transcription of endogenous and injected cytoskeletal actin genes during early embryonic development...

Differentiation (1990) 44: 11 1-121 Differentiat ion Ontogeny and Neoplasia 0 Springer-Verlag 1990

Transcription of endogenous and injected cytoskeletal actin genes during early embryonic development in Xenopus laevis Sean M. Brennan Department of Anatomy, University of Connecticut, School of Medicine, Farmington, CT 06032 USA

Accepted in revised form April 2, 1990

Abstract. The transcriptional regulation of a cytoskeletal actin gene during Xenopus laevis embryonic development has been investigated. New transcripts of this gene begin to accumulate at approximately the mid-gastrula stage, between 12 and 16 hours after fertilization, replenishing maternal supplies of this transcript. To study the molecular processes which act to determine the timing of transcriptional activation of this gene, a gene-injection assay was devised, utilizing a cloned copy of the gene which has been marked by a small DNA insertion. Accurately- initiated transcripts of the injected gene accumulate in concert with those of the endogenous gene, showing that injected genes can undergo developmental regulation. As little as 485 nucleotides of upstream sequence is sufficient for proper temporal control of activation of an injected gene. The results presented here demonstrate the feasibility of a microinjection assay for the identification of regulatory gene sequences and transacting regulatory factors in amphibian embryos. Such an assay will be useful in achieving an understanding of general transcriptional control mechanisms acting in early development, and should also provide a means to study certain aspects of long-standing developmental problems, such as cytoplasmic localization and embryonic induction.

Introduction

Following fertilization of the Xenopus laevis egg, there is very little transcription of the embryonic genome during the cleavage period of rapid DNA replication and cell division. After the 12th division, at the mid-blastula transition (MBT), the rate of transcription of the embryonic genome is greatly accelerated [ I , 8, 31, 321. None- theless, evidence is accumulating to show that there are low levels of transcription prior to the MBT, particularly by RNA polymerase I1 [19,40]. Genes encoding transfer RNA [38, 421, snRNA [lo, 23, 311, and 5s RNA [43, 461 are among the first to become actively transcribed

at MBT, followed shortly by ribosomal RNA genes [3, 391. Activation of a group of protein-coding genes transcribed by RNA Polymerase I1 also occurs at or shortly after MBT [2, 20, 35, 401. Transcription of mRNAs, whose genes are activated at MBT, is restricted to early stages of development and does not persist beyond meta- morphosis [ 5 ] , suggesting that a t least some of these transcripts may encode regulatory molecules that act during gastrulation and organogenesis.

Subsequent to the MBT, a new group of mRNAs appears at the gastrula stage; it has been suggested that their synthesis may be induced by regulatory factors which are the products of MBT-activated messages [6]. Among this collection of gastrula-activated genes are those for cytoskeletal proteins such as /?- and y-actin [27], vimentin [16, 371 and some of the cytokeratins [ l l , 261; as well as muscle proteins, such as a-actin [27], myosin heavy chain [34], and desmin [17]. Studies on the regulation of actin gene transcription in early embryogenesis have provided evidence that both cytoskeletal and muscle-specific actin genes are coordinately activated during gastrulation, to provide new muscle actin message and replenish the maternal store of cytoskeletal actin mRNA [4, 271. Upon activation, transcription of muscle actin genes is restricted to the somites [27], while the cytoskeletal actin genes are believed to be expressed in all cells of the embryo.

The mechanisms controlling sequential transcriptional activation of discrete batteries of genes during embryonic development are not well understood, nor is the process by which most transcription is repressed prior to MBT. As a first step towards understanding how these types of temporal regulation are exerted, I have studied the transcriptional regulation of the cytoskeletal actin gene. Here, I describe in detail the time- course of activation of a Xenopus /?-type cytoskeletal actin gene, comparing its activation to that of other genes whose embryonic transcription profiles have been well-characterized. In addition, I show that cloned cop- ies of this gene are correctly transcribed after injection into fertilized eggs. Transcription of injected genes is

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correct by two criteria: they remain transcriptionally quiescent until the time at which their endogenous coun- terparts are activated and, upon activation, accurate initiation occurs at the normal transcriptional startsite. Fi- nally, I show that no more than 485 nucleotides of 5'- flanking sequence are required for both of these manifes- tations of correct transcription.

Methods

Frogs and embryos. Adult Xenopus laeuis were purchased either from Scientific Animal Import (Glen Ridge, USA) or Xenopus I (Ann Arbor, USA), housed in stainless steel tanks and fed thrice weekly with Purina Trout Chow. Females were primed by injection, into the dorsal lymph sac, of 50 Units of Pregnant Mare Serum Gonadotropin (Calbiochem, La Jolla. USA) between 3 days and one week prior to use, and ovulation was induced by injection of 500 Units of Human Chorionic Gonadotropin (Sigma, St. Louis, USA) on the evening before eggs were required.

Eggs were fertilized by exposure to homogenized testis and cultured in 0.1X MBS [13] for approximately 30 min after contact with sperm. Fertilized eggs were dejellied with 2% (w/v) cysteine, pH 8 and placed into 1X MBS/5% (w/v) Ficoll 400 (Sigma, St. Louis, USA) in preparation for injection.

Microinjection and culture of embryos. Embryos were injected with DNA between the first and second cleavage divisions, using a gas pressure injection device (" Picospritzer", General Valve Corp., Farfield, USA). For all constructs, 250 pg of supercoiled DNA (in a volume of 15 nl) was deposited per blastomere. Injected embryos were cultured in 1X MBS/s0h (w/v) Ficoll400 until approximately the blastula stage, when they were transferred to 0.1X MBS for further incubation. At the appropriate developmental stages (see figure legends), individual embryos or groups of embryos were placed into plastic microcentrifuge tubes, frozen on dry ice and stored at -75" C. Embryonic stages were determined according to the Normal Table of Nieuwkoop & Faber [33].

Nucleic acid extraction from embryos. One hundred microliters of homogenization buffer [50 mM Tris-CI, pH 7.5, 1 mM EDTA, 0.5% (v/v) j-mercaptoethanol, 0.3 M NaCI, 1 YO (w/v) sodium dodecyl sulfate, 1 mg/ml Proteinase K] was added to a single frozen embryo (or 250 pl per group of 5 embryos) and the embryo was homogenized as it began to thaw by drawing into and out of a plastic micropipette tip ten times. The homogenate was incubated at 37" C for 30 min, extracted first with phenol and then with chloroform: isoamyl alcohol (24: 1 v/v). Nucleic acids were precipi- tated from the aqueous phase with ethanol and resuspended in 10 mM Tris-CI, pH 7.5,O.l mM EDTA.

Analyyis of DNA and RNA from injected embryos

DNA Analysis. One-fifth embryo equivalent of nucleic acid was subjected to electrophoresis on a 0.8% agarose gel and transferred to nylon (Nytran, Schleicher and Schuell, Keene, USA), using a vacuum transfer device (Vacugene, Pharmacia-LKB, Piscataway, USA). The blot was hybridized to an RNA transcript of pSP65 at 65" C in 5X SSPE, 5X Denhardt's solution, 100 lg/ml yeast tRNA, 0.1 YO (w/v) sodium dodecyl sulfate. After overnight hybridization, blots were washed twice with 0.1X SSPE/O.l% (w/v) sodium dodecyl sulfate at 55" C (30 min per wash) and subjected to autoradiography.

RNase protection. The synthesis of RNA probes and the conditions for annealing and RNase digestion followed those originally described [25, 471, with slight modifications. Probe synthesis was at 30" C for 30 min [21] in a volume of 10 pl. using 0.5 pg of

linearized template, 15 pM Z - ~ ~ P - U T P (specific activity 800 Ci/ mmol), and 7.5 units of SP6 RNA polymerase. One microgram of total RNA from an injected or uninjected embryo (one-fifth embryo equivalent) was incubated with 2 x lo5 cpm of probe in 50% (v/v) recrystallized formamide, 50 mM PIPES-CI, pH 7.0, 5 mM EDTA, 0.4 M NaCl in a volume of 20 pl. The mixture was heated to 85" C for 5 min, and annealing were conducted overnight at 45" C. The annealing mixture was then treated with 40 pg/ ml RNase A, 2 pg/ml RNase T1 and 10 Units/ml RNase T2 in 200 p1 of 10 mM Tris-Cl, pH 7.5, 5 mM EDTA, 0.3 M NaCl for 30 min at 37" C. Following ribonuclease treatment, the mixture was adjusted to 1 % (w/v) sodium dodecyl sulfate, 250 pg/ml Pro- teinase K and incubated a further 15 min at 37" C. Nuclease-resistant RNA was purified by phenol extraction and collected by ethanol precipitation after addition of 1 pg of glycogen as carrier. Pre- cipitated material was resuspended in 2 pl of 94% (v/v) recrystallized formamide, 1 mM EDTA, 0.05% (w/v) xylene cyanol, 0.05% (w/v) bromphenol blue, boiled for 3 min, fast-cooled, and subjected to electrophoresis on 0.5 mm gels containing 10% acrylamide (crosslinking ratio 1/20), 8.3 M urea, 50 mM Tris-borate, pH 8.3, 2.5 mM EDTA. Wet gels were subjected to autoradiography at -70" C using pre-exposed X-ray film and intensifying screens [22]. Densitometry of autoradiograms was performed on a Bio-Rad (Richmond, USA) Model 620 video densitometer.

DNA clones. A i genomic clone of a Xenopus laeuis 8-type cytoskel- eta1 actin gene was isolated, in collaboration with T. Mohun, from a genomic library provided by M. Bienz. A 5.5 kb Eco RI/Bam HI fragment of this clone, containing the entire j-actin gene plus 485 nucleotides of 5'-flanking- and 300 nucleotides of 3'-flanking sequences, was subcloned into pBR 322 to generate pCY-2.

A marked gene for use in microinjection experiments was constructed in the following fashion. An Eco RI/Hind 111 fragment of pCY-2 containing 485 nucleotides of upstream sequence, the entire 85-nucleotide first exon, and 780 nucleotides of the first intron was inserted into pUC 19. The resulting construct contains four Ava 11 sites: two in the vector, one in the first intron of the actin insert, and one in the first exon, 25 nucleotides downstream of the transcriptional startsite. DNA from this construct was subjected to partial digestion with Ava 11, the 5'-protruding ends were rendered blunt-ended with T4 DNA polymerase, and full-length linear molecules were purified from an agarose gel. These molecules were ligated at low concentration to favor recircularization and the ligation mixture was used to transform Escherichia coli. (Lin- earization and subsequent fill-in of the Ava 11 site in the first exon, followed by recircularization, will result in a 3-nucleotide insertion at this site, destroying the Ava I1 recognition sequence). DNA from individual colonies of transformed cells was analyzed by Ava 11 digestion for the loss of the Ava I1 site in the first exon. A clone with the expected properties was obtained and named pUC-p' . This clone was used in the construction of several further actin gene derivatives, as described below.

To complete the synthesis of a complete, marked actin gene, the Eco RI/Hind 111 insert-containing fragment from pUC-j', har- boring a marked first exon, was used to replace the corresponding region of pCY-2, generating pCY-10. This plasmid contains a complete fl-actin gene with 485 nucleotides of 5'-flanking sequence, a marked first exon. and 300 nucleotides of 3'-flanking DNA. To generate a construct with 3.5 kb of 5'-flanking sequence, pCY-10 was linearized at the unique Eco RI site defining the 5' border of the inserted Xenopus sequences, and a 3 kb Eco RI fragment containing the adjacent Xenopus genomic sequences was inserted. Clones with the correct orientation were identified by restriction enzyme digestion; one was selected and named pCY-11.

Hybridization probes. For analysis of persistence and replication of injected plasmid, RNA probes were synthesized from pSP65 [25] digested with Bgl I.

A template for the synthesis of RNase protection probes was constructed as follows. pUC-p+ (see above) was subjected to dele-

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Fig. 1 A-C. Developmental activation of cytoskeletal actin gene transcription in the Xenopus embryo. Embryos were collected at various times after fertilization and stored in groups of five embryos. Nucleic acids were extracted from pooled embryos and analyzed for the presence of cytoskeletal actin mRNA, GS 17 mRNA, ribosomal RNA and DNA. A One-half embryo equivalent of nucleic acid (approximately 2.5 pg RNA equivalent) from each time point was annealed to a probe complementary to part of the first exon of the Xenopus cytoskeletal actin gene, and RNase protection analysis was performed as described in Methods. An autoradiogram of a 10% denaturing acrylamide gel is shown. Developmental stages [33] are indicated below the gel. Also shown are lanes indicating background signal (annealing of probe with Drosophila RNA, Lane B) and input sample (probe plus Drosophila RNA, no RNase, Lane I). The position of the band representing undigested probe is shown at the right and the band representative of cytoskeletal actin mRNA is indicated at the left. B The same amount of nucleic acid as in A was incubated under annealing conditions with a probe homologous to the 5’-terminal 115 nucleotides of the first exon of the Xenopus GS 17 gene [20] and subjected to RNase protection analysis. The position of the protected fragment corresponding to GS 17 message is indicated at left. Developmental stages are shown above the gel. C One-fifth embryo equivalent (approximately 1 pg RNA equivalent) of nucleic acid, from the same samples that were analyzed in A and B above, was subjected to electrophoresis on a nondenaturing 1 % agarose gel, which was stained with ethidium bromide. Developmental stages are indicated at the top of the gel. The positions of embryonic DNA and ribosomal RNA are indicated to the right. Note the steadily increasing DNA levels and the relatively constant levels of rRNA throughout the period of analysis

A <-- ACTIN

A PROBE

2 6 9 9.5 10 10.5 11 12 12.5 13 14 15 17 18 20 22 24 26 C

tions at both ends of the insert, to generate a fragment of Xenopus DNA containing 340 nucleotides of 5’-flanking sequence, an 88- nucleotide marked first exon, and 19 nucleotides of the first intron. This fragment was inserted in the polylinker of pSP64 [25] such that the intron sequences were adjacent to the SP6 promoter. Di- gestion of this plasmid with Sau 3A1, followed by transcription with SP6 RNA polymerase, yields a 194-nucleotide runoff RNA consisting of 67 nucleotides of upstream sequence, the 88-nucleotide marked first exon, 19 nucleotides of first intron and 20 nucleotides of the pSP64 polylinker, with a polarity opposite to that of the actin niRNA.

Analysis of RNA from uninjected embryos (Fig. 1) utilized the same actin probe that was used for analysis of injected embryos. generating a 60-nucleotide signal homologous to part of the first exon of the gene (see Fig. 2). Identical results were obtained with a probe covering the entire first exon. A probe for the GS 17 gene [20] was generously provided by P. Krieg (Univ. of Texas, Austin) and contained sequences corresponding to the first 115 nucleotides of the transcript.

Enzymes and buffers. T4 DNA ligase and most restriction enzymes were from New England Biolabs (Beverley, USA). T4 DNA Poly- merase and Proteinase K were obtained from U.S. Biochemical (Cleveland, USA). Ribonucleases were obtained from Sigma (St.

Louis. USA) or Boehringer-Mannheim (Indianapolis, USA); formamide and glycogen were from Boehringer-Mannheim (Indianap- olis, USA). Radioisotopes were obtained from Amersham (Ar- lington Heights, USA).

5X SSPE contains 0.9 M NaCI, 50 mM sodium phosphate, 5 mM EDTA, pH 7.7. Denhardt’s solution (5 x ) consists of Ficoll 400, polyvinylpyrrolidone, and bovine serum albumin, each at a concentration of 0.1 % (w/v).

Results

Timing of actin gene activation in normal development

Previous studies have indicated that cytoskeletal actin genes are transcribed at least as early as the Stage 13 late gastrula [27], and that the transcription of cardiac muscle actin can first be detected in the Stage 11 mid- gastrula embryo [4]. A time-course analysis of the activation of the Xenopus P-cytoskeletal actin gene, using a sensitive RNase protection assay, was performed to

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confirm these results and to delineate more precisely the time of activation of endogenous cytoskeletal actin genes, for eventual comparison with injected genes. Transcripts were detected using a uniformly-labeled probe for the first exon of the chromosomal /?-actin gene. This analysis indicated that increased levels of cytoskeletal actin mRNA are first detectable between Stages 14 and 15 (Fig. 1 A). The level of maternal transcripts of the cytoskeletal actin gene remained constant or was sometimes observed to decline slightly until this time (Fig. 1 A, compare Stages 2-10 with Stages 10.5- 14). Following activation, transcripts continued to accumulate until at least Stage 26 (Fig. 1 A). From the analysis of 321 individual embryos from 8 batches of eggs, it appeared that there was a slight, batch-to-batch variation in the time of activation of the gene. The maximum variation observed was about two stages, representing a time of approximately two hours. In most experiments, activation occurred between Stages 12 and 14; in all cases, the gene was clearly activated by Stage 15. Cascio and Gurdon [4] were able to detect muscle actin gene transcription as early as Stage 11, in the absence of a background level of maternal transcript. It is perhaps likely that the cytoskeletal actin gene is activated in concert with its muscle-specific counterpart, but that its activation is more difficult to detect against the background of maternal cytoskeletal actin mRNA. A resolution to this question awaits methods for distinguishing newly- synthesized messages from maternal transcripts of the cytoskeletal actin gene. Alternatively, it is possible that the actin gene family is activated in a multi-step fashion, with different genes becoming activated at different times in development.

It is assumed that increased levels of transcript represent new message synthesis, although this is not easy to prove, given the difficulty of obtaining pure preparations of nuclei from Xenopus embryos. Nevertheless, data from nuclear transplantation experiments [ 141 have been interpreted to indicate transcriptional control of muscle actin gene activation; in the absence of data to the contrary, it is reasonable to suppose that increased levels of cytoskeletal actin RNA result from developmentally-controlled transcriptional activation of the gene.

I have compared the temporal activation of the cytoskeletal actin gene to that of the GS 17 gene [20], which is first transcribed at or shortly after the mid- blastula transition (MBT). Maximal levels of GS 17 mRNA are present during gastrulation; by the early neurula stage, transcription of this gene has ceased and existing transcripts are degraded, leading to a disappear- ance of GS 17 message from the steady-state RNA pop- ulation [20]. The RNA samples analyzed in Fig. 1 A were also tested for the presence of GS 17 transcripts, using an RNase protection probe which detects the transcriptional startsite and part of the first exon of the GS 17 gene (see Fig. 5 of Ref. 20). Figure 1 B shows that, in accordance with previously-described properties of GS 17 transcriptional activation and repression [20], accumulation of transcripts was first detected at Stage 9, with maximal levels present during gastrulation

(Stages 9.5 through 11) and decreasing through early neurula stages. With respect to actin gene activation, the GS 17 results showed that the low levels of actin message detected during blastula and early gastrula stages are not due to inefficient recovery of RNA; this conclusion was further strengthened by the observation that rRNA levels are roughly equivalent in all staged RNA samples (Fig. 1C). Moreover, the observation of correct developmental activation and repression of GS 17 transcription (Fig. 1 B) suggested that the method of nucleic acid extraction used here generates preparations which accurately reflect RNA populations at different stages of development. The steady increase of DNA level with developmental stage, also evident in Fig. 1 C, further attests to the authenticity of these nucleic acid preparations.

A gene injection assay for developmental regulatory sequences

A major objective of the experiments reported here was to begin to identify sequences which mediate the tempo- rally-controlled transcriptional activation of the cytoskeletal actin gene, which occurs at the early- to mid- gastrula stage in normal development. To facilitate eventual mutational analysis of gene regulatory sequences, a cloned copy of the complete gene, which can be easily mutagenized in vitro, was used for microinjection experiments. Initial experiments were designed to determine whether injected genes were capable of undergoing correct temporal transcriptional regulation ; thus, an assay for developmentally-regulated transcription of injected genes was required. The experimental design involved injection of cloned genes into 2-cell embryos (1.5-2 h post-fertilization), culture of injected embryos, and analysis of the transcription products of the injected gene at different stages of development.

RNA isolated from injected embryos may contain transcripts of both endogenous and injected genes, if the injected gene is transcriptionally active. To distinguish transcripts of injected genes from those of the endogenous, chromosomal gene, the cloned gene was marked by the insertion of three nucleotides within the 85-nucleotide first exon (see Methods) at an Ava I1 site lying 25 nucleotides downstream of the site of transcriptional initiation (Fig. 2). Total RNA from injected embryos was analyzed by RNase protection [25, 471, em- ploying a uniformly-labeled RNA probe complementary to the first exon of the injected, marked gene. Transcripts of the injected gene, which contain the same 3-nucleotide insertion as the probe, are complementary along the entire length of the marked first exon; thus, an 88-nucleotide protected product will remain after digestion of the hybrids with single strand-specific RNases (Fig. 2, left). Annealing of the endogenous transcript with this probe will, by contrast, generate a region of nonhomology at the site of the insertion, where the three extra nucleotides in the probe remain unpaired and susceptible to RNase digestion. Hence, protected probe fragments of 60 and 25 nucleotides will be generated (Fig. 2, right).

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I I 1 lNTRON1 + 3 EXON1

UPS t - 2 5 3 < 60 >

88 ntds A 88 ntds I r i 1 L N 1

1 88 ntds 25 ntds 60 ntds

I I 1 1 -- INJECTED GENE TRANSCRIPT ENDOGENOUS TRANSCRIPT

Fig. 2. An assay for cytoskeletal actin transcripts in injected embryos. The fop line shows the structure of the marked actin first exon present in the injected gene constructs and in the RNase protection probes. The 85-nucleotide wild-type exon (open bar) has been interrupted by a 3-nucleotide insertion (indicated by stippling) an an Ava I1 site located 25 nucleotides downstream from the transcription start (see Methods for details). Adjacent upstream (“UPS”) and first intron sequences present in the probe are repre- sented by thin lines. Numbers represent sizes, in nucleotides, of different regions of the first exon. Transcripts of the injected gene which are correctly initiated and spliced will contain the same 3- nucleotide insertion as the probe. They will thus protect the probe along the entire length of the first exon, generating an 88-nucleotide

Transcription of an injected actin gene

A prerequisite to the mutational analysis of the actin gene regulatory region is the demonstration that injected genes can be correctly regulated in the developing embryo. In an initial attempt to examine this question, I compared the ability of gene constructs containing different amounts of 5’-flanking sequence to undergo correct developmental activation. Two constructs, one containing 3,500, and the other containing 485 nucleotides of upstream sequence, were injected into both blastomeres of 2-cell embryos as supercoiled plasmid. Injected embryos were allowed to develop and individual embryos were sampled at late blastula (Stage 9) and again at late neurula (Stage 20). At the mid-blastula stage, transcription of the embryonic genome increases signifi- cantly [l , 8, 311, but transcription of the actin genes has not yet been activated [27] (see also Fig. 1 of this paper). Late blastula embryos are therefore referred to as “ pre-activation” samples. The cytoskeletal actin gene is activated at gastrulation, hence neurula stage embryos contain large amounts of message (e.g., Fig. 1) and are designated “ post-activation ” samples. The results shown in Fig. 3 represent analysis of the RNA content of individual injected embryos by RNase protection, which tests for correct formation of the first exon of the transcript, as described in the protocol outlined in Fig. 2. Both genes produced very low levels of transcript

protected product after annealing and RNase digestion, as shown in fhe middle and bottom lines on the left. By contrast, the endogenous actin gene produces a transcript which fails to protect the probe at the site of its insertion. This 3-nucleotide unpaired region in the probe is sensitive to RNase digestion. Thus, endogenous transcripts will generate two shorter products of 60 and 25 nucleotides, as shown schematically in the middle and bottom lines on the right. Open bars represent the labeled RNA probe, stippling represents the insertion used to mark the injected gene and the probe, cross-hatched bars represent actin gene transcripts, and the downward arrow signifies RNase digestion of RNA-RNA dup- lexes. Note that only the relevant portions of the probe and transcripts (i.e., the first exon) are diagrammed here

in the pre-activation samples (Figure 3, lanes 1-4 for the - 485 construct, lanes 12-1 5 for the - 3.5 kb construct). A similar low level of transcription of an injected muscle actin gene at mid-blastula was observed previously [45] and may reflect the normal low level of RNA polymerase I1 activity in the embryo prior to MBT [19, 401. By neurula stage, transcripts have accumulated to high levels (Fig. 3, lanes 5-11 and 1619). These results indicate that at least some of the injected genes undergo accurate transcriptional initiation as well as correct splic- ing, and are consistent with the idea that the injected genes are also undergoing correct temporal regulation.

A simple explanation for these results is that the increased transcript levels at neurula stage result from a gene dosage effect, as a consequence of continued replication of the injected DNA. For example, if injected DNA were replicated at a constant rate, a neurula embryo might be expected to contain tenfold more injected genes than a blastula, based on the number of cells per embryo at these two stages [12]. Assuming equivalent transcriptional rates at both developmental stages, a similar excess of injected gene transcripts might be anti- cipated.

To examine this possibility, the persistence of injected DNA in the embryos used for the RNA analyses shown in Fig. 3 was investigated. Figure 4 shows that injected supercoiled DNA became concatenated by neurula stage. In addition, the degree of replication of the in-

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- 485 - 3500

PRE POST PRE POST B I

INJ ->

ENDOG ->

1 2 3 4 5 6 7 8 9 1 0 1 1 > I I (16171819 20 21

12 13 14 15 Fig. 3. Transcription of marked cytoskeletal actin genes in injected embryos. Marked actin genes containing either 485 or 3,500 nucleotides of 5’-flanking sequence were introduced into2-cell embryos by microinjection. Injected embryos were cultured until either Stage 9 (6 h post-fertilization, lanes labeled PRE) or Stage 20) (24 h post-fertilization, lanes labeled POST). RNA was isolated from individual embryos and analyzed by RNase protection using the probe diagrammed in Fig. 2. Each lane shows the analysis of a single embryo, using 0.2 embryo equivalent of nucleic acid (containing approximately 1 kg total RNA). Lanes 1-1 1 contain RNA from embryos injected with a construct containing 485 nucleotides of 5’-flanking sequence (pCY-10); lanes 1-4 show samples from Stage 9 (late-blastula) embryos, lanes 5-11 are from Stage 20 (neurula) embryos. Lanes 12-19 contain RNA from embryos injected with a construct containing 3,500 nucleotides of 5’-flanking DNA

jected DNA varied from embryo-to-embryo. This variability was particularly pronounced for the -485 construct (Fig. 4, lanes 5-11) and may be a consequence of mosaic distribution of injected DNA in the embryo. Both of these features (concatenation of supercoiled molecules and embryo-to-embryo variability of replication) have also been observed by other investigators [7, 241. Despite the variable degree of replication, it is clear from examination of Figs. 3 and 4 (and has been con- firmed by densitometry) that several neurula embryos contained less injected DNA, yet produced more transcript, than blastula embryos (compare, for example, lanes I and 11 with lanes 1 4 and note that the embryo whose RNA is analyzed in a given numbered lane in Fig. 3 is the same embryo whose DNA is analyzed in the correspondingly numbered lane of Fig. 4). This suggests that amount of injected DNA is not the sole deter- minant of transcript levels for injected genes.

Nonetheless, it is reasonable to expect that, providing an injected gene is capable of being transcribed, its message levels may, in some cases, be proportional to the amount of template present. To investigate this question,

<- PROBE

(pCY-11); lanes 12-1 5 show late-blastula samples. lanes 16-19 contain RNA from neurula embryos. Lane 20 shows annealing and digestion of the probe in the presence of Drosophila RNA (Background), while lane 21 shows probe incubated with Drosophi- la RNA, but not subjected to nuclease digestion (Input). The positions of bands representing undigested probe and digestion products corresponding to transcripts of the injected and endogenous genes are indicated. The smaller of the two bands representing endogenous actin mRNA (25 nucleotides) is present on the original autoradiograph, but is not included in this figure. RNA molecular weight markers of 28, 59, 63, 83 and 99 nucleotides, obtained by SP6 RNA polymerase transcription of appropriately linearized templates, were run on adjacent lanes of the gel (not shown in figure)

the data from the post-activation samples in Figs. 3 and 4 were quantitated by densitometry to determine whether, subsequent to the time of activation, a correlation exists between injected DNA levels and amount of injected gene transcript. As shown in Fig. 5 , there was a correlation between amounts of injected DNA and transcript levels. Linear regression analysis of the data yielded a correlation coefficient of 0.62 with p =0.006, which indicated that the dependence of transcript levels on amount of injected DNA was significant. The degree of correlation was similar if one excludes supercoiled forms of injected DNA (the most rapidly-migrating bands in Fig. 4) from the analysis and relates transcript levels to amount of high molecular weight DNA present (correlation coefficient = 0.62, p = 0.006). This may indicate that all forms of injected DNA are equally compe- tent to serve as transcriptional templates. Occasional failure of an injected embryo to exhibit a correlation between DNA and transcript levels (e.g. lane 10 of Figs. 3 and 4) may plausibly be explained by an extreme degree of mosaicism of the injected gene in that particu- lar embryo. That is, if the injected gene is present in

A

117

D 12 14 17 18 20 B I

- 485

-3500

12 13 14 15 16 17 18 19

Fig. 4. Persistence of injected DNA in embryos. The injected embryos examined in the experiment described in Fig. 3 were analyzed for the amount and form of exogenous DNA. Lane M contains 250 pg of pCY-10, a marked actin gene containing 485 nucleotides of upstream sequence, cloned into pBR 322. This amount of DNA represents approximately half of the amount introduced into an injected embryo. The remaining lanes contain one-fifth embryo equivalent of nucleic acid, and each lane represents an individual injected embryo. Lanes 1-11 are from embryos injected with a construct containing 485 nucleotides of 5’-flanking sequence (pCY- 10): lanes 1 4 are from injected blastulae, lanes 5-11 are from injected neurulae. Lanes 12-19 show analysis of DNA persistence from embryos injected with an actin gene containing 3,500 nucleotides of 5’-flanking sequence (pCY-11): lanes 12-15 are from blastula embryos, lanes 16-19 are from neurulae. Lane numbers corre- spond exactly between this figure and Fig. 3, so that relative amounts of injected DNA and actin mRNA can be directly compared between individual injected embryos

301

l- 10

1

D 1 0 2 0 Injected DNA

Fig. 5. Correlation between degree of replication of injected DNA and levels of injected gene transcript. The autoradiograms shown in Figs. 3 and 4, as well as material from additional injected embryos, were analyzed by densitometry. Peak area representing DNA content of an individual injected embryo was plotted against the level of injected gene transcript in that embryo, for 18 post- activation embryos. Each point represents the analysis of an individual embryo

a z E 9 I-

.-

A

0 i v

ul 0 0 c W

8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 Stage

Fig. 6. Time-course of transcription of an injected actin gene. A plasmid containing a marked actin gene with 485 nucleotides of 5‘-flanking sequence (pCY-10) was introduced into two-cell embryos by microinjection as described in Methods. At various developmental stages, groups of 5 injected embryos were pooled and frozen for RNA analysis. The levels of transcript from the injected actin gene, as well as endogenous cytoskeletal actin mRNA, were determined by RNase protection. Panel A shows an autoradiogram of a 10% acrylamide gel. Developmental stages are indicated above the gel lanes; the lanes labelled “B” and “ I ” show background (hybridization and RNase digestion in the presence of Drosophila RNA), and input (a thousand-fold dilution of probe incubated under the same conditions but not subjected to RNase digestion), respectively. Positions of signals corresponding to injected gene transcript and endogenous transcript are shown to the left. Panel B shows quantitation of the results. Peak areas of bands representing injected and endogenous transcripts were obtained by densitometry of the autoradiogram shown in Panel A. DNA levels were estimated by measuring the peak area of the band running just ahead of undigested probe in each lane, which has been shown to be proportional to injected DNA levels in the embryo (unpublished). Levels of endogenous transcript were plotted versus developmental stage (filled diamonds); levels of injected gene transcript were normalized to DNA levels before plotting (open squares)

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only a few cells but has nevertheless undergone many rounds of replication, it may saturate the transcriptional machinery of those few cells in which it resides, leading to the production of fewer transcripts than might be expected based on the amount of DNA present. In sum- mary, these observations argue against a gene dosage effect being responsible for increased levels of injected gene transcript at neurula stage, and strongly suggest that injected genes are undergoing correct developmental regulation.

Timing of activation of injected genes

As a final test for correct developmental regulation of injected genes in the embryo, a time-course analysis was undertaken to determine whether injected actin genes are transcriptionally activated at the same time as the endogenous genes. As before, 500 pg of supercoiled pCY-10 DNA (containing 485 nucleotides of upstream sequence) was injected into 2-cell embryos, injected embryos were cultured, and groups of embryos were sampled for injected DNA and RNA content at several time points surrounding the normal time of activation. The results (Fig. 6A) show very low levels of transcript from the injected gene at Stage 9 (late blastula) and steadily increasing levels of transcript from Stage 12 (mid-gastrula) onward. When normalized to account for injected DNA levels, the accumulation profile of the injected transcript parallels that of the endogenous gene (Fig. 6B) which, in this experiment, began to undergo activation at approximately Stage 12 (note that the time of activation varies somewhat between different batches of embryos, see above). Accumulation of injected gene transcripts occurred at a constant rate until Stage 17 (mid-neurula), then began to plateau. Thus, injected actin genes are subjected to both negative and positive regulation, in that they remain transcriptionally inactive or only weakly active prior to gastrulation, and they are transcriptionally activated at approximately Stage 12. Thereafter, injected genes continue to be transcribed until at least Stage 20.

Discussion

A gene-injection assay has been developed for use in the eventual definition of sequences responsible for embryonic transcriptional regulation in Xenopus. A cloned cytoskeletal actin gene has been used as a model in these studies. The cloned gene differs from its endogenous counterpart only in the possession of three additional nucleotides in the first exon. This small amount of additional sequence serves to mark the injected gene, so that its transcripts can be distinguished from those of the endogenous gene, yet is unlikely to disrupt important regulatory elements, since it constitutes such a minor change in the structure of the gene. A marked gene of this sort, although somewhat more difficult to construct, has the advantage that it allows the detection of regulatory regions lying within the transcribed region of the gene. By contrast, fusion of actin upstream sequences

to a reporter gene would preclude detection of such downstream regulatory sequences, if they exist.

The marked gene was introduced into embryos and its transcriptional regulation assessed by RNase protection. The choice of this strategy, in contrast to a simpler fusion gene assay, reflects earlier observations on gene injections into Xenopus oocytes. In these earlier experiments, injection of ovalbumin gene plasmids into oocytes led to the production of immunoprecipitable ovalbumin protein of the correct molecular weight and iso- electric point, in the absence of any detectable correctly- initiated transcripts [44]. More recent studies have con- firmed this result by showing that, when vectors containing a chloramphenicol acetyltransferase (CAT) gene are injected into oocytes, production of CAT activity does not require the presence of a eukaryotic promoter [36]. These results strongly suggests that an incorrectly-initiated transcript can be correctly translated in the oocyte. This, in turn, indicates that experiments relying upon measurement of enzymatic activity of a protein reporter molecule (whose coding region has been fused to presumptive embryo-specific regulatory elements) in Xenopus embryos, may not be informative with respect to transcriptional regulatory mechanisms. Furthermore, such assays are unable to distinguish transcriptional from translational effects on steady-state levels of reporter molecule.

With these considerations in mind, the marked gene approach was developed. Correct transcriptional inita- tion on the injected gene, coupled with correct cleavage at the exon l/intron 1 border, generates a uniquely-sized fragment in an RNase protection assay (Fig. 2). The assay additionally detects endogenous cytoskeletal actin transcripts, which are clearly distinguished from the transcripts of the injected gene, and which provide an internal control for actin gene activation and RNA recovery (see Figs. 2, 3, and 5).

Using this assay, I demonstrate here that cloned cytoskeletal actin genes introduced into embryos by microinjection are capable of undergoing correct developmental regulation. This includes accurate transcription initiation, correct cleavage at the donor splice site of the first intron and proper timing of activation. Furthermore, the comparison of two constructs containing different lengths of 5’-flanking sequence, indicate that less than 500 nucleotides of upstream sequence suffice to direct both accurate initiation and correct temporal regulation. A low level of injected gene transcription is observed prior to the normal time of activation. This could be due to a small fraction of injected genes escaping the normal controls which maintain the inactive state of this gene, or to low-level, unregulated transcription of all injected genes. However, recent observations of low levels of RNA Polymerase I1 transcription prior to the Midblastula Transition [ 19, 401, suggest the additional possibility that the low-level transcription of injected genes observed prior to activation is a normal consequence of RNA Polymerase I1 activity during this period of development.

A potential source of artifact arises because individual injected embryos replicate their injected DNA to

119

different extents (Fig. 4), and the amount of injected DNA present at gastrulation can influence the amount of injected gene transcript that is produced by an active gene (Fig. 5) . It is important to stress that degree of replication can influence transcript levels only from active genes; comparison of pre-activation and post-activation embryos showed that extremely low levels of injected gene transcripts are observed in pre-activation embryos containing similar, or even higher amounts of injected DNA, than certain post-activation embryos (compare lanes 14 and 15 with 16-19 in Figs. 3 and 4). Fur- thermore, an extensive 5’-deletion mutant is able to replicate to high levels, but no transcripts from this gene are ever detected in injected embryos (unpublished results). Despite this complication, it is possible to correct for the variability of replication of injected DNA between different embryos, by measuring DNA levels and normalizing transcript levels to amount of injected DNA present (Fig. 6B).

Experiments related to the ones described here have recently been performed to identify sequences involved in controlling the expression of injected muscle actin genes. Regulation of these genes is more complex, as they are subject to both temporal and regional regulation in the developing embryo, being activated around the same time as cytoskeletal actin genes, but transcribed only in the myotome region of the mesoderm [27]. Con- sequently, analyses of mutants by microinjection are more difficult to interpret, since it is often difficult to determine whether a given mutation affects temporal or regional regulation of the gene. Nonetheless, several studies have attempted to address this question. Analysis of transcripts of a fusion gene in injected embryos indicated that a 4.5 kilobase (kb) fragment of the Xenopus borealis cardiac actin gene, containing 2.8 kb of 5’-flanking sequence, the first exon, the first intron, and the second exon was sufficient to direct both temporal and regional regulation [45]. Studies of X. laeuis cardiac actin-CAT fusion genes have shown that injected genes containing 3 kb of upstream sequence plus 24 nucleotides downstream of the transcriptional startsite undergo correct temporal regulation, as assayed by production of CAT activity [28]. In both of these sets of experiments, temporal control was not investigated for constructs containing less extensive amounts of upstream sequence.

In another set of experiments seeking to identify sequences involved in temporal and regional regulation of skeletal muscle actin gene transcription, fragments of the Xenopus laevis a-skeletal actin gene were injected into embryos and the RNA transcripts of injected gene fragments were characterized [41]. Two fragments were compared : one containing 680 nucleotides of upstream sequence, the 41 nucleotide first exon, the 1.5 kb first intron, the 91 nucleotide second exon and approximately the first 250 nucleotides of the second intron, and the other lacking all upstream sequences and the first 27 nucleotides of the first exon, but otherwise identical. RNA was assayed using a probe, homologous to part of the second exon, which detects both specific and non- specifically-initiated transcripts which are correctly

cleaved at the acceptor splice site of the first intron. Using this assay, correctly-cleaved products were not present at gastrula stage, but were detected at neurula from a complete gene (containing approximately 1.9 kb of upstream sequence), as well as from the gene fragment containing only 680 nucleotides of 5’-flanking sequence. In contrast, the gene fragment lacking all upstream sequences plus part of the first exon appeared to be activated prematurely, producing transcripts at the gastrula stage, the majority of which were incorrectly processed [41]. When a fragment containing only sequences between -680 and +27 was injected into embryos, and the resultant RNA was analyzed for correctly-initiated transcripts using a probe for the first exon, no RNA homologous to the injected gene was detected at gastrula stage, while correctly-initiated transcripts were present at neurula stage [41]. From these results it was concluded that temporal regulatory sequences of the skeletal muscle actin gene reside between 680 nucleotides upstream and 27 nucleotides downstream of the transcriptional startsite [41]. Analyses directed toward identification of regional regulatory sequences for cardiac actin [15, 28, 30, 451, skeletal muscle actin [41] and epidermal keratin [18] gene expression in the amphibian embryo have also been described.

The results reported here are in agreement with previous data and refine those earlier results by showing that at least some of the sequences participating in tem- porally-regulated embryonic transcriptional activation of Xenopus actin genes lie within 500 nucleotides upstream of the transcriptional startsite. The fact that temporal regulatory sequences appear to be situated in approximately the same general location in both cytoskeletal and muscle actin genes is consistent with the idea that these genes may be coordinately regulated with respect to their temporal activation.

The observation that injected cytoskeletal actin genes are subjected to correct regulation in vivo suggests that transcription factors are present in excess in the embryo. At Stage 20 (corresponding to the post-activation samples in Fig. 3), there is approximately a 100-fold excess of injected to endogenous genes in the embryo. While it is unlikely that every injected gene is serving as a transcriptional template, clearly there is sufficient excess of factors to support a significant degree of transcription from injected genes. it is interesting, in light of this large excess of exogenous genes, that transcription factors are not competed away from the endogenous genes. The normal activation of endogenous genes (Figs. 3 and 6), in the face of what appears to be a significant excess of competing injected genes, may be explained in one of several ways. It is possible that some injected genes are sequestered in regions of the cell where they do not have access to transcription factors ; only injected genes which gain entry to the nucleus would be able to com- pete, and this may represent a small fraction of the total injected DNA. Alternatively, genes injected into the two- cell embryo may not be equally distributed to all daugh- ter blastomeres. Others have presented evidence both for mosaic distribution of the products of injected genes in the late neurula stage embryo 1181, and for mosaicism

1 20

of exogenous sequences (that had been introduced by microinjection into fertilized eggs) in post-metamorphic frogs [9]. These results make it likely that injected genes are mosaically distributed in the embryo. In this case, it may be that in certain blastomeres, an excess of injected genes is able to block the activation of endogenous genes but that, in the whole embryo, sufficient blastomeres remain free of injected DNA and undergo normal activation of their endogenous genes. A third possibility is that some sort of stable transcriptional complex exists on endogenous genes, rendering them resistant to com- petition from exogenous genes. If such stable complexes exist, they may represent a vestige of the transcription of this gene that occurred during oogenesis. Studies on mosaicism and intracellular distribution of injected genes should shed further light on this question.

The ability to observe correct developmental regulation of an injected gene now permits the analysis of in vitro-constructed deletion and substitution mutants by the same type of microinjection assay. Such experiments are in progress and will lead to the identification of regulatory sequences which control the temporal activation of the cytoskeletal actin gene. One candidate for such a temporal regulatory site is a region, located approximately 75 nucleotides upstream of the transcriptional startsite, known as a Serum Response Element [29]. However, recent experiments in this laboratory have shown that the Serum Response Element is not necessary for correct temporal activation of the cytoskel- eta1 actin gene [Brennan S, Savage R, submitted for pub- lication). The eventual identification of temporal regulatory sequences will, in turn, facilitate a search for regulatory factors which act during the earliest stages of development to specify patterns of transcription in the embryo. Further study of such transcriptional regulatory molecules is likely to aid our understanding of earlier, maternal contributions to transcriptional regulation in the embryo, and may also shed light on the mechanisms of various inductive processes, since these often result in new transcriptional patterns in induced cells.

Acknowledgemenis. Construction of earlier versions of some of the plasmids used in this study, as well as my introduction to Xenopus biology, took place in the laboratory of J.B. Gurdon, whom I warmly thank for guidance and support. I thank J. Gerhart (UC Berkeley) and M. Danilchick (Wesleyan U.) for advice on establish- ing and maintaining an amphibian colony. The GS 17 gene probe was generously provided by P. Krieg (University of Texas, Austin). I thank E. Rocher and M. Davis for technical assistance, R. Kosher for use of the video densitometer, and J. Peluso for advice. Thanks also to B. White and G. Maxwell for discussions and G. Carmichael and B. White for comments on the manuscript. The research was supported by grants from the State of Connecticut and the Ameri- can Cancer Society.

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