JPH03172184A - Genome dna sequence - Google Patents

Abstract

NEW MATERIAL:Genome DNA sequence shown by the formula substantially containing a range controlling transfer and termination of LZ-8 structural gene. USE:Efficiently producing LZ-8. PREPARATION:The title sequence is extracted and purified from Ganoderma lucidum as a raw material by conventional procedures.

Description

[Detailed Description of the Invention] (Industrial Application Field) The present study relates to the genomic DNA of C. chinensis, which contains the structural gene of a novel protein with immunosuppressive ability derived from the mycelia of C. chinensis (CG*nodtrm*). It is.

(Prior Art and Problems to be Solved by the Present Invention) Cypress mushroom (1; xnoder*a lucidius)
Notebook Hidanashitake +4 (Pol
It is a basidiomycete that belongs to the genus yporx + es), and is also called reishi, and has been prized as a herbal medicine since ancient times, and is still used as an ingredient in Chinese herbal medicine. It is widely used as a food.

The Ministry of the Invention and others previously extracted a novel protein with immunosuppressive ability (hereinafter referred to as LZ-8) from the cephalothelium of C. chinensis, identified its amino acid sequence, and identified the cDN^ encoding the amino acid sequence. was cloned (Japanese Unexamined Patent Publication No. 1-2749, Medical Patent Application No. 83-274552, The
Journal of Bloglcal Che
*Istry 264. 472-478 (1989
) and The Journal of Biology
cal Chesstry 2B4. 16372
-16377 (1989)). LZ-8 is N
It consists of 110 amino acids in which Song Dynasty amino acids are acetylated, and the molecular weight is 1 including the N-terminal acetyl group.
2,420, and the physiological activity of lnvlvo is allergic type I! f! (anaphylaxis), isotype (insulin-dependent diabetes), isotype (Arthus reaction), and isotype (mixed lymphocyte reaction).
Ding. From this, it is considered that LZ-8 is effective in treating allergies.

In order to efficiently produce LZ-8 by genetic engineering, the present inventors further modified the LZ-8 coding region, including the 5° transcription-regulating t8 region and the 3' transcription termination region. We decided to clone the LZ-8 genome ON^ containing the LZ-8 genome and elucidate the structure involved in transcriptional control (means for solving the problem). It is extracted and produced by a conventional method using the stone mushroom described in Japanese Patent Application IV1 83-274552 as a raw material.

In other words, the mushrooms used in the present invention are published in the Illustrated Encyclopedia of Japanese Fungi (Yakushusha Edition) and the Journal of Japanese Mycology (I) written by Seiya Ito.
Any mycelium that has been identified according to the Kendo version) may be used.Since there may be variations in production depending on the strain, or there may be regional differences in the strains collected, this substance should not be used. In order to obtain it efficiently, the FERII IIP-1
828) is preferably used.

The present invention will be explained below using examples. (Example) <Preparation of the genome ON^ of Cinnamon lucidum> The Cinnamon mycelium (wet weight about 20 g) was mixed with a liquid 'g. Grind in the air,
10-ml extraction buffer (10mls-MCI p
H 7.5, 10 ml I N@CI, 2
5 All EDTA (hereinafter referred to as TEII street solution) is mixed with V, and then SOS is added so that the final concentration is 1%.
Next, if my protease K was added to this suspension and treated at 37°C for 2 hours.
After adding one liter of H NaCI, the phenol treatment was repeated five times.The aqueous layer was extracted twice with chloroform/isoamyl alcohol (24:I), and 2.5 times the amount of ethanol was added to precipitate the genome ON^. After washing this precipitate with 80% ethanol, it was dissolved in 10% TELLIi solution. This sample contains a large amount of RN^, so in order to remove it, 200 μg of bonuclease A was added to the solution. The RNA was degraded by incubation at ℃ for 30 minutes, followed by repeated phenol treatment and ethanol precipitation, and finally approximately 200 μg of genomic DNA was obtained.
Genome LoNA (1!8) Noku O-27g> 12 μg of the genome ON^ obtained above was completely digested with restriction dyes EcoR I and Sex I, and the DNA fragments were separated by 0.8% agarose gel electrophoresis. At this time, it was known that the LZ-8 genome DN^ was contained in a fragment of approximately 5 kb, so both DNA fragments of 4 to 5 kb were extracted from this gel 1]. Since Scx I cleavage produces blunt ends, EcoR I linkers (
pCCGAATTCGG; TAKAI＾ Company I! ) and then ligated the linker with EcoR I and separated the desired DNA fragment by agarose gel electrophoresis. Approximately 1/3 fil of the recovered DNA was transferred to RUG's λgtl07-me (manufactured by Stratagene). Gigapack Golds (Stra
Using Tagene's packaging ● kit),
This library contains approximately 250,000 clones. Approximately 25,000 plaques were packaged on 5 plates (90 am diameter) according to the method described in the attached protocol. and lifted onto a nitrocellulose filter. The previously obtained L was used as a probe for screening positive clones.
Cloned cDN of Z-8 (patent application 1986-27455)
This cDNA (520 bp)
was integrated into the EcoR I site of pUcI9, so we first digested it with EeoR I, fractionated it by agarose gel electrophoresis, and extracted the cDN^ from the gel.
Approximately 50 ng of DN^ was added to Amersham's Multl.
prlme DNA Labeling System
ms, “P-dCTP (3,000 CI/g
The aforementioned nitrocellulose filter labeled with aol) and immobilized with phage ON^ was then filtered with 4×S
SC (l x SSC is L 10 x F containing 0.15 M NaCI and 0.015 11 citric acid)
BP(l x FOP is 0.02% Ficoll, 0.0
2% BS^, 0.02% polyvinylpyrrolidone and 0.02% polyvinylpyrrolidone.
Prehybridize by treating with a solution containing 1% SOS at 65°C for over 1 hour, and then
X FOP. 0.1 at/-Sake Kenko ON^ and 2
．． Perform a hybridization scene overnight at 65°C with a solution containing 8 x 10'cps/d''P-cDN^.After the hybridization scene is completed, the filter is heated to 0.
After washing twice for 20 minutes at room temperature and 30 minutes at 65°C using a solution of 0.1% SOS, three strong signals were found on the autoradiogram. Finally, one clone was confirmed to contain the LZ-8 genome ON^ from analysis of its nucleotide sequence. Structure〉The restriction map of the ON^ fragment of the LZ-8 gene containing the LZ-8 gene obtained by the method described above is shown in Figure 1. The region of the base sequence shown in the figure is shown.

Furthermore, p
The nucleotide sequence was determined by subcloning into Ucl9 and using the Sanger method (2) while creating primers as necessary.The results are shown in Figure 2.In this figure, ... indicates rotational symmetry. Structures containing CCAAT-like sequences, are T^Ding^ boxes, are dots,
is the base sequence 1 that is thought to be related to splicing.
1 and ↓ indicate the BoliA addition sites, respectively.

The nucleotide sequence of the translated region of LZ-8Ffi white matter and the untranslated region on the 3° side is exactly the same as the cDNA nucleotide sequence described in the previously filed Tokuro Sho 63-274552, except for poly(^). It is. cDNA in the untranslated region on the 5I side
There is a missing sequence in A (base numbers 54 to 114 in Figure 2; all base numbers below are from Figure 2). This part is an intron, and the LZ-8 gene is transcribed into the mature ■RN^? spliced before cutting
It seems that it has disappeared from cDNA. The sequences GT on the 5° side and ^G on the 3° side of the splice are well conserved1.conser+sus in plants and vertebrates
There is also a branching site sequence that is consistent with the sequence and is common to vertebrates and viruses (base numbers 97 to 101).

However, the sequence 1 is the same as the base sequence of the branching site in yeast.
does not exist. Using the primer extension method using two types of oligonucleotides, the transcription start point is the base number! The nucleotides on both sides of this base were found to be (;ACAC
This sequence has a very characteristic feature of having a cluster of virimidine on the outside.
(R represents purine salt & Y represents pyrimidine base).

Approximately 80 bases upstream from this transcription start point is a sequence containing a TAT^ box, ccgtataaaagg, which seems to play an important role in the transcription of LZ-8 mRNA.

gcaattc, which contains a sequence similar to the CCAAT box, begins from base sequences -259 and -174. Furthermore, base sequences exhibiting rotational anti-fish characteristics, such as those found in the UAS (upstream transcriptional promotion region) of the yeast GAL gene group, start from salt identification numbers -406 and -168 (in Figure 2... Please indicate)●〉. These base sequences are also LZ
-8 It may have the effect of enhancing transcription of mRNA.

In animal cells, transcription of ■RN^ is known to occur far downstream from the poly(A) addition site1+, and in C. chinensis, the transcription of ■I
If transcription of IN^ is performed up to a point downstream of the poly(A) addition site, LZ-a mRNA is likely to form the secondary structure shown in FIG. 3. If such a structure is adopted, the addition site of poly(A) exists in the loop part, and is similar to AAUAA^●, which is said to be the poly(A) signal in animal cells, and is similar to ■IIN^ in yeast. UAAt often found near the 3′ end of
l will also be located in the loop portion. It is thought that by adopting such a secondary structure of Peng RNA, poly(A) is added at a precise position, and it becomes less susceptible to the action of degrading enzymes, and ■R
This may be related to NA stability and lifespan.

LZ-8af white matter is produced in large amounts in the medulla, and therefore, the LZ-8af white matter corresponds to the LZ-4ffi white matter.
The amount of N^ is also large. The reason for this may be that the characteristic nucleotide sequence on the 5° side of the LZ-8 structural gene mentioned above functions as a promoter, enhancer, or efficient transcription initiation site. Also, po1yO on the 3° side
) The base sequence downstream of the addition site is such that when it is transcribed into the mRNA precursor, it has the possibility of forming a secondary structure that has the OIF (A) addition site in the loop and is stable against degrading enzymes. Possible gender. Furthermore, what about introns? l
It is also thought that the nucleotide sequence immediately before the methionine codon that initiates translation has the effect of increasing the efficiency of the biosynthesis of the final product LZ-8 protein.

References 1. Recovery of DNA f
rom Low letlng Tem-pe
rate Agarose. p. I70 In''
Ilolecular Clone-ngt^ Iabo
ratory Manual” (ads. llan
latlg, T. , Frltsch, E. F. ，
l Sambrook, J. ) Cold Sprin
g Harbor Laboratory (1982
)2, Sanger, F. , Mlklen, S.
．． & Coulson, A. Il. (+977)
Proe. Natl. ＾cad. Scl. USA
74. 5483-3. RNA sp.clng
, pp. +31-206 In "Transcrl"
p-tronand Spllclng' (eds.
HaI+es, B. D. I Glover, D.
M. ) IRL Press (+988) 4.517 Ping and Primer Extension Method, PP241-25Fhr Follow-up Biochemistry Experiment Course I (Japan Biochemical Society IEI) Michiden Research Method ■, Recombinant DNA Technology” (Tokyo Kagaku Doujin) (19
1988) 5. Hahn. S. ．． }four, E. T.
& Guarenta, L. (+985) Pro
c. Natl. Acad. Scl. LIS＾8
2, 8582-6. Fukazawa et al. (+985) af white matter nucleic acid enzyme t. 30, 1491-? ．． Ter
mlnatlon and 3' Procassln
g of Euka-ryotlc l)IA p
p. 97-129 1n ″Dranscrlpt
lon and Splicing” (ads.N
ames, B. D. l Glover, 0.11
．． ) IRL Press (1988) 8, foychlk. R. P. , Lyons, R.
．． H. , Post, L. I.Rottman, F.
．． lI. (+984) Proc. Natl. A
cad. Scl. IJSA, 81. 3944
-39489. 8rake, A. J. , Jull
us, D. J. i Thorner, J. (+9
83) Ilol. Cell. Blol. 3.
Brief explanation of the 1440-1450 drawings Figure 1 shows the LZ-8 genome containing the LZ-8 gene.
A restriction enzyme map of the EcoR I and Sea I cleavage fragment of ^ is shown. FIG. 2 shows the base sequence of the genome ONA containing the structural gene of LZ-8. Figure m3 shows the estimated secondary structure of RNA transcribed from the LZ-8 gene.

Applicant Meiji Dairies Co., Ltd. Figure 1 Restriction enzyme map of the EcoRI/Sea I fragment of C. chinensis genome DNA containing the LZ-8 gene CIz8)) Figure 2 Estimated secondary structure of LZ8 mRNA 3' end Ltca ttcLggaggaact tgaa
Lgtcctscggca taccaagtggca
tit H tcagcagggg t tgca
tccag Lccacctgcggtgcas t
tcggttccatgtacttgcagggcc
tcgcacgtctt*tgcacctggttt
aam tea tcz ttaaaCggcgctc
cgg t tgccg t etas a agga
cggcaaagLgagccetcccccttea
mtaacccceLamCttctgccccgca
gc＾Ding CI+8＾TG TCC GAC ACT G
CC TTG ATC TTC AGG CTC GC
C TGG 164IktSerAspThrAle
LeuIIsPheArgLau▲laTrp+2GA
C GTG AAG AAG CTC TCG TTC
l;AC TAC ACC CCG AAC 19
0Asp N'al Lys Lys Leu Ser
Pha Asp Tyr Thr Pro Asn
24TGG Ili[;C CGC GGC AA
C CCC AAC^^C TTC ATC GAC
ACT 226Trp Gly Arg Gly A
sn Pro Asn Asr+ Phe Ile A
sp Thr 3BGTC ACC TTC
CCG ^^A GTC TTG ACC
GAC AAG GC (i ding^C 262V
al dinghr Phe Pro LyII V
al Leu Thr ^sp Lym Al
a Tyr 48ACG TAC CGC G
TC GCC (;TC TCC GGA CGG A
AC CTC GGC 298Thr Tyr＾rg
V@l^lm Vil 5er Gly^rt As
nLeu Gly 60[;TGAAACCCTCG
dingACGCGGTCGAGAGC(;ACGGCTCG
334▼ml Lys Pro Ser Tyr Al
aYal Glu Ser Asp Gly Ser
72Xho CAG^^G GTC AAC TTC CTC GA
G TAC AAC TCC GGG TAT 37
GGln LyIIIVml Asn Ph1Leu G
lu Tyr Asn Sar Gly Tyr 8
4GGC ATA GCG GAC ACG^^C A
CG ATC CAG GT (; TTC GTT
408(ily II@AI-▲in Thr Lan
Thr Ila Gln Val Phe V@1
98▼a1 ^mp Pro Asp Thr
▲sn Asn Asp Pha IIs
IIs AleCAG TGG^^C TAG
＾Gln Tr
p Asn End

Claims (1)

[Scope of Claims] 1. The following genomic DNA sequence substantially containing a region controlling transcription and termination of the LZ-8 structural gene. -446 ttcattctggagggaacttgaatgt
cctacggcataccaagtggcat-33
9 tatatcaccttcggatatcgccatc
cataaccaacgtctcaacccccg-35
2 agatggacgtcatctggaggtaacg
accaaggcggtcttccggcaac-30
5 tgtggtctcgaagacgtgaggcgtt
tacaaggttggacatctcgggg-25
8 caattctgccaaactcgcaaggaga
atgtaccgtcctatcacctgca-21
1 gtggtcagcaggggttgcatccagg
tccacctgcggtgcaattcggt-16
4 tccctgtggcttgcagggcctcgca
cgtcgtatgcaccctggtttac-11
7 atcatcgtgaaacggcgctccggtt
gccgtataaaaggacggcaaag-70 gcggccagtggacttcagcacctgc
tcttgtacccacttctagtaag-23
+124 tcgcataccacctctcctgacagCG
ACAGGTTCACTGACTAGTTCAT71 AGTCCACAGCTCTTTGCCTTACAAT
CAAGgtttgccgacaccctctt118 tgagccctcccccttcaataaccccc
ctaacttctgccccgcagCATC154 ATGTCCGACACTGCCTTGATCTTCA
GGCTCGCCTGGMetSerAspThrAl
aLeuIlePheArgLeuAlaTrp190 GACCTGAAGAAGCTCTCGTTCGACT
ACACCCCGAACAspValLysLysLe
uSerPheAspTyrThrProAsn228 TGGGGCCGCGGCAACCCCCAAACAACT
TCATCGACACTTrpGlyArgGiyAs
nProAsnAsnPheIleAspThr282 GTCACCTTCCCGAAAGTCTTGACCG
ACAAGGCGTACValThrPheProLy
sValLeuThrAspLysAlaTyr298 ACGTACCGCGTCGCCGTCTCCGGAC
GGAACCTCGGCThrTyrArgValAl
aValSerGlyArgAsnLeuGly334 GTGAAAACCCTCGTACGCGGTCGAGA
GCGACGGCTCGValLysProSerTy
rAlaValGluSerAspGlySer370 CAGAAGGTCAACTTCCTCGAGTACA
ACTCCGGGTATGlnLysValAsnPh
eLeuGluTyrAsnSerClyTyr408 GGCATAGCGGACACGAACACGATCC
AGGTGTTCGTTGlyIleAlaAsnTh
rAsnThrIleGlnValPheVal442 GTCGACCCCGACACCAACAACGACT
TCATCATCGCCValAspProAspTh
rAsnAsnAspPheIleIleAla485 CAGTGGAACTAGGAGGAGGCAGTGA
CTGACCCCTGGCGGTCTAGlnTrpA
snEnd 532 ATCTGCGGGCACTGTGGGGGAGG
GCATCGCCCATCAGCTTCCTTG579 TCTCATAATCATGCCCGTGTAACAA
TTGTAAATCGACCTTGTTGTAC626 CATGCAtccagcttttgtggtgtgc
cgtcttatgtttggttgaatgc