Polyoma genome transcription in transformed mouse cells growing in culture and as tumors in syngeneic mice

Grady, Leo J.; North, Arlene B.; Campbell, Wayne P.

doi:10.1002/ijc.2910190213

Intl Journal of Cancer

1977

DOI: 10.1002/ijc.2910190213

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Polyoma genome transcription in transformed mouse cells growing in culture and as tumors in syngeneic mice

Leo J. Grady

Arlene B. North

Wayne P. Campbell

Abstract: The strands of the polyoma genome coding for early and late viral RNA were separated by means of asymmetric cRNA synthesized under high salt conditions by Escherichia coli RNA polymerase. Each strand was then employed in RNA-DNA hybridization experiments to determine the degree to which it is transcribed in transformed cells under several conditions. Except for a concanavalin-A-selected revertant, approximately one-quarter of the early strand was expressed in all of the situations investigated. In contrast, wh… Show more

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Cited by 3 publications

(3 citation statements)

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“…That large T-antigen plays no role in maintaining the transformed phenotype was further suggested by other observations. First, several investigators failed to find mRNA sequences complementary to the distal portion of the polyoma viral early region in some transformed cell lines (2,14,25). Moreover, large T-antigen could not be detected in a number of cell lines transformed by the virus (19).…”

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confidence: 99%

Polyoma viral middle T-antigen is required for transformation

Mes¹,

Hassell²

1982

J Virol

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To determine whether small or middle T-antigen (or both) of polyoma virus is required for transformation, we constructed mutants of recombinant plasmids which bear the viral oncogene and measured the capacity of these mutants to transform rat cells in culture. Insertion and deletion mutations in sequences encoding small and middle T-antigens (79.7, 81.3, and 82.9 map units) rendered the DNA incapable of causing transformation by the focus assay. Similar mutations in sequences that encoded middle but not small T-antigen (89.7, 92.1, and 96.5 map units) generally abolished the transforming activity of the DNA. However, two mutants (pPdll-4 and pPdl2-7) that carried deletions at 92.1 map units retained the capacity to transform cells; pPdll-4 did so at frequencies equal to those of the parental plasmid, whereas pPdl2-7 transformed at 10% the frequency of its antecedent. From these studies we conclude that small T-antigen alone is insufficient to cause transformation and that middle T-antigen is required for transformation, either in combination with small T-antigen or by itself.

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mentioning

confidence: 99%

Polyoma viral middle T-antigen is required for transformation

Mes¹,

Hassell²

1982

J Virol

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“…Mutants containing lesions that map within the early region either fail to transform cells completely [the host-range transformation-defective mutants (1,2)] or are temperature sensitive for the establishment of transformation and in some cases its maintenance [the tsa mutants (3)(4)(5)(6)]. Moreover, RNA complementary to only the "E" strand of the early region of the viral DNA is always detected in transformed cells (7)(8)(9)(10), and these cells inevitably contain tumor antigens (T antigens), proteins encoded by the early region of the virus (11)(12)(13).…”

mentioning

confidence: 99%

“…Several analyses of virus-specific RNA in polyoma-transformed cells revealed that some lines either lack or contain smaller amounts of those RNA sequences that are transcribed from the distal part of the early region (7,9,10 (15). Transfections with viral and plasmid DNAs were performed as described (16), using intact plasmids purified on CsCl gradients.…”

mentioning

confidence: 99%

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

Hassell¹,

Topp²,

Rifkin³

et al. 1980

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

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Recombinant plasmids containing either the entire polyoma viral genome or one or the other of the two HindIl fragments of polyoma virus DNA were constructed and cloned in Escherichis coli X1776, and their DNAs were individually tested for the capacity to transform an established line of rat cells. The recombinant plasmids containing the entire polyoma genome and those containing the HindIII-1 fragment of polyoma DNA (45-1.4 map units) efficiently transform rat cells, whereas the plasmids containing the HindU-2. fragment (1.445.0 map units) do not. The properties of many independent transformed cell lines established by infection with the cloned HindIII-1 fragment were determined. In contrast to the parent cell line, rat cells transformed with the cloned HindIII-1 fragment grow to high saturation densities, form colonies with high efficiency in dilute agar suspension, produce high levels of plasminogen activator, and display a disorganized arrangement of actin cables. By all criteria examined, these cells transformed by fragments are indistinguishable from cells transformed by whole polyoma viral DNA. Cellular DNA prepared from many HindIII-1 fragment-transformed cell lines was analyzed for the presence and arrangement of polyoma viral sequences by Southern blot-hybridization. In all cases examined, only those viral sequences contained within the HindIII-1 fragment of polyoma DNA were detected. These data establish a strong correlation between polyoma DNA sequences mapping within a restricted portion of the early region and the induction and maintenance of the transformed phenotype. The transforming region of the polyoma viral genome has not been precisely defined, though a wealth of evidence links cell transformation to the early region (70-26 map units, Fig. 1 Fig. 1) enhances the tumorigenic capacity of the DNA in newborn hamsters. These observations suggest that not all of the early region may be required for transformation. To determine which segment of polyoma virus DNA is required to transform cells, we have measured the capacity of cloned subgenomic fragments of viral DNA to transform cells in culture. Using this approach, we have been able to localize the transforming gene(s) of polyoma virus to a restricted portion of the viral genome containing only part of the early region.MATERIALS AND METHODS Cell Culture and Transformation. The Rat-i cells used in this work were obtained from C. Basilico and recloned in our laboratory. Rat-i cells are a subclone of Fisher rat F2408 cells (15). Transfections with viral and plasmid DNAs were performed as described (16), using intact plasmids purified on CsCl gradients. The plasmids appeared as homogeneous monomeric molecules after gel electrophoresis, although the presence of small quantities of multimeric molecules is possible. After transfection with DNA, the cells were incubated for 2-3 weeks, and transformants were scored as dense foci. To isolate transformed cell lines, individual foci from separate Petri dishes were ringed with stainless steel cylinder...

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Complexity of poly(A⁺) and poly(A⁻) polysomal RNA in mouse liver and cultured mouse fibroblasts

1978

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RNA excess hybridization experiments were used to measure the complexity of nuclear RNA, poly(A+) mRNA, poly(A-) mRNA, and EDTA-released polysomal RNA sedimenting at less than 80 S in mouse liver and in cultured mouse cells. With both cell types, poly(A-) RNA was found to contain 30-40% of the sequence diversity of total mRNA. In the case of liver this represents 5,700 poly(A-) molecules and 8,600 poly(A+) molecules for a total of approximately 14,300 different mRNAs. Comparison of the complexity of mRNA with that of nuclear RNA revealed that in liver and in cultured cells, mRNA has only 10-20% of the sequence diversity present in nuclear RNA. This latter observation is consistent with existing data on mammalian cells from this and other laboratories.

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Polyoma genome transcription in transformed mouse cells growing in culture and as tumors in syngeneic mice

Cited by 3 publications

References 16 publications

Polyoma viral middle T-antigen is required for transformation

Polyoma viral middle T-antigen is required for transformation

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

Complexity of poly(A⁺) and poly(A⁻) polysomal RNA in mouse liver and cultured mouse fibroblasts

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Polyoma genome transcription in transformed mouse cells growing in culture and as tumors in syngeneic mice

Cited by 3 publications

References 16 publications

Polyoma viral middle T-antigen is required for transformation

Polyoma viral middle T-antigen is required for transformation

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

Complexity of poly(A+) and poly(A−) polysomal RNA in mouse liver and cultured mouse fibroblasts

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Product

Resources

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Complexity of poly(A⁺) and poly(A⁻) polysomal RNA in mouse liver and cultured mouse fibroblasts