A rapid, convenient and economical method for the hybridization of electrophoretically resolved RNA to DNA restriction fragments immobilized on nitrocellulose filters is described. DNA was digested, electrophoresed on agarose gels in a wide band and transferred to a nitrocellulose filter. The filter was then placed on the surface of a second gel containing radioactively labeled RNA electrophoresed under denaturing conditions in a similar way. The filter and gel were oriented so that the DNA and RNA bands were perpendicular to one another and the RNA was transferred from the gel through the filter under conditions which promote RNA-DNA hybridization. Following washing, the filter was autoradiographed. RNA-DNA sequence relationships could be conveniently determined from the spots produced at regions of intersection of homologous nucleic acids. The two dimensional array formed in this procedure fascilitates the rapid ordering of DNA restriction fragments. An example of its use for this purpose is presented.
Electron microscopic analysis of reassociated deoxyribonucleic acid (DNA) from the aquatic fungus Achlya bisexualis revealed details of the sequence arrangement of the inverted repeats and both the highly and moderately repetitive sequence clusters. We used the gene 32 protein-ethidium bromide technique for visualizing the DNA molecules, a procedure which provides excellent contrast between single-and double-stranded DNA regions. Long (>6-kilobase) DNA fragments were isolated after reannealing to two different repetitive Cot values, and the renatured structures were then visualized in an electron microscope. Our results showed that the inverted repeat sequences were short (0.5 kilobase, number-average) and separated by nonhomologous DNA of various lengths. These pairs of sequences were not clustered within the genome. Both highly repetitive and moderately repetitive DNA sequences were organized as tandem arrays of precisely paired, regularly repeating units. No permuted clusters of repeating sequences were observed, nor was there evidence of interspersion of repetitive with single-copy DNA sequences in the Achlya genome.Many features of fungal chromosome structure and function are similar to those of higher eucaryotes (5,7,18,24). However, the sequence arrangement of several fungus deoxyribonucleic acids (DNAs) has been found to differ significantly from either of the two general patterns of eucaryotic genome organization (1, 25). These are (i) short-period interspersion in which 0.2-to 0.4-kilobase pair (kbp) repetitive sequences are interspersed among single-copy sequences at intervals of<3 kbp (e.g., Xenopus, sea urchin) and (ii) long-period interspersion in which repetitive sequences averaging 6 kbp are contiguous with single-copy sequences of >10 kbp in length (e.g., Drosophila). Reassociation analysis of the nuclear DNA ofAspergillus (23), Neurospora (16), Saccharomyces (12), Phycomyces (11), Schizophillum (6), and Achlya (13) has shown that these organisms have very small genomes (<0.05 pg) and contain only limited amounts of repetitive DNA, most of which may be ribosomal DNA (rDNA). The repetitive DNA appears not to be interspersed with single-copy sequences at the longest DNA lengths studied (>10 kbp). In another mold, Dictyostelium, half of the repetitive sequences are interspersed in the short period mode described above, whereas the remaining half are organized as long blocks of >2 to 3 kbp in length (8). These latter sequences also contain rDNA (9).The average patterns of DNA sequence organization in the fimgus Achlya have been studied by Hudspeth et al. (13)
Electron microscopic analysis of reassociated deoxyribonucleic acid (DNA) from the aquatic fungus Achlya bisexualis revealed details of the sequence arrangement of the inverted repeats and both the highly and moderately repetitive sequence clusters. We used the gene 32 protein-ethidium bromide technique for visualizing the DNA molecules, a procedure which provides excellent contrast between single- and double-stranded DNA regions. Long (greater than 6-kilobase) DNA fragments were isolated after reannealing to two different repetitive C0t values, and the renatured structures were then visualized in an electron microscope. Our results showed that the inverted repeat sequences were short (0.5 kilobase, number-average) and separated by nonhomologous DNA of various lengths. These pairs of sequences were not clustered within the genome. Both highly repetitive and moderately repetitive DNA sequences were organized as tandem arrays of precisely paired, regularly repeating units. No permuted clusters of repeating sequences were observed, nor was there evidence of interspersion of repetitive with single-copy DNA sequences in the Achlya genome.
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