| The morphological and biochemical transforming properties of herpes simplex viruses type 1 and 2 were investigated by DNA transfection. Two regions within the U(,L) were found to initiate morphological transformation; mtr-I (HSV-1, BglII-I, 0.311-0.415, 15.8 kb), and mtr-II (HSV-2, BglII-N, 0.582-0.628, 7.8kb). Transformed lines established with either region displayed growth characteristic of the transformed state. The analysis of morphological transformants demonstrated that morphological and biochemical (Tk) transformation were separable and distinct.;Genomic DNA from mtr fragment transformants revealed, (i) homology of BglII-I to BALB/3T3 DNA, but no retained viral sequences and (ii) the presence of unique BglII-N integration in two mtr-II transformants tested. F4A1A1 (mtr-II transformed), possessed sufficient BglII-N sequences to be mapped by regular Southern blotting using cloned BglII-N as probe. This line had multiple integrations, which were labile through rearrangements and/or loss.;Biochemical (Tk) transformed lines also demonstrated sequence lability by integral loss of reiterated sequences but without rearrangements. The findings with biochemically transformed lines can be summarized: (i) multiple integration sites within one line are not uncommon, (ii) multi-copy repeats of integrated viral DNA occur but are unstable, (iii) extensive retention of sequences contiguous to Tk is possible and (iv) there is not a unique region for Tk integration.;The complete genomes of HSV-1 and 2 were cloned and a specific spontaneously deleting region (0.404-0.414) identified. This deletion, absent in wild type and defective viral genomes, occurred subsequent to cloning. Cloned Tk was active in biochemical transformation allowing functional mapping to establish colinearity of a 1.5 kb region for HSV-1 and 2 Tks.;In vitro manipulation of HSV-2 Tk produced a eukaryotic vector pGR18. A panel of cell lines were established containing different regions of the HSV genome. Analysis of ten lines generated with mtr-II cloned into pGR18 (pGR136) revealed retention of 50 percent of linked non-selected sequence compared to little, if any, in unlinked coselected lines.;The general usefullness of linked coselection was shown by construction of a hybrid Tk-human-(beta)-interferon gene in a plasmid with active Tk. Ltk('+) lines, although not constitutive, were poly rI:rC inducible and one line also HSV superinfection inducible. The viral ts nature of this phenomenon demonstrated that the Tk promoter regulated (beta)-interferon expression. |
