Identifying Cis-Regulatory Motif in Co-Expressed Beetle Genes

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Identifying cis-regulatory motif in co-expressed Tribolium castaneum genes and inspection of immunity conferring genes in females post-mating

Amrita Pal
(Master’s Non-thesis candidate)

Demuth Lab
April 18, 2011

Acknowledgement:

I am highly obliged to be able to perform tests and learn new techniques and concepts in the lab of Prof. Jeff Demuth, University of Texas at Arlington. He also allowed me work as a Research Assistant through a semester and was an excellent teacher. I express my gratitude towards the Biology Department at UTA which provided me assistantship during my stay. I sincerely want to thank Prof. Esther Betran, Andre Pires de Silva for serving my committee and being helpful teachers. Besides, I want to acknowledge the contribution of faculty especially Prof. Trey Fondon, Cedric Feschotte, Shawn Christensen, James Robinson, Ellen Pritham, postdocs Anna Williford, Miguel Gallach and students Prince Eldon, Marcel Guibot, Amrita Naidu, Eric Watson, Emma Mujica, Heath Blackmon, Heather Hawkins, Susana Domingues, Mahima Varma, Blaine Thompson, Jainy Tharun who through their examples gave me inspiration and guided me through their knowledge so that I could carry on graduate studies. My thanks to Prof Laura Mydlarz for allowing me conduct an experiment in her lab. I want to thank the Graduate School that provided me travel stipends for presenting at an International Conference.

Also I am indebted to my parents for everything they did and are still doing to support me. I am grateful for all the criticism and feedback I received from fellow people here at UT Arlington, this helped me become a better individual.

Abstract

Computational experimentation was done to find cis-regulatory motifs in co-expressed Tribolium casteneum Nimblegen microarray data (available from earlier work done in the same lab). The different categories of data available were male and female gonads, pupae and adults. The intent was to cluster data that had similar expression values across genome at different treatments like pupae-adult and male-female. Then for the co-expressed up-regulated genes, 2 kb sequences upstream were extracted for determining putative cis-regulatory motifs that had good hits with respect to homologous sequences in Drosophila. When double checked I did not find strong correlative evidence for the initially found 'Mad' motif in co-expressed target genes, so further loss-of-function and gain-of-function experimentation was not done. Later using the same method I found Transcription Factor 'Hunchback' motif in putative cis-regulatory sequences of co-expressed female biased genes in mated and virgin gonads. A functional annotation of these female biased genes was done. The data showed high expression of immunity conferring genes in the gonads of both virgin and mated females. Mated female gonad data showed up-regulation of serpins, and virgin female gonad data showed up-regulation of serine proteases suggesting generation of immune response in females post-mating via extracellular serine proteinase pathway. This was in alignment with previous work done by Begun et al, 2004 in Drosophila where they found immune related genes overrepresented among the mating-influenced candidates. Virgin and mated females of Tribolium castaneum GA-2 strain (corresponding to microarray data) were raised to see if indeed such immunity conferring genes were up-regulated in the haemolymph of females post mating. A PO-PPO test was conducted to confirm results, but no satisfactory result was obtained during test run. Further experimentation was then performed by other members of the lab using the same technique and results that they found seem to tally with microarray data.

Introduction
Tribolium castaneum genome has 16,437 ‘genes’ as shown by microarray data. This red flour beetle is raised on 5% yeast and grain flour. Egg production happens at 25 °C on seventh day...
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