An expression cDNA library for suppression cloning in yeast mutants, complementation of a yeast his4 mutant, and EST analysis from the symbiotic basidiomycete Hebeloma cylindrosporum

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Abstract:

An oriented expression library was constructed from the mycelia of the symbiotic model fungus Hebeloma cylindrosporum in the high-level yeast expression vector pDR196. DNA sequencing of ~500 expressed sequence tags (ESTs) showed that 15% correspond to known genes, two thirds contain sequences with unknown function, and the remaining 20% showed no significant similarity to any known genes. The ESTs had a GC content between 44 and 56%, with most of them having a GC content of 52–54%, which could be correlated with GC contents of fungal genes. The library was successfully used to identify the Hebeloma HIS4 gene by functional complementation of a yeast his4 mutant. Thus, the library may serve as a powerful tool for identification and characterization of mycorrhizal genes by EST analysis and for the identification of ectomycorrhizal genes by means of suppression cloning.Key words: ectomycorrhiza, functional cDNA library, Hebeloma cylindrosporum, yeast complementation.

Une banque d'expression orientée a été préparée à partir du mycélium du champignon symbiotique modèle Hebeloma cylindrosporum dans le pDR196, un vecteur permettant une forte expression chez la levure. Le séquençage d'environ 500 EST (« expressed sequence tags  ») a révélé que 15 % de ceux-ci correspondaient à des gènes connus, deux tiers étaient homologues à des gènes de fonction inconnue et le dernier 20 % ne montrait aucune homologie significative aux gènes clonés à ce jour. Les EST avaient un contenu en GC variant entre 44 et 56 %, la plupart se situant entre 52 et 54 %, ce qui pouvait être mis en lien avec le contenu en GC des gènes fongiques. La banque a été criblée avec succès pour le gène HIS4 du Hebeloma par complémentation fonctionnelle d'un mutant his4 de la levure. Ainsi, cette banque pourra servir d'outil puissant pour identifier et caractériser des gènes impliqués dans les mycorrhizes par analyse d'EST et pour l'identification de gènes ectomycorrhiziens par clonage suppressionnel.Mots clés : ectomycorrhizes, banque d'ADNc fonctionnelle, Hebeloma cylindrosporum, complémentation chez la levure.[Traduit par la Rédaction]

Document Type: Research Article

Publication date: April 1, 2003

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  • From its inception in 1957, this international cytogenetics journal has catered to the research areas of the members of the Genetics Society of Canada; traditionally, these have included agriculture, entomology, genetics/cytogenetics, and evolutionary mechanisms. The contents of the journal have evolved as contributors developed new technologies and interests. A 20-member Editorial Board is composed of scientists from around the world. Reviews and commentary from respected experts are often featured.
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