Transcriptional regulation and chromosomal assignment of the mammalian calbindin-D28k gene.
To determine whether 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] regulates transcription of the rat renal calbindin-D28k gene, the rate of calbindin-D28k mRNA synthesis was measured directly in nuclei using the in vitro nuclear transcription assay. Nuclei were prepared from kidneys of vitamin D-deficient rats at various times after a single ip injection of 1,25-(OH)2D3, and transcription was allowed to proceed in vitro in the presence of [32P]UTP for 30 min at 29 C, at which time the incorporation of UTP into trichloroacetic acid-precipitable material was optimal. Incorporation of UTP was decreased by 64.6% by alpha-amanitin, which selectively inhibits polymerase II. Purified [32P]RNA was analyzed for newly synthesized calbindin-D-28k gene transcripts by hybridization to calbindin-D28k cDNA immobilized on nitrocellulose filters. Using this assay we found that the first significant increase in calbindin-D28k gene transcription occurred at 1 h, and the peak of transcriptional activity occurred at 2 h. Within 12 h of 1,25-(OH)2D3 treatment, calbindin-D28k gene transcription returned to control levels. Using Northern blot analysis, a significant increase in calbindin-D RNA was first observed 2 h after hormone administration, reaching a maximum at 12 h. Renal calbindin-D28k protein levels are significantly increased by 3 h and reach a maximum value 48 h after hormone administration. Our results suggest that the early increase in renal calbindin-D28k may be due to transcriptional regulation. The long time lag between transcription and the peak of calbindin mRNA and calbindin protein accumulation may reflect the involvement of post-transcriptional mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)
Varghese
S
S
Department of Biochemistry, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Graduate School of Biomedical Sciences, Newark 07103.
Deaven
L L
LL
Huang
Y C
YC
Gill
R K
RK
Iacopino
A M
AM
Christakos
S
S
D000818:Animals; D064092:Calbindin 1; D064026:Calbindins; D002117:Calcitriol / Q000494:pharmacology; D002874:Chromosome Mapping; D002898:Chromosomes, Human, Pair 8; D015342:DNA Probes; D005796:Genes; D006801:Humans; D007668:Kidney / Q000378:metabolism; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D064030:S100 Calcium Binding Protein G / Q000235:genetics*; D014158:Transcription, Genetic
C576498:CALB1 protein, human; C576500:Calb1 protein, rat; D064092:Calbindin 1; D064026:Calbindins; D015342:DNA Probes; D064030:S100 Calcium Binding Protein G; D002117:Calcitriol