Identification of unintegrated forms of Kirsten murine sarcoma viral DNA and restriction endonuclease cleavage map of linear DNA.

We detected unintegrated linear 7.0-kilobase pair DNA and covalently closed circular DNA species in NIH3T3 cells recently infected with Kirsten murine sarcoma virus. Using the linear DNA, we constructed a restriction endonuclease cleavage map and compared it with the map of Harvey murine sarcoma virus. The restriction endonuclease maps of two segments, one 1.2 kilobase pairs (SmaI site) to 3.7 kilobase pairs (HindIII site) from the right end (corresponding to the viral 3' side) and the other 0.5 kilobase pair (SmaI and KpnI sites) to 0.9 kilobase pair (KpnI site) from the left end, were identical in the two virus types.     

Studies on the Nucleic Acid Sequences of Kirsten Sarcoma Virus: a Model for Formation of a Mammalian RNA-Containing Sarcoma Virus

The genetic information contained in the Kirsten and Moloney strains of mammalian RNA-containing sarcoma viruses has been analyzed by RNA . (3)H-DNA hybridization. Kirsten sarcoma virus has been found to possess two distinct sets of nucleic acid sequences. One set of sequences is contained in murine type C helper virus, and the other set is contained in rat type C helper virus. Moloney sarcoma virus contains sequences of murine type C helper virus but not of rat type C helper virus. The results indicate that Kirsten sarcoma virus arose through a process of recombination between Kirsten murine leukemia virus and nucleic acid sequences found in rat cells. A model is suggested for the formation of transforming type C viruses involving the transduction of oncogenic information.     

Selection of Revertants of Kirsten Sarcoma Virus Transformed Nonproducer BALB/3T3 Cells

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.     

Transfection of Fibroblasts by Cloned Abelson Murine Leukemia Virus DNA and Recovery of Transmissible Virus by Recombination with Helper Virus

A cloned, permuted DNA copy of the Abelson murine leukemia virus (A-MuLV) genome was capable of eliciting the morphological transformation of NIH/3T3 fibroblasts when applied to cells in a calcium phosphate precipitate. The efficiency of the process was extremely low, yielding approximately one transformant per microgram of DNA under conditions which give 10(4) transfectants per microgram of other DNAs (e.g., Moloney sarcoma virus proviral DNA). The DNA was able to induce foci, even though the 3' end of the genome was not present. The transforming gene was thus localized to the 5' portion of the genome. The transformed cells all produced viral RNA and the virus-specific P90 protein. Transmissible virus could be rescued from these cells at very low frequencies by superinfection with helper virus; the rescued A-MuLV virus had variable 3' ends apparently derived by recombination with the helper. Dimerization of the permuted A-MuLV cloned genome to reconstruct a complete provirus did not improve transformation efficiency. Virus could be rescued from these transformants, however, at a high efficiency. Cotransfection of the permuted A-MuLV DNA with proviral M-MuLV DNA yielded a significant increase in the efficiency of transformation and cotransfection of dimeric A-MuLV and proviral M-MuLV resulted in a high-efficiency transformation yielding several thousand more transformants per microgram than A-MuLV DNA alone. We propose that helper virus efficiently rescues A-MuLV from transiently transfected cells which would not otherwise have grown into foci. We hypothesize that multiple copies of A-MuLV DNA introduced into cells by transfection are toxic to cells. In support of this hypothesis, we have shown that A-MuLV DNA sequences can inhibit the stable transformation of cells by other selectable DNAs.     

Isolation of Temperature-Sensitive Mutants of Murine Sarcoma Virus

Three separate murine sarcoma virus nonproducer cell lines have been isolated which are temperature sensitive for the maintenance of transformation. In each case, a viral rather than a cellular genetic mutation is the reason for the temperature-sensitive effect. Superinfection of one of the mutants with murine leukemia virus overcomes the temperature-sensitive change in the transformed state.     

Relationship Between Moloney Murine Leukemia and Sarcoma Virus RNAs: Purification and Hybridization Map of Complementary DNAs from Defined Regions of Moloney Murine Sarcoma Virus 124

Complementary DNAs (cDNA's) specific for various regions of the Moloney murine sarcoma virus (MSV) 124 RNA genome were prepared by cross-hybridization techniques. A cDNA specific for the first 1,000 nucleotides adjacent to the RNA 3' end (cDNA 3') was prepared and shown to also be complementary to the 3'-terminal 1,000 nucleotides of a related Moloney murine leukemia virus (MLV) genome. A cDNA complementary to the "MSV-specific" portion of the MSV 124 genome was prepared. This cDNA was shown not to anneal to Moloney MLV RNA and to anneal to a portion of the viral RNA of about 1,500 to 1,800 nucleotides in length, located 1,000 nucleotides from the 3' end of MSV RNA. A cDNA common to the genome of MSV and MLV was also obtained and shown to anneal to the 5'-terminal two-thirds, as well as to the 3'-terminal 1,000 nucleotides, of the MSV RNA genome. This cDNA also annealed to the RNA from MLV and mainly to the 5'-terminal half of the MLV genome. It is concluded that the 6-kilobase Moloney MSV 124 RNA genome has a sequence arrangement that includes (i) a 3' portion of about 1,000 nucleotides, which is also present at the 3' terminus of MLV; (ii) an MSV-specific region, not shared with MLV, which extends between 1,000 and 2,500 nucleotides from the 3' terminus; and (iii) a second "common" region, again shared with MLV, which extends from 2,500 nucleotides to the 5' terminus. This second common region appears to be located in the 5' half of the 10-kilobase MLV genome as well. Experiments in which a large excess of cold MLV cDNA was annealed to (3)H-labeled polyadenylic acid-containing fragments of MSV RNA gave results consistent with this arrangement of the MSV genome.     

Genesis of Kirsten murine sarcoma virus: sequence analysis reveals recombination points and potential leukaemogenic determinant on parental leukaemia virus genome.

The genome of Kirsten murine sarcoma virus was formed by recombination between Kirsten murine leukaemia virus sequences, and rat sequences derived from a retrovirus-like '30S' (VL30) genetic element encompassing the Kras oncogene. Using cloned DNAs we have determined the nucleotide sequences of the long terminal repeats and adjacent regions, extending across the points of recombination on the sarcoma and leukaemia virus genomes. Our results suggest that discrete regions of homology and other cryptic sequence features, may have constituted recombinational hot-spots involved in the genesis of the Kirsten murine sarcoma virus genome. We have also compared the sequence of the Kirsten murine leukaemia virus p15 env and adjacent long terminal repeat with the corresponding regions of the AKV and Gross A murine leukaemia virus genomes. This comparison has identified a leukaemogenic determinant in the U3 domain of the long terminal repeat, possibly within a enhancer-like sequence element.     

Molecular cloning of the Harvey sarcoma virus circular DNA intermediates. II. Further structural analyses.

Three species of unintegrated supercoiled Harvey sarcoma virus DNA (6.6, 6.0, and 5.4 kilobase pairs) have been molecularly cloned from Harvey sarcoma virus-infected cells. On the basis of restriction enzyme analyses, the 6.6- and 6.0-kilobase pair viral DNAs contain two and one copies, respectively, of a 650-base pair DNA segment which contains sequences present at the 3' and 5' termini of the viral genome. R-loop structures formed between Moloney leukemia virus RNA and the cloned Harvey sarcoma virus DNA indicated that about 500 base pairs of the 650-base pair repeating segment was complementary to the 3' end of the viral RNA. During amplification in the Escherichia coli host, some recombinants containing the 6.6- or the 6.0-kilobase pair Harvey sarcoma virus DNA insert acquired or lost the complete 650-base pair DNA segment. These changes occurred in both recA+ and recA- E. coli.