Genomic organization of alpha satellite DNA on human chromosome 7: evidence for two distinct alphoid domains on a single chromosome.

A complete understanding of chromosomal disjunction during mitosis and meiosis in complex genomes such as the human genome awaits detailed characterization of both the molecular structure and genetic behavior of the centromeric regions of chromosomes. Such analyses in turn require knowledge of the organization and nature of DNA sequences associated with centromeres. The most prominent class of centromeric DNA sequences in the human genome is the alpha satellite family of tandemly repeated DNA, which is organized as distinct chromosomal subsets. Each subset is characterized by a particular multimeric higher-order repeat unit consisting of tandemly reiterated, diverged alpha satellite monomers of approximately 171 base pairs. The higher-order repeat units are themselves tandemly reiterated and represent the most recently amplified or fixed alphoid sequences. We present evidence that there are at least two independent domains of alpha satellite DNA on chromosome 7, each characterized by their own distinct higher-order repeat structure. We determined the complete nucleotide sequences of a 6-monomer higher-order repeat unit, which is present in approximately 500 copies per chromosome 7, as well as those of a less-abundant (approximately 10 copies) 16-monomer higher-order repeat unit. Sequence analysis indicated that these repeats are evolutionarily distinct. Genomic hybridization experiments established that each is maintained in relatively homogeneous tandem arrays with no detectable interspersion. We propose mechanisms by which multiple unrelated higher-order repeat domains may be formed and maintained within a single chromosomal subset.

Chromosome-specific alpha satellite DNA from the centromere of human chromosome 16.

To examine the molecular organization of DNA sequences located in the centromeric region of human chromosome 16 we have isolated and characterized a chromosome 16-specific member of the alpha satellite DNA family. The probe obtained is specific for the centromere of chromosome 16 by somatic cell hybrid analysis and by fluorescence in situ hybridization and allows detection of specific hybridizing domains in interphase nuclei. Nucleotide sequence analysis indicates that this class of chromosome 16 alpha satellite (D16Z2) is organized as a series of diverged 340-bp dimers arranged in a tandem array of 1.7-kb higher-order repeat units. As measured by pulsed-field gel electrophoresis, the total D16Z2 array spans approximately 1,400-2,000 kb of centromeric DNA. These sequences are highly polymorphic, both by conventional agarose-gel electrophoresis and by pulsed-field gel electrophoresis. Investigation of this family of alpha satellite should facilitate the further genomic, cytogenetic, and genetic analysis of chromosome 16.

Localization of 24 cosmid clones on the human Y chromosome.

Twenty-four novel cosmid clones were cloned and mapped on the human Y chromosome using a panel consisting of DNA from seven individuals each having a different segment of the Y chromosome. Eight were assigned to the short arm, 15 to the long arm and 1 to the both short and long arms.

Detection of restriction fragment length polymorphisms at the centromeres of human chromosomes by using chromosome-specific alpha satellite DNA probes: implications for development of centromere-based genetic linkage maps.

We describe a general strategy for the detection of high-frequency restriction fragment length polymorphisms in the centromeric regions of human chromosomes by molecular analysis of alpha satellite DNA, a diverse family of tandemly repeated DNA located near the centromeres of all human chromosomes. To illustrate this strategy, cloned alpha satellite repeats isolated from two human chromosomes, 17 and X, have been used under high-stringency conditions that take advantage of the chromosome-specific organization of this divergent repeated DNA family. Multiple high-frequency restriction fragment length polymorphisms are described for the centromeric region of both chromosome 17 and X chromosome. Mendelian inheritance of the variants is demonstrated. The X-linked alpha satellite polymorphisms in particular are highly informative and constitute a virtually unique centromeric DNA marker for each X chromosome examined. Since the strategy we describe is a general one, the alpha satellite family of DNA should provide a rich source of molecular variation in the human genome and should contribute to the development of centromere-based genetic linkage maps of human chromosomes.

Molecular analysis of muscular dystrophy.

It is now possible to map almost any disease locus to a chromosomal region in the human genome by family studies with restriction fragment length polymorphisms. Duchenne and Becker muscular dystrophies have been shown to be localized within the same small region of Xp21 on the human X chromosome. Myotonic dystrophy has been localized to a region close to the centromere of chromosome 19. Technologies are now available to identify candidate genes for the diseases. Autosomal recessive muscular dystrophies are more difficult to study, but even these will be amenable to analysis in the very near future. The next decade should witness some exciting advances in the molecular analysis and clinical management of human muscular dystrophies.

Very mild muscular dystrophy associated with the deletion of 46% of dystrophin.

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), a much milder form of the disease where the age of onset can sometimes be as late as the third or fourth decade of life, are caused by mutations in the same X-linked gene, a 14 kilobase (kb) transcript which is spread over more than 2 megabases of the human X chromosome. The corresponding protein, dystrophin, has a relative molecular mass of 400,000. Most mutations causing DMD and BMD are deletions and deletions associated with both phenotypes are observed throughout the gene sequence. This observation led to the suggestion that DMD patients possess deletions that disrupt the reading frame of the protein, whereas BMD patients have deletions that retain the translational reading frame and enable the muscle cells to produce altered dystrophin products. This theory is supported by immunoblotting studies, which show that DMD patients lack dystrophin in their muscle cells or that dystrophin is present at very low levels, whereas BMD patients produce a protein with reduced abundance or abnormal size. Here we describe a deletion of the dystrophin gene in a family segregating for very mild BMD, one member of which was still ambulant at age 61 years, which removes a central part of the dystrophin gene encompassing 5,106 base pairs of coding sequence, almost half the coding information. Immunological analysis of muscle from one of the patients demonstrates that this mutation results in the production of a truncated polypeptide localized correctly in the muscle cell. These results are particularly significant in the context of gene therapy which, if it is ever envisaged, would be facilitated by the replacement of the very large dystrophin gene with a more manipulatable mini-gene construct.

Sequences of junction fragments in the deletion-prone region of the dystrophin gene.

The Duchenne muscular dystrophy locus is remarkable in that it shows a high mutation rate and the majority of mutations found are deletions. These deletions are generated as meiotic as well as mitotic events and occur preferentially in the central region of the gene. Nothing is known so far about the mechanisms involved. This paper reports the first sequencing of deletion junctions in the dystrophin gene. The data from a study of two patients with deletions in the central region of dystrophin show the breakpoints to lie in regions of introns in which stretches of dA-dT are seen. The relationship between these observations and possible mechanisms for the mutations is discussed.