Introduction To Microbiology
Introduction To Microbiology BIOL 22100
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Date Created: 09/19/15
Bio 221 Fall 2011 Homework 2 This homework consists of 6 questions worth 1 point each Please type the answers on a separate sheet of paper double spaced Sketches may be drawn by hand There is no need to turn in the questions along with your answers If you use more than one sheet please staple them together There is not a stapler available in the lecture room Please remember to put your NAME at the UPPER RIGHT CORNER of your homework You can pick up the graded homework from in of ce G232 if you wish to have it back The key will be posted online immediately after lecture This homework is DUE W 1 For each of the three reactions in the central dogma complete the following sentence The process of 7replication7 begins when 7DNA 2017 binds to a 7origin ori17 sequence on a 7M7 template It then begins synthesizing 7M7 at the primer7 reading the template in the 73 to 5 7 direction until it reaches a 7terminator ter17 when it dissociates from the template and the process stops The process 0f7transcription7 begins when 7RNA pol 0 factor 7 binds to a promoter7 sequence on a 7M7 template It then begins synthesizing 7M7 at the 7amp7 reading the template in the 7m direction until it reaches a 7haiI1gin100127 when it dissociates from the template and the process stops The process 0f7translation7 begins when 721 ribosome7 binds to a 7ShineDalgarno sequence prok or 5 end euk7 sequence on a 7mRNA7 template It then begins synthesizing protein7 at the 7AUG start codon7 reading the template in the 7 direction until it reaches a 7stop codon7 when it dissociates from the template and the process stops 2 Sketch a bacterial DNA replication bubble At each replication fork label the 0 leading and lagging strands o 539 and 3 ends of the original DNA 0 primers o helicase o 0 DNA polymerase DNA gyrase helicase 3 A eukaryotic DNA template strand minus stmnd has the following sequence 539 GCTGCTCAATGGGGCCTTACAAACATAATG 3 39 RNA 3 39 CGACGAGUUACCCCGGAAUGUUUGUAUUAC 539 What peptide sequence would eventually be translated from the DNA ACC mRNA 39 CAUUAUGquyGUAAgg qgwusAGcAGc 3 39 ACA ribo binds here K start here MetPheValArgProHjs Stop here W CC urn cm gin Ifyou wanted to mutate a sing e base so that the 3ml amino acidin the peptide was changed as dmmatically as possible whichbase would you change What would be the new amino acid placed into the protein at that position 3rd codon is GUA aa is Valjne ihydrophobic Can change third base of codon but no effect wobble Can change lst base AU AIle h phobic CUALeu h phobic UUALeu h phobic Can change 2m1 base GAAGlu neg charge GCAAla h phobic GGAgly ts in both h phobic and h phjlic regions hunntton Yemunnuun Ofthese the greatest change is codon GUA Val to codon GAA Cl u So the DNA mutation would change 5 GCTGCTCAATGGGGCCTTQCAAACATAATG 3 0 5 GCTGCTCAATGGGGCCTTECAAACATAATG 3 4 For each of the following regulaton schemes sketch the transcriptional regulation when the ene is be39 tmnscribed is quotONquot Use an arrow to represent the promoter a circle for the regulatory protein and a triangle for the environmental inducer or co repressor Label the opemtor with an O and the activator binding site with anA example Positive control in an inducible o eron activator is produced in an inactive form activator binds vvhen inducer changes activators shape inducer present mRNAispmduced RNA p ulymerase A Positive control in a repressible operon O activator is producedin an active form activator binds in absence of corepressor corepressor NOT present mRNA is produced B Negative control in a repressible operon C repressor is produced in an inactive form i corepressor NOT present 1 repressor fails to bind without corepressor i I I r39 quotquotquotquotquotquot quot mRNA is produced C Negative control in an inducible operon transcription except quot I inducer binds to repressor and inactivates it 71 I I l repressor is produced in an active form and COULD bind to block inducer present l I mRNA is produced D Positive control in an inducible operon when the inducer remains outside the cell inducer present binds to sensor kinase p osp a v 2 is an activator protein which turns on the gen ON P sensMse autophospho39rwylem Pl hen transfers P to response regulator 5 In humans susceptibility to colon cancer can be greatly enhanced by a single mutation in a DNA sequence TTGCGA changing it to TCGCGA as shown below To test whether a patient will be susceptible to this disease a Southern blot can be done using the indicated probe The results of such a test are shown for two patients Does Patient 2 have enhanced susceptibility to colon cancer Explain thoroughly Patient 1 Patient 2 TCGCGA TTGCGA TCGCGA mutation probe TCGCGA TCGCGA TCGCGA R Yes mutation causes an extra restriction enzvme quot J 39 site to occur in the middl of the DNA sequence shown so that the RE will cut the DNA into 4 pieces 2 smal ends and 2 middle pieces rather than 3 The probe still binds to the same sequence in oth patients but now in the mutant this seguence is found on a smaller piece of M Patient 2 shows the smaller piece of DNA on the gel since smaller DNA sequences migrate further down the gel 6 There are sure to be many genes that change expression when a bacterium enters a human host Some will be turned on Type HI SS for example and some will be turned off agella perhaps A Set up an experiment to determine what genes are turned on and off when a bacterium enters a host You d use microarray hybridization to do this Isolate bacteria from a human host and the same bacteria from a lab culture Isolate mRNA from each bacterial population Label one mRNA set red and the other green Hybridize both red and green nucleic acids to a microarray that contains unique probes for each gene a few thousand in the bacterium Look for spots on the array that are green or red rather than yellow or dark These will be genes that are transcribed more in the host or more in the lab culture B How can a bacterium change expression of many genes all at the same time Several ways One use an alternate sigma factor that recognizes a promoter sequence different from the normal promoter the cell uses Two use the same regulatory protein binding site sequence and the same environmental inducer or corepressor to regulate multiple genes C You want to clone one of these genes called actl to study it further Describe how you would clone this particular gene Two options Digest the bacterial DNA with a restriction enzyme Separate the pieces by gel electrophoresis Use a probe complementary to the actl gene to find this gene on the gel Use a razor blade to cut out the portion of the gel containing this gene Digest a cloning vector plasmid with the same RE you used to cut the original DNA Use DNA ligase to insert the actl gene from the gel into the plasmid Insert transform the plasmid cloning vector into a host cell Or Make replication primers complementary to the 3 ends of the actl gene Use these primers in a PCR experiment to produce many copies of the actA gene Use RE and ligase to insert this PCR product into a plasmid cloning vector Insert transform the plasmid cloning vector into a host cell