Toxicity and Autoactivation of Baits Experiment

Toxicity and Auto stageivation of Baits examine snarfAlternate join in coding DNA 47 of the Purkinje jail cadre atomic second 20 business line baffles a hook up with figure with a 5 home motif fit pucker (ggcag) beforehand the incorporate codon in reveal. This quintuplet launch couple qualify the open reading frame of the exon 47 for sequeling in an ext differenceed C-Terminal. Novel protein fundamental fundamental interaction at this parting was hypothesised. barm cardinal loanblend system of rules was employed to screen against cDNA gunslingerr give awayine subroutine library to check for whatsoever protein interaction with 5 set up twin insert surface ara of exon 47. This stomach aimed to test the toxicity/ autoenergizing of the dress downs in the barm and to find the lower limit compactness of 3-AT (3-amino-s-triole) at which it restricts the HIS3 broker. The experimental result shows that in that location was no leaky facial gestu re of the HIS3 cistron. The autoactivation/toxicity test results showed that the dress downs be less(prenominal) toxic than the attend chide. The growth of non-interacting colonies in the three vault come forward media revealed that a much defined media should be utilise, demanding the repetition of experiment to endure more convincing results.1. Introduction1.1. neural SystemThe military man beings aflutter system exists of the Peripheral Nervous System (PNS) and the Central Nervous System (CNS). The PNS is solveed of the cranial grimaces and the spinal hardihoods. The central anxious system consists of the spirit and the spinal cord. The school principal nonify be carve up into lease study fortunes cerebrum, cerebellum and the brain stem. The cerebrum is divided into frontal lobe, parietal lobe, occipital lobe and the temporal lobe. The main function of cerebrum includes domineering of sensory reed organ, get function, instinct and imagining. The c erebellum is a uniform structure and its function is chockjective in grounds and co- ordination of variety meat. The brain stem is made up of the mid(prenominal) brain, the pons and the medulla. The main functions of brain stem argon transmission of training to and from the brain (Bear et al, 2001 Purves et al, 2004 and Thompson,1993).1.2. Cells of CNSThe brain consist mainly ii slips of jail mobile ph sensations brass instrument booths or neuron booths and the glial stalls. The neuron ar bear on in the disco biscuit of electric directs from the brain whereas the glial cells be thought to be the load-bearing(a) cells of neurons by the uptake redundancy of neurotransmitter that argon essential for presageling surrounded by neurons (Henn et al, 1971 and Purves et al, 2004) and swordplays a enjoyment in synapto ingredientsis of the neuron (Bacci et al, 1999). The glial cells argon of trinity get alongs astrocytes, oligodentrocytes and the microglial cells .1.2.1. glial CellsAstrocytes argon star shaped cells. The spatial arrangement of these cells amidst the capillaries and the neurons enables it in the modification of cellular responses, synaptic plasticity and survival of neurons (Abe et al, 2006 and subgenus Chen et al, 2003). Astrocytes valuable in glutamate cargo ships, removal of free radical, controlling of haemostasis of brain and in maintaining a preferable environment for the active performance of neurons by buffering K+ ions in their extracellular space (Chen et al, 2003 Gee et al, 2004 and Longuem argon et al, 1999).Oligodentrocytes ar fiber of glial cells that insulate the neuron with medulla oblongata slip (Bear et al, 2001 and Lubetzki et al, 1993. The myelin sheath is a tissue layer which is made up of lipid and cardinal proteins the proteolipoprotein (PLP) and the myelin primary protein (MBP). (Colman et al, 1982 and Boison et al, 1995). At regular intervals myelin sheath becomes thinner and is cognise as N odes of Ranvier (Peter et al, 1966). These regions atomic number 18 the billet for potential gated atomic number 11 stemmas and a yield of proteins. Microglial cells ar the macrophages of the brain, which atomic number 18 make in the b peerless ticker and ar wherefore transported to the brain by specialized protein c in alled chemokines (Khoury et al, 2008) The study of chemokine sense organs is atomic number 53 of the grave research argonas in the avenueo componentsis of Human Immuno Deficiency Virus. HIV muckle bottom microglial cells for their replication (Albright et al, 1999 Ghorpade et al, 1997 and Meer et al, 2000). Microglial cells be to a fault studied for their inflammatory responses in the brain. The realisation of role and mechanism by which microglial cells provoke inflammation has surface path for finding stubs and therapeutics for many an early(a)(prenominal) diseases.(Bhatia, 2008 Huang et al 2008 Hwang et al, 2008 and Kim et al, 2008).1.2.2. NeuronsNeuron or the nerve cells atomic number 18 unit of measurements of the nervous system come to in stir of galvanizing signal amongst each cutting(prenominal) and to the effector cells. There argon many types of nerve cells. Purkinje cells atomic number 18 one among them (Brown, 1991). The study of atomic rate 20 ion stock of Purkinje cell is the subject of this project.The basic parts of neuron consist of a soma or cell body, axonee, dendrites and neurites.All neurons atomic number 18 covered by the neuronic membrane. The soma or the cell body is alike(p) to any anformer(a)(prenominal) type of cell in the body. The axon is fibre that transport signal from the cell body to different neuron or to the target cell. The axons are covered by myelin sheath of the glial cells. The axon whitethorn be weapon systemed or unbranched. The main function of axon is to direct the electrical signal from the axon hillock of soma byout the axon cognise as the action potential and to transfer the signals to other cell in the form of chemical signal, the neurotransmitter (Purves et al, 2004 and Bear et al, 2001). The region of encounter with other cells where release of neurotransmitter takes place is cognize as the synapse. The release of neurotransmitter is facilitated by synaptic vesicles of the presynaptic oddment (one which release chemical signal). The neurotransmitters are released by the synaptic vesicle in the space between pre synaptic and post synaptic endpoint cognise as the synaptic cleft (Purves et al, 2004 and Brown et al, 1991). The neurotransmitters are then bring ind by detail receptors of the post synaptic magnetic pole which would componentrate an action potential in the cell. Apart from these receptors the ion transmit of the cell membrane of the synaptic term excessively respond in the transfer of signal. Dendrites are branched fibres that arise from the cell. Their surface is lined with number of receptor to rec eive signals for the neuron (Brown,1991., Purves et al, 2004., Thompson,1993 and Bear et al, 2001).Purkinje cells are one of the largest types of neurons on the brain. They are name in the cerebellar region of the brain. The study of atomic number 20 ion persuade of Purkinje cell is the subject of this project. Purkinje cells clear a number of branches dendrites that receive synaptic inputs. As the dendrites receive signals it initiates a Ca2+ signal, which are all substantial(predicate) secondary courier in the cells. The dendrites are the region for a atomic number 20 ion immersion through with(predicate) the atomic number 20 ion manoeuver. Similarly the soma contains K+ and Na+ bloods(Schutter et al, 1994). These ions are of particular(prenominal) sizeableness as their charge bod inside and outside the membrane set forth signalling in the cell. The transport of these ions is highly selective and they are well-kept by the ion deport proteins of the Purkinje cell membrane and other nervous membrane. These proteins form a center for the transport of ions. Techniques much(prenominal) as the Patch squeeze regularity choose made the study of these ion maneuvers easier (Bear et al, 2001).1.3. Ion roadIon conduct are glycoprotein complex that allow specialized ions through them. The proteins of ion line of products are work outd by antithetical divisor. More than 100 genes are cognise to code ion seams. The out-migration of ion is meaning(a) in generating action potential in the cell and is overly fundamental as the ions are second couriers in signalling. illnesss associated with the ion manoeuver are cognise as billetopathies. Ion carry raise be collar study types potency gated ion steer. Ligand gated ion driveway and the stretch and heat trigger off ion production line (Purves et al.,2004). potential difference gated ion conduct open and close on response to electrical potential. The voltage gated gives are m ade up of disparate protein sub unit. The fractional monetary units force out move to open or close the channel (Horn, 2002). Depending on the type of ions they conduct they are further divided into atomic number 20 channel, sodium channel and potassium channel. Ligand gated channels are those that respond to chemical signals. The ligand gated receptors are of quint types nicotinic acetylcholine receptor (AChR), glutamate receptor, -aminobutyric acid (GABA), glycine-activated Channels and the ryanodine receptor(Stroud et al, 1990). Each of these receptors bind to circumstantial ion and are rear in polar organs. The stretch and heat activated ion channel respond to heat or structural de validation of membrane (Purves et al, 2004).1.4. potential drop Gated atomic number 20 Channel (VGCC)Ca2+ ions are cardinal secondary messenger in cells and play important role in biochemical pathways of cell. The take aim and ingress of these Ca2+ ions in the cell is highly regulated. The regulations of these ions are controlled by the potency Gated atomic number 20 Channel (Gribkoff et al, 2006). These VGCC are mainly ground in excitatory cells such(prenominal) as the muscle builder cells and neurons. They exert their function by controlling muscle con pathwayion, neurotransmitter release, neuronal plasticity, synapses, and neuronal excit major power (Pietrobon, 2005 and Yang et al, 2005) . VGCC respond to membrane depolarisation facilitating Ca2+ entering into the cell and thereby activating the signalling cascade of the cell (Yang et al, 2005).The pattern functioning of the atomic number 20 channel protein is very important in a cell. magnetic variation in the gene cryptogram channel protein, give birth been know to ca example a number of diseases which include Timothy syndrome, Familial hemiplegic sick headache type 2, episodic ataxia type 2, spinocerebellar ataxia type 6 and autism spectrum disorderliness which are grouped under atomic number 20 c hannelopathies (Bidaud et al, 2006 and Jen et al, 1999). Calcium channels besides play a key role to mediate neuronal offend pathways (Gribkoff et al, 2006). A number of doses bring forth been cognise to block atomic number 20 channel and they are categorised as Calcium Channel Blockers. verapamil was the beginning(a) drug tack together to block Calcium Channel and subsequently dihydropyridines (DHPs) grade of drug was observe to act as calcium channel blocking agent (Dolphin, 2006). DHPs are of much importance in examine the channel properties of the Dihydropyridine rude(a) calcium channel. These DHP fine channels have dihydropyridine receptor for their attach (Campbell et al, 1988). Calcium Channel Blockers are now being bring potent in the handling of pain and hypertension (Atanassoff et al, 2000 Kize et al, 2001, and Thompson et al, 2001) but the question of stall hit in Coronary Heart Disease and the increased risk of malignant neoplastic disease in patients frame unanswered (Eisenberg et al, 2004 and Fitzpatrick et al, 1997).1.5. Calcium channel structureA calcium channel consists of five important fractional monetary units 1. 2, , and . The 1 subunit is cognise as the centralize forming complex (Yang et al, 2006).The 1 subunit is a whizz polypeptide and its functions mainly include voltage sensing, gating and selective permeation (Horn et al, 2000). The structure of 1 subunits consist of 24 surgical incisions (S1-S6) which constitute 4 domains, a C- terminal, N-terminal and Inter consorters. The linkers connecting domains are known as Loops and they are referred as loop I-II, loop II-III and loop III-IV estimateing on the domains they link (Dolphin, 2006).The intracellular loop of the 1 subunit has interaction point for the medical dressing subunit. The interaction elicit modulate the G- protein, an important second messenger in the cell (Dolphin, 1998). The detail binding of subunit to the tryptophan balance wheel is im portant for controlling the gating of 1 subunit of certain type of channels (Berrou, 2002). S4 is another(prenominal) important element of the calcium channel. It is the voltage sensitive region of the calcium channel. S4 segment moves outward make the channel to open by acquiring depolarised. S4 segment is positively charged due to the presence of arginine aminoacid reservation it voltage sensitive by translocation of the charges across the membrane (Sigworthl, 2003 and Horn et al, 2000). The S5, S6 and the linker connecting the S5 and S6 segment forms the boundaries ion conducting pore of the 1 subunit. The ion conductance partly depends on the rotational movement of the S4 segment which every cause the S6 segment to open or close the pore (Horn et al, 2000).The subunits of the calcium channel are thought to be tissue specific and organ specific. Primarily they are of 4 dissimilar types, 1, 2, 3 and 4. dissimilar isoforms of the subunits overly do exist which include (CaB2 a, CaB2b and CaB3) (Hullin et al, 1992 and Petegem et al, 2006). Their association with subunit is essential for flexion of VDI, CDI and CDF (Petegem et al, 2006). The 2 subunit is to a fault known as the 2/ subunit as both the subunits are product of a whizz gene (Petegem et al, 2006). The 2 and subunits are linked together by disulphide bonds. standardized other subunits 2/ besides exists as isoforms (Wang et al, 1999). They are known to play an important role in plasticity of neuron after(prenominal) a nerve injury and neuropathic pain butting (Luo et al, 2001). Gabapentin is a drug known to act on 2/ subunit, but their binding affinity varies with antithetic isoforms of the subunit (Luo et al, 2001 and Luo et al, 2002).The subunit is free-base exclusively in boney muscles. Their in operation(p) roles are yet to be detect (Petegem et al, 2006).The C- consummation of calcium channel is a settle for a number of protein- protein interactions in some channels. The e laborateness of the polyglutamine tract of the calcium channel is a major reason for the pathogenesis of the disease, Spino cerebellar dyssynergia 6 (SCA6). The cell death in SCA6 is thought to be ca utilize by the tipsiness of the nucleus by the localisation of C-terminal fragments (Kordasiewicz, 2006).1.6. Calcium Channel TypesCalcium channels key for the major amount of the calcium entry into the cell. The channel properties are tightly regulated to maintain Ca2+ concentration of the cell. The regulation was do through triplet well known summonses.Voltage Dependent defusing (VDI) obligated for preventing entry of calcium into the cell. Calcium Dependent Inactivation (CD1) responsible for(p) for preventing entry of calcium into the cell whereas Calcium Dependent Facilitation (CDF) allows for the entry of calcium for signalling (Petegem et al, 2006). ground on the amount of legitimate required to activate the channel the VDCC were termed both LVA channel (Low Voltage Activated) or HVA channel (High Voltage Activated). subsequent on due to the breakthrough of different current types, location of channel and sensitiveness to different types VDCC were broadly classified. Thus now 6 different types VDCC are known, in T type the current is transient, located in T-tubules and sensitive to dihydropyridine (DHP) (Dolphin, 2006). In L-Type the current is long lasting, found in neuron, touchwood and skeletal muscles and are sensitive to DHP. The N-Type stands for Non L Type or neural and they are sensitive to -conotoxin GVIA (Petegem et al, 2006). The current found in Purkinje cells of the cerebral cerebral cortex were P-Type, they were sensitive to -agatoxin IVA. The Q-Type current are found in granular cells, further scientist tump over P-Type and Q-Type to be same and are now term as P/Q- Type. The difference between the P Type and Q-Type is thought to depend on the subunit to which it is associated(Dolphin., 2006). Another type of Residual cu rrent was as well discovered which to exit is not sensitive to any of the known toxin, this current is known as R-Type (Dolphin, 2006 and Petegem et al, 2006).1.7. Calcium Channel GeneThe alpha sub unit of the calcium channel are coded by 10 genes, indeed 10 different 1 sub units are known. Of the ten types Cav 1.1 1.4 which is found in L-type, Cav 2.1 or the Cav1A is found in P/Q type channel, Cav2.2 is found in N type and Cav2.3 in R type channel. The Cav 3.1- 3.3 is found in T type channel. All these alpha subunit have one or more isoforms that would contribute to their useful diversity (Dolphin, 2006).The gene coding for the Cav 2.1, CACNA1A is found on the chromosome 19p13. This gene belongs to CACN family of gene that code for calcium channel. The gene characterised by the extension of CAG tri foot repeats. In humans the extension of the may interpolate from 4 to 18. Mutation of this gene cause diseases cause three major diseases FHM1 (Familial Hemiplegic sick headache 1), EA2 ( casual Ataxia 2) and SCA6 (Spino Cerebellar Ataxia 6).Familial Hemiplegic Migraine is an autosomal dominant type of migraine caused by the missense mutation in CACNA1A. one-third different mutations of CACNA1A cause FHM1 (Ducros et al, 1999). FHM1 strikes the channel deactivation and the energizings of the calcium channel (Kraus et al, 1997). The replacement of threonine with methionine is the mutation associated with FHM1. This mutation changes the channel structure do more flow of calcium into cell. This ultimately results in the release of excess neurotransmitter (Ophoff et al, 1998). Episodic Ataxia 2 (EA2) is neurological disorder affecting the cerebellum and causing ataxia. The drug acetozolamide is known to be effective on EA2 (Ophoff et al, 1998). This disease has been found to have piddling but stable trinucleotide expansion but the role of the expansion is unknown for this disease (Jodice et al, 1997). The mutation in EA2 causes truncation of 1A subunit whic h talent cause a complete loss of the function of the channel (Wappl et al, 2002).1.8. Spino Cerebellar Ataxia 6Spino Cerebellar Ataxia 6 is in like manner a neurodegenerative disease caused by the increase in number of CAG repeats in the CACNA1A gene (Tanaka et al, 2000). The number of trinucleotide repeat is between 22 and 28 in SCA6 (Riess, 1997). simply it is not only the CAG repeats that are causing the disease. The 1A have 6 isoforms and not all the isoforms are with the polyglutamine repeat. Therefore whether SCA6 is a channelopathy or Polyglutamine Disease remains a question among scientist (Frontali, 2006). The isoforms responsible for SCA6 is mainly special to the C-Terminal. As the C-terminal is site for protein- protein interaction, changes in strength of interaction or changes in interacting partners tremendously affect the channel kinetics and other functional modification. As polyglutamine disease it cause toxic effect considered through aggregate formation (Pril et al, 2004). affinity of number of repeats with other polyglutamine diseases where the repeat number is much high, the aggregate formation solo faecesnot account for pathogenesis (Matsuyama et al, 1999). As a channelopathy the degeneration of Purkinje cell is caused by the drunkenness of nucleus with the localised fragments of C-Terminal. The cleaved C terminal product is considered to have involved in signalling mechanism of the cell (Kordasiewcz et al, 2006). The isoforms of the C-Terminal of calcium channel are of great importance as the variation are found to be species specific (Kanumilli et al, 2005) and a a couple of(prenominal) of them do not code for polyglutamine repeats. This invokes an kindle in the C-terminal of the 1A subunit of the calcium channel. The isoforms are formed by a process known as the pre-mribonucleic acid parachute lap joint.1.9. spliceTranscription of messenger ribonucleic acid (mRNA) from DNA and reading of proteins from mRNA forms the cen tral dogma of molecular biology (Crick, 1970). These processes involves a series of important events, one among them is pre mRNA conjoin. sooner translation of protein, the mRNA needs to be processed by removing of non-coding introns. A human gene on an average consists of 8 introns. splicing stick out lead to more than one type of mRNA from a private gene and consequently different protein isoforms (Faustino et al, 2003). Many different proteins are involved in lap joint most importantly the spliceosome, a complex formed of small thermonuclear RNA (snRNA) and small nuclear ribonucleoproteins (Hagiwara et al, 2005 and Jurica et al, 2003). Small nuclear RNA can be of 5 important types U1, U2, U4, U5 and U6. All these in different combination target specific pre mRNA. The targeting is base on a number of factors which include phosphorylation of snRNAs, catalytic metal ions, enhancers, transcriptional coregulators and serine/arginine rich SR protein (Shi et al, 2006 Saba et al 2005 Auboeuf et al, 2007 Jurica et al, 2003 Hicks et al, 2005 and Manley et al, 2006).In general a spliceable introns has three regions splice donor, splice acceptor and a branch site. Most of the splice donor regions consist of AU nucleotide and the splice acceptor region consist of AG (Kenneth., 2005). Spliceosomes attach to these ends and by transesterification pull away the introns, followed by the ligation of the exon (Rio,1993). Several mRNA have inherent splicing mechanism that does not require any spliceosome as they can splice themselves known as self splicing (Herrin et al, 1990 and Landthaler et al, 1999). Though most of the splicing is limited within the same mRNA, splicing also finds between devil different mRNAs by trans-splicing mechanism. The 2 mRNA exons called the mini exons were transcribed in different gene and were then combined to translate for a integrity protein (Bonen, 1993 and Bonen, 2008). alternate(a) splicing is a mechanism by which a few genes find innumerable proteins that are diversified in structure and function. close to 75% of the human genes are involved in alternate splicing to relent different protein isoforms (Hagiwara et al, 2005 and Stamm et al, 2004). The needs to understand alternate splicing have arised in almost all fields of biology. In evolutionary harm alternate splicing has a major role in the functional information of species right from the times of RNA world. The importance of isoforms has been silent through a number of studies. The Active and inactive forms of Sex lethal protein isoform are the determinants of sex of Drosophila (Herbert et al, 1999 Irimia et al, 2007 and Poole et al, 1998). Many different isoforms of normal proteins are discovered in cancer cells. These studies of these isoforms and their role have revealed some important symptomatic approach and cancer cell biomarkers (Brinkman, 2004 Skotheim et al, 2007 and Pampalakis et al, 2008). In the drug discovery process it is indisp ensable to consider the mechanism of protein isoforms and pre mRNA splicing pathways and signalling molecules to recognise new targets for drugs (Levanon et al, 2003 and Hagiwara et al, 2005).Alternative splicing in ion channels alter the conductance and functional properties of the channel. Splicing has been known in voltage gated sodium channel, voltage gated calcium channel, ligand gated ion channel and in calcium gated potassium channel. Although the ion channels differ in their properties, all share some basic function. These ion channels have multiple splicing site through which their channelling properties are regulated based on the organs where these channels are located (Copley, 2004 Raymond et al, 2004 Sarao et al, 1991 and Schaller et al, 1992).1.10. CaV2.1 splice variantsVariants in calcium channel protein, in particular the 47 exon of the c-terminal is the basis of this study. Splicing in calcium channel occurs at distinct region such as the loops between the II-III do mains which is the major interacting site for ryodine receptor. Two isoforms BI and rbA are found in loop II-III of rat and rabbit. They differ in their interacting ability towards syntaxin and synaptotagmin proteins. These proteins can modulate the Ca+ influx of the neuron (Charvin et al, 1997 and Rettig et al, 1996). Site specific variations are found in exons 9, 31, 44, 46 and the extreme C terminus e47 (shown in Fig 3)(Kanumilli et al, 2005).The C- termini of calcium channels are involved in the passage of G-proteins, molecular switching of calmodulin and are the site for protein-protein interaction. So a wholeness amino acid change can potentially change the gating spot and other function of the channel and its interacting partners (Chaudhuri et al, 2004 Gray et al, 2007 Krovetz et al, 2000 and Ligon et al, 1997) splice variants were known to occur in the C- terminal the calcium channel. A 5 base couple launching (ggcag) was reported in pancreatic islets of rats a variant already known in human (Ligon et al, 1997). This 5 base oppose insertion is expected to alter the length on the c terminal and because channel property as it found before the stop codon, which manner a change in the reading frame. The existence of variants with and without the 5 base pair (ggcag) insert before the stop codon of rat Purkinje cell is sustain by Kanumilli et al (2005).Another independent study with hook by Tsunemi et al (2001) also confirmed the 5 base pair insert. In addition, variants without the stop codon and a ggcag insert, 150 nucleotide deletions in the 5- end of the C- terminal is reported in mouse (Kanumilli et al, 2005). The absence of stop codon was also discovered in the study by Tsunemi et al (2001) in mouse. Richards et al (2007) obtained similar results with rat Purkinje cell, the chronological sequence of exon 47 were same as the rat pancreatic cells except for variations in other exons. However variation in the number of amino acid (156 resid ues, 153 residues and 115 residues) coded by exon 47 were observed in different clones. The 156 amino acid length was also reported by Ligon et al (1998).These finding and most other results decipher the calcium channel properties in terms of activation or inactivation kinetics. However no protein- protein interaction study is available public treasury date for the exon 47 with five base pair (ggcag) inclusion before the stop codon. The need for studies at the protein-protein interaction level is necessary which is patent from the studies of Dolphin(2006), Richards et al (2007), Sandoz et al (2001) and Kanumilli et al (2005). This study was aimed at studying executable protein-protein interaction for exon 47 of rat Purkinje cell. Then linking the interacting the protein to already known biochemical pathway is expected to give more taste the channel and possibly a new perspective in the treatment of SCA6.1.11. Protein protein interaction studiesProtein-Protein interaction is a n important part in all biologic process. A protein- protein interaction can altogether change the binding characteristics, kinetic property and their catalytic ability (Eisenberg et al, 2000). A number of methods have been develop and used to study protein-protein interaction. These methods can be the detection and analysis of interaction or can be wake against a family of proteins. Detection methods are largely used to confirm and study known interaction. These methods include Protein phylogenetic relation chromatography, proportion Blotting, Coimmunoprecipitation and Cross- linking. The natural covering methods include protein probing, phage display and the barm Two Hybrid System (Y2H) (Phizicky et al, 1995). Bioinformatics tools such as protein docking are also important in predicting the protein interactions (Smith et al, 2002).1.12. yeast Two Hybrid System (Y2H)Yeast two loan-blend system is the most widely used protein screening methods. The requirement of an interac tion between two domains DNA Binding field of operation (DNA-BD) and energizing Domain (AD) for the facial expression of a newsperson gene (lac-z) in yeast is being exploited in Y2H. The lac-z gene expression gives our -galactosidase enzyme which can be observed by colour change confirming interaction (as shown in Fig 4) (Criekinge et al, 1999).The protein of interest ( trounce) is usually amalgamate with the BD and the interacting protein or the library protein is fused with activation domain. The protein of interest is normally termed as bait and the interacting protein is called a antecede. bacterial plasmid can be easily constructed to express alliance protein of interest. The bacterial shuttle vector can be disjunct and transfected into the yeast for their expression. On expression the DNA-BD fusion protein will bind to the upstream activation sequence of the newsperson gene. Two types of Y2H are known one is the congius4 based system and the other is the Lex A based s ystem. In Lex yeast two hybrid system the prey is fused with the Lex A binding domain. The specifically interacts with the Lex A operator upstream sequence which is the part of the takeoff booster for reporter gene. The prey will be fused with the GAL 4 protein. In the GAL 4 system instead of Lex A the GAL 4 promoter will be used. Both the systems have their advantages and their disadvantages (Criekinge et al, 1999 and Luban et al, 1995)The yeast caudex L40 is congruous with LexA operator and the GAL 4 promoter system. Most Y2H methods are make more than one reporter gene for more selectivity. HIS3 gene is one such reporter that is used for the nutritional selection of the cells. HIS3 reporter expression needs the interaction of proteins. So cells would not grow in a media lacking histidine if no interactions take place. Similar nutritional selections are also used in cell containing only the baits or only the prey. The nutritional selection for bait is tryptophan and for the pr ey is leucine. It is therefore important to use a defined media. A positive interaction between bait and the prey will allow growth in the Triple take down Out media (TDO/ -His/-Leu/-Trp) (Criekinge et al, 1999 and Luban et al, 1995)The use of histidine reporter gene can sometimes account for leaky expression. In which case 3-AT (3-amino-s-triole) a competitive inhibitor of histidine can be tried in various concentration to find a minimum concentration at which cells grow and the enzyme is inhibited. Cells growing concentration of 100mM concentration cannot be used as baits (Criekinge et al, 1999).Toxicity caused by bait can inhibit the growth of yeast (Zhong et al, 2003). Toxicity tests have to be carried out to after the baits are designed. Autoactivation of the baits should be checked before proceeding to the, library screening as nearly 5% of the protein can initiate transcription without an interactor (Criekinge et al, 1999). afterward the library screening the plasmids can b e isolated and used to exchange bacterial cells. The interaction also has to be confirmed and isolated by techniques such as coimmunoprecipitation.2. AimThis study was undertaken as a part of the project by Dr. Claire Palmer in finding novel protein-protein interaction for 5-base pair insert in exon 47 of rat cerebellar Purkinje cell(AF051526). Yeast 2 hybrid system was employed to study interaction. thence two protein baits 5inSER and NLSER were constructed by colleague Surya to screen against library protein.Baits5inSER is a 472 base pair length protein with ggcagNLSER is a 397 base pair length protein without the nuclear placement Sequence. It was constructed to find the significance of the nuclear localisation signal (Surya, 2008).The aims of the project areTo test for toxicity and autoactivation of baits. To determine the concentration of 3-AT at which the expression of Histidine gene is inhibited. Control uniting experiment.3. Materials and Methods3.1. Control Mating3.1.1. Control strainsThe control mating experiments were done prior to the library screen. The positive control yeast strains AH109 with the bait pGBKT7-53 and Y187 with the target pGADT7-T , glycerol stock were provided. For negative control the bait strain was L40 with bait pBTM116/GluR2 and the target was the same Y187pGADT7-T The negative control bait was obtained by the transformation of L40 with the plasmids isolated from provided E.Coli subtletys.3.1.2. Small Scale Yeast interlingual renditionA colony of Saccharomyces cerevisiae L40 yeast was inoculated into 10ml of YPAD media. It was left overnight in a shaking incubator (200rpm) at 30 C. The overnight culture was reduce in 50 ml of YPAD to an OD600Toxicity and Autoactivation of Baits ExperimentToxicity and Autoactivation of Baits ExperimentAbstractAlternate splicing in exon 47 of the Purkinje cell calcium channel generates a splice variant with a five base pair insert (ggcag) before the stop codon in rat. This five base pair change the open reading frame of the exon 47 for resulting in an extended C-Terminal. Novel protein interaction at this region was hypothesised. Yeast Two Hybrid System was employed to screen against cDNA library to check for any protein interaction with 5 base pair insert region of exon 47. This project aimed to test the toxicity/ autoactivation of the baits in the yeast and to find the minimum concentration of 3-AT (3-amino-s-triole) at which it inhibits the HIS3 gene. The experimental result shows that there was no leaky expression of the HIS3 gene. The autoactivation/toxicity test results showed that the baits are less toxic than the control bait. The growth of non-interacting colonies in the Triple Drop Out media revealed that a more defined media should be used, demanding the repetition of experiment to obtain more convincing results.1. Introduction1.1. Nervous SystemThe human nervous system consists of the Peripheral Nervous System (PNS) and the Central Nervous System (CNS). The PNS is formed of the cranial nerves and the spinal nerves. The central nervous system consists of the brain and the spinal cord. The brain can be divided into three major parts cerebrum, cerebellum and the brain stem. The cerebrum is divided into frontal lobe, parietal lobe, occipital lobe and the temporal lobe. The main function of cerebrum includes controlling of sensory organ, motor function, consciousness and imagining. The cerebellum is a uniform structure and its function is essential in movement and co- ordination of organs. The brain stem is made up of the mid brain, the pons and the medulla. The main functions of brain stem are transmission of information to and from the brain (Bear et al, 2001 Purves et al, 2004 and Thompson,1993).1.2. Cells of CNSThe brain consist mainly two types of cells nerve cells or neuron cells and the glial cells. The neuron are involved in the transport of electrical signals from the brain whereas the glial cells are thought to be the supporti ng cells of neurons by the uptake excess of neurotransmitter that are essential for signalling between neurons (Henn et al, 1971 and Purves et al, 2004) and plays a role in synaptogenesis of the neuron (Bacci et al, 1999). The glial cells are of three types astrocytes, oligodentrocytes and the microglial cells.1.2.1. Glial CellsAstrocytes are star shaped cells. The spatial arrangement of these cells between the capillaries and the neurons enables it in the modification of cellular responses, synaptic plasticity and survival of neurons (Abe et al, 2006 and Chen et al, 2003). Astrocytes important in glutamate transport, removal of free radical, controlling of haemostasis of brain and in maintaining a preferable environment for the active functioning of neurons by buffering K+ ions in their extracellular space (Chen et al, 2003 Gee et al, 2004 and Longuemare et al, 1999).Oligodentrocytes are type of glial cells that insulate the neuron with myelin sheath (Bear et al, 2001 and Lubetzki et al, 1993. The myelin sheath is a membrane which is made up of lipid and two proteins the proteolipoprotein (PLP) and the myelin basic protein (MBP). (Colman et al, 1982 and Boison et al, 1995). At regular intervals myelin sheath becomes thinner and is known as Nodes of Ranvier (Peter et al, 1966). These regions are the site for voltage gated sodium channels and a number of proteins. Microglial cells are the macrophages of the brain, which are formed in the bone marrow and are then transported to the brain by specialized protein called chemokines (Khoury et al, 2008) The study of chemokine receptors is one of the important research areas in the pathogenesis of Human Immuno Deficiency Virus. HIV can target microglial cells for their replication (Albright et al, 1999 Ghorpade et al, 1997 and Meer et al, 2000). Microglial cells are also studied for their inflammatory responses in the brain. The identification of role and mechanism by which microglial cells cause inflammation has pave d path for finding targets and therapeutics for many diseases.(Bhatia, 2008 Huang et al 2008 Hwang et al, 2008 and Kim et al, 2008).1.2.2. NeuronsNeuron or the nerve cells are units of the nervous system involved in transfer of electrical signal between each other and to the effector cells. There are many types of nerve cells. Purkinje cells are one among them (Brown, 1991). The study of calcium ion channel of Purkinje cell is the subject of this project.The basic parts of neuron consist of a soma or cell body, axon, dendrites and neurites.All neurons are covered by the neuronal membrane. The soma or the cell body is similar to any other type of cell in the body. The axon is fibre that transport signal from the cell body to other neuron or to the target cell. The axons are covered by myelin sheath of the glial cells. The axon may be branched or unbranched. The main function of axon is to transfer the electrical signal from the axon hillock of soma passim the axon known as the actio n potential and to transfer the signals to other cell in the form of chemical signal, the neurotransmitter (Purves et al, 2004 and Bear et al, 2001). The region of contact with other cells where release of neurotransmitter takes place is known as the synapse. The release of neurotransmitter is facilitated by synaptic vesicles of the presynaptic terminal (one which release chemical signal). The neurotransmitters are released by the synaptic vesicle in the space between pre synaptic and post synaptic terminal known as the synaptic cleft (Purves et al, 2004 and Brown et al, 1991). The neurotransmitters are then received by specific receptors of the post synaptic terminal which would generate an action potential in the cell. Apart from these receptors the ion channels of the cell membrane of the synaptic terminal also respond in the transfer of signal. Dendrites are branched fibres that arise from the cell. Their surface is lined with number of receptor to receive signals for the neuron (Brown,1991., Purves et al, 2004., Thompson,1993 and Bear et al, 2001).Purkinje cells are one of the largest types of neurons on the brain. They are found in the cerebellar region of the brain. The study of calcium ion channel of Purkinje cell is the subject of this project. Purkinje cells have a number of branches dendrites that receive synaptic inputs. As the dendrites receive signals it initiates a Ca2+ signal, which are important secondary messenger in the cells. The dendrites are the region for a calcium ion entry through the calcium ion channel. Similarly the soma contains K+ and Na+ channels(Schutter et al, 1994). These ions are of particular importance as their charge variation inside and outside the membrane trigger signalling in the cell. The transport of these ions is highly selective and they are maintained by the ion channel proteins of the Purkinje cell membrane and other neuronal membrane. These proteins form a pore for the transport of ions. Techniques such as the P atch clamp method have made the study of these ion channels easier (Bear et al, 2001).1.3. Ion channelIon channels are glycoprotein complex that allow specific ions through them. The proteins of ion channel are coded by different gene. More than 100 genes are known to code ion channels. The transportation of ion is important in generating action potential in the cell and is also important as the ions are second messengers in signalling. Diseases associated with the ion channel are known as channelopathies. Ion channels can be three major types voltage gated ion channel. Ligand gated ion channel and the stretch and heat activated ion channel (Purves et al.,2004).Voltage gated ion channels open and close on response to electrical potential. The voltage gated channels are made up of different protein sub unit. The subunits can move to open or close the channel (Horn, 2002). Depending on the type of ions they conduct they are further divided into Calcium channel, sodium channel and pota ssium channel. Ligand gated channels are those that respond to chemical signals. The ligand gated receptors are of five types nicotinic acetylcholine receptor (AChR), glutamate receptor, -aminobutyric acid (GABA), glycine-activated Channels and the ryanodine receptor(Stroud et al, 1990). Each of these receptors bind to specific ion and are found in different organs. The stretch and heat activated ion channel respond to heat or structural deformation of membrane (Purves et al, 2004).1.4. Voltage Gated Calcium Channel (VGCC)Ca2+ ions are important secondary messenger in cells and play important role in biochemical pathways of cell. The level and entry of these Ca2+ ions in the cell is highly regulated. The regulations of these ions are controlled by the Voltage Gated Calcium Channel (Gribkoff et al, 2006). These VGCC are mainly found in excitatory cells such as the muscle cells and neurons. They exert their function by controlling muscle contraction, neurotransmitter release, neuronal plasticity, synapses, and neuronal excitability (Pietrobon, 2005 and Yang et al, 2005) . VGCC respond to membrane depolarization facilitating Ca2+ entry into the cell and thereby activating the signalling cascade of the cell (Yang et al, 2005).The normal functioning of the calcium channel protein is very important in a cell. Mutation in the gene coding channel protein, have been known to cause a number of diseases which include Timothy syndrome, Familial hemiplegic migraine type 2, episodic ataxia type 2, spinocerebellar ataxia type 6 and autism spectrum disorder which are grouped under calcium channelopathies (Bidaud et al, 2006 and Jen et al, 1999). Calcium channels also play a key role to mediate neuronal pain pathways (Gribkoff et al, 2006). A number of drugs have been known to block calcium channel and they are categorised as Calcium Channel Blockers. Verapamil was the first drug found to block Calcium Channel and later dihydropyridines (DHPs) class of drug was discovered to a ct as calcium channel blocker (Dolphin, 2006). DHPs are of much importance in studying the channel properties of the Dihydropyridine sensitive calcium channel. These DHP sensitive channels have dihydropyridine receptor for their binding (Campbell et al, 1988). Calcium Channel Blockers are now being found effective in the treatment of pain and hypertension (Atanassoff et al, 2000 Kize et al, 2001, and Thompson et al, 2001) but the question of safety in Coronary Heart Disease and the increased risk of cancer in patients remains unanswered (Eisenberg et al, 2004 and Fitzpatrick et al, 1997).1.5. Calcium channel structureA calcium channel consists of five important subunits 1. 2, , and . The 1 subunit is known as the pore forming complex (Yang et al, 2006).The 1 subunit is a single polypeptide and its functions mainly include voltage sensing, gating and selective permeation (Horn et al, 2000). The structure of 1 subunits consist of 24 segments (S1-S6) which constitute 4 domains, a C- t erminal, N-terminal and Interlinkers. The linkers connecting domains are known as Loops and they are referred as loop I-II, loop II-III and loop III-IV depending on the domains they link (Dolphin, 2006).The intracellular loop of the 1 subunit has interaction site for the binding subunit. The interaction can modulate the G- protein, an important second messenger in the cell (Dolphin, 1998). The specific binding of subunit to the tryptophan residue is important for controlling the gating of 1 subunit of certain type of channels (Berrou, 2002). S4 is another important segment of the calcium channel. It is the voltage sensitive region of the calcium channel. S4 segment moves outward causing the channel to open by getting depolarised. S4 segment is positively charged due to the presence of arginine aminoacid making it voltage sensitive by translocation of the charges across the membrane (Sigworthl, 2003 and Horn et al, 2000). The S5, S6 and the linker connecting the S5 and S6 segment f orms the boundaries ion conducting pore of the 1 subunit. The ion conductance partly depends on the rotational movement of the S4 segment which either cause the S6 segment to open or close the pore (Horn et al, 2000).The subunits of the calcium channel are thought to be tissue specific and organ specific. Primarily they are of 4 different types, 1, 2, 3 and 4. Different isoforms of the subunits also do exist which include (CaB2a, CaB2b and CaB3) (Hullin et al, 1992 and Petegem et al, 2006). Their association with subunit is essential for modulation of VDI, CDI and CDF (Petegem et al, 2006). The 2 subunit is also known as the 2/ subunit as both the subunits are product of a single gene (Petegem et al, 2006). The 2 and subunits are linked together by disulphide bonds. Like other subunits 2/ also exists as isoforms (Wang et al, 1999). They are known to play an important role in plasticity of neuron after a nerve injury and neuropathic pain processing (Luo et al, 2001). Gabapentin i s a drug known to act on 2/ subunit, but their binding affinity varies with different isoforms of the subunit (Luo et al, 2001 and Luo et al, 2002).The subunit is found only in skeletal muscles. Their functional roles are yet to be discovered (Petegem et al, 2006).The C-terminus of calcium channel is a site for a number of protein- protein interactions in some channels. The expansion of the polyglutamine tract of the calcium channel is a major reason for the pathogenesis of the disease, Spino Cerebellar Ataxia 6 (SCA6). The cell death in SCA6 is thought to be caused by the poisoning of the nucleus by the localisation of C-terminal fragments (Kordasiewicz, 2006).1.6. Calcium Channel TypesCalcium channels account for the major amount of the calcium entry into the cell. The channel properties are tightly regulated to maintain Ca2+ concentration of the cell. The regulation was done through three well known processes.Voltage Dependent Inactivation (VDI) responsible for preventing entr y of calcium into the cell. Calcium Dependent Inactivation (CD1) responsible for preventing entry of calcium into the cell whereas Calcium Dependent Facilitation (CDF) allows for the entry of calcium for signalling (Petegem et al, 2006).Based on the amount of current required to activate the channel the VDCC were termed either LVA channel (Low Voltage Activated) or HVA channel (High Voltage Activated). Later on due to the discovery of different current types, location of channel and sensitiveness to different types VDCC were broadly classified. Thus now 6 different types VDCC are known, in T type the current is transient, located in T-tubules and sensitive to dihydropyridine (DHP) (Dolphin, 2006). In L-Type the current is long lasting, found in neuron, heart and skeletal muscles and are sensitive to DHP. The N-Type stands for Non L Type or Neuronal and they are sensitive to -conotoxin GVIA (Petegem et al, 2006). The current found in Purkinje cells of the cerebral cortex were P-Typ e, they were sensitive to -agatoxin IVA. The Q-Type current are found in granular cells, however scientist consider P-Type and Q-Type to be same and are now term as P/Q- Type. The difference between the P Type and Q-Type is thought to depend on the subunit to which it is associated(Dolphin., 2006). Another type of Residual current was also discovered which to date is not sensitive to any of the known toxin, this current is known as R-Type (Dolphin, 2006 and Petegem et al, 2006).1.7. Calcium Channel GeneThe alpha sub unit of the calcium channel are coded by 10 genes, therefore 10 different 1 sub units are known. Of the ten types Cav 1.1 1.4 which is found in L-type, Cav 2.1 or the Cav1A is found in P/Q type channel, Cav2.2 is found in N type and Cav2.3 in R type channel. The Cav 3.1- 3.3 is found in T type channel. All these alpha subunit have one or more isoforms that would contribute to their functional diversity (Dolphin, 2006).The gene coding for the Cav 2.1, CACNA1A is found on the chromosome 19p13. This gene belongs to CACN family of gene that code for calcium channel. The gene characterised by the extension of CAG trinucleotide repeats. In humans the extension of the may vary from 4 to 18. Mutation of this gene cause diseases cause three major diseases FHM1 (Familial Hemiplegic Migraine 1), EA2 (Episodic Ataxia 2) and SCA6 (Spino Cerebellar Ataxia 6).Familial Hemiplegic Migraine is an autosomal dominant type of migraine caused by the missense mutation in CACNA1A. Three different mutations of CACNA1A cause FHM1 (Ducros et al, 1999). FHM1 affects the channel inactivation and the kinetics of the calcium channel (Kraus et al, 1997). The replacement of threonine with methionine is the mutation associated with FHM1. This mutation changes the channel structure causing more flow of calcium into cell. This ultimately results in the release of excess neurotransmitter (Ophoff et al, 1998). Episodic Ataxia 2 (EA2) is neurological disorder affecting the cerebellum and causing ataxia. The drug acetozolamide is known to be effective on EA2 (Ophoff et al, 1998). This disease has been found to have small but stable trinucleotide expansion but the role of the expansion is unknown for this disease (Jodice et al, 1997). The mutation in EA2 causes truncation of 1A subunit which might cause a complete loss of the function of the channel (Wappl et al, 2002).1.8. Spino Cerebellar Ataxia 6Spino Cerebellar Ataxia 6 is also a neurodegenerative disease caused by the increase in number of CAG repeats in the CACNA1A gene (Tanaka et al, 2000). The number of trinucleotide repeat is between 22 and 28 in SCA6 (Riess, 1997). But it is not only the CAG repeats that are causing the disease. The 1A have 6 isoforms and not all the isoforms are with the polyglutamine repeat. Therefore whether SCA6 is a channelopathy or Polyglutamine Disease remains a question among scientist (Frontali, 2006). The isoforms responsible for SCA6 is mainly limited to the C-Terminal. As th e C-terminal is site for protein- protein interaction, changes in strength of interaction or changes in interacting partners tremendously affect the channel kinetics and other functional modification. As polyglutamine disease it cause toxic effect considered through aggregate formation (Pril et al, 2004). Comparison of number of repeats with other polyglutamine diseases where the repeat number is much high, the aggregate formation alone cannot account for pathogenesis (Matsuyama et al, 1999). As a channelopathy the degeneration of Purkinje cell is caused by the poisoning of nucleus with the localised fragments of C-Terminal. The cleaved C terminal product is considered to have involved in signalling mechanism of the cell (Kordasiewcz et al, 2006). The isoforms of the C-Terminal of calcium channel are of considerable importance as the variation are found to be species specific (Kanumilli et al, 2005) and a few of them do not code for polyglutamine repeats. This invokes an interest i n the C-terminal of the 1A subunit of the calcium channel. The isoforms are formed by a process known as the pre-mRNA alternate splicing.1.9. SplicingTranscription of messenger RNA (mRNA) from DNA and translation of proteins from mRNA forms the central dogma of molecular biology (Crick, 1970). These processes involves a series of important events, one among them is pre mRNA splicing. Before translation of protein, the mRNA needs to be processed by removing of non-coding introns. A human gene on an average consists of 8 introns. Splicing can lead to more than one type of mRNA from a single gene and consequently different protein isoforms (Faustino et al, 2003). Many different proteins are involved in splicing most importantly the spliceosome, a complex formed of small nuclear RNA (snRNA) and small nuclear ribonucleoproteins (Hagiwara et al, 2005 and Jurica et al, 2003). Small nuclear RNA can be of 5 important types U1, U2, U4, U5 and U6. All these in different combination target spec ific pre mRNA. The targeting is based on a number of factors which include phosphorylation of snRNAs, catalytic metal ions, enhancers, transcriptional coregulators and serine/arginine rich SR protein (Shi et al, 2006 Saba et al 2005 Auboeuf et al, 2007 Jurica et al, 2003 Hicks et al, 2005 and Manley et al, 2006).In general a spliceable introns has three regions splice donor, splice acceptor and a branch site. Most of the splice donor regions consist of AU nucleotide and the splice acceptor region consist of AG (Kenneth., 2005). Spliceosomes attach to these ends and by transesterification remove the introns, followed by the ligation of the exon (Rio,1993). Several mRNA have inherent splicing mechanism that does not require any spliceosome as they can splice themselves known as self splicing (Herrin et al, 1990 and Landthaler et al, 1999). Though most of the splicing is limited within the same mRNA, splicing also occurs between two different mRNAs by trans-splicing mechanism. The two mRNA exons called the mini exons were transcribed in different gene and were then combined to translate for a single protein (Bonen, 1993 and Bonen, 2008).Alternative splicing is a mechanism by which a few genes produce innumerable proteins that are diversified in structure and function. Nearly 75% of the human genes are involved in alternate splicing to give different protein isoforms (Hagiwara et al, 2005 and Stamm et al, 2004). The needs to understand alternate splicing have arised in almost all fields of biology. In evolutionary terms alternate splicing has a major role in the functional development of species right from the times of RNA world. The importance of isoforms has been understood through a number of studies. The Active and inactive forms of Sex lethal protein isoform are the determinants of sex of Drosophila (Herbert et al, 1999 Irimia et al, 2007 and Poole et al, 1998). Many different isoforms of normal proteins are discovered in cancer cells. These studies of these isoforms and their role have revealed some important diagnostic approach and cancer cell biomarkers (Brinkman, 2004 Skotheim et al, 2007 and Pampalakis et al, 2008). In the drug discovery process it is necessary to consider the mechanism of protein isoforms and pre mRNA splicing pathways and signalling molecules to identify new targets for drugs (Levanon et al, 2003 and Hagiwara et al, 2005).Alternative splicing in ion channels alter the conductance and functional properties of the channel. Splicing has been known in voltage gated sodium channel, voltage gated calcium channel, ligand gated ion channel and in calcium gated potassium channel. Although the ion channels differ in their properties, all share some basic function. These ion channels have multiple splicing site through which their channelling properties are regulated based on the organs where these channels are located (Copley, 2004 Raymond et al, 2004 Sarao et al, 1991 and Schaller et al, 1992).1.10. CaV2.1 splice variants Variants in calcium channel protein, in particular the 47 exon of the c-terminal is the basis of this study. Splicing in calcium channel occurs at distinct region such as the loops between the II-III domains which is the major interacting site for ryodine receptor. Two isoforms BI and rbA are found in loop II-III of rat and rabbit. They differ in their interacting ability towards syntaxin and synaptotagmin proteins. These proteins can modulate the Ca+ influx of the neuron (Charvin et al, 1997 and Rettig et al, 1996). Site specific variations are found in exons 9, 31, 44, 46 and the extreme C terminus e47 (shown in Fig 3)(Kanumilli et al, 2005).The C- termini of calcium channels are involved in the modulation of G-proteins, molecular switching of calmodulin and are the site for protein-protein interaction. So a single amino acid change can potentially change the gating property and other function of the channel and its interacting partners (Chaudhuri et al, 2004 Gray et al, 2007 Krov etz et al, 2000 and Ligon et al, 1997) splice variants were known to occur in the C- terminal the calcium channel. A 5 base pair insertion (ggcag) was reported in pancreatic islets of rats a variant already known in human (Ligon et al, 1997). This 5 base pair insertion is expected to alter the length on the c terminal and hence channel property as it found before the stop codon, which means a change in the reading frame. The existence of variants with and without the 5 base pair (ggcag) insert before the stop codon of rat Purkinje cell is confirmed by Kanumilli et al (2005).Another independent study with mouse by Tsunemi et al (2001) also confirmed the 5 base pair insert. In addition, variants without the stop codon and a ggcag insert, 150 nucleotide deletions in the 5- end of the C- terminal is reported in mouse (Kanumilli et al, 2005). The absence of stop codon was also observed in the study by Tsunemi et al (2001) in mouse. Richards et al (2007) obtained similar results with rat Purkinje cell, the sequence of exon 47 were same as the rat pancreatic cells except for variations in other exons. However variation in the number of amino acid (156 residues, 153 residues and 115 residues) coded by exon 47 were observed in different clones. The 156 amino acid length was also reported by Ligon et al (1998).These finding and most other results describe the calcium channel properties in terms of activation or inactivation kinetics. However no protein- protein interaction study is available till date for the exon 47 with five base pair (ggcag) inclusion before the stop codon. The need for studies at the protein-protein interaction level is necessary which is evident from the studies of Dolphin(2006), Richards et al (2007), Sandoz et al (2001) and Kanumilli et al (2005). This study was aimed at studying possible protein-protein interaction for exon 47 of rat Purkinje cell. Then linking the interacting the protein to already known biochemical pathway is expected to give more insight the channel and possibly a new perspective in the treatment of SCA6.1.11. Protein protein interaction studiesProtein-Protein interaction is an important part in all biological process. A protein- protein interaction can altogether change the binding characteristics, kinetic property and their catalytic ability (Eisenberg et al, 2000). A number of methods have been developed and used to study protein-protein interaction. These methods can be the detection and analysis of interaction or can be screening against a family of proteins. Detection methods are mostly used to confirm and study known interaction. These methods include Protein Affinity chromatography, Affinity Blotting, Coimmunoprecipitation and Cross- linking. The screening methods include protein probing, phage display and the Yeast Two Hybrid System (Y2H) (Phizicky et al, 1995). Bioinformatics tools such as protein docking are also important in predicting the protein interactions (Smith et al, 2002).1.12. Yeas t Two Hybrid System (Y2H)Yeast two hybrid system is the most widely used protein screening methods. The requirement of an interaction between two domains DNA Binding Domain (DNA-BD) and Activation Domain (AD) for the expression of a reporter gene (lac-z) in yeast is being exploited in Y2H. The lac-z gene expression gives our -galactosidase enzyme which can be observed by colour change confirming interaction (as shown in Fig 4) (Criekinge et al, 1999).The protein of interest (bait) is usually fused with the BD and the interacting protein or the library protein is fused with activation domain. The protein of interest is normally termed as bait and the interacting protein is called a prey. Bacterial plasmid can be easily constructed to express fusion protein of interest. The bacterial shuttle vector can be isolated and transfected into the yeast for their expression. On expression the DNA-BD fusion protein will bind to the upstream activation sequence of the reporter gene. Two types of Y2H are known one is the GAL4 based system and the other is the Lex A based system. In Lex yeast two hybrid system the prey is fused with the Lex A binding domain. The specifically interacts with the Lex A operator upstream sequence which is the part of the promoter for reporter gene. The prey will be fused with the GAL 4 protein. In the GAL 4 system instead of Lex A the GAL 4 promoter will be used. Both the systems have their advantages and their disadvantages (Criekinge et al, 1999 and Luban et al, 1995)The yeast strain L40 is compatible with LexA operator and the GAL 4 promoter system. Most Y2H methods are done more than one reporter gene for more selectivity. HIS3 gene is one such reporter that is used for the nutritional selection of the cells. HIS3 reporter expression needs the interaction of proteins. So cells would not grow in a media lacking histidine if no interactions take place. Similar nutritional selections are also used in cell containing only the baits or only the p rey. The nutritional selection for bait is tryptophan and for the prey is leucine. It is therefore important to use a defined media. A positive interaction between bait and the prey will allow growth in the Triple Drop Out media (TDO/ -His/-Leu/-Trp) (Criekinge et al, 1999 and Luban et al, 1995)The use of histidine reporter gene can sometimes account for leaky expression. In which case 3-AT (3-amino-s-triole) a competitive inhibitor of histidine can be tried in various concentration to find a minimum concentration at which cells grow and the enzyme is inhibited. Cells growing concentration of 100mM concentration cannot be used as baits (Criekinge et al, 1999).Toxicity caused by bait can inhibit the growth of yeast (Zhong et al, 2003). Toxicity tests have to be carried out to after the baits are designed. Autoactivation of the baits should be checked before proceeding to the, library screening as nearly 5% of the protein can initiate transcription without an interactor (Criekinge et al, 1999). After the library screening the plasmids can be isolated and used to transform bacterial cells. The interaction also has to be confirmed and isolated by techniques such as coimmunoprecipitation.2. AimThis study was undertaken as a part of the project by Dr. Claire Palmer in finding novel protein-protein interaction for 5-base pair insert in exon 47 of rat cerebellar Purkinje cell(AF051526). Yeast 2 hybrid system was employed to study interaction. Accordingly two protein baits 5inSER and NLSER were constructed by colleague Surya to screen against library protein.Baits5inSER is a 472 base pair length protein with ggcagNLSER is a 397 base pair length protein without the Nuclear Localisation Sequence. It was constructed to find the significance of the nuclear localisation signal (Surya, 2008).The aims of the project areTo test for toxicity and autoactivation of baits. To determine the concentration of 3-AT at which the expression of Histidine gene is inhibited. Control mating experiment.3. Materials and Methods3.1. Control Mating3.1.1. Control strainsThe control mating experiments were done prior to the library screen. The positive control yeast strains AH109 with the bait pGBKT7-53 and Y187 with the target pGADT7-T , glycerol stock were provided. For negative control the bait strain was L40 with bait pBTM116/GluR2 and the target was the same Y187pGADT7-T The negative control bait was obtained by the transformation of L40 with the plasmids isolated from provided E.Coli cultures.3.1.2. Small Scale Yeast TransformationA colony of Saccharomyces cerevisiae L40 yeast was inoculated into 10ml of YPAD media. It was left overnight in a shaking incubator (200rpm) at 30 C. The overnight culture was diluted in 50 ml of YPAD to an OD600