Levels of ornithine decarboxylase genomic sequences, heterogeneous nuclear RNA and mRNA in human myeloma cells resistant to alpha-difluoromethylornithine.
We previously isolated and characterized a human myeloma cell line overproducing ornithine decarboxylase (ODC) due to gene amplification [Leinonen, Alhonen-Hongisto, Laine, Jänne & Jänne (1987) Biochem. J. 242, 199-203]. We have now employed the PCR combined with reverse transcription to determine semiquantitatively ODC gene dosage and the amounts of heterogeneous nuclear (hn) RNA and of mature mRNA of the enzyme in parental and alpha-difluoromethylornithine-resistant human myeloma cells. Experiments with dilution series revealed that the ODC gene copy number and the amount of both hnRNA and mRNA were increased to the same extent (about 100-fold) in the resistant cells. Similar dot-blot analyses of ODC-specific genomic DNA and total RNA indicated that the ODC gene copy number was increased by a factor of 380 and the amount of ODC mRNA by a factor of 700. Our results indicate that the PCR combined with reverse transcription is at least as useful as blot analyses to give semiquantitative assessment of the amounts of specific DNA or RNA sequences. In addition, the use of the PCR enables the analysis of minute sample amounts in extremely short time.
Hyttinen
I M
IM
Department of Biochemistry and Biotechnology, University of Kuopio, Finland.
Halmekytö
M
M
Alhonen
L
L
Jänne
J
J
D001483:Base Sequence; D002460:Cell Line; D004351:Drug Resistance; D000518:Eflornithine / Q000494:pharmacology*; D006801:Humans; D008969:Molecular Sequence Data; D009101:Multiple Myeloma / Q000201:enzymology*; D009955:Ornithine Decarboxylase / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012332:RNA, Heterogeneous Nuclear / Q000032:analysis*; D012333:RNA, Messenger / Q000032:analysis*
D012332:RNA, Heterogeneous Nuclear; D012333:RNA, Messenger; D009955:Ornithine Decarboxylase; D000518:Eflornithine