Bacteriophage

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 gene of bacteriophage T4; Gutman PD et al.; Deinococcus radiodurans has 2 endonucleases that incise UV-irradiated DNA . UV endonuclease-alpha and UV endonuclease-beta, that are believed to functionally overlap . Both endonucleases must be mutationally inactivated to yield an incisionless, markedly UV-sensitive phenotype . denV, the bacteriophage T4 gene encoding pyrimidine dimer-DNA glycosylase (PD-glycosylase), was introduced and expressed via duplication insertion in D . radiodurans wild-type, and single and double UV endonuclease mutants . The strain deficient in UV endonuclease-alpha has wild-type UV resistance, and the expression of PD-glycosylase exerted no survival effect on this strain or wild-type . Expression of denV increased survival of both the markedly UV-sensitive double mutant and the moderately UV-sensitive strain deficient only in UV endonuclease-beta . In endonuclease-beta-deficient cells phenotypic complementation by denV was almost complete in restoring UV resistance to wild-type levels . These results suggest that UV endonuclease-alpha (which is present in the endonuclease-beta-deficient cells) does not recognize one or more types of cyclobutane dimer incised by the PD-glycosylase or UV endonuclease-beta.

Lab Invest, 1991 May, 64(5), 709 - 12
Transcription of cRNA for in situ hybridization from polymerase chain reaction-amplified DNA; Young ID et al.; The synthesis of cRNA probes for in situ hybridization is usually accomplished with transcription systems using cloning vectors that contain bacteriophage RNA polymerase promoters . In this report we describe an alternative technique for generating cRNA probes that obviates the need for cDNA cloning by using the polymerase chain reaction . The segment of DNA corresponding to the desired RNA sequence was amplified from genomic DNA by the polymerase chain reaction . The bacteriophage SP6 and T7 RNA polymerase promoters were incorporated into the amplified DNA by including the promoter sequences in the 5' termini of the oligonucleotides used to prime the polymerase chain reaction . The promoters permitted the subsequent transcription of sense and antisense cRNA from the amplified DNA . The antisense cRNA was radiolabeled to high specific activity for use as a probe in Northern and mRNA in situ hybridization analyses . This technique offers the advantages of speed and simplicity over conventional transcription systems, which require cDNA cloning . Bypassing the need for cloning in the synthesis of cRNA probes may facilitate the use of these probes in research and diagnostic applications of mRNA in situ hybridization.

FASEB J, 1991 May, 5(8), 2209 - 16
Expression of ion channels and receptors in Xenopus oocytes using vaccinia virus; Yang XC et al.; The cytoplasmic injection of mRNA synthesized in vitro into Xenopus oocytes is widely used for heterologous expression of ion channels and neurotransmitter receptors . We report two new methods for expression of ion channels and receptors in oocytes using vaccinia virus (VV) . 1) A recombinant VV carrying the Shaker H4 K+ channel cDNA driven by the VV P7.5 early promoter was injected into oocytes . 2) A recombinant VV containing the bacteriophage T7 RNA polymerase driven by the P7.5 promoter was coinjected along with plasmids containing a T7 promoter and cDNAs for channels and receptors . The functionally expressed proteins include a) voltage-gated ion channels: the Shaker H4 K+ channel and the rat brain IIA Na+ channel, b) a ligand-gated ion channel: the mouse muscle nicotinic acetylcholine receptor (AChR), and c) a G protein-coupled receptor: the rat brain 5HT1C receptor . After virus/cDNA injection into oocytes, these channels and receptors generally showed characteristics and expression levels similar to those observed in mRNA-injected oocytes . However, the AChR expressed at lower levels in virus/cDNA-injected oocytes than in mRNA-injected oocytes . Because our methods bypass mRNA synthesis, they are more rapid and convenient than the mRNA injection method . Potential applications to structure-function studies and expression cloning are discussed.

Mol Microbiol, 1991 May, 5(5), 1005 - 11
The tol gene products and the import of macromolecules into Escherichia coli; Webster RE; Genetic studies have identified a number of genes whose products appear to be required for the transport of the group A colicins and the single-stranded DNA of certain filamentous bacteriophages into Escherichia coli . Mutations in these genes allow normal binding of the colicins to their outer-membrane receptors and of the bacteriophage of the tip of specific conjugative pili, but do not allow translocation of the macromolecules to their target . These mutations have been designed 'tolerant' (tol) mutations and the protein products specified by these genes appear to comprise part of a transport system known as the Tol import system . Some of these genes have been isolated, sequenced and their protein products localized to the membranes or periplasm of E . coli . Information is also available regarding the domains of the colicins or phage proteins which interact with the Tol proteins . A preliminary model of the location and possible interactions of the Tol proteins is presented.

Nucleic Acids Res, 1991 Apr 25, 19 Suppl, 2023 - 43
Compilation of DNA sequences of Escherichia coli (update 1991); Kroger M et al.; We have compiled the DNA sequence data for E . coli available from the GENBANK and EMBL data libraries and over a period of several years independently from the literature . This is the third listing replacing and increasing the former listing roughly by one fifth . However, in order to save space this printed version contains DNA sequence information only . The complete compilation is now available in machine readable form from the EMBL data library (ECD release 6) . After deletion of all detected overlaps a total of 1 492,282 individual bp is found to be determined till the beginning of 1991 . This corresponds to a total of 31.62% of the entire E . coli chromosome consisting of about 4,720 kbp . This number may actually be higher by some extra 2.5% derived from lysogenic bacteriophage lambda and various DNA sequences already received for statistical purposes only.

Nucleic Acids Res, 1991 Apr 25, 19(8), 1759 - 66
A conserved sequence element in ribonuclease III processing signals is not required for accurate in vitro enzymatic cleavage; Chelladurai BS et al.; Ribonuclease III of Escherichia coli is prominently involved in the endoribonucleolytic processing of cell and viral-encoded RNAs . Towards the goal of defining the RNA sequence and structural elements that establish specific catalytic cleavage of RNase III processing signals, this report demonstrates that a 60 nucleotide RNA (R1.1 RNA) containing the bacteriophage T7 R1.1 RNase III processing signal, can be generated by in vitro enzymatic transcription of a synthetic deoxyoligonucleotide and accurately cleaved in vitro by RNase III . Several R1.1 RNA sequence variants were prepared to contain point mutations in the internal loop which, on the basis of a hypothetical 'dsRNA mimicry' structural model of RNase III processing signals, would be predicted to inhibit cleavage by disrupting essential tertiary RNA-RNA interactions . These R1.1 sequence variants are accurately and efficiently cleaved in vitro by RNase III, indicating that the dsRNA mimicry structure, if it does exist, is not important for substrate reactivity . Also, we tested the functional importance of the strongly conserved CUU/GAA base-pair sequence by constructing R1.1 sequence variants containing base-pair changes within this element . These R1.1 variants are accurately cleaved at rates comparable to wild-type R1.1 RNA, indicating the nonessentiality of this conserved sequence element in establishing in vitro processing reactivity and selectivity.

J Mol Biol, 1991 Apr 20, 218(4), 779 - 89
Sequence requirements for protein-primed DNA replication of bacteriophage PRD1; Yoo SK et al.; In vitro studies have demonstrated that linear duplex, protein-free DNA molecules containing an inverted terminal repeat (ITR) sequence of the PRD1 genome at one end can undergo replication by a protein-primed mechanism . No DNA replication was observed when the ITR sequence was deleted or was not exposed at the terminus of the template DNA . We have determined the minimal origin of replication by analyzing the template activity of various deletion derivatives . Our results showed that the terminal 20 base-pairs of ITR are required for efficient in vitro DNA replication . We have found that, within the minimal replication origin region, there are complementary sequences . A site-specific mutagenesis analysis showed that most of the point mutations in the complementary sequences markedly reduced the template activity . The analyses of the results obtained with synthetic oligonucleotides have revealed that the specificity of the replication origin is strand specific and even on a single-stranded template a particular DNA sequence including a 3'-terminal C residue is required for the initiation of PRD1 DNA replication in vitro.

J Mol Biol, 1991 Apr 20, 218(4), 705 - 21
Reiterated gene amplifications at specific short homology sequences in phage T4 produce Hp17 mutants; Wu DG et al.; Bacteriophage T4 gene 17 amplification mutants (Hp17) selected by growth of gene 17 amber mutants on ochre suppressor strains of Escherichia coli carry two to more than sixfold tandem head-to-tail repeats of the gene 17-18 region (Wu & Black, 1987) . We characterized the structures of Hp17 isolates by restriction enzyme mapping and Southern blot analysis . The left and right boundaries of the amplified sequences were mapped within genes 16 and gene 18 or 19, respectively . The TaqI-restriction fragments containing the novel junctions arising from fusion of the amplified gene were then cloned and sequenced . Three Hp17 mutants arose from rearrangement in one five base-pair (bp) block within a G + C-rich region of partial homology (24 bp with 4 mismatches) between genes 16 and 19 . Moreover, an oligonucleotide probe showed that 190/191 mutants isolated had recombined within the 5 bp block, and other rearrangements within this 24 bp region were not detected . Only one anomalous Hp mutant rearranged elsewhere between genes 16 and 18 in a 14 bp homology region with one mismatch . Elimination of gene alt of phage T4 is required for isolation of Hp17 mutants, apparently because more DNA can be packaged into alt- heads . Requirements for the dispensable replication and recombination genes of T4 were probed; T4 topoisomerase (39, 52, 60), primase (58/61), and uvsX are required, whereas the host recA gene and T4 denV gene do not appear to be required for isolation of the Hp17 mutants . The evidence suggests an initiating sequence-specific rearrangement leads to the T4 Hp17 amplification mutants.

J Mol Biol, 1991 Apr 20, 218(4), 723 - 33
Functional and structural elements of the mRNA of the cIII gene of bacteriophage lambda; Altuvia S et al.; The bacteriophage lambda cIII gene product is an early regulatory protein that participates in the lysis-lysogeny decision of the phage following infection . We have previously shown that the translation of the cIII gene is determined by two unique factors: (1) efficient expression is dependent upon the presence of RNaseIII in the cell; (2) alternative mRNA structures of the cIII coding region determine the rate of its translation initiation . In this study we demonstrate the presence of the alternative mRNA structures in vivo . The presence of minor RNaseIII cleavage sites within this region indicate that RNaseIII can differentiate between the two alternative structures . We localize by a deletion analysis the RNaseIII responsive element to the cIII coding region, and suggest that regulation of cIII translation by RNaseIII is achieved through binding to the alternative structures region of the mRNA.

Cell, 1991 Apr 19, 65(2), 259 - 69
Translational stimulation: RNA sequence and structure requirements for binding of Com protein; Wulczyn FG et al.; Translation of the bacteriophage Mu mom gene is positively regulated by the phage Com protein . We report here that purified Com protein specifically stimulates mom gene expression in vitro . Furthermore, Com is shown to bind a site in the mom translational initiation region (TIR) in a sequence-specific manner . In vitro RNA footprint experiments have been used to define the Com-binding site and to study mRNA secondary structure in the mom TIR . Com binding is shown to correlate with a conformational change in the mom TIR both in vivo and in vitro . The role of secondary structure was further examined by testing the effects of mutations in the TIR on translation and stimulation . The results support a model for translational stimulation in which Com binding induces a conformational change in the mom mRNA, thereby enhancing ribosome binding.

Nucleic Acids Res, 1991 Apr 11, 19(7), 1627 - 32
The molecular mechanism of inhibition of alpha-type DNA polymerases by N2-(butylphenyl)dGTP and 2-(butylanilino)dATP: variation in susceptibility to polymerization; Khan NN et al.; Calf thymus DNA polymerase alpha (pol alpha) and bacteriophage T4 DNA polymerase (pol T4) were exploited as model enzymes to investigate the molecular mechanism of inhibitory action of N2-(p-n-butylphenyl)dGTP (BuPdGTP) and 2-(p-n-butyl-anilino)dATP (BuAdATP) on the BuPdNTP-susceptible alpha polymerase family . Kinetic analysis of inhibition of pol alpha with mixtures of complementary and noncomplementary template:primers indicated that both nucleotides induced the formation of a polymerase: inhibitor:primer-template complex . Primer extension experiments using the guanine form as the model analog indicated that pol alpha cannot utilize these nucleotides to extend primer termini . In contrast, pol T4 polymerized BuPdGTP, indicating that resistance to polymerization is not a common feature of the inhibitor mechanism among the broad membership of the alpha polymerase family.

J Mol Biol, 1991 Apr 5, 218(3), 569 - 81
Generation of infectious nucleocapsids by in vitro assembly of the shell protein on to the polymerase complex of the dsRNA bacteriophage phi 6; Olkkonen VM et al.; A method for the in vitro uncoating of the phi 6 nucleocapsid (NC) was developed . The resulting particle, designated as the NC core, containing the genomic double-stranded (ds) RNA segments and the proteins P1, P2, P4 and P7, was not infectious but had a highly enhanced in vitro transcriptase activity compared to that of the intact NC . The NC shell protein P8 was purified by immunoaffinity chromatography, and it was shown to self-assemble to shell-like structures upon addition of calcium ions . The conditions for the self-assembly of the shell were optimized . Shell reassembly on to the NC cores restored the infectivity but resulted in a decrease of transcriptase activity . No reassembly of the shell on to RNA-less cores (procapsids) produced from a cDNA construction in Escherichia coli was observed . Our results suggest that the intracellular uncoating of the NC is the event activating the phi 6 dsRNA transcriptase and that the NC shell is necessary for infectivity, probably for the passage of the NC through the host cytoplasmic membrane . Packaging of the dsRNA segments into the procapsid appears to be a prerequisite for NC shell assembly.

J Biol Chem, 1991 Apr 5, 266(10), 6159 - 67
Steady-state kinetic analysis of ATP hydrolysis by the B protein of bacteriophage mu . Involvement of protein oligomerization in the ATPase cycle; Adzuma K et al.; The DNA strand-transfer reaction of bacteriophage Mu requires Mu B protein and ATP for high efficiency . These factors facilitate the capture of target DNA by the donor protein-DNA complex . To understand the mechanism of the Mu B ATPase cycle in the Mu DNA strand-transfer reaction, we undertook a steady-state kinetic analysis of Mu B ATPase . The results reveal complex properties of the ATPase activity; Mu B protein oligomerizes in the presence of ATP, and ATP hydrolysis by the Mu B ATPase is stimulated by the protein oligomerization and shows a positive cooperativity with respect to ATP concentration . Mu B ATPase activity is also modulated by DNA and Mu A protein . DNA alone suppresses the catalytic activity of Mu B ATPase, whereas DNA enhances the apparent binding affinity for ATP . In the presence of Mu A protein together with DNA, however, the catalytic activity is greatly stimulated . Based on these results, we propose a working hypothesis in which oligomerization of Mu B protein plays a key role in its ATPase cycle.

Appl Environ Microbiol, 1991 Apr, 57(4), 1218 - 22
Development and application of new positively charged filters for recovery of bacteriophages from water; Borrego JJ et al.; Electronegative and electropositive filters were compared for the recovery of indigenous bacteriophages from water samples, using the VIRADEL technique . Fiber glass and diatomaceous earth filters displayed low adsorption and recovery, but an important increase of the adsorption percentage was observed when the filters were treated with cationic polymers (about 99% adsorption) . A new methodology of virus elution was developed in this study, consisting of the slow passage of the eluent through the filter, thus increasing the contact time between eluent and virus adsorbed on the filters . The use of this technique allows a maximum recovery of 71.2% compared with 46.7% phage recovery obtained by the standard elution procedure . High percentages (over 83%) of phage adsorption were obtained with different filters from 1-liter aliquots of the samples, except for Virosorb 1-MDS filters (between 1.6 and 32% phage adsorption) . Phage recovery by using the slow passing of the eluent depended on the filter type, with recovery ranging between 1.6% for Virosorb 1-MDS filters treated with polyethyleneimine and 103.2% for diatomaceous earth filters treated with 0.1% Nalco.

Gene, 1991 Apr, 100, 195 - 9
Construction of versatile low-copy-number vectors for cloning, sequencing and gene expression in Escherichia coli; Wang RF et al.; Using the polymerase chain reaction and standard recombinant DNA techniques, a series of new multipurpose low-copy-number (lcn) vectors, pWSK29, pWKS30, pWKS129 and pWKS130, have been constructed . Plasmids pWSK29 and pWKS30 carry the ampicillin-resistance marker (ApR), 20 unique cloning sites flanked by T7 and T3 RNA polymerase promoters, the lacZ alpha gene and the bacteriophage f1 origin of replication (ori) for production of single-stranded (ss) DNA in the presence of a helper phage . Plasmids pWSK129 and pWKS130 carry the kanamycin-resistance marker (KmR) and have 16 unique cloning sites flanked by T7 and T3 RNA polymerase promoters positioned within the lacZ alpha gene . Plasmids pWSK129 and pWKS130 also contain the f1 ori for the generation of ss DNA in the presence of a helper phage . All of the plasmids have an lcn of six to eight per cell . Each vector can be used for: (i) complementation analysis, (ii) generating unidirectional deletions with exonuclease III/S1 nuclease, (iii) DNA sequencing, (iv) high-level gene expression using T7 RNA polymerase, and (v) run-off transcription . They are very useful for analyzing genes encoding proteins which are toxic in Escherichia coli in high copy number.

Mol Gen Genet, 1991 Apr, 226(1-2), 65 - 9
Primer protein of bacteriophage M2 exposes the RGD receptor site upon linking the first deoxynucleotide; Kobayashi H et al.; Primer protein (PP) of bacteriophages M2 and phi 29 contains an Arg-Gly-Asp (RGD) sequence . The RGD-mediated protein-protein interaction in protein-primed DNA replication of M2 was studied in vitro using three purified and indispensable components: PP, DNA polymerase (POL) and template DNA linked to terminal protein (TP) . PP competed with a synthetic RGD peptide for binding to the template DNA-TP complex (TP-DNA) . In addition, POL bound to template TP-DNA only when complexed with PP . These results indicate that the RGD sequence of PP is responsible for the interaction of the PP-POL complex with TP-DNA, which contains the initiation site for the protein priming of DNA synthesis . At the moment when PP converts to TP upon linking the first deoxynucleotide, a conformational change results in exposure of the RGD binding site.

Genetics, 1991 Apr, 127(4), 637 - 47
Fine structure genetic and physical map of the gene 3 to 10 region of the bacteriophage P22 chromosome; Casjens S et al.; The mechanism by which dsDNA is packaged by viruses is not yet understood in any system . Bacteriophage P22 has been a productive system in which to study the molecular genetics of virus particle assembly and DNA packaging . Only five phage encoded proteins, the products of genes 3, 2, 1, 8 and 5, are required for packaging the virus chromosome inside the coat protein shell . We report here the construction of a detailed genetic and physical map of these genes, the neighboring gene 4 and a portion of gene 10, in which 289 conditional lethal amber, opal, temperature sensitive and cold sensitive mutations are mapped into 44 small (several hundred base pair) intervals of known sequence . Knowledge of missense mutant phenotypes and information on the location of these mutations allows us to begin the assignment of partial protein functions to portions of these genes . The map and mapping strains will be of use in the further genetic dissection of the P22 DNA packaging and prohead assembly processes.

Proc Natl Acad Sci U S A, 1991 Apr 1, 88(7), 2859 - 63
Prediction of the thermodynamics of protein unfolding: the helix-coil transition of poly(L-alanine); Ooi T et al.; The method given earlier for predicting the thermodynamics of protein unfolding from the x-ray structure of a protein is applied here to the poly(L-alanine) helix . First, the fitting parameters derived earlier from a data base of 10 proteins were used to predict the unfolding thermodynamics of 4 other proteins . The agreement between the observed and predicted values is comparable to that found for the 10 proteins studied initially . Next, the temperature dependences of the Gibbs energy and enthalpy changes for unfolding of bacteriophage T4 lysozyme were predicted and compared with data in the literature . The predicted and observed temperature dependences are similar and the predicted results indicate that cold denaturation should be observed at low temperatures, as observed recently for a T4 lysozyme mutant . The fitting parameters derived from thermodynamic data for protein unfolding and for hydration of model compounds were used to predict the unfolding thermodynamics of the poly(L-alanine) helix . The results predict that helix formation is enthalpy-driven, and the predicted enthalpy change for unfolding (0.86 kcal per mol per residue) is close to the value found in a recent calorimetric study of a 50-residue alanine-rich helix.

Biotechniques . 1991 Apr;10(4):446, 448, 450.
Using sodium chloride step gradients to fractionate DNA fragments; Fink PS; A method is described for the separation of DNA fragments by ultracentrifugation through a sodium chloride step gradient . The gradients are quickly and easily prepared and require a five- to six-hour centrifugation . Fractionated samples of DNA may be directly examined by agarose gel electrophoresis, then further analyzed by Southern transfer and hybridization . A simple ethanol precipitation followed by several ethanol washes yields fragments that ligate efficiently to vector DNA . The method has been applied to separate chromosomal DNA restriction fragments as well as bacteriophage lambda arms from insert DNA.

Mol Microbiol, 1991 Apr, 5(4), 901 - 7
Regulation of the chlA locus of Escherichia coli K12: involvement of molybdenum cofactor; Baker KP et al.; The chlA locus encodes functions required for the biosynthesis of the molybdopterin part of the molybdenum cofactor . Mutants, carrying gene fusions at the chlA locus, which place beta-galactosidase expression under the control of the chlA promoter, have been isolated employing lambda placMu1 as the mutagen . The mutants exhibited beta-galactosidase expression which was greatly enhanced when grown anaerobically . Secondary mutations at the chlB, D, E or G loci did not affect the high level of expression . The fnr gene product was not required for the anaerobic expression . Bacteriophage lambda transducing phages were isolated which carried the phi(chlA-lac) mutations and were used to construct chlA+/phi(clA-lac) merodiploids . The merodiploids exhibited a much lower level of expression but showed the same characteristics as strains carrying lac fusions to the single chromosomal chlA locus . Genetic evidence is presented which strongly suggests that the molybdenum cofactor is a repressor of chlA expression . The anaerobic enhancement of chlA expression is mediated via a mechanism that is distinct from the molybdenum cofactor effect.

J Biomol Struct Dyn, 1991 Apr, 8(5), 953 - 65
The wac gene product of bacteriophage T4 contains coiled-coil structural patterns; Sobolev BN et al.; The bacteriophage T4 late gene wac (whisker antigen control) encodes the protein which forms the fibrous structure on the neck of the virion called whiskers . Amino acid sequence analysis of wac gene product, as deduced from the nucleotide sequence, indicate ten alpha-helical domains (19-40 residues long) with coiled-coil structural patterns . These regions comprise about 70% of the entire 486 amino acid sequence . The alpha-helices are separated by short stretches of polypeptide chain which are similar to the loop regions of the globular protein sequences . We propose a structural model for the dimer of wac gene product molecule, that we call fibritin in which two polypeptide chains associate in a parallel fashion and form a segmented alpha-helical coiled-coil rod similar to epidermal keratins.

Biotechniques, 1991 Apr, 10(4), 520 - 5
A new cationic liposome reagent mediating nearly quantitative transfection of animal cells; Rose JK et al.; One of the most efficient systems for the expression of genes in the cytoplasm of animal cells utilizes a recombinant vaccinia virus encoding the bacteriophage T7 RNA polymerase . Cells infected with this virus are transfected with plasmid DNAs containing the gene to be expressed under T7 promoter control . The major limitation of this system is the efficiency with which DNA is introduced into the cell . Recently, a cationic liposome-mediated transfection reagent has yielded transfection frequencies of greater than 80% . To determine if commercially available cationic lipids could form liposomes that would yield similar transfection efficiencies, we tested liposomes prepared with five different cationic lipids . When used at appropriate concentrations in liposomes that also contained a neutral lipid, four of the five cationic lipids were effective in the transfection of HeLa cells . However, liposomes formed with the neutral lipid and one of the cationic lipids, dimethyldioctadecylammonium bromide (DDAB), gave transfection frequencies of greater than 95% and had a broad spectrum of effectiveness on a variety of cell lines . Liposomes containing DDAB are an inexpensive, highly efficient and reproducible alternative for the transfection of animal cells and are well suited for use with the vaccinia virus/T7 expression system.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1991 Apr, 13(2), 79 - 84
{A study of protein engineering for human cardionatrin . II . Construction of two new human cardionatrin analog genes, RH-2 & RH-3 by intragenic recombination in vitro}; Ren H; Two new analog genes of the alpha-human atrial natriuretic polypeptide (alpha-hANP) were constructed by accurate intragenic recombination of analog RH-1 gene with ANP gene in vitro, both of which had been synthesized and reported . One recombined analog gene, RH-2, was designed to contain an equivalent of the alpha-hANP gene, capping the peptidase inhibitor SQ20881 sequence at its 5'end . The other analog gene, RH-3, was designed to contain an equivalent of the alpha-hANP gene, wedging in two proline codons CCG & CCG at the 3' end . The recombinants bearing the genes, bacteriophage RH-2/M13mp18 & RH-3/M13mp18, were identified by DNA domain combination analysis--in situ hybridization and dot blotting with three probes matching three specific DNA domains, and by restriction analysis . The DNA sequences of RH-2 and RH-3 were confirmed by the dideoxynucleotide chain termination method.

Mol Gen Genet, 1991 Apr, 226(1-2), 113 - 9
Heat-inducible reactivation of UV-damaged bacteriophage lambda; Calsou P et al.; Induction of the SOS response in UV-irradiated bacteria leads to an increase in the survival of an infecting irradiated bacteriophage lambda (Weigle 1953) . We report that a similar reactivation of irradiated phage lambda was induced by shifting the culture of recipient bacteria from 30 degrees to 47 degrees C . However, this repair process was nonmutagenic . The amplitude of the phenomenon was increased with the quantity of UV lesions in the phage DNA . It was present despite mutations affecting the SOS response or the heat shock response in the infected strains (recA, lexA, umuC or rpoH mutations respectively) . In contrast, the heat-inducible repair process was abolished in uvrA derivatives . Also, pretreatment with chloramphenicol largely enhanced phage reactivation after heat shock . Therefore, it appears that the excision repair mechanism of UV lesions was stimulated both by temperature shift-up and blockage of protein synthesis.

Virology, 1991 Apr, 181(2), 778 - 82
Vaccinia virus-mediated expression of African swine fever virus genes; Hammond JM et al.; Bacteriophage lambda and plasmid clones containing African swine fever virus (ASFV) DNA inserts, which together covered more than 90% of the genome of a Malawi ASFV isolate (LIL 20/1), were transfected into vaccinia virus (VV)-infected cells . Expression of ASFV-encoded proteins was assayed at late times after VV infection by immunoprecipitation of {35S}methionine-labeled proteins with hyperimmune serum from ASFV-infected pigs, separation of immunoprecipitated proteins by denaturing polyacrylamide gel electrophoresis, and detection by autoradiography . Synthesis of eight additional proteins not observed in control experiments was detected . Seven VV recombinants were constructed, each containing an ASFV DNA insert from a separate bacteriophage lambda clone ranging in size from 9 to 15 kb . BSC40 cells were infected with recombinant viruses and expression of ASFV-encoded proteins assayed at early and late times postinfection . Synthesis of additional proteins, not observed in control experiments, was detected by immunoprecipitation with ASFV antiserum both early and late postinfection with two of these recombinants . In these experiments VV promoters were not included upstream of individual ASFV genes.

J Allergy Clin Immunol, 1991 Apr, 87(4), 803 - 11
Antibody responses to protein, polysaccharide, and phi X174 antigens in the hyperimmunoglobulinemia E (hyper-IgE) syndrome; Sheerin KA et al.; To investigate whether an underlying defect in antibody (Ab)-forming capacity could contribute to the infection susceptibility of patients with hyper-IgE syndrome, we evaluated 11 such patients for their responses to bacteriophage phi X174 (phi X174), diphtheria and tetanus toxoids, and pneumococcal (Pneumovax) and Hemophilus influenzae vaccines . Three of nine patients immunized with phi X174 had normal primary and secondary Ab responses, five had accelerated declines in their titers after initially normal primary Ab responses and lower than normal secondary Ab responses, and two of the latter patients failed to switch normally from IgM to IgG Ab production . Only one of 10 patients tested had normal Ab responses to diphtheria toxoid, and postimmunization antitetanus titers were abnormally low in five of the 10 patients tested . Serum Abs to H . influenzae polyribose phosphate were protective in seven of the eight immunized patients . Five of the nine patients administered Pneumovax had poor Ab responses to at least one of the pneumococcal serotypes 7, 9, or 14 . Abnormal antipolysaccharide responses did not correlate with IgG2 deficiency . All patients responded with protective Ab levels to type 3 . Thus, patients with hyper-IgE syndrome are heterogeneous with respect to their Ab-forming capacities . Ab deficiency may contribute to infection susceptibility in some of these patients.

Proc Natl Acad Sci U S A, 1991 Apr 1, 88(7), 2874 - 8
Escherichia coli DnaJ and GrpE heat shock proteins jointly stimulate ATPase activity of DnaK; Liberek K et al.; The products of the Escherichia coli dnaK, dnaJ, and grpE heat shock genes have been previously shown to be essential for bacteriophage lambda DNA replication at all temperatures and for bacterial survival under certain conditions . DnaK, the bacterial heat shock protein hsp70 analogue and putative chaperonin, possesses a weak ATPase activity . Previous work has shown that ATP hydrolysis allows the release of various polypeptides complexed with DnaK . Here we demonstrate that the ATPase activity of DnaK can be greatly stimulated, up to 50-fold, in the simultaneous presence of the DnaJ and GrpE heat shock proteins . The presence of either DnaJ or GrpE alone results in a slight stimulation of the ATPase activity of DnaK . The action of the DnaJ and GrpE proteins may be sequential, since the presence of DnaJ alone leads to an acceleration in the rate of hydrolysis of the DnaK-bound ATP . The presence of GrpE alone increases the rate of release of bound ATP or ADP without affecting the rate of hydrolysis . The stimulation of the ATPase activity of DnaK may contribute to its more efficient recycling, and it helps explain why mutations in dnaK, dnaJ, or grpE genes often exhibit similar pleiotropic phenotypes.

J Struct Biol, 1991 Apr, 106(2), 93 - 101
Mass analysis of bacteriophage T4 proheads and mature heads by scanning transmission electron microscopy and hydrodynamic measurements; Baschong W et al.; Quantitative mass analysis of bacteriophage T4 proheads by scanning transmission electron microscopy (STEM) revealed a mass of 79.5 +/- 0.6 MDa, while hydrodynamic measurements yielded a prohead mass of about 80 MDa . This is 25% less than the prohead mass deduced from its polypeptide composition, and this finding implies that the bacteriophage T4 prohead is built of fewer polypeptide copies than previously reported . In contrast, the mass of mature heads measured by STEM, 194 +/- 2 MDa, is in agreement with previous mass measurements of DNA and protein content, and it is consistent with the previously determined stoichiometry . This good agreement of average STEM values for proheads and mature heads with corresponding hydrodynamic measurements suggests that STEM allows faithful evaluation of the masses of large supramolecular assemblies (i.e., greater than or equal to 200 MDa) such as whole viruses or cellular organelles.

Indian J Biochem Biophys, 1991 Apr, 28(2), 93 - 5
A bacteriophage M13 based sandwich hybridization assay for the detection of hepatitis B virus in human blood; Banerjee K et al.; A two step hybridization procedure was developed to detect the presence of hepatitis B virus in blood samples using bacteriophage M13 radiolabelled DNA as probe . During the first step of hybridization, single-stranded bacteriophage M13 tg 130 DNA, with 3.2 kb HBV DNA cloned into it, was hybridized to target HBV DNA immobilized on nitrocellulose membrane filter . In the second step of hybridization, M13 DNA annealed to HBV target is detected with the help of double stranded form of M13 DNA . The assay offers minimum 4- to 6-fold higher sensitivity in comparison to single-step conventional hybridization assays . Additionally M13 DNA offers itself as universal probe.

Biochem Biophys Res Commun, 1991 Mar 29, 175(3), 1112 - 8
Tolbutamide and phenytoin hydroxylations by cDNA-expressed human liver cytochrome P4502C9; Veronese ME et al.; A human cytochrome P4502C9 cDNA clone has been isolated from a human liver bacteriophage Lambda gt11 library using oligonucleotide probes . Expression of the 1762 base pair cDNA in COS cells demonstrated that the encoded enzyme has a molecular mass of 55 kDa as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis . The expressed enzyme catalysed the methylhydroxylation of tolbutamide with an apparent Km of 131.7 microM, similar to that observed in human liver microsomes . P4502C9 also catalysed the 4-hydroylation of phenytoin, and inhibition experiments demonstrated that phenytoin was a competitive inhibitor of tolbutamide hydroxylation with an apparent Ki of 19.1 microM . Sulphaphenazole was a potent inhibitor of the expressed enzyme with respect to both tolbutamide and phenytoin hydroxylations . These data demonstrate that a single isozyme can catalyse the hydroxylations of both tolbutamide and phenytoin, and suggest that both reactions are mediated by the same isozyme(s) of cytochrome P450 in human liver.

Biochim Biophys Acta, 1991 Mar 26, 1088(3), 345 - 58
Potassium salts influence the fidelity of mRNA translation initiation in rabbit reticulocyte lysates: unique features of encephalomyocarditis virus RNA translation; Jackson RJ; It is widely assumed that in vitro translation of mRNA is more efficient in the presence of potassium acetate rather than KCl, that the optimum concentration of potassium acetate is higher than for KCl, and that uncapped RNAs exhibit a lower optimum salt concentration than capped mRNAs . When these assumptions were examined using several different mRNA species in four batches of rabbit reticulocyte lysate, some notable exceptions were found . The translation of encephalomyocarditis virus (EMCV) RNA exhibited a salt optimum unusually high for an uncapped mRNA, and was very much more efficient and accurate with KCl rather than potassium acetate . It was also unique in being strongly activated by low concentrations (5-10 mM) KSCN in the presence of 90 mM potassium acetate . For the translation of other uncapped RNAs (poliovirus RNA, cowpea mosaic virus (CPMV) M RNA and bacteriophage MS2 RNA) amino acid incorporation at the optimum potassium acetate level was significantly greater than could be achieved using KCl . However, KCl was found to be restrictive and potassium acetate permissive for the synthesis of abnormal products thought to arise from initiation at incorrect sites, with the result that KCl gave a product pattern closer to that observed in vivo . In the particular case of the reticulocyte lysate system, accurate translation therefore requires the use of KCl rather than potassium acetate, but the choice of salt was found to be less critical in cell-free extracts from HeLa or L-cells.

Biochemistry, 1991 Mar 19, 30(11), 2772 - 82
Reversible denaturation of the gene V protein of bacteriophage f1; Liang H et al.; The guanidine hydrochloride (GuHCl)-induced denaturation of the gene V protein of bacteriophage f1 has been studied, using the chemical reactivity of a cysteine residue that is buried in the folded protein and the circular dichroism (CD) at 211 and 229 nm as measures of the fraction of polypeptide chains in the folded form . It is found that this dimeric protein unfolds in a single cooperative transition from a folded dimer to two unfolded monomers . A folded, monomeric form of the gene V protein was not detected at equilibrium . The kinetics of unfolding of the gene V protein in 3 M GuHCl and the refolding in 2 M GuHCl are also consistent with a transition between a folded dimer and two unfolded monomers . The GuHCl concentration dependence of the rates of folding and unfolding suggests that the transition state for folding is near the folded conformation.

Proc Natl Acad Sci U S A, 1991 Mar 15, 88(6), 2417 - 21
DNA polymerization in the absence of exonucleolytic proofreading: in vivo and in vitro studies; Reha-Krantz LJ et al.; Classical genetic selection was combined with site-directed mutagenesis to study bacteriophage T4 DNA polymerase 3'----5' exonuclease activity . A mutant DNA polymerase with very little (less than or equal to 1%) 3'----5' exonuclease activity was generated . In vivo, the 3'----5' exonuclease-deficient DNA polymerase produced the highest level of spontaneous mutation observed in T4, 500- to 1800-fold above that of wild type . The large reduction in 3'----5' exonuclease activity appears to be due to two amino acid substitutions: Glu-191 to Ala and Asp-324 to Gly . Protein sequence similarities have been observed between sequences in the Escherichia coli DNA polymerase I 3'----5' exonuclease domain and conserved sequences in eukaryotic, viral, and phage DNA polymerases . It has been proposed that the conserved sequences contain metal ion binding ligands that are required for 3'----5' exonuclease activity; however, we find that some proposed T4 DNA polymerase metal binding residues are not essential for 3'----5' exonuclease activity . Thus, our T4 DNA polymerase studies do not support the hypothesis by Bernad et al . {Bernad, A., Blanco, L., Lazaro, J.M., Martin, G . & Salas, M . (1989) Cell 59, 219-228} that many DNA polymerases, including T4 DNA polymerase, share an extensively conserved 3'----5' exonuclease motif . Therefore, extrapolation from E . coli DNA polymerase I sequence and structure to other DNA polymerases for which there is no structural information may not be valid.

J Biol Chem, 1991 Mar 15, 266(8), 4883 - 8
The characterization of a complex of three bacteriophage T4 recombination proteins, uvsX protein, uvsY protein, and gene 32 protein, on single-stranded DNA; Hashimoto K et al.; Three recombination proteins of bacteriophage T4, uvsX, uvsY, and gene 32 proteins, were examined for the formation of a complex with short single-stranded DNA (ssDNA) molecules containing either 24 or 69 nucleotides . Gel-shift assays revealed that either the uvsX or uvsY protein, when present alone, formed a stable complex only with the 69-mer, while the gene 32 protein bound stably to both ssDNAs . However, a characteristic stable complex formed on the 24-mer when both the uvsX and uvsY proteins were present, and the uvsY protein bound to this DNA in the presence of the gene 32 protein . Isolation of the complexes by centrifugation through a glycerol gradient revealed their protein constituents and showed that the uvsX protein-uvsY protein-24-mer ssDNA complex formed even in the presence of excess gene 32 protein . The possible biological significance of these protein-DNA complexes is discussed.

Biochem J, 1991 Mar 15, 274 ( Pt 3), 813 - 7
Regulated expression of cytochrome P-450scc (cholesterol-side-chain cleavage enzyme) in cultured cell lines detected by antibody against bacterially expressed human protein; Hu MC et al.; The first step in the synthesis of steroids is catalysed by cytochrome P-450ssc (cholesterol-side-chain cleavage enzyme) . We have investigated the synthesis of this enzyme in three cultured cell lines at the protein and hormone secretion levels . Hormone levels were measured by an enzyme immunoassay using a monoclonal antibody against progesterone . The protein level was detected using polyclonal antibodies directed against a P-450scc fusion protein overproduced in Escherichia coli . Utilizing a bacteriophage T7 promoter expression system, a large amount of human P-450scc fusion protein was produced and easily purified . P-450scc was synthesized in the mouse adrenal tumour cell line Y1 and human choriocarcinoma cell line JEG-3, but not in monkey kidney cell line COS-1 . The production of P-450scc in Y1 and JEG-3 cells was stimulated by 8-bromo cyclic AMP, the effect of which was not observed until 6 h after induction and was more pronounced at 24 h . Y1 and JEG-3 cells exhibited a difference in progesterone secretion after induction.

Gene, 1991 Mar 15, 99(2), 261 - 5
Expression of an immunogenic region of HIV by a filamentous bacteriophage vector; Tsunetsugu-Yokota Y et al.; Vectors derived from the Escherichia coli filamentous phage, fd-tet, expressing parts of the human immunodeficiency virus (HIV) gag genes were constructed and analyzed . The immunoreactive domain of HIV Gag antigens was produced in the form of a fusion protein, with a phage minor coat protein, called protein III, playing an important role in phage infectivity . A micropanning procedure, utilizing the strong affinity of biotinylated antibody to streptavidin, was applied for the selection of clones . A simple preparation procedure consisting of polyethyleneglycol precipitation of the recombinant phage from the E . coli supernatant allowed us to detect HIV antigens by enzyme-linked immunosorbent assay (ELISA) . Cloned FUSE-gag, as isolated using anti-Gag RL4.72.1 monoclonal antibody (mAb), contained a nucleotide sequence coding for 91 amino acids of HIV Gag p24 . It specifically reacted with the mAb in the ELISA . Construction of the mAb-selectable phages permitted localization of epitopes for mAb . Infectivity of the phage clone was specifically neutralized by the anti-HIV mAb . Immunoelectroblotting analysis of recombinant phages revealed the presence of an about 65-kDa band reacting with anti-HIV mAb . This Mr corresponded to the size of the fused form of the FUSE 1 protein III . Human sera from HIV-infected and uninfected individuals reacted with recombinant protein III, as well as the original form of protein III.

Nucleic Acids Res, 1991 Mar 11, 19(5), 1063 - 6
Identification and sequence determination of the host factor gene for bacteriophage Q beta; Kajitani M et al.; The host factor (HF-I) required for phage Q beta RNA-directed synthesis of complementary minus-strand RNA was purified to homogeneity from phage-infected Escherichia coli cells . The hfq gene encoding HF-I was cloned using synthetic probes designed based on the partial amino acid sequence of HF-I, and mapped at 94.8 min on the E . coli chromosome downstream of the miaA gene involved in 2-methylthio-N6-(isopentyl)-adenosine (ms2i6A) tRNA modification . Sequence determination of the cloned hfq gene indicated that HF-I is a small protein of Mr 11,166 consisting of 102 amino acid residues.

Cell, 1991 Mar 8, 64(5), 1007 - 15
Liquid-crystalline, phage-like packing of encapsidated DNA in herpes simplex virus; Booy FP et al.; The organization of DNA within the HSV-1 capsid has been determined by cryoelectron microscopy and image reconstruction . Purified C-capsids, which are fully packaged, were compared with A-capsids, which are empty . Unlike A-capsids, C-capsids show fine striations and punctate arrays with a spacing of approximately 2.6 nm . The packaged DNA forms a uniformly dense ball, extending radially as far as the inner surface of the icosahedral (T = 16) capsid shell, whose structure is essentially identical in A-capsids and C-capsids . Thus we find no evidence for the inner T = 4 shell previously reported by Schrag et al . to be present in C-capsids . Encapsidated HSV-1 DNA closely resembles that previously visualized in bacteriophages T4 and lambda, thus supporting the idea of a close parallelism between the respective assembly pathways of a major family of animal viruses (the herpesviruses) and a major family of bacterial viruses.

Biotechniques, 1991 Mar, 10(3), 366 - 74
Universal promoter for gene expression without cloning: expression-PCR; Kain KC et al.; We present a rapid and simple system called expression-PCR (E-PCR) for in vitro synthesis of functional protein from genomic or plasmid DNA . A universal promoter was developed containing an untranslated leader sequence from alfalfa mosaic virus directly downstream from the T7 bacteriophage promoter . When this universal promoter is spliced to a DNA segment, it produces a suitable template for in vitro transcription and translation . The DNA to be expressed is first amplified by the PCR using a 5'-primer that incorporates an area homologous to the 3'-end of the universal promoter . The universal promoter and this DNA fragment are mixed and re-amplified in a reaction analogous to splicing by overlap extension, generating a recombinant DNA template that can be transcribed and translated in vitro without further processing . Unlike standard methods for in vitro transcription and translation, E-PCR is not dependent upon specialized transcription vectors, cloning, plasmid isolation and purification, or restriction enzyme sites . This approach has been used to synthesize and examine the biological activity of malaria proteins that are vaccine candidates for Plasmodium falciparum . E-PCR represents a significant improvement over current in vitro expression systems, most notably in its time savings, versatility of gene expression and its compatibility with rapid PCR-based site-directed mutagenesis procedures.

Mol Microbiol, 1991 Mar, 5(3), 715 - 25
Gene 32 transcription and mRNA processing in T4-related bacteriophages; Loayza D et al.; We have analysed transcription and mRNA processing for the gene 32 region of five phages related to T4 . Two different organizations of gene 32 proximal promoters were found . In T4 and M1, middle- and late-mode promoters are separated by 50 nucleotides and located within an upstream open reading frame . In T2, K3, Ac3, and Ox2, the 626bp T4 sequence that includes these promoters is replaced by a 59bp sequence containing overlapping middle and late promoters . The RNase E-dependent processing of the g32 mRNAs is conserved in all of the phages . The processing site immediately upstream of g32 in T4 and M1 has been replaced in the other phages by a different sequence that is also cleaved by RNase E . The remarkable conservation of these regulatory features, despite the sequence divergences, suggests that they play an important role in the control of gene expression.

Mol Microbiol, 1991 Mar, 5(3), 685 - 94
Transcriptional analysis of the restriction and modification genes of bacteriophage P1; Sharrocks AD et al.; Bacteriophage P1 res and mod genes encode the restriction and modification polypeptides of the Type III restriction enzyme EcoP1 . Northern blot analysis using res- and mod-specific probes revealed the presence of two separate transcripts in strains harbouring the EcoP1 restriction and modification genes . Furthermore, by constructing a series of fusions with a promoter less lacZ gene, we show that both the res and mod genes are transcribed from separate promoters . A more detailed investigation of the mod promoter region revealed two promoters located some 70 and 140bp upstream from the translational start codon . In addition, another pair of promoters and a further separate promoter are located more than 500bp upstream from this start codon . Two short open reading frames are located between these distal and proximal promoter clusters . Transcription of the res gene is initiated from within the mod open reading frame from two adjacent promoters . In addition a functional promoter is located on the antisense strand close to the res promoter region . The relationship between the transcription units of the res and mod genes is discussed.

FEMS Microbiol Lett, 1991 Mar 1, 62(2-3), 305 - 13
Isolation and characterization of extragenic suppressor mutants of the tolA-876 periplasmic-leaky allele in Escherichia coli K-12; Amouroux C et al.; tolA mutants of Escherichia coli K-12 release periplasmic proteins into the extracellular medium; they are sensitive to growth inhibitors such as cholic acid and tolerant to group A colicins and filamentous bacteriophage . Suppressor mutants of the tolA-876 allele were isolated by selecting for cholic acid resistant clones that did not release periplasmic ribonuclease I . One class of tolA suppressor strains carried mutations in the staA gene (for suppressor of tolA) located a 41 min . tolA-876 staA strains partially recovered a wild-type phenotype: they exported alkaline phosphatase and beta-lactamase into the periplasm and only released very low amounts of periplasmic proteins; moreover, they were sensitive to E1 and A colicins and more resistant than tolA-876 staA+ strains to various growth inhibitors . Furthermore, tolA-876 staA-2 and tolA+staA-2 mutants were 10- to 2700-times more resistant than staA+ strains to bacteriophages TuIa, TuIb and T4, and TuII whose receptors are major outer membrane proteins OmpF, OmpC and OmpA, respectively . SDS-PAGE analysis suggested that cell envelopes of staA or staA+ strains contained similar amounts of these proteins but characterization of strains carrying ompF (or C or A)-phoA gene fusions showed that mutation stA-2 reduced ompF gene expression by a factor of two . Analysis of double mutants strains carrying mutation staA-2 and a tolA, tolB, excC or excD periplasmic-leaky mutation showed that staA suppression was allele specific which suggested that proteins TolA and StaA might directly interact.

Mol Gen Genet, 1991 Mar, 225(3), 427 - 34
Gene product dsbA of bacteriophage T4 binds to late promoters and enhances late transcription; Gansz A et al.; Gene product 33 of phage T4 is known to be essential in late transcription . Upstream from gene 33 and overlapping its 5' terminal sequence by 20 bp, we identified an open reading frame coding for a binding protein for double-stranded DNA (DsbA) . Gene product DsbA is composed of 89 amino acid residues with a Mr of 10376 kDa . We purified this protein to homogeneity from over-expressing cells . Gel retardation assays reveal that it binds to DNA and footprint analyses disclose that it interacts preferentially with T4 late promoter regions . At the sites of binding the protein introduces nicks in double-stranded DNA . In vitro transcription assays performed with T4 late modified RNA polymerase on restriction fragments harbouring a T4 late promoter region prove that gene product DsbA enhances transcription from these promoter regions in the presence of gene product 33 . Gene dsbA is distinct from gene das which maps close to this genomic region.

Mol Gen Genet, 1991 Mar, 225(3), 355 - 62
Structural and functional comparison between the stability systems ParD of plasmid R1 and Ccd of plasmid F; Ruiz-Echevarria MJ et al.; The stability determined by the systems ParD of plasmid R1 and Ccd of plasmid F is due to the concerted action of two proteins, a cytotoxin and an antagonist of this function . In this paper we report that CcdA and Kis proteins, the antagonists of the Ccd and ParD systems respectively, share significant sequence homologies at both ends . In Kis, these regions seem to correspond to two different domains . Despite the structural similarities, Kis and CcdA are not interchangeable . In addition we have shown that the cytotoxins of these systems, the Kid and CcdB proteins, do not share structural homologies . In contrast to CcdB, the Kid protein of the ParD system induces RecA-dependent cleavage of the cI repressor of bacteriophage lambda very inefficiently or not at all . The functional implications of these results are discussed.

J Bacteriol, 1991 Mar, 173(6), 2017 - 25
Cloning of Azorhizobium caulinodans nicotinate catabolism genes and characterization of their importance in N2 fixation; Buckmiller LM et al.; Twenty Azorhizobium caulinodans vector insertion (Vi) mutants unable to catabolize nicotinate (Nic- phenotype) were identified and directly cloned as pVi plasmids . These pVi plasmids were used as DNA hybridization probes to isolate homologous wild-type sequences . From subsequent physical mapping experiments, the nic::Vi mutants defined four distinct loci . Two, possibly three, of these loci are physically linked . A . caulinodans nic loci II and III encode the structural genes for nicotinate catabolism; nic loci I and IV encode nicotinate-driven respiratory chain components . Recombinant lambda bacteriophages corresponding to three of these loci were subcloned in pRK293; resulting plasmids were used for complementation tests with resolved nic::IS50 derivatives of the nic::Vi mutants . When wild-type A . caulinodans was cultured in defined liquid medium under 3% O2, nicotinate catabolism stimulated N2 fixation 10-fold . In these exponentially growing cultures, the entire (300 microM) nicotinate supplement was exhausted within 10 h . While nic::Vi mutants retained the ability to fix some N2, they did so at rates only 10% of that of the wild type: nitrogenase activity by nic::Vi mutants was not stimulated by 300 microM added nicotinate . Higher-level (5 mM) nicotinate supplementation inhibited N2 fixation . Because 5 mM nicotinate repressed nitrogenase induction in all nic::Vi mutants as well, this repression was independent of nicotinate catabolism . During catabolism, nicotinate is first oxidized to 6-OH-nicotinate by a membrane-bound nicotinate hydroxylase which drives a respiratory chain to O2 . In A . caulinodans wild-type cultures, added 300 microM 6-OH-nicotinate stimulated N2 fixation twofold better than did added 300 microM nicotinate . Likewise, nic::Vi mutant 61302, defective in nicotinate hydroxylase, fixed N2 at wild-type levels when supplemented with 300 microM 6-OH-nicotinate . Therefore, nicotinate catabolism stimulates N2 fixation not by nicotinate hydroxylase-driven respiration but rather by some subsequent aspect(s) of nicotinate catabolism.

Proc Natl Acad Sci U S A, 1991 Mar 1, 88(5), 1706 - 10
Energetics of repacking a protein interior; Sandberg WS et al.; To test whether interactions in the hydrophobic core of a protein can be adequately modeled based on the properties of a liquid hydrocarbon, we measured the unfolding free energies of the wild-type bacteriophage f1 gene V protein and 29 mutants with apolar substitutions at positions 35 and 47 . Stability changes arising from identical mutations at these two buried sites are quite different, suggesting that one site is more rigid than the other . Reversals of residues at positions 35 and 47 confirm that their environments are distinct . Mutants containing weakly polar residues at these two sites suggest that the protein interior is more polar than a liquid hydrocarbon . Interactions between residues at the two sites appear to be minimal . These observations are compatible with a view of protein interiors that incorporates properties of liquid hydrocarbons but also includes polar interactions and a site-dependent "packing energy" associated with changes in internal structure.

Virology, 1991 Mar, 181(1), 109 - 17
In vivo complementation of infectious transcripts from mutant tobacco mosaic virus cDNAs in transgenic plants; Holt CA et al.; A full-length cDNA clone of the U1 (common) strain of tobacco mosaic virus (TMV) was constructed, and highly infectious transcripts were produced in vitro using bacteriophage T7 RNA polymerase . Frameshift mutations designed to cause premature termination of translation were introduced into either the 30-kDa movement protein (MP) gene or the coat protein (CP) gene . The MP-frameshift mutant was unable to locally or systemically infect inoculated tobacco plants . However, inoculation of transgenic tobacco plants that expressed a wild-type TMV MP gene resulted in both local and systemic viral infection . The CP-frameshift mutant, although unable to move systemically in nontransformed tobacco, exhibited systemic movement in transgenic plants that expressed a wild-type TMV CP gene . Transgenic tobacco plants that expressed the appropriate wild-type TMV gene were thus able to complement, in trans, mutant viruses lacking a functional MP or CP gene.

Biopolymers, 1991 Mar, 31(4), 359 - 72
Conformational preference and ligand binding properties of DNA junctions are determined by sequence at the branch; Guo Q et al.; Four-arm DNA branched junctions are stable analogues of Holliday recombinational intermediates . A number of four-arm DNA junctions synthesized from oligonucleotides have now been studied . Gel mobility or chemical footprinting experiments on several immobile four-arm junctions indicate that in the presence of Mg2+, they assume a preferred conformation consisting of two helical domains, each formed by stacking a particular pair of arms on each other . We show here that a junction we designate as J1c that has the same chemical composition as one we have previously studied in detail, J1, but is formed from the four strands complementary to those of the latter, exhibits the reverse stacking preference . The pattern of self-protection of the strands of J1c exposed to Fe(II).EDTA-induced scission reveals that twofold symmetry is preserved, but the opposite pair of strands preferentially cross over . Moreover, the Fe(II).EDTA scission profiles of J1c indicate that this junction exhibits a weaker bias as to which strands cross over than is observed in J1 . The preference for the dominant species in J1 is 1.3 times greater than in J1c at 4 degrees C and in the presence of 10 mM Mg2+, based on chemical reactivity data . This is confirmed by a cleavage experiment using the resolvase enzyme, endonuclease I, from bacteriophage T7 . This difference could reflect either sequence-dependent differences in the equilibrium among isomers, or in the structure of these junctions . Chemical footprinting experiments using the probes MPE.Fe(II) and (OP)2Cu(I) show that the high-affinity ligand binding site in immobile junctions is determined by junction geometry.

Gene, 1991 Mar 1, 99(1), 31 - 7
Overproduction and characterization of the uracil-DNA glycosylase inhibitor of bacteriophage PBS2; Wang ZG et al.; A plasmid expression vector (pZWtac1) was constructed which allowed inducible overexpression of the uracil-DNA glycosylase (Ung) inhibitor (Ugi)-encoding gene (ugi) in Escherichia coli . In this plasmid, the ugi gene was under the control of both its own promoter and the tac promoter . Constitutive expression of the ugi was observed in the absence of isopropyl-beta-D-thiogalactopyranoside (IPTG) . In the presence of 1 mM IPTG, the Ugi protein was overproduced to an approx . 16-fold higher level, and accounted for approx . 19% of the total soluble cellular proteins . Following high-level production in E . coli, the Ugi protein was purified to apparent homogeneity . Using E . coli Ung, we observed that Ugi inactivated the enzyme in a noncompetitive manner . Kinetic studies revealed a Ki value (0.14 microM) of approx . twelve-fold lower than Km value (1.7 microM) of glycosylase . Ugi did not act synergistically with free uracil to inhibit E . coli Ung suggesting that uracil and Ugi could share a similar mode of inhibition.

J Radiat Res (Tokyo), 1991 Mar, 32(1), 1 - 12
Enhanced killing effect on 5-bromodeoxyuridine labelled bacteriophage T1 by monoenergetic synchrotron X-ray at the energy of bromine K-shell absorption edge; Furusawa Y et al.; 5-Bromo-2'-deoxyuridine labelled bacteriophage T1 was irradiated with monoenergetic X-rays obtained from synchroton radiation with the energies at 13.51 keV and 12.40 keV, just above and below the K-shell absorption edge (13.41 keV) of bromine, respectively . Phage samples were irradiated under three conditions, wet N2 gas, dry N2 gas and in vacuum, with water contents (g H2O/g sample) of 60, 2.6 and 0% . At 12.40 keV the D0 in kGy were 1.1 ("wet"), 1.9 ("dry") and 2.5 ("vacuum") for the Br-labelled phages and 1.6 ("wet"), 2.4 ("dry") and 6.2 ("vacuum") for unlabelled phages . The results clearly demonstrated that the X-ray sensitivities decreased with decrease in water contents . Br-enhancement ratios, ER = D0 (unlabelled)/D0 (Br-labelled), varied from 1.4 ("wet", 12.40 keV) to 2.5 ("vacuum", 13.51 keV) . Auger enhancements which were defined by energy-dependent enhancement factor, Fen = {ER(13.51 keV)-ER(12.40 keV)}/ER(12.40 keV), were 0.09 +/- 0.09, 0.29 +/- 0.07 and 0.02 +/- 0.03 under "wet", "dry" and "vacuum" conditions, respectively . The change in Auger enhancement under "dry" condition in comparison with "wet" condition could be explained due to less of water . However the Auger enhancement decreased sharply under "vacuum" condition as the water content was zero . The reason for the sharp decrease in Auger enhancement under "vacuum" condition is difficult to understand . A possible explanation is discussed in the text.

Biofizika, 1991 Mar-Apr, 36(2), 281 - 5
{Changes in membrane potential and transport of ions through the S . typhimurium LT2 membrane induced by bacteriophages}; Ter-Nikogosian VA et al.; Bacteriophages P22 and dp8 cause the membrane potential depolarization for 10-30 mV, reversal rapid H+ influx into bacteria and K+ exit from S . typhimurium LT2, these effects depend on infection plural and are observed only in the presence of Ca+2 in the medium . delta psi depolarization and K+ efflux induced by phage dp8 are increased with the growth of Mg+2 concentration from 0 to 2 mM . Changes of delta pH and also Na+,Ca+2 concentrations are not observed . In the presence of glucose phage infection leads to changes in H(+)-K(+)-exchange . The phages P22 and dp8 adsorption on bacteria causes changes in the form or turn of the channels in S . typhimurium membrane.

Genetics, 1991 Mar, 127(3), 453 - 62
The specificity of topoisomerase-mediated DNA cleavage defines acridine-induced frameshift specificity within a hotspot in bacteriophage T4; Masurekar M et al.; Acridine-induced frameshift mutations in bacteriophage T4 occur at the precise location in the DNA at which acridines stimulate DNA cleavage by the T4-encoded type II topoisomerase in vitro . The mutations are duplications or deletions that begin precisely at the broken phosphodiester bond . In vivo, acridine-induced frameshift mutagenesis is reduced nearly to background levels when the topoisomerase is genetically inactivated . These observations are consistent with a model in which cleaved DNA, induced by the topoisomerase and acridine, serves as the substrate for the production of frameshift mutations at the same site . Our model predicts that the specificity and frequency of cleavage direct the specificity and frequency of mutagenesis . This prediction was tested by examining the influence of DNA sequence changes on topoisomerase-mediated cleavage and on mutagenesis in the T4 rIIB gene . The model successfully predicted the results . When DNA sequence changes altered the position of acridine-induced, topoisomerase-mediated DNA cleavage in vitro, frameshift mutations were found at the new positions . DNA sequence changes that strongly decreased in vitro cleavage also reduced mutagenesis at that site . These results demonstrate that acridine-induced frameshift mutation specificity is directed by the characteristics of the acridine-topoisomerase reaction and do not suggest that slipped pairing in repeated sequences plays a major role in acridine-induced frameshifts in bacteriophage T4.

J Virol, 1991 Mar, 65(3), 1383 - 91
The amino-terminal half of rotavirus SA114fM VP4 protein contains a hemagglutination domain and primes for neutralizing antibodies to the virus; Lizano M et al.; We have previously reported the synthesis in Escherichia coli of polypeptide MS2-VP8', which contains the amino-terminal half of the SA114fM VP4 protein fused to MS2 bacteriophage polymerase sequences (C . F . Arias, M . Lizano, and S . Lopez, J . Gen . Virol . 68:633-642, 1987) . In this work we have synthesized the carboxy-terminal half of the VP4 protein also fused to the MS2 polymerase . This protein, designated MS2-VP5', was recognized by sera to the complete virion and was able to induce antibodies to the virus when administered to mice; however, these antibodies had no neutralizing activity . The two chimeric polypeptides were tested for their ability to agglutinate erythrocytes and to prime the immune system of mice . Bacterial lysates enriched for the MS2-VP8' hybrid polypeptide, but not those enriched for the MS2-VP5' protein or those containing proteins from the host E . coli strain, had hemagglutinating activity . This hemagglutination was inhibited by sera to SA114fM rotavirus . In addition, a single dose of the MS2-VP8' polypeptide was able to prime the immune system of mice for an augmented neutralizing antibody response when the animals were subsequently immunized with purified SA114fM virus.

J Virol, 1991 Mar, 65(3), 1263 - 72
Effect of marker distance and orientation on recombinant formation in poxvirus-infected cells; Parks RJ et al.; Little is known about the mechanism of poxvirus recombination even though construction of recombinant viruses by recombination-dependent methods is a widely adopted technique . We have shown previously that transfected DNAs are efficiently recombined while replicating in cells infected with Shope fibroma virus . Because recombinant DNA can be recovered from infected cells as a high-molecular-weight head-to-tail concatemer, it was possible to transfect genetically marked lambda DNAs into infected cells and assay recombinants as bacteriophage particles following in vitro packaging . This approach was used in this study to examine how marker distance and marker orientation influence recombination in Shope fibroma virus-infected cells . Simple two-factor crosses were readily modelled by using a mapping function derived from classical phage studies and showed low negative interference (I = -2.8 +/- 0.5) in crosses involving markers greater than 100 bp apart . More complex four- and five-factor crosses showed that the recombination frequency per unit distance was not constant (rising as the marker separation was reduced from 100 to 1 bp) and that crosses performed in poxvirus-infected cells are subject to high negative interference . One consequence is that marker orientation does not dramatically influence the outcome of most Shope fibroma virus-catalyzed crosses in clear contrast to what is observed in adenovirus or simian virus 40-infected cells . These results can be interpreted to indicate that similar statistical and physical constraints influence both viral and phage recombination and suggest that heteroduplexes may be important intermediates in the poxvirus recombination process.

FEMS Microbiol Lett, 1991 Mar 1, 62(2-3), 201 - 5
Cloning of xylanase gene of Streptomyces flavogriseus in Escherichia coli and bacteriophage lambda-induced lysis for the release of cloned enzyme; Srivastava R et al.; The xylanase gene of Streptomyces flavogriseus was cloned in pUC8 plasmid and expressed in Escherichia coli lysogenic for lambda cI857 . lambda-Induced lysis of E . coli at 42 degrees C allowed efficient release of cloned enzyme activity in extracellular environment . The xylanase gene was located in the 0.8-kb HindIII fragment and coded for 18,000 Mr xylanase.

J Bacteriol, 1991 Mar, 173(5), 1670 - 6
HU and integration host factor function as auxiliary proteins in cleavage of phage lambda cohesive ends by terminase; Mendelson I et al.; HU and integration host factor (IHF) are small, basic heterodimeric DNA-binding proteins which participate in transcription initiation, DNA replication, and recombination . We constructed isogenic Escherichia coli strains in which HU, IHF, or both proteins were absent . Bacteriophage lambda did not grow in hosts lacking both HU and IHF . Phage DNA replication and late gene transcription were normal in the double mutants, but packaging of lambda DNA was defective . Mature phage DNA molecules were absent, indicating that terminase was unable to linearize lambda DNA . Phage variants carrying a small substitution near cos or the ohm1 mutation in the terminase gene, Nul, formed plaques on HU- IHF- strains . We propose that HU or IHF is required to establish the higher-order DNA-protein structure at cos that is the substrate for lambda terminase.

Virology, 1991 Mar, 181(1), 353 - 8
Nucleotide sequence of the genes encoding the major tail sheath and tail tube proteins of bacteriophage P2; Temple LM et al.; The major structural components of the contractile tail of bacteriophage P2 are proteins FI and FII, which are believed to be the tail sheath and tube proteins, respectively . Both proteins were mapped previously to the P2 late gene F, based on the pattern of protein synthesis in various P2 amber mutants . In order to clarify the gene arrangement and to provide a basis for structural comparisons with other contractile phage tails, we have determined the nucleotide sequence of the region of the P2 genome encoding these two proteins . The coding regions were confirmed by location of the Fam4 mutation and by N-terminal amino acid sequencing of both proteins . The molecular weight and amino acid composition predicted by each of the coding regions correspond well to those determined experimentally for each protein . FII is encoded by a newly identified P2 late gene . These proteins bear little resemblance to their functional homologues in bacteriophage T4.

Virology, 1991 Mar, 181(1), 55 - 61
Biological characterization of infectious molecular clones derived from a human immunodeficiency virus type-1 isolate with rapid/high replicative capacity; Fredriksson R et al.; In order to molecularly characterize rapidly and slowly replicating HIV-1 variants, molecular clones were obtained from a rapid/high virus isolate . This isolate, 4803, had only been passaged in peripheral blood mononuclear cells (PBMC) prior to cloning . Molecular cloning was done in bacteriophage lambda-dash using high molecular weight DNA of isolate 4803 infected PBMC . Seven recombinant phages were identified . The clones were found to be related to each other and differed only at 1 or 2 restriction sites (out of 28) . The molecular clones were transfected into various cell types by electroporation . The phenotype of progeny viruses was found to be dependent on the cell type used for transfection . Progeny viruses produced by PBMC cultures differed from the parental isolate in that they did not form syncytia and lacked the capacity to replicate in cell lines . Since transfection of PBMC yielded progeny viruses within 1 week, this phenotype is considered to be the true phenotype of the clones . Transfection of the T-lymphoid HUT-78 cell line and of the monocytoid U937-2 cell line yielded progeny viruses after considerable delay (more than 1 month) . Progeny viruses from HUT-78 cells were similar to the parental isolate in that they formed syncytia in PBMC and replicated in all cell lines tested . Progeny viruses from U937-2 cells showed an intermediate phenotype in that they replicated in U937-2 but not in T-lymphoid cell lines . These results indicate that molecular clones of a rapid/high virus may have a restricted replicative capacity compared to the parental, genetically heterogenous virus isolate.

Virus Res, 1991 Mar, 18(2-3), 165 - 78
High level expression of the major core protein VP7 and the non-structural protein NS3 of bluetongue virus in yeast: use of expressed VP7 as a diagnostic, group-reactive antigen in a blocking ELISA; Martyn JC et al.; The major core protein VP7 and a non-structural protein NS3 of bluetongue virus serotype 1 have been synthesized from recombinant plasmids using both an in vitro transcription/translation system and a yeast expression system . Bluetongue virus genes were transcribed under the control of the bacteriophage SP6 promoter and the regulatable yeast metallothionein promoter . An indirect ELISA showed that expression of NS3 in yeast was inducible with 1 mM CuSO4 and VP7 synthesis was constitutive but could be further induced . The preferred procedure for antigen extraction from yeast was sonication for VP7 and SDS/NaOH treatment for NS3 . Yeast-expressed VP7 antigen and a monoclonal antibody were used in a blocking ELISA to distinguish sera raised against bluetongue virus serotypes from those generated to viruses of the epizootic haemorrhagic disease serogroup.

Nucleic Acids Res, 1991 Feb 25, 19(4), 751 - 7
Interaction of HIV-1 reverse transcriptase with a synthetic form of its replication primer, tRNA(Lys,3); Barat C et al.; Using synthetic oligonucleotides, a gene encoding the HIV-1 replication primer, tRNA(Lys,3), was constructed and placed downstream from a bacteriophage T7 promoter . In vitro transcription of this gene yielded a form of tRNA(Lys,3) which lacks the modified bases characteristic of the natural species and the 3' -C-A-dinucleotide . Synthetic tRNA(Lys,3) annealed to a pbs-HIV1 RNA template can prime cDNA synthesis catalysed by recombinant HIV-1 reverse transcriptase . Trans-DDP crosslinking indicates that this synthetic tRNA is still capable of interacting with HIV-1 RT via a 12-nucleotide portion encompassing the anticodon domain . Gel-mobility shift and competition analyses imply that the affinity of synthetic tRNA for RT is reduced . In contrast to earlier observations, synthetic tRNA is readily competed from RT by natural tRNA(Pro) . The reduced affinity of synthetic tRNA(Lys,3) for RT is not appreciably affected by mutations in positions within the loop of the anticodon domain . These results would imply that the overall structure of the anticodon domain of tRNA(Lys,3) is an important factor in its recognition by HIV-1 RT . In addition, modified bases within this, although not absolutely required, would appear to make a significant contribution to the enhanced stability of the ribonucleoprotein complex.

J Mol Biol, 1991 Feb 20, 217(4), 681 - 9
Circular dichroism and fluorescence analysis of the interaction of Pf1 gene 5 protein with poly(dT); Carpenter ML et al.; Circular dichroism (c.d.) and fluorescence spectroscopy have been used to investigate the interaction of the gene 5 protein of the filamentous bacteriophage Pf1 with single-stranded DNA . The c.d . spectrum of the Pf1 gene 5 protein is consistent with the absence of any significant alpha-helical content . The negative c.d . peak in the region of 210 nm, which arises from the protein, is diminished in the complex with poly(dT) . Likewise, the c.d . peak at 265 nm arising from the poly(dT) decreases when the Pf1 gene 5 protein is bound, c.d . titrations of poly(dT) with Pf1 gene 5 protein indicate strong binding with a stoichiometry (n) of four nucleotides per protein subunit . In contrast, when the titrations were done using fluorescence anisotropy or fluorescence spectral shifts to follow binding, apparent stoichiometries between n = 2 and n = 4 were observed, often in the same experiment, depending on precise conditions . The results are interpreted in terms of two distinct modes of binding, in which either one or two subunits of the protein dimer are bound to the polynucleotide lattice, but still retaining the same local interaction with the DNA, with each binding site covering four nucleotides . The apparent stoichiometry of 2 results from the interaction of only one subunit of the dimer with the nucleic acid lattice, when protein is in excess . The second, unfilled, subunit of the dimer is nevertheless incorporated into the complex, resulting in the maximum possible fluorescence change when only half the sites are filled, since the fluorescence properties of the complex arise from protein-protein contacts associated with co-operative binding to the lattice . Further experiments in which the order of addition of components is changed, and the concentration of MgCl2 is varied, show that both of these factors are important in determining the dominant binding mode . In the absence of salt, dissociation and redistribution of the polynucleotide can occur following the addition of excess protein . This transition is suppressed in the presence of greater than 3 mM-MgCl2.

J Mol Biol, 1991 Feb 20, 217(4), 599 - 602
Shared operator recognition specificity between Trp repressor and the repressors of bacteriophage 434; Somerville RL et al.; Trp repressor is the only DNA-binding regulatory protein having a helix-turn-helix motif that has been reported to engage its operator target by a mechanism termed indirect readout: the Trp repressor-DNA interface is replete with hydrogen bonds between amino acid residues and non-esterified oxygen atoms of the sugar-phosphate backbone, and contains numerous specifically positioned water molecules . In Escherichia coli mutants deleted for trpR, the immunity repressor of phage 434 led to an eightfold reduction in trp promoter utilization . The Cro434 repressor also inhibited transcription from the trp promoter . The 434 repressors, considered to interact directly with operator targets, carry recognition helices positioned near the N terminus of each protein . The DNA-recognizing elements of Trp repressor lie toward the C terminus . The trp operator thus appears to possess significant plasticity in terms of its ability to assume conformational states that allow complex formation with more than one class of regulatory protein.

Biochemistry, 1991 Feb 19, 30(7), 1887 - 91
A differential scanning calorimetric study of the thermal unfolding of seven mutant forms of phage T4 lysozyme; Connelly P et al.; High-sensitivity differential scanning calorimetry has been applied to the study of the reversible thermal unfolding of the lysozyme of T4 bacteriophage in which the threonine residue at position 157 has been replaced by seven different residues . High-resolution structures derived from X-ray crystallography have been reported for these and six other mutants by Alber et al . {Alber, T., Dao-Pin, S., Wilson, K., Wozniak, J . A., Cook, S . P., & Matthews, B . W . (1987) Nature 330, 41-46} . At pH 2.5 the changes relative to the wild-type protein in the standard free energy of unfolding produced by these mutations indicate apparent destabilizations of 0.6 kcal mol-1 (T157R) to 1.9 kcal mol-1 (T157I), whereas the changes in enthalpy of unfolding range from -5.8 kcal mol-1 (T157N) to 11.9 kcal mol-1 (T157E) . Since the denaturations are in all cases accompanied by large changes in heat capacity amounting to 2.5 kcal K-1 mol-1, both the free energies and enthalpies are functions of temperature . An intriguing feature of the present results is the relatively large enthalpy changes and the corresponding compensating entropy changes . Our present understanding of the intramolecular energetics of proteins is insufficient to account for these changes.

Nature, 1991 Feb 14, 349(6310), 627 - 30
A homologue of the bacterial heat-shock gene DnaJ that alters protein sorting in yeast; Blumberg H et al.; Heat-shock proteins have been implicated in assembly of protein complexes, correct protein folding and uptake of proteins into organelles . In Escherichia coli, the heat-shock protein DnaJ and the Hsp70 homologue, DnaK, act together to disassemble a protein complex involved in bacteriophage lambda replication . We report the identification of SCJ1, a gene in the yeast Saccharomyces cerevisiae that encodes a homologue of the bacterial DnaJ protein . SCJ1 was identified by a genetic screen in which increased expression of candidate genes results in missorting of a nuclear-targeted test protein . The predicted amino-acid sequence of SCJ1 is 37% identical to the entire E . coli DnaJ protein . Hybridization experiments indicate that there is a family of yeast genes related to SCJ1 . These findings suggest that the Hsp70 DnaK-DnaJ interaction is general to eukaryotes.

Eur J Biochem, 1991 Feb 14, 195(3), 841 - 7
Lys631 residue in the active site of the bacteriophage T7 RNA polymerase . Affinity labeling and site-directed mutagenesis; Maksimova TG et al.; A highly selective affinity labeling of T7 RNA polymerase with the o-formylphenyl ester of GMP and {alpha-32P}UTP was carried out . The site of the labeling was located using limited cleavages with hydroxylamine, bromine, N-chlorosuccinimide and cyanogene bromide and was identified as the Lys631 residue . Site-directed mutagenesis using synthetic oligonucleotides was used to substitute Lys631 by a Gly, Leu or Arg residue . Kinetic studies of the purified mutant enzymes showed alterations of their polymerizing activity . For the Lys----Gly mutant enzyme, anomalous template binding was observed.

Nucleic Acids Res, 1991 Feb 11, 19(3), 585 - 90
Ligation with T4 RNA ligase of an oligodeoxyribonucleotide to covalently-linked cross-sectional base-pair analogues of short, normal, and long dimensions; Petric A et al.; Compounds that are covalent analogues of nucleic acid base pairs of normal, long, and short C1' to C1' dimensions {B . Devadas and N.J . Leonard (1990) J . Am . Chem . Soc., 112, 3125-3135.} have been added to the oligodeoxyribonucleotide d(A)6 with bacteriophage T4 RNA ligase as a prelude to placing them at defined loci within nucleic acid duplexes . Analogue cross sections that represent a normal Watson-Crick base pair as well as a pyrimidine-pyrimidine and a purine-purine apposition were ligated in modest yields (approximately 20%) to the oligonucleotide . Ligation conditions were optimized for each analogue, and the cross section was joined to only a single oligonucleotide in each case . The structures of the ligated products were proved by HPLC, enzymatic degradation, and spectroscopic analyses.

Biochemistry, 1991 Feb 5, 30(5), 1425 - 32
Second-site revertants of an inactive T4 lysozyme mutant restore activity by restructuring the active site cleft; Poteete AR et al.; Substitution of Thr26 by Gln in the lysozyme of bacteriophage T4 produces an enzyme with greatly reduced activity but essentially unaltered stability relative to wild type . Spontaneous second-site revertants of the mutant were selected genetically; two of them were chosen for structural and biochemical characterization . One revertant bears (in addition to the primary mutation) the substitution Tyr18----His, the other, Tyr18----Asp . The primary mutant and both revertant lysozyme genes were reconstructed in a plasmid-based expression system, and the proteins were produced and purified . The two revertant lysozymes exhibit enzymatic activities intermediate between wild type and the primary mutant; both also exhibit melting temperatures approximately 3 degrees C lower than either the wild type or the primary mutant . Crystals suitable for X-ray diffraction analysis were obtained from both revertant lysozymes, but not the primary mutant . Structures of the double mutant lysozymes were refined at 1.8-A resolution to crystallographic residuals of 15.1% (Tyr18----His) and 15.2% (Tyr18----Asp) . Model building suggests that the side chain of Gln26 in the primary mutant is forced to protrude into the active site cleft, resulting in low catalytic activity . In contrast, the crystal structures of the revertants reveal that the double substitutions (Gln26 and His18, or Gln26 and Asp18) fit into the same space that is occupied by Thr26 and Tyr18 in the wild-type enzyme; the effect is a restructuring of the surface of the active site cleft, with essentially no perturbation of the polypeptide backbone . This restructuring is effected by a novel series of hydrogen bonds and electrostatic interactions that apparently stabilize the revertant structures.

J Biol Chem, 1991 Feb 5, 266(4), 2531 - 6
Resolution of branched DNA substrates by T7 endonuclease I and its inhibition; Lu M et al.; Endonuclease I is a multipurpose enzyme implicated in the breakdown of host DNA, packaging of phage DNA, and recombination during the lytic cycle of bacteriophage T7 . We investigate here some aspects of the substrate requirements for its activity in resolving branched intermediates similar to Holliday junctions (Holliday, R . (1964) Genet . Res . 5, 282-304) that arise in recombination . The enzyme is able to resolve branched substrates containing very short duplex arms: 4 base pairs suffice . It cleaves 5' to the branch, with a distinct preference for the non-crossover strands in Holliday-like model junctions . Ligands that interact strongly with the branch site can inhibit the enzyme, with KI values in the 10-50 microM range.

Mol Microbiol, 1991 Feb, 5(2), 373 - 9
Nucleotide sequence of the Escherichia coli thymidine kinase gene provides evidence for conservation of functional domains and quaternary structure; Black ME et al.; Using lambda bacteriophage clones from the Kohara Escherichia coli library spanning minutes 25.5 to 28.5 on the E . coli chromosome (strain W3110), two overlapping DNA fragments were identified which were able to confer thymidine kinase (TK) enzyme activity to a TK- strain of E . coli (KY895) . This genetic complementation assay was used in concert with subcloning procedures to identify the minimal region (a 900 bp EcoRI-SalI fragment) which contained the E . coli thymidine kinase gene (tdk) . The nucleotide sequence of the EcoRI-SalI fragment and a small portion of the adjoining downstream fragment was determined . Computer analysis of the derived sequence indicated the presence of a rightward-reading open reading frame of 615 bp which was capable of encoding a 205-amino-acid polypeptide with a predicted Mr of 23458 daltons . The in vivo transcriptional activity of this locus was confirmed by Northern blot hybridization analysis of RNA isolated from E . coli JM101 or KY895 which detected a 650-nucleotide RNA transcribed from this region . This places the tdk gene at approximately minute 27.35 on the E . coli W3110 chromosome, about 15 kb downstream from the narG locus and approximately 25 kb upstream of the trp operon . Although the predicted Mr of the E . coli TK protein was 23.5 kDa, gel-filtration analyses suggested that, like eukaryotic thymidine kinases, the active form of this enzyme is a multimeric complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene, 1991 Feb 1, 98(1), 83 - 8
High-level expression of a semisynthetic dam gene in Escherichia coli; Hulsmann KH et al.; We constructed a semisynthetic gene encoding a DNA-adenine-methyltransferase (Dam) that codes for the same amino acid sequence as the wild type (wt) Escherichia coli dam gene . Since for unknown reasons the entire wt sequence, from the start codon to the end of the gene, could not be cloned, a gene was constructed consisting of a chemically synthesized 5' portion and a 3' portion from the E . coli chromosome . Introduction of this semisynthetic gene into a suitable vector allows overproduction of E . coli Dam in mg amounts per liter E . coli culture, with optimum expression of the gene in the vector pJLA503 . This plasmid places the target gene under control of the strong, tandemly arranged pR pL promoters from bacteriophage lambda, regulated by a temperature-sensitive lambda repressor . A rapid, two-column purification protocol is described that allows for very fast purification of the protein . The 32-kDa recombinant protein methylates the sequence GATC.

Genomics, 1991 Feb, 9(2), 247 - 56
The Chinese hamster HPRT gene: restriction map, sequence analysis, and multiplex PCR deletion screen; Rossiter BJ et al.; The fine structure of the Chinese hamster hypoxanthine guanine phosphoribosyltransferase (HPRT) gene has been determined; the gene has nine exons and is dispersed over 36 kb DNA . Exons 2-9 are contained within overlapping lambda bacteriophage clones and exon 1 was obtained by an inverse polymerase chain reaction (PCR) . All the exons have been sequenced, together with their immediate flanking regions, and these sequences compared to those of the mouse and human HPRT genes . Sequences immediately flanking all exons but the first show considerable homology between the different species but the region around exon 1 is less conserved, apart from the preserved location of putative functional elements . Oligonucleotide primers derived from sequences flanking the HPRT gene exons were used to amplify simultaneously seven exon-containing fragments in a multiplex PCR . This simple procedure was used to identify total and partial gene deletions among Chinese hamster HPRT-deficient mutants . The multiplex PCR is quicker to perform than Southern analysis, traditionally used to study such mutants, and also provides specific exon-containing fragments for further analysis . The Chinese hamster HPRT gene is often used as a target for mutation studies in vitro because of the ease of selection of forward and reverse mutants; the information presented here will enhance the means of investigating molecular defects within this gene.

J Bacteriol, 1991 Feb, 173(4), 1536 - 43
Expression of gene 1.2 and gene 10 of bacteriophage T7 is lethal to F plasmid-containing Escherichia coli; Schmitt CK et al.; Plasmids expressing bacteriophage T7 gene 1.2 or gene 10 DNA transform F plasmid-containing strains of Escherichia coli only at low efficiency, though they transform plasmid-free strains normally . The gene products T7 gp1.2 and T7 gp10 appear to be the toxic agents, and their effects are directed towards the product of the F pifA gene, PifA . T7 gp1.2 and gp10 are also the two targets of the pif exclusion system of F, and their synthesis normally triggers the abortive infection of T7 in pifA+ hosts . The properties of plasmids containing T7 gene 1.2 or 10 suggest that they can be used to study the molecular mechanisms of phage exclusion in model systems that avoid the pleiotropic dysfunctions associated with an abortive infection.

J Bacteriol, 1991 Feb, 173(3), 947 - 54
Defective transcription of the right end of bacteriophage T7 DNA during an abortive infection of F plasmid-containing Escherichia coli; Beck PJ et al.; Transcription of T7 and mutant T3 DNA during infections of F plasmid-containing cells has been analyzed by using Southern hybridization . A transcriptional defect is apparent in these abortively infected cells that is most severe in the class III region of the phage genome . In particular, RNAs that are initiated from the gene 13 promoter are not elongated to give full-length molecules . It is suggested that the transcription defect results from positive supercoiling of the template DNA and that torsional constraints may even prevent the complete entry of the phage genome into an abortively infected cell.

Virology, 1991 Feb, 180(2), 709 - 15
Dissection of functional domains of the packaging protein of bacteriophage T3 by site-directed mutagenesis; Kimura M et al.; Intracellular phage T3 DNA is synthesized as a concatemer in which unit-length molecules are joined together in head-to-tail fashion through terminally redundant sequences . During packaging of DNA, mature monomers are cut from the concatemer . The cutting is obligatorily coupled to DNA packaging . The packaging of phage DNA is under the control of a pair of noncapsid proteins, called packaging proteins, gp 18 and gp19 . gp19 is an ATP-binding protein that plays multiple roles in DNA packaging . gp19 is predicted, from the sequence of its gene, to contain 586 amino acids, and has consensus sequences for an ATP binding site . To dissect structure-function relationships of gp19, mutations were introduced into the ATP binding domain and the mutant proteins were overproduced, purified and characterized . Mutant gp19 with a Gly-to-Asp mutation at amino acid 61 (gp19 G61D) was defective in DNA packaging due to an altered interaction with ATP . Gp19 G424E, with a change in another putative ATP binding domain, was active in DNA packaging but was defective in DNA cutting . A second mutation in the latter domain, gp19 K430T, and a mutation at 553 (to give gp19 H553L), within a putative Mg2+ binding domain, had only minor effects on gp19 activities.

Bioorg Khim, 1991 Feb, 17(2), 189 - 96
{Hybrid human lymphokine genes . I.Design of recombinant plasmids coding hybrids of immune interferon and human tumor necrosis factor}; Korobko VG et al.; Recombinant plasmids coding for hybrid proteins between human interferon gamma and human tumour necrosis factor alpha or beta have been constructed using site-directed mutagenesis . The genes were fused via a synthetic oligonucleotide linker coding for tetrapeptide Pro-Val-Gly-Pro . The fused genes were expressed in Escherichia coli under control of early promoters of bacteriophage T7 . E . coli cells harbouring the plasmids with the hybrid genes gave rather high level of the fused proteins biosynthesis . The hybrid recombinant proteins proved to be unstable in E . coli cells.

Mol Microbiol, 1991 Feb, 5(2), 299 - 306
Identification of gene products of the P1 operon of Mycoplasma pneumoniae; Sperker B et al.; Gene P1 of Mycoplasma pneumoniae, which codes for a major adhesin, is flanked by two sequences with open reading frames designated ORF4 and ORF6 (Inamine et al., 1988b) . In order to identify proteins translated from those ORFs, gene fusions between the N-terminus of the RNA replicase of the Escherichia coli bacteriophage MS2 and selected regions of ORF4 and ORF6 were constructed . The corresponding fusion proteins synthesized in Escherichia coli were used to immunize mice . Antisera directed against ORF4-related sequences did not recognize M . pneumoniae antigens in Western blot analysis, but antisera directed against ORF-6-derived fusion proteins reacted with two M . pneumoniae proteins of 40 kDa and 90 kDa . In addition, some of the antisera also recognized proteins that formed in a sodium dodecyl sulphate/polyacrylamide gel a protein ladder between 115 and 145 kDa.

J Bacteriol, 1991 Feb, 173(4), 1485 - 91
Analysis of the Escherichia coli nusA10(Cs) allele: relating nucleotide changes to phenotypes; Craven MG et al.; The Escherichia coli nusA gene product, known to influence transcription elongation, is essential for both bacterial viability and growth of lambdoid phages . We report the cloning and sequencing of the conditionally lethal nusA10(Cs) allele . Changes from nusA+ were observed at nucleotides 311 and 634 . Functional studies showed that both nucleotide changes are necessary for the cold-sensitive phenotype, although bacteria with the change at 634 grew more slowly at 30 degrees C than the nusA+ controls . The mutant nusA10(Cs) gene product is not as active as nusA+ in supporting transcription antitermination mediated by the N regulatory protein of bacteriophage lambda . The change at nucleotide 634 is shown to be solely responsible for this phenotype . Four differences were found between the nusA+ gene that we sequenced and the published nusA sequence . These changes alter the reading frame of nusA in a functionally important domain {as identified by the nusA1 and nusA11(Ts) mutations}, resulting in an arginine-rich region that may be involved with RNA binding.

J Bacteriol, 1991 Feb, 173(3), 1339 - 43
Specificity of mini-Mu bacteriophage insertions in a small plasmid; Castilho BA et al.; Target site selection for bacteriophage Mu transposition was studied in pools of over 10(7) independent mini-Mu insertions in pUC9, selected by transduction of the plasmid . Insertions in both orientations were clustered in three regions and, within these, at preferred sites.

J Bacteriol, 1991 Feb, 173(3), 1208 - 14
Transcription and initiation of ColE1 DNA replication in Escherichia coli K-12; Inoue N et al.; By S1 nuclease protection mapping, we characterized RNA transcripts and nascent ColE1 DNA synthesized in wild-type Escherichia coli cells after infection with lambda-mini-ColE1 hybrid bacteriophages . Transcription of the RNA II region of ColE1 DNA in vivo starts mostly from the RNA II promoter, which was identified by in vitro experiments, and ends at or near the ori site . Synthesis of the leading strand of ColE1 DNA was found to start at the ori site . Nevertheless, the molar ratio of the nascent DNA to the synthesized transcripts ending at the ori site was less than 0.05 . In bacterial rnh mutants whose RNase H activities were less than 0.06% of that of the wild type, transcription patterns, as well as nascent DNA synthesis, were still similar to those in rnh+ cells . However, in bacteria whose rnh gene was interrupted by insertion of a drug resistance gene, the number of transcripts ending at the ori site was much reduced and that of transcripts reading through the ori site was definitely increased relative to that observed in wild-type bacteria . These results suggested that cleavage of the RNA transcript at the ori site in vivo is dependent on RNase H activity, as demonstrated in the in vitro system, but most of the cleaved RNA is unable to prime initiation of ColE1 DNA synthesis efficiently.

Virology, 1991 Feb, 180(2), 716 - 28
Bacteriophage SPO1 middle transcripts; Scarlato V et al.; Phage SPO1 middle transcripts are known to fall into two classes, m and m1l . Class m1l transcripts continue to be made late in the viral infection, while the synthesis of class m transcripts ceases soon after the onset of replication and late transcription . The experiments that are reported here deal with the regulatory nature of this diversity . The accumulation of transcripts associated with eight middle promoters was analyzed by S1 nuclease mapping . DNA sequence surrounding these middle promoters was determined or redetermined, and the stability of RNA associated with most of these promoters was also analyzed . Class m1l transcription was shown to be associated with SPO1 middle promoters that remain active at late stages of viral development, when middle promoters of class m are repressed . The consensus sequences of class m and m1l middle promoters were found to be indistinguishable and the search for sequences consensual with late promoters yielded only divergent candidates . No other consensus sequence that is specific and exclusive to either class of middle promoters was detected within a hundred base pairs upstream or downstream of these promoters . Considerable variations in the stabilities of SPO1 middle transcripts were found . Two promoters that are only 71 base pairs apart yielded transcripts that had substantially different stabilities . The 5'-flanking segment of the transcript associated with the upstream promoter apparently conferred a high degree of stability on this RNA.

FEMS Microbiol Lett, 1991 Feb, 62(1), 43 - 7
A novel indicator system allows detection of point mutations conferring a Ner- phenotype of phage Mu; Mayer E et al.; A plasmid-based indicator system was constructed which allows the easy detection of point mutations within the ner gene of bacteriophage Mu and its regulatory region . Five mutants have been sequenced exhibiting point mutations within the ner gene itself or its upstream region . These data are discussed with respect to the activity of the Ner protein.

Genome, 1991 Feb, 34(1), 66 - 71
Abundance and DNA sequence of two-base repeat regions in tropical tree genomes; Condit R et al.; Tandem DNA repeats of two-base pairs are potentially important tools for population genetic studies because of their abundance and length variation . As part of our research into the ecology of tropical forest plants, we began a study of dinucleotide repeat regions in several genera of tropical trees . Genomic libraries in bacteriophage lambda were screened with the oligonucleotide probes poly(GT) and poly(AG) . Both types of repeat regions were abundant in the genomes of all six plant species examined . Using the size of inserts in the phage libraries and number of phage screened, we estimated that there were 5 x 10(3) to 3 x 10(5) poly(AC) sites per genome, with slightly more AG than AC sites . When libraries were made from smaller fragments of genomic DNA, abundance estimates were higher, suggesting that two-base repeat sites were clustered in the genome . Poly(AC) sites were 16-22 bp in length, and four of the five sequenced were adjacent to either poly(AG)or poly(AT) sites . Other repeat region s appeared in DNA flanking the AC sites . This further demonstrated that two-base repeats and other repetitive DNA were clustered in the genome . Two-base repeats are abundant in plant genomes and could provide a large number of polymorphic markers for studies of plant population genetics.

J Bacteriol, 1991 Feb, 173(3), 1287 - 97
Cloning, sequencing, and recombinational analysis with bacteriophage BF23 of the bacteriophage T5 oad gene encoding the receptor-binding protein; Krauel V et al.; Binding of bacteriophage T5 to its receptor, the Escherichia coli FhuA protein, is mediated by tail protein pb5 . In this article we confirm that pb5 is encoded by the T5 oad gene and describe the isolation, expression, and sequencing of this gene . In order to locate oad precisely, we analyzed recombinants between BF23, a T5-related phage with a different host range, and plasmid clones containing segments of the T5 chromosome . This analysis also showed that oad has little or no homology with hrs, the analogous BF23 gene . We were able to overproduce a protein that comigrates with pb5 after fusing a 2-kb segment containing oad to a phage T7 promoter . This segment contains an open reading frame that can encode a protein of the appropriate size . Its deduced amino acid sequence does not closely resemble that of any other protein in the database . The sequence upstream of the open reading frame shows typical characteristics of a promoter region with two overlapping, divergently orientated promoters.

Can J Microbiol, 1991 Feb, 37(2), 97 - 104
Colicinogeny of O157:H7 enterohemorrhagic Escherichia coli and the shielding of colicin and phage receptors by their O-antigenic side chains; Bradley DE et al.; Twenty O157:H7 enterohemorrhagic Escherichia coli strains from patients with different clinical conditions were tested for colicinogeny and the presence of Verotoxin (VT) genes . From bloody diarrhea cases, 7/8 isolates and from hemolytic uremic syndrome cases 3/5 isolates all synthesized what appeared to be colicin D . The remaining strains, which included 7 from asymptomatic sources, were noncolicinogenic . The plasmid determining the colicin was found to be 1.4 kb larger than the 5.2-kb pColD . The colicin D protein had a molecular weight of about 90,000, whereas the O157 colicin was 87,000 . The plasmid was designated pColD157 to reflect these differences . Of O157:H7 isolates 17/20 had genes for both of the Verotoxins VT1 and VT2, and the remaining 3/20 for VT1 only . There was no correlation between the presence of VT determinants and colicinogeny or symptoms . The O157:H7 strains exhibited significant resistance to other colicins and bacteriophages.

Int J Biol Macromol, 1991 Feb, 13(1), 14 - 6
Three-stranded alpha-fibrous proteins: the heptad repeat and its implications for structure; Conway JF et al.; Amino acid sequence data have been collected for the coiled-coil rod domains of three-stranded alpha-fibrous proteins--fibrinogen, laminin, tenascin, macrophage scavenger receptor protein and the leg fibre protein from bacteriophage . Such domains are characterized by a heptad substructure in which apolar residues occur alternately three and four residues apart . The distribution of residues in each position of the heptad has been analysed, and the results compared with those obtained for the two-stranded alpha-fibrous proteins, which include the intermediate filament and myosin families . Distinctions can be drawn between the sequences in two- and three-stranded coiled-coil structures and these provide criteria that will prove useful in predicting secondary and tertiary structure purely from sequence data.

J Bacteriol, 1991 Feb, 173(4), 1554 - 60
Characterization of the late-gene regulatory region of phage 21; Guo HC et al.; A segment of Escherichia coli bacteriophage 21 DNA encoding the late-gene regulator, Q21, and the late-gene leader RNA segment was sequenced; its structure is similar to those of the related phages lambda and 82 . The leader RNA is about 45 nucleotides long and consists essentially entirely of sequences encoding the p-independent terminator that is the putative target of the antitermination activity of Q21 . Like the corresponding regions of lambda and 82, the 21 late-gene promoter segment encodes an early transcription pause in vitro, at about nucleotide 18, during which Q21 presumably acts to modify RNA polymerase . The 21 Q gene, cloned in isolation, is active on the late-gene leader segment in trans, and its purified product is active as an antiterminator in vitro; Q21 represents a third late-gene antiterminator, in addition to those of lambda and 82 . There is little evident similarity in the primary sequences of the three Q genes.

J Bacteriol, 1991 Feb, 173(4), 1376 - 81
RNase T affects Escherichia coli growth and recovery from metabolic stress; Padmanabha KP et al.; To determine the essentiality and role of RNase T in RNA metabolism, we constructed an Escherichia coli chromosomal rnt::kan mutation by using gene replacement with a disrupted, plasmid-borne copy of the rnt gene . Cell extracts of a strain with mutations in RNases BN, D, II, and I and an interuppted rnt gene were devoid of RNase T activity, although they retained a low level (less than 10%) of exonucleolytic activity on tRNA-C-C-{14C}A due to two other unidentified RNases . A mutant lacking tRNA nucleotidyltransferase in addition to the aforementioned RNases accumulated only about 5% as much defective tRNA as did RNase T-positive cells, indicating that this RNase is responsible for essentially all tRNA end turnover in E . coli . tRNA from rnt::kan strains displayed a slightly reduced capacity to be aminoacylated, raising the possibility that RNase T may also participate in tRNA processing . Strains devoid of RNase T displayed slower growth rates than did the wild type, and this phenotype was accentuated by the absence of the other exoribonucleases . A strain lacking RNase T and other RNases displayed a normal response to UV irradiation and to the growth of bacteriophages but was severely affected in its ability to recover from a starvation regimen . The data demonstrate that the absence of RNase T affects the normal functioning of E . coli, but it can be compensated for to some degree by the presence of other RNases . Possible roles of RNase T in RNA metabolism are discussed.

Carcinogenesis, 1991 Feb, 12(2), 249 - 55
Construction of a recombinant adenovirus containing the denV gene from bacteriophage T4 which can partially restore the DNA repair deficiency in xeroderma pigmentosum fibroblasts; Colicos MA et al.; The denV gene from bacteriophage T4 encodes a pyrimidine dimer-specific endonuclease that has the capacity to initiate excision repair of DNA . Cells from excision repair-deficient xeroderma pigmentosum (XP) patients are able to carry out excision repair initiated by the denV gene product and introduction of the denV gene into XP cells results in the partial restoration of colony-forming ability after irradiation with UV light . In this work we have constructed a helper-independent recombinant human adenovirus, Ad5denV, which contains the denV gene . A 1.9 kb cartridge consisting of the denV gene flanked by the long terminal repeat (LTR) promoter from Rous sarcoma virus (RSV) and the simian virus 40 (SV40) polyadenylation (poly A) splice signals, was inserted into the E3 region of an E3 deletion mutant (Ad5d1E3) of adenovirus type 5 . Infection of human fibroblasts and other permissive human cells with Ad5denV resulted in lytic infection and expression of the denV gene was confirmed by primer extension of infected cell RNA . The ability of the denV gene to restore the DNA repair deficiency in XP fibroblasts was examined using host cell reactivation of viral structural antigen formation for UV-irradiated adenovirus . The control virus, Ad5VSV, was also a recombinant which contained the gene for vesicular stomatitis virus glycoprotein G inserted into the E3 region of Ad5d1E3 . UV survival of Ad5denV was similar to that of Ad5VSV following infection of two normal fibroblast strains and a Cockayne syndrome fibroblast strain, CS7SE, from complementation group B . In contrast, UV survival of Ad5denV was significantly greater than that for Ad5VSV after infection of three unrelated XP fibroblast strains from complementation groups A, C and E . However, UV survival of Ad5denV in the XP fibroblasts did not reach levels obtained in normal fibroblasts, indicating that restoration of the XP defect was partial.

Clin Genet, 1991 Feb, 39(2), 121 - 4
Using the polymerase chain reaction to maintain DNA probe inventories in clinical and diagnostic laboratories; Tarleton J et al.; Clinical and diagnostic DNA laboratories must maintain a large inventory of DNA probes for use in hybridization studies . The preparation of plasmid DNA and isolation of DNA fragments for use as probes in both expensive and time consuming . We present here a rapid and relatively inexpensive method of producing large amounts of DNA fragments from stocks, using the polymerase chain reaction (PCR) . Our experience over the past year using this technique has been very positive and we believe many laboratories could benefit by employing such a labor-saving approach to maintaining DNA probes . The technique uses the bacteriophage M13 DNA sequencing primers to amplify cloned inserts contained in commonly used plasmid vectors . As examples, we illustrate the use of DNA produced in this manner as probes for linkage analysis of the fragile X syndrome and for detection of deletions in the Duchenne muscular dystrophy gene . We have also found that at least two probes can be amplified in the same PCR reaction, allowing the detection of two different restriction fragment length polymorphisms (RFLP) simultaneously . It should be possible for laboratories to devise strategies particular to their individual needs using more than one DNA probe produced in the same PCR reaction to detect RFLP's . Such strategies would need only to consider that the predicted alleles of the multiple polymorphisms do not migrate to the same position during electrophoresis . Stocks of single or multiple probes produced by the PCR could then be maintained for more rapid Southern analyses.

Res Microbiol, 1991 Feb-Apr, 142(2-3), 295 - 9
Alkaline phosphatase fusions in the study of cell division genes; Barondess JJ et al.; Alkaline phosphatase fusions have been used to analyse plasmid- or phage-carried genes from the two-minute region of the Escherichia coli chromosome . These studies have revealed the following: 1) Bacteriophage lambda carries two genes for cell envelope proteins, lom and bor, that are expressed in lysogens and probably contribute to the pathogenicity of its E . coli host . 2) The ftsQ and ftsl gene products are integral proteins of the cytoplasmic membrane with small cytoplasmic domains and large periplasmic domains . 3) The ftsQ and ftsl gene products are made in very small amounts, on the order of 25 molecules per cell . 4) The ftsQ gene product is essential for cell growth and is required throughout the formation of the cell septum . 5) An open reading frame just upstream from ftsl, thought to be involved in cell division, is expressed and probably codes for a cytoplasmic membrane protein.

Res Microbiol, 1991 Feb-Apr, 142(2-3), 259 - 67
Synchronous division induced in Escherichia coli K12 by phage Mu: analysis of DNA topology and gene expression during the cell cycle; Ghelardini P et al.; Bacteriophage Mu mutants in gene gem (Mu gemts2) induce cycles of synchronous divisions after infection of a bacterial population in steady-state conditions . In this paper, two classes of gyrB mutants, synchronizable and non-synchronizable, are described . The existence of the non-synchronizable class suggests that the gyrase B subunit is involved with Gem in the process of synchronization . Cyclical variations in DNA topology of a resident multicopy plasmid occur during synchronous growth and correlate with a modulation of the chromosomal lacZ gene expression . Transcription data for the cell division genes, ftsZ and envA, obtained studying first steps in synchronous growth after infection, show that synthesis of the two mRNA is not constant . The specific mRNA of envA seems to be stimulated soon after infection, whereas the two transcripts initiating upstream from ftsZ apparently decrease to a basal level . In both cases, however, synthesis of the mRNA virtually doubles at the time of cell division.

Biochem Biophys Res Commun, 1991 Jan 31, 174(2), 1009 - 14
A quantifiable phenotype of viral propagation; Yin J; A system has been identified where a virus, replicating continuously on its host, displays a distinct and quantifiable phenotype, and thereby continuously reports on the state of the virus-host relationship . When bacteriophage T7 is plated out with its host, Escherichia coli, it establishes a constant-velocity infection wave, which is driven by an autocatalytic reaction-diffusion mechanism . The velocity--which is easily measured--continuously reflects the infection environment . The simplicity of the system extends to the investigator an unprecedented ability to monitor and control a viral infection process.

Biochemistry, 1991 Jan 29, 30(4), 1119 - 26
Stereochemical studies of the beta-elimination reactions at aldehydic abasic sites in DNA: endonuclease III from Escherichia coli, sodium hydroxide, and Lys-Trp-Lys; Mazumder A et al.; The DNA strand cleavage reaction catalyzed by endonuclease III from Escherichia coli (endo III) on the 3'-side of aldehyde abasic sites proceeds by a syn beta-elimination involving abstraction of the 2'-pro-S proton and formation of a trans alpha,beta-unsaturated aldose product; we previously reported the same stereochemical course for the reaction catalyzed by UV endonuclease V from bacteriophage T4 (UV endo V) {Mazumder, A., Gerlt, J . A., Rabow, L., Absalon, M . J., Stubbe, J., & Bolton, P . H . (1989) J . Am . Chem . Soc . 111, 8029-8030} . Since the UV endo V does not contain an 4Fe-4S center, the 4Fe-4S center present in endo III need not be assigned a unique role in the beta-elimination reaction . The beta-elimination reactions that occur under alkaline conditions (0.1 N NaOH) and in the presence of the tripeptide Lys-Trp-Lys proceed by anti beta-elimination mechanisms involving abstraction of the 2'-pro-R proton and formation of a trans alpha,beta-unsaturated aldose product . The different stereochemical outcomes of the enzymatic and nonenzymatic beta-elimination reactions support the hypothesis that the enzyme-catalyzed reactions may involve general-base-catalyzed abstraction of the 2'-pro-S proton by the internucleotidic phosphodiester leaving group.

J Biol Chem, 1991 Jan 25, 266(3), 1866 - 71
Role of a disulfide bond in the thermal stability of the LamB protein trimer in Escherichia coli outer membrane; Luckey M et al.; In order to understand the unusual heat resistance of LamB protein (the outer membrane component of the maltose transport system in Escherichia coli and its receptor for bacteriophage lambda), we investigated the role of its 2 cysteinyl residues . Our studies show that Cys22 and Cys38 form an intrasubunit disulfide bond which contributes to the heat stability of the LamB protein trimer . Physical evidence for the disulfide was obtained by using site-directed mutagenesis to convert Asn36 to Met, which allowed cyanogen bromide cleavage between the 2 cysteines . Upon reduction one of the N36M fragments migrated as two pieces, resolved by two-dimensional polyacrylamide gel electrophoresis . Other mutagenized LamB proteins, in which 1 or both Cys residues were converted to Ser, exhibited a sharp loss of thermal stability . In contrast to wild-type LamB protein trimer, which does not dissociate to monomers even after 60 min at 100 degrees C, only 10-15% of the mutant LamB proteins remain trimeric after boiling 10 min . The disulfide bond in LamB protein is not required for its transport function, since both mutagenized LamB protein and N-ethylmaleimide-labeled LamB protein exhibit normal uptake of sugars in proteoliposomes . Finally, the disulfide bond must not be between subunits of the LamB trimer since reversible dissociation of trimer is achieved by low pH or denaturants in the absence of reducing agent.

J Biol Chem, 1991 Jan 25, 266(3), 1830 - 40
Stimulation of the processivity of the DNA polymerase of bacteriophage T4 by the polymerase accessory proteins . The role of ATP hydrolysis; Jarvis TC et al.; In this paper we examine the role of the DNA polymerase accessory proteins in modulating the processivity of DNA synthesis by the bacteriophage T4-coded five protein "holoenzyme" replication complex in vitro . Primed single-stranded DNA was used as a template for the DNA synthesis reactions, and buffer conditions were chosen to mimic in vivo salt concentrations . We find that the accessory proteins significantly increase the DNA-bound lifetime of the holoenzyme complex but that the maximum lifetime of the complex is still less than 10 s at 22 degrees C . The accessory proteins greatly enhance the processivity of the holoenzyme relative to that of the polymerase alone . ATP hydrolysis catalyzed by the accessory proteins complex is required to achieve this enhancement . We have investigated the temporal relationship between ATP hydrolysis by the accessory proteins and primer elongation by the holoenzyme and find that ATPase activity is required for initial assembly of the holoenzyme complex but not for elongation per se . Thus we conclude that the increased processivity displayed by the holoenzyme in moving through regions of template secondary structure reflects the high intrinsic processivity of the holoenzyme complex itself rather than a requirement for a concomitant ATPase-driven helicase activity during elongation . We have also measured the ATPase activity of the accessory proteins as a function of polymerase concentration and find that the rate of ATP hydrolysis catalyzed by this complex decreases significantly when the accessory proteins are assembled (with polymerase and gene 32 protein) into the five-protein holoenzyme and coupled to primer elongation . Based on these results we discuss mechanisms by which the ATPase activity of the polymerase accessory proteins might stimulate the overall processivity of the holoenzyme.

J Biol Chem, 1991 Jan 25, 266(3), 1888 - 97
Bacteriophage T4 encodes an RNase H which removes RNA primers made by the T4 DNA replication system in vitro; Hollingsworth HC et al.; RNase H activity increases markedly after bacteriophage T4 infection of Escherichia coli MIC2003, an RNase H-deficient host . We have extensively purified the RNase H from these T4-infected cells and have shown that the RNase H activity copurifies with a 5' to 3' DNA exonuclease activity . The N-terminal sequence of a 35-kDa protein copurifying with the RNase H activity matches the terminus of the predicted product of an open reading frame (designated ORF A or 33.2) upstream of T4 gene 33, identified previously by Hahn and co-workers (Hahn, S., Kruse, U., and Ruger, W . (1986) Nucleic Acids Res . 14, 9311-9327) . Plasmids containing ORF A under the control of the T7 promoter express RNase H and 5' to 3' DNA exonuclease activities as well as a protein that comigrates on sodium dodecyl sulfate-polyacrylamide gels with the 35-kDa protein present in the RNase H purified from T4-infected cells . T4 RNase H removes the pentamer RNA primers from DNA chains initiated by the T4 primase-helicase (gene products 61 and 41) . Addition of T4 RNase H and T4 DNA ligase leads to extensive joining of discontinuous lagging strand fragments in the T4 DNA replication system in vitro.

J Mol Biol, 1991 Jan 20, 217(2), 223 - 7
Regulation of filamentous bacteriophage length by modification of electrostatic interactions between coat protein and DNA; Greenwood J et al.; Bacteriophage fd gene VIII, which encodes the major capsid protein, was mutated to convert the serine residue at position 47 to a lysine residue (S47K), thereby increasing the number of positively charged residues in the C-terminal region of the protein from four to five . The S47K coat protein underwent correct membrane insertion and processing but could not encapsidate the viral DNA, nor was it incorporated detectably with wild-type coat proteins into hybrid bacteriophage particles . However, hybrid virions could be constructed from the S47K coat protein and a second mutant coat protein, K48Q, the latter containing only three lysine residues in its C-terminal region . K48Q phage particles are approximately 35% longer than wild-type . Introducing the S47K protein shortened these particles, the S47K/K48Q hybrids exhibiting a range of lengths between those of K48Q and wild-type . These results indicate that filamentous bacteriophage length (and the DNA packaging underlying it) are regulated by unusually flexible electrostatic interactions between the C-terminal domain of the coat protein and the DNA . They strongly suggest that wild-type bacteriophage fd makes optimal use of the minimum number of coat protein subunits to package the DNA compactly.

Biochem Biophys Res Commun, 1991 Jan 15, 174(1), 323 - 30
Demonstration of extrinsic DNA from immune complexes in plasma of a patient with systemic lupus erythematosus; Terada K et al.; Antigen DNA isolated from immune complexes present in plasma of three patients with active systemic lupus erythematosus using an affinity column was cloned and sequenced . One clone, designated pKS7, was found to have a region homologous with that of the E . coli metK gene, and another, designated pKS8, had a region homologous with a sequence including the replication origin of bacteriophage f1 . Gel retardation assay revealed that pKS7 and pKS8 interacted with the patient's IgG fraction to form immune complexes, respectively . The affinity-purified antigen DNA was proved to be originated from bacteria or bacteriophage.

Proc Natl Acad Sci U S A, 1991 Jan 15, 88(2), 400 - 4
Location of the active site for enzyme-adenylate formation in DNA ligases; Tomkinson AE et al.; The enzyme-AMP reaction intermediate of the 102-kDa bovine DNA ligase I was digested with trypsin, and the adenylylated peptide was isolated by chromatography under conditions that maintain the acid-labile phosphoramidate bond . Microsequencing of the peptide showed that it contains an internal trypsin-resistant lysine residue, as expected for the site of adenylylation . Inhibition of DNA ligase I activity by pyridoxal 5'-phosphate also indicated the presence of a reactive lysine residue in the catalytic domain of the enzyme . Comparison of the known primary structures of several other DNA ligases with the adenylylated region of mammalian DNA ligase I allows their active sites to be tentatively assigned by sequence homology . The ATP-dependent DNA ligases of mammalian cells, fission yeast, budding yeast, vaccinia virus, and bacteriophages T3, T4, and T7 contain the active site motif Lys-Tyr/Ala-Asp-Gly-(Xaa)-Arg, with the reactive lysine residue flanked by hydrophobic amino acids . The distance between the postulated adenylylation site and the carboxyl terminus of the polypeptide is very similar in these ATP-dependent DNA ligases, whereas the size of the amino-terminal region is highly variable.

Biochemistry, 1991 Jan 15, 30(2), 589 - 94
Correlation between mutational destabilization of phage T4 lysozyme and increased unfolding rates; Klemm JD et al.; The thermodynamics and kinetics of unfolding of 28 bacteriophage T4 lysozyme variants were compared by using urea gradient gel electrophoresis . The mutations studied cause a variety of sequence changes at different residues throughout the polypeptide chain and result in a wide range of thermodynamic stabilities . A striking relationship was observed between the thermodynamic and kinetic effects of the amino acid replacements: All the substitutions that destabilized the native protein by 2 kcal/mol or more also increased the rate of unfolding . The observed increases in unfolding rate corresponded to a decrease in the activation energy of unfolding (delta Gu) at least 35% as large as the decrease in thermodynamic stability (delta Gu) . Thus, the destabilizing lesions bring the free energy of the native state closer to that of both the unfolded state and the transition state for folding and unfolding . Since a large fraction of the mutational destabilization is expressed between the transition state and the native conformation, the changes in folding energetics cannot be accounted for by effects on the unfolded state alone . The results also suggest that interactions throughout much of the folded structure are altered in the formation of the transition state during unfolding.

J Biol Chem, 1991 Jan 15, 266(2), 1348 - 53
Linker mutagenesis of a bacterial fatty acid transport protein . Identification of domains with functional importance; Kumar GB et al.; The product of the fadL gene (FadL) of Escherichia coli is a multifunctional integral outer-membrane protein required for the specific binding and transport of exogenous long-chain fatty acids {C12-C18} . FadL also serves as a receptor for the bacteriophage T2 . In order to define regions of functional importance within FadL, the fadL gene has been mutagenized by the insertion of single-stranded hexameric linkers into the unique SalI restriction site that lies towards the 3' end of the gene and into four HpaII restriction sites distributed throughout the coding region . The five insertion mutants were classified into three groups based on their specific growth rates (alpha) in minimal media containing the long-chain fatty acid oleate (C18:1) as a sole carbon and energy source: Oleslow, alpha = 0.035-0.045; Ole +/-, alpha = 0.020-0.035; and Ole-, alpha less than or equal to 0.005 (wild-type, alpha = 0.07-0.10) . The hexameric insertion at the SalI site (fadL allele termed S1; insertion after amino acid 410) conferred an Oleslow phenotype and resulted in a reduction of long-chain fatty acid transport (36% the wild-type level) . This insertion mutant, however, bound oleic acid at wild-type levels and was fully functional as a receptor for the bacteriophage T2 . The modified FadL-S1 protein did not have the heat-modifiable property characteristic of wild-type FadL . Insertions in the four HpaII sites (fadL alleles termed H1, H2, H3, and H5; after amino acids 41, 81, 238, and 389, respectively) resulted in all three classes of mutants . The fadL insertion mutant H5 was defective for long-chain fatty acid transport but bound oleic acid at significant levels . Together with the S1 allele, these data suggest that the carboxyl terminus of FadL is crucial for long-chain fatty acid transport . The insertion mutants H1 and H2 were defective for both oleic acid binding and transport suggesting that the amino terminus of FadL is important for long-chain fatty acid binding and transport . The fadL linker mutant H3 was defective in oleic acid binding yet had significant levels of oleic acid transport . These studies delineated for the first time different regions of the fadL gene that encode domains of FadL implicated in the binding and transport of long-chain fatty acids.

J Biol Chem, 1991 Jan 15, 266(2), 1269 - 75
The role of N3-ethyldeoxythymidine in mutagenesis and cytotoxicity by ethylating agents; Grevatt PC et al.; The significance of DNA ethylation at the central hydrogen-bonding site (N3) of thymine was investigated using an in vitro DNA replication system . The system utilized a primed template in which the 3'-end of the primer is eight nucleotides away from N3-ethyldeoxythymidine (N3-Et-dT), present at template position 26 from the 3'-end . The 34-nucleotide template corresponds to a specific DNA sequence at gene G of bacteriophage phi X174 . DNA synthesis products were quantitated by electrophoretic separation and autoradiography . At 10 microM dNTP and 0.5 mM Mn2+, N3-Et-dT blocked DNA synthesis by Escherichia coli polymerase I (Klenow fragment): 60% after incorporating a nucleotide opposite N3-Et-dT (incorporation-dependent blocked product) and 39% 3' to N3-Et-dT . DNA replication past the lesion (post-lesion synthesis) was negligible . Post-lesion synthesis increased using higher concentrations of dNTP, reaching 68% at 200 microM dNTP . DNA sequencing revealed that dA was incorporated opposite N3-Et-dT in the incorporation-dependent blocked product . In the post-lesion synthesis product, dT was exclusively incorporated opposite N3-Et-dT . Formation of the N3-Et-dT.dA base pair at the replication fork terminated DNA synthesis, while the N3-Et-dT.dT base pair formed at the 3'-end of the growing chain was extended, leading to an A.T----T.A transversion mutation . The results suggest a dual role for the N3-Et-dT lesion, contributing in part to the cytotoxicity and mutagenicity of ethylating agents . These studies provide a basis for understanding the activation of oncogene neu by A.T----T.A transversion mutation in rat neuroblastomas induced by N-ethyl-N-nitrosourea.

J Immunol, 1991 Jan 15, 146(2), 775 - 82
T cell receptor alpha and beta gene expression in a murine antigen-specific T suppressor lymphocyte clone with cytolytic potential; Heuer J et al.; The composition of alpha and beta TCR genes was analyzed in a murine BSA-specific Ts cell clone with cytolytic potential . The isolated poly(A)+ mRNA from Ts cell clone BVI/5 was used to construct a cDNA library in the bacteriophage lambda gt11 . Full-length cDNA clones specific for TCR alpha and TCR beta genes have been detected and isolated by hybridization with specific oligonucleotide probes . The functional rearranged TCR alpha gene is composed of a member of the V alpha 1 family, the junctional gene J alpha TT11 and C alpha . The gene segments V beta 13, D beta 2.1, J beta 2.4, and C beta 2 form the functional rearranged TCR beta gene . Furthermore, a nonfunctional TCR alpha gene transcript has been detected, where a V alpha 8 gene is rearranged to a so far not described J alpha gene segment (J alpha BVI) . A stop codon in its junctional region is responsible for this non-functional transcript . By using Southern blot analysis, the described rearranged TCR genes can be detected in the J alpha junctional region and in the J beta 2 cluster on the genomic DNA level . Immunoprecipitation studies with the KT3 anti-CD3 mAb and flow microfluorimetry analysis with the H57-597 anti-TCR-alpha/beta mAb show that TCR/CD3 complexes are synthesized and expressed on BVI/5 Ts cells . Taken together, the cDNA sequencing data, the protein studies, and the specificity of Ag recognition demonstrate that the BVI/5 Ts cell clone not only transcribes the TCR alpha and beta genes but also expresses a functional BSA-specific receptor.

Proc Natl Acad Sci U S A, 1991 Jan 15, 88(2), 588 - 92
A switch in the formation of alternative DNA loops modulates lambda site-specific recombination; Moitoso de Vargas L et al.; The virally encoded Xis protein is one of the components in the site-specific recombination reactions of bacteriophage lambda . It is required for excisive recombination and inhibits integrative recombination . The mechanism of Xis inhibition of the integration reaction was investigated by methylation protection assays (footprinting analyses) in conjunction with recombination assays . Xis is shown to mediate the formation of a specific attP looped structure involving cooperative and competitive long-range interactions among integrase, integration host factor, and Xis proteins . This higher-order structure precludes supercoiled attP from engaging in the productive partner interactions that lead to execution of the first strand exchange in integrative recombination . In addition to its previously characterized role in excision, Xis-induced DNA bending is postulated to act as a regulatory switch (in an alternative loop mechanism) that converts the attP intasome from an integrative-competent complex to a nonreactive one.

Cell, 1991 Jan 11, 64(1), 171 - 9
Kinetic and structural analysis of a cleaved donor intermediate and a strand transfer intermediate in Tn10 transposition; Haniford DB et al.; Tn10 transposes by a nonreplicative "cut and paste" mechanism . We describe here two protein-DNA complexes that are reaction intermediates in the Tn10 transposition process: a cleaved donor complex whose DNA component consists of transposon sequences cleanly excised from flanking donor DNA, and a strand transfer complex whose DNA component contains transposon termini specifically joined to a target site . The kinetic behavior of the first species suggests that it is an early intermediate in the transposition reaction . These two Tn10 complexes are closely analogous to complexes identified in the pathway for replicative "cointegrate" formation by bacteriophage Mu and thus represent intermediates that may be common to both nonreplicative and replicative transposition . These and other results suggest that the Tn10 and Mu reactions are fundamentally very similar despite their very different biological outcomes . The critical difference between the two reactions is the fate of the DNA strand that is not joined to target DNA.

Biochemistry, 1991 Jan 8, 30(1), 189 - 98
Preparation and characterization of N-(1-pyrenyl)iodoacetamide-labeled Escherichia coli RNA polymerase; Johnson RS et al.; N-(1-Pyrenyl)iodoacetamide has been used to introduce fluorescent probes into Escherichia coli RNA polymerase . After an incubation time of 15 min, approximately 2 pyrene equiv was introduced per enzyme molecule . There was no further increase in modification after more extended periods of incubation . Neither calf thymus DNA nor nucleotides protected the holoenzyme from modification . Thus, the sites of modification do not appear to involve the binding sites for polynucleotides or the ribonucleoside triphosphates . From the isolation and analysis of the individual subunits, it was found that sigma contained approximately 1 pyrene equiv, beta contained 0.6, beta' contained 0.6, and alpha less than 0.1 . Spectral and Stern-Volmer analyses indicate that the covalently attached pyrene molecules are in comparable apolar microenvironments . On the basis of CD analyses, the introduction of pyrene molecules into RNA polymerase alters its secondary structure . This alteration in secondary structure manifests itself by a reduction in overall enzymatic activity . Transcript analysis of the products obtained by using a linearized plasmid containing the A1 promoter and the Te terminator of bacteriophage T7 indicates that the pyrenyl derivative is capable of producing full-length transcripts and that it has an efficiency of chain termination comparable to the native enzyme . Analysis of tau plots for the interaction of the pyrenyl derivative and the native enzyme, respectively, with the A1 promoter yielded comparable values for the isomerization constant in the conversion of the closed complex to an open one . Comparable values were also obtained for the association constant . The rate of chain elongation for the pyrenyl derivative, however, is approximately 54% of that observed for the native enzyme . Thus, the decrease in overall transcriptional activity observed with the pyrenyl derivative is not due to a decrease in the efficiency of initiation or premature termination, but rather to a decrease in the rate of chain elongation.

J Biol Chem, 1991 Jan 5, 266(1), 645 - 51
Specific contacts between the bacteriophage T3, T7, and SP6 RNA polymerases and their promoters; Jorgensen ED et al.; The specificity and structural simplicity of the bacteriophage T3, T7, and SP6 RNA polymerases make these enzymes particularly well suited for studies of polymerase-promoter interactions . To understand the initial recognition process between the enzyme and its promoters, DNA fragments that carry phage promoters were chemically modified by three different methods: base methylation, phosphate ethylation, and base removal . The positions at which these modifications prevented or enhanced binding by the RNA polymerases were then determined . The results indicate that specific contacts within the major groove of the promoter between positions-5 and -12 are important for phage polymerase binding . Removal of individual bases from either strand of the initiation region (-5 to +3) resulted in enhanced binding of the polymerase, suggesting that disruption of the helix in this region may play a role in stabilization of the polymerase-promoter complexes.

Gene, 1991 Jan 2, 97(1), 13 - 9
Cloning and overexpression of the gene encoding bacteriophage T5 DNA polymerase; Chatterjee DK et al.; T5 DNA polymerase (T5Pol), an essential enzyme for bacteriophage T5 DNA replication, is unusual because of its high processivity and strand-displacing ability . These two properties in a single polypeptide make T5Pol an ideal candidate for structural and functional analysis . Therefore, the structural gene encoding the DNA polymerase of bacteriophage T5 (T5pol) has been cloned and overexpressed in Escherichia coli . Elimination of sequences upstream from the 5' end of the T5pol by exonuclease III digestion was necessary to obtain stable clones containing a full-length structural gene . Determination of the nucleotide (nt) sequence of the region deleted during clone construction revealed the presence of a promoter sequence having extensive homology with known T5 phage 'early' promoters . By primer extension of mRNA isolated from T5 phage-infected cells, two successive G residues located 6 and 7 nt downstream from the -10 region of this promoter were identified as the initiating nt at the 5' end of T5pol mRNA . T5Pol produced in E . coli from the cloned gene under control of a tac or phage lambda pL promoter represented as much as 40% of total cell protein . The majority of the T5Pol present in extracts of E . coli was insoluble . The amount of active enzyme present was estimated to be a maximum of tenfold higher than that found in extracts of T5 phage-infected cells.

Gene, 1991 Jan 2, 97(1), 7 - 12
Bacteriophage lambda promoters pL and pR: sequence determinants of in vivo activity and of sensitivity to the DNA gyrase inhibitor, coumermycin; Kincade JM et al.; Sequence encompassing the region between bp -43 and +8 of the pL and pR promoters of bacteriophage lambda, as well as sequence variants of these promoters, were compared with respect to their ability to drive a promoterless cat gene in vivo . For both pL- and pR-based promoters, variants with one nonconsensus bp rather than the consensus promoter were found to be maximally active . Determination of promoter function in CSH26 and C600 revealed a marked strain dependence in the activity of some promoter variants . In response to the antibiotic coumermycin, which effects a reduction in DNA superhelical density in vivo, promoters were found to be activated, inhibited or unaffected, depending on their sequence . No simple correlation between a particular response and sequence features of a promoter has become apparent.

Proteins, 1991, 10(1), 10 - 21
Comparison of the crystal structure of bacteriophage T4 lysozyme at low, medium, and high ionic strengths; Bell JA et al.; Crystals of bacteriophage T4 lysozyme used for structural studies are routinely grown from concentrated phosphate solutions . It has been found that crystals in the same space group can also be grown from solutions containing 0.05 M imidazole chloride, 0.4 M sodium choride, and 30% polyethylene glycol 3500 . These crystals, in addition, can also be equilibrated with a similar mother liquor in which the sodium chloride concentration is reduced to 0.025 M . The availability of these three crystal variants has permitted the structure of T4 lysozyme to be compared at low, medium, and high ionic strength . At the same time the X-ray structure of phage T4 lysozyme crystallized from phosphate solutions has been further refined against a new and improved X-ray diffraction data set . The structures of T4 lysozyme in the crystals grown with polyethylene glycol as a precipitant, regardless of the sodium chloride concentration, were very similar to the structure in crystals grown from concentrated phosphate solutions . The main differences are related to the formation of mixed disulfides between cysteine residues 54 and 97 and 2-mercaptoethanol, rather than to the differences in the salt concentration in the crystal mother liquor . Formation of the mixed disulfide at residue 54 resulted in the displacement of Arg-52 and the disruption of the salt bridge between this residue and Glu-62 . Other than this change, no obvious alterations in existing salt bridges in T4 lysozyme were observed . Neither did the reduction in the ionic strength of the mother liquor result in the formation of new salt bridge interactions . These results are consistent with the ideas that a crystal structure determined at high salt concentrations is a good representation of the structure at lower ionic strengths, and that models of electrostatic interactions in proteins that are based on crystal structures determined at high salt concentrations are likely to be relevant at physiological ionic strengths.

Gerontology, 1991, 37(1-3), 5 - 32
Drosophila to bacteriophage to erythrocyte: the erythrocyte as a model for molecular and membrane aging of terminally differentiated cells; Kay MM; Senescent cell antigen appears on old cells and marks them for death by initiating the binding of IgG autoantibody and subsequent removal by phagocytes in mammals and other vertebrates . Although the initial studies are done using erythrocytes as a model, senescent cell antigen has been found on all cells examined . Oxidation generates senescent cell antigen in situ . Senescent cell antigen is generated by the modification of an important structural and transport membrane molecule, protein band 3 . Band 3 is a ubiquitous protein . It is present in cell, nuclear, Golgi, and mitochondrial membranes . Band 3 is the most heavily used anion exchanger in the human body because of its crucial role in respiration and acid-base balance . Senescent cell antigen has been localized to band 3 residues 538-554 and 812-827, using competitive inhibition studies with synthetic peptides of band 3 to absorb the IgG isolated from senescent erythrocytes and immunoblotting studies . In mammalian brain, band 3 performs the same functions as that of erythroid band 3 . These functions are anion transport, ankyrin binding, and generation of senescent cell antigen, an aging antigen that terminates the life of cells . Our results suggest that the transport domain of erythroid and neural band 3 are similar functionally and structural . This supports the hypothesis that the immunological mechanism of maintaining homeostasis is a general physiologic process for removing senescent and damaged cells in mammals and other vertebrates.

Biopolymers, 1991 Jan, 31(1), 11 - 21
Variation of the permeability of bacteriophage T4: analysis by use of a protein-specific probe for the T4 interior; Griess GA et al.; The permeability of bacteriophage T4 and the change in T4 permeability caused by mutation to osmotic shock resistance are investigated here by quantification of the kinetics with which both a DNA-specific probe (ethidium) and a protein-specific probe {1,1'-bi(4-anilino)naphthalene-5,5'-di-sulfonic acid, or bis-ANS)} bind to T4 . In the case of an osmotic shock-resistant mutant, T40s41, both ethidium and bis-ANS bind with first order kinetics . The first-order rate constant (k*) for both bis-ANS and ethidium is a function of anion type and concentration . Adenosine triphosphate, phosphate, bisulfite, sulfate, and acetate anions all reduce k* below the k* observed when chloride is the only anion . When chloride is the only anion at 25 degrees C, k* values for binding to T40s41 are orders of magnitude above k* values for binding to wild-type T4 (T4wt) . At 25 degrees C, k* for T4wt is too small to measure but k* for T4wt increases at 50-55 degrees C to values approaching those measured for T40s41, without inactivating T4wt, when chloride is the only anion; during heating, T4wt is stabilized by both ethidium and bis-ANS . Binding to T4wt is reversible at 50-55 degrees C, but not at 25 degrees C . Equilibrium binding of bis-ANS to T40s41 reveals 112 +/- 24 sites per T4 capsid . Equilibrium binding of ethidium to T40s41 reveals both high- and low-affinity sites previously observed in the packaged DNA of other bacteriophages . The ATP-induced decrease in k* is not accompanied by a decrease in equilibrium binding . The following hypotheses are presented to explain the above data: (a) All detected bis-ANS binding sites on T4 are interior to the outer surface of T4 . (b) The value of k* for both bis-ANS and ethidium is controlled at the port(s) of passage through the outer shell of the T4 capsid . (c) The anions present control k* values at the port(s) of entry, probably by controlling the size of this port . The effects on k* of phosphate explain the otherwise paradoxical observation {P . J . McCall and V . A . Bloomfield (1976) Biopolymers 15, 2323-2336} that in a phosphate buffer the permeabilities of T4wt and T40s41 are the same.

Genetics, 1991 Jan, 127(1), 61 - 73
A novel recombinator in yeast based on gene II protein from bacteriophage f1; Strathern JN et al.; Interchromosomal mitotic recombination in yeast can be stimulated by the protein encoded by gene II of bacteriophage f1 . The normal role of the gene II enzyme is to make a site-specific cleavage of a particular strand of the duplex form of the bacteriophage DNA at the origin of DNA replication . The gene II protein was expressed in yeast in an attempt to determine the role of nicked DNA in the initiation of recombination . Stimulation of recombination in yeast by the gene II protein was dependent on the presence of a recognition site for gene II enzyme in the region being assayed . Recombination was stimulated in both directions from the gene II recognition site but showed a directional bias . The distribution of alleles among the recombinants indicated that the chromosome with the gene II recognition site acted as the recipient in gene conversion events.

Cytogenet Cell Genet, 1991, 56(1), 27 - 30
Painting of defined chromosomal regions by in situ suppression hybridization of libraries from laser-microdissected chromosomes; Lengauer C et al.; "Painting" of defined chromosomal regions provides a powerful tool for cytogenetic analyses . Here, we demonstrate that chromosomal in situ suppression (CISS)-hybridization of DNA libraries derived by microcloning laser-microdissected chromosomal regions can be applied to achieve this goal . As an example, we used unbanded metaphase spreads from a female patient carrying a balanced translocation . t(1;7)(1qter----1p36::7q11----7qter) . Fragments from the long arms of 130 translocation chromosomes were microdissected . After microcloning, human inserts with an average size of about 3 kb were pooled from 400 recombinant bacteriophage DNA clones and used as a complex probe set in CISS-hybridization experiments . This resulted in painting of the translocation chromosome along the region 7q35 to 1p31 . Painted chromosomal subregions in normal chromosomes 1 and 7 were consistent with this finding . This approach may be used to perform painting of any chromosome regions for which microlibraries can be established . Possible applications include the definition of marker chromosomes in clinical and tumor cytogenetics and studies of chromosomal evolution, as well as studies of nuclear chromosome topography in animal and plant species.

J Bacteriol, 1991 Jan, 173(1), 37 - 45
Mutational analysis of a bacteriophage P4 late promoter; Van Bokkelen GB et al.; Transcription from the late Psid promoter of satellite bacteriophage P4 is dependent on the bacterial RNA polymerase carrying the sigma 70 subunit and is positively regulated by the product of the P4 delta gene or the ogr gene of helper bacteriophage P2 . Through deletion and mutational analyses of the Psid promoter, we identified mutations in the -10 region and in a region of hyphenated dyad symmetry centered around position -55 that inactivate Psid . Most of these mutations alter base pairs that are highly conserved in the five other delta-activated P4 and P2 late promoters . We propose that the P4 delta and P2 ogr gene products bind the -55 region of the P4 and P2 late promoters.

J Bacteriol, 1991 Jan, 173(1), 353 - 64
Nucleotide sequence of the Escherichia coli recJ chromosomal region and construction of recJ-overexpression plasmids; Lovett ST et al.; The nucleotide sequence of the recJ gene of Escherichia coli K-12 and two upstream coding regions was determined . Three regions were identified within these two upstream genes that exhibited weak to moderate promoter activity in fusions to the galK gene and are candidates for the recJ promoter . recJ appeared to be poorly translated: the recJ nucleotide sequence revealed a suboptimal initiation codon GUG, no discernible ribosome-binding consensus sequence, and relatively nonbiased synonymous codon usage . Comparison of the sequence of this region of the chromosome with DNA data bases identified the gene immediately downstream of recJ as prfB, which encodes translational release factor 2 and has been mapped near recJ at 62 min . No significant homology between recJ and other previously sequenced regions of DNA was detected . However, protein sequence comparisons with a gene upstream of recJ, denoted xprB, revealed significant homology with several site-specific recombination proteins . Its genetic function is presently unknown . Knowledge of the nucleotide sequence of recJ allowed the construction of a plasmid from which overexpression of RecJ protein could be induced . Supporting the notion that translation of recJ is limiting, a strong T7 bacteriophage promoter upstream of recJ did not, by itself, allow high-level expression of RecJ protein . The addition of a ribosome-binding sequence fused to the initiator GTG of recJ in this construction was necessary to promote expression of high levels of RecJ protein.

J Bacteriol, 1991 Jan, 173(1), 150 - 5
Isolation of temperature-sensitive McrA and McrB mutations and complementation analysis of the McrBC region of Escherichia coli K-12; Piekarowicz A et al.; We isolated temperature-sensitive mcrA and mcrBC mutants of Escherichia coli . At 42 degrees C, they were unable to restrict the T-even bacteriophages T6gt and T4gt or plasmids encoding cloned DNA methylase genes whose specificities confer sensitivity to the McrA and McrBC nucleases . Complementation analysis of the McrBC region (mcrB251) with the complete cloned McrBC system or a derivative with mcrB alone indicated that the mutation shows an absolute defect for the restriction of DNA containing hydroxymethylcytosine and a thermosensitive defect for the restriction of DNA containing methylcytosine . The properties of the McrA temperature-sensitive mutants suggest that some of these mutations can also influence the restriction of DNA containing hydroxymethylcytosine or methylcytosine residues.

Proc Natl Acad Sci U S A, 1991 Jan 1, 88(1), 179 - 83
Functional division and reconstruction of a plasmid replication origin: molecular dissection of the oriV of the broad-host-range plasmid RSF1010; Honda Y et al.; Two single-stranded DNA initiation signals (designated ssi) present in the origin of vegetative DNA replication (oriV) of the broad-host-range plasmid RSF1010 are essential for the priming of replication of each complementary DNA strand of this plasmid in Escherichia coli . Each of the RSF1010 ssi signals, ssiA and ssiB, could be replaced by a primosome assembly site from plasmid pACY184 or from bacteriophage phi X174 . In these chimeric origins, replication of the strand complementary to that containing the primosome assembly site was no longer dependent on the RSF1010 primase, protein RepB', but required the E . coli primase, DnaG . If both ssiA and ssiB sites of RSF1010 were replaced by primosome assembly sites, protein RepB' was no longer essential for the replication at this origin, whereas proteins RepA and RepC of RSF1010 were still required . These results strongly suggest that the two ssi sites and the RepB' protein actually direct the priming of DNA synthesis in the replication of RSF1010, and the proteins RepA and RepC are involved in the prepriming events--i.e., the opening of the DNA duplex at oriV . It is evident that the origin of RSF1010 can be separated into three functional domains and reconstructed by replacing the ssi sites with heterologous elements.

Virology, 1991 Jan, 180(1), 372 - 9
Infectious in vitro transcripts from a cloned cDNA of barley yellow dwarf virus; Young MJ et al.; A full-length cDNA clone of barley yellow dwarf virus (BYDV-PAV serotype) has been constructed and fused to the bacteriophage T7 RNA polymerase promoter . RNA transcripts produced in vitro, either capped or uncapped, were infectious in Triticum monococcum protoplasts . Protoplasts inoculated with in vitro-transcribed BYDV RNA accumulated coat protein, synthesized new viral RNAs, and produced virus particles . Aphid feeding on extracts from protoplasts inoculated with in vitro RNA transcripts can be used to transfer the virus progeny to whole plants . Introduction of mutations which interrupt specific BYDV-PAV open reading frames (ORFs) V and VI eliminated infectivity while an ORF I-mutant remained infectious . Infectious RNA transcripts derived from BYDV cDNA clones will facilitate analysis of the molecular aspects of BYDV infection and further enhance our understanding of this economically important virus.

Anim Genet, 1991, 22(3), 285 - 9
DNA fingerprints of sheep using an M13 probe; Gatei MH et al.; The bacteriophage M13 DNA was used to detect hypervariable minisatellites in several families of Booroola sheep as well as Merino and Suffolk sheep . Digestion of sheep DNA gave rise to three to eight fragments with different restriction enzymes demonstrating considerable polymorphism between the different breeds . The length of informative DNA fragments varied in size from 6 to 20kb . The DNA fingerprints generated were individual specific and allowed for differentiation between closely related animals . The pattern obtained with sheep DNA was different from that observed with humans and other vertebrates in the proportion of high molecular weight DNA fragments present . Pedigree analysis of DNA patterns of dams and their offspring for several sets of twins and triplets showed a clear distinction between individuals and failed to reveal the presence of monozygosity.

NIDA Res Monogr, 1991, 105, 116 - 22
Tissue-specific splicing of pro-enkephalin mRNA; Howells RD et al.; A testis cDNA library derived from 4-6 week-old rats was screened using a 32P-labeled 435 base pair (bp) cDNA probe derived from exon 3 of rat brain proenkephalin (PE) . Two positive clones were isolated from an initial screening of approximately 20,000 recombinant bacteriophage plaques . The longest insert (1,500 bp) was sequenced and was found to contain a portion of intron A at the 5'-terminus . Intron B was sliced as it is in the brain, therefore, the domain structure of the cDNA was intron A-exon 2-exon 3, reading in the 5'-3' direction . Since the translational initiator codon is located at the 5'-terminus of exon 2, the reading frame of PE is unaltered, however, within intron A are 4 upstream AUG codons which exist in a favorable context for initiation of translation . It is probable that translation of PE would be inhibited by the presence of these upstream initiator codons, which may explain the discrepancy between the levels of PE mRNA and opioid peptides in the testis . Hypophysectomy prior to the onset of puberty was found to drastically reduce the level of PE mRNA in the testis and epididymus, suggesting that pituitary factors affect the expression of the PE gene in these tissues, either directly or indirectly.

Arch Virol, 1991, 117(3-4), 183 - 92
The derivation of a restriction endonuclease map for Alcelaphine herpesvirus 1 DNA; Bridgen A; The gammaherpesvirus Alcelaphine herpesvirus 1 (AHV-1), indigenous to the wildebeest (Connochaetes species), is a causative agent of the fatal lymphoproliferative disease malignant catarrhal fever in cattle and deer . The genome of the attenuated WC11 isolate of AHV-1 has previously been shown to possess a region of unique DNA together with multiple direct repeat sequences . Approximately 70% of the genome of WC11 has now been cloned into plasmid or bacteriophage vectors and these clones have been used in hybridisation experiments to construct a restriction endonuclease map of the WC11 unique DNA with respect to BamHI . EcoRI, SalI, SmaI and XhoI . The map allows the size of the unique region of the WC11 genome to be estimated as 130 kbp and thus the entire genome as 155-160 kbp . The results confirm a terminal location for the repeat sequences.

J Bacteriol, 1991 Jan, 173(2), 869 - 78
Deletion mutagenesis independent of recombination in bacteriophage T7; Scearce LM et al.; Deletion between directly repeated DNA sequences in bacteriophage T7-infected Escherichia coli was examined . The phage ligase gene was interrupted by insertion of synthetic DNA designed so that the inserts were bracketed by 10-bp direct repeats . Deletion between the direct repeats eliminated the insert and restored the ability of the phage to make its own ligase . The deletion frequency of inserts of 85 bp or less was of the order of 10(-6) deletions per replication . The deletion frequency dropped sharply in the range between 85 and 94 bp and then decreased at a much lower rate over the range from 94 to 900 bp . To see whether a deletion was predominantly caused by intermolecular recombination between the leftmost direct repeat on one chromosome and the rightmost direct repeat on a distinct chromosome, genetic markers were introduced to the left and right of the insert in the ligase gene . Short deletions of 29 bp and longer deletions of approximately 350 bp were examined in this way . Phage which underwent deletion between the direct repeats had the same frequency of recombination between the left and right flanking markers as was found in controls in which no deletion events took place . These data argue against intermolecular recombination between direct repeats as a major factor in deletion in T7-infected E . coli.

Nucleic Acids Symp Ser, 1991, (24), 181 - 4
Atomic structure of a pyrimidine-dimer specific excision-repair enzyme from bacteriophage T4; Morikawa K et al.; T4 endonuclease V is an enzyme responsible for the first step of a pyrimidine-dimer specific excision-repair process . The three-dimensional structure of this enzyme has been determined at 1.6A resolution by X-ray crystallography . The enzyme consists of one single compact domain classified into the all alpha structure . This single domain possesses two distinct catalytic activities, a pyrimidine-dimer glycosylase and an apurinic/apyrimidinic endonuclease . The backbone of the enzyme represents a unique folding scheme incompatible with close packing of alpha-helices . The refined structure suggests possible residues that participate in interactions with DNA.

Mol Biochem Parasitol, 1991 Jan, 44(1), 63 - 72
A sensitive repetitive DNA probe that is specific to the Leishmania donovani complex and its use as an epidemiological and diagnostic reagent; Howard MK et al.; Leishmania donovani (HU3 strain) metacyclic promastigotes generated in vitro were used to construct a cDNA library in the bacteriophage vector lambda gt10 . A cDNA clone (Lmet 2), was isolated by differential screening with metacyclic-derived or log-phase promastigote-derived cDNA . The clone insert was comprised predominantly of four copies of an imperfect 60-bp repeat motif, which was represented in the genome by multiple tandem repeats distributed among at least six chromosomes . The corresponding mRNA transcript was a developmentally regulated 12-kb doublet . The Lmet 2 sequence was entirely specific to L . donovani donovani, L . donovani (East Africa), L . donovani infantum and L . donovani chagasi, even when genomic Southern blots and slot blots of other Leishmania species were washed at low stringencies . Twenty-two strains of L . donovani were clearly detected by radiolabelled Lmet 2 cDNA probe, with signals of approximately equal intensity, irrespective of geographical origin, which encompassed widely dispersed endemic regions . The probe could detect DNA from fewer than 100 organisms and identified small numbers of promastigotes in infected sand flies . Amastigotes were also detected in impression smears of organs from infected hamsters . The Lmet 2 probe is likely to be a valuable reagent for clinical diagnosis and epidemiological investigations.

Acta Biochim Pol, 1991, 38(1), 191 - 200
Role of the Escherichia coli grpE heat shock protein in the initiation of bacteriophage lambda DNA replication; Osipiuk J et al.; Initiation of replication of lambda DNA requires assembly of the proper nucleoprotein complex consisting of the lambda origin of replication-lambda O-lambda P-dnaB proteins . The dnaJ, dnaK and grpE heat shock proteins destabilize the lambda P-dnaB interaction in this complex permitting dnaB helicase to unwind lambda DNA near ori lambda sequence . First step of this disassembling reaction is the binding of dnaK protein to lambda P protein . In this report we examined the influence of dnaJ and grpE proteins on stability of the lambda P-dnaK complex . Our results show that grpE alone dissociates this complex, but both grpE and dnaJ together do not . These results suggest that, in the presence of grpE protein, dnaK protein has a higher affinity for lambda P protein complexed with dnaJ protein than in the situation where grpE protein is not used.

Ciba Found Symp, 1991, 159, 145 - 51; discussion 151-5
Screening combinatorial antibody libraries for catalytic acyl transfer reactions; Sastry L et al.; A bacteriophage lambda vector system for the expression of Fab fragments from the mouse antibody repertoire in Escherichia coli has been described . We have used this system to generate a catalytic antibody from a combinatorial antibody library . Monoclonal antibody 43C9 was raised against a transition state analogue of the hydrolysis of carboxyamide . mRNA from hybridoma cells expressing this antibody was cloned into phage lambda and clones that expressed the mRNA for either the heavy or the light chain of the antibody were isolated . These individual libraries were then crossed to generate a combinatorial library in which clones coexpressed the heavy and light chains . This library was screened for antibodies/Fab fragments that bound to the original antigen with high affinity . DNA sequencing showed that these fragments were the same as those in antibody 43C9 . Three different clones were found to catalyse the hydrolysis of carboxyamide . More efficient expression vectors and improved screening techniques should lead to the isolation of many more catalytic antibodies from combinatorial antibody libraries.

Lasers Surg Med, 1991, 11(4), 380 - 4
In vitro production of viable bacteriophage in carbon dioxide and argon laser plumes; Matchette LS et al.; Both CO2 and argon laser ablation of an agar substrate containing high titres of bacteriophage phi X174 create plumes which disperse viable phage particles . Irradiances at the beam impact site ranged from 73 to 215 W/cm2 for the CO2 laser and from 40 to 227 W/cm2 for the argon laser . To increase the absorption of argon laser radiation, oxidized hemoglobin was added to the target material . Plume-borne viable phage were observed to be associated with particles large enough to settle out from the plume within 100 mm of the beam impact site . The ratio of the number of dispersed viable phage to the number of viable phage potentially dispersible by a single, 1-second laser exposure was on the order of 10(-6) to 10(-5).

EXS, 1991, 58, 70 - 84
DNA fingerprinting of the human intestinal parasite Giardia intestinalis with hypervariable minisatellite sequences; Upcroft P; Individual isolates of the Giardia duodenalis group of protozoan intestinal parasites were identified by DNA fingerprinting with hypervariable minisatellite sequences . A morphologically identical parasite is found in some forty different animal species . Although the species name intestinalis is reserved for the human isolates, electrophoretic karyotyping suggests that most duodenalis isolates fall into the same species grouping . Distinction based upon morphology, restriction endonuclease cleavage of genomic DNA or isoenzyme analysis has not been adequate to identify individual strains . The successful use of hypervariable sequences in the identification of individual human genomes encouraged us to examine the use of these same sequences for the possible identification of parasite isolates . We initially use as a fingerprinting probe the genome of the bacteriophage M13, which has repeated sequences recognising homologous hypervariable sequences in the human genome . The M13 probe recognises a weakly homologous set of hypervariable sequences in Giardia . The number of informative bands is comparable to those seen in mammals, since the lower molecular weight bands are also useful . There is considerable divergence in the sequences of individual Giardia minisatellites . Some cloned Giardia hypervariable sequences are more homologous to M13 than they are to each other . Similar results were observed with the hypervariable repeat sequences 3' to the human alpha-globin gene when they were used as a probe to distinguish Giardia isolates . The poly(dA-dC).poly(dG-dT) probe which recognises frequent TG tracts in a number of organisms also detects a few variable bands amidst a hybridisation background in the Giardia genome . Thus Giardia isolates which could not be distinguished by restriction endonuclease cleavage, antibody typing or isoenzyme analysis have been identified by DNA fingerprinting procedures . Detailed analysis of strain movement, resurgence, variation, host range and drug resistance is now possible . Similar families of sequences may be widespread in lower eukaryotes and useful for generating individual specific fingerprints . A procedure for detecting individual parasites is also presented . Since Giardia is regarded as the most ancient eukaryote before the occurrence of symbiosis with purple non-sulphur bacteria to generate mitochondria, the identification of hypervariable sequences in the Giardia genome should also aid in understanding the mechanism of generation and evolution of these sequences.

Res Microbiol, 1991 Jan, 142(1), 13 - 21
Global changes in gene expression in Escherichia coli K12 induced by bacteriophage Mu Gem protein; Butler RH et al.; We have studied the growth properties of some Mu lysogens with respect to the non-lysogenic strain and have observed that the division time in minimal medium was increased over 4-fold when the bacteria carried the prophage mutated in the gem gene (Mu gem3) . Since this phage gene has previously been shown to be involved in modulation of expression of host genes, we have analysed the proteins extracted from lysogens and non-lysogens as a rapid assay of global gene expression . The pattern of proteins extracted showed marked quantitative variations between non-lysogens, lysogens for wild-type Mu and lysogens for phage Mu gem3 . These effects were no longer as evident when the strains were grown in rich medium . This dramatic change in the physiological state of the lysogenic strain versus the non-lysogenic in particular growth conditions extends the concept of lysogeny . For many years, the prophage has been considered only as a potentially lethal factor, while here it also appears as a genetic element capable of profoundly modifying host biology.

Anal Biochem, 1991 Jan, 192(1), 17 - 22
A nonradioactive screening method for cloning genes encoding sequence-specific DNA binding proteins; Tan TH; We have developed a novel nonradioactive screening method for cloning genes encoding sequence-specific DNA binding proteins . This method is derived from previously described protocols developed for the same purpose by using radioactively labeled DNA probes containing protein recognition sequences . This nonradioactive strategy relies upon the use of a small hapten, digoxigenin . Fusion proteins expressed from the recombinant bacteriophage lambda gt11/lambda ZAP are immobilized on nitrocellulose filters and probed with digoxigenin-labeled double-stranded DNA as a ligand . The specifically bound DNA probes can be detected through sequential incubations with antibody-enzyme conjugate and enzyme substrates . This technique has been successfully utilized to isolate several cDNA clones encoding DNA binding proteins.

Mol Biochem Parasitol, 1991 Jan, 44(1), 43 - 9
Vaccination against Taenia taeniaeformis infection in rats using a recombinant protein and preliminary analysis of the induced antibody response; Ito A et al.; Primary screening of a cDNA expression library of Taenia taeniaeformis oncospheres in lambda gt11 bacteriophage was carried out using rabbit anti-T, taeniaeformis oncosphere serum affinity-purified from oncosphere pellets . From approximately 1.6 x 10(5) plaques, 21 single clones that were positive with the affinity-purified antibodies were isolated . Sibling analysis revealed that 17 clones out of the 21 could be assigned to five different antigen families . Only family 1 was strongly recognized by a serum prepared in a rabbit against a partially purified host-protective oncosphere antigen fraction . The fragments of lambda DNA were inserted into a pGEX plasmid vector that encodes glutathione S-transferase (GST) of Schistosoma japonicum . Clones designated TtO-18, -49.53 (family 1), 46 (family 2), 15 (family 3), 40 (family 4) and 66 (family 5) were established as subclones in pGEX-1 plasmid vectors which produced GST fusion proteins . All GST fusion proteins were soluble and recognized by anti-GST and anti-TtO sera . Three vaccination experiments with these fusion proteins using specific-pathogen-free Wistar rats revealed that all three fusion proteins of family 1 were exclusively effective against T . taeniaeformis oncosphere challenge with approximately 95% and 91% reductions in cystic metacestode and total metacestode recoveries, respectively . Rats vaccinated with fusion proteins of family 1 produced antibodies which reacted with a 21-kDa oncosphere antigen component which appeared to be a major oncosphere stage-specific antigen.

J Bacteriol, 1991 Jan, 173(2), 893 - 5
Complementation of the lytD1 mutation of Escherichia coli by either the cI or cro gene of bacteriophage lambda; Dai DX et al.; The lytD1 mutant of Escherichia coli exhibits temperature-sensitive growth which is attributed to cellular autolysis at the restrictive temperature . Either of two cloned phage lambda genes, identified as cI and cro, suppressed the lytD1(Ts) lysis phenotype, suggesting that lytD encodes a DNA-binding protein with a DNA-binding specificity similar to that of CI and Cro . LytD may be a repressor of a gene(s) involved in cellular autolysis.

J Bacteriol, 1991 Jan, 173(2), 810 - 5
Genetic analysis of the cIII gene of bacteriophage HK022; Kornitzer D et al.; The cIII gene product of lambdoid bacteriophages promotes lysogeny by stabilizing the phage-encoded CII protein, a transcriptional activator of the repressor and integrase genes . Previous works showed that the synthesis of the bacteriophage lambda CIII protein has specific translational requirements imposed by the structure of the mRNA . To gain insight into the mRNA structure and its role in regulating cIII translation, we undertook a mutational analysis of the cIII gene of the related bacteriophage HK022 . Our data support the hypothesis that in HK022, as in lambda, translation initiation requires a specific mRNA structure . In addition, we found that translation of HK022 cIII, like that of lambda, is strongly reduced in a host deficient in the endonuclease RNase III.

J Bacteriol, 1991 Jan, 173(2), 734 - 40
The early promoters of bacteriophage HK022: contrasts and similarities to other lambdoid phages; Cam KM et al.; The pL, pR and pM promoters of lambdoid phages direct the transcription of early phage genes and the prophage repressor gene . We have determined the start points of transcription for these three promoters in the lambdoid phage HK022 and have shown that the HK022 repressor represses the early promoters, pL and pR, and activates the repressor promoter, pM . HK022 resembles other phages of the lambda family in these respects, as it does in the functional organization of most of its early genes and sites . One exception is nun, the first gene of the HK022 pL operon, which is expressed in the presence of prophage repressor and thus differs from its lambda counterpart, gene N . We show that transcription of nun in a lysogen does not initiate at pL but instead starts upstream at the pM promoter . This difference in transcription fits the different roles of Nun and N proteins in the physiology of the two phages: Nun protects HK022 lysogens against superinfection with certain other lambdoid phages, while N promotes the transcription of early lambda genes.

J Bacteriol, 1991 Jan, 173(2), 609 - 17
Genetic analysis of Escherichia coli integration host factor interactions with its bacteriophage lambda H' recognition site; Lee EC et al.; The bacteriophage P22-based challenge phage system was used to study the binding of integration host factor (IHF) to its H' recognition site in the attP region of bacteriophage lambda . We constructed challenge phages that carried H' inserts in both orientations within the P22 Pant promoter, which is required for antirepressor synthesis . We found that IHF repressed expression of Pant from either challenge phage when expressed from an inducible Ptac promoter on a plasmid vector . Mutants containing changes in the H' inserts that decrease or eliminate IHF binding were isolated by selecting challenge phages that could synthesize antirepressor in the presence of IHF . Sequence analysis of 31 mutants showed that most changes were base pair substitutions within the H' insert . Approximately one-half of the mutants contained substitutions that changed base pairs that are part of the IHF consensus binding site; mutants were isolated that contained substitutions at six of the nine base pairs of the consensus site . Other mutants contained changes at base pairs between the two subdeterminants of the H' site, at positions that are not specified in the consensus sequence, and in the dA + dT-rich region that flanks the consensus region of the site . Taken together, these results show that single-base-pair changes at positions outside of the proposed consensus bases can weaken or drastically disrupt IHF binding to the mutated site.

J Bacteriol, 1991 Jan, 173(1), 391 - 3
A newly identified DNA replication terminus site, TerE, on the Escherichia coli chromosome; Hidaka M et al.; To search for heretofore unidentified DNA replication termination (Ter) sites on the Escherichia coli chromosome, we screened the entire Kohara lambda bacteriophage library using as probes the four known 22-bp Ter sequences . We found a Ter site, which we named TerE, located at 23.2 min on the linkage map . TerE inhibits only counterclockwise DNA replication . Macroscopically, five Ter sites are located in a periodic arrangement on the genome.

Ciba Found Symp, 1991, 161, 91 - 103; discussion 103-7
The application of computational methods to the study of enzyme catalysis by triose-phosphate isomerase and stabilities of variants of bacteriophage T4 lysozyme; Kollman PA et al.; We review our research on triose-phosphate isomerase and bacteriophage T4 lysozyme . In our studies over the last ten years we have used electrostatic potentials, computer graphics, quantum mechanics, molecular mechanics, molecular dynamics and free energy calculations to try to understand why triose-phosphate isomerase is such an efficient enzyme and why its efficiency is dramatically decreased by several site-specific mutations . For T4 lysozyme we have used free energy methods to analyse and try to understand why Thr-157----Val and Thr-157----Ala mutations decrease protein stability by about 1-2 kcal/mol.

Ciba Found Symp, 1991, 161, 63 - 74
Simulation analysis of the stability mutants R96H of bacteriophage T4 lysozyme and I96A of barnase; Karplus M et al.; Free energy simulation methods are used to analyse the effects of the mutation Arg-96----His on the stability of bacteriophage T4 lysozyme and of Ile-96----Ala on the stability of barnase . By use of thermodynamic integration, the contributions of specific interactions to the free energy change are evaluated . It is shown that a number of contributions that stabilize the wild-type or the mutant partially cancel in the overall free energy difference; some of these involve the unfolded state . Comparison of the results with conclusions based on structural and thermodynamic data leads to new insights into the origin of the stability difference between wild-type and mutant proteins . For the charged-to-charged amino acid mutation in T4 lysozyme, the importance of the contributions of more distant residues, solvent water and the covalent linkage involving the mutated amino acid are of particular interest . Also, the analysis of the Arg-96 to His mutation with respect to the interactions with the C-terminal end of a helix (residues 82-90) indicates that the nearby carbonyl groups (Tyr-88 and Asp-89) make the dominant contribution, that the amide groups do not contribute significantly and that the helix dipole model is inappropriate for this case . For the non-polar-to-non-polar amino acid mutation in barnase, the solvent contribution is unimportant, and covalent terms are shown to be significant because they do not cancel between the folded and unfolded state.

Ciba Found Symp, 1991, 161, 52 - 62
Structural and genetic analysis of electrostatic and other interactions in bacteriophage T4 lysozyme; Dao-pin S et al.; The lysozyme from bacteriophage T4 is being used as a model system to determine the roles of individual amino acids in the folding and stability of a typical globular protein . Such studies can provide quantitative information on the contributions made by different types of interactions including hydrogen bonds, hydrophobic interactions, salt bridges and disulphide bridges . To determine the contribution of long-range electrostatic interactions a combination of charge-change mutations was used to reduce the overall formal charge on T4 lysozyme at neutral pH from +9 to +1 units . Such changes in charge were found to have little effect on the stability of the molecule . Salt bridges engineered on the surface of the protein also were found to contribute little to stability . In contrast, the introduction of acidic groups designed to interact with the partial positive charges at the N-termini of alpha-helices consistently increased the stability of the protein . It is argued that this difference between electrostatic salt-bridge interactions and electrostatic 'helix-dipole' interactions lies in the entropic cost of bringing together the interacting partners . In an attempt to simplify the folding problem, and also to further investigate the helix propensity of different amino acids, a series of alanines was introduced within an alpha-helix of T4 lysozyme . The resultant protein not only folds normally but is also more stable than the wild-type enzyme, adding further support to recent evidence that alanine is a helix-favouring amino acid.

Biomed Biochim Acta, 1991, 50(4-6), 599 - 605
The human cystatin gene family: cloning of three members and evolutionary relationship between cystatins and Bowman-Birk type proteinase inhibitors; Saitoh E et al.; Three genes from the human cystatin gene family have been isolated from a bacteriophage lambda library containing Hind III digests of human genomic DNA . The cloned genes were identified with three DNA probes each containing exon 1, exon 2 and exon 3 of the CST1 gene for cystatin SN . The genes, which we name CST2B, CST4, and CST5, are 6.8 kb, 5.4 kb and 12.5 kb in size, respectively . Statistical analysis of DNA sequence homology elucidated that the second and third exons of cystatin (family II) genes and three cystatin (family II) gene like segments in the kininogen (family III) genes are significantly homologous to the gene segments coding for the inhibitory domains of Bowman-Birk type proteinase inhibitors.

DNA Seq, 1991, 1(5), 335 - 46
Sequence and structure of a phenylalanine ammonia-lyase gene from Glycine max; Frank RL et al.; The gene encoding a key enzyme in anthocyanin biosynthesis, phenylalanine ammonia-lyase (PAL), was cloned from soybean (Glycine max) . The purpose was to obtain a molecular probe to study the organization of this gene family in soybean and to examine novel regulatory mechanisms present in the anthocyanin biosynthetic pathway of this system . A soybean genomic library was constructed in the bacteriophage vector lambda Charon 35 . A PAL cDNA clone from Phaseolus vulgaris was used in screening the library, and two PAL genes were isolated . One gene was sequenced entirely and analyzed by sequence homology to the PAL2 gene of Phaseolus vulgaris . Genomic analysis indicates that PAL sequences of Glycine max exist as a small gene family consisting of only two to three members . The representative PAL gene sequenced (PAL1) has a coding region of 2142 basepairs divided among two exons . The single intron is 1519 basepairs and splits the 131st codon.

Acta Biochim Pol, 1991, 38(3), 311 - 9
Expression of bacteriophage T4 minor baseplate proteins in the bacteriophage T7 promoter/RNA polymerase expression system; Schlichtholtz B et al.; The central part of bacteriophage T4 baseplate is built of several proteins which are present in only a few copies per phage particle . Only some of these minor baseplate components have been identified previously as distinct protein species by biochemical analysis . We have used the bacteriophage T7 RNA polymerase expression system to identify and overexpress the minor baseplate proteins . The products of genes 25, 26 and 51 were identified on the autoradiographs after selective labelling with {35}S methionine . The overexpression of gene 25 and 51 products was high enough to make possible undertaking their purification and studies of their properties.

DNA Seq, 1991, 1(6), 389 - 94
Experience in shotgun sequencing a 134 kilobase pair DNA molecule; Davison AJ; Until now, large DNA sequences have been obtained by cloning fragments of the target molecule into plasmid, cosmid or bacteriophage lambda vectors . The 134 kbp DNA sequence of channel catfish virus was determined with relative ease by shotgun cloning of random fragments of genomic DNA directly into a bacteriophage M13 vector, sequencing by dideoxynucleotide chain termination, and compilation of the data using Staden's database handling programs . Experience gained during this endeavour indicates that sequences substantially larger than 134 kbp may be obtained using this approach.

Cytometry, 1991, 12(2), 167 - 71
Detection and analysis by dual-laser flow cytometry of bacteriophage T4 DNA inside Escherichia coli; Sanders CA et al.; Bacteriophage T4 DNA was detected and analyzed inside E . coli by dual-laser flow cytometry using a dye combination of Hoechst 33258 (H33258) and chromomycin A3 (CA3) which bind to A-T- and G-C-rich regions of DNA, respectively . An exponentially-growing culture of E . coli ATCC 11303 was infected with T4 bacteriophage at a 1:1 multiplicity of infection . Samples were taken immediately and at 5 min intervals and placed on ice to interrupt viral replication . The samples were then centrifuged, ethanol-fixed, stained with H33258 and CA3, and analyzed by flow cytometry . Twenty-five minutes post-infection, a population of cells which contained T4 DNA began to appear on both a bivariate contour plot and a frequency histogram plot of the data . By 35 min, T4 DNA-containing cells could be distinguished from E . coli cells containing little or no T4 DNA . The ratio of CA3:H33258 fluorescence was then used to calculate the % G + C value for T4 DNA inside E . coli . A value of 35.6 +/- 0.2% was obtained, which agrees with % G + C values determined by traditional methods . These results demonstrate that dual-laser flow cytometry can be used to study viral DNA inside the bacterial host.

Genomics, 1991 Jan, 9(1), 124 - 30
A yeast artificial chromosome contig encompassing the cystic fibrosis locus; Anand R et al.; The gene responsible for cystic fibrosis (CF) has recently been identified . Coding sequence for the cystic fibrosis transmembrane conductance regulator (CFTR) spans at least 230 kb of the human genome . Although all 27 exons of the gene are represented in cosmid or bacteriophage clones, there are still several gaps in the physical map of this region . It should be possible to complete the map and to clone the entire CFTR gene in a single fragment of DNA using a yeast artificial chromosome (YAC) vector . Herein we describe the construction and physical mapping of a 1.5-Mb YAC contig which encompasses D7S8 (J3.11) and D7S23 (KM19), two genetic loci flanking the CF locus . One of the clones in the contig, 37AB12, contains a 310-kb YAC which includes the entire CFTR gene and flanking sequence in both the 5' and 3' directions.

Biomed Sci, 1991, 2(6), 634 - 40
Construction and properties of fusion proteins between human interferon-gamma and human tumour necrosis factors alpha and beta; Korobko VG et al.; A number of recombinant plasmids coding for fusion proteins between human interferon-gamma (IFN-gamma) and human tumour necrosis factor alpha (TNF alpha) or beta (TNF beta) were constructed by using site-directed mutagenesis and ligation of the respective genes . In these proteins the whole IFN-gamma sequence of the molecule is linked at the N terminus via a short polypeptide linker to the TNF alpha sequence lacking two N-terminal amino acid residues or to the whole TNF beta sequence . A series of mutants with deletions in the interferon part of the fusion proteins were also produced . All the fusion genes obtained were efficiently expressed in Escherichia coli under the control of early promoters of bacteriophage T7 . The recombinant fusion proteins were found to be unstable inside bacterial cells . Bacterial cell lysates expressing these fusion genes or their deletion mutants showed both biological activities in vitro: the antiviral activity of IFN-gamma and the cytotoxic activity of TNF.

Dev Biol Stand, 1991, 75, 173 - 5
Viral contamination of fetal bovine serum used for tissue culture: risks and concerns; Erickson GA et al.; Four viral contaminants have been routinely detected in unprocessed and commercial lots of fetal bovine serum: bacteriophage, infectious bovine rhinotracheitis, parainfluenza-3 and bovine viral diarrhea virus (BVDV) . Of those, BVDV is consistently present in a majority of commercial lots of fetal bovine serum . Methods for BVDV detection and removal are reviewed . The tentative role of an unclassified pestivirus in microcephaly of infants has been reported . Its significance remains uncertain.

Eur J Biochem, 1990 Dec 27, 194(3), 889 - 96
Expression of human liver cytochrome P450 IIIA4 in yeast . A functional model for the hepatic enzyme; Renaud JP et al.; Cytochrome P-450 (P450) NF, a member of the P450 IIIA subfamily, is the major contributor to the oxidation of the calcium-channel blocker nifedipine in human liver microsomes . A cDNA clone designated NF25 encoding for human P450 NF was isolated from a bacteriophage lambda gt11 expression library {Beaune, P . H., Umbenhauer, D . R., Bork, R . W., Lloyd, R . S . & Guengerich, F . P . (1986) Proc . Natl Acad . Sci . USA 83, 8064-8068} . We have expressed NF25 cDNA in Saccharomyces cerevisiae using an expression vector constructed from pYeDP1/8-2 {Cullin, C . & Pompon, D . (1988) Gene 65, 203-217} . Yeast transformed with the plasmid containing the NF25 sequence (pVNF25) showed a ferrous-CO spectrum typical of cytochrome P-450 . Microsomal preparations contained a protein with an apparent molecular mass identical to that of P450-5 (a form isolated from human liver indistinguishable from P450 NF) that was not present in microsomes from control yeast (transformed with pYeDP1/8-2 alone), as revealed by immunoblotting with anti-P450-5 antibodies . On the other hand, antibodies raised in rabbits against human liver P450 IIC8-10 and rat liver P450 IA1 and P450 IIE1 did not recognize yeast-expressed P450 NF25 . The P450 NF25 content in microsomes was about 90 pmol/mg protein . Microsomal, yeast-expressed P450 NF25 exhibited a high affinity for different substrates including macrolide antibiotics, dihydroergotamine and miconazole as shown by difference visible spectroscopy . Microsomal suspensions containing P450 NF25 were also able to catalyze several oxidation reactions that were expected from the activities of the protein isolated from human liver, including nifedipine 1,4-oxidation, quinidine 3-hydroxylation and N-oxygenation, and N-demethylation of the macrolide antibiotics erythromycin and troleandomycin . The yeast endogenous NADPH-cytochrome P-450 reductase thus couples efficiently with the heterologous P450 NF25 though its level is far lower than that of its ortholog in human liver . Indeed addition of rabbit liver NADPH-cytochrome P-450 reductase increased the oxidation rates . Rabbit liver cytochrome b5 also caused a marked enhancement of catalytic activities, as had been noted previously for this particular P450 enzyme in a reconstituted system involving the protein purified from human liver . Furthermore, the level of the yeast endogenous cytochrome P-450 (lanosterol 14-demethylase) has been found to be negligible compared to the heterologously expressed cytochrome P-450 (30 times less) . Thus, yeast microsomes containing P450 NF25 constitute by themselves a good functional model for studying the binding capacities and catalytic activities of this individual form of human hepatic cytochrome P-450.

Biochemistry, 1990 Dec 25, 29(51), 11189 - 95
Arc repressor is tetrameric when bound to operator DNA; Brown BM et al.; The Arc repressor of bacteriophage P22 is a member of a family of DNA-binding proteins that use N-terminal residues in a beta-sheet conformation for operator recognition . Here, Arc is shown to bind to its operator site as a tetramer . When mixtures of Arc (53 residues) and an active variant of Arc (78 residues) are used in gel retardation experiments, five discrete protein-DNA complexes are observed . This result is as expected for operators bearing heterotetramers containing 4:0, 3:1, 2:2, 1:3, and 0:4 ratios of the two proteins . Direct measurements of binding stoichiometry support the conclusion that Arc binds to a single 21-base-pair operator site as a tetramer . The Arc-operator binding reaction is highly cooperative (Hill constant = 3.5) and involves at least two coupled equilibria . In the first reaction, two unfolded monomers interact to form a folded dimer (Bowie & Sauer, 1989a) . Rapid dilution experiments indicate that the Arc dimer is the kinetically significant DNA-binding species and allow an estimate of the equilibrium dissociation constant for dimerization {K1 = 5 (+/- 3) x 10(-9) M} . The rate of association of Arc-operator complexes shows the expected second-order dependence on the concentration of free Arc dimers, with k2 = 2.8 (+/- 0.7) x 10(18) M-2 s-1 . The dissociation of Arc-operator complexes is a first-order process with k-2 = 1.6 (+/- 0.6) x 10(-4) s-1 . The ratio of these kinetic constants {K2 = 5.7 (+/- 2.3) x 10(-23) M2} provides an estimate for the equilibrium constant for dissociation of the DNA-bound tetramer to two free Arc dimers and the operator . An independent determination of this complex equilibrium constant {K2 = 7.8 (+/- 4.8) x 10(-23) M2} was obtained from equilibrium binding experiments.

J Mol Biol, 1990 Dec 20, 216(4), 939 - 48
Bacteriophage T7 DNA packaging . III . A "hairpin" end formed on T7 concatemers may be an intermediate in the processing reaction; Chung YB et al.; An unusual left end (M-end) has been identified on bacteriophage T7 DNA isolated from T7-infected cells . This end has a "hairpin" structure and is formed at a short inverted repeat sequence centered around nucleotide 39,587 of T7, 190 base-pairs to the left of the site where a mature left end is formed on the T7 concatemer . We do not detect the companion right end that would be formed if the M-end is produced by a double-stranded cut on the T7 concatemer . This suggests that the hairpin left end may be generated from a single-stranded cut in the DNA that is used to prime rightward DNA synthesis . The formation of M-end does not require the products of T7 genes 10, 18 or 19, proteins that are essential for the formation of mature T7 ends . During infection with a T7 gene 3 (endonuclease) mutant, phage DNA synthesis is reduced and the concatemers are not processed into unit length DNA molecules, but both M-end and the mature right end are formed on the concatemer DNA . These two ends are also found associated with the large, rapidly sedimenting concatemers formed during a normal T7 infection while the mature left end is present only on unit length T7 DNA molecules . We propose that DNA replication primed from the hairpin end produced by a nick in the inverted repeat sequence provides a mechanism to duplicate the terminal repeat before DNA packaging . Packaging is initiated with the formation of a mature right end on the branched concatemer and, as the phage head is filled, the T7 gene 3 endonuclease may be required to trim the replication forks from the DNA . Concatemer processing is completed by the removal of the 190 base-pair hairpin end to produce the mature left end.

J Mol Biol, 1990 Dec 20, 216(4), 927 - 38
Bacteriophage T7 DNA packaging . II . Analysis of the DNA sequences required for packaging using a plasmid transduction assay; Chung YB et al.; Recombinant plasmids carrying a bacteriophage T7 origin of DNA replication and sequences from the T7 concatemer junction are efficiently packaged into transducing particles during phage infection . With some constructs, as many as 50 transducing particles are produced per infected cell . We have used this plasmid packaging system to determine which T7 DNA sequences are required for the processing and packaging of the plasmid concatemers and to investigate the effects of altering the spacing and orientation of the required sequences . An origin of T7 DNA replication is essential for high-efficiency transduction, presumably to form the plasmid concatemers that are the substrates of the packaging reaction . In addition, two short sequences from the concatemer junction are required, one flanking the site where the right end of T7 DNA is formed (pacR) and the other flanking the site for formation of the left end (pacL) . The spacing between pacR and pacL is not important, but the sequences must be positioned in the same orientation on the plasmid . With certain deletions of pacL, the specificity of end formation is reduced but the efficiency of packaging is near normal . Plasmids that contain only one of the two pac sites are packaged at about 10% of the efficiency of those with both sites . The residual packaging of these plasmids results from regeneration of the other packaging site by recombination with T7 phage DNA . To function in plasmid packaging, the sequences from the concatemer junction must be positioned on the plasmid in the same orientation relative to the T7 replication origin as is found in T7 DNA . This apparently results from a requirement for transcription through these sequences in the rightward direction from the T7 promoter that is associated with the replication origin . Such transcription from another T7 promoter (phi 10), that is not itself a replication origin, allows packaging when the origin is in the opposite orientation.

J Mol Biol, 1990 Dec 20, 216(4), 911 - 26
Bacteriophage T7 DNA packaging . I . Plasmids containing a T7 replication origin and the T7 concatemer junction are packaged into transducing particles during phage infection; Chung YB et al.; Bacteriophage T7 DNA is a linear duplex molecule with a 160 base-pair direct repeat (terminal redundancy) at its ends . During replication, large DNA concatemers are formed, which are multimers of the T7 genome linked head to tail through recombination at the terminal redundancy . We define the sequence that results from this recombination, a mature right end joined to the left end of T7 DNA, as the concatemer junction . To study the processing and packaging of T7 concatemers into phage particles, we have cloned the T7 concatemer junction into a plasmid vector . This plasmid is efficiently (at least 15 particles/infected cell) packaged into transducing particles during a T7 infection . These transducing particles can be separated from T7 phage by sedimentation to equilibrium in CsCl . The packaged plasmid DNA is a linear concatemer of about 40 x 10(3) base-pairs with ends at the expected T7 DNA sequences . Thus, the T7 concatemer junction sequence on the plasmid is recognized for processing and packaging by the phage system . We have identified a T7 DNA replication origin near the right end of the T7 genome that is necessary for efficient plasmid packaging . The origin, which is associated with a T7 RNA polymerase promoter, causes amplification of the plasmid DNA during T7 infection . The amplified plasmid DNA sediments very rapidly and contains large concatemers, which are expected to be good substrates for the packaging reaction . When cloned in pBR322, a sequence containing only the mature right end of T7 DNA is sufficient for efficient packaging . Since this sequence does not contain DNA to the right of the site where a mature T7 right end is formed, it was expected that right ends would not form on this DNA . In fact, with this plasmid the right end does not form at the normal T7 sequence but is instead formed within the vector . Apparently, the T7 packaging system can also recognize a site in pBR322 DNA to produce an end for packaging . This site is not recognized solely by a "headful" mechanism, since there can be considerable variation in the amount of DNA packaged (32 x 10(3) to 42 x 10(3) base-pairs) . Furthermore, deletion of this region from the vector DNA prevents packaging of the plasmid . The end that is formed in vector DNA is somewhat heterogeneous . About one-third of the ends are at a unique site (nucleotide 1712 of pBR322), which is followed by the sequence 5'-ATCTGT-3' . This sequence is also found adjacent to the cut made in a T7 DNA concatemer to produce a normal T7 right end.

J Mol Biol, 1990 Dec 20, 216(4), 873 - 82
DNA specificity of the Cre recombinase resides in the 25 kDa carboxyl domain of the protein; Hoess R et al.; The Cre protein of bacteriophage P1 is a 38.5 kDa site-specific recombinase that belongs to the Int family of recombination proteins . Cre acts by binding specifically to a 34 base-pair sequence, lox, where it carries out recombination . A limited chymotryptic digest of Cre resulted in two fragments of sizes 25 and 13.5 kDa, respectively . The sequence of the amino terminus of the purified 25 kDa peptide demonstrates that this peptide represents the carboxyl-terminal portion of the Cre protein . A truncated version of the cre gene was constructed which produces only the 25 kDa peptide . The 25 kDa peptide is capable of specific binding to the lox site, but binds at lower affinity than does wild-type Cre . Footprinting with Fe-EDTA indicates that the 25 kDa peptide protects the inverted repeats of the lox site but shows only partial protection of the spacer region . This is in contrast to the footprint obtained with wild-type Cre which protects the entire spacer region.

J Biol Chem, 1990 Dec 15, 265(35), 21430 - 2
Single crystals of a chimeric T7/T3 RNA polymerase with T3 promoter specificity and a nonprocessive T7 RNAP mutant; Sousa R et al.; Two RNA polymerases homologous to bacteriophage T7 RNA polymerase, bacteriophage Sp6 and T3 RNA polymerases, were screened for crystallization under conditions identical or similar to those reported for the growth of large single crystals of T7 RNA polymerase (Sousa, R., Rose, J . P., Chung, Y . J., Lafer, E . M., and Wang, B.-C . (1989) Proteins 5, 266; Sousa, R., Lafer, E . M., and Wang, B.-C . (1990) J . Crystal Growth, in press; Sousa, R., and Lafer, E . M . (1990) Methods 1, in press) . A number of mutant T7 RNAPs were also screened under these conditions as were three chimeric RNA polymerases consisting of T7 RNAP N-terminal and T3 RNAP C-terminal sequences . One chimeric polymerase and two mutant polymerases crystallized readily under T7 RNAP crystallization conditions . The chimeric polymerase crystallized in a space group different from T7 RNA polymerase: orthorombic with unit cell parameters a = 75 A, b = 98 A, c = 159 A; space group P2(1)2(1)2(1) and 4 molecules/unit cell . This chimeric enzyme exhibits T3 promoter specificity and will make it possible to investigate how structural differences between the T3 and T7 RNA polymerase promoter recognition domains determine their different promoter specificities . One of the mutant polymerases successfully crystallized was an enzyme which can carry out promoter recognition and abortive transcription but cannot carry out processive transcription . Its structure may provide information on the nature of the conformational changes undergone by T7 RNAP in the abortive-processive switch . Crystals of the second mutant T7 RNA polymerase were unsuitable for x-ray analysis.

Biochem Biophys Res Commun, 1990 Dec 14, 173(2), 748 - 55
Molecular cloning and sequence analysis of cDNA encoding human ferrochelatase; Nakahashi Y et al.; The cDNA encoding human ferrochelatase {EC 4.99.1.1} was isolated from a human placenta cDNA library in bacteriophage lambda gt11 by screening with a radiolabeled fragment of mouse ferrochelatase cDNA . The cDNA had an open reading frame of 1269 base pairs (bp) encoding a protein of 423 amino acid residues (Mr . 47,833) with alternative putative polyadenylation signals in the 3' non-coding regions and poly (A) tails . Amino acid sequencing showed that the mature protein consists of 369 amino acid residues (Mr . 42,158) with a putative leader sequence of 54 amino acid residues . The human enzyme showed an 88% identity to mouse enzyme and 46% to yeast enzyme . Northern blot analysis showed two mRNAs of about 2500 and 1600 bp for ferrochelatase in K562 and HepG2 cells . As full-length cDNA for human ferrochelatase is now available, molecular lesions related to erythropoietic protoporphyria can be characterized.

Biochem Biophys Res Commun, 1990 Dec 14, 173(2), 711 - 7
High level expression of porcine growth hormone in Escherichia coli from an expression vector containing bacteriophage lambda PL and N gene untranslated region; Hwang LH et al.; An Escherichia coli expression vector, pG408N containing a PL promoter and the upstream untranslated region of the N gene of bacteriophage lambda has been constructed . We have designed a PvuII site immediately behind the untranslated region . A DNA fragment starting with an initiation codon ATG could be inserted into this site for expression . This vector also contains 7 additional cloning sites downstream from the PvuII site . A gene could be cloned into one of these sites and the 5' sequence of this gene could be modified with synthetic oligonucleotides and ligated to the PvuII for the purpose of increasing gene expression . We have also cloned the lambda cl gene into a p15A plasmid . Cotransformation of this plasmid with the expression vector allows the cloning vector pG408N to be used in any E . coli strain . Using this system, we were able to express porcine growth hormone to approximately 35% of total proteins in E . coli DH5 alpha.

Nucleic Acids Res, 1990 Dec 11, 18(23), 7083 - 92
Binding of the bacteriophage T4 regA protein to mRNA targets: an initiator AUG is required; Unnithan S et al.; Bacteriophage T4 regA protein translationally represses the synthesis of a subset of early phage-induced proteins . The protein binds to the translation initiation site of at least two mRNAs and prevents formation of the initiation complex . We show here that the protein binds to the translation initiation sites of other regA-sensitive mRNAs . Analysis of mRNA binding by filtration and nuclease protection assays shows that AUG is necessary but not sufficient for specific binding of regA protein to its mRNA targets . Anticipating the need for large quantities of regA protein for structural studies to further define the regA protein-RNA ligand interaction, we also report cloning the regA gene into a T4 overexpression system . The expression of regA protein in uninfected E . coli is lethal, so in our system regA driven by a strong T7 promoter is sequestered in a T4 phage until 'induction' by phage infection is desired . We have replaced the regA sensitive wild-type ribosome binding site with a strong insensitive ribosome binding site at an optimal distance from the regA initiation codon for maximizing expression . We have obtained large amounts of regA protein.

Nucleic Acids Res, 1990 Dec 11, 18(23), 6903 - 7
Down modulation of HIV-1 gene expression using a procaryotic RNA-binding protein; Berkhout B et al.; The coat protein of the single stranded RNA bacteriophages acts as a translational repressor by binding with high affinity to a target RNA that encompasses the ribosomal binding site of the replicase gene . We have expressed this procaryotic RNA-binding protein in mammalian cells . Using the coat protein binding site attached to the HIV-1 5' leader RNA, we tested for the biological effect of co-expressed bacteriophage protein . We found that HIV-1 LTR-directed expression within this context was inhibited in trans by the coat protein . This example suggests the feasibility of using procaryotic RNA-binding proteins as genetic modulators in eucaryotic cells.

Science, 1990 Dec 7, 250(4986), 1400 - 3
Sequence requirements for coiled-coils: analysis with lambda repressor-GCN4 leucine zipper fusions; Hu JC et al.; A genetic system was developed in Escherichia coli to study leucine zippers with the amino-terminal domain of bacteriophage lambda repressor as a reporter for dimerization . This system was used to analyze the importance of the amino acid side chains at eight positions that form the hydrophobic interface of the leucine zipper dimer from the yeast transcriptional activator, GCN4 . When single amino acid substitutions were analyzed, most functional variants contained hydrophobic residues at the dimer interface, while most nonfunctional sequence variants contained strongly polar or helix-breaking residues . In multiple randomization experiments, however, many combinations of hydrophobic residues were found to be nonfunctional, and leucines in the heptad repeat were shown to have a special function in leucine zipper dimerization.

Nature, 1990 Dec 6, 348(6301), 552 - 4
Phage antibodies: filamentous phage displaying antibody variable domains; McCafferty J et al.; New ways of making antibodies have recently been demonstrated using gene technology . Immunoglobulin variable (V) genes are amplified from hybridomas or B cells using the polymerase chain reaction, and cloned into expression vectors . Soluble antibody fragments secreted from bacteria are then screened for binding activities . Screening of V genes would, however, be revolutionized if they could be expressed on the surface of bacteriophage . Phage carrying V genes that encode binding activities could then be selected directly with antigen . Here we show that complete antibody V domains can be displayed on the surface of fd bacteriophage, that the phage bind specifically to antigen and that rare phage (one in a million) can be isolated after affinity chromatography.

J Mol Biol, 1990 Dec 5, 216(3), 717 - 27
Complex between single-stranded DNA and gene 5 protein of bacteriophage M13 studied with linear dichroism and ultraviolet absorption; van Amerongen H et al.; We have studied complexes between the gene 5 protein (gp5) of bacteriophage M13 and various polynucleotides, including single-stranded DNA, using ultraviolet absorption and linear dichroism . Upon complex formation the absorption spectra of both the protein and the polynucleotides change . The protein absorption changes indicate that for at least two of the five tyrosine residues per protein monomer the environment becomes less polar upon binding to the polynucleotides but also to the oligonucleotide p(dT)8 . All gp5-polynucleotide complexes give rise to intense linear dichroism spectra . These spectra are dominated by negative contributions from the bases, but also a small positive dichroism of the protein can be discerned . The spectra can be explained by polynucleotide structures, which are the same in all complexes . The base orientations are characterized by a substantial inclination and propellor twist . The number of possible combinations of inclination and propeller twist values, which are in agreement with the linear dichroism results, is rather limited . The base orientations with respect to the complex axis are essentially different from those in the complex with the single-stranded DNA-binding protein gp32 of bacteriophage T4.

J Biol Chem, 1990 Dec 5, 265(34), 20966 - 9
The role of the bacteriophage T4 gene 32 protein in homologous pairing; Kodadek T; The gene 32 protein of the bacteriophage T4 is required for efficient genetic recombination in infected Eschericia coli cells and strongly stimulates in vitro pairing catalyzed by the phage uvsX protein, a RecA-like strand transferase . This helix-destabilizing factor is known to bind tightly and cooperatively to single-stranded DNA and to interact specifically with the uvsX protein as well as other phage gene products . However, its detailed role in homologous pairing is not well understood . I show here that when the efficiency of uvsX protein-mediated pairing is examined at different gene 32 protein and duplex DNA concentrations, a correlation between the two is found, suggesting that the two interact in a functionally important manner during the reaction . These and other data are consistent with a model in which the gene 32 protein binds to the strand displaced from the recipient duplex during pairing, thereby stabilizing the heteroduplex product . An alternative model in which the gene 32 protein replaces UvsX on the invading strand, thereby freeing the strand transferase to bind to the displaced strand, is also considered.

J Bacteriol, 1990 Dec, 172(12), 7098 - 103
Saturation and specificity of the Lon protease of Escherichia coli; Dervyn E et al.; Lon is an ATP-dependent protease of Escherichia coli . The lon mutation has a pleiotropic phenotype: UV sensitivity, mucoidy, deficiency for lysogenization by bacteriophage lambda and P1, and lower efficiency in the degradation of abnormal proteins . All of these phenotypes are correlated with the loss of protease activity . Here we examine the effects of overproduction of one Lon substrate, SulA, and show that it protects two other substrates from degradation . To better understand this protection, we mutagenized the sulA gene and selected for mutants that have partially or totally lost their ability to saturate the Lon protease and thus can no longer protect another substrate . Some of the SulA mutants lost their ability to protect RcsA from degradation but could still protect the O thermosensitive mutant protein (Ots) . All of the mutants retained their capacity to induce cell division inhibition . It was also found that deletion of the C-terminal end of SulA affected its activity but did not affect its susceptibility to Lon . We propose that Lon may have more than one specificity for peptide cleavage.

Proc Natl Acad Sci U S A, 1990 Dec, 87(23), 9173 - 7
In vitro assembly of infectious nucleocapsids of bacteriophage phi 6: formation of a recombinant double-stranded RNA virus; Olkkonen VM et al.; A system is described for assembling infectious bacteriophage phi 6 nucleocapsids in vitro . Procapsids encoded by cDNA copies of genomic segment L in Escherichia coli were used to package and replicate viral RNA segments . The resulting filled particles were shown to be capable of infecting host cell spheroplasts after incubation with purified nucleocapsid shell protein P8 . The infected spheroplasts yielded infectious virions . A modified cDNA-derived RNA segment was inserted into virions by this method . The resulting infectious virions contained the same 4-base-pair deletion as the modified cDNA . These findings support the contention that the preformed procapsids are the "machine" that replicates the phi 6 genome, by showing that the cDNA-derived procapsids are competent to package and replicate RNA properly.

Mol Cell Biol, 1990 Dec, 10(12), 6236 - 43
RIP60, a mammalian origin-binding protein, enhances DNA bending near the dihydrofolate reductase origin of replication; Caddle MS et al.; Replication of the Chinese hamster dihydrofolate (dhfr) gene initiates near a 281-bp HaeIII fragment of stably bent DNA that binds RIP60, a 60-kDa origin-specific DNA-binding protein that has been purified from HeLa cell nuclear extract (L . Dailey, M . S . Caddle, N . Heintz, and N . H . Heintz, Mol . Cell . Biol . 10:6225-6235, 1990) . Circular permutation assays showed that stable DNA bending in the dhfr origin region fragment was due to the presence of five oligo (dA)3-4 tracts, designated bend elements B1 to B5, that are spaced 10 bp apart . DNA bending directed by elements B1 to B5, as assessed by anomolous migration of DNA fragments on polyacrylamide gels, was accentuated at 4 degrees C . Bend element B5, which is in inverse orientation relative to elements B1 to B4, overlaps an ATT-rich motif that comprises the RIP60 protein-binding site . Gel mobility shift assays with circularly permuted bent DNA fragments and purified RIP60 showed that RIP60 markedly enhanced DNA bending of the dhfr origin region sequences . These results suggest that, as in many plasmids, bacteriophages, and eucaryotic viruses, mammalian DNA-binding proteins may enhance DNA bending near origins of replication during initiation of DNA synthesis.

What Is Bioengineering?, What Is Bioremediation?, What Is Anthrax?, What Is Molecular Biology?, What Is Botulism?, r, Bacteriology, o, Microorganism, a, Microbes, o, Microbiology, i, Bacteria, c, Bacteriological, n, Kluyveromyces, s, Escherichia coli, e, Bactericidal, c, Staphylococcus aureus, r, Escherichia coli, o, S. cerevisiae, a, Bacteriological, e, Bacillus, s, Ps. fluorescens, i, Salmonella, e, Pseudomonas aeruginosa, s, Salmonella, e, Bacteriological, n, Biodegradation, n, Escherichia coli, c, Antimicrobials, e, Yeasts, s, Bacteriological, c, Escherichia coli, a, S. cerevisiae

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005