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Biomedical subjects

P A Sharp

Publications and source records attributed to P A Sharp.

At least 235 records · Page 13Linked to original sources
PubMed1983

Expression of chimeric genes in the early region of SV40.

Chimeric genes have been constructed by inserting foreign gene sequences in the early region of SV40. The genes contained the first exon of the SV40 large T gene with 180 bp of its intron and either the third exon of the rat preproinsulin gene II with 488 bp of its large intron or the third exon of the mouse beta globin gene with 63 bp of its intron. The chimeric genes contained a 5' splicing site (SS) from SV40 and a 3' SS from the inserted gene. Both the preproinsulin and the globin insertions contained a polyadenylation signal. The SV40 early poly(A) addition signal was also retained. High-titer virus stocks were obtained when the recombinants, which contained SV40 origin of replication and the entire late region, were used to transfect a cloned line of COS cells (COS-M6). These stocks typically contained no detectable wild-type virus. RNA mapping demonstrated the following: (a) The SV40-rat preproinsulin chimeric RNA was initiated at the SV40 early promoter, spliced from the SV40 5' SS to the rat preproinsulin 3' SS, and polyadenylated solely at the SV40 poly(A) addition signal. (b) The SV40-mouse beta globin chimeric RNA was initiated at the SV40 early promoter, spliced from the 5' SS to the mouse beta globin 3' SS, and polyadenylated at the mouse beta globin poly(A) site. The chimeric RNAs were overproduced, owing to low levels of T antigen in the COS-M6 cells, which did not completely repress transcription from the early region. Fusion proteins of 15,500 molecular weight resulted from expression in vivo of the SV40-rat preproinsulin chimeric gene and of 11,500 molecular weight for the SV40-mouse beta globin chimeric gene. The molecular weights of the proteins suggested that they were initiated at the early SV40 AUG and that translation continued across the chimeric splice sites. The chimeric proteins were also overproduced.

Animals↗
PubMed1983

Malignant rabbit fibroma virus causes secondary immunosuppression in rabbits.

Shope fibroma virus (SFV) causes a localized, self-limited, fibroblastic proliferation in adult rabbits. Extracts of Shope fibroma tumors were found to contain a second virus that induces a rapidly progressive disseminated tumor. Dissemination of this malignant fibroma is associated with activation of commensal mucosal infection with Pasteurella multocida, causing purulent conjunctivitis and rhinitis and resulting in death from nasal obstruction. We have isolated this new agent by two cycles of plaque purification. It is a poxvirus that is antigenically virtually identical to SFV as measured by a plaque reduction assay, but behaves differently both in vivo and in vitro. We have called this virus malignant rabbit fibroma virus (MV). Electrophoresis of restriction digests made with HIND III indicates that despite the antigenic similarity of SFV and MV, the locations of HIND III sites in the two viral genomes are quite different. These experiments have enabled us to determine that MV was present in small quantities in our initial uncloned stock of Patuxent strain SFV. Lymphocytes from rabbits bearing MV-induced tumors responded poorly to both B and T lymphocyte mitogens. This nonspecific immunologic dysfunction is evident at or before the time when metastases and Gram-negative infection develop, and it becomes more profound as the disease progresses. MV-induced tumors may provide a model for Gram-negative infection and decreased immunologic responsiveness associated with malignancies.

Animals↗
PubMed1982

Separation and characterization of factors mediating accurate transcription by RNA polymerase II.

A whole cell extract of HeLa cells was resolved through two successive chromatographic steps using an extension of the procedure of Matsui et al. (Matsui, T., Segall, J., Weil, P. A., and Roeder, R. G. (1980) J. Biol. Chem. 255, 11992-11996). RNA polymerase II and three of the resulting fractions were necessary and sufficient for accurate transcription of the adenovirus major late promoter. This accurate transcription was quantitated as a function of each of the required fractions, polymerase, and DNA. A linear range of response was observed in each case. Using the linear ranges for assay, it was possible to calculate net purifications and yields for each of the required transcriptional activities after chromatography. These activities were each shown to sediment with a distinct peak on sucrose gradients. The effects of variations in salt concentration, magnesium concentration, temperature, and reaction time were determined. High resolution analysis of runoff transcripts showed that the reconstituted system initiated transcription precisely at the adenovirus major late and early region IV promoters.

DNA-Directed RNA Polymerases↗
PubMed1982

The sequences of an expressed rat alpha-tubulin gene and a pseudogene with an inserted repetitive element.

The rat genome contains two segments closely related to a rat alpha-tubulin mRNA. Both have been cloned and complete nucleotide sequences are presented. Analysis of the structure and sequence of one of these establishes it as a functional alpha-tubulin gene. The second segment is a processed alpha-tubulin pseudogene. Comparison of this pseudogene to the mRNA and gene coding for alpha-tubulin strongly suggests that a mature mRNA was involved in its origin. Features of the pseudogene and a dispersed repetitive element inserted within it possibly reflect a common RNA-mediated process of insertion.

Animals↗
PubMed1982

Expression of a X. laevis tRNATyr gene in mammalian cells.

Expression of a X. laevis tRNATyr gene has been studied in mammalian cells. This tRNATyr gene has a 13 base intervening sequence adjacent to its anticodon. A fragment containing the tRNATyr gene was cloned into the late region of SV40. Cells infected with a recombinant virus stock vastly overproduce a tRNATyr that is properly spliced, processed and modified. It was also found that the X. laevis tRNATyr is identical or nearly identical to an endogenous tRNATyr of monkey kidney cells. The possibility of using the X. laevis tRNATyr gene to create an amber suppressor for mammalian cells is discussed.

Animals↗
PubMed1982

Assembly of adenovirus major capsid protein is mediated by a nonvirion protein.

The assembly of hexon, the major capsid protein of adenovirus, was investigated with the use of conformation-specific monoclonal antibodies. The hexon capsomere is a trimer of three identical monomers folded into a highly conserved and stable structure. The unique nature of this structure is indicated by the lack of common antigenic determinants between the capsomere and either monomeric or denatured hexon. The assembly of the trimer requires the action of a nonvirion protein, the 100K protein. The virus-encoded 100K protein forms a tight complex with hexon polypeptides. This 100K-hexon complex can form on the polyribosomes while hexon is a nascent chain. Exclusion chromatography revealed that the complex has a molecular weight of 800,000. The complex contains only pretrimer hexon; no mature hexon capsomeres can be found bound to 100K. Kinetic analysis of hexon trimerization and hexon-100K binding indicated that trimerization and the release of hexon from the complex occur concomitantly.

Adenoviruses, Human↗
PubMed1982

Establishment of mammalian cell lines containing multiple nonsense mutations and functional suppressor tRNA genes.

We describe the generation of mammalian cell lines carrying amber suppressor genes. Nonsense mutants in the herpes simplex virus thymidine kinase (HSV tk) gene, the Escherichia coli xanthine-guanine phosphoribosyl transferase (Eco-gpt) gene and the aminoglycoside 3' phosphotransferase gene of the Tn5 transposon (NPT-II) were isolated and characterized. Each gene was engineered with the appropriate control signals to allow expression in both E. coli and mammalian cells. Expression in E. coli made possible the use of well developed bacterial and phage genetic manipulations to isolate and characterize the nonsense mutants. Once characterized, the nonsense mutants were transferred into mammalian cells by microinjection and used, in turn, to select for amber suppressor genes. Xenopus laevis amber suppressor genes, prepared by site-specific mutagenesis of a normal X. laevis tRNA gene, were microinjected into the above cell lines and selected for the expression of one or more of the amber mutant gene products. The resulting cell lines, containing functional amber suppressor genes, are stable and exhibit normal growth rates.

Animals↗
PubMed1982

An amber suppressor tRNA gene derived by site-specific mutagenesis: cloning and function in mammalian cells.

We describe the synthesis, cloning, expression, and in vivo function of a suppressor tRNA gene in mammalian cells. By using "primer-directed mutagenesis" on a Xenopus laevis tyrosine tRNA gene cloned into the recombinant single-strand phage M13mp5, we have generated an amber suppressor tRNA gene that has a nucleotide change--GTA leads to CTA--in the anticodon sequence. The suppressor (Su) tRNA gene was introduced into monkey kidney cells (CV-1) by using simian virus 40 (SV40) DNA as vector (SV40-tRNATyrSu+). CV-1 cells infected with virus containing the mutant, but not the wild-type, tRNA gene produce a functional amber suppressor tRNA as indicated by suppression of amber mutations in co-infecting adenovirus serotype 2-SV40 hybrids. Further evidence that suppression of these amber mutations is tRNA mediated was derived by isolation of total tRNA from CV-1 cells infected with the SV40-tRNATyr (Su+) recombinant and its use in demonstration of read through of an amber codon during in vitro translation of tobacco mosaic virus RNA in reticulocyte extracts. Interestingly, the amplification of an amber suppressor gene in CV-1 cells does not interfere with SV40 production, suggesting that suppression of amber codons may not be very deleterious to mammalian cell metabolism.

Animals↗
PubMed1982

Construction of a modular dihydrofolate reductase cDNA gene: analysis of signals utilized for efficient expression.

Dihydrofolate reductase (DHFR) modular genes have been constructed with segments containing the adenovirus major late promoter, a 3' splice site from a variable region immunoglobulin gene, a DHFR cDNA, and portions of the simian virus 40 (SV40) genome. DNA-mediated transfer of these genes transformed Chinese hamster ovary DHFR- cells to the DHFR+ phenotype. Transformants contained one to several copies of the transfected DNA integrated into the host genome. Clones subjected to growth in increasing concentrations of methotrexate eventually gave rise to lines containing several hundred copies of the transforming DNA. Analysis of the DHFR mRNA produced in amplified lines indicated the following. (i) All clones utilize the adenovirus major late promoter for transcription initiation. (ii) A hybrid intron formed by the 5' splice site of the adenovirus major late leader and a 3' splice site from a variable-region immunoglobulin gene is properly excised. (iii) The mRNA is not efficiently polyadenylated at sequences in the 3' end of the DHFR cDNA but rather uses polyadenylation signals downstream from the DHFR cDNA. Three independent clones produce a DHFR mRNA containing SV40 or pBR322 and SV40 sequences, and the RNA is polyadenylated at the SV40 late polyadenylation site. Another clone has recombined into cellular DNA and apparently uses a cellular sequence for polyadenylation. Introduction of a segment containing the SV40 early polyadenylation signal into the 3' end of the DHFR cDNA gene generated a recombinant capable of transforming cells to the DHFR+ phenotype with at least a 10-fold increase in efficiency, demonstrating the necessity for an efficient polyadenylation signal. Attachment of a DNA segment containing the transcription enhancer (72-base pair repeat) of SV40 further increased the biological activity of the modular DHFR gene 50- to 100-fold.

Adenoviridae↗
PubMed1981

A gene chimaera of SV40 and mouse beta-globin is transcribed and properly spliced.

A gene chimaera of the first exon from simian virus 40 (SV40) sequences coding for T antigen placed upstream from the third exon from mouse beta-globin, and separated by the resultant new chimaeric intron, was cloned into a bacterial plasmid. When transfected into monkey cells, the gene chimaera was transcribed, polyadenylated and spliced using the donor splice site from SV40 and the acceptor splice site from mouse beta-globin. This result suggests that a donor site from one gene can be spliced to an acceptor site from another gene.

Antigens, Neoplasm↗
PubMed1981

Transcription of Simian virus 40 DNA in a HeLa whole cell extract.

Extracts of HeLa cells containing RNA polymerase II and other factors recognize specific sites on linear simian virus 40 (SV40) DNA for initiation of transcription. The most prominent RNA products transcribed from the early region of SV40 and th E strand are initiated at sites 0.67 and 0.655 on the SV40 map. These two RNAs are synthesized by polymerase II. Their 5' termini were positioned by sizing transcripts that extend from the initiation site to the end of the template restriction endonuclease fragment, and by S1-nuclease mapping of unlabeled RNA using DNA probes labeled at their 5' termini. The limit of resolution of mapping of 5' termini is approximately 25 nucleotides. RNAs with 5' termini at similar positions have been found during characterization of mRNAs produced in infected cells. Thus, the whole cell extract is probably initiating transcription on linear SV40 DNA in vitro at the same sites as RNAs synthesized in vivo. Two other processes frequently involved in mammalian cell mRNA biosynthesis, creation of specific 3'-terminal polyadenine tracts and RNA splicing, were not detected during the course of these studies.

Cell Transformation, Viral↗
PubMed1981

Inhibition of transcription factor activity by poliovirus.

To study the poliovirus-induced inhibition of host-cell RNA synthesis, we prepared transcription extracts from mock-infected and poliovirus-infected HeLa cells. In contrast with the control extracts, poliovirus-infected cell extracts prepared 3 hr after infection were unable to transcribe specifically DNA templates recognized by RNA polymerase II. Accurate transcription by RNA polymerase III, however, was only slightly reduced. Supplementation of the infected cell extract with a crude preparation of transcription factors (S100) restored its ability to transcribe a polymerase II template specifically; supplementation with purified polymerase II had no effect. When the S100 was fractionated on a phosphocellulose column, the restoration activity eluted between 0.35 M and 1 M KCl. When we tested infected extracts for inhibitory activity by mixing uninfected and infected cell extracts, no in vitro inhibition of polymerase II transcription by the uninfected extract was evident. These results indicate that at least one factor required for specific transcription by polymerase II is deficient in extracts from poliovirus-infected cells.

HeLa Cells↗
PubMed1981

T antigen repression of SV40 early transcription from two promoters.

The SV40 early mRNAs encode large (T) and small (t) tumor antigens. During the lytic cycle, the 5' termini of the early mRNAs undergo a shift: shortly after infection only an initiation site downstream from the TATA box is utilized; later an upstream initiation site becomes prominent. Both initiation sites are utilized in an in vitro transcription extract. D2T, a T antigen analog, specifically represses transcription in vitro from both initiation sites, but at different concentrations. Binding of D2T to site I suppresses initiation from the site downstream of the TATA box; binding to sites II and I suppresses initiation from the upstream site. The role of T antigen binding in repression of transcription and in the shift of initiation sites on the early strand is discussed.

Antigens, Polyomavirus Transforming↗