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Biomedical subjects

C Tyler-Smith

Publications and source records attributed to C Tyler-Smith.

At least 73 records · Page 4Linked to original sources
PubMed1994

De novo formation of several features of a centromere following introduction of a Y alphoid YAC into mammalian cells.

The DNA sequence requirements for mammalian centromere function have been investigated by re-introducing human YAC clones containing either centromeric or non-centromeric sequences into hamster and human cells. All YACs integrated into the host chromosomes. In most cell lines produced by spheroplast fusion into hamster cells, intact copies of the YAC and a large amount of yeast DNA were found. Cell lines produced by lipofection into human cells usually contained simple structures without yeast DNA. YACs containing Y alphoid DNA reformed several of the properties of a centromere, including a cytogenetically visible constriction, CREST antiserum binding and disruption of anaphase chromosome movement. In contrast, YACs containing non-centromeric sequences produced none of these results. This work suggests that a few hundred kb of alphoid DNA is sufficient to reconstitute several important features of a centromere.

Anaphase↗
PubMed1993

Structure of the sequences adjacent to the centromeric alphoid satellite DNA array on the human Y chromosome.

Eighteen yeast artificial chromosome (YAC) clones containing alphoid satellite DNA and adjacent sequences from the human Y chromosome have been identified from three different YAC libraries. Restriction site mapping of the genomic alphoid arrays and the YACs has allowed seven of the alphoid clones to be positioned on the arrays. Three clones extend into flanking sequences. At one edge the alphoid DNA is highly diverged and is flanked by a small block of the 48 base-pair satellite, dispersed moderately repeated sequences and a separate short alphoid array. More distal sequences are Y-specific. At the other edge there is much less divergence and the alphoid DNA is flanked by an Alu sequence and the five base-pair satellite.

Centromere↗
PubMed1993

Mammalian chromosome structure.

The DNA sequences that are necessary for the formation of a functional mammalian chromosome are thought to be the origins of replication, the telomeres and the centromere. Telomere structure is now well understood, with the functional element characterized as the motif (TTAGGG)n. The structures of the DNA regions that contain origins of replication and a centromere are known, but the functionally important elements within these regions are still only poorly defined.

Animals↗
PubMed1993

Localization of DNA sequences required for human centromere function through an analysis of rearranged Y chromosomes.

We have localized the DNA sequences required for mitotic centromere function on the human Y chromosome. Analysis of 33 rearranged Y chromosomes allowed the centromere to be placed in interval 8 of a 24-interval deletion map. Although this interval is polymorphic in size, it can be as small as approximately 500kb. It contains alphoid satellite DNA and approximately 300kb of adjacent Yp sequences. Chromosomes with rearrangements in this region were analysed in detail. Two translocation chromosomes and one monocentric isochromosome had breakpoints within the alphoid array. Of 12 suppressed Y centromeres on translocation chromosomes and dicentric isochromosomes that were also analysed two showed deletions one of which only removed alphoid DNA. These results indicate that alphoid DNA is a functional part of the Y chromosome centromere.

Cell Line↗
PubMed1993

The major centromeric array of alphoid satellite DNA on the human Y chromosome is non-palindromic.

We have determined the orientation of the major centromeric alphoid array on the human Y chromosome. A PCR assay was used to analyse the vector-insert junctions of seven YAC clones previously positioned on two independent Y chromosomes. The orientation is the same at all 10 positions measured. This suggests that the alphoid array is a simple unidirectional repeat throughout and that human centromere structure thus differs from the palindromic organisation found in Schizosaccharomyces pombe.

Base Sequence↗
PubMed1993

Chromosomal localisation of a gene(s) for Turner stigmata on Yp.

Although recent cytogenetic and molecular studies in patients with Turner stigmata are consistent with a gene(s) for Turner stigmata being present on both Xp and Yp, the precise location has not been determined. In this report, we describe a phenotypically female infant with Turner stigmata and a partial Yp deletion and review genotype-phenotype correlations of the putative Turner gene(s) in non-mosaic patients with Y chromosome rearrangements resulting from chromosomal breakage at Yp or Yc (pericentromeric region). The results indicate that the putative Turner gene(s) on Yp is located in the Y specific region from interval 1A1A to interval 2B. In addition, assessment of ZFX/ZFY and RPS4X/RPS4Y in the context of the Turner gene(s) suggests that ZFX/ZFY rather than RPS4X/RPS4Y could be a candidate gene for the Turner stigmata.

Chromosome Deletion↗
PubMed1992

Structure of the pericentric long arm region of the human Y chromosome.

We have analysed the sequence organization of the DNA in the pericentric region of the long arm of the human Y chromosome. The structures of one cosmid and three yeast artificial chromosome clones were determined. The region consists of a mosaic of the known 5, 48 and 68 base-pair tandemly repeated sequences and at least five novel repeated sequence families. A long range-map of approximately 3.5 x 10(6) base-pairs of genomic DNA was constructed that placed the clones between about 500 x 10(3) and 850 x 10(3) base-pairs from the long arm edge of the centromeric alphoid DNA array.

Animals↗
PubMed1992

Dodeca satellite: a conserved G+C-rich satellite from the centromeric heterochromatin of Drosophila melanogaster.

To identify sequences from the centromeric region, we have constructed a Drosophila melanogaster yeast artificial chromosome (YAC) library and screened it with purified DNA from the minichromosome Dp(1;f)1187 derived from the X chromosome. We describe the structure of one clone isolated in this way. This YAC is structurally unstable and contains tandemly repeated G+C-rich 11-mer and 12-mer units, which we call dodeca satellite. Most of this satellite is located near the centromere of an autosome. Cross-hybridizing sequences are found in the genomes of organisms as distant as Arabidopsis thaliana and Homo sapiens.

Animals↗
PubMed1990

Structural instability of human tandemly repeated DNA sequences cloned in yeast artificial chromosome vectors.

The suitability of yeast artificial chromosome vectors (YACs) for cloning human Y chromosome tandemly repeated DNA sequences has been investigated. Clones containing DYZ3 or DYZ5 sequences were found in libraries at about the frequency anticipated on the basis of their abundance in the genome, but clones containing DYZ1 sequences were under-represented and the three clones examined contained junctions between DYZ1 and DYZ2. One DYZ3 clone was quite stable and had a long-range structure corresponding to genomic DNA. All other clones had long-range structures which either did not correspond to genomic DNA, or were too unstable to allow a simple comparison. The effects of the transformation process and host genotype on YAC structural stability were investigated. Gross structural rearrangements were often associated with re-transformation of yeast by a YAC. rad1-deficient yeast strains showed levels of instability similar to wild-type for all YAC clones tested. In rad52-deficient strains, DYZ5 containing YACs were as unstable as in the wild-type host, but DYZ1/DYZ2 or DYZ3 containing YACs were more stable. Thus the use of rad52 hosts for future library construction is recommended, but some sequences will still be unstable.

Chromosomes, Fungal↗
PubMed1989

Construction of yeast artificial chromosome libraries with large inserts using fractionation by pulsed-field gel electrophoresis.

A method for constructing yeast artificial chromosome (YAC) libraries with large insert sizes is reported. High molecular weight human DNA was partially digested with EcoRI and cloned in the vector pYAC4. When unfractionated DNA was used, the mean YAC size was 120kb. Fractionation by pulsed-field gel electrophoresis using a 'waltzer' apparatus to remove small DNA fragments increased the mean YAC size to congruent to 220kb or congruent to 370kb depending on the fractionation conditions. Ligated DNA prepared by this method was stable at 4 degrees C and routinely yielded transformation efficiencies of greater than 700 colonies/micrograms. It should be possible to extend the method to produce even larger inserts and to use high molecular weight DNA from any source.

Cell Line↗
PubMed1988

Structure of a hypervariable tandemly repeated DNA sequence on the short arm of the human Y chromosome.

The structure of a repeated DNA sequence located on the short arm of the human Y chromosome is described. Genomic mapping and cloning in lambda or cosmid vectors show that the repeated sequence consists of units 20.3 x 10(3) base-pairs long that contain the three previously described DNA sequences: Y-156, Y-190 and Y-223a. Analysis of male genomic DNA by pulsed-field gel electrophoresis shows that the units are tandemly arranged and are organized into two blocks. The major block is hypervariable in size and alleles in the range approximately 540 x 10(3) to 800 x 10(3) base-pairs were detected. The minor block is not variable in size and is approximately 60 x 10(3) base-pairs long. Analysis of rearranged Y chromosomes shows that both blocks are located on the short arm of the chromosome. Most commonly, the major block is distal to the minor block, but the opposite arrangement is also found.

Cell Line↗
PubMed1987

Structure of the major block of alphoid satellite DNA on the human Y chromosome.

Alphoid DNA is a family of tandemly repeated simple sequences found mainly at the centromeres of the chromosomes of many primates. This paper describes the structure of the alphoid DNA at the centromere of the human Y chromosome. We have used pulsedfield gradient gel electrophoresis, cosmid cloning and DNA sequencing to determine the organization of the alphoid DNA on each of the Y chromosomes present in two somatic cell hybrids. In each case there is a single major block of alphoid DNA. This is approximately 470,000 bases (475 kb) long on one chromosome and approximately 575 kb long on the other. Apart from the size difference, the structures of the two blocks and the surrounding sequences are very similar. However, one restriction enzyme, AvaII, detects two clusters of sites within one block but does not cleave the other. The alphoid DNA within each block is organized into tandemly repeating units, most of which are about 5.7 kb long. A few variant units present on one chromosome are about 6.0 kb long. These variants, like the AvaII site variants, are clustered. The 5.7 kb and 6.0 kb units themselves consist of tandemly repeating 170 base-pair subunits. The 6.0 kb unit has two more of these subunits than the 5.7 kb unit. Our results provide a basis for further structural analysis of the human Y chromosome centromeric region, and suggest that long-range structural polymorphisms of tandemly repeated sequence families may be frequent.

Base Sequence↗
PubMed1987

Structure of repeated sequences in the centromeric region of the human Y chromosome.

Alphoid satellite DNA is a family of sequences with an approximately 170 bp periodicity which is found near the centromere of all human chromosomes. The structure of the human Y-chromosome alphoid DNA has been studied in two somatic cell hybrids, 3E7 and 853 (Tyler-Smith & Brown, 1987). The 170 bp alphoid subunits are tandemly repeated and are organized into units approximately 5.7 kb long. A few variant units on the 3E7 Y chromosome contain two extra 170 bp subunits and are approximately 6.0 kb long; the variant units are present in two clusters at least 90 kb apart on the chromosome. On each Y chromosome there is a single major block of alphoid DNA: on the 3E7 Y chromosome it is approximately 440 kb long and on the 853 Y chromosome it is approximately 540 kb long. A long-range restriction map of the 853 block has been constructed covering approximately 1.1 mb of DNA. The distribution of restriction sites suggests that the sequences on one side of the alphoid block may be typical euchromatic DNA, while the sequences on the other side may be another satellite sequence.

Centromere↗