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 e