SELECTION FOR SPONTANEOUS NULL MUTATIONS IN A CHROMOSOMALLY-INTEGRATED HSV-1 THYMIDINE KINASE GENE YIELDS DELETIONS AND A MUTATION CAUSED BY INTRAGENIC ILLEGITIMATE RECOMBINATION

Spontaneous null mutations represent low frequency events that irrever sibly and completely inactivate a gene, and can often consist of major gene alterations. To study the molecular mechanisms leading to recess ive spontaneous null mutations in the human genome, we designed and te sted a selection procedure in cell culture to enrich for this rare cla ss of spontaneous mutations. The KT cell line contains the herpes simp lex virus type 1 (HSV-1) thymidine kinase (tk) gene and the neomycin-r esistance gene (neo), from plasmid pSV2neoKT, integrated as a single-c opy in the human tk- cell line 143B. The HSV-1 tk gene was the target for spontaneous gene inactivation, and antiviral drugs (acyclovir, tri fluorothymidine and ganciclovir) were used, in combination, to provide a selective enrichment for null mutations over the background of more frequent and revertible point mutations. The tk- mutations obtained w ith this multiple drug selection assay appeared at a very low frequenc y, rarely reverted to wild-type (tk+), and the TK protein was observed only in 4.8% of these null mutants. Deletions of the entire tk gene, or its 3' region, constituted the major class of DNA rearrangements se en in the null mutations. Additionally, one of the null mutants contai ned an intragenic 106-bp duplication within a 43-bp deleted region of the tk gene. We propose this mutation to be the outcome of an intragen ic gene conversion event which may have been facilitated by short regi ons of junctional homology.