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Biomedical subjects

P A Sharp

Publications and source records attributed to P A Sharp.

At least 127 records · Page 7Linked to original sources
PubMed1990

Identification and characterization of a HeLa nuclear protein that specifically binds to the trans-activation-response (TAR) element of human immunodeficiency virus.

Human immunodeficiency virus type 1 RNAs contain a sequence, trans-activation-response (TAR) element, which is required for tat protein-mediated trans-activation of viral gene expression. We have identified a nuclear protein from extracts of HeLa cells that binds to the TAR element RNA in a sequence-specific manner. The binding of this 68-kDa polypeptide was detected by UV cross-linking proteins to TAR element RNA transcribed in vitro. Competition experiments were performed by using a partially purified preparation of the protein to quantify the relative binding affinities of TAR element RNA mutants. The binding affinity of the TAR mutants paralleled the reported ability of those mutants to support tat trans-activation in vivo. We propose that this cellular protein moderates TAR activity in vivo.

Base Sequence↗
PubMed1990

Interactions of the Oct-1 POU subdomains with specific DNA sequences and with the HSV alpha-trans-activator protein.

Trans-activation by the herpes simplex virus (HSV) protein, alpha TIF (VP16), is dependent on an inducible enhancer sequence that contains a homolog of the octamer element. An ordered series of multiprotein complexes can be assembled on this enhancer, requiring the interactions of Oct-1, alpha TIF, and two additional cellular factors (C1 and C2). Oct-1 binds to the octamer homolog, whereas alpha TIF, also a sequence-specific DNA-binding protein, recognizes sequences within the HSV enhancer core. The partially purified C1 factor interacts directly with alpha TIF in the absence of DNA and is required to form a stabile Oct-1/alpha TIF/C1 factor complex. The POU domain of Oct-1 is a bipartite sequence recognition structure, as both the POU-specific box and the POU homeo box contribute directly to the recognition of the octamer element. Surprisingly, the POU homeo box alone is sufficient to direct the cooperative binding of alpha TIF and to assemble the Oct-1/alpha TIF/C1 factor complex.

Animals↗
PubMed1990

Transcription initiation complexes and upstream activation with RNA polymerase II lacking the C-terminal domain of the largest subunit.

RNA polymerase II assembles with other factors on the adenovirus type 2 major late promoter to generate pairs of transcription initiation complexes resolvable by nondenaturing gel electrophoresis. The pairing of the complexes is caused by the presence or absence of the C-terminal domain of the largest subunit. This domain is not required for transcription stimulation by the major late transcription factor in vitro.

Amino Acid Sequence↗
PubMed1990

A large protein containing zinc finger domains binds to related sequence elements in the enhancers of the class I major histocompatibility complex and kappa immunoglobulin genes.

A cDNA from a B-cell library was previously isolated that encodes a sequence-specific DNA-binding protein with affinities for related sites in a class I major histocompatibility complex (MHC) and kappa immunoglobulin gene enhancers. We report here approximately 6.5 kilobases of sequence of the MBP-1 (MHC enhancer binding protein 1) cDNA. MBP-1 protein has a molecular weight predicted to be greater than 200,000. A DNA-binding domain with high affinity for the MHC enhancer sequence TGGGGATTCCCCA was localized to an 118-amino-acid protein fragment containing two zinc fingers of the class Cys2-X12-His2. Analysis of expression of MBP-1 mRNA revealed relatively high expression in HeLa cells and in a human retinal cell line, with lower levels in Jurkat T cells and in two B-cell lines. Interestingly, expression of MBP-1 mRNA was inducible by mitogen and phorbol ester treatment of Jurkat T cells and by serum treatment of confluent serum-deprived human fibroblasts.

Amino Acid Sequence↗
PubMed1990

A helix-loop-helix protein related to the immunoglobulin E box-binding proteins.

A human cDNA encoding a novel protein in the helix-loop-helix family has been isolated by screening a bacteriophage expression library with a probe containing the binding site for major late transcription factor. The protein encoded by this cDNA, TFEB, probably recognizes E-box sequences in the heavy-chain immunoglobulin enhancer.

Amino Acid Sequence↗
PubMed1990

Inner ear autoantibodies in patients with rapidly progressive sensorineural hearing loss.

Recognition of immune-mediated sensorineural deafness that responds to immunosuppressive therapy has led to a search for a diagnostic assay to identify inner ear autoantibodies. Without a confirmed diagnosis of autoimmune disease, many patients have undergone inappropriate immunosuppressive treatment or developed irreversible inner ear damage. Serum from patients with progressive sensorineural hearing loss (n = 54), ulcerative colitis (N = 5), normal controls (N = 14), and animals with experimental autoimmune sensorineural hearing loss (EASNHL) were analyzed by Western blot against fresh bovine inner ear antigen preparations. The hearing loss group (19 [35%]) showed a single-or double-band migrating at 68,000 molecular weight (MW), differing from the normal group (1 of 14 [7%]) which showed a similar band (P = .031). Upon analysis by two-dimensional gel electrophoresis both the EASNHL guinea pigs and a patient reacted against identical components of inner ear antigen. These results suggest an autoimmune basis for disease in patients reacting against the 68,000 MW antigen.

Adult↗
PubMed1989

The octamer-binding proteins form multi-protein--DNA complexes with the HSV alpha TIF regulatory protein.

The herpes simplex virus transactivator, alpha TIF, stimulates transcription of the alpha/immediate early genes via a cis-acting site containing an octamer element and a conserved flanking sequence. The alpha TIF protein, produced in a baculovirus expression system, nucleates the formation of at least two DNA--protein complexes on this regulatory element. Both of these complexes contain the ubiquitous Oct-1 protein, whose POU domain alone is sufficient to allow assembly of the alpha TIF-dependent complexes. A second member of the POU domain family, the lymphoid specific Oct-2 protein, can also be assembled into similar complexes at high concentrations of alpha TIF protein. These complexes contain at least two cellular proteins in addition to Oct-1. One of these proteins is present in both insect and HeLa cells and probably recognizes sequences in the cis element. The second cellular protein, only present in HeLa cells, probably binds by protein-protein interactions.

Base Sequence↗
PubMed1989

Regulation by HIV Rev depends upon recognition of splice sites.

The ability of the Rev protein of HIV to regulate the cytoplasmic level of unspliced RNA from a beta-globin gene containing the Rev response element was dependent on the integrity of the 5' and 3' splice sites. A beta-globin pre-mRNA containing the Rev response element is not under regulation by Rev but is made Rev responsive by a mutation at either the 5' or 3' splice site. These mutant RNAs accumulated in the nucleus as unspliced precursors owing to recognition by splicing components. Only in the presence of Rev did these unspliced RNAs appear in the cytoplasm. Thus, regulation by Rev probably involves the dissociation of splicing components and pre-mRNA.

Animals↗
PubMed1989

Gene activation by induced DNA rearrangements.

A murine cell line (EN/NIH) containing the retroviral vector ZIPNeoSV(x)1 that was modified by deletion of the enhancer elements in the viral long terminal repeats has been used as an assay system to detect induced DNA rearrangements that result in activation of a transcriptionally silent reporter gene (neomycin phosphotransferase, neo) encoded by the viral genome. The spontaneous frequency of G418 resistance is less than 10(-7), whereas exposure to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) or the combination of UV irradiation plus TPA resulted in the emergence of drug resistant cell lines at a frequency of 5 per 10(6) and 67 per 10(6) cells, respectively. In several of the cell lines that were analyzed a low level of amplification of one of the two parental retroviral integrants was observed, whereas in others no alteration in the region of the viral genome was detected. To determine the effect of the SV40 large T antigen on induced DNA rearrangements, EN/NIH cells were transfected with a temperature sensitive (ts) mutant of SV40 T. Transfectants were maintained at the permissive temperature (33 degrees C) for varying periods of time (1-5 days) in order to vary SV40 T antigen exposure, after which they were shifted to 39.5 degrees C for selection in G418. The frequency of emergence of drug resistant cell clones increased with duration of exposure to large T antigen (9-52 per 10(6) cells over 1-5 days, respectively), and all cell lines analyzed demonstrated DNA rearrangements in the region of the neo gene. A novel 18-kilobase pair XbaI fragment was cloned from one cell line which revealed the presence of a 2.0-kilobase pair EcoRI segment containing an inverted duplication which hybridized to neo sequences. It is likely that the observed rearrangement was initiated by the specific binding of large T antigen to the SV40 origin of replication encoded within the viral genome. The investigations with phorbol esters, UV light, and the SV40 large T antigen demonstrate the utility of the EN NIH cell lines for the study of induced DNA rearrangements and support the future use of this system to investigate the mechanism by which varied stimuli or specific gene functions promote DNA rearrangements.

Animals↗
PubMed1989

Isolation of the gene encoding the yeast TATA binding protein TFIID: a gene identical to the SPT15 suppressor of Ty element insertions.

We report the cloning of the gene that encodes the yeast TATA binding protein TFIID. TFIID contains 240 amino acids and has no obvious sequence similarity to other known proteins. TFIID was synthesized in vitro and in two separate assays behaved identically to the protein purified from yeast. TFIID bound to TATA elements from the adenovirus major late promoter (TATAAAA) and the yeast LEU2 promoter (TATTTAA) and formed protein-DNA complexes stable to electrophoresis only in the presence of TFIIA. In vitro-synthesized yeast TFIID also complemented a mammalian in vitro transcription system that lacked TFIID. Comparison of the yeast TFIID gene with the yeast SPT15 gene (suppressor of Ty element insertions) showed that the two genes are identical. This finding indicates that the yeast TFIID activity defined in vitro is responsible for specific transcription in vivo.

Amino Acid Sequence↗
PubMed1989

Linkage between immunoglobulin E responses underlying asthma and rhinitis and chromosome 11q.

Family studies of IgE responses to common inhaled antigens have suggested dominant inheritance of atopy. Molecular genetic linkage analysis was used to confirm this proposal. In seven families the transmission of atopy was linked, with a maximum lod score of 5.58, to a DNA polymorphism defined by p lambda MS.51, which confirms dominant inheritance and assigns the gene locus to chromosome 11.

Asthma↗
PubMed1989

Replication of RNA by the DNA-dependent RNA polymerase of phage T7.

The DNA-dependent RNA polymerase of bacteriophage T7 utilizes a specific RNA as a template and replicates it efficiently and accurately. The RNA product (X RNA), approximately 70 nucleotides long, is initiated with either pppC or pppG and contains an AU-tich sequence. Replication of X RNA involves synthesis of complementary strands. Both strands are also significantly self-complementary, producing RNA with an extensive hairpin secondary structure. Replication of X RNA by T7 RNA polymerase is both template and enzyme specific. No other RNA serves as template for replication; neither do other polymerases, including the closely related T3 RNA polymerase, replicate X RNA. The T7 RNA polymerase-X RNA system provides an interesting model for studying replication of RNA by DNA-dependent RNA polymerases. Such a mechanism has been proposed to propagate viroids and hepatitis delta, pathogenic RNAs whose replication seems to depend on cellular RNA polymerases.

DNA-Directed RNA Polymerases↗
PubMed1989

Five intermediate complexes in transcription initiation by RNA polymerase II.

A native gel electrophoresis DNA binding assay was used to resolve complexes formed on the adenovirus Major Late Promoter by general transcription factors and RNA polymerase II. Five sets of complexes containing distinct components were identified. These complexes were generated by sequential binding of TFIID, TFIIA, TFIIB, RNA polymerase II, and TFIIE. The relative positions of each of the factors in the complexes were determined by DNAase I footprint analysis. TFIIA, derived from yeast or mammalian cells, formed a complex with yeast TFIID and the TATA element. TFIIB bound to this complex and probably acts as a "bridge" to the polymerase and the initiation site. The addition of ATP or dATP, necessary for "activation" of transcription, resulted in an alteration of the footprint in the +20 to +30 region, the same area protected upon addition of TFIIE to the initiation complex. Addition of ribonucleotide triphosphates generated new complexes that contained accurately initiated transcripts associated with the transcription machinery and the template DNA. A model for the interactions of components in initiation of transcription by RNA polymerase II is proposed.

Adenoviridae↗
PubMed1989

5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole inhibits transcription elongation by RNA polymerase II in vitro.

The purine nucleoside analog 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) is a selective inhibitor of transcription by RNA polymerase II. Although a wealth of in vivo studies have suggested that DRB inhibits transcription by enhancing the premature termination of elongating polymerase molecules, in vitro studies to date have been interpreted to suggest that DRB acts at the level of transcription initiation. We have analyzed the mechanism of DRB-mediated transcription inhibition in vitro both in HeLa whole cell extracts and in a partially purified transcription system. The results indicate that the extent to which DRB inhibits the synthesis of a RNA transcript is directly proportional to its length. For example, DRB was found to preferentially inhibit transcription in vitro of promoter-distal relative to promoter-proximal portions of the adenovirus major late transcription unit. A factor potentially involved in mediating this inhibitory effect is identified. We conclude that the mechanism of DRB inhibition of transcription in vivo and in vitro are similar.

Dichlororibofuranosylbenzimidazole↗
PubMed1989

A gamma-interferon-induced factor that binds the interferon response sequence of the MHC class I gene, H-2Kb.

Transcription of class I genes of the major histocompatibility complex (MHC) can be induced by interferons. Treatment of HeLa cells with interferon-gamma induces a DNA-binding factor, IBP-1, specific for a site within the interferon response sequence (IRS) of the H-2Kb promoter. The mol. wt of IBP-1, as estimated by photoactivated protein-DNA crosslinking analysis, is approximately 59 kd. Point-mutation of this binding site abolishes IBP-1 interaction and the ability of the MHC promoter to respond to interferon. Induction of this binding activity is rapid and closely parallels the previously reported time course of transcriptional activation of endogenous MHC class I genes. Treatment of cells with cycloheximide, a protein synthesis inhibitor, blocked the induction of the DNA-binding activity. An oligonucleotide derived from the virus- and double-stranded RNA-inducible promoter of the interferon-beta 1 gene is able to bind IBP-1. Sequences similar to the IBP-1 binding site are found upstream of many interferon-responsive genes.

Animals↗
PubMed1989

Identification of a yeast protein homologous in function to the mammalian general transcription factor, TFIIA.

The yeast homolog of the mammalian RNA polymerase II general transcription factor TFIIA has been identified by complementation of a mammalian in vitro transcription system depleted for TFIIA. Like the mammalian factor, the yeast protein does not bind DNA, alters the size of the TFIID DNase I footprint at the adenovirus major late promoter, and forms specific TFIIA-TFIID-DNA complexes which are stable during electrophoresis in native acrylamide gels. The partially purified yeast factor was used to investigate its effect on the binding of TFIID to the major late promoter. Contrary to earlier models, we find that TFIIA does not significantly change the affinity or kinetics of TFIID binding, suggesting that it acts by altering the conformation of TFIID and/or by serving as a bridge between TFIID and the other general transcription factors.

Adenoviridae↗