U.S. patent application number 12/356351 was filed with the patent office on 2009-08-20 for genes.
Invention is credited to Mitinori Saitou, Azim Surani.
Application Number | 20090210954 12/356351 |
Document ID | / |
Family ID | 9940726 |
Filed Date | 2009-08-20 |
United States Patent
Application |
20090210954 |
Kind Code |
A1 |
Saitou; Mitinori ; et
al. |
August 20, 2009 |
GENES
Abstract
The present invention relates to two primordial germ
cell-specific expressed genes, Fragilis and Stella. The sequences
and sues of human Stella and Fragilis are disclosed herein, as are
several mouse sequences related to Fragilis. The present invention
relates to the use of Stella and Fragilis as markers for primordial
germ cells and can be used to identify such cells. Additionally,
the present invention relates to the use of Stella and Fragilis for
the diagnosis, treatment and/or prevention of disease.
Inventors: |
Saitou; Mitinori;
(Cambridge, GB) ; Surani; Azim; (Cambridge,
GB) |
Correspondence
Address: |
WHYTE HIRSCHBOECK DUDEK S.C.;INTELLECTUAL PROPERTY DEPARTMENT
33 East Main Street, Suite 300
Madison
WI
53703-4655
US
|
Family ID: |
9940726 |
Appl. No.: |
12/356351 |
Filed: |
January 20, 2009 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11038676 |
Jan 18, 2005 |
7488803 |
|
|
12356351 |
|
|
|
|
PCT/GB03/03093 |
Jul 17, 2002 |
|
|
|
11038676 |
|
|
|
|
Current U.S.
Class: |
800/13 ; 435/29;
435/325; 435/6.18; 530/350; 536/23.5; 536/24.2 |
Current CPC
Class: |
A01K 2217/05 20130101;
C12N 15/8509 20130101; C07K 14/705 20130101; A01K 2267/0306
20130101; A61P 43/00 20180101; A01K 2217/075 20130101; A61P 35/00
20180101; A01K 67/0275 20130101; A01K 2227/105 20130101; C07K
2319/60 20130101; A01K 67/0276 20130101 |
Class at
Publication: |
800/13 ; 530/350;
536/23.5; 435/6; 435/29; 536/24.2; 435/325 |
International
Class: |
A01K 67/033 20060101
A01K067/033; C07K 14/00 20060101 C07K014/00; C07H 21/00 20060101
C07H021/00; C12Q 1/68 20060101 C12Q001/68; C12Q 1/02 20060101
C12Q001/02; C12N 5/00 20060101 C12N005/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 17, 2002 |
GB |
0216727.8 |
Claims
1-17. (canceled)
18. A polypeptide comprising a human Stella amino acid sequence as
shown in SEQ ID NO:4, or a sequence that is at least 95% identical
thereto, wherein the polypeptide can serve as a marker of
pluripotency.
19. A polynucleotide sequence that codes for the polypeptide of
claim 1, suitable for use as a marker of pluripotency.
20. The polynucleotide sequence of claim 19, wherein the sequence
is as shown in SEQ ID NO:3 or a sequence that is at least 95%
identical thereto.
21. A method for identifying a pluripotent cell comprising
detecting expression of a Stella nucleic acid sequence in the
cell.
22. The method of claim 21, wherein the nucleic acid sequence is as
shown in SEQ ID NO:3 or a sequence that is at least 95% identical
thereto.
23. A method for identifying a pluripotent cell comprising
detecting expression of a Stella polypeptide in the cell.
24. The method of claim 23, wherein the Stella polypeptide is as
shown in SEQ ID NO:4, or a sequence that is at least 95% identical
thereto.
25. A polynucleotide expression construct suitable for detecting
pluripotency in a cell, comprising: i) at least the upstream
sequences of a human Stella gene; and ii) the coding sequence of a
reporter gene, wherein expression of the coding sequence of the
reporter gene is in operable combination with and under the control
of the transcriptional control sequences of the human Stella gene,
and wherein induction of expression of the reporter gene in the
cell can be detected and thereby correlated with a pluripotent
phenotype in the cell.
26. The polynucleotide expression construct of claim 25, wherein
the upstream sequences of the Stella gene include the 5'
untranslated region of the Stella gene.
27. The polynucleotide expression construct of claim 25, wherein
the upstream sequences of the human Stella gene comprise up to
about 8.5 kb of the nucleic acid sequence located upstream of the
first exon of the human Stella coding sequence.
28. The polynucleotide expression construct of claim 25, wherein
the expression construct is in the form of a transgene that can be
integrated into the genome of the cell.
29. The polynucleotide expression construct of claim 25, wherein
the cell is a stem cell.
30. The polynucleotide expression construct of claim 25, wherein
the cell is selected from one of the group consisting of: a
pluripotent cell; an embryonic stem cell; an embryonic germ cell; a
germ cell tumour cell; and a primordial germ cell.
31. The polynucleotide expression construct of claim 25, wherein
the cell is a human cell.
31. The polynucleotide expression construct of claim 25, wherein
the reporter gene is a green fluorescent protein (GFP) gene.
32. A cell comprising a polynucleotide expression construct of
claim 25.
33. A non-human transgenic animal comprising a cell that comprises
a polynucleotide expression construct of claim 25.
Description
FIELD
[0001] The present invention relates to the fields of development,
molecular biology and genetics. More particularly, the invention
relates to genes which are expressed exclusively in the earliest
populations of primordial germ cells (PGCs) and the use of such
genes and the products thereof in identification of pluripotent and
multipotent cells such as PGCs, pluripotent embryonic stem cells
(ES) and pluripotent embryonic germ cells (EG), in cell
populations. They are also markers for a change in the state of
cells from being non pluripotent to becoming pluripotent, and in
being able to confer this state on a non pluripotent cell.
INTRODUCTION
[0002] Post fertilisation, the early mammalian embryo undergoes
four rounds of cleavage to form a morula of 16 cells. These cells,
following further rounds of division, develop into a blastocyst in
which the cells can be divided into two distinct regions; the inner
cell mass, which will form the embryo, and the trophectoderm, which
will form extra-embryonic tissue, such as the placenta.
[0003] The cells that form part of the embryo up until the
formation of the blastocyst are totipotent; in other words, each of
the cells has the ability to give rise to a complete individual
embryo, and to all the extra-embryonic tissues required for its
development. After blastocyst formation, the cells of the inner
cell mass are no longer totipotent, but are pluripotent, in that
they can give rise to a range of different tissues. A known marker
for such cells is the expression of the enzyme alkaline phosphatase
and Oct4.
[0004] Primordial germ cells (PGCs) are pluripotent cells that have
the ability to differentiate into all three primary germ layers. In
mammals, the PGCs migrate from the base of the allantois, through
the hindgut epithelium and dorsal mesentery, to colonise the
gonadal anlague. The PGC-derived cells have a characteristically
low cytoplasm/nucleus ratio, usually with prominent nucleoli. PGCs
may be isolated from the embryos by removing the genital ridge of
the embryo, dissociating the PGCs from the gonadal anlague, and
collecting the PGCs. The earliest PGC population is reported to
consist of a cluster of some 45 (forty-five) alkaline phosphatase
positive cells, found at the base of the emerging allantois, 7.25
days post-fertilisation (Ginsburg et al., (1990) Development
110:521-528).
[0005] PGCs have many applications in modern biotechnology and
molecular biology. They are useful in the production of transgenic
animals, where embryonic germ (EG) cells derived from PGCs may be
used in much the same manner as embryonic stem (ES) cells (Labosky
et al., (1994) Development 120:3197-3204). Moreover, they are
useful in the study of foetal development and the provision of
pluripotent stem cells for tissue regeneration in the therapy of
degenerative diseases and repopulation of damaged tissue following
trauma. Above all, PGCs while having some specialised properties,
retain an underlying pluripotency, which is lost from the
neighbouring cells that surround the founder population of PGCs
that acquire a somatic cell fate. PGCs and the surrounding somatic
cells share a common ancestry. However, the founder PGCs are few in
number and difficult to isolate from embryonic tissue and the
surrounding somatic cells, which complicates their study and the
development of techniques which make use thereof.
[0006] Little is known in the art about the expression of genes in
the founder population of PGCs and the relationship between
PGC-specific gene expression and the retention of pluripotency in
these cells. Certain markers for PGCs are known--for example, the
expression of tissue non-specific alkaline phosphatase (TNAP) has
been used as a marker for early PGCs (Ginsburg et al., (1990)
Development 110:521-528). Oct4 is known to be expressed in PGCs,
but not somatic cells (Yoem et al., (1996) Development
122:881-894). Other markers, such as BMP4, are known to be
expressed primarily in somatic tissues (Lawson et al., (1999) Genes
& Dev. 13:424-436). However, none of these genes is specific
for PGCs, since they are also expressed in other tissue types.
There is therefore a need in the art for the identification of
genes which may be used as markers for PGCs and which may provide
an insight into the biology of germ cell development and the nature
of the pluripotent state.
[0007] Our unpublished International Patent Application Number
PCT/GB02/00215, the contents of which are hereby incorporated by
reference, discloses sequences and uses of two rodent genes, Stella
and Fragilis. That document shows that Stella and Fragilis are
expressed by PGCs and other pluripotent cells. The sequences of
mouse Stella and Fragilis are also set out here as SEQ ID NO: 2 and
SEQ ID NO: 1 respectively.
SUMMARY
[0008] We now disclose human sequences of Stella and Fragilis, as
well as sequences of novel genes Fragilis 9-27, Fragilis 2,
Fragilis 3, Fragilis 4, Fragilis 5 and Fragilis 6 comprising
members of the Fragilis family of genes. We also disclose sequences
of human Fragilis homologues, Ifitm 1, Ifitm 2, Ifitm 3 and Human
ENSG142056. The genes and polypeptides described here, including
associated products such as antibodies, are useful in a number of
ways, for example, for diagnosis, treatment and/or prevention of
diseases such as cancer.
[0009] According to a first aspect of the present invention, we
provide a polypeptide comprising a human Stella amino acid
sequence, preferably a sequence as shown in SEQ ID NO: 4, or a
fragment, homologue, variant or derivative thereof.
[0010] There is provided, according to a second aspect of the
present invention, a polypeptide comprising a human Fragilis amino
acid sequence, preferably a sequence as shown in SEQ ID NO: 6, or a
fragment, homologue, variant or derivative thereof.
[0011] We provide, according to a third aspect of the present
invention, a polypeptide comprising an amino acid sequence selected
from the group consisting of: Fragilis 9-27 (preferably comprising
a sequence as shown in SEQ ID NO: 8), Fragilis 2 (preferably
comprising a sequence as shown in SEQ ID NO: 10), Fragilis 3
(preferably comprising a sequence as shown in SEQ ID NO: 12),
Fragilis 4 (preferably comprising a sequence as shown in SEQ ID NO:
14), Fragilis 5 (preferably comprising a sequence as shown in SEQ
ID NO: 16) and Fragilis 6 (preferably comprising a sequence as
shown in SEQ ID NO: 18), or a fragment, homologue, variant or
derivative thereof.
[0012] As a fourth aspect of the present invention, there is
provided a polypeptide consisting essentially of a sequence
selected from the group consisting of: (a) the sequence of human
Stella shown in SEQ ID NO: 4; (b) the sequence of human Fragilis
shown in SEQ ID NO: 6; (c) the sequence of human Fragilis 9-27
shown in SEQ ID NO: 8; (d) the sequence of mouse Fragilis 2 shown
in SEQ ID NO: 10; (e) the sequence of mouse Fragilis 3 shown in SEQ
ID NO: 12; (f) the sequence of mouse Fragilis 4 shown in SEQ ID NO:
14; (g) the sequence of mouse Fragilis 5 shown in SEQ ID NO: 16;
and (h) the sequence of mouse Fragilis 6 shown in SEQ ID NO:
18.
[0013] We provide, according to a fifth aspect of the present
invention, a polypeptide which has at least 50%, 60%, 70%, 80%, 90%
or 95% homology to a polypeptide according to any of the preceding
aspects.
[0014] The present invention, in a sixth aspect, provides a
polypeptide comprising a sequence of 15 or fewer contiguous amino
acids, preferably a sequence of fewer than 10 contiguous amino
acids, of a nucleic acid according to any of the preceding
aspects.
[0015] In a seventh aspect of the present invention, there is
provided a nucleic acid encoding a polypeptide according to any
preceding aspect.
[0016] According to an eighth aspect of the present invention, we
provide a nucleic acid sequence comprising a human Stella nucleic
acid sequence, preferably a sequence as shown in SEQ ID NO: 3, or a
fragment, homologue, variant or derivative thereof.
[0017] We provide, according to a ninth aspect of the invention, a
nucleic acid sequence comprising a human Fragilis nucleic acid
sequence, preferably a sequence as shown in SEQ ID NO: 5, or a
fragment, homologue, variant or derivative thereof.
[0018] There is provided, in accordance with a tenth aspect of the
present invention, a nucleic acid sequence comprising an Fragilis
nucleic acid sequence acid sequence selected from the group
consisting of: Fragilis 9-27 (preferably a sequence as shown in SEQ
ID NO: 7), Fragilis 2 (preferably a sequence as shown in SEQ ID NO:
9), Fragilis 3 (preferably a sequence as shown in SEQ ID NO: 11),
Fragilis 4 (preferably a sequence as shown in SEQ ID NO: 13),
Fragilis 5 (preferably a sequence as shown in SEQ ID NO: 15) and
Fragilis 6 (preferably a sequence as shown in SEQ ID NO: 17), or a
fragment, homologue, variant or derivative thereof.
[0019] As an eleventh aspect of the invention, we provide a nucleic
acid sequence consisting essentially of a sequence selected from
the group consisting of: (a) the sequence of human Stella shown in
SEQ ID NO: 3; (b) the sequence of human Fragilis shown in SEQ ID
NO: 5; (c) the sequence of human Fragilis 9-27 shown in SEQ ID NO:
7; (d) the sequence of mouse Fragilis 2 shown in SEQ ID NO: 9; (e)
the sequence of mouse Fragilis 3 shown in SEQ ID NO: 11; (f) the
sequence of mouse Fragilis 4 shown in SEQ ID NO: 13; (g) the
sequence of mouse Fragilis 5 shown in SEQ ID NO: 15; and (h) the
sequence of mouse Fragilis 6 shown in SEQ ID NO: 17.
[0020] We provide, according to a twelfth aspect of the invention,
there is provided a nucleic acid sequence which has at least 50%,
60%, 70%, 80%, 90% or 95% homology to a nucleic acid sequence as
disclosed.
[0021] According to a thirteenth aspect of the present invention,
we provide a nucleic acid comprising a sequence of 25 or fewer
contiguous nucleotides, preferably a sequence of 15 contiguous
nucleotides, of a nucleic acid as disclosed.
[0022] There is provided, according to a fourteenth aspect of the
present invention, a complement of a nucleic acid sequence as
disclosed.
[0023] We provide, according to a fifteenth aspect of the present
invention, a nucleic acid comprising a nucleic acid sequence as
disclosed, together with one or more nucleotide substitutions, in
which the one or more substitutions do not alter the coding
specificity of said nucleic acid as a result of the degeneracy of
the genetic code.
[0024] According to a sixteenth aspect of the present invention, we
provide a vector comprising such a nucleic acid sequence.
[0025] According to a seventeenth aspect of the present invention,
we provide a host cell comprising a nucleic acid sequence as
disclosed.
[0026] There is provided, according to a eighteenth aspect of the
present invention, a method of producing a polypeptide as
disclosed, the method comprising providing a nucleic acid as
disclosed, a vector as disclosed, or a host cell as disclosed, and
enabling the expression of a nucleic acid encoding the
polypeptide.
[0027] We provide, according to a nineteenth aspect of the
invention, a method for identifying a pluripotent cell, comprising
detecting such a polypeptide, or such a nucleic acid in the
cell.
[0028] Preferably, the method comprises the steps of amplifying
nucleic acids from a putative pluripotent cell using 5' and 3'
primers specific for Stella and/or Fragilis, and detecting
amplified nucleic acid thus produced. Preferably, the nucleic acid
sequence is detected by in situ hybridisation. Preferably, the
nucleic acid sequence is detected by detecting a protein product
encoded by the nucleic acid. Preferably, the polypeptide is
detected by immunostaining.
[0029] There is provided, according to a twentieth aspect of the
present invention, an antibody specific for a polypeptide as
disclosed. Preferably, the antibody is capable of specifically
binding to an extracellular domain of Fragilis.
[0030] We provide, according to a twenty-first aspect of the
present invention, use of such an antibody for the identification
and/or isolation of a pluripotent cell.
[0031] As a twenty-second aspect of the present invention, there is
provided pluripotent cell identified by a method as disclosed.
[0032] We provide, according to a twenty-third aspect of the
present invention, a method of treatment or prophylaxis of a
disease in an individual, the method comprising modulating the
expression and/or the activity of Stella and/or Fragilis in a cell
of the individual.
[0033] Preferably, the method comprises up-regulating the
expression and/or the activity of Stella and/or Fragilis.
[0034] The present invention, in a twenty-fourth aspect, provides a
Stella or Fragilis polypeptide or nucleic acid, or an antibody
against Stella or Fragilis, for use in a method of treatment or
prophylaxis of a disease in an individual.
[0035] In a twenty-fifth aspect of the present invention, there is
provided use of a Stella or Fragilis polypeptide or nucleic acid,
or an antibody against Stella or Fragilis, for the preparation of a
pharmaceutical composition for the treatment of a disease.
[0036] According to an twenty-sixth aspect of the present
invention, we provide a method of diagnosis of a disease, the
method comprising detecting a modulation of expression and/or
activity of Stella and/or Fragilis in a cell of an individual.
[0037] In preferred embodiments, the disease is selected from the
group consisting of: testis tumor, colon tumor, stomach, germ cell
tumors, choriocarcinoma, lung, large cell carcinoma, uterus, and
leiomyosarcoma Stella is therefore suitable for treatment or
diagnosis of any disease of or associated with such tissues, in
particular, cancer or tumours of such tissues.
[0038] We provide, according to a twenty-seventh aspect of the
invention, a method of identifying a molecule capable of binding to
Stella or Fragilis, the method comprising the steps of: (a)
providing a Stella or Fragilis polypeptide; (b) contacting the
Stella or Fragilis polypeptide with a candidate molecule; and (c)
detecting binding between the candidate molecule and the Stella or
Fragilis polypeptide, as the case may be.
[0039] There is provided, according to a twenty-eighth aspect of
the present invention, a method of identifying an agonist or
antagonist of Stella or Fragilis, the method comprising the steps
of: (a) providing a Stella or Fragilis polypeptide; (b) contacting
the Stella or Fragilis polypeptide with a candidate molecule; and
(c) detecting modulation of an activity of the Stella or Fragilis
polypeptide.
[0040] Preferably, the polypeptide is contacted with a plurality of
candidate molecules, preferably in the form of a library.
[0041] We provide, according to a twenty-ninth aspect of the
present invention, use of an antagonist of Stella or Fragilis,
preferably as identified by a method according to any of the
previous aspects, in a method of treatment or prophylaxis of a
disease in an individual.
BRIEF DESCRIPTION OF THE FIGURES
[0042] FIG. 1: Nucleotide and deduced amino acid sequence of mouse
Fragilis (SEQ ID NO: 1). Predicted positions of the two
transmembrane domains (TM I and TM II) are underlined and indicated
by bold letters. The poly(A) signal is underlined.
[0043] FIG. 2: Nucleotide and deduced amino acid sequence of mouse
Stella (SEQ ID NO: 2). Three nuclear localization signals are
underlined. A potential nuclear export signal is underlined twice,
and the hydrophobic residues are indicated in bold. Helical
structures in a motif with similarity to SAP domain (a.a.28 to
a.a.63) are underlined in red, and the conserved residues are
indicated by blue. A splicing factor-like motif is underlined and
the conserved residues are indicated in green. Poly(A) signals are
also underlined.
[0044] FIG. 3: Expression of mouse Fragilis in embryonic stem (ES)
cells. ES cells are fixed in 4% paraformaldehyde in PBS for 10 min.
at room temperature and processed for immunohistochemistry as
described by Saitou et al., (1998). J Cell Biol 141, 397-408.
(1998). Fragilis expression is similarly detected in E6.5 proximal
epiblast cells, which are germ cell competent cells, and in newly
specified germ cells. The expression declines after E8.5 following
completion of the specification of germ cells fate.
[0045] FIG. 4: Expression of mouse Stella in PGCs. PGCs from E12.5
genital ridges are fixed in 4% paraformaldehyde in PBS for 10 min.
at room temperature and processed for immunohistochemistry as
described by Saitou et al., (1998). J Cell Biol 141, 397-408.
(1998). Stella is detected in PGCs from E 7.25-13.5, as well as in
pluripotent ES cells and in EG cells. Stella is also detected in
the totipotent oocyte, zygote and in the totipotent and pluripotent
blastomeres during preimplantation development and in developing
gametes. When EG cells are derived from PGCs (Labosky et al.,
(1994) Development 120:3197-3204). Fragilis expression is again
detected in the pluripotent EG cells as it is in ES cells.
Therefore, Fragilis and Stella are also markers for the pluripotent
stem cells FIG. 5. Mouse Fragilis expression by whole-mount in situ
hybridization in E7.2 mouse embryos.
[0046] FIG. 6. Mouse Stella expression by whole mount in situ
hybridisation in E 7.2 mouse embryos.
[0047] FIG. 7. Mouse Stella expression in PGCs in the process of
migration into the gonads in E9.0 embryos.
[0048] FIGS. 8a and 8b. Expression of mouse Fragilis and mouse
Stella in single cells detected by PCR analysis of single cell
cDNAs. Numbers marked by symbol * in 8b are the PGCs. Note that
there are more single cells showing expression of Fragilis compared
to those showing expression of Stella. Only cells with the highest
levels of Fragilis expression were found to express Stella and
acquire the germ cell fate. Cells that express Stella were found
not to show expression of Hoxb1. Cells that express lower levels of
Fragilis and no Stella become somatic cells and showed expression
of Hoxb1. The founder population of PGCs also show high levels of
Tnap. Both the founder PGCs and the somatic cells show expression
of Oct4, T(Brachyury), and Fgf8.
[0049] FIG. 9. The Fragilis family cluster on mouse Chr7, and the
human homologues in the syntenic region on Chr11. In the mouse, the
five Fragilis genes are clustered within a 70 kb region. All genes
are encoded by two exons, and apart from fragilis2, they are
located on the minus strand. In human, the four homologous genes,
ENSG142056 and Ifitm1 (9-27), Ifitm2 (1-8D) and Ifitm3 (1-8U), are
clustered within a 25 kb stretch. The four human homologues are
each encoded by two exons, but the length of the intronic sequence
for Ifitm1 and Ifitm3 is not known. Apart Ifitm2, all human genes
are encoded on the minus strand. The green circles represent ISRE
consensus sequences.
[0050] FIG. 10. Protein alignment of the Fragilis family and their
homologues in human, cow and rat. Green bars indicate the location
of the two predicted transmembrane domains, of which the first as
well as the inter-domain stretch appear to be highly conserved
throughout the four mammalian species. Identical amino acids are
highlighted in dark grey, similar amino acids in light grey. The
alignment was done using ClustlW.
[0051] FIG. 11. Expression analysis of fragilis (a-f), fragilis2
(g-l) and fragilis3 (m-r) by whole mount in situ hybridisation.
Pictures are taken as lateral view unless otherwise stated, with
anterior to the left and posterior to the right. fragilis is
expressed throughout the epiblast in E5.5 embryos (a) and in the
region of germ cell specification at the base of the incipient and
early allantoic bud at E7.5 (b, b' posterior view, c). At E8.5,
signal is detected at the base and in the proximal third of the
allantois as well as in the latero-anterior aspects of the brain (d
superior view, e anterior view). At E9.5, fragilis appears
expressed in a population of cells at the beginning of the
invaginating hindgut (arrow in f), as well as in the pharyngeal
arches (f). fragilis2 is detected throughout the epiblast at E5.5
(g). Expression seems thereafter downregulated but becomes again
detectable in the posterior mesoderm and at the base of the
incipient and growing allantoic bud in E7.0 and E7.5 embryos (h, i,
i' posterior view). At E8.5, expression is seen in caudal mesoderm
(j, k posterior view), while at E9.5 expression is seen in the
tailbud, the mesoderm caudal to the 12.sup.th somite and the lung
primordia (arrow, l). fragilis3 is expressed throughout the
epiblast at E6.5 (m) and around E7.5 additionally in the region of
PGC specification (n, n' posterior view, o). At E8.5, fragilis2
expression is seen throughout the embryo, with exception of the
developing heart, and appears intense in single cells (arrow in q
posterior view) at the base and within the proximal region of the
allantois (p posterior view, q, r). asterix: allantois; black
arrowhead: allantoic bud; white arrowhead: developing heart; scale
bars: 100 .mu.m (a, b, g-i, m, n); 200 .mu.m (c-e, o-q); 400 .mu.m
(f, j-l, r).
[0052] FIG. 12. Expression analysis of fragilis2 by in situ
hybridisation on sections. (a-d) transverse sections through the
caudal region of an embryo at E9.5 (approx. 25 somites) at
progressively rostral levels. At most caudal levels, fragilis2
expression is seen in cells of the neural tube, in the presomitic
mesoderm, in single cells within the hindgut (arrowhead) and in the
body wall. (b) staining at approx. 23rd somite level is present
within the forming somite, the body wall mesoderm and cells within
the hindgut as well as the floorplate. (c) at approx. 21st somite
level, expression in the differentiating somites is reduced, while
cells in the floor plate and within the hindgut remain fragilis2
mRNA positive. (d) at approx. the 13th somite level, fragilis2
expression is absent from the somatic mesoderm as well as the
neural tube. (e) sagittal section through an E10.5 embryo shows
fragilis2 expression in developing lung tissue (asterix; higher
magnification in f) and migrating cells along the hindgut anterior
to the dorsal aorta (arrow). (g) shows a magnified view of
fragilis2 mRNA expressing, migrating cells. da: dorsal aorta; fp:
floor plate; g: gut; h: developing heart; nt: neural tube; s:
somite; bw: body wall; scale bars: 150 .mu.m (a-d); 1 mm (e); 400
.mu.m (f, g).
[0053] FIG. 13. Expression analysis of the Fragilis family genes in
single cells from the region of germ cell specification of E7.5
embryos. (a) shows PCR analysis of cDNAs from three nascent, stella
positive PGCs and three surrounding, stella negative somatic cells.
Note that fragilis, fragilis2 and fragilis3 are expressed in PGCs
and somatic cells, while fragilis4 and fragilis5 are not detected
in any of the cells. (b) shows expression of fragilis, fragilis2
and fragilis3 in single cell eDNAs using Southernblot analysis.
GAPDH was used as blotting control. (c) Semi-quantitative
expression analysis of the Southernblot data shows that all three
Fragilis genes are predominantly expressed in nascent PGCs compared
to the somatic cells within the region.
[0054] FIG. 14. Expression analysis of fragilis, fragilis2 and
fragilis3 at E11.5/E12.5 in single cells from the genital ridge and
by in situ hybridisation. (a) shows PCR analysis of cDNAs from
three gonadal stella-positive germ cells and three surrounding,
stella-negative somatic cells. While fragilis is detected only in
the three germ cell clones, fragilis2 and fragilis3 are expressed
in the germ cells as well as the somatic cells. (b) shows in situ
hybridisation of urogenital ridges of E11.5/E12.5 embryos. While
fragilis3 is expressed in the mesonephros as well as the genital
ridge, fragilis and fragilis2 are restricted to the genital ridge.
The staining pattern for fragilis appears punctuate and restricted
to single cells mimicking the pattern seen for the germ
cell-specific stella gene. asterix: genital ridge; black arrowhead:
mesonephros; scale bars: 400 .mu.m.
[0055] FIG. 15. Stella expression during preimplantation
development and evolutionary conservation. a-l, Confocal sections
of anti-stella (a,d,g,j) and propidium iodide (b,e,h,k) stained
embryos (c,f,i,l merged images). Maternal stella is stored in the
unfertilised egg (a-c) (arrow, exclusion of Stella from condensed
metaphase chromosomes) and localizes both to the cytoplasm and
pronuclei (PN) after fertilisation (d-f; PB, polar body). Also
during later stages (2-cell, g-i; 4-cell, j-l) it can be seen both
in the cytoplasm and the nucleus. Scale bar=20 .mu.m. Synteny (m)
of the stella gene in mouse, rat and human and close up view (n) of
stella and its neighbouring genes in mouse and human. Arrows
indicate the direction of transcription. a, Alignment of Stella
protein sequences. Identical amino acids have a black background
and similar amino acids a grey one. Putative nuclear export and
localisation signals are marked by red and black lines,
respectively. The red stars indicate conserved hydrophobic amino
acids, which are typical for nuclear export signals.sup.27. p,
RT-PCR analysis of STELLA-expression in human pluripotent cells and
reproductive organs. RPL32 was used as control. ES, embryonic stem
cells; EC, embryonic carcinoma-cells (nTera2); tet, testis tumor;
te, normal testis; ov, normal ovary; -Rt, without reverse
transcriptase; 0, water control.
[0056] FIG. 16. Knockout strategy of stella and confirmation of
correct targeting by Southern-blot and RT-PCR. a, The targeting
vector was designed to delete exon 2 and replace it with an
IRES-LacZ/MC-neo reporter-selection cassette. HSV-TK was used for
negative selection against non-homologous recombination. 5', 3' and
neo-probes were used to confirm correct targeting of ES-cells. b,
Southern blot analysis of genomic DNA derived from littermate mice
born from a stella.sup.+/- intercross. The example shows a NcoI
digest hybridised with the 3' probe, indicating the absence of the
wild-type allele in stella.sup.-/- mice. c, RT-PCR of testis (te)
or ovary (ov) RNA from male or female mice, respectively using exon
2-specific primers. The wild-type stella transcript is reduced in
stella.sup.+/- mice compared to stella.sup.+/+ mice and absent in
stella mice. Gapdh was used as a control for equivalent quality and
amount of RNA. -Rt, without reverse transcriptase; 0, water
control.
[0057] FIG. 17. Germ cell development in stella knockout mice. a,
Numbers of PGCs in wild-type (wt, n=9), stella.sup.+/- (n=14) and
stella.sup.-/- (n=7) embryos are not significantly different at
E8.5 (0-8 somites). The results are presented as means.+-.SEM. b-g,
Gonadal PGCs (E11.5) stained with anti-stella (b, e) and anti-SSEA1
(c, f) antibodies (d, g merge including Toto3 (blue) as DNA stain).
The PGC-marker SSEA1.sup.17 is coexpressed with stella in wild-type
PGCs (b-d) and also detectable in stella.sup.-/- animals (e-g),
showing that PGCs are present in knockout mice. Scale bar=10 .mu.m.
Sections of testes (h-j) and ovaries (k-m) of adult wild-type (h,
k), stella.sup.+/- (i, l) and stella.sup.-/- (j, m) mice Knockout
males show normal development of sperm (arrowheads) and knockout
females normal ovary morphology with follicles containing oocytes
of different stages (arrows). Scale bars in j (for h-j), m (for
k-m)=100 .mu.m.
[0058] FIG. 18. Maternal effect of the stella knockout and onset of
paternal expression of stella during preimplantation development.
a, 80% of matings with wild-type males resulted in pregnancies of
wild-type females, while in only 24% of the plugs stella.sup.-/-
females became pregnant. b, From these pregnancies, the littersize
was strongly reduced in knockouts compared to wild-type females.
c-i A stella-GFP reporter construct (c) was used to determine, when
the paternal allele of stella starts to be expressed. Zygotic
expression of the stella-GFP transgene begins at the 2-cell stage
(E1.5; e, h) and continues during later stages (E2.5, 4-8 cell; f,
i). d-f, GFP-fluorescence; g-i, brightfield merged with GFP-image;
arrowheads, non-transgenic embryos; arrows, transgenic embryos.
Scale bar in d (for d-i)=100 .mu.m. j-l, Confocal section through a
morula (E3.5) derived from a mating of a wild-type male with a
stella.sup.-/- female stained with anti-stella antibody (j) and
propidium iodide (k) (l, merge). Stella protein is made from the
paternal allele, but not sufficient to rescue the observed
phenotype. Scale bar in l (for j-l)=20 .mu.m
[0059] FIG. 19. Preimplantation development is perturbed without
Stella. a, The percentage of embryos developing in vivo to the
various stages are given for stella.sup.-/- (white bars) and
wild-type or stella.sup.+/- (black bars) mothers, respectively.
Total numbers of embryos examined at each timepoint are given in
parentheses. Development of knockout-derived embryos starts to be
affected from E1.5 onwards (2-cell stage) and only a low percentage
reach the blastocyst stage by E3.5 (b) compared to
wild-type-derived embryos (c). d-f, Distribution of stages of
embryos cultured in vitro from E1.5 until E4.5 (timepoint of
implantation). Similar as in vivo, most embryos from wild-type
mothers (black bars) develop to blastocysts (f), while many embryos
of stella knockout mothers (white bars) are delayed or show
abnormal morphology (e). Total number of embryos examined in d:
-/-mothers: 41, wt or +/-mothers: 36. Scale bar=100 .mu.m.
SEQUENCE LISTINGS
[0060] SEQ ID NO: 1 shows the nucleic acid sequence of mouse
Fragilis while SEQ ID NO: 2 shows the nucleic acid sequence of
mouse Stella.
[0061] SEQ ID NO: 3 shows the nucleic acid sequence of human
Stella; SEQ ID NO: 4 shows the amino acid sequence of human Stella;
SEQ ID NO: 5 shows the nucleic acid sequence of human Fragilis
1-8D; SEQ ID NO: 6 shows the amino acid sequence of human Fragilis
1-8D; SEQ ID NO: 7 shows the nucleic acid sequence of human
Fragilis 9-27/Leu13; SEQ ID NO: 8 shows the amino acid sequence of
human Fragilis 9-27/Leu13; SEQ ID NO: 9 shows the nucleic acid
sequence of mouse Fragilis 2; SEQ ID NO: 10 shows the amino acid
sequence of mouse Fragilis 2; SEQ ID NO: 11 shows the nucleic acid
sequence of mouse Fragilis 3; SEQ ID NO: 12 shows the amino acid
sequence of mouse Fragilis 3; SEQ ID NO: 13 shows the nucleic acid
sequence of mouse Fragilis 4; SEQ ID NO: 14 shows the amino acid
sequence of mouse Fragilis 4; SEQ ID NO: 15 shows the nucleic acid
sequence of mouse Fragilis 5; SEQ ID NO: 16 shows the amino acid
sequence of mouse Fragilis 5; SEQ ID NO: 17 shows the nucleic acid
sequence of mouse Fragilis 6; SEQ ID NO: 18 shows the amino acid
sequence of mouse Fragilis 6.
DETAILED DESCRIPTION
[0062] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of chemistry,
molecular biology, microbiology, recombinant DNA and immunology,
which are within the capabilities of a person of ordinary skill in
the art. Such techniques are explained in the literature. See, for
example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989,
Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3,
Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995
and periodic supplements; Current Protocols in Molecular Biology,
ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe,
J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing:
Essential Techniques, John Wiley & Sons; J. M. Polak and James
O'D. McGee, 1990, In Situ Hybridization: Principles and Practice;
Oxford University Press; M. J. Gait (Editor), 1984, Oligonucleotide
Synthesis: A Practical Approach, Irl Press; D. M. J. Lilley and J.
E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A:
Synthesis and Physical Analysis of DNA Methods in Enzymology,
Academic Press; Using Antibodies: A Laboratory Manual: Portable
Protocol NO. I by Edward Harlow, David Lane, Ed Harlow (1999, Cold
Spring Harbor Laboratory Press, ISBN 0-87969-544-7); Antibodies: A
Laboratory Manual by Ed Harlow (Editor), David Lane (Editor) (1988,
Cold Spring Harbor Laboratory Press, ISBN 0-87969-3142), 1855.
Handbook of Drug Screening, edited by Ramakrishna Seethala,
Prabhavathi B. Fernandes (2001, New York, N.Y., Marcel Dekker, ISBN
0-8247-0562-9); and Lab Ref: A Handbook of Recipes, Reagents, and
Other Reference Tools for Use at the Bench, Edited Jane Roskams and
Linda Rodgers, 2002, Cold Spring Harbor Laboratory, ISBN
0-87969-630-3. Each of these general texts is herein incorporated
by reference.
Stella and Fragilis
[0063] The disclosure provides generally for Fragilis and Stella
nucleic acids, polypeptides, as well as fragments, homologues,
variants and derivatives thereof. In particular, we provide for
human Stella and Fragilis nucleic acids, polypeptides, as well as
fragments, etc.
[0064] The names "Fragilis" and "GCR1" should be understood as
synonymous with each other, and likewise, "Stella" and "GCR2"
should be considered synonyms. In general, unless the context
requires otherwise, the term "Fragilis" should be taken to refer to
any member of the Fragilis family, including Fragilis itself,
Fragilis 9-27, Fragilis 2, Fragilis 3, Fragilis 4, Fragilis 5 and
Fragilis 6. In addition, Ifitm1, Ifitm2, Ifitm3 and ENSG142056 are
preferably included within the term "Fragilis". Preferably, the
Stella and Fragilis sequences disclosed are derived from higher
animals, for example primates, in particular, Homo sapiens
(man).
[0065] In preferred embodiments, therefore, Fragilis should be
taken to refer to the human nucleic acid sequence shown in SEQ ID
NO: 5, or the human amino acid sequence shown in SEQ ID NO: 6, as
the context requires. Furthermore, in preferred embodiments, Stella
should be taken to refer to the human nucleic acid sequence shown
in SEQ ID NO: 3, or the human amino acid sequence shown in SEQ ID
NO: 4, as the context requires.
[0066] Human Stella and Fragilis are related by homology and
function to the mouse Stella and Fragilis sequences disclosed in
PCT/GB02/00215, and also set out as SEQ ID NO: 1 and SEQ ID NO: 2
in this document. Fragilis and Stella are PGC-specific transcripts.
Fragilis is upregulated during the process of lineage commitment of
PGCs, while Stella is upregulated after Fragilis, and marks
commitment to the PGC fate.
[0067] Human Fragilis (also known as Germ cell restricted-1, GCR1),
encodes a 132 amino acid protein with a predicted molecular weight
of 14.5 kD. A best fit model of the EMBL program PredictProtein
predicts two transmembrane domains, both N and C terminus ends
being located outside. A BLASTP search revealed that Fragilis is a
novel member of the interferon-inducible protein family. One
prototype member, human 9-27 (identical to Leu-13 antigen), is
inducible by interferon in leukocytes and endothelial cells, and is
located at the cell surface as a component of a multimeric complex
involved in the transduction of antiproliferative and homotypic
adhesion signals (Deblandre, 1995). The BLASTN search revealed that
the Fragilis sequence was found in ESTs derived from many different
tissues both from embryos and adults, indicating that Fragilis may
play a common role in different developmental and cell biological
contexts, including cancer; this is described in further detail
below. Database searches reveal a sequence match with the rat
interferon-inducible protein (sp:INIB RAT, pir:JC1241) with unknown
function. The Fragilis sequence appears several times in our
screen, indicating high level expression in PGCs.
[0068] The second gene, human Stella, (Stella) encodes a 159 amino
acid protein, of 17.5 kD. It has no sequence homology with any
known protein, apart from murine and rodent Stella, contains
several nuclear localisation consensus sequences and is highly
basic pI, indicating a possible affinity to DNA. Furthermore a
potential nuclear export signal is identified, indicating that
Stella may shuttle between the nucleus and the cytoplasm. BLASTN
analysis reveals that the Stella sequence is found only in the
preimplantation embryo and germ line (newborn ovary, female 12.5
mesonephros and gonad etc.) ESTs indicating its predominant
expression in totipotent and pluripotent cells. Interestingly, we
found that Stella contains in its N terminus a modular domain which
has some sequence similarity with the SAP motif. This motif is a
putative DNA-binding domain involved in chromosomal organisation.
Furthermore, the SMART program reveals the presence of a splicing
factor motif-like structure in its C-terminus, These findings
indicate a possible involvement of Stella in chromosomal
organisation and RNA processing.
[0069] Antibodies may be raised against the Fragilis and/or Stella
polypeptides. In particular, antibodies may be raised against the
extracellular domain of Fragilis, which is a transmembrane
polypeptide.
[0070] Antibodies, polypeptides and nucleic acids disclosed here
are useful for the identification of PGCs in cell populations. The
methods and compositions described here therefore provide a means
to isolate PGCs, useful for example for the study of germ tissue
development and the generation of transgenic animals, and PGCs when
isolated by a method described here.
[0071] Homologues of Fragilis and Stella may also be used to
identify PGCs and other pluripotent cells, such as ES or EG
cells.
[0072] Furthermore, the methods and compositions described here are
useful for treating or preventing cancer, in view of the role of
Stella and Fragilis in development and cell fate control.
Human Stella and Fragilis
[0073] The murine Stella and Fragilis genes are isolated by the
methods shown in the Examples; these are disclosed in detail in our
International Application PCT/GB02/00215. The murine Fragilis
sequence isolated is shown as SEQ ID NO: 1, while the mouse Stella
sequence is shown as SEQ ID NO: 2.
[0074] Human homologues of Stella and Fragilis are described in
further detail in the following sections, and may (if necessary) be
identified and cloned by standard techniques.
Human Stella
[0075] Human Stella Nucleic Acid Sequence
TABLE-US-00001 AGCAATTTGAGGCTCTGTCATCAGTTTCTGCTACGTTTCAAAGATCCTGG
AGAAGCCTAGTGTTGTGTCAAGACGCCGATGGACCCATCACAGTTTAATC
CAACCTACATCCCAGGGTCTCCACAAATGCTCACCGAAGAAAATTCCCGG
GACGATTCAGGGGCCTCTCAAATCTCCTCCGAGACGTTGATAAAGAACCT
TAGTAACTTGACTATCAACGCTAGTAGCGAATCTGTTTCCCCTCTATCGG
AAGCTTTACTCCGTCGAGAGTCTGTAGGAGCAGCAGTCCTCAGGGAAATC
GAAGATGAGTGGCTTTACAGCAGGAGAGGAGTAAGAACATTGCTGTCTGT
GCAGAGAGAAAAGATGGCAAGATTGAGATACATGTTACTCGGCGGAGTTC
GTACGCATGAAAGAAGACCAACAAACAAGGAGCCTAAGGGAGTTAAGAAG
GAATCAAGACCATTCAAATGTCCCTGCAGTTTCTGCGTGTCTAATGGATG
GGATCCTTCTGAGAATGCTAGAATAGGGAATCAAGACACCAAGCCACTTC
AGCCATAAATCTTATTCTTGCACCTTTTTTTCTTGGTAGTAATTTTATAT
AGCAGGTTGAGAAAGCTACTCTATGCTAGTATAGACTATACACCAATAAT
TTTGATAATGAGTTCTAGGATGTATTTTTCTTGTATCTTTTTCTTCCTAC
TATGATACTAGTAATTCATAAGGGATCTGTGTAATCTGAATGTATTTGAA
AACTTTAGCTCTACTGTTTGATTTGACCCAAAGAAGCCAAGATGATATAA
TGTATTCCCATGTGTCTTAGAAGCCCAAAGTCAGTGAGATGAAACCCAAC
ATCAAGAAATTGAAGCAAAGTTACTTATGGATAAAGAAAGCATTAGGTAG
TTGGGCTATAGCATAATTAGATTTTCTGGCTTTCAAAAATTTGGATTGCA
ATCACAGCAAACTTTGTTATTTTTACAGTTTTCAGTACAAAAGTGTTTAT
ATAGAAACAATAAAGTTGACATTTGAGTACCTTTTAAAAA
[0076] Human Stella Amino Acid Sequence
TABLE-US-00002 MDPSQFNPTYIPGSPQMLTEENSRDDSGASQISSETLIKNLSNLTINASS
ESVSPLSEALLRRESVGAAVLREIEDEWLYSRRGVRTLLSVQREKMARLR
YMLLGGVRTHERRPTNKEPKGVKKESRPFKCPCSFCVSNGWDPSENARIG NQDTKPLQP
[0077] Human Stella Expression
[0078] Human Stella is expressed in a number of tissues, including:
testis, colon, lung, uterus, and germinal center B cell.
[0079] Human Stella Diseases
[0080] Diseases associated with abnormal expression of human Stella
include: testis tumor, colon tumor, stomach, germ cell tumors,
choriocarcinoma, lung, large cell carcinoma, uterus, and
leiomyosarcoma.
[0081] Human Stella is therefore suitable for treatment or
diagnosis of any disease of or associated with such tissues, in
particular, cancer or tumours of such tissues.
Human Fragilis
[0082] Human Fragilis is also referred to as Fragilis 1-8D.
[0083] Human Fragilis Nucleic Acid Sequence
TABLE-US-00003 TCCCGGTAACCCGATCACCGCTGGTCACCATGAACCACATTGTGCAAACC
TTCTCTCCTGTCAACAGCGGCCAGCCTCCCAACTACGAGATGCTCAAGGA
GGAGCAGGAAGTGGCTATGCTGGGGGTGCCCCACAACCCTGCTCCCCCGA
TGTCCACCGTGATCCACATCCGCAGCGAGACCTCCGTGCCTGACCATGTG
GTCTGGTCCCTGTTCAACACCCTCTTCATGAACACCTGCTGCCTGGGCTT
CATAGCATTCGCGTACTCCGTGAAGTCTAGGGACAGGAAGATGGTTGGCG
ACGTGACCGGGGCCCAGGCCTATGCCTCCACCGCCAAGTGCCTGAACATC
TGGGCCCTGATTTTGGGCATCTTCATGACCATTCTGCTCATCATCATCCC
AGTGTTGGTCGTCCAGGCCCAGCGATAGATCAGGAGGCATCATTGAGGCC
AGGAGCTCTGCCCGTGACCTGTATCCCACGTACTCTATCTTCCATTCCTC
GCCCTGCCCCCAGAGGCCAGGAGCTCTGCCCTTGACCTGTATTCCACTTA
CTCCACCTCCCATTCCTCGCCCTGTCCCCACAGCCGAGTCCTGCATCAGC
CCTTTATCCTCACACGCTTTTCTACAATGGCATTCAATAAAGTGTATATG TTT
[0084] Human Fragilis Amino Acid Sequence
TABLE-US-00004 MNHIVQTFSPVNSGQPPNYEMLKEEQEVAMLGVPHNPAPPMSTVIHIRSE
TSVPDHVVWSLFNTLFMNTCCLGFIAFAYSVKSRDRKMVGDVTGAQAYAS
TAKCLNIWALILGIFMTILLIIIPVLVVQAQR
[0085] Human Fragilis Genomic Location
[0086] Human Fragilis is located on chromosome 11, at chromosomal
location 11p15.5.
[0087] Human Fragilis Diseases
[0088] Human Fragilis 1-8D is expressed in a number of tissues,
including: stomach; adenocarcinoma; adenocarcinoma cell line;
adipose; adrenal cortico adenoma for cushing's syndrome; adrenal
gland; aorta; ascites; bone; bone marrow; brain; breast;
breast_normal; cartilage; cervical carcinoma cell line; colon;
colon_ins; colonic mucosa from 3 patients with crohn's disease;
cord blood; corresponding non cancerous liver tissue; embryonal
carcinoma; embryonal carcinoma, cell line; endometrium,
adenocarcinoma cell line; epitheloid carcinoma; epitheloid
carcinoma cell line; eye; foreskin; four pooled
poorly-differentiated adenocarcinomas; from acute myelogenous
leukemia; from chronic myelogenous leukemia; gall bladder;
head_neck; heart; hepatocellular carcinoma, cell line; human
retina; human skeletal muscle; hypemephroma; hypemephroma, cell
line; hypothalamus, cell line; insulinoma; iris; islets of
langerhans; kidney; kidney_tumor; large cell carcinoma; large cell
carcinoma, undifferentiated; leiomyosarcoma; leiomyosarcoma cell
line; lens; leukocyte; liver, lung; lung focal fibrosis;
lung_normal; lung_tumor; lymph; marrow; metastatic chondrosarcoma;
mixed (pool of 40 mas); mucoepidermoid carcinoma; muscle; muscle
(skeletal); neuroblastoma cells; normal epithelium; normal
head/neck tissue; optic nerve; osteosarcoma, cell line; ovary;
ovary (pool of 3); pancreas; papillary serous ovarian metastasis;
parathyroid; pheochromocytoma; pituitary; placenta;
placenta_normal; pool; pooled brain, lung, testis; pooled colon,
kidney, stomach; pooled lung and spleen; pooled pancreas and
spleen; primary b-cells from tonsils (cell line); primary lung
epithelial cells; primitive neuroectoderm; prostate;
prostate_normal; prostate-tumor; purified pancreatic islet; renal
cell adenocarcinoma; rpe and choroid; serous papillary tumor; skin;
spleen; squamous cell carcinoma, poorly differentiated (4 pooled
tumors, including primary and metastatic); stomach; subchondral
bone; testis; testis_normal; thyroid; tonsil; two pooled squamous
cell carcinomas; uterus; uterus_tumor; whole embryo.
[0089] Human Fragilis is therefore suitable for treatment or
diagnosis of any disease of or associated with such tissues, in
particular, cancer or tumours of such tissues.
Human Fragilis 9-27/Leu13
[0090] A sequence related to human Fragilis, i.e., human Fragilis
9-27 (also known as Leu13) is also identified. This sequence
comprises a member of the Fragilis family of proteins.
[0091] Human Fragilis 9-27/Leu13 Nucleic Acid Sequence
TABLE-US-00005 tgagaaactgaaacgacaggggaaaggAggtctcActgagcaccgtccca
gcatccggacaccacagcggcccttcgctccacgcagaaaaccacacttc
tcaaaccttcActcaacacttccttccccaaagccagaagatgcacaagg
aggaacatgaggtggctgtgctgggggcaccccccagcaccatccttcca
aggtccaccgtgatcaacatccacagcgagacctccgtgcccgaccatgt
cgtctggtccctgttcaacaccctcttcttgaactggtgctgtctgggct
tcatagcattcgcctactccgtgaagtctagggacaggaagatggttggc
gacgtgaccggggcccaggcctatgcctccaccgccaagtgcctgaacat
ctgggccctgattctgggcatccttatgaccattggattcatcctgtcac
tggtattcggctctgtgacagtctaccatattatgttacagataatacag
gaaaaacggggttactagtagccgcccatagcctgcaacctttgcactcc
actgtgcaatgctggccctgcacgctggggctgttgcccctgcccccttg
gtcctgcccctagatacagcagtttatacccacacacctgtctacagtgt
cattcaataaagtgcacgtgcttgtga
[0092] Human Fragilis 9-27/Leu13 Amino Acid Sequence
TABLE-US-00006 MHKEEHEVAVLGAPPSTILPRSTVINIHSETSVPDHVVWSLFNTLFLNWC
CLGFIAFAYSVKSRDRKMVGDVTGAQAYASTAKCLNIWALILGILMTIGF
ILSLVFGSVTVYHTMLQIIQFIKRGY
[0093] Human Fragilis 9-27/Leu13 Genomic Location
[0094] Human Fragilis 9-27/Leu13 is located on chromosome 11.
[0095] Human Fragilis 9-27/Leu13 Diseases
[0096] Human Fragilis 9-27/Leu13 is expressed in a number of
tissues, including: Stomach; adenocarcinoma; adenocarcinoma, cell
line; adipose; adrenal cortico adenoma for cushing's syndrome;
amnion normal; ascites; bone; bone marrow; brain; breast; cervical
carcinoma cell line; chondrosarcoma; colon; colon ins; cord blood;
ear; embryonal carcinoma; epid tumor; epitheloid carcinoma; eye;
eye anterior segment; from acute myelogenous leukemia; from chronic
myelogenous leukemia; germ cell; head neck; head normal; heart;
human retina; hypothalamus, cell line; insulinoma; kidney; kidney
tumor; large cell carcinoma; large cell carcinoma,
undifferentiated; leiomios; leiomyosarcoma; leukocyte; liver; lung;
lung normal; lymph; marrow; mixed (pool of 40 mas); mucoepidermoid
carcinoma; muscle; muscle (skeletal); nervous tumor; normal
head/neck tissue; nose; optic nerve; osteoarthritic cartilage;
ovary; pancreas; papillary serous ovarian metastasis; parathyroid;
pheochromocytoma; pituitary; placenta; placenta normal; pool;
pooled; pooled brain, lung, testis; pooled pancreas and spleen;
primary b-cells from tonsils (cell line); primitive neuroectoderm;
prostate; prostate normal; purified pancreatic islet; retinal
pigment epithelium sheets; rpe and choroid; skin; spleen; stomach;
testis, cell line; testis normal; tonsil; trabecular meshwork;
uterus; uterus tumor; whole embryo.
[0097] Human Fragilis 9-27/Leu13 is therefore suitable for
treatment or diagnosis of any disease of or associated with such
tissues, in particular, cancer or tumours of such tissues.
Mouse Fragilis 2
[0098] Furthermore, we have identified several mouse sequences
(namely, Fragilis 2, Fragilis 3, Fragilis 4, Fragilis 5 and
Fragilis 6) which are related to Fragilis and comprise Fragilis
family members.
[0099] Mouse Fragilis 2 Nucleic Acid Sequence
TABLE-US-00007 GCGGGTCTACAGAACCAGGATAGCAGCAGCCATCCTCCAGACGGGGCGAT
TGTTCCAGAGTCAGTACCATGAGCCACAATTCTCAAGCCTTCTTGTCCAC
CAATGCCGGGCTTCCTCCAAGCTATGAGACAATCAAAGAGGAGTACGGGG
TGACTGAGCTGGGGGAACCCAGCAACTCAGCTGTTGTGAGGACCACCGTG
ATCAACATGCCCAGAGAGGTGTCGGTGCCTGACCATGTGGTCTGGTCCCT
GTTCAATACACTCTTCTTCAACGCCTGCTGCCTGGGCTTCGTTGCCTATG
CCTACTCTGTGAAGTCTAGGGACAGGAAGATGGTGGGCGATGTGGTTGGA
GCCCAGGCCTACGCCTCCACTGCCAAGTGCCTGAATATCAGCTCCCTGAT
CTTCAGCATCCTTATGGTCATTATCTGCATCATTATTTTCTCTACCACCT
CTGTGGTAGTCTTTCAGTCTTTTGCACAAAGAACACCCCATTCTGGATTC
TAGCTGCCCTGTGCTCCACGGTCCACATCTGCCCCGCCCCTGCCCCGCCC
CCAGGCTCAAGCCTCGACCCTTTACCCTACGCGTATGCAAATGTTACCTT
CACCTATCTGTCCACAGTGGATTCAATAAAGTGCACGGGGTGGCAACTCT G
[0100] Mouse Fragilis 2 Amino Acid Sequence
TABLE-US-00008 MSHNSQAFLSTNAGLPPSYETIKEEYGVTELGEPSNSAVVRTTVINMPRE
VSVPDHVVWSLFNTLFFNACCLGFVAYAYSVKSRDRKMVGDVVGAQAYAS
TAKCLNISSLIFSILMVIICIIIFSTTSVVVFQSFAQRTPHSGF
[0101] Mouse Fragilis 2 Diseases
[0102] Mouse Fragilis 2 is expressed in a number of tissues,
including: adult; branchial arches; colon; embryo; embryonal
carcinoma; head; heart; hippocampus; liver; lymph; macrophage;
mammary gland; mandible; muscle; pancreas; pool; pooled lung
tumors; pooled mammary gland tumors; salivary gland; skin; small
intestine; spleen; spontaneous tumor, metastatic to mammary. stem
cell origin; stomach; subformical organ and postrema; t cell;
testis; thymus; tumor, biopsy sample; tumor, gross tissue; tumor,
metastatic to mammary; uterus; whole embryo.
[0103] Mouse Fragilis 2, as well as its human homologue, is
therefore suitable for treatment or diagnosis of any disease of or
associated with such tissues, in particular, cancer or tumours of
such tissues.
Mouse Fragilis 3
[0104] Mouse Fragilis 3 Nucleic Acid Sequence
TABLE-US-00009 TGGAGAAAAGGCCACTGCGCAAAGGGCTCTGGACTTCTCAGCTTGTACCA
CCATTCTCATTCCTTCCTTATTCTCAACTCTTCCAGCCTCAAAAACCAAG
AGATGCCTAAGGATCAGCATGAGGTGGTTGTAATGGGGACACCCCACACC
TCAACTTCTTCGACAACCACCATAATCACCATGCCTGAGATCTCGAAGCC
TGATTATGTGGTCTGGTCTCTGTTCAATACACTCTTCATGAACTTCTGCT
GCCTGGGTTTCATAGCCTATGCCTACTCTGTGAAGTCTAGGGACAGGAAG
ATGGTGGGTGATATGACTGGGGCCCAGGCCTTCGCCTCCACTGCCAGGTG
CCTGAACATCAGCTGCCTGATCCTCTCCGTCGTCATGGTCATCCTCTTCA
TCACTTTCTTTGCCACTAGAAGGTAGCCATCTTGTAGCATCTCACAGTAG
ATAACAGATTCTGGGGCCTTCCGGGCTTGCTATGTGTTCTATTGTCTATC
GCTGTCGCAAACCCTAGTCTTAGTCCTGACCATTTACCCCATACATATGC
AAATGTTACACTTGCATATCTGTTCATTCAATAAAGTGCA
[0105] Mouse Fragilis 3 Amino Acid Sequence
TABLE-US-00010 MPKDQHEVVVMGTPHTSTSSTTTIITMPEISKPDYVVWSLFNTLFMNFCC
LGFIAYAYSVKSRDRKMVGDMTGAQAFASTARCLNISCLILSVVMVILFI TFFATRR
[0106] Mouse Fragilis 3 Genomic Location
[0107] Mouse Fragilis 3 is located on chromosome 16.
[0108] Mouse Fragilis 3 Diseases
[0109] Mouse Fragilis 3 is expressed in a number of tissues,
including: adult; branchial round spermatids, pooled from multiple
mice; testis.
[0110] Mouse Fragilis 3, as well as its human homologue, is
therefore suitable for treatment or diagnosis of any disease of or
associated with such tissues, in particular, cancer or tumours of
such tissues.
Mouse Fragilis 4
[0111] Mouse Fragilis 4 Nucleic Acid Sequence
TABLE-US-00011 GATTCCTTCCTTATTCTCACTCTGCAGCTTCAAAAGCCGAGAGATGCCTA
AGGAGCAGCAAGAGGTGGTTGTACTGGGGTCACCCCACATCTCAACTTCT
GCGACAGCCACCACAATCAACATGCCTGAGATCTCCACGCCTGACCATGT
GGTCTGGTCCCTGTTCAATACACTCTTCATGAACTTCTGCTGCCTGGGCT
TCGTAGCCTATGCCTACTCCGTGAAGTCTAGGGACAGGAAGATGGTGGGT
GATACGACTGGGGCCCAGGCCTTCGCCTCCACCGCCAAGTGCCTGAACAT
CAGCTCCCTGTTCTTCACCATCCTCACGGCCATCGTCGTCATCGTTGTCT
GTGCCATTAGATGATGTGAGATGTCTTGCAACATCTCACAGTAGATAACA
GATTCTGGGGCCTCCCAGGCTTGCTATGTGTTTCCTTGTCTATCGCTGCC
CCAAACCCTAGACTTAGTCCTGACCATTTGCCCCATACATATGCAAATGT
GACACTCACAAATCTGTCCATGGTGGACTCAATAAAGTGCACGTGCTGTG
[0112] Mouse Fragilis 4 Amino Acid Sequence
TABLE-US-00012 MPKEQQEVVVLGSPHISTSATATTINIAPEISTPDHVVWSLFNTLFMNFC
CLGFVAYAYSVKSRDRKMVGDTTGAQAFASTAKCLNISSLFFTILTAIVV IVVCAIR
[0113] Mouse Fragilis 4 Diseases
[0114] Mouse Fragilis 4 is expressed in a number of tissues,
including: bowel; cerebellum; colon; embryo; head; heart; kidney;
liver; lung; lymph; macrophage; mammary gland; placenta; spleen;
spontaneous tumor, metastatic to mammary. stem cell origin;
stomach; t cell; thymus; tumor, biopsy sample; tumor, gross tissue;
tumor, metastatic to mammary; vagina.
[0115] Mouse Fragilis 4, as well as its human homologue, is
therefore suitable for treatment or diagnosis of any disease of or
associated with such tissues, in particular, cancer or tumours of
such tissues.
Mouse Fragilis 5
[0116] Mouse Fragilis 5 Nucleic Acid Sequence
TABLE-US-00013 CTCAGCTAGGAAGACACGGCGCTGGAACCCATGGACACTTCATATCCCCG
TGAGGACCCCCCGGCTCCATCATCCCGCAAGGCTGATGCTGCAGCCCACA
CAGCCCTCTCCATGGGAACACCTGGCCCTACACCACGAGATCACATGCTC
TGGTCTGTCTTCAGCACGATGTACCTGAATCTGTGCTGCCTTGGATTCCT
GGCGCTGGTCCACTCTGTCAAGGCCCGAGACCAGAAGATGGCTGGGAACT
TGGAGGCTGCAAGGCAGTATGGCTCCAAAGCCAAGTGCTACAACATCCTG
GCTGCAATGTGGACATTGGTGCCCCCATTGCTGCTCCTGGGACTGGTGGT
GACTGGCGCCTTGCACCTGTCCAAGTTAGCCAAAGACTCTGCGGCTTTCT
TCAGCACCAAGTTTGATGAGGAGGACTATAACTAAGAGTTCCGAGCCTGT
CCCTGAACCGAGGACAACGGGGCTAGAGCGGCCGCCACCGCGGTGGAGC
[0117] Mouse Fragilis 5 Amino Acid Sequence
TABLE-US-00014 MDTSYPREDPRAPSSRKADAAAHTALSMGTPGPTPRDHMLWSVFSTMYLN
LCCLGFLALVHSVKARDQKMAGNLEAARQYGSKAKCYNILAAMWTLVPPL
LLLGLVVTGALHLSKLAKDSAAFFSTKFDEEDYN
[0118] Mouse Fragilis 5 Genomic Location
[0119] Mouse Fragilis 5 is located on chromosome 6.
[0120] Mouse Fragilis 5 Diseases
[0121] Mouse Fragilis 5 is expressed in a number of tissues,
including: embryo; tumor, gross tissue.
[0122] Mouse Fragilis 5, as well as its human homologue, is
therefore suitable for treatment or diagnosis of any disease of or
associated with such tissues, in particular, cancer or tumours of
such tissues.
Mouse Fragilis 6
[0123] Mouse Fragilis 6 Nucleic Acid Sequence
TABLE-US-00015 TGAACTTCCTTGAAACAAGAGCTTCCTTGCTTCCTTTAAGCACAAAAACA
TGGTTAAGAGGGATCCTGACTCAGCTCCAGTGCCATCCACTGTGGTTTGC
ATCAACAGTGATGTTATCCAGCCGGATCACATTACCTGGTCTACATTTAA
CACAGTGTTCATGAATGGCTGCTGCCTGGGTTTCATTGCCTACATCTACT
CGGTGAAGTCCAGGGACCGGAAGATGGTGGGCGACATGACTGGGGCCCAA
TCCCATGCTTCAACCGCCAAGATTCTGAACATCCTTGCTCTGGTCATCTC
CCTCATCTTCTACATCATGCTTATCGTTTTATACAGCTTTAACTTACTAG
GTAACCAAAGATAATAGAACCACTAGTTAGGTACTAACTAGTTAGTTAGC
TAATTATTAATTAACTAAACTAGTACCGAATTTAGTATCTTTAGT
[0124] Mouse Fragilis 6 Amino Acid Sequence
TABLE-US-00016 MVKRDPDSAPVPSTVVCINSDVIQPDHITWSTFNTVFMNGCCLGFIAYIY
SVKSRDRKMVGDMTGAQSHASTAKILNILALVISLIFYIMLIVLYSFNLL GNQR
[0125] Mouse Fragilis 6 Genomic Location
[0126] Mouse Fragilis 6 is located on chromosome 7.
[0127] Mouse Fragilis 6 Diseases
[0128] Mouse Fragilis 6 is expressed in a number of tissues,
including: dbEST Library Tissue Type restricted to Spleen; cDNA
sources: in vitro fertilized eggs; lymph; spinal cord; spleen.
[0129] Mouse Fragilis 6, as well as its human homologue, is
therefore suitable for treatment or diagnosis of any disease of or
associated with such tissues, in particular, cancer or tumours of
such tissues.
Human Ifitm1, Ifitm2 Ifitm3 and ENSG142056
[0130] Human Ifitm1, Ifitm2 Ifitm3
[0131] We disclose four homologues of Fragilis genes, located on
Chromosome 11 (p15.5). This region is syntenic to the Fragilis
family locus on mouse Chr 7 (FIG. 9). Nucleic acids encoding each
of these proteins are also disclosed.
[0132] Three of these genes, Ifitm1 (9-27), Ifitm2 (1-8D) and
Ifitm3 (1-8U), share 58-65% similarity to the fragilis gene cluster
and are located within an 18 kb genomic stretch [AR Lewin, L E
Reid, M McMahon, G R Stark, I M Kerr: Molecular analysis of a human
interferon-inducible gene family. Eur J Biochem 1991, 199:
417-423].
[0133] They are responsive to type1/2 interferons and code for
interferon induced transmembrane (Ifitm) proteins, involved in
antiproliferative signalling and homotypic cell adhesion. See R L
Friedman, S P Manley, M Meahon, I M Kerr, G R Stark:
Transcriptional and posttranscriptional regulation of
interferon-induced gene expression in human cells. Cell 1984, 38:
745-755; J M Kelly, C S Gilbert, G R Stark, I M Kerr: Differential
regulation of interferon-induced mRNAs and c-myc mRNA by alpha- and
gamma-interferons. Eur J Biochem 1985, 153:367-371; S S Evans, D B
Lee, T Han, T B Tomasi, R L Evans: Monoclonal antibody to the
interferon-inducible protein Leu-13 triggers aggregation and
inhibits proliferation of leukemic B cells. Blood 1990, 76 (12):
2583-2593; and S S Evans, R P Collea, J A Leasure, D B Lee: IFN-a
induces homotypic adhesion and Leu-13 expression in human B
lymphoid cells J Immunol 1993, 150: 736-747.
[0134] Interferon stimulable response elements (ISREs,
GGAAAN(N)GAAAC) within the human Ifitm locus confer the
responsiveness of the three human Ifitm genes to interferons (A R
Lewin, L E Reid, M McMahon, G R Stark, I M Kerr: Molecular analysis
of a human interferon-inducible gene family. Eur J Biochem 1991,
199: 417-423; L E Reid, A H Brasnett, C S Gilbert, A C G Porter, D
R Gewert, G R Stark, I M Kerr: A single DNA response element can
confer inducibility by both alpha- and gamma-interferons. Proc Natl
Acad Sci USA 1989, 86: 840-844). Similar ISRE consensus sequences
are also found within the Fragilis family cluster in the mouse,
associated in particular with fragilis, fragilis 2 and fragilis5
(FIG. 9).
[0135] Human ENSG142056
[0136] We further disclose the fourth gene, ENSG142056, a novel
gene with two exons, is highly similar to mouse fragilis4 (83% DNA
sequence similarity) and neighbours Ifitm2. The human Fragilis
family homologues hence form a similar genomic cluster as the five
Fragilis genes in the mouse.
[0137] Other Fragilis Homologues
[0138] We also identified two Fragilis family-like genes in cow
(bovine 1-8U, bovine 9-27) and four genes in rat (P26376, JC1241,
NP110460, AAD48010). While the rat genes have been annotated as
putative interferon inducible, the two bovine genes that are
similar to the human Ifitm genes, have been reported to respond to
interferon signalling (D J Hayzer, E Brinson, M S Runge: A rat
beta-interferon-induced mRNA: sequence characterization. Gene 1992,
117 (2): 227-228; 17. J K Pru, K J Austin, A L Haas, T R Hansen:
Pregnancy and interferon-tau upregulate gene expression of members
of the 1-8 family in the bovine uterus. Biol Reprod 2001, 65 (5):
1471-1480).
Functions of Human and Stella
[0139] The functions of human Stella and Fragilis are disclosed
throughout this document. It is also expected that human Stella and
Fragilis will broadly display the properties of their mouse
counterparts. The functional homology of the human sequences
disclosed here to their mouse Stella and Fragilis counterparts may
be verified by a complementation test. An example of such a
complementation test is set out in the following paragraphs.
[0140] Mouse mutants for Stella and Fragilis are created by gene
knockout technology using ES cells, using standard techniques.
Further details are provided in the Examples, particularly Examples
11 to 15. Human STELLA and FRAGILIS clones may be isolated by
screening human genomic libraries, such as BAC libraries, or by
nucleotide synthesis or PCR of a human cDNA library using the
sequences disclosed here.
[0141] These BAC clones may then be introduced into the mouse
genome by microinjection of the genomic clones into the oocyte or
by transfection, lipofection or by using viral vectors, into ES
cells. Transgenic mice carrying human homologues of STELLA and
FRAGILIS are generated and crossed with mouse mutants for Stella or
Fragilis. If the human homologue contains appropriate regulatory
sequences and is therefore is expressed similarly to the mouse
gene, it will overcome phenotypic deficiencies in mutant mice, if
it is a true homologue. The wild type phenotype will be restored to
the transgenic knockout mouse.
[0142] BAC clones may be modified to contain reporter genes such as
GFP. When introduced into the mouse genome to generate transgenic
mice, the reporter constructs can be used to verify whether the
human homologue shows appropriate temporal and spatial patterns of
expression. This expression analysis combined with the phenotypic
rescue of mutants described above would verify if the human genes
are the homologues of murine genes.
[0143] There are other ways in which murine and human genes can be
tested for their effects on murine tumours. If the tumours are the
result of gain of function, it would be possible to use RNAi or
other antisense approaches to see if repression of modifies the
phenotype. If tumours result from loss of function, transgenes (as
BACs or under the control of appropriate tissue-specific regulatory
sequences) can be introduced into cells or mice to check if they
have an effect on tumours.
[0144] The procedure for BAC modifications and the generation of
transgenic mice may employ the technique described in Developmental
Biology, volume 236, 2001 by John, R M et al, or any other suitable
technique.
Stella Diseases and Fragilis Associated Diseases
[0145] Human Stella and Fragilis are expressed in a number of
tissues and cells, and in particular in a number of tumour or
cancerous cells and tissues, as described in further detail below.
Accordingly, the Stella and/or Fragilis polypeptides, nucleic
acids, antibodies, peptides, etc described here may be used in the
diagnosis and/or treatment or prevention of any diseases associated
with such tissues and cells, for example, tumours or cancers of the
tissues and cells.
[0146] Examples of diseases associated with Stella and Fragilis are
set out in the sections above relating to individual Stella and
Fragilis sequences.
[0147] Particular further examples of such tumours and cancers
include, but are not limited to, testis tumor, colon tumor,
stomach, germ cell tumors, choriocarcinoma, lung, large cell
carcinoma, uterus, and leiomyosarcoma. Alternatively or in
addition, Stella and/or Fragilis associated diseases include any
one or more of diseases of the following tissues, including:
adenocarcinoma; adrenal cortico adenoma for cushing's syndrome;
cervical carcinoma; Crohn's disease; embryonal carcinoma;
endometrium adenocarcinoma; epitheloid carcinoma;
poorly-differentiated adenocarcinomas; acute myelogenous leukemia;
chronic myelogenous leukemia; hepatocellular carcinoma;
hypernephroma; insulinoma; iris; kidney tumor, large cell
carcinoma; large cell carcinoma, undifferentiated; leiomyosarcoma;
lung focal fibrosis; metastatic chondrosarcoma; mucoepidermoid
carcinoma; neuroblastoma; papillary serous ovarian metastasis;
parathyroid; pheochrornocytoma; prostate tumor; serous papillary
tumor; squamous cell carcinoma, preferably poorly
differentiated.
Stella and Fragilis Polypeptides
[0148] It will be understood that polypeptide sequences disclosed
here are not limited to the particular sequences set forth in the
sequence listing, or fragments thereof, or sequences obtained from
Fragilis or Stella protein, but also include homologous sequences
obtained from any source, for example related cellular homologues,
homologues from other species and variants or derivatives thereof,
provided that they have at least one of the biological activities
of Stella or Fragilis (including related sequences such as Fragilis
2, 3, 4, 5 and 6), as the case may be.
[0149] This disclosure therefore encompasses variants, homologues
or derivatives of the amino acid sequences set forth in this
description, including in the sequence listings, as well as
variants, homologues or derivatives of the amino acid sequences
encoded by the nucleotide sequences disclosed here. In a preferred
embodiment, the homologues and variants of Stella and Fragilis do
not include the sequences disclosed in PCT/GB02/00215, in
particular do not include the mouse Stella and Fragilis sequences
set out in SEQ ID NO: 2 and 1 respectively. Furthermore, they
preferably do not include any sequence portion of these genes
previously disclosed without any indication of function, for
example, AC006927.26.74831.117191 (Chromosome 12, p12.3). In
particular, preferred embodiments do not comprise any EST
(Expressed Sequence Tag) which may have been disclosed, for
example, any one or more of the following ESTs:
gi|953020|gb|AA300687.1|AA300687 EST13535 Testis tumor cDNA 5' end;
gi|8149136|gb|AW959452.1|AW959452 EST371522 MAGE resequences, MAGF
cDNA (colon tumor); gi|10896747|gb|BF091037.1 |BF091037
MR3-SN0036-120900-007-h05 SN0036 cDNA (stomach normal); and
gi|3367222|gb|AI066520.1|AI066520 ov17h01.x1 NCI_CGAP_GC3 cDNA
clone IMAGE:1637617 3' (pooled germ cell tumors)
[0150] In further preferred embodiments, the variants, homologues,
etc of Stella and Fragilis do not include any one or more of the
following ESTs: gi|2335869|gb|AA564230.1|AA564230 nk43h01.s1
NCI_CGAP_GC2 cDNA clone IMAGE:1016305 3' (germ cell tumor);
gi|3076239|gb|AA927342.1|AA927342 orn69e05.s1 NCI_CGAP_GC4 cDNA
clone IMAGE:1552448 3' (pooled germ cell tumors);
gi|2959237|gb|AA864924.1|AA864924 oh44h08.s1 NCI_CGAP_GC4 cDNA
clone IMAGE:1469535 3' (pooled germ cell tumors);
gi|9512276|gb|BE466414.1|BE466414 hz21d11.x1 NCI_CGAP_GC6 cDNA
clone IMAGE:3208629 3' (pooled germ cell tumors);
gi|5837653|gb|AI990772.1|AI990772 ws23f02.x1 NCI_CGAP_GC6 cDNA
clone IMAGE:2498043 3' (pooled germ cell tumors);
gi|5741162|gb|AI948852.1|AI948852 wq37c09.x1 NCI_CGAP_GC6 cDNA
clone IMAGE:2473456 3' (pooled germ cell tumors);
gi|473-4510|gb|AI650531.1|AI650531 wa92b04.x1 NCI_CGAP_GC6 cDNA
clone IMAGE:2303599 3' (pooled germ cell tumors);
gi|5849349|gb|AW002433.1|AW002433 wu61h07.x1 NCI_CGAP_GC6 cDNA
clone IMAGE:2524573 3' (pooled germ cell tumors);
gi|5746921|gb|AI954611.1|AI954611 wq34b05.x1 NCI_CGAP_GC6 cDNA
clone IMAGE:2473137 3' (pooled germ cell tumors);
gi|4735956|gb|AI651977.1|AI651977 wb64h02.x1 NCI_CGAP_GC6 cDNA
clone IMAGE:2310483 3' (pooled germ cell tumors);
gi|19763950|gb|BQ028671.1|BQ028671 UI-1-EE0-ayz-b-02-0-UI.sl;
NCI_CGAP_P17 cDNA clone UI-1-EE0-ayz-b-02-0-UI 3'
(choriocarcinoma); gi|9890324|gb|BE619386.1|BE619386 601473204F1
NIH_MGC.sub.--68 cDNA clone IMAGE:3876145 5' (lung, large cell
carcinoma); gi|10344247|gb|BE888191.1|BE888191 601511709F1
NIH_MGC.sub.--71 cDNA clone IMAGE:3912866 5' (uterus,
leiomyosarcoma); and gi|1933621|gb|AA286758.1|AA286758 zs51a11.r1
NCI_CGAP_GCB1 cDNA clone IMAGE:700988 5' (germinal center B
cell).
Homologues
[0151] The polypeptides disclosed include homologous sequences
obtained from any source, for example related viral/bacterial
proteins, cellular homologues and synthetic peptides, as well as
variants or derivatives thereof. Thus polypeptides also include
those encoding homologues of Fragilis and/or Stella from other
species including animals such as mammals (e.g. mice, rats or
rabbits), especially primates, more especially humans. More
specifically, homologues include human homologues.
[0152] In the context of the present document, a homologous
sequence or homologue is taken to include an amino acid sequence
which is at least 60, 70, 80 or 90% identical, preferably at least
95 or 98% identical at the amino acid level over at least 30,
preferably 50, 70, 90 or 100 amino acids with Fragilis or Stella,
for example as shown in the sequence listing herein. In the context
of this document, a homologous sequence is taken to include an
amino acid sequence which is at least 15, 20, 25, 30, 40, 50, 60,
70, 80 or 90% identical, preferably at least 95 or 98% identical at
the amino acid level, preferably over at least 50 or 100,
preferably 200, 300, 400 or 500 amino acids with the sequence of
Fragilis or Stella, for example Fragilis (SEQ ID NO: 6) and Stella
(SEQ ID NO: 4), or the Fragilis family member sequences Fragilis
9-27 (preferably comprising a sequence as shown in SEQ ID NO: 8),
Fragilis 2 (preferably comprising a sequence as shown in SEQ ID NO:
10), Fragilis 3 (preferably comprising a sequence as shown in SEQ
ID NO: 12), Fragilis 4 (preferably comprising a sequence as shown
in SEQ ID NO: 14), Fragilis 5 (preferably comprising a sequence as
shown in SEQ ID NO: 16) and Fragilis 6 (preferably comprising a
sequence as shown in SEQ ID NO: 18).
[0153] Although homology can also be considered in terms of
similarity (i.e. amino acid residues having similar chemical
properties/functions), in the context of the present document it is
preferred to express homology in terms of sequence identity.
[0154] Homology comparisons can be conducted by eye, or more
usually, with the aid of readily available sequence comparison
programs. These commercially available computer programs can
calculate % homology between two or more sequences.
[0155] % homology may be calculated over contiguous sequences, i.e.
one sequence is aligned with the other sequence and each amino acid
in one sequence directly compared with the corresponding amino acid
in the other sequence, one residue at a time. This is called an
"ungapped" alignment. Typically, such ungapped alignments are
performed only over a relatively short number of residues (for
example less than 50 contiguous amino acids).
[0156] Although this is a very simple and consistent method, it
fails to take into consideration that, for example, in an otherwise
identical pair of sequences, one insertion or deletion will cause
the following amino acid residues to be put out of alignment, thus
potentially resulting in a large reduction in % homology when a
global alignment is performed. Consequently, most sequence
comparison methods are designed to produce optimal alignments that
take into consideration possible insertions and deletions without
penalising unduly the overall homology score. This is achieved by
inserting "gaps" in the sequence alignment to try to maximise local
homology.
[0157] However, these more complex methods assign "gap penalties"
to each gap that occurs in the alignment so that, for the same
number of identical amino acids, a sequence alignment with as few
gaps as possible--reflecting higher relatedness between the two
compared sequences--will achieve a higher score than one with many
gaps. "Affine gap costs" are typically used that charge a
relatively high cost for the existence of a gap and a smaller
penalty for each subsequent residue in the gap. This is the most
commonly used gap scoring system. High gap penalties will of course
produce optimised alignments with fewer gaps. Most alignment
programs allow the gap penalties to be modified. However, it is
preferred to use the default values when using such software for
sequence comparisons. For example when using the GCG Wisconsin
Bestfit package (see below) the default gap penalty for amino acid
sequences is -12 for a gap and -4 for each extension.
[0158] Calculation of maximum % homology therefore firstly requires
the production of an optimal alignment, taking into consideration
gap penalties. A suitable computer program for carrying out such an
alignment is the GCG Wisconsin Bestfit package university of
Wisconsin, U.S.A.; Devereux et al., 1984, Nucleic Acids Research
12:387). Examples of other software than can perform sequence
comparisons include, but are not limited to, the BLAST package (see
Ausubel et al., 1999 ibid--Chapter 18), FASTA (Atschul et al.,
1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparison
tools. Both BLAST and FASTA are available for offline and online
searching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60).
However it is preferred to use the GCG Bestfit program.
[0159] Although the final % homology can be measured in terms of
identity, the alignment process itself is typically not based on an
all-or-nothing pair comparison. Instead, a scaled similarity score
matrix is generally used that assigns scores to each pairwise
comparison based on chemical similarity or evolutionary distance.
An example of such a matrix commonly used is the BLOSUM62
matrix--the default matrix for the BLAST suite of programs. GCG
Wisconsin programs generally use either the public default values
or a custom symbol comparison table if supplied (see user manual
for further details). It is preferred to use the public default
values for the GCG package, or in the case of other software, the
default matrix, such as BLOSUM62.
[0160] Once the software has produced an optimal alignment, it is
possible to calculate % homology, preferably % sequence identity.
The software typically does this as part of the sequence comparison
and generates a numerical result.
Variants and Derivatives
[0161] The terms "variant" or "derivative" in relation to the amino
acid sequences as described here includes any substitution of,
variation of, modification of, replacement of, deletion of or
addition of one (or more) amino acids from or to the sequence.
Preferably, the resultant amino acid sequence retains substantially
the same activity as the unmodified sequence, preferably having at
least the same activity as the Fragilis and/or Stella polypeptides
shown in the sequence listings. Thus, the key feature of the
sequences--namely that they are specific for PGCs and other
pluripotent cells, such as ES or EG cells, and can serve as a
marker for these cells in a cell population--is preferably
retained.
[0162] Polypeptides having the amino acid sequence shown in the
Examples, or fragments or homologues thereof may be modified for
use in the methods and compositions described here. Typically,
modifications are made that maintain the biological activity of the
sequence. Amino acid substitutions may be made, for example from 1,
2 or 3 to 10, 20 or 30 substitutions provided that the modified
sequence retains the biological activity of the unmodified
sequence. Amino acid substitutions may include the use of
non-naturally occurring analogues, for example to increase blood
plasma half-life of a therapeutically administered polypeptide.
[0163] Natural variants of Fragilis and Stella are likely to
comprise conservative amino acid substitutions. Conservative
substitutions may be defined, for example according to the Table
below. Amino acids in the same block in the second column and
preferably in the same line in the third column may be substituted
for each other:
TABLE-US-00017 ALIPHATIC Non-polar G A P I L V Polar - uncharged C
S T M N Q Polar - charged D E K R AROMATIC H F W Y
Fragments
[0164] Polypeptides disclosed here and useful as markers also
include fragments of the above mentioned full length polypeptides
and variants thereof, including fragments of the sequences set out
in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID
NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 18.
[0165] Polypeptides also include fragments of the full length
sequence of any of the Fragilis and/or Stella polypeptides.
Preferably fragments comprise at least one epitope. Methods of
identifying epitopes are well known in the art. Fragments will
typically comprise at least 6 amino acids, more preferably at least
10, 20, 30, 50 or 100 amino acids.
[0166] Included are fragments comprising, preferably consisting of,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,
57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,
74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,
91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125,
130, 135, 140, 145 or 150, or more residues from a Fragilis and/or
Stella amino acid sequence.
[0167] Polypeptide fragments of the Stella/Fragilis proteins and
allelic and species variants thereof may contain one or more (e.g.
5, 10, 15, or 20) substitutions, deletions or insertions, including
conserved substitutions. Where substitutions, deletion and/or
insertions occur, for example in different species, preferably less
than 50%, 40% or 20% of the amino acid residues depicted in the
sequence listings are altered.
[0168] Fragilis and Stella, and their fragments, homologues,
variants and derivatives, may be made by recombinant means.
However, they may also be made by synthetic means using techniques
well known to skilled persons such as solid phase synthesis. The
proteins may also be produced as fusion proteins, for example to
aid in extraction and purification. Examples of fusion protein
partners include glutathione-S-transferase (GST), 6.times.His, GAL4
(DNA binding and/or transcriptional activation domains) and
.beta.-galactosidase. It may also be convenient to include a
proteolytic cleavage site between the fusion protein partner and
the protein sequence of interest to allow removal of fusion protein
sequences. Preferably the fusion protein will not hinder the
function of the protein of interest sequence. Proteins may also be
obtained by purification of cell extracts from animal cells.
[0169] The Fragilis and/or Stella polypeptides, variants,
homologues, fragments and derivatives disclosed here may be in a
substantially isolated form. It will be understood that such
polypeptides may be mixed with carriers or diluents which will not
interfere with the intended purpose of the protein and still be
regarded as substantially isolated. A Fragilis/Stella variant,
homologue, fragment or derivative may also be in a substantially
purified form, in which case it will generally comprise the protein
in a preparation in which more than 90%, e.g. 95%, 98% or 99% of
the protein in the preparation is a protein.
[0170] The Fragilis/Stella polypeptides, variants, homologues,
fragments and derivatives disclosed here may be labelled with a
revealing label. The revealing label may be any suitable label
which allows the polypeptide, etc to be detected. Suitable labels
include radioisotopes, e.g. .sup.125I, enzymes, antibodies,
polynucleotides and linkers such as biotin. Labelled polypeptides
may be used in diagnostic procedures such as immunoassays to
determine the amount of a polypeptide in a sample. Polypeptides or
labelled polypeptides may also be used in serological or
cell-mediated immune assays for the detection of immune reactivity
to said polypeptides in animals and humans using standard
protocols.
[0171] Fragilis/Stella polypeptides, variants, homologues,
fragments and derivatives disclosed here, optionally labelled, my
also be fixed to a solid phase, for example the surface of an
immunoassay well or dipstick. Such labelled and/or immobilised
polypeptides may be packaged into kits in a suitable container
along with suitable reagents, controls, instructions and the like.
Such polypeptides and kits may be used in methods of detection of
antibodies to the polypeptides or their allelic or species variants
by immunoassay.
[0172] Immunoassay methods are well known in the art and will
generally comprise: (a) providing a polypeptide comprising an
epitope bindable by an antibody against said protein; (b)
incubating a biological sample with said polypeptide under
conditions which allow for the formation of an antibody-antigen
complex; and (c) determining whether antibody-antigen complex
comprising said polypeptide is formed.
[0173] The Fragilis/Stella polypeptides, variants, homologues,
fragments and derivatives disclosed here may be used in in vitro or
in vivo cell culture systems to study the role of their
corresponding genes and homologues thereof in cell function,
including their function in disease. For example, truncated or
modified polypeptides may be introduced into a cell to disrupt the
normal functions which occur in the cell. The polypeptides may be
introduced into the cell by in situ expression of the polypeptide
from a recombinant expression vector (see below). The expression
vector optionally carries an inducible promoter to control the
expression of the polypeptide.
[0174] The use of appropriate host cells, such as insect cells or
mammalian cells, is expected to provide for such post-translational
modifications (e.g. myristolation, glycosylation, truncation,
lapidation and tyrosine, serine or threonine phosphorylation) as
may be needed to confer optimal biological activity on recombinant
expression products. Such cell culture systems in which the
Fragilis/Stella polypeptides, variants, homologues, fragments and
derivatives disclosed here are expressed may be used in assay
systems to identify candidate substances which interfere with or
enhance the functions of the polypeptides in the cell.
Fragilis/Stella Nucleic Acids
[0175] We provide generally for a number of Fragilis and Stella
nucleic acids, together with fragments, homologues, variants and
derivatives thereof. These nucleic acid sequences preferably encode
the polypeptide sequences disclosed here, and particularly in the
sequence listings. Preferably, the polynucleotides comprise Stella
and/or Fragilis nucleic acids, preferably selected from the group
consisting of: SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO:
9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 17 or
fragments, homologues, variants and derivatives thereof.
[0176] In particular, we provide for nucleic acids which encode any
of the Fragilis and/or Stella polypeptides disclosed here. Thus,
the terms "GCR nucleic acid", "Fragilis nucleic acid" and "Stella
nucleic acid" should be construed accordingly. Preferably, however,
such nucleic acids comprise any of the sequences set out as SEQ ID
NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ
ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 17 or a sequence encoding
any of the corresponding polypeptides, and a fragment, homologue,
variant or derivative of such a nucleic acid. The above terms
therefore preferably should be taken to refer to these
sequences.
[0177] In preferred embodiments, the Stella nucleic acid comprises
a nucleic acid having a sequence set out in SEQ ID NO: 3 and the
Fragilis nucleic acid comprises a nucleic acid having a sequence
set out in SEQ ID NO: 5. However, also included are nucleic acids
encoding any Fragilis family member, for example: Fragilis 9-27
(preferably a sequence as shown in SEQ ID NO: 7), Fragilis 2
(preferably a sequence as shown in SEQ ID NO: 9), Fragilis 3
(preferably a sequence as shown in SEQ ID NO: 11), Fragilis 4
(preferably a sequence as shown in SEQ ID NO: 13), Fragilis 5
(preferably a sequence as shown in SEQ ID NO: 15) and Fragilis 6
(preferably a sequence as shown in SEQ ID NO: 17).
[0178] As used here in this document, the terms "polynucleotide",
"nucleotide", and nucleic acid are intended to be synonymous with
each other. "Polynucleotide" generally refers to any
polyribonucleotide or polydeoxyribonucleotide, which may be
unmodified RNA or DNA or modified RNA or DNA. "Polynucleotides"
include, without limitation single- and double-stranded DNA, DNA
that is a mixture of single- and double-stranded regions, single-
and double-stranded RNA, and RNA that is mixture of single- and
double-stranded regions, hybrid molecules comprising DNA and RNA
that may be single-stranded or, more typically, double-stranded or
a mixture of single- and double-stranded regions. In addition,
"polynucleotide" refers to triple-stranded regions comprising RNA
or DNA or both RNA and DNA. The term polynucleotide also includes
DNAs or RNAs containing one or more modified bases and DNAs or RNAs
with backbones modified for stability or for other reasons.
"Modified" bases include, for example, tritylated bases and unusual
bases such as inosine. A variety of modifications has been made to
DNA and RNA; thus, "polynucleotide" embraces chemically,
enzymatically or metabolically modified forms of polynucleotides as
typically found in nature, as well as the chemical forms of DNA and
RNA characteristic of viruses and cells. "Polynucleotide" also
embraces relatively short polynucleotides, often referred to as
oligonucleotides.
[0179] It will be understood by a skilled person that numerous
different polynucleotides and nucleic acids can encode the same
polypeptide as a result of the degeneracy of the genetic code. In
addition, it is to be understood that skilled persons may, using
routine techniques, make nucleotide substitutions that do not
affect the polypeptide sequence encoded by the polynucleotides
described here to reflect the codon usage of any particular host
organism in which the polypeptides are to be expressed.
Variants, Derivatives and Homologues
[0180] The polynucleotides described here may comprise DNA or RNA.
They may be single-stranded or double-stranded. They may also be
polynucleotides which include within them synthetic or modified
nucleotides. A number of different types of modification to
oligonucleotides are known in the art. These include
methylphosphonate and phosphorothioate backbones, addition of
acridine or polylysine chains at the 3' and/or 5' ends of the
molecule. For the purposes of the present document, it is to be
understood that the polynucleotides described herein may be
modified by any method available in the art. Such modifications may
be carried out in order to enhance the in vivo activity or life
span of polynucleotides.
[0181] Where the polynucleotide is double-stranded, both strands of
the duplex, either individually or in combination, are encompassed
by the methods and compositions described here. Where the
polynucleotide is single-stranded, it is to be understood that the
complementary sequence of that polynucleotide is also included.
[0182] The terms "variant", "homologue" or "derivative" in relation
to a nucleotide sequence include any substitution of, variation of,
modification of, replacement of, deletion of or addition of one (or
more) nucleotides from or to the sequence providing the resultant
nucleotide sequence is specific for pluripotent cells, preferably
specific for PGCs, ES cells or EG cells. Most preferably, the
resultant nucleotide sequence is specific for PGCs.
[0183] As indicated above, with respect to sequence identity, a
"homologue" has preferably at least 5% identity, at least 10%
identity, at least 15% identity, at least 20% identity, at least
25% identity, at least 30% identity, at least 35% identity, at
least 40% identity, at least 45% identity, at least 50% identity,
at least 55% identity, at least 60% identity, at least 65%
identity, at least 70% identity, at least 75% identity, at least
80% identity, at least 85% identity, at least 90% identity, or at
least 95% identity to the relevant sequence shown in the sequence
listings.
[0184] More preferably there is at least 95% identity, more
preferably at least 96% identity, more preferably at least 97%
identity, more preferably at least 98% identity, more preferably at
least 99% identity. Nucleotide homology comparisons may be
conducted as described above. A preferred sequence comparison
program is the GCG Wisconsin Bestfit program described above. The
default scoring matrix has a match value of 10 for each identical
nucleotide and -9 for each mismatch. The default gap creation
penalty is -50 and the default gap extension penalty is -3 for each
nucleotide.
Hybridisation
[0185] We further describe nucleotide sequences that are capable of
hybridising selectively to any of the sequences presented herein,
or any variant, fragment or derivative thereof, or to the
complement of any of the above. Nucleotide sequences are preferably
at least 15 nucleotides in length, more preferably at least 20, 30,
40 or 50 nucleotides in length.
[0186] The term "hybridisation" as used herein shall include "the
process by which a strand of nucleic acid joins with a
complementary strand through base pairing" as well as the process
of amplification as carried out in polymerase chain reaction
technologies.
[0187] Polynucleotides capable of selectively hybridising to the
nucleotide sequences presented herein, or to their complement, will
be generally at least 70%, preferably at least 80 or 90% and more
preferably at least 95% or 98% homologous to the corresponding
nucleotide sequences presented herein over a region of at least 20,
preferably at least 25 or 30, for instance at least 40, 60 or 100
or more contiguous nucleotides.
[0188] The term "selectively hybridisable" means that the
polynucleotide used as a probe is used under conditions where a
target polynucleotide is found to hybridize to the probe at a level
significantly above background. The background hybridization may
occur because of other polynucleotides present, for example, in the
cDNA or genomic DNA library being screened. In this event,
background implies a level of signal generated by interaction
between the probe and a non-specific DNA member of the library
which is less than 10 fold, preferably less than 100 fold as
intense as the specific interaction observed with the target DNA.
The intensity of interaction may be measured, for example, by
radiolabelling the probe, e.g. with .sup.32P.
[0189] Hybridisation conditions are based on the melting
temperature (Tm) of the nucleic acid binding complex, as taught in
Berger and Kimmel (1987, Guide to Molecular Cloning Techniques,
Methods in Enzymology, Vol 152, Academic Press, San Diego Calif.),
and confer a defined "stringency" as explained below.
[0190] Maximum stringency typically occurs at about Tm-5.degree. C.
(5.degree. C. below the Tm of the probe); high stringency at about
5.degree. C. to 10.degree. C. below Tm; intermediate stringency at
about 10.degree. C. to 20.degree. C. below Tm; and low stringency
at about 20.degree. C. to 25.degree. C. below Tm. As will be
understood by those of skill in the art, a maximum stringency
hybridisation can be used to identify or detect identical
polynucleotide sequences while an intermediate (or low) stringency
hybridisation can be used to identify or detect similar or related
polynucleotide sequences.
[0191] In a preferred aspect, we disclose nucleotide sequences that
can hybridise to a Fragilis/Stella nucleic acid, or a fragment,
homologue, variant or derivative thereof, under stringent
conditions (e.g. 65.degree. C. and 0.1.times.SSC {1.times.SSC=0.15
M NaCl, 0.015 M Na.sub.3 Citrate pH 7.0}).
[0192] Where a polynucleotide is double-stranded, both strands of
the duplex, either individually or in combination, are encompassed
by the present disclosure. Where the polynucleotide is
single-stranded, it is to be understood that the complementary
sequence of that polynucleotide is also disclosed and
encompassed.
[0193] Polynucleotides which are not 100% homologous to the
sequences disclosed here but fall within the disclosure can be
obtained in a number of ways. Other variants of the sequences
described herein may be obtained for example by probing DNA
libraries made from a range of individuals, for example individuals
from different populations. In addition, other viral/bacterial, or
cellular homologues particularly cellular homologues found in
mammalian cells (e.g. rat, mouse, bovine and primate cells,
including human cells), may be obtained and such homologues and
fragments thereof in general will be capable of selectively
hybridising to the sequences shown in the sequence listing herein.
Such sequences may be obtained by probing cDNA libraries made from
or genomic DNA libraries from other animal species, and probing
such libraries with probes comprising all or part of SEQ ID NOs: 1
or 3 under conditions of medium to high stringency. Similar
considerations apply to obtaining species homologues and allelic
variants of Fragilis and Stella.
[0194] The polynucleotides described here may be used to produce a
primer, e.g. a PCR primer, a primer for an alternative
amplification reaction, a probe e.g. labelled with a revealing
label by conventional means using radioactive or non-radioactive
labels, or the polynucleotides may be cloned into vectors. Such
primers, probes and other fragments will be at least 15, preferably
at least 20, for example at least 25, 30 or 40 nucleotides in
length, and are also encompassed by the term polynucleotides as
used herein. Preferred fragments are less than 500, 200, 100, 50 or
20 nucleotides in length.
[0195] Polynucleotides such as a DNA polynucleotides and probes may
be produced recombinantly, synthetically, or by any means available
to those of skill in the art. They may also be cloned by standard
techniques.
[0196] In general, primers will be produced by synthetic means,
involving a step wise manufacture of the desired nucleic acid
sequence one nucleotide at a time. Techniques for accomplishing
this using automated techniques are readily available in the
art.
[0197] Longer polynucleotides will generally be produced using
recombinant means, for example using PCR (polymerase chain
reaction) cloning techniques. This will involve making a pair of
primers (e.g. of about 15 to 30 nucleotides) flanking a region of
the sequence which it is desired to clone, bringing the primers
into contact with mRNA or cDNA obtained from an animal or human
cell, performing a polymerase chain reaction under conditions which
bring about amplification of the desired region, isolating the
amplified fragment (e.g. by purifying the reaction mixture on an
agarose gel) and recovering the amplified DNA. The primers may be
designed to contain suitable restriction enzyme recognition sites
so that the amplified DNA can be cloned into a suitable cloning
vector
Nucleotide Vectors
[0198] The polynucleotides can be incorporated into a recombinant
replicable vector. The vector may be used to replicate the nucleic
acid in a compatible host cell. Thus in a further embodiment, we
provide a method of making polynucleotides by introducing a
polynucleotide into a replicable vector, introducing the vector
into a compatible host cell, and growing the host cell under
conditions which bring about replication of the vector. The vector
may be recovered from the host cell. Suitable host cells include
bacteria such as E. coli, yeast, mammalian cell lines and other
eukaryotic cell lines, for example insect Sf9 cells.
[0199] Preferably, a polynucleotide in a vector is operably linked
to a control sequence that is capable of providing for the
expression of the coding sequence by the host cell, i.e. the vector
is an expression vector. The term "operably linked" means that the
components described are in a relationship permitting them to
function in their intended manner. A regulatory sequence "operably
linked" to a coding sequence is ligated in such a way that
expression of the coding sequence is achieved under condition
compatible with the control sequences.
[0200] The control sequences may be modified, for example by the
addition of further transcriptional regulatory elements to make the
level of transcription directed by the control sequences more
responsive to transcriptional modulators.
[0201] Vectors may be transformed or transfected into a suitable
host cell as described below to provide for expression of a
protein. This process may comprise culturing a host cell
transformed with an expression vector as described above under
conditions to provide for expression by the vector of a coding
sequence encoding the protein, and optionally recovering the
expressed protein.
[0202] The vectors may be for example, plasmid or virus vectors
provided with an origin of replication, optionally a promoter for
the expression of the said polynucleotide and optionally a
regulator of the promoter. The vectors may contain one or more
selectable marker genes, for example an ampicillin resistance gene
in the case of a bacterial plasmid or a neomycin resistance gene
for a mammalian vector. Vectors may be used, for example, to
transfect or transform a host cell.
[0203] Control sequences operably linked to sequences encoding the
protein include promoters/enhancers and other expression regulation
signals. These control sequences may be selected to be compatible
with the host cell for which the expression vector is designed to
be used in. The term "promoter" is well-known in the art and
encompasses nucleic acid regions ranging in size and complexity
from minimal promoters to promoters including upstream elements and
enhancers.
[0204] The promoter is typically selected from promoters which are
functional in mammalian cells, although prokaryotic promoters and
promoters functional in other eukaryotic cells may be used. The
promoter is typically derived from promoter sequences of viral or
eukaryotic genes. For example, it may be a promoter derived from
the genome of a cell in which expression is to occur. With respect
to eukaryotic promoters, they may be promoters that function in a
ubiquitous manner (such as promoters of .alpha.-actin,
.beta.-actin, tubulin) or, alternatively, a tissue-specific manner
(such as promoters of the genes for pyruvate kinase). They may also
be promoters that respond to specific stimuli, for example
promoters that bind steroid hormone receptors. Viral promoters may
also be used, for example the Moloney murine leukaemia virus long
terminal repeat (MMLV LTR) promoter, the Rous sarcoma virus (RSV)
LTR promoter or the human cytomegalovirus (CMV) IE promoter.
[0205] It may also be advantageous for the promoters to be
inducible so that the levels of expression of the heterologous gene
can be regulated during the life-time of the cell. Inducible means
that the levels of expression obtained using the promoter can be
regulated.
[0206] In addition, any of these promoters may be modified by the
addition of further regulatory sequences, for example enhancer
sequences. Chimeric promoters may also be used comprising sequence
elements from two or more different promoters described above.
Expression of Stella and/or Fragilis Polypeptides
[0207] In order to express a biologically active Stella and/or
Fragilis, the nucleotide sequences encoding Stella and/or Fragilis
or homologues, variants, or derivatives thereof are inserted into
appropriate expression vector, i.e., a vector which contains the
necessary elements for the transcription and translation of the
inserted coding sequence.
[0208] Methods which are well known to those skilled in the art are
used to construct expression vectors containing sequences encoding
Stella and/or Fragilis and appropriate transcriptional and
translational control elements. These methods include in vitro
recombinant DNA techniques, synthetic techniques, and in vivo
genetic recombination. Such techniques are described in Sambrook,
J. et al. (1989; Molecular Cloning, A Laboratory Manual, ch. 4, 8,
and 16-17, Cold Spring Harbor Press, Plainview, N.Y.) and Ausubel,
F. M. et al. (1995 and periodic supplements; Current Protocols in
Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New
York, N.Y.).
[0209] A variety of expression vector/host systems may be utilized
to contain and express sequences encoding Stella and/or Fragilis.
These include, but are not limited to, microorganisms such as
bacteria transformed with recombinant bacteriophage, plasmid, or
cosmid DNA expression vectors; yeast transformed with yeast
expression vectors; insect cell systems infected with virus
expression vectors (e.g., baculovirus); plant cell systems
transformed with virus expression vectors (e.g., cauliflower mosaic
virus (CaMV) or tobacco mosaic virus (TMV)) or with bacterial
expression vectors (e.g., Ti or pBR322 plasmids); or animal cell
systems. Any suitable host cell may be employed.
[0210] The "control elements" or "regulatory sequences" are those
non-translated regions of the vector (i.e., enhancers, promoters,
and 5' and 3' untranslated regions) which interact with host
cellular proteins to carry out transcription and translation. Such
elements may vary in their strength and specificity. Depending on
the vector system and host utilized, any number of suitable
transcription and translation elements, including constitutive and
inducible promoters, may be used. For example, when cloning in
bacterial systems, inducible promoters such as the hybrid lacZ
promoter of the BLUESCRIPT phagemid (Stratagene, La Jolla, Calif.)
or PSPORT1 plasmid (GIBCO/BRL), and the like, may be used. The
baculovirus polyhedrin promoter may be used in insect cells.
Promoters or enhancers derived from the genomes of plant cells
(e.g., heat shock, RUBISCO, and storage protein genes) or from
plant viruses (e.g., viral promoters or leader sequences) may be
cloned into the vector. In mammalian cell systems, promoters from
mammalian genes or from mammalian viruses are preferable. If it is
necessary to generate a cell line that contains multiple copies of
the sequence encoding Stella and/or Fragilis, vectors based on SV40
or EBV may be used with an appropriate selectable marker.
[0211] In bacterial systems, a number of expression vectors may be
selected depending upon the use intended for Stella and/or
Fragilis. For example, when large quantities of Stella and/or
Fragilis are needed for the induction of antibodies, vectors which
direct high level expression of fusion proteins that are readily
purified may be used. Such vectors include, but are not limited to,
multifunctional E. coli cloning and expression vectors such as
BLUESCRIPT (Stratagene), in which the sequence encoding Stella
and/or Fragilis may be ligated into the vector in frame with
sequences for the amino-terminal Met and the subsequent 7 residues
of .beta.-galactosidase so that a hybrid protein is produced, pIN
vectors (Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem.
264:5503-5509), and the like. pOEX vectors (Promega, Madison, Wis.)
may also be used to express foreign polypeptides as fusion proteins
with glutathione S-transferase (GST). In general, such fusion
proteins are soluble and can easily be purified from lysed cells by
adsorption to glutathione-agarose beads followed by elution in the
presence of free glutathione. Proteins made in such systems may be
designed to include heparin, thrombin, or factor XA protease
cleavage sites so that the cloned polypeptide of interest can be
released from the GST moiety at will.
[0212] In the yeast Saccharomyces cerevisiae, a number of vectors
containing constitutive or inducible promoters, such as alpha
factor, alcohol oxidase, and PGH, may be used. For reviews, see
Ausubel (supra) and Grant et al. (1987; Methods Enzymol.
153:516-544).
[0213] In cases where plant expression vectors are used, the
expression of sequences encoding Stella and/or Fragilis may be
driven by any of a number of promoters. For example, viral
promoters such as the 35S and 19S promoters of CaMV may be used
alone or in combination with the omega leader sequence from TMV.
(Takamatsu, N. (1987) EMBO J. 6:307-311.) Alternatively, plant
promoters such as the small subunit of RUBISCO or heat shock
promoters may be used. (Coruzzi, G. et al. (1984) EMBO J.
3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and
Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105.)
These constructs can be introduced into plant cells by direct DNA
transformation or pathogen-mediated transfection. Such techniques
are described in a number of generally available reviews. (See, for
example, Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of
Science and Technology (1992) McGraw Hill, New York, N.Y.; pp.
191-196.).
[0214] An insect system may also be used to express Stella and/or
Fragilis. For example, in one such system, Autographa californica
nuclear polyhedrosis virus (AcNPV) is used as a vector to express
foreign genes in Spodoptera frugiperda cells or in Trichoplusia
larvae. The sequences encoding Stella and/or Fragilis may be cloned
into a non-essential region of the virus, such as the polyhedrin
gene, and placed under control of the polyhedrin promoter.
Successful insertion of Stella and/or Fragilis will render the
polyhedrin gene inactive and produce recombinant virus lacking coat
protein. The recombinant viruses may then be used to infect, for
example, S. frugiperda cells or Trichoplusia larvae in which Stella
and/or Fragilis may be expressed. (Engelhard, E. K. et al. (1994)
Proc. Nat. Acad. Sci. 91:3224-3227.)
[0215] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, sequences encoding Stella and/or Fragilis may be
ligated into an adenovirus transcription/translation complex
consisting of the late promoter and tripartite leader sequence.
Insertion in a non-essential E1 or E3 region of the viral genome
may be used to obtain a viable virus which is capable of expressing
Stella and/or Fragilis in infected host cells. (Logan, J. and T.
Shenk (1984) Proc. Natl. Acad. Sci. 81:3655-3659.) In addition,
transcription enhancers, such as the Rous sarcoma virus (RSV)
enhancer, may be used to increase expression in mammalian host
cells.
[0216] Thus, for example, the Stella and/or Fragilis proteins are
expressed in either human embryonic kidney 293 (HEK293) cells or
adherent dhfr CHO cells. To maximize receptor expression, typically
all 5' and 3' untranslated regions (UTRs) are removed from the
receptor cDNA prior to insertion into a pCDN or pcDNA3 vector. The
cells are transfected with individual receptor cDNAs by lipofectin
and selected in the presence of 400 mg/ml G418. After 3 weeks of
selection, individual clones are picked and expanded for further
analysis. HEK293 or CHO cells transfected with the vector alone
serve as negative controls. To isolate cell lines stably expressing
the individual receptors, about 24 clones are typically selected
and analyzed by Northern blot analysis. Receptor mRNAs are
generally detectable in about 50% of the G418-resistant clones
analyzed.
[0217] Human artificial chromosomes (HACs) may also be employed to
deliver larger fragments of DNA than can be contained and expressed
in a plasmid. HACs of about 6 kb 5 to 10 Mb are constructed and
delivered via conventional delivery methods (liposomes,
polycationic amino polymers, or vesicles) for therapeutic
purposes.
[0218] Specific initiation signals may also be used to achieve more
efficient translation of sequences encoding Stella and/or Fragilis.
Such signals include the ATG initiation codon and adjacent
sequences. In cases where sequences encoding Stella and/or Fragilis
and its initiation codon and upstream sequences are inserted into
the appropriate expression vector, no additional transcriptional or
translational control signals may be needed. However, in cases
where only coding sequence, or a fragment thereof, is inserted,
exogenous translational control signals including the ATG
initiation codon should be provided. Furthermore, the initiation
codon should be in the correct reading frame to ensure translation
of the entire insert. Exogenous translational elements and
initiation codons may be of various origins, both natural and
synthetic. The efficiency of expression may be enhanced by the
inclusion of enhancers appropriate for the particular cell system
used, such as those described in the literature. (Scharf, D. et al.
(1994) Results Probl. Cell Differ. 20:125-162.)
[0219] In addition, a host cell strain may be chosen for its
ability to modulate expression of the inserted sequences or to
process the expressed protein in the desired fashion. Such
modifications of the polypeptide include, but are not limited to,
acetylation, carboxylation, glycosylation, phosphorylation,
lipidation, and acylation. Post-translational processing which
cleaves a "prepro" form of the protein may also be used to
facilitate correct insertion, folding, and/or function. Different
host cells which have specific cellular machinery and
characteristic mechanisms for post-translational activities (e.g.,
CHO, HeLa, MDCK, HEK293, and W138), are available from the American
Type Culture Collection (ATCC, Bethesda, Md.) and may be chosen to
ensure the correct modification and processing of the foreign
protein.
[0220] For long term, high yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
capable of stably expressing Stella and/or Fragilis can be
transformed using expression vectors which may contain viral
origins of replication and/or endogenous expression elements and a
selectable marker gene on the same or on a separate vector.
Following the introduction of the vector, cells may be allowed to
grow for about 1 to 2 days in enriched media before being switched
to selective media. The purpose of the selectable marker is to
confer resistance to selection, and its presence allows growth and
recovery of cells which successfully express the introduced
sequences. Resistant clones of stably transformed cells may be
proliferated using tissue culture techniques appropriate to the
cell type.
[0221] Any number of selection systems may be used to recover
transformed cell lines. These include, but are not limited to, the
herpes simplex virus thymidine kinase genes (Wigler, M. et al.
(1977) Cell 11:223-32) and adenine phosphoribosyltransferase genes
(Lowy, I. et al. (1980) Cell 22:817-23), which can be employed in
tk.sup.- or apr.sup.- cells, respectively. Also, antimetabolite,
antibiotic, or herbicide resistance can be used as the basis for
selection. For example, dhfr confers resistance to methotrexate
(Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-70); npt
confers resistance to the aminoglycosides neomycin and G418
(Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14); and als
or pat confer resistance to chlorsulfuron and phosphinothricin
acetyltransferase, respectively (Murry, supra). Additional
selectable genes have been described, for example, trpB, which
allows cells to utilize indole in place of tryptophan, or hisD,
which allows cells to utilize histinol in place of histidine.
(Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci.
85:8047-51.) Recently, the use of visible markers has gained
popularity with such markers as anthocyanins, .beta.-glucuronidase
and its substrate GUS, and luciferase and its substrate luciferin.
These markers can be used not only to identify transformants, but
also to quantify the amount of transient or stable protein
expression attributable to a specific vector system. (Rhodes, C. A.
et al. (1995) Methods Mol. Biol. 55:121-131.)
[0222] Although the presence/absence of marker gene expression
suggests that the gene of interest is also present, the presence
and expression of the gene may need to be confirmed. For example,
if the sequence encoding Stella and/or Fragilis is inserted within
a marker gene sequence, transformed cells containing sequences
encoding Stella and/or Fragilis can be identified by the absence of
marker gene function. Alternatively, a marker gene can be placed in
tandem with a sequence encoding Stella and/or Fragilis under the
control of a single promoter. Expression of the marker gene in
response to induction or selection usually indicates expression of
the tandem gene as well.
[0223] Alternatively, host cells which contain the nucleic acid
sequence encoding Stella and/or Fragilis and express Stella and/or
Fragilis may be identified by a variety of procedures known to
those of skill in the art. These procedures include, but are not
limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay
or immunoassay techniques which include membrane, solution, or chip
based technologies for the detection and/or quantification of
nucleic acid or protein sequences.
[0224] The presence of polynucleotide sequences encoding Stella
and/or Fragilis can be detected by DNA-DNA or DNA-RNA hybridization
or amplification using probes or fragments or fragments of
polynucleotides encoding Stella and/or Fragilis. Nucleic acid
amplification based assays involve the use of oligonucleotides or
oligomers based on the sequences encoding Stella and/or Fragilis to
detect transformants containing DNA or RNA encoding Stella and/or
Fragilis.
[0225] A variety of protocols for detecting and measuring the
expression of Stella and/or Fragilis, using either polyclonal or
monoclonal antibodies specific for the protein, are known in the
art. Examples of such techniques include enzyme-linked
immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and
fluorescence activated cell sorting (FACS). A two-site,
monoclonal-based immunoassay utilizing monoclonal antibodies
reactive to two non-interfering epitopes on Stella and/or Fragilis
is preferred, but a competitive binding assay may be employed.
These and other assays are well described in the art, for example,
in Hampton, R et al. (1990; Serological Methods, a Laboratory
Manual, Section IV, APS Press, St Paul, Minn.) and in Maddox, D. E.
et al. (1983; J. Exp. Med. 158:1211-1216).
[0226] A wide variety of labels and conjugation techniques are
known by those skilled in the art and may be used in various
nucleic acid and amino acid assays. Means for producing labeled
hybridization or PCR probes for detecting sequences related to
polynucleotides encoding Stella and/or Fragilis include
oligolabeling, nick translation, end-labeling, or PCR amplification
using a labeled nucleotide. Alternatively, the sequences encoding
Stella and/or Fragilis, or any fragments thereof, may be cloned
into a vector for the production of an mRNA probe. Such vectors are
known in the art, are commercially available, and may be used to
synthesize RNA probes in vitro by addition of an appropriate RNA
polymerase such as T7, T3, or SP6 and labeled nucleotides. These
procedures may be conducted using a variety of commercially
available kits, such as those provided by Pharmacia & Upjohn
(Kalamazoo, Mich.), Promega (Madison, Wis.), and U.S. Biochemical
Corp. (Cleveland, Ohio). Suitable reporter molecules or labels
which may be used for ease of detection include radionuclides,
enzymes, fluorescent, chemiluminescent, or chromogenic agents, as
well as substrates, cofactors, inhibitors, magnetic particles, and
the like.
[0227] Host cells transformed with nucleotide sequences encoding
Stella and/or Fragilis may be cultured under conditions suitable
for the expression and recovery of the protein from cell culture.
The protein produced by a transformed cell may be located in the
cell membrane, secreted or contained intracellularly depending on
the sequence and/or the vector used. As will be understood by those
of skill in the art, expression vectors containing polynucleotides
which encode Stella and/or Fragilis may be designed to contain
signal sequences which direct secretion of Stella and/or Fragilis
through a prokaryotic or eukaryotic cell membrane. Other
constructions may be used to join sequences encoding Stella and/or
Fragilis to nucleotide sequences encoding a polypeptide domain
which will facilitate purification of soluble proteins. Such
purification facilitating domains include, but are not limited to,
metal chelating peptides such as histidine-tryptophan modules that
allow purification on immobilized metals, protein A domains that
allow purification on immobilized immunoglobulin, and the domain
utilized in the FLAGS extension/affinity purification system
(Immunex Corp., Seattle, Wash.). The inclusion of cleavable linker
sequences, such as those specific for Factor XA or enterokinase
(Invitrogen, San Diego, Calif.), between the purification domain
and the Stella and/or Fragilis encoding sequence may be used to
facilitate purification. One such expression vector provides for
expression of a fusion protein containing Stella and/or Fragilis
and a nucleic acid encoding 6 histidine residues preceding a
thioredoxin or an enterokinase cleavage site. The histidine
residues facilitate purification on immobilized metal ion affinity
chromatography (IMIAC; described in Porath, J. et al. (1992) Prot.
Exp. Purif. 3: 263-281), while the enterokinase cleavage site
provides a means for purifying Stella and/or Fragilis from the
fusion protein. A discussion of vectors which contain fusion
proteins is provided in Kroll, D. J. et al. (1993; DNA Cell Biol.
12:441-453).
[0228] Fragments of Stella and/or Fragilis may be produced not only
by recombinant production, but also by direct peptide synthesis
using solid-phase techniques. (Merrifield J. (1963) J. Am. Chem.
Soc. 85:2149-2154.) Protein synthesis may be performed by manual
techniques or by automation. Automated synthesis may be achieved,
for example, using the Applied Biosystems 431A peptide synthesizer
(Perkin Elmer). Various fragments of Stella and/or Fragilis may be
synthesized separately and then combined to produce the full length
molecule.
Recombinant Stella and Fragilis Proteins
[0229] We also provide for expression of Stella and Fragilis
proteins, for example, recombinant proteins. These may be expressed
by a number of methods as generally known in the art; the following
provides an example of how this may be achieved.
[0230] Nucleotide sequences of Stella and Fragilis are cloned into
a TRI-system vector (Qiagen). Stella sequence comprising the second
codon onwards (i.e., an N terminal fragment of Stella without the
first ATG codon) is cloned into a pQE vector using appropriate
restriction enzyme sites, and according to the manufacturers
instructions. QIAexpress pQE vectors enable high-level expression
of 6.times.His-tagged proteins in E. coli.
[0231] A His tag is placed in the N terminal portion of the Stella
gene. Recombinant protein is purified by affinity chromatography on
a Ni-NTA column, according to manufacturer's instructions. The His
tag is cleaved using a suitable protease.
[0232] Recombinantly expressed Stella and Fragilis protein are
found to be biologically active.
Modulators, Agonists and Antagonists
[0233] The methods and compositions described here rely, in some
embodiments, on blocking, reducing, or increasing the activity of
proteins such as Stella and Fragilis, for example, in methods of
treating or preventing a disease such as cancer. In general, the
methods employ modulators of Stella and/or Fragilis activity or
expression.
[0234] Agents which are capable of increasing the activity of a the
Stella and/or Fragilis protein are referred to as agonists of that
activity. Similarly, antagonists reduce the activity of the
relevant protein.
[0235] The term "antagonist", as used in the art, is generally
taken to refer to a compound which binds to an enzyme and inhibits
the activity of the enzyme. The term as used here, however, is
intended to refer broadly to any agent which inhibits the activity
of a molecule, not necessarily by binding to it. Accordingly, it
includes agents which affect the expression of a protein such as
Stella or Fragilis, or the biosynthesis of a regulatory molecule,
or the expression of modulators of the activity of the Stella or
Fragilis. The specific activity which is inhibited may be any
activity which is exhibited by, or characteristic of, the enzyme or
molecule, for example, any activity of Stella and/Fragilis as the
case may be, for example, a signal transduction activity or PGC
specification activity.
[0236] The antagonist may bind to and compete for one or more sites
on the relevant molecule preferably, the catalytic site of the
enzyme. Preferably, such binding blocks the interaction between the
molecule and another entity (for example, the interaction between a
enzyme and its substrate). However, the antagonist need not
necessarily bind directly to a catalytic site, and may bind for
example to an adjacent site, another protein (for example, a
protein which is complexed with the enzyme) or other entity on or
in the cell, so long as its binding reduces the activity of the
enzyme or molecule.
[0237] Where antagonists of a enzyme such as a enzyme are
concerned, an antagonist may include a substrate of the enzyme, or
a fragment of this which is capable of binding to the enzyme. In
addition, whole or fragments of a substrate generated natively or
by peptide synthesis may be used to compete with the substrate for
binding sites on the enzyme. Alternatively, or in addition, an
immunoglobulin (for example, a monoclonal or polyclonal antibody)
capable of binding to the enzyme may be used. The antagonist may
also include a peptide or other small molecule which is capable of
interfering with the binding interaction. Other examples of
antagonists are set forth in greater detail below, and will also be
apparent to the skilled person.
[0238] Blocking the activity of an inhibitor of the relevant Stella
and/or Fragilis protein may also be achieved by reducing the level
of expression of the protein or an inhibitor in the cell. For
example, the cell may be treated with antisense compounds, for
example oligonucleotides having sequences specific to the Stella
and/or Fragilis mRNA. The level of expression of pathogenic forms
of adhesion proteins may also be regulated this way.
[0239] In general, agonists, antagonists and modulators comprise
agents such as an atom or molecule, wherein a molecule may be
inorganic or organic, a biological effector molecule and/or a
nucleic acid encoding an agent such as a biological effector
molecule, a protein, a polypeptide, a peptide, a nucleic acid, a
peptide nucleic acid (PNA), a virus, a virus-like particle, a
nucleotide, a ribonucleotide, a synthetic analogue of a nucleotide,
a synthetic analogue of a ribonucleotide, a modified nucleotide, a
modified ribonucleotide, an amino acid, an amino acid analogue, a
modified amino acid, a modified amino acid analogue, a steroid, a
proteoglycan, a lipid, a fatty acid and a carbohydrate. An agent
may be in solution or in suspension (e.g., in crystalline,
colloidal or other particulate form). The agent may be in the form
of a monomer, dimer, oligomer, etc, or otherwise in a complex.
[0240] The terms "modulator", "antagonist" and "agent" are also
intended to include, a protein, polypeptide or peptide including,
but not limited to, a structural protein, an enzyme, a cytokine
(such as an interferon and/or an interleukin) an antibiotic, a
polyclonal or monoclonal antibody, or an effective part thereof,
such as an Fv fragment, which antibody or part thereof may be
natural, synthetic or humanised, a peptide hormone, a receptor, a
signalling molecule or other protein; a nucleic acid, as defined
below, including, but not limited to, an oligonucleotide or
modified oligonucleotide, an antisense oligonucleotide or modified
antisense oligonucleotide, cDNA, genomic DNA, an artificial or
natural chromosome (e.g. a yeast artificial chromosome) or a part
thereof, RNA, including mRNA, tRNA, rRNA or a ribozyme, or a
peptide nucleic acid (PNA); a virus or virus-like particles; a
nucleotide or ribonucleotide or synthetic analogue thereof, which
may be modified or unmodified; an amino acid or analogue thereof,
which may be modified or unmodified; a non-peptide (e.g., steroid)
hormone; a proteoglycan; a lipid; or a carbohydrate. Small
molecules, including inorganic and organic chemicals, which bind to
and occupy the active site of the polypeptide thereby making the
catalytic site inaccessible to substrate such that normal
biological activity is prevented, are also included. Examples of
small molecules include but are not limited to small peptides or
peptide-like molecules.
[0241] In a particular embodiment, the technique of RNA
interference (RNAi) may be used to abolish or knock out or reduce
gene activity, for example, Stella and/or Fragilis activity. The
overall strategy is to prepare double stranded RNA (dsRNA) specific
to each gene of interest and to transfect this into a cell of
interest to inhibit the expression of the particular gene.
[0242] The following protocol may be used: a sample of PCR product
is analysed by horizontal gel electrophoresis and the DNA purified
using a Qiagen QiaQuick PCR purification kit. 1 .mu.g of DNA is
used as the template in the preparation of gene specific single
stranded RNA using the Ambion T7 Megascript kit. Single stranded
RNA is produced from both strands of the template and is purified
and immediately annealed by heating to 90 degrees C. for 15 mins
followed by gradual cooling to room temperature overnight. A sample
of the dsRNA is analysed by horizontal gel electrophoresis, and
introduced into the relevant cell by conventional means.
Identifying Modulators, Agonists and Antagonists
[0243] Modulators, agonists and antagonists of Stella and/or
Fragilis activity or expression may be identified by any means
known in the art. Putative such molecules may be identified by
their binding to Stella and/or Fragilis, in an assay which detects
binding between Stella (or Fragilis as the case may be) and the
putative molecule.
[0244] Assays to detect modulators, agonists or antagonists
typically involve detecting modulation of any activity of Stella
and/or Fragilis in the presence, optionally together with detection
of modulation of activity in the absence, of a candidate molecule.
The assays involve contacting a candidate molecule with Stella or
Fragilis, whether in the form of a polypeptide, a nucleic acid
encoding the polypeptide, or a cell, organelle, extract, or other
material comprising such, with a candidate modulator. The relevant
activity of Stella or Fragilis (as described below) may be
detected, to establish whether the presence of the candidate
modulator has any effect. Promoter binding assays to detect
candidate modulators which bind to and/or affect the transcription
or expression of Stella and/or Fragilis may also be used. Candidate
modulators may then be chosen for further study, or isolated for
use. Details of such screening procedures are well known in the
art, and are for example described in, Handbook of Drug Screening,
edited by Ramakrishna Seethala, Prabhavathi B. Fernandes (2001, New
York, N.Y., Marcel Dekker, ISBN 0-8247-0562-9).
[0245] The screening methods described here preferably employ in
vivo assays, although they may be configured for in vitro use. In
vivo assays generally involve exposing a cell comprising Stella
and/or Fragilis to the candidate molecule. In in vitro assays,
Stella and/or Fragilis is exposed to the candidate molecule,
optionally in the presence of other components, such as crude or
semi-purified cell extract, or purified proteins. Where in vitro
assays are conducted, these preferably employ arrays of candidate
molecules (for example, an arrayed library). In vivo assays are
preferred. Preferably, therefore, the Stella and/or Fragilis is
comprised in a cell, preferably heterologously. Such a cell is
preferably a transgenic cell, which has been engineered to express
Stella and/or Fragilis.
[0246] It will be appreciated that any component of a cell
comprising Stella and/or Fragilis may be employed, such as an
organelle. A preferred embodiment utilises a nuclear preparation,
e.g., comprising a cell nucleus which comprises Stella and/or
Fragilis as described. The nuclear preparation may comprise one or
more nuclei, which may be permeabilised or semi-permeabilised, by
detergent treatment, for example.
[0247] Thus, in a specific embodiment, an assay format may include
the following: a multiwell microtitre plate is set up to include
one or more cells expressing Stella and/or Fragilis in each well;
individual candidate molecules, or pools of candidate molecules,
may be added to individual wells and modulation of Stella and/or
Fragilis activity measured. Where pools are used, these may be
subdivided in to further pools and tested in the same manner.
[0248] Alternatively or in addition to the assay methods described
above, "subtractive" procedures may also be used to identify
modulators, agonists or antagonists of Stella and/or Fragilis.
Under such "subtractive" procedures, a plurality of molecules is
provided, which comprises one or more candidate molecules capable
of functioning as a modulator (e.g., cell extract, nuclear extract,
library of molecules, etc), and one or more components is removed,
depleted or subtracted from the plurality of molecules. The
"subtracted" extract, etc, is then assayed for activity, by
exposure to a cell comprising Stella and/or Fragilis (or a
component thereof) as described.
[0249] Thus, for example, an `immunodepletion` assay may be
conducted to identify such modulators as follows. A nuclear extract
may be prepared from a pluripotent cell, for example, a pluripotent
EG/ES cell. The extract may be depleted or fractionated to remove
putative modulators, such as by use of immunodepletion with
appropriate antibodies. If the extract is depleted of a modulator,
it will lose the ability to affect Stella and/or Fragilis function
or activity or expression. A series of subtractions and/or
depletions may be required to identify the modulators, agonists or
antagonists.
[0250] It will also be appreciated that the above "depletion" or
"subtraction" assay may be used as a preliminary step to identify
putative modulatory factors for further screening. Furthermore, or
alternatively, the "depletion" or "subtraction" assay may be used
to confirm the modulatory activity of a molecule identified by
other means (for example, a "positive" screen as described
elsewhere in this document) as a putative modulator.
[0251] Candidate molecules subjected to the assay and which are
found to be of interest may be isolated and further studied.
Methods of isolation of molecules of interest will depend on the
type of molecule employed, whether it is in the form of a library,
how many candidate molecules are being tested at any one time,
whether a batch procedure is being followed, etc.
[0252] The candidate molecules may be provided in the form of a
library. In a preferred embodiment of the invention, more than one
candidate molecule is screened simultaneously. A library of
candidate molecules may be generated, for example, a polypeptide
library, a nucleic acid library, a library of compounds (such as a
combinatorial library), a library of antisense molecules such as
antisense DNA or antisense RNA, an antibody library etc, by means
known in the art. Such libraries are suitable for high-throughput
screening. Different cells comprising Stella and/or Fragilis may be
exposed to individual members of the library, and expression of the
reporter or reporters detected. Array technology may be employed
for this purpose. The cells may be spatially separated, for
example, in wells of a microtitre plate.
[0253] In a preferred embodiment, a small molecule library is
employed. By this term, we refer to a library of molecules, whose
molecular weights are individually less than about 50 kDa. In
particular embodiments, small molecule libraries comprise molecules
having molecular weights preferably less than about 30 kDa, more
preferably less than about 15 kDa, most preferably less than 10 kDa
or so. Such "small molecule" libraries may contain polypeptides,
small peptides, for example, peptides of 20 amino acids or fewer,
for example, 15, 10 or 5 amino acids, simple compounds, etc.
[0254] Alternatively or in addition, a combinatorial library, as
described in further detail below, may be screened for modulators,
antagonists or agonists of Stella and/or Fragilis.
[0255] Any of the activities of Stella and/or Fragilis may be used
as the basis of the assay. In particular, cellular activities
mediated by Stella and/or Fragilis may be assayed to identify
antagonists. For example, Fragilis family members are responsible
for homotypic adhesion between cells, and effects of the putative
antagonist or agonist on adhesion activity mediated by Fragilis
family members may be assayed using for example cell adhesion
assays as known in the art. Furthermore, we show that Fragilis
family members are capable of lengthening cell cycle times;
accordingly, the cell cycle period may be assayed in the presence
and absence of a candidate molecule to identify antagonists or
agonists of Fragilis activity.
Libraries
[0256] Libraries of candidate molecules, such as libraries of
polypeptides or nucleic acids, may be employed in the methods and
compositions described here. Such libraries are exposed to the
nucleic acid encoding the reporter (or cell comprising such a
nucleic acid), for the detection of expression of the reporter.
[0257] Selection protocols for isolating desired members of large
libraries are known in the art, as typified by phage display
techniques. Such systems, in which diverse peptide sequences are
displayed on the surface of filamentous bacteriophage (Scott and
Smith (1990 supra), have proven useful for creating libraries of
antibody fragments (and the nucleotide sequences that encoding
them) for the in vitro selection and amplification of specific
antibody fragments that bind a target antigen. The nucleotide
sequences encoding the V.sub.H and V.sub.L regions are linked to
gene fragments which encode leader signals that direct them to the
periplasmic space of E. coli and as a result the resultant antibody
fragments are displayed on the surface of the bacteriophage,
typically as fusions to bacteriophage coat proteins (e.g., pIII or
pVIII). Alternatively, antibody fragments are displayed externally
on lambda phage capsids (phagebodies). An advantage of phage-based
display systems is that, because they are biological systems,
selected library members can be amplified simply by growing the
phage containing the selected library member in bacterial cells.
Furthermore, since the nucleotide sequence that encodes the
polypeptide library member is contained on a phage or phagemid
vector, sequencing, expression and subsequent genetic manipulation
is relatively straightforward.
[0258] Methods for the construction of bacteriophage antibody
display libraries and lambda phage expression libraries are well
known in the art (McCafferty et al. (1990) supra; Kang et al.
(1991) Proc. Natl. Acad. Sci. USA., 88: 4363; Clackson et al.
(1991) Nature, 352: 624; Lowman et al. (1991) Biochemistry, 30:
10832; Burton et al. (1991) Proc. Natl. Acad Sci U.S.A., 88:10134;
Hoogenboom et al. (1991) Nucleic Acids Res., 19: 4133; Chang et al.
(1991) J. Immunol., 147: 3610; Breitling et al. (1991) Gene, 104:
147; Marks et al. (1991) supra; Barbas et al. (1992) supra; Hawkins
and Winter (1992) J. Immunol., 22: 867; Marlcs et al., 1992, J.
Biol. Chem., 267: 16007; Lerner et al. (1992) Science, 258: 1313,
incorporated herein by reference). Such techniques may be modified
if necessary for the expression generally of polypeptide
libraries.
[0259] One particularly advantageous approach has been the use of
scFv phage-libraries (Bird, R. E., et al. (1988) Science 242:
423-6, Huston et al., 1988, Proc. Natl. Acad. Sci. U.S.A., 85:
5879-5883; Chaudhary et al. (1990) Proc. Natl. Acad. Sci. U.S.A.,
87: 1066-1070; McCafferty et al. (1990) supra; Clackson et al.
(1991) supra; Marks et al. (1991) supra; Chiswell et al. (1992)
Trends Biotech., 10: 80; Marks et al. (1992) supra). Various
embodiments of scFv libraries displayed on bacteriophage coat
proteins have been described. Refinements of phage display
approaches are also known, for example as described in WO96/06213
and WO92/01047 (Medical Research Council et al.) and WO97/08320
(Morphosys, supra), which are incorporated herein by reference.
[0260] Alternative library selection technologies include
bacteriophage lambda expression systems, which may be screened
directly as bacteriophage plaques or as colonies of lysogens, both
as previously described (Huse et al. (1989) Science, 246: 1275;
Caton and Koprowski (1990) Proc. Natl. Acad. Sci. U.S.A., 87;
Mullinax et al. (1990) Proc. Natl. Acad. Sci. U.S.A., 87: 8095;
Persson et al. (1991) Proc. Natl. Acad. Sci. U.S.A., 88: 2432) and
are of use in the invention. These expression systems may be used
to screen a large number of different members of a library, in the
order of about 10.sup.6 or even more. Other screening systems rely,
for example, on direct chemical synthesis of library members. One
early method involves the synthesis of peptides on a set of pins or
rods, such as described in WO84/03564. A similar method involving
peptide synthesis on beads, which forms a peptide library in which
each bead is an individual library member, is described in U.S.
Pat. No. 4,631,211 and a related method is described in WO92/00091.
A significant improvement of the bead-based methods involves
tagging each bead with a unique identifier tag, such as an
oligonucleotide, so as to facilitate identification of the amino
acid sequence of each library member. These improved bead-based
methods are described in WO93/06121.
[0261] Another chemical synthesis method involves the synthesis of
arrays of peptides (or peptidomimetics) on a surface in a manner
that places each distinct library member (e.g., unique peptide
sequence) at a discrete, predefined location in the array. The
identity of each library member is determined by its spatial
location in the array. The locations in the array where binding
interactions between a predetermined molecule (e.g., a receptor)
and reactive library members occur is determined, thereby
identifying the sequences of the reactive library members on the
basis of spatial location. These methods are described in U.S. Pat.
No. 5,143,854; WO90/15070 and WO92/10092; Fodor et al. (1991)
Science, 251: 767; Dower and Fodor (1991) Ann. Rep. Med. Chem., 26:
271.
[0262] Other systems for generating libraries of polypeptides or
nucleotides involve the use of cell-free enzymatic machinery for
the in vitro synthesis of the library members. In one method, RNA
molecules are selected by alternate rounds of selection against a
target ligand and PCR amplification (Tuerk and Gold (1990) Science,
249: 505; Ellington and Szostak (1990) Nature, 346: 818). A similar
technique may be used to identify DNA sequences which bind a
predetermined human transcription factor (Thiesen and Bach (1990)
Nucleic Acids Res., 18: 3203; Beaudry and Joyce (1992) Science,
257: 635; WO92/05258 and WO92/14843). In a similar way, in vitro
translation can be used to synthesise polypeptides as a method for
generating large libraries. These methods which generally comprise
stabilised polysome complexes, are described further in WO88/08453,
WO90/05785, WO90/07003, WO91/02076, WO91/05058, and WO92/02536.
Alternative display systems which are not phage-based, such as
those disclosed in WO95/22625 and WO95/11922 (Affymax) use the
polysomes to display polypeptides for selection. These and all the
foregoing documents also are incorporated herein by reference.
[0263] The library may in particular comprise a library of zinc
fingers; zinc fingers are known in the art and act as transcription
factors. Suitable zinc finger libraries are disclosed in, for
example, WO 96/06166 and WO 98/53057. Construction of zinc finger
libraries may utilise rules for determining interaction with
specific DNA sequences, as disclosed in for example WO 98/53058 and
WO 98/53060. Zinc fingers capable of interacting specifically with
methylated DNA are disclosed in WO 99/47656. The above zinc finger
libraries may be immobilised in the form of an array, for example
as disclosed in WO 01/25417. Accordingly, preferred molecules
capable of altering the potency of a cell include zinc fingers.
Combinatorial Libraries
[0264] Libraries, in particular, libraries of candidate molecules,
may suitably be in the form of combinatorial libraries (also known
as combinatorial chemical libraries).
[0265] A "combinatorial library", as the term is used in this
document, is a collection of multiple species of chemical compounds
that consist of randomly selected subunits. Combinatorial libraries
may be screened for molecules which are capable of causing
expression of the reporter, and optionally of altering the potency
of the cell.
[0266] Various combinatorial libraries of chemical compounds are
currently available, including libraries active against proteolytic
and non-proteolytic enzymes, libraries of agonists and antagonists
of G-protein coupled receptors (GPCRs), libraries active against
non-GPCR targets (e.g., integrins, ion channels, domain
interactions, nuclear receptors, and transcription factors) and
libraries of whole-cell oncology and anti-infective targets, among
others. A comprehensive review of combinatorial libraries, in
particular their construction and uses is provided in Dolle and
Nelson (1999), Journal of Combinatorial Chemistry, Vol 1 No 4,
235-282. Reference is also made to Combinatorial peptide library
protocols (edited by Shmuel Cabilly, Totowa, N.J.: Humana Press, c
1998 Methods in Molecular Biology; v. 87). Specific combinatorial
libraries and methods for their construction are disclosed in U.S.
Pat. No. 6,168,914 (Campbell.TM., et al), as well as in Baldwin et
al. (1995), "Synthesis of a Small Molecule Library Encoded with
Molecular Tags," J. Am. Chem. Soc. 117:5588-5589, and in the
references mentioned in those documents.
[0267] In a preferred embodiment, the combinatorial library which
is screened is one which is designed to potentially include
molecules which interact with a component of the cell to influence
gene expression. For example, combinatorial libraries against
chromatin structural proteins may be screened. Other libraries
which are useful for this embodiment include combinatorial
libraries against histone modification enzymes (e.g., histone
acetylation or histone methylation enzymes), or DNA modification,
for example, DNA methylation or demethylation.
[0268] Further references describing chemical combinatorial
libraries, their production and use include those available from
the URL http://www.netsci.org/Science/Combichem/, including The
Chemical Generation of Molecular Diversity. Michael R. Pavia,
Sphinx Pharmaceuticals, A Division of Eli Lilly (Published July,
1995); Combinatorial Chemistry: A Strategy for the Future--MDL
Information Systems discusses the role its Project Library plays in
managing diversity libraries (Published July, 1995); Solid Support
Combinatorial Chemistry in Lead Discovery and SAR Optimization,
Adnan M. M. Mjalli and Barry E. Toyonaga, Ontogen Corporation
(Published July, 1995); Non-Peptidic Bradykinin Receptor
Antagonists From a Structurally Directed Non-Peptide Library.
Sarvajit Chakravarty, Babu J. Mavunkel, Robin Andy, Donald J.
Kyle*, Scios Nova Inc. (Published July, 1995); Combinatorial
Chemistry Library Design using Pharmacophore Diversity Keith Davies
and Clive Briant, Chemical Design Ltd. (Published July, 1995); A
Database System for Combinatorial Synthesis Experiments--Craig
James and David Weininger, Daylight Chemical Information Systems,
Inc. (Published July, 1995); An Information Management Architecture
for Combinatorial Chemistry, Keith Davies and Catherine White,
Chemical Design Ltd. (Published July, 1995); Novel Software Tools
for Addressing Chemical Diversity, R. S. Pearlman, Laboratory for
Molecular Graphics and Theoretical Modeling, College of Pharmacy,
University of Texas (Published June/July, 1996); Opportunities for
Computational Chemists Afforded by the New Strategies in Drug
Discovery: An Opinion, Yvonne Connolly Martin, Computer Assisted
Molecular Design Project, Abbott Laboratories (Published June/July,
1996); Combinatorial Chemistry and Molecular Diversity Course at
the University of Louisville: A Description, Arno F. Spatola,
Department of Chemistry, University of Louisville (Published
June/July, 1996); Chemically Generated Screening Libraries: Present
and Future. Michael R. Pavia, Sphinx Pharmaceuticals, A Division of
Eli Lilly (Published June/July, 1996); Chemical Strategies For
Introducing Carbohydrate Molecular Diversity Into The Drug
Discovery Process. Michael J. Sofia, Transcell Technologies Inc.
(Published June/July, 1996); Data Management for Combinatorial
Chemistry. Maryjo Zaborowski, Chiron Corporation and Sheila H.
DeWitt, Parke-Davis Pharmaceutical Research, Division of
Warner-Lambert Company (Published November, 1995); and The Impact
of High Throughput Organic Synthesis on R&D in Bio-Based
Industries, John P. Devlin (Published March, 1996).
[0269] Techniques in combinatorial chemistry are gaining wide
acceptance among modern methods for the generation of new
pharmaceutical leads (Gallop, M. A. et al., 1994, J. Med. Chem.
37:1233-1251; Gordon, E. M. et al., 1994, J. Med. Chem.
37:1385-1401.). One combinatorial approach in use is based on a
strategy involving the synthesis of libraries containing a
different structure on each particle of the solid phase support,
interaction of the library with a soluble receptor, identification
of the `bead` which interacts with the macromolecular target, and
determination of the structure carried by the identified `bead`
(Lam, K. S. et al., 1991, Nature 354:82-84). An alternative to this
approach is the sequential release of defined aliquots of the
compounds from the solid support, with subsequent determination of
activity in solution, identification of the particle from which the
active compound was released, and elucidation of its structure by
direct sequencing (Salmon, S. E. et al., 1993, Proc. Natl. Acad.
Sci. USA 90:11708-11712), or by reading its code (Kerr, J. M. et
al., 1993, J. Am. Chem. Soc. 115:2529-2531; Nikolaiev, V. et al.,
1993, Pept. Res. 6:161-170; Ohlmeyer, M. H. J. et al., 1993, Proc.
Natl. Acad. Sci. USA 90:10922-10926).
[0270] Soluble random combinatorial libraries may be synthesized
using a simple principle for the generation of equimolar mixtures
of peptides which was first described by Furka (Furka, A. et al.,
1988, Xth International Symposium on Medicinal Chemistry, Budapest
1988; Furka, A. et al., 1988, 14th International Congress of
Biochemistry, Prague 1988; Furka, A. et al., 1991, Int. J. Peptide
Protein Res. 37:487-493). The construction of soluble libraries for
iterative screening has also been described (Houghten, R. A. et al.
1991, Nature 354:84-86). K. S. Lam disclosed the novel and
unexpectedly powerful technique of using insoluble random
combinatorial libraries. Lam synthesized random combinatorial
libraries on solid phase supports, so that each support had a test
compound of uniform molecular structure, and screened the libraries
without prior removal of the test compounds from the support by
solid phase binding protocols (Lam, K. S. et al., 1991, Nature
354:82-84).
[0271] Thus, a library of candidate molecules may be a synthetic
combinatorial library (e.g., a combinatorial chemical library), a
cellular extract, a bodily fluid (e.g., urine, blood, tears, sweat,
or saliva), or other mixture of synthetic or natural products
(e.g., a library of small molecules or a fermentation mixture).
[0272] A library of molecules may include, for example, amino
acids, oligopeptides, polypeptides, proteins, or fragments of
peptides or proteins; nucleic acids (e.g., antisense; DNA; RNA; or
peptide nucleic acids, PNA); aptamers; or carbohydrates or
polysaccharides. Each member of the library can be singular or can
be a part of a mixture (e.g., a compressed library). The library
may contain purified compounds or can be "dirty" (i.e., containing
a significant quantity of impurities).
[0273] Commercially available libraries (e.g., from Affymetrix,
ArQule, Neose Technologies, Sarco, Ciddco, Oxford Asymmetry,
Maybridge, Aldrich, Panlabs, Pharmacopoeia, Sigma, or Tripose) may
also be used with the methods described here.
[0274] In addition to libraries as described above, special
libraries called diversity files can be used to assess the
specificity, reliability, or reproducibility of the new methods.
Diversity files contain a large number of compounds (e.g., 1000 or
more small molecules) representative of many classes of compounds
that could potentially result in nonspecific detection in an assay.
Diversity files are commercially available or can also be assembled
from individual compounds commercially available from the vendors
listed above.
Antibodies
[0275] Specific antagonists of Stella and/or Fragilis, which may be
used to regulate the activity of these proteins (for example, for
methods of treating or preventing diseases such as cancer) may
include antibodies against the protein(s).
[0276] Antibodies, as used herein, refers to complete antibodies or
antibody fragments capable of binding to a selected target, and
including Fv, ScFv, Fab' and F(ab').sub.2, monoclonal and
polyclonal antibodies, engineered antibodies including chimeric,
CDR-grafted and humanised antibodies, and artificially selected
antibodies produced using phage display or alternative techniques.
Small-fragments, such as Fv and ScFv, possess advantageous
properties for diagnostic and therapeutic applications on account
of their small size and consequent superior tissue
distribution.
[0277] The antibodies according described here are especially
indicated for the detection of PGCs and other pluripotent cells,
such as ES or EG cells. Accordingly, they may be altered antibodies
comprising an effector protein such as a label. Especially
preferred are labels which allow the imaging of the distribution of
the antibody in vivo or in vitro. Such labels may be radioactive
labels or radiopaque labels, such as metal particles, which are
readily visualisable within an embryo or a cell mass. Moreover,
they may be fluorescent labels or other labels which are
visualisable on tissue samples.
[0278] Recombinant DNA technology may be used to improve the
antibodies as described here. Thus, chimeric antibodies may be
constructed in order to decrease the immunogenicity thereof in
diagnostic or therapeutic applications. Moreover, immunogenicity
may be minimised by humanising the antibodies by CDR grafting [see
European Patent Application 0 239 400 (Winter)] and, optionally,
framework modification [EP 0 239 400].
[0279] Antibodies may be obtained from animal serum, or, in the
case of monoclonal antibodies or fragments thereof, produced in
cell culture. Recombinant DNA technology may be used to produce the
antibodies according to established procedure, in bacterial or
preferably mammalian cell culture. The selected cell culture system
preferably secretes the antibody product.
[0280] Therefore, we disclose a process for the production of an
antibody comprising culturing a host, e.g. E. coli or a mammalian
cell, which has been transformed with a hybrid vector comprising an
expression cassette comprising a promoter operably linked to a
first DNA sequence encoding a signal peptide linked in the proper
reading frame to a second DNA sequence encoding said antibody
protein, and isolating said protein.
[0281] Multiplication of hybridoma cells or mammalian host cells in
vitro is carried out in suitable culture media, which are the
customary standard culture media, for example Dulbecco's Modified
Eagle Medium (DMEM) or RPMI 1640 medium, optionally replenished by
a mammalian serum, e.g. foetal calf serum, or trace elements and
growth sustaining supplements, e.g. feeder cells such as normal
mouse peritoneal exudate cells, spleen cells, bone marrow
macrophages, 2-aminoethanol, insulin, transferrin, low density
lipoprotein, oleic acid, or the like. Multiplication of host cells
which are bacterial cells or yeast cells is likewise carried out in
suitable culture media known in the art, for example for bacteria
in medium LB, NZCYM, NZYM, NZM, Terrific Broth, SOB, SOC,
2.times.YT, or M9 Minimal Medium, and for yeast in medium YPD,
YEPD, Minimal Medium, or Complete Minimal Dropout Medium.
[0282] In vitro production provides relatively pure antibody
preparations and allows scale-up to give large amounts of the
desired antibodies. Techniques for bacterial cell, yeast or
mammalian cell cultivation are known in the art and include
homogeneous suspension culture, e.g. in an airlift reactor or in a
continuous stirrer reactor, or immobilised or entrapped cell
culture, e.g. in hollow fibres, microcapsules, on agarose
microbeads or ceramic cartridges.
[0283] Large quantities of the desired antibodies can also be
obtained by multiplying mammalian cells in vivo. For this purpose,
hybridoma cells producing the desired antibodies are injected into
histocompatible mammals to cause growth of antibody-producing
tumours. Optionally, the animals are primed with a hydrocarbon,
especially mineral oils such as pristane (tetramethyl-pentadecane),
prior to the injection. After one to three weeks, the antibodies
are isolated from the body fluids of those mammals. For example,
hybridoma cells obtained by fusion of suitable myeloma cells with
antibody-producing spleen cells from Balb/c mice, or transfected
cells derived from hybridoma cell line Sp2/0 that produce the
desired antibodies are injected intraperitoneally into Balb/c mice
optionally pre-treated with pristane, and, after one to two weeks,
ascitic fluid is taken from the animals.
[0284] The foregoing, and other, techniques are discussed in, for
example, Kohler and Milstein, (1975) Nature 256:495-497; U.S. Pat.
No. 4,376,110; Harlow and Lane, Antibodies: a Laboratory Manual,
(1988) Cold Spring Harbor, incorporated herein by reference.
Techniques for the preparation of recombinant antibody molecules is
described in the above references and also in, for example, EP
0623679; EP 0368684 and EP 0436597, which are incorporated herein
by reference.
[0285] The cell culture supernatants are screened for the desired
antibodies, preferentially by immunofluorescent staining of PGCs or
other pluripotent cells, such as ES or EG cells, by immunoblotting,
by an enzyme immunoassay, e.g. a sandwich assay or a dot-assay, or
a radioimmunoassay.
[0286] For isolation of the antibodies, the immunoglobulins in the
culture supernatants or in the ascitic fluid may be concentrated,
e.g. by precipitation with ammonium sulphate, dialysis against
hygroscopic material such as polyethylene glycol, filtration
through selective membranes, or the like. If necessary and/or
desired, the antibodies are purified by the customary
chromatography methods, for example gel filtration, ion-exchange
chromatography, chromatography over DEAE-cellulose and/or (immuno-)
affinity chromatography, e.g. affinity chromatography with Fragilis
or Stella, or fragments thereof, or with Protein-A.
[0287] Hybridoma cells secreting the monoclonal antibodies are also
provided. Preferred hybridoma cells are genetically stable, secrete
monoclonal antibodies of the desired specificity and can be
activated from deep-frozen cultures by thawing and recloning.
[0288] Also included is a process for the preparation of a
hybridoma cell line secreting monoclonal antibodies directed to
Fragilis and/or Stella, characterised in that a suitable mammal,
for example a Balb/c mouse, is immunised with a one or more
Fragilis or Stella polypeptides, or antigenic fragments thereof,
antibody-producing cells of the immunised mammal are fused with
cells of a suitable myeloma cell line, the hybrid cells obtained in
the fusion are cloned, and cell clones secreting the desired
antibodies are selected. For example spleen cells of Balb/c mice
immunised with Fragilis and/or Stella are fused with cells of the
myeloma cell line PAI or the myeloma cell line Sp2/0-Ag14, the
obtained hybrid cells are screened for secretion of the desired
antibodies, and positive hybridoma cells are cloned.
[0289] Preferred is a process for the preparation of a hybridoma
cell line, characterised in that Balb/c mice are immunised by
injecting subcutaneously and/or intraperitoneally between 10 and
10.sup.7 and 10.sup.8 cells expressing Fragilis and/or Stella and a
suitable adjuvant several times, e.g. four to six times, over
several months, e.g. between two and four months, and spleen cells
from the immunised mice are taken two to four days after the last
injection and fused with cells of the myeloma cell line PAI in the
presence of a fusion promoter, preferably polyethylene glycol.
Preferably the myeloma cells are fused with a three- to twentyfold
excess of spleen cells from the immunised mice in a solution
containing about 30% to about 50% polyethylene glycol of a
molecular weight around 4000. After the fusion the cells are
expanded in suitable culture media as described hereinbefore,
supplemented with a selection medium, for example HAT medium, at
regular intervals in order to prevent normal myeloma cells from
overgrowing the desired hybridoma cells.
[0290] Recombinant DNAs comprising an insert coding for a heavy
chain variable domain and/or for a light chain variable domain of
antibodies directed to Fragilis and/or Stella as described
hereinbefore are also disclosed. By definition such DNAs comprise
coding single stranded DNAs, double stranded DNAs consisting of
said coding DNAs and of complementary DNAs thereto, or these
complementary (single stranded) DNAs themselves.
[0291] Furthermore, DNA encoding a heavy chain variable domain
and/or for a light chain variable domain of antibodies directed to
Fragilis and/or Stella can be enzymatically or chemically
synthesised DNA having the authentic DNA sequence coding for a
heavy chain variable domain and/or for the light chain variable
domain, or a mutant thereof. A mutant of the authentic DNA is a DNA
encoding a heavy chain variable domain and/or a light chain
variable domain of the above-mentioned antibodies in which one or
more amino acids are deleted or exchanged with one or more other
amino acids. Preferably said modification(s) are outside the CDRs
of the heavy chain variable domain and/or of the light chain
variable domain of the antibody. Such a mutant DNA is also intended
to be a silent mutant wherein one or more nucleotides are replaced
by other nucleotides with the new codons coding for the same amino
acid(s). Such a mutant sequence is also a degenerated sequence.
Degenerated sequences are degenerated within the meaning of the
genetic code in that an unlimited number of nucleotides are
replaced by other nucleotides without resulting in a change of the
amino acid sequence originally encoded. Such degenerated sequences
may be useful due to their different restriction sites and/or
frequency of particular codons which are preferred by the specific
host, particularly E. coli, to obtain an optimal expression of the
heavy chain murine variable domain and/or a light chain murine
variable domain.
[0292] The term mutant is intended to include a DNA mutant obtained
by in vitro mutagenesis of the authentic DNA according to methods
known in the art.
[0293] For the assembly of complete tetrameric immunoglobulin
molecules and the expression of chimeric antibodies, the
recombinant DNA inserts coding for heavy and light chain variable
domains are fused with the corresponding DNAs coding for heavy and
light chain constant domains, then transferred into appropriate
host cells, for example after incorporation into hybrid
vectors.
[0294] Also disclosed are recombinant DNAs comprising an insert
coding for a heavy chain murine variable domain of an antibody
directed to Fragilis and/or Stella fused to a human constant domain
g, for example .gamma.1, .gamma.2, .gamma.3 or .gamma.4, preferably
.gamma.1 or .gamma.4. Likewise recombinant DNAs comprising an
insert coding for a light chain murine variable domain of an
antibody directed to Fragilis and/or Stella fused to a human
constant domain .kappa. or .lamda., preferably .kappa. are also
disclosed.
[0295] In another embodiment, we disclose recombinant DNAs coding
for a recombinant-polypeptide wherein the heavy chain variable
domain and the light chain variable domain are linked by way of a
spacer group, optionally comprising a signal sequence facilitating
the processing of the antibody in the host cell and/or a DNA coding
for a peptide facilitating the purification of the antibody and/or
a cleavage site and/or a peptide spacer and/or an effector
molecule.
[0296] The DNA coding for an effector molecule is intended to be a
DNA coding for the effector molecules useful in diagnostic or
therapeutic applications. Thus, effector molecules which are toxins
or enzymes, especially enzymes capable of catalysing the activation
of prodrugs, are particularly indicated. The DNA encoding such an
effector molecule has the sequence of a naturally occurring enzyme
or toxin encoding DNA, or a mutant thereof, and can be prepared by
methods well known in the art.
Anti-Peptide Stella and Fragilis Antibodies
[0297] Anti-peptide antibodies may be produced against Stella and
Fragilis peptide sequences. The sequences chosen may be based on
the mouse sequences as follow:
TABLE-US-00018 Fragilis: ASGGQPPNYERIKEEYE and RDRKMVGDVTGAQAYA
Stella: MEEPSEKVDPMKDPET and CHYQRWDPSENAKIGKN
[0298] Corresponding sequences from human Stella and Fragilis may
be chosen for use in eliciting anti-peptide antibodies from
immunised animals. Antibodies may be produced by injection into
rabbits, and other conventional means, as described in for example,
Harlow and Lane (supra).
[0299] Antibodies are checked by Elisa assay and by Western
blotting, and used for immunostaining as described in the
Examples.
Therapeutic Peptides
[0300] The polypeptides disclosed here, for example, Stella and
Fragilis polypeptides, may be used therapeutically for treatment of
various diseases, including cancer, in the form of peptides
comprising any portion of their sequence.
[0301] Where such Stella and/or Fragilis peptides are used
therapeutically, it is preferred to use peptides that do not
consist solely of naturally-occurring amino acids but which have
been modified, for example to reduce immunogenicity, to increase
circulatory half-life in the body of the patient, to enhance
bio-availability and/or to enhance efficacy and/or specificity.
[0302] A number of approaches have been used to modify peptides for
therapeutic application. One approach is to link the peptides or
proteins to a variety of polymers, such as polyethylene glycol
(PEG) and polypropylene glycol (PPG)--see for example U.S. Pat.
Nos. 5,091,176, 5,214,131 and U.S. Pat. No. 5,264,209.
[0303] Replacement of naturally-occurring amino acids with a
variety of uncoded or modified amino acids such as D-amino acids
and N-methyl amino acids may also be used to modify the Stella
and/or Fragilis peptides.
[0304] Another approach is to use bi-functional crosslinkers, such
as N-succinimidyl 3-(2 pyridyldithio) propionate, succinimidyl
6-[3-2 pyridyldithio) propionamido]hexanoate, and sulfosuccinimidyl
6-[3-(2 pyridyldithio) propionamido]hexanoate (see U.S. Pat. No.
5,580,853).
[0305] It may be desirable to use derivatives of the peptides
disclosed here which are conformationally constrained.
Conformational constraint refers to the stability and preferred
conformation of the three-dimensional shape assumed by a peptide.
Conformational constraints include local constraints, involving
restricting the conformational mobility of a single residue in a
peptide; regional constraints, involving restricting the
conformational mobility of a group of residues, which residues may
form some secondary structural unit; and global constraints,
involving the entire peptide structure.
[0306] The active conformation of the peptide may be stabilised by
a covalent modification, such as cyclization or by incorporation of
.gamma.-lactam or other types of bridges. For example, side chains
can be cyclized to the backbone so as create a L-.gamma.-lactam
moiety on each side of the interaction site. See, generally, Hruby
et al., "Applications of Synthetic Peptides," in Synthetic
Peptides: A User's Guide: 259-345 (W. H. Freeman & Co. 1992).
Cyclization also can be achieved, for example, by formation of
cystine bridges, coupling of amino and carboxy terminal groups of
respective terminal amino acids, or coupling of the amino group of
a Lys residue or a related homologue with a carboxy group of Asp,
Glu or a related homologue. Coupling of the alpha-amino group of a
polypeptide with the epsilon-amino group of a lysine residue, using
iodoacetic anhydride, can be also undertaken. See Wood and Wetzel,
1992, Int'l J. Peptide Protein Res. 39, 533-39.
[0307] Another approach described in U.S. Pat. No. 5,891,418 is to
include a metal-ion complexing backbone in the peptide structure.
Typically, the preferred metal-peptide backbone is based on the
requisite number of particular coordinating groups required by the
co-ordination sphere of a given complexing metal ion. In general,
most of the metal ions that may prove useful have a co-ordination
number of four to six. The nature of the coordinating groups in the
peptide chain includes nitrogen atoms with amine, amide, imidazole,
or guanidino functionalities; sulfur atoms of thiols or disulfides;
and oxygen atoms of hydroxy, phenolic, carbonyl, or carboxyl
functionalities. In addition, the peptide chain or individual amino
acids can be chemically altered to include a coordinating group,
such as for example oxime, hydrazino, sulfhydryl, phosphate, cyano,
pyridino, piperidino, or morpholino. The peptide construct can be
either linear or cyclic, however a linear construct is typically
preferred. One example of a small linear peptide is Gly-Gly-Gly-Gly
which has four nitrogen atoms (an N.sub.4 complexation system) in
the back bone that can complex to a metal ion with a co-ordination
number of four.
[0308] A further technique for improving the properties of
therapeutic peptides is to use non-peptide peptidomimetics. A wide
variety of useful techniques may be used to elucidating the precise
structure of a peptide. These techniques include amino acid
sequencing, x-ray crystallography, mass spectroscopy, nuclear
magnetic resonance spectroscopy, computer-assisted molecular
modelling, peptide mapping, and combinations thereof. Structural
analysis of a peptide generally provides a large body of data which
comprise the amino acid sequence of the peptide as well as the
three-dimensional positioning of its atomic components. From this
information, non-peptide peptidomimetics may be designed that have
the required chemical functionalities for therapeutic activity but
are more stable, for example less susceptible to biological
degradation. An example of this approach is provided in U.S. Pat.
No. 5,811,512.
[0309] Techniques for chemically synthesising therapeutic peptides
are described in the above references and also reviewed by Borgia
and Fields, 2000, TibTech 18, 243-251 and described in detail in
the references contained therein.
Transgenic Animals
[0310] We further describe transgenic animals capable of expressing
natural or recombinant Stella and/or Fragilis, or a homologue,
variant or derivative, at elevated or reduced levels compared to
the normal expression level. Included are transgenic animals
("Stella knockout"s or "Fragilis knockout"s) which do not express
functional Stella and/or Fragilis, as the case may be. The Stella
and Fragilis knockouts may arise as a result of functional
disruption of the Stella and/or Fragilis gene or any portion of
that gene, including one or more loss of function mutations,
including a deletion or replacement, of the Stella and/or Fragilis
gene. The mutations include single point mutations, and may target
coding or non-coding regions of Stella and/or Fragilis.
[0311] Preferably, such a transgenic animal is a non-human mammal,
such as a pig, a sheep or a rodent. Most preferably the transgenic
animal is a mouse or a rat. Such transgenic animals may be used in
screening procedures to identify agonists and/or antagonists of
Stella and/or Fragilis, as well as to test for their efficacy as
treatments for diseases in vivo.
[0312] Mice which are null for Stella and/or Fragilis may be used
for various purposes. For example, transgenic animals that have
been engineered to be deficient in the production of Stella and/or
Fragilis may be used in assays to identify agonists and/or
antagonists of Stella and/or Fragilis. One assay is designed to
evaluate a potential drug (aa candidate ligand or compound) to
determine if it produces a physiological response in the absence
Stella and/or Fragilis. This may be accomplished by administering
the drug to a transgenic animal as discussed above, and then
assaying the animal for a particular response.
[0313] Tissues derived from the Stella and/or Fragilis knockout
animals may be used in binding assays to determine whether the
potential drug (a candidate ligand or compound) binds to Stella or
Fragilis, as the case may be. Such assays can be conducted by
obtaining a first Stella and/or Fragilis preparation from the
transgenic animal engineered to be deficient in Stella and/or
Fragilis production and a second Stella and/or Fragilis preparation
from a source known to bind any identified ligands or compounds. In
general, the first and second preparations will be similar in all
respects except for the source from which they are obtained. For
example, if brain tissue from a transgenic animal (such as
described above and below) is used in an assay, comparable brain
tissue from a normal (wild type) animal is used as the source of
the second preparation. Each of the preparations is incubated with
a ligand known to bind to Stella and/or Fragilis, both alone and in
the presence of the candidate ligand or compound. Preferably, the
candidate ligand or compound will be examined at several different
concentrations.
[0314] The extent to which binding by the known ligand is displaced
by the test compound is determined for both the first and second
preparations. Tissues derived from transgenic animals may be used
in assays directly or the tissues may be processed to isolate
Stella and/or Fragilis proteins, which are themselves used in the
assays. A preferred transgenic animal is the mouse. The ligand may
be labeled using any means compatible with binding assays. This
would include, without limitation, radioactive, enzymatic,
fluorescent or chemiluminescent labeling (as well as other
labelling techniques as described in further detail above).
[0315] Furthermore, antagonists of Stella and/or Fragilis may be
identified by administering candidate compounds, etc, to wild type
animals expressing functional Stella and/or Fragilis, and animals
identified which exhibit any of the phenotypic characteristics
associated with reduced or abolished expression of Stella and/or
Fragilis function.
[0316] Methods for generating non-human transgenic animal are known
in the art, and are described in further detail in the Examples
below. Transgenic gene constructs can be introduced into the germ
line of an animal to make a transgenic mammal. For example, one or
several copies of the construct may be incorporated into the genome
of a mammalian embryo by standard transgenic techniques.
[0317] In an exemplary embodiment, the transgenic non-human animals
described here are produced by introducing transgenes into the
germline of the non-human animal. Embryonal target cells at various
developmental stages can be used to introduce transgenes. Different
methods are used depending on the stage of development of the
embryonal target cell. The specific line(s) of any animal used to
produce transgenic animals are selected for general good health,
good embryo yields, good pronuclear visibility in the embryo, and
good reproductive fitness. In addition, the haplotype is a
significant factor.
[0318] Introduction of the transgene into the embryo can be
accomplished by any means known in the art such as, for example,
microinjection, electroporation, or lipofection. For example, the
Stella or Fragilis transgene can be introduced into a mammal by
microinjection of the construct into the pronuclei of the
fertilized mammalian egg(s) to cause one or more copies of the
construct to be retained in the cells of the developing mammal(s).
Following introduction of the transgene construct into the
fertilized egg, the egg may be incubated in vitro for varying
amounts of time, or reimplanted into the surrogate host, or both.
In vitro incubation to maturity is also included. One common method
in to incubate the embryos in vitro for about 1-7 days, depending
on the species, and then reimplant them into the surrogate
host.
[0319] The progeny of the transgenically manipulated embryos can be
tested for the presence of the construct by Southern blot analysis
of the segment of tissue. If one or more copies of the exogenous
cloned construct remains stably integrated into the genome of such
transgenic embryos, it is possible to establish permanent
transgenic mammal lines carrying the transgenically added
construct.
[0320] The litters of transgenically altered mammals can be assayed
after birth for the incorporation of the construct into the genome
of the offspring. Preferably, this assay is accomplished by
hybridizing a probe corresponding to the DNA sequence coding for
the desired recombinant protein product or a segment thereof onto
chromosomal material from the progeny. Those mammalian progeny
found to contain at least one copy of the construct in their genome
are grown to maturity.
[0321] For the purposes of this document, a zygote is essentially
the formation of a diploid cell which is capable of developing into
a complete organism. Generally, the zygote will be comprised of an
egg containing a nucleus formed, either naturally or artificially,
by the fusion of two haploid nuclei from a gamete or gametes. Thus,
the gamete nuclei must be ones which are naturally compatible,
i.e., ones which result in a viable zygote capable of undergoing
differentiation and developing into a functioning organism.
Generally, a euploid zygote is preferred. If an aneuploid zygote is
obtained, then the number of chromosomes should not vary by more
than one with respect to the euploid number of the organism from
which either gamete originated.
[0322] In addition to similar biological considerations, physical
ones also govern the amount (e.g., volume) of exogenous genetic
material which can be added to the nucleus of the zygote or to the
genetic material which forms a part of the zygote nucleus. If no
genetic material is removed, then the amount of exogenous genetic
material which can be added is limited by the amount which will be
absorbed without being physically disruptive. Generally, the volume
of exogenous genetic material inserted will not exceed about 10
picoliters. The physical effects of addition must not be so great
as to physically destroy the viability of the zygote. The
biological limit of the number and variety of DNA sequences will
vary depending upon the particular zygote and functions of the
exogenous genetic material and will be readily apparent to one
skilled in the art, because the genetic material, including the
exogenous genetic material, of the resulting zygote must be
biologically capable of initiating and maintaining the
differentiation and development of the zygote into a functional
organism.
[0323] The number of copies of the transgene constructs which are
added to the zygote is dependent upon the total amount of exogenous
genetic material added and will be the amount which enables the
genetic transformation to occur. Theoretically only one copy is
required; however, generally, numerous copies are utilized, for
example, 1,000-20,000 copies of the transgene construct, in order
to insure that one copy is functional. There will often be an
advantage to having more than one functioning copy of each of the
inserted exogenous DNA sequences to enhance the phenotypic
expression of the exogenous DNA sequences.
[0324] Any technique which allows for the addition of the exogenous
genetic material into nucleic genetic material can be utilized so
long as it is not destructive to the cell, nuclear membrane or
other existing cellular or genetic structures. The exogenous
genetic material is preferentially inserted into the nucleic
genetic material by microinjection. Microinjection of cells and
cellular structures is known and is used in the art.
[0325] Reimplantation is accomplished using standard methods.
Usually, the surrogate host is anesthetized, and the embryos are
inserted into the oviduct. The number of embryos implanted into a
particular host will vary by species, but will usually be
comparable to the number of off spring the species naturally
produces.
[0326] Transgenic offspring of the surrogate host may be screened
for the presence and/or expression of the transgene by any suitable
method. Screening is often accomplished by Southern blot or
Northern blot analysis, using a probe that is complementary to at
least a portion of the transgene. Western blot analysis using an
antibody against the protein encoded by the transgene may be
employed as an alternative or additional method for screening for
the presence of the transgene product. Typically, DNA is prepared
from tail tissue and analyzed by Southern analysis or PCR for the
transgene. Alternatively, the tissues or cells believed to express
the transgene at the highest levels are tested for the presence and
expression of the transgene using Southern analysis or PCR,
although any tissues or cell types may be used for this
analysis.
[0327] Alternative or additional methods for evaluating the
presence of the transgene include, without limitation, suitable
biochemical assays such as enzyme and/or immunological assays,
histological stains for particular marker or enzyme activities,
flow cytometric analysis, and the like. Analysis of the blood may
also be useful to detect the presence of the transgene product in
the blood, as well as to evaluate the effect of the transgene on
the levels of various types of blood cells and other blood
constituents.
[0328] Progeny of the transgenic animals may be obtained by mating
the transgenic animal with a suitable partner, or by in vitro
fertilization of eggs and/or sperm obtained from the transgenic
animal. Where mating with a partner is to be performed, the partner
may or may not be transgenic and/or a knockout; where it is
transgenic, it may contain the same or a different transgene, or
both. Alternatively, the partner may be a parental line. Where in
vitro fertilization is used, the fertilized embryo may be implanted
into a surrogate host or incubated in vitro, or both. Using either
method, the progeny may be evaluated for the presence of the
transgene using methods described above, or other appropriate
methods.
[0329] The transgenic animals produced in accordance the methods
described here will include exogenous genetic material. As set out
above, the exogenous genetic material will, in certain embodiments,
be a DNA sequence which results in the production of a Stella
and/or Fragilis protein. Further, in such embodiments the sequence
will be attached to a transcriptional control element, e.g., a
promoter, which preferably allows the expression of the transgene
product in a specific type of cell.
[0330] It will be appreciated that it is possible to manipulate the
control elements (promoters or enhancers) to regulate the spatial
or temporal expression, or both, of Stella or Fragilis (as the case
may be). For example, specific control elements may be deleted from
the endogenous Stella and/or Fragilis locus so that expression is
restricted to only certain tissues. Alternatively, it is possible
to prepare transgenes which only contain one, some, or more, of the
control elements. Transgenic animals made this way for Stella
and/or Fragilis and having properties of ectopic expression,
temporally or spatially, or both, will be useful for investigation
of Stella and/or Fragilis gene function.
[0331] Retroviral infection can also be used to introduce transgene
into a non-human animal. The developing non-human embryo can be
cultured in vitro to the blastocyst stage. During this time, the
blastomeres can be targets for retroviral infection (Jaenich, R.
(1976) PNAS 73:1260-1264). Efficient infection of the blastomeres
is obtained by enzymatic treatment to remove the zona pellucida
(Manipulating the Mouse Embryo, Hogan eds. (Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, 1986). The viral vector
system used to introduce the transgene is typically a
replication-defective retrovirus carrying the transgene (Jahner et
al. (1985) PNAS 82:6927-6931; Van der Putten et al. (1985) PNAS
82:6148-6152). Transfection is easily and efficiently obtained by
culturing the blastomeres on a monolayer of virus-producing cells
(Van der Putten, supra; Stewart et al. (1987) EMBO J. 6:383-388).
Alternatively, infection can be performed at a later stage. Virus
or virus-producing cells can be injected into the blastocoele
(Jahner et al. (1982) Nature 298:623-628). Most of the founders
will be mosaic for the transgene since incorporation occurs only in
a subset of the cells which formed the transgenic non-human animal.
Further, the founder may contain various retroviral insertions of
the transgene at different positions in the genome which generally
will segregate in the offspring. In addition, it is also possible
to introduce transgenes into the germ line by intrauterine
retroviral infection of the midgestation embryo (Jahner et al.
(1982) supra).
[0332] A third type of target cell for transgene introduction is
the embryonal stem cell (ES). ES cells are obtained from
pre-implantation embryos cultured in vitro and fused with embryos
(Evans et al. (1981) Nature 292:154-156; Bradley et al. (1984)
Nature 309:255-258; Gossler et al. (1986) PNAS 83: 9065-9069; and
Robertson et al. (1986) Nature 322:445-448). Transgenes can be
efficiently introduced into the ES cells by DNA transfection or by
retrovirus-mediated transduction. Such transformed ES cells can
thereafter be combined with blastocysts from a non-human animal.
The ES cells thereafter colonize the embryo and contribute to the
germ line of the resulting chimeric animal. For review see
Jaenisch, K (1988) Science 240:1468-1474.
[0333] We also provide non-human transgenic animals, where the
transgenic animal is characterized by having an altered Stella
and/or Fragilis gene, preferably as described above, as models for
Stella or Fragilis function, as the case may be. Alterations to the
gene include deletions or other loss of function mutations,
introduction of an exogenous gene having a nucleotide sequence with
targeted or random mutations, introduction of an exogenous gene
from another species, or a combination thereof. The transgenic
animals may be either homozygous or heterozygous for the
alteration. The animals and cells derived therefrom are useful for
screening biologically active agents that may modulate Stella
and/or Fragilis function. The screening methods are of particular
use for determining the specificity and action of potential
therapies for Stella and/or Fragilis associated diseases, as
described above. The animals are useful as a model to investigate
the role of Stella and/or Fragilis proteins in the body.
[0334] Another aspect pertains to a transgenic animal having a
functionally disrupted endogenous Stella or Fragilis gene, or both,
but which also carries in its genome, and expresses, a transgene
encoding a heterologous Stella and/or Fragilis protein (i.e., a
Stella and/or Fragilis gene from another species). Preferably, the
animal is a mouse and the heterologous Stella or Fragilis is a
human Stella or Fragilis. An animal, or cell lines derived from
such an animal, which has been reconstituted with human Stella
and/or Fragilis, can be used to identify agents that inhibit human
Stella and/or Fragilis in vivo and in vitro. For example, a
stimulus that induces signalling through human Stella and/or
Fragilis can be administered to the animal, or cell line, in the
presence and absence of an agent to be tested and the response in
the animal, or cell line, can be measured. An agent that inhibits
human Stella and/or Fragilis in vivo or in vitro can be identified
based upon a decreased response in the presence of the agent
compared to the response in the absence of the agent.
[0335] We also provide for a Stella and/or Fragilis deficient
transgenic non-human animal (a "Stella/Fragilis knock-out" or a
"Stella/Fragilis null"). Such an animal is one which expresses
lowered or no Stella/Fragilis activity, preferably as a result of
an endogenous Stella or Fragilis (as the case may be) genomic
sequence being disrupted or deleted. The endogenous Stella or
Fragilis genomic sequence may be replaced by a null allele, which
may comprise non-functional portions of the wild-type
Stella/Fragilis sequence. For example, the endogenous
Stella/Fragilis genomic sequence may be replaced by an allele of
Stella/Fragilis comprising a disrupting sequence which may comprise
heterologous sequences, for example, reporter sequences and/or
selectable markers. Preferably, the endogenous Stella/Fragilis
genomic sequence in a Stella/Fragilis knock-out mouse is replaced
by an allele of Stella or Fragilis in which one or more, preferably
all, of the coding sequences is replaced by such a disrupting
sequence, preferably a lacZ sequence and a neomycin resistance
sequence. Preferably, the genomic Stella/Fragilis sequence which is
functionally disrupted comprises a mouse Stella/Fragilis genomic
sequence.
[0336] Preferably, such an animal expresses no Stella or Fragilis
activity, or both. More preferably, the animal expresses no
activity of the Stella or Fragilis proteins shown in the sequence
listings. Stella/Fragilis knock-outs may be generated by various
means known in the art, as described in further detail below. A
specific description of the construction of a Stella knock-out
mouse is disclosed in Example 20 et seq below.
[0337] We further disclose a nucleic acid construct for
functionally disrupting a Stella/Fragilis gene in a host cell. The
nucleic acid construct comprises: a) a non-homologous replacement
portion; b) a first homology region located upstream of the
non-homologous replacement portion, the first homology region
having a nucleotide sequence with substantial identity to a first
Stella/Fragilis gene sequence; and c) a second homology region
located downstream of the non-homologous replacement portion, the
second homology region having a nucleotide sequence with
substantial identity to a second Stella/Fragilis gene sequence, the
second Stella/Fragilis gene sequence having a location downstream
of the first Stella/Fragilis gene sequence in a naturally occurring
endogenous Stella/Fragilis gene. Additionally, the first and second
homology regions are of sufficient length for homologous
recombination between the nucleic acid construct and an endogenous
Stella/Fragilis gene in a host cell when the nucleic acid molecule
is introduced into the host cell. In a preferred embodiment, the
non-homologous replacement portion comprises an expression
reporter, preferably including lacZ and a positive selection
expression cassette, preferably including a neomycin
phosphotransferase gene operatively linked to a regulatory
element(s).
[0338] Another aspect pertains to recombinant vectors into which
the nucleic acid construct described above has been incorporated.
Yet another aspect pertains to host cells into which the nucleic
acid construct has been introduced to thereby allow homologous
recombination between the nucleic acid construct and an endogenous
Stella/Fragilis gene of the host cell, resulting in functional
disruption of the endogenous Stella/Fragilis gene. The host cell
can be a mammalian cell that normally expresses Stella/Fragilis
from the liver, brain, spleen or heart, or a pluripotent cell, such
as a mouse embryonic stem cell. Further development of an embryonic
stem cell into which the nucleic acid construct has been introduced
and homologously recombined with the endogenous Stella/Fragilis
gene produces a transgenic nonhuman animal having cells that are
descendant from the embryonic stem cell and thus carry the
Stella/Fragilis gene disruption in their genome. Animals that carry
the Stella/Fragilis gene disruption in their germline can then be
selected and bred to produce animals having the Stella/Fragilis
gene disruption in all somatic and germ cells. Such mice can then
be bred to homozygosity for the Stella/Fragilis gene
disruption.
Detection of Pluripotent Cells in Cell Populations
[0339] Polynucleotide probes or antibodies as described here may be
used for the detection of pluripotent cells such as primordial germ
cells (PGCs), stem cells such as embryonic stem (ES) and embryonic
germ (EG) cells in cell populations. As used herein, a "cell
population" is any collection of cells which may contain one or
more PGCs, ES or EG cells. Preferably, the collection of cells does
not consist solely of PGCs, but comprises at least one other cell
type.
[0340] Cell populations comprise embryos and embryo tissue, but
also adult tissues and tissues grown in culture and cell
preparations derived from any of the foregoing.
[0341] Polynucleotides as described here may be used for detection
of Fragilis and Stella transcripts in PGCs or other pluripotent
cells, such as ES or EG cells, by nucleic acid hybridisation
techniques. Such techniques include PCR, in which primers are
hybridised to Fragilis and/or Stella transcripts and used to
amplify the transcripts, to provide a detectable signal; and
hybridisation of labelled probes, in which probes specific for an
unique sequence in the Fragilis and/or Stella transcript are used
to detect the transcript in the target cells.
[0342] As noted hereinbefore, probes may be labelled with
radioactive, radiopaque, fluorescent or other labels, as is known
in the art.
[0343] The antibodies may also be used to detect Fragilis and/or
Stella. Fragilis, in particular, possesses an extracellular domain
which may be targeted by an anti-Fragilis antibody and detected at
the cell surface. Alternatively, intracellular scFv may be used to
detect Fragilis and/or Stella within the cell.
[0344] Particularly indicated are immunostaining and FACS
techniques. Suitable fluorophores are known in the art, and include
chemical fluorophores and fluorescent polypeptides, such as GFP and
mutants thereof (see WO 97/28261). Chemical fluorophores may be
attached to immunoglobulin molecules by incorporating binding sites
therefor into the immunoglobulin molecule during the synthesis
thereof.
[0345] Preferably, the fluorophore is a fluorescent protein, which
is advantageously GFP or a mutant thereof. GFP and its mutants may
be synthesised together with the immunoglobulin or target molecule
by expression therewith as a fusion polypeptide, according to
methods well known in the art. For example, a transcription unit
may be constructed as an in-frame fusion of the desired GFP and the
immunoglobulin or target, and inserted into a vector as described
above, using conventional PCR cloning and Ligation techniques.
[0346] Antibodies may be labelled with any label capable of
generating a signal. The signal may be any detectable signal, such
as the induction of the expression of a detectable gene product
Examples of detectable gene products include bioluminescent
polypeptides, such as luciferase and GFP, polypeptides detectable
by specific assays, such as .beta.-galactosidase and CAT, and
polypeptides which modulate the growth characteristics of the host
cell, such as enzymes required for metabolism such as HIS3, or
antibiotic resistance genes such as G418. In a preferred aspect,
the signal is detectable at the cell surface. For example, the
signal may be a luminescent or fluorescent signal, which is
detectable from outside the cell and allows cell sorting by FACS or
other optical sorting techniques.
[0347] Preferred is the use of optical immunosensor technology,
based on optical detection of fluorescently-labelled antibodies.
Immunosensors are biochemical detectors comprising an antigen or
antibody species coupled to a signal transducer which detects the
binding of the complementary species (Rabbany et al., 1994 Crit Rev
Biomed Eng 22:307-346; Morgan et al., 1996 Clin Chem 42:193-209).
Examples of such complementary species include the antigen Zif 268
and the anti-Zif 268 antibody. Immunosensors produce a quantitative
measure of the amount of antibody, antigen or hapten present in a
complex sample such as serum or whole blood (Robinson 1991 Biosens
Dioelectron 6:183-191). The sensitivity of immunosensors makes them
ideal for situations requiring speed and accuracy (Rabbany et al.,
1994 Crit Rev Biomed Eng 22:307-346).
[0348] Detection techniques employed by immunosensors include
electrochemical, piezoelectric or optical detection of the
immunointeraction (Ghindilis et al., 1998 Biosens Bioelectron
1:113-131). An indirect immunosensor uses a separate labelled
species that is detected after binding by, for example,
fluorescence or luminescence (Morgan et al., 1996 Clin Chem
42:193-209). Direct immunosensors detect the binding by a change in
potential difference, current, resistance, mass, heat or optical
properties (Morgan et al., 1996 Clin Chem 42:193-209). Indirect
immunosensors may encounter fewer problems due to non-specific
binding (Attridge et al., 1991 Biosens Bioelecton 6:201-214; Morgan
et al., 1996 Clin Chem 42:193-209).
Prophylactic and Therapeutic Methods
[0349] We provide methods of treating an abnormal conditions
related to both an excess of and insufficient amounts of Stella
and/or Fragilis activity. Examples of these include the Stella
associated diseases and Fragilis associated diseases disclosed
above.
[0350] If the activity of Stella and/or Fragilis is in excess,
several approaches are available. One approach comprises
administering to a subject an inhibitor compound (antagonist) as
described along with a pharmaceutically acceptable carrier in an
amount effective to inhibit activation by blocking binding of a
relevant molecule to the Stella and/or Fragilis, or by inhibiting a
second signal, and thereby alleviating the abnormal condition.
[0351] In another approach, where Stella and/or Fragilis act by
binding a ligand soluble forms of Stella and/or Fragilis
polypeptides still capable of binding the ligand in competition
with endogenous Stella and/or Fragilis may be administered. Typical
embodiments of such competitors comprise fragments of the Stella
and/or Fragilis polypeptide.
[0352] In still another approach, expression of the gene encoding
endogenous Stella and/or Fragilis can be inhibited using expression
blocking techniques. Known such techniques involve the use of
antisense sequences, either internally generated or separately
administered. See, for example, O'Connor, J Neurochem (1991) 56:560
in Oligodeoxynucleotides as Antisense Inhibitors of Gene
Expression, CRC Press, Boca Raton, Fla. (1988). Alternatively,
oligonucleotides which form triple helices with the gene can be
supplied. See, for example, Lee et al., Nucleic Acids Res (1979)
6:3073; Cooney et al., Science (1988) 241:456; Dervan et al.,
Science (1991) 251:1360. These oligomers can be administered per se
or the relevant oligomers can be expressed in vivo.
[0353] For treating abnormal conditions related to an
under-expression of Stella and/or Fragilis and its activity,
several approaches are also available. One approach comprises
administering to a subject a therapeutically effective amount of a
compound which activates Stella and/or Fragilis, i.e., an agonist
as described above, in combination with a pharmaceutically
acceptable carrier, to thereby alleviate the abnormal condition.
Alternatively, gene therapy may be employed to effect the
endogenous production of Stella and/or Fragilis by the relevant
cells in the subject.
[0354] For example, a polynucleotide as described in this document
may be engineered for expression in a replication defective
retroviral vector. The retroviral expression construct may then be
isolated and introduced into a packaging cell transduced with a
retroviral plasmid vector containing RNA encoding a Stella and/or
Fragilis polypeptide such that the packaging cell now produces
infectious viral particles containing the gene of interest. These
producer cells may be administered to a subject for engineering
cells in vivo and expression of the polypeptide in vivo. For
overview of gene therapy, see Chapter 20, Gene Therapy and other
Molecular Genetic-based Therapeutic Approaches, (and references
cited therein) in Human Molecular Genetics, T Strachan and A P
Read, BIOS Scientific Publishers Ltd (1996).
Formulation and Administration
[0355] Peptides and polypeptides, such as the Stella and/or
Fragilis peptides and polypeptides, and agonists and antagonist
peptides or small molecules, may be formulated in combination with
a suitable pharmaceutical carrier. Such formulations comprise a
therapeutically effective amount of the polypeptide or compound,
and a pharmaceutically acceptable carrier or excipient. Such
carriers include but are not limited to, saline, buffered saline,
dextrose, water, glycerol, ethanol, and combinations thereof.
Formulation should suit the mode of administration, and is well
within the skill of the art. We further describe pharmaceutical
packs and kits comprising one or more containers filled with one or
more of the ingredients of the aforementioned compositions.
[0356] Polypeptides and other compounds may be employed alone or in
conjunction with other compounds, such as therapeutic
compounds.
[0357] Preferred forms of systemic administration of the
pharmaceutical compositions include injection, typically by
intravenous injection. Other injection routes, such as
subcutaneous, intramuscular, or intraperitoneal, can be used.
Alternative means for systemic administration include transmucosal
and transdermal administration using penetrants such as bile salts
or fusidic acids or other detergents. In addition, if properly
formulated in enteric or encapsulated formulations, oral
administration may also be possible. Administration of these
compounds may also be topical and/or localize, in the form of
salves, pastes, gels and the like.
[0358] The dosage range required depends on the choice of peptide,
the route of administration, the nature of the formulation, the
nature of the subject's condition, and the judgment of the
attending practitioner. Suitable dosages, however, are in the range
of 0.1-100 .mu.g/kg of subject. Wide variations in the needed
dosage, however, are to be expected in view of the variety of
compounds available and the differing efficiencies of various
routes of administration. For example, oral administration would be
expected to require higher dosages than administration by
intravenous injection. Variations in these dosage levels can be
adjusted using standard empirical routines for optimization, as is
well understood in the art.
[0359] Polypeptides used in treatment can also be generated
endogenously in the subject, in treatment modalities often referred
to as "gene therapy" as described above. Thus, for example, cells
from a subject may be engineered with a polynucleotide, such as a
DNA or RNA, to encode a polypeptide ex vivo, and for example, by
the use of a retroviral plasmid vector. The cells are then
introduced into the subject.
Pharmaceutical Compositions
[0360] We also provide a pharmaceutical composition comprising
administering a therapeutically effective amount of the
polypeptide, polynucleotide, peptide, vector or antibody (such as a
Stella and/or Fragilis polypeptide, etc) and optionally a
pharmaceutically acceptable carrier, diluent or excipients
(including combinations thereof).
[0361] The pharmaceutical compositions may be for human or animal
usage in human and veterinary medicine and will typically comprise
any one or more of a pharmaceutically acceptable diluent, carrier,
or excipient Acceptable carriers or diluents for therapeutic use
are well known in the pharmaceutical art, and are described, for
example, in Remington's Pharmaceutical Sciences, Mack Publishing
Co. (A. R. Gennaro edit 1985). The choice of pharmaceutical
carrier, excipient or diluent can be selected with regard to the
intended route of administration and standard pharmaceutical
practice. The pharmaceutical compositions may comprise as--or in
addition to--the carrier, excipient or diluent any suitable
binder(s), lubricant(s), suspending agent(s), coating agent(s),
solubilising agent(s).
[0362] Preservatives, stabilizers, dyes and even flavoring agents
may be provided in the pharmaceutical composition Examples of
preservatives include sodium benzoate, sorbic acid and esters of
p-hydroxybenzoic acid. Antioxidants and suspending agents may be
also used.
[0363] There may be different composition/formulation requirements
dependent on the different delivery systems. By way of example, the
pharmaceutical composition as described here may be formulated to
be delivered using a mini-pump or by a mucosal route, for example,
as a nasal spray or aerosol for inhalation or ingestable solution,
or parenterally in which the composition is formulated by an
injectable form, for delivery, by, for example, an intravenous,
intramuscular or subcutaneous route. Alternatively, the formulation
may be designed to be delivered by both routes.
[0364] Where the agent is to be delivered mucosally through the
gastrointestinal mucosa, it should be able to remain stable during
transit though the gastrointestinal tract; for example, it should
be resistant to proteolytic degradation, stable at acid pH and
resistant to the detergent effects of bile.
[0365] Where appropriate, the pharmaceutical compositions can be
administered by inhalation, in the form of a suppository or
pessary, topically in the form of a lotion, solution, cream,
ointment or dusting powder, by use of a skin patch, orally in the
form of tablets containing excipients such as starch or lactose, or
in capsules or ovules either alone or in admixture with excipients,
or in the form of elixirs, solutions or suspensions containing
flavouring or colouring agents, or they can be injected
parenterally, for example intravenously, intramuscularly or
subcutaneously. For parenteral administration, the compositions may
be best used in the form of a sterile aqueous solution which may
contain other substances, for example enough salts or
monosaccharides to make the solution isotonic with blood. For
buccal or sublingual administration the compositions may be
administered in the form of tablets or lozenges which can be
formulated in a conventional manner.
Vaccines
[0366] Another embodiment relates to a method for inducing an
immunological response in a mammal which comprises inoculating the
mammal with the Stella and/or Fragilis polypeptide, or a fragment
thereof, adequate to produce antibody and/or T cell immune response
to protect said animal from Stella and/or Fragilis associated
disease.
[0367] Yet another embodiment relates to a method of inducing
immunological response in a mammal which comprises delivering a
Stella and/or Fragilis polypeptide via a vector directing
expression of a Stella and/or Fragilis polynucleotide in vivo in
order to induce such an immunological response to produce antibody
to protect said animal from diseases.
[0368] A further embodiment relates to an immunological/vaccine
formulation (composition) which, when introduced into a mammalian
host, induces an immunological response in that mammal to a Stella
and/or Fragilis polypeptide wherein the composition comprises a
Stella and/or Fragilis polypeptide or Stella and/or Fragilis gene.
The vaccine formulation may further comprise a suitable
carrier.
[0369] Since the Stella and/or Fragilis polypeptide may be broken
down in the stomach, it is preferably administered parenterally
(including subcutaneous, intramuscular, intravenous, intradermal
etc. injection). Formulations suitable for parenteral
administration include aqueous and non-aqueous sterile injection
solutions which may contain anti-oxidants, buffers, bacteriostats
and solutes which render the formulation instonic with the blood of
the recipient; and aqueous and non-aqueous sterile suspensions
which may include suspending agents or thickening agents. The
formulations may be presented in unit-dose or multi-dose
containers, for example, sealed ampoules and vials and may be
stored in a freeze-dried condition requiring only the addition of
the sterile liquid carrier immediately prior to use. The vaccine
formulation may also include adjuvant systems for enhancing the
immunogenicity of the formulation, such as oil-in water systems and
other systems known in the art. The dosage will depend on the
specific activity of the vaccine and can be readily determined by
routine experimentation.
[0370] Vaccines may be prepared from one or more polypeptides or
peptides as described here.
[0371] The preparation of vaccines which contain an immunogenic
polypeptide(s) or peptide(s) as active ingredient(s), is known to
one skilled in the art. Typically, such vaccines are prepared as
injectables, either as liquid solutions or suspensions; solid forms
suitable for solution in, or suspension in, liquid prior to
injection may also be prepared. The preparation may also be
emulsified, or the protein encapsulated in liposomes. The active
immunogenic ingredients are often mixed with excipients which are
pharmaceutically acceptable and compatible with the active
ingredient. Suitable excipients are, for example, water, saline,
dextrose, glycerol, ethanol, or the like and combinations
thereof.
[0372] In addition, if desired, the vaccine may contain minor
amounts of auxiliary substances such as wetting or emulsifying
agents, pH buffering agents, and/or adjuvants which enhance the
effectiveness of the vaccine. Examples of adjuvants which may be
effective include but are not limited to: aluminum hydroxide,
N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),
N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred
to as nor-MDP),
N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-s-
n-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred
to as MTP-PE), and RIBI, which contains three components extracted
from bacteria, monophosphoryl lipid A, trehalose dimycolate and
cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80
emulsion.
[0373] Further examples of adjuvants and other agents include
aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate
(alum), beryllium sulfate, silica, kaolin, carbon, water-in-oil
emulsions, oil-in-water emulsions, muramyl dipeptide, bacterial
endotoxin, lipid X, Corynebacterium parvum (Propionobacterium
acnes), Bordetella pertussis, polyribonucleotides, sodium alginate,
lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole,
DEAE-dextran, blocked copolymers or other synthetic adjuvants. Such
adjuvants are available commercially from various sources, for
example, Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.)
or Freund's Incomplete Adjuvant and Complete Adjuvant (Difco
Laboratories, Detroit, Mich.).
[0374] Typically, adjuvants such as Amphigen (oil-in-water),
Alhydrogel (aluminum hydroxide), or a mixture of Amphigen and
Alhydrogel are used. Only aluminum hydroxide is approved for human
use.
[0375] The proportion of immunogen and adjuvant can be varied over
a broad range so long as both are present in effective amounts. For
example, aluminum hydroxide can be present in an amount of about
0.5% of the vaccine mixture (Al.sub.2O.sub.3 basis). Conveniently,
the vaccines are formulated to contain a final concentration of
immunogen in the range of from 0.2 to 200 .mu.g/ml, preferably 5 to
50 .mu.g/ml, most preferably 15 .mu.g/ml.
[0376] After formulation, the vaccine may be incorporated into a
sterile container which is then sealed and stored at a low
temperature, for example 4.degree. C., or it may be freeze-dried.
Lyophilisation permits long-term storage in a stabilised form.
[0377] The vaccines are conventionally administered parenterally,
by injection, for example, either subcutaneously or
intramuscularly. Additional formulations which are suitable for
other modes of administration include suppositories and, in some
cases, oral formulations. For suppositories, traditional binders
and carriers may include, for example, polyalkylene glycols or
triglycerides; such suppositories may be formed from mixtures
containing the active ingredient in the range of 0.5% to 10%,
preferably 1% to 2%. Oral formulations include such normally
employed excipients as, for example, pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharine,
cellulose, magnesium carbonate, and the like. These compositions
take the form of solutions, suspensions, tablets, pills, capsules,
sustained release formulations or powders and contain 10% to 95% of
active ingredient, preferably 25% to 70%. Where the vaccine
composition is lyophilised, the lyophilised material may be
reconstituted prior to administration, e.g. as a suspension
Reconstitution is preferably effected in buffer
[0378] Capsules, tablets and pills for oral administration to a
patient may be provided with an enteric coating comprising, for
example, Eudragit "S", Eudragit "L", cellulose acetate, cellulose
acetate phthalate or hydroxypropylmethyl cellulose.
[0379] The polypeptides described here may be formulated into the
vaccine as neutral or salt forms. Pharmaceutically acceptable salts
include the acid addition salts (formed with free amino groups of
the peptide) and which are formed with inorganic acids such as, for
example, hydrochloric or phosphoric acids, or such organic acids
such as acetic, oxalic, tartaric and maleic. Salts formed with the
free carboxyl groups may also be derived from inorganic bases such
as, for example, sodium, potassium, ammonium, calcium, or ferric
hydroxides, and such organic bases as isopropylamine,
trimethylamine, 2-ethylamino ethanol, histidine and procaine.
Administration
[0380] Typically, a physician will determine the actual dosage
which will be most suitable for an individual subject and it will
vary with the age, weight and response of the particular patient.
The dosages below are exemplary of the average case. There can, of
course, be individual instances where higher or lower dosage ranges
are merited.
[0381] The pharmaceutical and vaccine compositions as disclosed
here may be administered by direct injection. The composition may
be formulated for parenteral, mucosal, intramuscular, intravenous,
subcutaneous, intraocular or transdermal administration. Typically,
each protein may be administered at a dose of from 0.01 to 30 mg/kg
body weight, preferably from 0.1 to 10 mg/kg, more preferably from
0.1 to 1 mg/kg body weight.
[0382] The term "administered" includes delivery by viral or
non-viral techniques. Viral delivery mechanisms include but are not
limited to adenoviral vectors, adeno-associated viral (AAV) vectos,
herpes viral vectors, retroviral vectors, lentiviral vectors, and
baculoviral vectors. Non-viral delivery mechanisms include lipid
mediated transfection, liposomes, immunoliposomes, lipofectin,
cationic facial amphiphiles (CFAs) and combinations thereof. The
routes for such delivery mechanisms include but are not limited to
mucosal, nasal, oral, parenteral, gastrointestinal, topical, or
sublingual routes.
[0383] The term "administered" includes but is not limited to
delivery by a mucosal route, for example, as a nasal spray or
aerosol for inhalation or as an ingestable solution; a parenteral
route where delivery is by an injectable form, such as, for
example, an intravenous, intramuscular or subcutaneous route.
[0384] The term "co-administered" means that the site and time of
administration of each of for example, the polypeptide and an
additional entity such as adjuvant are such that the necessary
modulation of the immune system is achieved. Thus, whilst the
polypeptide and the adjuvant may be administered at the same moment
in time and at the same site, there may be advantages in
administering the polypeptide at a different time and to a
different site from the adjuvant. The polypeptide and adjuvant may
even be delivered in the same delivery vehicle--and the polypeptide
and the antigen may be coupled and/or uncoupled and/or genetically
coupled and/or uncoupled.
[0385] The Stella and/or Fragilis polypeptide, polynucleotide,
peptide, nucleotide, antibody etc and optionally an adjuvant may be
administered separately or co-administered to the host subject as a
single dose or in multiple doses.
[0386] The vaccine composition and pharmaceutical compositions
described here may be administered by a number of different routes
such as injection (which includes parenteral, subcutaneous and
intramuscular injection) intranasal, mucosal, oral, intra-vaginal,
urethral or ocular administration.
[0387] The vaccines and pharmaceutical compositions described here
may be conventionally administered parenterally, by injection, for
example, either subcutaneously or intramuscularly. Additional
formulations which are suitable for other modes of administration
include suppositories and, in some cases, oral formulations. For
suppositories, traditional binders and carriers may include, for
example, polyalkylene glycols or triglycerides; such suppositories
may be formed from mixtures containing the active ingredient in the
range of 0.5% to 10%, may be 1% to 2%. Oral formulations include
such normally employed excipients as, for example, pharmaceutical
grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharine, cellulose, magnesium carbonate, and the like. These
compositions take the form of solutions, suspensions, tablets,
pills, capsules, sustained release formulations or powders and
contain 10% to 95% of active ingredient, preferably 25% to 70%.
Where the vaccine composition is lyophilised, the lyophilised
material may be reconstituted prior to administration, e.g. as a
suspension. Reconstitution is preferably effected in buffer.
Diagnostic Assays
[0388] We describe the use of Stella and/or Fragilis
polynucleotides and polypeptides (as well as homologues, variants
and derivatives thereof) for use in diagnosis as diagnostic
reagents or in genetic analysis. Nucleic acids complementary to or
capable of hybridising to Stella and/or Fragilis nucleic acids
(including homologues, variants and derivatives), as well as
antibodies against Stella and/or Fragilis polypeptides are also
useful in such assays.
[0389] We provide for a natural variant of Stella and/or Fragilis
polypeptide or nucleic acid, and the use of such a natural variant
in diagnosis of Stella and/or Fragilis associated disease. Stella
and/or Fragilis polymorphisms may include differences at the
nucleic acid level, which may or may not reflect differences in the
amino acid level. Preferably, such Stella and/or Fragilis variants
or mutants are such that they include changes in the amino acid
level. However, we also disclose Stella and/or Fragilis
polymorphisms which occur in non-coding regions, for example,
expression control regions such as promoters and enhancers.
[0390] Polymorphisms in Stella and/or Fragilis include deletions of
one or more nucleic acids, insertions of one or more nucleic acids,
inversions, etc. Preferably, Stella and/or Fragilis polymorphisms
comprise single nucleotide polymorphisms.
[0391] Polymorphisms in Stella and/or Fragilis may be identified by
comparing sequences at the appropriate level (whether nucleic acid
or protein) between individuals in a population. Differences in
sequences may be reflected in different physical properties, and
techniques for detecting these may rely on detection of changes in
physical properties. For example, single nucleotide polymorphisms
may be detected as restriction fragment length polymorphisms (i.e.,
difference in susceptibility to digestion by a restriction enzyme).
Furthermore, SNPS may affect the migration or mobility of a nucleic
acid fragment or protein fragment in a gel.
[0392] Non-coding polymorphisms in Stella and/or Fragilis may be
identified by sequencing non-coding regions of Stella and/or
Fragilis. For example, control regions of the Stella and/or
Fragilis gene, such as enhancers and promoters may be sequenced to
identify polymorphisms. The effect of such non-coding polymorphisms
on the expression level of Stella and/or Fragilis may be determined
by constructing transgenic mice (as described below) comprising the
mutant Stella and/or Fragilis sequences, or by generating
expression constructs and transfection into cell lines. In each
case, the expression level of Stella and/or Fragilis is detected,
by RT-PCR or antibody Western staining, to determine the effect of
the mutation in the control of expression of Stella and/or
Fragilis. Useful Stella and/or Fragilis polymorphisms are those
which modulate the level of expression, whether by up-regulation or
down-regulation of Stella and/or Fragilis levels.
[0393] Accordingly, we provide for a variant or mutant or
polymorphism in a non-coding region of Stella and/or Fragilis,
preferably in a control region of Stella and/or Fragilis,
preferably in a promoter and/or enhancer of Stella and/or Fragilis,
which is capable of modulating the level of expression of Stella
and/or Fragilis in an organism. We also provide for a set of two or
more of such mutants or variants or polymorphisms, preferably
non-coding polymorphisms. We also provide for the use of such
variants or polymorphisms or sets of variants to identify nucleic
acid and/or amino acid positions, in which changes to such
positions affect the level of expression of Stella and/or Fragilis.
We also provide for a transgenic animal comprising a variant or
mutant or polymorphism of Stella and/or Fragilis, preferably, a
non-coding polymorphism.
[0394] Detection of a mutated form of the Stella and/or Fragilis
gene associated with a dysfunction will provide a diagnostic tool
that can add to or define a diagnosis of a disease or
susceptibility to a disease which results from under-expression,
over-expression or altered expression of Stella and/or Fragilis.
Individuals carrying mutations in the Stella and/or Fragilis gene
(including control sequences) may be detected at the DNA level by a
variety of techniques.
[0395] For example, DNA may be isolated from a patient and the DNA
polymorphism pattern of Stella and/or Fragilis determined. The
identified pattern is compared to controls of patients known to be
suffering from a disease associated with over-, under- or abnormal
expression of Stella and/or Fragilis. Patients expressing a genetic
polymorphism pattern associated with Stella and/or Fragilis
associated disease may then be identified. Genetic analysis of the
Stella and/or Fragilis gene may be conducted by any technique known
in the art. For example, individuals may be screened by determining
DNA sequence of a Stella and/or Fragilis allele, by RFLP or SNP
analysis, etc. Patients may be identified as having a genetic
predisposition for a disease associated with the over-, under-, or
abnormal expression of Stella and/or Fragilis by detecting the
presence of a DNA polymorphism in the gene sequence for Stella
and/or Fragilis or any sequence controlling its expression.
[0396] Patients so identified can then be treated to prevent the
occurrence of Stella and/or Fragilis associated disease, or more
aggressively in the early stages of Stella and/or Fragilis
associated disease to prevent the further occurrence or development
of the disease. Stella and/or Fragilis associated diseases include
any cancer, for example, as described above.
[0397] We further disclose a kit for the identification of a
patient's genetic polymorphism pattern associated with Stella
and/or Fragilis associated disease. The kit includes DNA sample
collecting means and means for determining a genetic polymorphism
pattern, which is then compared to control samples to determine a
patient's susceptibility to Stella and/or Fragilis associated
disease. Kits for diagnosis of a Stella and/or Fragilis associated
disease comprising Stella and/or Fragilis polypeptide and/or an
antibody against such a polypeptide (or fragment of it) are also
provided.
[0398] Nucleic acids for diagnosis may be obtained from a subject's
cells, such as from blood, urine, saliva, tissue biopsy or autopsy
material. In a preferred embodiment, the DNA is obtained from blood
cells obtained from a finger prick of the patient with the blood
collected on absorbent paper. In a further preferred embodiment,
the blood is collected on an AmpliCard.TM. (University of
Sheffield, Department of Medicine and Pharmacology, Royal
Hallamshire Hospital, Sheffield, England S10 2JF).
[0399] The DNA may be used directly for detection or may be
amplified enzymatically by using PCR or other amplification
techniques prior to analysis Oligonucleotide DNA primers that
target the specific polymorphic DNA region within the genes of
interest may be prepared so that in the PCR reaction amplification
of the target sequences is achieved. RNA or cDNA may also be used
as templates in similar fashion. The amplified DNA sequences from
the template DNA may then be analyzed using restriction enzymes to
determine the genetic polymorphisms present in the amplified
sequences and thereby provide a genetic polymorphism profile of the
patient. Restriction fragments lengths may be identified by gel
analysis. Alternatively, or in conjunction, techniques such as SNP
(single nucleotide polymorphisms) analysis may be employed.
[0400] Deletions and insertions can be detected by a change in size
of the amplified product in comparison to the normal genotype.
Point mutations can be identified by hybridizing amplified DNA to
labeled Stella and/or Fragilis nucleotide sequences. Perfectly
matched sequences can be distinguished from mismatched duplexes by
RNase digestion or by differences in melting temperatures. DNA
sequence differences may also be detected by alterations in
electrophoretic mobility of DNA fragments in gels, with or without
denaturing agents, or by direct DNA sequencing. See, eg., Myers et
al, Science (1985)230:1242. Sequence changes at specific locations
may also be revealed by nuclease protection assays, such as RNase
and SI protection or the chemical cleavage method. See Cotton et
al., Proc Natl Acad Sci USA (1985) 85: 4397-4401. In another
embodiment, an array of oligonucleotides probes comprising the
Stella and/or Fragilis nucleotide sequence or fragments thereof can
be constructed to conduct efficient screening of e.g., genetic
mutations. Array technology methods are well known and have general
applicability and can be used to address a variety of questions in
molecular genetics including gene expression, genetic linkage, and
genetic variability. (See for example: M. Chee et al., Science, Vol
274, pp 610-613 (1996)).
[0401] Single strand conformation polymorphism (SSCP) may be used
to detect differences in electrophoretic mobility between mutant
and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad.
Sci. USA: 86:2766, see also Cotton (1993) Mutat Res 285:125-144;
and Hayashi (1992) Genet Anal Tech Appl 9:73-79). Single-stranded
DNA fragments of sample and control Stella and/or Fragilis nucleic
acids may be denatured and allowed to renature. The secondary
structure of single-stranded nucleic acids varies according to
sequence, the resulting alteration in electrophoretic mobility
enables the detection of even a single base change. The DNA
fragments may be labeled or detected with labeled probes. The
sensitivity of the assay may be enhanced by using RNA (rather than
DNA), in which the secondary structure is more sensitive to a
change in sequence. In a preferred embodiment, the subject method
utilizes heteroduplex analysis to separate double stranded
heteroduplex molecules on the basis of changes in electrophoretic
mobility (Keen et al. (1991) Trends Genet. 7:5).
[0402] The diagnostic assays offer a process for diagnosing or
determining a susceptibility to Stella and/or Fragilis associated
diseases, for example, as described above.
[0403] The presence of Stella and/or Fragilis polypeptides and
nucleic acids may be detected in a sample. Thus, infections and
diseases as listed above can be diagnosed by methods comprising
determining from a sample derived from a subject an abnormally
decreased or increased level of the Stella and/or Fragilis
polypeptide or Stella and/or Fragilis mRNA. The sample may comprise
a cell or tissue sample from an organism suffering or suspected to
be suffering from a disease associated with increased, reduced or
otherwise abnormal Stella and/or Fragilis expression, including
spatial or temporal changes in level or pattern of expression. The
level or pattern of expression of Stella and/or Fragilis in an
organism suffering from or suspected to be suffering from such a
disease may be usefully compared with the level or pattern of
expression in a normal organism as a means of diagnosis of
disease.
[0404] In general therefore, we describe a method of detecting the
presence of a nucleic acid comprising a Stella and/or Fragilis
nucleic acid in a sample, by contacting the sample with at least
one nucleic acid probe which is specific for said nucleic acid and
monitoring said sample for the presence of the nucleic acid. For
example, the nucleic acid probe may specifically bind to the Stella
and/or Fragilis nucleic acid, or a portion of it, and binding
between the two detected; the presence of the complex itself may
also be detected. Furthermore, we disclose a method of detecting
the presence of a Stella and/or Fragilis polypeptide by contacting
a cell sample with an antibody capable of binding the polypeptide
and monitoring said sample for the presence of the polypeptide.
This may conveniently be achieved by monitoring the presence of a
complex formed between the antibody and the polypeptide, or
monitoring the binding between the polypeptide and the antibody.
Methods of detecting binding between two entities are known in the
art, and include FRET (fluorescence resonance energy transfer),
surface plasmon resonance, etc.
[0405] Decreased or increased expression can be measured at the RNA
level using any of the methods well known in the art for the
quantitation of polynucleotides, such as, for example, PCR, RT-PCR,
RNase protection, Northern blotting and other hybridization
methods. Assay techniques that can be used to determine levels of a
protein, such as a Stella and/or Fragilis, in a sample derived from
a host are well-known to those of skill in the art. Such assay
methods include radioimmunoassays, competitive-binding assays,
Western Blot analysis and ELISA assays.
[0406] We also describe a diagnostic kit for a disease or
susceptibility to a Stella and/or Fragilis associated disease
(including an infection). The diagnostic kit comprises a Stella
and/or Fragilis polynucleotide or a fragment thereof; a
complementary nucleotide sequence; a Stella and/or Fragilis
polypeptide or a fragment thereof, or an antibody to a Stella
and/or Fragilis polypeptide.
Further Aspects of the Invention
[0407] We provide a nucleic acid molecule which is at least 90%
homologous to SEQ ID NO: 3 and a nucleic acid molecule which is at
least 75% homologous to SEQ ID NO: No. 5.
[0408] We disclose polynucleotides which comprise a contiguous
stretch of nucleotides from SEQ ID NO: 3 or SEQ ID NO: 5, or any of
SEQ ID NOs: 7, 9, 11, 13, 15 and 17, or of a sequence at least 90%
homologous thereto. Advantageously, this stretch of contiguous
nucleotides is 50 nucleotides in length, preferably 40, 35, 30, 25,
20, 15 or 10 nucleotides in length.
[0409] The human genes Fragilis and Stella encode novel
polypeptides, the sequences of which are set forth in SEQ ID NO: 4
and SEQ ID NO: 6. Other Fragilis genes are set out in SEQ ID NOs:
8, 10, 12, 14, 16 and 18. We therefore disclose polypeptides
encoded by the nucleic acids described here, as well as nucleic
acids encoding any of the polypeptides disclosed here. Preferably,
the polypeptides have the sequences set forth in SEQ ID NO: 4 and
SEQ ID NO: 6.
EXAMPLES
[0410] The following Examples 1 to 9 relate to cloning and
characterisation of rodent Fragilis and Stella genes, and are
included here for reference.
Example 1
Identification of Murine Genes Specific to the Earliest Population
of Primordial Germ Cells (PGCs) by Single Cell cDNA Differential
Screening
[0411] A method for single cell analysis is developed to identify
genes that are involved in the specification of the germ cell
lineage, which results in the establishment of a founder population
of Primordial Germ Cells (PGCs). It is determined that the lineage
specification of PGCs accompanies the expression of a unique set of
genes, which are not expressed in somatic cells.
[0412] The method for the identification of the genes is mainly
based on the differential screening of the libraries made from
single cells from day 7.25 mouse embryonic fragments that contain
PGCs. The single cell cDNA differential screen was originally
described by Brady and Iscove (1993), and subsequently modified by
Cathaline Dulac and Richard Axel which resulted in the successful
identification of the pheromone receptor genes from rat (Dulac, C.
and Axel, 1995). The method of Axel's group is employed, with
slight modifications as described.
[0413] Construction of Single Cell cDNAs from Embryonic Fragment
Bearing the Earliest Population of PGCs
[0414] In the mouse, the earliest population of the PGCs is
reported to consist of alkaline phosphatase positive cluster of
some 40 cells, at the base of the emerging allantois at day 7.25 of
gestation (Ginsburg, M., Snow, M. H. L., and McLaren, A. (1990)).
The precise location of the PGC cluster in the inbred 129Sv and
C57BL/6 strain is determined by microscopy using both whole-mount
alkaline phosphatase staining and semi-thin sections stained by
methylene blue. The earliest stage at which a cluster of PGcs can
be detected is at the Late Streak stage (Downs, K. M., and Davies,
T. (1993)), when a distinctively stained population of cells is
found just beneath an epithelial lining from which the allantoic
bud appears. This region is at the border between the
extraembryonic and embryonic tissues just posterior to and above
the most proximal part of the primitive streak. The cluster
persists at this position at least until Early/Mid Bud stage. In
the inbred 129Sv strain, the PGC cluster is found to contain a
slightly larger number of the cells, which are more tightly
packaged than in the C57BL/6 strain. The 129Sv strain is used for
subsequent experiments, as a better recovery of the earliest PGCs
is obtained.
[0415] 129Sv embryos are isolated at E7.5 in DMEM plus 10% FCS
buffered with 25 mM HEPES at room temperature and the developmental
stage of each embryo is determined under a dissection microscope.
The precise developmental stage can differ substantially even
amongst embryos within the same litter. Embryos that are at the no
bud or early bud (allantoic) stage are chosen for further
dissection, which in part is dictated by the ease of identification
of the region containing PGCs as seen under the dissection
microscope. The fragment that is expected to contain the PGC
cluster is cut out very precisely by means of solid glass needles.
This region is dissociated it into single cells using 0.25%
trypsin-1 mM EGTAJPBS treatment at 37.degree. C. for 10 min,
followed by gentle pipetting with a mouth pipette. The dissected
fragment usually contained between 250-300 cells. The procedure for
cell dispersal with this gentle procedure left the visceral
endoderm layer remained as an intact cellular sheet.
[0416] We picked single cells randomly from the cell suspension by
a mouth pipette and put individual single cells (but avoiding
generating air bubbles), into a thin-walled PCR tube containing 4
.mu.l of ice-cold cell lysis buffer (50 mM Tris-HCl pH8.3, 75 mM
KCl, 3 mM MgCl.sub.2, 0.5% NP40, containing 80 ng/ml pd(T)24, 5
.mu.g/ml prime RNase inhibitor, 324 U/ml RNA guard, and 10 mM each
of dATP, dCTP, dGTP, and dTTP). The volume of medium carried with
the single cell is less than 0.5 .mu.l. The tube is briefly
centrifuged to ensure that the cell is indeed in the lysis buffer.
During each separate experiment, we picked a total of 19 single
cells, and left one tube without a cell, to serve as a negative
control for the PCR amplification procedure. All the cells that are
collected in tubes are kept on ice before starting the subsequent
procedure.
[0417] The cells are lysed by incubating the tubes at 65.degree. C.
for 1 min, and then kept at room temperature for 1-2 min to allow
the oligo dT to anneal the to RNA. First-strand cDNA synthesis is
initiated by adding 50 U of Moloney murine leukaemia virus (MMLV)
and 0.5 U of avian myeloblastosis virus (AMV) reverse transcriptase
followed by incubation for 15 min at 37.degree. C. The reverse
transcriptases are inactivated for 10 min at 65.degree. C. This
reverse transcription reaction is restricted to 15 min, which
allows the synthesis of relatively uniform size cDNAs of between
500 base-1000 bases in length from the C termini. This enables the
subsequent PCR amplification to be fairly representative.
[0418] Next, in order to add the poly A tail to the 5 prime end of
the synthesised first-strand cDNA, 4.5 .mu.l of 2.times. tailing
buffer (200 mM potassium cacodylate pH7.2, 4 mM CoCl.sub.2, 0.4 mM
DTT, 200 mM dATP containing 10 U of terminal transferase) is added
to the reaction followed by incubation for 15 min at 37.degree. C.
The samples are heat inactivated for 10 min at 65.degree. C. The
reaction now contained synthesised cDNAs bearing poly T tail at
their C termini and poly A stretch at their N termini, ready for
the amplification by the PCR using the specific primer.
[0419] The contents of each tube is brought to 100 .mu.l with a
solution made of 10 mM Tris-HCl pH8.3, 50 mM KCl, 2.5 mM
MgCl.sub.2, 100 .mu.g/ml bovine serum albumin, 0-05% Triton-X 100,
1 mM of dATP, dCTP, dGTP, dTTP, 10 U of Taq polymerase, and 5 .mu.g
of the AL1 primer. The AL1 sequence is ATT GGA TCC AGG CCG CTC TGG
ACA AAA TAT GAA TCC (T).sub.24. The PCR amplification is performed
according to the following schedule: 94.degree. C. for 1 min,
42.degree. C. for 2 min, and 72.degree. C. for 6 min with 10 s
extension per cycle for 25 cycles. Five additional units of Taq
polymerase are added before performing 25 more cycles with the same
programme but without the extension time. Each tube at this point
contains amplified cDNA products derived from a single cell. The
protein contents of the solution are extracted by phenol/chloroform
treatment, and the amplified cDNAs are precipitated by ethanol and
eventually suspended in 100 .mu.l of TE pH8.0. 5 .mu.l of the cDNA
solution is run on a 1.5% agarose gel to check the success of the
amplification. Most of the samples show a very intense `smeared`
band ranging mainly between 500 bp to 1200 bp, indicating the
efficient amplification of the single cell cDNA. Only the
successfully amplified samples are used for the subsequent `cell
typing` analysis.
Example 2
Identification of PGCs by Examination of the Expression of Marker
Genes
[0420] The embryonic fragment which is excised theoretically
contains three major components: the allantoic mesoderm, PGCs, and
extraembryonic mesoderm surrounding PGCs. In order to identify the
single cell cDNA of PGC origin amongst these samples, positive and
negative selection of the constructed cDNAs is performed, by
examining the expression of four marker genes (BMP4, TNAP, Hoxb1,
and Oct4), which are known to be either expressed or repressed in
various cell types in this region.
[0421] At the No/Early Bud stage, BMP4 is reported to be expressed
in the emerging allantois and mesodermal components of the
developing amnion, chorion, and visceral yolk sac (Lawson, K. A.,
Dunn, N. R., Roelen, B. A. J., Zeinstra, L. M., Davis, A. M.,
Wright, C. V. E., Korving, J. P. W. F. M., and Hogan, B. L. M.
(1999)). The boundary of BMP4 expression is very sharp, and the
expression is completely excluded in the mesodermal region beneath
the epithelial lining continuous from the amnionic mesoderm where
the putative PGCs are determined. Therefore, BMP4 is used as a
negative marker for the selection. Primer pairs are designed for
amplifying the C terminal portion of BMP4 (5': GCC ATA CCT TGA CCC
GCA GAA G, 3': AAA TGG CAC TCA GTT CAG TGG G). The PCR
amplification is performed using 0.5 .mu.l of the cDNA solution as
a template according to the following schedule: 95.degree. C. for 1
min, 55.degree. C. for 1 min, and 72.degree. C. for 1 min for 20
cycles. Among 83 samples tested, 57 samples show the expected size
of bands, indicating expression of BMP4 these single cells. These
samples are considered to be of allantoic mesodermal origin, and
therefore excluded from amongst the candidates representing cells
of PGC origin.
[0422] The expression of tissue non-specific alkaline phosphatase
(TNAP), which has long been used as an early marker for PGCs
(Ginsburg, M., Snow, M. H. L., and McLaren, A. (1990)), is then
examined. Primer pairs are designed (5': CCC AAA GCA CCT TAT TTT
TCT ACC, 3': TTG GCG AGT CTC TGC AAT TGG) and the same PCR reaction
as above is performed. Amongst the 26 samples, 22 samples are
judged to be positive for TNAP. From the alkaline phosphatase
staining of the sectioned embryos, it is known that the somatic
cells surrounding PGCs also express some amount of TNAP, although
the level of expression is slightly lower than that in PGCs.
Therefore, amongst these 22 positive samples there should be still
be cells destined to become somatic cells as well as PGCs.
[0423] One of the genes known to be expressed in the totipotent
PGCs but not in somatic cells is Oct4 (Yoem, Y. II., Fuhrmann, G.,
Ovitt, C. E., Brehm, A., Ohbo, K., Gross, M., Hubner, K., and
Scholer, H. R. (1996)). To examine the possibility that Oct4 can be
used as a marker to distinguish PGCs from somatic cells at this
stage, Oct4 expression is checked in the 22 samples by PCR (5': CAC
TCT ACT CAG TCC CTT TTC, 3': TGT GTC CCA GTC TTT ATT TAA G). All
the 22 samples express Oct4 at comparable levels, indicating that
the somatic cells at this stage are still actively transcribing
Oct4 RNA.
[0424] The amount of expression of TNAP is quantitated in 22
samples by Southern blot analysis (reverse northern blot analysis).
Given the fairly representative amplification of the single cell
method, confirmed by amplifying single ES cell cDNA, Southern blot
analysis allows semi-quantitative measurement of the amount of the
genes expressed in the original single cells, although it does not
serve as a perfect indicator of cell identity. However, as a result
of this TNAP analysis, 10 samples out of 22 show relatively
stronger bands at an equivalent level, while the remaining 12
samples exhibit weaker signals. These results indicate that these
22 samples can be divided at least into two groups, one with
stronger TNAP expression (therefore from putative PGCs) and the
other with weaker TNAP.
[0425] The possibility that somatic cells surrounding PGCs start to
express Hoxb1, while PGCs do not (personal communication from Dr.
Kirstie Lawson) is also examined. Primer pairs are designed (5':
AAC TCA TCA GAG GTC GAA GGA, 3': CGG TGC TAT TOT AAG GTC TGC) and
the same PCR reaction as above is performed. Among the 22 samples
tested, 12 are positive, and more importantly, these 12 samples
perfectly match the ones which show weaker TNAP signals, by
Southern blot analysis.
[0426] Taking all these results into consideration, it is concluded
that 10 samples out of 83, which are Oct4 (+), TNAP (++), BMP4 (-),
and Hoxb1 (-), are of PGC origin. This ratio (10/83) is reasonable,
considering the number of the founding population of PGCs as 40 and
the number of cells in the fragment as 250-300.
Example 3
Differential Screening of Single Cell cDNA Libraries
[0427] As the efficiency of the amplification of cDNA differs in
each tube, it is very important to select the samples with the most
efficiently amplified cDNA for the construction of libraries. The
amplification of six different genes (ribosomal protein S112,
intermediate filament protein vimentin, .beta. tubulin-5, .alpha.
actin, Oct4, E-cadherin) is examined in the 10 PGC candidate
samples, by Southern blot analysis. Judging from the overall
profile of the amplification of all these six genes, three cDNA
preparations are selected for the construction of libraries.
[0428] To obtain the maximum amount of double strand cDNA, an
extension step is performed with 5 .mu.l of cell cDNA in 100 .mu.l
of the PCR buffer described as above (including 1 .mu.l of
AmpliTaq) according to the following schedule: 94.degree. C. for 5
min, 42.degree. C. for 5 min, 72.degree. C. for 30 min. The
solution is extracted by phenol/chloroform treatment, and the
amplified cDNAs are precipitated by ethanol, suspended in TE, and
completely digested with EcoRI. The PCR primer and excess amount of
dNTPs are removed by QIAGEN PCR Purification Kit, and all the
purified cDNAs are run on a 2% low melting agarose gel. cDNAs above
500 bp are cut and purified by QIAGEN Gel Purification Kit. The
purified eDNAs are precipitated by ethanol and suspended in TE and
ligated into .lamda. ZAP II vector arms. The ligated vector is
packaged, titered and the ratio of the successfully ligated clones
is monitored by amplifying the inserts with T3 and T7 primers from
20 plaques. More than 95% of the phage are found to contain
inserts.
[0429] The representation of the three genes, ribosomal protein
S12, .beta. tubulin-5, Oct4, is quantitated by screening 5000
plaques, and the library of the best quality among the three (S12
0.62%, .beta. tubulin 0.4%, Oct4 0.5%) is used for the differential
screening. As a comparison partner with the PGC probe, one of the
most efficiently amplified surrounding somatic cell cDNA (Oct4 (+),
TNAP(+/-), BMP(-), and Hoxb1(+)) is selected by the similar
Southern blot analysis.
[0430] The library is plated at a density of 1000 plaques per 15 cm
dish to obtain large plaques (2 mm diameter) and two duplicate
lifts are taken using Hybond N+ filters from Amersham. The filters
are prehybridized at 65.degree. C. in 0.5M sodium phosphate buffer
(pH7.3) containing 1% bovine serum albumin and 4% SDS. We prepared
the cell cDNA probes by reamplifying for 10 cycles 111 of the
original cell cDNA into 501 of total reaction with the AL1 primer,
in the absence of cold dCTP and with 100 .mu.Ci of newly received
.sup.32PdCTP, followed by the purification using Amersham Nick.TM.
Spin Column. The filters are hybridised for at least 16 hrs with
1.0.times.10.sup.7 cpm/ml (The first filter is hybridised with
somatic cell probe and the second filter is hybridised with the PGC
probe). After the hybridisation, the filters are washed three times
at 65.degree. C. in 0.5.times.SSC, 0.5% SDS and exposed to X ray
films until the appropriate signal is obtained (usually one to two
days).
[0431] The positive plaques in the two duplicate filters are
compared very carefully. Among 5000 plaques screened, 280 are
picked as candidates representing the differentially expressed
genes. The inserts of all the 280 plaques are amplified with T3 and
T7 primers, run on 1.5% gels, and double sandwich Southern blotted.
Each membrane is hybridised with the PGC and somatic cell probe,
respectively, using the same conditions as the screening. 38 clones
amongst the 280 are selected as differentially expressed genes.
These clones are next hybridised with the second PGC and somatic
cell cDNA probes, which resulted in 20 clones out of 38 to be
common in both PGC cDNAs but they are either not included or less
abundant in both somatic cell cDNAs. The sequences of all the 20
clones are determined.
[0432] Genes Highly Specific to the Earliest Population of PGCs
[0433] The 20 clones represent 11 different genes (two clones
appear two times, one clone appears three times, and one clone
appears 6 times). To further stringently check the specificity of
expression, primer pairs are designed for these 11 clones and their
expression checked in 10 different single PGC-candidate cDNAs and
10 different single somatic cell cDNAs by PCR. Two of them show
highly specific expression to PGC cDNAs.
[0434] The first gene, Fragilis (Germ cell restricted-1, Fragilis),
encodes a 137 amino acid protein with a predicted molecular weight
of 15.0 kD. The nucleic acid sequence of mouse Fragilis is set out
in SEQ ID NO: 2.
[0435] Fragilis, comprises two transmembrane domains, the amino and
carboxy terminus ends being located outside the cell. Fragilis is a
newly discovered member of the interferon (IFN)-inducible
transmembrane protein family (Deblandre, G. A. et al. Expression
cloning of an interferon-inducible 17-kDa membrane protein
implicated in the control of cell growth. J. Biol. Chem. 270,
23860-23866 (1995); Friedman, R. L., Manly, S. P., McMahon, M.,
Kerr, I. M. & Stark, G. R. Transcriptional and
posttranscriptional regulation of interferon-induced gene
expression in human cells. Cell 38, 745-755 (1984) and is detected
in expressed sequence tags (ESTs) derived from many different
embryonic and adult tissues, suggesting that it may have a common
role in different developmental contexts. One prototype member is
the IFN-inducible human 9-27 (identical to the Leu-13 antigen)
protein in leukocytes and endothelial cells, a cell surface
component of a multimeric complex involved in homotypic adhesion
and transduction of antiproliferative signals (Deblandre, G. A. et
al. Expression cloning of an interferon-inducible 17-kDa membrane
protein implicated in the control of cell growth. J. Biol. Chem.
270, 23860-23866 (1995); Evans, S. S., Collea, R P., Leasure, J. A.
& Lee, D. B. IPN-a induces homotypic adhesion and Leu-13
expression in human B lymphoid cells. J. Immunol. 150, 736-747
(1993); Evans, S. S., Lee, D. B., Han, T., Tomasi, T. B. &
Evans, R. L. Monoclonal antibody to the interferoninducible protein
Leu-13 triggers aggregation and inhibits proliferation of leukemic
B cells. Blood 76, 2583-2593 (1990)).
[0436] One prototype Fragilis family member, human 9-27 (identical
to Leu-13 antigen), is inducible by interferon in leukocytes and
endothelial cells, and is located at the cell surface as a
component of a multimeric complex involved in the transduction of
antiproliferative and homotypic adhesion signals (Deblandre,
1995).
[0437] The BLASTN search revealed that the Fragilis sequence was
found in ESTs derived from many different tissues both from embryos
and adults, indicating that Fragilis may play a common role in
different developmental and cell biological contexts. Database
searches reveal a sequence match with the rat interferon-inducible
protein (sp:INIB RAT, pir:JC1241) with unknown function. The
Fragilis sequence appears six times in our screen, indicating high
level expression in PGcs.
[0438] The second gene, Stella, encodes a 150 amino acid protein,
of 18 kD. The nucleic acid sequence of mouse Stella is set out in
SEQ ID NO: 1.
[0439] It has no sequence homology with any known protein, contains
several nuclear localisation consensus sequences and is highly
basic pI (pI=9.67, the content of basic residues--23.3%),
indicating a possible affinity to DNA. Furthermore a potential
nuclear export signal was identified, indicating that Stella may
shuttle between the nucleus and the cytoplasm. BLASTN analysis
revealed that the Stella sequence was found only in the
preimplantation embryo and germ line (newborn ovary, female 12.5
mesonephros and gonad etc.) ESTs indicating its predominant
expression in totipotent and pluripotent cells. Interestingly, we
found that Stella contains in its N terminus a modular domain which
has some sequence similarity with the SAP motif. This motif is a
putative DNA-binding domain involved in chromosomal organisation.
There is also apparently a splicing factor motif-like structure in
its C terminus. These findings suggest a possible involvement of
Stella in chromosomal organization and RNA processing.
Example 4
Identification of PGCs by Screening for Fragilis and Stella
Expression
[0440] Although PGCs are identified in Example 2 by analysis of
BMP4, TNAP, Hoxb1, and Oct4, no single one of these genes can be
taken as a marker for the PGC state. However, both Fragilis and
Stella may be used as such.
[0441] The expression of Fragilis is examined. Primer pairs are
designed (5': CTACTCCGTGAAGTCTAGG, 3': AATGAGTGTFACACCTGCGTG) and
the same PCR reaction as above is performed. Fragilis expression
was detected in germ cell competent cells. The definitive PGCs were
recruited from amongst this group of cells showing expression of
Fragilis.
[0442] The boundary of Stella expression in particular is
well-defined, and the expression is substantially limited to PGCs.
Therefore, Stella is used as a positive marker for the selection of
PGCs. Primer pairs are designed for amplifying the C terminal
portion of Stella (5': GCCATTCAGATGTCTCTGCAC, 3':
CTCACAGCTTGAGGCTTCTAA). The PCR amplification is performed using
0.5 .mu.l of the cDNA solution obtained from PGCs in Example 1 as a
template according to the following schedule: 95.degree. C. for 1
min, 55.degree. C. for 1 min, and 72.degree. C. for 1 min for 20
cycles. Among 83 samples tested, only those taken from PGCs show
expression of Stella. Hence, Stella is a positive marker for the
PGC fate.
[0443] Antibodies against Fragilis and Stella can be similarly used
to detect pluripotent cells. Preferably, antibodies against
Fragilis are used to detect germ cell competent cells, and
antibodies against Stella are used to detect PGCs.
[0444] Accordingly, both Fragilis and Stella are positive markers
for the PGC fate which can be used to positively identify PGC.
[0445] Identification of PGC by ISH
[0446] The in vivo expression of the two genes is examined by in
situ hybridisation. The expression of Fragilis starts very weakly
in the entire epiblast at E6.0-E6.5 (PreStreak stage) and becomes
strong in the few cell layers of the proximal rim of the epiblast.
BMP4 that is expressed in the extraembryonic ectoderm is one
signalling molecule that is important for the induction of germ
cell competence and expression of Fragilis. Other signals, such as
interferons are likely to be involved in the induction of Fragilis.
The expression becomes more intense at the proximo-posterior end of
the developing primitive streak at the Early/Mid Streak stage and
becomes very strong at this position from Late Streak stage onward.
The expression persists until Early Head Fold stage and eventually
disappears gradually. No expression is detected in the migrating
PGCs at E8.5.
[0447] The expression of Stella starts at the proximo-posterior end
of the developing primitive streak at Mid/Late Streak stage and
becomes gradually strong at the same position from the later stage
onward. The expression is specific and individual single cells
stained in a dotted manner can be seen in the region where PGCs are
considered to start differentiating as a cluster of cells. At Late
Bud/Early Head Fold stage, some cells considered to be migrating
from the initial cluster are stained as well as cells in the
cluster. At E8.5 and E9.5, a group of cells considered to be the
migrating PGCs are very specifically stained.
[0448] From these results, it is concluded that Fragilis is a gene
which is upregulated during the process of lineage specification
and germ cell competence, and subsequently of PGCs, when Stella is
turned on after Fragilis to fix the PGC fate.
[0449] Accordingly, expression of Fragilis may be detected in a
method of detecting lineage specification, and/or pluripotency,
such as germ cell competence. Similarly, expression of Stella may
be detected to detect commitment to cell fate, for example,
commitment to fate as a primordial germ cell.
Example 5
Expression of Fragilis and Stella During Germ Line Development
[0450] Antibodies against Stella and Fragilis are used to detect
expression of these genes in early embryos. It is found that each
of these genes is expressed in primordial germ cells. In
particular, we find that Fragilis is the first gene to mark PGC
competent cells at the time of germ cell allocation. Stella is
expressed only in the lineage-restricted founder PGCs and
thereafter in the germ cell lineage.
[0451] FIG. 3 shows expression of mouse Fragilis in embryonic stem
(ES) cells.
[0452] Fragilis is expressed in pluripotent ES and EG cells. During
the derivation of EG cells from PGCs, it is found that Fragilis
expression re-appears on EG cells. Late PGCs are negative for
Fragilis after specification of these cells is completed.
[0453] FIG. 5 shows expression of mouse Fragilis as detected by
whole-mount in situ hybridization in E7.2 mouse embryos.
[0454] There is strong Fragilis expression at the base of incipient
allantois where the founder PGC population differentiates in the
E7.25 embryos. Fragilis expression persisted until E7.5, but it was
not detected in migrating PGCs at E8.5. Fragilis is first detected
in germ cell competent proximal epiblast cells. Fragilis expression
can be induced in the epiblast cells when combined with the tissues
extraembryonic ectoderm tissues, which is the source of BMP4. In
the BMP4 mutant mice, there is no expression of Fragilis,
consistent with the absence of PGCs in these embryos (Lawson et
al., 1999).
[0455] FIG. 4 shows expression of mouse Stella in PGCs.
[0456] Stella expression which is strong in PGCs is downregulated
in EG cells. There is also low level expression of Stella in ES
cells. Stella and Fragilis are detectable in ES and EG cells by
Northern blot analysis. Stella is first detected at E7.0 in single
cells within the distinctive cluster of lineage-restricted PGCs,
and thereafter in migrating PGCs and subsequently when they enter
the gonads. FIG. 7 shows Stella expression in PGCs in the process
of migration into the gonads in E9.0 embryos. Stella is the only
gene so far known to be a definitive marker for the founder
population of PGCs.
[0457] FIG. 6 shows expression of mouse Stella as detected by
whole-mount in situ hybridization in E7.2 mouse embryos.
[0458] FIG. 8. Expression of mouse Fragilis and Stella in single
cells detected by PCR analysis of single cell cDNAs. Note that
there are more single cells showing expression of Fragilis compared
to those showing expression of Stella. Only cells with the highest
levels of Fragilis expression are found to express Stella and
acquire the germ cell fate. Cells that express Stella were found
not to show expression of Hoxb1. Cells that express lower levels of
Fragilis and no Stella become somatic cells and show expression of
Hoxb1. The founder population of PGCs also show high levels of
Tnap. Both the founder PGCs and the somatic cells show expression
of Oct4, T(Brachyury), and Fgf8.
Example 6
Expression of Fragilis and Stella in Individual Cells
[0459] Intracellular localisation of Stella and Fragilis is also
determined. Fragilis localised to a single cytoplasmic spot at the
Golgi apparatus, as well as in the plasma membrane. Stella
comprises a putative nuclear localisation signal and nuclear export
signal, and is localised in both the cytoplasm and nucleus.
[0460] Fragilis is observed in the Golgi apparatus as well as in
the plasma membrane of PGCs. The cell surface localization of
Fragilis is expected as a member of the interferon inducible gene
family [Deblandre, 1995]. Expression of Fragilis in the proximal
rim of the epiblast marks the onset of germ cell competence.
Fragilis has an IFN response element upstream of its exon 1, so it
is very likely to be induced by IFN after initial priming by BMP4
of the proximal epiblast cells. These IFN inducible proteins can
from a multimeric complex with other proteins such as TAPA1, which
is capable of transduction of antiproliferative signals, which may
be why the cell cycle time in founder PGCs increases from 6 to 16
hr, while the somatic cells continue to divide rapidly.
[0461] Stella, which has the putative nuclear localization signal
and a nuclear export signal, was observed in both the cytoplasm and
the nucleus. The onset of Stella is followed by the loss of
Fragilis expression by E8.5. Therefore, Fragilis expression marks
the onset of germ cell competence and Stella expression marks the
end of this specification process. Expression of Stella in the
founder PGCs marks an escape from the somatic cell fate and
consistent with their pluripotent state. These studies indicate
that specific set of genes are required to impose a germ line fate
on cells that may otherwise become somatic cells. Stella, with its
potential to shuttle between the nucleus and cytoplasm, could have
a role in transcriptional and translational regulation, since many
organisms possess elaborate transcriptional mechanisms to prevent
germ cells from becoming somatic cells. Expression of Stella in the
oocyte and preimplantation embryos indicates that it has a wider
role in totipotency and pluripotency.
Example 7
The Link Between Fragilis and Stella
[0462] Only some of the cells that express Fragilis, ended up
showing expression of Stella Only those cells with the highest
levels of Fragilis expression become PGCs and began to express
Stella. Furthermore, Stella positive PGCs never show expression of
Hoxb1. More importantly, only somatic cells with lower levels of
Fragilis expression, show Hoxb1 expression. Furthermore, only the
somatic cells show expression of two other homeobox-containing
genes, Lim1 and Evx-1. Therefore lack of expression of Hoxb1, Evx-1
and Lim1, appears to be important for the specification of germ
cell fate.
[0463] FIGS. 8a and 8b show expression of various genes in single
cell PGCs and somatic cells by PCR analysis.
[0464] Our experiments also show that Oct4 is not a definitive
marker of PGC, Previously, Oct4 expression is demonstrated in
totipotent and pluripotent cells [Nichols, 199, Pesce, 1998; Yeom,
1996]. However, we find that Oct4 is expressed to the same extent
in all PGCs and somatic cells. We do however find expression of T
(Brachyuri) and Fgf 8 in PGCs indicating that PGCs are recruited
from amongst embryonic cells that are initially destined to become
mesodermal cells.
Example 8
PGC Specification
[0465] The founder PGCs and their somatic neighbours share common
origin from the proximal epiblast cells. By analysing the founder
PGC and the somatic neighbour, a systematic screen for critical
genes for the specification of germ cell fate has been established.
Fragilis is an interferon (IFN) inducible gene that can promote
germ cell competence and homotypic association to demarcate
putative germ cells from their somatic neighbours, and such an
example may apply to other situation during development. Expression
of Stella occurs in cells with high expression of Fragilis.
Fragilis is no longer required once germ cell specification is
complete, but Stella expression continues in the germ cell lineage.
Stella may also be important throughout in the
totipotent/pluripotent cells since it is also expressed in oocytes
and early preimplantion development embryos.
Example 9
Germ Line and Pluripotent Stem Cells
[0466] PGCs can be used to derive pluripotent embryonic germ (EG)
cells. However, unlike EG cells, PGCs do not participate in
development if introduced into blastocysts. They either cannot
respond to signalling molecules, or that they are transcriptionally
repressed. PGCs once specified do not express Fragilis on their
cell surface. However, EG cells clearly show expression of Fragilis
on their cell surface as do ES cells. Both EG and ES cells express
Stella as judged by Northern analysis, although Stella is expressed
at a lower level in ES and EG cells than in PGCs. Fragilis and
Stella therefore have a role in pluripotent stem cells. These genes
are therefore markers of these pluripotent stem cells, where they
may also have a role in conferring pluripotency on these stem
cells.
Example 10
Proposed Roles of Fragilis and Stella in PGC Specification
[0467] Fragilis as a typical IFN-inducible cell surface protein,
probably shares certain properties common to all of these family
members (Deblandre, G. A. et al. Expression cloning of an
interferon-inducible 17-kDa membrane protein implicated in the
control of cell growth. J. Biol. Chem. 270, 23860-23866 (1995);
Evans, S. S., Collea, R. P., Leasure, J. A. & Lee, D. B. IFN-a
induces homotypic adhesion and Leu-13 expression in human B
lymphoid cells. J. Immunol. 150, 736-747 (1993); Evans, S. S., Lee,
D. B., Han, T., Tomasi, T. B. & Evans, R. L. Monoclonal
antibody to the interferoninducible protein Leu-13 triggers
aggregation and inhibits proliferation of leukemic B cells. Blood
76, 2583-2593 (1990)).
[0468] The acute but transient expression of fragilis is itself
consistent with the kinetics of IFN-inducible genes that can
increase by up to 40-fold within 1 h, and decline quickly after IFN
withdrawal (Friedman, R. L., Manly, S. P., McMahon, M, Kerr, I. M.
& Stark, G. R. Transcriptional and posttranscriptional
regulation of interferon-induced gene expression in human cells.
Cell 38, 745-755 (1984)). This Fragilis positive assembly of cells
could correspond to about 100 TNAP positive cells (Lawson, K. A.
& Hage, W. J. Clonal analysis of the origin of primordial germ
cells in the mouse. Ciba Found. Symp. 182, 68-84 (1994); Ginsburg,
M., Snow, M. H. & McLaren, A. Primordial germ cells in the
mouse embryo during gastrulation. Development 110, 521-528 (1990)),
which is larger than the number of stella positive cells.
[0469] According to our estimates, the stella positive cluster in
the 129/SvEv mouse strain consists of approximately 36-43 cells,
which is close to the expected 45 nascent PGCs. The fragilis
positive cells probably form a community of cells through homotypic
adhesion (Evans, S. S., Collea, R. P., Leasure, J. A. & Lee, D.
B. IFN-a induces homotypic adhesion and Leu-13 expression in human
B lymphoid cells. J. Immunol. 150, 736-747 (1993); Evans, S. S.,
Lee, D. B., Han, T., Tomasi, T. B. & Evans, R. L. Monoclonal
antibody to the interferoninducible protein Leu-13 triggers
aggregation and inhibits proliferation of leukemic B cells. Blood
76, 2583-2593 (1990)), from which the founder PGCs are recruited,
thus demarcating them from most of the cells destined for somatic
tissues. These IFN-inducible cell surface proteins are capable of
transduction of antiproliferative signals (Deblandre, G. A. et al.
Expression cloning of an interferon-inducible 17-kDa membrane
protein implicated in the control of cell growth. J. Biol. Chem.
270, 23860-23866 (1995)), which is a probable mechanism by which
the cell cycle time in the nascent PGCs increases from 6 to 16 h,
while the somatic cells continue to divide rapidly.
[0470] The induction of fragilis in epiblast cells may not by
itself be sufficient for the expression of stella, as shown by our
in vitro studies--induction may require a specific signal thought
to be within the niche, for PGC specification in vivo (Lawson, K.
A. et al. Bmp4 is required for the generation of primordial germ
cells in the mouse embryo. Genes Dev. 13, 4214436 (1999); McLaren,
A. Signaling for germ cells. Genes Dev. 13, 373-376 (1999)). This
signal could be a specific ligand that binds to fragilis during the
specification of germ cell fate. Once nascent PGCs are established,
expression of fragilis is diminished by E8.0, thus freeing the PGCs
from homotypic adhesion for their migration into the genital ridge
(Wylie, C. Germ cells. Cell 96, 165174 (1999); Gomperts, M.,
Garcia-Castro, M., Wylie, C. & Heasman, J. Interactions between
primordial germ cells play a role in their migration in mouse
embryos. Development 120, 135-141 (1994)). fragilis must have other
functions, as it is apparently expressed elsewhere in developing
embryos. In this context, we also note fragilis expression in
pluripotent ES and embryonic germ cells (data not shown), where it
may have a role in the propagation of the pluripotent state.
[0471] The role of stella may in part be regulated by its potential
to shuttle between the nucleus and cytoplasm. We have observed, for
example, that overexpression of stella in somatic cells causes the
protein to be retained in the cytoplasm and not in the nucleus, as
is predominantly the case in PGCs (data not shown). A particularly
critical event involved in the specification of PGCs is repression
of the region-specific homeobox genes, by which nascent PGCs escape
from the somatic cell fate. As the expression of stella is most
intimately connected with the generation of PGCs, this gene is a
chief candidate for either initiating or maintaining repression of
Hox genes in PGCs. The detection of stella in the oocyte and
through pre-implantation development (B. Payer et al., unpublished
data; Sato, M. et al. Identification of PGC7, a new gene expressed
specifically in preimplantation embryos and germ cells. Mech. Dev.
113, 91-94 (2002)) suggests that it may serve a critical role
during all the phases of totipotent/pluripotent states in mice.
Example 11
Fragilis 2, Fragilis 3, Fragilis 4 and Fragilis 5
[0472] Specification of primordial germ cells in mice depends on
instructive signalling events, which act first to confer germ cell
competence on epiblast cells, and second, to impose a germ cell
fate upon competent precursors. fragilis, an interferon-inducible
gene coding for a transmembrane protein, is the first gene to be
implicated in the acquisition of germ cell competence.
[0473] In this and the following Examples (Examples 11 to 20), we
describe four additional fragilis-related genes, fragilis2-5, which
are clustered within a 70 kb region in the vicinity of the fragilis
locus on Chr 7. These genes exist in a number of mammalian species,
which in the human are also clustered on the syntenic region on Chr
11. In the mouse, fragilis2 and fragilis3, which are proximate to
fragilis, exhibit expression that overlaps with the latter in the
region of specification of primordial germ cells. Using single cell
analysis, we confirm that all these three fragilis-related genes
are predominant in nascent primordial germ cells, as well as in
gonadal germ cells.
[0474] The Fragilis family of interferon-inducible genes is tightly
associated with germ cell specification in mice. Furthermore, its
evolutionary conservation suggests that it probably plays a
critical role in all mammals. Detailed analysis of these genes may
also elucidate the role of interferons as signalling molecular
during development.
Example 12
Background to Examples
[0475] Germ line determination in the mouse is thought to occur
through instructive signalling in the gastrulating
post-implantation embryo [1,2]. First, proximal epiblast cells
acquire germ cell competence at E6.5, partly in response to
extraembryonic ectoderm-derived signalling molecules. A subset of
these competent cells then acquire a primordial germ cell (PGC)
fate and a population of approximately 45 founder germ cells are
detected in the posterior proximal region of the embryo at the base
of the incipient allantoic bud on E 7.5 [1, 2]. The secreted
signalling molecules, BMP4, BMP8b and BMP2 as well as components of
the BMP signal transduction pathway, including Smad1 and Smad5,
appear to be involved in the specification of PGCs [3-7]. However,
in vitro culture studies and analysis of BMP4-deficient mice
suggest that an additional signal may also be required for the
acquisition of PGC fate, but its identity is yet unknown [2,3].
[0476] We have identified fragilis, a putative interferon-inducible
gene, which codes for a transmembrane protein that is apparently
associated with the acquisition of germ cell competence by epiblast
cells [8]. Extraembryonic ectoderm is able to induce fragilis
expression in epiblast tissue, and BMP4 is required for this
induction [8]. fragilis is expressed in proximal epiblast at E6.5,
the region in which PGC-competent cells reside according to clonal
analysis [1]. As these proximal cells move to the posterior
proximal region during gastrulation, fragilis expression increases
within a community of cells at the base of the incipient allantoic
bud. Cells with the highest expression of fragilis initiate the
germ cell-characteristic expression of TNAP and stella/PGC-7 [8, 9,
10]. These nascent PGCs with high expression of fragilis also show
repression of Hox genes, including Hoxb1 in nascent PGCs [8].
[0477] In view of the strong association of fragilis with PGC
specification, we have started to investigate further how this gene
may be regulated and what precise function it serves during germ
cell development. Towards this objective, we now report that
fragilis belongs to a novel murine gene family, comprising five
members, which code for five highly similar transmembrane proteins.
More importantly, the genes are clustered within a 70 kb genomic
region. As we found several homologues of the Fragilis family in
human, cow and rat, they seem to be evolutionarily conserved
amongst mammalian species. Most if not all homologous genes have
been reported to be responsive to interferon signalling, which is
in agreement with the presence of conserved interferon stimulable
response elements (ISREs) within at least the murine and human
loci. Furthermore, our in situ hybridisation and single cell
expression analysis reveal that the two members located close to
fragilis, fragilis2 and fragilis3, are also expressed in nascent
PGCs, although their overall expression pattern in
post-implantation embryos in other respects is distinct. Studies on
the Fragilis family of genes could therefore be crucial for our
understanding of PGC specification, especially since their
homologues have been implicated in mediating homotypic cell
adhesion and lengthening of the cell cycle time [14, 15]. These
studies may also show how interferons act as signalling molecules,
which has hitherto not been considered in the context of embryonic
development.
Example 13
Materials and Methods: Database Searches and Animals
[0478] Ensembl and NCBI genome browsers are used for data
retrieval.
[0479] Embryos and genital ridges used for in situ hybridisation
experiments came from 129.times.129 or F1xGoF1 mothers,
respectively. Embryos and genital ridges used for single cell
analysis came from 129.times.SvEv or Oct4GFP(129).times.MF1
mothers, respectively. The day of the vaginal plug was designated
as E0.5. Embryos were staged according to Downs and Davies
[22].
Example 14
Materials and Methods: In Situ Hybridisation
[0480] 3'-fragments of fragilis and fragilis2-5 cDNAs were PCR
amplified using the primers described below, and cloned into pGEMT
vector (Promega). DIG-labelled antisense RNA probes were
synthesized using DIG RNA labelling kit (Sp6/T7; Roche). In situ
hybridisation on embryos and urogenital ridges was performed as
described [23, 24]. Hybridisation was carried out using 1 .mu.g/ml
DIG-labelled RNA probe in hybridisation buffer (50% formamide,
1.3.times.SSC (pH 5), 5 mM EDTA (pH 8), 50 .mu.g/ml yeast RNA, 0.2%
Tween-20, 0.5% CHAPS, 100 .mu.g/ml heparin in DEPC treated
H.sub.2O) at 70.degree. C. over night. Hybridised probe was
detected using alkaline phosphatase conjugated anti-DIG Fab
fragments (Roche) and BM Purple alkaline phosphatase substrate
(Roche).
Example 15
Materials and Methods: Preparation, PCR and Southern Blot Analysis
of Single Cell cDNAs
[0481] Early bud stage embryos (E 7.5) and genital ridges (E 11.5)
were isolated in DMEM/10% fetal calf serum/25 mM HEPES (pH 7.4).
Fragments bearing primordial and gonadal germ cells, respectively,
were dissected out and dissociated into single cells. The latter
were picked using mouth pipettes and their cDNAs were amplified as
described previously [25]. The following primers were used in order
to PCR amplify stella cDNA and 3'-fragments of fragilis and
fragilis2-5 cDNAs (25 cycles of amplification):
TABLE-US-00019 stella: 5'CTCACAGCTTGAGGCTTCTAA3',
5'GCGATTCAGATGTCTCTGCAC3', fragilis: 5'GTTATCACCATTGTTAGTGTCATC3',
5'AATGAGTGTTACACCTGCGTG3'; fragilis3: 5'GATCTTCAGCATCCTTATGGTC3',
5'GAAGGTAACATTTGCATACGCG3'; fragilis2: 5'CCTTCCTTATTCTCACTCTG3',
5'GTTGCAAGACATCTCACATC3'; fragilis4: 5'AACTTGGAGGCTGCAAGGCAG3',
5'CTCGGAACTCTTAGTTATAGTC3'; fragilis5: 5'TGCTCTGGTCATCTCCCTCA3',
5'CAGGATAAGGGGCAACTCTG3'.
PCR products were run on 1.5% agarose/TBE electrophoresis gels. For
Southernblot analysis, single cell cDNAs were blotted onto
Hybond-N+ membranes (Amersham) and probed with .sup.32.alpha.P
dCTP-labelled DNA probes comprising the 3' regions of fragilis,
fragilis2 and fragilis3 cDNAs and full length stella cDNA. GAPDH
was used as loading control. Blotting signal was detected using a
Fuji film FLA 5000 scanner. Signal strength was quantified in
relation to GAPDH signal, whereby relative gene expression was
calculated as ratio of gene signal to GAPDH signal and this ratio
was subsequently normalized by division through the highest
hybridisation signal per blot. For dotblot analysis, full length
fragilis cDNAs were blotted and probed with .sup.32.alpha.p
dCTP-labelled 3' probes.
Example 16
The Fragilis Gene Family
[0482] Using the cDNA sequence of fragilis as a template to search
the ensembl genome browser (www.ensembl.org), we identified eight
mouse genes with moderate to high DNA sequence similarity to
fragilis (45-74%). ESTs from a variety of embryonic and adult
tissues have been reported for five of these genes, of which four
possess a two-exon structure similar to fragilis. Analysis of the
genomic location of the latter revealed that the four genes cluster
around the fragilis locus within a 70 kb region on the distal tip
of mouse Chr 7 (F5). We therefore named the four novel genes
fragilis2-5, reflecting their genomic location, similarity to
fragilis and germ cell associated expression pattern (see below;
FIG. 9). The four remaining putative genes that we detected have
few or mostly no reported ESTs and are coded by a single exon
unlike fragilis. We therefore consider them to be pseudogenes.
[0483] To determine whether the Fragilis genes are evolutionary
conserved, we have identified four homologues of mouse Fragilis in
the human genome on Chr 11 (p 15.5), a region which is indeed
syntenic to the Fragilis family locus on mouse Chr 7 (FIG. 9).
Three of these genes, Ifitm1 (9-27), Ifitm2 (1-8D) and Ifitm 3
(1-8U), share 58-65% similarity to the fragilis gene cluster and
are located within an 18 kb genomic stretch [11]. They are
responsive to type1/2 interferons and code for interferon induced
transmembrane (Ifitm) proteins, involved in antiproliferative
signalling and homotypic cell adhesion [12-15]. The fourth gene,
ENSG142056, a novel gene with two exons, is highly similar to mouse
fragilis4 (83% DNA sequence similarity) and neighbours Ifitm2. The
human Fragilis family homologues hence form a similar genomic
cluster as the five Fragilis genes in the mouse. Phylogenetic tree
analysis suggests however, that only two Fragilis genes, fragilis4
and either fragilis, fragilis2 or fragilis3, have been conserved
from mouse to human (data not shown). Subsequent gene duplications
may therefore have occurred independently in both species. We also
identified two Fragilis family-like genes in cow (bovine 1-8U,
bovine 9-27) and four genes in rat (P26376, JC1241, NP110460,
AAD48010). While the rat genes have been annotated as putative
interferon inducible, the two bovine genes that are similar to the
human Ifitm genes, have been reported to respond to interferon
signalling [16, 17]. Due to limited mapping information of the cow
and rat genomes, we cannot, at this stage, deduce whether these
homologous genes are also organised in a cluster. Interferon
stimulable response elements (ISREs, GGAAAN(N)GAAAC) within the
human Ifitm locus confer the responsiveness of the three human
Ifitm genes to interferons [11, 18]. Similar ISRE consensus
sequences are also found within the Fragilis family cluster in the
mouse, associated in particular with fragilis, fragilis 2 and
fragilis5 (FIG. 9).
[0484] The murine family of fragilis and related genes code for
five highly similar transcripts of 104 to 144 amino acids, each
containing two predicted transmembrane domains (FIG. 10). The
sequence similarity to human, cow and rat fragilis-like genes is
equally high (overall 68% amino acid similarity). It should be
noted, that the first transmembrane domain as well as the following
stretch to the beginning of the second transmembrane domain
constitute the regions of highest intra- and inter-species
conservation.
Example 17
Fragilis, Fragilis2 and Fragilis3 are Expressed During Early
Post-Implantation Development
[0485] We analysed the expression pattern of the five Fragilis
family genes by whole mount in situ hybridisation using probes that
span the 3' region (150-200 bp) of the corresponding mRNAs. These
probes show no significant cross-hybridization between members of
the Fragilis family as judged by dotblot analysis (data not shown).
As reported, we saw expression of fragilis restricted to the
epiblast at E5.5 and E6.5. More importantly, around E7.5,
expression of fragilis is intense within a population of cells at
the base of the allantois in the region where PGC specification
occurs (FIG. 11a-c) [8]. fragilis2 and fragilis3 are also expressed
within the epiblast of E5.5 embryos (FIG. 11g, data not shown).
While expression of fragilis2 is thereafter significantly
down-regulated, fragilis3 remains expressed at a similar level in
the embryonic tissues. At E7.5, fragilis2 is detected in the
posterior mesoderm, while fragilis3 expression is seen throughout
the epiblast. More significantly, like fragilis, both fragilis2 and
fragilis3 show high expression in the region where the cluster of
nascent PGCs originates (FIG. 11 i/i',n/n'). Thus, these three
members of the Fragilis family show significant expression at the
time and site of PGC specification.
[0486] At E8.5, fragilis expression is seen in cells at the base
and within the proximal third of the allantois (FIG. 11d).
Additionally, a signal is detected in the latero-anterior aspects
of the developing brain (FIG. 11e). At this stage, fragilis2 is
expressed in the mesoderm in the caudal half of the embryo (FIG.
11j,k), whereas fragilis3 appears present throughout the entire
embryo with the exception of the developing heart (FIG. 11p-r). It
is noteworthy, that expression seems significantly stronger in
single cells at the base and within the proximal third of the
allantois at this stage (FIG. 11q). At E9.5, when PGCs have started
to migrate along the hindgut, fragilis signal is seen in a
population of cells located at the beginning of the invaginated
hindgut. In addition, the signal appears enhanced in the pharyngeal
arches (FIG. 11f). At this stage, fragilis2 expression appears
restricted to the tailbud, the mesoderm caudal to the 12.sup.th
somite and the lung primordium (FIG. 11l).
[0487] In contrast to the first three members of the family,
neither fragilis4 nor fragilis5 showed expression at early
post-implantation stages (E7.0-E8.5, data not shown). Consequently,
only the three genes at the centre of the family cluster, that is
fragilis, fragilis2 and fragilis3 are expressed in the embryo
between E5.5 and E9.5. While their expression pattern is distinct,
there is a striking overlap within the region where founder germ
cells are located. This suggests that the three neighbouring genes,
fragilis, fragilis2 and fragilis3, may share regulatory elements
that are likely to be present within the cluster. These regulatory
elements may also be responsible for the genes' overlapping
expression pattern specifically around the region of nascent
PGCs.
Example 18
Single Cell Analysis of Fragilis, Fragilis2 and Fragilis3 in PGCs
and Somatic Neighbours
[0488] To obtain more precise information on the expression of the
new Fragilis family members in the context of germ cell
specification, we tested single cell cDNAs from PGCs and
surrounding somatic cells sited at the base of the incipient
allantoic bud in E7.5 embryos. Both fragilis2 and fragilis3 were
expressed in nascent PGCs, which show transcription of the germ
cell marker stella/PGC7 (FIG. 13a) [8, 10]. The two Fragilis family
members were also detected in surrounding somatic cells that lack
expression of stella/PGC7 [8]. Importantly, semi-quantitative
analysis using Southernblotting showed that fragilis2 and fragilis3
are expressed predominantly and at higher levels in nascent PGCs
compared to the neighbouring somatic cells (FIG. 13b,c). This
mimics the pattern seen for fragilis, although expression of the
latter is more specific to germ cells. Combined with the in situ
hybridisation data, these observations further support the notion
that certain common control elements may be involved in the
upregulated expression of the three Fragilis genes in the founder
PGCs.
[0489] During the developmental stages directly subsequent to PGC
specification, all three Fragilis family genes are expressed in a
population of cells associated with the allantois and in a location
where premigrating PGCs are thought to reside (FIG. 11d,k,q). The
precise gene expression during migration of PGCs is not clear at
this stage from our analysis. However, using in situ hybridisation
and PCR analysis of cDNAs from single cells within the genital
ridge, we found clear expression of fragilis, fragilis2 and
fragilis3 in the gonadal germ cells at E11.5-12.5 (FIG. 14). While
fragilis3 expression extends to the mesonephros, fragilis and
fragilis2 signal was restricted to the genital ridge. A punctuate
staining pattern was seen for fragilis, mimicking the germ cell
restricted expression of stella/PGC7 (FIG. 14b). This pattern in
addition to the PCR analysis suggests that fragilis is expressed
predominantly if not solely in germ cells at E11.5. As was the case
in earlier embryos, neither fragilis4 nor fragilis5 were detected
in gonadal germ cells (data not shown).
Example 19
Discussion
[0490] In this study we describe the identification of the murine
Fragilis gene family, which appears to be conserved amongst
mammalian species, and whose members code for five highly similar
transmembrane proteins. Three members of the Fragilis family,
fragilis, fragilis2 and fragilis3, exhibit expression, which is
associated with germ cell specification and development Located at
the cell membrane, the Fragilis proteins may be crucial for
mediating interactions amongst germ cells and their surrounding
neighbours. While the three genes are expressed earlier at E5.5 and
thereafter to a varying extent, they all show upregulation of
expression within nascent PGCs. It is likely that a cis control
element exists within the locus that is required for this
expression, which continues within gonadal PGCs. Future studies
will elucidate where these control elements are located and how
they regulate expression of the fragilis-related genes.
[0491] Although the five Fragilis family members are clustered
within a small genomic region, it appears that neither fragilis4 or
fragilis5 show expression in early embryos or embryonic germ cells.
It is striking that these two members are located at the periphery
of the cluster in contrast to the centrally located fragilis,
fragilis2 and fragilis3 genes. This lack of expression may be due
to the presence of boundary elements, which might restrict the
action of control elements to genes present within the centre of
the cluster. Since sequence comparison suggests that gene
duplications may have occurred independently in the two species, it
appears that a certain evolutionary constrain may exist on
duplication and maintenance of the duplicated genes within
immediate neighborhood. Since the four human homologues of the
Fragilis family in the syntenic region are also arranged in a
genomic cluster and are highly similar to the family genes, it is
tempting to suggest that they may also serve similar functions as
in the mouse.
[0492] The presence of several interferon stimulable response
element (ISRE) consensus sequences within the Fragilis locus,
together with the similarity of the genes to their
interferon-inducible human and bovine counterparts, suggest very
strongly that fragilis and the fragilis-related genes are
responsive to interferons. Indeed, the ISRE tandem repeat present
in the 5' flanking region of human Ifitm1, Ifitm2 and Ifitm3 genes
is also present in the 5' flanking region of fragilis exon 1 [11].
Interferons, as secreted signalling molecules, have so far been
implicated mainly in the process of immune response, the inhibition
of cellular growth and the control of apoptosis [19]. Although
interferons are expressed in the post-implantation embryo, their
role during development has not been addressed in detail [20, 21].
Our studies have pointed to a possible involvement of interferons
in germ cell development. Future work will determine whether the
Fragilis genes respond to interferon signals in all or some
instances where the genes are expressed, which we expect in view of
the presence of conserved ISRE elements in the mouse and human
loci.
Example 20
Conclusion
[0493] We have identified the Fragilis family of interferon
inducible genes, which code for transmembrane proteins. The five
members are arranged in a cluster within a genomic region of 70 kb
in the mouse that also contains ISRE elements. The centrally
located fragilis, fragilis2 and fragilis3 genes are of particular
interest, because they are expressed in the region where germ cell
specification occurs. The family is evolutionary conserved amongst
mammalian species where it may serve similar functions. Detailed
studies of the Fragilis family may also show what role interferons
have in embryonic development.
Example 21
Stella is a Maternal Effect Gene Required for Normal Early
Development in Mice
[0494] In this and the following Examples (Examples 21 to 25), we
have investigated the effects of a targeted mutation of stella in
mice. Maternal inheritance in mammalian oocytes includes proteins
important for totipotency and epigenetic modifications.sup.1, as
well as factors crucial for early development, which are
transcribed from so called maternal effect genes.sup.2-7.
[0495] Amongst these maternally inherited proteins is Stella, which
is also expressed in preimplantation embryos, primordial germ
cells, and pluripotent cells.sup.8,9. We show that while matings
between heterozygous animals resulted in the birth of apparently
normal stella-null offspring, stella-deficient females showed
severely reduced fertility, which is due to a lack of maternally
inherited Stella in their oocytes.
[0496] Stella is a maternal effect gene, as the phenotypic effect
on embryonic development is a consequence of the maternal stella
mutant genotype. Indeed, we demonstrate that embryos lacking
Stella-protein are compromised in preimplantation development and
rarely reach the blastocyst stage. Furthermore, we show that STELLA
that is expressed in human oocytes.sup.10 is also expressed in
human pluripotent cells and in germ cell tumours. Interestingly,
human chromosome 12p, which harbours STELLA is consistently
overrepresented in these tumours.sup.11. These findings suggest a
similar role for STELLA during early human development as in mice
and a potential involvement in germ cell tumours.
[0497] The aim of this study was to determine the role of stella by
loss of function analysis in mice. In our previous work, we have
shown that expression of stella (also called PGC7) is activated
during the process of germ cell specification at E7.25 specifically
in the founder population of lineage restricted primordial germ
cells (PGCs).sup.8,9. Thereafter it is expressed in the germ line
until about E15.5 in male and E13.5 in female gonads. Expression of
stella resumes in the immature oocytes in newborn ovaries, and it
is subsequently detected in maturing oocytes and in preimplantation
embryos (FIG. 15a-l).sup.8. Soon after the formation of the zygote,
Stella accumulates in the pronuclei, although it is also detected
in the cytoplasm (FIG. 15d-f). Both cytoplasmic and nuclear
staining continues during cleavage stages until the blastocyst
stage, after which Stella is downregulated (FIG. 15g-l and data not
shown).sup.8, until its re-appearance in the nascent
PGCs.sup.8,9.
Example 22
Materials and Methods
[0498] Immunofluorescence
[0499] Embryos were fixed in 4% paraformaldehyde for 15 minutes,
washed 3 times with PBS and permeabilised in AB-buffer (1%
Triton-X100, 0.2% SDS, 10 mg/ml BSA in PBS), which was also used
for the following antibody incubations and washes. They were then
incubated in primary antibody (anti-Stella.sup.9 1:200,
anti-PGC7.sup.8 1:2000) overnight at 4.degree. C., washed 3 times
and incubated with secondary antibody for 1-2 hours at
room-temperature (Alexa 564, Molecular probes, 1:500). After 3
further washes in AB-buffer, embryos were rinsed once in PBS and
incubated at 37.degree. C. with 0.1 mg/ml Rnase A (Roche) in PBS
for 30 minutes. Finally embryos were incubated for 10 minutes in
PBS with propidium iodide (2 .mu.g/ml) and mounted on slides in
Vectashield (Vector Laboratories) mounting medium, which also
contained propidium iodide.
[0500] For E11.5 PGC-stainings, genital ridges were washed in PBS,
treated for 10 minutes at 37.degree. C. with Trypsin/EDTA (Gibco),
diluted in PBS and dissociated into a cell suspension. Cells were
allowed to settle down on poly-L-lysine treated slides and fixed
with 3% formaldehyde for 15 minutes. After permeabilisation with
0.2% Triton X-100 in PBS and 3 washes in PBS cells were blocked
with 3% BSA in PBS (also used for subsequent washes and antibody
dilutions) for 40 minutes and incubated with primary antibodies
(anti-Stella 1:100, anti-SSEA1 (=TG1), P. Beverley 1:2) overnight
at 4.degree. C. Then the cells were washed and incubated with
secondary antibodies (Alexa 564, Alexa 488, Molecular probes,
1:500) for 1.5 hours. After washing, Rnase (0.1 mg/ml) treatment
was done for 1 hour at room temperature and the cells were mounted
with Vectashield containing Toto-3 (Molecular probes, 1:1000).
[0501] Immunofluorescence was visualized on a BioRad Radiance 2000
confocal microscope.
[0502] Identification of Stella-Homologues
[0503] Human STELLA was identified by blasting the mouse Stella
protein sequence against the translated human genome sequence using
the Ensembl server (http://www.ensembl.org). The only hit showing
the same intron-exon structure as the mouse gene is located on the
syntenic region (FIG. 15m,n) and was therefore considered to be the
human orthologue (hits without introns were considered as
pseudogenes). Three IMAGE-EST clones (Genbank IDs: AA927342,
AI066520, AA564230; UniGene cluster Hs.131358), which aligned to
the genomic region, were fully sequenced by us to confirm the
predicted sequence.
The putative rat-stella sequence was mapped as above and deduced
from the alignment of the mouse cDNA sequence with the syntenic rat
genome sequence.
[0504] RT-PCR Analysis of Human Tissues
[0505] 1 .mu.g total RNA of each human tissue (source: Ambion and
see acknowledgements) was reverse transcribed into 1.sup.st strand
cDNA with Superscript II reverse transcriptase (Gibco) for 1 hour
at 37.degree. C. 1 .mu.l of this cDNA was amplified by a 30 cycle
PCR-reaction using primers for human STELLA
(5'-CAATTTGAGGCTCTGTCATCAG-3',5'-TTCATCTCACTGACTTTGGGC-3') or
ribosomal protein L32
(5'-AGTTCCTGGTCCACAACGTC-3',5'-TGCACATGAGCTGCCTACTC-3').
[0506] ES-Cell Manipulation and Knockout Verification
[0507] The targeting construct consisted of 1.5 kb of upstream and
4.1 kb of downstream genomic sequence flanking the second exon of
stella. The 5' arm terminated after the first 32 bp of exon 2,
which was fused to an IRES lacZ reporter, followed by a promoted
neo selectable marker. The construct was linearized and
electroporated into CCB mouse embryonic stem (ES) cells which were
placed under selection. Individual G418-resistant clones were
picked and screened for correct integration of the targeting
construct by PCR using a vector primer and a primer external to the
5' arm. 288 clones were screened of which two exhibited the
expected size bands in the PCR. Homologous recombination was also
confirmed by Southern blot using 5', 3' and neo-probes on NcoI and
EcoRI digested genomic DNA. The correctly targeted ES-cell clone F4
was injected into MF1 and C57BL/6 blastocycsts to produce chimeric
mice. Germline transmission was achieved by breeding the male
chimeras with 129Sv/Ev females. All analysis was done on the inbred
129Sv/Ev background. To confirm that the stella gene was correctly
inactivated, mice were genotyped by Southern blot as above (FIG.
16b). Furthermore we performed RT-PCR (same protocol as for human
tissues--see above) on testis and ovary RNA of wt, heterozygous and
homozygous mice (FIG. 16c), using exon 2-specific primers
(5'-AGACGTCCTACAACCAGAAAC-3',5'-CCGAACAAGTCTTCTCATCT.TM.-3').
[0508] Counting of Primordial Germ Cells
[0509] Embryos of stella-heterozygous intercrosses were dissected
out at E8.5, fixed with 4% paraformaldehyde and stained for
TNAP-positive PGCs with .alpha.-naphthyl phosphate/fast red TR
solution (Sigma) as previously described.sup.20,26. The posterior
parts of the embryos were flattened under coverslips and used for
counting PGCs, while the anterior parts were used for genotyping by
PCR.
[0510] Histology
[0511] Testes and ovaries from adult mice were fixed in Bouin's
fixative at 4.degree. C. overnight and washed thoroughly in 80%
ethanol. After dehydration through an ethanol series they were
transferred into xylene and embedded in Paraplast Plus wax (Sigma).
8 .mu.m sections were cut, rehydrated and stained with Ehrlich's
Haematoxilin (BDH) and 1% eosin (Sigma) After dehydration, slides
were mounted with DPX (BDH).
[0512] Matings and In Vitro Culture
[0513] All studies for the assessment of fertility and embryonic
development were done using natural matings. Mice were kept on a
constant light/dark cycle and mating was assumed to have happened
in the middle of the dark period before a vaginal plug was detected
(E0.5=midday on day of plug). Embryos were collected by flushing
oviducts/uteri at the time of the observed stages (E0.5-E3.5) or at
E1.5, if they were cultured. Culturing was done under 5% CO.sub.2
in KSOM medium.
Work on animals was performed under Home Office project licenses
PPL80/1280 and PPL80/1706.
[0514] Generation of Stella-GFP Mice
[0515] Using the stella-cDNA as a probe, we screened a gridded
genomic 129 pBeloBAC library (Genome Systems St Louis, Mo.) to
identify a clone harbouring the stella locus. We subcloned 11.5 kb
of genomic sequence including about 8.5 kb upstream sequence and
exon 1, intron 1 and the start of exon 2 and fused it in frame to
eGFP (Clontech) and a SV40-polyadenylation signal. This sequence
was then injected into pronuclei of B6CBA F2 zygotes, to generate
transgenic mice. The transgene was maintained on the same genetic
background and the onset of expression of the paternal allele was
observed by mating stella-GFP transgenic males with non-transgenic
females.
[0516] The cDNAs of the Stella homologues mentioned in this study
have the following GenBank accession numbers: mouse Stella
(AY082485), rat Stella (BK001414, pending), human STELLA (AY317075,
pending).
Example 23
Stella Homologues
[0517] We have now identified stella homologues in the rat and
human genomes, which show the same exon-intron structure, and are
located within the syntenic chromosomal regions (see FIG. 15m,n).
The mouse gene is in position F2 of chromosome 6, the rat gene on
q42 of chromosome 4 and the human gene on p13.31 of chromosome 12.
Only one expressed-sequence tag (EST) (BI289609, aorta pool) was
found in the rat, while several human ESTs mainly from germ cell
tumour libraries (UniGene cluster Hs.131358) matched the genomic
sequence. The full-length amino acid sequences (FIG. 15o) of the
mouse and rat protein showed 70% identity (84% similarity), but the
mouse and human proteins shared only 35% identity (53% similarity).
While the Stella orthologues of rodents and humans have clearly
diverged, conserved sequence stretches are found in the centre and
the C-termini of the proteins. The biochemical function of these
motifs remains to be discovered, but some of the predicted nuclear
localisation and export signals reside within the regions of higher
conservation.
Example 24
Expression of Stella
[0518] To study the expression of human STELLA, we performed RT-PCR
analysis on pluripotent cell lines and reproductive organs (FIG.
15p). We detected STELLA in human embryonic stem (ES) cells and
embryonic carcinoma (EC) cells, as well as in normal testis and
ovary. The strongest expression was found in a testicular germ cell
tumour, which shows characteristics of pluripotency.sup.11.
Expression of STELLA in other tumours and somatic tissues was
either very low or undetectable (data not shown). Our findings
concur with a recent study.sup.10, where STELLA (termed fragment
7.1) was detected in human oocytes and in EC cells, in which it was
down-regulated after retinoic acid-induced differentiation. These
findings strengthen the hypothesis that STELLA might have a similar
role in humans as in mice. Furthermore, the short arm of chromosome
12 (12p) on which STELLA is located, is consistently
overrepresented in testicular germ cell tumours.sup.11.
Stella/STELLA resides within a conserved cluster of genes
consisting of nanog/NANOG.sup.12,13 and gdf.sub.3/GDF3.sup.14 (FIG.
15n), which are associated with pluripotency and germ cell tumours.
The conserved proximity in mice and humans and the overlapping
expression patterns of these genes suggest a possible co-regulation
at a transcriptional level.sup.15. Clearly, these findings prompt a
careful analysis of the functions of stella and its neighbours in
mouse and man.
Example 25
Stella Knockout Mice
[0519] To begin to address functions of stella, we generated stella
knockout (stella) mice (FIG. 16). Matings between heterozygous
(stella.sup.+/-) mice on the 129/SvEv background resulted in the
birth of 192 pups consisting of 56 (29.2%) wild-type, 81 (42.2%)
stella.sup.+/- and 55 (28.6%) stella.sup.-/- mice, in the
approximate mendelian ratio of 1:2:1. Therefore, stella.sup.-/-
deficient mice are viable and survive at a normal rate.
[0520] As stella is detected in the founder PGCs, we examined
stella.sup.-/- mice for any effects on development of germ cells.
Examination of germ cells at E8.5 in mutant embryos by tissue non
specific alkaline phosphatase (TNAP) activity, a marker of
PGCs.sup.16, revealed no significant differences in the numbers of
PGCs compared to those in wild-type embryos (FIG. 17a). Similarly
we found no effect on early gonadal PGCs (E1.5) in knockout
embryos, detected by the germ cell marker SSEA1.sup.17 (FIG. 17b).
Furthermore, histological examination of testes and ovaries of
adult mice showed no gross abnormalities in the development of
gametes in stella mutant animals (FIG. 3h-m). Indeed stella.sup.-/-
males showed normal fertility when mated with wild-type or
heterozygous females. In mutant females, we detected oocytes at all
stages of development and we found similar numbers of ovulated
oocytes compared to those from control animals
(stella.sup.-/-8.6.+-.1.0, n=9; wild-type or
stella.sup.+/-9.0.+-.0.4, n=9), suggesting that the loss of stella
has no gross effects on either germ cell determination or
development.
[0521] Next, we examined if development progressed normally from
oocytes of stella.sup.-/- females that lack maternal inheritance of
Stella. Despite the ovulation of normal numbers of Stella-deficient
oocytes, female stella-mice displayed a strongly reduced fertility.
When stella.sup.-/- females were mated with wild-type males, only a
low percentage of matings (detected by vaginal plugs) (24%, FIG.
18a) resulted in full pregnancy and live young. Those females,
which failed to become pregnant mated again after approximately 10
days, which reflects lack of embryo implantation in these females
and the consequent resumption of the estrous cycle after a period
of pseudopregnancy.sup.18. By contrast, 80% of wild-type females
(littermate controls), became pregnant and produced litters
following mating (FIG. 18a). Furthermore, even those stella.sup.-/-
females that became pregnant, produced considerably smaller litters
compared to the wild-type females (FIG. 18b). Preliminary results
also show reduced fertility in an outbred strain (129SvEv/C57BL/6),
although the effect is stronger in inbred 129Sv/Ev mice. This is
consistent with previous reports that genetic background can alter
the severity of knockout phenotypes.sup.19, including defects in
germ cell development.sup.20,21. These observations demonstrate
that embryos derived from Stella-depleted oocytes are affected in
development and that stella is a maternal effect gene, because the
oocytes were fertilised by normal sperm from wild-type males.
[0522] Next we wanted to know, if the Stella protein in
preimplantation embryos (FIG. 15).sup.8 is exclusively maternally
inherited and therefore absent in embryos derived from
stella.sup.-/- females, or if stella expression commences from the
paternal allele after fertilisation by wild-type sperm. For this
purpose, we made transgenic mice using a stella-GFP reporter
transgene (FIG. 18c-i). When a stella-GFP transgenic male was mated
with a non-transgenic female, we detected the transgene expression
as early as the 2-cell stage (E1.5, FIG. 18e, h), the time when the
bulk of embryonic transcription and translation begins.sup.22. This
indicates that the stella gene is transcribed very early during
preimplantation development. We confirmed this observation by
anti-Stella antibody stainings of E2.5 embryos (FIG. 18j-l), which
were derived from mating a wild-type male with a stella.sup.-/-
female. Therefore, Stella is clearly made in early embryos produced
by matings between stella.sup.-/- females and wild-type males. But
despite this, the majority of Stella-deficient oocytes did not
develop normally to term, demonstrating that the onset of stella
expression as early as the 2-cell stage from the paternal allele is
not sufficient to fully rescue the observed maternal effect
phenotype. By contrast, the maternally inherited Stella is
sufficient for normal development, as stella.sup.-/- mice are born
from heterozygous females mated with homozygous males at the same
frequency as wild-type mice (see above).
[0523] We then addressed the question concerning the embryonic
stages at which the absence of Stella affects development. As we
have so far not obtained any live young from matings between
stella.sup.-/- males and stella.sup.-/- females, we examined
embryos from these matings, and compared it with embryos from
matings between wild-type or stella.sup.-/- males with wild-type or
stella.sup.+/- females (FIG. 19). While fertilisation seems to
proceed normally in oocytes from stella.sup.-/- females, the
effects of lacking Stella become evident shortly thereafter, with
progressively fewer embryos exhibiting normal development at each
time point examined (FIG. 19a). The cumulative manifestation of
developmental anomalies are starkly obvious at E3.5, when most of
the embryos from controls (69%) reach the blastocyst stage, while
only 6% of embryos in stella.sup.-/- mothers do so (FIG. 19 a-c).
This observation was further supported by examination of similar
embryos cultured in vitro for 3 days until E4.5, when only 15% of
embryos from mutant oocytes reached the blastocyst stage compared
to 69% for controls. 49% of mutant embryos were still at the
single-cell stage, fragmenting or exhibiting asymmetric or abnormal
cleavage. The remainder were found at various stages including 10%
at the 2-cell stage and 27% at the morula stage (FIG. 5d-f). Since
uterine receptivity for blastocyst implantation is restricted to
late E3.5 to early E4.5, only those embryos that reach the
blastocyst stage by that time can implant.sup.23,24. This is
consistent with the observation that stella.sup.-/- females rarely
become pregnant and when they do, they produce very small litters.
In several cases, stella.sup.-/- females only become pseudopregnant
and resume mating after 10 days, which is indicative of a lack of
implanting blastocysts in these females.sup.18.
[0524] In conclusion, we demonstrate that the maternal inheritance
of Stella is needed for normal embryonic development. Depletion of
Stella from the oocytes compromises this process, resulting in a
progressive decline in the numbers of blastocysts, fewer implants
and a poor yield of viable young. Stella is a basic protein with a
SAP-like domain.sup.25 and a splicing factor-like motif and
therefore likely to have a role in chromosomal organisation or RNA
metabolism. We propose to look for the interacting partners and the
biochemical activity of the conserved domains of Stella to
elucidate its role in early development. Despite a lack of gross
abnormalities in germ cell development in stella.sup.-/- mice, we
cannot rule out subtle effects. One possibility is functional
redundancy through compensation by stella-related genes. There are
several stella-like sequences in the mouse genome, although these
are likely to be pseudogenes (data not shown). STELLA is also
expressed in human oocytes.sup.10, where it is likely to play a
similar role in early development as in mice. As the highest
expression of STELLA is in a human testicular germ cell tumour,
this could serve as a diagnostic marker or be of therapeutic value
in the future. The conservation of the syntenic chromosomal region
harbouring STELLA, together with NANOG and GDF3 on chromosome 12p
is noteworthy as it is associated with pluripotency,
teratocarcinomas and germ cell tumours in humans. The role of
likely coordinated regulation of all key genes within the region
may provide evolutionary insights into aspects of germ cell
development and germ cell tumours, as well as on pluripotency and
maternal effect genes.
REFERENCES
[0525] Brady, G. and Iscove, N. N. (1993). Construction of cDNA
libraries from single cells. Methods Enzymol. 225, 611-623. [0526]
Dulac, C. and Axel, R. (1995). A novel family of genes encoding
putative pheromone receptors in mammals. Cell 83, 195-206. [0527]
Ginsburg, M., Snow, M. H. L., and McLaren, A. (1990). Primordial
germ cells in the mouse embryo during gastrulation. Development
110, 521-528. [0528] Downs, K. M., and Davies, T. (1993). Staging
of gastrulating mouse embryos by morphological landmarks in the
dissecting microscope. Development 118, 1255-1266. [0529] Lawson,
K. A., Dunn, N. R., Roelen, B. A. J., Zeinstra, L. M., Davis, A.
M., Wright, C. V. E., Korving, J. P. W. F. M., and Hogan, B. L. M.
(1999). Bmp4 is required for the generation of primordial germ
cells in the mouse embryo. Genes & Dev. 13, 424-436. [0530]
Yoem, Y. II., Fuhrmann, G., Ovitt, C. E., Brehm, A., Ohbo, K.,
Gross, M., Hubner, K., and Scholer, H. R (1996). Germline
regulatory element of Oct4 specific for the totipotent cycle of
embryonal cells. Development 1996, 881-894. [0531] 1. Weismann, A.
Das Keimplasma. Eine theorie der Vereburg. Jenna. Gustav Fischer
(1892). [0532] 2. Eddy, E. M. Germ plasm and the differentiation of
the germ cell line. Int Rev Cytol 43, 229-80 (1975). [0533] 3.
Seydoux, G. & Strome, S. Launching the germline in
Caenorhabditis elegans: regulation of gene expression in early germ
cells. Development 126, 3275-83. (1999). [0534] 4. Wylie, C. Germ
cells. Cell 96, 165-74. (1999). [0535] 5. Lawson, K. A. et al. Bmp4
is required for the generation of primordial germ cells in the
mouse embryo. Genes Dev 13, 424-36. (1999). [0536] 6. Lawson, K. A
& Hage, W. J. Clonal analysis of the origin of primordial germ
cells in the mouse. Ciba Found Symp 182, 68-84 (1994). [0537] 7.
Tam, P. P. & Zhou, S. X. The allocation of epiblast cells to
ectodermal and germ-line lineages is influenced by the position of
the cells in the gastrulating mouse embryo. Dev Biol 178, 124-32.
(1996). [0538] 8. Yoshimizu, T., Obinata, M. & Matsui, Y.
Stage-specific tissue and cell interactions play key roles in mouse
germ cell specification. Development 128, 481-90. (2001). [0539] 9.
McLaren, A. Signaling for germ cells. Genes Dev 13, 373-6. (1999).
[0540] 10. Ying, Y., Liu, X. M., Marble, A., Lawson, K. A. &
Zhao, G. Q. Requirement of Bmp8b for the generation of primordial
germ cells in the mouse. Mol Endocrinol 14, 1053-63. (2000). [0541]
11. Ying, Y., Qi, X. & Zhao, G. Q. Induction of primordial germ
cells from murine epiblasts by synergistic action of BMP4 and BMP8B
signaling pathways. Proc Natl Acad Sci USA 98, 7858-7862. (2001).
[0542] 12. Ying, Y. & Zhao, G. Q. Cooperation of
endoderm-derived BMP2 and extraembryonic ectoderm-derived BMP4 in
primordial germ cell generation in the mouse. Dev Biol 232, 484-92.
(2001). [0543] 13. Chiquoine, A. D. The identification, origin and
migration of the primordial germ cells in the mouse embryo. Anat
Rec 118, 135-146 (1954). [0544] 14. Ginsburg, M., Snow, M. H. &
McLaren, A. Primordial germ cells in the mouse embryo during
gastrulation. Development 110, 521-8. (1990). [0545] 15. MacGregor,
G. R, Zambrowicz, B. P. & Soriano, P. Tissue non-specific
alkaline phosphatase is expressed in both embryonic and
extraembryonic lineages during mouse embryogenesis but is not
required for migration of primordial germ cells. Development 121,
1487-96. (1995). [0546] 16. Nichols, J. et al. Formation of
pluripotent stem cells in the mammalian embryo depends on the POU
transcription factor Oct4. Cell 95, 379-91. (1998). [0547] 17.
Pesce, M., Gross, M. K. & Scholer, H. R. In line with our
ancestors: Oct-4 and the mammalian germ. Bioessays 20, 722-32.
(1998). [0548] 18. Yeom, Y. I. et al. Germline regulatory element
of Oct-4 specific for the totipotent cycle of embryonal cells.
Development 122, 881-94. (1996). [0549] 19. Downs, K. M. &
Davies, T. Staging of gastrulating mouse embryos by morphological
landmarks in the dissecting microscope. Development 118, 1255-66.
(1993). [0550] 20. Brady, G. & Iscove, N. N. Construction of
cDNA libraries from single cells. Methods Enzymol 225, 611-23
(1993). [0551] 21. Dulac, C. & Axel, R. A novel family of genes
encoding putative pheromone receptors in mammals. Cell 83, 195-206.
(1995). [0552] 22. Frohman, M. A., Boyle, M. & Martin, G. R
Isolation of the mouse Hox-2.9 gene; analysis of embryonic
expression suggests that positional information along the
anterior-posterior axis is specified by mesoderm. Development 110,
589-607. (1990). [0553] 23. Deblandre, G. A. et al. Expression
cloning of an interferon-inducible 17-kDa membrane protein
implicated in the control of cell growth. J Biol Chem 270, 23860-6.
(1995). [0554] 24. Friedman, R. L., Manly, S. P., McMahon, M.,
Kerr, I. M. & Stark, G. R. Transcriptional and
posttranscriptional regulation of interferon-induced gene
expression in human cells. Cell 38, 745-55. (1984). [0555] 25.
Evans, S. S., Collea, R. P., Leasure, J. A. & Lee, D. B.
IFN-alpha induces homotypic adhesion and Leu-13 expression in human
13 lymphoid cells. J Immunol 150, 736-47. (1993). [0556] 26. Evans,
S. S., Lee, D. B., Han, T., Tomasi, T. B. & Evans, R. L.
Monoclonal antibody to the interferon-inducible protein Leu-13
triggers aggregation and inhibits proliferation of leukemic B
cells. Blood 76, 2583-93. (1990). [0557] 27. Aravind, L. &
Koonin, E. V. SAP--a putative DNA-binding motif involved in
chromosomal organization. Trends Biochem Sci 25, 112-4. (2000).
[0558] 28. Gurdon, J. B., Lemaire, P. & Kato, K. Community
effects and related phenomena in development. Cell 75, 831-4.
(1993). [0559] 29. Reid, L. E. et al. A single DNA response element
can confer inducibility by both alpha- and gamma-interferons. Proc
Natl Acad Sci USA 86, 840-4. (1989). [0560] 30. Kita, M. et al.
[Expression of cytokines and interferon-related genes in the mouse
embryo]. C R Seances Soc Biol Fil 188, 593-600 (1994). [0561] 31.
Gomperts, M., Garcia-Castro, M., Wylie, C. & Heasman, J.
Interactions between primordial germ cells play a role in their
migration in mouse embryos. Development 120, 135-41. (1994). [0562]
32. Herrmann, B. G., Labeit, S., Poustla, A., King, T. R. &
Lehrach, H. Cloning of the T gene required in mesoderm formation in
the mouse. Nature 343, 617-22. (1990). [0563] 33. Herrmann, B. G.
Expression pattern of the Brachyury gene in whole-mount TWis/TWis
mutant embryos. Development 113, 913-17 [0564] 34. Crossley, P. H.
& Martin, G. R. The mouse Fgf8 gene encodes a family of
polypeptides and is expressed in regions that direct outgrowth and
patterning in the developing embryo. Development 121, 439-51.
(1995). [0565] 35. Barnes, J. D., Crosby, J. L., Jones, C. M.,
Wright, C. V. & Hogan, B. L. Embryonic expression of Lim-1, the
mouse homolog of Xenopus Xlim-1, suggests a role in lateral
mesoderm differentiation and neurogenesis. Dev Biol 161, 168-78.
(1994). [0566] 36. Fujii, T. et al. Expression patterns of the
murine LIM class homeobox gene lim1 in the developing brain and
excretory system. Dev Dyn 199, 73-83. (1994). [0567] 37. Bastian,
H. & Gruss, P. A murine even-skipped homologue, Evx 1, is
expressed during early embryogenesis and neurogenesis in a biphasic
manner. Embo J 9, 1839-52. (1990). [0568] 38. Rogers, M. B.,
Hosler, B. A. & Gudas, L. J. Specific expression of a retinoic
acid-regulated, zinc-finger gene, Rex-1, in preimplantation
embryos, trophoblast and spermatocytes. Development 113, 815-24.
(1991). [0569] 39. Sutton, J. et al. Genesis, a winged helix
transcriptional repressor with expression restricted to embryonic
stem cells. J Biol Chem 271, 23126-33. (1996). [0570] 40. Cox, D.
N. et al. A novel class of evolutionarily conserved genes defined
by piwi are essential for stem cell self-renewal. Genes Dev 12,
3715-27. (1998). [0571] 41. Fujiwara, Y. et al. Isolation of a
DEAD-family protein gene that encodes a murine homolog of
Drosophila vasa and its specific expression in germ cell lineage.
Proc Natl Acad Sci USA 91, 12258-62. (1994). [0572] 42. Dixon, K.
E. Evolutionary aspects of primordial germ cell formation. Ciba
Found Symp 182, 92-110 (1994). [0573] 43. Mahowald, A. P. Assembly
of the Drosophila germ plasm. Int Rev Cytol 203, 187-213 (2001).
[0574] 44. Nieuwkoop, P. D. & Satasurya, L. A. Primordial germ
cells in the chordates. Cambridge University Press, Cambridge, UK
(1979). [0575] 45. Johnson, A. D., Bachvarova, R. F., Drum, M.
& Masi, T. Expression of axolotl dazl rna, a marker of germ
plasm: widespread maternal rna and onset of expression in germ
cells approaching the gonad. Dev Biol 234, 402-15. (2001). [0576]
46. Johnson, A. D., Bachvarova, R. F., Masi, T. & Drum, M.
Expression of nasa and Daz-like genes demonstrate that Axolotl
primordial germ cells (PGCs) are not predetermined. Germ cells Cold
Spring harbor laboratory, 61 (2000). [0577] 47. Toyooka, Y. et al.
Expression and intracellular localization of mouse Vasa-homologue
protein during germ cell development. Mech Dev 93, 13949. (2000).
[0578] 48. Saitou, M. et al. Occludin-deficient embryonic stem
cells can differentiate into polarized epithelial cells bearing
tight junctions. J Cell Biol 141, 397-408. (1998). [0579] 49.
Henrique, D. et al. Expression of a Delta homologue in prospective
neurons in the chick. Nature 375, 787-90. (1995). [0580] 50.
Wilkinson, D. G. & Nieto, M. A. Detection of messenger RNA by
in situ hybridization to tissue sections and whole mounts. Methods
Enzymol 225, 361-73 (1993). [0581] 51. Winnier, G., Blessing, M.,
Labosky, P. A. & Hogan, B. L. Bone morphogenetic protein-4 is
required for mesoderm formation and patterning in the mouse. Genes
Dev 9, 2105-16. (1995).
REFERENCES FOR EXAMPLES 11 TO 20
[0581] [0582] 1. K A Lawson, W J Hage: Clonal analysis of the
origin of primordial germ cells in the mouse. Germline development.
In Wiley, Chichester (Ciba Foundation Symposium 182) 1994, 68-91
[0583] 2. A McLaren: Signaling for germ cells. Genes Dev 1999, 13:
373-376 [0584] 3. K A Lawson, N R Dunn, B A J Roelm, L M Zeinstra,
A M Davies, C V E Wright, J P W F M Korving, B L M Hogan: Bmp4 is
required for the generation of primordial germ cells in the mouse
embryo. Genes Dev 1999, 13: 424-436 [0585] 4. Y Ying, X M Lui, A
Marble, K A Lawson, G Q Zhao: Requirement of Bmp8b for the
generation of primordial germ cells in the mouse. Mol Endocrinol
2000, 14: 1053-1063 [0586] 5. H Chang, M M Matzuk: Smad5 is
required for mouse primordial germ cell development. Mech Dev 2001,
104: 61-67 [0587] 6. K D Tremblay, N R Dunn, E J Robertson: Mouse
embryos lacking Smad1 signals display defects in extraembryonic
tissues and germ cell formation. Development 2001, 128: 3609-3621
[0588] 7. Y Ying, G Q Zhao: Cooperation of endoderm-derived BMP2
and extraembryonic ectoderm-derived BMP4 in primordial germ cell
generation in the mouse. Dev Biol 2001, 232 (2): 484-492 [0589] 8.
M Saitou, S C Barton, M A Surani: A molecular programme for the
specification of germ cell fate in mice. Nature 2002, 418: 293-300
[0590] 9. M Ginsburg, M H L Snow, A McLaren: Primordial germ cells
in the mouse embryo during gastrulation. Development 1990, 110:
521-528 [0591] 10. M Sato, T Kimura, K Kurokawa, Y Fujita, K Abe, M
Masuhara, T Yasunaga, A Ryo, M Yarnamoto, T Nakano: Identification
of PGC7, a new gene expressed specifically in preimplantation
embryos and germ cells. Mech Dev 2002, 113: 91-94 [0592] 11. A R
Lewin, L E Reid, M McMahon, G R Stark, I M Kerr: Molecular analysis
of a human interferon-inducible gene family. Eur J Biochem 1991,
199: 417-423 [0593] 12. R L Friedman, S P Manley, M Mcahon, I M
Kerr, G R Stark: Transcriptional and posttranscriptional regulation
of interferon-induced gene expression in human cells. Cell 1984,
38: 745-755 [0594] 13. J M Kelly, C S Gilbert, G R Stark, I M Kerr:
Differential regulation of interferon-induced mRNAs and c-myc mRNA
by alpha- and gamma-interferons. Eur J Biochem 1985, 153: 367-371
[0595] 14. S S Evans, D B Lee, T Han, T B Tomasi, R L Evans:
Monoclonal antibody to the interferon-inducible protein Leu-13
triggers aggregation and inhibits proliferation of leukemic B
cells. Blood 1990, 76 (12): 2583-2593 [0596] 15. S S Evans, R P
Collea, J A Leasure, D B Lee: IFN-a induces homotypic adhesion and
Leu-13 expression in human B lymphoid cells. J Immunol 1993, 150:
736-747 [0597] 16. D J Hayzer, E Brinson, M S Runge: A rat
beta-interferon-induced mRNA: sequence characterization. Gene 1992,
117 (2): 227-228 [0598] 17. J K Pru, K J Austin, A L Haas, T R
Hansen: Pregnancy and interferon-tau upregulate gene expression of
members of the 1-8 family in the bovine uterus. Biol Reprod 2001,
65 (5): 1471-1480 [0599] 18. L E Reid, A H Brasnett, C S Gilbert, A
C G Porter, D R Gewert, G R Stark, I M Kerr: A single DNA response
elemnt can confer inducibility by both alpha- and
gamma-interferons. Proc Natl Acad Sci USA 1989, 86: 840-844 [0600]
19. G R Stark, I M Kerr, B R G Williams, R H Silverman, R D
Schreiber: How cells respond to interferons. Annu Rev Biochem 1998,
67: 227-264 [0601] 20. D P Barlow, B J Randle, D C Burke:
Interferon synthesis in the early post-implantation mouse embryo.
Differentiation 1984, 27: 229-235 [0602] 21. M Kita, K Tanaka, K
Shinmura, Y Tanaka, Y Liu, J Imanishi: Expression of cytokines and
interferon-related genes in the mouse embryo. C. R. Seances Soc.
Biol. Fil. 1994, 188 (5-6): 593-600. [0603] 22. K M Downs, T
Davies: Staging of gastrulating mouse embryos by morphological
landmarks in the dissecting microscope. Development 1993, 118:
1255-1266 [0604] 23. D G Wilkinson, M A Nieto: Detection of
messenger RNA by in situ hybridisation to tissue sections and whole
mounts. Methods Enzymol 1993, 225: 361-373 [0605] 24. D Henrique, J
Adam, A Myat, A Chitnis, J Lewis, D Ish-Horowicz: Expression of a
Delta homologue in prospective neurons in the chick. Nature 1995,
375: 787-790 [0606] 25. G Brady, N N Iscove: Construction of cDNA
libraries from single cells. Methods Enzymol 1993, 225: 611-623
REFERENCES FOR EXAMPLES 21 TO 25
[0606] [0607] 1. Surani, M. A. Reprograming of genome function
through epigenetic inheritance. Nature 414, 122-8 (2001). [0608] 2.
Wu, X. et al. Zygote arrest 1 (Zar1) is a novel maternal-effect
gene critical for the oocyte-to-embryo transition. Nat Genet. 33,
187-91 (2003). [0609] 3. Tong, Z. B. et al. Mater, a maternal
effect gene required for early embryonic development in mice. Nat
Genet. 26, 267-8 (2000). [0610] 4. Howell, C. Y. et al. Genomic
imprinting disrupted by a maternal effect mutation in the Dnmt1
gene. Cell 104, 829-38 (2001). [0611] 5. Christians, E., Davis, A.
A., Thomas, S. D. & Benjamin, I. J. Maternal effect of Hsf1 on
reproductive success. Nature 407, 693-4 (2000). [0612] 6. Gurtu, V.
E. et al. Maternal effect for DNA mismatch repair in the mouse.
Genetics 160, 271-7 (2002). [0613] 7. Burns, K. H. et al. Roles of
NPM2 in chromatin and nucleolar organization in oocytes and
embryos. Science 300, 633-6 (2003). [0614] 8. Sato, M. et al.
Identification of PGC7, a new gene expressed specifically in
preimplantation embryos and germ cells. Mech Dev 113, 914 (2002).
[0615] 9. Saitou, M., Barton, S. C. & Surani, M. A. A molecular
programme for the specification of germ cell fate in mice. Nature
418, 293-300 (2002). [0616] 10. Goto, T. et al. Identification and
characterisation of known and novel transcripts expressed during
the final stages of human oocyte maturation. Mol Reprod Dev 62,
13-28 (2002). [0617] 11. Looijenga, L. H. et al. Role of gain of
12p in germ cell tumour development. Apmis 111, 161-71; discussion
172-3 (2003). [0618] 12. Mitsui, K. et al. The Homeoprotein Nanog
Is Required for Maintenance of Pluripotency in Mouse Epiblast and
ES Cells. Cell 113, 631-42 (2003). [0619] 13. Chambers, I. et al.
Functional expression cloning of nanog, a pluripotency sustaining
factor in embryonic stem cells. Cell 113, 643-55 (2003). [0620] 14.
Caricasole, A. A. et al. Human growth-differentiation factor 3
(hGDF3): developmental regulation in human teratocarcinoma cell
lines and expression in primary testicular germ cell tumours.
Oncogene 16, 95-103 (1998). [0621] 15. Spellman, P. T. & Rubin,
G. M. Evidence for large domains of similarly expressed genes in
the Drosophila genome. J Biol 1, 5 (2002). [0622] 16. Ginsburg, M.,
Snow, M. H. & McLaren, A. Primordial germ cells in the mouse
embryo during gastrulation. Development 110, 521-8 (1990). [0623]
17. Fox, N., Damjanov, I., Martinez-Hernandez, A., Knowles, B. B.
& Solter, D. Immunohistochemical localization of the early
embryonic antigen (SSEA-1) in postimplantation mouse embryos and
fetal and adult tissues. Dev Biol 83, 391-8 (1981). [0624] 18.
Johnson, M. H. & Everitt, B. J. Essential reproduction, xi, 377
(Blackwell Scientific, Oxford, 1988). [0625] 19. Montagutelli, X.
Effect of the genetic background on the phenotype of mouse
mutations. J Am Soc Nephrol 11 Suppl 16, S101-5 (2000). [0626] 20.
Lawson, K. A et al. Bmp4 is required for the generation of
primordial germ cells in the mouse embryo. Genes Dev 13, 424-36
(1999). [0627] 21. Winnier, G., Blessing, M., Labosky, P. A. &
Hogan, B. L. Bone morphogenetic protein4 is required for mesoderm
formation and patterning in the mouse. Genes Dev 9, 2105-16 (1995).
[0628] 22. Nothias, J. Y., Majurnder, S., Kaneko, K. J. &
DePamphilis, M. L. Regulation of gene expression at the beginning
of mammalian development. J Biol Chem 270, 22077-80 (1995). [0629]
23. Rugh, R. The mouse: Its Reproduction and Development, (Oxford
University Press, Oxford England; New York, 1990). [0630] 24.
McLaren, A. & Michie, D. Studies on the transfer of fertilized
mouse eggs to uterine foster-mothers. J Exp Biol 33, 394-416
(1956). [0631] 25. Aravind, L. & Koonin, E. V. SAP--a putative
DNA-binding motif involved in chromosomal organization. Trends
Biochem Sci 25, 112-4 (2000). [0632] 26. Chang, H. & Matzuk, M.
M. Smad5 is required for mouse primordial germ cell development.
Mech Dev 104, 61-7 (2001). [0633] 27. Fukuda, M. et al. CRM1 is
responsible for intracellular transport mediated by the nuclear
export signal. Nature 390, 308-11 (1997).
[0634] Each of the applications and patents mentioned in this
document, and each document cited or referenced in each of the
above applications and patents, including during the prosecution of
each of the applications and patents ("application cited
documents") and any manufacturer's instructions or catalogues for
any products cited or mentioned in each of the applications and
patents and in any of the application cited documents, are hereby
incorporated herein by reference. Furthermore, all documents cited
in this text, and all documents cited or referenced in documents
cited in this text, and any manufacturer's instructions or
catalogues for any products cited or mentioned in this text, are
hereby incorporated herein by reference.
[0635] Various modifications and variations of the described
methods and system of the invention will be apparent to those
skilled in the art without departing from the scope and spirit of
the invention. Although the invention has been described in
connection with specific preferred embodiments, it should be
understood that the invention as claimed should not be unduly
limited to such specific embodiments. Indeed, various modifications
of the described modes for carrying out the invention which are
obvious to those skilled in molecular biology or related fields are
intended to be within the scope of the claims.
Sequence CWU 1
1
671617DNAMus musculus 1gccgcagaaa gggcagaccc gcagcgcgct ccatcctttg
ccctccagtg ctgcctttgc 60tccgcaccat gaaccacact tctcaagcct tcatcaccgc
tgccagtgga ggacagcccc 120caaactacga aagaatcaag gaagaatatg
aggtggctga gatgggggca ccgcacggat 180cggcttctgt cagaactact
gtgatcaaca tgcccagaga ggtgtcggtg cctgaccatg 240tggtctggtc
cctgttcaat acactcttca tgaacttctg ctgcctgggc ttcatagcct
300atgcctactc cgtgaagtct agggatcgga agatggtggg tgatgtgact
ggagcccagg 360cctacgcctc cactgctaag tgcctgaaca tcagcacctt
ggtcctcagc atcctgatgg 420ttgttatcac cattgttagt gtcatcatca
ttgttcttaa cgctcaaaac cttcacactt 480aatagaggat tccgacttcc
ggtcctgaag tgcttcaccc tccgcagctg cgtccctcct 540tgcccctccc
tacacgcagg tgtaacactc atttatctat ccacagtgga ttcaataaag
600tgcacttgat aaccacc 6172823DNAMus musculus 2ggatcacaga ctgactgcta
attgggtctt ggttttaggt cttttcaaag actaagcaat 60cttgttccga gctagctttt
gaggcttctg cccatcgcat cgccatggag gaaccatcag 120agaaagtcga
cccaatgaag gaccctgaaa ctcctcagaa gaaagatgaa gaggacgctt
180tggatgatac agacgtccta caaccagaaa cactagtaaa ggtcatgaaa
aagctaaccc 240taaaccccgg tgtcaagcgg tccgcacgcc ggcgcagtct
acggaaccgc attgcagccg 300tacctgtgga gaacaagagt gaaaaaatcc
ggagggaagt tcaaagcgcc tttcccaaga 360gaagggtccg cactttgttg
tcggtgctga aagaccctat agcaaagatg agaagacttg 420ttcggattga
gcagagacaa aaaaggctcg aaggaaatga gtttgaacgg gacagtgagc
480cattcagatg tctctgcact ttctgccatt atcaaagatg ggatccctct
gagaatgcga 540aaatcgggaa gaattaggag cttacattgt acgctgccct
ggctgtcgac gatgccgcac 600agcagatgtg aaagctattt tttgtttaag
attaaacttt ttctggtgct gggaaatctt 660aacttgttaa cctttaaatt
gtagatagga tgcacaacga tccagattta tgtgaagttt 720agaagcctca
agctgtgagg cccagggctg aggaataaag taaatagaat ttggagtatg
780tacgttctaa tttccagaaa tttgtaataa aagcattttt gtt 82331040DNAHomo
sapiens 3agcaatttga ggctctgtca tcagtttctg ctacgtttca aagatcctgg
agaagcctag 60tgttgtgtca agacgccgat ggacccatca cagtttaatc caacctacat
cccagggtct 120ccacaaatgc tcaccgaaga aaattcccgg gacgattcag
gggcctctca aatctcctcc 180gagacgttga taaagaacct tagtaacttg
actatcaacg ctagtagcga atctgtttcc 240cctctatcgg aagctttact
ccgtcgagag tctgtaggag cagcagtcct cagggaaatc 300gaagatgagt
ggctttacag caggagagga gtaagaacat tgctgtctgt gcagagagaa
360aagatggcaa gattgagata catgttactc ggcggagttc gtacgcatga
aagaagacca 420acaaacaagg agcctaaggg agttaagaag gaatcaagac
cattcaaatg tccctgcagt 480ttctgcgtgt ctaatggatg ggatccttct
gagaatgcta gaatagggaa tcaagacacc 540aagccacttc agccataaat
cttattcttg cacctttttt tcttggtagt aattttatat 600agcaggttga
gaaagctact ctatgctagt atagactata caccaataat tttgataatg
660agttctagga tgtatttttc ttgtatcttt ttcttcctac tatgatacta
gtaattcata 720agggatctgt gtaatctgaa tgtatttgaa taactttagc
tctactgttt gatttgaccc 780aaagaagcca agatgatata agtattccca
tgtgtcttag aagcccaaag tcagtgagat 840gaaacccaac atcaagaaat
tgaagcaaag ttacttatgg ataaagaaag cattaggtag 900ttgggctata
gcataattag attttctggc tttcaaaaat ttggattgca atcacagcaa
960actttgttat ttttacagtt ttcagtacaa aagtgtttat atagaaacaa
taaagttgac 1020atttgagtac cttttaaaaa 10404159PRTHomo sapiens 4Met
Asp Pro Ser Gln Phe Asn Pro Thr Tyr Ile Pro Gly Ser Pro Gln1 5 10
15Met Leu Thr Glu Glu Asn Ser Arg Asp Asp Ser Gly Ala Ser Gln Ile
20 25 30Ser Ser Glu Thr Leu Ile Lys Asn Leu Ser Asn Leu Thr Ile Asn
Ala 35 40 45Ser Ser Glu Ser Val Ser Pro Leu Ser Glu Ala Leu Leu Arg
Arg Glu 50 55 60Ser Val Gly Ala Ala Val Leu Arg Glu Ile Glu Asp Glu
Trp Leu Tyr65 70 75 80Ser Arg Arg Gly Val Arg Thr Leu Leu Ser Val
Gln Arg Glu Lys Met 85 90 95Ala Arg Leu Arg Tyr Met Leu Leu Gly Gly
Val Arg Thr His Glu Arg 100 105 110Arg Pro Thr Asn Lys Glu Pro Lys
Gly Val Lys Lys Glu Ser Arg Pro 115 120 125Phe Lys Cys Pro Cys Ser
Phe Cys Val Ser Asn Gly Trp Asp Pro Ser 130 135 140Glu Asn Ala Arg
Ile Gly Asn Gln Asp Thr Lys Pro Leu Gln Pro145 150 1555653DNAHomo
sapiens 5tcccggtaac ccgatcaccg ctggtcacca tgaaccacat tgtgcaaacc
ttctctcctg 60tcaacagcgg ccagcctccc aactacgaga tgctcaagga ggagcaggaa
gtggctatgc 120tgggggtgcc ccacaaccct gctcccccga tgtccaccgt
gatccacatc cgcagcgaga 180cctccgtgcc tgaccatgtg gtctggtccc
tgttcaacac cctcttcatg aacacctgct 240gcctgggctt catagcattc
gcgtactccg tgaagtctag ggacaggaag atggttggcg 300acgtgaccgg
ggcccaggcc tatgcctcca ccgccaagtg cctgaacatc tgggccctga
360ttttgggcat cttcatgacc attctgctca tcatcatccc agtgttggtc
gtccaggccc 420agcgatagat caggaggcat cattgaggcc aggagctctg
cccgtgacct gtatcccacg 480tactctatct tccattcctc gccctgcccc
cagaggccag gagctctgcc cttgacctgt 540attccactta ctccacctcc
cattcctcgc cctgtcccca cagccgagtc ctgcatcagc 600cctttatcct
cacacgcttt tctacaatgg cattcaataa agtgtatatg ttt 6536132PRTHomo
sapiens 6Met Asn His Ile Val Gln Thr Phe Ser Pro Val Asn Ser Gly
Gln Pro1 5 10 15Pro Asn Tyr Glu Met Leu Lys Glu Glu Gln Glu Val Ala
Met Leu Gly 20 25 30Val Pro His Asn Pro Ala Pro Pro Met Ser Thr Val
Ile His Ile Arg 35 40 45Ser Glu Thr Ser Val Pro Asp His Val Val Trp
Ser Leu Phe Asn Thr 50 55 60Leu Phe Met Asn Thr Cys Cys Leu Gly Phe
Ile Ala Phe Ala Tyr Ser65 70 75 80Val Lys Ser Arg Asp Arg Lys Met
Val Gly Asp Val Thr Gly Ala Gln 85 90 95Ala Tyr Ala Ser Thr Ala Lys
Cys Leu Asn Ile Trp Ala Leu Ile Leu 100 105 110Gly Ile Phe Met Thr
Ile Leu Leu Ile Ile Ile Pro Val Leu Val Val 115 120 125Gln Ala Gln
Arg 1307677DNAHomo sapiens 7tgagaaactg aaacgacagg ggaaaggagg
tctcactgag caccgtccca gcatccggac 60accacagcgg cccttcgctc cacgcagaaa
accacacttc tcaaaccttc actcaacact 120tccttcccca aagccagaag
atgcacaagg aggaacatga ggtggctgtg ctgggggcac 180cccccagcac
catccttcca aggtccaccg tgatcaacat ccacagcgag acctccgtgc
240ccgaccatgt cgtctggtcc ctgttcaaca ccctcttctt gaactggtgc
tgtctgggct 300tcatagcatt cgcctactcc gtgaagtcta gggacaggaa
gatggttggc gacgtgaccg 360gggcccaggc ctatgcctcc accgccaagt
gcctgaacat ctgggccctg attctgggca 420tcctcatgac cattggattc
atcctgtcac tggtattcgg ctctgtgaca gtctaccata 480ttatgttaca
gataatacag gaaaaacggg gttactagta gccgcccata gcctgcaacc
540tttgcactcc actgtgcaat gctggccctg cacgctgggg ctgttgcccc
tgcccccttg 600gtcctgcccc tagatacagc agtttatacc cacacacctg
tctacagtgt cattcaataa 660agtgcacgtg cttgtga 6778125PRTHomo sapiens
8Met His Lys Glu Glu His Glu Val Ala Val Leu Gly Ala Pro Pro Ser1 5
10 15Thr Ile Leu Pro Arg Ser Thr Val Ile Asn Ile His Ser Glu Thr
Ser 20 25 30Val Pro Asp His Val Val Trp Ser Leu Phe Asn Thr Leu Phe
Leu Asn 35 40 45Trp Cys Cys Leu Gly Phe Ile Ala Phe Ala Tyr Ser Val
Lys Ser Arg 50 55 60Asp Arg Lys Met Val Gly Asp Val Thr Gly Ala Gln
Ala Tyr Ala Ser65 70 75 80Thr Ala Lys Cys Leu Asn Ile Trp Ala Leu
Ile Leu Gly Ile Leu Met 85 90 95Thr Ile Gly Phe Ile Leu Ser Leu Val
Phe Gly Ser Val Thr Val Tyr 100 105 110His Ile Met Leu Gln Ile Ile
Gln Glu Lys Arg Gly Tyr 115 120 1259651DNAMus musculus 9gcgggtctac
agaaccagga tagcagcagc catcctccag acggggcgat tgttccagag 60tcagtaccat
gagccacaat tctcaagcct tcttgtccac caatgccggg cttcctccaa
120gctatgagac aatcaaagag gagtacgggg tgactgagct gggggaaccc
agcaactcag 180ctgttgtgag gaccaccgtg atcaacatgc ccagagaggt
gtcggtgcct gaccatgtgg 240tctggtccct gttcaataca ctcttcttca
acgcctgctg cctgggcttc gttgcctatg 300cctactctgt gaagtctagg
gacaggaaga tggtgggcga tgtggttgga gcccaggcct 360acgcctccac
tgccaagtgc ctgaatatca gctccctgat cttcagcatc cttatggtca
420ttatctgcat cattattttc tctaccacct ctgtggtagt ctttcagtct
tttgcacaaa 480gaacacccca ttctggattc tagctgccct gtgctccacg
gtccacatct gccccgcccc 540tgccccgccc ccaggctcaa gcctcgaccc
tttaccctac gcgtatgcaa atgttacctt 600cacctatctg tccacagtgg
attcaataaa gtgcacgggg tggcaactct g 65110144PRTMus musculus 10Met
Ser His Asn Ser Gln Ala Phe Leu Ser Thr Asn Ala Gly Leu Pro1 5 10
15Pro Ser Tyr Glu Thr Ile Lys Glu Glu Tyr Gly Val Thr Glu Leu Gly
20 25 30Glu Pro Ser Asn Ser Ala Val Val Arg Thr Thr Val Ile Asn Met
Pro 35 40 45Arg Glu Val Ser Val Pro Asp His Val Val Trp Ser Leu Phe
Asn Thr 50 55 60Leu Phe Phe Asn Ala Cys Cys Leu Gly Phe Val Ala Tyr
Ala Tyr Ser65 70 75 80Val Lys Ser Arg Asp Arg Lys Met Val Gly Asp
Val Val Gly Ala Gln 85 90 95Ala Tyr Ala Ser Thr Ala Lys Cys Leu Asn
Ile Ser Ser Leu Ile Phe 100 105 110Ser Ile Leu Met Val Ile Ile Cys
Ile Ile Ile Phe Ser Thr Thr Ser 115 120 125Val Val Val Phe Gln Ser
Phe Ala Gln Arg Thr Pro His Ser Gly Phe 130 135 14011590DNAMus
musculus 11tggagaaaag gccactgcgc aaagggctct ggacttctca gcttgtacca
ccattctcat 60tccttcctta ttctcaactc ttccagcctc aaaaaccaag agatgcctaa
ggatcagcat 120gaggtggttg taatggggac accccacacc tcaacttctt
cgacaaccac cataatcacc 180atgcctgaga tctccaagcc tgattatgtg
gtctggtctc tgttcaatac actcttcatg 240aacttctgct gcctgggttt
catagcctat gcctactctg tgaagtctag ggacaggaag 300atggtgggtg
atatgactgg ggcccaggcc ttcgcctcca ctgccaggtg cctgaacatc
360agctgcctga tcctctccgt cgtcatggtc atcctcttca tcactttctt
tgccactaga 420aggtagccat cttgtagcat ctcacagtag ataacagatt
ctggggcctt ccgggcttgc 480tatgtgttct attgtctatc gctgtcccaa
accctagtct tagtcctgac catttacccc 540atacatatgc aaatgttaca
cttgcatatc tgttcattca ataaagtgca 59012107PRTMus musculus 12Met Pro
Lys Asp Gln His Glu Val Val Val Met Gly Thr Pro His Thr1 5 10 15Ser
Thr Ser Ser Thr Thr Thr Ile Ile Thr Met Pro Glu Ile Ser Lys 20 25
30Pro Asp Tyr Val Val Trp Ser Leu Phe Asn Thr Leu Phe Met Asn Phe
35 40 45Cys Cys Leu Gly Phe Ile Ala Tyr Ala Tyr Ser Val Lys Ser Arg
Asp 50 55 60Arg Lys Met Val Gly Asp Met Thr Gly Ala Gln Ala Phe Ala
Ser Thr65 70 75 80Ala Arg Cys Leu Asn Ile Ser Cys Leu Ile Leu Ser
Val Val Met Val 85 90 95Ile Leu Phe Ile Thr Phe Phe Ala Thr Arg Arg
100 10513550DNAMus musculus 13gattccttcc ttattctcac tctgcagctt
caaaagccga gagatgccta aggagcagca 60agaggtggtt gtactggggt caccccacat
ctcaacttct gcgacagcca ccacaatcaa 120catgcctgag atctccacgc
ctgaccatgt ggtctggtcc ctgttcaata cactcttcat 180gaacttctgc
tgcctgggct tcgtagccta tgcctactcc gtgaagtcta gggacaggaa
240gatggtgggt gatacgactg gggcccaggc cttcgcctcc accgccaagt
gcctgaacat 300cagctccctg ttcttcacca tcctcacggc catcgtcgtc
atcgttgtct gtgccattag 360atgatgtgag atgtcttgca acatctcaca
gtagataaca gattctgggg cctcccaggc 420ttgctatgtg tttccttgtc
tatcgctgcc ccaaacccta gacttagtcc tgaccatttg 480ccccatacat
atgcaaatgt gacactcaca aatctgtcca tggtggactc aataaagtgc
540acgtgctgtg 55014106PRTMus musculus 14Met Pro Lys Glu Gln Gln Glu
Val Val Val Leu Gly Ser Pro His Ile1 5 10 15Ser Thr Ser Ala Thr Ala
Thr Thr Ile Asn Met Pro Glu Ile Ser Thr 20 25 30Pro Asp His Val Val
Trp Ser Leu Phe Asn Thr Leu Phe Met Asn Phe 35 40 45Cys Cys Leu Gly
Phe Val Ala Tyr Ala Tyr Ser Val Lys Ser Arg Asp 50 55 60Arg Lys Met
Val Gly Asp Thr Thr Gly Ala Gln Ala Phe Ala Ser Thr65 70 75 80Ala
Lys Cys Leu Asn Ile Ser Ser Leu Phe Phe Thr Ile Leu Thr Ala 85 90
95Ile Val Val Ile Val Val Cys Ala Ile Arg 100 10515499DNAMus
musculus 15ctcagctagg aagacacggc gctggaaccc atggacactt catatccccg
tgaggacccc 60cgggctccat catcccgcaa ggctgatgct gcagcccaca cagccctctc
catgggaaca 120cctggcccta caccacgaga tcacatgctc tggtctgtct
tcagcacgat gtacctgaat 180ctgtgctgcc ttggattcct ggcgctggtc
cactctgtca aggcccgaga ccagaagatg 240gctgggaact tggaggctgc
aaggcagtat ggctccaaag ccaagtgcta caacatcctg 300gctgcaatgt
ggacattggt gcccccattg ctgctcctgg gactggtggt gactggcgcc
360ttgcacctgt ccaagttagc caaagactct gcggctttct tcagcaccaa
gtttgatgag 420gaggactata actaagagtt ccgagcctgt ccctgaaccg
aggacaaccg ggctagagcg 480gccgccaccg cggtggagc 49916134PRTMus
musculus 16Met Asp Thr Ser Tyr Pro Arg Glu Asp Pro Arg Ala Pro Ser
Ser Arg1 5 10 15Lys Ala Asp Ala Ala Ala His Thr Ala Leu Ser Met Gly
Thr Pro Gly 20 25 30Pro Thr Pro Arg Asp His Met Leu Trp Ser Val Phe
Ser Thr Met Tyr 35 40 45Leu Asn Leu Cys Cys Leu Gly Phe Leu Ala Leu
Val His Ser Val Lys 50 55 60Ala Arg Asp Gln Lys Met Ala Gly Asn Leu
Glu Ala Ala Arg Gln Tyr65 70 75 80Gly Ser Lys Ala Lys Cys Tyr Asn
Ile Leu Ala Ala Met Trp Thr Leu 85 90 95Val Pro Pro Leu Leu Leu Leu
Gly Leu Val Val Thr Gly Ala Leu His 100 105 110Leu Ser Lys Leu Ala
Lys Asp Ser Ala Ala Phe Phe Ser Thr Lys Phe 115 120 125Asp Glu Glu
Asp Tyr Asn 13017445DNAMus musculus 17tgaacttcct tgaaacaaga
gcttccttgc ttcctttaag cacaaaaaca tggttaagag 60ggatcctgac tcagctccag
tgccatccac tgtggtttgc atcaacagtg atgttatcca 120gccggatcac
attacctggt ctacatttaa cacagtgttc atgaatggct gctgcctggg
180tttcattgcc tacatctact cggtgaagtc cagggaccgg aagatggtgg
gcgacatgac 240tggggcccaa tcccatgctt caaccgccaa gattctgaac
atccttgctc tggtcatctc 300cctcatcttc tacatcatgc ttatcgtttt
atacagcttt aacttactag gtaaccaaag 360ataatagaac cactagttag
gtactaacta gttagttagc taattattaa ttaactaaac 420tagtaccgaa
tttagtatct ttagt 44518104PRTMus musculus 18Met Val Lys Arg Asp Pro
Asp Ser Ala Pro Val Pro Ser Thr Val Val1 5 10 15Cys Ile Asn Ser Asp
Val Ile Gln Pro Asp His Ile Thr Trp Ser Thr 20 25 30Phe Asn Thr Val
Phe Met Asn Gly Cys Cys Leu Gly Phe Ile Ala Tyr 35 40 45Ile Tyr Ser
Val Lys Ser Arg Asp Arg Lys Met Val Gly Asp Met Thr 50 55 60Gly Ala
Gln Ser His Ala Ser Thr Ala Lys Ile Leu Asn Ile Leu Ala65 70 75
80Leu Val Ile Ser Leu Ile Phe Tyr Ile Met Leu Ile Val Leu Tyr Ser
85 90 95Phe Asn Leu Leu Gly Asn Gln Arg 10019137PRTMus musculus
19Met Asn His Thr Ser Gln Ala Phe Ile Thr Ala Ala Ser Gly Gly Gln1
5 10 15Pro Pro Asn Tyr Glu Arg Ile Lys Glu Glu Tyr Glu Val Ala Glu
Met 20 25 30Gly Ala Pro His Gly Ser Ala Ser Val Arg Thr Thr Val Ile
Asn Met 35 40 45Pro Arg Glu Val Ser Val Pro Asp His Val Val Trp Ser
Leu Phe Asn 50 55 60Thr Leu Phe Met Asn Phe Cys Cys Leu Gly Phe Ile
Ala Tyr Ala Tyr65 70 75 80Ser Val Lys Ser Arg Asp Arg Lys Met Val
Gly Asp Val Thr Gly Ala 85 90 95Gln Ala Tyr Ala Ser Thr Ala Lys Cys
Leu Asn Ile Ser Thr Leu Val 100 105 110Leu Ser Ile Leu Met Val Val
Ile Thr Ile Val Ser Val Ile Ile Ile 115 120 125Val Leu Asn Ala Gln
Asn Leu His Thr 130 13520150PRTMus musculus 20Met Glu Glu Pro Ser
Glu Lys Val Asp Pro Met Lys Asp Pro Glu Thr1 5 10 15Pro Gln Lys Lys
Asp Glu Glu Asp Ala Leu Asp Asp Thr Asp Val Leu 20 25 30Gln Pro Glu
Thr Leu Val Lys Val Met Lys Lys Leu Thr Leu Asn Pro 35 40 45Gly Val
Lys Arg Ser Ala Arg Arg Arg Ser Leu Arg Asn Arg Ile Ala 50 55 60Ala
Val Pro Val Glu Asn Lys Ser Glu Lys Ile Arg Arg Glu Val Gln65 70 75
80Ser Ala Phe Pro Lys Arg Arg Val Arg Thr Leu Leu Ser Val Leu Lys
85 90 95Asp Pro Ile Ala Lys Met Arg Arg Leu Val Arg Ile Glu Gln Arg
Gln 100 105 110Lys Arg Leu Glu Gly Asn Glu Phe Glu Arg Asp Ser Glu
Pro Phe Arg 115 120 125Cys Leu Cys Thr Phe Cys His Tyr Gln Arg Trp
Asp Pro Ser Glu Asn 130 135 140Ala Lys Ile Gly Lys Asn145
15021133PRTHomo sapiens 21Met Ser His Thr Val Gln
Thr Phe Phe Ser Pro Val Asn Ser Gly Gln1 5 10 15Pro Pro Asn Tyr Glu
Met Leu Lys Glu Glu His Glu Val Ala Val Leu 20 25 30Gly Gly Pro His
Asn Pro Ala Pro Pro Thr Ser Thr Val Ile His Ile 35 40 45Arg Ser Glu
Thr Ser Val Pro Asp His Val Val Trp Ser Leu Phe Asn 50 55 60Thr Leu
Phe Met Asn Pro Cys Cys Leu Gly Phe Ile Ala Phe Ala Tyr65 70 75
80Ser Val Lys Ser Arg Asp Arg Lys Met Val Gly Asp Val Thr Gly Ala
85 90 95Gln Ala Tyr Ala Ser Thr Ala Lys Cys Leu Asn Ile Trp Ala Leu
Ile 100 105 110Leu Gly Ile Leu Met Thr Ile Leu Leu Ile Val Ile Pro
Val Leu Ile 115 120 125Phe Gln Ala Tyr Gly 13022132PRTHomo sapiens
22Met Asp Thr Ala Tyr Pro Arg Glu Asp Thr Arg Ala Pro Thr Arg Ser1
5 10 15Lys Ala Gly Ala His Thr Ala Leu Ile Leu Gly Ala Pro His Pro
Pro 20 25 30Pro Arg Asp His Leu Ile Trp Ser Val Phe Ser Thr Leu Tyr
Leu Asn 35 40 45Leu Cys Cys Leu Gly Phe Leu Ala Leu Ala Tyr Ser Ile
Lys Ala Arg 50 55 60Asp Cys Lys Val Val Gly Asp Leu Glu Ala Ala Arg
Arg Phe Gly Ser65 70 75 80Lys Ala Lys Cys Tyr Asn Ile Leu Ala Ala
Met Trp Thr Leu Val Pro 85 90 95Pro Leu Leu Leu Leu Gly Leu Val Val
Thr Gly Ala Leu His Leu Ala 100 105 110Arg Leu Ala Lys Asp Ser Ala
Ala Phe Phe Ser Thr Lys Phe Asp Asp 115 120 125Ala Asp Tyr Asp
13023146PRTBos taurus 23Met Asn Arg Thr Ser Gln Leu Leu Leu Thr Gly
Ala His Gly Ala Val1 5 10 15Pro Pro Ala Tyr Glu Val Leu Lys Glu Glu
His Glu Val Ala Val Leu 20 25 30Gly Ala Pro Gln Ser Gln Ala Pro Leu
Thr Thr Thr Val Ile Asn Ile 35 40 45Arg Ser Asp Thr Ala Val Pro Asp
His Ile Val Trp Ser Leu Phe Asn 50 55 60Thr Ile Phe Met Asn Trp Cys
Cys Leu Gly Phe Val Ala Phe Ala Tyr65 70 75 80Ser Val Lys Ser Arg
Asp Arg Lys Met Val Gly Asp Ile Thr Gly Ala 85 90 95Gln Ser Tyr Ala
Ser Thr Ala Lys Cys Leu Asn Ile Cys Ser Leu Val 100 105 110Leu Gly
Ile Leu Leu Thr Val Val Leu Ile Val Leu Val Ser Asn Gly 115 120
125Ser Leu Met Ile Val Gln Ala Val Ser Glu Leu Met Gln Asn Tyr Gly
130 135 140Gly His14524125PRTBos taurus 24Met Ile Lys Glu Glu His
Glu Val Ala Val Leu Gly Ala Pro Gln Ser1 5 10 15Arg Arg Pro Leu Thr
Thr Thr Val Ile Asn Ile Arg Ser Asp Thr Ala 20 25 30Val Pro Asp His
Ile Val Trp Ser Leu Phe Asn Thr Ile Phe Leu Asn 35 40 45Trp Cys Cys
Leu Gly Phe Val Ala Phe Ala Tyr Ser Val Lys Ser Arg 50 55 60Asp Arg
Lys Met Val Gly Asp Ile Thr Gly Ala Gln Ser Tyr Ala Ser65 70 75
80Thr Ala Lys Cys Leu Asn Ile Trp Ala Leu Val Leu Gly Ile Leu Leu
85 90 95Thr Ile Gly Ser Ile Val Leu Leu Ile Phe Gly Tyr Met Ala Val
Tyr 100 105 110Gln Thr Val Phe Leu Leu Met Gln Glu Lys Arg Gly His
115 120 12525137PRTRattus norvegicus 25Met Asn His Thr Ser Gln Ala
Phe Val Asn Ala Ala Thr Gly Gly Gln1 5 10 15Pro Pro Asn Tyr Glu Arg
Ile Lys Glu Glu Tyr Glu Val Ser Glu Leu 20 25 30Gly Ala Pro His Gly
Ser Ala Ser Val Arg Thr Thr Val Ile Asn Met 35 40 45Pro Arg Glu Val
Ser Val Pro Asp His Val Val Trp Ser Leu Phe Asn 50 55 60Thr Leu Phe
Met Asn Phe Cys Cys Leu Gly Phe Ile Ala Tyr Ala Tyr65 70 75 80Ser
Val Lys Ser Arg Asp Arg Lys Met Val Gly Asp Met Thr Gly Ala 85 90
95Gln Ala Tyr Ala Ser Thr Ala Lys Cys Leu Asn Ile Ser Ser Leu Val
100 105 110Leu Ser Ile Leu Met Val Ile Ile Thr Ile Val Thr Val Val
Ile Ile 115 120 125Ala Leu Asn Ala Pro Arg Leu Gln Thr 130
13526137PRTRattus norvegicus 26Met Asn His Thr Ser Gln Ala Phe Ala
Thr Val Ala Thr Gly Gly Gln1 5 10 15Pro Pro Asn Tyr Glu Arg Ile Lys
Glu Glu Tyr Glu Val Ser Glu Leu 20 25 30Gly Ala Pro His Gly Ser Ala
Ser Val Arg Thr Thr Val Ile Asn Met 35 40 45Pro Arg Glu Val Ser Val
Pro Asp His Val Val Trp Ser Leu Phe Asn 50 55 60Thr Leu Phe Met Asn
Phe Cys Cys Leu Gly Phe Ile Ala Tyr Ala Tyr65 70 75 80Ser Val Lys
Ser Arg Asp Arg Lys Met Val Gly Asp Met Thr Gly Ala 85 90 95Gln Ala
Tyr Ala Ser Thr Ala Lys Cys Leu Asn Ile Ser Ser Leu Val 100 105
110Leu Ser Ile Leu Met Val Ile Ile Thr Ile Val Thr Val Val Ile Ile
115 120 125Ala Leu Asn Ala Pro Arg Leu Gln Thr 130
13527144PRTRattus norvegicus 27Met Ser His Asn Ser Gln Ala Phe Leu
Pro Ala Asn Ala Gly Leu Pro1 5 10 15Pro Ser Tyr Glu Thr Ile Lys Glu
Glu Tyr Gly Val Thr Glu Leu Gly 20 25 30Glu Pro Asn Asn Ser Ala Val
Val Arg Thr Thr Val Ile Asn Met Pro 35 40 45Arg Glu Val Ser Val Pro
Asp His Val Val Trp Ser Leu Phe Asn Thr 50 55 60Leu Phe Phe Asn Ala
Cys Cys Leu Gly Phe Ile Ala Tyr Ala Tyr Ser65 70 75 80Val Lys Ser
Arg Asp Arg Lys Met Val Gly Asp Val Ile Gly Ala Gln 85 90 95Ala Tyr
Ala Ser Thr Ala Lys Cys Leu Asn Ile Ser Ser Leu Ile Phe 100 105
110Ser Val Leu Met Val Ile Ile Cys Ile Ile Ile Phe Ser Thr Thr Ser
115 120 125Ala Val Val Phe Gln Ser Leu Ser Gln Arg Thr Pro His Ser
Gly Phe 130 135 1402893PRTRattus norvegicus 28Met Asn His Thr Ser
Gln Ala Phe Val Asn Ala Ala Thr Gly Gly Gln1 5 10 15Pro Pro Asn Tyr
Glu Arg Ile Lys Glu Glu Tyr Glu Val Ser Glu Leu 20 25 30Gly Ala Pro
His Gly Ser Ala Ser Val Arg Thr Thr Val Ile Asn Met 35 40 45Pro Arg
Glu Val Ser Val Pro Asp His Val Val Trp Ser Leu Phe Asn 50 55 60Thr
Leu Phe Met Asn Phe Cys Cys Leu Gly Phe Ile Ala Tyr Ala Tyr65 70 75
80Ser Val Lys Val Ser Val Leu Glu Val Gly Val Met Val 85
9029156PRTArtificial sequenceConsensus 29Met Asn His Thr Ser Gln
Ala Phe Xaa Xaa Xaa Ala Xaa Gly Gly Gln1 5 10 15Pro Pro Asn Tyr Glu
Xaa Xaa Lys Glu Glu Tyr Glu Val Xaa Xaa Leu 20 25 30Gly Ala Pro Xaa
Xaa Arg Lys Ser Ala Xaa Xaa Xaa Thr Thr Val Ile 35 40 45Asn Met Xaa
Xaa Glu Xaa Ser Val Pro Arg Asp His Val Val Trp Ser 50 55 60Leu Phe
Asn Thr Leu Phe Met Asn Xaa Cys Cys Leu Gly Phe Ile Ala65 70 75
80Tyr Ala Tyr Ser Val Lys Ser Arg Asp Arg Lys Met Val Gly Asp Xaa
85 90 95Thr Gly Ala Gln Ala Tyr Ala Ser Thr Ala Lys Cys Leu Asn Ile
Xaa 100 105 110Xaa Leu Xaa Leu Xaa Ile Leu Met Xaa Ile Xaa Xaa Ile
Val Xaa Xaa 115 120 125Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 130 135 140Xaa Xaa Xaa Xaa Xaa Phe Asp Xaa Xaa
Asp Tyr Xaa145 150 15530158PRTRattus norvegicus 30Met Asp Glu Pro
Ser Glu Lys Val Asp Pro Val Val Asn Pro Glu Thr1 5 10 15Gln Met Tyr
Asp Gly Ser Gln Arg Glu Asp Glu Gly Asp Ser Pro Asp 20 25 30Asp Ser
Glu Ile Leu Gln Pro Glu Thr Leu Val Lys Val Met Lys Lys 35 40 45Leu
Thr Leu Asn Pro Ser Ala Lys Pro Thr Lys Tyr His Arg Arg Gln 50 55
60Arg Val Arg Leu Gln Val Lys Ser Gln Pro Val Glu Asn Arg Ser Glu65
70 75 80Arg Ile Met Arg Glu Val Gln Ser Ala Phe Pro Arg Arg Arg Val
Arg 85 90 95Thr Leu Leu Ser Val Leu Lys Asp Pro Ile Ala Arg Met Arg
Arg Phe 100 105 110Val Arg Ile Glu Gln Arg Gln Arg Gln Leu Glu Gly
Asn Glu Arg Arg 115 120 125Asp Glu Pro Phe Arg Cys Leu Cys Thr Phe
Cys His Tyr Gln Arg Trp 130 135 140Asp Pro Ser Glu Asn Ala Lys Ile
Gly Gln Asn Gln Lys Asn145 150 1553112DNAArtificial
sequenceInterferon stimulable response element 31ggaaanngaa ac
123211DNAArtificial sequenceInterferon stimulable response element
32ggaaangaaa c 113317PRTMus musculus 33Ala Ser Gly Gly Gln Pro Pro
Asn Tyr Glu Arg Ile Lys Glu Glu Tyr1 5 10 15Glu3416PRTMus musculus
34Arg Asp Arg Lys Met Val Gly Asp Val Thr Gly Ala Gln Ala Tyr Ala1
5 10 153516PRTMus musculus 35Met Glu Glu Pro Ser Glu Lys Val Asp
Pro Met Lys Asp Pro Glu Thr1 5 10 153617PRTMus musculus 36Cys His
Tyr Gln Arg Trp Asp Pro Ser Glu Asn Ala Lys Ile Gly Lys1 5 10
15Asn3760DNAArtificial sequencePrimer 37attggatcca ggccgctctg
gacaaaatat gaatcctttt tttttttttt tttttttttt 603822DNAArtificial
sequencePrimer 38gccatacctt gacccgcaga ag 223922DNAArtificial
sequencePrimer 39aaatggcact cagttcagtg gg 224024DNAArtificial
sequencePrimer 40cccaaagcac cttatttttc tacc 244121DNAArtificial
sequencePrimer 41ttggcgagtc tctgcaattg g 214221DNAArtificial
sequencePrimer 42cactctactc agtccctttt c 214322DNAArtificial
sequencePrimer 43tgtgtcccag tctttattta ag 224421DNAArtificial
sequencePrimer 44aactcatcag aggtcgaagg a 214521DNAArtificial
sequencePrimer 45cggtgctatt gtaaggtctg c 214619DNAArtificial
sequencePrimer 46ctactccgtg aagtctagg 194721DNAArtificial
sequencePrimer 47aatgagtgtt acacctgcgt g 214821DNAArtificial
sequencePrimer 48gccattcaga tgtctctgca c 214921DNAArtificial
sequencePrimer 49ctcacagctt gaggcttcta a 215021DNAArtificial
sequencePrimer 50ctcacagctt gaggcttcta a 215121DNAArtificial
sequencePrimer 51gcgattcaga tgtctctgca c 215224DNAArtificial
sequencePrimer 52gttatcacca ttgttagtgt catc 245321DNAArtificial
sequencePrimer 53aatgagtgtt acacctgcgt g 215422DNAArtificial
sequencePrimer 54gatcttcagc atccttatgg tc 225522DNAArtificial
sequencePrimer 55gaaggtaaca tttgcatacg cg 225620DNAArtificial
sequencePrimer 56ccttccttat tctcactctg 205720DNAArtificial
sequencePrimer 57gttgcaagac atctcacatc 205821DNAArtificial
sequencePrimer 58aacttggagg ctgcaaggca g 215922DNAArtificial
sequencePrimer 59ctcggaactc ttagttatag tc 226020DNAArtificial
sequencePrimer 60tgctctggtc atctccctca 206120DNAArtificial
sequencePrimer 61caggataagg ggcaactctg 206222DNAArtificial
sequencePrimer 62caatttgagg ctctgtcatc ag 226321DNAArtificial
sequencePrimer 63ttcatctcac tgactttggg c 216420DNAArtificial
sequencePrimer 64agttcctggt ccacaacgtc 206520DNAArtificial
sequencePrimer 65tgcacatgag ctgcctactc 206621DNAArtificial
sequencePrimer 66agacgtccta caaccagaaa c 216721DNAArtificial
sequencePrimer 67ccgaacaagt cttctcatct t 21
* * * * *
References