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Abstract

Alternate 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. Introduction

1.1. Nervous System

The 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 CNS

The 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 supporting 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 Cells

Astrocytes 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 paved 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. Neurons

Neuron 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 throughout the axon known 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 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 Patch clamp method have made the study of these ion channels easier (Bear et al, 2001).

1.3. Ion channel

Ion 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 potassium 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, γ-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 act 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 structure

A calcium channel consists of five important subunits α1. α2, β, δ and γ. The α1 subunit is known as the pore forming complex (Yang et al, 2006).

The α1 subunit is a single polypeptide and its functions mainly include voltage sensing, gating and selective permeation (Horn et al, 2000). The structure of α1 subunits consist of 24 segments (S1-S6) which constitute 4 domains, a C- terminal, 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 α1 subunit has interaction site for the binding β subunit. The interaction can modulate the G- protein, an important second messenger in the cell (Dolphin, 1998). The specific binding of β subunit to the tryptophan residue is important for controlling the gating of α1 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 forms the boundaries ion conducting pore of the α1 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 β subunits of the calcium channel are thought to be tissue specific and organ specific. Primarily they are of 4 different types, β1, β2, β3 and β4. Different isoforms of the β subunits also do exist which include (CaB2a, CaB2b and CaB3) (Hullin et al, 1992 and Petegem et al, 2006). Their association with α subunit is essential for modulation of VDI, CDI and CDF (Petegem et al, 2006). The α2 subunit is also known as the α2/δ subunit as both the subunits are product of a single gene (Petegem et al, 2006). The α2 and δ subunits are linked together by disulphide bonds. Like other subunits α2/δ 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 is a drug known to act on α2/δ subunit, but their binding affinity varies with different isoforms of the δ subunit (Luo et al, 2001 and Luo et al, 2002).The γ 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 Types

Calcium 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 entry 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 ω-conotoxin GVIA (Petegem et al, 2006). The current found in Purkinje cells of the cerebral cortex were P-Type, they were sensitive to ω -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 β 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 Gene

The alpha sub unit of the calcium channel are coded by 10 genes, therefore 10 different α1 sub units are known. Of the ten types Cav 1.1 - 1.4 which is found in L-type, Cav 2.1 or the Cavα1A 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 α1A subunit which might cause a complete loss of the function of the channel (Wappl et al, 2002).

1.8. Spino Cerebellar Ataxia 6

Spino 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 α1A 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 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). 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 in the C-terminal of the α1A subunit of the calcium channel. The isoforms are formed by a process known as the pre-mRNA alternate splicing.

1.9. Splicing

Transcription 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 specific 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; 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 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 studies

Protein-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. Yeast 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 β-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 prey. 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. Aim

This 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.

Baits

5inSER is a 472 base pair length protein with ggcag

NLSER 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 are

To 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 Methods

3.1. Control Mating

3.1.1. Control strains

The 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 Y187[pGADT7-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 Transformation

A colony of Saccharomyces cerevisiae L40 yeast was inoculated into 10ml of YPAD media. It was left overnight in a shaking incubator (200rpm) at 30⁰ C. The overnight culture was diluted in 50 ml of YPAD to an OD600 of 0.3. The cultures were grown an additional 3 hours. The cells were then pelleted by spinning at 2500 rpm for 5 minutes. Pellet was resuspended in 40 ml dH2O. Then cells were again centrifuged for pellet. The pellets were resuspended in 2ml of (LiAc)/0.5XTE and were incubated at room temperature for 10 minutes. One μg of plasmid DNA and 100μg of denatured, sheared salmon sperm DNA (ssDNA) were mixed in a tube. The plasmid isolation was done through miniprep (GenElute Kit) from E.coli. The transformation procedures are as described in appendix. The ssDNA was heated at 100⁰C in a water bath for 10 minutes and then leave on ice prior to mixing.

To the DNA 2 ml yeast suspension was added and mixed well. Then 700μl of 100mM LiAc/40% /PEG-3350/1X TE was added and incubated at 30⁰C for 30 minutes. After incubation 88μl of Dimethyl Sulphoxide (DMSO) was added and heat shocked at 42⁰ C for 7 minutes. It was centrifuged at maximum for 1 minute. The pellets were resuspended in 100 μl of dH2O and were plated on selective media (Single Drop Out (SDO)/- Trp).

The glycerol stock colonies were obtained by streaking on single drop out media plate. AH109 [pGBKT7-53] on SD/ -Trp agar and Y187 [pGADT7-T] on SD/ -Leu agar. All the plates were incubated at 30⁰C for 3 days to obtain culture. After cultures were obtained large colonies were picked from each of the plates for small scale mating. The mating were done between AH109 [pGBKT7-53] & Y187 [pGADT7-T] and L40 [pBTM116/GluR2] & Y187[pGADT7-T]. Then colonies were inoculated in 500μl of 2x YPAD and were mixed by vortexing. They were left 24 hours for mating in a 200rpm shaking incubator at 30⁰C .After 23 hours the cells were viewed under a 40X Phase Contrast Microscope for the formation of tri-lobed zygotes. The mated cultures were then serial diluted to concentrations of 1/10, 1/1000 and 1/1000 in 2xYPAD. The diluted cells were then plated on SD/ -Trp, SD/ -Leu, SD/-Trp/-Leu/-His. They were incubated at 30⁰C for 3 days. β- Galactosidase filter paper assay was done on 1/10 concentration plates for both positive and negative control. The plates were chosen as the growth of colonies in those plates was clear and without any background growth.

3.1.3. β- Galactosidase Filter Assay

Whatmann NO.1 filter paper was laid on the yeast plate. The filter paper was gently pressed to obtain close contact with yeast. The filter paper was lifted with forceps from the plate and was placed on a petri plate with colony side up. The cells were then lysed by incubated them at -80⁰C for 10 minutes.

In a lid of petridish 1.5 ml of the Z Buffer and 30μl of 40mg/ml X-gal, one Whatmann NO.1 filter paper and over that the filter paper with the colony side up were placed. The petridish was covered and incubated at 37⁰C for 30 minutes to overnight to look for colour development.

3.2. Toxicity/Auto Activation Test

Small scale transformation L40 yeast was done with all the baits (pBTM116, pBTM116/GluR2, pBTM116/5inSER and pBTM116/NLSER).

Prey plasmid (pGAD10) and the auto activation control plasmids (GluR2/NSF, GluR2/GRIP, GluR2/PICK1 and GluR2/pGAD10).

The transformation was done except for the doubling the volume of plasmid DNA, ssDNA and the final resuspension dH2O to obtain enough colonies for plating. All the baits were plated on Single Drop Out SDO/-Trp, SDO/-Leu and Triple Drop Out TDO/-Trp/-Leu/-His media. The prey plasmid was plated on SDO/-Trp, SDO/-Leu and TDO/-Trp/-Leu/-His media. The auto activation control plasmids were plated on Double Drop Out media DDO/-Trp/-Leu and on TDO/-Trp/-Leu/-His. The plates were then incubated at 30⁰C for 3 days. β- Galactosidase Filter Assay was done on all TDO plates.

3.3. Determining Concentration 3-AT for Histidine Inhibition

L40 colonies were transformed with bait plasmids (GluR2/pBTM116, 5inSER/pBTM116 and NLSER/pBTM116). The transformation was done except for the doubling the volume of plasmid DNA, ssDNA and the final resuspension dH2O to obtain enough colonies for plating. DDO media/-T/-H with different concentrations of 3-AT was used to plate the transformed cells. The concentrations used were 0mM, 5mM, 10mM, 50mM and 60mM. All the plates were incubated at 30⁰C for 3 days and observed for the growth of colonies.

4. Result and Discussion

4.1. Yeast Control Mating

The mating of yeast can occur only between the two opposite mating type MATa and MATα The type of yeast (a or α) is determined by the locus of the MAT gene (Montelone, 2002). Thus the strain used in the control mating are either a(AH109, L40) or α(Y187). The plasmid in the positive control strain code for a fusion protein that contain DNA-Binding Domain (DNA-BD) and the p53. The other strain of the positive control code for a fusion protein that contain the Activation Domain (AD) and the Simian Virus large T antigen. The role of p53 has been extensively studied right from their anti-apoptotic ability to the signalling property in all stages of cell cycle (Stewart et al, 2001). The role of SV large T antigen to bind and modulate p53 has been extensively studied and verified (Dobbelstein, 1998 et al and Mccarthy et al, 1993).Thus it was appropriate to choose their interaction as positive control for this yeast 2 hybrid study.

The negative control strain L40 plasmid (pBTM116/GluR2) code for a fusion protein containing the DNA-BD and Glutamate receptor 2. GluR2 belong to a group of receptor that are activated by the amino acid glutamate, a neurotransmitter. Their structure, function and interaction partners during synapse have been reviewed in detail by Palmer et al (2005) and Pin et al, (1994). But no interaction of GluR2 with SV large T antigen has ever been known. Thus making it as a negative control.

The mating of these strains gives rise to diploid zygotes as seen in Fig 7 and Fig 8 The mating culture when viewed after 22 hours of mating under a phase contrast microscope showed growth of trilobed diploid cells and mating cells.

Colony growth was observed in all the selective media plates including negative control TDO plates confirming the activation of the histidine gene. However background growth of very tiny cells was observed in all the plates. The dilution of the mated culture was done with 2x YPAD which is a rich media, this should probably account for the background growth.

The β-Galactosidase filter paper assay to determine the activation Lac z reporter gene was done on 1/10 dilution plates of the positive and negative control plates. The 1/10 dilution colonies of the triple drop out plates were chosen for the assay, as the formation of colonies were clear and there were no background growth. The positive control filter paper started to turn blue after 30 minutes confirming the interaction between p53 and SV large T antigen. The negative control filter paper did not show any colour change confirming no activation of the reporter gene even though there were colony growths on TDO plates. The colony growth in TDO plate can be due to the use rich media. A defined media is expected to eliminate any background growth.

4.2. Toxicity/Auto Activation Test

The toxicity test was done primarily to check the growth rate of colonies with the bait protein. The growth was compared with control L40 bait fusion protein GluR2/pBTM116, and L40 with plasmid pBTM116 that does not code any bait fusion protein. GluR2 is a well characterised protein and their interaction partners are found and studied mainly through the yeast two hybrid system.

The colony growth of test baits 5inSER and NLSER were same as pBTM116. After transformation the growth of colonies were checked next day. There were visible colony growth on 5inSER, NLSER and pBTM116 plates but no colony growth was observed in plates where the GluR2/pBTM116 transformed colonies are plated. Colony growth in GluR2/pBTM116 was observed only after 2 overnight growths. This result shows that the baits under study are less toxic than the GluR2 bait.

The autoactivation control study was done with GluR2 as bait and N-ethylmaleimide-sensitive fusion protein (NSF), Protein interacting with protein kinase C (PICK1) and Glutamate receptor-interacting protein (GRIP) proteins as targets .GluR2 is one of the four major subunits of the AMPA receptors. The role of AMPAR is to bind with the amino acid glutamate, an excitatory neurotransmitter (Palmer et al, 2005). NSF is an important fusion protein involved in the exocytosis of synaptic vesicles, its interaction with GluR2 is important in the current transmission of the AMPA receptors. Their interaction was studied through the yeast two hybrid system where NSF binds with the 21 amino acid region of AMPA C-termini (Osten et al, 1998). Dev et al (1999) reported that PICK1 interacts with the C-Terminal of the GluR2 receptor. PICK1 can modulate the AMPAR current by phosphorylation (Lin et al, 2001). The GRIP regulates the Ca+ impermeability of the AMPAR through its interaction with GluR2 (Srivastava et al, 1998). With these well known interactions, they were chosen for control autoactivation. The GluR2 baits were fused with the DNA Binding Domain binds to the Upstream Activation Sequence and the proteins PICK1, GRIP and NSF are fused with the Activation Domain, when these proteins interact activates the lacZ reporter gene for β-Galactosidase production. These three interactions were chosen as positive control.

For negative control the DNA-BD with GluR2 and no protein was fused with the AD. So that no protein interaction will take place and consequently no reporter gene expression.

The β-Galactosidase assay on the TDO plates (shown in Fig 8)showed that the reporter gene was activated in all two positive controls except for the GluR2+ NSF control where the interaction was found in very less number colonies. This was observed as the colour of the lysed colonies turned blue on treatment with X-gal. No colouration was observed with negative control, the bait colonies and colonies with just the bait vector and prey vector. However there were colony growth in all of the TDO plates including the ones with just the bait, vector with no bait (pBTM116) and the vector with no prey (pGAD10).The growth of these colonies were not expected as the bait strains cannot produce leucine and histidine amino acids. The growth was thought to be of two reason either there is an leaky expression of His3 gene that is providing the colonies enough nutrient or the colonies obtaining nutrients from other source possibly the media.To test the leaky expression of His3 gene a 3-AT test was done which showed that the His3 is not activated. Moreover the bait were found to be growing on media lacking Leucine confirming the other possibility that the media was nutrient rich, implies the need for a Defined Media to eliminate false positive results and contamination.

4.3. Determining Concentration 3-AT for Histidine Inhibition

In yeast two hybrid system which uses His3 marker, 3-AT (3-amino-s-triole) is used in the media to eliminate growth of cells in -His media. 3-AT is a competitive inhibitor of His3 (Walhout et al, 1999 and Barry et al, 2002). The baits were grown on DDO/-T/-H minimal media with varying concentration of 3-AT. The concentrations of 3-AT used are 0mM, 5mM, 10mM, 50mM and 60mM. The results showed no colony growth in any of the plate. This indicate that the His3 marker gene is not expressed or autoactivated by the bait.

This study is stopped at this stage due to time constraint. It is clear that His3 marker gene is not expressed in the L40 yeast. The auto activation/toxicity experiment needs to be repeated with a defined media to eliminate any non interacting colony growing on TDO and DDO plates. More convincing results are expected with the use of defined media. The next and the most important stage of the project, the library screening and to find interacting proteins should be carried out to on successful completion of autoactivation/toxicity test.

5. Conclusion

Before the genomic era scientists believed that 1,50,000 genes would be responsible for all biological activities of human. But the Human Genome Project reported, only 30,000 genes are responsible to regulate the central dogma of protein synthesis and all complex biological functions. Alternating splicing of mRNA was expected in about 59% of human genes which was once believed to be mere 5%. So considering the very low number of genes against the number of proteins, it is easy to assume the role of alternate splicing in determining protein complexity. The discovery of some 40,000 protein isoforms coded by a single gene of Drosophila implies the need for detailed understanding of alternate splicing (Modrek et al, 2002). Such large numbers of isoforms are also possible with human genes (Gray et al, 2007). The number of isoforms discovered in humans is only in the order of few hundreds. Therefore science is now facing the huge task of finding thousands of splice isoforms. More important is the task of understanding their functional role in normal condition and diseased condition. The future of drug discovery will be based on targeting domains in the isoforms (Kanumilli et al, 2005).

This project is one such step towards the understanding of the disease pathogenesis of Spino Cerebellar Ataxia 6 (SCA6). This study has reached an important step of library screening. The library screening of the bait proteins is expected to reveal novel protein-protein interaction in the exon 47 of Cav2.1 C-terminal. This interactions would give more detail about the role of the c-terminus splice variants in the intracellular signalling as these splicing have no effect on electrophysiological properties of the calcium channel (Kanumilli et al, 2005) The protein-protein interaction should be investigated with our knowledge of known cell signalling pathways. This would trigger more research to gain more insights into the pathogenesis of SCA6 and ultimately new target in designing of drugs. However before arriving at any conclusion it is important to test the protein-protein interaction with the human calcium channel as the length and nature of splicing are thought to be organ and species specific (Kanumilli et al, 2005).

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Appendix

SC-THULL MEDIA

Yeast Nitrogen Base 1.2 g

Ammonium Sulphate 5.0 g

-THULL amino acid mix 1.0 g

Bacto Agar 20 g

Bring to 900 ml with dH2O.

Sterilise by autoclaving at 120⁰C for 15 minutes. Let it cool to 55⁰C

Add

20% Glucose 100ml

YPAD

Yeast Extract 10 g

Bactopeptone 20 g

Adenine Sulphate 0.1 g

dH2O 900 ml

(For plates)

Bacto Agar 20g

Sterilise by autoclaving. Let it cool to 55⁰C

Add

20% Glucose 100 ml

0.1M(LiAc)/0.5XTE- 2000 μl

1M LiAc 200 μl

10 X TE 100 μl

dH2O 1700 μl

LiAc/40%/ PEG/1X TE- 700 μl

1M LiAc 70 μl

10 X TE 70 μl

50%PEG3350 560 μl

Z Buffer

60mM Na2HPO4

40mM NaH2PO4

10mM KCl

1mM MgSO4

(pH 7.0)


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