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3 Slide 3 / 7 1 The above diagrams illustrate the experiments carried out by Griffith and Hershey and Chaserespectively. Describe the hypothesis or conclusion that each set of experiments proved or set out to test. B Why were the Griffith experiments inconclusive as to the source of the genetic material?

4 Slide 4 / 7 2 If dividing cells are treated with the pyrimidine analog bromo- deoxyuridine(brdu), during DN replication, the cells are fooled into incorporating it instead of thymidine (thymine) into their DN. One of the properties of the resulting DN is that it fails to take up stain in a normal way. When cells are allowed to duplicate their chromosomes once in BrdU, the chromosome that appear at the next metaphase stain normally. However, when the cells duplicate their chromosomes a second time in BrdU, one of the sister chromatids that appears at the next metaphase stains normally, while its sister chromatid does not (Harlequin Chromosomes). Note that each chromatid is normally completely stained or not (circled). Thymine bromodeoxyuridin Using your knowledge of DN replication, describe a possible hypothesis of why only one sister chromatid gets stained darker than the other chromatid after the second round of cell division? What will you expect to see in the chromosome staining pattern if cells are allow to divide for one more round of B DN replication and cell division after the initial 2 rounds of cell division and DN replication?

5 Slide 5 / 7 3 Lactose metabolism is controlled by the lac operon, which consists of the lacz, lacy, and lac genes encoding β-galactosidase, lactose permease, and transacetylase, respectively. Expression of the operon is negatively regulated by a transcription factor, the lac repressor (LacI), which dissociates from its specific binding sequences of DN, the lac operators, in the presence of an inducer to allow transcription (Fig. ). The repressor LacI and permease LacY form a positive feedback loop. Expression of permease increases the intracellular concentration of the inducer lactose which causes dissociation of LacI (lactose repressor) from the promoter (Plac), leading to even more expression of permeases. Cells with a sufficient number of permeases will quickly reach a state of full induction (Figure B), whereas cells with too few permeases will stay uninduced. (B) fter 24 hours of growth in media containing 30 μm TMG, strain SX700 expressing a LacY-YFP fusion exhibits all-or-none fluorescence in a fluorescence-phase contrast overlay (bottom, image dimensions 31 μm 31 μm). Fluorescence imaging with high sensitivity reveals single molecules of permease in the uninduced cells (top, image dimensions 8 μm 13 μm). Stochastic Single-Molecule Event Triggers Phenotype Switching of a Bacterial Cell Paul J. Choi, Long Cai, Kirsten Frieda, and X. Sunney Xie Science 17 October 2008: 322 (5900), If there is no lactose present, why will the E. coli cells not produce the proteins involve in lactose metabolism? The boxed cells in figure B shows cells will single B molecules of permease present within the cells, why are these cells not fully fluorescent?

6 Slide 6 / 7 4 The diagram shows 2 possible pathways of polymerase exchanges that are supported by the results of observing changes in the fluorescence and bleaching (removing fluorescence) of single molecular fusions of polymerase III, the major DN polymerase in bacteria, within the replication fork of live E. coli cells. Polymerase Exchange During Okazaki Fragment Synthesis Observed in Living Cells Giuseppe Lia, Bénédicte Michel, and Jean-François llemand Science 20 January 2012: 335 (6066), Describe why so many proteins are required to carry out the process of DN replication? B Why does DN need to get copied? Select the model that you think that cells would favor and C provide a rationale for your choice.

7 Slide 7 / 7 5 For more than a decade, circadian (daily rhythms) researchers have examined the RN molecules produced cyclically in many tissues. Consistently, the rhythmically expressed genes exhibited expression peaks throughout the entire day, with an enrichment for transcripts peaking at the dawn and dusk transitions. The core transcriptional machinery of the mammalian circadian clock includes BML1, CLOCK, and NPS2 proteins that positively regulate gene targets that contain the circadian cis-regulatory sequence known as an enhancer box (E box), or a slight sequence variant the E box, in their promoters or enhancers. The circadian regulators PER1, PER2, PER3, CRY1, and CRY2 have a repressive role on these same targets. The transcriptional activity takes place in three phases (see the figure). First, a transcriptionally poised state occurs as both the repressor, CRY1, and the activators, BML1 and CLOCK, occupy regulatory sequences. s the amount of CRY1 binding slowly decreases, the transition to activation (the second phase) occurs as the coactivator protein p300 and marks of active chromatin (DN and protein) [histone acetylation (H3K9ac) and methylation (H3K4me1)] are established. In the final repressive state, BML1 and CLOCK occupancy decreases and accumulation of the repressive factors PER1, PER2, and CRY2 is observed on target genes. It will be interesting to see how this transition occurs at each individual promoter, whether the same DN region can retain both repressors and activators in the poised state, or whether this observation reflects an average of multiple cells in different states of transition. Circadian Surprise It's Not ll bout Transcription Colleen J. Doherty and Steve. Kay Science 19 October 2012: 338 (6105), Select one activity either performed by a cell or an organism that may be regulated by the circadian clock described in the above reading. Describe the rationale by your choice. Using the above graph, provide evidence to support the B idea that BML1 and CLOCK regulate daytime activity. Our day is 24 hours long; the length of day varies in other planets. How would a change in the length of the day C affect the circadian clock? How would the information be relayed to the transcription pathway that is the circadian clock?

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