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Biomedical subjects

P A Sharp

Publications and source records attributed to P A Sharp.

At least 253 records · Page 14Linked to original sources
PubMed1981

SV40 DNA transfection of cells in suspension: analysis of efficiency of transcription and translation of T-antigen.

A modification of the Graham and Van der Eb (1974) DNA-calcium phosphate coprecipitation technique is shown to routinely transfect 15% of CV-1 cells with SV40 DNA. The transfection is done in suspension after detachment of cells by trypsin digestion. Transfection efficiency was measured by staining cells for the presence of SV40 T-antigen by indirect immunofluorescence and by assaying for the presence of SV40 early message by the Berk and Sharp (1978) technique.

Animals↗
PubMed1981

Expression of early adenovirus genes requires a viral encoded acidic polypeptide.

Host-range mutants of adenovirus 5 that contain a defect in region E1A (0-4.5 units) fail to replicate in HeLa cells and to transform rodent cells. In HeLa cells, these mutants synthesize only the two RNAs from E1A that share the same 5' and 3' termini but differ in length by the amount of internal sequence removed by splicing. RNA from wild-type virus, selected by hybridization to DNA from region E1A, translates into polypeptides of Mr 51,000 and 48,000 that are highly acidic in isoelectric focusing gels. These acidic Mr 51,000 and Mr 48,000 polypeptides are encoded by the longer and shorter E1A RNAs, respectively. Two of the host-range mutants, H5hr1 and H5hr2, fail to synthesize the Mr 51,000 polypeptide but do produce the Mr 48,000 polypeptide and a novel polypeptide thought to be a truncated portion of the Mr 51,000 polypeptide. H5hr1 and H5hr2 are hypothesized to have termination codons in sequences found only in RNA encoding the Mr 51,000 polypeptide. This prediction is verified for H5hr1 by DNA sequence analysis. The other three host-range mutants (H5hr3-5) synthesize both acidic polypeptides and are predicted to be missense. These results strongly imply that the Mr 51,000 polypeptide, alone or in combination with the Mr 48,000 polypeptide, is needed to regulate expression of adjacent viral genes during the early phase of adenovirus infection.

Adenoviruses, Human↗
PubMed1981

In vitro transcription of adenovirus.

A series of recombinants of adenovirus DNA fragments and pBR322 was used to test the transcriptional activity of the nine known adenovirus promoters in a cell-free extract. Specific initiation was seen at all five early promoters as well as at the major late promotor and at the intermediate promoter for polypeptide IX. The system failed to recognize the two other adenovirus promoters, which were prominent in vivo only at intermediate and late stages in infection. Microheterogeneity of 5' termini at several adenovirus promoters, previously shown in vivo, was reproduced in the in vitro reaction and indeed appeared to result from heterogeneous initiation rather than 5' processing. To test for the presence of soluble factors involved in regulation of nRNA synthesis, the activity of extracts prepared from early and late stages of infection was compared on an assortment of viral promoter sites. Although mock and early extracts showed identical transcription patterns, extracts prepared from late stages gave 5- to 10-fold relative enhancement of the late and polypeptide IX promoters as compared with early promoters.

Adenoviruses, Human↗
PubMed1981

Sequential transcription-translation of simian virus 40 by using mammalian cell extracts.

Ribonucleic acids (RNAs) transcribed in vitro by using the whole-cell extract system of Manley et al. (Proc. Natl. Acad. Sci. U.S.A. 77:3855-3859, 1980) were tested for their efficiency and fidelity in directing protein synthesis in reticulocyte lysates. Simian virus 40 deoxyribonucleic acid (DNA), cleaved by various restriction endonucleases, was used as the template. Successful translation of the small tumor antigen t, as well as the capsid proteins VP1, VP2, and VP3, was detected by immunoprecipitation analysis. Although no synthesis of large T antigen was detected, use of this technology allows detection of large T synthesis resulting from the correct splicing of as little as 0.2% of the in vitro RNA transcripts, making it ideal for use as an in vitro splicing assay. Transcripts synthesized in vitro were used as messages at least as efficiently as were viral messenger RNA's (mRNA's) synthesized in vivo; and in the case of small t, there was more efficient translation of small t mRNA synthesized in vitro than of small t mRNA synthesized in vivo. The transcripts that served as mRNA's for the various polypeptides were identified by using the following two criteria. (i) The sensitivity of synthesis of a given protein to digestion of the template DNA with restriction enzymes allowed the localization of the promoter and coding regions. (ii) Translation of size-fractionated RNA allowed confirmation of the transcript-mRNA assignments. With these techniques we found that VP2, VP3 and, in some cases, VP1 synthesis resulted from the initiation of translation at internal AUG codons. In fact, families of polypeptides were produced by initiation of translation at AUG codons within sequences coding for VP1 and T, presumably as a result of transcription initiation events that generated 5' ends immediately upstream from these AUGs. Application of this technology for the identification of coding regions within cloned DNA fragments is discussed.

Animals↗
PubMed1980

DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract.

We have developed a cell-free system for studying the synthesis of mRNA in mammalian cells. The system consists of a dialyzed and concentrated whole-cell extract derived from HeLa cells, small molecules and cofactors needed for transcription, and exogenously added DNA. Accurate transcription by RNA polymerase II is entirely dependent upon addition of promoter-containing eukaryotic DNA. At optimal DNA and extract concentrations, transcription initiation from the adenovirus serotype 2 late promoter is readily detectable, and specific transcripts over 4000 nucleotides in length are observed. The RNA synthesized in vitro contains the same 5' capped RNase T1 undecanucleotide as does the in vivo transcript. RNA synthesis also initiates accurately at both an early and an intermediate adenovirus promoter site.

Adenoviruses, Human↗
PubMed1980

Regulation of adenovirus mRNA synthesis.

The lytic cycle of adenovirus is a tightly regulated sequence of stages. When this regulation is studied at the level of mRNA production, the most significant step in controlling gene expression is initiation of transcription. Thus in preceding from one stage of expression to another, viral factors seem to turn on transcription of new sets of genes. At the moment, it is thought that viral mRNA synthesis involves initiation of transcription at ten different promoter sites. It is likely that in some manner the frequency of an initiation of transcription at nine of these sites is affected by one or more viral gene products. With the recent development of soluble in vitro transcription systems that respond to exogenously added DNA, it should be possible to begin to study regulation of gene expression at this stage of transcription. At present, these systems yield the paradoxical observation that extracts prepared from uninfected human cells more efficiently recognize the late promoter as compared to the early promoter of adenovirus. As more is learned about regulation of synthesis of viral mRNAs, examples will surely be found where RNA processing and RNA turnover play a critical role in determining the level of mRNAs. Such cases are more likely to appear in the balancing of synthesis of different mRNAs derived from one transcriptional unit. Few experiments have been directed to this possibility and the study of adenovirus molecular biology is only now entering the age of maturity where these experiments are feasible.

Adenoviruses, Human↗
PubMed1980

Expression of early and late simian virus 40 transcripts in the absence of protein synthesis.

We examined the synthesis of early and late simian virus 40 (SV40) mRNA's in SV40-infected cells treated with two kinds of protein synthesis inhibitors. SV40 stimulated the synthesis of mRNA's for both large and small tumor antigens in cells pretreated with the drug emetine before the addition of virus. Emetine is a stringent inhibitor of protein synthesis and, thus, protein factors necessary for transcription and processing of these mRNA's probably preexist in the cell. Surprisingly, infection of cells pretreated with the protein synthesis inhibitor cycloheximide stimulated the synthesis of about 10-fold-higher levels of early viral mRNA's than did comparable infections of nontreated cells. This amplification of early viral mRNA steady-state levels is probably not due to inhibition of synthesis of the early A gene product since the same degree of drug-specific amplification was seen in SV40 tsA-infected cells that were cultured at the nonpermissive temperature. However, the most interesting effect of cycloheximide addition on viral mRNA synthesis was its stimulation of the appearance of late mRNA's in the cytoplasm of cells at early periods of infection. The synthesis of late mRNA's does not appear to require the A gene product as late RNAs can be found in the cytoplasm of cells infected with SV40 tsA mutants which have been maintained at 41 degrees C and continuously cultured in the presence of cycloheximide.

Animals↗
PubMed1979

Nucleotide sequence at the 5' terminus of adenovirus 2 late messenger RNA.

One major late promoter is transcriptionally active for the majority of adenovirus 2 late RNA synthesis. Nucleotide sequences proximal to this promoter are spliced onto the 5' termini of all messenger RNAs generated from it. We report here the complete nucleotide sequence of the capped 5-'terminal undecanucleotide common to these late mRNAs. The sequence m7G5'ppp5'm6AmCUCUCUUCCGp shows striking similarities to the 5'-terminal sequences of the eukaryotic alpha- and beta-globin mRNAs and contains the sequence 5'CUUCCG3' complementary to a six-base sequence near the 3' terminus of most eukaryotic 18 S ribosomal RNAs. On the basis of this sequence and the finding that this same sequence is present at the 5' end of adenovirus 2 late nuclear RNA precursor, it should now be possible to locate the major late promotor within the adenoviral genomic DNA sequence.

Adenoviruses, Human↗
PubMed1979

An MSV-specific subgenomic mRNA in MSV-transformed G8-124 cells.

An intracellular subgenomic RNA species from MSV-transformed G8-124 cells was characterized by electron microscopy of RNA:cDNA heteroduplexes using long cDNAs both MSV and MuLV. This subgenomic RNA, 3.1 kb long, consisted of 5'-derived sequences of about 0.4 kb joined to 2.7 kb of RNA derived from the 3' end of the RNA genome. The 3'-derived sequences included the residual sequences from the MuLV pol region and the acquired cellular sequences of MSV. The genome of MSV was shown to retain approximately 0.13 kb from the 5' end of the MuLV env region, including sequences which span the point in the MuLV env mRNA. No subgenomic MSV RNA could be detected, however, which consisted of a 5'-derived leader sequence spliced to the retained env region sequences. Nor could a subgenomic MSV RNA be detected in which a 5'-derived leader sequence was joined directly to the acquired cellular sequences. Although its translation products are unknown, the subgenomic MSV RNA was present in preparations of poly(A)+ polysomal RNA, consistent with this RNA functioning as a messenger. The structure of this 3.1 kb MSV subgenomic RNA suggests a possible role in the expression of 3'-encoded MSV information, possibly including transformation-specific sequences.

Cell Transformation, Viral↗
PubMed1979

RNA synthesis in isolated nuclei: in vitro initiation of adenovirus 2 major late mRNA precursor.

We have analyzed the RNA produced in vitro by incubating nuclei from HeLa cells infected with adenovirus serotype 2. Our results show that adenovirus-specific RNA is produced at a linear rate for up to 2.5 hr. Hybridization analysis of RNA produced in nuclei isolated 18 hr after infection indicated that transcription begins at the "late promoter" at map position 16.5. Sequence analysis of the 5' termini of the in vitro transcripts showed that this system initiates RNA chains de novo at the correct promoter and that the 5' terminus is capped.

Amanitins↗
PubMed1979

Cloning and nucleotide sequence of DNA coding for bovine preproparathyroid hormone.

We have cloned in Escherichia coli a DNA copy of mRNA coding for bovine preproparathyroid hormone. Double-stranded DNA was inserted into the Pst I site in plasmid pBR322 by using the poly(dG)-poly(dC) homopolymer extension technique to join the DNA molecules. Recombinant plasmids coding for preproparathyroid hormone were identified by the plasmid's ability to arrest specifically the translation of preproparathyroid hormone mRNA. The nucleotide sequence of the largest recombinant was determined by using both chemical and enzymatic techniques. The parathyroid insert contains 470 nucleotides--102 nucleotides from the 5' noncoding region of the mRNA, 345 nucleotides representing the entire coding region, and 23 nucleotides from the 3' noncoding region. The coding sequence clarifies the hormone's amino acid sequence, which has been disputed. Codon usage is discussed.

Amino Acid Sequence↗