Selection for spontaneous null mutations in a chromosomally-integrated HSV-1 thymidine kinase gene yields deletions and a mutation caused by intragenic illegitimate recombination

Abstract

Spontaneous null mutations represent low frequency events that irreversibly and completely inactivate a gene, and can often consist of major gene alterations. To study the molecular mechanisms leading to recessive spontaneous null mutations in the human genome, we designed and tested a selection procedure in cell culture to enrich for this rare class of spontaneous mutations. The KT cell line contains the herpes simplex virus type 1 (HSV-1)_thymidine kinase ( tk) gene and the neomycin-resistance gene ( neo), from plasmid pSV2 neoKT, integrated as a single-copy in the human tk − cell line 143B. The HSV-1 tk gene was the target for spontaneous gene inactivation, and antiviral drugs (acyclovir, trifluorothymidine 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 with this multiple drug selection assay appeared at a very low frequency, 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 seen in the null mutations. Additionally, one of the null mutants contained 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 intragenci gene conversion event which may have been facilitated by short regions of junctional homology.