Introduction
Proteomics is the study of proteins in terms of their structures and functions. The term proteome refers to the entire set of proteins modified of produced by the organism. However, the interactions and modifications tend to vary with time and their distinct requirements and environmental stresses that the organism undergoes. This is an interdisciplinary domain based on research studies originally on yeast, then mice. Experiments have been forwarded describing the ways of investigating genetic interactions and various aspects of different functional proteins in genes. This essay proposes experiments that would be used to investigate a particular gene (named Without Kicking Urge, WKU) in an experiment using mice as the subject. It provides a detailed description of the experiments and the meaning of the probable results.
Expression proteomics is the analysis of interactions between cellular proteins at large scale. The study helps in identification of major proteins in samples and the differential expression in other related samples. Examination of the differences in expression of differences in gene proteins provides significant differences between treatment and exposures. Previous experiments have based on multiple hypotheses to see the probability of having false positive results. Description of gene and protein interactions and functions introduces the field of functional genomics. This will focus on the aspects of genes such as translation, transcription and protein-protein interactions.
Protein interaction at large scale is termed as interaction proteomics, and is useful in determination of their functions and explaining the ways in which the proteins assemble to form larger complexes. Some of the techniques that can be used to study the functions of the gene proteins include mass spectrometry and protein profiling, DNA microarrays, and protein identification techniques.
DNA microarrays
In order to investigate the function of the WKU protein in the mouse, one of the experiments that can be used is the DNA microarray, which implies a collection of microscopic spots of DNA attached to a firm surface. This would be helpful in measuring the expression levels of the WKU in multiple regions of the genome; hence, explaining the function of the gene.
The key principle in the use of microarrays in hybridization between the DNA strands for formation of hydrogen bonds between the complimentary nucleotide bases. Washing off nonspecific bond sequences leaves only strongly paired strands hybridized. This experiment will help to understand the effect of the WKU protein in the mouse genetic sequence. The total strength of the signal in the genetic sequence depends upon the amount in the target sample. The intensity of the feature in the sample is then compared to the same feature in another control sample, which makes the experiment more quantitative than qualitative. In the case of the WKU, the phenotype in the mouse with the typical behavior will be the feature of comparison.
Microarray experiment will be applied in understanding the aspect of gene expression profiling in the mouse in which the WKU protein was encoded, then a comparative study taken with other specimen. Profiling will entail monitoring of expression levels of different sample genes simultaneously and monitoring the effects of the treatments such as those in the encoded proteins and their development stages. A comparative study between the genes of the mouse with the WKU and the other mice will provide conclusive results on the functions of the genes in question.
There are two types of arrays: spotted arrays and printed oligonucleotide chips. In preparing the arrays, the second type will be produced by light directed printing technology known as photolithography. The chips have oligonucleotides that have single strands containing 20 to 25 bases. In order to reduce false positive results, each sequence has a representation of a set of approximately 20 non-overlapping nucleotides with a high density of oligos in the sample version. In essence, such microarrays can be used in the study of gene expression patterns of different cell types through comparison. The analysis of the mouse gene protein expression will provide information about the expressed genes and the normal genes and the genes that are only expressed in the WKU protein. This will be helpful in identifying the genes.
The study of functional genetics in the mouse with WKU protein can be done through the tedious process of gene function determination involving cloning and mutating the gene from the mouse invitro before reintroducing the mutated gene into another host organism and analyzing its effects. This strategy is referred to as mutational genomics.
Systemic mutation of the genes in the genome will generate a number of mutant strains, although is dependent highly on the knowledge of the entire sequence of the genome. This approach will provide the research with the opportunity to exploit the homologous recombination in placing a selective marker gene within the gene of interest. Studies on the mutant genes will help to determine the functions of the missing genes.
Another method that would be useful in investigating the protein in the mouse is the use of immunoassays such as the ELISA procedure. This method can be used to detect and measure the levels of proteins in the sample. The principle behind this technique lies in the recognition of an analyte, which in this case is the WKU, in the sample under investigation. Elisa is a mapping method, which allows monitoring of hundreds of proteins function. The interactions can be examined in vivo or invitro.
The principle in proteomics is that the assays allow the identified proteins to link to genes that encode them. The ELISA will determine the protein to which the WKU gene is linked to, with the antibody serving as a probe.
Another technique that will be useful in this study involves the principle behind the yeast two-hybrid system where there is generation of a comprehensive library of baits and preys. There will then be an evaluation of the protein interaction in the library using different approaches such as the matrix approach and the random library method. Glutathione-S-transferase (GST-pull down) is an alternative technique where the bait protein is expressed as fusion with GST.
Mass spectroscopy and protein profiling
2-dimensional electrophoresis is a high-resolution technique that profiles unknown proteins, WKU, in the mouse. This method is applicable in separation of the different proteins from the sample. After separation, there will be selection and staining the proteins after which mass spectrometry will help in identification of the protein.
RNAi procedure is another study that can be used in understanding WKU gene functioning and validation. It will be a valuable tool in evaluating gene expression signatures in response to signal pathways. The technique will be applied in elucidating the functions of the gene, WKU, in expression of the said phenotypic characteristics. The principle in this technique is that it blocks the expression of a definite gene coupled by evaluation of its responses to chemical stimuli; hence, providing the functions and the properties of the gene under study.
The RNAi method uses that ability of the cell’s short interfering molecules to suppress the expression of a particular gene, which introduces the RNA-induced silencing complex and identifies the corresponding messenger RNA to cleave at a specific site. Such loss of function studies will help to outline the key functions of the gene protein Without Kick Urge, in the genetic makeup of the mouse.
Serial analysis of gene expression bases on the sequencing of the RNA rather than basing on hybridization. The principle is sequencing of 10-17 base pairs that are unique to each of the genes under study. This technique will provide an unbiased result as it does not use prior knowledge of the protein genes under study.
Conclusion
Studies have indicated that gene proteins rarely interact and act as a single species while performing their functions in vivo. However, proteins involved in the same functions interact. Therefore, one approach for understanding functions of unknown proteins and identification of other proteins with which it interacts in an organism. Protein-protein mapping plays an interesting role in elucidating protein function.
Genomic sequencing of the whole organism provides the genetic code that underlies the biological mechanisms in organisms and it is within the genetic code, which requires translating in understanding the gene functions and their regulations. Integration of computation and experiments helps in understanding the cellular processes. Application of the experiments as in the essay will elucidate on the functions of the gene, Without Kicking Urge, WKU.
