[0026] A preferred category of SCHAG
amino acid sequences are prion aggregation domains from
prion proteins. The term “prion-aggregation domain” is intended to define a subset of SCHAG
amino acid sequences that can exist in at least two conformational states, only one of which is typically found in the aggregated state. In one conformational state, proteins comprising the prion-aggregation domain or fused to the prion-aggregation domain perform their normal function in a
cell, and in another conformational state, the native proteins form aggregates (prions) that phenotypically alter the
cell, perhaps by sequestering the
protein away from its normal site of subcellular activity, or by disrupting the conformation of an
active domain of the protein, or by changing its activity state, or bay acquiring a new activity upon aggregation, or perhaps merely by virtue of a detrimental effect on the
cell of the aggregate itself. A hallmark feature of prion-aggregation domains is that the phenotypic alteration that is associated with prion formation is heritable and / or transmissible: prions are passed from mother to
daughter cell or to
mating partners in organisms such as in the case of
yeast Sup35, and Ure2 prions, perpetuating the [PSI+] or [URE3] prion phenotypes, or the prions are transmitted in an infectious manner in organisms such as in the case of PrP prions in mammals, leading to transmissible spongiform encephalopathies. This defining characteristic of prions is attributable, at least in part, to the fact that the aggregated prion protein is able to promote the rearrangement of unaggregated protein into the aggregated conformation (although chaperone-type proteins or other trans-acting factors in the cell may also assist with this
conformational change). It is likewise a feature of prion-aggregation domains that over-production of proteins comprising these domains increases the frequency with which the prion conformation and
phenotype spontaneously arises in cells.
[0036] To facilitate identification of cells that have been successfully transformed / transfected with the
polynucleotide of the invention, the
polynucleotide may further include a sequence encoding a
selectable marker protein. The
selectable marker may be a completely distinct
open reading frame on the
polynucleotide, such as an
open reading frame encoding an
antibiotic resistance protein or a protein that facilitates survival in a selective
nutrient medium. The
selectable marker also may itself be part of the chimeric polypeptide of the invention. In one embodiment, a
visual marker such as a
fluorescent protein (e.g.,
green fluorescent protein) is used that is distributed in the cell in a different manner when the protein is in the prion form than when the protein is in the non-prion form. In either case, cells comprising the selectable marker can be sorted, e.g., using techniques such as
fluorescence activated
cell sorting. Thus, this marker, in addition to permitting selection of transformed or transfected cells, also permits identification of the conformational state of the chimeric polypeptide. In another embodiment, the marker has two components: 1) a function that is changed when the protein is in a prion form and 2) a visual or selectable marker for that function. An example is the
glucocorticoid receptor, GR and a
reporter gene. GR is a
transcription factor that binds to a specific
DNA sequence to activate transcription. When this
DNA sequence is fused to the coding sequence for an easily detected protein such as β-galactosidase or
luciferase GR function can be easily assayed by the induction of the β-galactosidase or
luciferase proteins.
[0045] In yet another embodiment, the invention includes a host cell transformed or transfected with at least two polynucleotides encoding chimeric polypeptides according to the invention, wherein the at least two polynucleotides comprise compatible SCHAG
amino acid sequences and distinct polypeptides of interest. Such host cells are capable of producing two chimeric polypeptides of the invention, which can be induced
in vitro or
in vivo to aggregate with each other into higher ordered aggregates. As explained in greater detail below, such aggregates can be advantageously employed in multi-step chemical reactions when the two or more polypeptides of interest each participate in a step of the reaction. Experiments using
fluorescence resonance energy transfer (FRET) have demonstrated the
efficacy of heterogeneous polypeptide aggregation into co-polymers.
[0068] In still another variation, the invention provides various living cells with two or more customized, reversible phenotypes. For example, the invention provides a
living cell that comprises: (a) a first polynucleotide comprising a
nucleotide sequence encoding a polypeptide that comprises a prion aggregation domain and
a domain having transcription or translation modulating activity, wherein the
living cell is capable of existing in a first stable phenotypic state characterized by the polypeptide existing in an unaggregated state and exerting a transcription or translation modulating activity and a second phenotypic state characterized by the polypeptide existing in an aggregated state and exerting altered transcription or translation modulating activity; and (b) an exogenous polynucleotide comprising a
nucleotide sequence that encodes a polypeptide of interest, with the proviso that the sequence encoding the polypeptide of interest includes a
regulatory sequence causing
differential expression of the polypeptide in the first phenotypic state compared to the second phenotypic state. Exemplary prion aggregation domains are described with respect to Sup35, Rnq1, and Ure2. The first polynucleotide may itself be an endogenous (native) polynucleotide of the cell, such as the native
yeast Sup35 sequence in a
yeast cell, which comprises a prion aggregation domain fused to a
translation termination factor sequence. Alternatively, the first polynucleotide may be introduced into the cell (or a parent cell) using
genetic engineering techniques. The term “exogenous polynucleotide” is meant to encompass any polynucleotide sequence that differs from a naturally occurring sequence in the cell as a result of human genetic manipulation. For example, an exogenous sequence may constitute an expression construct that has been introduced into a cell, such as a construct that contains a
promoter, a foreign polypeptide-encoding sequence, a
stop codon, and a
polyadenylation signal sequence. Alternatively, an exogenous sequence may constitute an endogenous polypeptide-encoding sequence that has been modified only by the introduction of a
promoter, an
enhancer, or other
regulatory sequence that is not naturally associated with the polypeptide-encoding sequence. Introduction of a
regulatory sequence that is influenced by the aggregation state of the polypeptide encoded by the first polynucleotide is specifically contemplated. In one preferred variation, the cell further comprises a
nucleotide sequence that encodes a polypeptide that modulates the expression level or conformational state of the polypeptide that comprises the prion aggregation domain. Such a polynucleotide facilitates manipulation of the cell to switch phenotypes. Polynucleotides encoding chaperone proteins that influence prion
protein folding represent one example of this latter category of polynucleotide. In one specific variation, the invention provides a
living cell according to claim 97, wherein the first polynucleotide comprises a nucleotide sequence encoding a polypeptide that comprises a prion aggregation domain fused in-frame to a nucleotide sequence encoding a
translation termination factor polypeptide; and wherein the regulatory sequence comprises a
stop codon that interrupts translation of the polypeptide of interest.
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