U.S. patent application number 09/836077 was filed with the patent office on 2002-03-28 for human semaphorin l (h-semal) and corresponding semaphorins in other species.
Invention is credited to Ensser, Armin, Fleckenstein, Bernhard.
Application Number | 20020037851 09/836077 |
Document ID | / |
Family ID | 26038116 |
Filed Date | 2002-03-28 |
United States Patent
Application |
20020037851 |
Kind Code |
A1 |
Fleckenstein, Bernhard ; et
al. |
March 28, 2002 |
Human semaphorin L (H-SemaL) and corresponding semaphorins in other
species
Abstract
Human semaphorin L (H-SemaL) and corresponding semaphorins in
other species. The invention relates to novel semaphorins which are
distinguished by a particular domain structure and derivatives
thereof, nucleic acids (DNA, RNA, cDNA) which code for these
semaphorins, and derivatives thereof, and the use thereof. The
present invention relates to semaphorins which have a novel, as yet
undisclosed and unexpected domain structure and which possess a
biochemical function in the immune system (immunomodulating
semaphorins). The novel semaphorins are referred to as type L
semaphorins (SemaL). They comprise an N-terminal signal peptide, a
characteristic Sema domain and, in the C-terminal region of the
protein, an immunoglobulin-like domain and a hydrophobic domain
which represents a potential transmembrane domain.
Inventors: |
Fleckenstein, Bernhard;
(Wiesenthau, DE) ; Ensser, Armin; (Nurnberg,
DE) |
Correspondence
Address: |
William F. Lawrence
FROMMER LAWRENCE & HAUG LLP
745 Fifth Avenue
New York
NY
10151
US
|
Family ID: |
26038116 |
Appl. No.: |
09/836077 |
Filed: |
April 16, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09836077 |
Apr 16, 2001 |
|
|
|
09112904 |
Jul 9, 1998 |
|
|
|
Current U.S.
Class: |
435/69.1 ;
435/325; 514/19.1; 514/19.6; 530/350; 536/23.5 |
Current CPC
Class: |
C07K 14/4703 20130101;
A61K 48/00 20130101; C07K 2319/02 20130101 |
Class at
Publication: |
514/12 ; 530/350;
536/23.5; 435/69.1; 435/325 |
International
Class: |
C07K 014/435; C07H
021/04; C12P 021/02; C12N 005/06; A61K 038/17 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 9, 1997 |
DE |
19729211.9 |
Feb 11, 1998 |
DE |
19805371.1 |
Claims
1. An isolated semaphorin protein comprising an amino acid sequence
having an N-terminal signal peptide, a Sema domain and, in a
C-terminal region, an immunoglobulin-like domain and a
transmembrane domain.
2. The isolated semaphorin protein as claimed in claim 1, wherein
the amino acid sequence corresponds to SEQ ID NO.: 3, or a
derivative of SEQ ID NO.: 3.
3. The isolated semaphorin protein as claimed in claim 1, wherein
the amino acid sequence of the Sema domain of the semaphorin
protein is at least about 40% homologous to the Sema domain of SEQ
ID NO.: 3.
4. The isolated semaphorin protein as claimed in claim 1, wherein
the amino acid sequence of the protein is at least about 15 to 20%
homologous to SEQ ID NO.: 3.
5. The isolated semaphorin protein as claimed in claim 1,
comprising an amino acid sequence corresponding to SEQ ID NO.:
4.
6. An isolated nucleic acid molecule encoding a semaphorin protein
as claimed in claim 1.
7. The isolated nucleic acid molecule as claimed in claim 6,
comprising a nucleic acid sequence corresponding to SEQ ID NO.: 41,
or a derivative of SEQ ID NO.: 41.
8. The isolated nucleic acid molecule as claimed in claim 6 wherein
the nucleic acid molecule is a cDNA sequence coding for the
semaphorin protein.
9. The isolated nucleic acid molecule as claimed in claim 8,
wherein the cDNA sequence corresponds to SEQ ID NO.: 1, or to a
derivative of SEQ ID NO.: 1.
10. The isolated nucleic acid molecule as claimed in claim 8,
wherein the cDNA sequence corresponds to SEQ ID NO.: 2, or to a
derivative of SEQ ID NO.: 2.
11. The isolated semaphorin protein as claimed in claim 1, wherein
the protein is phosphorylated, glycosylated or myristylated.
12. A plasmid comprising the nucleic acid sequence as claimed in
claim 6.
13. A plasmid comprising a nucleic acid sequence corresponding to
SEQ ID NO.: 41, or a derivative of SEQ ID NO.: 41.
14. A plasmid comprising the nucelic acid sequence as claimed in
claim 8.
15. A vector comprising the nucleic acid sequence as claimed in
claim 6.
16. A vector comprising the nucleic acid sequence as claimed in
claim 8.
17. A process for preparing a semaphorin protein as claimed in
claim 1, which comprises the steps of: cloning a nucleic acid
sequence as claimed in claim 6 into an expression vector to form a
recombinant vector; transforming a cell with the recombinant
vector; and expressing the protein from the transformed cell.
18. The process as claimed in claim 17, wherein the transformed
cell is a eukcaryotic cell.
19. A process for preparing a nucleic acid molecule encoding a
semaphorin protein as claimed in claim 1, comprising amplifying the
nucleic acid sequence corresponding to SEQ ID NO.: 41, or a
derivative thereof, by the polymerase chain reaction using specific
primers.
20. A method for identifying immunomodulating agents, which
comprises incubating a semaphorin protein as claimed in claim 1
under defined conditions with an agent to be investigated, carrying
out a second batch in parallel without without the agent to be
investigated but under conditions which are otherwise the same, and
then determining the inhibiting or activating effect of the agent
to be investigated.
21. A method for identifying immunomodulating agents, which
comprises expressing a nucleic acid sequence as claimed in claim 6
under defined conditions and in the presence of an agent to be
investigated, and determining the extent of the expression.
22. An isolated semaphorin antibody which recognizes an epitope
corresponding to amino acids 179 to 378 of SEQ ID NO.: 3 or amino
acids 480 to 666 of SEQ ID NO.: 3.
23. A process for preparing an antibody to the semaphorin protein
as claimed in claim 1, which comprises the steps of: preparing a
recombinant plasmid with a fusion protein consisting of of a
semaphorin epitope and an epitope tag; transforming a suitable cell
with the recombinant plasmid; purifying the fusion protein from the
cells via the epitope tag; and using the purified fusion proteins
for immunization.
24. A method for preventing or treating an immunological disorder
which comprises administering to a host in need thereof a
pharmaceutical product containing an isolated semaphorin protein or
a derivative thereof, as claimed in claim 1.
25. A method for preventing or treating an immunological disorder
which comprises administering to a host in need thereof a
pharmaceutical product containing a nucleic acid sequence as
claimed in claim 6 or a derivative thereof.
26. A method for preventing or treating an immunological disorder
which comprises administering to a host in need thereof a
pharamceutical product containing a nucleic acid sequence as
claimed in claim 8.
27. The method according to claim 24 wherein the method is gene
therapy.
28. The method according to claim 25 wherein the method is gene
therapy.
29. A method for modulating an immune response or inhibiting
inflammation which comprises introducing the nucleic acid sequence
as claimed in claim 6 to a host cell.
Description
RELATED APPLICATIONS
[0001] This application claims priority to German Application Nos.
19729211.9 and 19805371.1, filed Jul. 9, 1997 and Feb. 11, 1998
respectively, each incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention relates to novel semaphorins which are
distinguished by a particular domain structure and derivatives
thereof, nucleic acids (DNA, RNA, cDNA) which code for these
semaphorins, and derivatives thereof, and the preparation and use
thereof.
[0004] 2. Description of the Related Art
[0005] The publications which are referenced in this application
describe the state of the art to which this invention pertains.
These references are incorporated herein by references.
[0006] Semaphorins were described for the first time by Kolodkin
{Kolodkin et al. (1993) Cell 75:1389-1399} as members of a
conserved gene family.
[0007] The genes or parts of the genes of other semaphorins have
now been cloned and, in some cases, characterized. To date, a total
of 5 human (H-Sema III, H-Sema V, H-Sema IV, H-SemaB and H-SemaE)
{Kolodkin et al. (1993); Roche et al. (1996) Onkogene 12:1289-1297;
Sekido et al. (1996) Proc. Natl. Acad. Sci. USA 93:41204125; Xiang
et al. (1996) Genomics 32:39-48; Hall et al. (1996) Proc. Natl.
Acad. Sci. USA 39:11780-11785; Yamada et al. (1997) (GenBank
Accession No. AB000220)}, 8 murine (mouse genes; M-Sema A to
M-Sema-H) {Puschel et al. (1995) Neuron 14:941-948; Messerschmidt
et al. (1995) Neuron 14:949-959; Inigaki et al. (1995) FEBS Letters
370:269-272; Adams et al. (1996) Mech. Dev. 57:33-45; Christensen
et al. (1996) (GenBank Accession No. Z80941, Z93948)}, 5 galline
(chicken) (collapsin-1 to -5) {Luo et al. (1993); Luo et al. (1995)
Neuron 14:1131-1140), and genes from rats (R-Sema-III) {Giger et
al. (1996) J. Comp. Neurol. 375:378-392}, zebra fish, insects
(fruit fly (Drosophila melanogaster: D-Sema I and D-Sema II),
beetles (Tribolium confusum: T-Sema-I), grasshoppers (Schistocerca
americana: G-Sema-I)) {Kolodkin et al. (1993)}, and nematodes
(C.elegans: Ce-Sema) {Roy et al. (1994) (GenBank Accession No.
U15667)} have been disclosed. In addition, two poxviruses (vaccinia
(ORF-A39) and variola (ORFA39-homologous)) {Kolodkin et al. (1993)}
and alcelaphine herpesvirus Type 1 (AHV-1) (AHV-Sema) {Ensser and
Fleckenstein (1995) Gen. Virol. 76:1063-1067} have genes homologous
to semaphorins.
[0008] Table 1 summarizes the semaphorins identified to date in
various species. Table 1 indicates the names of the semaphorins
(column 1), the synonyms used (column 2), the species from which
the particular semaphorin has been isolated (column 3) and, where
known, data on the domain structure of the encoded protein and on
the chromosomal location (column 4 in Table 1), the accession
number under which the sequence of the gene is stored in gene
databanks (for example in an EST (expressed sequence tags)
databank, EMBL (European Molecular Biology Laboratory, Heidelberg)
or NCBI (National Center for Biotechnology Information, Maryland,
USA), and the corresponding reference under which these data have
been published (column 5 in Table 1).
[0009] All the gene products (encoded semaphorins) of the
semaphorin genes disclosed to date have an N-terminal signal
peptide which has at its C-terminal end a characteristic Sema
domain with a length of about 450 to 500 amino acids. Highly
conserved amino acid motifs and a number of highly conserved
cysteine residues are located within the Sema domains. The gene
products (semaphorins) differ in the C-terminal sequences which
follow the Sema domains and are composed of one or more domains.
They have, for example, in these C-terminal amino acid sequences
transmembrane domains (TM), immunoglobulin-like domains (Ig)
(constant part of the immunoglobulin), cytoplasmic sequences (CP),
processing signals (P) (for example having the consensus sequence
(RXR) where R is the amino acid arginine and X is any amino acid)
and/or hydrophilic C termini (HPC). The semaphorins disclosed to
date can be divided on the basis of the differences in the domain
structure in the C terminus into 5 different subgroups (I to
V):
[0010] I Secreted, without other domains (for example ORF-A49)
[0011] II Ig Secreted (without transmembrane domain) for example
AHV-Sema)
[0012] III Ig, TM, CP Membrane-anchored with cytoplasmic sequence
(for example CD100)
[0013] IV Ig, (P), HPC Secreted with hydrophilic C terminus (for
example H-Sema III, M-SemaD, collapsin-1)
[0014] V Ig, TM, CP Membrane-anchored with C-terminal 7
thrombospondin motif (for example M-SemaF and G)
[0015] A receptor or extracellular ligand for semaphorins has not
been described to date. Intracellular, heterotrimeric GTP-binding
protein complexes have been described in connection with
semaphorin-mediated effects. One component of these protein
complexes which has been identified in chickens is called CRMP
(collapsin response mediator protein) and is presumed to be a
component of the semaphorin-induced intracellular signal cascade
(Goshima et al. (1995) Nature 376: 509-514). CRMP62, for example,
has homology with unc-33, a nematode protein which is essential for
directed growth of axons. A human protein with 98% amino acid
identity with CRMP62 is likewise known (Hamajima et al. (1996) Gene
180: 157-163). Several CRMP-related genes have likewise been
described in rats (Wang et al. (1996) Neurosci. 16: 6197-6207).
[0016] The secreted or transmembrane semaphorins convey repulsive
signals for growing nerve buds. They play a part in the development
of the central nervous system (CNS) and are expressed in particular
in muscle and nerve tissues (Kolodkin et al. (1993); Luo et al.
(1993) Cell 75:217-227).
[0017] Pronounced expression of M-SemaG has been observed not only
in the CNS but also in cells of the lymphatic and hematopoietic
systems, in contrast to the closely related M-SemaF {Furuyima et
al. (1996) J. Biol. Chem. 271: 33376-33381}.
[0018] Recently, two other human semaphorins have been identified,
H-Sema IV and H-Sema V, specifically in a region on chromosome
3p21.3, whose deletion is associated with various types of
bronchial carcinomas. H-Sema IV {Roche et al. (1996), Xiang et al.
(1996), Sekido et al. (1996)} is about 50% identical at the amino
acid level with M-SemaE, whereas H-Sema V {Sekido et al. (1996)} is
the direct homolog of M-SemaA (86% amino acid identity). Since
these genes (H-Sema IV and V) were found during DNA sequencing
projects on the deleted 3p21.3 loci, the complex intron-exon
structure of these two genes is known. Both genes are expressed in
various neuronal and non-neuronal tissues.
[0019] Likewise only recently, the cellular surface molecule CD100
(human), expressed and induced on activated T cells, has been
identified as a semaphorin (likewise listed in Table 1). It assists
interaction with B cells via the CD40 receptor and the
corresponding ligand CD40L. CD100 is a membrane-anchored
glycoprotein dimer of 150 kd (kilodaltons). An association of the
intracytoplasmic C-terminus of CD100 with an as yet unknown kinase
has been described {Hall et al. (1996)}. This means that CD100 is
the first and to date only semaphorin whose expression in cells of
the immune system has been demonstrated.
[0020] In the "transforming genes of rhadinoviruses" project, the
complete genome of alcelaphine herpesvirus Type 1 (AHV-1) has been
cloned and sequenced {Ensser et al. (1995)}. AHV-1 is the causative
agent of malignant catarrhal fever, a disease of various ruminants
which is associated with a lymphoproliferative syndrome and is
usually fatal. On analysis, an open reading frame was found, at one
end of the viral genome, having remote but significant homology
with a gene of vaccinia-virus (ORF-A39 corresponds to VAC-A39 in
Ensser et al. (1995) J. Gen. Virol. 76:1063-1067) which has been
assigned to the semaphorin gene family. Whereas the AHV-1
semaphorin (AHV-Sema) has a well-conserved semaphorin structure,
the poxvirus genes (ORF-A39 and ORF-A39-homologous, see Table 1)
have C-terminal truncations, i.e. the conserved Sema domain is
present in them only incompletely.
[0021] Databank comparison of the found AHV-Sema with dbEST (EST
(expressed sequence tags) databank (db)) provided in each case 2
EST sequences from 2 independent cDNA clones from human placenta
(accession numbers H02902, H03806 (clone 151129), accession numbers
R33439 and R33537 (clone 135941)). These display distinctly greater
homology with AHV-1 semaphorin than with the neuronal semaphorins
hitherto described.
SUMMARY OF THE INVENTION
[0022] The present invention relates to semaphorins which have a
novel, as yet undisclosed and unexpected domain structure and which
possess a biochemical function in the immune system
(immunomodulating semaphorins). The novel semaphorins are referred
to as type L semaphorins (SemaL). They comprise an N-terminal
signal peptide, a characteristic Sema domain and, in the C-terminal
region of the protein, an immunoglobulin-like domain and a
hydrophobic domain which represents a potential transmembrane
domain.
[0023] The amino acid sequence of the signal peptide may have fewer
than 70, preferably fewer than 60 amino acids and more than 20,
preferably more than 30 amino acids, and a particularly preferred
length is of about 40 to 50 amino acids. In a specific embodiment
of the invention, the signal peptide has a length of 44 amino
acids, i.e. a cleavage site for a signal peptidase is located
between amino acids 44 and 45.
[0024] The Sema domain may have a length of from 300 to 700 or
more, preferably of about 400 to 600, amino acids. Preferred Sema
domains have a length of 450 to 550 amino acids, preferably of
about 500 amino acids. In a preferred embodiment of the invention,
the Sema domain is joined to the signal peptide, in which case the
Sema domain preferably extends up to amino acid 545.
[0025] The immunoglobulin-like domain may have a length of about 30
to 110 or more amino acids, and preferred lengths are between 50
and 90, particularly preferably about 70, amino acids.
[0026] The transmembrane domain may have a length of about 10 to
35, preferably of about 15 to 30, particularly preferably of about
20 to 25, amino acids.
[0027] The invention relates to type L semaphorins from various
species, in particular from vertebrates, for example from birds
and/or fishes, preferably from mammals, for example from primates,
rat, rabbit, dog, cat, sheep, goat, cow, horse, pig, particularly
preferably from human and mouse. The invention also relates to
corresponding semaphorins from microorganisms, especially from
pathogenic microorganisms, for example from bacteria, yeasts and/or
viruses, for example from retroviruses, especially from
human-pathogenic microorganisms.
BRIEF DECEPTION OF THE DRAWING
[0028] The invention will be described in greater detail with the
aid of the following figures:
[0029] FIG. 1 is a Multiple tissue Northern blot for the
tissue-specific expression of H-SemaL.
[0030] FIG. 2 is a diagrammic representation of the cloning of the
H-SemaL cDNA and of the genomic organization of the H-SemaL
encoding sequence.
[0031] FIG. 3 is a phylogenetic tree.
[0032] FIG. 4 is a FACS analysis of H-SEMAL expression in various
cell lines.
[0033] FIG. 5 is a comparative analysis of CD 100 and H-SemaL
expression.
[0034] FIG. 6 is the expression of secretable human SEMA-L
(H-SemaL) in HiFive and SC3 cells.
[0035] FIG. 7 depicts the specificity of the antiserum.
[0036] FIG. 8 is a plasmid map of pMelBacA-H-SEMAL.
DETAILED DESCRIPTION OF THE INVENTION
[0037] One embodiment of the invention is a corresponding human
semaphorin (H-SemaL) which has a signal peptide, a Sema domain, an
immunoglobulin-like domain and a transmembrane domain. A specific
embodiment is the semaphorin which is given by the amino acid
sequence shown in Table 4.
[0038] Another embodiment of the invention comprises corresponding
semaphorins in other species which have, in the region of the Sema
domain, an amino acid identity greater than 40%, preferably greater
than 50%, particularly preferably greater than 60%, in relation to
the Sema domain of H-SemaL (amino acids 45 to 545 of the sequence
in Table 4). The corresponding semaphorins from closely related
species (for example primates, mouse) may perfectly well have amino
acid identities of greater than 70%, preferably greater than 80%,
particularly preferably greater than 90%. Percentage homologies can
be determined or calculated for example using the GAP program (GCG
program package, Genetic Computer Group (1991)).
[0039] Such an embodiment of the invention is a corresponding mouse
semaphorin (murine semaphorin (M-SemaL)). This contains, for
example, the partial amino acid sequence shown in Table 5 (murine
semaphorin (M-SemaL)).
[0040] The invention also relates to corresponding semaphorins
which have an amino acid identity (considered over the entire
length of the amino acid sequence of the protein) of only about 15
to 20% in the case of less related species (very remote from one
another phylogenetically), preferably 25 to 30%, particularly
preferably 35 to 40%, or a higher identity in relation to the
complete amino acid sequence of H-SemaL shown in Table 4.
[0041] The genes which code for type L semaphorins have a complex
exon-intron structure. These genes may have, for example, between
10 and 20 exons, preferably about 11 to 18, particularly preferably
12 to 16, exons and a corresponding number of introns. However,
they may also have the same number of exons and introns as does the
gene of H-SemaL (13 or 15 exons, preferably 14 exons). A particular
embodiment of the invention relates to the gene of H-SemaL. This
gene preferably has a length of 8888 to 10,000 or more nucleotides.
The human semaphorin gene preferably contains the nucleotide
sequence given in Table 14 or the nucleotide sequence which has
been deposited at the GenBank.RTM. databank under accession number
AF030697. These nucleotide sequences contain at least 13 introns.
In addition, the human semaphorin gene has at the 5' end an
additional sequence region. This region contains, where
appropriate, further coding and uncoding sequences, for example one
or two further introns or exons.
[0042] Attempts to locate the human type L semaphorin on the
chromosome revealed that the corresponding gene is located at
position 15q22.3-23. The gene for M-SemaL has correspondingly been
located at position 9A3.3-B.
[0043] As a consequence of the complex intron-exon structure, the
splicing of the primary transcript of the semaphorin mRNA may vary,
resulting in different splicing variants of the semaphorins. The
proteins translated from these splicing variants are derivatives of
the semaphorins according to the invention. They correspond in
their amino acid sequence and also substantially in their domain
structure to the described type L semaphorins according to the
invention, but are truncated by comparison with the latter where
appropriate. For example, splicing variants wholly or partly
lacking the transmembrane domain may be formed. A semaphorin
derivative which contains an incomplete, or no, transmembrane
domain, but contains a signal peptide, may be secreted and in this
way have effects outside the cell, locally or else over relatively
large distances, for example on other cells. Another splicing
variant may, for example, no longer contain a sequence which codes
for a signal peptide and, where appropriate, also no sequence which
codes for a hydrophobic amino acid sequence representing a
potential transmembrane domain. One consequence would be that this
semaphorin derivative is neither incorporated into the membrane nor
secreted (unless through secretory vesicles). Such a semaphorin
derivative may be involved in intracellular processes, for example
in signal transduction processes. It is possible in this way for a
wide variety of intra- and extracellular processes to be controlled
and/or harmonized with the same basic molecule (type L semaphorins)
and the derivatives derived therefrom (for example splicing
variants).
[0044] A particular embodiment of the invention relates to
semaphorin derivatives which are derived from the type L
semaphorins according to the invention but which contain an
incomplete, or no, transmembrane domain.
[0045] Another embodiment of the invention relates to semaphorin
derivatives which are derived from the type L semaphorins according
to the invention but which contain no signal peptide.
[0046] The signal peptide may also undergo post-translational
elimination. This forms a membrane-bound (with TM domain) or a
secreted (splicing variant without TM domain) semaphorin derivative
with truncated domain structure. A semaphorin derivative which has
undergone post-translational processing in this way now contains
only Sema domain, Ig domain and, where appropriate, transmembrane
domain. A signal peptide cleavage site can be located, for example,
right at the end of the signal peptide, but it may, for example, be
located 40 to 50 amino acids or more away from the amino
terminus.
[0047] A "truncated" (i.e. containing fewer domains) semaphorin L
derivative can be distinguished from other semaphorins which are
not derived from type L semaphorins in that there is a very great
(>90%) amino acid identity or an identical amino acid sequence
with the type L semaphorins in the domains which are present.
[0048] The semaphorins according to the invention may also have
undergone post-translational modification in other ways. For
example, they may be glycosylated (N- and/or O-glycosylated) once,
twice, three, four, five, six, seven, eight, nine, ten or more
times. The amino acid sequences of the semaphorins may then have an
equal number of or more consensus sequences for potential
glycosylation sites, preferably five such sites. One embodiment of
the invention relates to semaphorins in which the glycosylation
sites are located at positions which correspond to positions 105,
157, 258, 330 and 602 of the H-SemaL amino acid sequence (Table
4).
[0049] In addition, the semaphorins may be in the form of their
phosphorylated derivatives. Semaphorins may be the substrates of
various kinases, for example the amino acid sequences may have
consensus sequences for protein kinase C, tyrosine kinase and/or
creatine kinases. In addition, the amino acid sequences of the
semaphorins may have consensus sequences for potential
myristylation sites. Corresponding semaphorin derivatives may be
esterified with myristic acid at these sites.
[0050] The type L semaphorins according to the invention and their
derivatives may be in the form of monomers, dimers and/or
multimers, for example two or more semaphorins or their derivatives
can be linked together by intermolecular disulfide bridges. It is
also possible for intramolecular disulfide bridges to be
formed.
[0051] Further derivatives of the semaphorins according to the
invention are fusion proteins. A fusion protein of this type
contains, on the one hand, a type L semaphorin or parts thereof
and, in addition, another peptide or protein or a part thereof.
Peptides or proteins or parts thereof may be, for example, epitope
tags (for example His tag (6.times.histidine), Myc tag, flu tag)
which can be used, for example, for purifying the fusion proteins,
or those which can be used for labeling the fusion proteins, for
example GFP (green fluorescent protein). Examples of derivatives of
the type L semaphorins are given for example by the constructs
described in the examples. The sequences of these constructs can be
found in Tables 7 to 15, where appropriate taking account of the
annotations relating to the plasmids.
[0052] The invention further relates to nucleic acid sequences,
preferably DNA and RNA sequences, which code for the type L
semaphorins according to the invention and/or their derivatives,
for example the corresponding genes, the various splicing variants
of the mRNA, the cDNAs corresponding thereto, and derivatives
thereof, for example salts of the DNA or RNA. Derivatives for the
purpose of the inventions are sequences or parts thereof which have
been modified, for example, by methods of molecular biology and
adapted to the particular requirements, for example truncated genes
or parts of genes (for example promoter sequences, terminator
sequences), cDNAs or chimeras thereof, constructs for expression
and cloning and salts thereof.
[0053] One embodiment relates to the genomic sequences (genes) of
the type L semaphorins. The invention relates to the intron and
exon sequences and gene-regulatory sequences, for example promoter,
enhancer and silencer sequences.
[0054] This embodiment relates on the one hand to the gene of
H-SemaL or its derivatives. The invention relates on the one hand
to a gene which comprises the nucleotide sequence given in Table
14. The invention further relates to the gene which comprises the
nucleotide sequence which is deposited in the GenBank.RTM. databank
under accession number AF030697.
[0055] This embodiment further relates to the gene of M-SemaL and
its derivatives.
[0056] The invention further relates to the cDNA of H-SemaL or its
derivatives (for example parts of the cDNA). A particular
embodiment is the cDNA of H-SemaL according to the nucleotide
sequence in Table 2. The invention further relates to the cDNA of
H-SemaL which is deposited in the GenBank.RTM. databank under
accession number AF030698. The invention also relates to the mRNAs
corresponding to these cDNAs, or parts thereof.
[0057] The invention further relates to the cDNA of M-SemaL or its
derivatives (for example parts of the cDNA). A particular
embodiment is the partial cDNA sequence of M-SemaL shown in Table
3, and cDNA sequences which comprise this partial cDNA sequence.
Another embodiment of the invention relates to the cDNA of M-SemaL
which is deposited in the GenBank databank under accession number
AF030699. The invention also relates to the mRNAs corresponding to
these cDNAs, or parts thereof.
[0058] The invention also comprises alleles and/or individual
expression forms of the genes/mRNAs/cDNAs which differ only
slightly from the semaphorin sequences described herein and code
for an identical or only slightly modified protein (difference in
the amino acid sequence less than or equal to 10%) (further example
of derivatives). Further examples of the derivatives are given by
the constructs indicated in the examples. The sequences of these
constructs are depicted in Tables 7 to 14 and can be interpreted
taking account of the annotation for plasmids.
[0059] The invention further relates to plasmids which comprise DNA
which codes for the type L semaphorins or derivatives thereof.
Plasmids of this type may be, for example, plasmids with high
replication rates suitable for amplification of the DNA, for
example in E. coli.
[0060] A specific embodiment comprises expression plasmids with
which the semaphorins or parts thereof or their derivatives can be
expressed in prokaryotic and/or eukaryotic expression systems. Both
constitutive expression plasmids and those containing inducible
promoters are suitable.
[0061] The invention also relates to processes for preparing
nucleic acids which code for type L semaphorins or derivatives
thereof.
[0062] These nucleic acids, for example DNA or RNA, can be
synthesized, for example, by chemical means. In particular, it is
possible for these nucleic acids, for example the corresponding
genes or cDNAs or parts thereof, to be amplified by PCR using
specific primers and suitable starting material as template. (For
example cDNA from a suitable tissue or genomic DNA).
[0063] A specific process for preparing semaphorin L cDNA and the
H-SemaL gene is described in the examples.
[0064] The invention also relates to processes for preparing type L
semaphorins. For example, a semaphorin L or a derivative thereof
can be prepared by cloning a corresponding nucleic acid sequence
which codes for a type L semaphorin or a derivative thereof into an
expression vector and using the latter recombinant vector to
transform a suitable cell. It is possible to use, for example,
prokaryotic or eukaryotic cells. The type L semaphorins or
derivatives thereof may also, where appropriate, be prepared by
chemical means.
[0065] In addition, the type L semaphorins and derivatives thereof
can be expressed as fusion proteins, for example with proteins or
peptides which permit detection of the expressed fusion protein,
for example as fusion protein with GFP (green fluorescent protein).
The semaphorins may also be expressed as fusion proteins with one,
two, three or more epitope tags, for example with Myc and/or His
(6.times.histidine) and/or flu tags. It is correspondingly possible
to use or prepare plasmids which comprise DNA sequences which code
for these fusion proteins. For example, semaphorin-encoding
sequences can be cloned into plasmids which contain DNA sequences
which code for GFP and/or epitope tags, for example Myc tag, His
tag, flu tag. Specific examples thereof are given by the examples
and the sequences listed in the tables, where appropriate with the
assistance of the annotation relating to the plasmids.
[0066] The invention further relates to antibodies which
specifically bind or recognize the type L semaphorins, derivatives
thereof or parts thereof. Possible examples thereof are polyclonal
or monoclonal antibodies which can be produced, for example, in
mouse, rabbit, goat, sheep, chicken etc.
[0067] A particular embodiment of this subject-matter of the
invention comprises antibodies directed against the epitopes which
correspond to the amino acid sequences from position 179 to 378 or
480 to 666 of the H-SemaL sequence shown in Table 4. The invention
also relates to a process for preparing specific anti-semaphorin L
antibodies, using for the preparation antigens comprising said
epitopes.
[0068] The invention also relates to processes for preparing the
antibodies, preferably using for this purpose a fusion protein
consisting of a characteristic semaphorin epitope and an epitope
tag which can be used for the subsequent purification of the
recombinant fusion protein. The purified fusion protein can
subsequently be used for the immunization. To prepare the
recombinant fusion protein, a corresponding recombinant expression
vector is prepared and used to transform a suitable cell. The
recombinant fusion protein can be isolated from this cell. The
procedure can be, for example, like that described in Example
8.
[0069] These antibodies can be used, for example, for purifying the
corresponding semaphorins, for example H-SemaL and its derivatives,
for example on affinity columns, or for the immunological detection
of the proteins, for example in an ELISA, in a Western blot and/or
in immunohistochemistry. The antibodies can also be used to analyze
the expression of H-SemaL, for example in various cell types or
cell lines.
[0070] The cDNA of H-SemaL has a length of 2636 nucleotides (Table
2). The gene product of the H-SemaL cDNA has a length of about 666
amino acids (Table 4) and displays the typical domain structure of
a type L semaphorin. The gene product has an N-terminal signal
peptide (amino acids 1 to 44), Sema domain (amino acid 45 to
approximately amino acid 545), and Ig (immunoglobulin) domain
(approximately amino acids 550 to 620) and, at the C-terminal end,
a hydrophobic amino acid sequence which represents a potential
transmembrane domain. This domain structure has never previously
been described for semaphorins. It relates to a membrane-associated
glycoprotein which is probably located on the cell surface and
belongs to a new subgroup. On the basis of this previously unknown
domain structure, the semaphorins can now be divided into VI
subgroups:
[0071] I Secreted, without other domains (for example ORF-A49)
[0072] II Ig Secreted (without transmembrane domain) (for example
AHV-Sema)
[0073] III Ig, TM, CP Membrane-anchored with cytoplasmic sequence
(for example CD100)
[0074] IV Ig, (P), HPC Secreted with hydrophilic C terminus (for
example H-Sema-III, M-SemaD, collapsin-1)
[0075] V Ig, TM, CP Membrane-anchored with C-terminal 7
thrombospondin motif (for example M-SemaF and G)
[0076] VI Ig, TM Membrane-anchored (for example H-SemaL,
M-SemaL)
[0077] The unglycosylated, unprocessed form of H-SemaL has a
calculated molecular weight of about 74.8 kd (74823 dalton)
(calculated using Peptide-Sort, GCG program package). The
isoelectric point is calculated to be pH=7.56.
[0078] A possible signal peptide cleavage site is located between
amino acids 44 and 45 (Table 3; calculated with SignalP
(http.//www.cbs.dtu.dk/- services/Signal P), a program based on
neural networks for analyzing signal sequences {Nielsen H. et. al.
(1997) Protein Engineering 10:1-6}). This gives for the processed
protein (without signal peptide) a molecular weight (MW) of 70.3 kd
(70323 dalton) and an isoelectric point of pH=7.01.
[0079] The genomic structure is likewise substantially elucidated.
The H-SemaL gene has 13 or 15 or more exons, preferably 14 exons,
and 12 or 14 introns, preferably 13 introns. Because of this
complex exon-intron structure, various splicing variants are
possible. The mRNA of the transcribed H-SemaL gene is found in the
Northern blot particularly in placenta, gonads, thymus and spleen.
No mRNA has been detected in neuronal tissue or in muscle tissue.
There is evidence of specifically regulated expression in
endothelial cells.
[0080] Alternative splicing may also result in forms of H-SemaL
with intracytoplasmic sequences which are involved in intracellular
signal transduction, similar to, for example, CD100. It would
likewise be possible for alternative splicing to result in secreted
forms of H-SemaL, analogous to viral AHV-Sema.
[0081] Nucleotide and amino acid sequence analyses were performed
with the aid of the GCG program package (Genetics Computer Group
(1991) Program manual for the GCG package, Version 7, 575 Science
Drive, Wisconsin, USA 53711), FASTA (Pearson and Lipman (1988)
Proc. Natl. Acad. Sci. 85, 2444-2448) and BLAST program (Gish and
States (1993) Nat. Genet.3, 266-272; Altschul et al. (1990) J. Mol.
Biol. 215, 403410). These programs were also used for sequence
comparisons with GenBank (Version 102.0) and Swiss Prot (Version
34.0).
[0082] Post-translational modifications such as glycosylation and
myristylation of H-SemaL are likewise possible. Consensus sequences
for N-glycosylation sites were found with the aid of the Prosite
program (GCG program package) at positions 105, 157, 258, 330 and
602 of the amino acid sequence of H-SemaL (shown in Table 4), and
those for myristylation were found at positions 114, 139, 271, 498,
499, 502 and 654 (consensus sequence: G.about.(E, D, R, K, H, P, F,
Y, W).times.(S, T, A, G, C, N).about.(P)). In addition, the amino
acid sequence of H-SemaL contains several consensus sequences for
potential phosphorylation sites for various kinases. It can
therefore be assumed that H-SemaL can be the substrate of various
kinases, for example phosphorylation sites for creatine kinase 2,
protein kinase C and tyrosine kinase.
[0083] Predicted creatine kinase 2 phosphorylation sites (consensus
sequence Ck2: (S,T).times.2(D,E)) (Prosite, GCG) at positions 119,
131, 173, 338, 419 and 481 of the amino acid sequence.
[0084] Predicted protein kinase C phosphorylation sites (consensus
sequence PkC: (S,T).times.(R,K)) (Prosite, GCG) at positions 107,
115, 190, 296, 350, 431, 524 and 576 of the amino acid
sequence.
[0085] Predicted tyrosine kinase phosphorylation site (consensus
sequence: (R,K).times.{2,3}(D,E).times.{2,3}Y) (Prosite, GCG) at
position 205 of the amino acid sequence.
[0086] The consensus sequences are indicated in the single letter
code for amino acids.
[0087] An "RGD" motif (arginine-glycine-aspartic acid)
characteristic of integrins is located at position 267.
[0088] The glycosylation sites are highly conserved between viral
AHV-Sema, H-SemaL and (as far as is known) M-SemaL.
[0089] Di- or multimerization of H-SemaL is possible and has been
described for other semaphorins such as CD100 {Hall et al. (1996)}.
The CD100 molecule is likewise a membrane-anchored glycoprotein
dimer of 150 kd. However, CD100 is not closely related to the human
semaphorin (H-SemaL) according to the invention.
[0090] The partial cDNA sequence of M-SemaL has a length of 1195
nucleotides. This sequence codes for a protein having 394 amino
acids. These 394 amino acids correspond to amino acids 1 to 396 of
H-SemaL. The signal peptide in M-SemaL extends over amino acids 1
to 44 (exactly as in H-SemaL). The Sema domain starts at amino acid
45 and extends up to the end or probably beyond the end of the
sequence shown in Table 4.
[0091] Multiple alignments were carried out using the Clustal W
program (Thompson et al. (1994)). These alignments were processed
further manually using SEAVIEW (Galtier et al. (1996) Comput. Appl.
Biosci 12, 543-548). The phylogenetic distances were determined
using Clustal W (Thompson et al. (1994)).
[0092] Comparison of the protein sequences of the known and of the
novel semaphorins and phylogenetic analysis of these sequences
shows that the genes can be categorized according to their
phylogenetic relationship. The C-terminal domain structure of the
corresponding semaphorin subtypes is, of course, involved in this
as a factor deciding why semaphorins in the same subgroups are, as
a rule, also more closely related phylogenetically than are
semaphorins in different subgroups. The species from which the
semaphorin was isolated also has an influence, i.e. whether the
corresponding species are phylogenetically closely related to one
another or not.
[0093] A phylogenetic analysis (compare FIG. 3) of the known
semaphorin amino acid sequences (complete sequences and/or
part-sequences, using the amino acid sequences for H-SemaL and
M-SemaL shown in Tables 4 and 5 and for all other sequences the
sequences stored under the accession numbers or the encoded amino
acid sequences derived from these sequences) using the CLUSTAL W
program {Thompson J. D. et al. (1994) Nucleic Acids Res.
22:4673-4680} shows that the amino acid sequences of H-SemaL and
M-SemaL are phylogenetically closely related to one another and
form a separate phylogenetic group. H-SemaL and M-SemaL in turn are
phylogenetically most closely related to AHV-Sema and Vac-A39. The
are distinctly more closely related to one another than to any
other previously disclosed semaphorin. The analysis also shows that
other semaphorins are also phylogenetically closely related to one
another and form separate groups within the semaphorins. For
example, the semaphorins which are secreted, for example H-Sema
III, -IV, -V and -E belong in one phylogenetic group. Their
homologs in other species also belong to this subfamily, whereas
the human (transmembrane) CD100 belongs in one phylogenetic group
together with the corresponding mouse homolog (M-SemaG2) and with
Collapsin-4.
[0094] In relation to the complete amino acid sequences, the
observed homologies within the phylogenetic groups are between
about 90% and 80% amino acid identity in relation to very closely
related genes such as, for example, H- and M-SemaE or -III/D and
somewhat less than 40% in the case of less related genes of the
semaphorins. Within the Sema domain, the observed amino acid
identity is a few percent higher, and, owing to its great
contribution to the total protein (50-80% of the protein belong to
the Sema domain) of the amino acid sequence, this considerably
influences the overall identity.
[0095] H-SemaL is, calculated for the complete protein, 46%
identical with AHV-Sema, but if the Sema domain is considered on
its own, then the amino acid identity is 53%. This is higher than,
for example, between the related M-Sema-B and -C (37% identity in
relation to the complete protein, 43% identity in relation to the
Sema domain), similar to M-SemaA and -E (43% complete protein, 53%
Sema domain). The amino acid identity between the partial M-SemaL
sequence (Table 6) and H-SemaL (Table 5) in the region of the Sema
domain is 93% so that it can be assumed that the correspondingly
homologous mouse gene is involved.
[0096] Semaphorins corresponding to H-SemaL and M-SemaL in other
species may have an amino acid identity within the Sema domain of
more than 40% in relation to H-SemaL. In closely related
vertebrates (mammals, birds) amino acid identities above 70% may
even be found.
[0097] The semaphorins belong to a new subfamily with greater amino
acid identity to the viral AHV-Sema than to the previously
disclosed human and murine semaphorins, and with a C-terminal
structure not previously disclosed for human semaphorins. These
novel semaphorins (members of the subfamily) are distinguished by
belonging, because of their domain structure, to subgroup IV and/or
to the same phylogenetic group as H-SemaL and M-SemaL and/or have,
in relation to the complete amino acid sequence, an amino acid
identity of at least 30 to 40%, preferably 50 to 60%, particularly
preferably 70 to 80%, or a greater identity, to H-SemaL and/or
have, in relation to the Sema domain, an amino acid identity of at
least 70%, preferably greater than 80%, particularly preferably
greater than 90%, to H-SemaL.
[0098] The type L semaphorins also have a different type of
biochemical function. One novel function of these semaphorins is
modulation of the immune system.
[0099] The closest relative of H-SemaL is the viral AHV semaphorin
(AHV-Sema). The latter has a similar size but, in contrast to
H-SemaL, has no transmembrane domain. AHV-Sema is presumably
secreted by virus-infected cells in order to block the H-SemaL
equivalent receptor (type L semaphorin in the blue wildebeest) in
the natural host (blue wildebeest) and thus elude the attack of the
immune system. It is also conceivable that there is a function as
repulsive agent (chemorepellant) for cells of the immune
system.
[0100] The biochemical function of the novel type L semaphorins and
derivatives thereof is to be regarded as generally immunomodulating
and/or inflammation-modulating. They are able on the one hand
[0101] A) as molecules inhibiting the immune response to display
their effect as chemorepellant and/or immunosuppressant either
locally, for example as transmembrane protein on the surface of
cells, or else over larger distances, for example if they are
secreted due to processing (for example proteases) or alternative
splicing, for example by diffusion in the tissue.
[0102] For example, expression of these novel type L semaphorins
for example on the surface of the cells of the vascular endothelium
can prevent leukocyte attachment and migration thereof through the
vessel wall. The novel semaphorins may play a part in maintenance
of barrier effects, for example to prevent infections in
particularly "important" or exposed organs, for example to maintain
the blood-brain barrier, the placental circulation and/or other
immunologically privileged locations (for example pancreatic
islets) and/or in prevention of autoimmune diseases. In addition,
the novel semaphorins and/or their derivatives may also be involved
in repulsive signals in various tissues, for example for cells of
the immune system (for example leukocytes) to prevent inadvertent
activation of defense mechanisms.
[0103] B) In addition, the novel semaphorins and/or derivatives
thereof may have functions as accessory molecules. Expressed on the
cell surface, they may, for example, be involved in the interaction
with cells of the immune system as part of the activation of
defense mechanisms, for example in cases of virus infection.
[0104] This reveals several possible uses of the novel type L
semaphorins and derivatives thereof, and the nucleic acids coding
for these proteins.
[0105] Function A): This comprises an immunosuppressant and/or
anti-inflammatory principle: there are numerous potential
possibilities of use in the areas of organ transplantation, therapy
of inflammations, immunotherapy and gene therapy.
[0106] For example, nonhuman, transgenic animals can be produced
with the aid of the semaphorin-encoding DNA or derivatives
thereof.
[0107] One possible use of these animals is in the inhibition of
transplant rejection in transgenic models of organ
transplantations. For example, transgenic animal organs protected
against rejection can be produced for xenotransplantations. This
ought to be possible for example also together with other
transgenes (for example complement regulators such as DAF or CD59).
Another use is in the production of nonhuman knock-out animals, for
example knock-out mice ("Laboratory Protocols for Gene-Targeting",
Torres and Kuhn (1997) Oxford University Press, ISBN
0-19-963677-X): It is possible by knocking out the mouse M-SemaL
gene for example to find other functions of the gene. They also
represent potential model systems for inflammatory diseases if the
mice can survive without semaphorin gene. If M-SemaL is important
for immunomodulation, a plurality of such mice is to be expected.
In addition, nonhuman knock-in animals, for example mice, can be
produced. This entails, for example, replacing M-SemaL by
normal/modified H-SemaL or modified M-SemaL (for example
integration of the novel semaphorin subtypes under the control of
constitutive and/or inducible promoters). Animals of this type can
be used, for example, for looking for further functions of the
novel semaphorins, for example functions of the human gene or
derivatives of these genes, or be used for identifying and
characterizing immunomodulating agents.
[0108] Use of, for example, nucleic acids which code for type L
semaphorins or derivatives thereof for producing, for example,
recombinant immunosuppressants, other soluble proteins or peptides
derived from the amino acid sequence of type L semaphorins, for
example from H-SemaL or the corresponding nucleic acids, for
example genes. It is also possible in a similar way to produce
agonists with structural similarity. These immunosuppressant agents
or agonists may be used for autoimmune diseases and inflammatory
disorders and/or organ transplantations too.
[0109] Gene therapy with type L semaphorins, for example with
nucleic acids which code for H-SemaL or derivatives thereof, for
example using viral or nonviral methods. Use in autoimmune diseases
and inflammatory disorders, the transduction of organs and
before/during/after transplantations to prevent transplant
rejection.
[0110] It is particularly possible to employ the novel semaphorins
and/or the nucleic acids coding for these semaphorins, and
derivatives thereof, in particular H-SemaL, DNA coding for H-SemaL,
and derivatives thereof, in a method for screening for agents, in
particular for identifying and characterizing immunomodulating
agents.
[0111] Function B): H-SemaL is an accessory molecule which is
expressed on the cell surface and is involved in the interaction
with cells, for example of the immune system, for example as
accessory molecule in the activation of signal pathways. A viral
gene or the gene product of a viral or other pathogenic gene, for
example of microbiological origin, might act, for example, as
competitive inhibitor of this accessory molecule. One use of the
novel semaphorins with this function is likewise in the area of
organ transplantation, therapy of inflammation, immunotherapy
and/or gene therapy.
[0112] For example, the novel semaphorins can be used in a method
for screening for antagonistic agents or inhibitors. Agents
identified in this way can then be employed, for example, for
blocking the semaphorin receptor. Soluble and/or secreted H-SemaL
antagonists or inhibitors may be, for example, chemical substances
or the novel semaphorins or derivatives thereof themselves (for
example parts/truncated forms thereof, for example without membrane
domain or as Ig fusion proteins or peptides derived from the
latter, which are suitable for blocking the corresponding
receptor). Specific antagonists and/or inhibitors identified in
this way may, for example, have competitive effects and be employed
for inhibiting rejection, for example in transgenic models of organ
transplantations and for autoimmune diseases, inflammatory
disorders and organ transplantations. Nucleic acids, for example
DNA, which code for the novel semaphorins, or derivatives thereof
produced with the aid of methods of molecular biology, may be used,
for example, for producing nonhuman transgenic animals.
Overexpression of H-SemaL in these transgenic animals may lead to
increased susceptibility to autoimmune diseases and/or inflammatory
disorders. Such transgenic animals are thus suitable for screening
for novel specific immunomodulating agents.
[0113] Such nucleic acids can likewise be used to produce nonhuman
knock-out animals, for example knock-out mice in which the mouse
M-SemaL gene is switched off. Such knock-out animals can be
employed to search for further biochemical functions of the gene.
They also represent potential model systems for inflammatory
disorders if the mice are able to survive without the M-SemaL
gene.
[0114] This DNA can likewise be used to produce nonhuman knock-in
animals, for example mice. This entails the M-SemaL gene being
replaced by a modified M-SemaL gene/cDNA or an optionally modified,
for example mutated, type L semaphorin gene/cDNA of another
species, for example H-SemaL. Such transgenic animals can be used
to look for further functions of the semaphorins according to the
invention.
[0115] The invention also relates to the use of the type L
semaphorins and derivatives thereof, and of the nucleic acids
coding for these proteins, for example genes/cDNAs and derivatives
thereof and/or agents identified with the aid of these semaphorins
for producing pharmaceuticals. It is possible, for example, to
produce pharmaceuticals which can be used in gene therapy and which
comprise agonists and/or antagonists of the expression of the type
L semaphorins, for example of H-SemaL. It is possible to use for
this purpose, for example, viral and/or nonviral methods. These
pharmaceuticals can be employed, for example, for autoimmune
diseases and inflammatory disorders, organ transplantations before
and/or during and/or after the transplantation to prevent
rejection.
[0116] The nucleic acids coding for the novel semaphorins, for
example genes, cDNAs and derivatives thereof, can also be employed
as aids in molecular biology.
[0117] In addition, the novel semaphorins, especially H-SemaL and
nucleic acids, for example genes/cDNAs thereof can be employed in
methods for screening for novel agents. Modified proteins and/or
peptides derived, for example, from H-SemaL and/or M-SemaL can be
used to look for the corresponding receptor and/or its antagonists
or agonist in functional assays, for example using expression
constructs of H-SemaL and homologs.
[0118] The invention also relates to the use of a type L semaphorin
or a nucleic acid sequence which codes for a type L semaphorin in a
method for identifying pharmacological agents, especially
immunomodulating agents.
[0119] The invention also relates to methods for identifying agents
employing a type L semaphorin or a derivative thereof or a nucleic
acid sequence which codes for a type L semaphorin, or a derivative
thereof, in order to identify pharmacological agents, for example
immunomodulating agents. The invention relates, for example, to a
method in which a type L semaphorin is incubated under defined
conditions with an agent to be investigated and, in parallel, a
second batch is carried out without the agent to be investigated
but under conditions which are otherwise the same, and then the
inhibiting or activating effect of the agent to be investigated is
determined.
[0120] The invention also relates, for example, to methods for
identifying agents where a nucleic acid sequence which codes for a
type L semaphorin or a derivative thereof is expressed under
defined conditions in the presence of an agent to be investigated,
and the extent of the expression is determined. It is also
possible, where appropriate, in such a method to carry out two or
more batches in parallel under the same conditions but with the
batches containing different amounts of the agent to be
investigated.
[0121] For example, the agent to be investigated may inhibit or
activate transcription and/or translation.
[0122] The type L semaphorin can, like its viral homologs, bind to
the newly described receptor molecule VESPR (Comeau et al, (1998)
Immunity, Vol. 8, 473-482) and in monocytes can presumably cause
induction of cell adhesion molecules such as ICAM-1 and cytokines
such as interleukin-6 and interleukin-8. This may lead to
activation thereof and to cell aggregation. The expression pattern
of the VESPR receptor shows some interesting parallels with
H-SemaL, for example strong expression in placenta and pronounced
expression in spleen tissue. Interactions with other as yet unknown
receptors of the plexin family or other receptors are possible. It
may also interact with itself or other semaphorin-like molecules.
Interaction of the type L semaphorins may take place in particular
via a conserved domain in the C-terminal region of the Sema
domain.
[0123] Concerning the Annotation on Plasmids:
[0124] pMelBacA-H-SemaL (6622 bp) in pMelBacA (Invitrogen, De
Schelp, NL) (SEQ ID NO.42). Nucleotide 96-98 ATG--start codon,
nucleotide 96-168 mellitin signal sequence, nucleotide 168-173
BamHI cleavage site (PCR/cloning), nucleotide 171-1998 reading
frame SEMA-L amino acids 42-649 (without own signal sequence and
without transmembrane sequence), nucleotide 1993-1998 EcoRI
cleavage site (PCR/cloning) and nucleotide 1992-1994 stop
codon.
[0125] Plasmid pCDNA3.1-H-SemaL-MychisA (7475 bp) (SEQ ID NO. 35):
nucleotide 954-959 BamHI cleavage site (cloning), nucleotide
968-970 ATG SEMAL, nucleotide 968-2965 reading frame SEMAL,
nucleotide 2963-2968 Pml I cleavage site, nucleotide 2969-2974
HindIII cleavage site, nucleotide 2981-3013 Myc tag, nucleotide
3026-3033 6.times.His tag, nucleotide 3034-3036 stop codon,
[0126] Plasmid pCDNA3.1-H-SemaL-EGFP-MychisA (8192 bp):(SEQ ID NO.
36): nucleotide 954-959 BamHI cleavage site (cloning), nucleotide
968-970 ATG SEMA-L, nucleotide 968-2965 reading frame SEMA-L,
nucleotide 2963-2965 half Pml I cleavage site, nucleotide 2966-3682
reading frame EGFP (cloned in Pml I), nucleotide 3683-3685 half Pml
I cleavage site, nucleotide 3685-3691 HindIII, nucleotide 3698-3730
Myc tag, nucleotide 3743-3760 6.times.His tag, and nucleotide
3761-3763 stop codon.
[0127] Plasmid pIND-H-SemaL-EA (7108 bp) in vector pIND
(Invitrogen, De Schelp, NL) (SEQ ID No. 38): nucleotide 533-538
BamHI cleavage site (cloning), nucleotide 546-548 ATG SEMA-L,
nucleotide 546--reading frame SEMA-L, nucleotide 2542-2547 Pml I
cleavage site, nucleotide 2548-2553 HindIII cleavage site and
nucleotide 2563-2565 stop codon.
[0128] Plasmid pIND-H-SemaL-EE (total length 7102 bp) in vector
pIND (Invitrogen, De Schelp, NL) (SEQ ID No. 37): nucleotide
533-538 BamHI cleavage site (cloning), nucleotide 546-548 ATG
SEMA-L, nucleotide 546-reading frame SEMA-L, nucleotide 2542-2547
Pml I cleavage site, nucleotide 2548-2553 HindIII cleavage site,
nucleotide 2560-2592 Myc tag, nucleotide 2605-2622 6.times.His tag
and nucleotide 2623-2625 stop codon.
[0129] Plasmid pQE30-H-SemaL-179-378.seq (4019 bp) in vector pQE30
(Qiagen, Hilden) corresponds to pQE30-H-SemaLBH (SEQ ID No. 39):
nucleotide 115-117 ATG, nucleotide 127-144 6.times.His tag,
nucleotide 145-750 BamHI-HindIII PCR fragment SEMA-L amino acids
(aa) 179-378 and nucleotide 758-760 stop codon.
[0130] Plasmid pQE31-H-SemaL-(SH (3999 bp) in vector pQE31 (Qiagen,
Hilden) (SEQ ID No. 40): nucleotide 115-117 ATG, nucleotide 127-144
6.times.His tag, nucleotide (147-152 BamHI), nucleotide 159-729
Saci-HindIII fragment SEMA-L (C-terminal) aa480-666 and nucleotide
734-736 stop codon.
EXAMPLES
[0131] Experimental Conditions used in the Examples:
1 PCR programs used: Taq52-60 (with Ampli-Taq.sup.R polymerase,
Perkin Elmer, Weil der Stadt, Germany) 96.degree. C./60s 1 cycle
96.degree. C./15s-52.degree. C./20s-70.degree. C./60s 40 cycles
70.degree. C./60s 1 cycle Taq60-30 96.degree. C./60s 1 cycle
96.degree. C./15s-60.degree. C./20s-70.degree. C./30s 35 cycles
70.degree. C./60s 1 cycle Taq60-60 96.degree. C./60s 1 cycle
96.degree. C./15s-60.degree. C./20s-70.degree. C./60s 35 cycles
70.degree. C./60s 1 cycle Taq62-40 96.degree. C./60s 1 cycle
96.degree. C./15s-62.degree. C./20s-70.degree. C./40s 35 cycles
70.degree. C./60s 1 cycle
[0132] Reaction Conditions used for PCR with Taq Polymerase:
[0133] 50.mu.l reaction mixtures with 100-200 ng of template, 200
.mu.M dNTP, 0.2-0.4 .mu.M each primer, 2.5 U of Ampli-Taq.sup.R, 5
.mu.l of the 10.times. reaction buffer supplied
[0134] Programs used for:
2 XL62-6 (with expand-long template PCR System.sup.R, Boehringer
Mannheim, Germany) 94.degree. C./60s 1 cycle 94.degree.
C./15s-62.degree. C./30s-68.degree. C./6 min 10 cycles 94.degree.
C./15s-62.degree. C./30s-68.degree. C./(6 min + 15s/cycle) 25
cycles 68.degree. C./7 min 1 cycle XL62-12 (with expand-long
template PCR System Boehringer Mannheim, Germany) 94.degree. C./60s
1 cycle 94.degree. C./15s-62.degree. C./30s-68.degree. C./12 min 10
cycles 94.degree. C./15s-62.degree. C./30s-68.degree. C./(12 min +
15s/cycle) 25 cycles 68.degree. C./7 min 1 cycle
[0135] Reaction Conditions for PCR with Expand-long Template PCR
System
[0136] 50 .mu.l reaction mixtures with 100-200 ng of template, 500
.mu.M dNTP, 0.2-0.4 .mu.M each primer, 0.75 .mu.l of enzyme mix, 5
.mu.l of the 10.times. reaction buffer No. 2 supplied.
Example 1
[0137] Starting from AHV-Sema sequences (Ensser & Fleckenstein
(1995), J. General Virol. 76: 1063-1067), PCRs and RACE-PCRs were
carried out. The starting material used for this was human cDNA
from placental tissue onto which adaptors had been ligated for the
RACE amplification (Marathon.TM.-cDNA Amplification Kit, Clontech
Laboratories GmbH, Tullastra.beta.e 4, 69126 Heidelberg, Germany).
Firstly specific primers (No.121234+No. 121236, Table 6) were used
to amplify a PCR fragment with a length of about 800 bp (base
pairs) (PCR program: (Taq60-60)). This was cloned and sequenced
(Taq dye-deoxy terminator sequencing kit, Applied Biosystems,
Foster City, Calif., USA/Brunnenweg 13, Weil der Stadt). Sequencing
of the PCR product revealed a sequence which has a high degree of
homology with the DNA sequence of AHV-Sema, identical to the
sequence of the two ESTs.
[0138] A PCR fragment of 600 bp was identified using the primer
pair (No. 121237+No. 121239, Table 6). It emerged that they were
clones with DNA sequences from the same gene.
Example 2
[0139] The 800 bp PCR fragment from Example 1 was radiolabeled
(random priming by the method of {Feinberg (1983) Anal. Biochem.
132:6-13}, with .sup.32P-.alpha.-dCTP) and used as probe for a
multitissue Northern blot (Human Multiple Tissue Northern Blot II,
Clontech, Heidelberg, Germany) which contains mRNA samples from the
tissues spleen, thymus, prostate, testes, ovaries, small intestine,
large intestine and leukocytes (PBL). This clearly showed
expression of an mRNA with a length of about 3.3 kb in spleen and
gonads (testes, ovaries), and less strongly in the thymus and
intestine. Hybridization of a master blot (dot-blot with RNA from
numerous tissues (Human RNA Master Blot.TM., Clontech)) confirmed
this result and also showed strong expression in placental
tissue.
[0140] Hybridization was carried out under stringent conditions
(5.times.SSC, 50 mM Na phosphate pH 6.8, 50% formamide, 100
.mu.g/ml yeast RNA) at 42.degree. C. for 16 hours. The blots were
washed stringently (65.degree. C., 0.2.times.SSC, 0.1% SDS) and
exposed to a Fuji BAS2000 Phosphoimager.TM..
Example 3
[0141] A cDNA library from human spleen, cloned in the
bacteriophage Lambda gt10 (Human Spleen 5' STRETCH PLUS cDNA,
Clontech), was screened with this probe, and a lambda clone was
identified. The cDNA with a length of 1.6 kb inserted in this clone
was amplified by PCR (Expand.TM. Long Template PCR System,
Boehringer Mannheim GmbH, Sandhofer Stra.beta.ge 116, 68305
Mannheim) using the vector-specific primers No. 207608+No. 207609
(Table 6) (flanking the EcoRI cloning site), and the resulting PCR
fragment was sequenced. This clone contained the 5' end of the cDNA
and also extended the known cDNA sequence in the 3' direction.
Starting from the new part-sequences of the cDNA, new primers for
the RACE-PCR were developed (No. 232643, No. 232644, No. 233084,
Table 6). Together with an improved thermocycler technique (PTC-200
from MJ-Research, Biozym Diagnostik GmbH, 31833 Hess. Oldendort)
with distinctly better performance data (heating and cooling
rates), a 3' RACE-PCR product was amplified using the primers No.
232644 and No. 232643 and AP1, and was cloned into the vector
pCR2.1 (Invitrogen, De Schelp 12, 9351 NV Leek, The Netherlands).
The 3' RACE-PCR product was sequenced and the 3' end of the cDNA
was identified in this way. A RACE amplification in the 5'
direction (primers No. 131990 and No. 233084 and AP1) extended the
5' end of the cDNA by a few nucleotides and confirmed the amino
terminus of H-SemaL found in the identified lambda clone.
Example 4
[0142] Starting from a short murine EST (Accession No. AA260340)
and a primer derived therefrom, No. 260813 (Table 6) and the
H-SemaL specific primer No. 121234 (Table 6), PCR (conditions:
Taq52-60) was used to amplify a DNA fragment with a length of about
840 bp of murine cDNA, followed by cloning into the vector pCR2.1.
The gene containing this DNA fragment was called M-SemaL. The
resulting M-SemaL DNA fragment was used to investigate a cDNA bank
from mouse spleen (Mouse Spleen 5' STRETCH cDNA, Clontech),
identification of several clones being possible.
[0143] PCR (Taq60-30) with the primers No. 260812 and No. 260813
from murine endothelial cDNA provided a PCR fragment with a length
of 244 base pairs. The PCR results showed that there is distinct
baseline expression in murine endothelial cells which declines
after stimulation with the cytokine interferon-.gamma. and
lipopolysaccharides.
Example 5
[0144] Investigations on the location in the chromosome were
carried out by fluorescence in situ hybridization (FISH). For this
purpose, human and murine metaphase chromosomes were prepared
starting from a human blood sample and the mouse cell line BINE 4.8
(Keyna et al. (1995) J. Immunol. 155, 5536-5542), respectively
(Kraus et al. (1994) Genomics 23, 272-274). The slides were treated
with RNase and pepsin (Liehr et al. (1995) Appl. Cytogenetics 21,
185-188). For the hybridization, 120 mg of human nick-translated
semaphorin sample and 200 mg of a corresponding mouse sample were
used. The hybridization was in each case carried out in the
presence of 4.0 .mu.g of COT1-DNA and 20 .mu.g of STD at 37.degree.
C. (3 days) in a moistened chamber.
[0145] The slides were washed with 50% formamide/2.times.SSC (3
times for 5 min each time at 45.degree. C.) and then with
2.times.SSC (3 times for 5 min each time at 37.degree. C.), and the
biotinylated sample was detected using the FITC-avidin system
(Liehr et al. (1995)). The slides were evaluated using a
fluorescence microscope. 25 metaphases/sample were evaluated,
carrying out each experiment in duplicate. It emerged that H-SemaL
is located on chromosome 15q23. Located adjacent in the chromosome
is the locus for Bardet-Biedls syndrome and Tay-Sachs disease
(hexosaminidase A).
Example 6
[0146] The genomic intron-exon structure of the H-SemaL gene is for
the most part elucidated.
[0147] Genomic DNA fragments were amplified starting from 250 mg of
human genomic DNA which had been isolated from PHA-stimulated
peripheral lymphocytes (blood). Shorter fragments were amplified
using Ampli Taq.sup.R (Perkin Elmer), and longer fragments were
amplified using the expanded long template PCR System.sup.R
(Boehringer Mannheim).
[0148] It has been possible by PCR amplification to date to clone
and characterize almost the complete genomic locus of H-SemaL. It
has already been possible in total to determine more than 8888 bp
of the genomic sequence and thus substantially to elucidate the
intron-exon structure of the gene.
Example 7
[0149] Expression Clonings:
[0150] Since no complete clone of the semaphorin gene could be
isolated from the lambda-gt10 cDNA bank, and no complete clone was
obtainable by PCR either, the coding region of the cDNA was
amplified in 2 overlapping subfragments by PCR (XL62-6) using the
primers No. 240655 and No. 121339 for the N-terminal DNA fragment,
and the primers No. 240656 (contains HindIII and Pmel cleavage
sites) and No. 121234 for the C-terminal DNA fragment. The
resulting DNA fragments (subfragments) were cloned into the vector
pCR21. The two subfragments were completely sequenced and finally
the complete H-SemaL cDNA was prepared by inserting a 0.6 kb
C-terminal SstI-HindIII restriction fragment into the plasmid which
contained the N-terminal DNA fragment and had been cut with the
restriction enzymes SstI and HindIII. From this plasmid
pCR2.1-H-SemaL (sequence shown in Table 7, SEQ ID NO. 34), the
complete gene was cut out using the EcoRI cleavage site (in pCR2.1)
and HindIII cleavage site (in primer No. 240656, Table 6) and
ligated into a correspondingly cut constitutive expression vector
pCDNA3.1 (-)MycHisA (Invitrogen). The EcoRI-ApaI fragment (without
Myc-His tag) was cut out of the resulting recombinant plasmid
pCDNA3.1(-)H-SemaL-MycHisA (sequence shown in Table 8) and ligated
into the inducible vector pIND (Ecdysone-Inducible Mammalian
Expression System, Invitrogen) which had previously likewise been
cut with EcoRI-ApaI. The recombinant plasmid was called
pIND-H-SemaLEA (sequence shown in Table 11). An EcoRI-Pmel fragment
(with Myc-His tag) from pCDNA3.1(-)H-SemaL-Myc-HisA (sequence shown
in Table 9) was inserted into an EcoRI-EcoRV-cut vector pIND. The
recombinant plasmid was called pIND-H-SemaL-EE (sequence shown in
Table 10).
[0151] A fusion gene of H-SemaL with enhanced green fluorescent
protein (EGFP) was prepared by ligating the PCR-amplified EGFP
reading frame (from the vector pEGFP-C1 (Clontech), using the
primers No. 243068+No. 243069, Taq52-60) into the Pmel cleavage
site of the plasmid pCDNA3.1(-)H-SemaL-MycHisA, resulting in the
plasmid pCDNA3.1 (-)H-SemaL-EGFP-MycHisA (sequence shown in Table
9).
[0152] Small letters in Tables 7 to 13 and Table 15 denote the
sequence of H-SemaL, parts or derivatives thereof, and large
letters denote the sequence of the plasmid.
Example 8
[0153] To prepare H-SemaL-specific antibodies, cDNA fragments of
H-SemaL were integrated into prokaryotic expression vectors and
expressed in E. coli, and the semaphorin derivatives were purified.
The semaphorin derivatives were expressed as fusion proteins with a
His tag. Accordingly, vectors containing the sequence for a His tag
and permitting integration of the semaphorin cDNA fragment into the
reading frame were used. An N-terminal 6.times.histidine tag makes
it possible, for example, to purify by nickel chelate affinity
chromatography (Qiagen GmbH, Max-Volmer Stra.beta.e 4, 40724
Hilden):
[0154] 1. The part of the H-SemaL cDNA coding for amino acids
179-378 was amplified by PCR using the primers No. 150788 and No.
150789, and this DNA fragment was ligated into the vector pQE30
(Qiagen) which had previously been cut with the restriction enzymes
BamHI and HindIII (construct pQE30-H-SemaL-BH (sequence shown in
Table 12)).
[0155] 2. The section of the H-SemaL cDNA coding for the C-terminal
amino acids 480-666 was cut with the restriction enzymes SstI and
HindIII out of the plasmid pCR 2.1 and ligated into the vector
pQE31 (Qiagen) which had previously been cut with SstI and HindIII
(construct pQE31-H-SemaL-SH (sequence shown in Table 13)).
[0156] Correct integration of the sequences in the correct reading
frame was checked by DNA sequencing. The fusion proteins consisting
of an N-terminal 6.times.histidine tag and a part of the semaphorin
H-SemaL were purified by Ni.sup.2+ affinity chromatography. The
purified fusion proteins were used to immunize various animals
(rabbit, chicken, mouse).
Example 9
[0157] FACS Analysis of Various Cell Types (FIGS. 4 and 5)
[0158] The cells (about 0.2-0.5.times.10.sup.6) were washed with
FACS buffer (phosphate-buffered saline (PBS) with 5% fetal calf
serum (FCS) and 0.1% Na azide) and then incubated with the antisera
(on ice) for 1 hour in each case.
[0159] The primary antibodies used for the control (overlay chicken
preimmune serum (1:50)) and for the specific detection (specific
staining) comprised an H-SemaL-specific chicken antiserum (1:50).
The specific antiserum with antibodies against amino acids (Aa)
179-378 (with N-terminal His tag) of H-SemaL was generated by
immunizing chickens with the protein purified by Ni chelate
affinity chromatography (as described in Example 8). The second
antibody used was an FITC-labeled anti-chicken F(ab') antibody from
rabbits (Dianova Jackson Laboratories, Order No. 303-095-006,
Hamburg, Germany) (1 mg/ml). A rabbit anti-mouse IgG, FITC-labeled,
was used for the CD100 staining. The second antibody was employed
in each case in 1:50 dilution in FACS buffer.
[0160] The cells were then washed, resuspended in PBS and analyzed
in the FACS. The FACS analysis was carried out using a FACS-track
instrument (Becton-Dickinson). Principle: a single cell suspension
is passed through a measuring channel where the cells are
irradiated with laser light of 488 nm and thus fluorescent dyes
(FITC) are excited. The measurements are of the light scattered
forward (forward scatter, FSC: correlates with the cell size), and
to the side (sideward scatter, SSC: correlates with the granular
content: different in different cell types) and fluorescence in
channel 1 (FL 1) (for wavelengths in the FITC emission range, max.
at 530 nm). 10,000 events (cells) were measured in this way each
time.
[0161] The dot plot (FIGS. 4a-k) (figure on the left in each case):
FSC against SSC (size against granular content/scatter) with,
inside the boundary, the (uniform) cell population of similar size
and granular content analyzed in the right-hand window (relevant
right-hand figure in each case). The right-hand window shows the
intensity of FL 1 (X axis) against the number of events (Y axis),
that is to say a frequency distribution.
[0162] In each of these, the result with the control serum
(unfilled curve) is superimposed on the result of the specific
staining (filled curve). A shift of the curve for the specific
staining to the right compared with the control corresponds to an
expression of H-SemaL in the corresponding cells. A larger shift
means stronger expression.
[0163] Cell lines used for FACS analysis:
[0164] a) U937 cell line
[0165] American Type Culture Collection ATCC; ATCC number:
CRL-1593
[0166] Name: U-937
[0167] Tissue: lymphoma; histiocytic; monocyte-like
[0168] Species: human;
[0169] Depositor: H. Koren
[0170] b) THP-1 cell line
[0171] ATCC number: TIB-202
[0172] Tissue: monocyte; acute monocytic leukemia
[0173] Species: human
[0174] Depositor: S. Tsuchiya
[0175] c) K-562 cell line
[0176] ATCC number: CCL-243
[0177] Tissue: chronic myelogenous leukemia
[0178] Species: human;
[0179] Depositor: H. T. Holden
[0180] d) L-428 cell line
[0181] DSMZ-Deutsche Sammiung von Mikroorganismen und Zelikulturen
GmbH,
[0182] DSMZ No: ACC 197
[0183] Cell type: human Hodgkin's lymphoma
[0184] e) Jurkat cell line
[0185] DSMZ-Deutsche Sammiung von Mikroorganismen und zellkulturen
GmH,
[0186] DSMZ No: ACC 282
[0187] Cell type: human T cell leukemia
[0188] f) Daudi cell line
[0189] ATCC number: CCL-213
[0190] Tissue: Burkitt's lymphoma; B lymphoblast; B cells
[0191] Species: human
[0192] Depositor: G. Klein
[0193] g) LCL cell line
[0194] EBV-transformed lymphoblastoid B-cell line.
[0195] h) Jiyoye (P-2003) cell line
[0196] ATCC number: CCL-87
[0197] Tissue: Burkitt's lymphoma; B cells, B lymphocyte
[0198] Species: human
[0199] Depositor: W. Henle
[0200] i) CBL-Mix57
[0201] Human T-cell line (isolated from blood) transformed with
recombinant H. Saimiri (wild-type without deletion)
[0202] j) CBL-Mix59
[0203] Human T-cell line (isolated from blood) transformed with H.
Saimiri (deletion of ORF71).
Example 10
[0204] Protein Gel and Western Blot
[0205] Secretable human SEMA-L (amino acids 42-649 in Table 4
(without signal peptide and without transmembrane domain)) was
cloned into the plasmid pMelBac-A (Invitrogen, De Schelp, Leck, The
Netherlands, Cv 1950-20) and, in this way, the plasmid
pMelBacA-H-SemaL (length 6622 bp) was generated (FIG. 8). The
H-SemaL derivative was expressed in the baculovirus system
(Bac-N-Blue, Invitrogen). Expression was carried out in the cell
lines derived TM from insect egg cells Sf9 (from Spodoptera
frugiperda) and High Five.TM. (from Trichoplusia ni, U.S. Pat. No.
5,300,435, purchased from Invitrogen) by infection with the
recombinant, plaque-purified baculoviruses.
[0206] The expression was carried out in accordance with the
manufacturer's instructions.
[0207] The proteins were then fractionated in a gel, and the
H-SemaL derivative was detected in a Western blot. Detection was
carried out with H-SemaL-specific chicken antiserum (compare
Example 8 and FIG. 7) (dilution 1:100). The specific chicken
antibody was detected using anti-IgY-HRP conjugate (dilution:
1:3000, from donkey; Dianova Jackson Laboratories) in accordance
with the manufacturer's instructions.
Example 11
[0208] Preparation of pMelBacA-H-SEMAL
[0209] The recombinant vector (pMelBacA-H-SEMAL, 6622 bp) was
prepared by cloning an appropriate DNA fragment which codes for
amino acids 42-649 of H-SemaL into the vector pMelBacA (4.8 kb
Invitrogen) (compare annotation for pMelBacA-H-SEMAL). The cloning
took place via BamHI and EcoRI in frame behind the signal sequence
present in the vector ("honeybee melittin signal sequence"). A
corresponding H-SemaL DNA fragment was amplified using the primer
pair h-sema-1 baculo 5' and h-sema-1 baculo 3'.
[0210] Primers for amplification (TaKaRa Ex Ta9 polymerase) and
cloning: "h-sema-1 baculo 5'" for amplification without signal
sequence and for introducing a BamHI cleavage site
5'-CCGGATCCGCCCAGGGCCACCTAAGGAGCGG-3' (SEQ ID NO: 43) "h-sema-1
baculo 3'" for amplification without transmembrane domain and for
introducing an EcoRI cleavage site
5'-CTGMTTCAGGAGCCAGGGCACAGGCATG-3' (SEQ ID NO: 44).
DETAILED DESCRIPTION OF THE DRAWINGS
[0211] FIG. 1:
[0212] Tissue-specific expression of H-Sema-L
[0213] A) Multiple tissue Northern blot (Clontech, Heidelberg,
Germany). Loadings from left to right: 2 .mu.g in each lane of
Poly-A-RNA from spleen, thymus, prostate, testes, ovaries, small
intestine, large intestinal mucosa, peripheral (blood) leukocytes.
Size standards are marked.
[0214] The blots were hybridized under stringent conditions with an
H-SemaL probe 800 base-pairs long.
[0215] FIG. 2:
[0216] Diagrammatic representation of the cloning of the H-SemaL
cDNA and of the genomic organization of the H-SemaL encoding
sequences (H-SemaL gene)
[0217] Top: Location of the EST sequences (accession numbers;
location of the EST sequences is shown relative to the AHV-Sema
sequence).
[0218] Below: Amplified PCR and RACE products and the position of
the cDNA clones in relation to the location in the complete H-SemaL
cDNA and the open reading frame (ORF) for the encoded protein.
[0219] Bottom: Relative position of the exons in the H-SemaL gene
in relation to the genomic sequence. The position of the
oligonucleotide primer used is indicated by arrows.
[0220] FIG. 3:
[0221] Phylogenetic tree: Obtained by multiple alignment of the
listed semaphorin sequences. The phylogenetic relationship of the
semaphorins can be deduced from their grouping in the phylogenetic
tree.
[0222] FIG. 4:
[0223] FACS analysis of H-SemaL expression in various cell lines
and various cell types (compare Example 8).
[0224] FIG. 5:
[0225] Comparative analysis of CD100 and H-SemaL expression
(compare Example 9).
[0226] FIG. 6:
[0227] Expression of secretable human SEMA-L (H-SemaL) in HiFive
and Sf3 cells (compare Example 10).
[0228] Aa 42-649 in pMelBac-A (Invitrogen) in the baculovirus
system (Bac-N-Blue, Invitrogen)
[0229] Detection with specific chicken antiserum (1:100) and
anti-IgY-HRP conjugate (1:3000, from rabbits, Jackson Lab.)
[0230] 1,4,6 uninfected HiFive cells (serum-free)
[0231] 2,3,5,7,8 HiFive cells infected with recombinant baculovirus
(serum-free)
[0232] M Rainbow molecular weight marker (Amersham RPN756)
[0233] 9,10 infected Sf9 cells (serum-containing medium).
[0234] FIG. 7: Specificity of the antiserum
[0235] Lanes 1-3: chicken 1; lanes 4-6: chicken 2
[0236] Lanes 1 and 4: Preimmune serum
[0237] Lanes 2 and 5: 60.sup.th day of immunization
[0238] Lanes 4 and 6: 105.sup.th day of immunization
[0239] Immunization was carried out with amino acids 179-378 of
H-SemaL (with amino-terminal His tag) (compare Example 8, Section
1.)
[0240] FIG. 8: Depiction of the plasmid map of
pMelBacA-H-SEMAL.
[0241] The recombinant plasmid was prepared as described in Example
11.
TABLES
[0242]
3TABLE 1 Various subtypes of semaphorins from various species Name
Synonym Species Reference H-Sema III (H-SemaD) Human Sec. (Kolodkin
et al. 1993) CD-100 Human TM, IC; CD45 associated, expressed in T
cells (Hall et al. 1996) H-Sema V (H-SemaA) Human Sec.; Locus
3p21.3 (Sekido et al. 1996; Roche et al. 1996) H-Sema IV (H-Sema3F)
Human Sec.; Locus 3p21.3 (Xiang et al. 1996; Sekido et al. 1996)
H-SemaE Human Sec.; divergent from M-Sema-E at the 3' end AB000220
(Yamada 1997 unpublished) (alignment of reading frame improved)
H-SemaK KIAA0331 Human Sec.; (Nagase et al. 1997) H-SemaL SEMAL
Human TM, no IC This application M-SemaA Mouse Sec. (Puchel et al.
1995) M-SemaB Mouse TM, IC (Puchel et al. 1995) M-SemaC Mouse TM,
IC (Puchel et al. 1995) M-SemaD M-Sema III Mouse Sec. (Messersmith
et al. 1995; Puchel et al. 1995) M-SemaE Mouse Sec.; 5' partial
sequence (Puchel et al. 1995) M-SemaF1 M-SemaF Mouse TM, IC
(Inagaki et al. 1996) M-SemaG2 M-SemaG Mouse TM, IC; expressed in
lymphoid cells, mouse (Furuyama et al. 1996) homolog of CD100
M-SemaF2 M-SemaF Mouse TM, IC; Thrombospondin motif (Adams et al.
1996) M-SemaG1 M-SemaG Mouse TM, IC; Thrombospondin motif (Adams et
al. 1996) M-SemaH Mouse Sec. (Christensen 1996 unpub) Z80941 M-Sema
Via Mouse TM, IC (Zhou et al. 1997) M-SemaL Semal Mouse Partial
sequence This application Collapsin-1 Chicken Sec. (Luo et al.
1993) Collapsin-2 Chicken Sec. (Luo et al. 1995) Collapsin-3
Chicken Sec. (Luo et al. 1995) Collapsin-4 Chicken Partial sequence
(Luo et al. 1995) Collapsin-5 Chicken Sec. (Luo et al. 1995) R-Sema
III Rat Sec. (Giger et al. 1996) T-Sema I Tribolium TM, IC
(Kolodkin et al. 1993) confusum Ce-Semal C. elegans TM, IC U15667
(Roy 1994 unpublished) G-Sema I Fasciclin-IV Grasshopper TM, IC
(Kolodkin et al. 1992) D-Sema I Drosophila TM, IC (Kolodkin et al.
1993) D-Sema II Drosophila Sec. (Kolodkin et al. 1993) AHV-Sema
AHV-1 Sec. (Ensser and Fleckenstein, 1995) ORF-A39 Vaccinia Sec.
(Kolodkin et al. 1993) ORF-A39 Variola Sec.; (Kolodkin et al. 1993)
homologous TM: transmembrane domain Sec.: secreted IC: presumably
intracellular cytoplasmic sequence motif
[0243]
4TABLE 2 cDNA sequence of H-SemaL (2636 nucleotides) (SEQ ID NO.:
1) 1 cggggccacg ggatgacgcc tcctccgccc ggacgtgccg cccccagcgc 51
accgcgcgcc cgcgtccctg gcccgccggc tcggttgggg cttccgctgc 101
ggctgcggct gctgctgctg ctctgggcgg ccgccgcctc cgcccagggc 151
cacctaagga gcggaccccg catcttcgcc gtctggaaag gccatgtagg 201
gcaggaccgg gtggactttg gccagactga gccgcacacg gtgcttttcc 251
acgagccagg cagctcctct gtgtgggtgg gaggacgtgg caaggtctac 301
ctctttgact tccccgaggg caagaacgca tctgtgcgca cggtgaatat 351
cggctccaca aaggggtcct gtctggataa gcgggactgc gagaactaca 401
tcactctcct ggagaggcgg agtgaggggc tgctggcctg tggcaccaac 451
gcccggcacc ccagctgctg gaacctggtg aatggcactg tggtgccact 501
tggcgagatg agaggctacg cccccttcag cccggacgag aactccctgg 551
ttctgtttga aggggacgag gtgtattcca ccatccggaa gcaggaatac 601
aatgggaaga tccctcggtt ccgccgcatc cggggcgaga gtgagctgta 651
caccagtgat actgtcatgc agaacccaca gttcatcaaa gccaccatcg 701
tgcaccaaga ccaggcttac gatgacaaga tctactactt cttccgagag 751
gacaatcctg acaagaatcc tgaggctcct ctcaatgtgt cccgtgtggc 801
ccagttgtgc aggggggacc agggtgggga aagttcactg tcagtctcca 851
agtggaacac ttttctgaaa gccatgctgg tatgcagtga tgctgccacc 901
aacaagaact tcaacaggct gcaagacgtc ttcctgctcc ctgaccccag 951
cggccagtgg agggacacca gggtctatgg tgttttctcc aacccctgga 1001
actactcagc cgtctgtgtg tattccctcg gtgacattga caaggtcttc 1051
cgtacctcct cactcaaggg ctaccactca agccttccca acccgcggcc 1101
tggcaagtgc ctcccagacc agcagccgat acccacagag accttccagg 1151
tggctgaccg tcacccagag gtggcgcaga gggtggagcc catggggcct 1201
ctgaagacgc cattgttcca ctctaaatac cactaccaga aagtggccgt 1251
tcaccgcatg caagccagcc acggggagac ctttcatgtg ctttacctaa 1301
ctacagacag gggcactatc cacaaggtgg tggaaccggg ggagcaggag 1351
cacagcttcg ccttcaacat catggagatc cagcccttcc gccgcgcggc 1401
tgccatccag accatgtcgc tggatgctga gcggaggaag ctgtatgtga 1451
gctcccagtg ggaggtgagc caggtgcccc tggacctgtg tgaggtctat 1501
ggcgggggct gccacggttg cctcatgtcc cgagacccct actgcggctg 1551
ggaccagggc cgctgcatct ccatctacag ctccgaacgg tcagtgctgc 1601
aatccattaa tccagccgag ccacacaagg agtgtcccaa ccccaaacca 1651
gacaaggccC cactgcagaa ggtttccctg gccccaaact ctcgctacta 1701
cctgagctgc cccatggaat cccgccacgc cacctactca tggcgccaca 1751
aggagaacgt ggagcagagc tgcgaacctg gtcaccagag ccccaactgc 1801
atcctgttca tcgagaacct cacggcgcag cagtacggcc actacttctg 1851
cgaggcccag gagggctcct acttccgcga ggctcagcac tggcagctgc 1901
tgcccgagga cggcatcatg gccgagcacc tgctgggtca tgcctgtgcc 1951
ctggctgcct ccctctggct gggggtgctg cccacactca ctcttggctt 2001
gctggtccac tagggcctcc cgaggctggg catgcctcag gcttctgcag 2051
cccagggcac tagaacgtct cacactcaga gccggctggc ccgggagctc 2101
cttgcctgcc acttcttcca ggggacagaa taacccagtg gaggatgcca 2151
ggcctggaga cgtccagccg caggcggctg ctgggcccca ggtggcgcac 2201
ggatggtgag gggctgagaa tgagggcacc gactgtgaag ctggggcatc 2251
gatgacccaa gactttatct tctggaaaat atttttcaga ctcctcaaac 2301
ttgactaaat gcagcgatgc tcccagccca agagcccatg ggtcggggag 2351
tgggtttgga taggagagct gggactccat ctcgaccctg gggctgaggc 2401
ctgagtcctt ctggactctt ggtacccaca ttgcctcctt cccctccctc 2451
tctcatggct gggtggctgg tgttcctgaa gacccagggc taccctctgt 2501
ccagccctgt cctctgcagc tccctctctg gtcctgggtc ccacaggaca 2551
gccgccttgc atgtttattg aaggatgttt gctttccgga cggaaggacg 2601
gaaaaagctc tgaaaaaaaa aaaaaaaaaa aaaaaa
[0244]
5TABLE 3 Nucleotide sequence of the cDNA of M-SemaL (partial, 1195
nucleotides) (SEQ ID NO.: 2) 1 cggggctgcg ggatgacgcc tcctcctccc
ggacgtgccg cccccagcgc 51 accgcgcgcc cgcgtcctca gcctgccggc
tcggttcggg ctcccgctgc 101 ggctgcggct tctgctggtg ttctgggtgg
ccgccgcctc cgcccaaggc 151 cactcgagga gcggaccccg catctccgcc
gtctggaaag ggcaggacca 201 tgtggacttt agccagcctg agccacacac
cgtgcttttc catgagccgg 251 gcagcttctc tgtctgggtg ggtggacgtg
gcaaggtcta ccacttcaac 301 ttccccgagg gcaagaatgc ctctgtgcgc
acggtgaaca tcggctccac 351 aaaggggtcc tgtcaggaca aacaggactg
tgggaattac atcactcttc 401 tagaaaggcg gggtaatggg ctgctggtct
gtggcaccaa tgcccggaag 451 cccagctgct ggaacttggt gaatgacagt
gtggtgatgt cacttggtga 501 gatgaaaggc tatgccccct tcagcccgga
tgagaactcc ctggttctgt 551 ttgaaggaga tgaagtgtac tctaccatcc
ggaagcagga atacaacggg 601 aagatccctc ggtttcgacg cattcggggc
gagagtgaac tgtacacaag 651 tgatacagtc atgcagaacc cacagttcat
caaggccacc attgtgcacc 701 aagaccaagc ctatgatgat aagatctact
acttcttccg agaagacaac 751 cctgacaaga accccgaggc tcctctcaat
gtgtcccgag tagcccagtt 801 gtgcaggggg gaccagggtg gtgagagttc
gttgtctgtc tccaagtgga 851 acaccttcct gaaagccatg ttggtctgca
gcgatgcagc caccaacagg 901 aacttcaatc ggctgcaaga tgtcttcctg
ctccctgacc ccagtggcca 951 gtggagagat accagggtct atggcgtttt
ctccaacccc tggaactact 1001 cagctgtctg cgtgtattcg cttggtgaca
ttgacagagt cttccgtacc 1051 tcatcgctca aaggctacca catgggcctt
tccaaccctc gacctggcat 1101 gtgcctccca aaaaagcagc ccatacccac
agaaaccttc caggtagctg 1151 atagtcaccc agaggtggct cagagggtgg
aacctatggg gcccc
[0245]
6TABLE 4 Amino acid sequence of H-SemaL (666 amino acids) (SEQ ID
NO.: 3) 1 MTPPPPGRAA PSAPRARVPG PPARLGLPLR LRLLLLLWAA AASAQGHLRS 51
GPRIFAVWKG HVGQDRVDFG QTEPHTVLFH EPGSSSVWVG GRGKVYLFDF 101
PEGKNASVRT VNIGSTKGSC LDKRDCENYI TLLERRSEGL LACGTNARHP 151
SCWNLVNGTV VPLGEMRGYA PFSPDENSLV LFEGDEVYST IRKQEYNGKI 201
PRERRIRGES ELYTSDTVMQ NPQFIKATIV HQDQAYDDKI YYFFREDNPD 251
KNPEAPLNVS RVAQLCRGDQ GGESSLSVSK WNTFLKAMLV CSDAATNKNF 301
NRLQDVFLLP DPSGQWRDTR VYGVFSNPWN YSAVCVYSLG DIDKVFRTSS 351
LKGYHSSLPN PRPGKCLPDQ QPIPTETFQV ADRHPEVAQR VEPMGPLKTP 401
LFHSKYHYQK VAVHRMQASH GETFHVLYLT TDRGTIHKVV EPGEQEHSFA 451
FNIMEIQPFR RAAAIQTMSL DAERRKLYVS SQWEVSQVPL DLCEVYGGGC 501
HGCLMSRDPY GGWDQGRCIS IYSSERSVLQ SINPAEPHKE CPNPKPDKAP 551
LQKVSLAPNS RYYLSCPMES RHATYSWRHK ENVEQSCEPG HQSPNCILFI 601
ENLTAQQYGH YFCEAQEGSY FREAQHWQLL PEDGIMAEHL LGHACALAAS 651
LWLGVLPTLT LGLLVH
[0246]
7TABLE 5 (Partial) amino acid sequence of M-SemaL (394 amino acids,
corresponding to position 1-396 of H-SemaL) (SEQ ID NO.: 4) 1
MTPPPPGRAA PSAPRARVLS LPARFGLPLR LRLLLVFWVA AASAQGHSRS 51
GPRISAVWKG QDHVDFSQPE PHTVLFHEPG SFSVWVGGRG KVYHFNFPEG 101
KNASVRTVNI GSTKGSCQDK QDCGNYITLL ERRGNGLLVG GTNARKPSCW 151
NLVNDSVVMS LGEMKGYAPF SPDENSLVLF EGDEVYSTIR KQEYNGKIPR 201
FRRIRGESEL YTSDTVMQNP QFIKATIVHQ DQAYDDKIYY FFREDNPDKN 251
PEAPLNVSRV AQLCRGDQGG ESSLSVSKWN TFLKAMLVCS DAATNRNFNR 301
LQDVFLLPDP SGQWRDTRVY GVFSNPWNYS AVCVYSLGDI DRVFRTSSLK 351
GYHMGLSNPR PGMCLPKKQP IPTETFQVAD SHPEVAQRVE PMGP
[0247]
8TABLE 6 Synthetic oligonucleotides (Eurogentec, Seraing, Belgium)
Number of the primer/name Nucleotide sequence of the primer (of the
synthetic oligonucleotides) 91506/AP2 actcactatagggctcgagcggc (SEQ
ID NO.: 5) 121234 agccgcacacggtgcttttc (SEQ ID NO.: 6) 121235/Est 2
gcacagatgcgttcttgccc (SEQ ID NO.: 7) 121236/Est 3
accatagaccctggtgtccc (SEQ ID NO.: 8) 121237/Est 4
gcagtgatgctgccaccaac (SEQ ID NO.: 9) 121238 ccagaccatgtcgctggatg
(SEQ ID NO.: 10) 121239/Est 6 acatgaggcaaccgtggcag (SEQ ID NO.: 11)
131989/AP1 ccatcctaatacgactcactatagggc (SEQ ID NO.: 12) 131990/Est
7 aggtagaccttgccacgtcc (SEQ ID NO.: 13) 131991
gaacttcaacaggctgcaagacg (SEQ ID NO.: 14) 131992
atgctgagcggaggaagctg (SEQ ID NO.: 15) 131993 ccgccatacacctcacacag
(SEQ ID NO.: 16) 150788 ctggaagctttctgtgggtatcggctgc (SEQ ID NO.:
17) 150789 tttggatccctggttctgtttgaag (SEQ ID NO.: 18) 167579/cDNA
ttctagaattcagcggccgcttttttttttttttttttttttttttttttvn (SEQ ID NO.:
19) Synthesis primer 168421 ggggaaagttcactgtcagtctccaag (SEQ ID
NO.: 20) 168422 gggaatacacacagacggctgagtag (SEQ ID NO.: 21) 207608/
agcaagttcagcctggttaagt (SEQ ID NO.: 22) Amplification of
.gamma.gt10 insert 207609/ ttatgagtatttcttccaggg (SEQ ID NO.: 23)
Amplification of .gamma.gt10 insert 232643/Est 13
ccattaatccagccgagccacacaag (SEQ ID NO.: 24) 232644/Est 14
catctacagctccgaacggtcagtg (SEQ ID NO.: 25) 233084
cagcggaagccccaaccgag (SEQ ID NO.: 26) 240655/hs 5
gggatgacgcCtcctCcgCCcgg (SEQ ID NO.: 27) 240656/hs 3
aagcttcacgtggaccagcaagccaagagtg (SEQ ID NO.: 28) 240857/hs 3c
aagctttttccgtccttccgtccgg (SEQ ID NO.: 29) 243068
atggtgagcaagggcgaggagctg (SEQ ID NO.: 30) 243069
cttgtacagctcgtccatgccgag (SEQ ID NO.: 31) 260812
GGGTGGTGAGAGTTCGTTGTCTGTC (SEQ ID NO.: 32) 260813
GAGCGATGAGGTACGGAAGACTCTG (SEQ ID NO.: 33)
[0248]
9TABLE 7 Nucleotide sequence of the recombinant plasmid pCR2.1-H-
SemaL (SEQ ID NO.: 34) 1 AGCGCCCAAT ACGCAAACCG CCTCTCCCCG
CGCGTTGGCC GATTCATTAA 51 TGCAGCTGGC ACGACAGGTT TCCCGACTGG
AAAGCGGGCA GTGAGCGCAA 101 CGCAATTAAT GTGAGTTAGC TCACTCATTA
GGCACCCCAG GCTTTACACT 151 TTATGCTTCC GGCTCGTATG TTGTGTGGAA
TTGTGAGCGG ATAACAATTT 201 CACACAGGAA ACAGCTATGA CCATGATTAC
GCCaagcttc acgtggacca 251 gcaagccaag agtgagtgtg ggcagcaccc
ccagccagag ggaggcagcc 301 agggcacagg catgacccag caggtgctcg
gccatgatgc cgtcctcggg 351 cagcagctgc cagtgctgag cctcgcggaa
gtaggagccc tcctgggcct 401 cgcagaagta gtggccgtac tgctgcgccg
tgaggttctc gatgaacagg 451 atgcagttgg ggctctggtg accaggttcg
cagctctgct ccacgttctc 501 cttgtggcgc catgagtagg tggcgtggcg
ggattccatg gggcagctca 551 ggtagtagcg agagtttggg gccagggaaa
ccttctgcag tggggccttg 601 tctggtttgg ggttgggaca ctccttgtgt
ggctcggctg gattaatgga 651 ttgcagcact gaccgttcgg agctgtagat
ggagatgcag cggccctggt 701 cccagccgca gtaggggtct cgggacatga
ggcaaccgtg gcagcccccg 751 ccatagacct cacacaggtc caggggcacc
tggctcacct cccactggga 801 gctcacatac agcttcctcc gctcagcatc
cagcgacatg gtctggatgg 851 cagccgcgcg gcggaagggc tggatctcca
tgatgttgaa ggcgaagctg 901 tgctcctgct cccccggttc caccaccttg
tggatagtgc ccctgtctgt 951 agttaggtaa agcacatgaa aggtctcccc
gtggctggct tgcatgcggt 1001 gaacggccac tttctggtag tggtatttag
agtggaacaa tggcgtcttc 1051 agaggcccca tgggctccac cctctgcgcc
acctctgggt gacggtcagc 1101 cacctggaag gtctctgtgg gtatcggctg
ctggtctggg aggcacttgc 1151 caggccgcgg gttgggaagg cttgagtggt
agcccttgag tgaggaggta 1201 cggaagacct tgtcaatgtc accgagggaa
tacacacaga cggctgagta 1251 gttccagggg ttggagaaaa caccatagac
cctggtgtcc ctccactggc 1301 cgctggggtc agggagcagg aagacgtctt
gcagcctgtt gaagttcttg 1351 ttggtggcag catcactgca taccagcatg
gctttcagaa aagtgttcca 1401 cttggagact gacagtgaac tttccccacc
ctggtccccc ctgcacaact 1451 gggccacacg ggacacattg agaggagcct
caggattctt gtcaggattg 1501 tcctctcgga agaagtagta gatcttgtca
tcgtaagcct ggtcttggtg 1551 cacgatggtg gctttgatga actgtgggtt
ctgcatgaca gtatcactgg 1601 tgtacagctc actctcgccc cggatgcggc
ggaaccgagg gatcttccca 1651 ttgtattcct gcttccggat ggtggaatac
acctcgtccc cttcaaacag 1701 aaccagggag ttctcgtccg ggctgaaggg
ggcgtagcct ctcatctcgc 1751 caagtggcac cacagtgcca ttcaccaggt
tccagcagct ggggtgccgg 1801 gcgttggtgc cacaggccag cagcccctca
ctccgcctct ccaggagagt 1851 gatgtagttc tcgcagtccc gcttatccag
acaggacccc tttgtggagc 1901 cgatattcac cgtgcgcaca gatgcgttct
tgccctcggg gaagtcaaag 1951 aggtagacct tgccacgtcc tcccacccac
acagaggagc tgcctggctc 2001 gtggaaaagc accgtgtgcg gctcagtctg
gccaaagtcc acccggtcct 2051 gccctacatg gcctttccag acggcgaaga
tgcggggtcc gctccttagg 2101 tggccctggg cggaggcggc ggccgcccag
agcagcagca gcagccgcag 2151 ccgcagcgga agccccaacc gagccggcgg
gccagggacg cgggcgcgcg 2201 gtgcgctggg ggcggcacgt ccgggcggag
gaggcgtcat cccaagccga 2251 attcTGCAGA TATCCATCAC ACTGGCGGCC
GCTCGAGCAT GCATCTAGAG 2301 GGCCCAATTC GCCCTATAGT GAGTCGTATT
ACAATTCACT GGCCGTCGTT 2351 TTACAACGTC GTGACTGGGA AAACCCTGGC
GTTACCCAAC TTAATCGCCT 2401 TGCAGCACAT CCCCCTTTCG CCAGCTGGCG
TAATAGCGAA GAGGCCCGCA 2451 CCGATCGCCC TTCCCAACAG TTGCGCAGCC
TGAATGGCGA ATGGGACGCG 2501 CCCTGTAGCG GCGCATTAAG CGCGGCGGGT
GTGGTGGTTA CGCGCAGCGT 2551 GACCGCTACA CTTGCCAGCG CCCTAGCGCC
CGCTCCTTTC GCTTTCTTCC 2601 CTTCCTTTCT CGCCACGTTC GCCGGCTTTC
CCCGTCAAGC TCTAAATCGG 2651 GGGCTCCCTT TAGGGTTCCG ATTTAGAGCT
TTACGGCACC TCGACCGCAA 2701 AAAACTTGAT TTGGGTGATG GTTCACGTAG
TGGGCCATCG CCCTGATAGA 2751 CGGTTTTTCG CCCTTTGACG TTGGAGTCCA
CGTTCTTTAA TAGTGGACTC 2801 TTGTTCCAAA CTGGAACAAC ACTCAACCCT
ATCGCGGTCT ATTCTTTTGA 2851 TTTATAAGGG ATTTTGCCGA TTTCGGCCTA
TTGGTTAAAA AATGAGCTGA 2901 TTTAACAAAT TCAGGGCGCA AGGGCTGCTA
AAGGAACCGG AACACGTAGA 2951 AAGCCAGTCC GCAGAAACGG TGCTGACCCC
GGATGAATGT CAGCTACTGG 3001 GCTATCTGGA CAAGGGAAAA CGCAAGCGCA
AAGAGAAAGC AGGTAGCTTG 3051 CAGTGGGCTT ACATGGCGAT AGCTAGACTG
GGCGGTTTTA TGGACAGCAA 3101 GCGAACCGGA ATTGCCAGCT GGGGCGCCCT
CTGGTAAGGT TGGGAAGCCC 3151 TGCAAAGTAA ACTGGATGGC TTTCTTGCCG
CCAAGGATCT GATGGCGCAG 3201 GGGATCAAGA TCTGATCAAG AGACAGGATG
AGGATCGTTT CGCATGATTG 3251 AACAAGATGG ATTGCACGCA GGTTCTCCGG
CCGCTTGGGT GGAGAGGCTA 3301 TTCGGCTATG ACTGGGCACA ACAGACAATC
GGCTGCTCTG ATGCCGCCGT 3351 GTTCCGGCTG TCAGCGCAGG GGCGCCCGGT
TCTTTTTGTC AAGACCGACC 3401 TGTCCGGTGC CCTGAATGAA CTGCAGGACG
AGGCAGCGCG GCTATCGTGG 3451 CTGGCCACGA CGGGCGTTCC TTGCGCAGCT
GTGCTCGACG TTGTCACTGA 3501 AGCGGGAAGG GACTGGCTGC TATTGGGCGA
AGTGCCGGGG CAGGATCTCC 3551 TGTCATCTCG CCTTGCTCCT GCCGAGAAAG
TATCCATCAT GGCTGATGCA 3601 ATGCGGCGGC TGCATACGCT TGATCCGGCT
ACCTGCCCAT TCGACCACCA 3651 AGCGAAACAT CGCATCGAGC GAGCACGTAC
TCGGATGGAA GCCGGTCTTG 3701 TCGATCAGGA TGATCTGGAC GAAGAGCATC
AGGGGCTCGC GCCAGCCGAA 3751 CTGTTCGCCA GGCTCAAGGC GCGCATGCCC
GACGGCGAGG ATCTCGTCGT 3801 GATCCATGGC GATGCCTGCT TGCCGAATAT
CATGGTGGAA AATGGCCGCT 3851 TTTCTGGATT CAACGACTGT GGCCGGCTGG
GTGTGGCGGA CCGCTATCAG 3901 GACATAGCGT TGGATACCCG TGATATTGCT
GAAGAGCTTG GCGGCGAATG 3951 GGCTGACCGC TTCCTCGTGC TTTACGGTAT
CGCCGCTCCC GATTCGCAGC 4001 GCATCGCCTT CTATCGCCTT CTTGACGAGT
TCTTCTGAAT TGAAAAAGGA 4051 AGAGTATGAG TATTCAACAT TTCCGTGTCG
CCCTTATTCC CTTTTTTGCG 4101 GCATTTTGCC TTCCTGTTTT TGCTCACCCA
GAAACGCTGG TGAAAGTAAA 4151 AGATGCTGAA GATCAGTTGG GTGCACGAGT
GGGTTACATC GAACTGGATC 4201 TCAACAGCGG TAAGATCCTT GAGAGTTTTC
GCCCCGAAGA ACGTTTTCCA 4251 ATGATGAGCA CTTTTAAAGT TCTGCTATGT
CATACACTAT TATCCCGTAT 4301 TGACGCCGGG CAAGAGCAAC TCGGTCGCCG
GGCGCGGTAT TCTCAGAATG 4351 ACTTGGTTGA GTACTCACCA GTCACAGAAA
AGCATCTTAC GGATGGCATG 4401 ACAGTAAGAG AATTATGCAG TGCTGCCATA
ACCATGAGTG ATAACACTGC 4451 GGCCAACTTA CTTCTGACAA CGATCGGAGG
ACCGAAGGAG CTAACCGCTT 4501 TTTTGCACAA CATGGGGGAT CATGTAACTC
GCCTTGATCG TTGGGAACCG 4551 GAGCTGAATG AAGCCATACC AAACGACGAG
AGTGACACCA CGATGCCTGT 4601 AGCAATGCCA ACAACGTTGC GCAAACTATT
AACTGGCGAA CTACTTACTC 4651 TAGCTTCCCG GCAACAA1TA ATAGACTGGA
TGGAGGCGGA TAAAGTTGCA 4701 GGACCACTTC TGCGCTCGGC CCTTCCGGCT
GGCTGGTTTA TTGCTGATAA 4751 ATCTGGAGCC GGTGAGCGTG GGTCTCGCGG
TATCATTGCA GCACTGGGGC 4801 CAGATGGTAA GCCCTCCCGT ATCGTAGTTA
TCTACACGAC GGGGAGTCAG 4851 GCAACTATGG ATGAACGAAA TAGACAGATC
GCTGAGATAG GTGCCTCACT 4901 GATTAAGCAT TGGTAACTGT CAGACCAAGT
TTACTCATAT ATACTTTAGA 4951 TTGATTTAAA ACTTCATTTT TAATTTAAAA
GGATCTAGGT GAAGATCCTT 5001 TTTGATAATC TCATGACCAA AATCCCTTAA
CGTGAGTTTT CGTTCCACTG 5051 AGCGTCAGAC CCCGTAGAAA AGATCAAAGG
ATCTTCTTGA GATCCTTTTT 5101 TTCTGCGCGT AATCTGCTGC TTGCAAACAA
AAAAACCACC GCTACCAGCG 5151 GTGGTTTGTT TGCCGGATCA AGAGCTACCA
ACTCTTTTTC CGAAGGTAAC 5201 TGGCTTCAGC AGAGCGCAGA TACCAAATAC
TGTCCTTCTA GTGTAGCCGT 5251 AGTTAGGCCA CCACTTCAAG AACTCTGTAG
CACCGCCTAC ATACCTCGCT 5301 CTGCTAATCC TGTTACCAGT GGCTGCTGCC
AGTGGCGATA AGTCGTGTCT 5351 TACCGGGTTG GACTCAAGAC GATAGTTACC
GGATAAGGCG CAGCGGTCGG 5401 GCTGAACGGG GGGTTCGTGC ACACAGCCCA
GCTTGGAGCG AACGACCTAC 5451 ACCGAACTGA GATACCTACA GCGTGAGCAT
TGAGAAAGCG CCACGCTTCC 5501 CGAAGGGAGA AAGGCGGACA GGTATCCGGT
AAGCGGCAGG GTCGGAACAG 5551 GAGAGCGCAC GAGGGAGCTT CCAGGGGGAA
ACGCCTGGTA TCTTTATAGT 5601 CCTGTCGGGT TTCGCCACCT CTGACTTGAG
CGTCGATTTT TGTGATGCTC 5651 GTCAGGGGGG CGGAGCCTAT GGAAAAACGC
CAGCAACGCG GCCTTTTTAC 5701 GGTTCCTGGC CTTTTGCTGG CCTTTTGCTC
ACATGTTCTT TCCTGCGTTA 5751 TCCCCTGATT CTGTGGATAA CCGTATTACC
GCCTTTGAGT GAGCTGATAC 5801 CGCTCGCCGC AGCCGAACGA CCGAGCGCAG
CGAGTCAGTG AGCGAGGAAG 5851 CGGAAG
[0249]
10TABLE 8 Nucleotide sequence of the recombinant expression plasmid
pCDNA3.1(-)H-SemaL-MycHisA (SEQ ID NO.: 35) 1 GACGGATCGG GAGATCTCCC
GATCCCCTAT GGTCGACTCT CAGTACAATC 51 TGCTCTGATG CCGCATAGTT
AAGCCAGTAT CTGCTCCCTG CTTGTGTGTT 101 GGAGGTCGCT GAGTAGTGCG
CGAGCAAAAT TTAAGCTACA ACAAGGCAAG 151 GCTTGACCGA CAATTGCATG
AAGAATCTGC TTAGGGTTAG GCGTTTTGCG 201 CTGCTTCGCG ATGTACGGGC
CAGATATACG CGTTGACATT GATTATTGAC 251 TAGTTATTAA TAGTAATCAA
TTACGGGGTC ATTAGTTCAT AGCCCATATA 301 TGGAGTTCCG CGTTACATAA
CTTACGGTAA ATGGCCCGCC TGGCTGACCG 351 CCCAACGACC CCCGCCCATT
GACGTCAATA ATGACGTATG TTCCCATAGT 401 AACGCCAATA GGGACTTTCC
ATTGACGTCA ATGGGTGGAC TATTTACGGT 451 AAACTGCCCA CTTGGCAGTA
CATCAAGTGT ATCATATGCC AAGTACGCCC 501 CCTATTGACG TCAATGACGG
TAAATGGCCC GCCTGGCATT ATGCCCAGTA 551 CATGACCTTA TGGGACTTTC
CTACTTGGCA GTACATCTAC GTATTAGTCA 601 TCGCTATTAC CATGGTGATG
CGGTTTTGGC AGTACATCAA TGGGCGTGGA 651 TAGCGGTTTG ACTCACGGGG
ATTTCCAAGT CTCCACCCCA TTGACGTCAA 701 TGGGAGTTTG TTTTGGCACC
AAAATCAACG GGACTTTCCA AAATGTCGTA 751 ACAAGTCCGC CCCATTGACG
CAAATGGGCG GTAGGCGTGT ACGGTGGGAG 801 GTCTATATAA GCAGAGCTCT
CTGGCTAACT AGAGAACCCA CTGCTTACTG 851 GCTTATCGAA ATTAATACGA
CTCACTATAG GGAGACCCAA GCTGGCTAGC 901 GTTTAAACGG GCCCTCTAGA
CTCGAGCGGC CGCCACTGTG CTGGATATCT 951 GcAgaattcg gcttgggatg
acgcctcctc cgcccggacg tgccgccccc 1001 agcgcaccgc gcgcccgcgt
ccctggcccg ccggctcggt tggggcttcc 1051 gctgcggctg cggctgctgc
tgctgctctg ggcggccgcc gcctccgccc 1101 agggccacct aaggagcgga
ccccgcatct tcgccgtctg gaaaggccat 1151 gtagggcagg accgggtgga
ctttggccag actgagccgc acacggtgct 1201 tttccacgag ccaggcagct
cctctgtgtg ggtgggagga cgtggcaagg 1251 tctacctctt tgacttcccc
gagggcaaga acgcatctgt gcgcacggtg 1301 aatatcggct ccacaaaggg
gtcctgtctg gataagcggg actgcgagaa 1351 ctacatcact ctcctggaga
ggcggagtga ggggctgctg gcctgtggca 1401 ccaacgcccg gcaccccagc
tgctggaacc tggtgaatgg cactgtggtg 1451 ccacttggcg agatgagagg
ctacgccccc ttcagcccgg acgagaactc 1501 cctggttctg tttgaagggg
acgaggtgta ttccaccatc cggaagcagg 1551 aatacaatgg gaagatccct
cggttccgcc gcatccgggg cgagagtgag 1601 ctgtacacca gtgatactgt
catgcagaac ccacagttca tcaaagccac 1651 catcgtgcac caagaccagg
cttacgatga caagatctac tacttcttcc 1701 gagaggacaa tcctgacaag
aatcctgagg ctcctctcaa tgtgtcccgt 1751 gtggcccagt tgtgcagggg
ggaccagggt ggggaaagtt cactgtcagt 1801 ctccaagtgg aacacttttc
tgaaagccat gctggtatgc agtgatgctg 1851 ccaccaacaa gaacttcaac
aggctgcaag acgtcttcct gctccctgac 1901 cccagcggcc agtggaggga
caccagggtc tatggtgttt tctccaaccc 1951 ctggaactac tcagccgtct
gtgtgtattc cctcggtgac attgacaagg 2001 tcttccgtac ctcctcactc
aagggctacc actcaagcct tcccaacccg 2051 cggcctggca agtgcctccc
agaccagcag ccgataccca cagagacctt 2101 ccaggtggct gaccgtcacc
cagaggtggc gcagagggtg gagcccatgg 2151 ggcctctgaa gacgccattg
ttccactcta aataccacta ccagaaagtg 2201 gccgttcacc gcatgcaagc
cagccacggg gagacctttc atgtgcttta 2251 cctaactaca gacaggggca
ctatccacaa ggtggtggaa ccgggggagc 2301 aggagcacag cttcgccttc
aacatcatgg agatccagcc cttccgccgc 2351 gcggctgcca tccagaccat
gtcgctggat gctgagcgga ggaagctgta 2401 tgtgagctcc cagtgggagg
tgagccaggt gcccctggac ctgtgtgagg 2451 tctatggcgg gggctgccac
ggttgcctca tgtcccgaga cccctactgc 2501 ggctgggacc agggccgctg
catctccatc tacagctccg aacggtcagt 2551 gctgcaatcc attaatccag
ccgagccaca caaggagtgt cccaacccca 2601 aaccagacaa ggccccactg
cagaaggttt ccctggcccc aaactctcgc 2651 tactacctga gctgccccat
ggaatcccgc cacgccacct actcatggcg 2701 ccacaaggag aacgtggagc
agagctgcga acctggtcac cagagcccca 2751 actgcatcct gttcatcgag
aacctcacgg cgcagcagta cggccactac 2801 ttctgcgagg cccaggaggg
ctcctacttc cgcgaggctc agcactggca 2851 gctgctgccc gaggacggca
tcatggccga gcacctgctg ggtcatgcct 2901 gtgccctggc tgcctccctc
tggctggggg tgctgcccac actcactctt 2951 ggcttgctgg tccacgtgaa
gcttGGGCCC GAACAAAAAC TCATCTCAGA 3001 AGAGGATCTG AATAGCGCCG
TCGACCATCA TCATCATCAT CATTGAGTTT 3051 AAACCGCTGA TCAGCCTCGA
CTGTGCCTTC TAGTTGCCAG CCATCTGTTG 3101 TTTGCCCCTC CCCCGTGCCT
TCCTTGACCC TGGAAGGTGC CACTCCCACT 3151 GTCCTTTCCT AATAAAATGA
GGAAATTGCA TCGCATTGTC TGAGTAGGTG 3201 TCATTCTATT CTGGGGGGTG
GGGTGGGGCA GGACAGCAAG GGGGAGGATT 3251 GGGAAGACAA TAGCAGGCAT
GCTGGGGATG CGGTGGGCTC TATGGCTTCT 3301 GAGGCGGAAA GAACCAGCTG
GGGCTCTAGG GGGTATCCCC ACGCGCCCTG 3351 TAGCGGCGCA TTAAGCGCGG
CGGGTGTGGT GGTTACGCGC AGCGTGACCG 3401 CTACACTTGC CAGCGGCCTA
GCGCCCGCTC CTTTCGCTTT CTTCCCTTCC 3451 TTTCTCGCCA CGTTCGCCGG
CTTTCCCCGT CAAGCTCTAA ATCGGGGCAT 3501 CCCTTTAGGG TTCCGATTTA
GTGCTTTACG GCACCTCGAC CCCAAAAAAC 3551 TTGATTAGGG TGATGGTTCA
CGTAGTGGGC CATCGCCCTG ATAGACGGTT 3601 TTTCGCCCTT TGACGTTGGA
GTCCACGTTC TTTAATAGTG GACTCTTGTT 3651 CCAAACTGGA ACAACACTCA
ACCCTATCTC GGTCTATTCT TTTGATTTAT 3701 AAGGGATTTT GGGGATTTCG
GCCTATTGGT TAAAAAATGA GCTGATTTAA 3751 CAAAAATTTA ACGCGAATTA
ATTCTGTGGA ATGTGTGTCA GTTAGGGTGT 3801 GGAAAGTCCC CAGGCTCCCC
AGGCAGGCAG AAGTATGCAA AGCATGCATC 3851 TCAATTAGTC AGCAACCAGG
TGTGGAAAGT CCCCAGGCTC CCCAGCAGGC 3901 AGAAGTATGC AAAGCATGCA
TCTCAATTAG TCAGCAACCA TAGTCCCGCC 3951 CCTAACTCCG CCCATCCCGC
CCCTAACTCC GCCCAGTTCC GCCCATTCTC 4001 CGCCCCATGG CTGACTAATT
TTTTTTATTT ATGCAGAGGC CGAGGCCGCC 4051 TCTGCCTCTG AGCTATTCCA
GAAGTAGTGA GGAGGCTT1T TTGGAGGCCT 4101 AGGCTTTTGC AAAAAGCTCC
CGGGAGCTTG TATATCCATT TTCGGATCTG 4151 ATCAAGAGAC AGGATGAGGA
TCGTTTCGCA TGATTGAACA AGATGGATTG 4201 CACGCAGGTT CTCCGGCCGC
TTGGGTGGAG AGGCTATTCG GCTATGACTG 4251 GGCACAACAG ACAATCGGCT
GCTCTGATGC CGCCGTGTTC CGGCTGTCAG 4301 CGCAGGGGCG CCCGGTTCTT
TTTGTCAAGA CCGACCTGTC CGGTGCCCTG 4351 AATGAACTGC AGGACGAGGC
AGCGCGGCTA TCGTGGCTGG CCACGACGGG 4401 CGTTCCTTGC GCAGCTGTGC
TCGACGTTGT CACTGAAGCG GGAAGGGACT 4451 GGCTGCTATT GGGCGAAGTG
CCGGGGCAGG ATCTCCTGTC ATCTCACCTT 4501 GCTCCTGCCG AGAAAGTATC
CATCATGGCT GATGCAATGC GGCGGCTGCA 4551 TACGCTTGAT CCGGCTACCT
GCCCATTCGA CCACCAAGCG AAACATCGCA 4601 TCGAGCGAGC ACGTACTCGG
ATGGAAGCCG GTCTTGTCGA TCAGGATGAT 4651 CTGGACGAAG AGCATCAGGG
GCTCGCGCCA GCCGAACTGT TCGCCAGGCT 4701 CAAGGCGCGC ATGCCCGACG
GCGAGGATCT CGTCGTGACC CATGGCGATG 4751 CCTGCTTGCC GAATATCATG
GTGGAAAATG GCCGCTTTTC TGGATTCATC 4801 GACTGTGGCC GGCTGGGTGT
GGCGGACCGC TATCAGGACA TAGCGTTGGC 4851 TACCCGTGAT ATTGCTGAAG
AGCTTGGCGG CGAATGGGCT GACCGCTTCC 4901 TCGTGCTTTA CGGTATCGCC
GCTCCCGATT CGCAGCGCAT CGCCTTCTAT 4951 CGCCTTCTTG ACGAGTTCTT
CTGAGCGGGA CTCTGGGGTT CGAAATGACC 5001 GACCAAGCGA CGCCCAACCT
GCCATCACGA GATTTCGATT CCACCGCCGC 5051 CTTCTATGAA AGGTTGGGCT
TCGGAATCGT TTTCCGGGAC GCCGGCTGGA 5101 TGATCCTCCA GCGCGGGGAT
CTCATGCTGG AGTTCTTCGC CCACCCCAAC 5151 TTGTTTATTG CAGCTTATAA
TGGTTACAAA TAAAGCAATA GCATCACAAA 5201 TTTCACAAAT AAAGCATTTT
TTTCACTGCA TTCTAGTTGT GGTTTGTCCA 5251 AACTCATCAA TGTATCTTAT
CATGTCTGTA TACCGTCGAC CTCTAGCTAG 5301 AGCTTGGCGT AATCATGGTC
ATAGCTGTTT CCTGTGTGAA ATTGTTATCC 5351 GCTCACAATT CCACACAACA
TACGAGCCGG AAGCATAAAG TGTAAAGCCT 5401 GGGGTGCCTA ATGAGTGAGC
TAACTCACAT TAATTGCGTT GCGCTCACTG 5451 CCCGCTTTCC AGTCGGGAAA
CCTGTCGTGC CAGCTGCATT AATGAATCGG 5501 CCAACGCGCG GGGAGAGGCG
GTTTGCGTAT TGGGCGCTCT TCCGCTTCCT 5551 CGCTCACTGA CTCGCTGCGC
TCGGTCGTTC GGCTGCGGCG AGCGGTATCA 5601 GCTCACTCAA AGGCGGTAAT
ACGGTTATCC ACAGAATCAG GGGATAACGC 5651 AGGAAAGAAC ATGTGAGCAA
AAGGCCAGCA AAAGGCCAGG AACCGTAAAA 5701 AGGCCGCGTT GCTGGCGTTT
TTCCATAGGC TCCGCCCCCC TGACGAGCAT 5751 CACAAAAATC GACGCTCAAG
TCAGAGGTGG CGAAACCCGA CAGGACTATA 5801 AAGATACCAG GCGTTTCCCC
CTGGAAGCTC CCTCGTGCGC TCTCCTGTTC 5851 CGACCCTGCC GCTTACCGGA
TACCTGTCCG CCTTTCTCCC TTCGGGAAGC 5901 GTGGCGCTTT CTCAATGCTC
ACGCTGTAGG TATCTCAGTT CGGTGTAGGT 5951 CGTTCGCTCC AAGCTGGGCT
GTGTGCACGA ACCCCCCGTT CAGCCCGACC 6001 GCTGCGCCTT ATCCGGTAAC
TATCGTCTTG AGTCCAACCC GGTAAGACAC 6051 GACTTATCGC CACTGGCAGC
AGCCACTGGT AACAGGATTA GCAGAGCGAG 6101 GTATGTAGGC GGTGCTACAG
AGTTCTTGAA GTGGTGGCCT AACTACGGCT 6151 ACACTAGAAG GACAGTATTT
GGTATCTGCG CTCTGCTGAA GCCAGTTACC 6201 TTCGGAAAAA GAGTTGGTAG
CTCTTGATCC GGCAAACAAA CCACCGCTGG 6251 TAGCGGTGGT TTTTTTGTTT
GCAAGCAGCA GATTACGCGC AGAAAAAAAG 6301 GATCTCAAGA AGATCCTTTG
ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG 6351 AACGAAAACT CACGTTAAGG
GATTTTGGTC ATGAGATTAT CAAAAAGGAT 6401 CTTCACCTAG ATCCTTTTAA
ATTAAAAATG AAGTTTTAAA TCAATCTAAA 6451 GTATATATGA GTAAACTTGG
TCTGACAGTT ACCAATGCTT AATCAGTGAG 6501 GCACCTATCT CAGCGATCTG
TCTATTTCGT TCATCCATAG TTGCCTGACT 6551 CCCCGTCGTG TAGATAACTA
CGATACGGGA GGGCTTACCA TCTGGCCCCA 6601 GTGCTGCAAT GATACCGCGA
GACCCACGCT CACCGGCTCC AGATTTATCA 6651 GCAATAAACC AGCCAGCCGG
AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC 6701 TTTATCCGCC TCCATCCAGT
CTATTAATTG TTGCCGGGAA GCTAGAGTAA 6751 GTAGTTCGCC AGTTAATAGT
TTGCGCAACG TTGTTGCCAT TGCTACAGGC 6801 ATCGTGGTGT CACGCTCGTC
GTTTGGTATG GCTTCATTCA GCTCCGGTTC 6851 CCAACGATCA AGGCGAGTTA
CATGATCCCC CATGTTGTGC AAAAAAGCGG 6901 TTAGCTCCTT CGGTCCTCCG
ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG 6951 TTATCACTCA TGGTTATGGC
AGCACTGCAT AATTCTCTTA CTGTCATGCC 7001 ATCCGTAAGA TGCTTTTCTG
TGACTGGTGA GTACTCAACC AAGTCATTCT 7051 GAGAATAGTG TATGCGGCGA
CCGAGTTGCT CTTGCCCGGC GTCAATACGG 7101 GATAATACCG CGCCACATAG
CAGAACTTTA AAAGTGCTCA TCATTGGAAA 7151 ACGTTCTTCG GGGCGAAAAC
TCTCAAGGAT CTTACCGCTG TTGAGATCCA 7201 GTTCGATGTA ACCCACTCGT
GCACCCAACT GATCTTCAGC ATCTTTTACT 7251 TTCACCAGCG TTTCTGGGTG
AGCAAAAACA GGAAGGCAAA ATGCCGCAAA 7301 AAAGGGAATA AGGGCGACAC
GGAAATGTTG AATACTCATA CTCTTCCTTT 7351 TTCAATATTA TTGAAGCATT
TATCAGGGTT ATTGTCTCAT GAGCGGATAC 7401 ATATTTGAAT GTATTTAGAA
AAATAAACAA ATAGGGGTTC CGCGCACATT 7451 TCCCCGAAAA GTGCCACCTG
ACGTC
[0250]
11TABLE 9 Nucleotide sequence of the recombinant plasmid
pcDNA3.1-H- SemaL-EGFP-MychisA (SEQ ID NO.: 36) 1 GACGGATCGG
GAGATCTCCC GATCCCCTAT GGTCGACTCT CAGTACAATC 51 TGCTCTGATG
CCGCATAGTT AAGCCAGTAT CTGCTCCCTG CTTGTGTGTT 101 GGAGGTCGCT
GAGTAGTGCG CGAGCAAAAT TTAAGCTACA ACAAGGCAAG 151 GCTTGACCGA
CAATTGCATG AAGAATCTGC TTAGGGTTAG GCGTTTTGCG 201 CTGCTTCGCG
ATGTACGGGC CAGATATACG CGTTGACATT GATTATTGAC 251 TAGTTATTAA
TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA 301 TGGAGTTCCG
CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG 351 CCCAACGACC
CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT 401 AACGCCAATA
GGGACTTTCC ATTGACGTCA ATGGGTGGAC TATTTACGGT 451 AAACTGCCCA
CTTGGCAGTA CATCAAGTGT ATCATATGCC AAGTACGCCC 501 CCTATTGACG
TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA 551 CATGACCTTA
TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA 601 TCGCTATTAC
CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA 651 TAGCGGTTTG
ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA 701 TGGGAGTTTG
TTTTGGCACC AAAATCAACG GGACTTTCCA AAATGTCGTA 751 ACAACTCCGC
CCCATTGACG CAAATGGGCG GTAGGCGTGT ACGGTGGGAG 801 GTCTATATAA
GCAGAGCTCT CTGGCTAACT AGAGAACCCA CTGCTTACTG 851 GCTTATCGAA
ATTAATACGA CTCACTATAG GGAGACCCAA GCTGGCTAGC 901 GTTTAAACGG
GCCCTCTAGA CTCGAGCGGC CGCCACTGTG CTGGATATCT 951 GCAgaattcg
gcttgggatg acgcctcctc cgcccggacg tgccgccccc 1001 agcgcaccgc
gcgcccgcgt ccctggcccg ccggctcggt tggggcttcc 1051 gctgcggctg
cggctgctgc tgctgctctg ggcggccgcc gcctccgccc 1101 agggccacct
aaggagcgga ccccgcatct tcgccgtctg gaaaggccat 1151 gtagggcagg
accgggtgga ctttggccag actgagccgc acacggtgct 1201 tttccacgag
ccaggcagct cctctgtgtg ggtgggagga cgtggcaagg 1251 tctacctctt
tgacttcccc gagggcaaga acgcatctgt gcgcacggtg 1301 aatatcggct
ccacaaaggg gtcctgtctg gataagcggg actgcgagaa 1351 ctacatcact
ctcctggaga ggcggagtga ggggctgctg gcctgtggca 1401 ccaacgcccg
gcaccccagc tgctggaacc tggtgaatgg cactgtggtg 1451 ccacttggcg
agatgagagg ctacgccccc ttcagcccgg acgagaactc 1501 cctggttctg
tttgaagggg acgaggtgta ttccaccatc cggaagcagg 1551 aatacaatgg
gaagatccct cggttccgcc gcatccgggg cgagagtgag 1601 ctgtacacca
gtgatactgt catgcagaac ccacagttca tcaaagccac 1651 catcgtgcac
caagaccagg cttacgatga caagatctac tacttcttcc 1701 gagaggacaa
tcctgacaag aatcctgagg ctcctctcaa tgtgtcccgt 1751 gtggcccagt
tgtgcagggg ggaccagggt ggggaaagtt cactgtcagt 1801 ctccaagtgg
aacacttttc tgaaagccat gctggtatgc agtgatgctg 1851 ccaccaacaa
gaacttcaac aggctgcaag acgtcttcct gctccctgac 1901 cccagcggcc
agtggaggga caccagggtc tatggtgttt tctccaaccc 1951 ctggaactac
tcagccgtct gtgtgtattc cctcggtgac attgacaagg 2001 tcttccgtac
ctcctcactc aagggctacc actcaagcct tcccaacccg 2051 cggcctggca
agtgcctccc agaccagcag ccgataccca cagagacctt 2101 ccaggtggct
gaccgtcacc cagaggtggc gcagagggtg gagcccatgg 2151 ggcctctgaa
gacgccattg ttccactcta aataccacta ccagaaagtg 2201 gccgttcacc
gcatgcaagc cagccacggg gagacctttc atgtgcttta 2251 cctaactaca
gacaggggca ctatccacaa ggtggtggaa ccgggggagc 2301 aggagcacag
cttcgccttc aacatcatgg agatccagcc cttccgccgc 2351 gcggctgcca
tccagaccat gtcgctggat gctgagcgga ggaagctgta 2401 tgtgagctcc
cagtgggagg tgagccaggt gcccctggac ctgtgtgagg 2451 tctatggcgg
gggctgccac ggttgcctca tgtcccgaga cccctactgc 2501 ggctgggacc
agggccgctg catctccatc tacagctccg aacggtcagt 2551 gctgcaatcc
attaatccag ccgagccaca caaggagtgt cccaacccca 2601 aaccagacaa
ggccccactg cagaaggttt ccctggcccc aaactctcgc 2651 tactacctga
gctgccccat ggaatcccgc cacgccacct actcatggcg 2701 ccacaaggag
aacgtggagc agagctgcga acctggtcac cagagcccca 2751 actgcatcct
gttcatcgag aacctcacgg cgcagcagta cggccactac 2801 ttctgcgagg
cccaggaggg ctcctacttc cgcgaggctc agcactggca 2851 gctgctgccc
gaggacggca tcatggccga gcacctgctg ggtcatgcct 2901 gtgccctggc
tgcctccctc tggctggggg tgctgcccac actcactctt 2951 ggcttgctgg
tccacATGGT GAGCAAGGGC GAGGAGCTGT TCACCGGGGT 3001 GGTGCCCATC
CTGGTCGAGC TGGACGGCGA CGTAAACGGC CACAAGTTCA 3051 GCGTGTCCGG
CGAGGGCGAG GGCGATGCCA CCTACGGCAA GCTGACCCTG 3101 AAGTTCATCT
GCACCACCGG CAAGCTGCCC GTGCCCTGGC CCACCCTCGT 3151 GACCACCCTG
ACCTACGGCG TGCAGTGCTT CAGCCGCTAC CCCGACCACA 3201 TGAAGCAGCA
CGACTTCTTC AAGTCCGCCA TGCCCGAAGG CTACGTCCAG 3251 GAGCGCACCA
TCTTCTTCAA GGACGACGGC AACTACAAGA CCCGCGCCGA 3301 GGTGAAGTTC
GAGGGCGACA CCCTGGTGAA CCGCATCGAG CTGAAGGGCA 3351 TCGACTTCAA
GGAGGACGGC AACATCCTGG GGCACAAGCT GGAGTACAAC 3401 TACAACAGCC
ACAACGTCTA TATCATGGCC GACAAGCAGA AGAACGGCAT 3451 CAAGGTGAAC
TTCAAGATCC GCCACAACAT CGAGGACGGC AGCGTGCAGC 3501 TCGCCGACCA
CTACCAGCAG AACACCCCCA TCGGCGACGG CCCCGTGCTG 3551 CTGCCCGACA
ACCACTACCT GAGCACCCAG TCCGCCCTGA GCAAAGACCC 3601 CAACGAGAAG
CGCGATCACA TGGTCCTGCT GGAGTTCGTG ACCGCCGCCG 3651 GGATCACTCT
CGGCATGGAC GAGCTGTACA Aggtgaagct tGGGCCCGAA 3701 CAAAAACTCA
TCTCAGAAGA GGATCTGAAT AGCGCCGTCG ACCATCATCA 3751 TCATCATCAT
TGAGTTTAAA CCGCTGATCA GCCTCGACTG TGCCTTCTAG 3801 TTGCCAGCCA
TCTGTTGTTT GCCCCTCCCC CGTGCCTTCC TTGACCCTGG 3851 AAGGTGCCAC
TCCCACTGTC CTTTCCTAAT AAAATGAGGA AATTGCATCG 3901 CATTGTCTGA
GTAGGTGTCA TTCTATTCTG GGGGGTGGGG TGGGGCAGGA 3951 CAGCAAGGGG
GAGGATTGGG AAGACAATAG CAGGCATGCT GGGGATGCGG 4001 TGGGCTCTAT
GGCTTCTGAG GCGGAAAGAA CCAGCTGGGG CTCTAGGGGG 4051 TATCCCCACG
CGCCCTGTAG CGGCGCATTA AGCGCGGCGG GTGTGGTGGT 4101 TACGCGCAGC
GTGACCGCTA CACTTGCCAG CGCCCTAGCG CCCGCTCCTT 4151 TCGCTTTCTT
CCCTTCCTTT CTCGCCACGT TCGCCGGCTT TCCCCGTCAA 4201 GCTCTAAATC
GGGGCATCCC TTTAGGGTTC CGATTTAGTG CTTTACGGCA 4251 CCTCGACCCC
AAAAAACTTG ATTAGGGTGA TGGTTCACGT AGTGGGCCAT 4301 CGCCCTGATA
GACGGTTTTT CGCCCTTTGA CGTTGGAGTC CACGTTCTTT 4351 AATAGTGGAC
TCTTGTTCCA AACTGGAACA ACACTCAACC CTATCTCGGT 4401 CTATTCTTTT
GATTTATAAG GGATTTTGGG GATTTCGGCC TATTGGTTAA 4451 AAAATGAGCT
GATTTAACAA AAATTTAACG CGAATTAATT CTGTGGAATG 4501 TGTGTCAGTT
AGGGTGTGGA AAGTCCCCAG GCTCCCCAGG CAGGCAGAAG 4551 TATGCAAAGC
ATGCATCTCA ATTAGTCAGC AACCAGGTGT GGAAAGTCCC 4601 CAGGCTCCCC
AGCAGGCAGA AGTATGCAAA GCATGCATCT CAATTAGTCA 4651 GCAACCATAG
TCCCGCCCCT AACTCCGCCC ATCCCGCCCC TAACTCCGCC 4701 CAGTTCCGCC
CATTCTCCGC CCCATGGCTG ACTAATTTTT TTTATTTATG 4751 CAGAGGCCGA
GGCCGCCTCT GCCTCTGAGC TATTCCAGAA GTAGTGAGGA 4801 GGCTTTTTTG
GAGGCCTAGG CTTTTGCAAA AAGCTCCCGG GAGCTTGTAT 4851 ATCCATTTTC
GGATCTGATC AAGAGACAGG ATGAGGATCG TTTCGCATGA 4901 TTGAACAAGA
TGGATTGCAC GCAGGTTCTC CGGCCGCTTG GGTGGAGAGG 4951 CTATTCGGCT
ATGACTGGGC ACAACAGACA ATCGGCTGCT CTGATGCCGC 5001 CGTGTTCCGG
CTGTCAGCGC AGGGGCGCCC GGTTCTTTTT GTCAAGACCG 5051 ACCTGTCCGG
TGCCCTGAAT GAACTGCAGG ACGAGGCAGC GCGGCTATCG 5101 TGGCTGGCCA
CGACGGGCGT TCCTTGCGCA GCTGTGCTCG ACGTTGTCAC 5151 TGAAGCGGGA
AGGGACTGGC TGCTATTGGG CGAAGTGCCG GGGCAGGATC 5201 TCCTGTCATC
TCACCTTGCT CCTGCGGAGA AAGTATCCAT CATGGCTGAT 5251 GCAATGCGGC
GGCTGCATAC GCTTGATCCG GCTACCTGCC CATTCGACCA 5301 CCAAGCGAAA
CATCGCATCG AGCGAGCACG TACTCGGATG GAAGCCGGTC 5351 TTGTCGATCA
GGATGATCTG GACGAAGAGC ATCAGGGGCT CGCGCCAGCC 5401 GAACTGTTCG
CCAGGCTCAA GGCGCGCATG CCCGACGGCG AGGATCTCGT 5451 CGTGACCCAT
GGCGATGCCT GCTTGCCGAA TATCATGGTG GAAAATGGCC 5501 GCTTTTCTGG
ATTCATCGAC TGTGGCCGGC TGGGTGTGGC GGACCGCTAT 5551 CAGGACATAG
CGTTGGCTAC CCGTGATATT GCTGAAGAGC TTGGCGGCGA 5601 ATGGGCTGAC
CGCTTCCTCG TGCTTTACGG TATCGCCGCT CCCGATTCGC 5651 AGCGCATCGC
CTTCTATCGC CTTCTTGACG AGTTCTTCTG AGCGGGACTC 5701 TGGGGTTCGA
AATGACCGAC CAAGCGACGC CCAACCTGCC ATCACGAGAT 5751 TTCGATTCCA
CCGCCGCCTT CTATGAAAGG TTGGGCTTCG GAATCGTTTT 5801 CCGGGACGCC
GGCTGGATGA TCCTCCAGCG CGGGGATCTC ATGCTGGAGT 5851 TCTTCGCCCA
CCCCAACTTG TTTATTGCAG CTTATAATGG TTACAAATAA 5901 AGCAATAGCA
TCACAAATTT CACAAATAAA GCATTTTTTT CACTGCATTC 5951 TAGTTGTGGT
TTGTCCAAAC TCATCAATGT ATCTTATCAT GTCTGTATAC 6001 CGTCGACCTC
TAGCTAGAGC TTGGCGTAAT CATGGTCATA GCTGTTTCCT 6051 GTGTGAAATT
GTTATCCGCT CACAATTCCA CACAACATAC GAGCCGGAAG 6101 CATAAAGTGT
AAAGCCTGGG GTGCCTAATG AGTGAGCTAA CTCACATTAA 6151 TTGCGTTGCG
CTCACTGCCC GCTTTCCAGT CGGGAAACCT GTCGTGCCAG 6201 CTGCATTAAT
GAATCGGCCA ACGCGCGGGG AGAGGCGGTT TGCGTATTGG 6251 GCGCTCTTCC
GCTTCCTCGC TCACTGACTC GCTGCGCTCG GTCGTTCGGC 6301 TGCGGCGAGC
GGTATCAGCT CACTCAAAGG CGGTAATACG GTTATCCACA 6351 GAATCAGGGG
ATAACGCAGG AAAGAACATG TGAGCAAAAG GCCAGCAAAA 6401 GGCCAGGAAC
CGTAAAAAGG CCGCGTTGCT GGCGTTTTTC CATAGGCTCC 6451 GCCCCCCTGA
CGAGCATCAC AAAAATCGAC GCTCAAGTCA GAGGTGGCGA 6501 AACCCGACAG
GACTATAAAG ATACCAGGCG TTTCCCCCTG GAAGCTCCCT 6551 CGTGCGCTCT
CCTGTTCCGA CCCTGCCGCT TACCGGATAC CTGTCCGCCT 6601 TTCTCCCTTC
GGGAAGCGTG GCGCTTTCTC AATGCTCACG CTGTAGGTAT 6651 CTCAGTTCGG
TGTAGGTCGT TCGCTCCAAG CTGGGCTGTG TGCACGAACC 6701 CCCCGTTCAG
CCCGACCGCT GCGCCTTATC CGGTAACTAT CGTCTTGAGT 6751 CCAACCCGGT
AAGACACGAC TTATCGCCAC TGGCAGCAGC CACTGGTAAC 6801 AGGATTAGGA
GAGCGAGGTA TGTAGGCGGT GCTACAGAGT TCTTGAAGTG 6851 GTGGCCTAAC
TACGGCTACA CTAGAAGGAC AGTATTTGGT ATCTGCGCTC 6901 TGCTGAAGCC
AGTTACCTTC GGAAAAAGAG TTGGTAGCTC TTGATCCGGC 6951 AAACAAACCA
CCGCTGGTAG CGGTGGTTTT TTTGTTTGCA AGCAGCAGAT 7001 TACGCGCAGA
AAAAAAGGAT CTCAAGAAGA TCCTTTGATC TTTTCTACGG 7051 GGTCTGACGC
TCAGTGGAAC GAAAACTCAC GTTAAGGGAT TTTGGTCATG 7101 AGATTATCAA
AAAGGATCTT CACCTAGATC CTTTTAAATT AAAAATGAAG 7151 TTTTAAATCA
ATCTAAAGTA TATATGAGTA AACTTGGTCT GACAGTTACC 7201 AATGCTTAAT
CAGTGAGGCA CCTATCTCAG CGATCTGTCT ATTTCGTTCA 7251 TCCATAGTTG
CCTGACTCCC CGTCGTGTAG ATAACTACGA TACGGGAGGG 7301 CTTACCATCT
GGCCCCAGTG CTGCAATGAT ACCGCGAGAC CCACGCTCAC 7351 CGGCTCCAGA
TTTATCAGCA ATAAACCAGC CAGCCGGAAG GGCCGAGCGC 7401 AGAAGTGGTC
CTGCAACTTT ATCCGCCTCC ATCCAGTCTA TTAATTGTTG 7451 CCGGGAAGCT
AGAGTAAGTA GTTCGCCAGT TAATAGTTTG CGCAACGTTG 7501 TTGCCATTGC
TACAGGCATC GTGGTGTCAC GCTCGTCGTT TGGTATGGCT 7551 TCATTCAGCT
CCGGTTCCCA ACGATCAAGG CGAGTTACAT GATCCCCCAT 7601 GTTGTGCAAA
AAAGCGGTTA GCTCCTTCGG TCCTCCGATC GTTGTCAGAA 7651 GTAAGTTGGC
CGCAGTGTTA TCACTCATGG TTATGGCAGC ACTGCATAAT 7701 TCTCTTACTG
TCATGCCATC CGTAAGATGC TTTTCTGTGA CTGGTGAGTA 7751 CTCAACCAAG
TCATTCTGAG AATAGTGTAT GCGGCGACCG AGTTGCTCTT 7801 GCCCGGCGTC
AATACGGGAT AATACCGCGC CACATAGCAG AACTTTAAAA 7851 GTGCTCATCA
TTGGAAAACG TTCTTCGGGG CGAAAACTCT CAAGGATCTT 7901 ACCGCTGTTG
AGATCCAGTT CGATGTAACC CACTCGTGCA CCCAACTGAT 7951 CTTCAGCATC
TTTTACTTTC ACCAGCGTTT CTGGGTGAGC AAAAACAGGA 8001 AGGCAAAATG
CCGCAAAAAA GGGAATAAGG GCGACACGGA AATGTTGAAT 8051 ACTCATACTC
TTCCTTTTTC AATATTATTG AAGCATTTAT CAGGGTTATT 8101 GTCTCATGAG
CGGATACATA TTTGAATGTA TTTAGAAAAA TAAACAAATA 8151 GGGGTTCCGC
GCACATTTCC CCGAAAAGTG CCACCTGACG TC
[0251]
12TABLE 10 Nucleotide sequence of the recombinant plasmid pIND-H-
SemaL-EE (SEQ ID NO.:37) 1 AGATCTCGGC CGCATATTAA GTGCATTGTT
CTCGATACCG CTAAGTGCAT 51 TGTTCTCGTT AGCTCGATGG ACAAGTGCAT
TGTTCTCTTG CTGAAAGCTC 101 GATGGACAAG TGCATTGTTC TCTTGCTGAA
AGCTCGATGG ACAAGTGCAT 151 TGTTCTCTTG CTGAAAGCTC AGTACCCGGG
AGTACCCTCG ACCGCCGGAG 201 TATAAATAGA GGCGCTTCGT CTACGGAGCG
ACAATTCAAT TCAAACAAGC 251 AAAGTGAACA CGTCGCTAAG CGAAAGCTAA
GCAAATAAAC AAGCGCAGCT 301 GAACAAGCTA AACAATCTGC AGTAAAGTGC
AAGTTAAAGT GAATCAATTA 351 AAAGTAACCA GCAACCAAGT AAATCAACTG
CAACTACTGA AATCTGCCAA 401 GAAGTAATTA TTGAATACAA GAAGAGAACT
CTGAATACTT TCAACAAGTT 451 ACCGAGAAAG AAGAACTCAC ACACAGCTAG
CGTTTAAACT TAAGCTTGGT 501 ACCGAGCTCG GATCCACTAG TCCAGTGTGG
TGgaattcgg cttgggatga 551 cgcctcctcc gcccggacgt gccgccccca
gcgcaccgcg cgcccgcgtc 601 cctggcccgc cggctcggtt ggggcttccg
ctgcggctgc ggctgctgct 651 gctgctctgg gcggccgccg cctccgccca
gggccaccta aggagcggac 701 cccgcatctt cgccgtctgg aaaggccatg
tagggcagga ccgggtggac 751 tttggccaga ctgagccgca cacggtgctt
ttccacgagc caggcagctc 801 ctctgtgtgg gtgggaggac gtggcaaggt
ctacctcttt gacttccccg 851 agggcaagaa cgcatctgtg cgcacggtga
atatcggctc cacaaagggg 901 tcctgtctgg ataagcggga ctgcgagaac
tacatcactc tcctggagag 951 gcggagtgag gggctgctgg cctgtggcac
caacgcccgg caccccagct 1001 gctggaacct ggtgaatggc actgtggtgc
cacttggcga gatgagaggc 1051 tacgccccct tcagcccgga cgagaactcc
ctggttctgt ttgaagggga 1101 cgaggtgtat tccaccatcc ggaagcagga
atacaatggg aagatccctc 1151 ggttccgccg catccggggc gagagtgagc
tgtacaccag tgatactgtc 1201 atgcagaacc cacagttcat caaagccacc
atcgtgcacc aagaccaggc 1251 ttacgatgac aagatctact acttcttccg
agaggacaat cctgacaaga 1301 atcctgaggc tcctctcaat gtgtcccgtg
tggcccagtt gtgcaggggg 1351 gaccagggtg gggaaagttc actgtcagtc
tccaagtgga acacttttct 1401 gaaagccatg ctggtatgca gtgatgctgc
caccaacaag aacttcaaca 1451 ggctgcaaga cgtcttcctg ctccctgacc
ccagcggcca gtggagggac 1501 accagggtct atggtgtttt ctccaacccc
tggaactact cagccgtctg 1551 tgtgtattcc ctcggtgaca ttgacaaggt
cttccgtacc tcctcactca 1601 agggctacca ctcaagcctt cccaacccgc
ggcctggcaa gtgcctccca 1651 gaccagcagc cgatacccac agagaccttc
caggtggctg accgtcaccc 1701 agaggtggcg cagagggtgg agcccatggg
gcctctgaag acgccattgt 1751 tccactctaa ataccactac cagaaagtgg
ccgttcaccg catgcaagcc 1801 agccacgggg agacctttca tgtgctttac
ctaactacag acaggggcac 1851 tatccacaag gtggtggaac cgggggagca
ggagcacagc ttcgccttca 1901 acatcatgga gatccagccc ttccgccgcg
cggctgccat ccagaccatg 1951 tcgctggatg ctgagcggag gaagctgtat
gtgagctccc agtgggaggt 2001 gagccaggtg cccctggacc tgtgtgaggt
ctatggcggg ggctgccacg 2051 gttgcctcat gtcccgagac ccctactgcg
gctgggacca gggccgctgc 2101 atctccatct acagctccga acggtcagtg
ctgcaatcca ttaatccagc 2151 cgagccacac aaggagtgtc ccaaccccaa
accagacaag gccccactgc 2201 agaaggtttc cctggcccca aactctcgct
actacctgag ctgccccatg 2251 gaatcccgcc acgccaccta ctcatggcgc
cacaaggaga acgtggagca 2301 gagctgcgaa cctggtcacc agagccccaa
ctgcatcctg ttcatcgaga 2351 acctcacggc gcagcagtac ggccactact
tctgcgaggc ccaggagggc 2401 tcctacttcc gcgaggctca gcactggcag
ctgctgcccg aggacggcat 2451 catggccgag cacctgctgg gtcatgcctg
tgccctggct gcctccctct 2501 ggctgggggt gctgcccaca ctcactcttg
gcttgctggt ccacgtgaag 2551 cttGGGCCCG TTTAAACCCG CTGATCAGCC
TCGACTGTGC CTTCTAGTTG 2601 CCAGCCATCT GTTGTTTGCC CCTCCCCCGT
GCCTTCCTTG ACCCTGGAAG 2651 GTGCCACTCC CACTGTCCTT TCCTAATAAA
ATGAGGAAAT TGCATCGCAT 2701 TGTCTGAGTA GGTGTCATTC TATTCTGGGG
GGTGGGGTGG GGCAGGACAG 2751 CAAGGGGGAG GATTGGGAAG ACAATAGCAG
GCATGCTGGG GATGCGGTGG 2801 GCTCTATGGC TTCTGAGGCG GAAAGAACCA
GCTGGGGCTC TAGGGGGTAT 2851 CCCCACGCGC CCTGTAGCGG CGCATTAAGC
GCGGCGGGTG TGGTGGTTAC 2901 GCGCAGCGTG ACCGCTACAC TTGCCAGCGC
CCTAGCGCCC GCTCCTTTCG 2951 CTTTCTTCCC TTCCTTTCTC GCCACGTTCG
CCGGCTTTCC CCGTCAAGCT 3001 CTAAATCGGG GCATCCCTTT AGGGTTCCGA
TTTAGTGCTT TACGGCACCT 3051 CGACCCCAAA AAACTTGATT AGGGTGATGG
TTCACGTAGT GGGCCATCGC 3101 CCTGATAGAC GGTTTTTCGC CCTTTGACGT
TGGAGTCCAC GTTCTTTAAT 3151 AGTGGACTCT TGTTCCAAAC TGGAACAACA
CTCAACCCTA TCTCGGTCTA 3201 TTCTTTTGAT TTATAAGGGA TTTTGGGGAT
TTCGGCCTAT TGGTTAAAAA 3251 ATGAGCTGAT TTAACAAAAA TTTAACGCGA
ATTAATTCTG TGGAATGTGT 3301 GTCAGTTAGG GTGTGGAAAG TCCCCAGGCT
CCCCAGGCAG GCAGAAGTAT 3351 GCAAAGCATG CATCTCAATT AGTCAGCAAC
CAGGTGTGGA AAGTCCCCAG 3401 GCTCCCCAGC AGGCAGAAGT ATGCAAAGCA
TGCATCTCAA TTAGTCAGCA 3451 ACCATAGTCC CGCCCCTAAC TCCGCCCATC
CCGCCCCTAA CTCCGCCCAG 3501 TTCCGCCCAT TCTCCGCCCC ATGGCTGACT
AATTTTTTTT ATTTATGCAG 3551 AGGCCGAGGC CGCCTCTGCC TCTGAGCTAT
TCCAGAAGTA GTGAGGAGGC 3601 TTTTTTGGAG GCCTAGGCTT TTGCAAAAAG
CTCCCGGGAG CTTGTATATC 3651 CATTTTCGGA TCTGATCAAG AGACAGGATG
AGGATCGTTT CGCATGATTG 3701 AACAAGATGG ATTGCACGCA GGTTCTCCGG
CCGCTTGGGT GGAGAGGCTA 3751 TTCGGCTATG ACTGGGCACA ACAGACAATC
GGCTGCTCTG ATGCCGCCGT 3801 GTTCCGGCTG TCAGCGCAGG GGCGCCCGGT
TCTTTTTGTC AAGACCGACC 3851 TGTCCGGTGC CCTGAATGAA CTGCAGGACG
AGGCAGCGCG GCTATCGTGG 3901 CTGGCCACGA CGGGCGTTCC TTGCGCAGCT
GTGCTCGACG TTGTCACTGA 3951 AGCGGGAAGG GACTGGCTGC TATTGGGCGA
AGTGCCGGGG CAGGATCTCC 4001 TGTCATCTCA CCTTGCTCCT GCCGAGAAAG
TATCCATCAT GGCTGATGCA 4051 ATGCGGCGGC TGCATACGCT TGATCCGGCT
ACCTGCCCAT TCGACCACCA 4101 AGCGAAACAT CGCATCGAGC GAGCACGTAC
TCGGATGGAA GCCGGTCTTG 4151 TCGATCAGGA TGATCTGGAC GAAGAGCATC
AGGGGCTCGC GCCAGCCGAA 4201 CTGTTCGCCA GGCTCAAGGC GCGCATGCCC
GACGGCGAGG ATCTCGTCGT 4251 GACCCATGGC GATGCCTGCT TGCCGAATAT
CATGGTGGAA AATGGCCGCT 4301 TTTCTGGATT CATCGACTGT GGCCGGCTGG
GTGTGGCGGA CCGCTATCAG 4351 GACATAGCGT TGGCTACCCG TGATATTGCT
GAAGAGCTTG GCGGCGAATG 4401 GGCTGACCGC TTCCTCGTGC TTTACGGTAT
CGCCGCTCCC GATTCGCAGC 4451 GCATCGCCTT CTATCGGCTT CTTGACGAGT
TCTTCTGAGC GGGACTCTGG 4501 GGTTCGAAAT GACCGACCAA GCGACGCCCA
ACCTGCCATC ACGAGATTTC 4551 GATTCCACCG CCGCCTTCTA TGAAAGGTTG
GGCTTCGGAA TCGTTTTCCG 4601 GGACGCCGGC TGGATGATCC TCCAGCGCGG
GGATCTCATG CTGGAGTTCT 4651 TCGCCCACCC CAACTTGTTT ATTGCAGCTT
ATAATGGTTA CAAATAAAGC 4701 AATAGCATCA CAAATTTCAC AAATAAAGCA
TTTTTTTCAC TGCATTCTAG 4751 TTGTGGTTTG TCCAAACTCA TCAATGTATC
TTATCATGTC TGTATACCGT 4801 CGACCTCTAG CTAGAGCTTG GCGTAATCAT
GGTCATAGCT GTTTCCTGTG 4851 TGAAATTGTT ATCCGCTCAC AATTCCACAC
AACATACGAG CCGGAAGCAT 4901 AAAGTGTAAA GCCTGGGGTG CCTAATGAGT
GAGCTAACTC ACATTAATTG 4951 CGTTGCGCTC ACTGCCCGCT TTCCAGTCGG
GAAACCTGTC GTGCCAGCTG 5001 CATTAATGAA TCGGCCAACG CGCGGGGAGA
GGCGGTTTGC GTATTGGGCG 5051 CTCTTCCGCT TCCTCGCTCA CTGACTCGCT
GCGCTCGGTC GTTCGGCTGC 5101 GGCGAGCGGT ATCAGCTCAC TCAAAGGCGG
TAATACGGTT ATCCACAGAA 5151 TCAGGGGATA ACGCAGGAAA GAACATGTGA
GCAAAAGGCC AGCAAAAGGC 5201 CAGGAACCGT XAAAAGGCCG CGTTGCTGGC
GTTTTTCCAT AGGCTCCGCC 5251 CCCCTGACGA GCATCACAAA AATCGACGCT
CAAGTCAGAG GTGGCGAAAC 5301 CCGACAGGAC TATAAAGATA CCAGGCGTTT
CCCCCTGGAA GCTCCCTCGT 5351 GCGCTCTCCT GTTCCGACCC TGCCGCTTAC
CGGATACCTG TCCGCCTTTC 5401 TCCCTTCGGG AAGCGTGGCG CTTTCTCAAT
GCTCACGCTG TAGGTATCTC 5451 AGTTCGGTGT AGGTCGTTCG CTCCAAGCTG
GGCTGTGTGC ACGAACCCCC 5501 CGTTCAGCCC GACCGCTGCG CCTTATCCGG
TAACTATCGT CTTGAGTCCA 5551 ACCCGGTAAG ACACGACTTA TCGCCACTGG
CAGCAGCCAC TGGTAACAGG 5601 ATTAGCAGAG CGAGGTATGT AGGCGGTGCT
ACAGAGTTCT TGAAGTGGTG 5651 GCCTAACTAC GGCTACACTA GAAGGACAGT
ATTTGGTATC TGCGCTCTGC 5701 TGAAGCCAGT TACCTTCGGA AAAAGAGTTG
GTAGCTCTTG ATCCGGCAAA 5751 CAAACCACCG CTGGTAGCGG TGGTTTTTTT
GTTTGCAAGC AGCAGATTAC 5801 GCGCAGAAAA AAAGGATCTC AAGAAGATCC
TTTGATCTTT TCTACGGGGT 5851 CTGACGCTCA GTGGAACGAA AACTCACGTT
AAGGGATTTT GGTCATGAGA 5901 TTATCAAAAA GGATCTTCAC CTAGATCCTT
TTAAATTAAA AATGAAGTTT 5951 TAAATCAATC TAAAGTATAT ATGAGTAAAC
TTGGTCTGAC AGTTACCAAT 6001 GCTTAATCAG TGAGGCACCT ATCTCAGCGA
TCTGTCTATT TCGTTCATCC 6051 ATAGTTGCCT GACTCCCCGT CGTGTAGATA
ACTACGATAC GGGAGGGCTT 6101 ACCATCTGGC CCCAGTGCTG CAATGATACC
GCGAGACCCA CGCTCACCGG 6151 CTCCAGATTT ATCAGCAATA AACCAGCCAG
CCGGAAGGGC CGAGCGCAGA 6201 AGTGGTCCTG CAACTTTATC CGCCTCCATC
CAGTCTATTA ATTGTTGCCG 6251 GGAAGCTAGA GTAAGTAGTT CGCCAGTTAA
TAGTTTGCGC AACGTTGTTG 6301 CCATTGCTAC AGGCATCGTG GTGTCACGCT
CGTCGTTTGG TATGGCTTCA 6351 TTCAGCTCCG GTTCCCAACG ATCAAGGCGA
GTTACATGAT CCCCCATGTT 6401 GTGCAAAAAA GCGGTTAGCT CCTTCGGTCC
TCCGATCGTT GTCAGAAGTA 6451 AGTTGGCCGC AGTGTTATCA CTCATGGTTA
TGGCAGCACT GCATAATTCT 6501 CTTACTGTCA TGCCATCCGT AAGATGCTTT
TCTGTGACTG GTGAGTACTC 6551 AACCAAGTCA TTCTGAGAAT AGTGTATGCG
GCGACCGAGT TGCTCTTGCC 6601 CGGCGTCAAT ACGGGATAAT ACCGCGCCAC
ATAGCAGAAC TTTAAAAGTG 6651 CTCATCATTG GAAAACGTTC TTCGGGGCGA
AAACTCTCAA GGATCTTACC 6701 GCTGTTGAGA TCCAGTTCGA TGTAACCCAC
TCGTGCACCC AACTGATCTT 6751 CAGCATCTTT TACTTTCACC AGCGTTTCTG
GGTGAGCAAA AACAGGAAGG 6801 CAAAATGCCG CAAAAAAGGG AATAAGGGCG
ACACGGAAAT GTTGAATACT 6851 CATACTCTTC CTTTTTCAAT ATTATTGAAG
CATTTATCAG GGTTATTGTC 6901 TCATGAGCGG ATACATATiT GAATGTATTT
AGAAAAATAA ACAAATAGGG 6951 GTTCCGCGCA CATTTCCCCG AAAAGTGCCA
CCTGACGTCG ACGGATGGGG
[0252]
13TABLE 1 Nucleotide sequence of the recombinant plasmid pIN D-H-
SemaL-EA (SEQ ID NO.:38) 1 AGATCTCGGC CGCATATTAA GTGCATTGTT
CTCGATACCG CTAAGTGCAT 51 TGTTCTCGTT AGCTCGATGG ACAAGTGCAT
TGTTCTCTTG CTGAAAGCTC 101 GATGGACAAG TGCATTGTTC TCTTGCTGAA
AGCTCGATGG ACAAGTGCAT 151 TGTTCTCTTG CTGAAAGCTC AGTACCCGGG
AGTACCCTCG ACCGCCGGAG 201 TATAAATAGA GGCGCTTCGT CTACGGAGCG
ACAATTCAAT TCAAACAAGC 251 AAAGTGAACA CGTCGCTAAG CGAAAGCTAA
GCAAATAAAC AAGCGCAGCT 301 GAACAAGCTA AACAATCTGC AGTAAAGTGC
AAGTTAAAGT GAATCAATTA 351 AAAGTAACCA GCAACCAAGT AAATCAACTG
CAACTACTGA AATCTGCCAA 401 GAAGTAATTA TTGAATACAA GAAGAGAACT
CTGAATACTT TCAACAAGTT 451 ACCGAGAAAG AAGAACTCAC ACACAGCTAG
CGTTTAAACT TAAGCTTGGT 501 ACCGAGCTCG GATCCACTAG TCCAGTGTGG
TGgaattcgg cttgggatga 551 cgcctcctcc gcccggacgt gccgccccca
gcgcaccgcg cgcccgcgtc 601 cctggcccgc cggctcggtt ggggcttccg
ctgcggctgc ggctgctgct 651 gctgctctgg gcggccgccg cctccgccca
gggccaccta aggagcggac 701 cccgcatctt cgccgtctgg aaaggccatg
tagggcagga ccgggtggac 751 tttggccaga ctgagccgca cacggtgctt
ttccacgagc caggcagctc 801 ctctgtgtgg gtgggaggac gtggcaaggt
ctacctcttt gacttccccg 851 agggcaagaa cgcatctgtg cgcacggtga
atatcggctc cacaaagggg 901 tcctgtctgg ataagcggga ctgcgagaac
tacatcactc tcctggagag 951 gcggagtgag gggctgctgg cctgtggcac
caacgcccgg caccccagct 1001 gctggaacct ggtgaatggc actgtggtgc
cacttggcga gatgagaggc 1051 tacgccccct tcagcccgga cgagaactcc
ctggttctgt ttgaagggga 1101 cgaggtgtat tccaccatcc ggaagcagga
atacaatggg aagatccctc 1151 ggttccgccg catccggggc gagagtgagc
tgtacaccag tgatactgtc 1201 atgcagaacc cacagttcat caaagccacc
atcgtgcacc aagaccaggc 1251 ttacgatgac aagatctact acttcttccg
agaggacaat cctgacaaga 1301 atcctgaggc tcctctcaat gtgtcccgtg
tggcccagtt gtgcaggggg 1351 gaccagggtg gggaaagttc actgtcagtc
tccaagtgga acacttttct 1401 gaaagccatg ctggtatgca gtgatgctgc
caccaacaag aacttcaaca 1451 ggctgcaaga cgtcttcctg ctccctgacc
ccagcggcca gtggagggac 1501 accagggtct atggtgtttt ctccaacccc
tggaactact cagccgtctg 1551 tgtgtattcc ctcggtgaca ttgacaaggt
cttccgtacc tcctcactca 1601 agggctacca ctcaagcctt cccaacccgc
ggcctggcaa gtgcctccca 1651 gaccagcagc cgatacccac agagaccttc
caggtggctg accgtcaccc 1701 agaggtggcg cagagggtgg agcccatggg
gcctctgaag acgccattgt 1751 tccactctaa ataccactac cagaaagtgg
ccgttcaccg catgcaagcc 1801 agccacgggg agacctttca tgtgctttac
ctaactacag acaggggcac 1851 tatccacaag gtggtggaac cgggggagca
ggagcacagc ttcgccttca 1901 acatcatgga gatccagccc ttccgccgcg
cggctgccat ccagaccatg 1951 tcgctggatg ctgagcggag gaagctgtat
gtgagctccc agtgggaggt 2001 gagccaggtg cccctggacc tgtgtgaggt
ctatggcggg ggctgccacg 2051 gttgcctcat gtcccgagac ccctactgcg
gctgggacca gggccgctgc 2101 atctccatct acagctccga acggtcagtg
ctgcaatcca ttaatccagc 2151 cgagccacac aaggagtgtc ccaaccccaa
accagacaag gccccactgc 2201 agaaggtttc cctggcccca aactctcgct
actacctgag ctgccccatg 2251 gaatcccgcc acgccaccta ctcatggcgc
cacaaggaga acgtggagca 2301 gagctgcgaa cctggtcacc agagccccaa
ctgcatcctg ttcatcgaga 2351 acctcacggc gcagcagtac ggccactact
tctgcgaggc ccaggagggc 2401 tcctacttcc gcgaggctca gcactggcag
ctgctgcccg aggacggcat 2451 catggccgag cacctgctgg gtcatgcctg
tgccctggct gcctccctct 2501 ggctgggggt gctgcccaca ctcactcttg
gcttgctggt ccacgtgaag 2551 cttGGGCCCG AACAAAAACT CATCTCAGAA
GAGGATCTGA ATAGCGCCGT 2601 CGACCATCAT CATCATCATC ATTGAGTTTA
TCCAGCACAG TGGCGGCCGC 2651 TCGAGTCTAG AGGGCCCGTT TAAACCCGCT
GATCAGCCTC GACTGTGCCT 2701 TCTAGTTGCC AGCCATCTGT TGTTTGCCCC
TCCCCCGTGC CTTCCTTGAC 2751 CCTGGAAGGT GCCACTCCCA CTGTCCTTTC
CTAATAAAAT GAGGAAATTG 2801 CATCGCATTG TCTGAGTAGG TGTCATTCTA
TTCTGGGGGG TGGGGTGGGG 2851 CAGGACAGCA AGGGGGAGGA TTGGGAAGAC
AATAGCAGGC ATGCTGGGGA 2901 TGCGGTGGGC TCTATGGCTT CTGAGGCGGA
AAGAACCAGC TGGGGCTCTA 2951 GGGGGTATCC CCACGCGCCC TGTAGCGGCG
CATTAAGCGC GGCGGGTGTG 3001 GTGGTTACGC GCAGCGTGAC CGCTACACTT
GCCAGCGCCC TAGCGCCCGC 3051 TCCTTTCGCT TTCTTCCCTT CCTTTCTCGC
CACGTTCGCC GGCTTTCCCC 3101 GTCAAGCTCT AAATCGGGGC ATCCCTTTAG
GGTTCCGATT TAGTGCTTTA 3151 CGGCACCTCG ACCCCAAAAA ACTTGATTAG
GGTGATGGTT CACGTAGTGG 3201 GCCATCGCCC TGATAGACGG TTTTTCGCCC
TTTGACGTTG GAGTCCACGT 3251 TCTTTAATAG TGGACTCTTG TTCCAAACTG
GAACAACACT CAACCCTATC 3301 TCGGTCTATT CTTTTGATTT ATAAGGGATT
TTGGGGATTT CGGCCTATTG 3351 GTTAAAAAAT GAGCTGATTT AACAAAAATT
TAACGCGAAT TAATTCTGTG 3401 GAATGTGTGT CAGTTAGGGT GTGGAAAGTC
CCCAGGCTCC CCAGGCAGGC 3451 AGAAGTATGC AAAGCATGCA TCTCAATTAG
TCAGCAACCA GGTGTGGAAA 3501 GTCCCCAGGC TCCCCAGCAG GCAGAAGTAT
GCAAAGCATG CATCTCAATT 3551 AGTCAGCAAC CATAGTCCCG CCCCTAACTC
CGCCCATCCC GCCCCTAACT 3601 CCGCCCAGTT CCGCCCATTC TCCGCCCCAT
GGCTGACTAA TTTTTTTTAT 3651 TTATGCAGAG GCCGAGGCCG CCTCTGCCTC
TGAGCTATTC CAGAAGTAGT 3701 GAGGAGGCTT TTTTGGAGGC CTAGGCTTTT
GCAAAAAGCT CCCGGGAGCT 3751 TCTATATCCA TTTTCGGATC TGATCAAGAG
ACAGGATGAG GATCGTTTCG 3801 CATGATTGAA CAAGATGGAT TGCACGCAGG
TTCTCCGGCC GCTTGGGTGG 3851 AGAGGCTATT CGGCTATGAC TGGGCACAAC
AGACAATCGG CTGCTCTGAT 3901 GCCGCCGTGT TCCGGCTGTC AGCGCAGGGG
CGCCCGGTTC TTT1TGTCAA 3951 GACCGACCTG TCCGGTGCCC TGAATGAACT
GCAGGACGAG GCAGCGCGGC 4001 TATCGTGGCT GGCCACGACG GGCGTTCCTT
GCGCAGCTGT GCTCGACGTT 4051 GTCACTGAAG CGGGAAGGGA CTGGCTGCTA
TTGGGCGAAG TGCCGGGGCA 4101 GGATCTCCTG TCATCTCACC TTGCTCCTGC
CGAGAAAGTA TCCATCATGG 4151 CTGATGCAAT GCGGCGGCTG CATACGCTTG
ATCCGGCTAC CTGCCCATTC 4201 GACCACCAAG CGAAACATCG CATCGAGCGA
GCACGTACTC GGATGGAAGC 4251 CGGTCTTGTC GATCAGGATG ATCTGGACGA
AGAGCATCAG GGGCTCGCGC 4301 CAGCCGAACT GTTCGCCAGG CTCAAGGCGC
GCATGCCCGA CGGCGAGGAT 4351 CTCGTCGTGA CCCATGGCGA TGCCTGCTTG
CCGAATATCA TGGTGGAAAA 4401 TGGCCGCTTT TCTGGATTCA TCGACTGTGG
CCGGCTGGGT GTGGCGGACC 4451 GCTATCAGGA CATAGCGTTG GCTACCCGTG
ATATTGCTGA AGAGCTTGGC 4501 GGCGAATGGG CTGACCGCTT CCTCGTGCTT
TACGGTATCG CCGCTCCCGA 4551 TTCGCAGCGC ATCGCCTTCT ATCGCC1TCT
TGACGAGTTC TTCTGAGCGG 4601 GACTCTGGGG TTCGAAATGA CCGACCAAGC
GACGCCCAAC CTGCCATCAC 4651 GAGATTTCGA TTCCACCGCC GCCTTCTATG
AAAGGTTGGG CTTCGGAATC 4701 GTTTTCCGGG ACGCCGGCTG GATGATCCTC
CAGCGCGGGG ATCTCATGCT 4751 GGAGTTCTTC GCCCACCCCA ACTTGTTTAT
TGCAGCTTAT AATGGTTACA 4801 AATAAAGCAA TAGGATCACA AATTTCACAA
ATAAAGCATT TTTTTCACTG 4851 CATTCTAGTT GTGGTTTGTC CAAACTCATC
AATGTATCTT ATCATGTCTG 4901 TATACCGTCG ACCTCTAGCT AGAGCTTGGC
GTAATCATGG TCATAGCTGT 4951 TTCCTGTGTG AAATTGTTAT CCGCTCACAA
TTCCACACAA CATACGAGCC 5001 GGAAGCATAA AGTGTAAAGC CTGGGGTGCC
TAATGAGTGA GCTAACTCAC 5051 ATTAATTGCG TTGCGCTCAC TGCCCGCTTT
CCAGTCGGGA AACCTGTCGT 5101 GCCAGCTGCA TTAATGAATC GGCCAACGCG
CGGGGAGAGG CGGTTTGCGT 5151 ATTGGGCGCT CTTCCGCTTC CTCGCTCACT
GACTCGCTGC GCTCGGTCGT 5201 TCGGCTGCGG CGAGCGGTAT CAGCTCACTC
AAAGGCGGTA ATACGGTTAT 5251 CCACAGAATC AGGGGATAAC GCAGGAAAGA
ACATGTGAGC AAAAGGCCAG 5301 CAAAAGGCCA GGAACCGTAA AAAGGCCGCG
TTGCTGGCGT TTTTCCATAG 5351 GCTCCGCCCC CCTGACGAGC ATCACAAAAA
TCGACGCTCA AGTCAGAGGT 5401 GGCGAAACCC GACAGGACTA TAAAGATACC
AGGCGTTTCC CCCTGGAAGC 5451 TCCCTCGTGC GCTCTCCTGT TCCGACCCTG
CCGCTTACCG GATACCTGTC 5501 CGCCTTTCTC CCTTCGGGAA GCGTGGCGCT
TTCTCAATGC TCACGCTGTA 5551 GGTATCTCAG TTCGGTGTAG GTCGTTCGCT
CCAAGCTGGG CTGTGTGCAC 5601 GAACCCCCCG TTCAGCCCGA CCGCTGCGCC
TTATCCGGTA ACTATCGTCT 5651 TGAGTCCAAC CCGGTAAGAC ACGACTTATC
GCCACTGGCA GCAGCCACTG 5701 GTAACAGGAT TAGCAGAGCG AGGTATGTAG
GCGGTGCTAC AGAGTTCTTG 5751 AAGTGGTGGC CTAACTACGG CTACACTAGA
AGGACAGTAT TTGGTATCTG 5801 CGCTCTGCTG AAGCCAGTTA CCTTCGGAAA
AAGAGTTGGT AGCTCTTGAT 5851 CCGGCAAACA AACCACCGCT GGTAGCGGTG
GTTTTTTTGT TTGCAAGCAG 5901 CAGATTACGC GCAGAAAAAA AGGATCTCAA
GAAGATCCTT TGATCTTTTC 5951 TACGGGGTCT GACGCTCAGT GGAACGAAAA
CTCACGTTAA GGGATTTTGG 6001 TCATGAGATT ATCAAAAAGG ATCTTCACCT
AGATCCTTTT AAATTAAAAA 6051 TGAAGTTTTA AATCAATCTA AAGTATATAT
GAGTAAACTT GGTCTGACAG 6101 TTACCAATGC TTAATCAGTG AGGCACCTAT
CTCAGCGATC TGTCTATTTC 6151 GTTCATCCAT AGTTGCCTGA CTCCCCGTCG
TGTAGATAAC TACGATACGG 6201 GAGGGCTTAC CATCTGGCCC CAGTGCTGCA
ATGATACCGC GAGACCCACG 6251 CTCACCGGCT CCAGATTTAT CAGCAATAAA
CCAGCCAGCC GGAAGGGCCG 6301 AGCGCAGAAG TGGTCCTGCA ACTTTATCCG
CCTCCATCCA GTCTATTAAT 6351 TGTTGCCGGG AAGCTAGAGT AAGTAGTTCG
CCAGTTAATA GTTTGCGCAA 6401 CGTTGTTGCC ATTGCTACAG GCATCGTGGT
GTCACGCTCG TCGTTTGGTA 6451 TGGCTTCATT CAGCTCCGGT TCCCAACGAT
CAAGGCGAGT TACATGATCC 6501 CCCATGTTGT GCAAAAAAGC GGTTAGCTCC
TTCGGTCCTC CGATCGTTGT 6551 CAGAAGTAAG TTGGCCGCAG TGTTATCACT
CATGGTTATG GCAGCACTGC 6601 ATAATTCTCT TACTGTCATG CCATCCGTAA
GATGCTTTTC TGTGACTGGT 6651 GAGTACTCAA CCAAGTCATT CTGAGAATAG
TGTATGCGGC GACCGAGTTG 6701 CTCTTGCCCG GCGTCAATAC GGGATAATAC
CGCGCCACAT AGCAGAACTT 6751 TAAAAGTGCT CATCATTGGA AAACGTTCTT
CGGGGCGAAA ACTCTCAAGG 6801 ATCTTACCGC TGTTGAGATC CAGTTCGATG
TAACCCACTC GTGCACCCAA 6851 CTGATCTTCA GCATCTTTTA CTTTCACCAG
CGTTTCTGGG TGAGCAAAAA 6901 CAGGAAGGCA AAATGCCGCA AAAAAGGGAA
TAAGGGCGAC ACGGAAATGT 6951 TGAATACTCA TACTCTTCCT TTTTCAATAT
TATTGAAGCA TTTATCAGGG 7001 TTATTGTCTC ATGAGCGGAT ACATATTTGA
ATGTATTTAG AAAAATAAAC 7051 AAATAGGGGT TCCGCGCACA TTTCCCCGAA
AAGTGCCACC TGACGTCGAC 7101 GGATCGGG
[0253]
14TABLE 12 Sequence of the recombinant plasmid pQE30-H-SemaL-BH
(SEQ ID NO.:39) 1 CTCGAGAAAT CATAAAAAAT TTATTTGCTT TGTGAGCGGA
TAACAATTAT 51 AATAGATTCA ATTGTGAGCG GATAACAATT TCACACAGAA
TTCATTAAAG 101 AGGAGAAATT AACTATGAGA GGATCGCATC ACCATCACCA
TCACGGAtcc 151 ctggttctgt ttgaagggga cgaggtgtat tccaccatcc
ggaagcagga 201 atacaatggg aagatccctc ggttccgccg catccggggc
gagagtgagc 251 tgtacaccag tgatactgtc atgcagaacc cacagttcat
caaagccacc 301 atcgtgcacc aagaccaggc ttacgatgac aagatctact
acttcttccg 351 agaggacaat cctgacaaga atcctgaggc tcctctcaat
gtgtcccgtg 401 tggcccagtt gtgcaggggg gaccagggtg gggaaagttc
actgtcagtc 451 tccaagtgga acacttttct gaaagccatg ctggtatgca
gtgatgctgc 501 caccaacaag aacttcaaca ggctgcaaga cgtcttcctg
ctccctgacc 551 ccagcggcca gtggagggac accagggtct atggtgtttt
ctccaacccc 601 tggaactact cagccgtctg tgtgtattcc ctcggtgaca
ttgacaaggt 651 cttccgtacc tcctcactca agggctacca ctcaagcctt
cccaacccgc 701 ggcctggcaa gtgcctccca gaccagcagc cgatacccac
agaAAGCTTA 751 ATTAGCTGAG CTVGGACTCC TGTTGATAGA TCCAGTAATG
ACCTCAGAAC 801 TCCATCTGGA TTTGTTCAGA ACGCTCGGTT GCCGCCGGGC
GTTTTTTATT 851 GGTGAGAATC CAAGCTAGCT TGGCGAGATT TTCAGGAGCT
AAGGAAGCTA 901 AAATGGAGAA AAAAATCACT GGATATACCA CCGTTGATAT
ATCCCAATGG 951 CATCGTAAAG AACATTTTGA GGCATTTCAG TCAGTTGCTC
AATGTACCTA 1001 TAACCAGACC GTTCAGCTGG ATATTACGGG CTTTTTAAAG
ACCGTAAAGA 1051 AAAATAAGCA CAAGTTTTAT CCGGCCTTTA TTCACATTCT
TGCCCGCCTG 1101 ATGAATGCTC ATCCGGAATT TCGTATGGCA ATGAAAGACG
GTGAGCTGGT 1151 GATATGGGAT AGTGTTCACC CTTGTTACAC CGTTTTCCAT
GAGCAAACTG 1201 AAACGTTTTC ATCGCTCTGG AGTGAATACC ACGACGATTT
CCGGCAGTTT 1251 CTACACATAT ATTCGCAAGA TGTGGCGTGT TACGGTGAAA
ACCTGGCCTA 1301 TTTCCCTAAA GGGTTTATTG AGAATATGTT TTTCGTCTCA
GCCAATCCCT 1351 GGGTGAGTTT CACCAGTTTT GATTTAAACG TGGCCAATAT
GGACAACTTC 1401 TTCGCCCCCG TTTTCACCAT GGGCAAATAT TATACGCAAG
GCGACAAGGT 1451 GCTGATGCCG CTGGCGATTC AGGTTCATCA TGCCGTCTGT
GATGGCTTCC 1501 ATGTCGGCAG AATGCTTAAT GAATTACAAC AGTACTGCGA
TGAGTGGCAG 1551 GGCGGGGCGT AATTTTTTTA AGGCAGTTAT TGGTGCCCTT
AAACGCCTGG 1601 GGTAATGACT CTCTAGCTTG AGGCATCAAA TAAAACGAAA
GGCTCAGTCG 1651 AAAGACTGGG CCTTTCGTTT TATCTGTTGT TTGTCGGTGA
ACGCTCTCCT 1701 GAGTAGGACA AATCCGCCGC TCTAGAGCTG CCTCGCGCGT
TTCGGTGATG 1751 ACGGTGAAAA CCTCTGACAC ATGCAGCTCC CGGAGACGGT
CACAGCTTGT 1801 CTGTAAGCGG ATGCCGGGAG CAGACAAGCC CGTCAGGGCG
CGTCAGCGGG 1851 TGTTGGCGGG TGTCGGGGCG CAGCCATGAC CCAGTCACGT
AGCGATAGCG 1901 GAGTGTATAC TGGCTTAACT ATGCGGCATC AGAGCAGATT
GTACTGAGAG 1951 TGCACCATAT GCGGTGTGAA ATACCGCACA GATGCGTAAG
GAGAAAATAC 2001 CGCATCAGGC GCTCTTCCGC TTCCTCGCTC ACTGACTCGC
TGCGCTCGGT 2051 CTGTCGGCTG CGGCGAGCGG TATCAGCTCA CTCAAAGGCG
GTAATACGGT 2101 TATCCACAGA ATCAGGGGAT AACGCAGGAA AGAACATGTG
AGCAAAAGGC 2151 CAGCAAAAGG CCAGGAACCG TAAAAAGGCC GCGTTGCTGG
CGTTTTTCCA 2201 TAGGCTCCGC CCCCCTGACG AGCATCACAA AAATCGACGG
TCAAGTCAGA 2251 GGTGGCGAAA CCCGACAGGA CTATAAAGAT ACCAGGCGTT
TCCCCCTGGA 2301 AGCTCCCTCG TGCGCTCTCC TGTTCCGACC CTGCCGCTTA
CCGGATACCT 2351 GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC GCTTTCTCAA
TGCTCACGCT 2401 GTAGGTATCT CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT
GGGCTGTGTG 2451 CACGAACCCC CCGTTCAGCC CGACCGCTGC GCCTTATCCG
GTAACTATCG 2501 TCTTGAGTCC AACCCGGTAA GACACGACTT ATCGCCACTG
GCAGCAGCCA 2551 CTGGTAACAG GATTAGCAGA GCGAGGTATG TAGGCGGTGC
TACAGAGTTC 2601 TTGAAGTGGT GGCCTAACTA CGGCTACACT AGAAGGACAG
TATTTGGTAT 2651 CTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTT
GGTAGCTCTT 2701 GATCCGGCAA ACAAACCACC GCTGGTAGCG GTGGTTTTTT
TGTTTGCAAG 2751 CAGGAGATTA CGCGCAGAAA AAAAGGATCT CAAGAAGATC
CTTTGATCTT 2801 TVCTACGGGG TCTGACGCTC AGTGGAACGA AAACTCACGT
TAAGGGATTT 2851 TGGTCATGAG ATTATCAAAA AGGATCTTCA CCTAGATCCT
TTTAAATTAA 2901 AAATGAAGTT TTAAATCAAT CTAAAGTATA TATGAGTAAA
CTTGGTCTGA 2951 CAGTTACCAA TGCTTAATCA GTGAGGCACC TATCTCAGCG
ATCTGTCTAT 3001 TTCGTTCATC CATAGCTGCC TGACTCCCCG TCGTGTAGAT
AACTACGATA 3051 CGGGAGGGCT TACCATCTGG CCCCAGTGCT GCAATGATAC
CGCGAGACCC 3101 ACGCTCACCG GCTCCAGATT TATCAGCAAT AAACCAGCCA
GCCGGAAGGG 3151 CCGAGCGCAG AAGTGGTCCT GCAACTTTAT CCGCCTCCAT
CCAGTCTATT 3201 AATTGTTGCC GGGAAGCTAG AGTAAGTAGT TCGCCAGTTA
ATAGTTTGCG 3251 CAACGTTGTT GCCATTGCTA CAGGCATCGT GGTGTCACGC
TCGTCGTTTG 3301 GTATGGCTTC ATTCAGCTCC GGTTCCCAAC GATCAAGGCG
AGTTACATGA 3351 TCCCCCATGT TGTGCAAAAA AGCGGTTAGC TCCTTCGGTC
CTCCGATCGT 3401 TGTCAGAAGT AAGTTGGCCG CAGTGTTATC ACTCATGGTT
ATGGCAGCAC 3451 TGCATAATTC TCTTACTGTC ATGCCATCCG TAAGATGCTT
TTCTGTGACT 3501 GGTGAGTACT CAACCAAGTC ATTCTGAGAA TAGTGTATGC
GGCGACCGAG 3551 TTGCTCTTGC CCGGCGTCAA TACGGGATAA TACCGCGCCA
CATAGCAGAA 3601 CTTTAAAAGT GCTCATCATT GGAAAACGTT CTTCGGGGCG
AAAACTCTCA 3651 AGGATCTTAC CGCTGTTGAG ATCCAGTTCG ATGTAACCCA
CTCGTGCACC 3701 CAACTGATCT TCAGCATCTT TTACTTTCAC CAGCGTTTCT
GGGTGAGCAA 3751 AAACAGGAAG GCAAAATGCC GCAAAAAAGG GAATAAGGGC
GACACGGAAA 3801 TGTTGAATAC TCATACTCTT CCTTTTTCAA TATTATTGAA
GCATTTATCA 3851 GGGTTATTGT CTCATGAGCG GATACATATT TGAATGTATT
TAGAAAAATA 3901 AACAAATAGG GGTTCCGCGC ACATTTCCCC GAAAAGTGCC
ACCTGACGTC 3951 TAAGAAACCA TTATTATCAT GACATTAACC TATAAAAATA
GGCGTATCAC 4001 GAGGCCCTTT CGTCTTCAC
[0254]
15TABLE 13 Sequence of the recombinant plasmid pQE3I-H-SemaL-SH
(SEQ ID NO.:40) 1 CTCGAGAAAT CATAAAAAAT TTATTTGCTT TGTGAGCGGA
TAACAATTAT 51 AATAGATTCA ATTGTGAGCG GATAACAATT TCACACAGAA
TTCATTAAAG 101 AGGAGAAATT AACTATGAGA GGATCGCATC ACCATCACCA
TCACACGGAT 151 CCGCATGCga gctcccagtg ggaggtgagc caggtgcccc
tggacctgtg 201 tgaggtctat ggcgggggct gccacggttg cctcatgtcc
cgagacccct 251 actgcggctg ggaccagggc cgctgcatct ccatctaoag
ctccgaacgg 301 tcagtgctgc aatccattaa tccagccgag ccacacaagg
agtgtcccaa 351 ccccaaacca gacaaggccc cactgcagaa ggtttccctg
gccccaaact 401 ctcgctacta cctgagctgc cccatggaat cccgccacgc
cacctactca 451 tggcgccaca aggagaacgt ggagcagagc tgcgaacctg
gtcaccagag 501 ccccaactgc atcctgttca tcgagaacct cacggcgcag
cagtacggcc 551 actacttctg cgaggcccag gagggctcct acttccgcga
ggctcagcac 601 tggcagctgc tgcccgagga cggcatcatg gccgagcacc
tgctgggtca 651 tgcctgtgcc ctggctgcct ccctctggct gggggtgctg
cccacactca 701 ctcttggctt gctggtccac gtgaagcttA ATTAGCTGAG
CTTGGACTCC 751 TGTTGATAGA TCCAGTAATG ACCTCAGAAC TCCATCTGGA
TTTGTTCAGA 801 ACGCTCGGTT GCCGCCGGGC GTTTTTTATT GGTGAGAATC
CAAGCTAGCT 851 TGGCGAGATT TTCAGGAGCT AAGGAAGCTA AAATGGAGAA
AAAAATCACT 901 GGATATACCA CCGTTGATAT ATCCCAATGG CATCGTAAAG
AACATTTTGA 951 GGCATTTCAG TCAGTTGCTC AATGTACCTA TAACCAGACC
GTTCAGCTGG 1001 ATATTACGGC CTTTTTAAAG ACCGTAAAGA AAAATAAGCA
CAAGTTTTAT 1051 CCGGCCTTTA TTCACATTCT TGCCCGCCTG ATGAATGCTC
ATCCGGAATT 1101 TCGTATGGCA ATGAAAGACG GTGAGCTGGT GATATGGGAT
AGTGTTCACC 1151 CTTGTTACAC CGTTTTCCAT GAGCAAACTG AAACGTTTTC
ATCGCTCTGG 1201 AGTGAATACC ACGACGATTT CCGGCAGTTT CTACACATAT
ATTCGCAAGA 1251 TGTGGCGTGT TACGGTGAAA ACCTGGCCTA TTTCCCTAAA
GGGTTTATTG 1301 AGAATATGTT TTTCGTCTCA GCCAATCCCT GGGTGAGTTT
CACCAGTTTT 1351 GATTTAAACG TGGCCAATAT GGACAACTTC TTCGCCCCCG
TTTTCACCAT 1401 GGGCAAATAT TATACGCAAG GCGACAAGGT GCTGATGCCG
CTGGCGATTC 1451 AGGTTCATCA TGCCGTCTGT GATGGCTTCC ATGTCGGCAG
AATGCTTAAT 1501 GAATTACAAC AGTACTGCGA TGAGTGGCAG GGCGGGGCGT
AATTTTTTTA 1551 AGGCAGTTAT TGGTGCCCTT AAACGCCTGG GGTAATGACT
CTCTAGCTTG 1601 AGGCATCAAA TAAAACGAAA GGCTCAGTCG AAAGACTGGG
CCTTTCGTTT 1651 TATCTGTTGT TTGTCGGTGA ACGCTCTCCT GAGTAGGACA
AATCCGCCGC 1701 TCTAGAGCTG CCTCGCGCGT TTCGGTGATG ACGGTGAAAA
CCTCTGACAC 1751 ATGCAGCTCC CGGAGACGGT CACAGCTTGT CTGTAAGCGG
ATGCCGGGAG 1801 CAGACAAGCC CGTCAGGGCG CGTCAGCGGG TGTTGGCGGG
TGTCGGGGCG 1851 CAGCCATGAC CCAGTCACGT AGCGATAGCG GAGTGTATAC
TGGCTTAACT 1901 ATGCGGCATC AGAGCAGATT GTACTGAGAG TGCACCATAT
GCGGTGTGAA 1951 ATACCGCACA GATGCGTAAG GAGAAAATAC CGCATCAGGC
GCTCTTCCGC 2001 TTCCTCGCTC ACTGACTCGC TGCGCTCGGT CTGTCGGCTG
CGGCGAGCGG 2051 TATCAGCTCA CTCAPAGGCG GTAATACGGT TATCCACAGA
ATCAGGGGAT 2101 AACGCAGGAA AGAACATGTG AGCAAAAGGC CAGCAAAAGG
CCAGGAACCG 2151 TAAAAAGGCC GCGTTGCTGG CGTTTTTCCA TAGGCTCCGC
CCCCCTGACG 2201 AGCATCACAA AAATCGACGC TCAAGTCAGA GGTGGCGAAA
CCCGACAGGA 2251 CTATAAAGAT ACCAGGCGTT TCCCCCTGGA AGCTCCCTCG
TGCGCTCTCC 2301 TGTTCCGACC CTGCCGCTTA CCGGATACCT GTCCGCCTTT
CTCCCTTCGG 2351 GAAGCGTGGC GCTTTCTCAA TGCTCACGCT GTAGGTATCT
CAGTTCGGTG 2401 TAGGTCGTTC GCTCCAAGCT GGGCTGTGTG CACGAACCCC
CCGTTCAGCC 2451 CGACCGCTGC GCCTTATCCG GTAACTATCG TCTTGAGTCC
AACCCGGTAA 2501 GACACGACTT ATCGCCACTG GCAGCAGCCA CTGGTAACAG
GATTAGCAGA 2551 GCGAGGTATG TAGGCGGTGC TACAGAGTTC TTGAAGTGGT
GGCCTAACTA 2601 CGGCTACACT AGAAGGACAG TATTTGGTAT CTGCGCTCTG
CTGAAGCCAG 2651 TTACCTTCGG AAAAAGAGTT GGTAGCTCTT GATCCGGCAA
ACAAACCACC 2701 GCTGGTAGCG GTGGTTTTTT TGTTTGCAAG CAGCAGATTA
CGCGCAGAAA 2751 AAAAGGATCT CAAGAAGATC CTTTGATCTT TTCTACGGGG
TCTGACGCTC 2801 AGTGGAACGA AAACTCACGT TAAGGGATTT TGGTCATGAG
ATTATCAAAA 2851 AGGATCTTCA CCTAGATCCT TTTAAATTAA AAATGAAGTT
TTAAATCAAT 2901 CTAAAGTATA TATGAGTAAA CTTGGTCTGA CAGTTACCAA
TGCTTAATCA 2951 GTGAGGCACC TATCTCAGCG ATCTGTCTAT TTCGTTCATC
CATAGCTGCC 3001 TGACTCCCCG TCGTGTAGAT AACTACGATA CGGGAGGGCT
TACCATCTGG 3051 CCCCAGTGCT GCAATGATAC CGCGAGACCC ACGCTCACCG
GCTCCAGATT 3101 TATCAGCAAT AAACCAGCCA GCCGGAAGGG CCGAGCGCAG
AAGTGGTCCT 3151 GCAACTTTAT CCGCCTCCAT CCAGTCTATT AATTGTTGCC
GGGAAGCTAG 3201 AGTAAGTAGT TCGCCAGTTA ATAGTTTGCG CAACGTTGTT
GCCATTGCTA 3251 CAGGCATCGT GGTGTCACGC TCGTCGTTTG GTATGGCTTC
ATTCAGCTCC 3301 GGTTCCCAAC GATCAAGGCG AGTTACATGA TCCCCCATGT
TGTGCAAAAA 3351 AGCGGTTAGC TCCTTCGGTC CTCCGATCGT TGTCAGAAGT
AAGTTGGCCG 3401 CAGTGTTATC ACTCATGGTT ATGGCAGCAC TGCATAATTC
TCTTACTGTC 3451 ATGCCATCCG TAAGATGCTT TTCTGTGACT GGTGAGTACT
CAACCAAGTC 3501 ATTCTGAGAA TAGTGTATGC GGCGACCGAG TTGCTCTTGC
CCGGCGTCAA 3551 TACGGGATAA TACCGCGCCA CATAGCAGAA CTTTAAAAGT
GCTCATCATT 3601 GGAAAACGTT CTTCGGGGCG AAAACTCTCA AGGATCTTAC
CGCTGTTGAG 3651 ATCCAGTTGG ATGTAACCCA CTCGTGCACG CAACTGATCT
TCAGCATCTT 3701 TTACTTTCAC CAGCGTTTCT GGGTGAGCAA AAACAGGAAG
GCAAAATGCC 3751 GCAAAAAAGG GAATAAGGGC GACACGGAAA TGTTGAATAC
TCATACTCTT 3801 CCTTTTTCAA TATTATTGAA GCATTTATCA GGGTTATTGT
CTCATGAGCG 3851 GATACATATT TGAATGTATT TAGAAAAATA AACAAATAGG
GGTTCCGCGC 3901 ACATTTCCCC GAAAAGTGCC ACCTGACGTC TAAGAAACCA
TTATTATCAT 3951 GACATTAACC TATAAAAATA GGCGTATCAC GAGGCCCTTT
CGTCTTCAC
[0255]
16TABLE 14 (Partial) nucleotide sequence of the human semaphorin L
gene. (8888 nucleotides) (SEQ ID NO.:41):
GAGCCGCACACGGTGCTTTTCCACGAGCCAGGCAGCTCCTCTGTGTGGGTGGGAG- GACGT
GGCAAGGTCTACCTCTTTGACTTCCCCGAGGGCAAGAACGCATCTGTGCGC- ACGGTGAGC
CTCTCTCTTCCCCCAACACCCCCCCTACCCTCTTATCTCCCCTCTGGC- CCTGCCAAGGGT
CCTCAGGGAATCCGAGGGAGCTGGCTTCTCTTCCTAAACTGCCCC- CACCTCCGTATCCTA
TAAATGGCTCCTGGGGGAGGCTCCCTAAAGGTAGTCCAGATT- GGAGTGGGGAGCTGGGGC
GGTGTGGAGAAAAACAGGAGCTAATGGGCCTGGCCAGCT- GGGCAGCGCTGCTGCGGAAAG
CCCAGGCTGGAAGCTGGGCCCCAGAGCCCATGCCTG- GTCTTCTGAACCCTCTGGGCCTCA
GCTCTGGATATGAGACCCTGTTTGACCTCAGGT- AGATCACTCACCCTCTCAGAGCCCCAG
TTGCTCATCTGTCAGATGAGAATAATGGTT- GCTTCCTTTGGGGCTTATCCTGAGGCTGTG
TGGAAAGCATTTCAGGGGTACCTCACC- CCTGGCAGATTGAACTAATGCTTCTCCCCTTCC
CCAGGTGAATATCGGCTCCACAAA- GGGGTCCTGTCTGGATAAGCGGGTGAGCGGGGGAGG
GATCTGGAGGGGTCTGAGCCACTTGGTAAAGGGAGAGGAGACCCTGAGGGTCTAAGGAAG
GAAGCATGGCCCTGCCCCACGAGTCCCAGACTGATGGGGAGACGTGGTCCTCTGTGCTTA
GGGGATGGCGTCAGCTGCACACACTCTGGGCTGTCCCGGGAGGCTGTCACCTATGCTAAG
CCCTTCTGACACCTTCTTCCCTGATCCTGGGGGTCCTAGTGCTAGGCTTGCCAGGGCCTT
CCAGCAACCAATTTCTCTCCTCCCTTCTCTCTTCCCCGGGCAGGACTGCGAGAACTACAT
CACTCTCCTGGAGAGGCGGAGTGAGGGGCTGCTGGCCTGTGGCACCAACGCCCGGCA- CCC
CAGCTGCTGGAACCTGGTGAGAAGGCTGCTCCCCATGTGCCTGATCAGCTCACC- TTCTAC
TGCGTGGGCTTCTGCCCCTCATGGTGGGAAGGAGATGGCGAGACTCCAATG- CTGGCCTTG
CCCTGGGAGGATGGGGCTCCTGGCCGAGAAACTGGCCGTCATGGGAGG- CAGTGGCTGTGG
GATTATGTGGCCATCCAACCCTCTGGATCTCCCACAGGTGAATGG- CACTGTGGTGCCACT
TGGCGAGATGAGAGGCTACGCCCCCTTCAGCCCGGACGAGAA- CTCCCTGGTTCTGTTTGA
AGGTTGGGGCATGCTTCGGAACTGGGCTGGGAGCAGGAT- GGTCAGCTCTTTGTCCAGTGT
CCGGAGGAGGGACTTCCAGGAGCTGCCTGCCCTTAC- TCATTTCTCCCTCCCACTGACCCC
AGGGGACGAGGTGTATTCCACCATCCGGAAGCA- GGAATACAATGGGAAGATCCCTCGGTT
CCGCCGCATCCGGGGCGAGAGTGAGCTGTA- CACCAGTGATACTGTCATGCAGAGTGAGTC
AGGCTCCGGCTGGGCTGAGGGTGGGCA- AGGGGGTGTGAGCACTTAAGGTGGCAGATGGGA
TCCTGATGTTTCTGGGAGGGCTCC- CTGAGGGCCGCTGGGGCCATGCAGGAAAGCAGGACC
TTGGTATAGGCCTGAGAAGTTAGGGTTGGCTGGGAGCAGAGGAACAGACAAGGTATAGCA
GTGGGATGGGCCCAGCCCTCTTCAGGAACACAAACAGAGGGAGCCCCAGACCCAGTGCAG
GGTCCCCAGGAGCCAAAGTTTATCCTCTGCTGAGTTCACGTGGAGGCAGCCCCCCAACTC
CCTCCTCATCAGGGCTCTGCCAATTGAGCAGAAGTGACATAGGGGCCCCCAGGGACCTTC
CCCCACTCCCCAGGCATGAAGTCATTGCTCCTGGGCCGATGACATCTTTGTAGGAAGAGG
GCAAAACAGGTGTGGGGTGGAGGTGCAGGGTCTAGGGCCCCTCGGGGAGTTGGACCT- GAT
GTTATGAGTCCTATTCCAGATCTGATTTGCCATGGTTTGTGCAGACCCGAAGGA- GGGAGG
AGAGTGTGCAGGGTTGGAATGGTCTCCCGGGCAAGCTTCCCAGCCTTACGC- CCATTCGCT
TCTGTGCCCTGGCAGACCCACAGTTCATCAAAGCCACCATCGTGCACC- AAGACCAGGCTT
ACGATGACAAGATCTACTACTTCTTCCGAGAGGACAATCCTGACA- AGAATCCTGAGGCTC
CTCTCAATGTGTCCCGTGTGGCCCAGTTGTGCAGGGTGAACA- CGGGCGTGAGGGCTGCTG
GCTACGTGTCTGTGCATGAATAGGCCTGAGTGAGGGTGA- GTTCTGTGTGTCCGTGTGCAT
GTAGAAGTTGTGTGGATGTATGAGTGGGTCTGTGTC- AGGGACTGTGGGAGCAGCTGTGTG
TGCATGGAGCATCATGTGTCTGTGTGTGGGTAA- AGGTGGCTGAGCTCCTGTGCACGTATG
ATGGCGTGTGAGCGTGTGTATGATGGGGTG- TGTGTGTGTGTGTGTGTGTGTGTTTTGCCT
GTGTGAATGTGCTGTGCCACGTATGTG- GGTGCGTGAGTCAGTAAATGTGTGTCTGAGTCC
GTCTGCTCTGTGGGGACCTGGCAC- TCTCACCTGCCCTGACCCTGGGCACTGCTGGCCCTG
GGCTCTGGATCAGCCAGGCCTGCTTGCAGGAGTCTCATCTGGAGACCTGCCCTGAGTCCT
GGGGCACCCCCGGCAGGTCCTGGCCCCTCGCAGCCTGCCTTCCTCCTCTGGGCCCAGGTG
TTGATATTGCTGGCAGTGGTTTCCTGGGGTGTGTGGGGAAGCCCGGGCAGGTGCTGAGGG
GCCTCTTCTCCCCTCTACCCTTCCAGGGGGACCAGGGTGGGGAAAGTTCACTGTCAGTCT
CCAAGTGGAACACTTTTCTGAAAGCCATGCTGGTATGCAGTGATGCTGCCACCAACAAGA
ACTTCAACAGGCTGCAAGACGTCTTCCTGCTCCCTGACCCCAGCGGCCAGTGGAGGG- ACA
CCAGGGTCTATGGTGTTTTCTCCAACCCCTGGTGAGTGGCCCTTGTCCTGGGGC- CGGGGC
TGGCATTGGTTCAGTGTCCAGTAGGGACAGGAGGCCTTGGGCCCTGCTGAG- GGCCTCCCT
GGTGTGGCAGGAGCAGGGGCTGCAGGCTCAAGAGGCTGGGCTGTTGCT- GGGTGTGGGGTG
GGGGGACAGCCAGTGCGATGTATGTACTGTTGTGTGAGTGAGTCT- GCACTCATGGGTGTG
TGTGCATGCCCTATATGCACACTCATGACTGCACTTGTGCCT- GTGTGTCCCACCACCTGC
TTGTGCCGAGAGTGGACACTGGGCCCAGGAGGAAGCTGC- TGAAGCATCTCTCGGGGAGCT
GGGTGOTATTACACCTGCTCAGGCACTGCCTGAGCC- CGATAATTCACACTTCTTAATCAC
TCTCATTGATTGAACACACGGCAGGCGGAAGTG- TTGGGTGTGTGTGGGGAGAGTTAGGGA
TAGAGTGGAGGAAGCCAAGACCCTGCTCTG- TGGGTCCTGGGTGAGTGGGTCCCCCAGGCT
GGGAAGGGGTTGGGGGTCTGGCCTCCT- GGGGCATCAGCACCCCACAGCCTGTGCCCAGGG
AGGGCTAGAGAACTGCTCAGCCTA- TGATGGGGTTCCTCCTGCCTTGGGGTTGGGTAGAGC
AGATGGCCTCTAGACTCAGTGATTCTGTAACAGGATACAAGTTTGTGGTTTTAAATTGCA
GCACAAAGAAATTAGGCTGAACTCCTCTCCTTCCTCCTCTCCATCCCTCCCCAThTTCAG
TGGTGGTTGGCAACTCAGTGCCAGGCACAAGGCTGGCCTGGGTGAGTGGAGGTGGATGGG
TGGGTTCTGGGCCCCCCATTGAGCTGGTCTCCATGTCACTGCAGGAACTACTCAGCCGTC
TGTGTGTATTCCCTCGGTGACATTGACAAGGTCTTCCGTACCTCCTCACTCTTGGGCTAC
CACTCAAGCCTTCCCAACCCGCGGCCTGGCAAGGTGAGCGTGACACCAGCCGTGGCC- CAG
GCCCAGCCCTCCTTCTGCCTCACCTCCCACCACCCCACTGACCTGGGCCTGCTC- TCCTTG
CCCAGTGCCTCCCAGACCAGCAGCCGATACCCACAGAGACCTTCCAGGTGG- CTGACCGTC
ACCCAGAGGTGGCGCAGAGGGTGGAGCCCATGGGGCCTCTGAAGACGC- CATTGTTCCACT
CTAAATACCACTACCAGAAAGTGGCCGTCCACCGCATGCAAGCCA- GCCACGGGGAGACCT
TTCATGTGCTTTACCTAACTACAGGTGAGAGGCTACCCCGGG- ACCCTCAGTTTGCTTTGT
AAAAACGGGCATGAAAGGTGTAAGGAATAATGTAGTTAA- CATCTGGTTGGATCTTTACAT
GTGGAAGGAATAATTGAGTGACTGGAGTTGTCAGGG- GTTAATGTGTGTGGGTGTGGTTGA
GCCAGGCAGGGAGAGCTTCCTGGAGGAGGTAGG- GGCAAGAGGGAAAGGGGGATGGGAGAA
AAGCAAGCACTGGGATTTGGAGGCGGAAAT- CTGGAGAGTCTGAGCAAAGCCAGGTGCACC
TTTGGTCCAGATGTCTGACTCAGGGAA- GAAGATGGTAGGAAGAGACGTGGCAAATGAGGA
GGAGGGGCCTGAACCACAGGGATA- CTGGCCTCTGCCAGGCAGAATGAGGGAGTCAGGCCC
TGCGCCTGTCTTTGGGATTGTGCAGGTGAGAAGAAACATTTGAGGAGTTGATGGGGCACA
AATTAGGTATGGGGAAGGAGTTCCAGGGGGCAGAACCTTTGCCATCTCACAGAGGACAGG
GGCAGCTTCTCTTCTTCCCTGGAGTAGGCCCTGCTGGGGGAAGCTGGGTGGAATGCCGTG
GGAGATGCTCCTGCTTTCTGGAAAGCCACAGGACACGGAGGAGCCAGTCCTGAGTTGGGT
TTGTCGCAGCTTCCCATGCCAGCTGCCTTCCTTGAGACTGGAAAGGGCCTCTAGCACCCC
TGGGGCCATTCAATTCAGGCCCAGGCGCCCAACCTCAGTTGTTCACATTCCCCATGT- GAT
CTCCTGTTGCTGCTTCACCTTGGGACTGTCTCGGCTTTGGTGACCTTGTAGGAA- ACTGGA
ACCCCAGCACCATTGTTTGGCTCCTGGAAGCCTTGGGGAGAGGAATTTCCC- ACAGGGCAG
GGCCTGGGTCCTGATTCCCTGCCTCTTTACTCCCTATTCATCCCGGCT- ACACCCTTGGGC
CCCCATCCTTGCTTGGCTCCAGTACTGGCTGGCACAGCTGTTGTG- GTCATCCAGGGATGG
CAGGGCACTGGGGAACAGAAGAGAGAGGTCACACAGTGCGGA- ACTGGGAGCAGGAGCTAG
GACAAGGAAGGCTGGACTTGGGCCATGGATTCCCTTCCT- GCAGACTTGGGAAGTGAGCAC
ACTTGAGTGATTAGAGAAGGTGTCTTCGTTCTAAGG- GCAGTGGAGGAGGCACCATTTTGG
AGCCTGCATCATTCGTATTTGGGCTAGATTGAA- AAATAGAGCTTTCTAAGTCCTCTGCAG
AGAATGGGAGGCTCTCACAACTGGGAGAAG- TATTGGCTCTTTTCCTGAGAATTTTGCCTT
GGGTATGCTGTTACTGGGGCTGGTTTG- GAAGGAGTATAGGGCNTTATGTCTGTGAAGGCA
GTGGCTGGGGTGGGGCCTTATCAG- GCCCAAGGAGCATCTGGCCACATCTCAGAGTCCACA
GATGAGGATCACGGATGTGTAGAGGAAACATCCTAGGCAGGCAATCATCTGACTGCTTTT
TTGGGGCAGGTGATGCCCTGGGAAATTGGGAGGGAGGGAGAGAGGGAGGTAGGCTATTCT
AGAPACTGGGAGAGCAGGTGAGGTAGGATTGGGAGGACCAGGGGTCAGGGTCCCCATTGG
TCCCTAATTGAGAACGGAGAGAGCATTGGTCTAGGAGGCAGGCAGCTCGGTTATAAGACC
TTGGGAACTCTTGATTTAGAATCCAAGATCCTTTTTAGATCTAGGATTTTATAAAATTAA
GATATCCCCTAAGATCAAATGCTTCGTGGAGTCCTGAATTGGATCCTAGTTCAGTTG- TTG
GACATTTGTGGAAAAACTAGTGAAATCCAAATAAAGTCTGTAGTTTTGTTAATA- GTAATG
CACCAATGTCAGTTGCCTAGTTGTGACAAATATACCGTGGTTATGTAAGAT- GGTAACATT
AGGGGGAACTGGAGAAGGGTAGATTGGAGCTCTCTGTACTATCTTTGC- AACTTTTCTGGG
AATCTAAAATTACTCCAAAATAAAAAAAAAATGTATTTAAAGTAA- ATATATTCCCTTTGA
GTCCAGGAGGCAGGGGAGTTGTAGAAGCAGCTGAGTGGTTGG- GTTCTGACAGATTTGGTT
CCAACTCGGTCTCTGCTGCTCACCAGCTGTGTGACCTTG- AGCAAGTGGCTTAGCCTTTCT
GAGCCTGATTTCCTTATCTGTGGAGTGGGGAAGATG- ACAGCCACCTCGCAGGGCTGTGGA
GGGTTAAACGAGGTGATGCATGGACAGCAGCCG- CACTGACCTTGCTGGTGTGGGGCTCCT
GCTTCTGTTCTTCCCGTGCAGCCTTGGGAA- TGTTGGAGGCCGTATCCAGGGACCCCTGGG
CCTCCTGGGATGGCCTCTCTGGATCAG- CCTTGGAAGGTTCCAGGCTGCCCTTAGGCTCCC
ACATTCTTCCCCAGTCACGCTCTC- CTCGCCCTGCCCACACCAGTCCTGTGACCCTTGCCT
GAGTTGTGACTTCCCACCCCTCCCCGGCCTAGAGGAAAGCTGCCTGGCCCCTCAGTGGGA
CTCCCGCCCACTGACCCTCTGTCCACCATACACAGACAGGGGCACTATCCACAAGGTGGT
GGAACCGGGGGAGCAGGAGCACAGCTTCGCCTTCAACATCATGGAGATCCAGCCCTTCCG
CCGCGCGGCTGCCATCCAGACCATGTCGCTGGATGCTGAGCGGGTGAGCCTTCCCCCACT
GCGTCCCATGGGCTATGCAGTGACTGCAGCTGAGGACAGGGCTCCTTTGCATGTGATTTG
TGTGTTCTTTTAAGAGCTTCTAGGCCTTAGGGCCTGGACATTTAGGACTGAGTGTGG- GGT
GGGGCCCGGGCCTGACCCAATCCTGCTGTCCTTCCAGAGGAAGCTGTATGTGAG- CTCCCA
GTGGGAGGTGAGCCAGGTGCCCCTGGACCTGTGTGAGGTCTATGGCGGGGG- CTGCCACGG
TTGCCTCATGTCCCGAGACCCCTACTGCGGCTGGGACCAGGGCCGCTG- CATCTCCATCTA
CAGCTCCGAACGGTACGTTGGCCGGGATCCCTCCGTCCCTGGGAC- AAGGTGGGCATGGGA
CAGGGGGAGGTGTTGTCGGGCTGGAAGAGGTGGCGGTACTGG- GCCTTTCTTGTGGGACCT
CCTCTCTACTGGAACTGCACTAGGGGTAAGGATATGAGG- GTCAGGTCTGCAGCCTTGTAT
CTGCTGATCCTCTTTCGTCCTTCCCACTCCAGGTCA- GTGCTGCAATCCATTAATCCAGCC
GAGCCACACAAGGAGTGTCCCAACCCCAAACCA- GGTACCTGATCTGGCCCTGCTGGCGGC
TGTGGCCCAATGAGTGGGGTACTGCCCTGC- CCTGATTGTCCTGGTCTGAGGGAAACATGG
CCTTGTCCTGTGGGCCCCAGGTACATG- GGGCAGGATACAGTCCTGCAGAGGGAGCCCTCT
TGGTGGGATGAGCGAGACGGGAGA- AAAAAGGAGGACGCTGAGGGCTGGGTTCCCCACGTT
CATTCAGAAGCCTTGTCCTGGGATCCCAGTCGGTGGGGAGGACACATCCTCCCCTGGGAG
CTCTTTGTCCCTCCTCACGGCTGCTTCCCCACTGCCTCCCCAGACTTGGCCCCACTGCAG
AAGGTTTCCCTGGCCCCAAACTCTCGCTACTACCTGAGCTGCCCCATGGAATCCCGCCAC
GCCACCTACTCATGGCGCCACAAGGAGAACGTGGAGCAGAGCTGCGTTCCTGGTCACCAG
AGCCCCAACTGCATCCTGTTCATCGAGAACCTCACGGCGCAGCAGTACGGCCACTACTTC
TGCGAGGCCCAGGAGGGCTCCTACTTCCGCGAGGCTCAGCACTGGCAGCTGCTGCCC- GAG
GACGGCATCATGGCCGAGCACCTGCTGGGTCATGCCTGTGCCCTGGCCGCCTCC- CTCTGG
CTGGGGGTGCTGCCCACACTCACTCTTGGCTTGCTGGTCCACTAGGGCCTC- CCGAGGCTG
GGCATGCCTCAGGCTTCTGCAGCCCAGGGCACTAGTTCGTCTCACACT- CAGAGCCGGCTG
GCCCGGGAGCTCCTTGCCTGCCACTTCTTCCAGGGGACAGTTTTT- CCCAGTGGAGGATGC
CAGGCCTGGAGACGTCCAGCCGCAGGCGGCTGCTGGGCCCCA- GGTGGCGCACGGATGGTG
AGGGGCTGAGAATGAGGGCACCGACTGTGAAGCTGGGGC- ATCGATGACCCAAGACTTTAT
CTTCTGGAAAATATTTTTCAGACTCCTCAAACTTGA- CTAAATGCAGCGATGCTCCCAGCC
CAAGAGCCCATGGGTCGGGGAGTGGGTTTGGAT- AGGAGAGCTGGGACTCCATCTCGACCC
TGGGGCTGAGGCCTGAGTCCTTCTGGACTC- TTGGTACCCACATTGCCTCCTTCCCCTCCC
TCTCTCATGGCTGGGTGGCTGGTGTTC- CTGAAGACCCAGGGCTACCCTCTGTCCAGCCCT
GTCCTCTGCAGCTCCCTCTCTGGT- CCTGGGTCCCACAGGACAGCCGCCTTGCATGTTTAT
TGAAGGATGTTTGCTTTCCGGACGGAAGGACGGAAAAAGCTCTGAAAAAAAAAAAAAAAA
AAAAAAAA
[0256]
17TABLE 15 Nucleotide sequence of pMeIBacA-H-SEMAL (6622bp) (SEQ ID
NO:42) 1 GATATCATGG AGATAATTAA AATGATAACC ATCTCGCAAA TAAATAAGTA 51
TTTTACTGTT TTCGTAACAG TTTTGTAATA AAAAAACCTA TAAATATGAA 101
ATTCTTAGTC AACGTTGCCC TTGTTTTTAT GGTCGTATAC ATTTCTTACA 151
TCTATGCGGA TCGATGG gga tccgcccagg gccacctaag gagcggaccc 201
cgcatcttcg ccgtctggaa aggccatgta gggcaggacc gggtggactt 251
tggccagact gagccgcaca cggtgctttt ccacgagcca ggcagctcct 301
ctgtgtgggt gggaggacgt ggcaaggtct acctctttga cttccccgag 351
ggcaagaacg catctgtgcg cacggtgaat atcggctcca caaaggggtc 401
ctgtctggat aagcgggact gcgagaacta catcactctc ctggagaggc 451
ggagtgaggg gctgctggcc tgtggcacca acgcccggca ccccagctgc 501
tggaacctgg tgaatggcac tgtggtgcca cttggcgaga tgagaggcta 551
tgcccccttc agccGggaCg agaactccct ggttctgttt gaaggggacg 601
aggtgtattc caccatccgg aagcaggaat acaatgggaa gatccctcgg 651
ttccgccgca tccggggcga gagtgagctg tacaccagtg atactgtcat 701
gcagaaccca cagttcatca aagccaccat cgtgcaccaa gaccaggctt 751
acgatgacaa gatctactac ttcttccgag aggacaatcc tgacaagaat 801
cctgaggctc ctctcaatgt gtcccgtgtg gcccagttgt gcagggggga 851
ccagggtggg gaaagttcac tgtcagtctc caagtggaac acttttctga 901
aagccatgct ggtatgcagt gatgctgcca ccaacaagaa cttcaacagg 951
ctgcaagacg tcttcctgct ccctgacccc agcggccagt ggagggacac 1001
cagggtctat ggtgttttct ccaacccctg gaactactca gccgtctgtg 1051
tgtattccct cggtgacatt gacaaggtct tccgtacctc ctcactcaag 1101
ggctaccact caagccttcc caacccgcgg cctggcaagt gcctcccaga 1151
ccagcagccg atacccacag agaccttcca ggtggctgac cgtcacccag 1201
aggtggcgca gagggtggag cccatggggc ctctgaagac gccattgttc 1251
cactctaaat accactacca gaaagtggcc gttcaccgca tgcaagccag 1301
ccacggggag acctttcatg tgctttacct aactacagac aggggcacta 1351
tccacaaggt ggtggaaccg ggggagcagg agcacagctt cgccttcaac 1401
atcatggaga tccagccctt ccgccgcgcg gctgccatcc agaccatgtc 1451
gctggatgct gagcggagga agctgtatgt gagctcccag tgggaggtga 1501
gccaggtgcc cctggacctg tgtgaggtct atggcggggg ctgccacggt 1551
tgcctcatgt cccgagaccc ctactgcggc tgggaccagg gccgctgcat 1601
ctccatctac agctccgaac ggtcagtgct gcaatccatt aatccagccg 1651
agccacacaa ggagtgtccc aaccccaaac cagacaaggc cccactgcag 1701
aaggtttccc tggccccaaa ctctcgctac tacctgagct gccccatgga 1751
atcccgccac gccacctact catggcgcca caaggagaac gtggagcaga 1801
gctgcgaacc tggtcaccag agccccaact gcatcctgtt catcgagaaG 1851
ctcacggcgc agcagtacgg ccactacttc tgcgaggccc aggagggctc 1901
ctacttccgc gaggctcagc actggcagct gctgcccgag gacggcatca 1951
tggccgagca cctgctgggt catgcctgtg ccctggctgc ctgaattc 2001
AGCTTGGAGT CGACTCTGCT GAAGAGGAGG AAATTCTCCT TGAAGTTTCC 2051
CTGGTGTTCA AAGTAAAGGA GTTTGCACCA GACGCACCTC TGTTCACTGG 2101
TCCGGCGTAT TAAAACACGA TACATTGTTA TTAGTACATT TATTAAGCGC 2151
TAGATTCTGT GCGTTGTTGA TTTACAGACA ATTGTTGTAC GTATTTTAAT 2201
AATTCATTAA ATTTATAATC TTTAGGGTGG TATGTTAGAG CGAAAATCAA 2251
ATGATTTTCA GCGTCTTTAT ATCTGAATTT AAATATTAAA TCCTCAATAG 2301
ATTTGTAAAA TAGGTTTCGA TTAGTTTCAA ACAAGGGTTG TTTTTCCGAA 2351
CCGATGGCTG GACTATCTAA TGGATTTTCG CTCAACGCCA CAAAACTTGC 2401
CAAATCTTGT AGCAGCAATC TAGCTTTGTC GATATTCGTT TGTGTTTTGT 2451
TTTGTAATAA AGGTTCGACG TCGTTCAAAA TATTATGCGC TTTTGTATTT 2501
CTTTCATCAC TGTCGTTAGT GTACAATTGA CTCGACGTAA ACACGTTAAA 2551
TAAAGCCTGG ACATATTTAA CATCGGGCGT GTTAGCTTTA TTAGGCCGAT 2601
TATCGTCGTC GTCCCAACCC TCGTCGTTAG AAGTTGCTTC CGAAGACGAT 2651
TTTGCCATAG CCACACGACG CCTATTAATT GTGTCGGCTA ACACGTCCGC 2701
GATCAAATTT GTAGTTGAGC TTTTTGGAAT TATTTCTGAT TGCGGGCGTT 2751
TTTGGGCGGG TTTCAATCTA ACTGTGCCCG ATTTTAATTC AGACAACACG 2801
TTAGAAAGCG ATGGTGCAGG CGGTGGTAAC ATTTCAGACG GCAAATCTAC 2851
TAATGGCGGC GGTGGTGGAG CTGATGATAA ATCTACCATC GGTGGAGGCG 2901
CAGGCGGGGC TGGCGGCGGA GGCGGAGGCG GAGGTGGTGG CGGTGATGCA 2951
GACGGCGGTT TAGGCTCAAA TTGTCTCTTT CAGGCAACAC AGTCGGCACC 3001
TCAACTATTG TACTGGTTTC GGGCGTATGG TGCACTCTCA GTACAATCTG 3051
CTCTGATGCC GCATAGTTAA GCCAGCCCCG ACACCCGCCA ACACCCGCTG 3101
ACGCGCCCTG ACGGGCTTGT CTGCTCCCGG CATCCGCTTA CAGACAAGCT 3151
GTGACCGTCT CCGGGAGCTG CATGTGTCAG AGGTTTTCAC CGTCATCACC 3201
GAAACGCGCG AGACGAAAGG GCCTCGTGAT ACGCCTATTT TTATAGGTTA 3251
ATGTCATGAT AATAATGGTT TCTTAGACGT CAGGTGGCAC TTTTCGGGGA 3301
AATGTGCGCG GAACCCCTAT TTGTTTATTT TTCTAAATAC ATTCAAATAT 3351
GTATCCGCTC ATGAGACAAT AACCCTGATA AATGCTTCAA TAATATTGAA 3401
AAAGGAAGAG TATGAGTATT CAACATTTCC GTGTCGCCCT TATTCCCTTT 3451
TTTGCGGCAT TTTGCCTTCC TGTTTTTGCT CACCCAGAAA CGCTGGTGAA 3501
AGTAAAAGAT GCTGAAGATC AGTTGGGTGC ACGAGTGGGT TACATCGAAC 3551
TGGATCTCAA CAGCGGTAAG ATCCTTGAGA GTTTTCGCCC CGAAGAACGT 3601
TTTCCAATGA TGAGCACTTT TAAAGTTCTG CTATGTGGCG CGGTATTATC 3651
CCGTATTGAC GCCGGGCAAG AGCAACTCGG TCGCCGCATA CACTATTCTC 3701
AGAATGACTT GGTTGAGTAC TCACCAGTCA CAGAAAAGCA TCTTACGGAT 3751
GGCATGACAG TAAGAGAATT ATGCAGTGCT GCCATAACCA TGAGTGATAA 3801
CACTGCGGCC AACTTACTTC TGACAACGAT CGGAGGACCG AAGGAGCTAA 3851
CCGCTTTTTT GCACAACATG GGGGATCATG TAACTCGCCT TGATCGTTGG 3901
GAACCGGAGC TGAATGAAGC CATACCAAAC GACGAGCGTG ACACCACGAT 3951
GCCTGTAGCA ATGGCAACAA CGTTGCGCAA ACTATTAACT GGCGAACTAC 4001
TTACTCTAGC TTCCCGGCAA CAATTAATAG ACTGGATGGA GGCGGATAAA 4051
GTTGCAGGAC CACTTCTGCG CTCGGCCCTT CCGGCTGGCT GGTTTATTGC 4101
TGATAAATCT GGAGCCGGTG AGCGTGGGTC TCGCGGTATC ATTGCAGCAC 4151
TGGGGCCAGA TGGTAAGCCC TCCCGTATCG TAGTTATCTA CACGACGGGG 4201
AGTCAGGCAA CTATGGATGA ACGAAATAGA CAGATCGCTG AGATAGGTGC 4251
CTCACTGATT AAGCATTGGT AACTGTCAGA CCAAGTTTAC TCATATATAC 4301
TTTAGATTGA TTTAAAACTT CATTTTTAAT TTAAAAGGAT CTAGGTGAAG 4351
ATCCTTTTTG ATAATCTCAT GACCAAAATC CCTTAACGTG AGTTTTCGTT 4401
CCACTGAGCG TCAGACCCCG TAGAAAAGAT CAAAGGATCT TCTTGAGATC 4451
CTTTTTTTCT GCGCGTAATC TGCTGCTTGC AAACAAAAAA ACCACCGCTA 4501
CCAGCGGTGG TTTGTTTGCC GGATCAAGAG CTACCAACTC TTTTTCCGAA 4551
GGTAACTGGC TTCAGCAGAG CGCAGATACC AAATACTGTT CTTCTAGTGT 4601
AGCCGTAGTT AGGCCACCAC TTCAAGAACT CTGTAGCACC GCCTACATAC 4651
CTCGCTCTGC TAATCCTGTT ACCAGTGGCT GCTGCCAGTG GCGATAAGTC 4701
GTGTCTTACC GGGTTGGACT CAAGACGATA GTTACCGGAT AAGGCGCAGC 4751
GGTCGGGCTG AACGGGGGGT TCGTGCACAC AGCCCAGCTT GGAGCGAACG 4801
ACCTACACCG AACTGAGATA CCTACAGCGT GAGCTATGAG AAAGCGCCAC 4851
GCTTCCCGAA GGGAGAAAGG CGGACAGGTA TCCGGTAAGC GGCAGGGTCG 4901
GAACAGGAGA GCGCACGAGG GAGCTTCCAG GGGGAAACGC CTGGTATCTT 4951
TATAGTCCTG TCGGGTTTCG CCACCTCTGA CTTGAGCGTC GATTTTTGTG 5001
ATGCTCGTCA GGGGGGCGGA GCCTATGGAA AAACGCCAGC AACGCGGCCT 5051
TTTTACGGTT CCTGGCCTTT TGCTGGCCTT TTGCTCACAT GTTCTTTCCT 5101
GCGTTATCCC CTGATTCTGT GGATAACCGT ATTACCGCCT TTGAGTGAGC 5151
TGATACCGCT CGCCGCAGCC GAACGACCGA GCGCAGCGAG TCAGTGAGCG 5201
AGGAAGCATC CTGCACCATC GTCTGCTCAT CCATGACCTG ACCATGCAGA 5251
GGATGATGCT CGTGACGGTT AACGCCTCGA ATCAGCAACG GCTTGCCGTT 5301
CAGCAGCAGC AGACCATTTT CAATCCGCAC CTCGCGGAAA CCGACATCGC 5351
AGGCTTCTGC TTCAATCAGC GTGCCGTCGG CGGTGTGCAG TTCAACCACC 5401
GCACGATAGA GATTCGGGAT TTCGGCGCTC CACAGTTTCG GGTTTTCGAC 5451
GTTCAGACGT AGTGTGACGC GATCGGTATA ACCACCACGC TCATCGATAA 5501
TTTCACCGCC GAAAGGCGCG GTGCCGCTGG CGACCTGCGT TTCACCCTGC 5551
CATAAAGAAA CTGTTACCCG TAGGTAGTCA CGCAACTCGC CGCACATCTG 5601
AACTTCAGCC TCCAGTACAG CGCGGCTGAA ATCATCATTA AAGCGAGTGG 5651
CAACATGGAA ATCGCTGATT TGTGTAGTCG GTTTATGCAG CAACGAGACG 5701
TCACGGAAAA TGCCGCTCAT CCGCCACATA TCCTGATCTT CCAGATAACT 5751
GCCGTCACTC CAACGCAGGA CCATCACCGC GAGGCGGTTT TCTCCGGCGC 5801
GTAAAAATGC GCTCAGGTCA AATTCAGACG GCAAACGACT GTCCTGGCCG 5851
TAACCGACCC AGCGCCCGTT GCACCACAGA TGAAACGCCG AGTTAACGCC 5901
ATCAAAAATA ATTCGCGTCT GGCCTTCCTG TAGCCAGCTT TCATCAACAT 5951
TAAATGTGAG CGAGTAACAA CCCGTCGGAT TCTCCGTGGG AACAAACGGC 6001
GGATTGACCG TAATGGGATA GGTCACGTTG GTGTAGATGG GCGCATCGTA 6051
ACCGTGCATC TGCCAGTTTG AGGGGACGAC GACAGTATCG GCCTCAGGAA 6101
GATCGCACTC CAGCCAGCTT TCCGGCACCG CTTCTGGTGC CGGAAACCAG 6151
GCAAAGCGCC ATTCGCCATT CAGGCTGCGC AACTGTTGGG AAGGGCGATC 6201
GGTGCGGGCC TCTTCGCTAT TACGCCAGCT GGCGAAAGGG GGATGTGCTG 6251
CAAGGCGATT AAGTTGGGTA ACGCCAGGGT TTTCCCAGTC ACGACGTTGT 6301
AAAACGACGG GATCTATCAT TTTTAGCAGT GATTCTAATT GCAGCTGCTC 6351
TTTGATACAA CTAATTTTAC GACGACGATG CGAGCTTTTA TTCAACCGAG 6401
CGTGCATGTT TGCAATCGTG CAAGCGTTAT CAATTTTTCA TTATCGTATT 6451
GTTGCACATC AACAGGCTGG ACACCACGTT GAACTCGCCG CAGTTTTGCG 6501
GCAAGTTGGA CCCGCCGCGC ATCCAATGCA AACTTTCCGA CATTCTGTTG 6551
CCTACGAACG ATTGATTCTT TGTCCATTGA TCGAAGCGAG TGCCTTCGAC 6601
TTTTTCGTGT CCAGTGTGGC TT
[0257] The above description of the invention is intended to be
illustrative and not limiting. Various changes or modifications in
the embodiments described may occur to those skilled in the art.
These can be made without departing from the spirit or scope of the
invention. Accordingly, it is intended that the invention be
limited only to the extent required by the claims and the
applicable rules of law.
Sequence CWU 1
1
* * * * *