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Title: Molecular Mechanism Involved in Regulation of Serine Proteinase Isoforms Expression in Sistrurus Catenatus Edwardsii
Authors: Marwari, Subhi
Supervisor: Kini, R. Manjunatha
Reddy, M. S.
Keywords: Isoforms;Duplication
Issue Date: 12-Dec-2011
Abstract: The relationship between snakes and humans is a turbulent one that has evolved through the millenia. In some cultures, snakes are revered and worshipped, in others they are reviled. Snake venoms are cocktails of pharmacologically active proteins and peptides. Some of these toxins help us as highly specific molecular scalpels in deciphering molecular mechanisms of normal physiological processes. A number of these toxins could also help in developing therapeutic agents for the treatment or prevention of human diseases. A large proportion of the biologically active proteins and peptides present within snake venoms interact with components of the haemostatic system to promote or inhibit the normal sequence of events that lead to clot formation. The venom proteins achieve their effects through interaction with various components of the coagulation cascade, endothelial matrix and platelets. Within the latter group, a number of venom proteins target the interaction of platelets with the major adhesive proteins, Von Willebrand factor and collagen. Snake venoms which are complex mixtures of biologically active proteins and polypeptides, belong to a small number of superfamilies of proteins. The members in a single family show remarkable similarities in their primary, secondary and tertiary structures. At times, however, they differ from each other in their biological targeting and hence their pharmacological effects. Some of the well-recognized families of venom proteins are: (1) Phospholipase A2 (PLA2) (2) Serine Proteinases (3) Metallo Proteinase (4) Three-finger toxin (3FTs) (5) Proteinase Inhibitor and (6) lectin. Snake Venom Serine Proteinases (SVSPs) comprise an enzyme superfamily with multifunctional activities that may have diverged or have undergone gene duplication resulting in alteration of their biological properties during the process of evolution thus acquiring special functions (Calvete et al., 2005; Sanz et al., 2006). Although considerable amount of data is now available, no standardised grouping of these venom Serine Proteinases has yet been documented. However, in 2001 Wang et al compared sequences of 40 Serine Proteinases isolated from different snake venoms, using a constructed phylogramin which such sequences were clustered into three groups designated as coagulating enzymes, kininogenases, and plasminogen activators. No Serine Proteinases have yet been purified from venom of the Edward’s rattle snake Sistrurus catenatus edwardsii in particular or for members of the Echis genus in general. However, the fact that the Serine Proteinase superfamily was important in the venom of the Viperidae, suggested that such enzymes should be present in the venom of S. c. edwardsii and that Serine Proteinase-specific antibodies are likely to be an important factor in S. c. edwardsii envenoming. Therefore S. c. edwardsii cDNA library was screened in order to isolate and characterize different isoforms or variants of this enzyme. Proteins expressed in the venom gland are highly similar to the physiological protein, and yet are able to inhibit various physiological processes. Hence, it is a well knit mechanism which drives their differential expression in the venom gland. Gene expression is controlled in various levels; transcriptional, translational and post-translational. Transcriptional control resides on the highest in hierarchy of gene regulation as it aids the regulation in the copy of messenger RNA made. The mode of regulation involves the presence of cis/trans elements in the DNA which are recognized by the transcription factor to up/down regulate a particular gene. Total mRNA was isolated from the venom gland of Sistrurus catenatus edwardsii and cDNA of Sistrurus catenatus edwardsii (Dessert massasauga rattlesnake), reveals the expression of 11 isoforms of Snake Venom Serine Proteinase (SVSPs) were made. SVSPs perturb the hemostatic mechanism of prey. They act on diverse protein substrates such as fibrinogen, kininogen and platelet receptors. Some SVSPs exhibit more than one activity. Further, Real-Time polymerase chain reaction showed the differential expression of these genes. Thus, the present study focuses to understand the intricate mechanism which drives this expression pattern. This would lead to comprehend how genetic element control gene expression and evolution. Identification of 11 isoforms of Serine Proteinase and their differential expression in the venom gland has lead to speculate the presence of novel cis elements control the entire pattern. Control of gene transcription is commonly used in biological systems to regulate protein expression. Transcriptional regulation in eukaryotes depends upon a series of complex signal transduction networks that ultimately control gene promoter activity via cis-acting elements like enhancers, Matrix Attachment Regions (MARs), locus Control Regions (LCRs), and trans-acting elements (transcription factors). Promoters are generally defined as the region of a few hundred base pairs located directly upstream of the site of initiation of transcription. More distal regions and parts of the 5' UTR may also contain regulatory elements and may be part of the promoter. The start of the CDS (Coding Sequences) only corresponds to the translation start site and gives no hint on the localization of the promoter. Eukaryotic genes usually have 5' untranslated regions (5' UTRs) of variable length in a range of a few base pairs up to several kb. The five prime untranslated region (5' UTR) can contain elements for controlling gene expression by way of regulatory elements. It begins at the transcription start site and ends one nucleotide (nt) before the start codon (usually ATG) of the coding region. Our lab with the help of 5’RACE technique extensively identified the 5’ UTR region for all the 11 isoforms of Serine proteinases in S. c. edwardsii. Identification of 5’UTR region helps us to identify the transcription start site and provide us a better clue to understand the gene organization as well. Therefore, identification of the transcription start site directly leads to the location of the promoter of a gene. Multiple sequence alignment of all the 11 isoforms within their 5’UTR regions reveals the significant similarity and makes the basis for identifying the promoter region of the genes in order to study the varying expression pattern of these isoforms.
Description: M.Sc. (DBTES)
Appears in Collections:Masters Theses@DBT

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