The use of direct cDNA selection to rapidly and effectively identify genes in the fungus Aspergillus fumigatus.
Aspergillus fumigatus is one of the causes of invasive lung disease in immunocompromised individuals. To rapidly identify genes in this fungus, including potential targets for chemotherapy, diagnostics, and vaccine development, we constructed cDNA libraries. We began with non-normalized libraries, then to improve this approach we constructed a normalized cDNA library using direct cDNA selection. Normalization resulted in a reduction of the frequency of clones with highly expressed genes and an enrichment of underrepresented cDNAs. Expressed sequence tags generated from both the original and the normalized libraries were compared with the genomes of Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans, indicating that a large proportion of A. fumigatus genes do not have orthologs in these fungal species. This method allowed the expeditious identification of genes in a fungal pathogen. The same approach can be applied to other human or plant pathogens to rapidly identify genes without the need for genomic sequence information.
Kessler
Marco M
MM
Genome Therapeutics Corporation, Waltham, MA 02453, USA. marco.kessler@genomecorp.com
Willins
Debra Aker
DA
Zeng
Qiandong
Q
Del Mastro
Richard G
RG
Cook
Robin
R
Doucette-Stamm
Lynn
L
Lee
Hongmei
H
Caron
Anne
A
McClanahan
Terri K
TK
Wang
Luquan
L
Greene
Jonathan
J
Hare
Roberta S
RS
Cottarel
Guillaume
G
Shimer
George H
GH
D001232:Aspergillus fumigatus / Q000235:genetics*; D001483:Base Sequence; D002176:Candida albicans / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics*; D020224:Expressed Sequence Tags; D015723:Gene Library; D005800:Genes, Fungal; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012568:Schizosaccharomyces / Q000235:genetics; D013045:Species Specificity
D017931:DNA Primers; D018076:DNA, Complementary; D004271:DNA, Fungal