a mutant of tetrahymena thermophila with a partial mirror-image

/ . Embryol. exp. Morph. Vol. 49, pp. 203-227, 1979 2 0 3Printed in Great Britain © Company of Biologists Limited 1979

A mutant of Tetrahymena thermophilawith a partial mirror-image duplication of cell

surface pattern

II. Nature of genie control

By JOSEPH FRANKEL1 AND LESLIE M. JENKINS1

From the Department of Zoology, University of Iowa, Iowa City

SUMMARYThe 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 poreswith partial mirror-image reversal of asymmetry (Jerka-Dziadosz & Frankel, 1979), has beensubjected to breeding analysis. The progeny ratios obtained in various crosses indicate thatthe abnormalities of cell-surface asymmetry are brought to expression as a result of the actionof a recessive allele at a single gene locus, here named janus. When previously normal cellswere made homozygous for the jan allele, the cortical pattern characteristic of the CU-127clone came rapidly to expression, often without associated change in number of ciliarymeridians. 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 nor-mally located set of contractile vacuole pores while still retaining the high ciliary meridiannumber characteristic of the original CU-127 clone. The capacity for manifestation of theunique 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 ofsecondary 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 thepropagation and/or expression of a condition of reversed asymmetry in a precisely locatedcell region, and further indicates that the propagation and expression of this condition arelargely independent of the number and asymmetry of ciliary meridians.

INTRODUCTION

Little is known about the way in which genes influence patterns of symmetryat the level either of intracellular structure or multi-cellular organization. Mostanalyses of the problem of cellular and organjsmic symmetry have employedmicrosurgical interventions to obtain controlled reversals of asymmetry.Reversals of intracellular asymmetry have been studied almost exclusively withciliated protozoa. The best example is the reversal of intracellular asymmetryassociated 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 ofasymmetry of positioning of at least three structures situated outside of theciliary meridians: the contractile vacuole pore (Ng, 1977), the longitudinalmicrotubule band (Ng & Frankel, 1977; Ng, 1978), and the subcortically locatedmitochondria (Jerka-Dziadosz, personal communication; Aufderheide, 1978).Another well known example of a cortical asymmetry reversal on a larger scaleis the microsurgically induced reversal of the 'zone of stripe contrast' withinwhich the oral primordium of Stentor develops (Tartar, 1956, 1960). What isdistinctive about all pattern reversals in ciliates which have been described thusfar is that the event that generates the reversal is an abnormal juxtaposition ofcellular elements either deliberately created by the experimenter or else occurringfortuitously and then selected by the experimenter. In the case of the Parameciumciliary meridian inversion, Beisson & Sonneborn (1965) established that cellsthat possessed and transmitted the reversal did not differ in any relevant genesfrom those that did not. In the other examples, genetic analysis was not under-taken but the mode of origin of the reversals makes a genetic explanation hardlyconceivable (see Tartar, 1967, pp. 90-92). Strong evidence thus exists for theconclusion ' . . .that the "information" for the direction of asymmetry... residesin the cell cortex... ' (Tartar, 1967, p. 92), and no evidence has thus far beenadduced for a role of genes in influencing or controlling the 'direction of asym-metry'. It is the purpose of this communication to describe the existence andmanifestation of genie control in the first example in which reversal of corticalasymmetry arose following a chemical operation on the genome rather than astructural alteration of the cell surface.

MATERIALS AND METHODS

The CU-127 clone of Tetrahymena thermophila (formerly T. pyriformis syngen1; cf. Nanney & McCoy, 1976) was obtained in February 1977 from the labora-tory of Dr David L. Nanney, to whom it had earlier been sent by Dr Peter Brunsfor cryopreservation [this stock was designated as CU-127 (111.) in the previouspaper (Jerka-Dziadosz & Frankel, 1979) to distinguish it from another sampleof the same clone that had earlier been sent to us directly from the laboratoryof 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 cellspreviously subjected to mutagenesis in 10/^g/ml of jV-methyl-yV'-nitrosoguani-dine, followed by short-circuit genomic exclusion (Bruns, Brussard & Kavka,1976) with positive selection for mating (Bruns & Brussard, 1974). The geneticmarker being used in the above selection for mating was a dominant alleleconferring resistance to 6-me thy lp urine (Byrne, Brussard & Bruns, 1978), hencethe CU-127 clone possesses and (owing to the nature of short-circuit genomicexclusion) may be presumed to be homozygous for the Mpr allele. At the same

Genie control of symmetry in Tetrahymena 205time it is cycloheximide-sensitive and thus necessarily homozygous for therecessive wild-type allele conferring sensitivity at the locus (ChxA) which hasbeen characterized by two allelic dominant mutations (ChxAl and ChxA2) todrug resistance (Roberts & Orias, 1973; Bleyman & Bruns, 1977; Byrne et al.1978). The micronuclear genotype of the CU-127 clone at these two loci maythus be written as Mpr/Mpr, ChxA+/ChxA+. CU-127 was one of many clonesoriginally selected for lethality following a 7-day exposure to high temperature(Bruns & Sanford, 1978). This temperature-sensitivity was manifested inaxenic medium only after prolonged stationary phase at the restrictive tempera-ture (39-5 °C) (Jerka-Dziadosz & Frankel, 1979) but was sometimes expressedmore rapidly in bacterized medium. The abnormalities of cell patterning in theCU-127 clone have been described in the preceding paper (Jerka-Dziadosz &Frankel, 1979).

Other stocks used in the genetic analysis include the standard inbred 'wild-type' B strain (B-1975) of T. thermophila, a 'defective' A* (A-star) clone(Weindruch & Doerder, 1975) used in genomic exclusion crosses, plus cloneCU-329, a 'homozygous heterokaryon' [ChxA2/ChxA2(cycl. sens.,II)] suppliedby Dr Peter Bruns (see Bruns & Brussard, 1974, p. 838). Although now knownas 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 theResults.

General procedures for carrying out crosses are the same as described pre-viously (Frankel, Jenkins, Nelsen & Doerder, 1976). Most crosses were carriedout at room temperature (about 23 °C) in bacterized peptone medium (a 24 hculture of Enterobacter aerogenes in 1 % proteose peptone diluted 1/70 withdistilled water before use). These methods were adequate in reasonably fertilecrosses. However, in the original extraction of the allele responsible for thereversal of cortical pattern from the virtually sterile CU-127 clone, a differentset of procedures was followed. These methods are identical in principle, thoughnot in details of execution, to those described by Bruns & Sanford (1978). TheCU-127 clone was crossed to clone CU-329, the 'homozygous heterokaryon'which expresses cycloheximide sensitivity although its micronucleus is homo-zygous for cycloheximide resistance (cf. Table 1, and Results, section la). Thecross was carried out under axenic conditions in Dryl's salt solution made up asin Nelsen & DeBault (1978). Two days after initiation of the cross (day 2), themating mixture (containing both calls which had conjugated and those whichhad not) was combined with an equal amount of 2 % proteose peptone mediumcontaining 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 sur-viving cells were removed and used to establish clones that were maintained in1 % proteose peptone. Two days afterwards (day 7) replicas of the 60 clones wereexposed in 1 % proteose peptone to 6-methylpurine at a final concentration of15 /tg/ml for 5 further days. Penicillin (1-4 g/1) and streptomycin (2-2 g/1) were

14 E M B 49


present throughout to prevent bacterial contamination. All of the above pro-cedures were carried out in wells of 3-spot depression slides (Corning no. 7223)at room temperature. Replicas of the two clones that survived serial exposureto cycloheximide followed by 6-methylpurine were then maintained by routineprocedures (see below) and subjected to further crosses in bacterized peptonemedium.

Axenic media, both the standard medium and also an enriched iron-chelatemedium (see below for descriptions) were also employed in efforts to obtainviable exconjugants in crosses ofjanus homozygotes with each other.

The axenic medium utilized in stock maintenance and in virtually all experi-ments was '1 %-PPY', made up of 1 % proteose peptone (Difco) plus 01 %yeast extract (Difco). When cells were being transferred from bacterized toaxenic medium, 'pen-strep PPY' (Frankel et al. 1976) was used. Cells frombacterized peptone were passaged, one or two at a time, from one well of athree-spot depression slide filled with pen-strep 1 % PPY to another. After thecells in the last well had grown to a sufficient density, a loop transfer was madeto a culture tube containing 5 ml of 1 % PPY, with accompanying tests forbacterial contamination. It should be noted that these procedures have the effectthat even when both exconjugants of a mating pair survive, frequently progenyfrom only one of them are recovered for subsequent analysis. In two experimentsthe two exconjugants from a pair were deliberately isolated after separation ofthe pair but before the first division, and allowed to form exconjugant clones.

On a few occasions, a specially enriched medium containing 0-5 % dextroseas well as an Fe2+-EDTA complex in addition to 2 % proteose peptone and0-2 % yeast extract (Conner & Cline, 1964; Thompson, 1967) was used, bothfor experiments with vegetative cells and for maintenance of exconjugants. Inthe latter case, it was combined with penicillin and streptomycin as describedabove for 'pen-strep PPY'.

Axenic clones were maintained at 20-25 °C in tube cultures containing 5 mlof 1 % PPY, with transfer using a bacteriological loop. Frequency of transferdepended on the purpose for which the cultures were maintained, and rangedfrom 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 thecell surface. Testing of drug-resistance phenotypes of progeny of crosses wascarried out using essentially the same methods as in the positive selection formating described earlier: replicas of clones were maintained at room tempera-ture and exposed in parallel to cycloheximide (25 /tg/ml) for 2 days and to6-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 inorder properly to assess drug resistance in heterozygotes before phenotypicassortment to drug-sensitivity might have occurred. Viability of clones at hightemperature was scored by transferring subsamples in 1 % PPY to an incubator

Genie control of symmetry in Tetrahymena 207maintained at 39-5 °C. All other relevant phenotypes were scored in fixed sam-ples subjected to silver impregnation (see Jerka-Dziadosz & Frankel, 1979, forcitations).

Cultures were prepared for fixation and scoring of surface patterns by one ofthree procedures: the 'standard' method was to fix the first axenic tube culturegrown at room temperature, following the removal from bacteria as describedabove; such cultures were about 25 fissions removed from conjugation, andtended to bs in early stationary phase, when fixed. When a rapidly growingculture in exponential phase was desired, cells were inoculated from the tubesinto 50 ml batches of medium in 250 ml conical flasks, grown for about 15 hat 28 °C, and fixed at a density of about 5000 cells/ml as determined by Coultercounts. Finally, a 'quick' method was sometimes used, in which a sample of anexconjugant synclone in bacterized peptone was added to 2 % PPY medium ina 3-spot depression slide and allowed to grow at 28 °C for about 15 h. Thisresulted in a high density log-phase culture that could be fixed at about 15 fissionsafter conjugation, but the cells were full of bacteria and silver impregnation wassomewhat inferior in quality to that obtained with samples grown in pureaxenic media. More than one of these methods were sometimes used to scorethe same set of progeny.

Standard scoring of cell surface phenotype involved examination of at least20 silver-impregnated cells to ascertain the arrangement of contractile vacuolepores, followed by scanning at low power to search for cells with two oralapparatuses. Cross-checking by different observers as well as more extensiveexamination 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 ofthe CU-127 clone was the indication from conventional breeding that micro-nuclei of members of this clone are able to go through meiosis but unable tocarry out viable fertilization (see section 1 b). For this reason, the methods devisedby Bruns & Brussard (1974) for positive selection for mating were used. Themethod as applied here is essentially one of marker rescue under highly selectiveconditions. Instrumental to this technique is the mating of the clone containinggenetic markers to be 'rescued' (CU-127 in this case) to a specially constructedclone that has a pattern of drug responses complementary to that of CU-127and also possesses a special nuclear design such that all cells that fail to generatenew macronuclei at conjugation can readily be killed by a suitable drug. Thisclone, CU-329, is a 'homozygous heterokaryon' of genotype Mpr+/Mpr+,Chx/Chx (cycl.-S). The special feature of this 'homozygous heterokaryon' isthat its micronucleus is homozygous for the ChxA2 allele conferring cyclohexi-

14-2


mide resistance, whereas its macronucleus is heterozygous at this same locusand has undergone 'phenotypic assortment' (Nanney, 1964) to express only theallele conferring cycloheximide sensitivity (for full explanation, see Bruns &Brussard, 1974). This 'homozygous heterokaryon' thus expresses sensitivity tothe drug yet transmits only the allele conferring resistance to sexual progeny.Thus, any exconjugant of a cross involving this homozygous heterokaryon thathas formed a new macronucleus from a product of its micronuclear meiosis isresistant to cycloheximide, whereas all suspected progeny that have in fact re-tained their old macronuclei remain sensitive to this drug. It is important to note,however, that formation of a new macronucleus from a product of micronuclearmeiosis need not imply cross-fertilization: uniparental cycloheximide-resistantprogeny might arise in several ways, including self-fertilization (cytogamy) in theChx/Chx homozygous heterokaryon (Orias & Hamilton, 1977), development ofunfertilized 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 directlygenerate a new macronucleus in either or both partners without fertilization. Toselect for actual cross-fertilization, the two-step drug selection procedure pre-viously employed by Bleyman & Bruns (1977) and Bruns & Sanford (1978) wasapplied. 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 cycloheximide-resistant progeny must have formed new macronuclei from meiotic products ofthe micronucleus of the CU-329 clone, while the cycloheximide-resistant progenywhich are also 6-methylpurine-resistant must have undergone at least sufficientfertilization to bring the drug-resistant alleles from the two parents into the samemacronucleus (see Table 1).

Sixty cycloheximide-resistant clones were isolated from a culture derivedfrom a mass mating of CU-127 x CU-329, yet only two of these were also resis-tant to 6-methylpurine (generation 1, abbreviated as Gen. 1 in Table 1). Thisresult suggests that the majority of new macronuclei that arose from meioticproducts of the CU-329 partner were uniparental.1 If the two exceptional clonesthat were resistant to both drugs were indeed the outcome of conventionalfertilization, then their genotype should be Mpr/Mpr+, Chx/Chx+ and the clonesshould additionally be heterozygous for any allele(s) controlling the unusual cellsurface pattern of the CU-127 clone. To test foi this, the two doubly-resistantclones were carried through a mating protocol termed' genomic exclusion' (Allen,1967), in which the clone to be tested is deliberately crossed to a known sterileclone 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 manifestedsensitivity 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 assort-ment 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

CU-127 CU-329Gen. 0: (Mpr/Mpr, Chx+/Chx+,jan/?) x (Mpr+/Mpr+, Chx/Chx,jan+/jan+)

Cycloheximide screen II (must possess Chx)

6-methylpurine screen I1 (must possess Mpr)

Gen. 1: (2 clones) Mpr/ ?, Chx/ ?, ?/ ?Genomic exclusion I (produces homozygosity)

cross >l6-me-purine Cycloheximide Cortexf

Gen.

Thus

2:

, Gen.

CloneClone

1 is:

t

12

Scored

R S15 67 30(skewed

segregation)Mpr/Mpr+

R S21 037 0

(no segregation)Chx/Chx

in a subset of the progeny.

jan +2 102 10(skewed

segregation)jan/jan+

Weindruch & Doerder (1975)]. The end result is to bring about recovery ofalleles from only the fertile clone being tested. All such alleles will be in a homo-zygous state, with a 1:1 ratio of homozygotes for the two alleles at any originallyheterozygous locus. The results of the genomic exclusion cross are shown (asGen 2) in Table 1. Of the two drug-resistant markers, methylpurine resistancesegregated (though with skewed ratios in both crosses), while cycloheximideresistance surprisingly did not. Thus, even the two Gen. 1 'zygotes' recoveredfrom the CU-127 x CU-329 cross were probably not outcomes of completenuclear fusion, as the unselected Chx+ marker from the CU-127 partner was notincluded in a transmissible form. Fortunately, however, the putative allele under-lying the cortical abnormality of the CU-127 clone was recovered, as two of theexamined subset of the Gen. 2 progeny of each of the Gen. 1 clones manifesteddouble contractile vacuole pore (CVP) sets and secondary oral apparatuses(OAs) to the same or greater degree than did the CU-127 parent. This inheritablesyndrome will henceforth be referred to as the 'janus' phenotype (after the two-faced Roman god); details of its expression following conjugation will be con-sidered subsequently (Results, section 2 a). In view of the aberrant segregationratios of a known single gene marker (Mpr), the skewed segregation of the janusphenotype in this cross has little meaning (other examples of skewed segrega-tions in crosses of recently mutagenized clones have been reported by Byrneet at. 1978). All four janus clones were 6-methylpurine resistant. These fourGen. 2 progeny were thus provisionally assigned a genotype of Mpr/Mpr,


Chx/Chx,jan/jan. Further breeding indicated that the fertilization-block of theoriginal 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 lackedany indication of high-temperature lethality. We may therefore presume that atemperature-sensitive allele at a separate gene locus was not transmitted to theseprogeny. Further evidence for distinctness of the jan and lts' loci will be pre-sented below (Results, section 1 b).

Most of the breeding results were obtained with one of the Gen. 2 progenyfrom clone 1 (Table 1), and these results are presented in Table 2. In considera-tion of these results, the Gen. 2 clones are considered as 'parents', as they are thefirst jan/jan generation that yielded viable progeny without use of radicalmethods of positive selection. These parental clones, of presumed genotypeMpr/Mpr, Chx/Chx, jan/jan, were outcrossed to wild type (B-1975) cells toproduce presumed heterozygous Fl progeny. When two Fl clones resultingfrom the outcross of a janus 'parent' clone were crossed with each other, theyproduced a 3:1 segregation for all three markers, including jan (Table 2). Theobserved all-or-none expression of the janus phenotype in the different F2clones each arising from Fls that did not express the janus phenotype provesthat control of the janus phenotype is genie, and the 3:1 ratio strongly suggestsdetermination by a single recessive allele.

A genomic exclusion cross of one of the two Fl progeny ('a') used in cross 1yielded a good approximation of the expected 1:1 segregation for all threemarkers (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 progenythat expressed janus. Three of the four sets of crosses of this type yielded theexpected 1:1 segregation of janus (Table 2). The one that did not was the test-cross of the Fl ('b'), that had failed to produce genomic exclusion progeny, toan F2 derived from the ' a ' x ' b ' cross. Since this same Fl yielded an excellent1:1 segregation when testcrossed to a janus clone derived by genomic exclusionfrom the other, 'healthy' Fl ('a'), we conclude that the one discrepant ratio isdue to aberrant segregation in the ' b ' Fl somehow induced by mating with anF2 also carrying genetic material from that Fl. Cases of aberrant segregationare not infrequently encountered in crosses involving T. thermophila (e.g.Nanney, 1963; Allen & Lee, 1971; Frankei et al. 1976; McCoy, 1977; Byrneet al. 1978). Nonetheless, the predominant results of the crosses shown inTable 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, aresufficient to demonstrate that the janus character is controlled by a recessiveallele at a single gene locus.

Analysis of combinations of progeny phenotypes (not shown) indicates thatjan is not linked to either Chx or Mpr, and also (confirming Ares & Bruns,1978) that Chx and Mpr are unlinked to each other.

Tab

le 2

. Gen

etic

ana

lysi

s 0

/jan

Typ

e of

cro

ss

Ex-

Pro

geny

phe

noty

pes

Put

ativ

e pa

rent

al g

enot

ypes

Fl

pect

ed

6-m

e-pu

r.

Cyc

lohe

x.

Cel

l su

rfac

eut

iliz

ed

segr

e-

, *

*

, *

*

, A

in c

ross

ga

tion

R

S

R

S

+

jan

Num

ber

dead

-=-

tota

lco

nju-

gant

s

•s,

Flx

Fl

Fix

A*

(gen

omic

excl

usio

n)

Fl

x ja

n F

2(t

estc

ross

)F

l x

jan p

roge

nyof

Fla

x A

*(t

estc

ross

)

jan p

roge

ny o

f(F

la x

A*)

x A

*

(jan

x B

) x A

*

Mpr

/Mpr

+,

Chx

/Chx

+,

jan/

jan+

x

a x b

3

:1M

pr/M

pr+

, C

hx/C

hx+

,jan

/Jan

+

Mpr

/Mpr

+,

Chx

/Chx

+,

jan/

jan+

x A

* a b

jan/

jan+

x

jan/

jan

Mpr

/Mpr

+,

Chx

/Chx

+,

jan/

jan+

xM

pr+/M

pr+,

Chx

+/C

hx+

, jan

/jan

jan/

jan

x A

*

jan/

jan+

xA*-

\

1:1

1:1

1:1

1:1

1:1

1:1

All

jan

1:1

17 10 0 16 1529 22

18 9 0 23 14

10 0 22 23

19 13 0 21 31 37 18

19 0 17 13 30 19

>0-

25

>0-

25

>0-

25<

00

1

>0-

25>

0-25

_ _

_

_

0

28

—

—

—

—

2920

>0

10

6/30

25/5

730

/30

51/8

942

/86

14/8

111

/48

—

25/5

3

27/7

6

f O

ne ja

nu

s cl

one

from

am

ong

the

prog

eny

of th

e F

la x

A*

cros

s w

as o

utcr

osse

d to

wild

typ

e (B

-197

5). T

hree

of

the

prog

eny

of t

his

outc

ross

wer

e th

en c

ross

ed t

o A

*.

Dat

a fr

om s

epar

ate

cros

ses

wit

hin

the

sam

e ca

tego

ry h

ave

been

lum

ped.

The

se a

re in

all

cas

es h

omog

eneo

us.

The

tot

al n

umbe

r of

lin

es s

core

dfo

r dr

ug-r

esis

tanc

e ph

enot

ypes

is

in m

ost

case

s le

ss t

han

the

num

ber

scor

ed f

or t

he

cort

ical

phe

noty

pe b

ecau

se s

ome

cros

ses

wer

e sc

ored

onl

yfo

r co

rtic

al p

heno

type

s. D

rug-

resi

stan

ce p

heno

type

s co

uld

no

t m

eani

ngfu

lly

be a

sses

sed

in t

he

Fl

xja

n F

2 c

ross

bec

ause

no

jan

F2

s w

ere

sens

itiv

e to

bot

h dr

ugs.

H CD


As might be expected, when fully homozygous janus progeny of theFl(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 linewith another has yet succeeded in producing viable progeny, despite repeatedattempts 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 andalso in the Fe2+-EDTA enriched medium (cf. Methods) as well as in the standardbacterized medium. The successful outcome of the genomic exclusion cross ofhomozygous janus cells and the survival of both exconjugants in janus progenyof testcrosses (see section 2b) indicate that jan/jan micronuclei can undergosuccessful meiosis, that two jan pronuclei can fuse to form a viable jan/jansynkaryon, and that all steps of post-conjugation development can occur suc-cessfully in a conjugation partner that had previously expressed janus. Theunique element of the unsuccessful janus x janus crosses must thus relate to somedeficiency in the interaction of two conjugating cells both of which express janus.Two janus cells can form geometrically normal tight conjugating pairs thatproceed typically through the nuclear events of conjugation and form exconju-gants with the usual two new micronuclei and two macronuclear anlagen(Nanney, 1953). However, the oral replacement that normally takes place inexconjugants prior to the first post-conjugation division (Nelsen, personalcommunication, and unpublished observations by the authors) appears to bedefective in exconjugants derived from janus x janus crosses. Although the fateof these exconjugants has not been exhaustively studied, it is clear that the diffi-culty in these crosses is net in the mechanics of conjugation itself but rather inthe development of exconjugants.

As most recently reviewed by Sonneborn (1975), heterozygotes for all but oneof the known gene loci of T. thermophila undergo 'phenotypic assortment',whereby either allele at the locus may become permanently and exclusively ex-pressed in a vegetative lineage, with no relationship to conventional dominanceand with no effect on genetic transmission in crosses (Nanney, 1964). We thusexpected 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 ninenew heterozygous clones1 from an outcross of a janus clone derived from theFl(a) x A* cross, and analyzed samples fixed and silver stained at intervals of100 fissions over a total of 400 fissions of maintenance in 1 % PPY tube culturesat 25 °C with loop transfer thrice weekly. No clear manifestation of janus wasobserved. The probable reason for this failure became clear when we followedsimilarly maintained cultures derived from an equal mixture of the janus parentof the outcross with the wild type parent (under conditions in which conjugationcannot take place). The proportion of cells manifesting the janus phenotype

1 Heterozygosity of three of these clones was verified by genomic exclusion crosses, theaggregated results of which are given by the bottom line of Table 2.


Table 3. Characteristics of''pseudo-conjuganf offspring of CU-127 x B cross

Hightemperaturelethality in

Corticalreversal in

Probable source of new

i * * , * » Micro-Clone Cells Progeny Cells Progeny nucleus

Macro-nucleus Probable origin through

X No

Yes

Yes

Nonef

NoneJ

No Nonef

Yes Nonet

No

B B Short-circuit genomicexclusion

First round of typicalgenomic exclusion

(None)|| B/CU-127 B/CU-127 Atypical fertilization

B CU-127

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 15fissions after mixing, and to near-zero by 35 fissions. Cultures of this janusparent (unlike the original CU-127 clone) grew more slowly than did the wildtype. Hence, our failure to observe assortment of the janus phenotype injan/jan+ heterozygotes may be explained trivially by a selective disadvantageoften 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 theprevious section, the CU-127 clone had been outcrossed to fertile wild-type cellsof 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 thata new macronucleus was formed from a micronuclear derivative. All threeprogeny clones were substantially more fertile than the CU-127 parent (althoughstill less fertile than a typical wild type clone), suggesting the formation of a newmicronucleus. Nonetheless, the phenotypes of the three progeny clones and oftheir offspring following further genomic exclusion crosses indicate that noneof these clones were conventional Fls (Table 3). Clone X neither manifested nortransmitted the high-temperature lethality or the janus cell-surface phenotype(drug-resistance phenotypes were not assayed in this series of crosses). Thisclone was presumably uniparentally derived from the wild type partner, eitherthrough 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


phenotype, but transmitted neither. It may be presumed to have resulted fromthe first round of typical genomic exclusion (Allen, 1967) in which a meioticproduct of the wild type micronucleus generates new micronuclei for both part-ners while the old macronuclei are retained; in this case the only descendentsrecovered were those of the CU-127 partner, with a retained CU-127 macro-nucleus and a micronucleus derived from the B strain. The properties of clonesX and Y are thus readily explained as results of uniparental phenomena thathave received ample independent documentation in this organism, and are to beexpected from attempts to cross a virtually sterile clone1. Clone Z, however,both manifested high temperature lethality and transmitted it to all of its prog-eny, while it neither exhibited nor transmitted the janus cortical phenotype(although samples of only four progeny were checked in the latter case). Hencein this case two characteristics of the original CU-127 clone have become dis-sociated from one another. This combination is not easy to interpret in a straight-forward manner, particularly if it is accepted that the CU-127 clone arose fromshort-circuit genomic exclusion and is therefore homozygous at all loci2. Thestrong evidence for partial fertilization involving CU-127 presented in the pre-vious section, as well as parallel evidence reported by Kaczanowski (1975,pp. 637-638), suggests that clone Z might also have arisen from an aberrantfertilization. However this may be, the pattern of expression and transmissionof phenotypes by clone Z does establish that the janus cell surface phenotype isdissociable from the high-temperature lethality that provided the basis for theoriginal selection of the CU-127 clone.

Clone Z has been outcrossed to the wild-type B strain with subsequent analy-sis of high-temperature lethality in various types of progeny. The results (notshown) 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 pre-viously examined T. thermophila are (1) stable propagation of an unusually highnumber 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. Asprogeny of the first round of typical genomic exclusion retain their old macronuclei, theyshould be sexually mature (cf. Allen, 1967). We are therefore forced into the ad hoc postulatethat clone Y, for some unknown reason, did not conjugate when challenged with suitabletesters. The fact that clone Y, unlike the two others, retained its old mating type is consistentwith 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-circuitgenomic exclusion may be heterozygous for alleles newly arisen as a result of mutagenesis ofthe parental clone. But if the CU-127 clone were heterozygous for the janus allele, it wouldhave had to undergo phenotypic assortment to full expression of the janus allele. This is un-likely in view of our failure to achieve such assortment in known janus heterozygotes.


Table 4. Expression of the janus phenotype and ciliary meridian number in parentsand progeny of a genomic exclusion cross of a jan/jan+ heterozygote

Clone

Fl (jan/jan+)A* (jan+/jan+)

Progeny12345679

10111213141516171819

i

1 OA1 \Jr\

1 CVPset

100100

1001004957

10010043

1004242

1004153

10051373140

Phenotypet

1 OA2 CVP

sets

——

——4029——39—4744—4440—34525626

9 OAc

1 CVPset

——

——

48

——

9—

58

—65

—644

14

9 OA<?

2 CVPsets

——

——

76

——

9—

66

—92

—979

20

Over;

2OAs

00

00

111400

180

11140

1570

15111334

111 %

2 CVPsets

00

00

473500

480

53500

5342

043596546

Symmetryphenotype

++

++

janusjanus

++

janus+

janusjanus

+janusjanus

+janusjanusjanusjanus

Modalciliary

meridiannumber^

21§19-20§

2220

20-23||2111202120||2121191921211|19

1Q 011I

1 Qll

2120||

"Prpcn mpflX J wUlllvU

corticalparentage

——

Fl??7?F l?F lF lA*A*F l?A*7A*Fl?

t Based on tallies of 100 cells in each clone, scored from samples fixed at 40 fissions after 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: Fl : 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 contractilevacuole pores (CVPs), always located on the side of the cell that is to the rightof the normal OA and to the left of the secondary abnormal OA (Jerka-Dziadosz& Frankel, 1979). All progeny clones derived from the crosses described insection 1 of the Results were either indistinguishable from wild-type cells withrespect to features (2) and (3), or else were similar to the original CU-127 clonewith respect to these same features. The characteristic symmetry features ofwild-type and CU-127 respectively were expressed coordinately in progenyclones, justifying the notion of a 'janus' cortical syndrome. In the clones thatbecame homozygous for jan the expression of this syndrome was fully attainedwithin 25 fissions after conjugation despite the total absence of any micro-scopically visible trace of the syndrome in the heterozygous parents. In contrast,the number of ciliary meridians that were maintained by progeny clones other-


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 section2c).

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 underconditions of exponential growth that promote expression of secondary OAs.All clones grew at roughly the same rate. Phenotypic classes of OAs and CVPsets are presented in Table 4. Overall expression of secondary OAs and doubleCVP sets was (with one exception) homogeneous in all clones expressing yaw, ata level near 15 % and 50 % respectively. This is the same as observed underthese conditions in the original CU-127 clone (see the '1 % PPY-Log-280' linein Table 1 of Jerka-Dziadosz & Frankel, 1979). The one exception, the unusuallyhigh expression of secondary OAs in clone no. 19, will be considered more fullyin section 2(c). The expression of secondary OAs and of double CVP sets wasmutually independent in individual cells, as also observed in the originalCU-127 clone. A further common feature not shown in Table 4 is that dividingcells manifested numerous different combinations of traits in anterior andposterior moieties of the same cell, much as shown in Fig. 7 of the previouspaper. OAs on the 'primary axis' were always formed and always normal,whereas those on the secondary axis were sporadically generated and almostalways abnormal.

One respect in which the janus progeny resembled their cytoplasmic ancestorsmore than they did the original CU-127 clone was in the number of ciliarymeridians. For example, in clone 11 (Table 4) virtually all cells had 19 ciliarymeridians, suggesting cytoplasmic origin from the A* 'parent'. Most of thecells within this clone that expressed two OAs also possessed 19 ciliary meridians.Nonetheless, the relational geometry of positioning of OAs and CVPs withinsuch 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 ofcells of other janus clones with diverse ciliary meridian numbers indicated thatrelative positions of OAs and CVPs were much the same irrespective of thenumber of ciliary meridians. These results indicate that capacity to express thedistinctive janus symmetry pattern is not dependent on any particular numberof ciliary meridians, and strongly suggest that at least in some cases the ciliarymeridian numbers observed in the janus progeny clones were inertially carriedover from the cortical parents. Thus, when previously normal cells becomejan/jan they can express the new symmetry pattern within the old set of ciliarymeridians.

(b) Expression after loss of the jan allele

In the previous section the onset of expression of jan was examined in cellswith initial wild-type cortical symmetry patterns and ciliary meridian numbers.The appropriate reciprocal experiment would investigate loss of jan expression


Table 5. Expression of the janus phenotype and ciliary meridian numbers inparents and progeny of an outeross of a clone expressing jan

(Clone) 18

Parents B( + ) 16Progeny la —(all +) 2a 20

3a —4a —5a —6a —7a —8a —9a —

10a —

19

3420—1320192014202019

Ciliary

20

———

7—

1—

6——

1

meridian

(Clone)

Y(jan)lb2b3b4b5b6b7b8b9b

10b/ l laI lib

number

21

7t—

2—

3—

116——4

—

22

84

1820175

184

2079

—

23

3316———15

1——1376

24

2——————————14

1

25 +

————————————.19±

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 nomore than one CVP set in each segment between two OAs.

following introduction of a.jan+ allele into cells that initially possessed both thejanus symmetry pattern and the high ciliary meridian number of the originalCU-127 clone. This was accomplished with one of the three unconventionalprogeny clones described in section l(b) of the Results. Recall that clone Y inTable 3 was the direct offspring of a cross of CU-127 x B, and manifested thejanus symmetry pattern yet failed to transmit this pattern to any of its genomicexclusion progeny. As explained earlier, this clone probably possessed a.jan/janmacronucleus 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-127parent. Clone Y, then, was probably a CU-127 cell with an introduced wild-typemicronucleus. The new wild-type micronucleus of clone Y allowed moderatefertility on outcrosses; therefore, this clone was outcrossed to wild-type (B-1975)cells. Shortly after separation of conjugants but prior to the first post-conjuga-tion division the two exconjugants of each of 11 pairs were separated to generate22 initially immature clones. These clones were serially cultivated in 1 % PPYtube cultures and periodically stained and examined over an interval of 1000fissions. Over this entire interval not a single examined cell within any of theclones manifested any aspect of the symmetry pattern characteristic of the janusphenotype. Even at about 25 fissions after conjugation, when first examined, allcells observed had a single CVP set and no secondary OAs. Hence cells cyto-plasmically descended from a parent expressing jan lost every microscopically


visible trace of the janus symmetry features within 25 fissions after the formationof a new macronucleus, presumably containing jan+, at conjugation. Yet whenciliary meridian numbers were examined the result was different. In ten of theeleven pairs of clones, the clonal descendents of one exconjugant initially mani-fested the high ciliary meridian numbers characteristic of clone Y, while thedescendents of the other exconjugant exhibited the lower ciliary meridian num-bers characteristic of the wild-type B-1975 parent. These results are presentedin Table 5, arbitrarily arranged so that the exconjugant clone with the lowermeridian number is labelled 'a ' , whereas its sister clone with the higher numberis indicated as ' b ' . This general result is very similar to the transmission of pre-existing ciliary meridian number through conjugation demonstrated earlier byNanney (19666). In the one exceptional pair (no. 11), one clone (11 b) manifestedextremely high ciliary meridian numbers, but these were associated with thepresence within the clone of typical (wild-type) homopolar doublets with thegeometry described by Nanney, Chow & Wozencraft (1975), suggesting that thewild-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 theprevious section and summarized in Table 4. Whereas in the first case wild-typecells of normal ciliary meridian number that became homozygous for janrapidly expressed the new abnormal symmetry features while tending to retainthe pre-existing ciliary meridian numbers, in the second case janus cells withunusually high ciliary meridian numbers that acquired the jan+ allele soon re-verted to the normal symmetry pattern while retaining the pre-existing highciliary meridian number for some time.1 Ciliary meridian numbers and large-scale symmetry patterns are thus dissociable in both directions.

To provide a final test for this conclusion, in one set of test-crosses of thecategory Fl(a)x[janus progeny of Fl(a)xA*] (i.e. jan/jan+xjan/jan), bothexconjugants were separately isolated. In 13 out of 19 synclones both exconju-gants produced thriving clones. Within each synclone both exconjugant clonesare expected, to be genically identical, with one-half of the synclones heterozy-gous (jan/jan+) and the other half homozygous janus {jan/jan). However, withineach synclone the symmetry pattern of the pre-existing cell surface of one partnerhad 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 ofgenes controlling the wild-type cortical pattern was continued in the hithertonormal partner and newly initiated in the hitherto janus partner. Conversely,in those synclones that became jan/jan, expression of genes specifying the wild-type cell surface pattern was discontinued in the hitherto normal partner andnot initiated in the hitherto janus partner. Therefore, if there were any long-runtendency 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 backto the 'stability center' characteristic of the wild type. This will be described more extensivelyin a subsequent communication.


Table 6. Persistence of differences in expression of secondary OAs and ciliarymeridian numbers in serially propagated janus clones

Set Clone

A|| 310111419

B1f 512

40 fissions

y/o2OAf

1111147

34

25

t7

23

Merid.no.J s.D.

22-1 ±2-221-l±0-419-4±0-920-5 ±1-5201 ±10

fissions

18-5±l-023-5 + 0-8

250

y/o2 O A

1819141250

225

r1

14

fissions

Merid.no. s.D.

20-4 ±0-721-3 + 1-520-2 ±1020-2 ±2-421-7±2-4

fissions

18-6±l-424-4+1-3

/ o

525 fissions

Merid.2 OA no. s.D.

4349

26

116

20-7 ±0-419-9 ±0-919-3±0-420-0 ±1-421-8±l-9

500 fissions

19-6±0-723-0+1-4

650 fissions,

y/o2 O A

< 128

2256

- A ^

2OA(Fe2+)§

1317214483

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, differencesshould have been observed between the two exconjugant clones derived from asingle conjugating pair. No meaningful differences were detected in clones fixedabout 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 ofsecondary OAs than did any of the other janus clones. To find out whether thisdifference in expression was stable, five of the 11 janus clones as well as threeof the seven wild-type clones within this progeny set were maintained in con-tinuous growth in 1 % PPY at 25 °C by tri-weekly serial loop transfers of tubecultures. At intervals, subcultures were transferred into culture flasks containing1 % PPY, grown overnight at 28 °C, and fixed in early log phase at culture den-sities of close to 5000 cells/ml. The final fixation (at 650 fissions) was precededby subdivision of each janus clone into a portion grown as before in 1 % PPYand a portion cultivated for 2 weeks in the enriched Fe2+-EDTA medium,followed by growth in PPY and enriched-medium flask cultures respectively.


Silver-impregnated slides of the fixed samples were scored for expression ofsecondary OAs, double CVP sets, and ciliary meridian numbers. All five of thejanus clones continued to express the janus phenotype for the duration of theexperiment, whereas no cell in the three clones originally scored as wild-typeexhibited any of the diagnostic features of the janus phenotype. Among the fivejanus clones, the expression of double CVP sets remained fairly constant andhomogeneous. In contrast, as shown in Table 6 (set A), the expression ofsecondary OAs fluctuated, yet clone 19, which had the highest proportion ofsecondary OAs initially, consistently exhibited a frequency of secondary OAsabout triple that of any of the other clones. The Fe2+-EDTA medium enhancesexpression 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 andthe others persists; indeed, clone 19 was brought up to essentially completeexpression of secondary OAs (literal 100 % expression is probably impossible,due to the tendency of many secondary OAs to undergo resorption). This near-complete expression in Fe2+-EDTA medium was repeatedly observed forclone 19 but not for any of the others. Thus it is hard to avoid the conclusionthat clone 19 has an intrinsically higher capacity to express secondary oralstructures than do the other janus clones.

Somewhat surprisingly, the average ciliary meridian number of the janusclones of this set generally remained within the wild-type range of 18-21 ciliarymeridians (Table 6, set A); clone 19 became slightly higher than the others butstill did not attain the mean of 22-24 ciliary meridians characteristic of theoriginal CU-127 clone. However, cells with unusually high ciliary meridiannumbers (up to 31) appeared with greater or lesser frequency in janus cloneseven under the conditions of near-continuous growth that prevailed in thisexperiment. The proportion of such cells differed in different clones (highest inclone 19, also high in clone 14, and low in the others except for the first sampleof no. 3), suggesting possible differences among janus clones in the frequencywith which such high ciliary meridian numbers are generated and/or the stabi-lity with which they are propagated. To check on these possibilities, a subsequentprogeny set was searched for those janus clones initially manifesting the highestand the lowest prevailing ciliary meridian numbers. The two janus clones withextreme meridian numbers were then serially propagated and periodicallyexamined. The results, shown in Table 6 (set B), clearly indicate that the differ-ences are propagated. Ciliary meridian numbers in clone 5 slowly drifted up toa level similar to that in the majority of clones in set A, while expression, ofsecondary OAs remained low. Clone 12, on the other hand, maintained anaverage number of ciliary meridians and also of secondary OAs similar to thatfound 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 sub-clonal expansion was undertaken of clone 19, which manifested great internal

Genie control of symmetry in Tetrahymena 221variability of ciliary meridian numbers. The subclones differed substantially inciliary meridian number and also in expression of secondary OAs when firstfixed at 15 fissions after their establishment, but became homogeneous in ex-pression of secondary OAs and nearly so in ciliary meridian number by 100fissions (data not shown). These findings are consistent with the conclusion thatthe long-term differences observed between clones are inherent, and probablybased on genie differences.

Comparisons both between and within clones and subclones indicate apositive association between number of ciliary meridians and frequency of ex-pression of secondary OAs. However, there are at least two reasons for notconcluding that a high ciliary meridian number is a prerequisite for expressionof a secondary OA. First, cells with as few as 18 ciliary meridians that possessedsecondary OAs were commonly observed in clone 5 (set B, Table 6); the factthat no cells with fewer than 18 ciliary meridians have been found bearingsecondary OAs is probably simply due to the rarity of cells with 17 or fewerciliary meridians under our culture conditions. Hence there is no evidence for aminimum number of ciliary meridians required for expression of secondary OAs.Second, in clone 19 (set A, Table 6) a high frequency of secondary OAs becameestablished before the average number of ciliary meridians rose to a level some-what higher than that of the other janus clones in the set; further, the great in-crease in expression of secondary OAs brought about by the Fe2+-EDTAmedium was not accompanied by an increase in number of ciliary meridians(data not shown). Thus, if there is any causal relationship between number ofciliary meridians and expression of secondary OAs in janus cells, it is complexand indirect.

DISCUSSION

(A) Origin of janus

The preceding paper (Jerka-Dziadosz & Frankel, 1979) has demonstratedthat the CU-127 clone of T. thermophila possesses a unique phenotype stronglysuggestive of a geometrically reversed morphogenetic field that is reliably propa-gated within the clone. In this communication we have shown that a recessiveallele at a single gene locus (janus) determines the propagation and/or expressionof this reversed field. We do not know when this allele originated. The phenotypethat it controls first came to our notice in a clone that had been subjected tonitrosoguanidine mutagenesis followed by short-circuit genomic exclusion(Bruns et al. 1976). However, the selective procedure by which the clone waspicked out by Bruns & Sanford (1978) involved lethality at high temperature,and we have shown that this high-temperature lethality is separable from thecortical reversal (cf. Results, section 1 b). Hence it is likely that the jan mutationwas induced by the mutagen in the same micronucleus together with another,independent, temperature-sensitive mutation, and the two were both transmittedto the same progeny clone following short-circuit genomic exclusion. Selection

15 EMB 49


for the latter mutation then fortuitously brought about recovery of the former.Deliberate selection ofjanus would require a method for generating homozy-gosity following mutagenesis, such as the short-circuit genomic exclusionmethod of Bruns et ah (1976), coupled with a method for easily recognizingliving cells exhibiting the phenotype. As the somewhat flattened shape of knownjanus homozygotes tends to be accentuated in the Fe2+-EDTA medium, thephenotype might possibly be selectable in this medium. Only after such a selec-tion procedure is put into operation will it be possible to estimate the number ofloci 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 doubletsin possessing two oral apparatuses and two sets of CVPs, the geometrical arrange-ment of these structures is very different in janus and wild-type doublets. In thispaper, an equally fundamental difference in both the origin and time-course ofexpression of these two conditions is demonstrated. Wild-type homopolardoublets originate by lateral fusion of two cell-units. They, therefore, initiallymanifest a high number of ciliary meridians, which then declines. Concomitantlywith this decline, cells revert from the doublet back to the singlet state (Faure-Fremiet, 1948; Nanney et al. 1975). Intermediate states of expression of double-ness of oral structures and CVPs are uniquely associated with intermediatenumbers (22-27) of ciliary meridians (Nanney, 1966 a), which represent atransitional condition during the reversion from the doublet to the singletphenotype. The origin and dynamics of expression of the atypical doublet stateof janus clones differs from that in wild-type homopolar doublets in all of theabove respects. The janus doublet state becomes manifest only after cells con-taining a wild-type (jan+) allele become homozygous jan/jan, and disappearsonly after cells with jan/jan macronuclei acquire a jan+ allele. The change ofexpression in both directions is rapid, being completed by 25 fissions after con-jugation. Further, neither the appearance nor the loss of capacity to generatesecondary OAs or double CVP sets following the above-mentioned changes ingenotype need be associated with any change in number of ciliary meridians.Although there is some positive association between degree of expression ofabnormal secondary OAs and number of ciliary meridians, the association isfar less pronounced than that found in wild-type homopolar doublets: thesecondary oral structures can be expressed in cells with as few as 18 ciliarymeridians 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 anddouble CVP sets in janus cells are thus fundamentally different from thoseoperating in wild-type doublets. The difference is a result of a profound differ-ence in the basis of the two conditions, gene expression in the former and non-genic inheritance of large-scale cytoplasmic patterns in the latter.


(C) Subsidiary effects ofjanus

The janus allele has other effects in addition to maintaining the unique geo-metrical pattern of secondary OAs and double CVP sets. First, while not itselfinfluencing the modal number of ciliary meridians, thejanus allele when homo-zygous brings about a certain degree of instability in the propagation of ciliarymeridians, leading to the appearance of some cells with unusually high numbersof ciliary meridians. This instability appears to open a 'phenotypic window' forthe expression of modifying genes that can influence the modal number of ciliarymeridians. 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 progenyclone, these genes appear to stabilize the high ciliary meridian numbers thatappear sporadically in all janus clones. A second, more perplexing subsidiaryeffect of the janus allele relates to the capacity of exconjugants to generate viableclones. No viable progeny are produced when janus homozygotes are crossedwith each other, despite the demonstrated capacity of such homozygotes toundergo normal meiosis and fertilization. The crucial phenotypic defect in suchcrosses is not in conjugation itself, but in some step(s) in the development ofexconjugants. 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 genotypecan generate viable clones if either these cells or their mating partners had beenphenotypically wild-type prior to conjugation [the partner is effective in pro-moting a 'maternal effect' because macromolecules circulate freely betweenconjugating cells (McDonald, 1966)]. Curiously, a similar drastic yet maternallyrescuable effect on survival of exconjugants was also observed for one of thevery few other known ciliate genes that has a non-conditional effect on globalcell-surface pattern, 'basal body deficient' (bbd) in Euplotes minuta (Frankel,1973). The connexion between the respective gene products and the capacity toundergo post-conjugational development is obscure in both cases, although ahint may be provided by the fact that both bbd and jan bring about a corticalinstability that tends to be especially severe in young clones.

Additional modifying genes probably affect not only the stable number ofciliary meridians in janus cells, but also the extent of expression of secondaryOAs. The possible existence of one major gene enhancing expression of secon-dary OAs is currently under investigation.

(D) Mechanisms of pattern reversal

The actual mechanistic basis of the genically controlled expression of thereversed secondary morphogenetic field is unknown. This is the first examplein which a pattern reversal in ciliates is known to be the result of an action of anallele at a nuclear gene locus. Most earlier examples of mechanically inducedand non-genically propagated pattern reversals, such as the famous ciliary

15-2


meridian inversion in Paramecium (Beisson & Sonneborn, 1965) and its equiva-lent in Tetrahymena (Ng & Frankel, 1977), involve the ciliary meridians andadjacent organelles, and manifest no phenotypic effects of the kind seen in janusclones. However, there are a few examples of large scale reversals of asymmetrythat are more comparable to those brought about by the janus allele. Some ofthese are reviewed by Tartar (1967, pp. 90-92). In these cases, encountered inlarge ciliates such as Stentor, Blepharisma, and Condylostoma, microsurgicallyinduced large-scale pattern reversals have been propagated for a short durationonly. Tchang & Pang (1965), however, have described a microsurgical operationthat can bring about mirror-image doublet cells of the hypotrich ciliate Stylon-chia mytilus which propagate one normal and one reversed set of oral structuresfor hundreds of fissions. This finding suggests that although in the case of januscells the propagation and expression of a morphogenetic field of reversedasymmetry is dependent on the continuous action of a mutant allele, such depen-dence need not always be the case. An extreme possibility might be that thejanallele, rather than 'creating' a morphogenetic field of reversed asymmetry,brings a precisely positioned pre-existing field above a threshold of phenotypicmanifestation. However, as there is evidence that the janus condition can beexpressed 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 gene-controJled expression of pattern reversal in a unicellular organism, formallycomparable 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 specifica-tion of mirror-image double abdomens by the bicaudal mutant in Drosophila(Bull, 1966; Nusslein-Volhard, 1977), the genes act by maternal predetermina-tion of the egg cytoplasm. In the others, including visceral inversion in mice(Hummel & Chapman, 1959) and genically controlled mirror-image reduplica-tions such as those of the wing in duplicate fowl (Landauer, 1956) and of thedorsal thorax in wingless Drosophila (Sharma & Chopra, 1976), genes act afterzygote formation, presumably to influence the organization of organ anlage. Thecase best analyzed developmentally is the wingless allele in Drosophila, whichis known to act at an embryonic stage on the wing imaginal disc, probably tobring about an abnormal proximal-distal compartmentalization such that theportion of the disc that normally produces wing becomes mirror-image thoraxinstead (Babu, 1977; Morata & Lawrence, 1977). The janus phenotype ofTetrahymena could be viewed as a conversion of a 'dorsal' (aboral) cell surfaceto a mirror-image 'ventral' (oral) surface, in which case the janus phenotypecould be considered as analogous to the wingless condition in Drosophila. Thisformal resemblance leaves open the possibility that there might be some simi-larity in the underlying mechanisms of control of large scale asymmetry.

Genie control of symmetry in Tetrahymena 225The 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 apprecia-tion 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 sup-ported by grant no. HD-08485 from the U.S. National Institutes of Health.

REFERENCESALLEN, S. L. (1967). Genomic exclusion: a rapid means for inducing homozygous diploid

lines in Tetrahymenapyriformis, syngen 1. Science, N.Y. 155, 575-577.ALLEN, S. L. & LEE, P. H. T. (1971). The preparation of congenic strains of Tetrahymena.

J. Protozool. 18, 214-218.ARES, M. & BRUNS, P. J. (1978). Isolation and genetic characterization of a mutation affecting

ribosomal resistance to cycloheximide in Tetrahymena. Genetics 90, 463-474.AUFDERHEIDE, K. (1978). Specific associations of mitochondria with the cortex of Tetrahymena

thermophila. J. Protozool. 25 (suppl.), 7A.BABU, P. (1977). Early developmental subdivisions of the wing disc in Drosophila. Molec.

Gen. Genet. 151, 289-294.BEISSON, J. & SONNEBORN, T. M. (1965). Cytoplasmic inheritance of the organization of

the cell cortex in Paramecium aurelia. Proc. Natn. Acad. Sci. U.S.A. 53, 275-282.BLEYMAN, L. K. & BRUNS, P. J. (1977). Genetics of cycloheximide resistance in Tetrahymena.

Genetics 87, 275-284.BOYCOTT, A. E., DIVER, C, GARSTANG, S. L. & TURNER, F. M. (1930). The inheritance of

sinistrality in Limnaea peregra (Mollusca, Pulmonata). Phil. Trans. R. Soc. B 219,51-131.

BRUNS, P. J. & BRUSSARD, T. B. (1974). Positive selection for mating with functionalheterokaryons in Tetrahymena pyriformis. Genetics 78, 831-841.

BRUNS, P. J., BRUSSARD, T. B. & KAVKA, A. B. (1976). Isolation of homozygous mutantsafter induced self-fertilization in Tetrahymena. Proc. natn. Acad. Sci. U.S.A. 73,3243-3247.

BRUNS, P. J. & SANFORD, Y. M. (1978). Mass isolation and fertility testing of temperaturesensitive mutants in Tetrahymena. Proc. natn. Acad. Sci. U.S.A. 75, 3355-3358.

BULL, A. L. (1966). Bicaudal, a genetic factor which affects the polarity of the embryo ofDrosophila melanogaster. J. exp. Zool. 161, 221-242.

BYRNE, B. C, BRUSSARD, T. B. & BRUNS, P. J. (1978). Induced resistance to 6-methylpurineand cycloheximide in Tetrahymena. I. Germ line mutants of T. thermophila. Genetics89, 695-702.

CONNER, R. L. & CLINE, S. G. (1964). Iron deficiency and metabolism of Tetrahymena pyri-formis. J. Protczool. 11, 486-491.

FAURE-FREMIET, E. (1948). Doublets homopolaires et regulation morphogenetique chez leCilie Leuccphrys patula. Archs Anat. microsc. Morph. exp. 37, 183-203.

FRANKEL, J. (1973). A genically determined abnormality in the number and arrangement ofbasal bodies in a ciliate. Devi Biol. 30, 336-365.

FRANKEL, J., JENKINS, L. M., NELSEN, E. M. & DOERDER, F. P. (1976). Mutations affectingcell division in Tetrahymena pyriformis. I. Selection and genetic analysis. Genetics 83, 489-506.

HUMMEL, K. P. & CHAPMAN, D. Y. (1959). Visceral inversion and associated anomalies inthe mouse. / . Hered. 50, 9-13.

JERKA-DZIADOSZ, M. & FRANKEL, J. (1979). A mutant of Tetrahymena thermophila with apartial mirror image duplication of cell surface pattern. I. Analysis of phenotype. / . Em-bryol. exp. Morph. 49, 167-202.

KACZANOWSKI, A. (1975). A single gene dependent abnormality of adoral membranelles insyngen 1 of Tetrahymena pyriformis. Genetics 81, 631-639.


LANDAUER, W. (1956). Rudimentation and duplication of the radius and ulna in the duplicatemutant form of the fowl. / . Genet. 54, 199-218.

MCCOY, J. W. (1977). Linkage and genetic map length in Tetrahymena thennophila. Genetics87, 421-439.

MCDONALD, B. B. (1966). The exchange of RNA and protein during conjugation inTetrahymena. J. Prctozool. 13, 277-285.

MORATA, G. & LAWRENCE, P. A. (1977). The development of wingless, a homoeotic mutantof Drosophila. Devi Biol. 56, 227-240.

NANNEY, D. L. (1953). Nucleo-cytoplasmic interaction during conjugation in Tetrahymena.Biol. Bull. mar. biol. Lab., Woods Hole 105, 133-148.

NANNEY, D. L. (1963). Irregular genetic transmission in Tetrahymena crosses. Genetics48, 737-744.

NANNEY, D. L. (1964). Macronuclear differentiation and subnuclear assortment in ciliates.In The Role of Chromosomes in Development, (ed. M. M. Locke), pp. 253-273. New York:Academic Press.

NANNEY, D. L. (1966a). Corticotypes in Tetrahymena pyriformis. Amer. Nat. 100, SOS-SIS.

NANNEY, D. L. (19666). Corticotype transmission in Tetrahymena. Genetics, 54, 955-968.NANNEY, D. L., CHOW, M. & WOZENCRAFT, B. (1975). Considerations of symmetry in the

cortical integration of Tetrahymena doublets. / . exp. Zool. 193, 1-14.NANNEY, D. L. & MCCOY, J. W. (1976). Characterization of the species of the Tetrahymena

pyriformis complex. Trans. Am. microsc. Soc. 95, 664-682.NELSEN, E. M. & DEBAULT, L. E. (1978). Transformation in Tetrahymena pyriformis - des-

cription of an inducible phenotype. / . Protozool. 25, 113-119.NG, S. F. (1977). Analysis of contractile vacuole pore morphogenesis in Tetrahymena

pyriformis by 180°-rotation of ciliary meridians. / . Cell Sci. 25, 233-246.NG, S. F. (1978). Origin and inheritance of an extra band of longitudinal microtubules in

Tetrahymena cortex. Protistologia (In the Press.)NG, S. F. & FRANKEL, J. (1977). 180°-rotation of ciliary rows and its morphogenetical impli-

cations in Tetrahymena pyriformis. Proc. natn. Acad. Sci. U.S.A. 74, 1115-1119.NG, S. F. & WILLIAMS, R. J. (1977). An ultrastructural investigation of 180°-rotated ciliary

meridians in Tetrahymena pyrformis. J. Protozool. 24, 257-263.NUSSLEIN-VOLHARD, C. (1977). Genetic analysis of pattern formation in the embryo of

Drosophila melanogaster. Characterization of the maternal effect mutation bicaudal.Wilhelm Roux's Arch, devl Biol. 183, 249-268.

ORIAS, E. & HAMILTON, E. (1977). Induction of self-fertilization in conjugating Tetrahymenacells. Abstr. 5th Intl. Congr. Protozool., New York, p. 93.

PREPARATA, R. M. & NANNEY, D. L. (1977). Cytogenetics of triplet conjugation in Tetrahy-mena: origin of haploid and triploid clones. Chromosoma 60, 49-57.

ROBERTS, C. T. & ORIAS, E. (1973). A cycloheximide-resistant mutant of Tetrahymena pyri-formis. Expl. Cell Res. 81, 312-316.

SHARMA, R. P. & CHOPRA, V. L. (1976). Effect of wingless (wg) mutation on wing and halteredevelopment in Drosophila melanogaster. Devi Biol 58, 461-465.

SONNEBORN, T. M. (1963). Does preformed cell structure play an essential role in cell heredity?In The Nature of Biological Diversity (ed. J. M. Allen), 165-221. New York: McGraw-Hill.

SONNEBORN, T. M. (1975). Tetrahymena pyriformis. In Handbook of Genetics (ed. R. C.King), pp. 433-467. New York: Plenum Press.

STURTEVANT, A. H. (1923). Inheritance of the direction of coiling in Limnaea. Science, N.Y.58, 269-270.

TARTAR, V. (1956). Further experiments correlating primordium sites with cytoplasmicpattern in Stentor coeruleus. J. exp. Zool. 132, 269-298.

TARTAR, V. (1960). Reconstitution of minced Stentor coeruleus. J. exp. Zool. 144, 187-207.TARTAR, V. (1967). Morphogenesis in Protozoa. In Research in Protozoology (ed. T. T.

Chen), pp. 1-115. New York: Pergamon Press.

Genie control of symmetry in Tetrahymena 227TCHANG TSO-RUN, N. & PANG YAN-BIN (1965). Conditions for the artificial induction of

monster jumelles of Stylonychia mytilus which are capable of reproduction. Scientia Sinica14, 1332-1338.

THOMPSON, G. A. (1967). Studies on membrane formation in Tetrahymena pyriformis.I. Rates of phospholipid biosynthesis. Biochemistry 6, 2015-2022.

WEINDRUCH, R. H. & DOERDER, F. P. (1975). Age-dependent micronuclear deterioration inTetrahymena pyriformis, syngen 1. Mech. Ageing & Devel. 4, 263-279.

(Received 18 July 1978)

a mutant of tetrahymena thermophila with a partial mirror-image

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