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Similar times of differentiation of acid phosphatase heterozygotes in two syngens of Tetrahymena pyriformis
DEVELOPMENTAL
BIOLOGY
Similar
29,
65-72 (1972)
Times of Differentiation
Heterozygotes
of Acid Phosphatase
in Two Syngens
of Tetrahymena
pyriformis’ RUTH BROSI Departments
PHILLIPS
*
of Zoology, University of Illinois, Urbana and University of Wisconsin-Milwaukee, 53201 Accepted
Wisconsin
March 14, 1972
Genetic control of acid phosphatase isozymes has been described in two genetic species of Tetrahymena pyriformis: syngen 1 and syngen 7 (Allen et al., 1963a,b; Phillips, 1968). Previous work showed that the isozyme variations revealed by starch gel electrophoresis in syngen 1 were under the control of two alleles at a single locus and that phenotypic assortment takes place in heterozygotes during clonal development. Three allelic variants have been identified in syngen 7 using similar methods. The syngen 7 isozymes are different from those in syngen 1 in pattern, pH, and temperature stability. Despite these differences, heterozygotes in both syngens undergo phenotypic assortment at about the same time in the life cycle, so that within about 250 fissions after conjugation the majority of sublines resemble one or the other of the parental types.
isozymes in another genetic species, syngen 1 (Allen, et al. 1963,a. b. 1965, 1971). Previous work on the genetic control of acid phosphatase isozymes in syngen 1 of Tetrahymenu pyriformis revealed two different patterns controlled by alleles at a single locus or closely linked loci (Allen et al., 1963b). The hybrid has a unique pattern of three bands at first, and later up to five bands. Phenotypic assortment takes place in heterozygotes so that within about 250 fissions after conjugation most of the sublines resemble one of the two parents (Allen et al., 1963b; Allen, 1965, 1971) a minority having a unique hybrid type. Several lines of evidence, including the absence of mating and antigenic cross reactions between syngen 1 and syngen 7 suggest that the two syngens are not closely related (reviewed in Phillips, 1969). This report shows that although the acid phosphatase isozymes in syngen 7 are different in pattern, pH and temperature stability from those in syngen 1, phenotypic assortment occurs in heterozygotes in both syngens at approximately the same time in the life cycle.
INTRODUCTION
The development of an organism involves differential gene expression among genetically identical cells. In multicellular organisms it is characterized by progressive clonal determination of cells. Ciliated protozoa also undergo during their life cycles regular sequential changes that have been compared to developmental events in embryos. Ciliates are especially suitable for studies of gene expression because the external environment can be held constant in serial clonal transfer cultures. Sequential changes which take place under these conditions must involve internal control mechanisms. In this study acid phosphatase isozymes were examined in heterozygous clones of Tetrahymena pyriformis, syngen 7 at different times in the life cycle. The results are compared to a similar study on
‘Part of this work was supported by Grant GM07779 from the National Institutes of Health to Dr. D. L. Nanney. *Present address: Department of Zoology, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53201. 65 Copyright
0 1972 by Academic
All rights of reproduction
Press, Inc.
in any form resewed.
66
DEVELOPMENTALBIOLOGY MATERIALS
AND
METHODS
Wild strains of syngen 7 from Sault Saint Marie, Ontario (Phillips, 1969) and inbred derivatives were screened for acid phosphatases. The following strains inbred for three generations were used in the crosses: strain B (mtB/mtR, RB/RR, PA/PA), C2 (mtA/mtA, RA/RA, P”/P”) and strain D (mtA/mtA, R”/R’, PC/P”). Crosses were made in Cerophyl (0.15% w/v) inoculated with Aerobacter aerogenes and adjusted to pH 7, according to methods described previously (Phillips, 1969). Mating was obtained by centrifuging cultures and washing with demineralized water tc remove the Cerophyl. Several drops of the cultures resuspended in demineralized water were mixed in depression slides and single pairs isolated. Exconjugants were separated after conjugation, allowed to divide once, and the caryonides (first division products) were separated. Caryonidal clones were tested for the ability to mate and those few which mated were discarded since conjugants which have undergone a complete nuclear reorganization enter a period of sexual immaturity lasting about 120 fissions in syngen 7 (Phillips, 1967a; Phillips, 1969). Immature caryonidal clones were carried through a series of single cell isolation transfers in depression slides until they became sexually mature. Samples from initial and subsequent cultures were transferred to axenic peptone by a single cell washing procedure (Phillips, 1967a), Axenic cultures were inoculated into flasks containing proteose peptone (1 ml/ 100 ml) and allowed to grow at 30°C for 3 days before harvesting of cells and preparation of the enzyme extracts. The preparation of extracts, starch gel electrophoresis and identification of enzymes were very similar to those described by Allen et al., 1963a. Cells were concentrated by centrifugation at low speeds and washed twice with demineralized water. The crude extract was obtained by re-
VOLUME 29, 1972
peated freeze thawing of the concentrate. Horizontal electrophoresis was carried out in starch gels prepared in boric acid Tris buffer at pH 7.5 at 10 V/cm for 6 hr at 4°C. Gels were sliced and incubated in 50 mM acetate buffer at pH 5 containing sodium cu-naphthyl acid phosphate and Fast Garnet GBC dye. Gels were washed thoroughly and photographed with a Polaroid Land camera. RESULTS
Acid
Phosphatase
Patterns
in Syngen
7
Three major variations in patterns of acid phosphatase isozymes are found among the Sault Saint Marie strains (Phillips, 1968 and unpublished). Extracts of type A strains possess two intense bands, one at 2.7 cm and one at 4.0 cm toward the anode after 6 hr at 10 V/cm. Extracts of type B strains possess one fairly intense band at 2.7 cm, one intense band at 3.0 cm, and sometimes a faint band at 3.5 cm toward the anode. Extracts of type C strains possess one intense band at 4.0 cm and a less intense one at 3.5 cm toward the anode. All heterozygotes possess four bands at 2.7, 3.0, 3.5, and 4.0 cm toward the anode. Photographs of the phosphatases in different genotypes appear in Fig. 1. The acid phosphatases in syngen 7 share some properties with syngen 1. Both hydrolyze sodium cu-naphthyl acid phosphate and sodium fl-glycerophosphate, and both have similar, although not identical electrophoretic mobilities (Fig. 2). They differ in their stability to pH and temperature. Syngen 1 enzymes are progressively inactivated at pH’s higher than 7.5 and at temperatures of 50°C (Allen et al., 1963a), while syngen 7 enzymes are stable up to pH 9.0 and 68°C. The electrophoretic patterns of the inbred strains of syngen 7 are all distinguishable from those in syngen 1, and the patterns of hybrids within each
Acid Phosphutase
PHILLIPS
A B
67
Heterozygotes
C AB
-a AB Clones
-b FIG. 1. Acid phosphatases in different genotypes of syngen 7: (a) P&/P”, P”/P”, P“/P’ a PA/P” heterozygote. (b) PA/P’ heterozygous subclones at 120 fissions after conjugation.
syngen other.
are quite
Inheritance
of
different
Phosphatases
from
each
in Syngen
7
Three different patterns appear to be controlled by alleles at a single locus (P) or closely linked loci. The distribution of the phosphatase phenotypes in crosses
homozygotes
and
involving type A(P”/P”) and B(PB/PB) strains are shown in Table 1. (Data from crosses involving the type C strains will b e published later.) The crosses were carried out in inoculated Cerophyl. The caryonides were isolated and a sample from each clone was transferred to axenic peptone for preparation of an extract
68
DEVELOPMENTALBIOLOGY BP..,D
2 1' 1
SYNGEN 7
cm.from ORlGlNl
(a)
(b)
SYNGEN
(cl
Cd)
TABLE
x P”iPB x P”lP” xPB/PB
(e)
1
(f)
(CJ) 1
in different genotypes of syngen 7: (a) PA/Pa, (b) P”/P”, A (f) P-l’/p-1 ‘, (g) P-lA/P-I’.
DISTRIBUTION
PA/p” P”/p” pp /P
1 1
3.0 2.7 2.5
FIG 2. Acid phosphatases and of syngen 1: (e) P-l”/P-1
Parental genotypes
VOLUME 29. 1972
OF PHOSPHATASE
Phenotypes PA 8 26
(c) PC/P”, (d) P”/P”
1
PHENOTYPES IN F, AND TEST CROSS
of progeny
P”l” 18 34
12
60
(see Materials and Methods). In all cases the isozyme patterns of the four caryonides derived from a single pair were identical. Thus entries in the table represent phenotypes of pair cultures. The results are consistent with a 1: 2:l ratio in the F2 and a 1: 1 ratio in the backcross to PAlPA. Heterozygotes between PA and PBalways had four bands of equal intensity immediately after conjugation. (Heterozygotes between PAand PCwere somewhat variable and are the subject of a current investigation.) Phenotypic Assortment of Phosphutase Isozymes in Heterozygotes The P”lP” heterozygotes tested initially and up to 40 fissions after conjugation showed four intense bands. However, after about 70 fissions subclones began to lose certain bands and at 240 fissions after conjugation 75% of the subclones resembled one of the parents in enzyme type (Fig. lb shows subclones at 120 fissions). A similar phenotypic assortment has been described for syngen 1 enzymes. Table 2 shows the fraction of stabilized
Total
x2
P
38 60
0.95 1.07
0.7 > P > 0.5
P” P = 0.30
60
TABLE
2
TOTAL FRACTION STABILIZED CULTURES AT SUCCESSIVE FISSIONS AFTER CONJUGATION
Fissions 0 26 65 78 91 104 117 156 182 208 234
$t$~~$~~l 0.04 0.32 0.41 0.45 0.54 0.58 0.69 0.73 -
-
Syngen 7
Syngen lb
-
-
0.08 -
0.05 0.10
-
0.15 0.21
0.27 0.44 0.56 0.69 0.79
0.32 -
UData from a computer printout 1958). b Data from S. L. Allen (1971).
(Schensted,
cultures at different times after conjugation for both syngens. The data for syngen 7 was obtained from two experiments. In the first experiment PA/P” heterozygotes from a cross of inbred strains B (PA/PA) and C (PB/ P”) were analyzed. All four caryonides were obtained from 60 pairs. Each caryo-
PHILLIPS
Acid Phosphatase Heterorygotes
nide was carried through approximately 117 fissions by serial single cell transfers at 13 fission intervals (9 transfers). The phosphatases were examined in cultures tubed at 39 fissions (3rd transfer), 78 fissions (6th transfer), and 117 fissions (9th transfer). In the second experiment PA/P’ heterozygotes from a test cross of strain B x B/C2 Fl were analyzed. All four caryonides were obtained from 48 pairs. Each caryonide was carried through approximately 234 fissions (18 transfers). Cultures were examined for phosphatases at 156 fissions (12 transfers), 182 fissions (14 transfers), and 208 fissions (16 transfers) and 234 fissions (18 transfers). Data are shown in Table 2. The syngen 1 data are from a cross of inbred strains A and B (Allen, 1971). Four caryonides were analyzed from 25 pairs, carried through about 117 fissions (9 transfers). The enzymes were examined after each transfer. The time at which assortment begins and the rate of stabilization appear to be very similar for the two syngens. In both cases approximately equal numbers of sublines stabilize to the two parental types. In the syngen 7 experiments, 58% A, 42% B were found in the first experiment and 46% A, 54% B in the second experiment. Heterozygotes of type P”l P” and P”lP” also undergo assortment, although no clonal analyses have been done. A difference between the two syngens is the appearance of a third stable type with a hybrid phenotype much later at low frequency in syngen 1 (Allen, 1963b, 1971). Since Tetrahymena pyriformis has a compound macronucleus, there may be a phenotypic lag between the initial differentiation or repression of gene loci and the appearance of pure clones. Stabilization of pure types from heterozygotes occurs over a considerable time period at what appears to be a constant rate in several different genetic systems in syngen 1
69
(Nanney and Dubert, 1960; Phillips, 1967b; Allen, 1965, 1971). This has been explained by a model (reviewed in Nanney, 1964) involving differentiation of macronuclear subunits at a specific time followed by appearance of pure clones as the heterogeneous macronuclei become homogeneous during clonal multiplication (see Discussion). Schensted (1958) programmed a computer to simulate such a process. The data from the computer (MIDAC) for the case in which differentiation occurs immediately after conjugation and yields approximately equal numbers of the two pure types are shown in Table 2. Since the fraction of stabilized lines at 117 fissions is similar to that expected at 65 fissions after differentiation, the phosphatases differentiate at about 50 fissions after conjugation in both syngens according to this model. DISCUSSION
The results of this study show that the timing of differentiation in acid phosphatase isozyme heterozygotes is very similar in two different syngens which appear to be quite distantly related. The evidence for this distance is considerable. At the molecular level, the GC ratio (percent of total DNA bases) is 27% in syngen 1 and 25% in syngen 7, suggesting that perhaps these two genetic species are further removed from each other than the vertebrate classes, such as fishes and birds, are from each other. The antigenic and mating comparisons agree with this distance. In Paramecium aurelia, antigenie and mating type cross reactions often occur between different syngens which can be ordered with respect to each other by these data (reviewed in Sonneborn, 1957). There is no evidence for any antigens held in common or any mating cross reactions between the two syngens of Tetrahymena pyriformis. In addition, habitat and maturation time are quite different. Syngen 1 has been col-
70
DEVELOPMENTAL BIOLOGY
lected mainly from lakes, syngen 7 from running water (Gruchy, 1955). In syngen 1 cells become mature for mating at about 60-80 fissions after conjugation, while this occurs at 120-150 fissions in syngen 7. Are the phosphatase enzymes studied in the two syngens homologous enzymes, specified by genes which have diverged only slightly in the course of evolution? The answer to this question is by no means clear. Allen et al. (1963a) found up to 20 different phosphatase isozymes in syngen 1, probably specified by several different genes. The only reproducible variant in the strains available for genetic analysis in syngen 1 is that controlled by the P-l locus. Similarly in syngen 7, the phosphatase studied was the one for which phenotypic variation was found in the strains available for genetic analysis. Conditions of electrophoresis and mobilities of the enzymes are quite similar in both cases, although the patterns are different. Small alterations in the charge of enzyme molecules could of course account for the differences in pattern. If the enzyme loci are homologous, however, considerable change has occurred in the proteins, since the pH and temperature stabilities of the two groups of enzymes are quite different. The current analysis of the third isozymic variant in syngen 7 may elucidate the nature of these isozymes and aid in the comparison with syngen 1. A survey of phosphatase isozymes obtained under similar conditions in twelve different syngens of Tetrahymenu pyriformis (Allen and Weremiuk, 1971) showed that the enzymes are polymorphic with intrasyngenic differences occurring in almost every case, making it impossible to ascertain intersyngenic relationships. A number of possible mechanisms for the differentiations can be considered. An important point is whether they represent some type of chromosomal inac-
VOLUME 29, 1972
tivation or if they are restricted to a single locus. In other words, is the phenomenon similar to the X chromosome inactivation of mammals, or is allelic repression or differentiation involved? The evidence (reviewed in Nanney, 1964) is very strong for allelic differentiation. Differentiation of heterozygotes occurs in several genetic systems in syngen 1 and has been the subject of many detailed studies (Allen, 1965, 1971; Bleyman and Simon, 1968; Bleyman et al., 1966; Nanney and Dubert, 1960; Nanney et al., 1964; Phillips, 1967b). An important discovery was that the timing of differentiation is different for the various gene loci studied. In syngen 1, differentiation of heterozygotes occurs at 2, 30, 40, and 50 fissions after conjugation for the H, T, E-l, and P-l loci, respectively. In syngen 7, the phosphatase heterozygotes differentiate at about 50 fissions, whereas the antigenic heterozygotes studied do not appear to any differentiation (Phillips, undergo 1971). Shortly after phenotypic divergence has begun, the stabilization rates are similar for all systems in which this has been studied in syngen 1. The data from syngen 7 phosphatases, although not, precise enough to pinpoint a stabilization rate, shows that the stabilization process is similar to syngen 1. Since Tetrahymena has a compound macronucleus, these results suggest the presence of a number of subunits in the macronucleus which become determined for expression at each locus at a particular time and are then segregated or assorted into sublines. According to the model proposed by Nanney (1964) the macronucleus contains diploid subunits and differentiation of loci involves allelic repression. During the stabilization process, diploid subunits of a heterogeneous macronucleus are assorted to different daughter cells to yield pure clones (see Results). Another
PHILLIPS
Acid Phosphutase
model proposed by Allen and Gibson (1971) involves differential replication of chromosome fragments (masters) and subsequent segregation of nonreplicating copies (slaves). Recently experimental evidence of gene diminution during the formation of the macronucleus in certain ciliates has been presented (Bostock and Prescott, 1972). Further information on the structure of the macronucleus will be required to distinguish between these models. One way to investigate the mechanism of differentiation would be to attempt to change the timing. This has been unsuccessful where tried by environmental means, but mutants have been obtained which have different times for mating maturation in syngen 1 (Bleyman and Simon, 1967). The differentiation time for other loci was unaffected in these mutants, further supporting the independent nature of these events. If enzyme differentiations had been linked with maturation, one would expect the time of differentiation in the early maturing mutants to be moved up for the phosphatases. This was not the case (Bleyman, 1971). In summary, the search for the mechanism of timing of differentiation in Tetrahymena has been unsuccessful so far. Attempts at changing the timing by environmental modifications have failed. In one case (time of sexual maturation) Mendelian mutants have been obtained which are different from normal in their timing, but the time of differentiation at other loci was unaffected. Previous work elucidating the precision of timing for different loci and the extension of the sequence over relatively long time periods would seem to rule out explanations involving dilution of substances or accidental events. The fact that this precision in timing has been maintained over what appears to be a long period of
Heterozygotes
71
evolutionary time emphasizes the importance of this mechanism for the survival of the organism. REFERENCES ALLEN, S. L., (1965). Genetic control of enzymes in Tetrahymenn. Brookhaven Symp. Biol. 18, 27-54. ALLEN, S. L., (1971). A late-determined gene in Tetrahymena heterozygotes. Genetics 68, 415-433. ALLEN, S. L., and GIBSON, E. (1971). Genetics of Tetrahymena. In “Biology of Tetrahymena.” (A. M. Elliot, ed.). Appleton, Century Crofts, New York. ALLEN, S. L., and NANNEY, D. L. (1958). An analysis of nuclear differentiation in selfers of Tetrahymenu. Amer. Natur. 92, 139-160. ALLEN, S. L., and WEREMIUK, S. L. (1971). Intersyngenic variations in the esterases and acid phosphatases of Tetrahymena pyriformis. Biochem. Genet. 5, 119-133. ALLEN, S. L., MISCH, M. S., and MORRISON, B. (1963a). Variations in the electrophoretically separated acid phosphatases of Tetrahymenu. J. Histochem. Cytochem. 11, 706-719. ALLEN, S. L., MISCH, M. S., and MORRISON, B. (1963b). Genetic control of acid phosphatase in Tetrahymena: formation of a hybrid enzyme. Genetics 48, 1635-1658. BLEYMAN, L. K. (1971). Temporal patterns in the ciliated protozoa. In “Developmental Aspects of the Cell Cycle” (I. L. Cameron, G. M. Padilla and A. Zimmerman, eds), pp. 67-91. Academic Press, New York. BLEYMAN, L. K., and SIMON, E. M. (1967). Genetic control of maturity in Tetrahymena pyriformis. Genet. Res. 10, 319-321. BLEYMAN, L. K., and SIMON, E. M. (1968). Clonal analysis of nuclear differentiation in Tetrahymena. Deuelop. Biol. 18, 217-231. BLEYMAN, L. K., SIMON, E. M., and BROSI, R. (1966). Sequential nuclear differentiation in Tetrahymenn. Genetics 54, 277-291. BOSTOCK, C. J. and PRESCOTT,D. M. (1972). Evidence of gene diminution in the formation of the macronucleus in the protozoan Stylonychia. Proc. Nat. Acad. Sci. U.S. 69, 139-142. GRUCHY, D. F. (1955). The breeding system and distribution of Tetrahymena pyriformis. J. Protozool. 2, 178-185. NANNEY, D. L. (1964). Macronuclear differentiation and subnuclear assortment in Cilia&. Symp. Sot. Study Develop. 23, 253-273. NANNEY, D. L., and DUBERT, J. M. (1960). The genetics of the H serotype system in variety 1 of Tetrahymena pyriformis. Genetics 45, 1335-1349.
72
DEVELOPMENTALBIOLOGY
NANNEY, D. L., NAGEL, M. J., and TOUCHBERRY, R. W. (1964). The timing of H antigenic differentiation in Tetrahymenu. J. Exp. Zool. 155, 25-42. PHILLIPS, R. B. (1967a). Inheritance of T serotypes in Tetrahymena. Genetics 56, 667-681. PHILLIPS, R. B. (1967b). T serotype differentiation in Tetrahymena. Genetics 56, 683-692. PHILLIPS, R. B. (1968). Genetic control of acid phosphatase isoenzymes in syngen 7 of Tetrahymenu py$formis. Genetics 60, Suppl., 211-212 (Abstract). PHILLIPS, R. B. (1969). Mating type inheritance in syngen 7 of Tetrahymena pyriformis: intra- and interallelic interactions. Genetics 63, 349-359. PHII.LIP~, Ii B. (1971). Inheritance of immobilization
VOLUME 29, 1972
antigens in syngen 7 of Tetrahymena pyriformis: evidence for a regulatory gene. Genetics 67, 391-398. SCHENSTED, I. V. (1958). Appendix: Model of subnuclear segregation in the macronucleus of ciliates. Amer. Natur. 92, 161-170. SONNEBORN, T. M. (1957). Breeding systems, reproductive methods and species problems in Protozoa. In “The Species Problem” (E. Mayr, ed.), pp. 155-324. Amer. Ass. Advan. Sci., Washington, D. C. SUEOKA, N. (1961). Compositional correlation between deoxyribonucleic acid and protein. Cold Spring Harbor Symp. Quant. Biol. 26,35-43.
BIOLOGY
Similar
29,
65-72 (1972)
Times of Differentiation
Heterozygotes
of Acid Phosphatase
in Two Syngens
of Tetrahymena
pyriformis’ RUTH BROSI Departments
PHILLIPS
*
of Zoology, University of Illinois, Urbana and University of Wisconsin-Milwaukee, 53201 Accepted
Wisconsin
March 14, 1972
Genetic control of acid phosphatase isozymes has been described in two genetic species of Tetrahymena pyriformis: syngen 1 and syngen 7 (Allen et al., 1963a,b; Phillips, 1968). Previous work showed that the isozyme variations revealed by starch gel electrophoresis in syngen 1 were under the control of two alleles at a single locus and that phenotypic assortment takes place in heterozygotes during clonal development. Three allelic variants have been identified in syngen 7 using similar methods. The syngen 7 isozymes are different from those in syngen 1 in pattern, pH, and temperature stability. Despite these differences, heterozygotes in both syngens undergo phenotypic assortment at about the same time in the life cycle, so that within about 250 fissions after conjugation the majority of sublines resemble one or the other of the parental types.
isozymes in another genetic species, syngen 1 (Allen, et al. 1963,a. b. 1965, 1971). Previous work on the genetic control of acid phosphatase isozymes in syngen 1 of Tetrahymenu pyriformis revealed two different patterns controlled by alleles at a single locus or closely linked loci (Allen et al., 1963b). The hybrid has a unique pattern of three bands at first, and later up to five bands. Phenotypic assortment takes place in heterozygotes so that within about 250 fissions after conjugation most of the sublines resemble one of the two parents (Allen et al., 1963b; Allen, 1965, 1971) a minority having a unique hybrid type. Several lines of evidence, including the absence of mating and antigenic cross reactions between syngen 1 and syngen 7 suggest that the two syngens are not closely related (reviewed in Phillips, 1969). This report shows that although the acid phosphatase isozymes in syngen 7 are different in pattern, pH and temperature stability from those in syngen 1, phenotypic assortment occurs in heterozygotes in both syngens at approximately the same time in the life cycle.
INTRODUCTION
The development of an organism involves differential gene expression among genetically identical cells. In multicellular organisms it is characterized by progressive clonal determination of cells. Ciliated protozoa also undergo during their life cycles regular sequential changes that have been compared to developmental events in embryos. Ciliates are especially suitable for studies of gene expression because the external environment can be held constant in serial clonal transfer cultures. Sequential changes which take place under these conditions must involve internal control mechanisms. In this study acid phosphatase isozymes were examined in heterozygous clones of Tetrahymena pyriformis, syngen 7 at different times in the life cycle. The results are compared to a similar study on
‘Part of this work was supported by Grant GM07779 from the National Institutes of Health to Dr. D. L. Nanney. *Present address: Department of Zoology, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53201. 65 Copyright
0 1972 by Academic
All rights of reproduction
Press, Inc.
in any form resewed.
66
DEVELOPMENTALBIOLOGY MATERIALS
AND
METHODS
Wild strains of syngen 7 from Sault Saint Marie, Ontario (Phillips, 1969) and inbred derivatives were screened for acid phosphatases. The following strains inbred for three generations were used in the crosses: strain B (mtB/mtR, RB/RR, PA/PA), C2 (mtA/mtA, RA/RA, P”/P”) and strain D (mtA/mtA, R”/R’, PC/P”). Crosses were made in Cerophyl (0.15% w/v) inoculated with Aerobacter aerogenes and adjusted to pH 7, according to methods described previously (Phillips, 1969). Mating was obtained by centrifuging cultures and washing with demineralized water tc remove the Cerophyl. Several drops of the cultures resuspended in demineralized water were mixed in depression slides and single pairs isolated. Exconjugants were separated after conjugation, allowed to divide once, and the caryonides (first division products) were separated. Caryonidal clones were tested for the ability to mate and those few which mated were discarded since conjugants which have undergone a complete nuclear reorganization enter a period of sexual immaturity lasting about 120 fissions in syngen 7 (Phillips, 1967a; Phillips, 1969). Immature caryonidal clones were carried through a series of single cell isolation transfers in depression slides until they became sexually mature. Samples from initial and subsequent cultures were transferred to axenic peptone by a single cell washing procedure (Phillips, 1967a), Axenic cultures were inoculated into flasks containing proteose peptone (1 ml/ 100 ml) and allowed to grow at 30°C for 3 days before harvesting of cells and preparation of the enzyme extracts. The preparation of extracts, starch gel electrophoresis and identification of enzymes were very similar to those described by Allen et al., 1963a. Cells were concentrated by centrifugation at low speeds and washed twice with demineralized water. The crude extract was obtained by re-
VOLUME 29, 1972
peated freeze thawing of the concentrate. Horizontal electrophoresis was carried out in starch gels prepared in boric acid Tris buffer at pH 7.5 at 10 V/cm for 6 hr at 4°C. Gels were sliced and incubated in 50 mM acetate buffer at pH 5 containing sodium cu-naphthyl acid phosphate and Fast Garnet GBC dye. Gels were washed thoroughly and photographed with a Polaroid Land camera. RESULTS
Acid
Phosphatase
Patterns
in Syngen
7
Three major variations in patterns of acid phosphatase isozymes are found among the Sault Saint Marie strains (Phillips, 1968 and unpublished). Extracts of type A strains possess two intense bands, one at 2.7 cm and one at 4.0 cm toward the anode after 6 hr at 10 V/cm. Extracts of type B strains possess one fairly intense band at 2.7 cm, one intense band at 3.0 cm, and sometimes a faint band at 3.5 cm toward the anode. Extracts of type C strains possess one intense band at 4.0 cm and a less intense one at 3.5 cm toward the anode. All heterozygotes possess four bands at 2.7, 3.0, 3.5, and 4.0 cm toward the anode. Photographs of the phosphatases in different genotypes appear in Fig. 1. The acid phosphatases in syngen 7 share some properties with syngen 1. Both hydrolyze sodium cu-naphthyl acid phosphate and sodium fl-glycerophosphate, and both have similar, although not identical electrophoretic mobilities (Fig. 2). They differ in their stability to pH and temperature. Syngen 1 enzymes are progressively inactivated at pH’s higher than 7.5 and at temperatures of 50°C (Allen et al., 1963a), while syngen 7 enzymes are stable up to pH 9.0 and 68°C. The electrophoretic patterns of the inbred strains of syngen 7 are all distinguishable from those in syngen 1, and the patterns of hybrids within each
Acid Phosphutase
PHILLIPS
A B
67
Heterozygotes
C AB
-a AB Clones
-b FIG. 1. Acid phosphatases in different genotypes of syngen 7: (a) P&/P”, P”/P”, P“/P’ a PA/P” heterozygote. (b) PA/P’ heterozygous subclones at 120 fissions after conjugation.
syngen other.
are quite
Inheritance
of
different
Phosphatases
from
each
in Syngen
7
Three different patterns appear to be controlled by alleles at a single locus (P) or closely linked loci. The distribution of the phosphatase phenotypes in crosses
homozygotes
and
involving type A(P”/P”) and B(PB/PB) strains are shown in Table 1. (Data from crosses involving the type C strains will b e published later.) The crosses were carried out in inoculated Cerophyl. The caryonides were isolated and a sample from each clone was transferred to axenic peptone for preparation of an extract
68
DEVELOPMENTALBIOLOGY BP..,D
2 1' 1
SYNGEN 7
cm.from ORlGlNl
(a)
(b)
SYNGEN
(cl
Cd)
TABLE
x P”iPB x P”lP” xPB/PB
(e)
1
(f)
(CJ) 1
in different genotypes of syngen 7: (a) PA/Pa, (b) P”/P”, A (f) P-l’/p-1 ‘, (g) P-lA/P-I’.
DISTRIBUTION
PA/p” P”/p” pp /P
1 1
3.0 2.7 2.5
FIG 2. Acid phosphatases and of syngen 1: (e) P-l”/P-1
Parental genotypes
VOLUME 29. 1972
OF PHOSPHATASE
Phenotypes PA 8 26
(c) PC/P”, (d) P”/P”
1
PHENOTYPES IN F, AND TEST CROSS
of progeny
P”l” 18 34
12
60
(see Materials and Methods). In all cases the isozyme patterns of the four caryonides derived from a single pair were identical. Thus entries in the table represent phenotypes of pair cultures. The results are consistent with a 1: 2:l ratio in the F2 and a 1: 1 ratio in the backcross to PAlPA. Heterozygotes between PA and PBalways had four bands of equal intensity immediately after conjugation. (Heterozygotes between PAand PCwere somewhat variable and are the subject of a current investigation.) Phenotypic Assortment of Phosphutase Isozymes in Heterozygotes The P”lP” heterozygotes tested initially and up to 40 fissions after conjugation showed four intense bands. However, after about 70 fissions subclones began to lose certain bands and at 240 fissions after conjugation 75% of the subclones resembled one of the parents in enzyme type (Fig. lb shows subclones at 120 fissions). A similar phenotypic assortment has been described for syngen 1 enzymes. Table 2 shows the fraction of stabilized
Total
x2
P
38 60
0.95 1.07
0.7 > P > 0.5
P” P = 0.30
60
TABLE
2
TOTAL FRACTION STABILIZED CULTURES AT SUCCESSIVE FISSIONS AFTER CONJUGATION
Fissions 0 26 65 78 91 104 117 156 182 208 234
$t$~~$~~l 0.04 0.32 0.41 0.45 0.54 0.58 0.69 0.73 -
-
Syngen 7
Syngen lb
-
-
0.08 -
0.05 0.10
-
0.15 0.21
0.27 0.44 0.56 0.69 0.79
0.32 -
UData from a computer printout 1958). b Data from S. L. Allen (1971).
(Schensted,
cultures at different times after conjugation for both syngens. The data for syngen 7 was obtained from two experiments. In the first experiment PA/P” heterozygotes from a cross of inbred strains B (PA/PA) and C (PB/ P”) were analyzed. All four caryonides were obtained from 60 pairs. Each caryo-
PHILLIPS
Acid Phosphatase Heterorygotes
nide was carried through approximately 117 fissions by serial single cell transfers at 13 fission intervals (9 transfers). The phosphatases were examined in cultures tubed at 39 fissions (3rd transfer), 78 fissions (6th transfer), and 117 fissions (9th transfer). In the second experiment PA/P’ heterozygotes from a test cross of strain B x B/C2 Fl were analyzed. All four caryonides were obtained from 48 pairs. Each caryonide was carried through approximately 234 fissions (18 transfers). Cultures were examined for phosphatases at 156 fissions (12 transfers), 182 fissions (14 transfers), and 208 fissions (16 transfers) and 234 fissions (18 transfers). Data are shown in Table 2. The syngen 1 data are from a cross of inbred strains A and B (Allen, 1971). Four caryonides were analyzed from 25 pairs, carried through about 117 fissions (9 transfers). The enzymes were examined after each transfer. The time at which assortment begins and the rate of stabilization appear to be very similar for the two syngens. In both cases approximately equal numbers of sublines stabilize to the two parental types. In the syngen 7 experiments, 58% A, 42% B were found in the first experiment and 46% A, 54% B in the second experiment. Heterozygotes of type P”l P” and P”lP” also undergo assortment, although no clonal analyses have been done. A difference between the two syngens is the appearance of a third stable type with a hybrid phenotype much later at low frequency in syngen 1 (Allen, 1963b, 1971). Since Tetrahymena pyriformis has a compound macronucleus, there may be a phenotypic lag between the initial differentiation or repression of gene loci and the appearance of pure clones. Stabilization of pure types from heterozygotes occurs over a considerable time period at what appears to be a constant rate in several different genetic systems in syngen 1
69
(Nanney and Dubert, 1960; Phillips, 1967b; Allen, 1965, 1971). This has been explained by a model (reviewed in Nanney, 1964) involving differentiation of macronuclear subunits at a specific time followed by appearance of pure clones as the heterogeneous macronuclei become homogeneous during clonal multiplication (see Discussion). Schensted (1958) programmed a computer to simulate such a process. The data from the computer (MIDAC) for the case in which differentiation occurs immediately after conjugation and yields approximately equal numbers of the two pure types are shown in Table 2. Since the fraction of stabilized lines at 117 fissions is similar to that expected at 65 fissions after differentiation, the phosphatases differentiate at about 50 fissions after conjugation in both syngens according to this model. DISCUSSION
The results of this study show that the timing of differentiation in acid phosphatase isozyme heterozygotes is very similar in two different syngens which appear to be quite distantly related. The evidence for this distance is considerable. At the molecular level, the GC ratio (percent of total DNA bases) is 27% in syngen 1 and 25% in syngen 7, suggesting that perhaps these two genetic species are further removed from each other than the vertebrate classes, such as fishes and birds, are from each other. The antigenic and mating comparisons agree with this distance. In Paramecium aurelia, antigenie and mating type cross reactions often occur between different syngens which can be ordered with respect to each other by these data (reviewed in Sonneborn, 1957). There is no evidence for any antigens held in common or any mating cross reactions between the two syngens of Tetrahymena pyriformis. In addition, habitat and maturation time are quite different. Syngen 1 has been col-
70
DEVELOPMENTAL BIOLOGY
lected mainly from lakes, syngen 7 from running water (Gruchy, 1955). In syngen 1 cells become mature for mating at about 60-80 fissions after conjugation, while this occurs at 120-150 fissions in syngen 7. Are the phosphatase enzymes studied in the two syngens homologous enzymes, specified by genes which have diverged only slightly in the course of evolution? The answer to this question is by no means clear. Allen et al. (1963a) found up to 20 different phosphatase isozymes in syngen 1, probably specified by several different genes. The only reproducible variant in the strains available for genetic analysis in syngen 1 is that controlled by the P-l locus. Similarly in syngen 7, the phosphatase studied was the one for which phenotypic variation was found in the strains available for genetic analysis. Conditions of electrophoresis and mobilities of the enzymes are quite similar in both cases, although the patterns are different. Small alterations in the charge of enzyme molecules could of course account for the differences in pattern. If the enzyme loci are homologous, however, considerable change has occurred in the proteins, since the pH and temperature stabilities of the two groups of enzymes are quite different. The current analysis of the third isozymic variant in syngen 7 may elucidate the nature of these isozymes and aid in the comparison with syngen 1. A survey of phosphatase isozymes obtained under similar conditions in twelve different syngens of Tetrahymenu pyriformis (Allen and Weremiuk, 1971) showed that the enzymes are polymorphic with intrasyngenic differences occurring in almost every case, making it impossible to ascertain intersyngenic relationships. A number of possible mechanisms for the differentiations can be considered. An important point is whether they represent some type of chromosomal inac-
VOLUME 29, 1972
tivation or if they are restricted to a single locus. In other words, is the phenomenon similar to the X chromosome inactivation of mammals, or is allelic repression or differentiation involved? The evidence (reviewed in Nanney, 1964) is very strong for allelic differentiation. Differentiation of heterozygotes occurs in several genetic systems in syngen 1 and has been the subject of many detailed studies (Allen, 1965, 1971; Bleyman and Simon, 1968; Bleyman et al., 1966; Nanney and Dubert, 1960; Nanney et al., 1964; Phillips, 1967b). An important discovery was that the timing of differentiation is different for the various gene loci studied. In syngen 1, differentiation of heterozygotes occurs at 2, 30, 40, and 50 fissions after conjugation for the H, T, E-l, and P-l loci, respectively. In syngen 7, the phosphatase heterozygotes differentiate at about 50 fissions, whereas the antigenic heterozygotes studied do not appear to any differentiation (Phillips, undergo 1971). Shortly after phenotypic divergence has begun, the stabilization rates are similar for all systems in which this has been studied in syngen 1. The data from syngen 7 phosphatases, although not, precise enough to pinpoint a stabilization rate, shows that the stabilization process is similar to syngen 1. Since Tetrahymena has a compound macronucleus, these results suggest the presence of a number of subunits in the macronucleus which become determined for expression at each locus at a particular time and are then segregated or assorted into sublines. According to the model proposed by Nanney (1964) the macronucleus contains diploid subunits and differentiation of loci involves allelic repression. During the stabilization process, diploid subunits of a heterogeneous macronucleus are assorted to different daughter cells to yield pure clones (see Results). Another
PHILLIPS
Acid Phosphutase
model proposed by Allen and Gibson (1971) involves differential replication of chromosome fragments (masters) and subsequent segregation of nonreplicating copies (slaves). Recently experimental evidence of gene diminution during the formation of the macronucleus in certain ciliates has been presented (Bostock and Prescott, 1972). Further information on the structure of the macronucleus will be required to distinguish between these models. One way to investigate the mechanism of differentiation would be to attempt to change the timing. This has been unsuccessful where tried by environmental means, but mutants have been obtained which have different times for mating maturation in syngen 1 (Bleyman and Simon, 1967). The differentiation time for other loci was unaffected in these mutants, further supporting the independent nature of these events. If enzyme differentiations had been linked with maturation, one would expect the time of differentiation in the early maturing mutants to be moved up for the phosphatases. This was not the case (Bleyman, 1971). In summary, the search for the mechanism of timing of differentiation in Tetrahymena has been unsuccessful so far. Attempts at changing the timing by environmental modifications have failed. In one case (time of sexual maturation) Mendelian mutants have been obtained which are different from normal in their timing, but the time of differentiation at other loci was unaffected. Previous work elucidating the precision of timing for different loci and the extension of the sequence over relatively long time periods would seem to rule out explanations involving dilution of substances or accidental events. The fact that this precision in timing has been maintained over what appears to be a long period of
Heterozygotes
71
evolutionary time emphasizes the importance of this mechanism for the survival of the organism. REFERENCES ALLEN, S. L., (1965). Genetic control of enzymes in Tetrahymenn. Brookhaven Symp. Biol. 18, 27-54. ALLEN, S. L., (1971). A late-determined gene in Tetrahymena heterozygotes. Genetics 68, 415-433. ALLEN, S. L., and GIBSON, E. (1971). Genetics of Tetrahymena. In “Biology of Tetrahymena.” (A. M. Elliot, ed.). Appleton, Century Crofts, New York. ALLEN, S. L., and NANNEY, D. L. (1958). An analysis of nuclear differentiation in selfers of Tetrahymenu. Amer. Natur. 92, 139-160. ALLEN, S. L., and WEREMIUK, S. L. (1971). Intersyngenic variations in the esterases and acid phosphatases of Tetrahymena pyriformis. Biochem. Genet. 5, 119-133. ALLEN, S. L., MISCH, M. S., and MORRISON, B. (1963a). Variations in the electrophoretically separated acid phosphatases of Tetrahymenu. J. Histochem. Cytochem. 11, 706-719. ALLEN, S. L., MISCH, M. S., and MORRISON, B. (1963b). Genetic control of acid phosphatase in Tetrahymena: formation of a hybrid enzyme. Genetics 48, 1635-1658. BLEYMAN, L. K. (1971). Temporal patterns in the ciliated protozoa. In “Developmental Aspects of the Cell Cycle” (I. L. Cameron, G. M. Padilla and A. Zimmerman, eds), pp. 67-91. Academic Press, New York. BLEYMAN, L. K., and SIMON, E. M. (1967). Genetic control of maturity in Tetrahymena pyriformis. Genet. Res. 10, 319-321. BLEYMAN, L. K., and SIMON, E. M. (1968). Clonal analysis of nuclear differentiation in Tetrahymena. Deuelop. Biol. 18, 217-231. BLEYMAN, L. K., SIMON, E. M., and BROSI, R. (1966). Sequential nuclear differentiation in Tetrahymenn. Genetics 54, 277-291. BOSTOCK, C. J. and PRESCOTT,D. M. (1972). Evidence of gene diminution in the formation of the macronucleus in the protozoan Stylonychia. Proc. Nat. Acad. Sci. U.S. 69, 139-142. GRUCHY, D. F. (1955). The breeding system and distribution of Tetrahymena pyriformis. J. Protozool. 2, 178-185. NANNEY, D. L. (1964). Macronuclear differentiation and subnuclear assortment in Cilia&. Symp. Sot. Study Develop. 23, 253-273. NANNEY, D. L., and DUBERT, J. M. (1960). The genetics of the H serotype system in variety 1 of Tetrahymena pyriformis. Genetics 45, 1335-1349.
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DEVELOPMENTALBIOLOGY
NANNEY, D. L., NAGEL, M. J., and TOUCHBERRY, R. W. (1964). The timing of H antigenic differentiation in Tetrahymenu. J. Exp. Zool. 155, 25-42. PHILLIPS, R. B. (1967a). Inheritance of T serotypes in Tetrahymena. Genetics 56, 667-681. PHILLIPS, R. B. (1967b). T serotype differentiation in Tetrahymena. Genetics 56, 683-692. PHILLIPS, R. B. (1968). Genetic control of acid phosphatase isoenzymes in syngen 7 of Tetrahymenu py$formis. Genetics 60, Suppl., 211-212 (Abstract). PHILLIPS, R. B. (1969). Mating type inheritance in syngen 7 of Tetrahymena pyriformis: intra- and interallelic interactions. Genetics 63, 349-359. PHII.LIP~, Ii B. (1971). Inheritance of immobilization
VOLUME 29, 1972
antigens in syngen 7 of Tetrahymena pyriformis: evidence for a regulatory gene. Genetics 67, 391-398. SCHENSTED, I. V. (1958). Appendix: Model of subnuclear segregation in the macronucleus of ciliates. Amer. Natur. 92, 161-170. SONNEBORN, T. M. (1957). Breeding systems, reproductive methods and species problems in Protozoa. In “The Species Problem” (E. Mayr, ed.), pp. 155-324. Amer. Ass. Advan. Sci., Washington, D. C. SUEOKA, N. (1961). Compositional correlation between deoxyribonucleic acid and protein. Cold Spring Harbor Symp. Quant. Biol. 26,35-43.