Chicken single nucleotide polymorphism identification and selection for genetic mapping.

Single nucleotide polymorphisms (SNP) are the ideal markers for high-density genome wide mapping. A total of 327,000 expressed sequence tag (EST) sequences, obtained from the ChickEST project, were examined for the presence of SNP. A total of 32,268 potential chicken SNP were identified and stored in a customized Microsoft Access database and evaluated in silico for their usability for a high-density genetic map. Based on a minimum of 3 for the minor allele occurrence and a minimum of 30% for the minor allele frequency, 5,332 reliable SNP were selected, of which both SNP alleles were present in the database at a high frequency. To test the usefulness of the in silico SNP identification, 24 SNP affecting a BglII site were used for a genotyping study. A functional PCR assay could be designed for 21 of the 24 SNP. It was possible to validate 90% of this marker subset (21 SNP) by BglII restriction analysis. The high percentage of validated markers demonstrates the reliability of the 5,332 chicken SNP markers. Furthermore, the limited number of genomic DNA samples necessary to validate 90% of the SNP markers confirmed the prediction of the high frequency at which both alleles of the selected SNP were present in the tested chicken populations.

The SC15 protein of Schizophyllum commune mediates formation of aerial hyphae and attachment in the absence of the SC3 hydrophobin.

Disruption of the SC3 gene in the basidiomycete Schizophyllum commune affected not only formation of aerial hyphae but also attachment to hydrophobic surfaces. However, these processes were not completely abolished, indicating involvement of other molecules. We here show that the SC15 protein mediates formation of aerial hyphae and attachment in the absence of SC3. SC15 is a secreted protein of 191 aa with a hydrophilic N-terminal half and a highly hydrophobic C-terminal half. It is not a hydrophobin as it lacks the eight conserved cysteine residues found in these proteins. Besides being secreted into the medium, SC15 was localized in the cell wall and the mucilage that binds aerial hyphae together. In a strain in which the SC15 gene was deleted (DeltaSC15) formation of aerial hyphae and attachment were not affected. However, these processes were almost completely abolished when the SC15 gene was deleted in the DeltaSC3 background. The absence of aerial hyphae in the DeltaSC3DeltaSC15 strain can be explained by the inability of the strain to lower the water surface tension and to make aerial hyphae hydrophobic.

Cloning of a prolidase gene from Aspergillus nidulans and characterisation of its product.

Using EST sequence information available from the filamentous fungus Aspergillus nidulans as a starting point, we have cloned the prolidase-encoding gene, designated pepP. Introduction of multiple copies of this gene into the A. nidulansgenome leads to overexpression of an intracellular prolidase activity. Prolidase was subsequently purified and characterised from an overexpressing strain. The enzyme activity is dependent on manganese as a cofactor, is specific for dipeptides and hydrolyses only dipeptides with a C-terminal proline residue. Although these proline dipeptides are released both intracellularly and extracellularly, prolidase activity was detected only intracellularly.

Characterization of the kexin-like maturase of Aspergillus niger.

Secreted yields of foreign proteins may be enhanced in filamentous fungi through the use of translational fusions in which the target protein is fused to an endogenous secreted carrier protein. The fused proteins are usually separated in vivo by cleavage of an engineered Kex2 endoprotease recognition site at the fusion junction. We have cloned the kexin-encoding gene of Aspergillus niger (kexB). We constructed strains that either overexpressed KexB or lacked a functional kexB gene. Kexin-specific activity doubled in membrane-protein fractions of the strain overexpressing KexB. In contrast, no kexin-specific activity was detected in the similar protein fractions of the kexB disruptant. Expression in this loss-of-function strain of a glucoamylase human interleukin-6 fusion protein with an engineered Kex2 dibasic cleavage site at the fusion junction resulted in secretion of unprocessed fusion protein. The results show that KexB is the endoproteolytic proprotein processing enzyme responsible for the processing of (engineered) dibasic cleavage sites in target proteins that are transported through the secretion pathway of A. niger.