Mutat Res, 1991 Jul, 255(1), 19 - 29 Site-directed deletion mutagenesis within the T4 endonuclease V gene: dispensable sequences within putative loop regions; Dodson ML et al.; Endonuclease V from bacteriophage T4 may be one of the first DNA-repair enzymes to have its three-dimensional structure determined by X-ray crystallography (Morikawa et al., 1988) . However, since this structure is not yet available, analyses of the sequence of the protein were performed in order to guide site-directed mutational studies of enzyme structure-function relationships . The enzyme is predominantly alpha-helical, so that an algorithm which finds the locations of turns or loops in the structure would be expected to approximately locate the helices along the sequence . Two loop sites were identified which might be adjacent in the tertiary structure according to a model developed from the loop predictions and the derived secondary structure . Deletion of three residues at each loop site produced protein molecules which retained considerable in vitro enzyme activity and in vivo repair function . However, the mutant proteins did not accumulate as well within the cell as the wild-type enzyme, suggesting that the nascent molecules folded inefficiently . Combination of the two deletions yielded a molecule with activity enhanced over one of the individual mutants, a result which can be interpreted as a classic second-site mutational reversion . This result supports the hypothesis that these regions are adjacent in the enzyme tertiary structure. Mutat Res, 1991 Jul, 255(1), 1 - 9 Enhanced pyrimidine dimer removal in repair-proficient murine fibroblasts transformed with the denV gene of bacteriophage T4; Kusewitt DF et al.; The denV gene of bacteriophage T4, which encodes the pyrimidine dimer-specific repair enzyme endonuclease V, was introduced into murine fibroblasts with normal rodent pyrimidine dimer repair capabilities . Endonuclease V recognizes ultraviolet radiation (UVR)-induced pyrimidine dimers and produces single-strand breaks adjacent to the dimers . These nicks may serve as substrates to initiate excision repair of pyrimidine dimers by endogenous enzymes . In the present study, murine fibroblasts stably transfected with denV were able to remove 50-80% of UVR-induced pyrimidine dimers, while control cells removed only about 20% of dimers under the same conditions of pyrimidine dimer induction and repair . For both control and denV-transfected cells, repair continued for at least 24 h after exposure . When removal of UVR-induced photoproducts was initiated by endogenous excision repair mechanisms, an average of 38 nucleotides were replaced per dimer removed, as determined by bromouracil photolysis; denV-initiated excision repair, on the other hand, resulted in removal of an average of 6 nucleotides per dimer repaired . The enhanced pyrimidine dimer repair capabilities conferred by denV gene expression did not appear to improve post-UVR survival. J Bacteriol, 1991 Jul, 173(14), 4394 - 403 Internal deletions in the FhuA receptor of Escherichia coli K-12 define domains of ligand interactions; Carmel G et al.; The ferrichrome-iron receptor encoded by the fhuA gene of Escherichia coli K-12 is a multifunctional outer membrane receptor required for the binding and uptake of ferrichrome and bacteriophages T5, T1, phi 80, and UC-1 as well as colicin M . To identify domains of the protein which are important for FhuA activities, a library of 31 overlapping deletion mutants in the fhuA gene was generated . Export of FhuA deletion proteins to the outer membrane and receptor functions of the deletion proteins were analyzed . All but three of the deletion mutant FhuA proteins cofractionated with the outer membrane; no FhuA proteins were detected in outer membrane preparations or in cell extracts when the deletions spanned amino acids 418 to 440 . Most deletion proteins were susceptible to cleavage by endogenous proteolytic activity; some degradation products were detected on Coomassie blue-stained gels and on Western blots (immunoblots) . Receptor functions were measured with the mutated genes present on multicopy plasmids . Two deletion mutants, FhuA delta 060-069 and FhuA delta 129-168, conferred wild-type phenotypes: they demonstrated growth promotion by ferrichrome and the same efficiency of plating of bacteriophages as that of wild-type FhuA; killing by colicin M was also unaffected . For FhuA delta 021-128 and FhuA delta 406-417, reduced sensitivity to colicin M was detected; wild-type phenotypes were observed for all other FhuA functions . Deletions from amino acids 169 to 195 slightly reduced sensitivities to bacteriophages and to colicin M; ferrichrome growth promotion was unaffected . When deletions extended into the region of amino acids 196 to 405, all FhuA functions were either reduced or abolished . The results indicate that selected regions of the FhuA protein have receptor activities and demonstrate the presence of both shared and unique ligand-responsive domains. Biochim Biophys Acta, 1991 Jul 1, 1066(1), 102 - 8 A NMR investigation on the interactions of the alpha-oligomeric form of the M13 coat protein with lipids, which mimic the Escherichia coli inner membrane; Sanders JC et al.; The interaction of the M13 bacteriophage major coat protein in the alpha-oligomeric form with specifically deuterated phospholipid headgroups which mimic the Escherichia coli inner membrane, has been studied using NMR methods . As can be seen from the deuterium NMR spectra obtained with headgroup trimethyl deuterated DOPC, the coat protein in the alpha-oligomeric form does not give rise to trapped lipids as observed with M13 coat protein in the beta-polymeric form (Van Gorkom et al . (1990) Biochemistry 29, 3828-3834) . The quadrupolar splittings of the alpha headgroup methylene deuterons of deuterated phosphatidylcholine and phosphatidylethanolamine decrease, whereas the quadrupolar splittings of the beta headgroup methylene deuterons of the two lipids increase with increasing protein content . All deuterated segments in the phosphatidylglycerol headgroup show the same relative decrease of the NMR quadrupolar splittings . These results are interpreted in terms of a change in torsion angles of the methylene groups, induced by positive charges, probably lysine residues of the protein at the membrane surface . For all lipid bilayer compositions studied the head-group perturbations are similar . It is concluded that there is no strong specific interaction between one of the lipid types examined and the M13 coat protein . From the spin-spin (T2e) relaxation time and spin-lattice (T1z) relaxation time of all deuterated lipids it is concluded that at the bilayer surface only slow motions are affected by the M13 coat protein. Proc Natl Acad Sci U S A, 1991 Jul 1, 88(13), 5699 - 703 Specificity of the Mnt protein determined by binding to randomized operators; Stormo GD et al.; The relative binding affinities of Mnt protein from bacteriophage P22 are determined for each possible base pair at position 17 of the operator . These are determined from the partitioning of randomized operators into bound and unbound fractions; quantitation is provided by restriction enzyme analysis . Mnt protein is found to have an unusual specificity at this position: a C.G base pair (the wild-type operator) has the highest affinity, a G.C base pair has the lowest affinity, and both orientations of A.T base pairs are intermediate and nearly equivalent . A specific binding constant and specific binding free energy are defined and shown to be directly related to the information content of the operator sequences bound to the protein, taking into account the quantitative differences in binding affinities. Photochem Photobiol, 1991 Jul, 54(1), 99 - 107 Formation of single and double strand breaks in DNA ultraviolet irradiated at high intensity; Masnyk TW et al.; The induction of single-strand breaks (SSB) by two quantum processes in DNA is well established . We now report that biphotonic processes result in double-strand breaks (DSB) as well . pUC19 and bacteriophage M13 RF DNA were irradiated using an excimer laser (248 nm) at intensities of 10(7), 10(9), 10(10) and 10(11) W/m2 and doses up to 30 kJ/m2 . The proportion of DNA as supercoil, open circular, linear and short fragments was determined by gel electrophoresis . Linear molecules were noted at fluences where supercoiled DNA was still present . The random occurrence of independent SSB in proximity to each other on opposite strands (producing linear DNA) implies introduction of numerous SSB per molecule in the sample . If so, supercoiled DNA that has sustained no SSB should not be observed . A model accounting for the amounts of supercoiled, open circular, linear and shorter fragments of DNA due to SSB, DSB and Scissions (opposition of two independently occurring SSB producing an apparent DSB) was developed, our experimental data and those of others were fit to the model, and quantum yields determined for SSB and DSB formation at each intensity . Results showed that high intensity laser radiation caused an increase in the quantum yields for both SSB and DSB formation . The mechanism of DSB formation is unknown, and may be due to simultaneous cleavage of both strands in one biphotonic event or the biased introduction of an SSB opposite a preexisting SSB, requiring two biphotonic events. Mol Biol (Mosk), 1991 Jul-Aug, 25(4), 1080 - 9 {Complexity analysis of a genome . II . Extensive homology zones in bacteriophage lambda}; Gusev VD et al.; The suggested earlier complexity approach for detecting structural regularities in primary structures of nucleic acids is illustrated by using lambda phage as an example . Among the most interesting regularities detected in the lambda phage genome are the following: (a) the presence of "extended homology zones" i.e . fragments in which block transpositions of duplicative type predominate explicitly; (b) the abundance of palindrome-hairpin structures and duplications in the origins and termini of many genes; (c) the hierarchy in the repeats and inversions organization. Biotechniques, 1991 Jul, 11(1), 68 - 75 A simple method for direct automated sequencing of PCR fragments; Tracy TE et al.; A simple and rapid method for direct sequencing of PCR-generated fragments has been developed for use on Applied Biosystems 373A Automated DNA Sequencer utilizing the DyeDeoxy terminator chemistry . Standard PCR conditions are used to generate a DNA fragment, which is subsequently gel-purified to remove excess primers and unwanted PCR products . The sequencing reactions are carried out in a thermal cycler using the purified product as template DNA and the Dye-Deoxy terminators . The sequence of 500-bp region in the bacteriophage lambda genome and a 320-bp fragment of the human genomic erythropoietin gene were sequenced with greater than 99% accuracy using this method. J Basic Clin Physiol Pharmacol, 1991 Jul-Sep, 2(3), 223 - 31 Translation control of gene expression; Oppenheim A et al.; The bacteriophage lambda cIII gene product is an early regulator of the lysogenic pathway . The availability of a set of cIII expression mutants allowed us to establish the structure-function relationship of the cIII mRNA . We demonstrated, using defined in vitro systems, that the cIII mRNA is present in two conformations at equilibrium . Mutations that have been shown to lead to cIII overexpression were found to freeze the RNA in one conformation (structure B), and permit efficient binding to the 30S ribosomal subunit . Mutations that have been shown to prevent cIII translation cause the mRNA to assume the alternative conformation (structure A) . In this structure, the translation initiation region is occluded, thereby preventing 30S ribosomal subunit binding . Translation of the cIII gene is regulated by RNaseIII . We have localized the RNaseIII responsive element (RRE) to the cIII coding region . We suggest that the regulation of the equilibrium between the two mRNA conformations provides a mechanism for the control of cIII gene expression . The way in which RNaseIII participates in this regulation is as yet unknown. J Bacteriol, 1991 Jul, 173(13), 4171 - 81 Association of a retroelement with a P4-like cryptic prophage (retronphage phi R73) integrated into the selenocystyl tRNA gene of Escherichia coli; Sun J et al.; A new multicopy single-stranded DNA (msDNA-Ec73) was found in a clinical strain of Escherichia coli . Retron-Ec73, consisting of an msDNA-coding region and the gene for reverse transcriptase (RT), was found to be a part of a 12.7-kb foreign DNA fragment flanked by 29-bp direct repeats and integrated into the gene for selenocystyl-tRNA (selC) at 82 min on the E . coli chromosome . Except for the 2.4-kb retron region, the integrated DNA fragment showed remarkable homology to most of the bacteriophage P4 genome . Among the phage genes found in this element, however, the integrase gene had very low identity (40%) to P4 integrase, indicating that the cryptic prophage associated with the retroelement has its own unique site-specific integrase different from P4 integrase . Recently, we have shown that P2 phage can act as a helper to excise the cryptic prophage and to package its genome into an infectious virion . The newly formed phage (retronphage phi R73) can also lysogenize a new host strain, reintegrating its genome into the selC gene and enabling the newly formed lysogen to produce msDNA-Ec73 (S . Inouye, M . G . Sunshine, E . W . Six, and M . Inouye, Science 252:969-971, 1991). Mol Biol (Mosk), 1991 Jul-Aug, 25(4), 1024 - 32 {DNA-protein cross-links as a possible reason for genomic damage during its protonation}; Nasirov NG et al.; The change in survival of bacteriophages with DNA of different GC-contents after their incubation in media of different acidities with subsequent neutralization was studied . It was shown that the higher the GC-content, the more sensitive is the phage to the action of H(+)-ions . Evidence is presented that the acidic inactivation of virions is not connected with the helix-coil transition of the intraphage DNA due to its protonation . The extractability of DNA from phages subjected to different concentrations of H(+)-ions with subsequent neutralization of the medium to pH 8 was determined . The changes in: transfection ability, UV-spectra, the quantity of the residual proteins, and the contents of glutamic and lysine amino acid residues in these proteins were investigated . The effect of glutamic acid on the parameters of DNA melting curves was followed for different pH values . Proceeding from the data obtained, we concluded that acidification of the medium from neutral tp pH congruent to 4 leads to formation of non-covalent DNA-protein cross-links due to interaction of the GC base pairs of DNA with glutamic and aspartic amino acid residues, whereas acidification of the medium to pH less than 4 with subsequent neutralization to pH 8 results in the formation of covalent DNA-protein cross-links of Schiff base type . The influence of non-covalent DNA-protein cross-links on the properties of DNA and their regulatory role in genome functioning are discussed. J Gen Microbiol, 1991 Jul, 137 ( Pt 7), 1545 - 9 Induction and partial purification of bacteriophages from Desulfovibrio vulgaris (Hildenborough) and Desulfovibrio desulfuricans ATCC 13541; Seyedirashti S et al.; Bacteriophages were induced from cultures of Desulfovibrio vulgaris NCIMB 8303 and Desulfovibrio desulfuricans ATCC 13541 by UV light . The optimum time of UV exposure was 1 min and the maximum yield of phage was obtained 9-10 h after UV treatment . The two phage preparations were compared by restriction enzyme analysis and Southern blot hybridization . The nucleic acid from both phages was cut by restriction endonucleases specific for double-stranded DNA . The phage DNAs from D . vulgaris and D . desulfuricans showed different restriction enzyme cleavage patterns . No homology was observed between a 25 kb probe from the D . vulgaris phage DNA and the phage DNA from D . desulfuricans . Protein profiles of the phages from both sources were also studied; the D . vulgaris phage contained two major bands corresponding to Mr values of 37 000 and 56 000 while the D . desulfuricans phage contained only one major band, of Mr 38 000. Biotechnology (N Y), 1991 Jul, 9(7), 652 - 6 Phage vectors that allow monitoring of transcription of secondary metabolism genes in Streptomyces; Bruton CJ et al.; We describe a bacteriophage phi C31-based system that permits the transcriptional fusion of the convenient reporter gene xylE to chromosomally located promoters in Streptomyces hosts . Applicability of the system to genes for secondary metabolism is demonstrated in an experiment showing that transcription of genes for actinorhodin production in Streptomyces coelicolor A3(2) depends on a transfer RNA gene (bldA) for the rare UUA codon . Two other phi C31::xylE vectors are described that allow detection of promoter activity away from their natural location, either at single copy in a prophage or during lytic infections in plaques. Biochem Biophys Res Commun, 1991 Jun 28, 177(3), 1240 - 6 Amino acid sequence of the bacteriophage T5 gene A2 protein; Snyder CE Jr; The complete amino acid sequence of the bacteriophage T5-encoded gene A2 protein was determined by protein sequencing . The 134-residue sequence is closely similar to that reported for the product of gene A2-A3 of bacteriophage BF23 . Segments of the sequence are similar to segments of bacteriophage T4 gene 32 protein, bacteriophage T3 RNA polymerase, and the protein encoded by the host gene responsible for isoenzyme conversion of alkaline phosphatase . The similarity of residues 26-46 to a portion of a rabbit lipopolysaccharide binding protein is possibly relevant to the function of the A2 protein. Biochemistry, 1991 Jun 25, 30(25), 6305 - 13 Processivity of T7 RNA polymerase requires the C-terminal Phe882-Ala883-COO- or "foot"; Mookhtiar KA et al.; The role of the C-terminal Phe882-Ala883 residues of bacteriophage T7 RNA polymerase in specific transcription has been investigated by means of site-directed mutagenesis . A mutant enzyme that lacks the C-terminal Phe882-Ala883 residues, denoted the "foot" mutant, has been cloned and overproduced, and the effects of the deletion on promoter recognition, initiation, and elongation have been determined . Gel retardation assays and DNase I footprinting show that the foot mutant specifically recognizes and binds to T7 promoters, although this binding appears to be approximately 30-fold weaker than that of the wild-type enzyme . Transcription assays using oligonucleotide templates that contain the consensus T7 promoter show a dramatic decrease in transcriptional activity for the foot mutant . With templates whose coding region begins CCC..., the mutant synthesizes poly(G) products even in the presence of all four nucleotides . The synthesis of poly(G) products from such templates has previously been observed for the wild-type enzyme when GTP is the sole nucleotide present in the reaction and is thought to occur by a novel mechanism involving slippage of the RNA chain 3' to 5' relative to the template {Martin, C.T., Muller, D.K., & Coleman, J.E . (1988) Biochemistry 27, 3966-3974} . These data suggest that the loss in transcriptional activity by the foot mutant results from a severe decrease in processivity as well as catalytic efficiency of the enzyme . Removal of the C-terminal Phe and Ala residues from the wild-type enzyme with carboxypeptidase A generates the phenotype of the mutant precisely, proving that all of the properties of the foot mutant derive from the loss of the Phe-Ala-COOH moiety.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1991 Jun 25, 30(25), 6230 - 40 Approaches to predicting effects of single amino acid substitutions on the function of a protein; Zabin HB et al.; The relative activities of 313 mutants of the gene V protein of bacteriophage f1, assayed in vivo, have been used to evaluate two approaches to predicting the effects of single amino acid substitutions on the function of a protein . First, we tested methods that only depend on the properties of the wild-type and substituting amino acids . None of the properties or measures of the functional equivalence of amino acids we tested, including the frequency of exchange of amino acids among homologous proteins as well as changes in side-chain size, hydrophobicity, and charge, were found to be more than weakly correlated with the activities of mutants . The principal reason for this poor correlation was found to be that the effect of a particular substitution varies considerably from site to site . We then tested an approach using the activities of several mutants with substitutions at a site to predict the activity of another mutant, and we find that this is a relatively good indicator of whether the other mutant at that site will be functional . A predictive scheme was developed that combines the weak information from the models depending on the properties of the wild-type and substituting amino acids with the stronger information from the tolerance of a site to substitution . Although this scheme requires no knowledge of the structure of a mutant protein, it is useful in predicting the activities of mutants. Nucleic Acids Res, 1991 Jun 25, 19(12), 3403 - 8 p lambda Zd39: a new type of cDNA expression vector for low background, high efficiency directional cloning; Murphy AJ et al.; We have developed a new type of bacteriophage lambda vector which provides a strong biological selection against non-recombinants that is independent of the sequences immediately surrounding the cloning site . This system, which we call 'selective substitution', is ideally suited for cDNA expression vectors where it is necessary to flank the cDNA insert with sequence elements (promoters etc.) required to produce a biologically active mRNA in vivo . Selective substitution is a general method, which may be applied to many types of vectors . In this report, we have specifically applied selective substitution to the construction of a new mammalian retrovirus expression vector . The level of background obtained with this vector (that is, the number of plaques obtained when the vector is ligated in the absence of insert DNA) is 0.02% when compared to ligation with restriction fragments and 0.1% to 0.4% when compared to ligation with newly synthesized cDNA . These features have allowed us to easily and efficiently generate several large cDNA libraries using total and size selected cDNA. Biochemistry, 1991 Jun 25, 30(25), 6290 - 5 A specific, UV-induced RNA-protein cross-link using 5-bromouridine-substituted RNA; Gott JM et al.; The well-characterized RNA binding site of the bacteriophage R17 coat protein has been used to investigate the cross-linking of protein to 5-bromouridine (BrU)-substituted RNA using medium-wavelength UV light . We have demonstrated a specific RNA-protein cross-link and identified the site on the RNA of protein attachment . Formation of the covalent complex is dependent upon the presence of BrU at position -5 of the RNA and specific binding of the RNA by coat protein . The amount of cross-linking increases with time and depends on the light source and conditions used . Irradiations using a broad-spectrum UV transilluminator (peak at 312 nm) or monochromatic XeCl excimer laser (308 nm) gave levels of cross-linking exceeding 20 and 50%, respectively . The quantum yield of photo-cross-linking, determined with 308-nm excitation, was 0.003 . While little strand breakage or debromination of the RNA occurred, significant protein photodamage was observed. Eur J Biochem, 1991 Jun 15, 198(3), 783 - 7 Purification and characterization of a novel 5' exodeoxyribonuclease from the yeast Saccharomyces cerevisiae; Dolberg M et al.; We have isolated from yeast cells an exonuclease which preferentially attacks double-stranded DNA from the 5' ends producing 5'-mononucleotides as reaction products . A second typical product is a full-length single-stranded DNA complement, suggesting that the enzyme hydrolyzes one DNA strand in a processive manner before it associates with another DNA substrate to initiate a new reaction cycle . Its biochemical properties suggest that the enzyme is unlike the yeast exonucleases which have been reported so far . However, the new exonuclease is strikingly similar to the well characterized 5' exonuclease of bacteriophage lambda. Eur J Biochem, 1991 Jun 15, 198(3), 541 - 7 Single-step purification and structural characterization of human interleukin-6 produced in Escherichia coli from a T7 RNA polymerase expression vector; Arcone R et al.; Human interleukin-6 or B-cell stimulatory factor-2 is a cytokine involved in acute phase and immune response . Cloning of cDNA for human interleukin-6 in the pT7.7 expression plasmid under the control of a bacteriophage T7 RNA polymerase promoter system allows rapid production of the cytokine in Escherichia coli . Upon cell induction with isopropyl thiogalactopyranoside, recombinant human interleukin-6 is overexpressed and forms insoluble inclusion bodies . Solubilization of the protein with 6 M guanidine hydrochloride and refolding in the presence of a reduction/oxidation system results in a quantitative recovery of recombinant human interleukin-6 . This material is already 70% pure and can be further purified to homogeneity with a single passage over a weak anionic-exchange column . Extended structural characterization of the purified protein by electrospray mass spectrometry, automated Edman degradation and peptide mapping by high-pressure liquid chromatography/fast-atom-bombardment mass spectrometry demonstrates that recombinant human interleukin-6 is identical to the natural protein both in amino acid sequence and S-S bridge content . However, it contains a minor component accounting for about 20% of the entire translated protein which exhibits a Met-Ala dipeptide extension at the N-terminus . Purified recombinant human interleukin-6 is biologically active because it is able to induce at least 70-fold the human C-reactive promoter transfected in human hepatoma Hep 3B cells and is stable for several months in 10% glycerol at 4 degrees C . The expression system described in the present work has the main advantage of producing a high yield of recombinant human interleukin-6 (about 25 mg/l) combined with a very simple purification scheme. Proc Natl Acad Sci U S A, 1991 Jun 15, 88(12), 5217 - 21 The activity of the CIII regulator of lambdoid bacteriophages resides within a 24-amino acid protein domain; Kornitzer D et al.; The CIII protein of lambdoid bacteriophages promotes lysogeny by stabilizing the phage-encoded CII protein, a transcriptional activator of the repressor and integrase genes . We have isolated a set of missense mutations in the cIII gene of phage lambda and of phage HK022 that yield inactive CIII proteins . All the mutations are located in the relatively conserved central region of the protein . A comparative analysis of the CIII protein sequence in lambda, HK022, and the lambdoid bacteriophage P22 leads us to suggest that this central region assumes an amphipathic alpha-helical structure . This part of the lambda cIII gene was cloned within a fragment of the lacZ gene (the alpha-complementing fragment) . The resulting fusion protein displays CIII activity . Mutations that yield a nonfunctional fusion protein map within its CIII moiety . These results indicate that the central portion of the CIII protein is both necessary and sufficient for CIII activity. Biochim Biophys Acta, 1991 Jun 13, 1089(2), 183 - 92 Regulation of expression of the genome of bacteriophage M13 . Gene V protein regulated translation of the mRNAs encoded by genes I, III, V and X; Zaman G et al.; With the aid of a binary plasmid in vivo testsystem it was demonstrated that the single-stranded DNA binding protein encoded by gene V of bacteriophage M13 not only regulates the synthesis of its cognate DNA replication proteins at the level of translation, but also of the assembly proteins and the coat proteins encoded by genes I and II, respectively . Furthermore, gene V protein functions as a translational autoregulator of its own synthesis . Comparison of the mRNA levels of genes I and X in the presence and absence of wild-type gene V protein indicated that gene V protein augments the physical stability of these mRNAs . The expression of the Escherichia coli beta-galactosidase gene and of a gene X mutant containing a deletion in the nontranslated mRNA leader sequence was not influenced by gene V protein, lending support to the conclusion that gene V protein exerts its regulatory effect via a specific nucleotide sequence in the leader sequences of the respective M13 mRNAs . We conclude that gene V protein functions as a master regulatory protein of the expression and replication of the M13 genome. Nucleic Acids Res, 1991 Jun 11, 19(11), 3047 - 54 Effects of temperature on excluded volume-promoted cyclization and concatemerization of cohesive-ended DNA longer than 0.04 Mb; Louie D et al.; The 0.048502 megabase (Mb), primarily double-stranded DNA of bacteriophage lambda has single-stranded, complementary termini (cohesive ends) that undergo either spontaneous intramolecular joining to form open circular DNA or spontaneous intermolecular joining to form linear, end-to-end oligomeric DNAs (concatemers); concatemers also cyclize . In the present study, the effects of polyethylene glycol (PEG) on the cyclization and concatemerization of lambda DNA are determined at temperatures that, in the absence of PEG, favor dissociation of cohesive ends . Circular and linear lambda DNA, monomeric and concatemeric, are observed by use of pulsed field agarose gel (PFG) electrophoresis . During preparation of lambda DNA for these studies, hydrodynamic shear-induced, partial dissociation of joined cohesive ends is fortuitously observed . Although joined lambda cohesive ends progressively dissociate as their temperature is raised in the buffer used here (0.1 M NaCl, 0.01 M sodium phosphate, pH 7.4, 0.001 M EDTA), when PEG is added to this buffer, raising the temperature sometimes promotes joining of cohesive ends . Conditions for promotion of primarily either cyclization or concatemerization are described . Open circular DNAs as long as a 7-mer are produced and resolved . The concentration of PEG required to promote joining of cohesive ends decreases as the molecular weight of the PEG increases . The rate of cyclization is brought, the first time, to values that are high enough to be comparable to the rate observed in vivo . For double-stranded DNA bacteriophages that have a linear replicative form of DNA (bacteriophage T7, for example), a suppression, sometimes observed here, of cyclization mimics a suppression of cyclization previously observed in vivo . The PEG, temperature effects on DNA joining are explained by both the excluded volume of PEG random coils and an increase in this excluded volume that occurs when temperature increases. Nucleic Acids Res, 1991 Jun 11, 19(11), 2875 - 80 Identification of a plasmid-coded protein required for initiation of ColE2 DNA replication; Kido M et al.; The product of the rep gene of ColE2 is required for initiation of ColE2 DNA replication . The rep gene was placed under the control of the promoters, PL and PR, and the heat-labile cl857 repressor of bacteriophage lambda . The Rep protein was identified as a 35 Kd protein by the maxicell method in combination with heat-induced expression . The protein was efficiently expressed from these promoters in unirradiated cells and accumulated up to a few per cent of the total cellular proteins . It was partially purified (about 80% pure) and its properties examined . The amino acid sequence of the amino terminal portion of the partially purified protein agreed well with that predicted from the nucleotide sequence of the rep gene . One of the characteristic features of the rep gene is frequent usage of rare codons, especially those for arginine . The protein specifically stimulated replication of ColE2 DNA but not that of ColE3 DNA in crude cell extracts of Escherichia coli . Specific binding of the protein to plasmid DNA containing the origin region of ColE2 was demonstrated by the filter binding method . Neither endonuclease activity nor topoisomerase activity was detected by using ColE2 DNA. Nucleic Acids Res, 1991 Jun 11, 19(11), 2825 - 34 Analysis of the IHF binding site in the regulatory region of bacteriophage Mu; van Rijn PA et al.; In bacteriophage Mu the converging early and repressor transcriptions are both stimulated by binding of IHF to the same region, which is located just upstream of the early promoter (Pe) and 100 base pairs downstream of the repressor promoter (Pc) . Within this region two sequences are present (ihfa and ihfb) that match the consensus sequence for IHF binding . These sequences are partially overlapping and in inverted orientation . In this paper we describe the effect of mutations in the non-overlapping part of ihfa and ihfb on the binding of IHF . We show that IHF has a very strong preference to bind to ihfb even when a mutated ihfa has a better match with the consensus . A stretch of A residues located nine base pairs from the ihfb sequence appears to play an important role in the stability of the DNA-IHF complex, but not in the discrimination between the two putative binding sites . In addition we describe the effect of the mutations on the stimulation of early and repressor transcription . We show that for activation of the Pc promoter a stable complex between IHF and the DNA is required, whereas for normal Pe stimulation a much weaker DNA-IHF interaction is sufficient. Biochemistry, 1991 Jun 4, 30(22), 5429 - 37 3'-end formation at the phage lambda tR1 rho-dependent transcription termination site; Roberts EA et al.; The rho-dependent transcription terminator tR1 of bacteriophage lambda stops RNA synthesis downstream of the major rightward promoter, PR, shortly after the cro gene . Terminated transcripts produced in a purified in vitro transcription system display a heterodisperse set of 3' termini, occurring in clusters located at +290-300, 308-312, 340-345, 385-390, and 440-450 nucleotides from the transcription start site {Morgan, W.D., Bear, D.G., & von Hippel, P.H . (1983) J . Biol . Chem . 258, 9553-9564} . However, transcripts from the same promoter in vivo have been reported to end primarily at +310-312 {Court, D., Brady, C., Rosenberg, M., Wulff, D . L., Behr, M., Mahoney, M., & Izumi, S . (1980) J . Mol . Biol . 138, 231-254} . In order to understand the nature of this discrepancy, we have carried out a comparative analysis of lambda PR transcription products produced in translationally active S30 cell extracts, in a purified in vitro system and in vivo . RNAs from the cell extracts coupled to translation show primarily three PR-derived transcripts beginning at one predominant 5' end and terminating at +263, 308, and 318 . Sites +263 and +308 appear to be RNA processing sites . S1 nuclease mapping studies of RNAs produced in vivo show one 5' end and two 3' termini ending at +263 and 311; the +263 site is the predominant 3' end . When transcripts produced in a purified in vitro transcription system are incubated in the S30 cell extract under various conditions, the RNAs are degraded to two primary products with lengths of 263 and 308-311 nt.(ABSTRACT TRUNCATED AT 250 WORDS) Genomics, 1991 Jun, 10(2), 505 - 8 Yeast artificial chromosome vectors for efficient clone manipulation and mapping; Shero JH et al.; The yeast artificial chromosome (YAC) cloning system allows the cloning of exogenous DNA several hundred kilobases in length . To enhance the usefulness of this technology, yeast artificial chromosome vectors have been designed for efficient clone characterization, manipulation, and mapping . The vectors contain a polylinker with unique EcoRI, BglII, NotI, EagI, SacII, SalI, NruI, NheI, and ClaI cloning sites and T7 bacteriophage promoters positioned to allow the generation of riboprobes from the exogenous DNA ends . Centric and acentric vector arms were constructed as separate plasmids to allow the recovery of both ends of the YAC insert DNA directly in Escherichia coli . In addition, YACs generated using this vector system contain a yeast gene (SUP 11) that allows visual monitoring of YAC stability and copy number. Genetics, 1991 Jun, 128(2), 203 - 13 Mutational analysis of the mRNA operator for T4 DNA polymerase; Andrake MD et al.; Biosynthesis of bacteriophage T4 DNA polymerase is autogenously regulated at the translational level . The enzyme, product of gene 43, represses its own translation by binding to its mRNA 5' to the initiator AUG at a 36-40 nucleotide segment that includes the Shine-Dalgarno sequence and a putative RNA hairpin structure consisting of a 5-base-pair stem and an 8-base loop . We constructed mutations that either disrupted the stem or altered specific loop residues of the hairpin and found that many of these mutations, including single-base changes in the loop sequence, diminished binding of purified T4 DNA polymerase to its RNA in vitro (as measured by a gel retardation assay) and derepressed synthesis of the enzyme in vivo (as measured in T4 infections and by recombinant-plasmid-mediated expression) . In vitro effects, however, were not always congruent with in vivo effects . For example, stem pairing with a sequence other than wild-type resulted in normal protein binding in vitro but derepression of protein synthesis in vivo . Similarly, a C----A change in the loop had a small effect in vitro and a strong effect in vivo . In contrast, an A----U change near the base of the hairpin that was predicted to increase the length of the base-paired stem had small effects both in vitro and in vivo . The results suggest that interaction of T4 DNA polymerase with its structured RNA operator depends on the spatial arrangement of specific nucleotide residues and is subject to modulation in vivo. J Bacteriol, 1991 Jun, 173(12), 3872 - 8 Characterization of trbC, a new F plasmid tra operon gene that is essential to conjugative transfer; Maneewannakul S et al.; We have characterized a previously unidentified gene, trbC, which is contained in the transfer region of the Escherichia coli K-12 fertility factor, F . Our data show that the trbC gene is located between the F plasmid genes traU and traN . The product of trbC was identified as a polypeptide with an apparent molecular weight (Ma) of 23,500 that is processed to an Ma-21,500 mature protein . When ethanol was present, the Ma-23,500 polypeptide accumulated; the removal of ethanol resulted in the appearance of the processed mature protein . Subcellular fractionation experiments demonstrated that the processed, Ma-21,500 mature protein was located in the periplasm . DNA sequence analysis showed that trbC encodes a 212-amino-acid Mr-23,432 polypeptide that could be processed to a 191-amino-acid Mr-21,225 mature protein through the removal of a typical amino-terminal signal sequence . We also constructed two different Kmr gene insertion mutations in trbC and crossed these onto the transmissible F plasmid derivative pOX38 . We found that cells carrying pOX38 trbC mutant plasmids were transfer deficient and resistant to infection by F-pilus-specific phages . Transfer proficiency and bacteriophage sensitivity were restored by complementation when a trbC+ plasmid clone was introduced into these cells . These results showed that trbC function is essential to the F plasmid conjugative transfer system and suggested that the TrbC protein participates in F-pilus assembly. J Bacteriol, 1991 Jun, 173(12), 3770 - 5 Properties of the streptomycete temperate bacteriophage FP43; Hahn DR et al.; FP43 is a temperate bacteriophage for Streptomyces griseofuscus that forms plaques on many Streptomyces species . FP43 virions contain 56 kb of double-strand DNA that is circularly permuted and terminally redundant, and contains 65% G + C . A physical map of the FP43 genome was constructed, and the origin for headful packaging (pac) was localized to an 8.8-kb region of the genome (hft) that mediates high-frequency transduction by FP43 of plasmid pRHB101 . The phage attachment site (attP), a replication origin (rep), a region that inhibits plaque formation (pin), and a 3-kb deletion (rpt) that caused a 100-fold reduction in plasmid transduction were mapped. J Bacteriol, 1991 Jun, 173(11), 3500 - 6 ftsZ is an essential cell division gene in Escherichia coli; Dai K et al.; The ftsZ gene is thought to be an essential cell division gene in Escherichia coli . We constructed a null allele of ftsZ in a strain carrying additional copies of ftsZ on a plasmid with a temperature-sensitive replication defect . This strain was temperature sensitive for cell division and viability, confirming that ftsZ is an essential cell division gene . Further analysis revealed that after a shift to the nonpermissive temperature, cell division ceased when the level of FtsZ started to decrease, indicating that septation is very sensitive to the level of FtsZ . Subsequent studies showed that nucleoid segregation was normal while FtsZ was decreasing and that ftsZ expression was not autoregulated . The null allele could not be complemented by lambda 16-2, even though this bacteriophage can complement the thermosensitive ftsZ84 mutation and carries 6 kb of DNA upstream of the ftsZ gene. Infect Immun, 1991 Jun, 59(6), 1941 - 7 Molecular cloning and sequence analysis of antigen gene tdpA of Treponema denticola; Miyamoto M et al.; We isolated and characterized an immunogenic protein of an oral spirochete, Treponema denticola Johnson . A genomic DNA library constructed with bacteriophage lambda EMBL3 as a vector was immunologically screened with a rabbit antiserum against the whole cells . Using Western immunoblot analysis, we found a particular clone encoding an antigen with a molecular weight of 53,000; we designated the antigen as T . denticola protein A (TdpA) . Complete sequence determination revealed an open reading frame of 1,419 bp and a signal peptide sequence that was homologous to that of bacterial lipoprotein . Southern hybridization analysis revealed that the tdpA gene is highly conserved in six tested strains of T . denticola species . Furthermore, we found that sera from some periodontitis patients contained antibody against the TdpA protein, although the reactivities of the antibodies varied among individuals. J Virol, 1991 Jun, 65(6), 3219 - 26 A domain at the 3' end of the polymerase gene is essential for encapsidation of coronavirus defective interfering RNAs; van der Most RG et al.; Two murine hepatitis virus strain A59 defective interfering (DI) RNAs were generated by undiluted virus passages . The DI RNAs were encapsidated efficiently . The smallest DI particle, DI-a, contained a 5.5-kb RNA consisting of the following three noncontiguous regions from the MHV-A59 genome, which were joined in frame: the 5'-terminal 3.9 kb, a 798-nucleotide fragment from the 3' end of the polymerase gene, and the 3'-terminal 805 nucleotides . A full-length cDNA clone of the DI-a genome was constructed and cloned downstream of the bacteriophage T7 promoter . Transcripts derived from this clone, pMIDI, were used for transfection of MHV-A59-infected cells and found to be amplified and packaged . Deletion analysis of pMIDI allowed us to identify a 650-nucleotide region derived from the 3' end of the second open reading frame of the polymerase gene that was required for efficient encapsidation. Protein Eng, 1991 Jun, 4(5), 553 - 60 The role of tyrosine residues in the DNA-binding site of the Pf1 gene 5 protein; Plyte SE et al.; The 144 amino acid gene 5 protein of bacteriophage Pf1 binds tightly and cooperatively to single-stranded DNA during replication of the phage genome . It has been suggested that aromatic amino acid side chains are important for this interaction, probably through base stacking with the DNA . We have analysed the accessibility of tyrosine residues in the DNA-protein complex, and their importance to the DNA-binding activity of the protein, by chemical modification and protection experiments using tetranitromethane . Tyrosines 21, 30 and 55 are surface accessible in the free protein but are protected from modification in the complex with phage DNA . Moreover, modification of these residues in the free protein abolishes the ability to bind to DNA or oligonucleotides, as judged by fluorescence spectroscopy and gel retardation analysis . Modification of the protein also results in the formation of an intersubunit covalent cross-link between Tyr55 and Phe76, suggesting that Phe76 is located within the DNA-binding cleft of the protein . It is proposed that residues 17-34 of the Pf1 gene 5 protein form a beta-hairpin analogous to the 'DNA-binding wing' of the fd and Ike gene 5 proteins . We suggest the existence of a single-stranded DNA binding motif, in which Tyr30 of the Pf1 protein is equivalent to the functionally important Tyr26 of the fd gene 5 protein. Protein Eng, 1991 Jun, 4(5), 545 - 52 Intersubunit disulfide-bonded lambda-Cro protein; Shirakawa M et al.; Site-directed mutagenesis has been employed to substitute cysteine for valine at position 55, which is located on the dimer interface of the Cro protein of bacteriophage lambda . It has been found that the Cys55 Cro protein (Cro VC55) spontaneously forms a stable disulfide-bonded dimer in the absence of a reducing agent . UV-CD and NMR data showed that the mutant protein retains the conformation of the wild Cro protein and has acquired significant heat-stability . However, its specific DNA-binding activity is reduced several times compared with that of the wild Cro . Photochemically induced dynamic nuclear polarization (CIDNP) spectra demonstrated that a conformational change of Cro VC55 did not take place upon the formation of a complex with OR3, in contrast to the case of the wild Cro . These data suggest that the induced fitting, like loosening, of the two subunits of the wild Cro dimer contributes to the enhancement of its affinity to its operator DNA, which results in a specific interaction between Cro and OR3. Appl Environ Microbiol, 1991 Jun, 57(6), 1842 - 3 Dye to use with virus challenge for testing barrier materials; Lytle CD et al.; Can FD&C Blue no . 1 dye photoinactivate bacteriophages phi X174, T7, PRD1, and phi 6 under laboratory lighting conditions? At high levels of light, the dye (500 microM) photoinactivated only phi 6 . Thus, this dye can be used at concentrations up to 500 microM with bacteriophages phi X174, T7, and PRD1 to test barrier material integrity. Mol Gen Mikrobiol Virusol, 1991 Jun, (6), 13 - 6 {Survival curve during virus inactivation}; Davidovich IA et al.; The survival curves for bacteriophage lambda and vaccinia virus were shown theoretically and in experiments to have a plateau at prolonged inactivation by UV-irradiation or 8-methoxypsoralen . The level of the plateau is dependent of the accuracy of the repair process . The method for extrapolation of the survival curves is proposed. J Biomol Struct Dyn, 1991 Jun, 8(6), 1147 - 67 100ps molecular dynamic simulation of d(TATCACC)2; Mrigank et al.; A heptanucleotide sequence d(TATCACC)2 from OR3 region of bacteriophage lambda is considered sufficient for the recognition of Cro protein . We present here results on molecular dynamic simulations on this sequence for 100 ps in 0.02 ps interval . The simulations are done using computer program GROMOS . The conformational results are averaged over each ps . The IUPAC torsional parameters for 100 conformations are illustrated using a wheal and a dial systems . Several other stereochemical parameters such as H-bonding lengths and angles, sugar puckers, helix twist and roll angles as also distances between opposite strand phosphorus are depicted graphically . We find that there is rupture of terminal H-bonds . The bases are tilted and shifted away from the helix axis giving rise to bifurcated H-bonds . H-bonds are seen even in between different base pairs . The role of these dynamic structural changes in the recognition of OR3 operator by Cro protein is discussed in the paper. Proc Natl Acad Sci U S A, 1991 Jun 1, 88(11), 4996 - 5000 An additional function for bacteriophage lambda rex: the rexB product prevents degradation of the lambda O protein; Schoulaker-Schwarz R et al.; The rex operon of bacteriophage lambda excludes the development of several unrelated bacteriophages . Here we present an additional lambda rexB function: it prevents degradation of the short-lived protein lambda O known to be involved in lambda DNA replication . We have shown that it is the product of rexB that is responsible for the stabilization of lambda O: when a nonsense mutation is present in rexB, lambda O protein is labile; suppression of the mutation by the corresponding nonsense suppressor causes partial restabilization of lambda O . lambda rexB also stabilizes lambda O in trans . We discuss our results in relation to the function of rexB in lambda DNA replication and its role in the protein degradation pathways of bacteriophage lambda. Virology, 1991 Jun, 182(2), 534 - 44 Characterization of a versatile in vitro DNA-packaging system based on hybrid lambda/phi 29 proheads; Donate LE et al.; We have studied the assembly of bacteriophage lambda head proteins on the phage phi 29 connector to produce in vitro chimeric proheads, whose ability to package different types of DNA depends on the physical integrity of the phi 29 connector . Terminal protein-free phi 29 as well as nonviral DNAs have been shown to be efficiently packaged by this hybrid system . An RNA, that can be provided by any of the extracts used in the complementation mixture, was required for DNA packaging, both by the hybrid system as well as by the homologous lambda system . The DNA-packaging activity of RNase-treated proheads can be restored by adding a mixture of ribosomal RNAs . There is also a requirement for a minimal length of DNA to be stably packaged . The packaging protein p16 of phi 29 can replace the lambda terminase complex in the in vitro packaging system, both with the chimeric as well as genuine lambda proheads. Bioorg Khim, 1991 Jun, 17(6), 819 - 22 {RNA ligase from bacteriophage T4 . VIII . Solid phase enzymatic synthesis of oligoribonucleotides}; Mudrakovskaia AV et al.; A method of the solid-phase enzymic synthesis of oligoribonucleotides has been suggested . The donor is fixed through its 3'-end on a water-insoluble matrix followed by the stepwise RNA ligase- and T4 polynucleotide kinase-assisted coupling of trinucleoside diphosphates in the 5'-direction . As an example, (pA)6pAox was immobilised on Biogel P-300 hydrazine and the RNA ligase-catalyzed addition of acceptor ApApA to the donor gave (Ap)9 with the 50% yield. J Virol Methods, 1991 Jun, 33(1-2), 47 - 52 Characterization of two monoclonal antibodies to Epstein-Barr virus diffuse early antigen which react to two different epitopes and have different biological function; Tsai CH et al.; Five monoclonal antibodies (mAbs) were identified using immunofluorescence that were specific for the Epstein-Barr virus (EBV) encoded 52/50 kDa early antigen (EA-D) protein complex . Evidence to suggest that these mAbs react with the same 52/50 kDa EA-D protein was obtained by Western blotting, immunoprecipitation and ELISA . Two of the mAbs, 90E2 and 214A9, neutralized EBV DNA polymerase activity . The 214A9 mAb also inhibited the activity of bacteriophage T4 DNA polymerase while the 90E2 mAb did not . These data suggest that the 90E2 and 214A9 mAbs recognize two different epitopes on the 52/50 kDa EA-D protein . The high frequency of recovery of hybridomas producing anti 52/50 kDa EA-D mAbs suggest that this protein may have an important role in EBV pathogenesis/replication. J Virol, 1991 Jun, 65(6), 3227 - 37 Bacteriophage HK97 structure: wholesale covalent cross-linking between the major head shell subunits; Popa MP et al.; We describe initial genetic and structural characterizations of HK97, a temperate bacteriophage of Escherichia coli . We isolated 28 amber mutants, characterized them with respect to what phage-related structures they make, and mapped many of them to restriction fragments of genomic DNA . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of HK97 virions revealed nine different protein species plus a substantial amount of material that failed to enter the gel, apparently because it is too large . Five proteins are tail components and are assigned functions as tail fiber subunit, tail length template, and major shaft subunit (two and possibly three species) . The four remaining proteins and the material that did not enter the gel are head components . One of these proteins is assigned as the portal subunit, and the remaining three head proteins in the gel and the material that did not enter the gel are components of the head shell . All of the head shell protein species have apparent molecular masses well in excess of 100 kDa; they share amino acid sequence with each other and also with a 42-kDa protein that is found in infected lysates and as the major component of prohead structures that accumulate in infections by one of the amber mutants . We propose that all of the head shell species found in mature heads are covalently cross-linked oligomers derived from the 42-kDa precursor during head shell maturation. New Biol, 1991 Jun, 3(6), 615 - 25 Mutations altering chromosomal protein H-NS induce mini-Mu transposition; Falconi M et al.; Bacteriophage Mu is one of the most efficient transposons known, capable of moving a hundred viral copies to new positions in the bacterial chromosome in an hour . Mu also forms stable lysogens . In bacteria lysogenic for the defective protein fusion-forming phage MudII1681, which can transpose and replicate but does not encode genes for DNA packaging and cell lysis, the frequency of transposition changes as colonies age . To find host genes that alter the spontaneous Mu transposition frequency, we used a genetic screen with mini-MudlacZ fusion formation as an assay . H-NS (also called H1a and B1) is an abundant nonspecific DNA-binding protein localized to the bacterial chromosome . H-NS has an unusual structure of interspersed patches of acidic and basic residues reminiscent of eukaryotic HMG proteins . Mutations in hns caused an increase in Mu-specific transcription and a dramatic increase in MudII1681 transposition rates when cells were put under certain growth conditions . Purified H-NS stabilized Mu repressor-DNA complexes in vitro, suggesting that H-NS contributes to the organization of transcriptionally inactive DNA in vivo. J Bacteriol, 1991 Jun, 173(12), 3615 - 21 Cyclic AMP inhibits and putrescine represses expression of the speA gene encoding biosynthetic arginine decarboxylase in Escherichia coli; Moore RC et al.; The speA gene of Escherichia coli encodes biosynthetic arginine decarboxylase (ADC), the first of two enzymes in a putrescine biosynthetic pathway . The activity of ADC is negatively regulated by mechanisms requiring cyclic AMP (cAMP) and cAMP receptor protein (CRP) or putrescine . A 2.1-kb BamHI fragment containing the speA-metK intergenic region, speA promoter, and 1,389 bp of the 5' end of the speA coding sequence was used to construct transcriptional and translational speA-lacZ fusion plasmids . A single copy of either type of speA-lacZ fusion was transferred into the chromosomes of Escherichia coli KC14-1, CB806, and MC4100, using bacteriophage lambda . The speA gene in lysogenized strains remained intact and served as a control . Addition of 5 mM cAMP to lysogenic strains resulted in 10 to 37% inhibition of ADC activity, depending on the strain used . In contrast, the addition of 5 or 10 mM cAMP to these strains did not inhibit the activity of beta-galactosidase (i.e., ADC::beta-galactosidase) . Addition of 10 mM putrescine to lysogenized strains resulted in 24 to 31% repression of ADC activity and 41 to 47% repression of beta-galactosidase activity . E . coli strains grown in 5 mM cAMP and 10 mM putrescine produced 46 to 61% less ADC activity and 41 to 52% less beta-galactosidase activity . cAMP (0.1 to 10 mM) did not inhibit ADC activity assayed in vitro . The effects of cAMP and putrescine on ADC activity were additive, indicating the use of independent regulatory mechanisms . These results show that cAMP acts indirectly to inhibit ADC activity and that putrescine causes repression of speA transcription. Transfusion, 1991 Jun, 31(5), 415 - 22 Inhibition by albumin of merocyanine 540-mediated photosensitization of platelets and viruses; Prodouz KN et al.; The effect of the photosensitizer merocyanine 540 (MC 540) on platelets and on three marker viruses was examined to assess its potential in reducing virus transmission by blood products . The results demonstrated several deleterious effects of MC 540 (4-24 micrograms/mL) on platelet morphology and function in both the absence and presence of light (450-600 nm) . Treatment of washed platelets with MC 540 in the dark resulted in a significant release of serotonin in the absence of added agonist, as well as a diminished response to thrombin as measured in vitro . In addition, photosensitization caused spontaneous platelet aggregation and release of 92 percent of the releasable serotonin without the addition of an agonist . Because photo-treatment of blood products is likely to be performed in a protein-rich medium, the influence of albumin on the phototoxic effects on platelets was assessed . Albumin added to the suspension medium at concentrations greater than or equal to 1.0 percent protected the platelets against the effects of MC 540 in the dark, whereas 5-percent albumin was required for protection against the phototoxic effects of MC 540 on the platelet response to thrombin . The antiviral activity of MC 540 and light was examined by using the lipid-containing viruses herpes simplex virus (HSV) and bacteriophages phi 6 and PM2 . Of the lipid-enveloped viruses, HSV was 25 times more photosensitive to MC 540 than was phi 6 (15 micrograms/mL) . PM2, which has an internal lipid layer, was almost 300 times less sensitive to MC 540 and light than was HSV.(ABSTRACT TRUNCATED AT 250 WORDS) J Ind Microbiol, 1991 Jun, 7(4), 229 - 34 Transposition and transduction of plasmid DNA in Streptomyces spp; Hahn DR et al.; To expand the application of molecular genetics to many different streptomycete species, we have been developing two potentially widely applicable methodologies: transposon mutagenesis and plasmid transduction . We constructed three transposons from the Streptomyces lividans insertion sequence IS493 . Tn5096 and Tn5097 contain an apramycin resistance gene inserted in different orientations between the two open reading frames of IS493 . These transposons transpose from different plasmids into many different sites in the Streptomyces griseofuscus chromosome and into its resident linear plasmids . Tn5099 contains a promoterless xylE gene and a hygromycin-resistance gene inserted in IS493 close to one end . Tn5099 transposes in S . griseofuscus giving operon fusions in some cases that drive expression of the xylE gene product, catechol deoxygenase, giving yellow colonies in the presence of catechol . We have also developed plasmid vectors that can be transduced into many streptomycete species by bacteriophage FP43 . We describe the characterization of FP43 and mapping of several bacteriophage functions . The region of cloned FP43 DNA essential for plasmid transduction includes the origin for headful packaging. Biochemistry, 1991 May 21, 30(20), 4855 - 63 Differences in secondary structure between packaged and unpackaged single-stranded DNA of bacteriophage phi X174 determined by Raman spectroscopy: a model for phi X174 DNA packaging; Benevides JM et al.; The single-stranded packaged genome (ssDNA) of bacteriophage phi X174 is shown by Raman spectroscopy to lack both the ordered phosphodiester backbone and base stacking, which are demonstrated for unpackaged, protein-free ssDNA . In solutions of moderate ionic strength, unpackaged ssDNA contains 36 +/- 7% of deoxyribosyl phosphate groups with conventional B-type backbone geometry {i.e., gauche- and trans orientations, respectively, for the 5'O-P (alpha) and 3'O-P (zeta) torsions}, indicative of hairpin formation and intramolecular base pairing . Additionally, the bases of unpackaged ssDNA are extensively stacked . Estimates from Raman band hypochromic effects indicate that unpackaged ssDNA contains approximately 70% of the maximal base stacking exhibited in the linear, double-stranded, replicative form III of phi X174 DNA . Conversely, for the packaged phi X174 genome, ordered (B-type) phosphodiester groups are not present, and only 40% of the base stacking in RFIII DNA is observed . These results are interpreted as evidence that the substantial hairpin-forming potential of ssDNA is eliminated by specific and extensive ssDNA-protein interactions within the phi X174 virion . Comparison of the present results with studies of other packaged single-stranded nucleic acids suggests that proteins of the capsid shell (gpF + gpG + gpH) do not fully account for the conformational constraints imposed on ssDNA of phi X174 . Accordingly, we propose a model for ssDNA packaging in which the small basic gpJ protein, which is packaged along with the genome, is involved stoichiometrically in binding to the ssDNA (approximately 90 nucleotides per subunit) . The proposed gpJ-DNA interactions could prevent helical hairpin formation, restrict base stacking, and disfavor fortuitous base pairing within the capsid . The present analysis is based upon use of model nucleic acids of known conformation for calibration of the Raman intensity in the region 810-860 cm-1 in terms of specific secondary structures . The calibration curve allows quantitative determination of the percentage of ssDNA nucleotides for which the 5'O-P-O3' group is configured (g-,t) as in the B-form of DNA . The method proposed here is analogous to that employed by Thomas and Hartman (1973) for ssRNA and should be applicable to single-stranded DNA and to partially denatured forms of double- and multiple-stranded DNAs. Biochem Biophys Res Commun, 1991 May 31, 177(1), 305 - 11 Expression of human brain hexokinase in Escherichia coli: purification and characterization of the expressed enzyme; Liu F et al.; Human brain hexokinase (hexokinase I) was produced in Escherichia coli from a synthetic gene under control of the bacteriophage T7 promoter . The expressed coding region derives from a human cDNA clone thought to specify hexokinase I based on amino acid sequence identity between the predicted translation product and hexokinase I from rat brain . The open reading frame from this cDNA was fused to the promoter and 5' flanking region of T7 gene 10, and expressed in E . coli by induction of T7 RNA polymerase . Induced cells contained a hexokinase activity and an abundant protein of apparent molecular weight 100,000, neither of which was present in cells lacking T7 RNA polymerase . Enzyme purified to near homogeneity consisted of a 100,000 Da protein, the size predicted from the nucleotide sequence of the expressed cDNA . The purified enzyme had Michaelis constants of 32 microM and 0.3 mM for glucose and ATP, respectively, and bound to rat liver mitochondria in the presence of MgCl2 . Enzymatic activity was inhibited by glucose-6-P and this inhibition was relieved by inorganic phosphate . Deinhibition by phosphate is a property specific to brain hexokinase. Science, 1991 May 31, 252(5010), 1305 - 8 Membrane-mediated assembly of filamentous bacteriophage Pf1 coat protein; Nambudripad R et al.; Filamentous bacteriophage Pf1 assembles by a membrane-mediated process during which the viral DNA is secreted through the membrane while being encapsulated by the major coat protein . Neutron diffraction studies showed that in the virus most of the coat protein consists of two alpha-helical segments arranged end-to-end with an intervening mobile surface loop . Nuclear magnetic resonance studies of the coat protein in the membrane-bound form have shown that the secondary structure is essentially identical to that in the intact virus . A comparison indicates that during membrane-mediated viral assembly, while the secondary structure of the coat protein is largely conserved, its tertiary structure changes substantially. Science, 1991 May 31, 252(5010), 1303 - 5 NMR studies of the structure and dynamics of membrane-bound bacteriophage Pf1 coat protein; Shon KJ et al.; Filamentous bacteriophage coat protein undergoes a remarkable structural transition during the viral assembly process as it is transferred from the membrane environment of the cell, where it spans the phospholipid bilayer, to the newly extruded virus particles . Nuclear magnetic resonance (NMR) studies show the membrane-bound form of the 46-residue Pf1 coat protein to be surprisingly complex with five distinct regions . The secondary structure consists of a long hydrophobic helix (residues 19 to 42) that spans the bilayer and a short amphipathic helix (residues 6 to 13) parallel to the plane of the bilayer . The NH2-terminus (residues 1 to 5), the COOH-terminus (residues 43 to 46), and residues 14 to 18 connecting the two helices are mobile . By comparing the structure and dynamics of the membrane-bound coat protein with that of the viral form as determined by NMR and neutron diffraction, essential features of assembly process can be identified. Biochem Biophys Res Commun, 1991 May 31, 177(1), 140 - 4 Footprint of the sigma protein: a re-examination; Wellman A et al.; Escherichia coli RNA Polymerase is a multi-subunit enzyme that catalyzes RNA synthesis, using DNA as a template . The sigma subunit of this enzyme plays an important role in the recognition of promoter sites on DNA . Using DNase I footprinting, Utpala Ramesh and Claude F . Meares {(1989) Biochem . Biophys . Res . Comm . 160, 121-125} reported that in the absence of the other subunits, sigma binds specifically to the bacteriophage lambda PR promoter DNA sequence . We are unable to reproduce that result. J Biol Chem, 1991 May 25, 266(15), 9712 - 8 Inhibition of protein-mediated homologous pairing by a DNA helicase; Kodadek T; Protein-mediated exchange of homologous DNA strands is a central reaction in general genetic recombination and the mechanism by which proteins mediate this process in vivo is a topic of keen interest . The dda protein of the bacteriophage T4 is a DNA helicase that has been shown to accelerate branch migration catalyzed by the phage uvsX and gene 32 proteins in vitro (Kodadek, T., and Alberts, B.M . (1987) Nature 326, 312-314) . This study did not address the potential role of the helicase in protein-mediated homologous pairing, the first phase of the overall strand-exchange reaction . It is shown here that the dda protein inhibits uvsX protein-mediated pairing between homologous single and double-stranded DNAs . Experiments using deproteinized heteroduplex joints demonstrate that the dda helicase is capable of unwinding these structures to some extent and suggests that this activity may be responsible for the observed inhibition of pairing . It is found that the helicase also reduces the level of uvsX protein-mediated, single-stranded DNA-dependent ATP hydrolysis in the strand-exchange reactions, suggesting that the helicase may also act to destabilize the uvsX protein-DNA filaments that are important intermediates in the pairing reaction . Three other helicases are found to have no effect on the uvsX protein-mediated pairing reaction . A model rationalizing the ability of the dda protein to both inhibit homologous pairing and stimulate branch migration is presented and possible in vivo roles for this interesting activity are discussed. J Mol Biol, 1991 May 20, 219(2), 257 - 75 Isolation and in vitro characterization of temperature-sensitive mutants of the bacteriophage f1 gene V protein; Zabin HB et al.; In vivo selections were used to isolate 43 temperature-sensitive gene V mutants of the bacteriophage f1 from a collection of mutants constructed by saturation mutagenesis of the gene . The sites of temperature-sensitive substitutions are found in both the beta-sheets and the turns of the protein, and some sites are exposed to the solvent while others are not . Thirteen of the variant proteins were purified and characterized to evaluate their free energy changes upon unfolding and their affinities for single-stranded DNA, and eight were tested for their tendencies to aggregate at 42 degrees C . Each of the three temperature-sensitive mutants at buried sites and six of ten at surface sites had free energy changes of unfolding substantially lower (less stabilizing) than the wild-type at 25 degrees C . A seventh mutant at a surface site had a substantially altered unfolding transition and its free energy of unfolding was not estimated . The affinities of the mutant proteins for single-stranded DNA varied considerably, but two mutants at a surface site, Lys69, had much weaker binding to single-stranded DNA than any of the other mutants, while two mutants at another surface site, Glu30, had the highest DNA-binding affinities . The wild-type gene V protein is stable at 42 degrees C, but six of the eight mutants tested aggregated within a few minutes and the remaining two aggregated within 30 minutes at this temperature . Overall, each of the temperature-sensitive proteins tested had a tendency to aggregate at 42 degrees C, and most also had either a low free energy of unfolding (at 25 degrees C), or weak DNA binding . We suggest that any of these properties can lead to a temperature-sensitive gene V phenotype. Science, 1991 May 17, 252(5008), 969 - 71 Retronphage phi R73: an E . coli phage that contains a retroelement and integrates into a tRNA gene; Inouye S et al.; Some strains of Escherichia coli contain retroelements (retrons) that encode genes for reverse transcriptase and branched, multicopy, single-stranded DNA (msDNA) linked to RNA . However, the origin of retrons is unknown . A P4-like cryptic prophage was found that contains a retroelement (retron Ec73) for msDNA-Ec73 in an E . coli clinical strain . The entire genome of this prophage, named phi R73, is 12.7 kilobase pairs and is flanked by 29-base pair direct repeats derived from the 3' end of the selenocystyl transfer RNA gene (selC) . P2 bacteriophage caused excision of the phi R73 prophage and acted as a helper to package phi R73 DNA into an infectious virion . The newly formed phi R73 closely resembled P4 as a virion and in its lytic growth . Retronphage phi R73 lysogenized a new host strain, reintegrating its genome into the selC gene of the host chromosome and enabling the newly formed lysogens to produce msDNA-Ec73 . Hence, retron Ec73 can be transferred intercellularly as part of the genome of a helper-dependent retronphage. J Biol Chem, 1991 May 15, 266(14), 9180 - 5 Isolation and characterization of the mouse cardiac myosin heavy chain genes; Gulick J et al.; Two mouse genomic libraries were probed in order to isolate the murine cardiac myosin heavy chain (MHC) genes . Two overlapping cosmid clones that encode the cardiac genes were isolated . One of them encompasses the entire alpha-cardiac MHC gene, its 5'-flanking region and approximately 10 kilobase pairs (kb) of the 3'-end of the beta-cardiac MHC gene which we determined is located approximately 4 kb upstream of the alpha-cardiac MHC gene . Four clones isolated from a bacteriophage library were found to overlap with the 5'-region of the cosmid clones, and sequence analysis confirmed that the 5'-end of the beta-cardiac MHC gene was contained within one of the clones . Primer extension and polymerase chain reaction (PCR) analyses were used to define the transcriptional start site and the 5'-organization of the alpha-cardiac MHC gene . This region exhibits greater than 90% homology to the corresponding nucleotides of the rat alpha-cardiac MHC gene . To assess the importance of the intergenic region in directing expression of the alpha-cardiac MHC gene, a fragment containing the 3'-end of the beta-cardiac gene and the 5'-end of the alpha-cardiac gene was linked to a chloramphenicol acetyltransferase gene and used to generate transgenic mice . Analyses of the chloramphenicol acetyltransferase (CAT) activity in two lines indicate that the intergenic region is sufficient to properly direct expression in a tissue-specific manner. Biochemistry, 1991 May 14, 30(19), 4821 - 30 Fluorescence study of the topology of messenger RNA bound to the 30S ribosomal subunit of Escherichia coli; Czworkowski J et al.; Short RNAs (25-36 nucleotides in length) with sequences of the translational initiation region of bacteriophage R17 protein A mRNA were produced by chemical and in vitro transcription techniques and labeled at their 5' or 3' ends with fluorescent probes . The interaction of these labeled RNAs with the 30S subunit of Escherichia coli was studied by using fluorescence spectroscopic techniques . All the RNAs bound tightly to 30S subunits (Kd less than or equal to 200 nM) . Resonance energy transfer experiments demonstrated the proximity of the ends of the RNAs to each other and to two fluorescently labeled sites on the 30S subunit: the 3' end of 16S rRNA and the cysteine residue of ribosomal protein S21 . By using the distances calculated from energy transfer between the 3' end of 16S rRNA and the ends of RNAs of varying lengths, a topological map of this region of mRNA on the 30S subunit was constructed. J Mol Biol, 1991 May 5, 219(1), 61 - 8 Creation of a T7 autogene . Cloning and expression of the gene for bacteriophage T7 RNA polymerase under control of its cognate promoter; Dubendorff JW et al.; The coding sequence for bacteriophage T7 RNA polymerase has been cloned and expressed under control of a cognate T7 promoter, a configuration referred to as an autogene . Cloning a T7 autogene in a derivative of plasmid pBR322 in Escherichia coli was achieved by a combination of blocking initiation at the T7 promoter with bound lac repressor and inhibiting the polymerase itself by T7 lysozyme . Neither type of inhibition by itself was sufficient to control the autogene . Upon unblocking the T7 promoter with added inducer . T7 RNA polymerase produced its own mRNA, leading to autocatalytic production of polymerase protein . T7 autogenes may be useful for developing high-level gene expression systems in a variety of cell types, with little if any need for the host cell RNA polymerase. J Mol Biol, 1991 May 5, 219(1), 37 - 44 Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system; Studier FW; Bacteriophage T7 lysozyme, a natural inhibitor of T7 RNA polymerase, can reduce basal activity from an inducible gene for T7 RNA polymerase and allow relatively toxic genes to be established in the same cell under control of a T7 promoter . Low levels of T7 lysozyme supplied by plasmids pLysS or pLysL, which are compatible with the pET vectors for expressing genes from a T7 promoter, are sufficient to stabilize many target plasmids and yet allow high levels of target protein to be produced upon induction of T7 RNA polymerase . Higher levels of lysozyme supplied by plasmids pLysE or pLysH reduce the fully induced activity of T7 RNA polymerase such that induced cells can continue to grow and produce innocuous target proteins indefinitely . Different configurations of the expression system can maintain several different steady-state levels of target gene expression . The presence of T7 lysozyme has the further advantage of facilitating the lysis of cells in preparing extracts for purification of target gene products. J Biol Chem, 1991 May 5, 266(13), 8495 - 500 Combinatorial mutagenesis of the reactive site region in plasminogen activator inhibitor I; York JD et al.; Plasminogen activator inhibitor (PAI-I) rapidly inactivates tissue plasminogen activator (t-PA) and urokinase (UK) with nearly identical association rate constants . The contributions of Ser344, Ala345, and Arg346 (P3, P2, and P1 residues, respectively) in PAI-I to inhibition of UK and t-PA were evaluated using combinatorial mutagenesis of the human PAI-I cDNA . A bacteriophage lambda expression library potentially encoding the 8000 unique PAI-I species were screened for inhibitory activity against UK using a fibrin indicator gel . 390 plaques demarcated by zones of retarded fibrinolysis were analyzed to determine the DNA sequences of their associated active PAI-1 species . We found 134 unique PAI-1 variants that retained inhibitory activity towards UK; they contained a variety of amino acids in their P3 and P2 positions but only Arg or, infrequently, Lys in their P1 position . Each of the unique active PAI-1 were assayed for inhibitory activity towards UK or t-PA; many substitutions differentially affected the ability of the inhibitor to inactivate UK and t-PA . For example, replacement of Ser344 and Ala344 with Val and Pro, respectively, yielded a PAI-1 variant exhibiting an association rate constant that was unchanged for t-PA but decreased 23-fold for UK, relative to native PAI-1 . In general, the PAI-1 variants were more potent inhibitors of t-PA than UK . Hence, t-PA appears more tolerant than UK of structural diversity present in the P3 and P2 positions of the PAI-1 variants. J Biol Chem, 1991 May 5, 266(13), 8447 - 54 A 14-kDa Schistosoma mansoni polypeptide is homologous to a gene family of fatty acid binding proteins; Moser D et al.; The complete nucleotide sequence encoding a Schistosoma mansoni protein termed Sm14 was determined from cDNA clones propagated in bacteriophage lambda gt11 in Escherichia coli . The 14.8-kDa protein bears significant homologies with a family of related polypeptides which bind hydrophobic ligands . Members of this group of cytosolic proteins were originally identified based on their affinity for long chain fatty acids . The purified recombinant protein exhibited an affinity to fatty acids, in contrast to a mutant lacking 16 N-terminal amino acids . Immunofluorescence experiments show that tubercles, which are structures located on the dorsal surface of adult male schistosome and known to contain lipids, are stained using antibodies raised to the beta-galactosidase fusion protein . A regular staining pattern is also evident in the muscle layers as well as in the body of the parasite . As the schistosome cannot synthesize fatty acids de novo and is dependent on the uptake of lipids from serum, the available data support a role for Sm14 in the transport of fatty acids. J Biol Chem, 1991 May 5, 266(13), 7967 - 70 A retrovirus-like zinc domain is essential for translational repression of bacteriophage T4 gene 32; Shamoo Y et al.; Gene 32 protein (gp32), a single-stranded DNA-binding protein from bacteriophage T4, contains a zinc-binding subdomain with sequence homologies to the 3-cysteine/1-histidine zinc-binding motif found in a variety of retroviruses and plant viruses . In vitro studies suggest that autoregulation of gp32 occurs at the level of translation by gp32 specifically binding gene 32 mRNA at an unusual stem-loop structure that can be modeled as an RNA pseudoknot . Nucleation of gp32 binding via this pseudoknot is thought to be needed to facilitate cooperative binding of gp32 through a largely unstructured region that overlaps the ribosome binding site (McPheeters, D . S., Stormo, G . D., and Gold, L . (1988) J . Mol . Biol . 201, 517-535) . Removal of Zn(II) from gp32 results in a protein that retains the ability to bind single-stranded RNA with high affinity but is unable to specifically autoregulate itself at the level of translation . Deletion of the pseudoknot sequences from the gene 32 autoregulatory region results in an mRNA that cannot be repressed by gp32 . These results suggest that the zinc-binding subdomain of gp32 plays an essential role in autoregulation by providing a critical element necessary for nucleating cooperative binding at the gene 32 mRNA pseudoknot. J Mol Biol, 1991 May 5, 219(1), 45 - 59 Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor; Dubendorff JW et al.; Effects of placing a lac operator at different positions relative to a promoter for bacteriophage T7 RNA polymerase were tested . Transcription can be strongly repressed by lac repressor bound to an operator centered 15 base-pairs downstream from the RNA start, but T7 RNA polymerase initiates transcription very actively from this T7lac promoter-operator combination in the absence of repressor, or in the presence of repressor plus inducer . Sequence changes in the transcribed region were found to make transcription from some T7 promoters, including the T7lac promoter, more sensitive to inhibition by T7 lysozyme . The pET-10 and pET-11 series of plasmid vectors have been constructed to allow target genes to be placed under control of the T7lac promoter and to be expressed in BL21(DE3) or HMS174(DE3), which carry an inducible gene for T7 RNA polymerase . These vectors carry a lacI gene that provides enough lac repressor to repress both the T7lac promoter in the multicopy vectors and the chromosomal gene for T7 RNA polymerase, which is controlled by the lacUV5 promoter . Very low basal expression of target genes is achieved, but the usual high levels of expression are obtained upon induction . Addition of T7 lysozyme can reduce basal expression even further and still allow high levels of expression upon induction . Genes that are very toxic to Escherichia coli can be maintained and expressed in this system. J Biol Chem, 1991 May 5, 266(13), 8511 - 6 Gene characterization and promoter analysis of the human 5-lipoxygenase-activating protein (FLAP); Kennedy BP et al.; The human gene for the recently identified 5-lipoxy-genase-activating protein (FLAP) has been cloned . The gene was isolated from two different genomic libraries and is contained within four overlapping bacteriophage clones . The gene spans greater than 31 kilobases and consists of five small exons and four large introns . Southern blot analysis of human genomic DNA suggests the presence of a single FLAP gene per haploid genome . A restriction site polymorphism was identified in intron II of the gene . This restriction fragment length polymorphism appears to be present in the normal population at a fairly high frequency . The transcription initiation site was located, at an adenine residue, 74 base pairs upstream of the ATG initiation codon . Examination of the sequence of the gene 5' to the mRNA start site revealed the presence of a possible TATA box (TGTAAT) 22 base pairs upstream and potential AP-2 and glucocorticoid receptor binding sites . Functional analysis of the FLAP gene promoter was assayed by transient transfection of mouse P388D1 cells (macrophage) and human HepG2 cells (hepatoma) with 5'-flanking sequences of the FLAP gene fused upstream of the chloramphenicol acetyltransferase reporter gene . Expression in the mouse macrophage cell line of the various FLAP gene promoter constructs revealed both tissue specificity and enhancer-like activities whereas in the hepatoma cell line only a minimum level of activity was obtained. Mol Gen Genet, 1991 May, 227(1), 144 - 8 The two-step model of UV mutagenesis reassessed: deamination of cytosine in cyclobutane dimers as the likely source of the mutations associated with photoreactivation; Tessman I et al.; A large increase in the incidence of bacteriophage mutants is found after photoreactivation of UV-irradiated phage S13 . The increase was seen only when the irradiated phage were stored before they were photoreactivated; the maximum mutation frequency was achieved after storage for 2 h at 4 degrees C or 30 min at 37 degrees C . The mutations can be attributed entirely to deamination of cytosine in cyclobutane dimers . Naked S13 DNA was stored for 2 h at 37 degrees C after being irradiated with wavelengths greater than or equal to 290 nm in the presence of 0.2% acetophenone, which sensitizes the formation of thymine-thymine but not cytosine-containing dimers; the specific mutation frequency was 7.2-fold lower compared to the frequency produced by irradiation in the absence of the photosensitizer, confirming that cytosine dimers are a major source of mutations . These results undermine the basis for the two-step model of UV mutagenesis in which a distinctly separate misincorporation step is supposed to precede the lesion bypass step; instead the results support a different two-step model, in which a deamination step precedes the bypass . The S13 capsid appears to completely inhibit the putative deamination reaction at about 75% of the dimer sites. Biochem J, 1991 May 1, 275 ( Pt 3), 711 - 9 Interaction of recA protein with left-handed Z-DNA; Krishna P et al.; The ability of recA protein to interact with a Z-DNA polymer, Br-poly(dG-dC), or M13 bacteriophage single-stranded DNA was investigated . RecA protein binds more avidly to Z-DNA than to single-stranded DNA in the absence of a nucleotide cofactor . This binding pattern changes in the presence of adenosine 5'-(gamma-thio)triphosphate (ATP{S}), however, such that the binding to Z-DNA decreases while binding to single-stranded DNA increases roughly 2-fold . When present together, the two forms of DNA compete with each other in the presence of ATP{S} . Experiments involving recA protein binding to recombinant plasmids showed neither a preferential binding of recA protein to the plasmid containing Z-DNA nor a similar effect of ATP{S} to that observed with the Z-DNA polymer . In contrast, maximal binding was obtained with a plasmid (linear or supercoiled) containing a polypurine.polypyrimidine insert, thus suggesting that recA protein displays sequence preferences in its interaction with DNA . The results of the present study provide no evidence that recA protein specifically interacts with or stabilizes the Z-DNA insert of a recombinant plasmid in the left-handed conformation. Carcinogenesis, 1991 May, 12(5), 819 - 24 Induction of mutations by N-acetoxy-N-acetyl-2-aminofluorene modified M13 viral DNA; Gupta PK et al.; The specificity of N-(deoxyguanosin-8-yl)-N-acetyl-2-aminofluorene (G-8-AAF) adducts in double-stranded DNAs from M13mp8 and M13mp9 bacteriophage was determined following transfection of modified DNA with multiple adducts into competent JM103 cells . Mutant phages were selected by phenotypic screening for colorless or light blue plaques indicating a defective beta-galactosidase marker enzyme . Mutation frequencies of phage DNA with G-8-AAF adducts were increased up to 8-fold in SOS-induced host cells as compared to the uninduced JM103 host cells . DNA sequencing of mutants from SOS-induced host cells indicated approximately 52% frameshifts and 39% base substitutions in M13mp8 DNA and 65% frameshifts and 25% base substitutions in M13mp9 DNA . Mutation spectra exhibited mutations at many sites within the bp 6200-6400 region; one mutational hotspot at position 6343-6347 (5' GGGGG 3') for frameshifts was also observed . The G-8-AAF adduct induced mostly single base deletions at this site . In contrast, a deacetylated adduct, N-(deoxyguanosin-8-yl)-2-aminofluorene (G-8-AF) in our previous experiments induced mostly single base additions at the same position indicating the ability of adduct structure to modulate the specificity of frameshift mutations . A number of other frameshift mutations (11 out of 29) were observed within non-repetitive and non-palindromic sequences . Molecular mechanisms for the induction of these mutations by DNA perturbations produced by the G-8-AAF adducts are discussed. Virology, 1991 May, 182(1), 388 - 92 Red clover necrotic mosaic virus infectious transcripts synthesized in vitro; Xiong ZG et al.; The red clover necrotic mosaic dianthovirus (RCNMV) genome is split between two essentially nonhomologous ssRNAs of 3.9 kb (RNA-1) and 1.45 kb (RNA-2) which are each capped at the 5' terminus with m7GpppA . cDNA clones short of full length by several nucleotides at both termini have been generated to both RNAs . Oligonucleotide-directed mutagenesis was employed to generate a series of RNA-1 and -2 transcription vectors in which the bacteriophage T7 RNA polymerase promoter was fused to full-length cDNA clones . Yields of in vitro transcripts initiating with wild-type viral 5'-terminal adenosine were extremely low . Efficient transcription was achieved only when one, or alternatively two, nonviral guanosines were engineered 5' to the authentic viral sequence at the transcription start site . m7GpppG-capped or -uncapped RCNMV RNA-1 and RNA-2 transcripts were infectious and induced symptoms identical to those of wild-type virus infection when coinoculated on the systemic hosts Nicotiana benthamiana and N . clevelandii, and on the local lesion host Chenopodium amaranticolor . Uncapped in vitro transcripts were somewhat less infectious . Progeny virus derived from infectious transcript inoculum was as infectious as wild-type virus . Primer extension analysis indicated that the 5'-terminal nonviral guanosine residues were not maintained in the progeny virus. Proc Natl Acad Sci U S A, 1991 May 1, 88(9), 4010 - 4 Protein-protein interactions with the acidic COOH terminus of the single-stranded DNA-binding protein of the bacteriophage T4; Krassa KB et al.; The single-stranded DNA-binding protein of the bacteriophage T4 is encoded by gene 32 . Monoclonal antibodies were raised against intact gene 32 protein (gp32) . We mapped the epitopes recognized by 12 of these monoclonal antibodies; the epitopes are all within the COOH-terminal region of gp32 . As shown by others, removal of the COOH terminus of gp32 abolishes the ability of the intact protein to bind to many T4 proteins involved in replication, recombination, repair, and late transcription . These results suggest that the COOH terminus of gp32 is a protein-binding domain . The COOH terminus is attached to a DNA-binding domain that includes a zinc finger . We propose a model in which the DNA-binding and protein-binding domains are used in T4 replication, recombination, repair, and late transcription . The COOH terminus of gp32 is very acidic and may form four negatively charged amphipathic alpha-helices, which could fold into a four-helix bundle when associated with other proteins . At least six of the monoclonal anti-gp32 antibodies bind to the COOH terminus of gp32 and to DNA . Similarities between the COOH terminus of gp32 and DNA are explored. Proc Natl Acad Sci U S A, 1991 May 1, 88(9), 4001 - 4 Construction and characterization of a single-chain catalytic antibody; Gibbs RA et al.; The antigen-binding (Fab) fragment of the catalytic monoclonal antibody NPN43C9 has recently been cloned by using bacteriophage lambda . By inserting the variable regions of this Fab coding sequence into a (NH2)-VL-linker-VH-(COOH) construct (where VL and VH represent the heavy and light chain variable regions), we have assembled a recombinant gene encoding a catalytic single-chain antigen-binding protein . This protein has been expressed in Escherichia coli and exhibits the same catalytic parameters as the parent monoclonal antibody NPN43C9 . Single-chain forms of catalytic antibodies may prove valuable for structural and site-directed mutagenesis studies as well as for large-scale applications of catalytic antibodies. J Bacteriol, 1991 May, 173(10), 3170 - 6 Subcloning, nucleotide sequence, and expression of trkG, a gene that encodes an integral membrane protein involved in potassium uptake via the Trk system of Escherichia coli; Schlosser A et al.; The trkG gene encodes a component of the K+ uptake system Trk and is located at 30.5 min inside the lambdoid prophage region rac of the Escherichia coli chromosome . trkG was subcloned, its nucleotide sequence was determined, and its product was identified in a minicell system . The open reading frame of 1,455 bp encodes a hydrophobic membrane protein with a calculated molecular weight of 53,493 that is predicted to contain up to 12 transmembrane helices . The trkG gene product behaved as a hydrophobic membrane protein; it was found exclusively in the membrane fraction of the minicells and its migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis was anomalous, indicating an apparent molecular weight of 35,000 . The trkG gene contains an exceptionally high proportion of infrequently used codons, raising the question of the origin of this gene . trkG does not appear to be a prophage gene since no similarity was observed between the nucleotide sequence of trkG or the amino acid sequence of its product and the sequences of genes or proteins from bacteriophage lambda. J Bacteriol, 1991 May, 173(9), 2897 - 905 Dual start motif in two lambdoid S genes unrelated to lambda S; Bonovich MT et al.; The lysis gene region of phage 21 contains three overlapping reading frames, designated S21, R21, and Rz21 on the basis of the analogy with the SRRz gene cluster of phage lambda . The 71-codon S21 gene complements lambda Sam7 for lysis function but shows no detectable homology with S lambda in the amino acid or nucleotide sequence . A highly related DNA sequence from the bacteriophage PA-2 was found by computer search of the GenBank data base . Correction of this sequence by insertion of a single base revealed another 71-codon reading frame, which is accordingly designated the SPA-2 gene and is 85% identical to S21 . There are thus two unrelated classes of S genes; class I, consisting of the homologous 107-codon S lambda and 108-codon P22 gene 13, and class II, consisting of the 71-codon S21 and SPA-2 genes . The codon sequence Met-Lys-(X)-Met...begins all four genes . The two Met codons in S lambda and 13 have been shown to serve as translational starts for distinct polypeptide products which have opposing functions: the shorter polypeptide serves as the lethal lysis effector, whereas the longer polypeptide acts as a lysis inhibitor . To test whether this same system exists in the class II S genes, the Met-I and Met-4 codons of S21 were altered in inducible plasmid clones and the resultant lysis profiles were monitored . Elimination of the Met-1 start results in increased toxicity, and lysis, although not complete, begins earlier, which suggests that both starts are used in the scheduling of lysis by S21 and is consistent with the idea that the 71- and 68-residue products act as a lysis inhibitor and a lysis effector, respectively . In addition, the R gene of 21 was shown to be related to P22 gene 19, which encodes a true lysozyme activity, and was also found to be nearly identical to PA-2 ORF2 . We infer that the 21 and PA-2 R genes both encode lysozymes in the T4 e gene family . These three genes form a second class lambdoid R genes, with the lambda R gene being the sole member of the first class . The existence of two interchangeable but unrelated classes of S genes and R genes is discussed in terms of a model of bacteriophage evolution in which the individual gene is the unit of evolution. Environ Health Perspect, 1991 May, 92, 75 - 81 A possible role for chromium(III) in genotoxicity; Snow ET; Chromium is found in the environment in two major forms: reduced CrIII and CrVI, or chromate . Chromate, the most biologically active species, is readily taken up by living cells and reduced intracellularly, via reactive intermediates, to stable CrIII species . CrIII, the most abundant form of chromium in the environment, does not readily cross cell membranes and is relatively inactive in vivo . However, intracellular CrIII can react slowly with both nucleic acids and proteins and can be genotoxic . We have investigated the genotoxicity of CrIII in vitro using a DNA replication assay and in vivo by CaCl2-mediated transfection of chromium-treated DNA into Escherichia coli . When DNA replication was measured on a CrIII-treated template using purified DNA polymerases (either bacterial or mammalian), both the rate of DNA replication and the amount of incorporation per polymerase binding event (processivity) were greatly increased relative to controls . When transfected into E . coli, CrIII-treated M13mp2 bacteriophage DNA showed a dose-dependent increase in mutation frequency . These results suggest that CrIII alters the interaction between the DNA template and the polymerase such that the binding strength of the DNA polymerase is increased and the fidelity of DNA replication is decreased . These interactions may contribute to the mutagenicity of chromium ions in vivo and suggest that CrIII can contribute to chromium-mediated carcinogenesis. Vaccine, 1991 May, 9(5), 319 - 25 Principles of selective inactivation of viral genome . V . Rational selection of conditions for inactivation of the viral suspension infectivity to a given extent by the action of beta-propiolactone; Budowsky EI et al.; The influence of the initial concentration of beta-propiolactone, the composition of the solution, temperature, and pH on the bacteriophage MS2 infectivity inactivation kinetics has been studied . Rate constants have been determined for the infectivity inactivation and for the change in the concentration (the consumption) of the reactant under inactivation conditions . These constants have been shown to permit a sufficiently precise description of the phage MS2 survival curves under the action of beta-propiolactone . These data have been used to put forward a kinetic approach for the rational determination of conditions for inactivation of the viral infectivity to a required extent with agents whose concentration decreases during inactivation as a result of hydrolysis and reactions involving the medium components. Virology, 1991 May, 182(1), 34 - 46 Morphopoietic switch mutations of bacteriophage P2; Six EW et al.; During the growth of bacteriophage P4, for which the genome of bacteriophage P2 is needed as helper, the decision whether to make large, P2 size, heads or small, P4 size, heads depends on the size-directing function of P4's sid gene and on P2's "sid responsiveness." P2 mutants (=P2 sir) impaired in their response to P4's sid function are readily obtainable as one class of P2 plaque formers selected on certain P4 cl plasmid lysogens . We describe nine P2 sir mutants of independent origin . For eight we could assign their sir mutation to P2 gene N, which encodes the major capsid protein . DNA sequencing indicated an open reading frame of 357 codons for gene N and showed these sir mutations to affect only four codons within a 38-codon segment in the middle of N . Seven mutations are missense mutations (three of them identical); one is a deletion of one codon . There seems to be a correlation between the phenotypic "strength" of the sir mutations and the type of amino acid replacement by missense mutations . Although the weakest mutation, sir7, could not yet be assigned to any P2 gene, it appears clear from this work that P2's N gene product is the major (or only) target of P4's Sid gene function. Mol Microbiol, 1991 May, 5(5), 1265 - 72 The role of the OOP antisense RNA in coliphage lambda development; Krinke L et al.; We have made a derivative of bacteriophage lambda that makes no OOP antisense RNA . The mutant phage carries a point mutation that inactivates the OOP promoter, po . The phages lambda + and lambda po- have identical plaque morphologies, one-step growth curves, and frequencies of lysogenization of a sensitive host . OOP RNA synthesis is weakly repressed by the Escherichia coli LexA protein . Consonant with this inducibility of OOP RNA synthesis by ultraviolet light, we find a two-fold greater phage burst following ultraviolet induction of a lambda + than of a lambda po- prophage . In lambda + infections, OOP RNA causes two cleavage events in cll mRNA: one is in the 3'-end of the coding region, and the second is in the intercistronic region between the cll and O genes . The cll gene fragments are subject to additional hydrolytic events, and cll mRNA levels are several-fold lower in lambda + than in lambda po- infections late in the infection cycle . However, O mRNA levels are almost unaffected by the po- mutation. Mutagenesis, 1991 May, 6(3), 207 - 11 Damage and mutagenesis of bacteriophage lambda induced by high pH; Musarrat J et al.; Bacteriophage lambda-Escherichia coli complexes exhibited remarkable sensitivity to alkaline pH 10.0 at 37 degrees C . The decline in plaque forming units after alkali treatment was more pronounced in complexes with some of the radiation repair defective mutants of E . coli K-12, i.e . uvrArecA, recA, rer and lexA mutants as compared to those of uvrA, recB and wild-type strains . The red gene of lambda phage and recA gene of E . coli seem to have a complementary effect on the alkali induced lesions . Alkaline treatment to lysogenic lambda phage was also found to be mutagenic . An enhanced level of mutagenesis was observed when treated phage particles were allowed to adsorb on treated wild-type bacteria . Moreover, the alkali treatment to lysogen (lambda cI857-E . coli) resulted in prophage induction in nutrient broth even at 32 degrees C . Thus on the basis of these results the role of error prone SOS repair systems in the repair of alkali induced lesions in lysogenic bacteriophage lambda has been suggested. Genetics, 1991 May, 128(1), 7 - 22 Heteroduplex chain polarity in recombination of phage lambda by the red, RecBCD, RecBC(D-) and RecF pathways; Siddiqi I et al.; We have examined the chain polarity of heteroduplex DNA in unreplicated, bacteriophage lambda splice recombinants when recombination was by the RecBCD, RecBC(D-), or RecF pathway of Escherichia coli or the Red pathway of lambda . For each of these pathways, recombination is activated by the cutting of cos that accompanies chromosome packaging, and is effected by recombination enzymes acting at the right end created by that cutting . For exchanges occurring near cos, one parent makes a lesser physical and genetic contribution than does the other . For each pathway, when the phage carried standard cos, this minority contribution was predominantly on the r chain, ending 5' at the right end of lambda . When standard cos was replaced by a cloned inverted cos located centrally on the standard lambda genetic map, minority contribution was predominantly on the l chain . In each case, the polarity of the overlap was usually that formed by 3' overhangs of parental information and material . These results are discussed in the context of current models of recombination for the different pathways. Mol Gen Genet, 1991 May, 227(1), 1 - 8 Specificity of recognition sequence for Escherichia coli primase; Yoda K et al.; We have surveyed the frequency of each of 64 trinucleotide permutations at every nucleotide frame located from 1 to 15 nucleotides upstream of primer RNA-DNA transition sites mapped within a 1.5 kb region of the bacteriophage lambda genome and a 1.4 kb region of the Escherichia coli genome . We have demonstrated that in both systems initiation of DNA synthesis strongly correlates with a CAG sequence located 11 nucleotides upstream of the DNA start sites . Based on the examination of various reports of the priming reaction catalyzed by E . coli primase in vivo and in vitro, we propose that (i) E . coli primase itself recognizes a 3'GTC 5' sequence on the template strand, (ii) DnaB helicase releases the specificity of E . coli primase and, (iii) the consensus recognition sequence for E . coli primase associated with DnaB helicase is 3'PuPyPy 5'. Virology, 1991 May, 182(1), 351 - 2 Isolation and preliminary characterization of Escherichia coli mutants resistant to lethal action of the bacteriophage lambda P gene; Maiti S et al.; Both spontaneous and NTG-induced mutants of Escherichia coli 594 insensitive to the lethal action of lambda P gene were isolated and called rpl (resistant to P lethality) . These mutants were of two types, showing different phenotypes . On type I rpl mutants, lambda cl- and lambda v1v3 did not plate, while lambda vir, lambda cl- c17, lambda imm434, and lambda imm21 did; plasmid pMR45 carrying the lambda P gene could not complement lambda imm21P- phage in type I mutants . On the other hand, the type II rpl mutants support the growth of all the above phages including lambda cl- . Neither type of rpl mutation affects growth of the bacteria. Virology, 1991 May, 182(1), 324 - 35 Bacteriophage lambda P gene shows host killing which is not dependent on lambda DNA replication; Maiti S et al.; Bacteriophage lambda, having a mutation replacing glycine by glutamic acid at the 48th codon of cro, kills the host under N- conditions; we call this the hk mutation . In lambda N-N-cl-hk phage-infected bacteria, the late gene R is expressed to a significant level, phage DNA synthesis occurs with better efficiency, and the Cro activity is around 20% less, all compared to those in lambda N-N-cl-hk(+)-infected bacteria . Segments of lambda DNA from the left of pR to the right of tR2, carrying cro, cII, O, P, and the genes of the nin5 region from the above hk and hk+ phages, were cloned in pBR322 . Studies with these plasmids and their derivatives having one or more of the lambda genes deleted indicate that the hk mutation is lethal only when a functional P gene is also present . When expression of P from pR is elevated, due to the deletion of tR1, host killing also occurs without the hk mutation . We conclude that the higher levels of P protein, produced either (1) when cro has the hk mutation or (2) when tR1 is deleted, are lethal to the host . We also show that due to the hk mutation, the Cro protein becomes partially defective in its negative regulation at pR, resulting in the expression of P to a lethal level even in the absence of N protein-mediated antitermination . This P protein-induced host killing depends neither on lambda DNA replication nor on any other gene functions of the phage. Virology, 1991 May, 182(1), 316 - 23 Bacteriophage P22 accessory recombination function; Poteete AR et al.; The accessory recombination function (arf) gene of bacteriophage P22 is located immediately upstream of the essential recombination function (erf) gene . Three mutant alleles of arf were constructed and installed in P22 in place of the wild-type allele: an out-of-frame internal deletion, an in-frame internal deletion, and an amber mutation . The deletion mutant phages are partially defective in homologous recombination and plaque formation in wild-type and recA hosts; their defects are more severe in recB and recA recB hosts . The amber mutant phage exhibits the same growth phenotypes in nonsuppressing hosts, but not in an amber-suppressor host . Plasmids that express arf complement the growth defect of arf- phages . These plasmids stimulate erf-mediated recombination; they were also found to cause a small stimulation of recA-recBCD-mediated homologous recombination of phage lambda. J Bacteriol, 1991 May, 173(9), 2733 - 8 Interference with phage lambda development by the small subunit of the phage 21 terminase, gp1; Johnson G et al.; Bacteriophage lambda development is blocked in cells carrying a plasmid that expresses the terminase genes of phage 21 . The interference is caused by the small subunit of phage 21 terminase, gp1 . Mutants of lambda able to form plaques in the presence of gp1 include sti mutants . One such mutation, sti30, is an A . T-to-G.C transition mutation at base pair 184 on the lambda chromosome . The sti30 mutation extends the length of the ribosome-binding sequence of the Nul gene that is complementary to the 3' end of the 16S rRNA from GGA to GGAG . The sti30 mutation causes a approximately 50-fold increase in the level of expression of a Nul-lacZ reporter gene, indicating that the sti30 mutation overcomes the gp1 inhibition by increasing the level of expression of gpNul . Although the Nul and A genes of lambda overlap, the sti30 mutation has little effect on the level of gpA expression, indicating that translational coupling does not occur. Gene Expr, 1991 May, 1(2), 127 - 36 Analysis of bacteriophage T7 gene 10A and frameshifted 10B proteins; Sipley J et al.; Bacteriophage T7 capsid protein 10B has previously been proposed to arise by a translational frameshift near the 3' end of the capsid gene 10A coding sequence, adding an additional 53 amino acid residues to the carboxyl-terminal end of the protein . Here we show by peptide mapping experiments as well as by direct partial sequence analysis of an overlapping "junction" peptide, that 10B is in fact related to 10A by a -1 switch in reading frame in a narrow region near the carboxy terminus of 10A . Peptide mapping experiments demonstrate that 10A and 10B have the same amino terminus as well as virtually identical methionine-labeled peptide maps . However, the predicted unique carboxyl-terminal peptide from 10B was also identified . An overlapping peptide was isolated from 10B which spans the junction region in which the proposed translational frameshift is thought to occur . Partial sequencing of this junction peptide confirms a -1 frameshift within the last few codons of 10A. Mutat Res, 1991 May, 254(3), 207 - 15 Partial complementation of the UV sensitivity of Deinococcus radiodurans excision repair mutants by the cloned denV ge