Name: 1 2007 7.013 Problem Set 4 Due before 5 PM on WEDNESDAY, April 4, 2007. Turn answers in to the box outside of 68-120. PLEASE WRITE YOUR ANSWERS ON THIS PRINTOUT. Question 1. Working in the African Congo, you discover a new species of vertebrate in an isolated pond. The animal has no fur and seems equally comfortable on land or in the water. Wondering what type of species this might be, you decide to examine its DNA. First, you obtain a small tissue sample from the organism and extract its DNA. Next you clone a fragment of this DNA into a plasmid vector and sequence it. To identify your DNA sequence, you decide to do a series of analyses, using the BLAST tools available through the National Center for Biotechnology Information (NCBI). BLAST (Basic Local Alignment Search Tool) is a computational database search method for examining the hundreds of millions of known protein or DNA sequences in the world and rapidly identifying those relatively few sequences that are homologous to a given input sequence (the “query sequence”). BLAST essentially compares the query sequence to every other known sequence and identifies those that are most similar. Using such techniques, researchers can often infer the function of a given protein or DNA sequence simply based on the function of its closest known homologues. Your sequence (that is, the sequence that you have obtained from this “Mystery animal”) is available in the “DNA Sequence Text File” on the class website. Use the instructions in the file “Supporting File for Question 1 from Problem Set 4” to carryout a BLAST analysis of this sequence BLASTx is a tool that will first predict what kind of protein might be encoded by a segment of DNA (by electronically “translating” the sequence), and then it will compare the resulting protein sequence to all known protein sequences. To identify what kind of protein might be encoded by your sequence, carry out a BLASTx analysis. 1a. You find that your DNA sequence encodes part of a protein that is homologous to proteins found in many different organisms. Based on the reported names or functions of the homologous proteins, what is the likely name or function of the protein encoded by your sequence. Protein name or suspected function: 1b. From which organism comes the protein sequence that has the greatest homology to your sequence (i.e. what species provides the top hit in the BLASTx results table)? Species or common name:Name: 2 Puzzled by this outcome, you decide to examine the DNA sequence directly, rather than looking at the protein it encodes. To do this, you carry out a BLASTn of your sequence. 1c. From which organism comes the DNA sequence that has the greatest homology to your sequence (i.e. what species has the top hit in the BLASTn results table)? Species or common name: 1d. What principle of molecular biology or evolution allows the protein and DNA sequences to reveal homology to sequences from two such very different organisms? Explain your answer in 15 words or less.Name: 3 Question 2. What stage of the cell cycle best describes the following cells? 2a. A cell with condensed chromosomes aligned across the midpoint 2b. A cell with very active DNA polymerases 2c. A brain cell that has not undergone cell division in 19 years 2d. A cell with microtubules radiating from a centriole Above is a diagram of the cell cycle, displaying the interactions of several regulators. In your laboratory, you have identified several homozygous mutant cell lines, each carrying a temperature sensitive mutation in one of the above indicated factors. In each case, the protein in question becomes inactivated when the cells are grown at temperatures above 30ºC. In cells carrying temperature sensitive mutations for each of the following factors, at what point in the cell cycle would you expect to see cells arrest (halting further progression through the cell cycle) once you shifted their incubation temperature to 37ºC? 2e. Cyclin A 2f. Cyclin B 2g. Cyclin E 2h. RBName: 4 Question 3. PtsG is a membrane protein with an extracellular domain and an intracellular domain, and it is involved in bacterial uptake of the six-carbon sugar glucose. When there is no glucose in the culture medium, enzymes within the cell phosphorylate part of the PtsG. 3a. Which portion of PtsG is most likely phosphorylated by these enzymes? ! The extracellular domain ! The membrane-spanning domain ! The intracellular domain ! The tryptophan residue Phosphorylated PtsG translocates glucose into the cell. As it does so, it transfers its own phosphate group to the glucose molecule, leaving the protein unphosphorylated and producing glucose-phosphate, a form of the sugar that is activated for further metabolism. 3b. Which term best describes PtsG? ! Protein kinase ! Receptor ! Second messenger ! Responder Mlc is a regulator that can bind to the promoters of genes that are required for the cell to further metabolize glucose. When Mlc is bound to the promoter, RNA polymerase can no longer recognize the promoter. 3c. Which word best describes Mlc? ! Translational activator ! Translational attenuator ! Transcriptional Activator ! Transcriptional repressor ! Translation elongation factor 3d. Direct interactions between the proteins Mlc and PtsG are central to the regulation of glucose metabolism in bacterial cells. Given the above properties of Mlc and PtsG, is it more likely that Mlc binds the phosphorylated PtsG or the unphosphorylated PtsG? Briefly explain your answer.Name: 5 Question 4. Consider the following simplified diagram of a signal transduction pathway that regulates programmed cell death in mammalian cells. Assume that this diagram represents the behavior of a single cell within an organism. NucleusMitoSFRTNFRTNFINIBcl2EFFINHAKTActivates a protein or enzymeBlocks a function, pathway, or interactionSFCell DeathNucleusMitoMitoSFRTNFRTNFINIBcl2EFFINHAKTActivates a protein or enzymeBlocks a function, pathway, or interactionSFCell Death Key: TNF (small circles) and SF (small diamond shapes) are circulating in the plasma that bathes the surface of the cell; TNFR = TNF receptor; SFR = SF receptor; As indicated, arrowheads indicate an activating relationship between one protein and another, while the lines with perpendicular bars at their ends indicate a disruptive
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