Bulb1 and Bulb2 are transcription factors involved in the survival response of plants exposed to UV radiation. Wild-type (wt) and homozygous mutant (Bulb1-/-) Arabidopsis plants were exposed to UV-B for various times and their RNA extracted. RNA was dissolved in 20 µl of buffer. A 2 µl aliquot was taken from one of the stock solutions and diluted to 1 ml. The absorbance of the solution at 260 nm was found to be 0.046.
The RNA samples were used for measurement of bulb2 transcript levels by quantitative PCR (Figure 1A). In a different experiment, Bulb1 and Bulb2 protein levels in wild-type Arabidopsis, exposed to UV-B radiation for various times, were assessed (Figure 1B).
Q9.1. Bulb1 and Bulb2 are transcription factors involved in the UV-B survival response in Arabidopsis. RNA extracted from plant tissue was dissolved in 20 µl of buffer. A 2 µl aliquot was diluted to 1 ml, and its absorbance at 260 nm was 0.046. Given that a 40 µg/ml RNA solution gives an absorbance of 1.0 at 260 nm, calculate the concentration of the ORIGINAL (undiluted) stock solution, in µg/ml.
Q9.2. Each experiment requires 2 µg of RNA. What volume of the stock solution (920 µg/ml) would be needed? Give your answer in µl, to two decimal places.
Q9.3. How many experiments could the researchers conceivably carry out with the original 20 µl stock of RNA?
Q9.4 (box 1). The RNA samples were used to measure bulb2 transcript levels by qPCR in WT and Bulb1-/- plants exposed to UV-B for various times (Fig.1A), bulb2 transcript rises sharply with UV-B exposure in WT but stays flat in Bulb1-/- plants. Separately, Bulb1 and Bulb2 PROTEIN levels were assessed by blot in WT over a UV-B time-course (Fig.1B), Bulb1 protein stays constant throughout, while Bulb2 protein only appears after UV-B exposure and increases over time. Build a 3-step model: UV-B → [box 1] → [box 2] → [box 3] → UV protection. What is box 1?
Q9.4 (box 2). What is box 2 in the pathway (UV-B → Bulb1 nuclear translocation → [box 2] → [box 3] → UV protection)?
Q9.4 (box 3). What is box 3 in the pathway (UV-B → Bulb1 nuclear translocation → Bulb2 transcription → [box 3] → UV protection)?
Fig.1. Axis = Fold change relative to control. wt = Wild-type.