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Decreased adenosine triphosphatase activity of flagella from a paralyzed mutant of Chlamydomonas moewusii
C. J. Brokaw
430
either. The values obtained after the remainder of the treatments did not differ among themselves significantly. These results confirm the precedent work with the gametes of Rana pipiens, and indicate that the deleterious effect of chymotrypsin observed in that species at gastrulation probably affected cleavage stages also. We have earlier suggestedthat the initial effect of enzymes on the gametes is to prevent the proper combination of spermatozoa and egg cells necessaryto fertilization, possibly by an effect on combining sites of the gametes. But the separate effect noted on the post-gastrular development must be attributed to someother, probably internal, effect on the structure of the embryo, perhaps caused by the importation into the egg of the enzyme. REFERENCES 1. BARCH,
2. ~
S. H. and SHAVER, J. R., Acta Embryol. Morpkol. Expfl. Cell Research 17, 114 (1959).
3. SNEDECOR,
G. W., Statistical
Methods.
DECREASED ADENOSINE FLAGELLA FROM
Iowa
Exptl. 1, 211 (1958).
State CollegePress,1956.
TRIPHOSPHATASE ACTIVITY A PARALYZED MUTANT OF
CHLAMYDOMONAS
OF
MOEWUSII
C. J. BROKAW’ Biology&&ion, OakRictgeNationalLnboratory,’OakRidge,Tennessee, U.S.A. Received
January
1, 1960
!%VERAL ultraviolet-induced mutants of Chlamydomonas moewusii have been isolated by Lewin [3]. Some of these mutants are nonmotile and have been termed “paralyzed”. Movements of their flagella are restricted to an occasional slight twitching near their distal ends. The paralyzed flagella are sometimes slightly shorter than normal flagella, but otherwise appear normal by light and electron microscopy [2]. No serological differences were found between flagella from motile and nonmotile cells [5]. Techniques developed to study the movement and biochemistry of isolated flagella from Polyfoma uvella [l] have been used to compare flagella from normal cells of Chlamydomonas with flagella from one of Lewin’s paralyzed mutants, M 1002. The cellswere grown at 27” under approximately 500 ft-c of illumination, on a medium containing 1 g/liter KNO,, 0.2 g/l KH,PO,, 0.2 g/l K*HPO,= 3H,O, 0.1 g/l MgSO,. 7HI0, 0.05 g/l CaCl, and trace elements. The cells were harvested by centrifugation, washed once in 0.02 M tris-thioglycolate buffer, pH 7.8, and suspended in 3 to 4 1 Present ’ Operated
Experimental
address: Dept. of Zoology, Univ. by Union Carbide Corporation
Cell Research 19
of Minnesota, for the U.S.
Minneapolis. Atomic Energy
Commission.
Flagella
from a paralyzed
mutant
of Chlamydomonas
431
volumes of a solution containing 0.01 M tris-thioglycolate buffer, pH 7.8, 0.01 M MgCl, and 70 per cent v/v glycerol, at 0”. The mixture was rapidly cooled to -20” and subsequently kept near this temperature. After one hour the mixture was stirred vigorously and centrifuged for 15 min at 10,000 x g to remove the cell bodies.
Fig. I.-Dephosphorylation
of ATP by preparations of flagella from wild type cells (open circles)
and preparations
from
of flagella
paralyzed
cells (solid
circles).
Flagella in the resulting supernatant were examined for motility by suspending them in 10 volumes of a solution containing 0.02 M tris-thioglycolate buffer, pH 7.8, 0.05 A4 KCl, 0.004 M MgCl, and 2 x IO-’ M adenosine triphosphate (ATP). Observations were made at 10” to 15” with dark-field illumination at a magnification of 560 times. Flagella from normal cells responded to ATP under these conditions by beating and swimming through the medium as described for Polytoma flagella [l]. Flagella from the paralyzed mutant cells were motionless, and also could not be activated in the presenceof the supernatant from normal flagella. To measure ATPase activity, the flagella were collected by centrifugation for 6 hr at 36,000 x g, at - 10” or below, and resuspendedin a small volume of supernatant. Concentrated flagella suspension(0.05 ml) was added to 20 ml of the solution used to observe movement, and incubated at 18”. Three-milliliter aliquots were removed at 6-min intervals and assayed for inorganic phosphate by the standard Fiske-SubbaRow technique. The flagella concentrations in the sampleswere compared by counts of the flagella in a bacterial counting chamber, and by estimation of the protein by the method of Lowry et al. (41after the flagella were dissolved in 1 M NaOH. Results based on protein determinations are shown in Fig. 1. The results are similar when compared on the basis of flagellar counts; the mean activity of the normal flagella was 127 rg of phosphorus/hr/109flagella, and of the paralyzed flagella 40 pg of phosphorus/hr/lOO flagella. Since the lesion responsible for the paralysis of this mutant apparently alters the normal mechanism for converting the energy of ATP into flagellar movement, further study of these mutants should be profitable. The paralyzed flagella have 30-40 per cent of the ATPase activity of normal flagella, but their relative motility is very Experimental
Cell Research 19
Y. Endo much less. Paralysis may be caused by a deficiency in ATPase, but interpretation of the result is difficult because it is not known whether all the ATPase activity of normal flagella is concerned with movement. REFERENCES BROKAW, C. J., Submitted for publication (1960). GIBBS, S. P., LEWIN, R. A. and PHILPOTT, D. E., Ezptl. 3. LEWIN, R. A., .J. Gen. Microbial. 6, 233 (1952). 4. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. and 1. 2.
5.
MINTZ,
265 (1951). R. H. and
THE
R. A.,
LEWIN,
Canad.
J. Mierobiol.
Cell Research RANDALL,
R.
15, 619 (1959). J.,
.I. Biol.
Chem.
193,
1, 65 (1954).
FIRST CLEAVAGE FURROW IN SEA URCHIN NOT PASS THROUGH THE SPERM ENTRANCE
EGGS DOES POINT
Y. END0 Biological
Laboratory,
Keio Received
A~-rmwH
University, January
Yokohama-Hiyoshi.
Japan
20, 1960
the question whether the first cleavage furrow of sea urchin eggs passes through the entrance point of the spermatozoon or not is certainly not new, it yet has not been definitely settled (for references, see [2]). According to one opinion there is a coincidence between the entrance point of thk spermatozoon and the first cleavage furrow; this accordance is, however, also vigorously denied by others. This disagreement between research workers may be due to the fact that the method for determining the entrance point of the spermatozoon under normal conditions is not sufficiently conclusive. It is well known that the “entrance” or “fertilization cone” is formed at the attachment point of the spermatozoon soon after the fertilization membrane begins to be elevated. In most species of sea urchins, this entrance cone, with the lapse of time, gradually changes in form and finally becomes indistinguishable. In Pseudocentrotus depressus, however, the entrance cone remains for a long time and is possible to detect at the time of the first cleavage. Since the entrance cone is the most exact indication of the entrance point of the spermatozoon, Pseudocentrotus egg was used to test the relation between the cone and the first cleavage furrow. Immediately after membrane elevation in some of these eggs, the entrance cone can be seen in the optical section of the egg surface. Location of the cone is facilitated by the fact that cortical granule substance in the form of rods [l, 31 is distributed in the previtelline space following a gradient which is highest at the sperm entrance point (unpublished). At the time of cleavage, the entrance cone appears as a hyaline protuberance on the surface of the hyaline layer (Fig. 1). Experimental
Cell Research
19
430
either. The values obtained after the remainder of the treatments did not differ among themselves significantly. These results confirm the precedent work with the gametes of Rana pipiens, and indicate that the deleterious effect of chymotrypsin observed in that species at gastrulation probably affected cleavage stages also. We have earlier suggestedthat the initial effect of enzymes on the gametes is to prevent the proper combination of spermatozoa and egg cells necessaryto fertilization, possibly by an effect on combining sites of the gametes. But the separate effect noted on the post-gastrular development must be attributed to someother, probably internal, effect on the structure of the embryo, perhaps caused by the importation into the egg of the enzyme. REFERENCES 1. BARCH,
2. ~
S. H. and SHAVER, J. R., Acta Embryol. Morpkol. Expfl. Cell Research 17, 114 (1959).
3. SNEDECOR,
G. W., Statistical
Methods.
DECREASED ADENOSINE FLAGELLA FROM
Iowa
Exptl. 1, 211 (1958).
State CollegePress,1956.
TRIPHOSPHATASE ACTIVITY A PARALYZED MUTANT OF
CHLAMYDOMONAS
OF
MOEWUSII
C. J. BROKAW’ Biology&&ion, OakRictgeNationalLnboratory,’OakRidge,Tennessee, U.S.A. Received
January
1, 1960
!%VERAL ultraviolet-induced mutants of Chlamydomonas moewusii have been isolated by Lewin [3]. Some of these mutants are nonmotile and have been termed “paralyzed”. Movements of their flagella are restricted to an occasional slight twitching near their distal ends. The paralyzed flagella are sometimes slightly shorter than normal flagella, but otherwise appear normal by light and electron microscopy [2]. No serological differences were found between flagella from motile and nonmotile cells [5]. Techniques developed to study the movement and biochemistry of isolated flagella from Polyfoma uvella [l] have been used to compare flagella from normal cells of Chlamydomonas with flagella from one of Lewin’s paralyzed mutants, M 1002. The cellswere grown at 27” under approximately 500 ft-c of illumination, on a medium containing 1 g/liter KNO,, 0.2 g/l KH,PO,, 0.2 g/l K*HPO,= 3H,O, 0.1 g/l MgSO,. 7HI0, 0.05 g/l CaCl, and trace elements. The cells were harvested by centrifugation, washed once in 0.02 M tris-thioglycolate buffer, pH 7.8, and suspended in 3 to 4 1 Present ’ Operated
Experimental
address: Dept. of Zoology, Univ. by Union Carbide Corporation
Cell Research 19
of Minnesota, for the U.S.
Minneapolis. Atomic Energy
Commission.
Flagella
from a paralyzed
mutant
of Chlamydomonas
431
volumes of a solution containing 0.01 M tris-thioglycolate buffer, pH 7.8, 0.01 M MgCl, and 70 per cent v/v glycerol, at 0”. The mixture was rapidly cooled to -20” and subsequently kept near this temperature. After one hour the mixture was stirred vigorously and centrifuged for 15 min at 10,000 x g to remove the cell bodies.
Fig. I.-Dephosphorylation
of ATP by preparations of flagella from wild type cells (open circles)
and preparations
from
of flagella
paralyzed
cells (solid
circles).
Flagella in the resulting supernatant were examined for motility by suspending them in 10 volumes of a solution containing 0.02 M tris-thioglycolate buffer, pH 7.8, 0.05 A4 KCl, 0.004 M MgCl, and 2 x IO-’ M adenosine triphosphate (ATP). Observations were made at 10” to 15” with dark-field illumination at a magnification of 560 times. Flagella from normal cells responded to ATP under these conditions by beating and swimming through the medium as described for Polytoma flagella [l]. Flagella from the paralyzed mutant cells were motionless, and also could not be activated in the presenceof the supernatant from normal flagella. To measure ATPase activity, the flagella were collected by centrifugation for 6 hr at 36,000 x g, at - 10” or below, and resuspendedin a small volume of supernatant. Concentrated flagella suspension(0.05 ml) was added to 20 ml of the solution used to observe movement, and incubated at 18”. Three-milliliter aliquots were removed at 6-min intervals and assayed for inorganic phosphate by the standard Fiske-SubbaRow technique. The flagella concentrations in the sampleswere compared by counts of the flagella in a bacterial counting chamber, and by estimation of the protein by the method of Lowry et al. (41after the flagella were dissolved in 1 M NaOH. Results based on protein determinations are shown in Fig. 1. The results are similar when compared on the basis of flagellar counts; the mean activity of the normal flagella was 127 rg of phosphorus/hr/109flagella, and of the paralyzed flagella 40 pg of phosphorus/hr/lOO flagella. Since the lesion responsible for the paralysis of this mutant apparently alters the normal mechanism for converting the energy of ATP into flagellar movement, further study of these mutants should be profitable. The paralyzed flagella have 30-40 per cent of the ATPase activity of normal flagella, but their relative motility is very Experimental
Cell Research 19
Y. Endo much less. Paralysis may be caused by a deficiency in ATPase, but interpretation of the result is difficult because it is not known whether all the ATPase activity of normal flagella is concerned with movement. REFERENCES BROKAW, C. J., Submitted for publication (1960). GIBBS, S. P., LEWIN, R. A. and PHILPOTT, D. E., Ezptl. 3. LEWIN, R. A., .J. Gen. Microbial. 6, 233 (1952). 4. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. and 1. 2.
5.
MINTZ,
265 (1951). R. H. and
THE
R. A.,
LEWIN,
Canad.
J. Mierobiol.
Cell Research RANDALL,
R.
15, 619 (1959). J.,
.I. Biol.
Chem.
193,
1, 65 (1954).
FIRST CLEAVAGE FURROW IN SEA URCHIN NOT PASS THROUGH THE SPERM ENTRANCE
EGGS DOES POINT
Y. END0 Biological
Laboratory,
Keio Received
A~-rmwH
University, January
Yokohama-Hiyoshi.
Japan
20, 1960
the question whether the first cleavage furrow of sea urchin eggs passes through the entrance point of the spermatozoon or not is certainly not new, it yet has not been definitely settled (for references, see [2]). According to one opinion there is a coincidence between the entrance point of thk spermatozoon and the first cleavage furrow; this accordance is, however, also vigorously denied by others. This disagreement between research workers may be due to the fact that the method for determining the entrance point of the spermatozoon under normal conditions is not sufficiently conclusive. It is well known that the “entrance” or “fertilization cone” is formed at the attachment point of the spermatozoon soon after the fertilization membrane begins to be elevated. In most species of sea urchins, this entrance cone, with the lapse of time, gradually changes in form and finally becomes indistinguishable. In Pseudocentrotus depressus, however, the entrance cone remains for a long time and is possible to detect at the time of the first cleavage. Since the entrance cone is the most exact indication of the entrance point of the spermatozoon, Pseudocentrotus egg was used to test the relation between the cone and the first cleavage furrow. Immediately after membrane elevation in some of these eggs, the entrance cone can be seen in the optical section of the egg surface. Location of the cone is facilitated by the fact that cortical granule substance in the form of rods [l, 31 is distributed in the previtelline space following a gradient which is highest at the sperm entrance point (unpublished). At the time of cleavage, the entrance cone appears as a hyaline protuberance on the surface of the hyaline layer (Fig. 1). Experimental
Cell Research
19