Expression of adenovirus E1a and E1b gene products and the Escherichia coli XGPRT gene in KB cells.
The recombinant plasmid pSV2-gpt, which contains the Escherichia coli XGPRT gene under the control of a simian virus 40 early promoter, was modified to contain the type 2 adenovirus (Ad2) XhoI-C (0 to 15.5 map units) restriction endonuclease fragment. Plasmid (pLB206) DNA was introduced into human KB cells by Ca2+-mediated DNA transfection, and transformants were selected in medium containing xanthine, aminopterin, and mycophenolic acid, as a consequence of expression of the dominant, selectable XGPRT gene. A series of 13 gpt+ cell lines were isolated and tested for their ability to complement Ad5 deletion mutants in E1a (H5dl312) and E1b (H5dl315). Four classes of gpt+ KB cell lines were identified, including clones constitutively expressing both E1a and E1b, only E1a, or only E1b or not expressing either E1a or E1b. DNA and RNA filter transfer hybridization analysis substantiated the conclusions that those cell lines capable of complementing viral host range mutants contained the appropriate viral DNA sequences and cytoplasmic polyadenylated RNA species. DNA filter transfer hybridization studies also revealed that the transfected vector DNA was stably integrated into chromosomal DNA in the KB transformants and the number of integrated sites ranged from 1 to 3. The gpt+ KB cell line that only expressed E1b gene functions only contained viral E1b gene sequences; those cell lines that expressed neither E1a nor E1b gene function contained only small or no regions of Ad2 DNA. When weaned off the selective medium, transformed KB cell lines stably maintained their inserted DNA in the absence of selective pressure and could easily be adapted to growth in suspension culture.
D000260:Adenoviruses, Human / Q000235:genetics* / Q000502:physiology; D002460:Cell Line; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D005814:Genes, Viral; D005816:Genetic Complementation Test; D006801:Humans; D010430:Pentosyltransferases / Q000235:genetics; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D011995:Recombination, Genetic; D014162:Transfection; D014779:Virus Replication
D004274:DNA, Recombinant; D012333:RNA, Messenger; D012367:RNA, Viral; D010430:Pentosyltransferases; C024827:xanthine phosphoribosyltransferase