O

MDV
TIDT
SIS DURING EMBHIOGENESIS AND LATER DEVELOPMENT
RNA SYN
0Y
TRDT
-10
IN THE BARNACLE POLDIC
1
POLTMERUE
Tom Raffin
DT
HOPKINS MARINE STATION
p

LELAND STANFORD JUNIOA UNIVERSII.
June 6, 1967
INTRODUCTIO!
There has been alot of investigation recently, concerning
RNA synthesis in the indeterminant embryos of Echinoids (Glisin &
Glisin, 1964; Gross et al., 1964; Gross et al., 1965; Nemer, 1963;
Slater & Spiegelman, 1966a, 1966b; Stavy & Gross, 1967; Whiteley
et al., 1966; Wilt, 1963). Little experimentation has been carried
out with determinant, spiral cleaving embryos as in the barnacle
Pollicipes polymerus. The goose barnacle is distributed along
the exposed rocky coast of Western North America from British
Columbia to the middle of Baja, California (Cornwall, 1925). The
eggs of the barnacle pass down the oviducts to the atria, where they
receive a protective coating which glues them together into masses.
These masses eventually come to lie inâthe mantle cavity on either
side, where they are pressed flat to form the two ovigerous
lamellae (Hilgard, 1960). In a pair of ovigerous lamellae taken from
any one parent, embryos appear to be synchronous and develop until
they are hatched out as nauplius larvae. The technique employed
in this paper is radioactive lableing with C-Uridine with stages
ranging from cleavage to the second molt after hatching.
TEPT*
THODS AND MATERIALS
Preparation of Ovigerous Lamellae:
Pollicipes polymerus were collected from Mussel Point,
Pacific Grove, California. A pair of ovigerous lamellae, approximately
.5 grams, were removed from the same animal and placed in Millipore
Filtered Sea Water at 13° C. Care was taken to use ovigerous
lamellae of the same size, and experimentation was commenced within
4 hours of removal. Hatched nauplius larvae were obtained from
ovigerous lamellae that were placed in glass dishes of Millipore
Filtered Sea Water at 13 C (Hilgard, 1960).
RNA Extraction:
Ovigerous lamellae were incubated for one hour, before
C+"-Uridine (Calbiochemical Co.) was added, in sea water containing
508/ml of streptomycin (Sigma Chem. Co.) and 300 units/ml of
penicillin (Sigma Chem. Co.). Conditions for pulse lableing
zperiments are specified under the respective graphs.
The incorporation of C-Uridine was stopped by placing
the ovigerous lamellae in a homogenizer with 3.5 ml of homogenization
media and raoidly freezing in a dry ice-acetone bath. The homog.
media was composed of sodium acetate buffer,.O1 M, pH 5; Nacl, O.1M;
Mgclo, 10 2 M; with sodium lauryl sulfate (SLS), 0.5%; and
bentonite 1 mg ml.
ure was thawing, homogenization commencod, anda
As the mi
approximately 3 minutes mere required for disruption of the lamellae.
An equal volume of sodium acetate buffer-saturated purified phenol
(Allied Chem.) was then added to the homogenate and the mixture
19
vigorously shaken at regular intervals for 15 minutes at 4 C
(Gross et al., 1965; Slater and Spiegelman, 19660). It was
then placed in a Sorvall RC-2B Centrifuge at 12,500 rpm (20,000 g's)
for 3 minutes. The aqueous phase was collected and subjected
to 2 additional phenol extractions. The aqueous phase of the
3rd extraction was incubated for 15 minutes with.25 mg of
DNAase (Sigma Chem. Co.) before being extracted a Uth time
with phenol.
Two volumes of cold 75% ethanol were added to the aqueous
phase that was collected after the Ath phenol extraction and
then the precipitated RNA was stored for 1 hour at 3 C to allow
settling of the precipitate.
This precipitate was then centrifuged and washed with 75%
ethanol and dissolved in 3.5 ml of the original homogenization
media, andincubated for 15 minutes. This 2nd treatment with SLS
was to assure removal of lipids. The solution was then
subjected to a final phenol extraction and the collected aqueous
phase was precipitated with ethanol as before. After one hour
of refrigeration the precipitate was washed with ether and
then with 752 ethanol. It was then dissolved in.5 ml of sodium
acetate buffer (without SLS or Bentonite) and used for sedimentation
analysis.
Sedimentation Analysis:
A 20% - 5% sucrose gradient was used. The sucrose was
dissolved in sodium acetate buffer and stirred with bentonite,
l mgm/ml, overnight to remove any RNAase (Gross et al., 1964).
The .5 ml RNA sample was layered onto the top of the gradient by
18.
the method of Bri
ten & Roberts (1960). An I.E.C. model B-35
ultracentrifuge, with a SB-206 rotor, was used at 35,000 rpm
(206,000 g's) for 8 hours. The sucrose gradient was checked
for linearity with a sucrose refractometer (American Optical Co.).
Glucose Oxidase (Sigma Chem. Co.) was used as a sedimentation
velocity marker according to the method of Martin & Ames (1960).
A Yale Luer-Lok 216 hypodermic needle was used to
puncture the centrifuge tubes. Four-drop fractions were collected
(approximatelyl.3 ml), with 0.1 ml of each fraction used for
Optical Density measurements at 260mA3. Carrier RNA (---mgm)
was added to the remainderof the fraction and the RNA precipitated
by addition of 5 mls. of 5% TCA. The precipitate was collected
on a Whatman Glass Filter GF/B, washed two times with 752 ethanol.
— glued to an aluminum planchet, dried, and counted on a
Chicago Nuclear Gas Flow counter.
194
PTRIT
ADHV
BISLIOGR
n
tten, R.J., Roberts, R.B., (1960), Science, 131, 33
Cornwall, I.E., (1925), Cont
r. Canad. Biol., 2, 469-502
Glisin, V.R., Glisin, M.V.sProc. Natl.
Acad. Sci. U.S., 52, 1548

762
Gross, P.R., Malkin, L.1., Moyer, W.A.
Proc. Natl. Acad. Sci. U.S.,
51, 207
jross, P.R., Malking, L.I., Hubbard, M., J., (1965), J. Mol. Biol.,
13, 463
Hilgrad, G.H., (1960), Biol. Bull., 119, 169
Martin, R.B., Ames, B.N., (1960), J. Biol.
Chem., 236, 1372
Nemer, M., (1963), Proc. Natl. Acad. Sci. U.S.
50, 230
Slater, D.W., Spiegelman, S., (1966a), Proc.
atl. Acad.Sci. U.S.,
6. 164
later, D.W.,
Spiegelman, S., (1966b), Biophysical Journal,
385
javy, L., Gross, P.R., (1967), Proc. Natl. Acad. Sci. U.S.,,
57, 735
Whiteley, A.H., MoCarthy, B.J., Whiteley, H.R., (1966), Proc.
61.
5, 519
ads Sci. U.S., 5
Wilt, F.H., (1963), Biochem. Biophys. Res. Comm., 11, 447
195
0
ACKNOWI
EDGEMEN
1. MyCock and his manual dexterity in boring
out plastic.
tion from mixing
2. Big Bertha whose indiges
dyes with sugar lost me 240 hours.
helped
3. Corky Weaver uho
soothemy neurosis.
4. The Man for endless hours of aid, supervision...
-and of course-
5. To all the Anthopleuras in the world.
15