The application of databases and PCR in the cloning of glycosidase genes from the protozoan Tritrichomonas foetus.
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 beta-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 beta-D-galactosidase. CSA was also applied to the gene encoding the enzyme beta-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.
Vella
M
M
University of Malta.
Slater
E
E
Abu-Safieh
L
L
Hussein
A S
AS
Greenwell
P
P
D000118:Acetylglucosaminidase / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D017124:Conserved Sequence; D016208:Databases, Factual; D006026:Glycoside Hydrolases / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D017386:Sequence Homology, Amino Acid; D016835:Tritrichomonas foetus / Q000201:enzymology / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics
D006026:Glycoside Hydrolases; D001616:beta-Galactosidase; D000118:Acetylglucosaminidase