Amplification, Cloning And Characterization Of Genomic Sequences Coding For Rna Helicase Gene From Aedes aegypti
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Date
2010-07
Authors
H.Ibrahim, Ahmad
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Abstract
The mosquito Aedes aegypti is an important vector of arbovirus
pathogens, such as dengue fever and other diseases. About 2.5 billion people
live in regions where transmission of dengue virus occurs. This makes a vital
demand to control these diseases and their vectors. Studies on the genomics
of living organisms including Aedes aegypti can improve our understanding
of the disease vectors and help us to control these diseases. Extensive studies
have identified enzymes that are able to unwind complementary strands of a
duplex nucleic acid. These enzymes are known as helicases. RNA helicases
are proteins involved in several aspects of RNA metabolism, including
transcription, pre-mRNA splicing, ribosome biogenesis and cytoplasm
transport. A 1415 bp DNA fragment from Aedes aegypti that contained
sequences matching the 5' end of the mRNA coding for a putative RNA
helicase was amplified and cloned from Aedes aegypti. Sequences upstream
of the peR primer at the 3' end of our amplified fragment matches exactly
the first 346 nt of the 5' end of the Ae. aegypti DEAD box ATP- dependent
Rl'IA helicase (Ensembl accession no: AAEL004456-RA). The matched
sequence consists of 297 exon sequences and 49 untranslated sequences.
Upstream of the matching sequence is 1048 bp untranslated sequence that is
presumably the promoter of the gene. The promoter does not appear to
contain a T A TA box but at position -419 there is a sequence that matches a
TAT A box. The chromosome walking method was used to amplify sequence
3' of the initially cloned sequences. The PCR walking prhelgwO.72 DNA
fragment was cloned and the sequence confirmed the targeted selective
amplification of a RNA helicase region with 99% similarity (BLAST
analysis) to the mRNA sequence deposited in the ensembl data bank. This
new sequence was added to the sequence obtained from the initial
amplification to give a total of 2000 bp. Sequence alignment of the protein
sequence was performed with ClustalW program and showed that the amino
sequences was homologous to the RNA helicase gene of the mitochondrial
DEAD BOX 28 family [Drosophila melanogaster]. This leads to the
prediction that the encoded protein of the newly cloned gene is localized in
the mitochondria.
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Keywords
Characterization Of Genomic Sequences Coding , For Rna Helicase Gene