Article

Avian Tumor Virus RNA: A Comparison of Three Sarcoma Viruses and Their Transformation-Defective Derivatives by Oligonucleotide Fingerprinting and DNA-RNA Hybridization

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Abstract

Earlier electrophoretic analyses have shown that the 60-70S RNA of avian sarcoma viruses contains a characteristic subunit, termed class a subunit, which has a lower electrophoretic mobility than class b subunit found in transformation-defective derivatives of sarcoma viruses and in avian leukosis viruses. We have compared the RNAs of three nondefective avian sarcoma viruses, B77 and Prague and Schmidt-Ruppin strains of Rous sarcoma virus, with those of their transformation-defective (td) derivatives, td B77, td PR-C, and td SR-A, respectively, to determine the chemical basis for the difference between class a and b subunits. It was found by “fingerprinting” that (1) all (about 20-25) large T1 RNase-resistant oligonucleotides present in class b subunits of transformation-defective viruses have homologous counterparts in the class a subunits of corresponding nondefective sarcoma viruses and that (2) class a subunits contain a few (one or two) additional oligonucleotides that are not present in class b. By contrast the oligonucleotide fingerprints of avian tumor viruses of different strains and subgroups were very different. Cross hybridization of classes a and b RNA of sarcoma virus B77 with DNA transcribed from a corresponding transformation-defective virus td B77 showed that the two RNAs share at least 60% and differ by about 10% of their sequences. It is suggested that the structural relationship of class a and b subunits of corresponding viruses may be expressed as a = b + x, and that all the oligonucleotides present only in RNAs of sarcoma viruses but not in transformation-defective viruses of the corresponding strains are part of sequence(s) x. The possibility that x represents genetic information directly or indirectly involved in transformation of fibroblasts is discussed.

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... The discovery of dominant, retroviral oncogenes in the 1970s, beginning with the src gene of Rous sarcoma virus [Duesberg and Vogt, 1970;Martin, 1970;Lai et al., 1973], was also quickly adopted by the gene mutation hypothesis as a substitute for functional proof based on the following argument. The promoters of these oncogenes are shared with the virus, but their coding regions are derived from cellular genes by a conventional but rare process, termed transduction, which involves illegitimate recombination between viral and cellular DNAs [Duesberg, 1987;Goodrich and Duesberg, 1990;Schwartz et al., 1995]. ...
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The many complex phenotypes of cancer have all been attributed to “somatic mutation.” These phenotypes include anaplasia, autonomous growth, metastasis, abnormal cell morphology, DNA indices ranging from 0.5 to over 2, clonal origin but unstable and non‐clonal karyotypes and phenotypes, abnormal centrosome numbers, immortality in vitro and in transplantation, spontaneous progression of malignancy, as well as the exceedingly slow kinetics from carcinogen to carcinogenesis of many months to decades. However, it has yet to be determined whether this mutation is aneuploidy, an abnormal number of chromosomes, or gene mutation. A century ago, Boveri proposed cancer is caused by aneuploidy, because it correlates with cancer and because it generates “pathological” phenotypes in sea urchins. But half a century later, when cancers were found to be non‐clonal for aneuploidy, but clonal for somatic gene mutations, this hypothesis was abandoned. As a result aneuploidy is now generally viewed as a consequence, and mutated genes as a cause of cancer although, (1) many carcinogens do not mutate genes, (2) there is no functional proof that mutant genes cause cancer, and (3) mutation is fast but carcinogenesis is exceedingly slow. Intrigued by the enormous mutagenic potential of aneuploidy, we undertook biochemical and biological analyses of aneuploidy and gene mutation, which show that aneuploidy is probably the only mutation that can explain all aspects of carcinogenesis. On this basis we can now offer a coherent two‐stage mechanism of carcinogenesis. In stage one, carcinogens cause aneuploidy, either by fragmenting chromosomes or by damaging the spindle apparatus. In stage two, ever new and eventually tumorigenic karyotypes evolve autocatalytically because aneuploidy destabilizes the karyotype, ie. causes genetic instability. Thus, cancer cells derive their unique and complex phenotypes from random chromosome number mutation, a process that is similar to regrouping assembly lines of a car factory and is analogous to speciation. The slow kinetics of carcinogenesis reflects the low probability of generating by random chromosome reassortments a karyotype that surpasses the viability of a normal cell, similar again to natural speciation. There is correlative and functional proof of principle: (1) solid cancers are aneuploid; (2) genotoxic and non‐genotoxic carcinogens cause aneuploidy; (3) the biochemical phenotypes of cells are severely altered by aneuploidy affecting the dosage of thousands of genes, but are virtually un‐altered by mutations of known hypothetical oncogenes and tumor suppressor genes; (4) aneuploidy immortalizes cells; (5) non‐cancerous aneuploidy generates abnormal phenotypes in all species tested, e.g., Down syndrome; (6) the degrees of aneuploidies are proportional to the degrees of abnormalities in non‐cancerous and cancerous cells; (7) polyploidy also varies biological phenotypes; (8) variation of the numbers of chromosomes is the basis of speciation. Thus, aneuploidy falls within the definition of speciation, and cancer is a species of its own. The aneuploidy hypothesis offers new prospects of cancer prevention and therapy. Cell Motil. Cytoskeleton 47:81–107, 2000. © 2000 Wiley‐Liss, Inc.
... Thus the basis of the greater heterogeneity of small RNA molecules from heated older virus 60-70S RNA is the nicking of the RNA within the virion before its extraction. Fingerprint analysis has shown that most of the RNA sedimenting slower than 30-40S has the same composition as 30-40S RNA ( Lai et al. 1973;Duesberg et al. 1974 and this volume). Biologically consistent with this model for the structure of the 60-70S RNA is the observation that the infectivity of RNA tumor viruses has been shown to be proportional to the amount of 30-40S RNA obtainable from a virus preparation and not necessarily to the amount of 60-70S RNA ( Bader and Steck 1969). ...
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The authors report the use of the bacteriophage T4 gene 32 protein to mildly denature and extend the 60-70S RNA and the 30-40S RNA such that in conjunction with the protein monolayer spreading technique of Kleinschmidt, the structure of these RNA species can be visualized in the electron microscope. Under these conditions, the length of an RNA molecule is proportional to its molecular weight. By using double stranded DNA as an internal length standard, the determination of the molecular weight of RNA gene 32 protein complexes can be based on well characterized DNA standards rather than on a comparison with other RNA species.
... The non-defectiveness of the Prague strain of RSV was soon confirmed in avian cell culture [38]. RNA analysis also showed the Schmidt-Ruppin and Carr-Zilber strains of RSV and the B77 avian sarcoma virus to be non-defective [39,40]. ...
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Jan Svoboda triggered investigations on non-defective avian sarcoma viruses. These viruses were a critical factor in the genetic understanding of retroviruses. They provided the single and unique access to the field and facilitated the discovery of the first oncogene src and of the cellular origin of retroviral oncogenes. They continue to be of importance as singularly effective expression vectors that have provided insights into the molecular functions of numerous oncogenes. Combined with the contributions to the validation of the provirus hypothesis, Jan Svoboda’s investigations of non-defective avian sarcoma viruses have shaped a large and important part of retrovirology.
... Svoboda kindly sent me the Prague strain of Rous sarcoma virus, and I also obtained Schmidt-Ruppin, Carr-Zilber, and B77 viruses. I confirmed the nondefectiveness of these viruses: A single infectious virus particle could both transform cells and produce progeny virus (32)(33)(34). ...
Article
I always loved biology and to do experiments. This passion and a great deal of good fortune and serendipity landed me in the field of retrovirology at the time when it opened to experimental analysis. I became involved in viral replication, genetics, and viral oncogenes. In more recent years, I have applied what I learned in tumor virology to human cancer. The early years of my personal life were marked by displacements and migration: deportation into East Germany, escape to the West, and emigration to the United States. As a young man I faced heartbreaking personal tragedies but attained a peaceful and steady course in the second half of my life. I am fortunate to have found my home in Southern California and to continue in cancer research.
... RSV was understood to be an acutely transforming virus in chickens and was found to carry approximately 1.5kb of additional genomic sequence beyond that of its parent ALV genome. The oncogenicity of RSV was attributed to this extra sequence as mutation or loss of this region ablated its transforming capabilities (with no effect on virus replication) (98,111). The sequence was eventually identified as a mutant form of the cellular src gene, the first characterized protein-tyrosine kinase, which is involved in intracellular signaling in response to cell surface receptor stimulation (20,30). ...
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... Intensive investigations using Rous Sarcoma virus identified v-src as the gene that is required for cell transformation (Lai et al., 1973;Martin et al., 1971;Wang et al., 1975) and ...
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2009. Includes bibliographical references. Nearly 90% of cancer mortality from solid tumors is due to metastasis of malignant cells to the distant vital organs. It is now well established that a plethora of stromal cells are present within the tumor, and contribute in various ways to tumor initiation and progression, and plasma membrane proteins are the mediators for tumor-stromal communications. In this thesis, I focused on plasma membrane proteins that may contribute to tumor metastasis. I applied quantitative mass spectrometry technology to first identify plasma proteins that are expressed at different levels in melanoma cells with high versus low metastatic abilities. Using SILAC (stable isotope labeling with amino acids in culture) coupled with nano-spray tandem mass spectrometry, this work led to the discovery of C̲ub Ḏomain C̲ontaining Protein 1 (CDCP1) as one of those differentially expressed transmembrane proteins. We found that CDCP1 is not only a surface marker for cells with higher metastatic potential, it is also functionally engaged in enhancing tumor metastasis. When searching for the underlying mechanisms, we found that CDCP1 is important for soft agar colony-forming abilities, suggesting that CDCP1 might regulate the balance between cell proliferation and anoikis. Making use of 3D Matrigel culture system, we found that CDCP1 also regulates scattered growth of melanoma cells. We speculate these two factors may contribute to enhanced-metastatic ability observed in mice. (cont.) When investigating signaling pathways that may mediate the functions of CDCP1, we found that overexpression of CDCP1 correlates with hyper-activation of Src family kinases. While wild-type CDCP1 enhances SFK activation, point mutation that abolished CDCP1 functions (in scattered growth and in metastasis) also abolished SFK hyper-activation, suggesting that CDCP1 might function through the activation of SFKs. Such notion was further supported since pharmacological reagents PP2 and Dasatinib, which are two SFK inhibitors, blocked in vitro functions of CDCP1 in scattered growth. Thus the work in this thesis has identified a novel metastasis enhancer, CDCP1, and has gained insight into the mechanisms by which CDCP1 functions. by Hui Liu. Ph.D.
... The discovery of dominant, retroviral oncogenes in the 1970s, beginning with the src gene of Rous sarcoma virus [Duesberg and Vogt, 1970;Martin, 1970;Lai et al., 1973], was also quickly adopted by the gene mutation hypothesis as a substitute for functional proof based on the following argument. The promoters of these oncogenes are shared with the virus, but their coding regions are derived from cellular genes by a conventional but rare process, termed transduction, which involves illegitimate recombination between viral and cellular DNAs [Duesberg, 1987;Goodrich and Duesberg, 1990;Schwartz et al., 1995]. ...
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Full-text available
The many complex phenotypes of cancer have all been attributed to “somatic mutation.” These phenotypes include anaplasia, autonomous growth, metastasis, abnormal cell morphology, DNA indices ranging from 0.5 to over 2, clonal origin but unstable and non-clonal karyotypes and phenotypes, abnormal centrosome numbers, immortality in vitro and in transplantation, spontaneous progression of malignancy, as well as the exceedingly slow kinetics from carcinogen to carcinogenesis of many months to decades. However, it has yet to be determined whether this mutation is aneuploidy, an abnormal number of chromosomes, or gene mutation. A century ago, Boveri proposed cancer is caused by aneuploidy, because it correlates with cancer and because it generates “pathological” phenotypes in sea urchins. But half a century later, when cancers were found to be non-clonal for aneuploidy, but clonal for somatic gene mutations, this hypothesis was abandoned. As a result aneuploidy is now generally viewed as a consequence, and mutated genes as a cause of cancer although, (1) many carcinogens do not mutate genes, (2) there is no functional proof that mutant genes cause cancer, and (3) mutation is fast but carcinogenesis is exceedingly slow. Intrigued by the enormous mutagenic potential of aneuploidy, we undertook biochemical and biological analyses of aneuploidy and gene mutation, which show that aneuploidy is probably the only mutation that can explain all aspects of carcinogenesis. On this basis we can now offer a coherent two-stage mechanism of carcinogenesis. In stage one, carcinogens cause aneuploidy, either by fragmenting chromosomes or by damaging the spindle apparatus. In stage two, ever new and eventually tumorigenic karyotypes evolve autocatalytically because aneuploidy destabilizes the karyotype, ie. causes genetic instability. Thus, cancer cells derive their unique and complex phenotypes from random chromosome number mutation, a process that is similar to regrouping assembly lines of a car factory and is analogous to speciation. The slow kinetics of carcinogenesis reflects the low probability of generating by random chromosome reassortments a karyotype that surpasses the viability of a normal cell, similar again to natural speciation. There is correlative and functional proof of principle: (1) solid cancers are aneuploid; (2) genotoxic and non-genotoxic carcinogens cause aneuploidy; (3) the biochemical phenotypes of cells are severely altered by aneuploidy affecting the dosage of thousands of genes, but are virtually un-altered by mutations of known hypothetical oncogenes and tumor suppressor genes; (4) aneuploidy immortalizes cells; (5) non-cancerous aneuploidy generates abnormal phenotypes in all species tested, e.g., Down syndrome; (6) the degrees of aneuploidies are proportional to the degrees of abnormalities in non-cancerous and cancerous cells; (7) polyploidy also varies biological phenotypes; (8) variation of the numbers of chromosomes is the basis of speciation. Thus, aneuploidy falls within the definition of speciation, and cancer is a species of its own. The aneuploidy hypothesis offers new prospects of cancer prevention and therapy. Cell Motil. Cytoskeleton 47:81–107, 2000. © 2000 Wiley-Liss, Inc.
... The discovery of dominant, retroviral oncogenes in the 1970s, beginning with the src gene of Rous sarcoma virus [Duesberg and Vogt, 1970; Martin, 1970; Lai et al., 1973], was also quickly adopted by the gene mutation hypothesis as a substitute for functional proof based on the following argument. The promoters of these oncogenes are shared with the virus, but their coding regions are derived from cellular genes by a conventional but rare process, termed transduction, which involves illegitimate recombination between viral and cellular DNAs [Duesberg , 1987; Goodrich and Duesberg, 1990; Schwartz et al., 1995]. ...
... When 35 S RNA of RSV was digested with RNase Tl and subjected to two-dimensional electrophoresis and homochromatography, clusters of spots, representing large oligonucleotides that separated from the mass of smaller oligonucleotides, produced a specific pattern on the autoradiograms (Horst et al., 1972;Lai et al., 1973). The map of the spots was characteristic of the specific virus strain, but importantly some of the spots characteristic of nondefective sarcoma viruses were missing on the maps obtained with transformation-defective derivatives. ...
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Along with other carcinogens of physical or chemical origin, viruses are known to be associated with and to cause tumors in a variety of experimental animals. RNA tumor viruses in particular are widespread in many species of animals, and frequently cause sarcomas or leukemia. The isolation of fowl sarcoma-leukemia virus in the early 1910s by a number of investigators marked the first successful identification of such tumor viruses (Ellermann and Bang, 1909; Rous, 1911; Fujinami and Inamoto, 1914). The cell-virus systems used for the studies of basic aspects of the pathogenesis of avian tumor viruses illustrate the progress in methodology for studying animal viruses in general. Early work in animal hosts was gradually replaced by a system using the chorioallantoic membrane of eggs (Keogh, 1938), then by tissue culture cells (Manaker and Groupé, 1956). The establishment of an assay system for Rous sarcoma virus (RSV) in tissue culture cells (Temin and Rubin, 1958) eventually led to an era of quantitative studies on the mechanism of cellular alteration by viruses.
... With relatively high frequencies, these sarcoma viruses spontaneously convert to transformation defective (td) mutants which retain only replicating functions (5,6). The genomic RNA of the td mutants is about 15% smaller than the parental RSV RNA, corresponding to the loss of ~ 1,500 nucleotides (7)(8)(9)(10). The analysis of oligonucleotides obtained after hydrolysis of the RNAs of parental RSV and of td mutants showed that the deletions occur in a specific portion of the RSV genome, located near the 3' terminus (11,12). ...
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A map of the large T1 oligonucleotides of the RNA of Prague Rous sarcoma virus, strain B (Pr RSVb) has recently been established (Coffin and Billeter, submitted for publication). Since the RNA of Rous associated virus, type 1 (RAV-1) lacks many of the large 1 oligonucleotides of Pr RSV-B and contains others not present in the latter, the RNA of recombinants between RAV-1 and Pr RSV-B could be analyzed with regard to the origin of its sequences. Recombinants were selected for transforming capacity (characteristic for Pr RSV-B) and ability to grow on C/B chicken fibroblasts (characteristic for RAV-1). Four out of five recombinants examined had undergone at least two crossovers. The set of Pr RSV-B-specific oligonucleotides present in all recombinants defined an RNA region near the poly(A) segment; this must contain genetic information required for transformation required for transformation (the onc function). All recombinants lost a set of contiguous Pr RSV-B-specific oligonucleotides and concomitantly acquired a set of RAV-1-specific oligonucleotides. These define a region in the middle section of the oligonucleotide map, all or some of which must be required for determining growth capacity on C/B cells (the env function).
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We have prepared radioactive DNA (cDNAsarc) complementary to nucleotide sequences which represent at least a portion of the viral gene(s) required for neoplastic transformation of fibroblasts by an avian sarcoma virus. The genetic complexity of cDNAsarc (~1600 nucleotides) is sufficient to represent an entire cistron. The genomes of three independent isolates of avian sarcoma viruses share nucleotide sequences closely related to cDNAsarc, whereas the sequences are absent from transformation-defective mutants of avian sarcoma viruses, several avian leukosis viruses, a non-pathogenic endogenous virus of chickens (Rous-associated virus-O), sarcoma-leukosis viruses of mice and cats, and mouse mammary tumor virus. We conclude that the transforming gene(s) of all avian sarcoma viruses have closely related or common genetic lineages distinct from the transforming genes in sarcoma viruses of other species. Our results conform to previous reports that transformation-defective variants of avian sarcoma viruses are mutants with identical regions deleted from each subunit of a polyploid genome.
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RNA tumor viruses have been shown to contain two to three RNA subunits consisting of identical nucleotide sequences (Beemon et al., 1974; Billeter et al., 1974). It remained unclear whether these sequences were uniquely arranged or circularly permuted.The organization of the genome of Rous sarcoma virus (Prague strain, subgroup B) has now been investigated by locating 29 large oligonucleotides, arising by digestion of 36S viral RNA with RNAase T1, relative to the poly(A) segment of the RNA. A unique arrangement of the oligonucleotides was found, demonstrating that all 36S subunits are similar in their sequences. A physical map of the genome has been constructed and three oligonucleotides absent in a transformation-defective variant of the virus were located in a region near the 3′ end of the RNA.
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Genetic crosses were performed between a nonconditional transformation-defective mutant isolated from the Prague strain of Rous sarcoma virus and temperature-sensitive transformation-defective mutants isolated from the same strain. The results indicate that all of the temperature-sensitive mutants studied result from mutations within the segment of the viral genome which is deleted in the nonconditional mutant.
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INFECTION of fibroblasts by avian sarcoma virus (ASV) leads to neoplastic transformation of the host cell. Genetic analyses have implicated specific viral genes in the transforming process1-4, and recent results suggest that a single viral gene is responsible4. Normal chicken cells contain DNA homologous to part of the ASV genome5-8 moreover, embryonic fibroblasts from certain strains of chickens can produce low titres of infectious type C viruses either spontaneously9 or in response to various inducing agents10. None of the viruses obtained from normal chicken cells, however, can transform fibroblasts, and results with molecular hybridisation indicate that the nucleotide sequences responsible for transformation by ASV are not part of the genetic complement of the normal cell11. We demonstrate here that the DNA of normal chicken cells contains nucleotide sequences closely related to at least a portion of the transforming gene(s) of ASV; in addition, we have found that similar sequences are widely distributed among DNA of avian species and that they have diverged roughly according to phylogenetic distances among the species. Our data are relevant to current hypotheses of the origin of the genomes of RNA tumour viruses12 and the potential role of these genomes in oncogenesis13.
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Cells producing type C (avain sarcoma virus) or type B (mouse mammary tumor virus) RNA tumor viruses contain small amounts of RNA complementary to the viral genomes. The negative strands are complementary to at least 30 to 45% of the viral genomes and are found as RNA-RNA duplexes in the nucleus and cytoplasm of infected cells and in mature virions.
Article
The 50S-70S RNA of a Moloney sarcoma-leukemia virus [Mo-MSV(MLV)] complex produced by a particular mouse cell line was shown by gel electrophoresis to contain a major (97%) 30S sarcoma-specific subunit species and a minor (3%) 38S leukemia virus-specific subunit. On the basis of its sedimentation coefficient and known complexity, the 30S Mo-MSV RNA was estimated to be a unique RNA molecule of about 6000 nucleotides. Hybridization experiments using viral RNA and DNA complementary to viral RNA (cDNA) made by viral DNA polymerase indicated that the 30S Mo-MSV RNA shared 70% of its sequences with Mo-MLV, 30% with another MLV derived from Mo-MLV, and 30% with Kirsten sarcoma-xenotropic leukemia virus. The 30S Mo-MSV RNA sequences shared with these viruses were not additive. The Tm of a Mo-MSV RNA-MLV cDNA hybrid was 83 degrees C, indicating that large contiguous nucleotide sequences were shared between the two nucleic acids. Mo-MSV RNA and Mo-MLV RNA shared possibly seven of 20-30 RNAase T1-resistant oligonucleotides, while Mo-MSV RNA contained three, and Mo-MLV RNA contained at least five specific oligonucleotides. We conclude that the 30S Mo-MSV RNA molecule consists of approximately 70% (about 4200 nucleotides) Mo-MLV-specific sequences and of 30% (1800 nucleotides) Mo-MSV-specific sequences covalently linked. Our results favor the hypothesis that 30S Mo-MSV RNA was generated by recombination between Mo-MLV and other genetic elements. We discuss whether all or only the MSV-specific sequences of the 30S Mo-MSV RNA function as sarcoma genes. Mo-MLV cDNA was hybridized about 45% by unfractionated Mo-MSV (MLV) RNA at RNA/DNA ratios of up to 10, about 50% by electrophoretically purified 30S Mo-MSV RNA at RNA/DNA ratios up to 500, but close to 100% by unfractionated Mo-MSV(MLV) RNA at RNA/DNA ratios over 900. This indicated that unfractionated RNA of our Mo-MSV(MLV) contained a complete complement of Mo-MLV, albeit at a low ratio.
Article
The RNase-T1-resistant oligonucleotides of two Prague Rous sarcoma viruses with temperature-sensitive (ts) DNA polymerases (DNA nucleotidyltransferases), termed ts LA 337 and 335 of one leukosis virus, RAV-6, and 20 of their recombinant progeny have been mapped relative to the 3' poly (A) terminus of the viral RNA. The resulting oligonucleotide maps have been ocrrelated with markers of the four known viral genetic elements encoded in the RNA of 10,000 nucleotides. In accord with previous results recombinant RNAs contained (i) oligonucleotides characteristic of the src gene, coding for sarcoma formation, between the poly(A) end and 2000 nucleotides and (ii) olignucleotides characteristic of the env gene, coding for the envelope glycoprotein, between 2500 and 5000 nucleo tides from the poly(A) end. (iii) A cluster of four oligonucleotides that mapped between 6000 and 8000 nucleotides from the 3' poly(A) end of each RNA was shared by both parental viruses and all recombinants. Since all other map segments of our recombinants failed to segregate with the ts- or wild-type markers of the parental DNA polymerase gene (pol), it was concluded that the ts pol lesion maps in this RNA segment. (iv) The 5' segment of each recombinant RNA contained a cluster of four to five oligonucleotides whose parental origin correlated with an electrophoretic marker of one of the parental virion proteins, p27, a major product of the viral gag gene. The gene order 5'-gag-pol-env-src-poly(A) is consistent with our data.
Article
C-type RNA tumor viruses have been isolated from naturally occurring animal tumors and also from normal tissues. Infectious forms of these viruses carry an RNA-directed DNA polymerase. Virus-specific DNA can be detected by means of molecular hybridization techniques. Conclusive evidence about the synthesis and integration of full-length DNA copies of the viral genome has been obtained in transfection assays that show that chromosomal DNA of RSV-transformed cells is able to infect chicken cells. A possibility exists that virus-infected cells also contain, besides full-length viral DNA, partial transcripts that may escape the detection in transfection assays. Oncogenic C-type viruses, isolated for instance from cat, primate, and human tumors, lack genetic relatedness to their natural hosts and no endogenous counterparts of these viruses have so far been identified. Thus, a possibility is present that certain animal tumors are due to the products coded for by transforming genes of exogenous RNA tumor viruses.
Article
Nature is the international weekly journal of science: a magazine style journal that publishes full-length research papers in all disciplines of science, as well as News and Views, reviews, news, features, commentaries, web focuses and more, covering all branches of science and how science impacts upon all aspects of society and life.
Article
A new rifamycin derivative, rifazone-82 (R-82), an inhibitor of viral RNA-dependent DNA polymerase, is selectively toxic to transformed chicken cells in culture. R-82 has now been shown to possess antiviral activity as well. The relatively nontoxic properties of R-82 to nontransformed cells have permitted the execution of experiments examining the effect of a rifamycin derivative on virus reproduction. Addition of low concentrations of R-82 (15 mug/ml) to cultures soon after Rous sarcoma virus infection prevents the spread of infection thoroughout the culture. This inhibition is not dependent on concomitant cellular transformation as identical results were obtained with cells infected with a transformation-defective Rous sarcoma virus. Addition of R-82 to cultures in which all the cells are infected does not substantially affect the yield of physical particles as measured by RNA-dependent DNA polymerase activity and by (3H) uridine incorporation into viral RNA. However, the infectivity of the progeny virus, as measured by focus-forming ability, is decrreased 95 to 99% by R-82 treatment.
Article
RNA tumor viruses are useful tools for the study of oncogenesis because they rapidly induce tumors in animals and efficiently transform cells in culture. These viruses are distinguished by certain morphological features (Bernhard, 1960; Sarker et al., 1971a), an exceptionally large single-stranded RNA genome (about 30,000 nucleotides, Duesberg, 1970) and an RNA-directed DNA polymerase which transcribes the viral genome into single- and double-stranded DNA (Baltimore, 1970; Temin and Mizutani, 1970; Temin and Baltimore, 1972). This transcription, the mechanism by which it occurs, the fate of its products in the infected cell, and the role of the products in both the viral life cycle and virus-induced transformation of the host cell are the principal subjects of our discussion.
Chapter
The biological importance of virion polymerases to the infection process of viruses can be gauged from the fact that many groups of viruses possess virion nucleic acid polymerases of one form or another. A list of those animal RNA virus groups (and their members) shown to possess RNA-directed RNA polymerases (RNA transcriptases) is given in Table 1. Included are representatives of the arena-viruses, bunyaviruses (and bunyaviruslike viruses), orthomyxoviruses, paramyxoviruses, reoviruses (diplornaviruses), and rhabdoviruses. Oncornaviruses and similar virus types (e.g., visna) possess an RNA-and DNA-directed DNA polymerase, otherwise known as a reverse transcriptase (Table 2). Of the various DNA virus groups, the poxviruses and possibly the icosahedral cytoplasmic deoxyriboviruses possess a virion DNA-instructed RNA polymerase (Table 3). Virus isolated from patients with serum hepatitis appears to possess a DNA-directed DNA polymerase.
Chapter
The relationship between two types of retroviral onc genes and cellular structural homologs termed proto-onc genes was studied. The type I Rous sarcoma virus (RSV) src gene, which is unrelated to essential virion genes, was found to have a complete structural homolog in cloned chicken DNA based on fingerprinting RNA-DNA hybrids. By the same techniques only the specific part (mcv) of the type II MC29 virus onc gene, which is a hybrid that also includes part (Δ) of the structural gag gene of retroviruses (Δ gag-mcv), was found to have a structural homolog in the cell. Hence, the onc gene of MC29 does not have a complete homolog in the cell. Both onc-related cellular loci are not linked to any other virion sequences. Presumed host markers of certain viral src genes, said to be experimentally transduced from the cell, were not detected in the proto src-locus. The cellular mcv-locus was found to be interrupted by one sequence of non-homology relative to the viral counterpart; the src-locus is known to be interrupted by six. We deduce that there is a close qualitative sequence-homology between the virion gene-unrelated sequences of viral onc genes and cellular proto-onc genes. However, functional homology between viral one genes and proto-onc loci cannot be deduced due to the different arrangements of onc-related sequences in viruses and cells and to scattered single nucleotide differences in their primary structures and due to the lack of Δ gag in cellular prototypes of hybrid onc genes, such as Δ gag mcv. Considering the genetic structures of RSV and MC29 and those of the corresponding cellular DNA loci, it follows that the generation of viruses like RSV and MC29 by transduction of cellular sequences into the genome of a retrovirus must have involved rare, illegitimate recombinations and specific deletions.
Chapter
Type C RNA viruses (RNA tumor viruses, oncornaviruses, leukemia-sarcoma viruses, retraviruses) have been isolated from many species. In several species the viruses have been associated with leukemia, and sometimes the disease has been reproduced when the virus was inoculated into animals. In a few species the evidence seems to be conclusive that these viruses are a contributing factor in the etiology of the naturally occurring leukemia. This appears to be the case in chickens, cats, gibbons, cows, and some wild-type mice. There are three major problems in proving that type C viruses cause leukemia in some animal systems: (a) a long latent period for effect; (b) the fact that susceptibility appears to vary enormously among different members of a species; and (c) many type C viruses apparently are not leukemogenic.
Chapter
Endogenous retroviral genes are present in all rat species including the feral rats. This virus, termed RaLV, is produced spontaneously in cultures or after treatment of cells in vitro by various chemical or physical agents. The RaLV replicates to low levels in rat cells but does not cause tumors in experimentally inoculated rats. We have isolated an acutely transforming rat sarcoma virus (RaSV) by in vitro cocultivation of RaLV productive cells with that of chemically induced tumor cultures. Unlike the previously isolated murine sarcoma viruses that are hybrids of mouse virus and cellular ras gene, the RaSV contains exclusively rat derived sequences (i.e. rat virus and ras gene). The RaSV transformed nonproducer cells produce a protein of 29,000 daltons which is distinct from all other ras oncogene encoded products in that it is synthesized as a gag-ras fusion product. Thus, the rat-cell-rat-endogenous-virus system that we have established offers a unique model to study the interaction between the endogenous virogenes and cellular genes at various stages of tumor development.
Chapter
The earliest description of cancer of the breast and probably of cancer in any form dates back 3,000 years to the Egyptians. A recent report on cancer gives indirect evidence that the etiological agent in mammary carcinoma is a RNA tumor virus.
Article
Interactions between the viral and host genomes have been studied in numerous systems and in regard to different aspects. In the course of this interaction, viral genetic material often is inserted by covalent linkage into the host chromosome. One consequence of this insertion is the fixation of the viral genome or specific parts of it in the host cell. The effects of viral integration on host genetic functions are less clear and still require intensive investigations.
Chapter
In the early twentieth century, studies of the transmission of a tumor found in the breast of a Plymouth Rock hen formed the foundation for the discovery of Rous sarcoma virus (RSV). At the time, cancer was not believed to have a genetic basis and the idea that a virus could induce tumors was not accepted; however, those ideas would change. As RSV was studied further, it became clear that although it was an RNA virus, it had the property of transforming infected cells by altering their genetic composition. This key observation became the genesis of the “provirus hypothesis,” which led to the discovery of reverse transcription. Finding that RNA was converted to DNA through the activity of the viral reverse transcriptase enzyme was remarkable because the flow of genetic information had previously been thought to occur in only one direction, DNA to RNA. Further investigation of the nature of the integrated provirus and its transforming activity led to a second remarkable breakthrough – the identification of a cellular gene that was highly homologous to the viral transforming gene. The discovery of the src oncogene uncovered cellular pathways that control cell growth and differentiation and play a central role in malignant transformation.
Article
In 1954, the late Bjorn Sigurdsson drew attention to an unusual class of slow infection distinguished by a prolonged incubation period and a protracted symptomatic phase. Sigurdsson’s concept of slow infection resulted from his observations on the natural history of a group of diseases of Icelandic sheep. In this review, the focus is on one of these diseases, visna, as a prototype of slow infections caused by viruses, and as a paradigm of the challenging questions raised by slow infections: the persistence of virus in the face of the host’s defensive response, the strikingly slow pace of replication of virus, and the basis of the tissue lesions. Because visna virus is closely related to RNA tumor viruses, these central issues are discussed against the background of the structure and replication of RNA tumor viruses. In particular, the hypothesis is advanced here that the persistence of visna virus is the consequence of its ability, held in common with RNA tumor viruses, to transfer its genetic information to a DNA replica, or provirus, that resides in the cell and allows the virus to elude the host defensive response.
Chapter
This chapter provides description of the properties and classification of the oncornaviruses and related virus particles that possess RNA→DNA polymerase molecules. The chapter describes the properties of the DNA polymerase activities of oncornaviruses and related particles including the endogenous reaction, the reaction products, and the utilization of external templates. The chapter also deals with the studies on the inhibitors of the viral DNA polymerase, their mechanism of action and their effect on cell transformation and tumor induction. The properties of purified RNA→DNA polymerase and the mechanism of DNA synthesis are also explored. The chapter provides evidence for the in vivo function of the viral RNA→DNA polymerase and a concise description of the present understanding of the molecular events of oncornavirus replication and cell transformation. The chapter also discusses the analysis of viral related base sequences in normal and cancer cells by molecular hybridization with the viral DNA product of the RNA→DNA polymerase, and studies on RNA→DNA polymerase in normal and cancer cells.
Chapter
The genetic analysis of RNA tumor viruses has two main objectives: (1) to provide an understanding of virus replication and (2) to explain virus-induced transformation of the host cell. Virus replication results from a complex interaction of viral and cellular genomes. Viral genetics, however, considers only the virus side of this interaction; the numerous and specific cellular functions which are required for the synthesis of infectious virus will have to be defined by a genetic analysis of the host cell. In virus-induced transformation, viral genetic information presumably interferes with the genetic regulatory apparatus of the host cell, and here again it is important to realize that focusing on the viral information will reveal only part of a very complex interaction between two organisms. Despite these obvious limitations, the viral genome is at the moment that partner in this interaction which is more amenable to experimental study and offers realistic opportunities for an increase of our insight into virus replication and cellular transformation.
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Article
We have prepared DNA complementary to a sequence of 300–400 nucleotides adjacent to the poly(A) at the 3′-terminus of the genome of avian sarcoma virus (ASV) DNA was transcribed from denatured ASV RNA following initiation on the primer (dT)12–18. We demonstrated that homopolymer synthesis was not a significant feature of our enzymatic reaction, and we proved that most if not all the initiations on (dT)12–18 occurred at or near the 3'-terminus of the viral RNA. The presence of oligo(dT) on cDNA3, accelerates hybridization between cDNA3, and polyadenylated viral RNA; this artifact can be eliminated by including a competing homopolymer [poly(dT) or poly(A)l in the reaction mixtures. Nucleotide sequences complementary to cDNA3, occur only once or twice in the genome of avian sarcoma viruses, are present in the genomes of virus strains from subgroups A, B, C, D, E, and F of avian leukosis-sarcoma viruses, and are conserved even when large deletions affect an adjacent viral gene (src) By contrast, we find little or no complementarity between cDNA3, and the genomes of golden pheasant virus (subgroup G of avian leukosis-sarcoma viruses), mouse mammary tumor virus, and the Moloney strains of murine sarcoma virus and murine leukemia virus. cDNA3, can be used to identify the 3′-terminus of the avian sarcoma virus genome by molecular hybridization and will therefore be a useful reagent for the analysis of viral replication.
Article
We have investigated the RNAs of two avian sarcoma viruses recovered (rASV) from tumors induced in chickens by a deletion mutant of Schmidt-Ruppin Rous sarcoma virus (SR-RSV) that had lost part, but not all, of its sarcoma gene (src). The RNAs of the rASVs had the same size as SR-RSV RNA and were larger than the predominant RNA species of the partial src deletion mutant, if measured by electrophoresis in polyacrylamide gels. Fingerprinting of RNase T,-resistant oligonucleotides indicated that the rASVs shared one src gene oligonucleotide with SR-D which was also present in the partial src deletion mutant of SR-RSV. The two rASVs shared one other, probable src oligonucleotide, that was not found in SR-RSV, and SR-RSV contained a src oligonucleotide not found in the rASVs. However, the distinctive src oligonucleotide of the rASVs was structurally closely related to that of SR-RSV. We conclude that the src genes of the rASVs and that of SR-RSV are closely related. Possible mechanisms by which a partial src deletion may recover a complete src gene are discussed in view of our results.
Article
In avian sarcoma viruses a function required to transform cells is frequently lost giving rise to non-transforming virus. The genetic information for this function is localized near the 3′ end of the genome. Short 3′-terminal poly(A)-linked RNA fragments from the genomes of a transforming Rous sarcoma virus and its non-transforming derivative were isolated and gave rise to identical fingerprint patterns, suggesting that internal deletion rather than terminal elimination leads to non-transforming virus.
Article
Nature is the international weekly journal of science: a magazine style journal that publishes full-length research papers in all disciplines of science, as well as News and Views, reviews, news, features, commentaries, web focuses and more, covering all branches of science and how science impacts upon all aspects of society and life.
Article
This chapter focuses on the molecular studies and attempts particularly to interpret and correlate recent findings that bring us to a working model for their origin, evolution, function, and especially their relationship to neoplasia. RNA tumor viruses have been identified in many species, including birds, snakes, mice, rats, hamsters, pigs, cats, dogs, monkeys and possibly man. Many of these will induce leukemias, lymphomas, and sarcomas in their natural hosts. None are regularly derived from or cause carcinomas with the exception of the “type-B virus, the mouse mammary tumor virus (MMTV). Some of the viruses of the leukemialymphoma-sarcoma complex (type-C) have been isolated from spontaneous cancers. This is notable in birds, mice, cats, and monkeys. There are 4 types of primate RNA tumor viruses of special interest to us because of their relatedness to man and because they recently have been useful in showing the presence of RNA tumor virus information in human leukemic cells. The chapter also provides an overview on a new concept to virogenesis as described by the established oncogene-virogene and provirus-protovirus theories. Several observations suggest that RNA processing is a key event in the origination and replication of RNA tumor viruses. The RNA genomes of RNA tumor viruses resemble unprocessed nuclear RNA of normal cells. This raises the possibility, supported by several lines of evidence that an alteration in the normal processing of nuclear RNA in differentiated cells can result in the cytoplasmic appearance of RNA with the physical properties of type-C virus RNA. The coding potential of the unprocessed cytoplasmic RNA determines whether virus particles will form and determines the physiological effect of the RNA on cells.
Article
Normal cells in culture have membrane receptors for epidermal growth factor (EGF); EGF stimulates cells to divide by binding to these receptors. Cells transformed by murine and feline sarcoma viruses rapidly lose the ability to bind EGF, whereas cells transformed by the DNA tumour viruses, polyoma and SV40, or infected with non-transforming RNA tumour viruses have normal levels of functional EGF receptors. The results suggest that a product of the sarcoma virus genome specifically changes cell EGF receptors; the sarcoma gene product may, then, be functionally related to EGF.
Article
Full-text available
Heating the 60 to 70S ribonucleic acid (RNA) of Rous sarcoma virus (RSV) destroys both its subunit structure and its high template activity for RSV deoxyribonucleic acid (DNA) polymerase. In comparative analyses, it was found that the template activity of the RNA has a thermal transition of 70 C, whereas the 60 to 70S structure dissociates into 30 to 40S and several distinct small subunits with a T(m) of 55 C. Analysis by velocity sedimentation and isopycnic centrifugation of the primary DNA product obtained by incubation of 60 to 70S RSV RNA with RSV DNA polymerase indicated that most, but perhaps not all, DNA was linked to small (<10S) RSV RNA primer. Sixty percent of the high template activity of 60 to 70S RSV RNA lost after heat dissociation could be recovered by incubation of the total RNA under annealing conditions. The template activity of purified 30 to 40S subunits isolated from 60 to 70S RSV RNA was not enhanced significantly by annealing. However, in the presence of small (<10S) subunits also isolated from 60 to 70S RNA, the template activity of 30 to 40S RNA subunits was increased to the same level as that of reannealed total 60 to 70S RNA. It was concluded that neither the 30 to 40S subunits nor most of the 4S subunits of 60 to 70S RSV RNA contribute much as primers to the template activity of 60 to 70S RSV RNA. The predominant primer molecule appears to be a minor component of the <10S subunit fraction of 60 to 70S RSV RNA. Its electrophoretic mobility is similar to, and its dissociation temperature from 60 to 70S RSV RNA is higher than that of the bulk of 60 to 70S RSV RNA-associated 4S RNA. The role of primers in DNA synthesis by RSV DNA polymerase is discussed.
Article
Avian tumor viruses were induced in normal chicken cells treated with ionizing radiations or chemical carcinogens and mutagens. The induced leukosis viruses possess a buoyant density, DNA polymerase, polypeptides, and 70S RNA typical of avian tumor viruses. Induced leukosis viruses act as helper agents for the defective Bryan high titer strain of Rous sarcoma virus and with one exception belong to subgroup E as judged by host-range, interference, and neutralization patterns. Induction of leukosis viruses was successful in chicken cells lacking the “natural” group specific (gs) antigen of the avian tumor viruses as well as in cells carrying this antigen. This observation indicates that the viral genome is present in gs+ and in gs− cells. Therefore, the chromosomal locus which controls the presence of natural gs antigen in chicken cells does not represent the viral genome itself but regulates its expression in normal cells. These findings have implications for the origin of RNA tumor viruses and for theories of carcinogenesis.
Article
The action of γ-rays on the virus-producing and oncogenic capacities of clonal SR-RSV were studied in order to specify the relationships between these two functions. Two kinds of mutants were obtained from viral suspensions heavily irradiated: (1) virions still able to convert cells, but unable to give rise to progeny (sterile oncogenic virions), identified by their ability to produce nonproducing transformed cells; these cells, not activable by superinfection with RAV, were cancerous since they induced graft tumors on the egg CAM. (2) virions unable to transform chick embryo cells i.e., having lost the oncogenic capacity, but able to replicate at high titer and to induce a high level of gs antigen. Furthermore, this mutant, called the NT (γ) virus, confers to the cells a high resistance against challenge RSV belonging to the subgroups B and D of avian oncogenic viruses, and a slight resistance against challenge RSV belonging to the subgroup A. The meaning of these facts is discussed.Present results support the previous hypothesis of the independence of oncogenic and virus-producing capacities.
Article
Avian sarcoma virus strain Bratislava 77 (B77) was irradiated with ultraviolet light, and virus survivors were studied for their ability to transform cells and to reproduce. Two classes of radiation-damaged particles were found. One can still transform but fails to reproduce; the other reproduces but cannot transform. The first class of particles also fails to induce the synthesis of noninfectious virus. It cannot be rescued by superinfection of the transformed cells with an avian leukosis virus, and it is unable to maintain the transformed state during prolonged culture. The second class of particles is antigenically indistinguishable from B77, interferes with avian sarcoma viruses of subgroup C and produces pseudotypes with RSV(0).
Article
The 60-70S RNAs of several transforming and nontransforming avian tumor viruses have different electrophoretic mobilities. The RNA of transforming viruses contains two electrophoretically separable subunit classes: a and b. The relative concentrations of these subunits vary with the virus strain. Avian leukosis viruses and nontransforming derivatives of a sarcoma virus lack subunits of class a. It is suggested that the presence of the class a subunit is related to the transforming ability for fibroblasts of the virus.
Article
Ts 75 and ts 149 are two temperature-sensitive mutants of avian sarcoma virus B77 which fail to reproduce and to induce neoplastic transformation at 41°. Both mutants are indistinguishable from wild type in somatic properties of the virion: host range, type-specific antigenicity, and rate of inactivation at 41°. The temperature-sensitive step of ts 75 occurs late in the infectious cycle allowing the synthesis of group-specific antigen in increased amounts and of viral RNA under nonpermissive conditions. Ts 149 has an early temperature sensitive phase and does not produce group-specific antigen at 41°. The maintenance of some neoplastic properties in cells transformed by either ts 75 or ts 149 is continuously dependent on a temperature sensitive viral function: Shift of transformed cells to 41° results in disappearance of neoplastic traits. Double infection of cells at 41° with wild-type avian sarcoma virus and ts 75 or ts 149 results in the rescue of 3 markers derived from the mutant virus: temperature sensitivity, host range, and morphology of the transformed cell. Rescue of ts 75 or ts 149 with wild-type avian leukosis viruses at 41° has not been accomplished.
Article
Oncornavirus 60 to 70S ribonucleic acids (RNA), such as those from avian myeloblastosis virus, Schmidt-Ruppin virus, or mouse sarcoma-mouse leukemia viruses, isolated by conventional techniques, contain 4S transferlike RNA molecules that are released upon dissociation of the 60 to 70S RNA with heat. The 4S RNA represents 2.5 to 3.0% of the RNA in the 65S aggregate or 4 to 5 molecules per molecule of 35S RNA formed.
Article
Six genetically distinct lines of helper-independent avian sarcoma virus were cloned in chicken fibroblasts free of avian tumor virus group-specific (gs) antigen. The clones were then tested for the presence of nontransforming (NT) virus with a technique capable of detecting such agents in the presence of excess sarcoma virus. NT viruses were found in 6 out of 7 clones of avian sarcoma virus in concentrations ranging from 4 to 17% of the sarcoma virus. The 27 isolates of NT virus had the envelope characteristics of the sarcoma virus with which they were associated. These observations indicate that the occurrence of NT viruses in stocks of helper-independent avian sarcoma viruses is not the result of passage in gs positive chicken cells. Rather the NT viruses appear to be spontaneous segregants of sarcoma viruses.
Article
The two-dimensional fractionation patterns obtained by combined electrophoresis and homochromatography3 separate and reveal the large oligonucleotide fragments in enzymatic digests of 32P-labelled RNA with particular clarity. The patterns are characteristically different for each type of digest and for each RNA, so that large nucleic acids can readily be characterized and1 their interrelationships demonstrated.
Article
Two electrophoretically separable classes of subunits, a and b, are found after heat dissociation of the 60-70.S RNA obtained from several avian sarcoma viruses. Avian leukosis viruses yield only class b RNA subunits. The difference between sarcoma and leukosis viruses is not affected by the transformed or normal state of the host cell. Sarcoma viruses retain class a subunits even if grown in cells of normal phenotype. This fact is illustrated by T5, a temperature sensitive mutant of Schmidt Ruppin RSV (SR RSV A). T5 replicates at the non permissive temperature yet fails to transform the cells under these conditions. Class a subunits are found in the progeny particles. However, leukosis viruses released from sarcoma cells do not acquire class a subunits. Such a situation is seen in cultures transformed by the Bryan high titer strain of RSV (BH RSV), which release an excess of non focus forming Rous associated virus. Only small amounts of class a subunits are detectable in such viral harvests, despite the fact that the virus is produced by sarcoma cells. A similar preponderance of non focus forming virus may also explain the lack of class a subunits in avian leukosis virus strain MC29 which is capable of forming neoplastic foci in fibroblast cultures. A connection between the ability to form foci in fibroblast cultures and the presence of class a RNA subunits was further suggested by the observation that cloned stocks of SR RSV A segregate non focus forming virus which together with its transforming potential has lost class a subunits. The results are in agreement with the hypothesis that class a and b subunits of avian tumor virus RNA represent functionally specialized segments of the viral genome which may replicate independently.
Article
A two-dimensional fractionation procedure has been developed for separating radioactively-labelled oligonucleotides of up to 50 residues long, using uniformly 32P-labelled 5S RNA of Escherichia coli as a model compound. The method uses ionophoresis on cellulose acetate at pH 3.5 in the first dimension; and ascending chromatography with a concentrated mixture of oligonucleotides on thin layers of mixed DEAE-cellulose and cellulose in the second dimension.
Article
The isolation and sequence analysis of a series of oligonucleotides from complete ribonuclease T1 digests of bacteriophage R17 RNA have been reported (Jeppesen, 1971). We have obtained the homologous sets of large T1-resistant oligonucleotides from the related phages f2 and MS2. As expected from earlier findings (Nichols & Robertson, 1971), each RNA can be uniquely characterized by its ribonuclease T1 fingerprint.Comparison of nucleotide sequences from the sets of RNA fragments from R17, f2 and MS2 allows an estimate of the over-all degree of variation among the three RNA's (2.8 to 3.4%), and yields additional evidence that non-coding regions show significantly lower variation. R17, f2 and MS2 are apparently not linear descendants, but could have diverged from a common ancestor. Base substitutions involving U and C transitions occur at a higher frequency than would be expected on a random basis.It appears probable that a total of 15 to 20 base replacements in the phage coat protein cistron may accompany the single amino-acid difference between f2 coat protein and that of either R17 or MS2. We conclude that analysis of this sort should prove useful in determining the extent of variation among members of any set of closely related nucleic acid molecules.
Article
Three molecular-weight fractions of influenza virus RNA (SS1 SS2 and SS3) were prepared as described previously (Content & Duesberg, 1971) and subjected to digestion with RNase T1. Two-dimensional analyses of the digests by electrophoresis and homochromatography led to distinctive oligonucleotide patterns for each fraction of viral RNA. Almost all physically distinguishable large oligo-nucleotides of a given RNA species had a distinct base composition. It was concluded that each of the three fractions of viral RNA investigated contained different nucleotide sequences and presumably different genetic information.In the case of the smaller RNA fractions (SS2 and SS3) the presence of lesser amounts of oligonucleotides characteristic of the larger RNA fractions (SS1 and SS2, respectively) was observed. This was regarded as being due to contamination of the smaller RNA fractions by fragments of the larger RNA fractions. In contrast, the largest RNA species, SS1 contained very low concentrations of oligonucleotides characteristic of the intermediate RNA SS2.At least one large oligonucleotide occurred in most, perhaps all, species of influenza virus RNA. It was speculated that this oligonucleotide may be part of a common signal of all viral RNA's for the viral polymerase or for virus assembly.
Article
The rate of formation of complexes between T2 RNA and T2 DNA has been measured at various salt concentrations (0·2 to 1·5 M-KCl) and temperatures (50 to 85°C). With increasing temperature, the rate passes through a maximum which is higher the higher the salt concentration. In 0·5 M-KCI the optimum temperature is 67°C.The rate of reaction is proportional to both the RNA and DNA concentration. The apparent bimolecular constant for five different preparations of T2 RNA was 2 ml./μg DNA/minute, or 10 l./mole nucleotides/second (in 0·5 M-KCI, 67°C, pH 7·3). This rate is several orders of magnitude slower than that for the reaction between polyadenylic acid and polyuridylic acid. Differences and similarities between the two reactions are discussed.Annealing in the absence of RNA causes T2 DNA to lose its RNA-binding ability. The second-order rate constant for this process is approximately the same as that for the RNA-DNA reaction. Under the conditions used for their formation, RNA-DNA complexes are slowly destroyed. The fraction of complex breaking down in unit time increases as the concentration of RNA-DNA complex increases.RNA-DNA complexes were broken down completely by RNase at low salt concentrations. The resistance to RNase increased with increasing salt concentration but was not complete under any condition tested. The complex was destroyed completely by DNase.The extent of complex formation was measured using an excess of either reactant. At least 77% of RNA formed after T2 infection of Escherichia coli is capable of becoming bound to T2 DNA. The binding capacity of T2 DNA is approximately 0·3 μg RNA/μg DNA.
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