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- Dokumenttyp:
- Article
- Autor(en):
- Frey, UH; Nückel, H; Dobrev, D; Manthey, I; Sandalcioglu, IE; Eisenhardt, A; Worm, K; Hauner, H; Siffert, W
- Titel:
- Quantification of G protein G alpha s subunit splice variants in different human tissues and cells using pyrosequencing
- Abstract:
- The G protein Gas is derived from four alternatively spliced transcripts, two long variants (G alpha s(L)+CAG and G alpha s(L)-CAG), which include an extra 45-bp segment, and two short variants (G alpha s(S)+CAG and G alpha s(S)-CAG). The long and short forms differ in each case by splicing in or out of a serine residue encoded at the 3' end of the variable exon 3. The relative expression of all four variants in human tissues is poorly investigated due to experimental limitations. We therefore established a method for reliable relative mRNA quantification of these splice variants based on the Pyrosequencing technology, and determined Gas transcript ratios in various human tissues and cells. G alpha s(S)/G alpha s ratio was highest in blood mononuclear cells (0.84 +/- 0.02, n = 16) and lowest in the brain (0.51 +/- 0.14, n = 3). The different ranges resulted from differences in G alpha s(S)+CAG ratios, which ranged from a total G alpha s ratio of 0.32 +/- 0.07 (n = 12) in heart tissue to 0.57 +/- 0.03 (n = 16) in blood mononuclear cells (p < 0.0001), whereas the G alpha s(S)-CAG ratio was rather constant and ranged from 0.22 +/- 0.04 (n = 7) in retinoblastoma cells to 0.27 +/- 0.04 in lymphocytes (p = 0.19). The G alpha s(L)+CAG ratio ranged from 0.02 +/- 0.02 in heart tissue to 0.05 +/- 0.01 in retinoblastoma cells, with a varying proportion of G alpha sL-CAG, which ranged from 0.14 +/- 0.02 in blood mononuclear cells to 0.41 +/- 0.08 in heart tissue. Stimulation of immortalized B lymphoblasts with isoproterenol resulted in significant changes of splice variant ratios. Our data indicate that changes of long and short ratios of Gas in different tissues affected G alpha s(L)-CAG and G alpha s(S)+CAG rather than G alpha s(L)+CAG and G alpha s(S)-CAG. Furthermore, stimulation of cells seemed to affect splice variant ratios. These results are, therefore, suggestive of different biological functions of these variants.
- Zeitschriftentitel:
- Gene Expr
- Jahr:
- 2004
- Band / Volume:
- 12
- Heft / Issue:
- 2
- Seitenangaben Beitrag:
- 69-81
- Sprache:
- eng
- PubMed:
- http://view.ncbi.nlm.nih.gov/pubmed/15892449
- Print-ISSN:
- 1052-2166
- TUM Einrichtung:
- Else Kröner-Fresenius-Zentrum für Ernährungsmedizin - Klinik für Ernährungsmedizin
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