OPEN BOOKBIO 510 Mid-year Exam 2011 NAME (Please Print)___________________________

1( 20 pts) NOTE: This question is related to the basic cloning question from last year and draws directly from our lab experience in cloning, PCR and protein expression as well as from our class reading.

The sequence of the BFT1 gene is presented below. The bold and shaded region is the protein coding sequence. Show all steps needed to create a protein-fusion construct between the coding sequence of BFT1 and the maltose binding protein encoded by pMAL-p5X (page 345 in the NEB catalog; note that the sequences presented in the polylinker are already arranged in the correct reading frame. For instance, under the XmnI site, the GGA, AGG, ATT and TCA sequences represent codons that are in the same reading frame with the maltose binding protein). Assume that you start with one microgram of pMAL-p5X and an unlimited supply of the DNA presented below and any reagents or materials (e.g., enzymes, oligonucleotides) and machinery (e.g., PCR machine, incubators, etc) that you need. The only exception is that you cannot propose to chemically (that is, non-enzymatically) synthesize the entire gene. On the sequence below, the protein-coding region is shaded and the translational termination codon is underlined (a restriction map is provided below). The perfect clone (i.e., full-credit) is an in-frame fusion that includes all the BFT1 coding sequence and little else beyond the minimum sequence needed to fuse it to the maltose binding peptide.

Discuss every enzymatic step you need to perform in detail and state which specific enzyme you would use, what buffers, length of incubation, etc. Describe any cleanup steps needed after one step is completed before the next step is initiated. You need not discuss E. coli competent cell preparation or the transformation itself, simply every step needed to get to the point where you have DNA to be transformed into E. coli.

BFT1 1 ATTACGTCGC AAGACGATAC ATCTTTAACA ATGGCTAGAG GTAATCAAAG 51 AGACTTGGCA AGACAAAAAA ACTTGAAAAA ACAAAAGGAC ATGGCTAAGA 101 ACCAGAAAAA GAGTGGTGAT CCTAAGAAAA GAATGGAGTC AGACGCCGAA 151 ATCTTGAGAC AAAAGCAGGC CGCCGCAGAT GCTAGAAGAG AGGCTGAAAA 201 GCTTGAGAAG TTAAAAGCCG AAAAGACGAG AAGATAAGCA GACACTTTAT 251 ACCTGTCCTT TTATTGG

Briefly, the easiest way to do this is simply to PCR amplify the BFT1 coding sequence and drop it into the pMAL-p5X polylinker. This could be done as a blunt-ended PCR fragment inserted at the XmnI site after trimming the non-encoded 3’ A extension from the PCR fragment and phosphorylating its (i.e., the PCR fragment’s) 5’ ends. If you use this blunt-ended strategy, you should dephosphorylate the vector to prevent self-ligation and you must determine the orientation of fragment insertion after cloning (since only one direction gives you the appropriate protein fusion product). Alternatively, you can include restriction sites compatible with the pMAL-p5X polylinker (e.g., XmnI and NotI) in the BFT1 amplification by adding these to the 5’ ends of the PCR primers. With either cloning strategy, you have to adjust the PCR primer positions to retain the

translational reading frame from the maltose binding protein through the BFT1 coding sequence. Alternative (non-PCR) strategies that use existing restriction sites within the BFT1 for subcloning were acceptable but full credit requires that the full protein coding sequence be transferred.

2. (4 pts) NOTE: This question is related our lab experience in protein expression as well as from our class reading and lecture presentations.Your main goal is to purify large amounts of recombinant protein. Which of the two strains presented below would you choose for this experiment and why (marker descriptions on NEB 320-321)?c. ER3020 McrA+, McrBC-, EcoKR-, EcoKM- , lon-, ompT, sulA-, endAOnly this strain has the mutations needed to stabilize foreign proteins in E. coli (lon, ompT, sulA).

3. (5 pts) This question is related our lab experience in cloning as well as from our class reading and lecture presentations. Which enzyme pairs produce DNA with ends that are always compatible (i.e., ends that can be ligated together)?A) SspI, AAT/ATTD) CviKI-1, RG/CY

C) SalI, G/TCGACF) XhoI, C/TCGAG

G) BamHI G/GATCCj) Sau3AI /GATC

CLOSED BOOK1) (3 pts) Note: From radiation lecture and lab activities. The radioisotope we

used to prepare our plus and minus strand RNA hybridization probes was 32P (note, provide both the element and the atomic number of the isotope). This isotope is a beta particle emitter and that compared to tritium (3H) is a (high) energy emission.

2) (4 pts) Three steps relevant to laboratory radioisotope use were suggested in our radiation training to limit radiation exposure. These were to:

a. Reduce the time of exposureb. Increase the distance between the worker and the isotopec. Provide shielding between the worker and the isotoped. Following steps a through c above results in a use practice abbreviated

ALARA. The letters in ALARA stand for: As Low as Reasonably Achievable

3) (10 pts) From lectures, lab activities and reading assignments. Which of the following would be useful for (put letter alongside protein or process): 1) M13KO7 DNA polymerase D, 2) solubilizing E. coli, 3 A) lacZ gene expression E, 3) imaging DNA by florescence B, 4) selection of a chitin-binding protein C

A) B) C

D) E)

4) (4 pts) We did an in-class comparison between EtBr and SYBR safe for the detection of dsDNA. Add one or the other product name for each of the following blanks. We found that EtBr was more sensitive for detecting low levels of DNA under the conditions of use. EtBr was much less expensive to use when only the purchase price of the raw reagent was considered. EtBr has reported as a mutagen. EtBr must be disposed of as a hazardous material.

5) (10 pts) From lectures, lab activities and reading assignments. Describe in detail the substrate and describe products of:a. T4 polynucleotide kinase Substrate: 5’ hydroxyl on RNA or DNA (SS or DS

although 3’ recessed ends are modified slowly) plus ATP; product: 5’ phosphate group

b. Exonuclease III – Substrate: dsDNA with blunt or 5’ extended end; Product: removes nucleotides in a 3’-5’ manner leaving a partially ss substrate and released nucleotides

c. RNase H: Substrate: RNA in an RNA/DNA hybrid; Products, ssDNA and released RNA nucleotides monophosphates and small RNA fragments

d. Shrimp alkaline phosphatase: Substrate: 5’ phosphate on ss or dsDNA or RNA; Product: released inorganic phosphate & nucleic acid with 5’ hydroxyl

e. M13 K07 DNA polymerase Substrate: dsDNA with an appropriate viral replication origin plus dNTPs; Product: released ssDNA

f. β-galactosidase; Substrate: lactose (or X-gal); Product: glucose+ galactose (or chromogenic X-gal metabolite)

g. Reverse transcriptase: Substrate: primed ssRNA plus dNTPs; Product: Initially, a RNA/DNA hybrid

h. RISC complex: Substrate: single stranded mRNA (or other RNA) target; Product: cleaved DNA at the site of RISC binding leading to full mRNA degradation

i. RNase A: Substrate: Single stranded RNA; Products: cleaves adjacent to pyrimidines, released nucleotides monophosphates and RNA fragments

j. Mung bean nuclease: Suibstrate: Single stranded DNA, Products: released deoxynucleotide monophosphates

Single stranded Probe (5’->3’) Hybridizing Bands on Blot (all in kb)EcoRI –> HindIII 0 BamHI - >KpnI 1.4KpnI ->EcoRI 0.79, 1.8KpnI ->HindIII 0.79

k. Terminal deoxynucleotide transferase: Substrate: 3’ hydroxyl of DNA & dNTPs; Products: DNA chain extended on 3’ end by one or more non-encoded dNMPs

6) (5 pts) From lectures. Outline all steps needed to complete an RNase-protection assay to compare the mRNA abundance of the actin transcript in liver and heart tissue. Briefly, a synthetic antisense RNA is prepared (e.g., using a cloned copy of the gene in a vector like pTZ18u) and radiolabeled either during synthesis (like we did) or afterwards with T4 polynucleotide kinase. The labeled RNA is designed to be slightly longer that the natural transcript. The labeled RNA annealed to the mRNA population from the tissue of interest and subsequently treated with a single-stranded ribonuclease such as S1, T1 or RNase A. The products are then resolved by gel electrophoresis. The RNase destroys any unannealed probe and the resulting processed band intensities reflects the relative abundance of the mRNA in the two tissue preparations.

7) (12 pts) From last year’s exam, lab activities and reading assignments. Given the following hybridization data, determine the location and direction of transcription for each RNA transcript. Assume that no more than one transcript is present in the interval defined by two adjacent restriction sites (that is, EcoRI to HindIII, HindIII to BamHI, BamHI to KpnI) and that no transcripts extend into an adjacent fragment. Put an arrow head at the 3’ end of the transcript (5’ -> 3’) and be sure to indicate clearly by a solid line labeled with a transcript length where each transcript resides and its size.

EcoRI HindIII BamHI KpnI5’X------------- X------------------------ X------------------------X 3’

1.8 kb 0.79 1.4

8) (2 pts) From lab activities. Which of the following steps is expected to increase the number of E. coli transformants of pTZ19u (circle all that apply)

- Heat shock of the DNA + E.coli at 42C for 1.5 minutes- Addition of DMSO to the competent cell preparation

9) (2 pts) From lab activities. Which of the following steps is expected to increase the number of yeast transformants of YCplac33 (circle all that apply)

- Addition of excess denatured salmon sperm DNA during the transformation

- Washing the yeast culture with lithium acetate (LiOAc) prior to DNA addition

10) From lab activities. We prepared ssDNA for the recombinant pTZ18u and pTZ19u transformants and ran a gel containing the following lanes: 1) uncut recombinant pTZ18u plasmid isolated from E. coli, 2) single-stranded recombinant pTZ18u, 3) single-stranded recombinant pTZ19u, 4) the annealed mix of single-stranded recombinant pTZ18u and pTZ19u prepared in class.

a. (3 pts)Add bands to this mock gel that illustrate the relative mobilities of each form of DNA. Assume that the wells are on top and for lane 1 be sure to label the relative mobility of the supercoiled plasmid DNA, open circle (relaxed) plasmid DNA, and linear plasmid DNA.

b. (2 pts) Draw a free-hand image of the predicted configuration of the DNA in lane 4. That is, assume that you could directly see the shape of the DNA, for instance by using an electron microscope. What would it look like?

11) From lectures, lab activities and reading assignments. When using C. elegans in an RNAi experiment, we simply added E. coli to the nematode. a. (5 pts) How did the E. coli addition direct degradation of specific target

mRNAs (e.g., CLF1, DPY-1 etc) within the animal? Each E. coli strain was transformed with a plasmid that included the target gene flanked by T7 RNA polymerase promoters. The E. coli was plated on medium containing IPTG to

induce the expression of the T7 RNA polymerase gene which transcribed the target gene and produced dsRNA. The bacteria were fed to the C. elegans nematode where the microbially produced dsRNA was cleaved by DICER to ~22-24 nt segments. One strand of this RNA was then loaded onto RISC to target the corresponding endogenous mRNAs for destruction.

b. (3 pts) Unlike most RNAi approaches, the RNAi affect is passed on though generations of animal. That is, the offspring of treated animals still show knockdown of the targeted transcript. An RNA-dependent RNA polymerase is specifically responsible for this persistence across generations. Discuss how this might occur. C. elegans has a potent RNA-dependent RNA polymerase which is capable of using the “DICED” RNA from the initial infection as primers to synthesize the equivalent of full length antisense mRNA from the corresponding endogenous transcripts. These are passed on through the sex cells to continue the dsRNA triggered RNA interference.

12) (2.5 pts) From lab activities. Two students cultured recombinant pTZ19u for single stranded DNA production. Both students grew the cells properly and used identical amounts of all materials (e.g., cells, virus, reagents etc) except that student #1 failed to add kanamycin sulfate to the growing cells. While both cultures grew well, the yield of ssDNA from student culture #1 was only 1% of that for student culture #2. Offer an explanation. The M13K07 virus encodes the kanamycin resistance gene. If no kanamycin is added, uninfected cells continue to grow but since these lack the M13K07 DNA polymerase no ssDNA is made (lowering student #1’s yield).

13) From lab activities. The melting curve of a student’s real-time PCR experiment showed 2 peaks rather than one.

a. (2 pts) Offer an explanation for this result. Two different PCR fragments were generated.

b. (2 pts) Based on the threshold value, the student’s realtime PCR results suggest a concentration of 1.3 µg of viral DNA per ml of blood. Based on part “a” of this question, what caution do you offer this student concerning interpretation of her results? The data likely overestimate the viral load since the second PCR fragment (which contributes to the “threshold value”) may have no relationship to the virus but is included in the measurement.