Showing papers by "Cold Spring Harbor Laboratory published in 1980"
TL;DR: It is proposed that, upon transformation, the host cell ligates incorporated DNA into a large concatameric structure which may at times be as large as 2000 kilobases and from blotting data alone the authors cannot determine whether this structure is chromosomal or extrachromosomal in location.
415 citations
TL;DR: A possible role for the 130K protein in the organization of actin filaments at membrane attachment sites is suggested, and the use of microinjection of the protein covalently labeled with the fluorescent dye fluorescein isothiocyanate into living cultured mammalian cells is demonstrated.
389 citations
TL;DR: By examining both the transformation efficiency of yeast of various plasmids containing defined regions of the 2 mu circle genome and the characteristics of the resultant transformants, it is determined that efficient use of the2 mu circle origin requires some function or functions encoded in the molecule at a site away from the origin.
276 citations
TL;DR: It is shown that replication of the template per se is required for expression of late regions L2--L5 and that the accumulation of early gene products does not suffice and the promoter-proximal late gene block L1 appears to be subject to processing control.
217 citations
TL;DR: To clarify the action of T proteins in transformation, several monoclonal antibodies directed against different regions of the T molecule are prepared and one of these antibodies recognizes an antigenic determinant on T that is also present on a host cell protein of molecular weight 68,000.
Abstract: The large T protein coded by the early region of simian virus 40 (SV40) is involved in the induction and maintenance of cell transformation1. It is not clear which, properties of T are important in causing the transformation, since the protein is multifunctional2–9. To clarify the action of T proteins in transformation, we have prepared several monoclonal antibodies directed against different regions of the T molecule. One of these antibodies, DL 3C4, recognizes an antigenic determinant on T that is also present on a host cell protein of molecular weight 68,000. This cross-reactive 68K protein is located within the nucleus of all mammalian cell types examined and has a characteristic granular distribution as shown by immuno-fluorescence. The antigenic determinant recognized by DL 3C4 is resistant to denaturation. Studies on Adeno-SV40 hybrid viruses show that the antigenic site on T is coded for by sequences located between 0.44 and 0.29 on the SV40 map.
203 citations
TL;DR: A 606 base pair segment of adenovirus type 2 DNA which includes the gene for polypeptide IX and its flanking regions has been sequenced and direct analysis of its capped oligonucleotide show that the gene and its mRNA are colinear.
170 citations
TL;DR: The chicken thymidine kinase gene is obtained as a 2.2-kilobase EcoRI/HindIII insert in pBR322 and transforms tk− animal cells with an efficiency equal to that of the cloned herpes simplex virus-1 tk gene.
Abstract: We have used the bacterial plasmid pBR322 as a vehicle to isolate genes coding for selectable markers from higher eukaryotes. In this way, we have obtained the chicken thymidine kinase (tk) gene as a 2.2-kilobase EcoRI/HindIII insert in BR322. The cloned gene transforms tk- animal cells with an efficiency equal to that of the cloned herpes simplex virus-1 tk gene.
162 citations
TL;DR: The nucleotide sequences of eight origin-defective mutants of simian virus 40 have been determined and it is found that the 5' ends of viral mRNAs are located approximately the same distance from the A+T-rich region (A-T-T
Abstract: The nucleotide sequences of eight origin-defective mutants of simian virus 40 have been determined All of the mutants have suffered deletions, which range in size from 4 to 241 nucleotides Some of the mutants induce the synthesis of tumor (T) antigen, others do not Viral mRNA extracted from rat cells transformed by two of the T-antigen-positive mutants has been analyzed by the S1 nuclease technique of Berk and Sharp Irrespective of the size or the location of the deletions, the 5' ends of viral mRNAs are located approximately the same distance from the A+T-rich region (A-T-T-T-A-T) rather than at a specific site in the viral genome
161 citations
TL;DR: A new technique for the isolation and propagation of nonconditionally lethal mutants of SV40 that contain a defective origin of DNA replication is described, establishing that SV40 DNA replication and early transcription are functionally separate.
Abstract: We have described a new technique for the isolation and propagation of nonconditionally lethal mutants. We have used this method to generate mutants of SV40 that contain a defective origin of DNA replication. Using these mutants, we have established the following: (1) SV40 DNA replication and early transcription are functionally separate. (2) A functional viral origin of DNA replication is not necessary for the maintenance of transformation. (3) The lack of the origin of SV40 DNA replication does not affect the efficiency of transformation when nonpermissive cells are transfected by DNA using the calcium technique.
160 citations
TL;DR: These species presumably represent the subset of viral mRNAs that are "immediate early" products, requiring only host cell genes of their expression, and they do not include any of the conventionally recognized early m RNAs.
159 citations
TL;DR: A bacterial plasmid carrying the early region of SV40 (pOT) has been stably established in high molecular weight (hmw) DNA of mouse L cells by selection for the herpes virus thymidine kinase (tk) gene as mentioned in this paper.
TL;DR: These results argue strongly against the involvement of specific excision enzymes, and rather are discussed in terms of a model involving replication of the integrated viral DNA followed by recombination for release of integrated viral sequences.
TL;DR: Substantially different E1A and E1B proteins are encoded by RNA from cells infected with the adenovirus type 5 mutants, which are all defective for replication on human cells and, except for dl311, for transformation.
TL;DR: Analysis of the RNA produced in abortively infected monkey cells indicated that the depressed synthesis of many of the late proteins can be ascribed to the reduced concentration of their corresponding mRNAs, which suggest a role for RNA splicing in the control of gene expression.
TL;DR: Electron microscopy was used to investigate primary cilia in quiescent 3T3 cells, finding their basal centriole was found to be a focal point of numerous cytoplasmic microtubules which terminate at the basal feet.
TL;DR: The detection, by an immunocytochemical technique, of an enkephalin-like moiety which is localised in one of the 400 cells of each posterior midbody ganglion of the leech is reported.
Abstract: The small endogenous peptides, Met- and Leu-enkephalin, bind to the same specific receptors as opiate analgesics. They, and the larger endorphin peptides, have been widely found in mammals, where they seem to have a significant role in neuronal pathways mediating pain and emotional behaviour. Only recently has enkephalin-like activity been identified in an invertebrate, the earthworm1, although there is some preliminary evidence for opiate receptors in a marine mollusc2. Here I report the detection, by an immunocytochemical technique, of an enkephalin-like moiety which is localised in one of the 400 cells of each posterior midbody ganglion of the leech. The presence of enkephalin-like activity in an identifiable easily accessible neurone of a well characterised nervous system such as that of the leech3–5 could greatly facilitate elucidation of its mechanism of action.
TL;DR: It is demonstrated that the mature mRNAs originating in the major late transcription unit fall into five families, and the 5′-proximal RNAs within a family (such as those encoding polypeptides 52, 55 K, III, pVI, 100 K and 33 K) exhibit a broad distribution of molecular weights.
TL;DR: These isolates are distinguished by antigenic criteria, including hemagglutination and cross-reaction tests, as well as by independent properties including epidemiology, ability to induce tumors in experimental animals, and capacity to multiply in cultured cells.
Abstract: Adenoviruses have been isolated from species of animals as diverse as frogs, turkeys, and monkeys and from parts of the world as distant as Auckland and Anchorage. The adenovirus family has over 80 members, all with remarkably similar properties. In every case the infectious particle is about 70 nm in diameter and is composed of an inner DNA:protein core and an outer icosahedral capsid; it lacks a membranous envelope and is resistant to inactivation by both ether and trypsin. These structural features and other background information on adenoviruses have been reviewed in detail by Philipson et ~ 1 . ' ~ and by T o o ~ e . ~ ' The host range of any given adenovirus is restricted to one or, at most, a few closely-related species of animals. It is therefore possible to divide the family into groups of human. simian, bovine, canine, murine, porcine, tupian, avian, and frog strains. Furthermore many of the host-specific groups are represented by numerous serotypes. For instance, 36 human serotypes have been identified (TABLE 1) .9, 15. 4 7 . I S , 5 0 . 53, 9 2 . 102 These isolates are distinguished by antigenic criteria, including hemagglutination and cross-reaction tests, as well as by independent properties including epidemiology, ability to induce tumors in experimental animals, and capacity to multiply in cultured cells. During productive infection most adenoviruses, apart from those isolated from birds and cattle, induce the synthesis of a soluble, complement-fixing antigen, the major determinant of which is, in all probability, a portion of the major coat protein hexon. It cross-reacts with similar antigens from other members of the adenovirus family.\"I Notably, the gene for thc hexon protein from mouse adenovirus F L is related in DNA sequence to that from human adenovirus-2.58 All adenoviruses contain a chromosome of double-stranded DNA, about 36,500 base pairs in length, which has two unusual features. The 5'-end of each DNA strand both in the infected cell and in the mature virion is covalently attached to a pro tehF1 And the terminal DNA sequences of either strand are inverted duplications of one other for a hundred or more nucleotides.51 4 9 v
TL;DR: The observations suggest that the cytoplasmic matrix and the membranes of animal cells are so constructed as to express locally and autonomously any one of the elementary amoeboid movements listed above.
Abstract: Tiny fragments from the cytoplasm of human skin fibroblasts with about 2% of the original cell volume ("microplasts") were prepared by treatment with cytochalasin B, vigorous pipetting, and trypsinization of the attached fragments. They remained alive for 8 hr or longer. Some of the microplasts were able to produce and move filopodia, ruffle, or both; others blebbed continuously. Slow flattening was observed in the larger microplasts. In all cases tested, microplasts avoided contact with other cells or microplasts. The observations suggest that the cytoplasmic matrix and the membranes of animal cells are so constructed as to express locally and autonomously any one of the elementary amoeboid movements listed above. More importantly, whatever types of motile surface projections a microplast expressed, it continued to produce and move them in a stereotypical way as if there were long-lived structural or material determinants for each type. The microplasts were unable to locomote autonomously. Therefore, it is conceivable that directional movement of whole cells may require a supervising mechanism that confers a certain coordination and strategy on its component cytoplasmic bits. Otherwise they would continue to move in stereotypical and autonomous ways without ever displacing themselves, as suggested by the behavior of the microplasts.
TL;DR: Adenovirus 2 genes are introduced into high molecular weight DNA of permissive human cells by co-transformation of tk- human 143 cells with Ad2 restriction enzyme fragments and a cloned Bam HI fragment that carries the HSV-1 thymidine kinase gene.
TL;DR: Results presented here show that the VA RNAs bind to unfractionated late virus mRNA and to a cloned copy of a single mRNA species, but not to corresponding cloned segments of viral genomic DNA.
Abstract: Most, though not all, of the messenger RNAs of higher cells are composed of transcripts from two or more non-contiguous DNA segments that are 'spliced' together by mechanisms which are poorly understood. There has been recent speculation that small RNA molecules may play a part in the splicing reaction, acting as templates or adaptors to stabilize the appropriate conformation of a precursor RNA. Adenovirus-2 codes for two low molecular weight RNAs, the virus-associate (VA) RNAs I and II, major and minor species, respectively. These RNAs are about 160 nucleotides long and have both been sequenced. They originate from closely spaced genes which are transcribed by RNA polymerase III, but have not been definitively associated with any function. We have shown previously that a fraction of the VA RNA of infected cells is complexed with high molecular weight RNA in a denaturation-sensitive fashion. Results presented here show that the VA RNAs bind to unfractionated late virus mRNA and to a cloned copy of a single mRNA species, but not to corresponding cloned segments of viral genomic DNA. It is suggested that VA RNA may act as a template in the splicing reaction.
TL;DR: Primary hamster brain cells were transformed with four isolates of JC virus and JC virus DNA and several properties of these transformants were characterized and compared to those of simian virus 40 transformants isolated under identical conditions.
Abstract: We transformed primary hamster brain cells with four isolates of JC virus and JC virus DNA Several properties of these transformants were characterized and compared to those of simian virus 40 transformants isolated under identical conditions
TL;DR: The partial amino-terminal amino acid sequence has been determined for four adenovirus 2 proteins: hexon, fiber, component IX, and early protein E1B-15K, which has identified the initiation sites for protein synthesis.
TL;DR: The regions of the human adenoviral genome associated with the process of oncogenesis have been identified using several approaches and it is demonstrated that retention of the leftmost 14% of the genome is sufficient for the maintenance of the transformed growth properties of these cells.
Abstract: The regions of the human adenoviral genome associated with the process of oncogenesis have been identified using several approaches. Analysis of the viral DNAs contained in different lines of cells transformed by virus has demonstrated that retention of the leftmost 14% of the genome is sufficient for the maintenance of the transformed growth properties of these cells (Gallimore et al. 1974). The adenoviral mRNAs expressed in transformed cell lines are similar to those expressed from these DNA sequences during the early phase of the productive infection (Flint et al. 1975). The left end of the viral DNA contains at least two genes necessary for transformation, since two complementation groups of host-range mutants that map within this region (Frost and Williams 1978) are both defective for transformation (Graham et al. 1978). Transfection of cells with fragments of viral DNA has provided a direct means of determining the minimum amount of viral...
TL;DR: Advances in recombinant DNA technology have allowed the isolation of large numbers of biologically interesting fragments of DNA, so computer-assisted methods, with programs directed toward the manipulation of nucleic acid sequences, have become indispensable during the collection and analysis of that data.
Abstract: Advances in recombinant DNA technology have allowed the isolation of large numbers of biologically interesting fragments of DNA. Concomitant improvements in methods for nucleic acid sequencing have led many investigators to characterize their clones by sequencing them. This has resulted in the accumulation of such large amounts of sequence data that computer-assisted methods, with programs directed toward the manipulation of nucleic acid sequences, have become indispensable during the collection and analysis of that data.
TL;DR: Results favor the idea that the direction of switching is controlled by cell's mating phenotype rather than by the genetic content of MAT, and a molecular model based on a single-strand transfer is proposed.
TL;DR: The virus-cell interaction that leads cells that are nonpermissive for viral growth to become transformed and ultimately tumorigenic is complex, involving multiple viral stimuli and an array of host responses.
Abstract: The virus-cell interaction that leads cells that are nonpermissive for viral growth to become transformed and ultimately tumorigenic is complex, involving multiple viral stimuli and an array of host responses. The portion of the SV40 genome required for the induction of transformation (Graham et al. 1975) encodes two proteins, large T (m.w. 96,000) and small T (m.w. 17,000) (Prives et al. 1977). In response to these two proteins, cells acquire a variety of properties not demonstrated by their uninfected counterparts. These transformed cells proliferate at higher temperatures (Ossowski and Sachs 1967), frequently grow to much higher population densities, grow in medium supplemented with only low levels of serum factors, and exhibit anchorage-independent growth (Risser and Pollack 1974).
TL;DR: It is concluded that alpha-actinin is present in these membrane preparations, because it is bound to actin, and thatalpha- actinin does not form a direct link between actin and the membrane.
Abstract: The role of alpha-actinin in the attachment of actin to plasma membranes has been investigated. Specific antibody staining of SDS gels has indicated that alpha-actinin is a major component in isolated plasma membranes prepared from three different cell types by two different procedures. Using specific extraction conditions, most of the alpha-actinin can be selectively extracted from the membranes with relatively little parallel release of actin. This selective dissociation of alpha-actinin from the plasma membrane leads us to conclude that alpha-actinin is present in these membrane preparations, because it is bound to actin, and that alpha-actinin does not form a direct link between actin and the membrane.
TL;DR: It is reported here that H5 mRNA is polyadenylated, an H1-like tissue-specific histone occurring only in nucleated erythrocytes that is synthesised in the absence of DNA synthesis during maturation of the red blood cells.
Abstract: In most known systems1,2, histone mRNA lacks the poly(A) sequence at the 3′ end of the molecule typical of most mRNAs. Furthermore, the synthesis of histones, unlike that of most proteins, is tightly coupled to DNA synthesis3–5. Nevertheless, histone synthesis occurs in amphibian oocytes in the absence of DNA synthesis6. Moreover, it has recently been found that in amphibian oocytes most of the histone mRNA is polyadenylated7–9, and the polyadenylate is probably removed during maturation of the oocyte10. Histone H5, an H1-like tissue-specific histone occurring only in nucleated erythrocytes11, is also atypical in that it is synthesised in the absence of DNA synthesis during maturation of the red blood cells12,13. We report here that H5 mRNA is polyadenylated.