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Biomedical subjects

P A Sharp

Publications and source records attributed to P A Sharp.

At least 271 records · Page 15Linked to original sources
PubMed1979

Comparative study of different isolates of murine sarcoma virus.

The RNA genomes of a variety of murine sarcoma viruses (MSV) were compared by heteroduplex analysis. These viruses included the Moloney-derived isolates 124-MSV, m1-MSV, m3-MSV, HT1-MSV, and NP-MSV and also two independent isolates, Gazdar MSV and 1712-MSV. All of these viral genomes exhibited the acquired cellular sequences previously identified in 3124-MSV and thought to be responsible for transformation and sarcomagenesis. The location of the acquired cellular sequences within the envelope gene was variable in different MSV isolates, suggesting that the cellular sequences can be expressed in different positions relative to murine leukemia virus-derived information present in MSV. Deletions in the gag coding region of the different MSVs were consistent with their known gag-related gene products. Based on several features of the hetero-duplex analysis and the known genealogical relationships of the different MSVs, various possible mechanisms for the formation of MSV are considered.

Animals↗
PubMed1978

Structure of the adenovirus 2 early mRNAs.

We have defined the structure of adenovirus 2 (Ad2) cytoplasmic RNAs produced during the early phase of infection. Hybrids between cytoplasmic RNA and DNA restriction fragments of the viral genome were digested with endonuclease S1 or exonuclease VII, and the products were analyzed by gel electrophoresis. Seven abundant cytoplasmic RNAs (assumed to be mRNAs) were identified, and all have a spliced structure. Different mRNAs produced from a single transcriptional unit contain extensively overlapping sequences, and differ from each other by the pattern in which genome sequences are spliced together. The structures of the early Ad2 mRNAs are consistent with a model for mRNA biosynthesis in which an initial transcript is processed into a mature mRNA by "splicing out" internal sequences. The pattern of spliced mRNAs produced from the early region responsible for the transforming activity of Ad2 resembles the splicing pattern of the oncogenic early mRNAs of simian virus 40 (SV40). This fact, in conjunction with recent DNA sequencing results, leads us to suggest that, like the SV40 tumor antigens, the polypeptides encoded by these Ad2 mRNAs have an identical amino acid sequence at their N terminal ends, but have different C terminal sequences.

Adenoviruses, Human↗
PubMed1978

Heteroduplex analysis of the nonhomology region between Moloney MuLV and the dual host range derivative HIX virus.

The dual host range virus HIX has been previously characterized as an envelope gene recombinant between Moloney murine leukemia virus (Mo-MuLV) and an unidentified xenotropic murine leukemia virus. Using long reverse transcripts of Mo-MuLV, a region of nonhomology has been mapped by electron microscopic analysis of heteroduplexes formed with HIX 35S virion RNA. In this nonhomology region, the Mo-MuLV cDNA strand measured approximately 900 nucleotides, mapping between 1.6 and 2.5 kilobases from the 3' end. In a previous study, hybridization of Mo-MuLV 21S RNA with Mo-MuLV cDNA resulted in the formation of different heteroduplex structures diagnostic of a noncontiguously coded leader sequence at the 5' end of the 21S RNA. Following hybridization of poly(A)+ HIX 21S RAN with 8.2 kb Mo-MuLV cDNA, analogous heteroduplex structures were observed exhibiting the Mo-MuLV:HIX substitution loop in the DNA:RNA segment of the molecules. This analysis permitted more precise mapping of the nonhomology region with respect to the splice point in the 21S presumptive glycoprotein mRNA. The mapping of this nonhomology region in HIX virus provides an internal visual marker for the 3' end of the genome which may prove useful in future analyses of other deletion or substitution derivatives of Mo-MuLV.

Base Sequence↗
PubMed1978

Spliced early mRNAs of simian virus 40.

Biochemical methods are presented for determining the structure of spliced RNAs present in cells at low concentrations. Two cytoplasmic spliced viral RNAs were detected in CV-1 cells during the early phase of simian virus 40 (SV40) infection. One is 2200 nucleotides in length and is composed of two parts, 330 and 1900 nucleotides, mapping from approximately 0.67 to approximately 0.60 and from approximately 0.54 to approximately 0.14, respectively, on the standard viral map. The other is 2500 nucleotides long and also is composed of two parts, 630 and 1900 nucleotides mapping from approximately 0.67 to approximately 0.54 and from approximately 0.54 to approximately 0.14, respectively. Correlation of the structure of these mRNAs with the structure of the early SV40 proteins, small T antigen (17,000 daltons) and large T antigen (90,000 daltons), determined by others suggests that: (i) translation of the 2500-nucleotide mRNA yields small T antigen; (ii) translation of the 2200-nucleotide mRNA proceeds through the splice point in the RNA to produce large T antigen (and thus large T antigen is encoded in two separate regions of the viral genome); and (iii) the DNA sequences between approximately 0.67 and approximately 0.60 present in both mRNAs are translated in the same reading frame in both mRNAs to yield two separate gene products that have the same NH(2)-terminal sequence. Therefore, expression of the early SV40 genes is partially controlled at the level of splicing of RNAs.

Antigens, Neoplasm↗
PubMed1978

Replication of colicin E1 plasmid DNA in vivo requires no plasmid-encoded proteins.

A derivative of bacteriophage lambda containing a colicin E1 plasmid replicon was constructed by recombinant DNA techniques. This phage, lambdacol100, has two functional modes of DNA replication; it can replicate via either plasmid or phage replication systems. lambdacol100 has been used to introduce the colicin E1 plasmid replicon into Escherichia coli previously treated with chloramphenicol to block protein synthesis. Under these conditions, lambdacol100 DNA is replicated normally as a colicin E1 plasmid. This suggests that colicin E1 plasmid replication in vivo does not require any plasmid-encoded proteins.

Bacterial Proteins↗
PubMed1978

Construction of a hybrid bacteriophage-plasmid recombinant DNA vector.

A phage-plasmid hybrid was constructed for use as a recombinant DNA vector, allowing the propagation of cloned EcoRI restriction endonuclease fragments of about 2 X 10(6) to 11 X 10(6) daltons. The colicin E1 plasmid replicon was fused to the left arm of a lambdagt generalized transducing phage with a thermolabile repressor, yielding a genome which could be replicated either by phage lambda functions or via the colicin E1 plasmid replicon. At the nonpermissive temperature, phage functions were derepressed and phage growth occurred lytically. Alternatively, at the permissive temperature, lambda functions were repressed and the vector replicated as a covalently closed circular plasmid. The phage-plasmid hybrid vector could be maintained at a copy number determined by the colicin E1 plasmid replicon and was also sensitive to amplification after chloramphenicol treatment. An EcoRI fragment of Escherichia coli DNA encoding genes of the arabinose operon also was inserted into the central portion of the vector.

Chloramphenicol↗
PubMed1977

An improved bacteriophage lambda vector: construction of model recombinants coding for kanamycin resistance.

An attenuated bacteriophage lambda has been prepared for proposed use as an EK2 vector. This phage, designated lambdagt vir Jam27 Zam718-lambdaB' can accomodate up to 11.10(6) daltons of foreign DNA inserted through Eco RI ends. The virulence mutations and nin 5 reduce the frequency of lysogen and/or plasmid formation. The mutations Jam27 and Zam718 require a suppressor in the bacterial host. The phage recombination functions contained in the EcoRIlambdaC fragment have been deleted, and only the EcoRIlambdaB fragment remains (in reverse orientation) in the center portion of the vector. In addition, this phage adsorbs to sensitive bacteria at a significantly reduced rate, conferring another block to the escape of free phage. Model recombinants have been constructed by in vitro recombination with an EcoRI fragment coding for kanamycin resistance (originally derived from R-factor R6-5). This fragment of DNA is 4.6.10(6) daltons in size, contains an inverted repeat, and also appears to contain a promoter for the kanamycin resistance gene. Using this model recombinant, the rate of transfer of kanamycin resistance to permissive and nonpermissive strains of E. coli has been measured.

Coliphages↗
PubMed1977

Spliced segments at the 5' terminus of adenovirus 2 late mRNA.

An mRNA fraction coding for hexon polypeptide, the major virion structural protein, was purified by gel electrophoresis from extracts of adenovirus 2-infected cells late in the lytic cycle. The mRNA sequences in this fraction were mapped between 51.7 and 61.3 units on the genome by visualizing RNA-DNA hybrids in the electron microscope. When hybrids of hexon mRNA and single-stranded restriction endonuclease cleavage fragments of viral DNA were visualized in the electron microscope,branched forms were observed in which 160 nucleotides of RNA from the 5' terminus were not hydrogen bonded to the single-stranded DNA. DNA sequences complementary to the RNA sequences in each 5' tail were found by electron microscopy to be located at 17, 20, and 27 units on the same strand as that coding for the body of the hexon mRNA. Thus, four segments of viral RNA may be joined together during the synthesis of mature hexon mRNA. A model is presented for adenovirus late mRNA synthesis that involves multiple splicing during maturation of a larger precursor nuclear RNA.

Adenoviridae↗
PubMed1976

Characterization of single-stranded viral DNA sequences present during replication of adenovirus types 2 and 5.

Replication intermediates of adenovirus DNA apparently contain extensive stretches of single-stranded DNA. Such single-stranded viral DNA sequences homologous to different regions of the viral genome present in adenovirus-infected cells during viral DNA replication have therefore been characterized by hybridization to the separated strands of restriction endonuclease fragments of 32P-labeled adenovirus types 2 and 5 DNA. Saturation hybridization experiments with infected cell DNA extracted at late times suggest that all regions of the adenovirus genome are represented in the single-stranded fraction, but at unequal frequencies. This nonuniform representation has been characterized in more detail with self-annealed, total cell DNA extracted 18 hr after adenovirus type 2 infection: the concentration of single-stranded sequences homologous to different regions of the viral genome was determined by comparing the rates of hybridization of 32P-labeled, single-stranded DNA probes with such self-annealed 18 hr DNA to the rates of hybridization of the same probes with equal concentrations of their complements. This approach allows the concentration of single-stranded viral DNA sequences in excess of their complements to be determined. Such sequences can be represented by two concentration gradients across the viral genome: those homologous to the r strand increase in concentration from 27.8-40.9 units toward the right end, whereas sequences homologous to the 1 strand increase from an area 27.8-40.9 units toward the left end. The time course of synthesis of single-stranded viral DNA sequences relative to accumulation of total viral DNA during the productive cycle and their behavior following a shift of H5ts125-infected cells in which viral DNA replication has begun from a permissive to a nonpermissive temperature support the contention that these sequences are indeed generated as adenovirus DNA is replicated. These results are therefore discussed in terms of current models of adenovirus DNA replication.

Adenoviruses, Human↗
PubMed1976

Adenovirus transcription. IV. Synthesis of viral-specific RNA in human cells infected with temperature-sensitive mutants of adenovirus 5.

Cytoplasmic RNA sequences produced in HeLa cells infected with the adeno-virus 5 temperature-sensitive mutants ts1, ts2, ts9, ts17, ts18, ts19, ts20, ts22, ts49, ts36, and ts125 were characterized by hybridization to DNA probes generated by strand separation of restriction endonuclease fragments of adenovirus 5 DNA. Two ""early'' mutants defective in DNA synthesis, ts125 and ts36, fail to make wild-type levels of all previously reported classes of late RNA at the nonpermissive temperature. At 40.5 degrees C, both ts125 and ts36 synthesize a wild-type complement of early cytoplasmic RNA 16 h after infection. Under these conditions, no ""late'' cytoplasmic RNA sequences were observed. Similarly, nuclear RNA present in these cells resembled early cytoplasmic RNA rather than late nuclear RNA. All the late adenovirus 5 temperature-sensitive mutants synthesized normal wild-type levels of late cytoplasmic RNA at the nonpermissive temperature, except ts2, which appears to overproduce certain cytoplasmic species.

Base Sequence↗