John S. Waye

Hierarchical order in chromosome-specific human alpha satellite DNA

Abstract The alpha satellite DNA family consists of an extensive group of related, highly divergent repeats, each approximately 171 bp in length. Long tandem arrays of alphoid sequences at the centromeric region of each human chromosome are characterized by a multimeric higher-order repeat unit whose organization and nucleotide sequence is specific for a particular chromosome. Analysis of the sequence organization of these diverged chromosomal subsets provides a framework for considering mechanisms of generation of sequence diversity and for understanding evolutionary processes of DNA family homogenization and polymorphism.

Chromosome-specific subsets of human alpha satellite DNA: Analysis of sequence divergence within and between chromosomal subsets and evidence for an ancestral pentameric repeat

SummaryThe centromeric regions of human chromosomes are characterized by diverged chromosome-specific subsets of a tandemly repeated DNA family, alpha satellite, which is based on a fundamental monomer repeat unit ∼ 171 bp in length. We have compared the nucleotide sequences of 44 alphoid monomers derived from cloned representatives of the multimeric higher-order repeat units of human chromosomes 1, 11, 17, and X. The 44 monomers exhibit an average 16% divergence from a consensus alphoid sequence, and can be assigned to five distinct homology groups based on patterns of sequence substitutions and gaps relative to the consensus. Approximately half of the overall sequence divergence can be accounted for by sequence changes specific to a particular homology group; the remaining divergence appears to be independent of the five groups and is randomly distributed, both within and between chromosomal subsets. The data are consistent with the proposal that the contemporary tandem arrays on chromosomes 1, 11, 17, and X derive from a common multimeric repeat, consisting of one monomer each from the five homology groups. The sequence comparisons suggest that this pentameric repeat must have spread to these four chromosomal locations many millions of years ago, since which time evolution of the four, now chromosome-specific, alpha satellite subsets has been essentially independent.

Chromosome-specific alpha satellite DNA: Isolation and mapping of a polymorphic alphoid repeat from human chromosome 10

Distinct subsets of the human alpha satellite repetitive DNA family can be found in the centromeric region of each chromosome. Here we described the isolation and mapping of an alpha satellite repeat unit specific for human chromosome 10, using a somatic cell hybrid in which the only human centromere derives from chromosome 10. A hierarchical higher-order repeat unit, consisting of eight tandem approximately 171-bp alphoid monomer units, is defined by six restriction endonucleases. Under high-stringency conditions, a cloned representative of this 8-mer repeat family hybridizes to chromosome 10 only, both by Southern blot analysis of a somatic cell hybrid panel and by in situ hybridization. The probe furthermore detects a polymorphic restriction pattern of the alpha satellite array on chromosome 10. These features will make this probe a valuable genetic marker for studies of the centromeric region of chromosome 10.

Chromosome-specific alpha satellite DNA from human chromosome 1: Hierarchical structure and genomic organization of a polymorphic domain spanning several hundred kilobase pairs of centromeric DNA

The human alpha satellite repetitive DNA family is organized as distinct chromosome-specific subsets localized to the centromeric region of each chromosome. Here, we report he isolation and characterization of cloned repeat units which define a hierarchical subset of alpha satellite on human chromosome 1. This subset is characterized by a 1.9-kb higher-order repeat unit which consists of 11 tandem approximately 171-bp alpha satellite monomer repeat units. The higher-order repeat unit is itself tandemly repeated, present in at least 100 copies at the centromeric region of chromosome 1. Using pulsed-field gel electrophoresis we estimate the total array length of these tandem sequences at the centromere of chromosome 1 to be several hundred kilobase pairs. Under conditions of high stringency, the higher-order repeat probe hybridizes specifically to chromosome 1 and can be used to detect several associated restriction fragment length DNA polymorphisms. As such, this probe may be useful for molecular and genetic analyses of the centromeric region of human chromosome 1.

Organization and evolution of alpha satellite DNA from human chromosome 11.

The human alpha satellite repetitive DNA family is organized as distinct chromosomal subsets located at the centromeric regions of each human chromosome. Here, we describe a subset of the alpha satellite which is localized to human chromosome 11. The principal unit of repetition of this alpha satellite subset is an 850 bp XbaI fragment composed of five tandem diverged alphoid monomers, each ∼171 bp in length. The pentamer repeat units are themselves tandemly reiterated, present in ∼ 500 copies per chromosome 11. In filter hybridization experiments, the Alpha 11 probes are specific for the centromeric alpha satellite sequences of human chromosome 11. The complete nucleotide sequences of two independent copies of the XbaI pentamer reveal a pentameric configuration shared with the alphoid repeats of chromosomes 17 and X, consistent with the existence of an ancestral pentameric repeat common to the centromeric arrays of at least these three human chromosomes.

Chromosome specificity of satellite DNAs: short- and long-range organization of a diverged dimeric subset of human alpha satellite from chromosome 3.

The human alpha satellite DNA family, like many highly repeated satellite DNAs in eukaryotic genomes, is organized in distinct chromosome-specific subsets. As part of investigations into the molecular and evolutionary basis for the chromosome-specific nature of such subsets, we report the isolation and characterization of alpha satellite sequences specific for human chromosome 3. This subset is characterized by a predominant tandemly arranged ∼2.9 kb higher-order repeat unit which, in turn, consists of 17 tandem diverged monomer repeat units of ∼171 bp. Nucleotide sequence analysis reveals that the chromosome 3 higher-order repeat units are comprised, at least in part, of diverged dimeric (∼ 340 bp) sub-repeats and that this divergence accounts for the chromosome-specific behavior of this subset. Pulsed-field gel electrophoresis demonstrates that the chromosome 3 higher-order repeat units are localized in large domains, at least 1000 kb in length. Familial restriction fragment length polymorphisms associated with the satellite subset can be detected by pulsed field gel electrophoresis and may facilitate molecular analysis of interchromosomal variation.

Detection of novel centromeric polymorphisms associated with alpha satellite DNA from human chromosome 11.

SummaryThe pericentromeric region of human chromosomes is composed of diverse classes of repetitive DNAs, which provide a rich source of genetic variability. Here, we describe two novel centromeric polymorphisms associated with a subset of alpha satellite repetitive DNA, D11Z1, which is specific for human chromosome 11. Segregation and inheritance of the polymorphisms are demonstrated and their relative frequencies are determined. These polymorphisms may be useful genetic tools for distinguishing between individual chromosome 11 centromeres. In addition, these polymorphisms may be applied to the development of a centromerebased genetic linkage map of chromosome 11. Molecular models for the generation of these polymorphisms are discussed.

Isolation and expression in Escherichia coli of a cDNA clone encoding human β-glucuronidase

Abstract Mucopolysaccharidosis type VII is a lysosomal storage disease resulting from a deficiency of β-glucuronidase (BG) activity. To facilitate the investigation of mutation in the disease and provide molecular diagnostic tools for affected families, we have isolated human BG cDNA clones. The SV40-transformed human fibroblast cDNA library of Okayama and Berg [Mol. Cell. Biol. 3 (1982) 280–289] was screened with a fragment of a murine BG cDNA clone (pGUS-1). The 17 human cDNA clones (pHUG) isolated were identical by restriction mapping, varying only in length. The pHUG clones show 80% DNA sequence homology with pGUS-1 in a 198-bp PvuII-SstI restriction fragment. Both pGUS-1 and the pHUG clones contained an open reading frame (ORF) throughout the sequenced region with a predicted amino acid sequence homology of 73 %. Expression in Escherichia coli of a 1150-bp fragment of pHUG-1 subcloned in pUC9 resulted in an isopropylthio -β-galactoside (IPTG)-inducible 35-kDal fusion protein which was specifically immunoprecipitated by goat anti-human BG immunoglobulin G (IgG). This evidence provides direct confirmation that the pHUG cDNA clones correspond to human BG.

Organization and genomic distribution of “82H” alpha satellite DNA

SummaryWe have investigated the organization and genomic distribution of sequences homologous to p82H, a cloned human alpha satellite sequence purported, based on previous in situ hybridization experiments, to exist at the centromere of each human chromosome. We report here that, using Southern blotting analysis under conditions of high stringency, p82H hybridizes solely to a low-copy or single-copy alphoid domain located at or near the centromeric region of human chromosome 14. In contrast, conditions of reduced hybridization stringency permit extensive cross-hybridization with nonidentical, chromosome-specific alpha satellite subsets found elsewhere in the human genome. Thus, the previously described ubiquity of “82H” human centromeric sequences reflects the existence of diverse alpha satellite subsets located at the centromeric region of each human chromosome.