Proc. Nati. Acad. Sci. USA Vol. 73, No. 12, pp. 4608-4612, December 1976 Genetics

Modified recombination and transmission of mitochondrial genetic markers in rho minus mutants of Saccharomyces cerevisiae (yeast/petite colony/deletion mapping)

M. BOLOTIN-FUKUHARA* AND H. FUKUHARAt * Laboratoire de Biologie Generale, Universite Paris-Sud, Centre d'Orsay, Orsay, 91405, France; and t Fondation Curie, Institut du Radium, Section de Biologie, Orsay, 91405, France

Communicated by Hewson Swift, July 26, 1976

ABSTRACT A large number of primary petite (rho-) clones were isolated after ethidium bromide mutagenesis of various grande (rho+) strains of S. cerevisiae that contained the mitochondrial genetic markers, CR, ER, OIR (or OIR), and pR. From the frequency of coretention of markers in the petites, we have deduced a probable circular order of the markers in the grande mitochondrial genome. From these primary clones several series of pure and stable petite clones were obtained and analyzed genetically. (a) In general, the w allele is retained or lost together with the region carrying both CR and ER markers. (b)The petites that have retained only the- CR marker fall into two classes: some have kept the w allele of the grande strain they issued from; others exhibit a new w expression. (c) The proportion of diploid petites in petite X grande crosses is independent of the presence of.the w allele. (d) In most cases, the coordinated transmission of markers observed so far in all grande X grande nonpolar crosses does not exist anymore in petites.

of grande X petite cross (6). In parallel to standard crosses, all petites were checked for clonal purity by the replica-cross technique, and used only if the purity was more than 80%. Ethidium Bromide (EtdBr) Mutagenesis. Nongrowing grande strains were mutagenized essentially according to Deutsch et al. (9). The primary petite clones were replated and secondary clones isolated. The genotype test and subcloning were repeated until stable and pure clones were obtained (see ref. 10).

RESULTS Distribution of Genetic Markers in Primary Petite Clones after EtdBr Mutagenesis. Grande strains carrying multiple mitochondrial markers were mutagenized with EtdBr. Many independent petite isolates (randomly collected primary clones) were then tested for the presence of each genetic marker. Table I shows results of such experiments, using two grande strains, CR ER 1R pR? and two grande strains, CR ER 01R pR As summarized in the lower part of Table 1, all of the five markers could be rescued in petites with approximately the same frequency, i.e., there is no evidence for a selective loss of any specific markers. Among the 15 possible combinations of four markers (CR, ER, OIR or OIIR, and PR), some were very frequently found while others were very rare or absent (upper part, Table 1). The frequencies of association of markers in pairs are shown in Table 2. CR and ER were almost always found together, suggesting that these two markers are positioned very close to each other. This confirms the eArlier results of Deutsch et al. (9) and of Molloy et al. (11). All other pairs of markers were quite often dissociated from each other. The OilR marker was practically always found dissociated from the CR-ER block (see legends of Tables 1 and 2 for coretention in primary clones). This fact suggests that the distance between the CR-ER block and the O11R marker is greater than the distance between any other pair of markers. With the aim of ordering the four regions, C-E, P, O1, and OIn in a circular map, we examined the frequency of association between OI and OiI. Since OIR and OiR markers are not easily differentiated in the same grande strain because they have the same phenotype, we looked for the presence of the Oils allele in the O11-containing petites, on the one hand, and the presence of the OlS allele in the OiiR-containing petites, on the other (see ref. 7 for the technique of detection of Os alleles). Series of purified stable petites were used for this purpose. We found that (i) out of eleven OIR pR (COEO) petites examined, nine contained the Oi1s allele; (if) out of four 0IIR pR (COE0) petites, none contained the Ois allele; and (iii) out of eight OIR (COEOPO) petites, three contained the OiIS allele. Therefore, the most likely gene order is that which places OIj

The genetic properties of mitochondrial DNA in wild-type yeast (respiratory-sufficient, "grande") have been extensively studied and some rules governing exchange and segregation of mitochondrial genes have been described (for review see ref. 1). This paper describes the genetic properties of modified mitochondrial DNA in the respiratory deficient cytoplasmic petite mutants (rho-). We have isolated a large number of rho- clones with combinations of markers covering various parts of the grande genome, in an attempt'(i) to order the genetic markers on the mitochondrial DNA by means of deletion mapping, since deletions in petites can be "so large (2, 3) that two very distant markers that are not linked in recombination can be eliminated together 44, 5), and (ii) to detect modifications in the organization of sequences in petites by study of gene transmission and

recombination. We show that the combinations of various genetic markers retained in petites is confined to a limited pattern suggesting a circular arrangement of genes in mitochondrial DNA.- The genetic behavior of these genes is greatly modified in petites, as revealed by uncoordinated transmission of genes and by preferential loss of some markers with respect to others. MATERIALS AND METHODS Strains. Five grande strains were used for petite isolation; their genotypes are detailed in the legends of Tables 1 and 4. Media and Crosses. Normal and drug-containing agar media have been described (6-8). Replica-cross technique used to detect mitochondrial markers in petites has been detailed by Deutsch et al. (9). Standard-cross technique: when not specified otherwise, the results presented here are those implying the "quantitative random analysis" of the diploid grande progeny Abbreviation: EtdBr, ethidium bromide.

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Proc. Natl. Acad. Sci. USA 73 (1976)

Genetics: Bolotin-Fukuhara and Fukuhara Table 1. Marker retention in primary petite clones after EtdBr mutagenesis rho+ CRERORPR

Parental strains Mutation frequency

CRERORpR CREROR CRERPR CRORpR ERORpR CRER CROR CRPR EROR ERPR ORPR CR ER

OR

PR No marker Total

CR ER

OIR

01IR PR

rho+ CREROIRPR

MH41-7B TR3-15A MH32-12D FF1301-6B

Table 2. Frequency (%) of association of markers in petites Pair of markers

C-E 20%

62%

26%

Retained markers Number of clones scored 6* 166* 55* 5* 105 98 21 117 39 0 1* 2* 0 1* 4* 268 37 42 1* 7* 2* 1* 0 0 0 5 2 0 2* 0 67 43 68 9 3 2 2 7 0 134 167 98 141 89 104 706 1348 494 1495 1856 1118 % of clones carrying marker 27.6 12.1 31.4 26.6 12.7 31.1 13.1 39.0 16.9 _ 22.1 11.6 34.1

81%

C-01 CC-P

E-01

14* 18* 91 1* 4* 122 0 8* 0 8* 71 11 3 112

-121

595 1279 20.7 20.3 17.2 32.7

Four multimarker strains were used for EtdBr mutagenesis. C, E, 01, 0II, and P designate, respectively, the mitochondrial genetic loci conferring resistance or sensitivity to chloramphenicol, erythromycin, oligomycin (two loci), and paromomycin. The specific mutations used as markers for each locus are: MH41-7B, w+CR321ER514OR145(0I IS)PR454 (nuclear genotype a ade2hisL); TR315A, W-CR32iE2210IR(OIIS)PR454 (a try2his1); MH32-12D, c-CR321ER221OR144(OIS)PR454 (a ade2hisl); and FF 1301-6B, w+CR321ER5140R144(0IS)PR454 (a hisjura3). Superscripts R, S, and 0 mean resistance, sensitivity, and deletion, respectively. * These primary clones failed, when subcloned, to maintain the indicated genotype; in all other cases, the indicated genotypes were maintained after successive subclonings of the primary clones (several of each type were examined).

between OI and P, as shown in the following circular scheme:

P-(C-E)-0- OII-P The orientation of the C-E block cannot be decided from the mutagenesis data. We favor, however, the order suggested in parentheses in the scheme because (i) two stable petites havifg the configuration EROIR (COOIIOPO) have actually been isolated and (ii) our previous study (12) has shown that some petites of type CR (E0OI0OIIOPO) share some transfer RNA genes in common with the petites of type pR (COOIOII0). Behavior of Genetic Markers in Petite X Grande Crosses. Many of the primary clones isolated after EtdBr mutagenesis were unstable and represented mixed cells of varied genotypes. They had to be purified by subcloning before any genetic and molecular characterization of the petite genome was made. Among secondary and tertiary subclones, we have isolated pure

4609

Mutagenized ande

rho+

CRERO1RpR

OIIP

CREROuIRPR FF13016B

MH417B

TR315A

MH3212D

97.5 51.3

90.1 39.8 21.0 40.6 22.2 21.5 -

95.7

87.5

2.8* 19.8

19.4

1.7* 20.3

10.9

14.3

16.4

40.1 51.6

E-1-

E-P 0O-P

rho+

39.4 40.9 -

5.2*

8.1*

For a pair of markers, XR and yR, the frequency of the pair among petites is expressed as the ratio: (XRYR)/(XRYO + XOYR + XRYR). Values are calculated from the data of Table 1. * These apparent associations were all lost through further subcloning.

and stable (2) cell lines. In this way we have probably introduced some bias in the population of petite clones accessible to study, but, nonetheless, we consider these two factors as fundamental in order to work with a homogeneous DNA population (10). Our basic approach was to compare series of petites carrying the same set of genetic markers (i.e., representing the same regions of the grande genome); we thus expected to detect differences of internal organization of a given region leading to a modified behavior of the genetic markers. Since recombination and transmission of the C-E region in grande X grande crosses are controlled by the determinant w (13), all petites were crossed to both w+ and w- tester grande strains in order to determine if the properties of w were still present. Recombination Patterns for Multimarker Petites (rhoCREROR and rho- CRERPR). Representative results with the rho- clones carrying CRER segment are listed in Table 3 and allow us to point out the following traits: (a) The w allele from the original grande strain is maintained. All petites give one polar and one nonpolar pattern when crossed with an w+/Wcouple of rho+ tester strains, in the same way as the grande from which they are issued. This allows us to classify them as + or Wo rho- clones, as is done routinely with grande strains (13). The genotypic presence of a given w allele has been verified for four w+ CREl petites through meiosis after crosses with appropriate tester strains (data not shown). Therefore, it appears that a nonmodified w allele is present in those petites that have maintained the rgion C-E, regardless of other markers. (b) The general features of mitochondrial recombination are maintained. No striking changes in the frequency of recombination between C and E markers occur. Note also that the proportion of rho- among the diploid progeny is not correlated to w (data not shown). (c) Uncoordinated transmission of markers is observed in some petites. It is now well established that transmission values of various markers in the grande strains are statistically the same in nonpolar crosses (o- X o-; w+ X w+). This was defined as "coordinated output" by Dujon et al. (14). As shown in Table 3, such coordination is often lost in these multimarker petites. Significant differences in transmission values are observed for different markers. This differential transmission is usually seen for the third marker (OR or PR) relative to the CR-ER group, which behaves as a block. We examined whether the differential transmission could be correlated with a differential loss of genes after EtdBr mutagenesis

Genetics: Bolotin-Fukuhara and Fukuhara

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Pro'c. Natl. Acad. Sci. USA 73 (1976)

Table 3. Analysis of transmission and recombination in rho- CREROR and rho- CRERPR

Tested strain

Allele of rho+ tester

MH41-7B (rho+,w+)

-

W

+

MH41-7B/M534

+

MH41-7B/M422

-

MH41-7B/R12

-

+ +

MH41-7B/AB83

+

TR3-15A (rho+,w)

+

TR3-15A/K614

-

TR3-15A/K711

+

+

CR/total (%)

ER/total

98.4 55.7 90.1 62.7 84.7 24.2 55.8 14.4 72.5 17.2 61.1 17.9 63.9 7.8 55.9 3.8

78.2 62.4 63.5 65.0 39.6 32.9 33.9 15.4 43.1 24.7 57.7 58.2 61.9 53.4 54.2 43.4

(%)

OR/total (%)

PR/total (%)

74.2 58.3 33.4 16.1 49.6 43.7

57.4 56.1

61.9 76.9 30.0 41.5 51.2 56.7

33.4 27.8 3.5 6.4 58.7 78.0

-

Polarity of recombination CRES/CSER

Total recombinant (%)

61 0.3 78 0.64 123.3 0.31 6.2 0.8 62 0.3 2.1 0.1 1.5 0.01 1.4 0.003

20.5 11.8 26.7 10.6 45.9 16.4 30.3 8.5 30.4 12.7 9.4 49.1 10.7 46.5 10.1 39.5

All petites are derived from MH41-7B and TR3-15A, whose genotypes are given in the legend of Table 1. Tester strains were of the following genotype: IL125-1OC (aura1 w- rho+), DP1-lB (a hisl trp1 W+ rho+), IL166-2D/1 (a ural w+ rho+). A total of 11 petites were examined; those which are not listed gave results very similar to the examples shown. Values were obtained by quantitative random analysis; in each cross, an average of more than 600 colonies were counted. Total recombinants are the sum of CRES and CSER diploid colonies (in percent).

(Fig. 1). The clone MH41-7B/M534 transmits more of the C-E block than the OR marker, and the C-E block is more resistant to EtdBr than the OR marker. The situation is the inverse in the clone MH41-7B/M422, which transmits more of the OR marker than the C-E block. The clone TR3-15A/K711 shows no differential transmission and no differential loss of genes. Transmission Patterns for Petite Clones with Only One Marker (rho pR, rho OIR, rho OIIR, rho ER, rho CR). Knowing the results of the previous section, we looked at less complex petites and compared them with the three marker

1.5 C

F-OR 1.0

0.5

HOURS

FIG. 1. Differential loss of CRER markers and OR markers in CREROR petite clones during EtdBr mutagenesis. The three petite clones examined (all of them CREROR) were MH41-7B/M534 (0), MH41-7B/M422 (+), and TR3-15A/K711 (0). Abscissa: duration of EtdBr treatment (20,ug of EtdBr/ml of complete glucose liquid medium, 0.05 M Tris-maleate buffer, pH 6.5, 280). Ordinate: the ratio (2 CR)/(l OR) = (CROR petites + CRO0 petites)/(CROR petites + COOR petites). CR and ER markers behaved without disjunction; therefore CR also indicates the presence of ER marker in this experiment.

strains. Table 4 shows the properties of the petites that retained only one of the five available markers. Transmission of ER, oR, and pR markers is independent of W (Table 4, a, b, c, and d). The transmission values obtained are similar whatever the w allele of the partner. The slight variations observed are probably not significant since the grande strains are not isonuclear. This result is consistent with the location of W near the C region, as determined by recombination in grande X grande crosses (13, 15), and suggests that w had been deleted together with the CR marker in these petites. However, among 17 petites examined, two strains, MH41-7B/M92 and MH32-12D/K385, are exceptional: they distinguish between W+ and w- alleles in the grande partners. The number of diploid petites produced in all these crosses is independent of the w allele of the grande tester strain. There is no apparent correlation between the transmission of the gene and the production of diploid petites for a given rho- clone. However, since we have examined here only the transmission of markers into the diploid grandes, we ignore the frequency with which the marker is to be found in the diploid petites. Such analysis is now possible by the triploid cross technique (16). Petite clones with the same mitochondrial genotype and derived from the same grande can show great variation in transmission value. Some CR petite clones have a new w expression (Table 4, e). Two classes of CR petites appear: some rho- (D21H141, F21A15) recognize the w allele of the partner and give results comparable to the grande IL8-8C. The second class (D41H11, F21B13) behaves quite differently: although able to recognize the w allele of the partner, the polarity of transmission was the inverse. Taking advantage of the fact that all CR petite clones retained a spiramycin locus (unpublished data) that is very closely linked to the CR locus (15), we have analyzed polarity of recombination. The results of such studies confirmed the existence of inverse transmission and also an inversion of the polarity of recombination between these two loci. It has been shown (17) that there are two classes of CR petites,

Genetics: Bolotin-Fukuhara and Fukuhara

Proc. Natl. Acad. Sci. USA 73 (1976)

4611

Table 4. Transmission value of the unique marker retained in various sets of petite clones Crosses with w + strain

Crosses with w - strain

Petite tested (a) PR petites MH41-7B (w+) MH41-7B/P11 MH41-7B/P21 MH32-12D (w-) MH32-12D/H241 MH32-12D/L434 (b) ER petites IL8-8C (w +) IL8-8C/R53H231 IL8-8C/D21H121 (c) OIR petites MH41-7B (w+) MH41-7B/M92 MH41-7B/E91 (d) OIIR petites MH32-12D (& ) MH32-12D/K416 MH32-12D/K385 (e) CR petites IL8-8C (w+) IL8-8C/D41H11

IL8-8C/F21B13 IL8-8C/D21H141 IL8-8C/F21A15 TR3-15A (w-) TR3-15A/A115H112

Colonies tested

Transmission (%)

rho(%)

Colonies tested

38.8 25.2

298 1160 1400 262 885 787

56.1 28.3 20.8 33.0 98.1 82.4

33.3 53.6 56.0 45.8

271 709 1188 191 709 338

81.8 69.5 9.1

2.7 1.5

362 670 744

64.2 56.1 8.2

1.5 1.2

583 968 604

74.2 48.9 52.8

38.4 51.5

298 985 1218

58.3 3.3 27.8

40.6 38.2

271 1035 594

45.1 30.3 32.0

33.3 18.6

262 996 913

53.4 28.9 5.0

23.4 11.3

191 650 808

97.8 0.9 2.7 74.9 91.7

2.4 9.8 2.0 22.9

61.1 74.5

8.3

362 1184 1412 1028 1662 530 1789

61.4 83.6 80.3 49.7 71.8 17.9 12.6

Transmission (%)

rho-

57.4 50.9 37.8 37.0 96.1 88.6

27.2 41.7

(%)

3.3 3.9 1.8 4.9

583 919 823 1086 939 273 481

9.6

Mitochondrial genotypes of the parental grandes MH41-7B, TR3-15A, and MH32-12D have been described in the legend of Table 1; an additional series derived from IL8-8C (a his, trpi) w+ CR32jER5I4(0IS)(OIS)(PS). Data were obtained by quantitative random analysis. Tester strains (all drug-sensitive) were EL125-1OC (a ura: w- rho+) and DPi-lB (a his, trpL w+ rho+) for crosses involving MH41-7B, MH3212D, and their derivatives; IL125-lOC and IL166-2D/1 (a ura, w+ rho+) for crosses with TR3-15A and derivatives; IL125-2D (a ural w- rho+) and IL46-11C (a ura1 w+ rho+) for crosses involving IL8-8C and derivatives. A total of 22 strains were examined; results of only 15 strains are presented to avoid repetition of similar results. one capable of recombining with a Con grande strain (Cn is a new allelic form of that is unable to distinguish between w+ and w- alleles), another not. When we crossed the CR petites with a normal expression of with an WnCS grande strain, recombination occurred, whereas the CR petites with an inversed polarity did not recombine. When analyzed for genotype, a strain like F21A15, classified phenotypically as w+, indeed transmits its w+ allele to the progeny; in contrast, in a strain like F21B13, neither the modified allele nor the normal allele reappears in the progeny. w

w

example was given for the CRERPR petites. The loss appeared to begin almost always from the C-E region, thus accumulating new petites carrying only the pR marker (Table 5). The gene order proposed in Results has been deduced from Table 5. Selective loss of markers from petites CRERPR

w

w

Number of clones retaining

w

DISCUSSION Frequency of Marker Retention after EtdBr Mutagenesis. By petite mutation, any one of the mitochondrial markers could be rescued in primary petite clones with approximately the same frequency. This conclusion is different from that of Molloy et al. (11), who found that different regions of the genome may be preferentially retained in different strains. They compared the frequency of ER (00) with that of OR (EO) and not the frequency of all ER markers with that of all OR markers in the total petite population (regardless of other unselected

markers). In contrast to the grandes, unequal losses of markers were clearly observed in some multimarker petites. The most striking

Petite strains examined

CRERpR

pR

No CRER CR ER marker

111 3 0 176 MH41-7B/R12 321 0 54 0 MH41-7B/AB83 0 156 17 0 MH41-7B/AC21 0 35 0 MH32-12D/J4762 121 250 30 0 0 TR3-15A/F218 189 43 0 0 TR3-15A/F5262 210 37 0 0 TR3-15A/F857 Total: 1971 1358 3 392 0 (68.9) (19.9) (0.1) (0) (%)

0 0 0 0 0 0 0 0

36 20 18 19 42 63 20 218

(0) (11.1)

Seven petites containing CR, ER, and pR markers (deleted for and OI, regions) were examined for spontaneous and EtdBr induced loss of markers. Data were assembled from several. experiments. Markers that are not indicated mean deletion.

0O

4612

Genetics: Bolotin-Fukuhara and Fukuhara

the mutagenesis data only. All marker combinations found in stable pure petites fit with the suggested order. Note, however, an exception to this: some petites of OQRpR (OiOC0E0) type do exist, and this contradicts the map unless such petites should arise by a secondary deletion from the petites of ORO11SpR (COEO) type. Indeed, it is now known that secondary or multiple deletions can occur rather frequently in petites (5, 12, 18). The most meaningful result in support of the proposed gene order is the high frequency of retention of OiIs allele in the petites carrying both OiR and pR markers (and deleted for C-E region). Slonimski and Tzagoloff (19) have obtained a similar gene order by a different approach. Modification of Recombination Pattern Due to rhoMutation. Loss or retention of w. All the petites retaining the C-E region also retained a phenotypic expression of w; in contrast, the ER (CO) petite clones failed to distinguish phenotypically between the two allelic forms of w (see Results) and to transmit the original w allele (the one present in the grande from which they issued) (data not shown). Their behavior suggested a deletion of the c region closely linked to the C marker. Results obtained with the CR petites confirm this interpretation: some petites retain the phenotypic expression and the genotype of the original w allele (w+ or w-); others exhibit a new c expression that could be due to a modification of structure close to c. In the oR and pR petites, as in the grandes, these markers are not affected by the w allele. However, two petites (MH417B/M92, MH32-12D/K385) appear to be an exception to the rule. A physical linkage in these petites between oR markers and w or a new sequence with properties similar to co may be postulated. Relationship between physical and genetic properties. In-

dependent of the w effects, petite clones can be very different from each other in the transmission of genetic markers. Several explanations can be put forward. We know that petites correspond to deletions and repetitions of the maintained sequences (2) and that the kinetic complexity of the mitochondrial DNA of these clones varies greatly (3). Any kind of sequence repetition would vary the input of a marker in a cross and thus modify the output, i.e., the transmission value. Furthermore, the "transmission" of a marker in petites can be detected only through its integration into the grande DNA molecule; this integration may depend on the distance of the marker to the deletion point. Other modifications, such as the presence of tandem repeats or inverted repeats (20), or any internal repetition might also interfere with genetic properties. Among these possibilities, the effects of the sequence repetition on transmission seemed amenable to an experimental test. We found a differential loss of the group CRER and the OR marker during EtdBr mutagenesis of CREROR petites (see Results): the higher the transmission of a marker is, the more difficult it is to lose that marker. Such results could be explained if we assume that one of these markers was repeated more than the other. Although this explanation appears satisfactory for all CREROR petites examined, the situation is quite different for CRERPR petites. As shown in Table 5, the loss of the CRER region is almost 100 times more frequent than that of the pR marker, even when the transmission of CRER is higher than pR (compare Tables 5 and 3). It is clear that in petites there exist regions that are lost preferentially, while such is not the case for grandes. The hypothesis of differential internal repetition of sequences thus remains open.

Proc. Natl. Acad. Sci. USA 73 (1976)

Production of rho- Diploids in Petite X Grande Crosses. Perlman and Birky (21) proposed that a petite in which the distance between two maintained markers is identical to that in the grande would be neutral or weakly suppressive, while a petite in which this distance is reduced (due to internal deletions) would be highly suppressive. This model does not seem to explain our results: petites like MH41-7B/R12 or MH417B/AB83 (both containing all examined tRNA genes between C and P, and having a highly complex mitochondrial DNA) and a petite like MH41-7B/AC219 [known to be deleted for many of the tRNA genes (12)] all give approximately the same proportion of diploid petites (about 40%). We thank Drs. M. Rabinowitz and H. Swift who gave us facilities

for part of this work during a stay at the University of Chicago and Drs. J. C. Mounolou and B. Stevens for critical reading of the manuscript. Supported in part by NSF Grant GB 41555. 1. Gilham, N. (1974) Annu. Rev. Genet. 8, 347-391. 2. Faye, G., Fukuhara, H., Grandchamp, C., Lazowska, J., Michel, F., Casey, J., Getz, G. S., Locker, J., Rabinowitz, M., BolotinFukuhara, M., Coen,. D., Deutsch, J., Dujon, B., Netter, P. & Slonimski, P. P. (1973) Biochimie 55, 779-792. 3. Michel, F., Lazowska, J., Faye, G., Fukuhara, H. & Slonimski, P. P. (1974) J. Mol. Biol. 85,411-431. 4. Faye, G., Kujawa, C. & Fukuhara, H. (1974) J. Mol. Biol. 88, 185-203. 5. Morimoto, R., Lewin, A., Hsu, H., Rabinowitz, M. & Fukuhara, H. (1975) Proc. Natl. Acad. Sci. USA 72,3868-3872. 6. Coen, D., Deutsch, J., Netter, P., Petrochilo, E. & Slonimski, P. P. (1970) in Control of Organelle Development, Soc. Exp. Biol. Symp. ed. Miller, P. L. (Cambridge University Press, London), Vol. 24, pp. 449-496. 7. Avner, P. R., Coen, D., Dujon, B. & Slonimski, P. P. (1973) Mol. Gen. Genet. 125,9-52. 8. Wolf, K., Dujon, B. & Slonimski, P. P. (1973) Mol. Gen. Genet. 125,53-90. 9. Deutsch, J., Dujon, B., Netter, P., Petrochilo, E., Slonimski, P. P., Bolotin-Fukuhara, M. & Coen, D. (1974) Genetics 76, 195219. 10. Fukuhara, H., Faye, G., Lazowska, J., Michel, F., Deutsch, J., Bolotin-Fukuhara, M. & Slonimski, P. P. (1974) Mol. Gen. Genet. 130,215-238. 11. Molloy, P. L., Linnane, A. W. & Lukins, H. B. (1975) J. Bacteriol. 122, 7-18. 12. Fukuhara, H., Bolotin-Fukuhara, M., Hsu, H. & Rabinowitz, M. (1976) Mol. Gen. Genet. 145, 7-17. 13. Bolotin, M., Coen, D., Deutsch, J., Dujon, B., Netter, P., Petrochilo, E. & Slonimski, P. P. (1971) Bull. Inst. Pasteur Paris 69, 215-239. 14. Dujon, B., Slonimski, P. P. & Weill, L. (1974) Genetics 78, 415-437. 15. Netter, P., Petrochilo, E., Slonimski, P. P., Bolotin-Fukuhara, M., Coen, D., Deutsch, J. & Dujon, B. (1974) Genetics 78, 10631100. 16. Michaelis, G., Petrochilo, E. & Slonimski, P. P. (1973) Mol. Gen. Genet. 123, 51-65. 17. Dujon, B., Bolotin-Fukuhara, M., Coen, D., Deutsch, J., Netter, P., Slonimski, P. P. & Weill, L. (1976) Mol. Gen. Genet. 143, 131-165. 18. Faye, G., Kujawa, C., Dujon, B., Bolotin-Fukuhara, M., Wolf, K., Fukuhara, H. & Slonimski, P. P. (1975) J. Mol. Biol. 99, 203-217. 19. Slonimski, P. & Tzagoloff, A. (1976) Eur. J. Biochem. 61, 2741. 20. Locker, J., Rabinowitz, M. & Getz, G. S. (1974) Proc. Natl. Acad. Sci. USA 71, 1366-1370. 21. Perlman, P. S. & Birky, C. W., Jr. (1974) Proc. Natl. Acad. Sci. USA 71, 4612-4616.