Is the 5S RNA a Primitive Ribosomal RNA Sequence? (Ribosome/Evolutlon/Eukaryote/Gene Amplification) Ross N
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Proc. Nadl. Acad. Sci. USA Vol. 82, pp. 5608-5611, September 1985 Biochemistry Is the 5S RNA a primitive ribosomal RNA sequence? (ribosome/evolutlon/eukaryote/gene amplification) Ross N. NAZAR AND WILLIAM M. WONG Department of Molecular Biology and Genetics, University of Guelph, Guelph, Canada N1G 2W1 Communicated by J. Tuzo Wilson, April 22, 1985 ABSTRACT A tandemly arranged cluster of 5S RNA-like sequence. Further comparisons also suggest that similar sequences in the middle of ribosomal 26S to 28S rRNAs from sequences are found in all rRNAs, raising an intriguing divergent eukaryotic organisms raises the possibility that the question about the evolution of the larger ribosomal RNA larger ribosomal RNAs were built up, at least in part, by gene molecules. amplification events and suggests an intriguing evolutionary relationship between the 5S rRNA and the larger rRNA molecules. MATERIALS AND METHODS DNA Preparation and Cloning. Genomic DNA was extract- The advent of recombinant DNA technology and rapid gel ed and purified from Thermomyces lanuginosus (ATCC sequencing techniques has revolutionized our understanding 16455) as described (12); the DNA was isolated from mycelia of the eukaryotic genome. The organization and structure of essentially by using the method of Cryer et al. (13) and ribosomal genes has by no means been an exception; the purified on a cesium chloride/ethidium bromide gradient. genomic organization in many organisms has already been Saccharomyces cerevisiae (ATCC 26108) genomic DNA was determined (see ref. 1) and the primary nucleotide sequences also prepared by the method of Cryer et al. except that the of high molecular weight rRNA molecules (16S to 28S DNA was not repurified on a cesium chloride/ethidium rRNAs) appear almost monthly. More recently, comparative bromide gradient. A genomic library of EcoRI-digested T. studies have even given rise to working models for the lanuginosus DNA was prepared by using the X Charon 3A secondary structures ofthe larger rRNA molecules and have vector (12, 14). In addition, to isolate the intact repeating delineated both conserved and divergent regions or domains rDNA unit, a library in X Charon 4A was made by partially (e.g., see refs. 2-9). The results suggest a largely conserved digesting genomic DNA with EcoRI restriction endonuclease secondary structure core to which divergent domains have (digestion conditions, 0.5 unit of enzyme per ,g of genomic been added, especially in higher eukaryotes. DNA for 30 min at 37°C). Plaques were screened for frag- While this progress in our understanding of rRNA is very ments complementary to the cytoplasmic 5S rRNAs by impressive, little is yet known about the origins of the large plaque hybridization (15) and the complementary fragments RNA molecules. Indeed, the sequence comparisons that were subsequently subcloned into pBR322 (16). have been reported indicate relatively little sequence repe- DNA Sequence Analysis of Complementary Fragments. tition or duplication (10). Nevertheless, it is probably rea- Fragments that hybridized to the cytoplasmic SS rRNAs were sonable to speculate that the large RNAs must have gradually identified by electroblot hybridization techniques (17). DNAs built up in the course of evolution. Not surprisingly, se- from T. lanuginosus or S. cerevisiae were digested with quence comparisons between the smaller prokaryotic and EcoRI or HindIII restriction endonuclease, fractionated on larger eukaryotic species (e.g., see ref. 11) are consistent with 0.8% agarose gels (18), and electrophoretically transferred to this idea. nitrocellulose membranes or activated APT paper (15). The Recently, in attempting to isolate 5S rRNA genes from a 5S rRNA probe was prepared from whole cell RNA by eukaryotic thermophile (Thermomyces lanuginosus), we repeated purification using gel electrophoresis and end- found a tandemly arranged cluster of sequences that were labeled with [_y-32P]ATP by using polynucleotide kinase (19). '='50%o homologous in nucleotide sequence with the cytoplas- To prepare DNA for sequence analysis, hybrid plasmids mic 5S rRNA and selectable by hybridization techniques (12). containing complementary fragments were digested with Unlike typical pseudogenes, these sequences were not trun- EcoRI and the inserted DNA was purified on a 0.8% agarose cated but rather bore a limited sequence homology with the slab. The complementary fragments, identified by hybridiza- entire length of the 5S rRNA and were oriented end to end tion analysis, were further digested with restriction enzymes without significant intervening sequences. We suggested that (Taq I or Hinfl), 5' end-labeled with [y-32P]ATP and se- these were gene relic sequences that had been duplicated by quenced by the chemical degradation procedure of Maxam a rolling circle-like mechanism and had evolutionarily drifted and Gilbert (20). The fragments were ordered by using to their present state, perhaps playing the role of a DNA sequence overlaps in the two restriction enzyme digests and spacer. by comparisons with the published sequence for S. cerevisiae To more clearly define the role of these sequences, in the 26S rRNA (21). present study we determined the DNA sequence surrounding the cluster of 5S RNA-like sequences and examined their RESULTS AND DISCUSSION relationship to the other rRNAs. As might have been expect- ed, these 5S RNA-like sequences were present within the As shown in Fig. 1, hybridization experiments with T. highly repeated rDNA unit but, unexpectedly, they were not lanuginosus DNA indicate that an EcoRI digest of the situated in an intervening or spacer sequence region. Instead, genomic DNA contains three fragments, -6000, '-3000, and they have been localized in the middle of the 26S rRNA -900 base pairs, to which the cytoplasmic 5S rRNA readily hybridizes. These results are similar to those observed with as illustrated The publication costs of this article were defrayed in part by page charge DNA from other fungi except, with S. cerevi- payment. This article must therefore be hereby marked "advertisement" siae DNA (also shown in Fig. 1), hybridization to the in accordance with 18 U.S.C. §1734 solely to indicate this fact. 2.5-kilobase-pair (kbp) fragment predominates (23). In that 5608 Downloaded by guest on September 26, 2021 Biochemistry: Nazar and Wong Proc. Natl. Acad. Sci. USA 82 (1985) 5609 a b c d probe was twice repurified. Furthermore, sequence analyses of the more intense middle fragment with T. lanuginosus DNA did not reveal the 5S rRNA gene; instead, we found the unusual four-copy cluster of 5S RNA-like sequences (12). In our subsequent analyses, we have now shown that all three fragments are part of the rDNA in T. lanuginosus. As >20 indicated in Fig. 1, hybridization experiments have shown Wl f 6.0 that only one much larger (-20-kbp) fragment of a HindIII OM3.0 digest readily hybridizes with the cytoplasmic 5S rRNA, and ml 2.5 all three of the EcoRI-generated fragments could be isolated with the X Charon 4A vector from partially digested genomic DNA (X Ch 4A-TL rDNA). As indicated in Fig. 2, sequence analyses show that the two smaller fragments contained the 0.9 3' half of the 5.8S rRNA sequence and the entire 26S rRNA, while the largest fragment contained the 5' half of the 5.8S rRNA preceded by the entire 18S rRNA. More important, as indicated in Fig. 3, the cluster of 5S RNA-like sequences actually constitutes the middle portion of the 26S rRNA. Furthermore, the 26S rRNA sequence is a complementary copy to the mature 5S rRNA, the same orientation that is observed between the actual 5S rRNA gene and rDNA in yeast (24). In S. cerevisiae, these sequences are both present FIG. 1. Hybridization of cytoplasmic 5S rRNA to restriction in the same tandemly repeating unit. Unlike the yeast, a endonuclease EcoRI or HindIII digests of genomic or cloned DNAs from T. lanuginosus and S. cerevisiae. The DNA (0.2-2 ,ug) was strong signal for the actual 5S rRNA gene was not observed. digested with 1-5 units ofenzyme, fractionated on a 0.8% agarose gel Since the 5S rRNA genes of T. lanuginosus and some of the (18), and electrophoretically transferred to a nitrocellulose mem- other higher fungi (e.g., see refs. 25 and 26) have been shown brane or activated APT paper (15). The filters were hybridized (22) to be heterogeneous and dispersed, the actual copy number with 32P-labeled 5S rRNA at several different temperatures ofeach gene type is likely to be relatively low when compared (50°C-75°C) overnight in 50% formamide/0.75 M sodium to the four-copy cluster of 5S RNA-like sequences in the chloride/0.075 M sodium citrate, washed extensively with the same already highly repeated rDNA. Furthermore, the signals for solution and temperature, and washed twice with 0.30 M sodium 5S RNA-like sequences are particularly strong in T. chloride/0.030 M sodium citrate at room temperature prior to because of the G+C content in this autoradiography. Results for experiments at 50°C are shown. The lanuginosus higher molecular sizes (shown in kbp) of the fragments were determined thermophile. from marker fragments: lane a, EcoRI digest of yeast genomic DNA; Since the extended sequence homology and tandem ar- lane b, HindIlI digest of T. lanuginosus genomic DNA; lane c, EcoRI rangement make a coincidental relationship highly unlikely digest of T. lanuginosus genomic DNA; lane d, EcoRI digest of X Ch (12), we examined other known 23S to 28S rRNAs for similar 4A-TL rDNA. 5S RNA-like clusters (Fig. 3). Because the nucleotide se- quence of the rRNA from the thermophilic fungus is highly case, however, Rutter and co-workers subsequently found homologous (-90%) to that ofyeast, the entire cluster offour that this 2.5-kbp fragment contained the 5S rRNA gene and 5S RNA-like sequences could be easily identified in the yeast suggested that the other less intense signals from other sequence.