Cyclic AMP regulation of lactate dehydrogenase. Quantitation of lactate dehydrogenase M-subunit messenger RNA in isoproterenol-and N6,O2'-dibutyryl cyclic AMP-stimulated rat C6 glioma cells by hybridization analysis using a cloned cDNA probe.
We have cloned DNA complementary to mRNA coding for rat C6 glioma cell lactate dehydrogenase M-subunit. Double-stranded DNA complementary to a portion of lactate dehydrogenase mRNA was inserted into the Pst I site of plasmid pBR322 by the dC.dG tailing technique and amplified in Escherichia coli HB101. A recombinant plasmid containing lactate dehydrogenase cDNA was identified by colony hybridization to a cDNA prepared from partially purified lactate dehydrogenase mRNA and by hybridization-selected translation. The recombinant plasmid (pRLD42) contains a 680 nucleotide insert of lactate dehydrogenase mRNA. Hybridization of nick-translation pRLD42 to glioma cell poly(A)+RNA separated on agarose gel and transferred to nitrocellulose exhibited Mr = 5.9 X 10(5) for lactate dehydrogenase mRNA. Furthermore, Northern blot analysis of RNA from unstimulated and isoproterenol-stimulated glioma cells indicated a 2-fold increase of lactate dehydrogenase mRNA molecules in stimulated cells. The 2-fold increase of lactate dehydrogenase mRNA was confirmed by RNA-excess kinetic hybridization using pRLD42 DNA and poly(A)+RNA from unstimulated, isoproterenol-, and dibutyryl cAMP-stimulated glioma cells. These data demonstrate that isoproterenol and dibutyryl cAMP cause an increase of the number of lactate dehydrogenase M-subunit mRNA molecules in glioma cells which, in part, determines the extent of synthesis of the lactate dehydrogenase M-subunit.
D000818:Animals; D003994:Bucladesine / Q000494:pharmacology*; D002460:Cell Line; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism*; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant / Q000378:metabolism*; D005910:Glioma / Q000201:enzymology*; D007545:Isoproterenol / Q000494:pharmacology*; D007770:L-Lactate Dehydrogenase / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D014176:Protein Biosynthesis / Q000187:drug effects*; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification; D051381:Rats; D014158:Transcription, Genetic / Q000187:drug effects*
D004274:DNA, Recombinant; D012333:RNA, Messenger; D003994:Bucladesine; D000242:Cyclic AMP; D007770:L-Lactate Dehydrogenase; D004262:DNA Restriction Enzymes; D007545:Isoproterenol