Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptome analysis of the shoots of Arabidopsis wild-type and ammonium-insensitive mutant plants grown under 10 mM ammonium as sole nitrogen source

ABSTRACT: Abundant ammonium nutrition impairs plant growth, i.e. ammonium toxicity, the primary cause of which remains to be determined. To obtain a clue about the toxic mechanism, we performed microarray experiments and compared the expression of genes responsive to toxic levels of ammonium between the wild-type (Col) and an ammonium-insensitive mutant (ami2) shoots growing in media containing 10 mM ammonium.

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Takushi HACHIYA

PROVIDER: E-MTAB-10796 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Ammonium is a waste product that inhibits cell growth, recombinant protein production, and protein glycosylation in mammalian cell culture. Recent studies have demonstrated that ammonium adversely affects glycosylation-related gene expression in Chinese hamster ovary (CHO) cells. However, since the CHO cell line species has not been fully sequenced, a glycosylation transcriptome analysis is not possible in this cell line. Therefore, to further understand the effects of ammonium on glycosylation-related gene expression, NS0 cells, a mouse myeloma cell line, were cultured under elevated ammonium. NS0 cells are similar to CHO cells, in that the NS0 cells are anchorage-independent and commonly used to commercially produce recombinant proteins. Additionally, DNA microarrays containing all known mouse glycosylation-related genes were available to be used to examine gene expression. NS0 cells were cultured under normal (control), elevated ammonium, elevated salt, and elevated ammonium with proline. It was observed that the control and treatments culture growth rates were not significantly different; however, the final cell densities were significantly different. The DNA microarray data was analyzed using a Welch ANOVA test with a Benjamini and Hochberg false discovery rate correction for the multiple comparisons of the glycosylation-genes. No significant difference in gene expression levels between the four conditions examined were observed. The results of this study demonstrated that NS0 cells, at the gene expression level, are insensitive to ammonium. Thus, the decreased glycosylation observed in NS0 cell cultures at elevated ammonium is likely due to changes in synthesis and degradation enzyme activity. In contrast, CHO cells have decreased glycosylation levels due to decreased sialytransferase gene expression and not increased degradation enzyme activity. Therefore, even though NS0 and CHO cells are both commonly used recombinant hosts for glycoprotein synthesis, it appears that NS0 and CHO cells had different control mechanisms respect to glycosylation-related gene expression under elevated ammonium. NS0 cells (ECACC#85110503), originally from the European Collection of Cell Culture, were donated to Clemson University by Merck, Inc. NS0 cells are a mouse myeloma cell line with lymphoblast morphology, non-secreting clone, and cholesterol auxotroph. NS0 cells cultured under four conditions were examined: Control (C), Ammonium-Stressed (A), Salt-Stressed (S), and Ammonium-Stressed with Proline added to mitigate the negative effects of ammonium (P). Triplicates of each condition were used. The cultures were be monitored during the normal batch growth phase. To identify genes sensitive to ammonium in growing cultures, the 90-h time point was selected for RNA isolation and gene expression analysis. Other culture parameters that were monitored include: Cell density, viability, and glucose.