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Amino Acids

, Volume 12, Issue 2, pp 145–155 | Cite as

Expression of ornithine carbamoyltransferase gene in rat hepatoma-derived cell lines, H4-II-E and R-Y121B

  • M. Shinohara
  • R. Konno
  • S. Nagashima
  • Y. Imai
  • A. Niwa
Full Papers
  • 41 Downloads

Summary

The rat R-Y121B cell line is a unique cell line which has ornithine carbamoyltransferase (OCT, EC 2.1.3.3) and can be continuously cultured in a serum-free medium which lacks arginine but is supplemented with ornithine. The OCT gene expression was examined in R-Y121B cells and their parental H4-II-E cells. OCT activity in R-Y121B cells was about one-tenth of that of the adult rat liver while it was not detected at all in H4-II-E cells. Southern hybridization using an OCT cDNA probe containing the entire coding region showed that the OCT gene structure was apparently not different in R-Y121B cells, H4-II-E cells, or rat liver cells. Northern hybridization using the OCT cDNA probe detected a hybridizing signal in R-Y121B cells but not in H4-II-E cells. Reverse transcription-polymerase chain reaction (RT-PCR) showed that an expected size of the OCT cDNA fragment was amplified in R-Y121B cells but not in H4-II-E cells. The amplified fragment was confirmed to be a real rat OCT cDNA by the digestion of this fragment with two restriction enzymes and by the nucleotide sequencing of the fragment. These results indicated that OCT mRNA was present in R-Y121B cells but not in H4-II-E cells. Amino acid analysis showed that arginine was not present in the culture medium but was present in the hydrolysate of R-Y121B cells. The present experiments indicate that transcription, translation, and processing of OCT proceeds normally and the resultant OCT functions in R-Y121B cells, whereas the transcription does not occur in parental H4-II-E cells even though these cells have the normal gene.

Keywords

Amino acids Ornithine carbamoyltransferase Rat hepatoma cell line Southern hybridization Northern hybridization PCR Enzyme activity 

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Copyright information

© Springer-Verlag 1997

Authors and Affiliations

  • M. Shinohara
    • 1
  • R. Konno
    • 2
  • S. Nagashima
    • 2
  • Y. Imai
    • 1
  • A. Niwa
    • 2
  1. 1.Department of Oral SurgeryDokkyo University School of MedicineTochigiJapan
  2. 2.Department of MicrobiologyDokkyo University School of MedicineMibu, TochigiJapan

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