History Of The Existence Of Dna

Published Date: 02 Nov 2017

Introduction

The cellular processes in humans are regulated by a family of transcription factors known as Sp which are most preferentially expressed in human tissues. Sp1 was shown to activate the transcription of the early gene promoters of simian virus 40. Thus, it is regarded as the first member of the family. The Sp family comprises of eight proteins presently, designated as Sp1-8. The expression of genes involved in the apoptosis, cell cycle regulation and lipogenesis is regulated through Sp1 (Waby et al. 2010). Thus, Sp1 has a crucial role in embryogenesis. Furthermore, it has been indicated that Sp1 shows enhanced expression in a few cancers which reflect that’s tat Sp1 is switched on during cancer cell differentiation (Waby et al. 2008). An upregulation of genes at the end of Bak and p21 promoters is achieved when Sp1 is acetylated; this acetylation prevents Sp1 binding to the promoters. Sp1 and Sp3 are constitutively expressed in all tissues whereas the expression of Sp4 is restricted to developing testis and brain. In some cell lines, Sp2 expression has been seen and Sp7 is the bone specific transcription factor. The expression behavior of other transcription factors such s Sp5, Sp6 ad Sp 8 are not obvious (Waby et al. 2008). Sp1-4 have an identical domain configuration consisting of an N terminal activation domain and a C-terminal DNA binding domain, thus they are closely linked to each other (see Figure 1) (Suske 1999).

The structural characteristics of Sp-family members Sp1, Sp2, Sp3 and Sp4 are shown in figure 1. On the right side, the length of each protein is shown. A, B, C and D are the regions involved in the transcription mechanism of Sp1. The activation (AD) and inhibitory domain have been presented (Suske 1999).

The p21 protein causes obstruction of growth through impeding the activity of either proliferating cell nuclear antigen (PCNA) or cyclin-dependent kinases (CDK) which play a role in cell growth, differentiation and cell death (Gartel & Tyner, 1999). Completion of the cell cycle is essentially required for terminal differentiation and transcription of p21 gene is increased by treatment of myeloid cells along with differentiation inducers like okadaic acid or RA and PMA (Casini&Pelicci, 1999; Harper et al., 1993). There are some living systems where p21 impedes cell death (Gorospe et al., 1997), while in some others it promotes cell death (Gartel& Tyner, 1999). Physical or chemical DNA damaging agents like carcinogen and UV radiation activate transcription of p21 gene which is dependent on tumor suppressor p53 (Dulic et al., 1994; El-Deiry et al., 1994).

It is a well-established fact that the binding of transcription factors to the regulatory regions of genes leads to transcription regulation of gene expression. Thus, we can say that the defined gene expression program of the cell is under the control of the wide range of transcription factors residing within the nucleolus of the cell. Whether the factors are present or not depends upon the kind of cell being examined and the stimulus to which the cell was exposed. The expression of genes of interest largely relies upon the identification of the transcription factors which will facilitate a thorough understanding of the underlying molecular mechanisms (Smith and Delbary-Gossart, n.d.).

The interactions among the nucleic acids can be detected through a highly sensitive technique known as the Electrophoretic Mobility Shift Assay (EMSA). The assay is utilized for fulfilling qualitative needs like for the detection of proteins involved in DNA replication, repair and recombination mechanisms (Henderson, 2006; Hellman and Fried, 2007).

Nevertheless, under appropriate conditions, the technique may be employed in the investigation of quantitative characteristics such as affinities, kinetics and stoichiometry (Hellman and Fried, 2007). Moreover, the EMSA can also be employed in studying the assembly of the transcriptional complex (Travers and Buckle, 2000).

The principle of the EMSA is that a protein-DNA complex runs over an agarose gel or polyacrylamide under the electric field (Figure 2) which is actually an electrophoretic separation. Furthermore, the technique is dependent upon the molecular weight of the proteins incubated with a radio-labelled DNA probe or sequence so that complex binding could be achieved. In the electrophoretic step, the transfer of the DNA protein complexes is directly proportional to their molecular sizes. Moreover, the technique distinguishes the protein DNA complex from the unbound DNA. The protein complex in the gel moves as a single band as when the protein is bound to DNA, its migration speed is decreased (Becker, 2001).

The double stranded DNA probes in the EMSA are detected through a streptavidin–Horse Radish Peroxidase (HRP) conjugate. The DNA probes are labelled with biotin or radiolabelled. Streptavidin has a strong affinity for biotin, thus HRP reacts with a strong chemiluminiscent substrate to emit light.

Schematic overview of an idealized gel shift assay

Figure 2. The EMSA principle (Thermo Scientific, 2012).

The aim is to visualize proteins that bind to DNA and also visualize the change in signal in respective well after the addition of oligo. The tenet under which this is that protein bound DNA is slower in migration across polyacrylamide gel in comparison to unbound DNA.

Material and Method

Materials

Component was used for this experiment , 10 x binding buffer 1 x, 1μg/μL Poly dI.dC 50ng/μl;100mM MgCl2 5mM ;1% NP-40 0.05%;Molecular biology grade water;50% glycerol 2.5%;Unlabelled EBNA DNA 4pmol;EBNA extract 1 unit; Biotin-EBNA control DNA 20fmol;Unlabelled Sp Oligo 6pmol;TEST sample 10μg;Biotin Sp Oligo 10fmol ;Sp1 Antibody 0.1 μg/ μl. In addition to, TBE solution; electrophoresis unit; nylon membrane; bromophenol blue; UV machine ; 40 ml blocking buffer and wash buffer , 30ml substrate equilibrium buffer , 3 plastic trays .

Methods

First of all a master mix was made for the first four above reagents .After that make a 12x mix and then add 5μl to each labelled tube from 1 to 10. The other remaining components are followed according to the table offered by practical lab . After that, the tubes are incubated for 20 minutes. Prior running the gel 1 liter of 0.5x TBE have been made and placed 5% acrylamide 0.5x TBE gel and the blank plate on the electrophoresis unit and clamp it in place. This solution had then filled the inner chamber with 0.5x TBE and run the gel at 100volte for 20 minutes. The second part of the protocol was an electrophorus binding reaction, following switch of power and change the TBE, 5μl of 5x loading buffer to each binding reaction, hence 20 μl loaded of each sample onto the gel .The gel again runs at 100v however for 30 minutes. The Third part of the technique was an electrophoretic transfer of a binding reaction to nylon membranes .This part included the soak of nylon membrane in 0.5x TBE for at least 10 minutes then transferred at 100v for half hour after this period the membrane placed in bromophenol blue for 1 minutes. The last part was Crosslink transferred DNA to membrane at 120mj/cm2 (45-60 second exposure, and incubate the membrane dry at temperature overnight.

The next day practical lab was intended to detect Biotin-labelled DNA by chemiluminescence, this was done via series of washing buffer (4 x) and blocking buffer .The membrane was blocked in 20ml of blocking buffer for 15 min with shaking .Then the conjugated /blocking buffer was prepared (66.7μl Streptavdin-HRP conjugated to blocking solution).Dispose the blocking buffer and incubate the membrane with the conjugate /Blocking solution for 15 minute with shaking .preparation of 1x wash solution by diluting 40ml of 4x wash buffer to 120 ml of ultrapure water .Transfer membrane to a new container and rinse it briefly with 20mL of 1X wash solution .Wash membrane four times for 5 minutes each in 20mL of 1X wash solution with gentle shaking .Transfer to a new container, add 30ml of substrate equilibration buffer and incubate for 5 minutes with shaking .Prepare Substrate Working Solution by mixing 6ml Luminol/Enhancer Solution with 6ml Stable peroxide solution .Poor the Substrate Working Solution into the lid of the square dish and float membrane.

Result

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Figure 3: shows the expected result Figure 4: shows different protein band for practical class

Discussion

Transcription factor Sp1 selectively activates the mRNA synthesis from genes containing functional recognition sites. It is present in mammalian cells where it binds to the GC promoter elements to activate transcription. The cDNA encoding 696 C-terminal amino acid residues of human Sp1 was isolated. The truncated fragments of Sp1 were expressed in E.coli. Thus, the DNA binding activity to the C-terminal 168 amino acid residues was localized. This region of Sp1 contains three continual Zn (II) finger motifs which interact with DNA, being metalloprotein structures. It was determined that Zn (II) is required by Sp1 for sequence specific DNA binding. Thus, it is probable that Sp1 DNA interaction takes place through the Zn (II) fingers.

A bacterial colony assay was devised for the detection of Sp1 binding to DNA to aid the identification of the mutant variants of Sp1 that had a defective DNA binding property. Current literature provides substantial evidence indicating that the Sp-family proteins undergo several types of post translational modifications like glycosylation, phosphorylation, sumolation and acetylation. Since most of the research has been conducted on Sp1 and Sp3, most of the information for post translational modifications is linked to these proteins. A huge level of homology among members of the Sp - family, especially Sp 1-4, indicates that the effect of post translational modifications in regulating the transcription of a Sp protein can also affect other members of the Sp family. It seems that phosphorylation plays a crucial role in different biological processes through acting as a molecular ‘on-off’ switch. Significant amount of evidence is present verifying the involvement of phosphorylation in regulating transcription factors. During 1990, it was reported that SV40 infection stimulated phosphorylation of Sp2 which suggested for the first time that Sp family transcription factors were phosphorylated (Jackson et al., 1990). Further exploration of the role of phosphorylation in Sp-family transcription factors attracted researchers since that time. Most of the available literature indicates that dephosphorylation of Sp 1 leads to a reduction in DNA binding and decreased transcriptional activation. But numerous researches suggest this for Sp 1 phosphorylation only but are unable to determine the residues which undergo dephosphorylation (Ye et al, 2002). In addition to this, it has been shown that dephosphorylated Sp 1 has an elevated binding affinity for the inducible AAAT promoter region (Zhu et al, 2000). Over expression of Sp protein serves to be a negative prognostic factor for survival in most cancer patients, and it is not astounding that these transcription factors have contributed in the proliferative and metastatic tumor phenotype. Strategies aimed at inhibition of Sp-dependent pathways have taken into consideration several approaches including drugs which inactivate DNA motifs rich in GC, chemicals which regulate expression of Sp protein, and oligonucleotides and peptide nucleic acid-DNA chimeras which interact particularly with the Sp 1 binding motif (decoys). Mithramycin binds to DNA motif rich in GC and a number of researches indicate that when cells are treated with these compounds, both binding of Sp 1 with DNA and Sp 1-dependent gene expression get reduced (Arinze et al, 2003). Hedamycin, an antitumor drug, binds to the G-rich region of DNA and impedes existing transcription by interacting with a proximal GC-rich Sp1 binding site (_115 to _95) in the promoter region (Wu et al, 2005). In order to inhibit Sp-1 dependent gene expression, double stranded oligodeoxynucleotides with a consensus GC-rich Sp1 binding site (Sp 1 decoys) have been synthesized (Chae et al, 2004).

The existence of DNA binding proteins in vivo could be determined through electrophoretic mobility shift assay. Thus it is an efficient powerful and robust technique. The protocol of the assay is as follows: The cell or nuclear list is made which contains a recognized DNA binding protein. A DNA probe consisting of an associated nucleotide binding sequence of the protein of interest is mixed with the aliquot of the lysate. The probe is labelled for analytical reasons and the mixture is then electrophoresed through polyacrylamide gel. If the migration of DNA probe is retarded that will be an indication of specific DNA protein adduct being formed.

Here, the principle that applies is EMSA is simple and quick assay. It is worth noting that, for visualization of the gel shifts in the assay, it calls for various factors that impact upon the capability of various transcription factors (TF) to attach to their cognate sequences along the DNA. The conditions of gel electrophoresis and the coupled reaction involved in the binding are crucial in examination of every probe/ studied factor. The fundamental factors that must be established empirically in binding reactions are: concentration of divalent and monovalent ions; the quantity of nuclear extract added; pH; concentration of glycerol and concentration and type of non-specific competitor DNA (Garner and Revzin, 1981). The quantity of nuclear extract included in the reaction is worth to be taken into account in the process of examination of these parameters since the nuclear extract offers some bits of buffer. One of the commonest of the binding buffer that calls for titration is the concentration of Potassium Chloride. Usually its concentration ranges from 50-150 mM. Another fundamental aspect to take into consideration here is non-specific competitor DNA. The majority of the TFs has high affinities for their cognate DNA that is many times greater in comparison to that of DNA so to say. For a certain sample of nucleus extract, it is advisable to determine the amount of non-specific DNA added through titration. It is worth of note that both extremes in its concentration should be avoided as too low concentrations translates to no binding while too high translates to all probes attaching to non-specific DNA (Dagnam et al., 1983). It is worth that the kind of competitor DNA used is also crucial in this instance. Generally, simplistic copolymers e.g. poly (dI-dC) gives out the best outcome. Employing more complicated types of DNA for instance DNA from sonicated sperms from salmon risks giving the binding sites for the factors that are used in the process. On the other hand, factors in the non-denaturing gel stage which may be changed are: the % of acrylamide used in gel preparation which normally range from 4-6%, the composition of buffer and acryl amide to bis ratio. In most cases, Tris-borate (TBE) or Tris-Acetate (TAE) buffer at 0.25X to 0.5X are used. In other instances magnesium ions and or glycerol are included to advance the resolution of DNA/ Protein complex. Another factor to consider is also the preference of the particular DNA probe. DNA fragments of DNA of size 20 BP to 200 base pairs typically use in these assays (Garner and Revsin, 1981). Here, it is also good to note that the increases in length of DNA used are likely to culminate to many interactions between the DNA / protein in the process. Also, longer DNA probes makes telling a part of un-reacted probes from shifting complexes more difficult. The length of the Sp1 containing DNA fragment can be measured via inclusion of standard lines (with DNA segments of predetermined size). For further prove of peculiarity aspect of DNA-protein interactions or the specificity to certain sequence, researchers in this field carry out a competitive assay. These assays involve radiolabelled DNA fragments and also identical DNA fragments which are not radiolabelled. Here, fragments of identical non-radiolabelled DNA is aimed at replacing its radiolabelled counterpart as a transcription factor binding counterpart, in the process removes the detectable shift in band in the autoradiograph which only can detect the radiolabelled DNA. Reversing of the gel shift by non-radiolabelled DNA of predetermined sequence reiterates the importance of the very sequence in the binding of transcription factor. With respect to the results got from EMSA in this assay( figure 4) , in the lane one that contained Biotin-EBNA had no observable shift, this can be accounted for by the fact that there was no addition of nuclear extract in the reaction and hence the reaction has no protein to bind to DNA and result in a shift. In the case of the 2nd and the 3rd lane that contained control DNA, Biotin-EBNA and EBNA extract, there was enough protein extract aimed to bring about attachment to Biotin-EBNA DNA that resulted in a detectable shift in comparison to the position of the band observable in the reaction. In the case of lane 4, there was no observable change which can be attributed to excessive cold oligo DNA that brought about the competition for binding sites for the target protein available in the nuclear extract or could be error occurs in sample pipetting . It is also notable that the signal shift was controlled and distinguishable from the one in the second lane hence the proving the shift attributed to the interactions between DNA-protein.

On the 5th and 7th lanes, there was observation of great shift that was eminent from DNA binding to oligo plus nucleus extract. The oligo p21 promoter which had Sp1, 6 sites that recognize CC/GT boxes that enhanced further interactions with DNA (Safe and Abdelrahim., 2005). Also, Nuclear extract has got various nuclear proteins which enhances stable binding of DNA causing a stronger shift than the one produced in the 2nd lane. Notable also, there was no shift observable in the 6th lane, the sixth lane contained NE, oligo extract and cold oligo manifesting that the shift signal was hindered by the competition from the extremity in concentration of unlabeled short chain DNA hence no shift in production a case that applied also in lane 9.

Protein-DNA binding can also be stabilized by antibody (Ab) by strengthening the protein in binding-competent conformation. The result here is the super shifting of the resultant band a phenomenon manifest in 8th lane characterized with more abundance than initial band present in the case devoid of Ab. Abs are very important in unveiling the protein identities within a complex in EMSA. Notable here is that the use of Ab should be cautious since most monoclonal and polyclonal Abs are impure. The contaminants that accompany the impure Abs can lead to inhibition of the process of complex formation at the same magnitude as that of Ab-Ag interaction. Therefore, it is advisable to include designing controls in cases where Abs used are likely to result to inhibition rather than formation of super shifts of a complex. The most efficient control typical for polyclonal Abs are polyclonal preparations directed against a protein that is unrelated to it (foreign protein) or pre-immune serum. It is as important in testing the effects of Abs preparation on the resultant EMSA complex with a foreign probe attached to a protein that should not be detected by the Ab. The effects of the specificity of Ab can be greatly advanced via purification of Ab with Ag-Affinity chromatography or protein G-sepharose or protein A-sepharose chromatography (Harlow and Lane, 1999).

Conclusion

This experiment went successfully in all most reactions except in lane 4, for the reason discussed previously. The preliminary description of Sp-family proteins and other proteins which bind to GC/GT box provides significant information related to the possible roles played by these proteins. Notably, there are issues pertaining specific family members of SP3 and SP1. Differential expressions and the performance of the binding sites of these proteins don’t seem to instill specificity in the two of the proteins. Activation of any given promoter requires binding of multiple TFs which attach jointly to their respective sites or either act through other mechanisms. Transcription Factors (TFs) are now viewed as a rich target in establishment of drugs for curing cancer or stopping cancerous activity in a tissue (Darnell et al, 2002). The intricacy of regulation of genes that are SP-dependent in cancer has fundamentally been reported in Sp1 and to a small degree on Sp3.