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been worked out by Maniak eta/. 25 involves treatment of blots with guanidinium chloride, baking of the RNA, and hybridization with a 32p-labeled in vitro transcript. Acknowledgments Work on the described methods was supported by a grant of the Deutsche Forschungsgemeinschaft to Sonderforschungsbereich 266. 25 M. Maniak, U. Saur, and W. Nellen, Anal Biochem. 176, 78 (1989).

[29] Selection of Chlamydomonas Dynein Mutants By RITSU KAMIYA Introduction The inner and outer dynein arms, force generators in cilia and flagella, are complex molecular assemblies made up of different sets of more than 10 protein subunits each. The outer arms contain two or three and the inner arms five or more heavy chains with activities to hydrolyze ATP. 1,2 The molecular weights of these heavy chains are in excess of 400,0003,4; therefore, the total molecular weights of dynein arms can be as high as 1,500,000. The inner arms occur as three different subspecies, whereas there is only a single outer arm species. 5 Our understanding of the structure and function of the complex dynein arms has benefited greatly from the isolation of Chlamydomonas mutants that lack outer or inner dynein arms. 6 Chlamydomonas is uniquely suited for isolation of such mutants, because its flagellated vegetative cells are haploid and its mutants can be genetically analyzed by sexual crosses between different strains. The dynein-arm mutants so far reported are nonmotile strains lacking outer a r m s (pf13, 7 pf227) or m o s t of the inner F. D. Warner, P. Satir, and I. R. Gibbons (eds.), "Cell Movement." Alan R. Liss, New York, 1989. 2 K. A. Johnson, Annu. Rev. Biophys. Biophys. Chem. 14, 161 (1985). 3 A. L.-Eiford, R. A. Ow, and I. R. Gibbons, J. Biol. Chem. 261, 2337 (1986). 4 S. M. King and G. B. Witman, J. Biol. Chem. 262, 17596 (1987). 5 G. Piperno, Z. Ramanis, E. F. Smith, and W. S. Sale, J. CellBiol. 110, 379 (1990). 6 D. J. L. Luck, J. CellBiol. 98, 789 (1984). 7 B. Huang, G. Piperno, and D. J. L. Luck, J. Biol. Chem. 254, 3091 (1979).

METHODS IN ENZYMOLOGY, VOL. 196

Copyright © 1991 by Academic Press, Inc. All fights of reproduction in any form ~served.

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arm (pf237), motile strains lacking entire outer arms (odal- I0, s,9 pf281°) or subsets of inner arm heavy chains (idal-3, H,12 idbl, 12 pf3013), and a motile mutant with an aberrant outer arm heavy chain (supp/114). Considering the complexity of the dynein arms, however, we can expect to obtain more kinds of dynein mutants, which can further facilitate studies on how dynein forms and functions. This chapter describes strategies for isolation of outer or inner arm dynein mutants from Chlamydomonas. A comprehensive guide book on procedures for mutant isolation, genetic analysis, strain maintenance, and culture of this organism has been published. ~5 For isolation of Chlamydomonas flagella '6 and dynein, 17 see Vol. 134 of this series. Mutant and wild-type strains of Chlamydomonas can be obtained from the Chlamydomonas Genetics Center (Department of Botany, Duke University, Durham, NC).

General Strategies The mutants pfI3, pf22, and pf23, isolated before 1980, are all paralyzed and have short flageUa. However, later studies showed that there are motile mutants lacking the entire outer arm or partial structures of the inner arm, although their motility is only one-third to two-thirds of the normal value. Therefore, many kinds of dynein-arm mutants (except those missing a large portion of the inner arm, which may be nonmotile) can be obtained by selecting cells that are slow swimming rather than paralyzed. In fact, a large number of mutants missing the outer dynein arm have been isolated by just screening for poor motility. For isolation of mutants with inner arm defects, however, such a simple screening procedure does not work well. The reason for this is not clear but it may be that inner arm mutants generally can swim at a fairly high speed (one-half to two-thirds of the wild-type velocity). Therefore, another method of isolation was devised s R. Kamiya and M. Okamoto, J. CellSci. 74, 181 (1985). 9 R. Kamiya, J. Cell Biol. 107, 2253 (1988). ,o D. R. Mitchell and J. L. Rosenbaum, J. Cell Biol. 100, 1228 (1985). 11C. J. Brokaw and R. Kamiya, Cell Motil. Cytoskel. 8, 68 (1987). ~2R. Kamiya, E. Kurimoto, H. Sakakibara, and T. Okagakj, in "Cell Movement" (F. D. Warner, P. Satir, and I. R. Gibbons, eds.), p. 209. Alan R. Liss, New York, 1989. 13G. Piperno, J. Cell Biol. 106, 133 (1988). ~4B. Huan~ Z. Ramanis, and D. J. L. Luck, Cell (Cambridge, Mass). 28, 115 (1982). ~s E. H. Harris, "The Chlamydomonas Sourcebook." Academic Press, San Diego, California, 1989. ~6G. B. Witman, this series, Vol. 134, p. 280. t7 S. M. King, T. Otter, and G. B. Witman, this series, Vol. 134, p. 291.

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based on the assumption that a mutant with defects in both outer and inner arms would be nonmotile.

Isolation of Outer Arm Mutants The procedure used for isolation of outer arm mutants is essentially after that of Lewin, 18 which was devised for isolating paralyzed flagella mutants. Mutagenesis. UV light is used for mutagenesis to obtain the outer arm mutant. This appears to yield more outer dynein mutants than mutagenesis with nitrosoguanidine or ethane methyl sulfonate, although chemical mutagenesis tS,t9 can be more effective for obtaining certain types ofdynein mutants. Wild-type Chlamydomonas reinhardtii 137C (mating type either + or - ) is grown in Tris-acetate-phosphate (TAP) medium 2° (Tables I; III) to a cell density of about 1 × 106/ml under constant illumination. A cell suspension (15 ml) in a 9-cm petri dish is irradiated for 5 to 20 min with a 15-W UV lamp (GL-15; Toshiba, Tokyo, Japan) located 30 cm above it. The same is then divided into 8 to 12 1-ml aliquots and each is inoculated into 3 ml of medium in a test tube (l-cm diameter, 11 cm high) with an aluminum cap. The test tubes are kept in the dark for 12 hr to prevent photorepair. Enrichment of Motility-Deficient Cells. After dark incubation, the test tubes are left standing under 12 hr/12 hr, light/dark conditions. During the second light phase (illuminated from above), cells at the bottom of each test tube are carefully transferred using a Pasteur pipette to another test tube containing 4 ml of fresh medium. When the upper part of this culture becomes greenish after a few days, the cells growing on the bottom are transferred to a third test tube. This process is repeated two to three more times over a period of 1 - 2 weeks. The cells growing at the bottom of the last set of test tubes are saved, suspended in 0.5-1 ml of medium, and inoculated onto TAP/agar plates [TAP medium containing 1.5% (w/v) Bacto Agar; Difco, Detroit, MI] to obtain 5 0 - 100 colonies/plate. Selection of Slow Swimmers. The agar plates are kept under constant illumination for 3 - 5 days until single colonies grow. Colonies are then transferred with sterile toothpicks to 96-well cell culture plates (such as Coming #25860, Coming, NY), each well containing 200/d liquid medium. Usually 48 colonies are taken from a single test tube (i.e., 384-576 colonies altogether). Because mutants lacking the outer dynein arm are 18 R. A. Lewin, £ Gen. MicrobioL 11, 358 (1954). 19 B. Huang, M. R. Lifkin, and D. J. L. Luck, J. CellBiol. 72, 67 (1977). 20 D. S. Gorman and R. P. Levine, Proc. Natl. Acad. Sci. U.S.A. 54, 1665 (1965).

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TABLE I COMPONENTS FOR TAP MEDIUMa

Component NH+C1 MgSO+. 7H20 CaC12' 2H20 KzHPOdKH2PO 4 Trace metal stock solution b Glacial acetic acid Tris base

Stock solution (%, w/v)

Milliliters stock/ liter medium

20 5.0 2.5 4.7/3.1

2 2 2 2 1 1 10

24.2

Final concentration in medium (g/liter) 0.4 0.1 0.05 0.094/0.062

2.42

a Modified from Gorman and Levine.2° b See Table III.

frequently motile they tend to produce flat colonies like those of the wild type. Therefore, colonies should not be selected by their appearance. After the colony transfer, the 96-well plates are placed under constant illumination for 1 or 2 days, and then each well is observed with an inverted microscope. Cells in most wells usually display some abnormal motility. Because flagella are not clearly visible under these conditions, any interesting clones should be transferred onto a glass slide and examined under a dark-field microscope; in addition to slow swimmers which have apparently normal flagellar waveforms, there may be cells with paralyzed or short flagella, cells which swim backward, or cells that display aberrant waveforms. Cells that are judged to be slow swimmers are saved for further analysis. Mutants whose cell bodies appear to vibrate back and forth while swimming are most likely outer arm mutants, which are characterized by their low flagellar beat frequency (20-25 Hz, compared with 50-60 Hz of the wild type). Experience has shown that more than one-third of the slow swimmers are mutants lacking the entire outer arms, which are called oda (outer dynein arm deficient).9

Isolation of Inner Arm Mutants Inner arm mutations are first selected as those that produce a paralyzed (and short-flagellated) phenotype when present in combination with outer arm mutations. Thus the isolation consists of two steps: isolation of nonmotile double mutants from oda mutants and removal of the oda mutation by crossing the double mutants with the wild type. Mutagenesis and Screening. One of the oda strains of l0 different complementation groups9 (available from the Genetics Center) is cultured,

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TABLE II COMPONENTSFOR GAMETE-INDUCINGMEDIUMa Component

Amount/liter medium

MgSO4" 7H20 K2HPO4 KH2PO4 CaC12"2H20 Trace metal stock solutionb Trisodium-citrate dihydrate Na-HEPES

150 mg 20 mg 20 mg 10 nag 1 ml 60 nag 300 mg

a y. Tsubo, in "Methods in Microbial Genetics" (T. Ishikawa, ed.), p. 279. Kyoritsu Publishing Co., Tokyo, 1982 (in Japanese). b See Table IlL

mutagenized with UV light, screened for poor motility, and inoculated on agar plates, as described above. Colonies with heaped appearances are selected and transferred to 96-well cell culture plates. Clones that appear completely nonmotile 1 day after the transfer are saved and, after the cell density has increased in the wells, inoculated on TAP/agar plates. To economize medium, eight clones are usually streaked on a single 9-cm plate. Clones that appear to have lost flagella should also be saved because they may grow flagella when they differentiate into gametes. The agar plates are kept under constant illumination. Isolation of Inner Arm Mutations. One week after transfer to the agar plates, the cells are scraped off from the plates and suspended in 0.5 ml of gamete-inducing medium (Tables II; III) in test tubes. The cells differentiate into gametes after the test tubes have been kept shaking under light for 3- 6 hr. Each mutant must then be examined with a dark-field microscope, because many strains which initially appear paralyzed may become motile upon gametogenesis. Clones that show any motility are discarded. Nonmotile cells (including those missing flagella) are saved and mated with the wild-type gamete of the opposite mating type prepared in the same way as the mutant. A problem at this stage is that double mutants between outer arm and inner arm mutants usually have very short flagella (1 -3 ~tm) and only a small percentage of gametes undergo mating with the wild type. To circumvent this difficulty, a method that can induce mating in flagellaless gametes recently developed by Pasquale and Goodenough2~ may be useful. 21S. M. Pasquale and U. W. Goodcnough, J. CellBiol. 105, 2279 (1987).

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TABLE III TRACE METAL STOCK SOLUTIONS Stock solution (g/liter) Component

TAP ~

H 3BO3 ZnSO4" 7H~O MnC12" 4H20 MnSO4" H20 FeSO4.7H20 FeSO4(NH4)2SO4.6H20 CoC12.6H20 CoSO4" 7H20

11.4 22.0 5.0 -5.0 -1.6 -1.1 -1.6 50.0 - 16

(NH4)tMOTO24.4H20 Na2MoO4" 2H20 CuSO4- 5H20 Na2EDTA KOH

Gamete induction medium b 1.14 2.20 -0.58 0.57 -0.19 -0.15 0.16 5.00 - Ic

a From Gorman and Levine. 2° To make 1 liter of stock solution, dissolve all components but Na2EDTA in 550 ml of water and heat it to 100*. To this solution add 50 g Na2EDTA (dissolved in 250 ml of water), and adjust the pH with KOH to about 6.5 while keeping the temperature near 80*. Add water to make the total volume 1 liter. Filter out the precipitate after 2 or 3 weeks of maturation at room temperature. b y . Tsubo, see Table II legend. c Add until white precipitates dissolve. The final pH should be about 3.5.

One week after the mating, tetrads are obtained and analyzed according to standard procedures. 15,22 The desired mutants are isolated from a nonparental ditype tetrad, that is, a tetrad consisting of two oda phenotypes and two identical, possibly new, phenotypes. The latter two clones are saved and analyzed for the composition of the flagellar axoneme. If the two clones have paralyzed flagella, they can be mutants missing the central-pair or radial spokes; if their flagella look unusually stiff and straight, they are most likely missing the central pair. If, on the other hand, the two clones swim slowly, there is a good possibility that they are inner arm mutants. Previously isolated partial inner arm mutants swim slowly due to reduction in the flagellar bend angle rather than in the frequency.11 The motility characteristics of inner arm mutants make them appear similar to wild type, and care must be taken not to confuse them. 22 R. P. Levine and W. T. Ebersold, Annu. Rev. Microbiol. 11, 358 (1960).

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Analysis of Axoneme Compositions An easy way to detect lack of inner or outer dynein arms in the isolated mutants is to examine their dynein heavy chain composition by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of their axonemes. For this purpose, the mutants are cultured in 200 ml liquid medium in a 300-ml flask with aeration, and axonemes are isolated by the method of Witman.16 Axonemes are prepared from 6 strains at a time, and 12 samples are run in a single gel. A Laemmli gel made with a 3-5% (w/v) acrylamide and 3 - 8 M urea gradient, 22,23 and stained with silver,24 usually results in good resolution of the heavy chains. As described by Pfister et aL, 23 use of impure SDS and its omission from both running and stacking gels are important for good separation. Some inner arm heavy chains are difficult to identify in an SDS-PAGE pattern because of overlap with intense outer arm heavy chains. Thus, it is also desirable to analyze the axoneme composition in the background of an oda mutation, although this is sometimes difficult because the double mutants do not grow enough flagella. For this purpose, the axonemes are better isolated from gametes, which tend to grow flagella better than vegetative cells.

Genetic Analys& When mutants of interest are obtained, they are mated with wild-type cells to obtain daughter cells of the same phenotype and opposite mating types. These daughter cells are used for subsequent experiments. This step is necessary because the first generation mutants often carry second, undesirable mutations. The next step is to check the allelism with previously isolated mutants. This is most easily done by the so-called temporary dikaryon rescue experiments, z5 A newly isolated strain is mated with another mutant according to standard procedures. Any two flagellar mutants are judged to be nonallelic when quadriflagellated temporary dikaryons acquire a higher level of motility than that of their parents 1 - 2 hr after the onset of mating. If the motility of the temporary dikaryons does not improve with time, the two kinds of mutants can be allelic. However, this must be checked by examining the progeny, since some combinations of nonallelic dynein mutants do not undergo rescue in temporary dikaryons. 9 If the cross between the two strains yields only parental ditypes in more than 30 tetrads, they are most 23 K. K. Piister and G. B. Witman, CellMotil. 2, 525 (1982). 24 C. R. Merril, D. Goldman, S. A. Sedman, and M. H. Ebert, Science 211, 1437 (1981). 25 D. Starling and J. Randall, Genet. Res. 18, 107 (1971).

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likely allelic. For detailed methods of Chlamydomonas genetic analysis, including mapping, consult Refs. 15 and 22. Acknowledgments I would like to thank Professor Yoshihiro Tsubo of Kobe University for teaching me various techniques related to Chlamydomonasgenetics. The present work was supported by a grant-in-aid from the Ministry of Education, Science and Culture of Japan (No. 01657001).

[30] C l o n i n g a n d A n a l y z i n g G e n e s E n c o d i n g C y t o s k e l e t a l P r o t e i n s i n Y e a s t Saccharornyces cerevisiae

By TIM C. HUFFAKERand ANTHONY P. BRETSCHER Over the last 20 years, the eukaryotic cytoskeleton has been recognized as composing the framework around which a cell is built as well as providing the machinery for motile cellular events. During this period, a bewildering number of proteins from higher cells have been implicated in the organization of the cytoskeleton. To complicate the functional analysis further, many of these proteins appear to exist as a family of closely related species, sometimes with many variants being present in the same cell. One approach toward an understanding of the molecular function of cytoskeletal components is to seek a simple system in which the full power of genetics can be applied. The fungi, particularly the yeast Saccharomyces cerevisiae, fit this need. At first glance it may appear that this budding yeast is a poor choice: it has a closed nuclear division cycle, so microtubule organization might be different from higher eukaryotes; it has a cell wall that determines the shape of the cell and it is nonmotile, functions generally attributed to microfilaments in higher eukaryotes. However, microtubules are essential for chromosome segregation in yeast and their distribution is synchronized with the cell cycle, as it is in higher cells. Microfilaments have also been found to be vital to this organism. Given the presence of the cell wall and the lack of motility, this suggests that yeast may use microfilaments in a more restricted manner, perhaps in some previously unsuspected functions. A major advantage of S. cerevisiaeis that all the genes for cytoskeletal proteins so far examined are present in a single or at most two copies, making genetic analysis relatively straightforward. Additionally, most are devoid of introns simplifying cloning and analysis of these genes. Work over the last few years on the genetics and METHODS IN ENZYMOLOGY,VOL. 196

Copyright© 1991by AcademicPress,Inc. All rightsofreproductionin any formreserved.

Selection of Chlamydomonas dynein mutants.

348 MOLECULAR AND GENETIC APPROACHES [29] been worked out by Maniak eta/. 25 involves treatment of blots with guanidinium chloride, baking of the R...
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