The application of databases and PCR in the cloning of glycosidase genes from the protozoan Tritrichomonas foetus
- 55 Downloads
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.
Index EntriesTrichomonads glycosidases databases cloning
Unable to display preview. Download preview PDF.
- 1.Ncbi genbank. http://www.ncbi.nml.nih.gov/Google Scholar
- 2.Swissprot. http://expasy.ch/Google Scholar
- 3.Lindmark, D.J., Eckenrode, B.L., Halberg, L.A. and Dinbergs, I.D. (1989) Carbohydrate, energy and hydrogenosomal metabolism of Tritrichomonas foretus and Trichomonas vaginalis. Protozoology. 36, 214–216Google Scholar
- 5.Connaris, S. (1997) A study of the protozoan Tritrichomonas foetus with particular relevance to glycosidase production. PhD thesis. University of Westminster.Google Scholar
- 6.Vella, M. and Greenwell, P. (1996) Conserved sequence amplification (CSA) of the β-d-galactosidase gene from Tritrichomonas foetus. Second Electronic Glycoscience Conference. http://bellatrix.pcl.ox.ac.uk/egc/egc2/paper/.Google Scholar
- 10.Pedro’s biomolecular research tools. http://www.biophys.uni-duesseldorf.de/bionet/rt_1.htmlGoogle Scholar
- 12.Granger, B.L. and Warwood, S.J.. 1997 http://ncbi.nlm.nih.gov/cgi-bin/birx_doc?genbank+1521529.Google Scholar
- 14.Vella, M. (1998) Biochemical and molecular characterisation of an enzyme beat galactosidase from the protozoan Tritrichomonas foetus. PhD thesis. University of Westminster.Google Scholar