The third member of the Tetrahymena CCT subunit gene family, TpCCT alpha, encodes a component of the hetero-oligomeric chaperonin complex.
The sequence of a third member of the Tetrahymena pyriformis chaperonin CCT ('chaperonin containing TCP1') subunit gene family is presented. This gene, designated TpCCT alpha, is the orthologue of the mouse chaperonin gene TCP1/CCT alpha. To characterize the CCT complex in this ciliate, we have produced polyclonal antibodies against synthetic peptides based on C-terminal sequences deduced from the primary sequences of the TpCCT alpha, TpCCT gamma and TpCCT eta subunits. We have also used polyclonal antibodies produced against recombinant yeast CCT alpha and CCT beta subunits. Using these antibodies, we show that Tetrahymena cells contain a hetero-oligomeric CCT chaperonin comprising at least seven distinct subunits. Three of these were assigned to specific TpCCT genes, whereas a fourth was recognized by the polyclonal antibody against yeast CCT beta, suggesting that this gene is also present in the ciliate. The CCT complex also contains other unidentified proteins that were recognized by the polyclonal antibody UM-1, raised against the putative ATP binding domain of the chaperonin proteins. TpCCT alpha gene expression was shown in exponentially growing cells and cells regenerating their cilia for different periods to have a similar pattern to the previously identified genes TpCCT gamma and TpCCT eta, and also to tubulin genes.
Soares
H
H
Laboratório de Genética Molecular, Instituto Gulbenkian de Ciência, Oeiras, Portugal.
Cyrne
L
L
Casalou
C
C
Ehmann
B
B
Rodrigues-Pousada
C
C
D000595:Amino Acid Sequence; D000818:Animals; D000913:Antibodies, Protozoan / Q000737:chemistry; D056404:Chaperonin Containing TCP-1; D018833:Chaperonins / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D015180:Electrophoresis, Gel, Two-Dimensional; D017125:Genes, Protozoan; D051379:Mice; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D051381:Rats; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013769:Tetrahymena pyriformis / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification
D000913:Antibodies, Protozoan; D015800:Protozoan Proteins; C533315:Tcp1 protein, mouse; C533316:Tcp1 protein, rat; D056404:Chaperonin Containing TCP-1; D018833:Chaperonins