Muteins of the granulocyte colony stimulating factor

A granulocyte stimulating factor (G-CSF) or a G-CSF variant differs from natural G-CSF in that one or several amino acids of the sequence ##STR1## at position 50 to 56 of the mature G-CSF with 174 amino acids or at position 53 to 59 of the mature G-CSF with 117 amino acids or/and at least one of the 4 His residues at position 43, 79, 156 or 170 of the mature G-CSF with 174 amino acids position 46, 82, 159 or 173 of the mature G-CSF with 177 amino acids are mutagenized. It is suitable for immunotherapy.

DESCRIPTION 
The invention concerns muteins of the granulocyte stimulating factor G-CSF 
ill the sequence 
##STR2## 
at position 50 to 56 of the mature G-CSF with 174 amino acids or at 
position 53 to 59 of the mature G-CSF with 177 amino acids or/and at least 
one of the 4 His residues at positions 43, 79, 156 and 170 of the mature 
G-CSF with 174 amino acids or at positions 46, 82, 159 or 173 of the 
mature G-CSF with 177 amino acids. 
Lymphokines are involved in the maturation of blood cells. They stimulate 
the maturation of bone marrow stem cells to fully differentiated cells. 
G-CSF is synthesized by activated monocytes, macrophages as well as by a 
series of other cell lines. 
G-CSF was purified to homogeneity from cell culture supernatants of the 
human bladder carcinoma cell line 5637 (Welte et al., Proc. Natl. Acad. 
Sci 82 (1985), 1526). The sequence of the cDNA coding for native G-CSF is 
known from Souza et al., Science 232 (1986), 61. As a consequence of 
alternative splicing ill the second intron two naturally occurring forms 
of G-CSF exist with 204 or 207 amino acids of which the first 30 represent 
a signal peptide (Lymphokines, IRL Press, Oxford, Washington D.C., Editors 
D. Male and C. Rickwood). The mature protein has a molecular weight of 
ca.19.6 kD and has 5 cysteine residues which can form intermolecular or 
intramolecular disulphide bridges. Binding studies have shown that G-CSF 
binds to neutrophilic granulocytes. None or only slight binding is 
observed with erythroid, lymphoid eosinophilic cell lines as well as with 
macrophages. The G-CSF receptor consists of a single peptide chain with a 
molecular weight of 150 kD (Nicola, Immunol. Today 8 (1987), 134). The 
number of receptors per cell generally increases with the maturation of 
the cells and can amount to several hundred per cell. It is assumed that 
lymphokine receptors consist of an extracellular domain, which binds the 
ligands, a hydrophobic transmembrane region and an intracellular domain. 
Binding of lymphokines to their receptor can cause the synthesis of cyclic 
nucleotides, hydrolysis of phosphatidylinositol-4,5-biphosphate as well as 
the activation of protein kinase C and an increase in the intracellular 
calcium level. There is a great interest in how these processes effect the 
metabolism of the cell but at present they are hardly understood. A 
further result of the binding of a ligand to its receptor can be the 
migration of the receptor-ligand complex into the inside of the cell by a 
receptor-dependent endocytosis. This type of internalization apparently 
also occurs with lymphokines (e.g. G-CSF), however, the consequences for 
the metabolism of the cell are not yet understood. 
Since G-CSF is able to substantially increase the population of 
neutrophilic granulocytes within a short period, considerable therapeutic 
fields of application arise for G-CSF. Thus, G-CSF could be used e.g. 
after chemotherapy in cancer, in which the cells of the immune system are 
destroyed. In addition G-CSF could be used in bone marrow 
transplantations, in severe burn wounds, in opportunistic infections 
caused by immune deficiency and in leukemia. For the different types of 
therapy it would be desirable to develop more active and also less active 
forms of G-CSF. The object of the present invention is therefore to 
develop G-CSF molecules with a wide spectrum of activity by the specific 
introduction of point mutations. In this process the changes in activity 
should be achieved by changes in the amino acid sequence which are as 
small as possible. 
The object according to the present invention is achieved by a granulocyte 
stimulating factor (G-CSF) or a G-CSF variant, in which one or several 
amino acids of the sequence Leu-Gly-His-Ser-Leu-Gly-Ile at position 50 to 
56 of the mature G-CSF with 174 amino acids or at position 53 to 59 of the 
mature G-CSF with 177 amino acids or/and at least one of the 4 His 
residues at position 43, 79, 156 or 170 of the mature G-CSF with 174 amino 
acids or at position 46, 82, 159 or 173 of the mature G-CSF with 177 amino 
acids are mutagenized. 
Surprisingly the introduction of new amino acids yields G-CSF muteins which 
have a broad spectrum of activity. The determination of the activity can 
for example be carried out according to Biochem. J. 253 (1988) 213-218; 
Exp. Hematol. 17 (1989) 116-119; Proc. Natl. Acad. Sci. USA 83 (1986) 
5010. 
The term G-CSF or G-CSF variant according to the present invention includes 
all naturally occurring variants of G-CSF with or without a leader 
sequence as well as G-CSF proteins derived therefrom which are modified by 
recombinant DNA technology, in particular fusion proteins which contain 
further polypeptide sequences apart from the G-CSF moiety. In this sense a 
G-CSF mutein is particularly preferred with a N-terminal Met residue at 
position -1 which is suitable for expression in prokaryotic cells. Also 
preferred is a recombinant, methionine-free G-CSF variant which can be 
produced according to PCT/EP 91/00 192. The term "mutagenized" means that 
the respective amino acid is deleted or preferably substituted by another 
amino acid. 
In this sense G-CSF muteins are preferred in which one of the 7 amino acids 
of the sequence Leu-Gly-His-Ser-Leu-Gly-Ile is substituted by another 
amino acid. However, more than one, ill particular two amino acids, can 
also be replaced. 
A G-CSF mutein is particularly preferred ill which the Ser residue at 
position 53 of the mature G-CSF with 174 amino acids or at position 56 of 
the mature G-CSF with 177 amino acids is replaced by one of the other 19 
amino acids, in particular by Thr. 
Furthermore, it is preferred that the Leu residue at position 54 of the 
mature G-CSF with 174 amino acids or at position 57 of the mature G-CSF 
with 177 amino acids s substituted by one of the 19 other amino acids, in 
particular by Thr. By this means one obtains G-CSF muteins with a broad 
variation of G-CSF activity. 
In addition G-CSF muteins are preferred in which one of the 4 His residues 
at position 43, 79, 156 or 170 of the mature G-CSF with 174 amino acids or 
at position 46, 82, 59 or 173 of the mature G-CSF with 177 amino acids is 
substituted by another amino acid, in particular Gln. 
The invention also provides a recombinant DNA which codes for a G-CSF 
mutein according to the present invention. The invention also provides a 
recombinant vector which contains at least one copy of a recombinant DNA 
according to the present invention. In this connection a recombinant 
vector is preferred which is suitable for gene expression in prokaryotic 
cells. Vectors of this type are known to one skilled in the art. 
In addition the invention provides a cell which is transformed with a 
recombinant DNA according to the present invention or/and a recombinant 
vector according to the present invention. This cell is preferably a 
prokaryotic cell, particularly preferably an E. coli cell. 
The invention also provides a process for the production of a recombinant 
DNA according to the present invention in which a DNA sequence which codes 
for G-CSF or a G-CSF variant is site-specifically mutagenized. The usual 
molecular-biological methods for site-specific mutagenesis are known to 
one skilled in the art. The mutagenesis is preferably carried out by using 
synthetic oligonucleotides as mutagenesis primers on single-stranded DNA 
as the template. Common methods are for example described in Amersham No. 
1523 "Oligonucleotide-directed in vitro mutagenesis system"; Methods in 
Enzymology (Academic Press, Inc. Vol. 154, Part E, 367-382 (1987); 
Analytical Biochemistry 179 (1989) 309-311. 
In addition the invention provides a process for producing a G-CSF mutein 
according to the present invention in which a cell is transformed with a 
recombinant DNA according to the present invention or/and a recombinant 
vector according to the present invention, the transformed cell is 
cultured in a suitable medium and the protein is isolated from the cells 
or the medium. The methods usually used in molecular biology for the 
isolation of recombinant proteins from eukaryotic or prokaryotic cells are 
known to one skilled in the art and do not need to be elucidated in 
detail. 
Finally the invention also provides a pharmaceutical preparation based on a 
G-CSF mutein according to the present invention as the active substance, 
if desired, together with the usual pharmaceutical carrier, filling and 
auxiliary substances. Such a pharmaceutical preparation is particularly 
suitable for the therapeutic fields of application mentioned above and 
even for further therapeutic proceedures in which the formation of 
neutrophilic granulocytes is to be stimulated.

The following examples are intended to elucidate the invention without 
however limiting its scope. 
Example 1 
Production of the vector mgl-G-CSF-Bg 
The 554 bp long EcoRI/BamHI fragment from the vector pKK 177-3 G-CSF-Bg 
(DSM 5867) containing the Shine Dalgarno sequence, ATG codon and coding 
sequence for the G-CSF gene is cloned via a blunt-end ligation into the 
NcoI cleavage site of the vector pPZ 07-mgl lac (WO88/09373, FIG. 10). The 
ATG start codon of the lac Z gene, which is located in the protruding 
single strand after NcoI digestion, is digested beforehand by incubation 
with mung bean nuclease (Pharmacia). The resulting vector is denoted 
mgl-G-CSF-Bg. 
Example 2 
Mutagenesis of the amino acid Leu (X) in the sequence 
Gly-His-Ser-Leu-Gly-Ile 
The mutagenesis is carried out on the M13 template according to known 
techniques (Amersham No. 1523 "Oligonucleotide-directed in vitro 
mutagenesis system") . 
A 251 bp long G-CSF cDNA fragment is isolated via the cleavage site 
BstXI/AatII. The protruding single-strands are digested off by mung bean 
nuclease (Pharmacia) and the fragment is cloned into the vector M13mp19 
which was cleaved with EcoRI/SmaI (EcoRI protruding single strand was 
filled in for blunt-end cloning). After preparing single-stranded DNA, the 
oligonucleotide is hybridized to the single-stranded DNA and an elongation 
in the 5'.fwdarw.3' direction beyond the oligonucleotide is carried out 
using Klenow polymerase, ligase and the four nucleotide triphosphates 
(GTP, CTP, TTP, ATP). The DNA which is now double-stranded is transformed 
in E. coli cells which carry a F' episome so that infection by filamentous 
M13 phages is possible (e.g. JM101, obtainable from Stratagene, LaJolla, 
Calif.). Individual plaques are picked out and the mutagenized M13 phages 
contained therein are used for the preparation of single-stranded DNA. A 
DNA sequencing is carried out according to known techniques (e.g. dideoxy 
method according to Sanger) and the exact substitution to form the desired 
mutation is checked in this way. After preparing double-stranded DNA the 
mutated AvaI fragment of G-CSF is isolated and cloned in the expression 
vector mgl-G-CSF-Bg (cleaved with AvaI) . 
In order to reconstitute the complete G-CSF gene the DNA is subsequently 
cleaved with HindIII, the protruding ends are filled in with Klenow 
polymerase and afterwards partially digested with AvaI so that the 5' AvaI 
site in the G-CSF gene (at ca 130 bp) is not cleaved. This DNA is ligated 
with the approximately 240 bp G-CSF fragment AvaI/BamHI (BamHI site is 
filled in with Klenow polymerase) from the starting vector mgl-G-CSF-Bg. 
After transformation in E. coli JM83, the expression of G-CSF is carried 
out in the manner described in WO88/09373. 
The cDNA used has a sequence which codes for a G-CSF with 175 amino acids 
(without a signal sequence, but with a Met residue at position -1) so that 
the preferred mutation is located at Leu at position 54 of the G-CSF amino 
acid sequence (in this the N-terminal Met residue is not counted). 
The sequence of the cDNA (SEQ ID NO: 1) encoding G-CSF which codes for the 
amino acids 50 to 56 (with reference to the G-CSF with 174 amino acids) 
reads: 
##STR3## 
The corresponding complementary opposite strand (SEQ ID NO: 2) to be 
mutagenized reads: 
##STR4## 
The following 19 oligonucleotides corresponding to the opposite strand are 
used for site-directed mutagenesis: 
__________________________________________________________________________ 
Wild-type: (SEQ ID NO: 2) 5' .fwdarw. 3 
GAT GCC -----CAG 
AGA GTG TCC GAG 3' 
Met 
1. 
(SEQ ID NO: 3) 
5' GAT GCC CAT AGA GTG TCC GAG 3' 
Phe 
2. 
(SEQ ID NO: 4) 
5' GAT GCC GAA AGA GTG TCC GAG 3' 
Gln 
3. 
(SEQ ID NO: 5) 
5' GAT GCC CTG AGA GTG TCC GAG 3' 
Glu 
4. 
(SEQ ID NO: 6) 
5' GAT GCC CTC AGA GTG TCC GAG 3' 
Asp 
5. 
(SEQ ID NO: 7) 
5' GAT GCC GTC AGA GTG TCC GAG 3' 
Cys 
6. 
(SEQ ID NO: 8) 
5' GAT GCC GCA AGA GTG TCC GAG 3' 
Ala 
7. 
(SEQ ID NO: 9) 
5' GAT GCC GGC AGA GTG TCC GAG 3' 
Gly 
8. 
(SEQ ID NO: 10) 
5' GAT GCC AGG AGA GTG TCC GAG 3' 
His 
9. 
(SEQ ID NO: 11) 
5' GAT GCC GTG AGA GTG TCC GAG 3' 
Ile 
10. 
(SEQ ID NO: 12) 
5' GAT GCC GAT AGA GTG TCC GAG 3' 
Lys 
(SEQ ID NO: 13) 
5' GAT GCC CTT AGA GTG TCC GAG 3' 
Tyr 
(SEQ ID NO: 13) 
5' GAT GCC ATA AGA GTG TCC GAG 3' 
Asn 
(SEQ ID NO: 13) 
5' GAT GCC GTT AGA GTG TCC GAG 3' 
Pro 
(SEQ ID NO: 13) 
5' GAT GCC GGG AGA GTG TCC GAG 3' 
Arg 
(SEQ ID NO: 13) 
5' GAT GCC GCG AGA GTG TCC GAG 3' 
Ser 
(SEQ ID NO: 13) 
5' GAT GCC GGA AGA GTG TCC GAG 3' 
Thr 
(SEQ ID NO: 13) 
5' GAT GCC GGT AGA GTG TCC GAG 3' 
Val 
(SEQ ID NO: 13) 
5' GAT GCC GAC AGA GTG TCC GAG 3' 
Trp 
(SEQ ID NO: 13) 
5' GAT GCC CCA AGA GTG TCC GAG 3' 
__________________________________________________________________________ 
Example 3 
Production of a G-CSF with modified activity 
A G-CSF which is more enzymatically active compared to the wild-type can be 
produced by substituting serine at position 53 by a threonine at position 
53 of a G-CSF with 174 amino acids (serine in the sequence 
Gly-His-Ser-Leu-Gly). The following double-stranded oligonucleotide (SEQ 
ID NO: 22 and SEQ ID NO: 23 ) was used for the mutagenesis: 
##STR5## 
For the cloning, the G-CSF cDNA fragment (ca 300 bp, EcoRI/EcoRV) from the 
vector pKK 177-3 G-CSF-Bg (DSM 5867) was ligated into the EcoRI/SmaI 
cleavage site of the vector pUC19 (Yannish-Perron et al., (1985), Gene 33, 
103). 
This DNA is cleaved with AvaI/SacI and directly ligated with the primer 
pair described above according to the usual techniques. The mutated 
BstIX/SacI fragment can now be isolated from this costruct and cloned into 
the vector pKK 177-3 G-CSF-Bg (DSM 5867) (cleaved with BstXI/SacI). The 
final construction of the expression clone is carried out ill analogy to 
Example 1. The determination of activity is carried out as described in 
Example 5. 
Example 4 
Alteration of the enzymatic properties of G-CSF by mutation of amino acids 
which are not located in the active center. 
In analogy to known serine esterases it is assumed that the serine of the 
active centre interacts with histidine for the development of enzymatic 
activity. Four histidines which are present in the sequence of G-CSF 
(namely at positions 43, 79, 156 and 170, numbered from the 174 amino acid 
sequence without a signal peptide) are mutagenized. The histidine residue 
at position 52 (or at position 55 ill the 177 amino acid form) is left out 
of consideration in this mutagenesis. In this process His (CCA, CTA) is 
substituted by Gln (CAG). The sequence on the opposite strand 
corresponding to the codon coding for Gln is CTG. 
A G-CSF fragment is subcloned in M13mp19 as described in Example 1. 
The following oligonucleotides corresponding to the opposite strand are 
used for the mutagenesis: 
##STR6## 
The analytical procedure and the recloning in an expression vector is 
carried out in analogy to Example 1. 
Example 5 
Determination of the G-CSF activity 
The activity of G-CSF is tested with the murine leukaemia line NSF60 which 
is completely dependent on G-CSF as described in Biochem. J. 253 (1988) 
213-218, Exp. Hematol. 17 (1989) 116-119, Proc. Natl. Acad. Sci. USA 83 
(1986) 5010. In order that the factor-dependency of the cells is 
preserved, the medium (RPMI medium, Boehringer Mannheim GmbH, Order No. 
2099445 with 10% foetal calf serum) for the maintenance culture 
permanently contains 1000 U/ml G-CSF. 
The proliferation of the NSF60 cells stimulated by G-CSF is measured 
directly in this test by the incorporation of .sup.3 H thymidine. The test 
is carried out as follows: 
NSF60 cells which are in the exponential growth phase (cell density is 
maximally 1.times.10.sup.5 cells/ml) are transferred to microtitre plates 
(1.times.10.sup.4 cells/well) and cultured with a decreasing G-CSF 
concentration. The maximum dose of G-CSF in well 1 corresponds to the 
concentration in the maintenance culture (1000 U/ml, specific activity 
1.times.10.sup.8 U/mg proteins). The dilution is carried out in steps of 
ten. 
After about 24 hours incubation .sup.3 H thymidine (0.1 .mu.Ci/well) is 
added. Afterwards the cells are incubated for a further 16 hours. 
In order to evaluate the test the cells in the microtitre plates are frozen 
in order to lyse them. The cell lysate is aspirated on a glass fibre 
filter, rinsed, dried and measured in a scintillation counter. The 
incorporation of .sup.3 H thymidine is proportional to the G-CSF-induced 
proliferation of the NSF60 cells. 
Example 6 
Alteration in the activity of G-CSF by amino acid substitution in the 
active centre 
A G-CSF modified in amino acid position 54 can be produced by substitution 
of preferably one leucine at position 54 by a threonine at position 54 
(Leu in the sequence Gly-His-Ser-Leu-Gly) in correspondence with the 
procedure described in Example 3 using a suitable double-stranded 
oligonucleotide which contains a nucleic acid triplet (e.g. ACC) coding 
for the amino acid Thr at the appropriate position. In this connection 
position 54 of the 174 amino acid form of G-CSF corresponds to position 57 
of the 177 amino acid form. 
The activity of a mutant having 174 amino acids with Thr at position 54 is 
reduced in the NSF60 cell test (see Example 5) in comparison to the 
wild-type G-CSF with 174 amino acids. Moreover, the activity of this G-CSF 
mutant is reduced in comparison to a G-CSF mutant with an amino acid 
substitution of a serine by a threonine at position 53 (described in 
Example 3). 
TABLE 1 
______________________________________ 
Designation of the Mutants 
(mutants prepared analogously to examples 2 and 3) 
Position Designation Mutation Type 
______________________________________ 
WT Wild Type 
52 HY Histidine .fwdarw. Tyrosine 
52 HS Histidine .fwdarw. Serine 
52 HR Histidine .fwdarw. Arginine 
52 HE Histidine .fwdarw. Glutamine 
53 ST Serine .fwdarw. Threonine 
53 SA Serine .fwdarw. Alanine 
53 SY Serine .fwdarw. Tyrosine 
54 LT Leucine .fwdarw. Threonine 
54 LH Leucine .fwdarw. Histidine 
54 LK Leucine .fwdarw. Lysine 
52, 53 HY:ST Histidine .fwdarw. Tyrosine and 
Serine .fwdarw. Threonine 
52, 53, 54 
Del Histidine, Serine, and 
Leucine .fwdarw. deleted 
52, 53, 54 
3A Histidine, Serine, and 
Leucine .fwdarw. Alanine 
______________________________________ 
TABLE 2 
______________________________________ 
Activity of G-CSF Muteins 
(determined according to Example 5) 
Mutein Activity 
______________________________________ 
G-CSF not modified 
100 
HY 75 
HS 98 
HR 88 
HE 94 
ST 93 
SA 25 
SY 33 
LT 20 
LH 22 
LK 22 
Del 2 
3A 24 
HY:ST 42 
______________________________________ 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 27 
(2) INFORMATION FOR SEQ ID NO:1: 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
CTCGGACA CTCTCTGGGCATC21 
(2) INFORMATION FOR SEQ ID NO:2: 
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GATGCCC AGAGAGTGTCCGAG21 
(2) INFORMATION FOR SEQ ID NO:3: 
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(C) STRANDEDNESS: single 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
GATGCC CATAGAGTGTCCGAG21 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
GATGC CGAAAGAGTGTCCGAG21 
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GATG CCCTGAGAGTGTCCGAG21 
(2) INFORMATION FOR SEQ ID NO:6: 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
GAT GCCCTCAGAGTGTCCGAG21 
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GA TGCCGTCAGAGTGTCCGAG21 
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G ATGCCGCAAGAGTGTCCGAG21 
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GATGCCGGCAGAGTGTCCGAG21 
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GATGCCAGGAGAGTGTCCGAG21 
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GATGCCGTGAGAGTGTCCGAG21 
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GATGCCGATAGAGTGTCCGAG21 
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GATGCCCTTAGAGTGTCCGAG21 
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GATGCCATAAGAGTGTCCGAG21 
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GATGCCGTTAGAGTGTCCGAG21 
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GATGCCGGGAGAGTGTCCGAG21 
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(A) LENGTH: 21 base pairs 
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GATGCCGCGAGACTGTCCGAG21 
(2) INFORMATION FOR SEQ ID NO:18: 
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GATGCCGGAAGAGTGTCCGAG21 
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CCCGAGGAGCTGGTGCTGCTCGGACACACCCTGGGCATCCCCTGGACTCCCCTGAGC57 
(2) INFORMATION FOR SEQ ID NO:23: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 49 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
CAGGGGAGCCCAGGGGATGCCCAGGGTGTGTCCGAGCAGCACCAGCTCC49 
(2) INFORMATION FOR SEQ ID NO:24: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
GCTCCTGGGCTGGCACAGC19 
(2) INFORMATION FOR SEQ ID NO:25: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
GAAAAGGCCGCTCTGGAGTTGGCTC25 
(2) INFORMATION FOR SEQ ID NO:26: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 
GCTCTGCAGCTGGCCTAGCAACC23 
(2) INFORMATION FOR SEQ ID NO:27: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: 
GGGCTGCGCAAGCTGGCGTAGAACG25