Excerpt from Anual Report of the Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute: October 1, 1987 Through September 30, 1988

Previously, many neuroblastoma and related somatic hybrid cell lines were shown to acquire voltage-sensitive ion channels and other neuronal properties when intracellular cyclic amp levels were elevated for a number of days. Cells with elevated camp acquire new proteins such as the a-subunit of voltage - sensitive calcium channels and other proteins of unknown function. A cdna library was constructed from poly A] rna prepared from ng108 - 15 neuroblastoma-glioma hybrid cells that had been treated for 5 days with 1 mm dibutyryl camp. The library was screened and 17 cdna clones were obtained that correspond to species of rna that are 3 to 40 times more abundant in cells treated with dibutyryl camp than in cells cultured without this compound. Each cloned cdna was used as a probe with Northern blots to determine the number of species of poly A+ rna responsive to dibutyryl camp and the chain length of each species of rna. The results suggest that the 17 cdna clones correspond to species of rna transcribed from 10 genes. Partial nucleotide sequences of the cdna inserts from 3 clones were obtained. Clone ng-32 corresponds to mouse mitochondrial mrna for atp synthase subunit 6, a mitochondrial gene. This protein is part of the H+ channel portion of the mitochondrial atp synthase complex. Treatment of nglos - ls cells with dibutyryl camp results in an 8 fold increase in the abundance of mrna for this protein. The nucleotide sequence of clone NG - lo cdna was identified as part of the D-loop region of mouse mitochondrial dna that contains the origin of replication for the heavy strand of dna. The 5' - termina1 nucleotide sequence of some molecules of heavy strand mitochondrial dna is known to consist of a short segment of rna that is complimentary to a short light strand mitochondrial dna sequence nearby. Hence, ng-lo cdna may correspond to an rna transcript of the light strand of mitochondrial dna that serves as a primer for the initiation of heavy strand mitochondrial dna synthesis. Treatment of nglo8-15 cells with dibutyryl camp results in a 40-fold increase in this species of rna. These results show that treatment of ngio8-15 neuroblastoma-glioma cells with dibutyryl camp results in marked increases in the abundance of rna transcripts from heavy and light strands of mitochondrial dna. Further work is needed to determine whether camp regulates mitochondrial biogenesis or the ability to synthesize atp.

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