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Fully explain in the form of a detailed and annotated diagram how DnaK is involved in the heat shock response.
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- CTP synthetase catalyzes the glutamine-dependent conversion of UTP to CTP. The enzyme is allosterically inhibited by the product, CTP. Mammalian cells defective in this allosteric inhibition are found to have a complex phenotype: They require thymidine in the growth medium, they have unbalanced nucleotide pools, and they have an elevated spontaneous mutation rate. Explain the likely basis for these observations.Knowing that DNA cloning means making multiple identical copies (clones) of a DNA sequence of interest using target DNA, describe the process of amplification.Measure the uptake of leucine by epithetial cells of the mouse intestine. Measurements of the rate of update of L-leucine, D-Leucine, and L-valine , with and without Na+ in the assay were perform and yield different results (see table below). A) What can you conclude about the properties and mechanism of leucine transporter? B) Would you expect L-leucine uptake to be inhibited by Ouabain, which is a cardiac glycoside drug treatment?
- A chain of biochemical events is responsible for Aequorea victoria turning green. Firstly, the protein aequorin converts chemical energy into blue light. A second protein, known as GFP (green fluorescent protein), absorbs the light. Aequorin is a 21 kDa protein with a pI of 4.5 that generates blue light from an attached non-protein chromophore. GFP is a 27 kDa protein with a pI of 6.2. In order for it to fluoresce, 3 amino acids undergo a remarkable reaction when they are folded in the right way. Aequorin does not absorb light in the visible range, and GFP absorbs light at 395 nm and 475 nm. 1. Sketch what you would see if you ran a mixture of aequorin and GFP over a size-exclusion column. In your sketch, include absorbance data at 280, 395, and 475 nm.. Explain why DNA is stable in the presence of alkali (0.3 M KOH), while RNA is quantitatively degraded to 2'- and 3'-nucleoside monophosphates under these conditions.Rifamycins have been used for the treatment of many diseases, including HIV-related Tuberculosis. Explain how Rifamycins inhibit the activities of bacterial DNA dependent RNA polymerase.
- Certain hormones, such as epinephrine, can increase the levels ofcAMP within cells. Let’s suppose you pretreat cells with or withoutepinephrine and then prepare a cell extract that contains theCREB protein.You then use an electrophoretic mobility shift assay to analyzethe ability of the CREB protein to bind to a DNA fragmentcontaining a cAMP response element (CRE). Describe what theexpected results would be.CTP synthetase catalyzes the glutamine-dependent conversion of UTP to CTP. The enzyme is allosterically inhibited by the product, CTP. Mamma- lian cells defective in this allosteric inhibition are found to have a complex phenotype: They require thymidine in the growth medium, they have unbal- anced nucleotide pools, and they have an elevated spontaneous mutation rate. Explain the likely basis for these observations.Experiment: Heat shock factors (HSF) are transcription factors that induce genes encoding proteins that protect against cellular stress. Two distinct heat shock factors, called HSF1 and HSF2, recognize the same binding site in DNA. The figure shows the result of an experiment in which the effect of an anti-inflammatory drug, indomethacin, was studied. HeLa cells were cultured at 37°C (lane 1 [C, control] and lanes 3-10), or 42° C (lane 2; HS, heat-shocked). The cultures for samples 3- 10 were treated with various concentrations of indomethacin, indicated at the top of each lane. After treatment, nuclear protein extracts were prepared and mixed with 32P-labeled, double- stranded oligonucleotides containing the HSF binding site. Anti-HSF1 (samples 7 and 8) or anti- HSF2 (samples 9 and 10) antibodies were also added to some of the samples. The samples were subjected to electrophoresis in a nondenaturing polyacrylamide gel, and autoradiography was performed.
- Explain why aspartate residues play an important role on the active site of DNA polymerase.Mammalian cells growing in culture were labeled with [3H]-thymidineto estimate the rate of DNA synthesis. The thymidine administered had aspecific activity of 3000 cpm/pmol. At intervals, samples of culture weretaken and acidified to precipitate nucleic acids. The rate of incorporation ofisotope into DNA was 1500 cpm/106 cells /min. A portion of culture wastaken to determine the specific activity of the intracellular dTTP pool, whichwas found to be 600 cpm/pmol.(a) What fraction of the intracellular dTTP is synthesized from the exogenous precursor?(b) What is the rate of DNA synthesis, in molecules per minute per cell ofthymine nucleotides incorporated into DNA?(c) How could you determine the specific activity of the dTTP pool?The cyanobacterium Oscillatoria sancta appears reddish-brown when grown under green light but alters its gene expression patterns and becomes blue-green when grown under red light. Explain this observation.