In vitro BioID: mapping the CENP-A microenvironment with high temporal and spatial resolution

The centromere is located at the primary constriction of condensed chromosomes where it acts as a platform regulating chromosome segregation. The histone H3 variant CENP-A is the foundation for kinetochore formation. CENP-A directs the formation of a highly dynamic molecular neighborhood whose temporal characterization during mitosis remains a challenge due to limitations in available techniques. BioID is a method that exploits a “promiscuous” biotin ligase (BirA118R or BirA*) to identify proteins within close proximity to a fusion protein of interest. As originally described, cells expressing BirA* fusions were exposed to high biotin concentrations for 24 h during which the ligase transferred activated biotin (BioAmp) to other proteins within the immediate vicinity. The protein neighborhood could then be characterized by streptavidin-based purification and mass spectrometry. Here we describe a further development to this technique, allowing CENP-A interactors to be characterized within only a few minutes, in an in vitro reaction in lysed cells whose physiological progression is “frozen.” This approach, termed in vitro BioID (ivBioID), has the potential to study the molecular neighborhood of any structural protein whose interactions change either during the cell cycle or in response to other changes in cell physiology.

Centromere Repositioning in Cattle (Bos taurus) Chromosome 17

Eukaryotic organisms have developed a structure, called centromere, able to preserve the integrity of the genome during cell division. A young bull from the Marchigiana breed, with a normal external phenotype, underwent routine cytogenetic analysis to enter the reproduction center. All metaphases analyzed showed an unusual biarmed chromosome of medium size despite a diploid set of chromosomes (2n = 60,XY). FISH analysis excluded a pericentric inversion or a reciprocal translocation, but highlighted a repositioning of the centromere in BTA17. The satellite DNA was still in an acrocentric position. The telomeres were normally present. The primary constriction on the abnormal chromosome was C-band negative. Finally, the absence of a large genomic deletion in the BTA17 pericentromeric region was demonstrated by both array-CGH analysis and SNP array. To our knowledge, this is the first case of centromere repositioning reported in cattle.