Genetic Analysis of Long - Flagella Mutants of Chlamydomonas reinhurdtii Sara

The length of the flagella of Chlamydomonas reinhardtii cells is tightly regulated; both short-flagella and long-flagella mutants have been described. This report characterizes ten long-flagella mutants, including five newly isolated mutants, to determine the number of different loci conferring this phenotype, and to study interactions of mutants at different loci. The mutants, each of which was recessive in heterozygous diploids with wild type, fall into three unlinked complementation groups. One of these defines a new gene, lf3, which maps near the centromere of linkage group I. The flagellar length distributions in populations of each mutant were broad, with the longest flagella measuring four times the length of the longest flagella seen on wild-type cells. Each of the ten mutants had defective flagellar egrowth after amputation. Some of the mutants showed no regrowth within the time required for wild-type cells to regenerate flagella completely. Other mutants had subpopulations with rapid regeneration kinetics, and subpopulations with no observable regeneration. The mutants were each crossed to wild type to form temporary quadriflagellate, dikaryon cells; in each case the long flagella were rapidly shortened in the presence of the wild-type cytoplasm, demonstrating that the mutants were recessive, and that length control could be exerted on already assembled flagella. A number of lines of evidence indicate that the length of the flagella in the unicellular green alga Chlamydomonas reinhardtii is under active control. In a population every cell has two flagella of equal length, and the length of the flagella never exceeds a maximum length of 12 pm (for haploid cells of strain NO). T h e cell maintains a pool of assemblycompetent flagellar protein, as detected by deflagellating cells in the absence of protein synthesis, and observing the regrowth of new flagella which are approximately one-half of the pre-deflagellation length (ROSENBAUM, MOULDER and RINGO 1969). Thus some partitioning mechanism maintains a certain length of assembled flagella, and accumulates the remainder of the assembly-competent protein into an intracellular pool. T h e equality of flagellar length between the two flagella on a single cell is also regulated. For example, if one of the two flagella is amputated, the remaining flagellum rapidly shortens, in some cases all the way into the cell body. T h e two flagella then grow out together to pre-deflagellation length (ROSENBAUM, MOULDER and RINGO 1969; COYNE and ROSENBAUM 1970). Somehow the cell determines that the two flagella are not of equal length, and then repairs this defect. Flagellar length control and homeostasis clearly involve fundamental processes of intracellular signalling and macromolecular assembly and disassembly. Michigan State University, East Lansing, Michigan 48824-1312. Genetics 118: 637-648 (April, 1988) ' Current address: Department of Energy, Plant Research Laboratory, Chlamydomonas mutants with altered length control have been described, including short-flagella (shf) mutants in three different genes UARVIK et al. 1984; KUCHKA and JARVIK 1987), and long-flagella (Zf) mutants in two different genes (RANDALL et aE. 1967; RANDALL 1969; MCVITTIE, 1972a, b; JARVIK, LEFEBVRE and ROSENBAUM 1976; JARVIK, REINHART and ADLER 1980). In this report we analyze ten different long-flagella mutants, including the five previously described Zfmutants, and five newly isolated mutants. The new mutants were isolated in a screen for mutants with defective flagellar regeneration (LEFEBVRE et al. 1985). Complementation tests using stable diploids have been used to establish that the 10 If mutants represent 3 different genetic loci-If1 (1 allele), $2 (5 alleles), and a new locus described in this work, If3 (4 alleles). MATERIALS AND METHODS Strains: The wild-type strains of Chlumydomom reinhardtii used were the 137c derivatives NO m t f (Chlamydomonas Genetics Center number CC-620) and NO mt(CC-621); they were obtained from the Chlamydomonas Genetics Center, Duke University, Durham, North Carolina, as were the following strains: CC-48 (arg-2, mt+), CC-51 (arg-7, mt-) , CC-1709 (ery-3, pf-22, mt-) and the multiply marked strain CC-29 ( a 1 7 , can-1, nic-13, pf-2, y-1, pyr-1, mr-1, act2, sr-1, m t ) . Nitrate reductase deficient mutants nit-3 and nit-4, used for diploid construction, were provided by Dr. Emilio Fernindez, Department of Biochemistry, Cbrdoba, Spain. Sources of the long-flagella mutants used in this report are listed in Table 1. All lfmutants were backcrossed 638 S.-E. Barsel, D. E. Wexler and P. A. Lefebvre