| Summary: | 碩士 === 國立臺灣大學 === 醫事技術學研究所 === 87 === Abstract
Since the Human Genome Project officially launched in 1990, a large number of expressed gene sequences have been deposited in public databases, such as GenBank and in private databases. In order to cope with the rapid growth of sequence data, it is important to translate the stored information into biological significance with integrated bioinfor-
matic tools.
In our laboratory, we have developed an open reading frame (ORF)-trapping vectors system based on translational codes for restriction enzyme site (CRES) and "phenotypic reversion" for screening and isolating DNA fragments with protein-coding information. Validity of the system was tested previously with cDNA fragments containing an open translational frame. It is known that the majority of genes of unicellular organisms like Saccaromyces and Leishmania is intronless. In this thesis we intended to employ this vector system to isolate and identify ORF fragments directly from the genomic sequences.
Since the sequences of the yeast genome has been determined, it served as a reference system in our current study. The overall trapping efficiency of this system is about 70%. However, different ATG start codons were also found to be used for protein translation. Unexpected translation was terminated after the introduction of frame-shift mutation.
The trapping efficiency of the Leishmania genomic fragments was found similar to that of yeast. Of the 91 DNA fragments trapped, 70 sequences had no matches in public databases. We have submitted all the new data to
GenBank. In order to prove that these trapped inserts represent Leishmania genes, amplified DNA fragments were spotted on charged membrane in microarrays, and hybridized with Leishmania mRNA-derived probes. The results show that 7 of 21 ORF fragments with putative functions wereexpressed in Leishmania. 5 of 19 ORF fragments without database matches
expressed in Leishmania.
We demonstrated that the combined usage of the ORF-trapping vector system and microarray assay is a powerful tool high-throughput screening of DNA sequences with protein coding information.
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