Vol. 187, No. 3, 1992 September 30, 1992

BIOCHEMICAL

RESEARCH COMMUNICATIONS

AND BIOPHYSICAL

Pages

1493-1502

STRUCTURgOF TBE SUDANSPlIRHIDlXg/SPEBMHE N1-ACETYLTRANSFE GENE (Exon/intron

gene

I&dxiaof. Paul gthylin

Wang JabszB3,

organization

to Xp22.1)+

and localization

Celanol. Amy B. Msnk', Constance Griffi.n1,2, Anita L. Eawkinsl, and Bobert A. Casero.

Jr.'**

bhe Oncology Center, 'The Department of Medicine, and %e Department of Pediatrics, The Johns Hopkins University School of Medicine, 424 N. Bond Street, Baltimore, MD 21231 Received August 20, 1992 SUMMARY: The super inductionof speaidine/spemi.neH1-acetyltransferase (SSAT), has been implicated ia the cytotoxic response of human solid tumors to the bis(ethyl)polyamines. The SSAT response is a phenotype specific response and is wdulated at the level of increased steady-state mRHA levels and enzyme protein. Tha humangenomlc region (4,095 bases) containing the coding sequence of SSAThas been cloned and localised to the 4~22.1 region. Primer extension analysis indicates the transcription of SSAT starts 179 bases upstream from the translational start site and appears to be under the control of a "TATA-less" promoter. The availability of this human clone will facilitate the direct B 1492 Academic Press. Inc. functional examination of the SSAT gene.

ISTRODUCTIO~: The polyamines are polycationic

components of all

and are required

for cell

growth and, in some cases,

survival

of the polyamines

to bind DNA, RNA and other macromolecules

eukaryotic (1).

cells

The ability

led to investigations

which indicate the polyamines have a role in the regulation of gene expression (2). The intracellular concentration of the polyamines is highly regulated and is under the control of both synthetic and catabolic steps (3). decarboxylase and S-adenosylmethionine decarboxylase are the control the biosynthetic catabolic

pathway and are each highly

pathway

spermidine/spermine in

response

hormones,

to

is

under

control

of

N'-acetyltransferase several

polyamines

stimuli.

and polyamine

regulated the

(SSAT). including

toxic

analogues

(5-11)

and inducible

Ornithine points of (4).

The

tightly regulated enzyme, The SSAT activity is induced insults,

growth

or by thermal

factors,

stress

(12).

+SequencedatafromthisarticlehavebeendepositedwiththeEMBLJGenBankDataLibraries under AccessionNo.Zl4136HSSSPNlAG. *To whom correspondence should be addressed. Abbreviations used: BESpm, N1.N12-bis(ethyl)spermine; IPCR, inverse polymerase chain reaction; RT, reverse transcriptase; SSAT. spermidine/spermine N'acetyltransferase (EC 2.3.1.57); SSC, lx SSC is 0.15 l4 NaCl, 0.015 M Na,citrate.

1493

0006-291X/92 $4.00 Copyright 0 I992 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

187,

No.

Recently, and the

3,

1992

BIOCHEMICAL

BIOPHYSICAL

ve have demonstrated the association cytotoxic response to a class

bis(ethyl)polyamines this

AND

(13).

In an attempt

enzyme in response to treatment

RESEARCH

of the induction of polyamine

to investigate

vith

COMMUNICATIONS

the polyamine

of SSAT activity the analogues,

the regulation analogues,

of the

we initiated

the cloning and characterization of the human SSAT cDNA (14.15). This clone was useful in determining that an increase in SSAT activity in response to the bis(ethyl)polyamine

analogues

mRNA which

increase

codes for in polyamine

report

characterize

we present

location, transcriptional human SSAT gene. This regulation induction

with an increase in steady-state Since these studies indicated an

the SSAT protein (14). or polyamine analogue dependent transcription

gene, ve sought to further of its expression. In this

was associated

start information

of the SSAT

the SSAT gene to examine the regulation

the sequence, genomic structure, chromosomal cis-acting elements of the site, and putative should

facilitate

the further

study of the

of the human SSAT gene and aid in the study of the transcriptional in response to cellular exposure to the polyamine analogue, N1,N12-

bis(ethyl)spermine

(BESpm).

significance in determining regulation of gene expression

Additionally, the specific (16).

these role

that

data

may

polyamines

have play

general in

the

HATERIMS AND HXTRODS Analysis of Human Genomic DNA. Twenty pg each of genomic DNA isolated from either the human large cell lung carcinoma line (NC1 H157) or the human placenta was digested with different restriction endonucleases (GIBCO/BRL, Gaithersburg. MD). The digested DNAs were separated on 0.8% agarose gel, transferred onto the Zeta membrane (Bio-Rad. Richmond, CA), and hybridized with the human SSAT cDNA labeled by random priming (17).

Restriction

PCRGloning. Three pairs of oligonucleotides designed from the human SSAT cDNA were used as the primers to amplify the genomic counterparts by PCR (18). The PCR was performed with l-2 pg of the NC1 H157 genomic DNA or normal human placental genomic DNA in a total volume of 50 ~1 containing 67 mM Tris-Cl(pH8.8, at ZSOC), 16.6 mM N&SO,. 6.7 mMMgCl,. 10 mM 2-mercaptoethanol, 6.7 @i EDTA, 1.5 mM each dNTP, 10% DMSO, 10 pmol of each primer and 2 units of Amplitaq Taq DNApolymerase (Perkin-Elmer, Norwalk, CT). The 30 cycles of amplification consisted of denaturation at 94'C for 45 set, annealing at 53OC for 2 min, and extension at 7Z°C for 2 min. After the last cycle, samples were incubated at 72OC for 10 min. The PCR products vere purified by electroelution and blunt-end ligated into pBluescript SK plasmid (Stratagene, LaJolla, CA). For cloning the 5' flanking region of the SSAT gene, an inverse PCR strategy (IPCR) (19) was used. Briefly, aliquots of DNA were digested separately with BglII, HinfI, PstI. and Sac1 (GIBCO/BRL). The digested DNA was self-ligated for circularization at the final concentration of 1 pg/ml in ligation buffer (Nev England Biolabs, Beverly, MA). The ligated DNA was subjected to 35 cycles of PCRwith two oligonucleotides with opposite orientations. The sequences of the oligonucleotides used in cloning were FL1 (5'ATAGGMTTCCGTCACTCGCCGAGGTTCCT).P3 (5'GCATTGTTGGTTTTGCC). (5'TCATCACGMGAAGTCC).Pll(5'CGACTGCAGTGACATAC). P15 (5'CAGCTGCGAATCGGAGG), P:: (5'GCTAMTTCGTGATCCG), P23 (5'CCTTTGCCTCCATTTGG),XPl (5'CTCTTCTCAGTCAGGCTGGC). XP3 (5'CTCTAAGTACCAGTGTGTAC). XP4 (5'CCCCCGACAAGTGACCTTGC). XP5 (5'TACTGGGGGACACACCGGCT), XP6 (5'GCACTTCTGCAACCAGGCAG). and XP7 (5'AGACACGTTCAGATAAGCM) (Figure 1). 1494

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DEASequencing and Analysefs. The PCR-generated genomic clones were sequenced by the double-stranded dideoxynucleotide method (20) using the Sequenase 2.0 Kit (United States Biochemical Corporation, Cleveland, OH). The SSATgenomic sequence was derived from sequencing of both strands of each clone and overlapping clones with either universal or specific primers. PCR and in situ hybridization techniques were used to localize the human SSAT gene. For PCR, a complete somatic cell hybrid panel (BIOS Corporation, New Haven, CT) consisting of 25 Chinese Hamster Ovary (CHO)/human hybrids, each containing regions of one or more human chromosomes was used to determine the chromosomal localization of the human SSATgene. PCRwas performed with 1OOng of hybrid DNA and lOpmo1 of each human specific primer (P39: S'TCAGAAACCCACTATGATAC; and P40: S'GCTCTCCCCATTTGTTGAGT) as described above. The genomic probes LPSAT 1 and LPSAT3 (Figure 1) were nick-translated with biotin-14 substituted dATP (GIBCO/BRL). Slides with chromosome spreads were made fromnormalmale PRA-stimulated lymphocytes, cultured in the presence of S'bromodeoxyuridine (21) with slight modifications. Probe mix (2 x SSC, 50% formamide, 10% dextran sulfate. 4-6 ng/pl of each biotin-labelled probe, 400 x salmon sperm DNA. and 200 x Cot DNA) was denatured at 70°C for 5 min. placed on slides and hybridised at 37OC overnight. Slides were washed in 50% formamide/ x SSC at 37OC for 20 min. and twice for 4 min in 2 x SSC at 37OC. Biotinylated probe was detected with FITC-avidin following instruction of the manufacturer (Gncor, Inc.).

ChraroscmalLocali.zatian.

Primer

Extension Analysis. The transcription start site was determined by primer extension analysis as published (23.24) withmodification. Total cellular RWAwas isolated from the NC1 Ill57 cells treated for 24 h with 10 PM bis(ethyl)spermine (25). A 20-base oligonucleotide (XPl) complementary to the nucleotides between positions -93 and -112 relative to the translation initiation site was labeled using T4 polynucleotide kinase and [r-32P]ATP. Forty pg of total RNA and 1 pmol of r3'P] 5' end labeled primer were hybridized and extended with Moloney murine leukemia virus reverse transcriptase (GIBCO/BRL). The primer-extended products were separated on 69 polyacrylamide/S M urea gel and exposed to X-ray film for 2 h at -7OOC.

EESDLTS AND DISCDSSIOR

Several pairs of oligonucleotides designed from the human SSATcDNA sequence were used as the primers to define the

Cloning

of

the

Eman

SSAT Genumic

Sequence.

human SSAT genomic

constructs by PCR techniques. With primers FL1 and P8, corresponding to the most 5' non-coding region and amino acids 93-99 of the SSAT protein, a 2.2 Kb fragment was amplified and subcloned. This SSAT genomic

sequence contains

two complete exons and defines

three complete introns.

Primers

P3 and XP3, corresponding to amino acids 70-75 of the SSAT protein and the 3' non-coding region positioned at the 20 bp upstream of the polyadenylation signal These two genomic (MTAN e vere used to generate a 832 bp genomic fragment. constructs together span the intact coding region of human SSAT. Other primers designed from the genomic and cDNA sequence were used to generate a series of overlapping genomic clones to confirm the sequence information. The 1.1 Kb 5' region of the human SSAT gene was obtained using the inverse Two restriction endonucleases PstI (+210bp) and Sac1 PCR (IPCR) technique. (+252bp) were first selected to separately digest the NC1 II157 genomic DNA for 435 bp and 538 bp fragments from PCR IPCR. After 35 cycles of PCR amplification, 1495

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No.

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1992

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COMMUNICATIONS

E f I

I q u

- ;i ‘, no.

= 5 m

5: a-f

$$

b A s

- ---E m$ mu $2; T

7 f

-+

-3--L+ FL1

-= p

P3 -3

e

XP7

2

pll

t

+

XP6

~23

-5-t Pi5

t XP3

Pa

C LPSAT 1

500

BP

Pigaue 1. acetyltrmsferase

Exon-intron gene.

were obtained

from PstI-

of

structure

the

humsn

spermidine/spexmine

IV'-

Physical map of the SSAT gene. The six exons are sites are as indicated. numbered and representedby filled boxes. The restriction B. Strategy for PCR cloning: each arrow indicates the origin and direction of the Double-headed arrows 4 and b represent the core region and the primers. orientation of primers used in the inverse PCR. 4. XP4 and XPS; b. XPl and P16. Genomic probes LPSATl and LPSAT3. C. Probes used for in situ hybridization. A.

and SacI-digested

The sequence of these IPCR products

NC1 8157 genomic

demonstrated

DNA,

respectively.

sequence identity

in the 521 bp

overlapping region. Purther 5' "walking" was performed by IPCR after HinfI and BglII digestions. The subsequent overlapping IPCR clones supplied the 5'-flanking sequence information. The entire sequence of the human SSATgene is available

in the lMBL/GenBank

data base, accession numbers 214136. This sequence demonstrated no homology to any other sequence in the GenBank data base other than the SSAT cDNA (14.15). Organization SSAT

of

the

Ruman

S!WF

Gene.

The overall

genomic sequence from the above PCB clones

length

of the assembled human

is 4,095 bp (Figure

1). These

clones of the human SSAT gene span the entire cDNA coding region, define the first five complete axons and introns, and represent all but 20 bases of the sixth exon based on the cDNA sequence. The clones also define 1,097 bases upstream from the transcriptional start site. The physical map of this construct is consistent with the patterns human SSAT cDNA (Figure 2).

of genomic Southern

blotting

probed with

the

The sequences of the exon/intron junctions and sizes of the exons and introns for the human SSATgene are shown in Table I. All of the splice junctions in the SSATgene conform to the consensus sequence established for intronic donor (5' GT A/G

AGT 3') and acceptor

(5'

(Py)nN C/T 1496

AG

3')

splice

signals

(26).

Axon

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187,

No.

3,

1992

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Kb -23.1 -9.4 6.6 -4.3 -2.3 .2x)

,0.56

Figure 2. Bastriction analy8ls of human genaic DlJAs. Twenty pg of genomic DNA fromNC1 B157 cells was separately digested with restriction endonucleases BamlU (Be), BglII (Bg), EcoRI (II), Bind111 (H), PstI (Ps), PvuII (Pv), ScaI (SC), SphI to the human SSAT cDNA. (SP). and XbaI (X). The Southern blot was hybridized Molecular sizes sre es indicated in kilobases.

1 contains

sequence.

the

5'-untranslated

Exon 6 contains

and the

sequence the

sequence

coding

for

first the

SSAT, the stop codon (TGA). and the 3'-untranslated The sequences of the exons is identical

to that

last

66 bp of 56 amino

the

coding

acids

of the

sequence of the SSAT cDNA. of the cDNA previously

reported

(14.15). Chrowsomal

Ucalixation

of the Euman SSATGene.

using a complete somatic cell

Table Exon no. 1 2 3 4 5 6

Rxon size,

bp

238 50 03 102 41 >485

* Exon sequences

I.

Exon-Intron

Sequence 5' splice ATC MG AAAG GM G ACA G AGC

hybrid

CAG

Organization

at exon-intron donor gtagcg gtaatt gtaacc gtacga gtatgt

are in uppercase

--------------------letters,

The human SSAT gene was mapped

panel by PCR and in situ

of the Human

junction' acceptor

ccccq ttgcag ttacag ttttag taatag

GAG CTG AT CTG

1497

SSAT

GA CAC

GC TTT GTT GCA sequences

The

Gene

Intron

3' splice

and intron

hybridization.

siee, 239 89 1,444 06 117

Codons bp

interrupted Lys=Cl@ Asp" Gly" Gly"s Gln115-Va1116

are In lowercase

letters.

Vol.

187,

No.

Table II.

3,

1992

BIOCHEMICAL

Segregation

of the SAT

AND

BIOPHYSICAL

geac with human chromosomes

RESEARCH

in human-hamster

COMMUNICATIONS

(CHO)

somatic cell hybrids

Human chromosome

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

X

Y

Concordant hybrids* (+/+) 01 (-/-I

19

21

18

22

6

20

20

21

21

19

21

18

Ill

17

18

19

20

18

15

19

15

20

25

18

Discordant hybrids+ (+/-I 01 c-/+1

6

4

7

3

19

5

5

4

4

6

4

7

7

8

7

6

5

7

10

6

10

5

Q

7

24

16

28

12

76

20

20

16

16

24

16

28

28

32

28

24

20

28

40

24

40

20

Q

.78

% Discordancy Coocordant 1) contain 2) did not + Discordant 1) contain 2) did not l

hybrids are the number of the hybrids thati all or a portion of a particular human chromosome and produce the specific hare a particular chromosome or the PCR product. hybrids are the number of the hybrids thal: a particular human chromosome, hut not the PCR product; or contain a particular human chromosome, hut have the PCR product.

specific

PCR products

were only

present

with

contained the human X chromosome as determined (D. Dickerson, L. Weber. E. Hickey, personal 904). The scoring the specific

or

DNA from hybrid by cytogenetics communication.

for the SSAT gene was determined

PCR band which corresponds

PCR produc$

cell

lines

that

or PCR analysis regarding hybrid

by the presence or absence of

to a 354 bp human SSAT genomic fragment

in the third intron. The results are summarized in Table II. Chromosome X showed a 0% discordancy, whereas the discordancy for the other chromosomes ranged from 12% to 76%. On this

basis,

the human SSAT gene was assigned

to chromosome X.

Analysis of 292 metaphase cells showed 50 cells had at least one pair of signals involving both chromatids of a single chromosome; only paired signals were scored. Of fifty-one paired signals, 44 (86%) were at the approximate midpoint of a large C-group chromosome, and 7 (14%) were on various other chromosomes (none consistent). To identify the C-group specific band, the slide was G-banded by FPG (fluorescence with modifications)

(21).

After

banding,

39 (86%) were on chromosome X, primarily

40 of the 44 signals

chromosome and the plus Giesma method could be analyzed:

band ~22.1 or the interface

between p21

and ~22.1 (Figure 5); lwas on chromosome 6, and the remaining 4 chromosomes with signal were unidentifiable (banding quality was insufficient). We therefore conclude that this gene is localized to Xp22.1. Analyaia

of the 5' Flanking Region of the Ruman SSAT Gene. The 165 nucleotides upstream from the translation start site in the human SSAT cDRA corresponded to the sequence of the genomic DNA (15). Primer extension analysis using a primer within this sequence identified a major transcriptional start site and potentially a minor start site in the human SSAT gene at -179 and -160 relative to the translation start site, respectively (Figure 3). The minor extension product

(-160)

may be

the

result

of 1498

incomplete

elongation

by

reverse

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1992

AND

.

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1

ACGT

FQure3. Primer extension analysis. A synthetic oligonucleotide primer XPl complementary to the 5' region of the human SSAT mRNA vas 5' end-labeled, hybridized to the total RNA of BESpm-treated NC1 H157 cells, and extended. The major primer extension product is indicated by the arrow. The sequence ladder (dideoxynucleotide termination method), generated with the same primer on a cloned human SSAT genomic DNA template (PIPS-13). shown in lanes A, C, G, and T, vas used as size markers.

transcriptase

in this

previously site,

reported again,

step

possibility

that

cannot

flanking

from

due

this

site.

this

starting

at

-67.

starting

at

-136

area, -108 and

described

in TATA-less

sequence

of

the

transcriptional (30-32). site

-341

and

shown

and two

gene Spl.

a unique

bp and -910

has been

-125; Areas

bp,

Spl

three of

regions

site

respectively.

to be critically

putative

at the

1499

for

upstream is about

transcriptional

of the

the

previously

The 5'

sites

and CCMT

for

binding

are potential

transcription

repeats repeats

been

(28.29).

-41 bp and there

The presence important

promoter

G+C content

have

binding

CREB, OCT-NF3,

the

(GGGCGCC) direct

genes

5'

promoter,

(GAGAGG)tandem

pair pair

l,lll-bp

sequence

the

the start

of that

the

G+C content

of other

contains AP-4,

showed

The average

7-base

high

reverse

the putative

However,

6-base

start

the

transcriptional

-211 bp and -41 bp from

are

promoter

SSAT

is

is G&-rich.

there

-200.

factors, There

site between

We have

However,

sequence

region

(27).

of

region.

encompasses

of this

TATA box or CAAT box start

this

nucleotide

which

The sequence

in

-154).

translational

an authentic

The

SSAT gene,

170 nucleotides In

contributes

to the termination

structure

discounted.

the transcriptional the

premature

secondary also

4).

has no apparent

start

at

to

5'AGTCGCGGGCCGACT3'

has 165 bp 5'

potential

region

(Figure

(-168

cDNA (15)

the

of the human

was determined

70% over

structure

the

be presently

region

region

that

indicating

transcription site

hairpin

flanking several proteins

two CCMT boxes Spl-binding initiation

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-1097

gaatcaagtagggattacaacaacrttcaacagcaaccgaagtcagaagacagat~cc~t

-1037

ceatgtgctgsaagaggaaaacttcttctg*~cctcaaaatctata*~ctatga*at*aaccc

RESEARCH

COMMUNICATIONS

CCAAT-1 -977

ttcaagaattttaagatgggctggtccaasggtagtgagttatctc~ttcac

-917

aatCaPt~aCaCatCaBaCtCcttattrtactctgactactgcacttgactagtct~*a

-857

aaagaattstaagatgaattaaagacacgttcagstaagcaaaactgagaacgtttggct

-797

aaagcagatctttacteaaggaaattatgaagaatg~agaatgt~cctcgggc~gaagg~a~agtgt

-737

caagatcaagtgtcaeggsigattagagatttaagaaggtataagaaggttagaaaagta

-677

agsaasaaatatgccgtcaactctaaaaacracaagtatataaagaaaaaaaattattggga

-617

aatccaaataacctaccttspaaaattctaaagatatacgagctcacattccttggtcata

-557

agagcagtgtcatcattagacatcaggtcgcctctggaaaattccattgtgagtttaaa~ OCT-NF3/CREB ~caaataacntctataattctaaaactagttctcccaccgaaactctagaaaggatctt

-497 -437 -377 -317 -257 -197

gggatctgcagggtctcccggatcacactttgagaactttgagaactgctgctttagtcttcctcccat CCAAT-2 ctcctaaggccagagctc~ccaaggagaa*agagcaaggtc OCT-NF3 acttgtcggggggctgcagagggaattaccttctttc~tttgcaaatgtt*ctgggggac acaccggctcccagtagggtttccgccaaggctccgcgaaggctccgcgaaacgccactagagggcgccgc AP-4 tagcgaa~cccacaacgcgccccgctgcccccacttttgtcctccgggttcacacgggcg

*cwnHrf K*c*

-137 -77 -17 +44

cccgga~tggtg=~tg~~~~g=tgcgcct f* SPl tgga~cccacctcacggcc~ttctttgcgcttaaaagccgagc +1 x cgggccaatgttcaaatGCGCAGCTCTTAGTCGCCGGCCGACTGGTGTTTATCCGT~ACT CGCCGAGGTTCCTTGGGTCATGGTGCCAGCCTGACTGAGMGAGGACGCTCCCGGGAGAC **~**~~1~~~*~~~**1~~~

Figure 4. The 5' flauking region of the inmau SSAT gene. Nucleotide numbering is used for primer relative to the transcriptional start site. The oligonucleotide extensions is depicted by a jagged line. The major transcriptional start site is indicated es +l; the minor start site is shown with "W'. Potential binding sites for transcription factors (Spl, AP-4. OCT-NP3. and CREB) and CCMT boxes are double-underlined. Three tandem repeats of the sequence GAGAGG ere boxed; two 7base pair (GGGCGCC) repeats are shown by asterisks underneath the sequence. The transcribed sequence is shovn in uppercase letters with an underline.

directed

by the TATA-less promoter

(33-35).

Transcription

factors

such as Spland

CCAAT box transcription factor (CTP) have also been proposed to regulate transcription through coactivators or adaptors (32). However, the functional contribution human SSAT

of each of the above cis-acting gene remains to be determined.

The availability facilitate the further

elements

in the expression

of the

of the human genomic sequence and structures will now study of the regulation of the human SSAT gene by the

polyamines and their analogues. Additionally, such studies may yield information as to the general roles which polyamines may play in gene transcription (16). Finally, the examination of SSAT gene regulation in the differentially sensitive human lung tumors may yield interact with the polyamines

information to regulate 1500

on cell-type specific SSAT expression.

factors

which

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12 13

21

q

22 23

24 25 26 27 26

Pigrrre 5. Ideogram of human X chromosome. as

translated

to

20 G-banded

Each

dot

represents

a paired

signal

metaphases.

The authors vish to thank Drs. Kenneth W. Kinsler and Jon Oliner for helpful discussions, Jeff Smith for expert technical assistance. and Tammy Hess and Sandra Lund for their skilled administrative assistance. This vork was supported in part by NIH Grants CA51085. CA47492. GHlOlS-02, CA06973, and HG00373. A-.

REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Tabor, C. W. and Tabor. 8. (1984) Annu. Rev. Biochem., 53, 749-790. Eichler. W. and Corr. R. (1989) Biol. Chem. Hoppe Seyler, 370, 451-466. Pegg. A. E. and McCann, P.P. (1982) Am. J. Physiol., 243, C212-C221. Pegg, A. E. (1986) Biochem. J., 234, 249-262. Sailer. N. (1987) Can. J. Physiol. Pharmacol.. 65, 2024-2035. Della Ragione, F. and Pegg, A. E. (1983) Biochem. J., 213, 701-706. Persson, L. and Pegg, A. E. (1984) J. Biol. Chem., 259,12364-12367. Erwin, B. G. and Pegg, A. E. (1986) Biochem. J.. 238. 581-587. Casero, R. A., Celano, P., Ervin, S. J., Wiest. L. and Pegg, A. E. (1990) Biochem. J., 270, 615-620. Porter, C. W., Ganis, B., Libby, P. B. and Bergeron, B. J. (1991) Cancer Res., 51. 3715-3720. Shinki, T. and Suda, T. (1989) Eur. J. Biochem.. 183, 285-290. Puller, D. J. H.. Carper, S. W., Clay, L., Chen, J. R. and Gerner, E. W. (1990) Biochem. J.. 267, 601-605. Casero, B. A., Hank. A. B., Xiao, L., Smith, J., Bergeron, B. J. and Celano, P. (1992) Cancer Res., in press. 1501

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ACKNOWLEDGMENTS We have benefited from useful conversations with Michio D. Kuwabara. ‘Ihis research was supported by USPHS GM 2 I 199 and GM 39558.

1. :* 4: 2: 7. 8. 9. 10. 11. ii: 14. 15. 16. 17. 18.

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intron gene organization and localization to Xp22.1).

The super induction of spermidine/spermine N1-acetyltransferase (SSAT), has been implicated in the cytotoxic response of human solid tumors to the bis...
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