Localization of the ornithine aminotransferase gene and related sequences on two human chromosomes

Summary We have used a full length cDNA clone to determine the chromosomal location ofthegene encoding human ornithine aminotransferase (OAT), a mitochondrial matrix enzyme. Southern blot analysis of ScaI-digested DNA from 34 human-mouse somatic cell hybrids revealed 11 human fragments. Three fragments mapped to chromosome 10q23-10qter, confirming the previous provisional assignment of the functional gene to this autosome by analysis of OAT expression in somatic cell hybrids (O'Donnell et al. 1985). The remaining eight fragments were assigned to the X chromosome, and regionally assigned to Xp21-Xp11 by use of an X-chromosome mapping panel. These X chromosome sequences could represent pseudogenes, or related members of a multigene family. Two of the X chromosome fragments are alternate alleles of a restriction fragment length polymorphism (RFLP) making this OAT-related locus an excellent genetic marker. The RFLP may now be used to determine any possible relationship between this locus and several X-linked eye defects.     

Submicroscopic interstitial deletion of the X chromosome explains a complex genetic syndrome dominated by Norrie disease

Norrie disease (ND), an X-linked recessive disorder, is characterized by congenital blindness followed by bulbar atrophy. We have examined a three-generation family in which ND is part of a complex X-linked syndrome with severe mental retardation, hypogonadism, growth disturbances, and increased susceptibility to infections as additional features. This syndrome is apparently due to an interstitial deletion, as evidenced by the failure of the L1.28 DNA probe (DXS7 locus, Xp11.3) to detect complementary DNA sequences on the defective X chromosome of an affected male and of several obligatory heterozygotes. Attempts to further define this deletion with other DNA probes from the proximal short arm of the X chromosome or by prometaphase chromosome analysis were unsuccessful.     

Ornithine aminotransferase (OAT): recombination between an X-linked OAT sequence (7.5 kb) and the Norrie disease locus

A human ornithine aminotransferase (OAT) locus has been mapped to the Xp11.2, as has the Norrie disease locus. We used a cDNA probe to investigate a 3-generation UCLA family with Norrie disease; a 4.2-kb RFLP was detected and a maximum lod score of 0.602 at zero recombination fraction was calculated. We used the same probe to study a second multigeneration family with Norrie disease from Utah. A different RFLP of 7.5 kb in size was identified and a recombinational event between the OAT locus represented by this RFLP and the disease loci was observed. Linkage analysis of these two loci in this family revealed a maximum load score of 1.88 at a recombination fraction of 0.10. Although both families have affected members with the same disease, the lod scores are reported separately because the 4.2- and 7.5-kb RFLPs may represent two different loci for the X-linked OAT.     

Gyrate atrophy of the choroid and retina: assignment of the ornithine aminotransferase structural gene to human chromosome 10 and mouse chromosome 7.

Gyrate atrophy of the choroid and retina is an autosomal recessive, blinding human disease caused by a deficiency of the mitochondrial matrix enzyme ornithine aminotransferase (OAT). Since human OAT cDNA hybridizes to DNA sequences on both human chromosomes 10 and X, a locus coding for OAT enzyme activity may be present on one or both of these human chromosomes. We have used a series of mouse-human somatic cell hybrids, in combination with starch gel electrophoresis and a histochemical stain for OAT enzyme activity, to assign the structural gene for OAT to human chromosome 10. Our results suggest that the human X chromosome does not contain a locus coding for OAT enzyme activity. In addition, we have used a panel of Chinese hamster-mouse hybrids to assign the murine Oat structural gene to mouse chromosome 7. Our findings, combined with recent molecular studies, indicate that human OAT probes specific for chromosome 10 will be useful for the diagnosis and genetic counseling of individuals at risk for gyrate atrophy.     

Mapping of ornithine aminotransferase gene sequences to mouse Chromosomes 7, X,and 3

Ornithine aminotransferase (OAT), a mitochondrial matrix enzyme, is deficient in patients with gyrate atrophy of the choroid and retina. In human, the OAT structural gene maps to Chromosome (Chr) 10q26 and several OAT-related sequences, some of which are known to be processed pseudogenes, which map to Xp11.3–11.21. Here, we report chromosomal localization in the mouse of the OAT gene and related sequences. Genomic DNA blot analysis of a well-characterized panel of Chinese hamster x mouse somatic cell hybrids using a human OAT probe revealed two murine loci, one on mouse Chr 7 and the other on Chr X. In addition, segregation of restriction fragment length polymorphisms (RFLPs) detected by the OAT probe in recombinant inbred (RI) strains detected a third locus on Chr 3 and positioned the X locus near Cf-8 and Rsvp. Progeny of an intersubspecific backcross were used to map the Chr 7 locus between Tyr and Int-2, near Cyp2e-1.     

Ornithine aminotransferase-related sequences map to two nonadjacent intervals on the human X chromosome short arm.

Using a panel of human/rodent somatic cell hybrids segregating human X/autosome translocations and deletions, we have refined the localization of the X-linked sequences homologous to ornithine-delta-aminotransferase (OAT), the structural locus for which (OAT) maps to chromosome 10. OAT-related ("-like") (OATL) sequences mapped to two nonadjacent intervals: OATL1 mapped to Xp11.3-p11.23, while OATL2 mapped to Xp11.22-p11.21. X-linked OATL1 sequences polymorphic for ScaI and StuI map to the more distal interval in Xp11.3-p11.23. These results should help guide long-range cloning and mapping studies, as well as refine the genetic linkage map in this region of the X chromosome.     

The ornithine aminotransferase-encoding gene family of rat: cloning, characterization, and evolutionary relationships between a single expressed gene and three pseudogenes

As a first step towards understanding the molecular mechanisms through which the expression of the gene (OAT) encoding ornithine aminotransferase (OAT) is regulated in a tissue-specific manner, we have used a near full length OAT cDNA to isolate related sequences from a rat genomic DNA library. Twenty-one unique clones representing five contigs and spanning approximately 140 kb of genomic DNA were isolated and characterized. From these clones we have identified a single expressed OAT gene and three processed pseudogenes. The comparison of the EcoRI, BamHI, and HindIII fragments contained within these genomic clones with those detected in total genomic DNA by the cDNA probe suggests that essentially all of the OAT-related sequences in the rat genome have been isolated. Thus, the tissue-specific regulation of OAT gene expression appears to be effected through a single expressed gene. Data are presented which suggest that the OAT-1, OAT-2, and OAT-3 pseudogenes arose approximately 28.5, 7.3, and 25.1 Myr ago, respectively. Mutation rates are presented for each codon position of the expressed rat and human OAT genes. The region of the rat genome flanking the boundary of the OAT-3 pseudogene is of additional interest as it shares considerable identity to sequences contained within expressed genes and flanking other processed pseudogenes.     

The ornithine aminotransferase (OAT) locus: analysis of RFLPs in gyrate atrophy.

A cDNA probe (HOAT1) for ornithine aminotransferase (OAT) has recently been used to map (1) the structural gene for this enzyme to chromosome 10 and (2) several related DNA sequences to the X chromosome. We have defined six RFLPs for OAT, to explore its possible role in gyrate atrophy (GA) of the choroid and retina, an autosomal recessive genetic disorder associated with a deficiency of OAT activity. The RFLPs, which are detected by noncoding single-copy probes from the OAT gene and by subclones of the HOAT1 cDNA, all map on human chromosome 10, producing an overall level of heterozygosity for the OAT locus of 83%. Using the RFLPs, we have determined that the OAT locus segregates concordantly with GA in one available pedigree. Furthermore, the RFLPs display significant disequilibrium with GA, providing genetic evidence implicating a defect in the OAT structural gene as the cause of this disorder. The RFLPs for OAT are potentially applicable to prenatal diagnosis and carrier detection in families with a previous history of GA. They will also allow identification of specific haplotypes associated with GA chromosomes, as a guide for more detailed molecular-genetic investigations of the mutations underlying the disorder.     

Localization of the human thyroxine-binding globulin gene to the long arm of the X chromosome (Xq21-22).

Thyroxine-binding globulin (TBG) is the major thyroid-hormone transport protein in the plasma of most vertebrate species. A recombinant phage (lambda cTBG8) containing a cDNA insert of human TBG recently has been described. With the cDNA insert from lambda cTBG8 used as a radiolabeled probe, DNA from a series of somatic-cell hybrids containing deletions of the X chromosome was analyzed by means of blot hybridization. The results indicated that the TBG gene is located in the midportion of the long arm of the X chromosome between bands Xq11 and Xq23. The gene then was mapped to band region Xq21-22 by means of in situ hybridization to metaphase chromosomes. Sequences on the X chromosome that are homologous to the cDNA probe are contained within a single EcoRI restriction fragment of 12.5 kb in human DNA. On the basis of the intensity of the hybridization signal on Southern blots, it was determined that the human TBG cDNA probe used in the present study shares significant homology with hamster and mouse sequences. A single EcoRI restriction fragment was recognized in both hamster (8.0-kb) and mouse (5.1-kb) DNA.     

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.     

X-linked dominant Charcot-Marie-Tooth disease: Suggestion of linkage with a cloned DNA sequence from the proximal Xq

Summary A large kindred with the X-linked dominant form of peroneal muscular atrophy (Charcot-Marie-Tooth disease) was analyzed for individual variation in the length of DNA fragments after restriction endonuclease digestion. A systematic search was performed for linkage with a series of cloned single-copy DNA sequences of known regional assignment to the human X chromosome. Close linkage was found with the pDP34 probe (DXYS1 locus, Xq13-q21), suggesting that the gene responsible for the disease is located on the proximal long arm of the X chromosome.     

Close linkage between Norrie disease,a cloned DNA sequence from the proximal short arm,and the centromere of the X chromosome

Summary Norrie disease (ND) is an X-linked recessive disorder with congenital blindness (atrophia bulborum hereditaria, pseudoglioma). Six kindreds segregating for ND were studied for linkage with polymorphic markers of the human X chromosome. No recombination was observed between the ND-locus (NDP) and the DXS7 locus, the latter followed as a DNA-restriction fragment length polymorphism, detected by the recombinant DNA probe L1.28, and assigned to the region Xp11.2–Xp11.3. The maximum lod scores are at . Linkage data between NDP and the other genetic markers used in the present study are in keeping with this assignment of the mutation to the proximal Xp.     

Molecular cloning, primary structure and expression of the human X linked A1S9 gene cDNA which complements the ts A1S9 mouse L cell defect in DNA replication.

The temperature-sensitive ts A1S9 mutation of mouse L cells was previously shown to affect nuclear DNA replication and to be complemented by active and inactive human X chromosomes in human-ts A1S9 somatic cell hybrids. We report the isolation of cDNA clones which correct the ts A1S9 lesion, using as a probe a genomic fragment derived from the human A1S9 locus. The nucleotide sequence of the A1S9 cDNA encompasses a single open reading frame of 2409 bp which could encode a heretofore unreported protein of 90 393 daltons. Southern blot hybridization of the A1S9 cDNA probe with DNA from various species revealed homologous sequences in vertebrates but not in yeast. Northern blot analysis of serum-starved, synchronized cells demonstrated that the A1S9 gene was expressed at a relatively low level in quiescent cells and at a higher and constant level throughout the cell cycle. Human cell lines harbouring increasing numbers of inactive X chromosomes (47, XXX, 49, XXXXX) were found to express the A1S9 gene at the same level as control cells (45, X), suggesting that the gene does not escape X chromosome inactivation.     

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.     

The root-knot nematode resistance gene (Mi) in tomato: construction of a molecular linkage map and identification of dominant cDNA markers in resistant genotypes

A dominant allele at the Mi locus on chromosome 6 of tomato (Lycopersicon esculentum Mill) confers resistance to three species of root-knot nematodes (Meloidogyne). The resistance, which is associated with a localized necrotic response, was originally introduced into tomato from the wild species Lycopersicon peruvianum. As a step towards the molecular cloning of Mi, we have identified closely linked DNA markers from both cDNA and genomic DNA libraries as restriction fragment length polymorphisms (RFLPs). DNA from tomato populations segregating for nematode resistance was analyzed to generate a high-resolution genetic map of this region. Additional information on gene order was obtained by comparing the size of the introgressed L. peruvianum chromosomal segment within a collection of nematode-resistant tomato lines. Among the four cDNA markers that are tightly linked to Mi, three are dominant, i.e. L. peruvianum-specific. One cDNA marker corresponds to a gene family comprising 20-30 members, one of which is diagnostic for all nematode-resistant genotypes tested. The presence of non-homologous sequences around the Mi gene may contribute to the suppression of recombination in this region of the genome in crosses heterozygous for Mi. The potential of 'walking' from closely linked markers to Mi is discussed.     

First trimester prenatal diagnosis and detection of carriers of haemophilia A using the linked DNA probe DX13.

Although the use of a gene specific deoxyribonucleic acid (DNA) probe is the method of choice for detecting carriers of genes for rare genetic disorders, there will always be families in which such probes cannot be used because key subjects are not informative for restriction fragment length polymorphisms in or around the gene. In these cases closely linked DNA markers have to be used. An X chromosome specific DNA probe, DX13, which is closely linked to the haemophilia A locus on the X chromosome, was used for early prenatal diagnosis in two cases and to detect carriers in a series of nine possible heterozygote women. The first reported crossover between DX13 and the factor VIII:C locus was observed in this study. There are complexities inherent in using any linked DNA probe for assignment of genes, but such techniques are clinically important.     

Segregation of restriction fragment length polymorphism in an interspecies cross of laboratory and wild mice indicates tight linkage of the murine IFN-beta gene to the murine IFN-alpha genes.

Southern blot analysis with murine (Mu) interferon (IFN)-alpha cDNA of restricted genomic DNA of three inbred strains of mice belonging to the species Mus musculus domesticus (BALB/c, C57BL/6, and DBA/2) revealed only a limited degree of polymorphism. For example, with HindIII there were only two polymorphic bands out of 14 hybridizing fragments. With Mu IFN-beta cDNA there was no polymorphism at all between BALB/c and C57BL/6 in DNA restricted with seven different enzymes. In contrast, HindIII-restricted DNA of an inbred strain of wild mice (M. spretus Lataste) hybridized with the IFN-alpha probe displayed a high degree of polymorphism compared with the three strains of laboratory mice and was also polymorphic when probed with IFN-beta cDNA. Although M. musculus domesticus and M. spretus Lataste represent different species, certain interspecies crosses are possible in the laboratory. This enabled us to follow segregation of restriction fragment length polymorphism in HindIII-restricted DNA obtained from 18 backcross progeny of a (DBA/2 X M. spretus)F1 X DBA/2 interspecies cross. There was complete coincidence between the segregation of parental (DBA/2) and (DBA/2 X M. spretus)F1-type IFN-beta and IFN-alpha restriction fragment length polymorphism, indicating tight linkage of the IFN-beta and IFN-alpha genes. In addition, in 15 of 18 progeny the segregation coincided with that of the brown locus on chromosome 4, in accord with previous results obtained with the IFN-alpha probe in strains derived from crosses between BALB/c and C57BL/6 mice. Thus, the Mu IFN-beta gene is tightly linked to the Mu IFN-alpha gene cluster on chromosome 4 near the brown locus.     

Existence of glucose-6-phosphate dehydrogenase-like locus on chromosome 17.

Hybridization of DNA samples prepared from flow-sorted human chromosomes with a cDNA probe for the X-linked glucose-6-phosphate dehydrogenase (G6PD) suggested the existence of the G6PD-like locus on chromosome 17. Southern hybridization analysis of endonuclease-digested DNA samples from the human-mouse hybrid cell line with human chromosome 17, and from control human and mouse cells, proved that not only X chromosomes, but also chromosome 17, contain DNA sequences that are hybridizable with the G6PD cDNA probe. The G6PD-like locus on chromosome 17 could be a putative pseudogene or a functional gene for the fetal brain-specific G6PD isozyme or other protein.     

DNA polymorphisms, identified by an X-chromosome short-arm probe L 1.28 (DXS7), in different racial groups

Restriction fragment length polymorphisms of the L1.28 probe which is closely linked to X-linked disorders, retinitis pigmentosa and Norrie disease, were studied in samples from England, India and Nigeria. The frequency of the A2 allele (9-kb fragment) was 0.23, 0.55 and 0.46 in England, India and Nigeria, respectively. The differences between the English and Indian populations were highly significant.