* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Small-Subunit Ribosomal RNA Sequence from
Extrachromosomal DNA wikipedia , lookup
Nutriepigenomics wikipedia , lookup
Genome (book) wikipedia , lookup
Polyadenylation wikipedia , lookup
Bisulfite sequencing wikipedia , lookup
Transposable element wikipedia , lookup
Gene desert wikipedia , lookup
Genomic library wikipedia , lookup
RNA interference wikipedia , lookup
Pathogenomics wikipedia , lookup
Gene expression programming wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Gene expression profiling wikipedia , lookup
Point mutation wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
History of genetic engineering wikipedia , lookup
Non-coding DNA wikipedia , lookup
Genome evolution wikipedia , lookup
Human genome wikipedia , lookup
Short interspersed nuclear elements (SINEs) wikipedia , lookup
Epigenetics of human development wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
RNA silencing wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Nucleic acid tertiary structure wikipedia , lookup
Computational phylogenetics wikipedia , lookup
Designer baby wikipedia , lookup
History of RNA biology wikipedia , lookup
Genome editing wikipedia , lookup
Microevolution wikipedia , lookup
Primary transcript wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Non-coding RNA wikipedia , lookup
Metagenomics wikipedia , lookup
Helitron (biology) wikipedia , lookup
Epitranscriptome wikipedia , lookup
Small-Subunit Ribosomal RNA Sequence from Naegleria gruberi Supports the Polyphyletic Origin of Amoebas’ C. Graham Clark and George A. M. Cross Laboratory of Molecular Parasitology, The Rockefeller University We have sequenced the small-subunit xibosomal RNA gene of the amoebo-flagellate protozoan Naegleria gruberi. Comparison of this sequence with the rRNA sequences of other eukaryotes resulted in a phylogenetic tree that supports the suggested polyphyletic origin of amoebas and suggests a flagellate ancestry for Naegleria. Introduction Amoebas are a group of morphologically and biochemically very diverse protozoa and are most likely a polyphyletic aggregation of species (Bovee and Jahn 1973). The small number of classification criteria available-manner of locomotion and pseudopod appearance, presence or absence of flagellated stages, nuclear structure, and division patterns (Page 1976)-has led to the placing of all pseudopodal amoeboid organisms into the class Lobosea for convenience ( Lee et al. 1985 ) . Uncertainty about the value of these criteria as phylogenetic markers leaves the evolutionary relationships among groups of amoebas unclear. The use of small-subunit ribosomal RNA (rRNA) sequence comparison has greatly increased our understanding of evolutionary relationships among prokaryotes and, more recently, among eukaryotes. However, only one amoeba, Acanthamoeba, has so far been examined by this method (Gunderson and Sogin 1986). Within the class Lobosea, the order Schizopyrenida is distinguished by, among other characteristics, the presence of a number of amoebo-flagellate genera (Page 1976), but it is unclear whether it is closely related to any other order of amoebas within its class. We recently described the unique rRNA gene structure of the schizopyrenid amoeba Naegleria gruberi (Clark and Cross 1987). In this organism and in related genera (Clark and Cross 1988)) the rRNA genes ( rDNA) are carried only on multicopy circular plasmids-no chromosomal copy has been detected. Given the uniqueness of this gene structure and uncertainty about the organism’s phylogenetic relationship to other groups of amoebas, we decided to sequence its small-subunit rDNA. Our results show that the lobose amoebas are indeed polyphyletic in origin and that the Schizopyrenida are a relatively ancient eukaryotic group, with their closest relatives being flagellates. Material and Met hods The recombinant plasmid pNgex27 contains an entire Naegleria gruberi rDNA plasmid (Clark and Cross 1987). The small-subunit rRNA region was subcloned in 1. Key words: Naegleria gruberi, amoeba, 18s rRNA, eukaryote phylogeny. Address for correspondence and reprints: C. Graham Clark, Laboratory of Molecular Parasitology, The Rockefeller University, 1230 York Avenue, New York, New York 1002 l-6399; telephone (CGC): (212) 570-7574. Mol. Biol. Evol. 5(5):512-5 18. 1988. 0 1988 by The University of Chicago. All rights reserved. 0737-4038/88/0505-0004$02.00 512 Naegleria Small Subunit rRNA Sequence 5 13 Table 1 Structural Similarity and Distance Data from Eukaryotic Small-Subunit rRNA Sequence Comparisons Cr Cr.. . . . Zm . . . . AC..... Xl..... SC . . . . Tt.. . . . Pb..... Dd . . . . Tb .,.. Eg.. . . . Ng . . . EC. . . . . 0.076 0.094 0.155 0.118 0.185 0.185 0.211 0.327 0.319 0.282 0.720 Zm AC Xl SC Tt Pd Dd Tb Eg Ng Ec 0.928 0.912 0.928 0.860 0.864 0.853 0.891 0.904 0.899 0.853 0.836 0.867 0.860 0.812 0.856 0.836 0.839 0.837 0.783 0.832 0.807 0.816 0.820 0.844 0.797 0.820 0.796 0.793 0.735 0.737 0.734 0.713 0.726 0.702 0.700 0.728 0.740 0.740 0.731 0.7 13 0.734 0.710 0.710 0.705 0.728 0.765 0.754 0.763 0.752 0.762 0.732 0.755 0.737 0.691 0.697 0.537 0.525 0.535 0.5 17 0.528 0.5 11 0.521 0.522 0.505 0.5 19 0.520 0.076 0.150 0.103 0.146 0.181 0.181 0.324 0.319 0.298 0.752 0.164 0.108 0.155 0.184 0.175 0.329 0.333 0.285 0.726 0.164 0.216 0.256 0.237 0.362 0,362 0.301 0.775 0.160 0.190 0.206 0.341 0.328 0.286 0.744 0.223 0.238 0.380 0.367 0.325 0.791 0.242 0.383 0.367 0.297 0.763 0.338 0.375 0.324 0.761 0.338 0.398 0.809 0.388 0.769 0.766 NOTE.-The values above the diagonal are the structural similarities calculated according to the method of Elwood et al. (1985) for the regions of the sequence considered to be unambiguously alignable. Below the diagonal are the structural distances (substitutions/site) calculated according to the method of Jukes and Cantor (1969). For the Nuegleria gruberi (Ng) sequence, the base numbers included in the analysis were l-62,98- 130,373-547,597-7 17,1023- 1208,1245- 15 17, I60017 15, 1744- 1920, and 1937-20 19 for a total of 1,227. These bases were aligned with the corresponding regions from the small-subunit rRNAs of Chlurnydomonas reinhurdtii (Cr; Gunderson et al. 1987), Zeu muys (Zm; Messing et al. 1984), Acunthumoebu custellunii (AC; Gunderson and Sogin 1986), Xenopus luevis (X1; Salim and Maden 1981), Succhuromyces cerevisiue (SC; Mankin et al. 1986), Tetruhymenu thermophila (Tt; Spangler and Blackbum 1985), Plasmodium berghei (Pb; Gunderson et al. 1986), Dictyostelium discoideum (Dd; McCarroll et al. 1983), Trypanosomu brucei and Euglenu gracilis (Tb and Eg; Sogin et al. 1986), and Escherichia coli (EC; Brosius et al. 1978). both orientations into pGEM-4 (Promega Biotec). Following digestion with the restriction enzymes SphI and SalI, both of which cut only in the polylinker region of each clone, nested deletions were produced by the Exo III/S1 technique in the two small-subunit plasmids (pNgSS6 and pNgSS55 ) by using an Erase-a-Base@ kit (Promega Biotec) and the provided protocol. Deletions of - 200 bases in size were generated by sampling at 30-s time intervals. A small number of colonies from each time point were screened, and a complete nested set of deletion clones was obtained from both starting plasmids. DNA for sequencing was prepared from small-scale ( 5-40 ml) plasmid preparations by using the alkaline lysis method (Maniatis et al. 1982) but with an additional phenol-CHC13 extraction and ethanol precipitation following RNase treatment. After alkaline denaturation and hybridization of the SP6 promoter primer (Promega Biotec), deletion clones were sequenced by the dideoxynucleotide chain termination method with modified T7 DNA polymerase by using the Sequenase- kit and protocol from US Biochemical Corporation and [ 35S]a-dATP. dITP was included to eliminate compressions due to secondary structure (Barnes 1987 ) . Reactions were run on 6% acrylamide-urea sequencing gels, fixed in 10% acetic acid, dried, and exposed to Kodak XAR-5 film at room temperature. Complete overlapping sequence was obtained for both strands of the DNA. The Naegleria 18s rRNA sequence was deduced from the DNA sequence. The 5 ’and 3’ends of the 18s rRNA were inferred by sequence similarity, and the Naegleria 18s rRNA was aligned by eye to a number of other eukaryotic small-subunit rRNAs by using secondary structure as a guide. Similarity values were calculated for each pair of organisms by using the method described by Elwood et al. ( 1985 ) (see table 5 14 Clark and Cross TACCTGGTTG ATCCTGCCAG TACTATATGC TTGTCTCAAA GCCTAAGCCA AGATCAATCA TATGCGGTTT CGGCCGTGTA TUTGATAGT CTGTGGAAGG CTCATTATAA 121 CAGTTATACT CCTAGCCACT GGAAAGTTTA CAAGGATACC ACCGTTAACT GCAGCGATAT 1 61 TGCAA.ATGTA 181 ACTTGTTCCC TTCGGGGTGG TAATAGTATT TGTGCTGAAG CCTAGCTATT GTAACCTAGT 241 TTTTCGGGTG TGGCAACATA TTCGGGGGAT TAGGAATCGA CCGCTAGCAG GTGCCTTCGG 301 GCGCGGGAAA GTGAATTAAC AAGGTTTTCA TAAGGCCTTT CAGGTTTGCT TTTTCTAGTG 361 GCCAGGCAGA GGAGTTTCTT ACCTATCAGC TCGTTGTTTG TTTAAAGGAC AAACCAGGCT 421 TTGACGGGTA CGGGGAATCA GTGTTCGATT CCGGAGAGGG AGCCTGAGAA ATCGCTACCA 481 CATCTAAGGA CGGCAGCAGG CGCGCAAATT ACCCAATCTC AATACGAGGA GGTAGTGACA 5il AGCTATAGTG ACTCCACACC ATTCGGTGGG GAGGTATTGT CTTCTGACGA TTTTCCATGA 601 TTTGGGTGTA GATAACCCTT AGAGTAGCCA TTGGAGGAAA AGTCTGGTGC CAGCACCCGC 661 GGTAATTCCA GCTCCAAGAG CGTATATTAA TACTGCTGTA GTTAUACGC CCGTAGTATA 721 CCTAAGAGTG GGTGTGTAGT AATTAGTTTT ACCAGAGGAC GGTTGGCGAG AGTTTATCAC 781 TCTTGTTTGC CTACTTTTGG TAGACTTTAG TCGGGCTGGA TCTTTGGTCC TCGTCTGACA 841 GTTGCTACGT ACTTACTTAC CACGGTTCAT CCGTGAGGCC CTTGGCTTGC AACTGTAAAT 901 UTCGTTGT GCTTAAAGCG GGCTATGATA CTCTGCCAGA GCGATTTAGC ATGGGACTGC 981 AGAGTAGCTG TATTTGAGCG AAGGTTGCAC CTCGGTGTGG CTGGAGCTTG GTACAGCGCT 1021 TGTAATGGAG CTCAGGGTGA GGCCCCGGGT ACCATGAGGC TAGAGGTGAA ATTCTGAGAC 1081 CCTCATGTGA CCAACTAAGG CGAAAGCTGT CGTGGGCCAC CACAAGCTCG TCTATCAGGG 1141 ACAAAAGTTG GGGGATCGAA GACGATTAGA TACCGTCGTA GTCCCAACTA TAAACGATAC 1201 CAACCGAGTA TTTGGGAAGA CACTATCCCA GCCATCTTCT CAGAACTCAA GGGMACCTT 1261 AAGTCTTTGG GTTCTGGGGG GAGTATAGTC GCAAGACCGA AACTTAAAGG AATTGACGGA 1321 AAGGCACCAC CAGGAGTGGA GTCTGCGGCT TAATTCGACT CAACACGGGG AAACTCACCA 1381 GGTCAGGACA CAAGTTTGAT TGACAGGTTA ATAGCCCTTT CTTGATTGTG TGGTGGGTAG 1441 TGCATGGCCG TTTCCAGTTC GTGGAGTGAT CTGTCTTGTT AATTCAGATA ACGAACGAGA 1501 CCTAAGCCTT TAACTAGCCG TAGGCCTTTT CCTTCGGGGA AGGGTTAGTT TGTCGGAACA 1561 GGTTTCGGCC TGTTCCAAM CCTACGAGAC TTTTGTCAGC TTCTTAAAGG GACTTCATTC AGATGTCCTG 1021 GTAAACTAGG ATGAGGAAGA TTTAGGCCAT AACAGGTCTG TGATGCTCTT 1681 GGCTGCACGC GTACTACAAT AACGGTACCA GCGAGCGCTA TGGTTTTATA ACCCCTTATC 1741 CTAATAGGAT TGGGAAAACT TTTCAAACAC CGTTATGACA GGGATCGAGG ATTGGAACAT 1801 CCTCGTGAAC GAGGAATTCC TAGTAAGCGT GGTTCATCAT ACCACATTGA TTACGTCCCT 1861 GCCTTTTGTA CACACCGCCC GTCGCTCCTA CCGATGGGAC GAAGAGATGA ACCTGGCGGA 1921 CCGAlLCCGCA AGGTAAGGGA IuiCCAGTTAA ATCTCTTCGT CTGTAGGAAG GAAAAGTCGT 1981 AACAAGGTCT TCGTAGGTGA ACCTGCGTAG GGATCATTT of the small-subunit rRNA region of Nuegleriu gruberi rDNA. Unambiguous sequence FIG. 1.-Sequence was obtained at each of the 2,019 positions. (GenBank accession no.: M18732.) 1). A total of 1,227 positions were considered to be unambiguously alignable. The tree was constructed following the guidelines of Fitch and Margoliash (1967). Results and Discussion Sequencing of the Naegleria gruberi small-subunit rRNA gene gave an unambiguous sequence of 2,0 19 bases (fig. 1). The putative 5’ end is 173 bases upstream of the previously mapped PstI site, and the putative 3’ end is 63 bases upstream of the CZaI site in the 18S-28s spacer region (Clark and Cross 1987). All secondarystructure elements conserved among other eukaryotic small-subunit rRNAs were also found in the NaegZeria sequence. The size of this amoeba’s 18s rRNA is greater by -200 bases than those of many other eukaryotes, although it is smaller by 230-290 bases than those of Acanthamoeba, Euglena, and Trypanosoma. As might be predicted, the size differences reside in regions previously identified as variable in length (see table 2; discussed by Gunderson and Sogin [ 19861, Schnare et al. [ 19861, and Clark [ 19871). The tree constructed from the structural distance values (table 1) is shown in figure 2. This tree is very similar to others derived by this method, for example that of Gunderson et al. ( 1987). The node of the Naegleria branch is between those which gave rise to the euglenid and trypanosomatid flagellate groups on one side and Dic- Naegleria Small Subunit rRNA Sequence 5 15 Table 2 Position and Comparative Length of the Variable Regions of Nuegferiu 18s rRNA REGION LENGTH REGION, Ng LOCATION I, 143-361 ......... II, 606-630 ........ III, 713-1022 ....... IV, 1207-1247 ...... V, 1522-1610 ...... VI,1707-1769 ...... VII, 1886-1972 ..... Total length of 18s Ng Eg AC Tt EC 219 25 310 41 89 63 87 2,019 268 29 521 46 121 68 112 2,305 245 29 375 131 154 110 129 2,303 163 29 215 35 47 51 90 1,753 78 176 67 29 43 42 83 1,542 NOTE.-This table shows the location in N. gruberi of the seven major variable regions of 18s rRNA identified by Gunderson and Sogin ( 1986) and the comparative length of these regions in a selection of other species: Eg and AC are the two longest 18s rRNAs known; Tt is approximately average length for a eukaryote; and EC is prokaryotic (the secondary structure of Noller et al. [ 19851 was used). Abbreviations are as in table 1. tyostelium on the other. The NaegZeria sequence deviates at a number of previously universally conserved positions. The early branch point may explain this result, since a similar observation was made when the Euglena and Trypanosoma sequences were determined (Sogin et al. 1986). It must also be remembered that only a single, cloned copy of the approximately 4,000 genes per cell was sequenced and that variation between genes may exist, although we have no evidence for this. The unique structure of the Naegleria rDNA can be reassessed in light of the branch-point data. Both EugZena and the trypanosomes have tandemly repeated 18S5X3-28s rDNA units, in contrast to the extrachromosomal circular rDNA of Naegleria and the extrachromosomal linear rDNA of Dictyostelium (Cockburn et al. 1978). However, the 5s rRNA genes of Euglena are also located within the rDNA tandem array (Curtis and Rawson 198 1), while those of Trypanosoma are not (Cordingley 1985 ) . This may be an indication that rDNA tandem array formation, a process likely to have occurred numerous times in the eukaryotic lineage (Clark 1987)) occurred after divergence of the ancestors of these two flagellate organisms. Extrachromosomal rRNA genes may therefore be the ancestral eukaryotic condition rather than a lineagespecific peculiarity. The positioning of the NaegZeria branch node so close to that of Euglena and Trypanosoma suggests a flagellate ancestry for the Schizopyrenida. A close relationship of amoeba and flagellate is not without precedent: the amoeba Dientamoeba fragiZis is generally considered to be an aberrant member of the otherwise flagellate order Trichomonadida (Lee et al. 1985). The relative positioning of Naegleria and Acanthamoeba on the rRNA tree is supported by their respective forms of mitosis. Naegleria shares with EugZena and Trypanosoma a promitotic form of nuclear division (Schuster 1979)-persistence of the nucleolus and nuclear membrane through mitosis and the absence of centrioles. The metamitotic nuclear division of Acanthamoeba most closely resembles that of plants and animals-nuclear membrane and nucleolar disintegration and the presence of centrioles during mitosis. It is evident from the phylogenetic tree that the amoebas Acanthamoeba and Naegleria have separate origins and probably nonamoeboid ancestors. This confirms 5 16 Clark and Cross Chlamydomonas teinhardtii / brucei FIG. 2.-Eukaryotic phylogeny inferred from mutational distances between small-subunit rRNA sequences. With the data in table 1, the tree was constructed following the method of Fitch and Margoliash (1967). The % SD (Fitch and Margoliash 1967) for this tree is 4.24%, the lowest value obtained of the approximately 30 trees examined. The best-fit values for branch lengths and evolutionary distance between nodes are given alongside the relevant tree segment. the suggested polyphyletic origins of the class Lobosea (Bovee and Jahn 1973), while indicating that rRNA sequence comparison has potential as a method for producing a phylogenetically based classification of this diverse group. Acknowledgments C.G.C. is supported by the David C. Scott Foundation. LITERATURE CITED BARNES,W. M. 1987. Sequencing DNA with dideoxynucleotides as chain terminators: hints and strategies for big projects. Methods Enzymol. 152:538-556. BOVEE,E. C., and T. L. JAHN. 1973. Taxonomy and phylogeny. Pp. 38-82 in K. W. JEON, ed. The biology of amoeba. Academic Press, New York. BROSIUS,J., M. L. PALMER, P. J. KENNEDY, and H. F. NOLLER. 1978. Complete nucleotide Naegleria Small Subunit rRNA Sequence 5 17 sequence of a 16s ribosomal RNA gene from Escherichia coli. Proc. Natl. Acad. Sci. USA 75:480 l-4805. CLARK, C. G. 1987. On the evolution of ribosomal RNA. J. Mol. Evol. 25:343-350. CLARK,C. G., and G. A. M. CROSS. 1987. rRNA genes of Naegleria gruberi are carried exclusively on a 14 kilobase-pair plasmid. Mol. Cell. Biol. 7:3027-303 1. , 1988. Circular ribosomal RNA genes are a general feature of schizopyrenid amoebae. J. Protozool. 35:326-329. COCKBURN,A. F., W. C. TAYLOR, and R. A. FIRTEL. 1978. Dictyostelium rDNA consists of non-chromosomal palindromic dimers containing 5s and 36s coding regions. Chromosoma 70: 19-29. CORDINGLEY,J. S. 1985. Nucleotide sequence of the 5s ribosomal RNA gene repeat of Trypanosoma brucei. Mol. Biol. Parasitol. 17:321-330. CURTIS, S. E., and J. R. Y. RAWSON. 198 1. Characterization of the nuclear ribosomal DNA of Euglena gracilis. Gene 15~237-247. ELWOOD,H. J., G. J. OLSEN, and M. L. SOGIN. 1985. The small-subunit ribosomal RNA gene sequences from the hypotrichous ciliates Oxytricha nova and Stylonychia pustulata. Mol. Biol. Evol. 2:399-4 10. FITCH, W. M., and E. MARGOLIASH. 1967. Construction of phylogenetic trees. Science 155: 279-284. GUNDERSON,J. H., H. ELWOOD,A. INGOLD,K. KINDLE, and M. L. SOGIN. 1987. Phylogenetic relationships between chlorophytes, chrysophytes, and oomycetes. Proc. Natl. Acad. Sci. USA 84:5823-5827. GUNDERSON,J. H., T. F. MCCUTCHAN, and M. L. SOGIN. 1986. Sequence of the small subunit ribosomal RNA gene expressed in the bloodstream stages of Plasmodium berghei: evolutionary implications. J. Protozool. 33:525-529. GUNDERSON,J. H., and M. L. SOGIN. 1986. Length variation in eukaryotic rRNAs: small subunit rRNAs from the protists Acanthamoeba castellanii and Euglena gracilis. Gene 44: 63-70. JUKES, T. H., and C. R. CANTOR. 1969. Evolution of protein molecules. Pp. 2 l-l 32 in H. N. MUNRO, ed. Mammalian protein metabolism. Academic Press, New York. LEE, J. J., S. H. HUTNER, and E. C. BOVEE, eds. 1985. An illustrated guide to the protozoa. Society of Protozoologists, Lawrence, Kan. MCCARROLL,R., G. J. OLSEN,Y. D. STAHL, C. R. WOESE,and M. L. SOGIN. 1983. Nucleotide sequence of the Dictyostelium discoideum small-subunit ribosomal ribonucleic acid inferred from the gene sequence: evolutionary implications. Biochemistry 22:5858-5868. MANIATIS,T., E. F. F~ITSCH, and J. SAMBROOK.1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. MANKIN, A. S., K. G. SKRYABIN,and P. M. RUBSTOV. 1986. Identification of ten additional nucleotides in the primary structure of yeast 18s rRNA. Gene 44: 143-145. MESSING, J., J. CARLSON, G. HAGEN, I. RUBENSTEIN,and A. OLESON. 1984. Cloning and sequencing of the ribosomal RNA genes in maize: the 17s region. DNA 3:3 l-40. NOLLER, H. F., M. ASIRE, A. BARTA, S. DOUTHWAITE, T. GOLDSTEIN, R. R. GUTELL, D. MOAZED, J. NORMANLY,J. B. PRINCE, S. STERN, K. TRIMAN, S. TURNER, B. VAN STOLK, V. WHEATON, B. WEISER, and C. R. WOESE. 1985. Studies on the structure and function of ribosomal RNA. Pp. 143-163 in B. HARDESTYand G. KRAMER, eds. Structure, function and genetics of ribosomes. Springer, New York. PAGE, F. C. 1976. A revised classification of the Gymnamoebia (Protozoa: Sarcodina). Zool. J. Linn. Sot. 58:61-77. SALIM,M., and B. E. H. MADEN. 198 1. Nucleotide sequence of Xenopus laevis 18s ribosomal RNA inferred from the gene sequence. Nature 291:205-208. SCHNARE,M. N., J. C. COLLINGS,and M. W. GRAY. 1986. Structure and evolution of the small subunit ribosomal RNA gene of Crithidia fasciculata. Curr. Genet. 10:405-410. SCHUSTER,F. L. 1979. Small amebas and ameboflagellates. Pp. 2 15-285 in M. LEVANDOWSKY 5 18 Clark and Cross and S. HUTNER,eds. Biochemistry and physiology of protozoa. 2d ed. Vol. 1. Academic Press, New York. SOGIN, M. L., H. J. ELWOOD,and J. H. GUNDERSON. 1986. Evolutionary diversity of eukaryotic small-subunit rRNA genes. Proc. Natl. Acad. Sci. USA 83: 1383-1387. SPANGLER,E. A., and E. H. BLACKBURN.1985. The nucleotide sequence of the 17s ribosomal RNA gene of Tetrahymena thermophila and the identification of point mutations resulting in resistance to the antibiotics paromomycin and hygromycin. J. Biol. Chem. 260:63346340. WALTER M. FITCH, reviewing editor Received December 4, 1987; revision received March 2 1, 1988