J. Embryol. cxp. Morph., Vol. 14, Part 1, pp. 1-14. August 1965
Printed in Great Britain
An electrophoretic analysis of proteins of cellular
sap in normal and hybrid frog embryos
by RICHARD E. SHORE1
From the Department of Zoology, Duke University, and the Department of Biology, Saint
Louis University
T H E degree of abnormality found in amphibian interspecies hybrids varies from
one species pair to another, and may also differ between reciprocal matings
(Moore, 1955). Although some general ideas exist about the mechanism of
arrest and the course of abnormal development, our knowledge is far from
complete. One major aspect of development which has received attention only
at the gross level is the nature and diversity of those proteins which compose
the greater part of the cytoplasmic sap, the * soluble' proteins in the sense of this
paper (see below). This paper reports the results of studies on the development
of normal {Rana pipiens) and gastrula-arrested hybrid (R. pipiens $ x R. sylvatica
o) amphibian embryos.
The present work was undertaken (i) to provide a detailed survey of the salinesoluble proteins in R. pipiens during the embryonic period, and (ii) to apply
the same techniques to the gastrula-arrested hybrid with R. sylvatica. The findings do not indicate a major role in morphogenesis for any of the components
studied, but do provide some insight into degenerative changes in the hybrid
embryos. The principal technique was zone electrophoresis in a synthetic gel,
polyacrylamide, followed by protein staining.
Embryos of the two parent species are quite similar (Pollister & Moore,
1937; Shumway, 1940), but the hybrid embryos differ from both parents in
several ways. Morphologically they are arrested during gastrulation. They gain
functional cilia, swell, burst, and finally, about 3 days after arrest, they cytolize
(Moore, 1946). The inductive and differentiative capacities of hybrid tissues are
subnormal (Moore, 1947, 1948). Metabolic abnormalities of the hybrid
embryos include a depressed respiratory rate (Barth, 1946) and a depressed
respiratory potential (Gregg, 1960). Glycogen degradation apparently begins
earlier but continues at a very subnormal rate (Gregg, 1948). Glycolytic capacity
is subnormal (Gregg, 1962). Creatine phosphate under aerobiosis is present at
subnormal concentrations (Harrison, 1963). Acid phosphatase activity, at least
in the dorsal region, is subnormal (Mezger-Freed, 1953). DNA of whole
1
Author's address: Department of Biology, Saint Louis University, 1402 South Grand
Boulevard, Saint Louis, Missouri 63104, U.S.A.
1
2
R. E. SHORE
embryos increases normally to almost the end of maternal gastrulation, then
stops completely (Gregg & Lovtrup, 1960). During the period of arrest individual nuclei may increase their DNA content to an amount 'greater than
necessary for a subsequent mitosis but cell division has already ceased' (Moore,
1957). Cytoplasmic RNA also becomes quite subnormal and in the absence of
R. pipiens chromosomes the R. pipiens cytoplasm inhibits nucleolus formation in
R. sylvatica nuclei (Moore, 1957). An intact nucleolus appears to be required
for the normal production of ribosomes when this begins in tailbud-stage
embryos, but the lack of nucleoli in mutant Xenopus embryos does not prevent
the synthesis of messenger RNA (Brown & Gurdon, 1964).
The pattern of fast-green staining, believed to reflect changes in localized
histones, changes at the onset of gastrular movements in a variety of amphibian
embryos, but the hybrids of this paper cannot be distinguished from normal
embryos by this criterion (Horn, 1961).
Studies of amphibian development are approaching the molecular level in
descriptions of fine-structural changes during development (Karasaki, 1959a, b,
1963), and in descriptions of specific structural units such as yolk platelets
(Wallace, 1963a, b). Various methods reveal developmental changes in the
proteins of whole embryos: see, for example, reviews by Ranzi (1962) and by
Tyler (1957).
The present knowledge of mechanisms in protein synthesis suggests the
importance of genetic factors, and their unambiguous and controlled translation, in producing the normally occurring spectrum of proteins. One might
expect hybrid lethality to involve interference by the foreign genome in the
pattern of protein synthesis, either eliciting the production of some chemically
abnormal protein, or altering quantitatively or temporally the production of
some normal protein. Alterations in respiratory and DNA metabolism which
may express such an interference occur in a variety of hybrid embryos: see, for
example, Chen & Baltzer (1964). In the case of the hybrid R. pipiens tyxR.
sylvatica $ some kind of interference seems indicated since neither gynogenetic
nor androgenetic haploid embryos of R. pipiens are arrested at gastrulation nor
do they show the remainder of this arrest syndrome (Moore, 1955).
From this introduction it should be clear that we do not know the mechanism
of arrest. All of the factors studied (with the possible exception of glycolytic
metabolism) are normal until after one can already tell that hybrid embryos are
arrested because of their delay in forming a blastopore lip. There is as yet no
description of the course of development of the major cytoplasmic proteins,
and there is reason to suspect a causal abnormality in some aspect of protein
synthesis.
MATERIALS AND METHODS
Biological
Adult frogs of both sexes of the species Rana pipiens and adult males of the
species Rana sylvatica were obtained from C. H. Mumley, Alburg, Vermont.
Frog embryo protein electrophoresis
3
Normal (Rana pipiens) and hybrid (R. pipiens $xR. sylvatica $) embryos
were prepared by fertilizing ova stripped from pituitary-stimulated females
(Rugh, 1934; Hamburger, 1942). A sperm suspension of two testes macerated
in 40 ml. of a standard medium, bicarbonate-free 20 per cent. Holtfreter's
solution (Holtfreter, 1931), was made for each species. Ova from a single
female were stripped alternately into two dry dishes. Sperm suspension was
added by pipette. After 30 min. the dishes were rinsed and flooded with
medium. After the jelly swelled, the embryos were distributed in groups of
about fifty among several shallow dishes. Six different clutches were prepared
in this manner. All were reared at 18°C. in standard medium which was
changed at 2-day intervals. Fertilization exceeded 95 per cent.; normal
development of R. pipiens embryos to swimming tadpole stages exceeded 90 per
cent.
In this paper embryonic stages for R. pipiens embryos are those of Shumway
(1940). Hybrid embryos were staged by the morphology of half-siblings.
Developmental stages of hybrid embryos are designated by prefixing ' H ' to
the stage designation which would have applied to R. pipiens embryos which
had been reared identically. That is, hybrid embryonic stages were numbered
by reference to the morphology of a group of half-sibling R. pipiens embryos
which had been handled identically and reared simultaneously (Gregg, 1957).
From four clutches, groups of normal and hybrid embryos were harvested
at specified times after fertilization (6-, 12- or 24-hr, intervals) as shown in
Text-figs. 2 and 3. The jelly coats were removed with forceps. The developmental stage was noted. Groups of embryos, twenty-five each, were placed in
0-4 ml. polyethylene vials and rinsed in fresh rearing medium. The excess fluid
was withdrawn by a capillary pipette. During all subsequent operations the
embryonic material was chilled and remained in these capped, marked vials.
Embryos were homogenized without additional fluid by thrusting a platinum
wire into the storage vial and spinning the wire at 800-1000 r.p.m. for 30-60 sec.
Centrifugation at 5000-5500 g. for 30 min. at 0°C. produced a pellet and a clear
supernatant fraction of 20-50 /xl. The latter was removed and stored in a clean
vial at -20°C. The supernatant fraction so prepared is a whole-embryo
extract of the saline-soluble proteins, i.e., the proteins soluble in a small addition
of standard rearing medium (20 per cent. Holtfreter's). Samples from two
clutches were frozen prior to homogenization (pre-frozen); all other samples
were frozen only after transfer of the clear embryo extract to a clean storage
vial. The whole embryo extract was separated by zone electrophoresis without
prior dialysis.
Two clutches of R. pipiens embryos were reared for comparison of dorsal
and ventral fragments. Dorsal and ventral fragments were prepared from
stage 18 embryos by cutting them between two tungsten needles. Ventral
fragments consisted of the yolk-laden endoderm and the overlying belly epidermis. The remainder of the embryo, in front of the heart region and dorsal to
R. E. SHORE
the nephric prominence, was included in the dorsal fragment. Samples consisted
of the dorsal or ventral fragments of fifty embryos. Subsequent homogenization,
centrifugation and electrophoresis were accomplished in the manner described
for whole-embryo extracts.
Electrophoretic techniques
The method of electrophoresis was derived from methods for starch (Smithies,
1959) and for polyacrylamide gel (Raymond & Wang, 1960). Gel slabs were
wrapped in a vapor-barrier film (Saran-Wrap, T. M. Reg. U.S. Pat. Off.,
Dow Chemical Corp.) and laid horizontally between brine-cooled plates so that
their ends dipped directly into buffer reservoirs.
Acrylamide, JV,-/V',-rnethylenebisacrylamide, and N,N,N' ,Nr ,-tetT&methy\ethylenediamine (Davis, 1962), all from Distillation Products, Rochester, were
polymerized in 6-mm. thick sheets by ammonium persulfate. The gel was made
in tris-borate buffer, 10 mM. tris (hydroxymethyl) aminomethane (Sigma, Saint
Louis) and 10 mM. boric acid. The reservoir buffer was the same tris-borate
but with 3 mM. ammonium persulfate added, to avoid conductivity differences.
The pH in the gel was determined with a Beckman Zeromatic pH meter with
temperature compensation. Both temperature and voltage gradient were
monitored in the gel. During electrophoresis a mercury thermometer in contact
with the gel registered temperature; voltage gradient was monitored with a
voltmeter independent of the electrophoretic power source, measuring via
platinum electrodes across 20 cm. of gel. The conditions for electrophoresis
which gave the most satisfactory resolution were: 10 V./cm., pH 9-0, 3-0°C,
5 per cent, polyacrylamide gel (0-25 per cent, bisacrylamide), for 60 min.
At least three aliquots of the undialyzed embryo extract of each sample of
embryonic material were subjected to electrophoresis. Tabs of Whatman paper
41H (4x6 mm.) containing 5 yl. of extract were placed along the end of a slab
of gel and a second slab abutted against it. Such a set contained both hybrid
and normal material from two or more stages or two or more clutches. Three
such sets were placed in electrical series between the electrode reservoirs. There
was no evidence of regional effects on mobility with this arrangement.
Immediately after electrophoresis the gels were stained in amido black
(Matheson-Coleman and Bell, Cincinnati) 0 • 5 per cent, in glacial acetic acid:
methanol:water: 1:5:4 (Smithies, 1959). Excess dye was removed with changes
of 10 per cent, acetic acid. Electrophoretic destaining was found to produce
distortions in the banding pattern and therefore was not used. Except as
already noted chemicals were reagent grade from Mallinckrodt, New York.
Crude histone extracted from coelomic oocytes of Ranapipiens was prepared and
kindly provided by Professor E. C. Horn, Department of Zoology, Duke
University.
Protein concentration in the embryo extract was estimated according to
Frog embryo protein electrophoresis
5
Lowry et al. (1951), using bovine serum albumin as a standard. For both
normal and hybrid embryos to stage 12 (or H 12) protein concentration was
about 40 mg./ml. in the whole embryo extract, a value comparable to the value
for extractable protein calculated from the data of Gregg & Ballentine (1946).
Because of changes in the volume of the perivitelline fluid (normal) and blastocoelic fluid (hybrid), protein concentration in the embryo extract decreases
during neurula and tailbud stages (14 to 18). Older embryos were removed from
their vitelline membranes even though this involved rupture and loss of blastocoele fluid for the hybrid embryos. The volume of embryo extract was the same
for each electrophoresis; changes in total protein concentration were taken into
account in the interpretation of banding patterns.
Samples of embryo extract from several developmental stages or several
clutches were separated side by side in the same gel slab. The appearance of
novel bands in a few of the samples could not be blamed therefore on mobility
artifacts arising in the gel.
The destained gels were compared and enlarged scale drawings were made.
The prominent features described below were quite consistently seen, especially
from one sample to another within the same gel. Where differences are described they are supported by at least one simultaneous separation of the two
samples contrasted.
RESULTS
Nomenclature
The stained pattern resulting from electrophoresis was arbitrarily divided into
zones, as in Text-fig. 1, on the basis of band width, staining intensity and resolution. These zones were named from the anodic side (a) to the cathodic side of
the pattern (d). Each band was designated by a subscript numeral to the zone
name, thus c1? c2 and c3 were the bands in order from anodic side to cathodic
side of the c zone. Bands which were apparent only during a brief developmental interval were designated by adding + or — to the name of the nearest
band, to indicate a position on the anodic or cathodic side of that band,
respectively. Capital letters refer to hybrid material.
Major protein zones
Zone a, the most rapidly moving anodic zone, consisted of a pair of broad
bands, a1 and a2, which stained with a moderate intensity. The two were well
defined from each other and from the slower zone b by regions without
appreciable staining (i.e., there was no background).
Zone b stained with the least intensity and showed the poorest resolution of
any of the zones. There appeared to be from three to five resolvable bands.
The b bands were stained only slightly more intensely than the background.
6
R. E. SHORE
There was no sharp drop in the intensity of background stain between this zone
and the next slower anodic zone, c.
Zone c showed the most intense staining and the best resolution of any of the
zones. It usually included two or three very intensely stained, very sharply
defined and very thin bands. The background stain within the zone was
+20
Hours
@18°C
6
12
•15
+ 10
0
+5
-10
Stage
1-7
8
18
24
10
36
11
48
12
60
72
84
13
14-16
96
18
120
19
144
20
17
b4
b5
c, c2
c3
TEXT-FIG. 1. Development of proteins in normal embryos. Ordinate: embryonic stage and
equivalent age at 18°C. Abscissa: mobility in 5 per cent, gel expressed in mm. per
(V./mm.) per hr., cathode at right. Intensity of staining is shown by the degree of darkening
by stippling; horizontal widths represent actual width of stained region in gel.
moderately intense (usually much greater than in zone b), but between the
origin and the slowest of the sharp bends, c3, the background was negligible.
All of the cathodic bands were included in zone d. They were all moderately
broad. Two stained with moderate intensity; the remainder were usually faint.
Changes during embryogenesis
Text-fig. 1 presents a graphic summary of the electrophoretically resolvable
protein bands during early development. It is a plot of electrophoretic mobility
(abscissa) versus developmental age (ordinate) in which staining intensity is
indicated by the degree of darkening by the stippling. Horizontal width
represents the actual width of the stained region in the gel. The general picture
is one of slight change.
-15
Frog embryo protein electrophoresis
7
Zone a. Over the entire developmental period studied there was a slow
increase in the staining intensity of a± and a slight decrease in its width resulting
in an increase in the degree of resolution of ax. There was no demonstrable
change in the band designated a2 during early development.
Zone b. There appeared to be at least one change in zone b during development. The region containing bi and &3 began to increase in staining intensity
toward the end of gastrulation, and then the resolution increased to reveal
three discrete bands during the tailbud stages. The discreteness of the slowest
band of this zone, b5, appeared to decrease during gastrulation and again
toward the end of embryonic life. During these same periods the intensity of
the background staining increased, nearly obscuring b5.
Zone c. The changes in this slow anodic zone were fairly clear-cut. The
first major band (cj) was double during a short period of time; the extra band
(cj+) was demonstrated only at the end of gastrulation. When present, c± +
stained with as great an intensity and was as sharply defined as cx. The two
bands were very close together. The slowest band in this zone (c3) also appeared
to be double during part of development. Text-fig. 2 shows first a faster
(c 3 +) and then a slower (c3—) band close to c3. The former appeared during
the end of blastulation and persisted through most of gastrulation, while the
latter appeared in the neural tube stage and persisted at least to the establishment of circulation. Only c3 — was as sharply defined or stained as intensely
as the broader c3.
Zone d. In the cathodic zone two additional bands, d3 and d5, appeared
during gastrula and early neural stages. Then d5 reappeared in tailbud stages.
Both were always very diffuse and stained only faintly.
Comparison of normal and hybrid proteins
Proteins of the hybrid are summarized similarly in Text-fig. 2. The nybrid
material was found to differ slightly from the normal, and only in zones C
and D.
Zone C. There was no evidence of a split in Q at any time. Such a split
might have been partly obscured by the intensity of the background, but if
C\ + did occur, it was with much less intensity than c\ + (in normal). The
staining intensity of C2 appeared to be increasing toward the end of the period
studied, whereas c2 remained constant throughout the developmental period
studied. The staining intensity of C3 and its subsidiaries, C 3 + and C 3 - ,
decreased progressively in hybrids after stage H12, whereas c3 + , c3, and c 3 remained constant in the normal. The hybrid bands faded into the low intensity background in stage H20.
Zone D. The most dramatic difference between hybrid and normal protein
development was in the pattern of the cathodic components. The intermediate
8
R. E. S H O R E
minor band, D3, persisted from its appearance during the formation of the
blastoporal lip, instead of fading as d3 did after neural closure. In intensity,
width and resolution it did not distinguishably differ from a maximally intense
dy The fastest cathodic band in the normal material, d5, did not fade in the
hybrids, D5. From its appearance during gastrulation, D5 became more intense
until it approached in intensity and width both D4 and d4. An additional band,
D6, appeared late in the life of hybrid embryos. It migrated faster than any
mm.-} 20
+15
+ 10
-10
+5
15
Hours Stage
@18eC
6
Hl-7
12
H8
18
H9
24
H10
36
Hll
48
H12
60
H.I 3
72 H14-16
84
96
120
144
H17
HIS
H19
H20
Bi B2
TEXT-FIG.
'I (-2
(-3
D.,
D3 Dt
2. Development of proteins in arrested hybrid embryos. Represented as in
Text-fig. 1.
cathodic band demonstrated in the normal embryos. D6 appeared at about the
time normal embryos became motile. It was broad, diffuse and of low intensity,
resembling the slower D4.
Comparison of whole extract with crude histories of oocytes
Text-fig. 3 shows a direct comparison of the bands resolved from the saline
whole-embryo extract and from the crude histone fraction of Rana pipiens
oocytes, as revealed by simultaneous migration under the conditions already
described. Where a component of the histone extract appeared to have the
same mobility as a component of whole-embryo extract, the same designation
was applied to both. The previously described anodic component (Horn, 1961)
appears to be multiple in the gel. Two slow-moving components are seen in the
gel separation which were also apparently obscured by the initial boundary
during free electrophoresis. One was anodic and one cathodic at pH 9.
Frog embryo protein electrophoresis
9
Comparison of dorsal and ventral fragments of tailbud embryos
Electrophoresis of the dilute-saline extract of both dorsal and ventral embryonic
fragments resulted in patterns such as those illustrated in Text-fig. 2. There was
an extremely great similarity between patterns derived from dorsal fragment
extracts and those from ventral fragment extracts. Within the limits of the
technique there appears to be no significant difference between dorsal and
ventral fragments. In an attempt to provide further resolution of the proteins
of these two regions, some samples were subject to electrophoresis for twice the
time previously used. The relative position of the various bands still did not
differ from dorsal to ventral extracts.
0
a,
wo
a.,
^
b3
bj
:
b4
.
b5
c, c2
' II
c3
i
CH
dl
d2
d3
d4
d5
3. Partial fractionation of egg proteins: Crude histories. Abscissa: mobility as in
Text-fig. 1. WO: whole extract (stage 1 of Text-fig. 1). CH: crude histone extract of
coelomic oocytes.
TEXT-FIG.
Effects offreezing and thawing
For both normal and hybrid embryos the intensity of cathodic bands was
greater in the pre-frozen extracts than in post-frozen extracts of the same
development stage. The changes during development described above for normal
and for hybrid embryos were most clearly seen in the pre-frozen material, but
were also evident in post-frozen material. In no case did a d$ band appear in
pre-frozen material.
DISCUSSION
Migration in gel
Gel electrophoresis involves a sorting both by molecular size and by molecular
net charge (Smithies, 1959; Raymond & Nakamichi, 1964). Although no
rigorous theory of the sorting processes has been widely accepted, approaches
to such an understanding are discussed by Kunkel & Trautman (1959), with
proposals by Smithies (1959), Raymond & Nakamichi (1962), Ornstein (1962)
and Boyack & Giddings (1963).
10
R. E. SHORE
Comparison with other work
Spiegel (1960) analyzed a similar protein extract of Ranapipiens embryos at
several stages up to stage 21. His densitometric tracings of bromphenol-blue
stained protein after paper electrophoresis at pH 8-6 are very similar to what
would be expected from the present study. A pair of well separated anodic
bands was followed by a 'plateau' region containing a number of bands. He
distinguished only two cathodic components, possibly d2 and d4 of the present
work. A large amount of material remained at the origin, possibly an irreversible adsorption associated with the use of paper (Block et aL, 1958). Little if
any change was found in the embryo.
Denis (1961) analyzed a similar protein extract of Pleurodeles waltlii over the
entire embryonic period. His electrophoretic studies on cellulose acetate film
also reveal no major changes in number or intensity of bands until about the
time of blood circulation. His patterns are very different from those of the
present study. No correlation is evident.
Reports that the quantity or variety of soluble protein increases rapidly once
blood circulation is established have been interpreted as evidence of yolk
mobilization (Denis, 1961; Gregg & Ballentine, 1946). Direct evidence of
yolk platelet degradation (Karasaki, 1963) is consistent with such a view.
However, the present work does not reveal any changes in stage 19 through 21
that could be interpreted as yolk mobilization. This difference may result from
differences in sensitivity or operational definition of 'soluble'.
The proteins of zone d apparently correspond to most of the components of a
crude histone fraction previously described (Horn, 1961). Since the histone
fraction was prepared by extraction of washed yolk platelets with 0-1 N HC1,
the d bands represent the basic histone-like proteins of yolk platelets. The fact
that these proteins appear in extracts of all stages need not mean that this
material would be free in the cytoplasm under conditions which would prevent
yolk platelet damage. The enhancement of these d bands by a freeze-thaw cycle
prior to homogenization is consistent with a localization of these basic proteins
in the amorphous zone of yolk platelets (Karasaki, 1963). There is no direct
evidence of other locations for these proteins. The present study does not
illuminate the reported abrupt shift in fast-green affinity of yolk platelets and
nuclei (Horn, 1961), since the pattern of basic proteins in pre- and postgastrular stages changes only slightly and very slowly.
Discussion of the molecular size, shape or heterogeneity of components in
these or other bands is reserved until more direct evidence can be offered.
Comparison of normal and hybrid proteins
There are two zones showing differences between electrophoretic patterns of
normal and hybrid embryos. In zone C of hybrid material Cx + never appears,
Frog embryo protein electrophoresis
11
C2 becomes abnormally intense after stage HI7, and C3 and C3 — become abnormally faint after stage H14. In zone D, D3 remains visible after HI3, Z>5
intensifies after HI7, and D6 appears.
Since the C components are yet to be characterized or their position localized
intracellularly, a discussion of their role must be speculative. Furthermore,
since these differences occur most markedly well after morphological arrest,
they cannot represent primary defects. However, basic proteins, particularly
histones, can act as stabilizers of nucleic acid and may thus be involved in
genetic regulation and in control of protein synthesis (Irvin et al., 1963).
One interpretation of the appearance of the D bands is that subcytolytic
changes occur for some time prior to gross cytolysis and that these changes
allow the proteins represented in D bands to be more readily extracted. Karasaki
(1963) has clearly shown the presence of a limiting membrane surrounding
yolk platelets prior to degradation and elaborate concentric membranes during
their normal degradation. If pre-cytolytic degeneration involves a decrease in
the stability of cellular membranes in general, one could expect the amorphous
zone of yolk platelets to be more readily extractable and more readily liberated
by a freeze-thaw cycle. Those basic proteins found in yolk platelets are believed
to lie in the amorphous layer (Horn, 1961).
The intensity of the C2 band could be similarly interpreted if some identity
between C2 protein and either of the major proteins of yolk platelet central
region (Wallace, 1963a, b) were established. At present we have only the
suggestive evidence that the low mobility and high resolution of this band are
consistent with the large molecular size of yolk platelet proteins.
Comparison of dorsal and ventral fragments of tailbud embryos
The failure to demonstrate differences between dorsal and ventral fragments
of tailbud-stage embryos is surprising. The dorsal fragments represent tissues
engaged in relatively rapid differentiation in the sense of histogenesis and
organogenesis while the ventral fragments remain morphologically conservative. Other studies (see, for example, review by Boell, 1959) indicate the
existence of marked physiological differences between dorsal and ventral
fragments. Two factors may account for the similarity found in the present
study. First, enzyme changes are more readily detectable than are changes in
non-enzyme proteins. Therefore the enzymatic activity of an extract may in
fact change drastically without changes in the quantity of major constituent
proteins. Second, adsorption to particulate material, for which frog embryos
are notorious, could remove significant components from the embryo extract
during a centrifugal separation.
SUMMARY
1. Polyacrylamide gel electrophoresis of dilute saline extracts of embryonic
Rana pipiens resolved an array of six cathodic and at least seven anodic bands.
12
R. E. SHORE
2. During embryonic development changes in electrophoretic pattern were
restricted to intensities of a few of the bands.
3. The major differences between normally developing (R. pipiens) and
gastrula arrested hybrid embryos (R. pipiens $ x R. sylvatica <$) were the increased intensity of cathodic bands (probably histones) and the existence of one
extra cathodic band in the hybrid material.
4. No differences between extracts of dorsal and ventral fragments of tailbud
stage normal embryos were detected.
RESUME
Analyse par electrophorese des proteines du sue cellulaire d'embryons de grenouilles
normaux et hybrides
1. L'electrophorese, sur gel de polyacrylamide, d'extraits salins dilues
d'embryons de Rana pipiens a revele la presence de six bandes cathodiques et
d'au moins sept bandes anodiques.
2. Au cours du developpement embryonnaire, les modifications du type
electrophoretique ont ete restreintes a des variations d'intensite d'un petit
nombre de bandes.
3. Les principales differences entre les embryons a developpement normal
(R. pipiens) et les gastrulas hybrides bloquees (R. pipiens $ x R. sylvatica <£)
consistaient en un accroissement d'intensite des bandes cathodiques (probablement des histones) et en l'existence d'une bande cathodique supplemental dans
le materiel hybride.
4. On n'a pas decele de differences entre les extraits de fragments dorsaux
et de fragments ventraux d'embryons normaux au stade du bourgeon caudal.
ACKNOWLEDGEMENTS
It is a pleasure to acknowledge the guidance and helpful criticism of Professors J. R.
Gregg and E. C. Horn during the course of this research, and also the critical readings given
this manuscript by Professors Florence Moog and D. J. Fluke. This work was partially
supported by grants from the U.S. Public Health Service (GPM-16, 483) and from the
American Cancer Society (IN-63-C9). Portions of this work were done in partial fulfillment
of requirements for Ph.D. degree at Duke University.
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(Manuscript received 18th January 1965)