Your team has posed the question: Does MAD1 control the metaphase checkpoint? Th
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Question
Your team has posed the question: Does MAD1 control the metaphase checkpoint?
The hypothesis was: If MAD1 gives the signal to progress to anaphase after chromosomes have aligned, then a group of mutant MAD1 cells will more often have imperfect chromosome numbers after a series of cell divisions compared to normal cells.
You investigated this question by comparing the cell division of cells that have the normal, or “wildtype” MAD1 gene to the division of cells with the mutant MAD1 gene. In your experiments, you compared the number of “aneuploid” cells (imperfect chromosome numbers) to the number of “euploid” (correct chromosome numbers) in your experimental group of MAD1 mutant cells and your control group of MAD1 normal cells.
Results: Average Percent of Aneuploid Cells after 24 hrs
Control: Normal MAD1 cells had an average of 10 percent aneuploid cells after 24 hours.
Experimental: Mutant MAD1 cells had an average of 30 percent aneuploid cells after 24 hours
Question:
Develop a conclusion based on these results. Is the hypothesis supported or rejected? Explain your reasoning.
Percent Aneuploid cells in Sample (incorrect chromosome number) Sample 1 Sample 2 Sample 3 Sample 4 Average Standard Deviation Control: Normal MAD1 cells 7% 15% 8% 13% 10.75% 3.86 Experimental: Mutant MAD1 cells 30% 25% 37% 29% 30.25% 4.99Explanation / Answer
Mitotic Arrest Deficient (MAD) proteins forms component of mitotic spindle assembly checkpoint (SAC).
Before Anaphase can initiate and the duplicated sister chromatids can segregate into daughter cells, it is essential that all the chromosomes are attached to the spindle fibres and are in stable condition for proper segregation. The checkpoint components generate signals at the kinetochores (forming early prometaphase complex, that are displaced after the attachment and proper orientation of the microtubule), when the microtubules are not attached or stabilized at the kinetochores. Some of the checkpoint components include, MAD1 MAD2, BubR1, Bub3, and Mps1.
If kinetochores are unattached the SAC components mediates an anaphase inhibitor, so that a stable kinetochore-microtubule (K-MT) linkages. This would prevent aneuploidy and further complexities including metastasis or cancer.
In normal or wild type cells, during spindle assembly, MAD1 are activated by phosphorylation. MAD1 then associates with kinetochore, acts as receptor for MAD2. MAD2 binds to MAD1 to MAD1 (core complex), further catalysing MAD2-Cdc20 complexes formation, which act as anaphase inhibitor.
MAD1 (first described in yeast) are nuclear protein.
In normal or wild type cells, during spindle assembly, MAD1 are activated by phosphorylation. MAD1 then associates with kinetochore, acts as receptor for MAD2. MAD2 binds to MAD1 to MAD1 (core complex), further catalysing MAD2-Cdc20 complexes formation, which act as anaphase inhibitor. After proper spindle binding, promotes ubiquitination (by ubiquitin proteins), leading to degradation of anaphase inhibitor complex. This further, initiates formation of Anaphase Promoting Complex/ Cyclosome (APC/C), and anaphase is promoted. SAC also degrades cyclin and leads to APC/C degradation, if necessary
Mutant cells, with mutation in MAD1, affect the SAC activities. This would result in more anaphase defects, such as high frequencies of lagging chromosome, formation of K-MT complex, and causes increased mitotic error. This leads in increase in abnormal number of chromosomes or aneuploidy (more frequentlu nullisomic). Thus, when there is mutation in the MAD1 gene, there may be an increased percentage of aneuploidy in cells, in compared to the normal (where error may occur due to other genetic or physiological factors).
The experiment here depict the the percentage of abnormal chromosome in normal or wild type cells, compared to the mutant cells, with MAD1 mutation. It is observed that, in normal cells, the average percentage of aneuploidy is 10%, while in mutant cells, it icreases to 30%. Since this variation is caused by mutation of MAD1 gene, it is evedent that MAD1 plays an important role as checkpoint component. Thus, the theory of MAD1 as metaphase checkpoint is valid, and is supported by the increased aneuplidy percentage or deviation.
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