Identification of a heteromorphic microsatellite within the thymidine kinase gene in L5178Y mouse lymphoma cells

The objective of this work is to identify a heteromorphism within the thymidine kinase (Tk1) gene which can be used to assay for allele loss by means of PCR. Intron F of mouse Tk1 contains two (CA), microsatellite sequences separated by 107 bp of non-repetitive sequence. We tested this region for heteromorphism in L5178Y mouse lymphoma cells. A PCR primer pair designated Agl1 yielded products of 396 and 194 bp from L5178Y tk(+/-) genomic DNA. The 194-bp product resulted from a secondary binding site between the two (CA), repeats for the forward Agl1 primer and was not produced from tk(-/-) mutants that had lost the functional Tk1(b) allele. Agl2 primers produced two PCR products of 523 and similar to 440 bp and Agl3 primers produced products of 579 and similar to 500 bp. In both these cases, the difference in product size was approximately equal, indicating that Intron F is similar to 80 bp shorter in the non-functional Tk1(a) allele than in Tk1(b). This heteromorphism forms the basis for an assay for allele loss by means of PCR. Agl1 and Agl3 primers yielded additional products of 91 and 274 bp, respectively, consistent with sizes expected from the mouse Tk1 pseudogenes (Tk1-ps), Our conclusions drawn from an analysis of 122 mutants for Tk1(b) loss using Agl2 primers agreed with previous analysis of the NcoI heteromorphism. Thus, a simple PCR-based analysis can identify Tk1(b) loss in the L5178Y mouse lymphoma cells.