Gene, 139 (1994) l-7

0 1994 Elsevier Science B.V. All rights reserved. 0378-l Il9/94/$07.00

GENE 0763 1

1

Two homologous introns from related Saccharomyces species differ in their mobility

(Group-I introns; yeast; mitochondria; gene conversion)

Tomasz Szczepanek, Catherine Macadre, Brigitte Meunier* and Jaga Lazowska

Centre de GPnhique Molkulaire du C.N.R.S.. Laboratoire Propre Associt ri L’UniversitC Pierre et Marie Curie, 9119X Gif-sur-Yvette Cedex, France

Received by P. Slonimski: 5 August 1993; Revised/Accepted: 24 August/24 September 1993; Received at publishers: 30 September 1993

SUMMARY

We have studied gene conversion initiated by the ai intron of the Saccharomyces cerevisiae mitochondrial (mt) COXI

gene and its homologous intron (S.cap.ail) from Saccharomyces capensis. The approach used involved the measurement

of intron transmission amongst the progeny of crosses between constructed recipient and donor strains. We found that

the S. cerevisiae ai intron is extremely active as a donor in gene conversion, whereas its homologous S. capensis intron

is not. We have established the sequence of S.cap.ail and compared its open reading frame (ORF) with that of I-SceIII

encoded by the homologous S. cerevisiae intron. The two protein-coding intron sequences are almost identical, except

that the S. capensis ORF contains an in-frame stop codon. This finding provides a strong indication that the 3’ part of

the S. cerevisiae intron ORF encoding I-SceIII (which should not be translated in the S. capensis intron) must be critical

for function of mtDNA endonucleases to mediate intron mobility.

INTRODUCTION

Group-I and -11 introns have two remarkable features:

first, they are ribozymes that catalyze their own splicing

by different mechanisms and second, they have the ability

to function as mobile genetic elements (Dujon, 1989;

Correspondence to: Dr. J. Lazowska, Centre de GCnCtique Moltculaire

du C.N.R.S., 91198 Gif-sur-Yvette, France. Tel. (33-l) 69824356; Fax

(33-l) 69075539; e-mail: YAGA@frcgmSl.BITNET

*Present address: Unite de Gtnttique, Laboratoire de Genetique

Microbienne, Universitt Catholique de Louvain, B-1348 Louvain-la-

Neuve, Belgium. Tel. (32-10) 473404.

Abbreviations: aa, amino acid(s); bp, base pair(s); COXI, gene encoding

COXI (subunit I of cytochrome oxidase); CYTb, gene encoding apocy-

tochrome b; Er, erythromycin; I-Sce(I,II,III,IV), intron endonucleases

from S. cereuisiae; kb, kilobase or 1000 bp; LSrRNA, gene encoding

large ribosomal subunit RNA; mt, mitochondrial; nt, nucleotide(s);

01, oligomycin; ORF, open reading frame; S., Saccharomyces;

S.c.ai( 1,2,3,4,5a,5B,5y), introns of S. cerevisiae COXI; S.c.o, intron of S.

cerevisiar LSrRNA; S.cap.ai( 1,2,3,4,5), introns of S. capensis COXI;

Sd.ai2, intron of S. douglasii COXI.

SSDI 0378-l 119(93)E0627-P

Michel et al., 1989; Meunier et al., 1990). The first case

of a mobile group-I intron (the w system) had been de-

scribed by Slonimski and co-workers (Coen et al., 1970;

Bolotin et al., 1971) long before introns themselves were

discovered. The observation that some mutations in yeast

mtDNA, when used as genetic markers in crosses, showed

polar recombination with very high transmission of the

allele of one parent in the progeny, and the allele of other

parent being almost completely eliminated, was interpre-

ted as unidirectional gene conversion. Evidence is now

accumulating that several group-I introns in various or-

ganisms are mobile genetic elements (reviewed by

Saldanha et al., 1993; Lambowitz and Belfort, 1993).

It has been well documented that unidirectional gene

conversion in yeast mitochondria is initiated by endo-

nucleolytic cleavage of the intronless mtDNA by specific

proteins encoded by group-I introns. Because the double-

stranded break repair process depends on the homology

of flanking exons, the conversion is accompanied by

co-conversion of flanking genetic markers, if present, in

2

exons surrounding the intron insertion site. Group-I

introns mobility requires the action of a protein encoded

by the intron ORF, as demonstrated by blocking the

reaction upon truncation of the ORF and inferred from

the observation that an ORF transcript is translated into

a site-specific endonuclease in vitro or in vivo (Jacquier

and Dujon, 1985; Macreadie et al., 1985; Colleaux et al.,

1986; Delahodde et al., 1989; Wenzlau et al., 1989;

Sargueil et al., 1991; Seraphin et al., 1992; Moran et al.,

1992; Schapira et al., 1993).

Recently, we reported the first case of two group-I

homologous introns: the second intron (S.c.bi2) of the

CYTb gene from S. cerevisiue and its cognate intron from

S. cnpmsis, exhibiting drastically different behavior in

crosses (Lazowska et al., 1992). The S. capensis intron is

extremely mobile whereas its homologous S. cerevisiue intron is not, but functions as a maturase promoting pre-

mRNA splicing (Lazowska et al., 1980). Furthermore,

these two intron-encoded proteins differ only by five aa

one or more of which must therefore play a critical role

in the mobility vs. immobility of introns.

Here, we present another striking example of homolo-

gous group-I introns, S.c.ai3 and S.cap.ail of the COXI

gene, which also exhibit opposite behavior with regard

to their mobility. It should be remembered that the S.c.ai3

ORF encodes the I-SceIII endonuclease (Sargueil et al.,

1991; Schapira et al., 1993) which is supposed to promote

S.c.ai3 movement to the COXI gene, and that there is no

evidence whether it also has a maturase activity.

RESULTS AND DISCUSSION

(a) Polar transmission of the third intron (S.c.ai3) of S.

cevevisiae COXI gene

The acquisition of the intron by an intronless gene can

be easily detected by the quantitative analysis of the prog-

TABLE 1

Yeast strains

eny of crosses. In mt crosses, the transmission frequencies

of the alleles which are present in the progeny are similar

for all loci except polar regions such as mobile group-I

introns. The transmission frequency is a characteristic

feature of a given cross and appearance of nonpolar

markers is considered to be the input ratio (Dujon et al.,

1974), which reflects the contribution of each parental

mtDNA to the mating pool.

To study gene conversion of S.c.ai3 intron encoding

I-Scelll endonuclease we constructed strains in which

were deleted all group-I introns known to code for mt

endonucleases involved in intron mobility [I-SceI, o

intron in the LSrRNA gene (Jacquier and Dujon, 1985;

Macreadie et al., 1985; Colleaux et al., 1986); I-SceII, ai

intron of the COXI gene (Delahodde et al., 1989; Wenzlau

et al., 1989); I-SceIV, ai5a intron of the COXI gene

(Seraphin et al.. 1992; Moran et al., 1992)] as well as all

CYTb gene non-mobile introns (Meunier et al., 1990).

Two constructed strains are of particular interest in

this work. Strain WF3/5-3 (see Table I and Fig. lE), used

as recipient, has only one group-II intron (S.c.ai2) which

has been previously shown to be very active in gene con-

version (Meunier et al., 1990). Strain WF3/3-43 (Table I

and Fig. lF), used as donor, has the same group-II intron

(S.c.ai2), the next (S.c.ai3) and the last intron of the COXl

gene (S.c.aiSy). Thus, in this cross, both parents have

S.c.ai2, so that it will not be mobile and the S.c.aiSy pre-

sent in the donor mt genome can serve as a flanking

marker for S.c.ai3 movement. In addition we have intro-

duced genetic markers conferring resistance to Er and 01

(WF3/5-3) which are known to be transmitted in a non-

polar fashion (Dujon et al., 1974; Meunier et al., 1990).

The results of quantitative and qualitative analysis of

the progeny from this cross are shown, respectively, in

Table II and Fig. 1. The S.c.ai3 intron is transmitted at

very high frequency (94%), while the transmission of

markers located in nonpolar genes is much lower

Strain” Nuclear genotype Mitochondrial genotypeb

GF167-7B MATa, /4’s2 rlro+, SC. bi-, ai-, w- Er”Ols

CKYLZ M ATa, lrul, karl-1 rho+, SC. bi-, ai-, w- ErROIR

STl-24 MATa, his3 rho+, Scap. bi-, ai+, we ErROIR

WF3/5-3 MATa, led rho+, S.C. bi-, ai2+, o- ErROIR

WF3/3-43 MATa, Lys2 rho+. S.C. bi-, ai2, 3. 5y+, o- ErSOIS

“S. rereoisiue strains having only one (WF3/5-3; ai2’) or three introns (WF3/3-43; ai2, 3, 5y+) in their mt genomes were constructed by a series of

crosses between strains having no introns (GF167-7B or CKYL2) and strains having a reduced number (from 13 to 4) of them. The STl-24 strain,

having introns present only in the S. cnpensis COXl gene (ai’) was obtained by mating a strain with an intronless mt genome (CKYLZ) to the

previously constructed interspecific hybrid strain (FMD3; Lazowska et al., 1992) between S. crrrvisiae (nucleus) and S. capensis (mitochondria).

‘All SC. mt introns present in the constructed strains originated from strain 777-3A which has a complete set of 13 introns in its mt genome. All

strains are devoid of introns in CYTb (bi -) and LSrRNA (w-) genes. Each antibiotic-resistance marker (ErR and OIR) was initially obtained from

GF167-7B (Seraphin et al., 1987) by screening for spontaneous antibiotic-resistance mutations. The mutation sites were assigned to either LSrRNA

(ErR) or olil (OIR) by an allelic test using tester strains from laboratory collection.

3

2.0kb -)

1Akb +

123123456456M TABLE II

-2.32

-181

- 1.41

-105

- 0.68

0.5kb-,

F Donor genome

,”

Fig. 1. Qualitative analysis of the transmission of S. cereuisiae ai intron

to a S. cerevisiue COXI gene lacking it. After synchronous mating and

segregation of initially heteroplasmic to homoplasmic cells, the

mtDNAs from total progeny of the cross shown in Table II as well as

from the parental haploid strains were purified (according to the method

of Gargouri, 1989), digested with appropriate restriction endonucleases

and the digestion products subjected to Southern blot analysis

(Maniatis et al., 1982). mtDNAs from recipient (WF3/5-3, lanes 1 and

4), donor (WF3/3-43, lanes 3 and 6) and total progeny (lanes 2 and 5)

were digested with BstNI + NsiI (panels A and B) and BstNI +BamHI

(panels C and D). The digests were separated on a 1% agarose gel,

transferred to nitrocellulose and hybridized with COXI exons (panels

A and C) and ai intron-specific (panels B and D) probes. The COXI

exonic probe is recombinant plasmid pYGT21 which contains an

almost entire intronless S. cereuisiae COXl gene, a gift from G.-L. Tian.

Lane M corresponds to Raoul 1 DNA marker (from Appligene) and

numbers to the right indicate sizes (in kb) of some fragments. Maps in

E and F show the structure of the COXI genes present in the recipient

and donor strains, respectively. Each exon is depicted by differently

hatched boxes, intronic ORFs by open boxes and intronic non-coding

sequences by thick lines. The location of BstNI (B), NsiI (N) and BamHI

(Ba) sites, the lengths of fragments (in nt) and the location of molecular

probes are indicated. The BstNI (map E, 679 nt) and BstNI-NsiI (map

E, 469 nt) restriction fragments spanning the ai insertion site (indicated

by an arrow) in the recipient genome are underlined.

Polar transmission of the S.c.ai3 intron to the S. cerevisiue COXl gene

lacking it

Type of

colonies”

Marker?

Er 01 ai ai5y

Number

of colonies

Parent 1 R R _ _ 11

Parent 2 s s + + 25

Recombinant R R + + 30

Recombinant R R + _ 56

Recombinant S R + + 18

Recombinant S R + _ 8

Recombinant R S + + 12

Recombinant R S + _ 3

Recombinant S S + + 14

Recombinant S S + _ 5

Marker transmissionc: Ers= 38.5% (70/182)

Ols= 32.4% (59/182)

ai + = 94.0% (171/182)

ai5y’ = 54.4% (99/182)

‘Type of random diploid clones found amongst the progeny of the cross.

bPhenotypes of random diploid clones: R, resistance, S, sensitivity:

presence (+) or absence (-) of intronic markers.

“Transmission of genetic markers present in the donor genome to the

total progeny. Values in parentheses indicate the number of diploid

clones carrying each individual marker out of 182 colonies analysed.

Experimental design: Haploid cells with a homoplasmic genome con-

taining only the S.c.ai2 intron (Parent 1, recipient genome, strain

WF3/5-3, see Table I and Fig. 1E) were crossed to haploid cells whose

genome has only the S.c.ai2,3 and 5y introns (Parent 2, donor genome,

strain WF3/3-43, see Table I and Fig. 1F). Synchronous mating was

performed in a complete, non-selective medium (YPlO: 1% yeast

extract/l % peptone/lO% glucose). After about 15 generations in mini-

mum anaerobic medium (WOlO: 0.67% yeast nitrogen base without

aa, 10% glucose, non-selective for the mt genome), the progeny of the

cross were subjected to quantitative analysis. The cells were plated on

minimal medium (WO: as WOlO but supplemented with 2% glucose),

then 182 individual prototrophic diploid colones were picked at random

and the parental haploids (as controls) were grown on 96-well micro-

titration plates in 300 pi/well of complete medium (YPGA: as YPlO

but supplemented with 2% glucose and adenine at 20 pg/ml) for 2 days.

Plates were replicated on selective media WO, N3 (as YPlO but 2%

glycerol instead of glucose) and N3 supplemented either with Er or 01

and the random diploid clones were checked for phenotype. Total

DNAs were extracted in the plates in which the colonies were grown

(di Rago et al., 1990), loaded on Hybond filters and dot-blot hybridiza-

tions were performed using intron-specific probes to check the transmis-

sion of each intron. Molecular probes were: for ai3, recombinant

plasmid BV02, containing the S.c.ai3 fragment (Carignani et al., 1986)

and for ai5y, recombinant plasmid pYJL24 containing the TaqI-RsaI

fragment (825 nt) from S.c.aiSy cloned between the Hind111 and HincII

sites of pUC13.

(32-38%). Noticeably, the two major classes amongst the

recombinants (in bold in Table II) result only from the

S.c.ai3 intron transfer to the recipient genome. The largest

class of recombinants (56 out 146 recombinant colonies

analyzed) results from S.c.ai3 transfer without

co-conversion extending to S.c.aiSy while in the second

class (30 out 146 recombinants) the S.c.ai3 conversion

4

events are associated with co-conversion of the distant

(892 nt from the insertion site for S.c.ai3) S.c.aiSy intron

marker. The transmission of this intronic marker in the

overall population is higher than that of nonpolar genetic

markers indicating that the excess of S.c.aiSy transmis-

sion (about 20%) results from the mobility of S.c.ai3. The

extent of distant downstream co-conversion detected here

is comparable to that reported for co-conversion of sites

flanking the S.c.o (Jacquier and Dujon, 1985; Macreadie

et al., 1985), and S.c.ai4 (Wenzlau et al., 1989) and

S.c.aiScl (Moran et al., 1992) homing sites of yeast

mtDNA.

The extremely efficient unidirectional transfer of S.c.ai3

was confirmed by Southern blot analysis of mtDNAs

from total progeny and parental strains (Fig. 1). Both

restriction fragments (underlined in Fig. 1E) spanning the

S.c.ai3 insertion site in recipient mtDNA are absent in

mtDNA from total progeny (compare: in panel A, lanes

1 and 2 for the 0.5-kb BstNI-NsiI band, and in panel C,

lanes 4 and 5 for the 0.7-kb BstNI band). Moreover, the

BstNI-NsiI fragment of 538 nt (Fig. 1E) encompasing the

S.c.aiSy insertion point in the recipient genome, which

gives a very strong band with the exonic probe, is clearly

present but reduced in relative intensity in mtDNA from

progeny (panel A, lanes 1 and 2, compare the upper band

close to the 0.5-kb fragment), in agreement with the re-

sults of the quantitative analysis presented in Table II.

The S.c.ai3 intron is clearly present in total progeny

mtDNA in nearly the same proportion as in donor

genome (panel B, lanes 2 and 3; panel D, lanes 5 and 6).

The results presented here show that the group-1 ai

intron of the S. cerevisiae mt COXI gene is mobile in

crosses. The endonuclease activity of I-&e111 encoded by

this intron previously identified in vitro (Sargueil et al.,

1991) is probably responsible for initiating gene conver-

sion events. The S.c.ai3 intron transposes itself with very

high efficiency approaching the theoretical maximal value

of 100% as we can show by construction of strains in

which adjacent self-mobile and co-converting introns

(S.c.ail, 2, Meunier et al., 1990; S.c.ai4, Delahodde et al.,

1989; Wenzlau et al., 1989) were blocked or deleted from

the COXI gene.

(b) The first intron (S.cap.ail ) of S. cupensis COXI gene

homologous to the S.c.ai3 is transmitted in a nonpolar

fashion

S. capensis is a homothallic yeast belonging to the con-

specific taxonomic group of Saccharornyces. Wenzlau

et al. (1989) have described the gross features of S. ca-

pensis COXI gene with five introns and used it as an

acceptor gene in their studies on S.c.ai4 intron mobility

(S. capensis lacks this intron). No sequence data of S.

capensis genome have been reported until now. We have

addressed the question whether S. capensis introns are

mobile and can act as donors for gene conversion. For

this purpose we have previously constructed a respirato-

ry-competent interspecific hybrid strain (FMD3) having

the nuclear haploid genome of S. cerevisiae and the wild-

type mt genome of S. capensis (Lazowska et al., 1992).

Amongst the progeny of a cross between the interspecific

hybrid as donor and a S. cerevisiae strain with an intron-

less mt genome (CKYL2, Table I) as recipient we have

identified several novel strains having the nuclear genome

of S. cerevisiae and displaying various combinations of

S. capensis introns.

Strain STl-24 having in its mt genome only the introns

present in S. capensis COXI gene and two genetic nonpo-

lar markers (conferring resistance to Er and 01, Table I)

was used here as a donor. The results of quantitative

analysis of the progeny from a cross between this donor

strain and recipient strain GF167-7B (which has an in-

tronless mt genome, Table I) are shown in Table III.

Surprisingly, the S.cap.ail intron, which, as will be shown

by sequence determination (Fig. 2) is homologous to the

S. cerevisiae ai mobile intron, is transmitted in a nonpo-

lar fashion. It is therefore probable that none of the in-

trons of the COXI gene of S. capensis is mobile since the

two introns which were not tested here, S.cap.ai3 and

S.cap.ai4 (corresponding, respectively to 48 and 5(3 of

Wenzlau et al., 1989) are adjacent to S.cap.ai2 and

S.cap.aiS which showed uniform and nonpolar

transmission.

Comparing the function of introns present in the CYTb

and COXI genes from the closely related species S. cere-

visiae and S. capensis we made an interesting observation.

The S. cerevisiue CYTb introns essentially function as

maturases for their splicing from pre-mRNA (Lazowska

et al., 1980; 1989; Banroques et al., 1986; 1987) and are

not mobile elements (Meunier et al., 1990) but the second

intron of the S. capensis CYTb gene, homologous to the

S.c.bi2-maturase, is mobile (Lazowska et al., 1992).

Here, we compared the mobility of introns present in

COXI genes from two closely related yeasts. As shown

above none of the introns of the S. cupensis gene is mobile

whereas almost all protein-encoding introns (except ai5p)

whether belonging to group-l or -II introns of the S.

cerevisiae gene are mobile. However, the proposed mech-

anism of transposition of group-I introns (S.c.ai4,

S.c.aiSa, Delahodde et al., 1989; Wenzlau et al., 1989;

Straphin et al., 1992; Moran et al., 1992) differs from that

of group-II introns (S.c.ail and S.c.ai2, Meunier et al.,

1990). In contrast to the S. cerevisiae CYTh intron ORFs,

all acting as maturases, only one group-II intron of the

COXI gene (S.c.ail) encoding reverse transcriptase-like

protein has been demonstrated to be maturase (Carignani

et al., 1983) and one group-I intron (S.c.ai4) of this gene

TABLE III

Nonpolar transmission of the S.cap.ail intron to the S. cereoisiae COXI

intronless gene

Type of

colonies”

Marker?

Er 01 ail ai ai

Number

of colonies

Parent 1 s

Parent 2 R

Recombinant R

Recombinant R

Recombinant R

Recombinant R

Recombinant S

Recombinant S

Recombinant S

Recombinant S

Recombinant S

Marker transmissionc:

s - R +

R +

R _

S +

S _

R +

R _

S +

S +

S _

ErR=36.1%

OIR = 33.9%

ail + =42.8%

ai + = 42.2%

ai + = 42.2%

- -

+ + + - - -

+ + - -

+ + - -

+ + - - - +

(65/180)

(61/180)

(77/180)

(76/180)

(76/180)

16

31

1

4

16

13

16

9

12

1

1

a,b.cSee corresponding footnotes to Table II.

‘Quantitative analysis of the progeny from a cross between (Parent 1)

recipient strain GF167-7B (intronless mt genome, see Table I) and

(Parent 2) donor strain STl-24 (mt genome with all S. capensis COXI

gene introns, Table I), was carried out according to the experimental

design described in footnote b to Table II: ail corresponds to S.cap.ail

which is homologous to S.c.ai3; ai corresponds to S.cap.ai2 which is

homologous to the ai intron of the S. douglasii COXI gene (Tian et al.,

1993); ai corresponds to S.cap.aiS which is homologous to S.c.aiSy.

S.cap.ail, 2 and 5 were designated, respectively, as ai3a, ai3y and ai5y

by Wenzlau et al. (1989).

“The transmission of S. capensis introns was checked by dot-blot hybrid-

ization using intron-specific probes: for ail and ai5, recombinant plas-

mids BV02, and pYJL24, respectively (see footnote a to Table II) and

for ai2, recombinant plasmid pYJL26-2, containing an S.d.ai2 fragment

(Tian et al., 1991).

is latent maturase (Dujardin et al., 1982). There is no

evidence so far whether apart from the demonstrated en-

donuclease activity (Sargueil et al., 1991; Schapira et al.,

1993), S.c.ai3 ORF also has maturase activity. It has been

shown that S.c.ai3 is self-spliced in vitro, but in vivo, the

ai intron is not excised from precursor RNA in the ab-

sence of mt protein synthesis so a maturase activity may

be required (Tabak et al., 1987; Winter et al., 1992). So

far nothing is known about whether S. capensis intron

ORFs can act as maturases. From our previous study on

intron mobility, only the S.cap.bi2 ORF product is sus-

pected of being an endonuclease (Lazowska et al., 1992).

(c) The protein encoded by the S.cap.ail intron is identical

to the N-terminal 195 aa of the I&e111 endonuclease hut

terminates by a premature stop codon

We have established the nt sequence of the first intron

and its flanking regions of the S. capensis COXI gene.

N I

G I Y c

Fig. 2. The nt sequence of the first intron of S. capensis COXI gene

and its surrounding region. The COXI coding sequence starts with the

A of an ATG codon (position + 1). The exonic and intronic sequences

are indicated by upper and lower case letters, respectively. The exon-

intron junctions are indicated by downward arrows. Two conserved

dodecapeptide sequences Pl and P2 are underlined with broken lines.

The ochre codon (*) ending intron ORF is underlined. The S. cereoisiue

mt genetic code is used for translation. The nt sequence was determined

either from PCR-amplified products (using S. cupensis wild-type

mtDNA as template and a series of synthetic primers) or from the

Hpall-BumHI mt fragment (2.6 kb), which were cloned in pUC13,

transferred to Ml3mpl8 and Ml3mpl9 phages and sequenced by the

dideoxy chain termination method (Sanger et al., 1977) using the

Sequenase Version 2 kit (US Biochemical. Cleveland, OH. USA). This

sequence has been deposited in the GenBank database under accession

No. UOO801. The aa (intronic) are numbered in Fig. 3.

This sequence (Fig. 2) has been compared with the corre-

sponding region of S. cerevisiae COXI gene (data not

shown). The non-translated upstream regions of COXI

gene from two yeasts exhibit 92% identity, and differ only

by short additions/deletions. The first exon of the S. ca-

pensis gene is 240 nt long and its sequence corresponds

to that present in the first three exons (Al, 2 and 3, Bonitz

et al., 1980) of S. cerevisiae COXI gene. There are five

substitutions of nt between those exonic sequences. Two

of them are silent and occur at positions 140 and 198

(Fig. 2) in Gly and Met codons, respectively. Three other

changes lead to aa replacements. The Tyr5(j is Phe in S.

cereuisiae, the Ala62 is Gly and Met” is Ile. However, the

Tyr5’j and Ala6’ are conserved in all nine mt COXZ genes

6

compared (not shown) from diverse organisms except

S. cerevisiae.

The first intron of S. capensis COXI gene is inserted

at the same position (241) in the exonic sequence as

intron ai of the S. cerevisiae gene. These two group-I

introns are almost identical in length (S.cap.ail, 15 16 and

S.c.ai3, 1514 bp) and both have an ORF in phase with

the preceding exon characterized by a conserved LAGLI-

DADG motif (Hensgens et al., 1983; also referred to as

Pl and P2 by Michel et al., 1982, or dodecapeptide by

Waring et al., 1982). The main difference between them

is that the S.cap.ail ORF (195 aa) is shorter by 140 aa

when compared to the S.c.ai3 ORF because it contains

a premature stop codon replacing the GAA in S. cerevis-

iae by a TAAg26 codon (Figs. 2 and 3). A second in-phase

stop codon is present at position 1220 where Leu in S.

cerevisiae is replaced again by ochre. Beside these two nt

substitutions creating two stop codons in S. capensis in-

tronic ORF the two proteins are remarkably identical.

The third nt substitution we found at Trp1”4 is silent

(Fig. 3).

Thus, the S.cap.ail ORF displays 99.7% nt identity

with that of S.c.ai3 (strain 777-3A) that encodes the only

active form of I-SceIII actually known and displays 18

nt substitutions as compared to the ORF of S.c.ai3 pre-

sent in strain D273-10B (Bonitz et al., 1980) which appa-

rently lacks any functional I-SceIII (Schapira et al., 1993).

The immobility of the S. cupensis intron versus the

1

1

51

51

101

101

151

151

201

201

251

251

301

301

NQKRYESNNNNNQVMENKEYNLKLNYDKLGPYLAGLIEGDGTITVQNSSS S.Cap. IIIIIIIIIIIIIIlIIIIIlllllIIIIIIIIIIIIIIIIIIIIIIlII NQKRYESNNNNNQVMENKEYNLKLNYDkLGPYLAGLIEGDGTITVQNSSS S.C.

P1 MKKSKYRPLIVWFKLEDLELANYLCNLTKCGR'!&iiRNYVLWTIHDL S.CaP. IIIIIIlIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIlIIIIIIIIlIII MKKSKYP.PLIVWFKLEDLELANYLCNLTKCGKWKKINRNTIHDL S.C.

KGVYTLLNIINGYMRTPKYEAFVRGAEFMNNYINSTTITHNKLKNMDNIK S.Cap. IIIIIlIIIIIIlIIIIlIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII KGVYTLLNIINGYMRTPKYEAFVRGA!3FMNNYINSTTITHNKLKNMDNIK S.C.

IKPLDTSDIGSNAWLAGMTDADGNFSINLMNGKNRSSRAMN S.CaD. IIIIIIlIIIIIIIIIIIIIIIiIIIIIIIIIIIIIIIIIIllII llil - IKPLDTSDIGSNAWLAGMTDADGNFSINLMNGKNRSSRAMPYYCLELRQN S.C.

PZ YQKNSNNNNINFSYFYIMSAIATYFNVNLYSRERNLNLLVSTNNTYKTYY S.Cap. llIIlIi~lIlIIlllIIIIIIIIIIIlllIIlllIlllI~IIIIIIIII YQKNSNNNNINFSYFYIMSAIATYFNVNLYSRERNLNLLVSTNNTYKTYY S.C.

SYKVMVANTYKNIKVMEYFNKYSLLSSKHLDFLDWSKLVILINNEGQSMK S.cap. llIlIllIIIIIIlIIlIIIIIIIIIIlIIlIIIIIlIIIIIIIlIIIlI SYKVMVANTYKNIKVMEYFNKYSLLSSKHLDFLDWSKLVILINWEGQSMK S.C.

TNGSWELGMNLRKDYNKTMTTFTWSH*KNTYLENK 335 S.Cap. IlIIIlIlIIIIIIIlIIIIIlIIII IIIIIIlI TNGSWELGMNLJtKDYNKTMTTFTWSHLKNTYLENK 335 S.C.

Fig. 3. Comparison of the aa sequences of the putative protein encoded

by the S.cap.ail intron and of I-&VIII endonuclease encoded by the

homologous S.c.ai3 intron. The aa sequence of S.cap.ail intron corres-

ponds to translated sequence from nt +241 to + 1245 (Fig. 2). The

sequence data of the S.c.ai3 intron are taken from Schapira et al. (1993).

Sequences were aligned using the program CLUSTAL (Higgins and

Sharp, 1988). Identical residues are shown with vertical bars. Two con-

served dodecapeptide sequences (Pl and P2) are underlined (see Fig. 2).

Asterisks correspond to the stop codons. The first one (position 196)

ending the S.cap.ail ORF corresponds to the ochre codon underlined

in Fig. 2 (nt +826).

mobility of the S. cerevisiae intron is easily explained by

the presence of a stop codon in the I-SceIII endonuclease.

This result also supports the previous observation that

truncated mt LAGLI-DADG endonucleases resulting

from mutations have lost their activities (Jacquier and

Dujon, 1985; Macreadie et al., 1985; Wenzlau et al., 1989;

Moran et al., 1992) and that a 3’ end of intron ORF

rather than a 5’ part of protein could be involved in the

initiation of mt intron gene conversion. What is unex-

pected is the complete identity of primary sequence be-

tween the active and presumably inactive endonuclease

up to the truncation point (Fig. 3). Since the S.cap.ail

protein is totally devoid of all functions one would expect

to find several mutational changes accumulated as a

result of the genetic drift.

We believe that the study of cognate introns could help

in our understanding of the activity, and in particular the

dual function of their ORFs, maturase vs. endonuclease

(i.e., S.c.bi2 vs. S.cap.bi2; Lazowska et al., 1992) and the

evolution of group-I introns at both RNA and DNA

level. Mobile introns, like other selfish DNA, are under

selective pressure to control their movement to safeguard

the viability of their host. Even when the mobility is lost

like in the case of the S.cap.ail intron, the selective pres-

sures must be maintained because if the intronic RNA

were not correctly spliced the COXI protein would not

be made and the yeast would become respiratory defi-

cient. Thus, the complete preservation of the N-terminal

195 aa sequence (which includes the two critical dodeca-

peptides) upstream from the stop codon could be due to

such selective pressure implying that the S. cupensis in-

tron-encoded protein which has lost its DNA endonucle-

ase activity would still maintain its RNA maturase

activity.

ACKNOWLEDGEMENTS

We are grateful to Piotr P. Slonimski for his interest.

We thank Claude Jacq and Javier Perea for sharing their

results before publication. This work was supported by

C.E.E. grant No. 0290-C. T.S. is the recipient of a

Predoctoral Fellowship of Conseil General de 1’Essonne.

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