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Biomedical subjects

P A Sharp

Publications and source records attributed to P A Sharp.

At least 163 records · Page 9Linked to original sources
PubMed1988

Association of U2, U4, U5, and U6 small nuclear ribonucleoproteins in a spliceosome-type complex in absence of precursor RNA.

Small nuclear ribonucleoprotein particles (snRNPs) associate to form multi-snRNP complexes during splicing of mRNA precursors. A vast majority of the three snRNPs U4, U5, and U6 are present in a nuclear extract in a single complex, while U1 and U2 snRNPs exist as separate particles. Under conditions optimal for splicing in vitro the U4-U5-U6 (U4/5/6) complex dissociates to release free snRNPs, suggesting that the interactions between its components are dynamic. Several forms of splicing complexes assemble on precursor RNA during splicing in vitro. One of these forms, spliceosome B, contains U2, U4, U5, and U6 snRNPs bound to the precursor RNA. This same set of snRNPs associates efficiently in the absence of precursor RNA during incubation of the extract at high salt concentration. Formation of this U2-U4-U5-U6 (U2/4/5/6) complex, the pseudospliceosome, suggests that the basic structure of the spliceosome is specified by snRNP-snRNP interactions.

Electrophoresis, Polyacrylamide Gel↗
PubMed1988

Spliceosome assembly involves the binding and release of U4 small nuclear ribonucleoprotein.

Splicing complexes that form a rabbit beta-globin precursor mRNA (pre-mRNA) have been analyzed for their small nuclear RNA (snRNA) content by both affinity chromatography and specific probe hybridization of replicas of native electrophoretic gels. A pathway of spliceosome assembly was deduced that has at least three stages. (i) U2 small nuclear ribonucleoprotein (snRNP) alone binds to sequences of mRNA upstream of the 3' splice site. (ii) U4, U5, and U6 snRNPs bind, apparently simultaneously. (iii) U4 snRNP is released to generate a spliceosome that contains U2, U5, and U6 snRNPs together with the RNA intermediates in splicing. U1 snRNP was not detected in association with any of these complexes. A parallel analysis of the spliceosome found with an adenovirus precursor mRNA substrate yielded an identical snRNP composition with one additional, unidentified RNA species, called X. This latter RNA species was not detected in the spliceosome bound to the beta-globin substrate.

Cell Nucleus↗
PubMed1988

Two transcription factors, NF-kappa B and H2TF1, interact with a single regulatory sequence in the class I major histocompatibility complex promoter.

A sequence centered 166 nucleotides upstream of the mouse H-2Kb class I major histocompatibility gene binds a nuclear factor, H2TF1, found in many cell types. Previous studies have shown that binding of H2TF1 to this sequence stimulates class I gene expression. Furthermore, this factor binds a similar sequence in the 72-base-pair repeat enhancer element of simian virus 40. We show here that NF-kappa B, an inducible B-cell-specific factor that binds the kappa immunoglobulin light chain gene enhancer, also binds the H2TF1 regulatory sequence. Methylation-interference experiments demonstrate that NF-kappa B closely interacts with six of the eight symmetrically positioned guanines that contact H2TF1. These experiments suggest that NF-kappa B may play a role in class I major histocompatibility gene expression and that H2TF1 and NF-kappa B may be related DNA-binding proteins.

Animals↗
PubMed1988

Octamer-binding proteins from B or HeLa cells stimulate transcription of the immunoglobulin heavy-chain promoter in vitro.

The B-cell-type specificity of the immunoglobulin (Ig) heavy-chain and light-chain promoters is mediated by an octanucleotide (OCTA) element, ATGCAAAT, that is also a functional component of other RNA polymerase II promoters, such as snRNA and histone H2B promoters. Two nuclear proteins that bind specifically and with high affinity to the OCTA element have been identified. NF-A1 is present in a variety of cell types, whereas the presence of NF-A2 is essentially confined to B cells, leading to the hypothesis that NF-A2 activates cell-type-specific transcription of the Ig promoter and NF-A1 mediates the other responses of the OCTA element. Extracts of the B-cell line, BJA-B, contain high levels of NF-A2 and specifically transcribe Ig promoters. In contrast, extracts from HeLa cells transcribed the Ig promoter poorly. Surprisingly, addition of either affinity-enriched NF-A2 or NF-A1 to either a HeLa extract or a partially purified reaction system specifically stimulates the Ig promoter. This suggests that the constitutive OCTA-binding factor NF-A1 can activate transcription of the Ig promoter and that B-cell-specific transcription of this promoter, at least in vitro, is partially due to a quantitative difference in the amount of OCTA-binding protein. Because NF-A1 can stimulate Ig transcription, the inability of this factor to activate in vivo the Ig promoter to the same degree as the snRNA promoters probably reflects a difference in the context of the OCTA element in these two types of promoters.

B-Lymphocytes↗
PubMed1988

The B-cell-specific Oct-2 protein contains POU box- and homeo box-type domains.

Transcription of promoters of immunoglobulin genes is controlled by an octanucleotide sequence element. The sequence of a cDNA encoding a B-cell-specific protein, Oct-2, has been determined. This protein specifically recognizes the octanucleotide element and is part of the previously identified NF-A2 family of proteins. The DNA-binding domain of Oct-2 is structurally related to the homeo box consensus and thus contains a potential helix-turn-helix sequence. Oct-2 also possesses a potential 'leucine zipper' domain, where four leucines are each separated by exactly seven residues. Comparisons of Oct-2 with protein Oct-1, which also recognizes the octanucleotide element but is constitutively expressed in all cell types, show high sequence conservation through the 60-residue DNA-binding domain, as well as an adjacent tract of 75 residues. The latter conserved region is also found in regulatory genes expressed in pituitary cells and nematodes and has been termed a POU box. Because two different cDNAs were isolated, it is proposed that the oct-2 gene is expressed as multiple mRNAs that vary in splicing patterns. Most interestingly, the oct-2 cDNA contains a second overlapping open reading frame, 278 residues in length, which might also specify a protein important for B-cell development.

Amino Acid Sequence↗
PubMed1988

Ganciclovir prophylaxis for cochlear pathophysiology during experimental guinea pig cytomegalovirus labyrinthitis.

The effectiveness of the antiviral agent ganciclovir (9-[1,3-dihydroxy-2-propoxymethyl]guanine) against guinea pig cytomegalovirus was tested in vitro in guinea pig embryonic fibroblasts and in vivo in an experimental guinea pig cytomegalovirus labyrinthitis model. In vitro, ganciclovir completely prevented guinea pig cytomegalovirus infection of guinea pig embryonic fibroblasts at concentrations above 32.6 micrograms/ml. In vivo, antibody-negative animals had an average 17-dB elevation in their auditory nerve compound action potential thresholds (P less than 0.01, t test) and showed signs bilaterally of guinea pig cytomegalovirus labyrinthitis 8 days after intrathecal inoculation of virus. Ganciclovir administration starting 1 day before inoculation prevented the development of both cochlear histopathologic change and hearing loss. Guinea pig cytomegalovirus meningitis was observed in both the drug-treated and untreated groups. High-pressure liquid chromatography confirmed the presence of ganciclovir in the serum, perilymph, and cerebrospinal fluid of the drug recipients. Prophylactic ganciclovir thus can protect the cochlea from the histopathologic changes and hearing loss normally associated with experimental guinea pig cytomegalovirus labyrinthitis.

Acoustic Stimulation↗
PubMed1988

Sedimentation analysis of polyadenylation-specific complexes.

Precursor RNA containing the adenovirus L3 polyadenylation site is assembled into a 50S complex upon incubation with HeLa nuclear extract at 30 degrees C. The cofactor and sequence requirements for 50S complex formation are similar to those of the in vitro polyadenylation reaction. Assembly of this complex requires ATP but is not dependent upon synthesis of a poly(A) tract. In addition, a 50S complex does not form on substrate RNA in which the AAUAAA hexanucleotide upstream of the poly(A) site has been mutated to AAGAAA or on RNA in which sequences between +5 and +48 nucleotides downstream of the site have been removed. These mutations also prevent in vitro processing of substrate RNA. Kinetic studies suggest that the 50S complex is an intermediate in the polyadenylation reaction. It forms at an early stage in the reaction and at later times contains both poly(A)+ RNA as well as unreacted precursor. U-type small nuclear ribonucleoprotein particles are components of the 50S complex, as shown by immunoprecipitation with antiserum specific to the trimethyl cap of these small nuclear RNAs.

Adenoviruses, Human↗
PubMed1988

Saccharomyces cerevisiae SUP53 tRNA gene transcripts are processed by mammalian cell extracts in vitro but are not processed in vivo.

We describe the results of our studies of expression of a Saccharomyces cerevisiae amber suppressor tRNA(Leu) gene (SUP53) in mammalian cells in vivo and in cell extracts in vitro. Parallel studies were carried out with the wild-type (Su-) tRNA(Leu) gene. Extracts from HeLa or CV1 cells transcribed both tRNA(Leu) genes. The transcripts were processed correctly at the 5' and 3' ends and accurately spliced to produce mature tRNA(Leu). Surprisingly, when the same tRNA(Leu) genes were introduced into CV1 cells, only pre-tRNAs(Leu) were produced. The pre-tRNAs(Leu) made in vivo were of the same size and contained the 5'-leader and 3'-trailer sequences as did pre-tRNAs(Leu) made in vitro. Furthermore, the pre-tRNAs(Leu) made in vivo were processed to mature tRNA(Leu) when incubated with HeLa cell extracts. A tRNA(Leu) gene from which the intervening sequence had been removed yielded RNAs that also were not processed at either their 5' or 3' termini. Thus, processing of pre-tRNA(Leu) in CV1 cells is blocked at the level of 5'- and 3'-end maturation. One possible explanation of the discrepancy in the results obtained in vivo and in vitro is that tRNA biosynthesis in mammalian cells involves transport of pre-tRNA from the site of its synthesis to a site or sites where processing takes place, and perhaps the yeast pre-tRNAs(Leu) synthesized in CV1 cells are not transported to the appropriate site.

Animals↗
PubMed1988

Localization of a repressive sequence contributing to B-cell specificity in the immunoglobulin heavy-chain enhancer.

The immunoglobulin heavy-chain enhancer is a cis-acting element which activates transcription of nearby genes only in cells of the lymphoid lineage. To identify the minimal sequences necessary to impart cell type transcriptional specificity, we tested the activity of several deletions and internal mutations in the mu enhancer. Experiments involving measurement of both chloramphenicol acetyltransferase activity and RNA levels indicated the presence of a dominant repressor element within the mu enhancer. This repressive activity was detected in fibroblasts but not in myeloma cells. Removal or disruption of this repressor element revealed the presence of elements within the mu enhancer that activate transcription in fibroblasts. Thus, enhancer tissue specificity is in part due to the composite of both constitutive activation and cell-type-specific repressive activity. The possible biological roles of this phenomenon are discussed.

Animals↗
PubMed1988

Identification of two transcription factors that bind to specific elements in the promoter of the adenovirus early-region 4.

Two kinds of trans-acting factors that regulate transcription from the promoter of the adenovirus early-region 4 (E4) have been identified by reconstituting nuclear extracts of HeLa cells. They were designated E4TF1 and E4TF3 for E4 transcription factors. These factors were responsible for efficient and accurate transcription in vitro from the E4 promoter, as were another transcription factor, designated E4TF2, and a crude fraction containing endogenous RNA polymerase II. E4TF1 stimulated transcription from the E4 promoter but not from the major late promoter or the E4 mutant promoter lacking the E4TF1-binding site. Footprint analysis of E4TF1 revealed that it binds to a specific region, residing between 132 and 152 base pairs upstream from the initiation site of the E4 mRNA. E4TF3 also regulated transcription from the E4 promoter. E4TF3 protected four ca. 20-base-pair regions in a DNase I footprinting assay. They were located around 40, 160, 230, and 260 base pairs upstream from the initiation site of E4 mRNA. Specific inhibition of E4 transcription was observed by addition of DNA fragments covering one of the E4TF1- and E4TF3-binding sites to in vitro transcription assays. These results suggest that both E4TF1 and E4TF3 regulate E4 transcription by binding to the specific upstream elements in the E4 promoter. These factors may be involved in the E1A transactivation of E4 transcription.

Adenoviruses, Human↗
PubMed1987

The adenovirus major late transcription factor activates the rat gamma-fibrinogen promoter.

The major late transcription factor (MLTF) is a 46-kilodalton polypeptide that specifically binds to and activates transcription from the major late promoter of adenovirus. The presence of this promoter-specific transcription factor in uninfected HeLa cell extracts suggests that MLTF is also involved in the transcription of cellular genes. This report demonstrates that MLTF specifically stimulates transcription of the rat gamma-fibrinogen gene through a high-affinity binding site. Stimulation of transcription by MLTF was not dependent on the exact position of the MLTF binding site with respect either to the transcription initiation site or to adjacent promoter elements. These results suggest that one of the cellular functions of MLTF is to control gamma-fibrinogen gene expression.

Adenoviruses, Human↗
PubMed1987

An inducible mammalian amber suppressor: propagation of a poliovirus mutant.

We describe a general protocol for controlled gene amplification, which allows conditional expression of high levels of amber suppressor activity in monkey kidney cells, and we demonstrate its use in the genetic analysis of animal viruses by the generation and propagation of the first nonsense mutant of poliovirus. A human amber suppressor tRNASer gene linked to the SV40 origin of replication and a second DNA carrying a temperature-sensitive SV40 large T antigen gene were cotransfected into monkey cells. Cell lines having stably integrated the DNAs were isolated. Shifting the cells from the nonpermissive temperature to a lower permissive temperature caused the amplification of the suppressor tRNA gene, which resulted in suppression efficiencies at amber codons of 50%-70%, as measured by suppression of an amber codon in the E. coli chloramphenicol acetyltransferase gene. A mutant of poliovirus, in which a serine codon in the replicase gene was converted to an amber codon, was efficiently propagated on the suppressor-positive cell lines. The mutant virus reverted to wild-type by a single base change to a serine codon at a frequency of approximately 2.5 x 10(-6), surprisingly low for a RNA genome.

Animals↗
PubMed1987

Interactions between small nuclear ribonucleoprotein particles in formation of spliceosomes.

Electrophoretic separation of ribonucleoprotein particles in a nondenaturing gel was used to analyze the splicing of mRNA precursors. Early in the reaction, a complex formed consisting of the U2 small nuclear ribonucleoprotein particle (snRNP) bound to sequences upstream of the 3' splice site. This complex is modeled as a precursor of a larger complex, the spliceosome, which contains U2, U4/6, and U5 snRNPs. Conversion of the U2 snRNP-precursor RNA complex to the spliceosome probably involves binding of a single multi-snRNP particle containing U4/6 and U5 snRNPs. The excised intron was released in a complex containing U5, U6, and probably U2 snRNPs. Surprisingly, U4 snRNP was not part of the intron-containing complex, suggesting that U4/6 snRNP disassembles and assembles during splicing. Subsequently, the reassembled U4/6 snRNP would associate with U5 snRNP and participate in de novo spliceosome formation. U1 snRNP was not detected in any of the splicing complexes.

HeLa Cells↗
PubMed1987

Splicing of messenger RNA precursors.

A general mechanism for the splicing of nuclear messenger RNA precursors in eukaryotic cells has been widely accepted. This mechanism, which generates lariat RNAs possessing a branch site, seems related to the RNA-catalyzed reactions of self-splicing introns. The splicing of nuclear messenger RNA precursors involves the formation of a multicomponent complex, the spliceosome. This splicing body contains at least three different small nuclear ribonucleoprotein particles (snRNPs), U2, U5, and U4 + U6. A complex containing precursor RNA and the U2 snRNP particle is a likely intermediate in the formation of the spliceosome.

Animals↗