Question

4. In future lectures we will describe a technique known as Northern blotting that can be used to detect RNA transcribed from a particular gene. Briefly, in this method a specific RNA is detected using a short segment of cloned DNA as a probe. The DNA probe, which is radioactive, is complementary to the RNA that the researcher wishes to detect. After the radioactive probe DNA basepairs to the RNA, the RNA is visualized as a dark (radioactive) band on an X-ray film. 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 occurs in another cell, the band will appear twice as intense. Also, the method can distinguish if alternative RNA splicing has occurred to produce an RNA that has a different molecular mass. Northern blot 3 875 nucleotides 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 theRNA produced in nerve and kidney cells. 675 nucleotides Let's suppose a researcher was interested in the effects of mutations on the expression of a particular structural 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: An individual homozygous for a deletion that removes the -50 to -100 region of the gene that encodes this mRNA Lane 3: An individual heterozygous in which one gene is normal and the other gene had a deletion that removes the -50 to-100 region Lane 4: An individual homozygous for a mutation that introduces an early stop codon into the middle of the coding sequence of the gene Lane 5: An individual homozygous for a three-nucleotide deletion that removes the AG sequence at the 3' splice site

Answer 1

In Lane 1, the normal amound of RNA is detected.

In Lane 2, since the promoter sequences are deleted, no RNA is detected.

In Lane 3, as promoter sequence is deleted in one copy, lower RNA levels are detected.

In Lane 4, introducion of a STOP codon, does not affect RNA levels.

In Lane 5, loss of the 3' acceptor site prevent splicing from occuring, and the RNA detected is 400 bp longer (the size of the intron).

16oobe 1200 bp 7.8oobe 40obe 1oobp