Team:Fudan D/Modeling Result

As the figure showing above, we have successfully produced two over-elongated telomeres, which is highly TG repetitive. One is 450bp and the other is 600bp (the general length is 300bp). Both of the sequences are proved to be right according to the sequencing. We are now recombining them into the left arm of Chromosome Ⅶ in Saccharomyces cerevisiae and testing the decrease of the over-elongated telomere with the reproduction through the telomere PCR and sequencing.

For the telomere-intron construction, we tested three marker genes: HIS, ADE and URA in yeast, the 30bp, 60bp and 90bp 3’ sequence of the each gene was deliberated truncated relatively. The modified genes were transformed into p425-GPD-Pro vector together with the full length genes. The transformed yeast cell was incubated and transferred for dotting assay. The results are as follows that show the same growth rate for each construction. Our hypothesis is the dozen of base pairs loss may not be decisive for the genes’ function and more experiences are needed for further analysis.

Ribozyme:The construction of the ribozyme sequence and substrate sequence with a 600bp interval random sequence has been finished. Meanwhile, we have linked them after the reporter gene EYFP with the promoter ADH1. Then we are now transforming the whole construction into the Saccharomyces cerevisiae to examine the down-regulation effect of ribozyme to its target gene.

If inserted an over-elongated telomere into the wild type cell, the telomere may undergoes a rapid deletion instead of progressive erosion, a phenomenon call Telomere Rapid Deletion (TRD).

It is determined that the TRD rate to be 1.2x10^-3 and 5.4x10^-4/cell division in wild-type haploid and diploid cells, respectively. An ~840-bp TG1_3 tract, 540 bp larger than that found in the wild-type W303 background is sufficient to yield maximal TRD frequencies.

So we analyzed the possible influence of TRD in our system. In the analysis, we employ 100 generations as the standard lifespan with 10000 repeated trials and the results are as follows

It shows the TRD will not be a problem even after 100 generations and the main limit is the cleavage efficiency of ribozyme, which may repress 60%-80% gene expression.

To model the evolution of effective telomerase activity according to telomere length, Stephane Marcand et al. hypothesized that the elongation rate decreases with increasing length according to a linear function. They predicted the time course based on this assumption is indistinguishable from the experimental data. Therefore, although it is likely that the in vivo decline of telomere elongation obeys more complex functions, a linear model appears to be consistent with the in vivo behavior of an abnormally shortened telomere approaching the equilibrium in yeast. According at their model, the telomere length that balances the telomerase elongation activity and shortening rates is about 270bp, relatively near the normal length of telomeric repeats.

In our system, the basic telomere length is 600bp, which is enough to establish the over-elongated state of telomere. So in this condition, we expect that the shortening of telomere in accord with the linear model. With the data published in formal papers, the degradation rate of an over-elongated telomere in wild cells is about 2bp/generation (from 2.1bp/gen to 2.3bp/gen).

With this assumption, our system will have a capacity of counting up to 200 generations ideally. The functions are as follows:

, where L is the telomere length and Lori stands for the original telomere we insert into chromosome, the G is the generations after transformation, k0 is a constant determines the expected outcome of the system.