Post-transcriptional control of avian oncornavirus transforming gene sequences in mammalian cells

Krzyzek, Richard A.; Lau, Alan F.; Faras, Anthony J.; Spector, Deborah H.

doi:10.1038/269175a0

Cited by 33 publications

(33 citation statements)

References 27 publications

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“…[1][2][3][4][5][6][7][8][9][10]. Second, a portion of the 5' terminus (i.e., 21 nucleotides) is repeated at the 3' end of the viral genome and presumably is used to facilitate continued, uninterrupted transcription of the viral genome during proviral DNA synthesis (5,(11)(12)(13)(14). Finally, the initiator codon, AUG, and nucleotide sequences implicated in the binding of eukaryotic ribosomes to mRNA are present within this region of the genome, suggesting a possible role in the initiation of translation of the viral genome (6).…”

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confidence: 99%

Evidence for splicing of avian sarcoma virus 5′-terminal genomic sequences onto viral-specific RNA in infected cells

Krzyzek

Collett

Lau

et al. 1978

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

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The 5'-terminal nucleotide sequences of the avian sarcoma virus (ASV) genome are transcribed by the reverse transcriptase in vitro into a DNA transcript that represents the entire distance ('100 nucleotides) The 5' terminus of the avian sarcoma virus (ASV) genome appears to play an important role in the replication of the virus for several reasons. First, initiation of reverse transcription occurs on a tRNATrP primer molecule located near (s100 nucleotides) this end of the viral genome (refs. 1-10). Second, a portion of the 5' terminus (i.e., 21 nucleotides) is repeated at the 3' end of the viral genome and presumably is used to facilitate continued, uninterrupted transcription of the viral genome during proviral DNA synthesis (5,(11)(12)(13)(14). Finally, the initiator codon, AUG, and nucleotide sequences implicated in the binding of eukaryotic ribosomes to mRNA are present within this region of the genome, suggesting a possible role in the initiation of translation of the viral genome (6).In this communication we present a series of experiments indicating that the 5'-terminal genomic nucleotide sequences, or a portion thereof, are present on each size class of ASV-specific mRNA identified in infected cell cultures. Furthermore, we demonstrate that these nucleotide sequences are not present adjacent to the corresponding gene sequences in genomic RNA obtained from virus. These data suggest that the 5'-terminal nucleotide sequences of the ASV genome are somehow "spliced" onto the individual species of viral-specific mRNA present in infected cells to provide some as yet unknown (RSV) were propagated in avian embryo fibroblasts and purified as described (18).Purification of Viral RNA. RNA was extracted from purified virus with Pronase/sodium dodecyl sulfate (NaDodSO4)/ phenol and subsequently fractionated into 70S RNA and free low molecular weight RNA by rate-zonal sedimentation (18). 32P-Labeled RSV (B77 strain) was prepared and purified by described procedures (18).Synthesis of Viral-Specific Complementary DNA Transcripts. Virus-specific DNA (cDNA), representing the majority of viral genomic nucleotide sequences, was synthesized with detergent-activated RSV as described by Stehelin et al. (20).The specific activity of the [3H]cDNA probe was approximately 2.4 X 107 cpm/,ug. The [3HJcDNA probe specific for the transforming gene sequences (cDNAs,) of ASV was prepared as described (20,21).The enzymatic synthesis of cDNA transcripts-representing defined regions of the 5' terminus of the viral genome was performed in reconstructed reactions containing reconstituted 35s RNA-tRNATrP complexes (10 ,ug/ml) as template/primer and purified AMV DNA polymerase (30-60 units/ml) (19,22

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confidence: 99%

Evidence for splicing of avian sarcoma virus 5′-terminal genomic sequences onto viral-specific RNA in infected cells

Krzyzek

Collett

Lau

et al. 1978

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

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“…to detect env gene-specific 28S viral RNA in these cells (16). All other RSV-infected mammalian cells examined to date also appear to lack the 28S env gene RNA (2,22).…”

Section: Discussionmentioning

confidence: 99%

“…One of the interesting aspects of this particular cell system is that the reversion phenomenon, unlike that of other transformed mammalian cells, appears not to reflect a consequence of either an alteration in, or the loss of, the src gene sequence or its transcription (3). This suggested that posttkanscriptional changes must contribute to morphological reversion in these transformed vole cells.…”

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confidence: 96%

“…1 A -C ), whereas no detectable organization of the cytoskeleton components was observed in the transformed vole cell lines (Fig. 1 D and E (16), was indeed translated in these cells ( Fig. 2A) (17,18).…”

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confidence: 99%

“…For the past several years, we have been studying a mammalian fibroblast cell line (European field vole) that can not only be transformed by RSV but,-in some instances, has reverted morphologically to the normal phenotype (3,4). One of the interesting aspects of this particular cell system is that the reversion phenomenon, unlike that of other transformed mammalian cells, appears not to reflect a consequence of either an alteration in, or the loss of, the src gene sequence or its transcription (3).…”

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confidence: 99%

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Morphological revertants of an avian sarcoma virus-transformed mammalian cell line exhibit tumorigenicity and contain pp60src.

Lau¹,

Krzyzek²,

Brugge³

et al. 1979

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

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The biological and biochemical properties of Rous sarcoma virus-transformed and revertant field vole cells were investigated. Revertant vole cells appear morphologically similar to normal, uninfected cells, yet, like transformed vole cells, they are fully capable of growing in agar suspension and producing tumors in athymic nude mice. These highly tumorigenic, yet morphologically normal appearing, vole cells express viral-specific antigens such as the gag gene product (Pr76) but lack the env gene protein (gp85). Moreover, they contain the sre gene protein, pp6Osrc-These results support the concept of the pleiotropic nature of the src gene product and in addition suggest that pp6Osrc may have multiple mechanisms of action. With this revertant cell system it may be feasible to distinguish between those biochemical functions of the src gene product that are important for tumorigenicity in vivo and those that are related to in -vitro morphological transformation.The ability of Rous sarcoma virus (RSV) to transform avian and mammalian cells in culture and toinduce tumors in animals has been localized to one of the four genes of the RSV genome, designated src (1). Restriction of expression of the RSV genome in infected cells, particularly the src gene, has been examined by comparative molecular studies of transformed cells expressing the src gene product (pp6osrc) and their revertant counterparts that have lost the transformed phenotype and appear to be incapable of expressing this particular genetic sequence (2).For the past several years, we have been studying a mammalian fibroblast cell line (European field vole) that can not only be transformed by RSV but,-in some instances, has reverted morphologically to the normal phenotype (3,4). One of the interesting aspects of this particular cell system is that the reversion phenomenon, unlike that of other transformed mammalian cells, appears not to reflect a consequence of either an alteration in, or the loss of, the src gene sequence or its transcription (3). This suggested that posttkanscriptional changes must contribute to morphological reversion in these transformed vole cells. Moreover, viral-specific RNA, including src RNA, was detected in polyribosomes of revertant vole cells, suggesting that these viral mRNAs are actively translated in these cells. In this communication, we demonstrate that the viral proteins encoded for by these particular viral mRNAs, including pp60src, are present in revertant vole cells. Moreover, we demonstrate that although the presence of pp6osrc in these cells is not correlated with morphological transformation, it is correlated with tumorigenicity, because these normal-appearing vole cells are capable of producing tumors in athymic nude mice.MATERIALS AND METHODS Cells. Normal, uninfected field vole (Microtus agrestis) fibroblasts, established as described (5), were infected by the Schmidt-Ruppin (SR) (subgroup D) strain of RSV. Several transformed cell lines (clones 1, 21, 22) were isolated by cloning in agar. After four passage...

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Studies on the Structure and Function of the Avian Sarcoma Virus Transforming Gene Product

Erikson

Brugge

Erikson

et al. 1979

Modern Trends in Human Leukemia III

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Post-transcriptional control of avian oncornavirus transforming gene sequences in mammalian cells

Cited by 33 publications

References 27 publications

Evidence for splicing of avian sarcoma virus 5′-terminal genomic sequences onto viral-specific RNA in infected cells

Evidence for splicing of avian sarcoma virus 5′-terminal genomic sequences onto viral-specific RNA in infected cells

Morphological revertants of an avian sarcoma virus-transformed mammalian cell line exhibit tumorigenicity and contain pp60src.

Studies on the Structure and Function of the Avian Sarcoma Virus Transforming Gene Product

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