Histole Variant to Nucleosume Structure - Literature review Example

HISTOLE VARIANT TO NUCLEOSUME STRUCTURE al Affiliation) Aims of the experiment Investigate cloning and mutagenesis of novel histone variants and isoforms Preparation of histones and assembly into nucleosomes Objectives of the study Investigation of expression of H2AX mutants S139A, S139E and S139A Y142F Investigation of Cloning of Hydrastine echinata H2B.3 and H2B.6 histone variants  Mutagenesis of H2A isoform HIST1H2AE into HIST1H2AG  Expression and purification of variants, and assembly into nucleosomes  Comparison of nucleosomes by native polyacrylamide gel electrophoresis Materials and methods Materials used in the experiment E. coli strains  Bacterial culture Medias  Oligonucleotide primers  DNA methods  E. coli transformation  PCR amplification of DNA  Cloning by restriction enzymes and ligation  Site-directed mutagenesis  DNA midipreps  Protein methods  Histone expression tests  Introduction Genome packaging by chromatin in eukaryotes Chromatic eukaryotic cells contain uninterrupted molecules of DNA are tightly bound to a group of small proteins referred to as histones. Chromosomes constitute half proteins and half DNA. The materials are referred to as chromatin. The primary structure of histones contains amino acid with basic side chains. The chains contain nucleosomes which are further packed in high order supercoils (Abbott & Ausió, 2005). During the resting phase of the cell cycle, the chromosomes are predominantly unwound. As cell division methods, the chromosomes shrink and assume flexible rod-like shapes. This permits a more orderly apportionment of genetic material among the two daughter cells (Abbott & Ausió, 2005). The link that occur between histone core, linker DNA, core DNA and nucleosome can be illustrated using the image below Chromatin packing ratio can be represented below Eukaryotic chromosomes consist of a DNA-protein complex that is organized in a compact manner which permits the large amount of DNA to be stored in the nucleus of the cell. The subunit designation of the chromosome is chromatin. The fundamental unit of chromatin is the nucleosome (Bjergbæk, 2012). Chromatin is the unit analysis of the chromosomes that reflects the general structure of the chromosomes but they are not unique to any chromosomes. Nucleosome is the packing structure of DNA that is found in eukaryotic chromosomes (Calhoun, 2006). The current researches shows that there are minor changes in the primary conserved histones that may be determinants for the chromatin structure regulating gene expression and other DNA-related processes (Carrell & Aston, 2013). An analysis of the involvement of different core histone variants in different nuclear processes and the structure of different variant nucleosome cores shows that this may indeed be so. Histone variants may also be involved in demarcating functional regions of the chromatin (Carrell & Aston, 2013). Main histones include histone H2A variants which specialize the nucleosome for dedicated functions. Histone has variants in the sequence such as H1 and the sperm and testis-specific variants (Catalano, 2005). The sequences involved differ between the major histone subtypes and the variants in the N and C terminal of protein domains (Conerly, 2010). The abundance of the variants is in the cell types and during cell cycles, development and differentiation. Main histones and variants have distinctions biophysical properties. Histone H2A has variants including H2A.Z, MacroH2A, H2A-Bbd, H2AvD, and H2A.X. The conservation of some variants is during evolution while others are restricted to vertebrates and animals (Chakravarthy, 2004). Histone H2A shows an important role in major changes in the N and C terminal ends of their molecules with larger predominance of those that affect the carboxyl end. The application of the N-terminal heterogeneity is still unclear. Histone H2A variants and their involvement in nucleosome dynamics and conformation are also unclear. Amino acid sequence alignment of heteromorphous human H2A histone variants is involved (Forrey, 2008). There is displacement that occurs between H2A and H2B dimers that leads to an enhancement of nucleus activity in the sensitivity in the region that is core to the neuclosimal DNA (Goldman, 2010). Sperm histosomes shows great variation in contrast with conservation of most groups of somatic histosomes. DNA in human sperm is packed in nucleosomes and transmission of this fraction to the embryo potentially serves as a mechanism to facilitate paternal epigenetic programs during embryonic development (Goldman, 2010). This is about 15% to 30% of DNA in human cells. Sperm-derived nucleosome chromatin results to paternal zygotic chromatin, possibly portion as a template used in replication, when epigenetic information can be derivative (McLay, 2001). Hence, the implementation of epigenetic activities originating from transmitted paternal chromatin in the process of subsequent embryonic development is a logical consequence. A sperm chromatin is shown below Contribution of histone variants to nucleosome Histone proteins play important structural and functional roles in transmission between inactive and active chromatin states. Histones though have a high degree of conservation due to constraints to maintain the general structure of the neuclosimal octameric core; there have been changes in variants to take diverse roles in the regulation of genes and epigenetic silencing (McLay, 2001). The interactions that occur between histone variants and post-translational modifications remodel complexes that influence DMA modeling, replication, repair, transcription and recombination. The chromatin field has witnessed a renewed interest in histone variants as pertaining to their structural role, but mainly because of the functional specificity they impart to chromatin. In the review, the discussion is given on several of the most recent structural lessons on core histone (H2A.Z, H2A.X, H2A.Bbd, CENP-A, macroH2A, H3.3) and linker histone variants concentrating on their role in nucleosome constancy and chromatin fiber dynamics with special importance on their possible useful implications. The overall structure state of chromatin and its domains controls expression and replication of DNA of genes. The histones are characterized by the presence of N-terminals and of a histone fold domain of variable lengths that are the subjects of extensive post-translation modification. Chromatic eukaryotic cells contain uninterrupted molecules of DNA are tightly bound to a group of small proteins referred to as histones. Chromosomes constitute half proteins and half DNA. The materials are referred to as chromatin. The primary structure of histones contains amino acid with basic side chains. The chains contain nucleosomes which are further packed in high order supercoils. Histones and their variants are responsible for organizing the chromatin complex and within this context they can be classified into core histones (H2A/H2B, H3/H4) and linker histones (histones of the H1 family). Core histones consist of a demonizing central histone-fold domain which is flanked by N- and C-terminal unstructured domains commonly referred to as ‘tails’, and as their name indicate they are responsible for creating a protein ‘core’ around which 146 bp of DNA are wrapped in approximately two left-handed super helical turns giving rise to a particle that is known with the name of nucleosome core particle (NCP). Eukaryotic chromosomes consist of a DNA-protein complex that is organized in a compact manner which permits the large amount of DNA to be stored in the nucleus of the cell. The subunit designation of the chromosome is chromatin. The fundamental unit of chromatin is the nucleosome. The two major functions of the chromatin nucleoprotein complex are: folding of DNA to fit the genome within the limited space available in the nucleus of the eukaryotic cell and modulation of the chromatin metabolism. Implicit to these functions is the notion that this complex cannot be structurally rigid but rather a highly dynamic system. Thus, although a lot of the highly valuable structural information about the nucleosome has been obtained from the crystallographic analysis of NCP in recent years, this information is quite limited when trying to understand the dynamics of chromatin. Expression and purification of H2AX mutants The experiment reveals variety of biological systems that play regulatory roles for post-translation histone medication in cellular processes such as regulation of expression of genes, DNA damage response and recombination. Phosphorylation of histone H2AX at serine 139 is a critical event in the response to DNA damage, but the functional implications of this modification are not yet clear. To examine the activity of H2AX phosphorylation there is ectopically showed epitope-tagged H2AX or mutants at the phosphorylation place. Expression of H2AX Ser139Ala, which disrupts the phosphorylation site partially, suppressed early G (2)/M arrest following ionizing radiation, and cells expressing this mutant were more sensitive to DNA damage (Zheng, 2010). Conversely, expression of H2AX Ser139Glu, designed as phosphorylation mimic, induced a decrease in the number of cells in mitosis in the absence of DNA damage. Interestingly, this decrease induced by H2AX Ser139Glu was independent of the formation of 53BP1-containing foci and was partially suppressed in CHK2-deficient cells, suggesting a role for CHK2 in this process (Zheng, 2010). Site directed mutagenesis of H2A isoforms Mitochondrial integrity loss as a sequence of apoptogenic complexes formed on the outer membrane containing a main step in controlling progression of apoptotic cascades. In this situation, multiple members of the linker histone family of protein modify apoptotic cascades initiated by protein (Zunder, 2011). In mutants that mimic the constitutively phosphorylated form of H2A shown defected in chromatin structure, there are changes that occur. In the situation, both plasmid super helical density and micrococci nucleus digestion assays portrayed relaxed but not grossly altered structure of chromatin. The investigations also showed that Melc-dependent modification of H2AS129 is vital for break DNA repair in NHEJ pathway (Zunder, 2011). Repair of DNA may be facilitated by the phosphorylation of S129 to relax chromatic structure thereby allowing access of repair machinery to DNA. The N-terminal tail of H2A has been subjected to extensive mutation analysis and is necessary for transcription repression of dependent genes. Site directed mutagenesis of H2A Site directed mutagenesis of H2Ais a method that is used in molecular biology to make specific to the DNA sequences of a gene and all the gene products. The procedure requires synthesis of a short DNA primer. The primer contains the desire mutation and is also complementary to the template DNA around the mutation so it can hybridize with the DNA in the gene of interest. There are several approaches that can be given to the directed site mutagenesis including Kunkel’s method, cassette mutagenesis, PRC site directed mutagenesis and whole plasmid mutagenesis. The DNA segment to be mutagenized is inserted in the beta-galactosidase gene of a M13-lac vector, generally causing loss of beta-galactosidase function by generation of frame shifts or nonsense codons. Mutations in the inserted DNA which restore beta-galactosidase function are readily detected and analyzed. The application of the process to the agent of a eukaryotic tRNAPro gene has permitted the isolation of numerous mutants changed in record. Cloning of Hydratinia echitana sperm histones During the process of spermatogenesis, there is development of male germ cells into a series of essential development processes. The process involves differentiation of a stem cell population, meiosis and mitotic amplification. In the process, there is also morphological process in post-meiotic germ cells (Baldi & Muratori, 2013). References Abbott, D., & Ausió, J. (2005). The structural role of histone H2A variants in chromatin function. Baldi, E., & Muratori, M. (2013). Genetic Damage in Human Spermatozoa. Dordrecht: Springer. Bjergbæk, L. (2012). DNA repair protocols. Totowa, N.J.: Humana. Calhoun, S. (2006). Functional analysis of RNA interacting domains of Brome mosaic virus coat protein involved in genome packaging. Carrell, D., & Aston, K. (2013). Spermatogenesis. New York: Humana Press. Catalano, C. (2005). Viral genome packaging machines. Georgetown, Tex.: Landes Bioscience/Eurekah.com. Catalano, C. (2007). Viral Genome Packaging. Dordrecht: Springer. Chakravarthy, S. (2004). Structural and functional effects of histone variants on the nucleosome core particle. Conerly, M. (2010). Changes in histone variants, histone modifications, and DNA methylation during cancer progression. Craig, N. Molecular biology. Downey, M. (2008). Limiting the DNA damage checkpoint response at telomeres and DNA double-strand breaks. Forrey, C. (2008). Molecular modeling and Langevin dynamics simulations of viral genome packaging and DS-DNA translocation. Ghosh-Kumar, M. (2010). The headful packaging nuclease of bacteriophage T4. Goldman, J. (2010). Roles for histone H2A variants in gene regulation. Henderson, D. (2006). DNA repair protocols. Totowa, N.J.: Humana Press. King, R. (1976). Handbook of Genetics. Boston, MA: Springer US. Matulis, S. (2006). Cross-species comparison of spermatocyte responses to induced DNA damage. McLay, D. (2001). Developmental regulation and molecular nature of an activity in murine oocytes that transfers histones onto sperm DNA. Neurath, H. (1979). The Proteins Pt 4. Burlington: Elsevier Science. Resch, M. (2008). The influence of histone orthologues, histone variants and post-translational modifications on the structure and function of chromatin. Singh, S., & Rameshwar, P. (2014). MicroRNA in Development and in the Progression of Cancer. Dordrecht: Springer. Vyas, P. (2012). The contribution of terminal domains towards the differential chromatin binding characteristics of linker histone variants H1(0) and H1c. Zheng, G. (2010). The packaging of DNA in chromatin. Zunder, R. (2011). The Regulation of Chromatin Dynamics by Histone Chaperones and Variants. Berkeley, CA. Read More