Abstract
Proteins fromFusarium sporotrichioides M-1-1, a T2-toxin-producing strain, were separated by two-dimensional polyacrylamide gel electrophoresis. One thousand two hundred and forty-four protein spots were resolved and 103 protein spots were subjected to N-terminal sequencing. Fifty-eight protein spots were sequenced and 48 proteins were observed to have blocked N termini. Forty out of 58 sequenced proteins were identified by homology search against the PIR protein sequence data base and protein superfamily data base, while the residual 18 sequences were not identified. Twenty-seven of the N-terminal-blocked proteins were subjected to mild anhydrous hydrazine vapor deblocking. Twenty-four spots were not deblocked indicating the presence of acyl groups at the N termini, while 3 proteins were deblocked showing the blocked group to be pyrroglutamyl carboxylic acid residues. The results can provide a more global view of cellular genetic expression than any other technique. The created data may offer a unique opportunity to link information with DNA sequence data.
Similar content being viewed by others
References
Applied biosystem, Protein-peptide sequencing system Model 477, users manual. pp 3.38–3.39; Sequencing samples producing high lag.
Beremand MN, van Middlesworth F, Taylor S, Plattner RD, Weisleder D. Leucine autotroph specifically alters the pattern of trichothecene production in a T-2 toxin-producing strain ofFusarium sporotrichioides. Appl Environ Microbiol 54:2759–2766;1988.
Bhushan R, Goyal RN, Agarwal A. Complete amino acid sequence of an arachin subunit. Biochem Int 11:477–490;1985.
Bonnard G, Tinland B, Paulus F, Szegedi E, Otten L. Nucleotide sequence, evolutionary origin and biological role of a rearranged cytokinin gene isolated from a wide host range biotype IIIAgrobacterium strain. Mol Gen Genet 216:428–438;1989.
Booth C. The GenusFusarium. Common-wealth Agricultural Bureaux, 21;1971.
Bradshaw RE, Pillar TM. Isolation and nucleotide sequence of the ribosomal proteins S16-encoding gene fromAspergillus nudulans. Gene 108:157–162;1991.
Celis JE, Rasmussen HH, Olsen E, Madsen P, Leffers H, Honore B, Dejgaard K, Gromov P, Vorum H, Vassilev A, Baskin Y, Liu X, Celis A, Basse B, Lauridsen JB, Ratz GP, Andersen AH, Walbum E, Kajergaard I, Andersen Puype M, Van Damme J, Vandekerckhove J. The human keratinocyte two-dimensional protein database (update 1994): Towards an integrated approach to the study of all proliferation differentiation and skin diseases. Electrophoresis 14:1349–1458;1994.
Dailey DC, Alderete JE. The phenotypically variable surface protein ofTrichomonas vaginalis has a single tandemly repeated immunodominant epitope. Infect Immun 59:2083–2088;1991.
Ditlow C, Johansen JT, Martin BM, Svendsen I. The complete amino acid sequence of manganese-superoxide dismutase fromSaccharomyces cerevisiae. Carlsberg Res Commun 47:81–91;1982.
Ferea T, Contreras ET, Qung T, Bowman EJ, Bowman BJ. Characterization of the cit-1 gene fromNeurospora crassa encoding the mitochondrial form of citrate synthase. Mol Gen Genet 242:105–110;1994.
Fields S, Winter G, Brownlee GG. Structure of the neuraminidase gene in human influenza virus A/PR/8/34. Nature 290;213–217;1981.
Fukaya N, Chow LP, Sugiura Y, Tsugita A, Ueno Y, Tabuchi K. Partial amino acid sequences of peptidyl isomerases ofFusarium sporotrichioides. J Med Sci; submitted.
Goerg A, Postel W, Guether S. Two-dimension electrophoresis: The current state of two-dimensional electrophoresis with immobilized pH gradient. Electrophoresis 9;531–546;1988.
George DG, Barker WC, Mewes HW, Pfeiffer F, Tsugita A. The PIR International Protein Sequence Database. Nucl Acids Res 22:3569–3573;1994.
Giometti CS, Taylor J, Tollaksen SL. Mouse liver protein database: A catalog of proteins detected by two-dimensional gel electrophoresis. Electrophoresis 13:970–991;1992.
Haese A, Schubert M, Herrmann M, Zocher R. Molecular characterization of the enniatin synthetase gene encoding a multifunctional enzyme catalysing N-methyldepsipeptide formation inFusarium scirpi. Mol Microbiol 7:905–914;1993.
Heller J, Smith EL.Neurospora crassa cytochrome C. II. Chymotryptic peptides, tryptic peptides, cyanogen bromide peptides and the complete amino acid sequence. J Biol Chem 241:3165–3180;1966.
Hewick RM, Hunkapiller MW, Hood LE, Dreyer WJ. A gas-liquid solid phase peptide and protein sequenator. J Biol Chem 256:7990–7997;1981.
Hoffmann RJ, Boore JL, Brown WM. A novel mitochondrial genome organization for the blue mussel,Mytilus edulis. Genetics 131:397–412;1992.
Hohmann S, Cederberg H. Autoregulation may control the expression of yeast pyruvate decarboxylase structural gene PDCI and PDC5. Eur J Biochem 188:615–621;1990.
Holland MJ, Holland JP, Thill GP, Jackson KA. The primary structure of two yeast enolase genes. Homology between the 5′ noncoding regions of yeast enolase and glyceraldehyde-3-phosphate dehydrogenase genes. J Biol Chem 256:1385–1395;1981.
Horimoto K, Oshima K, Tsugita A, Otsuka J. Protein Superfamily Database. CODATA Proc Ser 4, in press.
Hohn T, Vanmiddlesworth F. Purification and characterization of the sesquiterpene cyclase trichodiene synthase fromFusarium sporotrichioides. Arch Biochem Biophys 251:756–761;1986.
Ishii K, Sato H, Ueno Y. Production of 3-acetyldeoxynivalenol in shake culture. Mycotoxin Res 1:19–24;1985.
Johnston PA, Perin MS, Reynolds GA, Wasserman SA, Suedhof TC. Two novel annexins fromDrosophila melanogaster: Cloning, characterization and differential expression in development. J Biol Chem 265:11382–11388;1990.
Kamo M, Kawakami T, Miyatake N, Tsugita A. Separation and characterization ofArabidopsis proteins by two-dimensional gel electrophoresis. Electrophoresis 16:423–430;1995.
Kawamura O, Nagayama S, Sato S, Ohtani K, Sugiura Y, Tanaka T, Ueno Y, Survey of T-2 toxin in cereals by an indirect enzyme-linked immunosorbent assay. Food Agric Immunol 2:173–180;1990.
Kopetzki E, Entian KD, Lottspeich F, Mecke D. Purification procedure and N-terminal amino acid sequence of yeast malate dehydrogenase isoenzymes. Biochim Biophys Acta 912:398–403;1987.
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685;1970.
Liu XQ, Gillham NW, Boynton JE. Chloroplast ribosomal protein gene rps 12 ofChlamydomonas reinhardtii: Wild-type sequence, mutation to streptomycin resistance and dependence, and function inEscherichia coli. J Biol Chem 264:16100–16108;1989.
Luke MM, Sutton A, Arndt KT. Characterization of SIS1, aSaccharomyces cerevisiae homologue of bacterial dnaJ proteins. J Cell Biol 114:623–628;1991.
Matsuda J, Okabe S, Hashimoto T, Yamada Y. Molecular cloning of hyoscyamine 6β-hydoxylase: A 2-oxoglutarate-dependent di-oxygenase from cultured roots ofHyoscyamus niger. J Biol Chem 266:9460–9464;1991.
Matsudaira P. Sequence from picomole quantities of protein electroblotted onto polyvinylidene difluoride membrane. J Biol Chem 262:10035–10038;1987.
Miyatake N, Kamo M, Satake K, Uchiyama Y, Tsugita A. Removal of N-terminal formyl groups and deblocking of pyrrolidone carboxylic acid of proteins with anhydrous hydrazine vapor. Eur J Biochem 212:785–789;1993.
O'Farrell PH. High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250:4007–4021;1975.
Ozkaynak E, Finley D, Varshavsky A. The yeast ubiquitin gene: Head-to-tail repeats encoding a polyubiquitin precursor protein. Nature 312:663–666;1984.
Palm D, Goerl R, Burger KJ. Evolution of catalytic and regulatory sites in phosphorylases. Nature 313:500–502;1985.
Perentesis JP, Phan LD, Gleason WB, LaPorte DC, Livingston DM, Bodley JW.Saccharomyces cerevisiae elongation factor 2: Genetic cloning, characterization of expression, and G-domain modeling. J Biol Chem 267:1190–1197;1992.
Pearson WR. Rapid and sensitive sequence comparison with FASTP and FASTA. In: Doolittle RF, ed. Methods in Enzymology. San Diego, Academic Press. 183:63–98;1990.
Ramagli LS, Rodriguez LV. Qunatitation of microgram amounts of protein in two-dimensional polyacrylamide gel electrophoresis sample buffer. Electrophoresis 6:559–563;1985.
Resnick S, Zimmer B, Pappagianis D, Eakin A, McKerrow J. Purification and amino-terminal sequence analysis of the complement-fixing and precipitation antigens fromCoccidioides immitis. J Clin Microbiol 28:385–388;1990.
Roux P, Labarere J. Isozyme characterization of dikaryotic strains of the edible basidiomyceteAgaricus bitorquis (Quel.) Sacc (Syn.Agaricus edulis). Exp Mycol 14:101–112;1990.
Russell PR. Transcription of the triose-phosphate-isomerase gene ofSchizosaccharomyces pombe initiates from a start point different from that inSaccharomyces cerevisiae. Gene 40:125–130;1985.
Russell PR, Hall BD. The primary structure of the alcohol dehydrogenase gene from the fission yeastSchizosaccharomyces pombe. J Biol Chem 258:143–149;1983.
Schwelberger HG, Kohlwein SD, Paltauf F. Molecular cloning primary structure and disruption of the structure gene of aldolase fromSaccharomyces cerevisiae. Eur J Biochem 180:301–308;1989.
Simpson RJ, Tsugita A, Celis JE, Garrels JI, Mewes HW. Workshop on two-dimensional gel protein database. Electrophoresis 13:1055–1061;1992.
Takemura M, Ota K, Yamato K, Ohta E, Nakamura Y, Nozato N, Akashi K, Ohyama K. Gene cluster for ribosomal proteins in the mitochondrial genome of a liverwort,Marchantia polymorpha. Nucleic Acids Res 20:3199–3205;1992.
Thomas D, Rothstein R, Rosenberg N, Surdin-Kerjan Y. SAM2 encodes the second methionine S-adenosyl transferase inSaccharomyces cerevisiae: Physiology and regulation of both enzymes. Mol Cell Biol 8:5132–5139;1988.
Tropschug M, Nicholson DW, Hartl FU, Koehler H, Pfanner N, Wachter E, Neupert W. Cyclosporin A-binding protein (cyclophilin) ofNeurospora crassa: One gene codes for both the cytosolic and mitochondrial forms. J Biol Chem 263:14433–14440;1988.
Tsugita A, Kawakami T, Uchiyama Y, Kamo M, Miyatake N, Nozu Y. Separation and characterization of rice proteins. Electrophoresis 15:708–720;1994.
Ueno Y. Mycotoxins. In: Miller K, ed. Toxicological Aspects of Food. London, Elsevier, 139–204;1987.
VanBogelen RA, Sankar P, Clark RL, Bogan JA, Neidardt FC. The gene-protein database ofEscherichia coli edition 5. Electrophoresis 13:1014–1054;1992.
Watanabe H, Hasumi K, Fukushima Y, Sakai K, Endo A. Cloning of two isozymes ofTrichoderma koningii glyceraldehyde-3-phosphate dehydrogenase with different sensitivity to koningii acid. Biochim Biophys Acta 1172:43–48;1993.
Wu M, Tzagoloff A. Mitochondrial and cytoplasmic fumarases inSaccharomyces cerevisiae are encoded by a single nuclear gene FUM1. J Biol Chem 262:12275–12282;1987.
Zuelli F, Weber H, Zuber H. Structure and function ofL-lactate dehydrogenases from thermophilic and mesophilic bacteria. VI. Nucleotide sequences of lactate dehydrogenase genes from the thermophilic bacteriaBacillus stearothermophilus, Bacillus caldolyticus andBacillus caldotenax. Biol Chem Hoppe-Seyler 368:1167–1177;1987.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Chow, LP., Fukaya, N., Miyatake, N. et al. Resolution ofFusarium sporotrichioides proteins by two-dimensional polyacrylamide gel electrophoresis and identification by sequence homology comparison in protein data base. J Biomed Sci 2, 343–352 (1995). https://doi.org/10.1007/BF02255221
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02255221