(Theme 5) You are testing the role of three proteins (Protein A, Protein B, and Protein C) which have potential roles in transcriptional regulation of a gene in a mammalian cell line. You do this using a gel shift assay. Here, purified proteins are mixed in a test tube with a labeled DNA fragment that contains a putative regulatory region found upstream of a protein-coding gene. Decrea in the mobility of the DNA (ie it cannot move as far through the gel from top to bottom as shown) can be inferred to reflect the effect of stable DNA-protein binding interactions. You perform s reactions, with the added proteins indicated above the gel (+ = protein present; - = protein absent). Low DNA Mobility High 1 2 3 4 + 5 + + 6 Protein A Protein B Protein C Lane a) From the data shown which protein(s) directly bind the DNA? Briefly justify your answer. b) Considering your answer in A, briefly explain the results in lanes 5 and 6. c) Additional genetic data indicates that two of these three proteins are both required to activate transcription of the gene. Briefly describe which two proteins this would be, and propose a model for the specific molecular role of each.

Through alternative splicing, eukaryotes (a) reinforce gene inactivation (b) prevent transcription of heterochromatin (c) produce related but different proteins in different tissues (d) amplify genes to meet the requirement of high levels of a gene product (e) bind transcription factors to enhancers to activate transcription

Chromatin decompaction is a preliminary step in gene expression (Figure 29.48). How is chromatin decompacted?

Transcriptional regulators are proteins that bind to promoters (the 5-flanking regions of genes) to regulate their transcription. Assume that a particular transcription regulator normally promotes transcription of gene X, a transport protein. If a mutation makes this regulator gene nonfunctional, would the resulting phenotype be similar to a mutation in gene X itself? Why or why not?

Two genes associated with breast cancer, BRCA1 and BRCA2, were discovered in 1994 and 1995, respectively, and shortly thereafter, were patented by Myriad Genetics, a company based in Utah. Under the patents, testing for mutations in these genes could only be performed by Myriad, at costs from 300 to 3,000. Myriad also patented the process of analyzing the results of such tests, preventing anyone who obtains the sequence of their BRCA genes by other means (which itself would probably be patent infringement) from interpreting the information. The idea that genes can be patented has been a contentious issue from the beginning. Patents are not granted for products of nature, meaning that genes inside the body are not patentable, but biotech companies successfully argued that by removing a gene from the human body, purifying it, and then obtaining its DNA sequence, they created something not found in nature, and which is therefore a patentable invention. The U.S. Patent Office found the argument persuasive, but opponents argue that genes are parts of our bodies and can be identified but not invented. Biotech companies argue that without the protection offered by patents, they would have no incentive for research and development of diagnostic tests. In Europe, patents for BRCA1 and BRCA2 were revoked in 2004 because they did not meet the standards for a patent. After more than a decade of legal disputes, the patents were partially restored in 2008 on a very restricted basis. In the United States, a lawsuit, focused on the patents for the BRCA genes, was filed in May 2009. The suit challenges the basic idea that genes are patentable. In November 2009, the judge ruled that the lawsuit can proceed, and the case is moving forward. In March 2010, a federal court invalidated Myriad Genetics patent on these genes. In August 2011, the U.S. Court of Appeals reversed the lower courts decision and ruled that gene sequences isolated from cells are not a product of nature and are therefore patentable. The case went to the U.S. Supreme Court, which ordered the appeals court to reconsider the case. The Federal Appeals Court did not change its decision, and the case once again, went to the U.S. Supreme Court. A unanimous decision in June 2013 invalidated Myriads patents on the basis that isolating a gene from nature does not make it patentable. This is a landmark decision on gene patenting with widespread ramifications for the biotechnoloogy industry. Will this decision reduce the incentives for companies to invest in new diagnostic tests that would be used by cancer victims or those with serious genetic disorders?

Discuss Concepts In a mutant strain of E. coli, the CAP protein is unable to combine with its target region of the lac operon. How would you expect the mutation to affect transcription when cells of this strain are subjected to the following conditions? Lactose and glucose are both available. Lactose is available but glucose is not. Both lactose and glucose are unavailable.

Below is a sequence of 540 bases from a genome. What information would you use to find the beginnings and ends of open reading frames? How many open reading frames can you find in this sequence? Which open reading frame is likely to represent a protein- coding sequence, and why? Which are probably not functioning protein-coding sequences, and why? Note: for simplicitys sake, analyze only this one strand of the DNA double helix, reading from left to right, so you will only be analyzing three of the six reading frames shown in Figure 19.4.

If the coding region of a gene (the exons) contains 2,100 base pairs of DNA, would a missense mutation cause a protein to be shorter, longer, or the same length as the normal 700 amino acid proteins? What would be the effect of a nonsense mutation? A sense mutation?

Control of gene expression in eukaryotic cells occurs at which level(s)? a. only the transcriptional level b. epigenetic and transcriptional levels c. epigenetic, transcriptional, and translational levels d. epigenetic, transcriptional, post-transcriptional, translational, and post-translational levels

Given the following tRNA anticodon sequence, derive the mRNA and the DNA template strand. Also, write out the amino acid sequence of the protein encoded by this message. tRNA: UAC UCU CGA GGC mRNA: protein: How many hydrogen bonds would be present in the DNA segment?