Transformation of rat embryo fibroblasts by cloned polyoma virus DNA fragments containing only part of the early region.

Hassell, John A.; Topp, William C.; Rifkin, Daniel B.; Moreau, Pierre

doi:10.1073/pnas.77.7.3978

Cited by 61 publications

(47 citation statements)

References 37 publications

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“…ld). The segment of the polyoma virus genome present in pgtlTK can malignantly transform mouse cells (18,27), and experiments suggest that the medium T function alone may be sufficient for malignant cellular 513 VOL. 3,1983 on April 30, 2019 by guest http://mcb.asm.org/ Downloaded from transformation (34).…”

Section: Resultsmentioning

confidence: 99%

Expression and Stabilization of Microinjected Plasmids Containing the Herpes Simplex Virus Thymidine Kinase Gene and Polyoma Virus DNA in Mouse Cells

1983

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To observe the effects of polyoma virus DNA on the expression of the herpes simplex virus (HSV) thymidine kinase (TK) 400 times as many molecules per cell were needed to cause early TK activity. The frequency of stable transformation observed 2 weeks after nuclear injection of 10 to 20 polyoma virus origin-containing plasmid molecules per cell was at least 2 orders of magnitude greater than with plasmids containing the TK gene alone. The greatest enhancement of stable TK transformation was obtained with plasmids containing the origin alone, when the maximum frequency of stable transformation was 5%. The addition of the coding regions for the small and medium T antigens or the entire early region significantly decreased TK transformation frequency in a copy-dependent fashion. The timing of stabilization of TK-positive transformation was analyzed by releasing hypoxanthine-aminopterin-thymidine selection pressure at various times after microinjection, culturing the cells in nonselective medium, and assaying for TK activity. Stabilization was found to occur between 3 and 6 days after nuclear injection. Cells injected with a plasmid containing the origin and the early region were examined for expression of the large T antigen with polyoma virus antitumor serum and immunofluorescent staining. The expression of the large T antigen was clearly associated with a cytopathic effect. TK-positive clones observed 2 weeks after injection of the plasmid were uniformly T antigen negative. Cytotoxicity may be the result of plasmid replication and toxic levels of T antigen or TK. In addition, expression of the large T antigen may block stabilization by preventing the integration of origincontaining plasmid molecules.Gene transfer techniques have been used to cells (2,23,36), frog oocytes (7,21,25), and introduce exogenous DNA into a variety of mammalian eggs (13). Somatic cells in vitro have recipient cell types, including cultured somatic been most commonly used as recipients because the methods for cell culture are well established

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Section: Resultsmentioning

confidence: 99%

Expression and Stabilization of Microinjected Plasmids Containing the Herpes Simplex Virus Thymidine Kinase Gene and Polyoma Virus DNA in Mouse Cells

1983

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“…The transforming potential of the virus resides in the early transcription unit which encodes three tumor antigens, large, middle, and small T antigen (28). Large T antigen facilitates the initiation of transformation (8, 9, 12), whereas middle T antigen is required to maintain the transformed phenotype (3,10,14,16,20,22,23,25,27,29). The role of small T antigen in transformation is not known.…”

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“…The two mutants derived from pPBR2, pPdl3, and pPdll5, carry deletions of about 15 and 10 bp that remove the PstI and SstI sites, respectively. DNA alone or in pairs, with calcium phosphate used to facilitate DNA uptake as described (14,20). The cultures were stained with Giemsa, and foci were counted after 10 to 14 days.…”

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Homologous recombination between transfected DNAs.

Pomerantz¹,

Naujokas²,

Hassell³

1983

Mol. Cell. Biol.

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An extensive analysis of the fate and structure of polyomavirus-plasmid recombinant molecules transfected into Rat-1 cells has revealed that the DNA often becomes integrated within transformed cell DNA in a head-to-tail tandem arrangement. This occurs independently of the replicative capacity of the transforming DNA and is facilitated by the use of large quantities of DNA during transfection. These observations have led us to suggest that head-to-tail tandems are formed by homologous recombination between transfected DNAs either before or after integration within cellular DNA. To test this hypothesis, we have measured the transforming activity of pairs of mutant, nontransforming, recombinant plasmid DNAs that carry different lesions in the transforming gene of polyomavirus. The results show that, although the individual mutant DNAs are incapable of transformation, transfection with pairs of mutant DNAs leads to the formation of transformed cells at high frequency. Moreover, there is a direct relationship between the distance between the lesions in pairs of mutant DNAs and their transforming activity. Finally, analyses of-the structures of integrated recombinant plasmid DNAs and the viral proteins within independent transformed cells prove that recombination occurs between the mutant genomes to generate a wild-type transforming gene.Polyomavirus, one member of the papovaviruses, is a small double-stranded DNA tumor virus that is capable of replicating in mouse cells and transforming other rodent cells in culture. The transforming potential of the virus resides in the early transcription unit which encodes three tumor antigens, large, middle, and small T antigen (28). Large T antigen facilitates the initiation of transformation (8, 9, 12), whereas middle T antigen is required to maintain the transformed phenotype (3,10,14,16,20,22,23,25,27,29). The role of small T antigen in transformation is not known. Stable cellular transformation is achieved by the integration of the viral sequences within cellular DNA. Recombination between the cellular and viral genomes does not appear to involve specific sites, nor are regions of extensive homology required (15). One distinguishing characteristic of polyomavirus-transformed cells, especially transformed rat cells, is the arrangement of the integrated viral sequences within transformed cell DNA in a headto-tail tandem array (1, 2,4,13,19). This arrangement of integrated viral sequences is not only unique to polyomavirus-transformed cells but also occurs in simian virus 40-transformed cells (5, 18) and in cells transformed with viral recombinant DNA (24). The formation of tandemly arranged, integrated viral DNA may be stimulated by large T antigen (7), perhaps indirectly, but is not absolutely dependent on the activity of this protein (24). We have suggested that tandemly integrated sequences are formed by recombination between transforming DNAs (24), and have now formally tested this possibility.To determine whether homologous recombination can occur after transfection of ma...

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“…An intact large T protein is not essential for maintenance ofthe transformed phenotype (4)(5)(6)(7)(8)(9). Small T and middle T (mT) antigens have been implicated in maintenance of the transformed state and in tumorigenicity by the virus.…”

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Carboxy terminus of polyoma middle-sized tumor antigen is required for attachment to membranes, associated protein kinase activities, and cell transformation.

Carmichael¹,

Schaffhausen²,

Dorsky³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

131

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We have constructed a transformation-defective polyoma virus mutant (Py 1387-T) that directs the synthesis of a normal small tumor antigen, a functional large tumor antigen, and a truncated (51,000-dalton) middle-sized tumor (mT) antigen that lacks 37 amino acids at its COOH terminus. The shortened mT polypeptide is missing the hydrophobic "tail" thought to be responsible for the anchorage of this protein into the plasma membrane and is in fact in cytosol fractions. This truncated mT polypeptide is inactive in an in vitro protein kinase assay and is altered in its phosphorylation in viva Mutant 1387-T differs from wildtype virus in having a T-A base pair instead of a C-G base pair at nucleotide position 1387. This change was introduced into viral DNA by using a synthetic undecanucleotide as a specific mutagen. Wild-type polyoma DNA was rendered single stranded by molecular cloning into coliphage M13. The oligonucleotide, which hybridizes with a mismatch at the site to be altered, was used to prime the synthesis ofdouble-stranded closed circular DNA. Progeny recombinant phages were screened by DNA sequence analysis for the desired base change. The polyoma mutant was reconstructed from recombinant phage replicative form DNA molecules containing the mutation.The early region of polyoma virus encodes three proteins, the small [22-kilodalton (kDal)], the middle-sized (56-kDal), and the large (100-kDal) tumor (T) antigens. The large T antigen is required for transformation by intact virus (1-3) and may function by promoting stable association ofthe viral genome with cellularDNA. An intact large T protein is not essential for maintenance ofthe transformed phenotype (4)(5)(6)(7)(8)(9). Small T and middle T (mT) antigens have been implicated in maintenance of the transformed state and in tumorigenicity by the virus. The nontransforming hr-t mutants (10-12) have lesions affecting both the small T and mT polypeptides but not large T protein (4,(13)(14)(15).The importance of the mT polypeptide in transformation has been demonstrated directly; DNA coding only for mT antigen can transform rat cells (16). Mutants in which mT, but not small T, antigen is altered are affected in their transformation properties (17-19). When deletions affecting the COOH-terminal region of mT antigen are created in cloned polyoma DNA, the ability of that DNA to transform cells is reduced (20).Phosphorylation is important to mT antigen function. A protein kinase activity that phosphorylates tyrosine residue 315 of mT antigen is present in T-antigen immunoprecipitates (21-25). Middle T antigen can be resolved into two species, 56 kDal and 58 kDal, that differ strikingly in activity in the in vitro kinase reaction (24,25). These species also differ in their patterns of phosphorylation in vivo. The high specific activity 58-kDal form is phosphorylated in vivo at different serine or threonine residues than is the 56-kDal form (24, 25). These results suggest that the activity of mT antigen may be regulated by cellular protein kinase(s).mT antig...

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Transformation of rat embryo fibroblasts by cloned polyoma virus DNA fragments containing only part of the early region.

Cited by 61 publications

References 37 publications

Expression and Stabilization of Microinjected Plasmids Containing the Herpes Simplex Virus Thymidine Kinase Gene and Polyoma Virus DNA in Mouse Cells

Expression and Stabilization of Microinjected Plasmids Containing the Herpes Simplex Virus Thymidine Kinase Gene and Polyoma Virus DNA in Mouse Cells

Homologous recombination between transfected DNAs.

Carboxy terminus of polyoma middle-sized tumor antigen is required for attachment to membranes, associated protein kinase activities, and cell transformation.

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