Role of perinuclear chromosome tethering in maintenance of genome stability
Introduction
This part of the paper is revealed in the first page of the study and it is designed to provide the readers with a more in depth discussion of the information that is revealed in the abstract section of the paper. Furthermore, the introduction is supposed to provide the readers with a firm discussion on why the topic is of great importance in the field of Biology. In other words, the introduction section of the paper is supposed to offer a well-justified discussion of the problem definition, the objectives of the study, and the significance of the paper to the scientific community and to the majority of the members of the study. By analysis, the authors are able to provide a more detailed discussion of the problem definition when they mentioned about the features of the eukaryotic ribosomal DNA (rDNA) tandem repetition for the formulation of the foundation of the nucleolus, one of the organelles of the cell that produces the ribosome (Mekhail et al., 2008). A specific example was also given with respect to the features of the yeast, Saccharomyces cerevisiae, where it was mentioned that the said fungus has about 100 200 rDNA units that are uniquely arranged on the chromosome XII (Nomura, 2010). The authors were also able to give an effective discussion of the intertwined ideas of the importance of certain cell processes and cell parts, such as the intergenic spaces1 and 2 (IGS), nucleolar protein complexes RENT (regulator of nucleolar silencing and telophase exit), and RNA polymerase II (Pol II). It was also said that the recruitment of Cohibin (Lrs4 and Csm with their monopolin proteins) along with the presence of the other cellular aggregates aid in the suppression of unequal recombinants at the repeat sequences of the genome (Mekhail et al., 2008). Such kind of suppression is needed in the preservation of the integrity and stability of the genome (Mekhail et al., 2008). Further laboratory investigations of Cohibin reveal that this substance tend to form an association with the inner membrane proteins of the cell to fulfill a function that is yet to be fully understood but is believed to be connected in the maintenance of genome stability. With this in mind, the objective of this paper is to describe the role of inner membrane proteins (INM) and that of the ribosomal DNA (rDNA) in the fulfillment of perinuclear chromosome tethering and the relevance of this event in the preservation of the integrity of the genome. The significance of the paper, on the other hand, was not discussed because the authors gave no explanation of the potential usage and promising effects of the incorporation of the conditions of the study to that of the general public.
Literature Review
In order to achieve an appropriate understanding of the aforesaid article or study it would be necessary to conduct a proper literature review. Thus, a search for relevant articles on the topic has been achieved through a search conducted on EBSCO. Specifically, among the keywords used for such purposes is genome stability. In total, three articles were acquired through the use of such keywords. The first article focuses on the function of base excision repair on the process of ensuring genome stability. To further expound, Baute and Depickers (2008) study establishes and delineates the necessity of having a means of repairing DNA damage based upon the base excision repair mechanism furthermore, the application of DNA glycosylases for such purposes have been explained and discussed as well. In general though, it would be most important for the purpose of this study to highlight the roles of base excision repair in maintaining genome stability so as to possible bring forth insights into the potential uses and similarities with perinuclear chromosome tethering.
Basically, the base excision repair mechanism has been found to be important in the following aspects first, in terms of preventing the occurrence of considerable genetic damage leading to cancer among humans, limiting the possibility of having significant oxidative damage throughout the brain, and lessening the extent of oxidative damage to plants such findings have been achieved through a review of the implications of genetic damage upon the gene which encodes for the presence of excision repair (Baute Depicker, 2008). In this sense, the means through which such roles have been determined is quite logical and appropriate. Expectedly though, despite the extent of information presented in the study, it is still undeniable that certain shortcomings are present. In particular, given the complexity of the process of repair and the lack of knowledge regarding the specific processes which occur among the proteins associated with the excision repair mechanism result in a limited capability to explain the process of repair in elaborate detail (Baute Depicker, 2008).
The second article found for review establishes further information regarding the process through which proteins initiate and facilitate repair. Specifically, Budd and colleagues (2005) attempted to determine the specific functions of each protein associated with the maintenance of genome stability. Instead of elucidating the specific action of the aforementioned proteins, the study instead resulted in further confirmation of the complexity of such. To expound, it has been found out that specific roles are not necessarily distributed among the proteins associated with the repair process instead, a multitude of potential functions, emphasizing flexibility in purpose, have been concluded for the interaction of each protein identified (Budd et al., 2005). Interestingly though, a unifying feature has been determined as well throughout the course of the study. The pathways linked to the repair, as well as Okazaki fragment facilitation and chromatin dynamics, have generally been identified to specifically need Dna2p (Budd et al., 2005). Thus, from the points discussed, it becomes apparent that maintaining genomic stability is indeed a considerable complex process which warrants further analysis.
The third article pertains to another process which supposedly facilitates genome stability. In particular, from the findings of Bakhoum and colleagues (2008) it is pointed out that throughout the process of mitosis, the capability to effectively and properly allow temporal control over the attachment of microtubules to chromosomes is vital to ensuring proper maintenance (Bakhoum et al., 2008). Similar to the previous articles discussed, the necessary tests and procedures to confirm such and to serve as evidences have been accomplished. In relation to this, induced alterations to the kinetochore-microtubule dynamics, regardless of the extent, have been known to result in considerable repercussions for genomic stability (Bakhoum et al., 2008). Therefore, the three articles found throughout the literature search for background information regarding the topic of genomic stability has highlighted the undeniable presence of a diverse means of ensuring the aforesaid stability, further emphasizing the essential need for genomic stability among organisms as significant repercussions may arise when it is compromised.
Methods
In order to fulfill the goals of this paper, the researchers executed a number of preparations to properly complete the methods of this study and some of the most essential steps employed are composed of the following strains and materials preparation, protein purification, mass spectrometry, fluorescent in-situ hybridization, imaging, three-dimensional reconstruction, employment of standard and modified ChIP, preparation of USCE assays, Southern blotting and CHEF procedure, whole cell preparation, and - factor arrest (Mekhail et al., 2008).
For the first step, strains that were used in this study came from the Jackson laboratories, Molecular probes, and Invitrogen (Mekhail et al., 2008). These institutions provided the researchers with strains that were free of endogenous genes and were proven to contain specific antibodies that are essential for the completion of specific steps of the experiment (Mekhail et al., 2008). Purification of the protein components was employed by performing standard assays, and by subjecting 10 to 50 of the cells to electrophoresissilver staining whole the rest was precipitated using tricholoroacetic acid (Mekhail et al., 2008). Next, mass spectrometry, was performed by subjecting the cells into trypsin digested mixtures to detect for false positive rates, and by utilizing the SEQUEST program for the detection of the methionine oxidation (). FISH, on the other hand, was accomplished by using labeled cells from Roche and Invitrogen and by exposing the cells to microscopic imaging (Mekhail et al., 2008). Imaging, which is one of the most essential step of the process, was done by using an Axiovert 200 microscope along with the application of several computer programs such as the Photoshop, Metamorph, and Office program (Mekhail et al., 2008). After imaging, reconstruction is an important part of the experiment that made use of the Zeiss LSM510 microscope which produced about four images that were needed for the reconstruction of the images (Mekhail et al., 2008). Next, protein cross-linkers were used for the ChIP imaging of the substances employed in the experiment which made use of the concepts of cell centrifugation, enzyme purification, and imaging (Mekhail et al., 2008). After this, assays for USCE were done by growing the cells in thick plates, incubating the cells for 5 days, and calculating for rates that will show the ratio of half-sectored to total colonies (Mekhail et al., 2008). CHEF and Sourthern blotting were also employed and it made use of overnight saturated cultures that were treated with EDTATris, Tris-HCl and other substances that were used to maintain the stability of the mixture (Mekhail et al., 2008). Next, whole cell preparation, was accomplished by bead-beating the samples, constant washing and rinsing, and by using chemicals such as Mercaptoethanol, and other elements to produce the treated protein aggregates (Mekhail et al., 2008). Lastly, factor arrest was employed by incubating cells that were grown for three weeks and by freezing these cells to -15 cells for proper liquid nitrogen effects (Mekhail et al., 2008). In general, the application of these steps justified the empirical nature of the experiment because the researchers employed an approach that made use of materials that were quantified and well calibrated. Furthermore, the materials utilized for the study were all of good quality and even the strains came from suppliers and sources that are of reputable nature. Hence, it can be said that the experimental biases regarding the controversies on the nature and dimensions of the materials used are clearly surpassed by this study as supported by the idea that all of the materials used were of great quality and appropriate quantity in relation to the demands of the experiment.
Investigations of the Study
After the discussion of the manner by which the materials in the study were utilized, it will then be necessary to focus on the specific experiments that were conducted for the completion of this study. Below is the discussion of the specific tests that were made in order to complete empirical phase of this paper.
Unequal sister-chromatid exchange (USCE)
The investigation of the unequal sister chromatid exchange was employed because the researchers wanted to investigate the association between the chromosome linkage inner membrane proteins (CLIP) and intergenic spacers (IGS1) in relation to the properties of the regulator of nuclear silencing and telophase exit (RENT) and Cohibin for the silencing of transcription and unequal rDNA recombination (Mekhail et al., 2008). To accomplish this goal, the researchers measured the loss of the marker gene, ADE2, from the repeats of rDNA (Mekhail et al., 2008). Figure 1 a and b showcases the cases by which the quantity of USCE can be quantified where it is stated in Fig. 1 A that the USCE rate has increased as a function of the increased deletion in the Sir2, Lrs4, and Csm1 while Fig. 1 B shows that the USCE count increase after the deletion of Heh1 or Nur1 happens (Mekhail et al., 2008).
Fig. 1 A B. Maker loss of ADE2 as depicted by wild-type (a) and representative colonies (Mekhail et al., 2008).
Other data suggest that the absence or presence of other genes, heh1 nur1, imply the participation of USCE in the activity of inner membrane proteins (Mekhail et al., 2008). Moreover, the involvement if Sir2 makes it possible for the CLIPCohibin complex to exist (Mekhail et al., 2008). Chromosome XII, on the other hand, was investigated using the contour-clamped homogenous electric field or CHEF, a process that requires the application of electric impulses to measure the base pairs of the cellular substances (Mekhail et al., 2008). Subsequent manipulations of these genetic segments affirm the role of the perinuclear network, including unequal sister-chromatid exchange, in the preservation of the integrity of the rDNA repeat stability (Mekhail et al., 2008).
By analysis, it can be said that this section of the paper is important and was ablt to provide the readers with a detailed explanation of the relationship of perinuclear protein network to genome stability. Although it must be emphasized that this section of the paper is filled with technical terms that will make it very hard for a person not adept in cellular biology to understand the meaning of the discussions.
Silencing of Pol-II transcribed mURA3 reporter gene
This part of the methodology was done to investigate the participation of the IGS1 and IGS2 in the silencing of Pol II-transcribed mURA3 reporter gene and it was done through the assessment of the growth of the cells in uracil deficient or 5-fluro-orotic acid-supplemented medium (Mekhail et al., 2008). It was discovered that silencing exists for Pol-II as a function of the deletion of Sir2, Lrs4 or Csm1 (Mekhail et al., 2008). Consequently, it has been proven in another study that RNA Pol-II play an important role in the interference pattern or silencing capabilities of cells as divctated by its subunit and other cellular aggregates (Djupedal et al., 2005). Chromatin silencing is actually the effect of the participation of RNA Pol-II in the interference pattern (Djupedal et al., 2005) and this is also in accordance to the findings that were formulated in this study except for the fact that the researchers believe that the disruption of IGS silencing is not sufficient for repeat size regulation (Mekhail et al., 2008, pg. 668).
Role of the tethering of the rDNA repeats to INM
As the name implies, this part of the experiment contains one of the most important section of the part since it will formulate the connection of perinuclear localization to the rDNA-associated activities. To address this issue, the researchers made use of Cohibin as the tethering mechanism to bind rDNA repeats to the inner nuclear membrane protein (Mekhail et al., 2008). Protein segments were tagged for proper observance during immunofluorescence and it was discovered that the a specific segment, Net1, recruits rDNA in the nucleolus during the entire cell cycle but the same event was absent when the media is supplied with additional Sir2 at the chromatin immunoprecipitation (ChIP) experiments (Mekhail et al., 2008). Additional studies were completed using the Net1 and green fluorescent (GFP) to demonstrate the coverage of such kind of localization on the cells being observed (Mekhail et al., 2008). Three zones were defined to properly quantify the limit of localization and these are zone II, II and III where zone I describes the least coverage and zone III means and wide localization (Mekhail et al., 2008). Figure 2 shows the three dimensional structure of the localization of rDNA with respect to the availability of Cohibin. It was proven in the literature that the utilization of green fluorescent pigment was able to aid researchers and scientists in the identification and description of a number of protein structures (Magliery Reagan, 2006). Moreover, it was proven through the years that GFP itself can be dissected to allow maximal analysis of certain unknown protein products (Magliery Reagan, 2006). With this in mind, the utilization of the GFP in this study further affirms the reliability and validity of the data and illustrations acquired.
Fig. 2. The three dimensional structure of the localization of rDNA with respect to the availability of Cohibin (Mekhail et al., 2008).
Effect of perinuclear network to subnuclear separation
Figure 3 depicts the structure of the DNA. It can be seen from this illustration that the rDNA is supposed to be intact with the entire structure but it recent reports show that the said structure is separated from the bulk or form the majority of the DNA components. The authors of this study speculate that the said event might be attributed to the activity of the perinuclear network where it is assumed that the said network might have produced a certain type of force that might have caused the detachment of the rDNA from the majority of the other nuclear components (Mekhail et al., 2008). To further investigate this issue, the researchers employed a fluorescence in situ hybridization (FISH) that will reveal rDNA activities and that of 4,6-diamino-2-phenylindole dihydrochloride (DAPI) that tracks for the behavior of the entire DNA aggregates (Mekhail et al., 2008). It was revealed from FISH that the force exerted by the perinuclear network might have caused the severe loss of inter-nuclear attraction between the holistic DNA structure and chromosome XII thereby suggesting subsequent detachment of the rDNA from the aggregate (Mekhail et al., 2008). Hence, this experiment provides substantial evidence in the role of perinuclear network in the separation of rDNA.
Fig.3. Nuclear organization of the DNA aggregates showing detachment of the rDBA as a function of perinuclear network (Mekhail et al., 2008).
rDNA repeats, the tetO array and the TetO-binding site (3 of 5)
It was revealed in the earlier part of the paper that pernuclear network affects the attachment of rDNA to the nuclear organization (Mekhail et al., 2008). In this part of the paper, the researchers investigated the participation of the perinuclear network in the suppression of recombination in the absence of Cohibin, a substance that is proven to be essential in the fulfillment of rDNA silencing (Mekhail et al., 2008). To investigate this issue, the researchers made use of strains wherein rDNA is linked to Heh1 via the connection in Sir2 (Mekhail et al., 2008). Results revealed that the rDNA silencing property is not hampered by the activity of the perinuclear network (Mekhail et al., 2008). It was even emphasized that the presence of the perinuclear network permits repeat stability that is independent of the silencing feature of the rDNA (Mekhail et al., 2008). Hence, it can be said that the perinuclear network and its tethering activity is not detrimental to the cell but actually renders positive effects because it is able to facilitate genome stability in the absence of Cohibin.
Findings, Conclusion Recommendations
Results of this study suggest that the inner membrane protein activity on the perinuclear chromosome is a more active way of ensuring genome stability as compared to the Sir-dependent siencing (Mekhail et al., 2008). It was also revealed that INM-mediated chromosome tethering supports the production of genome stability as a function of the prevention of the occurrence of unwanted crossovers (Mekhail et al., 2008). Consequently, this article emphasizes the importance of perinuclear networks in the preservation of the integrity of the genome through time. It can also be observed that the conclusions made by the researchers are based on empirically executed research method. The findings are actually based on a carefully prepared research experiment. Improvements of the study can be done through the utilization of other DNA aggregates for comparison purposes or the repetition of the methodology if this study to observe consistency.
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