Chromosomal site(s) of integration of herpes simplex virus type 2 thymidine kinase gene in biochemically transformed human cells

Abstract

AbstractTo investigate the chromosomal site(s) of integration of the herpes simplex virus type 2 (HSV‐2) thymidine kinase (TK) gene in biochemically transformed human cells, transformed human cells[HeLa (BU25)/HSV‐2‐6 Cl 4] were fused with TK‐deficient mouse fibroblast [LM(TK−)] cells and seven lines of human—mouse somatic cell hybrids [HL/1… HL/7] were isolated. Enzyme assays demonstrated that the HL somatic cell hybrid lines expressed significant TK activity. That the TK activity was HSV‐2 specific was verified by disc PAGE analyses, by superinfection experiments with TK‐negative HSV mutants, and by enzyme neutralization experiments with anti‐HSV‐2 TK immunoglobulins. The seven HL somatic cell hybrid lines contained a complement of mouse chromosomes and one or more human chromosomes. The sensitivities of the HL hybrid lines to poliovirus types 1 and 2 and to diphtheria toxin were also studied. The poliovirus challenge experiments showed that hybrid lines HL/1, HL/3, HL/4 and HL/5 were completely resistant to poliovirus and that less than 25% of the cells in line HL/2 were destroyed, indicating that with the exception of HL/2, human chromosome 19 was not retained in the human—mouse hybrid cells. Hybrid lines HL/1‐1, HL/2‐1, HL/3, HL/4, HL/5, HL/6 and HL/7 were insensitive to diphtheria toxin, indicating that human chromosome 5 was not retained in these human—mouse hybrid cells. Karyotype analyses showed that the only human chromosome found in hybrid lines HL/3, HL/4, HL/5 and HL/6 was marker chromosome M13; this chromosome was present in the majority of HL/3 cells and in all HL/4, HL/5, and HL/6 hybrid cells. The same chromosome was present in all cells of hybrid HL/7, in 17/19 analyzed mitosis of HL/2, and in all cells of HSV‐2 TK‐positive HL/2 subclones but, in addition, several other human chromosomes were found in the HL/7 and HL/2 hybrid lines. After counterselection with bromodeoxyuridine (Brd Urd), chromosome M13 was absent from all analyzed cells of HSV‐2 TK‐negative, Brd Urdcounterselected HL/2 subclones, but some of the other human chromosomes were still present. Morphology and G banding analyses suggest that M13 is an isochromosome formed from the short arm of human chromosome X. The only human chromosome present in cells of somatic hybrid HL/1 and four of its HSV‐2 TK‐positive subclones was chromosome 17 with a translocation on the short arm. This chromosome was present in all cells of hybrid HL/1 and its subclones and was absent in all cells of four analyzed HSV‐2 TK‐negative, Brd Urdcounterselected subclones. The preceding experiments demonstrate that the HSV‐2 TK gene is associated with human marker chromosome M13 and modified human chromosome 17 in HSV‐2‐transformed human line, HeLa(BU25)/HSV‐2‐6 Cl 4.