Patterns of DNA Variability at X-Linked Loci in Mus Domesticus

Introns of four X-linked genes (Hprt, Plp, Glra2, and Amg) were sequenced to provide an estimate of nucleotide diversity at nuclear genes within the house mouse and to test the neutral prediction that the ratio of intraspecific polymorphism to interspecific divergence is the same for different loci. Hprt and Plp lie in a region of the X chromosome that experiences relatively low recombination rates, while Glra2 and Amg lie near the telomere of the X chromosome, a region that experiences higher recombination rates. A total of 6022 bases were sequenced in each of 10 Mus domesticus and one M. caroli. Average nucleotide diversity (π) for introns within M. domesticus was quite low (π = 0.078%). However, there was substantial variation in the level of heterozygosity among loci. The two telomeric loci, Glra2 and Amg, had higher ratios of polymorphism to divergence than the two loci experiencing lower recombination rates. These results are consistent with the hypothesis that heterozygosity is reduced in regions with lower rates of recombination, although sampling of additional genes is needed to establish whether there is a general correlation between heterozygosity and recombination rate as in Drosophila melanogaster.     

Y chromosome variation of mice and men

DNA sequences from the nonrecombining portion of the Y chromosome were compared with autosomal and X-linked sequences from mice and humans to test the neutral prediction that ratios of polymorphism to divergence are the same for different genes. Intraspecific variation within Mus domesticus was compared with divergence between M. domesticus and Mus caroli for Sry, a region 5' to Sry, and four X-linked genes, Hprt, Plp, Amg, and Glra2. None of these comparisons revealed significantly reduced variation on the Y chromosome. Intraspecific variation within humans was compared with divergence between humans and chimpanzees for three Y-linked loci (Zfy, the YAP region, and the Sry region), seven X- linked loci (Il2rg, Plp, Hprt, Gk, Ids, Pdhal, and Dmd), and the beta- globin locus on chromosome 11. In these comparisons, the observed level of variation on the human Y chromosome was slightly lower than expected, but was significantly lower in only one case (Sry region vs. Dmd). These results suggest that the levels of variability on the Y chromosome in mice and humans are close to expected values given the effective population size and mutation rates for these loci. There is at most only a modest reduction in variability that may be attributed to natural selection (either genetic hitchhiking or background selection).      

Tempo and mode of concerted evolution in the L1 repeat family of mice

A 300-bp DNA sequence has been determined for 30 (10 from each of three species of mice) random isolates of a subset of the long interspersed repeat family L1. From these data we conclude that members of the L1 family are evolving in concert at the DNA sequence level in Mus domesticus, Mus caroli, and Mus platythrix. The mechanism responsible for this phenomenon may be either duplicative transposition, gene conversion, or a combination of the two. The amount of intraspecies divergence averages 4.4%, although between species base substitutions accumulate at the rate of approximately 0.85%/Myr to a maximum divergence of 9.1% between M. platythrix and both M. domesticus and M. caroli. Parsimony analysis reveals that the M. platythrix L1 family has evolved into a distinct clade in the 10-12 Myr since M. platythrix last shared a common ancestor with M. domesticus and M. caroli. The parsimony tree also provides a means to derive the average half-life of L1 sequences in the genome. The rates of gain and loss of individual copies of L1 were estimated to be approximately equal, such that approximately one-half of them turn over every 3.3 Myr.      

Higher differentiation among subspecies of the house mouse (Mus musculus) in genomic regions with low recombination

In the early stages of reproductive isolation, genomic regions of reduced recombination are expected to show greater levels of differentiation, either because gene flow between species is reduced in these regions or because the effects of selection at linked sites within species are enhanced in these regions. Here, we study the patterns of DNA sequence variation at 27 autosomal loci among populations of Mus musculus musculus, M. m. domesticus, and M. m. castaneus, three subspecies of house mice with collinear genomes. We found that some loci exhibit considerable shared variation among subspecies, while others exhibit fixed differences. We used an isolation-with-gene-flow model to estimate divergence times and effective population sizes (N(e) ) and to disentangle ancestral variation from gene flow. Estimates of divergence time indicate that all three subspecies diverged from one another within a very short period of time approximately 350,000 years ago. Overall, N(e) for each subspecies was associated with the degree of genetic differentiation: M. m. musculus had the smallest N(e) and the greatest proportion of monophyletic gene genealogies, while M. m. castaneus had the largest N(e) and the smallest proportion of monophyletic gene genealogies. M. m. domesticus and M. m. musculus were more differentiated from each other than either were from M. m. castaneus, consistent with greater reproductive isolation between M. m. domesticus and M. m. musculus. F(ST) was significantly greater at loci experiencing low recombination rates compared to loci experiencing high recombination rates in comparisons between M. m. castaneus and M. m. musculus or M. m. domesticus. These results provide evidence that genomic regions with less recombination show greater differentiation, even in the absence of chromosomal rearrangements.     

Functional respiratory chain analyses in murid xenomitochondrial cybrids expose coevolutionary constraints of cytochrome b and nuclear subunits of complex III

The large number of extant Muridae species provides the opportunity of investigating functional limits of nuclear/mitochondrial respiratory chain (RC) subunit interactions by introducing mitochondrial genomes from progressively more divergent species into Mus musculus domesticus mtDNA-less (rho0) cells. We created a panel of such xenomitochondrial cybrids, using as mitochondrial donors cells from six murid species with divergence from M. m. domesticus estimated at 2 to 12 Myr before present. Species used were Mus spretus, Mus caroli, Mus dunni, Mus pahari, Otomys irroratus, and Rattus norvegicus. Parsimony analysis of partial mtDNA sequences showed agreement with previous molecular phylogenies, with the exception that Otomys did not nest within the murinae as suggested by some recent nuclear gene analyses. Cellular production of lactate, a sensitive indicator of decreased respiratory chain ATP production, correlated with divergence. Functional characterization of the chimeric RC complexes in isolated mitochondria using enzymological analyses demonstrated varying decreases in activities of complexes I, III, and IV, which have subunits encoded in both mitochondrial and nuclear genomes. Complex III showed a striking decline in electron transfer function in the most divergent xenocybrids, being greatly reduced in the Rattus xenocybrid and virtually absent in the Otomys xenocybrid. This suggests that nuclear subunits interacting with cytochrome b face the greatest constraints in the coevolution of murid RC subunits. We sequenced the cytochrome b gene from the species used to identify potential amino acid substitutions involved in such interactions. The greater sensitivity of complex III to xenocybrid dysfunction may result from the encoding of redox center apoproteins in both nuclear and mitochondrial genomes, a unique feature of this RC complex.     

The complex history of a gene proposed to participate in a sexual isolation mechanism in house mice

Previous behavioral experiments showed that mouse salivary androgen-binding protein (ABP) was involved in interindividual recognition and might play a role in sexual isolation between house mouse (Mus musculus) subspecies. The pattern of evolution of Abpa, the gene for the alpha subunit of ABP, was found to be consistent with this hypothesis. Abpa apparently diverged rapidly between species and subspecies with a large excess of nonsynonymous substitutions, a lack of exon polymorphism within each of the three subspecies, and a lack of intron polymorphism in the one subspecies studied (M. musculus domesticus). Here we characterized the intron and exon sequence variations of this gene in house mouse populations from central Eurasia, a region yet unsampled and thought to be close to the cradle of the radiation of the subspecies. We also determined the intron and exon sequences in seven other species of the genus Mus. We confirmed the general pattern of rapid evolution by essentially nonsynonymous substitutions, both inter- and intraspecifically, supporting the idea that Darwinian selection has driven the evolution of this gene. We also observed a uniform intron sequence in five samples of M. musculus musculus, suggesting that a selective sweep might have occurred for that allele. In contrast to previous results, however, we found extensive intron and exon polymorphism in some house mouse populations from central Eurasia. We also found evidence for secondary admixture of the subspecies-specific alleles in regions of transition between the subspecies in central Eurasia. Furthermore, an abnormal intron phylogeny suggested that interspecific exchanges had occurred between the house mouse subspecies and three other Palearctic species. These observations appear to be at variance with the simple hypothesis that Abpa is involved in reproductive isolation. Although we do not rule out a role in recognition, the situation appears to be more complex than previously thought. Thus the selective mechanism behind the evolution of Abpa remains to be resolved, and we suggest that it may have changed during the recent colonization history of the house mouse.     

Testing for selection on the androgen-binding protein in the Danish mouse hybrid zone

The three peripheral subspecies of the house mouse Mus musculus have fixed specific variants of the androgen-binding protein (ABP) that have been proposed to be part of a recognition mechanism that could participate in sexual isolation between the subspecies. We tested for selection on Abpa by characterizing the pattern of Abpa introgression across a transect of the hybrid zone between M. m. musculus and M. m. domesticus in Jutland. On the musculus side, the cline for Abpa resembled that of a nearly neutral allozyme more than that of strongly selected X and Y chromosome markers. However, the high central step which displaces the tail of introgression of Abpa to higher frequencies was best accounted for by linkage to a locus under strong selection against hybrids. Still, we cannot exclude that this pattern results from weak selection against Abpa in the tail of introgression, which would be compatible with an assortative choice mechanism. On the domesticus side there was little introgression close to the hybrid zone, presumably due to a geographical barrier to migration. However, substantial frequencies of musculus alleles occurred further away, suggesting mixed colonization patterns as well as flow across the hybrid zone.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 84 , 447–459.     

Isolation and Characterization of a Mouse Y Chromosomal Repetitive Sequence

The Y chromosome plays a dominant role in mammalian sex determination, and characterization of this chromosome is essential to understand the mechanism responsible for testicular differentiation. Male mouse genomic DNA fragments, cloned into pBR322, were screened for the presence of Bkm (a female snake satellite DNA)-related sequences, and we obtained a clone (AC11) having a DNA fragment from the mouse Y chromosome. In addition to a Bkm-related sequence, this fragment contained a Y chromosomal repetitive sequence. DNA isolated from the XX sex-reversed male genome produced a hybridization pattern indistinguishable to that obtained with normal female DNA, suggesting that the AC11 sequence is not contained within the Y chromosomal DNA present in the sex-reversed male genome. Based on the hybridization patterns against mouse Y chromosomal DNA, AC11 classified 16 inbred laboratory strains into two categories; those with the Mus musculus musculus type Y chromosome and those with the M.m. domesticus type Y chromosome. Three European subspecies of Mus musculus (M.m. brevirostris, M.m. poschiavinus and M.m. praetextus) possessed the M.m. domesticus type Y chromosome, whereas the Japanese mouse, M.m. molossinus, had the M.m. musculus type Y chromosome. The survey was also extended to six other species that belong to the genus Mus, of which M. spretus and M. hortulamus showed significant amounts of AC11-related sequences in their Y chromosomes. The male-specific accumulation of AC11-related sequences was not found in M. caroli, M. cookii, M. pahari or M. platythrix. This marked difference among Mus species indicates that the amplification of AC11-related sequences in the mouse Y chromosome was a recent evolutionary event.     

Mitochondrial DNA Sequence Variation in the Eastern House Mouse, Mus Musculus: Comparison with Other House Mice and Report of a 75-Bp Tandem Repeat

The control region and flanking tRNAs were sequenced from 139 Mus musculus mitochondrial DNAs (mtDNAs) from mice collected at 44 localities extending from Germany to Japan. Among the 36 types of M. musculus mtDNA resolved, five have an added 75-bp direct repeat; the two copies within an individual differ by two to four base substitutions. Among 90 M. domesticus mtDNAs sequenced, 12 new types were found; 96 M. domesticus types have now been identified by sequencing this segment. Representative mtDNAs from M. castaneus, M. macedonicus, M. spicilegus and M. spretus were also sequenced. A parsimony tree for the M. musculus mtDNAs is about half as deep as the tree for the M. domesticus mtDNAs, which is consistent with the idea that M. musculus is genetically less diverse and younger than M. domesticus. The patterns of variation as a function of position are similar but not identical in M. musculus and M. domesticus mtDNAs. M. castaneus and M. musculus mtDNAs are allied, at a tree depth about three times as great as the start of intra-M. musculus divergence. The coalescence of the M. musculus and M. castaneus mtDNAs is about half as deep as their coalescence with the M. domesticus mtDNA lineages. The mtDNAs of the aboriginal M. macedonicus and M. spicilegus are each other's closest relatives, at a tree depth greater than the deepest intracommensal node. The mtDNA results support the view that the aboriginal M. spretus is the sister group of the other five species.     

Phylogenetic relationships in the genus mus,based on paternally,maternally, and biparentally inherited characters

Several species in the rodent genus Mus are used as model research organisms, but comparative studies of these mice have been hampered by the lack of a well-supported phylogeny. We used DNA sequences from six genes representing paternally, maternally, and biparentally inherited regions of the genome to infer phylogenetic relationships among 10 species of Mus commonly used in laboratory research. Our sample included seven species from the subgenus Mus; one species each from the subgenera Pyromys, Coelomys, and Nannomys; and representatives from three additional murine genera, which served as outgroups in the phylogenetic analyses. Although each of the six genes yielded a unique phylogeny, several clades were supported by four or more gene trees. Nodes that conflicted between trees were generally characterized by weak support for one or both of the alternative topologies, thus providing no compelling evidence that any individual gene, or part of the genome, was misleading with respect to the evolutionary history of these mice. Analysis of the combined data resulted in a fully resolved tree that strongly supports monophyly of the genus Mus, monophyly of the subgenus Mus, division of the subgenus Mus into Palearctic (M. musculus, M. macedonicus, M. spicilegus, and M. spretus) and Asian (M. cervicolor, M. cookii, and M. caroli) clades, monophyly of the house mice (M. m. musculus, "M. m. molossinus," M. m. castaneus, and M. m. domesticus), and a sister-group relationship between M. macedonicus and M. spicilegus. Other clades that were strongly supported by one or more gene partitions were not strongly supported by the combined data. This appears to reflect a localized homoplasy in one partition obscuring the phylogenetic signal from another, rather than differences in gene or genome histories.     

Adaptive introgression of anticoagulant rodent poison resistance by hybridization between old world mice

Polymorphisms in the vitamin K 2,3-epoxide reductase subcomponent 1 (vkorc1) of house mice (Mus musculus domesticus) can cause resistance to anticoagulant rodenticides such as warfarin [1-3]. Here we show that resistant house mice can also originate from selection on vkorc1 polymorphisms acquired from the Algerian mouse (M. spretus) through introgressive hybridization. We report on a polymorphic introgressed genomic region in European M. m. domesticus that stems from M. spretus, spans >10 Mb on chromosome 7, and includes the molecular target of anticoagulants vkorc1 [1-4]. We show that in the laboratory, the homozygous complete vkorc1 allele of M. spretus confers resistance when introgressed into M. m. domesticus. Consistent with selection on the introgressed allele after the introduction of rodenticides in the 1950s, we found signatures of selection in patterns of variation in M. m. domesticus. Furthermore, we detected adaptive protein evolution of vkorc1 in M. spretus (Ka/Ks = 1.54-1.93) resulting in radical amino acid substitutions that apparently cause anticoagulant tolerance in M. spretus as a pleiotropic effect. Thus, positive selection produced an adaptive, divergent, and pleiotropic vkorc1 allele in the donor species, M. spretus, which crossed a species barrier and produced an adaptive polymorphic trait in the recipient species, M. m. domesticus.     

Asymmetrical reproductive character displacement in the house mouse

Our study addressed reproductive character displacement between two subspecies of the house mouse, Mus musculus musculus and Mus musculus domesticus that hybridize in Europe along a zone where selection against hybridization is known to occur. Based on a multi-population approach, we investigated spatial patterns of divergence of mate preference in the two taxa. Mate preference was significantly higher in the contact zone than in allopatry in both subspecies, suggesting that reproductive character displacement occurs. Moreover, patterns of preference were stronger in M. m. musculus than in M. m. domesticus, indicating an asymmetrical divergence between the two. In the context of selection against hybridization, our results may provide empirical support for the hypothesis of reinforcement in a parapatric hybrid zone. We discuss factors that could explain the asymmetrical pattern of divergence and the possible impact of a unimodal structure on the maintenance of premating divergence between the two subspecies.     

Molecular cloning and long terminal repeat sequences of intracisternal A-particle genes in Mus caroli

We isolated DNA clones of intracisternal A-particle (IAP) genes from the genome of an Asian wild mouse, Mus caroli. A typical M. caroli IAP gene was 6.5 kilobase pairs in length and had long terminal repeat (LTR) sequences at both ends. The size of the LTR was 345 base pairs in clone L20, and two LTRs at both ends of this clone were linked to directly repeating cellular sequences of 6 base pairs. Each LTR possessed most of the structural features commonly associated with the retrovirus LTR. The restriction map of the M. caroli IAP gene resembled that of Mus musculus, although the M. caroli IAP gene was 0.4 kilobase pairs shorter than the M. musculus IAP gene in two regions. Sequence homology between the M. caroli and M. musculus IAP LTRs was calculated as about 80%, whereas the LTR sequence of the Syrian hamster IAP gene was about 60% homologous to the M. caroli LTR. The reiteration frequency of the M. caroli IAP genes was estimated as 200 to 400 copies per haploid genome, which is at least 10 times the reported value. These results suggest that the IAP genes observed in the genus Mus are present in multiple copies with structures closely resembling the integrated retrovirus gene.     

A TEST OF THE NEUTRAL MODEL OF EXPRESSION CHANGE IN NATURAL POPULATIONS OF HOUSE MOUSE SUBSPECIES

Changes in expression of genes are thought to contribute significantly to evolutionary divergence. To study the relative role of selection and neutrality in shaping expression changes, we analyzed 24 genes in three different tissues of the house mouse ( Mus musculus ). Samples from two natural populations of the subspecies M. m. domesticus and M. m. musculus were investigated using quantitative PCR assays and sequencing of the upstream region. We have developed an approach to quantify expression polymorphism within such populations and to disentangle technical from biological variation in the data. We found a correlation between expression polymorphism within populations and divergence between populations. Furthermore, we found a correlation between expression polymorphism and sequence polymorphism of the respective genes. These data are most easily interpreted within a framework of a predominantly neutral model of gene expression change, where only a fraction of the changes may have been driven by positive selection. Although most genes investigated were expressed in all three tissues analyzed, significant changes of expression levels occurred predominantly in a single tissue only. This adds to the notion that enhancer-specific effects or transregulatory effects can modulate the evolution of gene expression in a tissue-specific way.     

Insulin secretion and IP levels in two distant lineages of the genus Mus: comparisons with rat islets

Islet responses of two different Mus geni, the laboratory mouse (Mus musculus) and a phylogenetically more ancient species (Mus caroli), were measured and compared with the responses of islets from rats (Rattus norvegicus). A minimal and flat second-phase response to 20 mM glucose was evoked from M. musculus islets, whereas a large rising second-phase response characterized rat islets. M. caroli responses were intermediate between these two extremes; a modest rising second-phase response to 20 mM glucose was observed. Prior, brief stimulation of rat islets with 20 mM glucose results in an amplified insulin secretory response to a subsequent 20 mM glucose challenge. No such potentiation or priming was observed from M. musculus islets. In contrast, M. caroli islets displayed a modest twofold potentiated first-phase response upon subsequent restimulation with 20 mM glucose. Inositol phosphate (IP) accumulation in response to 20 mM glucose stimulation in [(3)H]inositol-prelabeled rat or mouse islets paralleled the insulin secretory responses. The divergence in 20 mM glucose-induced insulin release between these species may be attributable to differences in phospholipase C-mediated IP accumulation in islets.     

Polymorphism of C lambda genes and units of duplication in the genus Mus

The number of Ig C lambda genes in nine geographically widespread species from the four subgenera in the genus Mus was estimated from the number of Bam HI and Eco RI restriction fragments that hybridize under high stringency conditions to cDNA probes of BALB/c inbred mouse origin (Mus musculus domesticus). Three closely related species in the subgenus Mus, M. musculus, M. spretus, and M. spicelegus, show considerable variation in the number of C lambda genes. Estimates of gene numbers in these animals range from two C lambda genes in M. spretus from Puerto Real, Spain to 12 C lambda genes in M. musculus musculus from Studenec, Czechoslovakia. Strains of mice carrying either six or 10 C lambda genes were derived from a single population of M. musculus domesticus from Centreville, MD. The hybridization patterns of mice exhibiting C lambda gene amplification indicate that duplications are of relatively recent origin and probably occurred by reiteration of a DNA segment closely related to the 6.5 kb [C lambda 3 - C lambda 1] unit found in BALB/c inbred mice. Three more distantly related species in the subgenus Mus, and a species representing the Nannomys subgenus all appear to carry only four C lambda genes. DNA of species representing the Coelomys and Pyromys subgenera hybridized weakly to the C lambda cDNA probes, but these animals also have no more than four C lambda genes. Thus, there may be a base number of four C lambda genes in most species in the genus Mus. All inbred strains of mice so far examined also have only four C lambda genes, but no feral M. musculus examined have fewer than six C lambda genes. One explanation of the discrepancy in the number of genes between inbred and feral M. musculus is that C lambda genes were deleted during the process of inbreeding.     

A pronounced evolutionary shift of the pseudoautosomal region boundary in house mice

The pseudoautosomal region (PAR) is essential for the accurate pairing and segregation of the X and Y chromosomes during meiosis. Despite its functional significance, the PAR shows substantial evolutionary divergence in structure and sequence between mammalian species. An instructive example of PAR evolution is the house mouse Mus musculus domesticus (represented by the C57BL/6J strain), which has the smallest PAR among those that have been mapped. In C57BL/6J, the PAR boundary is located just ~700 kb from the distal end of the X chromosome, whereas the boundary is found at a more proximal position in Mus spretus, a species that diverged from house mice 2-4 million years ago. In this study we used a combination of genetic and physical mapping to document a pronounced shift in the PAR boundary in a second house mouse subspecies, Mus musculus castaneus (represented by the CAST/EiJ strain), ~430 kb proximal of the M. m. domesticus boundary. We demonstrate molecular evolutionary consequences of this shift, including a marked lineage-specific increase in sequence divergence within Mid1, a gene that resides entirely within the M. m. castaneus PAR but straddles the boundary in other subspecies. Our results extend observations of structural divergence in the PAR to closely related subspecies, pointing to major evolutionary changes in this functionally important genomic region over a short time period.     

Specificity of androgen resistance inMus caroli kidney

Androgen controls the expression of beta-glucuronidase and several other proteins in the kidney of the standard laboratory mouse, Mus musculus. Other species within the genus Mus exhibit a variety of response patterns for kidney beta-glucuronidase and other markers of androgen action. We have investigated the mechanism of androgen action in M. caroli, a Mus species that does not produce beta-glucuronidase in response to testosterone. The failure of testosterone to induce beta-glucuronidase in M. caroli females cannot be overcome by treatment with dihydrotestosterone, with pharmacological doses of testosterone propionate or dihydrotestosterone propionate, or with a variety of potent androgen analogues. All of these compounds induce kidney beta-glucuronidase in M. musculus females and kidney ornithine decarboxylase, submandibular gland renin, and submandibular gland epidermal growth factor in both M. caroli and M. musculus females. Furthermore, kidney androgen receptor proteins from M. caroli and M. musculus animals have the same sedimentation characteristics on sucrose density gradients. These data indicate that androgen resistance in M. caroli is not due to deficient 5 alpha-reductase or aberrant hormone metabolism producing suboptimal levels of functional androgen and is not caused by a defective androgen receptor. They suggest that the resistance of beta-glucuronidase in M. caroli kidney to induction by androgen occurs at the level of the beta-glucuronidase gene.