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
Vol.
187,
No.
3,
BIOCHEMICAL
1992
AND
BIOPHYSICAL
RESEARCH
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
Vol.
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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AND
.
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COMMUNICATIONS
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-.
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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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