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

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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

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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.

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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,

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(a)

Cml

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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).

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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.

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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 (