Analysis of the inducible MEL1 gene of Saccharomyces carlsbergensis and its secreted product, alpha-galactosidase (melibiase) |
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Authors: | M Sumner-Smith R P Bozzato N Skipper R W Davies J E Hopper |
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Institution: | 2. Allelix Inc., Division of Molecular Biology, 6850 Goreway Drive, Mississauga, Ontario Canada L4V 1P1 Tel. (416) 677-0831;1. Department of Biological Chemistry and the Cancer Research Center, The Milton S. Hershey Medical Center, Pennsylvania State University, Hershey, PA 17033 U.S.A. Tel. (717) 534-8590 |
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Abstract: | We have determined both the nucleotide sequence of the MEL1 gene of Saccharomyces carlsbergensis and the N-terminal amino acid (aa) sequence of its extracellular gene product, alpha-galactosidase (melibiase) (alpha-Gal). The predicted translation product of MEL1 is a pre-alpha-Gal protein containing an 18 aa N-terminal signal sequence for secretion. The purified enzyme is a dimer consisting of two 50-kDal polypeptides, each of which is glycosylated with no more than eight side chains. The 5'-flank of the MEL1 gene contains a region (UASm) having certain areas of sequence homology to similar sites found upstream of the structural genes GAL1, GAL7 and GAL10, which are also regulated by the action of the products of genes GAL4 and GAL80. There are three TATA boxes between UASm and the initiation codon of pre-alpha-Gal, as well as a typical yeast cleavage/polyadenylation sequence in the 3'-flank of the gene. |
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Keywords: | Yeast secretion signal sequence recombinant DNA sequencing upstream activating sequence regulation protein purification glycosylation aa amino acid(s) α-Gal α-galactosidase (melibiase) bp base pairs EndoH endo-β-acetylglucosaminidase medium I II see MATERIALS AND METHODS section b ORF open reading frame PAGE polyacrylamide gel electrophoresis pNP-gal PollK SDS sodium dodecyl sulfate U units(s) UAS upstream activating sequence UASg UASm YNM yeast nitrogen base medium without amino acids (Difco) |
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