Title: Cloning of Enhancer of Zeste Homolog 2 in forward orientation into Escherichia Coli using histidine-tagged pbluescript II KS+. Abstract
Enhancer of Zeste Homolog 2 locus is intensely over expressed in breast and prostate cancer and it’s been established that its promoter inhibition by p53 has led to reduced cell proliferation and invasion (Bracken, 2003; Xiao, 2011). Objective is to clone a forward orientated EZH2 insert into a his-tagged pbluescript. Cloning EZH2 into a histidine-tagged pbluescript in a forward orientation potentially allows isolation of protein via Affinity Chromatography or Chromatin Immunoprecipitation therefore its role, effects and targets in the genome can be established. Resultant Recombinant plasmids in this experiment had multiple inserts leading to inconclusive orientation of the inserts; however this can be tackled by Sanger or Maxam/Gilberts sequencing. Introduction
The capacity to segregate and amplify individual genes from an intricate genome using recombinant DNA technology technique like PCR has profoundly influenced ways scientists explored the obscurities in biology. Some of the major accomplishments of Recombinant DNA technology include the synthesis of medically essential proteins like insulin and developing new vaccines routes (Kingsman and Kingsman, 1988; Primrose et al., 2001). Gene Cloning allows the amplification of target sequences (e.g. Enhancer of Zeste Homolog 2/ insert) transferred as part of a vector (his- tagged pbluescript) into Escherichia Coli which reproduces asexually yielding progenies that contain the desired recombinant plasmid (Dale and Schantz, 2007). pbluescript is a bacterial based relaxed phagemid (~2.9kb), derived from pUC 19 which has 2 origins of replication (f1 and Col E1), 2 selectable marker Lac Z´ and ampR gene and due to its mutated RNA I it has a high copy number. It’s competent as cloning vector because it contains a multiple cloning site and transcribed by bacterial RNA polymerase. (Alting-Mees and Short, 1989) Histidine tags are amino acid motifs, consist of 5 or 6 strings of histidine which has high affinity for nickel, used for protein purification and detection. Transcription/Translation of the histidine-tagged EZH2 gene produces a his-tagged protein which can be used to extract a pure sample of the protein in a specially adapted column without the use of antibodies or probes, also using Chromatin Immunoprecipitation identifies the various sites of the genome where histone modification occurs as well as the targets (e.g. H3k27) of histone modifiers like EZH2 (Harlow and Lane, 1999). Homeotic genes such as Hox are amongst the best known targets of Polycomb group complexes. Homeotic genes are master genes whose expressions are involved in the segmentation and orientation of developing body parts. Polycomb recessive complex 1 and 2 are the two non interacting distinct forms of PcG complexes (Croonquist and Ness, 2005). EZH2 (insert) embedded in pGEM plasmid for this experiment is usually found in the nucleus, also known as ENX-1 or Lysine N-methyltransferase 6 and the catalytic subunit of PRC2, its presence on the complex induces the trimethylation of lysine 27 in histone 3. Its consecutive ability to methylate lysine 27 of histone 3 (mono, di and trimethylation) creates H3K27me1, H3K27me2 and H3K27me3, respectively (Li et al., 2010; UniProtkb, 2011). EZH2’s main role forms H3K27me3 which represses genes that would normally induce stem cell differentiation, also through its SET domain and methyltransferase activity it represses gene transcription at the chromatin level thereby silencing the targeted genes (Croonquist and Ness, 2005; Cao and Zhang, 2004). Subsequent histone methylation forms heterochromatin, hence the gene is switched off therefore no transcription occurs. EZH2 is involved in Hox gene silencing, genetic processes like X-inactivation, attenuated germ line development etc (Cao and Zhang, 2004). Over expression of EZH2...
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