Polymorphism and divergence at the 5' flanking region of the sex- determining locus, Sry, in mice

We have investigated patterns of evolution in the nonrecombining portion of the Y chromosome in mice by comparing levels of polymorphism within Mus domesticus with levels of divergence between M. domesticus and M. spretus. A 1,277-bp fragment of noncoding sequence flanking the sex determining locus (Sry) was PCR amplified, and 1,063 bases were sequenced and compared among 20 M. domesticus and 1 M. spretus. Two polymorphic base substitutions and two polymorphic insertion/deletion sites were identified within M. domesticus; nucleotide diversity was estimated to be 0.1%. Divergence between M. domesticus and M. spretus for this region (1.9%) was slightly lower than the average divergence of single-copy nuclear DNA for these species. Comparison of levels of polymorphism and divergence at Sry with levels of polymorphism and divergence in the mitochondrial DNA control region provided no evidence of a departure from the expectations of neutral molecular evolution. These findings are consistent with the presumed lack of function for much of the Y chromosome.      

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.     

LINE-1 repetitive DNA probes for species-specific cloning from Mus spretus and Mus domesticus genomes.

Mus domesticus and Mus spretus mice are closely related subspecies. For genetic investigations involving hybrid mice, we have developed a set of species-specific oligonucleotide probes based on the detection of LINE-1 sequence differences. LINE-1 is a repetitive DNA family whose many members are interspersed among the genes. In this study, library screening experiments were used to fully characterize the species specificity of four M. domesticus LINE-1 probes and three M. spretus LINE-1 probes. It was found that the nucleotide differences detected by the probes define large, species-specific subfamilies. We show that collaborative use of such probes can be employed to selectively detect thousands of species-specific library clones. Consequently, these probes could be exploited to monitor and access almost any given species-specific region of interest within hybrid genomes.     

In vivo interaction between mitochondria carrying mtDNAs from different mouse species

Mitochondrial disease model mice, mitomice, were created using zygotes of B6mtspr strain mice carrying mitochondrial DNA (mtDNA) from Mus spretus as recipients of exogenous mitochondria carrying wild-type and a deletion mutant mtDNA (DeltamtDNA) of M. musculus domesticus. In these experiments, mtDNAs from different mouse species were used for identification of exo- and endogenous wild-type mtDNAs in the mitomice. Results showed transmission of exogenous DeltamtDNA, but not exogenous wild-type mtDNA, of M. m. domesticus to following generations through the female germ line. Complete elimination of exogenous wild-type mtDNA would be due to stochastic segregation, whereas transmission of exogenous DeltamtDNA would be due to its smaller size leading to a propagational advantage. Tissues in mitomice of the F3 generation carrying exogenous DeltamtDNA showed protection from respiration defects until DeltamtDNA accumulated predominantly. This protection from expression of mitochondrial dysfunction was attained with the help of endogenous wild-type mtDNA of M. spretus, since mitomice did not possess exogenous wild-type mtDNA of M. m. domesticus. These observations provide unambiguous evidence for the presence of interaction between exogenous mitochondria carrying DeltamtDNA and endogenous mitochondria carrying M. spretus wild-type mtDNA.     

Selective and continuous elimination of mitochondria microinjected into mouse eggs from spermatids, but not from liver cells, occurs throughout embryogenesis

Exclusion of paternal mitochondria in fertilized mammalian eggs is very stringent and ensures strictly maternal mtDNA inheritance. In this study, to examine whether elimination was specific to sperm mitochondria, we microinjected spermatid or liver mitochondria into mouse embryos. Congenic B6-mt(spr) strain mice, which are different from C57BL/6J (B6) strain mice (Mus musculus domesticus) only in possessing M. spretus mtDNA, were used as mitochondrial donors. B6-mt(spr) mice and a quantitative PCR method enabled selective estimation of the amount of M. spretus mtDNA introduced even in the presence of host M. m. domesticus mtDNA and monitoring subsequent changes of its amount during embryogenesis. Results showed that M. spretus mtDNA in spermatid mitochondria was not eliminated by the blastocyst stage, probably due to the introduction of a larger amount of spermatid mtDNA than of sperm mtDNA into embryos on fertilization. However, spermatid-derived M. spretus mtDNA was eliminated by the time of birth, whereas liver-derived M. spretus mtDNA was still present in most newborn mice, even though its amount introduced was significantly less than that of spermatid mtDNA. These observations suggest that mitochondria from spermatids but not from liver have specific factors that ensure their selective elimination and resultant elimination of mtDNA in them, and that the occurrence of elimination is not limited to early stage embryos, but continues throughout embryogenesis.     

A Novel Mouse Chromosome 17 Hybrid Sterility Locus: Implications for the Origin of T Haplotypes

The effects of heterospecific combinations of mouse chromosome 17 on male fertility and transmission ratio were investigated through a series of breeding studies. Animals were bred to carry complete chromosome 17 homologs, or portions thereof, from three different sources-Mus domesticus, Mus spretus and t haplotypes. These chromosome 17 combinations were analyzed for fertility within the context of a M. domesticus or M. spretus genetic background. Two new forms of hybrid sterility were identified. First, the heterospecific combination of M. spretus and t haplotype homologs leads to complete male sterility on both M. spretus and M. domesticus genetic backgrounds. This is an example of symmetrical hybrid sterility. Second, the presence of a single M. domesticus chromosome 17 homolog within a M. spretus background causes sterility, however, the same combination of chromosome 17 homologs does not cause sterility within the M. domesticus background. This is a case of asymmetrical hybrid sterility. Through an analysis of recombinant chromosomes, it was possible to map the M. domesticus, M. spretus and t haplotype alleles responsible for these two hybrid sterility phenotypes to the same novel locus (Hybrid sterility-4). Previous structural studies had led to the hypothesis that the ancestral t haplotype originated through an introgression event from M. spretus or a related species. If this were true, one might expect that (1) M. spretus homologs would be transmitted at a non-Mendelian ratio within the M. domesticus background, and (2) t haplotypes would be transmitted at a ratio closer to Mendelian within the M. spretus background.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional constraints of nuclear-mitochondrial DNA interactions in xenomitochondrial rodent cell lines

The co-evolution of nuclear and mitochondrial genomes in vertebrates led to more than 100 specific interactions that are crucial for an optimized ATP generation. These interactions have been examined by introducing rat mtDNA into mouse cells devoid of mitochondrial DNA (mtDNA). When mtDNA-less cells derived from the common mouse (Mus musculus domesticus) were fused to cytoplasts prepared from Mus musculus, Mus spretus, or rat (Rattus norvegicus), a comparable number of respiring clones could be obtained. Mouse xenomitochondrial cybrids harboring rat mtDNA had a slower growth rate in medium containing galactose as the carbon source, suggesting a defect in oxidative phosphorylation. These clones respired approximately 50% less than the parental mouse cells or xenomitochondrial cybrids harboring Mus spretus mtDNA. The activities of respiratory complexes I and IV were approximately 50% lower, but mitochondrial protein synthesis was unaffected. The defects in complexes I and IV were associated with decreased steady-state levels of respective subunits suggesting problems in assembly. We also showed that the presence of 10% mouse mtDNA co-existing with rat mtDNA was sufficient to restore respiration to normal levels. Our results suggest that evolutionary distance alone is not a precise predictor of nuclear-mitochondrial interactions as previously suggested for primates.     

Mus Spretus Line-1s in the Mus Musculus Domesticus Inbred Strain C57bl/6j Are from Two Different Mus Spretus Line-1 Subfamilies

A LINE-1 element, L1C105, was found in the Mus musculus domesticus inbred strain, C57BL/6J. Upon sequencing, this element was found to belong to a M. spretus LINE-1 subfamily originating within the last 0.2 million years. This is the second spretus-specific LINE-1 subfamily found to be represented in C57BL/6J. Although it is unclear how these M. spretus LINE-1s transferred from M. spretus to M. m. domesticus, it is now clear that at least two different spretus LINE-1 sequences have recently transferred. The limited divergence between the C57BL/6J spretus-like LINE-1s and their closest spretus ancestors suggests that the transfer did not involve an exceptionally long lineage of sequential transpositions.     

Reduced nucleotide variability at an androgen-binding protein locus (Abpa) in house mice: evidence for positive natural selection.

Previous work has shown that the gene for the alpha subunit of androgen-binding protein, Abpa, may be involved in premating isolation between different subspecies of the house mouse, Mus musculus. We investigated patterns of DNA sequence variation at Abpa within and between species of mice to test several predictions of a model of neutral molecular evolution. Intraspecific variation among 10 Mus musculus domesticus alleles was compared with divergence between M. m. domesticus and M. caroli for Abpa and two X-linked genes, Glra2 and Amg. No variation was observed at Abpa within M. m. domesticus. The ratio of polymorphism to divergence was significantly lower at Abpa than at Glra2 and Amg, despite the fact that all three genes experience similar rates of recombination. Interspecific comparisons among M. m. domesticus, Mus musculus musculus, Mus musculus castaneus, Mus spretus, Mus spicilegus, and Mus caroli revealed that the ratio of nonsynonymous substitutions to synonymous substitutions on a per-site basis (Ka/Ks) was generally greater than one. The combined observations of no variation at Abpa within M. m: domesticus and uniformly high Ka/Ks values between species suggest that positive directional selection has acted recently at this locus.     

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.     

A Repeated Segment on the Mouse Y Chromosome Is Composed of Retroviral-Related, Y-Enriched and Y-Specific Sequences

We report the isolation and characterization of two recombinant clones containing DNA derived from the Y chromosome of the C57BL/10 inbred mouse strain. Both clones were isolated from a lambda phage library derived from a partial EcoRI digest of C57BL/10 male DNA using the murine retrovirus M720. Characterization of these clones showed they were derived from a repeated segment present on the C57BL/10J Y chromosome that contains sequences found elsewhere in the genome. In addition, one clone contained a sequence, designated YB10, that is unique to the Y chromosome and present in approximately 500 copies on the C57BL/10J Y chromosome. Analysis of Southern blots containing DNAs prepared from females and males of representative species from four subgenera of Mus probed with pYB10 and the 3'LTR from one of the Y-associated retroviruses (MuRVY) revealed that, with the exception of a single fragment observed in both female and male DNA of Mus saxicola, hybridization to pYB10 was observed only to male DNA of the species Mus spretus, Mus hortulanus, Mus musculus, Mus domesticus and Mus abbotti. In addition, the pattern and intensity of hybridization to YB10 and the MuRVY-LTR indicated that sequence of divergence was followed by amplification of Y chromosome sequences containing YB10 and MuRVY. The divergence and amplification occurred separately in each of the ancestral lineages leading to M. spretus, M. hortulanus, M. abbotti, M. musculus and M. domesticus. We suggest that acquisition and amplification of DNA sequences by the mammalian Y chromosome has contributed to its evolution and may imply that the mammalian Y chromosome is evolving at a faster rate than the rest of the genome.     

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.     

Phylogenetic distribution in the genus Mus of t-complex-specific DNA and protein markers: inferences on the origin of t-haplotypes

We have examined the phylogenetic distribution of two t-specific markers among representatives of various taxa belonging to the genus Mus. The centromeric TCP-1a marker (a testicular protein variant specific for all t-haplotypes so far studied) has also been apparently detected in several non-t representatives of the Mus IVA, Mus IVB, and probably M. cervicolor species. By contrast, a t-specific restriction- fragment-length polymorphism allele (RFLP) of the telomeric alpha- globin pseudogene DNA marker alpha-psi-4 was found only in animals belonging to the M. musculus-complex species either bearing genuine t- haplotypes or, like the M. m. bactrianus specimen studied here, likely to do so. This t-specific alpha-psi-4 RFLP allele was found to be as divergent from the RFLP alleles of the latter, non-t, taxonomical groups as it is from Mus 4A, Mus 4B, or M. spretus ones. These results suggest the presence of t-haplotypes and of t-specific markers in populations other than those belonging to the M. m. domesticus and M. m. musculus subspecies, implying a possible origin for t-haplotypes prior to the radiation of the most recent offshoot of the Mus genus (i.e., the spretus/domesticus divergence), some 1-3 Myr ago.      

Natural hybridization between 2 sympatric species of mice, Mus musculus domesticus L. and Mus spretus Lataste

Using protein loci and DNA markers, we show by a multilocus genetic analysis that certain populations of the two sympatric mouse species Mus musculus domesticus and Mus spretus show clear signs of partial introgression. Given the sterility of F1 males and the known partial genetic incompatibilities between the genomes of the two species, our finding does not invalidate the biological species complex, but allows to think that very limited genetic exchanges remain possible even long after the divergence of taxa. This may have some consequences on the dynamics of certain kinds of invasive or advantageous DNAs like transposable elements or pathogen resistance genes.     

Altered Turnover of Hypoxanthine Phosphoribosyltransferase in Erythroid Cells of Mice Expressing Hprt a and Hprt b Alleles

We have previously shown that mice expressing Hprt a allele(s) have erythrocyte hypoxanthine phosphoribosyltransferase (HPRT) levels that are approximately 25-fold (Mus musculus castaneus) and 70-fold (Mus spretus) higher than in mice that express the Hprt b allele (Mus musculus domesticus; C57BI/6J; C3H/HeHa), and that these differences in erythrocyte HPRT levels are due to differences in the turnover rates of the HPRT A and B proteins as reticulocytes mature to erythrocytes. We show here that: the taxonomic subgroups of the genus Mus are essentially monomorphic for the occurrence of either the Hprt a or the Hprt b allele, with Hprt a being common in the aboriginal species (M. spretus, Mus hortulanus and Mus abbotti) and in several commensal species (Mus musculus musculus, M. m. castaneus, Mus musculus molossinus), while Hprt b is common in feral M. m. domesticus populations as well as in all inbred strains of mice tested; in all these diverse Mus subgroups there is a strict association of Hprt a with high and Hprt b with low levels of erythrocyte HPRT; and, the association between the occurrence of the Hprt a allele and elevated erythrocyte HPRT levels is retained following repeated backcrosses of wild-derived Hprt a allele(s) into the genetic background of inbred strains of mice with the Hprt b allele. Collectively, these observations indicate that the elevated and low levels of erythrocyte HPRT are specified by differences in the Hprt a and b structural genes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Systematic identification of LINE-1 repetitive DNA sequence differences having species specificity between Mus spretus and Mus domesticus

LINE-1 is a family of repetitive DNA sequences interspersed among mammalian genes. In the mouse haploid genome there are about 100,000 LINE-1 copies. We asked if the subspecies Mus spretus and Mus domesticus have developed species-specific LINE-1 subfamilies. Sequences from 14 M. spretus LINE-1 elements were obtained and compared to M. domesticus LINE-1 sequences. Using a molecular phylogenetic tree we identified several differences shared among a subset of young repeats in one or the other species as candidates for species-specific LINE-1 variants. Species specificity was tested using oligonucleotide probes complementary to each putative species-specific variant. When hybridized to genomic DNAs, single-variant probes detected an expanded number of elements in the expected mouse. In the other species these probes detected a smaller number of matches consistent with the average rate of random divergence among LINE-1 elements. It was further found that the combination of two species-specific sequence differences in the same probe reduced the detection background in the wrong species below our detection limit.     

LINE-1 (L1) lineages in the mouse

Recently, a rapidly amplifying family of mouse LINE-1 (L1) has been identified and named T(F). The evolutionary context surrounding the derivation of the T(F) family was examined through phylogenetic analysis of sequences in the 3' portion of the repeat. The Mus musculus domesticus T(F) family was found to be the terminal subfamily of the previously identified L1Md4 lineage. The L1Md4 lineage joins the other prototypical mouse LINE-1 lineage (the L1MdA2 lineage) approximately 1 MYA at about the time of the common ancestor of M. m. domesticus, Mus spicilegus, and Mus spretus. However, the T(F) family from M. m. domesticus was found to join to the previously reported M. spretus Ms475 and Ms7024 LINE-1 families at just 0.5 MYA, indicating horizontal transfer. The T(F) family from M. m. domesticus was then found to be even more recently related to LINE-1's from another species, M. spicilegus. A separate spretus A2 lineage was found through a directed search of a PCR library. This lineage, in contrast to the spretus T(F) lineage, does join domesticus at about 1 MYA, as would be expected in the absence of horizontal transfer. A third major family was also found that splits off from the L1Md4 lineage shortly after its departure from the L1MdA2 lineage. The new family, named the Z family, was found to contain the de novo LINE-1 inserts causing the beige and med mutations. Whether the split with the Z family was before or after the recombination that introduced the F-type promoters and defined the inception of T(F) as a lineage is unclear. In enumerating copies of the various LINE-1 families, we found that T(F) 3' ends were not much more numerous than the reported number of 5' ends, suggesting that T(F) may not be subjected to the 90% truncation pattern typical of LINE-1 as a whole.     

Physical performance and soleus muscle fiber composition in wild-derived and laboratory inbred mouse strains.

We compared four inbred mouse strains in their physical performance, measured as a maximal treadmill running time, characteristics of soleus muscle, anatomic character, and growth. The strains used were Mus musculus domesticus [C57BL/6 (B6) and BALB/c], Mus musculus molossinus (MSM/Ms), and Mus spretus. Maximal running time was significantly different among these four mouse strains. Running time until exhaustion was highest in MSM/Ms and lowest in M. spretus. Maximal times for the laboratory mouse strains were nearly identical. Soleus muscle fiber type and cross-sectional area also differed significantly among the species. In particular, M. spretus was significantly different from the other inbred mouse strains. Growth in the wild-derived inbred mice appeared to be complete earlier than in the laboratory mice, and the body size of the wild strains was about half that of the laboratory strains. From these results, we propose that wild-derived inbred mouse strains are useful models for enhancing phenotypic variation in physical performance and adaptability.     

Molecular evolution of a Y-chromosomal repetitive sequence family in the genus Mus

A 522-base-long Y-chromosomal sequence was isolated from a BALB/c genomic library and was designated "BF046." It is repeated about 200 times in the male genome, and a difference was detected between the Mus musculus musculus and the M. m. domesticus type Y chromosomes. BF046- related sequences were present over the entire length of the Y chromosome as visualized by in situ hybridization. Southern blot analysis against DNAs isolated from eight species in the genus Mus showed that BF046-related sequences were amplified in the Y chromosomes of three closely related species: M. musculus, M. spicilegus, and M. spretus. To gain insight into the stability of the BF046 sequence family, we isolated 18 additional clones from these three mouse species and compared their sequences. The M. musculus sequences differed from the M. spicilegus and M. spretus sequences by two indels. The remaining parts of the sequences were very similar, but both parsimony and distance-based analytical methods divided the sequences into the same four subgroups, with each species having its own subgroup(s). Thus, the Y chromosomes of M. musculus, M. spicilegus, and M. spretus can be distinguished from one another.