CLONING AND SEQUENCING OF A CDNA-ENCODING GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE FROM TETRAHYMENA-THERMOPHILA - GROWTH-ASSOCIATED CHANGESIN ITS MESSENGER-RNA EXPRESSION

Glyceraldehyde-3-phosphate dehydrogenase is a key enzyme in the glycol ytic pathway. Since its transcript levels do not vary in most experime ntal conditions, it has been often used as a control in northern blot or reverse transcriptase-polymerase chain reaction analysis. We have c loned and sequenced a gene encoding glyceraldehyde-3-phosphate dehydro genase (Tthgapdh) from Tetrahymena thermophila cDNA library and determ ined whether the Tthgapdh mRNA is a loading control in gene expression studies of T. thermophila cell. The open reading frame encoded a prot ein of 341 amino acid residues (36.8 kDa) containing a nicotinamide ad enine dinucleotide-binding domain and a catalytic domain, which was hi ghly similar to those of other organisms. Its mRNA levels at different growth stages were examined by northern blot analysis. The fragment o f the isolated cDNA was hybridized to a 1.3-kb mRNA transcript. There was a marked increase in Tthgapdh mRNA level at the mid-exponential ph ase, followed by a gradual decrease. Therefore. much caution should be made to use Tthgapdh mRNA as an internal standard for northern blot a nalysis in Tetrahymena.