Nathan K Evanson and Dr. Daniel L Simmons, Chemistry and Biochemistry
Cyclooxygenases are proteins that make small molecules called prostaglandins, which relay signals from one cell to another, or from one cell to itself. Prostaglandins are involved in many different physiological processes such as inflammation, contraction of smooth muscle, and regulation of water balance in the kidney. Because prostaglandins are involved in so many different things, they are an important topic of research, and therefore, cyclooxygenases are a topic of intense research.
One of the cyclooxygenases that we have studied intensively is cyclooxygenase-2. It is considered to be the cyclooxygenase that is most responsible for inflammation. Because of its role in inflammation, inhibiting the activity of cyclooxygenase-2 often can alleviate inflammation, such as that experienced in rheumatoid arthritis. Indeed, drugs such as aspirin work by inhibiting cyclooxygenase-2.
In order to study cyclooxygenases more closely, it is important to have a source of the protein to study. This is often done using insect cells, because they are easy to grow and process proteins in much the same way that other animals do. However, for this work, I wanted to have a system that could express the protein in human cell lines, because these would process the protein in exactly the same way that other human cells would. In addition, I wanted to include various parts of the cyclooxygenase gene in order to test their effects on the protein.
Cyclooxygenase-2 RNA contains a large amount of untranslated region, which does not code for the amino acids in the protein that will be made. However, there are reports that the untranslated region can influence the amount of protein that is made from the cyclooxygenase-2 mRNA1. I therefore made constructs (artificial gene-like DNA) that would express cyclooxygenase-2 with varying portions of the 3´ untranslated region (see figure 1).
In order to make the constructs, I used standard recombinant DNA techniques. I used a base vector called pRc-RSV (Promega, Madison, WI) and removed the region containing a bovine growth hormone polyadenylation signal (marks the end of an RNA transcript). I then cut fulllength clones of human COX-2 with restriction enzymes that removed parts of the 3´ untranslated region and subcloned the COX-2 fragments into the modified pRc-RSV vector. I used restriction digesting to confirm the identity of the constructs (see figure 2 for example).
In conclusion, I was able to make a series of constructs containing the cyclooxygenase-2 coding region and various lengths of the 3´ untranslated region, which can now be used to study the effect of the 3´ untranslated region on translation of cyclooxygenase-2 in human cell lines.
Reference
- Cok SJ, Morrison AR (2001) The 3′-untranslated region of murine cyclooxygenase-2 contains multiple regulatory elements that alter message stability and translational efficiency. J. Biol. Chem. 276, 23179-23185.