Cell division is the most important phenomenon for reproduction and the continuity of the life. Each of the stages of the cell division (G1, G2, M, S) affects all the process and the importance of this process’s control is of vital importance.
Checkpoints regulated by proteins and different transcription factors are responsible for this process. The identification of any regulatory factor of the cell divison cycle is of great significance for achieving a deeper understand of the cell cycle, errors in the cell cycle (cancer mechanism) and complex processes, such as aging, senescence etc. Also a better understanding of this complex would garantuee an opportunity for the cure of retinablastoma . Our research group aimed the identification of the subunits of Mb304 which are involved in the cell growth/size regulation. Previous studies have shown that two of the subunits, reciprocally Mb304-a and Mb304-b of Mb304 complex are directly involved in the regulation of the checkpoint G1/S and in the editing of DNA errors. In our experiment, a change in the binding site of the subunit of this transciption factor was induced and changes on the reciprocal cell sizes were recorded. Initially the transciption factor was isolated by the method of Site Specific ChIP and it was confirmed by being visualized by Western Blotting. The separation of different subunits according to their differences in the specific masses was made by Mass Spectrometry. The subunits were confirmed by the technique of the Reverse Immunoprecipitation by usage of the specific antibodies for each of the subunits. Peptide sequences were determined by MS/MS Tandem Reverse Spectrometry. Subunits of an orthologous protein complex (in S. Pombe) were determined and sequenced for confirmation of our subunits in S. cerevisiae. Aftrards, the supression of function was realized by CRISPR-Cas9 and the identification of each subunit’s function was determined by the technique of Flow Cytometry.
2-Sbia M, Parnell EJ, Yu Y, Olsen AE, Kretschmann KL, et al. (2008) Regulation of the yeast Ace2 transcription factor during the cell cycle. J Biol Chem 283: 11135–11145