A cloning strategy for G-protein-coupled hormone receptors: the ovine beta 1-adrenergic receptor.
Regulation of beta 1-adrenergic receptors is unusual in developing animals. For example, glucocorticoid-and thyroid hormone-responsiveness for several genes is seen in animals treated during fetal life but beta 1-responsiveness is not seen until after birth. In order to investigate this at the transcriptional level, the ovine beta 1 receptor gene was cloned from a sheep genomic library. An approach using high-stringency screening with cDNA probes and oligonucleotides from regions of human and rat genes conserved but unique to the beta 1 receptor but not to other seven transmembrane, G-protein-coupled receptors. Over 800,000 clones were screened from which 40-50 positive clones were identified by each of the probes. There was, however, only a single clone which was recognized by each of the probes. A 5-kb insert was subcloned and shown to contain sequences which hybridized to each of the probes. Using the restriction map of the rat beta 1 receptor, a 1.0-kb Pst1 internal fragment was further subcloned for sequence identification. Confirmation of this fragment as the ovine beta 1 receptor was based on homology of the beta 1 receptor from other species and tissue distribution of mRNA. Nucleotide sequence homology was 93% with the human beta 1 receptor and 84% with rat. Amino acid sequence homology was > 75% and approached 100% in the transmembrane regions. The approach described represents a practical approach to cloning and identification of hormone receptors from the highly homologous members of the seven-transmembrane, G-protein-coupled receptors.
Padbury
J F
JF
Department of Pediatrics, Harbor-UCLA Medical Center, Torrance 90502, USA.
Tseng
Y T
YT
Waschek
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D015342:DNA Probes; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011943:Receptors, Adrenergic, beta / Q000737:chemistry / Q000235:genetics*; D017385:Sequence Homology; D012756:Sheep; D014018:Tissue Distribution
D015342:DNA Probes; D012333:RNA, Messenger; D011943:Receptors, Adrenergic, beta; D019204:GTP-Binding Proteins