Bot. Bull. Acad. Sin. (2001) 42: 101-107

Hwu et al. Ribotoxin gene of Penicillium

Nucleotide sequence and the action of ribotoxin gene (sar gene) of Penicillium isolates from Taiwan

Luen Hwu1, Chiou-Jau Cho2, S. S. Tzeanz3, and Alan Lin2,*

1Institute of Microbiology and Immunology, National Yang-Ming University, Taipei 112, Taiwan, ROC

2Institute of Genetics, National Yang-Ming University, Shih-Pai, Taipei 112, Taiwan, ROC

3Department of Plant Pathology and Entomology, National Taiwan University, Taipei, Taiwan, ROC

(Received February 16, 2000; Accepted September 25, 2000)

Abstract. Production of ribotoxin from species of Penicillium was reported. Among a total of fifty-six strains collected from Taiwan, six strains (P. resedanum, P. spinulosum, P. daleae, P. digitatum, P. aculeatum, and P. chermesinum) were found to carry ribotoxin gene (sar gene), but only four strains (P. resedanum, P. spinulosum, P. aculeaatum, and P. chermesinum) secreted the ribotoxic protein that cleaves ribosomes. The sar gene from the six strains was individually cloned and sequenced. These genes exhibit a typical genomic organization of the fungal-originated ribotoxin gene. The nucleotide and amino acid sequences of these sar genes from Penicillium are highly conserved and nearly identical. This is the first report that describes the production of ribotoxin by fungal species other than Aspergillus spp.

Keywords: Penicillium; Ribosome-inactivation; Ribotoxin; rRNA; sar gene.


Filamentous fungi of Aspergillus and Penicillium are important in modern medicine and molecular biology, because they produce a wide range of secondary metabolites. Some of the metabolites have antibiotic or toxic properties in plants or animals. Aspergillus giganteus carries the sar gene that encodes a ribotoxic a-sarcin protein which inhibits protein synthesis of cells. The inhibition results from endonucleolytic cleavage of an RNA domain in 23-28S rRNA (Endo et al., 1983; Wool, 1984). One molecule of a-sarcin could catalyze the cleavage of 5000 molecules of ribosomes. This action has made ribotoxin a potential protein drug in the fight against cancer (Wawrzynczak et al., 1991; Brinkmann and Pastan, 1994; Lin et al., 1994).

Apparently, Aspergillus spp. is the only fungal species definitely known to carry the sar gene (Lin et al., 1995). Expression of the sar gene by several species of Aspergillus has been observed (Fernandez-Luna et al., 1985; Lamy and Davies, 1991; Lamy et al., 1991; Huang et al., 1997). The major human pathogenic species of Aspergillus is A. fumigatus; responsible for a variety of allergenic (Arruda et al., 1990) and invasive diseases (Vanden Bossche et al., 1988). The major cause of aspergillosis is the secretion of a ribotoxic protein by A. fumigatus (Lamy et al., 1991). The sar gene displays a unique genomic organization, in which an intron is inserted between exons of the signal peptide and the mature ribotoxin gene (Lamy and Davies, 1991;

Lamy et al., 1991; Huang et al., 1997). The origin and the need of the sar gene in Aspergillus have been questioned (Wool, 1984) since the gene is neither an essentially functional nor a house-keeping gene. Furthermore, expression of sar gene in cells can stop their growth or cause their death (Miller and Bodley, 1988; Lin et al., 1991; Hwu et al., 2000). This report describes findings of an investigation of possible production of ribotoxin in fungi other than Aspergillus spp. Species of Penicillium from collections in Taiwan that carried sar gene and secreted ribotoxic proteins were identified, and the DNA sequences of the genes were determined and compared.

Materials and Methods

Fungal Species and Culture Conditions

Species of Penicillium, including anamorphs and teleomorphs (Table 1), were obtained from the culture collections of Taiwan (Culture Collection and Research Center, Hsinchiu, Taiwan). All fungi were grown in 500 ml of potato dextrose broth (Difco Lab, Detroit, USA) in one-liter flasks. They were maintained at 30C, with continuous agitation for 5 days. The mycelium were separated from the culture medium by filtering through Whatman No. 3 filter paper. Total DNA was extracted from the mycelium as follows: mycelium were frozen with liquid nitrogen and ground to a fine powder in a pre-chilled metal Waring blender cup. The powder was first suspended in a high salt containing buffer (1.5 M NaCl, 1.5 M EDTA, 100 mM Tris-HCl, pH 8.1), to which was added a 2% (the final concentration) cetyltrimethylammonium bromide (CTAB), reducing the salt concentration. The reduction

*Corresponding author. Tel: (02) 2822-5485; Fax: (02) 2826-4930; E-mail:

Botanical Bulletin of Academia Sinica, Vol. 42, 2001

Table 1. A survey on the production of ribotoxins from Penicellium spp.

Genus; Subgenus; Speciesa Immunoreactivityb Western blotting Ribosomeinactivation PCR with Primers


Penicillium bilaii (CCRC33169) _

P. chermesinum (CCRC32572) + + + +

P. citreonigrum (CCRC32574) _

P. glabrum (CCRC32585) _

P. lividum (CCRC33164) _

P. phoeniceum (CCRC32631) _

P. purpurescens (CCRC32570) _

P. resedanum (CCRC32037) + + + +

P. sclerotiorum (CCRC33160) _

P. spinulosum (CCRC32565) + +/_ +/_ +

P. vinaceum (CCRC33158) _


P. canescens (CCRC32050) + + + +

P. citrinum (CCRC33168)@ _

P. corylophilum (CCRC33167) _

P. daleae (CCRC32392) + +/_ +/_ +

P. digitatum (CCRC32571) + + + +

P. herquei (CCRC32610) _

P. janczewskii (CCRC32561) _

P. janthinellum (CCRC32644) _

P. melinii (CCRC32396) _

P. miczynskii (CCRC33163) _

P. oxalicum (CCRC4119) _

P. paxilli (CCRC33162) _

P. rolfsii (CCRC32036)* _

P. simplicissimum (CCRC33159) _


P. aculeatum (CCRC32621) + _ _ _

P. erythromellis (CCRC32044) _

P. formosanum (CCRC32654) _

P. islandicum (CCRC33165)@ _

P. minioluteum (CCRC32646) _

P. pinophilum (CCRC32388) + _ _ _

P. purpurogenum (CCRC32601)@ + _ _ _

P. rugulosum (CCRC33161) _

P. variabile (CCRC32651) _

P. verruculosum (CCRC32626) _

P. vulpinum (CCRC33157) _


Penicillium aurantiogriseum (CCRC32637) _

P. brevicompactum (CCRC32556) _

P. chrysogenum (CCRC32635) _

P. crustosum (CCRC33166) _

P. expansum (CCRC32629) _

P. griseofulvum (CCRC32638) _

P. hirsutum (CCRC32632) _

P. italicum (CCRC32630) _

P. olsonii (CCRC32049) _

P. ulaiense (CCRC32655) _

P. viridicatum (CCRC32022)@ _


Eupenicillium javanicum (CCRC32796) _

E. shearii (CCRC32431) _


Talaromyces assiutensis (CCRC32401) _

T. avellanus (CCRC33170) _

T. flavus (CCRC33156) _

T. stipitatus (CCRC32411) _

T. trachyspermus (CCRC32439) _

T. unicus (CCRC32703) _

T. wortmanii (CCRC32799) _

aThe number in parentheses is the public depository number from the Culture Collection and Research Center, Hsinchiu, Taiwan.

bTested results of culture medium that reacts to anti-a-sarcin antibody.

Remark: P. aculeatum (CCRC32621) fails to produce any products in RCR three times. The PCR product of P. chermesinum (CCRC32572) is larger than a-sar. The PCR product of is P. digitatum (CCRC32571) is smaller than a-sar. The protein of P. canescens (CCRC32050) produces a smaller sarcin-like protein. P. rolfsii (CCRC32036)* reacts positively to anti-tricholin antibody.

Hwu et al. Ribotoxin gene of Penicillium

in salt concentration resulted in the formation of CTAB/nucleic acid precipitates that were collected by centrifugation (6,000 g, 30 min). The pellet was resuspended in CsCl solution A (1 M CsCl, 50 mM Tris-HCl, pH 8, 5 mM EDTA, 50 mM NaCl). The fully resuspended DNA-CsCl solution (3.5-ml) was layered onto a 1.5-ml cushion of CsCl solution B (5.7 M CsCl, 50 mM Tris-HCl, pH 8, 5 mM EDTA, 50 mM NaCl) in an ultracentrifuge tube. After centrifugation at 32,000 g for 18 h, DNA fractions were collected from the interfaces and the refractive index was adjusted to 1.399 by adding CsCl solution B for DNA banding. DNA was recovered from equilibrium centrifugation and prepared for cloning.

Preparation of Extra-Cellular Proteins From Fungi and Detection of Immunological Cross Reactivity

The separated filtrate of culture medium was concentrated and dialyzed through a hollow fiber membrane filter (HIP10-20, cut off range of 10,000 daltons, Amicon Inc. MA. USA). The dialyzed filtrate was lyophilized, and its protein concentration was determined by Lowry reaction. The dialyzed filtrate was spotted on nitrocellulose paper (NC paper; Nitro Bind, MSI USA), and the immunological cross reactivity against anti-a-sarcin serum was tested (Lin et al., 1991). Briefly, the spotted NC paper was soaked with 3% (w/v) dried milk powder in phosphate buffer saline (PBS) to block the non-spotted paper area, then incubated with the anti-a-sarcin serum (diluted 1:5000 in PBS) for 1 h, followed by non-radioactive NBT/BCIP chromogen detection (Boehringer Mannheim Co. USA).

Assay of Ribotoxic Ribonuclease Activity

The ribotoxic nuclease activity was examined as follows: 5.7 OD260 units of rabbit reticulocyte lysates were treated with the same amounts of dialyzed filtrate in a buffer containing 20 mM Tris-HCl, pH 7.6, and 20 mM EDTA and incubated at 37C for 15 min. Total RNA was extracted from the reaction mixture by SDS-phenol and analyzed by 1.5% agarose gel electrophoresis in TBE buffer. Ribosomal RNA and a-fragment RNA resultant from ribotoxic action were visualized with ethidium bromide.

Polymerase Chain Reaction Amplification with Genomic DNA

Species that showed positive reactions on immuno dot blotting were selected and their total genomic DNA were prepared for polymerase chain reaction (PCR). Primers used for PCR amplification were derived from the published cDNA sequence of the a-sarcin gene (sar gene) (Oka et al., 1990). Two nucleotide sequences, 5'-CCATGGTT GCAATCAAAACCTTGTCCTGG-3' (primer 1), and 5'- GCAAGGAATTCTAATGAGAGCAG-3' (primer 2), were used: primer 1 is a 5' end sequence that matched the amino-terminal sequences of secretory signal peptide; primer 2 is a 3' end sequence, located at the carboxyl-terminal end of the mature a-sarcin and containing a stop codon and

non-coding sequence of nucleotides. The PCR amplification was performed according to the following scheme: denaturation at 95C for 1 min; annealing at 45C for 1 min; and extension at 72C for 1 min. After 20 cycles, the amplified products were analyzed by agarose gel electrophoresis, and verified by hybridization with radioactive a-sarcin cDNA (Lin et al., 1995). The sarcin cDNA probe was labeled at the 5' end with [g-32P] ATP using a procedure of T4 polynucleotide kinase (Pharmacia Co.). The hybridization temperature was 50C.

Results and Discussion

Ribotoxin has been known to be produced only by species of Aspergillus (Lin et al., 1995). In this study, six species of Penicillium were observed to carry the sar gene, and four of them produced ribotoxin that inactivated ribosomes. The initial detection was made by immuno dot

Figure 1. Analyses of polymerase chain reaction (PCR) of Penicillium species. The PCR amplification was conducted by using two primers that were described in the text. Total genomic DNA from P. chermesinum, P. resedanum, P. spinulosum, P. canescens, P. daleae, P. digitatum, P. aculeatum, P. pinophilum, and P. purpurogenum were subjected to amplification. Panel A gives the PCR products that are analyzed by 1.5% agarose gel electrophoresis in TBE buffer. Panel B contains the results of Southern hybridization with the [32P]labeled a-sarcin cDNA. The molecular weight markers are co-electrophoresed and sizes are indicated; a-sarcin cDNA was an amplified product from the constructed plasmid that contained the a-sarcin gene with intron.

Botanical Bulletin of Academia Sinica, Vol. 42, 2001

blot screening of fifty-six isolates of Penicillium (thirty-six anamorphs and twenty teleomorphs). The immuno dot blot assays (Table 1) indicated that extra-cellular proteins from nine species (P. chermesinum, P. resedanum, P. spinulosum, P. canescens, P. daleae, P. digitatum, P. aculeatum, P. pinophilum, and P. purpurogenum) reacted with anti-a-sarcin serum. When the Western blot for extra-cellular proteins of these nine species was done, four of themP. resedanum, P. spinulosum, P. aculeatum, and P. chermesinum tested positively to anti-a-sarcin antibody (data not shown).

To resolve the discrepancy between the immuno blots and Western blots, Southern blotting was applied to all

fifty-six isolates using radioactive cDNA derived from the sar gene of A. giganteus as probe (Lin et al., 1995). Instead of finding nine or four positive species, six (P. chermesinum, P. resedanum, P. spinulosum, and P. aculeatum, P. daleae, and P. digitatum) were observed to be hybridized by the sar-gene probe (Table 1). These six species, therefore, were genetically cloned and their DNA sequences determined. Prior to cloning the positive genes, the nine species that were positive in immuno-dot blot assay were subjected to PCR amplification. The six speciesP. chermesinum, P. resedanum, P. spinulosum, and P. aculeatum, P. daleae, and P. digitatumgenerated a comparably-sized PCR fragment that also hybridized with

Figure 2. Nucleotide sequences of six ribotoxin genes from Penecillium spp. and their comparison to that of a-sarcin of Aspergillus gigianteus. The sequences and the genomic organization of ribotoxin gene (sar gene) from P. resedanum (1), P. daleae (2), P. aculeatum (3), P. spinulosum (4), P. digitatum (5), P. chermesinum (6), are given, and their GenBank accession Numbers for each species are AF012812, AF012814, AF012816, AF012813, AF012815, and AF012817, respectively. These sequences were compared with the sequence of a-sarcin from Aspergillus gigianteus (a). The alignments are given and only the non-identical bases are in shaded boxes. These genes were cloned from their genomic DNA by PCR amplification using two primers (see text) that complement with both ends (underlined). The intron (shown in italics) and the suggested splicing signals (italic and underlined) are putatively based on the sequence of a-sarcin. The first amino acid of signal sequence and the mature protein elucidated are marked with *1 and +1, respectively. The last coding nucleotide is marked with two asterisks.

Hwu et al. Ribotoxin gene of Penicillium

Figure 3. The comparison of the deduced amino acid sequences of six ribotoxin proteins with that of a-sarcin. Protein sequences of A. gigianteus (a), P. resedanum (1), P. daleae (2), P. aculeatum (3), P. spinulosum (4), P. digitatum (5), and P. chermesinum (6) were aligned. The amino acid sequences of six ribotoxin proteins were deduced from their DNA sequence (Figure 2). The differing amino acids are shaded. The signal peptides are underlined. The first amino acid of the mature protein is marked with +1.

the a-sarcin cDNA probe (Figure 1). A lower degree of hybridization was observed in P. aculeatum, P. daleae, and P. digitatum, even through equal amounts of DNA were applied. Penicillium canescens and P. pinophilum, and P. purpurogenum failed to produce PCR-DNA fragments or to hybridize with a-sarcin cDNA (Figure 1).

The DNA that encoded for the sar gene of six positive strains of Penicillium were successfully cloned into pGEM(T) plasmid, and sequenced. The sequences of the six DNA were nearly identical (Figure 2), i.e. about 99% identity, indicating that the sar gene shared the same genomic organization for the signal peptide exon-intron-ribotoxin exon arrangement (Figure 2). This finding concurs with the previous suggestion for fungal ribotoxin (Lamy et al., 1991; Lin et al., 1995; Huang et al., 1997). The DNA and deduced amino acid sequences were compared with that of the a-sarcin (Oka et al., 1990), and a score of 93% identity was obtained. Ten to seventeen of 595 total nucleotides (plus or minus five nucleotides) were different (Figure 2). The deduced amino acid sequences also showed a 94% similarity to that of a-sarcin (Figure 3). All differences involved a conservative substitution. The Asn116 residue of a-sarcin was irreversibly replaced by asparatic acid in all sar gene isolates of Penicillium spp.

The potential expression of a ribotoxic protein from species carrying the sar-gene was further examined by the ribosome-inactivation assay using the rabbit reticulacyte lysate. The assay is sensitive and efficient. It detects the cytotoxic action of ribotoxin, which specifically cleaves

Figure 4. Ribotoxic action of extracellular proteins from Penicillium species. Each lane carried 5.7 OD260 units of rabbit reticulocyte lysates (RRL) that were treated with prepared extra-cellular protein (2 mg) from species as indicated at top of figure. The reaction occurred at 37C for 15 min, in a buffer containing 20 mM Tris-HCl, pH 7.6; and 20 mM EDTA. Total RNAs were extracted and analyzed by 1.5% agarose gel electrophoresis in TBE buffer and visualized with ethidium bromide. RRL is rabbit reticulocyte lysate without ribotoxic treatment. a-sarcin is RRL treated with 0.2 mg of a-sarcin and used as the positive control. Positions of a-fragment, 28S, 18S, and 5S rRNA are indicated.

Botanical Bulletin of Academia Sinica, Vol. 42, 2001

Hwu et al., 2000). The only advantage for fungi that have evolved a sar gene might be enhanced self-defense. Tricholin, a related ribotoxin from Trichoderma viride (Lin et al., 1991) that demonstrated an antibiotic effect against the fungi of Rhizotonia solani (Lin et al., 1994) in soil ecosystems could support this notion.

Acknowledgments. We would like to thank Mr. Dow-Tein Chen of our Institute for his preparation of multiple sequence alignments. This work was supported by a grant from the National Science Council, NSC85-2331-B010-007-BC, Taiwan, Republic of China. A. Lin was an award recipient of the Medical Research and Advancement Foundation in Memory of Dr. Chi-Shuen Tsou.

Literature Cited

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Berbee, M.L., A. Yoshimura, J. Sugiyama, and J.W. Taylor. 1995. Is Penicillium monophyletic? an evaluation of phylogeny in the family Trichocomaceae from 18S, 5.8S and ITS ribosomal DNA sequence data. Mycologia 87: 210-222.

Brinkmann, U. and I. Pastan. 1994. Immunotoxins against cancer. Biochim. Biophys. Acta 1198: 27-45.

Campos-Olivas, R., M. Bruix, J. Santoro, A. Martinez del Pozo, J. Lacadena, J.G. Gavilanes, and M. Rico. 1996. Structureal basis for the catalytic mechanism and substrate specificity of the ribonuclease a-sarcin. FEBS Lett. 99: 163-165.

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Endo, Y. and I.G. Wool. 1982. The site of action of a-sarcin on eukaryotic ribosomes. J. Biol. Chem. 257: 9054-9060.

Fernandez-Luna, J.L., C. Lopez-Otin, F. Soriana, and E. Mendez. 1985. Complete amino acid sequence of the Aspergillus cytotoxin mitogillin. Biochemistry 24: 861-867.

Huang, K.-C., Y.Y. Hwang, L. Hwu, and A. Lin. 1997. Characterization of a new ribotoxin gene (c-sar) from Aspergillus clavatus. Toxicon 35: 383-392.

Hwu, L., K.C. Huang, D-T. Chen, and A. Lin. 2000. The action mode of the ribosome-inactivating protein a-sarcin. J. Med. Sci. 7: 420-428.

Kao, R. and J. Davies. 1999. Molecular dissection of mitogillin reveals that the fungal ribotoxins are a family of natural genetically engineered ribonuclease. J. Biol. Chem. 274: 12576-12582.

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Lamy, B., M. Moutaouakil, J-P. Latge, and J. Davies. 1991. Secretion of potential virulence factor, a fungal ribonucleotoxin, during human aspergillosis infections. Mol. Microbiol. 5: 1811-1815.

Lin, A., C-K. Chen, and Y-J. Chen. 1991. Molecular action of

the sarcin domain of the large ribosomal RNA when ribosome is the substrate (Endo and Wool, 1982; Lin and Huang, 1994). The result is an a-fragment rRNA that contains a 469-nucleotide fragment from the 3' end of the large subunit ribosomal RNA (Wool, 1984). The action is distinguishable from that of other ribonucleases on ribosomes since treatment of ribosomes with other ribonucleases, such as RNase A, T2, or U2, caused all species of rRNA to be extensively digested without generating the a-fragment (Endo et al., 1983). When partially purified proteins prepared from species that carried the sar gene were tested for the ribosome-inactivation, it was rather surprising that only P. resedanum, P. spinulosum, P. aculeaatum, and P. chermesinum were able to hydrolyze ribosomes positively (Figure 4). Penicillium daleae and P. digitatum, also established sar gene carriers, were incapable of inactivating ribosomes (Figure 4) even if the amounts of extra cellular proteins were greatly increased (data not shown). The results concur with the findings of the Western blots, but raise the question as to why these species that contain the sar gene are unable to generate the gene product. Defects in the secretory mechanism are unlikely, simply because these sar genes carried a perfect secretory signal peptide. A defect at the transcriptional level is therefore suspected. Thus, an analysis of the upstream promoter region for these sar genes is currently in progress.

The sar gene of the filamentous fungi was speculated to originate from a single ancestor of ribonuclease T1. It has also been suggested that it is related to bacterial guanyl-specific ribonuclease (Kao and Davies, 1999). This was based on the close resemblance of the tertiary structure of two ribotoxins from Aspergillus (Campos-Olivas et al., 1996; Yang and Moffat, 1996) to that of the guanyl-specific ribonuclease T1 family (Sevcik et al., 1991; Pace et al., 1991). Both protein families share the common structure of a four-stranded b-pleated sheet. The b-pleated sheet structure is indispensable for ribotoxin to digest ribonucleic acids (Kao and Davies, 1999; Hwu et al., 2000).

In this study, species that exhibited ribosome-inactivation, except P. aculeatum, belong to the subgenus Aspergiloides, which is closely related to Aspergillus. The Penicillium species that carry sar gene, but fail to act in ribosome-inactivation, belong to the subgenus Furcatum and are taxonomically distant from Aspergillus. The observations of this study might help elucidate the evolutionary relationship between the genera Aspergillus and Penicillium (Berbee et al., 1995). On the other hand, the finding of near identical genes in Penicillium and Aspergillus suggests that a horizontal gene transfer might have occurred through the sexual life cycle, although most Aspergillus, except for the few that produce teleomorphs, have no sexual cycle. So far, there is no evidence supporting this. Reasons for the carrying of the sar gene in Aspergillus or in Penicillium have been questioned because the sar gene is not a constitutive gene for all species of Aspergillus (Lin et al., 1995) or Penicillium (this study). Moreover, the expression of the sar gene is lethal to the carrying host cell itself (Lamy and Davies, 1991;

Hwu et al. Ribotoxin gene of Penicillium

tricholin, a ribosome-inactivating protein isolated from Trichderma viride. Mol. Microbiol. 5: 3007-3013.

Lin, A., T.M. Lee, and J-C. Rern. 1994. Tricholin, a new antifugal agent from Trichoderma viride, and its action in biological control of Rhizotonia solani. J. Anitbiotics 47: 799-805

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Lin, A. and R.G. Huang. 1994. Hydrolysis of Ribosomes by electrophoretically blotted ribotoxin. BioTechniques 17: 636-637

Miller, S. P. and J.W. Bodley. 1988. The ribosomes of Aspergillus giganteus are sensitive to the cytotoxic action of a-sarcin. FEBS Lett. 229: 388-390.

Oka, T., Y. Natori, S. Tanaka, K. Tsurugi, and Y. Endo. 1990. Complete nucleotide sequence of cDNA for the cytotoxin alpha-sarcin. Nucleic Acids Res. 19: 1897.

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Wawrzynczak, E.J., R.V. Henry, A.J. Cumber, and G.D. Parnell. 1991. Biochemical, cytotoxic and pharmacokinetic properties of an immunotoxin composes of a mouse monoclonal antibody Fib 75 and the ribosome-inactivating protein alpha-sarcin from Aspergillus giganteus. Eur. J. Biochem. 196: 203-209.

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