* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download paramecium tetraurelia
Survey
Document related concepts
Koinophilia wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Polymorphism (biology) wikipedia , lookup
Gene therapy of the human retina wikipedia , lookup
Epigenetics of diabetes Type 2 wikipedia , lookup
Epigenetics in stem-cell differentiation wikipedia , lookup
Genetic drift wikipedia , lookup
Population genetics wikipedia , lookup
Hardy–Weinberg principle wikipedia , lookup
Quantitative trait locus wikipedia , lookup
Microevolution wikipedia , lookup
Dominance (genetics) wikipedia , lookup
Genomic library wikipedia , lookup
Star Wars: Episode II – Attack of the Clones wikipedia , lookup
Transcript
GENETIC ANALYSIS OF MATING-TYPE DIFFERENTIATION I N PARAMECIUM TETRAURELIA W E S BRYGOO Centre de Ge‘nktiqueMolkculaire du C.N.R.S., 91190 Gif-sur-Yvette,France. Manuscript received April 25, 1977 Revised copy received August 15,1977 ABSTRACT Whereas each of the two c9mplementary mating types, 0 and E, of Paramecium tetraulrelia normally shows cytoplasmic inheritance, an abnormal heredity of mating type was observed in the progeny of crosses between two stocks of different geographical origin of Paramecium tetraurelia (stock 51 and stock 32). The modified pattern of mating-type inheritance was shown to result from the interaction of the two wild-type alleles at the locus mtD (mtD51 and mtD32), leading to a new differentiated state O*, different from the normal 0 and E states observed in both stock 51 and stock 32 cells. The genetic analysis of O* clones showed that the O* phenotype involves both a new heritable cytoplasmic state and possibly a nuclear change which can be transmitted through conjugation and segregates in a Mendelian fashion. All the data can be interpreted if the assumption is made that mating-type determination is achieved only by the commitment or noncommitment t o the expression of mating-type E, and that this commitment may simply reflect the activation o r nonactivation of the locus mtD, under the influence of one or two “cytoplasmic factors” including the product of the gene mtD itself. I N metazoa, different cell types exhibit different functions specific for each tissue. As commonly defined, these terminal differences result from two SUCcessive processes: ( 1) determination (a limitation of devclopmental capacity) which occurs at an early stage of the development of the organism; once established determination is transmitted through cell divisions; (2) differentiation (the expression of the defined capacity). Determixition and differentiation are not restricted to metazoa. In unicellular organisms, too, some functions can be considered as differentiated functions in that they are not expressed throughout the entire cell cycle and occur only under particular physiological conditions, and also in that they correspond to functions for which the cell possesses two (or more) alternative genetic pathways, one of which is selectively expressed. This is the case for differentiation of mating type in some species of Paramecium. Although genetically competent for the expression of both mating types, called “odd” (0)and “even” ( E ) ,each cell is generally determined to express only one mating type. Mating type is expressed only under starvation conditions and its determination is inherited throuch vegetative multiplication. It is determbed at each sexual event (conjugation or autogamy) where the breakdown of the “old” macronucleus and the development of a “new” one from the newly formed diploid zygote nucleus occurs. During these nuclear Genetics 87: 633-653 December, 1977 634 Y. BRYGOO changes, the determination of mating type can be influenced by several factors. For instance, a rise in temperature favors determination towards the E mating type (for review, see SONNEBORN 1975). I n Paramecium tetraurelia, as in several other Paramecium species referred to as “group B” species (SONNEBORN 1947), the mating type is cytoplasmically inherited in crosses, and determination was shown to be predomir,antly under cytoplasmic control. A genetic dissection of the mechanisms underlying the differentiation of mating type has been under way for some time with the help of various mutations affecting determination and/or expression of mating type. So far, only “0-restricted” mutants, i.e., those unable to express mating-type E, have been found. This fact, as well as other physiological data, has led to the idea that the expression of the two mating types does not involve two separate pathways, but that the expression of mating-type E requires additional metabolic steps to those necessary for the expression of mating-type 0 (BUTZEL1955). With the exception of one mutation that seems to alter both the mechanism of determination and the capacity of expressing mating-type E (TAUB 1963), all the mutations studied thus far can be interpreted as blocking one of the steps necessary for the expression of mating-type E, without disturbing the process of determination. While it seems well established that mating-type determination in P. tetraure2ia results from an interaction between the cytoplasm and the developing new macronucleus after conjugation or autogamy, the mechanism of determination remains obscure, as it still does in higher organisms. I n this paper we describe a genetic situation found in P. tetraurelia in which the normal cytoplasmic inheritance of mating type is disturbed. This abnormal heredity was first observed in the progeny of crosses between two stocks of different geographical origin (stock 51 and stock 32). It is shown to result solely from the interaction of the two wild-type alleles at the locus mtD (mtD51 and mtD32) and to lead to a new differentiated state O*, which is different from the normal 0 and E states observed in both stock 51 and stock 32 cells. The genetic analysis of O* clones showed that the O* phenotype involves a new heritable cytoplasmic state and possibly also a nuclear change of unknown type which can be transmitted through conjugation and which segregates along with the mtD51 allele. All the data fit with the hypothesis that determination is achieved only by commitment o r noncommitment to the expression of mating-type E and that this commitment may simply reflect the activation of the locus mtD, under the influence of one or possibly two “cytoplasmic factors” including the product of the mtD gene itself. MATERIALS A N D METHODS Strains and culture conditions Two stocks of Paramecium tetraurelia, previously called Paramecium aurelia syngen 4 (see SONNEBORN’Snew nomenclature 1975), of different geographical origin were used: stock 32 -and stock d4-2, a derivative of stock 51, carrying the gene k in the genetic background of stock 51. For the sake of brevity, these two stocks will be designated st.51 or st.32 or E(51), 0(51), E(32) and O ( 3 2 ) according to their mating type. These two stocks interbreed and display little MATING-TYPE DIFFERENTIATION IN PARAMECIUM 635 or no lethality among the F, progenies, Two nuclear heat-sensitive markers were used in the course of the genetic analysis: t s l l l and ts401 (BEISSONand ROSSIGNOL1969). Two mitochondrial markers, C,R and E,R,(ADOUTTEand BEISSON1972) conferring resistance to chloramphenicol and to erythromycin, respectively, were used in some crosses to distinguish the two exconjugants. The general culture conditions were those described by SONNEBORN (1970). The growth medium was a Scotch grass infusion bacterized the day before utilization with Klebsiella pneumoniae. All the cultures were grown at 27" except when heat sensitivity was tested, in which case the cells were placed at 36". The cells were isolated in slides bearing three depressions. In each of these depressions, one cell can undergo approximately ten fissions (giving about IO3 daughter cells) in 1 ml of medium. Genetic analysis Crosses between cells of diferent mating types. The two mating types, 0 and E, have previously been named VI1 and VIII. Genetic analysis was carried out according to the methods developed and reviewed by SONNEBORN (1970). The two main features of the analysis are: (1) i n a cross A x B the two exconjugants of each pair represent respectively the two reciprocal crosses OB x $ A and $ B x OA; (2) : the F, generation is obtained directly from heterozygous F, clones by autogamy which yields homozygous F, clones. The parental strains were routinely marked by the recessive heat sensitive mutations ts111 or is401 in order to distinguish pairs that had undergone reciprocal fertilization from those that had not. The study of the sexual phenotype of the F, and F, progenies was carried out in two ways. (1) The first method, which is the usual one (SONNENBORN 1970), consisted in growing separately each exconjugant F, clone and each F, clone. Subsequently the mating type of the F, or F, clones was tested 10 fissions after conjugation or autogamy. ( 2 ) The second method, which is global, allows the testing of a larger number of clones. In the F, generation the two exconjugant cells were not separated from each other and were kept in the same depression, forming a synclone. If the two conjugants give rise to F, clones of opposite mating type, pairs will form a t the time of sexual reactivity, that is, when the food is exhausted. Under these conditions, it is impossible to analyze individually the heat sensitivity of the F, clones or to study the F, progeny. A similar method was also used to study the F, segregations. In this case, the autogamous cells issued from the F, clones analyzed by the first method, were kept together and grown to sexual reactivity. If the F, clones issued from the autogamous cells do not have the same mating type, pair formation will be observed. The number of pairs will increase as the ratio of 0 over E clones gets close to one. Regardless of the method used, the mating-type test itself was always carried out in the same way: the population to be tested was divided into three aliquots. One was mixed with reactive cells of mating-type 0, another with reactive cells of mating-type E, and the third served as a control. When reactive cells of complementary mating-type 0 and E are present, agglutination occurs. The populations were classified as 0, E or 0 E according to whether they agglutinated with only E, only 0 or both testers respectively. Populations in which a only few cells of opposite mating type were present were not classified as 0 -k E, but as 0 o r E according to the predominant response. In some experiments, stock d4-109 (see SONNEBORN 1975) was used as a source of sexually reactive cells for mating-type tests because of its very high and long lasting sexual reactivity. This stock was also used as a source of reactive cilia. Crosses between cells of identical mating type. One cross was carried out between two clones of the same mating type. In this case, conjugation was induced by the addition of reactive cilia isolated from cells of the opposite mating type. The preparation of reactive cilia was carried out according to the method of FUKUSHI and HIWATASHI (1970) using MnC1, (2g/l). To increase the efficiency of the method, a dense mixture of the cells to be crossed was used, and the relative proportion of the cells of the t w o strains was adjusted according to their respective sexual reactivity. In the best conditions, 50% of the pairs obtained were interstrain pairs. It must be stressed, however, that less than half of the interstrain pairs formed under these conditions underwent normal conjugation with reciprocal genetic exchanges. + 636 Y. BRYGOO Isohtion of isogenic clones of opposite mating type. Within an 0 or E population, a few cells of the opposite mating type can be observed. These cells constitute mating-type “revertants” resulting from a spontaneous switch in mating-type determination occurring at aut3gamy. These revertants were isolated by splitting the pairs formed in the control depression of the matingtype test; they provide isogenic clones of opposite mating type. Caryonidal and sub-caryonidal analysis. In one experiment the two products of the first postconjugal division (caryonides), then the four products of their next division (sub-caryonides), were isolated. The similarities or differences of the phenotypes among the 4 sub-caryonidal clones of a given F, clone should reveal whether thht.re is a correlation with the process of macronuclear development, since the 2 caryonides inherit two independently developed macronuclei, whereas the two sub-caryonides of a caryonide inherit the same macronucleus. RESULTS I n st.51 as well as in st.32 of P. tetraurelia, it is known that mating type shows cytoplasmic inheritance (SONNEBORN 1975). This is shown by the fact that each F, clone and its F, progeny usually retains the mating type of its cytoplasmic parent. A few exceptions occur which result from a spontaneous switch in matingtype determination a€ter conjugation or autogamy. Exceptions to the rule of cytoplasmic inheritance are also observed in crosses involving certain mutations. Figure 1 illustrates the inheritance of mating type in crosses between wild-type strains, or in crosses between a wild-type strain and one of the mutant strains. The following results deal with a new type of exception in the pattern of mating type inheritance. Occurrence of a new phenotype, O f , in the progeny of crosses between stocks 51 and 32 The F, progeny of crosses between st.51 and st.32 is analogous to the F, [+I [+I (a) Cml [+I c+1 Cml (b) FIGURE 1.-The different patterns of mating-type inheritance in Paramecium tetraurelia. (a) Cross between wild-type strains of complementary (0 and E ) mating type. (b) and (c) Cross between a wild-type strain of mating-type E and a mutant restricted to mating-type 0 (TAUB1963; BYRNE1973). In (b) the mutant strain is cytoplasmically E ; in (c) the mutant strain is cytoplasmically 0. (d) Cross between wild-type strain of mating-type 0 and mutant restricted to mating-type E. This latter type of strain was obtained twice but was almost inviable (BEISSON, personal communication; BRYGOO and KELLER,in preparation). 637 MATING-TYPE DIFFERENTIATION IN PARAMECIUM progeny of the intrastock crosses, i.e., it displays a majority of 0 : E pairs. However, in some of the F, progenies abnormalities in mating-type inheritance appeared which are formally similar to the situation depicted in Figure IC.While the F, progenies of the 0 F, clones were all normal, i.e., gave rise only to 0 F, clones, many 0 clones appeared in the F, generation of some E F, clones. The mating-type test by the global method of these abnormal F, progenies gave an 0 -I- E type of response. Table 1 indicates the frequency of such abnormalities in interstock crosses E(32) X O(51) o r 0 (32) X E(51). The results of intrastock crosses are given for comparison. This phenomenon has already been observed by SONNEBORN (personal communication). Despite the formal analogy between this situation and that of some 0-restricted mutants (Figure I C ) the abnormality is probably not due to a mutation, since it was not observed in the progeny of all pairs, but only in the progeny of a fraction of them. Furthermore it can be seen (Table 1) that the abnormal F, progenies are more frequent (20%) when the E cytoplasmic lines derives from st.32 than when it derives from stock st.51 ( 7 % ) . A number of these 0 F, clones appearing in E cytoplasmic lines of interstock crosses were isolated and shown to remain generally 0 through successive autogamies. These strains will be called O*. O* strains are characterized by a relative instability 04 their mating-type differentiation: each O* cell generally retains a pure 0 phenotype throughout its vegetative multiplication. However, all O* clones are characterized by a relative instability of their 0 phenotype at autogamy (This hollds true for conjugation, as will be shown later) : a relatively high proportion of E cells are present among the ex-autogamous cells derived from O* clones. The frequency of this O+E TABLE 1 Hereditary transmission of mating type in the F, generation following interstock and intrastock crosses Type of Fzprogeny E Crosses O(51) X E ( 3 2 ) $ O(mtDSl/mtD51) x E (mtD32/mtD32)$ ~ 3 2 x) ~ ( 5 1 ) ~ O(mtD32/mtD32) x E(mtDSl/mtDSl),$ ~ 5 1 x) E ( ~ I ) $ O W ) X E(32)$ “nomal-NS”* 80 63,5 93 92,5 100 100 O+E ‘L S 7 )*I 20 30,5 7 7,5 0 0 Number of F, clones analyzed 45 262 46 42 61 24 Frequencies (in ’%) of phenotypes in the F, generation from the E F, clones. * “normal-non-segregating”. t “S” = segregating. $ Crosses involving only the original wild stocks st.51 and st.32. $ Crosses involving various strains homozygous for the allele mtD51 or mtD32 at the m t D locus (see below). In each cross, the mating type in the F, progeny from each F, clone was analyzed by the global method. All the F, progenies presented in this table derive from the E F, clones of 0 : E pairs. In all crosses a minority of non 0 : E F, pairs (0f E : E, E : E , 0 :0 f E and 0 : 0) was found but the analysis of their F, progenies is not reported here. 638 Y. BRYGOO change at autogamy in O* clones was estimated to be about 1:30 from the number of pairs observed under these conditions. This frequency of change was compared to that of wild-type stocks under the same conditions: for 0 strains, the @E change has been observed only in very rare cases (<1 : 50,000), for E strains, the E+O change was estimated to about 1:3,000. In order to ascertain whether the cells expressing mating-type E were stable E , 44 pairs, formed in ex-autogamous populations from O* clones were isolated when it was still possible to separate the two conjugants (split pair method, KIMBALL1939). Each member of the split pairs was then grown and tested f o r mating type after the next autogamy. In 38 cases out of 44,the lines derived from the two split exconjugants both expressed mating-type 0. In only 6 pairs, one of the two potential conjugants was found to express mating-type E, while the other was 0. Therefore it can be concluded that although the great majority of O* cells remain 0 through autogamy, their mating-type determination is relatively unstable, since a notable proportion of cells switch to either stable o r transient E phenotypes. Genetic analysis of O* clones Different O* clones were crossed to st.51 and to st.32. The results obtained in the F, and F, generations are summarized in Tables 2 and 3. TABLE 2 Hereditary transmission of m a h g type in crosses O* x E(32) and O* x E ( 5 1 ) I. Frequencies (in %) of the observed combinations of co-conjugant phenotypes Phenotype of paLs of F, clones O:E O* x E (32)$ O * x E(mtD32/mtD32)$$ O*X E(51)$ O * x E(mtDSl/mtDSI)$$ + (O+E):E E:E 0 : ( 0 E) or 0 : 0 2 2,5 21 15 96 95 18 22 0 0 1 2 2 2,5 60 61 i\'o.,of pairs 58 189 67 113 $, $ as in Table 1. TABLE 3 Hereditary transmission of mating type in crosses O* x E(32) and Of x E ( 5 1 ) II. Frequencies (in %) of the phenotypes in the F, generation from the F, clones Phenotype of the F, clone Phenotype of the F, progeny O*x E(32)S O'X E(mtD32/mtD32)s O * x E(51)$ O * x E(mtDSl/mtD51)$$ 1 1,5 30,5 32 0 0 0 0 E O+E 0 0 O+E +E E 0 O f E E 0 0 0 0 0,5 6,5 6,5 0 0 0 O$ 95 3 92 5 2,5 7,5 48,5 3,5 7,5 50 0 0,5 0 1 1 3,5 0 0 0 E No. of F, clones analyzed 116 378 136 226 All the F, progenies presented in this Table derive from the F, clones of all the pairs described in Table 2. $, see Table 1. MATING-TYPE D I F F E R E N T I A T I O N IN P A R A M E C I U M 639 In the crosses O* X E(32), the mating type did not follow the rule of cytoplasmic inheritance: the pattern of mating-type inheritance was generally similar to that illustrated in Figure Ib. In the Flgeneration both the F, clones of 96% of the pairs express mating type E, regardless of their O* o r E cytoplasmic origin, (Table 2). In the F, generation (Table 3), the majority of the exconjugants (95%) which were E in the F, generation show an 0 4-E type of response, i.e., a “segregation” of mating type. A number of these 0 clones appearing in E cytoplasmic lines were isolated and shown to be O*, i.e., displayed the same unstable 0 phenotype as the parental O* strain. In contrast, in the crosses O* x E(51), the mating type was cytoplasmically inherited in the majority of cases, as seen in Figure la. In the Flgeneration (Table 2), most of the pairs (60 % ) were of the type 0 : E . However, exceptions were frequent, many O* parents producing E or 0 E clones (18% and 21 % respectively). These exceptions can be explained by the fact that O* is unstable and that the frequency of mating-type changes is known to be higher at conjugation than at autogamy. Among the F, progeny, no segregation was observed from the E F, clones: in nearly all pairs, the E F, clone gave rise to normal homogeneous E F, progenies. In F, progenies derived from O* parents, all the 0 F, clones isolated were O*. In other words, in the cross O* x E(51), the o* phenotype shows cytoplasmic inheritance. In conclusion, the O* phenotype behaves like a hereditary character when crossed to either st.32 or st.51 but, surprisingly, its pattern of transmission is different in the two crosses. The O* phenotype seems to segregate as a nuclear determined character in the first case (0*x st.32) but is generally cytoplasmically inherited in the second case (0*X st.51). Furthermore, in both types of crosses, although a predominant pattern of inheritance is observed, a minority of the F, or F, progenies display different features. This fact suggests that the inheritance of O* phenotype cannot be explained solely by either a cytoplasmic genetic difference with stock 51 or by a genic difference with stock 32. Identification of a one gene difference (mtD51/mtD32) between st.52 and st.32 The results obtained in the above crosses, 0’ x E(32) and O* x E(51), suggest the existence of a genetic difference between the strains E(51) and E(32), which is revealed only when they are crossed to O* This distinguishing property of O* clones was used to investigate the genetic basis of the difference between stocks 51 and 32. Segregations were sought in the F, generation of the crosses O(51) X E(32) or O(32) X E(51). As previously pointed out and shown in Table 1, in such crosses E exconjugants can yield two types of F, progenies: either a normal F, progeny called “NS”, or nonsegregating, in which no 0 clone appears, o r an abnormal F, progeny, called “S” for segregating, which yields a noticeable proportion of 0 clones. As a first step of the analysis, 14 clones of mating-type E isolated from two F, progenies of the “NS” type were crossed individually to O* clones. The results of the F, analysis of these 14 crosses are presented in Table 4.We observed two + 640 Y. BRYGOO classes of results indicating two categories among these E clones: clones 1 to 7 gave results similar to those from the cross O* x E(32) (Table 2), i.e., they gave principally E : E pairs of F, clones, whereas clones 8 to 14 gave results like those of the cross O* X E(51) (Table 2) , yielding principally 0 : E pairs of F, clones. These analogies are further supported by the F, progenies obtained from all the pairs of the 14 crosses which were studied in the F, generation. The 82 F, progenies derived from the E F, clones were mostly of the 0 f E type (97,5%) in crosses 1 to 7, and the 61 F, progenies studied in crosses 8 to 14 are mostly of the E type (82%). These features are quite comparable to those obtained in the crosses O* X E(32) and O* x E(51) respectively (Table 3). These two classes observed among the 14 E clones, either E (32)-like or E (51) like, most probably reflect the segregation of a pair of alleles which will be called mtD51 and mtD32. I n order to confirm this interpretation it was necessary to analyze more F, clones from st(51) X st(32) crosses, and the only method which enabled an extensive analysis consisted of taking advantage of the distinctive features of the F, generation of the crosses O* x E(32) and O* X E(51) : 95% of the synclones are E in the first case while 80% of the synclones are 0 fE in the second case. In a first experiment, 84 clones of mating-type E isolated from four F, progenies of the “NS” type were crossed individually to O* clones. (Three of these F, TABLE 4 Genetic analysis of different E clones: crosses to 0’ Phenotypes of F , clones E parent strain no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Types of pairs of E:E 6 3 3 5 8 8 7 1 4 1 1 E:O 0 0 0 0 0 1 0 6 6 4 0 6 4 0 1 2 13 Flclones E:(O+E) 0 1 1 0 1 0 0 0 0 0 0 0 1 3 0 :(OfE) 0:O 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 No. of pairs not analyzed+ 4 6 6 5 1 1 3 3 0 5 3 6 7 3 The figures indicate the number of each type of pair in each cross. The strains no. 5, 6, 7 and 14 came from one F, progeny from O(51) x E ( 3 2 ) and the other one from one F, progeny from E(51) x O(32). +: the “not analyzed” pairs correspond to pairs which failed to undergo genetic exchange o r in which one of the two exconjugants was lost. MATING-TYPE DIFFERENTIATION IN P A R A M E C I U M 2 0. 2 0- 10. IO. 0 0 20 40 60 80 100 64 1 0 FIGURE 2.-The two classes of F, clones from (a) crosses st.51 x st.32 (b) crosses st.51 X clones presumably homozygous mtD32/mtD32. Each F, clone is represented by the result of its cross to 0*,i.e., by the percentage of E synclones observed in each cross. Ordinate: number of clooes. Abscissa: % of E synclones. progenies were isolated in the cytoplasmic line of an E (51) parent, and one was isolated in the cytoplasmic line of an E ( 3 2 ) parent). For each cross, thirty p a h were studied in the F, generation by the global method which allows the determination of the E or 0 4- E mating type of each of the thirty synclones. Figure 2(a) sums up the results of these crosses. Each cross is represented by the frequency of pairs in which the two exconjugants were E ( E synclones) . It can be seen that the 84 crosses fall into t w o groups comprising 38 and 46 crosses, respectively. In the first group, the majority of the 38 crosses produced a frequency of E synclones higher than 70%; in the second group, this frequency was always lower than SO%, and contained between 0 and 30% in the majority of the cases. This 38 : 46 ratio (see Table 5) is equivalent to a 1 : 1 ratio. Therefore among the 84 F, clones studied, 38 can be considered as carrying the mtD32 allele and 46 as carrying the mtD51 allele. This conclusion was confirmed in the following way. One strain which was considered according to the above results to carry the mtD32 allele was crossed to st.51. Among the E F, clones, one new E (mtD32/mtD32) clone was identified by crossing to O*. This new E(mtD32/mtD32) clone was in turn crossed to O(51) leading to isolation of other strains carrying mtD32. This process was repeated eleven times. Altogether 47 new F, clones, isolated at various steps of this isogenization process, were studied. The results of these 47 crosses are given in Figure 2(b). It can be seen that all the clones fall into two distinct classes which correspond to those of Figure 2(a). Furthermore, as in the preceding analysis, the number of clones of each type, 20 and 27, (see Table 5 ) are close to a 1 : 1 ratio. It must be noted however that in the case of Figure 2(b), the difference between the two classes is more clearcut than in Figure 2(a). This is 642 Y. BRYGOO TABLE 5 Segregation of E(mtD32/mtD32) and E(mtDSl/mtDjl) in 9 progenies of the “NS” t y p e from uarious crosses mtD32/mtD32 X mtD5l/mtD51 F, no. 1 2 3 7 3 13 4 5 6 7 8 9 ~~ The figures indicate the number of F, clones. These segregations are deduced from the experimental data shown in Figure 2. The crosses 1, 2, 3 and 4 correspond respectively to three F, progenies from a cross E(32) X O(51) and one F, progeny from a cross E(51) x O(32). The experiments no. 5 to 9 correspond respectively io the F, progenies from the 2nd, 31d, 4th, 6 t h and 7th generations of isogenization. due to the fact that for the 47 crosses of Figure 2(b), each F, clone was analyzed separately for heat-sensitivity markers and mating type so that pairs that had undergone reciprocal genetic exchanges could be identified and were the only ones taken into account. In contrast, the crosses reported in Figure 2(a) were analyzed by the global method, which discloses only the E or E -t- 0 mating type of the F, synclones, regardless of the actual occurrence of reciprocal genetic exchange. The frequency of E 0 synclones was consequently overestim-ated, since in the pairs O* x E(32), the O* exconjugants which failed to undergo genetic exchanges remained 0. I n summary, the two stocks 51 and 32 differ by their allele at the locus mtD. Each allelic form (mtD51 or mtD32) can be identified by using the distinguishing property of Of strains. + Relationships between the mtD locus and the mtA, mtB, mtC loci The relationships between the mtD locus which we have just identified, and three other loci, m t A , mtB and mtC, which contain mutations restricting mating type to 0 (BYRNE1973), were analyzed. As all three mutants were isolated in stock 51, they can be assumed to carry the mtD51 allele. If m t D is not allelic with any of the other three genes, it should be possible to reisolate E ( m t D 5 I J mtD51) clones from the crosses E(32) x O ( m t A ) , E(32) X O ( m t B ) and E ( 3 2 ) x O ( m t C ) . These three crosses were carried out and in all three cases, E(mtD51lmtD51) F, clones were indeed recovered. Gene m t D is therefore not allelic to any of these three genes. The correlation between the segregation of the mtD alleles and the appearance of O* clones If the alleles mtD51 and mtD32 segregate in the “NS” F, progenies from the crosses st.51 X st.32, they can also be expected to segregate in “S” F, progenies. Two questions then arise: (1) does the appearance of O* clones result from the 643 M A T I N G - T Y P E D I F F E R E N T I A T I O N IN P A R A M E C I U M segregation of these alleles ? (2) are all the 0’ clones appearing in the “S” F, progenies capable of “distinguishing” between E (32) and E(51) ? As a first step towards answering these questions, 90 clones of 0 or E mating type were studied. They represented all of the viable clones isolated in the three F, progenies of the “S” type. Of these 90 clones, 38 were of mating type 0 and displayed the characteristic instability of O* strains. These 38 clones were therefore O*. As for the 52 clones of mating type E, when crossed to 0*,they turned out to belong to the two classes described above: 43 behaved like stock 32 and carried the mtD32 allele and 9 behaved like stock 51 and carried the mtD51 allele. Table 6 (columns 1,2 and 3) shows the distribution of the different types of clones among the three F, progenies. Results concerning six other F2progenies for which only a sample of thz E clones isolated were studied, have been included (columns 4-9) in the Table. It can be seen that among the E F, clones of columns 1-3, the respective proportions of mtD32 and mtD51 alleles, 43 : 9, differ significantly from the 1 : 1 ratio observed in the F, progenies of the NS type. However, if the O* clones are assumed to carry the mtD51 allele, then the proportions become 43 (mtD32) : 47 (mtD51) and are therefore quite compatible with a 1 : 1 segregation. To verify this hypothesis, it is necessary ( 1 ) to establish that all the O* clones carry the mtD51 allele and ( 2 ) to explain why some clones having this genotype display mating type E and do not have the O* phenotype. (1) The fact that O* clones do indeed have an mtD5Z/mtD51 genotype was demonstrated as follows. From 20 out of the 38 O* clones previously mentioned, an isogenic “revertant” of mating-type E was isolated (see MATERIAL A N D METHODS) and crossed to an O* clone. The result of each of these 20 crosses was TABLE 6 Segregation of 0 and E phenotypes in 9 F , progenies of the “S’ type from various crosses mtD32/mtD32 x mtD5l/mtD51 Phenotype 15 4 5 9 8 20 16 15 6 10 18 22 32 13 8 5 $2 6 10 7 5 3 1 0 3 2 2 0 Z=9 0 0 0 6 1 5 1 0 1 0 2 5 0 13 E 15 - 7 18 Z=38 21 Z Genotype mtD32/mtD32 of the mtD5l/mtD5l E F, clones ~ ~ _ _ _ _ _ _ _ not analyzed ~ 14 = 52 16 z =43 I __ ~ ~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ The figures indicate the number of F, clones. The crosses no. 1, 2 and 3 correspond respectively to two F, progenies from a cross E(32) X O(32). The experiments no. 4 to 9 correspond respectively to the F, progenies from the 2nd, 5th, Sth, 9 t h , 10th and 11th generation of isogenization. 644 Y. BRYGOO similar to that observed in an O f x E (51) cross (see Table 2). If one considers that the transformation O*+E in 0' clones does not lead to a modification of the allelic form of the gene mtD, and in particular does not lead to a transformation of the mtD32 allele into an mtD51 allele, it can be concluded that the 20 revertants from the 20 O* clones studied all carried the mtD51 allele. (2) As to the reason why some homozygous (mtD51/mtD51) clones are E and not O*, two factors may be considered. First, the known instability of the O* phenotype might account for a few early shifts towards E. Second, the fact that an 0' cell always switches to E when it becomes heterozygous mtD32/mtD51 suggests that the stability of the O* phenotype depends upon some interactions between the two alleles and/or their products. It seems therefore quite conceivable that the onset of the 0" phenotype could be more or less hampered by the product of gene mtD32, which is likely to be present in the heterozygotes mtD32/mtD51 from which the homozygotes O* (mtD51/mtD51) arise. Altogether, the data are consistent with the conclusions (1) that O* clones are mtD51/mtD51 and (2) that the appearance of O* clones is indeed correlated with the segregation of the pair of alleles mtD32/mtD51. As will be shown next, the appearance of O* clones, first observed in the progeny of interstock crosses, depends in fact only upon the interaction of the two alleles in any mtD51/mtD32 heterozygote. This is why the results obtained in various mtD51/mtD51 X mtD32/mtD32 crosses have been included in the data of Tables 1,2,3 in addition to the results obtained from interstock crosses. For this reason and to facilitate the understanding of the results presented below, Table 7 summarizes the results and conclusions concerning the origin and hereditary transmission of the 0' phenotype. Genetic determination of the O* phenotype Since the appearance of the O* phenotype in some F, progenies from crosses E(32) x O(51) or E(51) x O(32) is correlated with the segregation from mtD51/mtD32 heterozygotes, of clones homozygous for mtD51 in E cytoplasm, it remains to be understood why, in the progeny of the majority of pairs from the cross mtD51lmtD51 x mtD32/mtD32, the segregation is not expressed by the appertrance of O* clones. (1) T h e appearance of the O* phenotype results frcm a facultatiue interaction of the mtD51/intD32 alleles. The fact that the appearance of O* clones is reproducible but unpredictable suggests that they are not due to a mutation. The simplest hypothesis is to assume that they are due to the interaction between the two alleles mtD51 and mtD32. This hypothesis is strongly supported by the fact that the characteristics of the crosses st.51 x st.32 persist after elimination, as far as passible, of the other differences (nuclear and cytoplasmic) existing between st.51 and st.32. This elimination was obtained by the isogenization reported above, during which the mtD32 allele was introduced into the st.51 nuclear genetic background. The introduction of the mtD32 allele in st.51 cytoplasmic background was also undertaken at two steps of this isogenization by two M A T I N G - T Y P E D I F F E R E N T I A T I O N IN P A R A M ECIUM 0 Y 0 Y t t I 1 I I I T I I 0 0s. . C O Y Y ki Y 645 646 Y. BRYGOO crosses 0 (mtD32/mtD32) x E (5 1 ) , from which a new mtD32/mtD32 clone was isolated in the E cytoplasmic line of the parent E (5 1 ) . Therefore the appearance, even after the 11 steps of isogenization, of the O* phenotype in the F, progenies of the E (mtD51/mtD32) heterozygotes, supports the conclusion that it is indeed the mtD51/mtD32 heterozygous condition which is necessary for the onset of the O* phenotype. A question arises: when is the ability to yield O* F, clones determined, as soon as the heterozygote is formed or only when the heterozygote undergoes autogamy? I n order to answer this question, a selection of 158 E heterozygotes (mtD51/ mtD32) were grown vegetatively and two successive F, progenies were analyzed. The second F, progeny was made using a sub-clone derived from the clone which gave rise to the initial one. In this experiment, the appearance of O* clones in the F, generation was deduced only from the mating type test performed by the global method. The response 0 E was considered to indicate the presence of O* clones. I n most cases (150/158) the two F, progenies gave the same result: 69 heterozygous clones yielded O* in both F, progenies and 81 heterozygous clones did not yield any O* in either F, progeny. In 8 cases only, the two F, progenies were different: in 7 , 0 * appeared only in the second F, progeny and in 1 case, O* appeared only in the first one. From this experiment it can be concluded that the ability of a heterozygote to yield or not to yield O* clones is an intrinsic property of this heterozygote which is inherited through vegetative multiplication, although in a few cases this property can be modified. Furthermore, the determination of heterozygotes for the production of O* clones was shown to occur before the first division of these heterozygotes. A LLsubcaryonidal” analysis (see MATERIAL AND METHODS) of 92 E mtD51JmtD32 ex-conjugants was carried out. Table 8 indicates the results obtained. It can be seen that in the great majority of the cases, the four sub-caryonides were identical. This fact demonstrates that determination generally occurs before the first + TABLE 8 Correlation between the phenotypes of the 4 F , sub-caryonidesfromthe F , clones of a cross E(mtD32/mtD32) x O(mtD,,/mtD,,) ~ No. of E Phenotypes of the 4 sub-caryonides Caryonide Caryonide exconjugants 36 43 9 2 2 38 Total + indicates the presence of 0’ clones in the F, progeny and -indicates 130 their absence. + F, generation which failed to undergo genetic exchange or in which one of the subcaryonides was lost. MATING-TYPE DIFFERElYTIATION I N PARAMECIUM 64 7 division. The occurrence of a small number of cases in which differences between caryonides appeared (Table 8, line 3) suggests that the determination did not occur before conjugation and is probably based on macronuclear differentiation. The frequent identity between the caryonides indicates that the tn70 new macronuclei which develop in each exconjugant tend to be similarly determined and therefore that a “cytoplasmic factor” influencing macronuclear differentiation might be involved. (2) Factors influencing the appearance of the O* phenotype. O* clones appear in the progeny of some mtD51/mtD32 heterozygotes of E mating type. Such heterozygotes can be formed under different conditions: (a) in an ex-E(mtD51/ mtD51) cytoplasm or in an ex-E(mtD32/mtD32) cytoplasm; (b) in an ex-E (mtD32/mtD32) cytoplasm the male gametic nucleus carrying the allele mtD51 may come from either an 0 (mtD5l/mtD51) or an O* (mtD5l/mtD51) partner. It could be shown that although all three situations, 0 E(mtD5l/mtD51) X 8 0 (mtD32/mtD32), 0 E (mtD32/mtD32) x 8 0 (mtD5l/mtD51) and 0 E(mtD32/mtD32) x 8 O*(mtDSl/mtD51) can generate O* F, clones, the frequency of heterozygotes “detennined” for O* production is different. First, as already pointed out, mtD51/mtD32 heterozygotes are more frequently determined towards the production of O* clone when they develop in the cytoplasm of a n E(mtD32/mtD32) cell than when they develop in the cytoplasm of a n E(mtDSl/mtD51) cell (Table 1 and Table 7). This fact was confirmed as follows. A cross between clones E(mtDSl/mtD51) and E (mtD32/mtD32) was induced by the addition of cilia isolated from cells expressing mating type 0. Identification of reciprocal genetic exchange was made possible by the presence of the recessive nuclear markers (t.7401 and t s l l l ) in the two parental strains. An erythromycin-resistant marker was used to identify the cytoplasmic origin of each heterozygous clone. Of 71 E(mtD32/mtD51) exconjugants of mtD32/ mtD32 origin, 27 yielded O* clones, while of 67 E heterozygotes of mtD5l/mtD51 origin only two yielded O* clones. Secondly, the frequency of heterozygotes yielding O* clones in the F, generation is much higher (96%) in crosses O* (mtD5l/mtD51) X E(mtD32/mtD32) (Table 3) than in crosses 0 (ntDSl/mtD51) X E (mtD32/mtD32) which yield only 30% of “S” F, progenies. In order to analyze this difference more precisely, two isogenic O* and 0 clones were crossed to the same E(mtD32/mtD32) strain. Such isogenic clones were available because it had been observed several times that O* can yield normal 0 through the successive change O*+ [stable E] then E 3 0. I n order to identify and analyze only the F, progenies derived from the E(mtD32/mtD32) parent, this strain was marked by a mitochondrial mutation (ER).31/42 F, progenies of the “S” type were found in the cross O* X E and only 12/42 were found in the cross 0 X E (x2= 19, P < 1 % ) I n summary, regardless of the nature of the interaction taking place between mtD32 and mtD51 alleles in a heterozygote of E mating type which determines its ability to yield O* F, clones, the probability of this determination is influenced by both the properties-E(mtD32/mtD32) or E (mtD51/mtD51)- of the cyto- . 648 Y. BRYGOO plasmic parent and by those-O* (mtD5l/mtD51) or O(mtDSZ/mtDSl)-of the 0 parent. The most efficient situation is provided by the cross O* X E (mtD32/ mtD32) and the least efficient one by the cross 0 (mtD32/mtD32) X E (mtD51/ mtD51). ( 3 ) ConcZusicns. Since the onset and the heredity particularities of the O* phenotype do not result from a mutation, but from the interaction between the alleles mtD32 and mtD51, one must conclude that this interaction has modified, in a relatively stable way, some nuclear and/or cytoplasmic elements. First, the genetic determination of the O* phenotype certainly has a cytoplasmic basis, since the 0’ phenotype is cytoplasmically inherited in crosses O* X E ( m t D S l / m t D S l ) in the same way as the regular 0 phenotype is inherited in crosses O(mtD5l/mtD51) X E ( m t D S l / m t D 5 1 ) . Therefore 0*,0 and E can be considered as 3 different hereditary cytoplasmic states that can be associated with the mtD5l/mtD51 genotype. Besides this cytoplasmic determination, a nuclear determination may also be involved in the O* phenotype. Indeed, the difference observed between the two crosses, O* (mtD5l/mtD51) X E(mtD32/mtD32) and O ( m t D S l / m t D S l ) X E(mtD32/mtD32),can be explained in at least two ways. (1) It can be assumed that the O* and 0 strain differ by some “nuclear factor” transmitted to the mtD32/mtD32 partner along with the 8 gametic nucleus. (2) The results might also be explained by some interaction between the two conjugants such as: membrane interactions, migration of particular molecules through the membranes, migration of cytoplasmic elements accompanying the migratory nucleus, etc. favoring the onset of the (mtD32/mtD51) interaction which leads to O* clones production in the F, generation. DISCUSSION Two main problems are raised by the differentiation of mating type in P. tetraurelia: First, what is the mechanism for determination of the 0 or E mating type in cells of identical genotypes? Second, what is the relationship between determination towards either mating type and the actual expression (differentiation) of the corresponding phenotype? It has long been known that determination results from some interaction between a cytoplasmic state (determination towards 0 o r E is cytoplasmically inherited) and the nuclei. Determination takes place at nuclear reorganization, at an eirly stage of macronuclear development and is fixed in the macronucleus; it may be “preconditioned7’by the zygotic diploid micronucleus from which the 1954). I n turn, macronuclear determination macronucleus develop ( SONNEBORN towards 0 or E results in a cytoplasm endowed with the capacity of determining in the same way the new macronuclei at the next sexual generation. With respect to the second problem, genetic analysis of mating-type differentiation has provided tliree lines of information. (1) Determination and expression of mating type can be dissociated. Several mutations-“odd restricted”prevent the expression of the E mating type without modifying the mechanism MATING-TYPE DIFFERENTIATION I N PARAMECIUM 649 of determination: cells homozygous for the mutation and unable to express E, maintain equally an 0 or E determining cytoplasm according to their cytoplasmic origin (TAUB1963; BYRNE1973). (2) A correlation may exist between the cytoplasmic factor acting on the determination toward E and the gene products required for the expression of the E mating type (NANNEY 1957; TAUB 1963). (3) Regardless of the mechanism of determination, the expression of the E or 0 mating type does not seem to result from the functioning of two (alternative) mutually exclusive pathways: the expression of the E mating type seems to be branched on the pathway leading to the expression of the 0 mating type and involve additional metabolic steps (BUTZEL1955). This interpretation is based in part on the fact that only 0 restricted mutations have ever been found, the only two cases of E-restricted mutations obtained being lethal (BEISSON, personal communication; BRYGOO and KELLER,in preparation). All these data are consistent with the idea that mating-type determination does not bear on a E vs. 0 alternative but on a E vs. non-E alternative. I n other words, the “0-pathway” would be constitutively operative in any cell, while the “E-pathway” might or might not be induced to function. Before discussing to what extent this general scheme is supported by the data reported here and to what extent they specify the details of the scheme, the main results will first be summarized. (1) Systematic exceptions to the rule of cytoplasmic inheritance were observed in interstock crosses st.51 x st.32 of P. tetraurelia. These exceptions consist in the appearance, in some F, progenies derived from the E cytoplasmic line, of clones which express the 0 mating type (Table 7, interstock crosses). These clones, named O*, display a new phenotype characterized by a relative instability yielding cells expressing the E mating type either transiently or definitively. This modified heredity of mating type does not result from a mutation but from the interaction between two wild-type alleles, each one permitting a normal cytoplasmic inheritance of mating type when left in its original genetic background. (2) The O* phenotype behaves like a hereditary character when crossed to either st.32 or st.51, but its pattern of transmission is different in the two crosses. The Of phenotype seems to segregate as a nuclear determined character in the first case (0*x st.32), and is, in the majority of cases, cytoplasmically inherited in the second case (0*x st.51) (Table 7, crosses involving O*). (3) This differential breeding behavior of 0’ clones was used to analyze the basis of the difference between st.51 and st.32 and this difference was shown to result from a one gene difference at a new locus, mtD; st.51 and st.32 are respectively homozygous f o r the alleles mtD51 and mtD32. (4) Conversely, the properties of E (mtD51/mtD51) and E (mtD32/mtD32) cells were shown to be different: (mtD32/mtD51) heterozygotes yield O* clones in the F, generation more frequently when the heterozygote is formed in an E(mtD32/mtD32) cytoplasm than when it is formed in an E(mtD5l/mtD51) one (Table 7, interstock crosses). (5) Genetically, O* strains are nuclearly mtD5l/mtD51 and carry a particu- 650 Y. BHYGOO lar hereditary cytoplasmic state, different from the normal 0 or E hereditary cytoplasmic states. (6) I n contrast to the normal O ( m t D S l / n t D S l ) state, the O* cytoplasmic state is characterized in particular by its regular switching to the E phenotype upon introduction of an mtD32 allele. (7) Besides its particular cytoplasmic state, the O* phenotype seems to involve a particular nuclear “state” of unknown nature which is revealed only in crosses O* X E(mtD32/mtD32) by the transmission, along with the O* male gametic nucleus, of “something” which raises significantly (and to nearly 100%) the probability of obtaining O* F, clones in the progeny of the E(mtD32/mtD32) parent. (However, as previously pointed out, alternative explanations cannot be ruled out). Some of these results (in particular most aspects of points 1-5) can be explained in a rather simple way by assuming that the gene m t D is active in E cells and inactive in 0 and O* cells, and that the activity of this locus is selfinducible. (1) The mtD gene is active in E cells and inactive in 0 and O* cells. This is first suggcsted by the formal analogy, in the F, and F, progenies, of crosses O* X E(mtD32/mtD32) and of crosses between an 0 restricted mutant and a wild-type strain (Figure 2b). I n the latter situation, the pattern of mating-type inheritance can be interpreted by the presence in the mutant strain of an inactive gene product. Similarly, the pattern of mating-type inheritance in crosses O* X E(mtD32/mtD32) can be interpreted (1) by the absence of the product of the mtD5l gene m the O* cells and (2) the activity of the mtD32 allele in the E parent and in the two E exconjugants. That the mtD locus is active in E cells is also consistent with the existence of phenotypic differences between E (mtD32/ mtD32) and E ( m t D S l l m t D 5 1 ) cells. (2) The activity of the mtD locus is self-inducible. Since both E ( m t D active) and 0 ( m t D inactive) states are cytoplasmically inherited in both st.32 and st.51, one can assume that the activity of this locus is self-inducible, i.e., that the m t D product regularly induces or maintains the activity of the locus. Therefore the product of the m t D locus behaves as a cytoplasmically transmissible agent for determination towards the E phenotype. These two hypotheses can explain the properties of the two types of E (mtD32/ mtD51) heterozygotes as follows: in the heterozygotes which give rise to normal “NS” F,’s the two alleles, mtD51 and mtD32, are active, whereas in the heterozygotes which give rise to “S” F, progenies, i.e., with O* ( m t D 5 l / m t D 5 1 ) clones, the mtD32 allele only is induced and the mtD51 allele remains inactive. This situation is maintained in the F, generation: in mtD5l/mtD51 homozygotes these genes remain inactive and yield O* clones. The fact that the production of O* cells is more frequent in the progeny of E ( 3 2 ) cytoplasm than in the progeny of E(51) cytoplasm can be explained by assuming that the product of the mtD51 allele is more efficient than the product of the mtD32 allele in inducing the activity of the mtD51 allele. MATING-TYPE DIFFERENTIATION I N PARAMECIUM 65 1 The interpretation of other features of the system and in particular of points (6) and (7) is more difficult. ( 1 ) What is the nature of the cytoplasmic diflerence between E(mtD32/ mtD32), E(mtDSl/mtD51), O* and O? The difference may be based, as suggested by SONNEBORN (personal communication), on a quantitative difference in the concentration of the product-D-of the mtD locus. This concentration would be low (or nil) in 0 cells, high in the E(mtDSl/mtD51) cells and intermediate in O* and E(mtD32/mtD32) cells. Since these different states are cytoplasmically inherited, it must be assumed that the level of concentration of the D product would be cytoplasmically inherited. The difference may also be qualitative. In this hypothesis, the cytoplasmic difference between E(mtDSl/mtD51) and E (mtD?2/mtD?2) would be due to the qualitative difference of the respective products of the mtD51 and mtD32 alleles and the 0 or O* cells would not contain any D product. Therefore the cytoplasmic difference between 0 and O* cells would have to be explained by the occurrence of a second self-inducible factor, X , present in the Of strains and absent in the 0 cells. The properties of X can be deduced from the properties of the O* strains: the X factor would be generally incapable of triggering the expression oi the mtD51 allele but would be sufficient to trigger the activity of the mtD32 allele. With these two cytoplasmic factors, (D, and X ) 4 possible situations would be expected: the absence of any factor, the presence of either one or the other or the presence of both. The two stable phenotypes 0 and E might then correspond to the simultaneous absence or presence of both factors; the absence of one factor would result in a somewhat unstable phenotype: one of these unstable phenotypes would correspond to the O* phenotype ( X present and D absent) while the other phenotype ( X absent and D present) could correspond to the selfer er^" described by NANNEY (1957) which were cytoplasmically 0 and expressed E. The differential effect of the X product on the mtD alleles could correspond to a divergent evolution from a duplication of the mtD locus: the X product could still recognize the mtD32 allele but no longer recognize the mtD51 allele. (2) What is the “nuclear state” of O* strains? To account for the different results of O* X E (mtD32/mtD32) and O(mtD51/mtD51) X E(mtD32/mtD32) it may be assumed that the O* phenotype involves a r-uclear change of some unknown type which can be transmitted through conjugation by the male O* gametic nucleus. This nuclear particularity of O* cells might be the inactive state of the mtD51 allele which would be fixed in the micronucleus and maintained through conjugation in the migrating male pronucleus. This assumption is consistent with SONNEBORN’S data and interpretation of the mating type of F, clones in stock 51 for crosses between an 0 amicronucleate strain and a normal E strain or uice uersa (SONNEBORN 1954) : the amicronucleate conjugant yielded a F, clone displaying the same mating type ( E or 0) as the conjugant which supplied the micronucleus. A “fixed’’ inactive state of the mtD51 locus in o* cells, would resemble paramutation (BRINK,STYLESand AXTELL 1968). HOW- 652 Y. BRYGOO ever, in contrast to paramutation it must be pointed out that this “determined” state of the micronucleus is revealed only in O* x E(mtD32/mtD32) crosses, and is totally invisible under other nucleo-cytoplasmic conditions. Such non-autonomus functional states partly dependent upon cytoplasmic environment are likely to exist in higher organisms and may account for the stability of determined or differentiated states, which are also nevertheless susceptible to reprogramming under changing cytoplasmic conditions. Whatever the case may be, the interpretations concerning the cytoplasmic and nuclear states of O* cells can be experimentally tested: in particular, the existence and identification of the two cytoplasmic factors can be studied by microinjection experiments now under way, and the same technique will allow the investigation of whether 0*,0 and E nuclei carry particular nuclear states. I n conclusion, although it is not yet possible to propose a definitive interpretation of all the data on the origin and genetic behavior of the O* phenotype, our results at least suggest a general hypothesis concerning the basis of mating-type determination in Paramecium. In P. tetraurelia, as well as in other group B species, this determination could be equated to the early activation o r nonactivation of one locus, mtD, according to the presence or absence of its own product. This product would be required under physiological conditions suitable for the onset of sexual reactivity, for the expression of the functions leading to the E mating type. Although other species of Paramecium do not display the same pattern of mating-type inheritance, in all cases a locus with equivalent function might be involved and the differences in pattern of mating-type inheritance would result from differences in the regulatory properties of this gene. In group A species, the locus would not be self-inducible and would be randomly activated or not activated in each newly formed macronucleus, while in group C, the active form of the gene would be constitutively expressed. More generally this type of mechanism, based on the early activation of a dispensable function which later, under changed physiological conditions, might be necessary to trigger or permit the expression of other functions, could be involved in the determina tion processes occurring in the development of higher organisms. This hypothesis is quite similar to the model proposed by KAUFFMAN (1973) to explain determination and transdetermination in the development of Drosophila eggs. The author wishes to express his gratitude to DR. J. BEISSON for advice throughout this work and for many fruitful discussions concerning the manuscript. I thank DR. T. M. SONNEBORN for a thorough critical reading of the manuscript. I also thank A. M. KELLERfor her very valuable technical assistance and J. BRIZARD for typing the manuscript. This work was supported by the grants (ATP “Differentiation Cellulaire” n. 4317) from the Centre National de la Recherche Scientifique. LITERATURE CITED ADOUTTE, A. and J. BEISSON,1972 Evolution of mixed populations of genetically different mitochondria. Nature 235 : 393-396. MATING-TYPE DIFFERENTIATION I N PARAMECIUM 653 BEISSON,J. and M. ROSSIGNOL, 1969 The first case of linkage in Paramecium aurelia. Genet. Res. 13: 85-90. s J. D. AXTELL,1968 Paramutation: Directed genetic change. BRINK,R. A., E. D. S n ~ and Science 159: 161-170. BUTZEL,H. M., 1955 Mating type mutations i n variety 1 of Paramecium aurelia, and their bearing upon the problem of mating type determination. Genetics 40: 321-330. BYRNE,B. C., 1973 Mutational analysis of mating type inheritance in syngen 4 of Paramecium aurelia. Genetics 74: 63-80. FUKUSHI,T., and K. HIWATASHI, 1970 Preparation of mating reactive cilia from Paramecium cnudrrtum. J. Protozool. 17 (suppl.): 21. KAUFFMAN,S. A., 1973 Control circuits for determination and transdetermination. Science 181: 310-318. KIMBALL,R. F., 1939 A delayed change of phenotype following a change of genotype h Paramecium aurelia. Genetics 24: 49-58. NANNEY,D. L., 1957 Mating type inheritance at conjugation in variety 44 of Paramecium aurelia. J. Protozool. 4: 89-95. T. M., 1947 Recent advances in the genetics of Paramecium and Euplotes. Adv. SONNEBORN, 1954 Patterns of nucleo-cytoplasmic integration in ParaGenet. 1 : 263-358. --, mecium. Caryologia 6 (suppl.) 307-325. -- , 1970 Methods in Paramecium research. pp. 241-339. In: Methods in Cell Physiology. VQZ.4 . Edited by D. M. PRESCOTT. Academic Press, Inc., New York. -, 1975 Paramecium aurelia. pp. 469-594. In: Handbook of genetics, Vol. 2. Edited by R. C. KING.Plenum Press, New York. TAUB, S. R., 1963 The genetic control of mating type differentiation in Paramecium. Genetics 48: 815-834. Corresponding editor: S. L. ALLEN