Studies on the Amount and Location of the tRNA and 5-S rRNA Genes in Tetrahymena pyriformis GL

Abstract

The amount and location of tRNA and 5-S rRNA genes in the macronucleus of Tetrahymena pyrformis GL was investigated by DNA-RNA hybridization. Hybridization of 32P-labelled tRNA in excess of unlabelled rRNA (25-S + 1 7 4 + 5-S) showed that at saturation 0.021 % of the macronuclear DNA was complementary to tRNA. Hybridization of 32P-labelled 5-S rRNA in excess of unlabelled 25-S + 17-S rRNA and tRNA showed a saturation value of 0.017%.In contrast to the 25-S + 17-S rRNA genes, which are found in DNA of high buoyant density, tRNA and 5-S rRNA genes were distributed evenly throughout the main peak observed when bulk macronuclear DNA was fractionated by density centrifugation in CsCl gradients. Sucrose gradient analyses of total macronuclear DNA showed that tRNA and 5-S rRNA genes were found in DNA of all size classes but a significant enrichment in the slowly sedimenting DNA fraction was observed.Saturation hybridization of 5-S rRNA to purified rDNA showed that rDNA did not contain any 5-S rRNA genes.In the past few years, our knowledge about the organizational and informational complexity of the eukaryotic genome has increased rapidly and for some specific genes, e.g. the genes coding for tRNA and 5-S rRNA, their location and organization within the eukaryotic chromosomal DNA has been rather well established recently.The tRNA genes are repetitive in most eukaryotes. Clarkson et al. [l] showed that each tRNA repeating unit in Xenopus laevis contains in addition to the transcribed DNA, approximately 90 % non-transcribed spacer DNA of unknown function. These repeating units are tandemly arranged in clusters and the genes for different isoaccepting tRNA molecules are arranged in separate clusters containing different spacer sequences [l].Brown et al. [2] showed that the repetitive 5-S rRNA genes of Xenopus laevis are highly clustered, and each repeating unit contains about 85% untranscribed spacer sequences. By hybridization in situ Pardue et al. [3] have shown that the 5-S rRNA genes are clustered in separate linkage groups at the telomers Although 5-S rRNA is a component of ribosomes, the genes for this RNA are normally unlinked to those for 2 5 3 + 17-S rRNA [4,5]. In Xenopus laevis, for example, the 5-S rRNA genes are found in a light satellite band in isopycnic CsCl gradients, whereas the genes coding for 28-S + 18-S rRNA are found as a heavy satellite [5]. A physical linkage between the genes for 5-S rRNA and those for high-molecularweight rRNA genes has been found only in bacteria [6,7] and in the lower eukaryote Sacchuromyces cerevisiae [B, 91. To advance our knowledge about the genetic information and the structural organization of the macronuclear DNA in Tetrahymena pyrijormis we have investigated the genes corresponding to tRNA and 5-S rRNA. The information obtained may hopefully contribute to a better understanding of the structure and location of specific genes on eukaryotic chromosomes.

The Organisation of Genes for Transfer RNA and Ribosomal RNA in Amoebae and Plasmodia of Physarum polycephalum

“…Consequently each specific tRNA gene must be reiterated, on average, 24 times per haploid genome. A similar degree of reiteration is observed in Drosophila melanogaster [46], HeLa cells [43], Tetrahymena [44] and Saccharomyces cerevisiae [47], whereas in Xenopus it is 10 times greater [48]. Once again no tRNA genes could be detected in the extrachromosomal ribosomal DNA molecules, assuming that specific radioactivities of individual tRNA species do not vary greatly ( Table 2, Fig.4).…”

“…Consequently the 5-S rRNA genes are reiterated somewhat more than the 5.8-S, 19-S and 26-S rRNA genes, a property which has also been observed in several eucaryotic systems including HeLa cells [43], Xenopus [9] and Tetrahymena [44]. Unlike the 5.8-S rRNA genes, no 5-S genes could be detected in the extrachromosomal ribosomal DNA molecules of Physarum.…”

“…Instead it would appear that these genes are a component of the high-molecular-weight chromosomal DNA. A lack of linkage between the genes for 5-S rRNA and those for the large rRNA species has also been demonstrated in Tetrahymena [44] and several higher eucaryotic organisms [9-131. In contrast a physical linkage does exist in the lower eucaryotes Dictyostelium discoideum [49] [5] for plasmodia1 DNA.…”

Triphosphate residues at the 5' ends of rRNA precursor and 5S RNA from Dictyostelium discoideum.

“…In contrast, the 5'-end groups of several eukaryotic 5S RNAs have been shown to be triphosphate residues, almost always pppG- (26)(27)(28)36), although at least one algal 5S RNA is terminated with pppA- (37). In animals (9)(10)(11)(12), and also in the protozoan Tetrahymena (13), independent transcription of 5S RNA is insured by the complete lack of linkage between 5S DNA and rDNA. However, for the lower eukaryote yeast, as for Dictyostelium, the 5S RNA genes remain interspersed with the larger rRNA genes (38).…”

“…Similarly, the larger rRNAs of eukaryotes are synthesized together in a common transcription unit, with the 18S RNA sequences proximal to the initiation site sequences (4)(5)(6)(7)(8). However, in most eukaryotes, the 5S RNA is independently transcribed from DNA completely unlinked to the other rRNA genes (9)(10)(11)(12)(13).…”