RT-PCR and Northern blot analysis in search for a putative Paramecium beta-adrenergic receptor.
RT-PCR and Northern blot analysis were performed in order to search for a putative beta-adrenergic receptor (beta-AR) in Paramecium using several beta2-adrenergic-specific molecular probes. Under strictly defined RT-PCR conditions DNA species of expected molecular size about 360 bp were generated with the primers corresponding to the universal mammalian beta2-AR sequence tagged sites (located within the 4th and the 6th transmembrane regions of the receptor). This RT-PCR product hybridized in Southern blot analysis with the oligonucleotide probe designed to the highly conservative beta2-AR region involved in G-proteins interaction and located within the amplified region. Northern hybridization was performed on Paramecium total RNA and mRNA with human beta2-AR cDNA and two oligonucleotide probes: the first included Phe 290 involved in agonist binding (Strader et al., 1995) and the second was the backward RT-PCR primer. All these probes revealed the presence of about 2 kb mRNA which is consistent with the size of beta2-AR transcripts found in higher eukaryotes.
Płatek
A
A
Department of Cell Biology, Nencki Institute of Experimental Biology, Warszawa, Poland.
Wiejak
J
J
Wyroba
E
E
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D006801:Humans; D015345:Oligonucleotide Probes / Q000235:genetics; D010247:Paramecium / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D016053:RNA, Protozoan / Q000235:genetics / Q000302:isolation & purification; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D016053:RNA, Protozoan; D018343:Receptors, Adrenergic, beta-2