Multilocus molecular mapping of the mouse X chromosome

Using restriction fragment length polymorphisms (RFLPs) and enzymatic variants between distantly related mouse species, we have assigned three genes to the mouse X chromosome and concurrently mapped a total of eight genes spanning an estimated 50 cM of the chromosome. Segregation of RFLPs in over 200 male progeny from interspecies backcrosses between the inbred strain C57BL/6JRos and either wild-derived Mus musculus or Mus spretus was followed for the murine genes Timp (tissue inhibitor of metalloproteinases), Cf-8 (coagulation factor VIII), and Rsvp (red-sensitive visual pigment) and the known X-linked markers Otc, Hprt, Cf-9, G6pd, and Ags. From the centromere, the gene order was defined as Otc, Timp, Hprt, Cf-9, (Cf-8/Rsvp/G6pd), Ags, by minimizing the number of multiple recombinational events. No significant differences in map order or frequency of recombination were observed between the two backcross series studied. The use of Southern analysis has allowed us to add new genes to the map in a cumulative manner, and as probes become available, additional markers can be mapped, using the same set of mice, by utilizing existing blots or resampling the DNAs. The use of probes for functional genes has allowed us to directly compare the X chromosomes of mouse and man and has provided insight into chromosomal rearrangements which have occurred during the evolutionary divergence of these species, as well as to define the extent of linkage homologies.     

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.     

Mapping of Abll within a conserved linkage group on distal mouse chromosome 1 syntenic with human chromosome 1 using an interspecific cross

A human Abelson related gene (ABLL) cDNA clone was used to detect restriction fragment length polymorphisms (RFLPs) on mouse Southern blots. Abll was mapped to mouse chromosome 1 by analysis of segregation with other distal chromosome 1 genetic polymorphisms by using a panel of DNAs from [(C3H/HeJ-gld/gld x Mus spretus) F1 x C3H/HeJ-gld/gld] interspecific backcross mice. The data indicate the following gene order: (centromere)-CD45-6.5 cM-Lamb-2-1 cM-Abll-2 cM-At-3. The results extend the analysis of a large conserved linkage group spanning nearly 30 cM on distal mouse chromosome 1 syntenic with human chromosome 1q21-32. Within this linkage group similar relative positions have been characterized in both species for C4BP, REN, CD45, LAMB2, ABLL, AT3, APOA2, and SPTA.     

The properdin structural locus (Pfc) lies close to the locus for tissue inhibitor of metallothionine proteases (Timp) on the mouse X chromosome

We have confirmed the assignment of the structural locus of the complement factor properdin (Pfc) to the mouse X chromosome and mapped it between monoamine oxidase-A (Mao-a) and hypoxanthine phosphoribosyltransferase (Hprt) using a Mus spretus x Mus musculus interspecific backcross of 108 animals. The structural locus for murine tissue inhibitor of metallothionine proteases (Timp) could not be separated from properdin in a panel of 18 recombinant animals. By minimizing the number of double recombinants the following gene order was obtained: Otc-Mao-a-(Pfc, Timp)-Hprt-Cf-9. The implications for comparative mapping of human and mouse X chromosomes are discussed.     

Conservation and reorganization of loci on the mammalian X chromosome: a molecular framework for the identification of homologous subchromosomal regions in man and mouse

By means of cross-reacting molecular probes, some 18 loci specific for the X chromosome of both man and mouse have been localized on the mouse X chromosome using an interspecific mouse cross involving the inbred SPE/Pas strain derived from Mus spretus. Comparison of the localizations of these loci on the mouse X with their positions on the human X chromosome suggests that intrachromosomal rearrangements involving at least five X chromosome breakage events must have occurred during the period of evolutionary divergence separating primates from rodents. Within the five blocks of chromosomal material so defined, there is for the moment little or no evidence that either chromosomal inversion events or extensive rearrangements have occurred. These data confirm the remarkable evolutionary conservation of the X chromosome apparent in mammalian species, compared to autosomal synteny groups in which both inter- and intrachromosomal rearrangement events appear to have occurred frequently. The breakage events described here for the X chromosome should therefore provide a minimal estimate for the frequency of chromosomal rearrangement events, such as breakage and inversion, which have affected autosomal synteny groups during the evolutionary period separating man from mouse. The definition of the number of chromosome breakage events by which the X chromosomes of these species differ, together with their localization, provides a framework for the use of interspecies mouse crosses for further detailed mapping of particular subchromosomal regions of the human X chromosome and for defining loci in the mouse homologous to those implicated in human congenital diseases.     

A glutaminase (gis) gene maps to mouse chromosome 1, rat chromosome 9, and human chromosome 2

A rat cDNA clone encoding a portion of phosphate-activated glutaminase was used to identify DNA restriction fragment length polymorphisms (RFLPs) in sets of somatic cell hybrids and between wild-derived and inbred strains of mice. Segregation of rat and mouse chromosomes among somatic cell hybrids indicated assignment to rat chromosome 9 and mouse chromosome 1. Analysis of chromosome 1 alleles for several genes in an interspecific cross between Mus spretus and C3H/HeJ-gld/gld mice indicates that glutaminase can be positioned within 5.5 +/- 2.0 cM proximal to Ctla-4. Similarly, human-hamster somatic cell hybrids were examined for RFLPs, and four human EcoRI restriction fragments were found to hybridize with the rat glutaminase probe. Two of these restriction fragments cosegregated and mapped to human chromosome 2 in a region that is syntenic with mouse chromosome 1 and rat chromosome 9.     

Characterization of a set of X-linked sequences and of a panel of somatic cell hybrids useful for the regional mapping of the human X chromosome

Summary We have characterized 19 DNA fragments originating from the human X chromosome. Most of them have been isolated from an X chromosome genomic library (Davies et al. 1981) using a systematic screening procedure. These DNA probes have been used to search for restriction fragment length polymorphisms (RFLP). The frequency of restriction polymorphisms (1 per 350 bp analysed) was lower than expected from data obtained with autosomal fragments. The various probes have been mapped within 12 subchromosomal regions using a panel of human-rodent hybrid cell lines. The validity of the panel was established by hybridization experiments performed with 27 X-specific DNA probes, which yielded information on the relative position of translocation break-points on the X chromosome. The DNAs from the various hybrid lines are blotted onto a reusable support which allows one to quickly map any new X-specific DNA fragment. The probes already isolated should be of use to map unbalanced X chromosome aberrations or to characterize new somatic cell hybrid lines. The probes which detect RFLPs define new genetic markers which will help to construct a detailed linkage map of the human X chromosome, and might also serve for the diagnosis of carriers or prenatal diagnosis.     

cDNA sequence and chromosomal localization of the mouse parvalbumin gene, Pva

In the homozygous condition, the mutation adr (arrested development of righting response) of the mouse causes a myotonia and a drastic reduction of the Ca2+-binding protein parvalbumin (PV) in fast muscles. Using a rat PV probe, a mouse cDNA clone was isolated from a lambda gt11 wild-type fast-muscle library and its nucleotide sequence was determined. The protein coding and the 3' nontranslated regions of the mouse gene show extensive homology with the rat PV gene. The result of Southern blot hybridization is consistent with a single copy gene for parvalbumin. Restriction fragment length polymorphisms (RFLPs) between Mus musculus domesticus (e.g. C57BL/6) and Mus spretus (SPE) were detected with the enzymes Eco RI, Pst I, and Sst I. The restriction fragment patterns of DNA samples from 65 individual offspring of (C57BL/6 x SPE)F1 x C57BL/6 backcrosses were tested with the PV probe and matched, for linkage detection, to pre-existing patterns established with various RFLP probes on the same samples. A co-distribution of PV-RFLPs with Pvt-1 and Mlvi-2, which had been localized on chromosome 15, was detected. Thus, the structural gene for PV, designated Pva, maps to chromosome 15 of the mouse whereas the adr mutation shows no linkage with markers on this chromosome. Gene locus homology between chromosome 15 of the mouse and chromosome 22 of man (which carries the human PV gene) is discussed.     

Determination of a molecular map position for Hyp using a new interspecific backcross produced by in vitro fertilization.

We have established a Mus spretus/Mus musculus domesticus interspecific backcross segregating for two X-linked mutant genes, Ta and Hyp, using in vitro fertilization. The haplotype of the recombinant X chromosome of each of 241 backcross progeny has been established using the X-linked anchor loci Otc, Hprt, Dmd, Pgk-1, and Amg and the additional probes DXSmh43 and Cbx-rs1. The Hyp locus (putative homologue of the human disease gene hypophosphatemic rickets, HYP) has been incorporated into the molecular genetic map of the X chromosome. We show that the most likely gene order in the distal portion of the mouse X chromosome is Pgk-1-DXSmh43-Hyp-Cbx-rs1-Amg, from proximal to distal. The distance in centimorgans (mean +/- SE) between DXSmh43 and Hyp was 2.52 +/- 1.4 and that between Hyp and Cbx-rs1 was 1.98 +/- 1.39. Thus closely linked flanking markers for the Hyp locus that will facilitate the molecular characterization of the gene itself have been defined.     

Molecular genetic linkage maps of mouse chromosomes 4 and 6.

We have generated a moderate resolution genetic map of mouse chromosomes 4 and 6 utilizing a (C57BL/6J x Mus spretus) F1 x Mus spretus backcross with RFLPs for 31 probes. The map for chromosome 4 covers 77 cM and details a large region of homology to human chromosome 1p. The map establishes the breakpoints in the mouse 4-human 1p region of homology to a 2-cM interval between Ifa and Jun in mouse and to the interval between JUN and ACADM in human. The map for mouse chromosome 6 spans a 65-cM region and contains a large region of homology to human 7q. These maps also provide chromosomal assignment and order for a number of previously unmapped probes. The maps should allow the rapid regional assignment of new markers to mouse chromosomes 4 and 6. In addition, knowledge of the gene order in mouse may prove useful in determining the gene order of the homologous regions in human.     

Localization of the cryptdin locus on mouse chromosome 8

Cryptdin is a defensin-related peptide, and its mRNA accumulates to high abundance in epithelial cells of intestinal crypts beginning in the second week of postnatal development. The cryptdin (Defcr) locus was assigned to mouse chromosome 8 by Southern blotting of DNAs from mouse/hamster somatic hybrid cell lines. Analysis of somatic hybrid DNAs for mouse-specific restriction fragments showed zero discordance and perfect concordance with chromosome 8. The Defcr locus was localized on chromosome 8 by analysis of DNAs from recombinant inbred (RI) strains of mice after identification of three potential Defcr alleles based on restriction fragment length polymorphisms (RFLPs) in inbred strains. The strain distribution patterns of the Defcr locus were compared with those of chromosome 8 markers in five panels of RI strains. Analysis of cosegregation of Defcr with xenotropic proviral locus Xmv-26 and additional loci confirmed the chromosomal assignment and showed that Defcr is on proximal chromosome 8 within approximately 6 (1.3 to 21.3) cM of Xmv-26. The mouse Defcr locus and the human defensin gene(s) located on chromosome 8p23 appear to map to homologous regions.     

The construction of human somatic cell hybrids containing portions of the mouse X chromosome and their use to generate DNA probes via interspersed repetitive sequence polymerase chain reaction

Interspersed repetitive sequence polymerase chain reaction (IRS-PCR) has become a powerful tool for the rapid generation of DNA probes from human chromosomes present in rodent somatic cell hybrids. We have constructed a somatic cell hybrid containing a major portion of the mouse X chromosome in a human background (clone 8.0). IRS-PCR was developed for the specific amplification of mouse DNA using either of two primers from the rodent-specific portion of the murine B1 repeat. Amplification was subsequently performed with clone 8.0 and a subclone, 8.1/1, which retains a small murine X-chromosomal fragment including Hprt and the Gdx locus. A total of 15-20 discrete PCR products ranging in size from less than 500 to greater than 3000 bp were obtained from clone 8.0 with each primer. In clone 8.1/1, a subset of these bands plus some additional bands were observed. Nine bands amplified from clone 8.1/1 have been excised from gels and used as probes on Southern blots. All of the fragments behaved as single-copy probes and detected domesticus/spretus variation. They have been regionally mapped using an interspecific backcross. The probe locations are compatible with those of markers known to be present in clone 8.1/1. These results demonstrate the feasibility of this method as applied to the mouse genome and the high likelihood of generating useful DNA probes from a targeted region.     

Assignment of estradiol receptor gene to mouse chromosome 10

Differences in restriction fragment lengths were detected with murine estrogen receptor cDNA (clone MOR-100) between Chinese hamster and mouse. These were used to determine the chromosomal location of the estrogen receptor in the mouse by Southern blot analysis of DNAs obtained from a panel of mouse-Chinese hamster somatic cell hybrids. The mouse estrogen receptor gene was localized on mouse chromosome 10.     

High-density molecular map of the central span of the mouse X chromosome.

A total of 17 linking clones previously sublocalized to the central span of the mouse X chromosome have been ordered by detailed analysis through interspecific Mus spretus/Mus musculus domesticus backcross progeny. These probes have been positioned with respect to existing DNA markers utilizing a new interspecific backcross segregating for the Tabby (Ta) locus. The density of clones within this 11.5-cM interval is now, on average, one clone every 1000 kb. This high-density map provides probes in the vicinity of a number of important genetic loci in this region which include the X-inactivation center, the Ta locus, and the mottled (Mo) locus, and therefore provides a molecular framework for identification of the genes encoded at these loci.     

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.     

Efficient linkage of 10 loci in the proximal region of the mouse X chromosome

Interspecific Mus species crosses were used to construct a multilocus genetic map of the mouse X chromosome that extends for more than 50 cM. In these studies, we established the segregation of eight loci in more than 200 backcross progeny from crosses of M. musculus and M. spretus with a common inbred strain (C57BL/6JRos). Genetic divergence at the level of the nucleotide sequences makes these crosses a useful cumulative genetic resource for mapping additional genes defined by genomic or cDNA probes in a highly efficient manner. We have therefore devised a mapping strategy that uses a subset of these backcrosses that are recombinant between successive anchor loci to both localize and order an additional set of six genes without necessarily resorting to an analysis of the entire backcross series. Using this approach, we have defined the linkage of cytochrome b245 beta-chain (Cybb), synapsin (Syn-1), and two members of the X-linked lymphocyte-regulated gene family (Xlr-1, Xlr-2), as well as DXSmh141 and DXSmh172, two loci defined by random genomic probes. All six loci have been localized to the proximal portion of the mouse X chromosome and their order has been defined as Cybb, Otc, Syn-1/Timp, DXSmh141/Xlr-1, DXSmh172, Hprt, Xlr-2, Cf-9. Gene order was established by minimizing multiple recombination events across the region spanning an estimated 20 cM of the proximal X chromosome. The possible significance of the Xlr loci is discussed with respect to other X-chromosome loci that regulate the immune response.     

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.     

Construction and analysis of linking libraries from the mouse X chromosome

A hybrid cell line containing the mouse X chromosome on a human background has been used to construct linking libraries from the mouse X chromosome, and approximately 250 unique EagI and NotI clones have been identified. Seventy-three clones have been sublocalized onto the X chromosome using interspecific Mus spretus/Mus domesticus crosses and a panel of somatic cell hybrids carrying one-half of reciprocal X-autosome translocations. The average spacing of the linking clones mapped to date is about one every 2 Mb of DNA. Two clones from the central region of the chromosome have been physically linked by pulsed-field gel electrophoresis. A large number of clones contain conserved sequences, indicating the presence of CpG-rich island-associated genes. The clones isolated from these libraries provide a valuable resource for comparative mapping between man and mouse X chromosomes, isolation of X-linked disease loci of interest by reverse genetics, and analysis of the long-range structure and organization of the chromosome.     

Genetic dissection of X-linked interspecific hybrid placental dysplasia in congenic mouse strains.

Interspecific hybridization in the genus Mus results in male sterility and X-linked placental dysplasia. We have generated several congenic laboratory mouse lines (Mus musculus) in which different parts of the maternal X chromosome were derived from M. spretus. A strict positive correlation between placental weight and length of the M. spretus-derived part of the X chromosome was shown. Detailed analysis was carried out with one congenic strain that retained a M. spretus interval between 12.0 and 30.74 cM. This strain consistently produced hyperplastic placentas that exhibited an average weight increase of 180% over the weight of control placentas. In derived subcongenic strains, however, increased placental weight could no longer be observed. Morphometric analysis of these placentas revealed persistence of abnormal morphology. Fully developed placental hyperplasia could be reconstituted by recombination of proximal and central M. spretus intervals with an intervening M. musculus region. These results may suggest that placental dysplasia of interspecific mouse hybrids is caused by multiple loci clustered on the X chromosome that act synergistically. Alternatively, it is possible that changes in chromatin structure in interspecific hybrids that influence gene expression are dependent on the length of the alien chromosome.     

Construction of a detailed molecular map of the mouse X chromosome by microcloning and interspecific crosses.

A large number of microclones obtained by microdissection of the mouse X chromosome have been mapped using an interspecific Mus domesticus/Mus spretus cross. Clones displaying close linkage to a number of loci of known phenotype but unknown gene product, such as mdx (X-linked muscular dystrophy), have been obtained. Over a central 30 cM span of the mouse X chromosome, 17 clones have been mapped and ordered at a sufficient density to contemplate the complete physical mapping of this region that will aid in the isolation of a number of unidentified genes. Some of the mapped microclones detect moderately repetitive sequences that were clustered in several discrete regions of the mouse X chromosome.