Investigtion of Transcripional Control of genes
Having been provided with trays of pea seedlings which have been grown for 14 days in the light in the absence of NO3
-. 24 hours prior to the experiment they have been treated as follows:
Kept in Light Transferred to Dark
1 +NO3(14mM) 3 +NO3(14mM)
2 -NO3 4 -NO3
Remove approximately 0.5g of leaf tissue from each treatment (using approximately the same age of leaves where
possible) and weigh accurately. Keep the leaves cold on watch glasses in an ice bucket, and slice into thin (1-2mm)
sections with razor blade.Transfer the tissue to an ice-cooled side arm tube and add 5ml of the following chilled
reaction medium, pH 7.5, containing:
50 mm KH2PO4
100mm KCH3CO2 (potassium acetate)
1.5% v/v propan-1-ol
Attach the side arm to a vacuum line, place a bung over the top of the tube and evacuate it. Remove the bung
occasionally (4-5 times) over a total period of about 2-3 minutes and shake off any air bubbles that are trapped on
the surface of the leaf tissue.
Make sure all the leaf material is immersed, then cover the side arm tube in metal foil and incubate for 45 minutes at
30* C. After this time remove 1 ml from the tube and add to this 1ml reagent 1 (1%(w/v) sulphanilamide in 1.5M HCL
Caution- HARMFUL, IRRITANT, CORROSIVE) followed immediately by 1ml reagent II (0.02% (w/v) n-1(1-naphtylethylene
diamine – HCL Caution- HARMFUL, IRRITANT, CORROSIVE)). Mix by inversion and allow to stand for 20 minutes, then
determine its absorbance at 540 nm. What must you do if any of these readings exceed the upper range of your
calibration curve (below)?
Whilst the leaves are being incubated prepare a standard NO2 curve. From the 0.02 mM stock solution of NaNO2, make
approximate dilutions to produce 0.020, 0.015, 0.01, 0.005, 0.0025, and 0mM NaNO2. Take 1ml of each dilution, add 1ml
reagent I then 1ml reagent II, shake and allow to stand for 20 minutes. Read the absorbance at 540nm.
Tabulate all the results and use Excel to plot a standard curve of absorbance against nmoles NaN02.(10 marks). From
this determine the activity of NO3 reductase in leaf tissue as nmoles NO2- produced per minute per gram of tissue.
Show a single example calculation in full (10 marks)
What do the results suggest about the control systems that regulate the abundance of this enzyme?
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