Team:Fudan D/Background

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Background

BACKGROUND

Telomere

Overview

The telomere refers to the DNA sequence in the chromosome end. Telomeric DNA in most eukaryotes consists of short tandem repeats. In humans the telomeric repeat sequence is 5’- TTAGGG-3’, while in budding yeast telomeric DNA contains degenerate repeat sequences with the consensus 5’-(TG)0–6TGGGTGTG(G)-3’. The length of the duplex telomeric tract ranges from <30 bp in some ciliates, to 200–300 bp in budding yeast, to 5–15 kb in humans, and up to ~50 kb in mice.(Lingner 2006)

Function

Telomeres are critical at the cellular level for genome stability. The following roles of telomeres are well established:

First, telomeres can regulate the lifespan of cells. Telomere shortening of telomere that accumulate following an excessive number of cell division cycles induce cellular senescence.

Second, telomeres protect natural chromosome ends from unwanted DNA repair activities.

Third, telomeres establish a heterochromatic state at chromosome ends. Abnormal telomeric chromatin states have been linked to severe stochastic telomere sequence loss phenotypes, suggesting crucial roles of this structure during telomere replication.(Lingner 2009)

The DNA end replication problem

“All eukaryotes, and a few prokaryotes, keep their genomes in the form of linear DNA molecules. This structure requires special mechanisms to fully replicate DNA ends due to the following reasons: First, DNA replication is semiconservative; DNA polymerases use a parental template strand to synthesize a complementary daughter molecule. Eukaryotic telomeres end with 3’ protrusions at both chromosomal ends. Thus, the 5’ parental strand is resected and it cannot provide a template for the synthesis of a 3’ overhang. Semiconservative replication of the 5’-end- containing strand during leading strand synthesis should lead to loss of the 3’ overhang. However, the presumed blunt end intermediate is not detected and the templating strand is probably quickly resected to regenerate the 3’ overhang. Therefore, nucleolytic processing of telomere ends after semiconservative DNA replication contributes to telomere shortening. ”

Telomere counting mechanism

The telomeres tend to maintain a constant length in a certain cell. When the telomere is shorter than that of wild type cells, it will undergo a progressive elongation during each cell division; If there exists a over-elongated telomere (longer than normal), there will be a progressive degradation of several base pairs each cell division, a mechanism called Telomere counting mechanism. The counting mechanism is due to the interplay of telomerase activity and the DNA end replication problem.

At wild-type length in yeast (300 nucleotides), the frequency of telomere elongation was only around 6 –8%, while it increased steadily to nearly 50% upon shortening of telomeres to 100 nucleotides, which does not correlate well with telomere length. Thus, the extent of telomere elongation is not regulated in a length-dependent manner.

When telomerase is present but repressed at its site of action due to an over-elongated telomere, the telomere initially shortens at a constant pace with the successive generations. As it is approaching the vicinity of the regulated length, its pace of degradation progressively decreases and tends towards zero at equilibrium. In summary, when the telomere is over-elongated and the activity of telomerase maintain the same level, the degradation rate appears to be constant, independent of length.(Marcand, Brevet et al. 1999)

Reference

[1] Ancelin K, B. M., Bauwens S, Koering CE, Brun C, Ricoul M, Pommier JP, Sabatier L, Gilson E (2002). "Targeting assay to study the cis functions of human telomeric proteins: evidence for inhibition of telomerase by TRF1 and for activation of telomere degradation by TRF2." Mol Cell Biol 22: 3474–3487.

[2] Friedland, A. E., T. K. Lu, et al. (2009). "Synthetic gene networks that count." Science Signalling 324(5931): 1199.

[3] Dana, G. V., T. Kuiken, et al. (2012). "Synthetic biology: Four steps to avoid a synthetic- biology disaster." Nature 483(7387): 29-29.

[4] Blackburn, E. H. and C. W. Greider (1985). "Identification of a specific telomere terminal transferase activity in Tetrahymena extracts." Cell 43: 405-413.

[5] Hug, N. and J. Lingner (2006). "Telomere length homeostasis." Chromosoma 115(6): 413-425.

[6] Li, B. and A. J. Lustig (1996). "A novel mechanism for telomere size control in Saccharomyces cerevisiae." Genes & development 10(11): 1310-1326.

[7] Marcand, S., V. Brevet, et al. (1999). "Progressive cis-inhibition of telomerase upon telomere elongation." The EMBO Journal 18(12): 3509-3519.

[8] Burrill, D. R. and P. A. Silver (2010). "Making cellular memories." Cell 140(1): 13.

[9] Ulaner, G. A. and L. C. Giudice (1997). "Developmental regulation of telomerase activity in human fetal tissues during gestation." Molecular human reproduction 3(9): 769-773.

[10] Tham, W. H. and V. A. Zakian (2002). "Transcriptional silencing at Saccharomyces telomeres: implications for other organisms." Oncogene 21(4): 512.

APPLICATIONS

Biosafety

“Picture vast expanses of land, perhaps in the desert or near the sea, where giant ponds of synthetic microalgae pump out biofuels. Imagine seeds coated with engineered Escherichia coli bacteria that induce lateral root growth to reduce soil erosion, the goal of the Project Auxin team in the iGEM competition (see https://2011.igem.org).

These may sound like amazing innovations that have no downside, but what if some of the modified organisms persist and spread, despite efforts to control them? Could they disrupt the normal functions of ecosystems by transferring their altered DNA to other microbes? Might they, for instance, increase competition for resources, or disrupt crucial ecological functions?” (Genya V. Dana 2012)

While synthetic biology provides us with a promising future with endless possibilities, the biosafety remains The Sword of Damocles for all of us. These questions have been raised before and are always considered today but with no certain solution ahead. Unlike transgenic crops, synthetic microbes will be altered in more sophisticated and fundamental ways making them potentially more difficult to regulate, manage and monitor.

There are four areas of risk raised by Genya V. Dana et al.(Genya V. Dana 2012):

 First, differences in the physiology of natural and synthetic organisms will affect how they interact with the surround ing environment.

 Second, escaped  microorganisms have the potential to survive in receiving environments (for years if in a dormant state) and to compete successfully with non-modified counterparts.

 Third, synthetic organisms might evolve and adapt quickly, perhaps filling new ecological niches.

 Fourth, about gene transfer. Microorganisms are known for their ability to exchange genetic material with other organisms or to take up free DNA from the environment.

Cell memory

Biological memory can be defined as a sustained cellular response to a transient stimulus. There are several ways to accomplish this task. One way is through transcriptional states, which involve populations of molecules regulating gene expression. If the transcriptional response is bi-stable, a chemical state becomes defined as on or off and, given certain parameters, this state can be inherited through DNA replication and cell division. Another way is to utilize the DNA-based memory unit, which employs recombinase to modify DNA sequence in a site- specific manner.(Ari E. Friedland 2009; Silver 2009)

Several kinds of counters have been constructed nowadays; we will introduce two kinds of them. One is termed the riboregulated transcriptional cascade (RTC) counter, which is based on a transcriptional cascade with additional translational regulation. The other was built by chaining together modular DNA-based counting units, using recombinases, such as Cre and flpe, which can invert DNA between two oppositely oriented cognate recognition sites. Both the constructs were built to count up to three events, and said that they both should be extensible with the use of other unique polymerases or recombinases, of which many are known. (Ari E. Friedland 2009)

But in mathematic model, when the number of linear memory unit becomes larger, the noise will be severer and impede the efficiency of memorization.