Two-dimensional pulsed-field gel electrophoresis of yeast chromosomes: evidence for triplex-mediated DNA condensation.
The mobility of yeast chromosomes was analysed by two-dimensional pulsed-field gel electrophoresis. The first dimension was run at pH 8.0 in a 1% agarose gel. In the second dimension the electrophoresis conditions were identical, except that the pH was lowered and ethidium, spermine, or ionic detergents were added. Any mobility changes between the two dimensions could be identified as a deviation from the diagonal. At pH 6.0 the mobility of the chromosomes increases severalfold, whereas at pH 4.5 none of the chromosomes move into the agarose gel. The pH-induced mobility changes were reversed by the addition of 2 micrograms/mL of ethidium or 1% lauryl sarcosine. Alternatively, spermine at 1 microM enhanced the pH-mediated mobility changes. Hysteresis was also evident, since upon lowering the pH to 4.5 and then running the gel at pH 7 the mobilities were decreased. These results are interpreted in terms of pH-mediated triplex formation which causes chromosome condensation and thus mobility shifts. The effects of pH are reversed by ethidium which destabilizes triplexes, but enhanced by spermine which favours triplex formation. Therefore, chromosomes may be capable of spontaneous condensation which is mediated by tertiary interactions between appropriate duplex DNA sequences.
Hampel
K J
KJ
Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada.
Lee
J S
JS
D015825:Chromosomes, Fungal / Q000648:ultrastructure*; D004247:DNA / Q000378:metabolism*; D004271:DNA, Fungal / Q000378:metabolism*; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods*; D004996:Ethidium; D006863:Hydrogen-Ion Concentration; D008954:Models, Biological; D009068:Movement; D009690:Nucleic Acid Conformation; D011089:Polydeoxyribonucleotides / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D012521:Sarcosine
D004271:DNA, Fungal; D011089:Polydeoxyribonucleotides; C083473:poly(d(T-m(5)C)).poly(d(G-A)); C051802:triplex DNA; D004247:DNA; D004996:Ethidium; D012521:Sarcosine