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.     

Physical and genetic mapping of a novel chromosome 19 ERCC1 marker showing close linkage with myotonic dystrophy.

Recent genetic linkage analyses have mapped the myotonic dystrophy locus to the region of 19q13.2-13.3 lying distal to the gene for creatine kinase subunit M (CKM). The human excision repair gene ERCC1 has also been mapped to this region of chromosome 19. A novel polymorphic DNA marker, pEO.8, has been isolated from a chromosome 19 ERCC1-containing cosmid that maps to a 300-kb NotI fragment encompassing both CKM and ERCC1. Genetic linkage analysis reveals close linkage between pEO.8 and myotonic dystrophy (DM) (zmax = 19.3, theta max = 0.01). Analysis of two key recombinant events suggests a mapping of DM distal to pEO.8 and CKM.     

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. 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, χ² = 35.37, P < 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.     

Assignment of the gene encoding DNA ligase I to human chromosome 19q13.2-13.3.

The gene encoding DNA ligase I has been mapped on human chromosome 19 by analysis of rodent-human somatic cell hybrids informative for this chromosome and by two-color fluorescence in situ hybridization. The DNA ligase I gene (LIG1) is localized to 19q13.2-13.3 and is distal to ERCC1, the most telomeric of three DNA repair genes on this chromosome.     

Complementation of repair gene mutations on the hemizygous chromosome 9 in CHO: a third repair gene on human chromosome 19

A human DNA repair gene, ERCC2 (Excision Repair Cross Complementing 2), was assigned to human chromosome 19 using hybrid clone panels in two different procedures. One set of cell hybrids was constructed by selecting for functional complementation of the DNA repair defect in mutant CHO UV5 after fusion with human lymphocytes. In the second analysis, DNAs from an independent hybrid panel were digested with restriction enzymes and analyzed by Southern blot hybridization using DNA probes for the three DNA repair genes that are located on human chromosome 19: ERCC1, ERCC2, and X-Ray Repair Cross Complementing 1 (XRCC1). The results from hybrids retaining different portions of this chromosome showed that ERCC2 is distal to XRCC1 and in the same region of the chromosome 19 long arm (q13.2-q13.3) as ERCC1, but on different MluI macrorestriction fragments. Similar experiments using a hybrid clone panel containing segregating Chinese hamster chromosomes revealed the hamster homologs of the three repair genes to be part of a highly conserved linkage group on Chinese hamster chromosome number 9. The known hemizygosity of hamster chromosome 9 in CHO cells can account for the high frequency at which genetically recessive mutations are recovered in these three genes in CHO cells. Thus, the conservation of linkage of the repair genes explains the seemingly disproportionate number of repair genes identified on human chromosome 19.     

Homology of Melanoma-Inducing Loci in the Genus Xiphophorus

Several species of the genus Xiphophorus are polymorphic for specific pigment patterns. Some of these give rise to malignant melanoma following the appropriate crossings. For one of these pattern loci from the playfish Xiphophorus maculatus the melanoma-inducing gene has been cloned and found to encode a novel receptor tyrosine kinase, designated Xmrk. Using molecular probes from this gene in Southern blot analyses on single fish DNA preparations from 600 specimens of different populations of various species of the genus Xiphophorus and their hybrids, either with or without melanoma-predisposing pattern, it was shown that all individuals contain the Xmrk gene as a proto-oncogene. It is located on the sex chromosome. All fish that carry a melanoma-predisposing locus which has been identified by Mendelian genetics contain an additional copy of Xmrk, closely linked to a specific melanophore pattern locus on the sex chromosome. The melanoma-inducing loci of the different species and populations are homologous. The additional copy of Xmrk obviously arose by a gene-duplication event, thereby acquiring the oncogenic potential. The homology of the melanoma-inducing loci points to a similar mechanism of tumor suppression in all feral fish populations of the different species of the genus Xiphophorus.     

Localization of the nucleotide excision repair gene ERCC6 to human chromosome 10q11-q21.

We have cloned the human DNA excision repair gene ERCC6 by virtue of its ability to correct the uv sensitivity of Chinese hamster overy cell mutant UV61. This mutant is a member of complementation group 6 of the nucleotide excision repair-deficient rodent mutants. By means of in situ hybridization and Southern blot analysis of mouse x human somatic cell hybrids, the gene was localized to human chromosome 10q11-q21. An RFLP detected within the ERCC6 locus can be helpful in linkage analysis.     

Regional localization of the selenocysteine tRNA gene (TRSP) on human chromosome 19.

The human selenocysteine tRNA gene (TRSP) has been localized on chromosome 19q13.2-->q13.3 by in situ hybridization and ordered with respect to other genes and anonymous DNA markers in this region by linkage analysis in the forty CEPH pedigrees. These loci span only 10 cM in males and about 30 cM in females. The order of the loci is cen ... D19S7-D19S9-D19S47-CYP2A-CYP2F1-APOC2++ +-(TRSP, CKM). CYP2B flanks the CYP2A and CYP2F1 loci, but it cannot be determined whether it is proximal or distal to the other two cytochrome P450 loci with respect to the centromere.     

Linkage mapping of highly informative DNA polymorphisms within the human interferon-alpha receptor gene on chromosome 21.

Two polymorphic loci within the interferon-alpha receptor (IFNAR) gene on human chromosome 21 have been identified and mapped by linkage analysis in 40 CEPH families. These markers are (1) a multiallelic RFLP with an observed heterozygosity of 0.72 and (2) a variable (AT3)n short sequence repeat at the poly(A) tail of an Alu sequence (AluVpA) with an observed heterozygosity of 0.83. This locus is close to D21S58 (theta = 0.02, zeta = 36.76) and D21S17 (theta = 0.02, Zeta = 21.76) with chromosomal band 21q22.1. Multipoint linkage analysis suggests the most likely locus order to be 21cen-D21S58-IFNAR-D21S17-21qter. Given its high heterozygosity, the IFNAR gene can be used as an index marker on human chromosome 21.     

Linkage group VI of fish of the genus Xiphophorus (Poeciliidae): Assignment of genes coding for glutamine synthetase,uridine monophosphate kinase,and transferrin

Electrophoretic variation ascribable to three protein-coding loci, coding for glutamine synthetase (GS), uridine monophosphate kinase (UMPK), and transferrin (Tf), was observed in three species of fish of the genus Xiphophorus. Electrophoretic patterns in interspecific F1 hybrid heterozygotes suggested monomeric subunit structures of UMPK and Tf and a multimeric structure of undetermined subunit number of GS. Linkage analyses in backcross hybrids indicated a recombination map of GS-0%-Tf-10.8%-UMPK. This group (designated Xiphophorus linkage group VI) was shown to assort independently from the 14 enzyme loci assigned to linkage groups I-V and from 19 other informative markers within the limits of the data.     

Genetic linkage studies of autosomal dominant polycystic kidney disease: search for the second gene in a large Sicilian family

Summary Polycystic kidney disease (PKD) is a common autosomal dominant genetic disorder caused by mutation in at least two different gene loci. The PKD1 gene has been localized on the short arm of chromosome 16. The location of a second genetic locus in the human genome is not yet known. A large PKD kindred, unlinked to chromosome 16, with over 250 members was studied using both DNA and classical markers. In total, 29 informative marker loci on 11 autosomes have been analyzed for linkage with PKD. The data significantly exclude the linkage with disease locus from 17 marker loci and show no evidence of close linkage with the other loci.     

Synteny on mouse chromosome 5 of homologs for human DNA loci linked to the Huntington disease gene

Comparative mapping in man and mouse has revealed frequent conservation of chromosomal segments, offering a potential approach to human disease genes via their murine homologs. Using DNA markers near the Huntington disease gene on the short arm of chromosome 4, we defined a conserved linkage group on mouse chromosome 5. Linkage analyses using recombinant inbred strains, a standard outcross, and an interspecific backcross were used to assign homologs for five human loci, D4S43, D4S62, QDPR, D4S76, and D4S80, to chromosome 5 and to determine their relationships with previously mapped markers for this autosome. The relative order of the conserved loci was preserved in a linkage group that spanned 13% recombination in the interspecific backcross analysis. The most proximal of the conserved markers on the mouse map, D4S43h, showed no recombination with Emv-1, an endogenous ecotropic virus, in 84 outcross progeny and 19 recombinant inbred strains. Hx, a dominant mutation that causes deformities in limb development, maps approximately 2 cM proximal to Emv-1. Since the human D4S43 locus is less than 1 cM proximal to HD near the telomere of chromosome 4, the murine counterpart of the HD gene might lie between Hx and Emv-1 or D4S43h. Cloning of the region between these markers could generate new probes for conserved human sequences in the vicinity of the HD gene or possibly candidates for the murine counterpart of this human disease locus.     

Localisation of the myotonic dystrophy locus to 19q13.2–19q13.3 and its relationship to twelve polymorphic loci on 19q

Summary The order of fourteen polymorphic markers localised to the long arm of human chromosome 19 has been established by multipoint mapping in a set of 40 CEPH (Centre d'Étude de Polymorphisme Humain, Paris) reference families. We report here the linkage relationship of the myotonic dystrophy (DM) locus to twelve of these markers as studied in 45 families with DM. The resulting genetic map is supported by the localisation of the DNA markers in a panel of somatic cell hybrids. Ten of the twelve markers have been shown to be proximal to the DM gene and two, PRKCG and D19S22, distal but at distances of approximately 25 cM and 15 cM, respectively. The closest proximal markers are APOC2 (apolipoprotein C-II) and CKM (creatine kinase, muscle) approximately 3 cM and 2 cM from the DM gene respectively, in the order APOC2-CKM-DM. The distance between APOC2, CKM and DM (of the order of 2 million base pairs) and their known orientation should permit directional chromosome walking and jumping. The data presented here should enable us to determine whether or not new markers are distal to APOC2/CKM and thus potentially flank the DM gene.     

Isolation of the functional human excision repair gene ERCC5 by intercosmid recombination

The complete human nucleotide exicision repair gene ERCC5 was isolated as a functional gene on overlapping cosmids. ERCC5 corrects the excision repair deficiency of Chinese hamster ovary cell line UV135, of complementation group 5. Cosmids that contained human sequences were obtained from a UV-resistant cell line derived from UV135 cells transformed with human genomic DNA. Individually, none of the cosmids complemented the UV135 repair defect; cosmid groups were formed to represent putative human genomic regions, and specific pairs of cosmids that effectively transformed UV135 cells to UV resistance were identified. Analysis of transformants derived from the active cosmid pairs showed that the functional 32-kbp ERCC5 gene was reconstructed by homologous intercosmid recombination. The cloned human sequences exhibited 100% concordance with the locus designated genetically as ERCC5 located on human chromosome 13q. Cosmid-transformed UV135 host cells repaired cytotoxic damage to levels about 70% of normal and repaired UV-irradiated shuttle vector DNA to levels about 82% of normal.     

A Sex Chromosomal Restriction-Fragment-Length Marker Linked to Melanoma-Determining Tu Loci in Xiphophorus

In Xiphophorus, the causative genetic information for melanoma formation has been assigned by classical genetics to chromosomal loci, which are located on the sex chromosomes. In our attempts to molecularly clone these melanoma-determining loci, named Tu, we have looked for restriction-fragment-length markers (RFLMs) linked to the Tu loci. These RFLMs should be useful in obtaining a physical map of a Tu locus, which will aid in the cloning of the corresponding sequences. DNA samples from various Xiphophorus strains and hybrids including those bearing different Tu wild-type, deletion and translocation chromosomes, were screened for the presence of random RFLMs using homologous or heterologous sequences as hybridization probes. We find an EcoRI restriction fragment which shows limited crosshybridization to the v-erb B gene--but not representing the authentic c-erb B gene of Xiphophorus--to be polymorphic with respect to different sex chromosomes. Linkage analysis revealed that a 5-kb fragment is linked to the Tu-Sd locus on the X chromosome, a 7-kb fragment is linked to the Tu-Sr locus on the Y chromosome, both of Xiphophorus maculatus, and that a 12-kb fragment is linked to the Tu-Li locus on the X chromosome of Xiphophorus variatus. Using different chromosomal mutants this RFLM has been mapped to a frequent deletion/translocation breakpoint of the X chromosome, less than 0.3 cM apart from the Tu locus.     

Structural organization, mapping, characterization and evolutionary relationships of CDKN2 gene family members in Xiphophorus fishes

Xiphophorus fishes and their hybrids are used as models for the study of melanoma and other diseases. The cyclin-dependent kinase inhibitor gene family in humans is comprised of four members, including CDKN2A (P16), and dysregulation of this gene is implicated in numerous neoplasms including melanomas. We have investigated the status of the gene family in the southern platyfish X. maculatus. Xiphophorus harbors at least two such loci, which we now term CDKN2A/B and CDKN2D. Both loci map to Xiphophorus linkage group 5, a genomic area that has long been known to harbor the DIFF tumor suppressor locus. Within this report, we report on the complete cloning, genomic exon/intron boundary delineation, linkage mapping and expressional characteristics of Xiphophorus CDKN2D. We also compare and contrast this expression to that of the previously isolated CDKN2AB locus in normal and neoplastic tissues derived from non-hybrid and hybrid fishes. The hypothetical evolutionary relationships of gene family members and their involvement in melanoma is evaluated. In comparison to CDKN2A/B, the RNA expression of Xiphophorus CDKN2D differs in normal tissues and is not associated with melanotic/pathologic tissues, confirming functional divergence between obvious homologues.     

The predisposition to type 1 diabetes linked to the human leukocyte antigen complex includes at least one non-class II gene

The human leukocyte antigen (HLA) complex, encompassing 3.5 Mb of DNA from the centromeric HLA-DPB2 locus to the telomeric HLA-F locus on chromosome 6p21, encodes a major part of the genetic predisposition to develop type 1 diabetes, designated "IDDM1." A primary role for allelic variation of the class II HLA-DRB1, HLA-DQA1, and HLA-DQB1 loci has been established. However, studies of animals and humans have indicated that other, unmapped, major histocompatibility complex (MHC)-linked genes are participating in IDDM1. The strong linkage disequilibrium between genes in this complex makes mapping a difficult task. In the present paper, we report on the approach we have devised to circumvent the confounding effects of disequilibrium between class II alleles and alleles at other MHC loci. We have scanned 12 Mb of the MHC and flanking chromosome regions with microsatellite polymorphisms and analyzed the transmission of these marker alleles to diabetic probands from parents who were homozygous for the alleles of the HLA-DRB1, HLA-DQA1, and HLA-DQB1 genes. Our analysis, using three independent family sets, suggests the presence of an additional type I diabetes gene (or genes). This approach is useful for the analysis of other loci linked to common diseases, to verify if a candidate polymorphism can explain all of the association of a region or if the association is due to two or more loci in linkage disequilibrium with each other.     

Linkage of two enzyme loci in the fish genus Poecilia (Teleostei:Poeciliidae).

The linkage of loci coding for glucose-6-phosphate dehydrogenase (G6PD) and phosphogluconate dehydrogenase (PGD) is described in fish of the genus Poecilia (Teleostei:Poeciliidae) and designated Poecilia linkage group I. These two loci were shown to assort independently from six other informative markers (peptidase S, malate dehydrogenase 2 [soluble], mannose phosphate isomerase, parvalbumin 2, phosphoglucomutase, and glyceraldehyde-3-phosphate dehydrogenase 2) within the limits of the data obtained. Data for the linkage analyses were generated by scoring starch-gel electrophoretic phenotypes of the eight loci in reciprocal backcross hybrids obtained from matings between Poecilia perugiae and P. vittata. The linkage chi 2 for G6PD-PGD locus pairs was significant (P less than 0.001) in all reciprocal backcross hybrid broods (22.7% recombinants in the combined data), indicating linkage in both parental species. The linkage of G6PD and PGD in gene maps of the poeciliid genera Xiphophorus and Poeciliopsis documents homology of this linkage within the family. Linkages in salmonid and centrarchid fishes suggest conservation of this linkage group in most or all teleosts. The six additional indpendently assorting loci have been assigned to independent linkage groups in Xiphophorus; thus, no example of poeciliid linkage group divergence has yet been identified.     

Regional assignment of the gene for human liver/bone/kidney alkaline phosphatase to chromosome 1p36.1-p34

We have used three different methods to map the human liver/bone/kidney alkaline phosphatase (ALPL) locus: (1) Southern blot analysis of DNA derived from a panel of human-rodent somatic cell hybrids; (2) in situ hybridization to human chromosomes; and (3) genetic linkage analysis. Our results indicate that the ALPL locus maps to human chromosome bands 1p36.1-p34 and is genetically linked to the Rh (maximum lod score of 15.66 at a recombination value of 0.10) and fucosidase A (maximum lod score of 8.24 at a recombination value of 0.02) loci. These results, combined with restriction fragment length polymorphisms identified by ALPL DNA probes, provide a useful marker for gene mapping studies involving the short arm of chromosome 1. In addition, our results help to elucidate further the structure and evolution of the human alkaline phosphatase multigene enzyme family.     

Human monoamine oxidase gene (MAOA): chromosome position (Xp21-p11) and DNA polymorphism

An essentially full-length cDNA clone for the human enzyme monoamine oxidase type A (MAO-A) has been used to determine the chromosomal location of a gene encoding it. This enzyme is important in the degradative metabolism of biogenic amines throughout the body and is located in the outer mitochondrial membrane of many cell types. Southern blot analysis of PstI-digested human DNA revealed multiple fragments that hybridized to this probe. Using rodent-human somatic cell hybrids containing all or part of the human X chromosome, we have mapped these fragments to the region Xp21-p11. A restriction fragment length polymorphism (RFLP) for this MAOA gene was identified and used to evaluate linkage distances between this locus and several other loci on Xp. The MAOA locus lies between DXS14 and OTC, about 29 cM from the former.