Control Of Gene Expression By H Ns

The genome of E. coli 0157:H7 was recently sequenced and contains 1,387 new genes absent in non pathogenic strains. Analysis of the genome suggests that there are a variety of potential virulence genes that have yet to be explored, including fimbrial and other adhesions, secretion systems, and toxins. Many of the virulence genes are similar to those associated with pathogenesis in Salmonella and Shigella. Most of them are found on pathogenicity islands from viral DNA that is transmitted to the bacteria through the activity of a lysogenic phage. The two most important toxins, Stx1 and Stx2 (for shiga-toxin) are found on such islands and are composed of 5 B subunits surounding an active A component, which is transported into the cell after the

After recognizing the restriction sites, the restriction enzymes cut off the DNA strands at these points, leaving sticky ends on the original DNA fragment. The restriction enzymes can also travel to other cells to cut off DNA sequences, the sequences that can match the bases of the original sequence. The sticky end will find other sticky end to form a new recombined DNA sequence, obeying the law of base-pairing.

Sufu has the ability to bind to GLI1, GLI2 and GLI3 proteins. It is theorized that GLI proteins are binded to Sufu in a head to tail orientation. GL1 is solely found in the nucleus, whereas Sufu can be found expressed in both the nucleus and the cytoplasm. However when they are both expressed together, Sufu brings the GLI1 into the cytoplasm which forces the nucleus to limit its transcription activity. The cytoplasm anchoring model suggests that GLI1 is anchored to the cytoplasm with the help of Sufu. Likewise, Sufu plays a similar role in Drosophilia by halting nuclear processes. Thus, it is found that Sufu travels back and forth between the cytoplasm and nucleus in both Drosophilia and mammals. A second model of Sufu function in mammals suggests the Sufu gene holds the ability of repressing transcription of GLIs. A study found that Sufu restricts GLI transcription by enlisting the help of the mSin3A HDAC corepressor complex. Sufu was also found to increase the ability of GLI1 to bind to DNA. Therefore, simply repressing the Sufu gene is enough to activate GLI transcription, alluding to its key role in the negative regulation of the SHH signaling

Many people think of Escherichia coli as only a severe intestinal illness caused by eating uncooked, contaminated water, or unwashed fruits, but it is much more than only a negative (Amenu et al., 2014). The quick regeneration time of Escherichia coli makes it extremely useful in laboratory studies. In terms of research, a useful aspect of Escherichia coli is the extensive amount of information we have on them (Archer et al., 2011). Scientists have used Escherichia coli to host proteins, and it has been excellent resource for evolution studies as the bacteria has adapted frequently over the years (Archer et al., 2011). The non- pathogenic strains of Escherichia coli are frequently used in medical investigations involving diseases that are difficult to work with. Examples of these diseases include cancer or anthrax poisoning (Das et al., 2013).

When choosing the restriction enzymes to use, many factors are considered such as methylation of certain genes, which can influence the results of the experiment. Methylation will protect the gene from restriction enzymes, so in this experiment restriction enzymes that are not methylation sensitive are used (3). The wavelength of the UV Radiation can also have an impact on the rate of mutagenesis (4), which will be discussed more in the results. Restriction enzymes in this experiment such as Bam will cleave every six base pairs at a specific location on the target site (5). Bacteria such as E. Coli can naturally make restriction enzymes such as Eco, which is used naturally by the host cell to cut viral DNA (typically from phages) and make it non-functional in order to protect the integrity of its genome (6). In this discovery-based experiment, we will observe the changes in banding patterns of E. Coli DNA with multiple restriction endonucleases after UV induced mutation, and use these results against the control to determine if there are any changes in gene sequence by the number of base pairs in the observed DNA

This mutagenesis kit can make point mutations, replace certain amino acids, and delete or insert adjacent amino acids, making it an incredibly powerful tool. First, the mutant strand is synthesized by denaturing the DNA template and anneal the mutagenic primer(s) and then incorporated using DNA polymerase. A mutated plasmid is created and then digested by Dpn I endonuclease and transformed into competent cells for cell nick repair (Agilent, n.d.).

Essential to the pathogenesis and colonization of host tissues by S. typhimurium are virulence factors encoded on different regions of chromosome, known as Salmonella

E. coli ST131 is primarily an extraintestinal pathogenic E. coli (ExPEC) harbouring viru-lence genes required for successful pathogenic invasion of a human or animal host. These virulence genes allow the clone to do the following: to attach; to avoid and/or subvert host defense mechanisms within extraintestinal sites; to scavenge limiting nutri-ents, such as iron, from the host; and to incite a noxious host inflammatory response, cumulatively leading to extraintestinal diseases12.

Each pore complex is composed of approximately 1000 proteins called nucleoporins (18). Each pore is large enough to allow passage of ribosomal subunits, large protein-RNA complexes, and other materials that are necessary for gene expression and chromosomal maintenance. Larger molecules must be actively transported through the pore while small molecules and ions move freely between the nucleus and the cytosol. Therefore in addition to providing a protected compartment for the DNA, the nuclear envelope also regulates gene expression by controlling the movement of proteins, RNA and other regulatory molecules into or out of the nucleus

Restriction endonucleases are proteins that cleave DNA at specific restriction sites; the protein recognizes specific base pairs on the DNA and cuts

In bacteria and in eukaryotes, the continuing presence of the activator is required to keep the gene on. Scientists argue that active genes often associated with acetylated nucleosomes not because of epigenetic reasons but because of other reasons. One reason is that as genes are transcribed, their nucleosomes are removed and then replaced to make way for RNA polymerase which is involved in transcription. They argue that this nucleosome-replacement machinery works to replace one set of nucleosomes with others that are more highly acetylated. Therefore, nucleosome acetylation accompanies but does not cause transcription. (Ptashne, 2013)

It may be possible to correct an abnormality in a tumor suppressor gene such as P53 by inserting a copy of the wild-type gene; in fact, insertion of the wild-type P53 gene into P53-deficient tumor cells has been shown to result in the death of tumor cells (3). This has significant implications, since P53 alterations are the most common genetic abnormalities in human cancers. The over expression of an oncogene such as K-RAS can be blocked at the genetic level by integration of an antisense gene whose transcript binds specifically to the oncogene RNA, disabling its capacity to produce protein. Experiments in vitro and in vivo have demonstrated that when an antisense K-RAS vector is integrated into lung cancer cells that over express K-RAS their tumorigenicity is decreased (4).

Gene expression can be altered by different genetic and epigenetic mechanisms. These mechanisms can alter chromatin structures and the accessibility of DNA, thereby regulating patterns of gene expression (Wajed, S. A., Laird, P. W., & DeMeester, T. R. 2001). In the nucleus of the cell, chromatin is bound to a DNA-histone-protein complex in the form of chromosomes. Within the cell, chromatin modifications are made such as DNA methylation and histone modification in order to alter gene expressions. DNA Methylation involves the addition of a methyl group to the 5’ end of the cytosine ring within CpG dinucleotides. While most CpG dinucleotides are methylated, unmethylated CpGs are clustered together at promoter regions of many genes (Lim, D. H., & Maher, E. R. 2011). These promoter regions are essential for transcription to occur, however, DNA methylation can prevent transcription factors from binding leading to changes in chromatin structures. Another mechanism that can alter gene expression is histone modifications. Histone modification includes changing the composition of histones or the addition of acetyl, methyl or phosphate groups onto the amino acids of histone tails (Book). While histones are important proteins used to organize and wrap large amounts of DNA, when histones within these nucleosomes are modified, the mobility and positioning of DNA are affected resulting in gene promoters to be activated or repressed. In addition to both of

8A) (Coleman-Derr & Zilberman, 2012a; Sura et al., 2017; Zilberman et al., 2008). At highly expressed protein coding genes, H2A.Z is intensely enriched around the nucleosome-depleted region (NDR) at transcriptional start sites (TSSs), particularly at the +1 nucleosome (i.e. the first nucleosome downstream of the TSS) (Coleman-Derr & Zilberman, 2012a; Sura et al., 2017; Zhang et al., 2016; Zilberman et al., 2008) (Figure 8B). The +1 nucleosome can act as a barrier to transcriptional elongation by blocking and stalling RNA Polymerase II (Nock et al., 2012; Weber et al., 2014). H2A.Z incorporation moderates the nucleosomal barriers to RNA Polymerase II progression, likely by facilitating the loss

ZFN’s are chimeric proteins that are bound to “a modular array of Cys2-His2 DNA-binding zinc fingers” that are in turn bound to the FokI [1]. The TALEN’s are Transcription Activator-Like Effector (TALE) proteins that are secreted by bacteria that can control the gene expression of their host cell [1]. These particular bacterial proteins were first observed in plant cells [1]. TALENs assembly is less problematic but harder to synthesize than the ZFNs [1]. They both require formulating new DNA binding proteins for each gene [1].