Faculty Bibliography

Plasma non-HDL and HDL cholesterol levels are predictors of cardiovascular diseases. We carried out a genetic cross between two laboratory inbred mouse strains, C57BL/6J and CASA/Rk, to detect loci that control the plasma levels of non-HDL and HDL cholesterol. With regard to non-HDL cholesterol, chow-fed CASA/Rk males and females had 87% and 25% higher levels, respectively, than did C57BL/6Js. The levels of non-HDL cholesterol in F1s were similar to C57BL/6J. There was no strain difference in HDL cholesterol levels. An intercross between F1s was performed, and plasma non-HDL and HDL cholesterol was measured in 185 male and 184 female mice. In both male and female F2 mice, plasma non-HDL and HDL cholesterol levels were unimodally distributed; however, in both cases the values for females were significantly lower than for males. Therefore, linkage analysis was performed with sex as a covariate. Significant linkage for non-HDL cholesterol was found on chromosome 6 at 49 cM (LOD 5.17), chromosome 4 at 55 cM (LOD 4.22), and chromosome 8 at 7 cM (LOD 3.68). Significant linkage for HDL cholesterol was found on chromosome 9 at 14 cM (LOD 7.52) and chromosome 8 at 76 cM (LOD 4.69). A significant epistatic interaction involving loci on chromosomes 2 and 5 was also observed for non-HDL cholesterol. In summary, linkage analysis in these cross-identified novel loci confirmed previously identified loci in control of plasma non-HDL and HDL cholesterol and disclosed a novel interaction in controlling non-HDL cholesterol levels in the mouse.

A problem associated with automated analysis of fluorescently labeled fragments separated by slab gel or capillary electrophoresis is the doublet peak formed when Taq DNA Polymerase adds a nontemplated nucleotide (generally an adenosine) to the 3' end of the product.This nontemplated addition (plus A) is primarily dependent on the 5' sequence of the reverse primer and, to a lesser extent, polymerase chain reaction (PCR) conditions. Primers may amplify the true product, the plus A product or a doublet product comprised of both. When using markers based on dinucleotide repeats, this single base pair difference can make binning and accurate automated analysis problematic. To drive the PCR reaction consistently to the plus A product, the sequence of the nonfluorescent primer used in amplification can be modified by adding a 5' tail favoring the nontemplated addition. The present study, conducted by the Fragment Analysis Research Group (FARG) of the Association of Biomolecular Resource Facilities, provided researchers with an opportunity to compare normal products amplified with a dinucleotide marker to products amplified with the same primer to which a 5' tail designed to promote the plus A product had been added. The study also included a sample amplified with a tetranucleotide repeat marker for comparison. The results from this study were returned to the FARG for comprehensive analysis and are reported here.