Ancestral bias in the Hras1 gene and distal Chromosome 7 among inbred mice

Jennifer C. Drew 0 1 2 Andrew S. Kastenmeier 0 1 2 Norman R. Drinkwater 0 1 2 0 Present Address: A. S. Kastenmeier Department of Surgery, University of Utah Health Sciences Center , Salt Lake City, UT 84132, USA 1 Present Address: J. C. Drew Department of Microbiology and Cell Science, University of Florida , Gainesville, FL 32611, USA 2 J. C. Drew A. S. Kastenmeier N. R. Drinkwater (&) McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, 1400 University Avenue , Madison, Wisconsin 53706, USA Inbred strains of mice vary in their frequency of liver tumors initiated by a mutation in the Hras1 (H-ras) proto-oncogene. We sequenced 4.5 kb of the Hras1 gene on distal Chr 7 in a diverse set of 12 commonly used laboratory inbred strains of mice and detected no sequence variation to account for strain-specific differences in Hras1 mutation prevalence. Furthermore, the Hras1 sequence is essentially monoallelic for an ancestral gene derived from the M. m. domesticus species. To determine if the monoallelism and associated low rate of polymorphism are unique to Hras1 or representative of the general chromosomal locale, we extended the sequence analysis to 12 genes in the final 8 Mb of distal Chr 7. A region of at least 2.5 Mb that encompasses Nucleotide sequence data reported here are available in NCBI dbSNP build 127 under accession numbers listed in Supplementary Table 2. - several genes, including Hras1 and the H19/Igf2 loci, demonstrates virtually no sequence variation. The 12 inbred strains share one dominant haplotype derived from the M. m. domesticus allele. Chromosomal regions flanking the monoallelic segment exhibit a significantly higher rate of variation and multiple haplotypes, a majority of which are attributed to M. m. domesticus or M. m. musculus ancestry. Several papers have recently described the first inquiries into the single nucleotide polymorphism (SNP) and haplotype structure of the inbred mouse genome on a global scale (Lindblad-Toh et al. 2000; Pletcher et al. 2004; Tsang et al. 2005; Wade and Daly 2005; Wade et al. 2002; Wiltshire et al. 2003). This research verifies and details a patchwork pattern of variation first posited in 1987, in which pairs of laboratory inbred strains share large blocks derived from one ancestral strain, primarily M. m. domesticus or M. m. musculus (Bonhomme et al. 1987). Within any pairwise inbred strain comparison, there are haplotype blocks defined by a low or high SNP rate, and the average length of these blocks is estimated to be 1.21.4 megabases (Mb) (Frazer et al. 2004; Wade et al. 2002; Zhang et al. 2005). Genome-wide studies of a few inbred strains estimate that the average rate of polymorphism between two such strains is 0.5 SNP/10 kb in the low-SNP-rate blocks and 35 SNP/10 kb in SNP-dense blocks (Frazer et al. 2004). Blocks of limited diversity are attributed to a recent coalescence in which inbred strains inherited the same ancestral allele, while SNP-dense blocks reflect inheritance of divergent ancestral alleles. Laboratory inbred strains of mice vary in their frequency of Hras1 (H-ras) mutational activation during multistage hepatocarcinogenesis (Buchmann et al. 1991; J.C. Drew and N.R. Drinkwater, unpublished). Given its significant role in liver tumorigenesis and its strain-dependent frequency of activation, we tested the hypothesis that strainspecific polymorphisms in the Hras1 gene could account for variable frequencies of Hras1 initiation in liver tumors. We sequenced approximately 4.5 kb of the Hras1 gene from 12 diverse, yet commonly used laboratory inbred mouse strains and two distantly related inbred strains. The set includes representatives from all six families of inbred strains as defined by genome-wide parsimony analysis of simple sequence length polymorphisms (Witmer et al. 2003). In this article we report on the monoallelic inheritance of Hras1 and its neighbors on distal Chr 7 among 12 classic inbred strains. Sequence analysis revealed a region of remarkably low diversity among the strains. No strain-specific SNPs account for differences in frequency of Hras1 activation. Additional sequence analysis of surrounding genes in the final 8 Mb of distal Chr 7 exposed a unique 2.5-Mb block that is essentially devoid of any sequence variation among the 12 classic inbred strains. This block is flanked by regions with significantly greater diversity. Analysis of wild-derived inbred strains representing ancestral genomes of M. m. domesticus and M. m. musculus indicates that the 12 lab inbred strains have fixed alleles from the M. m. domesticus progenitor strain in the 2.5-Mb region on distal Chr 7 that includes the Hras1 gene. Considering the wellestablished role of Hras1 in cell signal transduction and of mutant Hras1 in tumor development, these results have important implications for the study of Hras1 in mouse models of neoplasia and contribute to the burgeoning understanding of laboratory inbred mouse genomes. Materials and methods The C57BL/6J (B6) Hras1 and additional 50 and 30 nucleotide sequence was originally obtained from GenBank (accession No. z50013) and the Trace Archive database (http://www.ncbi.nlm.nih.gov/Traces/trace.cgi). Primer sets for PCR were designed with Primer3 software (http:// www.frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) using the B6 sequence as a template to amplify a set of 400 500 bp of overlapping products. Genomic DNA from spleens was prepared according to a previously published protocol (Bilger et al. 2004) from the following strains housed in our colony: B6, C57BR/cdJ, C3H/HeJ, CBA/J, and SM/J. Genomic DNA from 129X1/SvJ, 129P3/J, A/J, AKR/J, BALB/cByJ, DBA/2J, SWR/J, CAST/EiJ, SPRET/EiJ, WSB/EiJ, and CZECHII/Ei was obtained from The Jackson Laboratory (Bar Harbor, ME). Primers for 11 additional genes on distal Chr 7 were designed from the published B6 sequence (Mouse Genome Consortium 2002; http://www.ensembl.org/mus_musculus). For each of the 11 genes, we sequenced a 300500-bp region. For 8 of 11 genes, the product spanned an intron/exon boundary. All primer sequences and accession numbers are listed in Supplementary Table 1. PCR reactions included 1020 ng of DNA, 62.5 lM dNTPs (Amersham, Piscataway, NJ), 15 pmol of forward and reverse primers (Integrated DNA Technologies, Coralville, IA), 0.3 U Taq DNA polymerase, and 1 PCR buffer (Roche, Indianapolis, IN) in a total reaction volume of 20 ll. The reactions were incubated in thermocyclers at 95 C for 3 min, followed by 40 cycles at 94 C for 15 sec, 60 C for 25 sec, 72 C for 90 sec, and a final step at 72 C for 7 min. Minor modifications to the standard protocol such as optimization of annealing temperatures and initial incubations at 95 C for 15 min with HotStar DNA Polymerase (Qiagen, Valencia, CA) were required for a small number of reactions. All PCR products were purified with a Qiagen PCR Purification Kit according to the recommended protocol, except fo (...truncated)