Integration of SV40 promoter/enhancer sequences in an anchorage independent clonal subline of SV40-infected human epidermal keratinocytes and alterations in the viral sequences induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).
Item
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Title
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Integration of SV40 promoter/enhancer sequences in an anchorage independent clonal subline of SV40-infected human epidermal keratinocytes and alterations in the viral sequences induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).
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Identifier
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AAI9707075
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identifier
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9707075
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Creator
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Chen, Guangtian.
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Contributor
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Adviser: Mark L. Steinberg
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Date
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1996
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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
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Abstract
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The work described in this paper was initiated with the goal using SV40-immortalized human keratinocytes as a system to study molecular alterations in the DNA of cells exposed to N-methyl-N{dollar}\sp\prime{dollar}-nitro-N-nitrosoguanidine (MNNG). Viral sequences in clonal sublines of SV40 transformed human keratinocytes which were exposed to MNNG at sublethal concentrations ranging up to 10 {dollar}\mu{dollar}g/ml for 15 minutes were examined by Southern blot hybridization using full length SV40 DNA as a probe. Of the clonal sublines tested, one (AG34) was found to exhibit certain consistent, dose dependent changes at 4 days post treatment: (a) loss of two EcoRI fragments of about 4.4 and 3 kb, and the appearance of two bands migrating between 1.8 and 2.3 kb and, (b) loss of a 1.5 kb fragment and the appearance of 2, 3.8 and 5 kb fragments in KpnI digests.;Two BamHI fragments of approximately 5 and 7 kb in length containing the viral sequences from MNNG-treated and untreated AG34 cells were cloned and sequenced. Sequence analysis revealed the larger fragment to be 6.93 kb in length and contained two subgenomic viral integrants with the origin/promoter of the virus: (a) a right hand integrant containing viral sequences 5051-1645 and 570 bp of 5{dollar}\sp\prime{dollar} flanking human sequence, and (b) a central integrant containing SV40 bases 5051-468 along with the leftward flanking human sequence was repeated with slight alterations in the same orientation. Northern blot hybridization followed by RT-PCR demonstrated that the early promoter in the central integrant was functionally active and gave rise to a fusion transcript containing a short segment of the viral early genes and the adjacent human sequences flanking the integration site. The 5.2 kb fragment was found to consist of full length BamHI-linearized SV40 genomic DNA with numerous point mutations throughout the early gene region. There was a nine nucleotide insertion which results in an insertion of the tripeptide Pro-Ser-Gln at residue 702 of the T antigen polypeptide.;Sequence analysis of the corresponding fragments from the MNNG-treated cells revealed that MNNG caused various alterations. Some of the alterations were clustered within the segment comprising the second T antigen binding site.
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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.
