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Summer Research Fellowship Programme of India's Science Academies

Construction of a phosphorylation resistant CUEDC2 gene

Subhadra Nandi1

14th Year of B.tech in Biotechnology, Techno India University, EM-4/1,Sector - V, Salt Lake, Kolkata 700091

Susanta Roychoudhury2, Somsubhra Nath3, Stuti Roy4

2PhD, Chief, Basic Research, Molecular Biology Research and Diagnostic Laboratory, Saroj Gupta Cancer Centre and Research Institute, Mahatma Gandhi Road, Thakurpukur, Kolkata 700063

3 PhD, Scientist, Basic Research, Molecular Biology Research and Diagnostic Laboratory, Saroj Gupta Cancer Centre and Research Institute,Mahatma Gandhi Road, Thakurpukur, Kolkata 700063

4M.Sc, Junior Research Fellow, Basic Research, Saroj Gupta Cancer Centre and Research Institute, Mahatma Gandhi Road, Thakurpukur, Kolkata700063

Abstract

CUE-domain-containing 2-protein, CUEDC2, is a cell cycle regulator that is phosphorylated by Cyclin dependent Kinase 1(CDK1) during metaphase of mitosis. It is an ubiquitin-binding motif – containing protein whose abnormal regulation might lead to tumor development by causing chromosomal instability. CUEDC2 downregulates Estrogen Receptor –alpha(ER-α) protein levels through the ubiquitin-proteasome pathway. Phosphorylation of CUEDC2 is necessary for metaphase to anaphase transition and ER-alpha degradation occurs during metaphase. Hence the aim of the project is to investigate if phosphorylation of CUEDC2 is necessary for ER-α degradation. In order to do so, there is a target to block phosphorylation of CUEDC2 by replacing the site of phosphorylation at 110th position Serine (Ser) with Alanine (Ala) (an amino acid that cannot undergo phosphorylation). To begin with, the Coding DNA Sequence (CDS) of CUEDC2 gene was searched in NCBI database and the codon, coding for S-110 was identified as "TCC". In order to change it for Alanine coding codon, nucleotide T was planned to be replaced with G to design a set of mutagenic primers. The manually designed mutagenic oligonucleotide primers were used to perform Site Directed Mutagenesis and the mutant CUEDC2 plasmid was then used to transform XL10 Gold competent cells. After transformation, single isolated colonies were picked and plasmid was isolated from them. The plasmid was subjected to sequencing PCR and thereafter Sanger’s sequencing was carried out for the mutational analysis whose result indicated a successfully created mutant CUEDC2 plasmid.

Keywords: ER-α, Ubiquitin-Proteasome Pathway, Primer, Site Directed Mutagenesis, Sanger’s Sequencing

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