SOLVED PROBLEM 1. In Chapter 9, we studied the structure of tRNA molecules. Suppose that you want to clone a fungal gene that encodes a certain tRNA. You have a sample of the purified tRNA and an E. coli plasmid that contains a single EcoRI cutting site in a tet^R (tetracycline-resistance) gene, as well as a gene for resistance to ampicillin (amp^R). How can you clone the gene of interest?

Solution

You can use the tRNA itself or a cloned cDNA copy of it to probe for the DNA containing the gene. One method is to digest the genomic DNA with EcoRI and then mix it with the plasmid, which you also have cut with EcoRI. After transformation of an amp^S tet^S recipient, select Amp^R colonies, indicating successful transformation. Of these Amp^R colonies, select the colonies that are Tet^S. These Tet^S colonies will contain vectors with inserts in the tet^R gene, and a great number of them are needed to make the library. Test the library by using the tRNA as the probe. Those clones that hybridize to the probe will contain the gene of interest.

Alternatively, you can subject EcoRI-digested genomic DNA to gel electrophoresis and then identify the correct band by probing with the tRNA. This region of the gel can be cut out and used as a source of enriched DNA to clone into the plasmid cut with EcoRI. You then probe these clones with the tRNA to confirm that these clones contain the gene of interest.

SOLVED PROBLEM 2. You have isolated a yeast gene that functions in the synthesis of the amino acid leucine, and you hypothesize that it has the same function as a related gene from E. coli. How would you use functional complementation/mutant rescue to test your theory?

Solution

First, let’s assume that the yeast gene of interest is on an EcoRI-digested fragment. You will use recombinant DNA techniques to insert this fragment into the polylinker site of a bacterial plasmid that has been cut with EcoRI and treated with ligase to reform the circular plasmid. This plasmid should also contain a selectable marker for antibiotic resistance, like amp^R (see Figure 10-9). Next, you need to transform this recombinant plasmid into leu⁻ E. coli mutants. However, because there are four genes needed for leucine biosynthesis and you don’t know which one your yeast gene may be, you need to test for complementation in four mutant E. coli strains, each with a mutation in a different one of the four genes (called leuA, leuB, leuC, and leuD). Grow the four E. coli strains separately, and perform a transformation experiment where the same recombinant plasmid is introduced into each of the four strains. Next plate out the transformants on agar plates that contain the antibiotic ampicillin (so only cells that have taken up the plasmid can grow) but do not contain leucine. If you see colonies growing on one of the four plates containing transformed E. coli cells, not only can you conclude that you isolated a yeast gene involved in leucine biosynthesis, but you can also determine which leu gene (leuA, leuB, leuC, or leuD). That is, if the yeast gene is leuA, it will only complement the leuA-E. coli mutant.

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