PubMed Health⌕ Search

Biomedical subjects

J D Parvin

Publications and source records attributed to J D Parvin.

34 records · Page 2Linked to original sources
PubMed1995

Pre-bending of a promoter sequence enhances affinity for the TATA-binding factor.

TATA-binding protein (TBP) binds the minor groove of the TATA element with the DNA bent 80 degrees towards the major groove. A constrained minicircle strategy has been used to test the effect of DNA topology on the affinity of TBP for the TATA element. We report here that TBP bound to DNA which was slightly pre-bent towards the major groove with 100-fold higher affinity than unbent (linear) DNA of identical sequence and 300-fold higher affinity than DNA pre-bent towards the minor groove. Similar discrimination was observed with the holo-TFIID transcription complex. DNA topology, particularly bending, is determined by many factors including chromatin in cells and may, through changes in the affinity of the TATA factor, be important in the control of transcription.

DNA↗
PubMed1994

Multiple sets of basal factors initiate transcription by RNA polymerase II.

The minimal requirements for transcription initiation from supercoiled templates were determined for the two major forms of TATA-binding factors found in cell extracts, the 300-kDa B-TFIID and the 1000-kDa D-TFIID complexes. As had been observed for the TATA-binding protein (TBP) subunit (Parvin and Sharp, 1993), transcription from the IgH promoter minimally requires TFIID activity plus TFIIB and RNA polymerase II. This minimal reaction is only active on negatively supercoiled template DNA. In contrast, the supercoiled templates encoding the adenovirus major late promoter (MLP), or several other promoters, require the addition of TFIIF to the minimal reaction. Further addition of TFIIE and TFIIH boosts the level of transcription from these latter promoters but is not required. In contrast to the complete reaction on linear template, transcription from supercoiled IgH or MLP templates does not require the hydrolysis of the beta-gamma bond of ATP. Fourteen different core promoters were compared in complete and minimal basal transcription reactions reconstituted with one of the three TATA activities: TBP, B-TFIID, and D-TFIID. Of these 14 promoters, only the IgH was active in the absence of TFIIF, and the other promoters demonstrated different levels of transcription depending on which basal factors were present in reaction. It is proposed that a significant level of basal transcription only requires a minimal set of factors, and stimulation by upstream activators may in part be mediated by the inclusion of additional basal factors into the initiation reaction.

Animals↗
PubMed1994

The interaction of GATA-binding proteins and basal transcription factors with GATA box-containing core promoters. A model of tissue-specific gene expression.

The core promoters of the rat platelet factor 4 (PF4), mouse erythropoietin and chicken beta globin genes contain a GATA motif in place of the consensus TATAAA site. In the case of the PF4 gene, this site has been shown to play a critical role in restricting transcription to the megakaryocyte lineage. In order to understand the mechanism of tissue specificity, we investigated the function of the GATA box-containing promoters in vitro. Our studies show that the TATA-binding protein of TFIID is required for initiation of transcription from the GATA box-containing promoters. GATA-1 interacts with the core promoter GATA motif and inhibits generation of preinitiation complexes. The functional significance of the inhibition of preinitiation complexes is supported by in vitro transcription assays in which transcription from the PF4 and erythropoietin core promoters is suppressed by GATA-1. We also demonstrate that GATA-2 inhibits initiation of transcription from the PF4 core promoter. Based on these results, we propose a model in which repression of PF4 expression in nonmegakaryocytes is mediated, in part, by competition between GATA-binding proteins and basal factors for the core promoter.

Animals↗
PubMed1993

DNA topology and a minimal set of basal factors for transcription by RNA polymerase II.

Immunoglobulin heavy chain (IgH) gene transcription in vitro can be reconstituted with a minimal reaction containing only TATA-binding protein (TBP), TFIIB, and RNA polymerase II (pol II) when the template is negatively supercoiled. Transcription from linear DNA templates containing either the IgH or the adenovirus major late promoters (MLPs) requires in addition TFIIF, TFIIE, TFIIH, and a fraction containing TFIIA and TFIIJ. Promoters vary in their activities in the minimal reaction. Initiation at the adenovirus MLP site was not observed in this reaction, even with templates containing negative superhelical density. When only TBP, TFIIB, and pol II were present in the reaction, the more negatively supercoiled the IgH template DNA was, the more active the transcription. It is suggested that the free energy of supercoiling promotes the formation of an open complex for initiation of transcription by the minimal set of transcription factors.

Cloning, Molecular↗
PubMed1992

Promoter specificity of basal transcription factors.

Regulation of expression of protein-encoding genes in eukaryotes is frequently mediated by sequence-specific transcription factors that control the activities of the basal factors and RNA polymerase II. Basal factors have been considered to be essential for all polymerase II promoters. Studies of the basal factor requirements for transcription from the immunoglobulin heavy chain gene (IgH) core promoter and the adenovirus major late gene core promoter (MLP) suggest that this paradigm is too simple. Basal transcription from the IgH promoter was reconstituted by TFIID, TFIIB, TFIIF, and polymerase, whereas basal transcription from the MLP is highly dependent upon TFIIE in addition to the above factors. Two novel protein activities, referred to as 700 kd and 90 kd, further stimulated the basal reaction from the MLP. Thus, these data indicate that not all basal factors are in fact general.

Base Sequence↗
PubMed1991

Identification of novel factors which bind specifically to the core promoter of the immunoglobulin heavy chain gene.

Nuclear extracts from HeLa cells and the B-cell line, BJA-B, generated two protein-DNA complexes which bound specifically to sequences in the TATA box of the immunoglobulin heavy chain gene (IgH) promoter. Complex A also bound the core promoter of a retroviral long terminal repeat but did not bind to five other promoters including the adenovirus major late promoter. Of these seven promoters, complex B bound only to the IgH promoter. Footprinting analysis revealed that both complexes A and B bound sequences which include the TATA element, and complex A additionally contacted sequences downstream to +28. Mutation of the IgH TATA element from ATTAATATA to GCTA-TAAAA, the optimal TATA sequence as found in the major late promoter, resulted in a 10-fold decrease in binding to complex A and a 25-fold decrease in binding complex B. Surprisingly, both transfection experiments in HeLa cells and in vitro transcription experiments with whole nuclear extract demonstrated that mutation of the TATA box in the core IgH promoter to this consensus sequence resulted in a 2-fold decrease in the level of transcription. These data suggest that the specific sequence of the TATA region is important, and factors which recognize these sequences, such as complex A and B, may modulate the level of transcription from the IgH promoter.

Base Sequence↗
PubMed1989

Amplification, expression, and packaging of foreign gene by influenza virus.

A system is described that allows use of recombinant DNA technology to modify the genome of influenza virus, a negative-strand RNA virus, and to engineer vectors for the expression of foreign genes. Recombinant RNA is expressed from plasmid DNA in which the coding sequence of the influenza A virus NS gene is replaced with that of the chloramphenicol acetyltransferase gene. When transfected with purified influenza A virus polymerase proteins--in the presence of helper virus--the recombinant RNA is amplified, expressed, and packaged into virus particles, which can be passaged several times. The data indicate that the 22 5' terminal and the 26 3' terminal bases of the influenza A virus RNA are sufficient to provide the signals for RNA transcription, RNA replication, packaging of RNA into influenza virus particles.

Animals↗
PubMed1989

Promoter analysis of influenza virus RNA polymerase.

Influenza virus polymerase, which was prepared depleted of viral RNA, was used to copy small RNA templates prepared from plasmid-encoded sequences. Template constructions containing only the 3' end of genomic RNA were shown to be efficiently copied, indicating that the promoter lay solely within the 15-nucleotide 3' terminus. Sequences not specific for the influenza virus termini were not copied, and, surprisingly, RNAs containing termini identical to those from plus-sense cRNA were copied at low levels. The specificity for recognition of the virus sense promoter was further defined by site-specific mutagenesis. It was also found that increased levels of viral protein were required in order to catalyze both the cap endonuclease-primed and primer-free RNA synthesis from these model templates, as well as from genomic-length RNAs. This finding indicates that the reconstituted system has catalytic properties very similar to those of native viral ribonucleoprotein complexes.

Base Sequence↗
PubMed1987

Genomic RNAs of influenza viruses are held in a circular conformation in virions and in infected cells by a terminal panhandle.

The viral RNA segments in influenza virions were shown to be circular in conformation by using psoralen crosslinking methods. Electron microscopy of purified RNA following treatment of virus with the psoralen reagent 4'-aminomethyltrioxsalen (AMT) revealed circles with lengths corresponding to the individual segments. RNA blot analysis using polyacrylamide gels demonstrated that RNA from AMT-treated virus had a slowed migration, consistent with it being a single-stranded circle. Furthermore, nuclease S1 protection assays indicated that the termini of the RNA segments form an approximately 15-base-pair-long panhandle. This structure is consistent with the partial sequence complementarity that has been observed for the termini of all influenza virus RNAs. By RNA blot analysis, circular structures of viral sense RNA were also found in influenza virus-infected cells at early and late time points. The circular RNA was the predominant species at the time when the major transcription product is message RNA. This finding and the observation that the termination signal for mRNA synthesis directly abuts the panhandle suggest that a panhandle in the template viral RNA is a cis regulatory signal promoting the synthesis of mRNA instead of plus-sense template. Also, since the panhandle is present in high concentration in virions, we suggest that it is required for packaging and that the input RNA after infection is in the proper conformation for synthesis of primary transcripts.

Cross-Linking Reagents↗
PubMed1986

Evolution of human influenza A viruses over 50 years: rapid, uniform rate of change in NS gene.

Variation in influenza A viruses was examined by comparison of nucleotide sequences of the NS gene (890 bases) of 15 human viruses isolated over 53 years (1933 to 1985). Changes in the genes accumulate with time, and an evolutionary tree based on the maximum parsimony method can be constructed. The evolutionary rate is approximately 2 X 10(-3) substitution per site per year in the NS genes, which is about 10(6) times the evolutionary rate of germline genes in mammals. This uniform and rapid rate of evolution in the NS gene is a good molecular clock and is compatible with the hypothesis that positive selection is operating on the hemagglutinin (or perhaps some other viral genes) to preserve random mutations in the NS gene.

Base Sequence↗
PubMed1986

Detection of single base substitutions in influenza virus RNA molecules by denaturing gradient gel electrophoresis of RNA-RNA or DNA-RNA heteroduplexes.

Single point mutations in the NS gene of influenza virus were detected by electrophoresis of double-stranded RNA heteroduplexes in denaturing gradient gels. The heteroduplex RNAs were made by hybridization of virion RNA with SP6-derived RNA probes of varying length. Mutations located at different positions along the NS gene (890 nucleotides long) were all detected in a predictable fashion. The method of heteroduplex analysis was also successfully used in detecting single point mismatches in DNA-RNA hybrids.

Base Sequence↗
PubMed1986

Rapid RNA sequencing using double-stranded template DNA, SP6 polymerase, and 3'-deoxynucleotide triphosphates.

A simple and efficient nucleic acid sequencing method is described in which RNA transcription by the SP6 polymerase is specifically terminated using 3'-deoxynucleotide triphosphates. Initial difficulties in resolving the RNA ladder were overcome by replacing guanosine triphosphate by inosine triphosphate in the reaction mixture and electrophoresing gels at high temperature (50 degrees C). This method presents advantages over current sequencing techniques: Unprocessed plasmid DNA is the template and preparation of inserts and/or single-stranded templates is unnecessary. Use of the specific promoter for SP6 polymerase removes the need for a primer in sequencing reactions.

Base Sequence↗
PubMed1986

Measurement of the mutation rates of animal viruses: influenza A virus and poliovirus type 1.

Epidemiologic and genetic evidence suggests that influenza A viruses evolve more rapidly than other viruses in humans. Although the high mutation rate of the virus is often cited as the cause of the extensive variation, direct measurement of this parameter has not been obtained in vivo. In this study, the rate of mutation in tissue culture for the nonstructural (NS) gene of influenza A virus and for the VP1 gene in poliovirus type 1 was assayed by direct sequence analysis. Each gene was repeatedly sequenced in over 100 viral clones which were descended from a single virion in one plaque generation. A total of 108 NS genes of influenza virus were sequenced, and in the 91,708 nucleotides analyzed, seven point changes were observed. A total of 105 VP1 genes of poliovirus were sequenced, and in the 95,688 nucleotides analyzed, no mutations were observed. We then calculated mutation rates of 1.5 X 10(-5) and less than 2.1 X 10(-6) mutations per nucleotide per infectious cycle for influenza virus and poliovirus, respectively. We suggest that the higher mutation rate of influenza A virus may promote the rapid evolution of this virus in nature.

Base Sequence↗
PubMed1984

Mechanisms for the generation of src-deletion mutants and recovered sarcoma viruses: identification of viral sequences involved in src deletions and in recombination with c-src sequences.

The precise src deletions in six transformation-defective (td) deletion mutants derived from the Schmidt-Ruppin strain of Rous sarcoma virus were determined by sequence analysis. Examination of the parental viral sequences neighboring the junctions of deletions in these td mutants revealed that these regions contained either directly repeated or inverted complementary sequences ranging from 9 to 28 nucleotides. Five td mutants were found to contain deletions flanked by directly repeated sequences, of which the 3' direct repeat was retained whereas the 5' direct repeat was deleted in the resulting td viral RNA. In the deletions of two td mutants where inverted complementary sequences were present at junctions of the deletions, both copies of the inverted complementary sequence were deleted in the td viral RNA. It is proposed from these observations that deletions of these mutants have been generated during the synthesis of minus-strand viral DNA by reverse transcriptase by jumping over a sequence flanked by direct repeats or by skipping a stem-and-loop structure formed via inverted complementary sequences on the viral RNA template. Data provide further information on the sequences in the td viral genome that are required for the generation of recovered sarcoma viruses (rASVs) by recombination with c-src. Sequence data of td viruses revealed that retaining as few as 82 nucleotides of the 3' src coding sequence is sufficient, whereas retaining as much as one-third of the 5' src but none of the 3' src coding sequences is not sufficient, for the generation of rASVs. Those that generate replication-competent rASVs retain, in addition to the 3' src region, a portion of the 5' src and/or its immediate upstream sequence that is homologous to exon 1 of the c-src DNA. These two sequence domains apparently provided 5' and 3' homologous regions for recombination between td viral genome and c-src DNA resulting in nondefective rASVs. Td109, which was shown previously to generate only replication-defective rASVs, retains 296 nucleotides of the 3' src sequence but lacks all the 5' src and 316 nucleotides of its immediate upstream region. It is concluded that the 5' src coding sequence and its immediate upstream region are not essential for the generation of rASVs. However, retaining a portion of those sequences is required for the generation of replication-competent rASVs.

Animals↗
PubMed1983

Nonsense mutations affecting the lengths of the NS1 nonstructural proteins of influenza A virus isolates.

The proteins from cells infected with influenza A virus field isolates were labeled with [35S]methionine and analyzed by SDS-polyacrylamide gel electrophoresis. By screening more than 100 field isolates, it was found that the NS1 proteins had the greatest mobility differences, far exceeding those observed among other corresponding viral polypeptides. Partial sequence determination of RNA segment eight from 12 viruses revealed the existence of nonsense mutations at six different positions in their NS1 coding regions. The termination codons consisted of opal, ochre, and amber mutations. The sizes predicted from these sequences of 202, 217, 219, 220, 230, and 237 amino acids were in agreement with the observed mobilities of the viral polypeptides on SDS-polyacrylamide gels. The observation of large deletions in the carboxy termini of the NS1 proteins of field virus isolates would suggest that a high degree of variation can be tolerated in this polypeptide without affecting its functional capability.

Base Sequence↗