15. Genomes and Genomics

Translational fusions between a protein of interest and a reporter protein are used to determine the subcellular location of proteins in vivo. However, fusion to a reporter protein sometimes renders the protein of interest nonfunctional because the addition of the reporter protein interferes with proper protein folding, enzymatic activity, or protein–protein interactions. You have constructed a fusion between your protein of interest and a reporter gene. How will you show that the fusion protein retains its normal biological function?

It can be said that modern biology is experiencing an 'omics' revolution. What does this mean? Explain your answer.

How would you perform a genetic screen to identify genes directing Drosophila wing development? Once you have a collection of wing-development mutants, how would you analyze your mutagenesis to learn how many genes are represented and how many alleles of each gene? How would you discover whether the genes act in the same or different pathways, and if in the same pathway, how do you discover the order in which they act? How would you clone the genes?

A 2-kb fragment of E. coli DNA contains the complete sequence of a gene for which transcription is terminated by the rho protein. The fragment contains the complete promoter sequence as well as the terminator region of the gene. The cloned fragment is examined by band shift assay. Each lane of a single electrophoresis gel contains the 2-kb cloned fragment under the following conditions:

Lane 1: 2-kb fragment alone

Lane 2: 2-kb fragment plus the core enzyme

Lane 3: 2-kb fragment plus the RNA polymerase holoenzyme

Lane 4: 2-kb fragment plus rho protein

Explain the relative positions of bands in lanes 1 and 4.

Metagenomics studies generate very large amounts of sequence data. Provide examples of genetic insight that can be learned from metagenomics.

A 3.5-kb segment of DNA containing the complete sequence of a mouse gene is available. The DNA segment contains the promoter sequence and extends beyond the polyadenylation site of the gene. The DNA is studied by band shift assay, and the following gel bands are observed.

Match these conditions to a specific lane of the gel.

3.5-kb fragment plus TFIIB and TFIID

A 3.5-kb segment of DNA containing the complete sequence of a mouse gene is available. The DNA segment contains the promoter sequence and extends beyond the polyadenylation site of the gene. The DNA is studied by band shift assay, and the following gel bands are observed.

Match these conditions to a specific lane of the gel.

3.5-kb fragment plus TFIIB, TFIID, TFIIF, and RNA polymerase II

A 3.5-kb segment of DNA containing the complete sequence of a mouse gene is available. The DNA segment contains the promoter sequence and extends beyond the polyadenylation site of the gene. The DNA is studied by band shift assay, and the following gel bands are observed.

Match these conditions to a specific lane of the gel.

3.5-kb fragment alone

A 3.5-kb segment of DNA containing the complete sequence of a mouse gene is available. The DNA segment contains the promoter sequence and extends beyond the polyadenylation site of the gene. The DNA is studied by band shift assay, and the following gel bands are observed.

Match these conditions to a specific lane of the gel.

3.5-kb fragment plus RNA polymerase II

A 3.5-kb segment of DNA containing the complete sequence of a mouse gene is available. The DNA segment contains the promoter sequence and extends beyond the polyadenylation site of the gene. The DNA is studied by band shift assay, and the following gel bands are observed.

Match these conditions to a specific lane of the gel.

3.5-kb fragment plus TFIIB

What are DNA microarrays? How are they used?

A 1.0-kb DNA fragment from the end of the mouse gene described in the previous problem is examined by DNA footprint protection analysis. Two samples are end-labeled with ³²P and one of the two is mixed with TFIIB, TFIID, and RNA polymerase II. The DNA exposed to these proteins is run in the right-hand lane of the gel shown below and the control DNA is run in the left-hand. Both DNA samples are treated with DNase I before running the samples on the electrophoresis gel.

Explain the role of DNase I.

A 1.0-kb DNA fragment from the end of the mouse gene described in the previous problem is examined by DNA footprint protection analysis. Two samples are end-labeled with ³²P, and one of the two is mixed with TFIIB, TFIID, and RNA polymerase II. The DNA exposed to these proteins is run in the right-hand lane of the gel shown below and the control DNA is run in the left-hand. Both DNA samples are treated with DNase I before running the samples on the electrophoresis gel.

Draw a diagram of this DNA fragment bound by the transcriptional proteins, showing the approximate position of proteins along the fragment.

A 1.0-kb DNA fragment from the end of the mouse gene described in the previous problem is examined by DNA footprint protection analysis. Two samples are end-labeled with ³²P and one of the two is mixed with TFIIB, TFIID, and RNA polymerase II. The DNA exposed to these proteins is run in the right-hand lane of the gel shown below and the control DNA is run in the left-hand. Both DNA samples are treated with DNase I before running the samples on the electrophoresis gel.

What length of DNA is bound by the transcriptional proteins? Explain how the gel results support this interpretation.

How would you design a genetic screen to find genes involved in meiosis?