the yeast nuclear gene nam2 is essential for mitochondrial dna integrity and can cure a...

Cell, Vol. 41, 133-143, May 1985, Copyright 0 1985 by MIT 0092-8874/85/050133-l 1 $02.0010

The Yeast Nuclear Gene NAM2 is Essential for Mitochondrial DNA Integrity and Can Cure a Mitochondrial RNA-Maturase Deficiency

Michel Labouesse, Genevieve Dujardin, and Piotr P. Slonimski Centre de GBnetique Mol6culaire du C.N.R.S. Laboratoire Propre Associe a I’Universit6 Pierre et Marie Curie 91190 GIF-sur-YVETTE France

Summary

Dominant mutations in the yeast nuclear gene NAM2 cure the RNA splicing deficiency resulting from the inactivation of the b14 maturase encoded by the fourth intron of the mitochondrial cytochrome b gene. This maturase is required to splice the fourth intron of this gene and to splice the fourth intron of the mitochondrial gene oxi encoding cytochrome oxidase subunit I. We have cloned the nuclear gene NAMP, which codes for two overlapping RNAs, 3.2 kb and 3.0 kb long, which are transcribed in the same direction but differ at their 5’ ends. NAM2 compensating mutations probably result from point mutations in the structural gene. integration of the cloned gene occurs at its homologous locus on the right arm of chromosome XII. Inactivation of the NAM2 gene either by transplacement with a deleted copy of the gene, or by disruption, is not lethal to the cell, but leads to the destruction of the mitochondrial genome with the production of 100% cytoplasmic petites.

Introduction

Although the mechanism of mitochondrial RNAsplicing is still not understood, it is apparent that the following two classes of transacting proteins are essential in the process: mitochondrially synthesized proteins, that is mRNA maturases, which are encoded in part by specific mitochondrial introns, and cytoplasmically synthesized proteins encoded in the nucleus. In yeast mitochondria, the involvement of a maturase has been demonstrated for three introns, the second (Lazowska et al., 1980; Guiso et al., 1984) and the fourth of the cob-box gene encoding apocytochrome b (Anziano et al., 1982; De la Salle et al., 1982; Weiss-Brtimer et al., 1982; Jacq et al., 1984) and the first of the oxi gene encoding cytochrome oxidase subunit I (COXI) (Carignani et al., 1983); it is believed that a similar situation occurs in other introns that contain an open reading frame (ORF). The involvement of proteins encoded in the nucleus has been demonstrated for the splicing of the COXI pre-mRNA (Faye and Simon, 1983) and for the excision of the third and the fifth introns of the cytochrome b pre-mRNA (Pillar et al., 1983; McGraw and Tzagoloff, 1983). Also, in Neurospora crassa, nuclear mutants defective in mitochondrial RNA splicing have been isolated (Bertrand et al., 1982).

Until now, the main approach was based upon the study of mutants that abolish splicing in mitochondria. We have introduced a different approach to studying genes, the

products of which are involved in mitochondrial RNA splicing. We searched for nuclear genes, which when mutated, compensate for a mitochondrial splicing deficiency (Du- jardin et al., 1980). Several such NAM (Nuclear Accommo- dation of Mitochondria) mutants have been isolated and have been characterized (Dujardin et al., 1980; Groudin- skyet al., 1981; Kruszewska et al., 1984). Among them, the nuclear gene NAM2 is particularly interesting.

Active alleles NAMP-7, NAM2-7, isolated either spon- taneously or after mutagenesis, are dominant over the wild-type allele nam2’. They were demonstrated to be al- lelic to a single gene located on the right arm of chromosome XII, 21 CM from UFfA4 (Dujardin et al., 1983). These active alleles specifically act on the mitochondrial mutations inactivating the maturase encoded by the fourth intron (b14) of the cob-box gene (b14 maturase). Interest- ingly, the b14 maturase is involved in the excision of two introns located in two different genes, the intron b14 and the fourth intron (a14) of oxi gene (Anziano et al., 1982; De la Salle et al., 1982; Labouesse et al., 1984).

To understand further the mechanism of splicing of mitochondrial introns and the function of the nuclear NAM2 gene, we have cloned the active NAM2-7 allele by transforming a b14 maturase-deficient mutant to respiratory competence with a genomic library constructed from the strain carrying the dominant compensating NAM2-7 allele. We have established the restriction map of the gene, have analyzed its transcription, and have shown that it codes for two poly(A)+-RNAs. Using the powerful techniques of yeast genetics, we have inserted various forms of the gene into its normal chromosomal locus and found that inactivation of the gene leads to a complete disintegration of the mitochondrial DNA.

Results

Characterization of the NA M2 Suppressor Active alleles of the NAM2 suppressor gene specifically act on mutations inactivating the b14 maturase (box7- mutations) and all mutations are suppressed by NAMP. Seven of these mutations have been sequenced (De la Salle et al., 1982; Anziano et al., 1982). They either create TAA, TAG stop codons, or missense mutations at various positions of the intron’s ORF. Similarly, an active NAM2 allele restores growth on respiratory substrates of the intron b14 deletion mutant (Jacq et al., 1982; Labouesse and Slonimski, 1983).

Figure 1 shows an example of suppression brought about by the NAM2-1 allele. When crossed to a rho0 strain carrying the wild-type noncompensating allele, the box7- mutant does not respire and hence does not grow on glycerol. When the same mutant is crossed to an iso- genie rho0 strain carrying the suppressor allele, the diploids respire and are able to grow on glycerol.

Figure 2 shows a Northern blot analysis of a b14 maturase mutant in the absence or in the presence of the NAM2-7 allele. In the presence of the NAM2-7 mutation,

Cdl 134

cob-box oxi 3

a bed e fgh with

suppressor

no suppressor

Figure 1. Example of Suppression by the NAMP-7 Allele

Diploid strains were selected cn minimal medium (WO) and were then streaked on glycerol medium (N3). The photograph was taken after five days incubation at 28%; (top) a diploid resulting from the mating of 777-3AlV328 (naml, rho*, box7-) with the suppressor carrying strain, S912/50 (NAMP-7, r/foe); (bottom) isogenic control, a diploid resulting from the mating of 777-3AlV328 with the strain S87/50 carrying no suppressor (namT, rboe). The haploid strains are listed in Table 2.

the box7- mutant (Figure 2, lanes c and g) is able to syn- thesize mature mRNAs for cytochrome b (2.2 kb RNA) and for COXI (2.1 kb RNA), although with a lower efficiency. In the presence of the nam2’ allele, the two messengers are not produced, and there is an accumulation of a precursor of cytochrome b mRNA containing the nonexcised intron bl4 and a precursor of COXI mRNA containing the nonexcised intron al4 (Labouesse et al., 1984).

These results demonstrate that the NAM2-7 mutation allows the correct splicing to occur in the absence of a functional bl4 maturase.

Isolation of a Recombinant Plasmid Able to Compensate for a b14 Maturase Deficiency To clone the NAM2 gene, we took advantage of the fact that the NAM2-7 allele is dominant over its wild-type allele, nam2+, and searched for a recombinant plasmid able to transform a yeast strain carrying a box7- mutation to respiratory competence, that is, to compensate for the b14 maturase deficiency.

We constructed a recombinant plasmid library from the yeast strain S912/50 in the shuttle vector YEpl3. This strain contains the NAMP-7 allele, which has the broadest action spectrum (Groudinsky et al., 1981; Dujardin et al., 1983) and is devoid of mitochondrial DNA (rho”), so that compensation of b14 maturase deficiency could not result from the cloning of a mtDNA fragment. DNA from the library was used to transform the yeast recipient strain CKG18 (see Experimental Procedures) carrying the box7- V328 mutation, which reverts at a low frequency (less than lo-‘). Among the 11,000 transformants recovered, two

were shown to have respiratory competence. Only one transformant (CKG18/2400) was shown by growth under nonselective conditions to lose or to retain, in a coordinate manner, leucine prototrophy and the ability to grow on glycerol.

The recombinant plasmid carried by transformant CKG18/2400 was recovered by transforming E. coli JA221 to ampicillin resistance. Plasmid DNA was shown by restriction analysis to contain a 15 kb insert with three internal Barn HI sites. This plasmid, named YEpGMCOOl, is

mit.genome + - - + +--+

NAM2 allele ++++ ++++ F 7 R 2 7 7 2T1 z

Figure 2. Action of the NAM2 Suppressor on RNA Splicing

Approximately 1 rg of mitochondrial RNA from cells grown on galactose medium (YPgal) was fractionated on 1% agarosel2.2 M formaldehyde gels, was transferred to nitrocellulose filters, and was hybridized with nick-translated radioactive probes prepared from the recombinant plasmid p14 containing the intron-free cob-box gene (A), or from the mitochondrial genome of rho- clone HLM 940-i/D8 the sequence of which is almost entirely restricted to the fourth exon of oxi gene (Net- ter et al., 1982) (B). (a and e) a diploid resulting from the mating of 777- 3A (nam2’, rho’, mif+) with S87/50 (nam2*, rhoo); (b and f) a diploid resulting from the mating of 7773AlV328 (nam2, ~/JO’, box7-) with S87/50; (c and g) a diploid resulting from the mating of 777.3A/V328 wrth S912/50 (NAMP-I, rboe); (d and h) a diploid resulting from the mating of 777.3A with S912/50. Size markers were obtained by running the 21s rRNA and 155 rRNA in parallel. Haploid strains are listed in Table 2.

able to transform CKG18 back to respiratory competence. Its restriction map is given in Figure 3.

To localize the NAM2 gene within the 15 kb fragment, we combined two approaches, cloning of random Sau3 Al fragments and construction of plasmids containing well- defined, overlapping fragments. As shown in Figure 3, the NAM2-7 gene should overlap the 2.35 kb Pst IlPst I fragment retained in YEpGMCO15 and should be smaller than the 4.1 kb fragment delimited by the Sac I site and the right-hand most Apa I site.

Demonstration that Plasmid YEpGMCOOl Carries the NAM2-7 Allele One would predict tha! a plasmid containing the cloned NAM2-7 allele would become integrated at the NAM2 locus on chromosome XII by homologous recombination and should be linked to the URA4 locus, known to be 21 CM from NAM2 (Dujardin et al., 1983). Two integrative plasmids, YlpGMC020 and YlpGMC026 (Figure 3), were cleaved at their unique Sac I site prior to transformation of CKG18 to leucine prototrophy, a treatment known to en- hance transformation efficiency and to direct the site of integration to the region homologous to the sequences cleaved by the restriction enzyme (Orr-Weaver et al., 1981).

Two transformants, obtained by integrating the two different plasmids, were crossed with two rho0 tester strains carrying either the nam2’ UK& or the NAMP-7 marker. Table 1 shows that the cloned gene and the LEUP plasmid-borne marker are linked to the ura4- marker, although the distances vary from 28 CM to 6 CM, depending

Yeast NAM2 and Mitochondnal DNA Integrity 135

r NAM%1 -----I --------

on the transformant. Table 1 also shows that the cloned gene is integrated near the NAM2 chromosomal marker (less than 1 CM or 8 CM, depending on the transformant). The differences observed between the two transformants may not be significant (ch? = 10.7 for n = lo), as only a small number of tetrads were examined in each case.

Finally, we have shown that the integrated cloned gene present in a rho0 derivative of the transformant CKG18/ GIT0042 (Table 2) exhibits, when crossed to mit- mutants, the same spectrum of suppression as the chromosomal NAM2-7 allele.

The fact that the cloned gene is integrated at or near the chromosomal locus of the NAM2 gene, and that it presents the same spectrum of action, demonstrates that we have cloned the NAM2-7 allele.

Transcription of the NAM2 Gene Poly(A)-containing RNA from strains carrying various forms of the gene (chromosomal nam2+, NAM2-7, and episomal NAM2-7) were analyzed by Northern blotting. Hybridization with the URA3 transcript was used as an internal standard. It appears from Figure 4 (lane a) that two

Frgure 3. Restriction Map of the Plasmid YEpGMCOOl and Localizatron of the NAM2 Gene within the Cloned Fragment

YEpGMCOOl is the original plasmid recovered from the leucine’ and glycerol* transformant CKG1812400 (see text). All other plasmids are derrved from it. The ability of the different plasmids to compensate the box%V328 mitochondrial mutation (right column; plasmids able to compensate are marked +) was ascertained by transforming CKG18 to leucine prototrophy and testing for the respiratory competence of several independent transformants. The de- duced posrtion of the NAM2 gene IS grven on the top line of the figure. Construction of plasmids was done using standard procedures (Maniatis et al., 1982). Dotted lines bracket the conserved fragment. Plasmid YEpGMC013 was constructed in the following way: DNA from plasmid YEpGMCOOl was partially digested with Sau 3A and was ligated with Barn HI-cleaved YEpl3 vector. This legation mrxture was used to transform the yeast strain CKG18 to respiratory competence; plasmrds were transferred from the respiration competent transformants to E. coli JA221 and then were tested for their ability to retransform CKG18 to respiratory competence Plasmrds YlpGMC- 020 and YlpGMC026 are integrative plasmrds constructed wrth the vector YlpGMCO19. Plas- mids YEpGMCOl6, YEpGMC015, YEpGMC024, and YEpGMC025 are replicative plasmrds constructed with the vector YEpl3. These plasmids were isolated by ligating appropriately digested YlpGMCO19 or YEpl3 with the fragment of interest and transforming E. coli to ampicillin resistance. The lower lines show the fragments that were subcloned In pBR322 to be used as molecular probes The different regrons of the plasmids are symbolrzed by an open bar (NAM2 chromosomal DNA), a boxed bar (2 t, DNA), a hatched bar (LEU2 chromosomal DNA), or a straight lkne (pBR322 DNA).

RNAs with a size of 3.0 kb and 3.2 kb are transcribed from the chromosomal NAM2 gene. The quantity of these two RNAs is comparable to the level of the URA3 transcript (representing 0.013% of poly(A)+ RNA; Bach et al., 1979) in a strain carrying the chromosomal namP (Figure 4, lanes b and d) or the chromosomal NAM2-7 allele (Figure 4, lanes c and e). When the cells contain a multicopy plasmid, the total amount of the two NAM2 transcripts is about 10 times higher (Figure 4, lanes f and g; and Figure 5, lane e) but their relative ratio is changed. When transcribed from the chromosomal gene, the 3.2 kb RNA is always 7-8 times less abundant than the 3.0 kb RNA, while the 3.2 kb is 1.5 times more abundant when transcribed from the plasmid. An interesting explanation is that the plasmid YEpGMCOOl contains a sequence recognized by a reg- ulatory factor repressing the accumulation of the 3.2 kb RNA, or increasing that of the 3.0 kb RNA, which would be titrated out. Comparison of RNAs from glucose-grown (fermentation) and glycerol-grown (respiration) cells indicates that fermentation does not repress the transcription of the NAM2 gene (Figure 4).

In conclusion two RNAs of 3.2 kb and 3.0 kb, respec-

Cell 136

Table 1. Genetic Linkage of the ura4 Marker and the NAM2 Marker in the Integrative Transformants

Parent MATa Distances (CM) his? /euP-V (LEUP’) nam2+V (NAMP-7) canR rho+ Parent MAT a Number of URA4-NAM2 URA4-LEU2 LEtJ2-NAM2 NAM2-NAM2

box7-V328 rho0 Tetrads 6) (1) CC) (1) 0) (1) CC) (1)

KM642-2C150 ade5 trpl ura4 leu2 canR 31 26 26 3 nam2

CKG18/GIT0021 S912/50 his4 NAM2-1 12 8

KM642-2C/50 18 8 6 3 CKG18/GIT0042

s912/50 24 0 Average 49 21 20 3

CC) (Cl

(“c,

CC) Control (from Dujardin et al., 31 21 52

1983)

For each cross, diploids were sporulated, tetrads were microdissected, and spores were analyzed by standard genetrc procedures. The map drs- tance between linked markers was calculated using the formula derived by Perkins (1949):

T + 8 NPD d (centimorgans) = x 100

2 (PD + NPD + T)

Transformants CKG18/GIT0021 and CKG18/GIT0042 were derived from CKG18 transformed either by the integrative plasmid YlpGMC020 or by YlpGMC026 carrying the LEU2 gene and the putative NAMP-7 allele. V indicates that this allele is integrated in the chromosome. C and I indicate that the gene is the resident chromosomal allele or is an integrated allele, respectively. See Table 2 for the genotype of the strains, and see Figure 3 for plasmid restriction maps.

tively, are transcribed from the NAM2 gene in strains carrying the noncompensating nama’ allele, as well as in strains carrying the compensating /VAMP-7 allele.

The position of the NAM2 RNAs and their direction of transcription were determined using the modification of the Sl nuclease protection procedure of Weaver and Weissman (1979). The plasmid taken as a probe contains the complete NAM2 gene. Figure 56 shows that when pGMCO31 is 5’ end-labeled, two protected fragments are detected by alkaline gel electrophoresis. They are 1.60 kb and 1.80 kb long with the Nco l-cleaved plasmid, or are 2.15 kb and 2.35 kb long with the Xho l-cleaved plasmid. Figure 5C shows that when pGMC031 is 3’ end-labeled, a single 1.25 kb protected fragment is detected with the Nco I cleaved plasmid. Labeling of the plasmid at its Xho I site shortens the length of the protected fragment by 0.55 kb (data not shown).

Taken together, these data indicate that transcription proceeds from upstream of the Nco I site towards the Xho I site (the two sites being 0.58 kb apart). Because the length of the protected fragments was similar as judged by alkaline or neutral gel electrophoresis (data not shown), we can exclude the presence of a large intervening sequence interrupting the gene but cannot exclude the pos- sibility of a small intron if it were located near the termini of the FlNAs.

Inactivation of the Chromosomal nam2’ Allele To gain insight into the function of the wild-type namP allele and into the mechanism of compensation, we decided to inactivate the namT allele at its chromosomal locus.

For this purpose, we transplaced (Scherer and Davis, 1979) the wild-type namT allele by a copy deleted for the internal 1.05 kb Barn HI/Barn HI fragment.

Plasmid YlpGMC023 (Figure 6) carrying an internally deleted NAM2 gene was used to transform the wild-type haploid strain, GRF18. Stable transformants were obtained that contained a functional copy of the wild-type namTgene, in addition to the internally deleted gene (Fig- ure 6, left, middle line). To select strains carrying only the deleted copy of the gene, we screened for leucine auxotroph segregants. After growth for about 10 generations in nonselective complete medium, four leucine auxotrophs, out of 240 segregants, were isolated from two independent transformants. Southern blotting experiments al- lowed us to determine which copy of the NAM2 gene (the complete or the deleted one) was maintained in the chromosome. Figure 6 shows that two segregants retained the complete copy carrying a 2.35 kb Pst IlPst I fragment (Fig- ure 6, lanes b and d), and two had a deleted copy of the NAM2 gene carrying a 1.30 kb Pst IlPst I fragment (Figure 6, lanes c and e). Characterization of the segregants has shown that those carrying the deleted copy of the NAM2 gene are unable to grow on glycerol medium, although they are able to grow on glucose medium. Therefore, inactivation of the nam2’ allele is not lethal to the cell but leads to a respiratory deficiency. This new allele has been termed nam2-88; we have shown that it is recessive in a diploid with respect to nam2’ and to NAMP-7, and that it segregates 2:2 at meiosis. Moreover, we have shown that plasmids carrying a complete NAM2-7 cloned gene can complement the nam2-A8 allele after transformation and

Yeast NAM2 and Mltochondrial DNA Integrity 137

Table 2. List of Strains

r

c

c

1

ae1-4il/s (2: 2 m m+ .I) &+ $&f Krusrewska and Srrresniak (1900:

ABI-43 (2) a 0~1 adel b1s4 nanZ+ -~ (11 m+ u+

777-3A (21 G' adel 0~1 namZ+ (1) a+ Q+ Kotylak and Slonimskl (1976)

777-31/1328 (2) 88 \I (11 a+ M-1328 De la Salle &al. (1902)

777-3A/G3659 (23 88 I* (1) &o+ &&-GM59 De la salle &al. (1982)

6430-20,1 (3) 2 M karl-1 namZ+ a+ a+ A. liinnen

643&2OA/50 (3) " " &o" this study

CKlOOO (3) II II (1) rho+ box7-V328 this study

CKlOOl (3) " " (11 rho+ bax7-G1659 this study

GRrl8 (4)M his3-11 hjs3-15 1~2-3 rho+ m1t+ A. liinnen

leuZ-112 canR nam2+

GRri8/50

CKGlO

CKG19

P5457-4

5912

S912/50

S87/50

5123

5911

51310

51010

liM51/24-1A

(4) IS I, m" this study

(4) " " (1: rho+ box7-V328 this study

(4) " " (1: rho+ box7-G1659 -- this study

(2) am &&+ (1) rho+ box7-G1659 Dujardln & 4. (1980)

(2) gm NAP12-1 II Groudlnsky &al. (1981)

(2) " " -0 II ,I

(2) j3u @I&+ !1 I, II

(2) ai NnM2-I (1: rh:,+ box7-G!659 -- DuJardln &A. (1980)

(2) ghlSq NAMZ-) I, II II

(2) gM NA112-4 II II 3,

(2) j3M m !I I, 1,

au NAM2-6 a+ intron free Lahouesse & Slonlmski (1983) cob-box gene

HPi54-12A

K11642-2C/50

a& NAM2-7 rho+ box7-G1659 this study

a ade5-7 w ura4 leu2 -- __ -0 this study

R m &+

CKGl8/2400 (4) strain CKG18 carrying (1) rho+ hox7-V328 -- this study the free plasmid YEpCllCOOl

CKG18/YEpGPIC013 (4) strain CKG18 carrying the free plasmid YCpGtlCO13

1, this study

CKG18/GIT0021 (4) strain CKGl8 with the 1, this study

CKG18/GIT0042

Integrated plasmid YIpGllCOZO

(4) strain CKGl8 with the 1, ,I this study lniegrated plasmld YIpGMCO26

E. coli strains

hame

m

a

Genotype

F- hsd520 (rk-, mk-) recA13 ara-14 ~raA2

lacy1 qalK2 rpsL2U (Sm") xyl-5 mtl-1

m, lamhda-

E& , m-, M', leuB6 & E5 .I -, lacY

All strains are isomitochondrial to 777-3A (1) except for the mltochondrial mutations Indicated, all strains are isonuclear to 777.3A (Z), 6430~20A (3), or to GRF16 (4) except for the nuclear mutations indicated.

Cell 138

A B

NAM2 allele 2-1 + 2-1 + 2-1

C

0.9- - rra~~w-lJRAI -

.a b c d * f B

glycerol glucose glucose

Figure 4. Transcripts of the nam2’ and the NAMP-1 Alleles Studied in Two Different Physiological Conditions

Approxrmately 2.5 rg of poly(A)* RNA extracted from cells grown on glycerol complete medium (N3) (lanes a, b. and c); on 10% glucose complete medium (YPlO) (lanes d and e); or on 10% glucose minimal medium (WOlO) supplemented wtth htstidine (lanes f and g); they were fractionated on 1% agarosel2.2 M formaldehyde gels, were transferred to nitrocellulose filters, and were hybridized wrth various nick-translated probes. (A) using a probe made from the recombinant plasmid pGMC003 (see Figure 3); (6) using a probe made from an equimolar mixture of the recombinant plasmids pGMCOO3 and Cl6 containing yeast URA3 sequences (see Experimental Procedures); (C) using a probe made from an equimolar mixture of the Inserts of the two above- mentioned plasmids, the 1.05 kb Barn HI/Barn HI fragment of pGMCOO3 and the 1.2 kb Hind Ill/Hind Ill fragment of Cl6, which were gel purified. The allele at the NAM2 locus of the strains examined (see table 2) IS indicated above each lane, the strains were as follows: (b and d) ABl-4A18; (a, c, and e) 5912; (f and g) CKG18/2400, which car- rres the multicopy plasmrd YEpGMCOOl (see text). The autoradiogram presented in lane f was exposed for 40 hr, whrle that presented in lane g was exposed for 4 hr.

reintroduction of a mitochondrial genome by cytoduction (Conde and Fink, 1976), demonstrating that the modified product of the NAMP-7 gene retains the nam2’ function.

Thus, in terms of formal genetics, the nam2-A8 mutant is equivalent to a nuclear pet- mutant (i.e. segregational petite, Chen et al., 1950). It is well known that some pet- mutants lose their mitochondrial genome at a much higher rate than do wild-type cells, and they become double mutants that are simultaneously deficient for a nuclear gene and for the mitochondrial genome (i.e. segregational and cytoplasmic petites, Chen et al., 1950). We have shown that the inactivation of nam2’ leads, in a compul- sory manner, to the destruction of the mitochondrial genome since all cells carrying the null allele of NAM2 are cytoplasmic petifes (rho0 or rho-). This conclusion is based on two observations. First, leucine auxotroph segregants in which the namP allele has been transplaced by the nam2-A8 allele are cytoplasmic petites. Sec- ond, all haploid glycerol-negative clones of monosporal origin, issued from the meiotic progeny of a respiratory competent nanQ+/nam2-A8 rho’ diploid, are cytoplasmic pefifes. The evidence demonstrating that all cells issued from a clone carrying the nam2-A8 allele are cytoplasmic petifes is as follows: none of them revert to respiratory competence, even when crossed to a rho0 strain carrying the wild-type nam2’ allele; most do not restore any of

Al. 5’ Xbl cut

‘. 5’

2.15

2.35

NC01 C”t 3’ .- 1.25

C I \

f M2a b c

2.91- ”

1.86. P cr: all:;;':;

1.06. " 0.9%

0.26-

Xhol Ncol Nco I Nco I

Figure 5. Sl Nuclease Mappmg of the NAM2 Transcripts

(A) Strategy for Sl nuclease mapping. A restriction map of the recombinant plasmid pGMC031 (see Figure 3) used as a hybridization probe is shown in the upper part. The strategy for, and the results of, the Sl nuclease protection experiments are summarized below. The wavy line represents the transcripts, the bars represent the protected fragment, and the asterisk represents the site of 5’ or 3’ end labeling. The thickness of the bar IS proportional to the intensity of the signal on the autoradiogram shown in B, lanes b and d. (B) Mapping the 5’ end of the transcripts. About 50 ng of plasmid pGMCO31 digested with Xho I (lanes a and b) or Nco I (lanes c, d, e. and f) and 5’ end labeled by polynucleotide kinase and yJ2P-ATP was hybridized with 2.5 pg of poly(A)’ RNA extracted from S912 (lanes b and d), or CKGlEIYEpGMC013 containing the multicopy plasmid YEpGMC013 (lane e), or without RNA (lanes a, c, and f). After digestion wrth Sl nuclease, the resistant products were fractionated on a 1.2% alkalrne-agarose gel that was dried before autoradiography. Size markers are pBR322 fragments labeled at their 5’ends after digestion with Bst NI (lane Ml) or with Ava I+ Hind Ill and Ava II+ Hind Ill (lane M2). (C) Mapping the 3’ end of the transcripts. 50 ng of plasmid pGMC031 drgested wrth Nco I and 3’ end labeled by the Klenow enzyme with aJ2P-dCTP and oJ2P-dATP was hybridized with 2.5 Kg of poly(A)’ RNA from CKG18/YEpGMC013 (lane a), or S912 (lane b), or without RNA (lane c) and was treated as described for 8.

several nam2+rho+ mit- mutants scattered over 65% of the mitochondrial genome (two oxil-, one oxP, one ox@, and two box- mutants); they display various degrees of zygotic suppressiveness (Ephrussi et al., 1955) ranging from neutral pefires (1 to 10% suppressive), which predominate, to highly suppressivepetites (92 to 98% suppressive), which are rare. On further subcloning, the nam2-A8 cells have a tendency to lose mtDNA fragments and suppressiveness; this is a well known feature of petites in general (cf.

Yeast NAM2 and Mitochondrial DNA Integrity 139

Figure 6. Transplacement of the nam2* Allele by a Gene Deleted for an Internal 1.05 kb Fragment

(Left) Strategy for the introduction of a 1.05 kb deletion within the chromosomal namT allele. The circle shows the structure of the plasmid YlpGMC023, which was constructed by removing the 1.05 kb fragment from plasmid YlpGMC020 (see Figure 3 for symbols). Below this is the restriction map of the NAM2 chromosomal gene. The middle line presents the expected structure of the NAM2 chromosomal regron after homologous integration of the YlpGMC023 plasmid following cleavage at its unique Sac I site: a wild-type copy of the NAM2 gene is separated from the nonfunc- tional copy presenting a 1.05 kb deletion, by the LEU2 gene and the pBR322 sequences. If recombination takes place between the two copies of the NAM2 gene, the LEUP gene will be lost, giving rise to leucine auxotrophic segregants that will contain a single copy of the NAM2 gene; some of these segregants (bottom line) will harbor the deletion. (Right) Identification of the NAM2 sequences retained in four leu2- segregants. Total DNA of /eu2- segregants, or of the wild-type strain was prepared, was drgested with Pst I, and was analyzed by fractionation through a 0.8% agarose gel, by blotting to a nitrocellulose filter, and by hybridization of the filter with nick-translated probes prepared from a mixture of plasmids pGMC002, pGMCOO3, pGMC004, and pGMC005 (see Figure 3). (a) wild type strain GRF18; (b) leucine- glycerol- segregant GRF181GIT0053/DIT3; (c) leucine- glycerol- segregant GRF18/GIT0051/DIT4; (d) leucine- glycerol* segregant GRF18/GIT0051/DIT43; (e) leucine- glycerol- segregant GRF18/GIT0051/DIT14. Segregant GRFlE/GIT0053/DIT3 has lost its mitochondnal genome but still keeps the wild-type copy of the NAM2 gene.

Gilham, 1978). The genetic data are consistent with biochemical data, which show that some cells retain small mtDNA fragments, while others do not. The destruction of mtDNA by the inactivation of NAM2 also occurs when the rho’ genomes are devoid of some mitochondrial introns. We have tested the omega- genome in which the large intron of the 21s rRNA gene is absent (Jacq et al., 1977) and a genome carrying an intron-free cytochrome b gene (Labouesse and Slonimski, 1983). In both instances the presence of the namPA8 allele leads to the formation of cytoplasmic petites. Disrupting the NAM2 gene in a manner similar to Shortle et al. (1982) gives identical results; all integrants are cytoplasmic petites (data not shown).

Discussion

Cloning of the NAM2 Gene This paper reports the first successful cloning of a mutated gene located in the nuclear DNA that compensates for a deficiency due to a mitochondrial mutation. It should be remembered that the wild-type gene does not possess this compensating function. We have cloned this nuclear gene by transforming a b14 maturase deficient mitochondrial mutant to respiratory competence with a plasmid li-

brary constructed from a strain carrying the compensating NAM2-7 allele.

Cosegregation of the two phenotypes associated with the plasmid, recovery of a plasmid able to transform the target mutation back, faithful integration of the cloned gene at the NAM2 locus on chromosome XII, and suppression of all b14 maturase mutants, demonstrate that we have cloned the NAMP-7 allele and not another gene.

Nature of the Compensating Mutations The results presented in Figure 4 demonstrate that transcription of the chromosomal NAM2 region in strains carrying either the nam2’ or the NAM2-7 allele is identical. Moreover, we have shown that the chromosomal restriction map of strains carrying the noncompensating nam2’ allele, or any one of the seven compensating alleles, is indistinguishable by hybridization (data not shown). Therefore it is logical to conclude that acquisition of the suppressor function does not result from a large chromosomal rearrangement, nor from switching on the expression of a silent gene (promoter up mutation). The NAMB7 mutation, as well as the other compensating mutations, is more likely to result from a point mutation or from a small rearrangement in the structural part of the

Cell 140

gene. Given the fact that the seven available alleles do not show the same efficiency in suppression (Dujardin et al., 1983) and that the NAMP-7 allele still retains the nam2 function (see below), it is probable that the NAM2 gene codes for a protein, and that acquisition of the suppressor function is due to a missense mutation and not to a non- sense one.

Transcription of the NAM2 Gene Two different methods (Figures 4 and 5) have shown that two poly(A)-containing RNA species, 3.0 kb and 3.2 kb, are homologous to the NAM2 gene. These two RNAs share a common 3’ end and are transcribed in the same direction from this gene. Assuming conventional 5’ and 3’ non- coding extensions, these two RNAs could specify two rather large proteins (ca. 90 kd and 96 kd). It appears that the gene is not interrupted by a large intervening sequence (see Figure 5), a conclusion that is further con- firmed by the fact that the size of the two RNAs, as calculated by Northern hybridization (see Figure 4) and Sl nuclease analysis (see Figure 5) is similar, if one as- sumes a length of 200 residues for the poly(A) tail (Dar- nell, 1982).

A priori, these two RNAs could be related to each other either by cotranscription from two different initiation points or by RNA splicing. The two RNAs may be cotranscribed from the NAM2 gene, as examples of cotranscription of several RNAs from a single gene are known in yeast, for example, the SUCP gene (Carlson and Botstein, 1982) and the GAL4 gene (Laughon and Gesteland, 1984). It could also be possible that the NAM2 gene possesses a small intron, (less than 150 bp) located near the 5’ end of the RNA; in this case, the precursor (3.2 kb RNA) would ap- pear to be unusually stable. Generally, unspliced precur- sors of yeast messenger RNAs are barely detectable (Rosbach et al., 1981). However, in the case of the precursor containing the second intron of the MATal gene (Miller, 1984), the precursor is clearly detectable.

Inactivation of the Nuclear Gene Destroys Mitochondrial DNA We have demonstrated that inactivation of the NAMPgene is not lethal to the cell, but is lethal to the mitochondrial genome. This inactivation, whether by disruption or deletion, leads to a respiration negative phenotype and to a rapid loss of the mitochondrial DNA. Therefore the wild- type product of the NAM2 gene is required for the respiratory process but not for cell viability. Moreover, the NAMP-7 mutation preserves this wild-type function, as a plasmid carrying the NAMP-7 allele is able to complement the respiratory deficiency due to the deleted nam2d8 allele. Be- cause the loss of the mitochondrial genome is rapid, we cannot determine whether this loss is a primary or a sec- ondary effect of the inactivation of the NAM2 gene. In other words, the products of the NAM2 gene could be required for the maintenance of the mitochondrial genome, in which case the respiration negative phenotype produced by inactivation of the gene would primarily be due to the loss of the mitochondrial genome. Alternatively, inactivation of the NAM2 gene product would render inoper-

ative a mitochondrial process, which, when impaired, would favor the formation of cytoplasmic petires. The accumulation of cytoplasmic petite mutants in strains carrying the nam2-A8 allele has prevented us from directly in- vestigating the cause of the loss of respiratory functions. We have attempted to construct a double mutant 0~7 and nam2-A8 by meiosis, but spores carrying the expected genotype did not germinate (0~7 is a nuclear mutation in the gene coding for adenine nucleotide translocase, which causes the petite mutation to be lethal to the cell Kovacova et al., 1968).

We have previously shown that the NAMP-7 mutated gene product requires a mitochondrial product (encoded by a region of the oxi gene encompassing the fourth exon and the al4 intron) for its compensating action. We have further argued that this mitochondrial product could be a cryptic maturase encoded by the open reading frame of the intron al4, which would become active in the presence of the NAM2-7 gene product (Dujardin et al., 1983). This is reminiscent of the activation of the al4 maturase by the mitochondrial mim2-7 mutation, where a single base sub- stitution (GAA-AAA) in the al4 ORF transforms a glu- tamic acid codon into a lysine codon. This change has been interpreted as the activation of a cryptic al4 maturase that substitutes for the deficient b14 maturase (Du- jardin et al., 1982). The NAMP-l-mediated activation of the alCencoded protein could involve either a translational or a posttranslational modification of a formerly inactive al4 ORFencoded protein, or a quaternary interaction, making the complex formed by the al4 product and the NAMP-7 gene product active in splicing. Thus, the nuclear product, which would have to be imported into the organelle, could be directly involved in splicing, but it would require a mitochondrial helper protein, or it could be indirectly involved by modifying the structure of a mitochondrial splicing protein.

Whatever the mechanism of suppression brought about by the NAMP-7 to NAM2-7mutations the main conclusion is that the NAM2 gene, present as a single copy, is essential not only for the expression of mtDNA but also for its integrity. The mitochondrial genome is lost when the nuclear gene is inactivated. Further analysis of the three forms of the gene, the wild type, which allows the maintenance of the whole mitochondrial genome but is ineffi- cient in correcting specific RNA splicing deficiencies, the suppressor form, which is active in both processes, and the null form, which leads to disintegration of mtDNA, should be of interest.

Experimental Procedures

Strains, Genetic Methods, and Plasmids Genetic techniques for handling yeast have been described elsewhere (Dujardin et al., 1980; Groudinsky et al., 1981). Minimal media (WO, WOlO) and complete media (N3, YPGA, and YPlO) are described in Dujardin et al. (1980); YPGal contains 2% galactose instead of glucose. E. coli amd S. cerevisiae strains are listed in Table 2. Five of the alleles of the NAM2 suppressor (NAM2-7 to NAM2-5) are isolated after ethyl methane sulfonate mutagenesis of strain PS457-4 (Dujardin et al., 1980). The other two alleles (NAM24 and NAM2-7) are spontaneous isolates that appeared as revertants of a strain carrying an intron-free cob-box gene (Labouesse and Slonimski, 1983) or a cob-box gene


lacking introns bl4 and b15 (Jacq et al., 1982; Dujardin et al., 1983). Strain CKG18 carrying the mitochondrial genome of 777-3AN328 in the nuclear background of GRF18, was constructed by cytoductlon (Dujardin et al., 1980) in a two step procedure. First, the mitochondrial genome of 777-3AN328 was transferred to 6430-20A/50 (Table 2), to obtain the cytoductant CKlOOO. Then, the mitochondrial genome of CKlOOO was transferred to GRF18/50 (Table 2) to obtain the cytoductant CKG18. Transformation of the strain CKG18 by the plasmid YEpl3, with selection for leucine prototrophy, appears to be as efficient as that of GRF18. Zygotic suppressiveness was determined in the followlng way (Ephrussi et al., 1955): cytoplasmic petite cells were crossed to o,ol rho* cells of strains 777-3A or ABl-4B according to the syn- chronous crosses procedure described in Dujardin et al. (1980); cells were then plated on minimal glucose or complete glycerol medium (WO and N3), allowing the differentiation of diploid rho* and diploid petite strains.

The plasmids YEpl3 and p14 were described by Broach et al. (1979) and Labouesse and Slonimski (1983). The integrative plasmid YlpGMCO19 was constructed by inserting the 2.2 kb Xho l/Sal I fragment of YEpl3, which contains the entire LEUZ gene of S. cerevisiae, into the Sal I site of pBR322, as the Sal I site remaining after the inser- tion is proximal to the Eco RI site of pBR322. The plasmid Cl6 is described in Bach et al. (1979) and was obtained from Dr. A. Sainsard; it contains the Hind Ill/Hind Ill fragment containing the yeast WA3 gene. Techniques for propagating E. coli are described in Maniatis et al. (1982).

Preparation of DNA, Enzymes, and Gel Electrophoresis Yeast nuclear DNA was prepared according to Cryer et al. (1975), with minor modifications. Quick preparations were made as described by Nasmyth and Reed (1980). Purification of bacterial plasmids was performed by the procedures described in Maniatis et al. (1982). Restric- tion enzymes and DNA-modifying enzymes were purchased from Boehringher Mannhein, New England Biolabs, or Bethesda Research Laboratories. Agarose gel electrophoresis of DNA fragments was carried out in 89 mM Tris, 89 mM boric acid, with 2 mM EDTA (Maniatis et al., 1982).

Transformation of Yeast and E. coli Yeast cells were transformed by the method of Hinnen et al. (1978), as modified by Ribes (Ph.D. thesis, Paris XI, 1983). As regeneration of spheroplasts is poor in a medium containing a nonfermentable carbon source such as glycerol, all transformation experiments were performed by selecting first for leucine prototrophy in a medium containing glucose.

E. coli cells were transformed either by24 hr incubation in CaCI, or by the CaCI,/RbCI procedures described in Maniatis et al. (1982), except that DMSO was omitted.

Construction of a Yeast Recombinant Plasmid Library and Selection of a Plasmid Carrying the NAM2.1 Gene Purified nuclear DNA from strain S912/50 (Table 2) was partially digested with Barn HI. The sample that gave the highest percentage of fragments in the lo-20 kb range was ligated with the plasmid YEpl3 digested with Barn HI. Prior to ligation, the plasmld was treated with bacterial alkaline phosphatase to prevent self-ligation. Ligation was carried out for 18 hr at 4%, with a yeast to plasmid DNA ratio of 90 pg to 25 pg in a total volume of 200 ~‘1. The ligated DNA was used to transform E. coli strain HBlOl to ampicillin resistance; about 36,000 independent transformants were recovered, out of which 93% were tetracycline-sensitive. Ampicillin resistant transformants were pooled, were respread on 40 ampicillin-selective plates, and were grown over- night at 37’C. Cells were collected from plates, and plasmid DNA was purified by CsCl gradient centrifugation. Growth on plates reduces the yield differences between slow- and fast-growing clones.

Yeast strain CKG18 was transformed to leucine prototrophy with a sample from this library. About 11,000 transformants were recovered by passing the regeneration agar through a syringe and eluting the cells in Ringer; these were then spread on a glycerol medium (N3) and were incubated at 28OC.

Construction of Subclones Relevant techniques for subcloning are described in Maniatis et al.

(1982). When specified, DNA fragments were purified by gel electrophoresis and electro-elution (Dretzen et al., 1981).

Preparation of Yeast RNA Mitochondrial RNA was prepared as described in Labouesse et al. (1984). Total yeast RNA was prepared as described by Carlson and Botstein (1982), except that the breakmg buffer was 0.1 M NaCI, 50 mM Tris (pH 7.5), 10 mM EDTA, and 5% SDS without diethyloxydiformate. Poly (A)-containing RNA was purified by chromatography on oligo(dT) cellulose (Pharmacia) as described in Maniatis et al. (1982).

Transfer of Nucleic Acids to Nitrocellulose Filters and Hybridization The transfer and hybridization of DNA to nitrocellulose filters were performed as described in Labouesse and Slonimski (1983), except that the prehybridization, hybridization, and washing were all carried out at 65’C. RNA was electrophoresed through agarose gels containing 2.2 M formaldehyde and then was transferred to nitrocellulose filters as described in Maniatis et al. (1982). Filters were hybridized according to Thomas (1980). Radioactive probes were prepared by nick-translation (Maniatis et al., 1982) in the presence of @P-dATP (Amersham).

Sl Nuclease Analysis Plasmid pGMCO31 (see Figure 3) was digested with Nco I or Xho I, the enzymes were heat denatured at 70°C and DNA was recovered by eth- anol precipitation. DNA was 5’ end-labeled with T4 polynucleotide kinase and Y-~~P-ATP (5000 Ci/mmol, Amersham) or was 3’end-labeled with the large fragment of DNA polymerase I and a-32P-dATP with U-~~P- dCTP (400 Cilmmol, Amersham) (Maniatis et al., 1982). Sl nuclease protection experiments were then performed essentially as described by Maniatis et al. (1982). The hybridization temperature was set at 46°C that is, 4% above the Tss of the pGMCO31 insert, which was determined by the method of Rosbach et al. (1979). In each experiment about 50 ng of plasmid was hybridized with at least two different amounts of RNA (1 pg-10 rg) to improve the reliability of the results. The optimal Sl nuclease concentration was established empirically in a pilot experiment.

Acknowledgments

This paper is dedicated to the memory of Murray Rabinowitz. We would especially like to acknowledge C. J. Herbert for a friendly col- laboration and discussion, communication of unpublished results, and critical reading of the manuscript. We thank B. Poirier for her very efficient help in the preparation of the manuscript and S. Robineau for artwork. This work was supported by grants from the CNRS, INSERM, Ligue Nationale Franqaise contre le Cancer, ATP “Biologie Mol&ulaire du GBne”.

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

Received December 18, 1984; revised February 26, 1985

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the yeast nuclear gene nam2 is essential for mitochondrial dna integrity and can cure a...

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