Combinatorial PCR approach to homology-based cloning: cloning and expression of mouse and human GM3-synthase.
GM3-synthase, also known as sialyltransferase I (ST-I), catalyzes the transfer of a sialic acid residue from CMP-sialic acid onto lactosylceramide to form ganglioside GM3. In order to clone this enzyme, as well as other sialyltransferases, we developed an approach that we termed combinatorial PCR. In this approach, degenerate primers were designed on the basis of conserved sequence motifs of the ST3 family of sialyltransferases (STs). The nucleotide sequence of the primers was varied to cover all amino acid variations occurring in each motif. In addition, in some primers the sequence was varied to cover possible homologous substitutions that are absent in the available motifs. A panel of cDNA from 12 mouse and 8 human tissues was used to enable cloning of tissue- and stage-specific sialyltransferases. Using this approach, the fragments of 11 new putative sialyltransferases were isolated and sequenced so far. Analysis of the expression pattern of a particular sialyltransferase across the panel of cDNA from the different tissues provided information about the tissue specificity of ST expression. We chose two new ubiquitously expressed human and mouse STs to clone full-length copies and to assay for GM3-synthase activity. One of the STs, which exhibited the highest homology to ST3 Gal III, showed activity toward lactosylceramide (LacCer) and was termed ST3 Gal V according to the suggested nomenclature [1]. The other ubiquitously expressed sialyltransferase was termed ST3Gal VI. All isolated sialyltransferases were screened for alternatively spliced forms (ASF). Such forms were found for both human ST3Gal V and ST3Gal VI in human fetal brain cDNA library. The detailed cloning strategy, functional assay, and full length cDNA and protein sequences of GM3 synthase (ST3Gal V, or ST-I) are presented.
Kapitonov
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Department of Biochemistry and Molecular Biophysics, Medical College of Virginia, Virginia Commonwealth University Richmond, 23298-0614, USA.
Bieberich
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Yu
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RK
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology; D002478:Cells, Cultured; D003001:Cloning, Molecular; D020650:Combinatorial Chemistry Techniques; D005333:Fetus; D005679:G(M3) Ganglioside / Q000378:metabolism*; D015723:Gene Library; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment; D012799:Sialyltransferases / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D005679:G(M3) Ganglioside; D012799:Sialyltransferases; C021343:haematoside synthetase