Somatic cell mapping and restriction fragment analysis of bovine genes for fibronectin and gamma crystallin

DNAs from cow-hamster and cow-mouse somatic hybrid cells segregating bovine chromosomes have been analyzed by Southern blotting and hybridization with human fibronectin and gamma crystallin probes. Concordancy of retention of these bovine genes was compared to cattle isozyme loci representing previously described syntenic groups. Bovine fibronectin (FNI) and gamma crystallin (CRYG) fragments were concordant with each other and with isocitrate dehydrogenase 1 (IDH1), representing the bovine syntenic group U17. The syntenic relationship of these genes is conserved on human chromosome 2q and also on mouse chromosome 1. In addition, bovine RFLPs were identified with both fibronectin and gamma crystallin probes. These polymorphisms will be used to study recombination between the syntenic loci in pedigreed herds and to mark a segment of the bovine genome that is likely homologous to the Lsh region of mouse chromosome 1, which confers resistance in mice to several intracellular parasites.     

Sheep linkage mapping: RFLP markers for comparative mapping studies

Restriction fragment length polymorphisms (RFLPs) detected using cDNA probes for conserved genes provide an important set of markers that anchor or link syntenic groups in a range of divergent mammalian species. DNA probes from sheep, cattle, pig, human and mouse were screened against sheep DNA samples and 24 new RFLP markers for sheep were identified. Among the loci tested, 22 had a homologue that has been mapped in humans. An RFLP for fibronectin (FN1) was linked to α-inhibin (INHA) at a distance of 5cM. The FN1 locus has been assigned to sheep chromosome 2q41–q44 and linkage between FN1 and INHA assigns INHA to the same chromosome in sheep. In addition to the new loci reported here, 28 RFLPs have been published previously by this group and these are collated together with RFLPs published from other laboratories. RFLPs have been reported for 86 loci in sheep. Fifty-four loci have been mapped to 16 different chromosomes.     

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.     

Regional localization of the fibronectin and gamma crystallin genes to mouse chromosome 1 by in situ hybridization

Genes for fibronectin, gamma crystallin, and isocitrate dehydrogenase-1 are syntenic in mouse, man, and cow. In an effort to physically locate this conserved chromosome region in the genomes of the respective species, we have localized the fibronectin and gamma crystallin genes to mouse chromosome 1, region C1-5 by in situ hybridization. In situ hybridization was conducted on metaphase chromosomes of bone marrow preparations of Rb 1.7 mice. These cells contain Robertsonian translocated chromosomes 1 and 7 as the only submetacentric chromosome in an otherwise acrocentric genome. Physically mapping these genes to mouse chromosome 1 now enables comparisons of the genetic map and the physical map on the proximal half of this chromosome. Genes in this conserved region of mouse chromosome 1 are also involved in resistance to intracellular pathogens, and the chromosomal localization of this region may facilitate the identification of homologous genes in other species.     

A genetic map of mouse chromosome 1 near the Lsh-Ity-Bcg disease resistance locus

Isozyme and restriction fragment length polymorphism (RFLP) analyses of backcross progeny, recombinant inbred strains, and congenic strains of mice positioned eight genetic markers with respect to the Lsh-Ity-Bcg disease resistance locus. Allelic isoforms of Idh-1 and Pep-3 and RFLPs detected by Southern hybridization for Myl-1, Cryg, Vil, Achrg, bcl-2, and Ren-1,2, between BALB/cAnPt and DBA/2NPt mice, were utilized to examine the cosegregation of these markers with the Lsh-Ity-Bcg resistance phenotype in 103 backcross progeny. An additional 47 backcross progeny from a cross between C57BL/10ScSn and B10.L-Lshr/s mice were examined for the cosegregation of Myl-1 and Vil RFLPs with Lsh phenotypic differences. Similarly, BXD recombinant inbred strains were typed for RFLPs upon hybridization with Vil and Achrg. Recombination frequencies generated in the different test systems were statistically similar, and villin (Vil) was identified as the molecular marker closest (1.7 +/- 0.8 cM) to the Lsh-Ity-Bcg locus. Two other DNA sequences, nebulin (Neb) and an anonymous DNA fragment (D2S3), which map to a region of human chromosome 2q that is homologous to proximal mouse chromosome 1, were not closely linked to the Lsh-Ity-Bcg locus. This multipoint linkage analysis of chromosome 1 surrounding the Lsh-Ity-Bcg locus provides a basis for the eventual isolation of the disease gene.     

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.     

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.     

Exclusion of stromelysin-1, stromelysin-2, interstitial collagenase and fibronectin genes as the mutant loci in a family with recessive epidermolysis bullosa dystrophica and a form of cerebellar ataxia

Summary The interstitial collagenase gene (CLG), one of the main candidates in severe generalized recessive epidermolysis bullosa dystrophica (SGREBD), is closely linked to the stromelysin-1 (STMY1) and stromelysin-2 (STMY2) genes. These three loci map on chromosome 11 (q21–q22.3), where they constitute a cluster of genes coding for metalloproteinases involved in the degradation of the extracellular matrix (ECM). A recessive form of cerebellar ataxia of post-puberal onset (CLA1) has also been assigned to chromosome 11 (q14–q21). Since useful restriction fragment length polymorphisms (RFLPs) for the CLG gene are not available, we have studied the inheritance of the marker TaqI RFLP of the STMY1 gene in a North Italian family with a child affected by SGREBD, and his two sisters showing cerebellar ataxia (CA) of post-puberal onset. We have also studied the MspI RFLP of the fibronectin gene (FN1), which is located on chromosome 2q34–q36, and which codes for non-collagenous matrix proteins. Since we did not observe the segregation of the pathological phenotypes with STMY1 and FN1 RFLPs, we excluded the involvement of these genes in both the SGREBD and CA present in this family. The exclusion of the STMY1 gene indicates that the mutation causing SGREBD cannot be located in the CLG and/or STMY2 genes because of their proximity to the STMY1 locus. These data also indicate that the CA form here reported is not attributable to alterations in regions close to the collagenase cluster on chromosome 11.     

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.     

Genetic polymorphism of the murine myogenic gene Myo-D1.

Polymorphism of the myogenic gene, Myo-D1, has been sought to examine genetic mechanisms which control skeletal muscle development. By Southern analysis, three restriction-fragment length polymorphisms (RFLPs) have been found in various mouse strains using the TaqI, SacI and BglII restriction endonucleases and a full-length cDNA Myo-D1 probe. Reference to the distribution of RFLPs in different mouse strains derived from Mus mus (M.m.) domesticus and M.m. musculus subspecies suggests that Myo-D1 rearrangements are subject to nonrandom association. The biological significance of RFLP of the Myo-D1 gene is yet to be determined.     

Human fibronectin gene (FN1) RFLPs: Mapping and linkage disequilibrium analysis

We report a mapping and linkage disequilibrium analysis of six restriction fragment length polymorphisms (RFLPs) of the human fibronectin gene. The polymerase chain reaction conditions are described for four of the RFLPs.     

Regional localization of the fibronectin and gamma-crystallin genes to bovine chromosome 8

The genes for isocitrate dehydrogenase-1, fibronectin, and gamma-crystallin are syntenic in man, mouse, and cow. In an effort to assign this bovine syntenic group to a specific chromosome and to allow a cytological comparison of the conserved chromosomal region containing these genes in their respective species, we have localized the fibronectin and gamma-crystallin genes to bovine chromosome 8, region 1.1-1.4. This study incorporates the techniques of hybrid somatic cell analysis and in situ hybridization and the use of a Robertsonian-translocated marker chromosome from a related species for regional assignment of genes to a specific bovine chromosome. The regions on human chromosome 2q, mouse chromosome 1, and cow chromosome 8 that contain these genes are cytologically similar, perhaps representing evolutionary conservation at the cytogenetic level as well as at the gene level for this group of loci.     

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 rat insulin I gene (INS1) to chromosome 1q55 by fluorescence in situ hybridization.

The rat insulin I gene (INS1) was assigned to chromosome 1q55 using fluorescent in situ hybridization. In addition, several RFLPs were detected among 11 inbred rat strains.     

Ethnic-specific allelic variation within the D1Z2 locus

DNAs from 122 individuals representing 5 ethnic groups (Black, Chinese, Japanese, Caucasian and Melanesian) were analyzed for restriction fragment length polymorphisms (RFLPs) with a hypervariable repeated sequence located uniquely on chromosome 1 (hMF No.1; is a component of the D1Z2 locus). When human genomic DNA is digested with a variety of enzymes (TaqI, EcoRI, SinI, PstI, HaeIII) the hMF No.1 probe reveals multiple RFLPs. Ethnic group differences were found in the frequencies of specific EcoRI bands. The most striking ethnic group variation was the presence of a unique fragment amongst the Japanese.     

Mapping Creb-1 to chromosome 1 in the mouse.

The gene CREB1 encoding the cyclic AMP response element DNA binding protein was previously assigned to human 2q32.3-q34. In this study, a panel of 207 backcross mice made between C57BL/10ScSn (=B10) females and (B10 x B10.L-Lsh)F1 males were used to map Creb-1 with respect to Cryg and Lsh/Vil on mouse chromosome 1. A reverse-transcribed, polymerase chain reaction-amplified cDNA probe covering bp 39 to 554 of the human sequence identified restriction fragment length polymorphisms with 7/18 restriction endonucleases used to digest whole genomic mouse DNA from the parental strains. BglII and DraI RFLPs for Creb-1 were scored on a subpanel of 16/207 known recombinants between Cryg and Lsh/Vil, yielding 2/16 recombinants between Cryg and Creb-1 and 14/16 recombinants between Creb-1 and Lsh/Vil. The 16/207 recombinants observed between Lsh/Vil and Cryg provide an estimated recombination frequency of 0.077 +/- 0.019, equivalent to a map distance of 7.7 +/- 1.9 cM. This is in good agreement with previously published map distances. The number of recombinants observed between Creb-1 and the other markers place Creb-1 approximately 1 cM distal to Cryg and 7 cM proximal to Lsh/Vil.     

Linkage of the Murine Transforming Growth Factor α Gene with Igk, Ly-2, and Fabp1 on Chromosome 6

TGFα is a mitogenic polypeptide found in the conditioned media of transformed cell lines as well as in various solid tumors. Although its physiological role in normal tissues is uncertain, the autocrine action of TGFα on the EGF receptor is postulated to play a role in tumorigenesis. To explore the possibility that pre-existing mouse mutants might have concordance with the mouse TGFα locus (Tgfa) we sought to establish the chromosomal localization of the murine TGFα gene. Using Southern analysis we have detected NcoI and PvuII RFLP in the TGFα gene of progenitor RI mouse strains. These RFLPs have been used to analyze four different RI sets of DNA and to assign Tgfa to the 35-cM region of chromosome 6. Linkage has been established and the data suggest that the distance between Igk and wa-1 anchor loci may be less than 8 cM and that the gene order for the proximal to mid region of mouse chromosome 6 may be: Ggc-Xmmv27-[Brp-1, Lvp-1, Ms6-4]-[Igk, Ly2, Ly3 Odc-rs5, Rn7s-6, Fabp1]-[Tgfa/wa-1]-IL5-R. Homology of synteny has been further defined between the proximal region of mouse chromosome 6 and with the 2p 13-p11 region of human chromosome 2 encompassing TGFA, IGK, CD8A, and FABP1 .     

Mapping of mouse gamma crystallin genes on chromosome 1

Restriction fragments analysis of DNA from mouse-hamster somatic-cell hybrid clones revealed that a mouse gamma crystallin cDNA hybridized to genomic sequences located on mouse chromosome 1. Identification of restriction fragment length polymorphisms (RFLPs) in the gamma crystallin sequences of inbred strains of mice permitted the further localization of the gamma crystallin genes (Cryg) to the proximal region of chromosome 1 closely linked to the loci encoding isocitrate dehydrogenase (Idh-1), a low molecular weight (LM) crystallin protein polymorphism (Len-1), and fibronectin (Fn-1). A single recombinant was observed betweenLen-1 and an RFLP in the gamma crystallin gene family, consistent with the hypothesis thatLen-1 is one of the several structural loci encoding gamma crystallin genes.Len-1 is probably located on the centromeric end of theCryg gene family. Linkage ofIdh-1, Cryg, andFn-1 in mice extends the syntenic relationship of those loci to the human, bovine, and rodent genomes and may define a chromosomal region that is generally conserved among mammals. The map position ofCryg, near the eye lens obsolescence (Elo) locus, was confirmed by the discovery that the restriction fragment patterns of gamma crystallin sequences differed between strain C3H/HeJ and the congenic anophthalmic mutant strain, C3H.Elo. Therefore, the gamma crystallin genes were contransferred with the mutantElo gene in the derivation of C3H.Elo. The results establish that LEN-1 is a marker for the gamma crystallin gene family, position the gamma crystallin gene family relative to other markers on mouse chromosome 1, and provide additional evidence that theElo mutation is encoded at a locus closely linked to the gamma crystallin gene cluster. This study found no evidence of recombination hot spots within the gamma crystallin gene cluster.     

Identification of the mouse and rat orthologs of the gene mutated in Usher syndrome type IIA and the cellular source of USH2A mRNA in retina,a target tissue of the disease

Usher syndrome type IIA (MIM: 27601) is an autosomal recessive disorder characterized by moderate to severe congenital deafness and progressive retinitis pigmentosa. We recently identified the human Usher syndrome type IIA gene (USH2A) on chromosome 1q41, which encodes a protein possessing 10 laminin epidermal growth factor and four fibronectin type 3 domains, both commonly observed in extracellular matrix proteins. To gain insight into the pathogenesis of Usher syndrome type IIA, we isolated and characterized the murine (Ush2a) and rat (rat Ush2a) orthologs of human USH2A. We mapped mouse Ush2a by fluorescence in situ hybridization to mouse chromosome 1 in the region syntenic to human chromosome 1q41. Rat Ush2a has been localized by radiation hybrid mapping to rat chromosome 13 between d13rat49 and d13rat76. The mouse and rat genes, similar to human USH2A, are expressed primarily in retina and cochlea. Mouse Ush2a encodes a 161-kDa protein that shows 68% identity and 9% similarity to the human USH2A protein. Rat Ush2a encodes a 167-kDa protein with 64% identity and 10% similarity to the human protein and 81% identity and 5% similarity to the mouse USH2A protein. The predicted amino acid sequence of the mouse and rat proteins, like their human counterpart, contains a leader sequence, an amino-terminal globular domain, 10 laminin epidermal growth factor domains, and four carboxy-terminal fibronectin type III motifs. With in situ hybridization, we compared the cellular expression of the USH2A gene in rat, mouse, and human retinas. USH2A mRNA in the adult rat, mouse, and human is expressed in the cells of the outer nuclear layer of the retina, one of the target tissues of the disease. In the developing rat retina, Ush2a mRNA expression appears in the neuroepithelium at embryonic day 17.     

Isolation of polymorphic DNA segments from human chromosome 21.

A somatic cell hybrid line containing only human chromosome 21 on a mouse background has been used as the source of DNA for construction of a recombinant phage library. Individual phages containing human inserts have been identified. Repeat-free human DNA subclones have been prepared and used to screen for restriction fragment length polymorphisms to provide genetic markers on chromosome 21. Nine independently isolated clones used as probes identified a total of 11 new RFLPs. Four of the DNA probes recovered from the library have been mapped unequivocally to chromosome 21 using a panel of somatic cell hybrid lines. A fifth probe detected an RFLP on chromosome 21 as well as sequences on other chromosomes. This set of RFLPs may now form the basis for construction of a genetic linkage map of human chromosome 21.