DATA ANALYSIS PROBLEM

Zhang, B., M. Gallegos, A. Puoti, E. Durkin, S. Fields, J. Kimble, and M.P. Wickens. 1997. A conserved RNA-binding protein that regulates sexual fates in the C. elegans hermaphrodite germ line. Nature 390:477–484.

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Question 22.13

The collaborative effort by Judith Kimble, Marvin Wickens, and colleagues, as described in their 1997 paper, resulted in discovery of the FBF proteins that bind the 3′UTR of the mRNA from the fem-3 developmental regulatory gene of nematodes. Compare the three-hybrid strategy used in this study (see Figure 1 in this chapter’s How We Know section) with the three-hybrid method presented in Chapter 7.

  1. How was the three-hybrid method modified in the Kimble and Wickens study?

Six different RNA sequences were screened for FBF-1 binding. The normal PME sequence (UCUUG) gave a positive response. The other five sequences were two 5 nucleotide sequences, an iron response element, a segment containing 30 consecutive A residues, and a 573 nucleotide RNA sequence derived from HIV.

  1. Of these controls, which would make the best case for specific binding of the PME by FBF-1?

  2. Why might the other controls be useful?

The investigators used immunofluorescence to detect expression of the FBF-1 protein in wild-type nematodes, as shown in the upper panel of Figure 1. All cells illuminated are in the germ line. The dark spots are cell nuclei.

  1. What conclusion can you draw from the protein expression pattern in the upper panel of the figure?

The lower panel in Figure 1 shows results for an animal treated with RNAi directed at the gene for FBF-1 (fbf). The expression of FBF-1 is essentially abolished.

  1. Given the function of FBF-1 in the germ line, what is the likely effect of this RNAi treatment on the germ line of the treated animals?

FIGURE 1