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Experimental

Cell Research 111 (1978) 37-46

MUTANTS OF CHLAMYDOMONAS REINHARDII ALTERED SENSITIVITY TO ANTIMICROTUBULAR J. R. WARR, DOROTHY Department

of Biology,

University

FLANAGAN of York,

and DIANA

Heslington,

York,

WITH AGENTS

QUINN YOl 5DD,

UK

SUMMARY Nine new colchicine-resistant, three vinblastine-resistant, two colchicine-sensitive and one colchicine-dependent mutant of Chlumydomonas reinhardii have been isolated. Some of the mutants have abnormal cell morphology in the absence of the drug. Some of the mutants have altered levels of resistance to puromycin and to caffeine, which may indicate that their phenotypes involve a non-specific permeability change. However, uptake of labelled colchicine is indistinguishable from wild type in all of these mutants except two. The discrepancy between these two results is discussed. All the resistant mutants except one behave as if they have a single gene defect in crosses to wild type, although zygote germination is consistently very poor. Strains carrying certain pairs of resistance mutations are much more resistant than those carrying single mutations indicating that gene effects are additive. Recombination frequencies between some genes have been measured. The colchicine-sensitive mutations are thought not to be cell wall deficient mutations because of their appearance in the electron microscope, growth on low agar concentrations and their colony morphology. The colchicine-dependent strain had a very low viability even in the presence of optimal concentrations of colchicine.

Colchicine-resistant mutants have been isolated in yeast [l], Chlumydomonas [2, 31 and in mammalian cell lines [4, 51. The Chlumydomonas mutants isolated in our laboratory have a very slow growth rate in drug-free medium and also have a very poor zygote germination, which makes genetic analysis difficult to perform [6]. Earlier work has suggested that increased resistance in the Chlamydomonas mutants is not due to a permeability defect [6] although, in contrast, extensive work by Till, Ling and associates have shown that membrane changes exist in colchicine resistant variants of Chinese hamster cells [7]. The present paper describes the isolation of further colchicine resistant mutants of Chlamydomonas reinhardii, some of which have clearly different phenotypes from

those which have previously been described. In order to broaden the search for mutants with altered sensitivity to antimicrotubular agents in Chlamydomonas, colchicine sensitive, colchicine-dependent and vinblastine-resistant strains have also been isolated. Genetic analysis of these mutants has been attempted and studies have been made to try to establish the biochemical nature of resistance in these strains. MATERIALS

AND METHODS

Culture conditions, mutagenesis and mutant isolation The wild tvne strain of Chlamvdomonas reinhardii used in this-work is strain 32C from the Culture Centre of Algae and Protozoa, Cambridge, UK. The culture medium is based on medium 1 described by Sager & Exp Cell

Res 11 I (1978)

38

Warr, Flanagan

and Quinn

Granick [8] in which ferric chloride was replaced by 0.01 g/l ferric citrate and 0.01 g/l citric acid. Culture conditions and techniques for crossing Chlamydomonas were as described in [6]. Nitrosoguanidine mutagenesis was as described in f2]. Ultraviolet mutagene& involved irradiation of logarithmic phase cultures for 170 set at a distance of about 28 cm from a 15 W Hanovia UV lamp delivering 350 pW/cm* at that distance, which resulted in about 5 % viability. Resistant mutants were selected by plating dense cell cultures directly onto 5 mM colchicine or onto 0.10 mglml vinblastine sulphate and incubating for 2 weeks. Colchicine-sensitive mutants were isolated following UV irradiation and plating at around 100 colonies/ dish on unsupplemented medium and then replica plating with a sterile velvet pad stretched over a wooden block onto medium containing 1 mM colchicine. After around 7 days’ incubation, colchicine-sensitive mutants can be detected by comparison of colchicinecontaining and drug-free plates. The colchicinedependent mutant was isolated by plating directly at around 100 cells/plate on medium containing 1 mM colchicine, following LJV irradiation and then replica plating onto unsupplemented medium.

for 1 h in the same buffer and then post-fixed in 1% osmium tetroxide in buffer. Sections embedded in Epon were stained with 2% uranyl acetate at pH 4.5 and Reynolds lead citrate and examined in a Hitachi BU-12A electron microscope.

Chemicals Colchicine was obtained from Hopkin & Williams, Chadwell Heath, Essex. Vinblastine sulphate (“Velbe”) was obtained from Eli Lilly & Co., Basingstoke. Puromycin and nalidixic acid were from the Sigma Chemical Co. Caffeine and streptomycin were from British Drug Houses, Poole. Ring-C[methoxyl-3H]colchicine was from The Radiochemical Centre. Amers-

RESULTS

Isolation and preliminary characterisation of mutants A total of nine new viable colchicineColchicine uptake experiments resistant mutants have been isolated by Colchicine uptake was measured in triplicate 5 ml single step selection on 5.0 mM colchicine shaken cultures containing an initial cell concentration following UV irradiation. These mutants of around 2.5Xl@/ml cells and 2.7 &i/ml [“Hlcolchicine (spec. act. of 5.9 Cilmmol). After 48 h 3 ml of are designated by the symbol col- with each of the cultures was collected at room temperature appropriate subscript. Several other muonto a pair of 1.8 cm diameter discs of Whatman GF/C glass fibre filters under suction. Each filter was then tants isolated on the basis of colchicine washed with 3x 10 ml quantities of medium; further washines contained no detectable radioactivitv above resistance had very low viability and could background. The pairs of filter discs bearing cells from not be maintained in stock culture. Three a single flask were then transferred to single scintillafurther new mutants were isolated by single tion vials and decolourised according to the procedure of Mahin Br Lofberg [91 by incubating for 1 h at 70°C step selection on 0.10 mg/ml vinblastine folwith 0.2 ml 68% perchloric acid and 0.4 ml 30% HzGz. lowing nitrosoguanidine mutagenesis (desAfter cooling, 10 ml of scintillation fluid containing equal volumes of toluene and Triton X-100 and 1% ignated by symbol vin-). With the addition PPG were added and the vials were counted after 5 mutants days when temporary chemiluminescence effects had of the five colchicine-resistant subsided. Quench corrections were applied using the whose isolation has been described previexternal standards method. Cells which were harously [2], a total of seventeen mutants with vested immediately after setting up the experiment contained negligible radioactivity. increased resistance to colchicine or vin1.5 ml of cell suspension from each 5 ml culture was used for protein estimation by the method of Lowry et blastine were available for study in this al. [lo] following extraction of chlorophyll with 80% work (table 1). acetone. The colchicine resistant mutants showed Purification of radioactive colchicine was performed as described in Results by thin-layer chromatography differing levels of resistance to this drug using chloroform/acetone/diethylamine [5 : 4 : l] as when streaked on solid medium (table 1). solvent. The position of the colchicine standard was observed under UV light. One cm bands of the chroNearly all the colchicine-resistant mutants matogram were scraped off and eluted in 3 x 1 ml volwere also slightly cross resistant to vinumes of methanol which was subsequently removed by rotary evaporation. blastine as determined by streaking on solid medium (table 1) and most of them were Electron microscopy as Pellets were prefixed for 1 h in 2% glutaraldehyde in slightly cross resistant to vinblastine 0.025 M sodium phosphate buffer at pH 7.0, washed measured by growth (expressed as cell proExp Cell Res I I I (1978)

Colchicine Table 1. Basic characterisation

of mutants resistant to colchicine

and vinblastine

39

and/or vinblastine

Mutagenesis and selection: (a) described in [2]; (b) UV mutagenesis followed by selection on 5.0 mM colchicine; (c) nitrosoguanidine mutagenesis followed by selection on 0.1 mg/ml vinblastine. Resistance has been scored by examining streaks after 8 days incubation, For colchicine resistance, + + + , + + and + represent good growth on 7.5, 5.0 and 4.0 mM colchicine, respectively; wild type only grows on 3 mM colchicine. For vinblastine resistance, + + +, + + and + represent good growth on 0.14, 0.12 and 0.10 mg/ml vinblastine respectively; wild type only grows on 0.08 mg/ml vinblastine. For puromycin, + + + and + + represent good growth on 0.5 and 0.4 mM puromycin, respectively; wild type only grows on 0.2 mM puromycin. Colony diameters are means of 50 measurements using a micrometer eyepiece, on colonies grown for 10 days on drug-free medium

Wild type col; col; col; col; col; ’ co& col; col, col, col, col, col, col, col, vin; vin; vin;

Muta-

Resistance levels

zesis selection

(a) colch.

(b) vinbl.

(cl pur.

a a a a

0 ++ ++ ++ ++

0 ++ ++ ++ ++

0 0 0 0 0

a b b

++ ++ +

++ 0 +

0 0 0

::

+++ +++

+++ +++

++ +++

b b

+ ++

++ ++

+++ ++

t:

+ +

0 ++

0 0

b

++

++

++

8

0 0

2.40

C

+ +

C

+++

+++

0

1.16

C

teinlml) in liquid culture. Marked cross resistance in liquid medium was only observed with col, and col,, although statistically significant slight cross resistance could be detected with col;, co&, col,, col,, and col, in liquid culture. Cross resistance of co& and cO1, could not be detected in liquid culture; cross resistance of co17 and col; has been described previously [6]. Two of the mutants isolated on vinblastine, vin; and vin;, similarly only have slight resistance to vinblastine, vin; is more strongly resistant to vinblastine and also is the only one of the three mutants isolated on vinblastine to show detectable cross resistance to colchicine (table 1).

Colony size diameter (mm)

Comments

2.49

1.19 1.66 1.32 1.45 1.06 1.90 1.85 1.62 1.04

Large cells Entirely non-flagellate. Tight palmella clumps

1.29 2.28 2.28

2.16 1.03 1.06

Large cells Low viability Low viability Low viability. Large roundish cells Low viability

Nearly all of the resistant mutants have slow growth rates in drug-free medium with doubling times approximately twice as long as that of wild type. The slow growth rates give rise to small colonies when plated on solid medium (table 1). In some cases, notably col,, vin;, vin; and viny the mutants have unusually low and rather variable plating efficiencies of between lO-30% compared with wild type plating efficiencies of around 75 % on our medium. Further differences can be seen between some of the mutants by direct visual observation of cells grown in drug-free medium. col, is always completely non-flagellate, with cells apparently blocked in the last stage of division. Two, four, eight or Exp Cd

Res 111 (1978)

40

Warr, Flanagan

and Quinn

: I ,

: : : \

2.0

4.0

6,O

Fig. 1. Abscissa: caffeine cont. (mM); ordinate: viability, expressed as proportion of cells plated which produce colonies and then converted to a percentage of viability on unsupplemented medium. O-O, Wild type; GO, COI& C!AI, ~01,; A---A, co&r; A---A, vin;. Cross resistance and sensitivity of mutants to caffeine.

more nuclei are often seen in apparently separated cells which are still tightly held together in the palmella envelope. Although cleavage furrows are often visible between these groups of cells, the cells do seem more tightly pressed against each other than cells of a wild type strain ready to be released at the end of a normal growth cycle. Some abnormal shaped cells are also seen. After prolonged incubation in liquid medium, some of the cells contain grey vacuolated regions and dead cells are also present. col;, col, and vin; are also distinguishable from wild type. In these cases, the cells are flagellated but are larger and more rounded than wild type when grown in drug free medium. For example, the mean length of vin; is 8.5 pm as opposed to 6.6 pm for wild type and the mean breadth is 7.5 pm as opposed to 5.0 pm. Cross resistance to apparently unrelated drugs One approach to establishing the mechanism by which resistance develops, which Exp Cell Res III

(1978)

has been employed in other systems [4], is to study patterns of cross resistance to unrelated drugs. If a mutation confers resistance not only to the drug used initially for mutant selection but also to functionally unrelated drugs, it suggests that resistance is more likely to arise by a fairly generalized membrane defect than by a more specific change at the site of action of the original drug. We therefore tested our mutants for cross resistance to streptomycin, benzimidazole, nalidixic acid, puromycin and caffeine. No cross resistance of any of the mutants was detected towards streptomycin, benzimidazole or nalidixic acid. Several of the mutants were slightly cross resistant to puromycin (table 1). Streak tests suggested that some of the mutants may be hypersensitive to caffeine and this unexpected observation was examined more carefully by plating at low cell densities and subsequently making colony counts on various concentrations of caffeine. At least two of the mutants, col, and

Table 2. [3H]Colchicine uptake into mutant strains of Chlamydomonas expressed as a percentage of uptake into wild type Each figure is a mean of three determinations and significance testing is by a t-test on the mean uptake into wild type compared with mean uptake into mutant

Strain and experiment number vinl expt expt col, expt expt C% expt

Uptake of rH] colchicine as % of uptake into wild type

Signiticance of difference between mean uptake of replicates into mutant compared with wild type

1 2

77.1 53.0

Significant at 5 % probability level

1 2

61.7 72.9

Not significant

1

102.9

Not significant

Colchicine

and vinblastine

41

around 5.0% of the radioactivity was present as an impurity with an Rf value close to 1.0 as opposed to the colchicine standard and the bulk of the hot colchicine which had an Rf value of 0.76. However, incubation of wild-type cells with this impurity under conditions used for the other uptake experiments gave no detectable uptake of radioactivity into cells, whilst incubation with the repurified colchicine gave normal uptake. In another control experiment, preincubation of colchicine containing medium for 48 h prior to the addition of cells did not raise the level of radioactive uptake, which would be predicted if the experiments were simply observing the uptake of light-induced breakdown products of colchicine, such as lumicolchicines. The concentration of radioactive colchicine used for these uptake experiments I.5 I.0 20 is around 1110000 the concentration of cold Fig. 2. Abscissa: colchicine cont. hM1: ordinate: viability expressed as a percentage‘of liability on uncolchicine used for selecting and scoring supplemented medium. O-O, Wild tvoe: 0-O. -. the growth of the resistant mutants. It’ is, coil; cl-o, cos,-. Sensitivity of colchicine-sensitive mutants to colhowever, not possible to carry out uptake chicine. experiments in the presence of concentrations of cold colchicine comparable to those used for selection of mutants, since under vin;, are hypersensitive to caffeine and one these conditions, uptake of label becomes other (~0112)appears slightly so. In contrast, indistinguishable from the background. In col, is cross resistant to the drug (fig. 1). an attempt to predict the uptake of colchicine into cells at the much higher concentrations used for mutant selection, we Uptake of [3H]colchicine by measured uptake into cells which had been resistant mutants incubated in a range of concentrations of The study of the uptake of labelled col- radioactive colchicine between 0.02 PM chicine should provide a more direct experi- and 0.34 PM and then estimated the uptake mental approach to the question of whether into cells which would be predicted at much our mutants are resistant due to a perme- higher external concentrations by extraability defect. Colchicine uptake into Chla- polation. This makes the assumption, which mydomonas is very slow and incubation for we are unable to test, that the relationship a period of around 48 h has to be carried out between uptake and external concentration to get sufficient drug into the cells for count- continues to be linear over the very wide ing. Purification of the hot colchicine by concentration range of 0.34 PM to 4.0 mM. thin layer chromatography indicated that Extrapolation predicts that the uptake of Exp Cell

Res II 1 (1978)

42

Warr, Flanagan

Table 3. Analysis

and Quinn

of progeny from pairwise

PD, parental ditype; NPD, non-parental scribed in Levine [21]

Cross

col, cog x co1:col; col, colt x COIBcol,

crosses

ditype; T, tetratype. Calculation

Res 111 (1978)

of recombination

No. of different types of tetrad

mutants frequency as de-

No. of zygotes dissected

No. of complete tetrads obtained

PD

NPD

T

Recomb. frequency

140 100

24 12

17 3

6 4

1 5

39.6 54.1

colchicine into cells growing in medium containing an external concentration of 4.0 mM would be sufftcient to give an internal concentration of 0.71 mM within the cells. Uptake of labelled colchicine after 48 h was indistinguishable from wild type in all of our resistant mutants except for vi&, which had a rate of uptake significantly less than wild type at the 5 % level of probability as judged by a t-test on the means of triplicate measurements in two experiments (table 2). col, also appeared to have a slightly reduced colchicine uptake during similar experiments but this was not significant by a t-test. col, produced very variable results during replicate experiments, on some occasions giving normal uptake and on others giving greatly increased uptake. This appeared to correlate with the presence of dead cells in some liquid cultures of this mutant and so we have not investigated the phenomenon further. It is immediately apparent that some of the mutants which have previously been found to be markedly cross-resistant to puromycin (table 1) appear to have quite normal uptake of labelled colchicine (col;, col,, ~01,). One hypothesis to resolve these two apparently conflicting results would be that our “uptake” experiments are not measuring the true uptake into the cell, but merely into the volume between the cell Exp Cell

of colchicine-resistant

wall and the cell membrane. If this were so, a mutant which is resistant due to a membrane change (which conferred cross resistance to puromycin) might still give quite normal “uptake” following incubation with labelled colchicine. To investigate this possibility we have compared the uptake of colchicine into a ‘cell wall less’ mutant of Chlamydomonas [ 1 l] with wild type. There was no detectable difference between the strains (table 2). Genetic analysis of resistant mutants

All the newly isolated mutants were backcrossed to wild type, except col, which being entirely non-flagellate, does not mate. All gave very poor zygote germination and incomplete tetrads from many of those zygotes which did germinate, as previously noted for mutants co&+, [6]. Several of the mutants were repeatedly back-crossed to wild type, selecting resistant progeny from complete tetrads each generation, in case the mutations had inadvertently been isolated in cells with chromosome abnormalities which were producing lethality in crosses. However, the viability of zygotes was in no way improved by this programme of back-crosses and which is consistent with the hypothesis that the poor viability is simply a pleiotropic effect of the original colchicine resistance mutation.

Colchicine Table 4. Viability of wild-type and mutant Chlamydomonas on 1.5 % and 0.8 % agar Viabilities expressed in terms of colonies produced following plating of loo0 cells on a total of 5 Petri dishes following haemocytometer counts

Strain Wild type c% cos; cos,

% viability on 1.5% agar (a)

%viability on 0.8% r

b/a

87 7 38 93

93 32 26 87

4.57 0.68 0.94

1.07

Analysis of those complete tetrads which were produced from crosses between individual mutants and wild type all yielded 2 : 2 ratios of resistant and wild-type progeny and hence were consistent with a single gene basis of the mutant defects. Pairwise crosses between mutants usually give even worse viability than crosses between an individual mutant and wild type. Mutant x mutant crosses typically give only around 30% viability of individual zygospores and around 10% complete tetrads (i.e. tetrads where all the four or eight spores produce viable clones). This makes genetic studies of these mutants by tetrad analysis extremely laborious and they have only been made in detail on two crosses (table 3). The data are consistent with free, or at least considerable, recombination between co& and colt and between co& and col,. Resistance levels from progeny from tetratype zygotes from both of these two crosses were studied in more detail. In both cases one-quarter (i.e. 2 out of 8) of the members of the tetrad had resistance similar to wild type, one quarter had resistance similar to each of the mutant strains which were used in the cross and one-quarter had resistance considerably greater than either

and vinblastine

43

of the mutant strains, being able to grow from streaks on 10 mM colchicine. This suggests that gene expression is additive in these pairwise combinations of mutations. Colchicine sensitive and dependent mutants Two colchicine sensitive mutants have been isolated by plating on drug-free medium and subsequently replica plating onto 1 mM colchicine as described in Materials and Methods. Viability platings (fig. 2) showed that one of the mutants, cos;, was considerably more sensitive to colchicine than the other, cos;. Both these strains, when grown in drug-free medium, contain cells which are more irregular in shape than wildtype cells. When crossed to wild type, both of these mutants segregated as single gene defects. During the course of this work, we have observed that a cell wall deficient mutant, CW,, isolated by Davies & Plaskitt [ll], does not grow when streaked on 1 mM colchicine, a drug concentration which permits vigorous growth of wild type. It therefore seemed possible that the coichicine sensitive mutants which were isolated here were cell wall deficient mutants similar to those studied by Davies & Plaskitt, and hence they were both examined in the electron microscope for the presence of a cell wall. An apparently normal cell wall was observed in both cases. However, Davies & Plaskitt report that a few cell wall deficient mutants (table 1, ref. [ll]) have apparently normal cell walls in the electron microscope and yet grow in a flat “amoeboid” colony characteristic of cell wall deficient mutants and also have a very poor viability on normal agar concentrations which can be improved by plating at reduced agar levels, which is another characteristic property of cell wall deficient mutants. We therefore Exp Cell Res 111 (1978)

44

Warr, Flanagan

and Quinn

compared the colony morphologies of the colchicine-sensitive mutants isolated here with wild type and with the cell wall deficient mutant CW, and observed that they were similar to wild type. These mutants also showed no tendency to be sensitive to normal agar concentrations (table 4) although we confirmed the observation by Davies & Plaskitt that cell wall mutant viability is improved by lowering the agar concentration. Both colchicine-sensitive mutants showed normal uptake of labelled colchicine. However, one of them, cos;, is hypersensitive to puromycin; it does not grow when streaked on 0.2 mM puromycin and only gives poor growth on 0.1 mM puromycin, whereas cos, and CW, give growth which is indistinguishable from wild type on both of these concentrations. This presents similar problems of interpretation as those already encountered with colchicine-resistant mutants which are crossresistant to puromycin and yet which appear to have normal colchicine uptake. We have also isolated one colchicinedependent mutant by replica plating from 1 mM colchicine onto drug-free medium after UV mutagenesis. Colchicine concentration below 0.75 mM or above 1.5 mM did not support its growth. However, even on 1.0 mM colchicine some cell death did occur. Inclusion of acetate in the medium and incubation in the dark did improve growth somewhat, but after a period of 3 months’ growth this mutant became progressively less viable and was lost.

DISCUSSION The principal aim of this work has been to understand the nature of the change which confers colchicine resistance in mutants of Exp Cell Res 111 (1978)

We have extended our earlier finding [6] that the uptake of labelled colchicine into mutants col; to col; inclusive is similar to the uptake into wild type by demonstrating that the same situation applies for ten other independently isolated colchicine or vinblastine resistant mutants. Two of the mutants, vin, and col,, appear to have slightly reduced levels of uptake of the drug, but this is not statistically significant in the case of col,. Two colchicine-sensitive mutants appear to have normal uptake. However, the observation that some of the mutants have altered levels of resistance to the apparently unrelated drugs caffeine and puromycin may be taken to suggest a generalised permeability defect in these mutants. This interpretation is contrary to the conclusion from the uptake experiments referred to in the previous paragraph and an explanation for the discrepancy should be sought. It is conceivable, although in our view unlikely, that caffeine is functionally related to colchicine since it is well established in other systems that caffeine inhibits phosphodiesterase and hence may regulate CAMP levels [12]. Evidence has been reported for the existence of CAMP in Chlamydomonas [ 131 and in other systems there is evidence for the involvement of CAMP in microtubule assembly [ 14, 151. Therefore it is just possible that caffeine may be exerting indirect effects on microtubule assembly in Chlamydomonas. However, it does not seem conceivable that puromycin has effects related to colchicine and hence the argument in favour of membrane changes can be based on the puromycin cross resistance data alone. It is necessary to use very high colchicine concentrations to select and study mutants of Chlamydomonas with altered resistance levels. and such colchicine concentrations Chlamydomonas.

Colchicine (of around 24 mM, cf table 1) are far higher than those used in our radioactive colchicine uptake experiments. (We have been unable to do these experiments in the presence of mM concentrations of cold colchicine, since the uptake of radioactive colchicine becomes very slight when diluted with these amounts of the cold drug.) Possibly at the very high concentrations of colchicine which we have used for the selection of drug resistant strains, there may be growth inhibition due to non-specific interaction with membrane proteins. Nonspecific binding to mammalian proteins has been reported by others at high colchicine concentrations [ 161. It is possible that our resistant mutants could have an altered membrane structure which reduces such non-specific interaction at high colchicine concentrations and also alters the permeability to puromycin and to caffeine, but which does not influence the uptake of colchicine at the low colchicine levels used in the radioactive uptake experiments. Such non-specific membrane effects should be distinguishable from microtubule description on the basis of the action of lumicolchicines, the structural isomers of colchicine which do not disrupt microtubules but which are potent inhibitors of membrane function [17]. We therefore hope to investigate the cross resistance of our mutants to lumicolchicines. Sato [18] has recently described an interesting mutant of Chlamydomonas which has a temperature sensitive defect in growth and is also resistant to colchicine during growth and flagellar regeneration. Sato points out that one possible explanation for the nature of the phenotype of his mutant is that it contains a defect in tubulin. Although this hypothesis is certainly very reasonable, we should at least point out that this data does not eliminate an alternative hypo-

and vinblastine

45

thesis, namely that the mutation causes a change in a membrane component, which reduces the uptake of colchicine (thus conferring resistance during growth and flagellar regeneration) and simultaneously destabilises the membrane (thus conferring temperature sensitivity upon the mutant). The use of colchicine binding studies to directly investigate the possibility of tubulin changes in resistant mutants is made very difficult by the very low level of colchicine binding activity in Chlamydomonas as in other plant material. Bums reported that no colchicine binding activity could be detected in post-ribosomal supematants from Chlamydomonas reinhardii [ 193. Very low levels of colchicine binding have recently been detected in Chlamydomonas cell extracts [20] and we are currently comparing the colchicine binding activity from wildtype and mutant strains of Chlamydomonas. We gratefully acknowledge financial assistance from The Science Research Council for part of this work. We also thank Mrs Monica Adams for isolating the three vinblastine-resistant mutants described here and Mr L. Fish for carrying out electron microscopy on the colchicine-sensitive mutants.

REFERENCES 1. Lederberg, S & Stetten, G, Science 168 (1970) 485. Adams, M & Warr. J R, EXD cell res 71(1972) 473. : : Flavin,.M & Slaughter; C, J bact 118 (1974) 59. 4. Beth-Hansen. N T. Till, J E & Lina.-. V, J cell physio188 (1976) 23: 5. Minor, P D & Roscoe, D H, J cell sci 17 (1975) 381. 6. Warr, J R & Gibbons, D, Exp cell res 85 (1974) 117. 7. Till, J E, Baker, R M, Brunette, D M, Ling, V, Thompson, L H &Wright, J A, Fed proc 32 (1973) 29. 8. Sager, R & Granick, S, J gen physio137 (1954) 729. 9. Mahin, D T & Lofberg, R T, Anal biochem 16 (1966) 500. 10. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193 (1951) 265. 11. Davies, D R & Plaskitt, A, Genet res 17 (1971) 33. 12. Wolfe, J, J cell physio182 (1973) 39. 13. Rubin, R W & Filner, P, J cell bio156 (1973) 628. Exp Cell

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14. Kram, R & Tomkins, G M, Proc natl acad sci US 70 (1973) 1659. 15. Puck, T T, Waldren, C A & Hsie, A W, Proc natl acad xi US 69 (1972) 1943. 16. Nunez, J, Fellows, A, Francon, J & Lennon, A M, Microtubules and microtubule inhibitors (ed M Borgers & M de Brabander) p. 269. NorthHolland, Amsterdam (1975). 17. Wilson, L, Bamburg, J R, Mizel, S B, Grisham, L M & Creswell, K M, Fed proc 33 (1974) 158.

Exp Ccl/ Res Ill

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18. 19. 20. 21.

Sato, C, Exp cell res 101 (1976) 251. Bums, R G, Exp cell res 81 (1973) 285. Flanagan, D & Warr, J R, FEBS lett 80 (1977) 14. Levine, R P, Genetics. Holt Rinehart & Winston, New York (1%2).

Received January 13, 1977 Revised version received August 23, 1977 Accepted August 3 1, 1977