Ha-ras polymorphisms in SV40-transformed human keratinocytes: Cloning and sequence analysis of putative regulatory elements adjacent to the human Ha-ras protooncogene.
Item
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Title
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Ha-ras polymorphisms in SV40-transformed human keratinocytes: Cloning and sequence analysis of putative regulatory elements adjacent to the human Ha-ras protooncogene.
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Identifier
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AAI9405513
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identifier
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9405513
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Creator
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Chen, Weiyi.
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Contributor
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Adviser: Mark L. Steinberg
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Date
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1993
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Cell | Biology, Molecular | Biology, Genetics
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Abstract
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Whereas much is known about the process of transformation by retroviruses and their transduced oncogenes, the mechanism of transformation by DNA viruses is still obscure. It is postulated that DNA viruses may cause oncogenic transformation by interacting with host cellular genes including oncogenes, resulting in a cooperative interaction leading to altered patterns of growth. However, very little research has been done. In this study, we used human cultured keratinocytes transformed by SV40 virus as an in vitro system to study structural changes in the cellular proto-oncogenes Ha-ras and myc resulting from infection by SV40. Southern blot analysis of ras sequences in the genomic DNAs of transformed cells using PTB-1, a probe which covers most 5{dollar}\sp\prime{dollar} region of the Ha-ras, revealed a polymorphic pattern of large restriction fragments of about 4-5 kb which were present in the normal keratinocytes but which were absent in both PstI and Sau3A digests of transformed cells. To determine the nature of the restriction fragment length polymorphisms (RFLP), we cloned and sequenced the 1.8 kilobases of the region upstream from the 6.4 kilobases BamHI fragment containing Ha-ras. In a further study, ras gene expression by northern blot analyses also indicated that ras proto-oncogenes in SV40 transformed cell lines were expressed at a level of at least five times higher than those from normal keratinocytes. Sequence homology analysis of the 1.8 kilobase upstream region by computer search of the Genbank DNA sequence database revealed: (1) two segments with homology to enhancer regions of previously studied genes and, (2) that the cloned segment contained a novel, previously uncatalogued nucleotide sequence which has been subsequently submitted to Genbank with the accession number L11526. These segments were also found to contain binding motifs for transcriptional activating proteins including T-ag, AP-1 and SP1. Nuclear proteins prepared from EP, HeLa and line 98 were found to bind specifically to subfragments of this region in gel mobility shift assays and it appears that the binding activity in 98 is slightly stronger than in EP. DNase I foot-printing experiments confirmed the existence of nuclease resistant nucleotide segments within the putative enhancer region of the upstream sequences (Fig. 31 and 32).
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
