Note: Descriptions are shown in the official language in which they were submitted.
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NEURONAL STEM CELL GENE
The present invention relates to a method of marking, selecting and generating
neuronal stem sells from tissues. In particular, the invention relates to the
use of
the Soxl gene for the generation of neuroblasts.
SOX proteins constitute a family of transcription factors related to the
mammalian
testis determining factor SRY through homology within their HMG box DNA
1o binding domains. In DNA binding studies, SOX proteins exhibit sequence
specific
binding; however, unlike most transcription factors, binding occurs in the
minor
groove resulting in the induction of a dramatic bend within the DNA helix. SOX
proteins can induce transcription of reporter constructs in vitro and display
properties of both classical transcription factors and architectural
components of
~5 chromatin (reviewed by Peveny and Lovell-Badge (1997) Curr. Opin. Genetics
and
Development, 7:338-344).
Members of the Sox gene family are expressed in a variety of embryonic and
adult
tissues, where they appear to be responsible for the development and/or
elaboration
20 of particular cell lineages. Sry is transiently expressed in the precursor
Sertoli cells
of the XY genital ridge and is responsible for triggering development of the
male
phenotype (reviewed by Lovell-Badge and Hacker, (1995) Phil. Traps. R. Soc.
Lond. B 350:205-2I4). Thus, the lack of Sry results in XY females and XX
males.
Sox9 is expressed in immature chondrocytes and male gonads; mutations in the
25 human SOX9 gene are associated with Campomelic Dysplasia, a human skeletal
malformation syndrome, and XY female sex reversal. Sox4 is expressed in many
tissues and a null mutation of the gene in mouse results in the absence of
mature B
cells and heart malformations. Xsoxl7 genes are involved in endoderm formation
in
Xenopus embryos. These functional analyses Suggest that Sox genes function in
3o cell fate decisions in diverse developmental pathways.
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2
A subfamily of Sox genes, that includes Soxl, Sox2 and Sox3, shows expression
profiles during vertebrate embryogenesis that suggest the genes could function
in
the control of cell fate decisions within the early developing nervous system.
Sox2
and Sox3 begin to be expressed at preimplantation and epiblast stages
respectively,
and are then restricted to the neuroepithelium. Soxl appears only at around
the
stage of neural induction.
The molecular mechanisms controlling neural induction and determination have
begun to be elucidated. The identification by cellular and biochemical
methods, of
to secreted molecules involved in neural induction illustrates the important
role of the
environment in specifying cell identity. In addition, a number of
transcription
factors have been isolated which play important roles in the specification and
differentiation of neural cell lineages. For example, the characterisation of
vertebrate homologues of Drosophila proneural and neurogenic genes, which
~5 control neural specification in the fly, has revealed analogous molecular
mechanisms in vertebrate neural cell fate determination and differentiation.
In an
Drosophila, the expression of basic helix-loop-helix transcription factors of
the AS-
C complex confirms neural potential on groups of ectodermal cells. Miss
expression of a transcription factors involved in a neural cell fate
determination is
20 observed to cause abnormalities in neural development.
It is known that Soxl expression appears only at around the stage of neural
induction in the embryo. The role of SOXl in embryogenesis is, however, not
known.
Summary of the Invention
It is shown herein that Soxl expression correlates with the formation of the
neural
plate. Moreover, the onset of Soxl expression in embryonal carcinoma cells is
3o shown to be dependent on neural induction. Upregulation of Soxl expression
is
itself sufficient to impart a neural fate on pluripotent cells.
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In a first aspect of the present invention, there is provided a method for
isolating a
neuroblastic cell from a population of cells comprising the steps of:
(a) detecting the expression of the Soxl gene in the cells; and
(b) sorting the cells to isolate those cells expressing the Soxl gene.
As set forth in the following description, the Soxl gene, which encodes SOX1,
is
responsible for the specification of neuroblast or neuronal stem cells, as
well as
acting as a marker for such cells. Expression of Soxl is responsible for the
to generation of the neuroblastic cell type, which in vivo is capable of
differentiating
into the many different cells and ganglia of the CNS. Moreover, Soxl is a
unique
marker for neuroblasts.
Cells which are identified as expressing this gene, for example by binding to
anti-
~5 SOX1 antibodies, by activation of SOX/ dependent ligand-receptor systems or
by
detection with antisense nucleic acids specific for Soxl mRNA, are pluripotent
neuroblasts. Such cells can be identified in early embryonic tissue or adult
CNS
material. Cells can be sorted by affinity techniques, or by cell sorting (such
as
fluorescence-activated cell sorting) where they are labelled with a suitable
label,
zo such as a fluorophore conjugated to or part of, for example, an antisense
nucleic
acid molecule or an immunoglobulin.
According to a second aspect of the invention, neuroblast cells can be
actively
sorted from other cell types by detecting the expression of SOX1 in vivo using
a
2s reporter system. Thus, for example, the invention provides a method for
isolating a
neuroblastic cell from a population of cells, comprising the steps of:
(a) transfecting the population of cells with a genetic construct comprising a
coding sequence encoding a detectable marker operatively linked to the Soxl
control
regions;
30 (b) detecting the cells which express the selectable marker; and
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(c) sorting the cells which express the selectable marker from the population
of cells.
As before, the selectable marker may be any selectable entity, but is
preferably a
s fluorescent or luminescent marker which may be detected and sorted by
automated
cell sorting approaches. For example, the marker may be GFP or luciferase.
Other
useful markers include those which are expressed in the cell membrane, thus
facilitating cell sorting by affinity means.
1o The genetic construct according to the invention may comprise any promoter
and
enhancer elements as required, so long as the overall control remains
sensitive to
SOX1; in other words, no expression of the marker coding sequence should take
place in the absence of SOX1. The regulatory sequences of the SOX1 gene are
known in the art and have been described in the literature cited herein and
~5 incorporated herein by reference; at least, however, the construct of the
invention
will comprise a SOXI binding site. Preferably, the SOX1 control elements are
used
in their entirety; however, other promoter and enhancer elements may be
substituted
where they remain under the influence of SOX1.
2o The selectable marker will only be expressed in neuroblastic cells because
only
these cells express SOX1, which is required for transcription from the Soxl
control
sequences. Preferably, therefore, the expression system used to express the
selectable marker is not leaky and expresses a minimal amount of the marker in
the
absence of SOX1. Techniques for transforming cells with coding genetic
constructs
25 according to the invention, detecting the marker and sorting cells
accordingly are
known in the art.
The present invention, in a third aspect, provides the use of the Soxl coding
sequence to transform precursor cells and thereby differentiate neuroblast
cells
30 therefrom. Accordingly, there is provided a method for differentiating one
or more
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neurobiastic cells from one or mores pluripotent precursor cell, comprising
the steps
of:
(a) transforming the pluripotent precursor cells) with a genetic construct
comprising a Soxl coding sequence operatively linked to suitable control
sequences;
5 and
(b) culturing the cells) so as to allow expression of the Soxl coding
sequence, thereby inducing the cell to differentiate into a neuroblast.
Suitable control sequences for use in the third aspect of the invention are
known in
1o the art and may include inducible or constitutive control sequences.
Inducible
control sequences have the advanta?e that Soxl expression may be switched off
when desired, for example once the cell is to be differentiated into another
neural
cell. Moreover, once the expression of exogenous Soxl has been switched off,
successfully differentiated neuroblasts may be identified by virtue of the
continued
~5 expression of the endogenous Soxl gene.
Precursor cells may be, for example, ES cells, such as human ES cells and
cells
with similar pluripotent properties derived from germ cells (EG cells). More
specific neuronal pluripotent precursors or direct neuroblast precursors may
also be
2o employed.
Neuroblasts obtained according to the invention may be employed in a number of
ways. Of course, the expression of Soxl has important implications for the
study of
neural differentiation; the generation and selection of neuroblasts will
provide
25 material for basic research.
a Moreover, the invention has medical and diagnostic applications. The
detection of
Soxl expressing cells is important in clinical neurology and in diagnosing and
treating cancers of the nervous system. Accordingly, the invention provides a
3o method for detecting the presence of a neuroblast as described above for
diagnostic
purposes.
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Neural stem cells are also useful for the treatment of neurological disorders,
especially for repair of accidentally induced trauma in the CNS or for the
correction
of congenital or pathological diseases of the CNS.
Moreover, in applications involving somatic gene therapy designed to correct a
genetic defect in nervous tissue, the removal, treatment and replacement of
pluripotent neuroblasts which are actively dividing has clear advantages,
providing a
constant source of modified neural cells to permanently treat the targeted
defect.
to Soxl control sequences may be used specifically to direct transgene
expression in
neuroblast cells where this is desired. Moreover, gene expression can be
directed
to neural cell types differentiated from neuroblasts by the use of other
control
sequences, such as NF-1 control sequences which direct expression of NF-1 in
mature neurons in vivo.
A significant advantage of the methods described herein is that a patient in
need of
treatment for a neurological disorder can act as a self donor. In other words,
cells
may be isolated from the patient and either sorted to extract neuroblasts, or
treated
in order to differentiate neuroblasts as described, from specific or general
precursors.
D_etaile Description of the Invention
The present invention relates to a method for isolating, or producing cells
which are
committed to the neural fate. Accordingly, the term neuroblast, as used
herein,
refers to any cell or cell line which has commenced di: wentiation along the
neural
pathways.
The isolation of neuroblastic cells from populations of cells is desirable, in
order to
obtain cells which are committed to neural pathways, but are not terminally
differentiated. Such cells are useful in the study of neuronal
differentiation, and in
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the treatment of diseases such as neurodegenerative diseases, and neural
damage,
for example occasioned by trauma. Thus, typical populations of cells from
which
neuroblastic cells may be differentiated include cell populations derived from
the
CNS of mammals, such as humans, including CNS from adult and foetal sources.
Moreover, cell populations derived from tissue cultures may be employed for
the
isolation of neuroblastic cells.
It has been determined that SOX1 expression is closely associated with the
acquisition of neural fate by the ectoderm, both in vitro and in vivo. In
vitro SOX1
to expression is initiated within 24 hours after the addition of retinoic acid
to
pluripotent EC cell aggregates coincident with the induction of
neuroepithelial
markers such as NESTIN, Mashl and Wntl. In mouse and rat embryos expression
is restricted to cells of the anteroldistal ectoderm. Previous fate mapping
studies
indicated that this region of the epiblast constitutes the promordium of the
nervous
system.
Expression of SOXI is detected throughout the cells of the neural plate and
early
neural tube along its entire anteroposterior axis. The early and uniform
expression
of SOX1 throughout the presumptive CNS indicates that SOX1 is activated by
2o neural inducing signals and lends support to the proposal of a two step
response of
the ectoderm to organiser signals in generating a nervous system:
neutralisation
followed by regionalisation.
Expression of this Sox gene subfamily has been evolutionarily conserved. The
Drosophila (Nambu and Nambu 1996; Russet et al., 1996) zebrafish (Vriz et al.,
1996) and avian (Unwanogho et al., 1995; Streit et al., 1997; Rex et al.,
1997)
putative orthologues of Soxl, Sox2 and Sox3 all show expression throughout the
neural primordium. Thus, this subfamily of Sox genes represents a novel group
of
transcription factors which can serve as general early neuroepitheiial
markers.
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In order to isolate neuroblastic cells, the present invention provides for the
detection
of Soxl therein. As used herein, Soxl may be derived from any source,
including
mammalian sources, avian sources and other vertebrate sources. Soxl may also
be
derived from invertebrate sources.
Soxl has been cloned from human, chicken and mouse. The sequence of chicken,
mouse and human Soxl is set forth in SEQ ID.s numbers 1 to 3 herein.
The preferred sequence encoding Soxl is that encoding human Soxl and having
to substantially the same nucleotide sequence as the sequence in SEQ ID No. 3,
with
the nucleic acid having the same sequence as the sequence in SEQ ID No. 3
being
most preferred. As used herein, nucleotide sequences which are substantially
the
same share at least about 90% identity. However, in the case of splice
variants
having e.g. an additional exon sequence homology may be lower.
The nucleic acids of the invention, whether used as probes or otherwise, are
preferably substantially homologous to the sequence of human Soxl as shown in
SEQ ID No. 3. As used herein, "homology" means that the two entities share
sufficient characteristics for the skilled person to determine that they are
similar in
origin and function. Preferably, homology is used to refer to sequence
identity.
Thus, Soxl sequences according to the invention preferably retain substantial
sequence identity human Soxl.
"Substantial homology", where homology indicates sequence identity, means more
2s than 40% sequence identity, preferably more than 45% sequence identity and
most
preferably a sequence identity of 50% or more, as judged by direct sequence.
alignment and comparison.
Sequence homology (or identity) may be determined using any suitable homology
3o algorithm, using for example default parameters. Advantageously, the BLAST
algorithm is employed, with parameters set to default values. The BLAST
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algorithm is described in detail at http://www.ncbi.nih.govlBLAST/blast
help.html,
which is incorporated herein by reference. The search parameters are defined
as
follows, and are advantageously set to the defined default parameters.
Advantageously, "substantial homology" when assessed by BLAST equates to
sequences which match with an EXPECT value of at least 7, preferablyat least 9
and most preferably 10 or more. The default threshold for EXPECT in BLAST
searching is ususally 10.
1o BLAST (Basic Local Alignment Search Tool) is the heuristic search algorithm
employed by the programs blastp, blastn, blastx, tblastn, and tblastx; these
programs ascribe significance to their findings using the statistical methods
of
Karlin and Altschul (see http:llwww.ncbi.nih.gov/BLAST/blast help.html) with a
few enhancements. The BLAST programs were tailored for sequence similarity
searching, for example to identify homologues to a query sequence. The
programs
are not generally useful for motif style searching. For a discussion of basic
issues in
similarity searching of sequence databases, see Altschul et al. (1994) Nature
Genetics 6:119-129.
The five BLAST programs available at http://www.ncbi.nlm.nih.gov perform the
following tasks:
blastp compares an amino acid query sequence against a protein sequence
database;
blastn compares a nucleotide query sequence against a nucleotide sequence
database;
blastx compares the six-frame conceptual translation products of a nucleotide
query
sequence (both strands) against a protein sequence database;
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tblastn compares a protein query sequence against a nucleotide sequence
database
dynamically translated in all six reading frames (both strands).
tblastY compares the six-frame translations of a nucleotide query sequence
against
5 the six-frame translations of a nucleotide sequence database.
BLAST uses the following search parameters:
HISTOGRAM Display a histogram of scores for each search; default is yes. (See
to parameter H in the BLAST Manual).
DESCRIPTIONS Restricts the number of short descriptions of matching sequences
reported to the number specified; default limit is 100 descriptions. (See
parameter V
in the manual page). See also EXPECT and CUTOFF.
ALIGNMENTS Restricts database sequences to the number specified for which
high-scoring segment pairs (HSPs) are reported; the default limit is 50. If
more
database sequences than this happen to satisfy the statistical significance
threshold
for reporting (see EXPECT and CUTOFF below), only the matches ascribed the
greatest statistical significance are reported. (See parameter B in the BLAST
Manual) .
EXPECT The statistical significance threshold for reporting matches against
database sequences; the default value is 10, such that 10 matches are expected
to be
found merely by chance, according to the stochastic model of Karlin and
Altschul
(1990). If the statistical significance ascribed to a match is greater than
the
EXPECT threshold, the match will not be reported. Lower EXPECT thresholds are
more stringent, leading to fewer chance matches being reported. Fractional
values
are acceptable. (See parameter E in the BLAST Manuai).
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CUTOFF Cutoff score for reporting high-scoring segment pairs. The default
value
is calculated from the EXPECT value (see above). HSPs are reported for a
database
sequence only if the statistical significance ascribed to them is at least as
high as
would be ascribed to a lone HSP having a score equal to the CUTOFF value.
s Higher CUTOFF values are more stringent, leading to fewer chance matches
being
reported. (See parameter S in the BLAST Manual). Typically, significance
thresholds can be more intuitively managed using EXPECT.
MATRIX Specify an alternate scoring matrix for BLASTP, BLASTX, TBLASTN
and TBLASTX. The default matrix is BLOSUM62 (Henikoff & Henikoff, 1992).
The valid alternative choices include: PAM40, PAM120, PAM250 and IDENTITY.
No alternate scoring matrices are available for BLASTN; specifying the MATRIX
directive in BLASTN requests returns an error response.
15 STRAND Restrict a TBLASTN search to just the top or bottom strand of the
database sequences; or restrict a BLASTN, BLASTX or TBLASTX search to just
reading frames on the top or bottom strand of the query sequence.
FILTER Mask off segments of the query sequence that have low compositional
2o complexity, as determined by the SEG program of Wootton & Federhen (1993)
Computers and Chemistry 17:149-163, or segments consisting of short-
periodicity
internal repeats, as determined by the XNU program of Claverie & States (1993)
Computers and Chemistry 17:191-201, or, for BLASTN, by the DUST program o~
Tatusov and Lipman (see http:/Iwww.ncbi.nlm.nih.gov). Filtering can eliminate
25 statistically significant but biologically uninteresting reports from the
blast output
(e.g., hits against common acidic-, basic- or proline-rich regions), leaving
the more
biologically interesting regions of the query sequence available for specific
matching
against database sequences.
3o Low complexity sequence found by a filter program is substituted using the
letter
"N" in nucleotide sequence (e.g., "NNNNNNNNNNNNN") and the letter "X" in
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protein sequences (e.g., "XXXXXXXXX"). Users may turn off filtering by using
the "Filter" option on the "Advanced options for the BLAST server" page.
Filtering is only applied to the query sequence (or its translation products),
not to
database sequences. Default filtering is DUST for BLASTN, SEG for other
programs .
It is not unusual for nothing at all to be masked by SEG, XNU, or both, when
applied to sequences in SWISS-PROT, so filtering should not be expected to
always
1o yield an effect. Furthermore, in some cases, sequences are masked in their
entirety,
indicating that the statistical significance of any matches reported against
the
unfiltered query sequence should be suspect.
NCBI-gi Causes NCBI gi identifiers to be shown in the output, in addition to
the
accession and/or locus name.
Most preferably, sequence comparisons are conducted using the simple BLAST
search algorithm provided at http://www.ncbi.nlm.nih.gov/BLAST.
2o Preferably, the invention makes use of fragments of the Soxl-encoding
sequence.
Fragments of the nucleic acid sequence of a few nucleotides in length,
preferably 5
to 150 nucleotides in length, are especially useful as probes.
Exemplary nucleic acids can alternatively be characterised as those nucleotide
sequences which encode a Soxl protein and hybridise to the DNA sequences set
forth SEQ ID No. 3, or a selected fragment of said DNA sequence. Preferred are
such sequences encoding Soxl which hybridise under high-stringency conditions
to
the sequence of SEQ ID No. 3.
3o Stringency of hybridisation refers to conditions under which polynucleic
acids
hybrids are stable. Such conditions are evident to those of ordinary skill in
the field.
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As known to those of skill in the art, the stability of hybrids is reflected
in the
melting temperature (Tm) of the hybrid which decreases approximately 1 to
1.5°C
with every 1 % decrease in sequence homology. In general, the stability of a
hybrid
is a function of sodium ion concentration and temperature. Typically, the
hybridisation reaction is performed under conditions of higher stringency,
followed
by washes of varying stringency.
As used herein, high stringency refers to conditions that permit hybridisation
of
only those nucleic acid sequences that form stable hybrids in 1 M Na+ at 65-68
°C.
to High stringency conditions can be provided, for example, by hybridisation
in an
aqueous solution containing 6x SSC, Sx Denhardt's, 1 % SDS (sodium dodecyl
sulphate), 0.1 Na+ pyrophosphate and 0.1 mglml denatured salmon sperm DNA as
non specific competitor. Following hybridisation, high stringency washing may
be
done in several steps, with a final wash (about 30 min) at the hybridisation
temperature in 0.2 - O. lx SSC, 0.1 % SDS.
Moderate stringency refers to conditions equivalent to hybridisation in the
above
described solution but at about 60-62°C. In that case the final wash is
performed at
the hybridisation temperature in lx SSC, 0.1 % SDS.
Low stringency refers to conditions equivalent to hybridisation in the above
described solution at about 50-52°C. In that case, the final wash is
performed at the
hybridisation temperature in 2x SSC, 0.1 % SDS. -
It is understood that these conditions may be adapted and duplicated using a
variety
of buffers, e.g. formamide-based buffers, and temperatures. Denhardt's
solution
and SSC are well known to those of skill in the art as are other suitable
hybridisation buffers (see, e.g. Sambrook, et al., eds. (1989) Molecular
Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, New York or Ausubel,
3o et al., eds. (1990) Current Protocols in Molecular Biology, John Wiley &
Sons,
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Inc.). Optimal hybridisation conditions have to be determined empirically, as
the
length and the GC content of the hybridising pair also play a role.
Advantageously, the invention moreover provides nucleic acid sequence which
are
capable of hybridising, under stringent conditions, to a fragment of SEQ. ID.
No. 3.
Preferably, the fragment is between 15 and 50 bases in length. Advantageously,
it
is about 25 bases in length.
As will be appreciated by those skilled in the art, the redundancy of the
genetic
to code allows the design of a large number of sequences encoding human Soxl.
Any
of these sequences may be useful for expressing SOX1 as described below. An
advantage of the use of a sequence encoding human SOX1 which is not the human
Soxl sequence is that the mRNA produced has a different sequence to that of
the
endogenous Soxl mRNA, and may thus be distinguished therefrom. Antisense
oligonucleotides may be designed which are capable of selectively inhibiting
the
expression of either endogenous or exogenous Soxl genes. Degenerate sequences
encoding human SOX1 are set forth in SEQ. ID. No. 5.
Given the guidance provided herein, nucleic acids encoding Soxl are obtainable
2o according to methods well known in the art. For example, a nucleic acid
encoding
Soxl is obtainable by chemical synthesis, using polymerase chain reaction
(PCR) or
by screening a genomic library or a suitable cDNA library prepared from a
source
believed to possess Soxl and to express it at a detectable level.
Chemical methods for synthesis of a nucleic acid of interest are known in the
art
and include triester, phosphite, phosphoramidite and H-phosphonate methods,
PCR
and other autoprimer methe,:v. as well as oligonucleotide synthesis on solid
supports.
These methods may be used if the entire nucleic acid sequence of the nucleic
acid is
known, or the sequence of the nucleic acid complementary to the coding strand
is
3o available. Alternatively, if the target amino acid sequence is known, one
may infer
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potential nucleic acid sequences using known and preferred coding residues for
each
amino acid residue.
An alternative means to isolate a gene encoding Soxl is to use PCR technology
as
5 described e.g. in section 14 of Sambrook et al., 1989. This method requires
the use
of oligonucleotide probes that will hybridise to Soxl nucleic acid. Strategies
for
selection of oligonucleotides are described below.
Libraries are screened with probes or analytical tools designed to identify
the gene
to of interest or the protein encoded by it. For cDNA expression libraries
suitable
means include monoclonal or polyclonal antibodies that recognise and
specifically
bind to Soxl ; oligonucleotides of about 20 to 80 bases in length that encode
known
or suspected Soxl cDNA from the same or different species; and/or
complementary
or homologous cDNAs or fragments thereof that encode the same or a hybridising
15 gene. Appropriate probes for screening genomic DNA libraries include, but
are not
limited to oligonucleotides, cDNAs or fragments thereof that encode the same
or
hybridising DNA; and/or homologous genomic DNAs or fragments thereof.
A nucleic acid encoding Soxl may be isolated by screening suitable cDNA or
2o genomic libraries under suitable hybridisation conditions with a probe,
i.e. a nucleic
acid disclosed herein including oligonucleotides derivable from the sequences
set
forth in SEQ ID NO. 3. Suitable libraries are commercially available or can be
prepared e.g. from cell lines, tissue samples, and the Like.
z5 As used herein, a probe is e.g. a single-stranded DNA or RNA that has a
sequence
of nucleotides that includes between 10 and 50, preferably between 15 and 30
and
most preferably at least about 20 contiguous bases that are the same as (or
the
complement ot~ an equivalent or greater number of contiguous bases set forth
in
SEQ ID No. 3. The nucleic acid sequences selected as probes should be of
3o sufficient Length and sufficiently unambiguous so that false positive
results are
minimised. The nucleotide sequences are usually based on conserved or highly
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homologous nucleotide sequences or regions of Soxl. The nucleic acids used as
probes may be degenerate at one or more positions. The use of degenerate
oligonucleotides may be of particular importance where a library is screened
from a
species in which preferential codon usage in that species is not known.
Preferred regions from which to construct probes include 5' and/or 3' coding
sequences, sequences predicted to encode ligand binding sites, and the like.
For
example, either the full-length cDNA clone disclosed herein or fragments
thereof
can be used as probes. Preferably, nucleic acid probes of the invention are
labelled
to with suitable label means for ready detection upon hybridisation. For
example, a
suitable label means is a radiolabel. The preferred method of labelling a DNA
fragment is by incorporating a32P dATP with the Klenow fragment of DNA
polymerase in a random priming reaction, as is well known in the art.
Oligonucleotides are usually end-labelled with y32P-labelled ATP and
polynucleotide
kinase. However, other methods (e.g. non-radioactive) may also be used to
label the
fragment or oligonucleotide, including e.g. enzyme labelling, fluorescent
labelling
with suitable fluorophores and biotinylation.
After screening the library, e.g. with a portion of DNA including
substantially the
2o entire Soxl-encoding sequence or a suitable oligonucleotide based on a
portion of
said DNA, positive clones are identified by detecting a hybridisation signal;
the
identified clones are characterised by restriction enzyme mapping and/or DNA
sequence analysis, and then examined, e.g. by comparison with the sequences
ses
forth herein, to ascertain whether they include DNA encoding a complete Soxl
(i.e.,
if they include translation initiation and termination codons). If the
selected clones
are incomplete, they may be used to rescreen the same or a different library
to
obtain overlapping clones. If the library is genomic, tl;en the cwerlapping
clones
may include exons and introns. If the library is a cDNA library, then the
overlapping clones will include an open reading frame. In both instances,
complete
3o clones may be identified by comparison with the DNAs and deduced amino acid
sequences provided herein.
r
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17
It is envisaged that Sox 1-encoding sequences can be readily modified by
nucleotide
substitution, nucleotide deletion, nucleotide insertion or inversion of a
nucleotide
stretch, and any combination thereof. Such mutants can be used e.g. to produce
a
SOX1 mutant that has an amino acid sequence differing from the SOX1 sequences
as found in nature. Mutagenesis may be predetermined (site-specific) or
random. A
mutation which is not a silent mutation must not place sequences out of
reading
frames and preferably will not create complementary regions that could
hybridise to
produce secondary mRNA structure such as loops or hairpins.
IO
Sorting of cells, based upon detection of expression of the Soxl gene, may be
performed by any technique known in the art, as exemplified above. For
example,
the cells may be sorted by flow cytometry or FACS. For a general reference,
see
Flow Cytometry and Cell Sorting: A Laboratory Manual (1992) A. Radbruch (Ed.),
I5 Springer Laboratory, New York.
Flow cytometry is a powerful method for studying and purifying cells. It has
found
wide application, particularly in immunology and cell biology: however, the
capabilities of the FACS can be applied in many other fields of biology. The
2o acronym F.A.C.S. stands for Fluorescence Activated Cell Sorting, and is
used
interchangeably with "flow cytometry". The principle of FACS is that
individual
cells, held in a thin stream of fluid, are passed through one or more laser
beams,
causing light to be scattered and fluorescent dyes to emit light at various
frequencies. Photomultiplier tubes (PMT) convert light to electrical signals,
which
25 are interpreted by software to generate data about the cells. Sub-
populations of cells
with defined characteristics can be identified and automatically sorted from
the
suspension at very high purity (--100%).
FACS machines collect fluorescence signals in one to several channels
3o corresponding to different laser excitation and fluorescence emission
wavelengths.
Fluorescent labelling allows the investigation of many aspects of cell
structure and
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18
function. The most widely used application is immunofluorescence: the staining
of
cells with antibodies conjugated to fluorescent dyes such as fluorescein and
phycoerythrin. This method is often used to label molecules on the cell
surface, but
antibodies can also be directed at targets within the cell. In direct
s immunofluorescence, an antibody to a particular molecule, the SOX1
polypeptide,
is directly conjugated to a fluorescent dye. Cells can then be stained in one
step. In
indirect immunofluorescence, the primary antibody is not labelled, but a
second
fluorescently conjugated antibody is added which is specific for the first
antibody:
for example, if the anti-SOXI antibody is a mouse IgG, then the second
antibody
could be a rat or rabbit antibody raised against mouse IgG.
FACS can be used to measure gene expression in cells transfected with
recombinant
DNA encoding SOX1. This can be achieved directly, by labelling of the protein
product, or indirectly by using a reporter gene in the construct. Examples of
reporter genes are ~3-galactosidase and Green Fluorescent Protein (GFP). ~i-
galactosidase activity can be detected by FACS using fluorogenic substrates
such as
fluorescein digalactoside (FDG). FDG is introduced into cells by hypotonic
shock,
and is cleaved by the enzyme to generate a fluorescent product, which is
trapped
within the cell. One enzyme can therefore generate a large amount of
fluorescent
2o product. Cells expressing GFP constructs will fluoresce without the
addition of a
substrate. Mutants of GFP are available which have different excitation
frequencies,
but which emit fluorescence in the same channel. In a two-laser FACS machine,
it
is possible to distinguish cells which are excited by the different lasers and
therefore
assay two transfections at the same time.
Alternative means of cell sorting may also be employed. For example, the
s~~vention comprises the use of nucleic acid probes complementary to Soxl
mRNA.
Such probes can be used to identify cells expressing Soxl individually, such
that
they may subsequently be sorted either manually, or using FACS sorting.
Nucleic
3o acid probes complementary to Soxl mRNA may be prepared according to the
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19
teaching set forth above, using the General procedures as described by
Sambrook et
al (1989).
In a preferred embodiment, the invention comprises the use of an antisense
nucleic
s acid molecule, complementary to Soxi mRNA, conjugated to a fluorophore which
may be used in FACS cell sorting.
Suitable imaging agents for use with FACS may be delivered to the cells by any
suitable technique, including simple exposure thereto in cell culture,
delivery of
to transiently expressing nucleic acids by viral or non- viral vector means,
liposome-
mediated transfer of nucleic acids or imaging agents, and the like.
The invention, in certain embodiments, includes antibodies specifically
recognising
and binding to SOX1. For example, such antibodies may be generated against the
15 SOX1 having the amino acid sequences set forth in SEQ ID No. 4.
Alternatively,
SOX1 or SOX1 fragments (which may also be synthesised by in vitro methods) are
fused (by recombinant expression or an in vitro peptidyl bond) to an
immunogenic
polypeptide and this fusion polypeptide, in turn, is used to raise antibodies
against a
SOXI epitope.
Anti-SOX1 antibodies may be recovered from the serum of immunised animals.
Monoclonal antibodies may be prepared from cells from immunised animals in the
conventional manner.
The antibodies of the invention are useful for identifying SOX1 in neural
cells
expressing Soxl , in accordance with the present invention.
Antibodies according to the invention may be whole antibodies of natural
classes,
such as IgE and IgM antibodies, but are preferably IgG antibodies. Moreover,
the
3o invention includes antibody fragments, such as Fab, F(ab')2, Fv and ScFv.
Small
fragments, such Fv and ScFv, possess advantageous properties for diagnostic
and
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therapeutic applications on account of their small size and consequent
superior
tissue distribution.
The antibodies may comprise a label. Especially preferred are labels which
allow
s the imaging of the antibody in neural cells in vivo. Such labels may be
radioactive
labels or radioopaque labels, such as metal particles, which are readily
visualisable
within tissues. Moreover, they may be fluorescent labels or other labels which
are
visualisable in tissues and which may be used for cell sorting.
1o Recombinant DNA technology may be used to improve the antibodies of the
invention. 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
~5 modification.
Antibodies according to the invention 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
2o established procedure, in bacterial or preferably mammalian cell culture.
The
selected cell culture system preferably secretes the antibody product.
Therefore, the present invention includes a process for the production of a~
antibody according to the invention comprising culturing a host, e.g. E. toll
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 prop~.r re:~::ing frame to a second
DNA
sequence encoding said protein, and isolating said protein.
3o 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
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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, Iow 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 x YT, or M9 Minimal Medium, and for yeast
in medium YPD, YEPD, Minimal Medium, or Complete Minimal Dropout
to Medium.
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.
Large quantities of the desired antibodies can also be obtained by multiplying
2o 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.
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22
The cell culture supernatants are screened for the desired antibodies,
preferentially
by immunofluorescent staining of cells expressing SOX1, by immunoblotting, by
an
enzyme immunoassay, e.g. a sandwich assay or a dot-assay, or a
radioimmunoassay.
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
to 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
SOX1
protein or with Protein-A.
The invention further concerns hybridoma cells secreting the monoclonal
antibodies
of the invention. The preferred hybridoma cells of the invention are
genetically
stable, secrete monoclonal antibodies of the invention of the desired
specificity and
can be activated from deep-frozen cultures by thawing and recioning.
2o The invention also concerns a process for the preparation of a hybridoma
cell line
secreting monoclonal antibodies directed SOX1, characterised in that a
suitable
mammal, for example a Balb/c mouse, is immunised with purified SOX1 protein,
an antigenic carrier containing purified SOXl or with cells bearing SOXl,
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 cells bearing SOX1 are fused with cells ~~f the myeloma
cell
line PAI or the myeloma cell line Sp2/0-Agl4, the obtained hybrid cells are
screened for secretion of the desired antibodies, and positive hybridoma cells
are
3o cloned.
..,..... r..
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23
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 107 and 108 cells of human tumour origin which express SOX1
containing 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
io 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.
The invention also concerns recombinant DNAs comprising an insert coding for a
heavy chain variable domain and/or for a light chain variable domain of
antibodies
directed to the extracelluiar domain of SOX1 as described hereinbefore. By
definition such DNAs comprise coding single stranded DNAs, double stranded
2o DNAs consisting of said coding DNAs and of complementary DNAs thereto, or
these complementary (single stranded) DNAs themselves.
Furthermore, DNA encoding a heavy chain variable domain and/or for a light
chain
variable domain of antibodies directed SOX1 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
3o acids. Preferably said modifications) are outside the CDRs of the heavy
chain
variable domain and/or of the light chain variable domain of the antibody.
Such a
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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
to light chain murine variable domain.
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.
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.
The invention therefore also concerns recombinant DNAs comprising an insert
coding for a heavy chain murine variable domain of an antibody directed SOX/
fused to a human constant domain g, for example yl, y2, y3 or y4, preferably
yl oY
y4. Likewise the invention concerns recombinant DNAs comprising an insert
coding for a light chain murine variable domain of an antibody directed to
SOX1
fused to a human constant domain K or ~., preferably K.
In another embodiment the invention pertains to recombinant nucleic acids
wherein
the heavy chain variable domain and the light chain variable domain are linked
by
3o way of a DNA insert coding for a spacer group, optionally comprising a
signal
sequence facilitating the processing of the antibody in the host cell andlor a
DNA
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coding for a peptide facilitating the purification of the antibody and/or a
DNA
coding for a cleavage site and/or a DNA coding for a peptide spacer and/or a
DNA
coding for an effector molecule, such as a label.
5 According to a further aspect, and as referred to above, neuroblastic cells
may be
actively sorted from other cell types by detecting Soxl expression in vivo
using a
reporter system. For example, such a reporter system may comprise a readily
identifiable marker under the control of a Soxl activated expression system.
Fluorescent markers, which can be detected and sorted by FACS, are preferred.
io Especially preferred are GFP and luciferase.
Alternatively, an in vivo construct expressing a reporter may be placed under
the
control of the Soxl control sequences themselves. These sequences are
activated at
the same time as Soxl expression is activated, and therefore mark the
transition into
15 the neural pathway with the same accuracy as Soxl. Advantageously, the Soxl
control sequences used are human Soxl control sequences. Preferably, they
comprise nucleotides 1 to 60 of SEQ. ID. No. 3.
In general, reporter constructs useful for detecting neural cells by
expression of a
2o reporter gene may be constructed the general teaching of Sambrook et al
(1989).
Typically, constructs according to the invention comprise a promoter by Soxl ,
and a
coding sequence encoding the desired reporter constructs, for example of GFP
or
luciferase. Vectors encoding GFP and luciferase are known in the art and
available
commercially.
SOX proteins bind to a sequence motif (A/T A/T CAA A/T G) (SEQ. ID. No. 6)
with high affinity. Accordingly, constructs according to the invention
advantageously comprise the above-recited motif, or a functional equivalent
thereof,
operably linked to a gene encoding a selectable marker.
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26
When transfected into cells which are potentially express Soxl, constructs
according
to the invention will be activated specifically by Soxl expression. Therefore,
the
selectable marker will be expressed once the cell enters the neural
differentiation
pathway and Soxl expression is induced. This allows cells entering the neural
differentiation pathway to be sorted by FACS.
In a still further aspect, the present invention relates to the transfection
of
pluripotent precursor cells, capable of differentiating into neural cells,
with a vector
expressing Soxl. By such means, pluripotent precursor cells may be induced to
t0 differentiate along the neural pathway, becoming precursor neurons capable
of
differentiating into a variety of neural tissues.
Herein, terms such as "transfection", "transformation" and the like are not
intended
to be significant, except to indicate that nucleic acid is transferred to a
cell or
organism in functional form. Such terms include various means of transferring
nucleic acids to cells, including transfection with CaP04, electroporation,
viral
transduction, Iipofection, delivery using liposomes and other delivery
vehicles,
biolistics and the like.
2o Suitable pluripotent precursor cells may be derived from a number of
sources. For
example, ES cells, such as human ES cells and cells derived from a Germ cells
(EG
cells) may be derived from embryonal tissue. Alternatively, pluripotent cells
may be
prepared by a retrodifferentiation, by the administration of growth factors or
otherwise (see WO 96/23870), or by cloning, such as by nuclear transfer from
an
adult cell to a pluripotent cell such as an ovum.
Human stem cells of neural lineage n::.y be isolated from human tissa~s
directly.
Alternatively, stem sells from non- human animals, such as rodents, may be
used.
3o Neural stem cells may also be propagated in vitro, for example as described
in
Snyder et al. (1996) Clinical Neuroscience 3: 310-3I6, and Martinez-Serrano et
al.,
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27
(1996) Clinical Neuroscience 3:301-309. Moreover, pluripotent cell lines such
as
the N-Tera II cell line which are capable of differentiating into neural cells
upon
stimulation with agents such as retinoic acid are also responsive to Soxl
stimulation.
The cDNA or genomic DNA encoding native or mutant SOX1, or a label under to
control of Soxl sequences or a sequence transactivatable by SOX1, can be
incorporated into vectors according too techniques known in the art. As used
herein, vector (or plasmid) refers to discrete elements that are used to
introduce
heterologous DNA into cells for expression. Selection and use of such vehicles
are
1o well within the skill of the artisan. The vector components generally
include, but
are not limited to, one or more of the following: an origin of replication,
one or
more marker genes, an enhancer element, a promoter, a transcription
termination
sequence and a signal sequence.
~5 Most expression vectors are shuttle vectors, i.e. they are capable of
replication in at
least one class of organisms but can be transfected into another class of
organisms
for expression. For example, a vector is cloned in E. coli and then the same
vector
is transfected into mammalian cells even though it is not capable of
replicating
independently of the host cell chromosome.
Advantageously, an expression and cloning vector may contain a selection gene,
also referred to as selectable marker, other than that intended for marking
Soxl-
expressing cells. This gene may encode a protein necessary for the survival
or'
growth of transformed host cells grown in a selective culture medium. Host
cells
not transformed with the vector containing the selection gene will not survive
in the
culture medium. Typical selection genes encode proteins that confer resistance
to
antibiotics and other toxins, e.~. ampicillin, neomycin, methotrexate or
tetracycline,
complement auxotrophic deficiencies, or supply critical nutrients not
available from
complex media.
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Since the replication of vectors is conveniently done in E. coli, an E. coli
genetic
marker and an E. coli origin of replication are advantageously included. These
can
be obtained from E. coli plasmids, such as pBR322, Bluescript° vector
or a pUC
plasmid, e.g. pUCl8 or pUCl9, which contain both E. coli replication origin
and
E. coli genetic marker conferring resistance to antibiotics, such as
ampicillin.
Expression vectors usually contain a promoter that is recognised by the host
organism and is operably linked to SOX/, or label-encoding, nucleic acid. Such
a
promoter may be inducible by factors which induce Soxl , or by Soxl itself.
The
to promoters are operably linked to DNA encoding SOX1 by removing the promoter
from the source DNA and inserting the isolated promoter sequence into the
vector.
Both the native SOX1 promoter sequence and many heterologous promoters may be
used to direct amplification and/or expression of SOX1 DNA. The term "operably
linked" refers to a juxtaposition wherein the components described are in a
i5 relationship permitting them to function in their intended manner. A
control
sequence "operably linked" to a coding sequence is ligated in such a way that
expression of the coding sequence is achieved under conditions compatible with
the
control sequences.
20 Control sequences, comprising a promoter and optionally enhancer(s), may be
derived from the human or other Soxl genes. Alternatively, any suitable
promoter
may be used, when placed under the control of a SOX1-inducible element. In
such
a construct, the promoter selected should have a low residual level of
activity, such
as to minimise expression of the Label in the absence of Soxl expression.
The vectors may also contain sequences necessary for the termination of
transcription and for stabilising the mRNA. Such se:.luences are commonly
available
from the 5' and 3' untranslated regions of eukaryotic or viral DNAs or cDNAs.
These regions contain nucleotide segments transcribed as polyadenylated
fragments
3o in the untranslated portion of the mRNA encoding SOX1 or the label.
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An expression vector includes any vector capable of expressing SOX1 or label-
encoding nucleic acids that are operatively linked with regulatory sequences,
such
as promoter regions, that are capable of expression of such DNAs. Thus, an
expression vector refers to a recombinant DNA or RNA construct, such as a
plasmid, a phage, recombinant virus or other vector, that upon introduction
into an
appropriate host cell, results in expression of the cloned DNA. Appropriate
expression vectors are well known to those with ordinary skill in the art and
include
those that are replicable in eukaryotic and/or prokaryotic cells and those
that remain
episomal or those which integrate into the host cell genome. For example, DNAs
1o encoding SOX1 may be inserted into a vector suitable for expression of
cDNAs in
mammalian cells, e.g. a CMV enhancer-based vector such as pEVRF (Matthias, et
al., (1989) NAR 17, 6418).
Particularly useful for practising the present invention are expression
vectors that
~5 provide for the transient expression of DNA encoding SOX1 or a label in
mammalian cells. Transient expression usually involves the use of an
expression
vector that is able to replicate efficiently in a host cell, such that the
host cell
accumulates many copies of the expression vector, and, in turn, synthesises
high
levels of SOXlor a label. For the purposes of the present invention, transient
2o expression systems are useful e.g. for identifying SOX1 expressing cells or
for
inducing a pluripotent cell to differentiate.
Construction of vectors according to the invention employs conventional
techniques,
for example as described in Sambrook et al. , 1989. Isolated plasmids or DNA
25 fragments are cleaved, tailored, and religated in the form desired to
generate the
plasmids required. If desired, analysis to confirm correct sequences in the
constructed plasmids is performed in a known fashion. Suitable methods for
constructing expression vectors, preparing in vitro transcripts, introducing
DNA
into host cells, and performing analyses for assessing gene expression and
function
3o are known to those skilled in the art. Gene presence, amplification and/or
expression may be measured in a sample directly, for example, by conventional
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Southern blotting, Northern blotting to quantitate the transcription of mRNA,
dot
blotting (DNA or RNA analysis), or in situ hybridisation, using an
appropriately
labelled probe which may be based on a sequence provided herein. Those skilled
in
the art will readily envisage how these methods may be modified, if desired.
s
The invention is described, for the purpose of illustration only, in the
following
examples.
MATERIAL AND METHODS
to Manufacture of SOX1 polyclonal antibodies: A 622bp HincII fragment encoding
sequences C-terminal of the HMG box of SOX1 (207 a.a.) is fused in frame to
the
bacterial GST gene in the construct pGEX3X. Fusion protein is induced and
purified as described by Smith and Johnson ( 1988) Gene 67:31-40. rabbits are
treated with a course of injections as recommended by Smith and Johnson
(1988):
15 each injection contains 250~.g of fusion protein. Two final bleeds, FB43
and FB44,
are obtained from the rabbits prior to the preparation of polyclonal sera.
Immunocytochemistry: Embryos, P19 cells and neural plate explants are examined
using standard techniques (Placzek et al., (1993) Development 117:205-218).
2o Antibodies are used at the following dilutions: anti-SOX1 PAb (1:500); K2
anti-
HNF3~3 MAb (1:40); 6G3 anti-FP3 MAb (1:10); anti-3A10 MAb (1:10); anti-
2H3(Neurofilament-160) MAb (1:10); 4D5 anti-Islet-1 MAb (1:1000); anti-SSEA1
MAb (1:80) (Hybridoma Bank); anti-NESTINE MAb (1:10) (Hybridoma Bank);
anti-BrDU MAb (1:500) (Sigma); Appropriate secondary antibodies (TAGO and
25 Sigma) are conjugated to fluorescein isothiocyanate (FITC), Cy2 or Cy3.
BrDU an:zlysis: Pregnant mice are injected intraperitoneally wi:_, SOp ~/g of
body
weight of 5-bromo-2deoxyuridine (BrDU) {Sigma) in 09. % NaCI and sacrificed
two
hours after injection. Embryos are fixed and sectioned as described above. The
3o slides are washed twice in PBS, and incubated in 0.2 % HCl at 37°C
for 30 minutes,
then rinsed thoroughly with PBS, followed by three rinses with PBS/0.1
,.
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31
Trinton/1 % heat inactivated goat serum (P-T-G). Monoclonal anti-BrDU (1:500
dilution in P-T-G) is applied to the sections and incubated at 4°C
overnight.
Sequential sections are incubated in SOX1 antibody (1:500 dilution in P-T-G)
at
4°C overnight. The slides are washed twice in P-T-G, then incubated in
the
appropriate secondary antibody for 30 minutes at room temperature, washed with
P-
T-G and mounted.
P19 cell cultured and retinoic acid treatment: P19 cells are cultured as
previously
described (Rudnichy and McBurney, 1987). To induce differentiation, cells are
to allowed to aggregate in bacterial grade petri dishes alone, in the presence
of lp,M
retinoic acid or in the presence of 1~M retinoic acid or in the presence of
SmM
IPTG. After 4 days of aggregation in the presence of inducing agents, cells
are
plated on tissue culture chamber slides. The cells are allowed to adhere and
grow
for 4-5 days, with media changes every 24 hours. For immunoflurescence, cells
are
grown on tissue culture chamber slides coated with 0.1 % gelatin, washed once
with
PBS, fixed at room temperature in lx MEMFA for I hour, washed in P-T-G twice;
then stained with appropriate antibody.
Cell counting analysis: For cell counting experiments P19 transfectant cell
lines
2o are induced to differentiate, plated on gelatine coated slides, fixed at
room
temperature in lxMEMFA for one hour at day 6-8 for neurons. Cells are stained
with Neurofilament (2H3) antibody and photographed using an Olympus
fluorescence microscope. Celi counts are expressed as percentages of total
cells in
a field. Eight fields from two different experiments are counted for each P19
clone.
Plasmids and transfection: To construct the SOX1 expression vector,
pRSVopSoxl, the POP113CAT operator vector (Stratagene) is digested with Notl,
end-filled Kpn/Stu (position 431-1694) fragment of the Soxl cDNA. The P3'SS,
eukaryotic Lac repressor expressing vector (obtained from Stratagene) is
transfected
3o into P19 cells by lipofection. Stable transformants are selected in 250
~Cg/mI of
hygromycin. Expanded clones {250) are isolated and examined for expression of
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32
the Lac repressor by indirect immunofluorescence with anti-lac PAb
(Stratagene).
Four cell lines are isolated (P3'SS-10, 13, 22 and 47) which show ubiquitous
and
constitutive expression of the Lac repressor. P3'SS-10 is chosen for the
subsequent
experiments. P3'SS-10 is then transfected with pRSVopSoxl by lipofection.
Stable
clones are selected using SOO~g/ml 6481. 250 clones are expanded and analysed
for inducible Soxl expression by RNase protection and immunocytochemistry with
SOX 1 antibody .
RNase protection assays: Total RNA is prepared from P19 cells and RNase
protection assays are carried out using Spg of P19 cell RAN as described by
Capel
et al., (1993) Cell 73:1019-1030. Anti-sense labelled probes are derived from
the
396 by SmaI-BspHl fragment (position 1467-1863) of the Soxl cDNA, a 215bp
Bsal exon 4 specific fragment of Wntl cDNA, a PvuII digest of the Mashl cDNA
(Johnson et al., (1992) Development 114:75-87) and a NotI digest of SAP D cDNA
is is used a loading control (Dresser et al., (1995) Hum. Moi. Genet. 4:1613-
1618).
RT-PCR: Total RNA is prepared from P19 cells as described by Capel et al.,
(1993). Reserve transcription, PCR reaction, and priming is performed as
described by Okabe et al., (1996).
Rat lateral neural piste explants: Lateral neural plates (LNP) are isolated
from
days 8.5-9.0 rat embryos from prospective hindbrain and spinal cord regions as
previously described (Placzek et al., 1993). Notochord explants are dissected
from
HH stage 608 chick embryos as previously described (Placzek et al., 1993).
2s Explants are embedded in collagen and cultured (Placzek et al.,1993) for
24, 48 and
~J6 hours Purified rat SHH-N (Ericson et al., (1996) Cell 87:661-673) is added
to
s;ultures at concentrations within the effe:cive ranges used in other assays
(Ericson
et al., 1996)
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33
EXAMPLE 1
SOXl IS EXPRESSED DURING EARLY NEURAL DEVELOPMENT
SOX1 expression during mouse and rat neurulation is analysed using a rabbit
polyclonal antibody against the SOX1 C-terminal region. In the mouse,
expression
of SOX1 is first detected at 7.5 days post coitum (dpc) in the anterior half
of the
late-streak egg cylinder. Cross-sections through the embryo at this stage
reveal
expression in columnar ectodermal cells, which appear to define the neural
plate,
while cells located more laterally are negative. Thus, SOX1 expression at this
stage
1o is specific to the neural plate. SOXI is maintained in all neuroepitheial
cells along
the entire anteroposterior axis as the neural pate bends (8.0-8.5 dpc, as
shown in
cross-sections of a 2 somite mouse embryos where Soxl expression is limited to
neural folds) and fuses to form the neural tube (9.0-9.5 dpc, where Soxl
labelling is
seen to be restricted to the neural tube in cross-sections of 10-12 somite
mouse
embryos). The pattern of expression of SOXl in the rat is similar to that in
the
mouse. The expression of SOXl throughout the neural plate and early neural
tube
implies a similarity amongst these cells.
After neural tube closure, neuroepithelial cells begin to differentiate into
defined
2o classes of neurons at specific dorsoventral (D/V) positions within the
spinal cord
(Altman and Bayer (1984) Adv. Anat. Embryol. Cell Biol. 85:32-46; Tanabe and
Jessell, (1996) Science 274:1115-1123). As development proceeds, Soxl is
downregulated in a stereotyped manner in cells alone D/V axis of the neural
tube.
In the spinal cord, expressions first downregulated in cells that occupy the
ventral
midline (cross-sections of the thoracic region of 20 somite mouse embryos
reveal a
lack of SOX1 staining in this area), then the ventral motor horns
(corresponding
lack of staining being visible in cross section of 30-35 somite embryos) and
subsequently the dorsal regions. These regions appear to correlate with floor
plate,
motor neurons and sensory relay interneurons, respectively.
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To ascertain this a series of antibody double-labelling experiments are
performed in
rat embryos. The SOX1 antibody is used in combination with a panel of
antigenic
markers which identify cells of the floor plate and mature neurons
(Neurofilament
(NF-1): labelled with contrasting colour markers and visualised in an E11 rat
embryo). Expression of SOX1 and expression of these markers is almost entirely
mutually exclusive. In the ventral spinal cord or the 10.0-12.0 dpc mouse
embryo,
SOXl expression is maintained only in 'region X' (Yamada et al., (1991) Cell
64:635-b47), as revealed by immunolabelling of two streams of cells located
between the differentiated floor plate and ventral motor horns in 30-35 somite
y 1o embryos. Eventually, by 13.5 dpc, SOXl expression is restricted to a thin
ventricular zone in the CNS. SOX1 expression in to detected in the peripheral
nervous system (PNS). These expression profiles suggest that SOX1 is expressed
by early neural cells in the CNS and is downregulated in the developing neural
tube
coincident with neural differentiation.
EXAMPLE 2
SOXl MARKS PROLIFERATION CELLS WITHIN THE EMBRYONIC
NEURAL TUBE
2o The uniform expression of SOX1 in the neural plate and early neural tube
followed
by its down regulation along the DIV axis and restriction to the ventricular
zone is
reminiscent of the pattern of cell proliferation in the developing central
nervous
system (Sauer, (1935) J. Comp. Neurol. 62:377-405; Fujita, (1963) J. Comp.
Neurol. 120: 37-42; Altman and Bayer, 1984). In the neural plate and early
neural
tube, proliferating progenitor cells are organised in a pseudostratified
epithelium in
a.-a ~ ~h the processes of these cells extend from the inner luminal to the
outer mantle
surface. At later stages the neural tube becomes progressively thicker and can
be
divided into different zones. The proliferating CNS progenitors are largely
restricted to the inner ventricular zone (VZ) around the lumen. They begin to
3o migrate away from the lumen while in S-phase, and after completing their
final
rt.
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WO 99/00516 PCT/GB98/01862
mitosis, migrate to the outer layer, the marginal zone (MZ). In the 10.5 dpc
mouse
embryo, SOX1 expression is detected, using an anti-SOX1 antibody, throughout
the
pseudostratified epithelium of the posterior neural tube and is restricted to
the
ventricular zone in more mature anterior region of the neural tube. In order
to
s evaluate the relationship between SOX1 expression and proliferating CNS
cells are
directly assayed proliferation by monitoring the incorporation of
bromodeoxyuridine
(BrDU) with an anti-BrDU antibody. Pregnant mouse females at 10.5 dpc are
injected with BrDU two hours prior to dissection to detect proliferating
cells.
Embryos are then fixed, sectioned and double-labelled for BrDU incorporation
and
to SOX1 expression. Similar to SOX1 expressing cells, those that incorporate
BrDU
are found throughout the posterior neural tube in 10.5 dpc mouse embryos and
lie
in the ventricular zone of the anterior neural tube. All cells that
incorporate BrDU
also express SOXl. SOX1-positive cells that do not incorporate BrDU are
restricted to the luminar surface of the ventricular zone. In contrast, no
SOX1 nor
~s BrDU-positive cells are detected in the outer marginal zone. These results
show
that SOX1 is expressed in dividing neuroepithelial cells within the embryonic
CNS.
EXAMPLE 3
SOXl IS DOWNREGULATED IN COMMITED CELLS
The mutual exclusion of SOX1 and markers of committed differentiated cells
such
as Isletl (Pfaff et al., (1996) Cell 84:1-20) raises the possibility that the
downregulation of SOX1 may be a pre-requisite step for the differentiation in
neural
plate explants in vitro. Isolated neural plates explants are cultured with
known
inducers of ventral neural cells, namely the notochord and purified Sonic
Hedgehog
protein. The expression of SOXI and incorporation of BrDU is then compared to
the expression of three markers of ventral cells, Isletl, FP3 and HNF3(3.
Consistent with our observations in vivo both the expression of SOXI and
Isletl as
well as SOX1 and FP3 is mutually exclusive in neural plate explants cultured
3o adjacent to notochord (n=8) or in the presence of purified Sonic Hedgehog
protein
as seen in E9 rat neural plate tissue cultured with Sonic Hedgehog protein for
48
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36
hours and stained with anti-SOX1 and anti-Isletl antibodies. Similarly, the
incorporation of both BrDU and Isletl as well as BrDU and FP3 (detected using
an
anti-FP3 antibody) is mutually exclusive. In contrast, the domain of
expression of
HNF3~3 is found to extend beyond that of FP3 and into the region of BrDU
positive
cells.
To determine whether a similar population of cells could be detected in vivo
embryos are analysed, and for co-expression of FP3 and HNF3~i and for co-
expression of BrDU and HNF3~3. We find that medial floor plate cells co-
express
to HNF3~3 and FP3 but do not incorporate BrDU, whereas lateral floor plate
cells
express only HNF3(3 and incorporate BrDU. HNF3~3 thus provides a marker for
cells that are mitotically active but have begun to differentiate.
These cells, occupying the medial regions of the floor plate, express HNF3(3
but not
SOXl. In contrast cells occupying lateral regions of the floor plate co-
express
HNF3(3 and SOX1. These observations, together with the mutually exclusive
expression of SOX1 with Islet 1 and FP3 in ventral neural cells provide
evidence
that SOX1 is downregulated as cells exit mitosis and not at the onset of cell
differentiation.
EXAMPLE 4
SOX1 EXPRESSION IS ASSOCIATED WITH NEURAL DIFFERENTIATION
Neural induction is accompanied by the onset of new gene expression which in
turn
enables the formation of neural rather than epidermal tissue. The early and
apparently uniform expression of SOX1 in neva-al cells, together with
observations
that Sox genes may affect cell lineage decisions, raises the possibility that
SOX1
expression is an early response to neural inducing signals and that its
expression
may be involved in directing cells towards a neural fate. To address whether
SOX1
3o plays a role in establishing neural fate in response to A P19 cell culture
system is
CA 02295305 1999-12-23
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37
used as an in vivo model system in which to analyse SOX1 expression and the
effects of its misexpression.
P19 cells are an embryona! carcinoma cell line with the ability to
differentiate into
all three germ layers (McBurney, (1993) Int. J. dev. Biol. 37:135-140). In the
undifferentiated state P19 cells morphologically resemble an uncommitted
primitive
ectodermal cell and express the cell surface antigen SSEA-1. These cells have
a
very low rate of spontaneous differentiation when grown in a monolayer in the
absence of chemical inducers. P19 cells grown as aggregates, however,
1o differentiate partially into endodermal cells. Furthermore, with the
addition of
retinoic acid, aggregated P19 cells differentiate into neuroepithelial-like
cells (Jone-
Villeneuve et al., {1982) J. Cell. Biol. 94:253-262). These express
neuroepithelial
markers such as NCAM, intermediate filament NESRIN, MASHI (Johnson et al. ,
1992) and WNT1 (St. Arnaud et al., (1989) Oncogene 4:1077-1080). When plated
onto a substrate, about 15 % of these cells differentiate into mature neurons
expressing Neurofilament. Thus, in this in vitro model system retinoic acid
acts as
a "neural inducer".
Initially, the expression of Soxl in P19 cells is examined by both RNase
protection
2o and immunocytochemistry. The features of Soxl expression in P19 cells are
similar
to those observed in prospective neural tissue in vivo. Soxl mRNA and protein
can
not be detected in undifferentiated P19 cells which express the cell-surface
antigen
SSEA1 when analysed using anti-SOX1 and anti-SSEA antibodies, and by RNase
protection. Similarly, when P19 cells are differentiated as aggregates without
the
addition of chemical inducers, SOX1 is not expressed as determined by RNase
protection. In contrast, SOX1 is rapidly induced during neural differentiation
when
aggregated P19 cells are differentiated in the presence of retinoic acid. Soxl
thus
behaves similarly to other neuroepithelial markers such as Mash 1 and Wnt l,
the
transcripts of which are detected in retinoic acid-treated P19 cells by RNase
3o protection.
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38
When retinoic acid-treated P19 cell a;~re~ates are plated onto tissue culture
substrate, about 15 % of the cells differentiate into mature process-bearing,
Neurofilament-expressing neurons. Double-label immunofluorescence is used to
simultaneously detect SOX1 and Neurofilament, to examine the expression of
SOX1
in P19 cells displaying a fully differentiated neuronal morphology. SOXl
immunoreactivity is not detected in process-bearing Neurofilament-positive
neurons.
Thus, as in vivo, SOX/ is expressed by P19 cells when they first assume a
neural
fate but it is then downreaulated with their differentiation.
i o EXAMPLE 5
USE OF SOXl TO DIRECT CELLS TO A NEURAL FATE
The previous data suggest that in P19 cells, as in vivo, SOX1 expression is
induced
at a time when neuroepithelial cells begin to differentiate. If SOX1 plays a
role in
directing cells towards the neural fate, expression of SOX/ in P19 cells may
be able
to substitute for retinoic acid to initiate neural differentiation. Endogenous
SOX1 is
accordingly activated in P19 cells using an inducibie eukaryotic lac repressor-
operator expression system. To establish this system a clonal line of PI9
cells is
generated which constitutively and ubiquitously expresses the lac repressor.
This
2o parent line (P3'SS-10) is transfected with pRSVopSoxl, a vector containing
the
Soxl cDNA under the regulation of an inducible RSV promoter and stable lines
are
established. In the uninduced state, without the addition of isopropyl-~3-d-
thiogalactase (IPTG) these lines express high levels of the lac repressor that
binds Io
operon sites upstream of the RSV promoter and thus blocks transcription of
Soxl.
Upon addition of IPTG a conformational change occurs, decreasing the affinity
of
the repressor and resulting in the activation of pRSVopSoxl. Approximately 250
clones of transfectants are isolated in the repressed state. Using RNase
protection
and immunocytochemistry assays three clones are selected (708-13, 708-16 and
708-
21) that express high levels of RSVopSoxl in response to IPTG.
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39
The pluripotentiality of these clones is not compromised by the transfection
and
selection. All three lines express SSEAl in the uninduced state. Furthermore,
when aggregated in retinoic acid the uninduced clones initiate expression of
endogenous Soxl and differentiate into mature Neurofilament-expressing neurons
after plating, in a manner similar to wild-type P19 untransfected cells.
In order to address whether expression of SOX1 can initiate neural
differentiation
and thereby substitute for the requirement of retinoic acid, it is determined
whether
the transient exposure of P19 aggregates to retinoic acid can be replaced by a
transient induction of RSVopSoxl, through addition of IPTG. Wildtype P19 cells
and transfected P19 clones (708-13, 708-16 and 708-21) are cultured as
aggregates
for 96 hours with or without the addition of IPTG. After 96 hours RNA is
isolated
from half of the aggregates for RNase protection and/or RT-PCR assays. The
remaining aggregates are plated onto tissue culture substrate, allowed to
differentiate for three days without further addition of IPTG and then scored
for the
expression of a panel of neuroepithelial and neuronal markers by
immunocytochemistry. These conditions are the same as those used for retinoic
acid-induced differentiation of wildtype P19 cells. After 96 hours the clones
induced to express RSVopSoxl with IPTG express endogenous Soxl and Mashl.
2o The expression of these two neuroepithelial markers is similar to that seen
in
wildtype cells induced with retinoic acid. In addition the IPTG induced clones
expressed NESTIN and Hoxa7 (Mahon et al., (1988) Development (Suppl.) 187-
195). Further differentiation of the transiently-induced clones on substrate
showed
the presence of mature neurons as demonstrated by Neurofllament-positive, 3A10-
2s positive and Isletl-positive cells. All three clones 708-13, 708-16 and 708-
21
differentiate in this matter although the number of mature neurons produced is
variable. The number of differentiated neurons formed in the IPTG induced
clones
is estimated by determining the number of Neurofilament-positive cells in a
given
field of cells. The number of neurons ranges from 6-8% for clone 708-13, 15-
20%
3o for clone 708-16 and 20-25 % for clone 708-21. The latter two clones show
uniform
and ubiquitous induction of SOX1 expression whereas expression in clone 708-13
is
CA 02295305 1999-12-23
WO 99/00516 PCT/GB98/01862
not in all cells. In addition, the transiently induced clones generate GFAP-
positive
cells indicating glial cell differentiation. None of these markers is detected
in
wildtype P19 cells cultured in the presence of IPTG or in clones 708-13, 708-
16,
and 708-21 cultured in the absence of IPTG. The expression of SOX1, both in
vivo
5 and in vitro, is mutually exclusive with mature neuronal markers such as
Neurofilament and Isletl. To examine SOX1 expression in the mature neurons
generated in the transiently-induced clones, double-label immunoflourescence
is
used to simultaneously detect SOX1 and Neurofilament. No SOX1 expression
could be detected in cells positive for Neurofilament in these cultures.
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41
SEQUENCE LISTING
(1) GENERAL
INFORMATION:
(i) APPLICANT:
(A) NAME: MEDICAL RESEARCH COUNCIL
(B) STREET: 20 PARK CRESCENT
(C) CITY: LONDON
(E) COUNTRY: UK
(F) POSTAL CODE (ZIP): W1N 4AL
(ii) TITLE OF INVENTION: NEURONAL STEM CELL GENE
15(iii) NUMBER OF SEQUENCES: 6
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO)
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2312 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Gallus gallus
(xi) SEQUENCE DESCRIPTION: SEQ ID
NO: 1:
CCGCAGAGCG CGGCAGGACG GGCACACGCC GGGCGCAGCACCGCGAAGCA CCGCGCAGCC60
CCGCGCAGCC CCGCACCTGT TTGCGCGCCC CGCGCCCGGAGCGGCCCCCG GCAGCGGGAG120
GACGCCGGCA GCGCCGCCGC CGCCGCTCCT CGCATGTGCGGTGCCTCCCC GCCGCCCGGC180
GCCGGAGGGA AGTGAGGAAG CCCCGTGAAT GTACAGCATGATGATGGAGA CGGACTTGCA240
CTCGCCCGGC GGAGCCCCGG CGCCCGGCGG CGGCCTCTCGGGGCAGAGCG GCGCGGGCGG300
CGGCGGCGGC GGCGGCGGCG GCGGCGGGGG CAAAGCGGGGCAGGACCGCG TGAAGCGCCC360
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42
CATGAACGCCTTCATGGTGTGGTCGCGGGGGCAGCGGCGGAAGATGGCCCAGGAGAATCC 420
CAAGATGCACAACTCGGAGATCAGCAAGCGGCTGGGCGCCGAGTGGAAGGTGATGTCGGA 480
GGCCGAGAAGCGGCCTTTCATCGACGAGGCGAAGCGGCTGCGGGCGCTGCACATGAAGGA 540
GCACCCGGATTATAAATACCGGCCCCGGCGGAAGACCAAGACGCTGCTCAAGAAGGACAA 600
GTACTCGCTGGCCGGAGGGCTGCTGGGCGCCGGCCCGGCCGCGGGCGGCCCTCCCGCCGT 660
CGGCGTGGGCATGGGCGTCGGCGTGATCCCCGGCGGAGTCGGGCAGCGGCTGGAGAGCCC 720
CGGCGGGGCGGCGGGCGGCGGCTACGCGCACATGAACGGGTGGGCCAACGGCGCCTACCC 780
GGGCTCGGTGGCGGCGGCGGCGGCGGCGGCGGCGATGATGCAGGAGGCGCAGCTCGCCTA 840
CGGGCAGCACCCGGGCGGCGGGGGGCACCCGCACCACCCGCACCCGCACCACCCGCACCA 900
CCCGCACAACCCGCAGCCCATGCACCGCTACGACATGGGCGCGCTGCAGTACAGCCCCAT 960
CTCCAACTCGCAGGGCTACGTGAGCGCCTCGCCCTCGGGCTACGGCGCGCTGCCCTACGG 1020
CTCGCAGCCCCACCAGAACTCGGCGGCCGCGGCGGCGGCGGCGGCGGCGGCGGCGGCCGC 1080
CTCGTCGGGCGCGCTGGGCGCGCTGGGCTCGCTCGTCAAGTCGGAGCCCAGCGTGAGCCC 1140
GCCCGTCACCTCGCACTCGCGGGCCCCGTGCCCCGGGGACCTGCGGGAGATGATCAGTAT 1200
GTACTTACCGGGCGGCGAGGGAGGCGACCCGGCGGCCGCCGCCGCCCAGAGCCGCCTGCA 1260
CTCCCTGCCCCAGCACTACCAGAGCGCCAGCACGGGGGTCAACGGCACCGTCCCCTTGAC 1320
GCACATCTGAGCGGCCCCGGAGCGGCCCCGGAGCGGCGCGGAGGGCCCCGGCCCGGGCCC 1380
CGCAGGACTGCGGCCCCGCCGCCGCCCCGCGCCCGCCGCCCCCCTTCGTTTTTGCCTTTC 1440
ATTCGGCTCCTTCCCGCCCTCCCCCTCCCTCCTTCCTTTTTTTGTTTTGTTTTGTTTTGT 1500
TTTTCTTTTCTTCCTTTTTGTACAGAAATGTTTTGATGTTCTTGTAATAATAATAAATAA 1560
TAATAATAATAATAACGAGAGAGAGAGAAAAAGAAGGTAACGGTGGCTTTACTGACCTTT 1620
TTGTTTTTAGGAGGACCAGATCCCGGGACTAGTTTTAGACTGAACTTCTGTGTTTTATCG 1680
AGACTTTTTGTACAGTATTTATCATTCACCCCAGAGACACAGAGCGTTTATTTGCAAAAG 1740
AGGAGAGAGAGAGGATTAAAAAACAAAAA'_'AAACAAACAAACAAACAAAAAAAGACGGCG 1800
ACGAAAAGACAAAACCATCGCCGCTGACACCCAAAGTTCGGGCGGGGCCAACTTTCGGGC 1860
TGCGCTTCGCCCCGCACCGCCTCACTGCAAACGGAGCCGACGGGGAGCGGTGCTCGTTCC 1920
TTCCTCGCACACCCCAAAACAGCACCACGAGTTTCCGTAGATGTTCTCGCGCTTTTCCTT 1980
TTTGGTTGGGTTATTTCGGCTGCTTTATTTATACAACTTTTTCTTCTTCTTCCTTTCTTC 2040
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CCGAGGTTGC AACGTTTGCT TGATTTTTAT TTTATTTTAT GGGTTATGTG2100
TTTTTTTTCT
AAACTTTACT GTATCTGCAT CATTTCGGTT TGTTTTCCTC CCTTTTTTTT2160
CCCCCCCCCC
TTTTTTTACA TTTTTTTGTA TCATCTCGTG TAAATGCATT TTTTATCTAG2220
GTGAAATAAT
GCGTGGCGAG GGAACCCAGA CTGTACATAG TTTACTAAAA CTAAACAGAA2280
AGCCTTTCTG
ACCCGAAGGA TGCGTTCCAT TTTGAGTTAA AT 2312
LO
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2376 base pairs
15(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
20
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
25(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus
30 (xi) SEQUENCE
DESCRIPTION:
SEQ ID
NO: 2:
ACAGGAACGGAGACTTCGAGCCGAGAAGAGGAGGCAGCGAACCCTGCGTCGGGCCCAGGG 60
GCACCGCTTCAGACCCAGAAAGTGGAGCCTCAACTTGGCCACGACTGCACCTGTTTGCAC 120
35
AGTTCAGCCCTGAGTGACCGGACGGCAGCAATCAACCTGGCCATCGGCCTCTTTGGCAAG 180
TGGTTTGTGCACCGGGAGAAACTTTCCACCTGCGAGCTGGACCCGCGCTAAGTGCGTGTG 240
40 CTTTTGCCTCTTTTTTGTTGTTGTTGTTGTTGTGGCCTCCACCCAACCCCCTTCTCTCCG 300
CTAGGCACCCACCGCACACACACCCCCCCCCCCAGTCTCTCTGGGCTGATCCTCTCTCCA 360
CCCACCCACCCCCACCCGGCCGTCTATGCTCCAGGCCCTCTCTTTGCGGTACCGGTGAAC 420
45
CCGCTAGCCGCCCAGATGTACAGCATGATGATGGAGACCGACCTGCACTCGCCCGGCGGC 480
GCCCAGGCGCCCACGAACCTCTCGGGCCCGGCCGGGGCGCGCGGGGGCGGCGGTGGGGGC 540
50 GGGGGCGGCGGCGGCGGCGGGGGCACCAAGGCCAACCAGGATCGGGTCAAGCGGCCCATG 600
AACGCCTTCATGGTGTGGTCCCGCGGACAGCGGCGCAAGATGGCCCAGGAAAACCCCAAG 660
ATGCACAACTCGGAGATCAGCAAGCGCCTCGGGGCCGAGTGGAAGGTCATGTCCGAGGCC 720
55
GAGAAGCGGCCGTTCATCGACGAGGCCAAGAGACTGCGCGCGCTGCACATGAAGGAACAC 780
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44
CCGGATTACAAGTACCGGCC TGCTCAAGAA 840
GCGCCGCAAG GGACAAGTAC
ACCAAGACGC
TCGCTGGCCGGCGGGCTGCT GCGGCGCGGCCGTGGCCATG 900
AGCGGCCGGC
GCGGGTGGCG
GGTGTGGGCGTGGGCGTCGGGGCGGCGGCG GTGGGCCAGCGCTTGGAGAGCCCAGGCGGC 960
GCGGCGGGAGGAGGCTACGCGCATGTCAAC GGCTGGGCTAACGGCGCCTACCCCGGCTCG 1020
GTGGCCGCGGCGGCTGCGGCCGCGGCCATG ATGCAGGAGGCACAGCTGGCCTACGGGCAG 1080
CACCCAGGCGCGGGCGGCCGGCACCCGCAC GCACACCCGGCGCACCCGCACCCGCACCAT 1140
CCGCACGCGCATCCTCACAACCCGCAGCCC ATGCACCGCTACGACATGGGCGCGCTGCAG 1200
TACAGCCCCATCTCCAACTCTCAGGGCTAC ATGAGCGCGTCGCCTTCGGGCTACGGCGGC 1260
ATCCCTTACGGCGCCGCGGCCGCCGCCGCC GCCGCTGCGGGCGGCGCGCACCAGAACTCG 1320
GCGGTGGCGGCAGCGGCAGCCGCGGCAGCC GCGTCGTCGGGGGCCCTGGGCGCCCTCGGA 1380
TCTCTGGTCAAGTCGGAGCCGAGCGGCAGT CCGCCGGCCCCGGCTCACTCACGGGCACCG 1440
TGTCCCGGGGACCTGCGCGAGATGATCAGC ATGTACCTGCCGGCCGGCGAGGGTGGCGAC 1500
CCGGCGGCGGCAGCGGCTGCGGCGGCCCAA AGCCGGCTGCACTCGCTGCCACAGCACTAC 1560
CAGGGCGCGGGCGCGGGCGTCAACGGCACG GTGCCCCTGACGCACATCTAGCGCCGCGGG 1620
GACGCCGGGGACACTGCGGCTTAAGGCCGG CGCCCCGGCGACGAAGAGCGAGGCCTGCGC 1680
CCCAGCCTCCAGAGCCCGACTTTGTACCGA GGTCCCCGCGCTCTCGATAAAAGGCCGCTC 1740
TGGAGAGCCGAGCGCCAGGTGACATCTGCC CCCATCACCTTCCCCAGGACTCCGAGGCGC 1800
TGACACCAGACTGGCCTCTTAGACTGAACT TTGGTGTTTTCATGAGACCTTTTGTACAGT 1860
ATTTATCGTCCGCAGAGGAGGCACACAGCG TTTTCTCGGCTTCGGAGGACAAAAGACAAA 1920
AACCCAGCGAGGCGATGCCAACTTTTGTAT GACTGCCGGCTCTGTAACTTTTTCCGGGGT 1980
TTACTTCCCGCCAGCTCTTCTGCCTGAGGC CGAGTGACGGACCTCGAGCCCTTCTCACTT 2040
GTTATAAATCTAAGTAAGGCAGATCCAAAC ATTTACAAGTTTTTTGTAGTTGTTACCGCT 2100
CTTTTGGGTTGGTTGGTTAATTTATACCGC AATCCCCTCTCAGACGGTGGAGTTATATTC 2160
TGGGTTTTGT ATCCGAGCAT TTCCAAT-TTTTGTTTTGTTTTGTATTATT 2220
AAATCTCTGT
TCTTGTAAAT ATTTTTATTT TAGGCGTTGC 2280
GCGTTGTGAC GATACGGGGG GAAGAGGAGT
CGGATGTTGT 2340
ACATAGCCTG
CAAGTCTTTC
ATCTAAAAGC
AAAAACAAAG
AGAGATACCC
CCAAAATGCA 2376
TCAAATTTGA
ACAATACATT
TAAGAG
(2) INFORMATION
FOR SEQ
ID NO:
3:
CA 02295305 1999-12-23
WO 99/00516 PCT/GB98/01862
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1542
base pairs
(B) TYPE: nucleic
acid
5 (C) STRANDEDNESS:single
(D) TOPOLOGY:
linear
(ii)MOLECULE TYPE: to
cDNA mRNA
10 ( iii)HYPOTHETICAL:
NO
(iv)ANTI-SENSE: NO
(vi)ORIGINAL SOURCE:
15 (A) ORGANISM: Sapiens
Homo
(ix)FEATURE:
(A) NAME/KEY:
CDS
(B) LOCATION:60..1223
20
(xi)SEQUENCE DESCRIPTION: EQ D
S I NO:
3:
CCGGCCGT CT ATCTCCAGGC CGGTGCCGGT GAACCCGCCA CCGCCCCG 59
CCTCTCCTCG G
25
ATG TACAGC ATG ATG ATG ACCGAC CTGCACTCG CCCGGCGGCGCC 107
GAG
Met TyrSer Met Met Met ThrAsp LeuHisSer ProGlyGlyAla
Glu
1 5 10 15
30 CAG GCCCCC ACG AAC CTC GGCCCC GCCGGGGCG GGCGGCGGCGGG 155
TCG
Gln AlaPro Thr Asn Leu GlyPro AlaGlyAla GlyGlyGlyGly
Ser
20 25 30
GGC GGAGGC GGG GGC GGC GGCGGC GGGGGCGCC AAGGCCAACCAG 203
GGC
35 Gly GlyGly Gly Gly Gly GlyGly GlyGlyAla LysAlaAsnGln
Gly
35 40 45
GAC CGGGTC AAA CGG CCC AACGCC TTCATGGTG TGGTCCCGCGGG 251
ATG
Asp ArgVal Lys Arg Pro AsnAla PheMetVal TrpSerArgGly
Met
40 50 55 60
CAG CGGCGC AAG ATG GCC GAGAAC CCCAAGATG CACAACTCGGAG 299
CAG
Gln ArgArg Lys Met Ala GluAsn ProLysMet HisAsnSerGlu
Gln
65 70 75 80
45
ATC AGCAAG CGC CTG GGG GAGTGG AAGGTCATG TCCGAGGCCGAG 347
GCC
Ile SerLys Arg Leu Gly GluTrp LysValMet SerGluAlaGlu
Ala
85 90 95
AAG CGGCCG TTC ATC GAC GCCAAG CGGCTGCGC GCGCTGCACATG 395
GAG
Lys ArgPro Phe Ile Asp AlaLys ArgLeuArg AlaLeuHisMet
Glu
100 105 110
AAG GAGCAC CCG GAT TAC TACCGG CCGCGCCGC AAGACCAAGACG 443
AAG
Lys GluHis Pro Asp Tyr TyrArg ProArgArg LysThrLysThr
Lys
115 120 125
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46
CTG CTC AAG GACAAGTAC CTG GGC GGGCTCCTG GCGGCC 491
AAG TCG GCC
Leu Leu LysLys AspLysTyr SerLeu Gly GlyLeuLeu AlaAla
Ala
130 135 140
GGC GCG GGTGGC GGCGGCGCG GCTGTGGCCATG GGCGTGGGC GTGGGC 539
Gly Ala GlyGly GlyGlyAla AlaValAlaMet G1yValGly ValGly
145 150 155 160
10GTG GGC GCGGCG CCCGTGGGC CAGCGCCTGGAG AGCCCAGGC GGCGCG 587
Val Gly AlaAla ProValGly GlnArgLeuGlu SerProGly GlyAla
165 170 175
GCG GGC GGCGCG TACGCGCAC GTCAACGGCTGG GCCAACGGC GCCTAC 635
15Ala Gly GlyAla TyrAiaHis ValAsnGlyTrp AlaAsnGly AlaTyr
180 185 i90
CCC GGC TCGGTG GCGGCCGCG GCGGCCGCCGCG GCCATGATG CAGGAG 683
Pro Gly SerVal AlaAlaAla AlaAlaAlaAla AlaMetMet GlnGlu
20 195 200 205
GCG CAG CTGGCC TACGGGCAG CACCCCGGCGCG GGCGGCGCG CACCCG 731
Ala Gln LeuAla TyrGlyGln HisProGlyAla GlyGlyAla HisPro
210 215 220
25
CAC CGC ACCCCG GCGCACCCG CACCCGCACCAC CCGCACGCG CACCCG 779
His Arg ThrPro AlaHisPro HisProHisHis ProHisAla HisPro
225 230 235 240
30CAC AAC CCGCAG CCCATGCAC CGCTACGACATG GGCGCGCTG CAGTAC 827
His Asn ProGln ProMetHis ArgTyrAspMet GlyAlaLeu GlnTyr
245 250 255
AGC CCC ATCTCC AACTCGCAG GGCTACATGAGC GCGTCGCCC TCGGGC 875
35Ser Pro IleSer AsnSerGln GlyTyrMetSer AlaSerPro SerGly
260 265 270
TAC GGC GGCCTC CCCTACGGC GCCGCGGCCGCC GCCGCCGCC GCGCAC 923
Tyr Gly GlyLeu ProTyrGly AlaAlaAlaAla AlaAlaAla AlaHis
40 275 280 285
CAG AAC TCGGCC GTGGCGGCG GCGGCGGCGGCG GCGGCCGCG TCGTCG 971
Gln Asn SerAla ValAlaAla AlaAlaAlaAla AlaAlaAla SerSer
290 295 300
45
GGC GCC CTGGGC GCGCTGGGC TCTCTGGTGAAG TCGGAGCCC AGCGGC 1019
Gly Ala LeuGly AlaLeuGly SerLeuValLys SerGluPro SerGly
305 310 315 320
50AGC CCG CCCGCC CCAGCGCAC TCGCGGGCGCCG TGCCCCGGG GACCTG 1067
Ser Pro ProAla ProAlaHis SerArgAlaPro CysProGly AspLeu
325 330 335
CGC ATG AGCATG TTGCCCGCC GAGGGGGGC GACCCG 1115
GAG ATC TAC GGC
55Arg MetIle SerMetTyr LeuProAla GiuGly AspPro
Glu Gly Gly
340 345 350
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GCG GCG GCA GCA GCG GCC GCG GCG CAG AGC CGG CTG CAC TCG 1163
CTG CCG
Ala Ala Ala Ala Ala Ala Ala Ala Gln Ser Arg Leu His Ser
Leu Pro
355 360 365
CAG CAC TAC CAG GGC GCG GGC GCG GGC GTG AAC GGC ACG GTG 1211
CCC CTG
Gln His Tyr Gln Gly Ala Gly Ala Gly Val Asn Gly Thr Val
Pro Leu
370 375 380
ACG CAC ATC TAG CGCCTTCGGG ACGCCGGGGA CTCTGCGGCG GCGACCCACG1263
Thr His Ile
385
AGCTCGCGGC CCGCGCCCGG CTCCCGCCCC GCCCCGGCGC GGCGTGGCTT 1323
TTGTATCAGA
CGTTCCCACA TTCTTGTCAA AAGGAAAATA CTGGAGACGA ACGCCGGGTG 1383
ACGCGTGTCC
CCCACTCACC TTCCCCGGAG ACCCTGGCGA CCGCCGGGCG CTGACACCAG 1443
ACTTGGTTTA
GACTGAACTT CGGTGTTTTC TTGAGACTTT TGTACAGTAT TTATCACCTA 1503
CGGAGGAAGC
GGAAGCGTTT TCTTTGCTCG AGGGACAAAA AATGCAAAA 1542
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 388 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Met Tyr Ser Met Met Met Glu Thr Asp Leu His Ser Pro Gly
Gly Ala
1 5 10 15
Gln Ala Pro Thr Asn Leu Ser Gly Pro Ala Gly Ala Gly Gly
Gly Gly
20 25 30
_
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ala Lys Ala
Asn Gln
35 40 45
Asp Arg Val Lys Arg Pro Met Asn Ala Phe Met Val Trp Ser
Arg Gly
50 55 60
Gln Arg Arg Lys Met Ala Gln Glu Asn Pro Lys Met His Asn
Ser Glu
65 70 75 80
Ile Ser Lys Arg Leu Gly Ala Glu Trp Lys Val Met Ser Glu
Ala Glu
85 90 95
Lys Arg Pro Phe Ile Asp Glu Ala Lys Arg Leu Arg Ala Leu
His Met
100 105 110
Lys Glu His Pro Asp Tyr Lys Tyr Arg Pro Arg Arg Lys Thr
Lys Thr
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115 120 125
Leu Leu Lys Lys Asp Lys Tyr Ser Leu Ala Gly Gly Leu Leu Ala Ala
130 135 140
Gly Ala Gly Gly Gly Gly Ala Ala Val Ala Met Gly Val Gly Val Gly
145 150 155 160
Val Gly Ala Ala Pro Val Gly Gln Arg Leu Glu Ser Pro Gly Gly Ala
165 170 175
Ala Gly Gly Ala Tyr Ala His Val Asn Gly Trp Ala Asn Gly Ala Tyr
180 185 190
Pro Gly Ser Val Ala Ala Ala Ala Ala Ala Ala Ala Met Met Gln Glu
195 200 205
Ala Gln Leu Ala Tyr Gly Gln His Pro Gly Ala Gly Gly Ala His Pro
210 215 220
His Arg Thr Pro Ala His Pro His Pro His His Pro His Ala His Pro
225 230 235 240
His Asn Pro Gln Pro Met His Arg Tyr Asp Met Gly Ala Leu Gln Tyr
245 250 255
Ser Pro Ile Ser Asn Ser Gln Gly Tyr Met Ser Ala Ser Pro Ser Gly
260 265 270
Tyr Gly Gly Leu Pro Tyr Gly Ala Ala Ala Ala Ala Ala Ala Ala His
275 280 285
Gln Asn Ser Ala Val Ala Ala Ala Ala Ala Ala Ala Ala Ala Ser Ser
290 295 300
Gly Ala Leu Gly Ala Leu Gly Ser Leu Val Lys Ser Glu Pro Ser Gly
305 310 315 320
Ser Pro Pro Ala Pro Ala His Ser Arg Ala Pro Cys Pro Gly Asp Leu
325 330 335 -
Arg Glu Met Ile Ser Met Tyr Leu Pro Ala Gly Glu Gly Gly Asp Pro
340 345 350
Ala Ala Ala Ala Ala Ala Ala Ala Gln Ser Arg Leu His Ser Leu Pro
355 360 365
Gln His Tyr ~:ln Gly Ala Gly Ala Gly Val Asn Gly Thr Val Pro Leu
370 375 380
Thr His Ile
385
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
CA 02295305 1999-12-23
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49
(A) LENGTH: 1161 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: RNA
(iii) HYPOTHETICAL: YES
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(7..9, "agy")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(28..30, "cun")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(34..36, "agy")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(64..66, "cun")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(67..69, "agy")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(148..150, "cgn")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(157..159, "cgn")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(184..186, "agy")
( ix) FEATURE
(A) NAME/KEY: variation
(B) LOCATION:replace(187..189, "cgn")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(196..198, "cgn")
(ix)
FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(199..201, "cgn")
CA 02295305 1999-12-23
WO 99/00516 PCT/GB98/01862
{ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(235..237,"agy")
5 (ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(250..252,"cgn")
(ix) FEATURE:
10 (A) NAME/KEY: variation
(B) LOCATION:replace(244..246,"agy")
(ix) FEATURE:
(A) NAME/KEY: variation
15 (B) LOCATION:replace(253..255,"cun")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(277..279,"agy")
20
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(291..294,"cgn")
25 ( ix FEATURE
)
{A) NAME/KEY: variation
(B) LOCATION:replace(316..318,"cgn")
(ix) FEATURE:
30 (A) NAME/KEY: variation
(B) LOCATION:replace(319..321,"cun")
(ix) FEATURE:
(A) NAME/KEY: variation
35 (B) LOCATION:replace(322..324,"cgn")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(328..330,"cun")
40
( ix) FEATURE
(A) NAME/KEY: variation
(B) LOCATION:replace(361..363,"cgn")
45 (ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(367..369,"cgn")
(ix) FEATURE:
50 {A) NAME/KEY: variation
(B) LOCATION:replace(370..372,"cgn")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(385..387,"cun")
CA 02295305 1999-12-23
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51
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(388..390,"cun")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(406..408,"agy")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(409..411,"cun")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(421..423,"cun")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(424..426,"cun")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(505..507,"cgn")
( ix) FEATURE
(A) NAME/KEY: variation
(B) LOCATION:replace(508..510,"cun")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(513..515,"agy")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(583..585,"agy")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(631..632,"cun")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(676..678,"cgn")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(742..744,"cgn")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(760..762,"cun")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(769..771,"agy")
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(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(778..780,
"agy")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(784..786,
"agy")
(ix) FEATURE:
(A) NAME/KEY: variation
{B) LOCATION:replace(799..801,
"agy")
(ix) FEATURE:
(A) NAME/KEY: variation
{B) LOCATION:replace(805..807, "agy")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(811..813, "agy")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(836..838, "cun")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace{871..873, "agy")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(907..909, "agy")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(910..912, "agy")
(ix) FEATURE:
(A) NAME/KEY: variation
(H) LOCATION:replace(919..921, "cun")
_
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(928..930, "cun")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(934..936, "agy")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(937..939, "cun")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(946..948, "agy")
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(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(955..957, "agy")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(96i..963, "agy")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(982..984, "agy")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(985..987, "cgn")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(1006..1008, "cun")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(1009..1011, "cgn")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(1021..1023, "agy")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(1030..1032, "cun")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(1084..1086, "agy")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(1087..1089, "cgn")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(1090..1092, "cun")
( ix) FEATURE
(A) NAME/KEY: variation
(B) LOCATION:replace(1096..1098, "agy")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(1099..1101, "cun")
(ix) FEATURE:
(A) NAME/KEY: variation
(B) LOCATION:replace(1150..1152, "cun")
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54
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
AUGUAYUCNA UGAUGAUGGA RACNGAYUUR CAYUCNCCNG GNGGNGCNCA 60
RGCNCCNACN
AAYUURUCNG GNCCNGCNGG NGCNGGNGGN GGNGGNGGNG GNGGNGGNGG 120
NGGNGGNGGN
GGNGGNGGNG CNAARGCNAA YCARGAYAGR GUNAARAGRC CNAUGAAYGC 180
NUUYAUGGUN
UCGUCNAGRG GNCAR.AGRAG RAARAUGGCN CARGARAAYC CNAARAUGCA 240
YAAYUCNGAR
AUHUCNAAR.A GRUURGGNGC NGARUCGAAR GUNAUGUCNG ARGCNGARAA 300
RAGRCCNUUY
G CNAARAGRW RAGRGCNUUR CAYAUGAARG ARCAYCCNGA YUAYAARUAY 360
AUHGAYGAR
ZS
ACNAA RACNUURUUR AARAARGAYA ARUAYUCNUU RGCNGGNGGN 420
GRAAR
.
AGRCCNAGRA
UURUURGCNG CNGGNGCNGG NGGNGGNGGN GCNGCNGUNG CNAUGGGNGU 480
NGGNGUNGGN
GUNGGNGCNG CNCCNGUNGG NCARAGRUUR GARUCNCCNG GNGGNGCNGC 540
NGGNGGNGCN
UAYGCNCAYG UNAAYGGNUC GGCNAAYGGN GCNUAYCCNG GNUCNGUNGC 600
NGCNGCNGCN
GCNGCNGCNG CNAUGAUGCA RGARGCNCAR UURGCNUAYG GNCARCAYCC 660
NGGNGCNGGN
GGNGCNCAYC CNCAYAGRAC NCCNGCNCAY CCNCAYCCNC AYCAYCCNCA 720
YGCNCAYCCN
CAYAAYCCNC ARCCNAUGCA YAGRUAYGAY AUGGGNGCNU URCARUAYUC 780
NCCNAUHUCN
AAYUCNCARG GNUAYAUGUC NGCNUCNCCN UCNGGNUAYG GNGGNUURCC 840
NUAYGGNGCN
GCNGCNGCNG CNGCNGCNGC NCAYCARAAY UCNGCNGUNG CNGCNGCNGC 900
NGCNGCNGCN
GCNGCNUCNU CNGGNGCNUU RGGNGCNUUR GGNUCNUURG UNAARUCNGA 960
RCCNUCNGGN
UCNCCNCCNG CNCCNGCNCA YUCNAGRGCN CCNUGYCCNG GNGAYUURAG 1020
RGARAUGAUH
UCNAUGUAYU URCCNGCNGG NGARGGNGGN GAYCCNGCNG CNGCNGCNGC 1080
NGCNGCNGCN
CARUCNAGRU URCAYUCNUU RCCNCARCAY UAYCARGGNG CNGGNGCNGG -1140
NGUNAAYGGN
1161
ACNGUNCCNU URACNCAYAU H
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 base pairs
(g) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
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5 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
7
m WWCAAWG
