Transcriptional regulation of rat calbindin expression during development determined by bacterially expressed protein.
Calbindin-D9k expression in intestinal mucosal cells reveals a specific pattern during development in rats. It shows a low basal level in suckling and adult rats, but after weaning at 21 d of age, increases to three times that of the basal level for several days only, around 24 d. We attempted to clarify whether the regulation of developmental change was at the transcriptional or post-transcriptional level. The calbindin-D9k protein and mRNA concentrations during pre- and postweaned development were determined by Western blot and Northern blot analysis, respectively, and compared with calcium binding activity by 45Ca. For Western blot analysis, a corresponding antibody was raised in rabbit using a bacterially expressed fusion protein, glutathione S-transferase (GST, EC 2.5.1.18), and calbindin-D9k. Calbindin-D9k cDNA was linked to a GST gene within a molecule of vector plasmid and a fusion protein was expressed in Escherichia coli. There were significant (p < 0.001) correlations between calbindin-D9k protein, mRNA concentrations and calcium-binding activity: r = 0.90 for protein vs. mRNA, r = 0.93 for protein vs. binding activity and r = 0.95 for mRNA vs. binding activity. These results indicate that calbindin-D9k expression during postnatal development is regulated at the transcriptional level.
Fukushima
A
A
Department of Biochemistry, Saitama Medical School, Moroyama, Japan.
Motohashi
Y
Y
Sakuma
K
K
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D064026:Calbindins; D002132:Calcium Radioisotopes / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005260:Female; D018507:Gene Expression Regulation, Developmental; D005982:Glutathione Transferase / Q000235:genetics; D008297:Male; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins; D064030:S100 Calcium Binding Protein G / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
D064026:Calbindins; D002132:Calcium Radioisotopes; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D064030:S100 Calcium Binding Protein G; C576508:S100g protein, rat; D005982:Glutathione Transferase