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Gene linkage by RNA-DNA hybridization

1968; Elsevier BV; Volume: 34; Issue: 3 Linguagem: Inglês

10.1016/0022-2836(68)90188-5

1089-8638

Donald D. Brown, Carl S. Weber,

RNA Research and Splicing

A method for measuring sequence homology between high specific activity RNA's and DNA of Xenopus laevis is described. A CsCl centrifugation step fractionates the DNA according to its buoyant density. The DNA in each fraction is denatured, immobilized on a nitrocellulose filter, and then hybridized with [3H]RNA. The technique combines specificity and versatility not afforded by other hybridization methods. Specificity arises from the fact that the DNA's homologous to different classes of RNA band at unique buoyant densities. Essentially all DNA homologous to 28 s and 18 s ribosomal RNA (rDNA) bands as a heavy satellite. The DNA homologous to 5 s RNA bands to the light side of the bulk DNA, and DNA homologous to 4 s RNA is distributed throughout the band of the bulk DNA and to the heavy side. Since the DNA's homologous to each of these RNA classes are separated by CsCl fractionation, competition experiments have increased specificity and have been used to demonstrate that the DNA's do not share common nucleotide sequences. The high-density rDNA comprises about 0.057% of the genome as determined by hybridization with saturating amounts of rRNA; this amounts to about 450 of the 28 s and 18 s genes for each haploid complement of chromosomes. This rDNA is clustered on a single chromosome, since more than 99% of it is lost by a deletion (the nucleolar mutation) which segregates as a simple Mendelian factor. This deletion does not affect the DNA homologous to 4 s and 5 s RNA, supporting the observation that these DNA sequences are not intermingled with the rDNA. Since a linear relationship exists between the amount of DNA immobilized on filters and the extent of hybridization with the three groups of [3H]RNA, the technique can be used to measure the amount of homologous sequences in unknown DNA preparations even when the RNA is used in subsaturating amounts. The relative abundance of nucleotide sequences homologous to rRNA and 5 s RNA in X. laevis liver, erythrocyte, and embryonic DNA has been found to be similar.