Localization of the gene coding for the mitochondrial 16 S ribosomal RNA using rho− mutants of Saccharomyces cerevisiae

J. Mol. Biol. (1975) 99, 203-217

Localization of the Gene Coding for the Mitochondrial 16 S Ribosomal R N A Using rho- Mutants of Saccharomyces cerevisiae G. FAYE, C. KUJAWA, B. DUJON, M. BOLOTIN-I~UKU1~ARA K. WOI~% H. FUXU--ARA and P. P. SLom~sxI

Gerdre de G$n~ique Mol~cuIaire du CNRS, 91190 Gif-sur. Yvette, France and ~In~$itut fi~r (Tenetil¢ der Universitgt Mi~nehen, Federal Re1~ublic of Germany (Received 4 Al~ril 1975) Series of genetically characterized cytoplasmic petite mutants of Saccharomyces eercvisiae presenting various combinations of five genetic loci: RI, R m , Oi, OH PI were used to try to locate the 16 S ribosomal RNA gene on the mitochondrial genome by RNA-DNA hybridization. It appeared that the 16 S rRNA gene lies in the vicinity of the PI locus. The two mitoehondrial rRNA genes (16 S aud 23 S) are therefore not adjacent. The 16 S rRNA gone seems to be transcribed in vice in some mutants, as in the case of the 23 S rRNA gene in mutants described earlier. Some petite genotypes could not arise from the deletion of a single continuous segment but could arise from a double deletion of the wild-type genome. The consequences of this result are discussed.

1. " I n t r o d u c t i o n I t has been established t h a t rho + mitochondrial DNA of Saccharomyces cerevisiae codes for 16 S and 23 S ribosomal RNAs (see recent review b y Mahler, 1973) and for at least 14 t!ansfer RNAs (Casey et al., 1974a,b). Numerous mitechondrial point mutations have been s~udied. T h e y confer resistance to specific antibiotics, for example chloramphenicol, erythromycin, spiramycin, paromomyein and oligomycin (see for references Dujon et al., 1974). Mapping of these mitochondrial mutations b y recombination between rho + strains, have been attempted. The chloramphenicol, spiramycin and erythromycin resistance markers are located in three ]inked loci and their relative order has been determined (Netter et aL, 1974). E x ~ n s i v e studies of multffactorial crosses have been made using two oligomycin loci (Avner et al., 1973) and a paromomycin locus (Wolf et ed., 1973). The results indicated t h a t these three loci were un]inl~ed b y recombination and not ]intred to the three preceding ones, therefore no respective order could be deduced. However, as shown b y the analysis of co-ordinated o u t p u t in multifactorial crosses all the six loci are carried b y the same molecule of mtDNA~ (Dujon et al., 1974). Cytoplasmic petite mutants (rho- mutants) are deletion mutants in the mitochondrial DNA (Faye e2 at., 1973). The deletions are compensated b y reiteration of non-deleted segments of the wild-type mtDNA. With the help of suitable genetic markers it is possible to isolate t h e - clones in which different parts of rho + m t D N A t Abbreviation used: mtDNA, mitochondrial DNA. 203

204

G. F A Y E E T A L .

are maintained and repeated (Deu~ch d a~., 1974). Extensive experiments of D N A R N A or I ) N A - D N A hybridization, denaturation-renaturation of D N A and electron microscopy of m t D N A have been published (Fukuhara et aJ., 1974; Lazowska et al., 1974; Michel et al., 1974; Locker e~ a/., I974~,b). Hybridization experiments of rho + mitochondrial r R N A with petite m t D N A s showed t h a t the 23 S r R N A gene was v e r y close to or overlapping with the chlor. amphenicol and erythromycin resistance m u t a t i o n (Faye e~ al., 1973; ~ u k u h a r a e~ ~d., 1974; Nagley e~ al., 1974). ~n vivo transcription studies of m t D N A in some spontaneous or ethidium bromide induced r h o - m u t a n t s (Faye e~ a[., 1973,1974; Faur~s-Renot e~ al., 1974) have shown t h a t r h o - strains carrying ch]oramphenicol and ery~hromycin markers contained t h e 2 3 S mitochondrial r R N A gene and t h a t this gene was transcribed. No r h o - clone in these series possessed a m t D N A sequence corresponding to the 16 S rRNA. W e suggested, therefore, t h a t 16 S and 23 S r R N A s genes might not be closed physically ]in~ed in the yeast mitochondrial genome. I n the present work new series of r h o - clones representing various combinations of deletion covering all available genetic loci have been used to a t t e m p t to locate the mitochondrial 16 S r R N A gene b y D N A - R N A hybridization. We show t h a t the 16 S r R N A gene lies in the vicinity of the p a r o m o m y c i n resistance-conferring marker. Tentative m a p s of the genetic m a r k e r are proposed on the basis of the hybridization data and the genetic pedigrees of the r h o - clones.

2. Materials and M e t h o d s (a) ~q~ains Table 1 gives t~e nuclear and the mitochondrial genotypes of the rho + strains (the R mitochondrial genetic markers are: Cs21, representative of the locus RI, E6~4 or E~ 1 of the locus Rm, O~44of the ~ocus On, P4~4 of the locus PI, O~6 or O~ of the locus Oi. I n the conventional nomenclature of Plisehke e~ aL (1975), R I ----[RIB1], R i n ------[RIB3], Oi ---- [OLI1], Oii -~ [OLI2] and PI ----[PARI]) f~om which the r h o - clones were derived. The genotypes of the rho + strains used as tester strains are also given. For the mlto. chondrial genotypes of the r h o - clones used in this work, see Table 2. All the r h o - clones were obtained b y ethidium bromide induction and successive subclonings according to the methodology described by Fukuhara e~ aL (1974). The numbering of successive subclones, which allows one to trace the hereditary ascendence of any clone under study, is TABLE 1

I ~ t of rM + haploid 8tra¢~ Name

KL14-~ IL836-6A§ PS228-1A§ KLS1-6A MH32-12D¶ MH41-7B¶ TR3-15A¶

Nuclear genotype

~ ~ trp~ ~ ura ~ ade2 = trp~ a his 1 ade~ a his 1 ade~ a his: trp2

Mitochondrial genotype

~ho÷ c~1 o~ P~, rho + O~ R R rho + C8~1 E~21 0144 rho + CsR21E~21 O1~4 R P4~4 R rh°+ Cs~l E~I 0144

rho + CR1 ~~.R nR ~ 514 v145 ~454 R ~R rho + C3R1~..R ~14 N~1 --454

Deutsch e~ aZ. (1974). Wolf e~aL (1973). § D. Coen e~ aZ. (manuscript in preparation). ¶ M. Bolotin-~lkut~ra & H. Fub~hara (manuscript in preparation).

ppp+

p-

pppppppppp-

p-

pppppppppp-

p-

ppp-

? 0 0 0 0 0 0 0 S R R R R ? 0 0 0 0 0 0 0 0 0 0 0 0 R R S

O~x 0 R R R R 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 R R R R R R R R S

0 0 0 0 0 0 0 0 0 0 0 0 0 R 0 R R R R R R R R R 0 0 0 0 R

Mitoohondrial genotype Px Rz ? 0 0 0 0 0 0 0 0 0 0 •0 0 ? R R R R R R S S S S 0 0 0 0 R

Rxxx 0 0 0 0 0 R R R R 0 0 0 0 R S R R R R 0 0 0 0 0 R R 0 0 S

Oz 0.8 96.0 104.0 0"7 0.7 3"4 3"9 2"6 1"1 0"6 1"5 1"7 0"6 2"9 6"0 10"0 5"0 3"1 1"3 5"0 3"6 4"9 5'1 3"7 83.0 84"0 3'0 9"0 100'0

0.1 2.7 2.8 0"1 0"3 3"3 8"0 1"3 1"0 0"5 0"8 1-5 1"0 22"0 100"0 114"0 108"0 102"0 93"0 107"0 72"0 82'0 95'0 78"0 2"4 4"6 4"4 12"0 100"0

Hybridization (% ) 16 S r R N A 23 S r R N A 99 99 99 99 63 65 98 81 99 69 92 96 23 -92 93 77 85 -77 13 73 69 75 76 83 74

Genetic purity (%)

1"684 1"684 1"685

-1"685 1"685 1"684 1"684 1"684

1"685 1"684 1"688 1"685 b

1"682

1.687 1.685 1-686 1-683 1"682

Buoyant density (g/ore 8)

Genetic p u r i t y indicates t h e percentage of t h e r h o - cells still possessing t h e desired mitoehondriM marker(s) conferring antibiotic resistance after a b o u t 40 gonerations, a n d is determined b y t h e replica cross t e c h n i q u e using a drug sensitive rho + tester (Deutsoh e~ al., 1974). T h e presence of a sensitive nn'toohondrial marker(s) is determined b y t h e s t a n d a r d cross technique (Coon e~ a~., 1970) using a drug resistant r h o + tester. T h e hybridization p e r cent is determined as described i n Materials a n d Methods. ?, N o t determined; R , resistance; S, sensitivity; 0, deleted. t F a y c eJ a~. (1974).

KL14-4A/F344 KL14.4A/A211 KL14-4A/E121 KL14.4A/A311 KL14-4AIEII2 KL14-4A/C211 KL14-4A/Clll MH41-7B/D42~ MH41-7B/C516 MH32-12D/K394 MH32-12D/K416 MH32-12D/K41~ MH32-12D/K385 KL14-4A/F331 IL8-8C/F12t TR3-15A/K711@ TR3-15A/K614~ TR3-15A/Kll7~ TR3-15A/K512 ILS-8G/D21t KL14-4A/F321 KLI4-4A/F34121 KL14-4A/F31111 KL14-4A/F34211 KL14-4A/F34131 TR3-16A/3412 M~H32-12D/C144 MH32-12D/T917~ IL8-SC

Strains

T~a.BLv. 2

Genetic properties and hybridization data of the different rIm- strains

206

G. F A Y E

ET

AL.

given by Fukuhara et aJ. (1974). The control of the purity of t h e - cultures was done by the "replica cross technique" (I)eutsch et at., 1974). Presence or absence of antibiotic sensitive alleles in rho- clones was determined by "standard crosses" (Coen et al., 1970) with appropriate rho + testers according to the methodology described by Bolotin et al. (1971). (b) IsolatAon of mitochondria and analysis of mitochondrial RIgA When studying the transcription products of some rho- clones, the total RNA was extracted from purified mitoehondria and analysed by acrylamide gel electrophoresis as described by Faye et al. (1974). (c) Preparation of mtDNA and analytical density gradient centrifugation The mtDNA was isolated from total cell DNA by fraetionation on hydroxyapatito columr~ according to Bernardi et al. (1970). Analytical density gradient centrifugation was carried out as described by Lazowska et a/. (1974). (d) R I g A - D N A hySr~d" ization OH.labelled mitochondrial RNA was prepared from the rnitochondria of the rho + strain IL8-8C. At 1 h before harvesting the cells, non-labelled adenine (final concentration 0.02%) was added to the [°H]adenine-containing culture. The purified OH-labelled mitochondrial RI~TAwas fractionated by gel electrophoresis. R N A - D N A filter hybridization was performed using urea. (For technical details see Faye et al., 1974.) A constant concentration of labelled RNA (about 0.6/,g/ml for 23 S rRNA and 0.3 #g/ml for 16 S rRNA; spec. act. 4100 cpm per/,g) was incubated with increasing amounts of filter-bound mtI)NA of rho- clones (10, 20, 30 or 40 ~g o f m t D N A per filter). The normalization of countings was done using hybridization with the rho + mtDNA as internal standard. For clones giving high levels of hybridization with 23 S or 16 S rRNA, values of 1/hybrid (cpm -1) were plotted on the ordinate and 1//~g DNA per filter on the abscissa, the curves obtained were extrapolated on the I/hybrid axis to evaluate "'~": the survival fraction of the unique sequence of 23 S or 16 S rRNA genes, for rho + : = 1, for rho- : 0 < ~ < 1 (Faye et al., 1974). For clones giving low levels of hybridization with 23 S or 16 S rRNA, we have divided the hybridized counts of each rho- I)NA filter carrying 40/~g of mtDNA by the hybridized counts of rho + DNA filber carrying the same quantity of DNA.

3. R e s u l t s (a) (Teneti~ 8taSility of t h e - donee The r h o - clones listed in the Table 2 were selected for the maintenance of various combinations of genetic markers at five loci: RI, Rm, Oi, O~ and PI. F a y e et al. (1974) have shown t h a t the 16 S r R N A gene is not situated in the Rx to P ~ region of the + DNA, thus we chose r h o - clones possessing genetic markers not genetically linked with this region, expecting t h a t this set of r h o - clones would cover the entire the + D N A sequence. The purity of cell populations used for the biochemical preparations was checked b y the replica cross technique. One can see, from Table 2, t h a t the genetic stability (Faye et al., 1973) was variable according to the r h o - clones. Some cultures were very pure: more than 95~/o of cells still possessed the desired mitochondrial markers after about 40 cell generations, others kept their markers in 60 to 85% of cells, and two clones were unstable, F331 and F34121. Stable and unstable subclones can emerge even from a common tertiary clone and, a fortiori, from secondary and primary clones after equivalent numbers of subclonings (example: F3 -> F34121 and F34131, F3 -> F31111 and F34121). The reason for such differences of the genetic stabilities

MITOCHONDRIAL

rRlgA GENES

207

is not understood at present, but it must reside in the organization of the mtDNA and not in the nucleus, for the clones have the same nuclear genetic background. (b) R N A - D N A hybr'utization We have isolated mtDNA from all the rho- clones listed. The absence of nuclear DNA in these preparations was ascertained by CsC1 isopycnic centrifugation. I t can be seen in Table 2 that the buoyant densities of mtDNA from the difference rhoclones are variable according to the clone. For example, clone K385 had a buoyant density higher than that of rho ÷ mtDNA, while C l l l and D42~ showed lower buoyant densities. We purified all-labelled 23 S and 16 S rRNA from the rho ÷ mitochondria and with each we titrated the rRNA gene sequences in the mtDNA of various rhoclones. The results are given in Table 2. They are expressed as survival fraction (~) of the unique rRNA sequences. Despite the limited precision of the extrapolation method, it is apparent that for each of the 16 S and 23 S rRNA genes, the results can be grouped into two distinct classes: high hybridization levels (72 to 114~ ofrho + hybridization) and low hybridization levels (0.1 to 22 ~ ofrho ÷ hybridization). The high levels of hybridization indicate that the mtDNA contains a large part of, or more likely all of, the rRNA gene sequences. It appears that the two clones pR: A211 and El21 and the two clones PaOIR: F34131 and J412 possess the 16 S rRNA gene in their mtDNA. A number of reasons may be envisaged to account for the low levels of hybridization (for example, with the mtDNA of C l l l , K711~, 1~12 or T917~) : only small fractions of rRNA genes may be present in some rho- clones, or 23 S rRNA or messenger RNA present as a contaminant of the 16 S rRNA may form hybrids with some rhomtDNA retainining corresponding sequences. The formation of poorly specific hybrids with variable stabilit5, cannot be excluded. Another possibility is that the low levels of hybridization are due to heterogeneity of rho- mtDNA molecules of some clones. The hybri~zation data with the 23 S rRNA confirm previous results described by Faye et at. (1974), which showed that the 23 S rRNA gene is present when the R~ to 1 ~ I region is kept. (e) Mitochondrial R1VA from some rho- clones We have analysed the in vivo transcription produc~ of different clones (Fig. 1). In the two rho- clones A211 and El21 having the 16 S rRNA gene, we detected two R i g a species by gel electrophoresis that migrated very similarly to the normal 16 S rRNA, but appeared as a double peak in both clones. The meaning of this result is not clear, one of the two peaks may be the 16 S rRNA. On the other hand, these clones transcribed a large amount of 4 S RNA. This may be correlated to the result that several tRNA hybridized in the PI region (H. Fukuhara et a/., manuscript in preparation). The clone F34131 presented a poor level of transcription (electrophoretic pattern not shown). The clones F31111 and F34211 which possess the RI to Rm region and present the mtDNA sequence corresponding to the 23 S rRNA gene transcribed the 23 S rRNA. This confirms our previous work on CRER rhoclones.

208

G. F A Y E

~T

A JS. A211

F34211

pR

p.RCR Es

I 1 i-Ii-

1

0

1

!

I

I

0

I

I

I

(o)

oo 8 JC~

i

I

!

I

I

I

I

0,

8

I

I

(c)

F31111

El21

pRCREs

pR

I

I

8

I

/ !

d

I

I

I

I

(b)

I

I

I

I, ,,,J

i

t

8 0 Distonce migroted (cm)

!

!

!

{d)

I

!

!

!

8

Fro. 1. Eleotrophoretic p a t t e r n s of mitoehondrial R N A of two P~CRE s 0 ° O°u and two pa~o~.onono v ~ v z v ~ t h e - clones. R N A s were extracted from the purified mitochondria of (a) F34211 (b} F S l l l l , (e) A211 a n d (d) El21. I n each electrophoresis, two reference tubes, one w i t h cell sap rRNAs, another with R N A s isolated from rho + mitochondria, were run in parallel w i t h R N A s isolated from the different r h o - . The two cytoplasmic r R N A s are indicated in the diagrams b y two broken lines a n d t h e two mitoohondrial r R N A s b y two solid lines. Electrophoresis was performed in 2.4% aorylamide gels (length 10 cm, diam. 0-6 om) for 8-4 h a t room temperature. The ele~rophoretic flow was from left t o right. Ordinate: absorbance a t 260 nm.

4. Discussion (a) Localization of the 16 S rRNA gene We will first consider the 26 new rho- clones analysed in this work and the nine clones presented in our previous paper (Faye et al., 1974) (the latter clones kept the genetic markers in the RI to R m region). The 16 S rRNA was hybridized with the mtDNA of these 85 rho- clones. The data are summarized in Table 3. It appears from part (a) of Table 3 that the P1 marker is present when the 16 S rRNA gene is present. I f we consider the clones carrying a single genetic marker, we may suppose that the probability for these clones to possess mtDNA sequences other than those located in the vicinity of their genetic marker is low. Then from part~ (b) and (d) of Table 3, it is clear that the 16 S rRNA gene lies in the vicinity of the genetic marker P~ but the locus PI does not seem to be in the sequence of the 16 S r RNA gene, because some rho- clones have kept the Pz locus but not the 16 S rRNA gene.

0 carries the 16 S rRNA gene

0 carries the 16 S rRNA gene

2 carry the 16 S rRNA gent

4 carry the 16 S rRNA gene

(e) Among the 12 clones carrying the the Pz marker Among the 12 clones carrying the Ox marker Among the 18 clones carrying the Rz or/and Rm markers Among the 7 clones carrying the On marker

carry Px carry Oz carries R1 or/and Rm carries Oix

4 2 0 0

(a) Among the 4 clones carrying the 16 S rRNA gene

Clones carrying one or several genetic markers

(d) Among the 4 clones carrying the P1 marker Among the 3 clones carrying the 0i marker Among the 8 clones carrying the RI and/or Rm markers Among the 4 clones carrying the 0ix marker

(b) Among the 2 clones carrying the 16 S rRNA gene

0 carries the 16 S rRNA gene

0 carries the 16 S rRNA gene

0 carries the 16 S rRNA gene

2 carry the 16 S rRNA gene

2 carry P1 0 carries 0i 0 carries RI or/and Rm 0 carries OIi

Clones carrying a single genetic marker

Relations between Chepresence of the genetic markers and the presence of the 16 S r R N A gene among the 35 rho- clones studied

TABLE 3

210

G. F A Y E

ET

.4L.

We have shown that the 23 S rRNA gene is closely associated with the R ~ region, which is not genetically ]inlred with the PI locus. Since the 16 S rRNA gene is close to the P1 marker, the 16 S and 23 S rRNA genes are far distant from each other. In E. coli the rRNA molecules are derived from six transcriptional units (Pace, 1973) containing adjacent genes for 16 S, 23 S and 5 S rRNAs which are read by the RNA polymerase in that order (Hackett & Sauerbier, 1974). In Bacillus subtili8 the transcription is probably similar. While it seems that in the yeast 8accharorayc~ cerevisiae the 5 S nuclear genes are interspersed with the 18 S and 28 S rRNA genes, i.e. are present in equal number (150 genes) and are closely ]lulled (Rubi~&Sulston 1973), in the higher eukaryotes the arrangements of the rRNA nuclear genes are different. The 28 S and 18 S rRNAs are transcribed in the nucleolus in the form of a large precursor RNA molecule. This transcriptional unit contains a copy of each of the two rRNA components and, in addition, stretches of non-ribosomal polynucleotide material (Planta et al., 1972). In all higher eukaryotes studied the 5 S genes are unlln~red to the 18 S and 28 S rRNA genes, for example in Drosophila and HeLa cells the 5 S genes are on different chromosomes; furthermore, the numbers of 5 S rRNA genes and of the high molecular weight rRNA genes are different: 25,000 and 450, respectively, in Xenopus laevis (Brown & Weber, 1968) 2000 and 280 in HeLa cells (Attardi & Amaldi, 1970). In mitechondria no 5 S rRNA component has been detected. According to Kuriyama & Luck (1973) Neurospora crazsa mitochondria synthesize a 32 S RNA precursor of the large and small subunit rRNA. In HeLa mitochondria, Wu et al. (1972) have mapped the positions of several 4 S RNA genes and of the 12 S and 16 S rRNA genes. The two rRNA genes are separated by a small spacer (160 nucleotides), which appears to bind one of the 4 S RNAs. It seems that these rRNAs derive from processing of larger molecules (Aloni & Attardi, 1972). From our results, it appears that the high molecular weight rRNA genes are well-separated. To our knowledge this is the first case of a relatively great distance separating the two large rRNA genes. A problem arises; how is the synthesis of these rRNA species co-ordinated? Perhaps a very large transcriptional unit exists or some regulation mechanism governs the transcription of the two rRNAs in yeast mitochondria. If we accept that Px is clearly outside the 16 S rRNA gene, we may suppose that Px muta~tion might effect a mitochondrial protein. Since it is known that paromomycin is an inhibitor of the initiation of prokaryotic RNA translation (see Vazquez, 1974) the Px mutation may be a mutation of an initiation factor or a structural protein of the 38 S ribosomal mitochondrial subunit (Schmltt, 1970). Messenger RNA transcribed from mtDNA is most likely translated by the mitochondrial ribosomes. The hypothetical protein product corresponding to the Px locus would be made also on the mitochondrial ribosomes. Although most mito. chondrial ribosomal proteins seem to be synthesized on the cytoplasmic ribosomes, it is possible that a few of these proteins are coded by the mtDNA and translated by mitechondrial ribosomes. Such a hypothesis could explain the generation of cytoplasmic petite mutants when growing cells are exposed to high concentrations of erythromyoin and ehloramphenieol (Williamson d a/., 1971). After about 15 generations in such a medium, some ribosomal protein will be completely diluted out, and when the antibiotic is removed the cells will not start their mitochondrial translation and will become

MITOCHONDRIAL

rRNA GENES

211

petite, the degradation of mtDNA being a consequence of this dilution phenomenon. Another possibility is that PI mutation might affect a transfer RNA gene. (b) Mapping oJ the rlm + mitovimndrial genera, by the use of various rim- clones (i) Multiple deletions and mapping The intact form of the wild-type mtDNA is thought to be a 25 ~m long circular molecule (Hollenberg et al., 1970). We have stated that recombination frequency studies made with a number of drug resistance genetic markers (Awaer et al., 1973; Wolf et al., 1973) did not allow us to deduce a circular (or linear) genetic map, since most of the known loci were found to be un|inlred by recombination. By means of the various deletion configurations present in rho- mutants, we attempted to see whether it was possible to establish a unique circular order of the five mitochondrial loci, RI to P ~ , Oi, Om P1 and 16 S rRNA gene. It has been shown that the deletions observed in rho- genomes are, in most cases, very large. Several lml~n~ed loci can be lost together b y these deletions. This situation offered a possibility of ordering distant loci b y comparison of different multisite deletions. To do this, two major prerequisites were to be considered. (1) Two genetic markers physically close to each other should have a high frequency of being lost together or being maintained together during various deletion events. This seems to happen in rho- mutations; the genetically ]~n~ed loci RrR n - R m (Netter et al., 1974) are lost or maintained together in most rho- clones, the dissociation between them by mutagenesis is a rather infrequent event (Deutsch et al., 1974). (2) Ideally, each deletion should represent the loss of a single continuous segment and the surviving sequence should remain unchanged. This seems to be true in many t h e - mutants so far examined (R~ to R ~ region) since the rho- DNA of a given genotype can be recognized by their genotype-speeific properties of sequences, as revealed by hybridization experiments and fine denaturation studies (Fukuhara et al., 1974; Lazowska e~ al., 1974; Michel et al., 1974). However, as shown below, the study of the five marker system revealed that some of the rho- DNA could not arise from the deletion of a single continuous segment. With five markers, there are 12 possible circular permutations, listed in the second row of Table 4. A number of one to five was assigned for each marker. 0 represents the absence of a marker, l~rom each permutation representing a hypothetical original map, we tried to derive all the observed rho- genotypes (third to eighth rows) with a m~n~r-um number of deletions. I t was found that whatever the order chosen, it was impossible to derive all six t h e - genotypes by single continuous deletions. Some mutant genotypes had to be made b y double deletion events. (This assertion is also true when we consider linear maps.) The question of how the multiple deletions are produced may now be considered. After mutagenesis, the cells are plated on agar media and primary clones are exs.m~ned for their genotype. Some 20 cell divisions occur with this stage. The primary clones are generally impure and unstable. Successive subclonings are made and finally stable clones of de~n]te genotype are obtained. For each subcloning, about 20 cell divisions occur. The clones used in this work have been obtained from two to five subclonings. Deletions may appear not only during the mutagenic treatment but also during subclonings by spontaneous events. The pedigrees represented in Figure 4

12345 21345 13245 12435 12354 52341 14523 53412 42351 31245 25134 13524

1 2 3 4 5 6 7 8 9 10 11 12

P, 16 00305 00305 03005 00035 00350 50300 00503 53000 00350 30005 05030 03500

R , Oz 10040 01040 10040 10400 10004 00041 14000 00410 40001 01040 00104 10004

P,R 00045 00045 00045 00405 00054 50040 04500 50400 40050 00045 05004 00504

P, Ov 16 10305 01305 13005 10035 10350 50301 10503 53010 00351 31005 05130 13500

P, O~ 02005 20005 00205 02005 02050 52000 00520 50002 02050 00205 25000 00520

P a t t e r n s of deletions for t h e different r h o - clones

Oz, Ozz 12000 21000 10200 12000 12000 02001 10020 00012 02001 01200 20100 10020

4 3 4 3 2 4 4 2 3 2 4 2

N u m b e r of double deletions

T h e presence of t h e m a r k e r Oz is designated as 1, On as 2, 16 S r R N A gene (16) as 3, 23 S R N A gene (Rz to R m sequence) as 4 a n d Pz as 5. The deletion of t h e corresponding m a r k e r is designated as 0.

Order

Order no.

PossibZe order8

TABLE 4

MITOCHONDRIAL

A

B

rRNA

GENES

213

C

(c)

•

®

(b)

(!) K

rl

I

B

G

,

~

® ~

@

H

L

®

(c)

K

NfAfLL~--,? A

H

N\

,j@or

(d) G

®

~K".... r J G J ~l t - ~

H

(I)

L (?-)

FIG. 2. Models of deleted m t D N A molecules. (a) rho + m t D N A molecule with 14 hypothetie genetic markers; (b) a t h e - m t D N A molecule generated b y a single deletion; (e) a r h o - m t D N A molecule generated by a second deletion non-contiguous to the first one. (d) Two non'2eontiguous deletions ((1) and (2)) occur simultaneously in one molecule of mtDNA, the two conserved D N A sequences are joined end ~o end. () Conservedsequences; ( -) deleted sequences ( ]-[ ) illegitimate recombination.

illustrate clearly the occurrence of such secondary deletions. When we examined the presence of various markers at successive stages of subcloning, we found that some markers that were present in the primary clone could be lost spontaneously at the second, third or later clonings. Pedigree studies suggest the following mechanism. A single continuous deletion appears in a circular molecule of a t h e + cell, then a reunion of two sequences that were not contiguous in the original molecule occurs (~'ig. 2(b)). This type of reunion may be called illegitimate recombination (FranMin, 1971). This molecule is sorted out during cellular division. A similar process may be repeated successively to form

214

G. F A Y E

R

R

Op~16 I O~

ET

AL.

R

R

R

~

Oii./"/~ P

R

011

OI

Fio. 3. Tentative orders of the mitochondrial genetic markers; R, O1, 16 S rRNA gene (16), P and Oxx.These six orders are the circular permutations where the 16 S rRNA gene is contiguous with the P* marker. The four orders that offer the minimal number of multiple deletions are shown in a square. R, R, to Rnx segment; 16, 16 S rRNA gene.

multiple deletions (Fig. 2(c)). Such a mechanism does not change the respective order of markers. B u t a multiple deletion could occur b y another mechanism. Two non-contiguous deletions appear simultaneously in a molecule, the two surviving sequences are joined end to end b y two illegitimate recombinations (Fig. 2(d)). I n such a process the respective order of markers m a y b e changed (Fig. 2(d)2) or not changed (Fig. 2(d)l). We will suppose t h a t this second mechanism is infrequent (arguments in favour of this idea are given below). I n the next part of the Discussion our a t t e m p t to order the five loci will be based on the simplest assumption, that each deletion removes a single segment and such deletions m a y successively occur more than once, the respective order of markers being kept. With five markers there are 12 possible orders on circular maps. Are some orders more probable in consideration of the hybridization data? Since the 16 S r R N A gene seems to be in the vicinity of the PI marker, we can exclude from the 12 possible orders shown in Table 4 the six orders in which P1 and the 16 S r R N A gene are not contiguous. The retained six orders are illustrated in Figure 3. I f we keep in mind t h a t the simplest events m a y be the most likely, we m a y choose the four orders t h a t offer the minimal number of multiple deletions (see Table 4 and Fig. 3) to construct all the r h o - genotypes observed. We can conceive that the molecular structures of some r h o - m t D N A might be complex. As a matter of fact, a yeast cell contains m a n y m t D N A molecules and it is probable t h a t differently deleted molecules could recombine, for it has been shown t h a t r h o - m t D N A can undergo recombination (i~chaelis ~ al., 1973). Another problem to be considered is the repetitive structure of r h o - m t D N A molecules (Faye et al., 1973, Locker et al., 19745), tandem repeats and inverted

MITOCHONDRIAL

rRNA

GENES

215

repeats. It is clear that secondary deletions of tandem repeats would not change the respective order of genetic markers, while secondary deletions of inverted repeats could lead to novel combination of the respective order of markers. :Despite these difficulties, it should be pointed out that the rho- I)NA are not the products of an extensive reshuffling of sequences. There are clearly severe restrictions on the number of possible combinations of various genetic markers. :For example, markers RI, R~ and Rm are maintained or deleted most frequently together (i)eutsch et aL, 1974); the marker Rm has never been dissociated from the 23 S rRNA gene (Faye e~ al., 1974 and this work). The 16 S RNA gene has not been observed in the absence of the P: locus (this work) and the combination of R I to Rm region and O~ region has never been found among some 6000 independent rho- clones analysed so far (unpublished results). I)ata pointing out that the selective retention of genetic markers is accompanied by the retention of specific mtDNA sequences have been given elsewhere (Lazowska et al., 1974; Michel et al., 1974).

(ii) Pedigree analysis Genetic data from the pedigree analysis are of help in deciding which of the four orders is most probable. Figure 4 illustrates that during the successive subclonings the genetic markers conferring the resistance to antibiotics were scored at the cell level, but the presence or absence of the antibiotic sensitive alleles or of the 16 S rRNA gene (silent markers) have only been scored in the last subclones.

R~P~b~

i

I ]I

R:P~ (0:)

F32 R:~ (0~)

R:P:O:

1

--. TIT

RIPI(01)

I "IV

IF541 I o,

RxPr(OI)

I F521

I

F5421

F3413

F3412

R:Pz(0:)

RIPI (0:)

o~P~(R~)

R~P[(0:)

F31111 R~P~(O~OnI6)

OIPI(RIRm)

I J412 OiP~16(RIRmOn)

I

F3111

I

R~Pxion%is)

J41

I

I

F34211

F54151 o~P~16{R:C~)

I F34121 1 R:P~(O~Ont6)

F i e . 4. Pedigrees of some r h o - olones. After mutagenesis t h e cells are spread on plates a n d a p r i m a r y clone is isolated. After a b o u t 20 generations this clone is suboloned to give secondary clones. This operation is repeated five times a n d constitutes a pedigree. A t each stage t h e presence of t h e genetic markers is assessed b y recombination with appropriate testers (see Materials a n d Methods). Markers n o t included in parentheses are those present in a given clone, those which are deleted are p u t in parentheses.

216

G. FAYE E T AL.

Since we have supposed that each deletion removes a single and continuous segment of mtDNA and such deletions may successively occur more than once, by studying the loss of the genetic markers during the successive subclonings from the primary clone, we may choose the orders that will be compatible with this hypothesis. Let us look at the pedigree derived from the F3 primary clone in which RI, Oi and Px markers were retained (Fig. 4). For a marker that has not been checked at every step of the pedigree, the presence of this marker in the last subclone demonstrates its presence in the first clone. On the contrary, when a m a r k e r is absent in the last subeloning, it is not certain at which subcloning this marker was lost. However, the absence of a marker in all the subclones tested suggests that the primary clone had probably lost this marker. We will suppose that On marker is lost in F3, because none of the subelones of F3 studied possesses the Oxi marker. On the other hand, during successive subclonings when Oz is lost, it appears that the 16 S rRNA gone is lost in the final clones. (This is ~rue with subclonmgs: F3 --> F31Ill, F 3 - > F 3 2 1 , F34-->F3421I and F341->F34121.) On the other hand, when the 16 S rRNA gone is kept, O~ is kept (this is true with F341 -> F34131). A similar situation is found in the pedigree J4 -> J412. So we will suppose that Oi and the 16 S rRNA gone are simultaneously lost or kept; these two genes were probably adjacent in the genomes of the primary clones ]~3 or 34. This interpretation may disfavour the map model number 8 (Fig. 3), which needs two non-contiguous deletions to lose 0i and the 16 S rRNA gone. Furthermore, when the 16 S rRNA gone is kept, the PI marker is kept. A more systematic pedigree analysis could be an effective approach to mapping the mitochondrial genes. A few examples are given; from the primary clones of the rho- mutants which possess a single marker: P1 for the clones A2 or A3, Oi for the clones C1, C2 or D4 and O~z for K3, K4 or Y4, it appears that a single clone, A211, has kept the 16 S rRNA gone after subcloning. This corroborates the fact that the 16 S rRNA gone lies in the vicinity of the genetic marker PI. We are grateful for the excellent technical assistance of Mrs M. O. Moss@and B. Duchaussoy. We also thank Dr J. Trowsdale for his critical reading of the manuscript. This work received financial support from Comm~esariat ~ l'Energie Atomiquo and Action Th~matique Programra~e, No. 4304 (Diff~ronciation cellulairo). REFERENCES Aloni, Y. & Attardi, G. (1972). J . Mol. Biol. 70, 363-373. Attardi, G. & Amaldi, F. (1970). A n n u . Roy. Biochern. 39, 183-226. Avnor, P. R., Coon, D., Dujon, B. & Slonimski, P. P. (1973). Mol. G~rb. Goner. 125, 9-52. Bernardi, G., Faures, M., Piporno, G. & Slonimski, P. P. (1970). J . Mol. Biol. 48, 23-42. Bolotin, M., Coen, D., Doutech, J., Dujon, B., Netter, P., Potrochilo, E. & Slonimski, P. P. (1971). Bull. In.st. Pasteur, 69, 215-239. Brown, D. & Weber, C. (1968). J . Mol. Biol. 34, 661-680. Casoy, J. W., Hsu, H. J., Rabinowitz, M., Getz, G. S. & Fukuhara, H. (1974a). J . Mol. Biol. 88, 717-733. Casoy, J. W., Hsu, H. J., Gotz, G. S., Rabinowitz, M. & Fukmhara, H. (1974b). J . Mol. Biol. 88, 735-747. Coon, D., Deutsch, J., Netter, P., Potrochilo, E. & Slonimski, P. P. (1970), In Gontrol of Organelle Development (Miller, P. L., ed.), Syrup. See. Exp. Biol. vol. 24, pp. 449-496. Cambridge University Press, London. Doutsch, J., Dujon, B., Netter, P., Petrochilo, E., Slonimski, P. P., Bolotin-Fukuhara, M. Coon, D. (1974}. Gcnvtics, 76, 195-219.

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