To make the production of A, B and C transcripts sensitive to eIF4E dependant mRNA export, the first thing which I will add is
1) A coding sequence of around discrete 50 nucleotide elements to their 3’UTR known as eIF4-4E sensitivity element (4E-SE).
• The presence of 4E-SE acts as a USER code for eIF4E sensitive transcripts and thus increase the mRNA export from nucleus to cytoplasm.
• eIF4E-SE containing mRNA is dependent on CRM1.
• To verify increased mRNA export, we can use an inhibitor like LeptomycinB (LMB), with appropriate amount of vehicle control (ethanol), to see whether 4E-SE and eIF4E dependent nuclear export of mRNA is increased compared to the inhibitor treated cells by separately collecting nuclear and cytoplasm fractions from these cells. Further detailed analysis can be done by qRT-PCR or by a Northern blot. (Culjkovic B, et al Cell Cycle. 2007, Journal of Oncology. 2009, Cell reports. 2012).
2) Secondly, I would add a complex coding sequence to the 5’UTR region thus making it an export sensitive mRNA and aids in easy translation.
• eIF4E sensitive transcripts will have a greater ratio of ribosomes/mRNA level, which helps in efficient translation.
• In the case of eIF4E, the complex 5_UTR and the 4E-SE in the 3_UTR can be regarded as USER codes for efficient translation and export.
• To verify increased translation efficiency, we can do polysomal loading analysis which will be associated with heavier polysomes.
b) (30 pts) If you wanted to suppress the production of A at the translational level, while increasing the levels of B and C mRNAs (post-transcriptionally), what might you do? (You are not allowed to use siRNA or miRNA strategies here; but you can engineer the constructs as you wish. You can also add oth...
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...ltiple polypeptides simultaneously from a single mRNA (Multiple ribosomal translations of a single mRNA). This can be verified by polysomal loading experiments. Polysomal loading experiments begin with isolation of mRNAs of interest from the cells by Immunoprecipitation with mRNA binding or with sucrose density gradient centrifugation based on the loading of ribosomes on mRNA. Untranslated and actively translated mRNAs can be separated by isolating monosomal and polysomal fractions by centrifugation through sucrose. If the mRNA of our interest has multiple ribosomes bound to it in the polysomal fraction, this can further be used for qRT-PCR and microarray analysis, by extracting RNA or by proceeding for Hybridization analysis of gradient fractions. The results might tell us the reason why there is an increased protein production of Z without changing RNA levels.
Miller, Kenneth R. and Joseph S. Levine. “Chapter 12: DNA and RNA.” Biology. Upper Saddle River: Pearson Education, Inc., 2002. Print.
Takahashi, Y., et al. “Analysis of Promoter binding by the E2F and pRB Families In Vivo: Distinct E2F Proteins Mediate Activation and Repression.” Genes 14 (2000): 804-816.
...s project is discovery of new high-penetrance genes as well as finding new low-penetrance gene modifiers of major genes in the human body.
Describe the steps of protein synthesis beginning with the attachment of a messenger RNA molecule to the small subunit of a ribosome and ending with the release of the polypeptide from the ribosomes. Include in your answer a discussion of how the different types of RNA function in this process.
Trafton, A. (2013, June 23). MIT News. Enhancing RNA Interference, pp. 1-2. Retrieved 12 16, 2013, from http://web.mit.edu/newsoffice/2013/enhancing-rna-interference-0623.html
If PGE4B is the target of miR-23b, we expect to see less amount of mRNA in the miR-treated cells than the scramble treated cells. We performed qPCR, then using the Ct values; we calculated the 2-∆∆Ct with the intention of comparing the level of RNA between treated cells and the control ones. We did the experiment twice and achieved two sets of data so that the result will not be products of randomness.
46- Kozak M. Rethinking some mechanisms invoked to explain translational regulation in eukaryotes. Gene. 2006; 382: 1-11.
...Glass, Non-coding RNAs as regulators of gene expression and epigenetics, 2011, oxford journals, 90 (3), p430-440
In this experiment, we are analyzing qualitive data of targeted characteristics of offspring Drosophila of a female Drosophila with a Gal 4 driver and male Drosophila with DNA containing UAS and genome needed for RNAi. If there is a deformation of such targeted characteristics in the offspring, in this experiment the eyes or wings, this indicates a possible disruption of metabolic genes.
Gene expression can be described as the conversion of information from genes into messenger RNA by way of transcription. Transcription happens in the nucleus, and is where RNA copies of DNA are produced. This process is facilitated by RNA polymerase, where one RNA nucleotide is added to an RNA strand. RNA polymerase is an enzyme used to produce transcripted RNA. It is responsible for constructing RNA chains, in the process previously described as transcription. RNA polymerase polymerizes the ribonucleotides and the 3’ end of RNA transcription. It is essential to life and found in all organisms. Also, it unwinds the DNA molecule, using it as a template, before synthesizing corresponding mRNA strands. mRNA, or messenger RNA, is part of a large group of RNA molecules that communicate information from DNA to ribosomes. mRNA contains adenine, uracil, guanine, and cytosine. Alternative to DNA which has thymine instead of uracil.
POLYSOME. "Analysis of ribosome loading onto mRNA species: implications for translational control." mRNA Formation and Function (1997): 305.
A USC researcher is interested in the roles of coregulators in steroid hormone regulation of transcription in cancer cells and would like to write a grant proposal on this topic. The two key coregulators of interests are Hic-5 and G9a. As he is preparing the proposal, he hopes to find information about the following questions:
The Gal4/UAS system is a common, powerful method used for studying gene expression, especially in model organisms (Lynd, 2012). This method can be used to directly study the phenotypes generated through transgene mis- or over-expression. It consists of two parts; the Gal4 gene that encodes the yeast transcription activator protein Gal4, and the UAS (upstream activation sequence) that is an enhancer to which Gal4 binds in order to activate gene transcription. This system was first found in the yeast Saccbaromyces cerevistae and has since been added into model organisms for the purpose of studying gene regulation (Laughon 1984). The Gal4 gene encodes for a protein that has been shown to be a regulator for Gal10 and Gal1 genes by binding to four loci located between the genes (Duffy 2002). The loci that the Gal4 binds to are defined as UASs, which are similar to the enhancers in eukaryotic organisms. Without the Gal4 binding, there would be no expression of the genes defined by the UAS (Giniger 1985).
The process of translation is a major part of protein synthesis. There are many different components related the process of protein synthesis which include the large ribosomal unit, 60S and the small ribosomal unit,40S. As well as these are the messenger RNA, “mRNA coding”, transfer RNA , tRNA for amino acids and finally greater than 12 of the catalytic proteins which have be found to be eIFs (eukaryotic initiation factors). (Norton and Layman, 2006)These initiation factors are quite important in relation to Protein Synthesis and translation initiation. ...“These initiation factors guide the assembly of the ribosome on the mRNA and are responsive to short-term changes in the availability of energy, amino acids, and growth factors. Initiation factors provide the cell with sensitivity to environmental factors, including changes in diet, such as leucine availability, and physical activity.”... As well as this they also enable the cell to become sensitive to factors like the availability of
Distinct characteristics are not only an end result of the DNA sequence but also of the cell’s internal system of expression orchestrated by different proteins and RNAs present at a given time. DNA encodes for many possible characteristics, but different types of RNA aided by specialized proteins sometimes with external signals express the needed genes. Control of gene expression is of vital importance for an eukaryote’s survival such as the ability of switching genes on/off in accordance with the changes in the environment (Campbell and Reece, 2008). Of a cell’s entire genome, only 15% will be expressed, and in multicellular organisms the genes active will vary according to their specialization. (Fletcher, Ivor & Winter, 2007).