- Email: [email protected]
Rate of RNA synthesis in haploid and diploid embryos of loach (Misgurnus fossilis)
274
SHORT COMMUNICATIONS
now remain to be further investigated, but it is very interesting that L-histidine acceptor tRNA's of bacteria have a peak of high specific activity in the early fractions from DEAL-cellulose. I am deeply indebted to Dr. WALTER S. McNuTT, with whom I worked under his suggestions, without which this work would have been impossible. I wish to thank Mrs. ROBERTA McDuFFIE for her skillful assistance.
Department o[ Biochemistry, School o/ Medicine, Tu/ts University, Boston, Mass. (U.S.A.)
ISAO TAKEDA*
1 T. YAMANE, T. Y. CHENG AND N. SOEOKA, Cold Spring Harbor Syrup. Quant. Biol., 28 (i963) 569. 2 S. G. NATHENSON, F. C. DOHAN, H. H. RICHARDS AND G. L. CANTONI, Biochemistry, 4 (1965) 2412. 3 T. P. BENNETT, J. GOLDSTEIN AND F. LIPMANN, Proc. Natl. Acad. Sci. U.S., 49 (1963) 850. 4 J. D. CHERAYIL AND R. M. BOCK, Biochemistry, 4 (1965) 1174. 5 ~N'. SUEOKA AND T. I(ANO-SUEOKA, Proc. Natl. Acad. Sci. U.S., 52 (1964) 1535. 6 G. HARTMAN AND U. COY, Biochim. Biophys. Acla, 47 (1961) 612. 7 J. APGAR, R. W. HOLLEY AND S. H. MERRILL, J. Biol. Chem., 237 (1962) 796. 8 H. G. ZACHAU, ~V[.TADA, W. LAWSON AND M. SCHWEIGER, Biochim. Biophys. Acta, 53 (1961) 221. 9 A. SAPONARA AND I{. M. BOCK, Federation Proc., 20 (1961) 356. io R. W. I-[OLLEY, J. APGAR, B. P. DOCTOR, J. FARROW, M. A. MARINI AND S. H. MERRILL, J . Biol. Chem., 236 (1961) 200. I I G. W. BEADLE AND I~. L. TATUM, Am. J. Botany, 32 (1945) 67812 C. KLEE AND M. STAEHELIN, Biochim. Biophys. Acta, 61 (1962) 668. 13 W. E. BARNETT AND K. B. JACOBSON, Proc. Natl. Acad. Sci. U.S., 51 (1964) 642.
Received July 3oth, 1968 * Present address: Asahi Chemical Co., Technical Research Laboratory, Nakadai 3-27, I t a bashi, Tokyo, Japan.
Biochim. Biophys. Acta, 169 (1968) 272-274
BBA 93367
Rote of RNA synthesis in hoploid ond diploid embryos of loach
fossnis)
(Misgurnus
In animal cells, the relationship between ploidy and biosynthetic capacity has been insufficiently studied from the quantitative point of view. In some ceils a direct proportionality is found between cell size and ploidy, suggesting the operation of a "gene dosage control" of biosynthetic activities in animal cellsZ,2; in others, however, no such correlation was revealed ~. On a more detailed level, there are examples of a "dosage compensation" phenomenon, e.g. compensatory inhibition of the activity of one or both allelic genes 4. Studies with embryes of sea urchins 5 and amphibia 1,8 point to the action of gene dosage control of biosynthetic capacities in developing cells. The evidence, however, is of an indirect nature. In the present work an attempt was made to employ a more direct approacb by comparing the rates of R N A synthesis in diploid (2n) and haploid (In) embryos Biochim. Biophys. Acta, 169 (1968) 274-277
SHORT COMMUNICATIONS
275
of loach (Misgurnus/ossilis). The dynamics of gene transcription in 2n embryos of loach have been quantitatively studied in earlier work 7 which revealed a sharp increase in the rate of RNA synthesis in the cell nuclei at mid-blastula stage; this is also the time of the onset of "morphogenetic activity" of the nuclei (i.e. nuclear function in supporting gastrulation and morphogenesis) s. Mature eggs of loach were obtained, fertilized, and allowed to develop in tap water at 21 ° as described elsewhere 8. Gynogenetic haploid embryos were obtained by fertilizing normal eggs (from the same lot as that used to obtain 2n embryos) with the X-ray-irradiated sperm (dose 80 kR). RNA synthesis was measured by the incorporation of 14C0~ which was introduced as described by COHEN9. Experiments consisted of pulsing (90-min pulses) whole eggs, at different stages of development, with ~4C02 under standard conditions (see the legend to Fig. I), and measuring specific radioactivities of RNA extracted from the egg.~. In order to avoid possible interference due Lo the terminal exchange in transfer RNA's the latter were removed from the preparations (together with DNA, protein and polysaccharide impurities) by precipitating the "salt-insoluble" RNA's with 2.0 M NaC1 in the cold (see details in the legend to Fig. I). The "salt-insoluble" RNA preparations thus obtained contain ribosomalRNA's and polydisperse RNA of the nuclei and cytoplasm 1°. Since rRNA's are not synthesized at the stages studied here l°,u, the label is incorporated exclusively into polydisperse RNA species 1°. It has been found weviously that the rate of 14C02 incorporation is not limited (in conditions specified here) by the incorporation into free (cold trichloroacetic acid-soluble) nucleotides of the eggs 7, and that the "salt-insoluble" RNA content of the eggs remains constant during the developmental period studied TM. Hence, it can be inferred that the variations in specific radioactivities of "salt-insoluble" RNA preparations obtained at different developmental stages after standard pulses with 14C0~, reflect the variations in the rates of synthesis of polydisperse RNA's (on a per embryo basis) and, consequently, can be regarded as an index of the changes in the overall rate (activity) of gene transcription in the developing embryos. Fig. I shows, in conformity with earlier observations 7, that incorporation of 14C02 into "salt-insoluble" RNA is very slow during the first 6 h of development (synchronous cleavage of the egg) but sharply increases after that time. The extent of activation is, however, markedly different in In and 2n embryos; the latter incorporates the label significantly more rapidly at corresponding stages of development. This difference becomes evident from the very beginning of the genetic activity of the cell nuclei which appears to imply that, in diploid embryos, both parental genomes are triggered to be transcribed at the same time. The significance of this finding can be realized in light of the fact that, as indicated by numerous embryological data (see, e.g. ref. I3), paternal characteristics are manifested in embryos of a number of animal species at the gastrula stage or even later. As the cleavage proceeds, the relative difference in RNA production between 2n and In embryos decreases. This could be due to the greater number of cells in In embryos, revealed by cell counts which were made 14 with tile two kinds of loach embryos. Fig. 2 shows changes in the rate of RNA synthesis calculated per cell, in In and 2n embryos. It is seen that the cells of 2n embryos produce about twice as much RNA per unit time as do the cells of In embryos. Biochim. Biophys. Acta, 169 (1968) 274-277
276
SHORT COMMUNICATIONS
12 2.C
x
m2n m2n D..
.c_ 8,
[]ln
~ln
E 1.5
8 < z
5 ca
~
4
.G 4o
o.
< z. r~
1.0
.>_ 0.5 .£
8
~J
._u ) 4 5 Time (h) of d e v e l o p m e n t at 21~
~3L 03
0
7 8 9 Time (h) of d e v e l o p m e n t ot 21°
Fig. I. C h a n g e s in t h e r a t e of 14CO2illcorporationinto 2 . o M N a C l - p r e c i p i t a t e d ( " s a l t - i n s o l u b l e " ) R N A of d e v e l o p i n g diploid (2n) a n d h a p l o i d (In) e m b r y o s of loach. A t s t a g e s indicated, e q u a l p o r t i o n s of eggs f r o m t h e s a m e lot of d e v e l o p i n g eggs were i n c u b a t e d in g l a s s - s t o p p e r e d flasks for 90 mill in a m e d i u m b u f f e r e d ( s o d i u m p h o s p h a t e , o.o2 M) a t p H 6.5, c o n t a i n i n g Ioo I.U. p e n icillin, 5 ° I.U. s t r e p t o m y c i n s u l p h a t e a n d 2o # C s o d i u m [14C]carbonate p e r I ml of final v o l u m e . T e m p e r a t u r e (21 °) a n d g e n t l e s h a k i n g were k e p t c o n s t a n t a t all stages. Nucleic acids were e x t r a c t e d a n d t h o r o u g h l y d e p r o t e i n i z e d w i t h s o d i u m d o d e c y l s u l p h a t e (0. 5 % final concn.) a n d p h e n o l in t h e cold, p r e c i p i t a t e d w i t h alcohol, d i s s o l v e d in t h e cold water, a n d s u b j e c t e d to t h r e e s u c c e s s i v e p r e c i p i t a t i o n s w i t h 2.o M NaC1 a t -- lO% T h e specific r a d i o a c t i v i t i e s of t h e " s a l t - i n s o l u b l e " R N A p r e p a r a t i o n s t h u s o b t a i n e d were d e t e r m i n e d b y m e a s u r i n g t h e R N A c o n t e n t in a l i q u o t s b y abs o r p t i o n a t 260 m # a n d c o u n t i n g cold 5 % trichloroacetic acid-insoluble r a d i o a c t i v i t y of precipit a t e s collected on m e m b r a n e filters, in a n a u t o m a t i c liquid scintillation s p e c t r o m e t e r A C E C (Belgium) u s i n g t h e P P O - P O P O P - t o l u e n e s c i n t i l l a t i o n m i x t u r e . T h e v a l u e s in t h e d i a g r a m repr e s e n t t h e m e a n of two parallel d e t e r m i n a t i o n s . Fig. 2. R a t e of 1*CO2 i n c o r p o r a t i o n into t h e " s a l t - i n s o l u b l e " R N A c a l c u l a t e d p e r one cell, in diploid (2n) a n d h a p l o i d (In) e m b r y o s of l o a c h a t m i d - to late b l a s t u l a s t a g e s . F o r t h e calculation, specific r a d i o a c t i v i t y v a l u e s t a k e n f r o m a n e x p e r i m e n t i l l u s t r a t e d in Fig. I were d i v i d e d b y t h e n u m b e r s of cells p r e s e n t in I n a n d 2n e m b r y o s of l o a c h (cell c o u n t s f r o m ref. 14) a t t h e t i m e of m e a s u r e m e u t of R N A s y n t h e s i s (e.g. to c a l c u l a t e p e r cell i n c o r p o r a t i o n into R N A p r e p a r a t i o n labelled 6 h 15 rain a n d 7 h 45 m i n of d e v e l o p m e n t , t h e cell c o u n t for t h e 7 t h h w a s e m p l o y e d ) .
Thus, RNA production in 2n cells appears to represent a simple sum of RNA productions directed b y the two parental sets of chromosomes. This conclusion is supported b y the results summarized from several experiments (Table I). It can be seen that the rate of RNA synthesis supported b y a single chromosome set is nearly equal in I n and 2n embryos and that, consequently, in 2n cells, the two sets of chromosomes are acting in RN'A synthesis in an additive manner. There is no significant "dose compensation" effect in the production of RNA in either kind of embryo. This allows the conclusion that, as far as the over-all quantitative ac;ivity is concerned, both sets of chromosomes function, independently of one another in early embryos of loach. The results suggest, furthermore, that in early embryos the Biochim. Biophys. Acta, I69 (1968) 2 7 4 - 2 7 7
277
SHORT COMMUNICATIONS TABLE
I
I N C O R P O R A T I O N OF 1 4 C O 2 I N T O BRYOS OF L O A C H A T D I F F E R E N T
"SALT-INSOLUBLE"
R~A
OF DIPLOID (2n) AND HAPLOID ( I n ) EMCALCULATED IN SPECIFIC RADIO-
S T A G E S OF B L A S T U L A F O R M A T I O N ,
ACTIVITY U N I T S P E R O N E C E L L A N D P E R O N E H A P L O I D S E T O F C H R O M O S O M E S
Expt. No.
Ploidy o/ the embryos (n)
Stages and time o/ development (2x °) Early-mid blastula (6th h)
Midblastula (7th h)
Mid-late blastula (8th h)
Late blastula (9th h)
Speci/ic radioactivity o/"salt-insoluble" R N A (counts/rain per rag) per cell
per set
per cell
per set
per cell
per set
per cell
per set
1
i 2
o.71 1.34
o.71 0.67
---
---
0.85 2.46
0.85 1.23
---
---
2
I 2
0.93 2.96
o.93 1.48"
---
---
1.75 3.84
1.75 1.93
---
---
3
I 2
---
---
0.26 0.55
0.26 0.27
0.59 1.43
0.59 o.71
0.90 1.7o
0.90 0-85
4
I 2
---
---
0.96 2.o8
0.96 1.o 4
2.35 5.o9
2.35 2-54
2.35 5.36
2-35 2.68
* I n s o m e e x p e r i m e n t s , t h e In/2n r a t i o of t h e r a t e o f R N A s y n t h e s i s t e n d s t o b e less t h a n 1/2 w h e n m e a s u r e d i n t h e v i c i n i t y of t h e 6 t h h o f d e v e l o p m e n t , a p p a r e n t l y d u e t o a d e l a y i n t h e a c t i v a t i o n o f 1RNA s y n t h e s i s i n t h e n u c l e i of I n c o m p a r e d t o 2 n e m b r y o s .
primary activation of gene transcription in the two parental sets of chromosomes depends on a common control mechanism which triggers them simultaneously at a definite moment of development. C. A. KAFIANI
Institute o/ Molecular Biology and Institute o/Developmental Biology, Academy o/Sciences o/the U.S.S.R., Moscow (U.S.S.R.)
IV[. J . T I M O F E E V A
N. L. MELNIKOVA A. A. NEYFAKH
G. FANKHAUSER, J. Exptl. Zool., i o o (1945) 445C. J . EPSTEIN, Proc. Natl. Acad. Sci. U.S., 57 (1967) 327. P. D. SCHINDLER, Acta Anat., 44 (1961) 273. A. S. ]}¢[UKHERGEE AND W . BEERMAN, Nature, 207 (1965) 785 . T H . BOVERY, Jen. Z. Naturwiss., 39 (19o5) 445. C. F. GRAHAM,Exptl. Cell Res., 41 (1966) 13. C. A. KAFIANI AND M. J. TIMOFEEVA, Dokl. Acad. Nauk SSSR, 154 (1964) 721. A. A. NEYFAKH,J. Embryol. Exptl. Morphol., 7 (1959) 173. S. COHEN, J. Biol. Chem., 211 (1954) 337. M. J. TIMOFEEVA AND C. A. KAFIANI, Dokl. Acad. Nauk S S S R , 165 (1965) 1183. M. A. AJTKHOZHIN, N . V. BELITSlNA AND A. S. SPIRIN, Biokhimiya, 29 (1964) 169. M. J . TIMOFEEVA AND C. A. KAFIANI, Biokhimiya, 29 (1964) i i o . P. BRIGGS AND T . J. K I N G , i n J. BRACHET AND A. E . MIRSKY, The Cell, Vol. I, A c a d e m i c P r e s s , N e w Y o r k , 196o, p . 53714 N . N. ROTT AND G. A. SHEVELEVA, Cytologia, 9 (1967) 1265.
I 2 3 4 5 6 7 8 9 io II 12 13
Received August 2nd, 1968 Biochim. Biophys. Acta,
169 (1968) 274 277
SHORT COMMUNICATIONS
now remain to be further investigated, but it is very interesting that L-histidine acceptor tRNA's of bacteria have a peak of high specific activity in the early fractions from DEAL-cellulose. I am deeply indebted to Dr. WALTER S. McNuTT, with whom I worked under his suggestions, without which this work would have been impossible. I wish to thank Mrs. ROBERTA McDuFFIE for her skillful assistance.
Department o[ Biochemistry, School o/ Medicine, Tu/ts University, Boston, Mass. (U.S.A.)
ISAO TAKEDA*
1 T. YAMANE, T. Y. CHENG AND N. SOEOKA, Cold Spring Harbor Syrup. Quant. Biol., 28 (i963) 569. 2 S. G. NATHENSON, F. C. DOHAN, H. H. RICHARDS AND G. L. CANTONI, Biochemistry, 4 (1965) 2412. 3 T. P. BENNETT, J. GOLDSTEIN AND F. LIPMANN, Proc. Natl. Acad. Sci. U.S., 49 (1963) 850. 4 J. D. CHERAYIL AND R. M. BOCK, Biochemistry, 4 (1965) 1174. 5 ~N'. SUEOKA AND T. I(ANO-SUEOKA, Proc. Natl. Acad. Sci. U.S., 52 (1964) 1535. 6 G. HARTMAN AND U. COY, Biochim. Biophys. Acla, 47 (1961) 612. 7 J. APGAR, R. W. HOLLEY AND S. H. MERRILL, J. Biol. Chem., 237 (1962) 796. 8 H. G. ZACHAU, ~V[.TADA, W. LAWSON AND M. SCHWEIGER, Biochim. Biophys. Acta, 53 (1961) 221. 9 A. SAPONARA AND I{. M. BOCK, Federation Proc., 20 (1961) 356. io R. W. I-[OLLEY, J. APGAR, B. P. DOCTOR, J. FARROW, M. A. MARINI AND S. H. MERRILL, J . Biol. Chem., 236 (1961) 200. I I G. W. BEADLE AND I~. L. TATUM, Am. J. Botany, 32 (1945) 67812 C. KLEE AND M. STAEHELIN, Biochim. Biophys. Acta, 61 (1962) 668. 13 W. E. BARNETT AND K. B. JACOBSON, Proc. Natl. Acad. Sci. U.S., 51 (1964) 642.
Received July 3oth, 1968 * Present address: Asahi Chemical Co., Technical Research Laboratory, Nakadai 3-27, I t a bashi, Tokyo, Japan.
Biochim. Biophys. Acta, 169 (1968) 272-274
BBA 93367
Rote of RNA synthesis in hoploid ond diploid embryos of loach
fossnis)
(Misgurnus
In animal cells, the relationship between ploidy and biosynthetic capacity has been insufficiently studied from the quantitative point of view. In some ceils a direct proportionality is found between cell size and ploidy, suggesting the operation of a "gene dosage control" of biosynthetic activities in animal cellsZ,2; in others, however, no such correlation was revealed ~. On a more detailed level, there are examples of a "dosage compensation" phenomenon, e.g. compensatory inhibition of the activity of one or both allelic genes 4. Studies with embryes of sea urchins 5 and amphibia 1,8 point to the action of gene dosage control of biosynthetic capacities in developing cells. The evidence, however, is of an indirect nature. In the present work an attempt was made to employ a more direct approacb by comparing the rates of R N A synthesis in diploid (2n) and haploid (In) embryos Biochim. Biophys. Acta, 169 (1968) 274-277
SHORT COMMUNICATIONS
275
of loach (Misgurnus/ossilis). The dynamics of gene transcription in 2n embryos of loach have been quantitatively studied in earlier work 7 which revealed a sharp increase in the rate of RNA synthesis in the cell nuclei at mid-blastula stage; this is also the time of the onset of "morphogenetic activity" of the nuclei (i.e. nuclear function in supporting gastrulation and morphogenesis) s. Mature eggs of loach were obtained, fertilized, and allowed to develop in tap water at 21 ° as described elsewhere 8. Gynogenetic haploid embryos were obtained by fertilizing normal eggs (from the same lot as that used to obtain 2n embryos) with the X-ray-irradiated sperm (dose 80 kR). RNA synthesis was measured by the incorporation of 14C0~ which was introduced as described by COHEN9. Experiments consisted of pulsing (90-min pulses) whole eggs, at different stages of development, with ~4C02 under standard conditions (see the legend to Fig. I), and measuring specific radioactivities of RNA extracted from the egg.~. In order to avoid possible interference due Lo the terminal exchange in transfer RNA's the latter were removed from the preparations (together with DNA, protein and polysaccharide impurities) by precipitating the "salt-insoluble" RNA's with 2.0 M NaC1 in the cold (see details in the legend to Fig. I). The "salt-insoluble" RNA preparations thus obtained contain ribosomalRNA's and polydisperse RNA of the nuclei and cytoplasm 1°. Since rRNA's are not synthesized at the stages studied here l°,u, the label is incorporated exclusively into polydisperse RNA species 1°. It has been found weviously that the rate of 14C02 incorporation is not limited (in conditions specified here) by the incorporation into free (cold trichloroacetic acid-soluble) nucleotides of the eggs 7, and that the "salt-insoluble" RNA content of the eggs remains constant during the developmental period studied TM. Hence, it can be inferred that the variations in specific radioactivities of "salt-insoluble" RNA preparations obtained at different developmental stages after standard pulses with 14C0~, reflect the variations in the rates of synthesis of polydisperse RNA's (on a per embryo basis) and, consequently, can be regarded as an index of the changes in the overall rate (activity) of gene transcription in the developing embryos. Fig. I shows, in conformity with earlier observations 7, that incorporation of 14C02 into "salt-insoluble" RNA is very slow during the first 6 h of development (synchronous cleavage of the egg) but sharply increases after that time. The extent of activation is, however, markedly different in In and 2n embryos; the latter incorporates the label significantly more rapidly at corresponding stages of development. This difference becomes evident from the very beginning of the genetic activity of the cell nuclei which appears to imply that, in diploid embryos, both parental genomes are triggered to be transcribed at the same time. The significance of this finding can be realized in light of the fact that, as indicated by numerous embryological data (see, e.g. ref. I3), paternal characteristics are manifested in embryos of a number of animal species at the gastrula stage or even later. As the cleavage proceeds, the relative difference in RNA production between 2n and In embryos decreases. This could be due to the greater number of cells in In embryos, revealed by cell counts which were made 14 with tile two kinds of loach embryos. Fig. 2 shows changes in the rate of RNA synthesis calculated per cell, in In and 2n embryos. It is seen that the cells of 2n embryos produce about twice as much RNA per unit time as do the cells of In embryos. Biochim. Biophys. Acta, 169 (1968) 274-277
276
SHORT COMMUNICATIONS
12 2.C
x
m2n m2n D..
.c_ 8,
[]ln
~ln
E 1.5
8 < z
5 ca
~
4
.G 4o
o.
< z. r~
1.0
.>_ 0.5 .£
8
~J
._u ) 4 5 Time (h) of d e v e l o p m e n t at 21~
~3L 03
0
7 8 9 Time (h) of d e v e l o p m e n t ot 21°
Fig. I. C h a n g e s in t h e r a t e of 14CO2illcorporationinto 2 . o M N a C l - p r e c i p i t a t e d ( " s a l t - i n s o l u b l e " ) R N A of d e v e l o p i n g diploid (2n) a n d h a p l o i d (In) e m b r y o s of loach. A t s t a g e s indicated, e q u a l p o r t i o n s of eggs f r o m t h e s a m e lot of d e v e l o p i n g eggs were i n c u b a t e d in g l a s s - s t o p p e r e d flasks for 90 mill in a m e d i u m b u f f e r e d ( s o d i u m p h o s p h a t e , o.o2 M) a t p H 6.5, c o n t a i n i n g Ioo I.U. p e n icillin, 5 ° I.U. s t r e p t o m y c i n s u l p h a t e a n d 2o # C s o d i u m [14C]carbonate p e r I ml of final v o l u m e . T e m p e r a t u r e (21 °) a n d g e n t l e s h a k i n g were k e p t c o n s t a n t a t all stages. Nucleic acids were e x t r a c t e d a n d t h o r o u g h l y d e p r o t e i n i z e d w i t h s o d i u m d o d e c y l s u l p h a t e (0. 5 % final concn.) a n d p h e n o l in t h e cold, p r e c i p i t a t e d w i t h alcohol, d i s s o l v e d in t h e cold water, a n d s u b j e c t e d to t h r e e s u c c e s s i v e p r e c i p i t a t i o n s w i t h 2.o M NaC1 a t -- lO% T h e specific r a d i o a c t i v i t i e s of t h e " s a l t - i n s o l u b l e " R N A p r e p a r a t i o n s t h u s o b t a i n e d were d e t e r m i n e d b y m e a s u r i n g t h e R N A c o n t e n t in a l i q u o t s b y abs o r p t i o n a t 260 m # a n d c o u n t i n g cold 5 % trichloroacetic acid-insoluble r a d i o a c t i v i t y of precipit a t e s collected on m e m b r a n e filters, in a n a u t o m a t i c liquid scintillation s p e c t r o m e t e r A C E C (Belgium) u s i n g t h e P P O - P O P O P - t o l u e n e s c i n t i l l a t i o n m i x t u r e . T h e v a l u e s in t h e d i a g r a m repr e s e n t t h e m e a n of two parallel d e t e r m i n a t i o n s . Fig. 2. R a t e of 1*CO2 i n c o r p o r a t i o n into t h e " s a l t - i n s o l u b l e " R N A c a l c u l a t e d p e r one cell, in diploid (2n) a n d h a p l o i d (In) e m b r y o s of l o a c h a t m i d - to late b l a s t u l a s t a g e s . F o r t h e calculation, specific r a d i o a c t i v i t y v a l u e s t a k e n f r o m a n e x p e r i m e n t i l l u s t r a t e d in Fig. I were d i v i d e d b y t h e n u m b e r s of cells p r e s e n t in I n a n d 2n e m b r y o s of l o a c h (cell c o u n t s f r o m ref. 14) a t t h e t i m e of m e a s u r e m e u t of R N A s y n t h e s i s (e.g. to c a l c u l a t e p e r cell i n c o r p o r a t i o n into R N A p r e p a r a t i o n labelled 6 h 15 rain a n d 7 h 45 m i n of d e v e l o p m e n t , t h e cell c o u n t for t h e 7 t h h w a s e m p l o y e d ) .
Thus, RNA production in 2n cells appears to represent a simple sum of RNA productions directed b y the two parental sets of chromosomes. This conclusion is supported b y the results summarized from several experiments (Table I). It can be seen that the rate of RNA synthesis supported b y a single chromosome set is nearly equal in I n and 2n embryos and that, consequently, in 2n cells, the two sets of chromosomes are acting in RN'A synthesis in an additive manner. There is no significant "dose compensation" effect in the production of RNA in either kind of embryo. This allows the conclusion that, as far as the over-all quantitative ac;ivity is concerned, both sets of chromosomes function, independently of one another in early embryos of loach. The results suggest, furthermore, that in early embryos the Biochim. Biophys. Acta, I69 (1968) 2 7 4 - 2 7 7
277
SHORT COMMUNICATIONS TABLE
I
I N C O R P O R A T I O N OF 1 4 C O 2 I N T O BRYOS OF L O A C H A T D I F F E R E N T
"SALT-INSOLUBLE"
R~A
OF DIPLOID (2n) AND HAPLOID ( I n ) EMCALCULATED IN SPECIFIC RADIO-
S T A G E S OF B L A S T U L A F O R M A T I O N ,
ACTIVITY U N I T S P E R O N E C E L L A N D P E R O N E H A P L O I D S E T O F C H R O M O S O M E S
Expt. No.
Ploidy o/ the embryos (n)
Stages and time o/ development (2x °) Early-mid blastula (6th h)
Midblastula (7th h)
Mid-late blastula (8th h)
Late blastula (9th h)
Speci/ic radioactivity o/"salt-insoluble" R N A (counts/rain per rag) per cell
per set
per cell
per set
per cell
per set
per cell
per set
1
i 2
o.71 1.34
o.71 0.67
---
---
0.85 2.46
0.85 1.23
---
---
2
I 2
0.93 2.96
o.93 1.48"
---
---
1.75 3.84
1.75 1.93
---
---
3
I 2
---
---
0.26 0.55
0.26 0.27
0.59 1.43
0.59 o.71
0.90 1.7o
0.90 0-85
4
I 2
---
---
0.96 2.o8
0.96 1.o 4
2.35 5.o9
2.35 2-54
2.35 5.36
2-35 2.68
* I n s o m e e x p e r i m e n t s , t h e In/2n r a t i o of t h e r a t e o f R N A s y n t h e s i s t e n d s t o b e less t h a n 1/2 w h e n m e a s u r e d i n t h e v i c i n i t y of t h e 6 t h h o f d e v e l o p m e n t , a p p a r e n t l y d u e t o a d e l a y i n t h e a c t i v a t i o n o f 1RNA s y n t h e s i s i n t h e n u c l e i of I n c o m p a r e d t o 2 n e m b r y o s .
primary activation of gene transcription in the two parental sets of chromosomes depends on a common control mechanism which triggers them simultaneously at a definite moment of development. C. A. KAFIANI
Institute o/ Molecular Biology and Institute o/Developmental Biology, Academy o/Sciences o/the U.S.S.R., Moscow (U.S.S.R.)
IV[. J . T I M O F E E V A
N. L. MELNIKOVA A. A. NEYFAKH
G. FANKHAUSER, J. Exptl. Zool., i o o (1945) 445C. J . EPSTEIN, Proc. Natl. Acad. Sci. U.S., 57 (1967) 327. P. D. SCHINDLER, Acta Anat., 44 (1961) 273. A. S. ]}¢[UKHERGEE AND W . BEERMAN, Nature, 207 (1965) 785 . T H . BOVERY, Jen. Z. Naturwiss., 39 (19o5) 445. C. F. GRAHAM,Exptl. Cell Res., 41 (1966) 13. C. A. KAFIANI AND M. J. TIMOFEEVA, Dokl. Acad. Nauk SSSR, 154 (1964) 721. A. A. NEYFAKH,J. Embryol. Exptl. Morphol., 7 (1959) 173. S. COHEN, J. Biol. Chem., 211 (1954) 337. M. J. TIMOFEEVA AND C. A. KAFIANI, Dokl. Acad. Nauk S S S R , 165 (1965) 1183. M. A. AJTKHOZHIN, N . V. BELITSlNA AND A. S. SPIRIN, Biokhimiya, 29 (1964) 169. M. J . TIMOFEEVA AND C. A. KAFIANI, Biokhimiya, 29 (1964) i i o . P. BRIGGS AND T . J. K I N G , i n J. BRACHET AND A. E . MIRSKY, The Cell, Vol. I, A c a d e m i c P r e s s , N e w Y o r k , 196o, p . 53714 N . N. ROTT AND G. A. SHEVELEVA, Cytologia, 9 (1967) 1265.
I 2 3 4 5 6 7 8 9 io II 12 13
Received August 2nd, 1968 Biochim. Biophys. Acta,
169 (1968) 274 277