In vitro transcription by RNA polymerase II in extracts of Xenopus oocytes, eggs, and somatic cells

In Vitro Transcription of Xenopus Oocytes, Tetsuya

Toyoda

and Alan

by RNA Polymerase II in Extracts Eggs, and Somatic Cells

P. Wolffe’

Laboratory of Molecular Enbrvolu~v. Health, Building 6, Room 13l:B&sda,

National Institute of Child Health and Human Development, Maryland 30892’

We describe procedures for preparing extracts of Xenopus oocytes, eggs, and somatic cells that will aceurately transcribe class II genes. A variety of viral and Xenopus promoters direct the accurate initiation of transcription by RNA polymerase II in these extracts. Optimal ionic conditions (100-200 rn~ KCl, 12 rn~ MgCl,), template concentration (20-40 &ml), incubation time (30-60 min), and temperature (25°C) for class II gene transcription are described. LI 1%~ kedemic mess, Ino.

Xenopus laeuis, the clawed frog, has become an established model system for research concerning the regulation of differential gene expression during development (1). Physical manipulations of Xenopus oocytes, eggs, and embryos are facilitated by their large size and accessibility (2). Xenopus eggs and oocytes contain rich stores of the components required for nuclear assembly, the transcription of genes, and the translation of messenger RNA (3-6). Cell-free preparations of oocytes and eggs that will assemble chromatin (7-91, assemble nuclei (lo), replicate DNA and nuclei (11,121, transcribe class I andclass III genes (13,141, and translate messenger RNA (15) have been established. These systems have proven extremely important for the molecular dissection of the process of interest and for understanding any potential developmental significance. Several genes in Xenopus are active during oogenesis, but are repressed in somatic cells. Among the best studied are the 5S RNA genes, which contain a large multigene family (oocyte-type) that are active in oocytes, but are repressed in somatic cells (16, 17). Utilization of oocyte and egg extracts capable of transcribing class III ’ To whom correspondence should be addressed.

National Institutes

of

genes has led to the establishment of detailed models describing the relative contributions of the association of transcription factors and histones to this regulated gene expression (18). Several related studies have used Xenopus extracts to examine the influence of chromatin assembly on class II gene transcription, but these have generally utilized heterologous transcription extracts derived from mammalian cells (19,201. Like the oocytetype class III genes, many class II genes are only actively transcribed in Xenopus oocytes and are relatively inactive or repressed in somatic cells. Such genes include those encoding heat shock protein (hsp)’ 70, transcription factor IRA, c-myc, and transcription factor FRG Y2 (21-241. The molecular mechanisms responsible for the selective inactivation of these genes in somatic cells remain unknown. Progress toward understanding the regulation of Xenopus class II genes during the developmental transition from oocyte to egg and to a somatic cell has been hindered by the lack of characterized in vitro transcription extracts. Xenopus oocytes, eggs, and embryos were among the first cells and organisms to have the activity of their RNA polymerase II characterized; however, this pioneering work was hindered at the time by the lack of specific cloned templates (56). In this report we describe methods for the preparation of extracts of Xenopcrs oocytes, eggs, and somatic cells that will transcribe class II genes accurately and efficiently. MATERIALS

AND

METHODS

Cell Culture

In order to obtain rapidly proliferating subclones of cells able to grow in suspension culture, A6 cells (ATCC, ‘Abbreviations used: hso, beat shock motein: DTT. &thiothreitol: PMSF, phenylmethylsulfa~yl fluoride; &IV, cytomegalovirus; HSV, herpes snmplex virus: tk, thymldine kinase: AdMLP. adenovims majm late promoter.

CLASS

II

GENE

TRANSCRIPTION

CCL 102) are passed at 1% confluence in 66% Leibovitz’s L-15 media (GIBCO) supplemented with 10% heat-inactivated fetal bovine serum (GIBCO), 100 U/ml penicillin G, and 100 pg/ml streptomycin at 25°C.

Somatic (A61 Cell ExtractsPreparati0n.s

Whole Cell and Nuclear

A6 MR, a rapidly proliferating subclone of A6 cells, were cultured at 5 X lO’/ml in the same media at 25°C until they grew to a density of approximately 107/ml. Whole cell extracts from A6 MR cells were prepared according to the method of Manley et al. (25) with some modifications. All of the extraction procedures were carried out at 4’C. Briefly, cells were washed in ice-cold 66% phosphate-buffered saline and the cell pellet was resuspended in four packed-cell volumes of 10 rn~ Tris-HCl, pH 7.9, 1 rn~ EDTA, 5 mu DTT, 1 rn~ PMSF, and 1 pg/ml leupeptin on ice. After 20 min, the cells were lyzed by homogenization in a Dounce homogenizer with the “B” pestle (6-10 strokes). Four packedcell volumes of 50 rn~ Tris-HCl, pH 7.9,lO rn~ MgCl,, 2 mM DTT, 1 mM PMSF, 1 pg/ml leupeptin, 25% sucrose, and 50% glycerol were then added to the suspension and the mixture was stirred gently. One packed-cell volume of saturated ammonium sulfate was added dropwise to this mixture. The cell lysate was then gently stirred for an additional 20 min. The cell lysate was then centrifuged at 50,000 rpm for 3 h in a Beckman 70 Ti rotor (170,OOOg) to remove insoluble debris and nucleic acid. The supernatant was decanted and the proteins potentially active in transcription were precipitated by

IN

Xempus

EXTRACTS

341

the addition of solid ammonium sulfate (0.33 g/ml of the supernatant). After the ammonium sulfate was dissolved, 0.01 ml of 1 M NaOH per 10 g of ammonium sulfate was added, and the suspension was stirred for an additional 30 min. The precipitate was collected by centrifugation at 15,000g for 20 min and resuspended with l/10 of the original volume of a HEMG(-)K buffer (25 mM Hepes-KOH, pH 7.6,12.5 mM M&l,, 1 mM DTT, 0.1 mM EDTA, 1 mM PMSF, 10 pg/ml leupeptin, 10% glycerol). This suspension was dialyzed for 4-8 h against two changes of 1 liter of HEMGO.lK (HEMG containing 0.1 M KCl). The lysate was centrifuged at 10,OOOg for 10 min and the supernatant was quickly frozen in small aliquots in a dry ice-methanol bath and stored at -80°C. Nuclear extracts from A6 MR cells were prepared by the method of Soeller et al. (26). The washed cells were suspended in 4 ml/g of cells in buffer A (0.35 M sucrose, 15 mM Hepes-KOH, pH 7.6, 10 mM KCl, 5 mM M&l,, 0.1 mM EDTA, 0.5 mM EGTA, 1 mM DTT, 1 mM PMSF, 1 pglml leupeptin, 0.1% Triton X-100) and were homogenized in a Dounce homogenizer with the B pestle (6-10 strokes). The homogenate was passed through three layers of cheese cloth to eliminate intact cells and then centrifuged at 4000 rpm in a Sorvall SS-34 rotor for 10 min (19OOg). Nuclear pellets were resuspended gently in buffer A (2 ml/g cells) and the nuclei were layered in 6-ml aliquots over an equal volume of buffer B (0.8 M sucrose, 15 ~IVI Hepes-KOH, pH 7.6,lO mM KCl, 5 mu M&l,, 0.1 rn~ EDTA, 0.5 mM EGTA, 1 mM PMSF, 10 pg/ml leupeptin) in 15.ml graduated polycarbonate tubes. Nuclei were pelleted by centrifugation in a Sorvail HB-4 rotor at 5000 rpm for 10 min (4000g).

342

TOYODA AND WOLFFE A6 Whole Cdl Eflracl

A6 Nuclear ExlnM

FIG. 2. In vitro transcription of the cytamegalovirus promoter wth Xenopus A6 whale cell and nuclear extracts: 0.5 +g of hCMVCAT was incubated with the indicated amount of each extract under standard conditions for 1 b. (Left) The initiation site of transcription of hCMVCAT: 0.5 pg of hCMVCAT was incubated with 40 cp of A6 and Drosophila nuclear extracts as indicated for 1 h. RNAs synthesized were annealed with 50 fmol of 3*P-labeled pruner ,200 kcpml (see Materials and Methods) The sequence of hCMVCAT mdicates the complimentary sequence of the RNA start site (A/+lGTCTAGl. (Right) Extract dependency of transcription with Xenopus A6 whole cell and nuclear extract: 0.5fig of hCMVCAT was incubated without extract (lane 11and with 10.?0.40.80, and 160pg of whole cell extract (lanes 2,3,4,5. and 6. respectiveIyJ or 10,20,40.80. and 160ergof nuclear extract (lanes 69. IO. 11, and I?, respectwely). n-Amanitin (4 &nll was added in the transcription reactions containing 80 pg of whole cell extract or nuclear extract ilanes 7 and 13, respectivelyl. M, DNA size marker; pBR322 digested with H&I.

The nuclear pellet was resuspended in 4 vol of buffer C (15 rn~ HepeseKOH, pH 7.6, 110 rn~ KCl, 3 mM MgCl,, 0.1 rn~ EDTA, 1 rn~ DTT, 1 rn~ PMSF, 10 fig/ml leupeptin) and transferred to Beckman 70 Ti D&carbonate tubes. Ammonium sulfate. 4.0 M (DH ?.6j, was added stepwise to a final concent;ation of 0.36 M. and lvsis of the nuclei was acconmlished bv eentle mixing using a rotary mixer for 30 min. The extract was clarified by centrifugation at 35,000 rpm for 1 h in a Beckman 70 Ti rotor (84,OOOg). Material floating on the supernatant surface was removed by aspiration. The supernatant was collected by decant&ion and precipitated through the stepwise addition of 0.3 g of solid ammonium sulfate per milliliter of supernatant. After stirring for 15 min, the precipitate was collected by centrifugation at 15,000 rpm for 15 min in an SS-34 rotor (27,OOOg). The pellet was gently resuspended with a glass rod in a equal volume of HEMG(-)K and dialyzed against each of two changes of 1 liter of HEMCO.lK for 4-8 h. Any residual insoluble precipitate was removed by centrifugation at 10,000 rpm for 10 min in an SS-34 rotor (12,OOOg) and the supernatant ”

was aliquoted and quickly frozen in a dry iceemethanol bath before storage at -80°C. Drosophila nuclear extract was purchased from Stratagene or Promega. Whole Cell Preparation

from

Eggs and Oocytes

I

Egg extract from X. laeuis was prepared according to the method of Lohka and Maui (ref. 27). Female X. laeuis were injected with 1000 U of human chorionic gonadotropin (Sigma) and left overnight at room temperature. Eggs were collected and dejellied in 2% cyst&e, 20% MBS (18 rn~ N&l, 0.2 rn~ KCl, 0.5 miv NaHCO,, 2 rn~ HepesmNaOH, pH 7.5, 0.15 mM M&l,, 0.05 mM Ca(NO,),, 0.1 nw CaCl,) and washed three times in 20% MBS, followed by washing twice in egg extract buffer (50 rn~ Hepes-KOH, pH 7.4,50 mM KCl, 2.5 rn~ MgCl,, 2 rn~ 2.mercaptoethanol, 1 rn~ PMSF, 2 rg/ml leupeptin). Eggs were crushed by centrifugation at 9OOOg for 15 min at 4°C. The combined light and heavy ooplasmic fractions above the yolk pellet and lipid pellicle were further fractionated by ammonium sulfate pre-

CLASS II GENE TRANSCRIPTION

IN Xenopus EXTRACTS

343

HEMGO.lK or by the use of a PG-10 column (Bio-Rad). The extract was aliquoted, quickly frozen, and stored at -80°C. Mature ovaries of X. la&s were excised, minced, and incubated in 200 ml of OR2 media (82.5 mu NaCl, 2.5 rn~ KCl, 1 mM C&l,, 1 rn~ MgCl,, 1 rn~ NaHCO,, 5 rn~ Hepes-NaOH, pH 7.8) containing 1 mg/ml collagenase (type II, for adipocyte isolation; Sigma) for 2 h at 25°C. After the removal of small oocytes and follicle cells, the oocytes were washed three times in 20% MBS and twice in egg extract buffer. Oocytes were crushed by

Promoter sensitivity ofXwwpus A6 and Drosophila nuclear extracts: 0.2 pm01 of hCMVCAT (CMV), pCAT control (SV40), pBLCAT2 (HSV tk), pXLlOXP (hap 701, pBl(m596+8)CAT Wit Bl), and pAdML (AdMLP) were incubated with 40 118of each extract under standard conditions for 1 h. RNAs were annealed with 50 fmol of each primer (200 kcpm) and reverse transcribed (see Materials and Methods). The dot indicates the major reverse transcripts. M, DNA size marker; pBR322 digested with HpaD. FIG. 3.

cipitation. Ooplasmic fractions were diluted with 4 vol of buffer C and 4.0 M ammonium sulfate (pH 7.6) was added to a final concentration 0.36 M and gently mixed for 30 min. The extract was clarified by centrifugation at 35,000 rpm in a Beckman 70 Ti rotor (84,OOOg). After the removal of material floating on the supernstant SW‘face, the supernatant was collected by decantstion and was precipitated as described for the cell extract. The crude extract was desalted by dialysis against

FIG. 4. Promoter sensitivity of Xenopus oocyte, egg, and A6 whole cell extracts: 0.2 pmo, of hCMVCAT (CMV), pCAT control (SV40), pBLCAT2 (HSV tk), pBl(-598+8)CAT Wit Bl), pXLlOXP (hsp 70), pKr (Krhppel), and pAdMLP (AdMLP) were incubated with 40 pg of each extract under standard canditions for I h. RNAs were annealed with 50 fmol of each primer (2M) kcpm) and reverse trsnscribed (see Materials and Methods). The dot indicates the major reverse transcripts. M, DNA size markers; pBR322 digested with HPdl

TOYODA

AND WOLFFE

pKr (two initiation sites are used, hence accurate reverse transcript lengths of 72 and 68 nt), pAdMLP (accurate reverse transcript length of 93 nt), and pXLlOXP (accurate reverse transcript length of 118 nt) reverse transcription were 5’.TATTACTCGCGGTTGTGTGTGGCACAAC-3’, !&TGACAATCTTAGCGCAGAAGTCATGCCC-3’, and Y-CTCCTTACAGTTTGCTTTTCGCTAGAATA-3’, respectively.

In Vitro

FIG. 5. Efkct of monovalent and divalent catim concentration on in U&O transcription: 0.5 pg of hCMVCAT was incubated with 40 pg of A6 nuclear extract at different concentrations of KC1 (8. 12.5,SO, 100, 150. and 200 rn~l or 50 rn~ N&l in 12.5 rn~ Hepes-KOH, pH 7.6. 6.25 rn~ M&b. 1 rn~ DTT. 0.05 rn~ EDTA. 10.5 no., ATP. 0.5 rn~ CTP, GTP. and UTP, 1 rn~ spermidine. 10 U RNase Inhibitor, and 5% glycerol. Parallel incubations examine the effect of varying concentrations ofMgC1, (1.5, $6, and 12 rn~) or 5.2 mu MnCI, and 1.1rn~ M&I,. Where indxated ATP was reduced to 0.5 or 5.5 mu. or spermidine was removed from the reaction mixture. RNAs were any nealed with 50 fmol of the primer (150 kcpmJ and reverse transcribed (see Materials and Methods). M. DNA size marker; pBR322 dlgested wth t&II. I

L

centrifugation at 150,OOOg for 1 h at 4’C and processed as described for preparation of the egg extract. The methodologies used to prepare all four transcription extracts are described in Fig. 1. Plasmids

and Synthetic

Oligonucleotides

The following plasmids were used in our experiments: pCMV1, hCMVCAT (cytomegalovirus (CMV) promoter and enhancer), pBLCAT2 (herpes simplex virus (HSV) thymidine kinase (tk) promoter), pBl(-596+8)CAT (Xenopus laeuis vitellogenin (Bl) promoter) and pXLlOXP (Xenopus la&s hsp 70 promoter) (21,28-30). pCMV1, hCMVCAT, pBLCAT2, pBl(-596+8)CAT, and pXLlOXP were the kind gifts of Drs. M. Stinsky, L. Henninghausen, B. Luckou, W. Wahli, and M. Bienz, respectively. pCAT control (SV40 early gene promoter and enhancer) was purchased from Promega. pKr (Drosophila Kriippel gene promoter) and pAdMLP (adenovirus major late promoter) were purchased from Stratagene (31,32). Plasmid DNA was purified by cesium chloride centrifugation or by Quiagen chromatography (Diagen). The primer for reverse transcription analysis of hCMVCAT (which would yield a reverse transcript length of 136 nt if transcription was accurately initiated), pBLCAT2 (accurate reverse transcript length of 128 nt), pCAT control (major reverse transcript length of 186 nt), and pBl(-596+8)CAT was 5’-GGTGGTATATCCAGTGATTTTTTTCTCCAT-3: and those of

Transcription

Reactions

The standard runoff assay was done in 25 pl final volume of a buffer consisting of 12.5 rn~ Hepes-KOH, pH 7.6, 6.25 mM M&l,, 50 rn~ KCl, 1 rn~ DTT, 0.05 m,v EDTA, 0.5 miv ATP, 0.5 rn~ CTP, 0.5 mu GTP, 50 FM UTP, 5 pCi [w3’P]UTP (3000 Ci/mmol; NEN), 1 rn~ spermidine (Sigma), 10 U RNase inhibitor (GIBCOIBRL), 5% glycerol, and the indicated amount of various extracts. The template was the plasmid pCMV1 at the indicated concentration after digestion with BstXI (New England Biolab) to produce 505.nt runoff transcripts. The transcription reaction mixture was incubated at the indicated temperature for various periods. The reaction was stopped through addition of 1 pl of diethyl pyrocarhonate (Sigma) or by phenol/chloroform extraction. Transcripts were precipitated with ethanol and washed with 70% ethanol, dried in 8 Speed Vat, and analyzed in a 4% polyacrylamide gel containing 8 M urea, followed by autoradiography at ~80°C with a DuPont Cronex screen. In vitro transcription assays by primer extension were carried out under conditions identical to those of the runoff assay except that no radiolabeled nucleoside triphosphate was present and supercoiled plasmids were used as templates. In vitro transcription assays using Drosophila extract were done according to the manufacturer’s instructions (Stratagem? or Promega). Transcripts were annealed with 0.05 pm01 of 5’-32Plabeled primer (2 X 10’cpm) in 10 ~1 of 20 mu Tris+HCl, pH 8.0,0.2 mu EDTA, 0.25 M KC1 at 65°C for 5 min and at 55°C for 20 min, before cooling to room temperature. The annealed primer was elongated using 2 U of avian myeloblastosis virus reverse transcriptase (Promegaj in 30 pl of 20 mu Tris-HCl, pH 8.0, 80 mM KCl, 8 IIIM MgCl,. 80 pglml actinomycin D (BMB), 10 mM DTT, 0.4 rn~ each of 4.deoxynucleoside 5’.triphosphates (BMB), and 10 U of human placental RNase inhibitor (GIBCO/BRL) at 37°C for 1 h. The reaction was stopped by the addition of 150 pl ethanol. The nucleic acid was precipitated and washed in 70% ethanol, dried, and dissolved in 10 pl formamide dye buffer. The solution was boiled for 2 min, chilled on ice, and then electrophoresed on a 6% polyacrylamide-urea gel.

CLASS II GENE TRANSCRIPTION

Oooyto

Extract

EQQ Extmct

345

IN Xenopus EXTRACTS

A6 Whole Call Extmci

FIG. 6. In vitro transcription ofXen.opm oocyte, egg, and A6 whole cell extracts. (Al pCMV1 (CMV) (0.5 cgl linearized with BstXI was incubated far 1 h with various amounts of each extract (10,20,40,80, and 160 rg) under standard conditions for runoff assays. (B) The same template was incubated with 20 ~g of each extract at the various concentrations of KC1 (6,12,25,50,100,150, and 200 rn~) or 50 mu NaCI. M, RNA size marker.

RESULTS

AND

DISCUSSION

In Vitro Transcription of Class II Gene Promoters Extracts of Oocytes, Eggs, and Somatic Cells

in

We determined that in vitro transcription ofthe cytomegalovirus promoter was accurately initiated and sensitive to a-am&tin (4 @g/ml). The initiation of transcription of the hCMV CAT construct in the nuclear extract of A6 cells was at the predicted site (33) (Fig. 2A, left) and identical to the site used by the Drosophila extract. Comparison of transcription initiation at various template concentrations in both A6 nuclear extracts and whole cell extracts revealed that the correct site of transcription initiation was used (Fig. 2, right). The appearance of transcript was dependent on the addition of template and was sensitive to the addition of low concentrations of a-amanitin (4 pg/ml). This indicates that a DNA-dependent transcription process was being driven by RNA polymerase II (5,34). Our next experiments compared the initiation of transcription in a wide variety of viral and cellular promoters (Fig. 3) using both Xenopw A6 nuclear extracts and the Drosophila extract. Both transcription extracts give accurate and efficient transcription initiation on the CMV, SV40 early, HSV tk, adenovirus MLP and

Xenopus vitellogenin Bl promoters. The Xenopus hsp 70 promoter, which is not normally active in somatic cells, did not generate any detectable initiation events. Relative transcription efficiencies very different from those observed in the somatic A6 cell nuclear extract were observed in the extracts of Xenopus oocytes and eggs (Fig. 4). Whereas transcription of the HSV tk and Xenopus vitellogenin Bl promoters remained strong, transcription from the SV40 early and CMV promoters was much reduced. Interestingly the Xenopus hsp 70 promoter, which is constitutively active in Xenopus oocytes (21), was accurately transcribed in oocyte and egg extracts. The somatic A6 whole cell extract gave results intermediate between those of the nuclear extract and the oocyte/egg extracts. Transcription from the SV40 early and CMV promoters was reduced relative to that observed in the nuclear extract. We conclude that a variety of promoters will direct the accurate initiation of transcription by RNA polymerase II in the various Xenopus extracts. We next sought to optimize in vitro transcription conditions. Optimization

of in Vitro

Transcription

Conditions

We varied monovalent and divalent cation concentrations to examine transcription of the CMV promoter

346

TOYODA AND WOLFFE

M Kb

FIG. 7, pCMV1 (CMV) (0.5 pgl linearized with BatXI was incubated with 20 ~g of each extract at the various concentration ofMgC1, or 6 nm M&I, in 12.5mu Hepes-KOH, pH 1.6.50 rn~ KCI, 1 rn~ DTT, 0.05 rn~ EDTA, 0.5 rn~ ATP, CTP, and GTP. 50 &M UTP, 50 pCi [w3’P]UTP. 1 rn~ spermidine, 10 U RNase inhibitor, and 5% zlvcerol for 1 h. Where indxated. template was removed or 4 eglml ofn-amanitin was added to the reaction mixture. M, RNA size marker

in the A6 nuclear extract (Fig. 5). Optimal transcription initiation is obtained with 100 rn~ KC1 and 12 rn~ MgCI,. Transcription is dependent on the addition of spermidine (1 mM). Having established these conditions using the primer extension assay and the CMV promoter, we next examined transcription of this promoter in the whole cell extracts from oocytes, eggs, and somatic A6 cells. Due to the low efficiency of CMV transcription in these extracts as assayed by the primer extension assay, we adopted a “runoff’ assay. Here accurately initiated RNA polymerase II transcribes a linearized template until it reaches the end of the DNA molecule. Thus in the presence of radiolabeled RNA precursors a defined length transcript is generated. In all of the whole cell extracts, transcription from the CMV promoter yields a major transcript of 505 nt (Fig. 6), which corresponds to accurately initiated RNA polymerase transcribing until it reaches the restriction endonuclease (BstXI) site at which the template is linearized. A minor transcript of 375 nt also accumulates. This corresponds to transcripts initiated at a secondary site 3’ to the correct promoter. Transcription of the CMV template in all three whole cell extracts is optimal at 20-40 @g/ml of extract. Once again optimal cation concentrations for specific transcription are 100-200 mM

KC1 and 12 mM MgCl, (Figs. 6 and 7). Transcription is dependent on the addition of DNA and is sensitive to low concentrations of a-am&tin (4 rg/ml) (Fig. 7). The addition of supplemental ATP and GTP (5 mu) reduced the specificity of transcription, although overall initiation events increased (not shown). The optimal temperature for both primer extension and runoff assays was 25°C. Our final general assay was to optimize the time period for transcription (Fig. 8). In all of the extracts. of which results for the oocyte, egg, and whole cell extracts are shown, detection of specific transcripts is easiest after 30-60 min. After this time, degradation products begin to accumulate. The transcription timecourse shown in Fig. 8 also reveals that a major degradation product of -300 nt accumulates at late times using the CMV template. In addition, large transcripts at the limit of mohility also accumulate as time progresses due to RNA polymerase II initiating transcription at the ends of the DNA fragments (35). CONCLUSIONS

We conclude that a broad spectrum of viral and&nopus promoters direct the accurate initiation of transcription by RNA polymerase II in extracts of Xenopw

CLASS II GENE TRANSCRIPTION

Oocyte

Extract

IN Xenopu EXTRACTS

347

Egg Extract

FIG. 8. Time-course of in vitro transcription: 0.5 pg of pCMV1 (CMV) linearized with BstXI was incubated with 40 pg of Xenopus oocyte or egg extract under standard conditions for the runoff assay for the indicated times at 25°C. M, RNA size marker.

oocytes, eggs, and somatic cells. These extracts should prove useful in the investigation of the molecular basis of differential class II gene expression through the transition from a germ cell (oocyte) to a somatic cell (A6). Use of homologous transcription and chromatin assembly systems should allow the influence of chromosomal architecture on gene expression to be more completely explored. ACKNOWLEDGMENTS We thank Drs. Caroline Schild and Genevike Almouzni for advice and Ms. Thuy Vo for preparing the manuscript. T.T. is a recipient of a scholarship from Uehars Science Foundation.

257.

18. 19. 20. 21. 22. 23. 24. 25.

REFERENCES 1. Wolffe, A. P. (1991) Bioehem. J, 278,313-324. 2. Gurdon, J. B., and Melton, D. A. (1981, Ann.

15. Patrick, T. D., Lewer, C. E., and Pain, V. M. (1969) Deoelopment 109, l-9. 16. Wakefield, L., and Gurdon, J. B. (1983) EMBOJ. 2, 1613-1621. 17. Wormington, W. M., and Brown, D. D. (1983) Dee. Biol. 99,248-

26. Reu.

Genet.

15,

189-218.

3. Davidson, E. H. (1986) Gene Activity in Early Development, 3rd ed., Academic Press, New York. 4. Forbes, D. J., Kirschner, M. W., and Newport, J. W. (1983) Cell 34.13-23. 5. Roeder, R. G. (1974) J. BioL Chem. 249, 241-248. 6. Roeder, R. G. (1974) J. BioL Chmn. 249, 249-256. 7. Laskey, R. A., andEamshaw, W. C. (1980) Nature 286,763-767. 8. Glikin, G. C., Rub&i, I., and Worcel, A. (1984) Cell 37, 33-41. 9. Almouzni, G., and MCchali, M. (1988) EMBO J 7,664-672. 10. Newport, J. (1987) Cell 48, 205-217. 11. Lohka, M. J., and Masui, Y. (1983) Science 220, 719-721. 12. Blow, J. J., and Leskey, R. A. (1986) Cell 47, 577-587. 13. Birkenmeier, E. H., Brown, D. D., and Jordan, E. (1978) CeU15, 1077-1086. 14. PUP% L. K., WindIe, J. J., Mougey, E. B., and Sollner-Webb, B. (1989) Mol. Cell. BioL 9,5093-5104.

21. 28. 29.

Wolffe, A. P., and Brown, D. D. (1988) Seienee241.1626-1632. Knezetic, J. A., and Luse, D. S. (1986) CeN45,95-104. Workman, J. L., and Roeder. R. G. (1987) Cell 51,613-622. Bienz. M. (1984) RMBOJ. 3,2477-2483. Hall, R. K., andTaylor, W. L. (1989) Mol. CeN.Biol. 9,5003-5011. Vriz, S., Taylor, M., and M&h&, M. (1989) EMBO J. 8, 40914097. Tafuri, S. R., and Wolffe, A. P. (1990) Pmt. NaK Acad. Sci. USA 87, 9028-9032. Manley, J. L., Fire, A.. Cane, A., Sharp, P. A., and Geftev, M. L. (1980) Proc. N&l. Acad. Sei. USA 77, 5706-5710. Soeller, W. C., Poole, S. J.. and Kornberg, T. (1988) GenesDe”. 2, 68-81. Lohka, M. J., and Masui, Y. (1984) J. Cell Btol 98, 1222-1230. Anderson, S., Davis, D. L., Dahlbsck, H., Jawall, H., and Russell, D. W. (1989) J. Biof. Chem. 264.8222-8229. Niller, H. H., and Hennighauaen, L. (1990) J. Viml. 64, 238% 2391.

30. Seiler-Tuyns, A., Walker, P., Martinez, E., Merillat, A. M., Givel, F., and Wahli, W. (1986) Nucleic Acids Rex. 14,8755-8770. 31. Matsui, T., &gall, J., Weil, P. A., and Roeder, R. G. (1980) J. Biol. Chem.

255,

11,892-11,996.

32. Rosenberg, U. B.. Schrbder, C., Preiss, A., Kienlin, A., C&b, S., Riede, I., and Jgckle, H. (1986) Nature 318, 336-339. 33. Boshart, M., Weber, F., Jahn, G., Dorsch-HP&r, K., Fleckenstein, B., and Schaffner, W. (1985) Cell 41,521-530. 34. Kamakaka, R. T., Tyree, C. M., and Kadonaga, J. T. (1991) Proc. Natl. Acad Sci. USA 88, 1024-1028. 35. Berg, P., Kornberg, R. D., Fan&w, H., and Dieekmann, M. (1965) Biochem. Biophys Res Commun. l&935-942.