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| Gene, 1987, 52(2-3), 207 - 14 Role of the merT and merP gene products of transposon Tn501 in the induction and expression of resistance to mercuric ions; Lund PA et al.; The bacterial transposon Tn501 carries, in addition to the genetic information for its own transposition, the genes of the mer operon (in the order merRTPAD), which code for resistance to Hg2+ ions . The basis for the resistance to Hg2+ is the enzymatic reduction of Hg2+ to Hg0 by mercuric reductase, the product of the Tn501 merA gene . We show here that deletion of the merT and merP genes from Tn501 leads to almost complete loss of the resistance phenotype, even if mercuric reductase is still present in the cytoplasm . Expression of the merT and merP genes in the absence of mercuric reductase gives a mercury-supersensitive phenotype . Mercury-dependent induction of transcription of the mer operon can occur in the absence of the merT and merP gene products . However, this induction is reduced by the presence of mercuric reductase in the cell . We conclude that one or both of the merT and merP genes of Tn501 are required for the expression of the mercury-resistance phenotype . They may in addition have a role in maximising the induction of the mer operon in the presence of Hg2+ ions . This is consistent with their proposed role in transport of Hg2+ into the cytoplasm. Gene, 1987, 52(2-3), 197 - 206 A family of lambda phage cDNA cloning vectors, lambda SWAJ, allowing the amplification of RNA sequences; Palazzolo MJ et al.; This paper describes the construction and characterization of a family of lambda phage cDNA cloning vectors that allows high-efficiency directional cDNA cloning and selective amplification of either sense or antisense cRNA sequences . These vectors contain several unique restriction sites (EcoRI, XbaI, and SacI) positioned between two specific phage promoters, SP6 and T7 . This system facilitates the in vitro preparation of single-stranded (ss) RNA molecules that should be useful in subtractive hybridization and in situ hybridization procedures . Using subtractive hybridization and this vector system, it should be possible to identify sequences present in one cDNA library and not another . In addition, it should be possible to construct subtracted cDNA libraries in these vectors and to generate high specific activity, ss, antisense cRNA probes directly from DNA prepared from the whole subtracted library or from individual clones. Gene, 1987, 52(2-3), 165 - 73 In vivo selection and characterization of internal deletions in the lamB::lacZ gene fusion; Benson SA et al.; Strain Pop3299 contains the lamB::lacZ42-12 gene fusion that encodes a hybrid protein that is efficiently exported to the cellular envelope of Escherichia coli . As a result of this efficient export, this strain is killed by the inducer maltose and unable to grow on minimal media supplemented with lactose . In an attempt to isolate mutants in which export of the hybrid protein is altered, we selected Lac+ mutants of strain Pop3299 on lactose tetrazolium media . Unlike mutants previously isolated on lactose minimal media, all the mutants we obtained carried large deletions within the lamB::lacZ gene fusion . Thus, it appears that the type of selection employed affects the type of mutations obtained . We have analyzed the nucleotide sequences of representative mutants, and demonstrate a correlation between the deletion size and the export-related maltose and lactose phenotypes . In addition, we demonstrate that the deletions do not appear to arise from regions of micro-homology. Gene, 1987, 52(2-3), 121 - 8 Physical characterization of katG, encoding catalase HPI of Escherichia coli; Triggs-Raine BL et al.; The gene encoding the bifunctional catalase-peroxidase HPI from Escherichia coli was located on a 3.8-kb HindIII fragment of the Clarke and Carbon plasmid pLC36-19 using transposon Tn5 insertions . This fragment was subcloned into the HindIII site of pAT153 to create pBT22 . The size of the insert was reduced by BAL 31 digestion of one end to an apparent minimum size for catalase expression of approx . 2.5 kb as determined by complementation and expression in maxicell strains . Further reduction in size or digestion from the opposite end inactivated the gene . The location and orientation of the promoter at the 0 kb end of the insert in pBT22 was confirmed by cloning a 320-bp BglII fragment into the promoter-cloning vector pKK232-8 . Differences in the Southern blots of genomic DNA from a wild-type strain and a katG17::Tn10 mutant digested with HincII and probed with pBT22 confirmed that the transposon previously mapped in katG was located in the 2.5-kb coding region for HPI. Gene, 1987, 53(1), 21 - 9 SV40-based Escherichia coli shuttle vectors infectious for monkey cells; Menck CF et al.; We describe SV40-based Escherichia coli shuttle vectors which can be packaged as pseudovirions without excision of plasmid sequences and which can be rescued in bacteria . These vectors replicate and are transmitted as virus in monkey COS cells without requiring a helper virus . Extrachromosomal vector DNA isolated from infected cells can be rescued in E . coli, so that DNA alterations can be easily screened . Indeed, some of the constructions give rise to very stable plasmids with no detectable rearrangements after multiple lytic cycles in COS cells . The spontaneous mutation frequency measured in bacteria, on the lacO target, is smaller than those usually found with shuttle vectors . We also constructed an expression vector derived from one of our infectious viruses by inserting the chloramphenicol acetyl transferase gene, expressed from the SV40 early promoter, which is efficiently transduced to cells by infection . In this system, the shuttle virus combines the convenience of plasmid rescue and analysis in bacteria, with the advantages of infectious virus. Gene, 1987, 52(1), 83 - 94 Hybrid protein thymidine kinase gene fusions: plasmid vectors for the study of transcription and translation initiation signals; Shapira SK et al.; The thymidine kinase (TK) gene (tk) from Herpes simplex virus type 1 has been used to form gene fusions encoding enzymatically active hybrid proteins . The promoter, translation initiation region, and the first three codons of the tk gene were removed and replaced with a series of DNA restriction sites . DNA fragments containing gene initiation regions were cloned into these sites and shown to synthesize enzymatically active proteins in Escherichia coli . These gene fusions were shown to complement an E . coli strain which is deficient in TK function . Gene initiation regions were used from the lac operon, the tnpR gene of Tn3, and the insA gene of ISl . TK synthesis was regulated by the control signals of the promoter fused to tk, and was dependent upon the phase alignment of the codons at the fusion joint . The size of the resulting protein was shown to be increased over the size of the original TK protein by the length of the coding region fused to TK . This demonstrated that the tk gene has non-essential N-terminal amino acids that can be replaced by other amino acid sequences with the retention of TK enzymatic activity . Such tk gene fusions are useful in situations where fusions with other genes cannot be conveniently selected or assayed. Gene, 1987, 52(1), 21 - 9 Phycocyanin alpha-subunit gene of Anacystis nidulans R2: cloning, nucleotide sequencing and expression in Escherichia coli; Lau RH et al.; The cloning and nucleotide sequence determination of the Anacystis nidulans R2 phycocyanin (PC) alpha-subunit gene are described . A 3.0-kb PstI fragment of Anacystis nidulans R2 genomic DNA cloned in plasmid pUC8 was found to hybridize with a heptadecameric oligodeoxynucleotide probe . Sequencing using synthetic primers revealed the presence of the PC alpha-subunit gene and the 3' proximal end of the beta-subunit gene . The alpha-gene is separated from the upstream beta-gene by a spacer length of 51 bp . The deduced amino acid (aa) sequence of the alpha-subunit protein is identical, except for 5 aa, to that of A . nidulans 6301 and is highly homologous (77%) to that reported for Agmenellum quadruplicatum PR6 . The 16-kDa alpha-subunit protein, detected by immunoadsorption, was fortuitously expressed in Escherichia coli from the lacZ promoter of the cloning vehicle pUC8. Gene, 1987, 52(1), 11 - 9 Use of a phage vector for rapid synthesis and cloning of single-stranded cDNA; Bellemare G et al.; We have developed a technique for synthesis of single stranded complementary DNA (ss cDNA) using specifically designed phage ssDNA as vector primer . This vector (pPBS27) was constructed by introducing a poly(dT) tail adjacent to the XbaI site of pTZ18R, which can exist either as a plasmid in Escherichia coli or as a ssDNA phage . The pPBS27 phage vector is linearized with XbaI using a restriction-site-directed fragment and used to anneal a mixture of poly(A) + RNA for cDNA synthesis by reverse transcriptase . The RNA is then hydrolysed with NaOH and a poly(dG) tail added to the 3' end of the vector-cDNA with terminal transferase . The linear hybrid ssDNA is then closed by annealing with a 15-mer site-directed fragment oligodeoxynucleotide molecule and ligated with T4 DNA ligase . Almost 10(5) E . coli transformants per microgram of vector primer can be obtained in two days. C R Seances Soc Biol Fil, 1987, 181(1), 52 - 4 {Endonuclease III of Escherichia coli and the repair of oxidized thymines and AP sites}; Bailly V; Escherichia coli endonuclease III is not an endonuclease . It breaks the C3'-O-P bond 3' to an AP site in DNA by catalysing a beta-elimination and not a hydrolysis . Therefore, it is a phosphoric monoester-lyase. Int J Immunopharmacol, 1987, 9(2), 243 - 53 Inhibition of delayed hypersensitivity reactions by a new agent, cis-1-methyl-4-isohexylcyclohexane carboxylic acid (IG-10)--II . The mechanism regarding the action on lymphokines; Nakatomi I et al.; A newly synthesized anti-allergic agent, cis-1-methyl-4-isohexylcyclohexane carboxylic acid (IG-10), has the capacity to inhibit the effector phase of delayed hypersensitivity reactions . In the present paper, the effect of IG-10 was studied on the generation of superoxide anion (O2) from macrophages, on macrophage chemotaxis, and on the activity of lymphokines such as skin reactive factor (SRF), macrophage migration inhibitory factor (MIF) and monocyte/macrophage chemotactic factor (MCF) in guinea pigs . Oral administration of IG-10 (50-200 mg/kg) inhibited SRF-induced skin erythema in a dose-dependent manner . In vitro, this agent (10(-7) -10(-5) g/ml) did not inhibit the generation of O2- from macrophages . The agent (10(-7) -10(-6) g/ml) significantly inhibited the activity of MIF and this inhibition was not due to the facilitation of normal migration . For macrophage chemotaxis, IG-10 (10(-8) -10(-6) g/ml) significantly inhibited MCF-induced chemotaxis . The agent also depressed the macrophage chemotaxis induced by N-formyl-methionyl-leucyl-phenylalanine but not the chemotaxis induced by E . coli culture filtrate . The inhibitory action of IG-10 on MCF activity was not influenced by antiglucocorticoid agents such as 17 alpha-methyltestosterone and androstenedione which reverse significantly the inhibitory action of glucocorticoids . The inhibitory action of IG-10 was relatively dependent on exogenous Ca2+ and Mg2+, and was antagonized by dbc-GMP. EMBO J, 1987 Jan, 6(1), 255 - 8 Titration of DnaA protein by oriC DnaA-boxes increases dnaA gene expression in Escherichia coli; Hansen FG et al.; Binding of the DnaA protein to its binding sites, the DnaA-boxes (TTATCCACA), was measured by a simple physiological approach . The presence of extra DnaA-boxes in growing cells leads to a derepression of dnaA gene expression, measured as beta-galactosidase activity of a dnaA-lacZ fusion polypeptide . Different DnaA-boxes caused different degrees of derepression indicating that the DnaA protein requires sequences in addition to the DnaA-box for efficient binding . The DnaA-boxes in oriC might act cooperatively in binding of the DnaA protein . The derepressed levels of DnaA protein obtained in a strain carrying an oriC+-pBR322 chimera were very high and sufficient to activate oriC on the chimeric plasmid, which was maintained at a copy number more than three times that of pBR322. EMBO J, 1987 Jan, 6(1), 133 - 8 Expression of the human papillomavirus type 18 E7 gene by a cassette-vector system for the transcription and translation of open reading frames in eukaryotic cells; Bernard HU et al.; We have constructed and functionally tested a cassette-vector-system for the transcription and translation of open reading frames (ORFs) in cells of higher eukaryotes . The vectors are derived from the plasmid pBR322 and can be selected and amplified in Escherichia coli . Alternative eukaryotic promoters can be inserted between the restriction sites SphI and KpnI, translation initiation motifs between KpnI and BglII, linkers for the adjustment of the translation reading frame and the insertion of genes or gene segments between BglII and HindIII, followed by a HindIII-EcoRI segment with splicing and polyadenylation signals derived from SV40 . A prototype vector system, pORFEX11, 12 and 13, contains the strong cytomegalovirus immediately early promoter and a 10-bp motif of the SV40 T-antigen translation start . Polylinkers derived from pUC18 permit the insertion of ATG-less ORFs downstream from the ATG of the vector . Either of the three alternative polylinkers adjusts the appropriate translation frame . A similar construct contains the regulatable promoter of the Drosophila heat shock gene 70 . We inserted genes or gene segments, that code for the bacterial chloramphenicol acetyltransferase, the bacterial gene conferring resistance against hygromycin, and the ORF E7 of the human papillomavirus type 18 into these vectors . After transfection of mouse L fibroblasts, all proteins and functions were expressed in accordance with the prediction . In transiently transfected L cells, the E7 protein expressed from pORFEX12 constitutes approximately 2.0% of total cell protein . This E7 protein could be localized by immunocytochemistry as a cytoplasmic component. Arch Virol, 1987, 94(3-4), 323 - 9 Effect of recombinant human interferon gamma against human cytomegalovirus; Yamamoto N et al.; Escherichia coli-derived human interferon-gamma (rIFN-gamma) inhibited the replication of human cytomegalovirus (HCMV) synergistically when combined with IFN-alpha . The induction of HCMV DNA polymerase was inhibited in rIFN-gamma-treated cells . It is suggested that the induction of 2-5 A synthetase does not play an important role in the anti-HCMV actions of IFNs. Mol Biol (Mosk), 1987 Jan-Feb, 21(1), 194 - 9 {Plasmid vector for cloning, determination of nucleotide sequence and directed assembly of DNA fragments}; Kravchenko VV et al.; A plasmid vector pNIMB has been constructed (starting) from the pUR222 plasmid as a result of substitution of the polylinker containing restriction sites: PstI, SalGI, AccI, HindII, BamHI EcoRI and by other synthetic linkers with additional sites for HindIII and HgaI . Plasmid pNIMB does not differ from the parent one phenotypically . Compared to pUR222 the vector contains an additional site for cloning HindIII fragments of DNA and allows to clone SalGI/BamHI- and PstI/SalGI-fragments . Cloning of DNA fragments in all seven unique sites of pNiMB gives the possibility for sequencing the fragments avoiding their isolation from the gel . Moreover, this vector may be useful for cloning and directed assembly of chemically synthesised DNA fragments when the endonuclease HgaI sites are used. Microbios, 1987, 49(199), 91 - 6 Effects of lipid A content, hydrocortisone and polymyxin B on endotoxin-induced decreases in in vivo drug metabolism; Abernathy CO et al.; Using hexobarbital sleeping and zoxazolamine paralysis time as indices of in vivo hepatic drug metabolism, the effects of endotoxin on drug action appear to be time- and dose-dependent and the lipid A moiety of endotoxin appears to be responsible for its inhibitory effects . These studies have also demonstrated that polymyxin B can ameliorate the adverse effects of endotoxin on drug metabolism . Since hydrocortisone protects mice from endotoxin lethality, but does not alter the prolongation of hexobarbital sleeping time caused by endotoxin, it is possible to separate the lethal effects of endotoxin from its effects on drug metabolism. Mol Gen Genet, 1987 Jan, 206(1), 95 - 100 Reduced transcription of the rnh gene in Escherichia coli mutants expressing the SOS regulon constitutively; Quinones A et al.; We have analysed the transcription levels for the convergently overlapping Escherichia coli genes for the DNA polymerase III proofreading function (dnaQ) and ribonuclease H (rnh) . The two tandem dnaQ promoters are about three times more active than the single rnh promoter as shown by analysing the level of in vivo transcription using dnaQ-galK and rnh-galK fusions . In E . coli mutants constitutively expressing the pleiotropic SOS response, which includes activities that enhance DNA repair, recombination and mutagenesis, a strong reduction in rnh transcription was observed . The lexA51 recA441 double mutant which fully expresses the SOS response shows the strongest reduction in rnh transcription and the highest increase in dnaQ transcription . Nuclease S1 mapping supported the finding that a constitutive expression of SOS function leads to a strong reduction in rnh transcription. Mol Gen Genet, 1987 Jan, 206(1), 9 - 16 Molecular cloning and nucleotide sequence of the mutT mutator of Escherichia coli that causes A:T to C:G transversion; Akiyama M et al.; The Escherichia coli mutator gene mutT, which causes A:T----C:G transversion, was cloned in pBR 322 . mutT+ plasmids carry a 0.9 kb PvuII DNA fragment derived from the E . coli chromosome . Specific labelling of plasmid-encoded proteins by the maxicell method revealed that mutT codes for a polypeptide of about 15,000 daltons . The protein was overproduced when the mutT gene was placed under the control of the lac regulatory region on a multicopy runaway plasmid . The nucleotide sequence of the mutT gene was determined by the dideoxy method. Mol Gen Genet, 1987 Jan, 206(1), 51 - 9 Overproduction of DnaA protein stimulates initiation of chromosome and minichromosome replication in Escherichia coli; Atlung T et al.; Increased synthesis of DnaA protein, obtained with plasmids carrying the dnaA gene controlled by the heat inducible lambda pL promoter, stimulated initiation of replication from oriC about threefold . The overinitiation was determined both as an increase in copy number of a minichromosome and as an increase in chromosomal gene dosage of oriC proximal DNA . The additional replication forks which were initiated on the chromosome did not lead to an overall increase in DNA content . DNA/DNA hybridization showed an amplification encompassing less than a few hundred kilobases on each side of oriC . Kinetic studies showed that the overinitiation occurred very rapidly after the induction, and that the initiation frequency then decreased to a near normal frequency per oriC . The results indicate that the DnaA protein is one important factor in regulation of initiation of DNA replication from oriC. Mol Gen Genet, 1987 Jan, 206(1), 161 - 8 Mobilization of the non-conjugative plasmid RSF1010: a genetic and DNA sequence analysis of the mobilization region; Derbyshire KM et al.; The entire region required for mobilization of the non-conjugative plasmid RSF1010 has been cloned into a mobilization-deficient pBR322 derivative . The segment of DNA cloned was approximately 1.8 kb and included the origin of conjugal DNA transfer (oriT) . The DNA sequence of the mobilization region has been determined, and revealed the presence of several overlapping reading frames . The isolation and mapping of both Tn1725 and BamH1-linker insertions and comparison with the DNA sequence data has allowed the identification of three genes required for mobilization . Two of these genes are overlapping and encode proteins of 16 kDa and greater than 65 kDa (although the truncated protein is functional, the gene extends outside the region cloned) . The third gene is transcribed in the opposite direction . Promoters capable of transcribing these genes were located by S1 mapping in the inter-cistronic region between these divergently transcribed genes . The oriT site is located in this region, and the transcriptional patterns observed for mob+ and mob- plasmids implied that the promoters may be regulated by two of the mobilization proteins binding to the oriT site. Mol Gen Genet, 1987 Jan, 206(1), 154 - 60 Mobilization of the non-conjugative plasmid RSF1010: a genetic analysis of its origin of transfer; Derbyshire KM et al.; The oriT site of the broad host-range multicopy IncQ plasmid RSF1010 was cloned onto the 2.2 kb pBR322-derived vector pED825 . By successive subcloning and construction of deletions, the oriT region was localised on an 80-88 bp segment of DNA . This segment was contained within the HaeII fragment of RSF1010 that is known to include the relaxation nick site . The oriT region was sequenced and inverted repeats and sequences homologous to the oriT regions of ColE1 and RK2 were identified . A striking 10 bp inverted repeat at one end of the 88 bp oriT segment may be important for recognition of oriT, and its possible role in transfer is discussed . As for other plasmids, the oriT region served as the site for recA-independent, transfer-dependent, site-specific recombination . This provides genetic evidence that strand breakage and re-joining occur at oriT during transfer . Mobilization was independent of transcription by RNA polymerase in the donor cell, as shown by the lack of effect of rifampicin . Inversion of the oriT site with respect to the plasmid oriV site showed that there was no functional dependence of oriT on oriV for synthesis of primers possibly involved in recipient conjugal DNA synthesis . Alternative mechanisms are discussed. Mol Gen Genet, 1987 Jan, 206(1), 141 - 3 Cloning in Escherichia coli of genes involved in the synthesis of proline and leucine in Desulfovibrio desulfuricans Norway; Fons M et al.; A library of Desulfovibrio desulfuricans Norway genomic DNA was constructed in Escherichia coli with pBR322 as vector and plasmids able to complement the proA and leuB mutations of the host were screened . It was observed that all the plasmids studied were highly unstable, the insert DNA being rapidly lost under non-selective growth conditions . A 2.75 kb DNA fragment of D . desulfuricans Norway was found to complement E . coli ProA, ProB and ProC deficiencies . From the results of restriction analysis and Southern hybridizations, it is proposed that the genes involved in proline and leucine biosynthesis are clustered on the chromosome of D . desulfuricans Norway. Mol Gen Mikrobiol Virusol, 1987 Jan, (1), 24 - 6 {The library of Rickettsia prowazekii genes}; Artem'ev MI et al.; The DNA of Rickettsia provazekii strain E was cleaved by PstI restriction endonuclease under the conditions of partial restriction . The fragments were inserted into the PstI site of pBR325 and cloned in this plasmid . E . coli strain HB101 was used as a recipient for cloning . 880 clones sensitive to ampicillin and resistant to tetracycline were selected from 5120 transformants . The cloning of rickettsial DNA has been confirmed by the blot hybridization technique . Analysis of individual and net probes of the hybrid DNA by gel electrophoresis makes it possible to conclude that 90% of the selected clones harbour hybrid plasmids, the size of the cloned fragments rangers from 0.9 to 10.4 Kb, the obtained library of clones contains 70% of the whole genome of Rickettsia provazekii. Mol Cell Biol, 1987 Jan, 7(1), 379 - 87 Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system; DuBridge RB et al.; We developed highly sensitive shuttle vector systems for detection of mutations formed in human cells using autonomously replicating derivatives of Epstein-Barr virus (EBV) . EBV vectors carrying the bacterial lacI gene as the target for mutation were established in human cells and later returned to Escherichia coli for rapid detection and analysis of lacI mutations . The majority of the clonal cell lines created by establishment of the lacI-EBV vector show spontaneous LacI- frequencies of less than 10(-5) and are suitable for studies of induced mutation . The ability to isolate clonal lines represents a major advantage of the EBV vectors over transiently replicating shuttle vectors (such as those derived from simian virus 40) for the study of mutation . The DNA sequence changes were determined for 61 lacI mutations induced by exposure of one of the cell lines to N-nitroso-N-methylurea . A total of 33 of 34 lacI nonsense mutations and 26 of 27 missense mutations involve G X C to A X T transitions . These data provide support for the mutational theory of cancer. Mol Cell Biol, 1987 Jan, 7(1), 26 - 32 A highly conserved endonuclease activity present in Escherichia coli, bovine, and human cells recognizes oxidative DNA damage at sites of pyrimidines; Doetsch PW et al.; We have compared the sites of nucleotide incision on DNA damaged by oxidizing agents when cleavage is mediated by either Escherichia coli endonuclease III or an endonuclease present in bovine and human cells . E . coli endonuclease III, the bovine endonuclease isolated from calf thymus, and the human endonuclease partially purified from HeLa and CEM-C1 lymphoblastoid cells incised DNA damaged with osmium tetroxide, ionizing radiation, or high doses of UV light at sites of pyrimidines . For each damaging agent studied, regardless of whether the E . coli, bovine, or human endonuclease was used, the same sequence specificity of cleavage was observed . We detected this endonuclease activity in a variety of human fibroblasts derived from normal individuals as well as individuals with the DNA repair deficiency diseases ataxia telangiectasia and xeroderma pigmentosum . The highly conserved nature of such a DNA damage-specific endonuclease suggests that a common pathway exists in bacteria, humans, and other mammals for the reversal of certain types of oxidative DNA damage. Genetics, 1987 Jan, 115(1), 51 - 63 Distribution and abundance of insertion sequences among natural isolates of Escherichia coli; Sawyer SA et al.; A reference collection of 71 natural isolates of Escherichia coli (the ECOR collection) has been studied with respect to the distribution and abundance of transposable insertion sequences using DNA hybridization . The data include 1173 occurrences of six unrelated insertion sequences (IS1, IS2, IS3, IS4, IS5 and IS30) . The number of insertion elements per strain, and the sizes of DNA restriction fragments containing them, is highly variable and can be used to discriminate even among closely related strains . The occurrence and abundance of pairs of unrelated insertion sequences are apparently statistically independent, but significant correlations result from stratifications in the reference collection . However, there is a highly significant positive association among the insertion sequences considered in the aggregate . Nine branching process models, which differ in assumptions regarding the regulation of transposition and the effect of copy number on fitness, have been evaluated with regard to their fit of the observed distributions . No single model fits all copy number distributions . The best models incorporate no regulation of transposition and a moderate to strong decrease in fitness with increasing copy number for IS1 and IS5, strong regulation of transposition and a negligible to weak decrease in fitness with increasing copy number for IS3, and less than strong regulation of transposition for IS2, IS4 and IS30. Genetics, 1987 Jan, 115(1), 41 - 9 Local DNA sequence control of deletion formation in Escherichia coli plasmid pBR322; DasGupta U et al.; The specificity of deletion formation was studied using tests involving reversion of palindromic insertion mutations . Insertions of a Tn5-related transposon at 13 sites in the ampicillin-resistance (amp) gene of plasmid pBR322 were shortened to a nested set of perfect palindromes, 22, 32 and 90 bp long . We monitored frequencies of reversion to Ampr, which is the result of deletion of the palindrome plus one copy of the flanking 9 bp direct repeats (which had been formed by transposition) . Revertant frequencies were found to depend on the location and the sequence of the palindromic insert . Changing a 45-kb interrupted palindrome to a 22-bp perfect palindrome stimulated deletion formation by factors of from fourfold to 545-fold among the 13 sites, while elongation of the perfect palindrome from 22 to 90 bp stimulated deletion formation by factors of from eight- to 18,000-fold . We conclude that deletion formation is strongly affected by subtle features of DNA sequence or conformation, both inside and outside the deleted segment, and that these effects may reflect specific interactions of DNA processing proteins with template DNAs. Genetics, 1987 Jan, 115(1), 33 - 40 Amplified RNase H activity in Escherichia coli B/r increases sensitivity to ultraviolet radiation; Bockrath R et al.; Strains of E . coli B/r transformed with the plasmid pSK760 were found to be sensitized to inactivation by ultraviolet radiation (UV) and to have elevated levels of RNase H activity . Strains transformed with the carrier vector pBR322 or the plasmid pSK762C derived from pSK760 but with an inactivated rnh gene were not sensitized . UV-inactivation data for strains having known defects in DNA repair and transformed with pSK760 suggested an interference by RNase H of postreplication repair: uvrA cells were strongly sensitized, wild-type and uvrA recF cells were moderately sensitized and recA cells were not sensitized; and minimal medium recovery was no longer apparent in sensitized uvrA cells . Biochemical studies showed that post-UV DNA synthesis was sensitized and that the smaller amounts of DNA synthesized after irradiation, while of normal reduced size as indicated by sedimentation position in alkaline sucrose gradients, did not shift to a larger size (more rapidly sedimenting) upon additional incubation . We suggest an excess level of RNase H interferes with reinitiation of DNA synthesis on damaged templates to disturb the normal pattern of daughter strand gaps and thereby to inhibit postreplication repair. Circ Shock, 1987, 21(1), 15 - 22 Inhibition of lipid peroxidation improves survival rate of endotoxemic rats; Kunimoto F et al.; The accumulation of lipoperoxide (LPO) is reported to occur in the organs of animals with endotoxemia, where it is accompanied by an activation of xanthine oxidase (XOD) and a depletion of superoxide dismutase (SOD) . In the present study, three measures of preventing LPO accumulation, ie, prior treatment with a XOD inhibitor, exogenous supply of enzymatic scavengers, and supplementation with chemical quenchers, were investigated to determine how to improve the survival rate of rats with lethal endotoxemia . Thirty minutes after treatment with various doses of allopurinol, SOD, catalase (CAT), vitamin E (VE), and reduced glutathione (GSH), adult male Wistar rats were subjected to endotoxemia by an intraperitoneal injection of 0.4 mg/100 g of Escherichia coli endotoxin . Allopurinol did not improve survival rates, denoting a lower level of XOD and an almost normal level of SOD in the liver . SOD (9,000 U/100 g) with or without CAT (4,000 U/100 g) markedly increased the survival rate of rats, with complete inhibition of hepatic LPO accumulation and suppression of XOD activity . CAT alone had no salutary effects on survival rate or hepatic LPO . Large amounts of VE (100 mg/100 g) or GSH (50 mg/100 g) slightly suppressed the accumulation of LPO in the liver but had no effect on survival rate . In that exogenous SOD has been considered not to penetrate the cellular membrane because of its high molecular weight, the results suggest that the extracellular spaces are the site of SOD action . Lipid peroxidation of the biomembrane initiated by oxygen free radicals released into extra-cellular space from phagocytes may play an important role in the development of lethality in experimental endotoxemia. J Gen Virol, 1987 Jan, 68 ( Pt 1), 19 - 38 DNA sequence and genetic content of the HindIII l region in the short unique component of the herpes simplex virus type 2 genome: identification of the gene encoding glycoprotein G, and evolutionary comparisons; McGeoch DJ et al.; The DNA sequence was determined of the HindIII l fragment of herpes simplex virus type 2 (HSV-2), which is located in the short unique region of the HSV-2 genome . HindIII l was found to comprise 9629 base pairs . Comparison with the previously determined corresponding sequence for herpes simplex virus type 1 (HSV-1), and limited mRNA mapping, showed that HindIII l contained six genes (termed US2 to US7) and part of another (US8) . The HSV-1 and HSV-2 sequences were found to be generally colinear, with one major exception: the HSV-2 DNA contained an extra sequence of about 1460 base pairs, in the coding region of gene US4 . By use of an antiserum raised against an oligopeptide representing amino acids near the C terminus of the predicted HSV-2 US4 polypeptide it demonstrated that this gene encodes the virion glycoprotein gG-2, while HSV-1 US4 encodes a much smaller virion glycoprotein with homology to the C-terminal portion of gG-2 . Quantitative comparisons of the HSV-2 HindIII l and corresponding HSV-1 sequences showed that they had diverged by point mutation and by local addition and deletion, as well as by the major change in genes US4 . It was found that within the HSV-2-specific part of gG-2 there was a locality showing sequence similarity to a glycoprotein of pseudorabies virus (gX), and weaker similarity to glycoproteins D of HSV-1 and HSV-2 . These data were interpreted to suggest, first, that HSV-2 US4 represents an ancient gene of alpha herpesviruses, and, more tentatively, that the evolution of the genes for gG and gD may have proceeded through a duplication event. Mutat Res, 1987 Jan, 183(1), 31 - 7 Mutagenic DNA repair in Escherichia coli . XIII . Proofreading exonuclease of DNA polymerase III holoenzyme is not operational during UV mutagenesis; Woodgate R et al.; We have introduced a mutD5 mutation (which results in defective 3'-5'-exonuclease activity of the epsilon proofreading subunit of DNA polymerase III holoenzyme) into excision-defective Escherichia coli strains with varying SOS responses to UV light . MutD5 increased the spontaneous mutation frequency in all strains tested, including recA430, umuC122::Tn5, and umuC36 derivatives . It had no effect on UV mutability or immutability in any strain or on misincorporation revealed by delayed photoreversal in UV-irradiated umuC36, umuC122::Tn5, or recA430 bacteria . It is concluded that the epsilon proofreading subunit of DNA polymerase III holoenzyme is excluded, inhibited, or inoperative during misincorporation and mutagenesis after UV. J Bacteriol, 1987 Jan, 169(1), 66 - 71 The signal sequence suffices to direct export of outer membrane protein OmpA of Escherichia coli K-12; Freudl R et al.; We studied whether information required for export is present within the mature form of the Escherichia coli 325-residue outer membrane protein OmpA . We had previously analyzed overlapping internal deletions in the ompA gene, and the results allowed us to conclude that if such information exists it must be present repeatedly within the membrane part of the protein encompassing amino acid residues 1 to 177 (R . Freudl, H . Schwarz, M . Klose, N . R . Movva, and U . Henning, EMBO J . 4:3593-3598, 1985) . A deletion which removed the codons for amino acid residues 1 to 229 of the OmpA protein was constructed . In this construct the signal sequence was fused to the periplasmic part of the protein . The resulting protein, designated Pro-OmpA delta 1-229, was processed, and the mature 95-residue protein accumulated in the periplasm . Hence, information required for export does not exist within the OmpA protein. J Bacteriol, 1987 Jan, 169(1), 42 - 52 Genetic and molecular characterization of the genes involved in short-chain fatty acid degradation in Escherichia coli: the ato system; Jenkins LS et al.; The structural organization and regulation of the genes involved in short-chain fatty acid degradation in Escherichia coli, referred to as the ato system, have been studied by a combination of classic genetic and recombinant DNA techniques . A plasmid containing a 6.2-kilobase region of the E . coli chromosome was able to complement mutations in the ato structural genes, atoA (acetyl-coenzyme A {CoA}:acetoacetyl {AA}-CoA transferase) and atoB (thiolase II), as well as mutations in the ato regulatory locus, atoC . Complementation studies performed with mutants defective in acetyl-CoA:AA-CoA transferase suggest that two loci, atoD and atoA, are required for the expression of functional AA-CoA transferase . The ato gene products were identified by in vitro transcription and translation and maxicell analysis as proteins of 48, 26.5, 26, and 42 kilodaltons for atoC, atoD, atoA, and atoB, respectively . In vitro and insertional mutagenesis of the ato hybrid plasmid indicated that the ato structural genes were arranged as an operon, with the order of transcription atoD-atoA-atoB . Although transcribed in the same direction as the atoDAB operon, the atoC gene appeared to use a promoter which was distinct from that used by the atoDAB operon . A delta atoC plasmid expressed the atoD, atoA, and atoB gene products only in strains containing a functional atoC gene . Although the exact mechanism of control was not evident from these studies, the data suggest that the atoC gene product is an activator which is required for the synthesis or activation of the atoDAB-encoded enzymes. J Bacteriol, 1987 Jan, 169(1), 386 - 93 Transcription control of the aroP gene in Escherichia coli K-12: analysis of operator mutants; Chye ML et al.; The nucleotide sequence of the region containing the promoter-operator for the aroP gene was determined . The start site of aroP transcription was identified by using S1 nuclease mapping and primer extension techniques . Examination of the nucleotide sequence revealed the presence of two "TYR R" boxes which are similar to those identified in the regulatory regions of other genes in the tyrR regulon . Bisulfite-induced aroP operator-constitutive mutants were analyzed, and the base-pair changes responsible for alterations in aroP regulation were located within these boxes. J Bacteriol, 1987 Jan, 169(1), 380 - 5 Characterization of the specific pyruvate transport system in Escherichia coli K-12; Lang VJ et al.; A mutant of Escherichia coli K-12 lacking pyruvate dehydrogenase and phosphoenolpyruvate synthase was used to study the transport of pyruvate by whole cells . Uptake of pyruvate was maximal in mid-log phase cells, with a Michaelis constant for transport of 20 microM . Pretreatment of the cells with respiratory chain poisons or uncouplers, except for arsenate, inhibited transport up to 95% . Lactate and alanine were competitive inhibitors, but at nonphysiological concentrations . The synthetic analogs 3-bromopyruvate and pyruvic acid methyl ester inhibited competitively . The uptake of pyruvate was also characterized in membrane vesicles from wild-type E . coli K-12 . Transport required an artificial electron donor system, phenazine methosulfate and sodium ascorbate . Pyruvate was concentrated in vesicles 7- to 10-fold over the external concentration, with a Michaelis constant of 15 microM . Energy poisons, except arsenate, inhibited the transport of pyruvate . Synthetic analogs such as 3-bromopyruvate were competitive inhibitors of transport . Lactate initially appeared to be a competitive inhibitor of pyruvate transport in vesicles, but this was a result of oxidation of lactate to pyruvate . The results indicate that uptake of pyruvate in E . coli is via a specific active transport system. J Bacteriol, 1987 Jan, 169(1), 283 - 90 Escherichia coli dnaK null mutants are inviable at high temperature; Paek KH et al.; DnaK, a major Escherichia coli heat shock protein, is homologous to major heat shock proteins (Hsp70s) of Drosophila melanogaster and humans . Null mutations of the dnaK gene, both insertions and a deletion, were constructed in vitro and substituted for dnaK+ in the E . coli genome by homologous recombination in a recB recC sbcB strain . Cells carrying these dnaK null mutations grew slowly at low temperatures (30 and 37 degrees C) and could not form colonies at a high temperature (42 degrees C); furthermore, they also formed long filaments at 42 degrees C . The shift of the mutants to a high temperature evidently resulted in a loss of cell viability rather than simply an inhibition of growth since cells that had been incubated at 42 degrees C for 2 h were no longer capable of forming colonies at 30 degrees C . The introduction of a plasmid carrying the dnaK+ gene into these mutants restored normal cell growth and cell division at 42 degrees C . These null mutants showed a high basal level of synthesis of heat shock proteins except for DnaK, which was completely absent . In addition, the synthesis of heat shock proteins after induction in these dnaK null mutants was prolonged compared with that in a dnaK+ strain . The well-characterized dnaK756 mutation causes similar phenotypes, suggesting that they are caused by a loss rather than an alteration of DnaK function . The filamentation observed when dnaK mutations were incubated at a high temperature was not suppressed by sulA or sulB mutations, which suppress SOS-induced filamentation.(ABSTRACT TRUNCATED AT 250 WORDS) J Bacteriol, 1987 Jan, 169(1), 205 - 9 Reconstitution of pyrroloquinoline quinone-dependent D-glucose oxidase respiratory chain of Escherichia coli with cytochrome o oxidase; Matsushita K et al.; D-Glucose dehydrogenase is a pyrroloquinoline quinone-dependent primary dehydrogenase linked to the respiratory chain of a wide variety of bacteria . The enzyme exists in the membranes of Escherichia coli, mainly as an apoenzyme which can be activated by the addition of pyrroloquinoline quinone and magnesium . Thus, membrane vesicles of E . coli can oxidize D-glucose to gluconate and generate an electrochemical proton gradient in the presence of pyrroloquinoline quinone . The D-glucose oxidase-respiratory chain was reconstituted into proteoliposomes, which consisted of two proteins purified from E . coli membranes, D-glucose dehydrogenase and cytochrome o oxidase, and E . coli phospholipids containing ubiquinone 8 . The electron transfer rate during D-glucose oxidation and the membrane potential generation in the reconstituted proteoliposomes were almost the same as those observed in the membrane vesicles when pyrroloquinoline quinone was added . The results demonstrate that the quinoprotein, D-glucose dehydrogenase, can reduce ubiquinone 8 directly within phospholipid bilayer and that the D-glucose oxidase system of E . coli has a relatively simple respiratory chain consisting of primary dehydrogenase, ubiquinone 8, and a terminal oxidase. J Bacteriol, 1987 Jan, 169(1), 184 - 8 Negative modulation of Escherichia coli NAD kinase by NADPH and NADH; Zerez CR et al.; NAD kinase was purified 93-fold from Escherichia coli . The enzyme was found to have a pH optimum of 7.2 and an apparent Km for NAD+, ATP, and Mg2+ of 1.9, 2.1, and 4.1 mM, respectively . Several compounds including quinolinic acid, nicotinic acid, nicotinamide, nicotinamide mononucleotide, AMP, ADP, and NADP+ did not affect NAD kinase activity . The enzyme was not affected by changes in the adenylate energy charge . In contrast, both NADH and NADPH were potent negative modulators of the enzyme, since their presence at micromolar concentrations resulted in a pronounced sigmoidal NAD+ saturation curve . In addition, the presence of a range of concentrations of the reduced nucleotides resulted in an increase of the Hill slope (nH) to 1.7 to 2.0 with NADH and to 1.8 to 2.1 with NADPH, suggesting that NAD kinase is an allosteric enzyme . These results indicate that NAD kinase activity is regulated by the availability of ATP, NAD+, and Mg2+ and, more significantly, by changes in the NADP+/NADPH and NAD+/NADH ratios . Thus, NAD kinase probably plays a role in the regulation of NADP turnover and pool size in E . coli. Blood, 1987 Jan, 69(1), 43 - 51 Purification and properties of bacterially synthesized human granulocyte-macrophage colony stimulating factor; Burgess AW et al.; Human granulocyte-macrophage colony stimulating factor (GM-CSF) has been synthesized in high yield using a temperature inducible plasmid in Escherichia coli . The human GM-CSF is readily isolated from the bacterial proteins because of its differential solubility and chromatographic properties . The bacterially synthesized form of the human GM-CSF contains an extra methionine residue at position 1, but otherwise it is identical to the polypeptide predicted from the cDNA sequence . The specific activity of 2.9 X 10(7) units/mg of protein for purified bacterially synthesized human GM-CSF indicates that despite the lack of glycosylation, the molecule is substantially in its native conformation . This molecule stimulated the same number and type of both seven- and 14-day human bone marrow colonies as the CSF alpha preparation from human placental conditioned medium . Human GM-CSF had no activity on murine bone marrow or murine leukemic cells . There was no detectable, direct stimulation of adult human erythroid burst forming units (BFU-E) by the bacterially synthesized human GM-CSF . Although impure preparations containing native human GM-CSF (eg, human placental conditioned medium) stimulated the formation of mixed colonies, even in the presence of erythropoietin, the bacterially synthesized human GM-CSF failed to stimulate the formation of mixed colonies from adult human bone marrow cells . The bacterially synthesized human GM-CSF increased N-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced superoxide production and lysozyme secretion . Antibody-dependent cytotoxicity and phagocytosis by human neutrophils was stimulated by the bacterially synthesized human GM-CSF and eosinophils were also activated in the antibody-dependent cytotoxicity assay. J Virol, 1987 Jan, 61(1), 50 - 9 Complete nucleotide sequence of wild-type hepatitis A virus: comparison with different strains of hepatitis A virus and other picornaviruses; Cohen JI et al.; The complete nucleotide sequence of wild-type hepatitis A virus (HAV) HM-175 was determined . The sequence was compared with that of a cell culture-adapted HAV strain (R . Najarian, D . Caput, W . Gee, S.J . Potter, A . Renard, J . Merryweather, G.V . Nest, and D . Dina, Proc . Natl . Acad . Sci . USA 82:2627-2631, 1985) . Both strains have a genome length of 7,478 nucleotides followed by a poly(A) tail, and both encode a polyprotein of 2,227 amino acids . Sequence comparison showed 624 nucleotide differences (91.7% identity) but only 34 amino acid differences (98.5% identity) . All of the dipeptide cleavage sites mapped in this study were conserved between the two strains . The sequences of these two HAV strains were compared with the partial sequences of three other HAV strains . Most amino acid differences were located in the capsid region, especially in VP1 . Whereas changes in amino acids were localized to certain portions of the genome, nucleotide differences occurred randomly throughout the genome . The most extensive nucleotide homology between the strains was in the 5' noncoding region (96% identity for cell culture-adapted strains versus wild type; greater than 99% identity among cell culture-adapted strains) . HAV proteins are less homologous with those of any other picornavirus than the latter proteins are when compared with each other . When the sequences of wild-type and cell culture-adapted HAV strains are compared, the nucleotide differences in the 5' noncoding region and the amino acid differences in the capsid region suggest areas that may contain markers for cell culture adaptation and for attenuation. J Virol, 1987 Jan, 61(1), 167 - 76 Characterization of the simian virus 40 late promoter: relative importance of sequences within the 72-base-pair repeats differs before and after viral DNA replication; Ernoult-Lange M et al.; We examined sequences involved in the simian virus 40 (SV40) late promoter in vivo, by using quantitative S1 nuclease analysis of a series of deletion mutants within the SV40 regulatory region . These mutants were constructed so as to place the altered promoter region in its normal position relative to the SV40 late genes . The effects of the deletions on late transcriptional activity were analyzed before and after viral DNA replication, by omitting or including SV40 large T antigen . The data show that (i) in the absence of large T antigen, the deletion of the 21-base-pair (bp) repeats results in a fourfold increase in late transcription, and (ii) the sequences within the 72-bp repeats are a component of the SV40 late promoter, acting not only before, but also after viral DNA replication . We identified two domains which contain sequences important for efficient late transcription . Domain I, at the late proximal end of each 72-bp repeat, was found to function before replication and was possibly also involved after replication . The contribution of domain II, at the late distal end of each 72-bp repeat, was much more significant after replication but only of minor importance before replication. C R Seances Soc Biol Fil, 1987, 181(5), 506 - 12 {Gene expression of D-beta-hydroxybutyrate dehydrogenase . III . Molecular cloning of a DNAc}; Bailly A et al.; In order to continue the molecular studies of D-beta-hydroxybutyrate dehydrogenase (BDH) undertaken in our laboratory for several years, we have initiated a genetic approach which consists in the BDH cDNA cloning from a rat liver cDNA library . The immunoscreening method allowed to isolate a clone which exhibits a DNA insert shorter than the expected full length BDH cDNA. Gene, 1987, 61(1), 103 - 12 High-level expression of alpha-human atrial natriuretic peptide from multiple joined genes in Escherichia coli; Lennick M et al.; A method is described which allows alpha-human atrial natriuretic peptide to be synthesized in stable form and with high yield in Escherichia coli . In the final expression system, eight copies of the synthetic alpha-hANP gene were linked in tandem, separated by codons specifying a 4-amino-acid (aa) linker with lysine residues flanking the authentic N and C termini of the 28-aa hormone . This sequence was in turn joined to the 3' end of a fragment containing the lac promoter and a leader sequence coding for the first seven N-terminal amino acids of beta-galactosidase . The expressed multidomain protein accumulated intracellularly into stable inclusion bodies and was easily purified by urea extraction of the insoluble cell fraction . The purified protein was cleaved into monomers by digestion with endoproteinase Lys-C, trimmed to expose the authentic C terminus by digestion with carboxypeptidase-B and a single disulfide bond was formed by gentle oxidation with potassium ferricyanide . The fully processed recombinant peptide was shown by reverse phase liquid chromatography to be indistinguishable from the chemically synthesized standard alpha-hANP in both the reduced and in the folded form. Gene, 1987, 60(2-3), 191 - 6 Molecular cloning of cDNA for human prostatic acid phosphatase; Yeh LC et al.; A human liver cDNA library in lambda gt11 was screened with polyclonal antiserum to human acid phosphatase isoenzyme 2a/4 . About eleven positive clones have been obtained . Two clones, lambda Hap21 and lambda Hap22 were further characterized: clone lambda Hap21 contained a 0.8-kb cDNA insert and clone lambda Hap22 a 1.8-2.0-kb insert . XbaI digestion of lambda Hap22 generated two fragments of 1.0 and 0.9 kb . BglII digestion resulted in a 1.2-kb fragment and several smaller fragments of undetermined size . Clone lambda Hap22 contained all the genes carried by lambda gt11(lac5cI857nin5Sam100) and the 2-kb insert . An Escherichia coli(lambda Hap22) lysogen was generated, and its acid phosphatase activity was approximately ten-fold higher than that in the control nonlysogenic lysate . Western-blot analysis of total proteins present in this E . coli(lambda Hap22) lysate revealed that the non-induced lambda Hap22 prophage directed the synthesis of an approx . 175-kDa protein . This protein was recognized by antibody to the human acid phosphatase isoenzyme 2a/4 and anti-beta-galactosidase and was produced only upon induction with IPTG . These results indicated that lambda Hap22 carried a major portion of the gene coding for the human acid phosphatase isoenzyme 2a and/or 4 and this protein fragment of acid phosphatase was sufficient to manifest enzymatic activity. J Basic Microbiol, 1987, 27(5), 263 - 73 Differential suppressor effects of the ssb-1 and ssb-113 alleles on uvrD mutator of Escherichia coli in DNA repair and mutagenesis; Quinones A et al.; We have constructed double mutants carrying either ssb-1 or ssb-113 alleles, which encode temperature-sensitive single strand DNA binding proteins (SSB), and the uvrD::Tn5 allele causing deficiency in DNA helicase II, and have examined sensitivity to ultraviolet light (UV), recombination and spontaneous as well as UV-induced mutagenesis . We have found in a recA+ background that (i) none of the ssb uvrD double mutants was more sensitive to UV than either single mutant; (ii) the ssb-1 allele partially suppressed the strong UV sensitivity of uvrD::Tn5 mutants; (iii) in the recA730 background with constitutive SOS expression, the ssb-1 and ssb-113 alleles suppressed the strong UV-sensitivity caused by the uvrD::Tn5 mutation; (iv) in ssb-113 mutants, the level of recombination was reduced only 10-fold but 100-fold in ssb-1 mutants, showing that there was no correlation between the DNA repair deficiency and the recombination deficiency; (v) the hyper-recombination phenotype of the uvrD::Tn5 mutant was suppressed by the addition of either the ssb-1 or the ssb-113 allele; (vi) no addition of the spontaneous mutator effects promoted by the uvrD::Tn5 and the ssb-113 alleles was observed . These results suggest a possible functional interaction between SSB and Helicase II in DNA repair and mutagenesis. J Hyg Epidemiol Microbiol Immunol, 1987, 31(4), 375 - 80 Screening the viricidal efficiency of antisepsis, disinfection and chemical sterilization--a draft methodology for practice; Bydzovska O; The paper documents the high resistance of E . coli phage phi X 174, one of the small non-enveloped viruses of icosahedral symmetry, vis-a-vis certain physico-chemical factors . It is this property that makes the phage a suitable model for evaluating in practical terms the efficiency of antisepsis, disinfection and chemical sterilization . The phage is detected in smears, prints and on carriers using the plaque method . The system E . coli--phage phi X 174 may serve as a bioindicator of the viricidal efficiency of chemical sterilization eventually of the measure of absorbance of sterilizing agents on the treated material. Gene, 1987, 57(1), 101 - 10 Isolation of a cDNA probe for a human jejunal brush-border hydrolase, sucrase-isomaltase, and assignment of the gene locus to chromosome 3; Green F et al.; We report the nucleotide sequence and derived amino acid sequence of a cDNA clone encoding most of the N-terminal, isomaltase region of human sucrase-isomaltase (SI) . A plasmid containing this cDNA, pS12, identifies a 6-kb mRNA found in human jejunum and the human colon carcinoma cell line Caco-2 . This human SI cDNA shows extensive overall homology with recently published rabbit SI cDNA . Using pS12 to probe DNA from a panel of somatic cell hybrids, we have assigned the gene encoding human SI to chromosome 3. Gene, 1987, 56(1), 145 - 51 Control of cloned gene expression by promoter inversion in vivo: construction of improved vectors with a multiple cloning site and the Ptac promoter; Hasan N et al.; We have constructed three gene-expression plasmids which contain (an) invertible promoter(s) and a multiple cloning site . We used either the plac promoter or the ptac-plac tandem promoters, the latter directing a more than fourfold increase in expression of the galK reporter gene in Escherichia coli host . All these plasmids were derived from the pNH7a expression plasmid of Podhajska et al . {Gene 40 (1985) 163-168} . Like pNH7a, these vectors have three novel properties: (i) in the 'OFF phase', the promoter is facing away from the gene to be expressed, (ii) the 'ON phase' is attained by the rapid and efficient inversion of the promoter mediated by the phage lambda Int product and the flanking attP and attB sites, which have a divergent orientation, and (iii) only a short heat pulse is required for the efficient inversion of the promoter and switching from the OFF to the ON phase . As for the pNH7 a vector, the present plasmids contain the nut-N transcriptional antitermination system, which permits efficient gene expression even if terminator(s) happen to be present between the promoter(s) and the expressed gene . The promoter inversion is rapid and over 95% efficient, as assayed by restriction analysis and galactokinase assay . Many genes could be conveniently cloned in the multiple cloning site, and then either kept totally silent or expressed in a rigidly controlled manner . Moreover, the pNH8, pNH16 and pNH18 plasmids, with already inverted promoters, could be used for expression of cloned genes, either in an unregulated manner or regulated by the lac repressor . They would be particularly useful for genes associated with terminators affecting their expression. Gene, 1987, 54(2-3), 285 - 90 The vitamin D-binding protein gene contains conserved nucleotide sequences that respond to heavy metal, adipocyte and mitotic signals; Yang F et al.; Characterization of the 5'-flanking region of the DBP genomic sequence is reported . Nucleotide sequences that serve as cis-regulatory elements for transcription in other genes have been found and include metal regulatory elements, viral enhancers, adipocyte and mitotic signals . The promoter region of DBP is not homologous to the 5'-flanking region of the albumin and alpha-fetoprotein genes despite the strong protein homology and evolutionary relationship among the three proteins. Gene, 1987, 54(1), 93 - 103 A library of trimethylguanosine-capped small RNAs in Physarum polycephalum; Adams DS et al.; We have constructed a cDNA library for the trimethylguanosine-capped small RNAs (sRNAs) in the acellular slime mold Physarum polycephalum . Capped sRNAs were purified from total cellular RNA of vegetative microplasmodia by preparative immunoprecipitation with anti-trimethylguanosine antibody . The purified RNA was analyzed by polyacrylamide gel electrophoresis . Approx . eleven different capped sRNAs were observed with a size range of 70-204 nucleotides (nt) . Based on their approximate sizes, the presence of trimethylguanosine cap, and the presence of a lupus type-Sm antigen, molecules U1-U7 (excluding U3) were identified . Further confirmation of the identity of molecule U1a was established by Northern hybridization, U4a by colony hybridization, and U6 and U7a by direct chemical sequence analysis . Purified capped sRNAs were tailed with oligo(A), and inserted into oligo(dT)-tailed plasmid pCDV1 . The cDNAs were used to transform Escherichia coli strain HB101 . Approx . 1.9 X 10(5) ampicillin-resistant (ApR) transformants were obtained per microgram of tailed sRNA . Dot-blot hybridization, using Physarum RNA precipitated with anti-cap antibody as a probe, indicated that approx . 94% of the ApR colonies contained recombinant DNAs . The library was screened by colony hybridization using heterologous sRNA probes . Clones hybridizing with heterologous sRNAs U1, U2, U4 and U7 were each represented in the library in approximately the same frequency as their relative abundance in the Physarum sRNA population they were derived from . The insert of one Physarum U4 clone was sequenced and was found to have 57.1% homology with nt 1-91 of the published sequence for rat U4 RNA . A 12-nt 'functional' subdomain of the rat U4 molecule was 83.3% conserved in Physarum U4. Gene, 1987, 52(1), 95 - 101 Human interleukin-1 beta gene; Bensi G et al.; We report the nucleotide sequence of the human chromosomal gene which encodes the interleukin-1 beta protein (IL-1 beta) . The gene spans a region of 7.5 kb and the coding part is divided into seven exons . Comparison with the homologous mouse gene reveals that the structural organization is conserved through evolution . In addition to this, human and murine IL-1 beta genes show extensive sequence homology within the intervening sequences. Gene, 1987, 51(1), 85 - 90 A cos-mini-Mu vector for in vivo DNA cloning; Gramajo HC et al.; Construction of a mini-Mu plasmid vector containing a cosmid replicon is described . Upon derepression of mini-Mu transposition, bacterial DNA sequences can be flanked by the integrated mini-Mu . These sequences can then be packaged into lambda heads by superinfection with a lambda helper phage . Cosmid clones carrying particular bacterial genes can be recovered by selection after infection of appropriate strains with the cosmid transducing lambda lysate . We report here the successful in vivo cloning of several Escherichia coli genes using the transposoncosmid vector. Z Naturforsch {C}, 1987 Jan-Feb, 42(1-2), 93 - 102 PAPS-reductase from Escherichia coli: characterization of the enzyme as probe for thioredoxins; Schwenn JD et al.; PAPS-reductase from Escherichia coli was employed to detect thioredoxins from pro- and eukaryotic organisms . A simple method for the isolation of this enzyme and properties of the enzymatic assay were described . A comparison between thioredoxins detected by the PAPS-reductase and the Fructose-bisphosphatase or NADP malate dehydrogenase was used to assess the validity of the test . The high cross-reactivity of the bacterial enzyme was useful in the purification of heterologous thioredoxins from spinach, Synechococcus, and Saccharomyces cerevisiae. Int Arch Allergy Appl Immunol, 1987, 82(3-4), 392 - 3 Recombinant human IgE; Gould HJ et al.; The gene for a human epsilon chain Fc fragment has been cloned and expressed at a high level in Escherichia coli, and its biological activity in binding to the high-affinity receptors on mast cells and basophils and mediating histamine release has been examined in a variety of assays, including the inhibition of passive cutaneous anaphylaxis in human skin, which was induced by ragweed immunoglobulin IgE antibody and antigen . The positive results obtained in these assays encouraged us to try to analyse the binding site on IgE by site-directed mutagenesis . We describe deletion mutants here that narrow down the binding site on IgE for the mast cell receptor to a stretch of 76 amino acids (residues 301-376 on the ND epsilon chain) spanning the CH2 and CH3 domains . This peptide displays activity in the human skin test indistinguishable from that of a myeloma IgE. Genetics, 1987 Jan, 115(1), 11 - 24 Genetic functions promoting homologous recombination in Escherichia coli: a study of inversions in phage lambda; Ennis DG et al.; We have studied homologous recombination in a derivative of phage lambda containing two 1.4-kb repeats in inverted orientation . Inversion of the intervening 2.5-kb segment occurred efficiently by the Escherichia coli RecBC pathway but markedly less efficiently by the lambda Red pathway or the E . coli RecE or RecF pathways . Inversion by the RecBCD pathway was stimulated by Chi sites located to the right of the invertible segment; this stimulation decreased exponentially by a factor of about 2 for each 2.2 kb between the invertible segment and the Chi site . In addition to RecA protein and RecBCD enzyme, inversion by the RecBC pathway required single-stranded DNA binding protein, DNA gyrase, DNA polymerase I and DNA ligase . Inversion appeared to occur either intra- or intermolecularly . These results are discussed in the framework of a current molecular model for the RecBC pathway of homologous recombination. J Bacteriol, 1987 Jan, 169(1), 117 - 25 Involvement of chlA, E, M, and N loci in Escherichia coli molybdopterin biosynthesis; Johnson ME et al.; All molybdenum enzymes except nitrogenase contain a common molybdenum cofactor, whose organic moiety is a novel pterin called molybdopterin (MPT) . To assist in elucidating the biosynthetic pathway of MPT, two MPT-deficient mutants of Escherichia coli K-12 were isolated . They lacked activities of the molybdenum enzymes nitrate reductase and formate dehydrogenase, did not reconstitute apo nitrate reductase from a Neurospora crassa nit-1 strain, and did not yield form A, a derivative of MPT . By P1 mapping, these two mutations mapped to chlA and chlE, loci previously postulated but never definitely shown to be involved in MPT biosynthesis . The two new mutations are in different genetic complementation groups from previously isolated chlA and chlE mutations and have been designated as chlM and chlN (closely linked to chlA and chlE, respectively) . The reported presence of Mo cofactor activity in the chlA1 strain is shown to be due to in vitro synthesis of MPT through complementation between a trypsin-sensitive macromolecule from the chlA1 strain and a low-molecular-weight compound from the nit-l strain. Vet Res Commun, 1987, 11(6), 509 - 18 Inheritance of K88-mediated adhesion of Escherichia coli to jejunal brush borders in pigs: a genetic analysis; Bijlsma IG et al.; The transmission and genetic organization of the adhesion of the serological variants of the K88 adhesin in the jejunum of the pig were investigated . The results of 28 matings of 5 boars with 15 sows are presented . On the basis of previous studies it has been accepted that the presence of specific receptor sites for K88ab and K88ac depends on a gene locus with 2 alleles S and s . The presence of additional receptor sites for K88ad is now presumed to depend on a separate locus with the alleles D and d . The expression of the alleles of the S and D loci is not always complete and is likely to be influenced by epistatic genes . Inhibition or modification of the expression of the receptor sites for K88 can result in intermediate phenotypes. Microbiol Immunol, 1987, 31(12), 1255 - 8 Hemagglutination by pilus antigen 987P of enterotoxigenic Escherichia coli; Ike K et al.; The hemagglutination (HA) by pilus antigen 987P of an enterotoxigenic Escherichia coli strain 987 was examined using fresh and glutaraldehyde (GA)-fixed erythrocytes (RBC) of various animals . Only when GA-fixed RBC was employed, a strain 987 exhibited striking HA activities . This was also demonstrated by using latex heads sensitized with the 987P antigen . The 987P-specific antiserum inhibited HA of strain 987 and 987P sensitized latex beads against GA-fixed RBC . We concluded that HA of strain 987 against GA-fixed RBC was specifically associated with the presence of 987P pilus antigen but do not exclude a possibility that adhesin is distinct from pili antigen. Enzyme, 1987, 38(1-4), 220 - 6 Molecular aspects of urea cycle enzymes and related disorders; Mori M et al.; Amino acid sequence of rat ornithine transcarbamylase (OTC) precursor containing an NH2-terminal presequence of 32 residues was deduced from the cDNA sequence . Comparison with the human and Escherichia coli enzymes indicated that regions containing the putative binding sites for the substrates are highly conserved among the three species . cDNA clones for rat and human liver arginase were isolated and predicted amino acid sequences were compared with that of the yeast enzyme . There are several regions highly conserved among the three species which may be important for catalysis . Several cases of carbamyl phosphate synthetase I and OTC deficiencies were analyzed for enzyme activity, enzyme amount and mRNA activity. Adv Biophys, 1987, 23, 1 - 37 Molecular biological studies on structure and mechanism of proton translocating ATPase (H+-ATPase, F0F1); Futai M et al.; Recent results on ATPase, mainly from E . coli, obtained by biochemical and molecular biological approaches are reviewed, with special emphasis on results obtained in this laboratory . The advantages of using E . coli in studies of this important enzyme in oxidative phosphorylation are indicated: variant enzymes with specific amino acid replacements can be obtained and their functions and structures can be compared with those of the wild-type enzyme . Structural aspects of this complex enzyme are discussed, including the primary amino acid sequences and molecular assembly of subunits, and mechanistic aspects of the catalytic mechanism and proton translocation. Pathol Immunopathol Res, 1987, 6(2), 103 - 16 Escherichia coli heat-stable enterotoxins and their receptors; Thompson MR; The family of ST has grown to include closely related toxins produced by a number of organisms . The core sequence of these toxins can bind specifically and reversibly to a receptor found in the microvillus membranes of the intestinal cell brush border . As a result of a specific binding event, the ST can stimulate fluid secretion via receptor-mediated stimulation of guanylate cyclase . The structure of the ST molecule has been partially obtained through a variety of techniques . It is apparent that in the near future, key amino acids and sequence regions of these toxins will be found that will identify the requirements for toxicity . The ST are ideal probes to study the secretory system of intestinal cells since they are not cytotoxic . The receptor for ST has been extensively studied and its purification and characterization will yield important insight into the reversal of toxin-mediated diarrheas, and possibly into the nonpathological secretory process . As yet, the nature of the 54,000-57,000 and 68,000-75,000 dalton binding proteins identified by cross-linking studies are not known . When purified ST-binding peptides and other components of the secretory cascade will become available, they will provide better insight into the actual dynamics of the ST receptor-guanylate cyclase complex secretory pathway. Microbiol Immunol, 1987, 31(5), 417 - 26 Incidence and some characteristics of fimbriae FY and 31A of Escherichia coli isolates from calves with diarrhea in Japan; Shimizu M et al.; Escherichia coli isolates from calves with diarrhea (1 day to 8 weeks old, 140 individuals) were surveyed for the three immunologically distinct fimbrial adhesins FY, 31A, and K99 . Of a total of 1,370 strains isolated, 96 (7.0%), 34 (2.5%), 75 (5.5%), and 13 (0.9%) were identified as FY+, 31A+, FY+.31A+, and K99+, respectively . The K99+ strains also manifested heat-stable enterotoxin production (ST+), while FY+, 31A+, and FY+.31A+ strains were ST- . Expression of FY and 31A was repressed at lower temperatures or poor aeration . The FY+ and 31A+ E . coli showed mannose-resistant hemagglutinating activity with bovine erythrocytes . Electron microscopy revealed that FY is a gently curled fimbria with a mean diameter of 4.2 nm, and 31A is a fimbria with a mean diameter of 5.1 nm . The molecular mass of protein subunits was found to be approximately 20 kilodaltons (Kd) and 19 Kd for FY and 31A, respectively . Lethal diarrhea of neonatal calves was induced by challenge with the combination of a K99+.ST+ E . coli strain and either a 31A+ E . coli strain or a 31A+ E . coli strain plus an FY+ E . coli strain under the experimental conditions in which lethal diarrhea was not induced by challenge with a K99+.ST+ E . coli strain alone. Mol Biol Rep, 1987, 12(1), 3 - 6 Microdissection and microcloning of the long arm of human chromosome 7; Kaiser R et al.; DNA-fragments from the region of the long arm of human chromosome 7 to which the CF-locus has been mapped recently were isolated by microdissection and microcloning . We developed a new fixation procedure resulting in inserts of 1.0-7.0 kb in length with a mean value of 2.9 kb . Regional mapping of three clones on 7q was carried out by the use of different hybrid cell lines containing fragments of human chromosome 7. Comp Biochem Physiol A, 1987, 87(4), 1017 - 20 Fever in snails, reflection on a negative result; Cabanac M et al.; 1 . Groups of aquatic snails (Limnaea auricularia) were placed in a temperature gradient and their thermopreferendum measured . 2 . Injected with various amounts of killed Escherichia coli, bacterial endotoxin, human interleukin, and prostaglandin E1, E2 and F2 alpha, they did not develop a fever . 3 . High doses of prostaglandins were toxic . 4 . These results suggest that fever appeared in the course of evolution after the emergence of molluscs and before that of arthropods. Comp Immunol Microbiol Infect Dis, 1987, 10(2), 117 - 24 Occurrence of mannose resistant hemagglutinins in Escherichia coli strains isolated from porcine colibacillosis; Truszczynski M et al.; Three-hundred and fifty-eight E . coli strains isolated from piglets were tested for the presence of hemagglutinins by the use of the active hemagglutination test with or without mannose . Additionally 86 strains from the mentioned number of strains were investigated for the presence of common fimbriae using the same method but growing the strains in media especially suited for the development of this kind of fimbriae . These 358 strains and additionally 202 E . coli strains were tested using antisera for 987P and K88 antigens . It was found, using the active hemagglutination test, that 51.4% of the strains were hemagglutinating . The hemagglutinating strains carried the K88 antigen . All these strains were isolated from new-born and weaned piglets with enterotoxic form of colibacillosis, called also E . coli diarrhea . From cases of this form of colibacillosis originated also 26.7% of the strains in which common fimbriae (type 1) were detected . This result was obtained when the BHI medium was used for cultivation . In case of TSA medium only 2.3% of strains were positive . No specific or common fimbriae were found in strains recovered from septic form of colibacillosis and oedema disease (called also enterotoxaemic form of colibacillosis) . No strain of 560 examined showed the presence of fimbrial 987P antigen. Gene, 1987, 53(2-3), 211 - 7 Nucleotide sequence of the glutamine synthetase gene and its controlling region from the acidophilic autotroph Thiobacillus ferrooxidans; Rawlings DE et al.; A 2089-bp chromosomal DNA segment containing the Thiobacillus ferrooxidans glnA gene has been sequenced . Putative glnAp1-type promoter sequences, a consensus ntrC-gene-product-binding site and a catabolite-activating protein consensus recognition sequence were detected upstream of the structural gene . The glnA gene was followed by a sequence resembling a Rho-independent termination sequence . The complete amino acid sequence (468 residues) of the glutamine synthetase (GS) has been deduced, and comparisons are made with reported amino acid sequences of GS from other organisms. Vet Microbiol, 1987 Jan, 13(1), 65 - 8 Features of enterotoxigenic Escherichia coli strains of porcine origin that express K88 and 987P fimbrial antigens; Suarez S et al.; Escherichia coli strains of porcine origin express K88 and 987P pilus-antigens in vitro . This study reports their enterotoxin producing ability, serological features and plasmid content . The bipiliated strains were enterotoxigenic and all contained a large plasmid of uniform size. J Bacteriol, 1987 Jan, 169(1), 80 - 6 Genes encoding the beta and epsilon subunits of the proton-translocating ATPase from Anabaena sp . strain PCC 7120; Curtis SE; The genes encoding the beta (atpB) and epsilon (atpE) subunits of the ATPase from the cyanobacterium Anabaena sp . strain PCC 7120 were cloned, and their sequences were determined . atpB and atpE are each single-copy genes in the Anabaena genome . The two genes are separated by a 96-base-pair intergenic spacer and transcribed as a single mRNA of 2.3 kilobases that initiates approximately 200 base pairs upstream of the atpB coding region . The predicted translation product of atpB has 81 and 68% amino acid identity with the corresponding proteins from spinach chloroplasts and Escherichia coli, respectively . The atpE gene product is less conserved, with 41 and 33% amino acid identity with the corresponding proteins from spinach chloroplasts and E . coli, respectively . The organization of the Anabaena atpB and atpE genes relative to adjacent genes differs from that of both E . coli and chloroplasts. Infect Immun, 1987 Jan, 55(1), 86 - 92 Identification of a new fimbrial structure in enterotoxigenic Escherichia coli (ETEC) serotype O148:H28 which adheres to human intestinal mucosa: a potentially new human ETEC colonization factor; Knutton S et al.; Three important fimbrial colonization factor antigens (CFAs) designated CFA/I, CFA/II, and E8775 were identified originally in some human enterotoxigenic Escherichia coli (ETEC) strains because of their mannose-resistant hemagglutination properties . To identify CFA, in strains lacking mannose-resistant hemagglutination properties we exploited the ability of human ETEC strains to adhere to human proximal small intestinal mucosa . ETEC strain B7A (O148:H28) was selected for study because it belongs to an epidemiologically important serotype and does not produce a known CFA, and yet it is known to be pathogenic and cause diarrheal disease in human volunteers . Results of an human enterocyte adhesion assay indicated that some bacteria in cultures of B7A produced adhesive factors . To select for such bacteria, cultured human duodenal mucosal biopsy samples were infected with B7A for up to 12 h, after which time a large percentage of the mucosal surface became colonized by bacteria . A new fimbrial structure morphologically distinct from CFA/I, CFA/II, and E8775 fimbriae and consisting of curly fibrils (approximately 3 nm in diameter) was readily identified when bacteria were subcultured from the mucosa and examined by electron microscopy . Identical fimbriae were produced by ETEC strain 1782-77 of the same serotype . Identification of these fimbriae only on bacteria subcultured from human intestinal mucosa strongly suggests that they promote mucosal adhesion of ETEC serotype O148:H28 and thus represent a potentially new human ETEC CFA. Infect Immun, 1987 Jan, 55(1), 78 - 85 Role of plasmid-encoded adherence factors in adhesion of enteropathogenic Escherichia coli to HEp-2 cells; Knutton S et al.; Plasmid-encoded adherence factors have been shown to be important for the full expression of enteropathogenic Escherichia coli (EPEC) pathogenicity and for EPEC adhesion to cultured HEp-2 cells . EPEC strain E2348 (O127) shows localized HEp-2 cell adhesion and possesses a 60-megadalton plasmid, pMAR2 . When E2348 is cured of pMAR2 it loses the ability to adhere to HEp-2 cells, while nonadherent E . coli K-12 strains P678-54 and HB101 acquire HEp-2 adhesiveness after they gain the plasmid . By electron microscopy, E2348 was seen to adhere to HEp-2 cells in a manner that closely resembled EPEC adhesion to intestinal mucosa; bacteria were intimately attached to projections of the apical HEp-2 cell membrane and caused localized destruction of microvilli . The plasmid-containing K-12 strains, on the other hand, did not show intimate attachment and there was no modification of cell surface architecture . It is concluded that plasmid pMAR2 codes for an adhesin, possibly fimbrial in nature, that promotes HEp-2 adhesion but that other chromosomally encoded factors are required for EPEC to achieve the characteristic mode of intimate cell attachment. Infect Immun, 1987 Jan, 55(1), 154 - 61 Cloning of the filamentous hemagglutinin of Bordetella pertussis and its expression in Escherichia coli; Brown DR et al.; Bordetella pertussis UT25 DNA was cloned into the kanamycin resistance gene of cosmid pCP13 to construct a genomic library in Escherichia coli LE392 . One clone containing plasmid pDB441 expressed the filamentous hemagglutinin (FHA) as identified by protein immunoblots with the use of rabbit anti-B . pertussis antiserum, rabbit anti-FHA antiserum, and a monoclonal antibody to FHA . FHA is a protein of 220 to 210 kilodaltons, but the immunoreactive FHA, as expressed in E . coli, was larger than that expressed in B . pertussis, suggesting that there was a difference in the processing of this protein between these two bacteria . The fha gene was mapped to a 6.5-kilobase pair DNA fragment by the use of various restriction endonucleases . The kanamycin resistance gene of pCP13 was found to provide the promoter function but probably not the translation start signal for the fha gene . Conjugative transfer of pDB441 to B . pertussis BP353, a transposon Tn5-induced FHA mutant, increased the expression of the FHA over that seen with wild-type B . pertussis. Ciba Found Symp, 1987, 131, 39 - 51 Human tumour necrosis factors: structure and receptor interactions; Aggarwal BB et al.; Activation of lymphoid and myeloid cells causes the production of factors cytotoxic to various tumour cell types in vitro and in vivo . We have investigated the biochemistry, molecular biology and mechanism of action of two such factors . The factor derived from a myeloid cell line was named TNF-alpha (previously referred to as TNF) and that derived from lymphoid cells named TNF-beta (previously called lymphotoxin) . Both proteins were purified from the conditioned media of the human cell lines and sequenced . Structural information revealed that TNF-alpha is 157 amino acid residues long and contains one disulphide bond . TNF-beta is a glycoprotein of 171 amino acids that contains no cysteine residues . Protein sequence information was used to isolate and characterize cDNAs for TNF-alpha and TNF-beta by recombinant DNA methods . The expression of the cDNAs in Escherichia coli made available large quantities of these proteins for biological studies . The two proteins are 31% identical and 52% homologous to each other . The genes for both cytokines are approximately three kilobases in size and are closely linked on human chromosome six . TNF-alpha and TNF-beta both bind to various cell types via a single class of high affinity receptors . On most cells the same receptor is recognized by both cytokines . The receptors for TNF-alpha can be up-regulated by both interferons and lectins . Up-regulation of receptors by interferons is accompanied by synergistic enhancement of the biological response whereas up-regulation by lectins results in an antagonistic response . Besides antiproliferative effects, both cytokines exhibit direct antiviral effects on infection by both DNA and RNA viruses. Curr Genet, 1987, 11(6-7), 491 - 8 The complex Arom locus of Aspergillus nidulans . Evidence for multiple gene fusions and convergent evolution; Hawkins AR; The physical positions of the DNA sequences encoding the five consecutive enzyme activities required to metabolise 3-deoxy-D-arabino-heptulosonic acid-7-phosphate to 5-enolpyruvyl-shikimate-3phosphate, which are encoded by the A . nidulans Arom polypeptide have been determined . Subfragments of the Arom locus encoding EPSP synthase and 3-dehydroquinase have been expressed in appropriate E . coli aro mutants . The DNA sequence of the A . nidulans Arom locus has been shown to have homology with the corresponding unlinked E . coli aro loci strongly suggesting (I) divergent evolution from common ancestral sequences and (II) that the complex A . nidulans Arom locus arose by multiple gene fusion . The DNA and protein sequence of the two 3-dehydroquinase isoenzymes of A . nidulans share no homology, strongly indicating separate phylogenetic origins and their convergent evolution . The 5' and 3' non-translated DNA sequence of the A . nidulans Arom locus is presented along with the presumed sites for transcription initiation and polyadenylation determined by S1 nuclease protection experiments. Curr Genet, 1987, 12(2), 135 - 9 Identification and isolation of a putative permease gene in the quinic acid utilization (QUT) gene cluster of Aspergillus nidulans; Whittington HA et al.; Mutations in the qutD gene of Aspergillus nidulans cause the loss of ability to grow upon quinic acid as sole carbon source in media at normal pH 6.5 and failure to induce three enzyme activities specifically required for metabolism to protochatechuic acid . All 9 qutD mutants recovered are recessive and have been found to be pH sensitive, growing strongly on quinic acid media at pH 3.5 and producing significant induced enzyme activities . These properties are consistent with the hypothesis that the QUTD gene encodes an essential component of a permease required for transport of quinate ion into mycelium at pH 6.5 . The QUTD gene has been located within the cloned QUT gene cluster of A . nidulans by transformation of qutD mutants with fragments of cloned sequences from phage lambda-Q1 . The QUTD locus is in a region distinct from other QUT genes and which contains sequences homologous to the QA-Y gene in the corresponding QA gene cluster of Neurospora crassa. Acta Microbiol Hung, 1987, 34(3-4), 241 - 5 Elimination of non-specific nucleases from restriction endonuclease preparations by different binding on free DNA ligand; Geck P et al.; In the purification of a novel restriction endonuclease (an AvaIII isoschizomer, isolated in this laboratory) standard methods were insufficient to eliminate non-specific nuclease contaminations . Taking advantage of the specific site recognition and binding of the restriction endonuclease on DNAs, a method is described for the simple extraction of non-specific nucleases . DNA substrates without recognizable sites do not bind the restriction endonuclease, while non-specific nucleases are absorbed to, and eliminated with, the DNA via gel filtration chromatography under special conditions. Proteins, 1987, 2(4), 283 - 9 Electrostatic calculations and model-building suggest that DNA bound to CAP is sharply bent; Warwicker J et al.; Two observations suggest that DNA, upon binding to E . coli catabolite gene activator protein (CAP), is sharply bent by a total angle of at least 100-150 degrees: (1) The electrostatic potential field of CAP shows regions of positive potential that form a ramp on 3 sides of the protein . (2) The DNA binding site size as determined by DNA ethylation interference with binding, (Majors: "Control of the E . coli Lac Operon at the Molecular Level." Ph.D . Thesis, Harvard University, Cambridge, 1977) and by relative affinities of DNA fragments of various lengths (Liu-Johnson et al.: Cell 47:995-1005, 1986) requires severe bending of the DNA to maintain its favorable electrostatic contact with the protein. Proteins, 1987, 2(4), 273 - 82 Clustering of null mutations in the EcoRI endonuclease; Yanofsky SD et al.; EcoRI endonuclease mutants were isolated in a methylase-deficient background following in vitro hydroxylamine mutagenesis of plasmid pKG2 (Kuhn et al.: Gene 44:253-263, 1986) . Mutants which survived high-level endonuclease expression (IPTG induction) were termed null mutants . Sixty-two of 121 null mutants tested by Western blot contained normal levels of endonuclease cross-reacting protein . The complete endonuclease gene was sequenced for 27 null mutants . This group was found to consist of 20 single base-change missense mutations, 6 double mutations, and 1 triple mutation . Ten of the 20 single mutations were clustered between residues 139 and 144 . When examined with respect to the structure of the EcoRI-DNA complex (McClarin et al.: Science 234:1526-1541, 1986), these alterations were found to fall predominantly into two classes: substitutions at the protein-DNA interface or substitutions at the protein-protein (dimer) interface . Protein from several of the mutants was purified and sized by using HPLC . Wild-type EcoRI endonuclease and protein from three of the DNA interface mutations (Ala139----Thr, Gly140----Ser, Arg203----Gln) appeared to be dimeric, while protein from subunit interface mutations (Glu144----Lys, Glu152----Lys, Gly210----Arg) migrated as monomers. Gene, 1987, 61(3), 277 - 89 Characterization of the Escherichia coli modified cytosine restriction (mcrB) gene; Ross TK et al.; The McrB restriction system of Escherichia coli K-12 is responsible for the inactivation of 5-methylcytosine-containing DNA . The mcrB mutation of E . coli strain K802 was complemented by hybrid plasmid pUC9-14 which consists of a 5.5-kb Bg/II-Eco RI fragment from the E . coli K-12 chromosome cloned in pUC9 (Ross and Braymer, 1987) . The limits of the mcrB gene within the 5.5-kb insert were defined by deleting portions the fragment and assaying for McrB restriction of M . AluI-methylated DNA . A 51-kDa polypeptide was identified as the mcrB gene product based on an analysis of maxicell-labeled polypeptides from pUC9-14 and deletion derivatives of this plasmid . Deletion analyses and transcription initiation assays enabled us to determine the direction of transcription and translation of mcrB . Transcription initiates approx . 710 bp beyond the end of the hsdS gene, and proceeds in the same direction as the transcription of the hsdR, hsdM, and hsdS genes, which is clockwise on the conventional E . coli map. Gene, 1987, 61(1), 91 - 101 Artificial transposable elements in the study of the ends of IS1; Prentki P et al.; We have constructed artificial IS1-based transposons by attaching synthetic oligodeoxynucleotides, corresponding to the sequence of the ends of IS1, to a selectable DNA segment {'omega' fragment; Prentki and Krisch, Gene 29 (1984) 303-313} . These transposons were used to examine the sequence requirements at the ends for IS1 transposition . We show here that a 24- to 28-bp sequence from the left or right ends of IS1 is capable of transposition when present at both ends of the omega fragment in the correct orientation . Transposition activity requires the presence of an intact IS1 in cis on the same plasmid molecule . In trans, however, neither resident genomic copies of IS1, nor copies carried by a compatible, high-copy-number plasmid present in the same cell, complement the artificial transposons efficiently . Transposition frequencies in the presence of a cis-complementing IS1 are, however, similar to those of the naturally occurring IS1-based transposon, Tn9 . In addition, transposition results in a 9-bp duplication in the target DNA molecule as is usually the case for insertion of the intact IS1 . Using this system, we have obtained evidence indicating that the activity of a synthetic IS1 end is not determined exclusively by its sequence, but can be strongly enhanced by a second, wild-type end used in the transposition event . The data also show that single base pair mutations can exhibit a cumulative effect in reducing transposition activity. Gene, 1987, 61(1), 21 - 30 Precise nucleotide sequence modifications with bidirectionally cleaving class-IIS excision linkers; Mormeneo S et al.; Bidirectionally cleaving blunt-ended DNA linkers have been constructed to generate defined nucleotide sequence modifications . The oligodeoxynucleotides (termed 'excision linkers'), contain two back-to-back recognition sites for class-IIS restriction endonucleases and provide a new instrument for modifying DNA primary structure . Following insertion of these linkers into host DNA, digestion with the cognate class-IIS enzyme results in a cleavage upstream and downstream from the adjoining enzyme recognition sites . Bidirectional cleavage efficiency can be improved by including spacer nucleotides between the two recognition sites . The number of nucleotides removed from or added to the host DNA depends upon the cleavage shift characteristic of the class-IIS enzyme, the design of the linker (including lateral spacer nucleotides to set the cleavage position), and the method used to make blunt ends from staggered ends following excision of the linker . BspMI linkers constructed in this study have been used to generate defined deletions in the ApR and TcR genes of pBR322 . BsmI excision linkers are also described. Gene, 1987, 60(2-3), 183 - 9 Purification of hepatitis B virus gene X product synthesized in Escherichia coli and its detection in a human hepatoblastoma cell line producing hepatitis B virus; Chisaka O et al.; A fused gene containing 94% of the hepatitis B virus (HBV) open reading frame X was expressed in Escherichia coli, and its 17-kDa product was purified by ion-exchange chromatography . Antibody elicited against the X-gene product reacted with materials proximal to the nuclear membrane of a human hepatoblastoma cell line producing HBV particles . No such reaction was observed with the same cell line that did not produce HBV particles. Gene, 1987, 59(2-3), 291 - 6 Genetic organization of insertion element IS2 based on a revised nucleotide sequence; Ronecker HJ et al.; We identified a transposable element resident in the chromosome of Escherichia coli K-12 strain HB101 . This is an approx . 4400-bp-long transposon flanked by two copies of insertion sequence (IS) 1 element in direct orientation . One of the IS1 elements was found to be integrated into an IS2 element between IS2 bp 139 and bp 140 with the large moiety of IS2 within the transposon . The sequence of this part of IS2 differs from the published sequence of galOP-308::IS2 at a number of positions . Restriction analysis of the published allele, however, indicated that both alleles may in fact be identical . Since six of the eight differences found alter open reading frames, the revised sequence presents a new outlook for the potential genetic organization of IS2. Gene, 1987, 59(2-3), 253 - 63 Organisation of the regulatory region of the Escherichia coli melibiose operon; Webster C et al.; The regulatory region of the Escherichia coli melibiose operon contains two divergent promoters . One promoter is responsible for the expression of the melR gene, that is essential for melibiose-dependent stimulation of the second promoter . Melibiose-induced transcription from this second promoter initiates at a start point 25 bp upstream from the start codon of the melA gene, encoding an alpha-galactosidase . The nucleotide sequence covering the divergent promoters and the melR gene is reported. Gene, 1987, 58(2-3), 305 - 9 Deletion of a repetitive extragenic palindromic (REP) sequence downstream from the structural gene of Escherichia coli glutamate dehydrogenase affects the stability of its mRNA; Merino E et al.; A deletion that removes one repetitive extragenic palindromic sequence downstream of the structural gene of Escherichia coli glutamate dehydrogenase, reduces twofold the half-life of gdhA mRNA. Gene, 1987, 58(2-3), 217 - 28 Isolation and structure of the replicon of the promiscuous plasmid pCU1; Kozlowski M et al.; Evidence is presented to indicate that a PvuII fragment of approx . 2 kb isolated from the 39-kb IncN-group plasmid pCU-1 contains all plasmid-borne determinants for stable maintenance as an extrachromosomal element in Escherichia coli K-12 . The fragment was sequenced . The features of this sequence include a group of 13 direct tandem repeats of 37 bp and a second group of two other direct repeats of 30 bp flanking a third partial member of this group . In addition, for a 19-bp sequence that overlaps a member of this second group, there are inverted repeats that straddle the members of the first group . There are three open reading frames within the fragment . We compare features of this sequence with that of other plasmid replicons and draw attention to similar and to dissimilar features. Gene, 1987, 58(2-3), 201 - 16 Plasmid construction by homologous recombination in yeast; Ma H et al.; We describe a convenient method for constructing new plasmids that relies on interchanging parts of plasmids by homologous recombination in Saccharomyces cerevisiae . A circular recombinant plasmid of a desired structure is regenerated after transformation of yeast with a linearized plasmid and a DNA restriction fragment containing appropriate homology to serve as a substrate for recombinational repair . The free ends of the input DNA molecules need not be homologous in order for efficient recombination between internal homologous regions to occur . The method is particularly useful for incorporating into or removing from plasmids selectable markers, centromere or replication elements, or particular alleles of a gene of interest . Plasmids constructed in yeast can subsequently be recovered in an Escherichia coli host . Using this method, we have constructed an extended series of new yeast centromere, episomal and replicating (YCp, YEp, and YRp) plasmids containing, in various combinations, the selectable yeast markers LEU2, HIS3, LYS2, URA3 and TRP1. Gene, 1987, 57(1), 89 - 99 Construction and characterization of plasmid and lambda phage vector systems for study of transcriptional control in Escherichia coli; Hirano M et al.; We constructed a family of lambda phage and plasmid vectors which facilitate cloning and quantitative analysis of transcriptional regulator in both single and multiple copies . Their expression system was modified from the ara-trp-lac fusion operon of plasmid pMC81 {Casadaban and Cohen, J . Mol . Biol . 138 (1980) 179-207}, which is designed to assay both promoters and terminators with a single vehicle . To eliminate transcriptional and translational polar effects liable to occur in the original fusion operon upon insertion of a foreign nucleotide sequence, intracistronic Rho-dependent terminators, that are present within the trpB gene and distal to the cloning site were deleted, and DNA spacers containing stop codons were introduced immediately before and after the cloning site . In analysis of the cloned trp regulatory region, the lambda phage system faithfully reproduced the tight regulation by tryptophan characteristic to the natural trp operon on the E . coli chromosome, whereas the plasmid counterpart exhibited a substantially relaxed response . Comparative studies on the relative strengths of various promoters and terminators have further demonstrated that the lambda phage vector system permits accurate assays of exceptionally strong promoters like Ptrp and lambda pL without disturbing the bacterial growth, while being sensitive enough for detecting low-level transcription under the control of weak promoters or potent terminators . Cloning with the lambda phage vector can be greatly facilitated by transferring the target regulatory site precloned with the plasmid onto the phage genome through in vivo recombination. Gene, 1987, 57(1), 11 - 9 Simultaneous deletion of the intervening sequences from the human interferon-gamma gene by oligodeoxynucleotide-directed mutagenesis; Shirai T et al.; Oligodeoxynucleotide (oligo)-directed mutagenesis was used to simultaneously delete the three intervening sequences (IVS) from the human interferon-gamma (IFN-gamma) gene . Prior to the deletion experiment, the two largest IVS of the native gene were shortened by restriction enzyme digestion and subsequent ligation (IVS-1 was reduced from 1239 bp to 399 bp and IVS-3 from 2425 bp to 183 bp) . This modified gene was cloned into vector M13mp9 . Three oligos {21-25 nucleotides (nt) long} were synthesized, one to bridge each of the three introns . A different set of three oligos was made for use as hybridization probes to identify colonies containing the correct deletion . A study was made of enzymatic reaction and primer annealing conditions needed to optimize intron deletions . The efficiency of the simultaneous deletions was higher than the product of the efficiencies of the deletions of the individual IVS, suggesting that the deletion events are not independent . The gene created by the simultaneous deletion was cloned into an Escherichia coli plasmid expression vector and IFN-gamma activity was produced. Nucleic Acids Symp Ser, 1987, (18), 233 - 6 Chemical-enzymatic synthesis, cloning and expression of a synthetic gene coding for an env protein fragment of the human T-cell leukemia virus type 1; Rosenthal A et al.; A synthetic gene for a 88 amino acid long env protein fragment of the human T-cell leukemia virus type 1 (HTLV1) has been assembled by ligation of 35 oligodesoxyribonucleotides, which were chemically synthesized by the phosphotriester segmental support method . After cloning into the pEX vector this HTLV1 env-protein fragment was expressed in E . coli. Circ Shock, 1987, 23(4), 263 - 9 Quantitative measurement of endotoxin in canine plasma using the new endotoxin-specific chromogenic test; Ikeda T et al.; Endospecy (a lyophilized mixture of factor G-free limulus coagulation enzymes and chromogenic substrate, Boc-Leu-Gly-Arg-pNA) coupled with modified perchloric acid (PCA) pretreatment was carried out for the quantitative measurement of endotoxin in canine plasma . The endotoxin recovery from normal canine plasma was 99.9 +/- 7.7% (n = 20) . The full recovery of endotoxin illustrated the applicability of the modified PCA pretreatment to the Endospecy in removal of interfering factors in a canine plasma . The normal canine plasma endotoxin level was less than 3.0 pg.ml-1 when Escherichia coli 0111:B4 endotoxin was used as a reference . The canine plasma endotoxin levels were markedly high(1-40 ng.ml-1) at 5 min after intravenous administration of 25 micrograms.Kg-1 (total 150-200 micrograms). Virchows Arch A Pathol Anat Histopathol, 1987, 412(1), 11 - 6 Acute, massive, haemorrhagic adrenal necrosis experimentally produced by the Shwartzman mechanism in rabbits; Aoyama H et al.; Acute and severe haemorrhagic necrosis of the adrenal was produced experimentally in rabbits by means of intravenous injection of endotoxin after pretreatment by adrenocorticotropic hormone (ACTH) administration . The change occurred mainly in the zona fasciculata of the adrenal cortex, and its pathology was quite similar to that of the Shwartzman reaction . Numerous microthrombi were found in and around the lesion, but no marked changes were seen in other parts of the body . Heparin administration was very effective in preventing the necrosis . The pathogenesis of this lesion was postulated to be a univisceral Shwartzman mechanism in the adrenal . This seems to be a good experimental model for massive haemorrhagic necrosis of the adrenal in man, for example in the Waterhouse-Friderichsen syndrome, the pathogenesis of which has been assumed to involve intravascular clotting . It is suggested that hyperfunction of the adrenal cortex caused by ACTH administration could be a preparative condition for the Shwartzman reaction. Gene, 1987, 56(1), 13 - 27 A genetic system for isolation and characterization of TaqI restriction endonuclease mutants; Barany F; The gene encoding TaqI restriction endonuclease has been subcloned downstream from an inducible phoA promoter . Certain strains of Escherichia coli remain viable when endonuclease is expressed, even in the absence of (protective) methylation . Infecting lambda phage DNA is not restricted in vivo . One E . coli strain, MM294, exhibited a temperature-sensitive phenotype when TaqI endonuclease was induced . This allowed for design of an in vivo plate assay for identification of specially constructed two-codon insertion mutants in the endonuclease gene . These mutants exhibited a wide range of in vitro activities, including wild-type activity, greater activity in low-salt buffer, and sequence-specific nicking activity. Gene, 1987, 56(1), 117 - 24 Transformation of Aspergillus based on the hygromycin B resistance marker from Escherichia coli; Punt PJ et al.; A new, heterologous, dominant marker for selection of Aspergillus transformants is described . This marker is based on the Escherichia coli hygromycin B (HmB) phosphotransferase gene (hph) . Expression of the hph gene is controlled by A . nidulans gpd and trpC expression signals . An Aspergillus transformation vector was constructed which contains this marker and confers HmB resistance to Aspergillus species . With both A . niger and A . nidulans, transformation frequencies of 5-20 transformants per micrograms vector DNA were obtained . Cotransformation with other vectors was shown to be very efficient in both species, when selection for HmB resistance was applied. Eksp Onkol, 1987, 9(4), 14 - 8 {Possible molecular mechanisms of limited or infinite division of cells}; Fradkin GE; A new approach to the possible molecular mechanisms which determine the ability of cells to the infinite or limited division is presented . The ideas proposed are based on the experimentally demonstrated premise that the realization of DNA reparation and replication depends on the maintenance of a strict balance between the growth rate of replication forks and SSB-protein synthesis within the cells . The limited ability of cells to the division is considered to be a result of the disorganization of DNA reparation and replication processes when the above-mentioned balance is disturbed . The infinite ability of cells to the division is interpreted as a result of the absence of the disorganization of the above-mentioned processes with disturbance of the same balance . The molecular mechanisms which determine either the presence or absence of the cell response to the disturbed balance between the growth rates of replication forks and SSB-protein synthesis are discussed in detail . The conclusion is drawn that the ssb-gene is a protooncogene converted into an oncogene as a result of the dotted mutation manifesting in the functional inactivation of the third domain of the SSB-proteins. Circ Shock, 1987, 23(2), 85 - 92 Effects of Escherichia coli lipopolysaccharide on the phosphoinositide metabolism and serotonin secretion in thrombin-activated platelets; Moscat J et al.; Lipopolysaccharide treatment of platelets in basal conditions does not produce any effect on serotonin secretion or on phosphatidic acid synthesis or arachidonic acid release . However, a brief exposure of platelets to endotoxin enhances the thrombin-stimulated activation of these parameters, whereas a more prolonged treatment with lipopolysaccharide impairs thrombin action . The results presented here also suggest that very short-term treatment of platelets with lipopolysaccharide activates the inositol 1,4,5-trisphosphate 3-kinase . The long-term effects of endotoxin could be mediated by protein kinase C activation. Arch Geschwulstforsch, 1987, 57(4), 275 - 81 Expression of simian virus 40 small t antigen in Escherichia coli and purification of the antigen; Ikeda S et al.; A simian virus 40 (SV 40) DNA fragment encoding small t antigen was cloned in expression vector pUC8 for the purification of the antigen . The SV40 Hind III B fragment was inserted into the Hind III site of pUC8 . A plasmid having the lacZ' and small t antigen genes in the same orientation was designated as pSVt . pSVt encodes the entire small t antigen and an extra 18 amino acids at the amino terminus of the antigen . E . coli transformed with pSVt produced hybrid small t antigen (22 kDa) which comprised about 6% of the total protein . The hybrid small t antigen reacted in immunoblot analysis with SV40-induced tumor-bearing hamster serum . Hybrid small t antigen was extracted from E . coli, and purified by preparative SDS-PAGE . The antigen was extracted from the gel using formic acid solution with high-yield . The gel-purified antigen showed the same antigenic reactivity as crude antigen. Mol Biol Rep, 1987, 12(2), 111 - 6 Alu-family repeat binding protein from HeLa cells which interacts with regulatory region of SV40 virus genome; Perelygina LM et al.; Using a gel retardation assay the protein which binds selectively to the Alu-family repeat (AFR) has been identified and partially purified from HeLa cell nuclear extract . The protein (AFR-binding protein, ABP) forms multiple discrete complexes with AFR even in the presence of 200 to 2000-fold excess of non-specific (E . coli) DNA . The most stable complex has a relative mobility in 4% polyacrylamide gel (as compared to the free Alu-fragment) of 0.54 . Heterogeneity of protein-DNA bands seen in the polyacrylamide gel suggests that ABP is able to form multimeric complexes with AFR . Competition experiments show that ABP does not interact with the RNA polymerase III promoter and with the TGGCA-sequence, but a high affinity binding site for ABP was found within a 660 bp restriction fragment containing the SV40 virus promoter and replication origin. Gene, 1987, 55(2-3), 157 - 67 Characterization of the terminal sequences flanking the transposon that carries the Escherichia coli enterotoxin STII gene; Hu ST et al.; The Escherichia coli enterotoxin STII gene is flanked by two repeat sequences, approx . 600 bp each and 8 kb apart . This 9-kb DNA fragment has been shown to transpose as a unit and is thus considered a transposon . It is presently designated as Tn4521 . In this study, the two terminal sequences of Tn4521 cloned in pPS1 were localized, isolated, and characterized . The two terminal sequences were found to be composed of IS2 sequences and were in an inverted repeat orientation . However, neither repeat contained a complete IS2 . The LTR contained bp 1-722, whereas the RTR contained bp 17-536 and 969-1327, all three of the IS2 sequence. J Gen Microbiol, 1987 Jan, 133 ( Pt 1), 119 - 26 Molecular cloning and isolation of a cyanobacterial gene which increases the UV and methyl methanesulphonate survival of recA strains of Escherichia coli K12; Geoghegan CM et al.; The unicellular cyanobacterium Gloeocapsa alpicola contains both photoreactivation and excision repair mechanisms for correcting UV-induced damage to its cellular DNA . An 11.5 kb EcoRI fragment was isolated from a cosmid bank of G . alpicola and was shown to complement a recA deletion in Escherichia coli S.17 and JC10289 . These recA strains showed increased survival to UV and methyl methanesulphonate (MMS) when transformed with the cyanobacterial DNA fragment, and also showed filamentation in response to UV irradiation . Preliminary analysis of the protein encoded by the cyanobacterial DNA fragment indicated a major protein of 39,000 Da; this is very similar in size to the recA protein of E . coli. J Cell Sci Suppl, 1987, 6, 215 - 23 Molecular signal for induction of the adaptive response to alkylation damage in Escherichia coli; Sedgwick B; Exposure of Escherichia coli to simple alkylating agents, such as methylnitrosourea, causes the induction of at least three DNA repair functions that are under the control of the ada gene . The ada gene product itself repairs several O-methylated lesions in DNA, including methylphosphotriesters and the mutagenic adduct O6-methylguanine . The methyl groups are transferred from these lesions on to two different cysteine residues within the Ada protein resulting in self-methylation . We have found that the Ada protein is converted to an activator of expression of genes involved in the adaptive response after accepting a methyl group from a methylphosphotriester, but not from O6-methylguanine . This was shown using the in vitro techniques of DNA-dependent protein synthesis and run-off transcription . Delayed electrophoretic migration and footprinting experiments have shown that the methylated activator of transcription binds to specific DNA sequences immediately upstream from the RNA polymerase binding sites in the promoter regions of the inducible genes . The Ada protein-binding sites contain the common sequence d(A-A-A-N-N-A-A-A-G-C-G-C-A). J Cell Sci Suppl, 1987, 6, 177 - 89 Molecular studies on the nature of the repair defect in ataxia-telangiectasia and their implications for cellular radiobiology; Debenham PG et al.; We have utilized DNA transfer and recombinant DNA techniques to probe DNA double-strand break repair in the human ionizing radiation-sensitive genetic syndrome ataxia-telangiectasia (A-T) . Using restriction enzyme-generated double-strand breaks in the coding sequence of a selectable gene we have detected a significantly greater frequency of mis-repair of such breaks in a permanent A-T cell line compared with cell lines of normal radiosensitivity . This mis-repair in A-T can plausibly explain many of the clinical features of the disease but was insufficiently detailed to address the broad problem of DNA repair mechanisms relevant to ionizing radiation-induced damage . To extend these observations of DNA double-strand break mis-repair we have now applied this type of repair assay to novel, de novo induced mammalian X-ray-sensitive cell lines and to appropriate Escherichia coli mutants . In both cellular systems we have now found some equivalence to the A-T repair defect . In particular, studies on one E . coli mutant have provided evidence suggesting an involvement of a topoisomerase activity in DNA double-strand break mis-repair, which may be relevant to the biochemical defect in A-T. J Cell Sci Suppl, 1987, 6, 1 - 23 The molecular biology of nucleotide excision repair of DNA: recent progress; Friedberg EC; Recent years have witnessed significant progress towards understanding the molecular mechanism of nucleotide excision repair in living cells . Biochemical studies in Escherichia coli, and genetic and molecular studies in lower and higher eukaryotes have revealed an unexpected complexity suggesting interesting protein-protein and protein-DNA interactions . This review considers selected aspects of nucleotide excision repair in E . coli, Saccharomyces cerevisiae and mammalian cells, with a particular emphasis on new observations and on models that may provide explanations for the complexity evident from genetic and biochemical studies. Gene, 1987, 54(2-3), 239 - 45 High level expression of the EcoP1 modification methylase gene and characterisation of the gene product; Hornby DP et al.; We have cloned the gene coding for the EcoP1 modification methylase in an expression system based on the phage lambda pL promoter and the cI857-coded thermoinducible repressor . We have used this system to purify the enzyme on the 20-30-mg scale and have examined some of its enzymatic properties . The enzyme is a tetramer of Mr 72,000 subunits and is approx . 40% alpha-helical . Experiments with the methyl donor, S-adenosyl methionine, radioactively labelled in different positions indicate that a methyl group is transferred to the enzyme during the reaction in what is most likely a covalent bond. Gene, 1987, 54(2-3), 229 - 38 The use of a selectable FokI cassette in DNA replacement mutagenesis of the R388 dihydrofolate reductase gene; Vermersch PS et al.; FokI, a class-IIS restriction endonuclease, cleaves double-stranded DNA to produce a protruding 5' end consisting of four nucleotides, 10-13 residues 3' from the nonpalindromic recognition sequence, GGATG . Cassettes which utilize this separation of cleavage and recognition site have been constructed for the purpose of linker mutagenesis and DNA replacement experiments . The cassettes are flanked by FokI recognition sequences oriented such that the FokI cleavage sites are several nucleotides beyond the cassette/vector fusion sites . FokI excises the cassette and several base pairs of the neighboring vector sequence . The ends produced in the vector by FokI cleavage are generally noncomplementary and suitable for the insertion of a segment of synthesized double-stranded replacement DNA . A cassette which contains a tyrosine tRNA suppressor gene (supF) is selectable by the suppression of amber mutations in the recipient host . A vector containing a pBR322-derived origin of replication, the Escherichia coli xanthine-guanine phosphoribosyl transferase gene as a selectable marker, and no FokI sites has been constructed for use with the FokI cassettes . An experiment which utilized the FokI/supF cassette to modify the N-terminal coding region of the R388 dihydrofolate reductase gene is described. Gene, 1987, 54(2-3), 221 - 8 Formation of MboII vectors and cassettes using asymmetric MboII linkers; Gayle RB 3rd et al.; Class-IIS restriction endonucleases such as MboII cleave DNA at a specified distance away from their recognition sequences . This feature was exploited to cleave DNA at previously inaccessible locations by preparing special asymmetric linker/adapters containing the MboII recognition sequence . These could be joined to DNA fragments and subsequently cleaved by MboII . Attachment of a 3' phosphate to one of the two different oligodeoxynucleotides comprising the asymmetric duplex prevented ligation at the improper end of the linker . Plasmids were constructed containing a unique BamHI or BclI site between the recognition and cleavage site of MboII . These sites were used to introduce a foreign fragment into the plasmid at a position permitting MboII to cleave within the newly inserted fragment . Once cleaved at the unique MboII site, another DNA fragment was inserted . DNA was thus inserted at a sequence not previously accessible to specific cleavage by a restriction enzyme . A cassette containing an identifiable marker, the lac operator, between two oppositely oriented MboII/BamHI linkers was made and tested in a random insertion linker mutagenesis experiment. Gene, 1987, 54(2-3), 175 - 84 Analysis of alpha-factor secretion signals by fusing with acid phosphatase of yeast; Sidhu RS et al.; In yeast, Saccharomyces cerevisiae, the PHO5 gene encodes the repressible acid phosphatase (APase) whose activity can be easily monitored by either the staining of colonies or by colorimetric assay . Therefore, gene fusions to PHO5 provide a convenient system for structural and functional analysis of yeast genes . We have constructed fusions of the PHO5 gene with a MF alpha 1 gene of yeast to delineate the secretion signal(s) in the alpha-factor leader peptide . Gene fusion between MF alpha 1 and PHO5 codes for a hybrid protein in which the alpha-factor leader peptide of 89 amino acids (aa) directed the export of APase, a periplasmic protein, into the medium . Since the hybrid gene is transcribed from the alpha-factor promoter, expression of the APase activity from these hybrid genes showed cell type-specific regulation . Further analyses of another MF alpha 1-PHO5 fusion showed that only the first 22 aa of the 89-aa alpha-factor leader peptide contained sufficient information for the secretion of APase into the medium . This shows that, in addition to the analysis of gene regulation, PHO5 fusions can be used to study signals involved in the proper localization of proteins. Acta Microbiol Hung, 1987, 34(1), 19 - 24 Nourseothricin (streptothricin) inactivated by a plasmid pIE636 encoded acetyl transferase: nature of the inactivated nourseothricin; Seltmann G et al.; Nourseothricin, a mixture of several streptothricins, is inactivated by an acetyl transferase produced by Escherichia coli containing the plasmid pIE636 . Nourseothricin inactivated in the presence of 14C-acetate was purified and submitted to partial hydrolysis . In the hydrolysate besides others a radioactive and ninhydrin-reactive substance moving only slightly towards the cathode was found . It proved to be {14C}-acetyl beta-lysine. Vet Med Nauki, 1987, 24(4), 55 - 8 {Total protein, urea and blood sugar in healthy calves and calves with diarrhea}; Abdelmalek B; Studied was the amount of the total protein, the urea, and the blood sugar in both normal calves and calves that were affected with enteritis with frequent isolates of pathogenic Escherichia coli strains and corona and rotaviruses . Stated are the normal values of these indices in calves aged from 1 to 6 days as well as from 7 to 15 days . It was found that with diarrhea-affected calves there were hypoproteinaemia, rise of urea, and very commonly encountered drop of blood sugar in the course of the diseases. Mem Inst Oswaldo Cruz, 1987, 82 Suppl 4, 111 - 4 GP38, P28-I and P28-II: candidates for a vaccine against schistosomiasis; Pierce RJ et al.; Three antigens protective against Schistosoma mansoni have been extensively characterized . The schistosomulum surface antigen GP38 possesses an immunodominant carbohydrate epitope of which the structure has been defined . Protection can be achieved via the transfer of monoclonal antibodies recognizing the epitope or by immunization with anti-idiotype monoclonal antibodies . The glycan epitope is shared with the intermediate host, Biomphalaria glabrata as well as being present on other molluscs, including the Keyhole Limpet . A group of molecules at 28 kDa were initially characterized in adult worms and shown to protect rats and mice against a challenge infection . One of these molecules, P28-I, was cloned and expressed in E . coli, yeast and vaccinia virus . The recombinant antigen significantly protected rats, hamsters and baboons against a challenge infection . P28-I is a glutathione-S-transferase and the recombinant antigen produced in yeast exhibits the enzyme activity and has been purified to homogeneity by affinity chromatography . A second P28 antigen, P28-II, has also been cloned, fully sequenced and expressed . This recombinant antigen also protects against S . mansoni infection. Curr Genet, 1987, 11(6-7), 529 - 36 Unexpectedly long 14S ribosomal RNA gene in Tetrahymena mitochondria; Labriola J et al.; Extraction of RNA from Tetrahymena mitochondrial ribosomes yields several RNA species, including a "large" 21S molecule, a "small" 14S molecule, a 7S molecule, and other smaller RNAs . The molecular weight of the 14S rRNA indicates that it is about 1,300 bases in length . We have sequenced the 14S rRNA gene and, by aligning our sequence with that of the corresponding small rRNA from E . coli, find that the 14S rDNA is at least 1,635 bases in length . We propose, based on the results of hybridization studies, that this unexpected length is due to the presence of 7S RNA sequence within the 14S gene sequence . The 7S region is apparently lost from the 14S rRNA, yet is still a component of the ribosome. Curr Genet, 1987, 11(5), 369 - 75 LEU2 directed expression of beta-galactosidase activity and phleomycin resistance in Yarrowia lipolytica; Gaillardin C et al.; The nucleotide sequence of a 968 bp DNA fragment spanning the promoter and the 5' upstream sequence of the LEU2 coding sequence of the yeast Yarrowia lipolytica has been determined . A LEU2::lacZ fusion has been constructed and expressed in transformed yeast cells, showing that as few as 232 bp of the LEU2 promotor were sufficient to direct gene expression . In order to develop new markers for transformation of this yeast, the LEU2 initiation codon was destroyed by in vitro mutagenesis and replaced by a cloning site . A gene conferring phleomycin resistance in E . coli was attached to the LEU2 promoter and shown to be efficiently expressed in yeast: direct selection of phleomycin resistant transformants was possible. Acta Microbiol Hung, 1987, 34(3-4), 219 - 24 Adsorption to Al(OH), gel of Escherichia coli is correlated with O and K antigens and with type of extraintestinal infection; Czirok E et al.; Living suspensions of 89 Escherichia coli strains were tested for adsorption to Al(OH)3 gel in the presence of phosphate ions . On the basis of AC50 (phosphate molarity inhibiting 50% adsorption of the strain examined), E . coli strains could be classified into two main groups . Forty-three strains belonged to group 1 (AC50, 0.01-0.04), and 42 of them fell into serogroups O1, O2, O5, O7, O18ac, O83 or were spontaneously agglutinable . One strain in group 1 was exceptional as it had antigen O4 . Of these 43 strains 33 had K antigen K1 . Serogroup distribution of 46 group 2 strains (AC50, 0.001-0.009) was O2, O4, O6, O18ac, O75 and O78; 20 out of 46 possessed antigen K5 . No correlation existed between H antigens or haemagglutinating capacity and AC50 of the strains . A close correlation was shown between AC50 pattern and the two main pathogenecity groups (i.e . "newborns' meningitis" and "sepsis and organotropic diseases") on one hand and between AC50 pattern and O, K serotypes on the other . The findings indicate that these E . coli strains with identical markers had a clonal connection. Gene, 1987, 61(1), 41 - 50 Transcription efficiency along the tissue-plasminogen-activator cDNA gene in Escherichia coli; Rothstein DM et al.; Transcription along the human tissue-plasminogen activator gene (tpa) cloned in Escherichia coli was studied by inserting the 4.5S RNA gene at various locations within tpa, and measuring the accumulation of 4.5S RNA . A six-fold decrease in transcription of 4.5S RNA was observed as the distance from the Ptrc promoter to the 4.5S RNA gene increased . Independent measurements indicated that the quantity of initiations from the Ptrc promoter could not account for the differences observed . Because the 4.5S RNA is stable, the decrease in transcription was assumed to be due to premature transcription-termination within the tpa gene . This polar effect could contribute to poor expression of tpa in E . coli. Gene, 1987, 59(1), 145 - 50 Use of outer membrane protein PhoE as a carrier for the transport of a foreign antigenic determinant to the cell surface of Escherichia coli K-12; Agterberg M et al.; PhoE protein is an abundant transmembrane protein from the Escherichia coli K-12 outer membrane . A synthetic oligodeoxynucleotide corresponding to an antigenic determinant of the C-terminal part of the VP1 protein of foot-and-mouth disease virus was inserted into the phoE gene in an area corresponding to a cell surface-exposed region of the PhoE protein . The level of expression of the hybrid protein was normal and the protein was incorporated into the outer membrane . The VP1-epitope was exposed at the cell surface since intact cells were recognized by a monoclonal antibody which was raised against the virus. Circ Shock, 1987, 23(3), 215 - 23 Endotoxin-induced hypotension in rats is not mediated by prekallikrein activation; Kaufmann U et al.; To test whether endotoxin decreases blood pressure acutely in rats by activating the plasma kinin-forming system, plasma kallikrein activity was determined in different experimental settings of endotoxemia . Conscious normotensive rats were infused for 45 min with endotoxin (LPS E . coli 0111:B4) at a dose (0.01 mg/min) which had no effect on blood pressure . Additional rats were infused with the vehicle of endotoxin . Plasma prekallikrein activity was measured at the end of the 45 min infusions . In other rats, a bolus intravenous injection of endotoxin (2 mg) was administered following the 45 min infusion of endotoxin or its vehicle . In these two latter groups of rats, plasma prekallikrein activity was determined 15 min after administration of the bolus dose of endotoxin . In rats pretreated with the endotoxin infusion, the bolus dose of endotoxin had no significant effect on blood pressure, whereas rats infused with the vehicle became and remained hypotensive up to the end of the experiment . There was however no significant difference in plasma prekallikrein activity within the different groups of rats . In another group of rats, dextran sulfate (0.25 mg i.v.), which activates factor XII and thereby the conversion of prekallikrein to kallikrein, induced a short-lasting fall in blood pressure . 15 min after administration of dextran sulfate, plasma prekallikrein activity was almost completely suppressed . These results obtained in unanesthetized rats strongly suggest that the blood pressure fall induced by E . coli endotoxin is not due to activation of prekallikrein and consequently of the kinin-forming system. Folia Haematol Int Mag Klin Morphol Blutforsch, 1987, 114(4), 480 - 1 Membrane properties and aggregation of human red blood cells after endotoxin treatment in vitro; Meier W et al.; Endotoxin treatment of blood in vitro (10(-11) mg/RBC, 30 min at 30 degrees C) leads to a significant increase of the membrane elastic shear modulus mu and membrane viscosities as well as of the aggregation index AI and of the velocity of doublet formation. Microbiol Immunol, 1987, 31(8), 809 - 20 Characterization of murine monoclonal antibodies to human interferon-gamma (IFN-gamma) and their application for sandwich enzyme-linked immunosorbent assay (ELISA); Jitsukawa T et al.; The three murine monoclonal antibodies (MAb), D1G2, D9D10, and D13C8, are specific for human interferon-gamma (IFN-gamma), but not human IFN-alpha and IFN-beta . They react weakly with heat-treated IFN-gamma . The three antibodies recognize different epitopes of the IFN-gamma molecule, as evaluated by antibody-binding inhibition experiments . We have used these three monoclonal antibodies to construct a sandwich enzyme-linked immunosorbent assay (ELISA) . The best result was obtained when we used D1G2 or D9D10 MAb as a solid-phase immunosorbent and D1G2 or D9D10 MAb as a tracer . When we measured IFN-gamma in sera by a combination of D1G2 (a solid-phase) and D1G2 (a tracer), a result similar to the one by a combination of D9D10 (a solid-phase) and D1G2 (a tracer), was obtained . This may suggest that human IFN-gamma exists in oligomeric form . Recombinant human IFN-gamma expressed in E . coli is detectable at a concentration of 1 ng/ml in this sandwich ELISA . This assay can be employed for the analysis of the structural characteristics of the human IFN-gamma molecule as well as measurement of IFN-gamma in human sera and tissue culture fluids. Gene, 1987, 56(2-3), 301 - 7 A rapid and improved method for generating cDNA libraries in plasmid and phage lambda vectors; Sartoris S et al.; We have developed a fast and efficient procedure for generating cDNA libraries in plasmid or phage lambda vectors . We used Mo-MuLV reverse transcriptase to synthesize the first strand and directly added Escherichia coli DNA polymerase I with RNase H to synthesize the second strand . A special advantage of our procedure is the use of oligodeoxynucleotide adapters to insert the cDNA into the vector, avoiding the use of methylating enzymes and subsequent digestion with massive amounts of restriction endonucleases . This also obviates the need to tail the cDNA molecules with homopolymers, simplifying subsequent procedures such as sequencing or transfer to other vectors . Finally, we have used a rapid screening procedure to isolate full-length clones with oligodeoxynucleotide probes recognizing conserved regions at the 5' termini of the mRNA . The system is ideal for cloning and analyzing polymorphic alleles of genes, such as those of the major histocompatibility complex. Gene, 1987, 56(2-3), 289 - 95 Expression of human papillomavirus type 6 E1, E2, L1 and L2 open reading frames in Escherichia coli; Thompson GH et al.; Open reading frame (ORF) fragments (putative gene fragments) from human papillomavirus type 6b (HPV-6b) were inserted into the bacterial expression vector pHK413 to provide viral antigenic determinants . Approximately 86% of the entire L1 ORF, 82% of the E2 ORF, and 52% of the L2 ORF were expressed in Escherichia coli . The E1 ORF was cloned as two fragments . The constructions containing E1n (coding for the N-terminal region) and E1c (coding for the C-terminal region) expressed 27% and 16% of the E1 ORF, respectively . Protein encoded by the L1 ORF, but not that encoded by the L2 ORF, reacted with antibodies elicited by disrupted bovine papillomavirus . These reagents will be extremely useful in unravelling the HPV-6b replication cycle. Comp Biochem Physiol B, 1987, 87(4), 961 - 7 The human Mr 90,000 heat shock protein and the Escherichia coli Lon protein share an antigenic determinant; Latchman DS et al.; 1 . A monoclonal antibody (TG7A) reacts with a Mr 90,000 mammalian protein, accumulating during virus infection and heat shock . 2 . This protein is encoded by a member of the Mr 90,000 heat shock gene family present in a range of organisms form yeast to man . 3 . The antibody also recognises a Mr 94,000 protein in E . coli which similarly accumulates in virus infection and heat shock . 4 . This protein has been identified as the Lon protease of E . coli . 5 . The shared epitope and similar stress inducibility of the two proteins suggests that a functional and/or evolutionary relationship exists between them. Circ Shock, 1987, 22(4), 291 - 301 Coagulation and fibrinolytic reactions in experimental porcine septic shock: pretreatment with different antiplatelet factors; Svartholm E et al.; The aim of this study was to investigate the influence on various hemostatic factors (alpha 2-macroglobulin, antiplasmin, antithrombin III, prothrombin-proconvertin activity, fibrinogen concentration, ethanol gelation test, and fibrinolytic activity on fibrin plates) of bacteremic shock in swine and the influence on these factors of drugs interfering with platelet function . Anesthetized pigs were given live Escherichia coli intravenously (n = 49) or Ringer's solution (n = 7) and were monitored for 3 hours . Pretreatment was given with indomethacin (n = 6), the TxA2 inhibitor UK 38 485 (n = 7), the prostacyclin analogue ZK 36 374 (n = 7), the 5HT antagonist ketanserin (n = 6), or ketanserin combined with UK 38 485 (n = 9) or dipyridamole (n = 8) . Septic shock developed in all E . coli animals . There were decreased levels of platelets and leukocytes and activation of the coagulation/fibrinolytic systems by E . coli . Except for a slight attenuating effect on the antithrombin III (ketanserin and dipyridamole) and alpha 2-macroglobulin (ketanserin) decreases, there were no significant effects of the drugs . It is concluded that live E . coli induced several changes within the coagulation and fibrinolytic systems . Only minor effects were seen when different drugs influencing platelet function were given. Acta Microbiol Pol, 1987, 36(1-2), 67 - 72 Penetration of nalidixic acid into Escherichia coli K-12 cells; Hrebenda J et al.; Transport of nalidixic acid (NAL) into Escherichia coli cells subjected to osmotic shock, permeabilised with toluene or treated with DNP, CCCP or EDTA, was studied . It was found that osmotic shock and protonophores do not inhibit the transport of {3H}NAL, however, the transport of {3H}DAP and {3H}glucose is reduced . EDTA and toluene enhance penetration of {3H}NAL . This effect is, however, abolished in the presence of Mg++ ions . It is suggested that NAL penetrates into the cell by simple or facilitated diffusion and that the outer membrane of E . coli is the penetration barrier for the drug. Acta Microbiol Pol, 1987, 36(1-2), 17 - 28 Two distinct types of mutations conferring to Escherichia coli K12 capability of D-tryptophan utilization; Wild J et al.; We showed that the ability of Escherichia coli K12 tryptophan auxotrophs to utilize D-tryptophan as a substitute for L-tryptophan may result from two types of mutations . The first type consisted in changes in the dadR regulatory site of the dad operon increasing the synthesis of D-amino acid dehydrogenase . The mutations of the second type mapped within the dad A structural gene . They changed the apparent substrate specificity of D-amino acid dehydrogenase . We suppose that the change may be due to an altered enzyme structure which make it more accessible to D-tryptophan. Vox Sang, 1987, 52(1-2), 79 - 82 Serological characterisation of a mouse monoclonal anti-P-like antibody; Inglis G et al.; An IgM mouse monoclonal antibody, LM 147/328, raised against an Escherichia coli immunogen, was found to possess anti-P-like specificity . It is proposed that this antibody recognises an epitope on a Gal beta 1-3GalNAc disaccharide structure (GalNAc = N-acetyl-D-galactosamine) . The haemagglutinating activity of this antibody was consistently strong with adult erythrocytes, but gave significantly weak reactions with about 7% of cord erythrocyte samples. Folia Haematol Int Mag Klin Morphol Blutforsch, 1987, 114(1), 78 - 83 The dose response of granulocyte-macrophage colony stimulating activity (GM-CSA) for sera of "Swiss" mice after endotoxin "lipopolysaccharide B" Escherichia coli (0127: B8) intraperitoneal administration; Chrostek ME et al.; The effect of different doses of endotoxin (Lipopolysaccharide B) on Granulocyte-Macrophage Colony Stimulating Activity (GM-CSA) in the sera of "Swiss" male mice was measured by granulocyte-macrophage "in vitro" colony formation . The highest GM-CSA was found to be present 3-4 hours after 200 micrograms of endotoxin intraperitoneal injection. Mol Biol (Mosk), 1987 Jan-Feb, 21(1), 93 - 101 {Photoaffinity modification of Escherichia coli ribosomes by fMet-tRNAf Met derivatives in the 70S initiation complex}; Babkina GT et al.; Photoaffinity labeling of E . coli ribosomes within the 70S initiation complex was studied by using photoreactive derivatives of fMet-tRNAfMet bearing arylazidogroups scattered statistically over guanosine residues . It is shown that fMet-azido-tRNAfMet-II bearing 2 moles of the reagent residues per mole of tRNA (modified in the conditions of stability of tRNA tertiary structure) is fully active in aminoacylation and in the factor-dependent binding with ribosomes to form the 70S initiation complex . Functional activity of fMet-azido-tRNAfMet-I bearing also 2 moles of the reagent residues per mole of tRNA (but modified in conditions of lability of tRNA tertiary structure) decreases up to approximately 45% in aminoacylation and up to 70% in IF-2 X GTP-dependent binding to the ribosomes . Irradiation of complexes 70S ribosome-MS2-RNA-fMet-azido-tRNAfMet results in covalent linking of the tRNA derivative to the ribosomes . Both subunits are labeled, the 30S to a larger extent than 50S . It is shown that fMet-azido-tRNAfMet-II labels proteins S1, S7, S9, L27 whereas fMet-azido-tRNAfMet-1--proteins S1, S3, S5, S9, S14, L1, L2, L7/L12. Mol Biol (Mosk), 1987 Jan-Feb, 21(1), 73 - 86 {Effectiveness of expression of the chloramphenicol acetyltransferase gene controlled by foreign regulator regions in Escherichia coli cells . II . Molecular cloning of promoters}; Mashko SV et al.; The trpOP, lacUV5, tacOP, PR and PL-promotors were cloned in the previously obtained pML4 vector plasmid . The expression of structural gene cat was studied by the chloramphenicolacetyltransferase determination in cell extracts . The level of protein synthesis by appropriate recombinant plasmids was analysed in vivo and in vitro . It was shown that the efficiency of the gene expression is determined by both the "strength" of the promotors and mRNA translation specificity . The obtained collection of the plasmids might be used for the determination of the promotor strength by the hybridization of pulse-labeled mRNA with DNA and an effective expression of the genes by means of "hybrid protein gene" and "hybrid operon" constructions. Mol Biol (Mosk), 1987 Jan-Feb, 21(1), 229 - 41 {Possible functional role of the DD-domain of RNA-dependent polymerases}; Zavriev SK et al.; An attempt to study the functional role of one of the most conservative domains found in all RNA-dependent RNA and DNA polymerases of plant and animal viruses (the so called "DD-domain") was made . A structure similar to the "DD-domain" was found in a minor T7 phage tail protein--gpII . Antibodies against this phage protein have been raised and used to probe "DD-domain" in molecules of avian myeloblastose virus reverse transcriptase and E . coli RNA-dependent RNA polymerase . The antibodies are shown to inhibit the activity of these enzymes under certain conditions . At the same time inhibition of the reverse transcriptase reaction causes the decrease in length of the most high molecular cDNA-products as well . The experimental data obtained are discussed in view of the suggested hypothesis on the probable functional role of the "DD-domain" of RNA-dependent polymerases. Bioorg Khim, 1987 Jan, 13(1), 69 - 81 {A new approach to DNA synthesis from synthetic oligonucleotides . Synthesis of DNA coding for the repeated antigenic determinant of foot and mouth disease virus}; Korobko VG et al.; A rapid method for assembly of DNA from synthetic oligodeoxynucleotides has been developed which involves separate ligation of top- and bottom-strand oligonucleotides followed by filling in 3'-ends of the duplex formed, blunt end cloning into a specialized vector pBBV, and recovery of the synthetic DNA from the recombinant plasmid by means of restriction nuclease BbvII . The method allows for many oligonucleotides to be ligated at once, with no intermediates being isolated, and any DNA to be recovered on cloning, no matter what the sequences of its termini are . Ten oligodeoxynucleotides (I)-(X) have been chemically synthesised and used to prepare, by this method, a 60-membered duplex with complementary tetranucleotide 5'-protrusions (DNA I) which comprises the cDNA sequence 3397-3456 of foot and mouth disease virus (FMDV) strain O1K . Self-ligation of the duplex in the head-to-tail manner yielded 120 to 900 bp long synthetic DNAs (DNA II-DNA XV) coding for oligomers of the major antigenic determinant (the amino acid sequence 141-160 of protein VP1) of FMDV . The synthetic hexamer (DNA VI) was fused to gene lacZ' on plasmid pBBV21 and expressed in E . coli . The fusion was found to complement the lacZ deletion M15, from which it follows that the fused protein associated with the alpha-deficient beta-galactosidase to yield a tetramer carrying, on its N-termini, 24 antigenic determinants of FMDV. J Oral Pathol, 1987 Jan, 16(1), 27 - 30 Differential expression of Class II (DR & DQ) antigens by human gingival Langerhans' cells and keratinocytes in vitro; Walsh LJ et al.; The expression of the Class II products DR and DQ on human gingival epithelium was examined using immunofluorescence and immunoperoxidase staining . Differential expression of Class II antigens was seen in chronic gingivitis in adults, with T6(+) DR(+) cells being more numerous than T6(+) DQ(+) cells . The periodontopathic organism Fusobacterium nucleatum (FN) induced DQ expression on Langerhans' cells (LC) during in vitro explant culture of gingival tissue . This effect was mimicked by endotoxin (LPS) from F . nucleatum and by E . coli LPS . These results indicate that differential expression of Class II products, a feature of chronic gingival inflammation, may result from the action of LPS on gingival LC. Biokhimiia, 1987 Jan, 52(1), 138 - 41 {Effect of 8-Br-ATP and 8-Oxy-ATP on RNA synthesis by RNA polymerase from Escherichia coli}; Kuriavyi VV et al.; The effect of 8-Br-ATP and 8-oxy-ATP on RNA synthesis on calf thymus DNA and on abortive synthesis of di- and trinucleotides on promoter AI of phage T7 delta DIII DNA in the case of an incomplete set of substrates was studied . It was shown that the ATP analogs used inhibit the RNA and di- and trinucleotide synthesis . In all cases, 8-oxy-ATP was a more effective inhibitor than 8-Br-ATP . Both analogs are incapable of being the primer and they do not replace ATP in the course of abortive initiation of pppApU synthesis. Infect Immun, 1987 Jan, 55(1), 258 - 62 Brucella outer membrane lipoprotein shares antigenic determinants with Escherichia coli Braun lipoprotein and is exposed on the cell surface; Gomez-Miguel MJ et al.; In an enzyme-linked immunosorbent assay (ELISA), purified Brucella abortus and Escherichia coli peptidoglycan-linked lipoproteins gave a strong cross-reaction with sera from rabbits hyperimmunized with the heterologous lipoprotein . When smooth E . coli cells were used as ELISA antigens, the immunological cross-reaction was not observed unless the cells were treated to remove lipopolysaccharide and other outer membrane components . In contrast, intact cells from smooth strains of B . abortus and Brucella melitensis bound anti-lipoprotein immunoglobulin G, and the controls performed by ELISA showed that this reaction was not due to antibodies to the lipopolysaccharide, group 3 outer membrane proteins, or porins . Electron microscopy of cells labeled with antilipoprotein serum and protein A-colloidal gold showed specific labeling of smooth cells from both B . abortus and B . melitensis, even though unspecific labeling by nonimmune serum was observed with rough B . abortus . These results confirm the close similarity between E . coli and Brucella peptidoglycan-linked lipoproteins and show that, in contrast to E . coli, the lipoprotein of B . abortus and B . melitensis is partially exposed on the surface of smooth cells. Biochem Biophys Res Commun, 1986 Dec 30, 141(3), 1099 - 103 Molecular cloning and expression in Streptomyces lividans of a proteinous alpha-amylase inhibitor (HaimII) gene from Streptomyces griseosporeus; Saito S et al.; The gene encoding a proteinous alpha-amylase inhibitor (HaimII) of Streptomyces griseosporeus YM-25 has been cloned in Escherichia coli K12 using a deoxyinosine-containing synthetic oligonucleotide as the probe . A 1.6 kilobases BamHI fragment was confirmed to hybridize with the probe and subcloned in an E . coli-S . lividans shuttle vector . The plasmid clone was transferred into S . lividans by transformation . An appreciable amount of alpha-amylase inhibitor activity was found in the culture medium of S . lividans harboring the plasmid . As the specificity was indistinguishable from that of HaimII produced by the original S . griseosporeus strain, we concluded that the HaimII protein was synthesized in S . lividans and excreted into the medium. Biochem Biophys Res Commun, 1986 Dec 30, 141(3), 986 - 92 Regulation of the nah and sal operons of plasmid NAH7: evidence for a new function in nahR; You IS et al.; The catabolism of naphthalene and salicylate is specified by two operons on an 80 Kb metabolic plasmid, NAH7 . These operons, nah and sal, are carried on the contiguous 30 Kb EcoRI-A, C fragments, and are under positive control of a regulator region, nahR . Five Nah Sal Tn5 insertion mutants form two complementation groups: A = nahR203, nahR204; and B = nahR201, nahR202, nahR205 . The physical and genetic maps assign the nahR location to the 15.7-17.2 Kb region of the EcoRI-A fragment, with suggestion of more than one control gene. Biochem Biophys Res Commun, 1986 Dec 30, 141(3), 1162 - 9 Maintenance of autonomous genetic elements in twelve transgenic mouse strains established after transfer of pPyLT1 DNA; Leopold P et al.; Maintenance and efficient meiotic segregation of autonomous genetic elements in four transgenic mouse strains established after micro-injection of plasmid pPyLT1 were previously reported . These findings are now extended to a total of twelve independent transgenic families . In spite of extensive rearrangements, half of these plasmids maintained the region of pBR322 necessary for shuttle transfer in E . coli . They all included mouse DNA sequences and the same, or closely related sequences were present in distinct mouse strains. J Chromatogr, 1986 Dec 26, 371, 353 - 60 Characterization of the reactivity of sulphydryl groups in tryptophanase by a dual-monitoring high-performance liquid chromatographic system with a site-directed fluorescent reagent; Honda T et al.; Sulphydryl groups of E . coli tryptophanase (L-tryptophan indole lyase, E.C . 4.1.99.1) were made to react with a fluorescent maleimide derivative, N-(4-anilino-1-naphthyl)maleimide(ANM) . By carefully controlling the reaction conditions it was possible to limit the extent of sulphydryl group modification . The modified enzyme was digested with (L-1-tosylamide-2-phenylethyl chloromethyl ketone)-trypsin . The fluorescent peptides obtained were analysed by reversed-phase high-performance liquid chromatography on a C18 column with a dual-monitoring system consisting of a UV and a fluorescence monitor connected in tandem . This was followed by the determination of the amino acid composition of the fluorescent peptides . Comparison of these results with the known, complete primary structure of tryptophanase from the K-12 strain of E . coli allowed the assignment of position 298 to the cysteine residue, which is more selectively modified by ANM under the conditions chosen and is involved in the maintenance of the catalytic activity. Cell, 1986 Dec 26, 47(6), 985 - 94 Transfer RNA shields specific nucleotides in 16S ribosomal RNA from attack by chemical probes; Moazed D et al.; Binding of tRNAPhe to ribosomes shields a set of highly conserved nucleotides in 16S rRNA from attack by a combination of structure-specific chemical probes . The bases can be classified according to whether or not their protection is strictly poly(U)-dependent (G529, G530, U531, A1408, A1492, and A1493) or poly(U)-independent (A532, G693, A794, C795, G926, 2mG966, G1338, A1339, U1381, C1399, C1400, and G1401) . A third class (A790, G791, and A909) is shielded by both tRNA and 50S ribosomal subunits . Similar results are obtained when the protecting ligand is tRNAPhe E . Coli, tRNAPhe yeast, tRNAPhe E . Coli lacking its 3' terminal CA, or the 15 nucleotide anticodon stem-loop fragment of tRNAPhe yeast . Implications for structural correlates of the classic ribosomal A- and P-sites and for the possible involvement of 16S rRNA in translational proofreading are discussed. J Biol Chem, 1986 Dec 25, 261(36), 16976 - 83 Cloning and sequence analysis of mRNA for mouse aspartate aminotransferase isoenzymes; Obaru K et al.; The nucleotide sequences of mRNAs for the mouse mitochondrial and cytosolic aspartate aminotransferase isoenzymes (mAspAT and cAspAT) (EC 2.6.1.1) were determined from complementary DNAs . The mAspAT mRNA comprises minimally 2460 nucleotides and codes for a polypeptide of 430 amino acid residues corresponding to the precursor form of the mAspAT (pre-mAspAT) . The cAspAT mRNA comprises minimally 2086 nucleotides and codes for a polypeptide of 413 amino acid residues . The region coding for the mature mAspAT and that for the cAspAT show about 53% overall homology . The former shares 49% and the latter 48% of homology, respectively, with that of the Escherichia coli aspC gene, which has been shown to code for the E . coli AspAT (Kuramitsu, S., Okuno, S., Ogawa, T., Ogawa, H., and Kagamiyama, H . (1985) J . Biochem . (Tokyo) 97, 1259-1262) . When the deduced amino acid sequence of the mouse pre-mAspAT was compared with that of the pig pre-mAspAT polypeptide, we found that they share a 94% homology and that the mouse pre-mAspAT yields a presequence consisting of 29 amino acid residues and a mature mAspAT, consisting of 401 amino acid residues . These numbers and the amino acid residues present at the putative cleavage site are all in complete agreement in these two species . The deduced amino acid sequence of the mouse cAspAT shares 91% homology with that of the pig cAspAT . Comparisons of the nucleotide and deduced amino acid sequences between the mouse and E . coli AspATs suggest that the mammalian mAspAT gene is more closely related to the E . coli aspC gene than is the mammalian cAspAT gene. J Biol Chem, 1986 Dec 25, 261(36), 17163 - 9 Assembly of the mitochondrial membrane system . Characterization of COR1, the structural gene for the 44-kilodalton core protein of yeast coenzyme QH2-cytochrome c reductase; Tzagoloff A et al.; The respiratory deficiency of yeast strains previously assigned to complementation group G7 has been ascribed to the absence in the mutants of functional cytochrome b . Since G7 mutants are capable of synthesizing the apoprotein, the primary effect of the mutations is to prevent maturation of this electron carrier . The recombinant plasmid pG7/T1 with a 6.7-kilobase pairs (kb) insert of wild type yeast nuclear DNA has been selected from a genomic library by transformation of a G7 mutant to respiratory competency . The genetically active region of the pG7/T1 insert has been subcloned on a 3-kb fragment of DNA which has been shown to contain an open reading frame encoding a protein of 50,236 Mr . In situ disruption of the reading frame causes a deficiency in cytochrome b . The strain with the disrupted gene fails to complement G7 mutants thereby confirming the correct identification of the gene henceforth referred to as COR1 . The carboxyl-terminal half of the COR1 gene has been fused to the amino-terminal half of the Escherichia coli trpE gene in the high expression vector pATH2 . This plasmid construct promotes a high level of expression of the trpE/COR1 hybrid protein . Antibodies against the purified hybrid protein react with a 44 kDa protein subunit of yeast coenzyme QH2-cytochrome c reductase corresponding to the largest core subunit of the complex . These data indicate that the yeast nuclear gene COR1 codes for the 44-kDa core protein and that the latter is required for the conversion of apocytochrome b to mature cytochrome b. J Biol Chem, 1986 Dec 25, 261(36), 17113 - 9 Interaction of a transcriptional activator, OmpR, with reciprocally osmoregulated genes, ompF and ompC, of Escherichia coli; Norioka S et al.; The ompB locus, comprised of the genes ompR and envZ, regulates the expression of the genes ompF and ompC that encode the major porin proteins in the outer membrane of Escherichia coli K-12 . OmpR is believed to activate transcription of ompF and ompC in a reciprocal manner depending upon the osmolarity of the culture medium . We were able to purify OmpR to homogeneity and to characterize its interaction with the porin gene promoters . The purified OmpR was shown to bind specifically to promoter fragments of both ompF and ompC . Deoxyribonuclease I footprinting was carried out in order to determine binding sites of OmpR in the promoter regions of the ompF and ompC . OmpR was found to protect the regions -105 to -60 of ompF and -102 to -78 of ompC, respectively . Two consensus sequences were found in these protected sites and are likely to play an important role in OmpR recognition of these promoter regions . The purified OmpR was also shown to be functionally active in transcription in vitro; OmpR activates the expression of ompF and inhibits the transcription of ompC from the most distal of its three tandem promoters, P3. Nucleic Acids Res, 1986 Dec 22, 14(24), 10009 - 26 Compilation and analysis of eukaryotic POL II promoter sequences; Bucher P et al.; A representative set of 168 eukaryotic POL II promoters has been compiled from the EMBL library and subjected to computer signal search analysis . Application of this technique to E . coli promoters as a control ensemble revealed the well known consensus sequences at -35 and -10 which indicates that the methods are adequate to approach problems of this kind . The results obtained from the eukaryotic promoter set can be summarized as follows: Common sequence features are confined to a region between -50 and +10 relative to the transcriptional initiation site . The only well conserved consensus sequence is TATAAA, centered at -28 . A weak motif, CA followed preferentially by pyrimidines, surrounds the cap-site . Two pentanucleotides which have been shown by experiments to stimulate transcription of certain genes, GGGCG and CCAAT, are moderately over-represented in the upstream region (between -129 and -50) . However, they occur at highly variable distances from the initiation site. Nucleic Acids Res, 1986 Dec 22, 14(24), 9713 - 28 D protein of miniF plasmid acts as a repressor of transcription and as a site-specific resolvase; Lane D et al.; Two activities of the D protein of the miniF plasmid have been found . Divergent promoters in ori-1 ("primary" replicative origin) of miniF are both repressed in cells which produce D protein . The mobilization of plasmids containing the ori-1 region by the F conjugation system is also repressed by D protein . In the former case D appears to act as a transcriptional repressor, whereas in the latter case D protein acts by resolving cointegrates of F and the mobilized plasmid . D protein resolves dimers whose monomer units contain the rfsF sequence needed for recA-independent, site-specific recombination of F . The nucleotide sequence of the D gene was determined . The D gene region contains two oppositely-oriented open reading frames which have the same reading phase and substantially overlap . Transposon insertion mutants were used to show that the gene for D protein occupies the top-strand (left-to-right) open reading frame. Nucleic Acids Res, 1986 Dec 22, 14(24), 9699 - 712 The miniF plasmid C protein: sequence, purification and DNA binding; Caughey PA et al.; The C (pifC) protein of miniF represses transcription of its own gene by binding to the pif operator (pifO); it is also needed for replication initiated from the miniF primary origin (ori-1) . We have determined the nucleotide sequence of the C gene . The gene has been inserted into an expression vector under Ptrp control where it is expressed at high levels . The C protein has been purified from cells carrying the Ptrp-C plasmid, and a preliminary study of C protein-DNA binding properties has been carried out . C protein binds strongly to pifO, and weakly to sequences in the ori-1 region. J Theor Biol, 1986 Dec 21, 123(4), 453 - 70 Analysis of the physiological control of replication of ColE1-type plasmids; Bremer H et al.; The physiology of ColE1-type plasmid replication in a growing host has been examined both theoretically, using computer simulation, and experimentally, by observing replication of the plasmid pBR322 after a nutritional shift-up from glycerol minimal medium (doubling time 71 min) to LB medium (doubling time 24 min) . The theory was based on a negative control model and uses three rate equations: for the accumulation of cell mass, for the accumulation of the replication inhibitor, and for the rate of plasmid synthesis . The implications of the theory were explored by simulating the effects of changes in the expression of replication control genes . The nutritional shift-up experiment showed that plasmid replication was blocked immediately after the shift for about half a mass doubling time; after that time, replication rapidly increased until plasmid numbers per unit volume of culture parallelled the increase in culture mass . After the establishment of steady-state growth in the post-shift medium, the plasmid concentration (plasmids per cell mass) was reduced in comparison to pre-shift growth in the same proportion as the culture doubling time . The results showed that plasmid replication factors are under metabolic control and that the changes in the control of these factors compensate one another during steady-state growth, but not immediately after the medium shift. EMBO J, 1986 Dec 20, 5(13), 3681 - 5 Both hydrophobic domains of M13 procoat are required to initiate membrane insertion; Kuhn A et al.; M13 procoat protein has two hydrophobic domains, one in the leader peptide and one which anchors the mature coat protein in the membrane . Disruption of the membrane anchor region by insertion of arginyl residues does not yield periplasmic coat protein . Instead, the rate of membrane assembly is slowed greater than 100-fold (t1/2 less than 5 s for wild-type, t1/2 greater than 10 min for mutant) . The hydrophobic region of mature coat protein not only functions as a membrane anchor, but has an important role in the membrane assembly process per se. EMBO J, 1986 Dec 20, 5(13), 3547 - 52 Molecular characterization of a karyophilic, histone-binding protein: cDNA cloning, amino acid sequence and expression of nuclear protein N1/N2 of Xenopus laevis; Kleinschmidt JA et al.; In the amphibian oocyte, most of the non-chromatin-bound histones are not free but form complexes with specific karyophilic proteins, the most prominent being nucleoplasmin and 'protein N1/N2' . Using antibodies against polypeptide N1 and N2 (Mr approximately 105,000 and approximately 110,000) we have isolated, from a Xenopus laevis ovary lambda gt11 expression library, several full length cDNA clones encoding one of the two closely related polypeptides N1 and N2 (these could not be distinguished by hybridization techniques) . The amino acid sequence deduced from one of these clones (N1/N2, lambda 106.2) defines a polypeptide of mol . wt 64,774 . The remarkably high difference between the value of Mr approximately 110,000 estimated from SDS-PAGE mobility and the true mol . wt has been found for (i) the cell protein, (ii) the polypeptide synthesized in vitro by transcription and translation and (iii) the fusion protein with beta-galactosidase expressed in Escherichia coli, indicating that the protein runs anomalously on SDS-PAGE . The amino and carboxy termini of the purified protein N1/N2 have been confirmed by direct amino acid sequencing of CNBr fragments . The amino acid sequence displays two glutamic acid-rich domains, which are probably involved in the interaction with the histones, and a putative nuclear targeting signal with high homology to that of the SV40 large T-antigen which is located near the carboxy terminus.(ABSTRACT TRUNCATED AT 250 WORDS) EMBO J, 1986 Dec 20, 5(13), 3697 - 703 Structures of mismatched base pairs in DNA and their recognition by the Escherichia coli mismatch repair system; Fazakerley GV et al.; The Escherichia coli mismatch repair system does not recognize and/or repair all mismatched base pairs with equal efficiency: whereas transition mismatches (G X T and A X C) are well repaired, the repair of some transversion mismatches (e.g . A X G or C X T) appears to depend on their position in heteroduplex DNA of phage lambda . Undecamers were synthesized and annealed to form heteroduplexes with a single base-pair mismatch in the centre and with the five base pairs flanking each side corresponding to either repaired or unrepaired heteroduplexes of lambda DNA . Nuclear magnetic resonance (n.m.r.) studies show that a G X A mismatch gives rise to an equilibrium between fully helical and a looped-out structure . In the unrepaired G X A mismatch duplex the latter predominates, while the helical structure is predominant in the case of repaired G X A and G X T mismatches . It appears that the E . coli mismatch repair enzymes recognize and repair intrahelical mismatched bases, but not the extrahelical bases in the looped-out structures. J Mol Biol, 1986 Dec 20, 192(4), 781 - 91 Transcriptional and translational initiation sites of IS50 . Control of transposase and inhibitor expression; Krebs MP et al.; We have examined transcriptional start sites responsible for expression of the transposase and transposition inhibitor proteins encoded by IS50R, and determined the likely translational start site of transposase . Amino-terminal analysis of a transposase-beta-galactosidase fusion protein gave the sequence Met-Ile-Thr-Ser-Ala, which corresponds to the predicted amino acid sequence starting at position 93 of IS50 . S1 nuclease mapping of IS50 RNA produced in vivo indicated that three transcripts, T1, T2 and T3, start near this position . Only T1 starts upstream from the transposase amino terminus . T2 corresponds to an in-vitro transcript described previously . Analysis of the transcripts and proteins produced from deletion derivatives of an IS50-lacZ construct suggested that the three transcripts initiate at independent but overlapping promoters clustered near the end of IS50 . This analysis confirmed that only T1 can encode transposase, and that T2 is largely responsible for expression of the inhibitor protein . The coding capacity of T3 was not determined . Finally, transcripts that originate outside of IS50 are prevented from expressing transposase because of a secondary structure that is present in these transcripts only. EMBO J, 1986 Dec 20, 5(13), 3673 - 9 Mutations affecting two distinct functions of the RNA component of RNase P; Shiraishi H et al.; The effect of structural changes on the functions of the RNA component (M1 RNA) of ribonuclease P (RNase P) of Escherichia coli has been studied using the thermosensitive mutants of the rnpB gene . One of the mutants, ts709, has two G--A substitutions at positions 89 and 365 from the 5' end of M1 RNA . Of these substitutions, the one at position 89 from the 5' end is responsible for the phenotype of this mutant . Although the RNase P activity of ts709 is thermosensitive, the mutant M1 RNA has the same catalytic activity as the wild-type RNA . M1 RNA of another mutant, ts2418, has a G--A substitution at position 329 . This mutant RNA has extremely low catalytic activity . The upstream mutational site of ts709 appears to play a role in the association with the protein subunit, whereas the mutational site of ts2418 is related to the catalytic function of M1 RNA. Biochim Biophys Acta, 1986 Dec 18, 868(4), 265 - 9 Escherichia coli ribosomal protein S1 does not protect the 49-nucleotide 3' terminal cloacin fragment of 16 S rRNA from nuclease S1; Wickstrom E; Footprinting of ribosomal protein S1 on the 49-nucleotide 3' terminal cloacin DF13 fragment of 16 S rRNA at physiological ionic strength, pH and temperature yielded no detectable protection of any nucleotides from subsequent attack by the single strand specific nuclease S1, even at large excesses of ribosomal protein S1. Biochemistry, 1986 Dec 16, 25(25), 8308 - 15 Dissociation of the lactose repressor protein tetramer using high hydrostatic pressure; Royer CA et al.; Dissociation of lac repressor tetramer by high hydrostatic pressures was monitored with intrinsic tryptophan fluorescence . With the assumption of complete dissociation to monomer, tryptophan polarization data gave delta V a approximately 170 mL/mol and the concentration for 50% tetramer dissociation, C1/2, was 3.8 X 10(-8) M . Upon addition of inducer, the calculated delta V a increased to approximately 220 mL/mol and the C1/2 decreased to approximately 1 X 10(-8) M, a free energy difference of approximately 0.7 kcal . These results indicate a modest stabilization of the tetramer by the presence of inducer . Monitoring the average energy of tryptophan emission demonstrated that tetramer dissociation takes place over the same range of pressures as evidenced by the polarization data and IPTG dissociation can be more or less superimposed upon tetramer dissociation depending upon the ligand concentration used . Although the two transitions cannot be separated entirely, the delta V a for the region of the pressure dependence dominated by ligand dissociation was 69 mL/mol, an unexpectedly large value . For tetramer modified with methyl methanethiosulfonate, subunit dissociation was shifted to much higher pressures and IPTG dissociation did not occur . The delta V a for subunit association was calculated as approximately 160 mL/mol, and the C1/2 was 3.5 X 10(-9) M . Interactions at the subunit interface of the modified protein are apparently stronger than in the unmodified protein . The absence of inducer dissociation from the MMTS-modified tetramer by the application of high hydrostatic pressure suggests that the volume change for inducer binding to the modified protein is much smaller than that observed for the unmodified repressor. Biochemistry, 1986 Dec 16, 25(25), 8230 - 4 Membrane disposition of the Escherichia coli mannitol permease: identification of membrane-bound and cytoplasmic domains; Stephan MM et al.; Two proteolytic fragments of the Escherichia coli mannitol permease (EIImtl) have been identified on autoradiograms of sodium dodecyl sulfate-polyacrylamide gels and mapped with respect to the membrane . EIImtl was selectively radiolabeled with either {35S}methionine or a mixture of 14C-labeled amino acids in E . coli minicells harboring a plasmid containing the mannitol operon . The intact permease (Mr 65,000) in everted vesicles derived from labeled minicells was cleaved by mild trypsinolysis into two smaller fragments (Mr 34,000 and 29,000) . The 34,000-dalton fragment remained in the membrane and was insensitive to further proteolysis by trypsin . This fragment was identified as the N-terminal half of the protein by comparing the amount of the original {35S}methionine label that it retained with the known differential distribution of methionine in the two halves of EIImtl . The 29,000-dalton fragment, which was released into the soluble fraction and was sensitive to further trypsinolysis, therefore corresponds to the C-terminal half of the mannitol permease . Both fragments were shown to be antigenically related to EIImtl by immunoblotting with anti-EIImtl antibody . The 34,000-dalton fragment was further shown to form an oligomer under conditions which allow the intact enzyme to dimerize, suggesting that this domain plays an important role in EIImtl subunit interactions . These results support a model in which EIImtl consists of two domains of approximately equal size: a membrane-bound, N-terminal domain with a tendency to self-associate, and a cytoplasmic C-terminal domain.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1986 Dec 16, 25(25), 8178 - 83 Identification of the high-affinity lipid binding site in Escherichia coli pyruvate oxidase; Hamilton SE et al.; Pyruvate oxidase from Escherichia coli is a peripheral membrane associated enzyme which is activated by lipids . We have investigated the high-affinity lipid binding site associated with lipid activation of pyruvate oxidase by covalent attachment of {14C}lauric acid to the enzyme . Lauric acid is bound stoichiometrically (1 mol/mol of active sites), and the enzyme is essentially irreversibly activated . Mild tryptic digestion of the modified enzyme shows that the lauric acid is bound within the last 100 residues of the 572-residue monomer . Digestion with thermolysin releases two closely related peptides, A and B, in approximately equal amounts . Comparison of the amino acid composition of peptide A with the entire sequence of the protein shows that peptide A corresponds to the sequence from Ala-543 to Ile-554 . The analysis of peptide B is very similar to that of A . Limited sequence analysis of peptide B shows that residue 1 is Ala and residue 2 is labeled . These results support the assignment of residue 1 in peptide B as Ala-543 and indicate that lauric acid is bound to Lys-544 . Previous work in this laboratory has shown that pyruvate oxidase may be activated independently of lipids by mild protease digestion . Proteolytic activation is accompanied by the release of a small peptide (residues 550-572) from the carboxyl terminus of the protein . The present work locates the lipid binding site very close to this peptide . The significance of these results for the mechanism of activation of pyruvate oxidase and other lipid-activated systems is discussed. Biochemistry, 1986 Dec 16, 25(25), 8167 - 72 Chemical modification of Escherichia coli RNA polymerase by diethyl pyrocarbonate: evidence of histidine requirement for enzyme activity and intrinsic zinc binding; Abdulwajid AW et al.; RNA polymerase (RPase) from Escherichia coli contains five subunits (alpha 2 beta beta' sigma) and two intrinsic Zn ions located in the beta and beta' subunits . This enzyme was rapidly inactivated by diethyl pyrocarbonate (DEP) at pH 6.0 and 25 degrees C . The difference spectrum of the DEP-inactivated and native RPases showed a single peak at 240 nm indicating the formation of N-carbethoxyhistidines . No decrease in absorbance at 278 nm, due to O-carbethoxytyrosine, or modification of amino and sulfhydryl groups was observed . Inactivated RPase with six to nine histidines being modified could be fully reactivated by incubation with 0.5 M hydroxylamine at pH 6.0 and room temperature for 1 h . No structural difference was detected between the native and modified enzymes as evidenced by UV/visible and fluorescence spectra, sodium dodecyl sulfate-polyacrylamide gel electrophoretic pattern, or gel filtration properties . Substrate ATP at 0.11 and 1.14 mM concentrations provided, respectively, 25% and 90% protection against DEP inactivation, while template DNA did not . These results suggest that one or more histidine residues is/are in close proximity to the substrate binding site . The pH dependence of the DEP inactivation of RPase suggested the modification of histidine at the active site with a pK value of 6.9 . The inactivation of RPase by DEP and the formation of N-carbethoxyhistidine displayed a similar second-order rate constant of approximately 0.9 mM-1 min-1.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1986 Dec 16, 25(25), 8163 - 6 Photochemical properties of Escherichia coli DNA photolyase: a flash photolysis study; Heelis PF et al.; Escherichia coli DNA photolyase contains a stable flavin neutral blue radical that is involved in photosensitized repair of pyrimidine dimers in DNA . We have investigated the effect of illumination on the radical using light of lambda greater than 520 nm from either a camera flash or laser . We find that both types of irradiations result in the photoreduction of the flavin radical with a quantum yield of 0.10 +/- 0.02 . While photoreduction with the camera flash is minimal in the absence of an electron donor (dithiothreitol), laser flash photolysis at 532 nm reduces the flavin to the same extent in the presence or absence or an electron donor . Thus, it is concluded that the primary step in photoreduction involves an electron donor that is a constituent of the enzyme itself . Laser flash photolysis produces a transient absorption band at 420 nm that probably represents the absorption of the lowest excited doublet state (2(1)IIII*) of the radical and decays with first-order kinetics with k1 = 0.8 X 10(6) s-1 . The photoreduction data combined with the results of recent studies on the activity of dithionite-reduced enzyme suggest that electron donation by excited states of E-FADH2 is the mechanism of flavin photosensitized dimer repair by E . coli DNA photolyase. Biochemistry, 1986 Dec 16, 25(25), 8274 - 7 Structure of unfolded and refolded recombinant derived {Ala125}interleukin 2; Arakawa T et al.; Naturally occurring interleukin 2 (IL-2) contains an odd number (three) of cysteinyl residues and thus is susceptible to the formation of a variety of intramolecular and intermolecular disulfide bonds . The cysteine at residue 125 has been replaced with an alanine residue by site-directed mutagenesis, and hence, this analogue can form only one intrachain disulfide bond . When expressed at high levels in Escherichia coli, this recombinant DNA derived IL-2 analogue is insoluble, reduced, and inactive . The protein was solubilized by denaturants and, after purification, was oxidized to form an intramolecular disulfide bond . Circular dichroism (CD) has been used to investigate the effects of various denaturants on the unfolding-refolding process of the purified, oxidized protein . A similar conformation is obtained when {Ala125}interleukin 2 {IL-2(Ala-125)} is refolded from 6 M guanidine hydrochloride, 8 M urea, or 5% acetic acid . The resultant protein, refolded from these denaturants, is monomeric and has activity comparable to or greater than that reported for naturally derived IL-2 . In addition to this form, aggregates, as evidenced from gel filtration, are obtained . The specific activities of these are greatly reduced, and CD spectra indicated that they have much less helical content than the monomeric form of the protein . CD spectra also showed that the tertiary structure of IL-2(Ala-125) is entirely different in the presence of sodium dodecyl sulfate (SDS) from that of the monomeric form in the absence of SDS. Biochemistry, 1986 Dec 16, 25(25), 8173 - 8 Dihydrolipoyl transacetylase of Escherichia coli . Formation of 8-S-acetyldihydrolipoamide; Yang YS et al.; The dihydrolipoyl transacetylase component (E2) of the pyruvate dehydrogenase complex catalyzes the reaction of acetyl coenzyme A (acetyl-CoA) with dihydrolipoamide, producing coenzyme A and S-acetyldihydrolipoamide . The acetyl group is shown by experiments reported herein to be bonded to S8 in the enzymatic product . 1H NMR analysis of synthetic samples of both structural isomers of S-acetyl-S-(phenylmercurio)dihydrolipoamide enabled structural assignments to be made . Reaction of 8-S-acetyl-6-S-(phenylmercurio)dihydrolipoamide with 3-mercaptopropionic acid in chloroform produced 8-S-acetyldihydrolipoamide which contained a small amount (5%) of the 6-S isomer . Reaction of 6,8-di-S-acetyldihydrolipoamide with NH2OH produced a 4:1 mixture of 6-S-acetyldihydrolipoamide and the 8-S isomer . These compounds did not isomerize at significant rates in chloroform but rapidly isomerized to the equilibrium mixture in aqueous solution (Keq = 3.4) . The second-order rate constants for the hydroxide-catalyzed isomerization were found to be kf = (1.15 +/- 0.07) X 10(6) M-1 X s-1 and kr = (3.36 +/- 0.20) X 10(5) M-1 X s-1 in the direction of the formation of the 8-S isomer . The enzymatic product was trapped by addition of phenylmercuric hydroxide within 15 s-30 min after starting the reaction . 1H NMR analysis of the products obtained at various times showed that the enzymatic product was 8-S-acetyldihydrolipoamide, which underwent progressive isomerization to the mixture of isomers within a few minutes . In the reaction of acetyl-CoA with dihydrolipoamide, the latter substrate reacts in place of enzyme-bound dihydrolipoyl moieties . Therefore, acetylation occurs at the 8-S position of bound lipoyl groups. Eur J Biochem, 1986 Dec 15, 161(3), 723 - 6 Tetracycline can inhibit tRNA binding to the ribosomal P site as well as to the A site; Geigenmuller U et al.; A and P sites of Escherichia coli ribosomes were titrated with AcPhe-tRNAPhe, in the absence or presence of tetracycline . The P-site location of the bound AcPhe-tRNA was assessed by means of a quantitative puromycin reaction . The results demonstrate that, in agreement with the generally held view, tetracycline exclusively inhibits the A-site binding, if the statistical number of bound acyl-tRNA molecules per ribosome does not exceed about 0.5 . However, above this value the P site becomes sensitive to tetracycline as well . It follows that the tightly coupled 70S ribosomes used in functional studies appear to be functionally heterogeneous, i.e . those P sites which cannot be affected by tetracycline are preferentially occupied by AcPhe-tRNA, whereas higher concentrations of this tRNA species are required to fill tetracycline-sensitive P sites . Furthermore, the results imply that under tRNA saturation conditions the tetracycline inhibition cannot be used as an indicator for the site location of bound tRNA. Biophys Chem, 1986 Dec 15, 25(2), 181 - 90 Effect of ethanol on the thermal stability of tRNA molecules; Beneventi S et al.; The thermal denaturation of E . coli unfractionated tRNA in ethanol/water mixtures has been studied as a function of alcohol concentration in the water-rich region (mole fraction of co-solvent chi 2 less than 0.2) . The results show that with increasing alcohol concentration the melting temperature of tRNA first reaches a minimum at an intermediate composition chi *2 approximately equal to 0.055 and then increases with increasing chi 2 . The value of chi *2 is close to that at which structural changes in the mixture occur as inferred from compressibility and optical absorption measurements . The present experimental data support the assumptions that the dominant mechanism by which ethanol affects the thermal stability of tRNA molecules is through its effect on the structure of water. J Biol Chem, 1986 Dec 15, 261(35), 16363 - 71 In vitro synthesis of alpha-carboxyl-linked folylpolyglutamates by an enzyme preparation from Escherichia coli; Ferone R et al.; Extracts of Escherichia coli contained an enzymatic activity which catalyzed the addition of L-glutamate to the alpha-carboxyl of various polyglutamate substrates, including folylpolyglutamates . Much of the enzyme activity was separated by DE52 chromatography and gel filtration from the enzyme which adds the first three glutamates in the biosynthesis of folylpolyglutamates, dihydrofolate synthetase-folylpolyglutamate synthetase . The two enzyme activities differed in many properties . Whereas dihydrofolate synthetase-folylpolyglutamate synthetase preferred pteroate or pteroylmonoglutamate substrates, the folylpoly-alpha-glutamate synthetase preparations effectively utilized tetrahydropteroylpolyglutamates, pteroylpolyglutamates, p-aminobenzoylpolyglutamates (pAB(Glu)n), and even a polyglutamate tripeptide . Several di- and triglutamyl peptides were inhibitory to folylpoly-alpha-glutamate synthetase activity, but not to dihydrofolate synthetase-folylpolyglutamate synthetase . Conversely, dihydropteroate noncompetitively inhibits the folylpolyglutamate synthetase reaction of the dihydrofolate synthetase-folylpolyglutamate synthetase protein, but did not inhibit the folylpoly-alpha-glutamate synthetase reaction . Potassium chloride was inhibitory to folylpoly-alpha-glutamate synthetase activity (as were other salts and several polyanions), in contrast to the absolute requirement of dihydrofolate synthetase-folylpolyglutamate synthetase activity for a monovalent cation such as K+ . Incubation of a folylpoly-alpha-glutamate synthetase preparation with (6S)-tetrahydropteroyltri(gamma)glutamate generated products which after chemical cleavage to pAB(Glu)n were identical to those from growing E . coli, in high performance liquid chromatography retention times and in pattern of digestion by alpha-COOH bond-specific carboxypeptidase Y . High performance liquid chromatography and mass spectral analysis of the products of the in vitro reactions of folylpoly-alpha-glutamate synthetase with several substrates also demonstrated the addition of glutamate residues via alpha-COOH linkages . Thus, there appear to be two folylpolyglutamate synthetase activities in E . coli, dihydrofolate synthetase-folylpolyglutamate synthetase which adds the first three glutamate residues by gamma-COOH linkages and the folylpoly-alpha-glutamate synthetase activity which extends the folylpolyglutamate chain via gamma-COOH peptide bonds. J Biol Chem, 1986 Dec 15, 261(35), 16356 - 62 alpha-Carboxyl-linked glutamates in the folylpolyglutamates of Escherichia coli; Ferone R et al.; Folate cofactors in most cells contain polyglutamate side chains, which since the late 1940s have been assumed to be linked via their gamma-COOH groups . We report here an investigation of the structure of the polyglutamate chain attached to the folates of Escherichia coli . Folates were extracted from E . coli grown with {7-14C} p-aminobenzoate and cleaved to p-aminobenzoyl polyglutamates of varying chain lengths (pAB(Glu)n) by the method of Foo et al . (Foo, S . K., Cichowicz, D . J., and Shane, B . (1980) Anal . Biochem . 107, 109-115) . The pAB(Glu)n derived from E . coli did not co-chromatograph with chemically synthesized pAB(gamma-Glu)n-Glu on several high performance liquid chromatography (HPLC) systems, except for the triglutamate which did elute with pAB(gamma-Glu)2-Glu . E . coli-derived pAB(Glu)3-8 were purified by HPLC on C18 columns eluted with acetonitrile/trifluoroacetic acid, and the structures were determined through mass spectrometry, chiral amino acid analysis, and peptidase digestion experiments . Molecular weight determinations on the methyl ester derivatives of E . coli-derived pAB(Glu)n by liquid secondary ion mass spectrometry and sequence analysis using collision-activated dissociation on a tandem mass spectrometer confirmed the structures as pAB(Glu)3-8 . Chiral HPLC of hydrolyzed and dansylated E . coli-derived materials, on a beta-cyclodextrin column, identified the glutamate as the L-enantiomer . pAB(Glu)n were digested with carboxypeptidase Y, which specifically cleaved glutamates linked at their alpha-carboxyls; E . coli-derived pAB(Glu)4-8 (but not synthetic pAB(gamma-Glu1-6-Glu) were sequentially digested to pAB(gamma-Glu)2-Glu . Thus, in E . coli folylpolyglutamates, glutamate residues 4-8 were each linked to the polyglutamate chain at the alpha-carboxyl of the preceding glutamate. Cancer, 1986 Dec 15, 58(12), 2764 - 72 Clinical effects and toxicity of interleukin-2 in patients with cancer; Lotze MT et al.; Interleukin-2 (IL-2) is a 15,000 dalton glycoprotein produced naturally by human T-cells during an immune response . IL-2 has been demonstrated to have substantial activity alone or in combination with the adoptive transfer of lymphokine-activated killer cells in murine tumor models . IL-2 derived from both natural (Jurkat human T-cell tumor) and recombinant (Escherichia coli) sources has been studied in Phase I protocols designed to evaluate toxicity in patients with a variety of solid tumors and to ascertain improvement in clinical parameters and immunologic status . A total of 16 patients (7 with acquired immune deficiency syndrome {AIDS} and 9 with non-AIDS malignancies) were treated with Jurkat derived IL-2 . The total maximum dose (1.3 X 10(5) U/kg) was limited only by supply of this reagent . A total of 25 patients have been treated with recombinant IL-2 (RIL-2) alone . Dose-limiting toxicity manifested by marked malaise and weight gain was achieved with doses of RIL-2 of 10(6) U/kg as a single bolus or 3000 U/kg/hr . IL-2 could be administered intraperitoneally with similar toxicity . Minimal renal or hepatic toxicity was demonstrated . Hematologic toxicity was limited to mild anemia (25/25), thrombocytopenia (10/25), and marked reversible eosinophilia (15/25) . Pronounced weight gain greater than 2 kg (16/25) occurred in most patients, primarily those who received cumulative doses of greater than 1-3 X 10(5) U/kg of IL-2 . The weight gain amounted to as much as 10% to 20% of the pretreatment weight over 3 weeks of treatment and limited our ability to give higher doses . Two partial responses (greater than 50% decrease in cross sectional diameters) were seen in two patients with melanoma metastatic to the lung. Chem Phys Lipids, 1986 Dec 15, 42(1-3), 137 - 51 Recent advances in identifying the functions of gangliosides; Fishman PH; The recent development of several new approaches has proven extremely useful in identifying functions for gangliosides, the sialic-acid containing glycosphingolipids . The first is the incorporation of exogenous gangliosides into the plasma membrane of ganglioside-deficient cells . Using this approach, specific gangliosides have been identified as the receptors for certain bacterial toxins and viruses and as important factors in the organization of fibronectin into an extracellular matrix . The second approach has been a ligand blotting technique which allows detection of ganglioside-binding proteins such as toxins and antibodies . Gangliosides are separated by thin-layer chromatography and overlain with the protein of interest . Specific binding of the ligand to gangliosides can then be detected by either direct or indirect methods . The third approach is the use of the B or binding subunit of cholera toxin as a specific probe for endogenous plasma membrane ganglioside function . The ability of the B subunit to alter the growth of cells directly demonstrates a role for gangliosides as biotransducers of signals for the regulation of cell growth. Biophys Chem, 1986 Dec 15, 25(2), 191 - 200 Dynamics of superhelical DNA studied by photon correlation spectroscopy; Langowski J et al.; We have conducted photon correlation spectroscopy (PCS) studies on the plasmid pUC8 (2717 bp) in order to elucidate the internal dynamics of this superhelical DNA . We confirm that the first-order autocorrelation function of the scattered light from pUC8 solutions can be separated into two distinct exponential decay components, as first shown by Lewis et al . (R . Lewis, J.H . Huang and P . Pecora, Macromolecules 18 (1985) 944) . A thorough analysis of the dependence on scattering vector K of the rates and amplitudes of the two components enables us to assign the slowly relaxing part to the center-of-mass diffusion of the DNA, while the faster component corresponds to rotational, bending and twisting motions of the superhelix . For larger K values the internal motions can be formally expressed in terms of an 'internal diffusion coefficient' Di, whose value of 2.0-2.5 X 10(-11) m2 s-1 is approximately equal to the translational diffusion coefficient predicted for a stiff DNA piece of the persistence length, 65 nm . Comparison of our measured Di values to those predicted from a recent theory of circular worm-like coils (K . Soda, Macromolecules 17 (1984) 2365) shows that the internal motions are faster than the theoretical values . One of the reasons for this discrepancy could be that the theory does not take into account torsional motions, which contribute significantly to the internal dynamics (J.C . Thomas, S.A . Allison, C.J . Appelof and J.M . Schurr, Biophys . Chem . 12 (1980) 177) . At low K values, the fast relaxation of superhelical pUC8 is no longer proportional to K2, but reaches a constant value as K approaches zero . This behavior, not seen for the linearized DNA, can be interpreted in terms of rotational diffusion of a flexible rod-like molecule (T . Maeda and S . Fujime, Macromolecules 17 (1984) 2381) and supports an interwound rod-like structure for pUC8 DNA with an average end-to-end distance of 220 nm. Eur J Biochem, 1986 Dec 15, 161(3), 715 - 21 Analysis of the puromycin reaction . The ribosomal exclusion principle for AcPhe-tRNA binding re-examined; Geigenmuller U et al.; The standard technique for determination of the ribosomal site location of bound tRNA, viz . the puromycin reaction, has been analyzed with regard to its applicability under tRNA saturation conditions . The criteria derived have been used to re-examine the exclusion principle for peptidyl-tRNA binding, which states that only one peptidyl-tRNA (AcPhe-tRNA) can be bound per ribosome although in principle two sites (A and P site) are available . The following results were obtained . The puromycin reaction is only appropriate for a site determination if the reaction conditions prevent one ribosome from performing more than one puromycin reaction . With an excess of AcPhe-tRNA over ribosomes, and in the absence of EF-G, this criterion is fulfilled at 0 degree C, where the P-site-bound material reacts with puromycin (quantitative reaction after 50 h), while the A-site-bound material does not . In contrast, at 37 degrees C the extent of the puromycin reaction can exceed the binding values by 2-4-fold ('repetitive reaction') . In the presence of EF-G a repetitive puromycin reaction is seen even at 0 degree C, i.e . EF-G can already promote a translocation reaction at 0 degree C . However, the extent of translocation becomes negligibly low for short incubation times (up to 60 min) at 0 degree C, if only catalytic amounts of EF-G are used . Using the criteria outlined above, the validity of the exclusion principle for Escherichia coli ribosomes was confirmed pursuing two different experimental strategies . Ribosomes were saturated with AcPhe-tRNA at one molecule per 70S ribosome, and a quantitative puromycin reaction demonstrated the exclusive P-site location of the AcPhe-tRNA . The same result was also found in the presence of viomycin, which blocks the translocation reaction . These findings also indicate that here nearly 100% of the ribosomes participate in AcPhe-tRNA binding to the P site . Precharging the P sites of 70S ribosomes with one Ac{14C}Phe-tRNA molecule per ribosome prevented additional Ac{3H}Phe-tRNA binding . In contrast, 70S particles carrying one molecule of {14C}tRNAPhe per ribosome were able to bind up to a further 0.64 molecule Ac{3H}Phe-tRNA per ribosome. J Biol Chem, 1986 Dec 15, 261(35), 16604 - 15 In vitro mutagenesis and overexpression of the Escherichia coli trpA gene and the partial characterization of the resultant tryptophan synthase mutant alpha-subunits; Milton DL et al.; A mutagenesis approach was initiated in order to examine further the folding behavior of the alpha-subunit of the Escherichia coli tryptophan synthase . A random single base pair saturation mutagenesis procedure (Myers, R.M., Lerman, L.S., and Maniatis, T . (1985) Science 229, 242-247) was applied in vitro to subcloned fragments of the trpA gene, which codes for this polypeptide . Mutagenesis plasmid vectors were constructed containing three fragments of the trpA gene which together code for about half of the total amino acid residues of the alpha-subunit . The vectors were constructed such that each strand of each trpA fragment could be altered . These trpA fragments were mutagenized in vitro (using nitrous acid, formic acid, hydrazine, and potassium permanganate), and several thousands of mutants have been isolated . Thirty-two mutants, contained within the first two trpA fragments (which encompass the first 206 base pairs of the trpA gene and encode the first 63 residues of the alpha-subunit) have been sequenced . Of these, 20 (63%) contained single base pair alterations, 12 (37%) contained multiple alterations, and 17 (53%) of these base pair alterations resulted in single amino acid substitutions . Selected mutant trpA fragments were subcloned into an overexpression vector in which the trpA gene is controlled by the tac promoter and is inducible by lactose . The kinetics and extent of induction show that after 22 h of induction, the wild-type alpha-subunit constituted about 30% of the total protein . A simple one-step purification procedure for the alpha-subunit is described in which 15 mg of alpha-subunit can be obtained from 200 ml of fully induced cultures . The mutant trpA genes were induced for mutant alpha-subunit expression, and an initial examination of their properties in crude extracts was performed . Of the 17 mutant proteins examined, most were overproduced to levels comparable to that for the wild-type alpha-subunit . An analysis of the apparent stability, beta 2-subunit-activating activity, and intrinsic activity of this group of mutant alpha-subunits suggests that many amino acid alterations have no apparent effect; there is a variety of novel functional defects; and a sequence located near residues 28 through 54 may be particularly critical for the normal folding of the polypeptide. J Biol Chem, 1986 Dec 15, 261(35), 16332 - 9 Structure-function analysis of three cAMP-independent forms of the cAMP receptor protein; Harman JG et al.; cAMP receptor protein (CRP)-dependent operon expression in Escherichia coli requires the CRP X cAMP complex form of wild-type CRP . One class of crp mutants (crp*) activates CRP-dependent promoters in strains (cya) incapable of endogenous cAMP synthesis . Of fundamental interest is the difference in regulatory properties exhibited by crp* mutant strains, some of which exhibit glucose-mediated repression of beta-galactosidase synthesis, some of which do not . To gain a better understanding of the mechanisms of cAMP-independent promoter activation and repression we have: determined through cloning and DNA sequence analysis the primary structure of three CRP* forms of CRP; purified the mutant proteins; characterized the effect of these mutations on CRP secondary structure; and studied CRP*-activated lac promoter regulation in a purified in vitro transcription system . The results of this study provide strong evidence that mutations in crp alter the conformation of CRP and result in cAMP-independent activation of CRP-dependent promoters in vitro . In addition, a CRP allele-specific inhibition of CRP* activity by spermidine was observed in vitro that parallels crp* strain-specific sensitivity to glucose-mediated repression of CRP-dependent enzyme synthesis in vivo . This observation provides evidence that catabolite repression in cells lacking cAMP may be mediated through a mechanism that inhibits CRP* activity. J Biol Chem, 1986 Dec 15, 261(35), 16722 - 6 Biologically active, recombinant DNA in clathrin-coated vesicles isolated from rat livers after in vivo injection of liposome-encapsulated DNA; Nandi PK et al.; DNA entrapped in liposomes containing lactosylceramide in the bilayers is found to be associated with clathrin-coated vesicles isolated from the rat livers after intravenous injection of these liposomes . The presence of the exogenous DNA in the coated vesicles was detected by Southern blotting . The amount of DNA present in the coated vesicles does not appear to vary up to 4 h after injection of the liposomes into the animals . The recognition of the lactosyl group present in the liposome by the galactose receptor present on the surface of the different liver cells may lead to their internalization in a way analogous to receptor-mediated endocytosis of various macromolecules . DNA present in the lumen of the coated vesicles is found to be biologically active as evidenced by its replication in bacterial cells and mouse fibroblasts. Nucleic Acids Res, 1986 Dec 9, 14(23), 9407 - 23 Xrep, a plasmid-stimulating X chromosomal sequence bearing similarities to the BK virus replication origin and viral enhancers; Riley DE et al.; The human X chromosome-linked fragment, "Xrep," was sequenced because it exerts a positive effect on plasmid growth in both E . coli and Saccharomyces cerevisiae . The sequence revealed three features similar to the human BK virus replication origin: Xrep has a true palindrome, CCTCC(T)3CCTCC, which is similar to "true" palindrome-like sequences found at the replication origins of polyoma {CCTC(T/C)10CTCC}, BK {CCTC(A/G)8CCTCC} and SV40 {CCTCC(A)6GCCTCC} viruses . Twenty nucleotides away from the true palindrome, Xrep has the sequence GAATCCTATTCACTTTT while BK virus, the human analogue of SV40, has GAAATCCCTATTCTTTT in exactly the same position relative to the true palindrome . These two 17-mers differ only in the positions of two nucleotides comparing Xrep and BK virus . Also similar to the replication origins of DNA viruses, Xrep appears to have a cluster of enhancers adjacent to the origin-like sequences . Potent enhancer-like activity was detected in pSV1 X CAT/Xrep constructs . Xrep may originate from an endogenous virus, or from an X chromosomal replication origin. J Biol Chem, 1986 Dec 5, 261(34), 16226 - 32 Induction of deletion and insertion mutations by 9-aminoacridine . An in vitro model; Conrad M et al.; The ability of 9-aminoacridine to induce mutagenic lesions during DNA replication in vitro was investigated . The ampicillinase gene of pBR322 was replicated in vitro in the presence of 9-aminoacridine . Transfection of the replicated DNA into Escherichia coli gave Amps mutants . Determination of the base changes in 76 of these mutants indicated that the spectrum of mutations induced by 9-aminoacridine was consistent with its action in vivo . Both large (407-base) and small (1- and 2-base) deletions were induced at repetitive sequences . The frequency of deletion mutations depended on the identity of the base deleted and sequences surrounding the deletions . The characteristics of the frameshift mutations induced were consistent with the interactions of 9-aminoacridine with DNA . These results establish that 9-aminoacridine can induce frameshift mutations during the replication process and provide an in vitro model of frameshift induction for mechanistic studies. J Biol Chem, 1986 Dec 5, 261(34), 16207 - 9 Crystallization and preliminary X-ray studies of recombinant murine interferon-beta; Matsuda S et al.; Recombinant murine interferon-beta produced in Escherichia coli was purified and crystallized in an orthorhombic space group C222(1) with a = 61.67 A, b = 55.62 A, and c = 92.16 A . The crystals with a slight tendency for orientational disorder around the c axis diffract at least up to 3.3-A resolution . The crystallizability and the fact that the crystallographic asymmetric unit contains only one molecule of murine interferon-beta strongly indicate that the present preparation (Matsuda, S., Utsumi, J., and Kawano, G . (1986) J . Interferon Res., in press) of recombinant murine interferon-beta is predominantly homogeneous with respect to chemical, tertiary, and quaternary structures. J Mol Biol, 1986 Dec 5, 192(3), 549 - 56 Nucleotide sequence of the phoR gene, a regulatory gene for the phosphate regulon of Escherichia coli; Makino K et al.; Genes in the phosphate regulon of Escherichia coli are positively regulated by the products of the phoB and phoR genes with limited phosphate, and negatively regulated by the product of the phoR gene with excess phosphate . We present here the complete nucleotide sequence of the phoR gene . Together with the DNA sequence of the upstream phoB gene that we determined previously, this region shows the following features . The flanking regions of the operon are abundant in A-T base-pairs . A possible stem-and-loop structure of the transcript followed by several U residues characteristic of rho-independent transcriptional terminators was distal to the phoR coding region . The operon is probably composed of only two cistrons . The nucleotide sequence of phoR indicates that its protein consists of 431 amino acid residues and has a molecular weight of 49,666 . The amino acid sequence of the PhoR protein has significant homology with that of the EnvZ protein, which is a regulator for the omp regulon . Therefore, the sequences of the PhoB and PhoR proteins have considerable homologies with those of the OmpR and EnvZ proteins, respectively, indicating that the two operons share a common ancestor . The PhoR protein contains an extensive hydrophobic region in the amino-terminal portion . Thus the protein may be a membrane protein and function as a component of a signal transducer. J Biol Chem, 1986 Dec 5, 261(34), 16078 - 81 Isolation of the structural gene encoding a mutant form of Escherichia coli phosphoenolpyruvate carboxylase deficient in regulation by fructose 1,6-bisphosphate . Identification of an amino acid substitution in the mutant; Sutton F et al.; The structural gene encoding a mutant Escherichia coli phosphoenolpyruvate carboxylase deficient in regulation by fructose 1,6-bisphosphate (Fru-P2) was isolated from total E . coli PpcI genomic DNA . This mutant gene is located on a 4.4-kilobase SalI DNA fragment which, when ligated to SalI-digested pBR322, resulted in the generation of the plasmid pFS16 . Detailed restriction mapping of the wild-type and mutant genes for phosphoenolpyruvate carboxylase revealed the presence of a ClaI restriction site at position 563 of the mutant gene only . This ClaI site is located on a 289 PvuII/DdeI fragment which codes for amino acid residues 174-270 of the phosphoenolpyruvate carboxylase enzyme . When this portion of the mutant gene is present in chimeras of the wild-type and mutant genes, the phosphoenolpyruvate carboxylase produced cannot be activated by Fru-P2 . The mutation resulting in the generation of the ClaI site in the mutant gene has also resulted in an amino acid substitution at residue 188; threonine in the wild-type enzyme has been replaced by isoleucine in the mutant enzyme . Comparison of the nucleotide sequence of this 289-base pair PvuII/DdeI region of the mutant gene with its homologous region in the wild-type gene verified that this mutation, which resulted in the generation of the ClaI site, is the only change that has occurred on this 289-base pair fragment of the mutant gene, and thus the amino acid replacement of threonine by isoleucine is the only change that could be linked to the inability of the mutant enzyme to be activated by Fru-P2. J Biol Chem, 1986 Dec 5, 261(34), 15906 - 9 Absence of a phosphorylated intermediate during ATP hydrolysis by Escherichia coli transcription termination protein rho; Stitt BL et al.; We have established the suitability of adenosine 5'-O-(gamma-thio)triphosphate(ATP gamma S) as an analog of ATP for the nucleoside triphosphatase activity of Escherichia coli transcription termination protein rho (EC 3.6.1.3) . Steady-state analysis gives a Vmax of 1.5 mumol min-1 mg-1, 9% of the value with MgATP as substrate, and indicates that ATP gamma S binds as tightly (based on Km and Ki versus ATP) to rho as does ATP . (gamma-S){beta gamma-17O,gamma-17O,gamma-18O}ATP gamma S was used as substrate to produce chiral product inorganic {17O,18O}thiophosphate and determine the stereochemical course of the hydrolysis . The results of this determination, inversion at the thiophosphoryl phosphorus, indicate that the enzymatic hydrolysis of ATP by rho consists of a direct transfer of the phospho group to water without the existence of a phosphoenzyme or phospho-RNA intermediate. J Biol Chem, 1986 Dec 5, 261(34), 15823 - 6 In vitro inactivation of methionine synthase by nitrous oxide; Frasca V et al.; Nitrous oxide (N2O) is commonly used as an anesthetic agent . Prolonged exposure to N2O leads to megaloblastic anemia in humans and to loss of methionine synthase activity in vertebrates . We now report that purified preparations of cobalamin-dependent methionine synthase (5-methyltetrahydrofolate-homocysteine methyltransferase, EC 2.1.1.13) from both Escherichia coli and pig liver are irreversibly inactivated during turnover in buffers saturated with N2O . Inactivation by N2O occurs only in the presence of all components required for turnover: homocysteine, methyltetrahydrofolate, adenosylmethionine, and a reducing system . Reisolation of the inactivated E . coli enzyme after turnover in the presence of N2O resulted in significant losses of bound cobalamin and of protein as compared to controls where the enzyme was subjected to turnover in N2-equilibrated buffers before reisolation . However, N2O inactivation was not associated with major changes in the visible absorbance spectrum of the remaining enzyme-bound cobalamin . We postulate that N2O acts by one-electron oxidation of the cob(I)alamin form of the enzyme which is generated transiently during turnover with the formation of cob(II)alamin, N2, and hydroxyl radical . Generation of hydroxyl radical at the active site of the enzyme could explain the observed irreversible loss of enzyme activity. Biochim Biophys Acta, 1986 Dec 5, 879(3), 322 - 9 Development of enzyme immunoassay for serum 13,14-dihydro-15-ketoprostaglandin F2 alpha; Yokota K et al.; An enzyme immunoassay was developed for a convenient and sensitive assay of 13,14-dihydro-15-ketoprostaglandin F2 alpha, a metabolite of prostaglandin F2 alpha appearing in human blood . The compound was chemically conjugated to beta-galactosidase from Escherichia coli . The enzyme-labeled antigen was mixed with a sample containing 13,14-dihydro-15-ketoprostaglandin F2 alpha, and the mixture was allowed to react competitively with the antibody immobilized in a polystyrene tube . The activity of beta-galactosidase bound to the antibody was assayed by fluorometry . The enzyme activity was plotted against the amount of authentic 13,14-dihydro-15-ketoprostaglandin F2 alpha to obtain a calibration curve, and the compound was detectable over a range of 10 fmol to 10 pmol . Prostaglandins were extracted from human serum by the use of an octadecylsilyl silica column, and the extract gave an abnormally high level of 13,14-dihydro-15-ketoprostaglandin F2 alpha by enzyme immunoassay due to the presence of unidentified interfering substance(s), which was removed by high-performance liquid chromatography (HPLC) . The purified material gave a value in the order of 0.1 pmol per ml of human serum . Validity of the enzyme immunoassay was confirmed by radioimmunoassay and gas chromatography/mass spectrometry (GC-MS) of a methyl ester n-butoximedimethylisopropylsilyl ether derivative. J Biol Chem, 1986 Dec 5, 261(34), 16043 - 8 Transcription initiation at the Escherichia coli galactose operon promoters in the absence of the normal -35 region sequences; Ponnambalam S et al.; The gal operon regulatory region contains two overlapping promoters, P1 and P2, regulated by cyclic AMP and the cyclic AMP receptor protein (cAMP X CRP) . Starting with a mutation that eliminated P1, the promoter that is usually dependent on cAMP X CRP, we constructed a series of deletions that substituted increasing amounts of DNA sequence from upstream of P2, the promoter that usually functions in the absence of cAMP X CRP . Expression from P2 in vivo was halved by deletions that replace the -35 region with unrelated sequences, showing that the -35 sequence participates in promoter function, but is not essential . In vitro studies show that replacement of the -35 sequence increases the time for open complex formation at P2, but does not alter the transcription start point . We examined the effects of the same deletions at the wild type gal promoter region: again, the deletion that replaces the -35 region halves expression in vivo . However, in this case, in the absence of cAMP X CRP, the deletion switches expression from the P2 promoter to P1, the promoter that is usually dependent on cAMP X CRP . Moreover, although the deletion also removes the specific cAMP X CRP binding site, this P1 activity is sharply inhibited in a crp+ background . We argue that this is due to a direct contact between CRP and RNA polymerase bound at the P1 Pribnow box, and we discuss the role of the -35 sequence at these and other promoters. J Biol Chem, 1986 Dec 5, 261(34), 15934 - 5 Preliminary X-ray diffraction studies of the putative catalytic domain of gamma delta resolvase from Escherichia coli; Abdel-Meguid SS et al.; Two crystal forms of the putative catalytic domain (residues 1-140) of gamma delta resolvase from Escherichia coli have been obtained . Type I is isomorphous with crystals of the intact protein, and type II is suitable for high resolution structure analysis . Type II crystals belong to the orthorhombic space group C222(1), with a = 76.8 A, b = 191.3 A, and c = 63.4 A . They contain two molecules (15,500 daltons each)/asymmetric unit and show diffraction beyond 2.7-A resolution . Calculation of a rotation function using 7-A data shows the orientation of the noncrystallographic axes. Biochemistry, 1986 Dec 2, 25(24), 7854 - 66 Structure of the toxic domain of the Escherichia coli heat-stable enterotoxin ST I; Gariepy J et al.; Active fragments of the heat-stable enterotoxin ST I of Escherichia coli were chemically synthesized with the sequence Cys-Cys-Glu-Leu-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-(Tyr) and studied by proton (1H NMR) and carbon-13 (13C NMR) nuclear magnetic resonance spectroscopy as a function of pH and temperature . All of the nonexchangeable protons in the 1H NMR spectrum were assigned . Although all amide protons were present at temperatures below 25 degrees C and and pH values below 6, some of the resonances are broad and could not be assigned . The temperature dependence of these broad resonances indicates a change in conformation that is localized in the N-terminus . Other amide protons disappear at higher temperatures owing to chemical exchange with the solvent . Sufficient resonance assignments can be made at high and low temperatures to permit structural conclusions to be made . The chemical shifts of the alpha-carbon protons indicate the presence of substantial structure, which was further defined with the observed pattern of nuclear Overhauser enhancements (NOEs), coupling constants, and exchange rates . The NMR data identify a turn from Ala-14 to Cys-18 . A second likely turn is centered around the proline residue . An interresidue NOE between the alpha-carbon protons of Asn-12 and Gly-17 indicates that the molecule folds back on itself . The NMR information is sufficient to define the structure of the C-terminal region of ST I . Manual model building then indicated that one arrangement of the three disulfides is particularly compatible with the NMR data and van der Waals constraints . A model incorporating the disulfide arrangement proposed by Houghten and his co-workers {Houghten, R.A., Ostresh, J.M., & Klipstein, F.A . (1984) Eur . J . Biochem . 145, 157-162} and the NMR constraints was derived with the programs PROTO {Frayman, F . (1985) Ph.D . Thesis, Northwestern University} and NOEMOT {Lane, A.N., Lefevre, J.-F., & Jardetsky, O . (1986) Biochim . Biophys . Acta 867, 45-56}. Biochemistry, 1986 Dec 2, 25(24), 8002 - 10 Reconstitution of Escherichia coli ribosomes containing puromycin-modified S14: functional effects of the photoaffinity labeling of a protein essential for tRNA binding; Kerlavage AR et al.; In previous work we have shown that puromycin photoaffinity labels two proteins, L23 and S14, from separate sites of high affinity on Escherichia coli ribosomes {Jaynes, E . N., Jr., Grant, P . G., Giangrande, G., Wieder, R., & Cooperman, B . S . (1978) Biochemistry 17, 561-569; Weitzmann, C . J., & Cooperman, B . S . (1985) Biochemistry 24, 2268-2274}, that puromycin-modified S14 is separable from native S14 by reverse-phase high-performance liquid chromatography (RP-HPLC), and that ribosomal proteins prepared by RP-HPLC can be reconstituted into active 30S subunits {Kerlavage, A . R., Weitzmann, C . J., & Cooperman, B . S . (1984) J . Chromatogr . 317, 201-212} . In this work we definitively identify puromycin-modified S14 by tryptic fingerprinting, an analysis that also provides evidence that the single tryptophan-containing peptide in S14 is the site of puromycin photoincorporation . We show that reconstituted 30S subunits, in which all of the S14 present is stoichiometrically modified with puromycin and all other ribosomal components are present in unmodified form, lack Phe-tRNAPhe binding activity and further that 70S ribosomes containing such reconstituted 30S subunits have substantially diminished binding activity to both the A and P sites, as differentiated through use of tetracycline . Suitable control experiments strongly indicate that this loss of activity is a direct consequence of puromycin photoincorporation. Biochemistry, 1986 Dec 2, 25(24), 7799 - 802 Escherichia coli single-strand binding protein forms multiple, distinct complexes with single-stranded DNA; Bujalowski W et al.; Four distinct binding modes for the interaction of Escherichia coli single-strand binding (SSB) protein with single-stranded (ss) DNA have been identified on the basis of quantitative titrations that monitor the quenching of the SSB protein fluorescence upon binding to the homopolynucleotide poly(dT) over a range of MgCl2 and NaCl concentrations at 25 and 37 degrees C . This is the first observation of multiple binding modes for a single protein binding to DNA . These results extend previous studies performed in NaCl (25 degrees C, pH 8.1), in which two distinct SSB-ss DNA binding modes possessing site sizes of 33 and 65 nucleotides per bound SSB tetramer were observed {Lohman, T.M., & Overman, L . B . (1985) J . Biol . Chem . 260, 3594-3603} . Each of these binding modes differs in the number of nucleotides occluded upon interaction with ss DNA (i.e., site size) . Along with the previously observed modes with site sizes of 35 +/- 2 and 65 +/- 3 nucleotides per tetramer, a third distinct binding mode, at 25 degrees C, has been identified, possessing a site size of 56 +/- 3 nucleotides per bound SSB tetramer, which is stable over a wide range of MgCl2 concentrations . At 37 degrees C, a fourth binding mode is observed, possessing a site size of 40 +/- 2 nucleotides per tetramer, although this mode is observable only over a small range of salt concentration.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1986 Dec 2, 25(24), 8057 - 62 Amino acid specific ADP-ribosylation: specific NAD: arginine mono-ADP-ribosyltransferases associated with turkey erythrocyte nuclei and plasma membranes; West RE Jr et al.; Turkey erythrocytes contain NAD:arginine mono-ADP-ribosyltransferases which, like cholera toxin and Escherichia coli heat-labile enterotoxin, catalyze the transfer of ADP-ribose from NAD to proteins, to arginine and other low molecular weight guanidino compounds, and to water . Two such ADP-ribosyltransferases, A and B, have been purified from turkey erythrocyte cytosol . To characterize further the class of NAD:arginine ADP-ribosyltransferases, the particulate fraction was examined; 40% of erythrocyte transferase activity was localized to the nucleus and cell membrane . Transferase activity in a salt extract of a thoroughly washed particulate preparation was purified 36,000-fold by sequential chromatography on phenyl-Sepharose, (carboxymethyl) cellulose, concanavalin A-Sepharose, and NAD-agarose . Subsequent DNA-agarose chromatography separated two activities, termed transferases C and A', which were localized to the membrane and nucleus, respectively . Transferase C, the membrane-associated enzyme, was distinguished from the cytosolic enzymes by a relative insensitivity to salt and histone; transferase C was stimulated 2-fold by 300 mM NaCl in contrast to a 20-fold stimulation of transferase A and a 50% inhibition of transferase B . Similarly, histones, which stimulate transferase A 20-fold, enhanced transferase C activity only 2-fold . Transferase A', the nuclear enzyme, was retained on DNA-agarose . It was similar to transferase A in salt and histone sensitivity . Gel permeation chromatography showed slight molecular mass differences among the group of enzymes: A, 24,300 daltons (Da); B, 32,700 Da; C, and A', 25,500 Da . The affinities of transferase C for NAD and agmatine were similar to those of the cytosolic transferases A and B.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1986 Dec 2, 25(24), 7812 - 21 Purification of a calf thymus DNA-dependent adenosinetriphosphatase that prefers a primer-template junction effector; Hockensmith JW et al.; A purification procedure has been developed that resolves four chromatographically distinct DNA-dependent ATPase activities from calf thymus tissue . One of these activities has been purified to a nearly homogeneous protein, as judged by polyacrylamide gel electrophoresis . This protein has a specific activity of 18 mumol of ATP hydrolyzed per minute per milligram of protein and is active only in the presence of a DNA effector . The DNA-dependent ATPase activity is greatest in the presence of DNA containing a 3'-hydroxyl primer-template junction with a segment of adjacent single strand, i.e., a DNA polymerase substrate . Primer-template effectors that have had the 3'-hydroxyl group eliminated by the addition of a dideoxyribonucleotide are less active as cofactors for ATP hydrolysis than effectors which retain the 3'-hydroxyl group . Other DNAs can serve as cofactors, but with a reduced rate of ATP hydrolysis . DNA cofactors which are single stranded are much more effective at promoting ATPase activity than completely double-stranded cofactors, although the effectiveness of single-stranded DNA decreases as the length of the oligonucleotide decreases . An RNA/DNA hybrid does not promote ATPase activity . These data suggest that ATPase A may be involved in the recognition of primer-template junctions and the elongation phase of DNA synthesis. Mol Biochem Parasitol, 1986 Dec, 21(3), 247 - 57 The biosynthesis of trypanothione and N1-glutathionylspermidine in Crithidia fasciculata; Fairlamb AH et al.; Studies on the biosynthesis of trypanothione {N1,N8-bis(glutathionyl)-spermidine} in the insect trypanosomatid Crithidia fasciculata have led to the discovery of an additional sulfur-containing peptide conjugated to spermidine . Labelling studies with {3H}spermidine show that 50% of the total intracellular spermidine is incorporated into peptide conjugates, the major component being N1-glutathionylspermidine . This compound has previously been identified in Escherichia coli, as the principal low molecular weight thiol in stationary phase, but not the logarithmic phase of growth . In contrast, in C . fasciculata, this compound is present in all phases of growth . In the presence of glutathione and ATP, extracts of C . fasciculata can catalyse conversion of spermidine to N1-glutathionylspermidine and trypanothione . Both N1- and N8-regioisomers of glutathionylspermidine will replace spermidine in the reaction, suggesting they may be intermediates in the biosynthetic pathway to trypanothione . The antiprotozoal drugs berenil, pentamidine, ethidium bromide, imidocarb, methylglyoxal-bis(guanylhydrazone) and 1,3-diacetylbenzene-bis(guanylhydrazone) had no effect on the synthesis of N1-glutathionylspermidine or trypanothione in vitro. J Bacteriol, 1986 Dec, 168(3), 1066 - 9 Mapping and complementation studies of the gene for release factor 1; Ryden M et al.; In Escherichia coli the release factor 1 protein (RF1) recognizes and terminates translation at UAG and UAA codons . Using the technique of ColE1 plasmid integration in polA strains, we have mapped the cloned gene for RF1 to 27 min on the E . coli chromosome . This is the same location as that of the uar gene in which temperature-sensitive mutations increase the suppression of UAG and UAA alleles . In this study we proved that the uar mutation lies in the gene for RF1 by complementation of the uar phenotype with plasmids carrying the RF1 gene and by cloning the uar allele onto the RF1 plasmid by means of homologous recombination . In addition, complementation and P1 mapping data suggest that sueB is also a mutation in the same position as the RF1 gene . We propose that the gene for RF1 be named prfA after protein release factor. Exp Cell Res, 1986 Dec, 167(2), 311 - 26 DNA strand breaks in rat tissues as detected by in situ nick translation; Iseki S; The nick-translation procedure without external addition of DNase was performed in situ on sections of various rat organs to detect possible DNA single-strand breaks (nicks) in normal tissues . The freshly frozen sections were briefly fixed in ethanol/acetone and nick-translated in the presence of E . coli DNA polymerase I . A significant difference in the amount of nuclear reaction was found among the different cell populations as detected by autoradiography following incorporation of tritiated TTP as well as by histochemical staining following incorporation of biotin-dUTP into nuclei . Such incorporation of triphosphates was localized in the DNA and was entirely dependent on E . coli DNA polymerase I . The nuclei with the highest reactivity were found in skeletal muscle cells, lymphocytes in various lymphatic organs, the proliferative cells in the gastrointestinal tract, stratified squamous epithelial cells, duct epithelial cells of salivary gland and the maturing spermatids in the seminiferous tubules . These results suggest that, under the conditions adopted, the cells in various tissues reveal different chromatin structures resulting in varying rates of nick translation reaction . Such difference(s) in chromatin structure, presumably including that in the number of DNA single-strand breaks or in the level of endogenous nuclease activity, may be associated with the mechanisms involved in cell growth and differentiation. J Biochem (Tokyo), 1986 Dec, 100(6), 1595 - 605 Analysis of Escherichia coli ribosomal proteins by an improved two dimensional gel electrophoresis . II . Characterization of four new proteins; Wada A; Four new proteins, A, B, C, and D, found in Escherichia coli ribosomes by an improved two dimensional gel electrophoresis were characterized by oxidation, reduction, and carboxymethylation of cysteine residues, and CsCl fractionation . The cysteine contents of proteins A, B, C, and D were determined to be 1 +/- 0, 3 +/- 1, 5 +/- 1, and 0 +/- 0 by carboxymethylation with iodoacetic acid . The components of protein complexes, which formed numerously under non-reducing conditions, were analyzed . Including protein A, B, and C, every ribosomal protein (r-protein) having cysteine residue(s) except unconfirmed S1 was proved to form such complexes with various combinations . The cysteine residue in protein A, in particular, was highly reactive to make intermolecular S-S bridges so that spot A almost disappeared on the second dimension gel under the non-reducing conditions . Proteins B and C shifted their spots by reduction towards upper left side as do all known r-proteins having plural cysteine residues except S1 . This suggests that proteins B and C change their conformation by intramolecular S-S bridges . The CsCl density gradient centrifugation of high salt washed 70S ribosomes showed that protein A belonged to the insoluble split proteins, proteins B and C to the core particles, and protein D and a small population of B to the soluble split proteins . The electrophoretic behaviors, CsCl fractionation and stoichiometry of the four new proteins suggested strongly that they were intrinsic ribosomal constituents different from known ribosomal proteins or factors. J Biochem (Tokyo), 1986 Dec, 100(6), 1583 - 94 Analysis of Escherichia coli ribosomal proteins by an improved two dimensional gel electrophoresis . I . Detection of four new proteins; Wada A; Kaltschmidt and Wittmann's two dimensional gel electrophoresis was improved in the following points . Preruns using radical scavengers were carried out to eliminate free radicals remaining in gels . Gelation of sample solutions was not performed to avoid immobilization of proteins in the sample gels . Instead, for preparing sample gels, prior to the first dimension (1-D) electrophoresis, another electrophoresis was performed to charge proteins into gel pieces polymerized previously . Proteins migrated together with charged reductants to avoid formation of artificial disulfide bridges during migration . The second dimension (2-D) electrophoresis was carried out at a more acidic pH, 3.6, to get better separation of very small and basic proteins . With these modifications, quantitative yield and reproducibility became better, and many faint spots disappeared not only at the high molecular weight side but also in the region containing primary spots of ribosomal proteins . The proportionality of the migration distance to the logarithm of molecular weight was also increased . On the improved 2-D electrophoretogram of Escherichia coli ribosomal proteins, four new spots, called protein A, B, C, and D, were found in the basic region . Proteins A, B, and C belong to 50S subunits but D to 30S . Their molecular weights were determined electrophoretically as 6,400, 4,900, 8,200, and 5,900, respectively . Their copy numbers in crude ribosomes were estimated to be 0.6, 0.4, 0.3, and 0.1, respectively, by using 14C-labeling. Mol Gen Genet, 1986 Dec, 205(3), 561 - 2 Spot-transformation with plasmids; Vogel W et al.; A method is described which allows transformation of bacterial cells on the surface of agar plates . It is suitable for transferring large numbers of different plasmids, such as gene libraries, into a new genetic background . One nanogram of plasmid DNA is sufficient for transformation. Mol Gen Genet, 1986 Dec, 205(3), 535 - 9 The Escherichia coli heat shock regulatory gene is immediately downstream of a cell division operon: the fam mutation is allelic with rpoH; Crickmore N et al.; Several mutations which affect critical cell functions in Escherichia coli map at 76 min on the chromosome . The genes which map in this region are the cell division genes fts Y, E, X and S, the heat shock regulatory gene rpoH/htpR/hin, the lipoprotein biogenesis gene fam and another essential gene dnaM . We determined the relative positions of most of these genes and show that the rpoH gene lies immediately downstream of the last gene (ftsX) of a cell division operon and is transcribed in the same direction . We also show that the fam-715 mutation is allelic with rpoH and so the conditional lipoprotein deficiency of the fam mutation must be due to the pleiotropic nature of the heat shock response. Mol Gen Genet, 1986 Dec, 205(3), 469 - 75 Cytosine-specific DNA modification interferes with plasmid establishment in Escherichia coli K12: involvement of rglB; Noyer-Weidner M et al.; Several chimeric pBR322/328 derivatives containing genes for cytosine-specific DNA methyltransferases (Mtases) can be transformed into the Escherichia coli K12/E . coli B hybrid strains HB101 and RR1 but not into other commonly used E . coli K12 strains . In vitro methylation of cytosine residues in pBR328 and other unrelated plasmids also reduces their potential to transform such methylation sensitive strains, albeit to a lesser degree than observed with plasmids containing Mtase genes . The extent of reduced transformability depends on the target specificity of the enzyme used for in vitro modification . The role of a host function in the discrimination against methylated plasmids was verified by the isolation of K12 mutants which tolerate cytosine methylated DNA . The mutations map in the vicinity of the serB locus . This and other data indicate that the host rglB function is involved in the discrimination against modified DNA. Mol Gen Genet, 1986 Dec, 205(3), 428 - 33 Organization of the ribosomal RNA genes in Mycoplasma hyopneumoniae: the 5S rRNA gene is separated from the 16S and 23S rRNA genes; Taschke C et al.; In order to study the organization of the ribosomal RNA genes of Mycoplasma hyopneumoniae the rRNA genes were cloned in phage vectors lambda EMBL3 and lambda EMBL4 . By subcloning the restriction fragments into various plasmids and analysing the resulting clones by Southern and Northern blot hybridization, a restriction map of the rRNA genes was generated and the organization of the rRNA genes was determined . The results show that the genes for the 16S and 23S rRNAs are closely spaced and occur only once in the genome, whereas the 5S rRNA gene is separated from the other two genes by more than 4 kb. J Antimicrob Chemother, 1986 Dec, 18(6), 661 - 6 Transport of tetracyclines into Escherichia coli requires a carboxamide group at the C2 position of the molecule; Chopra I; Structural features of the tetracycline molecule required for transport into Escherichia coli were investigated by examining the uptake of various tetracycline analogues . Uptake was assessed by determining the ability of analogues to induce Tn10 encoded resistance to tetracycline and by direct transport assays using tetracycline susceptible bacteria . Seven tetracyclines (including tetracycline itself) were studied . Two of these compounds, chlorotetracyclinonitrile and tetracyclinonitrile were not accumulated by E . coli, which suggests that the carboxamide group at the C2 position of the tetracycline molecule is required for transport into bacteria. EMBO J, 1986 Dec 1, 5(12), 3313 - 9 Facilitated diffusion of a DNA binding protein on chromatin; Hannon R et al.; Facilitated diffusion accounts for the rapid rate of association of many bacterial DNA binding proteins with specific DNA sequences in vitro . In this mechanism the proteins bind at random to non-specific sites on the DAN and diffuse (by 'sliding' or 'hopping') along the DNA chain until they arrive at their specific functional sites . We have investigated whether such a mechanism can operate in chromatin by using a bacterial DNA binding protein, Escherichia coli RNA polymerase, that depends on linear diffusion to locate initiation sites on DNA . We have measured the competition between chromatin and its free DNA for the formation of initiation complexes . Only the short linker segments exposed by the removal of histone H1 are available for interaction with the polymerase, but the sparsely distributed promoter sites on the linker DNA of such a polynucleosome chain are located at the same rate as those on DNA . We conclude that the polymerase is free to migrate between the separate linker DNA segments of a polynucleosome chain to reach a promoter site . This chain thus permits the 'hopping' of proteins between neighboring linker segments in their search for a target site on the accessible DNA. DNA, 1986 Dec, 5(6), 539 - 44 Increased amplification of plasmids pBR322 and pBR327 by low concentrations of chloramphenicol; Frenkel L et al.; The replication and amplification of plasmids pBR322 and pBR327 is maximal during partial inhibition of protein synthesis by low concentrations (10-20 micrograms/ml) of chloramphenicol in rich medium (LB) . In this manner, 5- to 10-fold greater yields of plasmid DNA at 2- to 5-fold greater initial purity (less protein and chromosomal DNA content) can be obtained than by using the standard high concentrations of chloramphenicol . These results have been obtained with an improved method of plasmid quantitation in unfractionated bacterial lysates. DNA, 1986 Dec, 5(6), 463 - 71 Rainbow trout growth hormone: molecular cloning of cDNA and expression in Escherichia coli; Agellon LB et al.; We have isolated several recombinant clones carrying the complementary DNA (cDNA) sequence of the rainbow trout (rt) Salmo gairdneri growth hormone (GH) mRNA by immunoblot screening using an antiserum to chum salmon (Oncorhyncus keta) GH . The nucleotide sequence of one of the rtGH cDNA clones (pAF51) was determined . The rt cDNA sequence in pAF51 encodes a hybrid polypeptide of 199 amino acid residues containing 9 amino acid residues of the bacterial beta-galactosidase, one residue from the codon at the junction of the beta-galactosidase gene, and the rtGH cDNA sequence, an additional residue from the presumptive signal peptide of the pre-rtGH and the entire sequence of the mature rtGH (188 amino acid residues) . Pairwise matrix comparisons of the hydropathy profiles of bovine, human, rat, and rainbow trout GH polypeptides indicate that regions of similarity exist between the rtGH and mammalian GH . In particular, there are two major regions of similarity found near the amino-terminal region and at the carboxy-terminal region . These regions correspond to hydrophilic domains of the GH molecules . The possible significance of these domains is discussed. Can J Microbiol, 1986 Dec, 32(12), 967 - 9 Lack of protection against ascending Escherichia coli pyelonephritis in diabetic rats following immunization with purified lipopolysaccharide; Bryan LE et al.; Purified lipopolysaccharide of Escherichia coli produced specific antibody when injected intraperitoneally or given to rats orally . Either route of immunization did not prevent ascending pyelonephritis in a diabetic rat model . The use of purified LPS excludes the potential contribution of other virulence factors of E . coli as protective antigens in the prevention of ascending pyelonephritis and confirms that anti-lipopolysaccharide antibody is not protective for ascending pyelonephritis. Arch Biochem Biophys, 1986 Dec, 251(2), 413 - 20 Polyamine binding sites on Escherichia coli ribosomes; Kakegawa T et al.; To determine the binding sites of polyamines on Escherichia coli ribosomes, ribosomal proteins crosslinked with polyamines through bifunctional reagents were analyzed . When 1,5-difluoro-2,4-dinitrobenzene was used as a crosslinking reagent, spermine was bound to S3, S8, S9, L1, L2, L3, L5, L6, L13, L18, L24, and L27 proteins . When dimethyl suberimidate was used, spermine was bound to S1, S3, S4, S5, S7, S8, S9, S15, L1, L2, L3, L6, L18, and L24 proteins . In addition to crosslinking with the above proteins, spermidine, when crosslinked with dimethyl suberimidate, bound to S2, S14, S20, L4, L5, L9, L13, and L16 proteins . The relationship between the binding site(s) of polyamines on ribosomes and the function of polyamines is discussed. Am J Vet Res, 1986 Dec, 47(12), 2554 - 65 Endotoxemia in neonatal calves given antiserum to a mutant Escherichia coli (J-5); Morris DD et al.; Endotoxemia was characterized in neonatal calves given a small amount of colostrum and smooth Escherichia coli endotoxin by small-dosage (0.5 microgram/kg of body weight), slow (5-hour) IV infusion to mimic natural conditions . Responses were compared among 22 calves freely allotted to groups treated with saline solution (group I), preimmunization plasma (PP, group II), or antiserum to the rough mutant of E coli O111:B4 (J-5, group III) before endotoxin was infused . Bovine J-5 antiserum was produced by immunization of 4 cattle with J-5 boiled cell bacterin . The antiserum titers of immunoglobulin (Ig) M, IgG1, and IgG2 to the J-5 boiled cells, as determined by enzyme-linked immunosorbent assay, were 240, 7,680, and 960, respectively . The PP had enzyme-linked immunosorbent assay titers to J-5 of 240, 480, and 60 of IgM, IgG1, and IgG2, respectively . Endotoxemia in the 3 groups was characterized by significant (P less than 0.05) time-related changes in rectal temperature, heart rate, respiratory rate, capillary refill time, oral mucous membranes, nose moistness, scleral injection, attitude, PCV, total plasma protein concentration, WBC count and differential, plasma glucose, and lactate concentrations . The only significant treatment effects on clinical or laboratory values were higher mean total plasma protein concentrations in groups II and III 10 to 30 hours after endotoxin infusion was started than that in group I and increasing mean most-severe attitude abnormality score in groups I, III, and II (P less than 0.05) . The administration of bovine J-5 antiserum to neonatal calves resulted in significantly higher serum IgG1 and IgG2 titers to J-5 boiled cells (P less than 0.05), and cross-reactive IgG2 to the challenge endotoxin (P less than 0.01) than did treatment with PP or saline solution; however, this antiserum did not mitigate the effects of sublethal endotoxemia . There was a significant negative correlation between IgG2 to J-5 at base line and the mean attitude abnormality score at 4.5 hours after infusion was started (P less than 0.05). Am J Vet Res, 1986 Dec, 47(12), 2520 - 4 Endotoxin-induced changes in plasma concentrations of thromboxane and prostacyclin in neonatal calves given antiserum to a mutant Escherichia coli (J-5); Morris DD et al.; Plasma concentrations of the stable hydrolysis products of thromboxane (Tx) A2, TxB2, and prostacyclin (6-keto-prostaglandin F1 alpha; PGF1 alpha) were evaluated in 22 neonatal calves given endotoxin (0.5 microgram/kg) by slow IV infusion over a 5-hour period . The effect of pretreatment with bovine antiserum to a mutant of Escherichia coli O111:B4 (J-5) on plasma concentrations of these eicosanoids was determined . Plasma concentrations of TxB2 and 6-keto-PGF1 alpha were increased in response to endotoxin infusion . The mean plasma concentrations of TxB2 peaked 1 hour after endotoxin infusion began and was significantly increased from base line through 8 hours . Mean plasma 6-keto-PGF1 alpha concentration increased more slowly and was increased from base line only at 2 hours after endotoxin infusion began . Administration of bovine J-5 antiserum had no significant effect on endotoxin-induced thromboxane and prostacyclin production. Am J Vet Res, 1986 Dec, 47(12), 2514 - 9 Endotoxin-induced changes in the hemostatic system in neonatal calves: the effect of antiserum to a mutant Escherichia coli (J-5); Morris DD et al.; Changes in the hemostatic system were studied in 22 neonatal calves given a small dosage of Escherichia coli endotoxin (0.5 microgram/kg) by slow (5-hour) IV infusion . The effect of pretreatment with an antiserum to mutant of E coli O111:B4 (J-5) was evaluated . The platelet count, plasma fibrinogen concentration, prothrombin time, and activated partial thromboplastin time changed significantly from base line during and after endotoxin infusions in all calves . The mean platelet count was significantly decreased from 1 through 24 hours after endotoxin infusion was started . Mean plasma fibrinogen was decreased 2 through 12 hours after endotoxin infusion was started . The mean prothrombin time and activated partial thromboplastin time were significantly greater than base line at 3 to 6 hours and 3 to 12 hours, respectively, after endotoxin infusion was started . Serum concentration of fibrinolytic degradation products remained less than 10 micrograms/ml . Bovine J-5 antiserum did not prevent the endotoxin-induced changes in the hemostatic system of these neonatal calves. Proc Natl Acad Sci U S A, 1986 Dec, 83(24), 9645 - 9 Positive and negative roles of an initiator protein at an origin of replication; Filutowicz M et al.; The properties of mutants in the pir gene of plasmid R6K have suggested that the pi protein plays a dual role; it is required for replication to occur and also plays a role in the negative control of the plasmid copy number . In our present study, we have found that the pi level in cell extracts of Escherichia coli strains containing R6K derivatives is surprisingly high (approximately equal to 10(4) dimers per cell) and that this level is not altered in cells carrying high copy number pir mutants . The wild-type and a high copy mutant (Cos405) pir gene were inserted downstream of promoters of different strengths to measure the copy number of an R6K gamma replicon as a function of a 1000-fold range of intracellular pi concentrations . The data demonstrate that reducing the intracellular level of pi to 5% of its normal value can result in a substantial increase in copy number of a gamma origin replicon and that a pi level less than 1% of normal is still permissive for replication . Conversely, increasing the pi level even a few-fold above normal results in a marked inhibition of replication of plasmids containing a single, two, or all three of the R6K origins (alpha, beta, and gamma) . We have also shown that the replication inhibition mediated by excess pi is greatly reduced by the pir405 Cos mutation . These results demonstrate that the total level of pi protein is not rate-limiting for a gamma replicon . We have also determined the sensitivity of the pir gene promoter to a wide range of pi concentrations . The activity of this promoter is stimulated by very low pi levels and is almost entirely inhibited when the protein is overproduced 2-fold. Proc Natl Acad Sci U S A, 1986 Dec, 83(24), 9289 - 93 Nucleotide binding by a 24-residue peptide from the RecA protein of Escherichia coli; Knight KL et al.; We have recently demonstrated that two ATP analog affinity labels, 8-azidoadenosine 5'-triphosphate (N3ATP) and 5'-p-fluorosulfonylbenzoyladenosine (5'FSBA), covalently modify RecA protein of Escherichia coli at a specific tyrosine residue (Tyr-264) located within a 24-residue tryptic peptide (T-31) spanning residues 257-280 . Here we show that N3ATP efficiently modifies purified peptide T-31 and show that the interaction is specific by the following criteria: photolabeling of peptide T-31 is saturable with respect to the N3ATP concentration; photolabeling is competitive with ATP and adenosine but not with adenine, UTP, or TTP; and other peptides derived from RecA protein were poor substrates for photolabeling except for one fragment that showed a nonspecific interaction with the photoaffinity analog . Analysis of N3ATP-modified T-31 shows that the photolabel attaches to more than one site within the peptide . These data argue that peptide T-31 contains some sites of contact for adenine and ribose moieties of ATP when it is bound to RecA protein. Parasitology, 1986 Dec, 93 ( Pt 3), 599 - 610 Immunochemical analysis of Taenia taeniaeformis antigens expressed in Escherichia coli; Bowtell DD et al.; Previously we reported the isolation of several Escherichia coli clones expressing fragments of Taenia taeniaeformis antigens as beta-galactosidase fused proteins (Bowtell, Saint, Rickard & Mitchell, 1984) . Here we describe the isolation of additional antigen-expressing clones from a larval cDNA library and the assignment of these clones to 7 antigen families . These were isolated with a polyspecific rabbit antiserum raised to the oncosphere . Since this serum was capable of reacting with a large number of antigens, it was important to develop techniques for rapidly determining the identity of the native T . taeniaeformis molecule corresponding to a cloned antigen gene . These included active immunization of rabbits with fused proteins and several techniques involving affinity purification on immobilized fused proteins . The reactivity of the antigen-positive clones with sera from humans infected with related parasites was also assessed . Finally, immunization of mice with several fused proteins failed to protect against subsequent infection, although antigens previously identified as candidate host-protective antigens (Bowtell, Mitchell, Anders, Lightowlers & Rickard, 1983) have yet to be identified in the expression library. Mol Cell Biol, 1986 Dec, 6(12), 4214 - 20 Activation of ras p21 transforming properties associated with an increase in the release rate of bound guanine nucleotide; Lacal JC et al.; An Ala-to-Thr substitution at position 59 activates the transforming properties of the p21ras protein without impairment of GTPase activity, a biochemical alteration associated with other activating mutations . To investigate the basis for the transforming properties of the Thr-59 mutant, we characterized guanine nucleotide release . This reaction exhibited a slow rate and stringent temperature requirements . To further dissect the release reaction, we used monoclonal antibodies directed against different epitopes of the p21 molecule . One monoclonal specifically interfered with nucleotide release, while others which recognized different regions of the molecule blocked nucleotide binding . Mutants with the Thr-59 substitution exhibited a three- to ninefold-higher rate of GDP and GTP release than normal p21 or mutants with other activating lesions . This alteration in the Thr-59 mutant would have the effect of increasing its rate of nucleotide exchange . In an intracellular environment with a high GTP/GDP ratio, this would favor the association of GTP with the Thr-59 mutant . Consistent with knowledge of known G-regulatory proteins, these findings support a model in which the p21-GTP complex is the biologically active form of the p21 protein. J Natl Cancer Inst, 1986 Dec, 77(6), 1273 - 9 Establishment of four mouse hybridoma cell lines producing monoclonal antibodies reactive with ras oncogene product p21; Kuzumaki N et al.; With the use of proteins derived from Escherichia coli cells expressing the v-H-ras gene product as immunogens and an enzyme-linked immunosorbent assay with whole cells for a screening method, 4 BALB/c mouse hybridoma cell lines (rp-12, rp-28, rp-35, and rp-38) were isolated that produced monoclonal antibodies (MoAbs) showing higher reactivity with murine ras gene-activated cell lines than with normal cell lines . All the MoAbs complexed p21ras from the ras gene-activated cell lines in Western immunoblot analysis and demonstrated a binding property of p21ras to guanine nucleotides . The indirect immunofluorescence assay revealed that MoAbs rp-12 and rp-28 stained the murine and human H- or K-ras-activated cell lines, and MoAbs rp-35 and rp-38 not only stained these cell lines but also weakly stained a human N-ras-activated cell line . All these MoAbs stained the murine fibroblast lines with lower intensity, but they did not stain a human fibroblast line . Further, positive reactions with MoAb rp-12 were seen against human melanomas, but there was no reaction against nevi . The rp-12, rp-28, rp-35, and rp-38 antibodies are useful additions to the MoAbs reacting with p21ras reported previously. J Biol Response Mod, 1986 Dec, 5(6), 527 - 38 Stimulation of human PMNs in vitro by a succinimide molecular complex of methylfurylbutyrolactone; Woolverton CJ et al.; The immunopotentiating effects on human neutrophils of a new synthetic immune biological response modifer were studied . The compound is a succinimide crystalline molecular complex of methylfurylbutyrolactone (MFBL) . The MFBL succinimide (MFBL-S) derivative was tested for its in vitro effects on polymorphonuclear leukocyte (PMN) functions . At microgram quantities, MFBL-S stimulated a twofold increase in: directed migration of PMNs; adherence to nylon; and uptake of E . coli lipopolysaccharide . The MFBL-S enhanced phagocytosis by PMNs of S . epidermidis and E . coli . Additionally, intracellular killing of S . epidermidis by MFBL-S treated PMNs was significantly increased at all doses studied, whereas killing of E . coli was only significantly different from controls at concentrations of 10 micrograms/ml. Cell Struct Funct, 1986 Dec, 11(4), 379 - 82 Ultrastructural modifications of the cavities formed by folliculo-stellate cells in chicken adenohypophysis under septic shock conditions; Fernandez AJ et al.; Effects of septic shock by repeated inoculations with Escherichia coli on the ultrastructure of the folliculo-stellate cells and cavities of the adenohypophysis of the chicken were investigated in order to determine the function of these cavities . The principal morphological modifications were dilation of the Golgi apparatus, endoplasmic reticulum and autophagic vacuoles, and necrosis phenomena in the stellate cells . The follicular cavities showed dilation, and there was heterogeneous dense material and granular elements in the follicular lumen . Based on results reported in the literature, the observations reported here are evidence of a "cleaning-role", for the removal of cell debris, when there is endocrine disfunction. Proc Natl Acad Sci U S A, 1986 Dec, 83(23), 8957 - 61 Anti-beta-interferon antibodies inhibit the increased expression of HLA-B7 mRNA in tumor necrosis factor-treated human fibroblasts: structural studies of the beta 2 interferon involved; May LT et al.; Recombinant Escherichia coli-derived human tumor necrosis factor (TNF) induces the 1.3-kilobase beta 2 interferon (IFN-beta 2) mRNA in human diploid fibroblasts (FS-4 strain) . IFN-beta 2 is serologically related to the well-characterized IFN-beta 1 (respective antisera cross-neutralize the heterologous protein) . Polyclonal and monoclonal anti-IFN-beta antibodies inhibit the increase in class I HLA gene expression (HLA-B7 mRNA) in TNF-treated FS-4 cells suggesting that TNF-induced IFN-beta 2 mediates the enhancing effect of TNF on HLA gene expression in human fibroblasts . The structure of this autocrine human interferon has been determined . A cDNA library was prepared from polyadenylylated RNA extracted from TNF-induced FS-4 cells, and eight IFN-beta 2 cDNA clones were isolated using a 21-nucleotide synthetic oligonucleotide probe . The 1128-nucleotide sequence of IFN-beta 2 mRNA and the 212-amino acid sequence of the IFN-beta 2 protein were deduced from these cDNA clones . The amino acid sequences of the serologically related human IFN-beta 1 and -beta 2 were compared using the Sellers TT metric algorithm for locating similarities and using the pattern scoring method for evaluating the observed similarities . IFN-beta 1 and -beta 2 each contain a segment that is approximately 100 amino acids including 39 amino acids that are aligned and identical in the two proteins . The hydropathic index plots across these segments in the two proteins are also strikingly similar . The region of similarity between IFN-beta 1 and -beta 2 includes a section that is also highly conserved in all IFN-alpha species sequenced . Thus IFN-beta 2 shares structural similarities with other human interferons that also preferentially increase class I HLA gene expression. Proc Natl Acad Sci U S A, 1986 Dec, 83(23), 8898 - 902 Oxidation of reduced menaquinone by the fumarate reductase complex in Escherichia coli requires the hydrophobic FrdD peptide; Cecchini G et al.; Plasmids carrying cloned segments of the frd operon of Escherichia coli have been used in genetic complementation studies to identify two independent mutants defective in the frdD gene, which encodes the hydrophobic FrdD polypeptide of the fumarate reductase complex . Mutations in the frdA and frdB genes have also been mapped by this technique . One of the FrdD peptide mutants, DW109 (frdD-109), showed that fumarate reductase was not as tightly bound to the membrane in this mutant . In addition, the mutation in the FrdD peptide caused an almost total loss of the ability of the enzyme to oxidize either menaquinol-6, a physiological donor for fumarate reduction, or reduced benzyl viologen . However, the mutation did not impair the ability of the membrane-bound fumarate reductase complex to function with succinate as substrate, as evidenced by unchanged turnover numbers for phenazine methosulfate and 2,3-dimethoxy-5-methyl-6-pentyl-1,4-benzoquinone (a quinone analogue) reductase activities . These data establish the essential role of the FrdD polypeptide both in the interaction of the enzyme with reduced menaquinone and thus in anaerobic respiration with fumarate as electron acceptor, and in binding the enzyme to the membrane. Mutat Res, 1986 Dec, 172(3), 211 - 22 Rhodium(III) complexes as genotoxic agents: photochemical effects and their implications; LaVelle JM et al.; The genetic toxicology of coordination compounds of transition metals has been of considerable interest since the application of cis-platinum(II) to the therapy of solid tumors . The nature of reactions of such compounds with DNA is still unclear, despite intensive investigation . In this study, several coordination compounds of rhodium(III) were tested for DNA-damaging activity and mutagenicity in bacterial assays in an attempt to understand both the chemical species involved in interactions with DNA and any structural requirements for such interactions . For several complexes it appears that dissociation of a ligand from the complex precedes reactions with DNA . This conclusion stems from the finding that photosensitive complexes of rhodium(III) are often many times more toxic to repair-deficient bacterial stains of E . coli K12 when incubated in the light than when incubated in the dark . Similar responses were seen for mutagenicity in S . typhimurium strain TA100 . However, reversion of strain TA102 was largely independent of light exposure . Comparisons between mutagenicity and DNA-damaging activity revealed that the 3 activities measured sorted with some independence among the different compounds tested . Thus, the profiles for crosslink formation and/or generation of oxidative mutagens (mutagenicity in S . typhimurium strain TA102), mutagenicity in TA100 and DNA-damaging activity for the various groups of complexes showed many of the theoretically possible combinations of response in the assays . It is possible, then, that there are different structural requirements for DNA-damaging activity and mutagenicity respectively . This may indicate that synthesis of coordination compounds with specific genotoxic properties is possible . Such syntheses may provide complexes for study of DNA-metal interactions and could, later, direct an approach to the design of new antitumor agents. Mutat Res, 1986 Dec, 163(3), 209 - 24 Mutagenesis by 8-methoxypsoralen plus near-UV treatment: analysis of specificity in the lacI gene of Escherichia coli; Yatagai F et al.; We have studied the specificity of mutation induced by PUVA treatment in the lacI gene of E . coli . Cells were exposed to near UV (approximately 365 nm) in the presence of 8-methoxypsoralen under conditions yielding about 7% survival and a 10-fold increase in mutation frequency . The cloning and sequencing of 131 mutants recovered following PUVA treatment revealed that almost all classes of mutation including base substitutions, frameshifts and deletions were induced . The distribution of mutations was non-random and a region of the lacI gene was found to be virtually silent for all classes of mutation . Intriguingly, the broad spectrum of mutation is accompanied by the recovery of mutation at two spontaneous hotspots . We observed a 7-fold increase at a frameshift hotspot involving the gain or loss of a tetramer tandemly repeated 3 times at this site and a 23-fold increase at an A:T----G:C transition hotspot located at the +6 mutational spectrum recovered following PUVA treatment was unique and a detailed analysis of the different classes of mutations indicates a role for DNA repair of both monoadducts and cross-links in the production of mutation. J Exp Med, 1986 Dec 1, 164(6), 1876 - 88 Adhesion-promoting receptors on human macrophages recognize Escherichia coli by binding to lipopolysaccharide; Wright SD et al.; We report here that human macrophages bind Escherichia coli by recognizing bacterial lipopolysaccharide (LPS) . Purified LPS was inserted into erythrocyte membranes, and the resulting LPS-coated red cells were bound by macrophages with the same temperature and cation dependence as observed for E . coli . When receptors for LPS were withdrawn from the plasma membrane by spreading the macrophages on LPS-coated surfaces, the binding of E . coli was blocked . We have also identified the receptors on macrophages that recognize LPS . Macrophages express three structurally homologous cell surface proteins, CR3, lymphocyte function-associated antigen (LFA-1), and p150,95 . We used surface-bound monoclonal antireceptor antibodies to selectively remove these proteins from the apical surface of macrophages . We found that each of these proteins mediated the binding of E . coli to macrophages. J Bacteriol, 1986 Dec, 168(3), 1491 - 4 Control of metF gene expression in maxicell preparations of Escherichia coli K-12: reversible action of the metJ protein and effect of vitamin B12; Emmett MR et al.; Expression of methionine regulon elements was controlled by the metJ protein gpMetJ . A maxicell system with cloned copies of the metF transcription unit allowed reversible action of gpMetJ . Expression of the metF transcription unit in maxicells was reduced by exogenous vitamin B12 at concentrations of 0.5 nM or greater. J Bacteriol, 1986 Dec, 168(3), 1487 - 90 Biological role of DNA methylation: sequence-specific single-strand breaks associated with hypomethylation of GATC sites in Escherichia coli DNA; Szyf M et al.; The effect of methylation of GATC sites in Escherichia coli DNA on the formation of single-strand breaks was studied with dam+, dam mutant, and Dam-overproducer strains . Single-strand breaks have been observed in dam mutant cells predominantly at TpT and, to a lesser extent, at CpC . In dam mutant cells harboring pTP166 (a plasmid containing the dam gene), no such nicks were observed. J Bacteriol, 1986 Dec, 168(3), 1479 - 83 Nucleotide sequence of the hag gene encoding flagellin of Escherichia coli; Kuwajima G et al.; We determined the DNA sequence of the hag gene of Escherichia coli K-12 and deduced the primary structure of the flagellin consisting of 497 amino acid residues . Comparison of the amino acid sequence with those of other bacterial flagellins revealed a high homology in the NH2- and COOH-terminal regions. J Bacteriol, 1986 Dec, 168(3), 1415 - 21 Three classes of Escherichia coli mutants selected for aerobic expression of fumarate reductase; Iuchi S et al.; Fumarate reductase (encoded by frd) and succinate dehydrogenase (encoded by sdh) of Escherichia coli are both known to catalyze the interconversion of fumarate and succinate . Fumarate reductase, however, is not inducible aerobically and therefore cannot participate in the dehydrogenation of succinate . Three classes of suppressor mutants, classified as frd oxygen-resistant {frd(Oxr)}, constitutive {frd(Con)}, and gene amplification {frd(Amp)} mutants, were selected from an sdh strain as pseudorevertants that regained the partial ability to grow aerobically on succinate . All contained increased aerobic levels of fumarate reductase activity . In frd(Oxr) mutants expression of the operon showed increased resistance to aerobic repression . Under anaerobic conditions expression of the operon became less dependent on the fnr+ gene product, a pleiotropic activator protein for genes encoding anaerobic respiratory enzymes . Exogenous fumarate, however, was still required for full induction, and repression by nitrate was undiminished . Thus, aerobic repression and anaerobic nitrate repression appear to involve separate mechanisms . In frd(Con) mutants expression of the operon became highly resistant to aerobic repression . Under anaerobic conditions expression of the operon no longer required the fnr+ gene product or exogenous fumarate and became immune to nitrate repression . In partial diploids bearing an frd(Oxr) or an frd(Con) allele and phi(frd+-lac) there was no mutual regulatory influence between the two genetic loci . Thus, the frd mutations act in cis and hence are probably in the promoter region . In frd(Amp) mutants the frd locus was amplified without significant alteration in the pattern of regulation. J Bacteriol, 1986 Dec, 168(3), 1408 - 14 Expression of the adenylate cyclase gene during cell elongation in Escherichia coli K-12; Utsumi R et al.; Expression of the adenylate cyclase gene (cya) in synchronized Escherichia coli cells was investigated by using the cya-lacZ protein and operon fusion plasmids . The regulation of cya expression during the cell cycle is characterized as follows: cya is expressed during cell elongation; expression is repressed during cell division; regulation is exerted at the transcriptional level . To test cya expression during cell elongation, we constructed a plasmid (pLCR1) in which the lacUV5 promoter operator was fused to the structural gene of cya and investigated the effect of cya expression by isopropyl-beta-D-thiogalactopyranoside (IPTG) on the cell division of cells containing pLCR1 . By the addition of IPTG, cell division was inhibited and filaments were formed . Such an inhibitory effect was antagonized by adding cyclic GMP to the culture medium and was not observed in the crp mutant. J Bacteriol, 1986 Dec, 168(3), 1315 - 8 Transcriptional control of flagellar genes in Escherichia coli K-12; Komeda Y; Autoregulation of the expression of flagellar genes was investigated by the technique of operon fusion . The results suggested that the flaU gene is a repressor and the flaD gene is an activator of transcription of the hag, flaS, and Mocha operons . The action of the putative flaU repressor appears to be masked by its interaction with other flagellar proteins during assembly; thus, repression is apparent only when the interacting proteins are absent . This hypothesis is supported by the phenotype of an unusual flaU mutant, which represses even though it is unable to promote flagellar assembly . Presumably, the mutant synthesizes a repressor whose activity is no longer masked by interaction with other flagellar proteins. J Bacteriol, 1986 Dec, 168(3), 1309 - 14 Interaction between two regulatory proteins in osmoregulatory expression of ompF and ompC genes in Escherichia coli: a novel ompR mutation suppresses pleiotropic defects caused by an envZ mutation; Matsuyama S et al.; The ompR and envZ genes, which together constitute the ompB operon, are involved in osmoregulatory expression of the OmpF and OmpC proteins, major outer membrane proteins of Escherichia coli . The envZ11 mutation results in the OmpF- OmpC-constitutive phenotype . A mutant which suppressed defects caused by the envZ11 mutation was isolated . The suppressor mutation also suppressed the LamB- PhoA- phenotype caused by the envZ11 mutation . The mutation occurred in the ompR gene and hence was termed ompR77 . The ompR77 mutation alone produced no obvious phenotype . Functioning of the ompR77 allele remained envZ gene dependent . Although the ompR77 mutation suppressed the envZ11 mutation, it did not suppress a mutation that occurred in another position within the envZ gene (envZ160) . These results indicate that OmpR and EnvZ, two regulatory proteins, functionally interact with each other. J Bacteriol, 1986 Dec, 168(3), 1197 - 204 Molecular cloning of an osmoregulatory locus in Escherichia coli: increased proU gene dosage results in enhanced osmotolerance; Gowrishankar J et al.; The proU locus in Escherichia coli encodes an important osmoregulatory function which mediates the growth-promoting effect of L-proline and glycine betaine in high-osmolarity media . This locus was cloned, in contiguity with a closely linked Tn10 insertion, onto a multicopy plasmid directly from the E . coli chromosome . For a given level of osmotic stress, the magnitude of osmoresponsive induction of a single-copy proU::lac fusion was reduced in strains with multiple copies of the proU+ genes; in comparison with haploid proU+ strains, strains with the multicopy proU+ plasmids also exhibited enhanced osmotolerance in media supplemented with 1 mM L-proline or glycine betaine . Experiments involving subcloning, Tn1000 mutagenesis, and interplasmid complementation in a deletion mutant provided evidence for the presence at this locus of two cistrons, both of which are necessary for the expression of ProU function . We propose the designations proU for the gene originally identified by the proU224::Mu d1(lac Ap) insertion and proV for the gene upstream (that is, counterclockwise) of proU. J Bacteriol, 1986 Dec, 168(3), 1165 - 71 cis-acting sites required for osmoregulation of ompF expression in Escherichia coli K-12; Ostrow KS et al.; OmpF and OmpC are major outer membrane proteins which form passive diffusion pores in Escherichia coli K-12 . The expression of the structural genes for these proteins, ompF and ompC, is influenced by medium osmotic strength and requires the products of two regulatory genes, ompR and envZ . We have constructed a series of ompF-lacZ fusions containing different regions of ompF to determine sites involved with osmoregulation . These fusions were crossed onto a specialized transducing phage and integrated into the bacterial chromosome in unit copy . By measuring the fluctuations of beta-galactosidase activity in lysogens grown in high versus low osmolarity, we have identified three regions which are necessary . Furthermore, we have determined that, although the OmpR activation site is not sufficient, OmpR is probably essential for ompF osmoregulation. J Bacteriol, 1986 Dec, 168(3), 1059 - 65 Repair response of Escherichia coli to hydrogen peroxide DNA damage; Hagensee ME et al.; The repair response of Escherichia coli to hydrogen peroxide-induced DNA damage was investigated in intact and toluene-treated cells . Cellular DNA was cleaved after treatment by hydrogen peroxide as analyzed by alkaline sucrose sedimentation . The incision step did not require ATP or magnesium and was not inhibited by N-ethylmaleimide (NEM) . An ATP-independent, magnesium-dependent incorporation of nucleotides was seen after the exposure of cells to hydrogen peroxide . This DNA repair synthesis was not inhibited by the addition of NEM or dithiothreitol . In dnaB(Ts) strain CRT266, which is thermolabile for DNA replication, normal levels of DNA synthesis were found at the restrictive temperature (43 degrees C), showing that DNA replication was not necessary for this DNA synthesis . Density gradient analysis also indicated that hydrogen peroxide inhibited DNA replication and stimulated repair synthesis . The subsequent reformation step required magnesium, did not require ATP, and was not inhibited by NEM, in agreement with the synthesis requirements . This suggests that DNA polymerase I was involved in the repair step . Furthermore, a strain defective in DNA polymerase I was unable to reform its DNA after peroxide treatment . Chemical cleavage of the DNA was shown by incision of supercoiled DNA with hydrogen peroxide in the presence of a low concentration of ferric chloride . These findings suggest that hydrogen peroxide directly incises DNA, causing damage which is repaired by an incision repair pathway that requires DNA polymerase I. Experientia, 1986 Dec 1, 42(11-12), 1197 - 205 Life without oxygen: what can and what cannot? Zehnder AJ, Svensson BH. The basic principles involved in the biotransformation of organic carbon compounds in the absence of molecular oxygen (dioxygen) are presented in this paper . The role of various electron acceptors during the breakdown of organic compounds is discussed and the metabolic end-products expected are summarized . The different biochemical possibilities and strategies for the anaerobic degradation of organic matter and the metabolic response of some organisms to anaerobiosis are elucidated . Positive and negative effects of anaerobiosis on environmentally relevant processes and their influence on man and on animals are reviewed . Finally, some examples of the biotechnological application of anaerobic processes are presented. Carcinogenesis, 1986 Dec, 7(12), 2081 - 4 Expression in mammalian cells of a truncated Escherichia coli gene coding for O6-alkylguanine alkyltransferase reduces the toxic effects of alkylating agents; Brennand J et al.; The lesion O6-alkylguanine (O6-AG) is produced in cellular DNA following exposure to monofunctional alkylating agents and its miscoding and mutagenic properties have been demonstrated in specific in vitro systems . In order to examine whether this lesion could be responsible for any of the biological effects of alkylating agents in mammalian cells, we have constructed a plasmid containing the O6-AG alkyltransferase (ATase) region of the gene from Escherichia coli, the product of which normally repairs both O6-AG and alkylphosphotriesters in DNA . We have transfected the construction into Chinese hamster fibroblasts which are deficient in endogenous ATase activity and selected a clone that expresses the truncated repair gene . We demonstrate that this protein is functional, acts on damage in host cell DNA and protects the cells from the toxic effects of those alkylating agents that react extensively at oxygen atom positions. Carcinogenesis, 1986 Dec, 7(12), 2077 - 80 Chinese hamster cells harbouring the Escherichia coli O6-alkylguanine alkyltransferase gene are less susceptible to sister chromatid exchange induction and chromosome damage by methylating agents; White GR et al.; Clones of Chinese hamster V79 cells harbouring the Escherichia coli O6-alkylguanine (O6-AG) alkylphosphotriester (AP) alkyltransferase (ATase) gene (clone 8) or a subclone of it that codes only for O6-AG ATase activity (clone SB) have been exposed to increasing doses of N-methyl-N-nitrosourea (MNU) or methylmethanesulphonate (MMS) and the frequencies of induced sister chromatid exchanges (SCEs) measured . In control (clone 2) cells, SCE induction was almost linearly proportional to dose of MNU or MMS and at the highest doses used (15 or 80 micrograms/ml) SCE frequencies were 6 or 8 times background levels, respectively . Slightly lower levels of MMS-induced SCEs were seen in clone 8 and clone SB cells whilst, in contrast, MNU-induced SCE levels in these two clones were drastically reduced being less than twice background levels at 15 micrograms/ml . After treatment with N-butyl-N-nitrosourea, SCE frequency was similar in all three clones . At higher doses, MNU treatment produced less chromatid aberrations and micronuclei in clone SB than in clone 2 cells . These results suggest that ATase-repairable damage is involved in the induction of SCE, chromosome aberrations and micronuclei in V79 cells. J Urol, 1986 Dec, 136(6), 1316 - 7 Transrectal longitudinal ultrasonography of prostatic abscess; Sugao H et al.; A definite diagnosis of prostatic abscess sometimes is difficult to make . We report 2 cases of prostatic abscess diagnosed with the aid of transrectal longitudinal ultrasonography by electronic linear scanning . Transperineal aspiration of the abscesses was performed easily and correctly with this echographic technique. Eur J Immunol, 1986 Dec, 16(12), 1577 - 81 Lymphoma models for B cell activation and tolerance . IV . Growth inhibition by anti-Ig of CH31 and CH33 B lymphoma cells; Pennell CA et al.; CH31 and CH33 are B cell lymphomas whose growth in vitro is inhibited by anti-Ig reagents, including both polyclonal and monoclonal anti-mu antibodies, and an anti-idiotype antiserum . Antibodies against class I or class II major histocompatibility complex antigens do not affect the growth of these cells . Inhibition is dependent on surface Ig cross-linking and does not require ligand binding to Fc receptors . Interestingly, the inhibition of growth by anti-mu is reversed in CH31 (but not CH33) by E . coli lipopolysaccharide . These lymphomas should provide excellent models to study the mechanisms of growth inhibition mediated by surface Ig cross-linking and the pathways of its reversal. J Pediatr Surg, 1986 Dec, 21(12), 1092 - 5 Plasma exchange therapy for endotoxin shock in puppies; Muraji T et al.; The efficacy of plasma exchange (PE) therapy for endotoxin (ET) shock has not been evaluated . The following experimental study was designed to evaluate the feasibility of PE in infants and children and determine the efficacy as a therapeutic modality for ET shock . A compact circuit for PE designed for this experiment consists of two pumps and a membrane plasma separator for a venovenous extracorporeal circulation with the limited prime volume of 100 mL . The capacity of this device for PE was tested . When a membrane separator of 0.3 m2 in surface area was used, a plasma flux of 5 to 13 mL/min was satisfactorily obtained with the blood flow rate of 25 to 40 mL/min, which was taken through centrally placed catheters of 18 gauge or larger . Transmembrane pressure was maintained in a safe range (0 to 50 mm Hg) in the first 90 minutes, during which time a PE of 100 mL/kg was completed with a sieving coefficient greater than 0.8 . In 14 puppies (1.8 to 3.5 kg), a shock state was produced by infusion of ET (Escherichia coli, 055:B5) . Eight puppies were resuscitated simply with lactated Ringer's solution and bicarbonate . Six puppies were treated with PE using 100 mL/kg of fresh-frozen plasma prepared from adult dogs . A significant decrease in the mean blood pressure, platelet and leukocyte counts, total protein, and CH50 was documented in all animals after ET administration . Seven of eight control animals died within 24 hours of post-ET infusion . All of the six PE-treated animals survived for 48 hours or longer.(ABSTRACT TRUNCATED AT 250 WORDS) Cancer Res, 1986 Dec, 46(12 Pt 1), 6029 - 33 Detection of activated Mr 21,000 protein, the product of ras oncogenes, using antibodies with specificity for amino acid 12; Wong G et al.; Antisera raised to a set of chemically synthesized peptides spanning position 12 of ras Mr 21,000 protein (p21) (residues 5 to 17) were able to distinguish between different forms of p21 according to the amino acid at the twelfth codon . The peptide immunogens differed in one amino acid corresponding to position 12 of the protein; the substitutions were valine, serine, arginine, aspartate, alanine, or cysteine at this position . Normal p21 contains glycine at position 12; the other amino acid substitutions are those which would result from a single base change in codon 12 and may therefore be the activating mutations most likely to occur in human tumors . The peptide antisera were evaluated by the Western immunoblot procedure for reactivity with v-ki-ras p21 expressed in Escherichia coli containing the corresponding position 12 mutations . Five of the antisera reacted with p21, and of these, anti-serine, -valine, -arginine, and -aspartate peptide antibodies were specific for their cognate protein . Similar analysis using mammalian cells as sources of position 12 variant forms of p21 demonstrated the ability of these antisera to distinguish among their oncogenic forms of p21 differing by single amino acid substitutions. Blood, 1986 Dec, 68(6), 1257 - 63 The influence of purified recombinant human heavy-subunit and light-subunit ferritins on colony formation in vitro by granulocyte-macrophage and erythroid progenitor cells; Broxmeyer HE et al.; Purified recombinant human heavy subunit (rHF, acidic) and recombinant human light subunit (rLF, basic) ferritins were assessed for their effects in vitro on colony formation by normal human granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) progenitor cells . The purity of the samples was confirmed by electrophoresis in both nondenaturing and denaturing conditions and silver staining . Concentrations of 10(-8) to 10(-10) mol/L rHF caused an approximately 40% significant decrease in colony formation . Some significant activity was detected at 10(-11) mol/L, and activity was lost at 10(-12) mol/L . In contrast, rLF had no significant activity at 10(-8) to 10(-16) mol/L . rHF was significantly active against mouse bone marrow CFU-GM to concentrations as low as 10(-8) to 10(-9) mol/L . The inhibitory activity of rHF was inactivated with three different monoclonal antibodies recognizing the heavy subunit of ferritin, but not with two monoclonal antibodies recognizing the light subunit of ferritin . The inhibitory activity of rHF was similar in the absence or presence of serum, monocytes, and T lymphocytes . We and others have shown an association of a glycosylated natural acidic isoferritin (AIF) with inhibitory activity, but since the rHF was expressed in Escherichia coli and did not bind to concanavalin A, glycosylation of AIF is not an absolute prerequisite for this activity . These results demonstrate that rHF has suppressive activity in vitro and substantiate our original observations using purified natural acidic isoferritins. Proc Natl Acad Sci U S A, 1986 Dec, 83(24), 9591 - 5 Rapid plasmid library screening using RecA-coated biotinylated probes; Rigas B et al.; A method for the rapid physical isolation of recombinant plasmids of interest from a mixture of plasmids such as a plasmid cDNA library is presented . This method utilizes the ability of RecA protein to form stable complexes between linear single-stranded and circular double-stranded DNA molecules sharing sequence homology, and procedures allowing isolation of biotinylated nucleic acid . Biotinylated linear DNA probes coated with RecA have been used to screen reconstituted plasmid libraries consisting of two plasmid species, one homologous and the other heterologous to the probe . When the link between biotin and the nucleotide base could be cleaved by reducing agents, the complex was purified by streptavidin-agarose chromatography and the recovered plasmid was propagated in Escherichia coli . When the link was not cleavable the complex was bound to avidin in solution and purified by cupric iminodiacetic acid-agarose chromatography . The complex was then dissociated and the plasmids were propagated in E . coli . With either protocol, homologous plasmid recovery was between 10% and 20%, and enrichment was between 10(4)- and 10(5)-fold . Potential applications and extensions of this method, such as plasmid, cosmid, and phage library screening and facilitation of physical mapping of macroregions of mammalian genomes are presented and discussed. Proteins, 1986 Dec, 1(4), 363 - 9 Primary structure of a precursor to the aspartic proteinase from Rhizomucor miehei shows that the enzyme is synthesized as a zymogen; Boel E et al.; In order to characterize the zymogen of the milk-clotting enzyme from Rhizomucor miehei, we constructed a cDNA library on pBR327 in Escherichia coli . Aspartic proteinase-specific recombinants were isolated by colony hybridization to a specific oligonucleotide mixture, and the cDNA sequence corresponding to a precursor form of the enzyme was determined . The deduced amino acid sequence shows that this secreted fungal proteinase is synthesized as a precursor . The first 22 amino acid residues in this precursor constitute a typical signal peptide . The amino acid sequence of the following 47-amino-acid-long prosegment shows homology to the prosegments from both the extracellular and intracellular vertebrate aspartic proteinases, and to the prosegments from the yeast and Mucor pusillus aspartic proteinases as well . These observations suggest that all aspartic proteinases are synthesized with a prosegment and that this prosegment is essential for the correct folding of all the mature enzymes . The active Rhizomucor miehei enzyme consists of 361 amino acid residues with a total molecular weight of 38,701 . Clusters of identities around the active site cleft support the assumption that these proteinases have a common folding of their peptide chains . The disulphide bridges were localized in the fungal enzyme, and 2 N-glycosylation sites were identified. J Gen Microbiol, 1986 Dec, 132 ( Pt 12), 3239 - 51 Transcript analysis of the citrate synthase and succinate dehydrogenase genes of Escherichia coli K12; Wilde RJ et al.; A transcript analysis of the citrate synthase and succinate dehydrogenase genes (gltA-sdhCDAB) of Escherichia coli was done by nuclease S1 mapping . Evidence was obtained for two monocistronic gltA transcripts extending anti-clockwise, to a common terminus, from independent promoters with start points 196 bp (major) and 299 bp (minor) upstream of the gltA coding region . Evidence was also obtained for two polycistronic sdh transcripts, sdhCDAB (minor) and sdhDAB (major), extending clockwise, from sites 219 bp upstream of sdhC and 1455 bp upstream of sdhD (i.e . within sdhC), to a common terminus . The synthesis of all of the transcripts was repressed by growth in the presence of glucose, and this is consistent with the well-established fact that both enzymes are subject to catabolite repression . Sequences resembling known binding sites for the cAMP-CRP (cyclic AMP-cyclicAMP receptor protein) complex occur in the vicinity of each promoter suggesting that they are activated by the cAMP-CRP complex. J Interferon Res, 1986 Dec, 6(6), 687 - 95 Structure and activity of glycosylated human interferon-gamma; Arakawa T et al.; Structural properties and activity of recombinant human interferon-gamma (IFN-gamma) purified from Chinese hamster ovary (CHO) cells or a natural source were determined and compared with those of Escherichia coli-derived IFN-gamma . One preparation of CHO-derived IFN-gamma showed three bands, with the middle band being a doublet, in a SDS-polyacrylamide gel . The two higher-molecular-weight bands were shown to be glycosylated . Western blot analysis indicated that the three bands are IFN-gamma and lack an intact carboxyl terminus . The circular dichroic (CD) spectra showed that conformation of the CHO-derived IFN-gamma is similar in the native state, in acid, and after renaturation from acid to the E . coli-derived IFN-gamma . These results indicate that neither glycosylation nor carboxy-terminal processing affects conformational properties of the protein, as detected by CD spectroscopy . However, the antiviral activity was fourfold lower for the preparation of CHO-derived IFN-gamma than for the E . coli-derived IFN-gamma . A different preparation or a natural IFN-gamma preparation with less extensive carboxy-terminal processing showed similar conformational properties and antiviral activity to the E . coli-derived IFN-gamma . These results indicate that the carboxyl terminus, but not glycosylation, plays an important role in the antiviral activity of IFN-gamma. Jpn J Cancer Res, 1986 Dec, 77(12), 1264 - 70 Characterization of polyethylene glycol-modified L-asparaginase from Escherichia coli and its application to therapy of leukemia; Yoshimoto T et al.; For the purpose of clinical application to the therapy of human leukemia and lymphosarcoma, L-asparaginase from Escherichia coli was modified with 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine (activated PEG2) by an improved method, which involves a purification step of activated PEG2 by gel filtration . The PEG2-modified asparaginase retained approximately 30% (73 IU/mg of protein) of the enzymic activity of the native enzyme, while it had almost wholly lost the immunoreactivity towards anti-asparaginase antibodies . The modified enzyme retained the characteristics of the native enzyme in terms of the pH- and temperature-dependencies of activity and stability, and the Km value for L-asparagine . Administration of the modified enzyme to a dog with spontaneous lymphosarcoma induced complete remission without any toxic side effects . Seven children with multiple relapses of acute leukemia were treated with a regimen of cycles of methotrexate and native asparaginase . Three of these children developed anaphylactic shock . In contrast to the native enzyme, the successive administration of PEG2-modified asparaginase to those three patients was therapeutically effective without causing any allergic reaction. EMBO J, 1986 Dec 1, 5(12), 3133 - 42 Use of a recombinant retrovirus to study post-implantation cell lineage in mouse embryos; Sanes JR et al.; We show that a gene introduced into cells of mouse embryos by a retrovirus can serve as a heritable marker for the study of cell lineage in vivo . We constructed a defective recombinant retrovirus in which the Escherichia coli beta-galactosidase (lacZ) gene is inserted in the genome of a Muloney murine leukemia virus (M-MuLV) . Expression of lacZ was detected with a histochemical stain that can be applied to cultured cells and embryonic tissue . Infection of cultured cells showed that lacZ has no detectable deleterious effects on cell viability or growth, that the enzyme is stably expressed in the progeny of infected cells for many generations in the absence of selective pressure, and that the virus can induce lacZ in a variety of cell types . Following injection of the virus into mid-gestation mouse embryos, clones of lacZ-positive cells were detected in skin, skull, meninges, brain, visceral yolk sac, and amnion . We identified the cell types comprising a series of lacZ-positive clones in the visceral yolk sac and skin to learn the lineage relationships of the labelled cells . In each tissue, we obtained evidence that several cell types have a pluripotential ancestor and that cell fate is progressively restricted as development proceeds. Biotechnol Appl Biochem, 1986 Dec, 8(6), 522 - 8 Design of a new automatic chromatography system for efficient enzyme purification equipped with a time-shared multiple activity analyzer; Tokushige M et al.; A new system equipped with a computer-controlled multiple activity analyzer has been developed for the efficient purification of multiple enzymes . The system consists of the following units: conventional enzyme fractionation system with a peristaltic pump, liquid chromatographic column, fraction collector, and uv monitor; computer-operated uv-vis spectrophotometer equipped with a thermo-regulated metal block and a flow-through type silica cuvette; personal computer; dot matrix printer; cooling facility; and automatic sampling-mixing system . The whole system is operated by a newly designed time-sharing computer program for periodic and repetitive sampling of the column eluants containing multiple kinds of enzymes and of designated assay mixtures for each enzyme and for measurement of the initial velocity of spectrophotometric signals . For example, a mixture of aspartase (EC 4.3.1.1) and malate dehydrogenase (EC 1.1.1.39) and also a mixture of these two enzymes and glutamate dehydrogenase (EC 1.4.1.3 or EC 1.4.1.4) were analyzed by the above system using gel permeation chromatography, and the two or three enzyme activities were repeatedly monitored within 4 min . Based on the above results further possibilities for the application of the system for a variety of purposes are discussed. Am Rev Respir Dis, 1986 Dec, 134(6), 1149 - 57 Endotoxin-induced changes in pulmonary hemodynamics and respiratory mechanics . Role of lipoxygenase and cyclooxygenase products; Ahmed T et al.; We investigated the role of leukotrienes and cyclooxygenase products in endotoxin-induced pulmonary vascular and airway changes . In 11 conscious sheep, measurements of pulmonary vascular resistance (PVR), lung resistance (RL), arterial PO2, leukocyte count (WBC), and plasma thromboxane B2 (TxB2), 6-keto-PgF1 alpha and PgF2 alpha were obtained, before and at predetermined intervals after a 10-min infusion of E . coli endotoxin (0.3 microgram/kg) . On a separate occasion, 5 sheep received an infusion of the leukotriene end-organ receptor antagonist FPL-57231 (0.7 to 1 mg/kg/min), before and for as long as 4 h after endotoxin infusion; and 6 sheep received a single injection of the cyclooxygenase inhibitor indomethacin (2 mg/kg) 1 h before endotoxin infusion . Endotoxin caused a biphasic response with an increase in mean PVR and RL to 441 and 353% of baseline, respectively, during the early phase (0 to 1 hr), and lesser increases to 168 and 195% of baseline during the late phase (1.5 to 4 h) . These changes were associated with mild hypoxemia, marked leukopenia, and marked increases in plasma TxB2, 6-keto-PgF1 alpha and PgF2 alpha . The FPL-57231 completely blocked the endotoxin-induced changes in PVR, RL, and PaO2 during both phases without preventing the increases in TxB2; however, it partly attenuated the increases in 6-keto-PgF1 alpha and enhanced the generation of PgF2 alpha . Indomethacin, which blocked the endotoxin-induced increases in TxB2, 6-keto-PgF1 alpha, PgF2 alpha, and RL, only partly blocked the increase in PVR during the early phase, followed by an exaggerated increase of PVR during the late phase.(ABSTRACT TRUNCATED AT 250 WORDS) Mutat Res, 1986 Dec, 164(6), 353 - 9 Kinetic determination of enzymatic activity and modification of the metabolic activation system in the SOS chromotest; Marzin DR et al.; The "SOS Chromotest" has recently been introduced by P . Quillardet et al . (1982; Quillardet and Hofnung, 1985), who use strain PQ37 of Escherichia coli K12 to test for genotoxicity . We have modified the procedure in order to optimize the determination of beta-galactosidase and alkaline phosphatase activities, and, where possible, to allow measurements to be made automatically . Kinetic determination is quicker, more sensitive and avoids interference by coloured compounds . Modification of the metabolic activation system increases the sensitivity of the test for progenotoxicity. J Bacteriol, 1986 Dec, 168(3), 1378 - 83 Mutation plus amplification of a transducer gene disrupts general chemotactic behavior in Escherichia coli; Park C et al.; Transducers are transmembrane receptor proteins that generate intracellular signals on stimulation and participate in adaptation by appropriate changes in the level of methylation . The transducer mutation trg-21 conferred a Trg- phenotype and defective taxis to galactose and ribose but a normal response to other attractants when present in a single chromosomal copy . Amplification of trg-21 by a multicopy plasmid made host cells generally nonchemotactic . The dominant phenotype resulted from a strong counterclockwise rotational bias of flagellar motors in Che- cells . Apparently, the Trg21 transducer sends a continuous counterclockwise signal to flagella independent of tactic stimulation . It appears that the cell has a homeostatic capacity that is sufficient to compensate for the effect of mutant transducers produced from a single chromosomal copy of trg-21, but the capacity is exceeded in cells that have multiple copies of the gene . The Trg21 protein did not have a significant effect on methylesterase activity, indicating that the two global effects of a stimulated transducer, that is, on flagellar rotation and on modification enzymes, can occur independently . The mutant protein exhibited essentially normal turnover of methyl groups but had a drastic defect in deamidation which thus reduced the number of methyl-accepting sites . The trg-21 mutation substitutes a threonine for Ala-419 . This alanine is a conserved residue in all sequenced transducers and is in a region of the carboxy-terminal domain in which homology among the transducers is very high . The Trg21 phenotype implicates this conserved region in the generation of the excitatory signal which is directed at the flagella. J Bacteriol, 1986 Dec, 168(3), 1319 - 24 Isolation and nucleotide sequence analysis of the ferredoxin I gene from the cyanobacterium Anacystis nidulans R2; Reith ME et al.; Two mixed oligonucleotide probes derived from conserved regions of the Synechocystis sp . strain PCC 6714 ferredoxin amino acid sequence were utilized to isolate an Anacystis nidulans R2 clone containing the ferredoxin I gene . Nucleotide sequence analysis revealed a 297-base-pair (bp) open reading frame with a deduced amino acid sequence having high homology to other cyanobacterial ferredoxins . Assuming proteolytic cleavage of the initial methionine residue, the molecular weight of the mature A . nidulans R2 ferredoxin was 10,370 . The initial methionine residue was preceded by a probable ribosome-binding site sequence, AGGA . Northern hybridization analysis with the cloned ferredoxin gene indicated an RNA transcript of approximately 450 bp . S1 nuclease mapping localized the transcription start site to a position 64 bases upstream from the initial methionine residue . The nucleotide sequence 14 to 8 bp preceding the transcription start site resembled a typical Escherichia coli promoter, but the sequence in the -35 region did not . Southern hybridization detected only a single copy of the ferredoxin sequence in the A . nidulans R2 genome. J Bacteriol, 1986 Dec, 168(3), 1272 - 6 Cloning and expression in Escherichia coli of the gene for an Arthrobacter beta-(1----3)-glucanase; Doi K et al.; When inserted in the correct orientation at the BamHI site of plasmid YRp7, an 8.6-kilobase BamHI fragment of Arthrobacter sp . strain YCWD3 DNA gave Escherichia coli HB101 cells harboring the recombinant plasmid pBX20 the ability to lyse bakers' yeast cell walls or bakers' yeast glucan in agar medium . An extract of the transformed E . coli cells contained an endo-beta-(1----3)-glucanase with the same activity pattern as that of glucanase I produced by Arthrobacter sp . strain YCWD3 . Although part of the glucanase activity was contributed by apparently defective molecules, two protein species were found which had high lytic activity on yeast cell walls and adsorbed to microcrystalline cellulose, and both had a single constituent polypeptide with a molecular weight of about 55,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . In these properties the protein species were indistinguishable from those glucanase I protein species of Arthrobacter sp . strain YCWD3 which we believe are nearly the intact molecule . We conclude that the cloned fragment of Arthrobacter sp . strain YCWD3 DNA contains the structural gene for glucanase I . A recombinant plasmid obtained by subcloning a PstI fragment of pBX20 into pBR322 caused the transformed E . coli cells to produce apparently defective glucanase molecules only . This observation serves as additional supporting evidence for our conclusion. Infect Immun, 1986 Dec, 54(3), 728 - 34 Influence of endotoxin-protein in immunoglobulin G isotype responses of mice to Brucella abortus lipopolysaccharide; Kurtz RS et al.; Brucella abortus endotoxin preparations, containing approximately 5 to 6% protein, induce strong immune and adjuvant immunoglobulin G (IgG) responses as compared with Escherichia coli endotoxin preparations, with equivalent amounts of protein, which induce responses in which IgM antibody predominates . Using an enzyme-linked immunoassay with isotype-specific conjugates, we found that antibody of all four subclasses of IgG were evoked during the course of the immune responses of C3H/HeAu mice to B . abortus endotoxin . Secondary responses of endotoxin-hyporesponsive C3H/HeJ mice were similar to those seen in C3H/HeAu mice, although lower levels of antibody were produced during their primary responses . The primary responses of BALB/c athymic mice consisted almost entirely of IgG3, and IgG1 appeared following a second injection . The effects of lipopolysaccharide (LPS)-associated protein on the immunogenic properties of B . abortus endotoxin were examined by comparing responses to endotoxin with those to a purified B . abortus LPS containing less than 1% protein . The endotoxin evoked strong primary and secondary responses in which antibody directed to LPS determinants consisted mainly of IgG3 and those to the protein determinants were largely IgG1 antibody . Primary and secondary responses to purified LPS consisted mainly of IgG3 antibody . The potential mechanism of the contribution of protein to the immunogenic properties of the endotoxin as well as possible immune mechanisms involved in these responses are discussed. J Biochem (Tokyo), 1986 Dec, 100(6), 1477 - 80 Characterization by EPR spectroscopy of cytochrome b-562 isolated from the cytochrome b-c1 complex of Rhodopseudomonas sphaeroides R-26; Iba K et al.; The EPR spectra of cytochrome b-562 isolated from the cytochrome b-c1 complex of Rhodopseudomonas sphaeroides were measured at liquid helium temperature . The purified cytochrome b-562 gives a high spin signal at g = 6.0 . Anaerobic titration of this signal confirmed the presence of two redox components with Em = 40 and -110 mV at pH 7.5 . These values are consistent with the published ones, Em = 55 and -100 mV at pH 7.0, that were optically estimated for the same type of preparation (Iba et al . (1985) FEBS Lett . 183, 151-154) . The power saturation behavior of the g = 6.0 signal at different redox potentials indicated a direct spin-spin interaction between these two redox centers. Mol Gen Genet, 1986 Dec, 205(3), 550 - 6 Analysis of Tn7 transposition; Rogers M et al.; Five gene products required for Tn7 transposition were identified using genetic complementation tests . Four of these (tnsA, tnsB, tnsC and tnsD) are essential for insertion into the attachment site, whereas the fifth (tnsE) is required for transposition to plasmids which lack this site . tnsD is not required for transposition to plasmids lacking the attachment site . This analysis used a chloramphenicol-resistant 'mini' transposon containing Tn7 termini but no complete Tn7 gene product and several compatible expression vectors containing cloned Tn7 fragments . The use of transcriptional and translational fusions allowed the identification of two promoters (P1 and P2) at the righthand end of the transposon and indicated that at least tnsA, tnsB and tnsC are translated in the same direction . Expression from P1 appears to be repressed by tnsB. Mol Gen Genet, 1986 Dec, 205(3), 540 - 5 Strains overproducing tRNA for histidine; Ulrich AK et al.; Hybridization analysis of total genomic DNA indicated that Escherichia coli K12 contains a single copy of the gene encoding the histidine-accepting tRNA . This gene was subcloned onto an inducible expression vector under the control of the tac promoter . Strains carrying the resulting plasmid showed five- to six-fold increased histidine-accepting activity after induction . This overproduction of tRNAHis did not effect the growth rate of the strain or lead to derepression of the histidine biosynthetic enzymes . Neither did it have an effect on mistranslation elicited by histidine starvation . However, in cells starved for histidine by the addition of alpha-methyl histidine, the overproduction of tRNAHis interfered with the ability of the cells to recover from starvation. Mol Gen Genet, 1986 Dec, 205(3), 515 - 22 The gene for Escherichia coli diadenosine tetraphosphatase is located immediately clockwise to folA and forms an operon with ksgA; Blanchin-Roland S et al.; The DNA sequences of the diadenosine tetraphosphatase gene (apaH) and of the flanking regions were determined . Three other genes were identified in the flanking regions: ksgA, apaG and folA encoding, respectively, a 16 S rRNA methyltransferase, an unidentified protein of Mr 13,826 and dihydrofolate reductase, with the order folA-apaH-apaG-ksgA . The apaH gene is thus located between folA and ksgA at 1 min on the Escherichia coli chromosome linkage map and folA is transcribed clockwise, whereas ksgA, apaG and apaH are transcribed in the opposite direction . It was shown that ksgA, apaG and apaH can be expressed from a polycistronic mRNA originating from a promoter (p1) located upstream of ksgA . However, another promoter (p2) was found within the ksgA structural gene . This promoter, active in vivo, can account for p1-independent expression of the two distal cistrons, apaG and apaH . Finally, the effect on diadenosine tetraphosphatase over-production of a frameshift mutation causing premature translational termination of apaG suggests that expression of apaG and apaH is coupled at the translational level. J Appl Bacteriol, 1986 Dec, 61(6), 535 - 40 Molecular relationships between trimethoprim R plasmids obtained from human and animal sources; Towner KJ et al.; A total of 65 trimethoprim R plasmids (35 obtained from human strains and 30 from animal strains of Escherichia coli) were examined by the technique of restriction endonuclease fingerprinting . Plasmids belonging to incompatibility groups B, FII and I delta obtained from human and animal sources showed close similarities within each group . Plasmids belonging to incompatibility groups I alpha and P were also obtained from both human and animal sources, but there was no conclusive evidence of close relationships within the groups . Restriction endonuclease fingerprinting was found to be useful for obtaining information about the epidemiology of R plasmids . Its main limitation in this study related to broad host range plasmids of the P incompatibility group, some of which contained very few sites susceptible to cleavage by the restriction endonucleases tested. AIDS Res, 1986 Dec, 2 Suppl 1, S71 - 8 Mechanism of the gene expression of HTLV-I and its association with ATL; Yoshida M et al.; Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia (ATL) and a trans-acting viral function was proposed to be involved in ATL development because of the non-specific provirus integration in leukemic cells and the frequent immortalization of helper T-cells by in vitro infection . An extra sequence "pX" in the HTLV-1 genome codes for three proteins, p40x-, p27x- and p21x-, and the p40x- is trans-activator of transcription from the viral LTR . A sequence of 21 bp repeats in the LTR was found to be an enhancer and respond to the trans-activation by p40x- . The transient expression of p40x- also activates a cellular gene for interleukin 2 receptor (IL-2R) in helper T-cell lines . This induction of IL-2R may explain the mechanism of preferential growth of HTLV-1 infected cells and may be an early event of ATL development . For practical purposes, the env gene fragments was expressed in E . coli as fusion proteins with beta-galactosidase . Using these fusion proteins, a diagnostic system detecting anti-env antibodies was developed . Immunization of monkeys with these envelope-fusion proteins protected the monkeys from the viral infection, suggesting possible usage of envelope proteins as vaccine. Anal Biochem, 1986 Dec, 159(2), 280 - 6 High resolution preparative gel electrophoresis of DNA fragments and plasmid DNA using a continuous elution apparatus; Hediger MA; An apparatus designed for preparative gel electrophoretic separation of proteins (M . A . Hediger, (1984) Anal . Biochem . 142, 445-454) has been used successfully for separating DNA restriction fragments . The apparatus displayed yields and resolutions that are higher than those obtainable with commercially available devices . The amounts of DNA applied to the column range from a few micrograms to milligram quantities . Restriction DNA fragments very similar in size were isolated in pure form with the apparatus . After ethanol precipitation, these fragments were successfully used for restriction enzyme cleavages, ligation, or chemical sequencing . Furthermore the apparatus provides a convenient method for the large-scale isolation of plasmid DNA . The method requires only 4 h of electrophoresis and therefore greatly reduces the preparation time compared with the conventional equilibrium centrifugation method which requires centrifugation times of up to 60 h . In contrast to the centrifugation method, contaminants such as RNA, proteins, and chromosomal DNA are efficiently removed by this technique.
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