini-cells has been shown to be localized mainly in DNA with a floating density in the CsCl-EtBr gradient different from the floating density of supercoil and open circle DNAs . During the chase of the pulse label, the DNA is transfered from the fraction with the intermediate floating density varying between the values for the supercoil and open circle DNA fractions to the fraction located below supercoil DNA in the equilibrium gradient and further to the open circle fraction . Electron microscopic analysis of the material with a higher floating density as compared to supercoil DNA has demonstrated the presence of "heavy" intermediates--covalently closed loosely supercoiled molecules . It is also supported by the sedimentation pattern of the characterized fraction in neutral and alkaline saccharose gradients . Molecules located in the CsCl-EtBr gradient between supercoil and open circle DNAs have the sedimentation constant characteristic for the elongation intermediates . It is suggested that NRL DNA molecules in E . coli mini-cells pass through all the basic stages of replication which results in the formation of open circle DNA or supercoil relaxation complexes.

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