Gene linkage by RNA-DNA hybridization: II. Arrangement of the redundant gene sequences for 28 s and 18 s ribosomal RNA

Abstract

The arrangement of the multiple sequences homologous with 28 s and 18 s ribosomal RNA has been studied in the genome of Xenopus laevis. Two tests were used to determine whether the nucleotide sequences homologous to 28 s and 18 s RNA are present on the same DNA molecule in DNA preparations of different molecular weights. First, in native DNA greater than 6 × 10 6 daltons, the sequences homologous to 28 s and 18 s RNA both band at 1.724 g/cm 3, whereas they separate in low-molecular weight DNA preparations banding at about 1.721 and 1.715 g/cm 3, respectively. The higher buoyant density of 28 s and 18 s DNA in higher-molecular weight preparations suggests the presence of non-homologous sequences intermingled with 28 s and 18 s DNA in the same molecules. Second, the DNA's were hybridized with 28 s or 18 s RNA, followed by centrifugation to equilibrium in CsCl. When the two genes were present on the same molecule of DNA, prehybridization with one gene product (28 s or 18 s RNA) altered the buoyant density of DNA homologous to the other gene product (18 s or 28 s RNA). The experiments reported here are sensitive enough to show that, on the average, sequences homologous to 28 s RNA do not extend for more than the length equivalent to two contiguous 28 s genes before a sequence homologous with 18 s RNA begins. Also, more than two complete repeating sequences of 18 s DNA do not occur before being interrupted by sequences homologous with 28 s RNA. Since DNA fragments large enough to code for a single 40 s rRNA precursor molecule contain sequences homologous to both 28 s and 18 s RNA, each precursor molecule must contain at least one 28 s and one 18 s RNA sequence. The linkage experiments described in this paper and the known mechanism of rRNA precursor metabolism in vivo support a model in which the redundant 28 s and 18 s genes alternate but are interspersed with DNA which is not homologous to either gene. There are 450 of these repeating units in each redundant cluster of the X. laevis genome. The 40 s rRNA is a transcript of one of these units. Similar analyses permit the conclusion that the 5 s ribosomal RNA is not transcribed as part of the same 40 s RNA precursor molecules. This physical method should be applicable to the measurement of linkage between any two genes the RNA products of which can be isolated.