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.     

Localization of the human X-linked gene for chronic granulomatous disease to the mouse X chromosome: implications for X-chromosome evolution

The gene encoded at the human X-linked chronic granulomatous disease locus (cytochrome b245 beta subunit) has been mapped to the mouse X chromosome using an interspecific Mus domesticus x M. spretus cross. The localization of this gene provides detailed information on one of the proposed ancestral breakpoints that account for the divergent evolution of the mouse and human X chromosomes.     

Paternal Transmission of X-Linked Placental Dysplasia in Mouse Interspecific Hybrids

It has previously been shown that abnormal placental development, i.e., hyper- and hypoplasia, occurs in crosses and backcrosses between different mouse (Mus) species. These defects are caused mainly by abnormal growth of the spongiotrophoblast. The precise genetic basis for these placental malformations has not been determined. However, a locus that contributes to the abnormal development (Ihpd: interspecific hybrid placental dysplasia) has been mapped to the X chromosome. The X-chromosomal location of Ihpd and its site of action, that is the spongiotrophoblast, mean that normally only the maternally inherited Ihpd locus is active even in female fetuses. However, by making use of the X-chromosomal inversion In(X)1H, we have produced interspecific hybrid X(p)0, in which the active X chromosome was inherited from Mus macedonicus males. In contrast to XX female and XY male conceptuses from this cross, which have hypoplastic placentas, the X(p)0 female conceptuses have hyperplastic placentas. This finding supports the view that it is expression of the M. macedonicus Ihpd locus in the spongiotrophoblast that leads to hyperplasia due to an abnormal interaction with M. musculus autosomal loci.     

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.     

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.     

Mapping of FMR1, the gene implicated in fragile X-linked mental retardation, on the mouse X chromosome

A genetic map of the Cf-9 to Dmd region of the mouse X chromosome has been established by typing 100 offspring from a Mus musculus x Mus spretus interspecific backcross for the four loci Cf-9, Cdr, Gabra3, and Dmd. The following order and genetic distances in centimorgans were determined: (Cf-9)-2.4 +/- 1.7-(Cdr)-2.0 +/- 1.4-(Gabra3)-4.1 +/- 2.0-(Dmd). Six backcross offspring carrying X chromosomes with recombination events in the Cdr-Dmd region were identified. These recombination events were used to define the position of Fmr-1, the murine homologue of FMR1, which is the gene implicated in the fragile X syndrome in man, and that of DXS296h, the murine homologue of DXS296. Both Fmr-1 and DXS296h were mapped into the same recombination interval as Gabra3 on the mouse X chromosome. These findings provide strong support for the concept that the order of loci lying in the Cf-9 to Gabra3 segment of the X chromosome is highly conserved between human and mouse.     

Localization of the rhodopsin gene to the distal half of mouse chromosome 6

We have assigned the mouse rhodopsin gene, Rho, to chromosome 6 using DNA from a set of mouse-hamster somatic hybrid cell lines and a partial cDNA clone for mouse opsin. This assignment rules out the direct involvement of the rhodopsin gene in the known mouse mutations that produce retinal degeneration, including retinal degeneration slow (rds, chromosome 17), retinal degeneration (rd, chromosome 5), Purkinje cell degeneration (pcd, chromosome 13), and nervous (nr, chromosome 8). Segregation of Rho-specific DNA fragment differences among 50 animals from an interspecific backcross (C57BL/6J X Mus spretus) X C57BL/6J indicates that the Rho locus is 4.0 +/- 2.8 map units distal to the locus for the proto-oncogene Raf-1 and 18.0 +/- 5.4 map units proximal to the locus for the proto-oncogene Kras-2. Linkage to Raf-1 was confirmed using four sets of recombinant inbred strains. The two loci RAF1 and RHO are also syntenic on human chromosome 3, but on opposite arms.     

Genetic mapping in the region of the mouse X-inactivation center

The mouse X-inactivation center lies just distal to the T16H breakpoint. Utilizing pedigree analysis of backcross progeny from a Mus domesticus/Mus spretus interspecific cross, we have mapped a number of genetic loci, gene probes, microclones, and EagI linking clones distal to the T16H breakpoint. The genetic analysis provides a detailed genetic map in the vicinity of the mouse X-inactivation center. Comparative mapping data from the human X chromosome indicate that the most probable location of the mouse X-inactivation center is distal to Ccg-1 and in the region of the Pgk-1 locus. We report the assignment of two new loci, EM13 and DXSmh44, to the Ccg-1/Pgk-1 interval.     

Genetic and developmental analysis of X-inactivation in interspecific hybrid mice suggests a role for the Y chromosome in placental dysplasia

It has been shown previously that abnormal placental growth, i.e., hyper- and hypoplasia, occurs in crosses and backcrosses between different mouse (Mus) species. A locus that contributes to this abnormal development has been mapped to the X chromosome. Unexpectedly, an influence of fetal sex on placental development has been observed, in that placentas attached to male fetuses tended to exhibit a more pronounced phenotype than placentas attached to females. Here, we have analyzed this sex dependence in more detail. Our results show that differences between male and female placental weights are characteristic of interspecific matings and are not observed in intraspecific Mus musculus matings. The effect is retained in congenic lines that contain differing lengths of M. spretus-derived X chromosome. Expression of the X-linked gene Pgk1 from the maternal allele only and lack of overall activity of two paternally inherited X-linked transgenes indicate that reactivation or lack of inactivation of the paternal X chromosome in trophoblasts of interspecific hybrids is not a frequent occurrence. Thus, the difference between male and female placentas seems not to be caused by faulty preferential X-inactivation. Therefore, these data suggest that the sex difference of placental weights in interspecific hybrids is caused by interactions with the Y chromosome.     

Establishment of the mouse chromosome 7 region with homology to the myotonic dystrophy region of human chromosome 19q

A number of genetic markers, including ATP1A3, TGFB, CKMM, and PRKCG, define the genetic region on human chromosome 19 containing the myotonic dystrophy locus. These and a number of other DNA probes have been mapped to mouse chromosome 7 utilizing a mouse Mus domesticus/Mus spretus interspecific backcross segregating for the genetic markers pink-eye dilution (p) and chinchilla (cch). The establishment of a highly syntenic group conserved between mouse chromosome 7 and human chromosome 19q indicates the likely position of the homologous gene locus to the human myotonic dystrophy gene on proximal mouse chromosome 7. In addition, we have mapped the muscle ryanodine receptor gene (Ryr) to mouse chromosome 7 and demonstrated its close linkage to the Atpa-2, Tgfb-1, and Ckmm cluster of genes. In humans, the malignant hyperthermia susceptibility locus (MHS) also maps close to this gene cluster. The comparative mapping data support Ryr as a candidate gene for MHS.     

Linkage analysis of the properdin deficiency gene: suggestion of a locus in the proximal part of the short arm of the X chromosome

Properdin is a component of the alternative pathway of complement activation. Inherited deficiency of the protein predisposes an individual to develop meningococcal disease. A family segregating for properdin deficiency (McKNo 31206), in a manner consistent with X-linked recessive inheritance, was studied by RFLP analysis using 24 X-chromosome-specific DNA probes of known regional assignments. Linkage was observed to the OTC locus and the DXS7 locus. These results suggest that the properdin gene is located on the short arm of the X chromosome in the region Xp21.1-Xcen.     

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.     

A new common integration region (int-3) for mouse mammary tumor virus on mouse chromosome 17.

Mus musculus subsp. musculus (Czech II) mammary tumor DNA frequently contains an integrated proviral genome of the mouse mammary tumor virus (MMTV) within a specific 0.5-kilobase-pair region of the cellular genome (designated int-3). Viral integration at this site results in activation of expression of an adjacent cellular gene. We mapped int-3 to mouse chromosome 17 by analysis of PstI-restricted cellular DNAs from mouse-hamster somatic cell hybrids. Restriction analysis of cellular DNA from (C3H/OuJ X Czech II) X Czech II backcross mice established the gene order T-H-2-int-3. These results demonstrated that the int-3 locus is distinct from two other common integration regions for mouse mammary tumor virus (designated int-1 and int-2) in mammary tumor DNA and suggest that several cellular genes may be at risk for virally induced activation during mammary tumor development.     

Neutron and X-ray scattering studies on the human complement protein properdin provide an analysis of the thrombospondin repeat

Properdin is a regulatory glycoprotein of the alternative pathway of the complement system of immune defense. It is responsible for the stabilization of the C3 convertase complex formed between C3b and the Bb fragment of factor B. Neutron and X-ray solution scattering experiments were performed on the dimeric and trimeric forms of properdin. These have RG values of 9.1 and 10.7 nm, respectively. The scattering curves were compared with Debye sphere modeling simulations for properdin. Good agreements were obtained for models similar to published electron micrographs showing that the properdin trimer has a triangular structure with sides of 26 nm. Such a structure also accounted for sedimentation coefficient data on properdin. Primary structure analyses for mouse and human properdin have shown that this contains six homologous motifs known as the thrombospondin repeat (TSR), which is the second most abundant domain type found in the complement proteins. Sequences for these 12 TSRs were aligned with 19 others found in thrombospondin and the late complement components. Three distinct groups of TSRs were identified, namely, the TSRs found in thrombospondin and properdin, the TSRs mostly found at the N-terminus of the late complement components, and the TSRs found at the C-terminus of the late components. Averaged secondary structure predictions suggested that all three groups contain similar backbone structures with two amphipathic turn regions and one hydrophilic beta-strand region. The mean dimensions of the TSRs of properdin in solution were determined to be approximately 4 nm X 1.7 nm X 1.7 nm, showing that these are elongated in structure.     

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.     

Mapping of mouse carbonic anhydrase-3, Car-3: another locus in the homologous region of mouse chromosome 3 and human chromosome 8

At least six separate genes determining tissue- and organelle-specific isoforms of carbonic anhydrase are known. We have determined the chromosome location of one of these genes, carbonic anhydrase-3 (Car-3), in the mouse and carried out a linkage analysis of Car-1, Car-2, and Car-3. Car-3 has been assigned to band 3A2 by in situ hybridization. We identified a PstI restriction fragment length polymorphism between Mus spretus and Mus mus domesticus and, by using an interspecific backcross, showed that Car-3 is 2.4 +/- 1.7% SE from both Car-1 and Car-2, calculating genetic distance as percentage recombination. No recombinants were found between Car-1 and Car-2 in 100 backcross offspring, and when these data are combined with earlier results, these two loci are estimated to be 1.2 cM from each other at the 95% confidence interval. The three homologous carbonic anhydrase loci in man had earlier been assigned to 8q22, and the finding of linkage of Car-3 to Car-1 and Car-2 in the mouse adds another locus to the conserved segments on mouse chromosome 3 and human chromosome 8.     

Mapping of the glycine receptor alpha 2-subunit gene and the GABAA alpha 3-subunit gene on the mouse X chromosome.

We have mapped the gene for the alpha 2-subunit of the inhibitory glycine receptor (Glra2) to the telomeric end of the mouse X chromosome by backcross analysis of a Mus musculus/Mus spretus interspecific cross. In addition, we have extended the mapping of the GABAA alpha 3-subunit receptor gene (Gabra3). A deduced gene order of cen-Cybb-Hprt-DXPas6-Gabra3-Rsvp-Gdx/Cf-8- Dmd-Pgk-1-DXPas2-Plp-DXPas1-Glra2-tel places Gabra3 proximal to the visual pigment gene Rsvp and Glra2 in the region of loci for hypophosphatemia (Hyp), steroid sulfatase (Sts), and the E1 alpha-subunit of pyruvate dehydrogenase (Pdha1). This establishes the XF region of the mouse X chromosome as homologous with the Xp22.1-p22.3 region of the human X chromosome and indicates the presence of an evolutionary breakpoint in the region of Xp21.3.     

New polymorphic markers in the vicinity of the pearl locus on mouse Chromosome 13

We have used a Mus domesticus/-Mus spretus congenic animal that was selected for retention of Mus spretus DNA around the pearl locus to create a highly polymorphic region suitable for screening new markers. Representation difference analysis (RDA) was performed with either DNA from the congenic animal or C57BL/6J as the driver for subtraction. Four clones were identified, characterized, and converted to PCR-based polymorphic markers. Three of the four markers equally subdivide a 10-cM interval containing the pearl locus, with the fourth located centromeric to it. These markers have been placed on the mouse genetic map by use of an interspecific backcross panel between Mus domesticus (C57BL/6J) and Mus spretus generated by The Jackson Laboratory. Received: 12 June 1995 / Accepted: 17 August 1995