A sequence-based variation map of 8.27 million SNPs in inbred mouse strains

A dense map of genetic variation in the laboratory mouse genome will provide insights into the evolutionary history of the species and lead to an improved understanding of the relationship between inter-strain genotypic and phenotypic differences. Here we resequence the genomes of four wild-derived and eleven classical strains. We identify 8.27 million high-quality single nucleotide polymorphisms (SNPs) densely distributed across the genome, and determine the locations of the high (divergent subspecies ancestry) and low (common subspecies ancestry) SNP-rate intervals for every pairwise combination of classical strains. Using these data, we generate a genome-wide haplotype map containing 40,898 segments, each with an average of three distinct ancestral haplotypes. For the haplotypes in the classical strains that are unequivocally assigned ancestry, the genetic contributions of the Mus musculus subspecies--M. m. domesticus, M. m. musculus, M. m. castaneus and the hybrid M. m. molossinus--are 68%, 6%, 3% and 10%, respectively; the remaining 13% of haplotypes are of unknown ancestral origin. The considerable regional redundancy of the SNP data will facilitate imputation of the majority of these genotypes in less-densely typed classical inbred strains to provide a complete view of variation in additional strains.     

The mosaic structure of variation in the laboratory mouse genome

Most inbred laboratory mouse strains are known to have originated from a mixed but limited founder population in a few laboratories. However, the effect of this breeding history on patterns of genetic variation among these strains and the implications for their use are not well understood. Here we present an analysis of the fine structure of variation in the mouse genome, using single nucleotide polymorphisms (SNPs). When the recently assembled genome sequence from the C57BL/6J strain is aligned with sample sequence from other strains, we observe long segments of either extremely high (approximately 40 SNPs per 10 kb) or extremely low (approximately 0.5 SNPs per 10 kb) polymorphism rates. In all strain-to-strain comparisons examined, only one-third of the genome falls into long regions (averaging >1 Mb) of a high SNP rate, consistent with estimated divergence rates between Mus musculus domesticus and either M. m. musculus or M. m. castaneus. These data suggest that the genomes of these inbred strains are mosaics with the vast majority of segments derived from domesticus and musculus sources. These observations have important implications for the design and interpretation of positional cloning experiments.     

Conservation of position and exclusive expression of mouse Xist from the inactive X chromosome

X-chromosome inactivation in mammals is a regulatory phenomenon whereby one of the two X chromosomes in female cells is genetically inactivated, resulting in dosage compensation for X-linked genes between males and females. In both man and mouse, X-chromosome inactivation is thought to proceed from a single cis-acting switch region or inactivation centre (XIC/Xic). In the human, XIC has been mapped to band Xq13 (ref. 6) and in the mouse to band XD (ref. 7), and comparative mapping has shown that the XIC regions in the two species are syntenic. The recently described human XIST gene maps to the XIC region and seems to be expressed only from the inactive X chromosome. We report here that the mouse Xist gene maps to the Xic region of the mouse X chromosome and, using an interspecific Mus spretus/Mus musculus domesticus F1 hybrid mouse carrying the T(X;16)16H translocation, show that Xist is exclusively expressed from the inactive X chromosome. Conservation between man and mouse of chromosomal position and unique expression exclusively from the inactive X chromosome lends support to the hypothesis that XIST and its mouse homologue are involved in X-chromosome inactivation.     

The origin of MHC class II gene polymorphism within the genus Mus

The I region of the major histocompatibility complex (MHC) of the mouse (H-2) contains a tightly-linked cluster of highly polymorphic genes (class II MHC genes) which control immune responsiveness. Speculation on the origin of this polymorphism, which is believed to be essential for the function of the class II proteins in immune responses to disease, has given rise to two hypotheses. The first is that hypermutational mechanisms (gene conversion or segmental exchange) promote the rapid generation of diversity in MHC genes. The alternative is that polymorphism has arisen from the steady accumulation of mutations over long evolutionary periods, and multiple specific alleles have survived speciation (trans-species evolution). We have looked for evidence of 'segmental exchange' and/or 'trans-species evolution' in the class II genes of the genus Mus by molecular genetic analysis of I-A beta alleles. The results indicate that greater than 90% (28 out of 31) of the alleles examined can be organized into two evolutionary groups both on the basis of restriction site polymorphisms and by the presence or absence of a short interspersed nucleotide element (SINE). Using this SINE sequence as an evolutionary tag, we demonstrate that I-A beta alleles in these two evolutionary groups diverged at least three million years ago and have survived the speciation events leading to several modern Mus species. Nucleotide sequence comparisons of eight Mus m. domesticus I-A beta alleles representing all three evolutionary groups indicate that most of the divergence in exon sequences is due to the steady accumulation of mutations that are maintained independently in the different alleles. But segmental exchanges between alleles from different evolutionary groups have also played a role in the diversification of beta 1 exons.     

Paternal inheritance of mitochondrial DNA in mice.

For nearly 20 years it has been assumed on the basis of low-resolution experiments that mitochondrial (mt)DNA, in contrast to the genes in the nucleus, has an exclusively maternal mode of inheritance in animals. Using the polymerase chain reaction, paternally inherited mtDNA molecules have now been detected in mice at a frequency of 10(-4), relative to the maternal contributions. These mice were hybrids between two inbred strains (C57BL/6J and Mus spretus) whose mtDNAs can be distinguished easily. This new mode of inheritance provides a mechanism for generating heteroplasmy and may explain mitochondrial disorders exhibiting biparental transmission.     

Wormy mice in a hybrid zone

As one approach to analysing the genetic barriers between species, we studied the numbers and types of parasitic worms in two species of house mice (Mus musculus and M. domesticus) and in their natural hybrids. Where the ranges of these two species meet in southern Germany, there is a zone of hybridization less than 20 kilometres across, in which about 98% of the mice have backcross genotypes. Fourteen of the 46 mice tested from within the zone have over 500 pinworms per gut, a number far exceeding the mean of 40 per gut for other mice inside and outside the zone. Other nematodes have a similar, non-random distribution. The number of mice bearing 9 or more tapeworms per gut is also excessive in the hybrid zone. These extraordinarily wormy mice may be unusually susceptible to parasitism; the different species may have different genes for resistance, and recombinant backcross animals may lose both. Our findings support the view that the hybrid populations may have reduced fitness and thereby act as a genetic sink, interfering with the flow of genes between the two species. The possibility that environmental or ecological peculiarites in the zone of hybridization make the mice more liable to infection is not supported.     

The mapping of a cDNA from the human X-linked Duchenne muscular dystrophy gene to the mouse X chromosome

The recent discovery of sequences at the site of the Duchenne muscular dystrophy (DMD) gene in humans has opened up the possibility of a detailed molecular analysis of the genes in humans and in related mammalian species. Until relatively recently, there was no obvious mouse model of this genetic disease for the development of therapeutic strategies. The identification of a mouse X-linked mutant showing muscular dystrophy, mdx, has provided a candidate mouse genetic homologue to the DMD locus; the relatively mild pathological features of mdx suggest it may have more in common with mutations of the Becker muscular dystrophy type at the same human locus, however. But the close genetic linkage of mdx to G6PD and Hprt on the mouse X chromosome, coupled with its comparatively mild pathology, have suggested that the mdx mutation may instead correspond to Emery Dreifuss muscular dystrophy which itself is closely linked to DNA markers at Xq28-qter in the region of G6PD on the human X chromosome. Using an interspecific mouse domesticus/spretus cross, segregating for a variety of markers on the mouse X chromosome, we have positioned on the mouse X chromosome sequences homologous to a DMD cDNA clone. These sequences map provocatively close to the mdx mutation and unexpectedly distant from sparse fur, spf, the mouse homologue of OTC (ornithine transcarbamylase) which is closely linked to DMD on the human X chromosome.     

Mating patterns in seminatural populations of mice influenced by MHC genotype

Because of the central role of major histocompatibility complex (MHC) genes in immune recognition, it is often assumed that parasite-driven selection maintains the unprecendented genetic diversity of these genes. But associations between MHC genotype and specific infectious diseases have been difficult to identify with a few exceptions such as Marek's disease and malaria. Alternatively, MHC-related reproductive mechanisms such as selective abortion and mating preferences could be responsible for the diversity. To determine both the nature and strength of selection operating on MHC genes by we have studied components of selection in seminatural populations of mice (Mus musculus domesticus). Here we assess MHC-related patterns of reproduction and early (preweaning) mortality by analysing 1,139 progeny born in nine populations, and 662 progeny from laboratory matings. Reproductive mechanisms, primarily mating preferences, result in 27% fewer MHC-homozygous offspring than expected from random mating. MHC genotype had no detectable influence on neonatal (preweaning) mortality. These mating preferences are strong enough to account for most of the MHC genetic diversity found in natural populations of Mus.     

Tumour necrosis factor and lymphotoxin genes map close to H-2D in the mouse major histocompatibility complex

Tumour necrosis factor (TNF-alpha) and lymphotoxin (TNF-beta) are related proteins, secreted by macrophages and lymphocytes respectively, which play a role in destruction of tumour cells and virally infected cells (for reviews see refs 1,2). TNF-alpha is a non-glycosylated protein of relative molecular mass 17,000 (Mr 17 K), whereas TNF-beta is a glycoprotein of Mr 25 K. Both TNF-alpha and TNF-beta aggregate into multimers and act through the same receptor molecule on target cells. Genes encoding these two TNF proteins have been cloned from mouse and man and in both are closely linked, being separated by approximately 1 kilobase (kb) of DNA. In the mouse these genes are located on chromosome 17, but in man they are on the short arm of chromosome 6. This segment of chromosome 6 also contains the genes of the major histocompatibility complex (MHC), as does chromosome 17 in the mouse. To find out whether the TNF genes are located within the MHC, we used polymorphic restriction sites to analyse a panel of MHC congeneic and intra-MHC recombinant mouse strains. Initially, we mapped the TNF genes the D or Qa region in the distal half of the mouse MHC. We then studied a gene cluster encompassing part of the D and Qa regions and found the TNF genes are located 70 kb proximal to the D gene.     

Endogenous mammary tumour virus DNA varies among wild mice and segregates during inbreeding.

Proviruses of the mouse mammary tumour virus (MMTV) endogenous to normal mice can be identified by molecular hybridisation and distinguished using restriction endonucleases. Feral mice display marked heterogeneity with respect to the number of copies and the sites of insertion of endogenous MMTV-specific DNA, with occasional mice apparently free of MMTV DNA. Several different MMTV proviruses present in laboratory mice have segregated like stable, independent genetic elements during the inbreeding which followed a cross between Bagg albino and DBA mice 60 years ago. The results favour the hypothesis that endogenous proviruses have been established by multiple, independent infections of germ cells rather than by somatic mutation of ancestral proviruses or of cellular genes.     

Conformational mutations in human mitochondrial DNA

Variation in the human mitochondrial DNA (mtDNA) sequence has been extensively analysed using restriction fragment length polymorphisms (RFLPs). MtDNA RFLPs have previously been attributed to nucleotide changes within restriction endonuclease recognition sites or to small insertion-deletion mutations. We now report that RFLPs detected by polyacrylamide gel electrophoresis can also result from single nucleotide substitutions which alter the mobility of small- to medium-sized restriction fragments that incorporate the sequence. We have defined the mutation responsible at two loci and have identified several possible additional loci. When screening human mtDNAs with multiple restriction endonucleases, such mutations can be misidentified as insertion-deletion mutations or counted as multiple polymorphic restriction sites. This can lead to errors in constructing restriction maps and estimating sequence diversity.     

Long-range restriction site mapping of mammalian genomic DNA

Molecular analysis of many problems in genetics would be facilitated by the ability to construct restriction site maps of long stretches of genomic DNA and to directly place genes on these maps. Pulsed-field gradient gel electrophoresis allows measurement of the size of DNA fragments up to at least 2,000 kilobase pairs (kb) long and we have used this technique here to map sites for one class of infrequently cutting restriction enzyme over a total of 1,500 kb of mouse genomic DNA. The sites for these enzymes tend to be clustered in the genome. These clusters may correspond to the short stretches of C + G-rich unmethylated DNA often associated with mammalian genes.     

The gene encoding the T-cell receptor alpha-chain maps close to the Np-2 locus on mouse chromosome 14

Serological and molecular genetic analyses of T-cell clones have shown that the T-cell antigen receptor apparently comprises two glycosylated, disulphide-linked polypeptide chains (alpha and beta), both of which span the cell membrane. Cloning of the genes encoding the two chains from mouse and human DNA has shown that the alpha- and beta-chains are composed of variable (V) and conserved (C) regions in agreement with peptide mapping data. Gene segments encoding variable and conserved domains of the beta-chain have been identified and undergo rearrangements during T-cell differentiation. The genes encoding the alpha-chain, so far described at the level of complementary DNA clones, also identify DNA rearrangements. Thus, the genes encoding the T-cell receptor show the same structure and dynamic behaviour as immunoglobulin genes, indicating that the two gene families belong to the same supergene family; this evolutionary relationship is supported by the fact that the genes encoding the beta-chain of the T-cell receptor are closely linked to immunoglobulin kappa light-chain genes on chromosome 6 in mouse. In man, however, the beta genes map to chromosome 7 (ref. 14) whereas the kappa-chain genes are located on chromosome 2, indicating that linkage between the two gene families is not needed for proper expression. Here we describe genomic clones encoding the constant portion of the T-cell receptor alpha-chain and map the gene to chromosome 14 in mouse, close to the gene for purine nucleoside phosphorylase (Np-2) which, in man, has been associated with T-cell immunodeficiencies.     

Mys, a family of mammalian transposable elements isolated by phylogenetic screening

It has recently been demonstrated both emperically and mathematically that transposable elements may spread rapidly throughout a population once introduced even when they dramatically reduce the fitness of individuals that carry them. Such events result in pronounced differences in the phylogenetic distribution of genetic elements capable of rapid genome invasion. Using a simple and general procedure to screen the genome of the white-footed mouse Peromyscus leucopus, we have isolated a family of retrovirus-like elements which is apparently absent from the genome of the house mouse Mus domesticus. Here, we report this procedure and an analysis of the organization, phylogenetic distribution and sequence of this family of transposable elements.     

Endogenous viral genes are non-essential in the chicken.

DNA sequences homologous to the genomes of type C retroviruses are widespread among vertebrates. Ten genetic loci containing endogenous viral DNA sequences have been documented in the white Leghorn chicken alone. Six of these genetic loci are associated with the production of virus or of viral proteins in embryonic fibroblasts (refs 2--4, and S.M.A., L, B. Crittenden and E.G.B., in preparation) and one of the loci may be expressed in the erythroblasts of 5-day-old embryos. The abiquitous presence of endogenous viral genes among vertebrate species and the association of their expression with development of the haematopoietic system in the mouse have led to the proposal that these genes are involved in ontogeny. In addition, the genes may be implicated in oncogenesis as in the case of the AKR mouse in which a high incidence of spontaneous leukaemia is associated with the expression of endogenous murine laukaemia virus genomes. We report here the production of a fertile rooster which lacks avian leukosis virus-related endogenous viral genes and which seems to be completely normal and healthy. Thus, endogenous viral genes are apparently not essential for the normal development of the chicken. An endogenous virus-free state has also been reported for three species of jungle fowl and for the B-type viral genes of the mouse.     

Amplification of immunoglobulin lambda constant genes in populations of wild mice

The lambda immunoglobulin light chain (Ig lambda) locus of BALB/c inbred mice consists of two variable region gene segments (V lambda)1-3, and four constant region gene segments (C lambda)1,2,4,5. Each C lambda gene segment is associated with a unique joining segment (J lambda)2,4-7, and they are organized in two paired units, J3C3-J1C1 and J2C2-J4C4 (refs 4, 8). Using cDNA probes specific for C lambda 1 and C lambda 2 (ref. 9) we have analysed the genomic organization of the C lambda gene segments in wild-derived and inbred strains of mice. Although Southern blots of the genomic DNA of inbred mice show a constant pattern of hybridization, wild-derived mice show a high degree of variation in the number, size and intensity of hybridizing fragments. We have now found that, per haploid genome, mice of a Mus musculus musculus stock isolated from Sladeckovce, Czechoslovakia (CzII) have at least 12 C lambda segments, and mice of a Mus musculus domesticus stock 'Centreville Lights' from Centreville, Maryland (CL) have at least 8 C lambda segments. There appears to have been relatively recent amplifications of the C lambda gene segments in wild mice.     

Resolution of quantitative traits into Mendelian factors by using a complete linkage map of restriction fragment length polymorphisms

The conflict between the Mendelian theory of particulate inheritance and the observation of continuous variation for most traits in nature was resolved in the early 1900s by the concept that quantitative traits can result from segregation of multiple genes, modified by environmental effects. Although pioneering experiments showed that linkage could occasionally be detected to such quantitative trait loci (QTLs), accurate and systematic mapping of QTLs has not been possible because the inheritance of an entire genome could not be studied with genetic markers. The use of restriction fragment length polymorphisms (RFLPs) has made such investigations possible, at least in principle. Here, we report the first use of a complete RFLP linkage map to resolve quantitative traits into discrete Mendelian factors, in an interspecific back-cross of tomato. Applying new analytical methods, we mapped at least six QTLs controlling fruit mass, four QTLs for the concentration of soluble solids and five QTLs for fruit pH. This approach is broadly applicable to the genetic dissection of quantitative inheritance of physiological, morphological and behavioural traits in any higher plant or animal.     

珠鸡mtDNA的限制性图谱

应用AvaⅠ，BamHⅠ,BglⅠ,DraⅠ,EcoRⅠ,HpaⅠ,PvuⅡ,SacⅠ,SalⅠ,ScaⅠ和StuⅠ共11种限制酶对珠鸡（Acryllium vulturinum)mtDNA进行单酶切分析，结果表明，珠鸡mtDNA的分子量为16.7kb，另以其中除AvaⅠ和StuⅠ外的9种限制酶进行双酶切分析，构建了珠鸡mtDNA的限制性图谱，并与家鸡（Gallus gallus domesticus)比较，二者mtDNA序列歧异值为0.073。     

Production of functional chimaeric mouse/human antibody

The availability of monoclonal antibodies has revived interest in immunotherapy. The ability to influence an individual's immune state by administering immunoglobulin of the appropriate specificity may provide a powerful approach to disease control and prevention. Compared with immunoglobulin from other species, human immunoglobulin (Ig) might be best for such therapeutic intervention; it might function better with the recipient's effector cells and should itself be less immunogenic. The success of the mouse hybridoma system suggests that immunoglobulin of virtually any specificity can be obtained from a properly immunized animal. In the human system, however, immunization protocols are restricted by ethical considerations, and it is not yet clear whether human antibody-producing cell lines of the required specificity can be obtained from adventitiously immunized individuals or from in vitro immunized cells. A method which might circumvent these difficulties is to produce antibodies consisting of mouse variable regions joined to human constant regions. Therefore, we have constructed immunoglobulin genes in which the DNA segments encoding mouse variable regions specific for the hapten trinitrophenyl (TNP) are joined to segments encoding human mu and kappa constant regions. These 'chimaeric' genes are expressed as functional TNP-binding chimaeric IgM. We report here some of the properties of this novel IgM.