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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
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Corresponding editor: S. L. ALLEN