Vol. 259. No. 8, Issue of April 2.5, pp. 5167-5172, 1984 Printed in U.S.A.

THE .JOURNAI. of BIOLOGICAL CHEMISTRY c 1984 hy The American Society of Biuloglcal Chemists, Inc.

Paramecium Mitochondrial Genes I. SMALL SUBUNIT rRNA GENE SEQUENCE AND MICROEVOLUTION*

(Received for publication, September 6, 1983)

Jeffrey J. Seilhamerz, Gary J. Olseng, and Donald J. CummingsV From the Uniuersit.y of Colorado Health Sciences Center, Departments of Microbiology and Immunology, 3.175, Denver, Colorado 86262

The sequences of the small subunit mitochondrial rRNA genes from two divergent species of Parame- cium (primaurelia and tetraurelia) were determined. The gene lies near the center of the linear mitochon- drial genome, on the same strand as are all other cur- rently identified genes. The sequences generally re- semble their counterparts found in cytoplasmic, pro- caryotic, and other mitochondrial sources. The rDNA gene boundaries were located by nuclease SI protec- tion. Small subunit rDNA spans about 1680 nucleo- tides, including an extraneous 83-base pair sequence very near the 3‘ end which is unique to Paramecium mitochondria. This “insert” occurs at the apex of the highly variable in length penultimate helix, according to proposed models for small subunit rRNA secondary structure. A discontinuity occurs in isolated rRNA near the start of the insert, resulting in a stable 13 S RNA species and a small segment containing the remaining 3‘ portion of the gene.

The overall rRNA gene sequence was 94% conserved between the two species, and the nucleotide differences consisted of 53% transitions, 37% transversions, and 9% insertions plus deletions. These substitutions were somewhat clustered, and the two most divergent re- gions coincided with the gene boundaries. The se- quence was aligned with Escherichia coli 16 S rRNA for direct comparison of sequence and structure.

In recent years, rRNA sequences from a wide variety of organisms have been determined (1-13). The increasing avail- ability of such sequences has allowed the construction of tent.ative models for both evolutionary pathways as well as ribosome structure (14-20). In particular, the structural evo- lution of small subunit rRNAs has been of recent interest and controversy concerning models for organelle evolution (19, 20). Within all proposed phylogenetic trees, a pivotal branch is that which gives rise to sequences found within organelles, and particularly mitochondria. Within the mit,ochondrial branch, however, published data exists for only two groups: mammals and fungi. In view of the large evolutionary gap

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adoertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

i Supported by National Institutes of Health Grant AG-05221. Present address, California Biotechnology, lnc., 2450 Bayshore Front- age Road, Mountain View, CA 94043.

S Supported by National Institutes of Heath Grant GM-23464 awarded to Mitchell Sogin. Present address, Department of Molecular and Cellular Biology, National Jewish Hospital, 3800 E. Colfax Ave. nue, Denver, CO 80206.

7 Supported by National Institutes of Health Grant GM-21948. To whom reprint requests should be addressed.

- .~ -~ ~ - ” - -~

between these two groups, we have investigated a mitochon- drial representative from a third group: the protozoa. Para- mecium, in particular, represents a rather unique third case in light of its atypical mitochondrial genome conformation (linear) and DNA replication mechanism (unidirectional rep- lication, initiating from one unique end; Refs. 21 and 22). Although we felt it inappropriate here to review in depth the phylogenetic relationships of all the other published se- quences with respect to Paramecium (e.g. Refs. 19 and no), these sequences could become an important evolutionary rep- resentative whenever models for evolution of mitochondrial DNAs are considered.

Also, we have examined the “microevolution” of small sub- unit rDNA sequence between two distantly related Parame- cium species, primaurdia (or species 1) and tetraurelia (or species 4). The Paramecium aurelia comp!ex contains some 14 distinct species, each of which contains a variety of differ- ent geographical isolates (or stocks) and mating types (for reviews, see Refs. 23-26). The degrees of inter-relatedness between these species have been studied using a variety of parameters. Such parameters include interspecific mating (251, interspecies mitochondrial microinjection compatibility (25), similarity of mitochondrial DNA restriction enzyme digestion patterns (26), and mitochondrial gene organization and cross-hybridization (27, 28). Generally speaking, these studies conclude that species 1, 5, and 7 are relatively closely related, as are species 4 and 8, and that the two groups are particularly divergent. Sequence comparisons between species 1 and 4, two of the most divergent examples relative to each other, should produce the greatest number of base changes. Such a comparison should therefore allow us to 2 ) determine the extent of genetic drift within Paramecium mitochondrial genes, and 2) draw stronger conclusions concerning evolution and/or relative functional importance of key gene regions.

In previous work, we aligned DNA restriction fragments from these two species and found that the rRNA genes were located similarly in both species (27, 28). It was not known, however, to what degree this similarity in gene organization extended. Presently, we find both here and in the accompa- nying paper (40) that although only 50% of mitochondrial DNA from species 1 and 4 cross-hybridizes even under non- stringent conditions (27), the overall gene order is retained in all of the species. Consequently, although small (2 bases or less) deletions and insertions do occur, particularly within intergenic regions, the evolution of the entire genome can be considered with respect to substitutions on a base by base level.

In addition, we have used nuclease S1 protection to deter- mine the approximate gene boundaries of rDNA. We exam- ined these end regions for conserved sequences that could possibly be involved in transcriptional regulation. Although no clear correlations of this nature were revealed, we did find,

5167

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5168 Paramecium Small Subuni t rRNA

surprisingly, that small subunit rDNA contains a t least one region near the 3‘ end which appears to be removed from isolated small subunit (13 S) rRNA. A shorter but analogous “deletable segment” was found within Paramecium mitochon- drial large subunit rDNA. These results are discussed in relation to proposed secondary structures for rRNA here and also in the accompanying paper (40).

MATERIALS AND METHODS Paramecium primaurelia (or species 1) stock 513 and Paramecium

tetraurelia (or species 4) stock 51 were obtained from the Edinburgh stocks and were grown in bacterial cultures of Scotch grass. Axenic strains of P. tetraurelia stock 51, used for most RNA analyses, were obtained from the laboratory of Edna Kaneshiro (University of Cin- cinnati, Cincinnati, OH), and were grown in Soldo’s axenic medium (29).

DNA isolation, RNA extraction, and RNA gel electrophoresis were performed as described previously (27, 35). RNA analysis was done by Northern blotting of 0.75 mM methyl mercuric hydroxide-agarose gels onto nitrocellulose filters using the Thomas method (30) with minor modifications (31). Specific DNA restriction fragments were obtained using previously isolated clones (27, 28). DNA fragments were sequenced using the Maxam and Gilbert method (32).

Nuclease SI protection mapping was performed as described by Berk and Sharp (33), and the precipitated reaction products were examined on lanes adjacent to sequence ladders for precise length determination. Optimal results were obtained using a 4-h hybridiza- tion and a 45-min digestion a t 37 “C in the presence of 5000 Boehrin- ger Mannheim S1 units of enzyme. Mitochondrial RNA for nuclease S1 protection experiments usually was obtained from species 4 stock 51 axenically grown cells; however, mitochondrial RNA from bacter- ized cultures of species 1 gave equivalent results. Gene 5’ ends were located using DNA probes which were 5’ end-labeled with polynu- cleotide kinase (Bethesda Research Laboratories). For 3’ ends, 3‘- labeled DNA probes were made by DNA polymerase fill-in with the appropriate [n-“P]dNTP(s) of 5”protruding ends left after restric- tion enzyme digestion. All enzymes were obtained from Bethesda Research Laboratories, except DNA polymerase I Klenow fragment and calf alkaline phosphatase (Boehringer Mannheim).

RESULTS AND DISCUSSION

Small Subunit rDNA Location-Paramecium mitochon- drial RNA obtained from cultures of species 4 (axenic) and species 1 (grown on Klebsiella) was examined by electropho- resis on both methyl mercury-agarose (Fig. 1) and urea- acrylamide (not shown) gels. rRNAs of Paramecium nuclei and mitochondria and of the food source bacteria are present and clearly distinguishable. When these RNAs were North- ern-blotted onto nitrocellulose and hybridized with appropri- ate DNA restriction fragment probes, transcripts of the small subunit rRNA gene could be identified. Note (Fig. 1, lane C) that one band was predominant, and that no larger sized precursor nor small sized discrete degradation products were readily visible. Paramecium small subunit rRNA (previously determined to be 13 S (27, 28)) migrated significantly ahead of E. coli 16 S rRNA (1542 bases (1)). Based on the observed mobilities of the procaryotic and known Paramecium rRNAs present, 13 S rRNA exhibited an empirical molecular size of -1400 nucleotides.

The small subunit rRNA gene region within the genome was more clearly defined using smaller DNA fragments as hybridization probes (Fig. 1, lower). The rDNA was found to be contained within HindIII fragments 7 and 17 (species 1) and 8 and 3B (species 4) with respect to previous restriction maps (27, 28). The genes were found to be located near the center of the genome in both species and to be transcribed in the same direction in which DNA replication proceeds.

Small Subunit rDNA Sequence-In Fig. 2, the complete rDNA sequences for species 1 and 4 are shown. Most regions of the sequence have been covered from a t least two different label points (both strands where possible); however, Some regions could be covered from only one label point due to lack

20s - I 1 16s-

13s-

I

Y

0 1 2 3 4

J I A

1 SSU rDNA-1

4 4 4H8 I 4H3B I H I Y

I I A

FIG. 1. Paramecium mitochondrial small subunit rRNA and rDNA map. Upper, total mitochondrial RNAs from P. primau- relia grown on Klebsiella (lane A ) and P. tetraurelia (axenic cultured, lane B ) were electrophoresed on a 0.75% methyl mercury, 1.5% agarose gel. Paramecium mitochondrial (20 and 13 S), Paramecium nuclear (unlabeled), and Klebsiella (23 and 16 S ) rRNAs are readily visible. A Northern blot of lane B was hybridized with labeled species 4 mitochondrial DNA HindIII fragment 8 (4H8) in lune C. Lower, simplified restriction map for HindIII ( H ) and XbaI (X) sites within these regions from species 1 and 4 is shown. Equivalent results were obtained with corresponding fragments (ZH7 and 1H17) from species 1.

of convenient restriction sites within the sequence. In the latter regions, the sequence integrity is supported by homology between the two species. All nucleotide differences between the two species have been rechecked.

The rRNA genes of both species are clearly homologous, allowing unambiguous alignment in all but a few short regions. The entire gene region contains 107 base differences between the two species (6.4% divergence), consisting of 53% transi- tions, 37% transversions, and 9% insertions/deletions. The

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differences priMUrelia tetraAUreliA E. c o l i

differences

E. coli tetraaurelia

differences priaaurelia

E. coli

differences primaurelia tetraaurelia E. coli

differences pri.arhe1ia tetraaurelia E. c o l i

differences primaurelia tetraaurelia E. coli

differences primurelia tetraaurelia E. col i

differences

tetraaure1ia E. coli

differences prlmurelia tetraaurelia E. coli

differences prlmurelia tetraaurelia E. coli

differences primaurelia

E. coli tetraaurelia

differences primaurelia tetraaurelia E. coli

dif f crences

PriUAUCelia

tCtCAaUCCliA

pri.aure1ia

PriMUCeliA tetrAaUreli0 E. coli

differences

Paramecium Small Subunit

A C G ' G C - - - T TGC GCC NG -GC ACT GTTITGTPGA TAGCCTAGGC TACTTCTAGC CTTAA-TA-A CTAAAATITA R m TAGGCCAG-C T-CTT-TACT TGCAGCCANG CT-WATACT

I - start

r A T CGGATGTAAG G A T I T W I T C TACGAGTGTG ATCTTAGCTT T G A m A A C G TCGATGTAAG AATWITi7T TAGGAGTGTG ATCTTAGCTT T G A m A A C G

AAAUU GAAGAGUUUG AUCAUGGCUC AGAUUGAACG

CTAATCGCGT GCATTACACA CGCAAGTTIT ATTIT-ACAC -AAGAACCTT CTAATCAAAT GCATTACACA CGCAAGTTIT ATTIT-ACAC -AAGAACCTT CUGGCGGCAG GCCUAACACA UCCAAGUCGA ACGGUAACAG GAAGAAGCUU

AAA

C T -CrlTXXC TTAAAATGCA AAG TCAAGT TITAAAATAG CGTATII'CTG

GCWCUWGC UGACG----- ---------- -----AGUC& CCGACGGGUG -cITcTITcc m m m A &ACT m A A A A T A G C G T A m T G

Bgl II G C

C G T W T A T G T C I T P l T A T TCCTATPGTA CCTTAGATAG GTTAGGlTlT CGTAAAATAT G T C l l T l T A T TCGTATETA CCTTAGATAG GCTAGGTITT AGUAAUCUCU GGGAAACUG- CCUGAUPGAG GG--GGAUM -CUACUCSA-

C CC AGA TG-- AA A T T A " T A - T ' t T C A W A G T l T A A ATCCCCACCC CCAAAAGACT TITAAAGAAA C T I T W I T C A A G A A G T l T M TGC-CACCC AAAAAMATT ITITAAAAAA ---MCGGUA GCUAAUACCC CAUAACGUCG CAAGACCMA GAGGCGGACC

A G T G C ATCGATCCIT AAG-ATTAAA GATCCAATAA GAAAAAAGAT ATCGATACTT A A G T A T K M CTC- GATCCAATAG G A M A M G A T UUCGGGCC-- - - - - - - - - - - ---------U CUUGCCAUCG GA-UGUGCCC

AA C C ATAGAGCGCT TCGTJTTCGA AGGCG----G G C C A m c G G C I T C C Z C G C A T A G A G C W TCGAATTCGA AGwic - - - -G GCCACTPGGG CCTCePGCGC AGAUWGAUU AGCUAGUAGG UGGWiUAACG GCUCACCUAG GCGACGAUCC

A TTAGCVZAIT TGEAGAAGA ATCAGCCACA TACAGGTATG TGA-ACGACC TTAGCEATT TGI'GAGAAGA ATCAGCCACA TACAGGTATG TGA-AAGACC CUAGCUGGUC UGAGAGGAUG ACCAGCCACA CU-GGAACUG AGACACCSUC

T T A T C D - - - ----CGACAG A A G W A A T l T I W X C A A TGCGCTTAAG = A m - - - ----CGACAG AAGTGCGGAA TPTTGGGCAA TGCGCTTAAG CMACUCCUA CGGGAGGCAG CAGUGGGGAA UAUUGCACAA UGGGCGCAAG

T C T CGTGACCCAG CAAATTCATG TTCCGGAAGA AAATAAGCGA GGGTACCTCG CGTCACCCAG CAAATIGATG TKCGGAAGA AAATAAGTGA GGGTGCCTTG CCUGAUGCAG CCAUGCCGCG UGUAUGAAGA ACGCCUUCGG GUUGUAAAGU

C CT C A A T G TAGCAATACG ACLTPAGCCT C m A A C T l T TTiTAAACGC AGGCCAGAAA CAGCAATCTG GGATATGCCT C m C A C T I T T T i T A M C G C AGGCCAGAAA ACulRlCAGCG GGGAGGAAGG GAGUAAAGUU ---------- -----AAUAC

CGAGTATAAT T M W A A G T T I T G l T A G A A G C T A " Z C A ATACATGTGC CGAGTATAAT T M W A A G T m C T T A C A A GCTAEGCCA ATACATGTGC CUUUGCUCAU UGACGUUACC CGCAGAAGAA GCACCGGCUA AC-UCCGUGC

C CAGCAGCCGC GGTAATACAT GAGTAWTAG CGTTAATCGT CTITAlTGAG CAGCAGCCGC GGTMTACAT GAGTAGCTAG CGTTAATCGT CClTAlTGAG CAGCAGCCGC GGUAAUACGG AGGGUGCMG CGUIJAAUCGG AAUUACUGGG

GT T CGTACAGAGT GAAGCGGCG- -GTCAGAACA ATTAATAALT ATITITAT" CGTACAGAGT GAGTCGGCG- -GTCATAACA ATTAATAACT AT IT ITAT" CGUAAAGCGC ACGCAGGCGG UUUGUUAAGU CAGAUGUGAA AUCCCCwjcc

G "_" "."

ATAAA AATATATTAA TAAAA-AGGA CTAGTGATIT T l ' I l l T A T G T ATAAA MGATATTAA T U - A G G A C T A G F A m TPTI?TATGT

UCAACCUGGG AACUGCAUCU GAUACUGGCA AGCUUGAGUC UCGUAGAGGG

A A G T I C A W TGTAAACTAG TGATAAAATA CAGGAGTIT ACAGG-AACA AAGTPGAGCT TGTAAACTAG TGATAAAATA CAGGGAGTIT ACAGG-AACA GGGUAGAAUU CCAGGUGUM CGGUGAAAUG CGUAGAGAUC UGGAGGAAUA

G A TITAACGCGA AAGCGACTAA CAATTAA-AA AATGACGCTC TATCACGAAA TITGACGCGA AAGCGACTAA CAATTAA-AA AATGACGCTA TATCACGAAA CCGGUGGCGA AGGCGGCCCC CUGGACGAAG ACUGACGCUC AGGUGCGALA

4 8 46

98 96 35

14h 144 85

195 193 115

245 243 160

294 291 207

34 3 34 1 235

389 387 285

438 436 334

481 479 384

531 529 434

581 579 469

b 3 1 b29 51R

a81 a79 568

727

618 725

771 769 668

818 820

718

869 86 7 768

differences prlmaurelia tetraaurelia E. coil

differences primaurclia tetraaurelia E. coli

differences primaurelia tetraaurella E. coli

differences prinaucelia tetraaurelia E. coli

differences primaurelia tetraaurelia E. coli

dlfferences primaurelia tetraaucelia E. coli

differences primaurelia tetraaur,elia E. coli

differences pri.ducella

E. coli tetrdaurelia

difterences priaaurelia

E. coll tetraaureii.3

dif ferencea prinaure1ia tetraaurelia E. coli

differences prinaurelia

E. coli tetraaurelia

dlfference?r priaaurelia tetraaurelia E. coli

differences primnurelia tetraallrelie E. coli

differences prlaaureiia

E. coli tetraaurelia

differences priaaurelia tetraaurelia E. coli

differences priaaurelia tetraaurelia E. coli

differences priaaurelia tetraaurelia E. coli

differences priaaurelia tetraaurelia E. coli

rRNA 5169

A GTATACGTAA TGAAAAGGCA TTITAACCCT AGTAGTCTAT ACTGTAAAAG GTATACGTAA TGAAAAGGCA TTTTAAACCT AGTAGTCTAT ACTGTAAAAG GCGUCXYXAG CAAACAGGAU UAGAUASCCU GGUAGUCCAC GCCGUAAACG

ATGAGTATIT wTA.-.... .....~.... ........cT MGTTAACAC A T G A G T A m AATA-..". ....._.... .......-CT AAGmAACAC AUGUCGACUU GGAGGUUGUG CCCUUGAGGC GUCGCUUCCG GAGCUAACGC

A AA-AAATACT CCGCTlr;GGT AGTAACGCCG CAAGTCAGAA AClTAACACA AA-AAATACT CCGCTEGGT AGTAAACCCG CAAGTCAGAA ACTTAACAGA GUUAACUCGA CCGCCUGCffi AGUACGGCCG CAAGGUUAAA ACUCAAAUGA

ATIYjGCGGGG ACTPGITCAA ACGGTGGAGC ATCTGGTITA ATGCGATAAT AlTGGCGGGG ACTKTTCAA ACGGTCGAGC ATG%TlTA ATGCGATAAT AUUGACGGGG GCCCGCACAA GCGGUCGAGC AUGUGGUUUA AUUCGAUGCA

c CCACGTAAAA CCTTACCAGC GlTAAATTTT TITCTITAAT T CCACGTAAAA CCTTACCAGC GTTAAATTIT T I T C m A G T TAAG CAT

- 9 L T

ACGCGAAGM CCUUACCUGG UCUUGACAUC CACGGAAGUU UUCAGAGAUG Hind 111

CGCFAAATT TITAGAAAAA MGAGAT-GG TATI'GCATGG CTGTCGTCAG GGCTAAAATT m A G A A A A A AAGAGAT-GG TATTGCATCX: CTGTCGTCAG AGAAUGUGCC UUCWCAACC GUGAGACAGG UGCUCX'AUGG CUGUCGUCAG

T T C G T G m T GAAATITAGA ATTAAGTTIT ATAAACGAAC GCAACCCTPG T K G T G T P I T GAAAWAGA ATTAAGTI1T ATAAACGAAC GCAACCCTTG CUCGUGUUGU GAAAUGUUGG GUUAAGUCCC GC-AACGAGC GCAACCCUUA

G TT ITGTATIT TTAATACMA ATAAATGATT AACCAATTAT TCAITITAGC T I T E T A K T TTAATACAAA ATAAATGATT AAGGAATTAT T C A m T A G C UCCUUUGUUG CCAGCGGUCC GGCCGGGAAC UCAAAGGAGA CUGCCAGUGA

G TAAAA--- - - --------GG CTCAAGTCAA AGTCCTTACG GWXGCGTAC TAGAA----- -------"X CTTXAGTCAA AGTCCTTACG GTCTGCCTAC UAAACUGGAG GAAGGUGGGG AUGACGUCA- AGUCAUCAUG GCCCUUACGA

GCTCGGCTAC ACACGTGTTA CAATGGTAAA AACAATAGM AAACM-- - - CCn%CCTAC ACACGTGTTA C A A m T A A A AACAATAGAA AAACAA----

CCAGGGCUAC ACACGUGCUA CAAUCGCGCA UACRA-AGAG AAGCGACCUC U

-AGCTCACCC m A G T I T I T AAAAAATTAC CACAGTACGA ATICI?TITCT -AGCTCAACC T I T A G T I T I T AAAAAATTAC CACAGTACGA ATTGTTITCT GCGAGAGCAA GCGGACCUCA UAAAGUGCGU CGUAGUCCGG AUUGGAGUCU

A GAAACTfCCA AGCATGAAGA AGAAATCGTT AGTAATCGCA TATAAGTATG GAAACITGCA AGCATGAAGA AGAAATCGTI AGTAATCGCA AATAAGTATG GCMCUCGAC UCCAUCAAGU CGGAAUCGCU AGUAAUCGUG GAUCAGAAUG

N T CTGCGGFAA ATAAAAGTCA AGTCCIGCAC ACACTCCCCA TCACGCTCAA N'IGCGGTGAA ATATAAGTCA AGTCCIGCAC ACACTGCCCA TCACGCTCAA CCACGGUGAA UACGUUCCCG GGCCUUGUAC ACACCGCCCG UCACACCAUG

C G C A - A A C A mAAAA AAATTCGCCA GTGCACAAGT GCGCTGGTGT TCTTAACAAG mAAAA AAACTCGCCG GTGCACCM- ACACCGGTGT TCTAAACMG

I.

C m C T E T T GTTAGCACAA AGAAGAGTAA ACGCGAAACC ATITGAAATG CTA-CTTGTT GTCAGCACAA AGAAGAGTAA ACACGAAACC ATITGAAATG """"" ."...... C GGGAGGGCGC UUACCACllUU GUGAUUCAUG

ATCGGAGTGA AGTEACACA AWTACCCGT ATGGGAACTT WCGGTGGAC ATCGGAGTGA AGTTGACACA AGTACCCGT ATGGCAACTT GCCGGTGGAC ACUGGGGUGA WUCGUAACA AGGUAACCGU AGGGGAACCU GCGGUUGGAU

A G GC TGCA G A GG C GGT CA-----TGA TGAACAGACC AATATAAATG CCCACA-CAC GGTGCATCTA CA-----AGG TGAACAGACC AATGCATCCA CCCAGAACGG G G m r C l T A CACCUCCWA * stop T

919 917 818

945 943 868

994 992 918

1044 LO42 q68

1094 1092 1018

1141 1143

1068

1193 1191 1117

1243 1241 llb7

1278 1280

1216

13Zh 1324 1265

1375 1373 1315

1425 1423 1365

1475 1473 1415

1925 1523 1451

1575 1572 1451

1621 lh25

1482

1075

1532 1671

1719 1716 1542

Hha I FIG. 2. Small subunit rDNA sequence. The sequence of small subunit rRNA and flanking regions was

determined using Maxam and Gilbert (32) chemistry. The identity strand sequences for P. aurelia species 1 and 4 are shown mutually aligned (rows 2 and 3, respectively), and the nucleotide differences are shown in row I. Regions containing the putative gene boundaries according to nuclease S1 data (see text) are indicated with horizontal arrows. Shown also (row 4 ) is the sequence for E. coli 16 S rRNA (l), aligned by both primary and secondary structures (see text).

overall A + T content is 63%, identical to genomic DNA. In general, the base changes are spread throughout the gene nonrandomly. There are three intragenic clusters containing a slightly higher rate of base substitution (bases 246-316,519- 556, and 1539-1588 in Fig. 2, species 1 numbers). These clusters fall within regions of little or no homology to E. coli, suggesting that primary structure within these regions is less constrained evolutionarily. The two most divergent regions (bases 1-50 and 1694-1717, species 1 numbers) lie immedi- ately adjacent to and include both ends of rDNA. These "hypervariable" blocks fall outside the boundaries of the E. coli 16 S rRNA sequence, but appear to contain the true ends of Paramecium mitochondrial rDNA based on nuclease S1

mapping data (see below). A proposed alignment with E. coli 16 S rRNA (1, 18) is also

presented in Fig. 2. The alignment was derived empirically using computer-assisted visual alignment of primary structure and by taking into account the constraints imposed by pro- posed models for small subunit rRNA secondary structure (14-18). We have used E. coli rDNAs as alignment models for two reasons. First, the E. coli sequences more than any other have been studied in detail with respect to secondary struc- ture, such that it is currently the standard with which most rDNAs are usually compared. Second, Paramecium genes in general exhibit a striking resemblance to E. coli counterparts, more so than they do to other mitochondrial counterparts

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5170 Paramecium Small Subunit rRNA

(34-36). Note that in the Fig. 2 alignment, the Paramecium sequence contains several groups of inserted and deleted bases relative to E. coli. Most of these involve 20 bases or less; however, one obvious inserted sequence in Paramecium rela- tive to E. coli begins a t base 1512 (species 1) and includes 83 bases which lie near the 3' end of small subunit rDNA. The latter region is discussed in detail below. Also note that the procaryotic Shine-Dalgarno sequence (CCUCCU, seen as bases 1535-1540 of the E. coli sequence) which is typically absent from mitochondrial sequences is also absent in Para- mecium. Tzagoloff (37) has proposed an alternative sequence of similar function for yeast mitochondria. Although the yeast sequence also is absent here, an analogous sequence could be present.

Nuclease SI Mapping of rDNA Boundaries-Approximate boundaries for both rDNA ends were determined by nuclease S1 protection of end-labeled DNA probes with total mito- chondrial RNA fractions. A fragment extending leftward from the BglII site a t base 167 was used as a probe for the 5' end, and the resulting digestion products were loaded into lanes adjacent to sequencing chemistries of the same fragment. Sequenced bases corresponding to the lengths observed fell into the large region labeled start in Fig. 2 (bases 27-44, species 4, S1 data not shown). Curiously, the protected bands appeared atypically faint, and most probe molecules were protected to full length (at least 2000 bases). Similar results were obtained with species 1. This result suggested small subunit rDNA could be part of a larger transcript or be contiguous with a separate upstream RNA initiated a great distance upstream. We were not able, however, to detect the presence of either a large stable precursor nor a discrete upstream RNA by Northern blot analysis. The latter, plus the proximity of the faint bands to the E. coli 16 S 5' end, suggested that 13 S rDNA begins somewhere within the bases indicated in Fig. 2 and that-apparent full length probe protec- tion was spurious.

In determining the small subunit rDNA 3' end, we sus- pected that the end would parallel the 3' end of the E. coli sequence. We prepared probes accordingly, using the fragment extending rightward from the XbaI site a t base 1485 in species 4. Indeed, S1 protection bands were observed corresponding to nucleotides 1706-1713. Again, the protected bands ap- peared faint, similar to the results at the 5' end, except that no full length protection was observed.

Unfortunately, these nuclease S1 data did not allow precise determinations of the rDNA ends; accurate placement will ultimately require sequence analysis of the rRNA itself. How- ever, using tentative assignments of base 30 (start) and 1710 (stop, species 1 numbers), small subunit rDNA spans about 1680 bases. This length was significantly greater than ex- pected, based upon electrophoretic mobility of the RNA ob- served (-1400 nucleotides; Fig. 1). Because of this discrep- ancy, we suspected that a segment of the nascent small subunit transcript was removed and that 13 S rRNA was the major cleavage product. The most likely cleavage site was at or within the 83-base "insert" mentioned above, lying within the gene near the 3' end. Therefore, we prepared a 3' probe as near as possible upstream of this region. The only conven- ient restriction site available was the HindIII site at base 1086, and the SI-protected bands are shown in Fig. 3. Indeed, protection bands were observed both at the gene terminus (bases 1703-1713, encompassing the HhaI site at base 1709, species 4) and at another site (about 20 bases beyond the XbaI site at base 1485) which corresponds exactly to the start of the 83-base insert. A third partial cleavage site was also detected near base 1325 (light arrow) which corresponds to another separate Paramecium insert relative to E. coli. Two

1 2 3 4 C T A G

b

FIG. 3. Small subunit rDNA 3' end determination. An end- labeled DNA fragment extending rightward from the HindIII site at base 1086 (Fig. 2) was used as a nuclease S1 probe against total mitochondrial RNA (lane 4 ) and was loaded in lanes adjacent to XbaI (lane 2 ) and HhaI (lane 1 ) digests of the same fragment, shown undigested in lane 3. Sequencing lanes (C, T, A, and G ) of a different DNA fragment were included to assist in length determination. Major S1 cleavage bands (solid arrows) appear at bases 1703-1713 (T) and 1505-1516 ( I ) , and a minor band (open arrow) appears around base 1325. The insert relative to E. coli 16 S rRNA is represented by the vertical arrow.

other much lighter bands were visible (not labeled) which probably represent minor degradation products. This experi- ment was done under conditions of relative RNA excess in order to detect the presence of internal cleavage sites; there- fore, the relative intensities of cleavage bands may not reflect the true amounts of the particular RNA species present. Also, note the major band representing full gene length rRNA (band T).

These results became more meaningful when compared to recent models for small subunit rDNA structure (e.g. Refs. 14-18). We propose in Fig. 4 a secondary structure model for the 3"terminal region of Paramecium small subunit rRNA based on these structures and our results. The Paramecium insert is contained within the penultimate stem and loop structure of the E. coli sequence (helix P in Fig. 4, bases 1409- 1445/1457-1491 of the E. coli structure (1, 18)). The insert is notably devoid of possible helices and therefore probably exists as a single stranded region in uiuo. We have therefore included these and neighboring nucleotides of undetermined structure (bases 1487-1618) as a block. Note that the insert

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Paramecium Small Subunit rRNA 5171

I4ii.C * ” - x

C 1 C U

4 c U S C ~ ~ C F U G A I U U U A 6 C A U 6 U 6 b C C C U ~ C C C A U U U U U U U U U U U U U Y ~ ~ A b I ~ L U U C e C C I C U G C A C A A ~ U 6 C C C U C 6 U ~ U U C U U C U U b b C A

r C C U U U C u U C U u e U ~ ~ C C A C A A b C A A C A 6 U b A A G 6 C 6 b C G ~ A b C C P’ A b U A Y I A A U

C * c 3

1.17“”

lip“ f l 7

1600

T : t.

FIG. 4. Small subunit rRNA 3’ end secondary structure model. A possible secondary structural model for the last 249 bases of Paramecium small subunit rRNA was generated by homology to E. coli (and other) small subunit rRNA sequences and structures (1- 20). The region shown is the sequence for Paramecium species 1 which corresponds to the boxed region (upper left) of one recently published structure (15, 18) for E. coli 16 S rRNA. The penultimate helix ( P ) is discussed in the text.

contains both the longest intragenic run of A and T (21 bases, nucleotides 1519-1540) and one of the three clusters of in- creased relative frequency of nucleotide substitutions men- tioned above. The discontinuity detected in Fig. 3 occurs 18- 24 nucleotides after the start of this unstructured region, as shown in Fig. 4.

The penultimate stem of small subunit rRNA exhibits great variability in length and structure in all published small subunit rRNA sequences. It can vary in length from -7 base pairs in mosquito mitochondria (13) to 49 base pairs in rat cytoplasmic 18 S rRNA (8). We suggest that the apical region of this stem and loop in Paramecium mitochondrial small subunit rRNA is particularly susceptible to endonucleolytic cleavage, probably due to the increased length of the single- stranded loop. Cleavage near the start of this loop, as observed (Fig. 3), would yield a stable 1476-nucleotide fragment, in good agreement with the observed mobility of 13 S rRNA (1400 nucleotides; Fig. 1) and presumably another small seg- ment containing the very 3”terminal portion of small subunit rRNA. We have not yet confirmed either the presence or the size of the latter; however, there are numerous small sized RNAs of similar size visible on urea-acrylamide gels.‘ We are currently attempting to isolate and identify these fragments. Note that the XbaI site used initially to locate the gene 3‘ end is located only -20 bases from the beginning of the excised region. Since this -20-base region probably could not form a stable hybrid under the S1 reaction conditions used, the observed faint S1 bands may have resulted from hybridization to the otherwise undetectably small amount of full-length small subunit rRNA present. The major $41-protected band in Fig. 3 representing full gene length RNA may have been protected by this minor species in addition to contiguous

’ J. J. Seilhamer, unpublished data.

cleavage products from this region. Thus, the products of cleavage within this region may be still present (i.e. stable).

We cannot determine from the current results whether discontinuous small subunit rRNA exists in ribosomes in uiuo or whether the discontinuity occurs as a consequence of cell disruption and/or RNA extraction. Clearly, the cleavage is specific to Paramecium rRNA, since the cleavage occurs only in Paramecium mitochondrial rRNAs and not in Klebsiella rRNAs present in mitochondrial fractions (Fig. 1, lane A). In rat liver, stable RNAs resulting from cleavage a t specific points within both the small subunit rRNA penultimate loop (38) and another loop near the 5‘ end (39) have been reported and were attributed similarly to increased susceptibility of these particular loops to endonucleolytic cleavage as a con- sequence of their topology within the ribosome. This could also be the case here, but the cleavage effect within the penultimate loop of Paramecium small subunit rRNA is much more specific. First, the cleavage event in Paramecium is a complete (as opposed to partial) effect since all small subunit rRNAs detectable by Northern hybridization (Fig. 1) are 13 S in size. Cleavage at the insert at base 1325, in contrast, does appear to be a partial effect. Second, the entire penultimate stem of rat liver small subunit rRNA, like most other small subunit rRNAs, can base pair to form a helix (8). Only the first portion of this stem is possible in Paramecium, leaving a large single-stranded region. Third, the cleavage point co- incides with a large inserted sequence relative to all other known small subunit rRNA sequences.

The separation of the 13 S and 3”terminal gene segments by such a large insert suggests the 3’ segment may be in the process of evolving into a separate gene segment, similar to the gene fragmentation seen in large subunit rRNAs. An analogous insert falls within the large subunit rRNA gene of Paramecium mitochondria. The evolutionary implications of these inserts, along with small subunit and large subunit rRNA transcription and structure, are discussed further in the accompanying paper (40).

Acknowledgment-We thank Robin Gutell and Harry Noller for help with secondary structure, homology studies, and referencing.

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J J Seilhamer, G J Olsen and D J Cummingsmicroevolution.

Paramecium mitochondrial genes. I. Small subunit rRNA gene sequence and

1984, 259:5167-5172.J. Biol. Chem. 

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