Patent ID: 6767701
Filing Date: 2004-07-27
Classification: C12N

Abstract:
A method of screening a library of polynucleotide sequences of interest having or encoding a desired activity or function in filamentous fungal cells, wherein the method comprises:(a) transforming the fungal cells with a population of DNA vectors, wherein each vector comprises: (i) a fungal selection marker polynucleotide sequence and a fungal replication initiating polynucleotide sequence, wherein the marker and the replication initiating sequence do not vary within the population; and wherein the replication initiating sequence is a nucleic acid sequence selected from the group consisting of: (1) a replication initiating sequence having at least 80% identity with the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:2, as determined using the GAP computer program with a GAP creation penalty of 5.0 and GAP extension penalty of 0.3, and is capable of initiating replication; and (2) replication initiating sequence which hybridises under low stringency conditions with (i) the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:2, or (ii) the respective complementary strands, wherein the low stringency conditions are defined by prehybridization and hybridization at 42Â° C. in 5Ã—SSPE, 0.3% SDS, 200 mg/ml sheared and denatured salmon sperm DNA, and 25% formamide, and wash conditions are defined at 50Â° C. for 30 minutes in 2Ã—SSC, 0.2% SDS; and (ii) a polynucleotide sequence of interest, wherein there are vectors in the population that vary from other vectors in the population by carrying different versions of the polynucleotide sequence of interest; (b) cultivating the cells in the presence of an effective amount of a selective agent or the absence of an appropriate selective agent; (c) selecting or screening for one or more transformants expressing the desired activity or function; and (d) isolating the transformant(s) of interest.