Cloning and caracterization of Toxoplasma gondii histones 2A

DALMASSO, MC; ECHEVERRIA, PC; HELLMAN, U; DUBREMETZ, JF; ANGEL, SO

Corsica - France

Congreso; Eighth International Congress on Toxoplasmosis.; 2005

In eukaryotes, chromatin showed to have many associated activities, such as transcription, DNA recombination, and repair. These chromatin-associated activities have been intimately correlated with posttranslational modifications of histone proteins. Although histone proteins are highly conserved, nonallelic variants have been identified for every class of histone, raising the possibility that different subtypes have different functions. Within H2A family, up to now there are 5 variants, among them: major H2A, H2A.X, and H2A.F/Z. H2A.X is related to repair mechanism after DNA damage. H2A.Z corelates with active chromatin and trasncriptional activity as well as regulation of heterochromatin silencing spreading. Here 3 genes were retrieved from www.toxodb.org database that encode for T. gondii H2As members. Based on sequence analysis and phylogenic studies they were identified as a major H2A, a H2A.X and a H2A.Z. Recombinant variant histones were expressed in E. coli, and a rabbit anti-H2A.Z was obtained. Indirect immunofluorescence was done on formaldehyde fixed tachyzoites infected human foreskin fibroblast using the polyclonal antibody and a nuclei located fluorescence was observed. By Western blot analysis a band of 16-kDa was detected either in tachyzoite or 4-days induced bradyzoite homogenate. Since the anti-H2A.Z showed crossreactivity with rH2A.X, it was not possible to identified both of them by this method. To obtain a better resolution, histones were purified from tachyzoites and electrophoresed by TAU-PAGE, according protocols kindly provided by Dr. W.J. Sullivan (Indiana University School of Medicine, USA). Fourteen bands were sliced and analyzed by MALDI-TOF and sequencing. Four of them were recognized as H2A proteins, whereas other corresponds to H2B, H3 or H4 histones.