nonconservative utilization of aldolase a alternative ...nonconservative utilization of aldolase a...

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THE JOURNAL. OF BIOLOGICAL CHEMISTRY Vol. 265, No. 20, Issue of July 15, pp. 11773-11782,199O 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in L’. S. A. Nonconservative Utilization of Aldolase A Alternative Promoters* (Received for publication, January 10, 1990) Jimmy K. Stauffer, Melissa C. Colbert, and Elena Ciejek-Baez From the Department of Biochemistry, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 Recently, analysis of the sequence and expression of the human aldolase A gene revealed the unique ar- rangement of three tandem promoters and exons pre- ceding a common coding sequence. A muscle-specific promoter (M) and two flanking widely used promoters (N and H) produce mRNA species which, in their ma- ture forms, differ only in the sequence of their 5’- untranslated regions. We have isolated and investi- gated the expression of a mouse aldolase A gene. This mouse gene represents a functional gene by sequence analysis, recombinational screening, and by transfec- tion into C&z cells. Although there is a high degree of sequence similarity between the mouse and the human gene in the region of the alternative first exons, we have been unable to detect a functional utilization of the 5’-most promoter (N) in the mouse. Steady state mRNAs isolated from a variety of adult tissues and cultured cells were analyzed by RNase protection and primer extension to identify first exon utilization. Con- sistent with previous reports, exon M is found only in skeletal muscle and exon H, the “housekeeping” exon, is utilized in every tissue where aldolase A is ex- pressed. Under identical conditions we fail to see any evidence of the N exon. Therefore, although sequence homology exists between rodents and primates in the N region, the absence of selective pressure to preserve its primate pattern of expression may have resulted in functional promoter extinction. Aldolase is a glycolytic enzyme whose function is indispen- sable to normal cellular metabolism. The aldolase isoenzymes, A, B, and C, and their respective mRNAs are each uniquely distributed in specific tissues in a strict developmentally programmed fashion (for a review see Ref. 41). In every tissue of the developing embryo, A and C are the only forms present (1, 27). Maturation of fetus to adult is accompanied by the establishment of tissue-specific expression of each isoenzyme. Aldolase B is found in the liver, kidney, and small intestine (34), and aldolase C is restricted to the tissues of the brain and central nervous system while aldolase A retains a some- what ubiquitous distribution. In addition, the A isoenzyme is exceptionally abundant in adult muscle where it constitutes over 5% of the soluble protein content (26). The mammalian isoenzymes are encoded by three evolutionarily related but unlinked genes (45). These genes have nearly identical intron- exon arrangements within and among species. The interspe- * This research was supported by Grant 5-560 from the March of Dimes. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in thispaper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) JO551 7. ties conservation of the protein coding sequence for each isoenzyme is also quite remarkable, particularly for aldolase A (22). Aldolase A, unlike aldolases B and C, exhibits an additional level of complexity in regulating its expression during devel- opment and differentiation. Multiple mRNA species for al- dolase A have been reported either in different tissues or in the same tissue at different times during development. Se- quence differences among these mRNAs occur only in their 5’-untranslated region. Subsequent sequence analysis of a rat aldolase A genomic clone revealed the presence of tandem promoters and exons whose alternative usage could account for the observed rat aldolase A cDNAs (20). The most 5’ promoter, promoter M, directs transcription of an exon se- quence found only in muscle tissue while the more 3’ or proximal promoter, promoter H, directs transcription of an alternate 5’ exon in all tissues and developmental stages (Fig. 1). Activation of the M promoter occurs during fetal myogen- esisand, as we have shown previously, an analogous activation of the muscle-specific promoter occurs during myogenesis of CZCIZ cells in culture (8). Surprisingly, analysis of the human aldolase A gene re- vealed the presence of a third promoter, promoter N, just. upstream of the muscle-specific promoter (Fig. 1) (18, 32). This promoter is utilized in a similar fashion to that of the most 3’ proximal promoter, H, but is much less active, pro- ducing lower amounts of steady state mRNA. Thus, the resultant human aldolase A promoter region consists of a muscle-specific promoter flanked by two relatively nonspecific or “housekeeping” promoters all clustered within 1.6 kbp’ of one another. In the present study we have isolated and analyzed the transcription of the mouse aldolase A gene. By utilizing two different screening regimens we have rigorously selected and verified the identity of the mouse aldolase A gene. The func- tionality of the putative promoter region was demonstrated by its ability to direct expression of a reporter gene in culture. Sequence comparisons show a high degree of similarity be- tween the mouse gene and the rat gene and somewhat less between the mouse and human aldolase A sequences. Expres- sion of the mouse A gene was found to be identical to the rat in the various mouse tissues assayed; however, no mRNAs were detected produced from the N promoter as seen in the human. This suggests that although sequence homology exists between rodents and primates in this particular promoter region, necessary selective pressures for conservation of expression could be absent. This, therefore, may be the first example of promoter extinction between two closely related species. 1 The abbreviations used are: kbp(s), kilobase pair(s); bp, base pair(s); SDS, sodium dodecyl sulfate; DTT, dithiothreitol; nt, nucleo- tide. 11773 by guest on May 10, 2020 http://www.jbc.org/ Downloaded from

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Page 1: Nonconservative Utilization of Aldolase A Alternative ...Nonconservative Utilization of Aldolase A Alternative Promoters* (Received for publication, January 10, 1990) Jimmy K. Stauffer,

THE JOURNAL. OF BIOLOGICAL CHEMISTRY Vol. 265, No. 20, Issue of July 15, pp. 11773-11782,199O 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in L’. S. A.

Nonconservative Utilization of Aldolase A Alternative Promoters*

(Received for publication, January 10, 1990)

Jimmy K. Stauffer, Melissa C. Colbert, and Elena Ciejek-Baez From the Department of Biochemistry, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

Recently, analysis of the sequence and expression of the human aldolase A gene revealed the unique ar- rangement of three tandem promoters and exons pre- ceding a common coding sequence. A muscle-specific promoter (M) and two flanking widely used promoters (N and H) produce mRNA species which, in their ma- ture forms, differ only in the sequence of their 5’- untranslated regions. We have isolated and investi- gated the expression of a mouse aldolase A gene. This mouse gene represents a functional gene by sequence analysis, recombinational screening, and by transfec- tion into C&z cells. Although there is a high degree of sequence similarity between the mouse and the human gene in the region of the alternative first exons, we have been unable to detect a functional utilization of the 5’-most promoter (N) in the mouse. Steady state mRNAs isolated from a variety of adult tissues and cultured cells were analyzed by RNase protection and primer extension to identify first exon utilization. Con- sistent with previous reports, exon M is found only in skeletal muscle and exon H, the “housekeeping” exon, is utilized in every tissue where aldolase A is ex- pressed. Under identical conditions we fail to see any evidence of the N exon. Therefore, although sequence homology exists between rodents and primates in the N region, the absence of selective pressure to preserve its primate pattern of expression may have resulted in functional promoter extinction.

Aldolase is a glycolytic enzyme whose function is indispen- sable to normal cellular metabolism. The aldolase isoenzymes, A, B, and C, and their respective mRNAs are each uniquely distributed in specific tissues in a strict developmentally programmed fashion (for a review see Ref. 41). In every tissue of the developing embryo, A and C are the only forms present (1, 27). Maturation of fetus to adult is accompanied by the establishment of tissue-specific expression of each isoenzyme. Aldolase B is found in the liver, kidney, and small intestine (34), and aldolase C is restricted to the tissues of the brain and central nervous system while aldolase A retains a some- what ubiquitous distribution. In addition, the A isoenzyme is exceptionally abundant in adult muscle where it constitutes over 5% of the soluble protein content (26). The mammalian isoenzymes are encoded by three evolutionarily related but unlinked genes (45). These genes have nearly identical intron- exon arrangements within and among species. The interspe-

* This research was supported by Grant 5-560 from the March of Dimes. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in thispaper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) JO551 7.

ties conservation of the protein coding sequence for each isoenzyme is also quite remarkable, particularly for aldolase A (22).

Aldolase A, unlike aldolases B and C, exhibits an additional level of complexity in regulating its expression during devel- opment and differentiation. Multiple mRNA species for al- dolase A have been reported either in different tissues or in the same tissue at different times during development. Se- quence differences among these mRNAs occur only in their 5’-untranslated region. Subsequent sequence analysis of a rat aldolase A genomic clone revealed the presence of tandem promoters and exons whose alternative usage could account for the observed rat aldolase A cDNAs (20). The most 5’ promoter, promoter M, directs transcription of an exon se- quence found only in muscle tissue while the more 3’ or proximal promoter, promoter H, directs transcription of an alternate 5’ exon in all tissues and developmental stages (Fig. 1). Activation of the M promoter occurs during fetal myogen- esis and, as we have shown previously, an analogous activation of the muscle-specific promoter occurs during myogenesis of CZCIZ cells in culture (8).

Surprisingly, analysis of the human aldolase A gene re- vealed the presence of a third promoter, promoter N, just. upstream of the muscle-specific promoter (Fig. 1) (18, 32). This promoter is utilized in a similar fashion to that of the most 3’ proximal promoter, H, but is much less active, pro- ducing lower amounts of steady state mRNA. Thus, the resultant human aldolase A promoter region consists of a muscle-specific promoter flanked by two relatively nonspecific or “housekeeping” promoters all clustered within 1.6 kbp’ of one another.

In the present study we have isolated and analyzed the transcription of the mouse aldolase A gene. By utilizing two different screening regimens we have rigorously selected and verified the identity of the mouse aldolase A gene. The func- tionality of the putative promoter region was demonstrated by its ability to direct expression of a reporter gene in culture. Sequence comparisons show a high degree of similarity be- tween the mouse gene and the rat gene and somewhat less between the mouse and human aldolase A sequences. Expres- sion of the mouse A gene was found to be identical to the rat in the various mouse tissues assayed; however, no mRNAs were detected produced from the N promoter as seen in the human. This suggests that although sequence homology exists between rodents and primates in this particular promoter region, necessary selective pressures for conservation of expression could be absent. This, therefore, may be the first example of promoter extinction between two closely related species.

1 The abbreviations used are: kbp(s), kilobase pair(s); bp, base pair(s); SDS, sodium dodecyl sulfate; DTT, dithiothreitol; nt, nucleo- tide.

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1 MOUSf2 aatTacCTgg

Rat . . . . . . . . . . HUllaIl . ..TggCTcc

CO~S~IISUS ---T--(-T--

101 "TATA" 150 ttCTTCCtCT CC.Cttccct gataaaCTAT TgtatGTgaG GtaGgatcga GaCatTGCTC ACC.CAggCA . . . . . . . . . . . . . ..cccgg gatgggCTAT TgaggGTgaG GatG... tag GaCatTGCTC ACC.CAggCA ccCTTCCcCT CCaCacgtca acgattCTAT TtgaaGTtgG GcaGgggggt GgCgcTGCTC ACCaCAcaCA -- - CT G-C---- ------CTAT T----GT--G G--G------ G-C--TGCTC ACC-q--CA

MOUSC? Rat

Human COnSenSUS

MOUS.2 Rat

HUllIan consensus

301 350 400 AA . . . . . . . . . . . . ..AAaa AGttCAaCCA CCagCAggca ccaGAGTCAA GGGAGGAG.. . . . . . . . . . . .GGAaCCAGa GgagGgcagt GGGAGGCaat AA . . . . . . . . . . . . ..AAaa AGtaCAaCCA CCggCAgaca c..GAGTCAA GGGAGGAG.. . . . . . . . ..~ .GGAcCCAGc Gga.Gtcagt GGGAGGCagc AAccagggct ccagagAAtc AGaaCAgCCA CCatCAccgc aggGAGTCAA GGGAGGAGgg agattagaga aGGAgCCAGg GaggGtggca GGGAGGCcac J#-------- ------mm- AG--(-A-CCA CC--CA---- ---aGTC&Z, GG(YjAGGAG-- ___------_ -aA-CCAG- G---G----- (T&Q+aC---

M0U.W Rat

Human COWSXlSUS

401 450 Exon N2 500 aTctagatgt TttCCcttCt tgTTctgcct aTctagatgt TttCCcttCt tgTTctgcct gTgatccgag TccCCtcaCc ccTTtccttc -T-------w T--CC---C- --TT ______

MOUS-2 Rat

Human COIX3enSUS

Mouse Rat

Human Consensus

601 650 700 GgaTGAATGT CAGGaGCcTC AGGTTTCcCT AAATATAGGT CCcgGCCgcg GGATtCGTGG tGGGgAAAGG GCAGgGGTtA .ccgagAaGg tCtGGGACAC GgaTGAATGT CAGGgGCcTc AGGTTTCaCT AAATATAGGT CCttGCCgcg GGATtCGTGG tGGGgAAAGG GCAGaGGTtA tggagaAgGt CcgGGGACAC GtgTGAATGT CAGGgGCtTC AGGTTTCcCT AAATATAGGT CCctGCCaga GGATcCGTGG cGGGaAAAGG GCAGgGGTcA ttagagAaGa tCgGGGACAC G--jXAATGT CAGG-GC-TC AGGTTTC-CT AAATATAGGT CC--GCC--- GGAT-CGTGG -G&Z-AAAGG GCAG-GGT-A ------A-G- -C-GGGXAC

MOUSC Rat

Human Consensus

701 "TATA" Exon M 800 GGTGcGGgG Gtgtgtwgg gaggggtggg gaGtAGGAGC TGCCTTAaAA cCCAGCCCtG Ga+i'WwG CTcac~~~i:~~~~~.:::~~~~EXT tgGTGcGGgG Gtgcgtaggg gaggggtgga gaGtAGGAGC TGCCTTAtAA cCCAGCCCtG GgccgCgggG CTCAC~~:'i&Q~~~~:..~Z~IX~GCT atGTGgGGcG G......... . . . . . ...*. ..GcAGGAGC TGCCTTAtAA .CCAGCCCgG GaaccCctaG CTCZCTC~.~~$~~~~~&G~f --GTG-G&G G--------- ______--__ --G-AGGAGC TGCCTTA-W -C~~CC-G (+---C---G cTcP;CT~~~;~~Cc~~.P~~~T

801 850 900 't.$$~~~:~~~~~~GT GGGCCgCTat CcGGAttG.. .caGGaTGGG aATGGGGgtT gcGGaTTgGG aCctgaGGaa aCtGa.ctGc TCtgagaGtt . . . . . ~~~~~~~~.;~~~~~~GT GGGCCgCTag CtGGAttG.. .gaGGaTGGG aATGGGGgtT gcGGaTTcGG gCctg.GGga aCtG.gatGc TCtgaaaGca ~~C!@j&$$~~~~~GT GGGCCcCTtg CaGGAccGgg ccgGGgTffiG gATGGGG.. T tgCGgTTgC% cCaacaGGqt cCaGatggGg TCcagghGag *. .r+? (-c-q-- C-Ga--G-- ---GG-pT&G -ATG(gg+-T --GG-TT-GG -c----g+- -C-G----G- TC-----G--

Mouse Rat

Human Consensus

901 950 1000 acaGGGtGac aagagaGcTc cGAGacgGa. . . .._..... . . . . . . . . . . . . . . . . . . . . . . ..tttTTT TaTTTTGGAG AAGGAAaTCA GGtTCggGaA acaGGGtG.. . atagaGcTc cGAGacaGa. . . . . . . . . . . . . . . . ..ttc tttttttttt ttttt.ttTTT TtTTTTGGAG AAGGAAaTCA GGtTCggGaA gagGGGaGat ttggacGaTa gGAGcagGgg gctcagcatc tgggaggcag atcagttcgg ggacggaTTT TcTTTTGGAG AAGGAAgTCA GGcTCaaGgA ---GGG-G-- ------G-T- -GAG---G-- ___-----__ ___-______ __________ -------pi T-TTTTGaG A&GGm.-TCA GG-TC--G-8

MOUSS Rat

Human Consensus

1001 1500 1550 AGACctgTct . . . ...//--) Approx. {-----//... tCgccaacCc ACATTCcGGC 'IGAGTCAC.. atgttccGcG CGCgccaGgC AggGgtTGGG AGACcggTct . . . ..//---I 480 bp. (----//.... tCgccaatCc ACATTCcGGC TGAGTCACga tgctcgcGcG CGCgccaGaC AggGacTGGG AGACgttTgg . . ..//----) consensust---//..... cCcattgcCa ACATTCtGGC TGAGTCACgg cgccccaGaG CGC....GcC A..GgcTGGG u---T-- ---,,-----)C-------->(--//--,,------ -C------C- ACATTC-G- TaG,'CAC-- -------G-G Cm----G-C A--G--TCXG

Mouse Rat

Human COIlSWlSUS

1551 1600 GGGCGG 1650 m%xxmootn ttooMhnd GoooTmXGA CCtGcGGGAT GTGGctCGAG TCACGTCCtA GcGGGGcGGa GGAGGGATCg tGTTCTaGcC GCttGtCtCC

-GA CCtGaGGGAT GTGGctCGAG TCACGTCCtA GcGGGGcGGa GGAGGGATCt aGTTCTaGcC GCttGtCtCC .----_ -;I---=-_ ;GoaaG GcccTaGcGA CCCGCGGGAT GTGGtcCGAG TCACGTCCqA GCIGGGGtGG. GGAGGGATCq tGTTCTcW GCccG.CcCC ~~~~ ~~ ~~ ~~, ~ ~~

GG-------- ----GG---G G---T-G-GA CC-G-GGGAT GTGG--CGAG TCACGTCC-A G-GGGG-GG- GGAGGGATC: -GTTCT-G-C GC--G-C-CC

MOUSe Rat

Human Consensus

1651 GGGCGG 1700 CAATT 1750 TcCCcAGtG. . ..CcgCCTC ctatCggagC atcttGGGGC GGt.CtgcGC aCAgTgC.cC aCcTtCaATT .GACGgttCc CGtcCCT..g cAAGGgaAAA TcCCcAGcG. . ..CccCCTC ctatCgtagC atcttGGGGC GGtgCcgcGC aCAaTgC.cC gCtTgCaATT gGACGgctCg ( :GtcCCT. .g cAAGGq.AAA TtCCtAGcG. . ..CggCCTC tgggCtggcC tctcgGGGGC GGccCgtaGC cCAgTcCgtC 9( :cTgCcATT gGACGccgCc CGctCCTcgt aAAGGadAA T-CC-AG-G- ---C--CCTC ---C--C ----aGGC G&-C---GC : -CA-T-C--C -C-T-C-ATT -GACG---C- CG--CCT--- -AAGG--AU

1751 GGGCGG "TATA" 1800 "TATA" 1850 . . ., .,. aacCtGCaGA GGGCGGaGc. . ..GGcGcCT TTAAAtGtCC GGGgcCCcgC cTCCg..... ._........ . pj q$qzpz. a*&$@@$ q$&$$qz.qg aacCtGCaGA GGGCGGgGc. . ..GGcG.CT TTAAAtGtCC GGGgcCCcgC cTCC...... .*........ cG~~~C~c+. :+$&$@%@&~~~~~ gctCgGCgGA GGGCGGaG.. ..tGGtGcCT TTAAAaG$C GGGcgCCgcC tTCCgcctgc ccgcctcctg c~~~~c~,~rCg~gfiCTa,tA'Pc~TO~ ---C-GC-~ G~CGG-(+- ---G&G-CT TT--G-CC GGG--CC--C -TCC------ _L-----___ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

Mouse Rat

HlJiVll-l COI-lSC?IlSUS

Nonconservation of Aldolase A Alternative Promoters

50 100 gttccttgAG ttCTCTgaaa gccagGattc tgAGagCCct TGGAccGCtg aAA.....AG GGcCTgaTGC TCtGGCcaGT GCCCctGCcT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..,....... . . . . . . . . . .

tgcagggcAG gaCTCTtggc cGgctGgacg gcAGctCCtc TGGAggGCca gAAaagagAG -------.wAG --CTCT---- -G---G---- --AG--CC-- TG@+--c-- -m-----AG

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GGgCTagTGC TCgGGCagGT GCCCtgGCtT GG-CT--TGC TC-GGC--GT GCCC--GC-T

" TA TA" 200 AcaG.TgTgG GAGGttTCTG cCaacct$i AcaG.TgTgG GAGGgcTCTG .Caacctq@ AgtGtTaTaG GAGGagTCTG $cctt&+ A--G-T-T-G GAGG--TCTG -C----+%4

300 cTGcCtGacT CTgCTCTCCT TtggaaaaAA cTGtCtGccT CTgCTCTCCT Tt......AA gTGcCaGtaT CTaCTCTCCT TcttaaaaAA

550 600 GAGTGagCAg tGGCacctcC tCC....... . . . . ..tCtC aAggCaaaCc aaagCTgCCt Cttcttcacc CCCcAcGcag

aaCAt tGGCa...cC tCC....... . . . . ..tCtC cAagCaaaCc taagCTtCCt Cttcttaccc CCCtAgGcag GAGTGggCAg gGGCtccctC CCCatcaata gggccgaCcC aAgtCttcCt ccccCTtCCc Ccatgccggg CCCcAcGata

--CA- -GC---m-C -CC------- -------C-C -A--C---C- ----CT-CC- C--------a CCC-A-G---

FIG. l--Left Panel

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Nonconservation of Aldolase A Alternative Promoters

promoter N Transcrbtion:

Promoter M TranscriDtion:

Promoter H Transcridion:

I 500 Bp.

FIG. 1. Generation of alternative transcripts from the aldolase A gene and the consensus sequence of the mammalian aldolase A promoter region. The left panel shows the consensus sequence of the mammalian aldolase A promoter region. Exons NL, NZ, M, and H are shaded and labeled accordingly. Double underlined sequences represent contiguous stretches of ~10 bp having 290% identity. Single underlined sequences show extensions of the double underlined domains only when contiguous regions of 237 bp with 279% identity can be formed. “TATA,” “GGGCGG,” and “CAATT” boxes are indicated as in Fig. 5 (see Miniprint Supplement). Upper case represents bases that are identical at a given position in all sequences compared. Dots indicate gaps, and dashes represent nonconserved bases in the consensus sequence. The figure in the right panel is drawn to scale. The boxes represent exons; stippled boxes (2-9) represent coding region exons. Angled arrows indicate transcriptional start sites, and angled lines show splicing patterns. Pertinent splice donor/acceptor site sequences are shown. Below each promoter-specified transcription unit is diagrammed the respective mature mRNA products. The open boxes represent alternative exon 1 sequences.

MATERIALS AND METHODS AND RESULTS*

Isolation of the Aldolase A Genes-As a primary step in analyzing the genomic structure of aldolase A in the mouse, we examined the hybridization pattern of mouse genomic DNA to a mouse brain aldolase A cDNA. Genomic DNA was digested with BumHI, EcoRI, and Hind111 and then hybridized to either an aldolase A or B cDNA clone as shown in Fig. 2 (see Miniprint Supplement). The aldolase B probe showed hybridization to single bands in each enzyme digest, consist- ent with the presence of that gene as a single copy as it is found within the human (46), rat (48), and chicken (6) ge- nomes. However, the same digests hybridized to an aldolase A cDNA (720-bp PstI coding region fragment) resulted in a much more complex pattern of bands indicative of multiple

’ Portions of this paper (including “Materials and Methods,” part of “Results,” part of “Discussion,” and Figs. 2-5) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

copies of aldolase A or aldolase A-like sequences. Identical results were obtained by repeating the analysis using exon 3- and exon g-specific probes (data not shown) suggesting a repeat is not associated with any particular region of the aldolase A cDNA.

The finding of multiple aldolase A-like sequences within the mouse genome is consistent with reports of multiple pseudogenes both in rat (20) and human (45). Therefore, some bands may be due to pseudogenes for mouse aldolase A. Nevertheless, the possibility still exists that mouse aldolase A is encoded by more than one gene, each with its own unique first exon, as opposed to being encoded by a single gene capable of producing all anticipated mRNA variants. In order to distinguish among these possibilities, a comprehensive approach to cloning the mouse aldolase A gene(s) was taken.

The possibility of multiple genes was explored by using ho.mologous recombination in vivo to preferentially select only those clones most similar to coding region probes (11, 23, 25, 43,47). A mouse genomic library was constructed in the vector Syrinx 2A and was allowed to undergo recombination between

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11776 Nonconservation of Aldolase A Alternative Promoters

TABLE I Hybridization patterns of homologous recombination clones with

exon-specific aldolase A probes

Recombination Number of clones showing hybridization with of genomic clone with Total Exon 3 Exon 9 No

probe plasmid Exon 3 Exon 9 hybridization hearing

only On” (background)

Exon 3 72 63 26 37 0 9 Exon 9 72 26 62 0 36 10

Totals 144 89 88 37 36 19

probe plasmids bearing exons 3 or 9. Recombinants were selected for on the proper hosts, and 72 plaques from each trial were picked and analyzed by hybridization to each probe. The results are shown in Table I. We further selected for clones that were most likely to span the entire gene by examining only those clones hybridizing to both exons 3 and 9. Forty-four were restriction-digested, and six sisters were identified. Three sisters predominated, constituting 34 of 44 cases. All three were found to have overlapping restriction maps which proved to encompass the entire aldolase A gene (Fig. 3A, Miniprint Supplement).

To assess the possibility of a single unique sequence, 70 aldolase A clones, isolated independently by hybridization to a mouse brain aldolase A cDNA probe, were screened with oligonucleotides specific to the optional exons N, M, and H (Fig. 3C, Miniprint Supplement). One clone, X16, showing hybridization to all three oligonucleotide probes was selected for further analysis. This clone proved identical by restriction mapping to the most frequently retrieved clones obtained by homologous recombination (Fig. 3A, Miniprint Supplement). In an effort to show these cloned sequences were representa- tive of a single locus, an internal restriction fragment corre- sponding to the intronic region between exons M and H was isolated and used as a hybridization probe against genomic DNA digested with EcoRI, HindIII, X&I, or SacI. In addition it was also hybridized against the three most prevalent recom- bination clones to further substantiate identity. As can be seen in Fig. 4 (Miniprint Supplement), a single genomic DNA fragment was identified in each digest, and, furthermore, the sizes of the Sac1 and Hind111 genomic fragments correspond exactly to the fragments produced from the recombinant clones. Therefore, we conclude that the aldolase A sequences cloned represent the genomic sequences most similar to a mouse brain aldolase A cDNA and contain all sequence infor- mation needed to encode anticipated alternate 5’ exons. Most importantly, these cloned sequences all overlap an identical single copy sequence.

N-specific Sequences Are Not Detected in a Variety of Mouse Tissues-In order to determine first exon utilization in steady state mRNA populations in the mouse, and in particular to look for the presence of the N exonic sequences, both RNase protections and primer extension analysis were performed. Initially, genomic fragments representing alternative exon sequences were subcloned into vectors containing T3 and T7 bacteriophage promoters such that uniformly labeled cRNA probes could be generated. These subclones, designated pAN2, PAM, and pAB, their relative lengths, positions in the ge- nomic clone, and the sizes of their anticipated protected fragments are shown in Fig. 6. A variety of mouse tissues was selected for analysis including those which correspond to tissues in the human where N was shown to be expressed at the highest levels, such as adult spleen and muscle. Mouse liver and mRNA from cultured hepatoma cells were also included as this was the initial site where N transcripts were first described in the human (40).

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a I 2 .l,LL7X ‘, IO I , I? 1.1 I4 1; 16 17 I S I’, ?I,

FIG. 6. First exon utilization in RNAs isolated from various mouse tissues. A, diagram of the 5’-noncoding alternative first exons of the aldolase A gene, the exon-specific subclones used to generate antisense RNA for RNase protection, and the resulting spliced mRNAs. Exons are indicated as open boxes on the first line (DNA) and at the bottom of the figure (RNA). Plasmid pAN2 is the probe for exon Nn and should yield a protected fragment (thick black lines) of 71 nucleotides. Probe PAM protects a 45-nucleotide fragment from exon M, and pAB protects two fragments of 141 and 94 nucleotides resulting from the two start sites of exon H. pAN2.:~, is a truncated version of pAN2 which is transcribed as unlabeled sense RNA and serves as a control. Protections of PAN~.~~ with pAN2 yield protected fragments of 130 nucleotides. B, RNase protection of mRNAs isolated from various mouse tissues. Analysis of RNAs isolated from brain (30 pg, lanes I, 9, and 14), muscle (20 pg, lanes 2, 10, and Z5), spleen (50 Kg, lanes 3, 1 I, and 16), hepatoma, BWTGS cells (30 /*g, lanes 4 and Z7), liver (50 pg, lanes 5 and Z8), or yeast (50 rg, lanes 6, 12, and 19) using pAN2 (lanes l-7), PAM (lanes g-12), and pAB (lanes I4- 19) as probes. Lane PAN?.~~ is an in uitro-generated sense RNA (10 ng) control for PAN? protection (lane 7). Undigested PAN,, PAM, and pAB are seen in lanes 8, 13, and 20, respectively. Lane M is molecular weight markers.

The H exon was specifically protected when hybridized to probe pAB and produced two sets of protected fragments in almost all tissues where the H exon is expressed (Fig. 6). This corresponds to the two transcriptional start sites, approxi-

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Nonconservation of Aldolase A Alternative Promoters 11777

mately 50 bp apart, previously identified by us in the mouse (8) and also seen in the rat (20) and human (18, 32). Bands of the greatest intensity were seen in mRNAs isolated from the brain (lane 14) and hepatoma cell line BWTG3 (44) (lane 17). Lower levels of exon H were detected in muscle (lane 15) and spleen (lane 16). Interestingly, we see in muscle a greater relative abundance of the second protected fragment size suggesting more frequent utilization of the second start within the H exon. This confirms our previous observation that a small portion of transcripts in adult mouse do indeed arise from the H promoter and use this second cap site (8). There also appears to be a preferential utilization of the 5’ start site in hepatoma in contrast to brain, where both starts are roughly equivalent. We were unable to detect the expression of H exon sequences by RNase protection using 50 pg of adult mouse liver (lane 18) where aldolase B is normally expressed. This corroborates Northern blot analysis which also fails to detect aldolase A mRNAs in adult mouse liver.”

In contrast with exon H, hybridization of mRNAs to probe PAM detected exon M only in muscle tissue (see lanes 9-13) as would be anticipated. From these results we conclude that our assay conditions, the probes, and the integrity of our RNA adequately represented the distribution of exons H and M. Conservation of this distribution of these sequences in the mouse was maintained as compared with the rat and human. However, such is not the case for expression of the N exon. Concurrent RNase protections utilizing aliquots of the same mRNA preparations and probe PAN, did not show protected fragments in either adult spleen (lane 3) or muscle (lane 2), where in similar amounts of mRNA, this exon was detected in the human. Furthermore, we failed to detect the presence of this sequence in adult brain (lane 1) or liver (lane 5) or in the mouse hepatoma cell line BWTG3 (lane 4). Additionally we have examined mRNA from fetal muscle and the mouse myoblastic cell line CC,, and again saw no evidence of any expression of the N exon (data not shown). In order to verify the competency of our probe pANZ to detect N exon sequences, we prepared an additional genomic subclone, designated PAN?.:~;, which included the last 37 nucleotides of the Nr exon along with the 3’-flanking sequence. In vitro-generated unla- beled mRNAs from PAN~.:~~ were hybridized to labeled pANr cRNAs as a control for RNase protection. The protection product, as seen in lane 7, was the appropriate size and was clearly detectable. In addition, titration studies (not shown) with similarly generated in vitro RNA from PAN~.:~; demon- strate that we can successfully detect a 1000-fold lower level of N-type transcripts than that shown in Fig. 6, lane 7. The above RNase protection experiments were repeated using a probe spanning exon N1 (data not presented). The findings corroborated the results obtained using probe PAN, and elim- inated alternative splicing as the determinant of N, absence.

Two different oligonucleotides were utilized for the primer extension assays to again check for the existence of N-specific exons within steady state mRNA. The first, IIeS, was designed to hybridize to sequences located downstream of the AUG sequence in exon 2 (Fig. 7) and thus should hybridize to any aldolase A mRNA irrespective of first exons. Using RNA from spleen, brain, and muscle as seen in lanes l-4 of Fig. 7, extension products from primer IIZR were detected in each tissue. Two start sites within exon H (extension products of 210 and 172 nucleotides long) were seen with spleen and brain mRNA confirming the RNase protection data. Although the levels of aldolase A were low in spleen, longer exposure of the autoradiographs (compare lanes 1 and 2) show the character-

“d. K. Stauffer, M. C. Colbert, and E. Ciejek-Baez, unpublished results.

B

210 - 172 -

1.37 - 119 -

FIG. 7. Identification of 5’ termini of mouse HNAs by primer extension. A, diagram of anticipated primer extension prod- ucts. open boxes represent mRNAs with numbered exons as indicated. Primers II,i or NXs hybridize to sequences within exons as indicated and are depicted as thick black lines. Their size, in nucleotides, is indicated as a subscript. Thin black lines represent extension prod- ucts, and the total extension size, in nucleotides, is indicated below the line. Numbers contained within the parenheses for the N, and Nz exons indicate the expected product size. R, analysis of extension products of ‘“P-end-labeled primers is also shown. II,i (lanes l-4) or Npg (lanes 6-9) hybridized to 2 pg of muscle (lanes 4 and 6), 5 pg of brain (lanes 3 and 7), or 5 fig of spleen (lanes 1, 2, and 8) poly(A)+ RNA. Lane pANY is in uitro-generated sense RNA (10 ng) used as a control for extension with primer Nz!,. Fragment sizes, indicated at the left, are given in bases. Markers (M) are in lanes 5 and 10.

istic double start sites. An additional extension product of 137 nucleotides was seen in the brain. We have not found that band to be consistently reproducible (see Ref. 8) and attribute it to “stuttering” of the reverse transcriptase caused by extensive secondary structure in the 5’-untranslated region of the mRNA. mRNAs isolated from muscle resulted in an extension product of 119 nucleotides, indicative of utilization

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11778 Nonconservation of Aldolase A Alternative Promoters

of the M exon. Although we detect small amounts of H exon- specific sequences with much longer exposures as seen previ- ously (not shown, Ref. 8), the major aldolase A mRNA species in muscle is associated with the M exon.

A second oligonucleotide, NZ9, was also hybridized to sam- ples of the same RNA preparations as IIzs. This oligonucleo- tide, previously utilized in our initial screening of X genomic clones to pick out those with homology to the N exons of the human gene, is specific for hybridization to exon NP. Consist- ent with our RNase protections, we see no extension products with any of the tissue RNAs (Fig. 7, lanes 6-8). As a further control for the competency of the NZ9 primer to hybridize to N-specific sequences, we synthesized exon NP mRNA in vitro from the PAN:! subclone containing the mouse NB exon. This mRNA gave an appropriately sized extension product (lane 9), demonstrating that the primer was both capable of hybrid- izing to N-specific sequences and of being extended.

From the above results we concluded that the N exon is not expressed in the mouse either in similar tissues or at similar levels as in the human. In fact, we have yet to detect expression of these sequences in any mouse tissues, cell lines, or at any time during development.

The Cloned Aldolase A Gene Is Transcriptionally Func- tional-In order to demonstrate that we had indeed cloned a functional gene, the putative promoter regions were subcloned into pBLCAT3 (30). As shown in Fig. 8, the construct 7K82CAT contained 7 kbp of the aldolase A sequence begin- ning 2 bp upstream of the ATG initiation codon in exon 2 and ending 5 kbp upstream of exon M. This construct allows any splice initiated by M, H, or putative N exons to be completed to the common splice acceptor found in exon 2. Additionally, an “H-less” construct was also created by delet- ing a region spanning from 195 bp 5’ to 500 bp 3’ of the H exon thus deleting the H exon and all proximal promoter elements including three “GC” boxes, one “CAATT” box, and all H-associated “TATA” boxes (12, 13, 21). The resultant construct contained 5 kbp of the 5’ exon M flanking region, exon M, a l.l-kbp hybrid intron, and 21 bp of exon 2 allowing splicing of exon M to exon 2. The myogenic cell line C&i2 was chosen as we have previously shown that both promoters are functional in these cells with the M promoter specifically activated as differentiation occurs (8).

Transient transfections were timed to coincide with myo- blast withdrawal from the cell cycle, just as myotube differ- entiation was beginning. As shown in Fig. 8, significant amounts of CAT activity were seen in all extracts at 48 h. The construct 7K82CAT was more active than 7KAH82CAT indicating promoter H is more active than promoter M at this stage. By 96 h, while SVSCAT (16) activity had fallen, 7K82CAT remained constant. Additionally an induction in

FIG. 8. Expression of aldolase A constructs during transient transfection into C#Zls cells. Constructs are schematized with open boxes representing exons as labeled from the mouse aldolase A gene and pBLCAT3. Arrows indicate cap sites, and angled lines show anticipated splicing patterns. Percent conversion was calculated as follows: {(cpm acetylated [“Clchloramphenicol (AC-CM))/(total cpm [“Clchloramphenicol (CM))] X 100 as excised from the thin layer chromatogram pictured to the right.

the activity of 7KAH82CAT was detected, possibly due to enhanced promoter M activity. This suggests the higher 7K82CAT activity may be the product of both “residual” promoter H activity and induced promoter M activity. These data demonstrate both that the cloned aldolase A promoters are transcriptionally active and that promoter M can function independently of promoter H.

DISCUSSION*

The aldolase A gene serves as an excellent system from which much can be learned about the developmentally regu- lated interactions of closely spaced promoter elements and multiple transcription initiation sites. The aldolase A gene in humans is known to have four and possibly as many as five such initiation sites regulated by three independent promoters producing four to five different mRNAs (18,32). To study the expression of this gene in the mouse we isolated an aldolase A cDNA from a mouse brain library (36). Subsequent use of this probe on genomic Southern blots shows that aldolase A in the mouse is part of a multisequence family. This correlates with reports of one to two aldolase A pseudogenes in the human (45) and four to five in the rat (20). It has not been conclusively proven, in these species, that all of these aldolase A-like loci are nonfunctional. We left open such a possibility in selecting for genomic sequences responsible for encoding the brain-specific cDNA by using two completely different independent approaches to isolate the gene. The homologous recombination screen, using coding region probes, unambig- uously selected the most aldolase A-like clones from a genomic library by virtue of their high relative recombination frequen- cies. Analysis of these clones identified the bulk (>90%) as carriers of a common genomic region. This sequence was shown to be single copy and identical to a clone isolated by oligonucleotide hybridization. That clone contained all nec- essary information needed to encode all aldolase A mRNA variants. The inherent stringency and thoroughness of the combined screening techniques ensure the cloned mouse al- dolase A sequence is the only mouse gene sequence capable of encoding the observed mouse aldolase A cDNAs (36, 38).

Recent reports of three functional promoters for the human aldolase A gene (18,32) led us to ask if such a situation existed in the mouse genome. Expression of the individual noncoding exons in a tissue-specific manner was investigated using both RNase protection and primer extension assays. Exon H was found in the highest levels in the brain followed by the hepatoma cell line BWTG3, muscle, and spleen. This exon shows two different “TATA”-directed start sites that are used differentially as is exemplified by muscle, hepatoma, and brain tissues. Exon M, as expected, was only found in muscle. Exons Ni and NZ were not found in mRNAs from any mouse tissues at any stage of development suggesting that promoter N is not functioning in mouse as it does in the human. The absence of N-containing mRNAs is surprising since critical homolo- gies such as the conserved splice junction consensus sequence GT-AG (5) and a “TATA” box exist in the N exon region along with other significant sequence similarities. Therefore, these sequences may represent “pseudoexons” similar to the IE-like sequence described for the aA-crystallin gene (19). Given that this assumption is correct, we can speculate as to how this situation may have arisen. Perhaps all three pro- moters were active prior to the rodent-primate divergence and due to increased fitness bestowed by promoters M and H, N received less selective pressure against deleterious promoter mutations. Alternatively, constitutive promoter N activity might have been down-regulating promoters M and H pas- sively by transcriptional interference (9). Thus selection

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Nonconservation of Aldolase A Alternative Promoters 11779

against the weaker 5’ N promoter allowed an overall net increase in aldolase A transcriptional output. As alluded to under “Results,” there are conflicting reports as to the exact start site of N transcripts (18, 32, 40). Interestingly, the “TATA” box corresponding to the extended N1 exon start site, indicated by the primer extension results of the human liver N-type cDNA (40), is highly conserved among all species. The more downstream “TATA” box, directing the transcrip- tion start site identified by Maire et al. (32), is not. Therefore, the exact contribution of such possible promoter mutations is hard to assess. The result in either case appears to be extinc- tion of N promoter function and the release of exons N1 and N2 to undergo a higher rate of sequence drift, as evidenced by the greater degree of divergence in these pseudoexons when compared with M and H exons. Last, we acknowledge that it is possible the promoter N has acquired a different tissue specificity, for which we have not yet properly assayed. How- ever, we believe that tissue-specific regulation would be un- likely given the ubiquitous distribution of N sequences in the human (32).

As a test for transcriptional competency of the cloned gene sequence, the promoter region was fused into a CAT expres- sion vector. Expression was obtained when 7K82CAT (the wild-type promoter) was transfected into the myogenic cell line C2C12. The H promoter was deleted from this construct, creating 7KAH82CAT, which also showed expression in C2C12 cells. Preliminary time course data (not shown) and the data shown suggest that the construct 7KAH82CAT contains those elements necessary for myotube specific expression and that promoter H may be down-regulated upon induction of pro- moter M during myogenesis. This supports the concept of promoter interference, which has been shown to occur be- tween the alternative promoters in the Drosophila melano- gaster alcohol dehydrogenase gene (9). Alternatively, down- regulation may be specifically mediated by negative factors. These constructs will be used for deletion analysis in con- junction with the C& cell line as a system to delineate the essential promoter elements regulating aldolase A expression and to subsequently identify and purify tram-acting factors that modulate aldolase A gene expression.

In summary, we have isolated a functional mouse aldolase A gene and have investigated both its structure and the pattern of expression of its alternative first exons. This in- vestigation has led us to conclude that the mouse aldolase A gene has two alternative functional promoters and exon one sequences and one set of pseudoexons. The pseudoexons appear to be the result of a promoter extinction event. Future experiments will address this more fully and should determine essential &-acting regions involved in promoter M and H utilization.

Acknowledgments-We thank Dr. B. Seed and Dr. C. T. Lutz for supplying the bacteriophage and hosts: Syrinx 2A, MC1061/p3, aAN13, LG75, and JF242; Dr. H. V. Huang for his advice on con- structing the Syrinx 2A genomic library; and Dr. S. Weaver for providing the XL47.1 genomic library.

;: 3.

it:

6. 7.

i: 10.

Adamson, E. D. (1976) J. Embryal. Exp. Morphal. 36,355-367 Angel, P., Imagawa, M., Chiu, R., Stein, B., Imbra, R. J., Rahmsdorf, H. J.,

Jonat, C., Herrlich, P.? and Karin, M. (1987) Cell 49, 729-739 Benton, W. D., and Davm, R. W. (1977) Science 196,180-182 Blau, H. M., Chiu, C.-P., and Webster, C. (1983) Cell 32,1171-1180 Breathnach, R., and Cbambon, P. (1981) Annu. Reu. Biochem. 50, 349-

383 Burgess, D. G., and Penhoet, E. E. (1985) J. Biol. &em. 260,4604-4614 Bohmann, D., Bos, T. J., Admon, A., Nishimura, T., Vogt, P. K., and Tjian,

R. (1987) Science 238, 1386-1392 Colbert, M. C., and Ciejek-Baez, E. (1988) Deu. Biol. 130,392-396 Corbin, V., and Maniatis, T. (1989) Nature 337, 279-282 Darn, A., Bollekens, J., Staub, A., Benoist, C., and Mathis, D. (1987) Cell

50,863-872 11.

12. 13.

14. 15. 16.

17. 18.

19. 20.

21.

22.

23.

24.

25.

26.

27. 28.

29. 30. 31.

32.

33.

DiMaio, D., Corbin, V., Sibley, E., and Maniatis, T. (1984) Mol. Cell. Biol. 4,340-350

Dynan, W. S. (1986) Trends Genef. 2,196-197 Evans, T., DeChiara, T., and Efstratiadis, A. (1988) J. Mol. BioL 199,61-

FeFAberg A. P. and Vogelstein B. (1983) Anal. Biaehem. 132 6-13 GardineryGardhn, M., and Fro&er, M. (1987) J. Mol. Biol. 196.261-282 German, C. M., Moffat, L. F., and Howard, B. H. (1982) Mol. Cell. Biol. 2,

1044-1051 Henikoff, S. (1984) Gene (Am&) 28,351-359 Izzo, P., Costanzo, P., Lupo, A., Rippa, E., Paolella, G., and Salvatore, F.

(1988) Eur. J. Biochem. 174.569-578 Jaworski, C. J., and Piatigorsky, J. (1989) Nature 337,752-754 Joh, K., Arai, Y., Mukai, T., and Hori, K. (1986) J. Mol. Biol. 190, 401-

410 Kadonaga, J. T., Jones, K. A., and Tjian, R. (1986) Trends Biochem. Sci.

11,20-23 Kelley, P. M., and Tolan, D. R. (1986) Plant Physiol. (Bethesda) 82,1076-

1080 Korczak, B., Zarain-Herzberg, A., Brandl, C. J., Ingles, C. J., Green, N. M.,

and MacLennan, D. H. (1988) J. Biol. Chem. 263,4813-4819 Kraft, R., Tardiff, J., Krauter, K. S., and Leinwand, L. A. (1988) Biotech-

niques 6,544-547 Leavitt, J., Gunning, P., Porreca, P., Ng, S.-Y., Lin, C.-S., and Kedes, L.

(1984) Mol. Cell. Biol. 4, 1961-1969 Lebherz, H. G., Petell, J. K., Shackelford, J. E., and Sardo, M. J. (1982)

Arch. Biochem. Biophys. 214,642-656 Lebherz, H. G., and Rutter, W. J. (1969) Biochemistry 8, 109-121 Lin, Y.-S., and Green, M. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 3396-

3400 Loenen, W. A. M., and Brammer, W. J. (1980) Gene (Am&) 10,249-259 Luckow, B., and Schiitz, G. (1987) Nucleic Acids Res. 15,549O Lutz, C. T., Hollitield, W. C., Seed, B., Davie, J. M., and Huang, H. V.

(1987) Proc. Natl. Acad. Sci. U. S. A. 84.4379-4383 Maire, P., Gautron, S., Hakim, V., Gregori, C., Mennecier, F., and Kahn,

A. (1987) J. Mol. Biol. 197, 425-438 Maniatis, T., Fritsch, E. F., an< Sambrook, J. (1982) Molecular, Cloning; A

Eratory Manual, Cold Sprmg Harbor Laboratory, Cold Sprmg Harbor, A. 1

2

36.

37. 38.

39.

40.

41.

Masters, C. J. (1968) Biochim. Biophys. Acta. 167,161-171 Melton, D. A., Krieg, P. A., Rebagliati, M. R., Man&is, T., Zinn, K., and

Green, M. R. (1984) Nucleic Acids Res. 12,703.S7056 Mestek, A., Stauffer, J., Tolan, D. R., and Ciejek-Baez, E. (1987) Nucleic

Acids Res. 15, 10595 Needleman, S. B., and Wunsch, 9. D. (1970) J. Mol. Biol. 48,443-453 Paolella, G.? Buono, P., Manc~m, F. P., Izzo, P., and Salvatore, F. (1986)

Eur. J. B&tern. 1 K(: VXLTK A. , “ , I . , ” ll””

Raymondjean, M., ( Cereghini, S., and Yaniv, M. (19a) Proc. Natl. Acad. Sci. (1. S. A. 85. 757-761

Sakakibara, M -_ hiukai, T., and Hori, K. (1985) Biochem. Biophys. Res. uommun. Idi, 413-420

Salvatore, F., Izzo, P., and Paolella, G. (1986) in Herman Genes and Diseases (Blasi, F., ed) pp. 611-655, John Wiley & Sons Ltd., Chichester, United Kingdom

42. Scbweighoffer, F., Maire, P., Tuil, D., Gautron, S., Daegelen, D., Bacbner,

43. 44. 45.

46. 47.

48.

49.

50. 51.

L., and Kahn, A. (1986) J. Biol. Chem. 261,10271-10276 Seed, B. (1983) Nucleic Acids Res. 11,2427-2445 Szpirer, C., and Szpirer, J. (1975) Differentiation 4,85-91 Tolan. D. R., Niclas, J.. Bruce. B. D.. and Lebo. R. V. (1987) Am. J. Hum.

-- --,--. _-. Tolan, D. R., and Penhoet, E. E. (1986) Mol. Biol. & Med. 3, 245-264 Townes, T. M., Shapiro, S. G., Wernke, S. M., and Lingrel, J. B. (1984) J.

Biol. Chem. 259,1896-1900 Tsutsumi, K., Mukai, T., Tsutsumi, R., Hidaka, S., Arai, Y., Hori, K., and

Ishikawa, K. (1985) J. Mol. Biol. 181, 153-160 Wigler, M., Pellicer, A., Silverstein, S., Axel, R., Urlaub, G., and Chasin,

L. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 1373-1376 Yu, Y-T., and Nadal-Ginard, B. (1989) Mol. Cell. Biol. 9,1839-1849 Grahlmann, R., and Kedes, L. (1989) J. CeN. Biochem. Suppl. 13E, 215

REFERENCES

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