Cloning and Characterizing the Promoter Region of Maternal Embryonic Leucine Zipper Kinase in Pigs
Date Issued
2010
Date
2010
Author(s)
Hsu, Chih-Feng
Abstract
Transition from morula to blastocyst is a critical step in early embryonic development of the pigs. This process coincides with the change in gene expression pattern. Thirteen differentially expressed candidate genes during this period were previously identified by cDNA microarray and quantitative PCR, among which the gene of maternal embryonic leucine zipper kinase (MELK) remains novel in the porcine genome. A better understanding of the regulatory mechanism of the gene may provide tools to improve the successful development of porcine embryos. The purpose of this study is to clone and verify the promoter regions of these candidate genes, specifically focusing on MELK which was previously shown in other species to regulate cell proliferation and cell-cycle progression.
Sequence alignment among porcine, human, murine and bovine to predict the promoter regions of MELK (453 bp), ataxia-telangiectasia mutated protein (1067 bp), F-box protein 32 (1133 bp) and beta-2-microglobulin (556 bp), and reporter analysis in vitro demonstrated their activities. Serial deletion analysis was applied to specifically investigate the regulatory activity of MELK promoter. The on-line prediction software (Genomatix) predicted several putative transcription factor binding elements present in this region with -129 bp to -56 bp harboring the basal transcription activity and also some other positive regulatory elements located from -348 bp to -272 bp. To elucidate their roles in transcriptional regulation, we created four constructs with point-mutation in different sets of potential transcription factor binding elements. Our results showed that mutations on the overlapping region of the estrogen response element, steroidogenic factor binding element and RXR heterodimer binding site, that of MEF3 binding site and heat shock factor binding site, or TATA box, significantly decreased the promoter activity. However, the cognate protein factors binding to these elements remains to be proven by chromatin immunoprecipitation (CHIP) and electrophoretic mobility shift assay (EMSA) in subsequent experiments.
In conclusion, we cloned and verified the promoters of several candidate genes. In addition, we completed their promoter-specific reporter constructs to be used for embryo microinjection and gene ontology study. We also characterized the promoter region of MELK gene and showed that there were potential transcription factors likely to positively regulate the MELK gene expression.
Subjects
Maternal embryonic leucine zipper kinase
promoter cloning
pig embryo
morula
blastocyst
transcription factor binding site
Type
thesis
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