Chapter 9

In a bacterial cell, two proteins, X and Y, are thought to have similar functions. Researchers genetically engineered each protein to fuse with a variant of the green fluorescent protein, one that glows red (X) and the other yellow (Y). Controls showed that both fusion proteins retained their activity, and both produced visible spots of light (foci) when expressed. To better understand the biological functions of the two proteins, the researchers expressed the fusion proteins in the same bacterial cell under two different conditions. Under nutrientrich conditions, distinct red and yellow puncta (well-defined clustering of foci) were distributed throughout the cell. One or two red puncta were typically found within the nucleoid (chromosomal DNA), whereas the multiple yellow puncta were distributed throughout the cell. However, under nutrient starvation, the yellow puncta migrated and colocalized (overlapped) with the red puncta. What might be concluded from these observations?

In a common protocol for immunofluorescence detection of cellular proteins, an investigator uses two antibodies. The first binds specifically to the protein of interest. The second is labeled with fluorochromes for easy visualization, and it binds to the first antibody. In principle, one could simply label the first antibody and skip one step. Why use two successive antibodies?

You are a researcher who has just discovered a new protein in a fungus. Design a yeast twohybrid experiment to identify the other proteins in the fungal cell with which your protein interacts, and explain how this could help you determine the function of your protein.

RNA-Seq is a next-generation sequencing method used to quantitatively profile the cellular transcriptome. Researchers use RNA-Seq to compare the expression of genes under different environmental conditions or between different types of cells. There are three general steps in an RNA-Seq workflow: 1\. Generate a cDNA library from cellular RNA. 2\. Add oligonucleotide adapters to the fragments of the cDNA library. 3\. Use next-generation sequencing to identify transcriptionally active genes from the cDNA library. What is the role of the enzyme reverse transcriptase in an RNA-Seq workflow?

Suppose that an investigative team conducted an RNA-Seq experiment on mouse liver cells. The team found many sequences that contained no open reading frames (Chapter 27) - long stretches of consecutive triplet codons that could be translated into a protein and therefore suggest the presence of a gene. Suggest a reason for this observed lack of ORFs.

Native American populations in North America and South America have mitochondrial DNA haplotypes that can be traced to populations in northeast Asia. The Aleut and Eskimo populations in the far northern parts of North America possess a subset of the same haplotypes that link other Native Americans to Asia, and the Aleut and Eskimo populations also have several additional haplotypes that can be traced to Asian origins but are not found in native populations in other parts of the Americas. Provide a possible explanation.

DNA (haplotypes) originating from the Denisovans can be found in the genomes of Indigenous Australians and Melanesian Islanders. However, the same DNA markers are not found in the genomes of people native to Africa. Explain.

You are a gene hunter, trying to find the genetic basis for a rare inherited disease. Examination of six pedigrees of families affected by the disease provides inconsistent results. For two of the families, the disease is co-inherited with markers on chromosome 7 . For the other four families, the disease is co- inherited with markers on chromosome 12. Explain how this difference might have arisen.