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320 Spring 2006 Exam 3 Review Practice Problems 1) Provided that a large number of individuals are involved, the kinetics of mutation can be usefully described by conventional mass-action kinetics. a. In a bacterial population of 1000 L of 3x107 cells/ml, the mutation of one gene g1 to a second type g2 occurs with a frequency of 10-8 per cell division, and the mutation from g2 to g1 occurs at a frequency 10-6 per cell division. Calculation the equilibrium concentrations of each species. b. In cells able to repair damaged DNA enzymatically, it is reasonable to suppose that, since the enzyme exists, there is an equilibrium between normal and damaged DNA:
Erepair
Suppose ultraviolet light opens a second forward path parallel to the first with rate constant k1. Show that the equilibrium population fraction of damaged DND rises from
k1 k1 + k 1
to
k1 + k
1
k1 + k + k 1
2) Repressor protein for the phage PR promoter can bind to three different operator sites designated OR1, OR2, and OR3. As indicated in the figure, binding of repressor to any site except OR3 blocks the PR promoter. Assume that repressor-operator binding is at equilibrium and that the equilibrium constants for repressor binding to the three sites are K1, K2, and K3, respectively.
PR
OR3
OR2
OR1
a. Develop a general equation for the fraction of PR promoters accessible for RNA polymerase binding in terms of the equilibrium constants and the total amount of repressor protein and PROR sequences in the cell b. Evaluate the fraction of accessible promoters for a cell containing 25 PROR sequences and 70 repressor molecules. K1, K2, and K3 are 125x108 M-1, 1 25x108 M-1, and 1x109 M-1, respectively. What is the physical significance of this result? 3) Determine both the maximum dilution rate and the dilution rate for maximum output for a CSTR with sterile feed and cells with growth kinetics according to the following equation (where Cs is the concentration of the limiting solute and has units of s-1): =kgrowthCs. Be sure to define all variables used. 4) For a specific type of cells after 3 hours, the concentration of cells per milliliter of solution is about 400/mL. After 10 hours, the concentration has gone up to 2000/mL. Determine the initial concentration of cells. 5) It is expected that stem cells provided a renewable tissue-culture source of cells that can be used to regrow whole organs such as hearts and livers. The cells will then be harvested for
applications in basic research or transplantation therapies. It is hypothesized that the undifferentiated proliferation of these cells follows a Monod growth model. Assume that the maximum growth rate (kp,max) of these cells is 0.3 hr-1 when in precense of a specific differentiation-inhibiting factor (DIF). The Monod constant, KM, is assumed to be 1.3 g/L and the cell yields in the presence of this factor (, is assumed to be 1.3 g/L and the cell yields in the presence of this factor (Yx/DIF) is 0.46 g cell/g of DIF. Assume steady-state operation for this question. An attempt is being made to use a 10L continuous reactor aerated with oxygen, using a sterile feed stream containing 5 g/L of DIF. What is the outlet concentration of the factor for a feed flow rate of 1L/hr? 6) Equal numbers of fibroblasts and endothelial cells are present initially in a culture. a. If endothelial cells double every 40 hours and fibroblasts double every 20 hours, draw a graph showing the percentage of cells in the culture that are fibroblasts as a function of time. b. What is the time required for the culture to contain 90% fibroblasts? Write an equation that describes this situation, and when solved for time, will provide the correct answer. 7) NR6 fibroblasts lack endogenous EGF receptors. Dr. L has transfected a mutant internalizationdeficient EGFR into NR6 cells, c973; the mutant is a deletion of a large portion of the cytoplasmic domain, leavning the extracellular domain intact. All other properties of the receptor (for example, affinity for EGF and other EGFR ligands) remain the same. The steady-state receptor number obtained in the transfected cells is 60,000 EGFR/cell. In culture medium containing 5,000 cells per ml, estimate the fraction of total receptors occupied when the EGF concentration in the medium is (a) 0.1 nM, (b) 1 nM; (c) 10 nM. Clearly state any assumptions or equations employed. 8) Consider a skin wound healing situation, for example, a thermal wound on pigs, in which epidermal growth factor (EGF) is applied in an attempt to hasten repopulation of the wound by dermal fibroblasts. The specific growth rate of the fibroblasts is defined as = (1/n)(dn/dt), where n is the cell number. A design correlation for fibroblast proliferation response to EGF has been found in which depends on the number of EGF-EGFR complexes per cell, C, according to the relationship (c) = maxC/(K+C) where max= 0.03 hr-L and K= 104 #/cell. Ligand-receptor kinetics have rate constants kf = 7x107 M-1min-1, kr = 0.35 min-1, ke = 0.2 min-1. a. If the dermal fibroblasts are found to possess 3x104 #/cell EGFR in the absence of EGF, determine the EGFR synthesis rate per cell, Vr b. Normal dermal tissue is approximately 10% fibroblasts by volume; wounded tissue initially contains no cells. Estimate the number of cells (and corresponding concentration) required to fill a wound bed of dimensions 0.1 x 1 x 1 cm. If the wound bed is initially invaded by migrating fibroblasts to a level of about 0.1% by volume, estimate the cell growth rate required to repopulate the entire bed to normal tissue cell concentration within a week. Is the number you obtain reasonable? c. Early attempts to provide EGF-enhanced skin wound healing involved inclusion of the peptide growth factor into a cream applied to the wound bed. Loss of EGF occurs by proteolytic degradation and by diffusion into the bloodstream, with a combined rate constant of approximately 0.01 min-1. It also occurs by EGFR-mediated uptake and degradation. Given the various parameter values above, calculate the transient EGF concentration in the wound bed following application for a series of possible initial doses. d. On the basis of your findings in part (c), predict the time needed for tissue regeneration as a function of applied EGF dose.