Dataset Information

Expression screening using a Medaka cDNA library identifies evolutionarily conserved regulators of the p53/Mdm2 pathway.

ABSTRACT:

Background

The p53 tumor suppressor protein is mainly regulated by alterations in the half-life of the protein, resulting in significant differences in p53 protein levels in cells. The major regulator of this process is Mdm2, which ubiquitinates p53 and targets it for proteasomal degradation. This process can be enhanced or reduced by proteins that associate with p53 or Mdm2 and several proteins have been identified with such an activity. Furthermore, additional ubiquitin ligases for p53 have been identified in recent years. Nevertheless, our understanding of how p53 abundance and Mdm2 activity are regulated remains incomplete. Here we describe a cell culture based overexpression screen to identify evolutionarily conserved regulators of the p53/Mdm2 circuit. The results from this large-scale screening method will contribute to a better understanding of the regulation of these important proteins.

Methods

Expression screening was based on co-transfection of H1299 cells with pools of cDNA's from a Medaka library together with p53, Mdm2 and, as internal control, Ror2. After cell lysis, SDS-PAGE/WB analysis was used to detect alterations in these proteins.

Results

More than one hundred hits that altered the abundance of either p53, Mdm2, or both were identified in the primary screen. Subscreening of the library pools that were identified in the primary screen identified several potential novel regulators of p53 and/or Mdm2. We also tested whether the human orthologues of the Medaka genes regulate p53 and/or Mdm2 abundance. All human orthologues regulated p53 and/or Mdm2 abundance in the same manner as the proteins from Medaka, which underscores the suitability of this screening methodology for the identification of new modifiers of p53 and Mdm2.

Conclusions

Despite enormous efforts in the last two decades, many unknown regulators for p53 and Mdm2 abundance are predicted to exist. This cross-species approach to identify evolutionarily conserved regulators demonstrates that our Medaka unigene cDNA library represents a powerful tool to screen for these novel regulators of the p53/Mdm2 pathway.

SUBMITTER: Zhang P

PROVIDER: S-EPMC4599741 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:BACKGROUNDEimeria maxima is one of the most prevalent Eimeria species causing avian coccidiosis, and results in huge economic loss to the global poultry industry. Current control strategies, such as anti-coccidial medication and live vaccines have been limited because of their drawbacks. The third generation anticoccidial vaccines including the recombinant vaccines as well as DNA vaccines have been suggested as a promising alternative strategy. To date, only a few protective antigens of E. maxima have been reported. Hence, there is an urgent need to identify novel protective antigens of E. maxima for the development of neotype anticoccidial vaccines.METHODSWith the aim of identifying novel protective genes of E. maxima, a cDNA expression library of E. maxima sporozoites was constructed using Gateway technology. Subsequently, the cDNA expression library was divided into 15 sub-libraries for cDNA expression library immunization (cDELI) using parasite challenged model in chickens. Protective sub-libraries were selected for the next round of screening until individual protective clones were obtained, which were further sequenced and analyzed.RESULTSAdopting the Gateway technology, a high-quality entry library was constructed, containing 9.2?×?106 clones with an average inserted fragments length of 1.63 kb. The expression library capacity was 2.32?×?107 colony-forming units (cfu) with an average inserted fragments length of 1.64 Kb. The expression library was screened using parasite challenged model in chickens. The screening yielded 6 immune protective genes including four novel protective genes of EmJS-1, EmRP, EmHP-1 and EmHP-2, and two known protective genes of EmSAG and EmCKRS. EmJS-1 is the selR domain-containing protein of E. maxima whose function is unknown. EmHP-1 and EmHP-2 are the hypothetical proteins of E. maxima. EmRP and EmSAG are rhomboid-like protein and surface antigen glycoproteins of E. maxima respectively, and involved in invasion of the parasite.CONCLUSIONSOur results provide a cDNA expression library for further screening of T cell stimulating or inhibiting antigens of E. maxima. Moreover, our results provide six candidate protective antigens for developing new vaccines against E. maxima.

Project description:BackgroundComparative genomic analysis using cDNA microarray is a new approach and a useful tool to identify important genetic sequences or genes that are conserved throughout evolution. Identification of these conserved sequences will help elucidate important molecular mechanisms or pathways common to many species. For example, the stockpiled transcripts in the oocyte necessary for successful fertilization and early embryonic development still remain relatively unknown. The objective of this study was to identify genes expressed in oocytes and conserved in three evolutionarily distant species.ResultsIn this study we report the construction of a multi-species cDNA microarray containing 3,456 transcripts from three distinct oocyte-libraries from bovine, mouse and Xenopus laevis. Following the cross-species hybridizations, data analysis revealed that 1,541 positive hybridization signals were generated by oocytes of all three species, and 268 of these are preferentially expressed in the oocyte. Data reproducibility analyses comparing same-species to cross-species hybridization indicates that cross-species hybridizations are highly reproducible, thus increasing the confidence level in their specificity. A validation by RT-PCR using gene- and species-specific primers confirmed that cross-species hybridization allows the production of specific and reliable data. Finally, a second validation step through gene-specific microarray hybridizations further supported the validity of our cross-species microarray results. Results from these cross-species hybridizations on our multi-species cDNA microarray revealed that SMFN (Small fragment nuclease), Spin (Spindlin), and PRMT1 (Protein arginine methyltransferase 1) are transcripts present in oocytes and conserved in three evolutionarily distant species.ConclusionCross-species hybridization using a multi-species cDNA microarray is a powerful tool for the discovery of genes involved in evolutionarily conserved molecular mechanisms. The present study identified conserved genes in the oocytes of three distant species that will help understand the unique role of maternal transcripts in early embryonic development.