Al-Harbi, Hamad (2010) Towards understanding latrophilin signalling in C.elegans. MRes thesis, University of Nottingham.
Latrophilin (LAT-1) is a G -protein-coupled receptor (GPCR), and mediates Black widow spider venom toxicity (Mee et al., 2004). A deletion in the lat-1(ok1465) gene (allele ok1465) in C. elegans causes 97% of worms to die during embryogenesis or early larval stages (Adenle, 2008). Thus latrophilin has an essential function in development, but it is not known how latrophilin signalling causes biological effects. The aim of this study is to identify genes that mitigate the effect of the lat-1(ok1465) deletion allele on offspring lethality, with a view to identifying the pathways involved in latrophilin signalling that mediate developmental lethality.
Brood size was determined for N2 worms (295 ±36; mean ± standard deviation), whereas lat-1(ok1465) worms produced only 4 ± 4 adult offspring (P<0.05). It was decided to undertake mutagenesis of the lat-1(ok1465) worms, but this requires bleaching of adult populations, to yield synchronously growing populations. While ~0% of wild type L1 died after bleaching, lat-1(ok1465) worms showed ~97% lethality. The recovery of worms after bleaching of lat-1(ok1465) worms was 0.07 offspring per input adult worm. The mutagenesis procedure involves three generations of worms with bleaching at each stage, and so around 6 million lat-1(ok1465) adult worms at P0 are required to get 20,000 F2 L4 worms. Liquid culture is required to grow this large number of worms, and worm growth in liquid media was optimised. A strategy for screening lat-1(ok1465) worms was devised; screening the brood size of 2x104 individual worms is prohibitively time-consuming, and so worms were screened in plates containing 20 lat-1(ok1465) worms. 20 lat-1(ok1465) worms would be expected to yield around 80 offspring, and experimental testing showed that there were 74 ± 14 worms per plate. To date, screening of 10000 F2 from mutagenised lat-1(ok1465) worms has been performed. Thirty independent plates were shown to have worms with the multi-vulva phenotype, a mutation rate of 0.6 per 1000 mutagenised genomes. The screen yielded two plates with >100 offspring, and these plates yielded two mutant lines whose brood size was statistically significantly increased (P<0.05) compared with the lat-1(ok1465) brood size.
This work shows that mutagenesis of lat-1(ok1465) worms was successful, and that the screening procedure can be used to yield lat-1(ok1465) mutants with increased brood size. Future work will involve mapping and identifying the role of these mutant genes that confer increased brood size to lat-1(ok1465) worms.
|Item Type:||Thesis (MRes)|
|Uncontrolled Keywords:||latrophilin, latrophilin signalling, cell receptors, secretin, C. elegans|
|Faculties/Schools:||UK Campuses > Faculty of Medicine and Health Sciences > School of Biology|
|Deposited By:||Dr David R Bell|
|Deposited On:||22 Feb 2011 11:33|
|Last Modified:||15 Dec 2011 12:47|
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