Chapter 12

A DNA fragment isolated from an EcoRI digest of genomic DNA was combined with a plasmid vector linearized by EcoRI digestion so that sticky ends could anneal. Phage \(T 4\) DNA ligase was then added to the mixture. List all possible products of the ligation reaction.

A vector has a polylinker containing restriction sites in the following order: Hind III, SacI, XhoI, BglII, XbaI, and ClaI. a. Give a possible nucleotide sequence for the polylinker. b. The vector is digested with Hind III and ClaI. A DNA segment contains a Hind III restriction site fragment 650 bases upstream from a ClaI site. This DNA fragment is digested with Hind III and \(C l a I,\) and the resulting Hind III-ClaI fragment is directionally cloned into the Hind III-Clal-digested vector. Give the nucleotide sequence at each end of the vector and the insert and show that the insert can be cloned into the vector in only one orientation.

Yeast (Saccharomyces cerevisiae) has a genome size of \(1.21 \times 10^{7}\) bp. If a genomic library of yeast DNA was constructed in a vector capable of carrying 16 -kbp inserts, how many individual clones would have to be screened to have a \(99 \%\) probability of finding a particular fragment?

Combinatorial chemistry can be used to synthesize polymers such as oligopeptides or oligonucleotides. The number of sequence possibilities for a polymer is given by \(x^{y}\), where \(x\) is the number of different monomer types (for example, 20 different amino acids in a protein or 4 different nucleotides in a nucleic acid) and \(y\) is the number of monomers in the oligomers. a. Calculate the number of sequence possibilities for RNA oligomers 15 nucleotides long. b. Calculate the number of amino acid sequence possibilities for pentapeptides.

Imagine that you are interested in a protein that interacts with proteins of the cytoskeleton in human epithelial cells. Describe an experimental protocol based on the yeast two-hybrid system that would allow you to identify proteins that might interact with your protein of interest.

Describe an experimental protocol for the preparation of two cDNA libraries, one from anaerobically grown yeast cells and the second from aerobically grown yeast cells.

Describe an experimental protocol based on DNA microarrays (gene chips) that would allow you to compare gene expression in anaerobically grown yeast versus aerobically grown yeast.

In your experiment in problem \(12,\) you discover a gene that is strongly expressed in anaerobically grown yeast but turned off in acrobically grown yeast. You name this gene nox (for "no oxygen"). You have the "bright idea" that you can engineer a yeast strain that senses \(\mathrm{O}_{2}\) levels if you can isolate the nox promoter. Describe how you might make a reporter gene construct using the nox promoter and how the yeast strain bearing this reporter gene construct might be an effective oxygen sensor.

Search the National Center for Biotechnology Information (NCBI) website at http://www. nchi.nlm. nih.gov/sites/entrez?db=Genome to discover the number of organisms whose genome sequences have been completed. Explore the rich depository of sequence information available here by selecting one organism from the list and browsing through the contents available.