Biomedical subjects

S S Broyles

Publications and source records attributed to S S Broyles.

28 records · Page 2Linked to original sources

PubMed1989

Vaccinia virus gene D12L encodes the small subunit of the viral mRNA capping enzyme.

Vaccinia virus gene D12L, which lies between nucleotides 14,350 and 13,487 in the HindIII D fragment, is transcribed at early times in infection and is capable of encoding a protein 287 amino acids in length with a predicted molecular mass of 33,331. A polyclonal antiserum was raised in rabbits to a fusion protein containing 279 amino acids of the D12L protein, and this serum was used to investigate both the time of synthesis and the function of the D12L protein. A combination of Western blot analysis and immunoprecipitation from pulse-labeled and pulse-chased cell extracts demonstrated that the synthesis of a 31-kDa protein begins early in infection, that it reaches a plateau by about 4 hr, and that it is stable in the infected cell. The D12L protein was localized by Western blot analysis of detergent-solubilized virions to the sodium deoxycholate soluble fraction which suggested that it may be a virion core-associated enzyme. Due to the similarity in apparent molecular weight between the D12L protein and the small subunit of the vaccinia mRNA capping complex the anti-D12L antiserum was employed in Western blot analysis of fractions generated during the purification of the virion mRNA capping enzyme. The 31-kDa D12L protein copurified with the virus capping enzyme through chromatography on heparin-agarose and phosphocellulose and also cosedimented with the capping enzyme through a glycerol density gradient. In addition, the anti-D12L antiserum coprecipitated the large subunit of the capping enzyme, confirming that gene D12L encodes the small subunit of the viral mRNA capping enzyme. An insertion mutation which destroys the gene D12L coding sequence was constructed in a plasmid containing a portion of both genes D11L and D12L and this plasmid was used to rescue a ts mutation, in a single step, in the adjacent gene D11L. Southern blot analysis of the re-plaque-purified virus permitted the identification of the mutant virus only when the mutant was propagated in the presence of wild-type helper virus. We concluded from these data that gene D12L is essential for virus propagation in tissue culture.

Base Sequence↗

PubMed1988

DNA-dependent ATPase activity associated with vaccinia virus early transcription factor.

Vaccinia virus early transcription factor (VETF) is required for efficient expression of the early class of viral genes in vitro. The factor copurified with an ATPase activity that was stimulated by DNA. In this report we show that the ATPase remains associated with the factor upon glycerol gradient sedimentation. Under these conditions VETF sedimented at a rate of 7.6 S suggesting that it may be a heterodimer of the Mr 82,000 and 77,000 polypeptides. Of the common nucleoside triphosphates, only ATP and dATP were hydrolyzed by the VETF-associated ATPase. The ATP analog gamma-thio ATP was not a substrate. The VETF-associated ATPase activity was stimulated up to 30-fold by the presence of polynucleotides. DNA was a much more effective cofactor for the ATPase than was RNA, and duplex polydeoxyribonucleotides were preferred. The enzymatic and physical properties of the VETF-associated ATPase distinguished it from all three vaccinia ATPase activities previously described, nucleoside triphosphate phosphohydrolases I and II, and capping enzyme. Except for the preference for double-stranded DNA, the substrate and cofactor requirements of the VETF-associated ATPase most closely resembled those of nucleoside triphosphate phosphohydrolase I. However, VETF-ATPase was not inhibited by polyclonal antibody to the latter enzyme. The association of an ATPase with an early gene transcription factor may explain the previously described requirement for ATP hydrolysis in transcription.

Adenosine Triphosphatases↗

PubMed1988

Purification of a factor required for transcription of vaccinia virus early genes.

Partially purified DNA-dependent RNA polymerase from infectious vaccinia virus particles exhibits the following two activities: 1) specific transcription of double-stranded DNA templates containing vaccinia early promoters and 2) nonspecific transcription of single-stranded DNA templates. After further purification of the RNA polymerase, specific transcriptase activity was selectively diminished suggesting the loss of a transcription factor. In agreement with the latter hypothesis, transcriptase activity could be reconstituted by mixing the purified RNA polymerase with certain column fractions. A quantitative complementation assay was developed and used to locate the transcription factor during successive column chromatography steps. The factor eluted as a single peak of activity from single strand DNA-cellulose and phosphocellulose columns. An observation that the transcription factor binds specifically to vaccinia early promoter sequences was exploited in the final affinity chromatography steps. The purified factor was separated from all previously identified vaccinia enzymes and contained two polypeptides of Mr 77,000 and 82,000. A DNA-dependent ATPase activity also copurified with the transcription factor. Although a single template was used for assays during isolation, the purified factor stimulated transcription of three other early genes by 20-30-fold suggesting that it has a general role in conferring promoter specificity for initiation of early transcription.

Chromatography, Affinity↗

PubMed1988

Characterization of human immunodeficiency virus gag/pol gene products expressed by recombinant vaccinia viruses.

Recombinant vaccinia viruses containing either the entire gag/pol gene or the reverse transcriptase (RT) domain of the human immunodeficiency virus (HIV) were constructed. In mammalian cells infected with the recombinant vaccinia virus containing the gag/pol gene, major and minor polypeptides of 55 and 41 kDa were made, but processed gag products (p24/p17/p15) were not detected. In addition, none of the products of the pol open-reading frame were seen. Both the 55- and 41-kDa gag proteins were post-translationally modified by addition of myristic acid residues in recombinant vaccinia-infected cells, and were immunoprecipitated by antiserum to p24 gag, as well as by antisera from HIV-infected patients. These results indicate that neither proteolytic processing nor other HIV proteins are required for myristilation, and suggest that the 55- and 41-kDa gag precursors share the same amino terminus as p17. Cells infected with a separate vaccinia recombinant containing a truncated piece of the gag/pol gene with added start and stop codons at the 5' and 3' ends of the RT reading frame synthesized a major 61-kDa and a minor 51-kDa protein product which reacted immunologically with both a monoclonal antibody to native HIV p66/51 and antisera from HIV-infected patients. These proteins were purified from recombinant vaccinia-infected mammalian cells, and their enzyme activity was found to be similar to that of authentic HIV RT. Cells infected with the vaccinia/RT vector contained approximately 200-fold more RT per milligram of protein than cells infected with HIV. Recombinant RT was inhibited by dideoxynucleoside triphosphates and should be useful in screening for specific inhibitors of this enzyme. Mice inoculated intradermally with 10(8) plaque-forming units of the vaccinia/RT vector developed specific antibodies to the p66/51 proteins of HIV, but anti-HIV antibodies were not detected in mice inoculated with the vaccinia/gag vector.

Antigens, Viral↗

PubMed1987

Purification and characterization of a transcription termination factor from vaccinia virions.

A DNA-dependent RNA polymerase that transcribes vaccinia virus early genes was partially purified from virus cores by deoxycholate extraction and DEAE-cellulose column chromatography. Accurately initiated and terminated RNAs were synthesized by this enzyme in the presence of a linear duplex DNA template. Glycerol gradient sedimentation resolved the in vitro transcription system into two components: fraction I, a rapidly sedimenting RNA polymerase that initiated transcription at an early promoter but transcribed beyond the in vivo 3' terminus to yield a run-off transcript, and fraction II, a more slowly sedimenting fraction, itself devoid of RNA polymerase, that restored efficient termination when added back to fraction I. The termination factor was heat-labile, resistant to N-ethylmaleimide, and did not exhibit endonucleolytic activity on run-off transcripts. Factor-dependent termination required specific sequence information upstream of the site of termination. The vaccinia termination factor was purified extensively by column chromatography on DEAE-cellulose, heparin-agarose, phosphocellulose, and DNA-agarose, and by velocity sedimentation in a glycerol gradient. At each step, termination factor copurified with the vaccinia mRNA capping enzyme. The preparation was well over 90% pure with respect to the latter enzyme, suggesting that termination activity was tightly associated with, if not intrinsic to, the capping enzyme. Nonetheless, formation of the 5'-cap structure did not appear to be a prerequisite for termination.

Adenine Nucleotides↗

PubMed1987

Identification of the vaccinia virus gene encoding nucleoside triphosphate phosphohydrolase I, a DNA-dependent ATPase.

Vaccinia virus encapsidates a DNA-dependent ATPase known as nucleoside triphosphate phosphohydrolase I (NPH I). A bacteriophage lambda gt11 expression library of poxvirus DNA was screened with antibodies specific for NPH I. Positive clones were used to probe restriction fragments of vaccinia virus genomic DNA to locate the NPH I gene. The identity of the open reading frame (ORF) was confirmed by placing it downstream of a bacteriophage T7 promoter, transcribing the ORF in vitro, and translating the RNA in a reticulocyte lysate. A polypeptide of the correct molecular weight, which was recognized by anti-NPH I antibody, was synthesized. Inspection of the deduced amino acid sequence of the NPH I ORF revealed consensus ATP-binding sites.

Adenosine Triphosphatases↗

PubMed1987

Sedimentation of an RNA polymerase complex from vaccinia virus that specifically initiates and terminates transcription.

A high-molecular-weight protein complex that is capable of accurate transcription initiation and termination of vaccinia virus early genes without additional factors was demonstrated. The complex was solubilized by disruption of purified virions, freed of DNA by passage through a DEAE-cellulose column, and isolated by glycerol gradient sedimentation. All detectable RNA polymerase activity was associated with the transcription complex, whereas the majority of enzymes released from virus cores including mRNA (nucleoside-2'-O)methyltransferase, poly(A) polymerase, topoisomerase, nucleoside triphosphate phosphohydrolase II, protein kinase, and single-strand DNase sedimented more slowly. Activities corresponding to two enzymes, mRNA guanylyltransferase (capping enzyme) and nucleoside triphosphate phosphohydrolase I (DNA-dependent ATPase), partially sedimented with the complex. Silver-stained polyacrylamide gels, immunoblots, and autoradiographs confirmed the presence of subunits of vaccinia virus RNA polymerase, mRNA guanylyltransferase, and nucleoside triphosphate phosphohydrolase I, as well as additional unidentified polypeptides, in fractions with transcriptase activity. A possible role for the DNA-dependent ATPase was suggested by studies with ATP analogs with gamma-S or nonhydrolyzable beta-gamma-phosphodiester bonds. These analogs were used by vaccinia virus RNA polymerase to nonspecifically transcribe single-stranded DNA templates but did not support accurate transcription of early genes by the complex. Transcription also was sensitive to high concentrations of novobiocin; however, this effect could be attributed to inhibition of RNA polymerase or ATPase activities rather than topoisomerase.

Adenosine Triphosphatases↗

PubMed1986

Interaction of the Escherichia coli HU protein with DNA. Evidence for formation of nucleosome-like structures with altered DNA helical pitch.

Nuclease digestion studies of DNA bound to the histone-like protein HU show that cuts in each strand of the DNA double helix are made with a periodicity of 8.5 base-pairs. By contrast, similar digestions of DNA in eukaryotic nucleosomes show a repeat of 10.4 base-pairs. This and other results (including circular dichroism studies) are consistent with the proposal that the pitch of the DNA double helix in the HU complex is reduced from a repeat length of 10.5 to 8.5 base-pairs per helical turn. Simultaneously, the DNA in the HU-DNA complex containing two dimers of HU per 60 base-pairs has its linking number decreased by 1.0 turn per 290 base-pairs. From these changes it is calculated that HU imposes a DNA writhe of 1.0 per three to four monomers of HU. The results suggest a model in which DNA is coiled in left-handed toroidal supercoils on the HU complex, having a stoichiometry resembling that of the half-nucleosome of eukaryotic chromatin. An important distinction is that HU complexes can restrain the same number of DNA superhelical turns as eukaryotic nucleosomes, yet the DNA retains more negative torsional tension, just as is observed in prokaryotic chromosomes in vivo. Another distinction is that HU-DNA complexes are less stable, having a dissociation half-life of 0.6 min in 50 mM-NaCl. This last property may explain prior difficulties in detecting prokaryotic nucleosome-like structures.

Bacterial Proteins↗

PubMed1986

Homology between RNA polymerases of poxviruses, prokaryotes, and eukaryotes: nucleotide sequence and transcriptional analysis of vaccinia virus genes encoding 147-kDa and 22-kDa subunits.

We have determined the nucleotide sequence of a region of the vaccinia virus genome encoding RNA polymerase subunits of 22 and 147 kDa and have mapped the 5' and 3' ends of the two mRNAs. The predicted amino acid sequence of the vaccinia 147-kDa subunit shows extensive homology with the largest subunit of Escherichia coli RNA polymerase, yeast RNA polymerases II and III, and Drosophila RNA polymerase II. The regions of homology between the five RNA polymerases are subdivided into five separate domains that span most of the length of each. A sixth domain shared by the vaccinia and the eukaryotic polymerases is absent from the E. coli sequence. In all specified regions, the vaccinia large subunit has greater homology with eukaryotic RNA polymerases II and III than with the E. coli polymerase. Vaccinia virus and eukaryotic RNA polymerases may therefore have evolved from a common ancestral gene after the latter diverged from prokaryotes.

Amino Acid Sequence↗

PubMed1983

Perfect palindromic lac operator DNA sequence exists as a stable cruciform structure in supercoiled DNA in vitro but not in vivo.

A perfect palindromic 66-base pair (bp) DNA sequence derived from the lac operator and cloned into plasmid pMB9 [Betz, J. L. & Sadler, J. R. (1981) Gene 13, 1-12] can exist in a 66-bp linear form or as two 33-bp cruciform arms. The fraction of the sequence in the cruciform depends on the superhelical density of the plasmid DNA. Relaxed DNA contains no cruciforms. The palindrome in the cruciform structure is cut by EcoRI endonuclease at the base of the cruciform arms, releasing 33-bp fragments; when in the linear form only 66-bp fragments are produced. The cruciform structure is fixed by trimethylpsoralen crosslinks in the cruciform arms. This together with the EcoRI cutting provides an assay for the cruciform structures in the DNA of living cells. Using this assay we show that the cruciform structure rarely if ever exists in vivo, but after DNA isolation greater than 90% of the sequence is in cruciforms. Results suggest that the plasmid DNA as organized in vivo either lacks sufficient torsional tension to form this cruciform or the palindrome is restrained in the linear form by other bound molecules.

Base Sequence↗