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P A Sharp

Publications and source records attributed to P A Sharp.

At least 217 records · Page 12Linked to original sources
PubMed1984

Messenger RNA splicing in vitro: an excised intervening sequence and a potential intermediate.

Four RNA products have been characterized from a soluble system that accurately splices purified substrate RNA. The labeled substrate RNA contained the first and second exons of the major late transcription unit of adenovirus 2 and a shortened form of the first intervening sequence. One of these RNA products was the excised intervening sequence which accumulated quantitatively with the level of splicing. This RNA species has an unusual structure and is modified at internal sites. A potential intermediate in the splicing pathway which is cleaved at the 5' splice site of the first exon has been isolated. This RNA species is also modified at sites within the first intervening sequence. These results suggest that the splicing of mRNA precursors may involve sites in the intervening sequence, cleavage at the 5' splice site, cleavage at the 3' splice site, and ligation of the two exons.

Adenoviruses, Human↗
PubMed1984

Regulation of transcription of the adenovirus EII promoter by EIa gene products: absence of sequence specificity.

During adenovirus infection, the EII promoter is positively regulated by products of the EIa region. We have studied this regulation by fusing a DNA segment containing the adenovirus EII promoter to a dihydrofolate reductase cDNA segment. Expression of this hybrid gene is stimulated in trans when cell lines containing an integrated copy are either transfected with plasmids carrying the EIa region or infected with adenovirus. This suggests that EIa activity regulates transcription of the EII promoter in the absence of other viral proteins and that this stimulation can occur when the EII promoter is organized in cellular chromatin. Transcription from the EII promoter is initiated at two sites in cell lines lacking EIa activity. Introduction of the EIa region preferentially stimulated transcription from one of these two sites. A sensitive, stable cotransfection assay was used to test for specific EII sequences required for stimulation. EIa activity stimulates all mutant promoters; the most extensive deletion retained only 18 base pairs of sequences upstream of the initiation site. We suggest that regulation of a promoter by the EIa region does not depend on the presence of a set of specific sequences, but instead reflects a characteristic of promoters that have been exogenously introduced into cells. Insertion of the 72-base-pair repeat of simian-virus 40 in cis enhances transcription from the EII promoter. The stimulatory effects of EIa activity and of the simian virus 40 sequence are additive and appear to differ mechanistically.

Adenoviridae↗
PubMed1984

Requirement for distal upstream sequences for maximal transcription in vitro of early region IV of adenovirus.

A series of deletion mutants spanning the adenovirus early region IV (EIV) promoter were tested for transcription activity in vitro. At least three elements were found to be important for maximal transcription in HeLa whole-cell extracts. Deletion of the TATA box drastically reduced the transcription activity from the EIV promoter. Sequences between nucleotides -58 and -44 are also important for efficient transcription since deletion of this region reduced activity by 50%. More importantly, sequences residing upstream from -140 critically influence the level of EIV transcription. Deletion of sequences between nucleotides -325 (the right terminus of adenovirus genome) and -140 reduced the level of transcription more than 10-fold. It is possible that a specific cellular factor stimulates EIV transcription by recognition of these upstream sequences. The dependence of transcription from the EIV promoter on a distal upstream element may explain some aspects of the regulation of this promoter.

Adenoviruses, Human↗
PubMed1983

Characterization of tRNA precursor splicing in mammalian extracts.

Transcription of a Xenopus laevis tRNATyr gene and splicing of the transcript have been studied in HeLa cell extracts. This tRNATyr gene has a 13-base intervening sequence and is expressed as mature tRNA when transfected into mammalian cells. The tRNATyr gene is transcribed under conditions of low concentrations of magnesium and ATP, but is processed by splicing only when both of these cofactors are added at higher concentrations. The endonucleolytic activity of the tRNA-splicing system in the HeLa extract produces exons with 3'-phosphate and 5'-hydroxyl groups. The 3'-phosphate is retained during the ligation reaction and forms the phosphodiester bond in the mature tRNA. Retention of the 3'-phosphate during tRNA splicing differs from the more extensively studied process in yeast extracts where a phosphate group from an ATP cofactor is used to form the phosphodiester bond joining the exons. Thus, eucaryotic organisms can splice tRNA precursors by at least two distinguishable mechanisms.

Animals↗
PubMed1983

Generation of adenovirus by transfection of plasmids.

Biologically active fragments of Adenovirus 5 (Ad5) DNA that span the entire genome have been cloned into plasmids. The covalently attached terminal protein was removed and Eco RI linkers added in a fashion that preserves the Ad5 terminal sequences. When plasmids containing overlapping fragments that represent the entire genome are cotransfected onto 293 cells, infectious virus is obtained. Generation of virus depends upon the release of the 0 or 100 mu Ad5 terminus from pBR322 DNA by Eco RI cleavage. During virus production the modified termini of the transfected fragments are corrected exactly to that of wt viral DNA. The above method for preparing adenovirus recombinants has been used to construct a mutant, Ad5 delta (78.9-84.3), lacking most of the non-essential EIII transcriptional unit. This mutant is phenotypically wild type with respect to burst size and kinetics of growth. Surprisingly, it inhibits wt viral growth upon mixed infections of HeLa or 293 cells, apparently at the level of DNA replication.

Adenoviruses, Human↗
PubMed1983

Measurement of suppressor transfer RNA activity.

Transfer RNA (tRNA) suppression of nonsense mutations in prokaryotic systems has been widely used to study the structure and function of different prokaryotic genes. Through genetic engineering techniques, it is now possible to introduce suppressor (Su+) tRNA molecules into mammalian cells. A quantitative assay of the suppressor tRNA activity in these mammalian cells is described; it is based on the amount of tRNA-mediated readthrough of a terminating codon in the influenza virus NS1 gene after the cells are infected with virus. Suppressor activity in L cells continuously expressing Su+ (tRNAtyr) was 3.5 percent and that in CV-1 cells infected with an SV40- Su+ (tRNAtyr) recombinant was 22.5 percent.

Animals↗
PubMed1983

Inhibition of adenovirus early region IV transcription in vitro by a purified viral DNA binding protein.

Adenoviruses depend on cellular mechanisms for the decoding of their genetic information, and so provide a useful and simple model system for the investigation of mammalian gene expression. The five regions transcribed early in adenovirus infection are termed EIa, EIb, EII, EIII and EIV. We report here that the primary product of the EII region, a 72,000 molecular weight DNA-binding protein (DBP), specifically represses transcription from the EIV promoter in an in vitro transcription system. Single-stranded DNA binds to the DBP with high affinity, and as a result inhibits its repressor activity. Our data extend previous genetic evidence that the DBP represses EIV transcription in vivo, and suggest that it acts directly by suppressing transcription from the EIV promoter.

Adenoviridae↗
PubMed1983

Sequences controlling in vitro transcription of SV40 promoters.

A series of deletion mutants of SV40 were tested for early and late promoter activity in vitro in a transcription extract prepared from HeLa cells. These mutants had previously been characterized for expression in vivo. Transcription in vitro from both the SV40 early and late promoters was strongly dependent on an upstream region of DNA that contains six direct GC repeats. Sequences spanning two or more of these repeats stimulated transcription in a bidirectional fashion, at distances of 50-200 bp. These sequences may function by mediating the activity of a specific transcriptional factor. Little effect on transcription in vitro was observed upon deletion of the 72-bp enhancer elements. With this exception, the sequence dependence of early and late transcription in vitro was similar to that observed previously in vivo, both of the region including the GC repeats and of the early TATA sequence.

Base Sequence↗
PubMed1983

Analysis of Ad5 hexon and 100K ts mutants using conformation-specific monoclonal antibodies.

Adenovirus type 5 ts mutants deficient in hexon metabolism were investigated using conformation-specific monoclonal antibodies directed against hexon capsomeres and the viral 100K protein. The ts mutants map either in the hexon structural gene or in the gene encoding the 100K protein, a major, late nonstructural protein. All of the mutants examined (ts1, ts2, ts3, ts4, ts17, and ts20 of J. F. Williams, M. Gharpure, S. Ustacelebi, and S. McDonald (1971). J. Gen. Virol. 11, 95-101) were unable to produce the capsomeric form of hexon (a trimer of three hexon monomers) at the nonpermissive temperature. However, all of the mutants retained the ability to produce a complex of 100K and hexon which has been demonstrated to play a major role in the assembly of hexon trimers. The mutants accumulated nontrimerized hexon in this ts complex in the perinuclear region of the cell. Several of the mutants (ts1, ts2, ts3) were found to successfully assemble hexon synthesized at the nonpermissive temperature upon shift down to the permissive temperature, even in the presence of a protein synthesis inhibitor. The mutant, ts2, which maps in the hexon structural gene, was found to be dependent on protein synthesis for transport of hexon trimers into the nucleus during temperature shift down, while the 100K ts mutants, ts1 and ts3, were independent of protein synthesis for both hexon assembly and transport.

Adenoviruses, Human↗
PubMed1983

Splicing of messenger RNA precursors is inhibited by antisera to small nuclear ribonucleoprotein.

A mouse monoclonal antibody and human autoimmune sera directed against various classes of small ribonucleoprotein particles have been tested for inhibition of mRNA splicing in a soluble in vitro system. The splicing of the first and second leader exons of adenovirus late RNA was inhibited only by those sera that reacted with U1 RNP. Both U1 RNP-specific human autoimmune serum and sera directed against the Sm class of small nuclear RNPs, including a mouse monoclonal antibody, specifically inhibited splicing. Antisera specific for U2 RNP had no effect on splicing nor did antisera specific for the La or Ro class of small RNPs. These results suggest that U1 RNP is essential for the splicing of mRNA precursors.

Adenoviruses, Human↗
PubMed1983

Splicing of adenovirus RNA in a cell-free transcription system.

A soluble whole-cell extract prepared accurately from HeLa cells splices 2-3% of the RNA transcribed from a DNA template containing the first and second leader exons of late adenovirus RNA. The spliced RNA was detected by a sensitive technique using hybridization to a single-stranded phage M13 cDNA clone, followed by binding to nitrocellulose filters. The identity of the spliced RNA was established by RNase T1 and pancreatic RNase two-dimensional peptide mapping. The bond formed during the in vitro splicing reaction appears to be a typical 3',5'-phosphodiester bond as judged by its sensitivity to RNase T1. The splicing reaction is specifically inhibited by KCl at concentrations greater than 50 mM and by the addition of cellular RNA. Three features of this system may account for the detection of splicing in a soluble extract: (i) the sensitive and unambiguous hybridization assay, (ii) the high transcriptional activity of the major late promoter of adenovirus, and (iii) the use of the first and second leader exon splice of adenovirus, which may be unusually rapid.

Adenoviruses, Human↗
PubMed1983

Adenovirus hexon monoclonal antibody that is group specific and potentially useful as a diagnostic reagent.

A monoclonal antibody to the adenovirus 2 hexon protein was produced and characterized as a group-specific antibody. Positive reactivity in immunoprecipitation, indirect immunofluorescence, and radioimmunoassays was observed with human, canine, swine, bovine, murine, and simian adenoviruses. This monoclonal antibody should provide a specific and sensitive diagnostic reagent for detection of all mammalian adenoviruses.

Adenoviridae↗
PubMed1983

Aberrant distribution of human adenovirus type 2 late proteins in monkey kidney cells.

Monkey kidney cells (CV-C) infected with adenovirus type 2 displayed an aberrant distribution of 100K, 100K-hexon complex, hexon monomers, hexon trimers, penton base, and fiber proteins, relative to the patterns observed in adenovirus type 2-infected human cells. Human cell patterns were observed in CV-C cells when mutants selected for growth on monkey cells were used.

Adenoviruses, Human↗
PubMed1983

Functional suppression in mammalian cells of nonsense mutations in the herpes simplex virus thymidine kinase gene by suppressor tRNA genes.

A nonsense mutation (UAG) in the thymidine kinase gene of herpes simplex virus type 1 can be suppressed in vivo to produce active thymidine kinase by prior infection with a defective simian virus 40 stock which acts as a vector to introduce a functional suppressor tRNA gene into mammalian cells in culture. The suppression is specific for UAG, but not UGA or missense, mutants and restores thymidine kinase activity to 20 to 40% of the wild-type level. These results suggest that many cell lines susceptible to simian virus 40 infection may be transiently converted to a suppressor-positive phenotype for use in the genetic study of mammalian viruses.

Animals↗
PubMed1983

Evolution of chromosomal regions containing transfected and amplified dihydrofolate reductase sequences.

A modular dihydrofolate reductase gene has been introduced into Chinese hamster ovary cells lacking dihydrofolate reductase. Clones capable of growth in the absence of added nucleosides contain one to five copies of the plasmid DNA integrated into the host genome. Upon stepwise selection to increasing methotrexate concentrations, cells are obtained which have amplified the transforming DNA over several hundredfold. A detailed analysis of the chromosomes in three clones indicated the appearance of cytologically distinct chromosomal regions containing the amplified plasmid DNA which differ in surrounding sequence composition, structure, and location. Two of the clones examined have extensive, homogeneously staining regions. The DNA in these homogeneously staining regions replicates in the early part of the S phase. The amplified plasmid DNA is found associated at or near the ends of chromosomes or on dicentric chromosomes. We propose that integration of DNA may disrupt telomeric structures and facilitate the formation of dicentric chromosomes, which may then undergo bridge breakage-fusion cycles. These phenomena are discussed in relation to DNA transfer experiments and modes of gene amplification and chromosome rearrangement.

Animals↗
PubMed1983

Growth-dependent expression of dihydrofolate reductase mRNA from modular cDNA genes.

Dihydrofolate reductase (DHFR) synthesis is regulated in a growth-dependent fashion. Dividing cells synthesize DHFR at a 10-fold-higher rate than do stationary cells. To study this growth-dependent synthesis. DHFR genes have been constructed from a DHFR cDNA segment, the adenovirus major late promoter, and fragments of simian virus 40 (SV40) which provide signals for polyadenylation. These genes have been introduced into Chinese hamster ovary cells. The DHFR mRNAs produced in different transformants are identical at their 5' ends, but differ in sequences in their 3' ends as different sites are utilized for polyadenylation. Three transformants that utilize either DHFR polyadenylation signals or the SV40 late polyadenylation signal exhibit growth-dependent DHFR synthesis. The level of DHFR mRNA in growing cells is approximately 10 times that in stationary cells for these transformants. This growth-dependent DHFR mRNA production probably results from posttranscriptional events. In contrast, three transformants that utilize the SV40 early polyadenylation signal and another transformant that utilizes a cellular polyadenylation signal do not exhibit growth-dependent DHFR synthesis. In these three cell lines, the fraction of mRNAs polyadenylated at different sites in a tandem array shifts between growing and stationary cells. These results suggest that the metabolic state of the cell is important in determining either the efficiency of polyadenylation at various sites or the stability of mRNA polyadenylated at various sites.

Animals↗