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D Tkachuk

Publications and source records attributed to D Tkachuk.

8 recordsLinked to original sources
PubMed1993

Libraries for each human chromosome, constructed from sorter-enriched chromosomes by using linker-adaptor PCR.

We describe here the production of complex libraries enriched in sequences from each human chromosome type, starting with only a few thousand sorter-purified chromosomes. In this procedure, DNA is extracted from the sorted chromosomes, digested to completion by using the frequently cutting restriction endonuclease Sau3A1, and ligated, on each end, to an adaptor oligonucleotide. These fragments are then amplified using PCR with a sequence homologous to the adaptor oligonucleotide as a primer. We have used this procedure to produce PCR libraries for each of the 24 human chromosomes. These libraries were characterized by gel electrophoresis and found to be composed of a continuum of sequences ranging in size from a few hundred to approximately 1,000 bp. The libraries, when used as probes for fluorescence in situ hybridization, stained the target chromosomes more or less continuously, even after PCR amplification for more than 200 cycles. These libraries are useful as hybridization probes to facilitate molecular cytogenetic studies and as sources of probes either for identification of polymorphic short tandemly repeated sequences or for development of sequence-tagged sites.

Base Sequence↗
PubMed1991

Applications of fluorescence in situ hybridization in biological dosimetry and detection of disease-specific chromosome aberrations.

Dual color FISH with whole chromosome and pan-centromere probes facilitates rapid detection of stable structural aberrations such as translocations. This approach should allow analysis of translocations for assessment of genetic damage at long times after exposure or as a result of chronic exposure during a long period of time. Multi-color FISH with locus specific probes allows assessment of the frequency of cells carrying specific aberrations known to be associated with tumorigenesis, analysis of the series of genetic changes that occur during tumor evolution and correlation between genotype and phenotype. The power of FISH for analysis of random and tumor related events will increase steadily as informative probes are developed during the course of the International Human Genome Project.

Chromosome Aberrations↗
PubMed1990

Analytical approaches to detection and characterization of disease-linked chromosome aberrations.

Flow karyotyping and FISH with chromosome specific or disease-locus-specific probes are powerful adjuncts to conventional cytogenetic analysis. Flow karyotyping is well suited to quantitative analysis of DNA content changes that occur during structural rearrangement. FISH with probes for repeated sequences allows ready detection of aneuploidy in interphase cells. FISH with whole chromosome composite probes to metaphase spreads facilitates detection of subtle structural changes and allows detection of structural aberrations that occur at frequencies as low as 10(-3). FISH with locus-specific probes facilitates diagnosis of specific genetic diseases, allows phenotype-genotype correlation on a cell-by-cell basis and may be developed into a sensitive method for detection of residual disease.

Chromosome Aberrations↗
PubMed1988

Sequence and organization of the human T cell delta chain gene.

A novel human T cell receptor (TcR) gene, located 85 kbp upstream to the C alpha coding regions, was isolated using human genomic clones to identify cDNA homologous to messages encoded by this region. The deduced protein sequence of this gene is highly homologous to that of the newly identified constant region found in the murine TcR alpha chain locus. This gene undergoes rearrangements and is expressed at the RNA level in human thymocytes, peripheral T cells and several leukemic T cell lines which have been shown to express the surface gamma-delta heterodimer, suggesting that this gene encodes the human T cell delta chain.

Amino Acid Sequence↗
PubMed1988

The human T cell receptor alpha-delta locus: a physical map of the variable, joining and constant region genes.

In this study a physical macro-restriction map of the entire human alpha locus that spans about 1000 kilobase pairs and includes the V alpha, J alpha and C alpha genes is presented. Evidence is provided that gene duplications were involved in the increase of genomic diversity of V alpha genes. In addition, we show a detailed map of a 40-kb region located approximately 100 kg upstream of the human C alpha gene. Direct evidence is provided to support that the human alpha chain locus, like the murine, also contains another T cell constant region gene in the alpha chain locus, the human delta chain gene. In addition, two J segments and one D segment have been identified. Using these genomic probes, we show that several T cell lines, including those known to express the surface gamma/delta heterodimer, have rearranged this region. The design of two separate centers of rearrangement within one locus that are involved in rearrangement events at different times, and the presence of high number of J segments in this region, may render the locus highly vulnerable to chromosomal translocation during T cell development.

Cell Line↗
PubMed1988

Sequence and organization of the diversity, joining, and constant region genes of the human T-cell delta-chain locus.

In this paper we describe the genomic organization and sequence of the human T-cell receptor delta-chain diversity, joining, and constant genes. There is one delta-chain constant region gene (C delta) located approximately equal to 85 kilobases (kb) upstream of the alpha-chain constant region. The delta-chain constant region consists of four exons, whose organization is very similar to that of the C alpha exons, suggesting that C alpha and C delta may have arisen from a gene duplication event. The first exon encodes most of the extracellular constant domain, the second encodes a hinge-like region, and the third encodes the entire transmembrane segment and intracytoplasmic portion, whereas the last exon contains exclusively 3' untranslated sequences. Three joining segments, J delta 1, J delta 2, and J delta 3, are found approximately equal to 12, approximately equal to 5.7, and approximately equal to 3.4 kb upstream of the first exon of C delta. Two functional diversity gene segments, D delta 1 and D delta 2, which can be productively translated in all three reading frames, are found 1 and 9.6 kb upstream of J delta 1. The presence of two D delta with such potential for diversity may offset the limited repertoire of the J delta and V delta genes. The spacer distribution in the recombinational signals flanking D delta and J delta segments allows recombination with V alpha gene segments; however, examination of delta-chain messages does not indicate that this is the case, suggesting that the delta chain uses unique variable gene segments and raising the question as to the reasons for this phenomenon.

Base Sequence↗
PubMed1982

Simple repeated sequences in human satellite DNA.

In an extensive analysis, using a range of restriction endonucleases, HinfI and TaqI were found to differentiate satellites I, II and III & IV. Satellite I is resistant to digestion by TaqI, but is cleaved by HinfI to yield three major fragments of approximate size 770, 850 and 950bp, associated in a single length of DNA. The 770bp fragment contains recognition sites for a number of other enzymes, whereas the 850 and 950bp fragments are "silent" by restriction enzyme analysis. Satellite II is digested by HinfI into a large number of very small (10-80bp) fragments, many of which also contain TaqI sites. A proportion of the HinfI sites in satellite II have the sequence 5'GA(GC)TC. The HinfI digestion products of satellites III and IV form a complete ladder, stretching from 15bp or less to more than 250bp, with adjacent multimers separated by an increment of 5bp. The ladder fragments do not contain TaqI sites and all HinfI sites have the sequence 5'GA(AT)TC. Three fragments from the HinfI ladder of satellite III have been sequenced, and all consist of a tandemly repeated 5bp sequence, 5'TTCCA, with a non-repeated, G+C rich sequence, 9bp in length, at the 3' end.

Base Composition↗