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a knockout of function would be a lethal event.
Therefore, the gene replacement should be done in
a diploid cell, which can then be sporulated to determine the phenotype of a knockout in a haploid
cell. To do a gene replacement with a nonfunctional gene, the gene should be disrupted by a selectable marker like KANR. The steps you would
take and the order in which you would take them
are as follows:
Clone KANR into the H2A gene on your plasmid
Transform diploid a/ cells (v).
Select KanR cells (iii).
Sporulate the transformed cells (vi).
b. The H2A protein may not be an essential protein
for cell viability or there may be another copy of
the H2A gene in yeast. har06584_refA_001-020 18 11/13/06 09:12 PM Page 18 Reference A Saccharomyces cerevisiae: Genetic Portrait of a Yeast To determine if there is another copy of the H2A
gene, the cloned H2A gene could be used as a
probe in a Southern hybridization using total yeast
d. The few base differences between the copies of the
H2A gene could be used to distinguish transcription of the two genes. Oligonucleotides that contain the speci...
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- Spring '08