Differential display identifies genes in Chinese hamster ovary cells sensitive to elevated ammonium.
Ammonium is a toxic waste product that has been reported to negatively inhibit cell growth and recombinant glycosylation in Chinese hamster ovary (CHO) cells; however, the effect of this toxicity on intracellular gene expression has received only limited investigation. We used a differential display method to identify genes in CHO cells that were affected by ammonium stress. Eight genes whose mRNA levels significantly changed in response to elevated ammonium were isolated and identified. Five of the genes were identified as having lower expression under the ammonium stress, whereas three genes were identified as having higher expression. Sequence homology with other mammalian organisms was used to attribute function to these newly identified genes. The identified ammonium-sensitive genes were grouped into three broad functional groups: cellular processes, energy metabolism, and genetic-information processing. The three cellular process-related genes had lower expression (anaphase-promoting complex subunit 5, eukaryotic initiation factor 5A II, KIAA1091 protein). The two energy-related genes had higher expression under ammonium stress (adenosine triphosphate synthase subunit C and mitofusin 1). Both of the genetic information-processing genes (endoplasmic reticulum [ER]-resident protein ERdj5 and structure-specific recognition protein 1) had lower expression under the ammonium stress, whereas the 26S proteasome subunit adenosine triphosphatase 3 gene had higher expression. These preliminary results indicate that ammonium stress lowers expression of genes controlling cell cycle, protein folding, and quality and raises genes that control energy metabolism and degradation. Our findings demonstrate the usefulness of mRNA differential-display techniques for the detection of CHO cell genes affected by ammonium stress.
Chen
Peifeng
P
Department of Chemical and Biomolecular Engineering, Clemson University, 125 Earle Hall, Clemson, SC 29634-0909, USA.
Harcum
Sarah W
SW
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D019149:Bioreactors; D001709:Biotechnology / Q000379:methods*; D015152:Blotting, Northern; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D017931:DNA Primers / Q000737:chemistry; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology*; D012313:RNA / Q000378:metabolism
D017931:DNA Primers; D000644:Quaternary Ammonium Compounds; D012313:RNA; D000251:Adenosine Triphosphatases