MOLECULAR AND CELLULAR BIOLOGY, Nov. 1984, P. 2428-2436 Vol. 4, No. 110270-7306/84/112428-09$02.00/0Copyright © 1984, American Society for Microbiology

Post-Transcriptional Regulation of the Abundance of mRNAsEncoding ot-Tubulin and a 94,000-Dalton Protein in Teratocarcinoma-

Derived Stem Cells Versus Differentiated CellsCHIN C. HOWE,* DAVID K. LUGG, AND G. CHRISTIAN OVERTON

The Wistar Institute ofAnatomy and Biology, Philadelphia, Pennsylvania 19104

Received 22 March 1984/Accepted 3 August 1984

Changes in the expression of the genes encoding a-tubulin and a 94,000-dalton protein (p94) specified by acDNA clone, p4-30, were examined in a differentiated teratocarcinoma-derived parietal endoderm cell line,PYS-2, and an undifferentiated teratocarcinoma stem cell line, F9. Relative to other proteins or mRNA species,the synthesis rate of the a-tubulins and of p94, as well as the levels of their corresponding cytoplasmic mRNAs,were lower in PYS-2 than in F9 cells. The decrease was greater for the relative abundance of cytoplasmica-tubulin mRNA than for p94 mRNA. Similarly, induction of differentiation of F9 cells by simultaneousexposure to retinoic acid (RA) and dibutyryl cyclic AMP resulted in reduced relative levels of the cytoplasmicmRNAs for these proteins. The reduction in abundance of the two RNA species was not due to a decrease ingrowth rate since the differentiated cells, PYS-2, RA-treated F9, and RA plus dibutyryl cyclic AMP-treated F9cells, grew at a rate similar to that of undifferentiated F9 cells. However, induction of differentiation of F9 cellsby treatment with RA alone did not cause down-regulation of the two RNA species. The relative levels of totalcellular RNA encoding ca-tubulin and p94 in PYS-2 cells were also lower than those in F9 cells to an extentcomparable to the decrease in the cytoplasmic RNAs. Since the apparent relative rates of RNA transcriptionwere similar in both cell types, we conclude that the reduction in relative levels of the a-tubulin and p94 RNAsin the cell depends largely on the relative stability of the two RNAs and not on the relative rates of transcription.The faster disappearance of the two RNA species relative to other cellular RNAs from actinomycin D-treatedPYS-2 compared with F9 cells is consistent with this interpretation.

Microtubules consist of two proteins, a- and ,B-tubulin,each shown in human, chicken, and Drosophila melanogas-ter to contain several distinct genes (7, 8, 10, 20, 37, 46). InD. melanogaster, members of the a- and P-tubulin multigenefamilies are differentially expressed during embryonic devel-opment and in different adult tissues (21, 23, 31, 35).However, little is known about the regulation of synthesis ofthese proteins during murine embryogenesis, though changesin protein synthesis have been demonstrated (17) to occurduring early embryonic development. A recent study (9)showed that treatment of Chinese hamster ovary cells withthe microtubule-depolymerizing drug colchicine resulted inthe specific and rapid loss of tubulin mRNA from thecytoplasm. It was suggested that this loss of tubulin mRNAwas probably not achieved through a transcriptionally regu-lated mechanism since the relative rates of tubulin RNAtranscription are essentially unchanged in isolated nucleiderived from colchicine-treated and untreated control cells(6). In contrast, expression of globin genes in a hemin-treated human erythroid cell line (5) and of a-fetoprotein andalbumin genes in liver from late prenatal to 1-month-postpar-tum mice (43) is regulated primarily at the transcriptionallevel.We report here that a-tubulin mRNA, assayed with the

cDNA clone pILaTl made from rat brain mRNA (27), is lessabundant in the murine teratocarcinoma-derived differenti-ated parietal endoderm-like cell line PYS-2 (26) than in theundifferentiated stem cell line (embryonal carcinoma cellline) F9 (3). These cell lines are developmentally related,both being derived from sublines of the transplantable tera-

* Corresponding author.

tocarcinoma, OTT6050, which itself is derived from a 6-daymale embryo transplanted into the testis (3). F9 cells resem-ble the inner cell masses of blastocysts morphologically andantigenically (16). F9 cells, at the time of isolation, candifferentiate and give rise to well-differentiated tumors invivo, but by now the spontaneous rate of differentiation inthese cells is very low. However, they can be induced withretinoic acid (RA) treatment to differentiate into cells whichsecrete basement membrane components and parietal endo-derm-specific plasminogen activator (18, 19, 40). The mag-nitude of such a response is increased when dibutyryl cyclicAMP (dbcAMP) is added to the culture medium (41). Fromthese results, it was concluded that RA treatment induces F9cells to differentiate to primitive endoderm which furtherdifferentiates into parietal endoderm-like cells upon theaddition ofdbcAMP (40, 41). PYS-2 cells, on the other hand,resemble parietal endoderm of embryos on the basis ofsynthesis of basement membrane components (15, 16, 18,19, 26). Parietal endoderm is the first terminally differenti-ated cell type to appear during early postimplantation devel-opment of murine embryo. Thus, F9 and PYS-2 represent asimple model system for undifferentiated and terminallydifferentiated cells during early embryonic development intoparietal endoderm. In this study, we have elucidated theexpression of a-tubulin mRNA in F9 and PYS-2 cells as astep to the future study of more complex systems, such asRA-induced F9 differentiation and, ultimately, normal em-bryonic development.

While screening a cDNA clone bank derived from PYS-2mRNA, we identified a clone, p4-30, which specifies a94,000-dalton protein (p94). The p94-specific mRNA is alsoless abundant in PYS-2 cells than in F9 cells. For compari-son, we include in this study the characterization of the p94

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gene. Analyses of the relative rate of transcription and therelative RNA concentration in cells indicate that post-tran-scriptional mechanisms underlie the differential abundanceof these mRNAs in F9 and PYS-2 cells.

MATERIALS AND METHODSCell culture and labeling. The F9 embryonal carcinoma cell

line was maintained in Dulbecco modified Eagle mediumcontaining 10% fetal bovine serum. The PYS-2 cells weremaintained in Eagle minimum essential medium containing10% fetal bovine serum. Actinomycin D (Sigma ChemicalCo.) was used at 5 ,ug/ml, all-trans-retinoic acid (RA) (East-man Kodak Co.) was used at 5 x 10-4 mM, and dbcAMP(Sigma) was used at 1 mM. dbcAMP was solubilized in waterand actinomycin D, and RA was solubilized in dimethylsulfoxide as 100x stock solutions.For [35S]methionine labeling, cultures were grown until

almost confluent and incubated in methionine-free minimumessential medium containing 10% fetal bovine serum and 500,uCi of [35S]methionine (>800 Ci/mmol; New England Nu-clear) per ml for 1 h. For [3H]uridine labeling, cultures werelabeled for 1 h in minimum essential medium containing 10%fetal bovine serum and 1 mCi of [3H]uridine (40 Ci/mmol;New England Nuclear) per ml.Two-dimensional gel electrophoresis. The [35S]methionine-

labeled proteins were prepared for two-dimensional gelelectrophoresis by the procedures of Peterson andMcConkey (34). Briefly, the metabolically labeled cells (5 x

105 cpm) or in vitro translated products (5 x 105 cpm) werelysed or dissolved, respectively, in 10 M urea-1 mM Tris-hydrochloride (pH 7.4)-0.1% sodium dodecyl sulfate (SDS)with the aid of sonication (probe sonicator; Ultrasonics).One-tenth volume of 0.3 M lysine (pH 3.8)-1% SDS-10 Murea-25 mM ZnCl2 was added to the lysate, and the lysatewas digested with S1 nuclease to remove the nucleic acid.The pH of the resulting lysate was neutralized with 0.1volume of 1 M Tris-hydrochloride (pH 7.4)-20% NonidetP-40-1% SDS, and mercaptoethanol, ampholine, and ureawere added to final concentrations of 5%, 2% (pH 5 to 7,1.6%; pH 3 to 10, 0.4%), and 10 M, respectively. Theprepared samples were analyzed by two-dimensional equi-librium pH gradient gel electrophoresis according toO'Farrell (32) or by two-dimensional non-equilibrium pHgradient gel electrophoresis according to O'Farrell et al.(33).

Isolation of cellular RNA. For preparation of polyadenyl-ated [poly(A)+] cytoplasmic mRNA, cells were washed withDulbecco modified phosphate-buffered saline, scraped fromthe plates, resuspended in lysis buffer (0.2 M Tris-hydrochlo-ride [pH 8.5, 4°C]-50 mM KCI, 15 mM MgCl2-50,ug ofheparin per ml-10 jig of polyvinyl sulfate per ml), and lysedwith 1% Nonidet P-40. The lysate was then underlaid with an

equal volume of the lysis buffer containing 24% (wt/vol)sucrose and 1% Nonidet P-40 and centrifuged at 2,000 rpm

for 5 min. The turbid upper layer was recovered; SDS,EDTA, and proteinase K were added to final concentrationsof 1%, 10 mM, and 100,ug/ml, respectively, and the solutionwas incubated at 37°C for 1 to 2 h. Cytoplasmic RNA was

then extracted with phenol-chloroform-isoamyl alcohol, pre-

cipitated with ethanol, and fractionated twice by oligo-deoxythymidylate-cellulose chromatography (2) to obtainpoly(A)+ RNA.

Total cellular RNA was isolated by disrupting cells with a

Polytron homogenizer (Brinkmann Instruments Inc.) inNETS buffer (0.1 M NaCl, 1 mM EDTA, 10 mM Tris-hy-drochloride [pH 7.4], 1% SDS) containing 100 jig of protei-

nase K per ml, incubating the lysate at 37°C for 1 to 2 h, andextracting the lysate with phenol-chloroform-isoamyl alco-hol. The resulting nucleic acid was precipitated with ethanoland dissolved in DNase digestion buffer (100 mM HEPES[N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; pH7.0], 10 mM magnesium-acetate, 25 mM NaCl, 2 mM CaC12);the DNA was digested with RNase-free DNase (50 ,ug/ml;Miles Laboratories), and the DNA fragments were removedby G-100 column chromatography (12). The RNA elutes inthe void volume, and the small DNA fragments elute in theincluded volume.

Preparation of cDNA from PYS-2 mRNA and constructionof p4-30 clone. The poly(A)+ RNA obtained from PYS-2 cellswas used for the production of cDNA. Double-strandedcDNA was constructed essentially as described previously(45). Avian myeloblastosis virus reverse transcriptase (J. W.Beard; Life Science) was used to synthesize the first strand,and DNA polymerase I (Boehringer Mannheim Biochemic-als) was used for the second-strand reaction. Double-stranded cDNA was treated with S1 nuclease (Miles) asdescribed before (38) to create blunt ends and was tailedwith cytosine residues, using terminal deoxynucleotidyltransferase (Bethesda Research Laboratories) according tothe protocol described previously (4). Tailed cDNA was sizefractionated on a polyacrylamide gel (29), and those largerthan 1.0 kilobase (kb) were excised, eluted, and inserted intopBR322 at the PstI site which was deoxyguanylate tailed.The recombinant plasmid was introduced into Escherichiacoli strain HB101 and transformants were screened bycolony hybridization (14). As a probe, PYS-2 poly(A)+ RNAwas randomly fragmented by incubating in 50 mM Na2CO3(pH 11.7) at 60°C for 15 min and labeled with [-y-32P]ATP(>5,000 Ci/mmol; Amersham Corp.) at the 5' ends with T4polynucleotide kinase (Bethesda Research Laboratories)(44). One of the clones positive for hybridization was alsofound to hybridize differentially to F9 and PYS-2 mRNAs bycolony hybridization. This clone, p4-30, has an insert ofabout 1.23 kb with an internal PstI site, yielding twofragments of about 1.0 and 0.23 kb. However, p4-30 does notcontain internal HindIll or EcoRI sites.

Agarose gel electrophoresis and transfer of nucleic acids tonitrocellulose. RNA was denatured with glyoxal and di-methyl sulfoxide, electrophoresed through 1.1% agarosegels, and transferred to nitrocellulose; the filters were bakedaccording to the method of Thomas (42).

Plasmid DNA (1 jig) was linearized with HindIll, whichdoes not cleavea-tubulin or p4-30 cDNA, and electrophor-esed through a 0.8% agarose gel in Tris-acetate buffer (40mM Tris, 8 mM sodium acetate [pH 8.0], 1 mM EDTA).Electrophoresis was carried out at 50 V until the dye frontmoved 6 cm. Plasmid DNA was transferred to nitrocelluloseas described by Southern (39). After transfer, the filters wererinsed briefly in 2x SSC (lx SSC = 0.15 M NaCl, 0.015 Msodium citrate), air-dried, and baked at 80°C for 2 h undervacuum.

Preparation of DNA dot filters. Plasmid DNA to be dot-blotted was first linearized by restriction with HindIIl,denatured in 0.2 M NaOH for 10 min at room temperature,neutralized by diluting into 6x SSC, and then spotted ontonitrocellulose filters. The filters were air-dried and baked asabove.

Preparation of labeled plasmid DNA probes and filterhybridization. Plasmid DNA was nick translated accordingto the procedure described before (28), using E. coli DNApolymerase I (Boehringer Mannheim), [a-32P]dCTP, and[a-32P]dTTP (>2,000 Ci/mmol; New England Nuclear).

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2430 HOWE, LUGG, AND OVERTON

Nitrocellulose filters containing bound RNA or DNA werewashed in Sx SSPE (1x SSPE = 0.18 M NaCl, 0.01 Msodium phosphate [pH 7.4], 1 mM EDTA), prehybridized for20 h at 37°C in 50% formamide-5 x SSPE-5 x Denhardtsolution (1 x Denhardt = 0.02% bovine serum albumin,0.02% Ficoll 400, 0.02% polyvinylpyrrolidone 360)-1%glycine-0.1 mg of sonicated, denatured salmon sperm DNAper ml-0.3% SDS, and then hybridized for 24 h at 37°C in50% formamide-5x SSPE-1x Denhardt's solution-0.1 mgof sonicated, denatured E. coli DNA per ml-0.3% SDS-1 x107 to 3 x 107 cpm of 32P-labeled denatured probe (100°C for10 min) per ml. The blots were washed at 37°C for 1.5 h in 2xSSC-0.3% SDS with several changes, dried, and exposed toX-ray film. To quantitate radioactivity in individual bands ofan autoradiogram, the blot was exposed for several differenttimes until the response curve of the film was in its linearrange. The individual bands in the autoradiogram werescanned with a densitometer (Clifford Instruments), and theareas under the curves were determined.

In vitro RNA synthesis in isolated nuclei and hybridizationto nitrocellulose-bound plasmid DNA. Nuclei were isolatedand in vitro nuclear transcription was performed by modifi-cations of the method of Groudine et al. (13). Cells werescraped off the plates, resuspended, allowed to swell inreticulocyte standard buffer (0.01 M Tris-hydrochloride [pH7.4], 0.01 M NaCl, 3 mM MgCI2), and homogenized in aDounce homogenizer. The nuclei were pelleted by centrifu-gation at 1,500 rpm for 5 min, and the pellets were resus-pended in reticulocyte standard buffer and homogenizedagain to disrupt unbroken cells. The suspension was thenunderlaid with buffer A (50 mM HEPES [pH 8.0], 5 mMMgCl2 40% glycerol, 0.5 mM dithiothreitol) and centrifugedat 1,500 rpm for 5 min, and the pelleted nuclei wereresuspended in buffer A at a DNA concentration of 2 mg/ml.

Nuclear transcription was carried out in a reaction volumeof 250 ,ul containing 100 ,ul of nuclei in buffer A, 114 ,ul of2x transcription buffer (2x transcription buffer = 0.1 MHEPES [pH 8.0], 0.3 M NH4Cl, 6 mM magnesium acetate,1% bovine serum albumin, 4 mM dithiothreitol), 12 ,u1 eachof 7.5 mM ATP, GTP, and CTP, and 200 ,LCi of [a-32P]UTP(>600 Ci/mmol; New England Nuclear). Nuclei were incu-bated for 15 min at 26°C, and the reaction was terminated bythe addition of DNase I (RNase-free; Miles) to 20 ,ug/ml andincubated further at 26°C for 5 min. The reaction mixturewas then deproteinized by incubation at 37°C for 1 h inNETS buffer containing 100 ,ug of proteinase K per ml andsubsequent phenol-chloroform extraction. Yeast tRNA wasadded to the aqueous phase to 100 ,ug/ml, and the macro-molecules were precipitated with cold 5% trichloroaceticacid containing 0.7% sodium pyrophosphate. The precipitatewas collected on a 25-mm (0.45-,um) nitrocellulose filter andwashed. The filter was again treated with DNase 1 (25 ,ug) for30 min at 37°C in DNase digestion buffer, and the reactionwas terminated by 1% SDS-15 mM EDTA. RNA (primarytranscript) was then eluted by incubating the filter in theabove solution at 65°C for 10 min and again in 10 mMTris-hydrochloride (pH 7.5)-S5 mM EDTA at 65°C for 10 min;it was then digested with proteinase K, extracted withphenol-chloroform, and precipitated with ethanol.The nitrocellulose filters containing bound plasmid DNA

were prehybridized at 37°C overnight in 50% formamide-50mM sodium phosphate (pH 6.5)-i x Denhardt's solution-4xSSC-0.1% sodium pyrophosphate-0.1 mM EDTA-250 ,ug ofsonicated, denatured salmon sperm DNA per ml. Hybridi-zation was carried out for 4 days in the same solutioncontaining 32P-labeled primary transcript. The same amounts

of labeled primary transcripts from F9 and PYS-2 cells wereused. The blot was washed at 37°C for 2 h in 2x SSC-0.3%SDS with several changes and then at 65°C for 30 min in0.3x SSC-0.1% SDS with one change. The blots wereexposed to X-ray films and the intensities of individualhybridization bands were quantitated as described above.

Hybridization-selection and in vitro translation. PlasmidDNA (10 ,ug) was linearized with HindIII, denatured in 0.2M NaOH, neutralized, and spotted onto a 25-mm nitrocel-lulose filter (BA85; Schleicher & Schuell) as describedabove. The filter was washed with 6x SSC, air-dried, andbaked in a vacuum oven at 80°C for 2 h. The filter wasprehybridized as described previously (36) in 50% formam-ide-0.01 M PIPES [piperazine-N, N'-bis(2-ethanesulfonicacid)] (pH 6.4)-0.4 M NaCl for 1 h at 40°C, and theprehybridization solution was replaced with 400 ,ul of thesame solution containing 10 ,ug of poly(A)+ RNA fromPYS-2 cells. Hybridization was allowed to proceed over-night at 40°C. The filter was then washed as follows: 10 timeswith lx SSC-0.5% SDS at 40°C and 3 times with 10 mMTris-hydrochloride (pH 7.9)-2 mM EDTA at room tempera-ture. Specifically hybridized mRNA was eluted by boilingeach filter in 300 ,ul of 1 mM EDTA for 60 s. Carrier tRNA(20 ,ug) was added, and the mRNA plus carrier was precip-itated with ethanol. The precipitate was collected, dried, andused for in vitro translation with rabbit reticulocyte lysate(New England Nuclear) according to the manufacturer'sprotocol, except that 0.15 M potassium acetate was used tofavor large-protein translation. The translated proteins werethen analyzed by one- (24) or two-dimensional gel electropho-resis as described above.

RESULTS

a-tubulin and p94 mRNA levels in the cytoplasm of undif-ferentiated and differentiated cells. We measured the concen-trations of a-tubulin mRNA and the p94 mRNA relative tototal mRNA in the cytoplasms of F9 and PYS-2 cells byhybridization of the specific probes to Northern blots andsubsequent densitometric analysis of the autoradiographs.The pILaTl and p4-30 probes hybridize to mRNAs whosesizes are 1.9 and 3.5 kb, respectively (Fig. 1). The relativeconcentration of the 1.9-kb a-tubulin mRNA is considerablylower in PYS-2 cells than in F9 cells (cf. lanes lb and 2b),whereas the concentration of the 3.5-kb p94 mRNA is onlyslightly lower in PYS-2 than in F9 cells (cf. lanes la and 2a).The densitometric scan from Fig. 1 indicated that the rela-tive concentrations of cytoplasmic a-tubulin and p94 mRNAsin PYS-2 cells were approximately 20 and 58%, respectively,of those found in F9 cells.To determine whether the relative levels of cytoplasmic

oa-tubulin and p94 mRNAs also decrease during RA-inducedF9 cell differentiation, the cytoplasmic mRNAs of unin-duced F9 cultures and F9 cultures induced with RA or RAplus dbcAMP were analyzed. The relative abundance of thetwo mRNA species was lower in the cytoplasm of the RAplus dbcAMP-treated F9 cultures than in that of the unin-duced undifferentiated cultures, but not in the cytoplasm ofthe RA-treated F9 cultures. In RA-treated F9 cells, therelative abundance of the two mRNAs is, in fact, higher thanin the untreated F9 cells (Fig. 2).The reduction in the mRNA levels is not due to a slower

growth rate of differentiated cultures versus undifferentiatedF9 cells. The growth rates of PYS-2 and F9 cells are similar(Fig. 3), as are those of untreated F9 cells and RA- or RAplus dbcAMP-treated F9 cells (40; our unpublished data). To

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a-TUBULIN AND p94 GENE EXPRESSION 2431

further test these results, we have examined the F9 cellscultured at 33°C, at which temperature the culture wasshown to grow more slowly than at 37°C (Fig. 3). Therelative levels of a-tubulin and p94 mRNAs in F9 cells areessentially identical at both temperatures (Fig. 4).

In vitro translation products from hybridization-selectedmRNA. In vitro translation of the PYS-2 poly(A)+ mRNAselected by hybridization to pILaTl cDNA yielded twoseries of polypeptide spots with slightly different molecularweights which can be resolved only by two-dimensional gelelectrophoresis (Fig. SD). These two series of polypeptidespots also cover a wide range of isoelectric points (Fig. 5,inset) and thus are evidently translated from different a-tubulin mRNAs. The same two a-tubulins were obtainedfrom in vitro translation of the hybrid-selected F9 mRNA(not shown). Analysis of metabolically labeled proteins bytwo-dimensional gel electrophoresis revealed the presenceof the same two a-tubulins with electrophoretic mobilitiesidentical to those of the in vitro translated products from thehybrid-selected mRNA (cf. Fig. 6C and SD) and showed areduced relative rate of synthesis of these polypeptides inPYS-2 cells as compared with F9 cells (Fig. 6, cf. C and D).Thus, both a-tubulins are synthesized by F9 and PYS-2 cellsand their synthetic rates in these cells are in agreement withthe abundance of the mRNAs (cf. Fig. lb and Fig. 6).

Translation of mRNA selected by p4-30 cDNA resulted ina polypeptide of about 94,000 daltons with an isoelectricpoint of approximately 7.0 (Fig. SB). A metabolically labeledpolypeptide with the same electrophoretic mobility as the in

kb23.5-

6.6-4.3-

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1.1-

1 2 1 21 2a b c

FIG. 1. pILaTl- and p4-30-specific mRNA in F9 and PYS-2cells. One microgram of poly(A)+ mRNA isolated from the cyto-plasm of PYS-2 (lane 1) and F9 (lane 2) cells was glyoxylated,fractionated by electrophoresis, transferred to nitrocellulose, andhybridized with 32P-labeled probes made from (a) p4-30 and (b)pILaTI. In (c), an example is shown of an ethidium bromide-stainedgel containing the fractionated poly(A+) mRNA (5 jig) from PYS-2(lane 1) and F9 (lane 2) cells. Molecular weight markers as obtainedfrom HindIll digests of phage X DNA and HaeIII digests of phage4XX DNA are shown on the left.

A1 2 3 1 2 3

B

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FIG. 2. pILaTl- and p4-30 specific mRNA in RA-treated and RAplus dbcAMP-treated cultures. One microgram of poly(A)+ mRNAisolated from cytoplasm of F9 (lane 1), RA-treated F9 (lane 2), andRA plus dbcAMP-treated F9 (lane 3) cells was electrophoresed andblotted as described in the legend to Fig. 1 and hybridized to 32p-labeled (A) pILaTl and (B) p4-30 plasmid DNA. Molecular weightmarkers (in kilobases) as obtained in Fig. 1 are shown on the right.

vitro translated product was also detected in the autoradio-grams of gels after two-dimensional electrophoresis (Fig. 6Aand B). Consistent with the mRNA levels, a slightly lowerrelative rate of p94 synthesis in PYS-2 cells compared withF9 cells was observed.

Relative rates of transcription. The relative amounts ofspecific primary transcripts synthesized in isolated F9 andPYS-2 cell nuclei were compared to determine whether thedifferent relative quantities of cytoplasmic a-tubulin or p94mRNA in F9 and PYS-2 cells resulted from different relativerates of transcription. In such a nuclear transcription sys-tem, elongation of in vivo initiated RNA polymerase IItranscripts occurs but reinitiation of RNA transcriptionprobably does not. Hence, the amount of radioactivityincorporated into transcripts that hybridize to a specificDNA fragment under conditions of DNA excess is propor-tional to the rate of transcription. No difference in theintensity of signals was observed when 1, 5, or 10 jig ofpILaT1 or p4-30 cDNA was hybridized to the labeledprimary transcripts (Fig. 7B), indicating that hybridization to1 ,ug of plasmid DNA occurred essentially under conditionsofDNA excess. To compare the relative rate of transcriptionof two different cell samples, equal amounts of labeledprimary transcripts isolated from F9 and PYS-2 nuclei werehybridized to 1 ,ug of plasmid DNA. The amounts ofoa-tubulin or p94 primary transcripts relative to other tran-scripts as measured by densitometry of the autoradiogram(Fig. 7A) are essentially identical, suggesting that the rela-tive rates of transcription for a-tubulin and p94 genes in F9and PYS-2 cells are about the same. No hybridization to thepBR322 vector was detected (not shown). Consistent withthe nuclear transcription rate data, the relative levels of[3H]uridine incorporation into the a-tubulin or the p94transcript during a 1-h pulse-labeling time in F9 and PYS-2

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2432 HOWE, LUGG, AND OVERTON

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DAYSFIG. 3. Growth curve of F9 and PYS-2 cells. F9 cells

plated at a density of 2 x 105 cells per 60-mm petri dish and culleither at 33 or 37°C. PYS-2 cells were also plated at the same deand cultured at 37°C. Each day, cells were harvested from dupldishes and counted. Symbols: 0, F9 cells cultured at 37°C; Ccells cultured at 33°C; A, PYS-2 cells cultured at 37°C.

cells are about the same: for PYS cells, 799 and 592remained hybridized (per 107 input cpm) to p4-30pILaTl DNA, respectively; for F9 cells, 781 and 622remained hybridized (per 107 input cpm) to p4-30pILaTl DNA, respectively. In this experiment, near-lfluent cells were labeled and lysed with NETS buffer,the RNA was isolated eluted and deproteinized as descrin Materials and Methods for isolation of nuclear transcifrom isolated nuclei. The RNA (107 cpm) was hybridizea dot blot containing 10 ,ug each of p4-30, pILaT1,pBR322 DNA. The dots were cut out and the radioactiwas counted. Little hybridization to the pBR322 DNAdetected. Data were corrected for background hybridizaby subtracting the radioactivity of pBR322 DNA from th,the p4-30 DNA and pILaTl DNA.

Relative levels of a-tubulin and p94 RNA in untreatedactinomycin D-treated cells. Total cellular RNAs (cytojmic and nuclear) were extracted from F9 and PYS-2untreated or treated with actinomycin D for various lenof time to block de novo RNA synthesis, and the relabundance of a-tubulin and p94 RNAs in the samplesdetermined by analysis of the RNA blot hybridizationtern (Fig. 8). Densitometric analysis of the autoradiog(cf. lanes 1 and 4 in Fig. 8A and B) showed that the a-tutand p94 RNA levels in untreated PYS-2 cells comparedtheir respective amounts in untreated F9 cells were onland 45%. These values are comparable to those observethe cytoplasm of the two cell types.The abundance of both a-tubulin and p94 transcripts ii

cells treated for 6 h with actinomycin D remained essentidentical to that in untreated F9 cells as indicated

densitometry of the autoradiogram (Fig. 8). However, thelevels of the two RNAs in actinomycin D-treated PYS-2 cellsare lower than in untreated control cells, and the reductionsbecome greater with longer treatment periods (Fig. 8).Analysis of the autoradiogram by densitometry revealed thehalf-lives of both a-tubulin and p94 RNAs to be >9 h in F9cells compared to 2 h for a-tubulin and 5 h for p94 RNA inPYS-2 cells (Fig. 8D).

DISCUSSION

Throughout our investigation of the expression of a-tubulin in undifferentiated F9 embryonal carcinoma cells andin differentiated PYS-2 cells, we have measured the rates ofprotein synthesis and RNA transcription as well as theabundance of RNA relative to that of the total population toallow comparison between the two cell types without com-plications arising from different growth or RNA synthesispatterns in the two cell types. We have assessed the sameparameters in the p4-30 gene which encodes a 94,000-daltonprotein for comparison. Although p94 has not been identi-fied, we know that it is not collagen, an intermediatefilament, a known actin-associated protein, or part of anintracisternal A-type particle RNA virus (unpublished data).The relative rate of a-tubulin synthesis is lower in PYS-2

cells than in F9 cells. Correspondingly, the relative abun-dance of a-tubulin cytoplasmic RNA is approximately five-fold lower in PYS-2 cells than in F9 cells. Similarly, the

were relative rate of p94 synthesis and the relative abundance oftured its cytoplasmic RNA are lower in the differentiated cells asi ty compared with F9 but only by a factor of <2. The reduction

)caF9 in the relative levels of cytoplasmic a-tubulin and p94mRNAs are also evident when F9 cells are treated simulta-neously with RA and dbcAMP. The reduced levels of thetwo mRNAs in differentiated cells compared with undiffer-

cpm entiated cells is not due to a slower growth rate in theand differentiated cells since PYS-2 as well as RA plus dbcAMP-cpmandcon-andibedriptsd toandivitywasttionat of

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FIG. 4. pILaTl- and p4-3-specific mRNA in F9 cells cultured at33°C. One microgram of cytoplasmic poly(A)+ mRNA isolated fromF9 cells cultured at 33°C (lane 2) and 37°C (lane 1) was electropho-resed and blotted to nitrocellulose as described in the legend to Fig.1 and hybridized to 32P-labeled (A) pILaT1 and (B) p4-30 plasmidDNA. Molecular weight markers (in kilobases) are shown on theright.

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FIG. 5. Two-dimensional gel electrophoresis of in vitro translation products from hybridization-selected mRNA. Polyadenylatedcytoplasmic RNAs from PYS-2 cells were hybridized to pILaTl or p4-30 cDNA, and the selected RNA was translated with rabbit reticulocytelysate. Non-equilibrium pH gradient two-dimensional gel electrophoresis of (A) background translation product from a reaction withoutexogenously added mRNA and (B) translation product from mRNA hybrid selected with p4-30 cDNA. Equilibrium pH gradienttwo-dimensional gel electrophoresis of (C) products from translation of the initial total mRNA and (D) translation product from mRNAhybrid-selected with pILaTl cDNA. Horizontal arrows indicate the locations of p4-30-specific polypeptides (A and B) and two a-tubulinpolypeptides (C and D). The right-hand side of the gel is the acidic end. The position of actin is indicated within each panel by "A" and thatof the endogenous product of the reticulocyte system is indicated by "E." (Inset) Enlargement of two a-tubulins from (D). Approximately5 x 105 cpm was loaded onto each gel. Molecular weight markers obtained from carbonic anhydrase, actin, albumin, and phosphorylase A(bottom to top) are shown on the left.

treated F9 cells have growth rates similar to that of F9 cells.To further test this conclusion, we have retarded the growthof F9 cells by culturing them at 33°C and analyzing them forcytoplasmic a-tubulin and p94 mRNA expression. The twomRNA species are equally abundant in 33 and 37°C cultures,indicating that the relative abundance of the two RNAspecies is unrelated to growth rate. Finally, the relativeabundance of the a-tubulin and p94 mRNAs differed inRA-treated F9 cells and in RA plus dbcAMP-treated F9cells, despite the similar growth rates in these cultures.The differences in the abundance of the two mRNAs are

also not correlated with the relative rates of transcription ofa-tubulin and p94 RNAs. Indeed, we found that the relativerates of transcription of these RNAs in nuclei isolated fromF9 and PYS-2 were essentially identical. Whereas the invitro transcription system appears to faithfully represent thein vivo situation for a variety of genes previously studied (5,43), it seems prudent to examine this relationship for eachspecific case. In view of the fact that the relative levels of themetabolically pulse-labeled a-tubulin and p94 transcripts arecomparable in F9 and PYS-2 cells, we are persuaded that invitro nuclear transcription closely resembles in vivo tran-scription in the present system. Both the metabolic labelingexperiments and in vitro nuclear transcription experimentspoint to similar transcription rates in the two cell types.Given that a-tubulin and p94 RNAs are synthesized at

approximately equal rates in the two cell types, the relativedecrease in abundance of the cytoplasmic a-tubulin and p94mRNAs in PYS-2 versus F9 can be caused by differences in

one or more of the three transcriptional processes, i.e., RNAprocessing, transport, and degradation. The concurrent de-crease in the abundance of total cellular a-tubulin and p94RNAs in PYS-2 cells compared with F9 cells indicates thatstability of RNAs plays a role in regulating the abundance ofthe two RNAs. Comparable levels of total RNA would beexpected in both cell types if degradation occurred at equalrates or did not occur at all. Our results, however, do notaddress the questions of RNA processing and of nuclear-to-cytoplasmic RNA transport since differences between F9and PYS-2 cells with respect to these two processes will notaffect the relative levels of total cellular oa-tubulin or p94RNA.To obtain additional evidence on the relative stability of

the two RNAs, we attempted to measure the stability of totalcellular oa-tubulin and p94 RNAs by a [3H]uridine pulsefollowed by a chase with an excess of unlabeled uridine andcytidine (1). However, in this system, the chase was inef-fective even in the presence of glucosamine, which shuntsUTP away from the pathway of RNA synthesis. After thechase, label continued to accumulate in RNA for 6 h in F9cells and for up to 10 h in PYS-2 cells (data not shown).As an alternative to the pulse experiment, we analyzed the

fate of a-tubulin and p94 RNAs in cells treated with acti-nomycin D to halt RNA synthesis: the half-life for a-tubulinRNA was more than fourfold shorter in PYS-2 cells com-pared with F9 cells, whereas that for p94 RNA was less thantwofold shorter in PYS-2 cells. Although the significance ofturnover rates of RNA is far from certain due to possible

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2434 HOWE, LUGG, AND OVERTON

artifacts in metabolism in the presence of actinomycin D, therates observed are entirely consistent with the reduction inthe relative levels of total cellular RNA found at steadystate: p94, present in PYS-2 cells at 45% of the level found inF9 cells, has a half-life in PYS-2 cells equal to 55% of that inF9 cells, whereas a-tubulin RNA, present in PYS-2 cells at18% of the level found in F9 cells, has a half-life in PYS-2cells equal to 22% of that in F9 cells. Thus, the turnoverrates in the presence of actinomycin D are in agreement withthe conclusion that a-tubulin and p94 RNAs are more stablein F9 than in PYS-2 cells.One plausible explanation for the different stabilities of F9

and PYS-2 a-tubulin RNAs might rest in the activation ofdifferent genes in the two cell types, reminiscent ofDrosoph-ila embryos in which different members of the a-tubulin genefamily are active at different stages. Analogous to findings inchickens (8), D. melanogaster (20), and rats (I. Lemishka,personal communication), we find that a-tubulins of micealso belong to a multigene family with 5 to 10 members whenassayed by Southern blot analysis. Based on two-dimen-sional gel electrophoresis of pulse-labeled proteins and invitro translation products, at least two different a-tubulinsare present in both F9 and PYS-2 cells. However, the twoa-tubulins in F9 cells were not distinguishable from those inPYS-2 cells. The two a-tubulins are evidently translatedfrom two different mRNAs rather than derived from post-translational modification which usually does not occurduring in vitro translation. In both F9 and PYS-2 cells, wealso detect only one RNA band with the same electrophor-etic mobility. The results were not entirely unexpected since

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different members of the a-tubulin gene family containingdifferent sequences of the 3' untranslated regions have beenreported to share similar sequences in the coding regions(21). Thus, it remains possible that different sets of a-tubulingenes are active in F9 cells and in PYS-2 cells and that thePYS-2 o-tubulin gene set is intrinsically less stable than theF9 set. We are in the process of isolating and sequencinga-tubulin cDNA clones derived from F9 and PYS-2 cDNAclone banks to identify the a-tubulin genes expressed in thetwo cell types. Like PYS-2 cells, the differentiated F9 cellsinduced by RA plus dbcAMP treatment also express a-tubulin mRNA at a lower level than the undifferentiated F9cells. Whether the expression of the a-tubulin RNA inRA + dbcAMP-treated F9 cells is governed by the same set ofgenes as those in PYS-2 cells remains to be elucidated. Thatinformation might in turn shed light on the correspondingmechanism during embryogenesis.

Post-transcriptional regulation of gene expression hasbeen demonstrated in many cases. The level of cytoplasmictubulin mRNA in colchicine-treated cells is regulated at thepost-transcriptional level (6), as is the histone mRNA levelin HeLa cells (30). The growth-dependent dihydrofolatereductase mRNA level is probably also determined bypost-transcriptional events since the relative level ofdihydrofolate reductase mRNA in growing cells is approxi-mately 10-fold that in stationary cells in contrast to the 2-foldchange in the relative transcription rate of the dihydrofolatereductase gene (22). It is of interest that the genes for thesethree proteins, all of which are controlled at the post-tran-scriptional level, encode housekeeping proteins. By con-

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cpm were analyzed by non-equilibrium pH gradient two-dimensional electrophoresis (A and B) and by equilibrium pH gradienttwo-dimensional electrophoresis (C and D). These gels were analyzed in the same batch as those in Fig. 5, so that the conditions of focusingand electrophoresis were the same. Thus it was possible to identify a-tubulins and p4-30-specific polypeptides in Fig. 6 by superimposing thisfigure on Fig. 5. The arrows indicate the p4-30-specific (A and B) and two a-tubulin-specific polypeptides (C and D). The right-hand side ofthe gel is the acidic end. The position of actin is indicated with "A." The molecular weight markers are shown on the left.

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a-TUBULIN AND p94 GENE EXPRESSION 2435

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FIG. 7. In vitro nuclear transcription. (A) The 32P-labeled pri-mary transcript from isolated F9 or PYS-2 nuclei was used forhybridization with 1 ,ug of pILaTl, p4-30, or pBR322 DNA, whichwas linearized, electrophoresed, and transferred to nitrocellulose.The same amount of label (approximately 107 cpm) from F9 andPYS-2 cells was used for hybridization. Lanes 5 and 6 show theethidium bromide-stained gel profile of pILaTT and p4-30 DNA,respectively, before transfer to nitrocellulose. pILaTT DNA hybrid-ized with primary transcripts from F9 and PYS-2 cells is shown inlanes 1 and 3, respectively. The p4-30 DNA hybridized with primarytranscripts from F9 and PYS-2 cells is shown in lanes 2 and 4,respectively. No hybridization was detected between pBR322 DNAand primary transcripts (not shown). (B) Hybridization of in vitronuclear transcript to various amounts of cloned plasmid DNA. The32P-labeled primary transcript from isolated F9 nuclei prepared in anexperiment parallel to but independent from those used in (A) washybridized to 1, 5, and 10 ,ug of linearized pILaTl DNA spottedonto nitrocellulose filters. Densitometry tracings of the autoradio-gram are shown. No increase in hybridization was detected withincreasing amounts of pILaTl DNA. The same results were ob-tained from the primary transcript from PYS-2 nuclei. Similarly,maximum hybridization was attained with 1 ,ug of p4-30 DNA.

trast, there is evidence suggesting that genes encodingspecialized proteins in differentiated cells are controlled atthe transcriptional level (11, 25). Investigations of regulatorymechanisms of genes encoding other housekeeping andspecialized proteins might reveal whether a general relation-ship exists between protein type and mode of control.

ACKNOWLEDGMENTS

We thank I. Lemischka for providing pILaTl, D. Solter forhelpful suggestions and discussion during the course of this study,Judith Singleton for technical assistance, and Marina Hoffman forskillful editorial advice in the preparation of this manuscript.

This work was supported by Public Health Service grants HD-17720 and HD-14915 from the National Institutes of Health.

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FIG. 8. Relative concentration of pILalT RNA and p4-30 RNAin F9 and PYS-2 cells untreated or treated with actinomycin D.Nearly confluent cultures were incubated in actinomycin D for 0, 3,or 6 h and total cellular RNAs were isolated. A 20-,ug portion of eachRNA sample was glyoxylated, electrophoresed, and transferred tonitrocellulose. A single blot was used for hybridization with the32P-labeled p4-30 probe (B) eluted as described by Thomas (42)followed by hybridization with the 32P-labeled pILaTl probe (A).Lanes 1 to 3 are RNA samples from F9 cells treated with acti-nomycin D for 0, 3, or 6 h, respectively, and lanes 4 to 6 are fromPYS-2 cells treated for 0, 3, or 6 h, respectively. An example of anethidium bromide-stained gel containing 5 ,ug of each RNA sample isshown in (C). Kinetics of loss of a-tubulin RNA and p4-30 RNA inF9 and PYS-2 cells treated with actinomycin D is shown in (D). Theautoradiograms shown in (A) and (B) were scanned by densitome-try, and the relative amount of remaining a-tubulin RNA or p4-30RNA was calculated. Symbols: 0, a-tubulin RNA in F9 cells; *,a-tubulin RNA in PYS-2 cells; 0, p4-30 RNA in F9 cells; O, p4-30RNA in PYS-2 cells.

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kb 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 66.6- kb4.3 - - 4.3

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