Field of Science

Analysis of purine gene regulation

Here is the analysis of expression of purine genes in the first microarray time course I did (data from expt. #911, done in 2002).  Because I sorted the genes alphabetically, this is all the genes with 'pur' names.  Four of these genes are not regulated by PurR (purA, purB, purR and purU), and some PurR-regulated genes may be missing.

The legend on the right shows the time each sample was collected from the culture.  In this time course, part of the culture was transferred to MIV at T=0 (green bars); the rest remained growing in sBHI for another 130 minutes (blue bars).  The first two bars (light blue) for each gene are cells in sBHI 70 minutes and 30 minutes before the transfer; the bars are the same for the two parts of the time course.

The Y-axis is the ratio in mRNA between RNA isolated at that time and a reference sample prepared by mixing the RNAs from all 9 time points.  This means that the height of the bars tells us nothing about the absolute expression level of the gene, just about whether its expression differs at different times. The genes not regulated by PurR have the same medium-high amount of RNA at all points (the high 30-minute MIV value for purU is due to an error in one spot of the array).  Genes whose expression is sometimes low and sometimes high have low ratios when their expression is low, and higher-than-average ratios when it is high.

The upper graph shows that, when cells were transferred into MIV, expression of the nine PurR-regulated genes was strongly induced.  This was known from the analysis we did in 2002 (see Redfield et al. 2005), and is also expected, because the cells have suddenly been moved from a medium with abundant purines to a medium with no purines, and the sudden lack of guanine and hypoxanthine will inactivate PurR.

The lower graph shows that, when the cells continued growing in sBHI, none of these genes were induced.  Even though the culture would have become quite dense by the last time point, the purine genes were just as repressed as they were when the culture was still very dilute.  This tells me that the medium still has lots of purines when cells stop growing (they must run out of something else), and that the moderate competence that develops at this late-sBHI stage doesn't depend on release of rec2 from PurR repression.

I didn't measure cell densities at these time points, but in similar experiments the density at T= -70 minutes would have been about 10^8, and at T=130 minutes about 7x10^9, only slightly below the stationary phase density of ~10^10.  The cells at T=130 would have just reached their peak transformation frequency of about 10^-4, lower than the ~3x10^-3 of MIV-induced cells but much higher than the 10^-8 of dilute cells in sBHI.


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