The construction and characterization of three genomic libraries of trichoderma virens strain Tv29-8

Abstract

Three genomic libraries (lambda, cosmid, and bacterial artificial chromosome) were constructed from Trichoderma virens strain Tv29-8. These libraries have been characterized and used to isolate a full-length copy of a large peptide synthetase, tex1, and genes involved in signal transduction. Isolation of large DNA for lambda and cosmid library construction was accomplished by lysing protoplasts, rather than by lyophilizing and grinding mycelia. The lambda library was constructed from genomic DNA partially digested with Mbo I and cloned into LambdaGem11®. Three ligation conditions yielded titers ranging from 10⁵ to 10⁶ plaques per packaging extract. The cosmid library was constructed with genomic DNA partially digested with Mbo I and dephosphorylated and cloned into pMOcosX. A total of 6400 clones were arrayed in 96-well plates, which were then converted to 384-well plates, representing approximately 6X genome coverage. High-molecular-weight (HMW) DNA for BAC cloning was obtained by embedding protoplasts in agarose plugs, which were subjected to pre-size selection to remove small DNA fragments. HMW DNA was partially digested with Hind III and fractionated on a contour-clamped homogeneous electrophoresis field (CHEF) gel. The region containing 100-450 kb fragments was excised and divided into three sections (small: 100-200 kb, medium: 200-300 kb, and large: 300-450 kb). DNA was recovered by electroelution and subjected to a second size selection. Insert DNA was ligated to pECBAC1 in a 6:1 molecular weight ratio. Random clones were analyzed for insert size by Not I digestion. As a large percentage of the clones analyzed from the medium and large ligations did not contain inserts, the BAC library was constructed from the small ligation. The average insert size was approximately 90 kb, but ranged from 10 kb to 170 kb. The library was arrayed as 12,243 clones in 32 384-well plates and immobilized on eight 8 cm x 12 cm nylon membranes. Genome representation was determined using two single-copy genes, arg2 and gpd, each hybridizing to approximately 25 clones. A full-length copy of tex1 was isolated on a 150 kb BAC clone. A contig of plasmids containing 24.5 kb of tex1 was constructed from a BAC clone for sequencing and analysis.