Characterization of the glyceraldehyde-3-phosphate dehydrogenase gene family from Kluyveromyces marxianus--polymerase chain reaction-single-strand conformation polymorphism as a tool for the study of multigenic families.
Three glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes from Kluyveromyces marxianus were identified and characterized. The coding region of two of them (GAP2 and GAP3) is very similar (99.6% homology). The other gene (GAP1) is only 86% homologous to GAP2 or GAP3 and is responsible for the expression of Gap1p. This protein is extremely homologous to the K. marxianus cell wall protein p37, presumably involved in flocculation. However, no leader sequence could be detected in this gene. The identification of the three genes was possible with the use of polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP), as it permits us to overcome the difficulties caused by the high homology amongst the genes. Expression of the GAPDH genes under different carbon sources (glucose or ethanol) was assessed either by Northern blot or reverse transcription-PCR-SSCP analysis, revealing that genes GAP1 and GAP2, but not GAP3, are transcribed. The results also indicate that the transcription of the gene encoding the cell wall protein p37 (Gap1p) is not dependent on the carbon source, in contrast with the expression of the gene GAP2, which is affected in cells growing in a glucose-depleted medium.
Fernandes
P A
PA
Centro de Citologia Experimental, Departamento de Biologia Molecular, Porto, Portugal.
Sena-Esteves
M
M
Moradas-Ferreira
P
P
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D015971:Gene Expression Regulation, Enzymologic; D005800:Genes, Fungal; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000235:genetics*; D007716:Kluyveromyces / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D005810:Multigene Family; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational
D005987:Glyceraldehyde-3-Phosphate Dehydrogenases