Proc. Natl. Acad. Sci. USAVol.'79, pp. 6332-6336, October 1982Genetics

Internal structure of a mitochondrial intron of Aspergillus nidulans(apocytobhrome b/DNA sequence analysis/RNA splicing/maturase)

RICHARD B. WARING*, R. WAYNE DAVIES*t, CLAUDIO SCAZZOCCHIOt, AND TERENCE A. BROWN**Applied Molecular Biology Group, Department of Biochemistry, University of Manchester Institute of Science and Technology, P.O. Box 88,Manchester M60 IQD, England; and MDepartment of Biology, University of Essex, Wivenhoe Park, Coichester C04 3SQ, Enland

Communicated by Frederick Sanger, July 21, 1982

ABSTRACT The intron of the mitochondrial apocytochromeb gene, cobA, of Aspergilus nidulans has been subjected to se-quence analysis. It contains an open reading frame of 957 basepairs contiguous with the preceding exon. Regions of the trans-lated open reading frames of cobA and the third intron of the cobgene in yeast show high amino acid homology. Comparison of thecobA intron with this and other yeast introns indicates that cobAcodes for a maturase protein that splices out the intron encodingit and possibly other mitochondrial introns. Two very similar de-camer peptides are found in the protein sequences of the cobAintron, four mitochondrial yeast introns, and the yeast mitochon-drial sequence reading frame 1 (RF-1) and may be diagnostic ofone class ofmaturase-coding introns. Four short DNA sequences,two ofwhich are in the region defined by box9 and box2 mutationsin the cob gene of yeast, are conserved in cobA and certain yeastintrons. Comparison with three yeast introns strongly suggeststhat the first 200 base pairs of the open reading frame of the cobAintron do not code for any amino acids present in the putativematurase protein but are required for splicing or the control ofsplicing, or both.

Conservation of DNA sequence or the amino acid sequenceencoded by related genetic elements ofdistantly related speciesis a strong indication that the sequence in question is of bio-logical significance. Within the gene in the Aspergillus nidulansmitochondrial genome coding for apocytochrome b we discov-ered (1) an intervening sequence (intron) in precisely the sameposition as one of the introns (cobI3) in the same gene of themitochondrial genome of Saccharomyces cerevisiae. This pro-vides a unique opportunity to identify regions of functional im-portance within mitochondrial introns. Some of these code fora class of proteins called maturases (2), which are involved inthe excision from the precursor mRNA of the very intron thatencodes them. This complements and supports the geneticanalysis that is being carried out in yeast.

Introns are found within a number of protein-coding genes,tRNA and rRNA genes in the nuclear genomes of eukaryotes(3-5). The primary transcript of these mosaic genes contains acopy of the intron, which is subsequently excised during thematuration of the RNA. The mechanism of intron excision andexon ligation is poorly understood in the case of mosaic genesthat code for proteins (5). Introns have also been found withincertain genes in the mitochondrial genomes of several lowereukaryotes [S. cerevisiae (2, 6), A. nidulans (1, 7), and Neu-rospora crassa (8)] and one plant species, Zea mays (9), but arecompletely absent from mammalian mitochondrial genes (10).In contrast to introns in nuclear genes, which do not containopen reading frames of significant length, some mitochondrialintrons of yeast have been shown by DNA sequence analysisto possess long open reading frames that are in phase with the

preceding exon. Because splicing and translation occur in thesame compartment in mitochondria, these open reading framescould be translated into proteins.

Introns of this sort include the second, third, and fourth in-trons of the cob gene which codes for apocytochrome b (2, 11,12), the first, second, third, and fourth introns of the oxi3 genewhich codes for subunit 1 of cytochrome oxidase (13), and anintron in the gene for the large rRNA (14). A large number ofcytochrome b-deficient mutants have been obtained, assignedto complementation groups, and mapped within this mosaicgene. The box7 class of mutations has been shown (15-17) todefine a transacting function encoded by intron 14 of the cobgene.

In the mitochondrial genome ofA. nidulans introns are foundin the same three genes as in yeast, the cobA gene (1), the oxiAgene (7), and the gene for the large rRNA (18). Therefore, wedetermined the complete nucleotide sequence of the cobA in-tron. The results suggest that the intron may be translated toproduce a protein that acts like the maturases of yeast. Mostimportant, comparison of the A. nidulans intron with other in-trons of yeast reveals a series of common features at the nu-cleotide and protein level that must be significant for the mech-anism of splicing or its control.

EXPERIMENTAL PROCEDURESDNA Preparation. Preparation ofA. nidulans mitochondrial

DNA, plasmid DNA, and restriction fragments as primers forDNA sequence analysis have been described (1). The M13mp6and M13mp7 cloning vectors (19) were used to obtain clonesfor sequence analysis.DNA Sequence Analysis. The chain-terminator method of

Sanger et aL (20) was followed as described, with materials asused before (1).

Separation of Mitochondrial DNA from M13mp6 DNA.Thirty micrograms of a clone of Bgl II fragment 5 inserted intothe M13mp6 vector was cut with the restriction enzyme EcoRIand loaded onto a 2-ml malachite green DNA affinity column(Boehringer Mannheim) in 1 mM EDTA/10 mM sodium phos-phate buffer, pH 6.0. The M13mp6 DNA was eluted from thecolumn with 20 ml of 0.96 M sodium perchlorate. The mito-chondrial DNA fragment was then eluted with =3 ml of 2.0 Msodium perchlorate, dialyzed overnight, and precipitated withethanol.

RESULTSDNA Sequence Determination. The cobA gene ofA . nidu-

lans lies predominantly in Bgl II fragments 4 and 5 (Fig. 1) onthe restriction map ofA. nidulans mtDNA. We had previouslydetermined the complete DNA sequence of both exons of the

Abbreviations: bp, base pair(s); RF-1, reading frame 1.t To whom reprint requests should be addressed.

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The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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S-rRNA oxiC L-rRNA oxi A cob A URF I URF4 oli AI- -~~~~~~~E= -II I I I I s___I

BlbB BH B BH1 kbp 1 2 4 5 3'

B BW Al A, AA,§ §E "l

_ _ -I_

1 1I i~~~~~~~~~~2:

5 ~~~~~~~~~~~~~~~~2'- 231 _.3 "---I 4

4 ----'

FIG. 1. The cobA gene ofA. nidulans with sequence analysis strategy. The top line shows the position of the cobA gene relative to other knownmitochondrial genes in A. nidulans (7). The genome is circular and is shown broken at a HindI site (as in ref 18). The Bgl II sites are labeled B,andtheBgll fragments are numbered. TwoHpaII sites are indicated (H). These defineHpa II fragment 5, which was cloned intoM13mp7 to providea suitable template for sequence determination across thejunction of Bgi I fragments 4 and 5. On the second line the region around the cobA geneis enlarged to show the sequence analysis strategy. Solid bar, exon region; hatched bar, open reading frame of intron; open bar, remainder of intron.The direction of transcription is from left to right. All sequence analysis was done by using the chain-terminator method of Sanger et al. (20). Tem-plates were single-stranded DNA derived from the phage of M13 clones. Unless otherwise indicated, a 30-base pair (bp) universal M13 primer wasused (21). Numbers at the tail of each arrow indicate the individual strategies used; 1, M13mp6 clones of BgI 11-cut mitochondrial fragments; 2,M13mp6 clones of Sau3A-cut mitochondrial fragments; 3, M13mp6 clones of Bgi H fragment 5 with Alu. I/Sau3A double-restricted fragments as"internal" primers; 4,. same as 3 but primers were pretreated with exonuclease m; 5, M13mp7 clones of Hpa II fragment 5 with an Rsa I/Dde Irestriction fragment as internal primer. The location of internal primers is shown by dotted lines. A, Alu I; B, Bg1 LI; D, Dde I; H, Hpa H; R, RsaI; S, Sau3A. kbp, Kilobase pair. S-rRNA, 16S ribosomal RNA; L-rRNA, 23S ribosomal rRNA.

100 bp

cobA gene (1). The-complete intron sequence was obtained bysequence analysis of the left-hand end of Bgl II fragment 5 andacross the junction of Bgl II fragments 4 and 5. The chain-ter-minator method (20) combined with the M13 cloning/se-quence analysis system (19) was used to determine the DNAsequence. Some ofthe sequence was obtained by using internalprimers hybridized to an M13mp6 clone of Bgl II fragment 5.The Bgl II site was crossed by using an Rsa I/Dde I fragmentas an internal primer on an M13mp7 clone ofHpa II fragment5 that contains the entire cobA gene.The cobA intron has a long open reading-frame in phase with

the first exon. The DNA sequence ofthe intron with its flankingexon sequences is presented in Fig. 2. The location ofthe splicepoints was determined previously (1, 11). The intron is 1,057bp long. The most obvious feature of the sequence is an openreading frame of 957 bp able to code for 319 amino acids andoccupying 91% of the intron. The open reading frame is con-tiguous and in phase with the exon sequence. It is 73% A+T-rich, which is the same as the exon regions of cobA. The prob-ability of a 957-bp open reading frame occurring randomly inan A+T-rich genome is extremely low. There are 23 and 40 stopcodons in the two other phases in which the sequence could beread in the direction of transcription of cobA. There are 29, 38,and 13 stop codons in the three phases in which the sequencewould be translated from the complementary strand. The pres-ence ofan open reading frame in the intron is clearly biologicallysignificant.The cobA Intron Shows Strong Amino Acid Homology with

Yeast cobl3. The cobA intron and the cobl3 intron ofyeast havebeen shown (1, 11) to be at precisely the equivalent point in theexon sequence and to have strong bp homology at the 5' and3' ends near the splice junctions. The DNA sequence of theyeast cobI3 intron has not been completed, but extensive se-quence information has been obtained by J. Lazowska and P.P. Slonimski (personal communication). A detailed comparisonofthe two sequences will be made by these authors, but we notethe main points here.

There is extremely strong amino acid homology between alarge portion of the translated open reading frames of the twointrons. In this region there are many base substitutions thatdo not alter the amino acid that is coded or lead to conservativeamino acid replacements. These results show that the cobA in-trons ofA. nidulans and cobI3 of yeast are closely related andsuggest that selection is acting to maintain homology primarilyat the protein level throughout a large part of the open readingframe.A 10-Amino Acid Sequence Characteristic of Many Matu-

rases. If maturases as a class of proteins have features in com-mon, the interspecies comparison should reveal any that canbe detected at the level ofprimary structure. Taking the proteinsequences as a whole, there was no strong homology betweenthe cobA intron protein sequence and maturases ofyeast. How-ever, we observed that the same 10-amino acid sequence, orclose permutations thereof, occurred twice in the amino acidsequence of the intron of cobA in A. nidulans, introns I3 andI4 of oxi3, and intron 14 ofcob ofyeast (Fig. 3). Such sequencesare also found in cobI3 of yeast (J. Lazowska, personal com-munication), in two introns in the oxiA gene of A. nidulans(unpublished data), and in reading frame 1 (RF-1), an openreading frame in yeast of unknown function (23) that has anintron-like codon usage but is not situated in a known structuralgene. There are always two ofthese sequences, spaced in a verysimilar manner relative to one another (Fig. 4). The peptidesequences in their first occurrence are very similar to those intheir second occurrence, the variation between the two groupsbeing only slightly greater than the variation within the groups.

Several lines of evidence indicate that these sequences arefound in the mature maturase proteins and that the first ofthemis at or very close to the NH2 terminus. In the case of yeastcob14, it is very clear that only part of the open reading frameis involved in coding for the maturase protein (15, 16). No trans-recessive mutations have been found in the first 300 bp of thisintron. The most upstream trans-recessive mutation, V328-1(defining the border of trans-acting maturase function), lies 55

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I V E F I W G G Liat~tgutgagtttatttgaggasgutt

Y T D E P Q C G D V L L K I L L N A G K0 ATACACAGATGAACCACAATGCGGTGACGTATTGTTAAAAATCCTGCTTMTGCTGGAAAA

----A----S P I L G F A Y D L F F I I V L L I G V

61 TCCCCAATCTTAGGATTTGCATACGACTTATTCTTTATAATAGTATTATTAATAGGCGTG

K I A M T R G K S A G V R S L H T S E A121 AAMTTGCAATGACACGGGGAAAATCAGCAGGGGTGAGAAGTTTACATACTTCAGAAGCC

----B--- -S Q R L H A G D L T Y A Y L V G L F E G

181 TCTCAGAGACTACATGCAGGAGATCTTACATATGCCTACTTAGTAGGATTGTTTGMGGT---"'Box9"1--- ------Decamer Peptide--

D G Y F S I T K K G K Y L T Y E L G I E241 GATGGTTATTTTTCCATTACAAAAAAAGGTAAATATTTAACCTATGMTTAGGTATTGM

L S I K D V Q L I Y K I K K I L G I G I301 TTATCAATTAAAGATGTACMTTGATTTACAAAATAAAAAAAATTTTAGGAATTGGTATT

V S F R K I N E I E M V A L R I R D K -N361 GTAAGTTTTAG.GAAAATATGAAATAGAAATGGTAGCCTTAAGAATAAGAGACAAAAAC

H L K S F I L P I F E K Y P M F S N K Q421 CATTTAAAAAGTTTCATTTTACCTATATTTGAGAAATATCCCATGTTTTCTMTMGCM

Y D Y L R F R N A L L S G I I S L E D L481 TATGACTATTTAAGATTTAGAAATGCATTACTTTCAGGTATTATTTCTTTAGAAGATTTA

P D Y T R S D E P L N S I E S 'I I N T S541 CCTGATTATACTAGAAGTGATGAACCTCTAAATTCTATAGAGTCTATTATTAATACATCT

Y F S A W L V G F I E A E G C F S V Y K601 TATTTTTCTGCTTGATTAGTAGGATTTATAGAAGCTGAAGGTTGTTTTAGCGTTTATAAA

-------Decamer Peptide-----L N K D D D Y L I A S F D I A Q R D G D

661 TTMATAAAGATGATGATTATTTAATAGCTAGTTTTGATATTGCTCAAAGAGATGGGGAT

I L -I S A I R K Y L S F T T K V Y L D K721 ATTTTAATATCAGCCATACGTAAATATTTATCTTTTACTACTAAAGTTTATTTAGATAAA

T N C S K L K V T S V R S V E N I I K F781 ACTAACTGTTCAAAGTTAAAAGTTACAAGTGTAAGATCAGTAGAAAATATTATTAAATTT

L Q N A P V K L L G N K K L Q Y L L W L841 TTACAA'ATGCACCTGTAAAATTACTAGGTAATAAAAAATTACAATATTTATTATGATTA

K Q L R K I S' R Y S E K I K I P S N Y ***901 AAACAGTTACGTAAAATATCTAGATATTCAGAAAAAATAAAAATACCTTCAAATTATTAA

961 AAGAGATCATGATATAGTCCGATCAATAAAGAAATTTATTGCGTATAGTMGAGGATTTA---"Box2"1---

| S V N N A T L1021 ATATTTATATTAAATCTGTAACTATCAACATAAATG ctctgtaaataatgcaacttta

FIG. 2. The sequence of the intron of cobA. The adjoining exonregions are shown in lowercase letters. The splice sites as determinedpreviously (1, 11) are marked with arrows. On the basis of previouswork (1) UGA codons have been translated as tryptophan. Regionsdescribed in the text are underlined (dashed lines).

bp downstream ofa group ofcis-acting mutations known as box9(15). The positioning of the conserved decamer. sequences inyeast cobI4 and oxi3I4 fits well with them being just within thematurase coding region ofthe intron (Fig. 4). Comparison ofthecobA intron and yeast cobI3 shows that the region of strongamino acid homology starts right at the first of the conserveddecamer sequences. Last, in RF-1 of yeast,;which, has beennoted to resemble closely intron open reading frames (23), thefirst amino acid of the first conserved decamer is the fMet ofthe NH2 terminus of the putative protein.

Introns of A. nidulans Possess Perfecedy Conserved cis-Act-ing DNA Sites. The cobI4 and oxi3I4 introns of S. cerevisiaehave strong DNA and amino acid homology. Mutations that mapin cobI4 can be divided into three complementation groupscalled box9, box7, and box2; box2 and box9 mutations are cis-dominant, whereas box7 mutations are trans-recessive. The lat-ter are defective in the splicing of oxi3I4 as well as their ownintron; whereas box9 and box2 mutations only prevent the splic-ing of cobI4.A short DNA sequence identical to the cob box9 sequence

S.cer. cobI4 DSNIRFNQ WLAGLIDGDG YFCITKNK

.S.cer. oxi3i4 SINKKFNQ WLAGLIDGDG YFGIVSKK

S.cer. oxi3I3 LNYDKLGP YLAGLIEGDG SITVQNSS

A.nid. cobA 'HAGDLTYA YLVGLFEGDG YFSITKKG

S.cer. RF1 fMISGFTDGDG SFYIKLND

S.cer. cobi4 IKLTKDNA WFIGFFDADG TINYYYSG

S.cer. oxi3I4 IKLTKDNS WFVGFFDADG TINYSFKN

S.cer. oxi3I3 TSDIGSNA WLAILTDADG NFSINLIN

A.nid. cobA .INTSYFSA WLVGFIEAEG CFSVYKLN

S.cer. RF1 PYDKINDY WILGFIEAE'G SFDTSPKR

FIG. 3. Conserved decamer-peptide sequences in proteins encodedby introns. The top group of five lists the first occurrences of the de-camer peptide; the bottom group of five lists the second occurrences.The neighboring amino acids in the proteins are shown on either sidein each case; Amino acids are symbolized by a single letter code (22)to clarify comparisons: A, Ala; D, Asp; E, Glu; F, Phe; G, Gly; I, Ile; L,Leu; M, Met; S; Ser; T, Thr; V, Val; W, Trp; Y, Tyr. S. cer., S. cerevisiae;A. nid., A. nidulans.

is found in the 5' part of the oxi3I4 intron and some mutationsthat are defective in splicing ofoxi3I4 have been shown by DNAsequence analysis to alter this sequence (24). We have founda 12-bp DNA sequence that is exactly homologous to the yeastbox9 sequence and that lies in an equivalent position in the cobAintron (Fig. 4). The finding of this sequence in A. nidulans isevidence that the mechanism of splicing of this intron is similarto that of the yeast introns.

Another important signal for efficient splicing ofthese intronsis located on the 3' side of the open reading frame within theblocked portion of the introns, as defined by DNA sequenceanalysis of box2 mutations (15). This sequence is present in asimilar position in yeast oxi3I4. A similar sequence can be foundnear the end of the cobA intron of A. nidulans and the cobI3gene (Fig. 4).

Other DNA Sequences Are Conserved Between Introns ofthe Two Species. Besides the box9 and box2.splicing signals,two other short DNA sequences that have not been recognizedbefore are conserved between the cobA intron and the yeastintrons cobI4 and oxi3I4. Both occur on the 5' or upstream sideof the box9 sequence and are 10 bp long. These have been tem-porarily called A and B and their positions and sequences areshown in Fig. 4. We note that 5 bp of sequences A and B areinverted repeats that could therefore base-pair with oneanother.

DISCUSSIONDNA sequence and phenotypic analysis of mutations has de-fined the position of cis-and trans-acting elements within cer-tain introns of yeast, particularly cobI2, cobI4, and oxi3I4. TheDNA sequence analysis results presented here reveal that pre-cisely those elements shown by genetic analysis to be importantin these yeast introns are strongly conserved in an intron in adistantly related species, A. nidulans, and two other conservedregions are added to the catalogue of important elements.

The cobA intron ofA. nidulans has the following features incommon with yeast introns that code for maturases (Fig. 4): (i)A long open reading frame is contiguous and in phase with thepreceding exon.and terminates before the 3' end of the intron.(ii) Similarities at the exon-intron junctions of the cobA intronand the yeast cobI3 intron have been noted previously (1, 11)(see also Fig. 4). (iii) Two lines ofevidence suggest that only the

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S. cer. cob 14

S.cer.oxi3 14

S.cer.oxi3 13

A. nid. cob A

1---* I r[-

2

Dii:-----

Intron 5' Splice Site Sequence A Sequence B "Box 9" "Box 2" 3' Splice Site

Sc cobI4 -UUAGGU CAAAAU-47-AUGCUGGAAA-192-AAUCAGCAGG-65-UCAGAGACUACA-1 043-AAGAUAUAGUCC-1 5-UAAAAG CAUCCU-

Sc oxi 3I4 -UUUGGU CAAACA-50-AUGCUGGAAA- 78-AAUCAGCAGG-50-UCAGAGACUACA- 747-AAGAUAUAGUCC-30-AACAAG CACCCU-

An cobA -AGGUUU AUACAC-44-AUGCUGGAAA- 74-AAUCAGCAGG-30-UCAGAGACUACA- 771-AUGAUAUAGUCC-70-UAAAUG CUCUGU-

Sc cobI3 -UGGGUU UAAUAU AAGAUAUAGUCC-61-UAAAAG CUCAGU-

FIG. 4. Conserved regions of mitochondrial introns. (Upper) Open bars represent the open reading frames of the introns; single lines, the closedremainder of the introns; dotted lines, the adjacent exons of the introns. The regions coding for the decamer peptide sequences are represented byshort solid blocks. A, B, 9, and 2 indicate the positions of the short DNA sequences of A, B, box9, and box2 (see text). The introns are I3 and I4 ofoxi3 (13), I4 of cob (12) in S. cerevisiae (S. cer.), and the cobA intron of A. nidulans (A. nid.). The long arrow indicates the direction of transcription.(Lower) The DNA sequences of the splice sites, the box9, box2, andA andB sequences are given. The number ofbp between each sequence is indicated.The available data for cobI3 of S. cerevisiae (S) are added (J. Lazowska, personal communication). An, A. nidulans.

downstream two-thirds of the cobA intron is important for pro-tein coding. First, amino acid homology with yeast cobI3 is verystrong in the downstream two-thirds but is weak in the 5' one-third. Second, the first of the conserved amino acid decamers,which seem to occur at the NH2 terminus ofmaturases, is foundat the beginning of the region of high homology with yeast cobI3(J. Lazowska, personal communication). (iv) The box9 and box2cis-acting sites are perfectly conserved in the cobA intron se-

quence and occur in positions within the introns correspondingto their locations in yeast introns (Fig. 4). De La Salle et aL (15)have noted that the box9 sequence could form a substantialbase-paired structure with a region of the small rRNA and thatthis potential pairing would be weakened by box9 mutationsthat are defective in splicing. This led them to suggest that theribosome not only synthesizes the maturase but also plays an

essential role in setting up tertiary structure or aligning RNAsequences to ensure accurate and efficient splicing. As shownin Fig. 5, the box9 sequence of the cobA intron pairs more

weakly with the equivalent sequence in the A. nidulans smallrRNA (ref. 25; unpublished data), suggesting that this may notin fact be the case.

Several new features of such introns are uncovered by com-

parison ofthe cobA intron sequence with the yeast introns. Twoother sequences, called A and B in Fig. 4, are strongly con-served throughout introns of the yeast cobI4 class. They areboth situated between the 5' exon-intron junction and the box9

A.nid cobA Intron S.cer cobi4 Intron S.cer Oxi3I4 Intron

5'

UCAGAGACUACAUGI 111111

CGAACCUGAUGAUC

3'

A.nid S-rRNA

3' 5'

iC UCAGAGACUACACGCJ

C GAUCUUUGAUGUGCUl

5' 3'

S.cer S-rRNA

3' 5' 3'

A UCAGAGACUACMGAAI 111111111 IIIU GAUCUUUGAUGUGCUU

5' 3' 5'

S.cer S-rRNA

FIG. 5. Potential base pairing between the box9 mitochondrial in-tron sequence and a region of the mitochondrial small rRNA. Thesmall rRNA sequence in yeast has been identified previously (15). Theequivalent region in the small rRNA of A. nidulans (A. nid.) was lo-cated by sequence comparison (ref 25; unpublished data). S. cer., S.cerevisiae.

sequence. The box9, box2, A, and B sequences are interrelatedand could pair in various combinations. Possible RNA secondarystructures and the implications for the mechanism ofRNA splic-ing await further investigation.

Most of the reading frame in the 5' end of the cobA intronis shifted by + 1 nucleotide with respect to the same region inthe homologous yeast cobI3 intron, although the putative ma-turase protein coding region is precisely in phase. Furthermore,the amino acids generated in the region of sequences A, B, andbox9 in cobI4 and oxi3I4 ofyeast are different from those in thesame regions of cobA, again due to a difference in phase. Thissupports the arguments, made from genetic data in yeast, thatthe 5' region of these introns-including the box9 region-isimportant as nucleic acid sequence not as protein coding se-

quence (15, 16).The comparisons have also revealed a new feature of certain

maturases themselves. A 10-amino acid sequence, which ispresent in two almost identical copies in the maturase proteinsequence coded for by the downstream two-thirds of the in-trons, is strongly conserved between the two species. We haveobserved no obvious sequence homology in the region lyingbetween the two conserved amino acid sequences except pos-sibly in the case of the cobA or cobI3 intron and the yeast RF-1 sequence. Jacq et aL (16) have identified a mutant (W300-1)in cob of yeast, in which the highly conserved glycine residuein the 10th position of the 2nd conserved decamer would bereplaced by an aspartic acid residue. They have suggested thatthis change produces a partially defective maturase protein.Clearly, these conserved amino acid blocks are biologically im-portant. Because they occur in maturases that as far as we can

tell are involved in splicing reactions with different specifica-tions, these sequences are more likely to play a role in inter-actions with elements that are always present-such as thesplice sites, the cis-acting signal sites, or some unidentifiednuclear-coded element.The cobI3 intron of yeast is an enigmatic intron, largely be-

cause only two trans-recessive mutations have been found inthis intron, and these are more leaky than trans-recessive mu-tations in cobI4 or cobI2 (16). On explanation is that cobI3 can

direct the splicing of its own intron, but so can something else.

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The excellent conservation of amino acid sequence despite nu-cleotide sequence drift between yeast cobI3 and A. nidulanscobA argues in fact that both code for a protein product thatperforms an important function. A function for the product ofthe cobA intron could be to coordinate expression of the cobAand oxiA genes; some ofthe oxiA introns possess the same splic-ing signals (unpublished results).The results of this work clearly support the maturase model

proposed by Slonimski and co-workers (2). Because A. nidulansis an obligate aerobe, it is clear that this kind of splicing mech-anism is of general importance and is not related to the unusualmitochondrial physiology of yeast. We expect that further ge-netic and genetic engineering work with the two systems willhelp us understand how introns are spliced out of precursormRNA, how this is controlled, and perhaps why introns arepresent at all.

We acknowledge the help of J. Cullum and B. Robson with computeranalysis. We thankJ. Lazowska and P. P. Slonimski for data on the cobI3intron prior to publication. This work was supported by project GrantG7904290CB to R.W.D. and C.S. from the Medical Research Council.

1. Waring, R. B., Davies, R. W., Lee, S., Grisi, E., McPhail Berks,M. & Scazzocchio, C. (1981) Cell 27, 4-11.

2. Lazowska, J., Jacq, C. & Slonimski, P. P. (1980) Cell 22, 333-348.

3. Crick, F. (1979) Science 204, 264-270.4. Gilbert, W. (1978) Nature (London) 271, 501.5. Breathnach, R. & Chambon, P. (1981) Annu. Rev. Biochem. 50,

349-383.6. Borst, P. & Grivell, L. A. (1981) Nature (London) 285, 439-440.7. Davies, R. W., Scazzocchio, C., Waring, R. B., Lee, S., Grisi,

E., McPhail Berks, M. & Brown, T. A. (1982) in MitochondrialGenes, eds. Slonimski, P. P., Borst, P. & Attardi, G. (ColdSpring Harbor Laboratory, Cold Spring Harbor, NY), pp.405-410.

8. Hahn, U., Lazarus, C., Lunsdorf, H. & Kuntzel, H. (1979) Cell17, 191-200.

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