In Vitro Transformation of Non-established Bovine Fibroblasts by BPV4 and Cooperating Factors

Jaggar, Rhys Trevor (1990) In Vitro Transformation of Non-established Bovine Fibroblasts by BPV4 and Cooperating Factors. PhD thesis, University of Glasgow.

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An in vitro assay system utilising non-established bovine fibroblasts derived from foetal palate was developed for the detection of BPV4-encoded transforming functions. This involved cotransfection of a dominant coselectable marker gene neo and recombinant BPV4 DNA linearised within the E1 ORF. The presence of BPV4 DNA resulted in the formation of colonies of diameter greater than 5mm (macrocolonies) which displayed a contact-inhibited phenotype. Cloning of regions of BPV4 into two vectors, pSV2neo and pZIPneoSV(X1), indicated that macrocolony formation could be induced by two regions of the genorpe. The first, a 2.0kb Xholl fragment (nts 6487-1275) encoded two complete ORFs, namely E7 and E8. A construct lacking 233bp (nts 906-1139), resulting in interruption of the E7 ORF, was inactive in this assay, indicating a requirement for the E7 ORF for induction of the phenotype. The second region, encoded by a 3.9kb Xholl fragment (nts 2597-6487), contained the E2, E3, E4, E5 and L2 ORFs. In this case, colonies contained cells showing a more elongated phenotype. Cotransfections of linear BPV4 with an activated ras gene did not lead to a loss of contact-inhibition. In contrast, overexpression of the 2.0kb fragment, in the presence of ras did lead to the formation of non-contact-inhibited neo r macrocolonies. An intact E7 ORF was again required for this activity. In the case of the 3.9kb construct, a single non-contact-inhibited colony was observed in constructs utilising the BPV4 promoters upon cotransfection with ras.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Developmental biology
Date of Award: 1990
Depositing User: Enlighten Team
Unique ID: glathesis:1990-78129
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 30 Jan 2020 15:39
Last Modified: 30 Jan 2020 15:39

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