Specific expression of activation-induced cytidine deaminase (AID), a novel member of the RNA-editing deaminase family in germinal center B cells.

We have identified a novel gene referred to as activation-induced deaminase (AID) by subtraction of cDNAs derived from switch-induced and uninduced murine B lymphoma CH12F3-2 cells, more than 80% of which switch exclusively to IgA upon stimulation. The amino acid sequence encoded by AID cDNA is homologous to that of apolipoprotein B (apoB) mRNA-editing enzyme, catalytic polypeptide 1 (APOBEC-1), a type of cytidine deaminase that constitutes a catalytic subunit for the apoB mRNA-editing complex. In vitro experiments using a glutathione S-transferase AID fusion protein revealed significant cytidine deaminase activity that is blocked by tetrahydrouridine and by zinc chelation. However, AID alone did neither demonstrate activity in C to U editing of apoB mRNA nor bind to AU-rich RNA targets. AID mRNA expression is induced in splenic B cells that were activated in vitro or by immunizations with sheep red blood cells. In situ hybridization of immunized spleen sections revealed the restricted expression of AID mRNA in developing germinal centers in which modulation of immunoglobulin gene information through somatic hypermutation and class switch recombination takes place. Taken together, these findings suggest that AID is a new member of the RNA-editing deaminase family and may play a role in genetic events in the germinal center B cell.

Apolipoprotein B mRNA editing is preserved in the intestine and liver of zinc-deficient rats.

Apolipoprotein B (apo B) mRNA editing is a site-specific, post transcriptional cytidine deamination reaction that generates apo B48 in the mammalian small intestine and in the liver of certain animals. This reaction is mediated by an enzyme complex that includes the catalytic subunit apobec-1, a zinc-dependent cytidine deaminase. To determine the importance of zinc status to apo B mRNA editing in vivo, we examined the effects of experimentally induced zinc deficiency in rats upon hepatic and serum lipid levels and several indices of apo B gene expression. Rats were either given unlimited access to or were pair-fed a semipurified zinc-supplemented (30 mg Zn/kg) diet or were fed a zinc-deficient diet (approximately 1 mg Zn/kg) for 17 d. Significant differences were detected in the ratio of serum apo B100/B48 in the unlimited access, zinc-supplemented group compared with either zinc-deficient rats or pair-fed controls. There were no alterations in hepatic triglyceride and cholesterol concentrations, hepatic apo B mRNA abundance or apo B mRNA editing in either the small intestine or liver. Taken together, these data suggest that the altered ratios of serum apo B isomorphs seen in zinc deficiency are not mediated through changes in hepatic or intestinal apo B mRNA editing.

K-ras mutations in 1,2-dimethylhydrazine-induced colonic tumors: effects of supplemental dietary calcium and vitamin D deficiency.

Recent studies from our laboratory have demonstrated that dietary supplemental calcium had no significant effect on the incidence of 1,2-dimethylhydrazine-induced colonic tumors, but did decrease the number of rats with multiple tumors and reduced tumor size. Moreover, concomitant vitamin D deficiency appeared to abolish these protective effects of calcium on colonic tumors in this experimental model. To date, however, the mechanism(s) involved in these phenomena remain unclear. In order to address these important issues, 1,2-dimethylhydrazine-induced colonic tumors from animals on control, Ca(2+)-supplemented, vitamin D-sufficient, and Ca(2+)-supplemented, vitamin D-deficient diets were examined for the presence of ras oncogene mutations. DNA was extracted from each of these tumors. Targeted areas of K-ras and H-ras genes were amplified by the polymerase chain reaction and analyzed for point mutations using allele-specific oligonucleotide hybridization and subsequent DNA sequencing. The results of these studies demonstrated that: (a) approximately one-third of 1,2-dimethylhydrazine-induced colonic carcinomas in the control group had K-ras G to A mutations; (b) no mutations, however, were detected in the cancers of the calcium-supplemented group; (c) concomitant vitamin D deficiency abolished the antimutagenic effect of dietary calcium supplementation (e.g., approximately one-third of cancers in this group again had detectable K-ras mutations); and (d) no H-ras point mutations were detected in colonic tumors from any group. These findings suggest that alterations in K-ras mutations may be one possible mechanism by which calcium and vitamin D status influence colonic carcinogenesis in this experimental model.