| First Author | Chapman HD | Year | 1994 |
| Journal | Mouse Genome | Volume | 92 |
| Issue | 4 | Pages | 677-682 |
| Mgi Jnum | J:22098 | Mgi Id | MGI:69989 |
| Citation | Chapman HD (1994) Polymorphisms in the dehydroepiandrosterone (DHEA) sulfotransferase (Std) gene on chromosome 7 distinguish the C57BL/6J and C57BL/6ByJ substrains. Mouse Genome 92(4):677-682 |
| abstractText | Full text of Mouse Genome contribution: Polymorphisms in the Dehydroepiandrosterone (DHEA) Sulfotransferase (Std) gene on Chromosome 7 distinguish the C57BL/6J and C57BL/6ByJ substrains. Harold D. Chapman and Edward H. Leiter, The Jackson Laboratory, Bar Harbor, ME 04609, USA INTRODUCTION Bailey has discussed the problem of substrain divergence which inevitably results as spontaneous mutations become differentially fixed (1). Although a variety of these polymorphisms distinguishing the various BALB/c substrains have been reported (2), few polymorphisms distinguishing the C57BL/6 substrains are known. We have discovered a mutation that occurred recently in the C57BL/6J (B6J) stock distributed by the Animal Resources Unit of The Jackson Laboratory. This mutation distinguishes the genome of current B6J mice from the closely related C57BL/6ByJ (B6ByJ) substrain in the Animal Resources Unit, as well as from B6J congenic stocks separated from the Animal Resources colony before 1970. This polymorphism was detected in the course of mapping the gene encoding a mouse 3Beta-hydroxysteroid sulfotransferase (Std, DHEA preferring) 2.2cm distal to the centromere on Chr 7 (3). METHODS AND RESULTS Mice. B6J and B6ByJ female mice between 6 to 8 weeks of age were provided by the Animal Resources Unit of The Jackson Laboratory or from the research colony of Dr. L. Mobraaten. B6J-+/ob females were also provided by the Animal Resources Unit. B6-m+/+db (N5F48) and B6.C-H2(bm1)/ByJ (F93) were produced in research colonies at The Jackson Laboratory. Southern blot analysis. High molecular weight DNAs were prepared from kidney tissue, or, in the case of the BXD RI lines, spleen DNAs were obtained from the institution's Mouse DNA Resource. SacI -digested fragments were electrophoretically separated in a 0.7% agarose gel. Blots were hybridized using a 1065 bp rat 3Beta-hydroxysteroid sulfotransferase cDNA probe (designated 2-4) described previously (3). A complex fragment pattern was observed, with at least 8 restriction fragments (2 - 20 kb in size) resolved for each DNA sample. DNA from B6J mice from the Animal Resources Unit was clearly distinguished from that of B6ByJ mice by the presence in the former of a ~4.9 kb fragment (Table I). Analysis of various B6/J stocks separated from the pure inbred B6J line for over 20 years provided insight as to when the mutation in B6J mice occurred. The B6 stock congenic for the ob (obese, Chr 6) mutation is maintained by mating current generation B6J-+/+ males to pseudopregnant females carrying transplanted ovaries from B6J-ob/ob donors. As expected, the 4.9 kb SacI fragment is present in DNA of +/ob lean mice produced in this fashion. In contrast, the B6 stock congenic for the db mutation lacks the 4.9 kb SacI fragment. This B6 congenic (N5) stock, has been separated from the standard B6J strain in Animal Resources since 1968-70. The B6 congenic (N10) stock carrying the H2bm1 mutation (Chr 17) was produced by Dr. Donald Bailey by transfer of the mutation from BALB/cBy onto B6J in the early 1970's. This congenic stock also lacks the 4.9 kb SacI fragment present in current B6J DNA. Finally, none of the 26 recombinant inbred (Rl) lines initiated by Dr. Benjamin Taylor in the early 1970Õs between B6J and DBA/2J exhibit the 4.6 kb fragment. From these data, we infer that B6ByJ mice (received in the Animal Resources Unit in 1977 at F111) carry the ancestral form of the gene, with the mutation in the Std region of B6J mice occurring after 1970. Northern blot analysis. Total RNA was extracted from fresh livers from similarly aged female mice (4). Total RNA (10 ug) was electrophoretically separated in a 1.25% agarose gel. The blotting, hvbridization, and wash conditions were as described previously (2). Hybridization with the rat 2-4 probe revealed a full length 1.1 kb Std mRNA in both the B6J and B6ByJ substrains. However, an additional ~0.7 kb unique RNA transcript (Fig. 1) was observed in B6J hepatic RNA that was not present in the other B6 substrains studied. Table 1. SacI Fragment Length Polymorphism Strain: C57BL/6J; 4.9 kb: +; Strain: C57BL/6J-ob stock; 4.9 kb: +; Strain: C57BL/6ByJ; 4.9 kb: -; Strain: C57BL/6-m+/+db; 4.9 kb: -; Strain: C57BL/6J-H2(bm1); 4.9 kb: -; Strain: BXD RI lines (all 26); 4.9 kb: -. Fig. 2 (Legend) Cytosolic DHEA ST assay. The Std gene is expressed in liver of mice of both sexes prior to puberty. After puberty, gene transcription and translation is limited to females. To determine whether the B6J-unique changes at the DNA and RNA levels affected DHEA sulfotransferase activity, enzyme specific activity in liver was assayed at either sub-optimal (0.2uM) or at saturating (10uM) substrate concentrations (2). Groups of 6 B6J and B6ByJ females (8-wk-old) were used, (Fig. 2). At both limiting and saturating substrate concentrations, a higher DHEA sulfotransferase specific activity was measured in the B6J preparations. Discussion The B6By stock was separated from the B6J line in the early 1960's and received by the Animal Resources Unit at F111 in 1977. We presume that the mutation in the Std region occurred sometime between the early 1970's and the present, since the DNA polymorphism distinguishing current B6J from B6ByJ is not carried by B6J progenitors used to produce the B6-rn+/+db congenic stock or the BXD recombinant inbred lines. The only other polymorphism distinguishing B6J and B6ByJ listed in The Jackson Laboratory database is Xmmv64, an unmapped xenotropic (MCF) proviral genome absent in B6ByJ and present in B6J. Distinct hydroxysteroid sulfotransferase cDNAs have been described in rats (5). In mice, the Std region may contain a ST gene family, since a comparison of Pvu II-endonuclease digested DNA from B6J versus SPRET/Ei hybridized with probe 2-4 revealed at least 16 restriction fragment length variants. These species-specific fragments segregated together, with no recombinations observed by Southern blot analysis of 94 Pvu II DNAs from a (B6 x SPRET)FI x SPRET backcross panel obtained from the Gene Mapping Resource of The Jackson Laboratory. We cannot resolve whether the mutation is a consequence of a partial gene duplication, an insertion event affecting alternative mRNA splicing, or a point mutation. The additional B6J-specific 0.7kb mRNA cannot encode a full-length sulfotransferase enzyme. The finding of a higher specific activity of the enzyme in liver of B6J versus B6ByJ mice may denote a longer messenger half-life, which might account for our ability to detect the truncated message in B6J, but not B6ByJ preparations. REFERENCES 1. Bailey, D.W. 1982. How pure are inbred strains of mice. Immunol. Today. 3: 210-214. 2. Roderick, T.H., S.H. Langley, and E.H. Leiter. 1985. Some unusual genetic characteristics of BALB/c and evidence for genetic variation among BALB/c substrains. Current Top. Microbiol. Immunol. 122: 9-18. 3. Leiter, E.H., and H.D. Chapman. 1994. Obesity-Induced Diabetes (Diabesity) in C57BL/KsJ Mice Produces Aberrant trans-Regulation of Sex Steroid Sulfotransferase Genes. J. Clin. Invest. 93: 2007-2013. 4. Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:152-159. 5. Falany, C.N. 1991. Molecular enzymology of human liver cytosolic sulfotransferases. Trends in Pharmacol. Sci. 12: 255-259. |
