The application of databases and PCR in the cloning of glycosidase genes from the protozoan Tritrichomonas foetus |
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Authors: | Marion Vella Emma Slater Leen Abu-Safieh Ayman S Hussein Pamela Greenwell |
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Institution: | (1) School of Biosciences, University of Westminster, 115, New Cavendish Street, W1M 8JS LONDON, UK;(2) University of Malta, MALTA, Malta;(3) Serotec, Oxford, U.K.;(4) School of Biosciences, University of Westminster, London, U.K.;(5) Dept. Biochemistry, Imperial College, London, U.K. |
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Abstract: | Conserved sequence amplification (CSA) has been used to obtain sequence data for two glycosidase genes from the primitive
eukaryote Tritrichomonas foetus. Few genes have been cloned from this organism, and there is little information concerning protein sequence. CSA is reliant
on the use of database searches to identify short sequences of 3–9 amino acids conserved within a protein across a wide range
of species. PCR primers are then constructed based on this sequence data and the DNA is amplified and sequenced. In the case
of the β-galactosidase gene, N-terminal amino acid sequence data were used to construct a primer that replaced the upstream
primer to ensure the amplified product was related to β-d galactosidase CSA was also applied to the gene encoding the enzyme β-N-acetyl-d-glucosaminidase from T. foetus, but in this case a segment of DNA was amplified, which, if correct, should contain a third conserved motif. The products
of the CSA were sequenced, and the data obtained were compared to data in the SwissProt database. The results obtained suggest
that this approach is useful for the cloning of genes to obtain novel sequence data from organisms where little genetic information
is available. |
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Keywords: | Trichomonads glycosidases databases cloning |
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