Interspecific recombinant congenic strains between C57BL/6 and mice of the Mus spretus species: a powerful tool to dissect genetic control of complex traits

Complex traits are under the genetic control of multiple genes, often with weak effects and strong epistatic interactions. We developed two new collections of mouse strains to improve genetic dissection of complex traits. They are derived from several backcrosses of the Mus spretus SEG/Pas or STF/Pas strains on the C57BL/6J background. Each of the 55 interspecific recombinant congenic strains (IRCSs) carries up to eight SEG/Pas chromosomal segments with an average size of 11.7 Mb, totalizing 1.37% of the genome. The complete series covers 39.7% of the SEG/Pas genome. As a complementary resource, six partial or complete interspecific consomic strains were developed and increased genome coverage to 45.6%. To evaluate the usefulness of these strains for QTL mapping, 16 IRCSs were compared with C57BL/6J for seven hematological parameters. Strain 66H, which carries three SEG/Pas chromosomal segments, had lower red blood cell volume and higher platelet count than C57BL/6J. Each chromosomal segment was isolated in a congenic strain to evaluate individual effects. Congenic strains were combined to assess epistasis. Our data show that both traits were controlled by several genes with complex epistatic interactions. IRCSs are therefore useful to unravel QTL with small effects and gene-by-gene interactions.     

Centimorgan-range one-step mapping of fertility traits using interspecific recombinant congenic mice

In mammals, male fertility is a quantitative feature determined by numerous genes. Until now, several wide chromosomal regions involved in fertility have been defined by genetic mapping approaches; unfortunately, the underlying genes are very difficult to identify. Here, 53 interspecific recombinant congenic mouse strains (IRCSs) bearing 1-2% SEG/Pas (Mus spretus) genomic fragments disseminated in a C57Bl/6J (Mus domesticus) background were used to systematically analyze male fertility parameters. One of the most prominent advantages of this model is the possibility of analyzing stable phenotypes in living animals. Here, we demonstrate the possibility in one-step fine mapping for several fertility traits. Focusing on strains harboring a unique spretus fragment, we could unambiguously localize two testis and one prostate weight-regulating QTL (Ltw1, Ltw2, and Lpw1), four QTL controlling the sperm nucleus shape (Sh1, Sh2, Sh3, and Sh4), and one QTL influencing sperm survival (Dss1). In several cases, the spretus DNA fragment was small enough to propose sound candidates. For instance, Spata1, Capza, and Tuba7 are very strong candidates for influencing the shape of the sperm head. Identifying new genes implied in mammalian fertility pathways is a necessary prerequisite for clarifying their molecular grounds and for proposing diagnostic tools for masculine infertilities.     

The Ig light chain restricted B6.kappa(-)lambda(SEG) mouse strain suggests that the IGL locus genomic organization is subject to constant evolution

In laboratory mice, the different Ig lambda light chain subtypes (lambda 1, lambda 2, lambda x and lambda 3) are expressed on 60, 16, 16 and 8%, respectively, of the lambda-positive peripheral B cells. Eighteen years ago, our laboratory characterized a lambda 1(-) wild mouse strain: SPE ( Mus spretus). In this report, we describe the characterization of another wild-derived Mus spretus inbred strain, SEG, that presents the same characteristic, namely the absence of lambda 1 expression. An almost congenic strain, B6.lambda(SEG), was detected in a series of recombinant congenic strains carrying 2% of SEG/Pas genome in a C57BL/6J background. This B6.lambda(SEG) strain was crossed to Igh (a) C kappa (-) mice in order to derive two different additional congenic strains: B6.kappa(-)lambda(SEG) Igh (a) and B6.kappa(-)lambda(SEG) Igh (b). In this paper, we characterize the genomic organization and the expression of the SEG IGL locus. Altogether, our data show that the SEG IGL locus is constituted by a single functional IGLJ2SEG-IGLC2SEG, two pseudo IGLJ4SEG1/2-IGLC4SEG1/2 gene clusters and two V gene segments: IGLV2SEG and IGLVXSEG. In particular, we show the absence of IGLV1 and IGLVSD26 gene segments. IGLVSD26 was reported to be present in some Mus m. musculus mice and absent in BALB/c. Here, we confirm its presence not only in other Mus m. musculus mice but also in Mus spretus mice. Consequently, we propose that IGLVSD26-related gene segments define a new family that we name V lambda 4. The study of the organization of different IGL loci, in addition to the V lambda 4(+) reported here, could elucidate questions concerning the evolution of the lambda locus.     

PCR-analyzed microsatellites: Data concerning laboratory and wild-derived mouse inbred strains

We have investigated 67 primers designed by Dr. J. Todd and co-workers to amplify microsatellites sequences in the mouse. We report on additional polymorphisms concerning seven laboratory inbred strains, complementary to those already published. We include the survey of three independently derived strains of Mus spretus: SPE/Pas, SEG/Pas and SPR/Smh. SPE/Pas and SEG/Pas are very close (3% polymorphism), whereas the third one, (SPR/Smh), is very different from the other two strains (33% polymorphism). Seventy-four to 84% of the microsatellites analyzed in this study are polymorphic between C57BL/6Pas and Mus spretus strains. By comparison, 36–46% are polymorphic between laboratory inbred strains involved in established sets of recombinant inbred strains. A strain derived from Mus musculus musculus (PWK/Pas) was found to be very different from both C57BL/6Pas (70% polymorphism) and SPE/Pas (82% polymorphism). These results emphasize the interest of using Mus musculus musculus inbred strains to establish interspecific crosses, particularly when considering their breeding performances.     

Hst-3: an X-linked hybrid sterility gene

A gene, Hst-3, responsible for sterility in F1 males from crosses between Mus spretus and laboratory strains of mice such as C57BL/6, has been localized on the distal part of the X chromosome, using both DNA probes and biochemical markers on a panel of F1(C57BL/6 x SEG) x C57BL/6 backcross males. This gene may be a model for studying mammalian hybrid sterility.     

Mus Spretus Line-1 Sequences Detected in the Mus Musculus Inbred Strain C57bl/6j Using Line-1 DNA Probes

The inbred mouse strain, C57BL/6J, was derived from mice of the Mus musculus complex. C57BL/6J can be crossed in the laboratory with a closely related mouse species, M. spretus to produce fertile offspring; however there has been no previous evidence of gene flow between M. spretus and M. musculus in nature. Analysis of the repetitive sequence LINE-1, using both direct sequence analysis and genomic Southern blot hybridization to species-specific LINE-1 hybridization probes, demonstrates the presence of LINE-1 elements in C57BL/6J that were derived from the species M. spretus. These spretus-like LINE-1 elements in C57BL/6J reveal a cross to M. spretus somewhere in the history of C57BL/6J. It is unclear if the spretus-like LINE-1 elements are still embedded in flanking DNA derived from M. spretus or if they have transposed to new sites. The number of spretus-like elements detected suggests a maximum of 6.5% of the C57BL/6J genome may be derived from M. spretus.     

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.     

Mapping of the mouse X chromosome using random genomic probes and an interspecific mouse cross.

Two libraries enriched in murine X chromosome material have been constructed in the lambda vector NM 1149 from flow-sorted chromosomes. Inserts of unique genomic sequence DNA were purified and their X chromosome specificity characterised by hybridisation to a panel of somatic cell hybrid lines. Of the first five such X chromosome-specific probes characterised, all detect restriction fragment length polymorphisms (RFLPs) between inbred mouse laboratory strains such as C57BL/6 and BALB/c and the SPE/Pas mouse strain established from a wild Mus spretus mouse, when their DNAs are digested with the restriction enzyme TaqI. Taking advantage of these RFLPs, all five probes have been localised on the X chromosome using an interspecific backcross between the B6CBARI and SPE/Pas mouse strains segregating the X chromosome markers hypoxanthine phosphoribosyl transferase (Hprt) and Tabby (Ta). Three of the probes map to the region between the centromere and Hprt, and two distal to Ta. Since such X-specific sequence probes detect RFLPs between M. spretus and M. musculus domesticus DNAs with high frequency, a large panel of well localised probes should soon be available for studies of biological problems associated with the X chromosome which can best be approached using the murine species.     

Mapping of alpha-spectrin on distal mouse chromosome 1

DNAs from different strains of inbred mice and feral Mus spretus were found to exhibit restriction fragment length polymorphisms (RFLP) when hybridized with a probe prepared from a c-DNA clone of the mouse alpha-spectrin (Spna-1) gene. Studies of five recombinant inbred strains and (C57BL/6 X M. spretus) F1 X C57BL/6 backcross mice demonstrated that these RFLPs were allelic and that Spna-1 is closely linked to Ly-9 and Ly-17 on the distal region of chromosome 1.     

DNA Sequence Analysis of Sry Alleles (Subgenus Mus) Implicates Misregulation as the Cause of C57bl/6j-Y(pos) Sex Reversal and Defines the Sry Functional Unit

The Sry (sex determining region, Y chromosome) open reading frame from mice representing four species of the genus Mus was sequenced in an effort to understand the conditional dysfunction of some M. domesticus Sry alleles when present on the C57BL/6J inbred strain genetic background and to delimit the functionally important protein regions. Twenty-two Sry alleles were sequenced, most from wild-derived Y chromosomes, including 11 M. domesticus alleles, seven M. musculus alleles and two alleles each from the related species M. spicilegus and M. spretus. We found that the HMG domain (high mobility group DNA binding domain) and the unique regions are well conserved, while the glutamine repeat cluster (GRC) region is quite variable. No correlation was found between the predicted protein isoforms and the ability of a Sry allele to allow differentiation of ovarian tissue when on the C57BL/6J genetic background, strongly suggesting that the cause of this sex reversal is not the Sry protein itself, but rather the regulation of SRY expression. Furthermore, our interspecies sequence analysis provides compelling evidence that the M. musculus and M. domesticus SRY functional domain is contained in the first 143 amino acids, which includes the HMG domain and adjacent unique region (UR-2).     

Mx1 causes resistance against influenza A viruses in the Mus spretus-derived inbred mouse strain SPRET/Ei

Inbred SPRET/Ei mice, derived from Mus spretus, were found to be extremely resistant to infection with a mouse adapted influenza A virus. The resistance was strongly linked to distal chromosome 16, where the interferon-inducible Mx1 gene is located. This gene encodes for the Mx1 protein which stimulates innate immunity to Orthomyxoviruses. The Mx1 gene is defective in most inbred mouse strains, but PCR revealed that SPRET/Ei carries a functional allele. The Mx1 proteins of M. spretus and A2G, the other major resistant strain derived from Mus musculus, share 95.7% identity. We were interested whether the sequence variations between the two Mx1 alleles have functional significance. To address this, we used congenic mouse strains containing the Mx1 gene from M. spretus or A2G in a C57BL/6 background. Using a highly pathogenic influenza virus strain, we found that the B6.spretus-Mx1 congenic mice were better protected against infection than the B6.A2G-Mx1 mice. This effect may be due to different Mx1 induction levels, as was shown by RT-PCR and Western blot. We conclude that SPRET/Ei is a novel Mx1-positive inbred strain useful to study the biology of Mx1.     

cDNA sequence of five mouse guanine deaminase (Gda) alleles and mapping to mouse chromosome 19.

Guanine deaminase catalyses the conversion of guanine to xanthine and ammonia, thereby irreversibly removing the guanine base from the pool of guanine-containing metabolites. We have identified five alleles at the mouse guanine deaminase locus by cDNA sequencing. These alleles were defined by single-nucleotide polymorphisms at a total of 19 positions. For each allele the representative strains are as follows: Gda(a), C57BL/6J and DBA/2J; Gda(b), A/J; Gda(c), MOLF/Ei; Gda(d), CAST/Ei; and Gda(e), SPRET-1. The only codon change resulting in an amino acid substitution was found at nucleotide 523, where GAT was replaced by AAT in Mus spretus resulting in the deduced substitution of Asp-174 by Asn. The single-nucleotide difference between the a and b alleles was also typed by allele-specific oligonucleotide amplification for 17 common strains of Mus musculus susbp. musculus. By typing the AxB and BxA recombinant inbred (RI) strain sets, Gda was mapped to mouse chromosome 19, a region syntenic with human chromosome 9q11-q22.     

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.     

Segregation of restriction fragment length polymorphism in an interspecies cross of laboratory and wild mice indicates tight linkage of the murine IFN-beta gene to the murine IFN-alpha genes.

Southern blot analysis with murine (Mu) interferon (IFN)-alpha cDNA of restricted genomic DNA of three inbred strains of mice belonging to the species Mus musculus domesticus (BALB/c, C57BL/6, and DBA/2) revealed only a limited degree of polymorphism. For example, with HindIII there were only two polymorphic bands out of 14 hybridizing fragments. With Mu IFN-beta cDNA there was no polymorphism at all between BALB/c and C57BL/6 in DNA restricted with seven different enzymes. In contrast, HindIII-restricted DNA of an inbred strain of wild mice (M. spretus Lataste) hybridized with the IFN-alpha probe displayed a high degree of polymorphism compared with the three strains of laboratory mice and was also polymorphic when probed with IFN-beta cDNA. Although M. musculus domesticus and M. spretus Lataste represent different species, certain interspecies crosses are possible in the laboratory. This enabled us to follow segregation of restriction fragment length polymorphism in HindIII-restricted DNA obtained from 18 backcross progeny of a (DBA/2 X M. spretus)F1 X DBA/2 interspecies cross. There was complete coincidence between the segregation of parental (DBA/2) and (DBA/2 X M. spretus)F1-type IFN-beta and IFN-alpha restriction fragment length polymorphism, indicating tight linkage of the IFN-beta and IFN-alpha genes. In addition, in 15 of 18 progeny the segregation coincided with that of the brown locus on chromosome 4, in accord with previous results obtained with the IFN-alpha probe in strains derived from crosses between BALB/c and C57BL/6 mice. Thus, the Mu IFN-beta gene is tightly linked to the Mu IFN-alpha gene cluster on chromosome 4 near the brown locus.     

Gene flow of unique sequences between Mus musculus domesticus and Mus spretus

Allelic diversity has been examined from a variety of Mus musculus subspecies and Mus spretus strains by sequencing at a 453-bp unique sequence locus. One M. m. domesticus classic inbred strain, C57BL/KsJ, contained a sequence identical to that in the M. spretus wild-derived inbred strain SEG, and other wild M. spretus isolates. Such a result should have been precluded by the expected divergence between the species unless there has been interspecies gene flow. Examination of C57BL/KsJ for M. spretus-specific repetitive sequences shows that it is neither a mis-identified spretus strain nor a domesticus/spretus hybrid. Thus, in addition to the previously reported presence of small amounts of Mus spretus-specific repetitive DNA in M. m. domesticus, there is a detectable flow of unique sequence between the two species. There was also ancestral polymorphism observed among the spretus alleles. The difficulty of distinguishing ancestral polymorphism from horizontal transfer is discussed. Received: 14 May 1999 / Accepted: 5 November 1999     

Developmental study of the different effects on the hybrid sterility of Kit and KitW-v alleles paired with Kit from Mus spretus

The combination of the KitW or KitW-n mutant alleles and KitS from Mus spretus results in male hybrid sterility with small testes. In the present study, reproduction of the combination between KitW-v and KitS alleles was examined. The KitW-v/KitS male was fertile and the histologic structure was normal; the seminiferous tubules showed all of the normal stages of spermatogenesis. The postnatal development of the testis at 8, 12, 16 and 20 days was also studied in the fertile +Kit/+Kit and KitW-v/KitS males and the sterile KitW/KitS. The results showed that at 8 days there was no noticeable difference among the three genotype combinations, while from 12 to 20 days spermatogenesis in the KitW/KitS male nearly stopped before the meiosis stage. The expression of Kit receptor protein from the KitS allele in the sterile testis of the KitW/KitS male was confirmed using western blot analysis. The Kit ligand derived from M. spretus showed two amino acid changes in the extracellular domain compared with that from C57BL and it appears that the ligand-receptor interaction between C57BL and SPR may influence the male hybrid sterility of KitW/KitS.     

Genetic variation in mouse apolipoprotein A-IV due to insertion and deletion in a region of tandem repeats.

We have detected three unique apolipoprotein A-IV (apoA-IV) charge isoforms in strains of commensal mice. The cDNA sequences for one representative of each isoform (Mus domestesticus strains C57BL/6J and 129/J and Mus castaneus) revealed a polymorphism within a series of four imperfect repeats encoding the sequence Glu-Gln-Ala/Val-Gln. Insertions or deletions of 12 nucleotides within this repetitive region have given rise to three genotypes characterized by three (129), four (C57BL/6), or five (M. castaneus) copies of the repeat unit. To ascertain the extent of this variation among other species of the Mus genus, we sequenced this region of apoA-IV cDNAs from eight additional M. domesticus inbred strains and from five wild-derived Mus species. All eight additional M. domesticus strains examined had four repeat units, as found in C57BL/6. Among wild-derived mice, however, one species (Mus spretus) had three repeats, two species (Mus cookii and Mus cervicolor) had four repeats, and two species (Mus hortulanus and Mus minutoides) had five repeats. A lack of correlation between the number of repeat units and the phylogeny of Mus species indicates that independent mutations may have occurred throughout the evolution of specific mouse lineages. We suggest that the repetitive nature of the polymorphic sequence may predispose this region to slippage errors during DNA replication, resulting in frequent deletion/insertion mutations.     

Novel carbonic anhydrase (Car-2) allele in Spanish mice

A unique electrophoretic form of carbonic anhydrase is characteristic of some laboratory-maintained mice of the wild mouse species Mus spretus. This isozyme has been characterized by cellulose acetate electrophoresis and by isoelectric focusing. It is proposed that this isozyme be called CAR-2C and that its encoding allele be designated Car-2c. Fertile hybrids of Mus spretus and C57BL/6J (Car-2a) show both CAR-2A and CAR-2C bands of approximately equal intensity. The CAR-2C isozyme is readily identified by electrophoresis on 75-mm cellulose acetate strips because it migrates significantly faster than the isozymes of inbred mice, the CAR-2A and CAR-2B that do not separate from one another under standard conditions. Isoelectric focusing cleanly resolves all three of these CAR-2 forms. Mus hortulanus, although closely related to Mus spretus in other biochemical-genetic characteristics, has a CAR-2-homologous isozyme that is distinctly different from the CAR-2C of Mus spretus and from the isozymes of the common inbred strains.     

A molecular genetic linkage map of mouse chromosome 4 including the localization of several proto-oncogenes

We have constructed a 64-cM molecular genetic linkage map of mouse chromosome 4 using interspecific backcross animals derived from mating C57BL/6J and Mus spretus mice. Several proto-oncogenes and common sites of viral integration have been assigned regional locations on chromosome 4 including Mos, Lyn, Jun, Lmyc, Lck, Fgr, and Dsi-1. Additional loci mapped in this study to chromosome 4 were Tsha, Mup-1, Rrm2-ps1, Ifa, and Anf. A comparison of our mapping data with inbred strain mapping data did not show any evidence for inversions or deletions on chromosome 4. New regions of synteny were defined between mouse chromosome 4 and human chromosomes 1 and 8; a region of homology was found between mouse chromosome 4 and human chromosome 6. This linkage map will provide a framework for identifying homologous genes in mice and humans that may be involved in various disease processes.     

Conservation of the immunoglobulin C lambda 5 gene in the Mus gene.

A gene encoding the lambda 5 light chain constant region was isolated from a genomic library from the SPE mouse strain (C lambda 5S). SPE is an inbred wild mouse strain belonging to the Mus 3 or Mus spretus group that has been genetically isolated from Mus 1 (the group to which laboratory mice belong) for a period of 1-3 million years. The sequence of the C lambda 5S gene shows strong homology to C lambda 5 of (C57BL/6J x DBA/2)F1 both in the coding region (98% identity) and in the 5'- and 3'-flanking regions (98 and 95% identity, respectively). Sequence comparison of C lambda 5 genes with C lambda 1 of BALB/c shows only few substitutions in the C lambda 5 coding regions and suggests that the three genes have a common ancestor. These data indicate that the C lambda 5 gene has evolved under strong selective pressure and probably encodes a functional gene product. The conservation of the C lambda 5 gene in various Mus species was observed by high stringency Southern blot analyses using a C lambda 5S probe on DNA sample from members of four different groups of wild mice. All the laboratory and wild mouse strains tested, including those with amplified sets of C lambda 1 and C lambda 2 hybridizing sequences, showed only single C lambda 5 hybridizing fragments. Little variation in size of restriction fragments detected with the C lambda 5 probe was seen in the different Mus species suggesting a high degree of conservation.(ABSTRACT TRUNCATED AT 250 WORDS)