Chapter 12, Problem 2EQ

Chapter 21 describes a technique known as Northern blotting that is used to detect RNA transcribed from a particular gene. In this method, a specific RNA is detected using a short segment of cloned DNA as a probe. The DNA probe, which is labeled, is complementary to the RNA that the researcher wishes to detect. After the probe DNA binds to the RNA, the RNA is run on a gel and then visualized as a labeled (dark) band. As shown here, the method of Northern blotting can be used to determine the amount of a particular RNA transcribed in a given cell type. If one type of cell produces twice as much of a particular mRNA as another type of cell does, the band will appear twice as intense. Also, the method can distinguish whether alternative RNA splicing has occurred to produce an RNA that has a different molecular mass.

Lane 1 is a sample of RNA isolated from nerve cells.

Lane 2 is a sample of RNA isolated from kidney cells. Nerve cells produce twice as much of this RNA as do kidney cells.

Lane 3 is a sample of RNA isolated from spleen cells. Spleen cells produce an alternatively spliced version of this RNA that is about 200 nucleotides longer than the RNA produced in nerve and kidney cells.

Let’s suppose a researcher is interested in the effects of mutations on the expression of a particular protein-encoding gene in eukaryotes. The gene has one intron that is 450 nucleotides long. After this intron is removed from the pre-mRNA, the mRNA transcript is 1100 nucleotides in length. Diploid somatic cells have two copies of this gene. Make a drawing that shows the expected results of a Northern blot using mRNA from the cytosol of somatic cells, which were obtained from the following individuals:

Lane 1: A normal individual

Lane 2: A homozygote for a deletion that removes the $-50$ to $-100$ region of the gene that encodes this mRNA

Lane 3: A heterozygote in which one gene is normal and the other gene has a deletion that removes the $-50$ to $-100$ region

Lane 4: A homozygote for a mutation that introduces an early stop codon into the middle of the coding sequence of the gene Lane

5: A homozygote for a two-nucleotide deletion that removes the AG sequence at the 3′ splice site