Satellite DNA is a known component of many genomes and can be found in both coding and noncoding DNA. When it is found in coding DNA, changes in the number of repeats can result in altered proteins. But the effect of these repeats in noncoding DNA is not as well understood. To determine whether repeats in the promoter region can alter gene expression and chromatin compaction, M. D. Vinces and colleagues (Vinces et al., 2009, Science 324:1213–1216) searched the Saccharomyces cerevisiae genome for the presence of repetitive DNA in promoters and examined how altering the number of repeats affected gene expression and DNA packaging.
a. The group first searched for satellite DNA in the genomes of various S. cerevisiae strains and found that 25 percent of promoters contained at least one repeat region. In addition, the same promoter in different strains often contained distinct numbers of repeats in each region of satellite DNA. What is the proposed mechanism by which the number of repeats in a given region of satellite DNA can increase?
_feedback: Slippage between the daughter and template strand during replication can result in an increase in repeats.
b. To determine whether there is a correlation between the number of repeats and gene expression, transcription at the SDT1 gene was analyzed. The SDT1 promoter contains satellite DNA, and the group modified the number of repeats in this region so that it ranged between 0 and 60 repeats. They discovered that SDT1 expression, analyzed by quantitative reverse transcriptase–PCR (RT-PCR , see Chapter 6), increased when the repeat number increased from 0 to 13 repeats. SDT1 expression then progressively decreased as the repeat number increased from 13 to 60. What conclusion can be drawn based on these experimental results? Explain how quantitative RT-PCR can be used to analyze SDT1 gene expression.
_feedback: An SDT1 promoter with 13 repeats results in the highest gene expression. Less than this results in lower expression as does more than 13 repeats. Q RT-PCR involves the extraction of mRNA from strains that contain varying promoters. The mRNA is then converted into cDNA, which is used as a template for a PCR reaction using SDT1 specific primers. If there is more gene expression, there will be more mRNA, which results in faster production of DNA in the Q PCR reaction.
c. The conclusion from part (b) then led the group to determine whether a cell can adapt to its surroundings by altering the number of repeats in promoter satellite DNA. They attached the promoter from the SDT1 gene (containing 48 repeats) to the URA3 open reading frame, a gene responsible for synthesis of the nucleotide uracil. Cells with the resulting hybrid protein were then placed on media lacking uracil or media containing uracil. After the cells were allowed to grow on each medium, the number of repeats in the promoter driving URA3 expression was analyzed. Those cells that were grown on the media lacking uracil showed a decrease in the number of repeats in the promoter, while those grown with uracil still had, on average, 48 repeats. What conclusion can be drawn from these results? Based on the data in part (b), how many repeats were probably found in the URA3 promoters for cells grown without uracil?
_feedback: This experiment assumes that under selective pressure, it is possible to obtain yeast that are capable of altering the number of repeats in the promoter. This is because repeat number affects gene expression. In the absence of uracil, those cells that are capable of increasing uracil production will have a growth advantage, which is evident from this experiment. It is most likely that somewhere in the neighborhood of 13 repeats would allow the most uracil synthesis.
d. The location of the repetitive DNA in the promoters was compared with the location of nucleosomes, and it was found that the nucleosome density in a promoter was inversely correlated with the number of repeats at that location of DNA. From this observation, what would you conclude about repetitive DNA and chromatin packaging? What effect would decreasing the number of repeats have on histone core binding at that DNA location?
_feedback: One would conclude that histones do not associate with repetitive DNA in the promoter region. By decreasing the repeats, you would be more likely to see histones associated with those areas of the promoter.
