A protein called CBF1 was identified in yeast as acentromere-binding protein. The CBF1 protein is essential for proper chromosome segregation during celldivision in yeast. You have identified a gene from thehuman genome that encodes a protein with somesimilarity in amino acid sequence with the yeastCBF1 protein.a. How could you establish whether the protein encoded by the human gene is associated with humancentromere regions? (Assume that you can make anantibody that can bind specifically to this protein.)Why would your test not be a FISH experiment?b. Describe two methods to test if this human proteinmight actually participate in centromere function(as opposed to just being present at centromeres).For the first method, assume that you can easilyproduce mutations in any given gene in a humantissue culture cell or even in a whole mouse.For the second method, you should use twodifferent recombinant YACs. YAC-1 contains theyeast CBF1+ gene and the yeast URA3+ gene thatallows URA3− yeast to grow in the absence of uracil. YAC-2 contains the wild type human CBF1-related gene and a TRP+ gene that allowsTRP− yeast cells to grow in the absence of theamino acid tryptophan. You will also want to use5-FOA, a chemical closely related to the URA3 enzyme’s normal substrate. Yeast that make theURA3 protein cannot grow in the presence of5-FOA because the enzyme will convert 5-FOA toa lethal toxin.
A protein called CBF1 was identified in yeast as a
centromere-binding protein. The CBF1 protein is essential for proper chromosome segregation during cell
division in yeast. You have identified a gene from the
human genome that encodes a protein with some
similarity in amino acid sequence with the yeast
CBF1 protein.
a. How could you establish whether the protein encoded by the human gene is associated with human
centromere regions? (Assume that you can make an
antibody that can bind specifically to this protein.)
Why would your test not be a FISH experiment?
b. Describe two methods to test if this human protein
might actually participate in centromere function
(as opposed to just being present at centromeres).
For the first method, assume that you can easily
produce mutations in any given gene in a human
tissue culture cell or even in a whole mouse.For the second method, you should use two
different recombinant YACs. YAC-1 contains the
yeast CBF1+ gene and the yeast URA3+ gene that
allows URA3− yeast to grow in the absence of uracil. YAC-2 contains the wild type human CBF1-
related gene and a TRP+ gene that allows
TRP− yeast cells to grow in the absence of the
amino acid tryptophan. You will also want to use
5-FOA, a chemical closely related to the URA3 enzyme’s normal substrate. Yeast that make the
URA3 protein cannot grow in the presence of
5-FOA because the enzyme will convert 5-FOA to
a lethal toxin.
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