U.S. patent application number 11/002458 was filed with the patent office on 2005-07-14 for peptides and polypeptides derived from the submaxillary gland of the rat, corresponding polyclonal and monoclonal antibodies, corresponding hybridomas and uses of these products for diagnosis, for detection or therapeutic purposes.
This patent application is currently assigned to INSTITUT PASTEUR. Invention is credited to Rosinski-Chupin, Isabelle, Rougeon, Francois, Seidah, Nabil, Tronik, Diana.
Application Number | 20050153374 11/002458 |
Document ID | / |
Family ID | 9370902 |
Filed Date | 2005-07-14 |
United States Patent
Application |
20050153374 |
Kind Code |
A1 |
Rosinski-Chupin, Isabelle ;
et al. |
July 14, 2005 |
Peptides and polypeptides derived from the submaxillary gland of
the rat, corresponding polyclonal and monoclonal antibodies,
corresponding hybridomas and uses of these products for diagnosis,
for detection or therapeutic purposes
Abstract
The invention relates to peptides having the formula (I):
X-His-Asn-Pro-Y wherein X is a rest Gln or pyro-Glu. Y is a group
OH or a rest of basic aminoacid.
Inventors: |
Rosinski-Chupin, Isabelle;
(Versailles, FR) ; Tronik, Diana; (Meudon, FR)
; Rougeon, Francois; (Poigny La Foret, FR) ;
Seidah, Nabil; (Montreal, CA) |
Correspondence
Address: |
BURNS DOANE SWECKER & MATHIS L L P
POST OFFICE BOX 1404
ALEXANDRIA
VA
22313-1404
US
|
Assignee: |
INSTITUT PASTEUR
Paris Cedex
FR
|
Family ID: |
9370902 |
Appl. No.: |
11/002458 |
Filed: |
December 3, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11002458 |
Dec 3, 2004 |
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09386850 |
Aug 31, 1999 |
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09386850 |
Aug 31, 1999 |
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08476120 |
Jun 7, 1995 |
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6025143 |
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08476120 |
Jun 7, 1995 |
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08153277 |
Nov 17, 1993 |
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5859189 |
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08153277 |
Nov 17, 1993 |
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07499276 |
Jul 19, 1990 |
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Current U.S.
Class: |
435/7.23 ;
424/155.1; 435/344; 530/330; 530/350; 530/388.8 |
Current CPC
Class: |
C07K 5/1024 20130101;
C07K 14/575 20130101; G01N 33/68 20130101; C07K 16/18 20130101;
A61K 38/00 20130101; C07K 5/1021 20130101; C07K 14/47 20130101 |
Class at
Publication: |
435/007.23 ;
530/350; 530/330; 530/388.8; 435/344; 424/155.1 |
International
Class: |
G01N 033/574; A61K
039/395; C07K 016/30; C12N 009/02; C12N 005/06 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 11, 1988 |
FR |
8813353 |
Oct 11, 1989 |
US |
PCTFR8800523 |
Claims
1-14. (canceled)
15. An isolated nucleic acid having the nucleotide sequence of
nucleotides 1 to 510 of SEQ ID NO: 7, or a sequence hybridizing
thereto, in a hybridization solution containing: 0.75 M sodium
phosphate, pH 7.2, 7% sodium dodecylsulfate, 1 mM EDTA, 1% bovine
serum albumin, and sonicated salmon sperm DNA: 100 mg/ml, at
65.degree. C.; and after washing four times for 10 minutes at
65.degree. C. with a solution containing: 40 mM sodium phosphate,
pH 7.2, 1% sodium dodccylsulfate, and 1 mM EDTA.
Description
[0001] The present invention relates to novel peptides which are
maturation products of polypeptides secreted by the submaxillary
gland of the rat and analogues of these peptides.
[0002] Many polypeptides having defined biological properties are
synthesized in large quantities in the submaxillary gland (SMG) of
rodents, and in particular in the SMG of the mouse. These proteins,
comprising the nerve growth factor (NGF), the epidermal growth
factor (EGF) and renin, have a certain number of properties in
common. They are all synthesized in the same type of cell, namely
the cells of the convoluted tubules GCT (granular convoluted
tubular), in response to various hormonal stimuli, in particular to
androgens. Furthermore, it is possible to observe the presence of
these secretory proteins in the saliva of the mouse, and they are
synthesized in the form of precursors which become active after
maturation processes which may involve proteases of the kallikrein
type. Some of these proteases of the kallikrein type are also
synthesized in the SMG under the control of androgens.
[0003] Attempts at the characterization of the genes controlled by
the androgens in the SMG of the rat have led the inventors to
analyse the electrophoretic profile of the in vitro translation
products of the mRNA of this tissue.
[0004] A specific mRNA of the submaxillary gland of the male rat
has been isolated. This mRNA corresponds to a polypeptide which has
been designated SMR1. This polypeptide gives maturation products
with physiological activity. The present invention relates mainly
to these peptides and to other peptides having similar
properties.
[0005] Thus the subject of the present invention is peptides of
formula:
X-His-Asn-Pro-Y I
[0006] in which:
[0007] X represents a glutamine (Gin) or pyroglutamic acid
(pyro-Glu) residue,
[0008] Y represents a OH group or a residue of a basic amino
acid.
[0009] The basic amino acids may be lysine or arginine.
[0010] More specifically, the subject of the present invention is
peptides of formula:
1 Gln-His-Asn-Pro II pyro-Glu-His-Asn-Pro III Gln-His-Asn-Pro-Arg
IV pyro-Glu-His-Asn-Pro-Arg V Gln-His-Asn-Pro-Lys VI
pyro-Glu-His-Asn-Pro-Lys VII
[0011] Another subject of the present invention is the polypeptide
SMR1 which gives the maturation products of formulae II, III, IV
and V. This polypeptide corresponds to the formula:
2 1 Met Lys Ser Leu Tyr Leu Ile Phe Gly Leu Trp Ile 20 Leu Leu Ala
Cys Phe Gln Ser Gly Glu Gly Val Arg 30 Gly Pro Arg Arg Gln His Asn
Pro Arg Arg Gln Gln 40 Asp Pro Ser Thr Leu Pro His Tyr Leu Gly Leu
Gln 50 60 Pro Asp Pro Asn Gly Gly Gln Ile Gly Val Thr Ile 70 Thr
Ile Pro Leu Asn Leu Gln Pro Pro Arg Val Leu 80 Val Asn Leu Pro Gly
Phe Ile Thr Gly Pro Pro Leu 90 Val Val Gln Gly Thr Thr Glu Tyr Gln
Tyr Gln Trp 100 Gln Leu Thr Ala Pro Asp Pro Thr Pro Leu Ser Asn 110
120 Pro Pro Thr Gln Leu His Ser Thr Glu Gln Ala Asn 130 Thr Lys Thr
Asp Ala Lys Ile Ser Asn Thr Thr Ala 140 Thr Thr Gln Asn Ser Thr Asp
Ile Phe Glu Gly Gly Gly Lys
[0012] Another subject of the present invention is monoclonal and
polyclonal antibodies directed against the peptides and the
polypeptide according to the invention.
[0013] Another subject of the present invention is hybridomas
producing monoclonal antibodies directed against the peptides I to
VII and the polypeptide according to the invention.
[0014] Another subject of the present invention is a procedure for
the assay or detection of the peptides and polypeptides according
to the invention in tissues and biological fluids which comprises
the utilization of monoclonal or polyclonal antibodies according to
the invention.
[0015] For this purpose, it is possible to utilize in particular a
method of the RIA type using a peptide labelled by a radioisotope,
and the competition between this peptide and the peptide to be
assayed (Niswender G. D. et al; Proc. Soc. Exp. Biol. 128, 807,
1968). It is also possible to utilize a method of the ELISA type
using, for example, a peptide bound to a support and the
competition between this peptide and the peptide to be assayed for
antibodies prepared against this peptide. The antibodies retained
by the peptide bound to the support are detected by antibodies
directed against the former and linked to an enzyme (method derived
from Avromeas J. and Guilbert B, C.R. Acad. Sci. Paris 1971, 273,
2305).
[0016] The peptides according to the present invention may be
prepared in a standard manner by peptide synthesis in liquid or
solid phase by successive couplings of the different amino acid
residues to be incorporated (from the N-terminal end toward the
C-terminal end in liquid phase, or from the C-terminal end to the
N-terminal end in solid phase) and the N-terminal ends and the
reactive side chains of which are blocked beforehand by standard
groupings.
[0017] For this synthesis on solid phase it is possible to utilize
in particular the technique described by Merrifield, in the article
entitled "Solid phase peptide synthesis" (J. Am. Chem. Soc., 85,
2149-2154).
[0018] In order to produce a peptide chain according to the
Merrifield procedure, recourse is had to a very porous polymeric
resin, to which the first C-terminal amino acid of the chain is
bound. This amino acid is bound to the resin through the
intermediary of its carboxyl group and its amino function is
protected, for example by the t-butoxycarbonyl group.
[0019] When the first C-terminal amino acid is thus bound to the
resin, the protecting group is removed from the amine function by
washing the resin with an acid. In the case in which the protecting
group for the amine function is the t-butoxycarbonyl group, it may
be removed by treatment of the resin with the aid of
trifluoroacetic acid.
[0020] The second amino acid, which provides the second residue of
the desired sequence, is then coupled to the deprotected amine
function of the first C-terminal amino acid bound to the chain.
Preferably, the carboxyl function of this second amino acid is
activated, for example, by means of dicyclohexylcarbodiimide, and
the amine function is protected, for example, by the
t-butoxycarbonyl.
[0021] In this way, the first part of the desired peptide chain is
obtained which contains two amino acids and the terminal amine
function of which is protected. As previously, the amine function
is deprotected and it is then possible to proceed to the attachment
of the third residue under similar conditions to those for the
addition of the second C-terminal amino acid.
[0022] In this manner, the amino acids which will constitute the
peptide chain are attached one after the other to the amine group,
deprotected beforehand each time, of the portion of the peptide
chain already formed and which is attached to the resin.
[0023] When the whole of the desired peptide chain is formed, the
protecting groups are removed from the different amino acids
constituting the peptide chain and the peptide is cleaved from the
resin, for example, with the aid of hydrogen fluoride.
[0024] The peptide thus obtained can be purified, for example by
means of column chromatography.
[0025] The SMR1 peptide or derivatives of this peptide can also be
obtained with the aid of the techniques of genetic engineering;
they can also be obtained by purification from biological material
by means of techniques of chromatography or precipitation similar
to those used, for example, for the purification of human growth
hormone starting from the hypophysis.
[0026] The monoclonal and polyclonal sera can be prepared according
to a standard technique. For this purpose the tetrapeptides or the
pentapeptides or multimeric derivatives of these peptides can be
coupled to immunogenic agents such as KLH (Keyhole Lympet
Hemocyanin), ovalbumin, bovine serumalbumin etc, by a coupling
agent such as glutaraldehyde. The SMR1 protein and the derivatives
of this protein (peptides derived from this protein, or hybrid
proteins containing a part or all of SMR1 linked to another protein
such as protein A) can also be injected directly.
[0027] The immunizations can be performed in a standard manner, for
example, in the rabbit and the mouse by injecting into the animal
100 micrograms, for example, of the coupling product in the
presence of Freund's adjuvant 3 to 4 times at intervals of 3 weeks.
It is thus possible to obtain polyclonal sera in the rabbit.
[0028] The hybridomas and the monoclonal antibodies can be obtained
by means of the standard procedures.
[0029] The isolation of the mRNA, which corresponds to the SMR1
polypeptide, and the properties of the polypeptide and the
maturation products will be described in more detail below.
[0030] 1) Materials and Methods
[0031] Animals and Hormonal Treatments
[0032] 10 weeks old male and female Wistar rats were obtained from
Iffa-Credo. The androgens were removed by castration and 10 days
later 35 mg of testosterone (Sterandryl retard, Roussel) were
injected by the intraperitoneal route in the cases indicated. In
the cases indicated, the same dose of testosterone was administered
to female rats. 8 weeks old DBA/2 and "Swiss" mice were obtained
from the Pasteur Institute.
[0033] Extraction of the RNA and In Vitro Translation
[0034] The RNA was prepared from rat and mouse tissues as described
in the literature (Tronik D. et al. 1987, EMBO J. 6, 983-987). The
in vitro translation of the RNAs was carried out by using the
mRNA-dependent translation system as a lysate in the presence of
mRNA-dependent reticulocytes (Pelham H. RB et al., 1976, Eur. J.
Biochem. 67, 247-256). The products were analysed by
electrophoresis on a denaturing polyacrylamide-NaDodSO.sub.4
gel.
[0035] Cloning and Characterization of the cDNA Coding for SMR1
[0036] Poly(A) RNA obtained from SMG of male Wistar-rats was used
as matrix for the reverse transcriptase, and the double-stranded
cDNAs were obtained by means of the DNA synthesis system of
Amersham, with the protocol supplied by the manufacturer. The
double-stranded cDNA was then inserted into the PstI site of pUC9
by the method of the oligo-d(C) ends (Maniatis, T. et al. (1982) in
Molecular Cloning: A Laboratory Manual (Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.) pp 241-242). The host
bacteria (bacterial strain DH 5-1 derived from DH1 and yielding
high efficiencies of transformation, see Hanatian DNA cloning vol.
1, p. 111) were transformed and the colonies were screened by
hybridization with the probes described below. In brief, the mRNA
derived from the SMG of males and females was fractionated in a
5-20% sucrose gradient. The fractions enriched in mRNA of low
molecular weight (which were shown by in vitro translation to
contain the mRNA coding for SMR1) were precipitated with ethanol,
and they were used as matrix for the reverse transcriptase in the
presence of dGTP and dCTP radiolabelled with .sup.32P. About 3000
recombinants were screened on filters in duplicate samples with the
radiolabelled cDNA obtained from the SMG of male and female rats.
The clones selected demonstrated a strong hybridization with the
male cDNA probe but a very weak hybridization with the female
probe. The recombinant clones were identified by experiments of
inhibition of translation of the mRNAs in a cell-free system by
DNA-mRNA hybridization (Paterson et al. (1977) Proc. Natl. Acad.
Sci. USA, 74, 4370-4374).
[0037] Sequencing of the cDNA Coding for SMR1
[0038] With the aid of various restriction enzymes, the cDNA coding
for SMR1 was cut and the fragments obtained were subcloned in the
vector M13 mp9. The sequencing of the DNA was carried out by means
of the method of termination with dideoxyribonucleotides (Sanger,
F. et al. (1977) Proc. Natl. Acad. Sci. USA, 74, 5463-5467).
[0039] 2) Results
[0040] Analysis of the In Vitro Translation of mRNA Prepared from
the SMG of Rats and Mice.
[0041] 5 .mu.g of total RNA obtained from the SMG of male and
female mice or male and female rats were translated in a cell-free
system of reticulocytes in the presence of .sup.35S-methionine. The
products of in vitro translation were subjected to electrophoresis
on a 12.5% sodium dodecylsulfate-polyacrylamide gel and
autoradiographed.
[0042] The comparison of the products of the in vitro translation
of RNA prepared from the SMG of male and female rats shows the
presence of several polypeptides (of apparent molecular weights of
18,000, 19,000, 35,000, 46,000) in larger amounts in the male rats
than in the females. These data show the existence of a sexual
dimorphism in the SMG of the rat.
[0043] Furthermore, the comparison of these products of translation
with those obtained with RNAs derived from SMG of mice shows that
most of the major polypeptides are different in the two species. In
particular, whereas sex-linked differences could also be observed
in the mouse, these latter did not relate to the same polypeptides
as in the rat. Conversely, the products of translation observed in
the rat to be specific for the male seem to be absent in the
mouse.
[0044] Isolation and Sequence of a cDNA Complementary to a Specific
mRNA of the Male Rat
[0045] In order to isolate mRNAs specific for the male from the SMG
of the rat, a bank of cDNA prepared from this tissue was
constructed in pUC9. The recombinant clones were screened by
utilizing a differential screening strategy such as that described
in the Materials and Methods section. The positive clones were
characterized by experiments of inhibition of translation of the
mRNAs in a cell system by DNA-mRNA hybridization. One category of
recombinant cDNAs suppressed the in vitro synthesis of a
polypeptide having an apparent molecular weight of 19,000 daltons.
This polypeptide, designated SMR1, is present in the products of in
vitro translation of RNAs from male rats but is not present in
those from females. The corresponding cDNA was utilized as probe in
RNA hybridization experiments with transfer to a nitrocellulose
membrane. For the hybridization a solution was used containing:
[0046] 0.5 M sodium phosphate, pH 7.2
[0047] 7% sodium dodecylsulfate (SDS)
[0048] 1 mM EDTA
[0049] 1% bovine serum albumin
[0050] sonicated salmon sperm DNA: 100 mg/ml
[0051] The experiment was done at 65.degree. C. for 16 to 20 h.
[0052] 4 washings of 10 minutes each were carried out at 65.degree.
C. with a solution containing:
[0053] 40 mM sodium phosphate, pH 7.2
[0054] 1% SDS
[0055] 1 mM EDTA
[0056] The cDNA is hybridized with a mRNA 700 nucleotides long,
present in large amounts in the SMG of male rats. This inserted
element of cDNA has been characterized in more detail.
[0057] The sequence of the cDNA coding for SMR1 and the sequence
deduced for the protein are represented below
3 1 2 3 4 5 6 7 8 9 10
[0058] The inserted cDNA has a length of 652 nucleotides, if the
poly(A) fragment is excluded. This sequence comprises an open
reading frame of 510 nucleotides. The only ATG which can serve as
initiation codon is situated 73 nucleotides downstream from the 5'
end of the clone of cDNA. The untranslated region at the 3' end
(142 nucleotides long) contains the consensus signal for
polyadenylation (AATAAA), 23 nucleotides upstream from the poly(A)
tail (nucleotides 625-630). The translation stop codon is found in
another AATAAA sequence (512-517) which apparently is not
recognized as polyadenylation signal.
[0059] The corresponding protein has a length of 146 amino acids,
which corresponds to a molecular weight of about 16,000 daltons.
This molecular weight is slightly lower than that determined from
the electrophoretic mobility of the in vitro translation product
(19,000 daltons). Such a difference between the molecular weight
calculated from the analysis of the sequence and that determined
from the electrophoretic mobility has already been described for
other proteins of the SMG of the rat or the mouse.
[0060] SMR1 has a relatively high content of glutamine and proline
residues, but does not contain a repetitive region. Hence, it does
not belong to the family of polypeptides "rich in proline" or "rich
in glutamine", which are essential proteins of the SMG.
Furthermore, the sequence of the mRNA does not contain at its 5'
end the sequence of 80nucleotides which is characteristic of this
family. However, like these proteins, SMR1 does not contain cystein
or methionine residues (except in its signal peptide).
[0061] The amino-terminal part of SMR1 is strongly hydrophobic,
which is characteristic of signal peptides of most of the secreted
proteins. Although the amino-terminal sequence of the mature
protein has not been determined directly, from the statistical
analysis made according to G. Von Heijne (Nucleic Acids Res. 14,
4683-4690, 1986) (rule "-3,-1"), it may be supposed that the
cleavage site of the signal peptide is located between the residues
18 and 19.
[0062] The protein SMR1 also shows certain features characteristic
of the glycoproteins. The presence of two potential glycosylation
sites linked to N are observed at the positions 139 and 136. The
protein is relatively rich in proline (12%), threonine (12%) and
glutamine (9.5%). Several glycosylation sites linked to 0 might
thus be present in the carboxy-terminal fragment of SMR1, since
regions rich in proline and threonine residues are usually present
in highly O-glycosylated proteins, such as the mucoproteins and the
sialoglyco-proteins.
[0063] An interesting characteristic is the presence of pairs of
basic amino acids Arg-Arg at positions 27-28 and 33-34. Such
dipeptides represent potential sites of cleavage by maturation
enzymes (Lazure, C., et al (1983) Can. J. Biochem., Cell Biol. 61,
501-515 and Docherty, K. et al (1982) Ann. Rev. Physiol. 44,
625-638). They flank a tetrapeptide Gln-His-Asn-Pro. The
tetrapeptide and its adjacent, sequences are located in a
hydrophilic environment which renders this region accessible to
possible maturation enzymes.
[0064] The cleavage of Arg-Arg linkages by a maturation enzyme
followed by the removal of the basic residues by carboxypeptidase E
(Fricker, L. D. et al (1983) J. Biol. Chem. 258, 10950-10955) and
possibly an aminopeptidase (Loh, Y. P. et al. (1984) Ann. Rev.
Neurosci. 7, 189-222) would produce a mixture of tetrapeptide
(Gln-His-Asn-Pro) and pentapeptide (Gln-His-Asn-Pro-Arg), since
"Pro-Arg" is not a good substrate for carboxypeptidase E. Other
post-translational modifications could also include the formation
of pyroGlu acid derivatives of these products, giving rise to a
mixture of pyroGlu-His-Asn-Pro-Arg and pyroGlu-His-Asn-Pro. These
structures recall those of thyroliberin (TRH).
[0065] Regulation of the Accumulation of mRNA Coding for SMR1 in
the SMG of the Rat by Androgens.
[0066] In order to study the regulation of the accumulation of mRNA
coding for SMR1 in the SMG of the rat by androgens, mRNA containing
a poly(A) sequence was prepared from the SMG of adult males, males
castrated 20 days previously, castrated males subjected to a
treatment with androgens, females and females treated with
androgens. 1 .mu.g of total RNA from male rats, female rats treated
with testosterone, castrated male rats, castrated male rats treated
with testosterone and female rats was subjected to electrophoresis
in a 1.4% agarose-formaldehyde gel, these RNAs were transferred to
a Nylon membrane and were hybridized with the cDNA probe coding for
SMR1. The time of exposure for autoradiography was 2 hours. The
results of the RNA analysis, by transfer of these mRNAs to solid
supports, by means of a SMR1 probe labelled with .sup.32P are shown
in the figure (part A). A considerable difference in the
accumulation of mRNA coding for SMR1 is observed in the SMG of male
rats and those of females.
[0067] Furthermore, various amounts (as indicated in the figure) of
RNA from the SMG of male rats and female rats were subjected to
electrophoresis on a 2% agarose-formaldehyde gel, they were
transferred to filters and hybridized with the cDNA probe coding
for SMR1. The film was exposed for 30 hours. As is apparent in part
B, the mRNA coding for SMR1 accumulates in very large quantities in
the SMG of male Wistar rats, since an amount as low as 1.5 ng of
total RNA was sufficient to give a hybridization signal.
Conversely, the level of accumulation of mRNA coding for SMR1 in
the SMG of female Wistar rats was about 1,000 to 3,000 times lower
than that in males.
[0068] In the castrated males, the quantity of mRNA coding for SMR1
was reduced 10 to 20 fold. The administration of testosterone to
these males restored the amount of mRNA coding for SMR1 to the same
value as that observed in the non-castrated males. Furthermore, the
administration of testosterone to adult female rats caused the
accumulation of mRNA coding for SMR1 in amounts similar to that
observed in the males.
[0069] A remarkable property of the mRNA coding for SMR1 is its
high degree of accumulation in the SMG of the rat in response to a
treatment with androgens. This strongly suggests that SMR1 is
synthesized in the GCT cells of the SMG (just like the EGF, NGF and
renin as well as other proteins under the control of androgens in
the SMG of the mouse). Furthermore, the difference in the level of
accumulation of mRNA coding for SMR1 in the male and in the female
is very great (greater than three orders of magnitude), in
comparison with that usually observed for other genes controlled by
antigens in the target organs (kidney, liver, SMG).
[0070] These results, and in particular the high degree of
induction of the SMR1 gene by androgens, suggest that SMR1 may
fulfil an important function specific for the male in the rat. SMR1
might be the precursor of a molecule (the tetra- or pentapeptides
or the C-terminal part of SMR1) controlling behavioural
characteristics in the male rat.
[0071] The products described in the present invention can be used
for therapeutic purposes or as laboratory reagents.
Sequence CWU 0
0
* * * * *