cDNA transformation markers from SV40-transformed human epidermal keratinocytes.
Item
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Title
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cDNA transformation markers from SV40-transformed human epidermal keratinocytes.
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Identifier
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AAI9510718
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identifier
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9510718
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Creator
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Shen, Huan.
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Contributor
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Adviser: Mark L. Steinberg
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Date
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1994
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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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Chemistry, Biochemistry | Biology, Molecular | Biology, Cell
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Abstract
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Candidate cDNAs representing sequences specifically induced by SV40 were selected by eliminating cDNAs hybridizing to labeled heterogeneous cDNA probes derived from normal human epithelial cells (previous work). From these candidates two cDNAs, E12 and E13, have been found to be markers of epithelial transformation in that the mRNAs show highly induced expression not only in SV40 transformants but also in cell lines derived from various squamous cell carcinomas.;Highly induced expression of E12 and E13 emerges early during SV40 transformation process and which are shared in common by transformed keratinocytes which arise independently from one another. These stable changes of expression are likely to play some significant role in transformation even if the genes are not themselves oncogenes. This indicates that the expression of the E12 and E13 may be transactivated directly or indirectly by SV40 virus.;A full-length cDNA of the E12 gene has been obtained by using the protocol of RACE (rapid amplification of cDNA ends). The complete nucleotide sequence of the E12 cDNA is 1251 bp in length and the deduced open reading frame sequence is 715 bp encoding a 235 amino acid protein. Computer analyses and sequence homology searches indicated that the E12 transcript contains a 5{dollar}\sp\prime{dollar} upstream Alu sequence. The Alu sequence is about 300 bp in length at the 5{dollar}\sp\prime{dollar} end of E12 and is apparently expressed in the open reading frame; the Alu sequences were found in many mRNA sequences catalogued in Genbank. The functions of the Alu sequences in mRNAs are not known. The remainder of the E12 gene showed no significant homology to any sequence currently cataloged in the Genbank, except for a piece of unannotated sequence from randomly amplified cDNA, HM02E05.;Two genomic libraries were created in the lambda phage vector and screened with E12 and E13 probes, respectively. Two positive clones, GE12 for the E12 cDNA and GE13 for the E13 cDNA were isolated from the libraries. GE12 contains a 9.2 kb insert and GE13 contains a 9.5 kb insert. Both GE12 and GE13 were subcloned into the plasmid PGEM3Z designated PGE12 and PGE13, respectively. Restriction maps and partial sequences of PGE12 and PGE13 were obtained. PGE12 and PGE13 were used for transfection focus-forming assays as an in vitro indication that these genes may carry oncogenic potential.
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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.
