Isolation and Characterization of a cDNA Clone That Codes for ...

4 downloads 6634 Views 4MB Size Report
From $The Johns Hopkins Oncology Center Laboratories, The Johns Hopkins School of Medicine, Baltimore, Maryland ... analogues, we sought to clone a cDNA that codes for the ... tory of Dr. Raymond J. Bergeron (University of Florida, Gainesville, ..... Mank for her technical expertise, and Tammy Hess and Sandra Lund.
THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 266, No. 2, Issue of January 15, pp. 810-814,1991 Printed in U.S.A.

Q 1991 by The American Society for Biochemistry and Molecular Biology, Inc

Isolation and Characterization of a cDNA Clone That Codes for Human Spermidine/Spermine N1-Acetyltransferase” (Received for publication, July 23, 1990)

Robert A. Casero,Jr.$§, Paul Celano$, Stephanie J. Ervin$, Nancy B. ApplegrenS, Laurie Wiestll, and AnthonyE.Peggll From $The Johns Hopkins Oncology Center Laboratories, The Johns HopkinsSchool of Medicine, Baltimore, Maryland 22231 and the TDepartments of Cellular and Molecular Physiology and Pharmacology, the Milton S. Hershey Medical Center, Pennsylvania State University, Hershey, Pennsylvania 17033

Spermidine/spermine W-acetyltransferase (Spa/ Spm acetyltransferase) is the rate-limiting enzyme in the catabolism of polyamines. This enzyme is highly inducible by several stimuli, including the natural polyamines and their structural analogues. To investigate the underlying mechanism responsible for the control of this enzyme a cDNA which codes for an active human Spd/Spmacetyltransferase has been isolated from a random primed cDNAlibrary constructed from mRNA of a polyamine analogue treated large cell lung carcinoma line, NCI H157. The 972-base pair cDNA was identified using a 32-fold degenerate, 20base oligomer probe to a 7-amino acid polypeptide sequence derived from the purified protein. The cDNA has a 513-base open reading frame that codes for a protein of 171 amino acids with a predicted molecular weight of 20,023. In vitro translation studies demonstrated the protein product ofthis cDNA to be a biologically active enzyme. The cDNA recognizes a 1.5-kilobase transcript in human cells which is highly induced in the human large cell lung carcinoma NCI H157 line following treatment with the polyamine analogue. The unusually high expression of Spd/Spm acetyltransferase mRNA by the NCI H157 cellsin response to treatment does not appearto be a result of an amplification of the Spd/Spm acetyltransferase gene.

Spermidine/spermine N’-acetyltransferase (Spd/Spm acetyltransferase)’ is the rate-limiting enzyme in the catabolic pathway of polyamine metabolism. It catalyzes the N’-acetylation of spermidine and spermine and, by the successive activity of polyamine oxidase, spermine can be converted to spermidine and spermidine to putrescine (1, 2). This enzyme is a cytosolic protein which is known to be inducible by a variety of toxic agents, hormones, and polyamine analogues

* This work was supported by National Institutesof Health Grants CA37606, CA51085, CA51068, and GM26290, a National Foundation for Cancer Research grant, The Wendy Will Case Cancer Fund, and The James S. McDonnell Foundation. 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 sequencefs)reported in thispaperhas been submitted totheGenBankTM/EMBLDataBankwith accession numberfs) M55580. To whom correspondence should be addressed: The Johns Hopkins Oncology Center Laboratories, 424 N. Bond St., Baltimore, MD 21231. I The abbreviations used in this paper are: Spd/Spm acetyltransferase, sperimidine/sperimine N’-acetyltransferase; SDS, sodiumdodecyl sulfate.

(1, 3-7). Additionally, this highly regulated protein is in a metabolic pathway which involves by two other highly regulated enzymes, ornithine decarboxylase and S-adenosylmethionine decarboxylase (2). The combination of these enzymes in the pathway allows for a fine attenuation of the intracellular polyamine concentration, underscoring the important role of these polycations in growth and, in some instances, cell survival (8-11). Recent evidence suggests that Spd/Spm acetyltransferase mayhavea role indetermining cellular sensitivity to a class of polyamine analogues (12). We have previouslyshown that a human large cell undifferentiated lung carcinoma line, NCI H157, responds to treatment with the polyamine analogue IP,W*-bis(ethyl)sperminewith rapid cell death (10-13). This cytotoxicity has only been observed in a few specific cell types (11, 14). Interestingly, those cells which respond in this unusual cytotoxic manner to bis(ethy1)spermine treatment also exhibit an unusually high induction of Spd/Spm acetyltransferase activity (12, 15). The study of the molecular mechanisms regulating ornithine decarboxylase and S-adenosylmethioninedecarboxylase has beengreatly facilitated by the availability of cDNA clones corresponding to these important proteins (16-19). To date, cDNA probes have not been available for the study of Spd/ Spm acetyltransferase. Therefore, to more fully understand the mechanismsof molecular control in the regulation of Spd/ Spm acetyltransferase activity and provide to a toolto further investigatethepotentialassociation of induced Spd/Spm acetyltransferase expression and sensitivity to the polyamine analogues, we sought to clonea cDNA that codes for the human protein. Wenow describe the isolation and characterization of one such cDNA derived from bis(ethy1)sperminetreated NCI H157 large cell lung carcinoma cells. The role of increased mRNA level in Spd/Spm acetyltransferase induction bypolyamineanalogues and the potential use of the cDNA in the studyof analogue sensitivity arediscussed. EXPERIMENTALPROCEDURES

Materials-N’,N*-Bis(ethy1)spermine was synthesized by previously published methods (20-22) and kindly provided by the laboratory of Dr. Raymond J. Bergeron (University of Florida, Gainesville, FL). [1-“C]Acetyl CoA (55 mCi/mmol), [r-”P]ATP (7000 Ci/mmol), [a-”S]ATP (1000 Ci/mmol), and [cY-~*P]CTP (3000 Ci/mmol) were purchased from ICN Radiochemicals,Irvine, CA. Restrictionand DNA modifying enzymes were purchased from Bethesda Research Laboratories. The X ZAPII cloning vector system was purchased from Stratagene, La Jolla,CA. Other biochemical reagents were purchased from Sigma. The human @-actin probewas provided by Dr. Donald Cleveland (Johns Hopkins University) and used as an indicator of equivalent RNA loading. Cell Culture and Treatment-The NCI H157 line of human large cell undifferentiatedlungcarcinoma was maintainedandtreated

810

cDNA

Human Spermidinelspermine N'-Acetyltransferase where indicated with 10 p~ bis(ethy1)spermine exactly as previously reported (12). Labeling of Oligomer Probe-A 20-base oligomer with 32-fold degeneracy which is complementary to allcoding possibilities of a portion of a peptide fragment from the previously purified human Spd/Spmacetyltransferaseprotein(fragment D in Ref. 15)was synthesized by the solid phase, P-cyanoethyl phosphoramide method on a Cyclone Plus DNA synthesizer (Millipore/Milligen, Burlington, MA) using the reagents and programs supplied by the manufacturer. One pg of this probe, designated Dl, was end-labeled with T, polynucleotide kinase and [y'"P]ATP. This probe was used for blotting procedures a t 2.0 X lo6cpm/ml of hybridization solutions as indicated below. Construction and Screeningof cDNA Library-Poly(A)+ RNA was isolated from bis(ethy1)spermine-treated NCI H157 cellsby the guanidinium thiocyanate procedure followed by spin column poly(dT)cellulose chromatography (23). cDNA was synthesized from 5 pg of RNA using the Bethesda Research Laboratoriesmodified protocol of Gubler and Hoffman (24) and prepared for ligation into the EcoRI site of the XZAPII cloning vector as described by Short et al. (25). Fifty ng of the cDNA was ligated into XZAPII, packaged, and titered using the XL1-Blue strain of Escherichia coli, yielding a recombinant frequency of -90%. The library was amplified once and plated for primary screeningat a density of 20,000 plaques/gO-mm dish. Initially 200,000 plaques were screened by transferring to duplicate NEF-978 nylon filters (Du Pont-New England Nuclear). The filterswere prepared for hybridization by amodification of themanufacturer's protocol. After transfer, filters were placed plaque side up on 0.5 N NaOH soaked paper for 1.5 min,thensequentiallytransferred to vessels containing 2 X SSC (1 X SSC is 0.15 M NaCI, 0.015 M Nan citrate), 0.5 M Tris, pH 8.0, for 5 min, 0.30 M NaCl 0.030 M Na:' citrate for 3 min, and a final wash in double-distilled HyO for 3 min. Filters were then dried a t 80 "C for 5 min, prehybridized 1 h a t 42 "C in Blotto (1% SDS, 0.5% powdered milk, 6% polyethylene glycol, 0.2 mg/ml salmon sperm DNA, 0.75 M NaCI, 50 mM NaHyP04.H&,pH 7.4, 5 mM EDTA Nay), andhybridized with 2 X lo6 cpm/5 ml Blotto containing the '"P-end-labeled 20-base oligomer D l probe a t 42 "C for 18-24 h. Filters werewashedtwicefor5 mineach a t room temperature in 3 X SSC, 0.1% SDS followed by one wash in3 X SSC, 0.1% SDS a t 42 "C for 30 min, blotted dry, andexposed to x-ray film with intensifying screens. Plaques that continued to hybridize to the D l oligomer through three levels of screening were selected and converted to Bluescript plasmids by thein vivo excision methods(25)accordingtothe protocolprovided by the manufacturer. Plasmids prepared in this manner were used for labeling and sequencing by the methods discussed below. Northern and SouthernAnalyses-Standard Northern and Southern analyses were performed as previously published (26-28). Where indicated the tetramethylammonium chloride protocol of Jacobs et al. (29) was used for the hybridization of end-labeled D l t oNorthern blots from control versus bis(ethy1)spermine treated NCI H157cells. Filters were prehybridized a t 50 "C in 3 M tetramethylammonium chloride, 0.1 M NaH2P04.Hy0, pH6.8, 1 mM EDTA Na4,0.6% SDS, 100 pg/ml denatured salmon sperm DNA, 0.5 g of Ficoll, 0.5 g of polyvinylpyrrolidone, 0.5 g of bovine serum albumin (fraction V). Hybridizations were performed in the same solution for 18-24 h, washed sequentially for 15 min a t room temperature and then at 50 "C for 1 h in 3 M tetramethylammonium chloride, 50 mM TrisHC1, pH 8.0, 0.2% SDS. Washed filters were exposed to x-ray film with intensifying screens. T o control for differences in RNA loading, the filters were rehybridized to the human &actinprobe. cDNA Sequence Determination-Double-stranded cDNA sequencing was performed directly from the Bluescript plasmid containing the pSAThl insert by the dideoxynucleotide methods of Sanger etal. (30, 31) using the Sequenase kit (United States Biochemical Corp.) following the protocol supplied by the manufacturer. I n Vitro Translation of SSAT cDNA-Ti RNA polymerase was used to produce sense transcripts from linearized pSAThl plasmid andanunrelatedplasmidas previously reported (19, 32) by the methods of Krieg and Melton (33), in the presence of 0.5 mM 3'-7methyl-G-5'ppp5'-G-3'. TheRNA producedwas thentranslated exactly aspreviously published, in a rabbit reticulocyte lysate system (Bethesda Research Laboratories) in the presenceof [:"S]methionine (15,34). The translated proteins were precipitated by specific antisera (35) and analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography. Spd/Spm acetyltransferase activity was meas-

811

ured by the formation of ['"C]N'-acetylspermidine as published (3, 15). RESULTS

Use of a Degenerate Oligomer for Northern Analysis and Library Screening-We have previously shownthat treatment of NCI H157 cells with 10 p~ bis(ethy1)spermine for 24 h results in a several orders of magnitude induction of Spd/ Spm acetyltransferase activity (12, 15). Furthermore, much of this increase appeared, from in vitro translation studies, to result from increasedsteady-statemRNA coding for the acetylase (15). Therefore a XZAP I1 cDNA library from similarly treated NCI H157 cells was constructed to screen for a Spd/Spm acetyltransferase coding cDNA. T o screen this library, an antisense,32-fold degenerate, 20-base oligomer designated D l correspondingtotheamino acid sequence EYMEEQV (15) was synthesized. This oligomer was first end-labeled with [-y-"''P]ATP and used to probe a Northern blot of bis(ethy1)spermine-treated and untreated control cells using the tetramethylammonium chloride protocol(Fig. 1). A significantincrease in a 1.5kilobase message that was homologous to the oligomer was onlyobserved in the bis(ethy1)spermine-treated NCI H157 cells. Since this result with the D l probe was consistent with previous information (15)suggesting that the acetylase induction involves increasedmRNA levels for this protein, the probe was used to screen the XZAP I1 cDNA library. The primary screen of -200,000 plaques yielded >lo0 prilifts. Ten of the most intense mary positives on duplicate signals were chosen for further screening. Three of these 10 produced intense signals through three levels of screening a t which point in vivo excision of the Bluescript plasmid was performed. The resulting plasmids wereindividually nicktranslated andused to probe Northern blots of control versus bis(ethy1)spermine-treated NCI H157cells usingstandard high stringency Northern techniques. One of these plasmids, designated pSAThl, recognized an mRNA which was identical in size and induction pattern to the mRNArecognized by t h e D loligomer. Sequence Determination of pSAThl-The entire sequence for the 972-base pair cDNA insert from pSAThl is shown in Fig. 2. The largest open reading frame is 513 bases and codes for a 171-amino acid protein witha predicted molecular weight of 20,023. I t isimportanttonotethatthereiscomplete identity with 81 of the 83 amino acids previously sequenced from endoproteinase Lys-C digests (15). Each of the amino acids that did not agree with the sequences derived from the Lys-C digests (amino acids69 and 84)were the secondresidue A

C A C W ' G ~ C A T R T A W3'

B

'

2

28s18s-

m

FIG. 1. a, sequence of D l oligomer. D l is a 32-fold degenerate 20base oligomer which is complementaryto the sequence coding for the Spd/Spm acetyltransferase-derived polypeptide EYMEEQV. Y repoccupied by either pyrimidinebase resents a base positionthat can be and R represents a baseposition that can be occupied by either purine. b, Northern blot usinga 32-fold degenerate probe torecognize spermidine/spermine N'-acetyltransferase transcripts. Five pg of total cellular RNA isolated from untreated NCI H1.57 cells (lane I ) or NCI H157 cells treated with 10 p~ N',N''-bis(ethyl)spermine for 24 h (lane 2 ) were separated by formaldehyde/agarose gel electrophoresis, transferred to a nylon membrane, and hybridized to the :12Plabeled D l 20-base oligomer. The relative migrations of the 28 S and 18 S ribosomal RNA are as indicated.

Human Spermidinelsperminte N*-Acetyltransferase cDNA

812 A PI RI PI

1

-"-H3 R I

RI

II

1

" " c "

1

1

(

1

1

0

3

4

c " ,

-

"

1

2

20092-

1

*

,

I

69

I

1.0

0.5

46-

Length(kilobose5)

B

~ vra m v7 H

h

,LM TIC CIC A= ax m ar m ccc X

F

V

I

P

ra- CAC nr ,cr m c ,\m cn: CT; cn: AX ~r.c : G\>

F

,

~

C

S

D

I

I

'XC A % GL\ GU OW

?

R

E

E

Q

"

,

.

R

P

GU CTC

L

I

A

T

13

A

11

c a ,va TAT

K

E

L

~

A X 7%\MT Gm MA C%T C E CPA CAB, : . I E Z D L L E

81 K 27 Y

30-

129 4,

TIT TAC W rrs' CR: CIT T U GU a 177 I l C P G e , i ~ ~ F Y I ! c i . v . \ E 5.3 i

CAT oil TIT G\GG CAC

,LL\GV CiiC % ACI. CxX F K C ! , u :

CO:

A%

"

TzW TATTI" E T : $ T Y Y Y I Y

G'A

CN Kc AT? CIT oil TIT CCZ

-1

p

~

.

C

G'S CxX To:ATT OT

D

P

W

I

C

n

s

J

y

LV ?TA TK TAT CIT K

L

L

Y

L

TX TIC DRi ATG NX G\T TAT PGI OT TIT OT ATA G A 73 D P Y Y ~ S P ~ R G F G I

CicI GW

E

&

14-

271 91

I

L

K

N

L

I

Q

Y

A

h

P

C

R

C

S

121

K c N G C M T I C T K C L % C C A W A ~ A 4 T W A O X T r X W C & % C T I C 417 S ~ ~ F L Y ~ E W ~ E 139 P

S

I

~

F

TA"AUACAACAcl7PaTlKTGWCETrXNXWAWWTo:PGI665

Y

K

R

B

D

R

S

R

L

S

I

E

E

5

W

P

155

C K " l C M A ~ W f J C W W K T K C T A A U A T C C C A K A W W 513 L F K I O X E Y L L ~ ~ A T E171

E

rn " 575

m

"

618

m

701 764

-w & ""100-fold over background). Essentially all of this activity was precipitable by the specific antisera. Translation of an unrelated

"RNA was transcribed from pSAThl or from pCM9 (a plasmid containing acDNA for S-adenosylmethionine decarboxylase (30) which was used as a negative control) and 1-pg aliquotswere translated in a reticulocyte lysate for 1 h a t 30 "C (13, 30). Aliquots were then removed and used for the assay of Spd/Spm acetyltransferase activity. In experiment R, the aliquots were treated overnight with rabbit antisera to rat Spd/Spm acetyltransferase, or to AGT (0"alkylguanine-DNA-alkyltransferase;used as a control) followed by precipitation with protein A priortodetermination of Spd/Spm acetyltransferase activity (15). One unit of Spd/Spm acetyltransferase activity represents the formation of 1 pmol of N'-acetylspermidine in 10 min. Results are shown as the meanof duplicate observations or as the meanf S.D. for more than three observations.

mRNA(CM9) produceda proteinthathadno acetylase activity. Northern and SouthernBlot Analysis Using pSAThl-We bis(ethy1)spermine-treatedNCI H157 next examined whether cells exhibited a time-dependent increasein steady-state Spd/ Spm acetyltransferase mRNA as our previous studies have suggested (15). Cells were exposed continuouslyto 10 p M bis(ethy1)spermine for increasing times, andmRNA was isolated for Northern blot analysis(Fig. 4). The results indicate a dramatic time-dependent increase in the Spd/Spm acetyltransferase steady-state mRNAlevels during treatment with the analogue. Since the Spd/Spm acetyltransferase mRNA level of the uninducedcells is so low, it isdifficult to estimate the precise fold increase which peaks 12 h after treatment and then appears to decline, consistent with the fact that a

H u m a n SpermidinelSpermine N'-AcetyltransferasecDNA

813

mer andpSAThl recognize the same size mRNA in bis(ethy1)spermine-induced NCI H157. The increase in mRNA levels corresponds favorably to the timecourse induc18s"Actin tion of enzyme activity we have previously reported for NCI -SSAT H157 cells induced with bis(ethy1)spermine (12). Theprotein sequence predicted by pSAThl agrees with 81 of the 83 amino acids determined from polypeptides produced by Lys-C digesFIG. 4. Steady-state spermidine/spermine N'-acetyltranstions which we have previously reported (15). The two misferase message levels in NCI H1.57 h u m a n large cell undifferentiated lung carcinoma after exposure to N',N''- matches (residues 69 and 84) are not unusualsince, as stated his(ethy1)spermine. NCIH157cellsweretreatedwith10 FM above, they were the second residue of sequential cycles (the l)is(ethyl)sperminefor 0 h, lane 1 ; 6 h, lane 2; 12 h, lane 3; 24 h, lane amino-terminal sequencing yielded Gly-Gly andPro-Pro 4 ; 48 h, lane 5 , RNA was then extracted and separatedhy formalde- whereas the cDNA codes for My-His and Pro-Trp). It is likely hyde/agarose gel electrophoresis and transferred to a nylon memthese errors in the amino-terminal sequencing procedure rehrane. This figure is a composite that represents a single blot, hybridized sequentially to pSAThl and then a human p-actin-specific sulted from carry-over between cycles. The mostconvincing evidence suggesting that pSAThldoes cDNA. Each lane was loaded with 5 p g of total cellular RNA. The relativemigration of the28 S and 18 S ribosomalRNA areas represent the authentic Spd/Spm acetyltransferase gene prodindicated. uct is provided by the translation of the T7 promoter transcripts from the pSAThl/Bluescript plasmid. The translation 1 2 products from pSAThl appear to be identical in size ( M , -20,000) to thepurified protein and to the in. vitro translation 12kbproducts from produced from poly(A)' mRNA isolated from 64" d bis(ethy1)spermine-treated cells (15). The pSAThl transla3d tion product is also recognized by the specific antisera. Most \ importantly, the translation product has the biological act.ivity to N'-acetylate spermidine in the standard in vitro assay system. It should be noted, however, that it is currently not known FIG. .i. Southern blot analysis of the human spermidine/ spermine N'-acetyltransferase gene. Five-figsamples of high whether pSAThl corresponds to the entire coding region of molecular weight human lung cancer DNAs were digested IScoRI with the Spd/Spm acetyltransferase gene, since it is possible that (lane 1, NCI H157, lane 2, NCI H82). Fragments were separated by additional in-frame protein translation start sites exist upelectrophoresis in a 1% agarose gel, transferred tonylon membranes, stream from the knownsequence. It is unlikely that more and hyhridized to the nick-translated plasmid pSATh1. Relative sizes than a few, if any, aminoacids are missing from the pSAThl in kilobases areas indicated. coded protein, since its translation product has activity and number of cells are actuallydying after extended exposure to appears tobe identical in size to both silver-stained authentic bis(ethy1)spermine (12). Reprobing with human @-actincon- human Spd/Spm acetyltransferase protein and to the transfirmed that unequal loading could not account for the ob- lated mRNA from bis(ethy1)spermine-treated cells (15). Additionally, the designated -5 to +1 nucleotidesequence of servedincreasein Spd/Spmacetyltransferasesteady-state pSAThl fit the most important criterionfor translation inimRNA. The nick-translated pSAThl clone was used to perform tiation as reportedby Kozak (40). The observed induction of Spd/Spm acetyltransferase acSouthern blotanalyses. DNA was isolated from the analoguetivity in NCI H157 cells in response to bis(ethy1)spermine sensitive NCI H157 and NCI H82 cells that do not respond to the analogue with cytotoxicity or significant induction of treatment (13) appears to resultfrom significant increases in acetylSpd/Spm acetyltransferase (12). The DNA was then digested steady-state message levels. ThismakesSpd/Spm transferase unique among the highly regulated enzymes inthe with the restriction enzyme EcoRI, and fragments were separated by agarose gel electrophoresis (Fig. 5). There are no polyamine metabolic pathway, since neither ornithine decarapparent differences in Spd/Spm acetyltransferasegene copy boxylase nor S-adenosylmethionine decarboxylase appear to be regulated similarly in response to this class of analogues number between the cell types. (41, 42). The availability of the cDNA probe will now allow DISCUSSION more indepth investigation intothe precise mechanisms Although the inducibility of Spd/Spm acetyltransferase by regulating the observed increase in Spd/Spm acetyltransferseveral stimuli has been known for several years, the mecha- ase mRNA. We havepreviously demonstrated that thedeplenisms underlying this process are only now coming to be tion of the natural polyamines canlead to profound changes in the transcription of important growth-related genes (43, understood (15, 35-39). The study of Spd/Spm acetyltransferase regulation has been limited by the lack of cDNA probes 44). Therefore, it is an intriguing possibility that the polyfor the mRNA responsible for the protein production. In the amines or polyamine analogues may directly or in combinacurrent studywe have isolatedand characterizeda cDNAthat tion with other factors lead to increased transcription of the codes for an active human Spd/Spm acetyltransferase protein. Spd/Spm acetyltransferase gene or changes in mRNA halfif the observed increases This process was facilitated by the availability of the human life. It will be of interest to determine large cell undifferentiatedcarcinomalineNCI H157 that of Spd/Spm acetyltransferase in the heat shockinduction responds treatment to with the polyamine analogue system (37) and the hormoneinduction system (7) are also a bis(ethy1)spermine with >l,OOO-fold induction of Spd/Spm result of increased Spd/Spm acetyltransferase mRNA. Although Spd/Spm acetyltransferase is known to be a rapacetyltransferase activity (12, 15). (6, 45), it is There are several lines of evidence presented here which idly inducible and rapidlydegradedprotein signal or PEST sequences desupport the hypothesis that pSAThl is an authentic cDNA important to note that the representing the transcript of the human gene for Spd/Spm scribed by Rogers et al. (46) for a majority of other rapidly acetyltransferase. Both the32-fold degenerate 20-base oligo- turning over proteins, are absent from pSAThl. Therefore, 1 2 3 4 5

28s-

814

SpermidinelSpermine Human

unlike ornithine decarboxylaseand S-adenosylmethionine decarboxylase, each which have PEST signal sequences (19,47), the Spd/Spm acetyltransferase protein appears to possess alternate mechanisms to signal for its rapid degradation. In summary, the availability of a cDNA clone which codes for an active human Spd/Spm acetyltransferase should provide a useful tool to examine the regulation of this important polyamine metabolic enzyme and potentially provide a model system for understanding at a molecular level how the polyamines can effect specific gene expression events. Acknowledgments-We wish tothank Drs. Stephen B. Baylin, Kenneth Kinzler, and Janice Nigro for helpful discussions, Amy Mank for her technical expertise, and Tammy Hess and Sandra Lund for their administrative assistance.

N'-Acetyltransferase cDNA 20. 21. 22.

23. 24. 25. 26. 27. 28.

REFERENCES 1. Seiler, N. (1987) Can. J. Physiol. Pharmacol. 65,2024-2035 2. Pegg, A. E. (1986) Biochem. J. 234,249-262 3. Matsui, I., Wiegand, L., and Pegg, A. E. (1981) J. Biol. Chem. 266,2454-2459 4. Della Ragione,F., and Pegg, A. E. (1982) Biochemistry 21,61526158 5. Della Ragione, R., and Pegg, A. E. (1983) Biochem. J. 2 1 3 , 701706 6. Persson, L., and Pegg, A. E. (1984) J. Biol. Chem. 2 5 9 , 1236412367 7. Shinki, T., and Suda, T. (1989) Eur. J. Biochem. 1 8 3 , 285-290 8. Mamont, P. S., Duchesne, M. C., Grove, J., and Bey, P. (1978) Biochem. Biophys. Res. Commun. 81,58-66 9. Luk, G. D., Goodwin, G., Marton, L. J., and Baylin, S. B. (1981) Proc. Natl. Acad. Sci. U. S. A. 78,2355-2358 10. Casero, R. A., Go, B., Theiss, H. W., Smith, J., Baylin, S. B., and Luk, G. D. (1987) Cancer Res. 47,3964-3967 11. Casero, R. A., Ervin, S. J., Celano, P., Baylin, S. B., and Bergeron, R. J. (1989) Cancer Res. 4 9 , 639-643 12. Casero, R. A., Celano, P., Ervin, S. J., Porter, C. W., Bergeron, R. J., and Libby, P. R. (1989) Cancer Res. 4 9 , 3829-3833 13. Denstman, S. C., Ervin, S. J., and Casero, R.A. (1987) Biochem. Biophys. Res. Commun. 1 4 9 , 194-202 14. Porter, C.W., Bernacki, R. J., and Bergeron, R. J. (1990) in Anticancer Drug Discovery (Valeriote, F., and Coebett, T., eds) Klowar AcademicPublishers, New York, in press 15. Casero, R. A., Celano, P., Ervin, S. J., Wiest, L., and Pegg, A. E. (1990) Biochem. J. 270,615-620 16. Berger, F. G., Szymanski, P., Read, E., and Watson, G. (1984) J. Biol. Chem. 259,7941-7946 17. Kahana, C., and Nathans, D. (1985) Proc. Natl. Acad. Sci. U. S. A. 82,1673-1677 18. Gupta, M., and Coffino, P. (1985) J. Biol. Chem. 260,2941-2944 19. Pajunen, A., Crozat, A., Janne, 0. A., Ihalainen, R., Laitinen, P.

29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47.

H., Stanley, B., Madhubala, R., and Pegg, A. E. (1988) J. Biol. Chem. 2 6 3 , 17040-17049 Bergeron, R. J., Garlich, J. R., and Stolowich, N. J. (1984) J. Org. Chem. 4 9 , 2997-3001 Bergeron, R. J., Neinns, A. H., McManis, J. S., Hawthorne, T. R., Vinson, J. R. T., Bartell, R., and Ingeno, M. J. (1988) J . Med. Chem. 3 1 , 1183-1190 Bergeron, R. J., Stolowich, N. J.,and Porter, C.W. (1982) Synthesis 3 9 , 689-692 Okayama, H., Kawaichi, M., Brownstein, M., Lee, F., Yokota, T., and Arai, K. (1989) Methods Enzymol. 154, 3-28 Gubler, U., and Hoffman, B. J. (1983) Gene (Amst.) 25,263-269 Short, J. M., Fernandez, J. M., Sorge, J. A,, and Huse, W. D. (1988) Nucleic Acids Res. 16, 7583-7600 Southern, E. M. (1975) J . Mol. Biol. 9 8 , 503-517 Thomas, P. S. (1980) Proc. Natl. Acad. Sci. U. S. A. 77,52015205 Casero, R. A., Baylin, S. B., Nelkin, B. D., and Luk, G. D. (1986) Biochem. Biophys. Res. Commun. 134,572-579 Jacobs, K.A., Rudersdorf, R., Neill, S. D., Doughtery, J. P., Brown, E. L., and Fritsch, E. F. (1988) Nucleic Acids Res. 16, 4637-4650 Sanger, F.,and Coulson, A. R. (1978) FEBS Lett. 87,107-110 Sanger, F., Coulson, A. R., Barrell, B.G., Smith, A. J. H., and Roe, B. A. (1980) J. Mol. Biol. 1 4 3 , 161-178 Stanley, B., Pegg, A. E., and Holm, I. (1989) J. Biol. Chem. 264, 21073-21079 Krieg, P. A., and Melton, D.A. (1984) Nucleic Acids Res. 12, 7057-7070 Kameji, T., and Pegg, A. E. (1987) J . Biol. Chem. 2 6 2 , 24272430 Pegg, A.E., Madhubala, R., Kameji, T., and Bergeron, R. J. (1988) J. Biol. Chem. 2 6 3 , 11008-11014 Libby, P. R., Bergeron, R. J., and Porter, R. W. (1989) Biochem. Pharmucol. 38,1435-1442 Fuller, D. J. M., Carper, S. W., Clay, L., Chen, J.-R., and Gerner, E. W. (1990) Biochem. J. 267, 601-605 Pegg, A. E., Wechter, R., Pakala, R., and Bergeron, R. J. (1989) J . Biol. Chem. 264, 11744-11749 Pegg, A. E., Pakala, R., and Bergeron, R. J. (1990) Biochem. J. 267,331-338 Kozak, M. (1984) Nucleic Acids Res. 1 2 , 857-872 Porter, C. W., Berger, F. G., Pegg, A. E., Ganis, B., and Bergeron, R. J. (1987) Biochem. J. 242,433-440 Pegg, A. E. (1988) Cancer Res. 48,759-774 Celano, P., Baylin, S. B., and Casero, R. A. (1989) J. Biol. Chem. 264,8922-8927 Celano, P., Baylin, S. B., Giardiello, F. M., Nelkin, B.D., and Casero, R. A., Jr. (1989) J. Biol. Chem. 263,5491-5494 Erwin, B. G., and Pegg, A. E. (1986) Biochem. J. 238,581-587 Rogers, S., Wells, S., and Rechsteiner, M. (1986) Science 2 3 4 , 364-368 Ghoda, L., van Daalen Weters, T., Macrae, M., Ascherman, D. and Coffino, P. '1989) Science 2 4 3 , 1493-1495