81
Technical Report Series
32
Number 74-3
/_
~N OUTLINE OF TECHNIQUES FOR STARCH
GEL ELECTROPHORESIS OF ENlYMES FROM
THE AMERICAN OYSTER CRASSOSTREA
VIRGINICA GMELIN
by
Barbara A. Schaal
and
Wyatt W. Anderson
31
31
Georgia Marine Science Center
University System of Georgia
Skidaway Island, Georgia
81
AN OUTLINE OF TECHNIQUES FOR STARCH GEL ELECTROPHORESIS OF
ENZYMES FROM THE AMERICAN OYSTER CRASSOSTREA VIRGINICA GMELIN
by
Barbara A. Schaal and
Wyatt W. Anderson*
Department of Zoology
University of Georgia
Athens, Georgia 30602
April 1974
The Technical Report Series of the Georgia Marine Science Center is issued
by the Georgia Sea Grant Program and the Marine Extension Service of the University of Georgia on Skidaway Island (P.O. Box 13687, Savannah, Georgia 31406).
It was established to provide dissemination of technical information and progress reports resulting from marine studies and investigations mainly by staff
and faculty of the University System of Georgia. In addition, it is intended
for the presentation of techniques and methods, reduced data and general information of interest to industry, local, regional, and state governments and the
public. Information contained in these reports is in the public domain. If
this prepublication copy is cited, it should be cited as an unpublished manuscript.
*Address correspondence to this author.
ABSTRACT
This bulletin describes techni ques for starch gel electrophoresis
of enzymes from the American oyster, Cras s ostrea virginica.
Procedures
for extraction of oyster tissue, preparation of gels, and staining of
twenty-five enzymes are outlined.
are included.
Recipes f or all necessary solutions
The use of these technique s for genetic analysis is briefly
discussed and illustrated with data f rom a s amp le of Georgia oysters.
INTRODUCTION
The biology of Crassostrea virginica has been intensively studied
(Galstoff, 1964), but its genetics has been neglected until the past
few years.
Our present knowledge of oyster genetics is largely the work
of Longwell and her colleagues (summarized in Longwell and Stiles, 1973),
who have published analyses of (1) cytogenetics of chromosome morphology
and number, gametogenesis, fertilization, and meiosis; (2) inbreeding
depression of fertility in crosses between sibs; (3) selective breeding
for growth rate and resistance to disease; (4) hybridization between
lines, subspecies, and full species ; and (5) irradiation and mutation.
These investigations have opened the way for a successful breeding program, but several important areas of research remain untouched.
In
particular, the genetic structure of natural oyster populations has not
been determined.
The genetic structure of a population would be completely known
if the genotype of each individual at each gene locus were known.
This
ideal has never been realized, of course, since an organism as complex
as an oyster has perhaps 10,000 different genes on its chromosomes.
However, a new technique
gel electrophoresis of proteins -- has in
the last decade permitted a direct attack on the problem of genetic population structure.
The ultimate product of gene activity is usually an
enzyme; different alleles at a particular gene locus will often specify
enzymes or other proteins differing in their net electrical charges.
In gel electrophoresis, an extract of tissue is embedded in a suitable
2
gel and placed in a strong electr i c field .
The different allelic forms of
each enzyme migrate different di stances in the gel, and selective histochemical staining permits local i zation of the enzyme forms representing the
alleles at a single gene locus.
forty or so enzymes.
Stains a r e presently available for some
In organisms where Mendelian breeding tests are pos s i -
ble, these allelic variants of enzymes have been proven to be codominant
alleles at single gene l ocus.
Thus , even in organisms not amenable to
"Mendelian" experiments, it is possible t o infer the genotype of an individual
at many loci from the enzyme variant s t hat individual carries.
The genes to
be studied are chosen by the avail ab ility of appropriate staining techniq ues
and not with regard to the functions they specify; hopefully, they will be a
nearly random sample from the entire set of genes.
In this way; the popula-
tion genetics of pl ants, f i sh, mammal s , insects, and other invertebrates
have been examined.
This report is an outline of the techniques we have developed for starch
gel electrophoresis of
f·
virginica f rom the Georgia coast.
These techniques
were modifi ed from a number of sour ces , including Brewer (1970)and Nicols
and Ruddle (1974).
We chose those l oci giving clear resolution in the oyster
and at the same time allowing economical and efficient analysis.
Twenty-
five enzyme systems were selected from a total of forty which we examined.
Several loci may be detected by a sing le enzyme assay; our banding patterns
indicate two loci for the following enzyme systems:
GOT, LAP, Leu leu, Leu
val, MDH, MGPI, PGM, and TO (see section G for explanation of the abbrev iations).
Thus, we can analyze for 31 dif f erent genes.
We have completed a preliminary anal ysis of these 31 loci in a sample
of 100 oysters from the mouth of the Dar i en River, Mcintosh County,
Georgia.
Thirteen of the thirty-one loci, or 42%, were
3
polymorphic; the average heterozygo sity per individual was 12%.
These
preliminary results indicate that gene t ic diversity in the American oyster is similar to that reported for many other invertebrates (Lewontin,
1974).
REFERENCES
Brewer, G. J.
1970.
Introduction to I so zyme Techniques.
Academic Press ,
New York.
Galstoff, P. S.
1964.
The American oyster Crassostrea virginica Gmelin.
Fisheries Bull. of the Fish and Wildlife Service, 64.
Lewontin, R. C.
1974.
The Genetic Basis of Evolutionary Change.
Columbia
University Press, New York.
Longwell, A. Crosby, and S. S. Stil es.
1973.
Oyster genetics and the
probable future role of genetics in aquaculture.
Malacological
Review, Jan. 1973.
Nicols, E. A., and F. H. Ruddle.
1974.
A review of enzyme polymorphism,
linkage and electrophoretic cond itions for mouse and somatic cell
hybrids i n starch gels.
Am. J . Human Genet.
(in press).
Figure 1.
Part of adductor muscle used for electrophoresis.
Figure 2.
Grinding oyster tissue.
4
PROCEDURES
A.
Preparation of gels
1.
Weigh starch, 40gm per gel and place in a 2 !.vacuum flask.
Electrostarch
(Otto Hiller, Madison, Wise.) gives the best results.
2.
Add 333 ml of the appropriate gel buffer solution; section E lists the
proper buffer for each enzyme.
3.
Simultaneously stir and heat the starch solution until it thickens and
reaches 80° C.
4.
Aspirate the starch . until all small bubbles disappear.
5.
Pour the starch into molds; remove any air bubbles with a Pasteur pipet.
6.
Allow the gels to cool to room temperat ure and then store them in a
refrigerator, preferably overnight, until use.
b.
Preparation of oysters
1.
Open oyster and cut out tissue to be analyzed.
We use the adductor muscle.
2.
Place tissue in a 2 m1 disposable beaker on ice.
3.
Add .5 ml cold gel buffer; section E lists the proper buffer for each
gel system.
4.
Grind the tissue thoroughly with the power mixer, using a glass rod that
just fits into the disposable plastic beakers.
5,
Keep extracts cool throughout; they may be frozen and stored for future
analysis.
C.
Loading and running gels
1.
Cut a slit in the gel for placement of samples, at the center for triacitrate and dehydrogenase gels, and at one-fourth the gel length for
Poulik gels.
2.
Absorb oyster extract onto filter paper strips (3 x 6 mm, Whatman #3)
and place in the slit.
Figure 3.
Loading oyster extracts into gel.
Figure 4.
Buffer tray used for electrophoresis.
5
3.
Place the gel on the buffer tray and fill tray with appropriate bu ff er as
listed in section F.
4.
Place sponge wicks into tray buffer a nd over ends of gel.
Cover gel with
Saran wrap.
5.
Turn on power supply; allow it towarmup for a minute
for all gels.
6.
Heathkit high voltage power supply IPW-17 is satisfactory.
Run dehydrogenase and Poulik gels 5 hours and tris-citrate gels 3 hours .
Gels are run in a low temperature i ncubator at 4
D.
and set at 30 rna
0
C.
Slicing and staining gels
1.
Indicate orientation of gel by punching holes . in one corner.
2.
Slice the gel into four layers with gel c utter.
steel wire stretched across a pl astic frame.
Our slicer is a stainless
Keep gel slices cool while
handling.
3.
Place gel slices in staining trays, add staining solutions, and incubate
as indicated in section G.
4.
Agar overlays:
place gel on a glass pl ate; combine equal parts of stain-
ing solution and 2% agar at 60
and incubate at 37° C.
0
C; pour over gel; cover with Saran wrap;
Agar overl a ys are generally used when expensive
chemicals are involved.
E.
Gel buffer systems
1.
Gel buffer system which gives best r esults for each enzyme assay:
enzymes run well in two buffer systems.
Some
Figu re 5 .
Electrophoresis in operation .
Figure 6.
Slicing gel for subsequent staining.
6
Poul ik
Tria-citrate
Dehydrogena se
AD KIN
ACP
HK
AD KIN
NSP
HK
ADH
LAP
ALKP
ODH
IDH
AD KIN
MDH
CAT
PGI
ME
ALKP
MPI
CK
PGM
NSP
CAT
NSP
GDH
PK
CK
PEP
GOT
6-PGDH
EST
PGI
MDH
XDH
G-6-PGDH PGM
MPI
SDH
GDH
PK
GOT
2.
F.
Gel buffer solutions
a.
Paulik gel:
use 333 ml of Paulik gel buffer.
b.
Dehydrogenase gel:
c.
Tris-citrate gel:
d.
Buffer recipes are given in sec tion F.
use 333 ml of dehydrogenase buffer.
add 11.7 ml tris-citrate buffer to 321 . 3 ml H2 0.
Buffer recipes
1.
Gel and tray buffers.
a.
Tris-citrate pH 5.8
(1)
Gel buffer:
162 gm tris
108.6 gm citric acid
6 1 H20
(2)
Tray buffer:
same as gel buffer
b.
Paulik discontinuous
7
(l)
Gel buffer pH 8.65:
37.2 gm tris
4. 202 gm citric acid
4.0 1 H2 0
(2)
Tray buffer pH 8.1:
76.44 gm boric acid
10.08 gm NaOH
4.2 1 H 0
2
c.
Dehydrogenase buffer
(1)
Gel buffer pH 9.0:
42.16 gm tris
2.16 gm boric acid
1.64 gm EDTA
4.0 1 H2 0
(2)
Tray buffer:
same as gel buffer
2.
Staining buffers
a.
The molarities and pH's of the various tris-HC1 and phosphate buffers
are given in section G on staining techniques.
b.
Each tris-HCl buffer is made from a solution of tris at the desired
molarity and adjusted to the appropriate pH with concentrated HCl.
c.
Each phosphate buffer is made from Na
HPo 4
2
and NaH
Po 4
2
solutions at
the desired molarity; pH is adjusted by mixi ng the two solutions.
G.
Staining techniques:
1.
These assays give consistently clear resolution.
Adenylate Kinase (ADKIN)
a.
Stain with:
8
900 mg glucose
160 units hexokinase
50 units glucose-6-phosphate dehydrogenase (G-6-PDH)
20 mg ADP
.5 ml 10% MgC1 2
25 mg NADP (TPN)
20 mg MTT (MTT tetrazolium)
5 mg PMS (phenazine methosulfate)
15 ml .5 M Tris-HCl pH 7.1
b.
2.
3.
Combine with 15 ml 2% agar; incubate at 37
0
C.
Catalase (CAT )
a.
Cover gel for one minute with .5% H o .
2 2
b.
Pour off and rinse with H20.
c.
Stain in · lOO ml .5% KI solution acidified with .5 ml acetic acid.
Creatine Kinase (CK)
a.
·stain with:
20 mg glucose
10 mg ADP
2 mg MgC1
2
10 units G-6-PDH
2 mg MTT
3 mg PMS
4 mg NADP
10 ml .1 M Tris-HCl pH 8.0
b.
Combine with 15 ml 2% agar and incubate at 37° C.
9
4.
Esterase (EST )
a.
Soak gel in .5M boric acid for 1 hr.
b.
Stain with:
2 ml esterase substrate solution
100
mg
fast garnet GBC
40 ml .1 MP0
4
pH 6.5
60 ml H2 0
c.
Incubate at room temperature in the dark .
d.
Substrate solution:
1 gm a.-naphthyl acetate
50 ml acetone
50 ml H20
5.
Glucose-6-phosphate dehydrogenase ( G-6-PDH)
a.
Stain with:
20 mg Na
2
glucose -6-phosphate
30 mg NADP
20 mg MTT
2 mg PMS
10 ml .1M Tris-HCl pH 7.0
b.
6.
Combine with 10 ml 2% agar and pour on gel; incubate at 37
Glutamate Dehydrogenase (GDH)
a.
Stain with:
5 ml substrate solution
50 mg NAD (DPN)
30 mg MTT
2 mg PMS
25 ml .5 M P0
70 ml H 0
2
4
buffer, pH 7.0
0
C.
b.
Incubate at 37° C.
c.
Substrate solution
10
4.25 g Na glutamate
100 ml .5 M P0
7.
4
pH 7.0
Glutamate-Oxaloacetate Transaminase
a.
Stain with:
50 ml substrate solution
250 mg fast blue BB
50 ml H 0
2
c.
b.
Incubate at 37°
c.
Substrate solution, pH 7.4:
.146 gm a-ketoglutaric acid
.532 gm L-aspartic acid
2.0 gm polyvinylpyrolidone
.2 gm EDTA
5.68 gm Na HPo 4
2
200 ml H20
8.
Hexokinase (HK)
a.
Stain with:
1 gm glucose
25 mg ATP
20 mg MgC1
2
80 units G-6-PDH
20 mg MTT
25 mg NADP
5 mg PMS
100 ml .1M Tris-HCl pH 7.1
b.
Incubate at 37° C.
<Gor)
11
9.
Isocitrate Dehydrogenase (IDH)
a.
Stain with:
150 mg Na 3 isocitric acid
20 mg MnC1 2
20
mg MTT
20 mg
NADP
5 mg PMS
100 m1 .1 M Tris-HCl pH 8.4
b.
10.
Incubate at 37° C.
Leucine Amino Peptidase (LAP)
a.
Soak gel in .5 M boric acid for 20 min.
b.
Stain with:
SO ml sol. A
10 ml sol. B
70 mg 1-leucyl-B-naphthylamide
30 mg fast black K salt
c. · Incubate at 37
d.
0
C.
Sol. A:
8 gm NaOH
19.6 gm maleic anhydride
e.
Sol. B:
12.8 gm NaOH
1.0 1 H 0
2
11.
Malic Dehydrogenase (MDH)
a.
Stain with:
12
10 ml malic substrate sol .
50 mg NAD
30 mg MTT
2 mg PMS
80 ml H 0
2
10 m1 .1M Tris-HCl pH 7.0
b.
Incubate at 37° C.
c.
Substrate solution, pH 7.0 :
13.4 gm Na-L-ma.lic acid
49 ml 2M Na
co3
2
51 ml H 0
2
Dissolve malic acid in H2 0; slowly add Na 2co sol. while swirling in
3
an i ce bath; adjust pH with con HCl.
12.
Malic Enzyme (ME)
a.
Stain with:
5 ml malic substrate (see MDH)
1 ml 10% MgC1 2
20 mg NADP
20 mg NBT (nitro blue tetrazol ium)
10 mg PMS
20 m1 .1 M Tris-HCl pH 8.4
75 m1 H20
b.
13.
Incubate gels at 37° C.
Mannose-6-Phosphate Isomerase (MPI)
a.
Stain with:
20 mg mannose-6-phosphate
10 mg NADP
13
10 mg MTT
1 mg PMS
10 units G-6-PDH
10 units phosphoglucose isomerase
15 ml .1 M Tris-HCl pH 8.0
b.
14.
Combine with 15 ml of 2% agar; pour on gel and incubat e at 37° C.
Nucleoside Phosphoralase (NSP)
a.
Stain with :
30 mg inosine
2 mg MTT
1 mg PMS
10 units xanthine oxidase
15 ml .1 M P0 4 buffer pH 6.5
b.
15.
Combi ne with 15 ml 2% agar ; pour on gel and incubate at 37
Octanol Dehydrogenase (ODH)
a.
Stain with:
1.0 ml octa nol-ethanol sol.
100 ml .1 M.Tris-HCl pH 7.4
mix well; then add
20 mg MI'T
25 mg NADP
5 mg PMS
b.
Incubate at 37° C.
c.
Octanol-ethanol solution: ·
20 ml octanol
80 ml 95% ethanol
0
C.
14
16.
Peptidase (named for peptide as listed below)
a.
Stain with:
20 mg peptide
5 mg amino acid oxidase
10 mg peroxidase
5 mg 3,3 diamine benzedine-4HC1
trace MnC1
2
15 m1 .1M P0
buffer pH 7.5
b.
Combine with 15 ml 2% agar and incubate at 37° C.
c.
Peptides:
d.
17.
4
1eucy1 tyrosine
Leu tyr
leucy1 proline
Leu pro
1eucy1 glycylglycine
Leu glygly
leucy1 valine
Leu val
leucyl leucine
Leu leu
Leu pro, Leu glygly, and Leu val may code for the same loci.
Phosphoglucose Isomerase (PGI)
a.
Stain with:
40 mg fructose-6-phosphate
2 mg NADP
2 mg MTT
1
mg PMS
7 units G-6-PDH
2 m1 MgC1
2
13 ml .1 M Tris-HC1 pH 8.0
b.
Combine with 15 m1 2% agar; incubate at 37
0
C.
15
18.
Phosphoglucomutase (PGM)
a.
Stain with:
500 mg glucose-1-phosphate
50
mg
EDTA
20 mg NBT
10
mg
NADP
2 ml 10% MgC1
2
80 units G-6-PDH
2 mg PMS
100 ml .1M Tris-HCl pH 7.1
b.
19.
Incubate at 37° C.
6-Phosphogluconate Dehydrogenase (6-PGDH)
a.
Stain with:
40 mg 6-phosphogluconic acid
20 mg NADP
25 mg MTT
2 mg PMS
15 ml .1M Tris-HCl pH 7.0
b.
Combine with 15 m1 2% agar; pour over gel and incubate at 37° C.
20·. · Pyruvate Kinase (PK)
a.
Stain with:
20 mg glucose
10 mg ADP
2 mg Mgcl
35
mg
2
phosphoenol pyruvate
10 units G-6-PDH
4 mg NADP
16
3 mg PMS
2 mg MTT
15 ml .1 M Tris-HCl pH 8.0
b.
21.
Combine with 15 ml 2% agar; incubate at 37
0
C.
Sorbitol Dehydrogenase (SDH)
a.
Stain with:
• 5 gm
sorbitol
10 mg NAD
15 mg MTT
2 mg PMS
100 ml .1 M Tris-HCl pH 8.0
b.
22.
Incubate at 37° C.
Tetrazolium Oxidase (TO)
This enzyme is scored on gels stained for CK.
on the blue background.
23.
Xanthine Dehydrogenase (XDH)
a.
Stain with:
100 mg hypoxanthine
10 ml .5 M Tris-HCl, pH 7.0
mix together; combine with
30 mg NAD
20 mg MTT
2 mg PMS
80 ml H 0
2
b.
Incubate at 37° C.
TO appears as white bands
17
H.
Staining techniques:
1.
these assays gi ve variable results.
Acid Phosphatase (ACP)
a.
Soak sliced gel for 1 hr. in . 5 M boric acid.
b.
Stain with:
100 mg a-naphthyl acid phospha t e
100 mg fast blue BB
100 ml
c.
2.
.05 M acetate pH 5.0
Incubate at 37° C.
Alcohol Dehydrogenase (ADH)
a.
Stain with:
5 ml 95 % ethanol
50 mg NAD
30 mg MTT
2 mg PMS
10 ml .5M P0
4 pH 7.0
90 ml H 0
2
b.
3.
Incubate at 37
0
C.
Alkaline Phosphatase (AKP)
a.
Stain with:
100 mg a -naphthyl acid phosphate
100 mg fast blue BB
• 6 ml 10% MgC1
2
100 ml .1M Tris-HCl pH 8.6
b.
Incubate at 37° C.
ACKNOWLEDGMENT
This report is the result of work sponsored jointly by the Office of
Sea Grant, NOAA, U. S. Dept. of Commerce under Grant Number 04-03-158-6, and
the University of Georgia, Athens, Georgia
30602.
The U. S. Government is
authorized to produce and distr ibute reprints for governmental purposes
notwithstanding any copyright notation that may appear hereon.