Investigating the Functions of Proteins
The steps made so far in the process of gene mapping has opened up ways in which more studies are done on the human DNA. ENCODE and Genome researchers have so far sequenced more than 90 genomes. With the understanding of genes and their loci, it has been easier to locate and carry out studies on a single gene. Most genes code for various proteins in the body of an organism; it has become paramount that the roles of these genes be identified and prescribed out. The knowledge of the functions of each gene, or the protein it codes for, will enable further research in the human genome. The research could be used effectively in managing and treatment of diseases and even controlling some genetic traits. In this case study, the function of the unknown gene had been found to be associated with constant kicking in the mouse.
The gene associated with kicking in mouse codes for some particular protein. This paper seeks to propose mechanisms by which this protein is investigated so as to come out with a conclusive idea on its function in mice. This paper gives a process through which a gene or its corresponding protein’s functions can be determined. It is based on a number of researches which have provided pathways through which the characteristics and functions of proteins are studied (Zhu, Bilgin, and Sydner, 2008).
Before the functions of a certain protein are studied, it is important to understand the nature and the identity of such proteins. The process of determining the identity of proteins is called protein profiling which can be done through mass spectroscopy, two-dimensional gel electrophoresis or antibody microarrays. Protein functioning depends on the levels at which it can get expressed in the phenotypes. There are also mechanisms through which levels of protein expression is measured. One of the methods of achieving this is through protein localization. Protein localization gives information which can be used to determine the function of a protein (Zhu, Bilgin, and Sydner, 2008).
The other important information which must be taken into consideration is the fact that most proteins do not function alone. Therefore, it is important to identify and understand the interactions between various proteins that lead to their expression. Protein-protein interactions can be studied using affinity tagging and mass spectroscopy mechanisms, among others. According to Travella, Klimm, and Keller (2005), the best approach determining the functions of genes in an organism’s genome is to reduce or knock out expression of the gene. The reduction induces a mutant phenotype that is indicative of the gene function. In this mechanism, a mutant gene is inserted in the DNA, a process called Insertional mutagenesis. This mechanism is a form of induced gene silencing. Another method involves RNA interference, which works on the messenger RNA. The functions of WKU would be determined by the use of either insertion mutagenesis or gene silencing methods. Gene silencing is be done through RNA interference.
In investigating the functions of WKU, the first step would be to identify the locus of that specific gene. After identification of its locus, a gene specific double-stranded RNA, (dsRNA), would be introduced into the cell of the mouse. This dsRNA used must be specific for this WKU gene.
Introduction of the double-stranded RNA into the cell is be done by microinjection or electroporation. The dsRNA then degrades homologous mRNA to form small interfering RNAs (siRNA) which are between 22 and 25 nucleotides long. The degradation is done by an RNAse III complex. The RNAse complex is then joined to the small interfering RNAs to form an RNA-induced silencing complex (RISC).This compound is very specific on its action, and it can only target a specific RNA for degradation (Shuey, McCallus, Giordano, 2000). Helicase enzyme is used to unwind the small interfering RNAs, thereby activating the RISC. The RNA-induced silencing complex is what degrades the messenger RNA. Other molecules that can be used instead of RNA interference instead include, RNA-dependent RNA polymerase, enzymes such as helicase, Dicers and dsRNA endonucleases. These alternatives are mainly used to process non-coding RNAs or microRNAs whose functions are similar to the RNAi. Degradation of messenger RNA takes place when the RISC complex comes into contact with the cognate mRNA to cause endonucleolytic cleavage of the messenger RNA. One dsRNA can mark more than one target mRNAs, and then; it is more economical to use this mechanism (Montgomery, 2013). In order to validate the expected results, other samples should be treated with negative control siRNA so that the results can be analyzed together. The two different treatments should not affect the viability of the cells or the organism as a whole (Al-Anouti, Quach and Ananvoranich, 2003).
While silencing the gene on one hand, on the other hand, it would be important to overexpress the gene because it will be able to produce two extreme sets of results for that gene. Overexpressing the gene is done by microinjection of the corresponding mRNA of this gene to the zygote of a mouse in its early stages. It is very hard to deliver mRNA into an adult mouse because it may be degraded, therefore giving wrong results (Anandalakshmi et al., 1998). Analysis of the overexpressed gene and the non-translated counterpart would give clearer results on the function of the gene.
Before the products are run through DNA microarray so as to measure the gene expression in the two sets of results, they are first subjected to mass spectroscopy mechanism. The expression helps to identify the protein in the two sets of results. Identification of the protein in the two sets is important to ensure that we are working on the right protein. In the mass spectroscopy method, the proteins are converted into volatile ions by a mechanism called matrix-assisted laser desorption ionization (Zhu, Bilgin, and Sydner, 2008). The protein samples from the two sets are first solidified in an acidified matrix. In the process; it ejects the analyses into a gas phase where they become charged. The difference in the electrical charge between the matrix-assisted laser desorption ionization plate and the mass spectroscopic tube makes the charged proteins analyses to land at different speeds. The whole process depends on their mass/charge ratios (Zhu, Bilgin, and Sydner, 2008). After identification of proteins in the two sets of results and confirming that they are both for WKU, they are then subjected to a DNA microarray analysis so as to determine the level of expression. The mice are then observed for a number of days to monitor the differences in their phenotypes.
At the end of the experiment, the mouse that had been given this treatment will not show the effects of the WKU gene if compared to the wild type.It would happen so because the protein coded for by this gene will not be synthesized in this mouse. The ones that had been treated with negative siRNA will show the effects of this gene.
Cleavage of the messenger RNA prevents translation of the RNA and subsequent protein synthesis. Without a translation and subsequent protein synthesis, the expression of the gene that was coded for by that particular protein is prevented. This specific type of gene silencing is called post-transcriptional silencing because it does the silencing after DNA transcription (Bass, 2000). Argrawal et al. (2003) says that the potential of siRNA technique has been demonstrated to be effective in targeting a sequence from hepatitis C virus and the FAS gene by RNA interference in mouse.
These results would confirm that this process can be specific in that it is possible to target a specific gene in an organism. This mechanism can as well be used to silence natural mutant genes with undesirable effects in organisms, thereby preventing their phenotypic expression. Most drugs used in the medical field are post-translational. Therefore, they target proteins hence the use of RNAis can control development of diseases in their early stages (Shuey, McCallus and Giordano, 2000). One important advantage of this mechanism is that the effects of RNAi are not inherited. The effects of RNAi are not inheritable because they do not in any way affect the DNA like other mechanisms such as knockout.
These experiments would reveal the important role that can be played by gene screening by the use of RNA interference method. The outcome would prove the specificity of the dsRNA to genes, and this knowledge would be crucial in the medical field. The ability to specifically identify a gene that may be coding for some undesired traits and preventing it from translation can be used to help individuals with such genes to avoid the adverse effects the genes. The situation can be specifically applied in the cases of mutation. Secondly, these procedures can be used to suppress the effects of diseases by preventing the synthesis of toxins by the disease causing microorganisms. The only disadvantage of this procedure is that their effects are not permanent and are not inheritable. So they can work well with diseases but not mutations.
References.
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Bass, B. L. (2000). Double-stranded RNA as a template for gene silencing. Cell 101: 235-238. CrossRef Medline.
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