Genetics, 1989 Mar, 121(3), 423 - 31 IS103, a new insertion element in Escherichia coli: characterization and distribution in natural populations; Hall BG et al.; IS103 is a previously unknown insertion sequence found in Escherichia coli K12 . We have sequenced IS103 and find that it is a 1441-bp element that consists of a 1395-bp core flanked by imperfect 23-bp inverted repeats . IS103 causes a 6-bp duplication of the target sequence into which it inserts . There is a single copy of IS103 present in wild-type E . coli K12 strain HfrC . In strain X342 and its descendents there are two additional copies, one of which is located within the bglF gene . IS103 is capable of excising from within bglF and restoring function of that gene . IS103 exhibits 44% sequence identity with IS3, suggesting that the two insertion sequences are probably derived from a common ancestor . We have examined the distribution of IS103 in the chromosomes and plasmids of the ECOR collection of natural isolates of E . coli . IS103 is found in 36 of the 71 strains examined, and it strongly tends to inhabit plasmids rather than chromosomes . Comparison of the observed distribution of IS103 with distributions predicted by nine different models for the regulation of transposition according to copy number and of the effects of copy number on fitness suggest that transposition of IS103 is strongly regulated and that it has only minor effects on fitness . The strong clustering of IS103 within one phylogenetic subgroup of the E . coli population despite its presence on plasmids suggests that plasmids tend to remain within closely related strains and that transfer to distantly related strains is inhibited. Biochem J, 1989 Mar 1, 258(2), 389 - 96 Dependence on pH of substrate binding to a mutant lactose carrier, lacYun, in Escherichia coli . A model for H+/lactose symport; Yamato I et al.; The lacYun gene, which encodes a lactose carrier showing the uncoupled phenotype of substrate transport in Escherichia coli {Wilson, Kusch & Kashket (1970) Biochem . Biophys . Res . Commun . 40, 1409-1414}, was cloned on a plasmid vector, pBR322 . The binding of a substrate, p-nitrophenyl alpha-galactoside, to the lacYun carrier in membranes from the strain harbouring the lacYun clone showed a pH-dependence different from its binding to the wild-type lactose carrier . This finding indicated that the lacYun mutation confers higher affinity for H+ on the carrier, exerting its effect on the less efficient dissociation of substrate inside cells . The result coincides with the proposal {Yamato & Rosenbusch (1983) FEBS Lett . 151, 102-104} that the proton affecting the substrate binding is the coupling proton of the proton/lactose symport reaction, which allows only the ordered mechanism of binding of substrate to an H+-carrier binary complex . From the simplest model of the symport reaction, constructed on the basis of these results, the coupling site of energy in the carrier cycle of the transport reaction can be identified at the substrate-dissociation step inside cells. Proc Natl Acad Sci U S A, 1989 Mar, 86(6), 1895 - 9 Site-specific antibodies against the PrlA (secY) protein of Escherichia coli inhibit protein export by interfering with plasma membrane binding of preproteins; Watanabe M et al.; Genetic evidence indicates that the PrlA (SecY) protein of Escherichia coli functions as a membrane integrated signal sequence receptor in protein "export"--i.e., in protein translocation across (or integration into) the plasma membrane . We have raised antibodies in rabbits against two synthetic peptides representing the hydrophilic N- or C-terminal region of PrlA . Using these antibodies as probes in cell fractionation experiments, we confirm that PrlA is an integral membrane protein of the plasma membrane of E . coli . Fab fragments prepared from each of the two antisera specifically inhibit protein export by interfering with the binding of preproteins to the plasma membrane . Inhibition of preprotein binding and export by Fab fragments was shown in a cell-free translocation system: precursors for LamB (an integral membrane protein) and for a truncated form of MalE (a periplasmic protein) were first synthesized in a membrane-depleted E . coli-derived cell-free translocation system followed by posttranslational incubation with inverted vesicles derived from the plasma membrane of E . coli . Our data thus indicate that the N and C termini of PrlA are exposed to the cytoplasm . We discuss the possibility that the transmembrane segments of PrlA could be arranged in the lipid bilayer in a cylindrical fashion, thereby delimiting a protein conducting channel, with a signal sequence binding domain represented, at least in part, by the N and C termini of PrlA . Such a channel would also contain a "stop-transfer" sequence binding domain that in response to a stop-transfer sequence would open the cylindrical channel to the lipid bilayer and permit displacement of the polypeptide from the channel to the lipid bilayer, resulting in membrane integration. Proc Natl Acad Sci U S A, 1989 Mar, 86(6), 1771 - 5 Conjugative transfer of promiscuous IncP plasmids: interaction of plasmid-encoded products with the transfer origin; Furste JP et al.; To characterize protein-DNA interactions involved in the initiation of conjugative transfer replication, we isolated and sequenced the transfer origins (oriT) of the promiscuous IncP plasmids RP4 and R751 . The central initiating event at the transfer origin of a conjugative plasmid is the cleavage at a unique site (nic) of the strand to be transferred to a recipient cell . This process can be triggered after the assembly of "relaxosomes" (plasmid DNA-protein relaxation complexes), requiring plasmid-encoded gene products . We analyzed the nicking reaction for plasmid RP4 and demonstrated that one of the plasmid strands is specifically cleaved within oriT . The fully functional oriT of RP4 represents an intergenic DNA region of approximately 350 base pairs . Dissection of oriT revealed that a portion carrying nic and symmetric sequence repeats determines oriT specificity . This part of oriT is contiguous to a region that is essential for efficient mobilization of oriT plasmids . In addition, oriT contains potential promoter sites allowing divergent transcription of two operons flanking oriT . We over-produced gene products and, from analyzing the products of defined deletion mutants, deduced the gene arrangements . Formation of RP4 relaxosomes is likely to depend on the presence of at least two plasmid-encoded components, which act in trans . Corresponding genes map on one side of oriT . Purification of the traJ product revealed it to be an 11-kDa polypeptide that binds to oriT DNA in vitro . The protein recognizes the part of oriT that is responsible for oriT specificity. Mutat Res, 1989 Mar-May, 220(2-3), 73 - 82 Studies on direct and indirect effects of DNA damage on mutagenesis in monkey cells using an SV40-based shuttle vector; Dixon K et al.; We are using an SV40-based shuttle vector, pZ189, to study mechanisms of mutagenesis in mammalian cells . The vector can be treated with mutagens in vitro and replicated in animal cells; resulting mutants can be selected and amplified in bacteria for DNA sequencing . This versatile vector system has allowed us to explore several different questions relating to the mutagenic process . We have studied the direct effects of template damage caused by UV or benzo{a}pyrene diolepoxide by treating vector DNA with these agents and then replicating the damaged DNA in monkey cells . Mutational mechanisms were deduced from the spectrum of mutations induced in the supF target gene of the vector DNA . To study the role of indirect effects of DNA damage on mutagenesis in mammalian cells, we have treated the cells and the vector DNA separately with DNA-damaging agents . We find that pretreatment of cells with DNA-damaging agents, or with conditioned medium from damaged cells, causes an enhancement of mutagenesis of a UV-damaged vector . Thus, DNA damage can act indirectly to enhance the mutagenic process . We also have preliminary evidence that pZ189 can be used in an in vitro DNA replication system to study the process of mutation fixation on the biochemical level . We believe that the pZ189 vector will prove to be as useful for in vitro studies of mutational mechanisms as it has been for in vivo studies. Mutat Res, 1989 Mar-May, 220(2-3), 55 - 60 The development of transient SV40 based shuttle vectors for mutagenesis studies: problems and solutions; Seidman M; Shuttle-vector plasmids would appear to provide a powerful technology for studying mutagenesis in mammalian (including human) cells . Recently, as described in this and other papers in this volume, several shuttle-vector systems have been described and applied . The development of the first shuttle vectors for these purposes was hindered by two major problems . The first of these was the 'poison' sequence present in many pBR322 based vectors . The second was the problem of spontaneous mutagenesis associated with transfection of the plasmids into mammalian cells . Effective solutions for both problems have been devised, and it is now possible to experimentally address a variety of questions concerning mutagenesis and repair in mammalian cells. Mutat Res, 1989 Mar-May, 220(2-3), 161 - 8 Repair of DNA damage in shuttle vectors, virus, and chromosomal DNAs may depend on their biological imprinting--a 'Pygmalion' effect; Cleaver JE et al.; We have created a cell line that can repair damage in chromosomal DNA and in herpes virus, while not repairing the same damage in shuttle vectors (pZ189 and pRSVcat) . This cell line, a xeroderma pigmentosum (XP) revertant, repairs the minor (6-4)-photoproducts, but not cyclobutane dimers, in chromosomal DNA . The phenotype of this revertant after irradiation with ultraviolet (UV) light is the same as that of normal cells for survival, repair replication, recovery of rates of DNA and RNA synthesis, and sister-chromatid exchange formation, which indicates that a failure to mend cyclobutane dimers may be irrelevant to the fate of irradiated human cells . The two shuttle vectors were grown in Escherichia coli and assayed during transient passage in human cells, whereas the herpes virus was grown and assayed exclusively in mammalian cells . The ability of the XP revertant to distinguish between the shuttle vector and herpes virus DNA molecules according to their 'cultural background', i.e., bacterial or mammalian, may indicate that one component of the repair of UV damage involves gene products that recognize DNA markers that are uniquely mammalian, such as DNA methylation patterns . This component of excision repair may be involved in the original defect and the reversion of XP group A cells. Mutat Res, 1989 Mar-May, 220(2-3), 101 - 6 SV40-based shuttle viruses; Menck CF et al.; We summarize in this paper the advantages of the shuttle virus system . These SV40-based vectors exhibit the unique properties of being packaged as SV40 pseudo-virions and of being able to infect host cells . Using these transient vectors, we show that their replication can be regulated in some monkey cell lines, in such a way that either low or very high amounts of plasmid DNA can be obtained . The stability of these infectious shuttle vectors in different conditions is analyzed by rescuing them in E . coli, using various gene mutation targets . Moreover, we describe a new series of vectors which can be produced as single-stranded DNA in bacteria . They allow the transfection of a plasmid genome into mammalian cells, as either single-stranded or double-stranded DNA. J Neurosci, 1989 Mar, 9(3), 959 - 66 The effects of dephosphorylation on the structure of the projections of neurofilament; Hisanaga S et al.; Carboxy-terminal tail domains of larger molecular mass subunits (NF-M and NF-H) of neurofilaments (NFs), which are the highly phosphorylated moieties, were observed as thin flexible filaments projecting from NF core filaments by rotary shadowing (Hisanaga and Hirokawa, 1988) . Dephosphorylation of NFs has been suspected to affect the structures and the functions of the carboxy-terminal tail projections . We report here the effects of the dephosphorylation on the structure of NFs studied by electron microscopy . (1) The structures of carboxy-terminal tail projections after dephosphorylation were compared with those of the control NFs by low-angle rotary shadowing . This was examined with 2 samples; the isolated neurofilaments and the short filaments assembled from NF-H . Both the dephosphorylated NFs and the short filaments showed many projections laterally extending from core filaments similar to those observed in the control samples . (2) With respect to the structure of NF in physiological solution, the density of NFs in the precipitates was examined by thin-section electron microscopy . No difference in the density was noted between control and dephosphorylated NFs . (3) The ability to form cross-bridges in vitro was examined by quick-freeze, deep-etch electron microscopy . The structure and frequency of cross-bridges appeared to be similar in both control and dephosphorylated NFs . (4) Phosphate determination revealed that about 90% of the phosphate groups of NF-H subunit were removed by treatment with E . coli alkaline phosphatase . These results indicated that the dephosphorylation of NF did not affect the structure and the ability to form cross-bridges of the carboxy-terminal tail projections in vitro. J Exp Med, 1989 Mar 1, 169(3), 737 - 54 Potent leukocidal action of Escherichia coli hemolysin mediated by permeabilization of target cell membranes; Bhakdi S et al.; The contribution of Escherichia coli hemolysin (ECH) to bacterial virulence has been considered mainly in context with its hemolytic properties . We here report that this prevalent bacterial cytolysin is the most potent leukocidin known to date . Very low concentrations (approximately 1 ng/ml) of ECH evoke membrane permeability defects in PMN (2-10 x 10(6) cells/ml) leading to an efflux of cellular ATP and influx of propidium iodide . The attacked cells do not appear to repair the membrane lesions . Human serum albumin, high density and low density lipoprotein, and IgG together protect erythrocytes and platelets against attack by even high doses (5-25 micrograms/ml) of ECH . In contrast, PMN are still permeabilized by ECH at low doses (50-250 ng/ml) in the presence of these plasma inactivators . Thus, PMN become preferred targets for attack by ECH in human blood and protein-rich body fluids . Kinetic studies demonstrate that membrane permeabilization is a rapid process, ATP-release commencing within seconds after application of toxin to leukocytes . It is estimated that membrane permeabilization ensues upon binding of approximately 300 molecules ECH/PMN . This process is paralleled by granule exocytosis, and by loss of phagocytic killing capacity of the cells . The recognition that ECH directly counteracts a major immune defence mechanism of the human organism through its attack on granulocytes under physiological conditions sheds new light on its possible role and potential importance as a virulence factor of E . coli. Virology, 1989 Mar, 169(1), 68 - 77 Polypeptide 2A of human rhinovirus type 2: identification as a protease and characterization by mutational analysis; Sommergruber W et al.; Evidence is presented that the protein 2A of human rhinovirus serotype 2 (HRV2) is a protease . On expression of the VP1-2A region of HRV2 in bacteria, protein 2A was capable of acting on its own N-terminus; derived extracts specifically cleaved a 16 amino acid oligopeptide corresponding to the sequence at the cleavage site . Cleavage of the oligopeptide substrate provides a convenient in vitro assay system . Deletion experiments showed that removal of 10 amino acids from the carboxy terminus inactivated the enzyme . Site-directed mutagenesis identified an essential arginine close to the C-terminus and showed that the enzyme was sensitive to changes in the putative active site . This analysis supports the hypothesis that 2A belongs to the group of sulfhydryl proteases, although sequence comparisons indicate that the putative active site of HRV2 2A is closely related to that of the serine proteases. Virology, 1989 Mar, 169(1), 236 - 8 The bovine papillomavirus type 1 transcriptional activator E2 protein binds to its DNA recognition sequence as a dimer; Moskaluk CA et al.; The transcriptional trans-activator E2 protein from bovine papillomavirus type 1 has been shown to bind to the DNA consensus sequence ACCN6GGT . We have produced the DNA-binding domain of the E2 protein as a recombinant protein in Escherichia coli . The E2 DNA-binding domain was purified as two different molecular weight forms . Using these purified proteins, we show that two molecules of the E2 protein bind to a single DNA consensus binding site. Mutat Res, 1989 Mar, 211(1), 51 - 64 Genotoxicity of monofunctional methanesulphonates in the SOS chromotest as a function of alkylation mechanisms . A comparison with the mutagenicity in S . typhimurium TA100; Eder E et al.; 17 monofunctional methanesulphonates of widely varying structures were investigated in the SOS chromotest using the E . coli strain PQ37 . All compounds tested were positive in this assay . The monofunctional methanesulphonates in general possess low SOSiP values . Five of the compounds tested i.e . iBMS, NpMS, 2 PhPMS, PkMS and 1,3-DC12PMS (for abbreviations see Table 1) did not show increasing beta-galactosidase activity and both the positive induction factors and the positive SOSiP values resulted from the toxicity correction as performed according to Quillardet and Hofnung (1985) . In general methanesulphonates with a higher SN1 reactivity, in particular the secondary compounds, showed clear genotoxic activities whereas those possessing low SN1 reactivities (primary compounds) induced a low SOS repair indicating that the alkylation of O-atoms in the DNA bases contributes more to the induction of SOS repair in strain PQ37 than N-alkylations . The only exception was methyl methanesulphonate (MMS) which possessed a very high SN2 reactivity but a rather low SN1 reactivity . It had the highest SOSiP value of all tested methanesulphonates . No dependence of the genotoxicity on the SN2 reactivity could be found in this series . In general the phenyl-substituted methanesulphonates showed higher SOSiP values, which is presumably due to their relatively high SN1 reactivities and their relatively long life times in aqueous systems . There is a clear relationship between SN1 reactivities and the SOSiP values: the SOSiP values increase with rising SN1 reactivities reaching a maximum at iPMS after which the genotoxicities decrease due to the decreasing life times . The compounds with very high SN1 reactivities also possess very high hydrolysis rates . A good correlation could be established between the mutagenicities in S . typhimurium TA100 and the SOS chromotest (strain PQ37) . Only 4 small deviations from this correlation could be found . The reasons for these deviations are discussed. J Bacteriol, 1989 Mar, 171(3), 1683 - 91 Isolation, characterization, and nucleotide sequence of appY, a regulatory gene for growth-phase-dependent gene expression in Escherichia coli; Atlung T et al.; A plasmid carrying a regulator gene, designated appY, was found in the screening of an Escherichia coli gene library for clones overproducing AppA, an acid phosphatase which is induced as a culture approaches the stationary phase . In cells containing multicopy plasmids carrying the appY gene, the expression of the chromosomal appY gene was stimulated 10- to 40-fold in the stationary phase and more than 100-fold during exponential growth . The appA plasmid also changed the rate of synthesis of more than 30 other proteins in a growth-phase-dependent way . The appY gene was mapped to 13 min on the E . coli genetic map . The position of the appY gene on the 4.9-kilobase HindIII fragment of the original clone was located by Tn5 mutagenesis and deletion analysis, and the nucleotide sequence of a 1.9-kilobase region containing the gene was determined . The appY gene product was identified as a weakly expressed 243-amino-acid polypeptide which contains a stretch of 20 amino acids showing very good similarity to the conserved DNA-binding domain of repressors and transcriptional activators. J Bacteriol, 1989 Mar, 171(3), 1623 - 30 Cyclic-AMP-dependent switch in initiation of transcription from the two promoters of the Escherichia coli gal operon: identification and assay of 5'-triphosphate ends of mRNA by GTP:RNA guanyltransferase; Irani M et al.; We have studied the initiation of transcription of the gal operon in Escherichia coli (i) by analyzing the 5'-triphosphate ends and (ii) by measuring the level of promoter-proximal gal mRNA made in vivo . The 5' termini were identified and quantified by capping with GTP:mRNA guanyltransferase, and the mRNA levels were determined by hybridization of pulse-labeled {32P}RNA with a specific DNA probe . Our results conclusively demonstrate the in vivo activities of two promoters, P1 and P2, with separate initiation sites (S1 and S2) as suggested before from in vitro and in vivo experiments (S . Adhya and W . Miller, Nature {London} 279:492-494, 1979; R . E . Musso, R . DiLauro, S . Adhya, and B . de Crombrugghe, Cell 12:847-854, 1977) . We have also studied the effect of cyclic AMP (cAMP) on in vivo gal transcription and found that whereas total gal transcription remains largely unchanged, the relative proportions of the S1 and S2 mRNAs are influenced by the level of cAMP in the cell . In strains devoid of cAMP (cya), transcription initiates equally at S1 and S2; in cAMP-proficient cells (cya+), the S1 initiation increases twofold with a concomitant decrease in S2 initiation . Addition of a saturating amount of exogenous cAMP to cya mutant cells results in a relatively larger switch from S2 to S1 . Our results clearly show that while cAMP is an inhibitor of S2, it is not an absolute requirement for transcription initiation at S1, but only acts to increase low-level transcription from the P1 promoter . Using these approaches, we have also studied gal promoter mutants (P211, P18, and P35) which show altered behavior in transcription initiations and in response to cAMP . On the basis of these results, we have discussed models by which transcription initiates at the two overlapping gal promoters (P1 and P2) and discussed how cAMP level modulates the switch between them. J Bacteriol, 1989 Mar, 171(3), 1476 - 84 Escherichia coli proteins inducible by oxidative stress mediated by the superoxide radical; Walkup LK et al.; Two-dimensional gel analyses were made of proteins synthesized in Escherichia coli during various O2- -generating conditions . Nine proteins were constitutively synthesized over wild-type levels in superoxide dismutase (sodA sodB) double mutants . Addition of redox cycling agents such as paraquat and plumbagin at various concentrations induced up to 13 proteins in wild-type cells . Among these 13 were 5 of the 9 constitutively synthesized in the sodA sodB double mutants . Addition of these agents to the superoxide dismutase mutants in low micromolar concentrations induced an additional set of 14 proteins . The proteins induced included only five proteins that have been previously associated with stress responses, consisting of endonuclease IV (Nfo), three oxyR-regulated proteins, and one heat shock protein . O2- -mediated induction of the superoxide inducible (Soi) proteins in the wild type was independent of the oxyR+ gene for all but the three oxyR-regulated proteins . Analyses of proteins from three soi::lacZ gene fusions previously isolated (T . Kogoma, S . B . Farr, K . M . Joyce, and D . O . Natvig, Proc . Natl . Acad . Sci . USA 85:4799-4803, 1988) indicated the specific loss of one of these induced proteins in each fusion strain and the constitutive expression of some Soi proteins. J Bacteriol, 1989 Mar, 171(3), 1386 - 93 Toluene transposons Tn4651 and Tn4653 are class II transposons; Tsuda M et al.; The toluene degradative transposon Tn4651 is included within another transposon, Tn4653, and both of these elements are members of the Tn3 family . The tnpA gene product of each element mediates formation of cointegrates as intermediate products of transposition, and the tnpS and tnpT gene products encoded by Tn4651 take part in resolution of both Tn4651- and Tn4653-mediated cointegrates . Sequence analysis demonstrated that Tn4651 and Tn4653 have 46- and 38-base-pair terminal inverted repeats, respectively, and that both elements generate 5-base-pair duplication of the target sequence upon transposition . Complementation tests of the Tn4651- and Tn4653-encoded transposition functions with those of Tn3, Tn21, and Tn1721 showed that (i) the trans-acting transposition functions encoded by Tn4651 were not interchangeable with those encoded by the four other transposons, (ii) the Tn4653 tnpA function was interchangeable with the Tn1721 function, and (iii) Tn4653 coded for a resolvase (tnpR gene product) that complemented the tnpR mutations of Tn21 and Tn1721 . The Tn4653 tnpR gene was located just 5' upstream of the tnpA gene and shared extensive sequence homology with the Tn1721 tnpR gene . The res region was located adjacent to the tnpR gene, and sequence analysis indicated that failure of the Tn4653 tnpR product to resolve the Tn4653-mediated cointegrates is ascribed to an incomplete structure of the res region. J Bacteriol, 1989 Mar, 171(3), 1314 - 9 Molecular cloning of gltS and gltP, which encode glutamate carriers of Escherichia coli B; Deguchi Y et al.; Two genes encoding distinct glutamate carrier proteins of Escherichia coli B were cloned into an E . coli K-12 strain by using a cosmid vector, pHC79 . One of them was the gltS gene coding for a glutamate carrier of an Na+-dependent, binding protein-independent, and glutamate-specific transport system . The content of the glutamate carrier was amplified about 25-fold in the cytoplasmic membranes from a gltS-amplified strain . The gltS gene was located in a 3.2-kilobase EcoRI-MluI fragment, and the gene product was identified as a membrane protein with an apparent Mr of 35,000 in a minicell system . A gene designated gltP was also cloned . The transport activity of the gltP system in cytoplasmic membrane vesicles from a gltP-amplified strain was driven by respiratory substrates and was independent of the concentrations of Na+, K+, and Li+ . An uncoupler, carbonylcyanide m-chlorophenylhydrazone, completely inhibited the transport activities of both systems, whereas an ionophore, monensin, inhibited only that of the gltS system . The Kt value for glutamate was 11 microM in the gltP system and 3.5 microM in the gltS system . L-Aspartate inhibited the glutamate transport of the gltP system but not that of the gltS system . Aspartate was taken up actively by membrane vesicles from the gltP-amplified strain, although no aspartate uptake activity was detected in membrane vesicles from a wild-type E . coli strain . These results suggest that gltP is a structural gene for a carrier protein of an Na+-independent, binding protein-independent glutamate-aspartate transport system. J Bacteriol, 1989 Mar, 171(3), 1254 - 61 Genetics and sequence analysis of the pcnB locus, an Escherichia coli gene involved in plasmid copy number control; Liu JD et al.; Mutations at the Escherichia coli pcnB locus reduce the copy number of ColE1-like plasmids . We isolated additional mutations in this gene and conducted a preliminary characterization of its product . F-prime elements carrying the pcnB region were constructed and used to show that the mutations were recessive . The wild-type pcnB gene was cloned into a low-copy-number plasmid, and its nucleotide sequence was determined . The sequence analysis indicated that pcnB is probably the first gene in an operon that contains one or more additional genes of unknown function . The pcnB locus should encode a polypeptide of 47,349 daltons (Da) . A protein of this size was observed in minicells carrying a pcnB+ plasmid, and transposon insertions and deletions that truncated this protein generally abolished pcnB function . One exceptional transposon insertion at the promoter-distal end of the pcnB gene truncated the 47-kDa protein by about 20% but did not abolish complementation activity, indicating that the C-terminus of the PcnB product is dispensable . The deduced amino acid sequence of PcnB revealed numerous charged residues and, with 10% arginines, an overall basic character, suggesting that PcnB might interact with DNA or RNA in a structural capacity . Disruption of the pcnB gene by insertional mutagenesis caused a reduction in growth rate, indicating that PcnB has an important cellular function. Infect Immun, 1989 Mar, 57(3), 791 - 7 Dissociation of cell-associated interleukin-1 (IL-1) and IL-1 release induced by lipopolysaccharide and lipid A; Cavaillon JM et al.; The capacities of lipopolysaccharide (LPS) and lipid A to trigger mouse BALB/c peritoneal macrophages and to induce the production of cell-associated interleukin-1 (IL-1) and membrane-associated IL-1 and IL-1 release have been compared . Bordetella pertussis lipid A was 1,000 to 10,000 times less efficient than the native LPS to induce IL-1 release by freshly isolated elicited macrophages . When resident macrophages were studied, lipid A, at high concentrations (greater than 2 micrograms/ml), induced significant levels of cell-associated IL-1 but little or no IL-1 release . With synthetic lipid A built up with the Escherichia coli lipid A structure (compound 506), IL-1 activity was present in the supernatants of elicited peritoneal macrophages and to a lesser extent in those of resident macrophages . However, the release of IL-1 induced by synthetic lipid A 506 remained much lower than those induced by rough LPS . Membrane-associated IL-1 could be induced on BALB/c macrophages with LPS and natural or synthetic lipid A, the LPS being the most active . In C3H/HeJ mice, neither natural nor synthetic lipid A could induce detectable cell-associated IL-1, whereas LPS could induce cell-associated and membrane IL-1 activity but no IL-1 release . Our results indicate that fragments of endotoxins may induce the production of IL-1 but the entire structure of the LPS molecule is the most effective to induce intracellular IL-1 production, expression of membrane IL-1, and release of IL-1. J Virol, 1989 Mar, 63(3), 1345 - 53 Identification of the major capsid protein gene of human cytomegalovirus; Chee M et al.; The coding region for the major capsid protein (MCP) of human cytomegalovirus (HCMV) was identified by comparing the protein sequence with the respective sequences of herpes simplex virus (HSV), Epstein-Barr virus, and varicella-zoster virus . The predicted length of the HCMV MCP was 1,370 amino acids . Comparison of the MCP sequences of the different human herpesviruses showed a homology of 25% to the MCP of HSV type 1, a homology of 29% to the MCP of Epstein-Barr virus, and a homology of 23% to the MCP of varicella-zoster virus . A subfragment of the HSV type 1 KpnI i fragment encoding the MCP VP5 cross-hybridized with the HCMV HindIII U fragment containing part of the MCP gene . Northern (RNA) blot analyses with subclones out of the coding region for the HCMV MCP detected one large transcript of about 8 kilobases . A portion of the open reading frame was expressed in Escherichia coli plasmid pBD2 IC2OH as a beta-galactosidase fusion protein and was used to generate polyclonal antibodies in New Zealand White rabbits . The obtained antisera reacted in Western immunoblots with the MCP of purified HCMV virions . A monoclonal antibody against the human MCP and a monospecific rabbit antiserum against strain Colburn of simian cytomegalovirus detected the fusion protein as well as the MCP of purified virions in immunoblots. Plant Cell, 1989 Mar, 1(3), 275 - 84 Regulation of the Aspergillus nidulans pectate lyase gene (pelA); Dean RA et al.; Aspergillus nidulans pectate lyase was purified from culture filtrates . The enzyme catalyzed a random eliminative cleavage reaction, had an apparent molecular weight of 40,000, and a pl of 4.2 . Pectate lyase antisera were produced and used to identify pectate lyase clones in a cDNA expression library . Thirteen of 14 clones identified immunologically cross-hybridized . The identity of the single-copy pectate lyase gene, which we designated pelA, was confirmed in two ways . First, several cDNA clones expressed pectate lyase activity in Escherichia coli . Second, targeted mutation of the gene in A . nidulans resulted in complete loss of enzyme activity . pelA encodes a 1,300-nucleotide mRNA that was present in cells grown with polygalacturonic acid as carbon source but absent from cells grown with glucose or acetate as carbon source . Thus, pectate lyase expression is regulated at the level of mRNA accumulation. Mol Gen Genet, 1989 Mar, 216(1), 120 - 5 Transcription of a region downstream from lambda ori is required for replication of plasmids derived from coliphage lambda; Hase T et al.; DNA replication of lambda phage depends on transcriptional activation at or around the lambda ori region by RNA polymerase . To elucidate the function of the transcriptional activation, we constructed several plasmids carrying lambda ori and lacP, whose relative locations and directions were different from each other, and studied replication activity of these recombinant plasmids . Transcription in a region immediately downstream from lambda ori, but not in the lambda ori region, was found to be essential for plasmid replication . Transcription proceeding over a certain minimal length was required and only rightward-directed transcription was effective for the activation. Mol Cell Biol, 1989 Mar, 9(3), 1271 - 6 Molecular cloning of a Neurospora crassa carotenoid biosynthetic gene (albino-3) regulated by blue light and the products of the white collar genes; Nelson MA et al.; The albino-3 (al-3) gene of Neurospora crassa, which probably encodes the carotenoid biosynthetic enzyme geranylgeranyl pyrophosphate synthetase, was cloned . The N . crassa triple mutant al-3 qa-2 aro-9 was transformed to qa-2+ with mixtures of plasmids bearing N . crassa DNA inserts, and the transformants were screened for the al-3+ phenotype . One al-3+ qa-2+ transformant (AL3-1) was examined in detail and shown to contain intact vector sequences integrated into the N . crassa genome . The vector and some flanking sequences were recovered from AL3-1 after restriction, ligation, and selection of chloramphenicol-resistant transformants of Escherichia coli . The flanking sequences were subsequently used to detect the al-3-containing plasmid in the mixture of about 1,800 plasmids . Restriction fragment length polymorphism mapping was carried out to confirm the identity of the cloned fragment . The level of the al-3 mRNA was shown to be increased 15-fold in light-induced (compared with that in dark-grown) wild-type mycelia . The light-dependent increase in al-3 mRNA levels was not observed in presumed regulatory mutant (white collar) strains. EMBO J, 1989 Mar, 8(3), 981 - 7 MalT, the regulatory protein of the Escherichia coli maltose system, is an ATP-dependent transcriptional activator; Richet E et al.; We show that MalT, the transcriptional activator of the Escherichia coli maltose regulon, specifically binds ATP and dATP with a high affinity (Kd = 0.4 microM) and exhibits a weak ATPase activity . Using an abortive initiation assay, we further show that activation of open complex formation by MalT depends on the presence of ATP in addition to that of maltotriose, the inducer of the maltose system . Similar experiments in which ATP was replaced by ADP or AMP-PNP, a non-hydrolysable analogue of ATP, demonstrate that this reaction does not require ATP hydrolysis . As revealed by DNase I footprinting, both ATP and maltotriose are required for the binding of the MalT protein to the mal promoter DNA. J Clin Invest, 1989 Mar, 83(3), 897 - 903 Molybdenum cofactor biosynthesis in humans . Identification of two complementation groups of cofactor-deficient patients and preliminary characterization of a diffusible molybdopterin precursor; Johnson JL et al.; Molybdenum cofactor deficiency is a devastating disease with affected patients displaying the symptoms of a combined deficiency of sulfite oxidase and xanthine dehydrogenase . Because of the extreme lability of the isolated, functional molybdenum cofactor, direct cofactor replacement therapy is not feasible, and a search for stable biosynthetic intermediates was undertaken . From studies of cocultured fibroblasts from affected individuals, two complementation groups were identified . Coculture of group A and group B cells, without heterokaryon formation, led to the appearance of active sulfite oxidase . Use of conditioned media indicated that a relatively stable, diffusible precursor produced by group B cells could be used to repair sulfite oxidase in group A recipient cells . Although the extremely low levels of precursor produced by group B cells preclude its direct characterization, studies with a heterologous, in vitro reconstitution system suggest that the precursor that accumulates in group B cells is the same as a molybdopterin precursor identified in the Neurospora crassa molybdopterin mutant nit-1, and that a converting enzyme is present in group A cells which catalyzes an activation reaction analogous to that of a converting enzyme identified in the Escherichia coli molybdopterin mutant ChlA1. Curr Genet, 1989 Mar, 15(3), 177 - 80 Expression of the Escherichia coli beta-glucuronidase gene in industrial and phytopathogenic filamentous fungi; Roberts IN et al.; A chimaeric beta-glucuronidase (GUS) gene has been created by ligating the Aspergillus nidulans glyceraldehyde 3-phosphate dehydrogenase promoter to the coding sequence of the E . coli uidA gene . Co-transformation of this vector into A . nidulans, A . niger and the tomato pathogen Fulvia fulva (syn . Cladosporium fulvum (Cooke} resulted in the expression of beta-glucuronidase . GUS activity was detected by growth on agar media containing X-gluc and by enzyme assays of mycelial extracts . Expression of the gene in F . fulva transformants was also easily detectable during growth in plants and did not affect pathogenicity . These results form the basis for a versatile and sensitive reporter gene system for industrial and phytopathogenic filamentous fungi. Arch Pharm (Weinheim), 1989 Mar, 322(3), 133 - 6 {Antimycobacterial properties of phenothiazine type compounds}; Meindl W et al.; Tests for antimycobacterial properties (Mycobacterium tuberculosis H37 Ra, Middlebrook-7H9-broth) of the phenothiazines 1-11 are described . The growth inhibition of these compounds can be blocked by the addition of histamine. Mol Gen Genet, 1989 Mar, 216(1), 60 - 9 Characterization of the ndhC-psbG-ORF157/159 operon of maize plastid DNA and of the cyanobacterium Synechocystis sp . PCC6803; Steinmuller K et al.; The ndhC and ORF159 genes of the maize plastid DNA (ptDNA) were sequenced and maize ORF159 was used to screen a library of genomic DNA of the blue-green alga Synechocystis sp . PCC 6803 . The cyanobacterial gene homologous to ORF159 (ORF157) was isolated and sequenced . In sequencing the region upstream of ORF157, reading frames with homology to the ndhC and psbG genes of maize ptDNA were identified . The ndhC and psbG genes overlap in the ptDNAs of maize, tobacco and Marchantia polymorpha, but are separated by a noncoding spacer in Synechocystis . Northern blot analysis showed that the ndhC, psbG and ORF157/159 genes are cotranscribed in maize and Synechocystis . The three genes occur in the same order in ptDNA of maize, tobacco, and M . polymorpha as in Synechocystis 6803 . The amino acid sequences of the NDH-C, PSII-G and the ORF157/159 proteins deduced from the maize genes are 65%, 52% and 53% homologous to those of Synechocystis . However, the cyanobacterial and higher plant NDH-C protein sequences are only 23% homologous to the mitochondrial NDH-3 protein . Protein products of in vitro transcription/translation of the Synechocystis transcription unit had apparent molecular masses of 6 kDa (NDH-C), 25 kDa (PSII-G) and 22 kDa (ORF157) on lithium dodecyl sulfate (LDS) polyacrylamide gel electrophoresis . If these are components of an NADH dehydrogenase, cyanobacteria appear to resemble mitochondria more than they do Escherichia coli and Rhodopseudomonas capsulata with regard to this enzyme complex. Mol Gen Genet, 1989 Mar, 216(1), 51 - 9 Mutagenesis by random cloning of an Escherichia coli kanamycin resistance gene into the genome of the cyanobacterium Synechocystis PCC 6803: selection of mutants defective in photosynthesis; Chauvat F et al.; Photosynthetic mutants of the cyanobacterium Synechocystis PCC 6803 were produced by a random cartridge mutagenesis method leading to gene inactivation . This procedure relies on random ligation of an Escherichia coli kanamycin resistance (Kmr) gene to restriction fragments of genomic DNA from the host . Then recombination occurring during transformation promotes integration of the marker gene into the genome of the recipient cells . Several mutants impaired in photosynthesis were obtained by this procedure . All are partially or totally defective in photosystem II activity and some of them also harbour a functionally modified photosystem I . Restriction and recombination data showed that one mutant (AK1) is best explained as an insertion of the Kmr gene into an AvaII restriction site of the gene psbD-1 . All others harbour a deletion, ranging from at least 1.15 kb (AK3) to more than 50 kb (AK9), which partly or fully overlaps the genes psbB and/or psbD-1, depending on the mutant . A genetic-physical map of the more than 60 kb region of the cyanobacterial genome harbouring the genes psbB, psbC and psbD-1 was constructed by combining published sequence data on these genes with the results of recombination and restriction mapping. Mol Gen Genet, 1989 Mar, 216(1), 25 - 30 Genes for the ribosomal proteins S12 and S7 and elongation factors EF-G and EF-Tu of the cyanobacterium, Anacystis nidulans: structural homology between 16S rRNA and S7 mRNA; Meng BY et al.; A 6.5 kb region from the genome of the cyanobacterium, Anacystis nidulans 6301 was cloned using the tobacco chloroplast gene for ribosomal protein S12 as a probe . Sequence analysis revealed the presence of genes for ribosomal proteins S12 and S7 and elongation factors EF-G and EF-Tu in this DNA region . The arrangement is rps12 (124 codons) - 167 bp spacer - rps7 (156 codons) - 77 bp spacer - fus (694 codons) - 26 bp spacer - tufA (409 codons), which is similar to that of the Escherichia coli str operon . The deduced amino acid sequences of the A . nidulans S12 and EF-Tu show high homology (72%-82%) with the E . coli and chloroplast counterparts while those of the A . nidulans S7 and EF-G give low homology (51%-59%) . Striking structural homology was found between the potential S7 binding region of 16S rRNA and the beginning of S7 mRNA, suggesting that feedback regulation of rps7 expression operates in A . nidulans. J Biochem (Tokyo), 1989 Mar, 105(3), 390 - 4 Isolation and sequence studies of cysteinyl peptides from Spirulina glutathione reductase: comparison of active site cysteine peptides with those of other flavoprotein disulfide oxidoreductases; Cui JY et al.; The amino acid sequences of the cysteinyl peptides of Spirulina sp . glutathione reductase were determined . Spirulina glutathione reductase was covalently bound to Thiopropyl-Sepharose 6B in the presence of 8M urea through thiol-disulfide exchange . After tryptic digestion, 4 distinct cysteinyl peptides were finally isolated from NADPH-reduced glutathione reductase and 2 from oxidized glutathione reductase . The amino acid sequences of the two cysteinyl peptides which could not be isolated from the oxidized glutathione reductase were very similar to those around the active site disulfide of the other flavoprotein disulfide oxidoreductases and a unique replacement of asparagine and valine by isoleucine and arginine between the two cysteine residues was found . The other two peptides isolated from both oxidized and reduced glutathione reductase also show considerable homology to the corresponding parts of human and Escherichia coli glutathione reductases. J Biochem (Tokyo), 1989 Mar, 105(3), 341 - 7 Molecular analysis by deletion and site-directed mutagenesis of the cis-acting upstream sequence involved in activation of the ompF promoter in Escherichia coli; Kato M et al.; Expression of the ompF gene coding for an outer membrane protein of Escherichia coli is regulated by a transcriptional activation mechanism that requires the ompR gene product that acts on nucleotides located upstream of the -35 and -10 regions of the ompF promoter . We previously demonstrated that this cis-acting upstream sequence displays a sequence-directed curvature of the DNA helix . To characterize the structure and function of this upstream sequence, a series of deletion mutants and base-substitution mutants of the upstream sequence of the ompF promoter were constructed, and their abilities as to OmpR-binding and activities of the ompF promoter were examined after they had been connected to the lacZ gene . The nucleotides extending from position -91 to -79 are essential not only for sequence-specific recognition of the ompF promoter by the OmpR protein, but also for OmpR-dependent activation of the ompF promoter . It was also demonstrated that the nucleotides extending from position -111 to -92 play a role in stimulation of the ompF expression . A local structural alteration in the ompF promoter was observed in some of the base-substitution mutants . Based on the results, the structure and function of the upstream sequence of the ompF promoter are discussed in relation to activation of the ompF promoter by the OmpR protein. EMBO J, 1989 Mar, 8(3), 903 - 11 Suppression in Drosophila: su(Hw) and su(f) gene products interact with a region of gypsy (mdg4) regulating its transcriptional activity; Mazo AM et al.; The gypsy (mdg4) mobile element of Drosophila contains two closely spaced regions which bind proteins from nuclear extracts . One of these is an imperfect palindrome having homology with the lac-operator of Escherichia coli; the other contains a reiterated sequence (5'PyPuT/C TGCATAC/TPyPy) homologous to the octamer that is the core of many enhancers and upstream promoter elements . Transient expression of deletion mutants has shown that these DNA regions are negative and positive regulators of transcription . As was demonstrated earlier by other authors, mutations induced by the presence of gypsy in different loci are suppressed owing to either repression or activation of gypsy transcription in Drosophila strains carrying unlinked mutations in su(Hw) or su(f) genes . We have shown that binding to a negative regulator (silencer) is weakened in nuclear extracts isolated from fly stocks carrying su(f) mutations which activate gypsy transcription; therefore the su(f) gene seems to code for a protein capable of gypsy repression . Furthermore, binding to a positive regulator is weakened in nuclear extracts isolated from fly stocks carrying su(Hw) gene mutations which decrease the level of gypsy transcription; therefore, the su(Hw) gene most likely encodes a protein which activates gypsy transcription. Vet Microbiol, 1989 Mar, 19(3), 263 - 73 Effects of exogenous amino acids on the multiplication of porcine Escherichia coli; Aning KG et al.; The multiplication rates of 70 porcine Escherichia coli strains were compared in minimal medium and in medium supplemented with aspartic acid, lysine, serine and threonine, which were the amino acids taken up during multiplication of porcine E . coli in a complex medium . The effects of these amino acids singly or in combinations and the amino acids norleucine and norvaline on the growth of porcine E . coli were studied . Together, aspartic acid, threonine, serine and lysine increased the multiplication rates of 42.9% of the strains, an effect traced to aspartic acid, but they had no effect on an equal number of strains . The rest were inhibited, and this effect was traced to serine . Cysteine, threonine, leucine and phenylalanine singly inhibited some or all of the strains tested . Norleucine and to a lesser extent, norvaline greatly prolonged the lag phase of culture in minimal medium . The inhibitory effect of norleucine was reversed by only methionine, although isoleucine, leucine and valine which were more effective in norvaline inhibition, also showed limited antagonism to norleucine inhibition. Biochem J, 1989 Mar 1, 258(2), 335 - 42 Interactions between inhibitors of dihydrofolate reductase; Bowden K et al.; The binding of substrates and inhibitors to dihydrofolate reductase was studied by steady-state kinetics and high-field 1H-n.m.r . spectroscopy . A series of 5-substituted 2,4-diaminopyrimidines were examined and were found to be 'tightly binding' inhibitors of the enzyme (Ki less than 10(-9) M) . Studies on the binding of 4-substituted benzenesulphonamides and benzenesulphonic acids also established the existence of a 'sulphonamide-binding site' on the enzyme . Subsequent n.m.r . experiments showed that there are two binding sites for the sulphonamides on the enzyme, one of which overlaps the coenzyme (NADPH) adenine-ring-binding site . An examination of the pH-dependence of the binding of sulphonamides to the enzyme indicated the influence of an ionizable group on the enzyme that was not directly involved in the sulphonamide binding . The change in pKa value from 6.7 to 7.2 observed on sulphonamide binding suggests the involvement of a histidine residue, which could be histidine-28. Mutat Res, 1989 Mar-May, 220(2-3), 61 - 72 Use of supF, an Escherichia coli tyrosine suppressor tRNA gene, as a mutagenic target in shuttle-vector plasmids; Kraemer KH et al.; The Escherichia coli tyrosine amber suppressor tRNA gene, supF, has been utilized as a mutagenic target in several shuttle-vector plasmids . Data on mutagenic inactivation of suppressor activity was obtained from induced mutagenesis experiments with plasmids pZ189 and p3AC, and from studies on alterations of the supF gene transduced into E . coli . 162 single or tandem base-substitution mutations that reduce or eliminate suppressor activity were identified at 86 sites within 158 base pairs . The 2 transition and 4 transversion mutations possible in double-stranded DNA were all detectable . At 56 sites two different inactivating mutations were found; and at 20 sites all 3 possible base substitution mutations inactivated suppressor function . Most of the mutations were clustered within the mature tRNA region: 144 of the base-substitution mutations were found at 74 sites within the 85-bp mature tRNA region . Insertions of 1 or 2 bases at 4 sites and deletions of 1 to 3 bases at 15 sites were found to inactivate supF function . A few silent mutations which do not inactivate suppressor function were found: single base-substitutions at 4 sites, 14 pairs of silent double mutations, and a large deletion including the promoter region . The supF gene is thus an extremely sensitive target for mutagenic inactivation in shuttle-vector plasmids. J Mol Evol, 1989 Mar, 28(3), 242 - 55 DNA sequence and secondary structures of the large subunit rRNA coding regions and its two class I introns of mitochondrial DNA from Podospora anserina; Cummings DJ et al.; DNA sequence analysis has shown that the gene coding for the mitochondrial (mt) large subunit ribosomal RNA (rRNA) from Podospora anserina is interrupted by two class I introns . The coding region for the large subunit rRNA itself is 3715 bp and the two introns are 1544 (r1) and 2404 (r2) bp in length . Secondary structure models for the large subunit rRNA were constructed and compared with the equivalent structure from Escherichia coli 23S rRNA . The two structures were remarkably similar despite an 800-base difference in length . The additional bases in the P . anserina rRNA appear to be mostly in unstructured regions in the 3' part of the RNA . Secondary structure models for the two introns show striking similarities with each other as well as with the intron models from the equivalent introns in Saccharomyces cerevisiae, Neurospora crassa, and Aspergillus nidulans . The long open reading frames in each intron are different from each other, however, and the nucleotide sequence similarity diverges as it proceeds away from the core structure . Each intron is located within regions of the large subunit rRNA gene that are highly conserved in both sequence and structure . Computer analysis showed that the open reading frame for intron r1 contained a common maturase-like polypeptide . The open reading frames of intron r2 appeared to be chimeric, displaying high sequence similarity with the open reading frames in the r1 and ATPase 6 introns of N . crassa. J Mol Evol, 1989 Mar, 28(3), 232 - 41 DNA sequence, structure, and phylogenetic relationship of the small subunit rRNA coding region of mitochondrial DNA from Podospora anserina; Cummings DJ et al.; DNA sequence analysis and the localization of the 5' and 3' termini by S1 mapping have shown that the mitochondrial (mt) small subunit rRNA coding region from Podospora anserina is 1980 bp in length . The analogous coding region for mt rRNA is 1962 bp in maize, 1686 bp in Saccharomyces cerevisiae, and 956 bp in mammals, whereas its counterpart in Escherichia coli is 1542 bp . The P . anserina mt 16S-like rRNA is 400 bases longer than that from E . coli, but can be folded into a similar secondary structure . The additional bases appear to be clustered at specific locations, including extensions at the 5' and 3' termini . Comparison with secondary structure diagrams of 16S-like RNAs from several organisms allowed us to specify highly conserved and variable regions of this gene . Phylogenetic tree construction indicated that this gene is grouped with other mitochondrial genes, but most closely, as expected, with the fungal mitochondrial genes. Ann Surg, 1989 Mar, 209(3), 334 - 40 A new model for studying nutrition in peritonitis . The adverse effect of overfeeding; Alexander JW et al.; In guinea pigs fed ad libitum, controlled intraperitoneal infusion of bacteria by an implanted 7-day osmotic pump resulted in peritonitis or abscess formation with a 50% survival 14-18 days after pump implantation . Administration of 125 kcal/kg/day of a diet found to be optimal for burned guinea pigs by continuous pump controlled feedings via a previously placed gastrostomy was well-tolerated, with a 62.5% mortality by Day 17 . Administration of only 100 kcal/kg/day caused weight loss of approximately 17% after 16 days, but fewer animals died (42.8%, p = NS) . Feeding either 150 kcal/kg/day or 175 kcal/kg/day caused death in all 25 animals (p less than 0.001) and their survival time was slightly shortened (p = NS) when compared with animals receiving 100 or 125 kcal/kg/day . This is the first animal model of peritonitis that permits incisive dissection of the relative influences of dietary composition on outcome, because survival can be extended to 2 weeks or more in the presence of continuing sepsis. Proc Natl Acad Sci U S A, 1989 Mar, 86(5), 1485 - 9 Mechanism-based inhibition of a mutant Escherichia coli ribonucleotide reductase (cysteine-225----serine) by its substrate CDP; Mao SS et al.; The B1 subunit of Escherichia coli ribonucleotide reductase (EC 1.17.4.1) has been overexpressed using the pT7-5/pGP1-2 system developed by Tabor and Richardson {Tabor, S . & Richardson, C . (1985) Proc . Natl . Acad . Sci . USA 82, 1074-1078} . This method has allowed the preparation of two mutant B1 subunits in which two of the four thiols postulated to be within the active site of the enzyme, Cys-225 and Cys-759, have been changed to serines . Incubation of the {Ser225}B1 mutant with the B2 subunit, {U-14C}CDP, and the allosteric effector ATP results in production of cytosine, destruction of the tyrosyl radical in B2, radiolabeling of the protein, and cleavage of the B1 subunit into two pieces of 26 and 61.5 kDa . This process is independent of the identity of reductant . The {Ser759}B1 mutant reduces CDP in the presence of thioredoxin/thioredoxin reductase at 7.7% the rate of wild-type B1 . When dithiothreitol is utilized as reductant, however, the rate of CDP reduction with {Ser759}B1 is identical to that observed with wild type. J Bacteriol, 1989 Mar, 171(3), 1535 - 43 Site-directed mutation of the Escherichia coli ada gene: effects of substitution of methyl acceptor cysteine-321 by histidine in Ada protein; Tano K et al.; Oligodeoxynucleotide-mediated mutagenesis of the ada gene of Escherichia coli was used to produce two mutant Ada proteins . In mutant I the methyl acceptor Cys-321 for O6-methylguanine was replaced by histidine; and in mutant II the positions of Cys-321 and His-322 of the wild-type protein were inverted . Neither mutant protein had O6-methylguanine-DNA methyltransferase activity, but both retained the phosphotriester-DNA methyltransferase activity involving methyl group transfer to Cys-69 . Under the control of the endogenous promoter, synthesis of mutant I protein was undetectable before or after adaptation treatment with promoter, synthesis of mutant I protein was undetectable before or after adaptation treatment with N-methyl-N'-nitro-N-nitrosoguanidine . This appeared to be due to both inhibition of transcription of the mutant gene and degradation of the synthesized protein . On the other hand, mutant II protein was inducible by N-methyl-N'-nitro-N-nitrosoguanidine, although to a smaller extent than the wild-type protein was, and the phosphotriester-DNA methyltransferase activity appeared to reside in 24- to 30-kilodalton cleavage products . Mutant I protein could be produced under lac promoter control, and its cleavage products, unlike those of mutant II protein, tended to aggregate . These results indicate that (i) Cys-321 cannot be replaced or transposed with the nucleophilic amino acid histidine for O6-methylguanine-DNA methyltransferase function, (ii) single amino acid replacement or transposition at the O6-methylguanine methyl acceptor site can have a profound effect on the in vivo stability and regulatory function of the Ada protein, and (iii) the integrity of the protein may not be absolutely needed for its transcription-activation function. Arch Biochem Biophys, 1989 Mar, 269(2), 612 - 22 Escherichia coli-derived human interferon-gamma with Cys-Tyr-Cys at the N-terminus is partially N alpha-acylated; Honda S et al.; Purified preparations of recombinant human interferon-gamma (rIFN-gamma) with Cys-Tyr-Cys at the N-terminus ({ Cys-Tyr-Cys}IFN-gamma) derived from Escherichia coli gave two closely migrating bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two peaks on reversed-phase high-performance liquid chromatography (rpHPLC) . In contrast, rIFN-gamma without Cys-Tyr-Cys and rIFN-gamma in which both Cys-1 and Cys-3 were substituted with serine behaved as a single species on both SDS-PAGE and rpHPLC . These results suggest that the N-terminal portion of rIFN-gamma is heterogeneous . To elucidate the structure of the N-terminal portion, the N-terminal peptide preparation was obtained by binding rIFN-gamma to thiopropyl-Sepharose 6B gel with disulfide linkage followed by trypsin digestion and elution with 2-mercaptoethanol . The preparation gave four peaks (NT-1, NT-2, NT-3, and NT-4, in order of elution) on rpHPLC; all four were found to be Cys-1-Lys-9 by amino acid analysis after acid hydrolysis . Various analyses indicate that NT-1 is the intact nonapeptide, that NT-3 and NT-4 are N alpha-formyl and N alpha-acetyl forms of NT-1, respectively, and that NT-2 may be S-blocked at Cys-1 . It is concluded that E . coli-derived {Cys-Tyr-Cys}IFN-gamma is partially N alpha-acylated . The data also suggest that N alpha-acylation does not affect the biological activity of {Cys-Tyr-Cys}IFN-gamma. Mutat Res, 1989 Mar, 217(2), 109 - 15 Expression of DNA damage-inducible genes of Escherichia coli upon treatment with methylating, ethylating and propylating agents; Volkert MR et al.; Several alkylation-inducible genes have been identified by construction of Mu-d1 (Apr lac) fusions to genes whose expression is increased in response to alkylation treatment, but not UV treatment . We have examined the induction of 4 different alkylation-inducible genes by treatment with a variety of methylating and ethylating agents, and a propylating agent . We have compared the induction of the alkylation-inducible genes with the induction of the sulA gene, which is a component of the SOS response to DNA damage . We find that the Ada-regulated adaptive response genes (ada-alkB, alkA and aidB) are induced primarily in response to methylation treatment . The ada-independent aidC gene is induced upon treatment with agents that alkylate predominantly by SN1 nucleophilic attack . aidC induction occurs only when cells are not aerated during treatment . The SOS response, as indicated by sulA induction, is strongly induced by all types of alkylating agents used. Neuron, 1989 Mar, 2(3), 1265 - 73 VAT-1: an abundant membrane protein from Torpedo cholinergic synaptic vesicles; Linial M et al.; Expression screening was used to isolate cDNA clones encoding a synaptic vesicle membrane protein, VAT-1, which is specifically expressed in the electric lobe of marine rays . The predicted protein has a molecular weight of 41,572 daltons and contains several hydrophobic regions . An antibody raised against a fusion protein synthesized in E . coli recognizes an abundant 42 kd protein that copurifies largely with synaptic vesicles . Trypsin digestion of intact and lysed vesicles as well as membrane extractions suggests that VAT-1 is an integral membrane protein . The VAT-1 RNA is localized to the electromotor nucleus, and the fusion protein antibody stains the electric organ, demonstrating that the protein is transported to nerve terminals . These studies define a novel synaptic vesicle protein that is likely to play a central role in the functions mediated by specific classes of synaptic vesicles. Chem Pharm Bull (Tokyo), 1989 Mar, 37(3), 680 - 3 Effects of photo-activated bleomycin on deoxyribonuclease I, exonuclease III and deoxyribonucleic acid polymerase I reactions; Mori H; The activity of bleomycin to break the strand of deoxyribonucleic acid (DNA) in the presence of 2-hydroxy-1-ethanethiol (2-mercaptoethanol) was enhanced by ultraviolet (UV) irradiation . Photo-activated bleomycin stimulated the action of deoxyribonuclease I (DNase I) to degrade DNA and the DNA synthesis by DNA polymerase I with DNase I . On the other hand, although UV-irradiated bleomycin scarcely broke the DNA strand in the presence of 1,2-benzenediol (catechol), it stimulated the action of DNase I to degrade DNA in the presence of catechol . In accordance with the inhibition by catechol, when DNA treated with UV-irradiated bleomycin in the presence of catechol was employed as a primer for the DNA synthesis, the incorporation of precursor into the acid-insoluble fraction by DNA polymerase I with exonuclease III was reduced to about one-half of the incorporation into DNA treated with unirradiated bleomycin . These findings suggest that the ability of bleomycin to bind to double-helical DNA forming regions sensitive to DNase I was increased by an appropriate dose of UV irradiation and that catechol inhibited the activity of the UV-irradiated bleomycin to break the DNA strand rather than to bind to DNA. Microb Pathog, 1989 Mar, 6(3), 227 - 31 Cloning of Plasmodium yoelii genes expressing three different sporozoite-specific antigens; Wortman A et al.; Genomic DNA isolated from Plasmodium yoelii (strain #17XNL) was prepared by partial digestion and cloned in Escherichia coli TB-1 with pUC18 plasmid . Antigen-producing recombinants were detected by a battery of monoclonal antibodies against antigens of the sporozoite stages . Four clones producing stage-specific sporozoite antigens were identified . One produced P . yoelii circumsporozoite protein, and three produced other P . yoelii sporozoite antigens. Mol Gen Genet, 1989 Mar, 216(1), 113 - 9 Characterization of cis-acting mutations which increase expression of a glnS-lacZ fusion in Escherichia coli; Plumbridge J et al.; glnS-lacZ fusions have been used to isolate mutations which enhance expression of the glnS gene . One mutation, acting at the level of transcription changes the -10 region of the promoter from GATCAT to TATCAT and produces a ten-fold increase in mRNA . Four other mutations which enhance expression three-fold to nine-fold fall within the transcribed region, but not within the Shine and Dalgarno sequence nor in the initiator codon . These mutations are shown to enhance translation specifically and different models are considered to explain their mode of action. J Gen Virol, 1989 Mar, 70 ( Pt 3), 543 - 55 Expression in Escherichia coli of seven DNA fragments comprising the complete L1 and L2 open reading frames of human papillomavirus type 6b and localization of the 'common antigen' region; Strike DG et al.; Molecular cloning was used to express human papillomavirus type 6b (HPV-6b) antigens in Escherichia coli . Seven genomic DNA fragments of HPV-6b which together comprise the complete L1 and L2 open reading frames, known to code for capsid proteins, were cloned and expressed in E . coli as both beta-galactosidase and TrpE fusion proteins . Western blots of HPV-6b beta-galactosidase fusion proteins using 'genus-specific' antisera produced by immunization of rabbits with disrupted bovine papillomavirus type 1 (BPV-1) showed that polypeptides encoded by two DNA fragments from the mid portion of L1 of HPV-6b were cross-reactive . Only one of these two polypeptides reacted with antisera raised against disrupted HPV-1, directly demonstrating that this polypeptide contains the papillomavirus 'common antigen' . The cross-reactive region was confirmed by reversing antigen and antibody . Polyclonal antisera were raised against the seven HPV-6b beta-galactosidase fusion proteins and tested against BPV-1 virion proteins on Western blots . Only antiserum against the mid portion of L1 of HPV-6b reacted with the BPV-1 major capsid protein . HPV-6b fusion proteins were also used to test human sera for antibodies reactive in Western blots . Serum samples from 38 patients with documented HPV-6 infections and from 22 presumably uninfected controls were tested . Antibodies were not detected in any of the sera to any of the seven fusion proteins . HPV-6b beta-galactosidase fusion proteins are antigenic and can be used on Western blots to localize immunologically reactive sub-regions of proteins by reacting protein fragments with antisera from immunized animals . However, alternative methods will be required to detect anti-HPV antibodies in human sera. J Biochem (Tokyo), 1989 Mar, 105(3), 351 - 7 Molecular cloning and expression of rat interleukin-1 alpha cDNA; Nishida T et al.; A cDNA sequence coding for rat interleukin-1 alpha (IL-1 alpha) has been isolated from a cDNA library that was prepared with mRNA derived from LPS-stimulated rat peritoneal macrophages by using human IL-1 alpha cDNA as a probe . The rat cDNA encodes a 270 amino acid residue protein which is homologous (65%) to human IL-1 alpha . The rat cDNA sequence under SV40 early promoter directed the synthesis of biologically active IL-1 in monkey COS-1 cells . Rat IL-1 alpha mRNA is not expressed in spleen, lung, liver or brain, and is also not expressed in these organs of LPS-treated rat except spleen . This suggests that IL-1 alpha is not produced constitutively in various tissues and LPS is not sufficient to induce IL-1 alpha in most tissues . Our data indicate that the IL-1 activities which have been reported to be produced in the brain are not of alpha type . We have constructed a plasmid expressing the carboxy terminal 156 amino acids in Escherichia coli . Recombinant rat IL-1 alpha produced in COS cells or E . coli has cytotoxic activity against the human melanoma cell line A375S1 (GIF activity), which has been reported to be sensitive to human IL-1 alpha and IL-1 beta . This suggests that GIF activity is common to IL-1s derived from various sources. Mol Cell Biol, 1989 Mar, 9(3), 965 - 73 Antibody to a human DNA repair protein allows for cloning of a Drosophila cDNA that encodes an apurinic endonuclease; Kelley MR et al.; The cDNA of a Drosophila DNA repair gene, AP3, was cloned by screening an embryonic lambda gt11 expression library with an antibody that was originally prepared against a purified human apurinic-apyrimidinic (AP) endonuclease . The 1.2-kilobase (kb) AP3 cDNA mapped to a region on the third chromosome where a number of mutagen-sensitive alleles were located . The cDNA clone yielded an in vitro translation product of 35,000 daltons, in agreement with the predicted size of the translation product of the only open reading frame of AP3, and identical to the molecular size of an AP endonuclease activity recovered following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Drosophila extracts . The C-terminal portion of the predicted protein contained regions of presumptive DNA-binding domains, while the DNA sequence at the amino end of AP3 showed similarity to the Escherichia coli recA gene . AP3 is expressed as an abundant 1.3-kb mRNA that is detected throughout the life cycle of Drosophila melanogaster . Another 3.5-kb mRNA also hybridized to the AP3 cDNA, but this species was restricted to the early stages of development. Infect Immun, 1989 Mar, 57(3), 983 - 8 Quantitative study of the binding and hemolytic efficiency of Escherichia coli hemolysin; Eberspacher B et al.; Mono- and polyclonal antibodies were used to construct a sandwich enzyme-linked immunosorbent assay that permitted quantitation of Escherichia coli hemolysin in soluble and membrane-bound forms . Toxin concentrations of 4 to 14 micrograms/ml were measured in culture supernatants of E . coli LE 2001 at times of peak hemolytic activity . Quantitative studies on the binding of E . coli hemolysin to rabbit erythrocytes were conducted at 0 and 37 degrees C . At 37 degrees C, 85 to 95% of bindable toxin was cell bound after 60 min, and no saturability of binding was observed in the studied range of concentrations, which resulted in deposition of approximately 100 to 50,000 toxin molecules per cell . Binding was slower and less effective at 0 degrees C; however, hemolysis did occur at low temperature . The number of cell-bound toxin molecules required to generate a hemolytic lesion within 60 min was estimated to be approximately 100 molecules per cell at 37 degrees C and 800 to 1,000 molecules per cell at 0 degrees C . Upon prolonged incubation (5 to 20 h, 37 degrees C), the number of molecules evoking a functional lesion decreased to approximately 5 to 20 per cell . These results are compatible with the concept that E . coli hemolysin first adsorbs to the cell surface, with membrane insertion and pore formation following in a second step that may be temporally dissociated from that of binding . The data support the pore concept of toxin action by showing that attachment of a low and finite number of toxin molecules to an erythrocyte will ultimately generate a cytolytic lesion. Infect Immun, 1989 Mar, 57(3), 907 - 11 Ganglioside epitope recognized by K99 fimbriae from enterotoxigenic Escherichia coli; Ono E et al.; The receptor structure recognized by Escherichia coli possessing K99 fimbriae and by isolated K99 fimbrial fractions was examined by using an equine erythrocyte hemagglutination inhibition test . Both K99-positive organisms (strain B41) and fimbrial preparations reacted with N-glycolylneuraminyl-lactosyl-ceramide (NeuGcLacCer) purified from equine erythrocytes with very high potency . Fimbrial preparations were 253 times more potent than intact organisms, indicating that isolated fimbriae more precisely recognize the structure of NeuGcLacCer than do fimbriae located on the bacterial cell wall . Structurally, the N-glycolyl group of the sialic acid was shown to be essential because substitution of the N-acetyl group for the N-glycolyl group caused the reactivity to completely disappear . The substitution of the O-acetyl group for the C4 hydroxyl group of the sialic acid (4-O-Ac-NeuGcLacCer) also diminished the reactivity by about 500 times, indicating that the fine structure of NeuGc is necessary for recognition . N-Glycolylneuraminyl-neolactotetraosyl-ceramide (NeuGcnLc4Cer) and N-glycolylneuraminyl-neolactohexaosyl-ceramide (NeuGcnLc6Cer), both of which have identical disaccharides at the nonreducing terminal and longer carbohydrate chains, showed reduced reactivity, indicating that the ceramide of NeuGcLacCer is also involved in the recognition . Indeed, NeuGcLac oligosaccharides altered by cleavage of the ceramide or the terminal sialic acid (NeuGc) showed dramatically reduced reactivities . Ten other E . coli strains (isolated from diseased calves) and two strains (isolated from diseased piglets) which possessed the same K99 antigen and various O antigens were used for the recognition test . The results obtained were similar to those mentioned above. Infect Immun, 1989 Mar, 57(3), 887 - 95 Pore formation by the Escherichia coli hemolysin: evidence for an association-dissociation equilibrium of the pore-forming aggregates; Benz R et al.; Lipid bilayer experiments were performed in the presence of hemolysin of Escherichia coli . The toxin had a rather low activity in membranes formed of pure lipids, such as phosphatidylcholine or phosphatidylserine . In membranes from asolectin, a crude lipid mixture from soybean, hemolysin was able to increase the conductance by many orders of magnitude in a steep concentration-dependent fashion, which suggested that several hemolysin molecules could be involved in the conductive unit . Furthermore, the much higher toxin activity in asolectin membranes would be consistent with the assumption that this lipid contains a receptor needed for membrane activity of the toxin . The results of single-channel records showed that the membrane activity of hemolysin is due to the formation of ion-permeable channels with a single-channel conductance of about 500 pS in 0.15 M KCl . The hemolysin channel seemed to be formed by a toxin oligomer which showed an association-dissociation reaction and had a mean lifetime of about 2 s at small transmembrane voltages . The conductance of the hemolysin channels was only moderately dependent on the salt concentration in the aqueous phase . Zero-current membrane potential experiments showed that the hemolysin channel is cation selective . The mobility sequence of the cations in the channel was similar to their mobility sequence in the aqueous phase, which was consistent with the assumption that the hemolysin channel is wide and that the interior field strength is not very high . From the single-channel conductance, a lower limit of about 1.0 nm for the effective channel diameter could be estimated. Biochem Biophys Res Commun, 1989 Feb 28, 159(1), 363 - 7 Opossum kidney contains a functional receptor for the Escherichia coli heat-stable enterotoxin; White AA et al.; The Escherichia coli heat-stable enterotoxin (ST1 or STa) binds to specific receptors on mammalian intestinal brush border membranes, and stimulates guanylate cyclase in those membranes . We have found a similar signal transduction system in brush border membranes prepared from kidney cortex of the American opossum (Didelphis virginiana, and in a cell line (OK cell) derived from that tissue . Activation of guanylate cyclase by ST1 is therefore not limited to intestinal cells . Furthermore, since it is unlikely that ST1 which is produced in the intestinal lumen, would have access to kidney receptors, this suggests the existence of an endogenous peptide resembling ST1, at least in marsupials. Biochem Biophys Res Commun, 1989 Feb 28, 159(1), 337 - 42 {Arg292----Val} or {Arg292----Leu} mutation enhances the reactivity of Escherichia coli aspartate aminotransferase with aromatic amino acids; Hayashi H et al.; Arg292 of E . coli aspartate aminotransferase was substituted with valine or leucine by site-directed mutagenesis . In comparison with the wild-type enzyme, either of the mutant enzymes showed a decrease by over 5 orders of magnitude of kcat/km values for aspartate and glutamate . This supports the contention that Arg292 is important for determining the specificity of this enzyme for dicarboxylic substrates . In contrast, mutant enzymes displayed a 5- to 10-fold increase in kcat/Km values for aromatic amino acids as substrates . Thus, introduction of an uncharged, hydrophobic side chain into position 292 leads to a striking alteration in substrate specificity of this enzyme, thereby improving catalytic efficiency toward aromatic amino acids. Biochem Biophys Res Commun, 1989 Feb 28, 159(1), 103 - 11 Mutagenesis of human granulocyte colony stimulating factor; Kuga T et al.; To define the structure-function relationship, we have made a number of mutants of human granulocyte colony-stimulating factor (hG-CSF) by in vitro mutagenesis . The results indicate that most of the mutations located in the internal and C-terminal regions of the molecule abolished the activity, whereas the mutants without N-terminal 4, 5, 7, or 11 amino acids retained the activity . N-terminal amino acids were also altered by cassette mutagenesis using a synthetic oligonucleotide mixture . Among them, KW2228, in which Thr-1, Leu-3, Gly-4, Pro-5 and Cys-17 were respectively substituted with Ala, Thr, Tyr, Arg and Ser, showed more potent granulopoietic activity than that of intact hG-CSF both in vitro and in vivo. FEBS Lett, 1989 Feb 27, 244(2), 383 - 7 Expression of ricin B chain in Escherichia coli; Hussain K et al.; DNA encoding ricin B chain was fused to that encoding the E . coli OmpA signal peptide using the expression secretion vector pIN-111-ompA . When induced, E . coli cells transformed with the recombinant plasmid express ricin B chain . The recombinant product accumulates in the periplasmic space in a soluble, biologically active form. Nucleic Acids Res, 1989 Feb 25, 17(4), 1649 - 63 Processing pathway of Escherichia coli 16S precursor rRNA; Srivastava AK et al.; Immediate precursors of 16S rRNA are processed by endonucleolytic cleavage at both 5' and 3' mature termini, with the concomitant release of precursor fragments which are further metabolized by both exo- and endonucleases . In wild-type cells rapid cleavages by RNase III in precursor-specific sequences precede the subsequent formation of the mature ends; mature termini can, however, be formed directly from pre-16S rRNA with no intermediate species . The direct maturation is most evident in a strain deficient in RNase III, and the results in whole cells are consistent with results from maturation reactions in vitro . Thus, maturation does not require cleavages within the double-stranded stems that enclose mature rRNA sequences in the pre-16S rRNA. Nucleic Acids Res, 1989 Feb 25, 17(4), 1427 - 43 Expression of biologically active middle T antigen of polyoma virus from recombinant baculoviruses; Forstova J et al.; Two different recombinant baculoviruses have been generated for expressing the middle T antigen (MT) of polyoma virus in insect (Sf9) cells . One (pAcI-PyMT) produces moderate levels of MT and the other (pVL-PyMT) high levels . Indirect immunofluorescence and cellular fractionation studies with pAcI-PyMT infected Sf9 cells give results similar to those observed with wild type polyoma virus infected mouse cells, and show MT to be mainly associated with cytoplasmic membranes in the insect cell . In the latter, a sub-population of MT is phosphorylated in in vitro protein kinase assays . The yields of MT from pVL-PyMT infected cells are high enough to suggest that this protein can now be produced by this method in sufficient amounts for definitive biochemical and crystallographic analyses. J Biol Chem, 1989 Feb 25, 264(6), 3583 - 7 Energy-dependent in vitro translocation of a model protein into Escherichia coli inverted membrane vesicles can take place efficiently in the complete absence of the cytosol fraction; Matsuyama S et al.; The translocation of the precursor of a secretory protein into Escherichia coli inverted membrane vesicles was demonstrated in the absence of the cytosol fraction . A small, hydrophilic chimeric protein, OmpF-Lpp, possessing an uncleavable signal peptide was used as the model protein . As much as 80% translocation of the precursor protein into membrane vesicles was observed within 6 min in the absence of the cytosol fraction, when the precursor protein purified by means of immunoaffinity chromatography was used . The translocation was dependent on both ATP and respiratory substrates such as succinate . ATP could be replaced by a higher concentration of CTP or UTP, whereas GTP was inactive . Trichloroacetic acid treatment of the precursor protein, which is reported to result in removal of the trigger factor that is attached to a precursor protein (Crooke, E., and Wickner, W . (1987) Proc . Natl . Acad . Sci . U.S.A . 84, 5216-5220), did not lower the translocation efficiency significantly . Finally, the precursor protein, which was highly purified by means of successive immunoaffinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, could still be efficiently translocated into the membrane vesicles . The precursor proteins purified in the presence and absence of bovine serum albumin were both active . Neither washing of the membrane vesicles for prevention of possible contamination by cytosolic factors nor the addition of the cytosol fraction to the reaction mixture affected the translocation efficiency . These results indicate that the in vitro translocation of the OmpF-Lpp precursor protein can take place in the complete absence of cytosolic soluble proteins. J Biol Chem, 1989 Feb 25, 264(6), 3057 - 60 Discrimination between oxygen and carbon monoxide and inhibition of autooxidation by myoglobin . Site-directed mutagenesis of the distal histidine; Springer BA et al.; Sperm whale myoglobin mutants were constructed using site-directed mutagenesis to replace the highly conserved distal histidine residue (His(E7)-64) . His-64 was substituted with Gly, Val, Phe, Cys, Met, Lys, Arg, Asp, Thr, and Tyr, and all 10 mutant proteins expressed to approximately 10% of the total soluble cell protein in Escherichia coli as heme containing myoglobin . With the exception of His-64----Tyr, which did not form a stable oxygen (O2) complex, all mutant proteins could be reduced and bound O2 and carbon monoxide (CO) reversibly . However, removal of the distal histidine increased the rate of autooxidation 40-350-fold . The His-64----Gly, Val, Phe, Met, and Arg mutants all showed markedly increased O2 dissociation rate constants which were approximately 50-1500-fold higher than those for wild-type myoglobin and increased O2 association rate constants which were approximately 5-15-fold higher than those for the native protein . All mutants studied (except His-64----Tyr) showed approximately 10-fold increased CO association rates and relatively unchanged CO dissociation rates . These altered O2 and CO association and dissociation rate constants resulted in 3-14-fold increased CO affinities, 10-200-fold decreased O2 affinities, and 50-380-fold greater M (KCO/KO2) values for the mutants compared to the wild-type protein . Thus, the distal histidine of myoglobin discriminates between CO and O2 binding by both sterically hindering bound CO and stabilizing bound O2 through hydrogen bonding . The increased autooxidation rates observed for the mutants appear to be due to a decrease in oxygen affinity and an increase in solvent anion accessibility to the distal pocket. Nucleic Acids Res, 1989 Feb 25, 17(4), 1283 - 98 A stable core region of the tra operon mRNA of plasmid R1-19; Koraimann G et al.; The degradation of the polycistronic tra-mRNA of the resistance plasmid R1-19 leads to the accumulation of a well defined series of stable mRNA species . The majority of the most stable mRNAs contains the message for the traA gene only . The differently sized stable mRNAs possess a common 3'terminus within the traL gene but vary at their 5' ends . The 3'terminus probably results from protection against exoribonucleases by a secondary structural feature . We propose that the 5' ends are generated by endoribonucleolytic cleavage . The stability of this part of the tra-mRNA exceeds 30 minutes and probably increases the rate of expression of the traA gene product propilin, the precursor of the sex pilus subunit . The expression of propilin and its processing into a protein of the molecular weight of mature pilin is demonstrated with the isolated gene . The sequence of the so far unknown genes traL and traE of R1-19 is presented. J Biol Chem, 1989 Feb 25, 264(6), 3341 - 6 Conversion of amino acid residues in proteins and amino acid homopolymers to carbonyl derivatives by metal-catalyzed oxidation reactions; Amici A et al.; A number of metal-catalyzed oxidation (MCO) systems mediate the oxidative inactivation of enzymes . This oxidation is accompanied by conversion of the side chains of some amino acid residues to carbonyl derivatives (for review, see Stadtman, E . R . (1986) Trends Biochem . Sci . 11, 11-12) . To identify the amino acid residues which are sensitive to MCO oxidation, several enzymes/proteins and amino acid homopolymers were exposed to various MCO systems . The carbonyl groups which were formed were converted to their corresponding 3H-labeled hydroxy derivatives . After acid hydrolysis, the labeled free amino acids were separated by ion exchange chromatography . Each protein or polymer gave rise to several different labeled amino acids . The elution profiles of the labeled amino acids obtained from preparations of Escherichia coli glutamine synthetase which had been oxidized by MCO systems comprised of either Fe(II)/O2 or ascorbate/Fe(II)/O2 both in the presence and absence of EDTA were qualitatively the same . From a comparison of the elution profiles of labeled amino acids from various proteins with those obtained from homopolymers, it is evident that the side chains of histidine, arginine, lysine, and proline are particularly sensitive to oxidation by the MCO systems . This conclusion is supported also by direct amino acid analysis of acid hydrolysates which shows that the oxidation of glutamine synthetase, enolase, and phosphoglycerate kinase is associated with the loss of at least 1 histidine residue per subunit . From the results of studies with homopolymers, it is apparent that glutamic semialdehyde is a major product of both proline and arginine residues . In addition, hydroxyproline and unlabeled glutamic acid were identified among the hydrolysis products of oxidized poly-L-proline, and unlabeled aspartic acid was identified as a product of poly-L-histidine oxidation. Nucleic Acids Res, 1989 Feb 25, 17(4), 1409 - 25 Binding of the herpes simplex virus type 1 UL9 gene product to an origin of viral DNA replication; Weir HM et al.; The binding of a herpes simplex virus type 1 (HSV-1) encoded polypeptide to a viral origin of DNA replication has been studied by using a gel retardation assay . Incubation of nuclear extract from HSV-1 infected cells with a labelled origin-containing fragment resulted in the formation of a specific retarded complex, the migration of which was further reduced in the presence of an antibody reactive with the UL9 gene product . Introduction of an additional copy of the UL9 gene, under the control of an immediate early (IE) promoter, conferred the ability to express origin binding activity at the non-permissive temperature upon an HSV-1 ts mutant blocked at the IE stage of infection . Endogenous or exogenous proteolytic activity revealed the presence of a relatively protease-resistant domain which retained sequence-specific DNA binding activity . The C-terminal 317 amino acids of the UL9 gene expressed as a fusion protein in Escherichia coli also bound to the origin . Our results demonstrate that the UL9 gene product binds to a viral origin and that sequence specific recognition and binding are specified by the C-terminal 37% of the polypeptide. J Biol Chem, 1989 Feb 25, 264(6), 3292 - 300 Proton translocation by the F1F0ATPase of Escherichia coli . Mutagenic analysis of the a subunit; Cain BD et al.; Cassette site-directed mutagenesis was employed to generate mutations in the a subunit (uncB (a) gene) of F1F0ATP synthase . Using sequence homology with similar subunits of other F1F0ATP synthases as a guide, 20 mutations were targeted to a region of the a subunit thought to constitute part of the proton translocation mechanism . ATP-driven proton pumping activity is lost with the substitution of lys, ile, val, or glu for arginine 210 . Substitution of val, leu, gln, or glu for asparagine 214 does not completely block proton conduction, however, replacement of asparagine 214 with histidine does reduce enzyme activity below that necessary for significant function . Two or three mutations were constructed in each of four nonpolar amino acids, leucine 207, leucine 211, alanine 217, and glycine 218 . Certain specific mutations in these positions result in partial loss of F1F0ATP synthase activity, but only the substitution of arginine for alanine 217 reduces ATP-driven proton pumping activity to undetectable levels . It is concluded that of the six amino acids studied, only arginine 210 is an essential component of the proton translocation mechanism . Fractionation of cell-free extracts of a subunit mutation strains generally reveals normal amounts of F1 specifically bound to the particulate fraction . One possible exception is the arginine 210 to isoleucine mutation which results in somewhat elevated levels of free F1 detectable in the soluble fraction . For nearly all a subunit mutations, F1F0-mediated ATP hydrolysis activity remains sensitive to inhibition by dicyclohexylcarbodiimide in spite of the fact that the mutations block proton translocation. Nucleic Acids Res, 1989 Feb 25, 17(4), 1379 - 93 Fidelity of human immunodeficiency virus type I reverse transcriptase in copying natural DNA; Weber J et al.; Reverse transcriptase from the human immunodeficiency virus type I (HIV-1) was expressed in E . coli and purified to near homogeneity . The enzyme was shown to contain reverse transcriptase, DNA polymerase and ribonuclease H activities . The DNA polymerase activity converted singly-primed phi X174 (+) DNA into the double-stranded form . Two third of the replication product is ligatable to covalently closed circular DNA (RFIV-form DNA) indicating that DNA synthesis by HIV reverse transcriptase can proceed until the enzyme matches the 5'-end of a pre-existing primer molecule . The in vitro accuracy of HIV reverse transcriptase was measured with the phi X174am16 reversion assay to be 1/7,400 . Reversion rates for the individual mispairs were determined from pool bias studies to be 1/8,000 for the dGMP:T template mismatch, 1/35,000 for the dGMP:A template mismatch, 1/45,000 for the dAMP:G template mismatch, 1/73,000 for the dCMP:T template mispair, 1/140,000 for the dCMP:A template mispair, and 1/180,000 for the dGMP:G template mismatch . The dTMP:T template mispair was below the detection limit of the assay indicating a reversion rate of less than 1/300,000 for this particular mispair. J Immunol Methods, 1989 Feb 24, 117(2), 247 - 55 Assays for total and antigen-specific polymeric IgA in serum based on binding to secretory component; Bartholomeusz RC et al.; Binding assays with secretory component (SC) were used to detect polymeric IgA antibody to E . coli lipopolysaccharide and to estimate total polymeric IgA in sera from 14 patients with alcoholic liver disease and eight normal controls . Radioiodinated human SC was shown to bind to polymeric IgA and IgM but not to monomeric IgA, secretory IgA or IgG . Serum aliquots (0.5 ml) were totally depleted of IgM using 2 ml anti-IgM affinity columns and the effluent sera were titrated in microtitre plates coated with lipopolysaccharide, the binding of polymeric IgA being detected by adding 10 ng radiolabelled SC . Total polymeric IgA was measured via its capacity to inhibit the binding of 5 ng labelled SC to IgM coated wells, quantitation being achieved by comparison with the inhibition produced by purified polymeric IgA . Total lipopolysaccharide-specific IgA antibody was detected by ELISA in sera from both patients and controls, 1185 +/- 793 and 56 +/- 19 U/100 microliters (mean +/- SD), respectively; but polymeric IgA antibody was detected only in patients' sera (131 +/- 214 U/100 microliters) . The concentration of total polymeric IgA was higher in patients' sera than in control sera (488 +/- 333 and less than 120 micrograms/ml respectively). Science, 1989 Feb 24, 243(4894 Pt 1), 1033 - 8 Reverse transcriptase in a clinical strain of Escherichia coli: production of branched RNA-linked msDNA; Lampson BC et al.; Branched RNA-linked multicopy single-stranded DNA (msDNA) originally detected in myxobacteria has now been found in a clinical isolate of Escherichia coli . Although lacking homology in the primary structure, the E . coli msDNA is similar in secondary structure to the myxobacterial msDNA's, including the 2',5'-phosphodiester linkage between RNA and DNA . A chromosomal DNA fragment responsible for the production of msDNA was cloned in an E . coli K12 strain; its DNA sequence revealed an open reading frame (ORF) of 586 amino acid residues . The ORF shows sequence similarity with retroviral reverse transcriptases and ribonuclease H . Disruption of the ORF blocked msDNA production, indicating that this gene is essential for msDNA synthesis. Biochemistry, 1989 Feb 21, 28(4), 1875 - 84 A covalent angiogenin/ribonuclease hybrid with a fourth disulfide bond generated by regional mutagenesis; Harper JW et al.; Human angiogenin is a blood vessel inducing protein whose primary structure displays 33% identity to that of bovine pancreatic ribonuclease A (RNase A) . Angiogenin catalyzes limited cleavage of 18S and 28S ribosomal RNA and is several orders of magnitude less potent than RNase A toward conventional substrates . A striking structural difference between angiogenin and RNase is the virtual absence of sequence similarity within the region of RNase that contains the Cys-65--Cys-72 disulfide bond . Indeed, angiogenin lacks this disulfide linkage . The present report describes the use of regional mutagenesis to generate a covalent angiogenin/RNase hybrid protein, ARH-I, where residues 58-70 of angiogenin have been replaced by the corresponding segment of RNase A (residues 59-73) . The protein expressed in Escherichia coli readily folds at pH 8.5 to form the four expected disulfide bonds . The in vivo angiogenic potency of ARH-I is markedly diminished compared with that of angiogenin when examined using the chick chorioallantoic membrane assay . In contrast, its enzymatic activity is dramatically increased . With high molecular weight wheat germ RNA and tRNA, ARH-I is 660- and 300-fold more active than angiogenin, respectively, while with poly(uridylic acid), poly(cytidylic acid), cytidylyl(3'----5')adenosine (CpA), and uridylyl(3'----5')adenosine (UpA) activity is enhanced by about 200-fold . In addition, the specificity of ARH-I toward dinucleoside 3',5'-phosphates is qualitatively similar to RNase A; while angiogenin prefers cytidylyl(3'----5')guanosine (CpG) to UpA, both RNase and the hybrid prefer UpA to CpG . ARH-I also displays greater than 10-fold enhanced activity toward rRNA in intact ribosomes, while abolishing the capacity of the ribosome to support cell-free protein synthesis . The enhanced enzymatic properties of ARH-I parallel a 2-fold increase in chemical reactivity of active-site lysine and histidine residues based on rates of chemical modification . The data indicate that introduction of a region of RNase A containing the Cys-65--Cys-72 disulfide bond into angiogenin dramatically increases RNase-like enzymatic activity while reducing its angiogenicity. Biochemistry, 1989 Feb 21, 28(4), 1923 - 9 Resonance Raman spectroscopy of ribonucleotide reductase . Evidence for a deprotonated tyrosyl radical and photochemistry of the binuclear iron center; Backes G et al.; Native ribonucleotide reductase from Escherichia coli exhibits a resonance-enhanced Raman mode at 1498 cm-1 that is characteristic of a tyrosyl radical . The Raman frequency as well as the absorption maximum at 410 nm identifies the radical as being in a deprotonated state . The B2 subunit of ribonucleotide reductase shows an additional resonance Raman mode at 493 cm-1 that has been assigned to the symmetric stretch of an Fe-O-Fe moiety . When samples of active B2 or metB2 are exposed to a tightly focused laser beam at 406.7 nm, there is a loss of intensity at 493 cm-1 and the appearance of a new peak at 595 cm-1 . Although the 595-cm-1 feature was previously assigned to an Fe-OH vibration on the basis of its 23-cm-1 shift to lower energy in H2(18)O and the apparent dependence of its intensity on pH {Sjoberg, B . M., Loehr, T . M., & Sanders-Loehr, J . (1987) Biochemistry 26, 4242}, the present studies indicate that the intensity of this mode is dependent primarily on input laser power . The peak at 595 cm-1 is more plausibly assigned to a new vs(Fe-O-Fe) mode in view of its lack of the deuterium isotope dependence expected for an Fe-OH mode and its resonant scattering cross section which is comparable to that of the 493-cm-1 mode . This new species has a calculated Fe-O-Fe angle of approximately 113 degrees compared to approximately 138 degrees calculated for the Fe-O-Fe unit in unmodified protein B2 . One possible explanation for the photoinduced vibrational mode is that a bridging solvent molecule has been inserted in place of a bridging carboxylate. Biochemistry, 1989 Feb 21, 28(4), 1892 - 7 Effects of single-base bulges on intercalator binding to small RNA and DNA hairpins and a ribosomal RNA fragment; White SA et al.; The way in which a single-base bulge might affect the structure of an RNA helix has been examined by preparing a series of six RNA hairpins, all with seven base pairs and a four-nucleotide loop . Five of the hairpins have single-base bulges at different positions . The intercalating cleavage reagent (methidiumpropyl)-EDTA-Fe(II) {MPE-Fe(II)} binds preferentially at a CpG sequence in the helix lacking a bulge and in four of the five hairpins with bulges . Hairpins with a bulge one or two bases to the 3' side of the CpG sequence bind ethidium 4-5-fold more strongly than the others . V1 RNase, which is sensitive to RNA backbone conformation in helices, detects a conformational change in all of the helices when ethidium binds; the most dramatic changes, involving the entire hairpin stem, are in one of the two hairpins with enhanced ethidium affinity . Only a slight conformational change is detected in the hairpin lacking a bulge . A bulge adjacent to a CpG sequence in a 100-nucleotide ribosomal RNA fragment enhances MPE-Fe(II) binding by an order of magnitude . These results extend our previous observations of bulges at a single position in an RNA hairpin {White, S . A., & Draper, D.E . (1987) Nucleic Acids Res . 15, 4049} and show that (1) a structural change in an RNA helix may be propagated for several base pairs, (2) bulges tend to increase the number of conformations available to a helix, and (3) the effects observed in small RNA hairpins are relevant to larger RNAs with more extensive structure . A bulge in a DNA hairpin identical in sequence with the RNA hairpins does not enhance MPE-Fe(II) binding affinity, relative to a control DNA hairpin . The effects of bulges on ethidium intercalation are evidently modulated by helix structure. Biochemistry, 1989 Feb 21, 28(4), 1617 - 26 A loop involving catalytic chain residues 230-245 is essential for the stabilization of both allosteric forms of Escherichia coli aspartate transcarbamylase; Middleton SA et al.; The allosteric transition of Escherichia coli aspartate transcarbamylase involves significant alterations in structure at both the quaternary and tertiary levels . On the tertiary level, the 240s loop (residues 230-245 of the catalytic chain) repositions, influencing the conformation of Arg-229, a residue near the aspartate binding site . In the T state, Arg-229 is bent out of the active site and may be stabilized in this position by an interaction with Glu-272 . In the R state, the conformation of Arg-229 changes, allowing it to interact with the beta-carboxylate of aspartate, and is stabilized in this position by a specific interaction with Glu-233 . In order to ascertain the function of Arg-229, Glu-233, and Glu-272 in the catalytic and cooperative interactions of the enzyme, three mutant enzymes were created by site-specific mutagenesis . Arg-229 was replaced by Ala, while both Glu-233 and Glu-272 were replaced by Ser . The Arg-229----Ala and Glu-233----Ser enzymes exhibit 10,000-fold and 80-fold decreases in maximal activity, respectively, and they both exhibit a 2-fold increase in the aspartate concentration at half the maximal observed velocity, {S}0.5 . The Arg-229----Ala enzyme still exhibits substantial homotropic cooperativity, but all cooperativity is lost in the Glu-233----Ser enzyme . The Glu-233----Ser enzyme also shows a 4-fold decrease in the carbamyl phosphate {S}0.5, while the Arg-229----Ala enzyme shows no change in the carbamyl phosphate {S}0.5 compared to the wild-type enzyme . The Glu-272 to Ser mutation results in a slight reduction in maximal activity, an increase in {S}0.5 for both aspartate and carbamyl phosphate, and reduced cooperativity . Analysis of the isolated catalytic subunits from these three mutant enzymes reveals that in each case the changes in the kinetic properties of the isolated catalytic subunit are similar to the changes caused by the mutation in the holoenzyme . PALA was able to activate the Glu-233----Ser enzyme, at low aspartate concentrations, even though the mutant holoenzyme did not exhibit any cooperativity, indicating that cooperative interactions still exist between the active sites in this enzyme . It is proposed that Glu-233 of the 240s loop helps create the high-activity-high-affinity R state by positioning the side chain of Arg-229 for aspartate binding while Glu-272 helps stabilize the low-activity-low-affinity T state by positioning the side chain of Arg-229 so that it cannot interact with aspartate.(ABSTRACT TRUNCATED AT 400 WORDS) Biochemistry, 1989 Feb 21, 28(4), 1488 - 92 Cytosine photoproduct-DNA glycosylase in Escherichia coli and cultured human cells; Weiss RB et al.; Ultraviolet irradiation of DNA produces a variety of pyrimidine base damages . The activities of Escherichia coli endonuclease III and a human lymphoblast endonuclease that incises ultraviolet-irradiated DNA at modified cytosine moieties were compared . Both the bacterial and human enzymes release this cytosine photoproduct as a free base . These glycosylase activities are linear with times of reaction, quantities of enzyme, and irradiation dosages of the substrates . Both enzyme activities are similarly inhibited by the addition of monovalent and divalent cations . Analysis by DNA sequencing identified loci of endonucleolytic incision as cytosines . These are neither cyclobutane pyrimidine dimers, 6-(1,2-dihydro-2-oxo-4-pyrimidinyl)-5-methyl-2,4(1H,3H)-pyrimidinediones, nor apyrimidinic sites . This cytosine photoproduct is separable from unmodified cytosine by high-performance liquid chromatography . This separation should facilitate identification of this modified cytosine and elucidation of its biological significance. Biochemistry, 1989 Feb 21, 28(4), 1917 - 22 Ordered disruption of subunit interfaces during the stepwise reversible dissociation of Escherichia coli phosphofructokinase with KSCN; Deville-Bonne D et al.; The reversible inactivation and dissociation of the allosteric phosphofructokinase from Escherichia coli has been studied in relatively mild conditions, i.e., in the presence of the chaotropic agent KSCN . At moderate KSCN concentration, the loss of enzymatic activity involves two separated phases: first, a rapid dissociation of part of the tetramer into dimers, second, a slower displacement of the dimer-tetramer equilibrium upon further dissociation of the dimer into monomers . These two reactions can no longer be distinguished above 0.3 M KSCN since complete inactivation occurs in a single reaction . Different changes are observed for the fluorescence and the activity of the enzyme in KSCN: the fluorescence is not affected by the dissociation into dimers which is responsible for inactivation . The decrease in fluorescence reflects the change in environment of the unique tryptophan residue, Trp 311, during the dimer to monomer dissociation . This residue belongs to the interface containing the regulatory site, and its native fluorescence indicates that this interface is still present in the dimer . The substrate fructose 6-phosphate protects phosphofructokinase from inactivation by binding to the tetramer and prevents its dissociation into dimers . The presence of phosphoenolpyruvate prevents the slow dissociation of the dimer into monomers, which shows the ability of the dimer to bind the inhibitor . Two successive processes can be observed during reassociation of the protein upon KSCN dilution . First, a fast reaction (k1 = 2 x 10(5) M-1.s-1) is accompanied by a fluorescence increase and results in the formation of the dimeric species.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1989 Feb 21, 28(4), 1912 - 6 Intermediates on the reassociation pathway of phosphofructokinase I from Escherichia coli; Teschner W et al.; The folding and association pathway of the allosteric phosphofructokinase from Escherichia coli has been investigated after complete denaturation of the protein in guanidine hydrochloride by spectroscopical methods, fluorescence and circular dichroism . Three successive processes can be observed during the renaturation of this protein . First, a fast reaction, detected by fluorescence, results in the formation of a (partially) structured monomer . Second, two monomers associate into a dimeric species . This step involves the shielding of the unique tryptophan residue, Trp 311, from the aqueous solvent, and it corresponds to the formation of the interface containing the effector binding site . The presence of ATP during renaturation increases the rate of formation of this dimeric species . The other ligands of the enzyme have no effect on this reaction as well as on the whole reactivation . Finally, the enzymatic activity is regained during the third slowest step . This last reaction is due to the association of two dimers into the native tetrameric structure . The presence of fructose 6-phosphate does not increase the rate of reactivation, even though this ligand strongly stabilizes the native enzyme against denaturation by bridging the interface corresponding to the active site . The self-assembly of phosphofructokinase from E . coli from its unfolded and separated chains follows a specific order in the formation of the interactions between subunits and involves a dimeric intermediate with a defined geometry. Biochemistry, 1989 Feb 21, 28(4), 1884 - 91 Purification and characterization of tissue plasminogen activator kringle-2 domain expressed in Escherichia coli; Cleary S et al.; We have expressed the 174-263 fragment (kringle-2 domain) of human tissue-type plasminogen activator (t-PA) in Escherichia coli by secretion into the periplasmic space using the alkaline phosphatase promoter and stII enterotoxin signal sequence . A large portion of the secreted protein is associated with an insoluble cellular fraction . This material can be solubilized by extraction with denaturant and reducing agent and then recovered in active form by refolding in the presence of reduced and oxidized glutathione . Kringle-2 is then easily purified by affinity chromatography on lysine-Sepharose followed by cation-exchange chromatography . The isolated protein has an amino acid composition and N-terminal sequence as expected for the 174-263 fragment of t-PA, indicating that the signal peptide has been properly removed . Circular dichroic spectra suggest that the protein is folded similar to the kringle-4 domain of plasminogen {Castellino et al . (1986) Arch . Biochem . Biophys . 247, 312-320} . Equilibrium dialysis experiments indicate a single binding site on kringle-2 for L-lysine having a KD of 100 microM . Using a method based on elution of kringle from lysine-Separose with omega-aminocarboxylic acids {Winn et al . (1980) Eur . J . Biochem . 104, 579-586}, we have shown the lysine binding site of t-PA kringle-2 to have a preference for a ligand with 8.8-A separation between amine and carboxylate functions . Charge interactions with the epsilon-amino group of L-lysine are important in binding since the affinities for N epsilon-acetyl-L-lysine, L-arginine, and gamma-guanidinobutyric acid are decreased greater than 2000-fold, 200-fold, and 12-fold, respectively, relative to the affinity for L-lysine.(ABSTRACT TRUNCATED AT 250 WORDS) J Mol Biol, 1989 Feb 20, 205(4), 783 - 5 Crystallization and preliminary X-ray studies of recombinant human granulocyte-macrophage colony stimulating factor; La Londe JM et al.; Human granulocyte-macrophage colony stimulating factor (hGM-CSF) is an important regulator of growth and differentiation for mononuclear and polymorphonuclear phagocytes . Here we report the crystallization and preliminary X-ray studies of Escherichia coli-expressed hGM-CSF . The crystals are orthorhombic, with the space group P212121, and have unit cell dimensions a = 46.62 A, b = 58.73 A and c = 126.42 A . Recombinant hGM-CSF crystals diffract X-rays to 2.4 A resolution and are thus suitable for X-ray structural studies. J Mol Biol, 1989 Feb 20, 205(4), 659 - 64 Spectrum of N4-aminocytidine mutagenesis; Bessho T et al.; N4-Aminocytidine, a nucleoside analog, is a potent mutagen towards phages, bacteria, Drosophila and mammalian cells in culture . In vitro, biochemical studies indicate that this reagent acts by being incorporated into DNA . To elucidate the mechanism of N4-aminocytidine mutagenesis, it is essential to identify the nature of DNA sequence alterations taking place during the mutagenesis . We have analyzed the nucleotide sequence changes in the lac promoter-lacZ alpha region of M13mp2 phage induced by treatment of phage-infected Escherichia coli with N4-aminocytidine . The sequence alterations of DNA samples from 89 mutants of the phage were determined . These mutants had single point mutations, except one mutant, in which a double point mutation was detected . Several hot spots were found: however, there are no apparent relations to particular DNA sequences regarding the locations of these spots . All the mutations are transitions; neither transversions nor deletions/insertions were found . A feature in these transitions is that the A/T to G/C and G/C to A/T changes occur at approximately equal rates . The overall picture of the mutagenesis is consistent with a scheme in which misincorporation and misreplication caused by the modified cytosine structure are the key steps in the DNA replication leading to transitions . Similar nucleotide alterations were found for the mutagenesis induced by an alkylated derivative, N'-methyl-N4-aminocytidine . N4-Aminocytidine also induced reversions of these mutants; both A/T to G/C and G/C to A/T transitions again took place. Gene, 1989 Feb 20, 75(2), 315 - 21 A vector for sequencing long (40-kb) DNA fragments; Ahmed A; An improved vector (pAA113M) has been constructed for sequencing long (40-kb) DNA fragments . The DNA fragment is cloned in the tet gene of the cosmid and subdivided into numerous overlapping segments by IS1-promoted deletions . Plasmids bearing these deletions are fractionated by gel electrophoresis, and shortened further from the opposite end by treatment with specific restriction endonucleases . Thus, a series of short overlapping segments, spread across the entire length of the fragment, become fused to IS1 . Each segment can then be sequenced