| Popis: |
I have cloned the chicken BubR1 cDNA and raised antibodies to the protein. The protein is highly conserved when compared to other vertebrate BubR1s with 55% identity and 70% similarity to human. The antibody recognises a protein of approximately 150 kDa and stains the kinetochores of chicken cells during prometaphase with the signal disappearing as they become attached to microtubules, a localisation typical of spindle checkpoint proteins. In order to further characterise the function of this protein in vertebrates, I have attempted to generate a chicken DT40 cell line conditionally null for BubR1. I have successfully targeted one allele of the BubR1 locus in DT40 cells. BubR1 (+/-) cells grow normally and have an intact checkpoint. Since I expect BubR1 to be essential, the gene must be expressed under the control of the tetracycline operator in the heterozygote cells. Attempts to target the second allele have failed thus far although the expected targeting frequency is 1/20 clones. It appears that expression of exogenous BubR1 is detrimental to these cells and they shut off expression when possible. It seems that expression levels of this essential checkpoint component are critical to avoid problems within the cell. In addition to this study in chicken cells I have cloned two other vertebrate checkpoint components from Xenopus laevis. The proteins Zw10 and Rod were initially identified in Drosophila and since no homologues have been identified in yeast these are considered as metazoan-specific components of the spindle assembly checkpoint. In order to study the checkpoint and the kinetochore, I have cloned partial Xenopus cDNA for Zw10 and Rod and raised antibodies to these proteins. Zw10 is localised to the kinetochores of metaphase Xenopus cells and along with Rod is present in a complex of approximately 11S in Xenopus egg extracts. |
