Genetic Screen For Zebrafish Digestive Organ Mutants And Prokaryotic Expression & Purification And Antibody Generation Of The Zebrafish Ubel Protein

This research paper is composed of two relatively independent parts. First, genetic screen for zebrafish digestive organ mutants; Second, prokaryotic expression and purification and antibody generation of the zebrafish Ube1 protein. Therefore, the two parts are explained separately below.The zebrafish, Danio rerio, a small subtropical freshwater animal, has proven to be a wonderful model system to study the vertebrate embryogenesis due to its characteristics. Zebrafish are easy to raise and reach sexual maturity at around three months; Females lay several hundreds of eggs per week; Zebrafish embryos are small, transparent and develop outside of the maternal body. These advantages make Zebrafsih suitable for large-scale genetic screen. Both the intestine and liver develop from the endoderm, yet little is known how these two digestive organs share and differ in their developmental programs at the molecular level. A classical forward genetic screen, with no gene bias, is an effective way to address the question by examining genetic mutants harboring developmental defects in the intestine and liver, followed by cloning and characterization of the mutated genes responsible for the phenotypes of either organ, or both. We report here such a screen in zebrafish. ENU was used as the mutagen because of its high mutagenic efficiency and no documented hotspots in mutagenesis site. Embryos were collected at 3.5dpf for RNA whole mount in situ hybridization with a cocktail probe of the intestine marker ifabp and the liver marker lfabp to determine their parental heterozygosis. The screen was first performed in F2 generation. Putative mutants with general developmental defects were aborted. To rule out non-inheritable phenotypes caused by high mutation background, putative F2 mutants were outcrossed with wild type fish and a re-screen in F3 generation was performed. A total of 37 F3 putative mutants originated from 22 F2 families were identified after screening 78 mutagenized genomes. Classification of mutant phenotypes indicated that 31 out of the 37 putative mutants showed defects in both the intestine and liver. In addition, four "intestine specific mutants" and two "liver specific mutants" showed selectively more severe phenotype in the intestine and liver respectively. These results suggested that the liver and intestine share a substantial number of regulatory genes during both organs development in zebrafish. Further studies of the mutants are likely to shed more insights into the molecular basis of the development of the zebrafish and vertebrate digestive system.Ubiquitination is a ATP-dependent process, which requires the cooperation of "ubiquitin activating enzyme" (E1), "ubiquitin conjugating enzyme" (E2) and "ubiquitin ligase" (E3). Monoubiquitination often leads to changes in subcellular localization or aggregation, while multiple ubiquitination cycles resulting in a polyubiquitin chain (K48) are required for targeting a protein to the proteasome for degradation. The multisubunit 26S proteasome recoganizes, unfolds and degrades polyubiquitinated substrates into small peptides. The ubiquitin proteasome pathway, conserved from yeast to mammals, is required for the targeted degradation of most short-lived proteins in the eukaryotic cell. This pathway is not only important in cellular homeostasis, but also in human disease. Since ubiquitin/proteasome-dependent degradation is often employed in the control of the cell cycle and cell growth, researchers have found that proteasome inhibitors hold some promises of being developed into potential cancer or leukemia therapeutic agents. Ube1, the most important El in ubiquitination pathway, is essential for the first step of ubiquitin proteasome pathway. Thus, the critical activities in vivo such as cell cycle, cell differentiation and homeostasis of the body will be perturbed without Ubel.Purification of Ube1 protein with high purity is a prerequisite for the molecular and biochemical research based on Ubel as well as for the study of ubiquitin proteasome pathway related enzymes and proteins. Firstly, the full length CDS of zebrafish ubel gene was cloned into the prokaryotic expression vector pSUMO (pET28a-SUMO) and the recombinant plasmid was successfully transformed into the E.coli strain BL21. Affinity chromatography was utilized to purify the Ubel protein. However, the Commassie Blue straining result indicated that a high proportion of unwanted proteins were included in purified protein solution. Western blot results revealed that most of those unwanted proteins may be cleavage pieces of the full length Ubel protein generated during Ubel protein purification. Then, the sequence coding the Ubel C’-terminal 105 aa was cloned into the pET28a expression vector instead. The perfect recombinant plasmid was transformed into the E.coli strain BL21 for prokaryotic expression, and followed by affinity purification of this Ubel truncated protein. High purity of the Ubel C’-terminal truncated protein was obtained and used as an antigen to generate Ube1 antibody in male rabbits. Western blot results demonstrated that the blood serum, after three or four times of immunization, worked pretty well as antibody. And the titer of this antibody was very high. Antibody affinity purification was committed to extract the Ubel antibody from the original serum. The specificity of Ubel antibody was improved to a large extent and the recovery rate of this antibody was higher than 25%.