The cloning and molecular analysis of pawn-B in Paramecium tetraurelia.
Pawn mutants of Paramecium tetraurelia lack a depolarization-activated Ca(2+) current and do not swim backward. Using the method of microinjection and sorting a genomic library, we have cloned a DNA fragment that complements pawn-B (pwB/pwB). The minimal complementing fragment is a 798-bp open reading frame (ORF) that restores the Ca(2+) current and the backward swimming when expressed. This ORF contains a 29-bp intron and is transcribed and translated. The translated product has two putative transmembrane domains but no clear matches in current databases. Mutations in the available pwB alleles were found within this ORF. The d4-95 and d4-96 alleles are single base substitutions, while d4-662 (previously pawn-D) harbors a 44-bp insertion that matches an internal eliminated sequence (IES) found in the wild-type germline DNA except for a single C-to-T transition. Northern hybridizations and RT-PCR indicate that d4-662 transcripts are rapidly degraded or not produced. A second 155-bp IES in the wild-type germline ORF excises at two alternative sites spanning three asparagine codons. The pwB ORF appears to be separated from a 5' neighboring ORF by only 36 bp. The close proximity of the two ORFs and the location of the pwB protein as indicated by GFP-fusion constructs are discussed.
Haynes
W J
WJ
Laboratory of Molecular Biology, University of Wisconsin, Madison, Wisconsin 53706, USA.
Ling
K Y
KY
Preston
R R
RR
Saimi
Y
Y
Kung
C
C
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D015870:Gene Expression; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics*; D008845:Microinjections; D009154:Mutation; D016366:Open Reading Frames / Q000235:genetics; D016805:Paramecium tetraurelia / Q000235:genetics* / Q000378:metabolism; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014162:Transfection
D015220:Calcium Channels; D008164:Luminescent Proteins; D008565:Membrane Proteins; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; C412164:pawn-B protein, Paramecium tetraurelia; D049452:Green Fluorescent Proteins; D002118:Calcium