Answer to Question 1 of 1997

Many of you turned this into a much more complicated question than necessary. I think that careful reading of the question (which has a lot of embedded hints) would have helped.

a. The simplest and most accurate way to determine the exon/intron boundaries would be to determine the nucleotide sequences of the genomic and cDNA clones. Comparison of the nucleotide sequences would immediately reveal the precise locations of the introns, which would be present only in the genomic clone.

b. There are numerous ways to create mutations throughout a cDNA clone. I gave credit for any reasonable approach, including (i) systematic "linker substitution;" (ii) PCR mutagenesis; (iii) cleavage at different sites with restriction enzymes, treatment with exonuclease to generate deletions, and ligation. Most of you forgot to point out that, whatever method of mutagenesis is used, the reading frame must be preserved. I did not deduct credit for this common omission.

Many of you neglected the next step. How can the mutated cDNAs be converted to mutated proteins, since only proteins can be tested for function? The simplest approach was discussed in class: in vitro transcription of the mutated cDNAs followed by in vitro translation to generate protein products that could then be tested in the in vitro assay for PSI protein function. An alternative approach, suggested by many of you, is to overexpress the mutated cDNAs in bacteria, purify the proteins from bacteria, and then test the purified proteins in the in vitro assay. The cDNAs could also have been overexpressed in other systems, such as insect cells.