Showing papers by "Phillip A. Sharp published in 1974"

Mapping of adenovirus 2 RNA sequences in lytically infected cells and transformed cell lines.

Abstract: The strands of the six EcoRI fragments and the HpaI fragments E and C of Ad2 DNA were separated by electrophoresis in agarose gels. Using 32P-labeled fragment strands in solution hybridization experiments, the fraction of each strand complementary to RNA extracted from infected or transformed cells was assayed by chromatography on hydroxylapatite. In this manner, a tentative map of the cytoplasmic RNA sequences has been constructed for viral RNA extracted from cells both early and late during infection (see Fig. 16; in the map shown, the two strands of Ad2 are named the r and l strands following the bacteriophage convention). Since early cytoplasmic RNA anneals to four distinct regions of the genome, Ad2 probably codes for at least four early gene functions. Summation experiments have shown that all RNA sequences found in the cytoplasm of cells early during infection are also present in the cells' cytoplasm at late times. Viral RNA sequences in five independently isolated and cloned transformed rat cell lines were also mapped on the Ad2 genome. One class of Ad2-transformed rat cells contains RNA sequences complementary to only the segment of Ad2 DNA from 0.03-0.10 on the physical map, and this corresponds to one of the four regions of the genome expressed early during infection. If a viral gene product is necessary to maintain the transformed phenotype of the cell or codes for the virus-specific tumor (T) antigen, this genetic information must be at the left end of the genome (see Fig. 16). The two other classes of Ad2-transformed rat cells contain viral RNA sequences complementary to two or three of the regions of the genome transcribed into early cytoplasmic RNA. At both early and late times during the lytic cycle, the nucleus of the infected cell contains viral RNA sequences that are not transported to the cell's cytoplasm, suggesting that RNA processing and selection may play a role in the regulation of viral mRNA production.

Viral DNA Sequences in Cells Transformed by Simian Virus 40, Adenovirus Type 2 and Adenovirus Type 5

Abstract: Until recently, the study of DNA tumor viruses has been an essentially syntactic subject. Those who have worked in the field commonly believe, for example, that there are interconnections between the expression of integrated viral genomes, the structure of cell surfaces and the growth properties of cells. On the whole, however, the anastomosis of various features has worked out well: it has resulted in a groundplan on which the apparently diverse elevations of a fast-growing field can quickly be sited with respect to one another.

Conditional Lethal Mutants of Adenovirus 2-Simian Virus 40 Hybrids I. Host Range Mutants of Ad2+ND1

Abstract: Human adenovirus type 2 (Ad2) grows poorly in monkey cells, although this defect can be overcome by co-infection with simian virus 40 (SV40). The nondefective Ad2-SV40 hybrid virus, Ad2(+)ND1, replicates efficiently in both human and African green monkey kidney cells, presumably due to the insertion of SV40 sequences into the Ad2 DNA. Several mutants of Ad2(+)ND1 have been isolated that grow and plaque poorly in monkey cells, although they retain the ability to replicate and plaque efficiently in HeLa cells. One of these mutants (H39) has been examined in detail. Studies comparing the DNA of the mutant with Ad2(+)ND1 either by the cleavage patterns produced by Escherichia coli R.RI restriction endonuclease digestion or by heteroduplexing reveal no differences. The pattern of protein synthesis of Ad2(+)ND1 and H39 in monkey cells is quite different with the mutant resembling Ad2, which is defective in the synthesis of late proteins. However, in human cells, the proteins synthesized by H39 and the parent Ad2(+)ND1 are very similar. The production of SV40 U antigen in H39-infected cells is different from that in Ad2(+)ND1-infected cells. Finally, the growth of H39 in monkey cells can be complemented by SV40.

Specific Fragmentation of DNA of Adenovirus Serotypes 3, 5, 7, and 12, and Adeno-Simian Virus 40 Hybrid Virus Ad2+ND1 by Restriction Endonuclease R· EcoRI

Abstract: The products of complete digestion of duplex DNA of each of seven human adenoviruses with restriction endonuclease R. EcoRI ranged from two fragments for adenovirus 7 DNA (Ad7) to six fragments for Ad12 and Ad2 DNA. Viral serotypes from the same subgroups appeared to have related cleavage sites; Ad3 DNA and Ad7 (cl E46-LL) DNA were each cleaved into three fragments, and Ad7 (cl 19) DNA lacked one of the cleavage sites present in Ad3 and Ad7 (cl E46-LL) DNA. One of the cleavage sites in Ad2 DNA was deleted in the DNA' of adeno-SV40 hybrid virus Ad2(+)ND1, and three of the cleavage sites in Ad2 DNA were missing in Ad5 DNA. Thus, Ad2(+)ND1 DNA was cleaved into five and Ad5 DNA into three fragments. Each fragment represented a unique segment of viral DNA since each fragment was obtained in equimolar amounts and since the sum of the molecular weights of the fragments equaled the molecular weight of the homologous intact adenovirus DNA.

Viral DNA in Transformed Cells. III. The Amounts of Different Regions of the SV40 Genome Present in a Line of Transformed Mouse Cells

Abstract: 32P-Labeled SV40 DNA was treated sequentially with restricting endonucleases EcoRI and Hpa I, and the resulting four fragments of DNA were separated by gel electrophoresis. The kinetics of renaturation of each of the fragments and of complete SV40 DNA were measured in the presence of DNA extracted from the SVT2 line of SV40-transformed mouse cells. It was found that these cells contain about six copies of a segment of DNA which includes the early region of the SV40 genome, and about one copy of the late viral sequences. To map the region of the viral genome which is transcribed in SVT2 cells, separated strands of each of the four fragments were prepared and hybridized to total transformed cell RNA. Part of the E strands of the two DNA fragments (A and C) which span the early region of the SV40 genome were found to enter the hybrid.

Studies on the transcription of simian virus 40 and adenovirus type 2.

Abstract: Both adenovirus 2 and simian virus 40 interact with cultured cells in two different ways. On the one hand, there is a productive or lytic response in which the great majority of the cells yield progeny virus and die, and on the other there is an incomplete infection in which little or no virus is produced and the cells survive. Some of these surviving cells assume a new set of stable properties that closely resemble the properties of cells derived from tumors. These cells are said to be “transformed.” Which consequence virus infection produces is solely determined by the species of the host cell. Table I lists the cells that are commonly used in studies of lytic infection and transformation by adenovirus 2 and SV40.