Efficacy of BTB Complex in Double Holliday Junction Dissolution - Essay Example

A Double Holliday Junction Dissolvasome Comprising BLM, Topoisomerase III alpha, and BLAP75 Bloom syndrome is a genetic disorder that occurs due to the inability to conserve the genomic structure of the body (Cohen, 2004). The mishap in genetics comes because of crossovers of sister chromosomes; the syndrome is brought by autosomal resseciveness of BML gene (Cohen, 2004). Bloom disease predisposes individuals to cancer as it causes pro activity in suppressor cells that alters the division of cells to a hyperactive state (Cox, 2009). This disease is common amongst the Jews; the casualties appear short in stature and are sensitive to sunlight. The study of the journal considered purposed to establish the efficacy of BTB complex in Double Holliday Junction dissolution in prevention of crossovers. Indeed the study shows that dissolution of the DHJ using BTB is possible only with the combination of energy provider, ATP that is hydrolyzed by BLM and the complex mixed with a protein complex, Fanconi ammonia. This article I have to admit that the article embraces all the scientific controls and procedures that were well programmed and followed to ensure that the objective and hypothesis of the study is tracked to produce results that satisfies both validity and reliability. Notwithstanding, the article is short of coherency and unequivocal state of the objectives of the study amongst other oversights. Introduction Bloom disease is one of the rarest diseases that are known today, it is a genetically disease that occurs in a homozygous recessive situation. The disease occurs due to the crossing over of the chromosomes during cell division, i.e. meiosis (Modrich, 2006). During this stage of cell division, fragment of the Deoxyribonucleic Acids (DNA) are realized due to the unwinding nature of the DNA at this stage. These fragments later rejoin randomly leading to alteration of the initial genomic structure and one resultant features is the bloom disease (Modrich, 2006). Specifically, bloom disease due to the changes discussed above occurs due to mutations at the BLM gene (Cohen, 2004). The disease is hereditary and can be passed to the offspring by the parents who are carriers. Bloom disease is known to be responsible for a number of misfortunes in human beings; the disease causes cancers as it makes the regulatory genes in the human body to be hyperactive leading to the uncontrolled proliferation of cells causing cancers (Tropp & Freifelder, 2008). The occurrence of cancers in this case is observed even in young children, this has made the disease to be known for causing old age diseases in children. The individuals with this disease also show a short a stature and may prompt acquisition of other medical conditions like diabetes type II, color change of the skin making the individuals to be vulnerable and sensitive to sunlight, and chronic pulmonary disease (Tropp & Freifelder, 2008). The prevalence of the disease is high amongst the Jews and that for every one hundred Jews, there is an individual with the bloom disease (Modrich, 2006). The article critique has revolved around the bloom disease in the capacity of mitigating the cause and finding the most appropriate item to be used in mitigation efforts. The article identifies the disease and looked at how it occurrence and the factors that accelerate its occurrence. The gene responsible for this disease is identified as BLM. BLM is found in the human genome and is one of the human RecQ genes (Waldman, 2004). RecQ genes are important in the maintenance of genetics in both the prokaryotes and the eukaryotes. In prokaryotes, the RecQ genes are important in ensuring that the structure of the genome is maintained despite the gene recombination that takes place in the bacteria (Waldman, 2004). There are about five RecQ genes in human of which all strive to maintain the originality of the genome in order to arrest erratic gene disruption that may cause genetic disorder of bloom’s caliber. In eukaryotes, RecQ genes are important during replication and normally replication without these genes is paralyzed (Wei, Li, Chen, & Chen, 2007). These genes also ensure that silencing, aging, recombination, and DNA repair are taken care of to avoid gene alteration and mutations. All the RecQ families show consistency in the sense that all shares certain regions that are conserved in proteins; N- terminal i.e. Helicase, middle part i.e. RecQ conserved and the C- terminal referred to as Helicase-and-RNase-D C-terminal (HRDC) domains. (Wei, Li, Chen, & Chen, 2007). In the article, the knowledge that the gene that is responsible for the occurrence of bloom syndrome exhibits higher levels of sister chromatids exchanges during meiosis forms the basis of the article. The ability demonstrated by the gene is a proof that regulation of crossover is not monitored and there is haphazard combination of these genes resulting in to their eventual alteration. In this case, such alteration is focused onto the occurrence of the bloom syndrome. The study in this article thus was intended to establish what is responsible for the regulation of this crossover that mutates the BLM gene resulting in to the genetic disorder, bloom syndrome (Waldman, 2004). The study exploits the fact that BLM is known to cooperate with other compounds like type 1A topoisomerase, Topo III α to initiate control of the haphazard gene regulation- homologous recombination (HR) intermediates that are linked to the Double Holliday Junction (DHJ) (Cohen, 2004). This dissolution activity of the BLM- Topo III α complex is known to hinder the crossover of the sister chromosomes during meiosis stage thus mitigating o the development of cancerous cells. In relation to the syndrome, another compound is also looked into regarding its effect on the regulation of the sister chromosomes crossover, BLAP75 which is a nuclear extract from Hela. BLAP75 following some other studies is known to deplete the levels of BLM and Topo III α in the cells (Modrich, 2006). Interestingly, reduction of these compound was found to increase the frequency of the sister chromosomes crossover consistent to those cells that have insufficient BLM. The BLAp75 together with BLM-Topo III α complex forms a complex called BTB that compounds the dissolution process of the DHJ. The involvement of the BTB in the dissolution of the DHJ forms the core of the study, the chemical properties of the BTB and its role in the DHJ dissolution is considered as the objective of the study in this article. BLAP75 is suspected to have effects on the formation of BLM-Topo III α in dissolution of the DHJ or plays a role in the formation of the complex (Cox, 2009). The formation and identification of a successful dissolution factor is crucial in the control of the uncontrolled cell division that causes bloom syndrome a prerequisite for the development and occurrence of cancer cells. Bloom syndrome is envisaged to increase the prevalence of cancer around the world as it is genetically inherited. Remedies do exist if the condition can be identified amongst the couples early in life. Thus, individuals are encouraged to go for bloom’s syndrome screening (Waldman, 2004). Even though the main objective of this study was to identify the role of the BLAP75 in dissolution process in DHJ, the BLAP75 is a cellular protein and is a component of BTB complex responsible for the dissolution of the DHJ. The study was designed to evaluate BLAP75 and other aspects together with which the intended objective would be reached. In line with this, many tests and processes were run to establish the objectives and consequently many results obtained as per the study. In the first place, the compounds to be involved in the study were to be purified and expressed to avail a pure form of the compounds. The first results obtained were that there is no functional difference between the non–cleaved BLAP75 and cleaved GST, this finding was arrived at following this procedure; BLAP75 cDNA containing carboxyl terminal together with GST (cleavable amino terminal) was constituted and expressed in the promoter T7 in the Escherichia Coli (IPTG- inducible). Cells that are rich in GST-BLAP75 plasmid were treated with IPTG in order to express the protein, upon staining of the bacterial cells with a staining agent coomassie blue, about 110kD of unexpressed protein were identified. Through immunoblast technique, these uninduced proteins were confirmed to be consistent with BLAP75 (Modrich, 2006). The immunoblast technique was done using the anti-GST antibodies. After the expression of the BLAP75 protein, what followed was the purification of the protein. BLAP75 was purified using E. coli sylate precipitated with ammonium sulfate followed by a chromatographic technique in two fractional steps and purification based on affinity. In this case, two compounds were used; glutathione-sepharose and Ni2+ nitrilotriacetate-agarose (Waldman, 2004). This experiment done confirmed that the functional difference does not exist between non-cleaved BLAP75 protein and the cleaved GST proteins (Wei, Li, Chen, & Chen, 2007). After the purification, the protein-protein interaction was investigated within the BTB complex in a consecutive of pulldown procedures. The pulldown experiment was guided by the initial studies that BLM and Topo III α through western analysis reported interactions, this was to be confirmed using another experiment as indicated in this article. The purified BLM and Topo III α were mixed and subjected to immunoprecipitation processes coupled with anti- BML antibodies (Cox, 2009). A controlled experiment was again run where Topo III α alone was taken and run through immunoprecipitation. The result indicated that the linkage between the two proteins, BLM and Topo III α was perfect and this confirmed the cooperation of the two proteins. Having confirmed this, the next was whether BLM interacts with BLAP75. Either GST-BLAP75 or GST was incubated with BLM and the conditions improved by addition of glutathione-sepharose beads for retention of the protein particles that might have been formed during the incubation period (Modrich, 2006). The experiment showed a perfect retention of the BLM protein in the GST- BLAP75 with glutathione-sepharose beads. Unfortunately, this retention was not observed in the GST alone. It was then construed that BLM-BLAP75 interaction specific unlike that of GST-BLAP75 that did not bind to the human WRN protein or the E.coli RecQ (Tropp & Freifelder, 2008). The next confirmation was if there were interactions between Topo III α by running a process-a pull down was performed using GST and Topo III α tagged with (His)6 in one set and another GST-BLAP75 as the second set in the pulldown without being tagged using (His)6 (Waldman, 2004). The purpose of running the two sets in this experiment was informed by the knowledge that both had the same mobility in gel (Wei, Li, Chen, & Chen, 2007). Visualization using immunoblotting technique was used together with anti-histidine antibodies and it was found that there was avidity between BLAP75 and Topo III α. The purpose streptavidin of doing the series of the experiments was occasioned by the fact that the complex BTB was constituted of three compounds; BLAP75, BLM, and Topo III α (Waldman, 2004). The entire component is suspected to trigger dissolution of the DHJ to prevent crossover. The hypothesis that the three compounds works together to bring about the dissolution had to be first proven that there indeed a close link between the two to form a complex that could occasion dissolution of the DHJ (Cox, 2009). The core of the findings was about the complex BTB and more specifically, the role of BLAP75 protein in dissolution of DHJ, initially it had been found that the presence of BML and Topo III α were capable of reducing crossover during homologous regulation (HR) (Modrich, 2006). The realization that BLAP75 could bind to the BLM-Topo III α led to the hypothesis that the linkage of BLAP75 to the already existing complex could have some influence on the dissolution effect of the DHJ. This hypothesis necessitated another series of experiment to establish the influence of the BLAP75 on BLM-Topo III α to form another complex called BTB (Tropp & Freifelder, 2008). A double Holliday junction substrate (DHJ) was constructed through ligation and annealing of two oligonucleotides (32p- labeled B1 and the unlabelled R1) (Wei, Li, Chen, & Chen, 2007). In this experiment it was realized that BLAP75 aggravated the dissolution of the DHJ unlike when BLM-Topo III α where used (Waldman, 2004). BLM-Topo III α complex could only dissolve less than ten percent of the DHJ while with the inclusion of the BLAP75 the dissolution rate increased to more than eighty percent (80%) (Waldman, 2004). The result was a vivid clarity that with the present of BLAST75 the level of dissolution of the DHJ is tremendously compounded hence enhancement of the correction of the crossover. The level of the dissolution even increased with the adjustment of the physiological factors such as salt. On the same breadth, it was noticed that none between the RecQ and the human WRN protein are capable of dissolution of the DHJ in the presence or absence of BLAP75 and Topo III α (Cohen, 2004). The limitation of crossover of sister choromatids had been reported in the earlier studies as successful roles played by the complex of BLM-Topo III α, there was need to show a replica of the same with the new complex of BLAP75, BLM, and Topo III α (BTB) (Waldman, 2004). The concept in the study was that dissolution process in non-crossover would untangle the two oligonucleotides used in this experiment. (32p that was labeled B1 oligonucleotides and the biotynilated oligonucleated labeled as R1). Streptavidin beards added was intended to deplete the Double Holliday Junction, DHJ and not the products that were radiolabeled (Cox, 2009). In this experiment, it became apparent that the radiolabeled oligonucleotides treated with BTB avoided depletion by streptavidin. This was an indication that the compounds were free oligonucleotide of 32P B1 formed because of the failure of the crossover in the DHJ (Wei, Li, Chen, & Chen, 2007). Following the success of the experiment, the configuration of the products that initiated dissolution of the substrate DHJ was analyzed using restriction method of analysis. In this case, the restriction enzymes Hhal and Rsal were used and the radiolabeled oligonucleotides demonstrated susceptibility to Hhal but resistance in the case of Rsal (Cox, 2009). This further informed that BTB initiated anon crossover in the DHJ substrate. The operation of the BTB in the avoidance of crossover required energy (Waldman, 2004). ATP that is the energy currency of a cell provides this energy. In this case, the BLM protein in human hydrolyses it. The study in this article thus construe that the BTB complex under the influence of BLAP75 necessitates Dissolution of DHJ in certain physiological states when BLM-Topo III α complex alone cannot do dissolution effectively (Tropp & Freifelder, 2008). The novel complex, BTB is applauded of maintaining the genomic integrity thus limiting cases of cancers in BS individuals. The critique In as much as the article has endeavored to give the insight of what to most people would be news i.e. the occurrence and circumstances that leads to the rare syndrome, bloom disease, it has also faulted in many areas. This is with regard to the coherency and the ease with which those who are not conversant with the scientific terms would find it. First, in the article, hypothesis is not clearly stated in the initial paragraph. Scientifically, the hypothesis or the objectives should precede every aspect of the study as it gives snapshot information about the intention of the article (Cohen, 2004). Stating the hypothesis or the objectives before the rest of the information on an article provides the readers with the interest to know more about what the study concluded on the objective (Wei, Li, Chen, & Chen, 2007). On the same note hypothesis should be categorically specified as to whether they are null-those that are of no difference or objective hypothesis-those of difference. Stating the two objectives is important in the final finding of the study as it gives an opportunity to the person who is carrying the research to opt for either of the hypothesis depending on the results of the findings (Tropp & Freifelder, 2008). In pursuant of the molecular theory of the central dogma, the information should flow from the DNA to the protein for expression as phenotypes (Wei, Li, Chen, & Chen, 2007). With regard to the article, chromosomal crossovers involves the DNA which can be tested by use of the southern blotting technique- this technique shows successful transfer of the DNA strand and is mostly used in the confirmation of strands generated through Polymerase Chain Reaction, PCR (Waldman, 2004). In the article, the Southern technique is used, but because the compound of interest in the study also constituted proteins e.g. BTB complex, BLM, and BLAP75 there ought to be the western blotting technique used in identification of specific protein molecules. In the purification of these compounds, it is not mentioned the procedure that were used to verify that after the expression the outcome was the intended product. The article also failed to clearly stipulates the variables involved in the study, reasons why they were used and the effect when the optimum range is not met. The variables were also supposed to be categorized into independent-those that can be changed or manipulated and the dependant-those that relies on the independent ones (Waldman, 2004). Independent variables in this article included time for incubation, amount of reagents used and the temperature. The dependant variable remains the purpose for which the experiment was done. Regarding variable, the purpose of choosing them should be stated to avoid speculation of the reason why they were chosen, this is lacking in the article. From the article, the aim of the study done was to establish whether in the dissolution of the Double Holliday Junction (DHJ), the complex BTB has a role to play. The objective then ought to have been clear regarding the association of the complex and the DHJ. From the article, it becomes very difficult to establish the objective as the preliminary information dominates the better part of the article. By reading the article, it is difficult to know whether the interest of the article was to ascertain the formation of the BTB complex from BLM, Topo III α, and BLAP75 or it was to know its role in the dissolution of the DHJ. ` In the article are also misinterpretation of the findings related to blooms syndrome, the article majorly is concerned with the crossovers in meiosis leading to increased development of cancers, one would the construe that the only problem with the bloom disease is transformation of the normal cells to cancerous cells, this is not true. The human body contains several genes that control many traits in human, if at all there is chromosomal crossover then they are most likely to affect a wide range of genes resulting to variation of the phenotypic nature of the alteration (Waldman, 2004). Unless in blooms syndrome during crossover, the genes that are affected are specific then it will be understood to be skewed only to those genes that are responsible for triggering cancer. However, if during crossover there are haphazard linkages, then it is inadequate to purport that in bloom syndrome cancer is the predominant result. The study in the article was based on the external environment of the body, in vitro. It does not show any indication that the system was improvised to mimic that of the body. The body of a living human is responsible for many physiological functions that may alter any experiment that is done in vitro (Waldman, 2004). In respect to this, the article fails to explicitly inform us how the information acquired following the study can be applied in real life to mitigate on the disease. In the experimental bit, it has been shown that the complex BTB can be used to reduce the crossover that is helpful to individuals with bloom syndrome. The article is also written in a manner that conceiving the ideas becomes a problem to those who are not proficient in scientific terms more so those related to biology like the case in this article. The article is starts on a high note ignoring the basics that would enable people to have the in depth understanding of what the article reveals. The core of the article relates so much to the human genetic factor, Deoxyribonucleic Acid (DNA) that harbors almost every trait in a living organism (Cox, 2009). The article then ought to have considered defining what the DNA is, the structure of the DNA and some features of the DNA that relates to the study. Amongst the salient features of the DNA that were not suppose to miss the article include, DNA replication, transcription to RNA (Ribonucleic Acid) and translation i.e. the flow of information from the DNA to protein that can be observed as phenotypes not genotypes (Wei, Li, Chen, & Chen, 2007). This preliminary information would have formed the basis for understanding issues with the bloom syndrome. Regarding the figures in the diagram, it is difficult to conceive and interpret the results that are shown, figure A shows the purification of BLAP75 but one cannot comment on the results as even the vertical and the horizontal axis of the figure are not labeled. Gauging that the expression processes is successful or not is also difficult as the bench marks are not given for comparison purposes. This is similar to figures B, C and D which also shows the purification of BLM, Topo III alpha. A similar ambiguity is observed in the set of diagram two that shows testing of crossover after the dissolution by BTB complex using streptavidin magnet beards. The flow of the diagrams is poor and depicts inconsistency and difficult to follow, results cannot be interpreted by just looking at the diagrams, one cannot deduce own results from just viewing the diagrams and figures. The figures ought to have shown consistency with the literature part but this is conspicuously missing. Despite the faults sited regarding this article, it also serves as a source of information. It brings to the limelight the rare disease, bloom syndrome that many individuals may have little information on. The article also discusses the circumstances under which the disease occurs, this dispels any wrong notion that some people might be nursing about the disease. The article clearly indicates that the disease is genetically inherited in an autosomal recessive situation, this informs that it is not a contagious disease. The knowledge about how a disease spreads is important as it helps avoids unnecessary stigma form the public. This article has also strived to bring a new concept; according to the article, dissolution was known to be done by BLM-Topo III alpha complex but the study has proven that under some physiological states, inclusion of the protein BLAP75 can help accelerate the dissolution of the DHJ to prevent crossovers in cases of bloom syndrome. The article has brought a new concept that will help in resolving cases of bloom syndrome. In the article are also several enzymes, reagents and terms that are introduced and needed to be expounded on for the information to reach the public without distortion of the intended meaning. The diagrams used in reinforcing the content in the article were haphazardly placed and lacked the coherency in relaying the correct flow of information to the readers. The study considered in this paper triggers quest in carrying another researches. These researches can aimed at identifying the predominant type of cancer elicited by bloom syndrome and whether they are mostly malignant or benign Reference List Cohen, P. E. (2004). Repair Proteins in Meiosis. Madison: Karger. Print. Cox, L. (2009). Molecular Themes in DNA Replication. Cambridge: Royal Society of Chemistry. Print. Modrich, P. (2006). DNA Repair: Methods in Enzymology. London: Academic Press. Print. Tropp, B. E., & Freifelder, D. (2008). Molecular Biology: Genes to Proteins. London: Jones & Bartlett Learning. Print. Waldman, A. S. (2004). Genetic Recombination: Reviews and Protocols. New Jersey: Humana Press. Print. Wei, Q., Li, L., Chen, D. J., & Chen, D. (2007). DNA Repair, Genetic Instability, and Cancer. London: World Scientific. Print. Read More