Figure 4.

(A) PCR genotyping of Poli codon 27. An 88 bp fragment from exon 2 was amplified from genomic DNA. A unique TaqI restriction enzyme site (TCGA) is centrally located within this 88 bp PCR fragment. Substitution of C→A within the Ser27 codon (TCG) changes it to an amber stop codon (TAG) and simultaneously destroys the TaqI site. DNA with a wild-type sequence is cut by TaqI to generate restriction fragments of 39 and 49 bp, while DNA containing the substitution remains uncut. The gel analysis reveals that C57BL/6J has two wild-type alleles, whereas 129/J, 129/SvJ and 129/ReJ contain the amber codon on both alleles. (B) Western analysis of Polι in testis extracts. Testis extracts from C57BL/6J and 129/SvJ mice were separated in a 10% polyacrylamide-SDS gel and transferred to an Immobilon P membrane. The membrane was cut in half, and the top half containing high molecular weight proteins was probed with polyclonal antisera to polι, and the bottom half containing lower weight proteins was probed with polyclonal antisera to β-actin. Cross-reacting proteins were visualized by chemiluminescence. The data shows that while both extracts contain similar levels of β-actin, polι can only be detected in C57BL/6J mice (genotypically Poli ^+/+) and not in 129/SvJ (genotypically Poli ^−/−) mice.