Expression of the rat brain creatine kinase gene in C6 glioma cells.
We have recently shown that while brain creatine kinase (CKB) mRNA was detectable in RNA from cultured primary rat brain neurons, CKB mRNA was about 15-fold higher in primary astrocytes and 17-fold higher in oligodendrocytes (Molloy et al., J Neurochem 59:1925-1932, 1992). To begin to understand the molecular mechanisms responsible for brain glial cells containing the highest levels of CKB mRNA in the body, we have examined the expression of rat CKB mRNA in established C6 glioma cells. RNase-protection analysis showed the endogenous CKB mRNA levels in exponentially growing C6 were high and measured 50% of that in total RNA from rat brain lysate and 60% of that in cultured primary astrocytes and oligodendrocytes. The 5' and 3' ends of CKB mRNA in C6 were mapped to the same nucleotides as CKB mRNA from rat brain, indicating that the sites of in vivo transcription initiation and termination/polyadenylation of CKB mRNA in C6 are the same as in total rat brain RNA. The level of CKB enzyme activity in C6 whole cell lysates was among the highest of the glial cell lines which we measured. All creatine kinase enzyme activity present in C6 was found in the dimeric CKB isoform (BB), which is characteristic of CKB expression in the brain. A 2.9 kb gene fragment containing the basal CKB promoter and far-upstream 5' sequences was cloned upstream of the chloramphenicol acetyltransferase (CAT) gene and transfected into C6 cells. CAT activity was readily detectable in C6 and mapping of the 5' end of the CAT mRNA showed that transcription was directed from the correct initiation site. Since we found C6 cells were difficult to transfect, conditions were established which both maximized transfection efficiency and maintained normal C6 cell morphology. These results should permit the future identification of the nuclear trans-acting factors and the cognate cis-acting regulatory elements responsible for high CKB mRNA expression in brain glial cells.
University of Delaware, School of Life and Health Sciences, Newark 19716.
C D
CD
Wilson
B
B
Parameswaran
G R
GR
Molloy
D000818:Animals; D016375:Antisense Elements (Genetics); D001483:Base Sequence; D001921:Brain / Q000201:enzymology*; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000302:isolation & purification / Q000378:metabolism; D003402:Creatine Kinase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D005910:Glioma; D007527:Isoenzymes; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D015347:RNA Probes; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D014162:Transfection; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D016375:Antisense Elements (Genetics); D007527:Isoenzymes; D015347:RNA Probes; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase; D003402:Creatine Kinase; D001616:beta-Galactosidase