Isolation, characterization, and chromosomal localization of the porcine calcitonin receptor gene. Identification of two variants of the receptor generated by alternative splicing.
The gene encoding the calcitonin receptor (CTR) was isolated from a porcine kidney epithelial cell line (LLC-PK1) genomic library and found to span approximately 70 kilobases. Analysis of the gene sequence revealed that the CTR mRNA encompasses 14 exons with 12 exons encoding the protein. Two splicing acceptor sites separated by 48 nucleotides were found in intron 7. The expression of two mRNA species in LLC-PK1 cells was subsequently confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and DNA sequencing. In LLC-PK1 cells the mRNA encoding the shorter CTR (CTR-1a) is approximately 1,000 times more abundant than the longer variant (CTR-1b), as estimated by the competitive RT-PCR. The transcription initiation site of the CTR gene was mapped by primer extension, S1 nuclease, and RT-PCR analysis. The proximal promoter region of 500 base pair is GC-rich (66%) and CpG-rich (CpG/GpC ratio 0.71). Transient transfection of CTR gene promoter-luciferase chimeras in LLC-PK1 cells led to the expression of luciferase activity. The CTR gene was mapped to chromosome band 9q11-q12.
Friedrich Miescher Institut, Basel, Switzerland.
S
S
Zolnierowicz
P
P
Cron
S
S
Solinas-Toldo
R
R
Fries
H Y
HY
Lin
B A
BA
Hemmings
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D005091:Exons / Q000235:genetics; D014644:Genetic Variation; D007438:Introns / Q000235:genetics; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D018003:Receptors, Calcitonin / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D013552:Swine; D014158:Transcription, Genetic
D012333:RNA, Messenger; D018003:Receptors, Calcitonin; D011993:Recombinant Fusion Proteins; D008156:Luciferases