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Colchicine-resistant mutants of Chlamydomonas reinhardi
Colchicine-resistant mutants
413
Stillmark, I-K Inaug .diss, Domat (1888) (as and commenced to study colchicine-resistant referenced in Makehi [12]). mutants of Chlamydomonas reinhardi. 26. Nicolson, G L & Blaustein, J, Biochim biophys
25.
acta. In press, Received November 15, 1971.
Colchicine-resistant mutants of Chlamydomonas reinhardi MONICA
ADAMS
and J. R. WARR, Department
of Biology, University of York, Heslington, York, UK Summary Five colchicine-resistant mutant strains of Chlamydomonas reinhardi have been isolated. In colchicine-free medium they all have abnormally long cell doubling times and tend to occur within palmella envelopes, rather than as free-swimming cells. Zygote germination of all the mutants is abnormal, but crosses with wild type suggest that resistance is in each case due to a Mendelian mutation. It is suggested, though not proved, that the mutations may affect microtubular structures.
Chlamydomonas reinhardi is convenient for a study of the genetic control of cell division since it is one of the few eucaryotic species with an extensively described cell division [3] which has also been widely studied by the techniques of microbial genetics [4]. A single gene mutation has previously been described in this species which causes asynchrony between nuclear and cell division [7,8]. In order to extend this work, it is necessaryto isolate mutants with other kinds of defects in the processes of cell division. One approach to this problem is to select mutants which are resistant to specific inhibitors of cell division in the hope that such mutant strains will have changes at the site of action of the inhibitor. For example, since it is well known that colchicine disrupts the mitotic spindle and other microtubular structures, some mutations altering such structures might be recognised in the first instance on the basis of their colchicine resistance. With these considerations in mind, we have now isolated
Materials and Methods Wild type strains of Chlamydomonas reinhardi of both mating tvoes were obtained from the Cambridge Colle&on’of Algae. Culture conditions and media have been described in 181.Colchicine 03.D.H. Laboratories, Poole, Do&j was dissolved in liquid medium and sterilised by filtration. Nitrosoguanidine mutagenesis was carried out as follows. Late logarithmic phase cultures of wild type in liquid medium were harvested by low speed centrifugation and resuspended in an equal volume of 0.0067 M pH 6.0 phosphate buffer. One ml of cell suspension was added to a final concentration of 0.005 % w/v Nmethyl-N-nitroso-N’nitroguanidine (Loch-Light Laboratories, Colnbrook, Mdx, UK) in a total volume of 10 ml phosphate buffer. The cells were incubated for 15 min then washed with 5 ml of ‘phosphate buffer and resuspended in a further 5 ml of the same buffer. They were then plated at 0.2 ml per dish on colchicine-containing medium. Control experiments had indicated that this treatment resulted in about 2 % survival. Growth curves were determined in 100 ml cultures grown on a rotary shaker revolving at 120 rpm. For protein determinations flasks were removed-in triplicate, harvested and treated as described in 181.The crossing technique and methods for maturation of zygotes were as described in [2].
Results and Discussion
of wild type Chlamydomonas reinhardi, when plated at high densities or streaked on solid medium was completely inhibited by 5 x 1O-3 M colchicine. Mutant isolation attempts were therefore made by first subjecting wild type mating type minus cultures to nitrosoguanidine mutagenesis and then plating at approx. 5 x lo5 total cells (lo4 viable cells) per plate on 10 plates of 6 x 1O-3 M and 10 plates of 5 X 1O-9 M colchicine. A total of five colonies were observed and subcultured from theseplates. The clones established from two colonies on 6 x 1O-3 M colchicine were designated col. 1 and col. 2 and the clones established from three colonies on 5 x 1O-3 M colchicine were designated col. 3, col. 4 and col. 5. Each of the five clones was repurified twice by plating at low cell Preliminary
tests indicated
that
growth
Exptl Cell Res 71
4’74 Monica Adams & J. R. Warr
Fig. I. Colchicine resistance of wild type and five mutants tested by streaking on solid medium. Small streaks of each of the mutants and of wild type have been placed on a plate containing 5 x lo-* M colchicine and incubated for 7 days. The faint patches at the wild type position are the original cells placed on the plate which have grown no further.
density on 6 x 1O-3M colchicine and restarting the culture from a single colony. Resistance levels of the mutants were then compared with wild type by plating at low cell densities on colchicine-containing medium. Percentage viabilities were then calculated from the numbers of colonies observed on colchicine-containing medium compared with numbers of colonies on unsupplemented medium. Wild type mating type minus showed a viability of nil on 4 x 1O-3M and 6 x 1O-3 Table 1. Cell and protein doubling times of colchicine-resistant mutants and wild type during logarithmic growth phase Strain
Cell doubling time (hours)
Protein doubling time (hours)
Wild type Cal. 1 Cal. 2 Cal. 3 Cal. 4 Cal. 5
174.: 13:90 15.46 17.17 14.58
8.49 17.78 17.22 15.17 14.93 15.89
Exptl Cell Res 71
M colchicine, whereas all of the mutants showed close to 100% viabilities on both these concentrations except col. 4, which showed 76 % on 4 x 1O-3 M but only 1.3 % on 6 X 1O-3 M. The mutants can also be distinguished from wild type by placing small streaks or patches of cells directly with a wire loop onto plates containing 5 x 1O-3M colchicine (fig. 1). In addition to their colchicine resistance, the mutants differ from wild type in two ways when grown in colchicine-free medium. Firstly, their cell and protein doubling times during the logarithmic phase of growth are about twice as long as wild type (table 1). This slow growth rate is also seen on solid medium, where the mutants form much smaller colonies than wild type. Secondly, logarithmic phase cultures can be seen to contain a very high proportion of cells held within palmella envelopes, either singly or in small groups. In wild type cultures such palmella clumps are seenonly as a temporary
Effects of liquid water phase transition on the growth of L929 cells
division stageprior to the liberation of daughter cells. Less than 5 % of wild type cells are normally seenin this form. In order to establish the genetic basis of the colchicine-resistantphenotype,wehavecrossed all the mutants to wild type. Germination of zygotes from these crosses is unusually rapid (usually occurring within 12 h instead of the normal period of 14 to 20 h.) The cells liberated are often unusually small and are frequently inviable. However, when the progeny clones from those zygotes giving all viable zoospores are classified for colchicine resistanceby making small streaks on 5 x 1O-3 M colchicine they consistently show a Mendelian segregation of resistanceversus sensitivity for all five mutants. Slow growth rate segregates with colchicine resistance, as would be expected if the two traits were pleiotropic effects of a single mutation in each strain. It is clearly of interest to us to establish the mechanism by which these mutations confer colchicine resistance.Sincethe mutants show abnormalities of cell growth in the absence of colchicine, we feel that it is unlikely that resistance is due to a simple permeability defect preventing the uptake of the substance but more probably due to a change at the site of action of colchicine. Naturally we speculatethat this change may be affecting some aspect of microtubule structure and we hope to test this by electron microscopy, by comparison of colchicine binding of cell proteins [l] and by investigating the effect of colchicine on flagella.regeneration [5, 61 in wild type and mutant strains. We gratefully acknowledge financial support from the Science Research Council for this work. References 1. Borisy, G C & Taylor, E W, J cell biol 34 (1967) CIC JL.J. 2. Ebersold, W T & Levine, R P, Z Vererblehre 90 (1959) 74. 3. Johnson, U G & Porter, K R, J cell biol 38 (1968) 403.
475
4. Levine, R P & Ebersold, W T, Ann rev microbial 14 (1960) 197. 5. Randall, J T, Cavalier-Smith, T, McVittie, A, Warr, J R & Hopkins, J M, Dev biol, suppl. 1 (Control mechanisms in developmental processes) (1967) 43. 6. Rosenbaum, J L, Moulder, J E & Ringo, D L, J cell biol41 (1969) 600. 7. Warr, J R, J gen microbial 52 (1968) 243. 8. Warr, J R & Durber, S, Exptl cell res 64 (1971) 463. Received December 21, 1971
Effects of liquid water phasetransition on the growth of L929 cells L. C. LAU, Departments of Biochemistry and Botany, University of Toronto, Toronto, Ont., Canada
Summary Alteration in the energetics of growth for L929 cells has been demonstrated at approx. 30.2”C which coincides with the temperature of phase transition for liquid water. Possible modification of solvation spheres and diffusion movement of all molecular species due to changes in the conformation of water are proposed to account for the six-fold change in the energy of activation.
Sufficient evidence has now accumulated indicating that liquid water undergoes higher order phase transition at specific temperatures namely 15”, 30”, 45” and 60°C (to within k2”C) [l-5]. Water is the material by which all molecular species are solvated and the medium in which all chemical reactions take place. By physical consideration alone, any changes in its structural conformation would correspondingly bring about a modification of the state of solvation to all molecules as well as an alteration of the conditions of reaction in the medium. Hence a change of energetics, or biological activities, could be expected together with the occurrence of a phase transformation in liquid water [6]. The present study is an attempt to detect the effect of one such change in a system of mammalian origin. Exptl Cell Res 71
413
Stillmark, I-K Inaug .diss, Domat (1888) (as and commenced to study colchicine-resistant referenced in Makehi [12]). mutants of Chlamydomonas reinhardi. 26. Nicolson, G L & Blaustein, J, Biochim biophys
25.
acta. In press, Received November 15, 1971.
Colchicine-resistant mutants of Chlamydomonas reinhardi MONICA
ADAMS
and J. R. WARR, Department
of Biology, University of York, Heslington, York, UK Summary Five colchicine-resistant mutant strains of Chlamydomonas reinhardi have been isolated. In colchicine-free medium they all have abnormally long cell doubling times and tend to occur within palmella envelopes, rather than as free-swimming cells. Zygote germination of all the mutants is abnormal, but crosses with wild type suggest that resistance is in each case due to a Mendelian mutation. It is suggested, though not proved, that the mutations may affect microtubular structures.
Chlamydomonas reinhardi is convenient for a study of the genetic control of cell division since it is one of the few eucaryotic species with an extensively described cell division [3] which has also been widely studied by the techniques of microbial genetics [4]. A single gene mutation has previously been described in this species which causes asynchrony between nuclear and cell division [7,8]. In order to extend this work, it is necessaryto isolate mutants with other kinds of defects in the processes of cell division. One approach to this problem is to select mutants which are resistant to specific inhibitors of cell division in the hope that such mutant strains will have changes at the site of action of the inhibitor. For example, since it is well known that colchicine disrupts the mitotic spindle and other microtubular structures, some mutations altering such structures might be recognised in the first instance on the basis of their colchicine resistance. With these considerations in mind, we have now isolated
Materials and Methods Wild type strains of Chlamydomonas reinhardi of both mating tvoes were obtained from the Cambridge Colle&on’of Algae. Culture conditions and media have been described in 181.Colchicine 03.D.H. Laboratories, Poole, Do&j was dissolved in liquid medium and sterilised by filtration. Nitrosoguanidine mutagenesis was carried out as follows. Late logarithmic phase cultures of wild type in liquid medium were harvested by low speed centrifugation and resuspended in an equal volume of 0.0067 M pH 6.0 phosphate buffer. One ml of cell suspension was added to a final concentration of 0.005 % w/v Nmethyl-N-nitroso-N’nitroguanidine (Loch-Light Laboratories, Colnbrook, Mdx, UK) in a total volume of 10 ml phosphate buffer. The cells were incubated for 15 min then washed with 5 ml of ‘phosphate buffer and resuspended in a further 5 ml of the same buffer. They were then plated at 0.2 ml per dish on colchicine-containing medium. Control experiments had indicated that this treatment resulted in about 2 % survival. Growth curves were determined in 100 ml cultures grown on a rotary shaker revolving at 120 rpm. For protein determinations flasks were removed-in triplicate, harvested and treated as described in 181.The crossing technique and methods for maturation of zygotes were as described in [2].
Results and Discussion
of wild type Chlamydomonas reinhardi, when plated at high densities or streaked on solid medium was completely inhibited by 5 x 1O-3 M colchicine. Mutant isolation attempts were therefore made by first subjecting wild type mating type minus cultures to nitrosoguanidine mutagenesis and then plating at approx. 5 x lo5 total cells (lo4 viable cells) per plate on 10 plates of 6 x 1O-3 M and 10 plates of 5 X 1O-9 M colchicine. A total of five colonies were observed and subcultured from theseplates. The clones established from two colonies on 6 x 1O-3 M colchicine were designated col. 1 and col. 2 and the clones established from three colonies on 5 x 1O-3 M colchicine were designated col. 3, col. 4 and col. 5. Each of the five clones was repurified twice by plating at low cell Preliminary
tests indicated
that
growth
Exptl Cell Res 71
4’74 Monica Adams & J. R. Warr
Fig. I. Colchicine resistance of wild type and five mutants tested by streaking on solid medium. Small streaks of each of the mutants and of wild type have been placed on a plate containing 5 x lo-* M colchicine and incubated for 7 days. The faint patches at the wild type position are the original cells placed on the plate which have grown no further.
density on 6 x 1O-3M colchicine and restarting the culture from a single colony. Resistance levels of the mutants were then compared with wild type by plating at low cell densities on colchicine-containing medium. Percentage viabilities were then calculated from the numbers of colonies observed on colchicine-containing medium compared with numbers of colonies on unsupplemented medium. Wild type mating type minus showed a viability of nil on 4 x 1O-3M and 6 x 1O-3 Table 1. Cell and protein doubling times of colchicine-resistant mutants and wild type during logarithmic growth phase Strain
Cell doubling time (hours)
Protein doubling time (hours)
Wild type Cal. 1 Cal. 2 Cal. 3 Cal. 4 Cal. 5
174.: 13:90 15.46 17.17 14.58
8.49 17.78 17.22 15.17 14.93 15.89
Exptl Cell Res 71
M colchicine, whereas all of the mutants showed close to 100% viabilities on both these concentrations except col. 4, which showed 76 % on 4 x 1O-3 M but only 1.3 % on 6 X 1O-3 M. The mutants can also be distinguished from wild type by placing small streaks or patches of cells directly with a wire loop onto plates containing 5 x 1O-3M colchicine (fig. 1). In addition to their colchicine resistance, the mutants differ from wild type in two ways when grown in colchicine-free medium. Firstly, their cell and protein doubling times during the logarithmic phase of growth are about twice as long as wild type (table 1). This slow growth rate is also seen on solid medium, where the mutants form much smaller colonies than wild type. Secondly, logarithmic phase cultures can be seen to contain a very high proportion of cells held within palmella envelopes, either singly or in small groups. In wild type cultures such palmella clumps are seenonly as a temporary
Effects of liquid water phase transition on the growth of L929 cells
division stageprior to the liberation of daughter cells. Less than 5 % of wild type cells are normally seenin this form. In order to establish the genetic basis of the colchicine-resistantphenotype,wehavecrossed all the mutants to wild type. Germination of zygotes from these crosses is unusually rapid (usually occurring within 12 h instead of the normal period of 14 to 20 h.) The cells liberated are often unusually small and are frequently inviable. However, when the progeny clones from those zygotes giving all viable zoospores are classified for colchicine resistanceby making small streaks on 5 x 1O-3 M colchicine they consistently show a Mendelian segregation of resistanceversus sensitivity for all five mutants. Slow growth rate segregates with colchicine resistance, as would be expected if the two traits were pleiotropic effects of a single mutation in each strain. It is clearly of interest to us to establish the mechanism by which these mutations confer colchicine resistance.Sincethe mutants show abnormalities of cell growth in the absence of colchicine, we feel that it is unlikely that resistance is due to a simple permeability defect preventing the uptake of the substance but more probably due to a change at the site of action of colchicine. Naturally we speculatethat this change may be affecting some aspect of microtubule structure and we hope to test this by electron microscopy, by comparison of colchicine binding of cell proteins [l] and by investigating the effect of colchicine on flagella.regeneration [5, 61 in wild type and mutant strains. We gratefully acknowledge financial support from the Science Research Council for this work. References 1. Borisy, G C & Taylor, E W, J cell biol 34 (1967) CIC JL.J. 2. Ebersold, W T & Levine, R P, Z Vererblehre 90 (1959) 74. 3. Johnson, U G & Porter, K R, J cell biol 38 (1968) 403.
475
4. Levine, R P & Ebersold, W T, Ann rev microbial 14 (1960) 197. 5. Randall, J T, Cavalier-Smith, T, McVittie, A, Warr, J R & Hopkins, J M, Dev biol, suppl. 1 (Control mechanisms in developmental processes) (1967) 43. 6. Rosenbaum, J L, Moulder, J E & Ringo, D L, J cell biol41 (1969) 600. 7. Warr, J R, J gen microbial 52 (1968) 243. 8. Warr, J R & Durber, S, Exptl cell res 64 (1971) 463. Received December 21, 1971
Effects of liquid water phasetransition on the growth of L929 cells L. C. LAU, Departments of Biochemistry and Botany, University of Toronto, Toronto, Ont., Canada
Summary Alteration in the energetics of growth for L929 cells has been demonstrated at approx. 30.2”C which coincides with the temperature of phase transition for liquid water. Possible modification of solvation spheres and diffusion movement of all molecular species due to changes in the conformation of water are proposed to account for the six-fold change in the energy of activation.
Sufficient evidence has now accumulated indicating that liquid water undergoes higher order phase transition at specific temperatures namely 15”, 30”, 45” and 60°C (to within k2”C) [l-5]. Water is the material by which all molecular species are solvated and the medium in which all chemical reactions take place. By physical consideration alone, any changes in its structural conformation would correspondingly bring about a modification of the state of solvation to all molecules as well as an alteration of the conditions of reaction in the medium. Hence a change of energetics, or biological activities, could be expected together with the occurrence of a phase transformation in liquid water [6]. The present study is an attempt to detect the effect of one such change in a system of mammalian origin. Exptl Cell Res 71