Cloning of rat olfactory bulb tubulin tyrosine ligase cDNA: a dominant negative mutant and an antisense cDNA increase the proliferation rate of cells in culture.
In this paper we describe the cloning of rat olfactory bulb tubulin tyrosine ligase (TTL) cDNA, and investigate the physiological role of TTL in cultured CHO-K1 cells. Comparison of the deduced amino acid sequence of rat TTL cDNA with those of bovine and pig showed approximately 90% of identity. Transient transfection of CHO-K1 cells with a dominant negative mutant of TTL that contains the binding site to the substrate (tubulin) but not the catalytic domain, significantly decreased the endogenous TTL activity as determined in vitro. Similar results were obtained using a construction encoding for the antisense sequence of TTL. The reduction in TTL activity is not accompanied by a decrease in the tyrosination levels of microtubules, as judged by immunofluorescence analysis. Strikingly, the number of cells in the plates transfected with the mutant TTL or the antisense TTL cDNA was, after 72 h of culture, two and three times higher, respectively, than the number of cells in the control plates. These results support the hypothesis that TTL may play a role in the regulation of the cell cycle in living cells.
Mas
Carlos R
CR
Centro de Investigaciones en Química Biológica de Córdoba, CIQUIBIC (UNC-CONICET), Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina.
Arregui
Carlos O
CO
Filiberti
Adrián
A
Argaraña
Carlos E
CE
Barra
Héctor S
HS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002455:Cell Division / Q000187:drug effects / Q000235:genetics*; D003001:Cloning, Molecular; D006224:Cricetinae; D016373:DNA, Antisense / Q000494:pharmacology; D018076:DNA, Complementary; D008870:Microtubules / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009830:Olfactory Bulb / Q000201:enzymology*; D010453:Peptide Synthases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D051381:Rats
D016373:DNA, Antisense; D018076:DNA, Complementary; D010453:Peptide Synthases; C023213:tyrosyltubulin ligase