/. Embryol. exp. Morph. Vol. 49, pp. 203-227, 1979
Printed in Great Britain © Company of Biologists Limited 1979
203
A mutant of Tetrahymena thermophila
with a partial mirror-image duplication of cell
surface pattern
II. Nature of genie control
By JOSEPH FRANKEL 1 AND LESLIE M. JENKINS 1
From the Department of Zoology, University of Iowa, Iowa City
SUMMARY
The CU-127 clone of Tetrahymena thermophila, which manifests an unusually high number
of ciliary rows plus a second set of abnormal oral structures and of contractile vacuole pores
with partial mirror-image reversal of asymmetry (Jerka-Dziadosz & Frankel, 1979), has been
subjected to breeding analysis. The progeny ratios obtained in various crosses indicate that
the abnormalities of cell-surface asymmetry are brought to expression as a result of the action
of a recessive allele at a single gene locus, here named janus. When previously normal cells
were made homozygous for the jan allele, the cortical pattern characteristic of the CU-127
clone came rapidly to expression, often without associated change in number of ciliary
meridians. Conversely, when cells previously expressing jan re-acquired the wild-type (jan+)
allele, they returned to the normal pattern of a single normal oral structure and a single normally located set of contractile vacuole pores while still retaining the high ciliary meridian
number characteristic of the original CU-127 clone. The capacity for manifestation of the
unique asymmetry pattern depends solely on homozygous expression of the janus allele,
whereas the stable number of ciliary meridians in janus clones and the degree of expression of
secondary OAs is modulated by other factors, probably at least in part genie. These results,
taken together with those of the preceding paper, indicate that the janus allele promotes the
propagation and/or expression of a condition of reversed asymmetry in a precisely located
cell region, and further indicates that the propagation and expression of this condition are
largely independent of the number and asymmetry of ciliary meridians.
INTRODUCTION
Little is known about the way in which genes influence patterns of symmetry
at the level either of intracellular structure or multi-cellular organization. Most
analyses of the problem of cellular and organjsmic symmetry have employed
microsurgical interventions to obtain controlled reversals of asymmetry.
Reversals of intracellular asymmetry have been studied almost exclusively with
ciliated protozoa. The best example is the reversal of intracellular asymmetry
associated with the 180° rotation of ciliary meridians in Paramecium (Beisson &
1
Authors' address: Department of Zoology, University of Iowa, Iowa City, Iowa 52242,
U.S.A.
204
J. FRANKEL AND L. M. JENKINS
Sonneborn, 1965) and Tetrahymena (Ng & Frankel, 1977; Ng & R. Williams,
1977). This rotation brings about, in Tetrahymena, a right-left reversal of
asymmetry of positioning of at least three structures situated outside of the
ciliary meridians: the contractile vacuole pore (Ng, 1977), the longitudinal
microtubule band (Ng & Frankel, 1977; Ng, 1978), and the subcortically located
mitochondria (Jerka-Dziadosz, personal communication; Aufderheide, 1978).
Another well known example of a cortical asymmetry reversal on a larger scale
is the microsurgically induced reversal of the 'zone of stripe contrast' within
which the oral primordium of Stentor develops (Tartar, 1956, 1960). What is
distinctive about all pattern reversals in ciliates which have been described thus
far is that the event that generates the reversal is an abnormal juxtaposition of
cellular elements either deliberately created by the experimenter or else occurring
fortuitously and then selected by the experimenter. In the case of the Paramecium
ciliary meridian inversion, Beisson & Sonneborn (1965) established that cells
that possessed and transmitted the reversal did not differ in any relevant genes
from those that did not. In the other examples, genetic analysis was not undertaken but the mode of origin of the reversals makes a genetic explanation hardly
conceivable (see Tartar, 1967, pp. 90-92). Strong evidence thus exists for the
conclusion ' . . .that the "information" for the direction of asymmetry... resides
in the cell cortex...' (Tartar, 1967, p. 92), and no evidence has thus far been
adduced for a role of genes in influencing or controlling the 'direction of asymmetry'. It is the purpose of this communication to describe the existence and
manifestation of genie control in the first example in which reversal of cortical
asymmetry arose following a chemical operation on the genome rather than a
structural alteration of the cell surface.
MATERIALS AND METHODS
The CU-127 clone of Tetrahymena thermophila (formerly T. pyriformis syngen
1; cf. Nanney & McCoy, 1976) was obtained in February 1977 from the laboratory of Dr David L. Nanney, to whom it had earlier been sent by Dr Peter Bruns
for cryopreservation [this stock was designated as CU-127 (111.) in the previous
paper (Jerka-Dziadosz & Frankel, 1979) to distinguish it from another sample
of the same clone that had earlier been sent to us directly from the laboratory
of Dr Bruns. Here, for simplicity, we refer to CU-127 (111.) simply as CU-127].
CU-127 was one of a set of clones (Bruns & Sanford, 1978) obtained from cells
previously subjected to mutagenesis in 10/^g/ml of jV-methyl-yV'-nitrosoguanidine, followed by short-circuit genomic exclusion (Bruns, Brussard & Kavka,
1976) with positive selection for mating (Bruns & Brussard, 1974). The genetic
marker being used in the above selection for mating was a dominant allele
conferring resistance to 6-me thy lp urine (Byrne, Brussard & Bruns, 1978), hence
the CU-127 clone possesses and (owing to the nature of short-circuit genomic
exclusion) may be presumed to be homozygous for the Mpr allele. At the same
Genie control of symmetry in Tetrahymena
205
time it is cycloheximide-sensitive and thus necessarily homozygous for the
recessive wild-type allele conferring sensitivity at the locus (ChxA) which has
been characterized by two allelic dominant mutations (ChxAl and ChxA2) to
drug resistance (Roberts & Orias, 1973; Bleyman & Bruns, 1977; Byrne et al.
1978). The micronuclear genotype of the CU-127 clone at these two loci may
thus be written as Mpr/Mpr, ChxA+/ChxA+. CU-127 was one of many clones
originally selected for lethality following a 7-day exposure to high temperature
(Bruns & Sanford, 1978). This temperature-sensitivity was manifested in
axenic medium only after prolonged stationary phase at the restrictive temperature (39-5 °C) (Jerka-Dziadosz & Frankel, 1979) but was sometimes expressed
more rapidly in bacterized medium. The abnormalities of cell patterning in the
CU-127 clone have been described in the preceding paper (Jerka-Dziadosz &
Frankel, 1979).
Other stocks used in the genetic analysis include the standard inbred 'wildtype' B strain (B-1975) of T. thermophila, a 'defective' A* (A-star) clone
(Weindruch & Doerder, 1975) used in genomic exclusion crosses, plus clone
CU-329, a 'homozygous heterokaryon' [ChxA2/ChxA2(cycl. sens.,II)] supplied
by Dr Peter Bruns (see Bruns & Brussard, 1974, p. 838). Although now known
as ChxA owing to the recent characterization of a ChxB locus (Ares & Bruns,
1978), for ease of presentation ChxA2 will be referred to simply as 'CV/x' in the
Results.
General procedures for carrying out crosses are the same as described previously (Frankel, Jenkins, Nelsen & Doerder, 1976). Most crosses were carried
out at room temperature (about 23 °C) in bacterized peptone medium (a 24 h
culture of Enterobacter aerogenes in 1 % proteose peptone diluted 1/70 with
distilled water before use). These methods were adequate in reasonably fertile
crosses. However, in the original extraction of the allele responsible for the
reversal of cortical pattern from the virtually sterile CU-127 clone, a different
set of procedures was followed. These methods are identical in principle, though
not in details of execution, to those described by Bruns & Sanford (1978). The
CU-127 clone was crossed to clone CU-329, the 'homozygous heterokaryon'
which expresses cycloheximide sensitivity although its micronucleus is homozygous for cycloheximide resistance (cf. Table 1, and Results, section la). The
cross was carried out under axenic conditions in Dryl's salt solution made up as
in Nelsen & DeBault (1978). Two days after initiation of the cross (day 2), the
mating mixture (containing both calls which had conjugated and those which
had not) was combined with an equal amount of 2 % proteose peptone medium
containing 50 /tg/ml of cycloheximide, to make a final concentration of 1 %
proteose peptone and 25 /^g/ml cycloheximide. Three days later (day 5) 60 surviving cells were removed and used to establish clones that were maintained in
1 % proteose peptone. Two days afterwards (day 7) replicas of the 60 clones were
exposed in 1 % proteose peptone to 6-methylpurine at a final concentration of
15 /tg/ml for 5 further days. Penicillin (1-4 g/1) and streptomycin (2-2 g/1) were
14
E M B 49
206
J. FRANKEL AND L. M. JENKINS
present throughout to prevent bacterial contamination. All of the above procedures were carried out in wells of 3-spot depression slides (Corning no. 7223)
at room temperature. Replicas of the two clones that survived serial exposure
to cycloheximide followed by 6-methylpurine were then maintained by routine
procedures (see below) and subjected to further crosses in bacterized peptone
medium.
Axenic media, both the standard medium and also an enriched iron-chelate
medium (see below for descriptions) were also employed in efforts to obtain
viable exconjugants in crosses ofjanus homozygotes with each other.
The axenic medium utilized in stock maintenance and in virtually all experiments was '1 %-PPY', made up of 1 % proteose peptone (Difco) plus 0 1 %
yeast extract (Difco). When cells were being transferred from bacterized to
axenic medium, 'pen-strep PPY' (Frankel et al. 1976) was used. Cells from
bacterized peptone were passaged, one or two at a time, from one well of a
three-spot depression slide filled with pen-strep 1 % PPY to another. After the
cells in the last well had grown to a sufficient density, a loop transfer was made
to a culture tube containing 5 ml of 1 % PPY, with accompanying tests for
bacterial contamination. It should be noted that these procedures have the effect
that even when both exconjugants of a mating pair survive, frequently progeny
from only one of them are recovered for subsequent analysis. In two experiments
the two exconjugants from a pair were deliberately isolated after separation of
the pair but before the first division, and allowed to form exconjugant clones.
On a few occasions, a specially enriched medium containing 0-5 % dextrose
as well as an Fe2+-EDTA complex in addition to 2 % proteose peptone and
0-2 % yeast extract (Conner & Cline, 1964; Thompson, 1967) was used, both
for experiments with vegetative cells and for maintenance of exconjugants. In
the latter case, it was combined with penicillin and streptomycin as described
above for 'pen-strep PPY'.
Axenic clones were maintained at 20-25 °C in tube cultures containing 5 ml
of 1 % PPY, with transfer using a bacteriological loop. Frequency of transfer
depended on the purpose for which the cultures were maintained, and ranged
from thrice weekly to once every 2 weeks.
Phenotypes assayed were resistance to cycloheximide and 6-methylpurine,
survival following exposure to high temperature, and structural patterns of the
cell surface. Testing of drug-resistance phenotypes of progeny of crosses was
carried out using essentially the same methods as in the positive selection for
mating described earlier: replicas of clones were maintained at room temperature and exposed in parallel to cycloheximide (25 /tg/ml) for 2 days and to
6-methylpurine (15/tg/ml) for 4-5 days, both in pen-strep proteose peptone.
Exposures to the two drugs were always carried out shortly after conjugation in
order properly to assess drug resistance in heterozygotes before phenotypic
assortment to drug-sensitivity might have occurred. Viability of clones at high
temperature was scored by transferring subsamples in 1 % PPY to an incubator
Genie control of symmetry in Tetrahymena
207
maintained at 39-5 °C. All other relevant phenotypes were scored in fixed samples subjected to silver impregnation (see Jerka-Dziadosz & Frankel, 1979, for
citations).
Cultures were prepared for fixation and scoring of surface patterns by one of
three procedures: the 'standard' method was to fix the first axenic tube culture
grown at room temperature, following the removal from bacteria as described
above; such cultures were about 25 fissions removed from conjugation, and
tended to bs in early stationary phase, when fixed. When a rapidly growing
culture in exponential phase was desired, cells were inoculated from the tubes
into 50 ml batches of medium in 250 ml conical flasks, grown for about 15 h
at 28 °C, and fixed at a density of about 5000 cells/ml as determined by Coulter
counts. Finally, a 'quick' method was sometimes used, in which a sample of an
exconjugant synclone in bacterized peptone was added to 2 % PPY medium in
a 3-spot depression slide and allowed to grow at 28 °C for about 15 h. This
resulted in a high density log-phase culture that could be fixed at about 15 fissions
after conjugation, but the cells were full of bacteria and silver impregnation was
somewhat inferior in quality to that obtained with samples grown in pure
axenic media. More than one of these methods were sometimes used to score
the same set of progeny.
Standard scoring of cell surface phenotype involved examination of at least
20 silver-impregnated cells to ascertain the arrangement of contractile vacuole
pores, followed by scanning at low power to search for cells with two oral
apparatuses. Cross-checking by different observers as well as more extensive
examination was carried out on some complete progeny sets.
RESULTS
1. Genetic analysis
(a) Breeding analysis of true conjugants
A major obstacle to genetic analysis of the unusual cortical phenotype of
the CU-127 clone was the indication from conventional breeding that micronuclei of members of this clone are able to go through meiosis but unable to
carry out viable fertilization (see section 1 b). For this reason, the methods devised
by Bruns & Brussard (1974) for positive selection for mating were used. The
method as applied here is essentially one of marker rescue under highly selective
conditions. Instrumental to this technique is the mating of the clone containing
genetic markers to be 'rescued' (CU-127 in this case) to a specially constructed
clone that has a pattern of drug responses complementary to that of CU-127
and also possesses a special nuclear design such that all cells that fail to generate
new macronuclei at conjugation can readily be killed by a suitable drug. This
clone, CU-329, is a 'homozygous heterokaryon' of genotype Mpr+/Mpr+,
Chx/Chx (cycl.-S). The special feature of this 'homozygous heterokaryon' is
that its micronucleus is homozygous for the ChxA2 allele conferring cyclohexi14-2
208
J. FRANKEL AND L. M. JENKINS
mide resistance, whereas its macronucleus is heterozygous at this same locus
and has undergone 'phenotypic assortment' (Nanney, 1964) to express only the
allele conferring cycloheximide sensitivity (for full explanation, see Bruns &
Brussard, 1974). This 'homozygous heterokaryon' thus expresses sensitivity to
the drug yet transmits only the allele conferring resistance to sexual progeny.
Thus, any exconjugant of a cross involving this homozygous heterokaryon that
has formed a new macronucleus from a product of its micronuclear meiosis is
resistant to cycloheximide, whereas all suspected progeny that have in fact retained their old macronuclei remain sensitive to this drug. It is important to note,
however, that formation of a new macronucleus from a product of micronuclear
meiosis need not imply cross-fertilization: uniparental cycloheximide-resistant
progeny might arise in several ways, including self-fertilization (cytogamy) in the
Chx/Chx homozygous heterokaryon (Orias & Hamilton, 1977), development of
unfertilized nuclei in asymmetric triplet conjugants (Preparata & Nanney, 1977),
or by a process known as 'short-circuit genomic exclusion' (Bruns et al. 1976)
in which a haploid meiotic product of a Chx/Chx micronucleus might directly
generate a new macronucleus in either or both partners without fertilization. To
select for actual cross-fertilization, the two-step drug selection procedure previously employed by Bleyman & Bruns (1977) and Bruns & Sanford (1978) was
applied. The CU-127 clone, of genotype Mpr/Mpr, Chx+/Chx+ and mating type
(m.t.) IV was crossed to clone CU-329 [Mpr+/Mpr+, Chx/Chx (cycl.-S, m.t.
II)]. Cycloheximide was added first, then 6-methylpurine. All cycloheximideresistant progeny must have formed new macronuclei from meiotic products of
the micronucleus of the CU-329 clone, while the cycloheximide-resistant progeny
which are also 6-methylpurine-resistant must have undergone at least sufficient
fertilization to bring the drug-resistant alleles from the two parents into the same
macronucleus (see Table 1).
Sixty cycloheximide-resistant clones were isolated from a culture derived
from a mass mating of CU-127 x CU-329, yet only two of these were also resistant to 6-methylpurine (generation 1, abbreviated as Gen. 1 in Table 1). This
result suggests that the majority of new macronuclei that arose from meiotic
products of the CU-329 partner were uniparental.1 If the two exceptional clones
that were resistant to both drugs were indeed the outcome of conventional
fertilization, then their genotype should be Mpr/Mpr+, Chx/Chx+ and the clones
should additionally be heterozygous for any allele(s) controlling the unusual cell
surface pattern of the CU-127 clone. To test foi this, the two doubly-resistant
clones were carried through a mating protocol termed' genomic exclusion' (Allen,
1967), in which the clone to be tested is deliberately crossed to a known sterile
clone with a defective micronucleus [we employed the A* (A-star) clone of
1
It is not, however, certain that all of the 58 cycloheximide resistant clones that manifested
sensitivity to 6-methylpurine on the subsequent test necessarily lacked the Mpr allele.
Mpr/Mpr+ heterozygotes are known to be unusually apt to undergo early phenotypic assortment to express only the allele conferring methylpurine sensitivity (Bleyman & Bruns, 1977).
Genie control of symmetry in Tetrahymena
209
Table 1. Rescue of the fan allele from CU-127 by positive selection
Gen. 0:
Gen. 1:
Gen. 2:
CU-127
CU-329
(Mpr/Mpr, Chx+/Chx+,jan/?) x (Mpr+/Mpr+, Chx/Chx,jan+/jan+)
Cycloheximide screen
I
I
(must possess Chx)
6-methylpurine screen
I
1
(must possess Mpr)
(2 clones)
Mpr/ ?, Chx/ ?, ?/ ?
Genomic exclusion
I
(produces homozygosity)
cross
>l
6-me-purine
Cycloheximide
Cortexf
Clone 1
Clone 2
Thus , Gen. 1 is:
t
R
S
15
6
7
30
(skewed
segregation)
Mpr/Mpr+
R
21
37
S
0
0
(no segregation)
Chx/Chx
jan
+
2
10
2
10
(skewed
segregation)
jan/jan+
Scored in a subset of the progeny.
Weindruch & Doerder (1975)]. The end result is to bring about recovery of
alleles from only the fertile clone being tested. All such alleles will be in a homozygous state, with a 1:1 ratio of homozygotes for the two alleles at any originally
heterozygous locus. The results of the genomic exclusion cross are shown (as
Gen 2) in Table 1. Of the two drug-resistant markers, methylpurine resistance
segregated (though with skewed ratios in both crosses), while cycloheximide
resistance surprisingly did not. Thus, even the two Gen. 1 'zygotes' recovered
from the CU-127 x CU-329 cross were probably not outcomes of complete
nuclear fusion, as the unselected Chx+ marker from the CU-127 partner was not
included in a transmissible form. Fortunately, however, the putative allele underlying the cortical abnormality of the CU-127 clone was recovered, as two of the
examined subset of the Gen. 2 progeny of each of the Gen. 1 clones manifested
double contractile vacuole pore (CVP) sets and secondary oral apparatuses
(OAs) to the same or greater degree than did the CU-127 parent. This inheritable
syndrome will henceforth be referred to as the 'janus' phenotype (after the twofaced Roman god); details of its expression following conjugation will be considered subsequently (Results, section 2 a). In view of the aberrant segregation
ratios of a known single gene marker (Mpr), the skewed segregation of the janus
phenotype in this cross has little meaning (other examples of skewed segregations in crosses of recently mutagenized clones have been reported by Byrne
et at. 1978). All four janus clones were 6-methylpurine resistant. These four
Gen. 2 progeny were thus provisionally assigned a genotype of Mpr/Mpr,
210
J. FRANKEL AND L. M. JENKINS
Chx/Chx,jan/jan. Further breeding indicated that the fertilization-block of the
original CU-127 clone was greatly attenuated in these progeny, two of which,
were used as the foundation stocks (parents) for further crosses.
It should parenthetically be noted that the four jan/jan Gen. 2 progeny lacked
any indication of high-temperature lethality. We may therefore presume that a
temperature-sensitive allele at a separate gene locus was not transmitted to these
progeny. Further evidence for distinctness of the jan and lts' loci will be presented below (Results, section 1 b).
Most of the breeding results were obtained with one of the Gen. 2 progeny
from clone 1 (Table 1), and these results are presented in Table 2. In consideration of these results, the Gen. 2 clones are considered as 'parents', as they are the
first jan/jan generation that yielded viable progeny without use of radical
methods of positive selection. These parental clones, of presumed genotype
Mpr/Mpr, Chx/Chx, jan/jan, were outcrossed to wild type (B-1975) cells to
produce presumed heterozygous Fl progeny. When two Fl clones resulting
from the outcross of a janus 'parent' clone were crossed with each other, they
produced a 3:1 segregation for all three markers, including jan (Table 2). The
observed all-or-none expression of the janus phenotype in the different F2
clones each arising from Fls that did not express the janus phenotype proves
that control of the janus phenotype is genie, and the 3:1 ratio strongly suggests
determination by a single recessive allele.
A genomic exclusion cross of one of the two Fl progeny ('a') used in cross 1
yielded a good approximation of the expected 1:1 segregation for all three
markers (Table 2). The genomic exclusion progeny of the other Fl ('b') all died.
The two Fl progeny were also testcrossed to F2 and genomic exclusion progeny
that expressed janus. Three of the four sets of crosses of this type yielded the
expected 1:1 segregation of janus (Table 2). The one that did not was the testcross of the Fl ('b'), that had failed to produce genomic exclusion progeny, to
an F2 derived from the ' a ' x ' b ' cross. Since this same Fl yielded an excellent
1:1 segregation when testcrossed to a janus clone derived by genomic exclusion
from the other, 'healthy' Fl ('a'), we conclude that the one discrepant ratio is
due to aberrant segregation in the ' b ' Fl somehow induced by mating with an
F2 also carrying genetic material from that Fl. Cases of aberrant segregation
are not infrequently encountered in crosses involving T. thermophila (e.g.
Nanney, 1963; Allen & Lee, 1971; Frankei et al. 1976; McCoy, 1977; Byrne
et al. 1978). Nonetheless, the predominant results of the crosses shown in
Table 2, as well as those of a few pilot Fl x Fl and genomic exclusion crosses
(not shown) involving descendants of two of the other 'parental' janus lines, are
sufficient to demonstrate that the janus character is controlled by a recessive
allele at a single gene locus.
Analysis of combinations of progeny phenotypes (not shown) indicates that
jan is not linked to either Chx or Mpr, and also (confirming Ares & Bruns,
1978) that Chx and Mpr are unlinked to each other.
+
jan/jan+xA*-\
jan/jan x A*
Mpr/Mpr+, Chx/Chx+, jan/jan+ x
Mpr+/Mpr+, Chx+/Chx+, jan/jan
jan/jan+ x jan/jan
Mpr/Mpr+, Chx/Chx , jan/jan x
axb
Mpr/Mpr+, Chx/Chx+,jan/Jan+
Mpr/Mpr+, Chx/Chx+, jan/jan+ x A* a
b
+
Putative parental genotypes
All
jan
1:1
1:1
1:1
1:1
1:1
1:1
1:1
3:1
—
_
_
—
29
22
16
15
10
0
17
—
_
23
14
9
0
18
—
_
22
23
10
0
29
0
21
31
37
18
13
0
19
Cyclohex.
,
*
* ,
R
S
+
20
28
17
13
30
19
19
0
jan
A
>010
—
>0-25
<001
>0-25
>0-25
>0-25
>0-25
Cell surface
Progeny phenotypes
27/76
25/53
51/89
42/86
14/81
11/48
25/57
30/30
6/30
Number
dead -=total
conjugants
f One janus clone from among the progeny of the F l a x A* cross was outcrossed to wild type (B-1975). Three of the progeny of this outcross
were then crossed to A*.
Data from separate crosses within the same category have been lumped. These are in all cases homogeneous. The total number of lines scored
for drug-resistance phenotypes is in most cases less than the number scored for the cortical phenotype because some crosses were scored only
for cortical phenotypes. Drug-resistance phenotypes could not meaningfully be assessed in the Fl xjan F2 cross because no jan F2s were
sensitive to both drugs.
F i x A*
(genomic
exclusion)
Fl x jan F2
(testcross)
Fl xjan progeny
of Flax A*
(testcross)
jan progeny of
(Fla x A*) x A*
(jan x B) x A*
FlxFl
Type of cross
ExFl
pected 6-me-pur.
*
*
utilized segre- ,
in cross gation
R
S
Table 2. Genetic analysis 0/jan
CD
H
•s,
212
J. FRANKEL AND L. M. JENKINS
As might be expected, when fully homozygous janus progeny of the
Fl(a) x A* genomic exclusion cross are again carried through genomic exclusion,
all of the progeny express janus. Curiously, however, no cross of one janus line
with another has yet succeeded in producing viable progeny, despite repeated
attempts with various janus clones derived from three of the four 'parental'
(Gen. 2, Table 1) janus lines with rearing of exconjugants in pen-strep PPY and
also in the Fe2+-EDTA enriched medium (cf. Methods) as well as in the standard
bacterized medium. The successful outcome of the genomic exclusion cross of
homozygous janus cells and the survival of both exconjugants in janus progeny
of testcrosses (see section 2b) indicate that jan/jan micronuclei can undergo
successful meiosis, that two jan pronuclei can fuse to form a viable jan/jan
synkaryon, and that all steps of post-conjugation development can occur successfully in a conjugation partner that had previously expressed janus. The
unique element of the unsuccessful janus x janus crosses must thus relate to some
deficiency in the interaction of two conjugating cells both of which express janus.
Two janus cells can form geometrically normal tight conjugating pairs that
proceed typically through the nuclear events of conjugation and form exconjugants with the usual two new micronuclei and two macronuclear anlagen
(Nanney, 1953). However, the oral replacement that normally takes place in
exconjugants prior to the first post-conjugation division (Nelsen, personal
communication, and unpublished observations by the authors) appears to be
defective in exconjugants derived from janus x janus crosses. Although the fate
of these exconjugants has not been exhaustively studied, it is clear that the difficulty in these crosses is net in the mechanics of conjugation itself but rather in
the development of exconjugants.
As most recently reviewed by Sonneborn (1975), heterozygotes for all but one
of the known gene loci of T. thermophila undergo 'phenotypic assortment',
whereby either allele at the locus may become permanently and exclusively expressed in a vegetative lineage, with no relationship to conventional dominance
and with no effect on genetic transmission in crosses (Nanney, 1964). We thus
expected cells expressing the janus phenotype to appear eventually in jan/jan+
clones. However, after we failed to find such cells in cultures of heterozygotes
' a ' and ' b ' (Table 2) fixed 250 fissions after conjugation, we constructed nine
new heterozygous clones1 from an outcross of a janus clone derived from the
Fl(a) x A* cross, and analyzed samples fixed and silver stained at intervals of
100 fissions over a total of 400 fissions of maintenance in 1 % PPY tube cultures
at 25 °C with loop transfer thrice weekly. No clear manifestation of janus was
observed. The probable reason for this failure became clear when we followed
similarly maintained cultures derived from an equal mixture of the janus parent
of the outcross with the wild type parent (under conditions in which conjugation
cannot take place). The proportion of cells manifesting the janus phenotype
1
Heterozygosity of three of these clones was verified by genomic exclusion crosses, the
aggregated results of which are given by the bottom line of Table 2.
Genie control of symmetry in Tetrahymena
213
Table 3. Characteristics of''pseudo-conjuganf offspring of CU-127 x B cross
High
temperature
Cortical
Probable source of new
lethality in
reversal in
i
*
* ,
*
» MicroMacroClone Cells Progeny Cells Progeny nucleus
nucleus Probable origin through
X
No
Nonef
No
Yes
NoneJ
Yes
Yes
No
Short-circuit genomic
exclusion
First round of typical
Nonet B
CU-127
genomic exclusion
(None)|| B/CU-127 B/CU-127 Atypical fertilization
Nonef
B
B
Characters resembling those of the CU-127 parent are indicated in italics.
All progeny are derived from genomic exclusion progeny (second round) following cross
with A*.
t Six immature progeny scored.
t Ten immature progeny scored.
§ Thirty-three immature progeny scored (in two separate crosses).
|| Subset of four progeny scored.
(double CVP sets) was reduced from about 30 % initially to 2-3 % within 15
fissions after mixing, and to near-zero by 35 fissions. Cultures of this janus
parent (unlike the original CU-127 clone) grew more slowly than did the wild
type. Hence, our failure to observe assortment of the janus phenotype in
jan/jan+ heterozygotes may be explained trivially by a selective disadvantage
often being encountered by cells expressing the janus phenotype.
(b) Breeding analysis of"'pseudo-conjugants"1
Prior to the extraction of t\vtjan allele by positive selection as described in the
previous section, the CU-127 clone had been outcrossed to fertile wild-type cells
of the B strain. Three immature progeny clones were obtained from 60 pairs
(the remainder were dead or mature non-conjugants). Two of these three
(designated X and Z in Table 3) manifested new mating types, confirming that
a new macronucleus was formed from a micronuclear derivative. All three
progeny clones were substantially more fertile than the CU-127 parent (although
still less fertile than a typical wild type clone), suggesting the formation of a new
micronucleus. Nonetheless, the phenotypes of the three progeny clones and of
their offspring following further genomic exclusion crosses indicate that none
of these clones were conventional Fls (Table 3). Clone X neither manifested nor
transmitted the high-temperature lethality or the janus cell-surface phenotype
(drug-resistance phenotypes were not assayed in this series of crosses). This
clone was presumably uniparentally derived from the wild type partner, either
through self-fertilization (cytogamy) or through the direct generation of new/
nuclei from haploid products of meiosis (short-circuit genomic exclusion).
Clone Y manifested both high-temperature lethality and the janus cortical
214
J. FRANKEL AND L. M. JENKINS
phenotype, but transmitted neither. It may be presumed to have resulted from
the first round of typical genomic exclusion (Allen, 1967) in which a meiotic
product of the wild type micronucleus generates new micronuclei for both partners while the old macronuclei are retained; in this case the only descendents
recovered were those of the CU-127 partner, with a retained CU-127 macronucleus and a micronucleus derived from the B strain. The properties of clones
X and Y are thus readily explained as results of uniparental phenomena that
have received ample independent documentation in this organism, and are to be
expected from attempts to cross a virtually sterile clone1. Clone Z, however,
both manifested high temperature lethality and transmitted it to all of its progeny, while it neither exhibited nor transmitted the janus cortical phenotype
(although samples of only four progeny were checked in the latter case). Hence
in this case two characteristics of the original CU-127 clone have become dissociated from one another. This combination is not easy to interpret in a straightforward manner, particularly if it is accepted that the CU-127 clone arose from
short-circuit genomic exclusion and is therefore homozygous at all loci2. The
strong evidence for partial fertilization involving CU-127 presented in the previous section, as well as parallel evidence reported by Kaczanowski (1975,
pp. 637-638), suggests that clone Z might also have arisen from an aberrant
fertilization. However this may be, the pattern of expression and transmission
of phenotypes by clone Z does establish that the janus cell surface phenotype is
dissociable from the high-temperature lethality that provided the basis for the
original selection of the CU-127 clone.
Clone Z has been outcrossed to the wild-type B strain with subsequent analysis of high-temperature lethality in various types of progeny. The results (not
shown) strongly suggest a recessive single-gene basis for this lethality.
2. Dynamics of expression of the janus phenotype
(a) Expression after acquisition of homozygosity for the jan allele
The three cortical features that distinguish the CU-127 clone from all previously examined T. thermophila are (1) stable propagation of an unusually high
number of ciliary meridians; (2) capacity to produce a second oral apparatus
(OA) with characteristic abnormalities at a position close to 180° to the cell's
1
The only anomaly inconsistent with this explanation is the immaturity of clone Y. As
progeny of the first round of typical genomic exclusion retain their old macronuclei, they
should be sexually mature (cf. Allen, 1967). We are therefore forced into the ad hoc postulate
that clone Y, for some unknown reason, did not conjugate when challenged with suitable
testers. The fact that clone Y, unlike the two others, retained its old mating type is consistent
with this postulate, though it is not compelling evidence for it as the mating type in question
(IV) is the commonest encountered in this species.
2
Recent results obtained by Ares & Bruns (1978) indicate that progeny of short-circuit
genomic exclusion may be heterozygous for alleles newly arisen as a result of mutagenesis of
the parental clone. But if the CU-127 clone were heterozygous for the janus allele, it would
have had to undergo phenotypic assortment to full expression of the janus allele. This is unlikely in view of our failure to achieve such assortment in known janus heterozygotes.
Genie control of symmetry in Tetrahymena
215
Table 4. Expression of the janus phenotype and ciliary meridian number in parents
and progeny of a genomic exclusion cross of a jan/jan + heterozygote
Phenotypet
Over;111 %
i
1 OA
\Jr\
1
1 OA
1 CVP 2 CVP
set
sets
Clone
+
9
OAc
9 OA<?
1 CVP 2 CVP
set
sets
X J wUlllvU
2OAs
Fl (jan/jan )
A* (jan+/jan+)
Progeny
100
100
—
—
—
—
—
—
0
0
1
2
3
4
5
6
7
9
10
11
12
13
14
15
16
17
18
100
100
49
—
—
40
29
—
—
39
—
47
44
—
44
40
—
34
52
56
—
—
4
—
—
7
6
—
—
9
—
6
6
—
9
2
—
9
7
0
0
11
14
57
100
100
43
100
42
42
100
41
53
100
51
37
31
8
—
—
9
—
5
8
—
6
5
—
Modal
ciliary "Prpcn mpfl
meridian cortical
number^ parentage
0
0
18
0
11
14
0
15
7
0
15
11
2 CVP Symmetry
sets phenotype
+
0
21§
0
+
19-20§
0
0
47
35
0
0
48
0
53
50
0
53
42
0
43
59
65
+
+
22
20
janus
janus
20-23||
2111
+
20
21
+
janus
+
20||
21
—
—
Fl
?
?
7
?
Fl
?
Fl
Fl
A*
A*
Fl
?
A*
21
janus
19
janus
19
+
21
janus
janus
211|
19
+
1Q 011I
janus
6
7
1 Qll
4
A*
janus
21
janus
13
Fl
4
9
?
19
20
34
40
14
46
26
janus
20||
t Based on tallies of 100 cells in each clone, scored from samples fixed at 40fissionsafter conjugation.
% Based on tallies of 50 cells in each of the two parent clones, and 30 or 40 cells in each progeny clone
scored from samples fixed at 25 and 40 fissions after conjugation.
§ Total distribution: F l : 19-1, 20-14, 21-35; A*: 17-1, 18-8, 19-22, 20-18, 21-1.
|| Includes a substantial proportion of cells with high (23-29) number of ciliary meridians.
right of the normal OA, and (3) frequent possession of two sets of contractile
vacuole pores (CVPs), always located on the side of the cell that is to the right
of the normal OA and to the left of the secondary abnormal OA (Jerka-Dziadosz
& Frankel, 1979). All progeny clones derived from the crosses described in
section 1 of the Results were either indistinguishable from wild-type cells with
respect to features (2) and (3), or else were similar to the original CU-127 clone
with respect to these same features. The characteristic symmetry features of
wild-type and CU-127 respectively were expressed coordinately in progeny
clones, justifying the notion of a 'janus' cortical syndrome. In the clones that
became homozygous for jan the expression of this syndrome was fully attained
within 25 fissions after conjugation despite the total absence of any microscopically visible trace of the syndrome in the heterozygous parents. In contrast,
the number of ciliary meridians that were maintained by progeny clones other-
216
J. FRANKEL AND L. M. JENKINS
wise similar to the CU-127 clone was only infrequently as high as in CU-127,
and more commonly resembled that found in the wild-type (see below and section
2c).
One set of progeny clones of the Fl(a) x A* genomic exclusion cross (Table 2)
was analyzed in more detail, after fixation of cultures grown in 1 % PPY under
conditions of exponential growth that promote expression of secondary OAs.
All clones grew at roughly the same rate. Phenotypic classes of OAs and CVP
sets are presented in Table 4. Overall expression of secondary OAs and double
CVP sets was (with one exception) homogeneous in all clones expressing yaw, at
a level near 15 % and 50 % respectively. This is the same as observed under
these conditions in the original CU-127 clone (see the '1 % PPY-Log-280' line
in Table 1 of Jerka-Dziadosz & Frankel, 1979). The one exception, the unusually
high expression of secondary OAs in clone no. 19, will be considered more fully
in section 2(c). The expression of secondary OAs and of double CVP sets was
mutually independent in individual cells, as also observed in the original
CU-127 clone. A further common feature not shown in Table 4 is that dividing
cells manifested numerous different combinations of traits in anterior and
posterior moieties of the same cell, much as shown in Fig. 7 of the previous
paper. OAs on the 'primary axis' were always formed and always normal,
whereas those on the secondary axis were sporadically generated and almost
always abnormal.
One respect in which the janus progeny resembled their cytoplasmic ancestors
more than they did the original CU-127 clone was in the number of ciliary
meridians. For example, in clone 11 (Table 4) virtually all cells had 19 ciliary
meridians, suggesting cytoplasmic origin from the A* 'parent'. Most of the
cells within this clone that expressed two OAs also possessed 19 ciliary meridians.
Nonetheless, the relational geometry of positioning of OAs and CVPs within
such cells was very similar to that found in cells of the original CU-127 clone,
in which few cells had less than 22 ciliary meridians. Subsequent analysis of
cells of other janus clones with diverse ciliary meridian numbers indicated that
relative positions of OAs and CVPs were much the same irrespective of the
number of ciliary meridians. These results indicate that capacity to express the
distinctive janus symmetry pattern is not dependent on any particular number
of ciliary meridians, and strongly suggest that at least in some cases the ciliary
meridian numbers observed in the janus progeny clones were inertially carried
over from the cortical parents. Thus, when previously normal cells become
jan/jan they can express the new symmetry pattern within the old set of ciliary
meridians.
(b) Expression after loss of the jan allele
In the previous section the onset of expression of jan was examined in cells
with initial wild-type cortical symmetry patterns and ciliary meridian numbers.
The appropriate reciprocal experiment would investigate loss of jan expression
Genie control of symmetry in Tetrahymena
217
Table 5. Expression of the janus phenotype and ciliary meridian numbers in
parents and progeny of an outeross of a clone expressing jan
Ciliary meridian number
Parents
Progeny
(all +)
(Clone)
18
19
20
(Clone)
21
22
23
24
25 +
B( + )
la
2a
3a
4a
5a
6a
7a
8a
9a
10a
16
—
20
—
—
—
—
—
—
—
—
34
20
—
13
20
19
20
14
20
—
—
—
7
—
1
—
6
—
—
1
Y(jan)
7t
—
8
4
18
20
17
5
18
4
20
7
9
—
33
16
—
—
—
15
1
—
—
13
7
6
2
—
—
—
—
—
—
—
—
—
—
14
1
—
—
—
—
—
—
—
—
—
—
—
—.
19±
20
19
lb
2b
3b
4b
5b
6b
7b
8b
9b
10b
/lla
I lib
2
—
3
—
1
16
—
—
4
—
t Includes one cell with 20 ciliary meridians.
% Distribution of ciliary meridian numbers: 24-1, 25-2, 26-7, 27-8, 28-2. Fewer than 1 %
of the cells are homopolar doublets with two normal OAs, 30-35 ciliary meridians, and no
more than one CVP set in each segment between two OAs.
following introduction of a.jan+ allele into cells that initially possessed both the
janus symmetry pattern and the high ciliary meridian number of the original
CU-127 clone. This was accomplished with one of the three unconventional
progeny clones described in section l(b) of the Results. Recall that clone Y in
Table 3 was the direct offspring of a cross of CU-127 x B, and manifested the
janus symmetry pattern yet failed to transmit this pattern to any of its genomic
exclusion progeny. As explained earlier, this clone probably possessed a.jan/jan
macronucleus carried over from the CU-127 parent together with a jan+/jan+
micronucleus introduced from its wild-type mate. Its ciliary meridian number,
with a mode of 23, was also consistent with direct derivation from the CU-127
parent. Clone Y, then, was probably a CU-127 cell with an introduced wild-type
micronucleus. The new wild-type micronucleus of clone Y allowed moderate
fertility on outcrosses; therefore, this clone was outcrossed to wild-type (B-1975)
cells. Shortly after separation of conjugants but prior to the first post-conjugation division the two exconjugants of each of 11 pairs were separated to generate
22 initially immature clones. These clones were serially cultivated in 1 % PPY
tube cultures and periodically stained and examined over an interval of 1000
fissions. Over this entire interval not a single examined cell within any of the
clones manifested any aspect of the symmetry pattern characteristic of the janus
phenotype. Even at about 25 fissions after conjugation, when first examined, all
cells observed had a single CVP set and no secondary OAs. Hence cells cytoplasmically descended from a parent expressing jan lost every microscopically
218
J. FRANKEL AND L. M. JENKINS
visible trace of the janus symmetry features within 25 fissions after the formation
of a new macronucleus, presumably containing jan+, at conjugation. Yet when
ciliary meridian numbers were examined the result was different. In ten of the
eleven pairs of clones, the clonal descendents of one exconjugant initially manifested the high ciliary meridian numbers characteristic of clone Y, while the
descendents of the other exconjugant exhibited the lower ciliary meridian numbers characteristic of the wild-type B-1975 parent. These results are presented
in Table 5, arbitrarily arranged so that the exconjugant clone with the lower
meridian number is labelled 'a', whereas its sister clone with the higher number
is indicated as ' b ' . This general result is very similar to the transmission of preexisting ciliary meridian number through conjugation demonstrated earlier by
Nanney (19666). In the one exceptional pair (no. 11), one clone (11 b) manifested
extremely high ciliary meridian numbers, but these were associated with the
presence within the clone of typical (wild-type) homopolar doublets with the
geometry described by Nanney, Chow & Wozencraft (1975), suggesting that the
wild-type conjugation partner was in this case a rare homopolar doublet cell.
The result shown in Table 5 is then the complement of that described, in the
previous section and summarized in Table 4. Whereas in the first case wild-type
cells of normal ciliary meridian number that became homozygous for jan
rapidly expressed the new abnormal symmetry features while tending to retain
the pre-existing ciliary meridian numbers, in the second case janus cells with
unusually high ciliary meridian numbers that acquired the jan+ allele soon reverted to the normal symmetry pattern while retaining the pre-existing high
ciliary meridian number for some time.1 Ciliary meridian numbers and largescale symmetry patterns are thus dissociable in both directions.
To provide a final test for this conclusion, in one set of test-crosses of the
category Fl(a)x[janus progeny of Fl(a)xA*] (i.e. jan/jan+xjan/jan), both
exconjugants were separately isolated. In 13 out of 19 synclones both exconjugants produced thriving clones. Within each synclone both exconjugant clones
are expected, to be genically identical, with one-half of the synclones heterozygous (jan/jan+) and the other half homozygous janus {jan/jan). However, within
each synclone the symmetry pattern of the pre-existing cell surface of one partner
had been normal at the time of the cross, while that of the other had been janus.
Hence in those synclones that became jan/jan+ after fertilization, expression of
genes controlling the wild-type cortical pattern was continued in the hitherto
normal partner and newly initiated in the hitherto janus partner. Conversely,
in those synclones that became jan/jan, expression of genes specifying the wildtype cell surface pattern was discontinued in the hitherto normal partner and
not initiated in the hitherto janus partner. Therefore, if there were any long-run
tendency for the pre-existing cortical configuration to perpetuate itself in the
1
This high ciliary meridian number was not retained indefinitely; there was a drift back
to the 'stability center' characteristic of the wild type. This will be described more extensively
in a subsequent communication.
Genie control of symmetry in Tetrahymena
219
Table 6. Persistence of differences in expression of secondary OAs and ciliary
meridian numbers in serially propagated janus clones
40 fissions
250 fissions
650 fissions
,
2 O/oA (Fe 2 +)§
y
/o
Set Clone 2OAf
Merid.
no.J s.D.
2 O/oA
Merid.
no. s.D.
2 OA
Merid.
no. s.D.
A||
22-1 ±2-2
21-l±0-4
19-4±0-9
20-5 ±1-5
201 ± 1 0
18
19
14
12
50
20-4 ±0-7
21-3 + 1-5
20-2 ± 1 0
20-2 ±2-4
21-7±2-4
4
3
4
9
26
20-7 ±0-4
19-9 ±0-9
19-3±0-4
20-0 ±1-4
21-8±l-9
3
10
11
14
19
11
11
14
7
34
y
525 fissions
25 fissions
B1f
5
12
t
7
23
18-5±l-0
23-5 + 0-8
/o
1
14
18-6±l-4
24-4+1-3
y
<1
2
8
22
56
^
2OA
13
17
21
44
83
500 fissions
225 fissions
r
-A
1
16
19-6±0-7
23-0+1-4
t Based on tallies of 100 cells in each sample.
% Based on tallies of 20 cells in each sample.
§ Maintained for 2 weeks prior to fixation in the Fe2+-EDTA enriched medium (see
Methods).
II Progeny of a genomic exclusion cross of a jan/jan+ heterozygote (same as in Table 4).
\ Two progeny clones from the Fib x [janus progeny of Fla x A*] testcross, selected for
low and high extremes of ciliary meridian number.
face of a gene expression incompatible with that configuration, differences
should have been observed between the two exconjugant clones derived from a
single conjugating pair. No meaningful differences were detected in clones fixed
about 25 fissions after conjugation: in eight synclones both clones were normal,
while in five both clones fully expressed janus. Hence a phenomiclag, if it exists,
is relatively short.
(c) Stable differences in expression of different janus clones
As already pointed out, in the progeny set that was examined in most detail
(Table 4), one clone (no. 19) manifested a considerably higher proportion of
secondary OAs than did any of the other janus clones. To find out whether this
difference in expression was stable, five of the 11 janus clones as well as three
of the seven wild-type clones within this progeny set were maintained in continuous growth in 1 % PPY at 25 °C by tri-weekly serial loop transfers of tube
cultures. At intervals, subcultures were transferred into culture flasks containing
1 % PPY, grown overnight at 28 °C, and fixed in early log phase at culture densities of close to 5000 cells/ml. The final fixation (at 650 fissions) was preceded
by subdivision of each janus clone into a portion grown as before in 1 % PPY
and a portion cultivated for 2 weeks in the enriched Fe2+-EDTA medium,
followed by growth in PPY and enriched-medium flask cultures respectively.
220
J. FRANKEL AND L. M. JENKINS
Silver-impregnated slides of the fixed samples were scored for expression of
secondary OAs, double CVP sets, and ciliary meridian numbers. All five of the
janus clones continued to express the janus phenotype for the duration of the
experiment, whereas no cell in the three clones originally scored as wild-type
exhibited any of the diagnostic features of the janus phenotype. Among the five
janus clones, the expression of double CVP sets remained fairly constant and
homogeneous. In contrast, as shown in Table 6 (set A), the expression of
secondary OAs fluctuated, yet clone 19, which had the highest proportion of
secondary OAs initially, consistently exhibited a frequency of secondary OAs
about triple that of any of the other clones. The Fe2+-EDTA medium enhances
expression of secondary OAs in all of the janus clones (Table 6, right column).
At this higher general level of expression, the difference between clone 19 and
the others persists; indeed, clone 19 was brought up to essentially complete
expression of secondary OAs (literal 100 % expression is probably impossible,
due to the tendency of many secondary OAs to undergo resorption). This nearcomplete expression in Fe2+-EDTA medium was repeatedly observed for
clone 19 but not for any of the others. Thus it is hard to avoid the conclusion
that clone 19 has an intrinsically higher capacity to express secondary oral
structures than do the other janus clones.
Somewhat surprisingly, the average ciliary meridian number of the janus
clones of this set generally remained within the wild-type range of 18-21 ciliary
meridians (Table 6, set A); clone 19 became slightly higher than the others but
still did not attain the mean of 22-24 ciliary meridians characteristic of the
original CU-127 clone. However, cells with unusually high ciliary meridian
numbers (up to 31) appeared with greater or lesser frequency in janus clones
even under the conditions of near-continuous growth that prevailed in this
experiment. The proportion of such cells differed in different clones (highest in
clone 19, also high in clone 14, and low in the others except for the first sample
of no. 3), suggesting possible differences among janus clones in the frequency
with which such high ciliary meridian numbers are generated and/or the stability with which they are propagated. To check on these possibilities, a subsequent
progeny set was searched for those janus clones initially manifesting the highest
and the lowest prevailing ciliary meridian numbers. The two janus clones with
extreme meridian numbers were then serially propagated and periodically
examined. The results, shown in Table 6 (set B), clearly indicate that the differences are propagated. Ciliary meridian numbers in clone 5 slowly drifted up to
a level similar to that in the majority of clones in set A, while expression, of
secondary OAs remained low. Clone 12, on the other hand, maintained an
average number of ciliary meridians and also of secondary OAs similar to that
found in the original CU-127 clone. Both clones manifested typical proportions
(50-70 %) of double CVP sets (not shown).
To help interpret the stability of differences observed between clones, a subclonal expansion was undertaken of clone 19, which manifested great internal
Genie control of symmetry in Tetrahymena
221
variability of ciliary meridian numbers. The subclones differed substantially in
ciliary meridian number and also in expression of secondary OAs when first
fixed at 15 fissions after their establishment, but became homogeneous in expression of secondary OAs and nearly so in ciliary meridian number by 100
fissions (data not shown). These findings are consistent with the conclusion that
the long-term differences observed between clones are inherent, and probably
based on genie differences.
Comparisons both between and within clones and subclones indicate a
positive association between number of ciliary meridians and frequency of expression of secondary OAs. However, there are at least two reasons for not
concluding that a high ciliary meridian number is a prerequisite for expression
of a secondary OA. First, cells with as few as 18 ciliary meridians that possessed
secondary OAs were commonly observed in clone 5 (set B, Table 6); the fact
that no cells with fewer than 18 ciliary meridians have been found bearing
secondary OAs is probably simply due to the rarity of cells with 17 or fewer
ciliary meridians under our culture conditions. Hence there is no evidence for a
minimum number of ciliary meridians required for expression of secondary OAs.
Second, in clone 19 (set A, Table 6) a high frequency of secondary OAs became
established before the average number of ciliary meridians rose to a level somewhat higher than that of the other janus clones in the set; further, the great increase in expression of secondary OAs brought about by the Fe2+-EDTA
medium was not accompanied by an increase in number of ciliary meridians
(data not shown). Thus, if there is any causal relationship between number of
ciliary meridians and expression of secondary OAs in janus cells, it is complex
and indirect.
DISCUSSION
(A) Origin of janus
The preceding paper (Jerka-Dziadosz & Frankel, 1979) has demonstrated
that the CU-127 clone of T. thermophila possesses a unique phenotype strongly
suggestive of a geometrically reversed morphogenetic field that is reliably propagated within the clone. In this communication we have shown that a recessive
allele at a single gene locus (janus) determines the propagation and/or expression
of this reversed field. We do not know when this allele originated. The phenotype
that it controls first came to our notice in a clone that had been subjected to
nitrosoguanidine mutagenesis followed by short-circuit genomic exclusion
(Bruns et al. 1976). However, the selective procedure by which the clone was
picked out by Bruns & Sanford (1978) involved lethality at high temperature,
and we have shown that this high-temperature lethality is separable from the
cortical reversal (cf. Results, section 1 b). Hence it is likely that the jan mutation
was induced by the mutagen in the same micronucleus together with another,
independent, temperature-sensitive mutation, and the two were both transmitted
to the same progeny clone following short-circuit genomic exclusion. Selection
15
EMB
49
222
J. FRANKEL AND L. M. JENKINS
for the latter mutation then fortuitously brought about recovery of the former.
Deliberate selection ofjanus would require a method for generating homozygosity following mutagenesis, such as the short-circuit genomic exclusion
method of Bruns et ah (1976), coupled with a method for easily recognizing
living cells exhibiting the phenotype. As the somewhat flattened shape of known
janus homozygotes tends to be accentuated in the Fe2+-EDTA medium, the
phenotype might possibly be selectable in this medium. Only after such a selection procedure is put into operation will it be possible to estimate the number of
loci that can give rise to the janus phenotype.
(B) Dynamics of expression of janus
It has been shown in the preceding paper (Jerka-Dziadosz & Frankel, 1979)
that, although janus cells superficially resemble wild-type homopolar doublets
in possessing two oral apparatuses and two sets of CVPs, the geometrical arrangement of these structures is very different in janus and wild-type doublets. In this
paper, an equally fundamental difference in both the origin and time-course of
expression of these two conditions is demonstrated. Wild-type homopolar
doublets originate by lateral fusion of two cell-units. They, therefore, initially
manifest a high number of ciliary meridians, which then declines. Concomitantly
with this decline, cells revert from the doublet back to the singlet state (FaureFremiet, 1948; Nanney et al. 1975). Intermediate states of expression of doubleness of oral structures and CVPs are uniquely associated with intermediate
numbers (22-27) of ciliary meridians (Nanney, 1966 a), which represent a
transitional condition during the reversion from the doublet to the singlet
phenotype. The origin and dynamics of expression of the atypical doublet state
of janus clones differs from that in wild-type homopolar doublets in all of the
above respects. The janus doublet state becomes manifest only after cells containing a wild-type (jan+) allele become homozygous jan/jan, and disappears
only after cells with jan/jan macronuclei acquire a jan+ allele. The change of
expression in both directions is rapid, being completed by 25 fissions after conjugation. Further, neither the appearance nor the loss of capacity to generate
secondary OAs or double CVP sets following the above-mentioned changes in
genotype need be associated with any change in number of ciliary meridians.
Although there is some positive association between degree of expression of
abnormal secondary OAs and number of ciliary meridians, the association is
far less pronounced than that found in wild-type homopolar doublets: the
secondary oral structures can be expressed in cells with as few as 18 ciliary
meridians yet is not invariably expressed in cells with as many as 29 meridians.
The rules governing both the origin and the expression of secondary OAs and
double CVP sets in janus cells are thus fundamentally different from those
operating in wild-type doublets. The difference is a result of a profound difference in the basis of the two conditions, gene expression in the former and nongenic inheritance of large-scale cytoplasmic patterns in the latter.
Genie control of symmetry in Tetrahymena
223
(C) Subsidiary effects ofjanus
The janus allele has other effects in addition to maintaining the unique geometrical pattern of secondary OAs and double CVP sets. First, while not itself
influencing the modal number of ciliary meridians, thejanus allele when homozygous brings about a certain degree of instability in the propagation of ciliary
meridians, leading to the appearance of some cells with unusually high numbers
of ciliary meridians. This instability appears to open a 'phenotypic window' for
the expression of modifying genes that can influence the modal number of ciliary
meridians. In these clones where such genes may be presumed to be present,
which include the original CU-127 clone and at least one known janus progeny
clone, these genes appear to stabilize the high ciliary meridian numbers that
appear sporadically in all janus clones. A second, more perplexing subsidiary
effect of the janus allele relates to the capacity of exconjugants to generate viable
clones. No viable progeny are produced when janus homozygotes are crossed
with each other, despite the demonstrated capacity of such homozygotes to
undergo normal meiosis and fertilization. The crucial phenotypic defect in such
crosses is not in conjugation itself, but in some step(s) in the development of
exconjugants. Thus, normal development of exconjugant cells requires jan+
gene product. Interestingly, a sufficient amount of this product can be supplied
'maternally', since exconjugant cells with a newly established jan/jan genotype
can generate viable clones if either these cells or their mating partners had been
phenotypically wild-type prior to conjugation [the partner is effective in promoting a 'maternal effect' because macromolecules circulate freely between
conjugating cells (McDonald, 1966)]. Curiously, a similar drastic yet maternally
rescuable effect on survival of exconjugants was also observed for one of the
very few other known ciliate genes that has a non-conditional effect on global
cell-surface pattern, 'basal body deficient' (bbd) in Euplotes minuta (Frankel,
1973). The connexion between the respective gene products and the capacity to
undergo post-conjugational development is obscure in both cases, although a
hint may be provided by the fact that both bbd and jan bring about a cortical
instability that tends to be especially severe in young clones.
Additional modifying genes probably affect not only the stable number of
ciliary meridians in janus cells, but also the extent of expression of secondary
OAs. The possible existence of one major gene enhancing expression of secondary OAs is currently under investigation.
(D) Mechanisms of pattern reversal
The actual mechanistic basis of the genically controlled expression of the
reversed secondary morphogenetic field is unknown. This is the first example
in which a pattern reversal in ciliates is known to be the result of an action of an
allele at a nuclear gene locus. Most earlier examples of mechanically induced
and non-genically propagated pattern reversals, such as the famous ciliary
15-2
224
J. FRANKEL AND L. M. JENKINS
meridian inversion in Paramecium (Beisson & Sonneborn, 1965) and its equivalent in Tetrahymena (Ng & Frankel, 1977), involve the ciliary meridians and
adjacent organelles, and manifest no phenotypic effects of the kind seen in janus
clones. However, there are a few examples of large scale reversals of asymmetry
that are more comparable to those brought about by the janus allele. Some of
these are reviewed by Tartar (1967, pp. 90-92). In these cases, encountered in
large ciliates such as Stentor, Blepharisma, and Condylostoma, microsurgically
induced large-scale pattern reversals have been propagated for a short duration
only. Tchang & Pang (1965), however, have described a microsurgical operation
that can bring about mirror-image doublet cells of the hypotrich ciliate Stylonchia mytilus which propagate one normal and one reversed set of oral structures
for hundreds of fissions. This finding suggests that although in the case of janus
cells the propagation and expression of a morphogenetic field of reversed
asymmetry is dependent on the continuous action of a mutant allele, such dependence need not always be the case. An extreme possibility might be that thejan
allele, rather than 'creating' a morphogenetic field of reversed asymmetry,
brings a precisely positioned pre-existing field above a threshold of phenotypic
manifestation. However, as there is evidence that the janus condition can be
expressed in cells of inbred strain A as well as B cytoplasmic ancestry (cf.
Table 4 and accompanying text), such a 'silent' pre-existing field, if it exists,
must be present in T. thermophila of diverse natural sources.
It is worth noting that although janus is the first known case of a genecontroJled expression of pattern reversal in a unicellular organism, formally
comparable examples are known in multicelJular organisms. In some of these,
such as the control of direction of coiling of the shell of the snail Limnaea
(Sturtevant, 1923; Boycott, Diver, Garstang & Turner, 1930) and the specification of mirror-image double abdomens by the bicaudal mutant in Drosophila
(Bull, 1966; Nusslein-Volhard, 1977), the genes act by maternal predetermination of the egg cytoplasm. In the others, including visceral inversion in mice
(Hummel & Chapman, 1959) and genically controlled mirror-image reduplications such as those of the wing in duplicate fowl (Landauer, 1956) and of the
dorsal thorax in wingless Drosophila (Sharma & Chopra, 1976), genes act after
zygote formation, presumably to influence the organization of organ anlage. The
case best analyzed developmentally is the wingless allele in Drosophila, which
is known to act at an embryonic stage on the wing imaginal disc, probably to
bring about an abnormal proximal-distal compartmentalization such that the
portion of the disc that normally produces wing becomes mirror-image thorax
instead (Babu, 1977; Morata & Lawrence, 1977). The janus phenotype of
Tetrahymena could be viewed as a conversion of a 'dorsal' (aboral) cell surface
to a mirror-image 'ventral' (oral) surface, in which case the janus phenotype
could be considered as analogous to the wingless condition in Drosophila. This
formal resemblance leaves open the possibility that there might be some similarity in the underlying mechanisms of control of large scale asymmetry.
Genie control of symmetry in Tetrahymena
225
The authors would like to thank Drs Peter J. Bruns, Anne W. K. Frankel, and David L.
Nanney for valuable advice with regard to the genetic analysis. We also express our appreciation for helpful criticisms of the manuscript provided by Drs Karl Aufderheide, Peter Bruns,
Anne W. K. Frankel, Maria Jerka-Dziadosz, and E. Mario Nelsen. This research was supported by grant no. HD-08485 from the U.S. National Institutes of Health.
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