Her Majesty the Queen in Right of Canada, As Represented by the National

Steve N Slilaty

Shi-Hsiang Shen

Susan Lebel

Pierre Chretien

Lison Bastien

The present invention relates to recombinant .beta.-1,3-glucanase essentially free of proteases. The enzyme is obtained through the use of a recombinant DNA expression vector which comprises a DNA sequence encoding the .beta.-1,3-glucanase gene or mutants and variants thereof placed under the control of an exogenous expression promoter, preferably a bacterial promoter. Also, the .beta.-1,3-glucanase gene may include sequences flanking the open reading frame of the native .beta.-1,3-glucanase gene. The present invention also relates to a microorganism transformed with a recombinant DNA expression vector comprising the .beta.-1,3-glucanase gene or mutants and variants thereof under the control of an exogenous expression promoter.

Lytic .beta.-1,3-glucanase gene

The present invention relates to an approach for conducting site-directed mutagenesis using double-stranded DNA templates. The approach involves the development of a method for generating structures capable of directing full-length complementary-strand synthesis from double-stranded plasmid DNA. These structures are formed following heat denaturation and cooling of linearized plasmid DNA molecules in the presence of what is referred to as a 'closing oligonucleotide'. A 'closing oligonucleotide' is a single-stranded oligonucleotide consisting of a sequence complementary to either or both free ends of one of the two plasmid DNA strands. The 'closing oligonucleotide' therefore functions as an agent for recircularization of a DNA strand and generation of a primer-circular template structure suitable for polymerase-dependent full-length complementary-strand synthesis and ligation into a covalently-closed heteroduplex DNA molecule. When combined with a mutagenic oligonucleotide and uracil-substituted DNA templates, this approach allows site-directed mutagenesis to be performed directly on double-stranded DNA with a mutant formation efficiency of about 50%, a level amenable to rapid screening by DNA sequencing.

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Ville St-Laurent, Canada

Steve N Slilaty