Miroslav Hill

Sarcoma and transformation-defective viruses produced with infectious DNA(s) from Rous sarcoma virus (RSV)-transformed chicken cells

Abstract Chicken cells were transformed with helper-independent avian sarcoma viruses, subgroup C and D, respectively. The DNA extracted from these cells gave rise, in infectivity assays, to both transforming and transformation-defective viruses. Both viruses were recovered with a similar frequency at the end-point dilution (0.05 μg DNA per 10 7 cells) of infectious DNA. Increasing the DNA concentration had little influence on the probability of successful recovery of a sarcoma virus. On the other hand, the probability of recovering a transformation-defective virus increased when chicken cells received higher concentrations of DNA. Transformation-defective viruses here recovered for the first time after transfection with DNA from RSV-transformed cells were further investigated. Interference assays showed that these transformation-defective viruses interfered with sarcoma viruses recovered with the same DNA or belonging to the same antigenic subgroup as the DNA parent. No interference was encountered between viruses produced with infectious DNAs of antigenically different parents. Interference patterns of a cloned transforming virus suggested that the transforming viral DNA progeny was independent of a helper virus. Both transformation-defective and transforming viruses possessed the same buoyant density of 1.168 g/cm 3 . Viral RNA of the transformation-defective viruses sedimented slower than that of the transforming virus, the sedimentation coefficients being 65 S and 71 S, respectively. In distinction to the sarcoma virus, a transformation-defective isolate did not give rise to tumors in bioassays. Chicken cells infected with the transformation-defective virus harbored infectious DNA which produced transformation-defective viruses in infectivity assays but was unable to generate a sarcoma virus. A transformation-defective virus isolated in these infections interfered with its sarcomatogenous ancestor. To explain these results it is suggested that RSV-transformed chicken cells may carry two species of infectious DNA belonging to the sarcoma virus and its transformation-defective segregant, respectively. RSV-transformed mammalian cells appear to harbor only sarcoma viral DNA.

Genetic Transformation of Animal Cells With Viral Dna of Rna Tumor Viruses

Publisher Summary 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.

Amplification of the provirus region in Rous sarcoma virus-transformed Chinese hamster cells and segregation of the amplified copies in somatic cell hybrids

Four independent clones of RSV-transformed Chinese hamster fibroblasts were isolated. Southern blots and dot hybridization studies showed that in three out of four clones there were four to eight times as many integrated proviruses as in the fourth clone which contained at least one complete provirus. Restriction mapping studies showed that although the integration site varied from clone to clone, all the proviral copies in the same clone shared the same flanking cellular sequences. In one clone there are at least two polymorphic proviral variants A and B, one with and one without a BglI site. Further experiments were performed to see if the variants could be physically separated. RSV-Transformed Chinese hamster cells resistant to thioguanine were fused with mouse cells to give somatic hybrids which preferentially segregate Chinese hamster chromosomes. Ten out of eleven hybrids positive for virus rescue have lost up to 90% of the parental provirus copies. Four of these hybrids were found to contain the provirus variant A alone, one the variant B alone, and the rest contained both variants. All the proviruses retained in somatic hybrids shared the flanking cellular sequences of the parental provirus. Provirus segregation in somatic hybrids confirms that multiple (about ten) copies of the provirus region are present in the karyotype of parental RSV-transformed cells and, furthermore, suggests that the amplified copies of this region are translocated to different chromosomes.

Inability of the nondefective Rous sarcoma provirus to generate, upon transfection, a transformation-defective virus

Abstract DNA preparations extracted from six out of eight clonal lines of RSV-transformed chicken cells are unable to generate td viruses. In this respect, these DNAs differed from those obtained from mass cultures of RSV-transformed chicken cells. These results rule out the possibility that an nd provirus can give rise to transformation-defective progeny. Deletions occurring in the nd provirus, if they exist, cannot account for the appearance of td proviruses that are generated during the replication cycle of the virus.

Infectivity of Rous Sarcoma Cell DNA: Comparison of Two Techniques of Transfection Assay

A DNA extracted from a clone of chicken cells transformed by the Schmidt-Ruppin strain of Rous sarcoma virus, subgroup D(SR-RSV-D), was assayed for infectivity by means of DEAE-dextran and calcium techniques. The calcium technique like the previously described DEAE-dextran procedure gave rise to viruses in transfection assays with both native and denatured (S1 nuclease susceptible) DNAs. The efficiency of these transfection techniques with native DNA was compared and found to be about the same provided that with the calcium technique carrier DNA was used to complement DNA concentrations lower than 2.5 mug/ml.

Identification of novel sequences in the repertoire of hypervariable TRE17 genes from immortalized nonmalignant and malignant human keratinocytes

The TRE17 oncogene, originally cloned from transfected DNA of Ewings sarcoma cells, maps to chromosome 17q and is expressed in a wide variety of human cancer cells. We recently detected the variants of this gene by using the polymerase chain reaction (PCR) and sequencing from the first exon to the third intron. Based on sequence homology scores, the variants could be grouped into three families, denoted alpha, beta, and gamma. Here, we used human keratinocytes from healthy skin which had been spontaneously immortalized and then rendered malignant by serum privation in vitro. Both immortalized and malignant cells expressed TRE17 sequences to the same extent, and, according to the restriction site analysis of cloned PCR products, both contained common and rare TRE17 variants in similar proportions. These variants, one of each from both cell types, were then sequenced and compared with those from the previous study. In the phylogenetic tree, they clustered with alpha and gamma at the most distant tree positions. The overall fraction of conserved sites in the whole TRE17 repertoire was 80%. An unexpected feature of the observed variability was that intronic sites were significantly better conserved than exonic sites. Members of TRE17 gamma detected in immortalized and malignant keratinocytes differed one from another, and both differed from the TRE17 gamma already identified in Ewings sarcoma. No TRE17 gamma has been found so far in healthy tissues, thus leaving open the possibility of its origin from TRE17 beta by somatic changes during tumor progression.

Nuclear localization and covalent linkage of infective virus DNA to chromosomal DNA of non-producer Rous sarcoma cells.

Summary The nuclei of Rous sarcoma cells were prepared from an established line of non-producer rat XC cells transformed with a Prague strain of Rous sarcoma virus (PR-RSV). Electron microscopic examination of the nuclear pellet showed a slight contamination with cytoplasmic debris and an absence of mitochondria. The DNA samples extracted from isolated nuclei and from whole XC cells were both assayed for infectivity in chicken cell cultures and found to contain about the same number of infective units per unit weight of DNA. Furthermore, the DNA from whole XC cells was set free by alkali and sedimented through an alkaline glycerol gradient in order to separate cellular DNA species according to sedimentation velocity. Under these conditions the infective RSV DNA consistently sedimented with the chromosomal 110S DNA and thus behaved as if covalently linked to the chromosomal DNA of XC cells. These results show that the infective virus DNA of non-producer RSV-transformed cells is carried in these cells as an integral part of the cellular chromosome.

Infectious DNA coding for a temperature-sensitive DNA polymerase of the coordinate LA335 mutant of Rous sarcoma virus (RSV)

Abstract The DNA extracted from chicken cells transformed by the ts-LA 335 mutant of Rous sarcoma virus (RSV) gave rise, in transfection experiments, to transforming viruses. These viruses exhibited a temperature-sensitive lesion in early functions similar to that characteristic of the LA 335 mutant. One of the recovered viruses was further examined for a DNA polymerase activity at permissive and nonpermissive temperatures and was found to carry an enzyme resembling, with respect to thermosensitivity, the DNA polymerase of the parent LA 335 mutant. It was concluded that the infectious viral DNA extracted from RSV-transformed cells carries the genetic information for the DNA polymerase specific for the parent virus. This information is transferred, upon transfection of permissive cells, to the progeny virus.

Ability of mammalian fibroblasts to grow in synthetic medium containing neither serum nor exogenously added macromolecules

SummaryRous sarcoma virus transformed Chinese hamster fibroblasts, clone CHR1-3, were established at high temperature, then subcloned. Six subclones with round and flat morphology harboring undeleted and partially deleted RSV proviruses, respectively, were seeded into serum-free synthetic medium with no macromolecular additives, and maintained for 2 mo. One flat subclone no. 14, fully designatedsfCHR1-3.14 for itsserum-free phenotype, was further propagated in the same medium. The cells grew exponentially in loosely attached monolayers and dould be serially passaged on bare polystyrene with an average population doubling time of 46 h. Cell attachment could be improved by using collagen-coated polystyrene or by adding a methionine supplement to the culture medium. Furthermore, thesfCHR1-3.14 cells could be subcloned and further grown in nonselective medium. The reversion rate of thesf phenotype was estimated to be 1 to 2%/cell generation. Evidence for an autocrinal stimulation was obtained by cloning efficiency assays showing a requirement for a threshold cell density. Slight growth stimulation could also be detected in assays using conditioned medium fromsfCHR1-3.14 cells and serum-restrictedwild-type (wt)NIH3T3, but notwtCHR1-3.14, cells as indicator cells. Finally,wtNIH3T3 cells used in these assays were assayed for serum-free growth and found to be able to develop their ownsf phenotype; in this respect they resemble the previously establishedsfCHR1-3.14 cells.

Continuous production of Rous sarcoma virus free of transformation defective virus in clones of established RSV-transformed quail cells.

Abstract Quail embryo fibroblasts were infected with a Schmidt-Ruppin strain RSV × chf recombinant virus. Virus-transformed cells were established as a permanent line and then cloned in methyl cellulose. Out of 140 clones isolated four clones were capable of indefinite growth. These clones were examined for (i) production of sarcoma and td virus particles, (ii) number of integrated virus genome equivalents, and (iii) deletions of the src gene in the provirus. We found that the clones yield about 106 focus-forming units of the sarcoma virus per milliliter of the culture medium. No td virus could be detected by plating of the virus at the endpoint dilution and no 35 S td virus RNA but only 38 S sarcoma virus RNA was found in virions. Hybridization kinetic studies indicated that three different clones contain about 2 virus genome equivalents, and one clone contains about 4 virus genome equivalents per diploid cell. Upon transfection the proviruses of different clones generated sarcoma viruses and no td viruses. Finally digestion with EcoRI restriction endonuclease released in all four clones a 1.9 × 106-dalton fragment characteristic of the complete src gene, while no 0.8 × 106-dalton fragment characteristic of a td provirus could be detected. We concluded that the clones of RSV-transformed quail cells contain only nondefective sarcoma proviruses and produce from these proviruses nondefective focus-forming virions in the absence of any segregant td virions.