RESUMO
Twenty-nine deletion breakpoints were mapped in 220 kb of the DXS164 locus relative to potential exons of the Duchenne and Becker muscular dystrophy gene. Four deletion junction fragments were isolated to acquire outlying Xp21 loci on both the terminal and centromere side of the DXS164 locus. The junction loci were used for chromosome walking, searches for DNA polymorphisms, and mapping against deletion and translocation breakpoints. Forty-four unrelated deletions were analyzed using the junction loci as hybridization probes to map the endpoints between cloned Xp21 loci. DNA polymorphisms from the DXS164 and junction loci were used to follow the segregation of a mutation in a family that represents a recombinant. Both the physical and genetic data point to a very large size for this X-linked muscular dystrophy locus.
Assuntos
Deleção Cromossômica , Fragilidade Cromossômica , Ligação Genética , Distrofias Musculares/genética , Cromossomo X , Mapeamento Cromossômico , Clonagem Molecular , Feminino , Marcadores Genéticos , Humanos , Masculino , Linhagem , Polimorfismo de Fragmento de RestriçãoRESUMO
A protocol for the rapid cloning of many DNA fragments from an amplified genomic region is described. The procedure is based on a modification of the phenol-emulsion reassociation technique (PERT) previously used to clone DNA fragments missing from a chromosomal deletion [Kunkel et al., Proc. Natl. Acad. Sci. USA 82 (1985) 4778-4782]. The procedure was used to construct recombinant libraries in the plasmid pBR322 which were highly enriched for amplified sequences from two neuroblastoma cell lines, CHP-126 and IMR-32. Many new amplified DNA fragments were isolated from these libraries, indicating that the PERT methodology should be of general use in isolating amplified DNA from other cell lines and tumors.
Assuntos
Clonagem Molecular/métodos , DNA de Neoplasias/isolamento & purificação , DNA Recombinante/isolamento & purificação , Neuroblastoma/genética , Hibridização de Ácido Nucleico , Sequência de Bases , Linhagem Celular , DNA de Neoplasias/genética , Emulsões , Humanos , Fenol , FenóisRESUMO
The inheritance of Duchenne muscular dystrophy in 25 families was studied with 13 X chromosome specific cloned DNA fragments from 10 loci in and surrounding Xp21. When multiple probes were informative, the meiotic exchange points for each meiosis were located in individual families. Neither genetic nor physical evidence indicates an unusually high recombination rate across Xp21 in these 25 families.
Assuntos
Distrofias Musculares/genética , Recombinação Genética , Cromossomo X , Deleção Cromossômica , Mapeamento Cromossômico , Feminino , Ligação Genética , Humanos , Masculino , Meiose , Polimorfismo de Fragmento de Restrição , SíndromeRESUMO
Duchenne muscular dystrophy (DMD) and the less severe Becker muscular dystrophy (BMD) are human X-linked muscle-wasting disorders that have been localized to the band Xp21 by genetic linkage analysis and cytologically detectable abnormalities. A cloned DNA segment, DXS164 (or pERT87), has been shown to detect deletions in the DNA of unrelated DMD and BMD males. Here we present the nucleotide sequence of two highly conserved DNA fragments from the DXS164 locus and their homologous sequences from the mouse X chromosome. One of the human conserved segments hybridized to a large transcript in RNA isolated from human fetal skeletal muscle and was used to isolate cDNA clones which cover approximately 10% of this transcript. The cDNA clones map to Xp21 and hybridize with a minimum of eight small regions that span 130 kilobases (kb) of the DXS164 locus. These expressed sequences are candidates for portions of the gene responsible for both DMD and BMD.