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