Localization of the multiple calmodulin messenger RNAs in differentiated PC12 cells.
Calmodulin, a ubiquitous calcium-binding protein which is involved in many biological processes, including cell proliferation and differentiation, has been shown to be encoded by three genes from which five calmodulin messenger RNAs are transcribed. In our previous studies, using the PC12 pheochromocytoma cell line as a model system for neuronal differentiation, all five calmodulin messenger RNAs were found to be present, and treatment with both nerve growth factor and dibutyryl cyclic AMP, which induce neurite outgrowth in these cells, increased the level of calmodulin and differentially increased the levels of the various calmodulin messenger RNAs. In an attempt to uncover the nature of the differential increase in the calmodulin messenger RNAs during neuronal differentiation, we examined here the subcellular distribution of the individual calmodulin messenger RNAs in PC12 cells treated with nerve growth factor and dibutyryl cyclic AMP by in situ hybridization cytochemistry, using radiolabeled oligodeoxynucleotide probes. Using an oligodeoxynucleotide probe which detects all of the calmodulin transcripts, the calmodulin messenger RNAs were found to be distributed throughout the cell bodies of differentiated PC12 cells; significant amounts of calmodulin messenger RNAs were also found in most neurites (approximately 70% of the total number). Using specific probes for the calmodulin messenger RNAs derived from each calmodulin gene, distinct patterns of localization of the different calmodulin messenger RNAs were revealed. The messenger RNAs from calmodulin genes I and II were readily detected in all cell bodies and in about one-half of the neurites. In contrast, a weak signal for the messenger RNAs from calmodulin gene III was associated with cell bodies, while no significant signal was found in neurites. A population distribution analysis of the labeling of individual PC12 cell bodies, as determined by counting autoradiographic grains, revealed differences in the relative abundance of each group of messenger RNAs derived from each of the three calmodulin genes. The order of relative abundance of the messenger RNAs in cell bodies was found to be: calmodulin gene II messenger RNA > calmodulin gene I messenger RNAs >> calmodulin gene III messenger RNAs. An analysis of the labeling density along neurites indicated a similar density of neuritic messenger RNAs from calmodulin gene I and calmodulin gene II, whereas there was no significant signal for the messenger RNAs from calmodulin gene III.(ABSTRACT TRUNCATED AT 400 WORDS)
Zhang
S P
SP
Department of Pharmacology, Medical College of Pennsylvania, Philadelphia 19129.
Natsukari
N
N
Bai
G
G
Nichols
R A
RA
Weiss
B
B
D000818:Animals; D001345:Autoradiography; D001483:Base Sequence; D015152:Blotting, Northern; D003994:Bucladesine / Q000494:pharmacology; D002147:Calmodulin / Q000096:biosynthesis*; D002454:Cell Differentiation / Q000187:drug effects; D015870:Gene Expression / Q000187:drug effects; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000494:pharmacology; D015345:Oligonucleotide Probes; D016716:PC12 Cells; D015347:RNA Probes; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis*; D014158:Transcription, Genetic / Q000187:drug effects; D014316:Tritium; D014529:Uridine / Q000378:metabolism
D002147:Calmodulin; D009414:Nerve Growth Factors; D015345:Oligonucleotide Probes; D015347:RNA Probes; D012333:RNA, Messenger; D014316:Tritium; D003994:Bucladesine; D014529:Uridine