Systems for sensitive detection of G-protein coupled receptor and orphan receptor function using reporter enzyme mutant complementation

Abstract

Methods for detecting G-protein coupled receptor (GPCR) activity; methods for assaying GPCR activity; and methods for screening for GPCR ligands, G-protein-coupled receptor kinase (GRK) activity, and compounds that interact with components of the GPCR regulatory process are described. Included are methods for expanding ICAST technologies for assaying GPCR activity with applications for ligand fishing, and agonist or antagonist screening. These methods include: engineering seronine/threonine phosphorylation sites into known or orphan GPCR open reading frames in order to increase the affinity of arrestin for the activated form of the GPCR or to increase the reside time of arrestin on the activated GPCR; engineering mutant arrestin proteins that bind to activated GPCRs in the absence of G-protein coupled receptor kinases which may be limiting; and engineering mutant super arrestin proteins that have an increased affinity for activated GPCRs with or without phosphorylation. These methods are intended to increase the robustness of the GPCR/ICAST technology in situations in which G-protein coupled receptor kinases are absent or limiting, or in which the GPCR is not efficiently down-regulated or is rapidly resensitized (thus having a labile interaction with arrestin). Included are also more specific methods for using ICAST complementary enzyme fragments to monitor GPCR homo- and hetero-dimerization with applications for drug lead discovery and ligand and function discovery for orphan GPCRs.

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods of detecting G-protein-coupled receptor (GPCR) activity, and provides methods of assaying GPCR activity, methods for screening for GPCR ligands, agonists and/or antagonists, methods for screening natural and surrogate ligands for orphan GPCRs, and methods for screening compounds that interact with components of the GPCR regulatory process.

2. Background of the Technology

The actions of many extracellular signals are mediated by the interaction of G-protein-coupled receptors (GPCRs) and guanine nucleotide-binding regulatory proteins (G-proteins). G-protein-mediated signaling systems have been identified in many divergent organisms, such as mammals and yeast. The GPCRs represent a large super family of proteins which have divergent amino acid sequences, but share common structural features, in particular, the presence of seven transmembrane helical domains. GPCRs respond to, among other extracellular signals, neurotransmitters, hormones, odorants and light. Individual GPCR types activate a particular signal transduction pathway; at least ten different signal transduction pathways are known to be activated via GPCRs. For example, the beta 2-adrenergic receptor (β2AR) is a prototype mammalian GPCR. In response to agonist binding, β2AR receptors activate a G-protein (Gs) which in turn stimulates adenylate cyclase activity and results in increased cyclic adenosine monophosphate (cAMP) production in the cell.

The signaling pathway and final cellular response that result from GPCR stimulation depends on the specific class of G-protein with which the particular receptor is coupled (Hamm, “The Many Faces of G-Protein Signaling.” J. Biol. Chem., 273:669-672 (1998)). For instance, coupling to the Gs class of G-proteins stimulates cAMP production and activation of the Protein Kinase A and C pathways, whereas coupling to the Gi class of G-proteins down regulates cAMP. Other second messenger systems such as calcium, phospholipase C, and phosphatidylinositol 3 may also be utilized. As a consequence, GPCR signaling events have predominantly been measured via quantification of these second messenger products.

The decrease of a response to a persistent stimulus is a widespread biological phenomenon. Signaling by diverse GPCRs is believed to be terminated by a uniform two-step mechanism. Activated receptor is first phosphorylated by a GPCR kinase (GRK). An arrestin protein binds to the activated and phosphorylated receptor, thus blocking G-protein interaction. This process is commonly referred to as desensitization, a general mechanism that has been demonstrated in a variety of functionally diverse GPCRs. Arrestin also plays a part in regulating GPCR internalization and resensitization, processes that are heterogenous among different GPCRs (Oakley, et al., J. Biol. Chem., 274:32248-32257 (1999)). The interaction between an arrestin and GPCR in processes of internalization and resensitization is dictated by the specific sequence motif in the carboxyl terminus of a given GPCR. Only a subset of GPCRs, which possess clusters of three serine or threonine residues at the carboxyl termini, were found to co-traffick with the arrestins into the endocytic vesicles after ligand stimulation. The number of receptor kinases and arrestins involved in desensitization of GPCRs is rather limited.

A common feature of GPCR physiology is desensitization and recycling of the receptor through the processes of receptor phosphorylation, endocytosis and dephosphorylation (Ferguson, et al., “G-protein-coupled receptor regulation: role of G-protein-coupled receptor kinases and arrestins.” Can. J. Physiol. Pharmacol., 74:1095-1110 (1996)). Ligand-occupied GPCRs can be phosphorylated by two families of serine/threonine kinases, the G-protein-coupled receptor kinases (GRKs) and the second messenger-dependent protein kinases such as protein kinase A and protein kinase C. Phosphorylation by either class of kinases serves to down-regulate the receptor by uncoupling it from its corresponding G-protein. GRK-phosphorylation also serves to down-regulate the receptor by recruitment of a class of proteins known as the arrestins that bind the cytoplasmic domain of the receptor and promote clustering of the receptor into endocytic vescicles. Once the receptor is endocytosed, it will either be degraded in lysosomes or dephosphorylated and recycled back to the plasma membrane as a fully-functional receptor.

Binding of an arrestin protein to an activated receptor has been documented as a common phenomenon of a variety of GPCRs ranging from rhodopsin to β2AR to the neurotensin receptor (Barak, et al., “A β-arrestin/Green Fluorescent Fusion Protein Biosensor for Detecting G-Protein-Coupled Receptor Activation,” J. Biol. Chem., 272:27497-500 (1997)). Consequently, monitoring arrestin interaction with a specific GPCR can be utilized as a generic tool for measuring GPCR activation. Similarly, a single G-protein and GRK also partner with a variety of receptors (Hamm, et al. (1998) and Pitcher et al., “G-Protein-Coupled Receptor Kinases,” Annu. Rev. Biochem., 67:653-92 (1998)), such that these protein/protein interactions may also be monitored to determine receptor activity.

Many therapeutic drugs in use today target GPCRs, as they regulate vital physiological responses, including vasodilation, heart rate, bronchodilation, endocrine secretion and gut peristalsis. See, e.g., Lefkowitz et al., Annu. Rev. Biochem., 52:159 (1983). Some of these drugs mimic the ligand for this receptor. Other drugs act to antagonize the receptor in cases when disease arises from spontaneous activity of the receptor.

Efforts such as the Human Genome Project are identifying new GPCRs (“orphan” receptors) whose physiological roles and ligands are unknown. It is estimated that several thousand GPCRs exist in the human genome.

Various approaches have been used to monitor intracellular activity in response to a stimulant, e.g., enzyme-linked immunosorbent assay (ELISA); Fluorescense Imaging Plate Reader assay (FLIPR™, Molecular Devices Corp., Sunnyvale, Calif.); EVOscreen™, EVOTEC™, Evotec Biosystems Gmbh, Hamburg, Germany; and techniques developed by CELLOMICS™, Cellomics, Inc., Pittsburgh, Pa.

Germino et al., “Screening for in vivo protein-protein interactions.” Proc. Natl. Acad. Sci., 90(3):933-937 (1993), discloses an in vivo approach for the isolation of proteins interacting with a protein of interest.

Phizicky et al., “Protein-protein interactions: methods for detection and analysis.” Microbiol. Rev., 59(1): 94-123 (1995), discloses a review of biochemical, molecular biological and genetic methods used to study protein-protein interactions.

Offermanns et al., “Gα 15 and Gα 16 Couple a Wide Variety of Receptors to Phospholipase C.” J. Biol. Chem., 270(25):15175-15180 (1995), discloses that Gα 15 and Gα 16 can be activated by a wide variety of G-protein-coupled receptors. The selective coupling of an activated receptor to a distinct pattern of G-proteins is regarded as an important requirement to achieve accurate signal transduction. Id.

Barak et al., “A β-arrestin/Green Fluorescent Protein Biosensor for Detecting G Protein-coupled Receptor Activation.” J. Biol. Chem., 272(44):27497-27500 (1997) and U.S. Pat. Nos. 5,891,646 and 6,110,693 disclose the use of a β-arrestin/green fluorescent fusion protein (GFP) for imaging protein translocation upon stimulation of GPCR with optical devices.

Each of the references described above has drawbacks. For example,

The prior art methodologies require over-expression of the proteins, which could cause artifact and tip the balance of cellular regulatory machineries.

The prior art visualization or imaging assays are low throughput and lack thorough quantification. Therefore, they are not suitable for high throughput pharmacological and kinetic assays.

In addition, many of the prior art assays require isolation of the GPCR rather than observation of the GPCR in a cell. There thus exists a need for improved methods for monitoring GPCR function.

SUMMARY OF THE INVENTION

The present invention provides modifications to the disclosure in U.S. application Ser. No. 09/654,499. In particular, the present invention is directed to modifications of the below aspects of the invention to further enhance assay sensitivity. The modifications include the use of genetically modified arrestins that exhibit enhanced binding to activated GPCR regardless of whether the GPCR is phosphorylated or non-phosphorylated; the use of a serine/threonine cluster strategy to facilitate screening assays for orphan receptors that do not possess this structural motif on their own; and the use of a combination of the above modifications to achieve even more enhanced detection.

A first aspect of the present invention is a method that monitors GPCR function proximally at the site of receptor activation, thus providing more information for drug discovery purposes due to fewer competing mechanisms. Activation of the GPCR is measured by a read-out for interaction of the receptor with a regulatory component such as arrestin, G-protein, GRK or other kinases, the binding of which to the receptor is dependent upon agonist occupation of the receptor. The present invention involves the detection of protein/protein interaction by complementation of mutant reporter enzymes.

Binding of arrestin to activated GPCR is a common process in the first step of desensitization that has been demonstrated for most, if not all, GPCRs studied so far. Measurement of GPCR interaction with arrestin via mutant enzyme complementation (i.e., ICAST) provides a more generic assay technology applicable for a wide variety of GPCRs and orphan receptors.

A further aspect of the present invention is a method of assessing GPCR pathway activity under test conditions by providing a test cell that expresses a GPCR, e.g., muscarinic, adrenergic, dopamine, angiotensin or endothelin, as a fusion protein to a mutant reporter enzyme and interacting a protein in the GPCR pathway, e.g., G-protein, arrestin or GRK, as a fusion protein with a complementing mutant reporter enzyme. When test cells are exposed to a known agonist to the target GPCR under test conditions, activation of the GPCR will be monitored by complementation of the reporter enzyme. Increased reporter enzyme activity reflects interaction of the GPCR with its interacting protein partner.

A further aspect of the present invention is a method of assessing GPCR pathway activity in the presence of a test arrestin, e.g., β-arrestin.

A further aspect of the present invention is a method of assessing GPCR pathway activity in the presence of a test G-protein.

A further aspect of the present invention is a method of assessing GPCR pathway activity upon exposure of the test cell to a test ligand.

A further aspect of the present invention is a method of assessing GPCR activity upon co-expression in the test cell of a second receptor. The second receptor could be the same GPCR or orphan receptor (i.e., homo-dimerization), a different GPCR or orphan receptor (i.e., hetero-dimerization) or could be a receptor of another type.

A further aspect of the present invention is a method for screening for a ligand or agonist to an orphan GPCR. The ligand or agonist could be contained in natural or synthetic libraries or mixtures or could be a physical stimulus. A test cell is provided that expresses the orphan GPCR as a fusion protein with a mutant reporter enzyme, e.g., a β-galactosidase mutant, and, for example, an arrestin or mutant form of arrestin as a fusion protein with a complementing mutant reporter enzyme, e.g., another β-galactosidase mutant. The interaction of the arrestin with the orphan GPCR upon receptor activation is measured by enzymatic activity of the complemented reporter enzyme. The test cell is exposed to a test compound, and an increase in reporter enzyme activity indicates the presence of a ligand or agonist.

A further aspect of the present invention is a method for screening a protein of interest, for example, an arrestin protein (or mutant form of the arrestin protein) for the ability to bind to a phosphorylated, or activated, GPCR. A test cell is provided that expresses a GPCR as a fusion protein with a mutant reporter enzyme, e.g., a β-galactosidase mutant, and contains arrestin (or a mutant form of arrestin) as a fusion protein with a complementing mutant reporter enzyme, e.g., another β-galactosidase mutant. The interaction of arrestin with the GPCR upon receptor activation is measured by enzymatic activity of the complemented reporter enzyme. The test cell is exposed to a known GPCR agonist and then reporter enzyme activity is detected. Increased reporter enzyme activity indicates that the β-arrestin molecule can bind to phosphorylated, or activated, GPCR in the test cell.

A further aspect of the present invention is a method to screen for an agonist to a specific GPCR. The agonist could be contained in natural or synthetic libraries or could be a physical stimulus. A test cell is provided that expresses a GPCR as a fusion protein with a mutant reporter enzyme, e.g., a β-galactosidase mutant, and, for example, an arrestin as a fusion protein with a complementing mutant reporter enzyme, e.g., another β-galactosidase mutant. The interaction of arrestin with the GPCR upon receptor activation is measured by enzymatic activity of the complemented reporter enzyme. The test cell is exposed to a test compound, and an increase in reporter enzyme activity indicates the presence of an agonist. The test cell may express a known GPCR or a variety of known GPCRs, or may express an unknown GPCR or a variety of unknown GPCRs. The GPCR may be, for example, an odorant GPCR or a βAR GPCR.

A further aspect of the present invention is a method for screening a test compound for GPCR antagonist activity. A test cell is provided that expresses a GPCR as a fusion protein with a mutant reporter enzyme, e.g., a β-galactosidase mutant, and, for example, an arrestin as a fusion protein with a complementing mutant reporter enzyme, e.g., another β-galactosidase mutant. The interaction of arrestin with the GPCR upon receptor activation is measured by enzymatic activity of the complemented reporter enzyme. The test cell is exposed to a test compound, and an increase in reporter enzyme activity indicates the presence of an agonist. The cell is exposed to a test compound and to a GPCR agonist, and reporter enzyme activity is detected. When exposure to the agonist occurs at the same time as or subsequent to exposure to the test compound, a decrease in reporter enzyme activity after exposure to the test compound indicates that the test compound has antagonist activity to the GPCR.

A further aspect of the present invention is a method of screening a sample solution for the presence of an agonist, antagonist or ligand to a GPCR. A test cell is provided that expresses GPCR as a fusion protein with a mutant reporter enzyme, e.g., a β-galactosidase mutant, and contains, for example, a β-arrestin as a fusion protein with a complementing reporter, e.g., another β-galactosidase mutant. The test cell is exposed to a sample solution, and reporter enzyme activity is assessed. Changed reporter enzyme activity after exposure to the sample solution indicates the sample solution contains an agonist, antagonist or ligand for a GPCR expressed in the cell.

A further aspect of the present invention is a method of screening a cell for the presence of a GPCR. According to this aspect, an arrestin fusion protein with a mutant reporter enzyme and a GPCR downstream signaling fusion protein with a mutant reporter enzyme are employed to detect GPCR action. A modification of this aspect of the invention can be employed to provide a method of screening a plurality of cells for those cells which contain a GPCR. According to this aspect, a plurality of cells containing a conjugate comprising a β-arrestin protein as a fusion protein with a reporter enzyme are provided; the plurality of cells are exposed to a GPCR agonist; and activity of reporter enzyme activity is detected. An increase in reporter enzymatic activity after exposure to the GPCR agonist indicates β-arrestin protein binding to a GPCR, thereby indicating that the cell contains a GPCR responsive to the GPCR agonist.

A further aspect of the invention is a method for mapping GPCR-mediated signaling pathways. For instance, the system could be utilized to monitor interaction of c-src with β-arrestin-1 upon GPCR activation. Additionally, the system could be used to monitor protein/protein interactions involved in cross-talk between GPCR signaling pathways and other pathways such as that of the receptor tyrosine kinases or Ras/Raf. According to this aspect, a test cell is provided that expresses a GPCR or other related protein with a mutant reporter enzyme, e.g., a β-galactosidase mutant, and contains a protein from another pathway as a fusion protein with a complementing mutant reporter enzyme, e.g., another β-galactosidase mutant. Increased reporter enzymatic activity indicates protein/protein interaction.

A further aspect of the invention is a method for monitoring homo- or hetero-dimerization of GPCRs upon agonist or antagonist stimulation. Increasing evidence indicates that GPCR dimerization is important for biological activity (AbdAlla, et al., “AT1-receptor heterodimers show enhanced G-protein activation and altered receptor sequestration.” Nature, 407:94-98 (2000); Bockaert, et al., “Molecular tinkering of G protein-coupled receptors: an evolutionary success.” EMBO J. 18:1723-29 (1999)). Jordan, et al., “G-protein-coupled receptor heterodimerization modulates receptor function.” Nature, 399:697-700 (1999), demonstrated that two non-functional opioid receptors, κ and δ, heterodimerize to form a functional receptor. Gordon et al., “Dopamine D2 receptor dimers and receptor blocking peptides.” Bioch. Biophys. Res. Commun. 227:200-204 (1996), showed different pharmacological properties associated with the monomeric and dimeric forms of Dopamine receptor D2. The D2 receptors exist either as monomers that are selective targets for spiperone or as dimer forms that are targets for nemonapride. Herbert, et al., “A peptide derived from a β2-adrenergic receptor transmembrane domain inhibits both receptor dimerization and activation.” J.B.C. 271:16384-92 (1996), demonstrated that the agonist stimulation was found to stabilize the dimeric state of the receptor, whereas inverse agonists favored the monomeric form. Indeed, the same study showed that a peptide corresponding to the sixth transmembrane domain of the β2-adrenergic receptor inhibited both receptor dimerization and activation. Further, Angers et al., Detection of beta-2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer, Proc. Natl. Acad. Sci. USA, 97(7):3684-3689, discloses the use of β2-adrenergic receptor fusion proteins (i.e., β2-adrenergic receptor fused to luciferase and β2-adrenergic receptor fused to an enhanced red-shifted green fluorescent protein) to study β2-adrenergic receptor dimerization.

GPCR dimerization in the context of cellular physiology and pharmacology can be monitored in accordance with the invention. For example, β-galactosidase complementation can be measured in test cells that co-express GPCR fusion proteins of β-galactosidase mutant enzymes, e.g., GPCR 1 Δα and GPCR 2 Δω (FIG. 27 ). According to this aspect, the interconversion between monomeric to dimeric forms of the GPCRs or orphan receptors can be measured by mutant reporter enzyme complementation. FIG. 27 illustrates a test cell co-expressing GPCR or an orphan receptor as a fusion protein with Δα form of β-galactosidase mutant (e.g., GPCR 1 Δα), and the same GPCR or orphan receptor as a fusion protein with Δω form of β-galactosidase mutant (e.g. GPCR 1 Δω). Formation of the GPCR homodimer is reflected by formation of an active enzyme, which can be measured by enzyme activity assays, such as the Gal-Screen™ assay. Similarly, hetero-dimerization between two distinct GPCRs, or two distinct orphan receptors, or between one known GPCR and one orphan receptor can be analyzed in test cells co-expressing two fusion proteins, e.g., GPCR 1 Δα and GPCR 2 Δω. The increased β-galactosidase activity indicates that the two receptors can form a heterodimer.

A further aspect of the invention is a method of monitoring the interconversion between the monomeric and dimeric form of GPCRs under the influence of agonist or antagonist treatment. The test receptor(s) can be between the same GPCR or orphan receptor (homodimer), or between two distinct GPCRs or orphan receptors (heterodimer). The increased β-galactosidase activity after treatment with a compound means that the compound binds to and/or stabilizes the dimeric form of the receptor. The decreased β-galactosidase activity after treatment with a compound means that the compound binds to and/or stabilizes the monomeric form of the receptor.

A further aspect of the invention is a method of screening a cell for the presence of a GPCR responsive to a GPCR agonist. A cell is provided that contains protein partners that interact downstream in the GPCR's pathway. The protein partners are expressed as fusion proteins to the mutant, complementing enzyme and are used to monitor activation of the GPCR. The cell is exposed to a GPCR agonist and then enzymatic activity of the reporter enzyme is detected. Increased reporter enzyme activity indicates that the cell contains a GPCR responsive to the agonist.

The present invention involves the use of a combination of proprietary technologies (including ICAST™, Intercistronic Complementation Analysis Screening Technology, Gal-Screen™, etc.) to monitor protein/protein interactions in GPCR signaling. As disclosed in U.S. application Ser. No. 09/654,499, the method of the invention in part involves using ICAST™, which in turn involves the use of two inactive β-galactosidase mutants, each of which is fused with one of two interacting target protein pairs, such as a GPCR and an arrestin. The formation of an active β-galactosidase complex is driven by interaction of the target proteins. In this system, β-galactosidase activity can be detected using, e.g., the Gal-Screen™ assay system, wherein direct cell lysis is combined with rapid ultrasensitive chemiluminescent detection of β-galactosidase reporter enzyme. This system uses, e.g., a Galacton-Star® chemiluminescent substrate for measurement in a luminometer as a read out of GPCR activity.

FIG. 23 is a schematic depicting the use of the complementation technology in the method of the present invention. FIG. 23 shows two inactive β-galactosidase mutants that become active when they are forced together by specific interactions between the fusion partners of an arrestin molecule and an activated GPCR or orphan receptor. This assay technology will be especially useful in high throughput screening assays for ligand fishing for orphan receptors, a process called de-orphaning. As illustrated in FIG. 28 , a β-galactosidase fusion protein of an orphan receptor (e.g., GPCR orphan Δα) is co-expressed in the test cell with a fusion protein of β-arrestin (e.g., β-ArrΔω). When the test cell is subjected to compounds, which could be natural or synthetic, the increased β-galactosidase activity means the compound is either a natural or surrogate ligand for this GPCR. The same assay system can be used to find drug leads for the new GPCRs. The increased β-galactosidase activity in the test cell after treatment indicates the agonist activity of the compound. The decreased β-galactosidase activity in the test cell indicates antagonist activity or inverse agonist activity of the compound. In addition, the method of the invention could be used to monitor GPCR-mediated signaling pathways via other downstream signaling components such as G-proteins, GRKs or the proto-oncogene c-Src.

The invention is achieved in part by using ICAST™ protein/protein interaction screening to map signaling pathways. This technology is applicable to a variety of known and unknown GPCRs with diverse functions. They include, but are not limited to, the following sub-families of GPCRs:

(a) receptors that bind to amine-like ligands-Acetylcholine muscarinic receptor (M1 to M5), alpha and beta Adrenoceptors, Dopamine receptors (D1, D2, D3 and D4), Histamine receptors (H1 and H2), Octopamine receptor and Serotonin receptors (5HT1, 5HT2, 5HT4, 5HT5, 5HT6, 5HT7);

(b) receptors that bind to a peptide ligand-Angiotensin receptor, Bombesin receptor, Bradykinin receptor, C—C chemokine receptors (CCR1 to CCR8, and CCR10), C-X-C type Chemokine receptors (CXC-R5), Cholecystokinin type A receptor, CCK type receptors, Endothelin receptor, Neurotesin receptor, FMLP-related receptors, Somatostatin receptors (type 1 to type 5) and Opioid receptors (type D, K, M, X);

(c) receptors that bind to hormone proteins-Follic stimulating hormone receptor, Thyrotrophin receptor and Lutropin-choriogonadotropic hormone receptor;

(d) receptors that bind to neurotransmitters-substance P receptor, Substance K receptor and neuropeptide Y receptor;

(e) Olfactory receptors-Olfactory type 1 to type 11, Gustatory and odorant receptors;

(f) Prostanoid receptors-Prostaglandin E2 (EP1 to EP4 subtypes), Prostacyclin and Thromboxane;

(g) receptors that bind to metabotropic substances-Metabotropic glutamate group I to group III receptors;

(h) receptors that respond to physical stimuli, such as light, or to chemical stimuli, such as taste and smell; and

(i) orphan GPCRs-the natural ligand to the receptor is undefined.

Use of the ICAST™ technology in combination with the invention provides many benefits to the GPCR screening process, including the ability to monitor protein interactions in any sub-cellular compartment-membrane, cytosol and nucleus; the ability to achieve a more physiologically relevant model without requiring protein overexpression; and the ability to achieve a functional assay for receptor binding allowing high information content.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . Cellular expression levels of β2 adrenergic receptor (β2AR) and β-arrestin-2 (βArr2) in C2 clones. Quantification of β-galactosidase (β-gal) fusion protein was performed using antibodies against β-gal and purified β-gal protein in a titration curve by a standardized ELISA assay.

FIG. 1A shows expression levels of β2AR-βgalΔα clones (in expression vector pICAST ALC).

FIG. 1B shows expression levels of 3Arr2-βgalΔω in expression vector pICAST OMC4 for clones 9-3,-7,-9,-10,-19 and -24, or in expression vector pICAST OMN4 for clones 12-4,-9,-16,-18,-22 and -24.

FIG. 2 . Receptor β2AR activation was measured by agonist-stimulated cAMP production. C2 cells expressing pICAST ALC β2AR (clone 5) or parental cells were treated with increasing concentrations of (−)isoproterenol and 0.1 mM IBMX. The quantification of cAMP level was expressed as pmol/well.

FIG. 3 . Interaction of activated receptor β2AR and arrestin can be measured by β-galactosidase complementation.

FIG. 3A shows a time course of β-galactosidase activity in response to agonist (−)isoproterenol stimulation in C2 expressing β2AR-βgalΔα (β2AR alone, in expression vector pICAST ALC), or a pool of doubly transduced C2 co-expressing β2AR-βgalΔα and βArr2-βgalΔω (in expression vectors pICAST ALC and pICAST OMC and clones isolated from the same pod (43-1, 43-2, 43-7 and 43-8)).

FIG. 3B shows a time course of β-galactosidase activity in response to agonist (−)isoproterenol stimulation in C2 cells expressing β2AR-βgalΔα alone (in expression vector pICAST ALC) and C2 clones co-expressing β2AR-βgalΔα and βArr1-βgalΔω (in expression vectors ICAST ALC and pICAST OMC).

FIG. 4 . Agonist dose response for interaction of β2AR and arrestin can be measured by β-galactosidase complementation.

FIG. 4A shows a dose response to agonists (−)isoproterenol and procaterol in C2 cells co-expressing β2AR-βgalΔα and βArr2-βgalΔω fusion constructs.

FIG. 4B shows a dose response to agonists (−)isoproterenol and procaterol in C2 cells co-expressing β2AR-βgalΔα and βArr1-βgalΔω fusion constructs.

FIG. 5 . Antagonist mediated inhibition of receptor activity can be measured by β-galactosidase complementation in cells co-expressing β2AR-βgalΔα and βArr-βgalΔω.

FIG. 5A shows specific inhibition with adrenergic antagonists ICI-118,551 and propranolol of β-galactosidase activity in C2 clones co-expressing β2AR-βgalΔα and βArr2-βgalΔω fusion constructs after incubation with agonist (−)isoproterenol.

FIG. 5B shows specific inhibition of β-galactosidase activity with adrenergic antagonists ICI-118,551 and propranolol in C2 clones co-expressing β2AR-βgalΔα and βArr1-βgalΔω fusion constructs in the presence of agonist (−)isoproterenol.

FIG. 6. C 2 cells expressing adenosine receptor A2a show cAMP induction in response to agonist (CGS-21680) treatment. C2 parental cells and C2 cells co-expressing A2aR-βgalΔα and βArr1-βgalΔω as a pool or as selected clones (47-2 and 47-13) were measured for agonist-induced cAMP response (pmol/well).

FIG. 7 . Agonist stimulated cAMP response in C2 cells co-expressing Dopamine receptor D1 (D1-βgalΔα) and β-arrestin-2 (βArr2-βgalΔω). The clone expressing βArr2-βgalΔω (Arr2 alone) was used as a negative control in the assay. Cells expressing D1-βgalΔα in addition to βArr2-βgalΔω responded agonist treatment (3-hydroxytyramine hydrochloride at 3 μM). D1(PIC2) or D1(PIC3) designate D1 in expression vector pICAST ALC2 or pICAST ALC4, respectively.

FIG. 8 . Variety of mammalian cell lines can be used to generate stable cells for monitoring GPCR and arrestin interactions.

FIG. 8A , FIG. 8 B and FIG. 8C show the examples of HEK 293, CHO and CHW cell lines co-expressing adrenergic receptor β2AR and arrestin fusion proteins of β-galactosidase mutants. The β-galactosidase activity was used to monitor agonist-induced interaction of β2AR and arrestin proteins.

FIG. 9 . Beta-gal complementation can be used to monitor β2 adrenergic receptor homo-dimerization.

FIG. 9A shows β-galactosidase activity in HEK 293 clones co-expressing β2AR-βgalΔα and β2AR-βgalΔω.

FIG. 9B shows a cAMP response to agonist (−)isoproterenol in HEK 293 clones co-expressing β2AR-βgalΔα and β2AR-βgalΔω. HEK293 parental cells were included in the assays as negative controls.

FIG. 10 A. pICAST ALC: Vector for expression of β-galΔα as a C-terminal fusion to the target protein. This construct contains the following features: MCS, multiple cloning site for cloning the target protein in frame with the β-galΔα; GS Linker, (GGGGS)n (SEQ ID NO:10); NeoR, neomycin resistance gene; IRES, internal ribosome entry site; ColE1ori, origin of replication for growth in E. coli; 5′MoMuLV LTR and 3′MoMuLV LTR, viral promoter and polyadenylation signals from the Moloney Murine leukemia virus.

FIGS. 10B-10P . Nucleotide sequence for pICAST ALC (SEQ ID NO:01).

FIG. 11 A. pICAST ALN: Vector for expression of β-galΔα as an N-terminal fusion to the target protein. This construct contains the following features: MCS, multiple cloning site for cloning the target protein in frame with the β-galΔα; GS Linker, (GGGGS)n (SEQ ID NO:10); NeoR, neomycin resistance gene; IRES, internal ribosome entry site; ColE1ori, origin of replication for growth in E. coli; 5′MoMuLV LTR and 3′MoMuLV LTR, viral promoter and polyadenylation signals from the Moloney Murine leukemia virus.

FIGS. 11B-11L . Nucleotide sequence for pICAST ALN (SEQ ID NO:02).

FIG. 12 A. pICAST OMC: Vector for expression of β-galΔω as a C-terminal fusion to the target protein. This construct contains the following features: MCS, multiple cloning site for cloning the target protein in frame with the β-galΔω; GS Linker, (GGGGS)n (SEQ ID NO:10); Hygro, hygromycin resistance gene; IRES, internal ribosome entry site; ColE1ori, origin of replication for growth in E. coli; 5′MoMuLV LTR and 3′MoMuLV LTR, viral promoter and polyadenylation signals from the Moloney Murine leukemia virus.

FIGS. 12B-12L . Nucleotide sequence for pICAST OMC (SEQ ID NO:03).

FIG. 13 A. pICAST OMN: Vector for expression of β-galΔω as an N-terminal fusion to the target protein. This construct contains the following features: MCS, multiple cloning site for cloning the target protein in frame with the β-galΔω; GS Linker, (GGGGS)n (SEQ ID NO:10); Hygro, hygromycin resistance gene; IRES, internal ribosome entry site; ColE1ori, origin of replication for growth in E. coli; 5′MoMuLV LTR and 3′MoMuLV LTR, viral promoter and polyadenylation signals from the Moloney Murine leukemia virus.

FIGS. 13B-13L . Nucleotide sequence for pICAST OMN (SEQ ID NO:04).

FIG. 14 . pICAST ALC βArr2: Vector for expression of β-galΔα as a C-terminal fusion to β-arrestin-2. The coding sequence of human β-arrestin-2 (Genebank Accession Number: NM — 004313) was cloned in frame to β-galΔα in a pICAST ALC vector.

FIG. 15 . pICAST OMC βArr2: Vector for expression of β-galΔω as a C-terminal fusion to β-arrestin-2. The coding sequence of human β-arrestin-2 (Genebank Accession Number: NM — 004313) was cloned in frame to β-galΔω in a pICAST OMC vector.

FIG. 16 . pICAST ALC βArr1: Vector for expression of β-galΔα as a C-terminal fusion to β-arrestin-1. The coding sequence of human β-arrestin-1 (Genebank Accession Number: NM — 004041) was cloned in frame to β-galΔα in a pICAST ALC vector.

FIG. 17 . pICAST OMC βArr1: Vector for expression of β-galΔω as a C-terminal fusion to β-arrestin-1. The coding sequence of human β-arrestin-1 (Genebank Accession Number: NM — 004041) was cloned in frame to β-galΔω in a pICAST OMC vector.

FIG. 18 . pICAST ALC β2AR: Vector for expression of β-galΔ(α as a C-terminal fusion to β2 Adrenergic Receptor. The coding sequence of human β2 Adrenergic Receptor (Genebank Accession Number: NM — 000024) was cloned in frame to β-galΔα in a pICAST ALC vector.

FIG. 19 . pICAST OMC β2AR: Vector for expression of β-galΔω as a C-terminal fusion β2 Adrenergic Receptor. The coding sequence of human β2 Adrenergic Receptor (Genebank Accession Number: NM — 000024) was cloned in frame to β-galΔω in a pICAST OMC vector.

FIG. 20 . pICAST ALC A2aR: Vector for expression of β-galΔα as a C-terminal fusion to Adenosine 2a Receptor. The coding sequence of human Adenosine 2a Receptor (Genebank Accession Number: NM — 000675) was cloned in frame to β-galΔα in a pICAST ALC vector.

FIG. 21 . pICAST OMC A2aR: Vector for expression of β-galΔω as a C-terminal fusion to Adenosine 2a Receptor. The coding sequence of human Adenosine 2a Receptor (Genebank Accession Number: NM — 000675) was cloned in frame to β-galΔω in a pICAST OMC vector.

FIG. 22 . pICAST ALC D1: Vector for expression of β-galΔα as a C-terminal fusion to Dopamine D 1 Receptor. The coding sequence of human Dopamine D1 Receptor (Genebank Accession Number: X58987) was cloned in frame to β-galΔα in a pICAST ALC vector.

FIG. 23. A schematic depicting use of the complementation technology in the method of the invention. FIG. 23 shows two inactive mutant reporter enzymes that become active when the corresponding fusion partners, GPCR and β-arrestin interact.

FIG. 24 . Vector for expression of a GPCR with inserted seronine/threonine amino acid sequences as a fusion with β-galΔα. The open reading frame of a known or orphan GPCR is engineered to contain additional seronine/threonine sequences, such as SSS (seronine, seronine, seronine), within the C-terminal tail. The engineered GPCR is cloned in frame with β-galΔα in a pICAST ALC vector. The pICAST ALC vector contains the following features: MCS, multiple cloning site for cloning the target protein in frame with the β-galΔα; GS Linker, (GGGGS)n (SEQ ID NO:10); NeoR, neomycin resistance gene; IRES, internal ribosome entry site; ColE1ori, origin of replication for growth in E. coli; 5′MoMuLV LTR and 3′MoMuLV LTR, viral promotor and polyadenylation signals from the Moloney Murine leukemia virus.

FIG. 25 . Vector for expression of mutant (R170E) β-arrestin2 as a fusion with β-galΔω. The open reading frame of β-arrestin2 is engineered to contain a point mutation that converts arginine 170 to a glutamate. The mutant β-arrestin2 is cloned in frame with β-galΔω in a pICAST OMC vector. The pICAST OMC vector contains the following features: MCS, multiple cloning site for cloning the target protein in frame with the β-galΔα; GS Linker, (GGGGS)n (SEQ ID NO:10); Hygro, hygromycin resistance gene; IRES, internal ribosome entry site; ColE1 ori, origin of replication for growth in E. coli; 5′MoMuLV LTR and 3′MoMuLV LTR, viral promotor and polyadenylation signals from the Moloney Murine leukemia virus.

FIG. 26 . Phosphorylation insensitive Mutant R170E β-Arrestin2Δω binds to β2ARΔα in Response to Agonist Activation. A parental β2ARΔα C2 cell line was tranduced with the Mutant R170E β-Arrestin2Δω construct. Clonal populations co-expressing the two constructions were plated at 10,000 cells/well in 96 well plates and treated with 10 μM (−)isoproterenol, 0.3 mM ascorbic acid for the indicated time period. β-galactosidase activity was measured by addition of Tropix Gal-Screen™ assay system substrate (Applied Biosystems) and luminescence was measured using a Tropix TR717™ luminometer (Applied Biosystems). Treatments were performed in triplicate. For comparison, a clonal cell line (43-8) co-expressing β2ARΔα and wild-type β-Arrestin2Δω was also plated at 10,000 cells/well and given the same agonist treatment regimen. Minutes of (−)isoproterenol treatment is shown on the X-axis and β-galactosidase activity indicated by relative light units (RLU) is shown on the Y-axis.

FIG. 27 . GPCR dimerization measured by β-galactosidase complementation. A schematic depicting the utilization of the invention for monitoring GPCR homo- or hetero-dimerization. One GPCR is fused to one complement enzyme fragment, while the second GPCR is fused to the second complement enzyme fragment. Interaction of the two GPCRs is monitored by complementation of the enzyme fragments to produce an active enzyme complex (i.e., β-galactosidase activity). GPCR homo- or hetero-dimerization can be monitored in the absence or presence of ligand, agonists, inverse agonists or antagonists.

FIG. 28 . Ligand fishing for orphan receptors by β-galactosidase mutant complementation in ICAST™ system. A schematic depicting the utilization of the invention for ligand fishing and agonist/antagonist screening for orphan GPCRs. As an example, a test cell expressing two β-gal fission proteins, GPCR orphan- Δα and Arrestin-Δω, is subjected to treatments with samples from natural or synthetic compound libraries, or from tissue extracts, or from conditioned media of cultured cells. An increased β-gal activity after treatment indicates the activation of the orphan receptor by a ligand in the testing sample. The readout of increased β-gal activity reflects the interaction of an activated GPCR orphan receptor with a β-arrestin. Therefore, a cognate or a surrogate ligand for the testing receptor is identified.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention provides a method to interrogate GPCR function and pathways. The G-protein-coupled superfamily continues to expand rapidly as new receptors are discovered through automated sequencing of cDNA libraries or genomic DNA. It is estimated that several thousand GPCRs may exist in the human genome. Only a portion have been cloned and even fewer have been associated with ligands. The means by which these, or newly discovered orphan receptors, will be associated with their cognate ligands and physiological functions represents a major challenge to biological and biomedical research. The identification of an orphan receptor generally requires an individualized assay and a guess as to its function. The present invention involves the interrogation of GPCR function by monitoring the activation of the receptor using activation dependent protein-protein interactions between the test GPCR or orphan receptor and a β-arrestin. The specific protein-protein interactions are measured using the mutant enzyme complementation technology disclosed herein. This assay system eliminates the prerequisite guessing because it can be performed with and without prior knowledge of other signaling events. It is sensitive, rapid and easily performed and is applicable to nearly all GPCRs because the majority of these receptors desensitize by a common mechanism.

The present invention provides a complete assay system for monitoring protein-protein interactions in GPCR pathways. The invention employs the complementation technology, ICAST™ (Intercistronic Complementation Analysis Screening Technology as disclosed in pending U.S. patent application Ser. No. 053,614, filed Apr. 1, 1998, the entire contents of which are incorporated herein by reference). The ICAST™ technology involves the use of two mutant forms of a reporter enzyme fused to proteins of interest. When the proteins of interest do not interact, the reporter enzyme remains inactive. When the proteins of interest do interact, the reporter enzyme mutants come together and form an active enzyme. According to an embodiment of the invention, the activity of β-galactosidase may be detected with the Gal-Screen™ assay system developed by Advanced Discovery Sciences™, which involves the use of Galacton-Star®, an ultrasensitive chemiluminescent substrate. The Gal-Screen™ assay system and the Galacton-Star® chemiluminescent substrate are disclosed in U.S. Pat. Nos. 5,851,771; 5,538,847; 5,326,882; 5,145,772; 4,978,614; and 4,931,569, the contents of which are incorporated herein by reference in their entirety. The invention provides an array of assays, including GPCR binding assays, that can be achieved directly within the cellular environment in a rapid, non-radioactive assay format. The methods of the invention are an advancement over the invention disclosed in U.S. Pat. Nos. 5,891,646 and 6,110,693 and the method disclosed in Angers et al., supra., which rely on microscopic imaging or spectrometry of GPCR components as fusion with Green-fluorescent-protein. The imaging technique disclosed in U.S. Pat. Nos. 5,891,646 and 6,110,693 and spectrometry-based technique in Angers et al. are limited by low-throughput and lack of thorough quantification.

The assay system of the invention combined with Advanced Discovery Sciences™ technologies provide highly sensitive cell-based methods for interrogating GPCR pathways which are amenable to high-throughput screening (HTS). Among some of the technologies developed by Advanced Discovery Sciences™ that may be used with the present invention are the Gal-Screen™ assay system (discussed above) and the cAMP-Screen™ immunoassay system. The cAMP-Screen™ immunoassay system provides ultrasensitive determination of cAMP levels in cell lysates. The cAMP-Screen™ assay utilizes the high-sensitivity chemiluminescent alkaline phosphatase (AP) substrate CSPD® (disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2′-(5′-chloro) tricyclo 3.3.1.1. 3,7 } decan-4-yl phenyl phosphate) with Sapphire-II™ luminescence enhancer.

Unlike yeast-based-two-hybrid assays used to monitor protein/protein interactions in high-throughput assays, the present invention (1) is applicable to a variety of cells including mammalian cells, plant cells, protozoa cells such as E. coli and cells of invertebrate origin such as yeast, slime mold (Dictyostelium) and insects; (2) detects interactions at the membrane at the site of the receptor target or in the cytosol at the site of downstream target proteins rather than a limited cellular localization, i.e., nucleus; and (3) does not rely on indirect read-outs such as transcriptional activation. The present invention thus provides assays with greater physiological relevance and fewer false positives.

The present inventors have developed modifications to the embodiment disclosed in U.S. patent application Ser. No. 053,614 described above in order to enhance the sensitivity of the inventive GPCR assay. According to an embodiment, the invention incorporates the use of serine/threonine clusters to enhance and prolong the interaction of GPCR with arrestin in order to make the detection more robust. The clusters can be utilized for orphan receptors or known GPCRs, which do not have this sequence motif. By adding this sequence to the C-terminal tail of the receptor, the activation of the receptor can be detected more readily by readouts of arrestin binding to GPCR, i.e., β-galactosidase complementation from fusion proteins of target proteins with β-galactosidase mutants.

According to another embodiment, the invention incorporates the use of arrestin point mutations to bypass the requirement of phosphorylation, by the action of specific GRK, on the C-terminal tail or intracellular loops of GPCR upon activation. The applications include i) wherein the cognate GRK for a particular GPCR or orphan receptor is unknown; and ii) wherein the specific GRK for the receptor of interest (or under test) may not be present or may have low activity in the host cell that is used for receptor activation assay.

According to another embodiment, the invention incorporates the use of a super arrestin to increase the binding efficiency of arrestin to an activated GPCR and to stabilize the GPCR/arrestin complex during GPCR desensitization. This application can be used to increase the robustness of ICAST/GPCR applications in cases where the GPCR is normally resensitized rapidly post desensitization.

Each of these methodologies is discussed below.

The invention will now be described in the following non-limiting examples.

EXAMPLE

According to an embodiment of the invention, GPCR activation is measured through monitoring the binding of arrestin to ligand-activated GPCR. In this assay system, a GPCR, e.g., β-adrenergic receptor (β2AR), and an arrestin, e.g., β-arrestin, are co-expressed in the same cell as fusion proteins with mutant forms of a reporter enzyme, e.g., β-galactosidase (β-gal). As illustrated in FIG. 23 , the β2AR is expressed as a fusion protein with Δα form of β-gal mutant (β2ARΔα) and the β-arrestin as a fusion protein with the Δω form of β-gal mutant (β-ArrΔω). The two fusion proteins, which at first exist in a resting (or un-stimulated) cell in separate compartments, i.e., the membrane for GPCR and the cytosol for arrestin, cannot form an active β-galactosidase enzyme. When such a cell is treated with an agonist or a ligand, the ligand-occupied and activated receptor becomes a high affinity binding site for arrestin. The interaction between an activated GPCR, β2ARΔα, and arrestin, β-ArrΔω, drives the β-gal mutant complementation. The enzyme activity can be measured by using an enzyme substrate, which upon cleavage releases a product measurable by colorimetry, fluorescence, or chemiluminescence (e.g., the Gal-Screen™ assay system).

Experiment Protocol

1. In the first step, the expression vectors for β2ARΔα and βArr2Δω were engineered in selectable retroviral vectors pICAST ALC, as described in FIG. 18 and pICAST OMC, as described in FIG. 15 .

2. In the second step, the two expression constructs were transduced into either C2C12 myoblast cells, or other mammalian cell lines, such as COS-7, CHO, A431, HEK 293, and CHW. Following selection with antibiotic drugs, stable clones expressing both fusion proteins at appropriate levels were selected.

3. In the last step, the cells expressing both β2ARΔα and βArr2Δω were tested for response by agonist/ligand stimulated β-galactosidase activity. Triplicate samples of cells were plated at 10,000 cells in 100 microliter volume into a well of 96-well culture plate. Cells were cultured for 24 hours before assay. For agonist assay (FIGS. 3 and 4 ), cells were treated with variable concentrations of agonist, for example, (−)isoproterenol, procaterol, dobutamine, terbutaline or L-L-phenylephrine for 60 min at 37° C. The induced β-galactosidase activity was measured by addition of Tropix Gal-Screen™ assay system substrate (Applied Biosystems) and luminescence measured in a Tropix TR717™ luminometer (Applied Biosystems). For antagonist assay (FIG. 5 ), cells were pre-incubated for 10 min in fresh medium without serum in the presence of ICI-118,551 or propranolol followed by addition of 10 micro molar (−)isoproterenol.

Serine/Threonine Cluster Strategy

Background

Based on structure-function relationship studies on β-arrestins, a large region within the amino-terminal half of β-arrestins (termed the activation-recognition domain) recognizes the agonist-activated state of GPCRs. This region of β-arrestin also contains a small positively charged domain (approximately 20 amino acids with net charge +7) called the phosphorylation-recognition domain, which appears to interact with the GRK-phosphorylated carboxyl termini of GPCRs.

GPCRs can be divided into two classes based on their affinities for β-arrestins. Oakley et al., “Association of β-Arrestin with G Protein-Coupled Receptors During Clathrin-Mediated Endocytosis Dictates the Profile of Receptor Resensitization.” J. Biol. Chem., 274(45):32248-32257 (1999). The molecular determinants underlying this classification appear to reside in specific serine or threonine residues located in the carboxyl-terminal tail of the receptor. The receptor class that contains serine/threonine clusters (defined as serine or threonine residues occupying three consecutive or three out of four positions) in the carboxyl-termini binds β-arrestin with high affinity upon activation and phosphorylation and remains bound with β-arrestin even after receptor internalization, whereas the receptor class that contains only scattered serine and threonine residues in the carboxy-terminal tail binds β-arrestins with less affinity and disassociates from the β-arrestin upon internalization. Several known GPCRs, such as vasopressin V2 receptor (Oakley, et al.), neurotensin receptor 1 and angiotensin II receptor type 1A (Zhang, et al., “Cellular Trafficking of G Protein-Coupled Receptor/β-Arrestin Endocytic Complexes.” J. Biol. Chem., 274(16):10999-11006 (1999)), which possess one or more of such serine/threonine clusters in their carboxyl-termini, were shown to bind β-arrestins with high affinity.

EXAMPLE

According to an embodiment of the invention, a serine/threonine cluster strategy is used to facilitate screening assays for orphan receptors that do not possess this structural motif of their own. The orphan receptors are easily classified by sequence alignment. Orphan receptors lacking the serine/threonine clusters are each cloned into an expression vector that is modified to introduce one or more serine/threonine cluster(s) to the carboxyl-terminal tail of the receptor (FIG. 24 ). The serine/threonine clusters enhance the receptor activation dependent interaction between the activated and phosphorylated receptor (negative charges) and β-arrestin (positive charges in the phosphorylation-recognition domain) through strong ionic interactions, thus prolonging interaction between the receptor and arrestin. The modification of the orphan receptor tail thus makes detection of receptor activation more robust.

Experiment Protocol

1. In a first step, the open-reading-frame (ORF) of an orphan receptor, which lacks the serine/threonine clusters, is cloned into a modified expression vector such as pICAST ALC described in FIG. 10 A. The modified pICAST ALC includes coding sequences for one or more sets of serine/threonine clusters (for example, SSS or SST) located downstream from the insert of the ORF of an orphan receptor (FIG. 24 ).

2. In a second step, chimeric orphan receptor, ORF orphan R -(SSS) n -Δα, is co-expressed in a mammalian cell with a β-arrestin chimera, such as βArr2Δω described in FIG. 15 .

3. In a third step, the cell is treated with an agonist or a ligand and the activated receptor with phosphorylated serine cluster(s) binds the β-arrestin with high affinity producing strong signals in readouts of β-gal complementation.

This assay, which provides a means for sensitive measurement of functional activation of the orphan receptors, can be used to screen for natural or surrogate ligands for orphan receptors, a process called de-orphaning or target discovery for new GPCRs (FIG. 28 ). Furthermore, this assay is also useful in screening for potential agonists and antagonists for lead discovery of GPCRs.

Enhanced Binding of Arrestin in the Presence and in the Absence of GPCR Phosphorylation

Background

Six different classes of G-protein coupled receptor kinases (GRKs) have been identified and each of these has been reported to be expressed as multiple splice variants. Krupnick et al., “The role of receptor kinases and arrestins in G protein-coupled receptor regulation.” Ann. Rev. Pharmacol. Toxicol., 38:289-319 (1998). Although many cell lines express a variety of GRKs, the specific GRK required for phosphorylation of a given GPCR may not always be present in the cell line used for recombinant GPCR and arrestin expression. This is particularly an issue for applications using orphan receptors, in which case the cognate GRK will likely be unknown. In other cases, the cell line used for recombinant expression work may have the required GRK, but may express the GRK at low levels. In order to bypass such caveats, genetically modified arrestins that bind specifically to activated GPCRs, but without the requirement of GRK phosphorylation are employed.

Mutagenesis studies on arrestins demonstrate that point mutations in the phosphorylation-recognition domain, particularly mutations converting Arg175 (of visual arrestin) to an oppositely charged residue such as glutamate (R175E mutation), result in an arrestin which specifically binds to activated GPCRs, but does so without the requirement for phosphorylation.

Numerous observations have led to the hypothesis that arrestin exists in an inactive state that has a low affinity for GPCRs. Once a GPCR is both activated and phosphorylated, the phosphorylated region of the GPCR C-terminus interacts with the phosphorylation-recognition domain of arrestin causing the arrestin to change conformations allowing the activation-recognition region to be exposed for binding to the activated/phosphorylated receptor. Vishnivetskiy et al., “How does arrestin respond to the phosphorylated state of rhodopsin?” J. Biol. Chem., 274(17):11451-11454 (1999); Gurevich et al., “Arrestin interactions with G protein-coupled receptors. Direct binding studies of wild-type and mutant arrestins with rhodopsin, beta 2-adrenergic and m2 muscarinic cholinergic receptors.” J. Biol. Chem., 270(2):720-731, (1995); Gurevich et al., “Mechanism of phosphorylation-recognition by visual arrestin and the transition of arrestin into a high affinity binding site.” Mol. Pharnacol., 51(1):161-169 (1997); Kovoor et al., “Targeted construction of phosphorylation-independent beta-arrestin mutants with constitutive activity in cells.” J. Biol. Chem., 274(11):6831-6834 (1999). In summary, binding studies of single mutation, double mutation, deletion, and chimerical arrestins with inactive, inactive and phosphorylated, activated but not phosphorylated, or activated and phosphorylated visual or non-visual GPCRs all support this model.

EXAMPLE

A phosphorylation insensitive mutant of arrestin fused to mutant reporter protein can be produced that will bind to activated GPCRs in a phosphorylation independent manner. As proof of concept, a point mutation for β-arrestin2, R170E β-arrestin2, has been produced and its interaction with β2AR has been analyzed in accordance with the invention.

Experimental Protocol:

1) In the first step, β-arrestin2 was mutated such that Arg170 was converted to Glu. This mutation is equivalent to the R175E mutation of visual arrestin. The mutant β-arrestin2 open reading frame was cloned in frame with Δω-β-galactosidase in the pICAST OMC expression vector to produce a modified expression vector R170E β-arrestin2 (FIG. 25 ).

2) In the second step, the R170E β-arrestin2 expression construct was transduced into a C2C12 myoblast cell line that had been engineered to express β2AR as a fusion to Δα-β-galactosidase as described in FIG. 18 of U.S. application Ser. No. 09/654,499. Following selection with antibiotic drugs, a population of clones expressing both fusion proteins was obtained.

3) In the last step, this population of cells expressing both R170E β-arrestin2Δω and β2AR Δα were tested for response by agonist/ligand stimulated β-galactosidase activity as demonstrated in FIG. 26 . The C2C12 clone 43-8 co-expressing β2ARΔα and wild-type 1β-arrestin2Δω ( FIG. 26 ) was used as reference control. Triplicate samples of cells were plated at 10,000 cells in 100 microliter volume into wells of a 96-well culture plate. Cells were cultured for 24 hours before assay. For agonist assay as in FIG. 26 , cells were treated with 10 μm (−)isoproterenol stabilized with 0.3 mM ascorbic acid 37° C. for 0, 5, 10, 15, 30, 45 or 60 minutes. The induced β-galactosidase activity was measured by addition of Tropix Gal-Screen™ assay system substrate (Applied Biosystems) and luminescence measured in a Tropix TR717™ luminometer (Applied Biosystems). As shown in FIG. 26 , the mutant arrestin interacts with β2AR in an agonist-dependent manner and was comparable with that of wild-type arrestin.

4) To expand the application of phosphorylation-insensitive arrestin, cell lines such as C2C12, CHO or HEK 293, are developed that express the R170E β-arrestin2Δω construction. These cell lines can be used to transduce orphan or known GPCRs as fusions with Δα-β-galactosidase in order to develop cell lines for agonist and antagonist screening and

Development of Super Arrestins:

Background

Attenuation of GPCR signaling by the arrestin pathway serves to ensure that a cell or organism does not over-react to a stimulus. At the same time, the arrestin pathway often serves to recycle the GPCR such that it can be temporarily inactivated but then quickly resensitized to allow for sensitivity to new stimuli. The down-regulation process involves phosphorylation of the receptor, binding to arrestin and endocytosis. Following endocytosis of the desensitized receptor, the receptor is either degraded in lysosomes or resensitized and sent back to the membrane. Resensitization involves release of arrestin from the receptor, dephosphorylation and cycling back to the membrane. The actual route a GPCR follows upon activation depends on its biological function and the needs of the organism. Because of these diverse pathways that may be required of the down-regulation pathway, arrestin affinities for activated GPCRs vary from receptor to receptor. It would thus be very advantageous to engineer super arrestins that have a higher affinity and avidity for activated GPCRs than what nature has provided.

Although mutational, deletion and chimerical studies of arrestins have focused on understanding regulatory switches in the molecule that respond to GPCR phosphorylation states, several of these altered recombinant forms of arrestin have resulted in molecules with enhanced binding to activated, phosphorylated GPCRs. Conversion of Arg175 to histidine, tyrosine, phenylalanine or threonine results in significantly higher amounts of binding to phosphorylated, activated rhodopsin than wild-type arrestin or R175E arrestin, although these mutations result in less binding to activated, non-phosphorylated receptor. Gurevich et al. (1997). In addition, conversion of Valine 170 to alanine increased the constitutive affect of the R175E mutation, but also nearly doubled the amount of interaction of wild-type arrestin with activated, phosphorylated rhodopsin. Gurevich et al. (1997).

Truncation of β-arrestin1 at amino acid 382 has been reported to enhance binding of both R169E (equivalent to arrestin R175E) and wild-type β-arrestin1 to activated or activated and phosphorylated receptor, respectively. Kovoor et al. Chimerical arrestins in which functional regions of visual arrestin were swapped with those of β-arrestin1 have been reported to be altered in binding affinity to activated, phosphorylated GPCRs. Gurevich et al. (1995). Several of these chimeras, such as β-arrestin1 containing the visual arrestin extreme N-terminus, show increased specific binding to phosphorylated activated GPCRs compared to wild-type β-arrestin1 (Gurevich et al. (1995)). Modifications that enhance arrestin affinity for the activated GPCR such as described above, whether phosphorylated or non-phosphorylated, could also enhance signal to noise of β-galactosidase activity since the arrestin/GPCR complex is stabilized and/or more long-lived. The use of mutant arrestins with higher activated-GPCR affinity would improve the inventive technology for GPCR targets, without compromising receptor/ligand biology.

In addition, this “super arrestin” approach can be combined with the use of arrestin point mutations to provide a stronger signal to noise with or without GRK requirements.

EXAMPLE

An arrestin mutant fused to mutant reporter protein can be produced to enhance binding of the arrestin to an activated GPCR to enhance sensitivity of detection.

Experiment Protocol

1) In the first step, mutant β-arrestin2 constructions will be generated which include R170E/T/Y/or H, V165A, substitution of a.a. 1-43 with a.a. 1-47 of visual arrestin, or deletion of the C-terminal and combinations of these alterations. The mutant β-arrestin2 open reading frames will be cloned in frame with Δω-β-galactosidase in the pICAST OMC expression vector similar to cloning of the R170E β-arrestin2 mutation shown in FIG. 25 .

2) In the second step, mutant expression constructs will be transduced into a C2C12 myoblast cell line that has been engineered to express β2AR as a fusion to Δα-β-galactosidase. Following selection with antibiotic drugs, a population of clones expressing both fusion proteins will be obtained. Wild type and R170E β-arrestin2 constructions will be transduced to generate control, reference clonal populations.

3) In the third step, populations of cells expressing both β-arrestin2Δω (mutant or wild type) and β2ARΔα will be tested for response by agonist/ligand stimulated β-galactosidase activity.

4) In the next step, mutant (super) β-arrestin2Δω constructions that show a significantly higher signal to noise ratio in the agonist assay compared with wild-type β-arrestin2Δω will be chosen. These constructions will be used to develop stable cell lines expressing the “super” β-arrestin2Δω that can be used for transducing in known or orphan GPCRs. Use of a super β-arrestin2Δω could increase the signal to noise of ICAST/GPCR applications allowing improved screening capabilities for lead and ligand discovery.

Super Arrestin is used to increase the binding efficiency of arrestin to an activated GPCR and to stabilize the GPCR/arrestin complex during GPCR desensitization. This application can be used to increase the robustness of ICAST/GPCR applications in cases where the GPCR is normally resensitized rapidly post desensitization.

The assays of this invention, and their application and preparation have been described both generically, and by specific example. The examples are not intended as limiting. Other substituent identities, characteristics and assays will occur to those of ordinary skill in the art, without the exercise of inventive faculty. Such modifications remain within the scope of the invention, unless excluded by the express recitation of the claims advanced below.

10 1 6700 DNA Unknown pICAST ALC. 1ctgcagcctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca 60gggccaagaa cagatggaac agctgaatat gggccaaaca ggatatctgt ggtaagcagt 120tcctgccccg gctcagggcc aagaacagat ggtccccaga tgcggtccag ccctcagcag 180tttctagaga accatcagat gtttccaggg tgccccaagg acctgaaatg accctgtgcc 240ttatttgaac taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga 300gctcaataaa agagcccaca acccctcact cggggcgcca gtcctccgat tgactgagtc 360gcccgggtac ccgtgtatcc aataaaccct cttgcagttg catccgactt gtggtctcgc 420tgttccttgg gagggtctcc tctgagtgat tgactacccg tcagcggggg tctttcattt 480gggggctcgt ccgggatcgg gagacccctg cccagggacc accgacccac caccgggagg 540caagctggcc agcaacttat ctgtgtctgt ccgattgtct agtgtctatg actgatttta 600tgcgcctgcg tcggtactag ttagctaact agctctgtat ctggcggacc cgtggtggaa 660ctgacgagtt ctgaacaccc ggccgcaacc ctgggagacg tcccagggac tttgggggcc 720gtttttgtgg cccgacctga ggaagggagt cgatgtggaa tccgaccccg tcaggatatg 780tggttctggt aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt 840cggtttggaa ccgaagccgc gcgtcttgtc tgctgcagca tcgttctgtg ttgtctctgt 900ccgactgtgt ttctgtattt gtctgaaaat tagggccaga ctgttaccac tcccttaagt 960ttgaccttag gtaactggaa agatgtcgag cggctcgctc acaaccagtc ggtagatgtc 1020aagaagagac gttgggttac cttctgctct gcagaatggc caacctttaa cgtcggatgg 1080ccgcgagacg gcacctttaa ccgagacctc atcacccagg ttaagatcaa ggtcttttca 1140cctggcccgc atggacaccc agaccaggtc ccctacatcg tgacctggga agccttggct 1200tttgaccccc ctccctgggt caagcccttt gtacacccta agcctccgcc tcctcttcct 1260ccatccgccc cgtctctccc ccttgaacct cctcgttcga ccccgcctcg atcctccctt 1320tatccagccc tcactccttc tctaggcgcc ggccgctcta gcccattaat acgactcact 1380atagggcgat tcgaatcagg ccttggcgcg ccggatcctt aattaagcgc aattgggagg 1440tggcggtagc ctcgag atg ggc gtg att acg gat tca ctg gcc gtc gtg gcc 1492 Met Gly Val Ile Thr Asp Ser Leu Ala Val Val Ala 1 5 10cgc acc gat cgc cct tcc caa cag tta cgc agc ctg aat ggc gaa tgg 1540Arg Thr Asp Arg Pro Ser Gln Gln Leu Arg Ser Leu Asn Gly Glu Trp 15 20 25cgc ttt gcc tgg ttt ccg gca cca gaa gcg gtg ccg gaa agc tgg ctg 1588Arg Phe Ala Trp Phe Pro Ala Pro Glu Ala Val Pro Glu Ser Trp Leu 30 35 40gag tgc gat ctt cct gag gcc gat act gtc gtc gtc ccc tca aac tgg 1636Glu Cys Asp Leu Pro Glu Ala Asp Thr Val Val Val Pro Ser Asn Trp45 50 55 60cag atg cac ggt tac gat gcg ccc atc tac acc aac gtg acc tat ccc 1684Gln Met His Gly Tyr Asp Ala Pro Ile Tyr Thr Asn Val Thr Tyr Pro 65 70 75att acg gtc aat ccg ccg ttt gtt ccc acg gag aat ccg acg ggt tgt 1732Ile Thr Val Asn Pro Pro Phe Val Pro Thr Glu Asn Pro Thr Gly Cys 80 85 90tac tcg ctc aca ttt aat gtt gat gaa agc tgg cta cag gaa ggc cag 1780Tyr Ser Leu Thr Phe Asn Val Asp Glu Ser Trp Leu Gln Glu Gly Gln 95 100 105acg cga att att ttt gat ggc gtt aac tcg gcg ttt cat ctg tgg tgc 1828Thr Arg Ile Ile Phe Asp Gly Val Asn Ser Ala Phe His Leu Trp Cys 110 115 120aac ggg cgc tgg gtc ggt tac ggc cag gac agt cgt ttg ccg tct gaa 1876Asn Gly Arg Trp Val Gly Tyr Gly Gln Asp Ser Arg Leu Pro Ser Glu125 130 135 140ttt gac ctg agc gca ttt tta cgc gcc gga gaa aac cgc ctc gcg gtg 1924Phe Asp Leu Ser Ala Phe Leu Arg Ala Gly Glu Asn Arg Leu Ala Val 145 150 155atg gtg ctg cgc tgg agt gac ggc agt tat ctg gaa gat cag gat atg 1972Met Val Leu Arg Trp Ser Asp Gly Ser Tyr Leu Glu Asp Gln Asp Met 160 165 170tgg cgg atg agc ggc att ttc cgt gac gtc tcg ttg ctg cat aaa ccg 2020Trp Arg Met Ser Gly Ile Phe Arg Asp Val Ser Leu Leu His Lys Pro 175 180 185act aca caa atc agc gat ttc cat gtt gcc act cgc ttt aat gat gat 2068Thr Thr Gln Ile Ser Asp Phe His Val Ala Thr Arg Phe Asn Asp Asp 190 195 200ttc agc cgc gct gta ctg gag gct gaa gtt cag atg tgc ggc gag ttg 2116Phe Ser Arg Ala Val Leu Glu Ala Glu Val Gln Met Cys Gly Glu Leu205 210 215 220cgt gac tac cta cgg gta aca gtt tct tta tgg cag ggt gaa acg cag 2164Arg Asp Tyr Leu Arg Val Thr Val Ser Leu Trp Gln Gly Glu Thr Gln 225 230 235gtc gcc agc ggc acc gcg cct ttc ggc ggt gaa att atc gat gag cgt 2212Val Ala Ser Gly Thr Ala Pro Phe Gly Gly Glu Ile Ile Asp Glu Arg 240 245 250ggt ggt tat gcc gat cgc gtc aca cta cgt ctg aac gtc gaa aac ccg 2260Gly Gly Tyr Ala Asp Arg Val Thr Leu Arg Leu Asn Val Glu Asn Pro 255 260 265aaa ctg tgg agc gcc gaa atc ccg aat ctc tat cgt gcg gtg gtt gaa 2308Lys Leu Trp Ser Ala Glu Ile Pro Asn Leu Tyr Arg Ala Val Val Glu 270 275 280ctg cac acc gcc gac ggc acg ctg att gaa gca gaa gcc tgc gat gtc 2356Leu His Thr Ala Asp Gly Thr Leu Ile Glu Ala Glu Ala Cys Asp Val285 290 295 300ggt ttc cgc gag gtg cgg att gaa aat ggt ctg ctg ctg ctg aac ggc 2404Gly Phe Arg Glu Val Arg Ile Glu Asn Gly Leu Leu Leu Leu Asn Gly 305 310 315aag ccg ttg ctg att cga ggc gtt aac cgt cac gag cat cat cct ctg 2452Lys Pro Leu Leu Ile Arg Gly Val Asn Arg His Glu His His Pro Leu 320 325 330cat ggt cag gtc atg gat gag cag acg atg gtg cag gat atc ctg ctg 2500His Gly Gln Val Met Asp Glu Gln Thr Met Val Gln Asp Ile Leu Leu 335 340 345atg aag cag aac aac ttt aac gcc gtg cgc tgt tcg cat tat ccg aac 2548Met Lys Gln Asn Asn Phe Asn Ala Val Arg Cys Ser His Tyr Pro Asn 350 355 360cat ccg ctg tgg tac acg ctg tgc gac cgc tac ggc ctg tat gtg gtg 2596His Pro Leu Trp Tyr Thr Leu Cys Asp Arg Tyr Gly Leu Tyr Val Val365 370 375 380gat gaa gcc aat att gaa acc cac ggc atg gtg cca atg aat cgt ctg 2644Asp Glu Ala Asn Ile Glu Thr His Gly Met Val Pro Met Asn Arg Leu 385 390 395acc gat gat ccg cgc tgg cta ccg gcg atg agc gaa cgc gta acg cga 2692Thr Asp Asp Pro Arg Trp Leu Pro Ala Met Ser Glu Arg Val Thr Arg 400 405 410atg gtg cag cgc gat cgt aat cac ccg agt gtg atc atc tgg tcg ctg 2740Met Val Gln Arg Asp Arg Asn His Pro Ser Val Ile Ile Trp Ser Leu 415 420 425ggg aat gaa tca ggc cac ggc gct aat cac gac gcg ctg tat cgc tgg 2788Gly Asn Glu Ser Gly His Gly Ala Asn His Asp Ala Leu Tyr Arg Trp 430 435 440atc aaa tct gtc gat cct tcc cgc ccg gtg cag tat gaa ggc ggc gga 2836Ile Lys Ser Val Asp Pro Ser Arg Pro Val Gln Tyr Glu Gly Gly Gly445 450 455 460gcc gac acc acg gcc acc gat att att tgc ccg atg tac gcg cgc gtg 2884Ala Asp Thr Thr Ala Thr Asp Ile Ile Cys Pro Met Tyr Ala Arg Val 465 470 475gat gaa gac cag ccc ttc ccg gct gtg ccg aaa tgg tcc atc aaa aaa 2932Asp Glu Asp Gln Pro Phe Pro Ala Val Pro Lys Trp Ser Ile Lys Lys 480 485 490tgg ctt tcg cta cct gga gag acg cgc ccg ctg atc ctt tgc gaa tac 2980Trp Leu Ser Leu Pro Gly Glu Thr Arg Pro Leu Ile Leu Cys Glu Tyr 495 500 505gcc cac gcg atg ggt aac agt ctt ggc ggt ttc gct aaa tac tgg cag 3028Ala His Ala Met Gly Asn Ser Leu Gly Gly Phe Ala Lys Tyr Trp Gln 510 515 520gcg ttt cgt cag tat ccc cgt tta cag ggc ggc ttc gtc tgg gac tgg 3076Ala Phe Arg Gln Tyr Pro Arg Leu Gln Gly Gly Phe Val Trp Asp Trp525 530 535 540gtg gat cag tcg ctg att aaa tat gat gaa aac ggc aac ccg tgg tcg 3124Val Asp Gln Ser Leu Ile Lys Tyr Asp Glu Asn Gly Asn Pro Trp Ser 545 550 555gct tac ggc ggt gat ttt ggc gat acg ccg aac gat cgc cag ttc tgt 3172Ala Tyr Gly Gly Asp Phe Gly Asp Thr Pro Asn Asp Arg Gln Phe Cys 560 565 570atg aac ggt ctg gtc ttt gcc gac cgc acg ccg cat cca gcg ctg acg 3220Met Asn Gly Leu Val Phe Ala Asp Arg Thr Pro His Pro Ala Leu Thr 575 580 585gaa gca aaa cac cag cag cag ttt ttc cag ttc cgt tta tcc ggg caa 3268Glu Ala Lys His Gln Gln Gln Phe Phe Gln Phe Arg Leu Ser Gly Gln 590 595 600acc atc gaa gtg acc agc gaa tac ctg ttc cgt cat agc gat aac gag 3316Thr Ile Glu Val Thr Ser Glu Tyr Leu Phe Arg His Ser Asp Asn Glu605 610 615 620ctc ctg cac tgg atg gtg gcg ctg gat ggt aag ccg ctg gca agc ggt 3364Leu Leu His Trp Met Val Ala Leu Asp Gly Lys Pro Leu Ala Ser Gly 625 630 635gaa gtg cct ctg gat gtc gct cca caa ggt aaa cag ttg att gaa ctg 3412Glu Val Pro Leu Asp Val Ala Pro Gln Gly Lys Gln Leu Ile Glu Leu 640 645 650cct gaa cta ccg cag ccg gag agc gcc ggg caa ctc tgg ctc aca gta 3460Pro Glu Leu Pro Gln Pro Glu Ser Ala Gly Gln Leu Trp Leu Thr Val 655 660 665cgc gta gtg caa ccg aac gcg acc gca tgg tca gaa gcc ggg cac atc 3508Arg Val Val Gln Pro Asn Ala Thr Ala Trp Ser Glu Ala Gly His Ile 670 675 680agc gcc tgg cag cag tgg cgt ctg gcg gaa aac ctc agt gtg acg ctc 3556Ser Ala Trp Gln Gln Trp Arg Leu Ala Glu Asn Leu Ser Val Thr Leu685 690 695 700ccc gcc gcg tcc cac gcc atc ccg cat ctg acc acc agc gaa atg gat 3604Pro Ala Ala Ser His Ala Ile Pro His Leu Thr Thr Ser Glu Met Asp 705 710 715ttt tgc atc gag ctg ggt aat aag cgt tgg caa ttt aac cgc cag tca 3652Phe Cys Ile Glu Leu Gly Asn Lys Arg Trp Gln Phe Asn Arg Gln Ser 720 725 730ggc ttt ctt tca cag atg tgg att ggc gat aaa aaa caa ctg ctg acg 3700Gly Phe Leu Ser Gln Met Trp Ile Gly Asp Lys Lys Gln Leu Leu Thr 735 740 745ccg ctg cgc gat cag ttc acc cgt gca ccg ctg gat aac gac att ggc 3748Pro Leu Arg Asp Gln Phe Thr Arg Ala Pro Leu Asp Asn Asp Ile Gly 750 755 760gta agt gaa gcg acc cgc att gac cct aac gcc tgg gtc gaa cgc tgg 3796Val Ser Glu Ala Thr Arg Ile Asp Pro Asn Ala Trp Val Glu Arg Trp765 770 775 780aag gcg gcg ggc cat tac cag gcc gaa gca gcg ttg ttg cag tgc acg 3844Lys Ala Ala Gly His Tyr Gln Ala Glu Ala Ala Leu Leu Gln Cys Thr 785 790 795gca gat aca ctt gct gat gcg gtg ctg att acg acc gct cac gcg tgg 3892Ala Asp Thr Leu Ala Asp Ala Val Leu Ile Thr Thr Ala His Ala Trp 800 805 810cag cat cag ggg aaa acc tta ttt atc agc cgg aaa acc tac cgg att 3940Gln His Gln Gly Lys Thr Leu Phe Ile Ser Arg Lys Thr Tyr Arg Ile 815 820 825gat ggt agt ggt caa atg gcg att acc gtt gat gtt gaa gtg gcg agc 3988Asp Gly Ser Gly Gln Met Ala Ile Thr Val Asp Val Glu Val Ala Ser 830 835 840gat aca ccg cat ccg gcg cgg att ggc ctg aac tgc cag ctg gcg cag 4036Asp Thr Pro His Pro Ala Arg Ile Gly Leu Asn Cys Gln Leu Ala Gln845 850 855 860gta gca gag cgg gta aac tgg ctc gga tta ggg ccg caa gaa aac tat 4084Val Ala Glu Arg Val Asn Trp Leu Gly Leu Gly Pro Gln Glu Asn Tyr 865 870 875ccc gac cgc ctt act gcc gcc tgt ttt gac cgc tgg gat ctg cca ttg 4132Pro Asp Arg Leu Thr Ala Ala Cys Phe Asp Arg Trp Asp Leu Pro Leu 880 885 890tca gac atg tat acc ccg tac gtc ttc ccg agc gaa aac ggt ctg cgc 4180Ser Asp Met Tyr Thr Pro Tyr Val Phe Pro Ser Glu Asn Gly Leu Arg 895 900 905tgc ggg acg cgc gaa ttg aat tat ggc cca cac cag tgg cgc ggc gac 4228Cys Gly Thr Arg Glu Leu Asn Tyr Gly Pro His Gln Trp Arg Gly Asp 910 915 920ttc cag ttc aac atc agc cgc tac agt caa cag caa ctg atg gaa acc 4276Phe Gln Phe Asn Ile Ser Arg Tyr Ser Gln Gln Gln Leu Met Glu Thr925 930 935 940agc cat cgc cat ctg ctg cac gcg gaa gaa ggc aca tgg ctg aat atc 4324Ser His Arg His Leu Leu His Ala Glu Glu Gly Thr Trp Leu Asn Ile 945 950 955gac ggt ttc cat atg ggg att ggt ggc gac gac tcc tgg agc ccg tca 4372Asp Gly Phe His Met Gly Ile Gly Gly Asp Asp Ser Trp Ser Pro Ser 960 965 970gta tcg gcg gaa ttc cag ctg agc gcc ggt cgc tac cat tac cag ttg 4420Val Ser Ala Glu Phe Gln Leu Ser Ala Gly Arg Tyr His Tyr Gln Leu 975 980 985gtc tgg tgt caa aaa aga tct gac tat aaa gat gag gac ctc gac cat 4468Val Trp Cys Gln Lys Arg Ser Asp Tyr Lys Asp Glu Asp Leu Asp His 990 995 1000cat cat cat cat cac cgg taataatagg tagataagtg actgattaga 4516His His His His His Arg1005 1010tgcattgatc cctcgaccaa ttccggttat tttccaccat attgccgtct tttggcaatg 4576tgagggcccg gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc 4636tcgccaaagg aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt 4696cttgaagaca aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg 4756acaggtgcct ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac 4816cccagtgcca cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg 4876tattcaacaa ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg 4936ggcctcggtg cacatgcttt acatgtgttt agtcgaggtt aaaaaacgtc taggcccccc 4996gaaccacggg gacgtggttt tcctttgaaa aacacgatga taataccatg attgaacaag 5056atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg 5116cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc 5176cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag 5236cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca 5296ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat 5356ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata 5416cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac 5476gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc 5536tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg 5596tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg 5656gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta 5716cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg 5776gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct 5836gagcgggact ctggggttcg catcgataaa ataaaagatt ttatttagtc tccagaaaaa 5896ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg 5956caaggcatgg aaaaatacat aactgagaat agagaagttc agatcaaggt caggaacaga 6016tggaacagct gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc 6076agggccaaga acagatggaa cagctgaata tgggccaaac aggatatctg tggtaagcag 6136ttcctgcccc ggctcagggc caagaacaga tggtccccag atgcggtcca gccctcagca 6196gtttctagag aaccatcaga tgtttccagg gtgccccaag gacctgaaat gaccctgtgc 6256cttatttgaa ctaaccaatc agttcgcttc tcgcttctgt tcgcgcgctt ctgctccccg 6316agctcaataa aagagcccac aacccctcac tcggggcgcc agtcctccga ttgactgagt 6376cgcccgggta cccgtgtatc caataaaccc tcttgcagtt gcatccgact tgtggtctcg 6436ctgttccttg ggagggtctc ctctgagtga ttgactaccc gtcagcgggg gtctttcatt 6496catgcagcat gtatcaaaat taatttggtt ttttttctta agtatttaca ttaaatggcc 6556atagttgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttggcgctct 6616tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 6676gctcactcaa aggcggtaat acgg 6700 2 1010 PRT Unknown pICAST ALC. 2Met Gly Val Ile Thr Asp Ser Leu Ala Val Val Ala Arg Thr Asp Arg1 5 10 15Pro Ser Gln Gln Leu Arg Ser Leu Asn Gly Glu Trp Arg Phe Ala Trp 20 25 30Phe Pro Ala Pro Glu Ala Val Pro Glu Ser Trp Leu Glu Cys Asp Leu 35 40 45Pro Glu Ala Asp Thr Val Val Val Pro Ser Asn Trp Gln Met His Gly 50 55 60Tyr Asp Ala Pro Ile Tyr Thr Asn Val Thr Tyr Pro Ile Thr Val Asn65 70 75 80Pro Pro Phe Val Pro Thr Glu Asn Pro Thr Gly Cys Tyr Ser Leu Thr 85 90 95Phe Asn Val Asp Glu Ser Trp Leu Gln Glu Gly Gln Thr Arg Ile Ile 100 105 110Phe Asp Gly Val Asn Ser Ala Phe His Leu Trp Cys Asn Gly Arg Trp 115 120 125Val Gly Tyr Gly Gln Asp Ser Arg Leu Pro Ser Glu Phe Asp Leu Ser 130 135 140Ala Phe Leu Arg Ala Gly Glu Asn Arg Leu Ala Val Met Val Leu Arg145 150 155 160Trp Ser Asp Gly Ser Tyr Leu Glu Asp Gln Asp Met Trp Arg Met Ser 165 170 175Gly Ile Phe Arg Asp Val Ser Leu Leu His Lys Pro Thr Thr Gln Ile 180 185 190Ser Asp Phe His Val Ala Thr Arg Phe Asn Asp Asp Phe Ser Arg Ala 195 200 205Val Leu Glu Ala Glu Val Gln Met Cys Gly Glu Leu Arg Asp Tyr Leu 210 215 220Arg Val Thr Val Ser Leu Trp Gln Gly Glu Thr Gln Val Ala Ser Gly225 230 235 240Thr Ala Pro Phe Gly Gly Glu Ile Ile Asp Glu Arg Gly Gly Tyr Ala 245 250 255Asp Arg Val Thr Leu Arg Leu Asn Val Glu Asn Pro Lys Leu Trp Ser 260 265 270Ala Glu Ile Pro Asn Leu Tyr Arg Ala Val Val Glu Leu His Thr Ala 275 280 285Asp Gly Thr Leu Ile Glu Ala Glu Ala Cys Asp Val Gly Phe Arg Glu 290 295 300Val Arg Ile Glu Asn Gly Leu Leu Leu Leu Asn Gly Lys Pro Leu Leu305 310 315 320Ile Arg Gly Val Asn Arg His Glu His His Pro Leu His Gly Gln Val 325 330 335Met Asp Glu Gln Thr Met Val Gln Asp Ile Leu Leu Met Lys Gln Asn 340 345 350Asn Phe Asn Ala Val Arg Cys Ser His Tyr Pro Asn His Pro Leu Trp 355 360 365Tyr Thr Leu Cys Asp Arg Tyr Gly Leu Tyr Val Val Asp Glu Ala Asn 370 375 380Ile Glu Thr His Gly Met Val Pro Met Asn Arg Leu Thr Asp Asp Pro385 390 395 400Arg Trp Leu Pro Ala Met Ser Glu Arg Val Thr Arg Met Val Gln Arg 405 410 415Asp Arg Asn His Pro Ser Val Ile Ile Trp Ser Leu Gly Asn Glu Ser 420 425 430Gly His Gly Ala Asn His Asp Ala Leu Tyr Arg Trp Ile Lys Ser Val 435 440 445Asp Pro Ser Arg Pro Val Gln Tyr Glu Gly Gly Gly Ala Asp Thr Thr 450 455 460Ala Thr Asp Ile Ile Cys Pro Met Tyr Ala Arg Val Asp Glu Asp Gln465 470 475 480Pro Phe Pro Ala Val Pro Lys Trp Ser Ile Lys Lys Trp Leu Ser Leu 485 490 495Pro Gly Glu Thr Arg Pro Leu Ile Leu Cys Glu Tyr Ala His Ala Met 500 505 510Gly Asn Ser Leu Gly Gly Phe Ala Lys Tyr Trp Gln Ala Phe Arg Gln 515 520 525Tyr Pro Arg Leu Gln Gly Gly Phe Val Trp Asp Trp Val Asp Gln Ser 530 535 540Leu Ile Lys Tyr Asp Glu Asn Gly Asn Pro Trp Ser Ala Tyr Gly Gly545 550 555 560Asp Phe Gly Asp Thr Pro Asn Asp Arg Gln Phe Cys Met Asn Gly Leu 565 570 575Val Phe Ala Asp Arg Thr Pro His Pro Ala Leu Thr Glu Ala Lys His 580 585 590Gln Gln Gln Phe Phe Gln Phe Arg Leu Ser Gly Gln Thr Ile Glu Val 595 600 605Thr Ser Glu Tyr Leu Phe Arg His Ser Asp Asn Glu Leu Leu His Trp 610 615 620Met Val Ala Leu Asp Gly Lys Pro Leu Ala Ser Gly Glu Val Pro Leu625 630 635 640Asp Val Ala Pro Gln Gly Lys Gln Leu Ile Glu Leu Pro Glu Leu Pro 645 650 655Gln Pro Glu Ser Ala Gly Gln Leu Trp Leu Thr Val Arg Val Val Gln 660 665 670Pro Asn Ala Thr Ala Trp Ser Glu Ala Gly His Ile Ser Ala Trp Gln 675 680 685Gln Trp Arg Leu Ala Glu Asn Leu Ser Val Thr Leu Pro Ala Ala Ser 690 695 700His Ala Ile Pro His Leu Thr Thr Ser Glu Met Asp Phe Cys Ile Glu705 710 715 720Leu Gly Asn Lys Arg Trp Gln Phe Asn Arg Gln Ser Gly Phe Leu Ser 725 730 735Gln Met Trp Ile Gly Asp Lys Lys Gln Leu Leu Thr Pro Leu Arg Asp 740 745 750Gln Phe Thr Arg Ala Pro Leu Asp Asn Asp Ile Gly Val Ser Glu Ala 755 760 765Thr Arg Ile Asp Pro Asn Ala Trp Val Glu Arg Trp Lys Ala Ala Gly 770 775 780His Tyr Gln Ala Glu Ala Ala Leu Leu Gln Cys Thr Ala Asp Thr Leu785 790 795 800Ala Asp Ala Val Leu Ile Thr Thr Ala His Ala Trp Gln His Gln Gly 805 810 815Lys Thr Leu Phe Ile Ser Arg Lys Thr Tyr Arg Ile Asp Gly Ser Gly 820 825 830Gln Met Ala Ile Thr Val Asp Val Glu Val Ala Ser Asp Thr Pro His 835 840 845Pro Ala Arg Ile Gly Leu Asn Cys Gln Leu Ala Gln Val Ala Glu Arg 850 855 860Val Asn Trp Leu Gly Leu Gly Pro Gln Glu Asn Tyr Pro Asp Arg Leu865 870 875 880Thr Ala Ala Cys Phe Asp Arg Trp Asp Leu Pro Leu Ser Asp Met Tyr 885 890 895Thr Pro Tyr Val Phe Pro Ser Glu Asn Gly Leu Arg Cys Gly Thr Arg 900 905 910Glu Leu Asn Tyr Gly Pro His Gln Trp Arg Gly Asp Phe Gln Phe Asn 915 920 925Ile Ser Arg Tyr Ser Gln Gln Gln Leu Met Glu Thr Ser His Arg His 930 935 940Leu Leu His Ala Glu Glu Gly Thr Trp Leu Asn Ile Asp Gly Phe His945 950 955 960Met Gly Ile Gly Gly Asp Asp Ser Trp Ser Pro Ser Val Ser Ala Glu 965 970 975Phe Gln Leu Ser Ala Gly Arg Tyr His Tyr Gln Leu Val Trp Cys Gln 980 985 990Lys Arg Ser Asp Tyr Lys Asp Glu Asp Leu Asp His His His His His 995 1000 1005His Arg 1010 3 6700 DNA Unknown pICAST ALC. 3gacgtcggac ttatacccgg tttgtcctat agacaccatt cgtcaaggac ggggccgagt 60cccggttctt gtctaccttg tcgacttata cccggtttgt cctatagaca ccattcgtca 120aggacggggc cgagtcccgg ttcttgtcta ccaggggtct acgccaggtc gggagtcgtc 180aaagatctct tggtagtcta caaaggtccc acggggttcc tggactttac tgggacacgg 240aataaacttg attggttagt caagcgaaga gcgaagacaa gcgcgcgaag acgaggggct 300cgagttattt tctcgggtgt tggggagtga gccccgcggt caggaggcta actgactcag 360cgggcccatg ggcacatagg ttatttggga gaacgtcaac gtaggctgaa caccagagcg 420acaaggaacc ctcccagagg agactcacta actgatgggc agtcgccccc agaaagtaaa 480cccccgagca ggccctagcc ctctggggac gggtccctgg tggctgggtg gtggccctcc 540gttcgaccgg tcgttgaata gacacagaca ggctaacaga tcacagatac tgactaaaat 600acgcggacgc agccatgatc aatcgattga tcgagacata gaccgcctgg gcaccacctt 660gactgctcaa gacttgtggg ccggcgttgg gaccctctgc agggtccctg aaacccccgg 720caaaaacacc gggctggact ccttccctca gctacacctt aggctggggc agtcctatac 780accaagacca tcctctgctc ttggattttg tcaagggcgg aggcagactt aaaaacgaaa 840gccaaacctt ggcttcggcg cgcagaacag acgacgtcgt agcaagacac aacagagaca 900gactgacaca aagacataaa cagactttta atcccggtct gacaatggtg agggaattca 960aactggaatc cattgacctt tctacagctc gccgagcgag tgttggtcag ccatctacag 1020ttcttctctg caacccaatg gaagacgaga cgtcttaccg gttggaaatt gcagcctacc 1080ggcgctctgc cgtggaaatt ggctctggag tagtgggtcc aattctagtt ccagaaaagt 1140ggaccgggcg tacctgtggg tctggtccag gggatgtagc actggaccct tcggaaccga 1200aaactggggg gagggaccca gttcgggaaa catgtgggat tcggaggcgg aggagaagga 1260ggtaggcggg gcagagaggg ggaacttgga ggagcaagct ggggcggagc taggagggaa 1320ataggtcggg agtgaggaag agatccgcgg ccggcgagat cgggtaatta tgctgagtga 1380tatcccgcta agcttagtcc ggaaccgcgc ggcctaggaa ttaattcgcg ttaaccctcc 1440accgccatcg gagctctacc cgcactaatg cctaagtgac cggcagcacc gggcgtggct 1500agcgggaagg gttgtcaatg cgtcggactt accgcttacc gcgaaacgga ccaaaggccg 1560tggtcttcgc cacggccttt cgaccgacct cacgctagaa ggactccggc tatgacagca 1620gcaggggagt ttgaccgtct acgtgccaat gctacgcggg tagatgtggt tgcactggat 1680agggtaatgc cagttaggcg gcaaacaagg gtgcctctta ggctgcccaa caatgagcga 1740gtgtaaatta caactacttt cgaccgatgt ccttccggtc tgcgcttaat aaaaactacc 1800gcaattgagc cgcaaagtag acaccacgtt gcccgcgacc cagccaatgc cggtcctgtc 1860agcaaacggc agacttaaac tggactcgcg taaaaatgcg cggcctcttt tggcggagcg 1920ccactaccac gacgcgacct cactgccgtc aatagacctt ctagtcctat acaccgccta 1980ctcgccgtaa aaggcactgc agagcaacga cgtatttggc tgatgtgttt agtcgctaaa 2040ggtacaacgg tgagcgaaat tactactaaa gtcggcgcga catgacctcc gacttcaagt 2100ctacacgccg ctcaacgcac tgatggatgc ccattgtcaa agaaataccg tcccactttg 2160cgtccagcgg tcgccgtggc gcggaaagcc gccactttaa tagctactcg caccaccaat 2220acggctagcg cagtgtgatg cagacttgca gcttttgggc tttgacacct cgcggcttta 2280gggcttagag atagcacgcc accaacttga cgtgtggcgg ctgccgtgcg actaacttcg 2340tcttcggacg ctacagccaa aggcgctcca cgcctaactt ttaccagacg acgacgactt 2400gccgttcggc aacgactaag ctccgcaatt ggcagtgctc gtagtaggag acgtaccagt 2460ccagtaccta ctcgtctgct accacgtcct ataggacgac tacttcgtct tgttgaaatt 2520gcggcacgcg acaagcgtaa taggcttggt aggcgacacc atgtgcgaca cgctggcgat 2580gccggacata caccacctac ttcggttata actttgggtg ccgtaccacg gttacttagc 2640agactggcta ctaggcgcga ccgatggccg ctactcgctt gcgcattgcg cttaccacgt 2700cgcgctagca ttagtgggct cacactagta gaccagcgac cccttactta gtccggtgcc 2760gcgattagtg ctgcgcgaca tagcgaccta gtttagacag ctaggaaggg cgggccacgt 2820catacttccg ccgcctcggc tgtggtgccg gtggctataa taaacgggct acatgcgcgc 2880gcacctactt ctggtcggga agggccgaca cggctttacc aggtagtttt ttaccgaaag 2940cgatggacct ctctgcgcgg gcgactagga aacgcttatg cgggtgcgct acccattgtc 3000agaaccgcca aagcgattta tgaccgtccg caaagcagtc ataggggcaa atgtcccgcc 3060gaagcagacc ctgacccacc tagtcagcga ctaatttata ctacttttgc cgttgggcac 3120cagccgaatg ccgccactaa aaccgctatg cggcttgcta gcggtcaaga catacttgcc 3180agaccagaaa cggctggcgt gcggcgtagg tcgcgactgc cttcgttttg tggtcgtcgt 3240caaaaaggtc aaggcaaata ggcccgtttg gtagcttcac tggtcgctta tggacaaggc 3300agtatcgcta ttgctcgagg acgtgaccta ccaccgcgac ctaccattcg gcgaccgttc 3360gccacttcac ggagacctac agcgaggtgt tccatttgtc aactaacttg acggacttga 3420tggcgtcggc ctctcgcggc ccgttgagac cgagtgtcat gcgcatcacg ttggcttgcg 3480ctggcgtacc agtcttcggc ccgtgtagtc gcggaccgtc gtcaccgcag accgcctttt 3540ggagtcacac tgcgaggggc ggcgcagggt gcggtagggc gtagactggt ggtcgcttta 3600cctaaaaacg tagctcgacc cattattcgc aaccgttaaa ttggcggtca gtccgaaaga 3660aagtgtctac acctaaccgc tattttttgt tgacgactgc ggcgacgcgc tagtcaagtg 3720ggcacgtggc gacctattgc tgtaaccgca ttcacttcgc tgggcgtaac tgggattgcg 3780gacccagctt gcgaccttcc gccgcccggt aatggtccgg cttcgtcgca acaacgtcac 3840gtgccgtcta tgtgaacgac tacgccacga ctaatgctgg cgagtgcgca ccgtcgtagt 3900ccccttttgg aataaatagt cggccttttg gatggcctaa ctaccatcac cagtttaccg 3960ctaatggcaa ctacaacttc accgctcgct atgtggcgta ggccgcgcct aaccggactt 4020gacggtcgac cgcgtccatc gtctcgccca tttgaccgag cctaatcccg gcgttctttt 4080gatagggctg gcggaatgac ggcggacaaa actggcgacc ctagacggta acagtctgta 4140catatggggc atgcagaagg gctcgctttt gccagacgcg acgccctgcg cgcttaactt 4200aataccgggt gtggtcaccg cgccgctgaa ggtcaagttg tagtcggcga tgtcagttgt 4260cgttgactac ctttggtcgg tagcggtaga cgacgtgcgc cttcttccgt gtaccgactt 4320atagctggca aaggtatacc cctaaccacc gctgctgagg acctcgggca gtcatagccg 4380ccttaaggtc gactcgcggc cagcgatggt aatggtcaac cagaccacag ttttttctag 4440actgatattt ctactcctgg agctggtagt agtagtagta gtggccatta ttatccatct 4500attcactgac taatctacgt aactagggag ctggttaagg ccaataaaag gtggtataac 4560ggcagaaaac cgttacactc ccgggccttt ggaccgggac agaagaactg ctcgtaagga 4620tccccagaaa ggggagagcg gtttccttac gttccagaca acttacagca cttccttcgt 4680caaggagacc ttcgaagaac ttctgtttgt tgcagacatc gctgggaaac gtccgtcgcc 4740ttggggggtg gaccgctgtc cacggagacg ccggttttcg gtgcacatat tctatgtgga 4800cgtttccgcc gtgttggggt cacggtgcaa cactcaacct atcaacacct ttctcagttt 4860accgagagga gttcgcataa gttgttcccc gacttcctac gggtcttcca tggggtaaca 4920taccctagac tagaccccgg agccacgtgt acgaaatgta cacaaatcag ctccaatttt 4980ttgcagatcc ggggggcttg gtgcccctgc accaaaagga aactttttgt gctactatta 5040tggtactaac ttgttctacc taacgtgcgt ccaagaggcc ggcgaaccca cctctccgat 5100aagccgatac tgacccgtgt tgtctgttag ccgacgagac tacggcggca caaggccgac 5160agtcgcgtcc ccgcgggcca agaaaaacag ttctggctgg acaggccacg ggacttactt 5220gacgtcctgc tccgtcgcgc cgatagcacc gaccggtgct gcccgcaagg aacgcgtcga 5280cacgagctgc aacagtgact tcgcccttcc ctgaccgacg ataacccgct tcacggcccc 5340gtcctagagg acagtagagt ggaacgagga cggctctttc ataggtagta ccgactacgt 5400tacgccgccg acgtatgcga actaggccga tggacgggta agctggtggt tcgctttgta 5460gcgtagctcg ctcgtgcatg agcctacctt cggccagaac agctagtcct actagacctg 5520cttctcgtag tccccgagcg cggtcggctt gacaagcggt ccgagttccg cgcgtacggg 5580ctgccgctcc tagagcagca ctgggtaccg ctacggacga acggcttata gtaccacctt 5640ttaccggcga aaagacctaa gtagctgaca ccggccgacc cacaccgcct ggcgatagtc 5700ctgtatcgca accgatgggc actataacga cttctcgaac cgccgcttac ccgactggcg 5760aaggagcacg aaatgccata gcggcgaggg ctaagcgtcg cgtagcggaa gatagcggaa 5820gaactgctca agaagactcg ccctgagacc ccaagcgtag ctattttatt ttctaaaata 5880aatcagaggt ctttttcccc ccttactttc tggggtggac atccaaaccg ttcgatcgaa 5940ttcattgcgg taaaacgttc cgtacctttt tatgtattga ctcttatctc ttcaagtcta 6000gttccagtcc ttgtctacct tgtcgactta tacccggttt gtcctataga caccattcgt 6060caaggacggg gccgagtccc ggttcttgtc taccttgtcg acttataccc ggtttgtcct 6120atagacacca ttcgtcaagg acggggccga gtcccggttc ttgtctacca ggggtctacg 6180ccaggtcggg agtcgtcaaa gatctcttgg tagtctacaa aggtcccacg gggttcctgg 6240actttactgg gacacggaat aaacttgatt ggttagtcaa gcgaagagcg aagacaagcg 6300cgcgaagacg aggggctcga gttattttct cgggtgttgg ggagtgagcc ccgcggtcag 6360gaggctaact gactcagcgg gcccatgggc acataggtta tttgggagaa cgtcaacgta 6420ggctgaacac cagagcgaca aggaaccctc ccagaggaga ctcactaact gatgggcagt 6480cgcccccaga aagtaagtac gtcgtacata gttttaatta aaccaaaaaa aagaattcat 6540aaatgtaatt taccggtatc aacgtaatta cttagccggt tgcgcgcccc tctccgccaa 6600acgcataacc gcgagaaggc gaaggagcga gtgactgagc gacgcgagcc agcaagccga 6660cgccgctcgc catagtcgag tgagtttccg ccattatgcc 6700 4 8518 DNA Unknown pICAST ALN. 4ctgcagcctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca 60gggccaagaa cagatggaac agctgaatat gggccaaaca ggatatctgt ggtaagcagt 120tcctgccccg gctcagggcc aagaacagat ggtccccaga tgcggtccag ccctcagcag 180tttctagaga accatcagat gtttccaggg tgccccaagg acctgaaatg accctgtgcc 240ttatttgaac taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga 300gctcaataaa agagcccaca acccgtcact cggggcgcca gtcctccgat tgactgagtc 360gcccgggtac ccgtgtatcc aataaaccct cttgcagttg catccgactt gtggtctcgc 420tgttccttgg gagggtctcc tctgagtgat tgactacccg tcagcggggg tctttcattt 480gggggctcgt ccgggatcgg gagacccctg cccagggacc accgacccac caccgggagg 540caagctggcc agcaacttat ctgtgtctgt ccgattgtct agtgtctatg actgatttta 600tgcgcctgcg tcggtactag ttagctaact agctctgtat ctggcggacc cgtggtggaa 660ctgacgagtt ctgaacaccc ggccgcaacc ctgggagacg tcccagggac tttgggggcc 720gtttttgtgg cccgacctga ggaagggagt cgatgtggaa tccgaccccg tcaggatatg 780tggttctggt aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt 840cggtttggaa ccgaagccgc gcgtcttgtc tgctgcagca tcgttctgtg ttgtctctgt 900ctgactgtgt ttctgtattt gtctgaaaat tagggccaga ctgttaccac tcccttaagt 960ttgaccttag gtaactggaa agatgtcgag cggctcgctc acaaccagtc ggtagatgtc 1020aagaagagac gttgggttac cttctgctct gcagaatggc caacctttaa cgtcggatgg 1080ccgcgagacg gcacctttaa ccgagacctc atcacccagg ttaagatcaa ggtcttttca 1140cctggcccgc atggacaccc agaccaggtc ccctacatcg tgacctggga agccttggct 1200tttgaccccc ctccctgggt caagcccttt gtacacccta agcctccgcc tcctcttcct 1260ccatccgccc cgtctctccc ccttgaacct cctcgttcga ccccgcctcg atcctccctt 1320tatccagccc tcactccttc tctaggcgcc ggccgctcta gcccattaat acgactcact 1380atagggcgat tcgaacacca tgcaccatca tcatcatcac gtcgactata aagatgagga 1440cctcgagatg ggcgtgatta cggattcact ggccgtcgtg gcccgcaccg atcgcccttc 1500ccaacagtta cgcagcctga atggcgaatg gcgctttgcc tggtttccgg caccagaagc 1560ggtgccggaa agctggctgg agtgcgatct tcctgaggcc gatactgtcg tcgtcccctc 1620aaactggcag atgcacggtt acgatgcgcc catctacacc aacgtgacct atcccattac 1680ggtcaatccg ccgtttgttc ccacggagaa tccgacgggt tgttactcgc tcacatttaa 1740tgttgatgaa agctggctac aggaaggcca gacgcgaatt atttttgatg gcgttaactc 1800ggcgtttcat ctgtggtgca acgggcgctg ggtcggttac ggccaggaca gtcgtttgcc 1860gtctgaattt gacctgagcg catttttacg cgccggagaa aaccgcctcg cggtgatggt 1920gctgggctgg agtgacggca gttatctgga agatcaggat atgtggcgga tgagcggcat 1980tttccgtgac gtctcgttgc tgcataaacc gactacacaa atcagcgatt tccatgttgc 2040cactcgcttt aatgatgatt rcagccgcgc tgtactggag gctgaagttc agatgtgcgg 2100cgagttgcgt gactacctac gggtaacagt ttctttatgg cagggtgaaa cgcaggtcgc 2160cagcggcacc gcgcctttcg gcggtgaaat tatcgatgag cgtggtggtt atgccgatcg 2220cgtcacacta cgtctgaacg tcgaaaaccc gaaactgtgg agcgccgaaa tcccgaatct 2280ctatcgtgcg gtggttgaac tgcacaccgc cgacggcacg ctgattgaag cagaagcctg 2340cgatgtcggt ttccgcgagg tgcggattga aaatggtctg ctgctgctga acggcaagcc 2400gttgctgatt cgaggcgtta accgtcacga gcatcatcct ctgcatggtc aggtcatgga 2460tgagcagacg atggtgcagg atatcctgct gatgaagcag aacaacttta acgccgtgcg 2520ctgttcgcat tatccgaacc atccgctgtg gtacacgctg tgcgaccgct acggcctgta 2580tgtggtggat gaagccaata ttgaaaccca cggcatggtg ccaatgaatc gtctgaccga 2640tgatccgcgc tggctaccgg cgatgagcga acgcgtaacg cgaatggtgc agcgcgatcg 2700taatcacccg agtgtgatca tctggtcgct ggggaatgaa tcaggccacg gcgctaatca 2760cgacgcgctg tatcgctgga tcaaatctgt cgatccttcc cgcccggtgc agtatgaagg 2820cggcggagcc gacaccacgg ccaccgatat tatttgcccg atgtacgcgc gcgtggatga 2880agaccagccc ttcccggctg tgccgaaatg gtccatcaaa aaatggcttt cgctacctgg 2940agagacgcgc ccgctgatcc tttgcgaata cgcccacgcg atgggtaaca gtcttggcgg 3000tttcgctaaa tactggcagg cgtttcgtca gtatccccgt ttacagggcg gcttcgtctg 3060ggactgggtg gatcagtcgc tgattaaata tgatgaaaac ggcaacccgt ggtcggctta 3120cggcggtgat tttggcgata cgccgaacga tcgccagttc tgtatgaacg gtctggtctt 3180tgccgaccgc acgccgcatc cagcgctgac ggaagcaaaa caccagcagc agtttttcca 3240gttccgttta tccgggcaaa ccatcgaagt gaccagcgaa tacctgttcc gtcatagcga 3300taacgagctc ctgcactgga tggtggcgct ggatggtaag ccgctggcaa gcggtgaagt 3360gcctctggat gtcgctccac aaggtaaaca gttgattgaa ctgcctgaac taccgcagcc 3420ggagagcgcc gggcaactct ggctcacagt acgcgtagtg caaccgaacg cgaccgcatg 3480gtcagaagcc gggcacatca gcgcctggca gcagtggcgt ctggcggaaa acctcagtgt 3540gacgctcccc gccgcgtccc acgccatccc gcatctgacc accagcgaaa tggatttttg 3600catcgagctg ggtaataagc gttggcaatt taaccgccag tcaggctttc tttcacagat 3660gtggattggc gataaaaaac aactgctgac gccgctgcgc gatcagttca cccgtgcacc 3720gctggataac gacattggcg taagtgaagc gacccgcatt gaccctaacg cctgggtcga 3780acgctggaag gcggcgggcc attaccaggc cgaagcagcg ttgttgcagt gcacggcaga 3840tacacttgct gatgcggtgc tgattacgac cgctcacgcg tggcagcatc aggggaaaac 3900cttatttatc agccggaaaa cctaccggat tgatggtagt ggtcaaatgg cgattaccgt 3960tgatgttgaa gtggcgagcg atacaccgca tccggcgcgg attggcctga actgccagct 4020ggcgcaggta gcagagcggg taaactggct cggattaggg ccgcaagaaa actatcccga 4080ccgccttact gccgcctgtt ttgaccgctg ggatctgcca ttgtcagaca tgtatacccc 4140gtacgtcttc ccgagcgaaa acggtctgcg ctgcgggacg cgcgaattga attatggccc 4200acaccagtgg cgcggcgact tccagttcaa catcagccgc tacagtcaac agcaactgat 4260ggaaaccagc catcgccatc tgctgcacgc ggaagaaggc acatggctga atatcgacgg 4320tttccatatg gggattggtg gcgacgactc ctggagcccg tcagtatcgg cggaattcca 4380gctgagcgcc ggtcgctacc attaccagtt ggtctggtgt caaaaaagat ctggaggtgg 4440tggcagcagg ccttggcgcg ccggatcctt aattaacaat tgaccggtaa taataggtag 4500ataagtgact gattagatgc attgatccct cgaccaattc cggttatttt ccaccatatt 4560gccgtctttt ggcaatgtga gggcccggaa acctggccct gtcttcttga cgagcattcc 4620taggggtctt tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc 4680agttcctctg gaagcttctt gaagacaaac aacgtctgta gcgacccttt gcaggcagcg 4740gaacccccca cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat aagatacacc 4800tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa 4860atggctctcc tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg taccccattg 4920tatgggatct gatctggggc ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa 4980aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgatgataa 5040taccatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct 5100attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct 5160gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga 5220actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc 5280tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg 5340gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc 5400aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca 5460tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga 5520cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc 5580cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga 5640aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca 5700ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg 5760cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct 5820tcttgacgag ttcttctgag cgggactctg gggttcgcat cgataaaata aaagatttta 5880tttagtctcc agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg caagctagct 5940taagtaacgc cattttgcaa ggcatggaaa aatacataac tgagaataga gaagttcaga 6000tcaaggtcag gaacagatgg aacagctgaa tatgggccaa acaggatatc tgtggtaagc 6060agttcctgcc ccggctcagg gccaagaaca gatggaacag ctgaatatgg gccaaacagg 6120atatctgtgg taagcagttc ctgccccggc tcagggccaa gaacagatgg tccccagatg 6180cggtccagcc ctcagcagtt tctagagaac catcagatgt ttccagggtg ccccaaggac 6240ctgaaatgac cctgtgcctt atttgaacta accaatcagt tcgcttctcg cttctgttcg 6300cgcgcttctg ctccccgagc tcaataaaag agcccacaac ccctcactcg gggcgccagt 6360cctccgattg actgagtcgc ccgggtaccc gtgtatccaa taaaccctct tgcagttgca 6420tccgacttgt ggtctcgctg ttccttggga gggtctcctc tgagtgattg actacccgtc 6480agcgggggtc tttcattcat gcagcatgta tcaaaattaa tttggttttt tttcttaagt 6540atttacatta aatggccata gttgcattaa tgaatcggcc aacgcgcggg gagaggcggt 6600ttgcgtattg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc 6660tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg 6720ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg 6780ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac 6840gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg 6900gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct 6960ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg 7020tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct 7080gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac 7140tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt 7200tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc 7260tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 7320ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat 7380ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac 7440gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttgcggc 7500cgcaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 7560agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 7620gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 7680ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 7740gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 7800cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 7860acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 7920cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 7980cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 8040ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 8100tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 8160atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 8220tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 8280actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 8340aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 8400ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 8460ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttc 8518 5 8518 DNA Unknown pICAST ALN. 5gacgtcggac ttatacccgg tttgtcctat agacaccatt cgtcaaggac ggggccgagt 60cccggttctt gtctaccttg tcgacttata cccggtttgt cctatagaca ccattcgtca 120aggacggggc cgagtcccgg ttcttgtcta ccaggggtct acgccaggtc gggagtcgtc 180aaagatctct tggtagtcta caaaggtccc acggggttcc tggactttac tgggacacgg 240aataaacttg attggttagt caagcgaaga gcgaagacaa gcgcgcgaag acgaggggct 300cgagttattt tctcgggtgt tggggagtga gccccgcggt caggaggcta actgactcag 360cgggcccatg ggcacatagg ttatttggga gaacgtcaac gtaggctgaa caccagagcg 420acaaggaacc ctcccagagg agactcacta actgatgggc agtcgccccc agaaagtaaa 480cccccgagca ggccctagcc ctctggggac gggtccctgg tggctgggtg gtggccctcc 540gttcgaccgg tcgttgaata gacacagaca ggctaacaga tcacagatac tgactaaaat 600acgcggacgc agccatgatc aatcgattga tcgagacata gaccgcctgg gcaccacctt 660gactgctcaa gacttgtggg ccggcgttgg gaccctctgc agggtccctg aaacccccgg 720caaaaacacc gggctggact ccttccctca gctacacctt aggctggggc agtcctatac 780accaagacca tcctctgctc ttggattttg tcaagggcgg aggcagactt aaaaacgaaa 840gccaaacctt ggcttcggcg cgcagaacag acgacgtcgt agcaagacac aacagagaca 900gactgacaca aagacataaa cagactttta atcccggtct gacaatggtg agggaattca 960aactggaatc cattgacctt tctacagctc gccgagcgag tgttggtcag ccatctacag 1020ttcttctctg caacccaatg gaagacgaga cgtcttaccg gttggaaatt gcagcctacc 1080ggcgctctgc cgtggaaatt ggctctggag tagtgggtcc aattctagtt ccagaaaagt 1140ggaccgggcg tacctgtggg tctggtccag gggatgtagc actggaccct tcggaaccga 1200aaactggggg gagggaccca gttcgggaaa catgtgggat tcggaggcgg aggagaagga 1260ggtaggcggg gcagagaggg ggaacttgga ggagcaagct ggggcggagc taggagggaa 1320ataggtcggg agtgaggaag agatccgcgg ccggcgagat cgggtaatta tgctgagtga 1380tatcccgcta agcttgtggt acgtggtagt agtagtagtg cagctgatat ttctactcct 1440ggagctctac ccgcactaat gcctaagtga ccggcagcac cgggcgtggc tagcgggaag 1500ggttgtcaat gcgtcggact taccgcttac cgcgaaacgg accaaaggcc gtggtcttcg 1560ccacggcctt tcgaccgacc tcacgctaga aggactccgg ctatgacagc agcaggggag 1620tttgaccgtc tacgtgccaa tgctacgcgg gtagatgtgg ttgcactgga tagggtaatg 1680ccagttaggc ggcaaacaag ggtgcctctt aggctgccca acaatgagcg agtgtaaatt 1740acaactactt tcgaccgatg tccttccggt ctgcgcttaa taaaaactac cgcaattgag 1800ccgcaaagta gacaccacgt tgcccgcgac ccagccaatg ccggtcctgt cagcaaacgg 1860cagacttaaa ctggactcgc gtaaaaatgc gcggcctctt ttggcggagc gccactacca 1920cgacgcgacc tcactgccgt caatagacct tctagtccta tacaccgcct actcgccgta 1980aaaggcactg cagagcaacg acgtatttgg ctgatgtgtt tagtcgctaa aggtacaacg 2040gtgagcgaaa ttactactaa agtcggcgcg acatgacctc cgacttcaag tctacacgcc 2100gctcaacgca ctgatggatg cccattgtca aagaaatacc gtcccacttt gcgtccagcg 2160gtcgccgtgg cgcggaaagc cgccacttta atagctactc gcaccaccaa tacggctagc 2220gcagtgtgat gcagacttgc agcttttggg ctttgacacc tcgcggcttt agggcttaga 2280gatagcacgc caccaacttg acgtgtggcg gctgccgtgc gactaacttc gtcttcggac 2340gctacagcca aaggcgctcc acgcctaact tttaccagac gacgacgact tgccgttcgg 2400caacgactaa gctccgcaat tggcagtgct cgtagtagga gacgtaccag tccagtacct 2460actcgtctgc taccacgtcc tataggacga ctacttcgtc ttgttgaaat tgcggcacgc 2520gacaagcgta ataggcttgg taggcgacac catgtgcgac acgctggcga tgccggacat 2580acaccaccta cttcggttat aactttgggt gccgtaccac ggttacttag cagactggct 2640actaggcgcg accgatggcc gctactcgct tgcgcattgc gcttaccacg tcgcgctagc 2700attagtgggc tcacactagt agaccagcga ccccttactt agtccggtgc cgcgattagt 2760gctgcgcgac atagcgacct agtttagaca gctaggaagg gcgggccacg tcatacttcc 2820gccgcctcgg ctgtggtgcc ggtggctata ataaacgggc tacatgcgcg cgcacctact 2880tctggtcggg aagggccgac acggctttac caggtagttt tttaccgaaa gcgatggacc 2940tctctgcgcg ggcgactagg aaacgcttat gcgggtgcgc tacccattgt cagaaccgcc 3000aaagcgattt atgaccgtcc gcaaagcagt cataggggca aatgtcccgc cgaagcagac 3060cctgacccac ctagtcagcg actaatttat actacttttg ccgttgggca ccagccgaat 3120gccgccacta aaaccgctat gcggcttgct agcggtcaag acatacttgc cagaccagaa 3180acggctggcg tgcggcgtag gtcgcgactg ccttcgtttt gtggtcgtcg tcaaaaaggt 3240caaggcaaat aggcccgttt ggtagcttca ctggtcgctt atggacaagg cagtatcgct 3300attgctcgag gacgtgacct accaccgcga cctaccattc ggcgaccgtt cgccacttca 3360cggagaccta cagcgaggtg ttccatttgt caactaactt gacggacttg atggcgtcgg 3420cctctcgcgg cccgttgaga ccgagtgtca tgcgcatcac gttggcttgc gctggcgtac 3480cagtcttcgg cccgtgtagt cgcggaccgt cgtcaccgca gaccgccttt tggagtcaca 3540ctgcgagggg cggcgcaggg tgcggtaggg cgtagactgg tggtcgcttt acctaaaaac 3600gtagctcgac ccattattcg caaccgttaa attggcggtc agtccgaaag aaagtgtcta 3660cacctaaccg ctattttttg ttgacgactg cggcgacgcg ctagtcaagt gggcacgtgg 3720cgacctattg ctgtaaccgc attcacttcg ctgggcgtaa ctgggattgc ggacccagct 3780tgcgaccttc cgccgcccgg taatggtccg gcttcgtcgc aacaacgtca cgtgccgtct 3840atgtgaacga ctacgccacg actaatgctg gcgagtgcgc accgtcgtag tccccttttg 3900gaataaatag tcggcctttt ggatggccta actaccatca ccagtttacc gctaatggca 3960actacaactt caccgctcgc tatgtggcgt aggccgcgcc taaccggact tgacggtcga 4020ccgcgtccat cgtctcgccc atttgaccga gcctaatccc ggcgttcttt tgatagggct 4080ggcggaatga cggcggacaa aactggcgac cctagacggt aacagtctgt acatatgggg 4140catgcagaag ggctcgcttt tgccagacgc gacgccctgc gcgcttaact taataccggg 4200tgtggtcacc gcgccgctga aggtcaagtt gtagtcggcg atgtcagttg tcgttgacta 4260cctttggtcg gtagcggtag acgacgtgcg ccttcttccg tgtaccgact tatagctgcc 4320aaaggtatac ccctaaccac cgctgctgag gacctcgggc agtcatagcc gccttaaggt 4380cgactcgcgg ccagcgatgg taatggtcaa ccagaccaca gttttttcta gacctccacc 4440accgtcgtcc ggaaccgcgc ggcctaggaa ttaattgtta actggccatt attatccatc 4500tattcactga ctaatctacg taactaggga gctggttaag gccaataaaa ggtggtataa 4560cggcagaaaa ccgttacact cccgggcctt tggaccggga cagaagaact gctcgtaagg 4620atccccagaa aggggagagc ggtttcctta cgttccagac aacttacagc acttccttcg 4680tcaaggagac cttcgaagaa cttctgtttg ttgcagacat cgctgggaaa cgtccgtcgc 4740cttggggggt ggaccgctgt ccacggagac gccggttttc ggtgcacata ttctatgtgg 4800acgtttccgc cgtgttgggg tcacggtgca acactcaacc tatcaacacc tttctcagtt 4860taccgagagg agttcgcata agttgttccc cgacttccta cgggtcttcc atggggtaac 4920ataccctaga ctagaccccg gagccacgtg tacgaaatgt acacaaatca gctccaattt 4980tttgcagatc cggggggctt ggtgcccctg caccaaaagg aaactttttg tgctactatt 5040atggtactaa cttgttctac ctaacgtgcg tccaagaggc cggcgaaccc acctctccga 5100taagccgata ctgacccgtg ttgtctgtta gccgacgaga ctacggcggc acaaggccga 5160cagtcgcgtc cccgcgggcc aagaaaaaca gttctggctg gacaggccac gggacttact 5220tgacgtcctg ctccgtcgcg ccgatagcac cgaccggtgc tgcccgcaag gaacgcgtcg 5280acacgagctg caacagtgac ttcgcccttc cctgaccgac gataacccgc ttcacggccc 5340cgtcctagag gacagtagag tggaacgagg acggctcttt cataggtagt accgactacg 5400ttacgccgcc gacgtatgcg aactaggccg atggacgggt aagctggtgg ttcgctttgt 5460agcgtagctc gctcgtgcat gagcctacct tcggccagaa cagctagtcc tactagacct 5520gcttctcgta gtccccgagc gcggtcggct tgacaagcgg tccgagttcc gcgcgtacgg 5580gctgccgctc ctagagcagc actgggtacc gctacggacg aacggcttat agtaccacct 5640tttaccggcg aaaagaccta agtagctgac accggccgac ccacaccgcc tggcgatagt 5700cctgtatcgc aaccgatggg cactataacg acttctcgaa ccgccgctta cccgactggc 5760gaaggagcac gaaatgccat agcggcgagg gctaagcgtc gcgtagcgga agatagcgga 5820agaactgctc aagaagactc gccctgagac cccaagcgta gctattttat tttctaaaat 5880aaatcagagg tctttttccc cccttacttt ctggggtgga catccaaacc gttcgatcga 5940attcattgcg gtaaaacgtt ccgtaccttt ttatgtattg actcttatct cttcaagtct 6000agttccagtc cttgtctacc ttgtcgactt atacccggtt tgtcctatag acaccattcg 6060tcaaggacgg ggccgagtcc cggttcttgt ctaccttgtc gacttatacc cggtttgtcc 6120tatagacacc attcgtcaag gacggggccg agtcccggtt cttgtctacc aggggtctac 6180gccaggtcgg gagtcgtcaa agatctcttg gtagtctaca aaggtcccac ggggttcctg 6240gactttactg ggacacggaa taaacttgat tggttagtca agcgaagagc gaagacaagc 6300gcgcgaagac gaggggctcg agttattttc tcgggtgttg gggagtgagc cccgcggtca 6360ggaggctaac tgactcagcg ggcccatggg cacataggtt atttgggaga acgtcaacgt 6420aggctgaaca ccagagcgac aaggaaccct cccagaggag actcactaac tgatgggcag 6480tcgcccccag aaagtaagta cgtcgtacat agttttaatt aaaccaaaaa aaagaattca 6540taaatgtaat ttaccggtat caacgtaatt acttagccgg ttgcgcgccc ctctccgcca 6600aacgcataac cgcgagaagg cgaaggagcg agtgactgag cgacgcgagc cagcaagccg 6660acgccgctcg ccatagtcga gtgagtttcc gccattatgc caataggtgt cttagtcccc 6720tattgcgtcc tttcttgtac actcgttttc cggtcgtttt ccggtccttg gcatttttcc 6780ggcgcaacga ccgcaaaaag gtatccgagg cggggggact gctcgtagtg tttttagctg 6840cgagttcagt ctccaccgct ttgggctgtc ctgatatttc tatggtccgc aaagggggac 6900cttcgaggga gcacgcgaga ggacaaggct gggacggcga atggcctatg gacaggcgga 6960aagagggaag cccttcgcac cgcgaaagag tatcgagtgc gacatccata gagtcaagcc 7020acatccagca agcgaggttc gacccgacac acgtgcttgg ggggcaagtc gggctggcga 7080cgcggaatag gccattgata gcagaactca ggttgggcca ttctgtgctg aatagcggtg 7140accgtcgtcg gtgaccattg tcctaatcgt ctcgctccat acatccgcca cgatgtctca 7200agaacttcac caccggattg atgccgatgt gatcttcttg tcataaacca tagacgcgag 7260acgacttcgg tcaatggaag cctttttctc aaccatcgag aactaggccg tttgtttggt 7320ggcgaccatc gccaccaaaa aaacaaacgt tcgtcgtcta atgcgcgtct ttttttccta 7380gagttcttct aggaaactag aaaagatgcc ccagactgcg agtcaccttg cttttgagtg 7440caattcccta aaaccagtac tctaatagtt tttcctagaa gtggatctag gaaaacgccg 7500gcgtttagtt agatttcata tatactcatt tgaaccagac tgtcaatggt tacgaattag 7560tcactccgtg gatagagtcg ctagacagat aaagcaagta ggtatcaacg gactgagggg 7620cagcacatct attgatgcta tgccctcccg aatggtagac cggggtcacg acgttactat 7680ggcgctctgg gtgcgagtgg ccgaggtcta aatagtcgtt atttggtcgg tcggccttcc 7740cggctcgcgt cttcaccagg acgttgaaat aggcggaggt aggtcagata attaacaacg 7800gcccttcgat ctcattcatc aagcggtcaa ttatcaaacg cgttgcaaca acggtaacga 7860tgtccgtagc accacagtgc gagcagcaaa ccataccgaa gtaagtcgag gccaagggtt 7920gctagttccg ctcaatgtac tagggggtac aacacgtttt ttcgccaatc gaggaagcca 7980ggaggctagc aacagtcttc attcaaccgg cgtcacaata gtgagtacca ataccgtcgt 8040gacgtattaa gagaatgaca gtacggtagg cattctacga aaagacactg accactcatg 8100agttggttca gtaagactct tatcacatac gccgctggct caacgagaac gggccgcagt 8160tatgccctat tatggcgcgg tgtatcgtct tgaaattttc acgagtagta accttttgca 8220agaagccccg cttttgagag ttcctagaat ggcgacaact ctaggtcaag ctacattggg 8280tgagcacgtg ggttgactag aagtcgtaga aaatgaaagt ggtcgcaaag acccactcgt 8340ttttgtcctt ccgttttacg gcgttttttc ccttattccc gctgtgcctt tacaacttat 8400gagtatgaga aggaaaaagt tataataact tcgtaaatag tcccaataac agagtactcg 8460cctatgtata aacttacata aatcttttta tttgtttatc cccaaggcgc gtgtaaag 8518 6 8175 DNA Unknown pICAST OMC. 6ctgcagcctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca 60gggccaagaa cagatggaac agctgaatat gggccaaaca ggatatctgt ggtaagcagt 120tcctgccccg gctcagggcc aagaacagat ggtccccaga tgcggtccag ccctcagcag 180tttctagaga accatcagat gtttccaggg tgccccaagg acctgaaatg accctgtgcc 240ttatttgaac taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga 300gctcaataaa agagcccaca acccctcact cggggcgcca gtcctccgat tgactgagtc 360gcccgggtac ccgtgtatcc aataaaccct cttgcagttg catccgactt gtggtctcgc 420tgttccttgg gaggytctcc tctgagtgat tgactacccg tcagcggggg tctttcattt 480gggggctcgt ccgggatcgg gagacccctg cccagggacc accgacccac caccgggagg 540caagctggcc agcaacttat ctgtgtctgt ccgattgtct agtgtctatg actgatttta 600tgcgcctgcg tcggtactag ttagctaact agctctgtat ctggcggacc cgtggtggaa 660ctgacgagtt ctgaacaccc ggccgcaacc ctgggagacg tcccagggac tttgggggcc 720gtttttgtgg cccgacctga ggaagggagt cgatgtggaa tccgaccccg tcaggatatg 780tggttctggt aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt 840cggtttggaa ccgaagccgc gcgtcttgtc tgctgcagca tcgttctgtg ttgtctctgt 900ctgactgtgt ttctgtattt gtctgaaaat tagggccaga ctgttaccac tcccttaagt 960ttgaccttag gtaactggaa agatgtcgag cggctcgctc acaaccagtc ggtagatgtc 1020aagaagagac gttgggttac cttctgctct gcagaatggc caacctttaa cgtcggatgg 1080ccgcgagacg gcacctttaa ccgagacctc atcacccagg ttaagatcaa ggtcttttca 1140cctggcccgc atggacaccc agaccaggtc ccctacatcg tgacctggga agccttggct 1200tttgaccccc ctccctgggt caagcccttt gtacacccta agcctccgcc tcctcttcct 1260ccatccgccc cgtctctccc ccttgaacct cctcgttcga ccccgcctcg atcctccctt 1320tatccagccc tcactccttc tctaggcgcc ggccgctcta gcccattaat acgactcact 1380atagggcgat tcgaatcagg ccttggcgcg ccggatcctt aattaagcgc aattgggagg 1440tggcggtagc ctcgagatgg gcgtgattac ggattcactg gccgtcgttt tacaacgtcg 1500tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc 1560cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tacgcagcct 1620gaatggcgaa tggcgctttg cctggtttcc ggcaccagaa gcggtgccgg aaagctggct 1680ggagtgcgat cttcctgagg ccgatactgt cgtcgtcccc tcaaactggc agatgcacgg 1740ttacgatgcg cccatctaca ccaacgtgac ctatcccatt acggtcaatc cgccgtttgt 1800tcccacggag aatccgacgg gttgttactc gctcacattt aatgttgatg aaagctggct 1860acaggaaggc cagacgcgaa ttatttttga tggcgttaac tcggcgtttc atctgtggtg 1920caacgggcgc tgggtcggtt acggccagga cagtcgtttg ccgtctgaat ttgacctgag 1980cgcattttta cgcgccggag aaaaccgcct cgcggtgatg gtgctgcgct ggagtgacgg 2040cagttatctg gaagatcagg atatgtggcg gatgagcggc attttccgtg acgtctcgtt 2100gctgcataaa ccgactacac aaatcagcga tttccatgtt gccactcgct ttaatgatga 2160tttcagccgc gctgtactgg aggctgaagt tcagatgtgc ggcgagttgc gtgactacct 2220acgggtaaca gtttctttat ggcagggtga aacgcaggtc gccagcggca ccgcgccttt 2280cggcggtgaa attatcgatg agcgtggtgg ttatgccgat cgcgtcacac tacgtctgaa 2340cgtcgaaaac ccgaaactgt ggagcgccga aatcccgaat ctctatcgtg cggtggttga 2400actgcacacc gccgacggca cgctgattga agcagaagcc tgcgatgtcg gtttccgcga 2460ggtgcggatt gaaaatggtc tgctgctgct gaacggcaag ccgttgctga ttcgaggcgt 2520taaccgtcac gagcatcatc ctctgcatgg tcaggtcatg gatgagcaga cgatggtgca 2580ggatatcctg ctgatgaagc agaacaactt taacgccgtg cgctgttcgc attatccgaa 2640ccatccgctg tggtacacgc tgtgcgaccg ctacggcctg tatgtggtgg atgaagccaa 2700tattgaaacc cacggcatgg tgccaatgaa tcgtctgacc gatgatccgc gctggctacc 2760ggcgatgagc gaacgcgtaa cgcgaatggt gcagcgcgat cgtaatcacc cgagtgtgat 2820catctggtcg ctggggaatg aatcaggcca cggcgctaat cacgacgcgc tgtatcgctg 2880gatcaaatct gtcgatcctt cccgcccggt gcagtatgaa ggcggcggag ccgacaccac 2940ggccaccgat attatttgcc cgatgtacgc gcgcgtggat gaagaccagc ccttcccggc 3000tgtgccgaaa tggtccatca aaaaatggct ttcgctacct ggagagacgc gcccgctgat 3060cctttgcgaa tacgcccacg cgatgggtaa cagtcttggc ggtttcgcta aatactggca 3120ggcgtttcgt cagtatcccc gtttacaggg cggcttcgtc tgggactggg tggatcagtc 3180gctgattaaa tatgatgaaa acggcaaccc gtggtcggct tacggcggtg attttggcga 3240tacgccgaac gatcgccagt tctgtatgaa cggtctggtc tttgccgacc gcacgccgca 3300tccagcgctg acggaagcaa aacaccagca gcagtttttc cagttccgtt tatccgggca 3360aaccatcgaa gtgaccagcg aatacctgtt ccgtcatagc gataacgagc tcctgcactg 3420gatggtggcg ctggatggta agccgctggc aagcggtgaa gtgcctctgg atgtcgctcc 3480acaaggtaaa cagttgattg aactgcctga actaccgcag ccggagagcg ccgggcaact 3540ctggctcaca gtacgcgtag tgcaaccgaa cgcgaccgca tggtcagaag ccgggcacat 3600cagcgcctgg cagcagtggc gtctggcgga aaacctcagt gtgacgctcc ccgccgcgtc 3660ccacgccatc ccgcatctga ccaccagcga aatggatttt tgcatcgagc tgggtaataa 3720gcgttggcaa tttaaccgcc agtcaggctt tctttcacag atgtggattg gcgataaaaa 3780acaactgctg acgccgctgc gcgatcagtt cacccgtgtc gatagatctg aacagaaact 3840catttccgaa gaagacctag tcgaccatca tcatcatcat caccggtaat aataggtaga 3900taagtgactg attagatgca tttcgactag atccctcgac caattccggt tattttccac 3960catattgccg tcttttggca atgtgagggc ccggaaacct ggccctgtct tcttgacgag 4020cattcctagg ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa 4080ggaagcagtt cctctggaag cttcttgaag acaaacaacg tctgtagcga ccctttgcag 4140gcagcggaac cccccacctg gcgacaggtg cctctgcggc caaaagccac gtgtataaga 4200tacacctgca aaggcggcac aaccccagtg ccacgttgtg agttggatag ttgtggaaag 4260agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg aaggatgccc agaaggtacc 4320ccattgtatg ggatctgatc tggggcctcg gtgcacatgc tttacatgtg tttagtcgag 4380gttaaaaaac gtctaggccc cccgaaccac ggggacgtgg ttttcctttg aaaaacacga 4440tgataatacc atgaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga 4500aaagttcgac agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt 4560cagcttcgat gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt 4620ctacaaagat cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt 4680gcttgacatt ggggaattta gcgagagcct gacctattgc atctcccgcc gtgcacaggg 4740tgtcacgttg caagacctgc ctgaaaccga actgcccgct gttctgcagc cggtcgcgga 4800ggccatggat gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg 4860accgcaagga atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc 4920ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc 4980tctcgatgag ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc 5040ggatttcggc tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg 5100gagcgaggcg atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc 5160gtggttggct tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc 5220aggatcgccg cggctccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag 5280cttggttgac ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt 5340ccgatccgga gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg 5400gaccgatggc tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc 5460gagggcaaag gaatagagta gatgccgacc gggatctatc gataaaataa aagattttat 5520ttagtctcca gaaaaagggg ggaatgaaag accccacctg taggtttggc aagctagctt 5580aagtaacgcc attttgcaag gcatggaaaa atacataact gagaatagag aagttcagat 5640caaggtcagg aacagatgga acagctgaat atgggccaaa caggatatct gtggtaagca 5700gttcctgccc cggctcaggg ccaagaacag atggaacagc tgaatatggg ccaaacagga 5760tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc 5820ggtccagccc tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc 5880tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc 5940gcgcttctgc tccccgagct caataaaaga gcccacaacc cctcactcgg ggcgccagtc 6000ctccgattga ctgagtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat 6060ccgacttgtg gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccgtca 6120gcgggggtct ttcattcatg cagcatgtat caaaattaat ttggtttttt ttcttaagta 6180tttacattaa atggccatag ttgcattaat gaatcggcca acgcgcgggg agaggcggtt 6240tgcgtattgg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct 6300gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga 6360taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc 6420cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg 6480ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 6540aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt 6600tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt 6660gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg 6720cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact 6780ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt 6840cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct 6900gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac 6960cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc 7020tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg 7080ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta 7140aaaatgaagt ttgcggccgc aaatcaatct aaagtatata tgagtaaact tggtctgaca 7200gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 7260tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc 7320ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa 7380accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 7440agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca 7500acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat 7560tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag 7620cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac 7680tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt 7740ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt 7800gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc 7860tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat 7920ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca 7980gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 8040cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg 8100gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg 8160ttccgcgcac atttc 8175 7 8175 DNA Unknown pICAST OMC. 7gacgtcggac ttatacccgg tttgtcctat agacaccatt cgtcaaggac ggggccgagt 60cccggttctt gtctaccttg tcgacttata cccggtttgt cctatagaca ccattcgtca 120aggacggggc cgagtcccgg ttcttgtcta ccaggggtct acgccaggtc gggagtcgtc 180aaagatctct tggtagtcta caaaggtccc acggggttcc tggactttac tgggacacgg 240aataaacttg attggttagt caagcgaaga gcgaagacaa gcgcgcgaag acgaggggct 300cgagttattt tctcgggtgt tggggagtga gccccgcggt caggaggcta actgactcag 360cgggcccatg ggcacatagg ttatttggga gaacgtcaac gtaggctgaa caccagagcg 420acaaggaacc ctcccagagg agactcacta actgatgggc agtcgccccc agaaagtaaa 480cccccgagca ggccctagcc ctctggggac gggtccctgg tggctgggtg gtggccctcc 540gttcgaccgg tcgttgaata gacacagaca ggctaacaga tcacagatac tgactaaaat 600acgcggacgc agccatgatc aatcgattga tcgagacata gaccgcctgg gcaccacctt 660gactgctcaa gacttgtggg ccggcgttgg gaccctctgc agggtccctg aaacccccgg 720caaaaacacc gggctggact ccttccctca gctacacctt aggctggggc agtcctatac 780accaagacca tcctctgctc ttggattttg tcaagggcgg aggcagactt aaaaacgaaa 840gccaaacctt ggcttcggcg cgcagaacag acgacgtcgt agcaagacac aacagagaca 900gactgacaca aagacataaa cagactttta atcccggtct gacaatggtg agggaattca 960aactggaatc cattgacctt tctacagctc gccgagcgag tgttggtcag ccatctacag 1020ttcttctctg caacccaatg gaagacgaga cgtcttaccg gttggaaatt gcagcctacc 1080ggcgctctgc cgtggaaatt ggctctggag tagtgggtcc aattctagtt ccagaaaagt 1140ggaccgggcg tacctgtggg tctggtccag gggatgtagc actggaccct tcggaaccga 1200aaactggggg gagggaccca gttcgggaaa catgtgggat tcggaggcgg aggagaagga 1260ggtaggcggg gcagagaggg ggaacttgga ggagcaagct ggggcggagc taggagggaa 1320ataggtcggg agtgaggaag agatccgcgg ccggcgagat cgggtaatta tgctgagtga 1380tatcccgcta agcttagtcc ggaaccgcgc ggcctaggaa ttaattcgcg ttaaccctcc 1440accgccatcg gagctctacc cgcactaatg cctaagtgac cggcagcaaa atgttgcagc 1500actgaccctt ttgggaccgc aatgggttga attagcggaa cgtcgtgtag ggggaaagcg 1560gtcgaccgca ttatcgcttc tccgggcgtg gctagcggga agggttgtca atgcgtcgga 1620cttaccgctt accgcgaaac ggaccaaagg ccgtggtctt cgccacggcc tttcgaccga 1680cctcacgcta gaaggactcc ggctatgaca gcagcagggg agtttgaccg tctacgtgcc 1740aatgctacgc gggtagatgt ggttgcactg gatagggtaa tgccagttag gcggcaaaca 1800agggtgcctc ttaggctgcc caacaatgag cgagtgtaaa ttacaactac tttcgaccga 1860tgtccttccg gtctgcgctt aataaaaact accgcaattg agccgcaaag tagacaccac 1920gttgcccgcg acccagccaa tgccggtcct gtcagcaaac ggcagactta aactggactc 1980gcgtaaaaat gcgcggcctc ttttggcgga gcgccactac cacgacgcga cctcactgcc 2040gtcaatagac cttctagtcc tatacaccgc ctactcgccg taaaaggcac tgcagagcaa 2100cgacgtattt ggctgatgtg tttagtcgct aaaggtacaa cggtgagcga aattactact 2160aaagtcggcg cgacatgacc tccgacttca agtctacacg ccgctcaacg cactgatgga 2220tgcccattgt caaagaaata ccgtcccact ttgcgtccag cggtcgccgt ggcgcggaaa 2280gccgccactt taatagctac tcgcaccacc aatacggcta gcgcagtgtg atgcagactt 2340gcagcttttg ggctttgaca cctcgcggct ttagggctta gagatagcac gccaccaact 2400tgacgtgtgg cggctgccgt gcgactaact tcgtcttcgg acgctacagc caaaggcgct 2460ccacgcctaa cttttaccag acgacgacga cttgccgttc ggcaacgact aagctccgca 2520attggcagtg ctcgtagtag gagacgtacc agtccagtac ctactcgtct gctaccacgt 2580cctataggac gactacttcg tcttgttgaa attgcggcac gcgacaagcg taataggctt 2640ggtaggcgac accatgtgcg acacgctggc gatgccggac atacaccacc tacttcggtt 2700ataactttgg gtgccgtacc acggttactt agcagactgg ctactaggcg cgaccgatgg 2760ccgctactcg cttgcgcatt gcgcttacca cgtcgcgcta gcattagtgg gctcacacta 2820gtagaccagc gaccccttac ttagtccggt gccgcgatta gtgctgcgcg acatagcgac 2880ctagtttaga cagctaggaa gggcgggcca cgtcatactt ccgccgcctc ggctgtggtg 2940ccggtggcta taataaacgg gctacatgcg cgcgcaccta cttctggtcg ggaagggccg 3000acacggcttt accaggtagt tttttaccga aagcgatgga cctctctgcg cgggcgacta 3060ggaaacgctt atgcgggtgc gctacccatt gtcagaaccg ccaaagcgat ttatgaccgt 3120ccgcaaagca gtcatagggg caaatgtccc gccgaagcag accctgaccc acctagtcag 3180cgactaattt atactacttt tgccgttggg caccagccga atgccgccac taaaaccgct 3240atgcggcttg ctagcggtca agacatactt gccagaccag aaacggctgg cgtgcggcgt 3300aggtcgcgac tgccttcgtt ttgtggtcgt cgtcaaaaag gtcaaggcaa ataggcccgt 3360ttggtagctt cactggtcgc ttatggacaa ggcagtatcg ctattgctcg aggacgtgac 3420ctaccaccgc gacctaccat tcggcgaccg ttcgccactt cacggagacc tacagcgagg 3480tgttccattt gtcaactaac ttgacggact tgatggcgtc ggcctctcgc ggcccgttga 3540gaccgagtgt catgcgcatc acgttggctt gcgctggcgt accagtcttc ggcccgtgta 3600gtcgcggacc gtcgtcaccg cagaccgcct tttggagtca cactgcgagg ggcggcgcag 3660ggtgcggtag ggcgtagact ggtggtcgct ttacctaaaa acgtagctcg acccattatt 3720cgcaaccgtt aaattggcgg tcagtccgaa agaaagtgtc tacacctaac cgctattttt 3780tgttgacgac tgcggcgacg cgctagtcaa gtgggcacag ctatctagac ttgtctttga 3840gtaaaggctt cttctggatc agctggtagt agtagtagta gtggccatta ttatccatct 3900attcactgac taatctacgt aaagctgatc tagggagctg gttaaggcca ataaaaggtg 3960gtataacggc agaaaaccgt tacactcccg ggcctttgga ccgggacaga agaactgctc 4020gtaaggatcc ccagaaaggg gagagcggtt tccttacgtt ccagacaact tacagcactt 4080ccttcgtcaa ggagaccttc gaagaacttc tgtttgttgc agacatcgct gggaaacgtc 4140cgtcgccttg gggggtggac cgctgtccac ggagacgccg gttttcggtg cacatattct 4200atgtggacgt ttccgccgtg ttggggtcac ggtgcaacac tcaacctatc aacacctttc 4260tcagtttacc gagaggagtt cgcataagtt gttccccgac ttcctacggg tcttccatgg 4320ggtaacatac cctagactag accccggagc cacgtgtacg aaatgtacac aaatcagctc 4380caattttttg cagatccggg gggcttggtg cccctgcacc aaaaggaaac tttttgtgct 4440actattatgg tactttttcg gacttgagtg gcgctgcaga cagctcttca aagactagct 4500tttcaagctg tcgcagaggc tggactacgt cgagagcctc ccgcttctta gagcacgaaa 4560gtcgaagcta catcctcccg cacctataca ggacgcccat ttatcgacgc ggctaccaaa 4620gatgtttcta gcaatacaaa tagccgtgaa acgtagccgg cgcgagggct aaggccttca 4680cgaactgtaa ccccttaaat cgcrctcgga ctggataacg tagagggcgg cacgtgtccc 4740acagtgcaac gttctggacg gactttggct tgacgggcga caagacgtcg gccagcgcct 4800ccggtaccta cgctagcgac gccggctaga atcggtctgc tcgcccaagc cgggtaagcc 4860tggcgttcct tagccagtta tgtgatgtac cgcactaaag tatacgcgct aacgactagg 4920ggtacacata gtgaccgttt gacactacct gctgtggcag tcacgcaggc agcgcgtccg 4980agagctactc gactacgaaa cccggctcct gacggggctt caggccgtgg agcacgtgcg 5040cctaaagccg aggttgttac aggactgcct gttaccggcg tattgtcgcc agtaactgac 5100ctcgctccgc tacaagcccc taagggttat gctccagcgg ttgtagaaga agacctccgg 5160caccaaccga acatacctcg tcgtctgcgc gatgaagctc gcctccgtag gcctcgaacg 5220tcctagcggc gccgaggccc gcatatacga ggcgtaacca gaactgcttg agatagtctc 5280gaaccaactg ccgttaaagc tactacgtcg aacccgcgtc ccagctacgc tgcgttagca 5340ggctaggcct cggccctgac agcccgcatg tgtttagcgg gcgtcttcgc gccggcagac 5400ctggctaccg acacatcttc atgagcggct atcacctttg gctgcggggt cgtgagcagg 5460ctcccgtttc cttatctcat ctacggctgg ccctagatag ctattttatt ttctaaaata 5520aatcagaggt ctttttcccc ccttactttc tggggtggac atccaaaccg ttcgatcgaa 5580ttcattgcgg taaaacgttc cgtacctttt tatgtattga ctcttatctc ttcaagtcta 5640gttccagtcc ttgtctacct tgtcgactta tacccggttt gtcctataga caccattcgt 5700caaggacggg gccgagtccc ggttcttgtc taccttgtcg acttataccc ggtttgtcct 5760atagacacca ttcgtcaagg acggggccga gtcccggttc ttgtctacca ggggtctacg 5820ccaggtcggg agtcgtcaaa gatctcttgg tagtctacaa aggtcccacg gggttcctgg 5880actttactgg gacacggaat aaacttgatt ggttagtcaa gcgaagagcg aagacaagcg 5940cgcgaagacg aggggctcga gttattttct cgggtgttgg ggagtgagcc ccgcggtcag 6000gaggctaact gactcagcgg gcccatgggc acataggtta tttgggagaa cgtcaacgta 6060ggctgaacac cagagcgaca aggaaccctc ccagaggaga ctcactaact gatgggcagt 6120cgcccccaga aagtaagtac gtcgtacata gttttaatta aaccaaaaaa aagaattcat 6180aaatgtaatt taccggtatc aacgtaatta cttagccggt tgcgcgcccc tctccgccaa 6240acgcataacc gcgagaaggc gaaggagcga gtgactgagc gacgcgagcc agcaagccga 6300cgccgctcgc catagtcgag tgagtttccg ccattatgcc aataggtgtc ttagtcccct 6360attgcgtcct ttcttgtaca ctcgttttcc ggtcgttttc cggtccttgg catttttccg 6420gcgcaacgac cgcaaaaagg tatccgaggc ggggggactg ctcgtagtgt ttttagctgc 6480gagttcagtc tccaccgctt tgggctgtcc tgatatttct atggtccgca aagggggacc 6540ttcgagggag cacgcgagag gacaaggctg ggacggcgaa tggcctatgg acaggcggaa 6600agagggaagc ccttcgcacc gcgaaagagt atcgagtgcg acatccatag agtcaagcca 6660catccagcaa gcgaggttcg acccgacaca cgtgcttggg gggcaagtcg ggctggcgac 6720gcggaatagg ccattgatag cagaactcag gttgggccat tctgtgctga atagcggtga 6780ccgtcgtcgg tgaccattgt cctaatcgtc tcgctccata catccgccac gatgtctcaa 6840gaacttcacc accggattga tgccgatgtg atcttcttgt cataaaccat agacgcgaga 6900cgacttcggt caatggaagc ctttttctca accatcgaga actaggccgt ttgtttggtg 6960gcgaccatcg ccaccaaaaa aacaaacgtt cgtcgtctaa tgcgcgtctt tttttcctag 7020agttcttcta ggaaactaga aaagatgccc cagactgcga gtcaccttgc ttttgagtgc 7080aattccctaa aaccagtact ctaatagttt ttcctagaag tggatctagg aaaatttaat 7140ttttacttca aacgccggcg tttagttaga tttcatatat actcatttga accagactgt 7200caatggttac gaattagtca ctccgtggat agagtcgcta gacagataaa gcaagtaggt 7260atcaacggac tgaggggcag cacatctatt gatgctatgc cctcccgaat ggtagaccgg 7320ggtcacgacg ttactatggc gctctgggtg cgagtggccg aggtctaaat agtcgttatt 7380tggtcggtcg gccttcccgg ctcgcgtctt caccaggacg ttgaaatagg cggaggtagg 7440tcagataatt aacaacggcc cttcgatctc attcatcaag cggtcaatta tcaaacgcgt 7500tgcaacaacg gtaacgatgt ccgtagcacc acagtgcgag cagcaaacca taccgaagta 7560agtcgaggcc aagggttgct agttccgctc aatgtactag ggggtacaac acgttttttc 7620gccaatcgag gaagccagga ggctagcaac agtcttcatt caaccggcgt cacaatagtg 7680agtaccaata ccgtcgtgac gtattaagag aatgacagta cggtaggcat tctacgaaaa 7740gacactgacc actcatgagt tggttcagta agactcttat cacatacgcc gctggctcaa 7800cgagaacggg ccgcagttat gccctattat ggcgcggtgt atcgtcttga aattttcacg 7860agtagtaacc ttttgcaaga agccccgctt ttgagagttc ctagaatggc gacaactcta 7920ggtcaagcta cattgggtga gcacgtgggt tgactagaag tcgtagaaaa tgaaagtggt 7980cgcaaagacc cactcgtttt tgtccttccg ttttacggcg ttttttccct tattcccgct 8040gtgcctttac aacttatgag tatgagaagg aaaaagttat aataacttcg taaatagtcc 8100caataacaga gtactcgcct atgtataaac ttacataaat ctttttattt gtttatcccc 8160aaggcgcgtg taaag 8175 8 8161 DNA Unknown pICAST OMN. 8ctgcagcctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca 60gggccaagaa cagatggaac agctgaatat gggccaaaca ggatatctgt ggtaagcagt 120tcctgccccg gctcagggcc aagaacagat ggtccccaga tgcggtccag ccctcagcag 180tttctagaga accatcagat gtttccaggg tgccccaagg acctgaaatg accctgtgcc 240ttatttgaac taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga 300gctcaataaa agagcccaca acccctcact cggggcgcca gtcctccgat tgactgagtc 360gcccgggtac ccgtgtatcc aataaaccct cttgcagttg catccgactt gtggtctcgc 420tgttccttgg gagggtctcc tctgagtgat tgactacccg tcagcggggg tctttcattt 480gggggctcgt ccgggatcgg gagacccctg cccagggacc accgacccac caccgggagg 540caagctggcc agcaacttat ctgtgtctgt ccgattgtct agtgtctatg actgatttta 600tgcgcctgcg tcggtactag ttagctaact agctctgtat ctggcggacc cgtggtggaa 660ctgacgagtt ctgaacaccc ggccgcaacc ctgggagacg tcccagggac tttgggggcc 720gtttttgtgg cccgacctga ggaagggagt cgatgtggaa tccgaccccg tcaggatatg 780tggttctggt aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt 840cggtttggaa ccgaagccgc gcgtcttgtc tgctgcagca tcgttctgtg ttgtctctgt 900ctgactgtgt ttctgtattt gtctgaaaat tagggccaga ctgttaccac tcccttaagt 960ttgaccttag gtaactggaa agatgtcgag cggctcgctc acaaccagtc ggtagatgtc 1020aagaagagac gttgggttac cttctgctct gcagaatggc caacctttaa cgtcggatgg 1080ccgcgagacg gcacctttaa ccgagacctc atcacccagg ttaagatcaa ggtcttttca 1140cctggcccgc atggacaccc agaccaggtc ccctacatcg tgacctggga agccttggct 1200tttgaccccc ctccctgggt caagcccttt gtacacccta agcctccgcc tcctcttcct 1260ccatccgccc cgtctctccc ccttgaacct cctcgttcga ccccgcctcg atcctccctt 1320tatccagccc tcactccttc tctaggcgcc ggccgctcta gcccattaat acgactcact 1380atagggcgat tcgaacacca tgcaccatca tcatcatcac gtcgacgaac agaaactcat 1440ttccgaagaa gacctactcg agatgggcgt gattacggat tcactggccg tcgttttaca 1500acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc 1560tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttacg 1620cagcctgaat ggcgaatggc gctttgcctg gtttccggca ccagaagcgg tgccggaaag 1680ctggctggag tgcgatcttc ctgaggccga tactgtcgtc gtcccctcaa actggcagat 1740gcacggttac gatgcgccca tctacaccaa cgtgacctat cccattacgg tcaatccgcc 1800gtttgttccc acggagaatc cgacgggttg ttactcgctc acatttaatg ttgatgaaag 1860ctggctacag gaaggccaga cgcgaattat ttttgatggc gttaactcgg cgtttcatct 1920gtggtgcaac gggcgctggg tcggttacgg ccaggacagt cgtttgccgt ctgaatttga 1980cctgagcgca tttttacgcg ccggagaaaa ccgcctcgcg gtgatggtgc tgcgctggag 2040tgacggcagt tatctggaag atcaggatat gtggcggatg agcggcattt tccgtgacgt 2100ctcgttgctg cataaaccga ctacacaaat cagcgatttc catgttgcca ctcgctttaa 2160tgatgatttc agccgcgctg tactggaggc tgaagttcag atgtgcggcg agttgcgtga 2220ctacctacgg gtaacagttt ctttatggca gggtgaaacg caggtcgcca gcggcaccgc 2280gcctttcggc ggtgaaatta tcgatgagcg tggtggttat gccgatcgcg tcacactacg 2340tctgaacgtc gaaaacccga aactgtggag cgccgaaatc ccgaatctct atcgtgcggt 2400ggttgaactg cacaccgccg acggcacgct gattgaagca gaagcctgcg atgtcggttt 2460ccgcgaggtg cggattgaaa atggtctgct gctgctgaac ggcaagccgt tgctgattcg 2520aggcgttaac cgtcacgagc atcatcctct gcatggtcag gtcatggatg agcagacgat 2580ggtgcaggat atcctgctga tgaagcagaa caactttaac gccgtgcgct gttcgcatta 2640tccgaaccat ccgctgtggt acacgctgtg cgaccgctac ggcctgtatg tggtggatga 2700agccaatatt gaaacccacg gcatggtgcc aatgaatcgt ctgaccgatg atccgcgctg 2760gctaccggcg atgagcgaac gcgtaacgcg aatggtgcag cgcgatcgta atcacccgag 2820tgtgatcatc tggtcgctgg ggaatgaatc aggccacggc gctaatcacg acgcgctgta 2880tcgctggatc aaatctgtcg atccttcccg cccggtgcag tatgaaggcg gcggagccga 2940caccacggcc accgatatta tttgcccgat gtacgcgcgc gtggatgaag accagccctt 3000cccggctgtg ccgaaatggt ccatcaaaaa atggctttcg ctacctggag agacgcgccc 3060gctgatcctt tgcgaatacg cccacgcgat gggtaacagt cttggcggtt tcgctaaata 3120ctggcaggcg tttcgtcagt atccccgttt acagggcggc ttcgtctggg actgggtgga 3180tcagtcgctg attaaatatg atgaaaacgg caacccgtgg tcggcttacg gcggtgattt 3240tggcgatacg ccgaacgatc gccagttctg tatgaacggt ctggtctttg ccgaccgcac 3300gccgcatcca gcgctgacgg aagcaaaaca ccagcagcag tttttccagt tccgtttatc 3360cgggcaaacc atcgaagtga ccagcgaata cctgttccgt catagcgata acgagctcct 3420gcactggatg gtggcgctgg atggtaagcc gctggcaagc ggtgaagtgc ctctggatgt 3480cgctccacaa ggtaaacagt tgattgaact gcctgaacta ccgcagccgg agagcgccgg 3540gcaactctgg ctcacagtac gcgtagtgca accgaacgcg accgcatggt cagaagccgg 3600gcacatcagc gcctggcagc agtggcgtct ggcggaaaac ctcagtgtga cgctccccgc 3660cgcgtcccac gccatcccgc atctgaccac cagcgaaatg gatttttgca tcgagctggg 3720taataagcgt tggcaattta accgccagtc aggctttctt tcacagatgt ggattggcga 3780taaaaaacaa ctgctgacgc cgctgcgcga tcagttcacc cgtgtcgata gatctggagg 3840tggtggcagc aggccttggc gcgccggatc cttaattaac aattgaccgg taataatagg 3900tagataagtg actgattaga tgcatttcga ctagatccct cgaccaattc cggttatttt 3960ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct gtcttcttga 4020cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg 4080tgaaggaagc agttcctctg gaagcttctt gaagacaaac aacgtctgta gcgacccttt 4140gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat 4200aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg atagttgtgg 4260aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg 4320taccccattg tatgggatct gatctggggc ctcggtgcac atgctttaca tgtgtttagt 4380cgaggttaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac 4440acgatgataa taccatgaaa aagcctgaac tcaccgcgac gtctgtcgag aagtttctga 4500tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc ggagggcgaa gaatctcgtg 4560ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg 4620gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc ccgattccgg 4680aagtgcttga cattggggaa tttagcgaga gcctgaccta ttgcatctcc cgccgtgcac 4740agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg 4800cggaggccat ggatgcgatc gctgcggccg atcttagcca gacgagcggg ttcggcccat 4860tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc gcgattgctg 4920atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc 4980aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc 5040acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg 5100actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga 5160ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc 5220ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc 5280agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa 5340tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg 5400tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc 5460gtccgagggc aaaggaatag agtagatgcc gaccgggatc tatcgataaa ataaaagatt 5520ttatttagtc tccagaaaaa ggggggaatg aagaccccaa cctgtaggtt tggcaagcta 5580gcttaagtaa cgccattttg caaggcatgg aaaaatacat aactgagaat agagaagttc 5640agatcaaggt caggaacaga tggaacagct gaatatgggc caaacaggat atctgtggta 5700agcagttcct gccccggctc agggccaaga acagatggaa cagctgaata tgggccaaac 5760aggatatctg tggtaagcag ttcctgcccc ggctcagggc caagaacaga tggtccccag 5820atgcggtcca gccctcagca gtttctagag aaccatcaga tgtttccagg gtgccccaag 5880gacctgaaat gaccctgtgc cttatttgaa ctaaccaatc agttcgcttc tcgcttctgt 5940tcgcgcgctt ctgctccccg agctcaataa aagagcccac aacccctcac tcggggcgcc 6000agtcctccga ttgactgagt cgcccgggta cccgtgtatc caataaaccc tcttgcagtt 6060gcatccgact tgtggtctcg ctgttccttg ggagggtctc ctctgagtga ttgactaccc 6120gtcagcgggg gtctttcatt catgcagcat gtatcaaaat taatttggtt ttttttctta 6180agtatttaca ttaaatggcc atagttgcat taatgaatcg gccaacgcgc ggggagaggc 6240ggtttgcgta ttggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc 6300ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag 6360gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa 6420aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc 6480gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc 6540ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 6600cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt 6660cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc 6720gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 6780cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 6840agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg 6900ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 6960ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 7020gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 7080cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttgc 7140ggccgcaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta 7200atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc 7260cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg 7320ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga 7380agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt 7440tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt 7500gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc 7560caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc 7620ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca 7680gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag 7740tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg 7800tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa 7860cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa 7920cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga 7980gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga 8040atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg 8100agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt 8160c 8161 9 8161 DNA Unknown pICAST OMN. 9gacgtcggac ttatacccgg tttgtcctat agacaccatt cgtcaaggac ggggccgagt 60cccggttctt gtctaccttg tcgacttata cccggtttgt cctatagaca ccattcgtca 120aggacggggc cgagtcccgg ttcttgtcta ccaggggtct acgccaggtc gggagtcgtc 180aaagatctct tggtagtcta caaaggtccc acggggttcc tggactttac tgggacacgg 240aataaacttg attggttagt caagcgaaga gcgaagacaa gcgcgcgaag acgaggggct 300cgagttattt tctcgggtgt tggggagtga gccccgcggt caggaggcta actgactcag 360cgggcccatg ggcacatagg ttatttggga gaacgtcaac gtaggctgaa caccagagcg 420acaaggaacc ctcccagagg agactcacta actgatgggc agtcgccccc agaaagtaaa 480cccccgagca ggccctagcc ctctggggac gggtccctgg tggctgggtg gtggccctcc 540gttcgaccgg tcgttgaata gacacagaca ggctaacaga tcacagatac tgactaaaat 600acgcggacgc agccatgatc aatcgattga tcgagacata gaccgcctgg gcaccacctt 660gactgctcaa gacttgtggg ccggcgttgg gaccctctgc agggtccctg aaacccccgg 720caaaaacacc gggctggact ccttccctca gctacacctt aggctggggc agtcctatac 780accaagacca tcctctgctc ttggattttg tcaagggcgg aggcagactt aaaaacgaaa 840gccaaacctt ggcttcggcg cgcagaacag acgacgtcgt agcaagacac aacagagaca 900gactgacaca aagacataaa cagactttta atcccggtct gacaatggtg agggaattca 960aactggaatc cattgacctt tctacagctc gccgagcgag tgttggtcag ccatctacag 1020ttcttctctg caacccaatg gaagacgaga cgtcttaccg gttggaaatt gcagcctacc 1080ggcgctctgc cgtggaaatt ggctctggag tagtgggtcc aattctagtt ccagaaaagt 1140ggaccgggcg tacctgtggg tctggtccag gggatgtagc actggaccct tcggaaccga 1200aaactggggg gagggaccca gttcgggaaa catgtgggat tcggaggcgg aggagaagga 1260ggtaggcggg gcagagaggg ggaacttgga ggagcaagct ggggcggagc taggagggaa 1320ataggtcggg agtgaggaag agatccgcgg ccggcgagat cgggtaatta tgctgagtga 1380tatcccgcta agcttgtggt acgtggtagt agtagtagtg cagctgcttg tctttgagta 1440aaggcttctt ctggatgagc tctacccgca ctaatgccta agtgaccggc agcaaaatgt 1500tgcagcactg acccttttgg gaccgcaatg ggttgaatta gcggaacgtc gtgtaggggg 1560aaagcggtcg accgcattat cgcttctccg ggcgtggcta gcgggaaggg ttgtcaatgc 1620gtcggactta ccgcttaccg cgaaacggac caaaggccgt ggtcttcgcc acggcctttc 1680gaccgacctc acgctagaag gactccggct atgacagcag caggggagtt tgaccgtcta 1740cgtgccaatg ctacgcgggt agatgtggtt gcactggata gggtaatgcc agttaggcgg 1800caaacaaggg tgcctcttag gctgcccaac aatgagcgag tgtaaattac aactactttc 1860gaccgatgtc cttccggtct gcgcttaata aaaactaccg caattgagcc gcaaagtaga 1920caccacgttg cccgcgaccc agccaatgcc ggtcctgtca gcaaacggca gacttaaact 1980ggactcgcgt aaaaatgcgc ggcctctttt ggcggagcgc cactaccacg acgcgacctc 2040actgccgtca atagaccttc tagtcctata caccgcctac tcgccgtaaa aggcactgca 2100gagcaacgac gtatttggct gatgtgttta gtcgctaaag gtacaacggt gagcgaaatt 2160actactaaag tcggcgcgac atgacctccg acttcaagtc tacacgccgc tcaacgcact 2220gatggatgcc cattgtcaaa gaaataccgt cccactttgc gtccagcggt cgccgtggcg 2280cggaaagccg ccactttaat agctactcgc accaccaata cggctagcgc agtgtgatgc 2340agacttgcag cttttgggct ttgacacctc gcggctttag ggcttagaga tagcacgcca 2400ccaacttgac gtgtggcggc tgccgtgcga ctaacttcgt cttcggacgc tacagccaaa 2460ggcgctccac gcctaacttt taccagacga cgacgacttg ccgttcggca acgactaagc 2520tccgcaattg gcagtgctcg tagtaggaga cgtaccagtc cagtacctac tcgtctgcta 2580ccacgtccta taggacgact acttcgtctt gttgaaattg cggcacgcga caagcgtaat 2640aggcttggta ggcgacacca tgtgcgacac gctggcgatg ccggacatac accacctact 2700tcggttataa ctttgggtgc cgtaccacgg ttacttagca gactggctac taggcgcgac 2760cgatggccgc tactcgcttg cgcattgcgc ttaccacgtc gcgctagcat tagtgggctc 2820acactagtag accagcgacc ccttacttag tccggtgccg cgattagtgc tgcgcgacat 2880agcgacctag tttagacagc taggaagggc gggccacgtc atacttccgc cgcctcggct 2940gtggtgccgg tggctataat aaacgggcta catgcgcgcg cacctacttc tggtcgggaa 3000gggccgacac ggctttacca ggtagttttt taccgaaagc gatggacctc tctgcgcggg 3060cgactaggaa acgcttatgc gggtgcgcta cccattgtca gaaccgccaa agcgatttat 3120gaccgtccgc aaagcagtca taggggcaaa tgtcccgccg aagcagaccc tgacccacct 3180agtcagcgac taatttatac tacttttgcc gttgggcacc agccgaatgc cgccactaaa 3240accgctatgc ggcttgctag cggtcaagac atacttgcca gaccagaaac ggctggcgtg 3300cggcgtaggt cgcgactgcc ttcgttttgt ggtcgtcgtc aaaaaggtca aggcaaatag 3360gcccgtttgg tagcttcact ggtcgcttat ggacaaggca gtatcgctat tgctcgagga 3420cgtgacctac caccgcgacc taccattcgg cgaccgttcg ccacttcacg gagacctaca 3480gcgaggtgtt ccatttgtca actaacttga cggacttgat ggcgtcggcc tctcgcggcc 3540cgttgagacc gagtgtcatg cgcatcacgt tggcttgcgc tggcgtacca gtcttcggcc 3600cgtgtagtcg cggaccgtcg tcaccgcaga ccgccttttg gagtcacact gcgaggggcg 3660gcgcagggtg cggtagggcg tagactggtg gtcgctttac ctaaaaacgt agctcgaccc 3720attattcgca accgttaaat tggcggtcag tccgaaagaa agtgtctaca cctaaccgct 3780attttttgtt gacgactgcg gcgacgcgct agtcaagtgg gcacagctat ctagacctcc 3840accaccgtcg tccggaaccg cgcggcctag gaattaattg ttaactggcc attattatcc 3900atctattcac tgactaatct acgtaaagct gatctaggga gctggttaag gccaataaaa 3960ggtggtataa cggcagaaaa ccgttacact cccgggcctt tggaccggga cagaagaact 4020gctcgtaagg atccccagaa aggggagagc ggtttcctta cgttccagac aacttacagc 4080acttccttcg tcaaggagac cttcgaagaa cttctgtttg ttgcagacat cgctgggaaa 4140cgtccgtcgc cttggggggt ggaccgctgt ccacggagac gccggttttc ggtgcacata 4200ttctatgtgg acgtttccgc cgtgttgggg tcacggtgca acactcaacc tatcaacacc 4260tttctcagtt taccgagagg agttcgcata agttgttccc cgacttccta cgggtcttcc 4320atggggtaac ataccctaga ctagaccccg gagccacgtg tacgaaatgt acacaaatca 4380gctccaattt tttgcagatc cggggggctt ggtgcccctg caccaaaagg aaactttttg 4440tgctactatt atggtacttt ttcggacttg agtggcgctg cagacagctc ttcaaagact 4500agcttttcaa gctgtcgcag aggctggact acgtcgagag cctcccgctt cttagagcac 4560gaaagtcgaa gctacatcct cccgcaccta tacaggacgc ccatttatcg acgcggctac 4620caaagatgtt tctagcaata caaatagccg tgaaacgtag ccggcgcgag ggctaaggcc 4680ttcacgaact gtaacccctt aaatcgctct cggactggat aacgtagagg gcggcacgtg 4740tcccacagtg caacgttctg gacggacttt ggcttgacgg gcgacaagac gtcggccagc 4800gcctccggta cctacgctag cgacgccggc tagaatcggt ctgctcgccc aagccgggta 4860agcctggcgt tccttagcca gttatgtgat gtaccgcact aaagtatacg cgctaacgac 4920taggggtaca catagtgacc gtttgacact acctgctgtg gcagtcacgc aggcagcgcg 4980tccgagagct actcgactac gaaacccggc tcctgacggg gcttcaggcc gtggagcacg 5040tgcgcctaaa gccgaggttg ttacaggact gcctgttacc ggcgtattgt cgccagtaac 5100tgacctcgct ccgctacaag cccctaaggg ttatgctcca gcggttgtag aagaagacct 5160ccggcaccaa ccgaacatac ctcgtcgtct gcgcgatgaa gctcgcctcc gtaggcctcg 5220aacgtcctag cggcgccgag gcccgcatat acgaggcgta accagaactg gttgagatag 5280tctcgaacca actgccgtta aagctactac gtcgaacccg cgtcccagct acgctgcgtt 5340agcaggctag gcctcggccc tgacagcccg catgtgttta gcgggcgtct tcgcgccggc 5400agacctggct accgacacat cttcatgagc ggctatcacc tttggctgcg gggtcgtgag 5460caggctcccg tttccttatc tcatctacgg ctggccctag atagctattt tattttctaa 5520aataaatcag aggtcttttt ccccccttac tttctggggt ggacatccaa accgttcgat 5580cgaattcatt gcggtaaaac gttccgtacc tttttatgta ttgactctta tctcttcaag 5640tctagttcca gtccttgtct accttgtcga cttatacccg gtttgtccta tagacaccat 5700tcgtcaagga cggggccgag tcccggttct tgtctacctt gtcgacttat acccggtttg 5760tcctatagac accattcgtc aaggacgggg ccgagtcccg gttcttgtct accaggggtc 5820tacgccaggt cgggagtcgt caaagatctc ttggtagtct acaaaggtcc cacggggttc 5880ctggacttta ctgggacacg gaataaactt gattggttag tcaagcgaag agcgaagaca 5940agcgcgcgaa gacgaggggc tcgagttatt ttctcgggtg ttggggagtg agccccgcgg 6000tcaggaggct aactgactca gcgggcccat gggcacatag gttatttggg agaacgtcaa 6060cgtaggctga acaccagagc gacaaggaac cctcccagag gagactcact aactgatggg 6120cagtcgcccc cagaaagtaa gtacgtcgta catagtttta attaaaccaa aaaaaagaat 6180tcataaatgt aatttaccgg tatcaacgta attacttagc cggttgcgcg cccctctccg 6240ccaaacgcat aaccgcgaga aggcgaagga gcgagtgact gagcgacgcg agccagcaag 6300ccgacgccgc tcgccatagt cgagtgagtt tccgccatta tgccaatagg tgtcttagtc 6360ccctattgcg tcctttcttg tacactcgtt ttccggtcgt tttccggtcc ttggcatttt 6420tccggcgcaa cgaccgcaaa aaggtatccg aggcgggggg actgctcgta gtgtttttag 6480ctgcgagttc agtctccacc gctttgggct gtcctgatat ttctatggtc cgcaaagggg 6540gaccttcgag ggagcacgcg agaggacaag gctgggacgg cgaatggcct atggacaggc 6600ggaaagaggg aagcccttcg caccgcgaaa gagtatcgag tgcgacatcc atagagtcaa 6660gccacatcca gcaagcgagg ttcgacccga cacacgtgct tggggggcaa gtcgggctgg 6720cgacgcggaa taggccattg atagcagaac tcaggttggg ccattctgtg ctgaatagcg 6780gtgaccgtcg tcggtgacca ttgtcctaat cgtctcgctc catacatccg ccacgatgtc 6840tcaagaactt caccaccgga ttgatgccga tgtgatcttc ttgtcataaa ccatagacgc 6900gagacgactt cggtcaatgg aagccttttt ctcaaccatc gagaactagg ccgtttgttt 6960ggtggcgacc atcgccacca aaaaaacaaa cgttcgtcgt ctaatgcgcg tctttttttc 7020ctagagttct tctaggaaac tagaaaagat gccccagact gcgagtcacc ttgcttttga 7080gtgcaattcc ctaaaaccag tactctaata gtttttccta gaagtggatc taggaaaacg 7140ccggcgttta gttagatttc atatatactc atttgaacca gactgtcaat ggttacgaat 7200tagtcactcc gtggatagag tcgctagaca gataaagcaa gtaggtatca acggactgag 7260gggcagcaca tctattgatg ctatgccctc ccgaatggta gaccggggtc acgacgttac 7320tatggcgctc tgggtgcgag tggccgaggt ctaaatagtc gttatttggt cggtcggcct 7380tcccggctcg cgtcttcacc aggacgttga aataggcgga ggtaggtcag ataattaaca 7440acggcccttc gatctcattc atcaagcggt caattatcaa acgcgttgca acaacggtaa 7500cgatgtccgt agcaccacag tgcgagcagc aaaccatacc gaagtaagtc gaggccaagg 7560gttgctagtt ccgctcaatg tactaggggg tacaacacgt tttttcgcca atcgaggaag 7620ccaggaggct agcaacagtc ttcattcaac cggcgtcaca atagtgagta ccaataccgt 7680cgtgacgtat taagagaatg acagtacggt aggcattcta cgaaaagaca ctgaccactc 7740atgagttggt tcagtaagac tcttatcaca tacgccgctg gctcaacgag aacgggccgc 7800agttatgccc tattatggcg cggtgtatcg tcttgaaatt ttcacgagta gtaacctttt 7860gcaagaagcc ccgcttttga gagttcctag aatggcgaca actctaggtc aagctacatt 7920gggtgagcac gtgggttgac tagaagtcgt agaaaatgaa agtggtcgca aagacccact 7980cgtttttgtc cttccgtttt acggcgtttt ttcccttatt cccgctgtgc ctttacaact 8040tatgagtatg agaaggaaaa agttataata acttcgtaaa tagtcccaat aacagagtac 8100tcgcctatgt ataaacttac ataaatcttt ttatttgttt atccccaagg cgcgtgtaaa 8160g 8161 10 5 PRT Artificial Sequence GS Linker 10Gly Gly Gly Gly Ser1 5

Claims

1. A method of assessing the effect of a test condition on G-protein-coupled receptor (GPCR) pathway activity, comprising:a) providing a cell that expresses a GPCR as a fusion protein to a first mutant form of a reporter enzyme and an arrestin as a fusion protein to a second mutant form of the reporter enzyme complementary to the first mutant form of the reporter enzyme, wherein the arrestin is modified to enhance binding of said arrestin to the GPCR, wherein said enhanced binding between said arrestin and the GPCR increases sensitivity of detection of said effect of the test condition; b) exposing the cell to a ligand for said GPCR under the test condition; and c) monitoring activation of said GPCR by complementation of the first and second mutant forms of the reporter enzyme; wherein increased reporter enzyme activity in the cell compared to that which occurs in the absence of the test condition indicates increased GPCR interaction with the modified arrestin compared to that which occurs in the absence of the test condition, and decreased reporter enzyme activity in the cell compared to that which occurs in the absence of the test condition indicates decreased GPCR interaction with the modified arrestin compared to that which occurs in the absence of the test condition; and wherein the GPCR and the first mutant form of reporter enzyme are linked together by a polypeptide linker represented by the formula —(GGGGS)n— (SEQ ID NO:10).

2. The method of claim 1, wherein the modified arrestin exhibits enhanced binding to activated, phosphorylated GPCR.

3. The method of claim 1, wherein the modified arrestin comprises conversion of Arg169 to an amino acid selected from the group consisting of histidine, tyrosine, phenylalanine and threonine.

4. The method of claim 1, wherein the modified arrestin comprises conversion of Val170 to alanine.

5. The method of claim 1, wherein the arrestin is selected from the group consisting of β-arrestin1 and β-arrestin2, and wherein the β-arrestin1 or the β-arrestin2 is truncated for all or part of a carboxyl-terminal half of the β-arrestin1 or the β-arrestin2.

6. The method of claim wherein the 5, wherein the β-arrestin1 or the β-arrestin2 is truncated from amino acid 190 of the β-arrestin1 or the β-arrestin2 to the carboxyl-terminal end of the β-arrestin1 or the β-arrestin2.

7. The method of claim 1, wherein the arrestin is a chimera of β-arrestin1, β-arrestin2 and/or visual arrestin.

8. The method of claim 1, wherein the modified arrestin comprises conversion of Arg170 to an amino acid selected from the group consisting of histidine, tyrosine, phenylalanine and threonine.

9. The method of claim 1, wherein n is 2 or more.

10. The method of claim 1, wherein n is 4.

11. The method of claim 1, wherein the second mutant form of the reporter enzyme is linked to the C-terminal of the arrestin.

12. A method of assessing the effect of a test condition on G-protein-coupled receptor (GPCR) pathway activity, comprising:a) providing a cell that expresses a GPCR as a fusion protein to a first mutant form of a reporter enzyme and an arrestin as a fusion protein to a second mutant form of the reporter enzyme complementary to the first mutant form of the reporter enzyme, wherein the arrestin is modified by introducing a point mutation in a phosphorylation-recognition domain to remove a requirement for phosphorylation of the GPCR for arrestin binding to permit binding of the arrestin to said GPCR in the cell regardless of whether the GPCR is phosphorylated, b) exposing the cell to a ligand for said GPCR under the test condition; and c) monitoring activation of the GPCR by complementation of the first and second mutant forms of the reporter enzyme; wherein increased reporter enzyme activity in the cell compared to that which occurs in the absence of the test condition indicates increased GPCR interaction with the modified arrestin compared to that which occurs in the absence of the test condition, and decreased reporter enzyme activity in the cell compared to that which occurs in the absence of the test condition indicates decreased GPCR interaction with the modified arrestin compared to that which occurs in the absence of the test condition; and wherein the GPCR and the first mutant form of reporter enzyme are linked together by a polypeptide linker represented by the formula —(GGGGS)n—(SEQ ID NO:10).

13. The method of claim 12, wherein the arrestin is mutated to increase a property selected from affinity and avidity for activated, non-phosphorylated GPCR.

14. The method of claim 13, wherein the arrestin is β-arrestin2 and wherein the β-arrestin2 is mutated to convert Arg169 to an oppositely charged residue.

15. The method of claim 14, wherein the oppositely charged residue is selected from the group consisting of histidine, tyrosine, phenylalanine and threonine.

16. The method of claim 15, wherein the arrestin is a chimera of β-arrestin1, β-arrestin2 and/or visual arrestin.

17. The method of claim 14, wherein the arrestin is a chimera of β-arrestin1, β-arrestin2 and/or visual arrestin.

18. The method of claim 13, wherein the arrestin is a chimera of β-arrestin1, β-arrestin2 and/or visual arrestin.

19. The method of claim 13, wherein the arrestin is β-arrestin2 and wherein the β-arrestin2 is mutated to convert Arg170 to an oppositely charged residue.

20. The method of claim 12, wherein the arrestin is mutated to increase a property selected from affinity and avidity for activated and phosphorylated GPCR.

21. The method of claim 12, wherein n is 2 or more.

22. The method of claim 12, wherein n is 4.