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The adaptation of cellulose acetate electrophoresis to fish enzymes
Comp. Biochem. Physiol., VoL 6111, pp. 421 to 425 © Peryamon Press Ltd I978. Printed in Great Britain
0305-0491/78/1015-0421502.00/0
THE ADAPTATION OF CELLULOSE ACETATE ELECTROPHORESIS TO FISH ENZYMES R. W. GAULDIE and P. J. SMITH Fisheries Research Division, P.O. Box 19062, Wellington, New Zealand
(Received 7 March 1978) Abstraet--l. Fast, inexpensive, cellulose acetate strips allowed screening of five species a day for 22 enzymes. 2. Serum of Cheilodactylus macropterus from 2700 individuals were screened in 7 days for allelic variation of phosphoglucomutase.
INTRODUCTION
The use of protein polymorphisms as genetic tags in the separation of fish stocks is a well-described biological tool (Jamieson, 1974; Utter et al., 1974). The laboratory procedure for this technique can be divided into two parts: (a) screening for polymorphic loci over a few small samples and (b) examining the polymorphic loci in a large n u m b e r of samples. This second stage can prove labour intensive and also costly when the enzymes under investigation require expensive substrates a n d cofactors in the staining preparation. Cellulose acetate has been widely used to examine fish proteins but was superseded by the starch and acrylamide support media which gave greater resolution of protein b a n d s [see for instance Jimenez & Planas (1973)1. We have adopted a standardized cellulose acetate m e t h o d presented by the Helena c o m p a n y to provide a cheap and easily reproducible technique for the separation of teleost enzymes. This
paper describes the buffer systems and enzymes tested in a n u m b e r of teleosts and invertebrates. MATERIALS AND METHODS
(a) Gel plates We used mylar backed Titan III and Titan IIl-lso Zip Zone Cellulose Acetate Plates catalogue nos. 3023 and 3905, respectively, manufactured by Helena Laboratories, P.O. Box 752, Beaumont, TX, U.S.A. Four batches of 3023 plates (7573023, 7673023, 4023023 and 1873023) were used. One batch of 3905 plates (18073905) was used. (b) Tank and 9el buffers The different buffers tested are shown in Table 1, along with their resolution for phosphoglucomutase and esterase; representative slow and fast migrating enzymes. Gel buffer strength was kept below 0.025 M to prevent hysteresis. (c) Sample material and sample preparation A complete list of species surveyed including common names and families is given in Table 2. Livers of the tarak-
Table 1. Buffers tested. Buffer ionic strength is given in moles Buffer
Cathode Anode *gel pH PGM
1. Helena HR Tris-barbital sodium barbital 2. 0.378 Tris, 0.165 M atric acid 3. Sodium acetate, sodium barbitone, barbitone 4. Sodium barbitone, barbitone 5. Tris, EDTA, boric acid 6. Tris 0.1 M, maleic 0.1 M, 0.1 M EDTA 7. K2HPO,, 0.214 M, citric acid 0.027 M 8. Tris-HCl 0.5 M 9. Tris 0.155 M, citric acid 0.043 M
EST
9.6
9.2
9.2
+ +
+ +
6.0
6.0
6.0
0
0
8.6
8.6
8.6
+ +
+
8.6
8.6
8.6
+
+
9.0
9.0
9.0
+
+
7.4
7.4
7.4
0
0
7.0 9.0
7.0 9.0
7.0 9.0
0 0
0 0
7.0
7.0
7.0
0
0
Buffers 3, 4 and 5 were taken from Sargent & George (1975). Buffers 2, 6, 7, 8 and 9 were taken from Shaw & Prasad (1970). Buffer 1 is a commercial preparation. Good resolution is shown as + +, reaction showing presence of enzyme but poor resolution is shown as +. No enzyme reaction, or extensive smearing is shown as 0. * Gel ionic strength was adjusted below 0.25 M to prevent hysteresis. 421
422
R.W. GAULDIE and P. J. SMITH Table 2. List of species tested giving common and scientific names and families Common name a b d e f g h i j k 1 m n o p q r s t u v w x
Tarakihi Trevally Barracouta Gurnard Jack mackerel Opal fish Sardine Warehou Parore Silver dory John dory Kingfish Kahawai Porae Snapper Hake Ling Leatherjacket Blue mackerel Southern kingfish Elephant fish Jack mackerel Short-fin eel Invertebrates
Scientific name
Family
Cheilodactylus macropterus Caranyx georgianus Thyrsites atun Trigla kumu Trachurus declivis Hemerocoetes sp. Sprattus antipodum Seriolella brama Girella tricuspidata Cyttus australis Zeus.japonicus Seriola grandis Arripis trutta Cheilodactylus douglasi Chrysophrys auratus Merluccius australis Genypterus b l a c o d e s Novodon scaber Scomber australasicus Rexea solandri Callorhynchus milii Trachurus novaezelandiae Anguilla australis
Cheilodactylidae Carangidae Gempylidae Triglidae Carangidae Percophididae Clupeidae Centrolophidae Kyphosidae Zeidae Zeidae Carangidae Arripidae Cheilodactylidae Sparidae Merluccidae Genypteridae Balistidae Scombridae Gempylidae Callorhynchidae Carangidae Anguillidae
X enostrobus pulex Aulacomya maoriana Paphies australis Stichopus mollis Pagurus sp. Jasus edwardsii
Mytilidae Mytilidae Mesodesmatidae Holothuridae Paguridae Palinuridae
ihi (Cheilodactylus macropterus) were collected both at sea and from ungutted fish in fish packing and processing sheds. It is customary in most New Zealand fisheries to bring whole ungutted fish on ice into the packing sheds. The muscle tissue of the trevally (Caranyx georgianus), snapper (Chrysophrys auratus) and the marine crayfish (Jasus edwardsii), as well as the molluscs, the pagurid and holothurians surveyed, were collected at sea into liquid nitrogen, and stored at - 7 0 ° C in the laboratory. All of the remaining teleosts were surveyed from liver samples taken from packing sheds. Livers taken from the packing sheds were individually stored in labelled plastic bags, frozen in liquid nitrogen and were maintained at -20°C in the laboratory. No differences in resolution or mobility were noted between fresh livers and those collected in packing sheds. Samples were collected during the winter months and it is possible that samples collected in the summer may suffer some spoilage with resulting loss of resolution. Samples were prepared for electrophoresis by grinding pieces of frozen tissue in equal volumes of homogenizing buffer in glass test tubes with a glass rod. Four homogenizing buffers were tested: 0.1 M phosphate buffer pH 7.0, distilled water, distilled water with approximately 10 mM mereaptoethanol and distilled water with 0.5~o sucrose. No differences were detected among these homogenizing buffers; nonetheless, distilled water with approximately 10 mM mercaptoethanol was used routinely in sample preparation. The resolution of certain enzymes, esterases, for example, was greatly improved by extraction (simple shaking) with carbon tetrachloride. Samples were not centrifuged. Samples were printed on to the gel plates using the Helena Zip Zone Applicator following the instructions issued by the Helena company. In some cases multiple applications could be made on the same plate (Fig. l a).
(d) Stain buglers and procedures Stain recipes were taken from Shaw & Prasad (1970) and Harris & Hopkinson (1976). Stains were applied to the gels with a paint brush. Stain volumes were consequently very low and 5 ml of stain was sufficient to process up to 600 samples. Stain materials were usually made fresh each day, but were occasionally kept for up to 36hr in the dark without loss of reactivity. (e) Voltage and current conditions Most gels were run for about 20 rnin at 200 V at 1-3 mA/gel. Jack mackerel enzymes (Trachurus declivis and T. novaezelandiae) migrated very slowly and required up to 45 rain for clear resolution. All electrophoresis was carried out at room temperature in standard Helena electrophoresis tanks. RESU LTS A range of buffers was tested against resolution of a slowly migrating enzyme, phosphoglucomutase, and a rapidly migrating enzyme, esterase (Table 2). The best buffer system proved to be the Helena HR buffer adjusted to p H 9.6 at the cathode a n d pH 9.2 at the anode with gel buffer at pH 9.2 (buffer n u m b e r 1 in Table 1). This buffer proved capable of resolving a wide range of enzymes. Table 3 (teleost enzymes) a n d Table 4 (invertebrate enzymes) gives the results of a screening experiment undertaken to detect polymorphisms in 22 enzymes in 22 teleosts and one elasmob r a n c h (Callorhynehus milii) and in five enzymes in six invertebrates which were all resolved using the HR buffer. A n u m b e r of enzymes (see Table 3) were detected in some fish and not in others. We did not explore a full range of tissues (all of the results in
The adaptation of cellulose acetate electrophoresis
423
a
0
0
-f} ..--m
PGM
•q.-- $
O
Fig. I. (a} The fast (fk medium (m) and slow (sl alleles of tarakihi (Cheilodactylus macropterus) liver phosphoglucomutase. The upper and lower printations are identical while the centre are of another s~,mple set. The lower printation has migrated cathodally in the usual manner while the upper printation although maintaining the fast, medium and slow configuration, has moved anodally due to electroendosmosis.
b
=
PGM2
4
e
4
f
4
m
GPI
S
PGM~ -.---'- 0
, kig. I. (b} Four alleles e, f, m and s of the jack mackerel (Trachurus declivis} liver glucose phosphate isomerase are shown in addition to the two monomorphic phosphoglucomutase loci. The gel plate was stained with a mixture of GPI and P G M substrates.
424
R. W. GAULD1E and P. J. SMITH Table 3. Results of screening for polymorphism in teleost enzymes using the Helena HR buffer described in the text
¢~
u.1
~'~
~ ~
?~ ~ ~ r~
~
Teleost enzymes 1. Sorbitol dehydrogenase 2. Fumarase 3. Glutamate-oxaloacetate transaminase
~
~
0 MMMMMM 0MMMMS
=
6
~
6" ," ~
P
M
MMMMMMM MM--
M 0
M--MMM 0
0
0
0 0 - - 0 0
o 0
O0 O0
0--MMMMM
M
M--MMM--
00
MMM--M
M
MMM--
M
MOMMM--
OM
000--M
M
MMM----M
0
M
0--00
M
MMMM,
0
0
0
--0
M M 0
MMMMM 0 MMM MMM 0 M 0 M 0 0 0 0M 0 0 0 0 0 0
M
MM
M
M--MMM--O
P
MMMMM--
P M P M M 0 0 0
P-P MM 0 M--MMM P P P P M M P M M M M M 0 0 0 0 0 0 0 0 P 0 P 0 0 0 "O'M P M M M M MMMMPMO 0 0 0 0 0 00 00----M 00 MP OMMMPMMM P 0 00MO PM
~
"4 ~
~
0 --
S 0
0
--
M
M
M
--
0
--
M
-
0
4. Malate dehydrogenase
5. 6. 7. 8. 9. 10. 11.
12. 13. 14. 15.
16. 17. 18. 19. 20. 21. 22.
Malic enzyme Xanthine dehydrogenase 7-Glycerophosphate dehydrogenase Hydroxybutyrate dehydrogenase Alcohol dehydrogenase Glutamate dehydrogenase Hexose-6dehydrogenase Lactate dehydrogenase A B C Superoxide dismutase Isocitrate dehydrogenase 6-Phosphogluconate dehydrogenase Glucose-6-phosphate dehydrogenase Phosphoglucomu- A tase B Glucosephosphate A isomerase B Hexokinase Adenylate kinase Creatine kinase Esterase A B Leucine amino peptidase
M--
M
0
SMM
0 0 MM 0
MMMMMPM
O--S--
--
0
0 M--M 0 0--0 0 00
--
MMM--MMM
0
PM
0 MMMMM--
--
--0
--
0 --M------
0
0
0
S .
.
.
M 0
000--M----M
M
P
M0--0--0
0 MPMMMM S
MMM M
.....
.
.
0 0 0
----
M
--
0
0 M
0
--M
M
MP
P--MPM
M
MMM--MMM
M
MMM--M P M M0 P--PMM 0M------0M M 0 P PP P P P ----0 0 0 0 0 0 0 - - - S
.... P 0 P 0
S S
M 0
S
-
M 0 0 M M 0 0
M 0 0 0
0 M
M M
S MMPM OM 0 0
0
M
--
S --M
S
--
P 0
S S
M 0 M 0 0
Species and tissue type are shown. Enzyme status, polymorphic or monomorphic is shown as P or M. Enzymes that showed no activity are indicated as 0. Enzymes not tested for are shown as --. Enzymes that show activity but are too streaky to score are shown as S. Isozymes are indicated A, B and C with A as most cathodal and C most anodal.
T a b l e 3 c a m e from liver tissue except (c) muscle of Caranyx georgianus, consequently, we c a n n o t tell w h e t h e r the absence of e n z y m e activity was a result of i n a p p r o p r i a t e buffer or due to the p r e p a r a t i o n of the s a m p l e or tissue specificity. T w o enzymes, hexose6 - d e h y d r o g e n a s e and creatine kinase, were not
detected in any species. Again, we d o n o t k n o w w h e t h e r the lack of activity was due to i n a p p r o p r i a t e buffer or s a m p l e p r e p a r a t i o n . Loci were classed p o l y m o r p h i c if the frequency of h e t e r o z y g o t e s w a s greater t h a n one in eight, i.e. comm o n allele <0.94. F o r s o m e species only eight fish
The adaptation of cellulose acetate electrophoresis
425
Table 4. Results of screening for polymorphisms in invertebrate enzymes using Helena HR buffer described in the text
Invertebrate enzymes I. Phosphoglucomutase A B
2. Glucose phosphate isomerase 3. Esterase 4. Lactate dehydrogenase 5. Glucose-6-phosphate dehydrogenase
M 0 P
M M P
M 0 P
--
--
--
--
--
--
0
0
0
P 0
S S
M
M
M
M
M
--
S M
0
0
M
Legend as for Table 3.
were used as our interest in polymorphism extends only to those in excess of 5%. (Lower frequencies would require unmanageably large samples for effective analysis of isolated stocks of fish.) We noted some variation in the resolution obtained using different types of gel plates, and also noted variation between batches of the same type. These latter differences were overcome by minor changes in the pH of the tank buffers but the differences between plate types was very great. We found that the best general resolution was obtained with type 3023 plates. The resolution of alleles was clear with a great difference in relative mobilities between bands (Figs la and b). The electroendosmosis in Fig. l(a) appeared to affect phosphoglucomutase and not other enzymes. The effect could be overcome by increasing the gel buffer ionic strength.
CONCLUSION While the cellulose acetate technique is well established in clinical biochemistry it has made little impact on the comparative studies of allozymes in fish. It is a fast and relatively cheap technique. We were able to screen five species a day for some 22 enzymes in the search for polymorphisms, and with the tarakihi (Cheilodactylus macropterus) we were able to process a New Zealand-wide sample of 2700 individuals for allelic variation at the phosphoglucomutase locus in 7 working days. It is possible to stain simultaneously for more than one locus but this usually decreases the number of samples per plate.
Cellulose acetate gels do not require cooling during electrophoresis nor do samples require centrifugation. This minimal preparation and the rapidity of the electrophoresis itself, combined with high resolution, make this technique particularly suited to fisheries research which often requires the processing of large samples within a short period of time. Acknowledgements--We are indebted to Dr Barry Richardson of the Australian National University for his generous advice and encouragement in the application of cellulose acetate gels. Dr P. J. Smith was supported by an New Zealand National Research Advisory Council postdoctoral fellowship. R E F E R E N C E S
HARRISH. & HOPKINSOND. A. (1976)Handbook of Enzyme Electrophoresis in Human Genetics. North-Holland. JAMIESON A. (1974) Genetic tags for marine fish stocks. In Sea Fisheries Science (Edited by HARDEN-JONES F. R.), pp. 91-99: Elk Science, London. JIMENEZ M. & PLANAS J. (1973) Plasma proteins of the goosefish Lophius piscatorius (L.). J. Fish. Biol. 5. 125-130. "SARGENT J. R. & GEORGE S. G. (1975) Methods in Zone Electrophoresis, 3rd edition, 219 pp. BDH Chemicals, Poole. SHAW C. R. & PRASAD R, (1970) Starch gel electrophoresis of enzymes--a compilation of recipes. Biochem. Genet. 4, 297-320. UTTER F. M., HODGINS H. D. & ALLENDORF F. W. (1974) Biochemical genetic studies of fishes: potentialities and limitations. In Biochemical and Biophysical Perspectives in Marine Biology, Vol. 1 (Edited by SARGENT J. R.), pp. 213 238. Academic Press, New York.
0305-0491/78/1015-0421502.00/0
THE ADAPTATION OF CELLULOSE ACETATE ELECTROPHORESIS TO FISH ENZYMES R. W. GAULDIE and P. J. SMITH Fisheries Research Division, P.O. Box 19062, Wellington, New Zealand
(Received 7 March 1978) Abstraet--l. Fast, inexpensive, cellulose acetate strips allowed screening of five species a day for 22 enzymes. 2. Serum of Cheilodactylus macropterus from 2700 individuals were screened in 7 days for allelic variation of phosphoglucomutase.
INTRODUCTION
The use of protein polymorphisms as genetic tags in the separation of fish stocks is a well-described biological tool (Jamieson, 1974; Utter et al., 1974). The laboratory procedure for this technique can be divided into two parts: (a) screening for polymorphic loci over a few small samples and (b) examining the polymorphic loci in a large n u m b e r of samples. This second stage can prove labour intensive and also costly when the enzymes under investigation require expensive substrates a n d cofactors in the staining preparation. Cellulose acetate has been widely used to examine fish proteins but was superseded by the starch and acrylamide support media which gave greater resolution of protein b a n d s [see for instance Jimenez & Planas (1973)1. We have adopted a standardized cellulose acetate m e t h o d presented by the Helena c o m p a n y to provide a cheap and easily reproducible technique for the separation of teleost enzymes. This
paper describes the buffer systems and enzymes tested in a n u m b e r of teleosts and invertebrates. MATERIALS AND METHODS
(a) Gel plates We used mylar backed Titan III and Titan IIl-lso Zip Zone Cellulose Acetate Plates catalogue nos. 3023 and 3905, respectively, manufactured by Helena Laboratories, P.O. Box 752, Beaumont, TX, U.S.A. Four batches of 3023 plates (7573023, 7673023, 4023023 and 1873023) were used. One batch of 3905 plates (18073905) was used. (b) Tank and 9el buffers The different buffers tested are shown in Table 1, along with their resolution for phosphoglucomutase and esterase; representative slow and fast migrating enzymes. Gel buffer strength was kept below 0.025 M to prevent hysteresis. (c) Sample material and sample preparation A complete list of species surveyed including common names and families is given in Table 2. Livers of the tarak-
Table 1. Buffers tested. Buffer ionic strength is given in moles Buffer
Cathode Anode *gel pH PGM
1. Helena HR Tris-barbital sodium barbital 2. 0.378 Tris, 0.165 M atric acid 3. Sodium acetate, sodium barbitone, barbitone 4. Sodium barbitone, barbitone 5. Tris, EDTA, boric acid 6. Tris 0.1 M, maleic 0.1 M, 0.1 M EDTA 7. K2HPO,, 0.214 M, citric acid 0.027 M 8. Tris-HCl 0.5 M 9. Tris 0.155 M, citric acid 0.043 M
EST
9.6
9.2
9.2
+ +
+ +
6.0
6.0
6.0
0
0
8.6
8.6
8.6
+ +
+
8.6
8.6
8.6
+
+
9.0
9.0
9.0
+
+
7.4
7.4
7.4
0
0
7.0 9.0
7.0 9.0
7.0 9.0
0 0
0 0
7.0
7.0
7.0
0
0
Buffers 3, 4 and 5 were taken from Sargent & George (1975). Buffers 2, 6, 7, 8 and 9 were taken from Shaw & Prasad (1970). Buffer 1 is a commercial preparation. Good resolution is shown as + +, reaction showing presence of enzyme but poor resolution is shown as +. No enzyme reaction, or extensive smearing is shown as 0. * Gel ionic strength was adjusted below 0.25 M to prevent hysteresis. 421
422
R.W. GAULDIE and P. J. SMITH Table 2. List of species tested giving common and scientific names and families Common name a b d e f g h i j k 1 m n o p q r s t u v w x
Tarakihi Trevally Barracouta Gurnard Jack mackerel Opal fish Sardine Warehou Parore Silver dory John dory Kingfish Kahawai Porae Snapper Hake Ling Leatherjacket Blue mackerel Southern kingfish Elephant fish Jack mackerel Short-fin eel Invertebrates
Scientific name
Family
Cheilodactylus macropterus Caranyx georgianus Thyrsites atun Trigla kumu Trachurus declivis Hemerocoetes sp. Sprattus antipodum Seriolella brama Girella tricuspidata Cyttus australis Zeus.japonicus Seriola grandis Arripis trutta Cheilodactylus douglasi Chrysophrys auratus Merluccius australis Genypterus b l a c o d e s Novodon scaber Scomber australasicus Rexea solandri Callorhynchus milii Trachurus novaezelandiae Anguilla australis
Cheilodactylidae Carangidae Gempylidae Triglidae Carangidae Percophididae Clupeidae Centrolophidae Kyphosidae Zeidae Zeidae Carangidae Arripidae Cheilodactylidae Sparidae Merluccidae Genypteridae Balistidae Scombridae Gempylidae Callorhynchidae Carangidae Anguillidae
X enostrobus pulex Aulacomya maoriana Paphies australis Stichopus mollis Pagurus sp. Jasus edwardsii
Mytilidae Mytilidae Mesodesmatidae Holothuridae Paguridae Palinuridae
ihi (Cheilodactylus macropterus) were collected both at sea and from ungutted fish in fish packing and processing sheds. It is customary in most New Zealand fisheries to bring whole ungutted fish on ice into the packing sheds. The muscle tissue of the trevally (Caranyx georgianus), snapper (Chrysophrys auratus) and the marine crayfish (Jasus edwardsii), as well as the molluscs, the pagurid and holothurians surveyed, were collected at sea into liquid nitrogen, and stored at - 7 0 ° C in the laboratory. All of the remaining teleosts were surveyed from liver samples taken from packing sheds. Livers taken from the packing sheds were individually stored in labelled plastic bags, frozen in liquid nitrogen and were maintained at -20°C in the laboratory. No differences in resolution or mobility were noted between fresh livers and those collected in packing sheds. Samples were collected during the winter months and it is possible that samples collected in the summer may suffer some spoilage with resulting loss of resolution. Samples were prepared for electrophoresis by grinding pieces of frozen tissue in equal volumes of homogenizing buffer in glass test tubes with a glass rod. Four homogenizing buffers were tested: 0.1 M phosphate buffer pH 7.0, distilled water, distilled water with approximately 10 mM mereaptoethanol and distilled water with 0.5~o sucrose. No differences were detected among these homogenizing buffers; nonetheless, distilled water with approximately 10 mM mercaptoethanol was used routinely in sample preparation. The resolution of certain enzymes, esterases, for example, was greatly improved by extraction (simple shaking) with carbon tetrachloride. Samples were not centrifuged. Samples were printed on to the gel plates using the Helena Zip Zone Applicator following the instructions issued by the Helena company. In some cases multiple applications could be made on the same plate (Fig. l a).
(d) Stain buglers and procedures Stain recipes were taken from Shaw & Prasad (1970) and Harris & Hopkinson (1976). Stains were applied to the gels with a paint brush. Stain volumes were consequently very low and 5 ml of stain was sufficient to process up to 600 samples. Stain materials were usually made fresh each day, but were occasionally kept for up to 36hr in the dark without loss of reactivity. (e) Voltage and current conditions Most gels were run for about 20 rnin at 200 V at 1-3 mA/gel. Jack mackerel enzymes (Trachurus declivis and T. novaezelandiae) migrated very slowly and required up to 45 rain for clear resolution. All electrophoresis was carried out at room temperature in standard Helena electrophoresis tanks. RESU LTS A range of buffers was tested against resolution of a slowly migrating enzyme, phosphoglucomutase, and a rapidly migrating enzyme, esterase (Table 2). The best buffer system proved to be the Helena HR buffer adjusted to p H 9.6 at the cathode a n d pH 9.2 at the anode with gel buffer at pH 9.2 (buffer n u m b e r 1 in Table 1). This buffer proved capable of resolving a wide range of enzymes. Table 3 (teleost enzymes) a n d Table 4 (invertebrate enzymes) gives the results of a screening experiment undertaken to detect polymorphisms in 22 enzymes in 22 teleosts and one elasmob r a n c h (Callorhynehus milii) and in five enzymes in six invertebrates which were all resolved using the HR buffer. A n u m b e r of enzymes (see Table 3) were detected in some fish and not in others. We did not explore a full range of tissues (all of the results in
The adaptation of cellulose acetate electrophoresis
423
a
0
0
-f} ..--m
PGM
•q.-- $
O
Fig. I. (a} The fast (fk medium (m) and slow (sl alleles of tarakihi (Cheilodactylus macropterus) liver phosphoglucomutase. The upper and lower printations are identical while the centre are of another s~,mple set. The lower printation has migrated cathodally in the usual manner while the upper printation although maintaining the fast, medium and slow configuration, has moved anodally due to electroendosmosis.
b
=
PGM2
4
e
4
f
4
m
GPI
S
PGM~ -.---'- 0
, kig. I. (b} Four alleles e, f, m and s of the jack mackerel (Trachurus declivis} liver glucose phosphate isomerase are shown in addition to the two monomorphic phosphoglucomutase loci. The gel plate was stained with a mixture of GPI and P G M substrates.
424
R. W. GAULD1E and P. J. SMITH Table 3. Results of screening for polymorphism in teleost enzymes using the Helena HR buffer described in the text
¢~
u.1
~'~
~ ~
?~ ~ ~ r~
~
Teleost enzymes 1. Sorbitol dehydrogenase 2. Fumarase 3. Glutamate-oxaloacetate transaminase
~
~
0 MMMMMM 0MMMMS
=
6
~
6" ," ~
P
M
MMMMMMM MM--
M 0
M--MMM 0
0
0
0 0 - - 0 0
o 0
O0 O0
0--MMMMM
M
M--MMM--
00
MMM--M
M
MMM--
M
MOMMM--
OM
000--M
M
MMM----M
0
M
0--00
M
MMMM,
0
0
0
--0
M M 0
MMMMM 0 MMM MMM 0 M 0 M 0 0 0 0M 0 0 0 0 0 0
M
MM
M
M--MMM--O
P
MMMMM--
P M P M M 0 0 0
P-P MM 0 M--MMM P P P P M M P M M M M M 0 0 0 0 0 0 0 0 P 0 P 0 0 0 "O'M P M M M M MMMMPMO 0 0 0 0 0 00 00----M 00 MP OMMMPMMM P 0 00MO PM
~
"4 ~
~
0 --
S 0
0
--
M
M
M
--
0
--
M
-
0
4. Malate dehydrogenase
5. 6. 7. 8. 9. 10. 11.
12. 13. 14. 15.
16. 17. 18. 19. 20. 21. 22.
Malic enzyme Xanthine dehydrogenase 7-Glycerophosphate dehydrogenase Hydroxybutyrate dehydrogenase Alcohol dehydrogenase Glutamate dehydrogenase Hexose-6dehydrogenase Lactate dehydrogenase A B C Superoxide dismutase Isocitrate dehydrogenase 6-Phosphogluconate dehydrogenase Glucose-6-phosphate dehydrogenase Phosphoglucomu- A tase B Glucosephosphate A isomerase B Hexokinase Adenylate kinase Creatine kinase Esterase A B Leucine amino peptidase
M--
M
0
SMM
0 0 MM 0
MMMMMPM
O--S--
--
0
0 M--M 0 0--0 0 00
--
MMM--MMM
0
PM
0 MMMMM--
--
--0
--
0 --M------
0
0
0
S .
.
.
M 0
000--M----M
M
P
M0--0--0
0 MPMMMM S
MMM M
.....
.
.
0 0 0
----
M
--
0
0 M
0
--M
M
MP
P--MPM
M
MMM--MMM
M
MMM--M P M M0 P--PMM 0M------0M M 0 P PP P P P ----0 0 0 0 0 0 0 - - - S
.... P 0 P 0
S S
M 0
S
-
M 0 0 M M 0 0
M 0 0 0
0 M
M M
S MMPM OM 0 0
0
M
--
S --M
S
--
P 0
S S
M 0 M 0 0
Species and tissue type are shown. Enzyme status, polymorphic or monomorphic is shown as P or M. Enzymes that showed no activity are indicated as 0. Enzymes not tested for are shown as --. Enzymes that show activity but are too streaky to score are shown as S. Isozymes are indicated A, B and C with A as most cathodal and C most anodal.
T a b l e 3 c a m e from liver tissue except (c) muscle of Caranyx georgianus, consequently, we c a n n o t tell w h e t h e r the absence of e n z y m e activity was a result of i n a p p r o p r i a t e buffer or due to the p r e p a r a t i o n of the s a m p l e or tissue specificity. T w o enzymes, hexose6 - d e h y d r o g e n a s e and creatine kinase, were not
detected in any species. Again, we d o n o t k n o w w h e t h e r the lack of activity was due to i n a p p r o p r i a t e buffer or s a m p l e p r e p a r a t i o n . Loci were classed p o l y m o r p h i c if the frequency of h e t e r o z y g o t e s w a s greater t h a n one in eight, i.e. comm o n allele <0.94. F o r s o m e species only eight fish
The adaptation of cellulose acetate electrophoresis
425
Table 4. Results of screening for polymorphisms in invertebrate enzymes using Helena HR buffer described in the text
Invertebrate enzymes I. Phosphoglucomutase A B
2. Glucose phosphate isomerase 3. Esterase 4. Lactate dehydrogenase 5. Glucose-6-phosphate dehydrogenase
M 0 P
M M P
M 0 P
--
--
--
--
--
--
0
0
0
P 0
S S
M
M
M
M
M
--
S M
0
0
M
Legend as for Table 3.
were used as our interest in polymorphism extends only to those in excess of 5%. (Lower frequencies would require unmanageably large samples for effective analysis of isolated stocks of fish.) We noted some variation in the resolution obtained using different types of gel plates, and also noted variation between batches of the same type. These latter differences were overcome by minor changes in the pH of the tank buffers but the differences between plate types was very great. We found that the best general resolution was obtained with type 3023 plates. The resolution of alleles was clear with a great difference in relative mobilities between bands (Figs la and b). The electroendosmosis in Fig. l(a) appeared to affect phosphoglucomutase and not other enzymes. The effect could be overcome by increasing the gel buffer ionic strength.
CONCLUSION While the cellulose acetate technique is well established in clinical biochemistry it has made little impact on the comparative studies of allozymes in fish. It is a fast and relatively cheap technique. We were able to screen five species a day for some 22 enzymes in the search for polymorphisms, and with the tarakihi (Cheilodactylus macropterus) we were able to process a New Zealand-wide sample of 2700 individuals for allelic variation at the phosphoglucomutase locus in 7 working days. It is possible to stain simultaneously for more than one locus but this usually decreases the number of samples per plate.
Cellulose acetate gels do not require cooling during electrophoresis nor do samples require centrifugation. This minimal preparation and the rapidity of the electrophoresis itself, combined with high resolution, make this technique particularly suited to fisheries research which often requires the processing of large samples within a short period of time. Acknowledgements--We are indebted to Dr Barry Richardson of the Australian National University for his generous advice and encouragement in the application of cellulose acetate gels. Dr P. J. Smith was supported by an New Zealand National Research Advisory Council postdoctoral fellowship. R E F E R E N C E S
HARRISH. & HOPKINSOND. A. (1976)Handbook of Enzyme Electrophoresis in Human Genetics. North-Holland. JAMIESON A. (1974) Genetic tags for marine fish stocks. In Sea Fisheries Science (Edited by HARDEN-JONES F. R.), pp. 91-99: Elk Science, London. JIMENEZ M. & PLANAS J. (1973) Plasma proteins of the goosefish Lophius piscatorius (L.). J. Fish. Biol. 5. 125-130. "SARGENT J. R. & GEORGE S. G. (1975) Methods in Zone Electrophoresis, 3rd edition, 219 pp. BDH Chemicals, Poole. SHAW C. R. & PRASAD R, (1970) Starch gel electrophoresis of enzymes--a compilation of recipes. Biochem. Genet. 4, 297-320. UTTER F. M., HODGINS H. D. & ALLENDORF F. W. (1974) Biochemical genetic studies of fishes: potentialities and limitations. In Biochemical and Biophysical Perspectives in Marine Biology, Vol. 1 (Edited by SARGENT J. R.), pp. 213 238. Academic Press, New York.