Cloning Of Swine Maternal Embryonic Leucine Zipper Kinase (MELK)

As a unique member of AMPK-related protein Ser/Thr kinases, maternal embryonic leucine zipper kinase (MELK) is also known as murine protein kinase38(MPK38) in mice. Unlike other members of the AMPK-related protein kinase family which are thought to be important regulators of cell energy homeostasis, MELK was shown to be involved in the control of cell proliferation, regulation of apoptosis, cell cycle and tumorigenesis. Although MELK is conserved across animal kingdom, its expression is restricted to some proliferating cells comprising epithelium, hemopoietic stem cells, embryonic cells. Also, MELK was overexpressed at a significantly high level in multiple cancer cells, particularly in undifferentiated and malignant tumor cells, but was not expressed in normal vital organs (heart, liver, brain and kidney), or was expressed at a faint and undetectable level.Although MELK has been implicated in such a large variety of biological processes, its precise function remains elusive. As there is no literature suggesting the cloning of swine MELK, it is very necessary to clone and express the swine Melk gene for further investigation of its intricate roles in sus scrofa model. Firstly, we use human maternal embryonic leucine zipper kinase transcript variant1(NM₀14791) as a initiating sequence to align with expression sequence tags (ESTs) in sus scrofa and assemble into a novel cDNA contig as a result of repetitive hand assembly through BLAST. And in the support of alignment with sequences in NCBI Genomes (Chromosome), the novel sequence is referred to as putative complete swine Melk coding sequence. The length of the sequence is2636bp, comprising open reading frame (ORF) of1953bp which encodes a peptide including650amino acids. Besides, the sequence shares88.2%and91.1%identity with human and cattle respectively, and its loci is in the chromosome1of swine. The encoding peptide contains a S_TKc domain, kinase catalytic domain, in the N-terminal and KA1domain in the opposite C-terminal in accordance with the report concerning the MELK structure of other species. The analysis of3D-structure indicates that there is a very high similarity between human and swine MELK, both containing a leucine zipper motif. Further verification was conducted through RT-PCR proliferating a2100bp DNA fragment as long as the predicted swine Melk cDNA. These results demonstrate that the in silico cloning of swine Melk is reliable which undoubtedly pave a solid way for further investigation of its vital biological roles.