between subspecies of tetrahymena pzgmentosa - genetics

MATING-TYPE INHERITANCE AND MATURITY TIMES IN CROSSES BETWEEN SUBSPECIES OF TETRAHYMENA PZGMENTOSA*

ELLEN M. SIMON

Department of Genetics and Deuelopment, University of Illinois, Urbana 61801

Manuscript received October 2,1978 Revised copy received April 16, 1979

ABSTRACT

Subspecies 6 and 8 of T . pigmentosa (formerly syngens 6 and 8 of T . pyriformis) share a mating-type system controlled by three alleles with “peck-order’’ dominance at a single locus. The system is apparently closed and limited to three mating types that are homologous, but not identical, in the subspecies. These relationships are reflected in new mating-type designations. -The viability in some intersyngenic crosses is excellent, and the inheritance of major mating types in first-generation hybrids and their progeny follows the pattern of subspecies 8.-The period of immaturity is shorter than that previously reported for subspecies 8, with 50% of the subclones maturing between 46 and 100 fissions after conjugation. Maturity curves are generally sigmoid, but some are apparently biphasic. The onset of maturity in triplicate sublines from the same synclone is usually highly correlated.

ETRAHYMENA pigmentosa was originally described ( GRUCHY 1955) as Ttwo separate “varieties” of T . pyriformis on the basis of mating tests carried out with cells washed from peptone medium into distilled water. The strains identified as variety 6 would not mate with those of variety 8 under these circumstances. Later studies by ORIAS (1959a) demonstrated that mating could be achieved between these groups of strains if they were grown in bacterized Cerophyl (dried rye grass) medium prior to mixture. The matings between groups were weaker, less reliable, and were usually delayed in comparison to mixtures within groups. Nevertheless, the F, pairs were fully viable and vigorous, and F, survival was excellent. The progeny were not characterized as to mating types.

ORIAS (1963) studied mating-type inheritance in the group of strains by then called “syngen” 8 (substituting SONNEBORN’S 1957 term for the older and ambiguous term variety). He demonstrated that mating types were usually synclonally determined and suggested a simple genetic basis for mating types, particularly a system of “peck-order” dominance. Allele mtA determined type I and was dominant over mlB and mtc. MtB was associated with type I11 and was dominant over mtc, which defined type I1 in the homozygous state. Unexplained departures from this interpretation included an excess of type I1 segregants in crosses and a small fraction (5%) of selfing clones.

This work was supported by Public Health Service grants GM-7779 and AG-00010 to D. L. NANNEY.

Genetics 94: 93-113 January, 1980.

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94 E. M. SIMON

GRUCHY (1955) had previously reported the results of a cross between two strains of syngen 6, with results that can be interpreted in retrospect as similar to some of those in syngen 8, but he had insufficient data for a full genetic interpretation.

When the syngens of the T. pyriformis complex were assigned Latin binomials (NANNEY and McCoy 1976), syngens 6 and 8 were both placed in T. pigmentosa on the basis of their genetic compatibility, in spite of the fact that they were readily distinguished by isozyme mobilities (BORDEN et al. 1977). In fact, the large molecular differences between the two groups of compatible strains provide one reason for undertaking breeding analyses with them. The preliminary studies suggested that only one-third of the isozyme mobilities were alike in the two groups. Such differences should provide genetic markers for a wide variety of cell structures and functions and should facilitate mapping studies.

Genetic studies on T. pigmentosa are also of interest because of the possibility of large differences in genetic strategies between T. pigmentosa and T. thermophila (see SONNEBORN 1957, 1974). T . pigmentosa (syngen 8 ) was reported to have a long immaturity period (120-150 fissions) and synclonal mating-type determination (ORIAS 1959b, 1963), while T. thermophila has a short immaturity period and caryonidal mating-type inheritance. These features indicated, according to SONNEBORN’S (1957) criteria, that T. thermophila is a relative inbreeder and T. pigmentosa a relative outbreeder. Preliminary observations in our laboratory suggest that coincident with the different breeding patterns are differences in macronuclear organization. Particularly, the phenotypic assortment characteristic of nearly all traits in T . thermophila (ALLEN and GIBSON 1973; SONNEBORN 1974) does not occur in T. pigmentosa, at least not in the same way. The first example is the behavior of the immobilization antigens. Young heterozygous synclones of T. pigmentosa commonly react with only one. rather than both, parental antisera. Furthermore, sublines of rare synclones that do express both specificities may alternately react with either or both (or occasionally neither) of the antisera for as long as 200 fissions following conjugation. T. thermophila sublines, o n the contrary, generally begin clonal life expressing both antigens but become monospecific by approximately 100 fissions. A second example is the stabilization of sublines from selfing caryonides. Subclones pure for mating type have been obtained from a number of selfers, but the rate at which stabilization occurs may differ by as much as seven-fold in different selfers, even after 200 to 300 fissions. For a third example, instability in mating-type expression, see DISCUSSION.

In this paper, the terms syngen and subspecies are used synonymously for the sake of convenience, although neither term is exactly appropriate. The other syngens of the T. pyriformis complex are now recognized as separate species and work in progress in this and other laboratories suggests that syngens 6 and 8, despite their lack of total genetic isolation, are too divergent to be considered semispecies (MAYR 1970; WHITE 1978) or even to belong to one species. While awaiting resolution of these relationships, we will retain for historical reasons

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TETRAHYMENA MATING-TYPE INHERITANCE 95

the arabic numerals 6 and 8 although this may incorrectly suggest that at least 6 other subspecies of T . pigmentosa are known.

Studies on T . pigmentosa were reinitiated a few years ago, but the available strains of both subspecies appeared to be senescent (NANNEY 1974 and unpublished). Recent collections of D~ERDER and NYBERG (unpublished) have greatly improved the opportunities to make crosses and begin analyses. The new strains have been stored in liquid nitrogen (SIMON and FLACKS 1975) so that their breeding performance should not deteriorate.

In this initial presentation of new observations, we describe the mating-type homologies between syngens 6 and 8, and demonstrate the existence of three equivalent mating-type alleles in both subspecies. Recognition of the homologies between subspecies necessitates switching some mating-type designations. One of these changes results in switching I11 for I1 (and I1 for 111) in syngen 8. In the new designations, the 6 or 8 following the roman numeral indicates the pattern of reactions in intersyngenic matings. We also propose a new designation of the alleles that corresponds to mating-type designation and conforms to general microbial practice. Allele mtA becomes mat-I, mtB becomes mat-2 and mP becomes mat-3. Finally, we present evidence that, under our growth conditions, most synclones derived from either subspecies or from matings between them have immaturity periods shorter than those previously reported for syngen 8.

MATERIALS A N D METHODS

The strains from which viable progeny were obtained and their sources are listed in Table 1. Strains belonging to different subspecies do not mate, even if they are of different mating

types, when washed from axenic peptone into DRYL'S (1959) salt solution or deionized water. Therefore, most of the manipulations and observations here reported were conducted in bacterized Cerophyl medium. Even in this medium, the mating responses between subspecies are generally weaker, frequently delayed and very rare in some combinations.

The stanciard methods of culture and breeding analysis usually employed for bacterized Tetrahymena (NANNEY and CAUCHEY 1955) were followed with some modifications. Cultures were maintained at room temperature (about 23") or at 15" in a 0.15% (w/v) infusion of Cerophyl (Cerophyl Laboratories, Inc., Kansas City, Missouri) previously inoculated with Klebsiella pneumoniae (designated in earlier publications from this laboratory as Aerobacter aerogenes or Enterobacter aerogenes) . Batches of bacterized medium were sometimes used €or several days if stored at 4" after the first day.

Washing of glassware was reduced by isolating pairs either into drops of Cerophyl on the bottom of new plastic petri dishes or in the small conical wells of #23&72 Histocompatibility Plates (Cooke Laboratory Products Division of Dynatech Laboratories, Inc., Alexandria Virginia).

Pairs from intrasyngenic crosses were isolated 18 to 30 hr after parental mixtures were made (one to five after refeeding to eliminate uncommitted pairs). Depressions containing intersyngenic mixtures were examined three or four times daily. If mating had not occurred by the third day, fresh bacterized Cerophyl was qdded, with or without first decanting the slides. Developing synclones were examined at two, three and four days for mating, indicating failure to complete conjugation. Surviving negative cvnclones were tested for maturity to eliminate nonconjugants with only one surviving parent. Assurance of a new genetic constitution in the progeny is provided by the usual onset of sexual immaturity following conjugation. Under some conditions a€ genetic-environmental incompatibility, this period of immaturity may be greatly

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96 E. M. SIMON

TABLE 1

Strains from which viable progeny were obtained

Subspecies mahng type’

Syngen 6 I6

I16

1116

Syngen 8 I8

I18

I118

Time of origin Strain and collector

U M 1091 FA 8 U M 1060 HG 2 UM 1147 U1 7152 HG 8

Alpena 6 IL 3 Alpena 15 AB 6-7

XE 8 UM 1286 Alpena 2

1954 Gruchy 1975 Nyberg 1953 Gruchy 1975 Nyberg 1956 Gruchy 1971 Doerder 1975 Nyberg

1957 Nanney 1955 Nanney 1957 Nanney 1959 Orias

1976 Nyberg 1953 Gruchy 1957 Nanney

Place of origin

Mariana FL Somme Woods, Northbrook, IL Waterloo Recreation Area, M I Riverdale, Cook Co., IL “F1” U M 1091 x U M 1060 Rocky Mtn., Nad., Pk., CO Riverdale, Cook Co., IL

Lake Huron, Alpena, MI Indian Lake, Upper Pen., M I Lake Huron, Alpena, MI Progeny of cross between

Ramsey Lake, Sudbury, Ontario Clear Lake, Jackson, M N Lake Huron, Alpena, MI

Alpena strains

* New mating-type designations (see Table 4).

shortened in T. thermophila (NANNEY and MEYER 1977), but immaturity is a reliable indication of completion of reorganization. The following strains were selected as standard testers: I6 (UM 1091), I16 (HG2), 1116 (HG 8, U1 7152), I8 (IL 3), I18 (AB 6-7), I118 (UM 1286). In 6 X 6 and 8 x 8 crosses, the three homologous testers were used. Since mating reactions even between nonhomologous mating types of the two subspecies are inconsistent (see Table 3) testers of the three mating types for 6 x 8 crosses each consisted of mixtures oE homologous strains of the two subspecies (I6 and 18, etc.). Maturity tests were made by mixing one drop of the unknown culture with one drop of each of three standard testers, then feeding 0.4 ml of medium to each mixturc. Observations for mating were made on the following day, and in the case of weak or negative responses, were continued for an additional three days. Mixtures that did not mate well were decanted and fed again after they had starved, usually following the second reading, and were observed again on the two following days.

The immature synclones were now usually expanded to three sublines (only one in some later experiments), which were carried to maturity by single cell transfers. Dead cells were replaced only from the backup culture of the same subclone. After checking the viability of the new transfer, the onset of maturity was monitored in most experiments by mixing portions of leftover cultures with the three testers, so that for each transfer one subline was mixed with each tester, and the testers were rotated to permit mixtures of each subline with all testers over an interval of three transfers (approximately 13 fissions per transfer). ’rests were examined, USU- ally only once, the next day, then discarded.

The mating types of the progeny of 6 x 6 and 8 x 8 crosses were established during the repeated testing just described or by the method used to test new synclones for immaturity. The major mating types (I, TI, 111) of 6 x 8 hybrids could be determined by using the mixed testers. However, if the complete set of 6 and 8 testers was used individually, variations in the expression of mating type were found among hybrids and their F, progeny (to be reported in another communication).

Although immaturity is good evidence for the formation of new macronuclei, it does not certainly establish that the new macronuclei are derived from a normal synkaryon. In T

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thermophila, haploid micronuclei sometimes give rise to macronuclei, with or without diploidi- zation (ALLEN 1 x 7 ; PREPARATA and NANNEY 1977), and cytogamy may bring together t w o pronuclei from the same parent (ORIAS, HAMILTON and FLACKS 1979). For this reason the information derived from immobilization tests with antiserum (LOEFER, OWEN and CHRISTENSEN 1958; NANNEY and DUBERT 1960) was valuable in providing evidence of biparental contributions. Cerophyl cultures of each of the original parental strains were used to provoke an immune response in rabbits; the procedures were similar to those described by PHILLIPS (1967). These antisera could in each case distinguish the parental cultures by immobilization at titers that did not noticeably affect the other parent. Although the basis for the immobilization responses has not yet been thoroughly analyzed, the appearance of new combinations of mating types and antibody responses in the first generation is consistent with a normal synkaryon formation.

RESULTS AND ANALYSIS

Crosses within the subspecies Crosses within subspecies 8: Seven strains of syngen 8 were still available from

the collection of the 1950’s or from progeny of ORIAS’ experiments. Fifteen crosses among these strains were studied with samples of 18 pairs. Only seven crosses produced any viable progeny, and the mean survival among these was 32% (range 7 to 70%). Relatively long periods of immaturity (40 to >I50 fissions) occurred in 68% of the clones, and three out of the 14 clones showing earlier maturity were selfers. Generally the mating types of the progeny obtained were consistent with the observations and genetic interpretations of ORIAS, except for a somewhat higher frequency of selfing. Nineteen percent of the pairs (8) were selfers. Two of these seven old strains have been successfully crossed to syngen 6. IL 3 appears to have the genotype mat-l/mat-3 and AB 6-7 is mat-2/mat-2 (data not shown). A larger cross between these two strains yielded 64/105 pairs dead and an additional two with incomplete conjugation.

Several new strains of syngen 8 were collected in 1975 and 1976. All of these are of mating type 11. One (XE 8) was crossed to IL 3, but produced only 17% viable progeny, in contrast to the 37% obtained in the cross with AB 6-7 as a parent.

Crosses within subspecies 6: Crosses involving three of the more recently collected strains of subspecies 6 resulted in mating-type distributions compatible with the system described for subspecies 8 (Table 2). HG 2 (11) behaves as if it has the genotype mat-2/mat-3 and U1 7152 (111) and HG 8 (111) are apparently homozygous for mat-3.

In contrast, all possible crosses involving the six strains available early in this

TABLE 2

Results of crosses among recently collected strains of syngen 6

Parents Mating types of progeny % +able* I16 I116 Died Aborted I I1 I11 conlugants

HG2 U17152 42 3 0 29 29 5’7 HG2 H G 8 33 1 0 33 36 68

* The mating types of two pairs from each cross were not determined.

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98 E, M. SIMON

study (HG 2 was acquired recently) gave anomalous results-virtually all of the progeny expressed mating type 111. Only eight of 119 synclones gave rise to sublines manifesting non-I11 mating types; selfing was observed in sublines of five of these and sublines pure for either of two mating types were derived from the other three synclones. At least five of these eight were atypical in other respects; they matured precociously, showed symptoms of micronuclear loss [the semi-amicronucleate syndrome (NANNEY 1959)] or both. Since type 111, in the new nomenclature, is the lowest type in the peck-order (at least in syngen 8), one of the parents (either U1 71 52 or HG 8 was a parent of 1 13 of the 1 19 synclones) should be homozygous for this type, but the other parent must have carried at least one allele for another mating type, and this allele should have been transmitted to some of the progeny in a normal conjugation. Since such transmission was not regularly observed, the possible kinds of abnormal events must be considered. Possibilities include (1 ) lethality associated with certain phenotypes or (2) failure to complete conjugation with retention of the old macronucleus by one parent. The viability in these crosses was very poor (80% died, 76% of those surviving retained old macronuclei) making it impossible to rule out death associated with non-I11 mating types. Arguing against macronuclear retention are the regular occurrence of an immaturity p e ~ o d of 40 to >130 fissions in 80% of the progeny and the fact that some progeny manifested responses to antisera (unpublished) different from those of the type 111 parent (see DISCUSSION for other possibilities).

Extensive efforts were made to obtain syngen 6 breeding stocks by intercrosses among the progeny of the first matings. Each line selected for these crosses showed evidence that nuclear reorganization of some kind had occurred in the pair from which it was derived. Either the subclone was immature for 80 to 150 fissions, or i t was a stabilized selfer, or it expressed a nonparental mating type. Each of the five non-I11 lines was crossed to either ten or 12 different type I11 clones, and an average of 17 pairs per cross was studied. The results were similar to those obtained in the first series. fighty-four percent of the pairs died, 1% showed macronuclear retention. Of 199 viable synclones carried to maturity, only six were non-111 in mating type. Again the non-111 progeny were atypical in having a shorter immaturity period and/or some other abnormality. A few crosses gave high frequencies of survivors; 100% in three cases and 82-96% in six other crosses, but the same mating-type results were obtained. Differential viability of non-I11 progeny in these crosses cannot explain their absence. Some abnormal process must be invoked (see DISCUSSION).

We conclude from these experiments that mating-type inheritance in syngen 6 is comparable to that of syngen 8, except that strains of 6 capable of transmit- ting the mating-type I allele (mat-1) are not available at the present time.

Crosses between syngens 6 and 8

Mating reactions and homologies between mating types of subspecies 6 and 8: Intersyngenic matings have been attempted in several circumstances, but have never been observed with washed axenic cultures, and very rarely with cultures

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grown in bacterized peptone medium (GRUCHY 1955; ORIAS 1959a; unpublished) though these conditions are perfectly satisfactory for T. thermophila matings and for matings within either subspecies of T. pigmentosa. The advantages of the use of diluted bacterized peptone over bacterized Cerophyl in genetic experiments with T. thermophila were outweighed by disadvantages when its use was attempted with T. pigmentosa. Both the growth rate and the final population density of T. pigmentosa in Cerophyl exceed that in bacterized peptone. In addition, mating reactions even in intrasyngenic mixtures are less dependable and, with one possible exception, the intersyngenic mating reactions of Cerophyl- grown cells disappeared after a few cell divisions in bacterized peptone.

Even when the cultures are grown in bacterized Cerophyl medium, the mating reactions between strains of the two subspecies are weaker than those observed in intrasyngenic crosses. Moreover, the reactions among the mating types differ in systematic ways. Even though the number of strains examined is small and differences occur among strains of the same mating type, some generalizations about the mating reactions may be supported (Table 3 ) . Strong and consistent matings with tight pairs that, like those in 6 x 6 and 8 x 8 matings do not separate for several hours when refed, occur between I6 and 1118, if UM 1091 is the I6 parent. Matings between I8 and 1116, and between I18 and 1116, occur after a delay of 24 hours or more, may include many “loose” pairs, but do provide some tight pairs under appropriate conditions. The crosses with I16 are the most difficult to achieve. Pairs are never observed in most mixtures with 18; single pairs in rare mixtures may result from technical accidents, i.e., micro- droplets from an aerosol reaching the wrong depression. The pairs with I118 are not quite so rare; attempts to isolate these pairs have failed, however, because the cell unions are very weak. The matings between I6 and I18 are also rare but a few tight pairs have been isolated. Most of the subsequent studies of intersyngenic crosses are necessarily based on the kinds of matings that can be regularly achieved.

Because of the irregularity of intersyngenic matings, the homologies among the mating types of the two subspecies were not previously established. We have

TABLE 3

Characterization of mating reactions between strains of syngens 6 and 8 under optimal conditions after growth in bacterized Cerophyl medium

I no reactions rare reactions stronger reactions; tight few pairs pairs with UM 1091

I1 rare reactions no reaction rare reactions (if ever?) no tight pairs

I11 delayed-weak delayed-weak no reaction reactions reactions

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100 E. M. SIMON

TABLE 4

Reassignment of mating type designations

Original syngen 6 Original syngen 8 New T. pigmentosa designation designation designation

I1 I 16, I8 I 111 116, I18

111 I1 1116, 1118

found, however, that three combinations of the syngen 6 and syngen 8 mating types never result in conjugation: using old designations 61 with 8111, 611 with 81 and 6111 with 811. We conclude, therefore, that these are the homologous combinations and propose that the homologies should be reflected in new designations. ORIAS showed a peck-order dominance pattern in which three alleles mtA, mtB, mtc corresponded to mating types I, 111, and 11, respectively. Since some mating-type designations must be changed in any case, a transformation that brings genic designations into seriation with mating-type designations makes the correspondences easy to remember. We have, therefore, arbitrarily renamed the mating types of both syngens as shown in Table 4. In the new designations, the 6 or 8 following the roman numeral indicates the pattern of reactions in intersyngenic matings. The fact that three and only three types are present in both instances supports the idea that the T . pigmentosa mating system is a limited three-type system rather than an open multiple mating system with additional types yet to be discovered. We will return to this matter in the

F , uiability: The viability of the progeny of intersyngenic crosses was highly variable. Strains that performed poorly in intrasyngenic crosses was usually poor parents here also. However, some intersyngenic crosses gave better progeny survival than intrasyngenic crosses involving the same strains. In particular two strains of each syngen were identified through progeny tests as suitable for study- ing the genetics of intersyngenic crosses: HG 8, mat-3/mat-3 (IIIG), U1 7152, mat-3/mat-3 (1116), IL 3, mat-l/mat-3 (IS) and AB 6-7, mat-2/mat-2 (118).

DISCUSSION.

TABLE 5 Results of intersyngenic crosses that provided first-generation hybrids used in breeding analysis

Clones Mating selected for

Mating type types not % Viable further Parents Died Aborted I I1 I11 Selfers determined coniucants analvsui

HG8 (1116) x 12 3 9 0 10 2* 102 89 18,27 (I) ; IL3 (18) 30,32 (111)

U17152 (1116) x 9 1 0 24 0 2 0 72 21,22,34, AB6-7 (118) 36 (11)

AB6-7 (118) 60 (11) HG8 (1116) x 16 4 0 2 6 0 0 26 72 48,49,50,

* Selfing was not observed in transfers of nine sublines from one synclone, but these sublines expressed either I1 or I11 when mature.

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The viabilities in the permitted crosses among these strains (Table 5) are above 70%, except IL3 x U1 7152; this cross formed no pairs that could be isolated. The other three crosses provided the strains used in the breeding analysis.

F , viability: As noted above, some crosses between syngens 6 and 8 gave higher frequencies of normal progeny than most of the available crosses within the syngens. ORIAS (1959a) had reported good viability in both the Fl and F2, and this latter observation was also verified.

The Fl clones to be crossed (listed in Table 5) were chosen because of their immaturity period of at least 65 fissions, their possession of recombinations of parental phenotypes for mating type and serotype (in eight of 12 cases), and/or alterations in the pattern of matings in mixtures with syngenic testers.

A total of 1199 pairs from crosses among the 12 intersyngenic clones were screened for maturity. The proportion of F, pairs that survived and completed conjugation (82.2%) was close to the average observed in the Fl generation; 16.5% died and 1.2% apparently aborted their macronuclei. However, the distribution of pairs that died was not entirely random. When the 8 type I1 Fl’s derived from U1 7152 X AB 6-7 were crossed to F,’s of mating types I and 111, 25% (136/539) died; when the 8 type I1 Fl’s derived from HG 8 x AB 6-7 were crossed to the same type I and I11 clones, only 8% died.

Cytological observations on conjugation: Feulgen preparations were made of conjugating pairs from two intersyngenic crosses: HG 8 X IL 3 and HG 8 X AB 6-7. All major stages of conjugation, including macronuclear anlagen formation, were observed and compared with similar stages in normal intrasyngenic crosses. No obvious differences were observed in nuclear behavior or morphology. Apparently the cytogenetic proccsses occur normally in intersyngenic matings, provided that tight pairs can be found.

Mating type distribution among F , progeny: The mating types of the progeny of the first generation hybrids are shown in Table 6. The occurrence of synclones yielding sublines of more than one mating type (except selfers) cannot be determined since only one subclone from each pair was transferred to maturity in this experiment. The crosses between types I and I11 (crosses 1-4) yield approximately equal frequencies of types I and I11 with a slight excess of type 111. ORIAS’ (1963) results with comparable crosses within subspecies 8 are given for comparison. His explanation of the genetic basis for his results is equally appli- cable to the present data. The type I clone is heterozygous (mat-l/mat-3) and the type I11 clone is the homozygote mat-3/mut-3. Both the selfer lines and the shift toward type I11 are related to an instability at the mat locus, in either the macronucleus or the micronucleus. It has been characterized as a tendency for the mat-I or mat-2 allele to shift to the mat-3 state.

The crosses between types I and I1 (crosses 5 to 20) similarly repeat the pattern demonstrated by ORIAS. About half the progeny are type I, a quarter are I1 and a quarter are 111. This result is again consistent with a genotype of mat-I/ mat-3 for the type I parent and a genotype of mat-2/mat-3 for the type I1 parent. Two anomalous crosses occurred among the 16 studied. Crosses 7 and 15 produced no type I1 progeny. They are responsible for the slight overall excess of

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102 E. M. SIMON

TABLE 6

Mating-type distribution among the progeny of first-generation hybrids

Parental mating type and Cross Parental Phenotypes of progeny Total

postulated genotype No. clonesf I I1 111 Selfer tested. - I x I11 mat-i/mat-3 x mst-3/mat-3

I x I1 mat-l/mat-3 x mat-2/matJ

I1 x I11 mat-2/mat-3 x mat-3/mat-3

1 1 8 x 3 0 2 32 3 27 x 30 4 32

Total ORIAS’ (1963) results

5 18 x 21 6 22 7 34 8 36 9 48

10 49 11 50 12 60 13 27 x 2 1 14 22 15 34 16 36 17 48 18 49 19 50 20 60

Total Excluding 7 and 15

ORIAS’ (1963) results

21 3 0 x 2 1 22 22 23 34 24 36 25 48 26 49 27 50 28 60 29 3 2 x 2 1 30 22 31 34 33 36 33 48 34 49 35 50 36 60

Total Excluding 23 and 31 ORIAS’ (1963) results

18 3

13 13 47 72

3 6 8 8 7 6 5 5 4 9 4 5 7 5 7 7

96 84 83

0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0

-

0 a 0 0 0 0

3 3 0 2 2 4 6 2 3 2 0 2 1 2 4 3

39 39 39

7 4 1 4 4 5 7 6 2 7 0 3

13

7 5

75 74

173

-

12 10 15 19 56 88

4 3 4 2 3 2 1 4 3 0 8 4 3 4 1 2

48 36 45

5 8

11 8 8 7 5 6 6 5

12 9 5

5 7

107 84

173

-

0 30 0 13 1 29 0 32 1 104 1 161

2 0 0 0 0 0 0 1 1 0 0 1 1 0 0 0 6 6 7

0 0 0 0 0 0 0 0 0 8 0 0 0 0 18

0 0 0 182 0 158 5 350

- -

11 11

11

189 165 174

* N=12 unless otherwise noted. + Derivation shown in Table 5.

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type 111 progeny. They may represent instances of rnicronuclear shift from the mat-2 allele to mat-3.

The 15 crosses between type I1 and type I11 are consistent with genotypes already assigned to the parents in the previous crosses. Genotype mat-3/mat-3 X mat-2/mat-3 yields 50% type I1 (mat-2/mat-3) and 50% type I11 (mat-3/ mat-3). Again, one anomalous cross was observed (cross 31), which produced no type I1 off spring. This cross had the same type I1 parent (clone 34) as crosses 7 and 15, which also produced no type I1 progeny. Note also cross 23, involving the same type I1 parent and only one type I1 offspring. This result suggests that clone 34 might be mixed, with a few of the cells still having a micronucleus of type mat-2/mat-3, even though most have become homozygous mat-3/mat-3. One possible explanation for the extreme deficiency of II’s in crosses involving clone 34 might be differential survival, since 33% (47/136) of the pairs isolated in these four crosses died. Even if present in this instance, differential survival is not a general phenomenon, since 33% and 28% of the progeny of clones 21 and 22, respectively, died but no deficiency of mating type I1 survivors occurred.

The behavior of clone 34 is similar to that of the old syngen 6 strains. Test crosses of progeny of I x 34 to 111, which should distinguish between several hypotheses (see DISCUSSION) regarding this phenomenon, have not yet been performed. We conclude from these experiments that the mat alleles from subspecies 6 behave in intersyngenic crosses as alleles to those of subspecies 8, and their interactions at a first approximation are equivalent to those of subspecies 8 alleles among themselves.

The length of the immaturity period The length of the immaturity period was monitored at each transfer in many

of these crosses. Most of the maturation curves obtained were approximately S-shaped whether the cumulative totals of mature sublines were based on one (Figure 1) or three (Figure 2) subclones from each synclone. In these experiments, 100% of the lines were mature in 90 to 156 fissions following conjugation.

Inspection of the six curves involving 6 x 6 crosses (Figure 1, Figure 2 A and B) reveals that the length of the immaturity periods varies among crosses involving different parental strains. The time at which 50% of the subclones derived from crosses between HG 2 and HG 8 or U1 7152 were mature was approximately 50 fissions following conjugation (Figure 1, curves A and B) . Curves C and D include the pooled data from simultaneous crosses between two derived lines ( N ~ c , curve C; A13a, curve D) and four other wild or derived lines. The 50% points on these curves at about 82 and 100 fissions, respectively, indicate that a difference in the time of maturation of about 13 fissions between N4c and A13a was inherited. In each curve in Figure 1, a relatively linear increase in the cumulative number of mature lines lasted for 50 fissions.

Curves A and B in Figure 2, like A and B in Figure 1, depict the results of two 6 X 6 crosses performed simultaneously and involving one common parent, and reveal possible differences in addition to the average length of the immaturity period. (1) The slope of the linear portion of A with 70% of the lines

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104 E. M. SIMON

I I I I I I I I I I I I

Transfer Number FIGURE 1 .-Maturation curves of progeny of crosses among recently collected strains (curves

A and B) and progeny of crosses involving older strains (curves C and D); all of subspecies 6. (A) HG 2 x HG 8, N = 54; (B) HG 2 x U1 7152, N ’ = 54; (C) N4c (I from FA 8 X HG 8) x 4 mating type I11 lines (HG 8; U1 7152; B2b, an “F:’ from HG 2 x HG 8; A13b, an “Fir from CUM 1060 x UM 10911 x [UM 1060 x U1 7152]), N = 59; (D) A13a7 sister of A13b, x the same four I11 lines, N = 51. One subline from each synclone.

I I I I I I I I I ‘ I

Transfer Number FIGURE 2.-Maturation curves of progeny of crosses involving older strains of 2‘. pigmentosa.

(A) UM 1091 x U1 7152, syngen 6, N = 90; (B) UM 1060 x U1 7152, syngen 6, N = 146; (C) pooled data from crosses among Gve strains of subspecies 8, N = 114. Values of N include three sublines per synclone.

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maturing within 26 fissions is steeper than that of B with only 60% maturing in 52 fissions. (2) Between 90 and 115 fissions, no new mature lines appear in curve A, but a relatively steep increase occurred in the next transfer. This same plateau phenomenon occurred between 65 and 80 fissions in at least two of the three intersyngenic crosses, when 70 to 80% of the lines were mature (50% mature points were between 48 and 54 fissions). In crosses among hybrid Fl’s ( 1 to 19 in Table 6) maturity screening for the first 65 fissions indicates an average 507; maturity at about 70 fissions, which is almost identical with the mean 50% point in both Figures 1 and 2.

Curve C in Figure 2, derived from the pooled data on crosses among the old stocks of syngen 8 is also biphasic with a break lasting from 65 to 80 fissions, but in this case fewer than 45% of the lines had matured. Further experiments are required to determine the basis for these departures from a sigmoid distribution, and for the variation in the average length of the immaturity period. Segregation is probably occurring for genetic factors affecting maturation.

In order to investigate the homogeneity of individual synclones, data on three subclones from each of the 46 synclones derived from a cross between the syngen 6 strains UM 1060 and U1 7152 were analyzed (see also curve B, Figure 2). In Figure 3, we have plotted for each pair the transfer in which the first subclone tested mature on the x axis and the transfer in which the third subclone was mature on the y axis. The visually apparent correlation was confirmed by the calculated I value of 0.89. The corresponding value for the first us. second subline is 0.88 and the second us. third, 0.94.

Although a strong correlation in the onset of maturity among the triplet subclones pertains to 43 of the 46 pairs in this experiment, more heterogeneity was observed in 8 X 8 and 6 X 8 crosses: in 20% (18/92) of the pairs more than 50 fissions separated the initial observations of maturity in the first and third subclones. Furthermore, a study of 30 sublines of one hybrid synclone suggested that the onset of maturity even among the sublines of a heterogeneous synclone is not randomly distributed in time. The capacity to mate was observed in 28 of the lines between the third and the seventh transfers, but the remaining two lines showed no inclination to mate until the 15th transfer (200 fissions). Lines from the same synclone that manifest significant differences off er the possibility of additional tests on the heritability and control of the immaturity period.

As noted for syngen 6, a correlation was found between early maturity and abnormal behavior. In these crosses, 70% of the subclones giving strong mating reactions before 40 fissions grew slowly, showed the semi-amicronucleate syndrome and/or were difficult to maintain by single cell isolations. Only 5% of the cultures maturing later showed these symptoms. Although exceptions have been noted, these growth peculiarities and associated early maturity tend to be synclonal, i.e., the subclones of the same abnormal synclone tend to behave alike.

DISCUSSION

Mating type homologies in the subspecies and the question of open vs. closed multiple allelic systems: The homologies of the 6 and 8 mating types are readily demonstrated by the interactions observed in Cerophyl cultures. Each

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106 E. M. SIMON

Transfer Number FIGURE 3.-Correlation in the onset of maturity among triplicate sublines of 46 synclones

from UM lG60 x U1 7152. The transfer following which the first subline mated with a tester is plotted on the .z axis, that of the third subline on the y axis. One transfer = approximately 13 fissions. Numerals in ( ) indicate the number of coincident points. A circle indicates the mean value of y for each 2.

mating type of syngen 6 never mates with one of the types of syngen 8. We have simply declared the identity and established a uniform mating-type nomenclature (Table 4).

The fact that three and only three mating types have been discovered in each subspecies, combined with the conclusion that these mating types are homologous and controlled by equivalent alleles, despite evidence of considerable evolutionary distance between the subspecies (BORDEN et aZ. 1977), leads to the conclusion that this species probably has only three mating types. Previous hypotheses regarding the mating system of syngen 8 were based chiefly on the analysis of three clones isolated from the same sample in Michigan (ORIAS 1963). GRUCHY (1955) crossed two strains of syngen 6, one from Michigan and ope from Florida. With information on such a limited collection, it was necessary to consider the possibility of an open-ended series of peck-order alleles. The probability of iden- tifying precisely the same three alleles in strains of two subspecies isolated in geographically separate habitats is very small. To be sure, additional alleles may

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appear, but the expectation of many more is greatly reduced. The ternary nature of the mating system should be considered likely, and its closed nature a virtual certainty.

WELLS’ (1961 ) conclusion that mating-type inheritance in European syngen 6 (see below) was cytoplasmically controlled as in the B systems in Paramecium was probably based on pairs that had failed to complete conjugation.

Control of mating type specificities: In a vast majority of the synclones derived from 6 x 8 and 8 x 8 matings, the inheritance of mating type is readily explained by simple genic control involving a series of three alleles with peck-order dominance. However, the selfers and the small excess of mating type 111 progeny which appeared in these crosses, as well as in ORIAS’ (1963) syngen 8 crosses, and the apparent lack of transmission of the mat-I allele from I6 parents to normal progeny indicate that some epigenetic process or some instability in the DNA (see below) may be superimposed on that system. In contrast to the situ- ation in subspecies 6, the mat-I allele in subspecies 8 has maintained its dominance over mat-2 and mat-3 in both intra- and intersyngenic crosses. ORIAS (1963) interpreted the appearance of selfers and an excess of mating type 1118 synclones in syngen 8 as the result of a “mutation” or a micronuclear instability. His analysis of a selfer (ORIAS 1959b) revealed aberrant but indistinguishable ratios of mating types among the progeny of stable sublines expressing different mating types when testcrossed. We have found (unpublished) that, among stabilized lines from selfers, the majority mating type is almost always the one determined by the more dominant allele. A common factor in all of these observations is that the instability involves a change from a relatively dominant mating type to a more recessive one.

Examples of other systems with phenotypic instability in strains with identical genomes include mating-type interconversion in Saccharomyces cerevisiae (HICKS, STRATHERN and HERSKOWITZ 1977), phase variation in Salmonella (SILVERMAN et al. 1979) and trichocyst discharge in Paramecium tetraurelia (SONNEBORN and SCHNELLER 1979). Whether or not the mechanism responsible for the changes in T . pigmentosa is similar to that bringing about insertions and inversions of segments of DNA implicated or suggested in these other systems is unknown. The apparent unidirectionality of the changes in T. pigmentosa is seen also in Paramecium, but not in yeast or Salmonella.

Selfing synclones occur in several species of ciliates, but those most pertjnent to this discussion are those of Paramecium bursaria, in which mating types are directly controlled by two loci with two alleles each (JENNINGS 1942; SIEGEL and LARISON 1960). Several workers have considered that the ciliate mating-type loci may be regulatory as well as structural (see NANNEY 1977). Further analysis of a number of selfers, which will be considered in a separate communication, may contribute to our understanding of these heritable, but somewhat unstable, elements.

The nearly universal expression of mating type I11 by the progeny of 6 X 6 crosses poses questions whose resolution will require further studies, using appropriate genetic markers. Three possible explanations are considered here.

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108 E. M. SIMON

One is macronuclear retention. Arguing against the interpretation. however, are the regular occurence of immaturity, which never follows macronuclear retention in T. thermophila, and the appearance of antiserum responses different from those of the type I11 parent.

Another possibility is cytogramy in which the non-I11 exconjugant (in these experiments usually from the old clone) died or generated a sick clone. ORIAS (1959b) showed that, if cytogamy occurred at all in his 8 X 8 crosses, it was not sufficiently frequent to explain the increase in type I118 progeny. ORIAS, HAM- ILTON and FLACKS (1 979) have demonstrated cytogamy in T. thermophila but only in a small percentage of the pairs unless specifically induced.

A third possible interpretation of these events is genomic exclusion (GE) (ALLEN 1967), well documented for T. thermophila, in which the micronuclear contributions of one parent are lost entirely, but different homozygous progeny may be obtained from the same heterozygous parent. GE can thus yield immature and recombinant progeny. The older syngen 6 strains are possibly behaving much like the * (star) strains of T. thermophila. As descrihed by ALLEN (1967) , GE requires an aborted conjugation with micronuclear replacement, then a second conjugation with macronuclear replacement. This two-step process is excluded here because pairs were usually isolated within five to ten hours of the onset of mating, i.e., before second-round pairing could begin, and yet their progeny were immature. Moreover, pairs isolated from one 6 x 8 mixture at seven different times during a period of 67 hours were essentially alike in viability and immaturity of the synclones. These observations exclude two- step GE as observed in T. thermophila. Nevertheless, one should not insist on uniformity of cytogenetic practice in these distantly related species. More- over, one-step GE has been described in T. thermophila (BRUNS, BRUSSARD and KAVKA 1976). It occurs in a frequency of less than 5%, and its evocation to account for the syngen 6 survivors is not unreasonable. In syngen 6, however, if present, it is probably a common mechanism rather than an exception.

WELLS (1961) reported the results of a cross between two strains of European syngen 6 (see below) that are similar to those reported here for strains of Ameri- can syngen 6. Using new mating-type des:gnations, a I x I1 mating produced only I1 progeny. Again the presumably dominant allele was “lost.” An inversion in the order of dominance might be involved in WELLS’ cross, but it is ruled out in our study by the results of the intersyngenic crosses.

The period of immaturity: The period of immaturity found in this series of experiments is shorter than the 120 to 150 fissions following conjugation reported for syngen 8 by ORIAS (1 95913). Whether the parental strains were old or recently isolated from natural sources and whether they belonged to subspecies 6 or 8 or both, more than 98% of subclones were mature before 120 fissions. The mean 50% point for 11 experiments, each involving from 30 to 146 subclones (mean = 71), was 66.6 fissions, which is almost identical with that for inbred families A and B and for a hybrid in T. thermophila (BLEYMAN 1971). Exceptional subclones of T. pigmentosa, however, did not mature until between 150 and 200 fissions; 140 fissions was the latest included in the T . thermophila data. The

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conditions of growth and testing may influence the apparent length of the immature period. Two facets of our testing procedure that would have delayed recognition of maturity in some lines are (1) the practice of reading the screening tests only once, possibly missing mature lines that had not completed their preparation for mating (see BRUNS and PALESTINE 1975 for discussion of this process in T. thermophila) and (2) the use of one tester per subline per transfer, resulting in the mixing of some just-matured lines with the tester of the same mating type. The new maturity data do not support the contention that T. pigmentosa is more of an outbreeder than T. thermophila.

The few synclones manifesting early maturity share other characteristics with those in T. thermophila (BLEYMAN and SIMON 1967; BLEYMAN 1971,1972; NANNEY and MEYER 1977). Before being tested for maturity, many could be identified as early mature because of their slow growth rate, especially in the early fissions after conjugation. The heterogeneity of onset of maturity sometimes observed among sublines of the same synclone (some sublines of three synclones were mature before 25 fissions, but other sublines were immature until 90 or 105 fissions) was also reported by BLEYMAN (1971) for four of 19 pairs in vegetative pedigrees. The shape of the early portions of most of the maturity curves in Figures 1 and 2, and others not reproduced here, suggests that lines mature in the second transfer are not a discrete population. In five of the seven curves (plus three from 6 X 8 crosses not shown) 3% to 13% of the subclones were mature at this time, and new mature lines appeared at each subsequent transfer. In curves A and B of Figure 1 and in 6 X 8 crosses, the cumulative totals increase from this point almost linearly. In the other curves, a break occurs at transfer 4, 5 or 6, and the rate of appearance of new mature lines increases abruptly.

Plateaus similar to that between transfers 7 and 9 in curve A, Figure 2 occurred in four other experiments after 70 to 90% of the subclones were mature. Fur- ther study will be required to elucidate the basis for this behavior, but several phases of maturation show differences that might be exploited as genetic markers.

Special features of intersyngenic mating reactions: We are including in this category three phenomena that may or may not be directly connected. First, the differences between mating reactions in inter- and intrasyngenic mixtures. Why, in the former, do very few pairs form? Why is there usually a 24- to 48- hour delay before mating, in addition to that observed in intrasyngenic mixtures? Why does the mating reaction in many of the 6 X 8 mixtures consist of small clumps of cells from which pairs may emerge? Some pairs never form tight unions, but others may. Similar transient pair formation was described by ELLIOTT, ADDISON and CAREY (1962) with strains of Tetrahymena collected in Europe. It is not clear whether transient interactions were limited to the mixtures of European and American syngen 6 strains (see below). ELLIOTT, ADDISON and CAREY’S interpretation was that strains reacting this way were not of complementary mating types, and the reactions were recorded as negative. However, the apparently normal flow of genetic information in some of our intersyngenic crosses in which this type of mating occurred supports the hypothesis that the

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110 E. M. SIMON

mating types are complementary. Evolutionary divergence has probably affected macromolecules directly involved in pair formation, as it has affected the iso- zymes (BORDEN et al. 1977) and the rDNA (WILD and GALL 1979).

The delayed reactions suggest that for mating to occur between subspecies much more prolonged proximity, or contact, of cells is required than for 6 x 6 or 8 x 8 crosses. This period of interaction may permit the induction of changes in whatever substance or structure is directly involved in the adhesion of cells of complementary types (see MIYAKE 1978). Transient pair formation may indicate a stereochemical incompatability-only a portion of each “active site” being involved-somewhat analogous to that of an enzyme and antimetabolite or weakly cross-reacting antigens and antibodies.

The second type of irregularity occurs in a small fraction of intersyngenic progeny, and only preliminary unpublished observations corxerning it are available. Although wild strains mate strongly with the two permissible testers of their own subspecies, matings with heterologous testers are inconsistent and weaker. When mixed with the complete array of testers, the progeny may give strong mating without unusual delay with one, two, three or all four permissible testers. In addition a few subclones change their patterns with fission age. This instability is not equivalent to mating-type assortment in T . thermophila (ALLEN and NANNEY 1958) in which two or more specificities are expressed in early fissions with later loss of all but one in individual sublines. Nor have we observed the age-related decrease (or loss) in mating reactivity reported by ELLIOTT, ADDISON and CAREY (1962) in syngen 6.

A third irregular aspect of intersyngenic mating concerns the effect of the medium in which the cells have been grown. ELLIOTT’S group (ELLIOTT, ADDISON and CAREY 1962) was unable to confirm ORIAS’ mating between strains of the subspecies 6 and 8 grown in a water suspension of bacteria washed from nutrient agar. However, their report erroneously states that ORIAS used “bacterized water.” We failed to obtain mating in dilute bacterized peptone, which is similar to ELLIOTT et al.’s medium. The Cerophyl rye grass infusion itself, or the bacteria grown in it, supplies some factor necessary for initiation of intersyngenic mating that is not required for intrasygenic mating, or bacterized peptone contains some inhibitory factor.

The possibility of other subspecies of T. pigmentosa: The European (EU) syngen 6 described by ELLIOTT, ADDISON and CAREY (1962) displayed several characteristics suggesting a subspecific relationship with GRUCHY’S American (A) syngen 6. Mating was observed in some mixtures of EU and A strains, even following axen;c growth and washing in distilled water; for this reason both groups were included in variety 6. However, pairs isolated from crosses between A 6 strains of mating-type I and EU-type I1 gave rise to viable Fl’s, but in crosses among these porgeny and lethality was 100%. We do not know of any EU 6 strains available for further study of their relationship to A 6 and to syngen 8. If any are acquired, attempts should be made to isolate pairs from transient matings, since the fertile F1’s in the present study were almost entirely derived

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from matings characterized by few pairs, many of which manifest unstable unions.

Clonal age and senility: According to ELLIOTT (1973), the mating reactivity of wild strains of syngen 6 declined over a period of years and sometimes disappeared completely. The old strains in our study, originally from the same collection (GRUCHY 1955), mated strongly and consistently if mixed from healthy cultures. Nor has a decline in mating been observed in the new collections.

The instability of the expression of mating reactivity mentioned here for intersyngenic hybrids and their progeny resulted in the loss of some specificities and a fluctuating reactivity with some testers. But no instance of loss of ability to mate with all testers has been observed.

Although the 20-year-old stocks of syngen 6 are senescent, io the sense that they can produce few viable progeny, evidence of recombination of mating type and antigenic markers appeared when some were mated with recently collected strains. Moreover, at least two of the 20-year-old stocks of syngen 8 are still capable of producing a high proportion of viable progeny, especially when mated to new stocks of syngen 6. This variation among strains in respect to aging has also been noted in inbred strains of T . thermophila (NANNEY 1974; SIMON and NANNEY 1979) and negates the supposition that all stocks of Tetrahymena lose their breeding potential in about two years or less of laboratory cultivation.

I am grateful to D. L. NANNEY for helpful suggestions and constant encouragement through- out the experimental phases of this work and in the preparation of the manuscript; H.-M. SEYFERT for the cytological study; D. A. SIMON, S.-S. CHEN and L. E. COOPER for technical assistance with a few of the experiments; and the referees for their comments and suggestions.

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between subspecies of tetrahymena pzgmentosa - genetics

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