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Temperature induced isozyme variants in individuals of the sea urchin, Arbacia punctulata
Comp. Biochem. Physiol., 1977, Vol. 58 I~ pp. 109 to 113. Pergamon Press Printed in Great Britmn
TEMPERATURE INDUCED ISOZYME VARIANTS IN INDIVIDUALS OF THE SEA URCHIN, A R B A C I A P U N C T U L A T A NANCY H. MARCUS* Yale University, Department of Biology, Osborn Memorial Laboratories, New Haven, CT 06520, U.S.A.
(Received 4 January 1977) Abstract--I. The technique of microacrylamide slab gel electrophoresis was used to repeatedly monitor the qualitative isozyme composition in the tube feet of the sea urchin, Arbacia punctulata exposed to different temperature regimeg 2. Four enzyme systems were assayed. Three (MDH, HK, and ACPH) were monomorphic for each animal studied and no change in band migration pattern was observed. Banding patterns for the fourth system (EST) were observed to vary in the same individual. 3. The reversible induction of esterase variants in the tube feet of the same individual is reported. 4. The change was temperature dependent, and a period of 7-14 days acclimation was required to induce the pattern alteration.
INTRODUCI'ION
Do organisms have the capability of responding to environmental fluctuations by permanently or temporarily altering their cellular isozyme composition qualitatively? Because such a response is affected by the entire genetic repertoire of an organism, it is essential that repeated monitoring of the same genome's response he conducted so that individual variations due to epistatic interactions are eliminated. The sea urchin, Arbacia punctulata is a widely distributed benthic marine invertebrate, reported to occur from Woods Hole, MA, U.S.A. south to the Yucatan, Mexico, with occasional specimens being found as far south as the northern coast of Venezuela (Harvey, 1956). Throughout this range, environmental conditions vary a great deal between regions and also seasonally within the same region. The present paper reports a procedure, developed for the repeated monitoring of the isozymal phenotype of the tube feet of individual adult Arbacia punctulata. Repeated monitoring of the same individual is possible because the animal is not killed by the initial analysis. An individual urchin possesses hundreds of tube feet, each of which it is capable of regenerating. Removal of the tube feet in the laboratory does not appear to adver~ly affect the animal. In the present study, the isozyme composition was determined for individual urchins exposed to alternate temperature regimes for different periods of time. The procedure I developed for the present research may be applied to further studies directed towards determining the biochemical and/or physiological response of an organism to a change in external parameters.
tuses were constructed and gels prepared according to the general method of Ogita (1975).
Gel preparation Gels were prepared from the following stock solutions: 47.5 g acrylamide, 2.5g N,N'-methylene bisacrylamide in 250.0mi deionized water; gel buffer (see below); 1.0ml TEMED in 100.0 ml deionized water; 240.0 rag ammonium persulfate in 100.0ml deionized water; and deionized water. Stock solutions were mixed in the following proportions to obtain a 6.5% acrylamide gel, 6.5:5:5:2.5:1. This gel concentration was used for all enzyme assays. Gel and electrode buffers Three buffer systems were used: gel buffer--0.11 M Tris` 0.062 M boric acid, 0.008 M NaCI pH 8.65 (Ogita, personal communication), electrode buffer---0.3 M borate pH 8.0 (Shaw & Prasad, 1970); gel buffer--0.32 M Tris glycine pH 8.8, electrode buffer---0.1 M glycine pH 8.6; and gel buffer--0.25 M Tris HC! pH 9.0, electrode buffer---0.5 M Tris HCI pH 9.0 (modified Shaw & Prasad, 1970).
Preparation of tissue samples All urchins studied were collected by the Marine Biological Laboratory Supply Department from the waters off Woods Hole, MA. The urchins were shipped to Yale University, New Haven, CT and were held there in recirculating salt water aquaria. The urchins were kept in individual plastic containers within the tanks. Prior to and during all experiments, the urchins were starved. Under such circumstances urchins can survive for several months. Samples of individual tube feet, chosen without regard to position on a single urchin, were prepared by grinding each foot separately in small wells in a Teflon block, with a machine driven Plexiglass rod. The tissue was homogenized over ice in 5.0/d of extraction buffer pH 7.0 (0A M Tris` pH 7.0--1.0raM EDTA--25.0mM 2-Mercaptoethanol--50.0/~M NADP ÷) (Levin et al., 1972). The homogenate was transferred to a gel slot by micropipette. Sixteen tube feet were analyzed for each enzyme assay. Bulk samples of tube feet were prepared by grinding MATERIALS AND METHODS 15-20 tube feet from a single urchin in a 0.4 ml polyethyElectrophoresis of tissue samples was conducted on a lene microcentrifuge tube with a machine driven Plexiglass horizontal microacrylamide gel apparatus. The appara- rod. The tissue was homogenized over ice in 50.0 ~1 of extraction buffer pH 7.0 (Levin et al., 1972). The homogenate was centrifuged in a refrigerated Sorvall centrifuge * Present address: Woods Hole Oceanographic Insti- at 3%100 0 for 5 rain. A portion of the clear supernatant tution, Woods Hole, MA 02543, U.S.A. was transferred to a gel slot by micropipette. 109
110
NANCY H. MARCUS
Each gel was filled with eight samples. Each slot held 2.5/al of sample. The gel was then inverted over buffer trays and electrophoresed, in a refrigerator at 2°C. At the conclusion of electrophoresis the gels were removed and stained.
less of position on the test. Because of the homogeneous nature of the tissue, bulk preparations were used in all the acclimation experiments and in vitro incubations. Following exposure to different temperature regimes Enzyme assays the isozymal phenotype (interpreted as the number Each gel was stained for a single enzyme. Individual and position of bands) was not changed in samples stained for either M D H , ACPH, or HK. Differences tube feet provide enough material for two enzyme assays. in the number of band variants were noted for the Bulk preparations provide enough material for at least a dozen assays. The following four assays (modified from fourth system, EST. The esterase assays using :t-napShaw & Prasad, 1970) were conducted in this study: thyl acetate, ~-napthyl acetate, and :t-napthyl butyrmalate dehydrogenase (MDH), buffer system two, three ate indicated a general reactivity with all substrates. hours at 40 V, stain~40.0 mg L-malic acid, 10 mg fl-NAD, Similar migration patterns were obtained with each 5 mg NBT, 1.5 mg PMS, in 25 ml 0.05 M Tris buffer pH substrate. Staining for esterase revealed three zones 9.2; acid phosphatase (ACPH), buffer system two, 2½hrof activity (EST-I, EST-2, and EST-3). in order ot at 40 V, stain--25 mg Fast Blue BB, 25 mg a-napthyl acid decreasing mobility towards the anode (Fig. IA, BI. phosphate, 200 mg polyvinylpyrrolidine in 25 ml 0.125 M During the study, EST-I was often blurred and EST-2 acetate buffer pH 5; hexokinase (HK), buffer system three, did not undergo a change in band phenotype. The 2hr at 40V, stain---40mg glucose, 10mg ATP, 5rag NADP ', 5 mg MTT, 20 units glucose-6-phosphate deresults and discussion below, are based solely on the hydrogenase, 1.5 mg PMS, 20 mg MgCI 2 in 25 ml 0.05 M most cathodal zone, EST-3. EST-3 on day one of the Tris buffer pH 8; and esterase (EST), buffer system one, study was represented by a single slow band (EST-3a) 1 hr at 70 V, stain--20 mg Fast Garnet GBC salt, 0.8 ml in all individuals sampled (Figs I A and 2). Shown substrate solution, in 25 ml 0.1 M phosphate buffer pH 6.5. in Fig. I A are the band phenotypes of urchins I .~ Substrate solutions for esterase were prepared by adding 1 g of either ~-napthyl acetate, fl-napthyl acetate, or ~t-nap- (corresponding to slots I -8, left to right) sampled on day one. The gel was stained for esterase, using//-napthyl butyrate to 100 ml of acetone and water (1:1). thyl acetate as the substrate. The other eight urchins Gel fixation (not shown) manifested identical patterns. Shown in After Staining, the gel was washed in water, then soaked Figs 1B and 2B are the band phenotypes of urchins for l hr in 5% glycerine. Permanent preservation was I-8, after 25 days acclimation. Urchins 1--6 (slots 1-.6) achieved by drying the gel between two sheets of cello- which were placed at 25°C developed a second faster phane (Ogita, 1975). band (EST-3b) in all but one case. Urchin 2 did not develop a new band. The double banded phenotypc Acclimation experiments was observed only in urchins acclimated to 25~C. The On day one, 16 urchins were sampled. The urchins had four control urchins kept at 19°C did not develop been acclimated to 19°C for more than 4 weeks. Bulk tube a new band. The original pattern was not altered. feet tissue samples were prepared from each animal and assayed for the enzymes discussed above. After electro- The four urchins acclimated to 13'C (9a-12a) maniphoresis of the samples, urchins 1-6 were placed at 25°C; fested patterns like the urchins acclimated to 1 9 C urchins 9-14 were placed at 2°C, and individuals 7, 8, 15, i.e. the EST-3a band only (Fig. 2B). After 14 days and 16 were placed back at 19°C. The latter four specimens acclimation to the alternate temperature regime these served as.controls. After 25 days, bulk samples were pre- patterns were reversed (Fig. 2C). Urchins I-6 which pared from the urchins held at 19 and 25°C. The animals had developed the double banded pattern characterat 2~C had died. Additional samples were prepared from four urchins which had been acclimated previously to 13°C istic of 25°C, reverted to the single slow banded for a period of 28 days. These four animals were labelled phenotype (EST-3a) after 14 days acclimation to 13°C. On the other hand, the urchins held at 1 3 C 9a-12a. After analysis of the urchins the animals were transferred to a new temperature regime. Urchins I-6 were for 28 days originally, developed the second faster placed at 13°C; urchins 9a-12a were placed at 25°C; and band (EST-3b), in addition to EST-3a, when held for the controls (7, 8, 15 and 16) were placed back at 19°C. 14 days at 25°C. No change was observed in the conAfter 14 days, the urchins were monitored again, and then trol urchins. Reversal of the two patterns was not switched to the alternate temperature regime. Urchins 1-6 observed in animals acclimated for 7 days, but was were placed at 25°C; urchins 9a-12a were placed at 13°C, observed in animals acclimated for 14 days or longer. and the controls were kept at 19°C. After exposure to the Incubation of the samples at 25"C did not alter altered conditions for one week, and a final electrophoretic the band pattern. Identical phenotypes were observed analysis, the experiment was terminated. in samples incubated as long as one hr. The patterns In vitro incubation were similar to those observed for the control .sample The supematant from bulk tube feet samples from a held at 2"~C. The single slow band. EST-3a, was single urchin, acclimated to 19°C for 6 weeks, was divided observed in all samples. into four aliquots. Four aliquots were incubated in an oven at 25°C for I hr, 45 rain, 30min, and 15min respectively. DISCUSSION The fifth aliquot, the control, was kept in the refrigerator at 2~C for I hr. After incubation, the samples were electroThe phenotype of an organism is the culmination phoresed and stained for esterase using ~-napthyl acetate of a series of interactive events between the indivias the substrate. dual's genotype and the environment. Identical genotypes may give rise to quite different phenotypes RESULTS dependent upon the environmental conditions. This Analysis of individual tube feet from a single urchin study suggests that the isozyme phenotype of indivireflected similar band patterns for all tube feet regard- duals is not stable, and under certain conditions may
111
Isozyme induction in Arbacia :..
..
EST-1
EST-2 EST-3a Origin ;;
..:...
t
Fig. 1. (A) Esteras¢ isozymes m the tube feet of urchins 1-8 after 28 days acclimation to 19°C. (B) Esterase isozymes in tube feet of urchins 1-6 after 25 days acclimation to 25°C, and urchins 7, 8 (the controls) retained at 19°C. Activity zones depicted are EST-I, EST-2, and EST-3 in order of decreasing mobility towards the anode, and bands EST-3a and EST-3b in order of increasing mobility towards the anode (+). change following a fluctuation in environmental parameters. The results indicate that not all enzyme systems respond to temperature changes in the same manner. Three systems (MDH, HK, and ACPH) did not undergo a phenotypic change in band migration pattern. However, alteration of the esterase pattern was observed and the change was reversible. This phenotypic response was dependent upon the period of acclimation. Appearance or disappearance of EST-3b required 7-14 days of acclimation.
Different mechanisms may be postulated to explain this phenomenon. The faster band (EST-3b) may be the product of a gene distinct from that which directs the synthesis of the slower variant (EST-3a). This induction mechanism referred to as the "On-Off' hypothesis by Hochachka & Somero (1973) has been noted in populations of trout (Baldwin & Hochachka, 1970), in the creek chub (Kent & Hart, 1976), and in barnacles (Flowerdew & Crisp, 19761 On the other hand, the faster band (EST-3b) may be a conforma-
112
NANCY H.
r.~C°ntr°. ~ 7,B,15,16~
14days [~ 7days [~ EST-So 19" EST-Sa 19" " EST-3a
EST-Sa
Urchins 9a-12a 20days
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MARCUS
13 ~J EST-Sa "-'~-~'~
EST-3a
m JJ
14days EST-3a 13 I IEST-Sa H Esr-5o (b) (c) (d)
Fig. 2. Flow diagram of acclimation experiments. tional variant (Hochachka & Somero, 1973) resulting from the breakdown or alteration of the already existing form (EST-3a). Some of the organisms for which the interconversion of isozyme variants has been observed are the Alaskan king crab for pyruvate kinase (somero, 1969), and the snail, Cepaea nemoralis for esterase (Oxford, 1975). The present data are best explained by the "On-Off" hypothesis. The variant (EST-3a) typifying the single slow band phenotype migrated the same distance as the slower band of the double banded phenotype (EST-3a/3b). If the double banded phenotype was representative of the breakdown products of the structure represented by the single banded phenotype, it is likely that the migration distances of the breakdown products would be distinct from that of the single form. If, on the other hand, an equilibrium condition is postulated, then the conformational hypothesis could explain the observed pattern changes. Such conditions would require that at cool temperatures the single slow band form predominates, but that as the temperature is raised some of the molecules are converted to the alternate form (EST-3b), producing a double banded phenotype. A change of this nature could occur in the protein itself or perhaps in an associated molecule, such as a lipid. However, the fact that urchin 2 did not undergo a band pattern change, despite the fact that it manifested the slow variant (EST-3a) singly, suggests that the appearance or disappearance of EST-3b was not due to a structural alteration. The results of the in vitro study indicated no breakdown of the single slow variant at an incubation temperature of 25°C. These facts lead me to conclude that the "'On-Off" switching of genes is indeed the mechanism responsible for the appearance or disappearance of the fast variant, EST-3b. Furthermore the results suggest that these bands (EST-3a and 3b) represent two distinct enzymes (produced by different loci), rather than two allozymes (at a single locus). If the variants were allozymcs, it would follow that all, but one of the urchins were heterozygotes. This seems unlikely. The time delay, 7--14 days, observed in this study must represent the time required to perceive the external fluctuation and the time needed to activate a
mechanism to cope with the change. Synthesis of new proteins may be energetically costly to the overall metabolism of the cell, so that the duration of the perturbation may have to reach a threshold value, before a response mechanism is triggered. In a constantly fluctuating environment the "On-Off" mechanism is not a rapid enough response. Arbacia punctulata may not be capable of adjusting to sudden dramatic shifts as indicated by the deaths of those animals exposed to 2°C after previous acclimation to 19°C. Natural populations frequently encounter temperatures of 2°C at Woods Hole in the winter (Bumpus, 1957), but gradual adjustment is possible over several months. Mass mortality has been observed when temperatures drop suddenly (Allee, 1923). Such rapid fluctuations, however, are not common in the subtidal marine environment because the water medium acts as a buffer against such shifts. The induction and repression of genes under different environmental regimes has important implications in both the fields of developmental and population genetics. Although this adaptive mechanism may not typify all enzyme systems, nor all organisms, it should be dealt with and elucidated. The methods presented in this paper provide an ideal system for ascertaining the phenotypic plasticity of the sea urchin and similar organisms. The results suggest that for an accurate assessment of the true genetic variation present within and between species, consideration must be given to the possible existence of temporary isozymes induced by variations in environmental conditions.
Acknowledoements.. I thank J. Ramus, M. Clutter, J. Powell, J. Grassle, L. Murphy, and A. Stone Ament for their helpful comments and advice concerning the manuscript. This work was supported by a Sigma Xi grant and U.S.P.H.S. grant HD 00032 awarded to the author and N.S.F. grant BMS 75-00436 awarded to J. Ramus. REFERENCES ALLEE W. C. (1923) Studies in marine ecology--IV. The effect of temperature in limiting the geographical range of invertebrates of the Woods Hole littoral. Ecoloyy 4, 341 •354.
lsozyme induction in Arbacia BALDWIN J. & HOCHACHKAP. (1970) Functional significance of isoenzymes in thermal acclimation. Biochem. J. 116, 883-887. BUMPUSD. (1957) Surface water temperatures along Atlantic and Gulf coasts of the United States. U.S. Fish Wildl. Sert,. Spec. Sci. Rep. 214, 1-153. FLOWERDEW M. W. ~. CRISP D. J. (1976) Allelic esterase isozymes, their variations with season, position on the shore, and stage of development in the Cirripede, Balanus balanoides. Mar. Biol. 35, 319-325. HARVEY E. B. (1956) The American Arbacia and otlter sea urchins. Princeton University Press, Princeton. HOCHACHKAP. & SOMEROG. (1973) Strategies of Biochemical Adaptation. W. B. Saunders Co,, Philadelphia. KENT J. D. & HART R. G. (1976) The effect of temperature and photoperiod on isozyme induction in selected tissues of the creek chub, Semotilus atromaculatus. Comp. Biochem. Physiol. 54B, 77-80.
C BP
~ . IB - - H
I13
LEVtN D., HOWl_ANDP. & STONER E. (1972) Protein polymorphism and genie heterozygosity in a population of the permanent translocation heterozygote Oenothera biennis. Proc. Natn Acad. $ci. U.S.A. 69, 1475-1477. O<;IT^ Z. (1975) Genetic control and epigenetic modification of human serum cholinesterase. In lsozymes--ll. Physiological function. (Edited by MARKERT C.), pp. 289-314. Academic Press, New York. OxeogD G. S. (1975) Food induced esterase phenocopies in the snail Cepaea nemoralis. Heredity 35, 361-370. SHAW C. R. & PRA,~D R. (1970) Starch gel electrophoresis of enzymes--a compilation of recipes. Biochem. Genet. 4, 297-320. SOM~ROG. (1969) Pyruvate kinase variants in the Alaskan king crab. Biochem. J. 114, 237-241.
TEMPERATURE INDUCED ISOZYME VARIANTS IN INDIVIDUALS OF THE SEA URCHIN, A R B A C I A P U N C T U L A T A NANCY H. MARCUS* Yale University, Department of Biology, Osborn Memorial Laboratories, New Haven, CT 06520, U.S.A.
(Received 4 January 1977) Abstract--I. The technique of microacrylamide slab gel electrophoresis was used to repeatedly monitor the qualitative isozyme composition in the tube feet of the sea urchin, Arbacia punctulata exposed to different temperature regimeg 2. Four enzyme systems were assayed. Three (MDH, HK, and ACPH) were monomorphic for each animal studied and no change in band migration pattern was observed. Banding patterns for the fourth system (EST) were observed to vary in the same individual. 3. The reversible induction of esterase variants in the tube feet of the same individual is reported. 4. The change was temperature dependent, and a period of 7-14 days acclimation was required to induce the pattern alteration.
INTRODUCI'ION
Do organisms have the capability of responding to environmental fluctuations by permanently or temporarily altering their cellular isozyme composition qualitatively? Because such a response is affected by the entire genetic repertoire of an organism, it is essential that repeated monitoring of the same genome's response he conducted so that individual variations due to epistatic interactions are eliminated. The sea urchin, Arbacia punctulata is a widely distributed benthic marine invertebrate, reported to occur from Woods Hole, MA, U.S.A. south to the Yucatan, Mexico, with occasional specimens being found as far south as the northern coast of Venezuela (Harvey, 1956). Throughout this range, environmental conditions vary a great deal between regions and also seasonally within the same region. The present paper reports a procedure, developed for the repeated monitoring of the isozymal phenotype of the tube feet of individual adult Arbacia punctulata. Repeated monitoring of the same individual is possible because the animal is not killed by the initial analysis. An individual urchin possesses hundreds of tube feet, each of which it is capable of regenerating. Removal of the tube feet in the laboratory does not appear to adver~ly affect the animal. In the present study, the isozyme composition was determined for individual urchins exposed to alternate temperature regimes for different periods of time. The procedure I developed for the present research may be applied to further studies directed towards determining the biochemical and/or physiological response of an organism to a change in external parameters.
tuses were constructed and gels prepared according to the general method of Ogita (1975).
Gel preparation Gels were prepared from the following stock solutions: 47.5 g acrylamide, 2.5g N,N'-methylene bisacrylamide in 250.0mi deionized water; gel buffer (see below); 1.0ml TEMED in 100.0 ml deionized water; 240.0 rag ammonium persulfate in 100.0ml deionized water; and deionized water. Stock solutions were mixed in the following proportions to obtain a 6.5% acrylamide gel, 6.5:5:5:2.5:1. This gel concentration was used for all enzyme assays. Gel and electrode buffers Three buffer systems were used: gel buffer--0.11 M Tris` 0.062 M boric acid, 0.008 M NaCI pH 8.65 (Ogita, personal communication), electrode buffer---0.3 M borate pH 8.0 (Shaw & Prasad, 1970); gel buffer--0.32 M Tris glycine pH 8.8, electrode buffer---0.1 M glycine pH 8.6; and gel buffer--0.25 M Tris HC! pH 9.0, electrode buffer---0.5 M Tris HCI pH 9.0 (modified Shaw & Prasad, 1970).
Preparation of tissue samples All urchins studied were collected by the Marine Biological Laboratory Supply Department from the waters off Woods Hole, MA. The urchins were shipped to Yale University, New Haven, CT and were held there in recirculating salt water aquaria. The urchins were kept in individual plastic containers within the tanks. Prior to and during all experiments, the urchins were starved. Under such circumstances urchins can survive for several months. Samples of individual tube feet, chosen without regard to position on a single urchin, were prepared by grinding each foot separately in small wells in a Teflon block, with a machine driven Plexiglass rod. The tissue was homogenized over ice in 5.0/d of extraction buffer pH 7.0 (0A M Tris` pH 7.0--1.0raM EDTA--25.0mM 2-Mercaptoethanol--50.0/~M NADP ÷) (Levin et al., 1972). The homogenate was transferred to a gel slot by micropipette. Sixteen tube feet were analyzed for each enzyme assay. Bulk samples of tube feet were prepared by grinding MATERIALS AND METHODS 15-20 tube feet from a single urchin in a 0.4 ml polyethyElectrophoresis of tissue samples was conducted on a lene microcentrifuge tube with a machine driven Plexiglass horizontal microacrylamide gel apparatus. The appara- rod. The tissue was homogenized over ice in 50.0 ~1 of extraction buffer pH 7.0 (Levin et al., 1972). The homogenate was centrifuged in a refrigerated Sorvall centrifuge * Present address: Woods Hole Oceanographic Insti- at 3%100 0 for 5 rain. A portion of the clear supernatant tution, Woods Hole, MA 02543, U.S.A. was transferred to a gel slot by micropipette. 109
110
NANCY H. MARCUS
Each gel was filled with eight samples. Each slot held 2.5/al of sample. The gel was then inverted over buffer trays and electrophoresed, in a refrigerator at 2°C. At the conclusion of electrophoresis the gels were removed and stained.
less of position on the test. Because of the homogeneous nature of the tissue, bulk preparations were used in all the acclimation experiments and in vitro incubations. Following exposure to different temperature regimes Enzyme assays the isozymal phenotype (interpreted as the number Each gel was stained for a single enzyme. Individual and position of bands) was not changed in samples stained for either M D H , ACPH, or HK. Differences tube feet provide enough material for two enzyme assays. in the number of band variants were noted for the Bulk preparations provide enough material for at least a dozen assays. The following four assays (modified from fourth system, EST. The esterase assays using :t-napShaw & Prasad, 1970) were conducted in this study: thyl acetate, ~-napthyl acetate, and :t-napthyl butyrmalate dehydrogenase (MDH), buffer system two, three ate indicated a general reactivity with all substrates. hours at 40 V, stain~40.0 mg L-malic acid, 10 mg fl-NAD, Similar migration patterns were obtained with each 5 mg NBT, 1.5 mg PMS, in 25 ml 0.05 M Tris buffer pH substrate. Staining for esterase revealed three zones 9.2; acid phosphatase (ACPH), buffer system two, 2½hrof activity (EST-I, EST-2, and EST-3). in order ot at 40 V, stain--25 mg Fast Blue BB, 25 mg a-napthyl acid decreasing mobility towards the anode (Fig. IA, BI. phosphate, 200 mg polyvinylpyrrolidine in 25 ml 0.125 M During the study, EST-I was often blurred and EST-2 acetate buffer pH 5; hexokinase (HK), buffer system three, did not undergo a change in band phenotype. The 2hr at 40V, stain---40mg glucose, 10mg ATP, 5rag NADP ', 5 mg MTT, 20 units glucose-6-phosphate deresults and discussion below, are based solely on the hydrogenase, 1.5 mg PMS, 20 mg MgCI 2 in 25 ml 0.05 M most cathodal zone, EST-3. EST-3 on day one of the Tris buffer pH 8; and esterase (EST), buffer system one, study was represented by a single slow band (EST-3a) 1 hr at 70 V, stain--20 mg Fast Garnet GBC salt, 0.8 ml in all individuals sampled (Figs I A and 2). Shown substrate solution, in 25 ml 0.1 M phosphate buffer pH 6.5. in Fig. I A are the band phenotypes of urchins I .~ Substrate solutions for esterase were prepared by adding 1 g of either ~-napthyl acetate, fl-napthyl acetate, or ~t-nap- (corresponding to slots I -8, left to right) sampled on day one. The gel was stained for esterase, using//-napthyl butyrate to 100 ml of acetone and water (1:1). thyl acetate as the substrate. The other eight urchins Gel fixation (not shown) manifested identical patterns. Shown in After Staining, the gel was washed in water, then soaked Figs 1B and 2B are the band phenotypes of urchins for l hr in 5% glycerine. Permanent preservation was I-8, after 25 days acclimation. Urchins 1--6 (slots 1-.6) achieved by drying the gel between two sheets of cello- which were placed at 25°C developed a second faster phane (Ogita, 1975). band (EST-3b) in all but one case. Urchin 2 did not develop a new band. The double banded phenotypc Acclimation experiments was observed only in urchins acclimated to 25~C. The On day one, 16 urchins were sampled. The urchins had four control urchins kept at 19°C did not develop been acclimated to 19°C for more than 4 weeks. Bulk tube a new band. The original pattern was not altered. feet tissue samples were prepared from each animal and assayed for the enzymes discussed above. After electro- The four urchins acclimated to 13'C (9a-12a) maniphoresis of the samples, urchins 1-6 were placed at 25°C; fested patterns like the urchins acclimated to 1 9 C urchins 9-14 were placed at 2°C, and individuals 7, 8, 15, i.e. the EST-3a band only (Fig. 2B). After 14 days and 16 were placed back at 19°C. The latter four specimens acclimation to the alternate temperature regime these served as.controls. After 25 days, bulk samples were pre- patterns were reversed (Fig. 2C). Urchins I-6 which pared from the urchins held at 19 and 25°C. The animals had developed the double banded pattern characterat 2~C had died. Additional samples were prepared from four urchins which had been acclimated previously to 13°C istic of 25°C, reverted to the single slow banded for a period of 28 days. These four animals were labelled phenotype (EST-3a) after 14 days acclimation to 13°C. On the other hand, the urchins held at 1 3 C 9a-12a. After analysis of the urchins the animals were transferred to a new temperature regime. Urchins I-6 were for 28 days originally, developed the second faster placed at 13°C; urchins 9a-12a were placed at 25°C; and band (EST-3b), in addition to EST-3a, when held for the controls (7, 8, 15 and 16) were placed back at 19°C. 14 days at 25°C. No change was observed in the conAfter 14 days, the urchins were monitored again, and then trol urchins. Reversal of the two patterns was not switched to the alternate temperature regime. Urchins 1-6 observed in animals acclimated for 7 days, but was were placed at 25°C; urchins 9a-12a were placed at 13°C, observed in animals acclimated for 14 days or longer. and the controls were kept at 19°C. After exposure to the Incubation of the samples at 25"C did not alter altered conditions for one week, and a final electrophoretic the band pattern. Identical phenotypes were observed analysis, the experiment was terminated. in samples incubated as long as one hr. The patterns In vitro incubation were similar to those observed for the control .sample The supematant from bulk tube feet samples from a held at 2"~C. The single slow band. EST-3a, was single urchin, acclimated to 19°C for 6 weeks, was divided observed in all samples. into four aliquots. Four aliquots were incubated in an oven at 25°C for I hr, 45 rain, 30min, and 15min respectively. DISCUSSION The fifth aliquot, the control, was kept in the refrigerator at 2~C for I hr. After incubation, the samples were electroThe phenotype of an organism is the culmination phoresed and stained for esterase using ~-napthyl acetate of a series of interactive events between the indivias the substrate. dual's genotype and the environment. Identical genotypes may give rise to quite different phenotypes RESULTS dependent upon the environmental conditions. This Analysis of individual tube feet from a single urchin study suggests that the isozyme phenotype of indivireflected similar band patterns for all tube feet regard- duals is not stable, and under certain conditions may
111
Isozyme induction in Arbacia :..
..
EST-1
EST-2 EST-3a Origin ;;
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Fig. 1. (A) Esteras¢ isozymes m the tube feet of urchins 1-8 after 28 days acclimation to 19°C. (B) Esterase isozymes in tube feet of urchins 1-6 after 25 days acclimation to 25°C, and urchins 7, 8 (the controls) retained at 19°C. Activity zones depicted are EST-I, EST-2, and EST-3 in order of decreasing mobility towards the anode, and bands EST-3a and EST-3b in order of increasing mobility towards the anode (+). change following a fluctuation in environmental parameters. The results indicate that not all enzyme systems respond to temperature changes in the same manner. Three systems (MDH, HK, and ACPH) did not undergo a phenotypic change in band migration pattern. However, alteration of the esterase pattern was observed and the change was reversible. This phenotypic response was dependent upon the period of acclimation. Appearance or disappearance of EST-3b required 7-14 days of acclimation.
Different mechanisms may be postulated to explain this phenomenon. The faster band (EST-3b) may be the product of a gene distinct from that which directs the synthesis of the slower variant (EST-3a). This induction mechanism referred to as the "On-Off' hypothesis by Hochachka & Somero (1973) has been noted in populations of trout (Baldwin & Hochachka, 1970), in the creek chub (Kent & Hart, 1976), and in barnacles (Flowerdew & Crisp, 19761 On the other hand, the faster band (EST-3b) may be a conforma-
112
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14days [~ 7days [~ EST-So 19" EST-Sa 19" " EST-3a
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Urchins 9a-12a 20days
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MARCUS
13 ~J EST-Sa "-'~-~'~
EST-3a
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14days EST-3a 13 I IEST-Sa H Esr-5o (b) (c) (d)
Fig. 2. Flow diagram of acclimation experiments. tional variant (Hochachka & Somero, 1973) resulting from the breakdown or alteration of the already existing form (EST-3a). Some of the organisms for which the interconversion of isozyme variants has been observed are the Alaskan king crab for pyruvate kinase (somero, 1969), and the snail, Cepaea nemoralis for esterase (Oxford, 1975). The present data are best explained by the "On-Off" hypothesis. The variant (EST-3a) typifying the single slow band phenotype migrated the same distance as the slower band of the double banded phenotype (EST-3a/3b). If the double banded phenotype was representative of the breakdown products of the structure represented by the single banded phenotype, it is likely that the migration distances of the breakdown products would be distinct from that of the single form. If, on the other hand, an equilibrium condition is postulated, then the conformational hypothesis could explain the observed pattern changes. Such conditions would require that at cool temperatures the single slow band form predominates, but that as the temperature is raised some of the molecules are converted to the alternate form (EST-3b), producing a double banded phenotype. A change of this nature could occur in the protein itself or perhaps in an associated molecule, such as a lipid. However, the fact that urchin 2 did not undergo a band pattern change, despite the fact that it manifested the slow variant (EST-3a) singly, suggests that the appearance or disappearance of EST-3b was not due to a structural alteration. The results of the in vitro study indicated no breakdown of the single slow variant at an incubation temperature of 25°C. These facts lead me to conclude that the "'On-Off" switching of genes is indeed the mechanism responsible for the appearance or disappearance of the fast variant, EST-3b. Furthermore the results suggest that these bands (EST-3a and 3b) represent two distinct enzymes (produced by different loci), rather than two allozymes (at a single locus). If the variants were allozymcs, it would follow that all, but one of the urchins were heterozygotes. This seems unlikely. The time delay, 7--14 days, observed in this study must represent the time required to perceive the external fluctuation and the time needed to activate a
mechanism to cope with the change. Synthesis of new proteins may be energetically costly to the overall metabolism of the cell, so that the duration of the perturbation may have to reach a threshold value, before a response mechanism is triggered. In a constantly fluctuating environment the "On-Off" mechanism is not a rapid enough response. Arbacia punctulata may not be capable of adjusting to sudden dramatic shifts as indicated by the deaths of those animals exposed to 2°C after previous acclimation to 19°C. Natural populations frequently encounter temperatures of 2°C at Woods Hole in the winter (Bumpus, 1957), but gradual adjustment is possible over several months. Mass mortality has been observed when temperatures drop suddenly (Allee, 1923). Such rapid fluctuations, however, are not common in the subtidal marine environment because the water medium acts as a buffer against such shifts. The induction and repression of genes under different environmental regimes has important implications in both the fields of developmental and population genetics. Although this adaptive mechanism may not typify all enzyme systems, nor all organisms, it should be dealt with and elucidated. The methods presented in this paper provide an ideal system for ascertaining the phenotypic plasticity of the sea urchin and similar organisms. The results suggest that for an accurate assessment of the true genetic variation present within and between species, consideration must be given to the possible existence of temporary isozymes induced by variations in environmental conditions.
Acknowledoements.. I thank J. Ramus, M. Clutter, J. Powell, J. Grassle, L. Murphy, and A. Stone Ament for their helpful comments and advice concerning the manuscript. This work was supported by a Sigma Xi grant and U.S.P.H.S. grant HD 00032 awarded to the author and N.S.F. grant BMS 75-00436 awarded to J. Ramus. REFERENCES ALLEE W. C. (1923) Studies in marine ecology--IV. The effect of temperature in limiting the geographical range of invertebrates of the Woods Hole littoral. Ecoloyy 4, 341 •354.
lsozyme induction in Arbacia BALDWIN J. & HOCHACHKAP. (1970) Functional significance of isoenzymes in thermal acclimation. Biochem. J. 116, 883-887. BUMPUSD. (1957) Surface water temperatures along Atlantic and Gulf coasts of the United States. U.S. Fish Wildl. Sert,. Spec. Sci. Rep. 214, 1-153. FLOWERDEW M. W. ~. CRISP D. J. (1976) Allelic esterase isozymes, their variations with season, position on the shore, and stage of development in the Cirripede, Balanus balanoides. Mar. Biol. 35, 319-325. HARVEY E. B. (1956) The American Arbacia and otlter sea urchins. Princeton University Press, Princeton. HOCHACHKAP. & SOMEROG. (1973) Strategies of Biochemical Adaptation. W. B. Saunders Co,, Philadelphia. KENT J. D. & HART R. G. (1976) The effect of temperature and photoperiod on isozyme induction in selected tissues of the creek chub, Semotilus atromaculatus. Comp. Biochem. Physiol. 54B, 77-80.
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LEVtN D., HOWl_ANDP. & STONER E. (1972) Protein polymorphism and genie heterozygosity in a population of the permanent translocation heterozygote Oenothera biennis. Proc. Natn Acad. $ci. U.S.A. 69, 1475-1477. O<;IT^ Z. (1975) Genetic control and epigenetic modification of human serum cholinesterase. In lsozymes--ll. Physiological function. (Edited by MARKERT C.), pp. 289-314. Academic Press, New York. OxeogD G. S. (1975) Food induced esterase phenocopies in the snail Cepaea nemoralis. Heredity 35, 361-370. SHAW C. R. & PRA,~D R. (1970) Starch gel electrophoresis of enzymes--a compilation of recipes. Biochem. Genet. 4, 297-320. SOM~ROG. (1969) Pyruvate kinase variants in the Alaskan king crab. Biochem. J. 114, 237-241.