THE
JOURNAL OP
BIOLOGICAL CHEMISTRY
THEJOURNAL
OF BIOLOGICAL
0
1988 by
The American Society
Society for Biochemistry
Biochemistry and Molecular Biology.
Biology, Inc.
© 1988
hy Tbe
Vol. 263,
7, IIseue
3202-3207,1988
263. No.
No. 7.
••ue of March 5. pp. 3202-3207.
1988
p,.inted in U.
S.A.
Printed
U.S.A.
Characterization of aa cDNA Clone for the Nonspecific
Nonspecific Cross-reacting
(NCA) and a Comparison of NCA and Carcinoembryonic
Antigen (NCA)
Antigen*
Antigen*
(Received
(Received for publication, August 28,
28, 1987)
1987)
Michael NeumaierS,
Neumaier:j:, Wollgang
Michael
WolfgangZimmermann§,
Zimmermanns, Louise Shively, Yuji Hinoda,
Hinoda, Arthur D. Riggs, and
E. Shively
John E.
From the
the Division of
o{ Immumbgy,
Immunology, Beckman Research Institute of
o{ the City
City of
of Hope,
Hope, Duarte, California 91010 and the SIrntitut
§lnstitut
ffür
u r Immunbiologie der Universität,
Uniuersitat, 07800
o{ Germany
D7800 Freiburg,
Freiburg, Federal Republic of
~·
••
•
."
Section 1734
1734 solely
solelyto indicate this fact.
The
sequence(s) reported in
The nucleotide sequencds)
in this paper has
hos been submitted
to the
with accession
the GenBank™/EMBL
GenBankTM/EMBL Data Bankwith
accession number(s)
J03550.
503550.
:t$ Recipient of fellowship
fellowship support from
from the Deutsche Forschungsgemeinschaft.
gemeinschaft.
1 The abbreviations used are: CEA,
CEA, carcinoembryonic
carcinoembryonic antigen;
antigen;
nonspecific cross-reacting antigen; kb,
kilobase; BGP, biliary
NCA, nonspecific
kb, kilobase;
glycoprotein;
bp, base
isopropyl-1-thio-0-o-galactopyranoside;
bp,
glycoprotein; IPTG, isopropyl-l-thio-ß-D-galactopyranoside;
pairs.
3202
Downloaded from www.jbc.org at UBM Bibliothek Grosshadern on August 4, 2008
i
t:
antigen), aa glycoNCA (nonspecific
(nonspecific cross-reacting antigen),
glycoCarcinoembryonic antigen (CEA)l
(CEA)’ is a 180-kDa
180-kDa highly glyglyprotein found
found in normal lung and spleen, is immunoimmuno- cosylated glycoprotein.
Gold and
glycoprotein. CEA was
was first described by Gold
logically
logically related to carcinoembryonic antigen (CEA),
(CEA), Freedman inin 1965
1965 (1)
(1) as aa colon tumor-associated antigen.
which is found
found in over
over95%
95%of colon
colon adenocarcinomas.
adenocarcinomas. Immunoassays for CEA have found wide acceptance as a
genomic library, we previously cloned diagnostic tool in primary elinical
From a human genomic
clinical diagnosis of colon cancer
part of an NCA gene and showed that the
the amino-ter- as well
weIl as in the long term monitoring of patients following
following
minal region has extensive
mind
extensivesequence
sequence homology to CEA colorectal
colorectaI tumor resection. The original concept that CEA is
(Thompson,
(Thompson, J. A.,
A., Pande, H., Paxton, R. J., Shively, aa tumor-specific antigen was abandoned when smaH
small amounts
L., Padma, A., Simmer, R. L., Todd,
Riggs, A.
Todd, Ch. W., Riggs,
of CEA were found in normal adult colon (2,
3). In addition,
addition,
(2,
n.,
E. (1987)
Natl. Acad. Sei.
D., and Shively,
Shively,J. E.
(1987) Proc. Natl.
Sei. U.
U. several CEA-related antigens expressed in normal
and
maJignormal
maligS.
S. A. 84,2965-2969).
84,2965-2969). We now present the
the nucleotide
have
been
described.
the
nonspecific
nant
tissues
described.
NCA,
nonspecific
crosssequence
clone, containing the
theentire coding
coding
sequence of aa cDNA clone,
normallung
lung and spleen
region of NCA (clone
(clone 9).
9). The clone
clone was obtained from reacting antigen, was first described in normal
1972 (4,5).
(4, 5). Using monoclonal antibodies, Buchegger et al.
aa X
~gtiO
g t l O Iibrary
library made from the colon carcinoma cell in 1972
(6) have differentiated between two molecular weight forms
forms
line SW 403; the
theclone
clone contains a a34-amino acid leader (6)
NCA: NCA-55,
NCA-55, a 55-kDa glycoprotein
glycoprotein found
found in granuloamino acids for
forthe
themature
mature
protein,
sequence, 310 amino
sequence,
protein,
andandof NCA
ceHs, and NCA-95, a 95-kDa glycoprotein
glycoprotein
1.4 kilobases of 3'
-untranslated region of the NCA cytes and epithelial cells,
3’-untranslated
found only in granulocytes.
granulocytes. Other CEA-like
CEA-like antigens include
gene. A comparison
comparison of the NCA sequence
sequence to the
the CEA found
(7), two
128-kDa colon tumor-associated antigen (7),
sequence
sequence (Oikawa, S., Nakazato, H., and Kosaki, G.
G . a unique 128-kDa
weights 160
meconium antigens of molecular weights
160 kDa (NCA-2)
(NCA-8)
(1987)
Biochem. Biophys.
Biophys. Res. Commun.
(1987) Biochem.
Commun. 142,
142, 511- meconium
518;
Zimmerman, W.,
W., Ortlieb, B., Friedrich, R., and and 100
(7, 8), and an
100 kDa (7,8),
618; Zimmerman,
an85-kDa biliary glycoprotein
glycoprotein (BGP
(BGP
S. (1987)
(1987) Proc. Natl.
U.S.
von Kleist, S.
Natl. Acad. Sci.
Sei. U.
S. A. I)
I) (9).
(9). CEA-like
CEA-like antigens in the serum of normal blood donors
84, 2690-2694)
2690-2694) shows that both proteinscontain
contain have been isolated and characterized with monoclonal
antimonoclonal antidoublets
doublets of an immunoglobulin-like
immunoglobulin-like domain,
domain, of which bodies
bodies to CEA (10).
(10). Four antigens with molecular weights
weights of
there are one copy in NCA and three
threecopies
copies in CEA, aa 200,
200, 180, 114, and 85
85 kDa were found.
found. The 180180- and 85-kDa
108-amino
108-amino acid amino-terminal domain with no cyscys- antigens correspond to CEA and BGP
respectively. The
BGP I, respectively.
a carboxyl-terminal hydrophobie
teine residues, and a
hydrophobic identities of the remaining antigens are unknown, but these
domain of sufficient length
lengthto anchor the glycoproteins and the
meconium antigens may arise from
themeconium
from CEA,
CEA, BGP I, and
in the cell membrane. Overall, the corresponding
correspondingcodcod- NCA by
byproteolytic eleavage
cleavage or may be
bedistinct gene products.
85% sequence
sequence homology at the All of the antigens are obviously
ing regions possess 85%
obviously related by sharing common
amino acid level and 90%
90% homology at the nucleotide
antigenic determinants
determinants but,
but,in most cases,
cases, each has also been
level. Forty nucleotides 3'
3’ of their stop codons,
codons, the
shown
to
antigenic
determinants. For CEA
possess
unique
CEA and NCA cDNAs
cDNAs become dissimilar. The 108108sequence information has demonstrated
amino acid amino-terminal region together with part and NCA, amino acid sequence
homology between the two antigens and
of the leader
leader peptide sequence
sequence corresponds
corresponds exactly
exactly to extensive sequence homology
immunoglobulin supergene
supergene family
family
aa single exon described in our previous work. The
Thedata placed them within the immunoglobulin
(11).
(11).
presented here
herefurther demonstrate the
thelikelihood that
Earlier, we reported the sequences
sequences of a partial
partial genomic
CEA recently evolved from NCA by gene duplication,
NA clones for CEA (13).
(13). These
NCA (12)
(12) and cD
cDNA
including two duplications of the immunoglobulin-like
immunoglobulin-like clone for NCA
data demonstrated that CEA and NCA each contain aa 108108domain
domain doublet of NCA.
amino acid amino-terminal domain which has no cysteine
copies of a
residues. In addition, CEA contains multiple copies
178 amino acid residues,
residues, each of which has four
*“This
This workwas
was supported by National Cancer Institute
Grant domain of 178
Institute Grant
disulfide loops.
loops. Oikawa
Oikawa
Scheel Stiftung für
f(lr cysteine residues and presumably, two disulfide
CA37808
from the Dr. Mildred Scheel
a grant from
CA37808 and agrant
al. (14)
Krebsforschung.
(14) obtained aanear
nearfull-Iength
full-length clone for CEA,
CEA, extending
Krebaforachung.The costs
costa of publication of this article were defrayed
defrayed et at.
in part by the payment of page charges.
charges. This articIe
article must therefore our data and
copies of this
andconfirming the occurrence of three copies
be hereby marked "aduertisement"
U.S.C.
“aduertisemnt” in accordance
accordance with 18
18 U.S.C.
178-amino
178-amino acid immunoglobulin-like
immunoglobulin-like domain in CEA.
cDNA
3203
NCA
NCA cDNA Clone
Clone
ble-stranded
ble-stranded sequencing.
sequencing.Commercially
Commerciallyavailable
available Bluescript sequencsequencing
ing primers were
were used (Stratagene)
(Stratagene) or,
or, alternatively, 1717-or 18-residue
18-residue
oligonucleotides
oligonucleotides were designed
designed from
from known sequences
sequences of the insert
DNA.
(2 /lg)
pg) was
wasalkaline-denatured in 100
100
DNA. Supercoiled
Supercoiledplasmid DNA
DNA (2
mM
mM NaOH in
in aa total
total volume
volume of 22 /ll
pl and heated to
to 65°C
65 "C for
for 5 min.
min.
After addition
50 ng of the respective
respective primer, the sampie
sample was
was
addition of 50
neutralized by addition of 2 /ll
pl of 11M
M sodium
sodium acetate,
acetate, pH
pH4.5, quickly
quickly
preeipitated
precipitated with 2.5
2.5 volumes
volumes of 100%
100%ethanol and
and kept on solid
solid dry
dry
ice
ice for
for about 20
20 min.
min. The
The DNA
DNA was then pelleted by centrifugation
centrifugation
MATERIALS
MATERIALS AND
AND METHODS
METHODS
once with
in a microfuge
microfuge for
for 10
10 min at
a t 4°C,
4 "C, washed
washed once
with 70%
70% ethanol,
ethanol,
Chemicals-Nitrocellulose
Chemicals-Nitrocellulose filters
filters were purchased from
from Schleicher and dried.
dried. Annealed
Annealed primer/template hybrids
hybrids could
could be
be kept up to 2
&
& Schuell.
Schuell. Restriction enzymes
enzymes were
were from
from Boehringer Mannheim and weeks
weeks at -20°C.
-20 "C.
Bethesda Research Laboratories. S,
SI nuclease,
nuclease, T.
T, polymerase,
polymerase, T.
T,
For sequencing
sequencing with avian
avian myeloblastosis
myeloblastosis virus reverse
reverse transcriptranscripkinase,
kinase, and
and T.
T, Iigase
ligase were from
from Bethesda Research Laboratories. tase and Klenow
Klenow polymerase,
polymerase, nucleotide
nucleotide working
working solutions
solutions were
were as
as
Avian
myeloblastosis
virus
reverse
transcriptase
was
from
Life
SeiAvian myeloblastosis
reverse
was from Life Sci- given
given by
byZagurski
Zagurski et al.
al. (22)
(22) and Strauss etetal.
al. (23).
(23). The
The dried primer/
primer/
ences
ences and deoxy
deoxy and dideoxy
dideoxy nucleotides
nucleotides were from
from Pharmacia LKB
LKB template
template hybrids were resuspended
resuspended in deionized
deionizedwater and the
theapproapproBiotechnology
7 polymerase,
36
BiotechnologyInc. For sequeneing
sequencing with T
T7
polymerase, the Sequenase
Sequenase priate reaction buffer to
S1
to give
give aa volume
volume of 15
15 /ll.
pl. Four /ll
pl of [a[a-35S]
kit from
from United States
States Biochemicals
Biochemicals were
were used.
used.Sequeneing
Sequencing of singlesingle- dATP
dATP (500
(500 Ci/mmoi)
Ci/mmol) were
were added
added and the
thesequencing
sequencing reactions were
stranded DNA
DNA was
was performed using
using the Amersham
Amersham Corp.
Corp. sequencing
sequencing carried out as
23). For sequencing
as described
described (22,
(22.23).
sequencing with
with the Sequenase
Sequenase
3
32
kit. [a-"'PjdATP,
P1ATP, [aßPldCTP,
and [a·S1dATP
were
[a-32P]dATP,(-y-[y3'P]ATP,
[LY-~'P]~CTP,
and
[(u-~'S]~ATP
kit the primer/template hybrids were resuspended
resuspended in aa total
total volume
volume
from
England Nuclear.
from Du Pont-New England
Nuclear. All
All other reagents were
were of of 15
15 /ll
pl consisting
consisting of the reagents supplied
supplied with the kit and 10
10 /lCi
pCi oe
of
analytical
36
analytical grade.
grade.
[aSjdATP. The
[a-36S]dATP.
The sequeneing
sequencing reactions were then performed
performed accordaccordConstruction
Construction and
and Screening
Screening of the
the cDNA
cDNA Library-Total
Library-Total cytocyto- ing
ing to
to the
theUnited States
States Biochemicals
Biochemicalsprotocol.
protocol. Sampies
Samples were run on
on
plasmic
SW 403
403 cells
cells according
according to
to Dashai
Dashal 4-8% polyacrylamide
plasmic RNA
RNA was prepared from
from SW
polyacrylamide gels
gels (0.4
(0.4 mm),
mm), using
using standard size
size gels
gels as
as weil
well
et
of poly(A)+
RNA as 85-cm-Iong gels with up to two loadings per sampie .
et al.
al. (15)
(15) and
and cDNA
cDNA was
was 8ynthesized
synthesized from
from 55 /lg
pg of
poly(A)' RNA
as 85-cm-long gels with up totwo loadings
sample.
essentially
essentially as
as described
described by Maniatis et
et al.
al. (16).
(16). Double-strand cDNA
cDNA
was
T, polymerase
polymerase and then
thentreated with
was rendered blunt-ended witb
with T.
RESULTS
RESULTS
T.
ends of tbe
NA moleeules
were
T, kinase to
to ensure
ensure that all
all 5' ends
the cD
cDNA
molecules were
phosphorylated.
A cD
NA library was constructed in Xgt10
cDNA
X g t l O using mRNA
mRNA
For convenient cloning
cloning into
into >.gtlO,
XgtlO, asymmetrical
asymmetrical EcoRI
EcoRI adapters,
adapters, isolated from
403 and
from the
the colon
colon carcinoma
carcinoma cell
cell line
line SW
SW403
also
NA by
ClaI site,
site, were
were added
added to
to ends of the cD
cDNA
byblunt screened with a 1400-bp
also containing aaCIaI
1400-bp EcoRI
EcoRI probe
probe from
from a genomic
genomic NCA
end ligation
ligation (16).
(16). The
The sequence
sequence of the adaptor was
was
Since
itsrelationships
relationships to
Since the biological
biological role
role of CEA
CEA and its
other members
not clear,
members of its gene
gene family
family is
is still not
clear, we have
have
continued our efforts to isolate
isolate and characterize CEA-like
CEA-like
NA clone
genes.
genes. In this
thisreport, we present data on
on an NCA cD
cDNA
clone
isolated from
from a colon
colon tumor cell
cell line and a comparison
comparison of
NCA with CEA.
CEA.
••
•
."
clone
200,000 clones
clone (12).
(12). Seven
Seven positives
positives out of200,000
clones were
were obobtained, one
9) also
also hybridized with a probe
one of which (clone
(clone 9)
specific
specific for
for the amino
amino terminus of NCA. Restriction analysis
analysis
clone with EcoRI
EcoRI revealed
revealed two
two fragments of size
size 2.1
2.1
The
ClaI site
site is
is underlined. Only
Only the 5' end ofthe
of the shorter strand (14
(14 of this clone
The CIaI
1.4 kb.
kb. When the
the clone
clone was
was cut with ClaI
ClaI the fuIl-Iength
full-length
nucleotides)
nucleotides) was
was phosphorylated, and a 200-fold
200-fold excess
excess of adaptor and 1.4
over
over calculated cDNA
cDNA ends
ends was
was used during the ligation
ligation reaction (16).
(16). 3.5-kb
3.5-kb insert was
was obtained.
obtained. The
The 2.1-kb
2.1-kb fragment hybridized
After
After size
size selection
selection by gel
gel electrophoresis and
and phosphorylation of the to probes from
EcoRI
from the
the coding
coding region
region of NCA or CEA. Both EcoRI
5'
fragments larger than 600
were c10ned
5' ends,
ends, fragments
600 bp were
cloned into
into dephosphodephospho- fragments
the 3.5-kb
fragments as
as weIl
well as
as the
3.5-kb ClaI
ClaI insert were subcloned
subcloned
rylated arms
arms of >'gtl0
X g t l O (Stratagene,
(Stratagene, San
San Diego,
Diego, CA).
CA). For in vitro
vitro
linker sites of the phagemid Bluescript.
into the
therespective
respective poly
polylinker
Bluescript.
packaging,
(Stratagene) were
used. A into
packaging, Gigapack
Gigapack packaging
packaging extracts (Stratagene)
were used.
-tracking of recombinant clones
T-tracking
clones
total
total of 55 XX 10·
10' independent recombinant
recombinant clones
clones were
were obtained. The
The Restriction analysis and T
2.1-kb fragment showed
showed that only
only one
one orienorienlibrary was
was amplified
amplified using CES
CES 200
200 cells
cells (rec
(rec BC-), which
which reduces
reduces containing the 2.1-kb
loss
were
loss of clones
clones containing repetitive sequences
sequences (17).
(17). Approximately
Approximately 2 tation of the insert was
was obtained when the colonies
colonieswere
X
X 10·
lo5clones
clones were screened
screened according
according to
to the method of Benton and grown
grown in the presence of IPTG
IPTG and X-ga!.
X-gal. However,
However, by first
Davies
(18)with aa 1400-bp
1400-bp EcoRI
EcoRI fragment from
from the genomic
genomic NCA growing
Davies (18)
growing the cells
cells on nitrocellulose
nitrocellulose filters
filters without induction
induction
clone
NCA
clone >'39.2,
X39.2, which codes
codes for
for a portion of the Ig-like
Ig-like domain of NCA
the
opposite
orientation
ofthe
insert
was
obtained.
by
IPTG,
IPTG,
the
opposite
of
the
insert
obtained.
(12).
(12). Seven
Seven strong
strong positives
positives were
were obtained. Rescreening
Rescreening was
was done
done
We also
also observed
observed aa considerable
considerable improvement
improvement of color
color develdevelwith
with a 561-bp
561-bp Sau3A
SalL3A fragment of the
the same
same genomic
genomic clone
clone that codes
codes We
fully grown
grown colonies
colonies were
were induced
induced by transferfor
for a portion of the
the amino-terminal
amino-terminal domain
domain of NCA.
NCA. One
One clone
clone (clone
(clone opment when fully
9)
9) was
was found
found to
to be positive with
with this
this probe. Labeling
Labeling of the
the probes ring the filters
filters to plates containing IPTG and
and X-ga!.
X-gal.
was
was performed as
as described
described elsewhere
elsewhere (19),
(191,and
and hybridization
hybridization condicondiThe
The 1.4-kb
1.4-kb fragment was
was sequenced
sequenced by standard dideoxy
dideoxy
tions were
were as
as given
given in the protocol
protocol by Carni
Cami and
and Kourilsky
Kourilsky (20).
(20).
methodology
However, the
methodology using single-stranded DNA (24). However,
5'
-AATTCCGTATCGATGTGC
5"AATTCCGTATCGATGTGC
GGCATAGCTACACG-5'
GGCATAGCTACACG-5'
Subckming
Subcloning into a Phagemid
Phugemid Vector-Restriction
Vector-Restriction analysis
analysis of clone
clone
99 showed
showed that the
theinsert was
was 3.5 kb
kb and contained an
an EcoRI
EcoRI site.
site. The
The
two
two EcoRI
EcoRI fragments
fragments of the insert were
were 2.1
2.1 and
and 1.4
1.4 kb.
kb. The
The 3.5-kb
insert obtained by CIaI
ClaI digestion
digestion and
and the
the EcoRI
EcoRI fragments
fragments were
were gelgelpurified and subcloned
subcloned into the phagemid
phagemid Bluescript (Stratagene),
(Stratagene),
which
be used to
to produce
produce either single-stranded DNA
DNA or supersuperwhich can
can be
coiled
coiled double-stranded DNA.
Because
Because color
color discrimination was
was poor and only one
one orientation of
the insert DNA
DNA could
could be obtained in recombinant clones,
clones, a procedure
procedure
involving
developed. In some
involving plating on
on nitrocellulose
nitrocellulose was
was developed.
some experiexperiments the transformation mix was
wasspread directly onto
onto nitrocellulose
nitrocellulose
filters
plates lacking
filters on LB/amp plates
lacking IPTG and X-gal.
X-gal. After overnight
growth
growth of the transformed bacteria, the
the nitrocellulose
nitrocellulose filters
filters were
transferred onto
onto plates containing
containing 55 mM
mM IPTG and
and 40
40 /lg/ml
pg/ml X-gal.
X-gal.
Excellent color
color discrimination was
was obtained 2 to
to 4 hhafter
aftertransfer
transferto
to
the plates containing
containing inducer and substrate. Plasmid DNA from
from
clones was
was prepared by the method of Hattori
Hattori and
recombinant clones
and
Sakaki
Sakaki (21)
(21) or by banding in cesium
cesium chloride
chloride gradients (16).
(16).
DNA Sequence
Sequence Determination-The
Determination-The 1.4-kb
1.4-kb fragment
fragment was
was sesequenced
quenced by standard
standard M13
M13 single-stranded DNA
DNA methods.
methods. The
The 2.12.1and
and 3.5-kb
3.5-kb fragments
fragments were
were sequenced
sequenced by using
using double-stranded plasmid
mid DNA. Three
Three different enzymes
enzymes and
and procedures
procedures were
were used
used at
at
various
various times.
times. Sequencing
Sequencing with T,
T7polymerase
polymerase was
was performed with
the
the Sequenase
Sequenase kit from
from United States
States Biochemicals
Biochemicals adapted
adapted to
to doudou-
;;:
g
w
I
..,c
-46
i
i
I
. .
<
8
Ul
I
..,
.0
x
I
C
i
;;:
i
0
,;J
I I
I
r=-
~
200
2W bp
bp
FIG.
FIG.1.
1. Restrietion
Restriction map
map and
and sequencing
sequencing strategy
strategy for
for NCA
cDNA
contains 1.0
-flanking
cDNA clone
clone 9.
9. The
The 3.5-kb
3.5-kb insert contains
1.0 kbofof 5'
5"flanking
-untranslated region,
bp of an open
region,
region, 1.5
1.5 kb
kb of 3'
3'-untranslated
region, and 1032
1032bp
open
reading
boxes, contains
reading frame.
frame. The
The open
open reading frame,
frame, shown
shown by boxes,
contains aa
leader sequence
sequence (L,
(L,102
102 bp),
bp), an amino-terminal
amino-terminaldomain
domain (N-term,
(N-term, 324
324
bp),
bp), an Ig-like
Ig-likerepeat (repeat,
(repeat,528
528bp), and a acarboxyl-terminal
carboxyl-terminal domain
domain
(C,
(C, 78
78 bp). The
The internal EcoRI
EcoRI site
site begins
begins at
at nucleotide
nucleotide 2108.
2108. HoriHorizontal
arrows indicate
indicate regions
regions and orientation of sequence
sequence analysis.
analysis.
zontal arrows
The
The vertical
vertical bar indicates the position at which
which a cloning
cloning artifact in
in
clone
may have
have occurred
occurred resulting
resulting in
in the fusion
fusion of the NCA
NCA gene
clone 99 may
gene
to
to an
an unrelated gequence
sequence of 974
974 bp 5' of nucleotide
nucleotide -46.
-46.(See
(See text for
for
-flanking region.)
aa discussion
discussion of the
the 5'
5"flanking
region.)
Downloaded from www.jbc.org at UBM Bibliothek Grosshadern on August 4, 2008
'·
I
t:
3204
NCA cDNA
cDNAClone
a
~
I
.'~~ TJ~~(jl~.lC~ !ß}.I~AGAA TGa: TTCGOC Tce TTTCOCCOC eGGT'" ffOGGTGTATA r~ TceeAC TC TC"'C fClCA,. AGGA~t'" TC TCfAAGl AGA TCA' ATA(:ACCAGM TACC&GCGGCG'A
GGAIZCGAAGC TCAAGGGTMA~AGT AGI.M TA TU. TTCAGTMACAA TM JGTGHiMC lrTT.fiOCA TG~ TM r NiGOC'" TGGAC TGolG Ta: TOC TA TC r TGMA TGra:ACAGGfACACTTAteT T
100
TTl TTn
THTTT TTTMGT Tl TTCCCA TTCAGGA TAAtAACA TT&TGA TC TGTAC TACAGGM,CCM,A
1GfC" TGeerCA TACATGTGGC
TA TMAGJACATMAAU.
TI. TC TMe u r TCA TAATGTGG
~
_
.
~
GGI GGGTAA TAC TGe JGTCiMA TM TGI AAGMGC TnTCA(' TAJN.MAA TGe'" TTAC TlTC"'C TTMeAC TACiACACCAGGTCGAAAA TT TTCAAGGTTATAGrAC. n ... TTle"ACAA.TTCT TAGAGl
TQ: T.AGe T"GTGYT GlAtZ. TTMM TAOC ITl AT TAß. TOCTGM T'GTGA TTl Tl TTA TOCCMM TTl TTTT
m
TerM TCA TTGA TG\ r "ocr TGGAM TMIt TM TTA r OCCA TGOCA Tl TGt.C ACT
ICAn Ancc IA !MGM Tl... TlGAG TTTAGAGAGAA ,GGiil2IGTl ... Ge lGA TlA Tl MCAGI IAC lGAM ICW IM IAn ATllGT IACAnA I ICCA Tl IGIA I I Tl AGGT TTCC I Tl TACA I
IC I I TlA I AICJ:I!IflIC ''''CA TlACA lAI TTT TTMGAC TA IGGW IM TTl...... Tl IMGe TC IGGIGGA 'GA TTA TC lGe IMGI lAGTC lGAW IGIM JA TlT
TG TC.ACACMA TIX 1 n TC TU. TeTA Tl AACC n GAGTA Tl OC.AGTT Ge TCl: Tl TGI
bb
I AA TAC TGTM TA TACC
F
-46
-46
C 1 t A A ~ T C C T C T U : M A U G G T G U C A U U A U C A ~ A U U C CATG
ATG GGII.
G U cce
CCC cce
CCC TCA
1CA ocr
( E CC (CI
Cl
crCAAa:TccrcrAC.u~CÄiGTGGACAGAGAAGACAOC"'GAGACC
]": G
P PP
S P
A PS A P
cce Ta: AGA TlG (AT ere cce H'aG ANj GAG GTC eTC CTC -.cA OCC TCA eH OA Ace He teiG AM. CCA cce Ace ACT100OCC ~
JE, CTC ...Cl ATT GM Tee
PCRlHYPWKEVllTASllTfWNPPYTlKl
TIES
•
200
ACG (CA
cu He
TCI
MT Grc
G r c a:.A
m GAG
UG GGG
~ G GAAG
AIG GAG 6fT
GIT elT
c n c u. crc
crc occ
at (cACl t AN:.
uc (TG
C T G cce
ccc (AG
cu MT
MT (Cil
ctr ATT
iTr
G G ~TA(
r A c AC[
Aa2%
T ~ CAAA
uL GCG
tcG W
w ACA
AU GIG
GTG
AeG
AAT
GGI
TGG TAC
1
P r
FN
N V
V A
A E
E G
G K
K E
i V
V L
L L
L L
L A
A H
H
N
l P
P Q
Q H
N R
R Il G
G Tl SS W
Y T
t lKG
Gr R
i
R
V
T
P
N
l
V
w
2:
Ga Me
uc AGI
ACT (TA
C I A An
ATT eTA
t u G~
GU TAT
TAT GTA ATA
AT& GGA
G U "'1
ACT CM
CAA CM ocr
at ACe
Act (CA GGG
GtG (ce
ccc OCA
a A TN:
r e "GT GGT
GGT (GA
cu ~ lc~ ATA
A T 1 TAt
m c cce
ccc ,ur OCA
a h Tce
Tcc
~r GGe
D
G
N S
S lI
L V
I G
V fV
G I~GVT Q
I
G
T
~ PPGAP A
T T
P S
G G~ RAE T
~ I~' f P
G N
R A
E S~
D
G
N
Q
A
J
w
tu
cu
'00OCA
CTG ClG
ClG Are
A l C (Al>
C I G AK.
ALc GlC
GTC Ace
ACC CAG
ChG AlT
A T 1 GA(
W AO.
ACA GGA
G U I TTe
l C 1 TAT
A l ACC
ACC crA
C I A CA}.
C M GTe
GTC ATA
A l AA UG
I G Tc.1U GAY
U T CTl
C l l GTG
GlG MT
U T rN.
UL GU
d %
A ACe
ACC GGA
G U CA{;
C I G He
1TCCAT
CTG
(AT
L lI
L Q
l N
Q V
N T
V
l Q~ H
N O
O T
l G
G fFYYTll Q
L Y
P IVKI SKOSL OVLM
V [[
U A
E T
i G
A Q
T JGM
Q
l
I
V
~
H
AM AGG
AGG AN:.. GAT
U T IXA
a AGeiA
GU Tec
TCC TAl
111 GM
GU HiT
167 GM
W ATA
AlA eAG
C I G Me
M C co.
CCA GeG
KG AGT
AGT oce
OtC AJC.
AU eo:
C C AGT
A G l GtC
Ut ceA
CCA GTe
G l C ..ACC
C f G MT
Ml GlC
GTC700
C E TAT
TAT Ga:
GC ceA
CCA rAT
U T
AM
cc eTG
Cle
K
RR f l t
D O
A A
GG
S SVYt EtC~E lI Q
Q H
N P
P A
A SSAAI (NRRS O
S P
D V
~ T
V lH
l
lVNl Y
V G
L P
V D
G
P
K
O
~
P
N
500
GTA TA( eCG GAG ClG CCC AAG CCC lec ATe Tee All: Me Me Tee AAC CCC GTG GAG !i'.C A,~ GA.T (Lr (iT(i oce Tl, ACe TGT GM CCI GAG COl1 ( AG
VYPElPkP
S
JSSNNSNPY[OKDAYArTCEP(YO
600
~c ACA
ACA ACe
y c TAC
~ l eTG
CTG
t TeiG
TGG TGG
IGG
GTA .....
UT T Gel
CGT CAG
CIG A9:
A a Cle
CTC eeG
CCG Gle
GTC AGl
ACT cec
ccc ~
IGC eTG
C T G CAG
CIG (TG
CTGTCC
AAT ca:
Ga AM.
AAC ATG
ATG ACe
ACC eTe
€02 Aer
crc
ACI eTA
C T A erc
crc A([
h a eTe
GTC
Me
GYA
lee MT
L W
Y W
Y
VY
N N
G 6
OQ
S SL l P
P Y
V S
S P
P A
R lLQ
Ql S
l H
S G
N
G "N
n
ll ll
ll l
l
l S S Y V
H TT T V' r l
"
T
700
800
ou
GE cce
ccc Ace
ACC ATT
111 Tce
rcc cee
ccc TCA
TU AN:,
AIG OCC
EC MT
M T TAC
TIC CGT
ccr CCA
c u GGG
G ~ W.
GGU MT
MTCTG
CTG AN:.
AU CTC
crt TCC
Tcc TCG
TCG (A(
CAC OCA OCC
at TCT
TCT Me
MCceA
CCA ceT
ccr 8w
CA(AG
c y . TAC
r A c TeT
T C T lGG
TGG
GGC
OCA
G P
P
Tl II
SS PP SS K K AA I H Y Y , R. R P P GG ( E HW l L fit N L L S S CC HAI..
H
A
A SS I(N PP PP AA QQ 'f'1
Y
G
SS
W'
.
~
111 ATe
A l C MT
Ml GGG
GEG ACIi
ACG TlC
1TC CAG
CAG CA}.
C M Tce
TCC ACA
ACA CAA
C M GHi
WG erc
C l C TTT
111ATC
A l C CCC
CCC AN:.
AM ATC
ATC ACT
ACT GTG
GlG MT
MT MT
Ml A(I:
Aa G~
G U Tce
TCC TAl
TAT ATG
AlG Ta:
l a CM
C
900
M OC(
E C CAl'
C I l Aß(
AU T1CA
tA
TTl
I H
U G
G T
T F
F O
Q Q
Q S
S T
T Q
P E
E lr
L
FI P
1 IP( {
N Tl V
T Jl
Y tN
I
N G
S S
G ,"
S C
V O
~ A
C H
~ N
A S
H
U
I
S
rT
1000
EC ACT
A c r Ga:
Ga eTC
CTC MT
MT AGG.
AU ACC
ACC N:,J..
IU Grc
GTC AC6
ACG .H
AG
X ATe
CAI
AU Gle
GTC TCT
TCT GGA
GU ACT
KT ocr
a1 CCl
CCT GTC
GIC eTC
CTC TCA
ru OCT
DT GTG
GTG OCC
ac Ace
ACC GTe
GTC GOC
ca ATC
ATC
1 AeG
ACG AH
111 G~
GU GTG
GTG
Ge(
ACA
A
TT GGLLNNR R
TT
TT
VV
T lM
I
I II T
I V
V S
S G
( S
G A
A P
P V
V l
l S
SA
A Y
V A
I T
T Y
V
CGI I T
T I
I G
G V
V
A
~
S
wo
Downloaded from www.jbc.org at UBM Bibliothek Grosshadern on August 4, 2008
FIG.
FIG. 2.
2. a,
a, nucleotide
nucleotide sequenee
sequence of
of the
the
974 bp that flank
flank the
the NCA gene
gene at its
ita 5'
5'
end in elone
9. (For
(Foraa diseussion
discussion see
see
clone 9.
text.)
text.) The
The cloning
cloning adaptor
adaptor is
is shown
shown in
in
broken
broken lines.
lines. b,
b, nucleotide
nucleotide sequenee
sequence of
of
the NCA eDNA
cDNA gene.
gene. The open
open reading
frame
1, indieated
indicated
frame begins
begins at
a t nucleotide
nucleotide 1,
by
by the
the first
first Iwrizontal
horizontal arrow.
arrow. The
The second
second
Iwrizontal
horizontal arrow
arrow indieates
indicates the
the amino
amino terterminus
minus of
of the
the mature
mature protein. The
The third
third
horizontal
horizontal arrow
arrow indieates
indicates the
the beginning
beginning
of
of the
the Ig-like
Ig-like repeat,
repeat, and
and the
thefourth
fourth the
the
beginning
beginning of
of the
the hydrophobie,
hydrophobic, earboxylcarboxylterminal
terminal domain_
domain. The
The stop
stop eodon
codon and
and
internal EcoRI
EcoRI site
site are
are boxed.
boxed. The
The uervertical
indicates the
the proposed
proposed polypolytical arrow
arrow indieates
adenylation
adenylation site,
site, followed
followedbyby aa 14-nu14-nuc1eotide
(underlined). The
The
cleotide poly(A)
poly(A) tail
tail (underlined).
asymmetrie
asymmetric cloning
cloning adaptor
adaptor is
is shown
shown by
by
broken
brokenlines.
lines.
m
C l G GeC
CCC Nil:;
AGG GlC
GTC Gel
a1 ClG
CTG ATA
A T A~AOCCCIGGIGlAlTTTCGA'~TTIC"'&lAGAC
~ A ~ C C l G G ~ G l A l l T T C U l A l l l C f f IGGeA&lTTGGACCAGACCC~~CT'GeTCCTCCAATCCCAlTITATCCATGGMCCACl
iUAUCTG~AUTlGUCCAUCCC~~ClA~~CCTCCMlCCCAlTllAlCCAlGWCCACl
ClG
L
L A
A R
R V
V A
A L
L )1
1200
A M L I C M C G TTGC
C T TC
K TIGelce
ClETC
C l U L G TA
t C TA
C TTGC
A TTGG,AGA
A l G C TG(iACAACTCAA
T~UTGWCMC
l C M T1PW
U A M T l T W ~ C C C T C l G t C TGAGG
C l U GTGTGTGCCACTC"GAGAC
G l G l G l G C C L C T C ATG
L U CTlM
T ~e ATAGAGACAGGeM
CClMClAGLUCffi~I
MAAACMGGTC
IGMGCCC
TGMAA
TTTMAGGCä.U..U.eCCTCAGGCC
TC.ACC
1100
A CTGCMACeA
T E M C A l GTGGTGAGMA
G l U t l M l lTtGAeGAAC
U C ~ l l C1300
ATTeACAC
C A C T A TA
l GTGGACAGClfT
W C A G t l T T TCCeAAGA
T C C C M U TTGTCMAAeAAGACTCC
G T C ~ C M G L C T C C TTCltCATGA
C A I C A l UTMGGe.
l M ~ T CTClTTTAAceCCC
C C C C CnT
~ TTM
I MTlTITCTCC
T G T tHGC
C l TTCTACToce
~ l ATGCCTC
T O C C lTE C T C l
AC
1400
l T C ( + T l G C C A G U T U l E l G l U l T A G T A l T C A U A U A G l A ( E T l C A W G G G T M C T f M C A ~ G T A ~ C A U T T C 1 A l C l T G l C M l C C C M C C f1~
lT~~~lMlAAGIUlCCTlTAGT~ACCC
"00
TTCGC TTGGCAGG\ TG\ TOC TGTCA TI AGT AHCACAAGAAGT AOC TTCAGAGGGTMeTT MCAGAGTA TCA~ TlCTA TCHG reM rCCCUCG r TT TACA TA._.u TAAGA~ TCC n rAGT a:Aece
1600
A G l ~ C TGACA
~ C A TTTTAGCAOCA
A ~ A O C A Je
l CTTlMCACAOCCGTGTGTTCMA
T l l M C A C I O C C G l G T G T T UTGT
U T G.ACAGTG6TCC
l A C A G l G GTTTClTCAGA6T
C l T l l C ATGG\C
M G ~ lTTe
~ ~TlAGAC
l C l ATCAce
U C l UTGTTe
C C I GTCle
T l C Tcee
T C A CTGTTTTM
f C C C T G Tl
l lTCAAece
T l M T T fAOCCA
C M C CTCOCM
A ~ C AT(reA
T(EMItCA
AGTGAC
1100
A ATM.
T MTAGAA
T A WTll Ge
l GTeec
C T TACCAG(
C C C T ATGMCAGGGAGGAGTC
C C A G C f ~ ~ ~1700
G WTGTGCAGTTTCfGACACTTGTTGTTGMCATGOC
G l C l G l ~ A G T T T C l ~ C A C T T G TTAM
l G ~fACM
l ~ CTGGGTA
A l ~ TTCGe
~ I ATGAGAe
C M T fTAAGTTG
f i G lTAGAU.
A l C tTtTAACMA
l ~ ~ C fGTGe
l M G lTa:
l GTGT A ~ l l M ~ l G l ~ I ~
AI..
1800
I_
1600
CGT~TGttTACACTCATCTUCTCAllC~llAlIClAllllAGTlGGlTlGTAlCTlUClMffilEGlAGlCCMClCllGGlATTACCClCClMlAGTCAl~~~GTAGlCAlACTCCCTCGlGT
TAAAA TGGC
' ACAeTtA TC TGAC TCA TlC nTA He TA TfTTAGTTGGT nGU TC HOCC TAAGGTGCGTACT(CAA(TC TTGG TA TI Aece Tce TM TAGTeA TAC TAGTAG reATAe Teec TGGTGT
2000
~ G l GAlTlTC\JC
T lTAAAAOC
C I C l UT'I ATAAA
O C TT6lTC
l WTOCA
T G ITOCAOCCAOCCA
C l E A T G t A ~TeAM
C A & CUGlGI.A
A l U U TTGGTC
A G l WTCTTCTT
G G lTGOC
C T C lGW
I C l TTTACMMC
T G l i T G UTCAGAGAA.A
L ~ ~ M C l CTGTGT(A
A U U M TICAGGAGl.ACA
G l G l ~ T C h GTCA
~ WTMceeA
C A l C AJlGUGAA
M C C C ATAl U A ~ T A
AGTGT
2100
21AwC A C l C T C l C A C C l A t G T U G t G C A T T U t C C I t T G G f t C T A M l G C l A C A l A C l C C M C T U M T G T T A I G U l U f f i
I G Ge
C CCCceCAM.
I M TIfce
f i TlGeT
G G TAAC
M CTGA
lUlM
l ATGAGeAC
C A C lTM
M l ETGC
l l MTl
U AAGA
l l l f f i lTCTTGGTC
AI ......
TAl
ACAC TCTCTCA" TAGe TGAGC GeA TTGAGC eAGT GGT Ge TAM TGe TACA TAC TC e ...... c TGAM TGT TAAGGMC'aUG
22m
2200
2300
~ A ~TeCM
~ C C TTA.MMU.A
M T T U U " L Tl
~ TAMA((MTTT
T A U I C C M ~GAACJlAGGAGA
T T T U A U M P I U I W C A C ~ TCCCAGTC
~ ~ U U ~ CTACl
C C ATGAGfT
G ~ C AGCATAA
~ A C ~ ~ TACA!iMGTCCCC
U ~ T T A G C A ~ TC
M TTACT
A C TTAA(
A G U GTTTT
~ CAeAAAMAG
C C C ~2C3W
~ TAACCTGAAC
A C T ~ ~ A A TM
C~TTTAC~GTAACCT
AAT,a,GA
2~
~ C G.A
l ~TlGTT
G lMT eCM
I C C MTG'A
l C ITTT
A TATT ~HAClTGTGGTTCT
l T C I G I CGTGTlCC
T T CTTGTTCC""
T G l T l C CTTlGACMMCCCACT
l l G l l C C A A l l lGTTCTTGT"
U C ~ C C C ATTGTA
C l C lna:;CAGGGGGGAOC
T C l l G l A l T G lTA
A lTCAC
~ ~ ~TCT
~ UAC~ lnGr
I T CAGAGTGG'
A C l G l Aocr
C lOC
T GTlTTAAAU ~TC~ G G l ~ l ~ l T l ~ l C
Tet
2S00
2500
CA
C TAAJ,
I lreAcm
~ l CTAAMf:CAAT
A C U ITAGe
l TC~ TAT
E Me
A TAM/!.AAAAMAAAMGCAeATCGA
l T A C C l C I ATACGGM
I M Tl
C l ~ ~ ~ ~ ~ ~ ~ ~ ~ A ~ ~ ~ ~ ~
rI"
2.1-kb
2.1-kbEcoRI
EcoRI fragment
fragment and
and the
the3.5-kb
3.5-kb full-Iength
full-length insert
insert were
were
directly
directly sequenced
sequenced using
using double-stranded
double-stranded supercoiled
supercoiled plasplasmids
mids (22,
(22, 23).
23).For
For sequencing
sequencing of
of double-stranded
double-stranded DNA,
DNA, best
best
results
DNA polymerase.
polymerase. For
For the
the
results were
were obtained
obtained with
with the
the TT,7 DNA
3.5-kb
3.5-kb subclone,
subclone, only
only the
the region
region around
around the
the internal
internal EcoRI
EcoRI
site
site was
was sequenced,
sequenced, demonstrating
demonstrating that
that no
no small
small fragment
fragment
was
was lost
lost during
during subcloning.
subcloning.The
The restriction
restriction map
map and
and sequencsequencing
-flanking
ing strategy
strategy isisshown
shown in
in Fig.
Fig. 1.1.Clone
Clone99 has
has aalong
long5'
5'-flanking
region
regionof
of 1020
1020bp,
bp, followed
followedby
byan
anopen
openreading
readingframe
frameencoding
encoding
34
34 amino
amino acids
acids of
of aa leader
leader peptide,
peptide, 310
310 amino
amino acids
acids of
of the
the
mature
-untranslated region
mature protein,
protein, and
and 1430
1430 bp
bp of
of aa 3'
3"untranslated
region
ending
ending in
in aa poly(A)
poly(A) tail
tail of
of 14
14 adenine
adenine residues.
residues. The
The entire
entire
sequence
sequence of
of clone
clone 99 isis shown
shown in
in Fig.
Fig. 2.
2. Recently,
Recently, aa partial
genomic
clone for
genomicclone
for NCA
NCA has
has been
been reported
reported by
by us
us (12)
(12) and
and
Oikawa
. The
Oikawaet
et al.
al. (31)
(31).
Thegenomic
genomicclones
clonescontain
contain almost
almost 600
600bp
bp
of
ofsequences
sequencesupstream
upstreamof
of the
thestart
startof
of the
the NCA
NCAsignal
signalpeptide.
peptide.
/ -flanking region of clone 9
The
of the
the 55'-flanking
region of clone 9
The nucleotide
nucleotide sequence
sequenceof
(the
cDNA
containing
the
NCA
(the cDNA containing the NCA gene)
gene) diverges
diverges from these
these
genomic
genomicsequences
sequencesat
at position
position -46
-46 upstream
upstream of
of the
the translatranslational
tional start.
start. The
The genomic
genomic sequences
sequences surrounding
surrounding nucleotide
nucleotide
-46
. To
-46 could
couldbe
beinterpreted
interpreted asasan
an acceptor
acceptor splice
splicesignal
signal (32)
(32).
To
test
due to
test the
the hypothesis,
hypothesis, whether
whether the
the divergence
divergence was due
to an
an
alternative
alternative splicing
splicing event
event in
in the
the precursor
precursor mRNA,
mRNA, we
we rerescreened
screened the
the cDNA
cDNA library
library with
with aa probe
probe specific
specificfor
for the
the 5'5'flanking
flanking region
region of
of clone
clone 9.9. Two
Two independent
independent clones
clones (cDNA
(cDNA
clones
of 1.9
1.9and
and 1.5
1.5kb
kb were
were
clones13
13and
and2-3)
2-3)with
with insert
insert DNAs
DNAsof
-------------------
obtained
obtained and
and partially
partially sequenced
sequenced (Fig.
(Fig. 3).
3). They
They match
match the
the
sequence
-flanking region
sequenceof
of the
the 5'
5'-flanking
region of
of clone
clone 99 (containing
(containing the
the
NCA
NCA gene)
gene) only
only upstream
upstream of
of nucleotide
nucleotide -46.
-46. While
While there
there isis
no
no homology
homology to
to the
the NCA
NCA gene
gene downstream
downstream of
of this
this position,
position,
clones
show identical
ich makes
clones 13
13 and
and 2-3
2-3show
identical sequences,
sequences, wh
which
makes
them
them likely
likely to
to be
be transcripts
transcripts of
of the
the same
same gene.
gene. Both
Both clones
clones
have
have the
the polyadenylation
polyadenylation signal
signal AATAAA,
AATAAA,and
and clone
clone 13
13pospossesses
sesses also
also 23
23 adenine
adenine residues.
residues. In
In addition,
addition, the
the sequences
sequences
surrounding
surrounding the
the position
position of
of divergence
divergence from
fromclone
clone99 show
show no
no
indication
indication for
foraa donor
donor splice
splicesite.
site. Furthermore,
Furthermore, Northern
Northernblot
blot
analysis
analysis of
of total
total RNA
RNAobtained
obtained from
from SW
SW403
403cells
cellsshows
showstwo
two
NCA
NCA clone
clone 99 AATGCTTT'l'CTAATG'l'AT'l'AACCTTGlIGTATTGCAG'1"l'GCTGCTT'l'
AATGCTlTTCTAATDGCXTTIETGCTTT
clone
clone 13
13 AATGCTTTTCTAATG'l'AT'l'AACC'l.'TGAG'rATTGCAGTTGCTGCTTT
AATGCT'ITTCTAATGTATTAACCCTAATDTATTAACTTPGAOPATNXlRGPTT
clone
23
AATGCTTTTCTAATG'l'AT'l'AACCTTGAGTAT'1'GCAG'1"l'GGCTTT
clone 2-3 AATGCTTTTCTAATGTATTAACCTTVGTA!lTCXAGTTGCTGCTTT
11
·46
-46
NCA
NCA clon.
clone 99 ~~~
G
K
T
C
N
I
I
X
T
Z
C
X
T
~
~
clone
clone 13
13 ~~ITCJ\ITAAN::cTlWW\AAAA
~
~
A
clone
clone 2-3
2-3 GrN:.Af'ACGIT~ITCJ\ITAAAa::r
C X A C V A ~ A ~ ~ T W T T ~ C C l
10
NCA
NCA clone
clone 99
cclone
l one 13
13
GACCCCCCTCAGCC
R4R4pR4R4ApAAA
FIG.
FIG.3.3. Comparison
Comparisonof
of the
the clones
clones2-3
2-3 lind
and 13
13with
with clone
clone 99
containing
containingthe
theNCA
NCA gene.
gene.Sequenees
Sequencesupstream
upstream of
of nucleotide
nucleotide-46
-46
are
areshared
sharedamong
amongthe
thethree
threeclones
clones(boldface
(boldfaceIA!tters).
letters).Sequenees
Sequencesunique
unique
to
lightface!etters.
letters.The
Theputative
putative polyadpolyadtothe
therespeetive
respectiveclones
clonesare
are inin lightface
enylation
enylation signals
signalsare
are underlined.
underlined. Numbering
Numberingrefers
refers to
to clone
clone 9.9. The
The
translation
al start
underlinedboldface
boldfaceIA!tters_
letters.
translational
startsite
sitefor
forNCA
NCAisisshown
shownin
inunderlined
~
~
3205
3205
NCA
NCA cDNA
cDNA Clone
Clone
••
•
."
r--N-lcrminal ioomain . . -
20
30
40
DISCUSSION
Characterization of a cD
NA clone
clone containing the
cDNA
the entire
coding
NCA makes possible
coding region
region of NCA
possible a detailed
detailed comparison
comparison
of NCA
NCA to
to CEA,
CEA, the cDNA
cDNA of which was recently cloned
cloned by
Zimmermann
al. (13)
(13) and Oikawa
Oikawa et al.
al. (14).
(14). A high degree
degree
Zimmermann et al.
of sequence
sequence homology
homology was
was previously
previously noted by amino-terminal sequence
sequence analysis (11,
(11, 25)
25) and by the extensive
extensive immunoimmunological
logical cross-reactions between
between CEA
CEA and NCA. Although
amino acid
acid differences
differences between
between CEA and NCA occur throughthroughout their corresponding
corresponding sequences
sequences (Fig.
(Fig. 4), there are regions
regions
of perfeet
perfect homology
homology (e.g.
(e.g. residues
residues 45-77) and regions
regions with
multiple
(e.g. residues
residues 164-196). The
The 4 cysteine
cysteine
multiple substitutions (e.g.
residues
residues of the Ig-like
Ig-like domain
domain are
are conserved,
conserved, as are several
several
residues
invariant in many immunoglobulin
residues that are
are invariant
immunoglobulin supersupergene
gene family
family sequences
sequences (11).
(11).
There are
arealso
also differences
differences in glycosylation
glycosylation sites.
sites. NCA concontains 12
12 potential glycosylation
glycosylation sites,
sites, whereas
whereas CEA
CEA contains
13
potential glycosylation
13potential
glycosylation sites
sites for
for the corresponding
corresponding sesequence.
12 sites
sites in NCA,
NCA, 88 are
are conserved
conserved in CEA;
CEA, the
quence. Of the
the 12
unique
unique sites
sites in NCA occur
occur at residue
residue 77
77 in the amino-terminal
amino-terminal
domain
domain and residues
residues 139
139 and 190
190 in
in the
the Ig-like
Ig-like domain.
domain. The
The
five
five glycosylation
glycosylation sites
sites unique
unique to CEA
CEA occur
occur at residues
residues 148,
148,
170,
170, 174,
174, 212, and
and 240,
240, al!
all within the
the repeated domain.
domain. From
the number of potential glycosylation
glycosylation sites
sites ininNCA,
NCA, it cannot
cannot
be concluded
concluded whether this clone
clone is
is equivalent to NCA-95
NCA-95 or
NCA-55.
NCA-55. However,
However,the similar amino acid compositions
compositions (Ta(Table I)
and amino-terminal
I) and
amino-terminal sequences
sequences (11)
(11)for
for both suggest
suggest
that the
the protein sequences
sequences are
are the same
same for
for both species
species of
NCA.
NCA
NCA. Limited studies
studies on the glycosylation
glycosylation patterns of NCA
indicate a mixture of bi-,
Asn-linked
bi-, tri-,
tri-,and
andtetraantennary
tetraantennary
Asn-linked
carbohydrate chains (26).
12 biantennary chains
chains
(26). Assuming
Assuming 12
(M,
(Mr=
= 2,200/chain),
2,20O/chain), NCA could have aa minimum
minimum molecular
weight
weight of 60,000.
60,000. Assuming
Assuming 12
12 tetraantennary chains
chains (M,
(M,=
=
4,400/chain)
weight
4,40O/chain) NCA could
could have
have a maximum
maximum molecular weight
of 86,000.
86,000. Thus,
Thus, ititis
is possible that the
thetwo
two forms
forms of NCA are
are
different only in their glycosylation
glycosylation patterns.
The
The regions
regions of amino acid sequence
sequence differences,
differences, especially
especially
those which produce
produce different glycosylation
glycosylation patterns, are
aregood
candidates for
for antigenie
antigenic differences
differences between CEA
CEA and NCA.
NCA.
The
The length of the carboxyl-terminal domains
domains are
are the
the same
same
between CEA
CEA and NCA and both
both are
areof sufficient hydrophohydropho-
~ KLTIESTPFNVAEGKEVLLL~HNLP~GYSWVKGEIlVD
NCA K L T1ES TP F N VA EG KE V LLL~HN LP~ V S WV KGEIl VD
41
50
60
70
80
~ G NfRQ/I/ilG V V I G TQQA TPG P A Y S G Il Efilu P /LA S LLI QNJiI):l
NCA G N~It1G V V I G TQQATPG PA VS G Il ElIIlYPI1AS LLIQ~Q
---Firsl Repe.,·,,,
81
90
100
110
120
r
~ I1DTGFYT4mVIKSDLVNEEATGQF~VVPELPKPSISSNNS
NCA I1DTG FY T4gJV IKS DLV N EEA TG QFl!!JV V PELPKPS ISS N N S
121
130 •
140
150
160
V E D K D A V A F TC E P F../'i1QIi5AlT V L W W V N/mQ S L P v S P Il L Q L S
NCA I!:1P v ED K D A V A F TC EP F.t1~TV L WW V N[gQ S LP V S P IlLQLS
~
fKlp
161
170
180
190
200
~ N G ~ L T LjF]jV~1l11 DfTAlS v!Klc E/i'lQ N pfVJs AfRJR S ofSlvfi]L N V L
NCA N G lil!1T L T ~v I!9R 11 D~S VI§lC Ei.!jQ N ~S A/!jJR S ~VI:!JL N V L
201
210
V G P D!AlPTI S ~LltT
NCA Y G P DI2IPTI S S KA
~
24 I
250
220
230
240
10.0
N PP A Q/YJS W~
V at!lG E N LI1LS C H AAS N pp A Q\!!IS WF\!lIi
~V tt/SlG EN LI1LS C H AAS
260
270
280
~ GTFQQSTQELFIPILITVILNSGSymCQAHNS~TGLILRTTVT
NCA G TFQQS TQEL F IP ILITV /LN S G S VIMICQA H N S~TG LILR TTV T
--- Membrane Se,menl--~
[
281
290
300
~ fi'lITV Y A
S GffSjPf3JLS AfGlA TV G I/MJ IG V L vfGlv ALl
8.7
NCA ~ITV. .
S~~LSA!::1ATVGII!JIGV LAl!JVALI
FIG.
FIG. 4. Compari80n
Comparison of
of CEA
CEA and
and NCA amino
amino acid
acid sequences.
sequences.
the NCA sequence.
sequence. Only
Onlv the first Ig·like
Ie-like repeat
reueat of
Numbers refer to the
CEA is
is shown.
shown. Amino
Amino acid
acid differences
differences are
are baxed.
boxed. Cysteine
Cysteine residues
residues
CEA
3.2
are
sites
are indicated by asolid
a solid circle. Asparagine-linked
Asparagine-linked glycosylation
glycosylation
sites
are
are underlined.
underlined. The
The three domains
domains are
are indicated
indicated by arrows.
arrows.
TABLE
II
TABLE
Amino acid
acid compositions of NCA-95,
NCA-95, NCA-55,
NCA-55,and NCA
NCA cDNA
cDNA
Oata
Data for
for NCA-95
NCA-95 and
and NCA-55
NCA-55 are
are taken from
from Paxton et al.
al. (11).
(11).
Oata
Data for
for Trp
Trp are taken
taken from
from Engvall
Engvall et al.
al. (25).
(25). Oata
Data for
for NCA cONa
cDNa
are
are calculated from
from the
the nucleotide
nucleotide sequence.
sequence. The
The predicted molecular
molecular
weight
weight for
for the mature protein
proteinis
is 33,619, based
based on ONA
DNA sequence
sequencedata.
data.
Amino
NCA·95
NCA-55
NCA cDNA
Amino acid
acid
NCA-95
NCA-55
mol %
%
'l2Cys
1.4
%Cy5
Asx
Asx
Thr
Ser
Glx
Glx
11.6
Pro
Gly
GlY
Ala
Val
Val
Met
He
Ile
8.6 Leu
4.8 Tyr
TYr
Phe
Phe
His
His
Lys
LY8
2.8 Arg
Arg
Trp
Tn,
2.3
1.6
1.6
12.7
12.7
8.4
9.4
9.4
11.3
11.3
7.7
7.1
6.9
6.9
6.2
7.0
1.0
1.0
4.4
8.8
4.8
2.7
2.1
1.9
1.9
2.8
2.6
2.6
1.4
12.2
8.3
9.3
9.3
11.6
7.4
8.1
8.1
6.2
7.0
7.0
1.3
4.3
8.6
4.8
2.5
1.3
3.2
2.8
2.0
2.0
1.3
11.3
8.7
9.4
10.0
7.1
6.8
7.1
7.1
8.1
8.1
1.0
1.0
5.5
5.5
8.7
3.9
2.3
1.6
1.6
2.6
3.2
1.6
1.6
-"
Downloaded from www.jbc.org at UBM Bibliothek Grosshadern on August 4, 2008
'·
I
t:
different size
size messages,
messages, depending
depending on
on which fragments of
clone
clone 9 are used as
as probes.
probes. A 795-bp
795-bp EcoRI/BsmI fragment
from
from the 5' end of clone
clone 9 identities
identifies a distinct
distinct 7-kb
7-kb message;
message;
while
2.5while using the coding
coding region of NCA as
as aa probe,
probe, only
only a 2.5kb band is
is seen,
seen, consistent with the length of the actual NCA
gene
(data
gene in clone
clone 99(data
notnot shown).
shown). For these reasons we
conclude
conclude that the first 974
974 nucleotides in clone
clone 9 may be a
product of a splicing
cloning
cloning artifact
artifact rather
ratherthan
thanthethe
splicing event in
the 5'
-untranslated region
NCA andrepresent
represent the
3'5"untranslated
region ofNCA
the 3'untranslated sequence
sequence of an
an unrelated gene
gene which
which has been
-untranslated region
fused
fused to
to the
the5'
5"untranslated
region of the NCA gene.
gene.
The
The amino-terminal
amino-terminal sequences
sequences of NCA-55
NCA-55 and NCA-95
NCA-95 and
a number of sequences
sequences from
from internal peptides of NCA-95
NCA-95 (11)
(11)
are
NA sequence.
are identical to that predicted by the cD
cDNA
sequence. The
The
directly determined amino
amino acid composition
composition of NCA
NCA sequence
sequence
contains 12
12 potential N-glycosylation
N-glycosylation sites,
sites, some
some of which
were
were previously
previously sequenced
sequenced by Paxton et al.
al. (11).
(11).
A comparison
comparison of the
the NCA
NCA sequence
sequence to
to CEA
CEA (14)
(14) is
is shown
shown
in Fig.
Fig. 4.
4. CEA
CEA contains three
three copies
copies of the
the immunoglobulinimmunoglobulinlike
like repeat domain,
domain, only
only one
one of which
which is
is shown
shown here.
here. Three
domains
domains are depicted:
depicted: a 108-amino
108-amino acid
acid amino-terminal
amino-terminal dodomain wh
ich contains no cysteine
which
cysteine residues,
residues, a 176-amino
176-amino acid
acid
immunoglobulin-like
immunoglobulin-like domain containing two
two disulfide
disulfide loops
loops
(4
(4 cysteine residues),
residues), and aa 26-amino
26-amino acid hydrophobie
hydrophobic dodothe amino-terminal
main.
main. The
The first
first domain is
is identical to
to the
amino-terminal
107-amino
107-amino acids
acids of genomic
genomic NCA
NCA shown
shown by Thompson et al.
al.
(12).
(12). In the
the domains
domains shown,
shown, the
the homology
homology is
is 85%
85% at the
theamino
acid
acid level
level and 90%
90% at the
the nucleotide
nucleotide level.
level. A comparison of
homologies
homologies at the
the nucleotide
nucleotide level
level of the three
three copies
copies of the
repeated domain in CEA
CEA to
to the
the single
single copy
copy in NCA reveals
reveals
the highest homology
homology for
for the first
firstcopy
copy (90%)
(90%)compared
compared to 86
86
and 83% for
third copies,
for the second
second and third
copies, respectively.
respectively. SeSequence
quence homology
homology at the
the nucleotide
nucleotide level
level continues to
to 40
40 bp
beyond the stop
stop codon,
codon, after which there isis no apparent
homology
homology between
between both messages.
messages. The
The nucleotide sequence
sequence
of approximately 800
-untranslated region
800 bp of the 3'
3"untranslated
region of CEA
CEA
has been reported. CEA
CEA contains aatruncated
truncatedAlu sequence
sequence in
-untranslated region which
its 3'
3"untranslated
which is
is lacking in NCA.
NCA.
3206
3206
••
•
."
bieity
bicity and length to
to funetion
function in membrane insertion. These
hydrophobie
hydrophobic sequenees
sequences are
are reminiseent
reminiscent of the
the earboxyl-tercarboxyl-terminal domain
domain found in
in the
theThy-1
Thy-1antigen (27).
(27).
Prior to
to amino aeid
acid sequenee
sequence analysis of CEA
CEA (11),
( l l ) , there
was
was no indieation
indication that CEA
CEA or CEA-like
CEA-like antigens were
were memmembers of the
the immunoglobulin
immunoglobulin gene
gene superfamily.
superfamily. Indeed,
Indeed, the
biological
biological funetion
function of CEA
CEA is
is still unknown.
unknown. The
The diseovery
discovery of
the
aHows the
the Ig-like
Ig-like domains
domains in CEA
CEA allows
the predietion
prediction that these
domains may fold like
like adjaeent
adjacent domains
domains of immunoglobulin
immunoglobulin
(i.e.
(ie.CH2
CH2 and CH3).
CH3). With respeet
respect to the
the observed
observed homology,
homology,
aa similar predietion
prediction ean
can be made
made for
for the Ig-like
Ig-like domain
domain in
NCA.
NCA. Cleavage
Cleavage of the
the disulfide
disulfide bonds whieh
which eonneet
connect the
the two
two
sets
sets of antiparaHel
antiparallel ß-sheets
@-sheetsmay lead to the
theloss
loss of antigenie
antigenic
aetivity
(28). Thus,
activity in redueed
reduced and S-alkylated
S-alkylated CEA
CEA or NCA (28).
the
the identifieation
identification of immunologieally
immunologically reaetive
reactive epitope-eonepitope-containing domains within CEA
CEA and NCA
NCA should be assisted by
the
above,
the predieted
predicted strueture
structure of immunoglobulin.
immunoglobulin. As noted above,
eaeh
each eopy
copy of the
the repeat domains
domains in
in CEA
CEA and NCA
NCA aetually
actually
eontain
contain two
two Ig-like
Ig-like domains
domains (equivalent
(equivalent to
to CH2
CH2 and
and CH3),
CH3),
which would
would be
be interactive, and
and also
also likely
likely to
to form
form dimerie
dimeric
two-chain
Thus, it should now
two-chain structures.
structures.Thus,
now be possible
possible to
express
CEA or
express the
the amino-terminal
amino-terminal and Ig-like
Ig-like domains of CEA
NCA independently and to
to test the potential for
for an immuimmunoglobulin-like
noglobulin-like fold
fold in these proteins directly.
directly. If the folding
folding
pattern is
is indeed
indeed like
like Ig,
Ig, then the glyeosylation
glycosylation sites should
should
be on the
We are currently
currently using
the surfaee
surface of the moleeule.
molecule. We
moleeular
molecular graphie
graphic teehniques
techniques to simulate
simulate the structure of CEA
CEA
and NCA. Preliminary results are in eoncordance
concordance with this
model.
model. Evolutionary relationships
relationships between
between CEA
CEA and NCA
can be dedueed
deduced from
from a eomparison
comparison of their struetures.
structures. First,
First,
it is
is likely
likely that CEA
CEA evolved
evolved from
from the
the simpler NCA gene
gene by
a proeess
process of gene
gene duplication and subsequent divergence
divergence (Fig.
(Fig.
5).
would
5). In this
this view, the
the Ig-like
Ig-like domain doublet in NCA
NCA would
have been copied
may also be expected
expected that
copied twice
twice in CEA.
CEA. It mayaiso
some
some other member of the CEA
CEA gene
gene family
family would
would have
have two
two
copies
copies of this repeated domain. The
The molecular
molecular weight of the
128
128 kDa antigen (7)
(7) is
is of the
the right size
size for
for containing two
two but
not three
threecopies. We
We are currently
currently sequeneing
sequencing this antigen to
test this
comparison of the
the degree
degree of homology
homology
thishypothesis.
hypothesis. A comparison
for
for each copy
copy of these repeats
repeatsrelative to
to eaeh
each other and
andNCA
would
would reveal
revealthe
the approximate time
time of gene
gene duplication.
duplication. Since
Since
NCA
NCA but not
notCEA
CEA is
is found
found in
in lower
lower mammals
mammals by immunologimmunological
(29),
the duplication
ical tests(29),
it it is
is likely
likely that the
duplication and gene
gene
....
0
Primitive
Ig - liJee gene
Primitive
lg-like
gene
° n
&
+---ddlsulllde
brldge
~
dl sulfide Oridge
!
N-term
N-term.. domoin
domain
!
==-
1
•
~
hydrophobIe
hydrophobic dameln
dorneln
.“:c
C
•
1. A_.filL .ro-la I ~srwlY .P"ilJl.~
VArtc~&V~~T~W~V.~s~~
t.bbbbb
bbbbbtt t
t tt ttbb b
r b b b b b b
b b
b bbbbbb
bb
b b
b bbtbtttt t tbb
bb
bb
b bbbbbb
tb
tt
t ttt tt ttttt t tt
t tttLt t
bbbbb
bbbbb
t tfbt b
bb
b b
bb
b b
b tt t
t ttttbbbb b
bb
b b
bb
t t ttttt tt b
b bbbb
bb
b b
b bbbb
tb
t t ttttt t ttttt
LIVGYVIGTQQATF~'Ars
LIVGYVIGTOPATPGPAYS
b
tt
b bbb b
b b
b bbb b
to
t tt t
t ttttt t
t tt ttttt t
L I
Y C
L
~TmY~A~.~Q.~~o~~~r~~L~IXSDLV~Z~GOrB~rp
~N~M~T~Ls~a.~A~S~.C~Q.PASA~. D.V~L.~Lr
b
bbbbb
bbbbb
t tttht t
bbbbb
btttttttt
b b
bb
bb
t ttttbt b
b b
bb
bb
b b
t tb
t L
bb
bb
Db
b b
bb
b tbttttttbt b
b b
b bbbbbb
bb
bb
b b
~
b b b b b b tttbbbbbbb
t t t b b b b b b b bt
b t ttttbbb
t t c t b b b bbbbttttbbbbbbbbbtt
b b b b t t t t b b b b b b b b b t t tt
t t
FIG.
FIG. 6.
6. Alignment of the
the amino-terminal
amino-terminal and
and Ig-like
Ig-like dodomains in
in NCA.
NCA. The
The first two
two lines compare
compare the amino
amino acid
acid sequences
sequences
of the two
two domains,
domains, starting
starting the
thealignment 66 residues
residues within the IgIglike
were obtained by dot matrix alignlike domain.
domain. Homologies
Homologies which were
alignments are indicated with ooxes,
boxes, allowing
allowing conservative
conservative replacements
replacements
for
I,V,L,M and Y,F,W.
Y,F,W.
for the groups
groups S,T,G,A,P;
S,T,G,A,P; Q,N,D,E;
Q,N,D,E; H,R,K;
H,R,K; I,V,L,M;
Possible
Possible positional displacements
displacements are also
also shown.
shown. Maximal
Maximal homology
requires the separate consideration of residues
residues 44-62 (see
(see Oikawa
Oikawa et
al.
al. (30».
(30)).The
The secondpair
second pair oflines
of lines compares
compares ß-strand
@-strandpredictions using
the Chou-Fasman algorithm and aligning
aligning the Ig-like
Ig-like domain with the
CH2
CH2 domain
domain of immunoglobulin.
immunoglobulin.Predicted ß-strands
@-strandsare
areshown
shown with
a band
b and interconnecting loops
loops or turns
turns are
are shown
shown with
with a t.t. Residues
Residues
44-62 may
may correspond
correspond to
to an extra ß-strand
,%strand not found
found in immunoimmunoglobulin.
globulin. The
The size
size of the homologous
homologous domains
domains roughly
roughly corresponds
corresponds to
one
immunoglobone immunoglobulin
immunoglobulin domain. Invariant residues
residues of the immunoglobulin supergene
supergene family
family members
members are shown
shown above
above the amino
amino acid
acid
comparisons.
comparisons.
expansion
expansion event occurred
occurred recently,
recently, along
along with the divergence
divergence
of higher primates.
The amino-terminal
amino-terminal domain
domain has low
low but significant hohomology to the
6), suggesting
suggesting that itit also
also
the Ig-like
Ig-like domain
domain (Fig.
(Fig. 6),
is
the
is related to immunoglobulin.
immunoglobulin. The
The homology
homology is
is low at the
nucleotide
level
nucleotide level
level (30%),
(30%), but higher at the amino
amino acid level
(40%),
(40%), if one
one allows
allows for
for conservative
conservative replacements and pospossible
are reasonable,
sible positional shifts.
shifts. The
The positional shifts
shifts are
reasonable,
given
given the
the overall
overall ß-sheet
@-sheetstructure prediction for
for this domain.
domain.
The
The size
size of the amino-terminal
amino-terminal domain
domain may indicate that it
corresponds
corresponds to one-half the
the Ig-like
Ig-like domain
domain doublet
doublet or the
equivalent of a single
single CH2
CH2 or CH3
CH3 domain
domain in immunoglobuimmunoglobulins.
lins. Alignment
Alignment of CH2
CH2 or CH3
CH3 to
to one-half of this NCA
domain
domain results in the
the assignment of seven
seven ß-strands.
P-strands. If the
amino-terminal
the Ig-like
amino-terminal domain
domain is
is aligned
aligned to the
Ig-like domain
domain in
NCA,
NCA, seven
seven ß-strands
@-strandsalso are
are found.
found. The
The low
low homology,
homology,lack
lack
of the
the size
the disulfide
disulfide loops,
loops, and
and the
size difference
difference suggest
suggest that
extensive
extensive divergence
divergence would have had to occur
occur to
to explain its
origin
origin from
from the
the Ig-like
Ig-like domains
domains in this moleeule.
molecule. More
Moreimporimportantly,
tantly, the
the dissimilarity
dissimilarity of the
the amino-terminal
amino-terminal domain
domain from
from
the
to its
its probable
the Ig-like
Ig-like domains
domains ealls
calls attention to
probable different
funetional
functional aspeet.
aspect. Sinee
Since the
the biological
biological funetion
function of these molmolecules
ecules is
is still
still unknown,
unknown, we
we await the
the results of further experexperiments to
to assign
assign functions
functions to
to individual
individual domains
domains..
°n °n °n °n
n n
1128
2B JekDa
D0 An
t i ge n
Antigen
°n °n °n °n
I
REFERENCES
1.
1. Gold,
Gold, P.,
P., and Freedman, S. O.
0.(1965)
(1965) J.
J.Exp.
Exp. Med.
Med. 121,439-462
121,439-462
1
‘I
0
AekrwwledgmentsWewould
would like
Acknowledgments-We
like to
to thank
thankDrs.
Drs. Raymond
Raymond Paxton
and
and John
JohnThompson for
for many helpful
helpful suggestions
suggestions and discussions
discussions in
preparing this manuscript.
NCA
.,...=
r:l!l"·ZlciWii!lSWBl,'c::::::r::::::::I::==r1
===-m
,’ , :
Q
.'~.D
Ig-IiJee
I g - l i k e domoin
domain
_
O
I
_ aa::::::O::::r::
,
'r
1XI~'l' r !,JiijVJAr:1:la!:~lo L
ELP@.~ ~
!
-
CEA
CEA
FIG.
FIG.5.
5. P08tulated
Postulated evolution
evolutionofofNCA and CEA
CEA genes
genes from
from aa
primitive ancestor.
ancestor. The
The ancestral gene
gene may
may have
have ansen
arisen from
from a
single
single Ig-like
Ig-like gene
gene and
and duplicated several
several times
times to
to produce
produce this
this gene
gene
family.
family. CEA
CEA would
would be
be the latest member
member of the family.
family.
2.
W., and Go, V. L.
2. Egan,
Egan, M. L.,
L.,Pritchard, D. G., Todd, C. W.,
L. W.
(1977)
(1977) Cancer
Cancer Res.
Res. 37,2638-2643
37,2638-2643
3.
3. Fritsche, R.,
R.,and Mach,
Mach, J.·P.
J.-P. (1977)
(1977) Immunochemistry
Immunochemistry 14,
1 4 , 119127
127
4. von Kleist,
Kleist, S.,
S., Chavanel,
Chavanel, G., and Burtin, P.
P. (1972)
(1972) Proc. Natl.
Aead. Sei.
Acad.
Sei. U.
U.SS.. A.
A. 69,
69, 2492-2494
5.
.-P., and Pusztaszeri,
5. Mach,
Mach. JJ.-P..
Pusztaszeri. G. (1972)
(1972)
Immunochemistry
9,
.
. Immunoehemistry
- 9,
10311034
1031-1034
6. Buchegger,
Buchegger, F., Schreyer, M., Carrel,
Carrel, S., and Mach, J.-P.
J.-P. (1984)
(1984)
Int. J.
643-649
J. Cancer
Cancer 33,
33,643-649
7.
7. Neumaier,
Neumaier, M., Fenger, U., and
and Wagener,
Wagener, C. (1985)
(1985) J.
J.Immunol.
Immunol.
135,
3604-3609
136,3604-3609
Downloaded from www.jbc.org at UBM Bibliothek Grosshadern on August 4, 2008
'·
I
t:
NCA
NCA cDNA
cDNAClone
Clone
cDNA Clone
NCA cDNA
~·
••
•
."
20.
Res. 5,
20. Cami, B., and Kourilsky,
Kourilsky, P. (1978)
(1978)Nucleic Acids Res.
6. 23812390
2390
M., and Sakaki, Y.
Y. (1986)
Anal. Biochem.
Biochem. 152,
232-238
21.
21. Hattori, M.,
(1986)Anal.
162,232-238
Zagursky, R.
R. J., Baumeister, K., Lomax, N., Berman, M. L. (1985)
22.
(1985)
22. Zagursky,
Gene Anal. Teehn.
2, 89-94
Techn. 2,89-94
Hood, L. E.
Anal.
23.
Kobori, J.
J. A., Siu, G.,
G., and Hood,
E. (1986)
(1986)Anal.
23. Strauss, E. C., Kobori,
353-360
Bioehem. 154,
Biochm.
164,353-360
F., Nicklen, S., and Coulson,
Proe. Natl.
Natl.
24.
24. Sanger, F.,
Coulson, A. R. (1977)
(1977)Proc.
Aead. Sci.
Sei. U.
5463-5467
Acad.
U. S.
S. A. 74,
74,5463-5467
Engvall, E.,
E., Shively,
Proe. Natl.
Natl.
25.
25. Engvall,
Shively, J.
J. E., and Wrann, M.
M. (1978)
(1978)Proc.
S. A.
A. 76,1670-1674
75, 1670-1674
Aead. Sci.
Sei. U.
Acad.
U. S.
Kessler, M. J., Shively,
C. W.
26.
26. Kessler,
Shively, J. E., Pritchard, D. G.,
G., and Todd, C.
(1978)
Res. 38,
1041-1048
(1978)Cancer
Cancer Res.
38,1041-1048
27.
Williams, A. F., and Gagnon,
Seience 216,
696-703
27. Williams,
Gagnon, J. (1982)
(1982)Science
216,696-703
J. H., and Thomas, P. (1975)
70828.
28. Westwood, J.
(1975)Brit. JJ.. Cancer 32,
32,708715
715
29. Haagensen, D. E.,
Jr., Metzgar, R. S.,
29.
E., Jr.,
S., Swenson,
Swenson, B., Dilley,
Dilley, W. G.,
C.,
Cox, C.
C. E., Davis,
J., Zamcheck,
Davis, S.,
S., Murdoch,
Murdoch, J.,
Zamcheck, N., and Wells,
S.
(1982) J.
J. Natl. Cancer Znst.
Inst. 69,
S. A., Jr. (1982)
69, 1073-1076
1073-1076
Oikawa, S.,
30.
S., Imajo,
Imajo, S.,
S., Noguchi,
Noguchi, T.,
T., Kosaki, G.,
G., and Nakazato, H.
30. Oikawa,
(1987)
Bioehem. Bwphys.
Biophys. Res.
Res. Commun.
(1987)Biochem.
Commun. 144,634-642
144, 634-642
31.
Oikawa, S., Kosaki, G.,
Bioehem. Bio31. Oikawa,
G., and Nakazato, H. (1987)
(1987)Biochem.
Res. Commun.
phys. Res.
Commun. 146,464-469
146,464-469
32.
Y., and Gotoh, Y.
Y. (1987)
J. Mol.
Mol. Biol.
Biol. 195,247-259
(1987)J.
195,247-259
32. Ohshima, Y.,
Downloaded from www.jbc.org at UBM Bibliothek Grosshadern on August 4, 2008
i
t:
8.
Burtin, P..
P., Chavanel.
Chavanel,. G.,
J. Immu8. Burtin.
G... and Hirsch-Marie,
Hirsch-Marie. H. (1973)
(1973)J.
nol. 111,
n01.
ili, 1926-1928
9.
T. (1976)
Int. J.
588-596
9. Svenberg,
Svenberg, T.
(1976)Znt.
J . Cancer
Cancer 17,
17,588-596
Mol. Immuol.
Immunol.
10.
10. Neumaier, M., Fenger, U., and Wagener, C. (1985)
(1985)Mol.
22,1273-1277
22,1273-1277
11.
Puton, R. J., Mooser,
11. Paxton,
Mooser, G., Pande, H., Lee, T. D., and Shively,
Shively, J.
E. (1987)
Proc. Natl.
Natl. Acad.
Aead. Sei.
Sei. U.
A. 84,920-924
E.
(1987)Proc.
U. S.
S. A.
84,920-924
12.
J. A., Pande, H., Puton,
Paxton, R. J., Shively,
Shively, L., Padma,
12. Thompson, J.
J.
A., Simmer, R. L., Todd, Ch. W., Riggs, A. D., and Shively,
Shively, J.
Proc. Natl.
Natl. Acad.
Aead. Sci.
Sei. U.
A. 84,
2965-2969
E. (1987)
(1987)Proc.
U. S.
S. A.
84,2965-2969
13. Zimmermann, W., Ortlieb, B., Friedrich, R., and von Kleist, S.
13.
(1987)
Proc.
Aead. Sci.
Sei. U.
A. 84,
2960-2964
(1987)P
m . NatL Acad.
U. S.
S. A.
84,2960-2964
Bioehem. BwBio14.
14. Oikawa,
Oikawa, S.,
S., Nakazato, H., and Kosaki, G.
G . (1987)
(1987)Biochem.
phys. Res.
Res. Commun.
phys.
Commun. 142,511-518
142,511-518
15.
H., Wu, B.,
15. Dashal, I., Ramirez, S. A., Ballal, R. N., Spohn, W. H.,
Res. 36,1026-1034
and Busch, H. (1976)
(1976)Cancer
Cancer Res.
16.
Fritsch, E. F., and Sambrook, J.
Moleeular
16. Maniatis, T., Fritach,
J. (1982)
(1982)Molecular
Cloning,
Manual, Cold Spring Harbor Laboratory,
Cloning, a Laboratory Manual,
Cold Spring Harbor,
Harbor, NY
17.
F.,
T. D., Huettermann,
17. Nader, W. F
.,Edlind,
Edlind, T.
Huettermann, A., and Sauer, H. W.
(1985)
Proc. Natl.
NatL Acad.
Aead. Sei.
2698-2702
(1985)Pnx.
Sci. U.
U. S.
S. A. 82,
82,2698-2702
Seience 196,
180-182
18.
18. Benton, W. D., and Davies, R. W. (1977)
(1977)Science
196,180-182
19.
Vogelstein, B. (1983)
Anal Biochem.
Biochem. 132,
19. Feinberg, A. P., and Vogelstein,
(1983)AnaL
132,
6-13
6-13
3207