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Name: __________________________ Genetics 314 – Spring 2004 First Exam – 100 points 1. You are asked to determine whether three unknown DNA samples are from a virus, prokaryotic organism or a eukaryotic organism. You decide to produce Cot curves for the three samples and get the following results: B 1.0 C/Co 0.5 C A 0.0 Cot Based on the Cot curve information what type of organism is represented by each sample and briefly explain your reasoning. Sample A. Virus or phage DNA based on having the smallest amount of total genomic DNA made up of unique sequences of DNA. Sample B. Prokaryotic or bacterial DNA based on having the next largest amount of total genomic DNA made up of primarily unique sequences of DNA. Sample C. Eukaryotic DNA based on the presence of highly repetitive and moderately repetitive DNA sequences plus having the largest amount of total genomic DNA and the largest amount of unique sequences based on the length of time needed to get reannealing of the DNA strands. 1 Name: __________________________ 2. The Mars rovers make a surprising discovery, not only did they find evidence of water they also found evidence of life on Mars! Surprisingly Mars life also had DNA but the DNA replicated differently. It was found to replicate in a conservative manner. How does this differ from life here on earth and how could you demonstrate the difference? (you can use diagrams if you wish) To determine method of replication you would need to label the old and new strands in such a way as to tell if the DNA molecules or strands stay intact after replication. For conservative you would observe the old strands still together after replication and the new strands together. For semi-conservative you would observe that the covalent bonds between the nucleotides of a strand stay intact but the hydrogen bonds between the strands are broken resulting in DNA having one old strand and one new strand after replication 3. You are asked to determine the amino acid sequence for one of the Mars DNA samples. You are given the following DNA sequence but are told that the sequence is complementary to the template DNA strand for the polypeptide. 5’ ATG GGG ACC CCT AGA TTT TGC AAA CCA AGG TGA 3’ Assuming the genetic code is truly universal: a. What is the template DNA sequence? 3’TAC CCC TGG GGA TCT AAA ACG TTT GGT TCC ACT 5’ b. What is the mRNA sequence? 5’ AUG GGG ACC CCU AGA UUU UGC AAA CCA AGG UGA 3’ 2 Name: __________________________ c. What is the polypeptide sequence coded for by this DNA sequence? met gly thr pro arg phe cys lys pro arg stop d. You observe that you have two of the same amino acids in the polypeptide but no duplication in the DNA or mRNA sequence. How is this possible? Due to the degeneracy of the genetic code where more than one codon codes for an amino acid. 4. You would like to produce a large supply of this protein for study so you want to have the gene be transcribed and translated. a) You decide to put the gene into bacteria. What other DNA sequences would you need to add to your DNA fragment to insure expression of the gene in bacteria? Diagram your ‘complete’ gene and briefly explain what the purpose of each sequence. promoter seq. leader seq. gene seq. termination seq. promoter sequence – needed to provide a binding site for RNA polymerase to start transcription leader sequence – needed as a binding site for the small sub-unit of the ribosome to start translation termination sequence – needed for the termination of transcription b) NASA decides it only wants its DNA in eukaryotic organisms. What would you need to change in your ‘complete’ gene to get expression in a eukaryote? Briefly explain your reasons. You would need to change the promoter and termination sequence for transcription and the leader sequence for translation. Enzymes in eukaryotes recognize different base sequences than the enzymes needed for transcription and translation in prokaryotes. 5. You have been so successful with your first Mars sample NASA gives you a complete sample from the first organism found on Mars. You isolate the DNA polymerase from the sample and discover that it does not need an open 3’ end to start replication. How would this change how DNA replicates? 3 Name: __________________________ You would not need to have primase or RNA nucleotides to start replication. You would still get Okazaki fragments due to the anti-parallel nature of DNA and the fact that DNA polymerase can only synthesize DNA in one direction. In addition, if you are dealing with a eukaryotic system you would need to add telomerase to insure that the ends of any linear DNA were properly replicated. 6. You decide to replicate the Mars DNA so you buy a DNA replication kit. It supposedly carried all the components for replication. In replicating the DNA you discover a couple of problems: a) The DNA sequences are not complete; on one strand there appear to be fragments instead of one continuous strand. What is the problem and how would you fix it? The synthesized pieces of the lagging strand are not being connected (so the Okazaki fragments are remaining as fragments) indicating that ligase is missing. The solution then is to add ligase. b) If you had the unique Mars DNA polymerase (from problem 5) would this problem have occurred? Briefly explain your reasoning. Yes, DNA polymerase does not have the ability to connect the fragments so even without needing an open 3’ end the enzyme could not make the necessary connection and ligase would still need to be added. 7. You next try to transcribe your replicated Martian DNA so you buy a transcription kit. You appear to get only random lengths of mRNA that do not appear to code for anything. a) What is causing the production of random lengths of mRNA and how would you solve the problem? If random lengths of mRNA are being produced it means the RNA polymerase is not binding in the correct location. This indicates that the proper sigma factor is not present or the sigma factor is completely missing. The sigma factor is necessary to properly orientate the core enzyme of the RNA polymerase in the proper location (in the promoter region). Solution, add the sigma factor that corresponds to the sequence in the promoter region. 4 Name: __________________________ b) If you solve the first problem you start getting transcripts of the same length but they all appear to be too long. What could be causing the production of extremely long transcripts and how could it be repaired? The problem is either with the termination sequence for transcription being missing or having the wrong sequence or that the termination for transcription of this gene is rho dependent and rho is missing. Depending on the problem the solution would be to either change/add the proper termination sequence or add the missing rho factor. 8. You now translate your mRNAs even though they are too long. Would you expect to get a functional polypeptide? Briefly explain your answer? Yes, if the stop codon is in the correct location translation will function normally and produce the proper length polypeptide even if the transcript is too long. 9. You get tired of working on extraterrestrial life so you decide to work on eukaryotic genes. You isolate two genes and put them into a prokaryote. For the first sample you discover you get a non-functional protein but for the second sample you do get a functional protein. a) Why would translation of the first gene produce a non-functional protein? There are introns present that the prokaryote can not process out. This results in the transcript having unnecessary bases resulting in a polypeptide with undesired amino acids resulting in a non-functional protein. b) What is different with the second gene? This is a case of a eukaryotic gene that does not have any introns. So when the gene is transcribed and translated in a prokaryote the message has no extra bases resulting in a polypeptide that does hot have any extra amino acids. Extra credit PCR (polymerase chain reaction) is a method to rapidly replicate small pieces of DNA using a single enzyme. Please explain how this is possible and what is replacing the other enzymes needed for normal DNA replication. Heat is used to separate the DNA strands replacing the functions of helicase, gyrase, topoisomerase and the single-strand binding proteins. Primers are then added so primase is not needed. The DNA being replicated is small so no fragments are produced so ligase is not needed. All that is needed is a heat tolerant DNA polymerase. 5 Name: __________________________ RNA Codons UUU UUC UUA UUG phe phe leu leu UCU UCC UCA UCG ser ser ser ser UAU UAC UAA UAG tyr tyr stop stop UGU cys UGC cys UGA stop UGG trp CUU CUC CUA CUG leu leu leu leu CCU CCC CCA CCG pro pro pro pro CAU CAC CAA CAG his his gln gln CGU CGC CGA CGG arg arg arg arg AUU AUC AUA AUG ile ile ile met ACU ACC ACA ACG thr thr thr thr AAU AAC AAA AAG asn asn lys lys AGU AGC AGA AGG ser ser arg arg GUU GUC GUA GUG val val val val GCU GCC GCA GCG ala ala ala ala GAU GAC GAA GAG asp asp glu glu GGU GGC GGA GGG gly gly gly gly 6