Use of the polymerase chain reaction for the detection of alternatively spliced mRNAs of plasma membrane calcium pump.
Polymerase chain reaction [PCR, reverse transcriptase-PCR (RT-PCR)] has been used to amplify the mRNA subspecies of the plasma membrane calcium pump isoform 1 (PMCA1) in total RNA extracted from hamster tissues. Two primers were synthesized that encompass the site at which a 154-bp exon is included totally (PMCA1a), partially (PMCA1c and d), or completely excluded (PMCA1b) in the carboxy-terminal regulatory region. PCR amplification revealed two bands (PMCA1b and 1c) that are more abundant in various tissues, while Southern hybridization of the samples after PCR amplification has detected two additional mRNA variants corresponding to PMCA1a and 1d. The distribution of these mRNA variants are tissue specific and correlate well with the pump protein distribution patterns on immunoblot. Since these multiple bands on the immunoblot are not derived from proteolysis, it is suggested that they represent the PMCA1 isozymes encoded by these alternatively spliced mRNAs. To our knowledge, this is the first report to show all four alternatively spliced mRNAs that are simultaneously detected in one single RNA sample using PCR technique. Since these isozymes are different in their regulatory domain, their tissue-specific expression may be physiologically important.
Sarkar
F H
FH
Department of Pathology, Wayne State University School of Medicine, Detroit, MI 48201.
Ball
D E
DE
Tsang
W
W
Li
Y W
YW
Kuo
T H
TH
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D000252:Calcium-Transporting ATPases / Q000235:genetics*; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D004277:DNA, Single-Stranded; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics*
D004277:DNA, Single-Stranded; D012333:RNA, Messenger; D000252:Calcium-Transporting ATPases