Hahn, S., Hoar, E. T., and Guarente, L. (1985) Proc. Natl. Acud. Sci. U. S. A. 82,8562-8566. 38. Struhl, K. (1987) Cell 49, 295-297. 39. Dean-Johnson, M., and ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.
Val. 266, No. 4, Issue of February 5,pp. 24862493,1991 Printed in U.S.A.
Molecular Cloning of a cDNA and a Gene Encoding an Immunomodulatory Protein, Ling Zhi-8, from a Fungus, Ganoderma lucidurn” (Received for publication, July 31, 1990)
Akira Murasugi, Shizuko Tanaka, Naoki Komiyama, Noriko Iwata, Kohsuke Kino, Hajime Tsunoo, and Sadatoshi Sakuma From the Molecular Oncology Division and the Biochemical Genetics Division,Me@ Institute of Health Science, Odawara, Kanagawa 250, Japan
A large amount of the novel immunomodulatoryprotein Ling Zhi-8 (LZ-8) is synthesized in the myceliaof Ganodermalucidum (Kino, K., Yamashita, A., Yamaoka, K., Watanabe, J., Tanaka, S., KO,K., Shimizu, K., and Tsunoo, H. (1989) J. Biol. Chem. 264, 472478). A cDNA and a gene for LZ-8 were isolated and characterized. The mixed oligonucleotide probes for LZ-8 cDNA were designed from the results of protein sequencing (Tanaka, S., KO, K., Kino, K., Tsuchiya, K., Yamashita, A., Murasugi, A., Sakuma, S., and Tsunoo, H. (1989) J. Biol. Chem. 264, 16372-16377) and were used for screening the mycelial cDNA library. The nucleotide sequence of the cloned cDNA confirms the amino acid sequence of LZ-8 that was previously determined by protein sequencing. The clones containing the LZ-8 gene (12-8) were obtained from the mycelial genomic DNA library using the cDNA probe. Two CCAAT-like sequences and one TATA box were found at the upstream region of the postulated transcription initiation site of 12-8. A small intron (61 nucleotides long) divided 12-8 into two exons at the 6”untranslated region. The other characteristic sequences were alsofound around the postulated transcription initiation site and around the poly(A) additional site.
A novel immunomodulatory protein, LZ-8,’ has been isolated from the mycelial extract of an Oriental medicinal fungus, Ling Zhi (Ganoderma lucidum) (1).LZ-8 has mitogenic capacity toward mouse spleen cells and human peripheral lymphocytes in vitro. It agglutinates sheep redblood cells but does not agglutinate human redbloodcells. LZ-8 also functions as a potent suppressor of bovine serum albumininduced anaphylaxis in CFWmice in vivo (1). The protein LZ-8 consists of 110 amino acid residues including an acetylated N-terminal serine and has a molecular mass of 12,420 Da (2). LZ-8 shows considerable similarity to the variable region of the immunoglobulin heavy chain both
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. has been submitted The nucleotide sequence(s)reported in this paper to the GenBankTM/EMBL Data Bank with accession numbeds) 505735. ‘The abbreviations used are: LZ-8, Ling Zhi-8; kbp, kilobase pair(s); bp, base pair(s); kb, kilobases(s); SDS, sodium dodecyl sulfate; R, purine; Y, pyrimidine; N, any nucleotide; UAS, upstream activating site.
in its amino acid sequence and in its predicted secondary structure. It may be possible to consider that LZ-8 is related to an ancestral protein of the immunoglobulin superfamily and that thesimilar structure of LZ-8 to theimmunoglobulin heavy chain is responsible for its immunomodulating function. A cIoned cDNA or a cloned gene for LZ-8 is necessary for mutagenesis studies to examine changes in hemagglutinating activity when a particular amino acid residue is replaced by another. This is also true for the mitogenic or the immunomodulating activity. A fairly large amount of LZ-8 protein, at least 5%of the totalsoluble protein calculated from the results of the purification (I), is synthesized in the mycelia. It is expected that theLZ-8 gene is actively transcribed in mycelia andthatthereare some indications from the nucleotide sequence of the gene for an efficient transcription initiation, a strong promotor, and so on. Therefore, we cloned a cDNA and a gene encoding the LZ-8 protein and have characterized them. EXPERIMENTAL PROCEDURES*
RESULTS AND DISCUSSION
Isolation of cDNA Encoding LZ-%”he aminoacid sequence of LZ-8 was determined completely by protein sequencing (2). The four sets of oligonucleotide mixtures were designed from the amino acid sequence data and synthesized as the probes for LZ-8 mRNA or cDNA (Fig. 1M). Theprobe, T13b23M, was expected to contain an oligonucleotide that would efficiently hybridize to LZ-8 mRNA, or cDNA, even if it did not contain a perfectly matched oligonucleotide for the target. Onthe other hand,one set of the threeoligonucleotide sets, T14c17A, T14c17G, and T14c17T, should contain one oligonucleotide completely matched with LZ-8 mRNA, because these contain all possible sequences deduced from the amino acid sequence. Northern blot analysisof poly(A)+RNA from mycelia of G.lucidum was performed using 32P-labeled four sets of oligonucleotide probes, as shown in Fig. 7. The strong signals were given byT13b23M and by T14c17G. It is concluded that T14c17A or T14c17T gave a weak signal because of a single base mismatch, A:C or T:C, at the third base from the 5’ end of the probe, and that thecodon for Ile in the peptide is AUC. Therefore, T14c17G and T13b23M were used for screening the cDNA library. In Fig. 7, the Portions of this paper (including “Experimental Procedures,” Table lM, and Figs. 1M-6M) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal thatis available from Waverly Press.
2486
LZ-8
A Gene Encoding Immunomodulator, an 2
1
3
4
2487 TABLE 2
5
Codon usage of LZ-8 cDNA
rn a
-7.6
-4.* -2.4 -1.8 -1.3
-0.70 -0.63
Codon
Phe
uuu uuc
Leu
(%)of
codon use
UUA UUG
cuu
-Os!@
FIG.7. Northen blot analysis of mycelial poly(A)* RNA. About 5 pg of total RNA from mycelia ( l a n e 1 ) or 4.7 pg of poly(A)+ RNA (lanes 2-5) was electrophoresed on a 1.5% agarose gel in a denaturing condition, transferred to a Genescreen filter (Du PontNew England Nuclear), and fixed by baking at 80 “C for 2 h. The filter containing lane 1 was cut out andstained with methylene blue (8). The fractionated poly(A)+ RNA was probed with 32P-labeled T13b23M ( l a n e 2), T14c17A ( l a n e 3), T14c17G ( l a n e 4 ) , or T14c17T ( l a n e 5). The sizes of the markers are shown in kb.
Number
Amino acid
Ile Met Val
Ser
CUC CUA CUG AUU AUC AUA AUG GUU GUC GUA GUG
ucu
signals were observed at about 600 bases of RNA, and any ucc UCA other signals were not detected. The 600 bases of mRNA may UCG have enough length for coding a protein that is made of 110 AGU amino acids. The large amount of LZ-8 mRNA was expected AGC in the poly(A)+RNA from the signal strength. The lengths of ccu Pro the ribosomal RNAsof G. lucidurn are approximately 3.5 and ccc CCA 1.9 kb. The probes usedin Northern blot analysis are specific CCG enough to detect the sequences in a genomic DNA using a Thr ACU dried agarose gel as shown in Fig.2M. The signals were ACC observed at about 8.4 kbp with the two oligonucleotide probes, ACA when the genomic DNA was digested with EcoRI. The cDNA ACG library constructed from poly(A)+RNA of well growing myAla GCU GCC celia was screened with the two probes described above.The GCA signals were clear and strong enough even when the 17-mer GCG probe was used, as shown in Fig.3M. When the signals UAU Tyr obtained on duplicate filters with T13b23M and with UAC T14c17Gwere compared, almost all positive signals were TeP UAA common (Fig. 3M). Approximately 200 positive clones were UAG UGA obtained when 2.5 x lo4 clones of the partial library were His CAU screened. Finally, seven positives were purified. The nucleoCAC tide sequence of the longest cDNA (520 bp) was determined, CAA Gln as shown in Fig. 4M. The cDNA sequencecan be seen in the CAG genomicDNAsequence (see Fig. 11). The longest cDNA Asn AAU AAC contained 27 bp of 5’-noncoding region, 333 bp of entire AAA LYS coding region, 133 bp of 3’-noncoding region including the AAG termination codon, and 27 bp of poly(A) tract. The amino GAU ASP acid sequence previously determined (2) was completely the GAC same as thatdeduced fromthe nucleotide sequenceof cloned Glu GAA cDNA except that the first Met was removed in the LZ-8 GAG UGU protein. The matured intact mRNA might havean additional CYS UGC 30 nucleotides at the 5’-noncoding region fromthe results of UGG Trp analysis on the transcription initiation site, described later. CGU Arg The codon usageof LZ-8 is shown in Table 2. It is obvious CGC that for the third letter of the codons, G or C is favored. About CGA CGG 93% of the time the third letteris G or C. It is not similar to AGA the codon usagesof highly or lowly expressed genes fromyeast AGG (5) nor those of genes from filamentous fungi (17), the taxoGGU G ~ Y nomically related organisms. The codons used in highly exGGC pressed genes might mainly depend on the availability of GGA tRNAs (18). Therefore, this organism might have the major GGG tRNAs that efficiently recognize those codons. One of the a Ter, termination codon. oligonucleotide probes used for screening the LZ-8 cDNA, T13b23M, does not contain all possible sequences deduced The consensus sequence of the ATG context for the transfrom the correspondingamino acid sequence,as shown inFig. lation initiation in highly expressed yeast gene proposed by 1M. Fortunately, the third letter for the Phe, Asp, and Tyr Hamilton et al. (19) is 5’ (A/U)A(A/C)A(A/C)AAUGUC(U/ codonsis C. One of the 16 kinds of oligonucleotides in C) 3’. The AUG context of LZ-8 mRNA, 5‘ AGCAUCAUGT13b23M matches completely with the targetsequence. UCC 3’, does not fit completely withthe consensus sequence
2488
Encoding A Gene
an Immunomodulator, LZ-8
above, but some similarity is observed. The downstream tri- genomic DNA from mycelia using a cDNA probe are shown plet next toAUG and the third, fourth, and sixth nucleotidesin Fig. 9. When the genomic DNA was digested with KcnRI, upstream from AUG fit with the yeast consensussequence. a single band at about 8.4 kbp was observed. The entire 12-8 According to the results obtained by Guan and Weiner (20) should be included in the 8.4-kbp fragment.In lanes of other using the in vitro translation systemof rat liver, the secondary digestions, one major band and minor bands were observed. structure formed around the AUG start codon inhibits the These resultssuggest the presenceof homologous sequence(s) translation. Therefore, A-rich in an untranslated region up- in the genomic DNA or that the recognition sites of the stream of the AUG start codon might be preferable for the restriction enzymes are present in the 12-8 sequence in spite initiation of the translation (19). In LZ-8 mRNA, a very tight of the absence of these recognition sites in the cDNA sesecondary structure cannot be formed around this region. quence. The former is the case with lz-t? (data not shown). The hydrophobicity of LZ-8 protein is shown together with When 12-8 and its homologous sequence(s) are presentin the the expected secondary structures in Fig. 8. Many parts of same X clone, the gene may be gradually lost in the cloning LZ-8 arehydrophilic, but the extreme N-terminalregion and processes because of the unequal crossingover in a wild type the regions of @-sheetswere relatively hydrophobic. The sim- Escherichia coli host (27-29). Therefore, we decided to clone ilarity of the amino acid sequence between LZ-8 and the a 5.1-kbp genomic DNA fragment that occurred in a double immunoglobulin heavy chain variable region was discussed digestion with EcoRI and ScaI. The library of the genomic previously (2). In the immunoglobulin heavy chain variable DNA was screened with "'P-labeled cDNA probe. Three posresidues, and theseform itives were obtained from 1'25,000 clones. One of the positive region, B and F @-sheets contain Cys S-S linkage. This should be important to keep the proper clones was further analyzed. Therestrictionmap of this tertiary structure of the protein. LZ-8 does not contain the cloned genomic DNA is shown in Fig. 10. A part of the cloned Cys residue. Some other interactions, such as the hydrophobic DNA, which involved the entire LZ-8 gene, was sequenced. in Fig. 11. All of the interactions between &sheets, may be necessary to maintain The nucleotidesequenceisshown the proper structure. Thehydrophilic loop region near the C sequence in LZ-8 cDNA is found in genomic DNA except for terminus contains the sequence Val-Asp-Pro-Asp-Thr-Asn- the poly(A) tract, but the 5"untranslated region of the geAsn-Asp-Phe. This is very similar to the sequences having nomic DNA is divided by a 61-bp sequence that shouldbe an Ca2+ binding capacity (23). However, the biological activity intron. The oligonucleotide probes were prepared to confirm of LZ-8 relating to Ca2+ is not yet known. the presenceof an intron. One (IN-1) is specific to the putative Recently, a similar hemagglutinating protein (FVA) from intron sequence, and the other (EX1-2) is specific to the the fruitbody of Flammulina velutipes (24) was reisolated and junction of the first and the second putative exons (Table characterized (25), and this protein was also analyzed by x- 1M). The three longer cloned cDNAs out of seven could be ray crystallography(26). The resultsof further investigations hybridized with an EX1-2 probe, as shown in Fig. 5M. The on FVA should be important for the studies on the function four negative cDNAs should not have enough length for 5'and structureof LZ-8. Isolation of a Genomic DNA Clone for LZ-B-The relation1 2 3 4 5 6 7 8 ship between the structure and thebiological activity of LZ-8 can be studied using a cDNA by mutagenesis experiments. We are also interested in the control mechanisms of the highly a genomic DNA expressed LZ-8 gene (12-8).We tried to clone containing lz-8. The results of Southern blot analysis of the 2.32.0-
FIG. 9. Southern blot analysis of themycelialgenomic DNA. The genomicDNA was digestedwith &YJRI (lane I ) . with HamHl (lane Z ) , with KcoRI and HamHl (lane .'I), with HnmHl and SphI (lane 4 ) . with EcoRI and S p h l (lone 5). with EroHl and Scnl (lane 6 ) , with EcoRI and Hglll (lnnr 7). or with Sphl and &/I1 (lane 8 ) . The fractionated DNAs fixed on a GeneScreen filter were probed with :"P-laheled LZ-8 cDNA. The sizes of markers are shown in khp.
S ,,'
FIG.8. Hydrophobicityprofileandpredictedsecondary structure of LZ-8. Hydrophobicity or hydrophilicity for each part o f 1.7,-8 ( A ) was calculated with the program of GENETYX (Software Development. Co. Ltd., Tokyo) using the parameters of Kyte and Doolittle (21). The secondary structure of LZ-8 ( H ) was predicted according to Gamieret al. (22) also using theprogram of GENETYX with a decision constant of 0 for n-helices or @-conformation.0, nhelix; V, turn: A,&sheet.
P
FIG. 10. The restriction map and the strategy for sequencing a genomic DNA harboring LZ-8 gene. The hoxes show the exons of LZ-8 gene. The a r r o m show the extent and the directiono f the sequence reading. The small closed circlr at the end of the arrow shows the positionof the UTR-3 oligonucleotide (Table 1 M ) used as the sequencing primer. K R , kilobase pair(s); 15'. EeoRI: X H . X h ~ l : X R , XhaI; S,Smol; 1'. Pstl; SI.:,Sprl;SC, Sacll: SI,. S n l l ; N . Nhrl: R, RamHI; R t , RglIl; S A , Scal.
A Gene Encoding Immunomodulator, an
LZ-8
2489
TABLE 3 Sequence elements in pre-mHNA introns The nucleotides,the degree of consensus over SOr;. are shown exceDt for the seuuence elements of G . lucidum.
38 78 I17
156 185
234
Internal cnnsensus sequence
5' site
273
Hases IO 3' splice site
3'
site
312
G. lucidum GUUUGC Neurospora GUANGU crassa Saccharomyces GUAUGU cerevisiae Plant GURNR Vertehrate GURRC
35 I 390 428 488 gacaCCCtCtttgagCCCtCccccttcaataacccccta
507
a c t t c t g c c c c g c w C A T C ATC TCC GAC ACT CCC Met Ser Asp Thr A l e
541 5
TTO ATC lTC A00CTC Gu: .n;G GAC Glyj AAG Leu Ile Phn ArR Leu A l a T r o Asp Val LYS
571 15
M G CTC TCG l T C GAC TAC ACCCCGAAC .n;G LyS Leu Ser Phe Asp Tyr Thr P r o A m T r p
60I 25
GGC CCC CCC M C CCC AAC M C T T C ATC GAC C l y A r g Cly A m P r o A m A m P h e Ile A8p
83 I 35
ACT CTC ACC T T C CCG AAA CTC TTO ACCCAC T h r V a l T h rP h eP r o LyS V R I Leu T h r Asp
88 I 45
AAG CCC TAC ACO TAC UX CTC GCC CTC TCC Lys A l a Tyr Thr Tyr A r g V a l A l a V a l S e r
681
GOA COG AAC Cn: COC Glyj AAA CCC TCG TAC C l y Are Asn Leu C l y V a l Lys Pro Ser Tyr
721
Oco CTC GAG AOC GAC GGC TCG CAG AAG CTC A l a V a l GI" Ser Asp Gly S e r Gln Lys V a l
751 75 781 811 85
CIT GTC CAC CCC GAC ACCAACAACGAC TTC Val Val Asp P r o Asp Thr A m A m Asp P h e
841 105
ATC ATC OcC CAG TCG AAC TAG CAGCAGCCAGT Ile Ile A l a Gln T r p A m End
873 912
- 80
GCAGGCGCATCOCUXTCACCITC~~TAATCA
851
-so
-40
- I90 - 180
1
"
- 170 - 1eo - so - 140 1
2
FIG. 12. Mapping the LZ-8 gene transcriptional start site by primer extension analysis. The mycelial total RNA was hy-
880
OU\tccaRcttttRT88tgtgccgtcttatrRtttggtta
2
YAG Y.,NYA(;
-210
-eo
1
15-:16
-200
-70
111
16-49
YAG
B
85
GAClCACCCCTOCXXITCTAAl'Cr~CCA~
r(jCCajn;TAACAATrGTAAATVZACClT-ACCAT I wIY(A)
YUNA YNYURAY
85
CTC GAG TAC AAC TCC GGC TAT o(jc Tyr Asn Ser GlyTyrCly
AB^ P h e Leu G l u
9-138
UACUAAC
A
55
ATA Oco CAC ACG AAC ACG ATC CAG lTC Ile A l a Asp Thr Aen Thr Ile G l n V e l P h e
AAC R ' C
CCCUAAC RCURAC
IO28
FIG. 11. Nucleotide sequence of LZ-8 gene and the flanking in the region. Thenucleotidesequencesthatshouldbeinvolved matured LZ-8 mRNA are in capital letters. * partial palindromic a,
-
the sequence involving CCAAT-like sequence; sequence; -, putative TATA hox; p,putative transcription start site; poly(A) additional site.
4.
untranslated regions. The cloned genomic DNA containing 12-8 should be negative in hybridization with EX1-2. By the use of the IN-1 probe, only the genomic DNA was positive. The results of Northern blot hybridization analysesof mycelial poly(A)+ RNA withIN-1and with EX1-2probesare shown in Fig. 6M. Obviously, the strong signalwas observed only with the EX1-2 probe. The same results were obtained when Southernblotanalyses were doneusing adoublestranded cDNA synthesized from themycelial poly(A)+ RNA (data not shown). Therefore, the deleted sequence in a cDNA should be an intron, and the deletion should not be a result of a cloning artifact. Thenucleotide sequence of the 5' splice site, a putative internal consensus sequence, or a 3' splice site in k - 8 may be similar to thoseof Neurospora (17, 30), vertebrate (31, 32), and plant cells(33).However, the putative internal consensussequence in 12-8 is obviously different from that of Saccharomyces cerevisiae (32,34). Theconsensus sequences are summarized in Table 3. The splicing of this small intron in 12-8 might have a role for the formation of the stable mRNA (35, 36) to produce a lot of LZ-8 protein. The primer extension analyses were performed with the oligonucleotidesLZ8-1 and UTR-3 (Table 1M) to presume the transcription start site of 12-8. The results shown in Fig. 12. A single majorspecific band was observed at 53 bases long with UTR-3 and 168 bases long with LZ8-1, respectively. These results suggest that the transcription of 12-8 initiates at the C residue, the nucleotide number 409 in Fig. 11. The results of S1 mapping analyses alsosuggested the same initi-
bridized with :"I'-laheled UTR-3 primer ( h e I of panrl A 1 or with :"P-labeled LZ8-1primer (lane I of panel H ) . and the primers were extended withreverse transcriptase. Simultaneously,yeast tRNA was hybridized with "9-laheled UTR-3 (lane 2 of panel A ) or with "1'laheled LZ8-1 (lane 2 of panel H ) , and the primers were extended in the same way for the negative controls. The sizes of markers are in bases.
ation site (data not shown). The C residue is between two direct repeats of GACAG, and therefore thesequence around the C residue is YYYYYYYYRRYRRYRRYRRRYYY. The sequence RRYRR isknown as one of the consensussequences observed at the transcription sites of yeast (37). The translation initiation codon AUG might be the first AUC sequence a ribosome would encounter in the matured LZ-8 mRNA. I t should be favorable for the high efficiency of the translation initiation. The TATA sequence (TATAAA) begins at 88 nucleotides upstream from the putative transcription initiation point. This distance is similar to that of yeast (40-120 bp) (38) or of filamentous fungi (31-112 hp)(17)ratherthan those of the higher eukaryotes (2.5-30 bp) (38). Upstream of the TATA sequence, two direct repeats of GCAATTC. which include the CCAAT-like sequence, are found. The distance between the TATA sequence and the first GCAATTC to the upstream direction, andbetween both GCAATTC sequences, is about 80 nucleotides. It is not yet known whether these CCAAT-like sequences are functionalin the mycelia for modulatingtheLZ-8mRNAtranscription,butthe sequence GCAAT was also found a t about 20 bases upstream from the TATA sequence of the inducibly expressed I N 0 1 gene from yeast (39) and a t about 60 bases upstream from the TATA sequence of the inducibly highly expressed glucoamylase gene from Aspergillus awamori (40). In the genes of filamentous fungi, the sequence CAAT can often he found upstream of theTATAsequence(17).Twopartially palindromic sequences are alsofound upstream of the T A T A sequence in It8. The lengths of the sequences are 15 and 27 nucleotides.
Encoding A Gene
2490 C A
>
Immunomodulator, an
cCUu
REFERENCES
G
C
C
A
1. Kino, K., Yamashita, A., Yamaoka, K., Watanabe, J., Tanaka, S., KO, K., Shimizu, K., and Tsunoo, H. (1989) J. Biol. Chem. 264, 472-478 2. Tanaka, S., KO, K., Kino, K., Tsuchiya, K., Yamashita, A., Murasugi, A., Sakuma, S., and Tsunoo, H. (1989) J. Biol. Chem. 264, 16372-16377 3. Cathala, G., Savouret, J.-F., Mendez, B., West, B. L., Karin, M., Martial, J. A., and Baxter, J. D. (1983) DNA (N. Y.)2, 329335 4. Davis, L. G., Dibner, M. D., and Battey, J. F. (1986) Basic
1000
C
A
C U
U
A C C
A
C U A A
C
G C
C
gucuua
LZ-8
U C -950
I
POly(A)
FIG. 13. The putative secondary structures formed at the 3”untranslated region of the mycelial LZ-8 mRNA. The nucleotides involved in the matured LZ-8 mRNA are in capital letters. The nucleotide numbers correspond to those in Fig. 11.
The partially palindromic sequences have also been known as the UAS of GAL operon genes in yeast (41) and again may be the UAS of the genes from filamentous fungi (17). The sequence specifying the true termination of transcription byRNA polymerase I1 is not clear yet in yeast or in mammalian cells. In mammalian cells, the distances between the poly(A) additional site and the downstream true termination site are surprisingly variable (42-45), and thesequence near the poly(A) site might affect the proper polyadenylation (46,47). Also in yeast, removing or changing the sequence in the 3”untranslated region of mRNA affected the proper poly(A) addition and the stability of the mRNA (48). When the primary transcripts of LZ-8 mRNA contain at least 40 nucleotides beyond the poly(A) site, putativestem-loop structures can be formed, as shown in Fig. 13. In yeast, upstream of the poly(A) addition site, the TAA sequence has often been found (49). It maybe possible to consider that the TAA sequence is a signal for polyadenylation. In this mRNA, four TAAs exist between the termination codon and poly(A) site. The sequence AUAA, which appeared in the 3”untranslated region of glucoamylase mRNAfrom A . awamori (40), was also found about 50 bases preceding the polyadenylation site. The functional significance of these sequences remains unclear. However, the structures like those shown in Fig. 13 may be important for the proper nuclease attack to thepoly(A) site and the poly(A) addition and, therefore, for the stability of the mRNA (50). Such a secondary structureas described above should be taken into consideration when the relationship between the proper termination, or the polyadenylation, and its nucleotide signal is analyzed. On the other hand, it may also be possible to consider that a protein factor binds to a stem-loop structure in the 3”untranslated region and regulates the mRNA stability (51, 52).
Methods in Molecular Biology, Elsevier Science Publishers B.V., Amsterdam 5. Pesole, G., Attimonelli, M., and Liuni, S. (1988) Nucleic Acids Res. 1 6 , 1715-1728 6. Ikuta, S., Takagi, K., Wallace, R.B., and Itakura, K. (1987) Nucleic Acids Res. 1 5 , 797-811 7. Uhlenbeck, 0.C., Martin, F. H., and Doty, P. (1971) J. Mol. Biol. 57,217-229 8. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular C1oning:A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 9. DiLella, A. G., and Woo, S. L. C. (1987) Methods Enzymol. 152, 447-451 10. Conner, B. J., Reyes, A. A., Morin, C., Itakura, K., Teplitz, R. L., and Wallace, R. B. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 278-282 11. Southern, E. M. (1975) J . Mol. Biol. 98,503-517 12. Perbal, B. (1985) A Practical Guide to Molecular Cloning, pp. 221226, John Wiley and Sons, Inc., New York 13. Sanger, F., Nicklen, S., and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74,5463-5467 14. Chen, E. Y., and Seeburg, P. H. (1985) DNA (NY) 4, 165-170 15. Lim, H. M., and PBne, J. J. (1988) Gene Anal. Tech. 5,32-39 16. de la Peiia, P., and Zasloff, M. (1987) Cell 50,613-619 17. Gurr, S. J., Unkles, S. E., and Kinghorn, J. R. (1987) in Gene Structure in Eukaryotic Microbes (Kinghorn, J. R., ed) pp. 93139, IRL Press Ltd., Oxford 18. Ikemura, T., and Ozeki, H. (1982) Cold Spring Harbor Symp. Quant. Biol. 47,1087-1097 19. Hamilton, R., Watanabe, C. K., and de Boer, H. A. (1987) Nucleic Acids Res. 1 5 , 3581-3593 20. Guan, K., and Weiner, H. (1989) J. Biol. Chem. 2 6 4 , 1776417769 21. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132 22. Gamier, J., Osguthorpe, D. J., and Robson, B. (1978) J. Mol. Biol. 120,97-120 23. Kligman, D., and Hilt, D. C. (1988) Trends Biochem. Sci. 13, 437-443 24. Tsuda, M. (1979) J. Biochem. (Tokyo) 86,1463-1468 25. Yatohgo, T., Nakata, M., Tsumuraya, Y., Hashimoto, Y., and Yamamoto, S. (1988) Agric. Biol. Chem. 5 2 , 1485-1493 26. Hirano S., Matsuura, Y., Kusunoki, M., Kitagawa, Y., and Katsube, Y. (1987) J. Biochem. (Tokyo) 102, 445-446 27. Lauer, J., Shen, C.-K. J., and Maniatis, T. (1980) Cell 20, 119130 28. Taub, R. A., Hollis, G. F., Hieter, P. A., Korsmeyer, S., Waldmann, T. A., and Leder, P. (1983) Nature 304, 172-174 29. Bell, G. I., Selby, M. J., and Ruttler, W. J. (1982) Nature 2 9 5 , 31-35 30. Drygas, M. E., Lambowitz, A. M., and Nargang, F. E. (1989) J . Biol. Chem. 264,17897-17906 31. Padgett, R. A., Grabowski, P. J., Konarska, M. M., Seiler, S., and Sharp, P. A. (1986) Annu. Reu. Biochem. 55, 1119-1150 32. Krainer, A. R., and Maniatis, T. (1988) in Transcription and Splicing (Hames, B. D., and Glover, D. M., eds) pp. 131-206, IRL Press Ltd., Oxford 33. Brown, J. W. S. (1986) Nucleic Acids Res. 14, 9549-9559 34. Langford, C. J., and Gallwitz, D. (1983) Cell 33, 519-527 35. Hamer, D. H., and Leder, P. (1979) Cell 18,1299-1302 36. Gruss, P., and Khoury, G. (1980) Nature 286,634-637 37. Hahn, S., Hoar, E. T., and Guarente, L. (1985) Proc. Natl. Acud. Sci. U. S. A. 82,8562-8566 38. Struhl, K. (1987) Cell 4 9 , 295-297 39. Dean-Johnson, M., and Henry, S. A. (1989) J. Biol. Chem. 264, 1274-1283
A Gene Encoding an Immunomodulator, LZ-8 40. Nunberg, J. H., Meade, J. H., Cole, G., Lawyer, F. C., McCabe, P., Schweickart, V., Tal, R., Wittman, V. P., Flatgaard, J. E., and Innis, M. A. (1984) Mol. Cell. Biol. 4 , 2306-2315 41. Nogi, Y.,and Fukasawa, T. (1983) NucleicAcidsRes. 11, 85558568 42, Hagenbiichle, o.,wellauer, p, K., cribbs, D. L., and Schibler, u, (1984) Cell 38,737-744 43. Whitelaw, E., and Proudfoot, N. (1986) EMBO J. 5, 2915-2922 44. FraYne, E. G., and Kellems, R. E. (1986) Nucleic Acids Res. 14, 4113-4125 45. Pribyl, T. M., and Martinson, H. G. (1988) Mol. Cell. Biol. 8, 5369-5377
46. McDevitt, M. A., Imperiale, M. J., Ali, H., and Nevins, J. R. (1984) Cell 37,993-999 47. Woychik, R. P., Lyons, R. H., Post, L., and Rottman, F. M. (1984) Proc. Nutl.Acad.Sci. U. S. A. 8 1 , 3944-3948 48. Zaret, K. S., and Sherman, F. (1984) J.Mol. Biol. 1 7 6 , 107-135 49. Brake, A. J.9 Julius, D. J., and Thorner, J. (1983) Mol. Cell. B i d . 3,1440-1450 50. Drummond, D. R., Armstrong, J., and Colman, A. (1985) Nucleic Acids Res. 13, 7375-7394 51. Casey, J. L., Hentze, M. W., Koeller, D. M., Caughman, S. W., Rouault, T. A,, Klausner, R. D., and Harford, J. B. (1988) Science 240,924-928 52. Mullner, E. W., and Kiihn, L. C. (1988) Cell 53,815-825
SUPPIIFMENTAL
EXPERIMENTAL
2491
MATERlAL
TO
PROCEDURE
Preparation of poly(A)tRNA and genomic DNA from mycelia - SU8Pe"SiOn culture ofG. lucidum mycellawas described prev10USlY (11. TO DPepBre total RNA. the myceliawere collected at B logarlthmic W W t h phase by centrifugation at 13.000 x g for 10 min. washed three times wlth PBS (10 phosphate-buffered saline SolutionIPH 7.211. frozen. and stored at -8O'C. a pestle In a mDFtFX The mycelia (14 g in wet uelghtl were ground finely with partially filled with liquld mtrogen. The resulting powderW B S suspended i n 26 ml of a buffer containing 0. 5 M NeC1. 0. 2 M Tris-HCI IUH 7. 51. 10 mM EDTA. end 1% SDS. and extracted five tlmes withphen01/chIOrOf0~16OBmyl SlCOhOl (25:24:1). The liquid phase was extrected with ethylether. and RNA end DNA were precipitated by the sddltlon Of 2.5 volume Of ethanol. About 20 mg of the crude totel RNA was obtained. A part Of the crude RNA (4 mgl was purified further by the seiectlve precipitation with LiCl (31, and by the with phenol. Approximately 3 mg of total RNA wes obtalned. F r m this total RNA. 42 pg Of paly(A1iRNAwas purified Using Olig~(dTl-CellUlOee et 81. 141. (PL. type 7) according to the method described Davis by F O ~the preparetlon of genomic DNA, the mycelia I 2 0 g in w e t weight1 was ground finely with a pestle i n B moptar partially filled with liquid nitrogen. The resulting pOrrder was suspended in 10 ml Of B buffer containing I O mM Tris-HCL (pH 7. 51. 10 mM NBCI. and 25 mM EDTA. TO this 6u8pension.1. 1 ml of 10% SDS and 1 1 w of proteinaseK (Boehringer) were added. After proteolysis at 37OC for2 h. 1 ml Of 5M NaCl was added to the SUBPenSion. This 8u8pension was extracxed five times with phenol. and twice with chloroform/isoamyl sloohol(24:ll. Nucleic acids in the a9ueou6 phase were precipitated with 2.5volume of ethanol. washed with 70% ethanol. and dissolved in 10 m l Of TE (10 mM Trim-HC1 (pH 7. 51. I mM EDTA). TO the mlution. ZOO Pg of RNase A I S z m , boiled for 15 minl was added. and the contaminated RNAwas digested at 3T°C for 3 0 mi". RNase we6 denatured and removed f r m this DNASOiUflOn by the extractions twlcewlth phenol, and twice with chloroform/isaamyl alcohal(24:I). Finally. 200 rg Of the purified genomic DNAwee obtained.
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Oligonucleotides The probes for the screening Of cDNA library were designed f r m the le8Ult8 Of the protein sequencing of LZ-8 (21. Two tryptic peptldes were chosen. One peptide was T13b. F r m the amino 8Cldsequence of T13b. the mixture Of T13b23M was designed and synthesized~FiK.lM~A11. We imagined that the gene LZ-8 Of is highly expressed in themycelia from the mount Of LZ-8 protein ina cell 111. In the highly expressed gene Of yeast that may be taxOnanically relatedto G. lucidum C residue 18 favorable in the third letters Ofthe codons for Phe. Asp. W r , and Asn 151. O n the other hand. a C:T or G:U mismatched bese pair is much lessUnstable thana A:C mlsmatch ( 6 . 7). Therefore. we chose G reeidueset the corresponding positions in T13b23M. The Other peptide Chosen for the probedesle was all gOeSible sequences. These were Ti4c. The designed probe contained dlvided intothree groups. and Synthesized8 6 sh-n in Figure 1MIB). All other oligonucleotides had e single Sequence (TableL M I . IN-I and EX1-2 were used 8 6 the hybridizationprObe.9. and LZ8-1 and UTR-3 were used 88 the primers forthe mapping Of the transcription start site. UTR-3 was also used 88 the primer for the DNA sequencing. All oligonucleotide8 except for IN-I were complementary toLZ-8 mRNA. ( A B 1 1 DNA DligOnUCleOtldeB were synthesized with Applied Biosystems gel electrophoresis in Synthesizer (A3801. and purlfled wlth polyacrylamide denaturing condition. or with the DPC colwnns purchased from A B I . In order to prepare the hybridization probes. oligonucleotides were labeled at their 5' ends using (T-JZPIATP (7.000 C i h o l e . ICNI and T4-polynucleotide kinase (Takaral. and thenpurified wlth DE-52 l1Yhetmanl. The speciflC were radioaetivities Of the labeled and purified oligonucleotides approximately 1 x 109 c W g .
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Northern blot anslysib RNA was denatured in I O Pl Bolutlon Of 50% formmide, 2.2 M formaldehyde, and the gel buffer I20 mM 3-IN-m0~phol1noI propanesnlfonlr: acid.5 mM sodium acetate. 1 mM EDTA (pH 7.011 14. 81 at 55OC for 15 min. andto this RNA2 p l Of the loading buffer containing 50% glycerol. 1 mM EDTA. 0 . 4 % branophenol blue. and 0.4% xylene cyano1 was added (81. The samples were applied O n the horizontal 1.5% agaro8e gel 10.4 x 1 1 x 13.5 cml contalnlng 2.2 M formaldehyde and the gel buffer (4. 81. The electrophoresis was done In the gel bufferat 100 V fop 2 to 4 h. The fractionated RNAwas transferred OYeFnight directly from gel the to a GeneScreen filter INEN1 with 1 x SSC 10.15 M NaC1. 0.015 M Sodlum citrate (pH 7 . 0 1 ) by the capillary method. The fllter was baked at 80°C for 2 h. RNA ladders (BRL) o r the mycelial total RNA fixed on the filterwas Stained with were prehybridized at 65OC fop 4 methylene blue (8). The filters for probing
SePUenciiig Of a CDNA for LZ-8One of the longest CDNA Obtained van Cloned intoM I 3 Vector, and the nucleotide Sequence was determined by the chain termination method (131. The CDNA insert was Cut Out Of the 1 DNA with E c m I . and directly ligated to PUCIS that had been digested Ecml with and dephosphorylated with alkaline pho8phatese f r m calf intestine 141. a p e t e n t E. coli cells (HB101) Were transformed with the ligated DNA. pUCl8-=DNA IpCLZ81 we8 thus obtalned.
Ecdll was directly llgatedto M I 3 RF DNA pCLZ8 DNA digested with (M13mp191 which had been digested with EcdlI end dephosphoryiated. The resulted DNA w 8 8 transfected to competent E. Coli cell6 l J M l O 9 1 . The slngle-stranded templates for both strands of LZ-8 CDNA were obtelned. one Xhol slte at the center as shown in Figure 4M. The =DNA Of LZ-8 has pCLZB DNA dxgesied withE c a l end XhOl Was dlreotly ligated to MI3 RP DNA (M13mp181 which had been digested with &dl1 and Sa11. and dephoaphor'Yi*ted. Both clones containing e 5' half region and a 3' half Pegionwere obtelned. The sequencing Of the cDNA was performed with these single-stranded templater U81ng K l e n m f r m e n t .and I-deaza-dmP In place Of dGTP. Sequencing was done as shown l n Figure 4M. Soufnern blot a n e l y s i ~Of senmic DNA -The mycelial genmlc DNA, 2 pg. was drgested with the restriction enzyme(slll0 Units each1 at 37% for 2 h. The samples were loaded on 0.7% agaroee gel 10.4 x 13. 5 x 1 1 MI and electrophresed at 25 V overnight. The fraorionated DNA w a s transferred
2492
A Gene Encoding an Immunomodulator, LZ-8
