Cloning and sequencing of a cDNA encoding glyceraldehyde-3-phosphate dehydrogenase from Tetrahymena thermophila: growth-associated changes in its mRNA expression.
Glyceraldehyde-3-phosphate dehydrogenase is a key enzyme in the glycolytic pathway. Since its transcript levels do not vary in most experimental conditions, it has been often used as a control in northern blot or reverse transcriptase-polymerase chain reaction analysis. We have cloned and sequenced a gene encoding glyceraldehyde-3-phosphate dehydrogenase (Tthgapdh) from Tetrahymena thermophila cDNA library and determined whether the Tthgapdh mRNA is a loading control in gene expression studies of T. thermophila cell. The open reading frame encoded a protein of 341 amino acid residues (36.8 kDa) containing a nicotinamide adenine dinucleotide-binding domain and a catalytic domain, which was highly similar to those of other organisms. Its mRNA levels at different growth stages were examined by northern blot analysis. The fragment of 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 phase, followed by a gradual decrease. Therefore, much caution should be made to use Tthgapdh mRNA as an internal standard for northern blot analysis in Tetrahymena.
Zhao
Y
Y
Department of Biochemistry, Gifu Univérsity School of Medicine, Japan.
Nakashima
S
S
Andoh
M
M
Nozawa
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D017125:Genes, Protozoan / Q000235:genetics; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis; D016053:RNA, Protozoan / Q000032:analysis; D017422:Sequence Analysis, DNA; D016808:Tetrahymena thermophila / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development
D018076:DNA, Complementary; D016054:DNA, Protozoan; D012333:RNA, Messenger; D016053:RNA, Protozoan