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Copyright 0 1983 by the Genetics Society of America

COMPLEMENTATION AND PRELIMINARY LINKAGE ANALYSIS OF ZYGOTE MATURATION MUTANTS OF THE HOMOTHALLIC ALGA, CHLAMYDOMONAS MONOlCA KAREN P. VANWINKLE-SWIFTA N D CYNTHIA G. BURRASCANO Deportment of Biology, San Diego State University, Son Diego, Colifornio 92282 Manuscript received September 27, 1982 Revised copy accepted November 17,1982 ABSTRACT

Sexual reproduction in Chlamydomonas monoica is homothallic: pair formation and cell fusion occur in clonal culture and give rise to a heavily walled diploid zygospore. During maturation of the young zygote, a distinctive “primary zygote wall” is released before the development of the highly reticulate zygospore wall. Using ethyl methanesulfonate and ultraviolet irradiation as mutagens, we have isolated 19 maturation-defective (zym) mutant strains which upon self-mating produce inviable zygotes. These zygotes fail to release a primary zygote wall, fail to develop the normal zygospore wall, and eventually undergo spontaneous lysis. In nearly all cases, the mutations appear to be expressed only in the diploid zygote; pleiotropic effects on vegetative cell testing performed growth or morphology are not evident.-Complementation on 17 of these mutants indicates that all are recessive and that they define seven distinct complementation groups. Preliminary tetrad analysis of two-factor and multifactor zym crosses provides no evidence for physical clustering of the maturation genes, and instead suggests that they are widely distributed throughout the nuclear genome.

exual reproduction in Chlamydomonas provides a potentially useful experS imental system for the study of regulated gene expression during cellular development. The sexual cycle of this unicellular eucaryotic alga comprises a precise sequence of cellular events that must involve interactions between regulatory genes and numerous structural genes expressed only in sexual cells (i.e., gametes or zygotes). Progression of the sexual cycle involves the differentiation of vegetative haploid cells into sexually active gametes, the development of a diploid zygote through fusion of compatible gamete pairs, the maturation of the young zygote into an elaborately walled zygospore, entrance into and maintenance of the dormant state, and, finally, breaking of dormancy and the release of haploid asexual zoospores through meiotic germination of the zygospore in response to specific environmental conditions (LEVINEand EBERSOLD 1960).

Our present understanding of sexual differentiation in Chlamydomonas is based primarily upon ultrastructural, biochemical, and genetic studies on the heterothallic species, C . reinhardtii. In this species, the differentiation of a vegetative cell into a sexually active form (gametogenesis) occurs in response to Genetics 103 429-445 March, 1983

430

K. P.

VANWINKLE-SWIFT

AND

C. G.

BURRASCANO

nitrogen starvation (SAGER and GRANICK 1954), and involves extensive intracellular changes (JONES, KATESand KELLER 1968; SIERSMA and CHIANG 1971; MARTIN and GOODENOUGH 1975; WEEKSand COLLIS1979). Differentiated gametes of opposite mating-type differ from one another in terms of surface flagellar 1976; RAY,SOLTER and GIBOR1978), and proteins (BERGMANet al. 1975; SNELL specialized mating structures (FRIEDMANN, COLWIN and COLWIN 1968; CAVALIERSMITH1975; TRIEMER and BROWN1975; GOODENOUGH and WEISS1975). These flagellar differences promote agglutination between flagella of opposite mating types, bringing the partners into close contact. Flagellar agglutination, in turn, triggers the release of an autolytic enzyme facilitating shedding of the gametic TAMAKI and TSUBO1978) cell wall (GOODENOUGH and WEISS1975; MATSUDA, and activates the mating structures, which upon contact produce a cytoplasmic connection between the two cells promoting cell fusion (CAVALIER-SMITH 1975; WEISS,GOODENOUGH and GOODENOUGH 1977). Genetic studies on sexual reproduction in C. reinhardtii have involved analysis of mutants defective in these early stages. Thus, mutants showing condi1975), as well as mutants with tional gametogenesis (FORESTand TOGASAKI specific flagellar defects preventing agglutination between opposite mating HWANGand WARREN types (GOODENOUGH and JURIVICH 1978; GOODENOUGH, 1978) or interferring with normal cell fusion (GOODENOUGH and WEISS 1975; GOODENOUGH, HWANGand MARTIN1976) have been described. Mutations linked to the mating-type locus have been identified, whereas others are unlinked to and TOGASAKI 1975; mating type but show sex-limited expression (FOREST GOODENOUGH, HWANGand MARTIN1976; GOODENOUGH, HWANGand WARREN 1978; HWANG,MONKand GOODENOUGH 1981). Although genetic analyses of events unique to the diploid zygote have not been reported for C. reinhardtii, this period in the sexual cycle is of obvious interest to developmental geneticists. During the period of zygote development (maturation), novel proteins appear, several of which are components of the protective zygospore wall (MINAMIand GOODENOUCH 1978). Nuclear fusion occurs early in zygote maturation, followed soon after by plastid fusion (CAVALIER-SMITH 1970, 1975; BLANK,GROBEand ARNOLD1978), and provides the opportunity for recombination between parental DNAs. Dedifferentiation of the zygotic plastid (CAVALIER-SMITH 1976) and selective methylation and degrada1979) also occur in the tion of chloroplast DNAs (BURTON, GRABOWY and SAGER maturing zygote. The highly resistant, albeit dormant, zygospore provides for species survival under harsh environmental conditions which would be lethal to vegetative cells (cf. LEWIN1951, and VANWINKLE-SWIFT 1977). Thus events occurring after gamete fusion have major consequences in terms of species survival and adaptability. These events appear to occur in a precise sequential pattern suggesting a carefully regulated mechanism(s) for gene expression, and most probably involve interacting signals initially received from the individual parental gametes. The apparent absence of zygote maturation mutants of C. reinhardtii undoubtedly reflects technical problems inherent in the use of an obligately heterothallic species. In such species, the diploid zygote results only from the

MATURATION MUTANTS OF C. MONOICA

431

union of cells of stable opposite mating types, i.e., from interclonal matings. Recessive mutations in genes expressed only in the diploid zygote cannot be readily identified since their effects will be masked by the corresponding wildtype alleles in the heterozygous zygotes. In contrast, recessive mutations causing defective zygote maturation should be easily detected in homothallic species since homozygous mutant zygotes will be produced by self-mating within the mutant clone. This point is well illustrated by the successful application of homothallic yeast strains to genetic studies on yeast sporulation (analogous to and EGEL zygote maturation in Chlamydomonas) and meiosis (BRESCH, MULLER 1968; ESPOSITO and ESPOSITO1974). For these reasons, we have begun studies on the homothallic alga Chlamydomonas monoica (VANWINKLE-SWIFT 1979, 1980; VANWINKLE-SWIFT and BAUER1982). We report here the isolation of mutant strains (zym) exhibiting abnormal zygote maturation. The responsible mutations are expressed only in the diploid zygotes produced by self-mating within the mutant clones. Complementation testing has identified seven “cistrons” involved in zygote maturation, and verifies that all of the maturation mutations analyzed are recessive lethals. Preliminary tetrad analysis has provided no evidence for physical clustering as a mechanism for coordinated expression of these maturation genes. A brief and VANWINKLE-SWIFT abstract of this work has been published (BURRASCANO 1982). MATERIALS AND METHODS Strains. The mutant strains of C. monoica utilized in this study were derived from the wild-type strain maintained by The Cambridge Culture Collection and provided to us by Dr. RALPHA. LEWIN. 1979) and ger-1 (germinaThe origins of the spr-fd-1 (spectinomycin-resistant; VANWINKLE-SWIFT tion-defective) mutant strains within which the maturation mutations (zym) were induced are depicted in Figure 1.The ger-1 strain was originally isolated as a high efficiency mating subclone of wild type and was only later found to be carrying the spontaneous mutation interfering with normal zygote germination. Culture conditions. Vegetative cultures of all strains were routinely grown on agar solidified Bold’s basal minimal medium (BM) (BISCHOFFand BOLD1963) and were maintained under continuous cool white fluorescent light (3000-5000 lx) at 20-25’. To induce mating, vegetative cells were transferred to a low phosphate-low nitrate liquid medium (LPN) as described previously (VANWINKLE-SWIFT and BAUER1982) and were incubated at 19-21O under continuous illumination (30005000 Ix) for 3-7 days. Aliquots of 7-day-old LPN cultures containing mature zygospores were plated on BM agar and were incubated in the dark for 1-3 days at ambient room temperature. At the end of the dark incubation period, unmated cells were killed by inverting the plate over a dish of chloroform for 15 sec, and the zygospores were induced to germinate by returning the plates to continuous illumination. Mutagenesis. The procedure for ethylmethanesulfonate (EMS) mutagenesis of vegetative cells of the spr-fd-1 strain has been described previously (VANWINKLE-SWIFTand BAUER1982). For mutagenesis by ultravioltet irradiation (UV), vegetative cells of the ger-1 strain were removed from BM agar plates and were resuspended in LPN mating-induction liquid medium (CO. IO6 cells/ml). The culture was maintained under continuous illumination (3000 lx) for 18 h r after which 10-ml aliquots were removed and placed in small sterile Petri dishes. The aliquots were stirred continuously and were irradiated using a General Electric 30W germicidal UV lamp held at 10 cm from the surface of the cultures. After irradiation for 0,30, 60 or 90 sec, the cultures were placed in darkness for 6 h r to prevent photoreactivation of the UV-induced damage. At the end of the dark incubation period, several 0.03-ml aliquots from each UV dose were plated separately on BM agar at 0, 0.1 and 0.01

432

K. P. VANWINKLE-SWIFT A N D C. G. BURRASCANO

Cmonoicu

spont. __I

ger-l I

(Cambridge 11/70)

I

FdURD

1

uv

spr - fd / EMS

f6-2/)

spr-fd / zymo-5)

I

X

fspr-fd /'zym-3'ger-/+zym-6"/ FIGURE1.-Derivation of strains. Abbreviations: FdURD = 5-fluorodeoxyuridine; UV = ultraviolet irradiation; EMS = ethyl methanesulfonate; spont. = spontaneous mutation; spr-fd-l = high level spectinomycin resistant mutant (VANWINKLE-SWIFT 1980); ger-1 = germination-defective mutant; zym = zygote maturation-defective mutants; wtl5c = wild-type recombinant tetrad product from the cross: spr-fd-l zym-3 x ger-1 zym-6. dilutions. The plated cells were allowed to grow for 7-10 days under standard conditions. Dilution plates were used to assay plating efficiency and percent survival in the same way as previously and BAUER1982). Cells derived from the 30-sec described for EMS mutagenesis (VANWINKLE-SWIFT UV dose (yielding 28% survival) were chosen for the mutant search. The cells obtained after postmutagenesis growth of the undiluted aliquots were removed from the BM plates and suspended in 10 ml BM liquid in standard test tubes. The tubes were illuminated from above for 30 sec after which the top few milliliters, containing motile phototactic cells, were removed and streaked onto several BM agar plates to permit isolation of individual post-mutagenesis progeny clones. From 200 to 400 clones derived from each of four aliquots of the original mutagenized culture were picked and transferred to BM agar for 5 days further growth before screening for mutants. Identification of zym mutants. The methods for screening for zygote maturation mutants (zym) have been described previously (VANWINKLE-SWIFT and BAUER 1982).Briefly, each post-mutagenesis progeny clone was suspended separately in a 0.3-nil aliquot of LPN mating-induction liquid medium, incubated under continuous illumination at 21' for 3-4 days, and then sampled for inspection by phase contrast microscopy. For a wild-type strain, a 3- to +day incubation in LPN liquid medium is sufficient to allow for gametic differentiation, pair formation, cell fusion, release of a primary zygote wall, and nearly complete development of the highly reticulate zygospore wall. The LPN cultures of post-mutagenesis progeny clones were assayed for the presence of the discarded primary

MATURATION MUTANTS OF C. MONOICA

433

zygote wall and for zygote morphology. Additional details of the screening procedure can be found in the text or in VANWINKLE-SWIFT and BAUER 1982. Maturation mutants (zym) were assigned sequential identification numbers indicating their order of isolation. Because of possible complications in the interpretation of complementation data (see DISCUSSION), renaming of the mutants to indicate locus position is not yet warranted. Complementotion analysis. Visual inspection: The maturation-defective mutant clones were suspended in LPN medium in all possible pairwise combinations and were allowed to undergo gametogenesis, mating and zygote maturation (4days). Clonal cultures of each parental strain were set up at the same time to verify that each strain had become sexually active. Aliquots were removed from each pairwise culture and inspected by phase contrast microscopy to determine whether normal, fully matured zygospores were produced (by crossing) in addition to the maturation-defective zygotes resulting from continued self-mating within each strain. The presence of normal fully matured zygospores and discarded primary zygote walls in a mixed culture was taken as an indication that the parent strains carried recessive complementary zym mutations. See text for further discussion. Chloroform selection: Each UV-induced zym mutant was incubated in LPN induction medium with the EMS-induced zym-1 strain. After 7 days the culture was plated on BM agar and placed in darkness for 1-3 days. The plates were then removed from the dark, inverted over a dish of chloroform for 10-20 sec and then placed under continuous illumination for 7-10 days. Growth after chloroform treatment indicated the presence of normal fully matured zygospores in mixed cultures and thus served to identify UV-induced zym mutants complementary to zym-1. Application of the chloroform selection method for complementation analysis to pairwise cultures of UV-induced mutants could not be undertaken until the ger-l marker had been removed from each of the UV-induced zym strains via tetrad analysis of EMS-zym X UV-zym crosses (see below). Tetrad analysis. The procedures for maturation of zygotes, induction of zygote germination and dissection of tetrads have been described previously (VANWINKLE-SWIFT and BAUER1982). Crosses between the EMS-induced zym-1 strain (carrying the spr-fd-l marker) and each complementary UV-induced zym strain were performed and, whenever possible, 20-30 complete tetrads were isolated. Each tetrad product was scored for spectinomycin resistance by transfer to minimal medium supplemented with 40 pg/ml spectinomycin, and for zym marker genotype by inspection of 4-day-old LPN cultures containing each tetrad product singly, as well as mixed cultures containing the tetrad product and each of the parental zym strains (i.e., zym marker composition was determined by complementation testing of each tetrad product). Tetrads were classified as parental ditypes (PD), nonparental ditypes (NPD), or tetratypes (T) with respect to each possible marker pair and linkage was assayed by the PD:NPD ratio in each case. A small random sample of single UV-induced and double zym mutant tetrad products from each of these initial crosses was scored for the presence of the ger-l marker by crossing each product to a complementary UV-induced zym mutant carrying ger-1. The resultant zygotes were matured and induced to germinate following standard procedures. Successful germination, as evidenced by growth after chloroform selection, indicated that the tetrad product did not carry the ger-l marker. Germination-proficient tetrad products carrying each of the UV-induced zym markers alone and in combination with zym-l were saved for use in further constructions of multiply marked strains. The procedures for the analysis of multifactor zym crosses were identical to those described above for two-factor zym crosses. Centromere-linked markers were identified as those which in combination yielded a low frequency of tetratype tetrads. Gene-to-centromere distances (percent recombination or map units) for other markers were then estimated as one-half the tetratype frequency obtained for the new 1965; HAWTHORNE and marker and a centromere-linked marker included in the cross (cf. GOWANS 1960). Alternatively, for multifactor crosses in which three markers segregated indeMORTIMER pendently of one another, centromere distances to each of these markers were calculated from the various tetratype tetrad frequencies according to the formulae developed by WHITEHOUSE (1950; see also GOWANS 1965). RESULTS

ldentification of zygote maturation-defective (zym) mutant strains. Zygotes produced by self-mating within individual post-mutagenesis clones recovered

K.

434

P. VANWINKLE-SWIFT AND C. G. BURRASCANO

by EMS or UV mutagenesis were examined by phase contrast microscopy. In a standard mating, pair formation occurs within 36 hr after inoculation of nutrient-limited liquid medium (LPN) with vegetative cells. At the end of a 3day incubation, most zygotes are fully rounded and a distinctive “primary zygote wall” has been released into the culture medium. We have used the presence of this discarded primary zygote wall as an indicator of normal zygote maturation in clonal cultures. As summarized in Table 1, observations on 3700 individual clonal mating cultures revealed 19 maturation-defective mutants. The majority of these mutants exhibit the same general phenotype: failure to release a primary zygote wall, a tendency to produce zygotes that are not uniformly spherical, extensive bleaching of the zygote, and eventual disintegration of the immature zygote without development of the reticulate zygospore wall. In addition to this general phenotype, the zym-4 and zym-17 mutants show a reduction in self-mating efficiency, and the latter strain also exhibits a reduced vegetative growth rate. In contrast, the zym-16 and zym-18 mutants (which may in fact be duplicates of the same original mutation; see Table 1) are less stringent: apparently mature zygospores and discarded primary zygote walls appear at a low frequency in clonal LPN cultures of these strains, although maturation-defective zygotes are more common. Complementation analysis. Interstrain matings between mutant strains carrying complementary zym markers result in normal fully matured zygospores TABLE 3 Recovery of maturatioIi-defective (zym) mutants after induced mutagenesis Mutagen

50 mM EMS

% Survival

100

(90min)

100 mM EMS (60 min)

100 mM EMS (90 min)

30 sec UV

(6 hr dark)

37

25

28

Zvm mutants

Aliquot No. clones

A B C D E

137 120 155

A B C D E

230 248 120 120 120

A B C D E

368

120

170 zym-3 zym-2

zym-1

zym-4

178 247 70 146

zym-5

A B

300 240

C D

300 360

zym-10 zym-6, zym-7, zym-9, zym-11, zym-13, zym20, zym-21 zym-14, zym-15, zym-16, zym-17, zym-18 zym-8

MATURATION MUTANTS OF C. MONOICA

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(and release of the primary zygote wall). Pairwise mixed mutant cultures will thus contain normal zygotes (zygospores) as well as maturation-defective zygotes (resulting from continued intrastrain pairings within the mixed cultures) if the mutants carry recessive and complementary markers, but will contain only maturation-defective zygotes if either marker is dominant or if the two mutations are within the same cistron. Typical observations for a pair of complementary zym strains are illustrated in Figure 2. The results of observations on all pairwise combinations of 17 zym strains are summarized in Figure 3 and define seven cistrons affecting zygote maturation. As is typical of Chlamydomonas species, the normal mature zygospore of C. monoica is resistant to brief exposure to acetone or chloroform vapor, whereas unmated gametes and vegetative cells are not (LEWIN1951). Zym zygotes, if plated on standard medium before their spontaneous lysis in liquid LPN cultures, do not germinate although they remain intact for at least several days. Thus zym zygotes do not produce viable progeny whether or not chloroform

FrcuRe 2.-Phase contrast microscopy .. of in1 I- and inter-strain mated cultures of the complementary zym-6 and eym-13 maturation mutants. (a) clonal w115c culture: (b) clonal zym-6 mutant culture: (c) clonal zym-13 culture: (d)mixed culture containing both the zym-6 and zym-13 strains. Abbreviations: Z = normal fully matured zygospore: YZ = young immature zygote: W = discarded “primary zygote wall”; M = maturation-defective zygotes. Scale Bar = 10 pM.All observations are on cultures maintained in mating-induction medium (LPN) for 4 days. The smallcr size of the zym6 and zym-13 zygotes is a consequence of a pleiotropic effect of the ger-1 marker present in these strains (see Figure 1 ) and affecting both vegetative haploid and zygotic cell size.

436

K. P. VANWINKLE-SWIFT AND C. C. BURRASCANO

1 2 3 5 6 I 2 ...

0 9 10 I I 13 14 15 16 I8 2 0 2

I

? 4

-

3 ...... 5 ......... 6 ............ 7 ............... 8 ............... 9 ............... IO ............... 11 ............... 13 ............

L

14

I

............... 15 . . . . . . . . . . . . . . . 16 ............... 18 ............... 20 ............................................. 21

4

I

................................................

A

B

C

0

E

F

G

- - w - - - 1,2,3,5,10, 6.9 7 14,15,21

8

II, 13

16,18

20

RCURE3.-Complementation analysis of maturation-defective mutants. Numbers correspond to the I.D.numbers of the various zym mutants. Shaded squares indicate complementary pairs of mutant strains which produced normal zygospores in mixed cultures: open squares show mutant combinations for which marker complementation was not observed. Zym strains excluded from and BAUER1982) to be this analysis include the zym-4 strain, previously found (VANWINKLE-SWIIT noncomplementary to the other EMS-induced zym strains (zym’s 1-3, 5). but which shows such poor mating efficiency that accurate testing against the UV-induced mutants has not been possible, and the zym-17 mutant which shows similarly poor mating. Strains initially designated zym-I2 and zym-19 were discarded as unstable “phenocopies” which later showed normal zygote maturation.

selection has been employed. However, unmated zym haploids (vegetative cells or gametes) remain viable for prolonged periods in mating-induction medium, and chloroform exposure after plating is necessary to select heterozygous zygospores. Complementation analysis is readily accomplished by exposing plated aliquots from mixed mated cultures to chloroform and assaying for

MATURATION MUTANTS OF C. MONOICA

437

subsequent growth (indicating the presence of fully matured heterozygous zygospores carrying complementary zym markers). The application of this technique was initially hampered by the presence of the spontaneous ger-1 mutation (a recessive mutation that allows normal zygote maturation but blocks subsequent germination of the zygospore) in all of the AND METHODS, Fig. 1).Thus UV-induced zym mutant strains (see MATERIALS the chloroform selection assay for complementation could be applied initially only to mutant combinations including one EMS-induced zym strain (germination-proficient) and one UV-induced zym strain (germination-defective), as illustrated in Figure 4. Removal of the ger-2 marker through tetrad analysis of these two-factor zym crosses (see below) eventually allowed us to use the chloroform selection assay to verify all of the original complementation data (Fig. 3) obtained by visual inspection of mixed cultures.

FIGURE 4.-Chloroform selection assay for complementation analysis of maturation-defective strains. The wild-type vegetative cells and zygotes are derived from strain wtl5c (see Figure 1).The aliquot of vegetative cells was taken from a standard liquid culture (asynchronous): all other aliquots were taken from 7-day-old LPN cultures containing a high proportion of zygotes (normal or maturation-defective). See MATERIALS AND METHODS, and text, for further details. Growth within the central spot-derived from a mixed zym-l/zym-6 mating exposed to chloroform-indicates complementation between zym-1 and zym-6. The same result was obtained after the ger-1 marker had been removed from the zym-6 strain (not shown).

438

K . P. VANWINKLE-SWIFT AND C. G. BURRASCANO

Tetrad analyses. To determine linkage relationships between the UV-induced zym mutations, and to permit construction of multiply-marked mapping stocks, it was first necessary to remove the ger-2 mutation from the UV-induced mutants. This was accomplished by crossing each to the EMS-induced zym-2 strain (which also carried the additional Mendelian marker, spr-fd-2, conferring resistance to the antibiotic spectinomycin). The ger-2 marker was scored (see AND METHODS) in a small number of randomly selected zym tetrad MATERIALS products (parental zym products and double zym recombinants) to allow recovery of germination-proficient mapping stocks, but no attempt was made to determine the linkage relationships between ger-2 and the various zym loci. From these preliminary crosses, complete tetrads were analyzed for progeny genotypes with respect to the spr-fd-2, EMS-induced zym-2, and the various UV-induced zym markers (Table 2). Although evidence was obtained initially for linkage between zym-2 and zym-13 (Table 2 ) , analysis of subsequent multifactor crosses (Table 3) failed to support this conclusion. The earlier interpretation was also contradicted by the apparent absence of linkage between zym1 and zym-22 which maps to the same cistron as zym-13 by complementation analysis (Table 1,Figure 3). Although sample sizes are too small to rule out very loose linkage, the data from these preliminary crosses provide no evidence for tight linkage between zym-2 and any of the UV-induced zym markers, or between spr-fd-2 and any of the zym loci. However, analysis of multifactor crosses suggests that zym-6 and zym-8 are loosely linked since we have found a consistent excess of parental ditype tetrads relative to nonparental ditypes in all crosses involving these TABLE 2 Tetrad analysis of crosses of the general type: spr-fd-1 zym-1 X ger-1 UV-zym Marker pairs zym-VJV-zym UV-zym

P

NP

zym-6 zym-7 zym-8 zym-9 zym-11 zym-13 zym-16 zym-18 zym-20

7 3

8 5 5 6 10 8

4

8 13 18 3 2

8

11

5 15

T

7 11

13 7 5 9 8 4 12

spr-fd-l/zym-l

spr-fd-l/UV-zym

PNP

P

NP

T

P:NP

P

NP

T

P:NP

0.88 0.60 0.57 1.33 1.30 2.25* 0.27 0.40 0.53

5 5

13 7 3

4 7 13 4 4 5

0.38 0.71 2.67 0.42 1.00

6 6

1.58

12

15

8

2.25

7

8

9 9 5 7 4 9 10

0.86 1.00 1.38

5 13

7 6 8 9 12

5

0.20

4

2

5

2.00

12

0.92

7

13

10

0.54

71

80

68

0.89

8

5 13 19 9 1 11

12

13 13 4 5 12

11

0.56

1.08 0.80 0.88

Abbreviations: P = parental ditype tetrad; NP = non parental ditype; T = tetratype tetrad; UVzym = zym mutant induced by UV irradiation of the ger-1 strain (see Figure 1).* Indicates that the excess of parental ditype tetrads is statistically significant (p = 0.05). For a given cross, the numbers of tetrads reported for the various marker pairs are not always equal because of occasional ambiguities in scoring a particular marker.

439

MATURATION MUTANTS OF C. MONOICA

markers, and a tetratype:nonparental ditype ratio exceeding 4:l (Table 3; see discussion by GOWANS 1965). Tetrad analysis also revealed that three of our markers-spr-fd-1, zym-6 and zym-13-may be relatively close to their respective centromeres since in pairwise combinations these markers yield low tetratype frequencies (Tables 2, 3). By including these “centromere-linked markers” in crosses we can estimate other gene-to-centromere distances as one-half the tetratype frequency for the centromere-linked marker and the second marker under analysis. This assumes that no crossing-over occurs between the centromere and the gene being used as a centromere-marker (see GOWANS 1965 for discussion). Alternatively, the formulae derived by WHITEHOUSE (1950), which utilize tetratype tetrad frequencies from crosses involving three independently segregating markers, can be applied to estimate gene-to-centromere distances for several gene loci. The derivation of these formulae has been discussed in detail by GOWANS(1965). Table 4 compares the gene-to-centromere distances for several zym gene loci calculated by the various methods. These distances have been calculated and compared according to the data from the multifactor crosses of Table 3 in which the tetratype tetrad frequencies for the spr-fd-I/ zym-6 marker combination is somewhat higher than we typically observe. However, analysis of this marker combination in a total of 14 crosses yielding a pooled sample of 280 tetrads (unpublished data) gives a tetratype frequency of 0.08. Thus we feel application of methods assuming centromere linkage for the spr-fd-1 or zym-6 loci (methods A1 and AZ, Table 4) can provide reasonably accurate estimates of gene-to-centromere distances for other markers. Although there is relatively close agreement between the independent estimates for a given gene-to-centromere distance (Table 4), the differences in centromere TABLE 3 Pooled data from tetrad analysis of multifactor zym crosses ~~

P

Marker pair ~~

spr-fd-l/zym-l spr-fd-l/zym-6 spr-fd-l/zym-8 spr-fd-l/zym-13 zym-l/zym-6 zym-l/zym-8 zym-l/zym-13 zym-6/zym-8 zym-6/zym-13 zym-8/zym-13

NP

T

PNP

T:NP

%T

24 28 15 13

23

0.83 0.96 0.53

0.96

1.38

0.23 1.22 3.64 0.60

0.34 0.14 0.64 0.09 0.35 0.69 0.26 0.71 0.03 0.79

~

20

27 8 18 22 7 10 14 15 3

9 41 3

18 11

22

1.22

40

0.64

15 5 17 4

9

0.67 2.80* 0.88 0.75

46 1

26

0.32 2.73

9.20**

0.06 6.50

Crosses analyzed: spr-fd-1 zym-6 x zym-1 zym-8; spr-fd-1 zym-1 x zym-6 zym-8 zym-13; spr-fd1zym-6 zym-8 X zym-1 zym-7; spr-fd-l zym-1 zym-20 X zym-6 Zym-8 zym-13.

Abbreviations: P = parental ditype tetrad; NP = nonparental ditype; T = tetratype tetrad. * Statistically significant (p = 0.05) excess of parental ditype tetrads relative to nonparental ditypes suggests linkage. * * T:NP ratio statistically greater (P = 0.05) than 4:l provides additional support for hypothesis of loose linkage between zym-6 and zym-8. Because of smaller pooled sample sizes, for the zym-7 and zym-20 markers, data for these loci have been excluded from this analysis.

440

K . P.

VANWINKLE-SWIFT

A N D C. G. BURRASCANO

TABLE 4 Estimated centromere-to-gene distances (map units) Centromere (C)-to-gene interval

Method

C1-spr-fd-1

A. 1.

C2-zym-6

2.

0" 7.0

0"

4.5 1.5

3.

4.5

1.5

0"

3.3

4.2

B. 1. 2.

(i) (ii) (iii)

7.0

C3-zym-13

C4-zym-1

17.2 17.7 13.2

C2-zym-8

32.0 35.4 39.4

14.5 b b

1.3

35.8 35.4 31.9

Methods: A = use of "centromere-linked markers", including spr-fd-1 (A. I.), zym-6 (A. 2,), or zym-23 (A. 3.); B = application of the formulae derived by WHITEHOUSE (1950) using tetratype tetrad frequencies for crosses in which three markers (spr-fd-1, zym-1, and zym-6) are segregating independently (B. l.),or simplified by incorporating a previously determined (by Method B. 1.)geneto-centromere distance (see GOWANS1965) such as C4-zym-I (B. 2. i.), C2-zym-6 (B. 2. ii.), or C1spr-fd-l (B. 2. iii.). All estimates are based on data from Table 3. a Value is assumed by the method. * Negative values were obtained.

distances obtained for the several zym loci provide further evidence that the maturation genes are not tightly clustered, but rather reside at well-separated sites within the nuclear genome. A complete description of the linkage groups involved will require continued mutant searches and more extensive analyses of additional multifactor crosses (in progress). DISCUSSION

The successful isolation of maturation-defective mutants of C. monoica verifies our initial assumption that this self-mating species could provide a unique opportunity for the identification of recessive mutations in genes expressed only in the transient diploid stage (i.e., the zygote) of the Chlamydomonas life cycle. The identification of seven complementation groups within a sample of 17 mutant strains analyzed suggests that many genes are required for normal zygote development and that mutations in several genes can confer an identical maturation-defective (zym) phenotype. Preliminary genetic analyses of multifactor crosses reported here, as well as additional unpublished data, further suggest that these maturation genes are scattered throughout the nuclear genome, since no conclusive evidence for tight linkage between the zym markers has yet been found. Although no recombination has been detected between the complementary zym-13 and zym-20 markers (data not shown), we will present evidence elsewhere that the zym-20 mutant carries a major chromosomal aberration (translocation or inversion) because meiotic lethality is common in crosses involving this strain. Thus, the apparent linkage between zym-13 and zym-20 may be an artifact created by selection against recombinant meiotic products.

MATURATION MUTANTS OF C. MONOICA

441

Our two mutagenesis experiments yielded very different results both in terms of the spectrum of mutants recovered (i.e., the number of complementary classes) and the overall frequency of independently induced zym mutations. In both respects, UV mutagenesis appeared to be more effective than treatment with EMS. All zym mutants recovered after EMS mutagenesis proved to be members of the same complementation group, whereas those isolated after UV mutagenesis defined seven complementation groups. However, even in the latter case, independently arising mutations within the complementation group defined by the EMS-induced mutants were common. The apparent site-specific mutagenesis obtained after EMS treatment cannot, however, be directly correlated to the choice of mutagen because the two mutant searches differed in other details of experimental protocol as well. In particular, the EMS mutagenesis was performed on vegetative cells, whereas UV mutagenesis was conducted on cells grown for 18 hr in mating-induction medium and thus undergoing gametic differentiation. Furthermore, different strains were utilized in the two experiments (see Figure 1).Preliminary results from UV mutagenesis of a third strain (the wild-type C. monoica strain maintained by The University of Texas Culture Collection of Algae) suggest that additional zym loci (i.e., new complementation groups not described here) exist and that use of vegetative cells does not preclude isolation of mutants from several complementation groups, nor does it necessitate excessive recovery of mutants within the cistron defined by the original EMS mutagenesis. Thus the peculiar results reported here may be specific to EMS mutagenesis and/or to the particular strain utilized. Our initial screening method for the identification of zym mutants involved visual inspection of cultures by light microscopy and relied, first of all, upon the absence of the discarded primary zygote wall in mated cultures. Thus we have selected for mutants with defects expressed relatively early in the maturation process. In renewed mutant searches (in progress), we are now using chloroform selection as the first step in our screening process; preliminary results indicate that mutants with maturation defects expressed after primary zygote wall release (but before induction of germination) can also be readily obtained from C. monoica. The zym markers identified thus far are expressed only in the zygote and act there as recessive lethal mutations. Thus they have proved invaluable for the selection of heterozygous zygotes from the background of self-matings characteristic of homothallic strains and typically hampering genetic analyses. Incorporation of the zym markers into strains carrying other mutations of interestsuch as the mutations to antibiotic resistance (VANWINKLE-SWIFT 1979, 1980), auxotrophy (VANWINKLE-SWIFT 1979), or self-sterility (VANWINKLE-SWIFT and BAUER1982) previously isolated in our laboratory-will greatly facilitate our efforts to map the nuclear genome of this species. However, because production of a normal viable zygote in crosses between zym strains requires the use of complementary zym parents, we cannot directly assess the linkage relationships between independently isolated zym markers within a given complementation group by standard tests for allelism.

442

K. P. VANWINKLE-SWIFT AND C. G . BURRASCANO

Successful completion of the Chlamydomonas sexual cycle involves a complex progression of cellular events that must occur in an ordered sequence. Thus, for example, cell fusion must occur before nuclear fusion, and nuclear fusion before the induction of meiosis. Similarly, zygospore wall development is restricted to the product of mating events and does not occur in the haploid gametes. We have considered the possibility that mating and zygote maturation may be coordinately regulated in a complex fashion, and that such coordinate regulation could lead to a misinterpretation of complementation data for zygote maturation genes. Suppose, for example, that each cell from a homothallic species such as C. monoica carries copies of two mating-type “genes” analogous to the mt+ and mt- mating-type alleles of heterothallic species, and that pair formation in a clonal culture results from the expression of the mt+ locus in some cells and the mt- locus in others (a situation similar to that described for homothallic strains of the yeast Saccharomyces cerevisiae; HERSKOWITZ et al. 1980; cf. VANWINKLESWIFTand BAUER1982 for further discussion). Suppose also that these loci are regulatory and control the expression of structural genes-some of which are required for a particular mating behavior and others of which take part in zygote maturation. Cells expressing the mt+ gene are hypothesized to produce one set of zygote maturation gene products whereas cells expressing the mtlocus produce a second set. Maturation per se might require the interaction of these two subsets of gene products and would thus begin only after fusion of the mt+ and mt- -acting gametes. If such a situation were to exist, an absence of complementation between two zym markers could occur for mutations in separate genes which are exclusively expressed in the same mating-type-these mutations need not be physically linked. Table 5 summarizes the effects of various types of coordinate regulation on the outcome of a standard complementation test. The failure of two point mutations to complement one another may not necessarily indicate allelism and does not necessarily imply dominance of the mutant allele(s); instead it may be a consequence of similar sex-limited expression for the gene loci involved (model I, Table 5). Note also that a positive complementation test could be obtained even if one of the mutations involved were “dominant” to its wild-type allele (models 11, 111, Table 5 ) . In fact, at present we have no means of accurately assessing dominance if a maturation marker shows mating-type-specific expression, because the wild-type and mutant alleles would never be jointly expressed in the zygote. Our recovery of more than two zym complementation groups suggests that not all maturation genes show “mating-type-specific’’ expression, but the possibility remains that one or more of the zym loci identified thus far could be coordinately expressed with “mating-type”. In the yeast S. cerevisiae, the mating-type alleles (MATa and MATa) not only determine haploid mating behavior, but are also involved in the control of meiosis and sporulation in diploids produced by MATa X MATa matings (KASSIRand SIMCHEN 1976; KLAR, FOGEL and RADIN1979; HERSKOWITZ et al. 1980; SPRAGUE, RINEand HERSKOWITZ 1981). The existence of similarly complex loci in C. monoica might allow the recovery of mutants with mating-type-specific defects that allow normal haploid

443

MATURATION MUTANTS OF C. MONOICA

TABLE 5 The effect of coordinate regulation of mating and zygote maturation on the results obtained from a stondord complementation test" Genotypes Haploid gamete types Model

Strain

mt+

ZymA ZymB

AB' A+B

ZymA ZymB

(111)

(IV)

mt-

Diploid zygotes produced by Selfing

Crossingh

Apparent complementation

~

(I) (11)

A B+/A+ B/-

-

--

A B+ A+B

- B+ -B

A B+/A+ B/-

Bt B

?4 A B+/-

ZymA ZymB

AA+-

- Bt -B

A -/A' -/-

B+ B

ZymA ZymB

AB+ A+B

AB+ A+B

--e

-

A B+/A B+ A+ B/A+ B

- (Z) - (Z)

No No

% A+ B/-

B (Z) B+ (WT)

No Yesd

?hA -/?4A+ -/-

B (Z) B+ (WT)

No Yes

'h A B+/?hA'

B/-

A+ B/A B+ (WT)

Yesd

a Main assumptions: (a) Homothallic strains differentiate into two gamete types analogous to the mt+ and mt- mating types of heterothallic species; (b) Certain genes required for zygote maturation may be specifically induced (expressed) in one gamete type only. Alternative models: (I) Zym genes A and B are both induced only in mt+-acting gametes; (11) ZymA is mt+-specific but zymB is expressed in both gamete types; (111) ZymA is mt+-specific and zymB is mt--specific; ( I V ) ZymA and zymB are both expressed in both gamete types. 'Phenotypes of zygotes produced by inter-strain matings are shown in parentheses: Z = maturation-defective: WT = normal, fully matured zygotes. = Unexpressed gene locus. Mutant alleles at non-mating-type-specific loci are assumed to be recessive.

-

mating but interfere with zygote maturation. Thus it is also conceivable that some of our zym markers might reside at one or the other of the hypothesized "mating-type loci". As mentioned previously, such speculation cannot be directly evaluated by allelism testing of noncomplementing mutants since pairing between such zym strains does not produce viable zygotes. However, less direct approaches are feasible and might include the identification of nonselective markers closely linked to a given zym marker that could then be mapped to other zym markers within the same complementation group, or the isolation of conditional zym mutants or zym suppressor mutations that would allow the recovery of viable zygotes from crosses between noncomplementary maturation-defective strains. Continued genetic analysis of maturation mutants, and the extension of the work to include ultrastructural and biochemical studies, should provide clues to the underlying molecular mechanisms that insure progression of zygote development from initial cell fusion through full development of the mature zygospore. We would like to thank Drs. C. A. BARNETT, R. L. WEISSand F. AWBREY for providing access to needed laboratory space and equipment, and Drs. A. BAERand D. SHORTfor additional research support. K. V.W.-S. wishes to thank Dr. R. A. LEWINfor first introducing her to Chlamydomonas

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monoica and for encouragement and helpful discussions throughout the course of this work. This study was made possible by a grant from the National Science Foundation (PCM-8010026) to K.V.W.-S. LITERATURE CITED

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