Name ___Datoya Brown___________ BIOL 430 Assignment 4 Due Thursday, October 25th 25 points 1.

You are interested in a gene called coeur, which you think may be involved in heart development in Drosophila. a. You perform in situ hybridization (ISH) on Drosophila embryos, using a probe that specifically recognizes the coeur mRNA, and observe staining specifically in the embryonic heart. What can you conclude about coeur mRNA expression based on this ISH experimental result? (3 points) Based on the in situ hybridization (ISH) experimental results the conclusion is that the gene coeur mRNA is localized within the tissue (Drosophila embryonic heart). This is stated because obvious staining in the localized region provides insight as to the genes specific designation for activation in Drosophila species.

b. You then perform antibody staining using an antibody that specifically recognizes the Coeur protein, and are surprised to find that you do not see staining anywhere in the Drosophila embryo, not even in the heart. What can you conclude about Coeur protein expression based on this antibody staining experimental result? (3 points) Based on the antibody staining experimental results, the conclusion is that there is no visible staining all over due to the technique used. Antibody staining is a method used to label a specific protein (in this case, the protein encoded by the gene coeur) in a cell or tissue to determine whether it is a present or absent.

c. Propose at least one possible explanation for your observed experimental results. (6 points) A possible explanation for the observed experimental results is that in the Drosophila embryo the coeur mRNA observed for expression in the heart has not been translated into the protein. In the central dogma the process is DNA RNA Protein at this point in the experiment has not processed thoroughly. In the translation stage, this is the actual synthesis of a protein, which occurs under the direction of mRNA. In this particular Drosophila embryo, mRNA has not matured to express the protein needed for heart development.

Name ___Datoya Brown___________ 2. What are the 5 and 3 UTR of an mRNA? How are they involved in regulating gene expression? (8 points) Gene expression is unusually regulated at the post-transcriptional level. UTRs are known to play crucial roles in the post-transcriptional regulation of gene expression, including modulation of the transport of mRNAs out of the nucleus and of translation efficiency, subcellular localization and stability. The 5 UTR of an mRNA is any coding sequence just before the stop codon and the 3 UTR is any coding sequence just after the start codon.

3. The nervy mRNA is expressed everywhere in the nervous system. However, the nervy mRNA contains two binding sites within its 3UTR for a microRNA called miR-Brain. The miR-Brain microRNA is expressed in the brain, but nowhere else within the nervous system. Based on this information, predict the expression pattern of the Nervy protein and explain your answer. (5 points) MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression by binding to the 3UTRs of target mRNAs, thereby preventing translation of that mRNA (and preventing expression of that protein). Based on the information given, the prediction for the expression pattern of the Nervy protein is that it will be expressed everywhere in the nervous system. However, with the binding of the miR-Brain microRNA in the brain, only specific protein made for the brain will be expressed, and all other nonspecific types will be inhibited, therefore not allowed to be translated. The mRNA will still be accessible however, and will not be degraded.