Document Detail

Quantification of G protein Gaalphas subunit splice variants in different human tissues and cells using pyrosequencing.
MedLine Citation:
PMID:  15892449     Owner:  NLM     Status:  MEDLINE    
The G protein Galphas is derived from four alternatively spliced transcripts, two long variants (Galphas(L)+CAG and Galphas(L)-CAG), which include an extra 45-bp segment, and two short variants (Galphas(S)+CAG and Galphas(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 Galphas transcript ratios in various human tissues and cells. Galphas(S)/Galphas 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 Galphas(S)+CAG ratios, which ranged from a total Galphas 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 Galphas(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 Galphas(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 Galphas(L)-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 Galphas in different tissues affected Galphas(L)-CAG and Gas(S)+CAG rather than Galphas(L)+CAG and Galphas(S-)CAG. Furthermore, stimulation of cells seemed to affect splice variant ratios. These results are, therefore, suggestive of different biological functions of these variants.
Ulrich H Frey; Holger Nückel; Dobromir Dobrev; Iris Manthey; I E Sandalcioglu; Andreas Eisenhardt; Karl Worm; Hans Hauner; Winfried Siffert
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Gene expression     Volume:  12     ISSN:  1052-2166     ISO Abbreviation:  Gene Expr.     Publication Date:  2005  
Date Detail:
Created Date:  2005-05-16     Completed Date:  2005-06-16     Revised Date:  2009-11-19    
Medline Journal Info:
Nlm Unique ID:  9200651     Medline TA:  Gene Expr     Country:  United States    
Other Details:
Languages:  eng     Pagination:  69-81     Citation Subset:  IM    
Department of Pharmacology, University Hospital Essen, D-45122 Essen, Germany.
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MeSH Terms
Alternative Splicing / genetics*
Base Sequence
Brain / metabolism,  pathology
Breast / metabolism,  pathology
Cells, Cultured
GTP-Binding Protein alpha Subunits, Gs / classification,  genetics*,  metabolism
Gene Expression*
Genetic Variation
Isoproterenol / pharmacology
Lymphocytes / drug effects
Molecular Sequence Data
RNA, Messenger / analysis
Retinoblastoma / metabolism,  pathology
Sequence Analysis, DNA / methods*
Tissue Distribution
Transcription, Genetic
Urinary Bladder Neoplasms / metabolism,  pathology
Reg. No./Substance:
0/RNA, Messenger; 7683-59-2/Isoproterenol; EC Protein alpha Subunits, Gs

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