BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 645-65]
Vo1.180, No. 2,1991 October 31,1991
cDNA CLONING AND R E G U L A T I O N OF A NOVEL RAT CYTOCHROME P450 OF THE 2C GENE SUBFAMILY (P450IIC24) Peter G. Zaphiropoulos Department of Medical Nutrition, Huddinge U n i v e r s i t y Hospital, F60, NOVUM, K a r o l i n s k a Institute, S-141 86 Huddinge, Sweden Received September 9, 1991 A novel member of the cytochrome P450 2C gene subfamily was identified by screening rat prostate cDNA libraries. Two independent clones were isolated. Clone prosl was 1031 bp long and c o n t a i n e d a bizarre replacement in place of putative exon i. Clone pros2 was 1755 bp long, c o n t a i n e d a complete 3' end, and also had bizarre sequences in place of exon i, which in this case were compatible with an u n s p l i c e d intron. Northern analysis revealed m R N A expression in the liver and the kidney. Interestingly, although livers of mature rats of both sexes have comparable amounts of P4502C24 mRNA, a dramatic sex difference is seen in the kidney where only males express detectable levels of this mRNA. © 1991 Academic
Press,
Inc.
P450 cytochromes represent a wide class of enzymes that are capable of a variety drug
of m e t a b o l i c
detoxification,
and
reactions
including
carcinogen
steroid
activation
synthesis,
(1-3).
The
2C
subfamily is the largest known group of P450s that have relatively similar primary structures but quite different and expression patterns In order whether cDNA
to
further
additional
libraries
investigate forms
are
this
group
expressed
from the prostate,
were screened with a P4502C7 probe, two independent
catalytic profiles
(4-13).
in
a known
of P450s
and examine
extrahepatic source
tissues,
of P450s
(14),
resulting in the isolation of
clones coding for a new member of this subfamily,
P4502C24.
M A T E R I A L S AND METHODS Isolation and c h a r a c t e r i z a t i o n of p r o s t a t e cDNA clones A random p r i m e d and an oligo dT p r i m e d rat prostate cDNA library in IZAP and ~gtl0 respectively, kindly p r o v i d e d by Drs Stefan Andersson and Maria Str~mstedt, were screened with a 5' to n u c l e o t i d e 1013 (SphI site) P4502C7 cDNA fragment (6,15), under conditions of reduced stringency as d e s c r i b e d p r e v i o u s l y (ii). About 200.000 Rlaque ~ o r m i n g units from each of the two libraries were screened and this r e s u l t e d in the isolation of clone prosl from the random p r i m e d and clone pros2 from the oligo dT p r i m e d
645
0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
library. The inserts were subcloned into the pGEM3Z vector (Promega) and sequenced using Sequenase (U.S. Biochemicals) in the dideoxy nucleotide chain t e r m i n a t i o n protocol with the use of universal as well as c u s t o m - s y n t h e s i z e d o l i g o n u c l e o t i d e primers b a s e d on p r e v i o u s l y d e t e r m i n e d sequence.
Northern analysis RNA from different tissues of mature S p r a g u e - D a w l e y rats and RNA from liver of rats at different d e v e l o p m e n t a l stages were isolated by the m e t h o d of Chomczynski and Sacchi (16). Tissue from three animals were p o o l e d for each RNA preparation. N o r t h e r n analysis was p e r f o r m e d as before (11,15) using as a probe an AvaII - AvaII fragment (position 165 - 982) of prosl.
RESULTS The
screening
sequences prosl
1031) of
pros2.
into
two
Prosl
two
is highly
the
the
prostate
cDNA
has
segments
an
insert
(Fig.
i).
1 to
Clone
2C
gene
subfamily,
in
1 - 124), which is not.
exon
2
(19),
pros2
into two
junctions
has
an
segments
insert (Fig.
to
nucleotide
substitution
that
end
and
untranslated 7 - 124) but
is
also
codon and
in
furthermore,
(T/C) nN(C/T)AG (Fig. of
with
from to
the
the
(20),
the
is
segment prosl
pros2
The
amino
deduced
nucleotides identical
125 to
with that
acids
which
T~G,
(18),
and
be
divided
125 - 1755)
is
of
results
a
in an
it further extends to
the A,
acid
A
at
the
codon,
prosl has
3'
2C P450s
clone.
This
a termination of segment
splice
junction
a
(nucleotides
1 of the
sequence
intron/exon
represent
can
that
3'
makes
A
structure.
Segment
of
-
with the
exception
of exon
A
125
B
sequence,
with
segment
B
it likely that the segment A
part
of
an
of
clone
unspliced
intron,
i.
acid 1426
sequence was
of P450s are
the
segment
amino
present
might
of intron
P4502C12
termination
sequence
i). This last o b s e r v a t i o n
clone
with
(A)+ tail.
be
segment
coincides
(nucleotides
translation
deduced
to
again
813,
can
the p o s i t i o n of
which
In addition,
consensus
specifically
Amino
a
to the
different
contrast
gene
B
cDNA clones, which
region of the members
(17),
prosl,
and a poly
contrast
in frame
Segment
P4502C7
(nucleotides
a known
bp
at p o s i t i o n
is d i s s i m i l a r
segment,
1755
Ile~Ser.
B
sequences
with
clone
contains
region,
of
i).
of
amino acid replacement, 3'
with
bp
Interestingly,
of P4502CII
the rat 2C P450s
identical
the
1031
Segment
this switch from similar to d i s s i m i l a r
P4502C13
of
similar to the c o r r e s p o n d i n g
P450
(nucleotides
exon
libraries
r e s u l t e d in the isolation of two independent
and
divided
of
found to be IIC6,
conserved
IIC7,
in these
646
72,
pros2, 66,
IICII, five
75,
deduced 63,
IICI2, rat
and
from 65 %
and IICI3.
P450s
are,
in
Vol. 180, No. 2, 1991
1
50
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pros2: Prosl:
ArgIleProArgAsnSerAspLeuLysLeuTrpLeuAlaAsn GCGGATTCCGCGGAATTCCGATTTGAAGTTGTGGTTGGCAAAC GCAGCTTTCTTGCAGCA-A--A-TT-G-C-T-CC-GTG-TCCC-AGGCT GlnLeuSerCysSer LysPheGly ThrCysGlyProGluAla
TyrArgCysGlyGlu***AspAlaHisIleIleLeuAsnProSerSerAlaThrHisPheLeuSerTyrPheGln TACAGATGTGGAGAGTAAGATGCTCACATTATCCTAAATCCTTCTTCTGCTACTCATTTTCTTTCTTATTTCCAG .... G-C-GGAA--C-T-TGlnPheThrAspLysLeuThr LysCysHisSerSerVal LeuHisIleAspLeuProGlyAsnLeuLeu C-GTTCACA-ATA-ACTTAC-G-AA-GTGCCA-AGTTC-GTCAG--TGCAC-T-G
125
PheSerLysIleTyrGlyProValPheThrLeuTyrPheGlyProLysProThrValValValHisGlyTyrGlu TTCTCAAAAATTTATGGCCCTGTGTTCACTCTGTACTTTGGTCCGAAGCCTACTGTGGTGGTACATGGATATGAA ...........................................................................
200
AlaValLysGluAlaLeuAspAspLeuGlyGluGluPheSerGlyArgGlySerPheProIleValGluArgMet GCAGTAAAGGAAGCCCTGGATGATCTTGGAGAGGAGTTTTCTGGGAGGGGTAGTTTCCCAATTGTTGAAAGAATG ...........................................................................
275
AsnAsnGlyLeuGlyLeuIlePheSerAsnGlyThrLysTrpLysGluLeuArgArgPheSerLeuMetThrLeu AATAATGGCCTTGGGCTCATTTTCAGCAATGGAACAAATGGAAGGAGCTTCGGCGTTTCTCACTTATGACCTTG ...........................................................................
350
ArgAsnPheGlyMetGlyLysArgSerIleGluAspArgIleGlnGluGluAlaSerCysLeuValGluGluLeu AGAAATTTTGGGATGGGGAAGAGGAGCATCGAGGATCGCATTCAAGAGGAAGCCTCCTGTCTTGTGGAAGAGCTA ...........................................................................
425
ArgLysThrAsnGlySerLeuCysAspProThrPheIleLeuSerCysAlaProSerAsnValIleCysSerVal AGAAAAACAAATGGCTCACTCTGTGATCCCACATTCATCCTGAGCTGTGCTCCCTCCAACGTGATCTGCTCAGTT ...........................................................................
500
ValPheHisAsnArgPheAspTyrLysAspGluAsnPheLeuAsnLeuMetGluLysLeuAsnGluAsnPheLys GTTTTCCATAATCGTTTTGATTATAAAGATGAGAATTTCCTTAACTTGATGGAGAAACTCAATGAAAACTTTAAA ...........................................................................
575
IleLeuAsnSerProTrpMetGlnValCysAsnAlaLeuProAlaPheIleAspTyrLeuProGlySerHisAsn ATCTTGAACTCCCCATGGATGCAGGTTTGCAATGCTCTCCCTGCCTTCATCGATTATCTCCCAGGAAGCCATAAT ...........................................................................
650
ArgValIleLysAsnPheAlaGluIleLysSerTyrIleLeuArgArgValLysGluHisGlnGluThrLeuAsp AGAGTAATTAAAAATTTTGCTGAAATAAAAAGTTATATTTTGAGACGAGTGAAAGAACATCAGGAAACACTGGAC ...........................................................................
725
MetAspAsnProArgAspPheIleAspCysPheLeuIleLysMetGluGlnGluLysHisAsnProArgThrGlu ATGGACAATCCTCGGGACTTTATTGACTGTTTCCTGATCAAAATGGAACAGGAAAAACACAATCCCCGTACTGAG ...........................................................................
800
PheThrIleGluSerLeuMetAlaThrValSerAspValPheValAlaGlySerGluThrThrSerThrThrLeu TTTACTATTGAAAGCTTGATGGCTACTGTAAGTGATGTATTTGTAGCTGGATCAGAAACCACAAGCACTACCCTG ............. T ............................................................. Ile
875
ArgTyrGlyLeuLeuLeuLeuLeuLysHisIleGluValThrAlaLysValGlnGluGluIleAspHisValIle AGATATGGACTCTTGCTCCTACTGAAACACATAGAGGTCACAGCTAAAGTCCAGGAAGAGATTGATCACGTGATT ...........................................................................
950
GlyArgHisArgArgProCysMetGlnAspArgThrArgMetProTyrThrAspAlaMetValHisGluIleGln GGCAGACACAGGAGGCCCTGCATGCAGGACAGGACCCGCATGCCCTACACAGATGCGATGGTGCACGAAATCCAG ...........................................................................
1025
ArgTyrIleAsnLeuIleProAsnAsnValProHisAlaAlaThrCysAsnValArgPheArgAsnTyrValIle AGATATATTAACCTCATCCCCAACAATGTGCCCCATGCAGCTACCTGTAATGTTAGATTCAGAAATTATGTAATT
1100
ProLysGlyThrAspLeuLeuThrSerLeuThrSerValLeuHisAspAspLysGluPheProAsnProGluVal CCCAAGGGCACGGACTTACTAACATCACTGACTTCTGTGCTACATGATGACAAAGAATTTCCCAACCCAGAAGTA
1175
PheAspProGlyHisPheLeuAspGluAsnGlyAsnPheLysLysSerAspTyrPheMetProPheSerThrGly TTTGACCCAGGCCATTTTCTGGATGAGAATGGGAACTTTAAGAAGAGTGACTACTT~TATGCCTTTCTCAACAGGA
1250
LysArgMetCysValGlyGluAlaLeuAlaArgMetGluLeuPheLeuLeuLeuThrThrIleValGlnAsnPhe AAGCGAATGTGCGTGGGAGAGGCCCTGGCTCGCATGGAGCTGTTTTTGCTTCTGACCACCATTGTACAGAATTTT
1325
AsnLeuLysSerPheValAspThrLysAspIleAspThrThrProMetAlaAsnThrPheGlyArgValProPro AACCTGAAATCTTTTGTTGATACAAAGGACATTGACACTACTCCAATGGCTAATACCTTTGGCCGTGTACCACCT
1400 1475 1550 1625 1700
SerTyrGlnLeuCysPheIleProArg*** TCATACCAGCTGTGCTTCATTCCTCGTTAAAGCAGAGCACACTGGGCTGTTGCTATGCTGGTGTCTGTGACTAAT CAGGGGCAATCCAGTTTCCACTGTTAGGAACATCTCTCTCAATTCTCCTCTCACATCTCTTCATTCTCTCACAAT ACAGTTATCATCCATCTTTGTTTAAGAGGTTTTCCCAGAGTTATCTCGCAAATCTATCCTTTGTCTCCCACAGTC TATAACACTTATATTGACTGTGAACTGTACTAAGACATGTGCTGGGTTACTAATATGTTATGGGTGTAATACAGA ATAGTTCAACTGAGAGCCATATCTTCTTTGCTTGATTCAAAATAAAAGGAGTTATTAACTGA
Figure I. N u c l e o t i d e and deduced amino acid sequence of clones prosl and pros2. The nucleotide sequence of the two p r o s t a t e clones is shown. Identical n u c l e o t i d e s in the prosl sequence are indicated by bars. The deduced amino acid sequence of pros2 and prosl is shown above and b e l o w the respective nucleotide sequence. Only amino acids which are different in prosl codons, the conserved 3' intron polyadenylation signal are underlined.
647
are shown. The dinucleotides,
termination and the
Vol. 180, No. 2, 1991
2C24 2Cli 2C6 2C7 2C13 2C12
51
i01
151
201
251
301
351
401
451
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FSKI GQSIKKFSKV TQSLTSFSKV SQSLTKFSKT RQSLTNFSKT RQSISNFSKT
YGPVFTLYFG YGPIFTLYLG YGPVFTLYFG YGPVFTLYLG YGPVYTLYVG YGPVFTLYFG
PKPTVVVHGY MKPFVVLHG¥ TKPTVILHG¥ SQPTVILHGY SQPTVVLHGY SQPTVVLHGY
EAVEXALDDL EAVKEALVDL EAVKEALIDH EAIKEALIDN EALKEALVDH EAVKEALIDY
GEEFSGRGSF GEEFSGRGSF GEEFAERGSF GEKIrSC.-P.GSY GEEFSGRGRL GEEFSGRGRM
PIVERMNNGL PVSERVNKGL PVAEKINKDL PMIENVTKGF PICEKVAKGQ PVFEKATKGL
GLIFSNGTKW GVIFSNGMQW GIVFSHGNRW GIVFSNGNRW GIAFSHGNVW GISFSRGNVW
KELRRFSLMT KEIRRFSIMT KEIRRFTLTT KEMRRFTIMN KATRHFTVKT RATRHFTVNT
LRNFGMGKRS LRTFGMGKRT LRNLGMGKRN FRNLGIGKRN LRNLGMGKGT LRSLGMGKRT
IEDRIQEEAS IEDRIQEEAQ IEDRVQEEAR IEDRVQEEAQ XEDKVQEEAK IEIKVQEEAE
CLVEELRKTN CLVEELRKSK CLVEELRKTN CLVEELRKTK WLVKELKKTN WLVMELKKTK
GSLCDPTFIL GAPFDPTFIL GSPCDPTFIL GSPCDPSLIL GSPCDPQFIM GSPCDPKFII
SCAPSNVICS W F H N R F D Y K GCAPCNVICS IIFQNRFDYK GCAPCNVICS IIFQNRFDYK NCAPCNVICS ITFQNHFDYK GCAPGNVICC IILQNRFDYE GCAPCNVICS IIFQNRFDYK
DENFLNLMEK DPTFLNLMHR DQDFLNLMEK DKEMLTFMEK DKDFLNLIEK DKDFLSLIEN
LNENFKILNS FNENFRLFSS LNENMKILSS VNENLKIMSS VNEAVKIISS VNEYIKIVST
PWMLVCNALP PWLQVCNTFP PWTQFCSFFP PWMQVCNSFP PGIQVFNIFP PAFQVFNAFP
AFIDYLPGSH AIIDYFPGSH VLIDYCPGSH SLIDYFPGTH ILLDYCPGNH ILLDYCPGNH
NRVIKNFSEI NQVLKNFFYI TTLAKNVYHI HKIAKNINYM NIYLKNYTWV KTHSKHFAAI
KSYILRRVKE KNYVLEKVKE RNYLLKKIKE KSYLLKKIEE KSYLLEKIKE KSYLLKKIKE
HQETLDMDNP HQESLDKDNP HQESLDVTNP HQESLDVTNP HEESLDVSNP HEESLDVSNP
RDFIDCFLIK RD~IDCFLNK RDFIDYYLIK RDFVDYYLIK RDFIDYFLIE RDFIDYFLIQ
MEQEKHNPRT MEQEKHNPQS WKQENHNPHS QKHANNIEHS RNQENANQWM RCQENGNQQM
EFTIESLMAT EFTLESLVAT EFTLENLLIT EYSHENLTCS NYTLEHLAIM NYTQEHLAIL
VSDVFVAGSE VTDMFGAGTE VTDLFGAGTE IMDLIGAGTE VTDLFFAGIE VTNLFIGGTE
TTSTTLRYGL TTSTTLRYGL TTSTTLRYAL TMSTTLRYAL TVSSTMRFAL TSSLTLRFAL
LLLLKHIEVT LLLLKHVDVT LLLLKCPEVT LLLMKYPHVT LLLMKYPHVT LLLMKYPHIT
AKVQEEIDHV AKVQEEIERV AKVQEEIDRV AKVOEEIDRV AKVQEEIDHV DKVQEEIGQV
IGRHRRPCMQ IGRNRSPCMK VGKHRSPCMQ IGRHASPCMQ IGRHRSPSMQ IGRHRSPCML
DRTRMPYTDA DRSQ~YTDA DRSRMPYTDA DRKHMPYTDA DRSHMPYTNA DRIHMPYTNA
MVHEIQRYIN VVHEIQRYID HDHEVQRFID HDHEVQRFIN MVHEVQRYID MIHEVQRYID
LIPNNVPHAA LVPTNLPHLV LIPTNLPHAV FVPTNLPHAV IGPNGLLHDV LAPNGLLHEV
TCNVRFRNYV TRDIKFRNYF TCDIKFRNYL TCDIKFRNYL TCDTKFRNYF TCDTKFRDYF
IPKGTDLLTS IPKGTNVIVS IPKGTTIITS IPKGTKVLTS ZPF~TAVLTS IPKGTAVLTS
LTSVLHDDKE LSSILHDDKE LSSVLHDSKE LTSVLWnSKE LTSVLHDSKE LTSVLHDSKE
FPNPEVFDPG FPNPEKFDPG YPDPEIFDPG FPNPEMFDPG FPNPEMFDPG ¥PNPEMFDPG
HFLDENGNFKKSDYFMPFST HILDERGNFKKSDYFMPFSA HFLDGNGKFK KSDYFMPFSA HFLDENGNFKKSD~LPFSA HFLDENGNFKKSDYFIPFSA HFLDENGNFKKSDYFMPFSA
GERMCVGEALARMELFLLLT GKRICAGEAL ARTELFLFFT GKRMCAGEGL ARMELFLFLT GERACVGEGLARMQLFLFLT GERMCLGESL ARMELFLFLT GKP.KCVGEGL ASMELFLFLT
TIVQNFNLKS TILQNFNLKS TILQHFKLKS TILQNFNLKS TILQNFKLKS TILQNFKLKS
FVDTEDIDTT LVDVEDIDTT VLHPKDIDTT LVHPKDXDTM LVDPEDINTT LSDPEDXDIN
PSYQLCFIPR PFYEACFIPV QRADSLSSHL PFYELCFIPL PTYQLCFIPS PTFQMRFIPL PTFQLCFIPV
PMANTFGRVP PAISGFGHLP PVFNGFASLP PVLNGFASLP PICSSLSSVP SIRSEFSSIP
Figure 2. C o m p a r i s o n of t h e amino acid sequence of P 4 5 0 2 C 2 4 , deduced from nucleotides 125 to 1 4 7 6 of p r o s 2 , w i t h t h a t of P 4 5 0 s 2CII, 2C6, 2C7, 2 C 1 3 , a n d 2 C 1 2 . I d e n t i c a l a m i n o a c i d s a r e i n d i c a t e d by bold letters. The numbering corresponds t o t h a t of a t y p i c a l 2C P450.
general, 302
also c o n s e r v e d in this new form, including Cys 435 and Thr
(Fig. 2) and according to the
this P450 is termed IIC24. 648
standardized nomenclature
(21)
Vol. 180, No. 2, 1991
1
2
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
3
4
5
6
7
8
1
®
2
3
4
5
6
7
8
®
Figure 3. Northern analysis of total RNA from different rat tissues probed with CYP2C24 sequences (see Materials and Methods). The individual lanes are as follows: lane i, ovary; lane 2, female lung; lane 3, female kidney; lane 4, female liver; lane 5, prostate; lane 6, male lung; lane 7, male kidney; lane 8, male liver. Figure 4. Northern analysis of total liver RNA from rats at different developmental stages probed with CYP2C24 sequences (see Materials and Methods). The individual lanes are as follows: lanes 1 to 4; 8, 4, 2, and 1 week old females, lanes 5 to 8; 8, 4, 2, and 1 week old males.
Northern
analysis
of a b o u t
1800
not
apparently
sex
difference
P4502C24
indicated
detectable is
mRNAs
developmental
analysis
that,
comparable
amounts
express
in
the
females
do
of
the
although of
with a typical
in t h e
liver
in the p r o s t a t e .
observed
while
indicated
weeks
that mRNAs
bp are e x p r e s s e d
this
significant
where
mRNA,
only
amounts
males
do
3).
In
of
2C24
in
animals
of
males
of t h i s
both
of
P450
age
size
but
are
a dramatic
(Fig.
expression
mature
sexes
Furthermore
kidney not
2C P450
of b o t h
express
addition, the
liver
sexes two
(Fig.
have
and
four
4).
DISCUSSION cDNA
clones
have
been
do
not
P450
contain I.
of
a the
termination
the
region
Instead,
segment deduced codons
absence
might
that
clone
would
pros2
of u n s p l i c e d
that
has
P450IIC24
no
these
correspond
a typical
a
intron
termination amino
subfamily
Interestingly,
contains
part
2C g e n e
acid
to
segment
I, w h i l e codons
sequence.
exon
represent
of an i n i t i a t o r
an
1 te e x o n 2 j u n c t i o n alternatively
clone
in
methionine
649
codon
in t h i s
2C
with
lack
of
segment
that
clone
transcript. alternate
is
prosl
frame
This
suggests
spliced
clones
which
as w e l l as the fact t h a t t h i s d i s s i m i l a r
at t h e p u t a t i v e
prosl
of the P 4 5 0
from the prostate.
to r e p r e s e n t
contains
ends
for a n e w m e m b e r
isolated
exon
likely
that
coding
The
exon
1
Vol. 180, No. 2, 1991
sequence
(Fig.
produce
I) indicates
a protein
to the
5'
P4502C6,
end.
protein
A
that
exon (22).
similar
8, are In
of mature
case
still
male
and
the
prostate
libraries,
prosl
and
to
does not
is not fully extended splicing
of synthesizing
2C24,
female
rats
2C6
involves
pros2
expressed
in the
levels,
also
expression but in favor of
clones
observed in the Northern analysis,
also
a microsomal
at comparable
is expressed
no significant
rare events.
transcript
having an alternate instead of the
in developmental
and furthermore
Although
represent
this
of differential
capable
similarity
shows a sex difference the female,
either
or that the cDNA sequence
where mRNA transcripts,
canonical liver
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in the female brain have
signals
been
(22).
isolated
from this tissue
from were
indicating that such transcripts
In agreement with that,
is the large number
of Rlaque ~orming units that had to be screened in order to isolate the
two
kidney
clones.
Furthermore,
and the liver,
1800 bp are seen, the
cloning
characterization
be
P4502C24
speculated
from mRNAs
containing
Verification of
can
signals
transcripts
1 might predominate. await
where
it
of the exon 1 region
in
the
of a size of about
a typical
of this hypothesis from
that
these
P450-1ike
tissues
of the P4502C24
Acknowledqments: This research was supported by Swedish Medical Research Council (No 03X-06807) Work Health Fund.
exon
would have to and
the
gene.
grants from the and the Swedish
REFERENCES I. Gonzalez, F.J. (1988) Pharmacol. Rev. 40, 243-288. 2. Guengerich, F.P. (1991) J. Biol. Chem. 266, 10019-10022. 3. Porter, T.D. and Coon, M.J. (1991) J. Biol. Chem. 266, 1346913472. 4. Leighton, J.K. and Kemper, B. (1984) J. Biol. Chem. 259, 11165-11168. 5. Tukey, R.H., Okino, S., Barnes, H., Griffin, K.J., and Johnson E.F. (1985) J. Biol. Chem. 260, 13347-13354. 6. Gonzalez, F.J., Kimura, S., Song, B.-J., Pastewka, J., Gelboin, H.W., and Hardwick, J.P. (1986) J. Biol. Chem. 261, 10667-10672. 7. Yoshioka, H., Morohashi, K.-I., Sogawa, K., Miyata, T., Kawajiri, K., Hiroshi, T., Inayama, S., Fujii-Kuriyama, Y., and Omura, T. (1987) J. Biol. Chem. 262, 1706-1711. 8. Umbenhauer, D.R., Martin, M.V., LLoyd, R.S., and Guengerich, F.P. (1987) Biochemistry 26, 1094-1099. 9. Zaphiropoulos, P.G., Mode, A., M611er, C., Fernandez, C., and Gustafsson, J.-~. (1988) Proc. Natl. Acad. Sci. 85, 42144217. i0. McClellan-Green, P.D., Linko, P., Yeowell, H.N., and Goldstein, J.A. (1989) J. Biol. Chem. 264, 18960-18965.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ii
Zaphiropoulos, P.G., Str6m, A., Robertson, J.A., and Gustafsson, J.-A. (1990) Mol. Endocrinol. 4, 53-58. 12 Pendurthi U.R., Lamb, J.G., Nguyen, N., Johnson E.F., and Tukey, R.H. (1990) J. Biol. Chem. 265, 14662-14668. 13 Romkes, M., Falleto, M.B., Blaisdell, J.A., Raucy, J.L., and Goldstein, J.A. (1991) Biochemistry 30, 3247-3255. 14 Sundin, M., Warner, M., Haaparanta, T., and Gustafsson, J.-~. (1987) J. Biol. Chem. 262, 12293-12297. 15 Westin, S., Str6m, A., Gustafsson, J.-A., and Zaphiropoulos, P.G., (1990) Mol. Pharmacol. 38, 192-197. 16 Chomczynski, P. and Sacchi, N. (1987) Anal. Biochem. 162, 156-159. 17 Morishima, N., Yoshioka, H., Higashi, Y., Sogawa, K., and Fujii-Kuriyama, Y. (1987) Biochemistry 26, 8279-8285. 18 Zaphiropoulos, P.G., Westin, S., StrUm, A., Mode, A., and Gustafsson, J.-A. (1990) DNA Cell Biol. 9, 49-56. 19 Eguchi, H., Westin, S., Str6m, A., Gustafsson, J.-A., and Zaphiropoulos, P.G. (1991) Biochemistry, in press. 20 Green, M. R. (1986) Ann. Rev. Genet. 20, 671-708. 21. Nebert, D.W., Nelson, D.R., Coon, M.J., Estabrook, R.W., Feyereisen, R., Fujii-Kuriyama, Y., Gonzalez F.J., Guengerich, F.P., Gunsalus I.C., Johnson, E.F., Loper, J.C., Sato, R., Waterman, M.R., and Waxman, D.J. (1991) DNA Cell Biol. i0, 1-14. 22. Kimura, H., Sogawa, K., Sakai, Y., and Fujii-Kuriyama, Y. (1989) J. Biol. Chem. 264, 2338-2342.
651
Vo1.180, No. 2,1991 October 31,1991
cDNA CLONING AND R E G U L A T I O N OF A NOVEL RAT CYTOCHROME P450 OF THE 2C GENE SUBFAMILY (P450IIC24) Peter G. Zaphiropoulos Department of Medical Nutrition, Huddinge U n i v e r s i t y Hospital, F60, NOVUM, K a r o l i n s k a Institute, S-141 86 Huddinge, Sweden Received September 9, 1991 A novel member of the cytochrome P450 2C gene subfamily was identified by screening rat prostate cDNA libraries. Two independent clones were isolated. Clone prosl was 1031 bp long and c o n t a i n e d a bizarre replacement in place of putative exon i. Clone pros2 was 1755 bp long, c o n t a i n e d a complete 3' end, and also had bizarre sequences in place of exon i, which in this case were compatible with an u n s p l i c e d intron. Northern analysis revealed m R N A expression in the liver and the kidney. Interestingly, although livers of mature rats of both sexes have comparable amounts of P4502C24 mRNA, a dramatic sex difference is seen in the kidney where only males express detectable levels of this mRNA. © 1991 Academic
Press,
Inc.
P450 cytochromes represent a wide class of enzymes that are capable of a variety drug
of m e t a b o l i c
detoxification,
and
reactions
including
carcinogen
steroid
activation
synthesis,
(1-3).
The
2C
subfamily is the largest known group of P450s that have relatively similar primary structures but quite different and expression patterns In order whether cDNA
to
further
additional
libraries
investigate forms
are
this
group
expressed
from the prostate,
were screened with a P4502C7 probe, two independent
catalytic profiles
(4-13).
in
a known
of P450s
and examine
extrahepatic source
tissues,
of P450s
(14),
resulting in the isolation of
clones coding for a new member of this subfamily,
P4502C24.
M A T E R I A L S AND METHODS Isolation and c h a r a c t e r i z a t i o n of p r o s t a t e cDNA clones A random p r i m e d and an oligo dT p r i m e d rat prostate cDNA library in IZAP and ~gtl0 respectively, kindly p r o v i d e d by Drs Stefan Andersson and Maria Str~mstedt, were screened with a 5' to n u c l e o t i d e 1013 (SphI site) P4502C7 cDNA fragment (6,15), under conditions of reduced stringency as d e s c r i b e d p r e v i o u s l y (ii). About 200.000 Rlaque ~ o r m i n g units from each of the two libraries were screened and this r e s u l t e d in the isolation of clone prosl from the random p r i m e d and clone pros2 from the oligo dT p r i m e d
645
0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
library. The inserts were subcloned into the pGEM3Z vector (Promega) and sequenced using Sequenase (U.S. Biochemicals) in the dideoxy nucleotide chain t e r m i n a t i o n protocol with the use of universal as well as c u s t o m - s y n t h e s i z e d o l i g o n u c l e o t i d e primers b a s e d on p r e v i o u s l y d e t e r m i n e d sequence.
Northern analysis RNA from different tissues of mature S p r a g u e - D a w l e y rats and RNA from liver of rats at different d e v e l o p m e n t a l stages were isolated by the m e t h o d of Chomczynski and Sacchi (16). Tissue from three animals were p o o l e d for each RNA preparation. N o r t h e r n analysis was p e r f o r m e d as before (11,15) using as a probe an AvaII - AvaII fragment (position 165 - 982) of prosl.
RESULTS The
screening
sequences prosl
1031) of
pros2.
into
two
Prosl
two
is highly
the
the
prostate
cDNA
has
segments
an
insert
(Fig.
i).
1 to
Clone
2C
gene
subfamily,
in
1 - 124), which is not.
exon
2
(19),
pros2
into two
junctions
has
an
segments
insert (Fig.
to
nucleotide
substitution
that
end
and
untranslated 7 - 124) but
is
also
codon and
in
furthermore,
(T/C) nN(C/T)AG (Fig. of
with
from to
the
the
(20),
the
is
segment prosl
pros2
The
amino
deduced
nucleotides identical
125 to
with that
acids
which
T~G,
(18),
and
be
divided
125 - 1755)
is
of
results
a
in an
it further extends to
the A,
acid
A
at
the
codon,
prosl has
3'
2C P450s
clone.
This
a termination of segment
splice
junction
a
(nucleotides
1 of the
sequence
intron/exon
represent
can
that
3'
makes
A
structure.
Segment
of
-
with the
exception
of exon
A
125
B
sequence,
with
segment
B
it likely that the segment A
part
of
an
of
clone
unspliced
intron,
i.
acid 1426
sequence was
of P450s are
the
segment
amino
present
might
of intron
P4502C12
termination
sequence
i). This last o b s e r v a t i o n
clone
with
(A)+ tail.
be
segment
coincides
(nucleotides
translation
deduced
to
again
813,
can
the p o s i t i o n of
which
In addition,
consensus
specifically
Amino
a
to the
different
contrast
gene
B
cDNA clones, which
region of the members
(17),
prosl,
and a poly
contrast
in frame
Segment
P4502C7
(nucleotides
a known
bp
at p o s i t i o n
is d i s s i m i l a r
segment,
1755
Ile~Ser.
B
sequences
with
clone
contains
region,
of
i).
of
amino acid replacement, 3'
with
bp
Interestingly,
of P4502CII
the rat 2C P450s
identical
the
1031
Segment
this switch from similar to d i s s i m i l a r
P4502C13
of
similar to the c o r r e s p o n d i n g
P450
(nucleotides
exon
libraries
r e s u l t e d in the isolation of two independent
and
divided
of
found to be IIC6,
conserved
IIC7,
in these
646
72,
pros2, 66,
IICII, five
75,
deduced 63,
IICI2, rat
and
from 65 %
and IICI3.
P450s
are,
in
Vol. 180, No. 2, 1991
1
50
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pros2: Prosl:
ArgIleProArgAsnSerAspLeuLysLeuTrpLeuAlaAsn GCGGATTCCGCGGAATTCCGATTTGAAGTTGTGGTTGGCAAAC GCAGCTTTCTTGCAGCA-A--A-TT-G-C-T-CC-GTG-TCCC-AGGCT GlnLeuSerCysSer LysPheGly ThrCysGlyProGluAla
TyrArgCysGlyGlu***AspAlaHisIleIleLeuAsnProSerSerAlaThrHisPheLeuSerTyrPheGln TACAGATGTGGAGAGTAAGATGCTCACATTATCCTAAATCCTTCTTCTGCTACTCATTTTCTTTCTTATTTCCAG .... G-C-GGAA--C-T-TGlnPheThrAspLysLeuThr LysCysHisSerSerVal LeuHisIleAspLeuProGlyAsnLeuLeu C-GTTCACA-ATA-ACTTAC-G-AA-GTGCCA-AGTTC-GTCAG--TGCAC-T-G
125
PheSerLysIleTyrGlyProValPheThrLeuTyrPheGlyProLysProThrValValValHisGlyTyrGlu TTCTCAAAAATTTATGGCCCTGTGTTCACTCTGTACTTTGGTCCGAAGCCTACTGTGGTGGTACATGGATATGAA ...........................................................................
200
AlaValLysGluAlaLeuAspAspLeuGlyGluGluPheSerGlyArgGlySerPheProIleValGluArgMet GCAGTAAAGGAAGCCCTGGATGATCTTGGAGAGGAGTTTTCTGGGAGGGGTAGTTTCCCAATTGTTGAAAGAATG ...........................................................................
275
AsnAsnGlyLeuGlyLeuIlePheSerAsnGlyThrLysTrpLysGluLeuArgArgPheSerLeuMetThrLeu AATAATGGCCTTGGGCTCATTTTCAGCAATGGAACAAATGGAAGGAGCTTCGGCGTTTCTCACTTATGACCTTG ...........................................................................
350
ArgAsnPheGlyMetGlyLysArgSerIleGluAspArgIleGlnGluGluAlaSerCysLeuValGluGluLeu AGAAATTTTGGGATGGGGAAGAGGAGCATCGAGGATCGCATTCAAGAGGAAGCCTCCTGTCTTGTGGAAGAGCTA ...........................................................................
425
ArgLysThrAsnGlySerLeuCysAspProThrPheIleLeuSerCysAlaProSerAsnValIleCysSerVal AGAAAAACAAATGGCTCACTCTGTGATCCCACATTCATCCTGAGCTGTGCTCCCTCCAACGTGATCTGCTCAGTT ...........................................................................
500
ValPheHisAsnArgPheAspTyrLysAspGluAsnPheLeuAsnLeuMetGluLysLeuAsnGluAsnPheLys GTTTTCCATAATCGTTTTGATTATAAAGATGAGAATTTCCTTAACTTGATGGAGAAACTCAATGAAAACTTTAAA ...........................................................................
575
IleLeuAsnSerProTrpMetGlnValCysAsnAlaLeuProAlaPheIleAspTyrLeuProGlySerHisAsn ATCTTGAACTCCCCATGGATGCAGGTTTGCAATGCTCTCCCTGCCTTCATCGATTATCTCCCAGGAAGCCATAAT ...........................................................................
650
ArgValIleLysAsnPheAlaGluIleLysSerTyrIleLeuArgArgValLysGluHisGlnGluThrLeuAsp AGAGTAATTAAAAATTTTGCTGAAATAAAAAGTTATATTTTGAGACGAGTGAAAGAACATCAGGAAACACTGGAC ...........................................................................
725
MetAspAsnProArgAspPheIleAspCysPheLeuIleLysMetGluGlnGluLysHisAsnProArgThrGlu ATGGACAATCCTCGGGACTTTATTGACTGTTTCCTGATCAAAATGGAACAGGAAAAACACAATCCCCGTACTGAG ...........................................................................
800
PheThrIleGluSerLeuMetAlaThrValSerAspValPheValAlaGlySerGluThrThrSerThrThrLeu TTTACTATTGAAAGCTTGATGGCTACTGTAAGTGATGTATTTGTAGCTGGATCAGAAACCACAAGCACTACCCTG ............. T ............................................................. Ile
875
ArgTyrGlyLeuLeuLeuLeuLeuLysHisIleGluValThrAlaLysValGlnGluGluIleAspHisValIle AGATATGGACTCTTGCTCCTACTGAAACACATAGAGGTCACAGCTAAAGTCCAGGAAGAGATTGATCACGTGATT ...........................................................................
950
GlyArgHisArgArgProCysMetGlnAspArgThrArgMetProTyrThrAspAlaMetValHisGluIleGln GGCAGACACAGGAGGCCCTGCATGCAGGACAGGACCCGCATGCCCTACACAGATGCGATGGTGCACGAAATCCAG ...........................................................................
1025
ArgTyrIleAsnLeuIleProAsnAsnValProHisAlaAlaThrCysAsnValArgPheArgAsnTyrValIle AGATATATTAACCTCATCCCCAACAATGTGCCCCATGCAGCTACCTGTAATGTTAGATTCAGAAATTATGTAATT
1100
ProLysGlyThrAspLeuLeuThrSerLeuThrSerValLeuHisAspAspLysGluPheProAsnProGluVal CCCAAGGGCACGGACTTACTAACATCACTGACTTCTGTGCTACATGATGACAAAGAATTTCCCAACCCAGAAGTA
1175
PheAspProGlyHisPheLeuAspGluAsnGlyAsnPheLysLysSerAspTyrPheMetProPheSerThrGly TTTGACCCAGGCCATTTTCTGGATGAGAATGGGAACTTTAAGAAGAGTGACTACTT~TATGCCTTTCTCAACAGGA
1250
LysArgMetCysValGlyGluAlaLeuAlaArgMetGluLeuPheLeuLeuLeuThrThrIleValGlnAsnPhe AAGCGAATGTGCGTGGGAGAGGCCCTGGCTCGCATGGAGCTGTTTTTGCTTCTGACCACCATTGTACAGAATTTT
1325
AsnLeuLysSerPheValAspThrLysAspIleAspThrThrProMetAlaAsnThrPheGlyArgValProPro AACCTGAAATCTTTTGTTGATACAAAGGACATTGACACTACTCCAATGGCTAATACCTTTGGCCGTGTACCACCT
1400 1475 1550 1625 1700
SerTyrGlnLeuCysPheIleProArg*** TCATACCAGCTGTGCTTCATTCCTCGTTAAAGCAGAGCACACTGGGCTGTTGCTATGCTGGTGTCTGTGACTAAT CAGGGGCAATCCAGTTTCCACTGTTAGGAACATCTCTCTCAATTCTCCTCTCACATCTCTTCATTCTCTCACAAT ACAGTTATCATCCATCTTTGTTTAAGAGGTTTTCCCAGAGTTATCTCGCAAATCTATCCTTTGTCTCCCACAGTC TATAACACTTATATTGACTGTGAACTGTACTAAGACATGTGCTGGGTTACTAATATGTTATGGGTGTAATACAGA ATAGTTCAACTGAGAGCCATATCTTCTTTGCTTGATTCAAAATAAAAGGAGTTATTAACTGA
Figure I. N u c l e o t i d e and deduced amino acid sequence of clones prosl and pros2. The nucleotide sequence of the two p r o s t a t e clones is shown. Identical n u c l e o t i d e s in the prosl sequence are indicated by bars. The deduced amino acid sequence of pros2 and prosl is shown above and b e l o w the respective nucleotide sequence. Only amino acids which are different in prosl codons, the conserved 3' intron polyadenylation signal are underlined.
647
are shown. The dinucleotides,
termination and the
Vol. 180, No. 2, 1991
2C24 2Cli 2C6 2C7 2C13 2C12
51
i01
151
201
251
301
351
401
451
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FSKI GQSIKKFSKV TQSLTSFSKV SQSLTKFSKT RQSLTNFSKT RQSISNFSKT
YGPVFTLYFG YGPIFTLYLG YGPVFTLYFG YGPVFTLYLG YGPVYTLYVG YGPVFTLYFG
PKPTVVVHGY MKPFVVLHG¥ TKPTVILHG¥ SQPTVILHGY SQPTVVLHGY SQPTVVLHGY
EAVEXALDDL EAVKEALVDL EAVKEALIDH EAIKEALIDN EALKEALVDH EAVKEALIDY
GEEFSGRGSF GEEFSGRGSF GEEFAERGSF GEKIrSC.-P.GSY GEEFSGRGRL GEEFSGRGRM
PIVERMNNGL PVSERVNKGL PVAEKINKDL PMIENVTKGF PICEKVAKGQ PVFEKATKGL
GLIFSNGTKW GVIFSNGMQW GIVFSHGNRW GIVFSNGNRW GIAFSHGNVW GISFSRGNVW
KELRRFSLMT KEIRRFSIMT KEIRRFTLTT KEMRRFTIMN KATRHFTVKT RATRHFTVNT
LRNFGMGKRS LRTFGMGKRT LRNLGMGKRN FRNLGIGKRN LRNLGMGKGT LRSLGMGKRT
IEDRIQEEAS IEDRIQEEAQ IEDRVQEEAR IEDRVQEEAQ XEDKVQEEAK IEIKVQEEAE
CLVEELRKTN CLVEELRKSK CLVEELRKTN CLVEELRKTK WLVKELKKTN WLVMELKKTK
GSLCDPTFIL GAPFDPTFIL GSPCDPTFIL GSPCDPSLIL GSPCDPQFIM GSPCDPKFII
SCAPSNVICS W F H N R F D Y K GCAPCNVICS IIFQNRFDYK GCAPCNVICS IIFQNRFDYK NCAPCNVICS ITFQNHFDYK GCAPGNVICC IILQNRFDYE GCAPCNVICS IIFQNRFDYK
DENFLNLMEK DPTFLNLMHR DQDFLNLMEK DKEMLTFMEK DKDFLNLIEK DKDFLSLIEN
LNENFKILNS FNENFRLFSS LNENMKILSS VNENLKIMSS VNEAVKIISS VNEYIKIVST
PWMLVCNALP PWLQVCNTFP PWTQFCSFFP PWMQVCNSFP PGIQVFNIFP PAFQVFNAFP
AFIDYLPGSH AIIDYFPGSH VLIDYCPGSH SLIDYFPGTH ILLDYCPGNH ILLDYCPGNH
NRVIKNFSEI NQVLKNFFYI TTLAKNVYHI HKIAKNINYM NIYLKNYTWV KTHSKHFAAI
KSYILRRVKE KNYVLEKVKE RNYLLKKIKE KSYLLKKIEE KSYLLEKIKE KSYLLKKIKE
HQETLDMDNP HQESLDKDNP HQESLDVTNP HQESLDVTNP HEESLDVSNP HEESLDVSNP
RDFIDCFLIK RD~IDCFLNK RDFIDYYLIK RDFVDYYLIK RDFIDYFLIE RDFIDYFLIQ
MEQEKHNPRT MEQEKHNPQS WKQENHNPHS QKHANNIEHS RNQENANQWM RCQENGNQQM
EFTIESLMAT EFTLESLVAT EFTLENLLIT EYSHENLTCS NYTLEHLAIM NYTQEHLAIL
VSDVFVAGSE VTDMFGAGTE VTDLFGAGTE IMDLIGAGTE VTDLFFAGIE VTNLFIGGTE
TTSTTLRYGL TTSTTLRYGL TTSTTLRYAL TMSTTLRYAL TVSSTMRFAL TSSLTLRFAL
LLLLKHIEVT LLLLKHVDVT LLLLKCPEVT LLLMKYPHVT LLLMKYPHVT LLLMKYPHIT
AKVQEEIDHV AKVQEEIERV AKVQEEIDRV AKVOEEIDRV AKVQEEIDHV DKVQEEIGQV
IGRHRRPCMQ IGRNRSPCMK VGKHRSPCMQ IGRHASPCMQ IGRHRSPSMQ IGRHRSPCML
DRTRMPYTDA DRSQ~YTDA DRSRMPYTDA DRKHMPYTDA DRSHMPYTNA DRIHMPYTNA
MVHEIQRYIN VVHEIQRYID HDHEVQRFID HDHEVQRFIN MVHEVQRYID MIHEVQRYID
LIPNNVPHAA LVPTNLPHLV LIPTNLPHAV FVPTNLPHAV IGPNGLLHDV LAPNGLLHEV
TCNVRFRNYV TRDIKFRNYF TCDIKFRNYL TCDIKFRNYL TCDTKFRNYF TCDTKFRDYF
IPKGTDLLTS IPKGTNVIVS IPKGTTIITS IPKGTKVLTS ZPF~TAVLTS IPKGTAVLTS
LTSVLHDDKE LSSILHDDKE LSSVLHDSKE LTSVLWnSKE LTSVLHDSKE LTSVLHDSKE
FPNPEVFDPG FPNPEKFDPG YPDPEIFDPG FPNPEMFDPG FPNPEMFDPG ¥PNPEMFDPG
HFLDENGNFKKSDYFMPFST HILDERGNFKKSDYFMPFSA HFLDGNGKFK KSDYFMPFSA HFLDENGNFKKSD~LPFSA HFLDENGNFKKSDYFIPFSA HFLDENGNFKKSDYFMPFSA
GERMCVGEALARMELFLLLT GKRICAGEAL ARTELFLFFT GKRMCAGEGL ARMELFLFLT GERACVGEGLARMQLFLFLT GERMCLGESL ARMELFLFLT GKP.KCVGEGL ASMELFLFLT
TIVQNFNLKS TILQNFNLKS TILQHFKLKS TILQNFNLKS TILQNFKLKS TILQNFKLKS
FVDTEDIDTT LVDVEDIDTT VLHPKDIDTT LVHPKDXDTM LVDPEDINTT LSDPEDXDIN
PSYQLCFIPR PFYEACFIPV QRADSLSSHL PFYELCFIPL PTYQLCFIPS PTFQMRFIPL PTFQLCFIPV
PMANTFGRVP PAISGFGHLP PVFNGFASLP PVLNGFASLP PICSSLSSVP SIRSEFSSIP
Figure 2. C o m p a r i s o n of t h e amino acid sequence of P 4 5 0 2 C 2 4 , deduced from nucleotides 125 to 1 4 7 6 of p r o s 2 , w i t h t h a t of P 4 5 0 s 2CII, 2C6, 2C7, 2 C 1 3 , a n d 2 C 1 2 . I d e n t i c a l a m i n o a c i d s a r e i n d i c a t e d by bold letters. The numbering corresponds t o t h a t of a t y p i c a l 2C P450.
general, 302
also c o n s e r v e d in this new form, including Cys 435 and Thr
(Fig. 2) and according to the
this P450 is termed IIC24. 648
standardized nomenclature
(21)
Vol. 180, No. 2, 1991
1
2
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
3
4
5
6
7
8
1
®
2
3
4
5
6
7
8
®
Figure 3. Northern analysis of total RNA from different rat tissues probed with CYP2C24 sequences (see Materials and Methods). The individual lanes are as follows: lane i, ovary; lane 2, female lung; lane 3, female kidney; lane 4, female liver; lane 5, prostate; lane 6, male lung; lane 7, male kidney; lane 8, male liver. Figure 4. Northern analysis of total liver RNA from rats at different developmental stages probed with CYP2C24 sequences (see Materials and Methods). The individual lanes are as follows: lanes 1 to 4; 8, 4, 2, and 1 week old females, lanes 5 to 8; 8, 4, 2, and 1 week old males.
Northern
analysis
of a b o u t
1800
not
apparently
sex
difference
P4502C24
indicated
detectable is
mRNAs
developmental
analysis
that,
comparable
amounts
express
in
the
females
do
of
the
although of
with a typical
in t h e
liver
in the p r o s t a t e .
observed
while
indicated
weeks
that mRNAs
bp are e x p r e s s e d
this
significant
where
mRNA,
only
amounts
males
do
3).
In
of
2C24
in
animals
of
males
of t h i s
both
of
P450
age
size
but
are
a dramatic
(Fig.
expression
mature
sexes
Furthermore
kidney not
2C P450
of b o t h
express
addition, the
liver
sexes two
(Fig.
have
and
four
4).
DISCUSSION cDNA
clones
have
been
do
not
P450
contain I.
of
a the
termination
the
region
Instead,
segment deduced codons
absence
might
that
clone
would
pros2
of u n s p l i c e d
that
has
P450IIC24
no
these
correspond
a typical
a
intron
termination amino
subfamily
Interestingly,
contains
part
2C g e n e
acid
to
segment
I, w h i l e codons
sequence.
exon
represent
of an i n i t i a t o r
an
1 te e x o n 2 j u n c t i o n alternatively
clone
in
methionine
649
codon
in t h i s
2C
with
lack
of
segment
that
clone
transcript. alternate
is
prosl
frame
This
suggests
spliced
clones
which
as w e l l as the fact t h a t t h i s d i s s i m i l a r
at t h e p u t a t i v e
prosl
of the P 4 5 0
from the prostate.
to r e p r e s e n t
contains
ends
for a n e w m e m b e r
isolated
exon
likely
that
coding
The
exon
1
Vol. 180, No. 2, 1991
sequence
(Fig.
produce
I) indicates
a protein
to the
5'
P4502C6,
end.
protein
A
that
exon (22).
similar
8, are In
of mature
case
still
male
and
the
prostate
libraries,
prosl
and
to
does not
is not fully extended splicing
of synthesizing
2C24,
female
rats
2C6
involves
pros2
expressed
in the
levels,
also
expression but in favor of
clones
observed in the Northern analysis,
also
a microsomal
at comparable
is expressed
no significant
rare events.
transcript
having an alternate instead of the
in developmental
and furthermore
Although
represent
this
of differential
capable
similarity
shows a sex difference the female,
either
or that the cDNA sequence
where mRNA transcripts,
canonical liver
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in the female brain have
signals
been
(22).
isolated
from this tissue
from were
indicating that such transcripts
In agreement with that,
is the large number
of Rlaque ~orming units that had to be screened in order to isolate the
two
kidney
clones.
Furthermore,
and the liver,
1800 bp are seen, the
cloning
characterization
be
P4502C24
speculated
from mRNAs
containing
Verification of
can
signals
transcripts
1 might predominate. await
where
it
of the exon 1 region
in
the
of a size of about
a typical
of this hypothesis from
that
these
P450-1ike
tissues
of the P4502C24
Acknowledqments: This research was supported by Swedish Medical Research Council (No 03X-06807) Work Health Fund.
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