Note: Descriptions are shown in the official language in which they were submitted.
213~5~ .
I-- wo 93/2~331 i Pcr/uss3/o3936
HUMAN CRABP-I AND CRABP-II
FIELD OF THE INVENTION
The present invention relates g~nerally to cellular retinoic acid binding proteins
(CRABPs) and, more specifically, to human CRABP-I and CRABP-II and the sequences5 encoding them, and their use in various assay systems for screening and diagnostic
applications and for Ulerapeutic purposes.
RELATED APPI ICl~TlONS
This is a continuation of U.S. Application Serial No. 07/874,847, entitled "Human
CRABP-I and CRABP~ a filad April 28,1992 by Voorhees et al. which is a continuation-in-
10 part of co-pending U.S. application Serial No. 751,8~3 entitl0d ~Human CRABP-I and
CRA3P-IIR, filed August 30,1991 by Voorhees et al., all her~in incorporated by reference.
GENE!~ANK ACCESSION INFORMATlON
GENE ACCESSiON NO.
human CRABP-II M68867
E~ACKGROUND OF THE INVENTION
Retinoids are essential regulators of epithelial cell growth and cellular dfflerentiation,
skin being a major target in both normal and pathological states. Spom, M.B. et al.,
Cancer Res. 43:3034 3040 (1983); Kopan, R. ~t al., J. Cell BioL 109:29~307 (1989);
Asselineau, D. at al., Dcv. Bio/. 133:3æ-3~5 (1989); and Uppman, S.M. ~t al., Pharmacol.
ZO Th~r. 40:107-122 (1989).1t has been shown that retinoids prevent cancer in skin and have
efficacy as agents Tn human malignant and premalignant cutaneous disorders. Asselineau,
D. et al., Dev. Biol. 133:322-335 (1989). It has also been shown that retinoids cause
growth ir.hibition in many hyper-proliferating cell lines, a feature that makes the compounds
of fundamental interest as anti-tumor and anti-psoriatic agents. Sporn, M.B. st aî., Cancer
25 Res. 43:303~3040 ~1983); and Asselineau, D. et al., Dev. Biol. 133:322-335 (1989).
Retinoids also play fundamental roles in directins the spatial organization of cells during
development and the generation of vertebrate limbs. Eichele, G. Trends Genet. 5:24~251
~1989); and Summerbell, D. et al., Trends Neurosci. 13:142-147 (1990).
The elucidation of tlie function of retinoids in the complex biologicai processes
30 involved in cell growth and differentiation requires the identffication of the specific
components of the retir, ~ signal transduction system as well as the genes directly
regulated by this system. ~everal intraccllular reUnoid-binding proteins have already bcen
identified, including cel!ular retinol-binding proteins (CRBP), nuclear retinoic acid receptors
(RAR), cellular retinoic acid binding proteins (CRABP) and, most recentiy, RXRs, also
35 belonging to the nuclear receptor superfamily of genes. See Sundelin, J. et al., J. Biol.
Chem. 260:6488 6493 (1986); Li~ E. et al., PNAS (IJSA) 83:577~5783 (1986); Nilsson,
M.H.L et al., Eur. J. Biochem. 173:4~51 (1988); Stoner, C.M. et al., Cancer Res.
213~S'~
WO 93/22331 . ~ PCI~US93/03936 ~--
2 -
49:1497-1504 (1989); Giguere, V. et al., PNAS (USA) 87:6233 6237 (1990); Petkovieh, M.
et al., Naturs 330:444450 ~1987); Brand, N. et al., Nature 332:850 853 (1988); Benbrook,
D. et al., Nature 333:66~672 (1988); Zelent, A. et al., Nature 339:71~717 (1989); Krust, A.
et al., PNAS ~USA) 86:531~5314 ~1989); and Mangelsdo~, D.J. et al., Nature 345:22~229
5 (1 990).
Cellular retinoic acid binding proteins (CRABP) ar~ low molecular weight protsins
prasent in human skin with increased levels found in psoriatic lesions and aftcr external
and systemic retinoid ~reatment. Sieganthaler, a. et al., J. Inv~st. DermatoJ. 86:4245
(198~); Hirschel-Scholz, S. et al., Eur. J. Clin. Invest. 19:22~227 (1989); and Siegenthaler,
10 G. et al., Arch. De~matol. 123:169~1692 (1987). Although unde~ctable in keratinocytes
grown in low c~lciurn medium, CRABP is expressed when a more dl~ferentiated phenotype
is induced by growth to confluence in the presence of elevated extracellular calcium
concentrations. Siegenthaler, G. et al., Exp. CellRes. 178:114126 (1988).
Although th~ precise role of CRABP in retinoic acid ~RA) action has not been
15 determined, it has been suggested tha~ CRABP might act as a shuttle protein, facilitaUng
tha movement of RA to th~rnucleusl or that CRABP might sequester RA, thereby
decreasing the cellular response. Takæe, S. et al., Arch Biochem. Riop~fs. 247:~28 334
~1986); Maden, M. at al., Natu~e 335:733 735 (1988). In a recent stuciy, }t was found that
all biologically active RA analogs in F9 cells bound to RARs, while h~o of them did not bind
20 to CRABP. 3enbrook, D. et al., Nature 333:66~672 (1988). This suggests that retinoid
binding to RA, but not necessarily to CRABP is necessary to induce cell dfflerentiation.
The effects of retinoic acid on gene transcription can be mediated by retinoic acid
receptors (RARs) and retinoid X receptors (R,YRs). Leed, M. et ai., Cell 68:377-395 (1992);
Mangelsdorf, D.J. et al., Genes DeY. 6:329 344 (1992). RARs have been shown to bind
25 retinoic acid (RA) with high affinity, while iV(Rs apparentiy have no affinity for this ligand.
Mangelsdort, D.J. et ai., Natv~ 345:~229 (1990). However, it was recently
demonstrated that ~cis RA can bind to PXR - ~ with high affinity. Levin, A. A. et al., N2ture
35~: 35~361 (1992~; Heyman, R.A. et al., Cell 68:397406 (1992). CRABPs have beenshown to bind PA with high affinity, but thair ~unction is pooriy understood. However, it
30 was recently demonstrated that CRABP may be invoived in cytochrome P 450 metabolism
of i~ Fiorella, P.D. et al., J. 8iol. Chem. 266:16572-16579 (1991).
Two isofonns of CRABP, CRA8P-I and ll, have been identifi~d and cloned In the
mouse. Stoner, C.M. et al., Cancer Res. 49:1497-1504 (1989); Giguere, V. et al., PNAS
(USA) 87:6233 6237 (1990); Nilsson, M.H.L ~t ai., Eur. J. Biochem. 173:45-51 (1988). By
35 isoform is meant two amino acid sequences with substantial sequence idantity. Bovine
- W 0 93/22331 ~ 21~4~50 PCT/US93/03936
CRABP-I has also been sequenced, and the NH2 terminalregions ofrat and chicken
C:RABP-lhave also been recently determined. Bailey, J.S. etal.,J. Biol. Chem. 263:932
9332 (1988); K~amoto,J. et al., Biochem. Biophys. Res. Comm. 157:1302-1308 (1988).
Human CRABP has,however, not been pre~ous~ isolated or cloned.
SUMMARY OF THE INVENTION
The sequences encoding h~o isoforms of human c~llular retinoic acid binding
proteins, designatad CRA~P-I and CRABP-il! and the gene for CRABP-II, have been cloned
and sequenc~d. llleir nucleic acid and corresponding amino acid s~quences are sat ~orth
in the sequence listing preceding the claims. Expression ot human CRA13P-II, but not
CRABP-I was markedly increased in human skin in vivo and in skin fibroblasts fn vitro a~t~r
treatment with retinoic acid (RA) and a~ter trea~ments which induoe keratinocytedfflerentiation. The importance of RA dependent mRNA stabilization tor ksying CRABP 11
message at induced levels once transcription has occurrsd is also described.
The cloning and sequencing of human CRABP provides the basis for the
construction of hurnan CRABP-I and 11 viral, prokaryotic and eukaryotic expression vactors
and recombinant expression constructs. Hurnan CRABP can now also be produced
synthetically or ex vfvo (outside thc human body), for example, through the production of
h sion proteins in bacteria and later cleavage and purification of CRABP therefrom.
Ugand binding studies utilizing human CRABP sequenc~s can determine ligand
20 binding amnity and the interaction of human CRABP with other human retinoid-binding
proteins, and can be used to better identi~y tissu~speciffc drugs for pathologies in which
retinoid are implicated. Various ass~v schemes, including reporter assay systems, direct
and competition hybridization and Dinding assays employ the nucleic and amino acid
sequenc~s hersin described. Artibodies or binding fragments ther60f produced to human
25 CB~BP can also be used in immunoassays of patient tissues for CRABP levals for
diagnosis and the monitoring of treament. Purified or synthetic human CRABP can also
be used for supplementaUon therapy.
Other ~atures and advantages of the present invention will become apparent from
the following description and appended claims, taken in conjunction with the
30 accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 gives the sequences of the fo~ward and reverse degenerate primers used
in cloning human CRABP.
Figure 2 compares human, mouse, rat and chicken CRAaP. Panel A is a
35 comparison of the amino acid sequences of human (h) and mouse (m) CRABP. Dashes
213455G
WO g3~22331 ' PCI'/US93~03936 ~,
4- --
represent sequence identity; the asterisk at residue 118 represents a gap introduced in the
CRABP-I sequence for ma~imum alignment. Panel B is a sequence comparison of the
NH2-terrninai ends of hurnan (h), mouse (m), rat (r) and chicken (c) CRABP. Boxed
residues represent those dissimilar to human CRABP-II.
Figure 3 is an autoradiogram of RNA blots deriv~d trom ~hree individuals which
illustrates induction of CRABP-II mRNA in human skin by topically applied retinoic acid
(RA). Lane (a) represents no treatments; lane (b) RA vehicle; and lane (c) 0.1% RA cream
in RA vehicle under occlusion.
Figure 4 is a bar graph of the RNA blot hybridkation results (quanfflated by laser
densitometry) of nine independent axperiments involvin0 five dzrmai fibroblast lines
prepared from three individuals and three diploid human lung fibroblast lines. The results
iHustrate the induction of human CRABP-II m~NA in human darmal fibroblasts compared
to lung fibroblasts. Ths inset shows the relevant autoradiographic bands trom hvo
representati~ s experiments comparing dermal and lung fibroblasts.
Figure 5 illustrates the expression of CRABP-II mRNA in cultured k~ratinocytes
under various conditions. Figure 5A is a bar graph of the r~suts obtained tor seven
independent keratinocyte strains and illustrates the effects of confluence on human
CRABP-II mP~NA levels. The error bars rapresent ~ SE ~p<0.005, ~*~p~0.0005 relative
to cells 2 days preconfluence. Figure 5B shows tha effects of RA and increased calcium
23 concentration on human CRABP-II mRNA levels in postconfluant keratinocytes, with
treatment tor prolonged periods with low concentrations of RA (3x109M~ having nodetectable effect. Figure SC illustrates that prolonged treatment of pos~confluent
keratinocytes with higher concentrations of RA (3x10~M) reduced Ci~9BP-II mRNA to
undetectable levels.
Figure 6 is a schematic diagram of a retinoic acid receptor (RAR)-CRAi3P or RXR-CilABP cotransfection assay.
Figure 7 is a restriction map of a bacteriophage lambda clone (A2.1 ) isolated from
a human placenta genomic library of the human CRABP-II gene is provided with the exons
indicated as filled boxes numbered I to iV.
Figure 8 illustrates the nucleotide sequence and shown above the nucleotide
sequence is the deduced amino acid sequence at the human CRABP-II gene.
Figure 9 illustrates the transcripUon start sites in the human CRAi3P-II gene asdetermined by primer extension anaiysis, with thQ major transcripbon site indicated by an
arrow and the corresponding base in the sequenc~ indicated by an asterisk.
I -; W093/22331 2 1 ~ ~ 5 5 0 PCr/USs3/03936
Figure 10 illustrates a nuclear run~ff assay using nuclei isolated from cu~ured
human skin ffbroblasts treated with retinoic acid.
Figure 11 illustrates the mRNA lavel and transcription tate o~ C:RABP-II after
quantitation by phospho~maging and normalization to cyclophilin.
Figure 12 is an autoradiogram demonstrating the effects ot cycloheximide or
actinomycin D on the induction of CRABP-II mRNA expression.
Figure 13 is a bar graph demonstrating the effec~s of eycloheximide on CRABP-II
transcription rates determined by nuclear run~n assays.
DETAILED DESCRIPTION OF THE PREFERP~ED EMBODIMENTS
1 0 OVERVIEW
The cloning and sequencing of two human CRABP cDNAs revealed one with a
predicted amino acid sequence 99.3% identical to tha mouse and bovine CRABP-I, and
a second with a predicted amino acid sequence, 93.5% identical to the mouse CRABP-II.
The CRABP-II described herein appears to be the human homolog to rnouse CRABP-II,
15 since both are expr6ssed in adult skin, and is therefor dasignatad as such. The high
amino acid homstogy seen behNeen boYine, mouse and human CRABP-I indicates that this
isofonn has been more conssrved throughout 0volution than th3 CRABi'-ll (Figure 2A
described below). This was especially evident when the NH2-~erminal sequence between
rat, chicken, mouse and human CRABP-i and CRABP-II were compared. No dfflerences20 were seen between the CRABP-ls, while several dfflerences sxist betYeen the CRA8P-lls.
CRABP-I transcripts were undetectable in adult human epidermis by RNA blot
hybr;dkation, while the CRABP-II cDNA probe d2tected an approximately 1.2 kilobase (kb)
mRNA transcript. External applicaUon o~ 0.1% retinoic acid cream in vivo for 16 hours
resulted in a 1 ~fold induction of CRABP-II, but not CRABP-I. CRABP-II mRNA, was aiso
25 markedly increased (>1 ~fold) by retinoic acid treatment of fibroblast cultured from human
skin, whereas no significant induction of CRABP-II mRNA was observed in human lung
fibroblast. Human CRABP-II, but not CRABP-I mRNA was significantly induced by
treatrnents which induce keratinocyte dfflerentiaUon in vitr~. Highly increased levels of
CRABP-II RNA were found in psoriatic apidermis when compared to normal epidermis for
30 10 dfflerent patients. CRABP-I message levels were on the other hand very low or
undetectable in both normai and psoriatic skin ~data not presentsd in Figures). Previous
studies Ot the induction of CRABP by natural and syntheUc retlnoTds and their increased
Ievels during keratinocyte dfflerenffation are consist~r,t with the ~xpression of CRABP-II
message seen in the present investigation, but not with the exprsssion of CRABP-I. These
213~550 - ;
WO93~22331 ` ; PCI`~US93/03936 ~`,
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studies thus identify CRABP-II as the isoforrn likely to be expressed and regulated by RA
in adult human skin.
Since mRNA isolated from skin biopsiçs used in this study is approximately 95%
derived from keratinocytes (see Voorhees, J.J. et al., Arch. Dermatol. 105:69~701 (1972)),
5 the majority of RA induetion of CRABP-II seen in vivo.~(Figura 3 deserlbed below) cannot
be explained by the presence of mRNA derived from dermal fibroblasts. This is in contrast
to the reduction of CRABP-II transcripts observed in response to high concentrations of
RA in keratinocytes in vi~o (Figure 5C describ~d below). When grown to postconfluence
under the same conditions used hsrein, human keratinocytes undergo coordinate
10 increases in involucrin content and transglutaminase activity, h~o key determinants of
comffled envelope formation during terminal dfflerentiation. Pillai, S. et al., J. Cell. Physiol.
143:294302 (1990). Moreover, these events are accelerabd and began to occur prior to
confluence when the extracellular calciurn concentrations were increased to 1.2 or 2.4 mM.
Pillai, S. et al., J. Cell. Physiol. 1~3:29~302 (1990). CRABP-il mRNA levels responded to
15 confiuence and external calcium in an identical fashion (Figure 5 described below). These
results strongly favor the concept that the stratified structure of th~ epidermis and/or the
presence of dermis is an important detsrminan~ of CRABP-II rsgulation in vivo, and may
help to account for ths dfflerenUal rssponsiveness of keratlnocytes to RA in w~o and /n
vivo. The nsgative eflect of high concsntrations of RA on C:RABP-II exprsssion seen in
20 vitro is similar to the effect of this compound on transglutaminase type I mRNA levels in
cultured keratino~tes. See Floyd, E.E. et al., Mol. Cell. BioL 9:4846~851 (1989).
Whether RA inductlon of tne human CRABP-II gene occurs at the level of
transcription and whetherthis regulaUon is mediated by specffic nuclear receptors remains
to bs investigated. It has been shown that human skin as well as cultured human skin
25 fibroblasts express RAR~. Krust, A. et al., PNAS (USAJ. 86:531~5314 (1989); and Elder,
J.T. et al., J. Invest. Dermatol. 96:425 433 (1991). However, this does not explain the lack
of RA induction ot CRAi3P-II mRNA seen in cuitured human lung fibroblasts and
keratinocytes, also known to express RAR~. Elder, J.T. et al., J. Inves~ Dermatol.
96:425 433 (1991). H the CRABP-II gene is reguiated by the RARs, additional Ussue or
30 cell-specific factors may be required for RA induction.
Members o~ the nuclear superfamiiy of receptors have been shown to interact withtheir responsive elements as dimers. 61ass, C.K et al., Cen 59:697-708 (1989). RARs
have also been shown to Interact ~vith other members of this tamily (I.e., the thyroid
hormone receptors) forming heterodimers. Glass, C.~ et al., Cea 59:697-708 (1989). One
35 of the most exclting findings recently Ts that the thyroid hormone recsptors require an
WO 93/22331 ~ 2 1 3 ~ 5 5 0 Pcr/l~ss3/o3936
- 7 -
auxiliary protein (T~AP) to interact wth the thyroid hormone responsive element in the
growth hormone gene. O'Donell, A.L et al., Mol. Endocrinol. 5:9~99 (1991). TRAP
apparently is forming a heterodimer w~h the thyroid hormone receptor on the responsive
element. Such dimerization betw0en nuclear receptors and oth~r transcription factors
5 could explain tissue-specific regulation. ff there is a skin-specific factor forming a
neterodimer with the RARs, that could explain why the C:RABP-II gene is induced by RA
in skin fibroblasts, but not lung fibroblasts.
In summa~, we have demonstrated that CRABP-II is expres~ed in human skin in
vwo and the CRABP-II gene appears regulated by RA in skin in YiVo and in cultured skin
1 O fibroblasts in V~D. CRABP-II was not, however, inducsd by RA in cu~ured lung fibroblasts,
demonstrating cell-specific regulation of this gene. CRABP-I, on the other hand, does not
appear regulated by RA and is found at very low or undetectable levels in human skin in
vivo, as wall as in keratinocytes and fibroblasts. This suggests that CRABP-II may
participate in a rsgulato~ feedback mechanism to control the ac~ion of RA on cell
1~ dfflerentiation in skin. The identification of human CRABPs, RARs and RXRs now allows
studies, such as those describ~d below, on intera~ons between members of these
families in Ule complex molecular and cellular mechanisms of RA action.
The identificaUon of the nucleic and amino acids sequences of human CRABP-I and
CRABP-II provTde tha basis ~or a variety of recombinant products, including vectors
20 carrying the human CRA3P cDNA secuences and expression constrwts cotransfected or
infected w th such vectors. For example, plasmid or viral vectors carrying human CRABP
cDNA have been constructed and are used to cotransfect or coinfe~t receptor-deficient
CY-1 monkey kidnsy cells. Reporter assay systems utilizing CV-1 recombinant expr~ssion
constructs which include human CRABP cDNA, a reporter element containing a retinoid
responsive element and a reporter gene, and preferably internal control sequences, such
as generaliy descrlbed in Astrom, A. et al., Biochem. Biophys. Res. Comm. 173:339 34S
~- 390), oan also now be constructed. Chimeric receptor prot~ins such as those described
in U.S. Patent No. 4,981,784 to Evans et al., can aiso be synthesized and utilized in
rsport6r assays.
Assay systems emp'-\ ing human CRABi' in conjunction with additional retinoid-
binding proteins are aiso coMemplated as within the scope of the Inven~on. i~c ~ example,
as shown schemaffcally in Figure 6, viral (e.g. SV40) vector carrying a human CRABP, a
reporter plasmid carrying t5~-~ retinoid responsn~e element (e.g. RRE3~-CAT), a viral (e.g.
SV40) vector carrying a human RAR or i~R of interest and a ~-galactosidase vector
(pcH110) for an internal control are coinfected or cotransfected into CV-1 cells. As shown
~1345~
WO 93/~2331 ' ` PCr/US93/03936 ~'~
- 8 - ~
in Figure 6, the recombinant CV-1 construct is exposed to the binding ligand (e.g. retinoid)
of interest. By ligand is meant a molecule which binds to the receptor binding protein and
induces the expression ot the gene of interest. In this assay system, induction of the
reporter gene is used to assay for ligand binding to receptor protein. Absent any
5 additional regulatory requirements, functional ligand would bind RAR or i~(R, sUmulat~n~
the expression (and tran~lation) of the reporter gene ~CAT). By including human CRABP
in the system, the interaction between human CRA8P and RAR or i~YR or other binding
receptors can be determined. For example, if CRABP sequesters PA in the cytoplasm, less
ligand wili reach the nucleus, thereby reducing RAR or ~XR-mediated stimulation of the
10 reporter gene.
The sequencing of human CRABP also allows the raising or production of
antibodies or binding *agments, e.g. F~ab), which can be used in immunoassays tofurther characterize binding or for diagnostic purposes and to monitor the course of patient
treatment. For example, patient tissue can be assayed for the pressnce and levels of
15 human CRABP to diagnose particular conditions where retinoids are implicated and to
monitor the effectiveness of drugs and other treatments in aitering patient levels of CRABP.
CRABP purified directiy from human tissue or cells, in cuiture or human CRABP produced
synthetically or ex vivo will also provide CRABP ~or supplementation therapy where
needed.
The gene for human cellular retinoic acid-binding protein ll (CRABP-II) has beencloned and sequenced. It was isolated from a human placenta genomic library and is
contained within one bacteriophage clone. The gena spans 6 kilobases and cons~sts of
four exons and three introns as do other m0mbers of the hydrophobic ligand binding
protein sens family. Wei, LN. et ai., DNA Cell Biol. 9:471~78 (1990). The mouse CRABP-I
25 gene has previousiy been cloned, demonstrating a dfflerent intron, but similar exon
arrangement. Wei, L.N. et al., DNA Cel/ Biol. 9:471478 (1990). One major transcription
site was mapped to an A residue 137 nucleotides upstream of the ATG initiation codon.
The sequence of the upstream region of the CRABP-II gene is rather GC rich and has a
TATA box at ~i, and severai possible binding sites for transcription tactors.
Of spedal interest is the presence ot potantiai AP2 (CCC/GCA/GGGC) sites in the
5' flanking region. AP2 has been shown to be idenUcal to the transcription factor KER1
which has been suggest0d to bs gen~rally invoived in e~idermal gene regulaUon. L~ask,
A. et ai., PNAS ~JSA) 88: 794~7952 (1991). CRABP-II is predominanUy expressed in the
skin of aduit mice and we have recentiy demonstrated that CRABP-II, but not CRA8P-I is
expressed in human skin. Giguere, V. et al., PNAS ~JSA) 87:6233 6237 (1990); Astrom, A.
2~3455 0
WO g3/2~331 PCr/US93/03936
g
et al., J. Biol. Chem. 266:17662-17666 (1991). In addition, both AP2 and CRAi3P-II mRNA
have been shown to bs induced by RA. Astrbm, A. et al., J. ~iol. Chem. 266:17662-17666
(1991); Luscher, B. et al., Genes Dev. 3: 1507-1517 ~1989). Whather AP2 is involved Tn
skin-spec~ic expression and ~A Induction of the CRABP-II gene remains to be datermined.
5 As shown in Fi~ure 8, the upstream region also contains a high amnity Spl bindiny site
(Gt3GGCGGAGC) close to tha TATA box, and two sequences (GCGGGGGCG) identical
to ~ox-~4 binding sites. Kadonaga, J. T. et al., rrends E~lchem. Scl. 11:20 23 (1986).
Lemaire, P. et al., MoL C~ll. Biol. 10:3456 3467 (1990). K~ox-24 is a member of the early
responsive gene family, suggested to be Tnvoived in regulation of cell proliferation and
i O dfflerentiation. Lemaire, P. et al., Mol. Cell. Biol. 10:345~3467 (1990); Edwards, S.A. et al.,
Dev. Biol. 148:16~173 (1991). It was recently shown that Egr-1 Kox-24 was induced by
RA in embryonal P19 cells. The induction of Egr-1 probin by RA in these cells,
demonstrated~a transient-5 fold increase, peaking around 30-60 minutss and returned to
basic levels between 60-90 minutes. Thus, as shown in Figure 11, the induction of Egr-1
15 protein by RA just precedes CRABP-II gene transcription. However, the invoivement of
Egr-1 In RA induced transcriptional regulation ot the CRABP-II gene in skin fibroblasts is
unlikely, since Egr-1 was found to be induced by treatment of the cells with cycloheximide
while CRABP-II genB expression in this study was found to be inhibited by this compound
as demonstrated in Figures 12 and 13. Edwards, S.A. et al., Dev. Biol. 148:16~173 (1991).
20 The upstream region of the CRABP-II gene also contains a direct repeat (G/AGTTCA)
- spaced with one nucleoUde with homology to the RARE found in tne RAR-~2 promoter,
except that the RAR-~2 RAREIs spaced by ~ve nucleoUdas. Umesono, K et al., Cell
65:1255 1266 (1991). It was recently shown tnat ~NO other memb~rs ot the hydrophobic
ligand-binding family of genes contain a RARE(CRBP-I) and a RXRE(CRBP-II) in their
25 promoters. Smith, W.C. et al., EMBO J. 1 0:2223-2230 (1 991 ); Mangelsdorf, D.J. et al., Cell
68:5~561 (1991). Whetner this direct repeat Is functional remains to be determined. To
determine whether RA inductlon of CRABP-II Is transcriptional, nuclear run-on assays were
performed. As can be seen in Figures 10 and 11, treatment of cuitured human skinfibroblasts with RA resuited in a rapid transient ~fold increase of transcription, tollowed by
30 a ~fold inductlon of CRABP-II mRNA levels. lllus, the CRABP-II gene Is mainlytranscriptionally activated by RA. The CRABP-II mRi~lAwas rapidiy induced within 2~ hours
In cultured human skln flbroblasts by retinolc acid, reaching a plateau after 6 hours of
treabnent. However, removai of reUnoic acld from tha medlum aRer 12 hours caused a
sharp decline in CRABP-II mRNA levels. The rapid increase of GRABP-II message was
35 mainiy due to an increased rate of transcription as determined by the nuclear run-on
i 5 3
wos3/2233l ~ ~ Pcr/~iS93/03936 ;~
- ~0 - ,,
experiments as shown in Figure 10. Increased transcription could be detected as early as
1 hour aftsr addition of RA, peakad at 2 hours and returned to basal levels within 6 hours.
in addition, both the accumulation of rnessage, as shown in Figure 12, and the inductlon
of transcription, as shown in Figure 13, by RA was inhibited by cycloheximide, sugges~ng
5 that the CRABP-II gene is transcriptionally regulated by a newly synthesized protein. Once
transcription had occurred the message reached a plateau and did not decline until RA
was removed from th~ medium. On-going protein synthesis was required for the transient
inoreas~ in transcription, sinca the induction was blocked by cycloheximide. ~ is vely
unlikely that the dacrease of CRABP-II mRNA seen was a result o~ reduced transcription
10 of the gene, since the RA induction of transcription was transient an~ back to control
levels within 6 hours. A more likaly explanation would be that RA was involved in
stabilkation of the ~:RABP-II message. The CRABP-II mRNA is likely to be unstable, since
it contains AU rich sequences in the 3' untranslated region. Giguere, V. et al., PNAS (USA)
87:6233 6237 (1990); Astr~m, A. et al., J. Bfol. Chem. 266:17662-t7666 tl991). ~t has been
15 shown that many transiently expressed genes, including Iymphokine genes, c-myc, and
c-fos contain AU rich sequences in their 3' untranslated regions, and that the presence of
these sequences correlates with rapid mRNA degradation. Cleveland, D.W. et al., New
BJol. 1:121-126. Mechanisms by which CRABP-II mRNA stabilization could occur, may
involve the production of a factor after treatment ot the cells with RA, or interaction of RA
20 with a preexisting factor, stabilizing the message. Since cycloheximide blocked the RA
induced transcripUon of the gene and as a consequence, the induction of the message,
the possibilities were not distTnguishable. It has been demonstrated that the human
CRABP-il yene is transientlytranscriptionally induced by RA in human skin fibroblasts, and
that this induction Ts dependent on on~oing protein synthesis. llle eariy induction of
25 transcription is followed by a rapid increase in CRABP-II message lavels that does not
decrease until RA is removed from the medium, suggesting RA dependent mRNA
stabilization.
It will be appreciated that the nwleotide and amino acid sequences of the present
invention can include some variaUon from the sequences represented by and
30 complementary to the sequences set forth in the Sequenc~ Usting, but must be
substanUally tepresentcd by or complementary to thosQ set forth therein. By ~substantially I
represented by" or "substantially complementary to~ is meant that any variation therein
does not impair the functionality of the sequence to any significant degree. As used
herein, A rapresents adenine; T represents thymine; G repr~sents guanine; and C
35 represer~s cytosine; except where otherwise indicated.
- ~ WO 93/22331 '~ 1 3 4 5 ~ ~ PCI/US93/03936
SPEC:IFIC: EXAMPLES
SPECIFIC EXAMPLE 1. Clonin~ and Sequencln~ of Human CRABP
MATERIALS AND METHODS
Clonl.~g of CRABP ~rom human skln FtNA by Potymerase Chaln Reactlon (PCR)
Total RNA was isolated *om human keratoma biopsies as describad in Elder, J.T.
et al., J. Invest. Derma~ol. 94:1~25 (1990) and c5NA was ~ynthesized by reverse
transcription as described in Maniatis, T. et al., Molecular Cloning: A Labof~tory Manual,
Cold Spring Harbor Labor~tory, Cold Spring Harbor, NY (t982). Degenerate primersderived from mouse and bovine CRABP-I were designed so as to amplify the coding
10 region. Xbal and ~amHI sites w~re contained in the forward and reverse primers,
respectively, which are shown in Figure lA.
Tha cl:)NA was used as a template for PCR (2 minutss d~naturation at 92~C, 2
minutes, annealing at 4~C, 2 minutes amplffication at 72C and final ext3nsion for 10
rninut~s). After 40 cycles, th6 leng~h of the amplffied DNA was determined on a 1.5%
15 agarose gel. The amplified 43~bp region of CRABP was isolated ~rom the gel, subclone~
in~ Bluescnpt phagsmid and seguenced as described below.
Scr~ening ~nd sequencing of cDNA cion~s
Pcly A+ RNA was prepared from human skin as dascribed in Elder, J.T. et al., J.
Invest. Dennatol. 94:1~2~ (1990) and used to prepare a cDNA library in i~mbda Zapll
20 (Stratagene Inc., La Jolla, CA). The library containsd 1.0 x 107 primary recombinants. The
4~bp PCR product was labeled by random hexamer priming (Boeringer Mannheim,
Indianapolis, IN) and used to screen the adult human whole skin cDNA library and a
human skin ~broblast cDNA library in Agt11 (Clontech, Palo Alto, CA). Duplicate
nitrocellulose filters wera hybridized for 16 hours in 50% forrnamide containing 5 x SSC t1
25 x SSC = 150 mM NaCI, 15 mM sodium citrate), 1 x Denhardt's (0.02% Ficoll/0.02% bovine
serum albumin/0.02% polyvinylpyrrolidone), 0.1% SDS (sodium dodecyl suifate) and 200
~glml tRNA. The fli~ers were washed tWG ~,mes for 20 minutes in 0.2 x SSC, 0.2% SDS and
one Ume for 20 minutes in 0.2 x SSCI 0.1% SDS at 55C. Two positive clones were
isolated from the skin library and flve clones from the skin flbroblast library. llle clones
30 from the skin library were rescued, while the clones from the ffbroblast library were
subcloned into Bluescript phagemlds (Stratagene Inc., La Jolla, CA).
DNA sequence anaiysis was performed on both strands by dideoxy chain
- termination as generally described in Sanger, F. et al., PNAS ~JSA) 74:5463 5467 (1977),
using mod-ffied 17 polymerase (Sequenase, U.S. Biochemical Corp.) and synthetic
3~ oligonucleot~des.
213 ~S a ~ -
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Cell cuftur~
Primary cultures of normal human keratinocytes were prepared as described in
Boyce, S.T. et al., in In Vitro Models for Cancer Research (Weber, M.M., and Sekely, Ll.,
eds) Vol. 3, pp. 24~274. CRC Press, Boca Raton, FL (1 9a6). Subcuitures were axpandad
5 in keratinocyte growth medium (KGM) (l:~lonetics, San Diego, CA). Human dermalfibroblast cultures were prepared from punch biopsies of buttock skin (see Harper, R.A.
et al., Science 204:52~527 (t979)) and propagated in modffled McCoy's 5A medium
containing 10% caH serum. Human lung fibroblasts were obtained from the American Type
Cultura Collection (ATCC) (Rockville, MD) and grown in the same medium.
10 Nor~hem analysls o~ mRNA
RNA was isolated from keratorne biopsies and cultured cells by guanidinium
isothiocyanate iysis and uitracentri~ugation as previQus~y described in Elder, J.T. et al., J.
Invest. Dermatol. 94:1~25 (1990). For studies involving retinoic acid treatment, 0.1% RA
cream (Retin-A, Ortho Pharmaceutical Corp. Raritan, NJ) was applied once to skin and
15 maintained under plastic wrap for 4 hours to 96 hours prior to biopsy. Adjacent sites were
treated with Retin-A vehicle or left untreated. After 4 hours, 12 hours, 16 hours or 96
hours, keratome biopsies were obtained and used for RNA isolation.
RiW~ concentrations were determined by absorbance at 260 nm and verified by
nondenaturing agarose gel electrophoresis and ethidium bromide stainin~ as described
20 in mompson, C.B. et al., Natur~ 314:36~366 (1985). Equal quantities ot total Ri~lA were
electrophoreticaliy separated in 1% formaldehyde-agarose gels containing 0.5 l~g/ml
ethidium bromide and transferred to derivatized nylon membranes (Zeta-Probe, BioRad,
Richmond, CA) as described in Elder, J.T. et al., J. Invest Oermatol. 94:1~25 (1990) and
Maniatis, T. et ai., Molecul~r Cloning: A Laboratory Manual, Cold Spring HarborLaboratory,
25 Cold Spring Harbor, NY (1982). Fiiters were baked 2 hours at 80C in vacuo, then
prehybridized for 2-4 hours at 42C in 50% formamide, 5 x SSC, 50 mM sodium phosphate,
pH 7.0, 1 x Denhardt's solution, 250 ;-~g/ml yeast tRNA, 100 ~g/ml sonicated herring sperm
DNA and 1% SDS. Hybridiza~ion was carried out for 1~24 hours at 42C in the same i
buffer containing 1096 dextran suifate. Fiiters ware washed twic~ tor 10 minutes in 2 x
30 SSC, 0.1% SDS at room t2mperature, then twice for 20 mlnutes in 0.1 x SSC, 0.1% SDS
at 56C. Autoradiography was performed using Intensi~yTng screens at -70C. Fliters ware
stripped by boiling 2 x 10 minutes In 0.1 x SSC, 0.5% SDS. Hybridization probes were
prepared by random primlng (see Feinberg, A.P. et ai., Anal. E~loch~m. læ:~l 3 (1983))
of low meiting agarose-purdi6d insert fragments from the human CRABP-I PCR product,
213~5(~
Wo 93/22331 ' PCr/Uss3/~3s36
- 13-
the CRABP-I cDNA (Af1.1) and rat cyclophilin. Sea Danielson, P.E. et al., DNA 7:261-267
(1 988).
Autoradiograms were quantitated using a laser densitom~tsr (LK8 Modal 2202)
coupled to a Hewlett-Packard 3390 A integrator. On~ exposurec which were In the linear
5 ran~e of densitometric response were usad. When appropriate, autoradiographic
intensities were normalized to cyclophilin. See Elder, J.T. et al., J Invest. Derrnatol.
94:1~25 (1990). Statistical analysis of data was per~ormed by one way analysis of
variance using Scheffes correction for mufflple comparison and a ~No-tailed hypothesis.
RESlJLTS
t O Clonin~ of human C:RABP~I and CRABP~II
Using PCR and degenerate primers derhred from bovine and mouse CRABP-I, a
436 base pair (bp) product was obtained, subcloned and s0quenc*d. The predicted
amino acid sequence of the PCR product was found to be 99.3% homologous to the
mouse and bovine CRABP-I sequences. Sae Nilsson, M.H.L et al., Eur. J. Biochem.
15 173:4~51 (1988); and Stoner, C.M. et al., Cancer Res. 49:1497-1504 (198g). Ths PCR
product was used to screen a human skin library and a human skin fibroblast library. Five
dones wers isolated from the skin fibroblast library (lf1.1, 1f3.1, lf5.1, A~5.4 and Af5.6).
Two clones (~f1.1, 1f3.1) were sequenced and found to be identieal. The predicted a nino
acid sequences ot these clones wera found to be 77.4% similar to human CRABP-I and
20 93.5 % similar to the recently cloned mouse CRABP-I. See Giguere, V. et al., PNAS (USAJ
87:6233 6237 (1990). ~Because of the high homology to the mouse CRABP-II, clone lf1.1
wæ designated as human CRABP-II. A third clone Af5.1 was partially sequenced andfound to be a shorter clone, with a sequancs identical to human CRABP-II. No CRA8P-I
clones were isolated from the skin fibroblast library. Two clones were isolated from the
25 skin library and ~ound to contain shorter inserts, one with a s~quence identical to human
ORABP-II (As~1) and one with a sequence identical to human CRABP-II (ls3.1).
SEa ID NOS. 1 and 2 In the Sequence i isting represent the cDNA nucleoffde and
predict0d arnino acid sequences of human CRABP-II. The ~anslaffon initiaffon sRe was
assigned to the flrst methionine codon corresponding to nucleoffdes 9~101. An
30 open-reading frame of 138 amino acids was found, predicting a poiypeptide oi Mr 15,693.
The 3' untranslated region was tound to contain a poiyadenylaffon signai (Al~MA) and
a poly(A) tract of 1 (lfl.1) to 25 (Af5.t). SEQ ll:~ NOS. 3 and 4 represent the cDNA
nucleotide and predicted animo acid sequence of CRABP-I resp~ctveiy.
~ 1 3 4 ~O 93/22331 P~/US93/03936 ~
- 14- ~
Comparison of amino ~cJd s~quences of CRABP
The amino acid sequence comparison of CRABP-I and CRABP-llis presented in
Figure 2. Panel A is a comparison of the amino acid sequenees for human th) and mouse
~m) CRA8P. The predictsd amino acid seguences of mouse and human CRABP-I and
5 mouse CRABP-II were aligned with human CRABP-II. Dashes (--) represent identity to
human CRABP-II. One gap as indicated by an asterisk (~) was introduced in the human
and mouse C:RABP-I sequences for ma~dmum alignment. Panel B o~ Figure 2 is the
sequence comparison of the Nl 12-terminal ends of human (h), mouse (m), rat (r), CRABP-I
and CRABP-II. Residuesdissimilarto human CRABP-II are boxed.
The amino acid sequsnce comparison presented in Figur~ 2, Panel A, reveals a
73.7% overall degree of identity between mouse CRABP-I (Stoner, C.M. et al., Cancer P~es.
49:1497-1504 (1989); Giguere, V. et al., PNAS (USA) 87:6233 6237 (1990)) and between
human CRA8P-I and human CRABP-II. Human CRABP-I and mouse CRABP-I displayed
an overall identny of 99.3%, with a single amino acid substit~ion (amino acid residu0 86,
15 Ala instead of Pro), while human CRABP-II and mouse CRABP-II were 93.5% identical.
Human and mouse CRABP-II displayed 9 amino acid dmerences with the following amino
acids in the human sequence: residue 19 ~ Leu, 2~ - Val, 27 - Val, 29 ~ Leu, 48 - Gly, 68
- Val, 91 - Glu, 99 - Lys and 111 - Thr. In 6 of the 9 amino acid dfflerences seen
bet Neen human CRABP-II and mouse CRABP-II (residues 19, 29, 48, 68, 91 and 111), the
20 human ssquence was idantical to CRABP-I.
The NH2-terminal amino acid sequences of twoCRABPs from neonatal rat (Bailey,
J.S. et al., J. Blol. Chem. 263:932~9332 (1988)) and chicken embryos (Kitamoto, T. et al.,
Biochem Bioptrys. Res. Comm. 157:1302-1308 (1988) have been reported and were also
compared to human CRABP-I and CRABP-II. As shown in Figure 2, Panel B, there were
25 no amino acid dfflarences between the NH~terminal regions of rat, chicken, mouse and
human CRABP-I, whereas several differences appeared between the CRABP-lls.
SPECIFIC EXAMPiLE 2. iExpre~sion of Human CRABP
Exprcsslon of human CR~4~P In human skln In v~o
Volunteers were treated with 0.1% RA cream or control treatments under occlusion30 for various time intervals. Keratome biopsies consisting mostly of epldermis (see
Voorhees, J.~l. et al., Arch Dermatol. 105:695-701 (1972)) were obtained, and used to
prepare total RNA which was subsequently analyzed by blot hybridkation. The
autoradiograms of the RNA blots derived from thres individuais are shown in Figure 3.
Total RNA (40 ~g per lane) was hybridized against the human CRABP-II or cyclophilin
35 cDiYA probes as described above. Each volunteer was treated topically for 16 hours prior
2134550 ~ ;
~- WO 93~22331 ' PCr/US93/03936 ~ -
- -15- f
to biopsy as follows: ~a) no treatment, (b~ RA vehicle or (c) 0.1% RA cream in RA vehicle
under occlus~on with plastic wrap. Mobilities of ribosomal RNAs are indicated to the left
of the blots.
CRABP-II transcripts were detectable in untreated skin as well as skin traated with
5 vehicle, a shown in Figure 3. CRABP-II transcripts were marked~ (16.1-~old) and
significantly (p<0.004, n=4) induced in RAtreated relative to untreatad skin. Similar, albeit
less markad, inductions were observed after 4 days of RA treatment (8.3 ~ 2.~fold, n =
6). Induction did not occur after 4 hoursj but was evident after 12 hours of treatment. I
Consistent with our ability to ampl~y CRABP-I from human skin F~NA, faint CRABP-I probe
10 hybridization was observed in some but not ali blots ot human skin RNA samples alter
prolonged autoradiographic exposure (data not presented in Figures). Howevar, CRABP-I
transcripts were usually undetectable under exposure conditions sufficiently sensitNa to
detect single copy DNA sequences *om 10 ~^~g human genomic DNA. CRABP-I and
CRABP-II cDNA probes datect~d distinct band pattems using genomic DNA digested with
15 BamHI, EcoRI, Hindlll and Pstl, demonstrating the specHicity of these probes under our
hybridizaUon condiUons (data not presented in Figures).
Expresshn of CR4BP ln human fblobl2sts
Treatment of human de~nal fibroblasts with RA resulted in a marked (approximately
1~fold) and significant (P ~0.04) induction of human CRABP-II mRNA after treatment of
20 five independent dermal fibroblast stralns with 3 x 1~7 or 3 x 10~ M RA for 24 or 48 hours
as shown in Figure 4. Resuits shown are derived from nine independent experiments
invo~ving five dermai fibroblast lines prepared from three individuais as described above
and three diploid human lung fibroblast lines (LL47, CCD-1 8Lu, and CC~1 6Lu). RNA blot
hybridizations (20 1~9 total R~lane) were quantitated by laser densitometry and
25 normalized to the control gene, cy_~ophilin, as described in Elder, J. T. et al., J. Invest.
Dermatol. 94:1~25 (1990). At confluence, medium was changed and cells were treated
with RA dissoived in dimethyl suifoxide at the concentrations and for the times indicated
beneath the Figure. Data are expressed as fold induction ~ SEM, relative to the average
,
of duplicate dishes treated with dimethyl suifoxide aione for 4 hours *p~0.06, **p~0.005.
The relevant autoradiographic bands from two representative experiments comparing
~ermal and lung fibroblasts are, shown in the inset. ~
As shown in Figure 4, CRABP-II mRNA was not slgnificantiy induced by PA in three~trains of human lung fibroblasts, suggesUng that ~Is response may be Ussua specific.
Induc~ion of human CRABP-II transcripts by RA was dos~dependent over the range of 3
36 x 10-1 to 3 x 1~7 M RA (data not presented in Figures). In contrast, CRAPP-I transcripts
~1~453~
WO 93/22331 ~ . PCr/US93/03936 ~--~
` -16- ~;
were undetectable in dermal and lung fibroblasts and were not induced by RA, whereas
genomic DNA blots hybridked in parallel were positive.
Expression of CRd~P In human keratinocytes
As shown in Figure 5, ::RABP-II mRNA was markedly and significantly inducad in
5 cultured adult human keratinocytes when cells reached confluence, and remained elevatsd
in the postconfluent ~tate. Figure 5A summarizes the r~suits obtain~d for seven
independent keratinocyte strains. Third passage norrnal aduit human keratinoc~tes were
grown in KGM containing 0.15 mM CaCI2, and medium was chang~d every other day. At
the indicatsd number of days pr~ or post~o~uence, total RNA was prepared and
10 analyzed for human CRA~P-II mRNA by blot hybridization and densitometry. Data are
expressed as percent ma~amal expression for any given strain ot keratinocytes, as ths
absolute level of human Ci~ABP-II mRNA was variable from strain to strain. Error bars
represent ~ SEM. **p<.005, ***p<0.0005 relative to cells at 2 days preconFluence.
CRABP-II mRNA was also markedly induced in subconfluent cultures by raising the
15 caleium concentration in the medium from 0.15 mM to 2 mM, as illustrated ~or a
r~presentative strain of keratinocytes in Figure 5B. At 2~30% confluence, the medium was
changed to KGM or KGM containing 2 mM CaCI2 in the presence or absence of 3 x 10-9
M RA and maintained for the Indicatad number of days, with medium change every other
day. Mobilities of 28S and 1 8S ribosomal RNAs are indicated to the left. Figure 5C shows
~0 the effects of prolonged treatment with high concentrations of RA on CRABP-II mRNA
levels. The same experiment described for Figure 5B was conducted above, except that
cells were treated with or wlthout 3 x 1 o~6 M RA for 2 to 5 days, as indicaSed above the
autoradiograrns. Mobilities of 28 and 18S ribosomal RNAs are indicated to the left.
Figures 5A and B show the results representative of four independent experiments.
2~ As shown in Figure 5B, treatment of keratinocytes for prolonged periods of time
with low concentrations o~ RA (3 x 10-9 M) had no detectabls effect on CRABP-II mRNA
levels. Howevef, as shown in Figure 5C, prolonged treatment of subconfluent
keratinocytes with high concentrations of ~A (3 x 10~ M) reducsd CRABP-II mRNA to
undet~ctable l~v~ls. Hybridkation of the same blot against CRABP-I prior to its
30 hybridization against CRABP-II failed to detect CRABP-I transcripts, whereas genomic DNA
blots hybridized in parallel were positive (data not presented in Figures).
SPECIFIC EXAMPLE 3. Alternate Clonin~ Scheme
The CRABP-II cDNA probe was cloned by PCR from retinoic acid (RA) treated
human skin using the CRABP-II degenerate primers shown in Figure 1B, derNed frem35 mouse CRABP-II mRNA sequ~ncs. See Giguere, V. 0t al., PNAS ~VSAJ 87:6233 6237
- W09 . 21345~0
3/22331 PCI`/US93/03936
- 17 - ;
(1990). BamHI restriction sites were included in the forward and r~verss primers to aid in
subsequent cloning.
RA-treated human skin was used asthe source of RNAfor r~verse transcri~tion and
PCR amplification. Total RNA was extracted from epidermal ksratom~s as describ~d in
5 Elder, J.T. et al., J. Invest. Dermatol. 94:1~25 (1990), and reverse transcribed as described
in Maniatis, T. et al., Molecular Cloning: A Labo~atory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY (1982). During each PCR cycl~, samples were heated
to 94C in 1 minute and maintained for 30 seconds, cooled to 5~C for primer annealing,
and then heated in 1 minute duration to 72C and kept at th~t temperature for a further 30
10 seconds to allow template extension. The PCR product was further purified (Geneclean,
BIO 101 Inc.), digested with BamHI and cloned into BamHJ digested PGEM 3Z plasmid
(Promega inc., Madison, Wl). The PCR ~ragment was iniffally identified as CRABP-II by
digestion with Pstl restriction enzyme that cleaved the DNA at the same position as that
described in the mouse CRABP-II mRNA. The clone was s~quenced and found to
15 represent the major por~ion of the mature human analogue of the mouse CRABP-II. The
cDNA probe was hybridized to human P~NA prepared from skin of human volun~eers
topically ~reated with RA for 4 and 12 hours. Indwtion of CRABP-II mRNA was observed
in skin which had been RA-treated for 12 hours, but not skin treatad for only 4 hours (data
not illustrated in Figures).
20 SPECIFIC EXAMPLE 4. CRABP Mamn alian Expres~ion Vectors and Con~truc~
CRABP-I was excised from the Bluescript phagemid (Stratagene) cloning vector andinserted between the Xbal and BamHI sites of the eukalyotic expression vector pSVL
(Pharmacia) in forward ori~ntation (pSVLCRABP-I). CRABP-II was excised from the
BiuescrTpt phagemid (Stratagene) cloning vector and inserted into the EcoRI site of the
25 eukaryotic expression vector pSG5 (Stratagene) in forward (pS~;5CRABP-II) or reverse
oriontation (pSG5CRABP-llAS). The orien~ation of the insert was datermincd by restriction
analysis.
CV-1 cells are grown in Dulbecco's modified Eagles medium (DMEM) containing
10% fetal caff serum. CV-1 are monkey kidney cells derived from the CV-1 cell line which
30 do not express T cell antigen. Th8 day beforfl transfection, cells are seeded on tissus
culture dishes in DMEM containing 10% charcoal treated fetal calt serum (ChFCS). Cells
are contransfected using the calcium phosphate co precipitation tachnique essentially as
described in Rosenthal, N., Mc~7. Enzymol. 152:704 720 (1987) with CRABP-I (pSVLCRABP-
I) or CRABP-II (pSGSCRABP-II) expression vector. 24 hours after transfection, cells are
35 t~ypsinked and suspended in medium, pelleted and washed once with 40mM Tris-C1, pH
~,~ . . . -
213~ C~SJ
WQ 93/22331 ' pcr~lJss3/o3~36 l
18 - ~-
7.6 containing 150 mM NaCI and 1 mM EDTA. A cytosolic fraction is prepared on cell
Iysates by centrifugation at 100,000 x 9 for I hour.
SPECIFIC EXAMPLE 5. Bacterlal Production of Human CR~4BP
For CRABP-I, the nucleotide sequence of CRABP-I is changed at position 9 (T to
5 G) and 13 (C to T) ~o create a Stul site, using synth~tic oiigonucleotides and the
polymerase chain r~action. For CRABP-II, the nucleotid~ sequ~nce of C:RABP-II ischanged at position 100 (T to G) and 104 (C to T) to create a Stul site, using syntheUc
oligonucleotides and the polymerase chain reaction. The mutated CRABP cDNAs are then
cut with Stul and ligated into the Stul and EcoRI sites of the bacterial expression vactor
10 pMAL-c (New Engiand Biolabs). Bacteria is transformed and th~ maltosa-binding protein
(MBP)-C~ABP fusion protains are expressed in large guantities. The MBP-Cf~ABP fusion
protein is purffied by amnity chromatography on any amylos~ column (New England
Biolabs). CRABP lacking the first methionine is released from MBP by digestion with factor
X~ (New England Biolabs) and purified from MBP by a second passage over an amylose
1 5 column.
Spectrofluorimatric methods are used to study the amnities and binding
stoiclliometries ~f purified human CRABP-I and CRABP-II for a variety of iigands. The
capacity of ligands to bind to CRABP-I or CRABP-II is assessed by monitoring their ability
to queneh the native fluorescence of this protein.
2Q SPECIFIC EXAMPLE 6. Production of Antibodle~
Peptides correspondin~ to amino acids 94 to 104 in the CRABP-I and CRABP-II
pro~eins have been synthesized using the multiple antigenic peptide method as generally
desbribed in Posnett, D.N. et al., Met~7. En~ymol. 178:73~746 (1989), eliminating the need
for conjugation to a carrier protein. This region of the ~vo CRABPs has a low homology
25 and is most probably situated on the outside of the proteins based on hydrophobicity and
surface probability calculations.
The peptides were injectesi ~to chickens for production of egg IgY. llle resulting
antibodies, if not specfflc, are adsorbed to peptides (CRABP-I antibodies to CRABP-II
peptides and the reverse) irnmobilked on a sapharose gel to enhance specificity. Once
30 monospecific antibodies are obtained, the expression and regulation of CRABP-I and
Ci~ABP-II proteins In human skin and skin cells is examined quanUtativeiy by Western blot
anaiysis and seml~uantitativeiy by immunocytochemistry. The pattem o~ expression and
regu!ation of Ci~ABP-II (and CPABP-I) by PA in normal and psoriatic skin will provide
insight into the function of CRABPs.
WO 93/22331 2 1 ~ 4 5 S ~ P~/US93/03936
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Antibodies to ::RABP-I and CRABP-II, or binding fragments (e.g. F~(ab)) thereof,and the purified proteins obtained as deseribed above can be us~d to monitor levels of
CRABP-I and CRABP-II in normal and pathologicai states by using immunological
techniques known to those skilled in the art. For exampls, patient skin tissue can be
assayed with antibody specific for human C:~ABP-I or CRABP-II to determine the presence
and levels of CRABP by ELISA, Western blot analysis or immunochemistly essentially as
desc!ibed in Busch, C. et al., Meth. Enzymol. 189:316 324 (1990).
SPECIFIC EXAMPLE 7. Report~r As~ay Sy~tem
CV-1 cells ar~ grown in Dulbecco's mod~ied eagies medium ~DMEM) containing
10% fetal calf serum. The day before transfection, cells are seeded on tissue cultured
dishes in DMEM containing 10% charcoal treated fetal calf serum (CHFCS). Cells are
cotransfectsd using the calcium phospha~e co-precipitation technique with 0.6 mg of
human retinoic acid receptor (hRAR) expression vectors (hRARaO, hRAR~O or hRARAO),
a reporter plasmid and a ~alactosidase expression vec~or (pcH110, Pharrnacia) used as
an internal control to normalize tor variaHons in transfection emciency essentially as
dascribed in Astrom, A. et al., B~ochem. Biophys. Res, Comm. 173:33~345 (1990). Cells
are also cotransfscted wiUl CRABP-i (pSVLCRABP-I) or CRABP-II (pSG~CRABP-II)
expression vectors of pSVL rPharmacia) as a control. The reporter plasmid (TRE)~-CAT
is constructed by ligating synthetic oli~onucleotides encoding three palindromic thyroid
hormone rssponsiva elements (TRE) ((TCAGGTCATGACCTGA)3) flanked by Hindlll and
BamHI sites on the 5' and 3' ends respectively and cloned into the Hindlll - BamHI cloning
sites of the plasmid pBLCATæ 24 hours after transfection, cells are washed once with
DMEM, and medium (DMEM, 10%ChFCS) containing different concentrations of ligandsars added to the cells~ 24 hours later the cells are 'aypsinized and sucpended in medium,
pelleted and washed once with 40mM Tris-CI, pH 7.6 containing 1 50mM NaCI and 1 mM
EDTA. C:ell Iysates are prepared by Ulree consecutive freeze-thaw cycles and ~-
galactosidase and CAT-activities are determined by a xylene extraction method. The effect
of coexpression of CRAi3P-I or CRABP-II on RAR transcriptional activaUon for dfflerent
ligands can now be deterrnined as described below.
An aiternative reporter assay in which a recombinant adenovirus system is used to
coinfect cells in Gulture may aiso be employed to measure transcripUonal activation by
retinoids essentially as described in Shih, E~ et ai., MoL Endocrinol. 5:300 309 (1991). In
- such a system, two mutually dependent viruses, one containing a receptor transcripUon
unit and the second containing a gene responsive element are coinfected into receptor-
35 deficient cells such as CV-1. For example, hvo mutualiy dependent adenoviruses, one
w0~3~2 ~1345~0
2331 ` : PCI'/US93~03~36 ~^
- ` - 20 -
containing a human glucocorticoid receptor transcription unit and the other a
glucocorticoid responsive element linked to the firefly luciferase gane, or one con~aining
rat thyroid hormone receptor a and the other the luciferase gene, can be utilized in the
practice of the assay. Hormons-induc2d transcription is then quantitated a~ter infection
5 from cells coinfected with the complementary virus pair.
SPECIFIC EXAMPLE 8. Llgand Bind~n~A~say~ ;
Since the amino-acid homology between CRABP-I and CRAB~-II Ts on~ 77%, it is
important to determine the ligand binding properties of thas~ two protelns. There are
ssveral reports on the lack of correlation betwe0n biological activity and affini~y of retinoids
10 to CRABP. Darmon, M. et al., Skin Pharmacol. 1:161-175 (1988). Most of these studies
have used extracts from rat testis containing mostly rat CRABP-I as the source of CRABP.
In contrast, we are expressing the CRABP-I and CRABP-II cDNAs can now be expressed
in mammalian cells.
Availability of cloned human CRABP-I and 11 thus improves previous systems for
15 identification of tissue-specific ligands. This also makes identification of ligands interacting
with CRABP possible, in addition to nuclear receptors. Ugands for testing include, but are
not limited to didehydr~RA, RA and its metabolites, 4hydroxy-RA, 4 oxo-RA, and 5,~
epoxy-RA æ well as compounds previous reported not to bind to CRABP, e.g. CD 394.
Darmon, M. et al., Shn Phsn77acol. 1:161-175 (1988). Availabilty o~ CRA8P amin~acid
20 sequences or proteins provide the msans to select more tissue-specific drugs for repair
of photoaging skin, psoriasis, acne, skin cancer, leukemia, diseases of keratinization,
osteoperosis, rheumatoid sclerosis, and other conditions.
Soluble protein from CV-1 cells transfected with CRABP-I (pSVLCRABP-I) or
CRABP-II (pSG5CRABP-II) expression vectors obtained as described above is incubated
25 with [3H3 retinoic acid at 4C overnight. Free ligand is separat~d from bound ligand by
size fractionation on a GF 250 column (DuPont Pharrnaceuticals) connected to a FPLC
system (i'harmacia). The amount of specific binding is determined by incubating samples
with an excess of cold retinoic acid. The amnity of CRABP-II for retinoic acid can be
determined by titrating with increasing amounts of i~3H~ Retinoic acid.
The relative amnity for other ligands is deterrnined in competition assays in the
same system. A fixed amount of CRABP-I or i~H] reffnoic acid is Incubated with soluble
proteln from CV-1 cells expresslng CRABP-II together with increasing amounts of othsr
posslble ligands. -
WO 93/22331 2~ 1 ~ 4 ~ ~ O PCI~US93/0393~ i
`
SPEC:IFIC EXAMPLE 9. Hybridkation Assay
Availability of cloned human CRABP-I and CRABP-II cDNAs makes it possible to
determine the relative expression of Ulese two genes in norrnal and pathological states.
For exampla, RNA i5 isolated from tissue biopsies and cultured cells by guanidinium
5 isothiocyanate Iysis and ultrac~ntri~gation as previously described in Elder, et al., J.
Invest. Dermatol. 94:1~25 t1990). Equal quantities of total RNA can be separated on 1%
formaldehyde-agarose gels and transferred to nylon membranes. Aiter baking for 2 hours
at 80C, filters can be hybridized to agarose-purffled t::RABP-I or CRABP-II cDNAs labeled
by random priming. The amount of CRABP mRNA in a tissue can be detsrmined after
10 quanfftation of autoradiograms by laser-densitometry as described (Elder, et al., J. Invest.
Derma~ol. 94:19-25). It will be appreciated that CRABP^I and ll oligonucleotides (typically
~15 residues long) may also be utilized as probes in hybridization assays if o~ a sufficient
length to bind complementary sequences.
SPECIFIC EXAMPLE 10. Examination of Function of Human CRAE~P
15 Receptor assay
The most important and difficult issue to resolve regarding the CRABPs is their
function. It has been suggested that CPABP transports RA to the nucleus ~rakase, S. et
al.,Arch Biochem. f~iophys. 247:328 334 (1986)) or that CRABP remains in the cytoplasm,
thereby prevenUng RA from movinQ to the nucleus. Maden, M. et al., Nature 335:733 7~5
20 (1988). One way of addressing tnis issue is to express increasing amounts of CRABP-I
and CRABP-II in CV-1 cells tog~ther with the RARs and a reporter gene containing a
retinoic acid responsive element, as described above. if CRABP transports RA to the
nucleus an enhancement ot reporter gene activity at low concentrations of RA may be
seen. On the other hand, H CRABP sequesters RA a decrease in response will be seen.
25 Overexpression
CRABP-I and CRABP-llis overexpressed in fibroblasts and keratinocytes. Cells will
be transfected with CRABP expression vectors (pSVLCRABP-I or pSG5CRABP-II) as
described above, using Upofectin ~BRL) essentialiy as described by the manufacturer In
fibroblasts the effect of CRABP overèxpression on induction of til~ RAR-~ gen~ by i-~ is
30 studied. It has been shown that RAR-i3 mRNA is induced by RA in skin fibroblasts, and
it is known that this gene Is directly regulated by the RARs. See DeThe, H. et ai., NahJre
343:177-180 (19gO). The i~AR-~ gene acts as an ~endogenous reporter gene~ for RAR
induction. In prslHerating keratinocytes the effect of overexpr3ssion of CRABPs on
markers of dmerentiation is studied.
~,, .. ,. ~ . .. . . . . . . . . . . . .
wo g3/Z23312 1 3 4 5 S '~ ` PCI`/US93tO3936,~
TranslaUon blocking
Translation of CRABP-II is blocked in fibroblasts and keratinocytes by transfecting
the cells with an axpression vector construct having th~ CRABP-II cDNA in a reverse
orientation. This produc~s an anti-sense mRNA that jQ able to hybridize to the
5 endogenous CRABP-II mP~NA, thareby blo~king translation. The effect of antisense
expression in fibroblasts and keratinoc~tes is studied the same way as describad for
overexpression.
Effsct of CRAE~P on RA metabolJsm
The involvement of the cellular retinol binding protein (CRBP) in delivering retinol
10 to the appropriate m3tabolic enzyme in rat liver microsomes has been described. Ong,
D.E. et al., J. Biol. Chem. 263:578~5796 (1988). Since th~ CRABPs belong to the same
family of binding protein as CP~BP, it is possible that they also are involved in the
metabolism of their ligands. RA Ts metabolized by the cytochrome P~50 monooxygenase
system present in the endoplasmic reticulum. Bossche, H. et al., Skin Pharrnacol. 1:17~
15 185 (1988). CRABP may function by increasing the interaction of RA with cytochrome P-
450. Another possibility is that ~ is not the natural ligand for CRABP-II, but rather one
of the metabolites. If CRABP-II for example, bind-~ ~hydroxy-RA (as deterrnined by ligand-
binding studies) with a higher affinity than RA, it is possible that CRABP-II increases
cytochrome P~50 mediated metabolism by decreasing produc~ concentraUon. Whether
20 the CRABPS are involved in RA metabolism or not is tested by incubating skin microsomes
with increasin3 amounts of RA and of expressed and purified CRABP-I or CRABP-II. The
effects of CRABPs on RA metabolism is assayed by HPLC.
SPEC1FiC EXAMPLE ~1. Clonlng and Sequencing of Human CRABP-II Gene and
Transcription Studies
MATERIALS AND METHODS
ClonJng of U~e human CRA~P-~I gene
To isolate the CRABP-II gene, approximateiy 6 x 105 recombinants lrom a human
placenta genomic DNA libraly in ~ ambda FiX ll (Stratagene Inc., LaJolla, CA) was screened
using the human CRABP-10 cDNA as a probe. Astrom, A. et al., J. Biol. Chem. 266:17662-
17666 (1991). One positive clone was isolated, purified and restriction mapp~d. Parts ot
the insert were subcloned into Bluescript phagemid SK (Stratagene Inc., L~Jol.a, CA). The
sequence of the gene was determined on both strands after subcloning into M13 vectors
by dideoxy chain terrninaUon using modified T7 poiymerase (Sequenase, U.S. Biochemlcal
Corp.) and synthetic oligonucleoUdes. Sanger, F. et al., P~S ~US4J 74:5463 5467 (1~77).
35 Exon positions were determined by restriction mapping and sequencing.
- wo93/22331 2134550 :~cr/us93/03936 t
- 23 -
Cell culture
Human dermal fibroblast cultures were prepared from punch biopsies of buttock
skin and propagated in Dulbecco's modffled Eagle's medium containing 10% calf serum.
Harper, R.A. et al., Science 204:52~527 (1979).
5 Northem analysls of mRNA
RNA was isolated from cuitured human skin fibroblasts by guanidinium
isothiocyanata Iysis and uitracentrifugation as previously described. Elder, J.T. ~t al., J.
Invest. Dermatol 94:1~25 ~1990). RNA concentrations were determined by absorbance
at 260 nm and equal quantities of total RNA were electrophoretically separatad in 1%
10 formaldehyd~agaross gels containing 0.5~g/ml ethidium bromide. The RNA was
transferred to nylon membranes (Zeta-Probe, BioRad, Richmond, CA) as described. Elder,
J.T. et al., J. Inv~st. Dermatol 94:1~25 (1990); Maniatis, T. et al., Molecular Cloning: A
LaboratoryMamJal (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) (1982). Filters
were basked 2 hours at 80C in vacuo, then prehybridized for 24 hours at 42C in 50%
15 formamide, 5 x SSC (1 x SSC = 150mM NaCI, 15 Mm sodium citra~e), 50 mM sodiumphosphate, pH 7.0, 1 x Denhardt's solution, 250~g/ml yeast tRNA, 100~g/ml sonicated
herring sperm DNA and 1% SDS. Hybridization was carried out for 1~24 hours at 42C
in the same bu~fen Filters were washec once in 0.2 x SSC, 0.1% SDS at room
temperature, then twice for 20 min in 0.2 x SSC, 0.1% SDS at 56C, and final~ once for 20
20 min in 0.1 x SSC, 0.1% SDS at 56C. Autoradiography was performed using intensifying
screens at -70C. Filters were stripped by boiling 2 x 10 min in 0.1 x SSC, 0.1% SDS.
Hybridization probes were prepared by random primTng (Boeringsr Mannheim) ot purified
insert fragments from human CRA8P-II cDNA tlfl .1 ) rat cyclophilin and RAR-y, Astrom, A.
et al., J. E~lol. Chem. 266:17662-17666 (1991); Dani~lson, P.E. et al., DNA 7:261-267; Elder,
25 J.T. et al., J. Invest Derrnatol 96:425 433 (1991). Quantitation of mRNA levels were
performed using a phosphorimagar (Molecular Dynamics).
Primer ~xtensJon analy~is
RNA was isolated from untreated and RA treated skin fibroblasts as described.
Astrom, A. et ai., J. Biol. Chem. 266:17662-17666 (1991). A synthetic oligonucleotide 5'
30 CTAGGCTGGAGCACTGGACACTGTC 3' complementary to position 80-104 in the gene
was used as an ext~nsion primer. 1 O~g of totai RNA was heated for 10 minutes at 70C
togsther w~th 32p 5~ end-labeled primer. The mixture was allow~d to cool to 30C over
- 30 minutes and then kept at 30C for an additional 30 minutes. To the mixtures were
added (final concentration) 50mM Tris-HCI (pH 8.3), 75 mM KCI and 3mM MgCI2, 5mM35 DDT and 250 mM of dATP, dCTP, dGTP, drrP in 40~1 total volume. The reactions were
21345S~
WO 93/22331 . ~ ` PCI'/US93/03936 .---
- 24 - t, _,
started by the addition of 200 u Superscript RNase H (GIBCO, Bethesda Research
Laboratories) and incubated for 90 minutes at 42C. Samples were phenol/chloroform-
extracted, ethanol-precipitated, resuspended in formamide dye and after hsating to 75C
~or 3 minutes, separated on a 6% saquencing gel.
5 Transcr}ptJonal analysJs
Cuitured human skin hbroblasts were grown to confluency on 150 mm tissue
cuiture dishes as described. Astrom, A. et al., J. Biol. Chem. 266:17662-17666 (1991).
Cslls wsre then treated for various time-points with 1 ~i RA in pr~warm0d and equilibrat~d
Dulbecco's modified Eagle's medium containing 10% fstal calf serum. Nuciei for each
10 time-point were isolated from four dishes after incubation in Iysis buffer containing 0.596
NP40 as described. Greenberg, M.E. et al., Natur~ 311:433 438 (19~4). Nuclear run-on
experiments were performed with la-32P] UTP (DuPont-New England Nuclear, 800
Ci/mmol) as described. Antras, J. et al., J. Biol. Chem. 266:1157-1161 ~1991). Equal
amounts of radioactivity (0.5-1 x 107 cpm) were hybridized to nHrocellulose fiiters
15 containing 5 ~9 of each plasmid. After hybridization for 72 hours at 42C, the fiiters were
washed twice with 2 x SSC at 37C for 15 minutes and treated for 30 minutes at 3PC in
2 x SSC containing 5 ~g/ml RNase A. The fiiters were then washed twi,ce for 15 minutes
in 2 x SSC, 0.5% SDS at 42C and once for 30 minutes in 0.5 x SSC, 0.5% SDS at 42C.
A final wash was carried out in 0.1 x SSC, 0.1% SDS for 30 rninutes at 55C. The amount
20 of radioactivity present in each slot was determined using a phosphorimager (Molecular
Dynamics) after over-night exposure and autoradiograms were exposed for ~5 days at -
70C with intensifying screens.
RESULTS
Clon~ng and char~cter~zaUon of th~ human CR4BP-II gen~
Using the human CRABP-II cDNA as a probe, one bacteriophage lambda clonewas
isolated (Aæ1) from a human placenta genomic library. Restriction mapping and
sequencing ravealed that this clone contained the entire CRABP-II gene. A restriction map
of 12.1 is shown in Figure 7. The gene is composed of four exons, interrupted by one
. . , !
large and two small introns. The overall ske of the gene is approximately 6 kb.
30 DNA SQquenc~ of the human CR~4BP-II gene
To characterke the CRABP-II g~ne, fragments spanning the ~ntire gene, except forthe first intron were subcloned into M13 vectors for sequence analysis. The nucleotide
sequence and the deduced amino-acid sequence is presented in Figure 8. The first three -
sxons are small and range in size from 117 to 207 bp, with the fourth exon being the
35 largest at 466 bp as d~monstrated in Figurs 8. The exon sequences were found to be
_, . .. .... .. . . . ... . . . . . .. . . . .... . . . ..
- - wo 93~22331 , 2 1 3 ~ 5 ~ O PCr/US93/03936
-25-
identical to the published cDNA sequence. Astrom, A. et al., J. Biol. Chem. 266:17662-
17666 (1931). All splice junctions contained the expected GT splice donor and AT splice
acceptor.
Analysfs of the 5 ' end of U~e human CRABP II gene .
The 5~ bounda~ ot the first exon was determined by p~mer extension ana~sis.
Using an oligonucleotide primer complimentary to position 8~104, a predorninant reaction
product was ident fied when mRNA from untrea~ed cultured human skin fibroblasts were
used, see Figure 9. Comparison of the ex~ension product with sequencing reactions 7
originating from the same prim~r indicated that the major transcription initiation s~e should
be assigned to the A r~sidue 137 bp upsteam of ~he ATG. When ~broblasts were treated
with 1 ~M RA for 24 hours an increase in initiation at the A residu3 could ~e seen. In
addition a second initiation product with the same intensi~y could be seen at ~
Sequence analysis of the upstream region revealed a TATA box (TAT,MA) at -31
and severai potential regulatory elements including ~wo potential AP2 binding si~es at -631
and ~02, and one potential SP1 site at ~9 as demonstratsd in Figure 8. Mitchell, P.J. et
al., Cell 50:847-861 (1987);, Kadonaga, J. T. et al., Jrends 3ichem. Sci. 11:2~23 (1986).
In add~ion hvo sequences can be found that exactly match a binding site for the early
growth response gene ~ox-24 (Egr-1, zi~26B or NGFI-A) at -579 and -116. L0maire, P. et
al., Mol. CelL Biol. 10:3456~67 (1~90). In addition a direct repeat spaced by one bp
(AGl~CAgGGrrCA) can be found in the upstream region of the gene at 454, with
homology to the retinoic acid responsive element found in the RAR-~2 gene. l~mesono,
~ et al., Cell 65:12551266.
Effe~ts of RA on c~q~P-n tr~nscrfpUon rat~s and mRNA levels
To determine whsther RA induction of CRABP-II mRNA seen in cultured human skin
25 fibroblasts was dus to inaeased transcription, nuclsar run~n assays were performed
using nuclei isolated *om cultured human skin fibroblasts traated wKh RA for various
periods of Ume. A~ seen in Figure 10, RA caused a rapid transient increase in CRABP-II
transcription, peaking at 2 hours and being almost back to control levels at 6 hours.
However, there was no change u~ RAR~ and cyclophilin transcripUon, two genes known
30 to be unaffected by RA in skin fibroblasts as demonstrated by Figur~ 10. Edler, J.T. et al.,
.~. Invest. Dennatol. 96:425433 (1991). QuantRaffon and normallzation to cyclophilin
shows U~at CRABP-II transcription is induced approximately ~told aner 2 hours of RA
treatment. See Figure 11.
This increase in transcription can account tor the ~fold induction of CRABP-II
35 mRNA after 24 hours of treatment, as seen in Figure 11. Induction of CRABP-II mRNA can
i S f~
WO 93/~2331 ` PCI`/US93/03936 ~
- 26 - ~ ,
be detected as early as 2 hours a~ter addition of RA to cGlls, leveling out between 6 to 24
hours of treatment. However, a smaller increase can be ssen between 24 and 48 hours.
To determine whether RA is required to be present in the medium for maintaining the
induced CRABP-II message levels after the transcription has occurred, human skin5 fibroblasts were treated with RA for 12 hours, after which the cells were washed two times
and replaced wHh medium without any addition of RA. As can be seen in Figure 11,removal of RA from ths medium causes a decline of the CRABP-II messaga compared to
cells traated with RA for the entire experiment.
Effccts of cyclohexlmlde or actlnomycln 1~ on the fnducUon o~ CRABP-U message
Experiments were per~ormed to determine whether the addition of inhibitor-e of
transcription or translation could inhibit the increase of GRABP-II mRNA produced by RA.
Cycloheximide (10 ~g/ml) or actinomycin D (2 ~uglm!) were added simultaneously with RA
M) and CRABP-II levels were determined a~tsr 2 hours. As can be seen In Figure 12,
cycloheximide down-r~gulated basal CRABP-II exprassion and blocked the RA induction.
15 Cycloheximide had no effect on RAR~ expression or RA induction of RAR-,~ ~Figure 12),
as previously reported. Nenfl,~. et al., Cell Gr~wth Dffl 1:53~542 (1990); DeThe, H. et
al., FM80 J. 8:429 433. Actinomycin D, had no effect on basal levels of CRABP-IImessage, but complebly blocked induction by RA. As controls, RAR~ was found to be
down-regulated by actinomycin D, and RA induction of RAR-,B was compl~tely blockad in
20 agreement with previous reports. Nervi, C. et al., Cell Growth Diff. 1:53~542 (19~0);
DeThe, H. et al., EMBO J. 8:429 433`(1989).
Effects of cyclohe)dmlde on CRABP-II transcrlpUon rates
Since cyloheximide blocked RA induction of CRABP-II mRNA, run-on experiments
were performed to sae if protein synthesis was required for the induction of transcription.
25 As can be seen in Figure 13, addition of Cycloheximide at time 0 completaiy blocked RA
induction of Ci~ABP-II g~ne transcripUon.
The foregoing discussion discloses and describes mereiy exemplary embodiments
of the present invention. One skilled in the art will readily recognize trom such discussion,
and from the accompanying drawings and claims, that various changes, mod-ffications and
30 variations can be made therein without departing from the spirit and scope of the invention
as defined in the tollowing claims.
All publicatlons and applications cited herein are Incorporated by reference.
2134~S~ i
- WO 93/22331 PCI`~US~3/03936
27 -
SEQUENCE LI5TIING .
~1~ GENERAL INFORMATION:
(i3 APPLICANT: Yoorhees, ~ohn J~
Astrom, Anders .
Pattersson, Ul ri ka
Tavakkol, Ami r
(ii) TITLE OF INVENTION: HUMAN CRABP-I A~D CRABP-II
(iii) NUMBER OF SEQUENCFS: 6 1:
(iv) CORRESPONDENCE ADDRESS:
tA) ADDRESSEE: Harness, Dickey & Pierce
(B3 STREET: PD Box 828
(C) CITY: Bl oomfi el d Hi l l s
(D) STATE: Michigan
(E) COUNTRY: United States of Anerica
(~) ZIP: 48013
~v) CDMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
~B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release ~1.0, Version ~1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
~ J v
w o s3/2z331 ~ P ~ /US93/0393
- 28 -
(viii~ ATTORNEY/AGENT INFORMATION:
(A) NAME: Lewak, Anna M.
(B) REGISTRATION NUMBER: 33,006
(C) REFERENGE/DOCKET NUMBER: 211500676POB
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (313) 641-1600
(8) TELFFAX: (313) 641-0270
(C) TELEX: 287637
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHA~ACTERISTICS:
(A3 LENGTH: 924 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: NO
(iv~ ANTI~SENSE: NO
(vi) ORIGINAL 50URCE:
~A~ ORGANISM: Homo sapien
(F) TISSUE TYPE: skin
(G) CELL TYPE: fibroblast
(vii) IMMEDIATE SOURCE:
(A3 LIBRARY: HUMAN SKIN FIBROBLAST LAMBDA GT11
(B) CLONE: LAMBDA F1.1
,~ WO93/~2331 213~SO PCI'/US93/03936
- 29 -
( i x) FEATURE:
~A) NAME/KEY: CDS
(B) LOCATION: 99..515
(D) OTHER INFORMATION: /codon start~ 99/citation- ( El] )
(ix) FEATURE:
(A) NAME/KEY: polyA_site
(B~ LOCATION: 924 ¦
(D) OTHER INFORMATION: /citation= ([1])
(ix) FEATURE: -
(A) NAME/KEY: 5'UTR
~8~ LOCATION: 1..98
(D~ OTHER INFORMATION: /citation~
(ix~ FEATURE:
(A) NAME/KEY: 3'UTR
(8~ LOCATION: 516..924
(D) OTHER INFORMATION: /citation- ([1])
(ix) FEATURE:
(A) NAME/KEY: polyA signal
(B) LOCATION: 911..916
(D) OTHER INFORMATIO~: /citation~ (~1])
(ix) FEATURE:
(A) NAME/KEY: terminator
(B) LOCATION: 513..515
(D) OTHER INFORMATION: /citations (tl])
WO 93/22331 2 13 ~ 3 ~ ~ . P~/US93~03936 k--
30 -
~x) PUBLICATION INFORMATION:
(A) AUTHORS: Astrom, Anders . .
Tavakkol, Ani r
Pettersson, Ul ri ka
Cromi e, Matthe~
Elder, J~mes T.
Voorhees, John J.
(B) TITLE: Molecular Cloning of Two Human Cell~lar
Reti noi c Aci d- Protei ns ~CRABP)
(C) JûURNAL: J. Biol. Chem.
(G) DATE: 1991
(I() RELEVANT RESIDUES IN SEQ ID NO:1: FROM 1 TO 924
(x) PUBLICATION INFORMATION:
(A) AUTHORS: Astrom, Anders
Tavakkol, Ani r
~ Elder, James T.
Pettersson, Ulrika
Cromie, Matthew
~- Voorhees, John J.
(B) TITLE: Cloning of CRABPII cDNA from Human Skin:
Retinoic Acid Induces Expression of CRABPII but
Not CRABPI in Human Skin in Vivo and in Dermal but
Not Lung Fibroblasts in Vitro
(C) JOURNAL: J. Invest. Dermatol.
(D) VOLUME: 96
(F~ PAGES: 547-547
(G) DATE: April-1991
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
CCTGACGACC CGGCGACGGC GACGTCTCTT TTGACTAAM 6ACAGTGTCC AGTGCTCCA6 60
CCTAGGAGTC TACGGGGACC GCCTCCCGCG CCGCCACCAT GCCCAACTTC TCT6GCMCT 120
GGAAAATCAT CCGATCGGM MCTTCGAGG AATTGCTCM AGTGCTGGGG GTGMTGTGA 180
TGCTGAGGM GATTGCTGTG GCTGCAGCGT CCMGCCAGC AGTGGAGATC AAACAGGAGG 240
GAGACACm CTACATCMA ACCTCCACCA CCGTGCGCAC CACAGAGATT MCTTCMGG 300
TTGGGGAGGA GmGAGGAG CAGACTGTGG ATGGGAGGCC CTGTMGAGC CTGGTGAAAT 360
213~0
- Wo 93/22331 PCr/VS93/03936
- 31 -
GGGAGAGTGA GMTAMATG GTCT6TGAGC AGMGCTCCT GMGGGAGA~ GGCCCCMGA 420
CCTCGTGGAC CAGAG M CT6 ACC M CGATG GGG M CTGAT CCTGACCATG ACGGCGGAT6 480
ACGTTGTGTG CACCAGGGTC TACGTCCGAG AGTGAGTGGC CACAGGTAGA ACCGCG6CCG 540
M GCCCACCA CTG~CCATGC TCACCGCCCT GCTTCACTGC CCCCTCCGTC CCACCCCCTC 600
CTTCTAGGAT AGCGCTCCCC TTACCCCAGT CACTTCTG6G GGTCACTGGG ATGCCTCTTG 660
CAGGGTCTTG C m C m GA CCTCTTCTCT tCTCCCCTAC AGC M CAAA6 AGGAAT6GCT 720
GCAAGAGCCC AGATCACCCA TTCCGGGTTC ACTCCCCGCC TCCCC M GTC AGCAGTCCTA 780
GCCCCAAACC AGCCCAGAGC AGGGTCTCTC TAAAGGGGAC TTGAGGGCCT GAGCAGGAAA 840
GACTGGCCCT CTAGCTTCTA CCC m ~TCC CTGTAGCCTA TACAG m AG AATArrTATT 900
TGTT M TTTT ATTAAAATGC TTTA 924
(2) INFORMATION FOR SEQ ID NO:2:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 138 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: si ngl e
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: homo sapien
(x) PUBLICATION INFORMATION:
(A) AUTHORS: Astrom, Anders
Tavakkol, Amir
Pettersson, Ulrika
Cromie, Matthew
Elder, James T.
Voorhees, John J.
J
wos3/22331 :: : pcrJuss3/o393b
- 32 -
(B) TITLE: Mol eeul ar Cl oni ng of Two Human Cel l ul ar
Retinoic Acid-Proteins (CRABP~
(C) JOURNAL: J. Biol. Chem.
(G~ DATE: 1991
(K) RELEYANT RESIDUES IN SEQ ID NO:2: FRGM 1 TO 138
~x) PU~LICATION INFORMATION:
(A) AUTHORS: Astrom, Anders
Tavakkol~ Amir
Elder, James T.
Pettersson, Ulrika
Cromie, Matthew
Voorhees, 30hn J.
(B~ TITLE: Cloning of CRABPII eDNA from Human Sktn:
Retinoic Aeid Induces Expression of CF~BPII but
Not CRABPI in Human Skin in Vivo and in Dermal but
Not Lung Fi brobl asts i n Vi tro
(C) JOURNAL: J. Invest. Dermatol.
(D) VOLUME: 96
~F) PAGES: 547-547
- (G) DATE: April-1991
txi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
M~t Pro Asn Phe Ser Gly Asn Trp Lys Ile Ile Arg Ser Glu Asn Phe
5 10 15
Glu Glu Leu Leu Lys Yal Leu l;ly Val Asn Val Met Leu Arg Lys Ile
20 25 30
Ala Val Ala Ala Ala Ser Lys Pro Ala Val Glu Ile Lys Gln Glu Gly
35 ~ qO 45
Asp Thr Phe Tyr Ile Lys Thr Ser Thr Thr Val Arg Thr Thr Glu Ile
50 55 60
Asn Phe Lys Val Gly Glu Glu Phe Glu Glu Gln Thr Val Asp Gly Arg
65 70 75 80
Pro Cys Lys Ser Leu Val Lys Trp Glu Ser Glu Asn Lys Met Val Cys
8~ ~0 95
Glu Gln Lys Leu Leu Lys Gly Glu Gly Pro Lys Thr Ser Trp Thr Arg
100 105 110
213~Sg~
- ` WO 93~;!2331 PCI/US93/03936
- 33 -
Glu Leu Thr Asn Asp 6ly Glu Leu Ile Leu Thr Met Thr Ala Asp Asp
115 12~ 125
Val Val Cys Thr Arg Val Tyr Val Arg Glu
130 135
(2) INFDRMATION FOR SEQ ID ~0:3:
(i3 SEQUENCE CHARACTERISTICS: !
(A) LENGTH: 525 base pairs
(B) TYP: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo Sapien
(F) TISSUE TYPE: skin
(G) CELL TYPE: fibroblast :
(vii) IMMEDIATE SOURCE:
(A) LIBRARY: human skin Lambda ZapII
: (8) CLONE: la~bda s3.1
(ix) FEATURE:
(A) NAME/KEY: CDS
(~) LOCATION: 8..418
(D) OTHER INFORMATION: /codon_start~ 8
/citatlon- (tl])
~13~3~ '{
WO~3/2~331 PCl'/US93/~)3936
- 34 -
( i X) FEATURE ~
(A) NAME/KEY: 5 ' UTR
(B) LOCATION: 1. . 7
(D) OTHER INFORMATION: /ci tati on~
( i x) FEATURE:
(A) NAME/KEY: 3 ' UTR
~B) LOCATION: 419..525
(D) OTHER INFORMATION: /citation~ (~1])
(i x) FEATURE:
(A) NAME/KEY: termi nator
(B) LOCATION: 419 . . 421
(D) OTHER INFORMATION: /citation~
(x) PUBLICATION INFORMATION:
(A) AUTHORS: Astrom, Anders
Tavakkol, Amir
Pettersson, Ulrika
Cromie, Matthew
Elder, ~ames T.
Voorhees, John J.
- (B) TITLE: Molecular Cloning of Two Human Cellular
Reti noi c Aci d-Protei ns (CRABP)
(C) JOURNAL: J. Biol. Chem.
~G~ DATE: 1991
(K) RELEVANT RESIDUES IN SEQ ID NO:3: FROM 1 TO 525
(x) PVBLICATION INFORMATION: ¦
~A) AUTHORS: Astro~, Anders
Tavakkol, Amir
Elder, James T.
Pettersson, Ul ri ka
Cromie, Matthew
Voorhees, John J.
w o 93/22331 - ~2 1 3 4 ~ 5 0 P~/uss3/03s36
~B) TITLE: C.loning of DRABPII cDNA from Human Skin:
Retinoic Acid Induces Expression of CRABPII but
Not CRABPI in Human Skin in ViYo and in Dennal but
Not Lung Fi brobl asts i n Vi tro
(C) JOURNAL: 3. Invest. Denmatol.
(D) VOLUME: 96
(F) PAGES: 547-547
(G) DATE: April-1991
(xi~ SEQUENCE DESCRIPTION: SEQ ID NO:3:
TGCCACCATG CCCMCTTCG CCGGCACCTG 6MGATGCGC AGCAGCGAGA AmCGACGA 60
GCTGCTCMG GBACTGGGTG TGMCGCCAT GCTGAGGMG GT6GCCGTAG cGGclrGcGTc 120
CMGCCGCAC GTGGAGATCC GCCAGGACGG GGATCAGl~C TACATCMGA CATCCACCAC 180
GGTGCGCACC ACTGAGATCA ACTTCMGGT CGGAGMGGC mGAGGAGG AGACCGTGGA 240
CGGACGCMG T6CAEGAGTT TAGCCACTTG 6GA6MTGAG MCMGATCC ACTGI:ACGCA 300
MCTCTTCTT GAAGGGGACG GCCCCMAAC CTACTGGACC CGTGAGCT6G CCAACGATGA 360
ACTTATCCTG ACGmGGCG CCGATGACGT 6GTCTGCACC AGMmATG TCCGA6AGTG 420
M6GCAGCTG 6CTTGCTCCT ACI I ICAGGA A6GGATGCAG GCTCCCCTGA GGMTATGTC 480
ATAGT~CT6A 6CTGCCAGTG GACCGCCCTT TTCCCCTACC MTAT 525
(2) INFORMATION FOR SEQ ID NO:4
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 137 amino acids
(B) TYPE- amino acid
~C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: YES
~1~ 4a5 '~7
W(~ 93/22331 PCl`/U!~93~03936
- 36 -
~v;) ORIGINAL SOURCE:
(A) ORGANISM: homo sapien
~",
(x) PUBLICATION INFORMATION:
(A) AUTHORS: Astrom, Anders
Tavakkol, Amir
Pettersson, Ulrika
Cromie, Matthew
Elder, James T.
Voorhees, John J.
~B) TITLE: Molecular Cloning of Two Human Cellular
Retinoic Acid-Proteins (CRABP)
(C) JOURNAL: J. Biol. Chem.
(G) DATE: 1991
~K) RELEVANT RESIDUES IN SEQ ID NO:4: FROM 1 TO 137
(x) PU8LICATION INFORMATION:
(A) AUTHORS: Astrom, Anders
Tavakkol, Amir
Elder, James T.
Pettersson, Ulrika
Cromie, Matthew
Voorhees, John J.
(B) TITLE: Cloning of CRABPII cDNA from Human Skin:
Retinoic Acid Induces Expression of CRABPII but
Not CRABPI in Human Skin in Vivo and in Dermal but
Not Lung Fibroblasts in Vitro
(C) JOURNAL: J. Invest. Denmatol.
(D) VOLUME: 96
(F) PAGES: ~47 547
(G) DATE: April-1991
. .
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Pro Asn Phe Ala Gly Thr Trp Lys Met Arg Ser Ser Glu Asn Phe
1 5 10 15
Asp Glu Leu Leu Lys Ala Leu 61y Val Asn Ala Met Leu Arg Lys Val
~- ~Y0 93/~331 2 1 :~ 1 5 5 ~ PCI /U593/03936
- 37 -
Ala Val Ala Ala Ala Ser Lys Pro His Val Glu Ile Arg Gln Asp 61y
4~ 45
Asp Gln Phe Tyr Ile Lys Thr Ser Thr Thr Val Arg Thr Thr Glu Ile
~0 55 60
Asn Phe Lys Val Gly Glu Gly Phe Glu Glu Glu Thr Val Asp Gly Arg
~0
Lys Cys Arg Ser Leu Ala Thr Trp Glu Asn Glu Asn Lys Ile His Cys
Thr Gln Thr Leu Leu Glu Gly Asp Gly Pro Lys Thr Tyr Trp Thr Arg
100 105 11~
Glu Leu Ala Asn Asp Glu Leu Ile Leu Thr Phe Gly Ala Asp Asp Yal
115 120 125
Val Cys Thr Arg Ile Tyr Yal Arg Glu
130 135
(2~ INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1322 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLEGULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv~ ANTI-SENSE:~NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(F) TISSUE TYPE: Placenta
'2 13 45;3 `~
W093/22331 ' PCI/US93/03936
- 38 -
(Vi i ) IMMEDIATE SOURCE:
~A) LIBRARY: human placenta genomic library
(B) CLONE: lambda 2.1
,. ;~.
(ix) FEATURE:
(A) NAME/KEY: TATA signal
(B~ LOCATION: 1008~.1013
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 1039..1245
(îx) FEATURE:
(A~ NAME/KEY: intron
- (8) LOCATION: 1246.. 1322
(x) PUBLICATION INFORMATION:
(A) AUTHORS: Astrom, Anders
Pettersson, Ulrika .
Voorhees, John J
(B) TITLE: Structure of the human cellular retinoic -
acid-binding protein II (CRABP-II) gene: Early
transcri pti onal regul ati on by reti noi c aci d
(C) JOURNAL: J. Biol. Chem.
(G) DATE: 1992
(K) RELEVANT RESIDUES IN SEQ ID NO:5: FROM 1 TO 1322
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
CT6CAGGAAG CCGTGCCCTC CTCCCACCCT C m GATCTC CCG m CAAA 6CCGCTCTCC 60
AAGGGAGGGG`AGGTCGCTCC TTCCGCCC6T m ACAGCTC A6GATG6T6A CACCTGAGAC 120
CCTGCTCCGC CTTCTCCCCC GGCACCCATC CTCCCGCCTA TCTAGGTGGT GGCGCAGCTC 180
GCCAGGGCTC CGCGCCTCTG TCCCCGCCTC CCTCCCTTCC CCCTACTGAG ACCCCTCGGG 240
-- wo 93/2~331 2 1 3 4 5 ~ () PcrllJS93/03936
- 39 -
GTCTCGGGAG TGMGCGACA GAGMAGC6T TTTMTMAG ACCTTGCGTC MGTGATTGG 300
CTGTGACCTC TGCCCTCCCA GCCTCGCGCC CTGGGCTCCT GCTTMCCCT TCMTGTCCG 360 ~ -
CCCAGCGBAT T M GGGGAGC GAGTCGCCTG GCGACTACTT CCAGAGTCCC CAGGCATTAC 420
GT6AGCCCGA AGCAGGGTGG AGGGGTGGGG GGACCGT6CC GCCCCCGCCC AGCCTCTCCG 480
AGTTGTTCCA GCAGGGGGCG CCGTTGCCTC ACTTAGATCC CTMCCCCCG GMCCCCGCA 540 3
GCTCCCMGC CCCTCTCTGA GTACGGAGTG GTCCCACTGG ATCCAGTTCA GGGTTCAATG 600
GAGCTAGGGC CAGCTACGGC TCMGATCTG GGGTCCGCCT GCGGTGGGGT CGCCAGGT6T 660
CCGGCACC M GGAGTTG M T GCACCGAGTC AGGTTGGGGA TGG6TGGGGA ACA~GCGAGA 720
CGTGAGGAAC TCGGGTGGGG GACAGCCATA CACGAGCCCT GAGCATCTGC GCCCEiCAGCT 780
AGCTCCCCCC GCCTCTGCGG AGAGCGCGAT TCMGT&CTG GCTTTGCGTC CGCTTCCCCA 840
TCCACl~ACT A6CGCAGGA6 MGGCTATCT CGGTCCCCAG AGAAGCCTGG ACCCACACGC 900
GGGCTAGATC CA6AGGTT6G T6GCGGGGGC GCAG6GCCCC AGGTGGGGGG GGGCGGAGBG 960
GGAGGCGGGG CCACTTCMT CCTGG6~AGG GGCGGTTCCG TACAGGGTAT AAMGCTGTC 1020
CGCGCGGGAG CCCAGGCCAG CmGGGGTT GTCCCTGGAC TT6TCTTGGT TCCAGMCCT 1080
GACGACCCGG C6ACGGCGAC GTCTCI 1116 ACTAMAGAC AGTGTCCAGT GCTCCAGCCT 1140
AGGA6TCTAC GG6GACCGCC TCCCGCGCCG CCACCATGCC CMCl~CTCT GGCAACTGGA 1200
AAATCATCCG ATCGGMAAC TTCGAG6MT TGCTCMAGT GCTGGGTMG GAAATGTTCG 1260
AGGGCCCAGG TGGGGCA~GG GGG6GCTCTG 6AGTCCTCGA AGTTGGGGAT GAGAMGACA 1320
6C ` 1322
(2) INFORMATION FOR SEQ ID NO:S:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1?17 base pairs
(B) TYPE: nucl ei c aci d
(C) STRANDEDNESS: doubl e
~D) TOPOLOGY: l ~ near
(ii) MOLECULE TYPE: DNA (genomic)
213 ~5 ~ J
WO ~3~22331 ~ PCI`/US93/0393~
- 4~ - :
(i i i ) HYPOTHETICAL: NO
.
(iv) ANTI-SENSE: NO ~
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(F3 TISSUE TYPE: Placenta
(vii) IMMEDIATE SOURCE:
(A) LIBRARY: human placenta genomic
(B3 CLONE: lambda 2.1
(ix~ FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 178..356
(ix) FEATURE:
(A) NAME/KEY: intron
(B) LOCATION: 3~7..571
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 572,.6B8
(ix) FEATURE:
(A) NAME/KEY: intron
(B) LOCATION: 699..1152
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 1153..1618
~- W0~3/22331 21345~i0 PCI/US93/03936
~x) PUBLiCATION INFORMATION:
(A) AUTHORS: Astrom, Anders
Pettersson, Ulrika
Voorhees, John J
(B) TITLE: StructurR of the human cellular retinoic-acid
binding protein (CRABP-II) gene: Early
transcrip~ional regulation by retinoic ac~d
(C) JOURNAL: J. Biol. Chem.
(6) DATE: 1992
(K3 RELEVANT RESIDUES IN SEQ ID NO:6: FROM 1 TO 1717
'
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
CGCCTACCGT CTCCTTCMG GCACmCTT AGACACCCGG GCACCAGGCA GATGSACCCC 60
CCMCACACC CACCC~MGC AAGTCACA~A TCAGCCTGCT CCMCTGTCT TATGGGGAGG 120
GTGTGAGAGA GGTE. CAAA 6GCCCCTAAA AGGTGAGCCT CTCCTCTCTC CCCACAGGGG 180
TGMTGTGAT GCTGAGGMG ATTGCTGTGG CTGCAGCGTC CAAGCCAGCA GTGGAGATCA 240
AACAGGAGGG AGACACmC TACATCMM CCTCCACCAC CGTGCGCACC ACAGAGAl~A 300
ACTTCAAGGT TGG&GAGGAG mGAGGAGC AGACTGTGGA TGGGAGGCCC TGTMGGTGA 360
GTGCCAGAAG GGGCTCCAGG GTCATGGCGT CATTGCCCTG CCTCTCMCC T6CCA I I I I C 420
CAGGCTAGCA GTTAACTCCT AGCTTCTCTC TGTCCCAGTA GGGAAAATCC CTAGGTAGTG 480
GTGGGGGCTA GAMG6GGCT CTCTCCCl~A TCCCTCTCAC TGCATT6CCC CTGCTATGGG 540
CCCA5CTCAC TTGGCCACCT GTCTCTTGCA GAGCCTGGTG AAATGGGAGA GTGAGAATM 600
MTGGTCTGT GA6CAGM6C TCCTGAAGGG AGAGGGCCCC MGACCTCGT GGACCAGAGA 660
ACTGACCAAC GATGGG6AAC TGATCCTGGT AAGTCCTGCC TCCTCCCCAC TMTAGCAAt~ 720
CCCAGTGCTA CCTTCCMGA TTCTCTGGGA GACCCCAGGG TGCAGGAGAC TCMGAACM 780
CCATGGtT6G ACTCCGCACC CTGCTGATGG GACTGCTTGA ACAGMCTAA G6TGTCCCTA 840
TCCCATACAG TGCCCTGTGT 6MTTAGAAA TGGTGTTCCT mATGCMG CMAGGGCAT 900
GTACTGAGGG ATCCCAGCAG TTCTTCA6GG AGATCTTCCT GGCTTGAGGA GGAGGACGGG 960
CCCCAGGGCT CTATTGCTAT CCTCCCTCCA TTGATGCCTG GGCATTCTGG GACCAGCTCC 1020
TGCCTGTTGG TCTTGAGCCA AGMGCAGGT TTGGACCT6G AG5CCMGCA GAGTACCTCC 1080
wo 93/2~331 ' Pcr/usg3/o39
- 42 -
ATTCMCCCT CCTCTCCMA GCCACAGGAC CCCAGGGGCC TCTCAGGCTA ACMCTACTT 1140
CTGTCCTTCC AGACCATGAC GGCGGATGAC GTTGTGTGCA CCAGGGTCTA CGTCCCAGAG 12Q0
T6A6TGGCCA CAGGTAGMC CGCGGCCGM GCCCACCACT GGCCATGCTC ACCGCCCTGC 1260
TTCACTGCCC CCTCCGTCCC ACCCCCTCCT TCTAGGATAG CGCTCCCCTT ACCCCAGTCA 1320
CTTCTGGGGG TCACTGGGAT GCCTCTTGCA GGGTCTTGCT TTCmGACC TCTTCTCTCC 1380
TCCCCTACAC CAACAAA6AG GMTGGCTGC MGAGCCCAG ATCACCCATT CC6GGTTCAC 1440
TCCCCGCCTC CCC M GTCAG CAGTCCTAGC CCCAAACCAG CCCAGAGCAG GGTCTCTCTA 1500
M GGGGAGTT GAGGGCCTI;A GCAGGAMGA CTGGCCCTCT AGCllCTACC Cl I IGTCCCT 1560
GTA6C-,TATA CA&~I IAGM TAI I IAmG TTMl~TTAT TAMATGCTT TMMA~ATA 1620
MACCTGTCT CTGGCTCATT GGGCAGGTAG ATMGTCACC TGAGTTCMC CTTGCCrCTG 1680
AAAT6TAGTA TGGGAAAGAC TTGTGIIICT GCAGCAT 1717
