Identification of two differentially expressed mitochondrial genes in a methotrexate-resistant Chinese hamster cell strain derived from v79 cells using RNA fingerprinting by arbitrary primed polymerase chain reaction.
To identify genes that are differentially expressed in a methotrexate (MTX)-resistant cell strain designated as M5 that exhibits resistance to gamma radiation and a number of chemotherapeutic drugs compared to the parental Chinese hamster V79 cells, we used RNA fingerprinting by arbitrary primed polymerase chain reaction (RAP-PCR). By comparative analysis, we identified six differentially expressed transcripts that were cloned and sequenced. Two of these partial cDNA clones showed high homology to the mitochondrial genes NADH dehydrogenase subunit 1 and subunit 4. The steady-state mRNA level of both the NADH dehydrogenase subunits was about twofold higher in the M5 cell strain compared to V79 cells. Moreover, the expression of both the subunits decreased in gamma-irradiated Chinese hamster V79 cells. Cytochrome oxidase, another enzyme of the mitochondrial electron transport chain encoded in the mitochondrial genome, was also found to be overexpressed in M5 cells. All three genes are under the control of the same promoter. However, no amplification of DNA was observed. These data indicate that the alterations in mitochondrial gene expression may be involved in the recovery of irradiated cells, which may arise from transcriptional modulation of the mitochondria from the nucleus.
Chaudhuri
Keya
K
Human Genetics and Genomics Group, Indian Institute of Chemical Biology, 4 Raja S C Mullick Road, Kolkata-700032, India.
Banerjee
Ramanuj
R
Pandit
Bhaswati
B
Mukherjee
Aparna
A
Das
Soumita
S
Sengupta
Sharmila
S
Roychoudury
Susanta
S
Bhattacharyya
Nitai P
NP
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D015152:Blotting, Northern; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival; D003001:Cloning, Molecular; D006224:Cricetinae; D004247:DNA / Q000378:metabolism; D004272:DNA, Mitochondrial; D019008:Drug Resistance, Neoplasm; D005720:Gamma Rays; D005786:Gene Expression Regulation; D005978:Glutathione / Q000378:metabolism; D007150:Immunohistochemistry; D007700:Kinetics; D008727:Methotrexate / Q000494:pharmacology*; D009245:NADH Dehydrogenase / Q000378:metabolism; D016133:Polymerase Chain Reaction; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D014162:Transfection
D004272:DNA, Mitochondrial; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D012313:RNA; D004247:DNA; D009245:NADH Dehydrogenase; D000469:Alkaline Phosphatase; D005978:Glutathione; D008727:Methotrexate