Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF)- Based Cloning of Enolase, ENO1, from Cryphonectria parasitica

  • Kim, Myoung-Ju (Institute for Molecular Biology and Genetics, Basic Science Research Institute, Chonbuk National University) ;
  • Chung, Hea-Jong (Institute for Molecular Biology and Genetics, Basic Science Research Institute, Chonbuk National University) ;
  • Park, Seung-Moon (Institute for Molecular Biology and Genetics, Basic Science Research Institute, Chonbuk National University) ;
  • Park, Sung-Goo (Korea Research Institute of Bioscience and Biotechnology) ;
  • Chung, Dae-Kyun (School of Biotechnology and Institute of Life Science and Resources, Kyung Hee University) ;
  • Yang, Moon-Sik (Institute for Molecular Biology and Genetics, Basic Science Research Institute, Chonbuk National University) ;
  • Kim, Dae-Hyuk (Institute for Molecular Biology and Genetics, Basic Science Research Institute, Chonbuk National University)
  • Published : 2004.06.01
Download PDF
⟨ Previous Next ⟩

Abstract

On the foundation of a database of genome sequences and protein analyses, the ability to clone a gene based on a peptide analysis is becoming more feasible and effective for identifying a specific gene and its protein product of interest. As such, the current study conducted a protein analysis using 2-D PAGE followed by MALDI- TOF and ESI-MS to identify a highly expressed gene product of C. parasitica. A distinctive and highly expressed protein spot with a molecular size of 47.2 kDa was randomly selected and MALDI-TOF MS analysis was conducted. A homology search indicated that the protein appeared to be a fungal enolase (enol). Meanwhile, multiple alignments of fungal enolases revealed a conserved amino acid sequence, from which degenerated primers were designed. A screening of the genomic $\lambda$ library of C. parasitica, using the PCR amplicon as a probe, was conducted to obtain the full-length gene, while RT-PCR was performed for the cDNA. The E. coli-expressed eno 1 exhibited enolase enzymatic activity, indicating that the cloned gene encoded the C. parasitica enolase. Moreover, ESI-MS of two of the separated peptides resolved from the protein spot on 2-D PAGE revealed sequences identical to the deduced sequences, suggesting that the cloned gene indeed encoded the resolved protein spot. Northern blot analysis indicated a consistent accumulation of an eno1 transcript during the cultivation.

Keywords

References

  1. Science v.215 Biological control of chesnut blight Anagnostakis,S.L. https://doi.org/10.1126/science.215.4532.466
  2. Mol. Cell. Biol. v.10 Multiple factors bind the upstream activation sites of the yeast enolase genes ENO1 and ENO2:ABFI protein,like repressor activator protein RAP1,binds cis-acting seqeunces which modulate repression or activation of transcription Brindle,P.K.;J.P.Holland;C.E.Willett;M.A.Innis;M.J.Holland https://doi.org/10.1128/MCB.10.9.4872
  3. Gene Structure in Eukaryotic Microbes The structure and expression of nuclear gene in Saccharomyces cerevisiae Brown,A.J.P.;G.J.Lithgow;J.R.Kinghorn(ed.)
  4. Proc. Natl. Acad. Sci. v.93 Fungal gene transcript accumulation and elevation of G-protein-regulated cAMP levels by a virulence-attenuating hypovirus Chen,B.;S.Gao;G.H.Choi;D.L.Nuss https://doi.org/10.1073/pnas.93.15.7996
  5. Proc. Natl. Acad. Sci. v.92 Virus-mediated or transgenic suppression of a G-protein a subunit and attenuation of fungal virulence Choi,G.H.;B.Chen;D.L.Nuss https://doi.org/10.1073/pnas.92.1.305
  6. Mol. Plant-Microbe Interact. v.5 Molecular analysis of the laccase gene from the chestnut blight fungus and selective suppression of its expression in an isogenic hypovirulent strain Choi,G.H.;T.G.Larson;D.L.Nuss https://doi.org/10.1094/MPMI-5-119
  7. Nucleic Acid Res. v.18 Nucelotide sequence of the glyceraldehyde-3-phosphate dehydrogenase gene from Cryphonectria parasitica Choi,G.H.;D.L.Nuss https://doi.org/10.1093/nar/18.18.5566
  8. Gene v.125 Molecular analysis and overexpression of the gene encoding endothiapepsin,an aspartic protease from Cryphonectria parasitica Choi,G.H.;D.M.Pawlyk;B.Rae;R.Shapira;D.L.Nuss https://doi.org/10.1016/0378-1119(93)90320-3
  9. Curr. Genet, v.17 Transformation of the fungal pathogen Cryphonectria parasitica with a variety of heterologous plasmids Chruchill,A.C.L.;L.M.Ciufette;D.R.Hansen;H.D.Van Etten;N.K.Van Alfen https://doi.org/10.1007/BF00313245
  10. Protein Structure:A Practical Approach Creighton,T.E.
  11. Microbiology v.149 An ordered collection of expressed sequences from Cryphonectria parasitica and evidence of genomic microsynteny with Neurospora crassa and Magnaporthe grisea Dawe,A.L.;V.C.McMains;M.Panglao;S.Kasahara;B.Chen;D.L.Nuss https://doi.org/10.1099/mic.0.26371-0
  12. Phytopathology v.75 Characteristics of dsRNA-free and dsRNA-containing strains of Endothia parasitica in relation to hypovirulence Elliston,J.E. https://doi.org/10.1094/Phyto-75-151
  13. Chemosphere v.40 Fungal degradation of fluorene Garon,D.;S.Krivobok;F.Seigle Murandi
  14. Gene Structure in Eukaryotic Microbes The structure and organization of nuclear gene in filamentous fungi Gurr,S.J.;S.E.Unkles;J.R.Kinghorn;J.R.Kinghorn(ed.)
  15. Physiol. Plant Pathol. v.23 Oxalate production by virulent but not by hypovirulent strains of Endothia parasitica Havir,E.A.;S.L.Anagnostakis https://doi.org/10.1016/0048-4059(83)90021-8
  16. Biochemistry v.17 Isolation and identification of yeast messenger ribonucleic acids coding for enolase,glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate kinase Holland,M.J.;J.P.Holland https://doi.org/10.1021/bi00616a007
  17. J. Biotechnol. v.40 Self-cloning in filamentous fungi:Application to the construction of endothipepsin overproducers in Cryphonetria parasitica Jara,P.;P.Delmas;V.Razanamparany;L.Olsen;P.Dupin;A.Bayol;J.Begueret;G.Loison https://doi.org/10.1016/0168-1656(95)00036-P
  18. Mol. Plant Microbe Interact. v.10 Targeted disruption of a fungal G-protein β subunit gene results in increased vegetative growth but reduced virulence Kasahara,S.;D.L.Nuss https://doi.org/10.1094/MPMI.1997.10.8.984
  19. Proc. Natl. Acad. Sci. v.97 Identification of bdm-1,a gene involved in G protein β-subunit function and a subunit accumulation Kasahara,S.;P.Wang;D.L.Nuss https://doi.org/10.1073/pnas.97.1.412
  20. Mol. Plant-Microbe Interact. v.8 A new extracellular laccase of Cryphonectria parasitica is revealed by deletion of Lacl. Kim,D.H.;D.Rigling;L.Zhang;N.K.Van Alfen https://doi.org/10.1094/MPMI-8-0259
  21. Mol. Microbiol. v.45 Characterization of a fungal protein kinase from Cryphonectria parasitica and its transcriptional upregulation by hypovirus Kim,M.J.;J.W.Choi;S.M.Park;B.J.Cha;M.S.Yang;D.H.Kim https://doi.org/10.1046/j.1365-2958.2002.03079.x
  22. J. Microbiol. Biotechnol. v.10 Expression of murine GM-CSF from recombinant Aspergillus niger. Kim,M.J.;T.H.Kwon;Y.S.Jang;M.S.Yang;D.H.Kim
  23. Microbiol. Rev. v.47 Comparison of initiation of protein synthesis in procaryotes,eucaryotes, and organelles Kozak,M.
  24. Curr. Genet. v.30 Molecular cloning of a cDNA encoding enolase from the filamentous fungus,Aspergillus oryzae Machida,M.;Y.C.Chang;M.Manage;M.Yasukawa;S.Kunihiro;Y.Jigami https://doi.org/10.1007/s002940050152
  25. Biosci. Biotech. Biochem. v.60 Molecular cloning of a genomic DNA for enolase from Aspergillus oryzae Machida,M.;T.V.J.Gonzalez;L.K.Boon;K.Goni;Y.Jigami https://doi.org/10.1271/bbb.60.161
  26. Mol. Cell. Biol. v.11 Yeast glycolytic mRNAs are differentially regulted Moore,P.A.;F.A.Sagliocco,R.M.Wood;A.J.Brown https://doi.org/10.1128/MCB.11.10.5330
  27. Curr. Genet. v.37 Comprehensive cloning and expression analysis of glycolytic genes from the filamentous fungus Nakajima,K.;S.Kunihiro;M.Sano;Y.Zhang;S.Eto;Y.C.Chang;T.Suzuki;Y.Jigami;M.Machida https://doi.org/10.1007/s002940050534
  28. Mol. Cell. Biol. v.6 Cloning and characterization of the gene for btubulin from a benomyl-resistant mutant of Neurospora crassa and its use as a dominant selectable marker Orbach,M.J.;E.B.Porro;C.Yanofsky https://doi.org/10.1128/MCB.6.7.2452
  29. Mol. Microbiol. v.51 Characterization of HOGI homologue,CpMK1,from Cryphonectria parasitica and evidence for hypovirus-mediated peturbation of its phosphorylation in response to hypertonic stress Park,S.M.;E.S.Choi;M.J.Kim;B.J.Cha;M.S.Yang;D.H.Kim https://doi.org/10.1111/j.1365-2958.2004.03919.x
  30. J. Bacteriol. v.169 Two nonhomologus viruses of Cryphonectria(Endothia)parasitica reduce accumulation of specific virulence-associated polypeptides Powell,W.A.J.;N.K.Van Alfen https://doi.org/10.1128/jb.169.11.5324-5326.1987
  31. Curr. Genet. v.21 Cloning and mutation of the gene encoding endothiapepsin from Cryphonectria parasifica Razanamparany,V.;P.Jara;R.Legoux;P.Delmas;F.Msayeh;M.Kaghad;G.Loison https://doi.org/10.1007/BF00351655
  32. Phytopathology v.79 Reduction of laccase activity in dsRNA-containing hypovirulent strains of Cryphonectria(Endothia)parasitica Rihling,D.;U.Heininger;H.R.Hohl https://doi.org/10.1094/Phyto-79-219
  33. J. Bacteriol. v.173 Regulation of laccase biosynthesis in the plant-pathogenic fungus Cryphonectria parasitica by double-stranded RNA Rigling,D.;N.K.Van Alfen https://doi.org/10.1128/jb.173.24.8000-8003.1991
  34. Molecular Cloning:A Laboratory Manual Sambrook,J.;E.F.Fritsch;T.Maniatis
  35. Rapid Commum. Mass Spectrom. v.11 Rapid 'de novo'peptide sequencing by a combination of nanoelectrospray,isotopic labeling and a quadrupole/time-of-flight mass spectrometer Shevchenko,A.;I.Chernushevich;W.Ens;K.G.Standing;B.Thompson;M.Wilm;M.Mann https://doi.org/10.1002/(SICI)1097-0231(19970615)11:9<1015::AID-RCM958>3.0.CO;2-H
  36. J. Bacteriol. v.174 Molecular cloning of cDNA and analysis of protein secondary structure of Candida albicus enolase,an abundant,immunodominant glycolytic enzyme Sundstrom,P.;G.R.Aliaga https://doi.org/10.1128/jb.174.21.6789-6799.1992
  37. Curr. Genet. v.40 Deletion analysis of the enolase gene(enoA)promoter from the filamentous fungus Aspergillus oryzae Toda,T.;M.Sano;M.Honda;O.J.Rimoldi;Y.Yang;M.Yamamoto;K.Takase;K.Hirozumi;K.Kitamoto;T.Minetoki;K.Gomi;M.Machida https://doi.org/10.1007/s00294-001-0258-7
  38. Mol. Cell. Biol. v.12 Role of GCR2 in transcriptional activation of yeast glycolytic genes Uemura,H.;Y.Jigami
  39. Science v.189 Chestnut blight:Biological control by transmissible hypovirulence in Endothia parasitica Van Alfen,N.K.;R.A.Jaynes;S.L.Anagnostakis;P.R.Day https://doi.org/10.1126/science.189.4206.890
  40. Plant Cell v.3 Plant enolase:Gene structure,expression,and evolution Van Der Straeten,D.;R.A.Rodrigues Pousada;H.M.Goodman;M.Montagu https://doi.org/10.1105/tpc.3.7.719
  41. Mol. Cell. Biol. v.12 Cutinase in Cryphonectria parasitica,the chestnut blight fungus:Suppression of cutinase gene expression in isogenic hypovirulent strains containing double-stranded RNAs Varley,D.A.;G.K.Podila;S.T.Hiremath https://doi.org/10.1128/MCB.12.10.4539
  42. Life Sci. v.55 DNA sequences encoding enolase are remarkably conserved from yeast to mammals Verma,M.;S.K.Dutta https://doi.org/10.1016/0024-3205(94)00534-6
  43. Mol. Cell. Biol. v.13 Hypovirulence-associated traits induced by a mycovirus of Cryphonectria parasitica are mimicked by targeted inactivation of a host gene Zhang,L.;A.C.Churchill;P.Kazmierczak;D.H.Kim;N.K.Van Alfen https://doi.org/10.1128/MCB.13.12.7782
  44. Gene v.139 Virus-associated down-regulation of the gene encoding cryparin,an abundant cell-surface protein from the chestnut blight fungus Zhang,L.;D.Villalon;Y.Sun;P.Kazmierczak;N.K.Van Alfen https://doi.org/10.1016/0378-1119(94)90523-1