265

Biochimica et Biophysica Acta, 565 (1979) 2 6 5 - - 2 7 4 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press

BBA 9 9 5 6 4

ISOLATION AND CHARACTERIZATION OF cDNA COMPLEMENTARY TO TRANSIENT MATERNAL POLY(A) ÷ RNA FROM THE DROSOPHILA OOCYTE

E L L I O T T S. G O L D S T E I N * and C H A R L E S G. A R T H U R **

Arizona State University, Department of Zoology, Tempe, AZ 95281 (U.S.A.) (Received F e b r u a r y 19th, 1979) (Revised m a n u s c r i p t received May 29th, 1979)

Key words: Embryogenesis; Complementary DNA; Poly(A) ÷RNA; Oocyte; (Drosophila)

Summary cDNA complementary to total oocyte poly(A) ÷ RNA from Drosophila melanogaster was enriched for sequences complementary to transient maternal sequences; that is, those sequences which disappear from the oocyte during subsequent. A seven- to ten-fold enrichment factor was obtained, from 5.3% to about 50% of the total cDNA. Kinetic analysis of this enriched fraction indicates that the transient maternal sequences include 44 ± 14 different sequences.

Introduction The oocyte of Drosophila melanogaster, the fruit fly, develops as a 16 cell cluster containing the oocyte and 15 nurse cells. This development requires 80 h and has been divided into 14 sequential stages [1]. As soon as the cluster has formed, the oocyte enters the meiotic pathway and is soon arrested during prophase of the first meiotic division. The nurse cells synthesize most of the material found in the mature oocyte. Toward the end of development they begin to shrink; material can be seen streaming into the oocyte, which continues until the nurse cells disappear. Follicle cells, which surround the cluster and secrete the egg envelope, die prior to release of the oocyte from the ovary. Meiosis is completed as the egg passes down the oviduct.

* To w h o m reprint requests should be addressed. ** Present address: Zoology Department, University of Texas, Austin, U.S.A. Abbreviations: Hepes, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; SDS, sodium dodecyl sulfate; cDNA0 c o m p l e m e n t a r y DNA; AMV, avian myeloblastosis virus; RNPs, ribonucleoProtein particles.

266 The mature stage 14B oocyte of D. rnelanogaster contains approx. 14 500 different poly(A) containing RNA sequences (poly(A) ÷ RNA) averaging 2017 nucleotides each in length. These sequences fall into three frequency classes consisting of 102 high abundant, 2877 middle abundant, and 11 500 rare, sequences [2]. Of all poly(A) ÷ RNA sequences, 40% are found in polysomes, the remainder in free ribonucleoprotein particles (RNPs) [3]. During embryonic development, there is an increase in the proportion of poly{A) ÷ sequences on polysomes. However, in oocytes and prehatching embryos, there are essentially no differences in the sequences found in the free RNPs as compared to those found on polysomes (at approx. 1% resolution}, indicating that the poly(A) ÷ RNA sequences in the free RNPs consist of a surplus of the same sequences found on polysomes [4]. In the period of time from oocyte to prehatching embryo (19 h), 5.3% of the mature oocyte sequences are lost. Furthermore, 7.7% of gastrular and 11.0% of late prehatching embryo sequences are not found in the oocyte [2]. Our previous studies involved the analysis of hybridization kinetics of cDNA to poly(A) ÷ RNA from total cytoplasmic extracts. The number of sequences lost or gained could not be determined since the frequency class from which the poly(A) ÷ RNA was derived was not known. Because of the difficulty of interpreting kinetic measurements involving total cellular poly(A) ÷ RNA, we have begun fractionating the system into less complex, more easily analyzed populations. We report here that the transient maternal sequences (i.e. the 5.3% which disappear during embryonic development) are composed of approx. 44 different sequences. The implications of these results are discussed in a model of embryogenesis in Drosophila. Materials and Methods

Isolation o f oocyte and embryonic p o l y ( A ) ÷ R N A D. melanogaster derived from an Oregon R strain was used t h r o u g h o u t this investigation [5]. Oocytes were prepared en masse from three- to five-day-old flies by homogenization in a Waring blender. The homogenate was sifted through sieves and the oocytes further purified by differential settling and adhesion to glass. Final purity of mature stage 14B oocytes was greater than 95% [3]. Fertilized eggs were collected for 4 h and aged for 17 h to provide uniform populations of 19 + 2 hours-old late prehatching embryos. Oocytes and embryos were homogenized in extraction buffer (35 mM K Hepes, pH 7.4, 75 mM KC1, 5 mM Mg acetate, 4 mM dithiothreitol) containing 2--4% diethylpyrocarbonate. Post-mitochondrial supernatant fractions were prepared as previously described [5,6]. RNA was extracted with phenol/ chloroform and precipitated with ethanol. Poly(A) ÷ RNA was isolated using oligo(dT) cellulose chromatography (Collaborative Research) [ 3,7 ]. Quantification o f p o l y ( A ) ÷ R N A Quantification of poly(A) ÷ RNA was based on the hybridization of [3H]poly(U) {Schwarz-Mann; 22 Ci/mM), followed by digestion of single stranded material with pancreatic RNAase (Sigma) [8]. This reaction was standardized by titration of the [3H]poly(U) with known quantities of p o l y ( A ) t o establish

267

the cpm [3H]poly(U) bound per ng of poly(A). The size of the poly(A) ÷ RNA was determined by SDS sucrose gradient centrifugation followed by hybridization of each fraction to [3H]poly(U) [3,4]. The distribution of [3H]poly(U) binding across the gradient served as a basis for the calculation of numberaverage molecular weight of the R N A in each fraction. No difference in sedimentation of poly(A) ÷ R N A relative to the 19 S ribosomal RNA was observed following centrifugation in 70% formamide (8--23% sucrose} for 48 h at 37 000 rev./min in a Spinco SW 41 rotor. The size of the poly(A) tails was measured on 10% acrylamide-SDS gels using poly(A) standards averaging 120, 54 and 28 nucleotides in length (Miles Biochemicals) [2]. Synthesis and characterization o f cDNA Drosophila cDNA was made using o o c y t e poly(A) ÷ RNA as a template for Avian Myeloblastosis Virus (AMV) reverse transcriptase (the generous gift of Dr. J.W. Beard). Globin cDNA was synthesized from rabbit globin m R N A (Miles Biochemical). The reaction mixture consisted of 500 t~Ci of [3H]dCTP (20.9 Ci/mmol, ICN), 10 mM dithiothreitol, 50 mM Tris-HC1 (pH 8.3), 6 mM MgC12, 5t~g/ml oligo{dT),2-1s (Collaborative Research), 10 gg/ml actinomycin D, 1 mM each of dATP, dGTP, dTTP (Sigma), in a final reaction volume of 2.0 ml. 20 pg of poly(A) ÷ RNA and 150 units of AMV reverse transcriptase were added to the reaction vial and the mixture incubated at 37°C for 1 h [4,9]. cDNA length was determined by centrifugation on a linear 8--18% (w/v) sucrose gradient in 0.1 N NaOH at 37 000 rev./min for 24 h at 5°C in a Spinco SW 41 rotor, with a Hind III (Miles Biochemicals) digest of phage k as a size marker. A separate analysis using 1.4% agarose gel electrophoresis and the Hind III digest was also performed. All cDNA reactions were carried out in siliconized glassware. Hybridization o f cDNA to poly(A) ÷R N A A. Isolation o f transient sequences. Isolation of transient sequences was carried o u t b y three hybridization reactions modified from a previously described procedure [4]. The first hybridization reaction was carried o u t in a 0.24 M phosphate equimolar buffer, 0.2% SDS, and 2 mM EDTA (hybridization buffer) at 70°C in siliconized reactivials (Pierce Chemical Co.) overlaid with paraffin oil. A 500--1000-fold excess of embryonic poly(A) ÷ R N A was mixed with cDNA complementary to o o c y t e R N A and heated to 100°C to begin the reaction. Hybridization was allowed to proceed to a total R N A Rot o f 150--300 which represents a 5--10-fold excess of that required for plateau. The extent of hybridization was determined by sensitivity to S, nuclease (Sigma) [8]. The single stranded cDNA enriched for transient sequences remaining after hybridization was isolated by Chromatography on 500 mg hydroxylapatite columns (BioRad HTP DNA grade) at 60°C. The reaction mixture was diluted in 2.0 ml to a final concentration of 0.12 M phosphate equimolar buffer and applied to a column equilibrated in the same buffer. Most but n o t all of the single stranded material eluted in the first 5 ml. The cDNA fraction was treated with 0.3 N NaOH to hydrolyze RNA and dialyzed extensively into 100 mM NaC1, 10 mM Tris-HC1, pH 7.4. It was then made 200 mM in sodium-acetate,

26~ pH 5.4, precipitated with ethanol and recovered by ultra-centrifugation for 1.5 h at 37 000 rev./min in a Spinco SW 41 rotor in polyallomer tubes. '[he cDNA was resuspended in a small volume of hybridization buffer by incubation at 37°C for several hours. For the second hybridization step, a 500--1000 fold excess of oocyte RNA in hybridization buffer was added to the cDNA and the reaction was can'ied out as above. The double-stranded complexes formed during the reaction were isolated by chromatography on hydroxyapatite. Since a marked trailing of single-stranded material occurred, it was np.cessary to wash the column with 20--40 ml of 0.12 M phosphate equimolar buffer until the cpm showed no further decrease. Double-stranded material was eluted with 5 ml of 0.5 M phosphate equimolar buffer and treated as above. For the third hybridization step the enriched cDNA was hybridized to embryonic RNA as described in the first step. Several modifications of the original analytical procedure [2] were necessary to maximize recovery. Dialysis proved to be a better m e t h o d for changing salt concentration than gel filtration, since cDNA had a tendency to stick to gel filtration beads from Pharmacia, LKB, and BioRad. The major portion of the cDNA would elute in the void volume of the gel but a skewed peak trailed into the elution volume. This was not due to size separation of the cDNA. Dialysis, by comparison, resulted in quantitative recovery. The omission of SDS from the buffer used for h y d r o x y a p a t i t e chromatography resulted in faster elution of single-stranded material but did not alter the phosphate concentration required. We also found that SDS was extremely difficult to eliminate between rounds of hybridization and tended to cause elution of trace amounts of CaPO4 from the column which would then accumulate in the cDNA preparations. It was occasionally necessary to tre~t column elutes with a-amylase (Sigma) to prevent glycogen accumulation derived from the RNA preparation. No size change in the cDNA was observed during the isolation procedure. B. Reassociation kinetics hybridization. The reassociation kinetics of cDNA and poly(A) ÷ RNA were performed as described above. Aliquots of sufficient size to contain approx. 2000 cpm were withdrawn at appropriate times and assayed with $1 nuclease to determine the percentage of double stranded material formed. The RNA concentration used to calculate the Rot (total) was determined from the poly(A) content assuming the poly(A) represented 2.6% of the poly(A) ÷ RNA. The Rot for the transient maternal sequences was calculated from the total RNA Rot by multiplying by 5.3%, the proportion of RNA that disappears during development [2].

Results

Characterization o f poly(A) ÷RNA and cDNA The size of the poly(A) ÷ RNA fractions used in this study was characterized by sucrose gradient sedimentation. The distribution of A260 material and the distribution of poly(A) as determined by hybridization to [3H]poly(U) (Fig. 1A) indicated that the poly(A)* RNA preparations were 1500 to 2000 nucleotides in length. Any preparations showing visible rRNA contamination

269

Also

19 S

26S

I

I

cpm 5OO

00;

250

1

cpm

B I00C

2

3

4

6 7 8 g 5 Froction number

10

12 S

I

18 Froc--tion number Fig. I . C h a r a c t e r / z a t i o n o f p o l y ( A ) + R N A a n d e D N A . (A) T h e s e d i m e n t a t i o n p r o f i l e o n 1 5 - - 3 0 % S D S s u c r o s e g r a d i e n t s o f t h e p o l y ( A ) + R N A f r a c t i o n t h a t b i n d s t o o l i g o ( d T ) c e l l u l o s e as d e s e r i b e d i n Mateldals and Methods. An aliquot of each gradient fraction was analyzed for poly(A) content by hybridization to [3H]poIy(U). The location of the 19 S and 26 S markers were determined in parallel gradients with the m a t e r i a l t h a t d o e s n o t b i n d t o o l i g o ( d T ) cellulose, c p m o f [ 3 H ] D o l y ( U ) h y b r i d i z e d i n e a c h f r a c t i o n , b a r graph; A260 ( ), (B) A l k a l i n e s u c r o s e g r a d i e n t c e n t r i f u l a t i o n o f e D N A p r e p a r e d f r o m o o c y t e p o l y ( A ) + R N A as d e s c r i b e d i n M a t e r i a ] s a n d M e t h o d s . T h e e D N A w a s a u l y z e d o n 8 - - 1 8 % s u c r o s e g r a d i e n t s i n 0 . 1 N N a O H . T h e 1 2 S r n ~ k e r i n d i c a t e s t h e p o s i t i o n o f t h e p h a g e ~ 2 3 5 0 b a s e p a i r s ( a v e r a g e ) H i n d III restriction fragments.

as determined by A260, or poly(A) * R N A degradation as determined by the distribution of poly(A) ÷ R N A after hybridization with [3H]poly(U), were not used. Although ribosomal R N A contamination can not be rigorously excluded, it would not affect the Rot value since the assay only detects poly(A) containing RNA. Quantification o f the poly(A) ÷ R N A was based on the binding o f [3H]poly(U) to the RNA, with a poly(A) length of 52 nucleotides [2i. The cDNA synthesized from oocyte poly(A)+ R N A showed a broad sedimentation pattern on alkaline sucrose gradients (Fig. 1B). The c D N A had a number average o f 620 nucleotides. The 2.2 and 2.5 kilobase Hind III fragments Of

270 phage ,~ are indicated as a size marker of 12 S (average). A separate analysis using agarose gel electrophoresis, where the 490 base Hind III fragment of phage k is visible, confirmed the cDNA length.

Isolation and characterization of transient oocyte sequences The cDNA sequences complementary to poly(A) ~ RNA which disappears during development (i.e., transient maternal RNA) were isolated by a modification of the original analytical procedure [2]. The cDNA made from total oocyte poly(A) ÷ RNA was hybridized in three successive stages: first to total late embryonic, then to mature oocyte, and finally to total late embryonic poly(A) ÷ RNA. Both hybridizations to embryonic RNA depleted the cDNA sequences in common, while enriching for the transient oocyte sequences. Typically, 65% hybridization was reached in the first stage of hybridization. The second stage, hybridization to homologous oocyte RNA resulted in approx. 40% hybridization and was necessary so that the 35% nonhybridizing cDNA originally present does not accumulate. This hybridization does not affect the population of cDNA used for later complexity studies. Since the reaction is assumed to be random, no specific sequence will be favored or omitted. The third round of hybridization to RNA from 19 h embryos was 10--20% of the input cDNA. The extent of hybridization in each step was monitored by sensitivity to $1 nuclease and found to be identical to that in the analytical reactions [2]. Final recovery of cDNA complementary to transient maternal RNA was 10% of the sequences theoretically present in the mature o o c y t e and represents 0.5% of the initial cDNA input. Significant losses occurred during chromatography on HAP and recovery of ethanol precipitates. Three separate preparations of cDNA were used in this study (Table IA). Before enrichment, the cDNA hybridized almost as well to embryonic RNA as it did to oocyte RNA. Afterward there was an approximate 7--10 fold enrichm e n t from 7% to 47% for sequences originally present in the oocyte, but missing by 19 h (Table IB). The 7% value is in good agreement with the more accurate, previous determination of 5.3% [2]. Following enrichment, the level of hybridization to embryonic RNA decreased from 68% to 23%, while hybridization to oocyte RNA remained almost unchanged. The three differnt preparations of cDNA enriched for transient maternal sequences (Table I) were analyzed separately for sequence complexity by hybridization to total oocyte poly(A) ÷ RNA. The curve constructed from the kinetic analysis of one of the preparations is shown in Fig. 2. Both the total RNA Rot and the Rot of the transient maternal RNA sequences (5.3% of the total) are indicated on the abscissa. As expected, the curve shows two components. The main c o m p o n e n t with a plateau at about 45% hybridization corresponds to cDNA complementary to the transient sequences. The second component, 20 to 25% of the total, represents contamination by sequences which hybridize to embryonic RNA. These values are consistent with the data shown in Table IB, which indicate that hybridization to oocyte RNA is the result of hybridization to both transient and embryonic sequences. Table II is a summary of the sequence complexity data derived from kinetic analysis of the three enriched cDNA preparations described in Table I. Sequence complexity is calculated from the Rot~/2 real value based on the

271 TABLE I H Y B R I D I Z A T I O N OF e D N A C O M P L E M E N T A R Y TO OOCYTE P O L Y ( A )÷ TO M A T U R E OOCYTE AND LATE EMBRYONIC POLY(A)+ RNA A. B e f o r e E n r i c h m e n t Poly(A)+ R N A

S e q u e n c e s n o t in c o m m o n

(%) Oocyte

Embryo

(%)

(%)

Expt. 1 Expt. 2 Expt. 3

77 69 78

67 63 75

Average

75 + 5

68 ± 6

7

B. A f t e r E n r i c h m e n t S e q u e n c e s n o t in c o m m o n

Poly(A)+ RNA

(%) Oocyte

Embryo

(%)

(%)

Expt. 1 Expt. 2 Expt. 3

62 85 64

13 34 22

Average

70 • 13

23 ± 10

47

7C

5C 0 0

0 0

0

3( 0

0

0

0

0

0

0

10

-I

6

LgRet(transient)

÷2

.1

LgRot (total)

Fig. 2. H y b r i d i z a t i o n o f e D N A c o m p l e m e n t a r y t o t r a n s i e n t m a t e r n a l R N A w i t h t o t a l o o c y t e p o l y ( A ) + R N A . H y b r i d i z a t i o n is e x p r e s s e d as a p e r c e n t a g e o f S I r e s i s t a n t m a t e r i a l . A b l a n k o f 2% S 1 r e s i s t a n c e a f t e r 5 r a i n a t 1 0 0 ° C h a s b e e n s u b t r a c t e d . T h e Rot v a l u e s s h o w n are f o r t o t a l p o l y ( A ) + R N A , a n d t h e 5.3% o f t h e t o t a l p o l y ( A ) ÷ R N A w h i c h d i m ~ p e a r s d u r i n g d e v e l o p m e n t .

272 T A B L E II S E Q U E N C E C O M P L E X I T Y OF c D N A C O M P L E M E N T A R Y T O T H E T R A N S I E N T M A T E R N A L P O L Y (A)+ R N A OF T H E M A T U R E OOCYTE C o m p u t a t i o n o f t h e n u m b e r o f s e q u e n c e is as follows: R0 t l / 2 H b s t a n d a r d

1 = -- w h e r e the R 0 t 1/2 of the Hb s t a n d a r d is c o r r e c t e d for salt c o n c e n t r a t i o n a n d

RO tl /2 Drosophila

X

l e n g t h of e D N A a n d m R N A u s e d in e a c h i n d i v i d u a l p r e p a r a t i o n [ 1 4 , 1 5 ] such t h a t : (6-10-4) a.

2.9 (~)b.

1560 (1~)c.

470 d (~) = 6 . 4 8 . 10 -4

w h e r e : a, R 0 t 1/2 of H b s t a n d a r d ( a a n d ~); b, Salt c o n c e n t r a t i o n c o r r e c t i o n ; c, R N A l e n g t h c o r r e c t i o n ; d, e D N A l e n g t h c o r r e c t i o n .

Expt. 1 Expt. 2 Expt. 3 Average

R 0 tl/2 a p p a r e n t

RO tl/2 real

N u m b e r of different sequences

0.076 0.035 0.070

0.035 0.018 0.032

54 28 49 4 4 +_ 14

transient RNA Rot. The Rotl/2 apparent is converted to Rotl/2 real by multiplying that value by the fraction of the total cDNA represented in the major transition. This is then converted to the number of different sequences by comparison to the Rotl/: of the globin standard. The data presented in Table II indicate that the major transient RNA c o m p o n e n t is represented by 44 + 14 different sequences of 2000 nucleotides each.

Discussion The procedures described in this paper allow the isolation of specific enriched cDNA subfractions. These procedures can be adapted to any system where two different cell types can be isolated. Hybridization depletion of sequences in c o m m o n results in a cDNA fraction enriched for non-shared sequences. The major limitation in the procedure does not seem to be due to the hybridization procedures themselves, but rather to separation on hydroxyapatite which does n o t eliminate very low levels of single-stranded contamination of the double stranded material. There are two major advantages to this procedure for estimating sequence complexity differences between two cell types using an isolation procedure, as compared to the derivation of this value from the difference between two kinetic assays. First, it is more accurate; second, and more importantly, the isolated sequences are then available for further study. We are now using fractionated cDNAs to identify appropriate recombinant DNA clones. The results reported here would be sensitive to contamination by hnRNA, whether by leakage or by h n R N A synthesized in the oocyte at an earlier stage and transferred to the cytoplasm. Leakage would n o t be of concern in oocyte RNA preparations since the haploid nucleus represents an insignificant a m o u n t of the oocyte volume and little if any transcription is occurring at that stage [ 10]. Visual observations of 19-h preparations after homogenization reveals no

273 significant nuclear disruption. If h n R N A contamination were present, an increase in poly(A) ÷ RNA size would be expected, as h n R N A is larger than cytoplasmic RNA. However, no such increase was observed in any preparation. Contamination of 19-h R N A with younger or older stage material was eliminated by the techniques used to collect the embryos. Embryos derived from eggs laid over a 4 h interval and aged for 17 h must be at least 17 hours-old. No larvae were detected; this confirms an upper age limit of 22 h, the time at which hatching occurs. Previous results have shown that depletion of sequences in c o m m o n between mature oocytes and late embryos results in a fraction of cDNA enriched for the 5.3% ± 0.7% (n -- 3) of the sequences which complement poly(A) ÷ R N A from the mature o o c y t e [2]. These sequences are referred to as transient maternal sequences becuase they disappear during embryogenesis. Half of them are lost by gastrulation. We show here that a fraction of cDNA enriched 7--10 fold for these transient sequences can be prepared for further analysis. Kinetic analysis of this preparation shows two components. The data in Table IB is consistent with the interpretation that the main fraction cDNA is complementary to transient maternal sequences. We cannot rule out, however, the possibility that a few percent of the transient sequences are in the second class. The results presented here demonstrates that these sequences correspond to an even smaller fraction of the estimated number of different poly(A) ÷ R N A sequences. Since these 44 sequences comprise 5.3% of the o o c y t e sequences, they must be derived from a prevalent class. Thus, an overwhelming number o f different sequences are retained throughout development. The only other organism for which there is comparable data is in the sea urchin [11,12]. The sequence complexity of total cytoplasmic R N A is 37 • 106 nucleotides or approx. 19 000 different sequences. Development is accompanied by a severe drop in sequence complexity, both for total cytoplasmic and polysomal sequences. By the pluteus stage, the sequence complexity of both total cytoplasmic and polysomai RNA is a b o u t 15 • 106 nucleotides or a b o u t 7500 sequences. Extensive changes also take place in the prevalent class of poly(A) ÷ RNA between the blastula and pluteus [13]. From the available data, it appears that Drosophila uses a different design for building an o o c y t e than do sea urchins. The total cytoplasmic poly(A) ÷ RNA sequence complexity is approx. 14 500 different sequences [2]. No difference is observed between polysomai and nonpolysomal sequences throughout early development [4]. We show here that during the entire period of early embryogenesis only 44 maternal sequences are lost. Development is apparently accompanied by an increase in the number of zygotic sequences. We do n o t y e t know the sequence complexity involved in the increase, b u t 7.7% of the gastrular sequences and 11.0% of the late embryonic sequences are n o t present in the o o c y t e [2].

Acknowledgements We would like to thank Winifred Doane, Christine M. Beiswanger, Clarice Weide, and Waiter Vincent, III, for their advice and criticism. This research was supported in part by N.I.H. Grant No. HD 09157.

274

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

King, R.C. (1970) Ovarian Development in Drosophila melanogaster, Academic Press, N e w York Arthur, C.G., Weide0 C.M., Vincent, III,W.S. and Goldstein, E.S. (1979) Exp. Cell Res. 121, 87--94 Lovett, J.A. and Goldstein, E.S. (1977) Dev. Biol. 61, 70--78 Goldstein, E.S. (1978) Dev. Biol. 63, 59--66 Goldstein, E.S. and Snyder, L.A. (1973) Exp. Cell. Res. 81, 47--56 Goldstein, E.S. and Snyder, L.A. (1972) Biochim. Biophys. Acta 281, 130--139 Kaufman, Y., Goldstein, E.S. and Penman, S. (1976) Proc. Natl. Acad. Sci. U.S. 73, 1834--1838 Milcsxek, C., Price, R. and Penman, S. (1974) Cell 3, 1--10 Williams, J.G. and Penman, S. (1975) Cell 6,197--206 McKnight, S.L. and Miller, O.L. (1976) Cell 8, 305--319 Hough-Evans, B.R., Wold, B.J., Ernst, S.G., Britten, R.J. and Davidson, E.H. (1977) Dev. Biol. 60, 258--277 Davidson, E.H. (1977) Gene Activity in Early Development, 2nd edn., Academic Press, N e w York McColl, R.S. and Aronson, A.I. (1978) Dev. Biol. 65, 120--188 Bishop, J.O., Morton, J.G., Rosbach, M. and Richardson, M. (1974) Nature 250, 199--204 Hereford, L.M. and Rosbach, M. (1977) Cell 10, 453--462