Edward W. Holmes. .... I * t P r o L e u P h a y s L e u T h r G l y G l ~ G l ~ y s G l n l I c A s p A s p A l a I * t ..... Fishhein, W. N., Armhrustmacher, V. W., and Griffin.
Communication
OF BIOLOGICAL CHEMISTRY THE JOURNAL Vol. 262 No. 26 Issue of September 15 pp. 12397-12400 1987 0 1987 by The America; Societ; for Biochemistry andMolecular Biolok, Inc. Printed in 11.S A .
Cloning andSequence of Rat Myoadenylate Deaminase cDNA EVIDENCEFORTISSUE-SPECIFICAND DEVELOPMENTALREGULATION* (Received for publication, May 26, 1987) Richard L. Sabina*$n, Rainer Marquetantg, Nalini M. Desai$, Krystian Kalethag, and Edward W. Holmes.$$ From the $Howard Hughes Medical Institute Laboratories and §Departments of Medicine and Biochemistry, Duke University, Durham, North Carolina 27710
Myoadenylate deaminase is the muscle-specific isoform ofAMP deaminase (EC 3.5.4.6), an enzyme which plays a special role in energy metabolism in skeletal muscle. A 2.3-kilobase cDNA encoding this enzyme has been cloned from a XgtlO library prepared from rat skeletal muscle using oligonucleotide probes designed from AMP deaminase peptide sequences. This cDNA was sequenced, and the amino acid sequence of this isoform of AMP deaminase was deduced. Sequences homologous to this cDNA are identified in the genome of eukaryotes as diverse as yeast andman. Tissuespecific expression of a 2.5-kilobase AMP deaminase transcript is demonstrated, and the abundance of this transcript as well as the 80-kDa adult, muscle-specific peptide of AMP deaminase increase in parallel during postnatal skeletal muscle development. In the adult animal, the abundance of this transcript and AMP deaminase activity are differentially expressed in various skeletal muscle fiber types. We conclude that AMP deaminase sequences have been highly conserved during evolution, and in mammals there is developmental and tissue-specific control of expression of this gene.
AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) is a ubiquitousenzymefoundinalleukaryotes. Tissue-specific isoforms of AMP deaminase have been found inseveral organs of adultvertebrates (1-3), anddifferentAMPdeaminase isoforms are produceda t various stagesof organ development (1,4-6). AMP deaminase, and the purinenucleotide cycle of which it is onecomponent, playsa critical role in energy metabolism in skeletalmuscle (for review, see Ref. 7). Skeletal muscle of the postnatal animal contains a unique AMP deaminase peptide (6), and the amount of this peptide varies depending on the maturity of the animal (6), fiber type (8lo), and conditioning of the muscle (9). Myopathic patients * This work was supported inpart by National Institutes of Health Grant AM12413, Postdoctoral Fellowship AM07097, and the Muscular Dystrophy Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s)reported in this paper has been submitted to the GenBankTM/EMBL Data Bank withaccessionnumber(s)
with inherited deficiency of AMP deaminase are unable to produce the skeletal muscle-specific peptide found in postnatal muscle (11), yetprimarycultures of myocytesfrom these patients retain the ability to produce an embryonic isoform found in undifferentiated myoblasts (12). To define the molecular mechanisms which regulate tissue-specific expression of different AMP deaminase peptides and control the amount of AMP deaminase produced in different fiber types of skeletal muscle in response to developmental changes, muscle stimulation, and pathological states, we initiated the studies described in this report. We report here the cloning and sequence of a rat skeletal muscle cDNA for AMP deaminase. A 2.5-kb’ transcript which hybridizes with this cDNA is detected only in skeletal muscle, and the amount of this transcript varies in parallel with the amount of AMP deaminase skeletal muscle-specific peptide in different fiber types and at different stages of skeletal muscle development. MATERIALSANDMETHODS
Peptide Isolation and Sequencing-AMP deaminase was purified from adult rat skeletal muscle by the method of Smiley et al. (13). Peptides were produced by cyanogen bromide cleavage and isolated by reverse phase Chromatography. Sequencing was performed by Dr. Richard Cook of the Microbiology Department at Baylor College of Medicine as described elsewhere (14). Oligonucleotide Synthesis-Oligodeoxyribonucleotides were synthesized by the phosphoramidate method (15) on an Applied Biosystems 380 DNA synthesizer by Dr. Richard Randall in the Howard Hughes Medical Institute Laboratory at Duke. Isolation and Sequencing of cDNA-A cDNA library prepared from adult rat soleus muscle in the X g t l O vector (obtained from Dr. F. Schachat, Department of Anatomy, Duke University) was used for screening with oligonucleotide probes. 3 X lo5 plaques were transferred to nitrocellulose filters in duplicate and filters were prepared for hybridization with 32P-labeledoligonucleotides (>2 X 10’ dpm/ pg). Filters were hybridized in 6 X SET (1 X SET is 30 mM Tris, pH 8.0, containing 150 mM sodium chloride and 2 mM EDTA), 10 X Denhardt’s solution, 0.1% sodium dodecyl sulfate (SDS),0.5% sodium pyrophosphate, and 50 pg/ml sonicated herring sperm DNA at 42 “C for 16 to 20 h, washed three times for 15 min in 6 X SET, 0.1% SDS, 0.5% sodium pyrophosphate at 42 “C, and exposed to Kodak XAR-2 film for 2 to 3 days. Plaques which were positive on both filters were isolated and passed sequentially until all plaques hybridized with the probe. cDNAs were subcloned into theBlue M13.M plasmid vector (Stratagene Cloning Systems, San Diego, CA) or M13 bacteriophage for double-stranded or single-stranded DNA sequencing, respectively, by the dideoxy chain termination method (16). All DNA sequences were confirmed by sequencing of both strands. Isolation and Analysis of DNA and RNA-Genomic DNA was isolated from rat tissues or cultured cell lines by the method of Blin and Stafford (17). Human, mouse,dog, rabbit, chicken, and yeast DNA were gifts from investigators at this institution. Genomic DNA was digested with BamHI, and restriction fragments were resolved on 0.8% agarose gels. DNA was denatured and transferred to Nytran filters (Schleicher & Schuell) as described by Southern (18).Filters were hybridized with lo7 dpm of 32P-labeledcDNA (2 X IO9 dpm/pg; Multi-prime kit, Amersham Corp.) in 5 X SSC (1 X SSC is 150 mM sodium chloride, 15 mM sodium citrate,pH 7.5), 5 X Denhardt’s solution, 35% deionized formamide, 0.5% SDS, and 50 pg/ml sonicated herring sperm DNA at 42 “C for 16to 20 h. Filters were washed and exposed to x-ray film as described in the text. Total cellular RNA was extracted in guanidine isothiocyanate and isolated by cesium chloride gradient centrifugation (19).Poly(A)+
502811.
ll To whom reprint requests should be addressed Box 3491, Duke University Medical Center, Durham, NC 27710.
The abbreviations used are: kb, kilobase; SDS, sodium dodecyl sulfate.
12397
12398
Cloning and Sequenceof Rat Myoadenylate Deaminase cDNA 10 #) Jo 70 W I10 CCRGTC~TGCCTCTGTTCAAAtTTRCAGFTCRRGG4ARACRRRTT~TGT~TGCGT~TTTGCT~TRTTTFCCTCfl~TC~T~TC~AC~ I*tProLeuPhaysLeuThrGlyGl~Gl~ysGlnlIcAspAspAlaI*tRrgSc~RlaGl~~l~Rla~~Gl~lLysA~pGl~Gl~l~~~~Gl~ 130 150 170 190 2 10 233 RTCTCCCCCTTCGACGTU3RT~TCTGCCCRflTTl~CClTCGT~TGC~CCACRTflTTC~AT~CTGTCCflTGTCCflTGGT~~GCTTCC~ """-+""""-+""""-+""""-,-""""+""""-+""""-+""""-+""""-+""""~+""""~+""""-+" I leScrProPheAspUalAspGGlulIeCysProl leSerLeuArgOlutletGlr#31aHisl l e P h a H i s H e t G l L n r r l e S ~ I * t S e r ~ t A ~ p C l ~ ~ ~ y ~ f f ~ ~ ~ G l n
250 270 290 350 3 10 330 GG(ICGOIIXIACTGTTflRTTTGTCCATTtCGC~AGTGAFRCGTClTCTRCC~CTGTCCC~flTl~TTTRlTTCTTCA1CCCCGACCTRTGATGT~CT~TTCC~ """-+""""-+""""-+"""".+""""~+""""-+"-"""+""""-+""""~+""""~+""""-+""""-+"
Gly~hgCy~ThrValAsnCeuSgIleProGlnSerGluT~SerSerThrLyjLRISerHislleGluGluPhelleSerSerSerProThTyGluSprUalP~~hffil~g
"""~+""""~+""""~+""""~+""-""+""""~+""""-+"".""+""""~+""""~+""""~+""""~+" UaIGInIIeThrGI~spTyAIaSecGIyUoIThr~IGIuAspPhCGIuUoIUaICysLysGIylcuTyrffgRIdeuCysIIeR.PC,IuLysTyrllctGInLysSerPheGInRrg
4w 5 10 530 550 570 590 TTCCCC~ACCCCCTCCRTACCTO1GO1flCATCGRffiGC~TTTGCTRFCAATCGARRGCTTCTRTCCffiTATTTACCCCTCCTCCG~GARGGG~GRCCCCTTTCGC~ """-+""""~+""""-+""""~+""-~"~+""""~+""""-+"""".+""""~+""""~+""""~+""""~+" PheProCy~ThrProSerLy~T~L~r~snlleRspClyGluAldeu(lalAlalloGluSerPheTyrPr~IPhaThrProProProLy~LysG1~luA~pPr~h~~g 6 IO 630 650 670 690 7 10 ~~CTTCCCGC~~CTGGGCTflTCRCCTCRflGRTGffiGGlGGTGTGlTl~RTCl~~CTO1TG~GC~~C~GCffiffiflT~CCCRAGCCCTflCCClTACCC~TCTG """-+""""-+""""~+"""".+""-""+""""~+""""-+"""".+""""~+""""~+""""~+"""".+" G l ~ s p l e u P r o A I l d s n C e u G I y T y r H I s L e u L y s M e t L y s G I y G I ~ a I I l e T y l I e l y r P r o R s pR l uI ~ l c A l a S e r f f ~ s p G l u P ~ ~ L y ~ ~ o T y - P ~ ~ T ~ ~ o A s n L ~ ~
7P) 750 770 790 8 10 830 GTGACTTCCTGGRTGRCRTG~T~TTTTGCTTGCTCT~TTGCRCAffiGGCCTGTGRAGRCTTRCRCTCACCGCCGTCTGRRCTTCCTCTCCTCCRffilTCCRGGTCCflTCAORTGCTG """-+"""".+""""~+""""+-"--""*"""".+""""-+""""~+""""~+""""~+""""~*""""~+" RspclspPheLeuRspRsp~tRs"PheLeuLeuRIaLeu1leRlaGlnGlrJProUalLysThrTyrThrHisAr(P~euCysPheleuSerSerLysPheGlnUalH~sGlnnetLeu 850 870 890 9 10 930 950 iY(T(iffiATG(T(tGRGCT~RACi~CTO1A~CRRCCCCCRCCGGGRCTTTTATRRCTGCAGGRRGGTG~TflCTCRCRTCCRCGCRGCffiCTGCCTGCRT~RCClYIAAGCRCCTGCT~GC """-*"""".+"""""-"""".+""""~+""""~+""""-+""""~+""""~+""""~+""""~+"""".+" A s n G l u l l e t R s p G l l l e u L y r G l u L e l l y s A s n R s n P r ~ ~ s A r g R s p P h e T y r R s n C y s A r ~ y s U a l R s ~ Tl ~ aH Hl~ssl R l ~ l a R I ~ y s M c t A s n O l n L y s H i s l e v L c u A r ~ 970 990 1010 I 033 1050 1070 TTTRTT~RARTCTTACCRTATTGflTGCTG(ICRGRGT~TCTACRGCACCfl~~RAG~~lGRCCCTGflffiG~TlTTTGCTCRflTT~TRl~RTCCRTAl~C1GRCTGTT """-+""""-+""""~+"""".+""""~+""""~+""""-+""""~+""""~+""""~+""""~+"""".+" Phel l e L ~ s L y ~ S e r T y r H ~ s I l e A s p A l o A s p R r g U s l U o l T ~ S e r T h r L y ~ G l ~ L ~ ~ ~ A s ~ e u T ~ L e u L y s G l ~ e u P h e R l ~ l n L e u R ~ ~ M e t H ~ ~ P ~ o T ~ f l ~ ~ u T h r U ~ ~
1090 I 1 IO 1130 I150 I170 I I90 GRCTCTCTGORTGTTCRTFCTG~UiCRGACGTTCCRRCGCTTCGRTRA(jTTCRATGRCA~T~~TCCGGTGGGTFCGffiTGffiCTTC~GACCTCTRCCTR~ffiRCRRCT~ """~+""""-+""""~+"""-"""""+-""~"~+""""~+""""~+""""~+""""~+""""~~"""".+" RspSerLeuAspUalH~sAl~GlyR~gCl~lh~PheGl~~gPh~R~pLy~P~R~nR~pLy~TyAsnP~~UolGl~l~SerGluL~~ffgA~pLeuTyrLeuLy~ThrA~pR~~Ty~
1210 1230 1250 12x1 1290 13 IO RTT~GGffiRGTRTTTTGCCACTRTCRlC~GGRGGTGGGTGCAGCT00TO~GCCRffiTATCflGCRTGC~ffiCCCCGClTGT~TCTACGGlCGCffiTCC~flTGffiTGGflGC """~+""""~+""""~+""""~+""""~+""""~+""""-+""""~+""""-+""""-+""""~""""-+-Il~nGlyGluTyrPheRloTh~IlelleLysGluUalGlyRlaAspLe~~olA~pRlaLYsTyrGl~H~sRl~luPro~~euSerllcTyGl~gSerPro~rpGluTrPSer
1330 1350 1390 1370 14 10 1430 ~TCTCCTCTTGGTTTGTCG0t~GTATTTRTTGCCCC~CRTGRCATMTGATCC~TCCCCffiG$TCTATGF(TGTGlTCCGRTCCRACi~TTTCC~GCCRC~~TTGC~~G
1450 1470 1490 1510 1530 1550 RTGCTG~CRRTRTTTTCCTTCCAGTGTTlGAGGCCACCRTCRACCCCCflGCTCflTCCflG~CTCAGTGTCTT~TCRRGCflTRTC~TGGCTTT~CffiTGTGGAT~lGGRGTCCARR MctLeuGluAsnllePhcLeuProUalPheFluRlaThrIleRsnProGlnThrH~sProAspLeuSerUalPheleuLy~H~sl IeThrGIyPhCRspSerUOIRspAspGIuSerLys
1570 1590 16 IO 1630 1650 1670 CRCffiTGGTCRCATGTTTTCCTCCRRGRGTCCC~CCT~ffiT~RC~T~R~C~TCTTACRCflTRTTRTGCCTACTACRTGTRTGC~RCRTCATGGTGCTCRRCTGC """-~""""-+"""""-""""-+""""-+""""-+"-~""~+""""-+""""-+""""-*""""-~""""-+"
H ~ s S e r G l y H ~ s M a t P h e S e r S e r L y s S a r P r o L l ) s P r o Ty-RIaAsnIIeHetUL,ILeuAsnCys
1690 17 10 1730 1750 1770 1790 CTUR+)RRGGRACGffiGCATGAlTACRTTTTTGTTTCGCCCCCRTTGTWT~T~GCTCTC~TCACCTCflT~A~CTTlRTGRTRGCGGACflRTRTTTCGCRCUjCCTGRRC L e u ( k g L y ~ O l u A r g C I y J n P t A s n T h r P h a L e u P h ~ A r ~ ~ ~ ~ s C y ~ G l ~ l ~ R l a G l ~ l a L ~ u l h ~ H ~ s L e ~laRlaAspRsnl c t T h r R l o P hleSerHisGlyleuRsn eMetl
1930 1950 19x1 1990 20 10 2030 CTCCRORRRGGCCTCRTGRTCTCGCTGlCGACCGRTGACCCG(ITGCAGTTCCRTTTCRCCR~GffiCC~CTORT~~GTACGC~RTCGCR~CCRGGlCTTC~ClG~~1TGT
2050 2070 2090 21 10 2 130 2 150 GACR7GTGTWWjGTUiCRRGGAlCRGTGTCCTGCAGTGT~TTlCT~AT~~GC~GTTlCTGGG~C~TTACCTTGAGCRAGGTCCTGTT~RARTGRCflTCCGRRGG As~~tCy~GIuUaIAIaffgAsnScrU~ILcuGInCysOIyIIeSerHisGIuOIuLysRIdysPhcLeuGI~snRsnTyLe~IuGIuGIyPr~aIGIyAsnRspIIeAr9Rrg
2 170 2190 2210 2230 2250 2270 R C ~ T G T G G C T C ~ T T C G C A T ~ C C T R T C G T T R ~ G A ~ C T T ~ T G T T R T G ~ T C ~ ~ ~ T ~ T T O C T ~ G G ~ C T T A R R T C ~ C ~ ~ ~ ~ T R ~ G T G ~ C ~ R R
ThrRsnUolRIaGlnl I e R g M e t A l a T y - ~ ~ T y G I u T h r T r p C y 4 T y r G l u l r u A I c R l a G l u G l y l a u L y r S r T h ~ G l u E n d
FIG. 1. Nucleotide and deduced amino acid sequence for AMP deaminase cDNA. The sequence of the cyanogen bromide peptide determined experimentally is underlined. The complement of the synthetic oligonucleotide used in library screening is also underlined. RNA was selected by hybridization to oligo(dT)+columns (19). RNA samples of the indicated amount were resolved on 1.2% formaldehydeagarose gels and transferred to Nytran filters for Northern blotting (19). Filters were hybridized with lo7 dpm of 32PMulti-prime labeled cDNA in 4 X SSC, 0.8 X Denhardt's solution, 40% deionized formamide, 10% dextran sulfate, 40 m M sodium dihydrogen phosphate, 0.4% SDS, and 50 +g/ml sonicated herring sperm DNA a t 42 "cfor 16 to 20 h. Filters were washed four times for 5 min a t room temperature in 2 X SSC, 0.1% SDS, and twice for 15 min in 0.1 X SSC, 0.1% SDS a t the temperature described in the text. Protein Analyses-Isolation of AMP deaminase, assay of AMP deaminase activity, and Western blotting of tissue extracts for AMP deaminase immunoreactivity have been reported previously ( 6 ) .
RESULTSANDDISCUSSION
AS reported previously ( Z O ) , and confirmed by US, the amino terminus of rat skeletal muscle AMP deaminase is blocked. The longest cyanogen bromide peptide sequenced contains 13 residues (underlined in Fig. and this Peptide was used to synthesize a series of oligonucleotides. One oligonucleotide of 14 residues (the complement of the sequence underlined in Fig. 1, 1614 to 1627 base pairs) hybridized strongly toa single 2-5-kb transcript which was only detected in RNA from adult ratskeletal muscle (datanotshown).This oligonucleotide
Cloning and Sequence of Rat Myoadenylate DeaminasecDNA
A B C
12399
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ORIGIN -
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6.7 FIG.2. Amplification of the AMP deaminase gene in L-6 myoblasts which overexpress AMP deaminase by growth in media containing coformycin(21). 10 pg of DNA from wild-t.ype cells (Inn? A ) and cells with increases in AMP deaminase activity were analyzed hv Southern blotting andprobed with AMP deaminase cDNA. Densitometric analyses of these blots indicate AMP deaminase gene copy number is 4- to 7-fold greater in the cells which overproduce AMP deaminase. The filter was washed twice a t 65 "C in 3 X SSC. 0.srE SDS followed by two additional washes at 65 "C in 0.1 X SSC, 0.5'E SDS. This filter was secondarily probed with a rat creatine kinase cDNA and signals of equivalent intensity were obtained for DNA samples from all three cell lines (data not shown).
A.
FIG.4. Tissue-specific expression of AMP deaminase peptide and mRNA. A. 100 pg of protein from the indicated tissues of an adult rat was resolved on 8"; denaturing SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and prohed with antisera specific for the AMP deaminase peptide produced in adult rat skeletal muscle (6). R. 20 pg of total RNA from adult skeletal muscle and 40 pg of polv(A)+RNA from brain, liver, and heart were Northern blotted and prohedwith AMPdeaminase cDNA. The filter was washed in 0.1 X SSC.0.1% SDS at 50 "C.
B. DAYSAFTER
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FIG.5 . Changes in AMP deaminase activity and mRNA acRats were cumulationduringskeletalmuscledevelopment. 2.020killed at birth and at the times indicated postnatally; skeletal muscle was harvested for assay of AMP deaminase activity and isolation of RNA. A, changes in AMP deaminase activity over this interval of time aredepicted. R, 20 p g of total RNA from skeletal muscle ohtained each time was analyzed by Northern blotting and probedwith .
