Screening method for therapeutic agents against Alzheimer's disease

A preventive or therapeutic agent for Alzheimer's disease which comprises a substance exhibiting an inhibitory action to tau-protein kinase I as an effective component is provided. A pharmaceutical composition comprising said agent and a method of inhibiting neuronal cell death in the brain are also provided.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to a preventive or a therapeutic agent for 
Alzheimer's disease, a method of screening Alzheimer's disease and 
tau-protein kinase I which is originated from human being. More 
particularly, it relates to a preventive or a therapeutic agent for 
Alzheimer's disease using a tau-protein kinase I inhibitor; a method of 
screening a preventive or a therapeutic agent for Alzheimer's disease 
utilizing an amyloid beta-protein; a human-originated tau-protein kinase I 
which phosphorylates tau-protein, partial peptides thereof or peptides 
similar thereto; a gene which encodes the kinase; and a method of 
producing the same. 
2. Description of Related Art 
Alzheimer's disease is a progressive dementia which develops in late middle 
ages (45 to 65 years old) and its etiological changes are shrinkage of 
cerebral cortex due to a neuronal cell loss and degeneration of the 
neurons while, from the pathological view, many senile plaques and 
neurofibrillary tangles are noted in the brain. There is no pathologically 
substantial difference between the disease and senile dementia caused by 
the so-called natural aging which develops in the senile period of 65 
years and older ages and, therefore, this is called senile dementia of 
Alzheimer's type. 
Numbers of the patients of this disease are increasing with an increase of 
population of aged people and disease is becoming serious in the society. 
There are various theories on the cause of this disease but, the cause has 
been still ambiguous and, accordingly, there has been a demand for prompt 
clarification. 
It has been known that the quantities which appear in the two pathological 
changes which are characteristic to Alzheimer's disease and to senile 
dementia of Alzheimer type are well correlated with the degree of 
cognitive impairment. Accordingly, studies for clarifying the cause of 
those diseases by clarifying, in a molecular level, the accumulated 
insoluble substances resulting in those two pathological changes have been 
carried out since the first half of the 1980's. 
It has been clarified already that a main component of the senile plaques 
which is one of those pathological changes is amyloid beta-protein 
(hereinafter, it may be abbreviated as "A.beta.P") [Annu. Rev. Neurosci., 
12, 463-490 (1989)]. A neurofibrillary tangle which is another 
pathological change is due to an accumulation of a double-stranded fibrous 
substance called PHF (paired helical filament) in the neurons and, 
recently, the components thereof have been identified as ubiquitin and 
tau-protein which is one of the microtubule-associated proteins 
characteristic to brain [J. Biochem., 99, 1807-1810 (1986); Proc. Natl. 
Acad. Sci. USA, 83, 4913-4917 (1986)]. 
It is believed now that, in Alzheimer's disease, the amyloid beta-protein 
is extremely accumulated in the neurons and that, as a result of its 
correlation with the formation of PHF, death of the neurons is resulted. 
It has been known that the tau-protein (hereinafter, the protein may be 
abbreviated as a "tau") is usually a series of related proteins forming 
several bands at the molecular weights of 48-65 kd on SDS polyacrylamide 
gel electrophoresis and that it promotes the formation of microtubules. 
It has been proved already by the use of polyclonal antibody to PHF 
[anti-ptau: J. Biolchem. 99, 1807-1810 (1986)] and also of monoclonal 
antibody [tau-1 antibody; Proc. Natl. Acad. Asic. USA, 83, 4913-4917 
(1986)] that the tau which is incorporated in the PHF of the brain of 
Alzheimer's disease is extremely phosphorylated as compared with the 
normal one. 
The present inventors have isolated an enzyme which catalyzes such an 
abnormal phosphorylation, named it "tau-protein kinase I" (hereinafter, it 
may be abbreviated as "TPK-I") and clarified its biochemical properties 
[Seikagaku, vol. 64, no. 5, page 308 (1992)]. The inventors have further 
cloned the cDNA of rat TPK-I from the cDNA library of cerabral cortex of 
rats based upon the partial amino acid sequence of TPK-I, whereby the base 
sequence has been determined and the amino acid sequence has been proposed 
(Seq. ID No. 2 in the Sequence Listing; Japanese Patent Application 
177241/92, FEBS Lett., 325, 167-172 (1993)). 
As a result thereof, it has been confirmed that the primary structure of 
the rat TPK-I is identical with that of the enzyme which is known as a rat 
GSK-3.beta.(glycogen syntase kinase 3.beta.) [EMBO J., 9, 2431-2438 
(1990)]. 
However, in finding the drugs which are effective for the prevention or the 
therapy of human diseases, the primary structure which are targets for the 
drug usually vary depending upon the animal species. Therefore, there are 
many cases that the interaction between the drug and the protein (in other 
words, sensitivity and effectiveness of the drug) greatly differs 
depending upon the animal species [e.g. Nature, 360, 161 (1992)]. Thus, in 
order to find drugs which are really effective to human being, it is 
desired that the investigation is carried out using proteins which are 
originated from human being. Particularly in the case of finding the drugs 
effective for the diseases which have not been found in animals other than 
human being such as Alzheimer's disease, it is believed to be essential to 
use proteins originated from human being. However, there has been no 
report on the separation and purification of TPK-I (or GSK-3.beta.) from 
human tissues and, moreover, there has been no report on gene (cDNA) which 
encodes human TPK-I (or GSK-30.beta.). 
SUMMARY OF THE INVENTION 
An object of the present invention is to clarify the correlation between 
the death of neurons and accumulation of PHF and amyloid beta-protein 
characteristically found in the brains of Alzheimer's disease and also to 
apply it to the clarification of the cause of Alzheimer's disease and 
further to the investigation to the preventive or therapeutic agents 
therefor. 
Another object of the present invention is to clarify the structure of the 
human-originated TPK-I (which is essential for the progress of 
investigations for such drugs) on the molecular biological basis and to 
offer a method of producing it by means of gene technology. 
The present inventors have carried out the investigations for achieving the 
above-given objects and confirmed that, when amyloid beta-protein acted to 
the neurons in the brain, activity of TPK-I significantly increases 
whereupon the extremely phosphorylated tau-protein found in PHF of the 
brains of Alzheimer's disease is resulted and, moreover, the neurons are 
killed, and that the above-mentioned increase in the TPK-I activity and 
neuronal cell death in the brain is inhibited by the treatment with the 
antisense oligonucleotide of the TPK-I. 
In addition, with a view that the accumulation of PHF results in the 
degeneration of neurons in the brains of Alzheimer's disease and 
successively induces the death, the present inventors have for the first 
time cloned the gene (which encodes the human-originated TPK-I which is 
thought to be a key enzyme for the PHF formation) from the cDNA library of 
human fetus brain, whereupon its primary structure is determined and a 
method for constant supply (or production) of the human-originated TPK-I 
has been established. 
The present invention has been achieved as a result of the above-given 
findings followed by further investigations, and its characteristic 
features are as follows: 
(1) a preventive or a therapeutic agent for Alzheimer's disease, which 
comprises a substance exhibiting an inhibitory action to tau-protein 
kinase I as an effective component; 
(2) a preventive or a therapeutic agent for Alzheimer's disease, which 
comprises an antisense oligonucleotide capable of hybridizing with mRNA or 
DNA of tau-protein kinase I as an effective component; 
(3) a pharmaceutical composition for prevention or therapy of Alzheimer's 
disease, which comprises a substance exhibiting an inhibitory action to 
tau-protein kinase I and a pharmaceutically acceptable carrier; 
(4) a pharmaceutical composition for prevention or therapy of Alzheimer's 
disease, which comprises an antisense oligonucleotide capable of 
hybridizing with mRNA and DNA of tau-protein kinase I; 
(5) a method of screening a preventive or a therapeutic agent for 
Alzheimer's disease in which, when amyloid beta-protein, nerve cells and a 
drug which is presumed to be effective as a preventive or a therapeutic 
agent to Alzheimer's disease are incubated and the death of said nerve 
cells is inhibited, then said drug is judged to be effective as a 
preventive or a therapeutic agent for Alzheimer's disease; 
(6) a method of inhibiting the death of neurons in the brain, characterized 
in that a substance which exhibits an inhibitory action to tau-protein 
kinase I to the neuron in the brain is applied; 
(7) a method of inhibiting the death of neurons in the brain, characterized 
in that an antisense oligonucleotide which is capable of hybridizing with 
mRNA or DNA of tau-protein kinase I is applied to the cranial nerve cells; 
(8) human-originated tau-protein kinase I characterized in being 
represented by an amino acid sequence given in the Seq. ID No. 1 of the 
attached Sequence Listing or a partial sequence thereof; 
(9) gene which encodes the human-originated tau-protein kinase I which is 
represented by the amino acid sequence given in the Seq. ID No. 1 of the 
attached Sequence Listing or a partial sequence thereof; 
(10) recombinant human-originated tau-protein kinase I; 
(11) recombinant vector which is capable of expressing the recombinant 
human-originated tau-protein kinase I; 
(12) transformant which is obtained by a transformation of the host cells 
by a recombinant vector which is capable of expressing the recombinant 
human-originated tau-protein kinase I; and 
(13) a method of producing a recombinant human-originated tau-protein 
kinase I, characterized in that a transformant obtained by a 
transformation of host cells by a recombinant vector which is capable of 
expressing the recombinant human-originated tau-protein kinase I is 
incubated and then the recombinant human-originated tau-protein kinase I 
is collected from said culture. 
The present invention will be further illustrated as hereunder.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT(S) 
With regard to the substance which exhibits an inhibitory action to 
tau-protein kinase I in the present invention, any substance will do 
provided that, when said substance is incubated together with nerve cells 
and amyloid beta-protein, death of said nerve cells is inhibited. For 
example, it is chemically-synthesized substance, a substance which is 
extracted from living cells of microorganisms, etc. 
Further, in accordance with the present invention, an antisense 
oligonucleotide (hereinafter, it may be abbreviated as "TPK-I antisense 
oligonucleotide") which is capable of hybridizing with mRNA or DNA of 
TPK-I is used for prevention or therapy of Alzheimer's disease. 
Antisense oligonucleotide is capable of inhibiting the protein synthesis in 
a level of gene and, therefore, it has been receiving attention in the 
medical field as a synthetic inhibitor for the proteins causing the 
disease. The principle is that, when the antisense RNA or the antisense 
DNA forms a base pair with mRNA in a sense sequence, spread of the gene 
information is interrupted and synthesis of protein which is the final 
product is inhibited [Igaku no Ayumi, vol.162, no.13, 909-911 (1992)]. 
With regard to the TPK-I antisense oligonucleotide applied in the present 
invention, anything will do provided that it is capable of hybridizing 
with mRNA or DNA of TPK-I and that it has a sequence for inhibiting the 
synthesis of TPK-I by, for example, inhibition of transcription, 
inhibition of splicing of pre-mRNA, inhibition of mRNA septum 
transmission, inhibition of translation, etc. Usually, that comprising 
about 15 to 30 nucleotides is used. 
Furthermore, the antisense oligonucleotides applicable are a 
phosphorothioate type in which an oxygen atom which is bonded by means of 
a double bond with a phosphorus atom at the phosphodiester bond connecting 
deoxyribonucleosides is substituted with a sulfur atom; a methyl phosphate 
type in which methyl group is introduced instead of the sulfur atom; a 
phosphonate type without substitution; and an alpha-oligonucleotide type 
[Anticancer Drug Des. 6 (66), 606-646 (1991); Anticancer Research, 10, 
1169-1182 (1990)]. In addition, in the present invention, it is not always 
necessary to use a nucleotide type in which a nucleoside derivative is 
bonded provided that the substance can form a hybrid with the aimed 
sequence. For example, the antisense compounds which are described in 
Antisense Research and Development, 1, 65-113 (1991), etc. may be used as 
well. 
Specific examples of the TPK-I antisense oligonucleotides used in the 
present invention are TPK-I antisense oligonucleotide chain: 
5'-TCTCGGTCGCCCCGACAT-3' (Seq. ID No. 5 of the Sequence Listing) which is 
complementary to TPK-I sense oligonucleotide chain: 
5'-ATGTCGGGGCGACCGAGA-3' (Seq. ID No. 4 of the Sequence Listing) 
corresponding to the first six amino acid residues: Met Ser Gly Arg Pro 
Arg in the translation initiating domain of TPK-I in the primary structure 
of the rat GSK-3.beta. [same as the primary structure of the rat TPK-I 
(Seq.ID No. 2 of the Sequence Listing) described in the above-referenced 
EMBO J., 9, 2431-2438(1990)]; the TPK-I antisense oligonucleotide chain: 
5'-TCTGGGCCGCCCTGACAT-3' (Seq. ID No. 7 of the Sequence Listing) which is 
complementary to the TPK-I sense oligonucleotide chain: 
5'-ATGTCAGGGCGGGCCCAGA-3' (Seq. ID No. 6 of the Sequence Listing) 
corresponding to the first six amino acid residues: Met Ser Gly Arg Pro 
Arg in the translation initiating domain of TPK-I in the primary structure 
of human TPK-I (Seq. ID No. 1 of the Sequence Listing; refer to the 
examples which will given later); and the like. 
The above-mentioned TPK-I sense oligonucleotide and TPK-I antisense 
oligonucleotide can be easily synthesized by means of 
commercially-available automatic DNA synthesizers such as a DNA 
synthesizer manufactured by Applied Biosystems, that manufactured by 
MilliGen, etc. As mentioned already, the TPK-I antisense oligonucleotides 
of the present invention are not particularly limited to those having the 
above-given sequences provided that they are capable of hybridizing with 
mRNA or DNA of TPK-I and, so far as the hybrid-forming ability is not 
deteriorated, a part of the sequence may be substituted with any base. In 
addition, the antisense oligonucleotides which are changed or modified for 
passing through a blood-brain barrier as described in Science, 259, 
373-377 (1993) are included in the coverage of the present invention as 
well. 
When the TPK-I antisense oligonucleotides or the substances having an 
inhibitory action to TPK-I as mentioned above are used as preventive or 
therapeutic agents for Alzheimer's disease, they may be made into 
preparations meeting with the particular administering route together with 
usual carriers. For example, in the case of oral administration, 
preparations in the form of tablets, capsules, granules, diluted powder, 
liquid, etc. are prepared. 
In preparing solid preparations for oral use, commonly-used fillers, 
binders and lubricants as well as colorants, disintegrating agents, etc. 
may be used. Examples of the fillers are lactose, starch, talc, magnesium 
stearate, crystalline cellulose, methyl cellulose, carboxymethyl 
cellulose, glycerol, sodium alginate, gum arabic, etc. Examples of the 
binders are polyvinylalcohol, polyvinyl ether, ethyl cellulose, gum 
arabic, shellac, white sugar, etc. Examples of the lubricants are 
magnesium stearate, talc, etc. Besides those, commonly-used ones may be 
used for the colorants, disintegrating agents, etc. as well. Tablets may 
be coated by known methods. Liquid preparations may be aqueous or oily 
suspensions, solutions, syrups, elixiers and the like and may be 
manufactured by commonly-used methods. In preparing injections, 
pH-adjusting agents, buffers, stabilizers, isotonic agents, local 
anesthetics, etc. may be added to TPK-I antisense oligonucleotides or the 
substances having an inhibitory action to TPK-I and subcutaneous, 
intramuscular or intravenous injections may be prepared by common methods. 
With regard to the bases for the manufacture of suppositories, oily ones 
such as cacao butter, polyethylene glycol, Witepsol (registered trade mark 
of Dynamite Nobel) may be used. 
Doses of the preparations manufactured as such are not always the same but 
vary depending upon the symptoms, body weights, ages, etc. of the 
patients. Usually, however, the amount corresponding to about 1 to 1,000 
mg/kg of said drug per day for adults will do and it is preferred to 
administer by dividing that for 1 to 4 times daily. In some instances, the 
administration may be carried out once daily to every several or more 
days. 
Examples of the nerve cells used in the present invention are the neuron in 
the brain collected from mammals and the neuronal cell lines in which the 
nerve projections are extended by the induction of growth factors such as 
NGF (nerve growth factor; neurotrophic factor), IGF (insulin-like growth 
factor), etc. An example of the former is a culture prepared by incubation 
of tissues of hippocampus of mammals (such as rat) in a complete culture 
medium. Examples of the latter are PC 12 cells induced by NGF, FGF 
(fibroblast growth factor), EGF (epidermal growth factor), interleukin 6, 
etc. [Ann. Rev. Pharma col. Toxicol., 31, 205-228 (1991)]; SH-SY5Y cells 
induced by IGF [The Journal of Cell Biology, 102, 1949-1954 (1986)]; and 
those which are disclosed in Cell Culture in the Neurosciences, New York: 
Plenum Press, pages 95-123 (1955) such as NGF-induced MJB cells, NMB 
cells, NGP cells, SK-N-SH-SY5Y cells, LAN-1 cells, KA-9 cells, IMR-32 
cells and 5-bromodeoxyuridine-induced IMR-32 cells, NMB cells, NGP cells, 
etc. 
Amyloid beta-protein is a main component of senile plaques of Alzheimer's 
disease and it has been known that said substance is composed of a peptide 
comprising the following 43 amino acid residues [Science, 250, 279-282 
(1990) and Proc. Natl. Acad. Sci. USA, 87, 9020-9023 (1990)]. 
Amino Acid Sequence of Amyloid beta-Protein (Seq. ID No. 3 of the Sequence 
Listing): 
EQU Asp Ala Glu Phe Arg His Asp Aer Gly Tyr Alu Val His His Gln Lys Leu Val Phe 
Phe Ala Glu Asp Val Gly Ser Asn Lys Gloy Ala Ile Ile Gly Leu Met Val Gly 
Gly Val Val Ile Ala Thr 
The present invention will be further illustrated as hereinafter by an 
example on the behavior of hippocampus cells of rats and the 
phosphorylation activity of TPK-I when the hippocampus cells were treated 
with a certain amount of A.beta.P and also with TPK-I sense 
oligonucleotide (hereinafter, referred to as "TPKI-sense") and TPK-I 
antisense oligonucleotide (hereinafter, referred to as "TPKI-antisense") 
as controls under certain conditions. When the present invention is 
carried out as a method of screening the preventive and the therapeutic 
agent of Alzheimer's disease, hippocampus cells of rat are used as the 
neurons and, as the agent presumed to be the preventive or the therapeutic 
agent, TPKI-sense or TPKI-antisense is used. 
Certain amount of TPKI-antisense was added to the culture of the 
hippocampus cells at certain temperature, then certain aount of A.beta.P 
was added thereto, the mixture was kept at certain temperature and the 
living cell numbers with an elapse of time were measured by a method 
described in the examples which will be given later. For comparison, the 
living cell numbers were measured for the case in which only A.beta.P was 
added followed by the same treatments and the case in which TPKI-antisense 
and A.beta.P were added followed by the same treatments. The result showed 
that, as given in the examples later, the living cell numbers when 
TPKI-antisense and A.beta.P were added were significantly more than those 
when only A.beta.P was added and when TPKI-sense and A.beta.P were added 
and that the TPKI-antisense has an action of inhibiting the death of the 
cells by A.beta.P. 
The results of the observations of the samples using a phase contrast 
microscope (magnifying power: 400) when TPKI-antisense and A.beta.P were 
added to the cell culture followed by allowing to stand for 24 hours, when 
only A.beta.P was added followed by allowing to stand for the same time 
and when TPKI-sense and A.beta.P were added followed by allowing to stand 
for the same time showed that the cell toxicity by A.beta.P was little 
being similar to the controls only when TPKI-antisense was acted. 
Further, the phosphorylation activities of tau-protein by TPKI after 24 
hours were measured by the method given in the examples when only A.beta.P 
was added and allowed to stand and when TPKI-antisense and A.beta.P were 
added and allowed to stand same as above. The result was that, as shown in 
the examples given later, the phosphorylation activity of TPK-I when 
TPKI-antisense and A.beta.P were added was about one half of that when 
only A.beta.P was added and that TPKI-antisense exhibits an activity of 
inhibiting the phosphorylation activity of TPK-I. 
Out of the above results, it may be concluded that, when the present 
invention is carried out as a method of screening the preventive and the 
therapeutic agent for Alzheimer's disease, TPKI-antisense is effective as 
said preventive and therapeutic agent. Incidentally, the effectiveness of 
the agents other than the TPK-I antisense oligonucleotide can be evaluated 
similarly. 
Now, the method of obtaining the human-originated TPK-I and the method of 
production thereof will be illustrated as hereunder. 
The TPK-I originated from human being of the present invention may, for 
example, be manufactured as follows. Thus, microtubule fractions were 
obtained from an extract of human brain immediately after death by means 
of temperature-depending polymerization and depolymerization and then, 
operations such as phosphocellulose column chromatography, gel filtration, 
hydroxyapatite column chromatography, S-Sepharose column chromatography, 
heparin column chromatography, etc. are combined according to a method by 
Uchida, et al. [Seikagaku, vol.64, no.5, page 308 (1992)] whereby pure 
protein is obtained. The (partial) primary structure of such a pure 
protein may be determined by conducting a conventional amino acid 
analysis. It is not easy to obtain the human brain tissues in large 
quantities and it is difficult to purify the human TPK-I and, therefore, 
it is also possible that, by a method which will be given later, gene is 
previously cloned and the amino acid sequence is deduced therefrom whereby 
the primary structure is determined. 
The human TPK-I of the present invention prepared as such is a protein in 
which the primary structure is represented by the amino acids described in 
the Seq. ID No. 1 of the Sequence Listing (420 amino acid residues; 
molecular weight: 46,719; isoelectric point: 9.21) and alterations such as 
removal, substitution, modification or addition of some amino acids may be 
carried out within such a range that the functions (action, substrate 
specificity, etc.) will not be deteriorated. 
The gene (cDNA) which encodes the above TPK-I may be cloned by such a 
method that in which the corresponding protein is purified from natural 
material, its partial amino acid sequence is determined and the DNA probe 
corresponding thereto is utilized; that in which homology with the protein 
of the same species or the corresponding protein of the different animal 
species is utilized; that in which an antibody which is specific to the 
corresponding protein is utilized; that in which a detection of the 
specific function of the protein is utilized; etc. The present inventors 
have previously purified TPK-I from an extract of brain of rat or bovine 
and, depending upon the information of the partial amino acid sequence 
thereof, they cloned the rat TPK-IcDNA from the rat brain cDNA library 
(Seq. ID No. 2 of the Sequence Listing; Japanese Patent Application No. 
177241/92, FEBS Lett., 325, 167-172 (1993)). 
Usually, however, the homology of the primary structures of rats with human 
beings in the same protein is, in most cases, around 90% or more and, 
therefore, it is possible to clone human TPK-IcDNA from rat TPK-IcDNA by 
utilizing said homology. Thus, lamda-phage is infected to Escherichia coli 
by a method of Tomizawa, et al. ["Experiments in Bacteriophage" (Iwanami 
Shoten), pages 99-174 (1970)] from cDNA library containing the gene which 
encodes human TPK-I such as human fetus brain cDNA library followed by 
culturing. The plaques formed thereby were selected by a plaque 
hybridization method ["Molecular Cloning" Cold Spring Harbor Laboratory, 
pages 320-328 (1982)] using a rat TPK-IcDNA or DNA fragments having a 
partial structure thereof as a probe. The phage is promulgated from 
positive plaques by a method of Tomiza wa, et al., then DNA is prepared by 
a method of T. Maniatis, et al. ["Molecular Cloning", Cold Spring Harbor 
Laboratory, page 85 (1982)] or after subjecting to a subcloning if 
necessary, cleaved by a suitable restriction enzyme such as EcoRI and 
cloned to a plasmid such as pUC18 or pUC19. As such, cDNA of human TPK-I 
is prepared and its base sequence can be determined, for example, by a 
dideoxy method of Sanger, et al. [Proc. Natl. Acad. Sci. USA, 74, 5463 
(1977)]. 
An example of the above-mentioned gene (cDNA) encoding the human-originated 
TPK-I is that which has a base sequence given in the Seq. ID No. 1 of the 
Sequence Listing. 
In the human TPK-I prepared by the present invention, 5 amino acid residues 
were different out of the 420 amino acid residues in the amino acid 
sequence of the rat TPK-I and the homology in the bases in the translation 
domain was 92.5%. 
The above-prepared human TPK-I or its partial peptides can be expressed and 
generated by means of gene recombination. Thus, the 5'-terminal of the 
human TPK-IcDNA or its fragment is modified or added with precursor gene, 
inserted into the downstream of the promoter of the expressed vector and 
then the vector is induced into host cells such as bacteria, yeasts, 
insect cells, animal cells, etc. The transformed host cells as such is 
cultured under a suitable condition whereby the recombinant human TPK-I is 
produced in or outside the cells. 
Examples of the transformable host cells are bacteria (procaryotic cells) 
such as Escherichia coli (K-12 strain), Bacillus subtitlis, etc.; yeasts 
such as Saccharomyces cerevisiae; insect cells such as ovary-originated 
cells (Sf9 cell strain) of Spodoptera spp.; and (mammalian) animal cells 
such as ovary-originated cells (CHO cells) of Chinese hamster, mouse C127 
cells, kidney-originated cells (COS cells) of African green monkey, mouse 
L cells, mouse FM3A cells, kidney-originated cells (HEK cells, 293 cells) 
of human fetus, etc. 
The expression vector which is suitably used is that which contains a 
promoter at the position where a gene (cDNA) coding the human TPK-I or the 
DNA fragment threreof can be transcribed. For example, when cells are used 
as a host, it is preferred that the expression vector is composed of 
promoter, ribosome binding (SD) sequence, human TPK-I-encoding gene or 
fragment thereof, transcription terminating factor and promoter-controling 
gene. Even when eucaryonic cells such as (mammalian) animal cells, insect 
cells, yeasts, etc. are used as host cells, the fundamental unit 
comprising the expression vector is the same as that in the case of the 
above-mentioned bacteria. 
Examples of the promoter when bacteria are used as host cells are those 
originated from Escherichia coli, phage, etc. such as lactose operon 
(lac)., tryptophan-synthesizing enzyme (trp), lamda-phage P.sub.L, E. coli 
T7 polymerase promoter and tac (hybrid promoter originated from trp and 
lac UV5). The examples in the case of yeasts are promoters of gene of 
enzymes such as phosphoglyceric acid kinase (PGK), 
glyceraldehyde-3-phosphoric acid dehydrogenase (GPD), repressible acidic 
phosphatase (PHO5) and alcohol dehydrogenase 1 (ADH1). The examples in the 
case of insect cells are used as the host are promoter of polyhedron gene 
of baculovirus, etc. The examples in the case of (mammalian) animal cells 
are SV40 initial promoter, SV40 late promoter, apolipoprotein E gene 
promoter, etc. 
Examples of the ribosome binding sequence are those which are originated 
from E. coli, phage, etc. and those which are partially complementary to 
the base sequence of the 3'-terminal domain of 16S ribosome RNA. 
Though the transcription terminating factor is not always necessary, it is 
preferred to have that which is rho.rho.-independent such as lipoprotein 
terminator, trp operon terminator, etc. 
With regard to the sequence of those factors necessary for the expression 
on the expression plasmid (vector), it is desired that they are placed in 
the order of promoter, SD sequence, human TPK-I-encoding gene or fragment 
thereof and transcription terminating factor from the 5'-upstream side. 
Specific examples of the expression vectors satisfying those requirements 
in case that bacteria are used as the host are pKK233-2 (manufactured by 
Farmacia) and pET3C [Gene, 56, 125 (1987)]. Expression vector pGEX series 
(Farmacia) which are expressed as fused protein may be used as well in the 
same manner. When yeasts are used as a host, the vector in which the 
above-mentioned promoter (and, further, gene which complements the 
auxotropic mutant as a selected marker such as trp1 and leu2) is 
incorporated into YEp vector having a replication origin of 2 micron DNA 
is suitably used. In the case of insect cells, an example is Maxbac 
(trademark) which is a baculovirus expression system manual version 1.4 of 
Invitrogen. In the case of animal cells, those having the above-mentioned 
promoter and selective marker gene such as neomycin-resisting gene (Neo) 
and dihydrofolic acid reductase gene (DHFR) are suitably used. When 
eucaryonic cells are used as a host, a shuttle vector to E. coli may be 
used as well. 
Transformation of host cells may be carried out by conventional manner. 
Culture of the transformant may be carried out by a method depending upon a 
method of T. Maniatis, et al. described in "Molecular Cloning" Cold Spring 
Harbor Laboratory, 1982. Though the culture temperature is not always the 
same depending upon the conditons such as the host cells, a temperature of 
about 25 to 40.degree. C. is suitable. 
The human TPK-I produced by such host-vector systems can be purified by a 
series of purifying steps corresponding to hosts and culturing conditions 
such as extraction from the host, salting-out and chromatography using 
various columns. In the case of column chromatography, the suitably-used 
ones are phosphocellulose column chromatography, hydroxyapatite column 
chromatography, S-Sepharose column chromatography, heparin column 
chromatography, blue Sepharose column chromatographny, etc. 
The recombinant human TPK-I prepared as such is capable of phosphorylating 
proteins such as tau-protein, glycogen-synthesizing enzyme, protooncogene 
product c-jun, etc. and partial peptides thereof and the progress of the 
phosphorylation can be confirmed by, for example, the conditions as given 
below. Thus, the recombinant human TPK-I of the present invention is 
added, together with a suitable amount of the substrate protein, to a 
buffer of pH 5.0-8.0 containing 0.2-4.0 mM magnesium acetate and 0.2-4.0 
mM adenosine triphosphate, the mixture is incubated at the room 
temperature to 40.degree. C. and the phosphorylation of the substrate 
protein is checked and determined by radiochemical, proteinochemical or 
immunochemical means. Consequently, when an agent is added to this 
reaction system and the resulting promotion or inhibition of the 
phosphorylation reaction is checked, it is possible to find the agent 
having a physiologically important meaning whereby the investigation on 
the agent which is effective for the prevention or the therapy of human 
disease is now possible. 
EXAMPLES 
The present invention will be illustrated by way of the following examples 
though the present invention is not limited to those examples so far as 
they are not out of the characteristic feature of the present invention. 
Incidentally, judgement of the cytotoxity, measurement of the 
phosphorylation of tau-proten and immunohistochemistry by Alz-50 antibody 
were carried out in accordance with the following methods. Further, in 
each of the following examples, at least three independent experiments 
were carried out and the data were given by their average values. 
Judgement of Cytotoxity 
Numbers of many normal and healthy cells were counted by a phase contrast 
microscope as an index of the living cells after the treatment. Normal 
cells mean those which have morphologically flat circumference and many 
nerve cell projections while the degenerated cells were judged by checking 
the irregular shape, degeneration of the neural projections, etc. Numbers 
of the living cells were counted in a well. In the standard culture 
liquid, the cell numbers were not less than 400 per well. The result was 
confirmed by an immunohistochemical means. 
Measurement of Degree of Phosphorylation of tau-Protein 
Hippocampus cells were collected from the culture medium by washing with an 
ice-cooled phosphate buffer for three times. The cells were suspended in a 
buffer A (pH: 6.8) which contained 1 mM EGTA, 0.5 mM magnesium acetate and 
20 mM 2-(N-morpholino)-ethanesulfonic acid containing a phosphatase 
inhibitor (1 mM okadaic acid; manufactured by Seikagaku Kogyo) and a 
protease inhibitor (1 mM phenylmethylsulfonyl fluoride and each 1 micro 
gram/ml of leupeptin, pepstatin and aprotinin), homogenized and 
centrifuged at 14,000 rpm for one hour and the supernatant liquid was used 
for checking the phosphorylation. 
The rat tau-protein expressed in E. coli BL21 by a gene recombination was 
purified by a method described in J. Biol. Chem., 267, 10897-10901 (1992). 
The hippocampus extract (1 microliter) was added to a solution of the rat 
tau-protein (400 micrograms/ml) dissolved in a buffer A containing 1 mM 
[.tau.-.sup.32 P]ATP (10-20 Ci/mmole) and then 10 micromoles of okadaic 
acid was added to make the final volume 10 microliters. This was incubated 
at 37.degree. C. for three hours and the reaction was stopped by adding a 
buffer for electrophoresis. After subjecting to a 10% polyacrylamide gel 
electrophoresis, the .sup.32 P in the tau-protein was observed by a laser 
image analyzer (Fuji BAS 2000). 
Immunohistochemistry by an Alz-50 Antibody 
The cultured medium of the hippocampus cells was fixed in a phosphate 
buffer for ten minutes using 4% paraformaldehyde. The fixed culture liquid 
was incubated for 30 minutes in a Tris buffer containing 0.2% Triton X-100 
so that the cells were made permeable. 
Then this culture medium was subjected to an immunolabelling using a 1:5 
diluted Alz-50 mouse monoclonal antibody [Science, 232, 648-650(1986)], 
Vectastain ABC avidin-biotin-enzyme peroxide detector kit (manufactured by 
Vector Laboratory) and diaminobenzidine tetrahydrochloride as a dye. 
Example 1 
Preparation of Culture Medium of Cells 
The primary culture medium of hippocampus of rats was prepared in 
accordance with a method described in Brain Res., 126, 397-425 (1977). 
Thus, the hippocampus tissues were collected from embryo of the rats of 18 
days after fertilization and digested in papain (protease) (10 U/ml) at 
37.degree. C. for 20 minutes. The resulting cells were added to a 
Dulbecco's modified Eagle's medium supplied with 5% bovine fetus serum, 5% 
horse serum, 10 micrograms/ml insulin, 0.1 mg/ml transferrin, 1 
microgram/ml aprotinin, 1 mM sodium pyruvate and 84 micrograms/ml 
gentamycin. This was planted to a well for tissue culture covered with 
poly-L-lysine at the density of 2.times.10.sup.5 cells/cm.sup.2, cultured 
for three days and treated with 1 micromole of 
cytosine-beta-arabinofuranoside for 24 hours and the cells of the fifth 
day of the culture were used. 
Preparation of A.beta.P 
A.beta.P peptide (Seq. ID No. 3 of the Sequence Listing) comprising the 
already-mentioned 43 amino acid residues was synthesized by a method which 
was described in Science, 250, 279-282(1990) and Proc. Natl. Acad. Sci. 
USA, 87, 9020-9023(1990) and, after being purified, it was dissolved in 
35% acetonitrile to prepare a stock solution of 2 M. 
Preparation of TPKI-Sense and TPKI-Antisense 
Rat GSK-3.beta. [EMBO J., 9, 2431-2438 (1990)], i.e. the TPKI-sense 
comprising the followng 18 bases corresponding to the translation 
initiating domain of the primary structure of rat TPK-I (FEBS Lett., 325, 
167-172 (1993)) and the TPKI-antisense which is complementary thereto were 
synthesized using an automatic DNA synthesizer (MilliGen), recovered from 
20% acrylamide-urea gel and purified by means of an ethanol precipitating 
method and the precipitate was dissolved in water to adjust to a 
concentration of 1 micromole. 
TPKI-Sense: 5'-ATGTCGGGGCGACCGAGA-3' (Seq. ID No. 4 of the Sequence 
Listing) 
TPKI-Antisense: 5'-TCTCGGTCGCCCCGACAT-3' (Seq. ID No. 5 of the Sequence 
Listing) 
Inhibiting Action for the Death of Cranial Nerve Cells: 
The culture medium of the hippocampus prepared by the above-mentioned 
method was subjected to the following treatments (b) to (d), numbers of 
the living cells with an elapse of time were counted and the result is 
given in Table 1. 
(a) Nontreated culture medium (control): 
(b) TPKI-antisense (1 micromole) was added to 1 ml of the cell culture 
medium and, after five minutes, 20 micromoles of A.beta.P was added 
followed by keeping at 37.degree. C. for 24 hours. 
(c) A.beta.P (20 micromoles) was added to 1 ml of the cell cuture medium 
followed by keeping at 37.degree. C. for 24 hours. 
(d) TPKI-sense (1 micromole) was added to 1 ml of the cell culture medium 
and, after five minutes, 20 micromoles of A.beta.P was added followed by 
keeping at 37.degree. C. for 24 hours. 
TABLE 1 
______________________________________ 
Numbers of Living Cells 
(%) After 
Treating Agents 6 hours 21 hours 
______________________________________ 
(Control) 100 100 
A.beta.P + TPKI-Antisense 
83.0 72.6 
A.beta.P 25.4.3 
A.beta.P + TPKI-Sense 
49.5 
17.1 
______________________________________ 
Table 1 shows the numbers of the living cells with an elapse of time after 
the above-mentioned tretments (b), (c) and (d) and the numbers are given 
in terms of percentages to the control. 
As shown in Table 1, the numbers of the living cells after 6 and 21 hours 
of the treatment of the hippocampus cells with TPKI-antisense and A.beta.B 
(b) were significantly more than those of the case treated only with 
A.beta.B (c) and of the case treated with TPKI-sense and A.beta.P (d). 
This fact clearly shows that the TPKI-antisense significantly inhibits the 
death of the cells by A.beta.P. 
Further, it was clarified by the observations of the above-mentioned cases 
of (b) to (d) after 24 hours using a phase contrast microscope (magnifying 
power: 400) that, only in the case of (b) where TPKI-antisense and 
A.beta.P were acted to the hippocampus cells, the cytotoxity by A.beta.P 
was little and similar to the case of the control. 
Phosphorylation of tau-Protein 
Phosphorylating activity of the TPK-I was measured by the above-mentioned 
method for the samples of (1) untreated cell culture medium (control); (2) 
a sample in which 1 micromole of TPKI-antisense was added to 1 ml of the 
cell culture medium followed by adding 20 micromoles of A.beta.P after 5 
hours; and (3) a sample in which 20 micromoles of A.beta.P was added to 1 
ml of the cell culture medium and the result is given in Table 2. The 
phosphorylating activity of TPK-I in Table 2 shows that (units/mg protein) 
per mg of the protein in the supernatant liquid wherein one unit is 
equivalent to the intensity of the radioactivity measured by a laser image 
analyzer (BAS 2000; Fuji). 
TABLE 2 
______________________________________ 
Phosphorylating Activity of TPKI 
Treating Agent (unit/mg protein) 
______________________________________ 
(Control) 39.6 
A.beta.P + TPKI-Antisense 
31.6 
A.beta.P 66.2 
______________________________________ 
As shown in Table 2, the phosphorylating activity of the case (2) in which 
TPKI-antisense and A.beta.P were acted on the cell culture medium was only 
about one-half of that of the case (2) in which only A.beta.P was acted. 
Thus, it is clear that the TPKI-antisense significantly inhibits the 
phosphorylating activity of TPK-I by A.beta.P. 
Example 2 
Cloning of Human TPK-IcDNA 
Commercially-available human fetus brain cDNA library (prepared by 
inserting a 1:1 mixture of cDNA synthesized from mRNA of human fetus brain 
using oligo dT and ramdom primer to lamda-ZAPII; manufactured by 
Strategen) was infected to a host which was E. coli XL1-blue [W. O. 
Bullock, et al: Biotechnique, 5, 376-379 (1987)] to form plaques. The 
plaques (numbers: 450,000) were screened using a probe which was prepared 
by a part of the translation domain (170 base pair from the 1137th HindIII 
site to the 1306th A; Seq. ID No. 8 of the Sequence Listing) of the rat 
TPK-IcDNA (Seq. ID No. 2 of the Sequence Listing) whereby 19 positive 
clones were obtained. Among those, two clones were subcloned to Bluescript 
SK (Strategen) and then EcoRI fragments which hybridize with the above 
probe were subcloned to vector pUC19 [C. Yanisch-Perrou, et al: Gene, 33, 
103 (1985)]. The restriction enzyme map of the EcoRI fragments of the two 
clones prepared as such is given in FIG. 1. 
Base sequence was determined for entire domains of the clone #1 by a 
dideoxy method and it lacked the N-terminal moiety of human TPK-I protein. 
Total length of clone #2 was 2.2 kilobases and, out of a comparison with 
the restriction enzyme map, it was presumed to probably contain clone #1. 
Therefore, the base sequences corresponding to 5'-untranslated domain and 
N-terminal domain mostly comprising human TPK-I protein were determined by 
a dideoxy method of Sanger, et al. The base sequence of cDNA out of the 
both results and the amino acid sequence of TPK-I supposed therefrom are 
given in Seq. ID No. 1 of the Sequence Listing. 
Further, comparison with the amino acid sequence of the rat TPK-I is given 
in FIG. 2. 
Example 3 
Expression of Human TPK-I by Insect Cells 
NruI-EcoRI fragments containing entire length of translation domain of 
human TPK-IcDNA were inserted to a SmaI-EcoRI part of transfer vector 
PVL1392 [Invitrogen; N. R. Webb and M. D. Summers: Technique, 173-188 
(1990)] prepared by insertion of virus-originated DNA fragments containing 
baculovirus (nuclear polyhedrosis virus) polyhedron gene and promoter 
thereof into vector PUC8 (E. coli-hosted plasmid vector) whereupon an 
expression vector PVL-TPKI was prepared. 
Cell strain Sf9 originated from ovalium cells of Spodoptera spp. was 
cultured in a medium for insect cells FNM-FH [This was prepared as 
follows; thus, 0.35 mg/lit of sodium bicarbonate (manufactured by Wako 
Pure Chemical), 3.3 mg/ml of TC lactalbumin hydrolysate (manufactured by 
Difco) and 3.3 mg/ml of TC yeast late were added to a Grace's insect 
medium (Sigma), adjusted to pH 6.2, sterilized and then a heat-processed 
10% bovine fetus serum, 50 micrograms/ml of gentamycin sulfate and 2.5 
micrograms/ml of amphotericin B were added thereto.] and co-infected with 
wild baculovirus DNA and vector DNA whereupon a homogeneous recombination 
between them took place to some extent and, as a result, recombinant virus 
having a TPK-I expressing system was prepared. 
Selection of the wild virus-infected cells and the recombinant 
virus-infected cells was conducted visually and, as a result of repeated 
selections for three times, the recombinant virus-infected cells were 
separated. The virus-containing liquid with high infectivity obtained from 
the supernatant of the cells was further infected to Sf9 cells and 
cultured for 72 hours to recover 5 .times.108 cells. They were suspended 
in 30 ml of a buffer A [comprising 10 mM sodium phosphate (pH: 7.05), 1 mM 
ethylenediaminetetraacetic acid, 5 mM ethyleneglycol bis(2-amino-ethyl 
ether) tetraacetate, 2 mM dithiothreitol, 10 mM magnesium chloride, 0.1 mM 
sodium orthovanadate, 40 micrograms/ml phenyl methanesulfonyl fluoride, 1 
microgram/ml leupeptin, 1 microgram/ml pepstatin and 1 microgram/ml 
antipaini], homogenized and centrifuged at 105 G to recover the 
supernatant liquid. Then the supernatant was subjected to a 
phosphocellulose column chromatography (filled with P-11; Whatman) and 
fractionated with a buffer B [comprising 25 mM 
tris(hydroxymethyl)aminomethane hydrochloride (pH: 7.5), 1 mM 
ethylenediaminetetraacetic acid, 1 mM dithiothreitol, 0.1% 
beta-mercaptoethanol, 5% glycerol and 50 mM sodium chloride] with a 
gradient of sodium chloride concentrations of 50 mM to 250 mM. Each 
fraction of the eluate was analyzed by means of an electrophoresis and an 
immunoblot technique and an anti-TPK-I antibody positive fraction which 
cross-reacts with an anti-rat TPK-I amino terminal antibody [rabbit 
antiserum obtained as an antigen by expressing rat TPK-I amino terminal 36 
residues (Seq. ID No. 9 of the Sequence Listing) as an E. coli-hosted 
vector system as its fused protein with beta-galactosidase] was pooled. 
This was concentrated by means of an ultrafiltration, subjected to a blue 
sepharose column chromatography (filled with Blue-Sepharose CL-6B of 
Farmacia) and fractionated by a buffer C [comprising 20 mM 
N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (pH: 7.5), 1 mM 
ethylenediaminetetraacetic acid, 0.1 mM dithiothreitol and 5% glycerol] 
with a sodium chloride concentration gradient of 0 to 1M. Anti-TPK-I 
antibody positive fraction was pooled by analysis of immunoblotting and 
electrophoresis for each fraction, subjected to an ultrafiltration and 
dialyzed against a buffer D [comprising 100 mM 2- (N-morpholino) 
-ethanesulfonic acid (pH: 6.5), 0.5 mM magnesium acetate, 1 mM 
ethyleneglycol bis(2-aminoethyl ether)tetraacetate, 10% glycerol, 0.02% 
polyoxyethylenesorbitan monolaurate (Tween 20), 0.1 mM 
phenylmethanesulfonyl fluoride, 1 microgram/ml pepstatin, 1 microgram/ml 
antipain, 1 microgram/ml leupeptin and 5 mM beta-mercaptoethanol] to give 
1 ml of enzyme liquid. Total protein obtained was 0.4 mg. 
Progress of phosphorylation was checked using this enzyme solution by the 
following two methods. 
(Phosphorylating Method I) tau-Protein extracted from bovine brain followed 
by purification (2 microliters; 1.5 mg/ml concentration) and 1 microliters 
of the above partially-purified enzyme solution were mixed. To the mixture 
was added a solution containing 2 mM adenosinetriphosphate and 2 mM 
magnesium acetate and [gamma-32P]adenosinetriphosphate so that the 
phosphorylation of tau-protein was conducted at room temperature for 20 
hours whereby the amount of phosphoric acid incorporated in tau-protein 
was evaluated. 
(Phosphorylating Method II) 
Phosphorylation reaction which was the same as in the method I was 
conducted with an exception that no [gamma-32P]adenosinetriphosphate was 
contained followed by subjecting to an SDS electrophoresis to blot to 
nitrocellulose. The blotted tau-protein was subjected to an immunodyeing 
with anti-tau antibody (rabbit antiserum to chicken fetus brain-originated 
tau-protein) and anti-p-tau antibody [Ihara, et al: J. Biochem., 99, 
1807-1910 (1986)]. 
As a result of the method I, incorporation of tau-protein into phosphoric 
acid was confirmed while the result of the method II was that: 
1) mobility of tau-protein after the reaction was less than that of 
tau-protein which was not phosphorylated; and 
2) tau-protein which was not phosphorylated did not react with anti-p-tau 
antibody while tau-protein after the reaction reacted with anti-p-tau 
antibody. 
Those results indicate that the outcome was the same as that in the 
phosphorylation of tau-protein using the TPK-I purified from animal brain. 
Example 4 
Phosphorylation of Peptide by Recombinant Human TPK-I 
Peptide (hereinafter, abbreviated as "K2") represented by the amino acid 
sequence described in the Seq. ID No. 10 of the Sequence Listing was 
synthesized. This peptide was phosphorylated by the same manner as in the 
phosphorylating method II in Example 3 with an exception that tau-protein 
kinase II (TPK-II) purified from bovine brain microtubule was used instead 
of TPK-I whereupon phosphorylated peptide (hereinafter, abbreviated as 
"p-K2") was obtained. 
Phosphorylations of K2 and p-K2 were conducted according to a 
phosphorylating method I of Example 3 using human-originated partially 
purified TPK-I prepared in Example 3 whereupon the progress of 
phosphorylation of p-K2 was clearly noted while phosphorylation of K2 was 
slow and its initial speed was about one-tenth of that of p-K2. 
The result shows that the outcome was the same as the phosphorylation of K2 
and p-K2 using TPK-I purified from animal brain. 
Example 5 
Expression of Recombination of Human TPK-I by Escherichia coli. 
SacI-EcoRI fragment of human TPK-IcDNA clone #2 obtained in Example 2 was 
introduced into an SacI-EcoRI part of vector PUC19 [C. Yanisch-Perrou, et 
al: Gene, 33, 103 (1985)] to prepare pUSE2. In the meanwhile, in order to 
prepare an NdeI part in an oligonucleotide from 598th to 629th members of 
cDNA represented by the base sequence described in the Seq. ID No. 1 of 
the Sequence Listing, a plus strand oligonucleotide (Seq. ID No. 11 of the 
Sequence Listing) wherein CAT was inserted between 613th and 615th member 
and a minus strand oligonucleotide (Seq. ID No. 12 of the Sequence 
Listing) from 1076th to 1047th members were syn thesized and a cDNA 
fragment (Seq. ID No. 13 of the Sequence Listing) ranging from 598th to 
1076th members and having NdeI part duplicating with the initiation codon 
was obtained by a PCR method [Saiki, et al: Nature, 324, 126 (1986)]. 
Fragments ranging from a 5'-terminal of the cDNA fragment obtained by a PCR 
method to a SacI part were inserted to SmaI-SacI part of pUSE2. The 
NdeI-EcoRI fragment of the plasmid vector was introduced into NdeI-BamHI 
part of pET3C [A. H. Rosenberg, et al: Gene, 56, 125(1987)] which is one 
of the vectors having E. coli T7 polymerase promoter to construct 
pET3C/TPKI. 
pET3C/TPKI was transformed by conventional means using E. coli BL21 (DE3) 
[F. W. Studier and B. A. Moffatt: J. Mol. Biol. 189, 113 (1986)] as a host 
to prepare a recombinant. The resulting recombinant E. coli was cultured 
at 37.degree. C. until the middle stage of the logarithmic growth phase, 
kept at 21.degree. C., 0.3 mM (final concentration) of IPTG 
(isopropyl-beta-D(-)-thiogalactopyranoside) was added and cultured for 
four hours more. The living cells (5 g) were suspended in 50 ml of a 
buffer E [comprising 20 mM of 2-(N-morpholino)-ethanesulfonic acid (pH: 
6.5), 1 mM of ethylenediaminetetraacetic acid, 5 mM of 
beta-mercaptoethanol and 50 mM of sodium chloride], disintegrated with 
ultrasonic wave and centrifuged at 100,000 g for one hour. The supernatant 
was subjected to a phosphocellulose column chromatography (filled with 
P-11; Whatman) which was balanced with a buffer E and then subjected to a 
gradient elution with sodium chloride concentrations of 50 to 500 mM 
whereby the fraction which was positive to anti-TPK-I antibody was pooled 
and concentrated. This was dialyzed against a buffer F [comprising 20 mM 
of N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (pH: 7.2), 1 mm of 
ethylenediaminetetraacetic acid and 5 mM of beta-mercaptoethanol], 
subjected to a blue sepharose column chromatography (filled with 
Blue-Sepharose CL-6B of Farmacia) and eluted with a concentration gradient 
of 0 to 1M of sodium chloride. Anti-TPK-I antibody positive fractions were 
collected and dialyzed against a buffer D. 
Phosphorylation of tau-protein was conducted by the phosphorylating methods 
I and II by the same manner as in Example 3 using the resulting 
partially-purified TPK-I. It was found that, as a result of the 
phosphorylating method I, 1.2 moles of phosphoric acid was incorporated 
into one molecule of tau-protein while, as a result of the phosphorylating 
method II, the mobility of electrophoresis of tau-protein after the 
reaction became small and the reaction with anti-ptau antibody became 
positive. 
When the partially-purified TPK-I was used for phosphorylation of the 
peptides K2 and p-K2 by the same manner as in Example 4, the 
phosphorylation of p-Ka proceeded while that of K2 hardly proceeded. 
Those results show that the recombinant TPK-I prepared in this example had 
the same property as that of TPK-I purified from animal brain and of 
recombinant TPK-I prepared in Example 3. 
(Merit of the Invention) 
In accordance with the preventive and the therapeutic agent of Alzheimer's 
disease of the present invention, the phosphorylating activation of 
tau-protein kinase I by amyloid beta-protein was inhibited whereby the 
death of the neuron in the brain can be inhibited. Further, it is possible 
to conduct a screening of the preventive or the therapeutic agent of 
Alzheimer's disease utilizing the above mechanism. 
Moreover, the human-originated TPK-I of the present invention is an enzyme 
which specifically acts to tau-protein which is suggested to be related to 
Alzheimer's disease and also to senile dementia of Alzheimer's disease 
type and, therefore, its application to clarification of cause of those 
diseases and to the investigations for the agents for the prevention and 
the therapy thereof can be expected. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- (1) GENERAL INFORMATION: 
- (iii) NUMBER OF SEQUENCES: 13 
- (2) INFORMATION FOR SEQ ID NO:1: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 2088 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA to genomic RN - #A 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: human bei - #ng 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- TTACAGGTGT GAGCCACCTC GCCCAGCTGA GTTCAGTATA ATTTTCAATG AG - #AAACTGAA 
60 
- ATTCAGTTTT ATAATCAAAG AGCATGTTTG CTGAAGCCAT CATTCTCAGC AA - #ACTAATAC 
120 
- AGGGACAGAA AACCAAACAC CGCATGTTCC ACTCATAAGT GGGAGTTGAA CA - #ATGAGAAC 
180 
- ACACGGACAC AGGGAGGGAA ACATCACACA CCAGGGCCTG TCAGGCGGTC AG - #GGGTAAGG 
240 
- GGAGAGAGAG CATCGAGACA AATATCTAAG GTATGCGGGG CTTAAAACCT AG - #ATGATGGT 
300 
- TGATAGGTGC AGCAAACCAC CATGGCACAT GTATACCTGT GTAACAAACC CG - #CACGTCCT 
360 
- GCACATGCAT CCCACAACTT AAAGCAAAAT AAAAATATAT ATATTTTTCA TA - #TTTTCATA 
420 
- TATAATATAT AAATATATAA TTAAGATAAA ATATTACATA TTACATATGT AT - #AAATTCAT 
480 
- ATATAACATA TAAAATATAT AATATTATAT ATTATATACA TGTGTATATA AA - #ATCTGGCT 
540 
- GCGGAGTTTT TGATCTATAC ATTGAACAAA TTGTCTCACC TACTGATGAA AA - #GGTGATTC 
600 
#ACC TCC TTT GCG GAG 651GG CGG CCC AGA ACC 
#Ser Gly Arg Pro Arg Thr Thr Ser Phe Ala G - #lu 
# 10 
- AGC TGC AAG CCG GTG CAG CAG CCT TCA GCT TT - #T GGC AGC ATG AAA GTT 
699 
Ser Cys Lys Pro Val Gln Gln Pro Ser Ala Ph - #e Gly Ser Met Lys Val 
# 25 
- AGC AGA GAC AAG GAC GGC AGC AAG GTG ACA AC - #A GTG GTG GCA ACT CCT 
747 
Ser Arg Asp Lys Asp Gly Ser Lys Val Thr Th - #r Val Val Ala Thr Pro 
# 40 
- GGG CAG GGT CCA GAC AGG CCA CAA GAA GTC AG - #C TAT ACA GAC ACT AAA 
795 
Gly Gln Gly Pro Asp Arg Pro Gln Glu Val Se - #r Tyr Thr Asp Thr Lys 
# 60 
- CTC ATT GGA AAT GGA TCA TTT GGT GTG GTA TA - #T CAA GCC AAA CTT TGT 
843 
Leu Ile Gly Asn Gly Ser Phe Gly Val Val Ty - #r Gln Ala Lys Leu Cys 
# 75 
- GAT TCA GGA GAA CTG GTC GCC ATC AAG AAA GT - #A TTG CAG GAC AAG AGA 
891 
Asp Ser Gly Glu Leu Val Ala Ile Lys Lys Va - #l Leu Gln Asp Lys Arg 
# 90 
- TTT AAG AAT CGA GAG CTC CAG ATC ATG AGA AA - #G CTA GAT CAC TGT AAC 
939 
Phe Lys Asn Arg Glu Leu Gln Ile Met Arg Ly - #s Leu Asp His Cys Asn 
# 105 
- ATA GTC CGA TTG CGT TAT TTC TTC TAC TCC AG - #T GGT GAG AAG AAA GAT 
987 
Ile Val Arg Leu Arg Tyr Phe Phe Tyr Ser Se - #r Gly Glu Lys Lys Asp 
# 120 
- GAG GTC TAT CTT AAT CTG GTG CTG GAC TAT GT - #T CCG GAA ACA GTA TAC 
1035 
Glu Val Tyr Leu Asn Leu Val Leu Asp Tyr Va - #l Pro Glu Thr Val Tyr 
125 1 - #30 1 - #35 1 - 
#40 
- AGA GTT GCC AGA CAC TAT AGT CGA GCC AAA CA - #G ACG CTC CCT GTG ATT 
1083 
Arg Val Ala Arg His Tyr Ser Arg Ala Lys Gl - #n Thr Leu Pro Val Ile 
# 155 
- TAT GTC AAG TTG TAT ATG TAT CAG CTG TTC CG - #A AGT TTA GCC TAT ATC 
1131 
Tyr Val Lys Leu Tyr Met Tyr Gln Leu Phe Ar - #g Ser Leu Ala Tyr Ile 
# 170 
- CAT TCC TTT GGA ATC TGC CAT CGG GAT ATT AA - #A CCG CAG AAC CTC TTG 
1179 
His Ser Phe Gly Ile Cys His Arg Asp Ile Ly - #s Pro Gln Asn Leu Leu 
# 185 
- TTG GAT CCT GAT ACT GCT GTA TTA AAA CTC TG - #T GAC TTT GGA AGT GCA 
1227 
Leu Asp Pro Asp Thr Ala Val Leu Lys Leu Cy - #s Asp Phe Gly Ser Ala 
# 200 
- AAG CAG CTG GTC CGA GGA GAA CCC AAT GTT TC - #G TAT ATC TGT TCT CGG 
1275 
Lys Gln Leu Val Arg Gly Glu Pro Asn Val Se - #r Tyr Ile Cys Ser Arg 
205 2 - #10 2 - #15 2 - 
#20 
- TAC TAT AGG GCA CCA GAG TTG ATC TTT GGA GC - #C ACT GAT TAT ACC TCT 
1323 
Tyr Tyr Arg Ala Pro Glu Leu Ile Phe Gly Al - #a Thr Asp Tyr Thr Ser 
# 235 
- AGT ATA GAT GTA TGG TCT GCT GGC TGT GTG TT - #G GCT GAG CTG TTA CTA 
1371 
Ser Ile Asp Val Trp Ser Ala Gly Cys Val Le - #u Ala Glu Leu Leu Leu 
# 250 
- GGA CAA CCA ATA TTT CCA GGG GAT AGT GGT GT - #G GAT CAG TTG GTA GAA 
1419 
Gly Gln Pro Ile Phe Pro Gly Asp Ser Gly Va - #l Asp Gln Leu Val Glu 
# 265 
- ATA ATC AAG GTC CTG GGA ACT CCA ACA AGG GA - #G CAA ATC AGA GAA ATG 
1467 
Ile Ile Lys Val Leu Gly Thr Pro Thr Arg Gl - #u Gln Ile Arg Glu Met 
# 280 
- AAC CCA AAC TAC ACA GAA TTT AAA TTC CCT CA - #A ATT AAG GCA CAT CCT 
1515 
Asn Pro Asn Tyr Thr Glu Phe Lys Phe Pro Gl - #n Ile Lys Ala His Pro 
285 2 - #90 2 - #95 3 - 
#00 
- TGG ACT AAG GTC TTC CGA CCC CGA ACT CCA CC - #G GAG GCA ATT GCA CTG 
1563 
Trp Thr Lys Val Phe Arg Pro Arg Thr Pro Pr - #o Glu Ala Ile Ala Leu 
# 3150 
- TGT AGC CGT CTG CTG GAG TAT ACA CCA ACT GC - #C CGA CTA ACA CCA CTG 
1611 
Cys Ser Arg Leu Leu Glu Tyr Thr Pro Thr Al - #a Arg Leu Thr Pro Leu 
# 330 
- GAA GCT TGT GCA CAT TCA TTT TTT GAT GAA TT - #A CGG GAC CCA AAT GTC 
1659 
Glu Ala Cys Ala His Ser Phe Phe Asp Glu Le - #u Arg Asp Pro Asn Val 
# 345 
- AAA CTA CCA AAT GGG CGA GAC ACA CCT GCA CT - #C TTC AAC TTC ACC ACT 
1707 
Lys Leu Pro Asn Gly Arg Asp Thr Pro Ala Le - #u Phe Asn Phe Thr Thr 
# 360 
- CAA GAA CTG TCA AGT AAT CCA CCT CTG GCT AC - #C ATC CTT ATT CCT CCT 
1755 
Gln Glu Leu Ser Ser Asn Pro Pro Leu Ala Th - #r Ile Leu Ile Pro Pro 
365 3 - #70 3 - #75 3 - 
#80 
- CAT GCT CGG ATT CAA GCA GCT GCT TCA ACC CC - #C ACA AAT GCC ACA GCA 
1803 
His Ala Arg Ile Gln Ala Ala Ala Ser Thr Pr - #o Thr Asn Ala Thr Ala 
# 395 
- GCG TCA GAT GCT AAT ACT GGA GAC CGT GGA CA - #G ACC AAT AAT GCT GCT 
1851 
Ala Ser Asp Ala Asn Thr Gly Asp Arg Gly Gl - #n Thr Asn Asn Ala Ala 
# 410 
#CCGAGCAGCC AGCTGCACAG 1904ACC TGA ACAGTC 
Ser Ala Ser Ala Ser Asn Ser Thr 
# 420 
- GAAAAACCAC CAGTTACTTG AGTGTCACTC AGCAACACTG GTCACGTTTG GA - #AAGAATAT 
1964 
- TAAAAAGAGA AAAAAATCCT GTTCATTTTA GTGTTCAATT TTTTTATTAT TA - #TTGTTGTT 
2024 
- CTTATTTAAC CTTGTAAAAT ATCTATAAAT ACAAACCAAT TTCATTGTAT TC - #TCACTTTG 
2084 
# 2088 
- (2) INFORMATION FOR SEQ ID NO:2: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 1972 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA to genomic RN - #A 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: rat 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
- GGCCAAGAGA ACGAAGTCTT TTTTTTTTTT TTCTTGCGGG AGAACTTAAT GC - #TGCATTTA 
60 
- TTATTAACCT AGTACCCTAA CATAAAACAA AAGGAAGAAA AGGATTAAGG AA - #GGAAAAGG 
120 
- TGAATCGAGA AGAGCCATC ATG TCG GGG CGA CCG AGA ACC - # ACC TCC TTT GCG 
172 
# Met Ser Gly Arg Pro Arg Thr Thr Ser - # Phe Ala 
# 10 
- GAG AGC TGC AAG CCA GTG CAG CAG CCT TCA GC - #T TTT GGT AGC ATG AAA 
220 
Glu Ser Cys Lys Pro Val Gln Gln Pro Ser Al - #a Phe Gly Ser Met Lys 
# 25 
- GTT AGC AGA GAT AAA GAT GGC AGC AAG GTA AC - #C ACA GTG GTG GCA ACT 
268 
Val Ser Arg Asp Lys Asp Gly Ser Lys Val Th - #r Thr Val Val Ala Thr 
# 40 
- CCT GGA CAG GGT CCT GAC AGG CCA CAG GAA GT - #C AGT TAC ACA GAC ACT 
316 
Pro Gly Gln Gly Pro Asp Arg Pro Gln Glu Va - #l Ser Tyr Thr Asp Thr 
# 55 
- AAA GTC ATT GGA AAT GGG TCA TTT GGT GTG GT - #A TAT CAA GCC AAA CTT 
364 
Lys Val Ile Gly Asn Gly Ser Phe Gly Val Va - #l Tyr Gln Ala Lys Leu 
# 75 
- TGT GAC TCA GGA GAA CTG GTG GCC ATC AAG AA - #A GTT CTT CAG GAC AAG 
412 
Cys Asp Ser Gly Glu Leu Val Ala Ile Lys Ly - #s Val Leu Gln Asp Lys 
# 90 
- CGA TTT AAG AAC CGA GAG CTC CAG ATC ATG AG - #A AAG CTA GAT CAC TGT 
460 
Arg Phe Lys Asn Arg Glu Leu Gln Ile Met Ar - #g Lys Leu Asp His Cys 
# 105 
- AAC ATA GTC CGA TTG CGG TAT TTC TTC TAC TC - #G AGT GGC GAG AAG AAA 
508 
Asn Ile Val Arg Leu Arg Tyr Phe Phe Tyr Se - #r Ser Gly Glu Lys Lys 
# 120 
- GAT GAG GTC TAC CTT AAC CTG GTG CTG GAC TA - #T GTT CCG GAA ACA GTG 
556 
Asp Glu Val Tyr Leu Asn Leu Val Leu Asp Ty - #r Val Pro Glu Thr Val 
# 135 
- TAC AGA GTC GCC AGA CAC TAT AGT CGA GCC AA - #G CAG ACA CTC CCT GTG 
604 
Tyr Arg Val Ala Arg His Tyr Ser Arg Ala Ly - #s Gln Thr Leu Pro Val 
140 1 - #45 1 - #50 1 - 
#55 
- ATC TAT GTC AAG TTG TAT ATG TAC CAG CTG TT - #C AGA AGT CTA GCC TAT 
652 
Ile Tyr Val Lys Leu Tyr Met Tyr Gln Leu Ph - #e Arg Ser Leu Ala Tyr 
# 170 
- ATC CAT TCC TTT GGG ATC TGC CAT CGA GAC AT - #T AAA CCA CAG AAC CTC 
700 
Ile His Ser Phe Gly Ile Cys His Arg Asp Il - #e Lys Pro Gln Asn Leu 
# 185 
- TTG CTG GAT CCT GAT ACA GCT GTA TTA AAA CT - #C TGC GAC TTT GGA AGT 
748 
Leu Leu Asp Pro Asp Thr Ala Val Leu Lys Le - #u Cys Asp Phe Gly Ser 
# 200 
- GCA AAG CAG CTG GTC CGA GGA GAG CCC AAT GT - #T TCA TAT ATC TGT TCT 
796 
Ala Lys Gln Leu Val Arg Gly Glu Pro Asn Va - #l Ser Tyr Ile Cys Ser 
# 215 
- CGG TAC TAC AGG GCA CCA GAG CTG ATC TTT GG - #A GCC ACC GAT TAC ACG 
844 
Arg Tyr Tyr Arg Ala Pro Glu Leu Ile Phe Gl - #y Ala Thr Asp Tyr Thr 
220 2 - #25 2 - #30 2 - 
#35 
- TCT AGT ATA GAT GTA TGG TCT GCA GGC TGT GT - #G TTG GCT GAA TTG TTG 
892 
Ser Ser Ile Asp Val Trp Ser Ala Gly Cys Va - #l Leu Ala Glu Leu Leu 
# 250 
- CTA GGA CAA CCA ATA TTT CCT GGG GAC AGT GG - #T GTG GAT CAG TTG GTG 
940 
Leu Gly Gln Pro Ile Phe Pro Gly Asp Ser Gl - #y Val Asp Gln Leu Val 
# 265 
- GAA ATA ATA AAG GTC CTA GGA ACA CCA ACA AG - #G GAG CAA ATT AGA GAA 
988 
Glu Ile Ile Lys Val Leu Gly Thr Pro Thr Ar - #g Glu Gln Ile Arg Glu 
# 280 
- ATG AAC CCA AAT TAT ACA GAA TTC AAA TTC CC - #C CAA ATC AAG GCA CAT 
1036 
Met Asn Pro Asn Tyr Thr Glu Phe Lys Phe Pr - #o Gln Ile Lys Ala His 
# 295 
- CCT TGG ACG AAG GTC TTT CGG CCC CGA ACT CC - #A CCA GAG GCA ATC GCA 
1084 
Pro Trp Thr Lys Val Phe Arg Pro Arg Thr Pr - #o Pro Glu Ala Ile Ala 
300 3 - #05 3 - #10 3 - 
#15 
- CTG TGT AGC CGT CTC CTG GAG TAC ACG CCG AC - #C GCC CGG CTA ACA CCA 
1132 
Leu Cys Ser Arg Leu Leu Glu Tyr Thr Pro Th - #r Ala Arg Leu Thr Pro 
# 330 
- CTG GAA GCT TGT GCA CAT TCA TTT TTT GAT GA - #A TTA CGG GAC CCA AAT 
1180 
Leu Glu Ala Cys Ala His Ser Phe Phe Asp Gl - #u Leu Arg Asp Pro Asn 
# 345 
- GTC AAA CTA CCA AAT GGG CGA GAC ACA CCT GC - #C CTC TTC AAC TTT ACC 
1228 
Val Lys Leu Pro Asn Gly Arg Asp Thr Pro Al - #a Leu Phe Asn Phe Thr 
# 360 
- ACT CAA GAA CTG TCA AGT AAC CCA CCT CTG GC - #C ACC ATC CTT ATC CCT 
1276 
Thr Gln Glu Leu Ser Ser Asn Pro Pro Leu Al - #a Thr Ile Leu Ile Pro 
# 375 
- CCT CAC GCT CGG ATT CAG GCA GCT GCT TCA CC - #G CCT GCA AAC GCC ACA 
1324 
Pro His Ala Arg Ile Gln Ala Ala Ala Ser Pr - #o Pro Ala Asn Ala Thr 
380 3 - #85 3 - #90 3 - 
#95 
- GCA GCC TCA GAT ACT AAT GCT GGA GAC CGT GG - #A CAG ACC AAT AAC GCC 
1372 
Ala Ala Ser Asp Thr Asn Ala Gly Asp Arg Gl - #y Gln Thr Asn Asn Ala 
# 410 
- GCT TCT GCA TCA GCC TCC AAC TCT ACC TGA AC - #AG CCCCAAGTAG CCAGCTGCGC 
1426 
Ala Ser Ala Ser Ala Ser Asn Ser Thr 
415 4 - #20 
- AGGGAAGACC AGCACTTACT TGAGTGCCAC TCAGCAACAC TGGTCACGTT TG - #GAAAGAAA 
1486 
- ATTAAAAAGA GGAAAACAAA AACAAAAACA AAAAACCCCG GCTTTGGTTT GT - #TTCTTCTT 
1546 
- TCTTCTTTTC CTCTATTTTC TTTTTTAAAA ATCTGTTTCT CCTTTTAAAA AA - #ATTAAGAT 
1606 
- GAAGTCAAGT CTGATGTCAT GGGTAACCCC ACCTACTTGG AAGGCTGAGT CT - #AGAGGTTT 
1666 
- ACAGCTCAAG CCCATGCTGG ACTACAGTGG GAGTCCAAGG CCAGCNTGGG CA - #ACTTAAAA 
1726 
- AGAACTTGTT TCAAAAACGA CAAAGTTGGC TGATAATATG GCTCTCCAAG AG - #CCACAATA 
1786 
- AATAAATATG TAAATAAACT CAAATAAGTC TTGTAATTTA AATTACACTA AA - #CTAGGTTA 
1846 
- ACTTTTAAAC TCTCATCTTT AAGAACTACA GGTTTAAAAA CCCAACGGTT GT - #TTTATGTA 
1906 
- TTAGGGAAAA ATGAAAAATC TAATATAAAA AGAAGCAGCA ACAGCAGCAG GA - #GCCAACCA 
1966 
# 1972 
- (2) INFORMATION FOR SEQ ID NO:3: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 43 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
- Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Gl - #u Val His His Gln 
# 15 
- Lys Leu Val Phe Phe Ala Glu Asp Val Gly Se - #r Asn Lys Gly Ala 
# 30 
- Ile Ile Gly Leu Met Val Gly Gly Val Val Il - #e Ala Thr 
# 40 
- (2) INFORMATION FOR SEQ ID NO:4: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
synthetic DNA (senseYPE: other nucleic aci - #d 
primer) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
# 18 GA 
- (2) INFORMATION FOR SEQ ID NO:5: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
synthetic DNA (antisense other nucleic aci - #d 
primer) 
- (iv) ANTI-SENSE: yes 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
# 18 AT 
- (2) INFORMATION FOR SEQ ID NO:6: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
synthetic DNA (senseYPE: other nucleic aci - #d 
primer) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
# 18 GA 
- (2) INFORMATION FOR SEQ ID NO:7: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
synthetic DNA (antiTYPE: other nucleic aci - #d 
sense pri - #mer) 
- (iv) ANTI-SENSE: yes 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
# 18 AT 
- (2) INFORMATION FOR SEQ ID NO:8: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 170 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic aci - #d 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: rat 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
- AAGCTTGTGC ACATTCATTT TTTGATGAAT TACGGGACCC AAATGTCAAA CT - #ACCAAATG 
60 
- GGCGAGACAC ACCTGCCCTC TTCAACTTTA CCACTCAAGA ACTGTCAAGT AA - #CCCACCTC 
120 
# 170ATCCCT CCTCACGCTC GGATTCAGGC AGCTGCTTCA 
- (2) INFORMATION FOR SEQ ID NO:9: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 36 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: rat 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
- Met Ser Gly Arg Pro Arg Thr Thr Ser Phe Al - #a Glu Ser Cys Lys Pro 
# 15 
- Val Gln Gln Pro Ser Ala Phe Gly Ser Met Ly - #s Val Ser Arg Asp Lys 
# 30 
- Asp Gly Ser Lys 
35 
- (2) INFORMATION FOR SEQ ID NO:10: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 34 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
- Ser Gly Asp Arg Ser Gly Tyr Ser Ser Pro Gl - #y Ser Pro Gly Thr Pro 
# 15 
- Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Th - #r Pro Pro Thr Arg Glu 
# 30 
- Pro Lys 
- (2) INFORMATION FOR SEQ ID NO:11: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 32 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
synthetic DNA (senseYPE: other nucleic aci - #d 
primer) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
# 32 ATAT GTCAGGGCGG CC 
- (2) INFORMATION FOR SEQ ID NO:12: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
synthetic DNA (antisense other nucleic aci - #d 
primer) 
- (iv) ANTI-SENSE: yes 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
# 30 CTCG ACTATAGTGT 
- (2) INFORMATION FOR SEQ ID NO:13: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 479 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
#RNA (ii) MOLECULE TYPE: cDNA to genomic 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: human bei - #ng 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
- TTCGCGAAGA GAGTGCATAT GTCAGGGCGG CCCAGAACCA CCTCCTTTGC GG - #AGAGCTGC 
60 
- AAGCCGGTGC AGCAGCCTTC AGCTTTTGGC AGCATGAAAG TTAGCAGAGA CA - #AGGACGGC 
120 
- AGCAAGGTGA CAACAGTGGT GGCAACTCCT GGGCAGGGTC CAGACAGGCC AC - #AAGAAGTC 
180 
- AGCTATACAG ACACTAAACT CATTGGAAAT GGATCATTTG GTGTGGTATA TC - #AAGCCAAA 
240 
- CTTTGTGATT CAGGAGAACT GGTCGCCATC AAGAAAGTAT TGCAGGACAA GA - #GATTTAAG 
300 
- AATCGAGAGC TCCAGATCAT GAGAAAGCTA GATCACTGTA ACATAGTCCG AT - #TGCGTTAT 
360 
- TTCTTCTACT CCAGTGGTGA GAAGAAAGAT GAGGTCTATC TTAATCTGGT GC - #TGGACTAT 
420 
- GTTCCGGAAA CAGTATACAG AGTTGCCAGA CACTATAGTC GAGCCAAACA GA - #CGCTCCC 
479 
__________________________________________________________________________