Abstract
Myrosinase isoenzymes are known to be encoded by two different families of genes denoted MA and MB. Nucleotide sequence analysis of a Brassica napus genomic clone containing a gene for myrosinase revealed it to be a pseudogene of the MA family. The gene spans more than 5 kb and contains at least 12 exons. The exon sequence of the gene is highly similar to myrosinase cDNA sequences. However, the gene displays three potential or actual pseudogene characters. Southern blot analysis using probes from the 3′ portions of the genomic and B. napus MA and MB cDNA clones showed that MA type myrosinases are encoded by approximately 4 genes, while MB type myrosinases are encoded by more than 10 genes in B. napus. Northern blots with mRNA from seeds and young leaves probed with the MA-and MB-specific probes showed that the MA and MB myrosinase gene families are differentially expressed. Myrosinases are highly similar to proteins of a β-glycosidase enzyme family comprising both β-glycosidases and phospho-β-glycosidases of as diverged species as archaebacteria, bacteria, mammals and plants. By homology to these β-glycosidases, putative active site residues in myrosinase are discussed on the basis of the similarity between β-glycosidases and cellulases.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Baird SD, Hefford MA, Johnson DA, Sung WL, Yaguchi M, Seligy VL: The Glu residue in the conserved Asn-Glu-Pro sequence of two highly divergent endo-β-1.4-glucanases is essential for enzymatic activity. Biochem Biophys Res Commun 169: 1035–1039 (1990).
Boll W, Wagner P, Mantei N: Structure of the chromosomal gene and cDNAs coding for lactase-phlorizin hydrolase in humans with adult-type hypolactasia or persistence of lactase. Am J Hum Genet 48: 889–902 (1991).
Bones A, Slupphaug G: Purification, characterization and partial amino acid sequencing of β-thioglucosidase from Brassica napus L. J Plant Physiol 134: 722–729 (1989).
BreidtJr F, Stewart GC: Nucleotide and deduced amino acid sequences of the Staphylococcus aureus phosphobeta-galactosidase gene. Appl Environ Microbiol 53: 969–973 (1987).
Cubellis MV, Rozzo C, Montecucchi P, Rossi M: Isolation and sequencing of a new beta-galactosidase-encoding archaebacterial gene. Gene 94: 89–94 (1990).
Durham PL, Poulton JE: Enzymic properties of purified myrosinase from Lepidium sativum seedlings. Z Naturforsch 45: 172–178 (1990).
Ellerström M, Josefsson L-G, Rask L, Ronne H: Cloning of a cDNA for a plant enzyme by complementation in yeast. Plant Mol Biol 18: 557–566 (1992).
Falk A, Xue J, Lenman M, Rask L: Sequence of a cDNA clone encoding the enzyme myrosinase and expression of myrosinase in different tissues of Brassica napus. Plant Sci 83: 181–186 (1992).
Fenwick GR, Heaney RK, Mullin WJ: Glucosinolates and their breakdown products in food plants. CRC Crit Rev Food Sci Nutr 18: 123–201 (1983).
Gräbnitz F, Seiss M, Rucknagel KP, Staudenbauet WL: Structure of the β-glucosidase gene bglA of Clostridium thermocellum: Sequence analysis reveals a superfamily of cellulases and β-glycosidases including human lactase/phlorizin hydrolase. Eur J Biochem 200: 301–309 (1991).
Henicoff S: Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28: 351–359 (1984).
Henrissat B, Claeyssens M, Tomme P, Lemesle L, Mornon J-P: Cellulase families revealed by hydrophobic cluster analysis. Gene 81: 83–95 (1989).
Höglund AS, Lenman M, Falk A, Rask L: Distribution of myrosinase in rapessed tissues. Plant Physiol 95: 213–221 (1991).
Höglund AS, Lenman M, Rask L: Myrosinase is localized to the interior of myrosin grains and is not associated to the surrounding tonoplast membrane. Plant Sci (in press).
Jakobsen KS, Breivold E, Hornes E: Purification of mRNA directly from crude plant tissues in 15 minutes using magnetic oligo dT microspheres. Nucl Acids Res 18: 3669 (1990).
Josefsson L-G, Lenman M, Ericson M, Rask L: Structure of a gene encoding the 1.7S storage protein, napin, from Brassica napus. J Biol Chem 262: 12196–12201 (1987).
Lenman M, Rödin J, Josefsson L-G, Rask L: Immunological characterization of rapeseed myrosinase. Eur J Biochem 194: 747–753 (1990).
Little S, Cartwright P, Campbell C, Prenneta A, McChesney J, Mountain A, Robinson M: Nucleotide sequence of a thermostable beta-galactosidase from Sulfolobus solfataricus. Nucl Acids Res 17: 7980 (1989).
Lönnerdal B, Janson J-C: Studies on myrosinase; II. Purification and characterization of a myrosinase from rapeseed (Brassica napus L.). Biochim Biophys Acta 315: 421–429 (1973).
Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982).
Mantei N, Villa M, Enzler T, Wacker H, Boll W, James P, Hunziker W, Semenza G: Complete primary structure of human and rabbit lactase-phlorizin hydrolase: implications for biosynthesis, membrane anchoring and evolution of the enzyme. EMBO J 7: 2705–2713 (1988).
Oxtoby E, Dunn A, Pancoro A, Hughes MA: Nucleotide and derived amino acid sequence of the cyanogenic β-glucosidase (linamarase) from white clover (Trifolium repens L.) Plant Mol Biol 17: 209–219 (1991).
Rodman JE: Divergence, convergence and parallelism in phytochemical characters: The glucosinolate-myrosinase system. In: Young DA, Siegler DA (eds) Phytochemistry and Angiosperm Phylogeny, pp.43–79. Praeger, New York (1981).
Rodman JE: Phenetic and cladistic studies of plants producing glucosinolates and myrosinase. Abstracts 14th International Botany Congress 280 (1987).
Rödin J, Sjödahl S, Josefsson L-G, Rask L: Characterization of a Brassica napus gene encoding a cruciferin subunit: estimation of sizes of cruciferin gene families. Plant Mol Biol (in press).
Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA: Primer directed amplification with a thermostable DNA polymerase. Science 239: 487–491 (1988).
Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467 (1977).
Sekar V: A rapid screening procedure for the identification of recombinant bacterial clones. Biotechniques 5: 11–13 (1987).
Thangstad OP, Iversen T-H, Slupphaug G, Bones A: Immunocytochemical localization of myrosinase in Brassica napus L. Planta 180: 245–248 (1990).
Thangstad OP, Evjen K, Bones A: Immunogold-EM localization of myrosinase in Brassicaceae. Protoplasma 161: 84–93 (1991).
Tsuruo I, Hata T: Studies on myrosinase in mustard seed. Part V. On the β-glucosidase activity of myrosinase and the interaction of ascorbate with myrosinase. Agric Biol Chem 32: 1425–1431 (1968).
Wakarchuk WW, Greenberg NM, Kilburn DG, MillerJr RC, Warren RAJ: Structure and transcription analysis of the gene encoding a cellobiase from Agrobacterium sp. strain ATCC 214400. J Bact 170: 301–307 (1988).
Xue J, Lenman M, Falk A, Rask L: The glucosinolatedegrading enzyme myrosinase in Brassicaceae is encoded by a gene family. Plant Mol Biol 18: 387–398 (1992).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Lenman, M., Falk, A., Xue, J. et al. Characterization of a Brassica napus myrosinase pseudogene: myrosinases are members of the BGA family of β-glycosidases. Plant Mol Biol 21, 463–474 (1993). https://doi.org/10.1007/BF00028804
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00028804