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To achieve the results, genetic analysis will be done by aligning the clinically relevant human genes with anole genome to find out homologous sequences. These genes from anole genome will be isolated using PCR and cloned into a suitable plasmid for various downstream processes such as protein expression and purification, gene sequencing, assays, crystal structure studies and structure and activity relationships.
The study will lead to the identification of medically relevant genes and their products' homologyÂ between humans and anole lizard.. High level of similarity between maximum number of genes in humans and anole lizard will confirm that various diseases will progress, regress and become dormant similarly in both organisms, inferring that the drugs affecting anole will have almost same affects on humans as well. This will make anole an attractive model to study human diseases and for clinical trials to study potential drug candidates.
Review of Literature (1200-1800 words)
Human diseases and genetics
Mendel proved that traits are carried over from one generation to other and present under specific genetic combinations. There are several data resources for human phenotypic information such as Online Mendelian Inheritance in Man (OMIM),
(Hamosh A, Scott AF, Amberger JS, Bocchini CA, McKusick VA (2005) Online Mendelian Inheritance in Man (OMIM), a knowledgebase of human genes and genetic disorders. Nucleic Acids Res 33: D514-D517.)
But there is a dearth of similar databases for physiological and genetic information. Human genes, apart from phenotypic characteristics also carry genetic disorders or genes responsible for susceptibility to an ailment, to generations. There are about 4000 diseases that are caused by single gene defects and mechanism of progression of most of them is unknown. To understand the mechanism of development of various human genetic diseases animal models very close to human genetic makeup are commonly used. Genetic mutations can be induced in models to see phenotypic, physiological and behavioural changes in them. Although, the conclusions made from these studies had been very significant in past, all of the results can not be completely accepted as apparent from failed phase two or three clinical trials for diseases like Alzheimer's etc.
Also, there are many limitations such as age, apparatus, number of animals in a cage (cage density), experimenter, litter size, season, month and hour with most common models such as mice and ethical issues with monkeys.
Limitations with rats http://www.stats.ox.ac.uk/__data/assets/pdf_file/0011/4610/Flint.pdf
Fruit flies, mice, and zebrafish are very useful as animal models for studying gene functions as they are relatively easy to grow, genetically manipulate and dissect in the laboratory. By examining mutations in these organisms, one can identify candidate genes that cause disease in humans, and develop models to better understand human disease and gene function
Despite the pre-eminence of the mouse in modelling human disease, several aspects of murine biology limit its routine use in large-scale genetic and therapeutic screening. Many researchers who are interested in an embryologically and genetically tractable disease model have now turned to zebrafish. Zebrafish biology allows ready access to all developmental stages, and the optical clarity of embryos and larvae allow real-time imaging of developing pathologies. Sophisticated mutagenesis and screening strategies on a large scale, and with an economy that is not possible in other vertebrate systems, have generated zebrafish models of a wide variety of human diseases. This Review surveys the achievements and potential of zebrafish for modelling human diseases and for drug discovery and development.
A number of reptile lineages serve as important models for developmental biology, neurobiology, physiology, endocrinology and behavior. Furthermore, the availability of a reptilian genome sequence will play an important role in understanding the evolution of mammalian genomes as an important branch of the evolutionary tree of vertebrates.
This lizard species is a model organism for laboratory-based studies of organismal function and for field studies of ecology and evolution. This species was chosen for genome sequencing in part because of the ease and low expense of captive breeding, well studied brain, and sophisticated color vision. It is also well suited for studies involving the role of hormones in development and adult nervous system plasticity. http://www.ncbi.nlm.nih.gov/genomeprj?term=anolis
Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Lepidosauria; Squamata; Iguania; Iguanidae; Polychrotinae; Anolis; Anolis carolinensis
Lovern MB et al., "The green anole (Anolis carolinensis): a reptilian model for laboratory studies of reproductive morphology and behavior.", ILAR J, 2004;45(1):54-64
Lovern MB et al., "Sex steroids in green anoles (Anolis carolinensis): uncoupled maternal plasma and yolking follicle concentrations, potential embryonic steroidogenesis, and evolutionary implications", Gen Comp Endocrinol, 2003 Nov; 134(2):109-15
Aims (100-150 words)
Identification of genes responsible for various clinical conditions such as Alzheimer's and diabetes in humans.
Screening the genome of Anolis carolinensis for identification of homologous sequences.
PCR amplification of these genes from A. carolinensis genome for cloning.
Sequencing of these genes.
Expression, purification and characterization of proteins coded by these genes.
Structure and function similarity studies to identify applicability of anole as an animal model to study human diseases.
Experimental design methods (400-800 words)
Identification and annotation of genes in A.carolinensis
Genes responsible for either activation of a disease or causing a disease due to mutations in them have been reported in literature (examples in introduction). These gene sequences will be used as templates and aligned with the sequence available for Anole to find any homologous sequences present therein. Blast is one of the best programmes/algorithms available online to establish quantitative similarity between two/more sequences.
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local
alignment search tool. J Mol Biol 215: 403-410.
Washington NL, Haendel MA, Mungall CJ, Ashburner M, Westerfield M, et al. (2009) Linking Human Diseases to Animal Models Using Ontology-Based
Phenotype Annotation. PLoS Biol 7(11): e1000247. doi:10.1371/journal.pbio.1000247
Multiple sequence alignment can be used to align any number to sequences with each other.
Characterization of genes
Genes can be amplified into millions of copies using polymerase chain reaction (Karry Mullis in 1983). This will require a set of components as they are required in vivo too, dNTPs, forward and reverse primers, a DNA polymerase (Taq polymerase), suitable buffer, MgCl2 and a source of gene to be amplified (genomic DNA of an organism).
Primer design and amplification
A set of primers, forward and reverse, is required to initiate the reaction. These primers are 18-15 bp long oligomers, which give polymerase a base to attach further nucleotides complementary to the template strand.
Primers will be designed so as to facilitate restriction digestion at the ends of the PCR product. Restriction sites will be chosen first (compatible to the cloning vector) and then attached at the 5' end of the primers followed by 8-10 more bases at the same end to help restriction endonucleases to sit on the amplified DNA.
Extended sequence restriction site complementary primer
Cloning vectors possess a multiple cloning site with various sites for restriction enzymes. Suitable restriction enzymes will be chosen to cut both insert and the vector. As shown in the diagram similarly cut sites anneal to each other during ligation. The genes will be cloned into the vector so that the expressed protein will have a tag attached to either N or C-terminal for simplifying the protein purification steps.
Vector amplified gene with restriction sites at the ends
Ligation of insert and vector
Cloned gene into a vector plasmid
The plasmid will be sequenced for the gene inserted using the primers for the promoter and terminator sequences allowing only the gene to be sequenced.
Expression and purification
The chimera will be used to transform an expressing E.coli strain so that the cloned gene could be over-expressed.
This will involve identifying the crystal structures of various human homologs from anole and comparing for the structural similarity and mechanism of action. Deciphering the crystal structure could give much insight into how mutations alter the working of the proteins. The enzymatic activity of the proteins can be identified by calculating Km, Kd, Vmax values by performing enzyme assays. Affinity of the enzymes from anole could be tested for known ligands of human homolog using techniques such as Isothermal Titration Calorimetry (ITC) and circular dichroism. Based on the similarity of structure, function and mode of action we will be able to conclude weather or not these represent good models for the study of disease development, progression and control of human clinical conditions.