Satellite DNA is a known component of our genome and can be found in both coding and noncoding DNA. When it is found in coding DNA, the number of repeats can result in altered proteins. Bur the effect of these repeats in noncoding DNA is nor as well understood. To determine whether repeats in the promoter region can alter gene expression and chromatin compaction, Vinces et al. (Vinces et al., 2009, Science 324: 1213-1216) searched the Sacharomyces cereuisiae 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 genome of various S. cerevisiae strains and found that 25 percent of promoters contained at least one repeat region. In addition, a single 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?

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 number of repeats in this region was modified, ranging between 0 repeats to 60 repeats. It was discovered that SDT1 expression, analyzed by quantitative reverse transcriptase PCR (Q RT-PCR, see Chapter 5), 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 made based on these experimental results? Explain how Q RT-PCR can be used to analyze SDT1 gene expression.

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. The promoter from the SDT1 gene (containing 48 repeats) was attached to the URA3 open reading frame, a gene responsible for synthesis of the nucleotide uracil. Cells with this hybrid protein were then placed on media lacking uracil or media containing uracil. After growth on each media, the number of repeats in the promoter driving URA3 expression was analyzed. Those 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 made from these results? Based on the data in part (b), how many repeats were likely found in the URA3 promoters for cells grown without uracil?

d. The location of the repeat DNA in the promoters was compared ro 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, what would you conclude about repetitive DNA and chromatin packaging? What effect would decreasing the repeat regions have on histone core binding' at that DNA?