عنوان

Cloning and expression of the Aspergillus oryzae alpha-amylase gene

TLpq303461126

انگلیسی

Cloning and expression of the Aspergillus oryzae alpha-amylase gene

[Thesis]

M. J. Gines

Carleton University (Canada)

1987

1

Ph.D.

Carleton University (Canada)

1987

The filamentous fungus Aspergillus oryzae secretes large amounts of usd\alphausd-amylase upon growth in starch medium. Highest levels of usd\alphausd-amylase specific mRNA measured by in vitro translation (IVT) were found between 6 to 15h of growth on starch. Poly(A) RNA isolated from starch cultures at 10h was used as template for cDNA synthesis and construction of three cDNA libraries using a M13 phage vector and E.coli JM103 as host. The libraries were screened for usd\alphausd-amylase sequence using a mixture of synthetic 20-mer oligodeoxyribonucleotides specified by back translation of the Taka-amylase (TAA) sequence (Toda et al. 1982). Of the 5 positive recombinant clones initially detected by probe hybridization, two of these clones, M71 and M91, were demonstrated by sequencing to be usd\alphausd-amylase specific. Clone M71, was used as a probe for further identification of chromosomal sequences. Two genes were identified (designated amylase I and II) by genomic Southern blots and subsequent recovery from a Lambda Charon A-A.oryzae genomic bank. Restriction endonuclease and hybridization analysis of these genes subcloned into pUC8 revealed a high degree of homology between them. The nucleotide sequence of a 2.85 kb EcoRI/HindIII region of Amylase II was obtained and the predicted protein sequence compared with the TAA. The gene contains eight intervening sequences within the coding region, ranging from 55 to 86bp in length. The precursor amylase has 499 amino acids, 21 residues larger than the published sequence of TAA. Only 9 differences in amino acids were found. Little or no homology was found when the signal peptide and mature protein sequence as well as the nucleotide sequence were compared to those available of other genes, such as the glucoamylases of the yeast Saccharomyces diastaticus and the fungi Aspergillus awamori and A.niger. Nucleotide sequences of the 5 and 3 untranslated regions (UTR) of amylase I were obtained and compared to those of amylase II and to several additional sequences recovered from the cDNA libraries. These studies revealed that the 5 regions were identical and that both genes, which differ in their 3 UTRs are transcribed. The measured amylase mRNA content of mycelia during growth on starch agrees with the IVT estimates and indicates that the RNA is rapidly degraded after 15 h of growth.

Biological sciences

Biology

M. J. Gines

p

[Thesis]

276903

a

Y