Mutations of the chloramphenicol acetyl transferase transgene driven by the immunoglobulin promoter and intron enhancer.

Transgenic mice containing the chloramphenicol acetyl transferase (CAT) gene driven by the immunoglobulin (Ig) VH promoter and the heavy chain intron enhancer were prepared to increase our understanding of the mechanism responsible for somatic mutation. The transgene showed similar tissue specificity in terms of expression as endogenous Ig genes. Hybridomas were prepared after multiple immunization of a transgenic mouse with (4-hydroxy-3-nitrophenyl)acetyl-chicken gammaglobulin, a thymus-dependent antigen. We analyzed mutations in the CAT coding region as well as in the region 5' upstream of the promoter after amplification of DNA using the PCR followed by sequencing of cloned DNA. Mutation detection enhancement gel electrophoresis was also used to detect mutations. Only a single band was observed in PCR products from the region 5' upstream of the promoter and from the enhancer, whereas in those from the CAT coding region, three out of 11 hybridomas showed multiple bands. In DNA sequences of the CAT coding region from these three hybridomas, a total of six nucleotide substitutions were observed, but none in the region 5' to the promoter. The results of DNA sequencing and the electrophoresis were in good agreement, suggesting that mutation occurred only in the CAT gene but not in the region 5' to the promoter or the enhancer. The rate of mutation in the CAT gene was estimated to be 1.6 x 10(-5) per base pair per cell division, which was a lower limit of the rate for somatic mutation reported for Ig genes. Location and distribution of mutations were similar to that of the Ig gene, suggesting that (i) mutation in the CAT gene was induced by a mechanism similar to that occurring in the Ig gene and (ii) the mutator mechanism can operate in a non-Ig gene provided it is flanked by the VH promoter and heavy chain intron enhancer.