 So I was working for the summer in Dr. Richard Bierstra's lab in the Department of Genetics, specifically working on plant genetics in Arabidopsis thaliana, and the role of the BTB E3 ubiquitin ligase family in Arabidopsis. So the first question I'd like to address today is why would we choose Arabidopsis thaliana as a model organism? And there are several reasons for that. The first one being that it has a really short generation time of just 60 days. So to go from one generation to the next doesn't take us very long. It's a very small and compact plant and so a lot of plants can be grown at the same time in a small area. It's self-pollinating, which saves us a lot of trouble when it comes to actually going from one generation to the next and generating seeds because a plant just takes care of that by itself. And its genome has been sequenced. The genome was completed in 2000 and since then it's been maintained and updated by the Arabidopsis information resource. The system I'm working on is a system that is involved with proteolysis inside the cell. So what are the roles of proteolysis inside a cell? There are several reasons why a particular cell may want to degrade a protein and some of those reasons are that the protein may be incorrectly made and so errors in the protein sequence itself or the protein may be misfolded. Incorrect folding which will mean that it cannot perform its function. Or the cell may need to regenerate amino acids for re-dropping into other pathways for survival and so it may reduce the supply of a particular protein. Or in certain cases a cell would want to regulate the amounts of certain key proteins and regulatory enzymes and so proteolysis can be involved in regulation as well. In regulation particularly the cell usually tags the protein with a molecule such as ubiquitin and then marks it for degradation. Ubiquitin is a 76 amino acid long reusable polypeptide tag that is used by cells to direct selective protein removal among other things. It is remarkably conserved throughout evolution and it's restricted to eukaryotes and archaea. And typically when a cell is ubiquitinated with a single molecule of ubiquitin the function of the protein is modified but when a poly ubiquitin changes form the protein is usually marked down for degradation. Now I'll come into the system which we're working on. It's the ubiquitin 26S proteasome system and this system is one of the major ways in which a cell regulates the amount of protein it has. So in this pathway the first step is ubiquitization of an E1 enzyme which is called the ubiquitin activating enzyme. So this is the only step in the reaction which requires an ATP. So the ubiquitin moiety first binds to an AMP, the ATP degrades to form an AMP. And then it forms a thioester bond between the ubiquitin activating enzyme, the E1 and the ubiquitin molecule. Then the E1 transfers the ubiquitin to an E2 which is a ubiquitin conjugating enzyme and this happens to a transesterification reaction. Now the E2 is usually the protein which transfers the ubiquitin to the target molecule. It usually does this with the help of an E3 enzyme which is a ubiquitin ligase. And the E3 usually serves along with two other proteins as a scaffold which brings the target and the E2 close together so that the ubiquitin can be transferred. The specificity for this entire reaction is also provided by the E3. Now once the polycubic ubiquitin chain is formed on a target which needs to be degraded by the cell, the cell can either remove that ubiquitin chain by using the ubiquitylation enzymes in which the protein will not be degraded or it can send the target to a 26S proteasome which will then degrade the target and release the ubiquitin which will then regenerate single ubiquitin molecules using the ubiquitylation enzymes. Now why are we concentrating on E3 ligases? Because if we analyze the number of genes which are present in the ubiquitin 26S proteasome system, we notice that the highest number of genes are the E3 genes and these are the genes which provide specificity and selectivity to the pathway and it accounts for nearly 5% of the entire proteome of Arabatopsis which means that it is a critical pathway which is involved in a lot of selective protein degradation. Now coming to the BTB family which is a subfamily of the E3 ligases which we work on it consists of about 80 genes and it is essential for survival. This was discovered when this is a scaffold which is used by the E3 in this case the BTB and the E2 to transfer ubiquitin to the target. So when the BTB can only function when either one of two isoforms of column 3 are present column 3A or column 3B and when a double mutant was made with both columns knocked out it was discovered that none of the plants ever survived. So this led to the conclusion that the BTB set of E3 ligases are critical for plant survival. Phylogenic analysis of the BTB family is complete and there are several genes which are discovered which have similar suspected functions but the real functions of most genes are still unknown. And this is the phylogenic analysis of the BTB family and this is the basis for the study which we are doing in our lab right now which involves creating mutants or double mutants of certain BTB genes especially related ones. So the genes for which we have made mutants or double mutants are indicated at the bottom with these U shaped marks and on the basis of this we expect to see phenotypes depending on whether how critical that particular gene is for the cell and its development. Now another question I'd like to address is how Arabidapsis thaliana is mutated. So the mutation is done using a particular bacterium called agrobacterium tumor fashions which has a plasmid in it called the Ti plasmid or the tumor inducing plasmid. So when a plant is infected with agrobacterium the Ti plasmid has the property to transfer a small part of its DNA into the plant genome and it integrates in a random place in the plant genome. The DNA which does integrate is called the T DNA or transfer DNA and once it has integrated we can just sequence the T DNA and some other reason beyond it in the genome to find out where it has integrated in the genome and depending on whether it has knocked out our gene of interest we can use that mutant for the study. So the first objective of my work was to characterize an interesting double mutant phenotype that was discovered in the lab earlier. It showed a pin like phenotype which is clearly observable here and it also never showed more than a single cotyledon while it was being grown. So this is a wild type plant which is grown for the same time as this and this shows two cotyledons and two true leaves whereas this just shows a single cotyledon and isn't growing very well at all. So to characterize this the first step that we thought was appropriate was to genotype the plants that showed that phenotype to see whether the double mutant genotype is actually present and whether that might be what is causing the phenotype. So for this we set up a PCR reaction in which we use three primers. So the left and right primer together were used to amplify a small part of the gene itself so this is the left primer and the right primer and we set it up so that this region of the gene is about 1.2 kilobases in length. At the same time we use the third primer which is called the left border primer which binds to one end of the TDNA. So in case the TDNA is present in the genome then instead of the larger product only the shorter product will form which is about 700 bases in length and depending on which product we see in the gel after the PCR we can conclude which genotype is present. So when we did genotype this what we saw was that when the DNA from a wild type plant was used we saw the expected wild type length which is around 1.2 kilobases. Then when DNA from a mutant in the other locus is used we again see wild type length in the locus for which we are genotyping right now. When DNA from the mutant in the locus which we are genotyping for was done we see bands at 700 bases which is again the expected length. But then when we ran DNA from our suspected mutants which we expect have a double mutant genotype so we should expect mutants we should see mutant band at this location. We see a wild type band instead. We see only a wild type band. We should typically see only this band because that's the genotype we were expecting a homozygous mutant genotype but instead we see only a wild type genotype which indicates that the interesting pin life genotype we saw was not because of the double mutant genotype and this led us to conclude that there may be an extra transfer DNA insertion somewhere else in the genome and one of the ways to find that out is tail PCR. So, since that didn't work out we decided to screen for phenotypes in the F2 generation of crosses between phylogenically related BTP gene pairs in that the phylogenic tree I showed you earlier we picked out closely related genes from that main double mutants and in the F2 generation of those we expected segregation and in plants from the F2 generation we looked for some interesting phenotypes. So, some of the ways in which we did look for phenotypes are these are the cotyledons of a this is a wild type seedling of this these are the cotyledons so we look for differences in cotyledons size, number or the annulate which the cotyledons open or their pigmentation. We looked at this is the hypercotal so we looked at differences in hypercotal length or thickness and this is the root of the seedling we looked for differences in root length or even presence or absence of root. So, the method followed for the screen was in each cross we we plated out more than 50 seeds and looked for phenotypes that were showed by multiple plants multiple because we expected a Mendelian segregation ratio of about 1 is to 16 in the F2 generation and then for the suspected mutants that we picked out we genotype them and a few siblings which showed the wild type phenotype and the result we expected was that the suspected mutants should be homozygous for the two mutant alleles and siblings should segregate again. So, from the screen the results were that we when we genotype each cross with the two individual loci. In some cases we found plants which are heterozygous in both loci which again showed us that the phenotype we saw was not because of our mutant genotype. In some plants we found that one of the loci just gave us wild type plants and there was not even a single heterozygote which tells us that the phenotype was probably because of natural variation from wild type and that the cross that was done earlier did not work. A few of the crosses that we picked up we had to not pursue because they've already been characterized by other labs and in some cases we found plants which are homozygous for one locus and heterozygous for the other which means that when they segregate in the next generation they should give us a 50% double homozygous mutant phenotype which is what we are looking for because that's what we expect will show us the phenotype. Another reason why we could not have picked out certain phenotypes in this case is because several phenotypes are very subtle and we could only pick out very obvious phenotypes so if you notice here most of our phenotypes are smaller in size but there can be more subtle phenotypes which would be much more easily picked out in the next generation when we have 50% double homozygous mutants. In the future direction for this objective is to follow the plans which are homozygous at one locus and heterozygous at the other because they will segregate to give us 50% double homozygous mutants. And some crosses which did not segregate and gave us only wild types at one locus they need to be remade. In our lab recently we found that one of the crosses in which we had found a plant which was homo-head and did segregate in the next generation when we planted the next generation out in which we expected 50% double homozygous mutants we saw a few very interesting phenotypes again which are the cotyledons that we that I've shown you in the wild type seedling which would look like this typically in these plants we see fused cotyledons so cotyledons haven't actually separated after they came out of the seedling and they show us either a spoon-like or a cup-like phenotype so you can see the cup here as well and in the same cross in one particular plant we noticed that it had three cotyledons and we still have to characterize this so we will go ahead and characterize this in the lab and one more learning from that is that this phenotype that I showed you right here is as interesting as the one we saw right in the beginning the pin-like phenotype but we've learned through experience that we shouldn't get ahead of ourselves and make sure that the genotyping is right and the genotype is causing the phenotype before we go ahead with anything else in summary we proved that the pin-like phenotype that was observed was not because of the expected WU-2 genotype and we discovered a few homo-head or double-head plants that should segregate to give double-head roots in the next generation I'd like to acknowledge Dr. Arichar D. Viersha who is lab by Warden, Dr. Matthew Christians who was my mentor and guide and all the members of the Viersha lab who made the summer a great experience for me and special thanks to the Indo-U.S. Science and Technology Forum the Department of Biotechnology, Government of India and UW-Madison for finding an organizing platform