NERVE-SPECIFIC EXPRESSION OF NONMUSCLE MYOSIN HEAVY CHAINS

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Previous work has shown that the MRNA encoding the myosin heavy chain-B (MHC-B) isoform of brain and spinal cord contains insertions of 30 nucleotides and 63 nucleotides near the 25-50 Kd and 50-20 Kd domain boundaries, respectively, in the head region. In this project, we focused on the function of these insertions. Our results are as follows. 1) We screened 6 different kinds of human cancer cell lines, using the reverse transcription-competitive polymerase chain reaction, with a set of primers outside of each insertion. Only neuroblastoma cell lines (SK- NSH and SK-NMC) showed the 25-50 Kd junction insertion in their MRNAS and no cell line showed the 50-20 Kd junction insertion. 2) PC12 cells (rat pheochromocytoma cell line), prior to stimulation with NGF, showed neither insertion. After stimulation with 50 ng/ml NGF, these cells showed an increase in the ratio of MRNA containing the 25-50 Kd junction insertion to the total NMMHC-B MRNA (1 wk- 12%, 2 wks- 38%). Morphologically, they underwent massive neurite outgrowth (1 wk) and, ultimately, a rich network of processes was formed (3 wks). 3) After cessation of NGF stimulation, the amount of the insert decreased and disappeared as did the neurites within one week. 4) I subcloned a PCR product corresponding to the head region of rat NMMHC-B and -A and sequenced. Deduced amino acid sequences corresponding to the insertion were conservative among different species (rat, human, avian) and all contained a putative sequence (-S-P-K-) which could serve as a substrate for cyclin-p34-cdc2 (cdc2) and cdc2-like kinases. 5) 50 fM of antisense oligonucleotide corresponding inserted sequence inhibit the morphological change of PC12 cells stimulated by NGF and the same concentration of sense oligonucleotide did not inhibit morphological change. However, the inserted form of MRNA level did not change significantly. We are presently making an effort to quantitate the amount of MHC. A correlation between the differentiation of PC12 cells to a neuronal phenotype and expression of the 10 amino acid insertion in the NMMHC-B isoform is postulated.