Genome mapping: PCR based meiotic and somatic cell hybrid analysis.
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Animals↗
Biomedical subjects
H Lehrach
Publications and source records attributed to H Lehrach.
Huntington disease-linked locus D4S111 exposed as the alpha-L-iduronidase gene.
alpha-L-Iduronidase (IDUA) has been intensively studied due to its causative role in mucopolysaccharidosis type I (Hurler, Scheie and Hurler/Scheie syndromes). The recent cloning of a human IDUA cDNA has resulted in a reevaluation of the chromosomal location of this gene. Previously assigned to chromosome 22, IDUA now has been localized to 4p16.3, the region of chromosome 4 associated with Huntington's disease (HD). The existence of a battery of cloned DNA, physical map information, and genetic polymorphism data for this region has allowed the rapid fine mapping of IDUA within the terminal cytogenetic band of 4p. IDUA was found to be coincident with D4S111, an anonymous locus displaying a highly informative multiallele DNA polymorphism. This map location, 1.1 X 10(6) bp from the telomere, makes IDUA the most distal cloned gene assigned to 4p. However, it falls within a segment of 4p16.3 that has been eliminated from the HD candidate region, excluding a role for IDUA in this disorder.
Alleles↗
New DNA markers in the Huntington's disease gene candidate region.
The search for the Huntington's disease (HD) gene has prompted construction of a complete long-range restriction map of a 2.5-Mb candidate region, distal to the DNA marker D4S10. To facilitate the procurement of cloned DNA from this candidate region, we have augmented the existing regional mapping panel of somatic cell hybrids with hybrid HHW1071 containing a t(4p16;12) chromosome from a patient with Wolf-Hirschhorn syndrome. This translocation maps between D4S180 and D4S127, subdividing the HD candidate region and setting a proximal limit to the Wolf-Hirschhorn syndrome region. Using the expanded mapping panel, we have regionally assigned 14 independently cloned cosmids, five proximal to the t(4;12) breakpoint in the same region as D4S10 and nine distal to the breakpoint. By a combination of overlap with previously mapped cosmids and pulsed-field gel analysis, each of these cosmids has been positioned on the long-range restriction map of 4p16.3, increasing the clone coverage of the candidate region to approximately 40%. Single-copy probes from mapped cosmids were used to identify eight new DNA polymorphisms spanning the HD candidate region. These new DNA markers should prove valuable for analysis of recombination and linkage disequilibrium in HD, as well as for preclinical diagnosis of the disorder.
Alleles↗
Genetic mapping of the t-complex region on mouse chromosome 17 including the Hybrid sterility-1 gene.
t-haplotypes occupy a region on chromosome (Chr) 17 which slightly overlaps the ends of the T-H-2 interval. The wild-type form of this 14 centi-Morgan (cM) region was mapped in a multilocus backcross (C57BL/10-T x C3H)F1 x C57BL/10 using 15 DNA probes on Southern blots of the DNA extracted from 53 animals which were recombinants in the T-H-2 interval. Each recombinant was also progeny-tested to ascertain its Hybrid sterility-1 (Hst-1) genotype by crossing to PWB/Ph, a Mus musculus-derived inbred strain. The limit of resolution of the cross was 0.27 cM. The map distances have been determined for the DNA loci in the T-H-2 interval and the Hst-1 gene was mapped in close vicinity to the D17Rp17 locus.
Animals↗
Hybridization analyses of arrayed cDNA libraries.
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Base Sequence↗
Isolation of the human sex determining region from a Y-enriched yeast artificial chromosome library.
We describe the isolation and characterization of yeast artificial chromosome (YAC) clones spanning the male sex determining region on the short arm of the human Y chromosome. The clones were isolated by hybridizing probes in the interval between the genes MIC2 and ZFY to a Y chromosome-enriched YAC library. The YAC clones were consistent with the order of probes established for this interval and may be useful for functional studies of the region in male sex determination. However, many of the YAC clones from this library carried only one arm of the vector ("half-YACs"), deleted sequences from one end, and contained much smaller inserts (148 kb average) than the size of ligated fragments selected by pulsed-field gel electrophoresis (greater than 440 kb). These problems were overcome by protecting DNA with polyamines during YAC library construction and a second Y-enriched YAC library was constructed with an average insert size of 627 kb.
Cell Line↗
Interspersed repetitive element polymerase chain reaction product mapping using a mouse interspecific backcross.
We have developed a rapid method of generating and simultaneously mapping interrepeat polymerase chain reaction products using DNA from interspecific backcross animals derived from mating C57BL/6J and Mus spretus mice. This method is based on the high degree of B1, B2, and L1 dispersed repeat position polymorphism found between these two species of mouse. We have mapped 13 new loci to 9 different chromosomes and have found no evidence of clustering among these loci. The advantages of this approach are that no prior knowledge of sequence is required, a single PCR reaction generates many markers which can be mapped simultaneously, and only 50 ng of each backcross DNA (a finite resource) is required. We anticipate that many more markers remain to be characterized in this valuable new source of polymorphism.
Animals↗
Complex patterns of linkage disequilibrium in the Huntington disease region.
The genetic defect causing Huntington disease (HD) has been mapped to 4p16.3 by linkage analysis using DNA markers. Two apparently contradictory classes of recombination events in HD kindreds preclude precise targeting of efforts to clone the disease gene. Here, we report a new recombination event that increases support for an internal candidate region of 2.5 Mb between D4S10 and D4S168. Analysis of 23 DNA polymorphisms in 4p16.3 revealed a complex pattern of association with the disease gene that failed to narrow the size of the candidate region. The degree of linkage disequilibrium did not show a continuous increase across the physical map, nor was a region of extreme disequilibrium identified. Markers displaying no association with the disorder were interspersed with and, in many cases, close to markers displaying significant disequilibrium. Comparison of closely spaced marker pairs on normal and HD chromosomes, as well as analysis of haplotypes across the HD region, suggest that simple recombination subsequent to a single original HD mutation cannot easily explain the pool of HD chromosomes seen today. A number of different mechanisms could contribute to the diversity of haplotypes observed on HD chromosomes, but it is likely that there has been more than one and possibly several independent origins of the HD mutation.
Alleles↗
Defined physical limits of the Huntington disease gene candidate region.
The Huntington disease (HD) gene has been mapped 4 cM distal to D4S10 within the telomeric chromosome band, 4p16.3. The published physical map of this region extends from D4S10 to the telomere but contains two gaps of unknown size. Recombination events have been used to position the HD mutation with respect to genetic markers within this region, and one such event places the gene proximal to D4S168, excluding the distal gap as a possible location for the defect. One previously published recombination event appeared to have excluded the proximal gap. We have reassessed this event and have moved the proximal boundary for the HD candidate region centromeric to the gap within a "hot spot" for recombination between D4S10 and D4S125. We have closed the proximal gap and report here the complete physical map spanning the HD candidate region from D4S10 to D4S168, the maximum size of which can now be placed accurately at 2.5 Mb.
Chromosome Mapping↗
Easy removal of oil from microtiter dishes following PCR.
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Freezing↗
Quantitative measurements on the duplex stability of 2,6-diaminopurine and 5-chloro-uracil nucleotides using enzymatically synthesized oligomers.
2,6-Diaminopurine and 5-chloro-uracil 2'-deoxynucleoside 5'-triphosphates were synthesized from their 2'-deoxynucleosides. Using a method of creating oligonucleotides by enzymatic primer extension, dodecanucleotides representing an XbaI/SalI site and the complementary SalI/XbaI site were generated containing these base modifications. Their duplex stability was quantitatively compared by thin-layer chromatography to oligomers containing 2'-deoxyadenosine and 2'-deoxythymidine. The two unmodified oligomers already showed significant differences in dissociation temperature and binding equilibrium. Substitution with 5-chloro-2'-deoxyuridine did not affect the dissociation temperature of either oligomer, the 2,6-diaminopurine, however, led to an increase of 1.8 degrees C or 1.5 degrees C per modified base, respectively. While in the XbaI/SalI oligomer both base modifications changed the binding equilibrium, the 2,6-diaminopurine by a factor of 1.32, the 5-chloro-uracil by 0.65, no such effect was found with the complementary oligomer.
Base Sequence↗
Effect of 5-bromo- and 5-methyldeoxycytosine on duplex stability and discrimination of the NotI octadeoxynucleotide. Quantitative measurements using thin-layer chromatography.
Octadeoxynucleotides based on the recognition sequence of the restriction endonuclease NotI were synthesized containing unmodified nucleotides and nucleotides with methyl and bromide additions at the C5 position of the pyrimidine ring of deoxycytosine. On annealing to single-stranded DNA bearing one NotI site, thin-layer chromatography (TLC) of the different oligonucleotides was used quantitatively to determine differences in dissociation temperature (Td) and binding equilibrium. Buffers used in filter hybridization experiments could be used in this TLC system. In addition, actual hybridizations were carried out to filter-bound DNA with and without a NotI site. The incorporation of 5-methyldeoxycytosine and 5-bromodeoxycytosine led to a significant increase in stability of homoduplex formation during hybridization, due to a shift in the binding equilibrium and an increase of the Td, thereby improving discrimination considerably. Some implications of the results for several techniques involving oligomer hybridization are discussed.
5-Methylcytosine↗
Ordering of cosmid clones covering the herpes simplex virus type I (HSV-I) genome: a test case for fingerprinting by hybridisation.
To allow the efficient construction of ordered clone libraries, we have been investigating the use of 'oligonucleotide fingerprinting' as an approach to identify overlapping clones, and ultimately restore the linear order of the clone set. To test the effectiveness of the procedure, we have constructed a cosmid library from the genome of the human DNA virus HSV-I and used hybridisation to multiple oligonucleotides selected from the nucleotide sequence to reconstruct the order of clones and oligonucleotides on the genome.
Base Sequence↗
Cloning of the T gene required in mesoderm formation in the mouse.
The murine developmental mutation T identifies an essential gene in mesoderm formation. Embryos lacking normal gene activity fail to form the notochord, the entire posterior region and the allantois, and die at about 10 days of gestation. We have isolated the T gene using a combination of molecular and genetic techniques, thus making molecular tools available to study processes underlying mesoderm formation in the mouse.
Alleles↗
The murine genes Hox-5.1 and Hox-4.1 belong to the same HOX complex on chromosome 2.
Two different loci of Antennapedia-related homeobox-containing genes have been shown to map to mouse chromosome 2: the HOX-5 complex and the Hox-4.1 gene. These independently derived loci are likely to be parts of a single gene complex, although their close linkage has not yet been demonstrated. Since cosmid walks to extend the HOX-5 cluster and to potentially link the two loci were unsuccessful, we have used large restriction fragments separated by pulsed-field gel electrophoresis to demonstrate the linkage between probes from the HOX-5 region and sequences near Hox-4.1. To further define the distance between the two linked loci, we screened a NotI jumping library with sequences near the Hox-5.1 gene to obtain a marker within the region predicted to contain Hox-4.1. The jumping endpoint lies within genomic clones from a lambda phage walk extending from the 5' end of Hox-4.1, and thus provides clear evidence of linkage between the two Hox loci. Our results demonstrate that Hox-4.1 lies approximately 35 kb downstream of the Hox-5.1 gene and that the two loci do indeed thus constitute parts of the same HOX complex.
Animals↗
The HOX-5 and surfeit gene clusters are linked in the proximal portion of mouse chromosome 2.
Using an interspecies backcross, we have mapped the HOX-5 and surfeit (surf) gene clusters within the proximal portion of mouse chromosome 2. While the HOX-5 cluster of homeobox-containing genes has been localized to chromosome 2, bands C3-E1, by in situ hybridization, its more precise position relative to the genes and cloned markers of chromosome 2 was not known. Surfeit, a tight cluster of at least six highly conserved "housekeeping" genes, has not been previously mapped in mouse, but has been localized to human chromosome 9q, a region of the human genome with strong homology to proximal mouse chromosome 2. The data presented here place HOX-5 in the vicinity of the closely linked set of developmental mutations rachiterata, lethargic, and fidget and place surf close to the proto-oncogene Abl, near the centromere of chromosome 2.
Animals↗
The alpha-A-crystallin and cystathionine beta-synthase genes are physically very closely linked in proximal mouse chromosome 17.
Murine genes homologous to those contributing to the Down syndrome (DS) phenotype in man are currently of interest because of their potential for providing animal models for the study of specific DS symptoms. Most of the genes mapping to human chromosome 21q22, where the DS genes are concentrated, are related to sequences located on mouse chromosome 16. Others, however, are known to map to mouse chromosome 10, and two genes, cystathionine beta-synthase (Cbs) and alpha-A-crystallin (Crya-1), have been localized to the proximal portion of mouse chromosome 17. In this paper, we show that the two genes mapping to human chromosome 21q22 and mouse chromosome 17 are very tightly linked in mouse, being separated by at least 70 kb, but not more than 130 kb. The very close physical linkage of mouse Cbs and Crya-1, combined with data that localize homologs of the closely flanking markers H2k and Pim-1 to human chromosome 6, suggests that the human 21q22/mouse chromosome 17 conserved segment is of a very limited total physical size and is likely to contain a relatively small number of genes.
Animals↗
A yeast artificial chromosome containing the mouse homeobox cluster Hox-2.
We have isolated two genes, Hox-2.8 and Hox-2.9, from the mouse homeobox cluster Hox-2, located on chromosome 11. A 120-kilobase yeast artificial chromosome (YAC) containing a large region of the murine Hox-2 cluster, including 45 kilobases of sequence upstream of the most 5' gene, was cloned. The DNA sequence of the YAC is unrearranged relative to the genomic map. We have subcloned from the YAC insert a homeobox gene, Hox-2.8, whose homeodolmain is highly related to that of the Drosophila homeotic gene proboscopedia (pb). The expression pattern of Hox-2.8 during embryogenesis extends the trend established by genes from Hox-2.5 to -2.7 of successively anterior domains of expression in the neural tube. We have also subcloned and sequenced from a cosmid the labial (lab)-related Hox-2.9, the most 3' member of the cluster to date. These data lend further support to the idea of a common evolutionary origin of the mouse Hox and Drosophila HOM clusters. The YAC will enable us to construct modified forms of the Hox-2 cluster in yeast and to identify their effect on the phenotype of the animal in transgenic mouse strains.
Amino Acid Sequence↗