. Mutants of Neurospora crassa that lack carbamoyl phosphate syn- thetase I (CPS I) require arginine in the medium in order to grow, whereas mutants that lack carbamoyl-phosphate synthetase II (CPS II) require a pyrimidine, such as uracil. A priori, one would expect the active CPS II in the arginine mutants to provide sufficient carbamoyl phosphate for arginine synthesis, and the active CPS I in the pyrimidine mutants to "feed" the pyrimidine pathway. Explain these observations.
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- A classic way to isolate thymidylate synthase–negative mutants of bacteriais to treat a growing culture with thymidine and trimethoprim. Most ofthe cells are killed, and the survivors are greatly enriched in thymidylatesynthase–negative mutants.(a) What phenotype would allow you to identify these mutants?(b) What is the biochemical rationale for the selection? (That is, why are themutants not killed under these conditions?)(c) How would the procedure need to be modified to select mammalian cellmutants defective in thymidylate synthase?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.In the "biochemical assay of b-galactosidase activity", what is o-nitrophenyl-b-D-galactosidase (ONPG) used for? O When ONPG is cleaved by b-galactosidase, we are able to assay b-galactosidase activity. O It provides essential nutrients for bacterial growth. It partially disrupts the cell membrane to allow cellular proteins to diffuse out of the cell. O ONPG cleaves the b-galactosidase that is made by the lac operon so we can see how much activity there is in the cell. ONPG cleaves galactose so we can measure how much lactose there is in the cell.
- 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.Various antimicrobial drugs to treat microbial infection have diverse mechanism of action. Consider the following antimicrobial drugs: A. Seconeolitsine, known as DNA topoisomerase I inhibitor in bacteria. (i) Explain briefly how inhibiting DNA topoisomerase I is a good mechanism of action for an antibiotic, include possible molecular machineries being targeted. (ii) What would be an appropriate response if seconeolitsine works well by stating the state of supercoiling in bacteria. (iii) To prove your answer (ii), you test the condition of bacterial DNA by running gel electrophoresis, one has been treated with seconeolitsine (+ sample) and the other one is not (- sample). Explain the position of each + sample and – sample band on the gel in reference to the point of origin (where you load your samples) or how far each DNA sample travel across agarose gel. (iv) Explain why you would expect answer (iii) for each + sample and – sample. B.…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?
- Consider the biochemical pathway shown here. Suppose that a strain of bacteria must synthesize compound 4 to survive and divide. Successful survival and division of bacteria is observed as growth of colonies on an agar plate. This strain of bacteria can grow colonies on minimal medium as long as it is supplemented with compound 1. You are in a lab that has isolated several mutants of this strain. You find that these mutants cannot grow on minimal medium supplemented with compound 1, though they can grow colonies if supplemented with compound 4. Considering what you know about the Beadle-Tatum experiments, which of the following statements would be one that should be true?. 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.A classic way to isolate thymidylate synthase-negative mutants of bacteria is to treat a growing culture with thymidine and trimetho- prim. Most of the cells are killed, and the survivors are greatly enriched in thymidylate synthase-negative mutants. (a) What phenotype would allow you to identify these mutants? (b) What is the biochemical rationale for the selection? (That is, why are the mutants not killed under these conditions?) (c) How would the procedure need to be modified to select mam- malian cell mutants defective in thymidylate synthase?
- For E. coli strains with the lac genotypes show below, use a plus sign (+) to indicate the synthesis of β-galactosidase and permease and a minus sign (–) to indicate no synthesis of the proteins.(A) strain A strain B pro-Pase protease DOCKING pro-Pase protease SNARES exist as complementary partners that carry out membrane fusions between appropriate ves- icles and their target membranes. In this way, a vesicle with a particular variety of v-SNARE will fuse only with a membrane that carries the complementary t-SNARE. In some instances, however, fusions of identical membranes (homotypic fusions) are known to occur. For example, when a yeast cell forms a bud, vesicles derived from the mother cell's vacuole move into the bud where they fuse with one another to form a new vacuole. These vesicles carry both v-SNARES and t-SNARES. Are both types of SNARES essential for this homotypic fusion event? FUSION Pase (B) 100 75 To test this point, you have developed an ingenious assay for fusion of vacuolar vesicles. You prepare vesicles from two different mutant strains of yeast: strain B has a defective gene for vacuolar alkaline phosphatase (Pase); strain A is defective for the protease…A series of auxotrophic mutants were isolated in Neurospora. Examination of fungi containing these mutations revealed that they grew on minimal medium to which various compounds (A, B, C, D) were added; growth responses to each of the four compounds are presented in the following table. Give the order of compounds A, B, C, and D in a biochemical pathway. Outline a biochemical pathway that includes these four compounds and indicate which step in the pathway is affected by each of the mutations. Compound Mutation number А в С A D 134 276 987 773 772 146 333 123 + + I +