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Volume 142, Issue 9

4-Dihydromethyltrisporate dehydrogenase from , an enzyme of the sexual hormone pathway: purification, and cloning of the corresponding gene Free

Abstract

We have purified the NADP-dependent 4-dihydromethyltrisporate dehydrogenase from the zygomycete The enzyme is involved in the biosynthesis of trisporic acid, the sexual hormone of zygomycetes, which induces the first steps of zygophore development. Protein was obtained from the (−) mating type of after induction with trisporic acid, and purified by gel filtration and affinity chromatography steps. On SDS-PAGE a band with an apparent molecular mass of 33 kDa was ascribed to the enzyme. After transferring onto PVDF membranes the protein was digested with endoprotease Lys-C, and several peptides were sequenced. Oligonucleotides derived from protein sequence data were used for PCR amplification of genomic DNA. The PCR fragment was used as probe for isolation of the corresponding cDNA and complete genomic DNA clones. Comparison of protein and DNA sequence data showed that the cloned fragment corresponded to the purified protein. Search for similarity with protein sequences of the Swiss-Prot database revealed a relationship to enzymes belonging to the aldo/keto reductase superfamily. Southern-blot analysis of genomic DNA with the labelled cloned fragment detected a single-copy gene in both mating types of PCR with genomic DNA from other zygomycetes gave rise to several fragments. Hybridization analysis with the cloned fragment showed that a fragment of similar length cross-hybridized in (Choanephoraceae) as well as in and (Mucoraceae). The promoter region of the gene contains DNA elements with similarity to a cAMP-regulated gene of

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Keyword(s): aldo/keto reductase superfamily , mating type , Mucor mucedo , oxidoreductase and trisporic acid
© Society for General Microbiology 1996
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1996-09-01
2024-05-14
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References

  1. Amore R., Kötter P., Küster C., Ciriacy M., Hollenberg C. P. 1991; Cloning and expression in Saccharomyces cerevisiae of the NAD(P)H-dependent xylose reductase-encoding gene (XYL1) from the xylose-assimilating yeast Pichia stipitis . Gene 109:89–97
     [Google Scholar]
  2. Bartels D., Engelhardt K., Roncarati R., Schneider K., Rotter M., Salamini F. 1991; An ABA and GA modulated gene expressed in the barley embryo encodes an aldolase reductase related protein. EMBO J 10:1037–1043
     [Google Scholar]
  3. Benito E. P., Díaz-Mínguez J. M., Iturriaga E. A., Campuzano V., Eslava A. P. 1992; Cloning and sequence analysis of the Mucor circinelloides pyrG gene encoding omtidine-5′-monophosphate decarboxylase: use of pyrG for homologous transformation. Gene 116:59–67
     [Google Scholar]
  4. Blakeslee A. F., Cartledge J. L. 1927; Sexual dimorphism in Mucorales. IT. Interspecific reactions. Bot Gaz 84:51–57
     [Google Scholar]
  5. Braun H. P., Kruft V., Schmitz U. K. 1994; Molecular identification of the ten subunits of cvtochrome-c reductase from potato mitochondria. Planta 193:99–106
     [Google Scholar]
  6. Bruce N. C., Willey D. L., Coulson A. F. W., Jeffery J. 1994; Bacterial morphine dehydrogenase further defines a distinct super-family of oxidoreductases with diverse functional activities. Biochem J 299:805–811
     [Google Scholar]
  7. Bullock W. O., Fernandez J. M., Short J.M. 1987; XI1-blue: a high efficiency plasmid transforming recA Escherichia coii strain with β-galactosidase selection. Biotechniques 5:376–379
     [Google Scholar]
  8. Bu’Lock J. D., Jones B. E., Winskill N. 1976; The apocarotenoid system of sex hormones and prohormones in mucorales. Pure Appl Chem 47:191–202
     [Google Scholar]
  9. Burgeff H. 1924; Untersuchungen über Sexualität und Parasitismus bei Mucorineen. Bot Abh 4:5–135
     [Google Scholar]
  10. Burmester A. 1995; Analysis of the gene for the elongation factor lα from the zygomycete Absidia glauca. Use of the promoter region for constructions of transformation vectors. Microbiol Res 150:63–70
     [Google Scholar]
  11. Burmester A., Czempinski K. 1994; Sequence comparison of a segment of the gene for 3-hydroxy-3-mcthylglutaryl-coenzyme A reductase in zygomycetes. Eur J Biochem 220:403–408
     [Google Scholar]
  12. Burmester A., Wöstemeyer J. 1986; Cloned mitochondrial DNA from the zygomycete Absidia glauca promotes autonomous replication in Saccbaromyces cerevisiae . Curr Genet 10:435–441
     [Google Scholar]
  13. Chung S., LaMendola J. 1989; Cloning and sequence determination of human placental aldose reductase gene. J Biol Chem 264:14775–14777
     [Google Scholar]
  14. Díaz-Mínguez J. M., Iturriaga E. A., Benito E. P., Corrochano L. M., Eslava A. P. 1990; Isolation and molecular analysis of the orotidine-5′-phosphate decarboxylase gene (pyrG) of Phycomyces biakesleeanus . Mol Gen Genet 224:269–278
     [Google Scholar]
  15. van den Ende H. 1968; Relationship between sexuality and carotene synthesis in Blakeslea trispora . J Bacteriol 96:1298–1303
     [Google Scholar]
  16. van den Ende H., Stegwee D. 1971; Physiology of sex in Mucorales. Bot Rev 37:22–36
     [Google Scholar]
  17. Eslava A. P., Alvarez M. I., Cerda-Olmedo E. 1974; Regulation of carotene biosynthesis in Phycomyces by vitamin A and α-ionone. Eur j Biochem 48:617–623
     [Google Scholar]
  18. Gooday G. W. 1994; Hormones in mycelial fungi. In The Mycota I pp. 401–411 Edited by Wessels J. G. H., Meinhardt F. Berlin: Springer-Verlag;
     [Google Scholar]
  19. Grindley J. F., Payton M. A., van de Pol H., Hardy K. G. 1988; Conversion of glucose to 2-keto-L-gulonate, an intermediate in l-ascorbate synthesis, by a recombinant strain of Erwinia citreus . Appl Environ Microbiol 54:1770–1775
     [Google Scholar]
  20. Haberstroh L., Firtel R. A. 1990; A spatial gradient of expression of a cAMP-regulated prespore cell-type-specific gene in Dictyostelium . Genes Dev 4:596–612
     [Google Scholar]
  21. Hager D. A., Burgess R. R. 1980; Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: results with Sigma sub-unit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes. Anal Biochem 109:76–86
     [Google Scholar]
  22. Hasegawa M., Hashimoto T., Adachi T. 1992; Origin and evolution of eukaryotes as inferred from protein sequence data. In The Origin and Evolution of the Cell pp. 107–130 Edited by Hartman H., Matsuno K. Singapore: World Scientific;
     [Google Scholar]
  23. Horiuchi H., Takaya N., Yanai K., Nakamura M., Ohta A., Takagi M. 1995; Cloning of the Rhizopus niveus pyr4 gene and its use for the transformation of Rhizopus delemar . Curr Genet 27:472–478
     [Google Scholar]
  24. Kellner M., Burmester A., Wöstemeyer A., Wöstemeyer J. 1993; Transfer of genetic information from the my coparasite Parasitella parasitica to its host Absidia glauca . Curr Genet 23:334–337
     [Google Scholar]
  25. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
     [Google Scholar]
  26. Nachlas M. M., Margulies S. I., Goldberg J. D., Seligman A. M. 1960; The determination of lactic dehydrogenase with a tetrazolium salt. Anal Biochem 1:317–326
     [Google Scholar]
  27. Nieuwenhuis M., van den Ende H. 1975; Sex specificity of hormone synthesis in Mucor mucedo . Arch Microbiol 102:167–169
     [Google Scholar]
  28. Oechsner U., Magdolen V., Bandlow W. 1988; A nuclear yeast gene (GCY) encodes a polypeptide with high homology to a vertebrate eye lens protein. FEBS Lett 238:123–128
     [Google Scholar]
  29. Pawlowski J. E., Huizinga M., Penning T. M. 1991; Cloning and sequencing of the cDNA for rat liver 3α-hydroxysteroid/ dihydrodiol dehydrogenase. J Biol Chem 266:8820–8825
     [Google Scholar]
  30. Samaras N., Spithill T. W. 1989; The developmentally regulated P100/11E gene of Leishmania major shows homology to a superfamily of reductase genes. J Biol Chem 264:4251–4254
     [Google Scholar]
  31. Southern E. M. 1975; Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517
     [Google Scholar]
  32. Stoschek C. M. 1990; Quantitation of a protein. Methods Enjymol 182:50–67
     [Google Scholar]
  33. Sundstrom P., Lira L. M., Choi D., Linz J. E., Sypherd P. S. 1987; Sequence analysis of the EF-lα gene family of Mucor racemosus . Nucleic Acids Rex 15:9997–10006
     [Google Scholar]
  34. Vetter M., Wöstemeyer J., Burmester A. 1994; Characterization of specific cDNA clones of the zygomycetes Parasitella parasitica, derived from mRNAs which are regulated by the pheromone trisporic acid. Microbiol Res 149:17–22
     [Google Scholar]
  35. Werkman B. A. 1976; Localization and partial characterization of a sex-specific enzyme in homothallic and heterothallic mucorales. Arch Microbiol 109:209–213
     [Google Scholar]
  36. Werkman T. A., van den Ende H. 1973; Trisporic acid synthesis in Blakeslea trispora. Interaction between plus and minus mating types. Arch Microbiol 90:365–374
     [Google Scholar]
  37. Willey D. L., Caswell D. A., Lowe C. R., Bruce N. C. 1993; Nucleotide sequence and over-expression of morphine dehydrogenase, a plasmid-encoded gene from Pseudomonas putida M10. Biochem J 290:539–544
     [Google Scholar]
  38. Wilson D. K., Bohren K. M., Gabbay K. H., Quiocho F. A. 1992; An unlikely sugar substrate of the human aldose reductase holoenzyme implicated in diabetic complications. Science 257:81–84
     [Google Scholar]
  39. Wöstemeyer J. 1985; Strain-dependent variation in ribosomal DNA arrangement in Absidia glauca . Eur J Biochem 146:443–448
     [Google Scholar]
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4-Dihydromethyltrisporate dehydrogenase from Mucor mucedo, an enzyme of the sexual hormone pathway: purification, and cloning of the corresponding gene
Microbiology 142, 2647 (1996); https://doi.org/10.1099/00221287-142-9-2647
/content/journal/micro/10.1099/00221287-142-9-2647
/content/journal/micro/10.1099/00221287-142-9-2647
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