Gene, 80 (1989) 293-304

Elsevier 293

GENE 03070

Regulation of chicken a and /I actin genes and their hybrids inserted into myogenic mouse cells

(Recombinant DNA; gene transfer; G418 and neomycin antibiotics; development; post-transcriptional regu- lation; exons; differentiation; muscle; BC,Hl cell line)

Sandra B. Sharp”, Thomas A. Kostb, Stephen H. Hughes’ and Norman Davidson”

a Division of Chemistty, Calgomia Institute of Technology, Pasadena, CA 91125 (U.S.A.) Tel. (818)356-6055; b Glaxo Research Laboratories, Biochemtitry Department, Chapel Hill, NC27514 (U.S.A.) Tel. (919)490-3929, and c Frederick Cancer Research Facility, Frederick, MD 21701 (U.S.A.) Tel. (301)698-1619

Received by J. Piatigorsky: 15 August 1988 Revised: 22 February 1989 Accepted: 24 February 1989

SUMMARY

We have investigated the regulation of intact non-muscle (B) and muscle-specific (skeletal a) chicken actin genes and of hybrids of these two genes (a5’$3’ and fi5’-a3’) transferred into the mouse myogenic non-fusing cell line BC,Hl. BC,Hl cells express members of the actin multigene family in a differentiation-dependent manner. When proliferating, the cells accumulate large amounts of non-muscle actin mRNA; when the cells are induced to differentiate, the amount of non-muscle actin mRNA decreases and the amount of muscle-speci- fic actin mRNA increases. The transferred /?-a&in gene is efficiently expressed in undifferentiated cells and appropriately down-regulated upon differentiation. In contrast, the transferred cr-actin gene is inefficiently expressed and not consistently up-regulated. Results with the intact and hybrid genes, taken together, are consistent with the hypothesis that both 5’ and 3’ halves of these genes contain sequences important in regulating the efficiency and/or developmental timing of their expression in BC,Hl cells. By nuclear run-on experiments we found no evidence for gene-specific changes in the rate of transcription of the transferred actin genes during myogenesis. We conclude that the differentiation-dependent changes in expression of the intact j?-actin gene in BC,Hl cells must be regulated at the post-transcriptional level.

Correspondence to: Dr. S.B. Sharp at her present address: Biology Department, California State University Los Angeles, 5151 State University Drive, Los Angeles, CA 90032 (U.S.A.) Tel. (213)343-2050; Fax (213)343-3039.

NeoR, neomycin or G418 resistance determinant; nt, nucleo- tide(s); p, plasmid; Pn, penicillin; PolIk, Klenow (large) fragment of E. coli DNA polymerase I; SDS, sodium dodecyl sulfate; Sm, streptomycin; ss, single strand(ed); SSC, 0.15 M NaCl/O.OlS M Na, . citrate pH 7.6; TCA, trichloroacetic acid; tk, gene coding

Abbreviations: cpm, counts/min; ddATP, dideoxyadenosine tri- phosphate; DMEM, Dulbecco’s modified Eagle’s medium; dT, deoxythymidine; DTT, dithiothreitol; FBS, fetal bovine serum;

for thymidine kinase; Tn, transposon; tsp, transcription start point; UTR, untranslated.

0378-l 119/89/$03.50 0 1989 Elsevier Science Publishers B.V. (Biomedical Division)

294

INTRODUCTION

Vertebrates express at least six different actin pro- tein isoforms, each one encoded by a separate gene (for review, see Buckingham and Minty, 1983;

Chang et al., 1984). Two of these isoforms, the non- muscle or cytoskeletal actins (B and y), are found in the cytoskeletons of all cells. Four others are found in the contractile apparati of the various muscle tissues. When cells destined to become muscle cells undergo differentiation, non-muscle actin gene ex- pression decreases and muscle-specific actin gene expression increases. We are interested in the me- chanisms by which the developmentally timed ex- pression of the actin isogenes is regulated, and have therefore carried out experiments to locate &-acting control sequences in chicken non-muscle /I-actin and skeletal muscle a-actin genes and to determine whether these sequences exert their control at the transcriptional or post-transcriptional level. We have introduced cloned intact and hybrid chicken actin genes into the genome of the myogenic non- fusing mouse cell line BC,Hl (Schubert et al., 1974) and determined the patterns of transcription and of accumulation of mRNAs expressed from these and endogenous actin genes during differentiation. In ad- dition, we have asked whether the polyadenylation site of the mRNA transcribed from a given actin gene changes in a differentiation-dependent way. Our re- sults suggest that, at least in the cell line studied, regulation of accumulation levels is post-tran- scriptional.

MATERIALS AND METHODS

(a) Enzymes, chemicals, and cell culture supplies

Proteinase K and Sal1 were from Boehringer- Mannheim. Other restriction enzymes, T4 DNA ligase, and PolIk were from Bethesda Research La- boratories, Boehringer-Mannheim, or New England BioLabs. Terminal transferase was from RatliBe Biochemicals; guanidinium thiocyanate from Fluka; RNasin from Promega Biotec; and DNase I from Worthington Biochemicals. All radionucleotides were from Amersham. All ingredients in cell-culture

media were from Irvine Scientific except G418, which came from Gibco. Cell-culture supplies were from Corning or Falcon. Authentic chick mRNAs were a gift from S. Capetenaki in the laboratory of E. Lazarides; plasmid pNeo3 from B. Wold; pNeo3dRI from N. Fregien (Fregien and Davidson, 1986); and a mouse skeletal muscle actin Ml3 clone from M. Hu (also with N. Davidson). All these labo- ratories are at Cahech. pGia-actin 1, pa-actin 2, and pDdeC were from C. Ordahl, UCSF.

(h) The myogenic cell line BC,Hl

This is a non-fusing mouse line derived from a cranial tumor. The differentiated cells possess some biochemical, morphologic, ultrastructural, and elec- tric properties characteristic of smooth muscle (Schubert et al., 1974), and express vascular smooth muscle actin (Strauch and Rubenstein, 1984). How- ever, they also express high levels of the nicotinic acetylcholine receptor (cf., Boulter and Patrick, 1977) which is skeletal muscle-specific, as well as skeletal muscle actin mRNA (Sharp et al., 1987). Differentiation is reversible (Munson et al., 1982).

(c) Cell culture

Cell-culture conditions were based on those pre- viously described (Schubert et al., 1974). For collec- tion of RNA over a time course of differentiation, cells were induced to differentiate by one of two protocols, either starvation or a switch to low serum medium. In the first case, cells were plated on day 0 at 2.6 x 105/T150 flask in growth medium (20 ml DMEM + 100 u Pn/ml, 100 pg Sm/ml + 20% heat- treated FBS) and were never refed. Cells were har- vested up to eight days after plating. In the second case, cells were plated at the same density in 15cm dishes in 20 ml of DMEM + 20% FBS and were refed daily with a switch on day 4 to 20 ml of DMEM + 2% FBS. In this case, cells were collected up to twelve days after plating. All flasks and dishes were coated with a 0.2% gelatin solution. Cells either were kept in a 37°C incubator with a humidified atmosphere of 5% CO, in air or were first equili- brated in such an incubator and subsequently kept in airtight flasks in a room maintained at 37°C.

295

(d) Gene transfer methods are available from the corresponding author upon request.

Gene transfer was performed by a modification of the Ca * phosphate-precipitated DNA method (Graham and Van der Eb, 1973; Frost and Williams, 1978), using G418 (a neomycin analog) resistance (Davies and Jimenez, 1980) to select for transfor- mants. Cells were plated at 1.5 x 106/T75 flask on day 0. On day 1, 6.2 x lo-’ pmol carrier-free Ca * phosphate-precipitated closed circular plasmid

DNA was applied to the cells in a volume of 1.4 ml and cells were incubated for 30 min at 37 ‘C. Growth medium (13 ml) was added and incubation con- tinued for another 6 h. After aspiration of the DNA- containing medium and a single rinse with serum- free medium, cells were subjected to shock for 3 min at 37” C with 12 ml 15% glycerol in Hepes-buffered isotonic saline. The glycerol solution was aspirated and the cells were fed. Cells were refed on day 2, placed under G418 selection at 600 pg/ml on day 3, and refed every other day with selective growth medium. Surviving colonies were pooled around day 13. Efficiency of transformation was approx. 1 in lo4 starting cells and cell pools comprised 30-200 colonies.

(e) Plasmid constructions for gene transfer

Constructions were made by inserting chicken genomic fragments into vector pNeo3S at the unique Sal1 site in both possible orientations (same and opposite) relative to the direction of transcription of the NeoR gene, as depicted and further described in Fig. 1.

(f) Determination of fraction of poly(A)+RNA in

total cellular RNA

An excess of 32P-labeled poly(dT) was hybridized to total RNA spotted onto nitrocellulose. Spots contained either 12.5 or 25 ng of total RNA which had been collected on each of eight days of differen- tiation by starvation. Experiments were run with RNA from two different transformant cell pools. Spots on the filter were cut out and counted in scin- tillation fluid.

All common procedures not specifically refer- enced were similar to those described and referenced in Maniatis et al. (1982). Further details for all

RESULTS AND DISCUSSION

(a) Diierentiation-dependent actin message accu-

mulation as analyzed by blot hybridization

The dilferentiation-dependent transition in ex- pression of mouse actin mRNAs from non-muscle to predominantly muscle isoforms in BC,Hl cells is demonstrated in Fig. 2A. Although the RNA used in the experiment was collected from cells transformed with the chick /?-a&in gene, the results are virtually identical to those previously described by us (Sharp et al., 1987) for endogenous mRNAs in untrans- formed BC,Hl cells and are included here for ease of reference. The mouse finon-muscle- and a muscle- specific mRNAs have molecular lengths of 1850 nt and 1450 nt, respectively.

Hybridization analysis of chick /I-actin mRNA from the transferred b-actin gene is shown in Fig. 2,B and C. Both blots were analyzed using a 3’-UTR probe from chick #?-a&in cDNA. The blot for Fig. 2B is the same as that used for Fig. 2A. The fractional amount of chick fi-actin mRNA decreased five- to tenfold over the eight-day differentiation period. Comparison of Figs. 2B and 2C shows that down- regulation was similar whether the cells were trans- formed with the plasmid construction in which the /I-actin gene had been inserted in the s or o orienta- tion relative to the NeoR gene (Fig. 1). Thus, the down-regulation of chick /I-actin mRNA clearly mimics that of the endogenous non-muscle actin mRNA. In contrast to the five- to tenfold down- regulation of expression of the /I-actin mRNA, message accumulation from the NeoR gene in either construction decreased only two- to fourfold over the same period (Fig. 2D).

Regulation of expression of the transferred a-actin genes did not mimic that of the endogenous mouse skeletal a-actin gene (Fig. 3). Whereas the endo- genous a-actin gene was up-regulated normally (Fig. 3C), the chick transcripts were apparent to some extent at all times, that is, before and after differentiation, in cells transformed with either con- struction (Fig. 3,A and B). Expression from the

296

Chick p-ActIn Gene (5.9 kb)

Bgl II (Hybnd Junction)

CAAT 41 pNeo 3s I 1 121 267 327 I 1 I I pNeo 3s ------ -----

TPTA I

pNeo 3s --- _

I I I I

CBAT I I pNeo 3s I II I

TATA ; I50 203 2;7 317 I

Bgl II (Codon 851

Chick Skeletal a-Actin Gene (6.2 kb)

Fig. 1. Constructs containing a and pactin genes. (A) Schematic ofcloned constructions containing intact and hybrid chicken actin genes. Closed line, pBR322 sequence; hatched box, HSVrk promoter and polyadenylation sites; blackened box, E. coli Tn5 sequence. Arrows indicate the direction oftranscription. The dotted line parallel to the map indicates region used for hybridization in nuclear run-on assays. (B) Maps of the chicken actin genes. Open boxes denote untranslated exons; blackened boxes, translated exons. The vertical dashed line indicates the junction position of the hybrid genes. Numbers specify the last codon in each exon. Dotted lines parallel to the maps indicate regions used for hybridization in nuclear run-on assays. Plasmid pNeo3S was made from pNeo3 (Bond and Wold, 1987) by converting the unique EamHI site to a Sal1 site. pNeo3 includes 200 nt 5’ of the HSVtk tsp. The intact &actin insert was the genomic XhoI-EcoRI fragment from pbAct-1 (Kost et al., 1983). The intact a-actin insert was the entire a-actin genomic fragment from pGa-actin 1 (Fomwald et al., 1982). The B5’-a3’ and a5’$3’ hybrids had as junction site the conserved BglII site at codon 85.

NeoR gene transferred in the cr-actin gene constructs was down-regulated two- to fourfold in a manner similar to that of the same gene in the /3-actin gene constructs (Figs. 3D and 2D). Comparison of message accumulation from the two different trans- ferred actin genes, chick a and chick b, shows that a-actin gene expression was regulated differently from /Sactin gene expression; the message from the a-actin gene never underwent the obvious net de- crease in accumulation which was characteristic of the /Sactin message. Therefore, in view of the consis- tent regulation of the NeoR gene and the contrasting

regulation of the a- and /Sactin genes, we conclude that the #Igene contains sequences sticient to signal a significant level of gene-specific down-regulation.

Inasmuch as the transferred intact a and fi genes were differentially regulated - 8, always down; a, never-we made a pair of converse hybrids, a5’$3 and /S’-a3’, using as junction the conserved BglII site at codon 85 (Fig. l), and used the hybrids to ask whether the regulatory sequences were located in the 5’ or 3’ portions of the genes. Expression from the hybrid actin gene consisting of the 5’ half of the a

gene and the 3’ half of the Bgene was down-regulated

291

A 0, 12345678

Fig. 2. RNA blot hybridization analysis shows that expression of the transferred intact chick /I-actin gene is appropriately down- regulated. Total cellular RNA was prepared using modifications of a guanidinium thiocyanateCsC1 cushion procedure (Chirgwin et al., 1979) and final concentrations were determined spectro- photometrically. Total RNA (12 pg) from differentiating trans- formant cells were loaded in each lane. (There is no direct de- monstration that the same amount of RNA was loaded in each lane. However, all blots were reprobed with an actin coding region probe, a mouse skeletal or-actin 3’ untranslated region probe, and in ah but two cases, with a probe for the NeoR gene. The results from these hybridizations were essentially consistent from one blot to another, as can be seen by comparison of Figs. 2-4.) Lanes are numbered for days in culture using the starvation protocol. Gel blot analyses were performed essentially as described by Fyrberg et al. (1983), with final rinses at 55°C to minimize cross-species hybridization of isoform specific probes (Ordahl and Cooper, 1983; Ponte et al., 1983). The species specificity of the isoform specific probes under the condi- tions used has been previously demonstrated (Sharp et al., 1987). In panel A, the blot was probed with mouse skeletal muscle actin coding region sequence, which at the stringency used hybridizes with all actin mRNAs. The probe was the internal BnmHI-PvuII fragment of pJ, a cDNA clone from a library constructed by R. LaPolla, Caltech, which has an insert virtually identical to, but shorter than, that described by Minty et al. (1982). The lower M, actin species present on days l-3 is not skeletal a-actin (cf. Fig. 3C). In panels B and C, blots were probed with a gene-speci- tic chick p-actin 3’-UTR cDNA probe provided by D. Cleveland

Fig. 3. Expression from the transferred skeletal muscle actin gene is not down-regulated during the later days of differen- tiation. In panels A and B, the probe was the gene-specific chick skeletal muscle actin 3’-UTR, consisting of the BclI-EcoRI frag- ment ofpa-actin 2 (Ordahl et al., 1980). In panel C, the probe was a gene-specific mouse skeletal muscle 3’-UTR, consisting of either the 3’ PstI fragment or the longer 3’ HgiA-PstI fragment from pJ (see legend to Fig. 2). The specificities of these probes are discussed in the legend to Fig. 2. In panel D, the probe was the Neo%oding region described in Fig. 2, and the blot was the same as that shown in panels B and C. All symbols are as for Fig. 2. Cells were differentiated using the starvation protocol.

(Cleveland et al., 1980). In panel D, the blot was probed with a NeoR coding region probe, consisting of the BglII-SmaI frag- ment from pNeo3 (Bond and Wold, 1987). All probes were gel isolated, nick translated fragments. Autoradiograms were quan- titated using an LKB densitometer. Marker lanes indicate po- sition of human 28s and 18s ribosomal RNAs labelled in vivo in HeLa cells. o, actin and NeoR genes transcribed in opposite direction; s, same direction (see Fig. 1); m, mouse; c, chick.

298

12345678 A %

- 5090 nt

-178Ont

B 9%

@h&- -1760 I-It -tlW

C

-i?60 nt

0

- I760 nt

Fig. 4. Expression from the a5’-83’ hybrid gene is down-

regulated; expression From the 85’-a3’ hybrid gene is complex. The blot in panel A was probed with the chick j?-actin 3’JJTR probe (see Fig. 2); in panel B, with the mouse skeletal muscle actin-coding region probe (see Fig. 2); in panel C, with the NeoR- coding region probe (see Fig. 2); in panel D, with the chick skeletal muscle actin 3’-UTR probe (see Fig. 3). All symbols are as for Fig. 2. Cells were induced to differentiate by starvation.

fourfold, as shown in Fig. 4A. Clearly, this down- regulation was not as great as that of the intact fl gene, and indeed, did not differ significantly from

that of the NeoR gene (Fig. 4C), which was regulated in a manner similar to that observed with the intact actin gene constructs (Figs. 2D and 3D). However, in view of the fact that intact a did not undergo net down-regulation, the result must mean either that sequences present in the 3’ half of the /I gene confer down-regulation, or that sequences present in the 3’ half of the o! gene prevent down-regulation; the opposing regulatory trends of the two genes in question make either influence theoretically possible. These cells underwent normal differentiation (Fig. 4B).

We next tested the converse hybrid, B5’-~3’ (Fig. 4D). As we described previously (Sharp et al.,

1987), if only early and late time points were compared, expression from this gene appeared to be essentially constitutive (cf., Fig. 5B, the second four lanes). However, time-course analyses of both pools and clones (not shown) revealed that the amount of transcript from this gene decreased during the first live days of differentiation and then showed either no further decrease or, in some experiments, an increase at later time points (Fig. 4D). We observed that star- vation-induced differentiation resulted in a live- to sevenfold initial decrease; however, differentiation induced by constant refeeding with a switch to low serum medium, as described in MATERIALS AND

METHODS, section c, resulted in a decrease of only threefold or less (not shown). Because the /l5’-a3’ gene was not down-regulated to the same extent as was the intact figene, the result substantiates the role of sequences in the 3’ half of one or the other gene in controlling the extent of down-regulation. In the control experiments, endogenous a-actin gene ex- pression in the /35’-a3’ transformants was up-regu- lated normally, and NeoR gene expression was down-regulated in a manner similar to that described above for cells transformed with other actin con- structs (data not shown).

(b) Sl nuclease analysis of the polyadenylation sites of mRNAs from transferred actin genes

To determine whether or not the end of the 3 ’ -UTR in transcripts from a given transferred actin gene is (1) the same as that for authentic chick actin mRNA; (2) the same in undifferentiated vs. differen- tiated BC,Hl cells, and (3) the same in transcripts from intact vs. hybrid genes, Sl nuclease digestion assays were performed on the actin mRNAs from each of the different transformant pools. The results of these experiments (Fig. 5) show that BC,Hl cells utilize the same polyadenylation sites for chicken actin mRNAs as do native chicken cells and that neither the stage of cellular differentiation nor the hybrid sequence of the messages affects the choice of polyadenylation site.

In terms of message accumulation, the results of the S 1 assays concur with those from blot hybridiza- tion analysis. The j&a&in gene is down-regulated; the a-actin gene is not; the a5’-/?3’-actin gene is down-regulated; the /35’-a3’-actin gene appears nearly constitutive. The mRNA for the Sl assays

299

A

a, 4 @I a$ UDUDUDUD

622 nt -

527 nt -

403 nt -

‘I 309 nt -

242 nt -

i33‘UTR

*

b 266 nt

Q3’UTR

L 392 nt

- 403 nt

309 nt

Fig. 5. S 1 endonuclease analyses oftranscripts from transferred actin genes confirm regulatory trends seen in blot hybridization analysis and show faithful use of a single polyadenylation site. Analyses were performed according to the method of Berk and Sharp (1978), on urea 4% polyacrylamide sequencing gels. In panel A, the probe for chick j and as’-83 transcripts was a 600-bp Ban1 fragment isolated from a chick j-actin subclone and labeled by end-filling with PolIk using four radiolabeled nucleotides. The RNA in the second last lane of panel A was from undifferentiated, untransformed BC,Hl cells, indicating the specificity of this probe for chicken transcripts. In panel B, the probe for chick CL and j?5’-a3’ transcripts was a 2330-bp ScaI-Sal1 fragment isolated from the chick a-actin genomic construct (Fig. 1) and end-labeled with [a-32P]ddATP and terminal transferase (Yousaf et al., 1984). This probe is specific for chicken a-actin mRNA, because the differences in sequence between the 3’-UTR of chick and mouse skeletal a-actin mRNAs (Hu et al., 1986) would make a 409-nt fragment resulting from hybridization with mouse mRNA impossible. Furthermore, this probe gave no signal with mRNA from differentiated rat L6E9 cells (data not shown), which express skeletal a-actin mRNA nearly identical in sequence to mouse skeletal a-actin mRNA from BC,Hl (Hu et al., 1986). Diagrams show the probes and the lengths of the fragments protected from digestion by hybridization with mRNA. U, undifferentiated; D, differentiated. All other symbols are as described for Fig. 2. Cells were induced to differentiate by a switch to low-serum medium.

was collected from cells differentiated by switch to low-serum medium rather than by starvation. Thus, the results from the blot hybridization and S 1 assays taken together demonstrate that the net change in expression from uninduced to induced cells is similar in the two feeding protocols. In addition, the results (Fig. 5A) show that in undifferentiated cells the amount of mRNA expressed from the transferred a5’-83 gene is substantially less than the amount of mRNA expressed from the intact B-actin gene.

(c) Nuclear run-on analysis of the transcriptional activity of endogenous and transfected genes

We next asked whether the differentiation-depen- dent changes in the levels of accumulated actin mes- sage could be attributed to changes in transcriptional activity. We used nuclear run-on assays to measure the relative transcription rates in nuclei of undiffer- entiated and differentiated cells transformed with the different actin gene constructs (Fig. 6). Densitomet-

300

P 0

U D

Neo

ma

ca

P QO

U D

ma

a

ma

CP

P S

U D

Fig. 6. Nuclear run-on analyses of transcriptional activity of transferred genes in undifferentiated and differentiated nuclei. The procedures used were modifications of those used by Konieczny and Emerson (1985). Undifferentiated nuclei were collected after three or four days in growth medium and differentiated nuclei after eight days in differentiation medium, as per the refeeding protocol in MATERIALS AND METHODS, section c. Undifferentiated cells from which nuclei were collected had approximately the same amount of non-muscle actin mRNA as cells collected either the day before or after and had no detectable actin mRNA migrating at the position for muscle specific actin mRNA. The final concentration of nuclei in storage buffer was 6-12 x 10’ nuclei/ml, as determined by light microscopy. Each nuclear run-on reaction was done as described by Konieczny and Emerson (1985). except that 400 PCi [a-32P]UTP

tic analysis showed no evidence of gene-specific, differentiation-dependent changes in transcriptional activity of any of the transferred actin genes. In light

of the fact that the results of RNA blot hybridization analyses for expression from the intact /I and B5’-a3’ genes differed clearly from each other (B down-regu- lated; /35’-~3’ more nearly constitutive overall), it is particularly significant that the transcriptional ac- tivities for both these genes are constitutive. Thus, we can not invoke transcriptional regulation for the differences in regulation of these genes. In contrast, nuclear transcripts from the transferred NeoR gene decreased upon differentiation 1.5-fold or more in all cases tested except for 8,; and the endogenous mouse skeletal muscle actin gene consistently show- ed a six- to ninefold differentiation-dependent in- crease in transcriptional activity. We do not know whether the very faint band of hybridization for skeletal muscle actin in undifferentiated cells repre- sents a small amount of true activity or non-specific binding. No mRNA corresponding to this gene was apparent in analyses of RNA from the same cells used for nuclei collection. The relative levels of actin message accumulation in total RNA collected from cells on the same day as nuclei were collected were in agreement with those reported in RESULTS AND

DISCUSSION, section a. We were unable to study the transcription of the endogenous /I-actin gene because we had no gene-specific probe.

301

(d) Analyses of general cellular trends in tran- scription and in accumulation of mature RNA in B&H1 cells

To interpret our results with the actin genes more accurately and to place them into the context of general cellular activity, we measured relative levels of both total transcription and message accumula- tion in undifferentiated vs. differentiated cells. First, the ratio of total cellular RNA to DNA decreases more than twofold in the course of differentiation (Table I). Second, the fraction of poly(A) + RNA in total cellular RNA is the same in undifferentiated and differentiated cells. We determined this as de- scribed in MATERIALS AND METHODS, section f, and found that the amount of radiolabeled poly(dT) hybridized to 12.5 ng mRNA was essentially identi- cal on all eight days of differentiation. The average dpm/12.5 ng mRNA in the four trials described were 2255 f 542, 2106k 315, 241Ok 316 and 2273 2 269 (n = 8, one sample for each of the eight days of differentiation). There were no differen- tiation-dependent trends. Third, nuclear transcrip- tion rate decreases at least twofold during differen- tiation (Table II), in agreement with the twofold de- crease in ratio of RNA to DNA.

When these factors are taken into account, the differentiation dependent decrease in amount per cell of mature chick fl-actin message is not just five-

were included, fmal concentration of DTT was 1 mM, and incubation was for only 25 min. Isolation of transcript from the reaction required two steps of DNase I digestion, extraction and precipitation to sufficiently reduce viscosity for accuracy in handling. For hybridization, transcripts radiolabeled in vitro were hybridized with 7.5 pg filter-affixed Ml3 ssDNA carrying the complementary inserts designated in Fig. 1 and described further below. The filter-affixed DNA was both calculated and empirically determined to be in excess of transcripts. Nitrocellulose filter paper rectangles, each bearing a complementary ssDNA, were prehybridized overnight in 2 ml hybridization buffer (see below) at the same temperature to be used for hybridization (see below). The solution was replaced with a second 2-ml buffer and l-4 x 10’ dpm transcripts (equal counts in any one assay), and incubation continued for 64 h. Hybridization buffer for mouse skeletal muscle actin nRNA was 1 M NaCl/40 mM Tris pH 7.4/2 mM EDTA/lO x Denhardts/O.S% SDS/O.2 mM Na . pyrophosphate/0.25 mg/ml heparin/2.5 mM ATP/ZO pg/ml each poly(A) and poly(C), and 50 j&ml of fish sperm DNA. For all other nRNAs, buffer was 0.4 M NaCl/20% formamide/ mM Pipes pH 6.6, with everything else the same. After hybridization, all filters were rinsed for several hours in 0.1 x SSC, 0.1 y0 SDS. Filters for mouse skeletal muscle actin and NeoR were rinsed at 60°C the others at 70°C. Fragments cloned into Ml3 complementary to nRNA from transfected genes were the 1150-bp EcoRI-SmaI fragment from pNeo3dRI (Fregien and Davidson, 1986); the 1274-bp 5’ XhoI-EcoRI fragment from the chick /I-actin gene; and the 350-bp 5’ SmaI-Hind111 fragment from pDdeC, a chick skeletal a-actin subclone contributed by C. Ordahl. To probe for endogenous skeletal muscle actin mRNA, we used a previously constructed clone (Hu et al., 1986) containing a 688-bp SmaI fragment from the mouse skeletal muscle actin gene; the fragment includes the first exon and over half the first intron of the gene. Autoradiograms were quantitated by densitometry. U, undifferentiated; D, differentiated. Creek letters above the different sets of filters designate the gene present in the cells from which nuclei were collected. 0 and S are as in Fig. 2. The abbreviation at the left side of each row designates which complementary ssDNA was attached to the filter papers shown in that row: ~5, chick B; ma, mouse a; ca, chick a.

302

TABLE I

The ratio of total RNA to DNA decreases more than twofold during differentiation of BC,Hl cells”

Assay(s) IRNAl~[DNAl

U five days eight days

Change in ratios

U/five days U/eight days

A 260 4.7 3.0 i.8 1.6 2.4 0 and DPA 5.1 3.8 2.0 1.3 2.6

a Alkali-hydrolyzed RNA and hot perchloric acid-hydrolyzed DNA fractions were isolated sequentially from cells kept undifferentiated by refeeding with growth medium for three days and from cells differentiated for either five or eight days by starvation. [DNA] and [RNA] were measured on duplicate samples using absorbance at 260 nm or the diphenylamine (Burton, 1956) and orcinol (Mejbaum, 1939) methods, respectively. U, undifferentiated cells; DPA, diphenylamine; 0, orcinol.

to tenfold, but ten- to 20-fold or more. The nuclear transcriptional activity of the chick /Sactin gene, though not constitutive, decreases only two- to threefold. From these results we conclude that the decrease in amount of chick &actin mRNA upon differentiation cannot be accounted for principally by a decrease in transcriptional activity of the /I-actin gene. In contrast, the decrease in transcriptional ac- tivity noted in the nuclear runons for the NeoR gene could account for the decrease in amount of corre- sponding mRNA.

(e) Conclusions and discussion

(1) We have observed development~y timed, gene-specific down-relation of a transfected chicken fi-actin gene in the course of differentiation of BC,H 1 cells from the myoblast-like phenotype to the muscle cell-like phenotype. This down-regulation

TABLE II

The transcription rate of BC,HI cells decreases more than twofold during differentiation”

Cell

Pool

dpm per nucleus

U D

U/D

2

38 17 2.2

47 14 3.4

Ba, 34 16 2.1

a The total number of TCA-precipitable dpm from a single nu- clear run-on reaction was divided by the number of nuclei per reaction. Each reaction contained l-2.5 x 10’ nuclei (see legend to Fig. 6). U, undifferentiated; D, differentiated. 0 and S are as in Fig. 2.

could not be accounted for at either the level of transcription initiation or of polyadenylation, show- ing that in BC,Hl cells, postranscriptional mecha- nisms play a major role in regulation of transfected p-actin gene expression.

(2) We can make a number of observations re- garding the location of G-acting sequences impor- tant in differential and/or differentiation-dependent expression of the transferred actin genes in our system. The transfected cloned chicken @actin gene contains sequences sufficient for both efficient ex- pression and appropriate down-regulation. Thus, these sequences lie between a point 350 nt 5’ of the tsp and about 1.3 kb 3’ of the polyadenylation site. We can further conclude that the regulation con- ferred by these sequences is gene-specific, because expression of the transferred /I-actin gene was regu- lated differently than that of either the a-actin or the NeoR gene.

Our work strongly implicates regulatory function for sequences in the 3’ half of either the CI or /I gene. Work from other laboratories, using other cell lines,

is consistent with the conclusion that the 83’ end causes down-regulation (DePonti-Zilli et al., 1988; Melloul et al., 1984 and personal communication; Lohse and Arnold, 1988). The work from these labo- ratories does not, however, rule out a regulatory role for the a3’ end.

Our data also provide evidence for the role of 5’ sequences. First, the results with the /I5’-a3’ hybrid, some down-regulation during the early stages of dif- ferentiation, followed by no further don-regulation and sometimes up-regulation, suggest that the 5’ end of the fi-actin gene contains sequences sufficient for signalling initial down-regulation. This suggestion is

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speculative in view of the fact that the three- to sevenfold down-regulation at the level of message

accumulation of j?5’-a3’ did not differ significantly from the two- to fourfold down-regulation of the NeoR gene. It is, however, noteworthy, because at the level of transcription changes were not observed for /Z’-a3’, but they were for NeoR. Furthermore, the fact that in undifferentiated cells accumulation of intact /I mRNA is greater than that of a5’-83’ mRNA strongly suggests that sequences in the 5’ region of one or both of these genes or their tran- scripts are important in determining ‘efficiency’ of expression or accumulation in the undifferentiated state. Our data cannot distinguish between a positive role of /IS’ sequences and a negative role of a5’ sequences. With respect to /IS’ sequences, Fregien and Davidson (1986) have shown that the chick /I-actin promoter has enhancer-like properties. With respect to a5’ sequences, other workers, working with other cell lines and genes from other species, have shown that the 5’ flanking region of the skeletal muscle actin gene contains signals controlling differ- entiation specific up-regulation (Melloul et al., 1984; Grichnik et al., 1986; Muscat and Kedes, 1987). With reference to this point, we do not know why regulation of the intact chicken a-actin gene was inappropriate in our system. The results with the skeletal a gene have been inconsistent from one laboratory to another. Paterson and coworkers saw inefficient, unregulated expression of the chick skele- tal a gene in mouse myogenic cells (Seiler-Tuyns et al., 1984), whereas Yaffe and coworkers reported up-regulation of the chick skeletal a gene in rat myogenic cells (Nude1 et al., 1985). Both Melloul and colleagues and Grichnik and colleagues used homologous systems, the former in rat cells and the latter in chicken cells. Muscat and Kedes (1987) used transient expression in a heterologous system. There appears to be no single explanation for the observed differences.

(3) The results of our transcriptional analyses ditfer from those obtained by one laboratory and are similar to those from another. In contrast to our observations in the BC,Hl line, Paterson and co- workers have reported down-regulation at the tran- scriptional level of a pSV2NE03’ p-chicken actin hybrid in the C2C12 line (DePonti-Zilli et al., 1988). However, the data from Lohse and Arnold (1988), also in the C2C12 line, agree with ours, i.e., no evi-

dence of transcriptional regulation. In conclusion, we suggest that the mechanisms and/or timing of regulation of the same actin gene may differ in ditfer- ent cellular or genetic contexts, and point out that what actually happens in vivo remains, of course, an open question.

ACKNOWLEDGEMENTS

We thank Marie Krempin for invaluable help with the cell culture, Terry Snutch for radiolabeled human ribosomal RNA, and Hermann Lnppert for radio- labeled poly(dT). We thank C. Ordahl, S. Konieczny, B. Bond Matthews, M. Hu, N. Fregien, B. Paterson, H. Arnold, and S. Sharp for helpful discussion.

This work was supported by NIH post-doctoral fellowship No. 5 F32 GM08634-03 to S.B.S., by NIH grant GM55432 to N.D., and by an award from the School of Natural and Social Sciences at CSULA to S.B.S.

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