Direct end labelling of telomeres.
A novel approach of direct end labelling of telomeres is presented. Chromosome-sized, agarose-embedded DNA was treated with T4 DNA polymerase to remove protruding 3' end of telomeres and to generate single-stranded 5' ends. The DNA was then labelled by the same enzyme in the presence of [alpha-32P]dGTP and cold dATP and dTTP. Labelled yeast chromosomes separated by pulsed field gel electrophoresis maintained their integrity. Digestion of yeast chromosomes separated in pulsed field gels with a restriction nuclease (HinfI), followed by conventional electrophoresis in the second dimension, resulted in a fingerprint-like pattern of labelled telomeres. This was very similar to the hybridization pattern of a similar two-dimensional gel probed with cloned yeast telomeric sequence. The same approach enabled us to label telomeres in soybean, determine their size, and to reveal polymorphisms in the length of telomeres between the closely related subspecies Glycine max (soybean) and Glycine soja.
University of Tennessee, Knoxville 37901-1071.
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Kolchinsky
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PM
Gresshoff
D017360:Arabidopsis / Q000235:genetics; D001483:Base Sequence; D015825:Chromosomes, Fungal; D004277:DNA, Single-Stranded; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific; D005821:Genetic Techniques; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013025:Glycine max / Q000235:genetics; D016615:Telomere
D004277:DNA, Single-Stranded; D004259:DNA-Directed DNA Polymerase; D015252:Deoxyribonucleases, Type II Site-Specific; C032229:GANTC-specific type II deoxyribonucleases