DIRECT END LABELING OF TELOMERES

A novel approach of direct end labelling of telomeres is presented. Ch romosome-sized, agarose-embedded DNA was treated with T4 DNA polymeras e to remove protruding 3' end of telomeres and to generate single-stra nded 5' ends. The DNA was then labelled by the same enzyme in the pres ence of [alpha-P-32]dGTP and cold dATP and dTTP. Labelled yeast chromo somes separated by pulsed field gel electrophoresis maintained their i ntegrity. Digestion of yeast chromosomes separated in pulsed field gel s with a restriction nuclease (HinfI), followed by conventional electr ophoresis in the second dimension, resulted in a fingerprint-like patt ern of labelled telomeres. This was very similar to the hybridization pattern of a similar two-dimensional gel probed with cloned yeast telo meric sequence. The same approach enabled us to label telomeres in soy bean, determine their size, and to reveal polymorphisms in the length of telomeres between the closely related subspecies Glycine max (soybe an) and Glycine soja.