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1.(3 pts) Study Figure 2: Northern analysis of YAR1 expression, and then answer the following questions.

a. What does a comparison of lanes 1 and 4 tell you?

Lane one represents RNA isolated from a haploid strain of S. cerevisiae and lane four represents RNA isolated from a diploid strain. Comparison of the lanes shows that Yar1 RNA is expressed in both haploid and diploid cells. Judging by the band intensity, it appears that YAR1 may be expressed in slightly greater amounts in the diploid strain. [Although it does appear this way at first, comparison with the ACT1 loading control suggests that there is more total RNA in lane 4 than in lane 1, so expression of YAR1 appears to be similar in haploids and diploids.]

b. What do lanes 2 and 3 show, and why might this be important?

Lane 2 shows that YAR1 RNA is present in yeast cells arrested in G1 phase, and lane 3 shows that YAR1 RNA is present in cells arrested in S phase. [The level of YAR1 RNA is not significantly different in either of these cell cycle phases than it is in a mixed culture of cells, shown in lane 1.] Evidence that YAR1 RNA is present [at comparable levels] during both of these cell cycle phases suggests that the Yar1 protein product does not serve a function that is specific to either phase.

[This was an important issue to address because regions of the YAR1 sequence--specifically the ankyrin repeat regions and the PEST sequences--suggest that the Yar1 protein may be involved in regulation of the cell cycle. One way in which this could be accomplished is regulation of YAR1 transcription or mRNA stability at particular stages of the cell cycle. Figure 2 demonstrates that this type of regulation of YAR1 expression is not occuring, at least not in either G1 or S.]

[NOTE that this does not demonstrate that Yar1 protein is not involved in cell cycle regulation! There are many ways in which cell cycle regulatory proteins can function, and many ways in which their function can be regulated. Altering the levels of mRNA during specific cell cycle phases in only one mechanism.]

c. How is lane 5 different from the other lanes? What does this lane show?

Lane five contains RNA that was isolated on the basis of having a polyA tail, while the other lanes were run with total RNA isolated from a cell, polyA tail or not. This lane shows that YAR1 mRNA is present in the cells in a processed form with a polyA tail. This suggests that Yar1 protein is being expressed in these cells.

2. (3 pts) The GAL1 promoter is tightly regulated. Genes under the control of this promoter are expressed at very low levels when cells are grown in glucose, and strongly induced in the presence of galactose.

a. Why was the YAR1 gene cloned backward after the GAL1 promoter as well as forward? What would be the effect of expressing an "antisense" mRNA at high levels? (You may want to consult your textbook to answer this question.)

The purpose of encoding antisense mRNA was to create an RNA complementary to the YAR1 mRNA. These complementary RNA's hybridize. This prevents the Yar1 protein product from forming and thus from functioning. If the Yar1 protein function cannot be carried out, and the function not performed as a result of its absence could be characterized, it could (and did) reveal more about Yar1p's role in the cell.

b. What was the effect of overexpression of YAR1? Why might this be important?

Overexpression of YAR1 did not inhibit the cell cycle. If YAR1 encodes a cell cycle inhibitor [i.e. a protein with CDK-inhibitor activity analogous to the human ankyrin repeat proteins p15 and p16] then overexpression of YAR1 can be expected to inhibit the cell cycle. Since this did not occur, it suggests that Yar1 protein is not a cell cycle inhibitor.

c. What was the effect of overexpression of antisense YAR1 mRNA? What does this finding suggest?

The overexpression of antisense YAR1 mRNA caused a dramatic reduction in cellular growth. This strongly suggests that Yar1 protein is involved in cellular processes involved in cell division and/or growth.

[NOTE that this does not suggest that Yar1 protein is involved in the cell cycle per se. It suggests that Yar1 protein is necessary for a normal rate of progression through the cell cycle, but this could be due to any function related to growth or division.]

3. (2 pts) Explain why the findings discussed in section d, page 37, had at least two possible interpretations: slow germination or slow mitotic growth rate. How were these two possible interpretations tested? What was the result? (Germination of a spore refers to its initial mitotic division after exposure to a rich growth medium.)

The small colony phenotype was said to be due either to a defect in germination or to a slower growth rate of cells after germination. This means that either the spores took a very long time to achieve their first mitotic division once introduced to a growth medium, or that once germinated the cells grew slower than normal [or both]. It was determined that with or without a functional YAR1 gene spores germinated at around the same time. It was found, however, that strains with the wildtype YAR1 genotype had a doubling time of only 2.8 hours [at 23 deg C] whereas the strains without YAR1 doubled in 3.9 hours, indicating that it was slow mitotic growth and not a germination defect that was responsible for the small colony phenotype.

4. (2 pts) Look up the regulation of trasncription factor NFkappaB in your textbook. Briefly explain the role of IkappaB in regulation of NFkappaB, and how IkappaB is in turn regulated. What is the connection between this system and YAR1?

NFkappaB is a transcription factor that is regulated by the protein IkappaB. IkappaB regulates the transcription factor by binding to sites on NFkappaB that are necessary for nuclear localization. This binding is mediated by the ankyrin repeats on IkappaB. With its nuclear localization sites blocked, NFkappaB is retained in the cytoplasm, where it cannot activate transcription. However, in response to extracellular signals, a protein kinase phosphorylates N-terminal amino acids on IkappaB, which targets it for degradation in the proteosome. This degradation appears to also depend on the C-terminal PEST sequences of IkappB. Once IkappB is degraded, NFkappaB can enter the nucleus, bind to DNA, and activate transcription.

The connection between this system and YAR1 is that YAR1 has sequences in common with IkappaB that are important to IkappaB's function: ankyrin repeats (which mediate the interaction between IkappaB and NFkappaB) and C-terminal PEST sequences (which play a role in IkappaB's regulated degradation.)

To answer this question, you needed to integrate the information provided in your textbook with the information in the paper's introduction and first section. (The fact that I asked this question last does NOT mean that it relates to the paper's last figure--an assumption that got some of you in trouble on this one.)

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