Development and analysis of an expressed sequence tag database from Fusarium sporotrichioides.

Abstract

The comparison of four other fungal cDNA libraries sequenced in our lab, F. sporotrichioides library, two Neurospora crassa libraries and one Aspergillus nidulans library, shows that the percentage of biological function divisions in each library is remarkably similar in spite of the diverse nature of the libraries. Using the group of ESTs that have no significant homologues to GenBank non-redundant protein database from F. sporotrichioides database to perform tBlastX against dbEST, it was found that there are 27 singlets/clusters that have homologs with both A. nidulus and N. crassa ESTs. These 2-7 new genes that present in all four libraries are valuable candidate unknown genes for further studies.

To date, twelve genes in the trichothecene biosynthesis pathway have been identified in F. sporotrichioides. Eleven of the twelve genes products were found in the F. sporotrioides EST database. In total, 541 ESTs, 7.22% of the total 7495 ESTs in the database, represented genes involved in trichothecene biosynthesis pathway. Two of the twelve genes are genes newly defined during this EST projects by our collaborators and three other genes that have the subtle Tri patterns are being studying further.

During the F. sporotrichioides EST project, 7495 high quality ESTs were obtained. The high quality ESTs were assembled using Phrap and then analyzed by a BlastX homology search against the GenBank non-redundant protein database. In total, 2181 singlets and 1057 contigs were obtained in the assembled database and 2139 genes were represented in this database. Several computer programs have been used to enable a semi-automated process of biological function assignments for each F. sporotrichioides EST database member that has a significant BlastX homologue. 50% of the ESTs that had significant homologues in GenBank non-redundant protein database were placed into the seven Riley categories. 50% of the ESTs had no significant homologues in GenBank non-redundant protein database. They may represent undiscovered genes.

A Fusarium sporotrichioides Tri10 over-expressed cDNA library was sequenced and its EST database was constructed in this dissertation research. The EST database was made publicly available by submission to GenBank dbEST and publication on the ACGT web site. This database serves as a foundation for annotating and understanding gene expression in F. sporotrichioides and related fungi.

In this dissertation research, seven BACs, PAC and cosmids from the Human Genome Project also were sequenced to contiguity with an individual base error rate of less than 1 error every 10,000 bases. Among them, two BAC sequences were thoroughly analyzed. Sequence of BAC 3220 revealed the sequence of the 9098 bp absent in the Ig lambda region of the reference sequence for human chromosome 22. The discovery of this phenotypically silent inborn gap was a trigger to make a plea to search for deletion polymorphism through genome scans in population. BAC 239c 10 covering the William Syndrome region encoded three genes, human neutrophil cytosol factor 1 (NCF1) gene, human hPMS gene and human Bruton's tyrosine kinase-associated protein-135 (BAP-135) gene. NCF1 gene and hMSP gene have been mapped to 7q 11.23 before, but BAP-135 has not been mapped to 7q11.23 in the previous studies. A pseudogene of the human prohibitin gene, which is related to breast cancer and has been mapped to 17q21, also was found in BAC 239c 10 sequence.