Two-dimensional DNA gel electrophoresis as a method for analysis of eukaryotic genome structure: evaluation using Tetrahymena thermophila DNA.
There is growing interest in mapping and analyzing complete eukaryotic genomes. Yee and Inouye (in Experimental Manipulation of Gene Expression, pp. 279-290, Academic Press, New York) demonstrated that bacterial chromosomes can be resolved into interpretable patterns of DNA fragments by means of restriction enzyme digestion and electrophoresis in two dimensions. We have begun to explore applications of this procedure to analysis of eukaryotic genomes, which are far more complex. Tetrahymena thermophila was selected as a model organism because its genome is small, roughly equivalent to that of a single human chromosome. In addition, each Tetrahymena cell contains two nuclei which differ in sequence composition and methylation. Our results demonstrate that the Tetrahymena genome can be resolved into complex patterns of fragments in two dimensions. Hybridization to Southern blots of these gels with a multiply repeated sequence probe yielded analyzable patterns of a subset of the genome. The blots reveal alterations in genome structure due to methylation and rearrangement. Future extensions of the method are discussed.
Hüvös
P E
PE
Department of Biology, Brandeis University, Waltham, MA 02254.
Aquiles Sanchez
J
J
Kramer
K M
KM
Karrer
K M
KM
Wangh
L J
LJ
D000818:Animals; D004247:DNA / Q000032:analysis*; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005784:Gene Amplification; D008745:Methylation; D012091:Repetitive Sequences, Nucleic Acid; D013768:Tetrahymena / Q000235:genetics*
D004247:DNA; D004262:DNA Restriction Enzymes