Learning Objectives:
- Record Experimental results of RNAi experiment carried out in the last lab.
- Interpret Experimental results of RNAi experiment carried out in the last lab.
- Make a quantitative assessment of the RNAi effect on phenotype
Understanding how mutants are named in C. elegans:
- Unlike other model genetic organisms the names given to mutants in C. elegans are standardized and usually refer to their phenotype. They are all given a name that is 3 letter followed by a number. The number refers to the order in which the genes were discovered. This avoids the confusion that often occurs in other model organisms where mutant can be given any name.
- For example, mutants with the name "rol", like rol-3 and rol-6 move in a circular motion. In other words, they roll.
- Similarly mutants with the name "sma" are all small and skinny.
- We carried out RNAi against bli-1. All Bli mutants have blisters.
-We also carried out RNAi against dpy-11. All Dpy mutants are dumpy - that is they are short and plump.
- Bli-1, the protein product of the bli-1 gene is a structural component of the collagen and cuticulin-based cuticle.
- It is involved in cuticle development
- According to Wormbook "The nematode cuticle is an extremely flexible and resilient exoskeleton that permits locomotion via attachment to muscle, confers environmental protection and allows growth by molting. It is synthesized five times, once in the embryo and subsequently at the end of each larval stage prior to molting. It is a highly structured extra-cellular matrix (ECM), composed predominantly of cross-linked collagens, additional insoluble proteins termed cuticlins, associated glycoproteins and lipids. The cuticle collagens are encoded by a large gene family that are subject to strict patterns of temporal regulation. Cuticle collagen biosynthesis involves numerous co- and post-translational modification, processing, secretion and cross-linking steps that in turn are catalyzed by specific enzymes and chaperones. Mutations in individual collagen genes and their biosynthetic pathway components can result in a range of defects from abnormal morphology (dumpy and blister) to embryonic and larval death, confirming an essential role for this structure and highlighting its potential as an ECM experimental model system."
- Bli-1 is also involved in the cuticle molting cycle.
- As a result of the inactivation of the bli-1 gene by RNAi the worms have a hard time shedding their cuticle and this results in the formation of blisters appearing as either a small or large gap between their body and their cuticle. These blisters can be seen in Figure 8.1.
- Bli-1 is an ortholog of human COL27A1 (collagen type XXVII alpha 1 chain) and COL9A1 (collagen type IX alpha 1 chain).
- In Humans this gene is implicated in Stickler syndrome and multiple epiphyseal dysplasia 6.
- Dpy 11, the protein product of the dpy-11 gene enables the activity of the protein-disulfide reductase enzyme.
- This enzyme is involved in several processes, including cuticle development and the cuticle molting cycle.
- It is also involved in post-embryonic body morphogenesis and protein folding.
- Although it is active in the same place that bli-1 is active in, it has a different phenotype.
- In this case they have trouble molting their cuticle at all. The worms that continue to grow but are stuck inside a cuticle that is too small for them.
- This results in worms that are short and plump. This phenotype can be seen in Figure 8.2.
- Dpy-11 is an ortholog of the human TMX1 (thioredoxin related transmembrane protein 1) .