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Transcriptional Regulation and Chromosomal Assignment of the Mammalian Calbindin-D_28k Gene

Department of Biochemistry, University of Medicine and Dentistry of New Jersey-New Jersey, Medical School, Graduate School of Biomedical Sciences Newark, New Jersey 07103
Experimental Pathology Group, Los Alamos National Laboratory Los Alamos, New Mexico 87545

Address requests for reprints to: Sylvia Christakos, Ph.D., Department of Biochemistry, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, 185 South Orange Avenue, Newark, New Jersey 07103-2757.

Abstract

To determine whether 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] regulates transcription of the rat renal calbindin-D_28k gene, the rate of calbindin-D_28k 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 -amanitin, which selectively inhibits polymerase II. Purified [³²P]RNA was analyzed for newly synthesized calbindin-D-_28k gene transcripts by hybridization to calbindin-D-_28k cDNA immobilized on nitrocellulose filters. Using this assay we found that the first significant increase in calbindin-D_28k 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-D_28k gene transcription returned to control levels. Using Northern blot analysis, a significant increase in calbindin-D RNA was first observed 2h after hormone administration, reaching a maximum at 12 h. Renal calbindin-D_28k 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-D_28k 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. These findings indicate that renal calbindin-D_28k, similar to other steroidinduced proteins, is regulated by hormone at the transcriptional and posttranscriptional levels. The calbindin-D_28k cDNA was also used to assign the corresponding human gene to chromosome 8 from data obtained by hybridization of the calbindin probe to spots of individual human chromosomes that were flow sorted onto nitrocellulose filters. Results of Southern blot analysis are consistent with a single calbindin gene in the human genome.

FOOTNOTES

This work was supported in part by NIH Grants DK-38961 and NS20270 (to S.C.).

Received for publication October 10, 1988. Revision received December 12, 1988. Accepted for publication December 12, 1988.

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