Genetic mapping on the mouse X chromosome of human cDNA clones for the fragile X and Hunter syndromes.

Murine X-linked genes corresponding to the human Fragile X (FMR1) and Hunter syndrome (IDS) loci have been mapped in an interspecific backcross between B6CBA-Aw-J/A-Bpa and Mus spretus using human cDNA clones. Pedigree analysis of recombinants from a total of 248 backcross progeny favors a gene order of (Cf-9, Mcf-2)-(Fmr-1)-Ids-Gabra3-Rsvp. Gene order is conserved between the species, although no fragile site has been detected in the mouse in this region of the murine X chromosome.     

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 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.     

Extension of the physical map in the region of the mouse X chromosome homologous to human Xq28 and identification of an exception to conserved linkage.

We have extended our pulsed-field gel map of the region of the mouse X chromosome homologous to human Xq28 to include the loci Gdx (DXS254Eh), P3 (DXS253Eh), G6pd, Cf-8, and F8a. Gdx, P3, and G6pd are demonstrated to be physically linked to the X-linked visual pigment locus (Rsvp) within a maximal distance of 340 kb, while G6pd and Cf-8 are approximately 900 kb apart. These studies favor a gene order of cen-Rsvp-Gdx-P3-G6pd-(Cf-8)-tel and extend the physical map of this region to 5 million bp. In conjunction with previous physical mapping studies in both mouse and human, the results suggest conserved linkage for loci in this region of the mouse X chromosome and human Xq28. However, employing pulsed-field gel electrophoresis and genetic pedigree analysis of interspecific backcross progeny, we have found close linkage of a clone encoding a mouse homolog for human factor VIII-associated gene A (F8A) to DXPas8, thus revealing the first exception to conserved gene order between murine and human loci in the region.     

Comparison of the physical and recombination maps of the mouse X chromosome

The locations of five random mouse genomic DNA markers and five cloned genes, including the genes for clotting factors VIII and IX (Cf-8 and Cf-9), Duchenne muscular dystrophy (Dmd), phosphoglycerate kinase-1 (Pgk-1), and alpha-galactosidase (Ags), on the mouse X chromosome were determined by in situ hybridization. The five random DNA markers provide new genetic loci with useful restriction fragment length polymorphisms between mouse strains and species, including one locus close to the centromeric region of the mouse X chromosome. The physical map and the recombination map of these loci on the X chromosome were compared. There was good agreement in the order of loci. Relative distances between loci were consistent along the X chromosome, with the exception of the telomeric end of the long arm, where the recombination fraction observed between loci closely associated on the physical map was higher than that between similarly spaced markers located in the proximal region of the X chromosome. These results are discussed in comparison to the human X-chromosome map.     

Mapping of aminoacylase-1 and beta-galactosidase-A to homologous regions of human chromosome 3 and mouse chromosome 9 suggests location of additional genes.

Conserved linkage groups have been found on the X and autosomal chromosomes in several mammalian species. The identification of conserved chromosomal regions has potential for predicting gene location in mammals, particularly in humans. The genes for human aminoacylase-1 (ACY1, N-acylamino acid aminohydrolase, E.C.3.5.1.14), an enzyme in amino acid metabolism, and beta-galactosidase-A (GLB1, E.C.3.2.1.23), deficient in GM1-gangliosidosis, have been assigned to human chromosome 3. Using human-mouse somatic cell hybrids segregating translocations of human chromosome 3, expression of both ACY1 and GLB1 correlated with the presence of the p21 leads to q21 region of chromosome 3. In a previous study, assignment of these genes to mouse chromosome 9 used mouse-Chinese hamster somatic cell hybrids, eliminating mouse chromosomes. To approximate the size of the conserved region in the mouse, experiments were performed with recombinant inbred mouse strains. An electrophoretic variant of ACY-1 in mouse strains was used to map the Acy-1 gene 10.7 map U from the beta-galactosidase locus. These data suggest that there is a region of homology within the p21 leads to q21 region of human chromosome 3 and a segment of mouse chromosome 9. Since the mouse transferrin gene (Trf) is closely linked to the aminoacylase and beta-galactosidase loci, we predict that the human transferrin (TF) gene is on chromosome 3.     

Comparative mapping of Z-orthologous genes in vertebrates: implications for the evolution of avian sex chromosomes

Sex chromosomes of birds and mammals are highly differentiated and share several cytological features. However, comparative gene mapping reveals extensive conserved synteny between the chicken Z sex chromosome and human chromosome 9 but not the human X sex chromosome, implying an independent origin of avian and mammalian sex chromosomes. To better understand the evolution of the avian Z chromosome we analysed the synteny of chicken Z-linked genes in zebrafish, which is the best-mapped teleost genome so far. Existing zebrafish maps do not support the existence of an ancestral Z linkage group in the zebrafish genome, whereas mammalian X-linked genes show at least some degree of synteny conservation. This is consistent with in situ hybridisation mapping data in the freshwater pufferfish, Tetraodon nigroviridis where mammalian X-linked genes show a much higher degree of conserved synteny than human chromosome 9 or the avian Z chromosome. Collectively, these data argue in favour of a more recent evolution of the avian Z chromosome, compared with the mammalian X.     

Linkage assignment of a DNA sequence (ERCC2L1) homologous to a human DNA repair gene in Xiphophorus fishes: implications for the evolutionary derivation of human chromosome 19.

Fish gene mapping studies have identified several syntenic groups showing conservation over more than 400 million years of vertebrate evolution. In particular, Xiphophorus linkage group IV has been identified as a homolog of human chromosomes 15 and 19. During mammalian evolution, loci coding for glucosephosphate isomerase, peptidase D, muscle creatine kinase, and several DNA repair genes (ERCC1, ERCC2, and XRCC1) appear as a conserved syntenic group on human chromosome 19. When X. clemenciae and X. milleri PstI endonuclease-digested genomic DNA was used in Southern analysis with a human ERCC2 DNA repair gene probe, a strongly cross-hybridizing restriction fragment length polymorphism was observed. Backcrosses to X. clemenciae from X. milleri x X. clemenciae F1 hybrids allowed tests for linkage of the ERCC2-like polymorphism to markers covering a large proportion of the genome. Statistically significant evidence for linkage was found only for ERCC2L1 and CKM (muscle creatine kinase), with a total of 41 parents and 2 recombinants (4.7% recombination, chi 2 = 35.37, P less than 0.001); no evidence for linkage to GPI and PEPD in linkage group IV was detected. The human chromosome 19 synteny of ERCC2 and CKM thus appears to be conserved in Xiphophorus, while other genes located nearby on human chromosome 19 are in a separate linkage group in this fish. If Xiphophorus gene arrangements prove to be primitive, human chromosome 19 may have arisen from chromosome fusion or translocation events at some point since divergence of mammals and fishes from a common ancestor.     

X-linked genetic homologies between mouse and man

X-linked genes are conserved among all mammalian species, but the organization of genes on the X chromosome varies from one species to another. This review summarizes the evidence for established gene homologies between mice and human beings. It also describes genes that are possible homologies because of their locations in the human and murine X chromosomes and similarities in the phenotypes they produce. Based on current knowledge of homologous gene location, the human and murine X chromosomes appear to contain four highly conserved segments and differ in organization by only three to four simple chromosomal rearrangements.     

Pulse-field linkage of the P3, G6pd and Cf-8 genes on the mouse X chromosome: demonstration of synteny at the physical level.

Utilising pulse-field gel electrophoresis physical linkage between three mouse X-linked genes has been demonstrated. The three genes, P3, G6pd and Cf-8 all lie within 400 Kb of DNA. This physical linkage mirrors the situation on the human X chromosome, representing the first demonstration of mouse/human synteny at the physical level. A detailed physical map encompassing 1.6 Mbp of this region is presented. A number of the rare cutter restriction enzyme sites within this map are partially blocked on the inactive X chromosome, presumably due to the methylation of CpG rich islands. Pulsed field gel electrophoresis therefore provides a useful tool for the study of X-inactivation over large regions of the X chromosome.     

Localization by in situ hybridization of the coagulation factor IX gene and of two polymorphic DNA probes with respect to the fragile X site

Summary The coagulation factor IX gene and two other polymorphic loci corresponding to DNA probes 52 A and St 14 have been previously localized in the q27 to qter region of the human X chromosome. In order to study their localization with respect to the fragile site at Xq27-28, we have hybridized the three DNA probes to metaphase chromosomes of a boy with fragile X mental retardation. We show that probe 52A is located in the proximal part of the Xq27 band, while the coagulation factor IX gene is on the distal part of this band, but proximal to the fragile site. The very polymorphic St 14 probe is located in the distal part of the Xq28 band, on the other side of the fragile site.     

Comparative mapping of mouse and rat chromosomes by fluorescence in situ hybridization

Comparative fluorescence in situ hybridization mapping using DNA libraries from flow-sorted mouse chromosomes and region-specific mouse BAC clones on rat chromosomes reveals chromosomal homologies between mouse (Mus musculus, MMU) and rat (Rattus norvegicus, RNO). Each of the MMU 2, 3, 4, 6, 7, 9, 12, 14, 15, 16, 18, 19, and X chromosomes paints only a single rat chromosome or chromosome segment and, thus, the chromosomes are largely conserved between the two species. In contrast, the painting probes for MMU chromosomes 1, 5, 8, 10, 11, 13, and 17 produce split hybridization signals in the rat, disclosing evolutionary chromosome rearrangements. Comparative mapping data delineate several large linkage groups on RNO 1, 2, 4, 7, and 14 that are conserved in human but diverged in the mouse. On the other hand, there are linkage groups in the mouse, i.e., on MMU 1, 8, 10, and 11, that are disrupted in both rat and human. In addition, we have hybridized probes for Nap2, p57, Igf2, H19, and Sh3d2c from MMU 7 to RNO 1q and found the orientation of the imprinting gene cluster and Sh3d2c to be the same in mouse and rat. Hybridization of rat genomic DNA shows blocks of (rat-specific) repetitive sequences in the pericentromeric region of RNO chromosomes 3-5, 7-13, and 20; on the short arms of RNO chromosomes 3, 12, and 13; and on the entire Y chromosome.     

The genes of the lysosomal cysteine proteinases cathepsin B, H, L, and S map to different mouse chromosomes

The mouse genes for the lysosomal cysteine proteinases cathepsin B, H, L, and S were mapped to Chromosomes (Chrs) 14, 9, 13, and 3, respectively. Two of the DNA probes used in this study detected an additional, independently segregating locus. The cathepsin B-specific probe hybridized to a locus on Chr 2, and the cathepsin H probe to a locus on the X Chr. These loci either correspond to pseudogenes or to cathepsin B- and cathepsin H-related genes. The four cysteine proteinases mapped in this study lie within known regions of conserved synteny between mouse and human chromosomes, when compared with the corresponding positions of their human homologs. Assuming that the genes of the cysteine proteinase gene family arose from a common ancestral gene, our results suggest that these four cysteine proteinases had been dispersed over different chromosomes before separation of mouse and human in evolution. Received: 22 August 1996 / Accepted: 20 November 1996     

Definition of mouse chromosome 1 and 3 gene linkage groups that are conserved on human chromosome 1: Evidence that a conserved linkage group spans the centromere of human chromosome 1

Comparative mapping between the human and the mouse genomes allows characterization of linkage groups that have been conserved over evolution. In this study, genes previously localized to adjacent regions of human chromosome 1 were mapped to discrete regions on distal mouse chromosomes 1 and 3 using an interspecific cross. Linkage analysis in mouse defined two groups in which the gene order appears to be the same as that in humans: 15 genes localized between human chromosome 1q21 and 1q32 were found to span 29.5 cM on distal mouse chromosome 1; 6 genes localized between human chromosome 1q21 and 1p22 spanned 15.6 cM on distal mouse chromosome 3. These data suggest that gene order within large chromosome segments may remain stable over long periods of evolution and that the position of the centromere may reflect a late event in the evolution of higher eukaryotic organisms. These studies provide a model for examination of specific evolutionary events.     

The pronatriodilatin gene is located on the distal short arm of human chromosome 1 and on mouse chromosome 4.

Atrial natriuretic factors (ANF) are polypeptides having natriuretic, diuretic, and smooth muscle-relaxing activities that are synthesized from a single larger precursor: pronatriodilatin. Chromosomal assignment of the gene coding for human pronatriodilatin was accomplished by in situ hybridization of a [3H]-labeled pronatriodilatin probe to human chromosome preparations and by Southern blot analysis of somatic cell hybrid DNAs with normal and rearranged chromosomes 1. The human pronatriodilatin gene was mapped to the distal short arm of chromosome 1, in band 1p36. Southern blot analysis of mouse X Chinese hamster somatic cell hybrids was used to assign the mouse pronatriodilatin gene to chromosome 4. This assignment adds another locus to the conserved syntenic group of homologous genes located on the distal half of the short arm of human chromosome 1 and on mouse chromosome 4.     

Linkage of a gene for neural cell adhesion molecule, L1 (CamL1) to the Rsvp region of the mouse X chromosome

L1 is a glycoprotein with an apparent molecular weight of 200 kDa in the developing fetus and adult central nervous system. In the peripheral nervous system, it has a molecular weight of 230 kDa. The L1 protein appears to be encoded by a single gene that has been located on the human X chromosome by in situ hybridization. In this paper we describe restriction variation in genomic DNA Southern analysis between Mus species for the K13 cDNA probe for the L1 neural cell adhesion molecule. We have designated the locus described by this variation as cell adhesion molecule L1, CamL1. The X chromosome linkage and the relative position on the X chromosome coincident with the genes Rsvp/G6pd/Cf-8 were defined in backcross matings involving M. spretus and M. musculus.     

Gene-based anchoring of the rat genetic linkage and cytogenetic maps: new regional localizations, orientation of the linkage groups, and insights into mammalian chromosome evolution

In order to generate anchor points connecting the rat cytogenetic and genetic maps, the cytogenetic position of 62 rat markers (including 55 genes) already localized genetically was determined by fluorescence in situ hybridization. Whenever possible, markers located near one end of the linkage groups were included. These new localizations allowed us to unambiguously orient the 20 autosomal and the X chromosome linkage groups. The position of the centromere in the linkage map could also be determined in the case of several metacentric chromosomes. In addition, the regional localization of 15 other rat genes was determined. These new data bring useful information with respect to comparative mapping with the mouse and the human and to mammalian evolution. They illustrate, for instance, that groups of genes can remain syntenic during mammalian evolution while being subjected to intrachromosomal rearrangements in some lineages (synteny is conserved while gene order is not). This analysis also disclosed cases of synteny conservation in one the two rodent species and the human, while the synteny is split in the other rodent species: such configurations are likely examples of lineage-specific interchromosomal rearrangements associated with speciation. Received: 20 April 1998 / Accepted: 26 May 1998     

Chromosomal mapping of four different integration sites of Moloney murine leukemia virus including the locus for alpha 1(I) collagen in mouse

Infection of mouse embryos with Moloney murine leukemia virus (M-MuLV) has yielded several mouse substrains with stable germ line integration of retroviral DNA at distinct chromosomal loci (Mov loci; Jaenisch et al., 1981). There is evidence that flanking DNA sequences can have an effect on virus expression and, conversely, inserted viral DNA may affect the expression of adjacent host genes. As part of our studies on the interaction of inserted M-MuLV with the mouse genome, we have chromosomally mapped four different Mov loci by hybridizing single-copy mouse sequences, flanking the proviral DNA, to interspecies somatic cell hybrids. Furthermore, these sequences were assigned regionally by in situ hybridization to mouse metaphase chromosomes. In Mov-13 mice, M-MuLV had inserted into the alpha 1(I) collagen gene leading to early embryonic death in homozygotes. We have assigned this locus to the distal region of chromosome 11. Thus, the alpha 1(I) collagen gene is part of an evolutionarily conserved linkage group with the homologous genes on human chromosome 17. Three other proviral integration sites were mapped to chromosome 1, bands BC (Mov-7), chromosome 11, bands BC (Mov-9), and chromosome 3, bands FG (Mov-10). The Mov-10-specific probe detects an EcoRI-specific restriction fragment length polymorphism, which can make this probe a useful genetic marker.     

Comparative mapping using somatic cell hybrids

Summary Comparative mapping, or ascertaining the gene linkage relationships between different species, is rapidly developing. This is possible because new techniques in chromosome identification and somatic cell hybridization, such as the generation of hybrids preferentially segregating chromosomes of any desired species including rodents, and the development of gene transfer techniques have yielded new information about the human and rodent gene maps. In addition, the discovery and characterization of mouse subspecies has generated new mouse sexual genetic linkage data. The following picture is emerging. Several X-linked genes in man are X-linked in all mammalian species tested. The linkage relationships of several tightly linked genes, less than 1 map unit apart, are also conserved in all mammalian species tested. Ape autosomal genes are assigned to ape chromosomes homologous to their human counterparts indicating extensive conservation in the 12 million years (MYR) of evolution from apes to man. Similarly, mouse and rat, 10 MYR apart in evolution, have several large autosomal synteny groups conserved. In comparing the mouse and human gene maps we find that human genes assigned to different arms of the same human chromosome are unlinked in the mouse; mouse genes large map distances (20 to 45 cM) apart are very likely to be unlinked in the human. However, several autosomal synteny groups 10 to 20 cM apart, including thePgd, Eno-1, Pgm-1 group on human chromosome arm lp, are conserved in mice and man. This suggests that homology mapping, the superimposition of one species gene map on the homologous conserved portion of another species genome may be possible, and that ancestral autosomal synteny groups should be detectable. Presented in the formal symposium on Somatic Cell Genetics at the 27th Annual Meeting of the Tissue Culture Association, Philadelphia, Pennsylvania, June 7–10, 1976.