Chlamydomonas reinhardtii

A mutation at the PFlO locus of the unicellular green alga Chlamydomonas reinhardtii leads to abnormal cell motility. The asymmetric form of the ciliary beat stroke characteristic of wild-type flagella is modified by this mutation to a nearly symmetric beat. We report here that this abnormal motility is a conditional phenotype that depends on light intensity. In the absence of light or under low light intensities, the motility is more severely impaired than at higher light intensities. By UV mutagenesis we obtained 11 intragenic and 70 extragenic strains that show reversion of the pflO motility phenotype observed in low light. The intragenic events reverted the motility phenotype of the p f l 0 mutation completely. The extragenic events define at least seven suppressor loci; these map to linkage groups IV, VII, IX, XI, XI1 and XVII. Suppressor mutations at two of the seven loci (LIS1 and LIS2) require light for their suppressor activity. Forty-eight of the 70 extragenic suppressors were examined in heterozygous diploid cells; 47 of these mutants were recessive to the wild-type allele and one mutant (bop5-I) was dominant to the wild-type allele. Complementation analysis of the 47 recessive mutants showed unusual patterns. Most mutants within a recombinationally defined group failed to complement one another, although there were pairs that showed intra-allelic complementation. Additionally, some of the mutants at each recombinationally defined locus failed to complement mutants at other loci. They define dominant enhancers of one another. T HE structure and function of the eukaryotic flagellum are complex (GIBBONS 1981). Historically, two complementary genetic approaches have been used to dissect complex systems. T h e first is pseudoreversion or suppressor analysis, in which extragenic mutations that relieve or suppress the mutant phenotype are isolated (HARTMAN and ROTH 1973; JARVIK and BOTSTEIN 1975; HALL and GREENSPAN 1979; BOTSTEIN and MAUER 1982; LAKIN-THOMAS and BRODY 1985). Extragenic suppressors fall into two broad classes based on the interaction between the mutant and the suppressor. Interactive suppressors act by changing proteins that normally form complexes and bypass suppressors act by circumventing the actual defect. T h e second approach is enhancement analysis, in which extragenic mutations that accentuate or enhance the original mutant phenotype are isolated (LEWIS 1945; WELSHONS 1971; SATO, RUSSELL and DENELL 1983; ATKINSON 1985; KUSCH and EDGAR 1986; RINE and HERSKOWITZ 1987; STEARNS and BOTSTEIN 1988). Previous studies have demonstrated that these types of genetic analyses are powerful tools in understanding flagellar assembly (LUCK 1984) and function (HUANG, RAMANIS and LUCK 1982; SEGAL et al. 1984; HOOPS et al. 1984; MITCHELL and ROSENBAUM 1985; JAMES et al. 1988). T h e PFlO (paralyzed flagella) gene product of Chlamydomonas reinhardtii is required for wild-type Genetics 120: 965-976 (December, 1988) flagellar motility (LEWIN 1954). Only a single mutant allele, which displays an ineffective flagellar beat and abnormal swimming (RANDALL and STARLING 1971; INWOOD 1985), has been isolated a t this locus. Consequently, cells with the pfl0 mutation settle to form a pellet in liquid culture. This mutation maps to an unusual linkage group (HUANG et al. 1982), which was recently described genetically by DUTCHER ( 1 986) and RAMANIS and LUCK (1986). All mutations that map to linkage group XIX were identified because they affect flagellar assembly, function or microtubules. To approach the question of the role of the PFlO gene product in flagellar function, we isolated revertants of the mutant pfl0. These revertants define seven loci that can be mutated to act as extragenic suppressors of pfl0. Two of the extragenic suppressor mutations define a light-dependent pathway of suppression. We found that a subset of the mutants act as dominant enhancers of each other in diploid strains. MATERIALS AND METHODS Mutant strains: The original p f l 0 mutant was isolated by LEWIN (1 954) using the techniques he reported for isolating motility mutants in Chlamydomonas moewusii. The original pfl0 strain has been backcrossed to a wild-type strain (1 37c mt-) three times to remove modifiers of the pflO phenotype and to assure that the pflO phenotype segregated in a Mendelian fashion as a single gene mutation. Single colonies of the p f l 0 isolate p f l 0 1-1 mt+ were used in this reversion 966 S. K. Dutcher. W. Gibbons and W. B. Inwood analysis. Strains for mapping were provided by HARRIS (1982) of the Chlamydomonas Stock Center. Several of these strains were found to contain two mutations that each have a phenotype. Strains that contain single scorable mutations were recovered through backcrosses of the mutant strains to wild-type cells (1 37 mt+ and 137 mt-). The ac3l strain (cc977) contained two mutations, each conferring an acetate requirement. The ac31 mutation was identified by phenotypic analysis and linkage to p f l . The ac6 strain (cc665) also contained two mutations, each conferring an acetate requirement. The ac6 mutation was identified by linkage to pf l7 . The crl strain (cc40) contained two mutations and the crl mutation was identified by a cold-sensitive, acetate-requiring phenotype. The y l mutation (ccl168) was assayed after the following sequence of growth conditions; seven days in the dark, one day in the light, and two days in the dark. The arg2 and arg7 strains were obtained from F. LUX. The meiotic viability of the strains used for construction of the diploid strains in Figure 1 is between 90 and 98%. Culture media: General growth medium is medium I described by SAGER and GRANICK (1953) except that the K2HP04 concentration has been raised to 1.0 mM. Strains with arg mutations are grown on medium I that contains 200 pg/ml of arginine and has one-tenth the concentration of ammonium nitrate that is present in medium I. Genetic analysis: Standard techniques were used for mating and for tetrad analysis (LEVINE and EBERSOLD 1960). The methods of WHITEHOUSE (1950) and PERKINS (1 952) were used for determination of centromere distances and for linkage analysis, respectively. Only complete tetrads are included in this analysis; between 90-95% of the dissected tetrads were complete. Diploid strains were selected as described by EBERSOLD (1967). The reliability of scoring of tetrads from backcrosses and mapping crosses that involve the suppressors of PflO was insured in several ways. First, the phenotypes of some suppressed strains change with increasing cell density; therefore, all crosses were scored on several consecutive days at densities of less than 5 X lo5 cells/ml. Whenever data were questionable, the tetrads were transferred to new medium and rescored. Sensitivity to cell density makes it possible to identify the double mutant ( p f l 0 ; suppressor) in liquid culture as well as by microscopic examination. The double mutant, in all cases examined and tested, will form a pellet before wild-type cells or suppressor cells will. Second, some of the suppressors are affected by illumination conditions. All crosses reported in this paper were performed under constant light conditions (3100 ergs/cm* sec) in a temperature-controlled walk-in incubator. Light intensities were measured by a Minolta light intensity meter. During the initial backcrosses, the isolates bopl-1 and lisl22 produced significantly unequal numbers of parental and nonparental ditype tetrads. This increase in the PD:NPD ratio could result from the presence of two, unlinked suppressor mutations in the original strains. We have tested this hypothesis by crossing the original isolate to a PflO strain to ask if suppression segregates 2+:2-, where + designates suppression and designates the lack of suppression. More than one functional suppressor in the original isolate would produce 4+:0and 3+:1tetrads, as well as 2+:2tetrads. For each isolate only 2+:2segregation was observed (see Table 2). The number of extragenic suppressor isolates mapped in RESULTS is only 69 not 70. The suppressor in strain 58 has not been mapped with respect to the seven loci discussed. Strain J8 proved refractory to analysis; most matings involving JS cells, whether from the original isolate or from the progeny of the first backcross of the isolate failed to sporulate. The results of the initial backcross are shown at the end of Table 1. In the cross to lis2-1 pf l0 , the ratio of tetrad types was 6: 10: 10. In the other fifteen attempted crosses, matings appeared to proceed normally. Quadriflagellate cells were produced; these cells became zygotes that appeared normal, but did not sporulate. Complementation results were not conclusive because of the noncomplementation seen for many of the mutations; it is likely that J8 is an allele of l i s l . Complementation analysis was performed in stable diploids that were selected by arginine prototrophy using arg2 and arg7 mutations. In general, for each suppressor that was tested, a strain containing arg2 and a strain containing arg7 was constructed. Both arg sup pf l0 strains were tested for dominance of the suppression phenotype in heterozygotes. In the complementation analysis all the pairwise crosses were performed with both sets of parents except for lisl-4, and lis2-7 where only one arg strain was constructed. UV mutagenesis: The procedure for mutagenesis of pf l0 cells with UV light and selection for reversion to a motile phenotype was that of LUCK et al. (1 977) with the following modifications. Single colonies of pfl0 1-1 cells were selected for consistency of the pflO phenotype; cultures showed nearly 100% nonmotile cells at low density. Cells were irradiated under a General Electric 30 W germicidal lamp (G3T08) for 45 sec at 9 cm from the UV source. The concentration of irradiated cells varied between 5 X 1 O6 and lo’ cells/ml. Survival rates varied from 20% to 80%. Enrichment for motile cells following mutagenesis was carried out four times by selectively transferring cells in the upper one-sixth of liquid cultures. Following the last transfer, diluted samples were plated onto solid medium and 12-20 single colonies were picked from each plate for examination of phenotype. Only about one-half of the colonies picked from each independent culture contained motile cells; the remainder still showed the pflO phenotype. Where differences in swimming behavior were observed between colonies derived from a single irradiated culture, colonies exhibiting these differences were saved as potentially independent events. Where more than one colony was saved, the one containing the best suppressor, as measured by the closest approach to wild-type behavior, was designated by an “A” following the culture number. The other colonies saved from the same culture were designated “B” or “C.” In 16 of 22 cases where more than one isolate was saved from the same culture, the isolates mapped to different recombination groups. In the remaining six cases, two additional arguments for independence of the l i s l alleles from the same culture are presented. First, because at least 42 of the 55 original cultures contained lisl suppressors, calculations based on the Poisson distribution suggest that at least 23 of these tubes should have contained a second, independent lisl suppressor mutation and ten should have contained two or more additional lisl suppressor mutations. Therefore, the finding of two lisl suppressors in five tubes and three in one tube is certainly a likely occurrence. Second, in each of the cases in which two or more lisl isolates were chosen from a single original culture, this choice was based on distinct swimming patterns. The method used to screen for suppressors of PflO does not permit a determination of the number of reversion events represented in the original irradiated cultures. HOWever, some measure of the reversion frequency can be gleaned from the number of original isolation tubes that produced no revertant cells. If one assumes that distinct reversion events are divided among the original isolation tubes according to a Poisson distribution, the mean number Light-Conditional Mutations 967 of events per irradiated culture was calculated to be 4.04 based on 1/57 of the cultures that produced no revertants. The average number of cells surviving mutagenesis in each test tube culture was, on the average, 4.7 X lo6 and therefore the reversion frequency was about Because the number of tubes that have mutations is so high, it is likely that this estimate is poor. No spontaneous revertants were recovered in a parallel experiment involving 3 X 10' cells. Microscopic techniques: Samples for observation were taken from logarithmically growing cultures and were observed at a low cell concentration ( lo4 cells/ml). Microscope slides and coverslips were prepared by washing with 7X detergent, rinsing with deionized distilled water, air-drying, and wiping with 95% ethanol on lens paper. A 15-111 sample of suspended cells was placed on a clean slide and a streak of vacuum grease was placed to either side of the drop. A coverslip was gently placed over the drop and sealed. Observations of gross swimming behavior were carried out using a Zeiss Universal microscope, with a xenon lamp and with 16X or 40X Plan objectives.