Large scale isolation of nuclei from oocytes of Xenopus laevis

ANALYTICAL

BIOCHEMISTRY

(1989)

180,177-180

Large Scale Isolation of Nuclei from Oocytes of Xenopus laevis’ Ida Ruberti,

Elena

Beccari,

Elisabetta

Bianchi,

and Francesca

Centro di studio per& Acidi Nucleici de1 C.N.R. c/o Dipartimento Uniuersitci di Roma La Sapienza, Rome, Italy

Received

December

di Genetica

Carnevali e Biologia

Molecolare,

28,1988

We describe an improvement on the procedure of Scalenghe et al. (F. Scalenghe, M. Buscaglia, C. Steinheil, and M. Crippa (1978) Chromosoma 66, 299-308) for the large scale isolation of nuclei from Xenopus laevis oocytes. The nuclear extract obtained was tested for its ability to transcribe a cloned Xenopus 5 S RNA gene and for the presence of nuclear factors interacting with a X. laevis ribosomal protein gene promoter. Efficiency of accurate transcription and of factor binding is comparable with that of an extract prepared from manually isolated nuclei. Cm19SS Academic Press, Inc.

The biosynthesis of the ribosome is a complex process involving the coregulated production of ribosomal RNAs and ribosomal proteins. The mechanism involved in this regulatory system is still poorly understood in eukaryotes. For our studies on the transcriptional regulation of these genes we chose the oocyte of Xenopus laevis because during oogenesis large amounts of ribosomes are accumulated and it therefore seemed a promising system to search for nuclear transcription factors. Study of the promoter of the gene for the ribosomal protein L14 from X. laevis indicated that the nuclear extract, obtained from manually isolated X. laevis oocyte nuclei, contains two different sequence-specific factors that interact with two sequence elements, one located between -53 and -47 and the other between -27 and -19 upstream to the cap site. Deletion of the distal element abolished the expression of a linked reporter gene when the deleted mutant was injected into X. laevis oocytes. This element seems to be essential for the efficient use of the L14 promoter in an homologous system (F. Carnevali, C. La Porta, V. Ilardi, and E. Beccari, unpub’ This work was supported by the “Istituto Pasteur-Fondazione Cenci Bolognetti,” Universiti di Roma La Sapienza and by a grant from C.N.R., Progetto Bilaterale (I.R.). 0003.2697/89 $3.00 Copyright 0 1989 by Academic Press, All rights of reproduction in any form

lished work). In order to purify the factor interacting with this element and isolate its relative gene large amounts of nuclear extract are necessary since transcription factors are present only at low concentration in the cell. Scalenghe et al. (1) have described a method for the large scale isolation of nuclei from oocytes of X. laevis. Following this procedure, however, we were not able to reproducibly obtain a high yield of nuclei. Here we describe some modifications to this method in order to minimize the yield variability. The ability of these extracts to transcribe a cloned Xenopus 5 S RNA gene and their capacity to bind to the X. laevis L14 promoter were tested and compared with transcription efficiency and binding activity from extracts of manually isolated nuclei. METHODS

Large Scale Isolation of Nuclei Buffers used for nuclei isolation are designed as follows: Barth’s solution (2): 88 mM NaCl, 1 mM KCl, 2.4 mM NaHC03, 0.82 mM MgSO,, 0.33 mM Ca(NO& ,0.41 mM CaClz, 7.5 mM Tris base, 10 pg/ml benzylpenicillin, 10 pg/ml streptomycin sulfate, pH 7.4; buffer A: 5 mM Tris-HCl, pH 7.8, 10 mM MgC&, 20 mg/ml BSA’ (bovine serum albumin, Sigma); buffer B: 70 mM NH&l, 7 mM MgCIP, 0.1 mM EDTA, 2.5 mM DTT (dithiothreitol), 10 mM Hepes (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid), pH 7.4, 20 mg/ml BSA; buffer C: 70 mM NH,Cl, 7 mM MgClz, 0.1 mM EDTA, 2.5 mM DTT, 10 mM Hepes, pH 7.4, 5% glycerol; buffer J (3): 70 mM NH,Cl, 7 mM MgCl*, 0.1 mM EDTA, 2.5 mM DTT, 10 mM Hepes, pH 7.4, 10% glycerol; lysis medium: 70 mM NH&l, 7 mM MgC12, 0.1 mM EDTA, 2.5 mM DTT, 10 ‘Abbreviations used: BSA, bovine serum albumin; DTT, threitol; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic EGTA, ethylene glycol bis(S-aminoethyl ether) NJ’-tetraacetic SDS, sodium dodecyl sulfate.

dithioacid; acid;

177 Inc. reserved.

178

RUBERTI ET AL.

mM Hepes, pH 7.4,0.3% Nonidet-P 40; priming medium (3): 5 mM Tris-HCl, pH 7.8, 10 mM MgC&. TWO ovaries (corresponding to 12-15 g of wet tissue), obtained from adult X. laeuis female frogs (NASCO), were washed in Barth’s solution and digested for 15 h at 18°C in 150 ml of the same solution containing 0.15% collagenase type II (Sigma). Complete removal of follicle cells during the collagenase digestion of ovaries was particularly important for the subsequent isolation of nuclei. The oocytes were then washed 10 times by decantation in Barth’s solution to remove completely the collagenase. During the wash most of stage I, II, and III oocytes are discarded and the remaining large oocytes represent about BO-85% in weight of the starting material. In the original method the oocytes were then directly treated with Pronase. We noted a great variability in the response to Pronase by oocytes even from the same batch and this appeared to be the reason for the low yield in the subsequent isolation of nuclei. Pretreating of oocytes with priming medium minimized the observed variability. However, we noted that, in a few instances, perhaps correlated with the reproductive cycle, the oocytes were definitely resistant to Pronase thus preventing nuclei isolation. Oocytes were processed in petri dishes (5.5 cm) in batches of 2 ml each. The oocytes were washed 3 times in priming medium and incubated for 30 min in the same medium at room temperature. Two milliliters of packed oocytes was resuspended in 0.7 ml of priming medium containing 1 mg/ml Pronase B (Calbiochem). The incubation was continued for lo-15 min at room temperature until the oocytes became flattened and soft. Particular care was taken to remove Pronase by washing extensively (5 times) with successive 5-ml aliquots of buffer A. The oocytes were then washed once in cold buffer B and once, very briefly, in lysis medium. The treated oocytes were subsequently incubated for 10 min at 0°C under mild shaking in 2 ml of lysis medium. The nuclei floating to the surface of the medium were collected and further purified. Several sterile 12-ml tubes were filled with 10 ml of cold buffer J overlayed with 1 ml of buffer C. The tubes were kept in ice and about 0.1 ml of nuclei was transferred to the top of each tube. The nuclei were then allowed to sediment by gravity for 15 min. Buffer was withdrawn with a Pasteur pipet, and the nuclei were collected and stored intact at -70°C in buffer J, 0.5-l ~1 for each nucleus. The nuclear protein content was determined with the Bio-Rad microassay (cat. 500-0006) using different batches of large scale or manually isolated nuclei. In the first case the average value of 3.2 yg protein/nucleus was obtained. From manually isolated nuclei we found an average value of 5 pg protein/nucleus in agreement with the data reported in the literature (4). Several reasons could account for the difference in protein content/nucleus obtained with the two methods. First, we prepared manually isolated nuclei only from stage VI oocytes whereas for the large scale isolation of nuclei we used

stage IV, V, and VI oocytes. Second, the possibility of contamination by cytoplasm is higher for manually isolated nuclei since they are not washed and are not further purified by sedimentation through a glycerol cushion. Finally, we cannot exclude that the lower protein content of the large scale isolated nuclei might be caused by a loss of protein occurring at the moment of the oocyte lysis when glycerol is absent from the medium (4). We isolated nuclei from at least 30 females whose ovaries varied in weight and maturation stages. From two ovaries (12-15 g) we usually obtain about 10,000 large oocytes. After lysis an average of 7000-7500 nuclei are recovered. Manual

Isolation of Nuclei

Nuclei were prepared from large oocytes essentially as described by Birkenmeier et al. (3). Nuclei were stored intact at -70°C in buffer J, 0.5-l ~1 for each nucleus. In Vitro Transcription Plasmid DNA. Plasmid pXbsF201 carries the 240bp X. borealis somatic 5 S RNA gene inserted in the HindIII-BamHI site of pUc9 (5). Preparation of nuclear extract. Extract was prepared exactly as described by Birkenmeier et al. (3). Transcription reactions. Assays for transcription of the 5 S RNA gene were performed as described by Shimamura et al. (6) with slight modifications. The reaction mixture contained different amounts of DNA (as specified under Results and Discussion), 1 mM ATP, 6 mM MgClz, 1 mM DTT, 35 mM disodium creatine phosphate, 1 pg/ml creatine phosphokinase and nuclear extract (to a final reaction volume of 20%) at 700 to 750 pg/ml total proteins. All the reagents and the DNA were dissolved in 20 mM Hepes (pH 7.4), 1 mM EGTA (ethylene glycol bis(P-aminoethyl ether) N,N’-tetraacetic acid), 10% glycerol, 10 mM /3-glycerophosphate. After preincubation at 30°C for 2 h, a l/10 vol of a mixture containing 6 mM each UTP and CTP, 0.25 mM GTP, 2.5 &i of [LY32P]GTP, and 25 units of ribonuclease inhibitor RNasin (Promega) was added to the reactions. Transcription was continued at 30°C for 30 min. Reactions were stopped by adding a l/4 vol of a mixture containing 0.4 M Tris-HCl, pH 6.8, 50% glycerol, 0.02 M EDTA, 10% SDS (sodium dodecyl sulfate), 0.5 M DTT, 0.1% bromophenol blue and by boiling for 3 min (D. Brow, personal communication). The RNA was analyzed by electrophoresis in SDS-polyacrylamide gels (7) (12% polyacrylamide; acrylamide:bisacrylamide, 30:0.75) containing 4 M urea (D. Brow, personal communication). Gels were run at 30-mA constant current for 2 h. The gels were fixed in 10% acetic acid/45% methanol for 1 h and in 7.5% acetic acid/lo% methanol for 30 min. The gels were dried and autoradiographed at -70°C with an in-

NUCLEI

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2

3

4

5

6

7

ISOLATION

8

9

FROM

IO

FIG. 1. The 5 S Ri%A transcription on pXbsF201 DNA in uitro. Transcription reactions were performed with the large scale extract at 700 pg/ml total proteins (lanes l-5) and with the small scale extract at 720 pg/ml total proteins (lanes 6-10). Transcription reactions contained no DNA (lanes 1 and 6) or cloned 5 S DNA (pXbsF201) added at 5 pg/ml (lanes 2 and i), 7.5 pg/ml (lanes 3 and 8), 10 pg/ml (lanes 4 and 9), and 15 @g/ml (lanes 5 and 10). After preincubation for 2 h at 3O”C, transcription was continued for 30 min at 30°C in the presence of nucleoside triphosphates and [n-“‘PIGTP. The figure shows the autoradiograph resulting from the electrophoretic analyses of in vitro synthesized 5 S RNA.

tensifying screen. The amount of transcript obtained from different extracts was quantitated by scanning of the X-ray films with a laser densitometer (Ultroscan XL, LKB) and normalized to the amount of protein used.

Binding

Xenonus

laeuis

179

OOCYTES

Gel retardation assay. The labeled DNA probe was incubated with the nuclear extract in the presence of the nonspecific competitor DNA and the resulting DNAprotein complexes were analyzed by native polyacrylamide gel electrophoresis. Binding reactions, containing 0.5-2 ng (5000 cpm) of the “‘P-labeled fragment, nuclear proteins, and the competitor DNA (as specified under Results and Discussion) in 20 ~1 of binding buffer (15 mM Tris-HCl, pH 7.5, 0.1 mM EGTA, 5 mM MgC12, 0.1 M NaCl, 0.5 mM DTT, and 5% glycerol), were incubated for 30 min on ice. At the end of the incubation period, 2 ~1 of the dye mixture (0.25% bromophenol blue and 30% glycerol) was added to each tube and the samples were run on a 4% polyacrylamide (acrylamide:bisacrylamide, 29:0.8) gel in 0.25X TBE buffer (22.5 mM Tris, 22.5 mM boric acid, 0.25 mM EDTA). Gels were prerun in the cold room at 10 V/cm for 30 min. Electrophoresis was run under the same conditions for 90 min. The gels were dried and autoradiographed at -70°C with an intensifying screen. RESULTS

AND

DISCUSSION

Following our modification to the method of Scalenghe et al. (1) we have been able to eliminate most of the yield variability in nuclei isolation (see Methods).

1

Assay

2

3

4

Preparation of nuclear extract. Extract was prepared, just before use, disrupting nuclei in 0.5-l pl/nucleus buffer J by pipetting with yellow Gilson tips (five to seven times) in the presence of high salt (0.4 M NaCl) and incubating the extract on ice for 20 min. The resulting lysate was centrifuged for 10 min at 500g at 4°C and the supernatant was used or quickly frozen in dry ice and stored at -70°C. Preparation of radioactive DNA probe. A DNA fragment containing the promoter region (from -63 to $16 nucleotides relative to the cap site) of the gene coding for the L14 ribosomal protein in X. laevis was used to probe for the presence of promoter binding factors in the X. laeuis crude nuclear extracts. The lOO-bp-long DNA fragment containing the Ll4 proximal promoter region was 5’ labeled with T4 polynucleotide kinase and [r-32P]ATP and separated by 5% polyacrylamide gel electrophoresis. The required band was located by short exposure, cut out from the gel, and extracted overnight at room temperature with 400 ~1 of 1 M NaCl in TE (10 mM Tris-HCl, pH 7.5, 1 mM EDTA) and 400 ~1 of TEsaturated phenol. The aqueous phase was then extracted twice with an equal volume of chloroform, precipitated with 2 vol of ethanol at -70°C for 15 min, washed in 70% ethanol, dried, counted, and taken up in a small volume of sterile bidistilled water.

F

FIG. 2. Binding of nuclear factors to L14 promoter region and binding competition. The gel mobility shift assay was performed as described under Methods. The 32P-labeled probe, 0.5 ng (5000 cpm), was incubated with the large scale extract (720 Kg/ml total proteins) (lanes 1 and 2) or with small scale extract (720 fig/ml total proteins) (lanes 3 and 4). Binding reactions (20 ~1) contained 750 ng of sonicated E. colt’ chromosomal DNA as the nonspecific competitor (lanes 1 and 3) or 625 ng of the same nonspecific DNA plus 125 ng of the homologous unlabeled DNA fragment (lanes 2 and 4). The positions of the specific bound DNA-protein complexes (Cl and C2) and the freely migrating probe (F) are indicated.

180

RUBERTI

The extract obtained was then tested for functionality using two assays: 5 S RNA transcription and factor binding to the Ll4 promoter. 5 S RNA transcription in vitro. We assayed the extracts prepared from large scale and manually isolated nuclei for their ability to transcribe 5 S RNA using as template the recombinant plasmid pXbsF201 containing the somatic 5 S RNA gene from X. borealis. Figure 1 shows the results obtained when 0,5, 7.5, 10, and 15 pg/ ml plasmid DNA were transcribed in the extract prepared from large scale isolated nuclei (large scale extract, lanes l-5) and in the extract obtained from manually isolated nuclei (small scale extract, lanes 6-10). Both extracts accurately transcribed the 5 S RNA gene with similar efficiency. In the experiment shown in Fig. 1 the difference in the amount of transcript obtained with the two extracts, normalized for the amount of protein used, is less than 5%. However, comparison of several batches of large scale extracts showed slight differences (10-20s) in the efficiency of the 5 S RNA transcription. Binding in vitro of nuclear factors to a ribosomalprotein genepromoter. We used a gel retardation assay to compare the protein-DNA complexes formed between an Ll4 promoter fragment and the nuclear proteins of the two types of extract. The gel retardation assay, based on the altered mobility of protein-DNA complexes during gel electrophoresis, has been extensively used in equilibrium and kinetic analyses of purified prokaryotic gene regulatory proteins (8,9) and more recently to identify eukaryotic transcription factors present in crude nuclear extracts (10). Using crude extracts, an excess of heterologous competitor DNA (for instance sonicated Escherichia coli, salmon sperm, or equivalent heterologous chromosomal DNA) must be included with the specific probe fragment to bind the more abundant nonspecific DNA binding proteins. Figure 2 shows the results obtained when the lOO-bp radioactive DNA probe, derived

ET

AL.

from the upstream region of the L14 gene promoter, was incubated with the large scale extract (lanes 1 and 2) and with the small scale extract (lanes 3 and 4) in the presence of sonicated E. coli DNA used as the nonspecific competitor. Two retarded bands (Cl and C2) were resolved from the unbound DNA (lanes 1 and 3). Comparison of the complexes generated by the large scale extract with those produced by the small scale extract shows that both had the same electrophoretic mobility. To test the specificity of the Cl and C2 complexes we competed the probe binding with a 25-fold molar excess of the same unlabeled DNA. Keeping constant the concentration of total DNA the addition of the homologous DNA fragment effectively abolished the formation of both Cl and C2 complexes (lanes 2 and 4). Competition results confirm that the two extracts contain the same factors. Variability in the relative abundance of these two factors was sometimes observed from batch to batch in both extracts. ACKNOWLEDGMENTS We thank Marcella technical assistance.

Marchioni

and Angelo

Di Francesco

for skilled

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