frankel (heckmann frankel

PROPAGATION OF CORTICAL DIFFERENCES IN TETRAHYMENA

JOSEPH FRANKEL

Department of Zoology, University of Iowa, Iowa City, Iowa 52242

Manuscript received May 14, 1979 Revised copy received October 1, 1979

ABSTRACT

Progeny clones were derived froin crosses arranged so that the number of ciliary meridians (corticotype) was unusually high in one partner, and normal in the other. An analysis of the propagation of corticotypes during maintenance of these clones for up to 1,000 fissions indicated that corticotypos above 21 undergo a rapid downward shift, while corticotypes in the range of 18 to 21 change slowly. Although these observations are consistent with NANNEY’S earlier deduction of a “stability center” at corticotype 19, there appears to be little if any difference in the stability of perpetuation of corticotypes 18, 19 and 20. Within this “stability range,” the inertia of maintenance of pre-existing corticotypes is sufficiently strong that sister clones derived from an exconjugant pair can remain different for 1,000 fissions. These findings are consistent with observations made earlier, and those in the present study, indicating that cells in stock cultures express a substantial range of corticotypes even when maintained with frequent transfer. The results suggest that mechanisms of spatially ordered structural assembly within the cell can show sufficient fidelity to allow long-term vegetative perpetuation of phenotypic differences without artificial selection.

ILIATED protozoa are classic organisms for demonstrating hereditary diver- ‘ sity in cell structure that is maintained despite genic identity (e.g., SONNE- BORN 1963, 1970; NANNEY 1968b). One example of such inheritable diversity is variation in the number of ciliary meridians, which is typically polymorphic within strains (WILLIAMS 1961; NANNEY 1966a, 1968a; FRANKEL 1972,1973a) and sometimes differs between strains (HECKMANN and FRANKEL 1968; GENER- MONT, MACHELON and TUFFRAU 1976). Consistent differences between strains allow demonstration of genic control of the permitted range of ciliary meridian number (HECKMANN and FRANKEL 1968). The intra-strain polymorphism allows an assessment of the stability of vegetative propagation of phenotypic differences that is largely uncomplicated by problems of natural o r artificial selection, since small differences in number of ciliary meridians probably have negligible impact on cell-generation time and are thus far impossible to detect in living cells.

The vegetative stability of number of ciliary meridians has previously been analyzed in Tetrahymena by NANNEY (1966a,b, 1968a). The standard method was a ‘‘cross-sectional” analysis of the perpetuation of differences in number Genetics 94: 607-623 March, 1980.

608 J. FRANKEL

of ciliary meridians (termed “corticotype” by NANNEY) in a large number of clones. Substantial variation of corticotype was first generated by allowing cultures to remain in stationary phase for a long period. Single cells were then removed from these parent cultures and allowed to grow exponentially for 20 fissions. The “20 fission clones” were then fixed and silver impregnated. Com- parison of the patterns of corticotypic variation encountered in different “20 fission clones’’ to each other and to those of the parent cultures from which they were derived allowed NANNEY to draw two complementary conclusions: (1) that different pre-existing corticotypes tend to be propagated actively even under conditions of known genic homogeneity and (2) that corticotypes modulate in the direction of a “stability center” of 18 or 19 ciliary meridians, with the apparent rate of such modulation being proportional to the initial distance from the center. This form of analysis can not, however, reveal the degree to which differences in corticotype, especially near the stability center, might be perpetu- ated over a long term. An alternative approach designed to overcome this limita- tion is a “longitudinal” analysis, with frequent serial transfer of a more limited number of clones.

We have adopted this approach, and, in addition, designed our experiment so that it would commence with a substitution in a gene affecting the “permitted” corticotypic range. The results are consistent with earlier findings in Euplotes of a genic control of this permitted range. More important, however, they dem- onstrate that, within this genically permitted range, differences in pre-existing cortical phenotype can be maintained vegetatively for long periods without selection. The very similar stability of corticotypes near the “stability center” allows for a perpetuation of phenotypic differences in the face of genic identity.

MATERIALS A N D METHODS

Stocks and growth media: The organisms used in this investigation were Tetrahymena thermophila (formerly T. pyriformis, syngen 1, see NANNEY and McCoy 1976) of inbred strains A, B and D (for derivations of these strains, see Table 2 of ALLEN and GIBSON 1973). Two clones, manifesting mating types VI and VII, respectively, of strain B-1868 (18th generation of inbreeding, established in 1968) were obtained in 1972 from D. L. NANNEY. These two clones were crossed with each other in 1975 to yield B-1975. Two clones exhibiting mating types 111 and V, respectively, of this 19th inbred generation were used.

In certain crosses, defective A * (A-star) clones (mating types 111 and V) supplied by F. P. DOERDER (see WEINDRUCH and DOERDER 1975) were used. Other crosses involved clone CU-329, a “homozygous heterokaryon” (BRUNS and BRUSSARD 1974, p. 838) of genetic constitution ChzA2/ChzA2 (cycloheximide sensitive, mating type 11), provided by P. BRUNS. This clone is of strain B-1868 genetic background (BRUNS and SANFORD 1978), and will be referred to as “Chx” in the RESULTS.

Other stocks used in this investigation are homozygous for the janus mutation (FRANKEL and JENKINS 1979) or are progeny of crosses involving a janus homozygote. The CU-127 (Ill.) stock of jan/jan genotype, henceforth called “CU-l27”, was sent to us by D. NANNEY in February, 1977, after original selection in the laboratory of P. BRUNS (see FRANKEL and JENKINS 1979 for further particulars). Like CU-329, it also has a B-1868 genetic background. Clones “X” and “Y” were derived from outcrosses of CU-127 to B-1975 (V), made in February, 1977. These clones were used in further crosses, to be described below.

The culture medium used in most of this investigation was “l%-PPY,’’ made up of 1%

CORTICOTYPE PROPAGATION IN TETRAHYMENA 609

proteose peptone (Difco) plus 0.1% yeast extract (Difco). However, crosses were carried out in bacterized peptone medium (a 24-hr culture of Klebsiella pneumoniae i n 1% proteose peptone diluted 1/70 with distilled water before use). For transfer of cells from bacterized peptone to 1%-PPY, “pen-strep PPY” (FRANKEL et al. 1976) was employed. Some of the data in Table 1 from 1976 and earlier involved cells cultivated in tryptone-based media (“TDVS” and “TGVS”) or in a rich proteose-peptone yeast medium (2%-PPY). These media are described in an earlier communication (FRANKEL et al. 1976).

Stock maintenance and growth: “Back-cultures” of stocks were maintained at 20” in tube cultures containing 5 ml of l%-PPY medium, with loop transfer every second week. However, all material used in this investigation was maintained for a t least one month prior to use in “fonvard-stocks,” generally kept in an incubator a t 28” with transfer two or three times weekly. The medium used for these cultures was TDVS through the summer of 1974 and 1% PPY thereafter. These “forward cultures’’ were used for inoculation of experimental flask cultures, with TDVS tube cultures used to inoculate TGVS flasks and 1% PPY tubes used to inoculate 1% PPY or 2% PPY flasks. The procedures far setting up such flask cultures have been described previously (FRANKEL, NELSEN and JENKINS 1977) ; in some experiments, smaller volumes (50 instead of 150 ml) of medium were employed. The medium used in these flask cultures varied according to the date of the experiment: TGVS through 1-74, 2% PPY from 9-74 through 4 7 6 and 1 % PPY in 9-76 and thereafter.

The method of maintenance of the CU-127, X, and Y forward stocks was somewhat different. I%-PPY tube cultures were kept in the laboratory, a t approximately 22”, and loop- transferred weekly.

The bulk of this investigation was based on clonal isolates maintained in l%-PPY tube cultures kept in a 25” constant-temperature room and transferred with a bacteriological loop on Monday, Wednesday and Friday of each week. Cells were generally in the deceleratory phase of culture growth when the Wednesday and Friday transfers were made, and in early stationary phase at the Monday transfer. Approximately 103 cells were transferred, which inoculum grew to about 5 x 105 cells by the time of the next transfer. Hence, the culture went through about 9 fissions in the 2- to 3-day interval between one transfer and the next. At every 11th transfer, i.e., every 100th fission, the cells in a tube were fixed immediately after the transfer. This procedure was followed from the 25th (or 50th) fission onward. The procedures used in the inception of these clones are described below.

Crosses and initiation of clones: The general procedures used in carrying out crosses were the same as described previously (FRANKEL et al. 1976). Only true progeny, as demonstrated by sexual immaturity, were retained, However, one set of crosses (series 111) utilized a “positive selection for mating” as described by BRUNS and BRUSSARD (1974). The CU-127 clone was crossed to clone CU-329, the Chx “homozygous heterokaryon,” which expresses cycloheximide sensitivity although its micronucleus is homozygous for cycloheximide resistance. The selective agent was cycloheximide (15 pg/ml), and details of the procedure were carried out as described by FRANKEL and JENKINS (1979), except that the second drug exposure, to 6-methylpurine, was omitted. The survivors of this procedure were cyclolieximide-resistant clones that must have generated new macronuclei from micronuclear material derived wholly or in part from the Chx parent.

Following a conventional cross, cells were transferred from bacterized to axenic medium by being passaged, one or two a t a time, from one well of a 3-spot depression slide filled with pen-strep 1%-PPY to another. After the cells in the last well had grown to sufficient density, a loop transfer was made to a culture tube containing 5 m l of IX-PPY, with accompanying tests for bacterial contamination. This was the first culture tube of the clonal series; after two days of growth a loop transfer was made to a new tube and the remainder of the cells were fixed. This first fixation was estimated to take place about 25 fissions after conjugation. It should be noted that in one series of crosses (series 11), the two exconjugants from a pair were delib- erately isolated after separation of the pair, but before the first division, and these exconjugant clones were then made axenic as described above. In another series (series I), exconjugants were not individually isolated and synclones were carried through the transfer procedure. HOW-

610 J . FRANKEL

ever, the unimodal corticotype distributions observed in all but one clone after the first fixation strongly suggested that the great majority of these clones were actually derived from one or the other, but not both, of the two exconjugants, presumably because, commonly, only a single cell was transferred from one well of pen-strep PPY to the next.

The cycloheximide-resistant survivors of the CU-127 x Chx cross were isolated and tubed after a short period of growth in depression slides. In this case, the first fixation did nomt take place until about 50 fissions after conjugation. The selection procedure is inherently designed to generate clones, but in two cases strongly bimodal corticotype distributions appeared at the first fixation. These were assumed to originate from the accidental isolation of two cells, and were excluded from consideration.

The three series of crosses used to generate material for clonal analysis were as follows: I: Crosses involving clones X and Y, with probably random choice of a single exconjugant

for clonal analysis: A. X x A* (V) : 6 clones B. Y x A*(III): 10 clones C. X x Y: 12 clones

11: A backcross of clone Y to B-1975 (111), with deliberate selection of both exconjugants for clonal analysis; 22 clones were derived from 11 conjugating pairs, plus 4 clones from single exconjugants whose mates had died.

111: A cross of CU-127 to Chx, with selection of single surviving cells following exposure to cycloheximide; 28 clones were analyzed.

All clones, except the four single exconjugants from series 11, were serially propagated as described above, for a minimum of 125 to 150 fissions. They were sampled and fixed at 25 and 125 fissions (series I and 11) or at 50 and 150 fissions (series 111) after establishment (the single exconjugants of series I1 were sampled only at 25 fissions). The series IA and IB clones, plus all pairs from series I1 and an arbitrary subset of 9 clones from series 111, were carried to 225 fissions (series I and 11) or 250 fissions (series 111) and fixed a third time. At this time, all clones were discarded except for 10 clones that had been derived from 5 conjugating pairs of series 11. This limited subset was carried to 1000 fissions, with periodic sampling and fixation. Thus, a total of 82 clones were fixed at 25-50 fissions after establishment, 78 at 125-150 fissions, 47 at 225-250 fissions and 10 at intervals thereafter.

Cytology, scoring and analysis: Silver impregnatio,n was performed according to the procedure of FRANKEL and HECKMANN (1968), with slight modifications as described by NELSEN and D~BAULT (1978). Ciliary meridian counts were performed twice on each cell, going in opposite directions around the cell; if tlie two counts did not agree, two additional counts were made. With few exceptions, 50 cells were counted in samples from stock cultures, summarized in Table 1. Twenty cells were tallied from each sample of the serially cultivated clonal cultures. These tallies were carried out at scparate times in sets of 10 each time. For the longest-term experiment, graphs comparable to Figure 2 were drawn from the results obtained from the two separate sets of counts of 10. These two graphs were rery similar to each other. The sample size of 20 was therefore judged to be adequate.

Statistical evaluation was carried out according to procedures described by SOKAL and ROHLF (1969).

RESULTS A N D ANALYSIS

Corticotypic distributions of stock cultures Corticotypic distributions of samples of frequently transferred “forward

stocks” are given in Table 1. All samples were characterized by a range of Corti- cotypes rather than by a single, unique corticotype. In stocks sampled repeatedly, the distributions meandered within a broad but definite range. In wild-type stocks (other than stock X, considered below) this range was 1 7 to 21 ciliary meridians, with higher and lower corticotypes encountered only rarely. The

CORTICOTYPE PROPAGATION I N TETRAHYMENA

TABLE 1

Corticotype distributions of frequently transferred T. thermophila stocks

61 1

janus Date of Ciliary meridian number (corticotype) Stockf phenotype fixation Medium Monthss 16 17 18 19 20 21 22 23 23 25-k

B-1868 + 6-72 TGVS 0 1 12 37 (VW 11-72 TGVS 5 1 3 28 18

6-73 TGVS 12 3 12 23 12 1-74 TGVS 19 1 4 4 3 2 9-74 2%-PPY 27 1 8 3 9 2 1-75 2%-PPY 31 1 1 4 1 6 2 7 1

3-1975 f 10-75 2%-PPY 0 12 27 5 6 (111) 4-76 2%-PPY 6 1 3 1 6 2 4 3 3

9-76 l%-PPY 11 21 29 4-77* 1%-PPY 18 16 34 3-78 l%-PPY 29 3 19 15 4 9

Chx + 5-77* IX-PPY 0 1 13 33 3 3-78 l%-PPY 10 4 4 6 1

A-star f 8-72 TGVS 1 5 26 17 1 (111) 5-77* i%-PPY 0 2 29 19

12-77 l%-PPY 7 1 8 22 18 1

CU-127 janus 2-77* 1%-PPY 0 10-77 1%-PPY 8 3-78 l%-PPY 13 6-78 IX-PPY 16

x +$ 3-77* l%-PPY 0 5-77 l%-PPY 2

Y janus 3-77* l%-PPY 0 5-77* I%-PPY 2 9-77 l%-PPY 6 3-78 l%-PPY 12

2 3 8 5 5 1' 2 14 29 4

2 16 32 4 5 3 7 4

1 19 1 22 26 1

19 1 1 6 8 3 3 2

17 32 1 3 26 17 4

* Samples fixed close to the time of initiation of progeny clones. + See MATERIALS AND METHODS for details of derivation of stocks. $ Probably derived from janus cytoplasmic parent (see MATERIALS AND METHODS).

Months since inception of forward cultures; there are 50 to 100 fissions per month.

same was true of other stocks of strain B and strain D genetic background that are not included in Table 1. For unknown reasons, the B-1868 (VII) clone tended to exhibit corticotypes mostly in the upper portion of the prevalent range of corticotypic expression, whereas its progeny clone, B-1975 (III), favored the lower portion of this range. Changes of culture medium during the maintenance of these clones, and also of A* (111), had no impact on the corticotypic distributions. The characteristic meandering of corticotypes within a defined range has been observed previously in T . pyriformis sensu stricto (strain GL-C) and in a natural isolate of T . thermophila (strain WH-6) (FRANKEL 1972). The only

612 J. FRANKEL

discrepancy between the present observations and the earlier ones is that the corticotypic range currently observed in inbred strains of T. thermophila (1 7-21) is somewhat higher and broader than that (16-19) found earlier in strain WH-6.

Stocks homozygous for two recessive mutations manifest unusual corticotypes. Conical ( C O ) stocks tend toward abnormally low corticotypes (DOERDER et al. 1975), while janus ( jan) homozygotes sometimes, though not invariably, express atypically high corticotypes (FRANKEL and JENKINS 1979). The original janus clone, CU-127, is one of those that has characteristically expressed a range of corticotypes significantly higher than that of wild-type cells ( JERKA-DZIADOSZ and FRANKEL 1979). At the time when the experimental crosses were carried out (spring of 1977), the modal corticotype of the CU-127 (Ill.) stock was 23 (Table 1). Cells in later samples manifested lower corticotypes (20-23), but these were still distinctly higher than those of wild-type cells. The Cu-127 (Ill.) stock can therefore be assumed to have a higher “stability center” than wild type, a fact important in the subsequent analysis.

As reported earlier, an early cross of CU-127 X B-1975 (V) yielded three surviving “progeny,” all highly unconventional. Two of these, arbitrarily called X and Y (see FRANKEL and JENKINS 1979; Table 3) , manifested an unusually high corticotype when sampled shortly after the cross (Table 1, fixations of 3-77). Clone X was phenotypically wild type (non-janus) and its corticotype rapidly declined to a wild-type level (Table 1 ) . Clone Y was phenotypically janus, yet its corticotype also declined, but more slowly (note the persisting high value at the fixation of 5-77).

Clonal analysis of inheritance of corticotype Preliminary description of crosses: Three series of crosses were carried out,

including outcrosses of CU-127 and further breeding of two of the three surviving progeny clones of the CU-127 x B-1975 cross (see MATERIALS AND METHODS).

The original purpose of these crosses was genetic analysis of the janus phenotype manifested by the CU-127 clone. However, few progeny were produced, and subsequent genetic analysis of these progeny revealed no trace of the unique janus doublet organization [see FRANKEL and JENKINS (1979), section 1 (b) of RESULTS and Table 31. It was concluded that the X and Y clones were probably uniparental “progeny,” carrying micronuclei exclusively €rom the B-I 975 parent, and macronuclei €rom the B parent (clone X) or the CU-127 parent (clone U); the janus allele was later extracted from CU-127 by other means that are not relevant here. Both the X and Y clones, however, initially manifested high corticotypes, suggesting that they were cytoplasmically derived from the CU-127 partner. These clones, therefore, transmitted a combination of wild-type nuclear genes and abnormally high corticotypes to their progeny following mating.

The situation described above, together with the fact that the fertility of clones X and Y was substantially greater than that of the CU-127 parent (FRANKEL and JENKINS 1979), provided a good opportunity for clonal analysis of the vegetative propagation of corticotypes. All of the clones to be considered were wild type

CORTICOTYPE PROPAGATION IN T E T R A H Y M E N A 613

(non-janus) in phenotype, and were derived from crosses of one clone (X, Y or CU-127) of high corticotype with another (B-1975) of normal corticotype, except for one series (IC) in which two clones of high corticotype (X and Y) were crossed with each other (see MATERIALS AND METHODS for detailed pro- tocol). It is important to note that all crosses involved exclusively strain B genetic background, so that changes due to “transitory heterosis” (NANNEY and DOERDER 1972) were avoided.

The results will be presented and analyzed in three ways: first, by compari- sons of intraclonal variances of the first set of samples; next, by an analysis of regression of the change between the first and second samples on the corticotypes observed in the first sample; and finally, by a longitudinal analysis of long-term changes in periodically sampled material.

Intraclod variance of first samples: Intraclonal variances were computed as described by NANNEY (1 968a). For each clone, the mean and the variance of the corticotype in each first sample were calculated. Clones were then grouped into interval classes according to their mean corticotypes, so that clones with a mean of 17.50 to 18.45 were placed in the “18” class, clones with a mean of 18.50 to 19.46 were placed in the “19” class, etc. The separate intraclonal variances of the different clones within each corticotypic class are independent, so that it is possible to compute the mean, the standard error and 95% confidence intervals of the variances. This allows a comparison of the average dispersion of sets of corticotypic distributions with different means. The smaller this dispersion (i.e., the lower the clonal variances), the greater may be assumed to be the stability of the corticotype in ques ti0n.l

Table 2 shows the results of this type of analysis of o w clones, and also NANNEY’S (1968a) total of the corticotypic classes in his syngen 1 material (taken from the right-hand column of Table 1 of NANNEY 1968a, with com- putation of mean intraclonal variances and conversion of standard-errors to 95 % confidence intervals). We found no significant differences among the groups of clones derived from OUT three sets of crosses; these have been lumped in the left half of Table 2. (However, sample sizes were so small that the lack of statistical significance argues only weakly against the possibility of real differences among progeny sets.) Our results are in accord with NANNEY’S more extensive data in indicating minimum variances, and hence suggesting maximum stabilities, at corticotypes of 18 and 19. Clonal variances tend to be higher at the higher mean corticotypes, suggesting lesser stability of these higher corticotypes. Our results differ from NANNEY’S only in the less steep rise of clonal variances with corticotype, especially near the top of the portion of the corticotypic range that we were able to examine (22 and 23). This difference may be related to the different ways in which the clones were initiated: NANNEY’S with cells from extremely variable late stationary phase cultures (NANNEY 1966a), ours with cells from frequently transferred stocks, those of high corticotype having expressed janus prior to the cross.

This procedure of statistical analysis, as well as that of the following section, handles discrete distributions as if they were continuous. Hence, the results of the analysis must of necessity be considered as approximate rather than exact.

614 J. FR.4NKEL

TABLE 2

Intraclonal variance of first samples

20-50 Fission clones of T. ihermophilu Comparison

Intraclonal variance Intraclonal variance between the Midpoint 95%l;zLdence Number 95% confidence Number two studies of class Mean of clones Mean limits of clones ( t tests) p

This study (25-50 fissions) NANNEY 19G8 (20 fissions)

18 0 O*

19 0.092 0.012-0.172 (0.053) t (O.OO5-0.101)

(0.078)t (0.001-0.155) 20 0.180 0.080-0.2-80 21 0.170 0.082-0.258 22 0.152 0.074-0.230 23 0.194 0.082-0.305

0.037 0.019-0.055 26 <O.OOl

0.040 0.011-0.om 22 >0.05

0.209 0.085-0.333 13 >0.05 0.240 0.175-0.305 39 >0.05 0.317 0.229-0.4-05 14 <O.Ol 0.566 0.447-0.685 20 <O.OOl

w . 0 5 )

( x . 0 5 )

* All six clones identical, with zero variance. t Means and confidence limits resulting from transfer of one clone with corticotype midpoint

$ All are 25-fission clones. Four clones, belonging to classes with midpoints of 17,24,24 and 26, respectively, are excluded

of 18.50 from the 19 class to the 18 class.

from this analysis. For further explanation, see the text.

Change between first and second sample: The changes in corticotype between the first and second samples were analyzed as a problem in regression (SOKAL and ROHLF 1969, Sec. 14.6). The mean corticotype of each clone at the time of the first sampling, 25 to 50 fissions after conjugation, was taken as the independent variable. The difference between the mean corticotype of each clone at the first sampling and the mean corticotype of that same clone at the second sampling, 100 fissions later, was taken as the dependent variable; a negative value of this variable indicates a decrease in mean corticotype, a positive value an increase. The change in mean corticotype (ordinate) was then plotted against the starting mean corticotype (abcissa), each point on the graph representing a single clone (Figure 1). Different types of symbols represent different sets of clones (see Fig- ure 1 legend). I t can be seen at a glance that the relationship between change in mean corticotype and initial mean did not differ appreciably in the different sets of clones. Hence, data from different experiments were not distinguished in the analysis. When the total data set was analyzed, there was a highly significant regression of change in mean corticotype upon initial mean corticotype, with a negative slope. There was, however, also a significant deviation from linearity of regression. The regression was, therefore, reanalyzed, with the range of the independent variable (initial corticotype) broken up into two intervals, one from 17.2 to 21.0, the other from 21.2 to 26.4. A partially independent

FIGURE 1 .-Regression of change in mean corticotype between first and second sample (ordinate) on mean corticotypc in first sample (abscissa). (0) series IA; (0) series IB; ( A ) series IC; ( ) series 11; (x) series 111. The heavy solid line is the least-squares linear regression line for values of initial mean rorticotype of 21.2 and above; the heavy dashed line is the least-squares linear regression line €or values oE initial mean corticotype of 21.0 and below.

CORTICOTYPE PROPAGATION IN TETRAHYMENA

CHANGE IN SUBSEQUENT 100 FISSIONS

P VI 0

I I I I + + - - - 1

ru in 0 : 0 0 I I I I I

X

O D

X

615

616 J. FRANKEL

rationale for this is provided by the fact that the lower interval includes the range of corticotypes commonly found in frequently transferred wild-type stock cultures (Table l ) , while the upper interval is above the range of common expression.

In the upper range of corticotypes, a regression with a slope of -0.86 (95% confidence interval = -0.78 to -0.94) is obtained, with no significant deviation from linearity (Figure 1, solid line). This implies that when the initial corticotype was above 21, the corticotype modulated downward at an overall aggregate rate that was linearly proportional to the difference between the initial corticotype and a “target” corticotype near 21 (the extrapolated X intercept of the regression line). This, however, is only a rough approximation, as information concerning change over time within the 1 OO-fission interval is lacking.

I n the lower range, the slope of the regression line is much less, estimated at -0.19 (95% con€idence interval = -0.12 to -0.26). Although the slope of the regression line differs significantly from zero, there is, in this case, also a significant deviation from linearity of regression. Examination of the relationship of the data points to the computed regression line (Figure 1, dashed line) suggests that this deviation results from random scatter rather than from systematic curvilinearity, although the issue cannot be resolved decisively with this small data set. Nonetheless, the significant non-zero slope, plus the large cluster of “stationary” ( y = 0) points at x = 19, do suggest that corticotypes near 19 are maximally stable. However, rates of corticotypic change in the range of 17 to 21 ciliary meridians appear to be slow and the direction of change not always uniform. Therefore, one would predict that clones that had modulated rapidly from corticotypes of 22-26 down to 21 during the first 100-fission interval should sub- sequently continue to change in a downward direction, but much more slowly.

Serial progression of corticotypes: The expected marked deceleration in rate of change of corticotypes with approach to the apparent stability center was in fact observed. Results for the 47 clones that were followed over a span of 200 fissions are presented in Table 3, where they are grouped according to corticotypic interval classes in the first sample. (For example, 7 of the 47 clones had a mean corticotype between 22.5 and 23.45 in the first sample. The actual average of the 7 means and the standard deviation(s) are then given for the first sample and for the following two samples of these same 7 clones.) As already shown (in another way) in Figure 2, clones with initial corticotypes above 21 in the 25-to-50 fission sample all regressed to a corticotype near 21 in the next 100 fissions. Virtually all of these clones (21 out of 24) underwent further declines in mean corticotype in the next 100 fissions but the changes were mostly slight, with the new mean near 20.5. Clones initially in the range of 18 to 20 corticotypes underwent, on the average, no change between the second and third sampling, though fluctuations in different directions were encountered in indi- vidual clones (see below). No unique stability center was attained by 200 to 250 fissions after the inception of the clones.

Ten clones from set I1 were followed further, fo r a predetermined period of 1,000 fissions. The trajectories of mean corticotype of these clones are presented

CORTICOTYPE PROP.4GATIOh- IN T ETRAIIYMENA

TABLE 3

Corticotype progression during 200 fissions of clonal growth

61 7

Corticotype Nominal First sample Second sample Third sample midpoint I & 11-25 fissions I & 11-125 fissons I & 11-225 fissions

of first No. of 111-50 fissions 111-150 fissions 111-250 fissions sample class‘ clones Mean+ &S.D.$ Mean fS.D. Mean 2S .D.

18 6 18.00 0 18.36 k0.22 18.35 k0.29 19 14 19.09 k0.16 19.06 a0.15 19.09 CO.29 20 3 19.90 i0 .17 19.67 20.53 19.68 20.46 21 5 21.07 a0.29 20.71 k0.45 20.64 k0.38 22 11 21.98 k0.23 21.00 k0.27 20.48 20.32 23 7 23.01 a0.43 20.94 k0.27 20.59 C0.37 26 1 26.4 21.4 21 .o

* Classes constructed as in Table 2; e.g., class 18 includes all clones whose mean corticotype at the time of the first sample were between 17.5 and 18.46; the remaining classes are constructed in an analogous fashion.

t The actual means of all clones falling within the corresponding classes. Standard deviation of sample means.

in Figure 2. The ten clones were vegetative progeny of exconjugants from 5 mating pairs, each between a janus cell of high corticotype and a B-1975 cell of lower (normal) corticotype. By the time of the first sample, 25 fissions after conjugation, the abnormalities in cortical asymmetry characteristic of janus had been lost, but the high corticotype persisted in one exconjugant clone from each pair, presumably the one derived from the janus (clone-Y) partner (see also section 2(b) of FRANKEL and JENKINS 1979). As already described, these initially high corticotypes declined, at first rapidly and then more slowly, in the 200 fissions following the initial sample. From 225 to 1,025 fissions, statistically significant overall declines in mean corticotype were observed in clones lb, 4b and 5b, but not in 2b and 3b. In the latter two clones, the modal corticotype remained 20 (except in the last sample of 3b) and cells with 21 ciliary meridians kept reappearing.

The “normal” partners, presumably descended from B-1975 cells, started out with 18 of 20 ciliary meridians, predominantly 19. Two ( la and Sa) remained essentially constant, with corticotypic means at or near 19, although cells with 18 and 20 ciliary meridians appeared in both clones at one time or another. One (3a) continued to fluctuate between mean cortocotypes in the range of 19 to 20 ciliary meridians. Interestingly, one clone (2a) that had started at 18 ciliary meridians steadily increased to 19, while another (4a) that had started at 19 declined and then fluctuated between 18 and 19. The choice between 18, 19, or 20 ciliary meridians appears not to be under any uniform constraint in these clones.

Another perspective can be gained if we compare the “high” and OW^' progeny clone within each cross. Although genetic markers to exclude possible uniparental phenomena were not included in this cross, the two progeny clones of each pair were likely to be genically identical, In no case did the 95 % confidence limits of corticotypic distributions of the two clones within each pair overlap

618

23

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F I S S I O N S

Z.-Corticotype progressions of exconjugant clones derived from five conjugating Series 11. The pairs are numbered 1 through 5 . Each panel shows the urogression - - -

of mean corticotypes of a single pair. Within each panel, the open circles indicate the mean corticotypes of the clone (a) presumed to be initiated from B-1975 (111) parent, while the closed circles indicate the mean corticotypes of the clone (b) presnmed to be initiated from the clone-Y (janus) parent. The horizontal bars above and below each point show 95% confidence intervals. Where no bars are shown, all cells tallied in the sample were of the same corticotype.


(Figure 2). However, in synclone 5 they approached very closely in the last sample, while in synclones 3 and 4 the 95% confidence limits of one or more samples of the ‘‘low’’ clone overlapped that of another sample in the “high” clone; in that sense, the corticoltypic distributions may be said to have converged. In synclones 1 and 2, however, corticotypes of the “high” and “low” sister clones remained quite distinct throughout the experiment. It is apparent that the rela- tive stabilities of propagation of corticotypes 18, 19 and 20 are sufficiently high, and the random transition probabilities sufficiently low, to allow the maintenance of vegetatively propagated differences over an extremely large number of cell divisions.

DISCUSSION

The results of this investigation are consistent with earlier findings on Euplotes (HECKMANN and FRANKEL 1968; FRANKEL 1973a,b) suggesting that the “permitted” range of corticotypes is controlled by genes. Although naturally occurring intraspecific variation in genes affecting corticotype, as was found in Euplotes, has not yet been detected in Tetrahymena, some stocks homozygous for the nitrosoguanidine-induced janus gene do characteristically manifest an elevated range of ciliary meridian numbers ( JERKA-DZIADOSZ and FRANXEL, 1 979). It is this property that allowed us to initiate clones with cells that expressed corticotypes well above those commonly found in wild-type cells. When geno- typic support for the high ciliary meridian number was removed, the corticotypes of the exconjugant clones modulated downwards to the wild-type range. This implies a genic control of that range. However, in one of the clones expressing janus (clone U), the corticotype also drifted downwards during the period of this investigation, thus compromising somewhat the clarity with which the present data support this conclusion.

The most striking new finding of this investigation is the long-term perpetuation of differences in corticotype within the genically permitted range. Although the present results are consistent with NANNEY’S early inference (NANNEY 1966b) that the “stability center” in wild-type T. thermophila (T. pyriformis, syngen 1) is at corticotype 19, all three types of analysis of serially transferred clones carried out during this study indicate that the stabilities of maintenance of corticotypes 18, 19 and 20 are all very high, and barely, if at all, distinguish- able from each other. Differences within this range can be propagated for a very large number of fissions, conceivably indefinitely, without artificial selection. These results are in agreement with the earlier conlusion, based on less complete evidence in both Euplotes (FRANKEL 1973a) and Tetrahymena (FRANKEL 1972), that it is meaningful to think of corticotypes in terms of a “stability range.” This concept emphasizes the capacity for long-lasting propagation of differences by purely vegetative means, a possibility supported by the present results. This conclusion is also consistent with NANNEY’S results with inbred strains of T . thermophila, as the clonal variances that he observed at corticotypes 18 and 19 were indistinguishable, although those for corticotype 20 were distinctively higher (NANNEY 1968a; cf., Table 2 of this paper).

620 J. FRAPU‘KEL

The patterns of corticotypic modulation observed in this investigation can explain why, even in Tetrahymena cultures maintained under continuous (or near-continuous) exponential phase, a fairly broad range of corticotypes is always found (FRANKEL 1972; Table 1 of this paper). This would be expected if the corticotypes within this range are of equal, or very nearly equal, stability. The corticotypic range commonly expressed in stock cultures is 17-21. In the analytical portion of this study, we found that corticotypes of 18 to 20 are of nearly equal stability, those of 21 are only slightly less stable, whereas those of 22 and above are comparatively unstable. Hence, the upper bound of high stability revealed by clonal analysis is in agreement with the upper limit of corticotypes commonly expressed in growing stocks. Less can be said about the lower limit. One subclone with 17 ciliary meridians selected from clone 4a (Figure 2) increased up to 18 within 100 fissions (NELSEN, personal communication). A lower limit near or above 17 is consistent with NANNEY’s profile of clonal variances ( NANNEY 1968a, Table 1 ) .

What is somewhat more surprising is the degree to which the range of expressed corticotypes can meander over time, both in frequently transferred stock cultures (FRANKEL 1972; Table 1 of this paper) and also in the 1000-fission clonal lines (Figure 2). The simplest way of thinking about corticotypic transitions analytically is in terms of a matrix of transition probabilities, in which each corticotype has unique probabilities of perpetuating itself and of changing to any other corticotype. A cell of corticotype 19, for example, might have a probability of 0.994 of remaining 19 at the next generation, 0.004 of going down to 18 and 0.002 of going up to 20. If these probabilities are independent across generations (i.e., if previous history is irrelevant), then the assumptions of a Markov chain process (KEMANY, SNELL and THOMPSON 1956) are satisfied. Unfortunately, it is impossible to deduce the transition-probability matrix from any eventual distribution of corticotypes, as more than one matrix might yield the same distribution. However, if we make three assumptions, of (1) irrelevance of prior history, (2) nonzero probabilities of change in both directions (i.e., a finite probability of increase and a finite probability of decrease) and ( 3 ) constancy of transition probabilities over time, then we would expect some unique eventual distribution of corticotypes. This follows from the behavior of a regular Markov chain (KEMANY, SNELL and THOMPSON 1956). Yet, it has repeatedly been shown that no such unique distribution is attained. The assumptions stated above might, nonetheless, be correct, and the observed drift could be due to sampling fluctuations during transfer; however, in the present study about lo3 cells were transferred, while in the earlier study (FRANKEL 1972), in which transfer was by drops from a Pasteur pipette rather than by a bacteriological loop, about 5 X lo3 cells were transferred. I consider it more likely, therefore, that transition probabilities may change slightly in different phases of culture growth, and/or that the recency of one transition might affect that probabilities of the next.2

NANNEY (1966a,b) has deduced that “secondary transfomabons” (transitions) occur at a lower rate than “primary transformations ” This analysis of rates of transition involved use of the Polsson distnbution, w i d the fraction of cells


Corticotypes, then, go through a very slow “random walk” within genically circumscribed limits. The “cortical inertia” that makes the walk so slow can be understood mechanistically on the basis of the normal development of new ciliary units along the axis of the pre-existing row (WILLIAMS 1964; ALLEN 1969; PERLMAN 1973; KACZANOWSKI 1978). Occasional losses of ciliary rows may result from local failures of basal body proliferation (CHEN-SHAN 1969, 1970; NANNEY 1972; FRANKEL 1973b; SUHAMA 1975), gains from formation of new ciliary units at a right angle to the axis of the pre-existing ciliary row (FRANKEL 1973b) or by rupture of rows followed by lateral displacement of the free ends (KACZANOWSKA 1975; NELSEN, personal communication). All of these mechanisms assume a fundamental continuity of ciliary rows, and there is no reason for doubting such continuity under such conditions of continuous culture in nutrient medium as were employed in this investigation. However, NELSEN has recently discovered a special mechanism by which a new ciliary row can be added without any direct continuity with pre-existing ciliary rows (NELSEN and FRANKEL, 1979). This mechanism appears to be called into play only under conditions of shift-down to non-nutrient medium. Such conditions, though not relevant to the present study, may frequently be encountered in nature, making the extrapolation of the present conclusions to Tetrahymenas in nature uncertain. (See NELSEN and FRANKEL 1979 for further discussion of this point).

The expert assistance of LESLIE M. JENKINS was indispensible for the successful pursuit of this project; I am particularly grateful to him for the great care that he took in the maintenance and accurate transfer of the clonal isolation lines. JOSEPH P. HEGMANN and STEPHEN P. HUBBELL provided indispensible instruction and advice on statistical evaluation and mathematical models, respectively. The manuscript was read and useful comments provided by KARL AUFDERHEIDE, ANNE W. K. FRANKEL, MICHAEL A. GATES, DAVID L. NANNEY, E. MARLO NELSEN, STEPHEN NG and two reviewers. The research was supported financially by grant No. HD-OS485 from the Public Health Service.

LITERATURE CITED

ALLEN, R. D., 1969

ALLEN, S. L. and I. GIBSON, 1973

BRUNS, P. J. and T. B. BRUSSARD, 1974

BRUNS, P. J. and Y. M. SANFORD, 1978

The morphogenesis of basal bodies and accessory structures of the cortex of the ciliated protozoan Tetrahymena pyriformis. J. Cell Biol. 40: 716-733.

Genetics of Tetrahymena. pp. 307-373. In: The Biology of Tetrahymena. Edited by A. M. ELLIOTT. Dawden, Hutchinson and Ross, Stroudsberg, Penn.

Positive selection for matinq with functional hetero- karyons in Tetrahymena pyriformis. Genetics 78: 831-841.

Mass isolation and fertility testing of temperature sensitive mutants in Tetrahymena. Proc. Natl. Acad. Sci. U.S. 75: 3355-3358.

within the 20 fission clones that remain in an initial cortcotypic class being equated to the first Poisson term, e-@. How- ever, these clones are the outcome of numerous cell generations, and transitions may occur early or late during the growth of the clone and might be compounded by an unknown number of cell divisions. This circumstance violates the condition of independence of rare events, which is indispensable for application of the Poisson distribution. The first Poisson tenn, e-=, can legitimately be equated only to the fraction of clones in which no cell has undergone a transition. This would require enumeration of the entire culture (cf., LURIA and DEDRUCK 1943, p. 507) and even then would yield an estimate only of the average total number of primary transitions. The very existence of secondary transitions, as well as the possibility of changes of corticotype in both directions, makes the analytical strategy of LURIA and DELBXUCK (based OD

an extremely low probability of forward mutation, with back-mutation neglected) inapplicable to the present situation.

622 J. FRANKEL

CHEN-SIXAN, L., 1969 Cortical morphogenesis in Paramecium aurelia following amputation of the posterior region. J. Exp. Zool. 170: 205-227. - , 1970 Cortical morphogenesis in Paramecium aurelia follow-ing amputation of the anterior region. J. Exp. Zool. 174: 463478.

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A simplified Chatton-Lwoff silver impregnation procedure for use in expcrimental studies with ciliates. Trans. Amer. Microsc. SOC. 87: 317-321.

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A mutant of Tetrahymena with a partial mirror image duplication of cell surface pattern. I. Analysis of the phenotype. J. Embryol. Exp. Morph. 49: 167-202.

KACZANOWSKA, J., 1975 Shape and pattern regulation in regenerants of Chilodonella cucullulus (O.F.M.). Acta Protozoal. 30: 343-360.

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FRANKEL, J., E. M. NELSON and L. M. JENXINS, 1977

GENERMONT, J., V. MACHELON and M. TUFFRAU, 1976

HECKMANN, K. and J. FRANKEL, 1968

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resistance. Genetics 28: 491-511. Mutations of bacteria from virus sensitivity to virus

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Regulation of corticotype through kinety insertion in

Basal body addition in ciliary rows of Tetrahymena pyriformis. J. Exp.

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Corresponding editor: SALLY ALLEN

frankel (heckmann frankel

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