Isolation and DNA sequence analysis of distantly related fatty acid and retionol binding protein gene of Setaria digitata

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Date
2003
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Publisher
University of Colombo: UC(MED).
Abstract
Previously sequenced cDNA clone (designated PSD38) of Seteria digitata revealed a 102 bp region that had high homology (76 percent) to Fatty Acid and Retinol binding protein (FAR) gene of Brugia malayi. Rapid Amplification of 5' cDN Ends (RACE) method was performed on mRNA of Seteria digitata using primers derived from the above sequence. To obtain the promoter region of that gene, Genome Walker DNA Walking method was used. cDNA synthesized from Seteria digitata mRNA gave a 700 base pair fragment designated race-1. This fragment (race-1) was completely sequenced by PCR cycle sequencing. The sequence showed (64 percent) AT richness. When this sequence was aligned with NCBI GeneBank data a region of 109 bp showed a high degree (100 percent) of homology to Brugia malayi FAR protein gene sequence. Amino acid sequence deduced from this region (36 amino acids) had 100 percent similarity with the Brugia malayi FAR protein sequence. Single open reading frame of the race-1 sequence could not be obtained due to the presence of stop codons. The contig (cl) constructed from the race-1 sequence and the PSD38 sequence showed two regions that had high homology to Brugia nalayi FAR protein gene. A 109 bp (towards the 5' end) and a 102 bp (towards the 3' end) respectively. Seteria digitata genome walker library was constructed by cleaving genomic DNA with restriction enzymes. PCR amplification of this library using gene specific primers revealed a 636 bp region in the Dra 1 library. This fragment was cloned in pGEM T easy vector system 11 and recombinant clone designated DL-1 GW was sequenced completely. The sequence showed 74 percent AT richness displaying the characteristic observed in nematode genomes. The cloned sequenced was aligned with NCBI GenBank data showed a region of 100bp having 84 percent homology with B.malayi FAR protein gene sequence. The sequence contained ATG initiation codon and putative TATAA box indication the promoter region in the 5' end. When the DL-1 GW sequence was aligned with race-1 sequence a 100 bp region in the center and a 37bp region at the 3' end showed 100 percent homology. The resultindicate that the characterized sequences of Seteria digitata showed regions of sililary to the Brugia malayi FAR protein gene. The amino acids deduced from the homologous regions constitute 40 percent of the Brugia malayi FAR protein.
Description
Dissertation: M.Sc., University of Colombo: UC(MED), 2003.
Keywords
Filarioidea
Citation
YATAWARA, MDMLDK, Isolation and DNA sequence analysis of distantly related fatty acid and retionol binding protein gene of Setaria digitata, University of Colombo UC(MED), 2003: 92p.