Showing papers by "Cold Spring Harbor Laboratory published in 1974"
TL;DR: From the order of the fragments formed by EcoRI and Hpa I on the adenovirus 2 map, it is concluded that these cell lines contain only the segment of viral DNA that stretches from the left-hand end to a point about 14% along the viral genome, which must be coded by any viral function expressed in transformed cells.
400 citations
TL;DR: Analysis of the growth properties of 40 randomly selected colonies arising after SV40 infection of 3T3 cells revealed that only 5 of the clones were indistinguishable from 3T2 cells; the remaining 35 clones differed from 3 T3 cells in that they grew as rapidly in 1% calf serum as standard SV40 transformed cells.
235 citations
TL;DR: A tentative map of the cytoplasmic RNA sequences has been constructed for viral RNA extracted from cells both early and late during infection, suggesting that RNA processing and selection may play a role in the regulation of viral mRNA production.
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.
185 citations
TL;DR: Cytoplasmic RNA extracted from human tissue culture cells infected with adenovirus type 2 was used to program protein synthesis in a cell-free system derived from mammalian cells, demonstrating seven size classes of RNA each of which programmed the synthesis of only one or two virus-specific polypeptides.
Abstract: Cytoplasmic RNA extracted from human tissue culture cells infected with adenovirus type 2 was used to program protein synthesis in a cell-free system derived from mammalian cells. Analysis of the protein product by polyacrylamide gel electrophoresis revealed ten adenovirus-specific polypeptides. Five of these were further identified by analysis of tryptic peptides. Translation of RNA fractionated by sedimentation through sucrose gradients containing formamide demonstrated seven size classes of RNA, each of which programmed the synthesis of only one or two virus-specific polypeptides. Six of the virus-specific polypeptides were translated from RNAs much larger than expected for the size of the polypeptide.
134 citations
TL;DR: The folded Escherichia coli chromosome has been analyzed in the electron microscope following the Kleinschmidt spreading technique and the intact molecules which can be found show the DNA concentrated in few nueleation areas, with one or a few interconnecting DNA fibers.
96 citations
TL;DR: By choosing a suitable upper limit to the concentration gradient, the gel system provides a method for estimating approximate molecular weights of unknown DNA fragments, by comparing their mobilities to known standards.
82 citations
TL;DR: It would appear that the conditions of denaturation used do not equally denature parainfluenza virus RNA and other RNAs, such as cellular rRNA, 45S rRNA precursor, and R17 RNA.
Abstract: The molecular weights of Sendai and Newcastle disease virus RNA were estimated by sedimentation in sucrose gradients and by length measurements in the electron microscope under both denaturing and nondenaturing conditions. Sedimentation analyses under denaturing conditions yielded molecular weight estimates of 2.3 x 10(6) to 2.6 x 10(6), whereas length measurements yielded estimates of 5.2 x 10(6) to 5.6 x 10(6) for both denatured and nondenatured viral RNA. It would appear that the conditions of denaturation used (99% dimethyl sulfoxide at 26 C, and reaction with 1.1 M formaldehyde for 10 min at 60 C) do not equally denature parainfluenza virus RNA and other RNAs, such as cellular rRNA, 45S rRNA precursor, and R17 RNA.
81 citations
TL;DR: Renaturation kinetics indicate that this repeat of Bovine satellite I DNA is itself internally repetitious, arguing in favour of a DNA replication mechanism for the evolution of this satellite.
Abstract: Bovine satellite I DNA was shown by its susceptibility to cleavage by restriction endonucleases to consist of direct tandem repeats 1,400 base pairs in length. Renaturation kinetics indicate that this repeat is itself internally repetitious. These data argue in favour of a DNA replication mechanism for the evolution of this satellite.
81 citations
78 citations
TL;DR: The pattern of protein synthesis 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.
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.
75 citations
TL;DR: Some promoters for E. coli RNA polymerase contain either of two nucleotide sequences recognised by Hind endonucleases, GTCGAC or GTTAAC, which are found in the DNA of bacteriophages and mammalian viruses.
Abstract: Some promoters for E. coli RNA polymerase contain either of two nucleotide sequences recognised by Hind endonucleases, GTCGAC or GTTAAC. Such promoters are found not only in the DNA of bacteriophages (λ and T7) but also in that of mammalian viruses, including the simian virus 40 and the human adenovirus 2.
TL;DR: A single endonuclease from H. influenzae, Hin -II, is shown to cleave the early leftward and rightward promoters, p L and p R, at the sites of cleavage of the operators, O L and O R, because the corresponding cleavage sites are specifically protected by the DNA-dependent RNA polymerase.
TL;DR: The products of complete digestion of duplex DNA of each of seven human adenoviruses with restriction endonuclease R were analyzed in this article, and the products ranged from two fragments for Adenovirus 7 DNA (Ad7) to six fragments for ad12 and Ad2 DNA.
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.
TL;DR: Mutants in a tRNA methylase gene, supK, of Salmonella typhimurium give suppression of UGA and certain frameshift mutants, and suppressors which do not suppress UGA, but which suppress frameshIFT mutants of opposite (effective) signs are described.
Abstract: Mutants in a tRNA methylase gene, supK, of Salmonella typhimurium give suppression of UGA and certain frameshift mutants. In addition suppressors which do not suppress UGA, but which suppress frameshift mutants of opposite (effective) signs are described.
TL;DR: Four discrete species of DNA that appear after the infection of a sensitive strain of Escherichia coli K12 with temperate phage Mu1 have been identified by neutral and alkaline sucrose gradient centrifugation, CsCl-ethidium bromide equilibrium gradient centrifugalation, electron microscopy, differential labeling and DNA-DNA hybridization.
TL;DR: Temperature-sensitive mutants of bacteriophage Mu, which grow at 32 C but not at 42 C, have been isolated and apparently belong to the cistrons mapping to the left of gene C, whereas the group 2 mutants have lesions in various genes between D and S.
Abstract: Temperature-sensitive mutants of bacteriophage Mu, which grow at 32 C but not at 42 C, have been isolated These mutants fall into two groups Group 1 mutants fail to lyse host cells at nonpermissive temperatures, whereas lysis occurs normally with the group 2 mutants All of the group 1 mutants apparently belong to the cistrons mapping to the left of gene C, whereas the group 2 mutants have lesions in various genes between D and S
TL;DR: Infection by murine leukemia virus rescues Kirsten sarcoma virus from only the concanavalin-A-selected variants, though all the revertants are susceptible to infection by leukemia virus.
Abstract: Revertants of Kirsten sarcoma virus transformed nonproducer BALB/3T3 cells (KA31 cells) were isolated after exposing the transformed cells to 5-fluorodeoxyuridine at high cell density, or when suspended in methylcellulose. Revertants were also isolated by treating KA31 cells with the lectin, concanavalin A, which is manyfold more toxic to transformed cells than for normal cells. The revertants resemble BALB/3T3 cells in their morphology and growth characteristics in that they have a low saturation density, fail to grow in 1% calf serum or when suspended in methylcellulose, and cease to synthesize DNA after reaching their saturation density. Infection by murine leukemia virus rescues Kirsten sarcoma virus from only the concanavalin-A-selected variants, though all the revertants are susceptible to infection by leukemia virus. The concanavalin A revertants also become transformed after infection with murine leukemia virus. All the revertants can be transformed by Kirsten sarcoma virus but not by simian virus 40.
TL;DR: The two major capsid proteins of simian virus 40 (SV40) have been purified to homogeneity by electrophoresis in sodium dodecyl sulfate polyacrylamide gels and no amino-terminal residue was found for VP3 and this capsid polypeptide may, therefore, have a blocked amino terminus.
TL;DR: Recently it has become possible to study viral RNAs functionally by assaying their ability to program the synthesis of their respective proteins in cell-free systems and from such studies, the pattern of DNA expression is emerging.
Abstract: Lytic infection of hum an cells by adenovirus proceeds by a temporal expression of genes. C lassi cally two phases have been defined: an early phase, which includes events occurring before the onset of DNA synthesis (8 hr), and a late phase, including events whose existence depends on the onset of DNA synthesis. During the late phase of infection, host cell macromolecular synthesis is progressively in hibited so that eventually only virus-specific macro m olecules are synthesized (for a review, see Philip son and Pettersson 1973). Adenovirus DNA is transcribed in the nucleus of the infected cell by an a-am anitin-sensitive poly merase that is undoubtedly a host enzyme (Price and Penm an 1972; W allace and Kates 1972). At early tim es both DNA strands are transcribed, whereas at late times there is a bias towards tran scription of the L strand (Green et al. 1970; Tibbetts et al. 1974). The sequences of RNA that appear in the cytoplasm constitute a specific subset of those found in the nucleus (Sharp et al.; Philipson et al., both this volume), im plying that extensive process ing of the RNA occurs. The details of th is selection process and of the mechanism for the switch in strand bias remain obscure. The processed RNA that does appear in the cyto plasm can be isolated as a ribonucleoprotein com plex (Lindberg and Sundquist 1974). As described elsewhere in this volume (Sharp et al.; Philipson et al.; Craig et al.), nucleic acid reassociation tech niques have been used to size the RNA species and to map the location of their corresponding DNA sequences. From such studies, the pattern of DNA expression is emerging. Recently it has become possible to study viral RNAs functionally by assaying their ability to program the synthesis of their respective proteins in cell-free systems. A comparison of the cell-free products with those synthesized in vivo provides a method for the positive identification of specific messages.
TL;DR: The susceptibility of two classes of revertants of Simian virus 40 (SV40)-transformed 3T3 cells to retransformation by SV40 or murine sarcoma virus (MSV) was studied.
Abstract: The susceptibility of two classes of revertants of Simian virus 40 (SV40)-transformed 3T3 cells to retransformation by SV40 or murine sarcoma virus (MSV) was studied. Both serum-sensitive and density-sensitive revertants are not retransformable by SV40. MSV can transform both types of revertants. The MSV-transformed revertants grow to high cell densities and form colonies when suspended in semi-solid methylcellulose medium, but are unable to grow in 1% calf serum. The MSV-transformed revertants produce infectious MSV and murine leukemia virus and possess the same number of chromosomes as the untransformed revertants.
TL;DR: Electron microscopic examination of linear heteroduplex molecules was used to investigate the position of deletions and substitutions in SV40 DNA, and rearrangements seemed to be located throughout the genome.
TL;DR: This chapter describes the three systems based upon the ability of the transformed cells to grow in conditions where the normal cells cannot, and three different selective assays for reversion are described.
Abstract: Publisher Summary This chapter describes the three systems for the isolation of transformed cells based upon the ability of the transformed cells to grow in conditions where the normal cells cannot. These three assays measure (1) the maximum cell density attained by a line in excess serum, (2) the ability of a cell to establish an isolated colony suspended in agar or Methocel, and (3) the ability of a cell line to grow in limiting or depleted sera. In the case of assay (1), most normal cell lines grow in an oriented fashion and exhibit a density-dependent cessation of cell division. In the case of assay (2), a revertant cell line lacks at least one of the properties of a transformed cell line from which it is descended. A revertant may be selected by a modification of the protocol used to isolate the transformed parent. Three different assays for selecting transformants are in current use; therefore, three different selective assays for reversion are described.
TL;DR: The P1 restriction endonuclease (EcoP1) prepared from a P1 lysogen of Escherichia coli makes one double-strand break in simian virus (SV40) DNA, which protects most of the molecules from further cleavage.
TL;DR: Competition hybridization experiments show that most of the viral sequences which are represented in stable RNA late during lytic adenovirus infection are transcribed by E. coli RNA polymerase in vitro.
TL;DR: This restriction in the established mouse cell line 3T3, in a polyoma virus-transformed subline Py3T3 and in enucleated cytoplasms prepared from these lines by cytochalasin B treatment is quantitatively examined.
TL;DR: A protein methylase, which methylates the e-amino group of lysine residues, was found in soluble of extracts of chicken embryo nuclei and was capable of endogenous methylation (methylation without added acceptor molecules).