cloning of plasmid-EZH2 Gene into pBluescript II KS+ plasmid with corresponding Histidine tag for potential analysis Abstract The EZH2 (enhance of zeste homolog2) is an enzyme that in humans is fixed by EZH2 and its supply the information’s about making of enzyme called a histone methyltransferase In this experiment PCR2 was examined whilst the EZH2 contributes to chemical modification. This resulted in repression. The aim of the study was to re-clone the EZH2 gene from 5’ to 3’ into another plasmid with pBluescript II KS fusing together with EZH2 moving forward. The histidine tags originate in the plasmid. Methods used encountered E.coli transformation, PCR, restriction enzymes, plasmid mini-preps and agarose gel electrophoresis. The end result lead to a 3’ to 5’ of EZH2 inserts. There was a good link which showed that the returning agar plate ensured that the collection of colonies consisted of forward oriented inserts. INTRODUCTION The EZH2 (enhance of zeste homolog2) is an enzyme that in humans is fixed by EZH2 and its supply the information’s about making of enzyme called a histone me-thyltransferase. The ploycomb group (PcG) is a protein with the catalytic subunit of the PRC2 (polycomb repressive complex) which the transcriptional involved in maintaining the repressive of genes cells and also the EZH2 mainly performs as a silence gene. The EZH2 performs the role by addition of methyl groups on the ly-sine 27 of histone H3, a modification leading chromatin
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
134). They are loops of DNA that are separate from the chromosomal DNA and can self-replicate in a cell, found mostly in bacteria (Brown, 2011; Addgene, 2015). Lederberg and William Hayes discovered that plasmids were being transferred from one cell to another, not the chromosomal DNA (Brown, 2011, p. 135). This discovery lead to plasmids being an essential tool for scientists. Scientists can engineer plasmids to have specific genes to introduce into new cells (Brown, 2011, p. 134). On a plasmid loop there will be an origin of replication (ORI) and a multiple cloning site (MCS) where the gene of interest is inserted (Bio-Rad, 2015). This region has specific restriction enzyme recognition sites, which are cut by the enzymes to open up the DNA where the new gene will be inserted (Jove Science Education Database, 2015). Most plasmids will also contain an antibiotic resistance gene allowing cell survival in environments containing antibiotics (Jove Science Education Database, 2015).
This lab is about moving genes from one thing to another using plasmids. Plasmid has the ability to replicate, so it replicates independently, and separately from the chromosomal DNA. Plasmid are one or more small piece of DNA and they enter cells as a double strand DNA. When they enter the cell as a doubke strand they do not invade he chromosomal DNA. We will also transform bacteria into GFP which is mainly from the jelly fish Aequorea Victoria. The GFP causes the the jelly fish to fluorescent and glow in the dark. After the transformation, bacteria starts to make the GFP which causes them to glow a green color under a ultraviolet light.
The purpose of the experiment was to isolate plasmid DNA, followed by restriction digestion using restriction endonucleases and then visualizing the digested fragments after subjecting to gel electrophoresis. Plasmid DNA (pSP72 DNA) was isolated from Escherichia coli KAM32 (E.coli) cultures using the QIA prep miniprep kit and then subjected to restriction digestion by EcoRI and HindIII. The restriction digested DNA was then loaded into the wells of 0.7% agarose gel and subjected to electrophoresis. It can be concluded from our results that our plasmid DNA isolation was successful and the restriction digestion results were partially in agreement with our hypothesis.
Methylation is the process where methyl groups are attached to the sequence of DNA code letter and to the histone groups that the DNA encircle; whereas, acetylation is the process where acetyl groups are attached to the histone tails. The methylation decreases gene expression while the acetylation increases gene expression. Epigenetic inheritance mentions in the article supports the content that we cover in class, in which epigenetic inheritance means modification to DNA other than sequence that can be altered by environment or diet and can be passed on to next
The experiment was conducted in two main parts: in the first part, the single and double restriction digests were run to cut the plasmids using combinations of three different enzymes. In the second part, the fragments resulting from each reaction were separated using gel electrophoresis. 1) Restriction Digests In this part of the experiment, seven different samples were prepared with different combination of reactions. Given the unknown plasmid and three restriction enzyme (HindIII, BglI and PvuII), seven different Eppendorf tubes were prepared to account for 3 single digests, one with each of the three restriction enzymes, 3 double digests combining two enzymes at a time and alternating between all the possible combinations, and also for the control reaction where no enzymes were added to the plasmid.
One of the main conclusions drawn from this experiment was that the cis memory system is the one used to inherit modified histones because they concluded that epigenetic memory is stored locally in FLC gene. One of the main pieces of evidence to support this is in Figure 3 of this paper. This figure describes heritable expression states, and it is concluded that the cis memory system is used because mitotic stability of all four possible combinations (ON/ON, ON/OFF, OFF/ON, and OFF/OFF) are observed. ‘Mixed’ ON/OFF and OFF/ON states are only seen in cis systems, thus the trans system cannot be implemented. Using this they were also able to show that two identical DNA sequences can have different transcriptional states in the same nucleus, which is only demonstrated by cis memory. Another conclusion they were able to make was that PRC2 is necessary for histone
During methylation, methyl groups (CH3) are attached to histone tails with the aid of histone methyltransferases (HMTs). These proteins transfer one, two, or three methyl groups from S-adenosyl-L-methionine to lysine or arginine residues. Lysine methylation involves a lysine methyltransferase that specifically targets and methylates lysine within the N-terminal tails. Similarly, arginine methylation occurs through the transfer of methyl groups from SAM to the ɯ-guanidino group of arginine. Although histone methylation does not change the charge of residues and does not greatly impact nucleosome interactions for chromatin folding, it results in binding sites for certain enzymes, which enables the regulation of chromatin condensation and nucleosome mobility. Methylation can also block the binding of proteins that interact with unmethylated histones and inhibit other modifications. Therefore, histone methylation regulates genes by promoting the binding of positive transcription factors and by blocking the attachment of negative transcription factors. However, some methylations can prevent gene transcription by mediating heterochromatin formation. For example, H3-K9 and H3-K27 methylation silences gene expression at certain euchromatic
The replacement of somatic histones with testis-specific variants in early haploid germ cells is undoubtedly an essential step for promoting chromatin remodeling during spermiogenesis, and it is believed to involve the histone code. The histone code hypothesis proposes that combinations of histone modification could serve as specific signals that recruit protein domains to alter chromatin structure to either promote or suppress gene transcription (Turner, 2002). There is evidence for the involvement of the histone code in chromatin remodeling as stage-specific histone modifications have been reported to occur during spermiogenesis, namely histone H4 acetylation, histone H3 methylation and phosphorylation, and histone H2A ubiquitination (Song
Scientists have known for several decades that cancer may be caused by mutations in the DNA of cells. These mutations may result from exposure to certain substances (e.g. radiation, benzene) or they may occur spontaneously in the process of cell division, especially in the context of aging. Recently, researchers have discovered another level of inherited cellular information separate from the genes themselves. Epigenetics is the study of modifications to genes that change their patterns of expression. Epigenetic processes can silence a gene or even an entire chromosome. They can cause normally silent genes to be expressed, and can change the process of transcription so that the nucleotides are transcribed in a different order. Normally, epigenetic information is stabilized early in development and is maintained as cells divide. However, over time, mutations or epigenetic drift may change the inherited pattern. This type of event often results in disease such as cancer (Jones & Baylin 2002).
Investigation into the regulation of covalent histone modifications at enhancers and promoters has become a popular research interest due to the important role these modifications have on altering gene expression and modulating cell fate specification. Polycomb repressive complex 2 (PRC2) is a transcriptional repressive complex that consists of three components: enhance zeste 2 (EZH2), embryonic ectoderm development (EED) and suppressor of zeste 12 (SUZ12). The catalytic subunit, EZH2, can methylate lysine 27 of histone 3 (H3K27) to promote transcriptional silencing by facilitating chromatin compaction. Alterations of this complex have been
Acetyl plays role can start the transcription. Methyl groups binds to a specific amino acid in a specific histone type. When the CH3 are added methylation spreads from the tail of one histone to the adjacent histone. The addition of phosphate is example of epigenetic changes. The addition of these three groups should be balanced.
The ability to control expression of DNA at the level of transcription is vital for cells not only to have the correct proteins expressed when they are needed, but also to save on resources and energy by only making proteins and RNA needed at the correct time, rather than beginning the process of expressing DNA and degrading the results later on. Though there are several different ways that DNA transcription can be controlled, histone deacetylation is the one focused on in the Vershon lab. DNA, which has a negative charge, is stored wrapped around proteins called histones. Histones can have negative acetyl groups on them, contributing to the overall charge of the protein. Removal of the acetyl groups makes the histone more
DNA was quantified and a luminometric methylation assay was performed. This assay is based upon
Histones are proteins found in the nucleus of eukaryotic cell, and are positively charged. The positive charge is what allows them to bind tightly to DNA (that are negatively charged because of the phosphate group). DNA needs histones, during the initiation of DNA replication, to allow the DNA polymerase move along the DNA and replicate the right amount of genetic material required for a daughter cell. However, as all proteins, histones need DNA and has to go through the process of transcription and translation to be formed. Histones are responsible for the packaging of DNA and play an important role in the regulation of gene expression.