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In order to isolate acid Phosphatase mutants in the green alga Chlamy- domonas reinhardi, a staining method for detecting the enzyme activity in colonies has ...




Laboratory of Molecular Genetics, Department of Botany, Uniuersity of, Sart Tilman, Liige, Belgium Manuscript received July 5, 1973 ABSTRACT

In order to isolate acid Phosphatase mutants in the green alga Chlamydomonas reinhardi, a staining method for detecting the enzyme activity in colonies has been developed. The occurrence of more than one acid phosphatase brought about some difficulty in the selection of mutants. W e have, however, found an original method of selection based on the differential heat sensitivity of the enzymes. After treatment of the wild-type strain with N-methy1-N'nitro-N-nitrosoguanidine, two types of mutants were recovered, then analyzed by biochemical and electrophoretic methods. In the first class of mutants ( P I , P,, P , . . .) a heat-stable acid phosphatase bound to cellular debris of the crude extract was missing. The mutant Pa, representing the second class of mutations, was lacking a soluble heat-sensitive enzyme. These mutations were genetically different and exhibited mendelian inheritance.

ERY little work has been devoted to the genetic control of protein synthesis vand function in the green alga Chlamydomonas reinhardi. This gap probably results from the fact that very few genes of this organism satisfy the two primary requisites to gene action studies-the availability of mutations in this gene and the possibility of studying the protein coded by this gene. The scarcity of amino acid-requiring mutants (only arginine auxotrophs have been found by now in this organism) for example, constitutes a serious handicap in the study of the genetic control of enzymes involved in the metabolism of amino acids in Chlamydomonas. We were interested in finding a convenient system for the study of geneenzyme relationships in Chlamydomonas. In this respect, we have been looking for mutants impaired in acid phosphatase activity. In various organisms, as yeast (SCHURR and YAGIL1971) and fungi (DORN 1965), the activity of acid phosphatases may be measured rapidly in extracts but also, very simply, in cell suspensions. Furthermore, a very accurate study of these enzymes may be done thanks to the methods of specific staining for acid phosphatase activity after separation of the enzymes by gel electrophoresis. * Chercheur qualifid du Fonds National Belge de la Recherche Scientifique Genetics 7 5 : 591-604 December. 1973



In this paper we describe an original method which allowed for isolating twa types of mutants defective for acid phosphatase. These mutants will be characterized by biochemical, electrophoretic and genetic analyses. MATERIALS A N D METHODS

Strain: The wild-type (WT) strain 137 C, mating type plus or minus, received from R. P. LEVINE(Harvard University), has been used throughout this study. Medium: The algae were routinely grown in minimal medium containing 6.75 mMole phosphate per liter (SURZYCKI 1971). For solid media, 15 g/l Difco agar were added. Mutagenesis: WT cells from a late log phase culture in liquid minimal medium were treated with 50 ,pg/ml N-methyl-N-Nitro-N'-Nitrosoguanidine(MNNG) for 30 minutes, 25", as described earlier (LOPPES1970). Cells were plated on minimal medium and grown in the light for one week. Colonies were assayed for acid phosphatase activity as hereafter described. Genetic analysis: Crosses, maturation of zygotes and genetic analysis were carried out according to the methods of LEVINEand EBERSOLD (1958). Assays: Cultures were grown in Erlenmeyer flasks aerated with sterile air at a light intensity of 5000 lux, at 25". Cells were harvested by centrifugation, washed and resuspended in distilled water. The assays were carried out either in suspensions of whole cells, o r in crude extracts obtained after disruption of the cells with ultrasound (MSE Ultrasonic Disintegrator). In some cases, extracts were prepared in 0.1 M acetate buffer, pH 4.8. Acid phosphatase: As stated in other systems, a-naphthylphosphate constitutes a good substrate for acid phosphatases. The a-naphthol which is released is coupled to a diazonium salt, giving a red complex insoluble in water, but soluble in sodium dodecylsulfate (SDS). The assay was carried out according to a method described by MACINTYRE (1971) and modified as follows: 1.1 ml 0.05 M acetate buffer (pH 4.8, optimal pH) and 0.2 ml 2 mg/ml a-naphthylphosphate in acetate buffer were added to 0.2 ml of cell suspension o r extract. The mixture was incubated a t 37" f o r 30 minutes after which 1 ml of the post coupling solution (4% SDS and 0.2% tetrazotizedo-dianisidine in 0.2M acetate buffer, pH 4.8) was added. The extinction at 541) n m was read against a blank in which the substrate was added after incubation. The standard curve prepared with a-naphthol was found to be linear up to 6 pg naphthol in the assay; over this range, 1 pg naphthol corresponds to 0.16 optical density units a t 540 nm (CE 202 Cecil Spectrophotometer, 1 cm light path). Throughout this work, the naphthylphosphate method has been generally preferred to the commonly used nitrophenylphosphate method. This choice resulted from the fact that we made use of the same substrate, a-naphthylphosphate, for assaying acid phosphatases in colonies and in polyacrylamide gels after electrophoresis. Proteim Protein was determined by the method of LOWRY d al. (1951), using crystalline egg white lysozyme as a standard. Determination of acid phosphatase in colonies: Acid phosphatase activity in yeast colonies may be easily detected by spraying plates with a mixture of a-naphthylphosphate and tetrazotizedo-dianisidine. This method was unsuccessful in Chlamydomonas, mainly because sprayed colonies immediately flow over the whole surface of agar. The following method overcame this difficulty and had further advantages which will be discussed later in this paper. Six- to seven-day-old colonies (about 2 mm diameter) were replica-plated onto sterile filter paper (Macherey-Nagel and Co., type M N 615). About 90% of the cells forming the colony are transferred in this way on the paper. The plates were put back in the light and after two days, the colonies were again visible. The papers were allowed to dry at room temperature and the colonies became firmly fixed in the paper fibres. This fixation is almost irreversible and is not affected, for example, by prolonged soakings in acetate buffer. The papers were immersed in a solution containing 1 mg/ml a-naphthylphosphate and 2 mg/ml tetrazotized-o-dianisidine in 0.05 M acetate buffer at pH 4.8, placed on wet paper and incubated for 30 minutes at 37". Deep



brown red coloration of the colonies indicated acid phosphatase activity (Figure 5 ) . The assay remained positive on papers kept dried f o r several hours a t 25" o r for several days at -15". Electrophoresis: Cell extracts were prepared in distilled water or 0.1 M acetate buffer p H 4.8. They were centrifuged at 27,000 x g f o r 20 min and the supernatants, containing 15-20 mg protein/ml for water extracts or 3-4 mg protein/ml for acetate buffer extracts, were used for electrophoresis (10-15 pl of extract per assay). The acid phosphatase of WT and mutants were separated on polyacrylamide gel using the Beckman microzone apparatus. p-alanine (31.2 g/l)-glacial acetic acid (8 ml/l) (pH 4.5) was 1962). The gel buffer, modified from used as tray buffer (REISFELD,LEWIS and WILLIAMS REISFELD, LEWISand WILLIAMS (1962), was prepared as follows: acetic acid (glacial) 17.2 ml; TMED 8 ml; KOH 201% to make pH 4.8; water to make 100 ml. I t was diluted 8-fold before use. Gel wag prepared with 7.1 % acrylamide (5% methylene-bis-acrylamide cross-linking agent in 95% acrylamide monomer) dissolved in gel buffer. Ammonium persulfate was added as gelling accelerator (1.35 mg per ml of gel). Electrophoresis was carried aut by applying a current of 50-60 mA (160 V) for 4-5 hr, with migration toward cathode (migration indicator: basic fuchsin). After electrophoresis, the gel was stained for acid phosphatases in a solution of 0.05 M acetate buffer p H 4.8 containing 1 mg/ml Na l-naphthylphosphate and 1 mg/ml Fast Garnet GBC (o-amino azotoluene) as a diazonium salt. Staining was generally performed overnight at room temperature. Stained gels were stored in 5% acetic acid at 4". RESULTS

1. Attempts at isolating mutants lacking acid phosphatase activity Wild-type (-) cells were treated with 50 ,pg/ml MNNG for 30 minutes and plated on NI medium. The survival was about 25 % in these conditions which are particularly favorable for inducing forward mutations in Chlamydomonas (LOPPES 1970). After 7 days, colonies were assayed for acid phosphatase activity, as described in MATERIAL AND METHODS. In three independent experiments, more than 11,000 colonies were analyzed, out of which no mutant was found. As a control of one of these experiments, part of MNNG-treated cells were plated o n a medium allowing the recovery of arginine-requiring mutants (LOPPES 1969). Three arg- mutants were isolated in this experiment among 3.100 colonies, which indicated that cells had been properly mutagenized. One of the most plausible hypotheses which might account for the lack of acid phosphatase mutants in these experiments is that the wild-type strain of Chlamydomonas produces more than one acid phosphatase species and, accordingly, that a t least two mutations are needed for full suppression of enzyme activity. The probability of simultaneous mutation at two (or more) sites being very rare indeed, one should not expect to isolate mutants in one step. 2. More than one acid phosphatase species in the wild-type strain That WT cells of Chlamydomonas reinhardi contain several different acid phosphatases is suggested by the following experiment. WT (+) cells grown in liquid minimal medium were washed with and resuspended in 0.05 M acetate buffer, pH 4.8. The suspension was exposed to 60"; samples were removed at various intervals of time and transferred into cold test tubes. The remaining acid phosphatase activity in whole cells was assayed at 37" using naphthyl phosphate as a substrate (see MATERIALS AND METHODS).



z LL

0 I-

t K




FIGURE 1.-Decrease of acid phosphatase activity in a suspension of wild-type cells in relation to the time of treatment at 60".

It was found in this experiment (Figure 1) that about 40% of the total acid phosphatases were rapidly inactivated, whereas the remaining 60% displayed a much greater resistance to heat treatment. This result strongly suggested that W T cells contained two main acid phosphatase species differing in their sensitivity to heat. This curve is very reminiscent of that described by BENNUNand BLUM (1966) for acid phosphatase C of Euglena; these authors state that "Because of the difference in sensitivity to heat of these two constitutive enzymes, it is possible to selectively destroy all the thermolabile component without appreciable loss of the thermostable compound". If, in Chlamydomonas, the structure of the two phosphatases is actually coded by two different genes, then the only way to detect and isolate mutants is to get a mutation in one gene and to destroy by other means the enzyme dependent on the other gene. From Figure 1, it is obvious that a mutation which would make enzyme B inactive should be easily detected if the cells are heated at 60" for 6 minutes, treatment which completely inactivates enzyme A. 3. Isolation of mutants Colonies from MNNG-treated W T cells were replicated on filter papers. These were first dried at room temperature to allow strong fixation of the cells into the fibres. They were then soaked in 500 mlO.05 M acetate buffer pH 4.8, maintained at 60". After 6 minutes, the papers were removed, rapidly dried, then immersed in the phosphatase reagent mixture and incubated as described in MATERIAL A N D METHODS. Twenty-one colonies failing to stain after exposure to heat were found among 4,670 colonies tested, whereas no mutant was detected in untreated con-



trol (5,620 colonies). The suspected mutants were isolated from the plates two days later and maintained on minimal medium for further studies. They were called P,, P,, P , and so on.

4.Acid phosphatases in the P mutants and in the WT strain I n order to know more about the differences existing between W T and P mutants, namely whether one particular enzyme was actually missing in the mutants, we have determined the activity and analyzed some properties of acid phosphatases in different fractions of the crude extracts. In the experiments to be described now, crude extracts were prepared by sonication of the cells suspended in distilled water. The extract was centrifuged at 1000 g for 5 minutes to eliminate undestroyed cells; the supernatant was then centrifuged at 27,000 g for 20 minutes. The pellet was resuspended in 2 ml distilled water; this suspension was syringed several times for homogenization. The acid phosphatase activity was measured in supernatants and pellets. Table 1 presents acid-phosphatase-specific activity data obtained on the supernatant and pellet fractions of water extract of the algae. It can be seen that both the supernatant activity and the pellet activity were lower in the mutants than in the corresponding wild-type fractions. Further, unlike the wild type, each mutant had lower activity in the pellet fraction than in its supernatant fraction. There is heterogeneity among the supernatant and pellet activities of the mutants although they all fail to stain with the naphthylphosphate reagent after heat treatment. Figure 2 presents thermolability results obtained for wild type, P, and P,. The specific activity of acid phosphatase in the pellets is far higher in WT than in P, and P,. The major part of this enzyme is thermostable. There is almost no thermostable enzyme in the pellets of the mutants. The supernatants of WT, as well as mutants, contain high amounts of thermolabile enzyme and some thermostable enzyme. Similar results have been obtained with P,, P, and P,, although in these strains the remaining activity in pellets after heat treatment was somewhat higher than in P, and Ps. (The fact that, as a rule, WT has more thermostable enzyme in the supernatant than the mutants have will be discussed later.) The results of these experiments strongly support the idea that the P mutants lack a thermostable acid phosphatase located in the insoluble fraction of crude extracts. TABLE 1 Specific aciivities of acid phosphatase ( a mole naphthol/mg protein, hr, 37") in supernatants and pellets of water extracts in WT and several P mutants __


Supernatant Pellet




__ -____ 0.72 0 47 0.66 0.82 0.05 0.18

All cultures were grown in liquid minimal medium for 3 days.






_________ 0.44 0.31

0.54 0.37



FIGURE 2.-Decrease of the specific activity of acid phosphatases in crude extract (pellet or supernatant) of WT, P, and P, strains in relation to the time of treatment at 60”.

5. Selection of a mutant lacking the soluble thermolabile acid phosphatase Mutant P, has been chosen for further experiments by virtue of its “extreme” phenotype : in repeated experiments, the values of specific enzyme activity in the pellets were always about 10% of the corresponding values in the wild-type strain. It may be assumed that without heat treatment the staining of P, colonies is merely due to the activity of the thermolabile soluble enzyme (see Figure 2). Accordingly, it should be possible to isolate, from P,, mutants impaired in the activity of this enzyme. These expected double mutants, if they are viable, are predicted to have very low enzyme activities and not to stain without heat treatment. P, cells were treated with MNNG in the usual conditions and plated on minimal medium. The resulting colonies were examined for acid phosphatase activity without heat treatment. Only one mutant ( P , Pa) was found out of more than 12,000 colonies analyzed. The activity measured in cell suspensions was, in P, Pa, 12 times and 9 times lower than in the WT and P, strains, respectively. 6. Comparison of acid phosphatase activities in W T ,P, and PzPa Acid phosphatase activities have been measured in supernatants and pellets of crude extracts prepared either in distilled water as usual or in 0.1 NI acetate buffer pH 4.8. The results of these experiments are given in Table 2. The P, and P, Pa mutants differ from the W T strain by their very low activity in the pellet. P, Pa, moreover, differs from P, and WT by a low activity in the supernatant. I n the WT strain, the specific activity of acid phosphatase is higher in supernatants and lower in pellets of acetate buffer extracts than in corresponding frac-



TABLE 2 Specific acid phosphatase activities ( p mote naphthoE/mg protein, hr, 37") in the supernatant and pellet fractions of W T , P, and P , Pa Supernatant

Extract in



p2 p a


Pellet p2

p2 p,








Acetate buffer+ (PH 4.8)







* Mean of 5 independent experiments.

t Mean of 2 independent experiments. tions of water extracts. This results from the fact that a lot of proteins and particles present in the supernatant precipitate in acetate buffer, whereas acid phosphatases are not precipitated to the same extent (in contrast with water supernatants which are dark green, acetate buff er supernatants are fully transparent; the ratio : protein content in supernatant/protein content in pellet, was 2-4 f o r water extracts and 0.15-0.25 for acetate buffer extracts). As far as the supernatants of water extracts are concerned, the specific activity was always higher i n W T than in P,. This could be related to the fact that particles of the pellet, to which thermostable acid phosphatases are linked (see Figure 2), contaminate the supernatant. These particles are visible under the microscope. In acetate buffer extracts, the supernatants were found to be much less contaminated with particles and no difference in acid phosphatase activity between W T and Pa could be detected in the supernatants. All these results taken together strongly suggest that P, carries a mutation controlling a thermostable enzyme of the insoluble fraction and that P, Pa, derived from P,, is, moreover, mutated in a gene controlling a thermosensitive soluble enzyme. This conclusion has been confirmed by the following electrophoretic study. 7. Electrophoretic analysis

The separation of acid phosphatases by electrophoresis is mostly performed in gels prepared with neutral or basic buffers (ALLEN,MISCHand MORRISON 1963; EFRON1971; SHAW and PRASAD 1970; MACINTYRE 1971). Numerous attempts were made in our laboratory using acrylamide gels of Tris-Glycine or Tris-HC1 buffers at p H ranging from 7.0 to 8.9 and acetate buffers at pH 4.5 to 6.0. The best resolution was found to take place when the electrophoresis was carried out at low pH. This last technique was used throughout the present work (see MATERIAL AND METHODS). Moreover, it allows the gel to be stained directly after the electrophoresis without prolonged washing in acid medium, as is required for gels prepared with alkaline buffers. I n water extracts of W T cells, seven distinct acid phophatases were found migrating to the cathode (Figure 3 ) . The most active band was the fastest one. A deeply stained spot developed at the starting line and did not migrate whatever

600 .,-_


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