Team:Valencia/prion

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(Experimental procedure)
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==Yeastworld==
==Yeastworld==
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A model population of yeast containing the prionic switch growing in optimal conditions (we consider especially the standard yeast growth temperature: 30ºC) would contain a pool of [psi -] (black) cells and a minimal amount of [PSI+] (white) cells. The high proportion of black yeasts would promote the increase of the planet mean temperature (as can be deduced by the ALBEDO-TEMPERATURE TESTING section of the project). When the temperature is high enough, the prionic switch will tend to become activated. Then, the [psi-]/[PSI+] cell ratio will rapidly decrease. In addition to the underlying molecular tendency, the black cells will be selected against in this situation. This is because of the increasing of their inner temperature, in comparison to the white cells, that will provoke a decrease of their growth rate. The white cells, on the other hand, have an intrinsic protection against overheating due to their higher albedo. Thus, these cells will grow faster and become more and more abundant. The eventual high proportion of white cells will lead to an increase of the planetary albedo effect and a decrease the mean temperature. The dynamics of this process can be regarded with much more detail in the MODELLING section of the project, but the conclusion is quite interesting: an equilibrium temperature of about 30ºC.
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A model population of yeast containing the prionic switch growing in optimal conditions (we consider especially the standard yeast growth temperature: 30ºC) would contain a pool of [psi -] (black) cells and a minimal amount of [PSI+] (white) cells. The high proportion of black yeasts would promote the increase of the planet mean temperature (as can be deduced by the '''ALBEDO-TEMPERATURE TESTING''' section of the project). When the temperature is high enough, the prionic switch will tend to become activated. Then, the [psi-]/[PSI+] cell ratio will rapidly decrease. In addition to the underlying molecular tendency, the black cells will be selected against in this situation. This is because of the increasing of their inner temperature, in comparison to the white cells, that will provoke a decrease of their growth rate. The white cells, on the other hand, have an intrinsic protection against overheating due to their higher albedo. Thus, these cells will grow faster and become more and more abundant. The eventual high proportion of white cells will lead to an increase of the planetary albedo effect and a decrease the mean temperature. The dynamics of this process can be regarded with much more detail in the '''MODELLING''' section of the project, but the conclusion is quite interesting: an equilibrium temperature of about 30ºC.
==Experimental procedure==
==Experimental procedure==

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Regulating Mars temperature

using a prion switch


Contents

Prions

In 1982 Stanley B. Prusiner created the term “prion” (or proteinacius infectious particle) to name the exclusively proteic infectious agent responsible of the transmissible spongiform encephalopathies (TSEs), a group of mammalian neurodegenerative disorders. According to the widely supported “protein-only” model, the prion mechanism of transmissibility arise from the ability of the prion form of the protein to promote the conformational change of the normal cellular form to the infectious prion forms (Prusiner, 1998). The infectious forms are mis-folded proteins that induce by polymerization the formation of an amyloid fold constituted by tightly packed beta sheets. These aggregates are insoluble fibrils that display resistance to proteolytic digestion and have affinity for aromatic dyes.

Fungal prions

In 1994 Reed Wickner proposed the prion nature of Ure2, a protein involved in the nitrogen metabolism of the yeast Saccharomyces cerevisae, to explain the unusual dominant and cytoplasmatic inheritance of the phenotype [URE3] first described by Cox (1965). In later years a wide array of genetic and biochemical evidence have supported that the prionic behaviour is present in other proteins of the yeast such as Sup35, Rnq1 and Swi1 and in HET-s, a protein involved in the mechanism of genetic incompatibility between strains of Podospora anserina.

These prions can produce amyloid-like fibrils similar to those associated with the mammalian prions. The molecular architecture of these amyloids have been studied using solid-state NMR spectroscopy and it has been found that the fibrils formed by Ure2p, Rnq1, and Sup35 share a common parallel and in-register β-structure (Fig.1) (Wickner et al. 2008). But these fungal prions have no sequence similarity with their mammalian counterparts. Besides they are not generally pathogenic and might have a beneficial role providing an evolutionary advantage to their hosts (). The prion domains of these fungal proteins have an exceptionally high content (~40%) of two polar and amyloidogenic amino acids, glutamine and asparagine.


Sup35p

[PSI+] is a non-Mendelian trait of Saccharomyces cerevisae that supress nonsense codons. This phenotype is due to a self-replication conformation (prion state) of a protein encoded by the gene Sup35. This protein, Sup35p, is the yeast eukariotyc release factor 3 (eRF3) and forms the translation termination complex with Sup45p (eRF1). The function of Sup45p is releasing the nascent polypeptide chain from the ribosome through GTP hydrolysis when Sup45p recognize a stop codon.

Sup35p is 685 amino acids long and has three distinct parts (Fig.2). The NH2-terminus (N) is termed the prion-forming domain (PrD) because plays a critical role in Sup35p’s changes in proteic conformation and it is responsible for its prion behaviour. This domain is 114 amino acids long and has a high content in glutamine and asparagine. The middle region (M) provides a solubilizing and/or spacing function. Finally, the COOH-terminus (C) is responsible for the translation-termination activity.

Figure 2. The prion domain of Sup35 is Q/N Rich and has the ability to propogate the corresponding prion in the absence of the rest of the molecule. Source: Wickner et al. 2008.

In [PSI+] cells, most Sup35p is insoluble and nonfunctional, causing an increase in the translational read-through of stop codons. This trait is heritable because Sup35p in the amyloid state as every prion influences new Sup35p to adopt the same conformation and passes from mother cell to daughter. In [psi-] cells, the translation-termination factor Sup35p is soluble and functional.

Prion switch

Components

The switch is formed by two different parts: the activator and the reporter. The activator part is a construction of two fragments: the NM domains of the protein Sup35, which confers to the protein the prionic activity, and the GR526 portion, which contains the DNA-binding and transcription-activation domains. The ligand-binding domain of the protein GR was eliminated, decoupling the response of the protein to the presence of glucocorticoids, and thus generating a constitutive transcription activation factor. The normal activity of this protein results in the activation of the genes preceded by the GRE (Glucocorticoid Response Element). When exposed to heat shock or other stress conditions, the NM domains start the prionic activity, eventually inhibiting the activation of transcription.

This part was amplified by using the primers indicated by Lindquist et al (?), together with the sequence recommended to use for the ligation protocol with the plasmid pSB1C3. Those primers are: forward actagtagcggccgctgcagATGTCGGATTCAAAC and reverse tctagaagcggccgcgaattcTCCTGCAGTGGCTTG (again, capital letters represent the region that pairs with the coding sequence of NMGR526).

The second part consists of the GRE followed by the reporter gene. In our experiments, we used LacZ for this purpose. The amplification of this part could not be made because of some problems found when trying to find the sequence of the GRE.

Behaviour

Sup35p is a subunit of the translation termination complex. Its prionic nature has been proposed to have some effect on the stress response, as a possible mechanism to obtain modified genetic expression products. When the prionic conformation is activated, the termination of translation is less effective and thus new longer proteins form (True and Lindquist, 2000, Nature, 407: 477-483; Tyedmers et al., 2008, PLoS Biology, 6: e294). When the sequence corresponding to the NM domains of Sup35p is fused to other gene, the protein resulting of this construction acquires the prionic behaviour (Li and Lindquist, 2000, Science, 287: 661-664). On the other side, GR (Glucocorticoid Receptor) activates the transcription of genes preceded by GRE (Glucocorticoid Receptor Element) when steroid hormones are present (ARTICLE NEEDED). However, it becomes a constitutive transcription activator when it lacks its C terminal ligand-binding domain (Schena and Yamamoto, 1988, Science, 241: 965-967). Because of the length of amino acids of the cut protein, this short version of GR is named GR526.

Li and Lindquist (2000) showed that the fused protein (NMGR526) is a functional constitutive transcription activator. In addition, when the prionic conformation is reached because of the presence of a certain stimulus, NMGR526 is no longer capable of inducing the activation of the gene preceded by GRE. Tyedmers et al. (2000) checked the conditions that trigger the prionic conformation and they found that heat shock is a significantly relevant factor. The cells in which the prionic conformation is induced, the process is promoted in an autocatalytic manner and all the protein is found in the prionic conformation. The cells resulting show the phenotype [PSI+].

It is important to note that the rate of the change of conformation is not equal to zero at optimal growth conditions, and that not all the cells become [PSI+]. This will have very important implications for the yeastworld model that we describe in the following section.

Yeastworld

A model population of yeast containing the prionic switch growing in optimal conditions (we consider especially the standard yeast growth temperature: 30ºC) would contain a pool of [psi -] (black) cells and a minimal amount of [PSI+] (white) cells. The high proportion of black yeasts would promote the increase of the planet mean temperature (as can be deduced by the ALBEDO-TEMPERATURE TESTING section of the project). When the temperature is high enough, the prionic switch will tend to become activated. Then, the [psi-]/[PSI+] cell ratio will rapidly decrease. In addition to the underlying molecular tendency, the black cells will be selected against in this situation. This is because of the increasing of their inner temperature, in comparison to the white cells, that will provoke a decrease of their growth rate. The white cells, on the other hand, have an intrinsic protection against overheating due to their higher albedo. Thus, these cells will grow faster and become more and more abundant. The eventual high proportion of white cells will lead to an increase of the planetary albedo effect and a decrease the mean temperature. The dynamics of this process can be regarded with much more detail in the MODELLING section of the project, but the conclusion is quite interesting: an equilibrium temperature of about 30ºC.

Experimental procedure

Plasmids and strains

As we mentioned in the Structure of the prionic switch section, the switch is formed by two different parts, which were handled independently. The prionic transcription activator was inserted into a multicopy (2μ) constitutive vector: PG1-NMGR526 (Fig 1: Schena and Yamamoto, Science, 241: 965-967).

The GRE+reporter portion was cloned into the XXXXXX plasmid (Fig)

The plasmids were cloned into BL21 Star E. coli cultures. The cultures were grown overnight at 37ºC and then the plasmids were isolated. Finally, yeast strain 5523 ([psi-]) was transformed using the lithium acetate protocol we describe in XXXXXX.

Results

References