1.
RNA polymerase II
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RNA polymerase II is protein-protein complex. It is one of the three RNAP enzymes found in the nucleus of eukaryotic cells and it catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA. A550 kDa complex of 12 subunits, RNAP II is the most studied type of RNA polymerase, a wide range of transcription factors are required for it to bind to upstream gene promoters and begin transcription. Early studies suggested a minimum of two RNAPs, one of which synthesized rRNA in the nucleolus while the remaining enzyme synthesizes other RNA in the nucleoplasm, part of the nucleus but outside the nucleolus. In 1969, science experimentalists Robert Roeder and William Rutter definitively discovered an additional RNAP that was resonablable for transcription of some kind of RNA in the nucleoplasm, the finding was obtained by the use of DEAE-Sephadex ion-exchange chromatography. The technique separated the enzymes by the order of the elutions, Ι, ΙΙ, ΙΙΙ. The enzymes were named according to the order of the elutions, RNAP I, RNAP II and this discovery demonstrated that there was an additional enzyme present in the nucleoplasm, which allowed for the differentiation between RNAP II and RNAP III. The eukaryotic core RNA polymerase II was first purified using transcription assays, the purified enzyme has typically 10-12 subunits and is incapable of specific promoter recognition. DNA-directed RNA polymerase II subunit RPB1 – an enzyme that in humans is encoded by the POLR2A gene, RPB1 is the largest subunit of RNA polymerase II. It contains a carboxy terminal domain composed of up to 52 heptapeptide repeats that are essential for polymerase activity, the CTD was first discovered in the laboratory of C. J. Ingles at the University of Toronto and by JL Corden at Johns Hopkins University. In combination with several other subunits, the RPB1 subunit forms the DNA binding domain of the polymerase. Exists as a heterodimer with another polymerase subunit, POLR2J forming a core subassembly, RPB3 strongly interacts with RPB1-5,7, 10-12. RNA polymerase II subunit B4 – encoded by the POLR2D gene is the fourth-largest subunit, RPB5 – In humans is encoded by the POLR2E gene. Two molecules of this subunit are present in each RNA polymerase II, RPB5 strongly interacts with RPB1, RPB3, and RPB6. RPB6 – forms a structure with at least two other subunits that stabilizes the transcribing polymerase on the DNA template, RPB7 – encoded by POLR2G and may play a role in regulating polymerase function. RPB7 interacts strongly with RPB1 and RPB5, RPB8 – interacts with subunits RPB1-3,5, and 7. RPB9 – The groove in which the DNA template is transcribed into RNA is composed of RPB9, RPB10 – the product of gene POLR2L. It interacts with RPB1-3 and 5, and strongly with RPB3, RPB11 – the RPB11 subunit is itself composed of three subunits in humans, POLR2J, POLR2J2, and POLR2J3
2.
Escherichia coli
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Escherichia coli is a gram-negative, facultatively anaerobic, rod-shaped, coliform bacterium of the genus Escherichia that is commonly found in the lower intestine of warm-blooded organisms. Most E. coli strains are harmless, but some serotypes can cause food poisoning in their hosts. The harmless strains are part of the flora of the gut, and can benefit their hosts by producing vitamin K2. E. coli is expelled into the environment within fecal matter, the bacterium grows massively in fresh fecal matter under aerobic conditions for 3 days, but its numbers decline slowly afterwards. E. coli and other facultative anaerobes constitute about 0. 1% of gut flora, cells are able to survive outside the body for a limited amount of time, which makes them potential indicator organisms to test environmental samples for fecal contamination. A growing body of research, though, has examined environmentally persistent E. coli which can survive for extended periods outside of a host, the bacterium can be grown and cultured easily and inexpensively in a laboratory setting, and has been intensively investigated for over 60 years. E. coli is a chemoheterotroph whose chemically defined medium must include a source of carbon, under favorable conditions, it takes only 20 minutes to reproduce. E. coli is a Gram-negative, facultative anaerobic and nonsporulating bacterium, cells are typically rod-shaped, and are about 2.0 μm long and 0. 25–1.0 μm in diameter, with a cell volume of 0. 6–0.7 μm3. E. coli stains Gram-negative because its cell wall is composed of a peptidoglycan layer. During the staining process, E. coli picks up the color of the counterstain safranin, the outer membrane surrounding the cell wall provides a barrier to certain antibiotics such that E. coli is not damaged by penicillin. Strains that possess flagella are motile, the flagella have a peritrichous arrangement. E. coli can live on a variety of substrates and uses mixed-acid fermentation in anaerobic conditions, producing lactate, succinate, ethanol, acetate. Optimum growth of E. coli occurs at 37 °C, and it uses oxygen when it is present and available. It can, however, continue to grow in the absence of oxygen using fermentation or anaerobic respiration, the ability to continue growing in the absence of oxygen is an advantage to bacteria because their survival is increased in environments where water predominates. The bacterial cell cycle is divided into three stages, the B period occurs between the completion of cell division and the beginning of DNA replication. The C period encompasses the time it takes to replicate the chromosomal DNA, the D period refers to the stage between the conclusion of DNA replication and the end of cell division. The doubling rate of E. coli is higher when more nutrients are available, However, the length of the C and D periods do not change, even when the doubling time becomes less than the sum of the C and D periods. At the fastest growth rates, replication begins before the round of replication has completed, resulting in multiple replication forks along the DNA
3.
Entrez
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The name Entrez was chosen to reflect the spirit of welcoming the public to search the content available from the NLM. Entrez Global Query is a search and retrieval system that provides access to all databases simultaneously with a single query string. Entrez can efficiently retrieve related sequences, structures, and references, the Entrez system can provide views of gene and protein sequences and chromosome maps. Some textbooks are available online through the Entrez system. The Entrez front page provides, by default, access to the global query, all databases indexed by Entrez can be searched via a single query string, supporting boolean operators and search term tags to limit parts of the search statement to particular fields. This returns a unified results page, that shows the number of hits for the search in each of the databases, Entrez also provides a similar interface for searching each particular database and for refining search results. The Limits feature allows the user to narrow a search a web forms interface, the History feature gives a numbered list of recently performed queries. Results of previous queries can be referred to by number and combined via boolean operators, search results can be saved temporarily in a Clipboard. Users with a MyNCBI account can save queries indefinitely and also choose to have updates with new search results e-mailed for saved queries of most databases and it is widely used in the field of biotechnology as a reference tool for students and professionals alike. Entrez searches the following databases, PubMed, biomedical literature citations and abstracts, including Medline - articles from journals, in addition to using the search engine forms to query the data in Entrez, NCBI provides the Entrez Programming Utilities for more direct access to query results. The eUtils are accessed by posting specially formed URLs to the NCBI server, there was also an eUtils SOAP interface which was terminated on July 2015. In 1991, entrez was introduced in CD form, in 1993, a client-server version of the software provided connectivity with the internet. In 1994, NCBI established a website, and Entrez was a part of initial release. In 2001, Entrez bookshelf was released and in 2003, the Entrez Gene database was developed, Entrez search engine form Entrez Help
4.
Protein Data Bank
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The Protein Data Bank is a crystallographic database for the three-dimensional structural data of large biological molecules, such as proteins and nucleic acids. The PDB is overseen by a called the Worldwide Protein Data Bank. The PDB is a key resource in areas of structural biology, most major scientific journals, and some funding agencies, now require scientists to submit their structure data to the PDB. Many other databases use protein structures deposited in the PDB, for example, SCOP and CATH classify protein structures, while PDBsum provides a graphic overview of PDB entries using information from other sources, such as Gene ontology. By 1971, one of Meyers programs, SEARCH, enabled researchers to access information from the database to study protein structures offline. SEARCH was instrumental in enabling networking, thus marking the beginning of the PDB. Upon Hamiltons death in 1973, Tom Koeztle took over direction of the PDB for the subsequent 20 years, in January 1994, Joel Sussman of Israels Weizmann Institute of Science was appointed head of the PDB. In October 1998, the PDB was transferred to the Research Collaboratory for Structural Bioinformatics, the new director was Helen M. Berman of Rutgers University. In 2003, with the formation of the wwPDB, the PDB became an international organization, the founding members are PDBe, RCSB, and PDBj. Each of the four members of wwPDB can act as deposition, data processing, the data processing refers to the fact that wwPDB staff review and annotate each submitted entry. The data are automatically checked for plausibility. The PDB database is updated weekly, likewise, the PDB holdings list is also updated weekly. As of 14 March 2017, the breakdown of current holdings is as follows,103,514 structures in the PDB have a structure factor file,9,057 structures have an NMR restraint file. 2,826 structures in the PDB have a chemical shifts file, therefore, the final conformation of the protein is obtained, in the latter case, by solving a distance geometry problem. A few proteins are determined by cryo-electron microscopy, the significance of the structure factor files, mentioned above, is that, for PDB structures determined by X-ray diffraction that have a structure file, the electron density map may be viewed. The data of such structures is stored on the electron density server, however, since 2007, the rate of accumulation of new protein structures appears to have plateaued. The file format used by the PDB was called the PDB file format. This original format was restricted by the width of computer punch cards to 80 characters per line, around 1996, the macromolecular Crystallographic Information file format, mmCIF, which is an extension of the CIF format started to be phased in
5.
National Center for Biotechnology Information
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The National Center for Biotechnology Information is part of the United States National Library of Medicine, a branch of the National Institutes of Health. The NCBI is located in Bethesda, Maryland and was founded in 1988 through legislation sponsored by Senator Claude Pepper, the NCBI houses a series of databases relevant to biotechnology and biomedicine and is an important resource for bioinformatics tools and services. Major databases include GenBank for DNA sequences and PubMed, a database for the biomedical literature. Other databases include the NCBI Epigenomics database, all these databases are available online through the Entrez search engine. NCBI is directed by David Lipman, one of the authors of the BLAST sequence alignment program. He also leads a research program, including groups led by Stephen Altschul, David Landsman, Eugene Koonin, John Wilbur, Teresa Przytycka. NCBI is listed in the Registry of Research Data Repositories re3data. org, NCBI has had responsibility for making available the GenBank DNA sequence database since 1992. GenBank coordinates with individual laboratories and other databases such as those of the European Molecular Biology Laboratory. Since 1992, NCBI has grown to other databases in addition to GenBank. The NCBI assigns a unique identifier to each species of organism, the NCBI has software tools that are available by WWW browsing or by FTP. For example, BLAST is a sequence similarity searching program, BLAST can do sequence comparisons against the GenBank DNA database in less than 15 seconds. RAG2/IL2RG The NCBI Bookshelf is a collection of freely accessible, downloadable, some of the books are online versions of previously published books, while others, such as Coffee Break, are written and edited by NCBI staff. BLAST is a used for calculating sequence similarity between biological sequences such as nucleotide sequences of DNA and amino acid sequences of proteins. BLAST is a tool for finding sequences similar to the query sequence within the same organism or in different organisms. It searches the query sequence on NCBI databases and servers and post the results back to the browser in chosen format. Input sequences to the BLAST are mostly in FASTA or Genbank format while output could be delivered in variety of such as HTML, XML formatting. HTML is the output format for NCBIs web-page. Entrez is both indexing and retrieval system having data from sources for biomedical research
6.
UniProt
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UniProt is a freely accessible database of protein sequence and functional information, many entries being derived from genome sequencing projects. It contains an amount of information about the biological function of proteins derived from the research literature. The UniProt consortium comprises the European Bioinformatics Institute, the Swiss Institute of Bioinformatics, EBI, located at the Wellcome Trust Genome Campus in Hinxton, UK, hosts a large resource of bioinformatics databases and services. SIB, located in Geneva, Switzerland, maintains the ExPASy servers that are a resource for proteomics tools. In 2002, EBI, SIB, and PIR joined forces as the UniProt consortium, each consortium member is heavily involved in protein database maintenance and annotation. Until recently, EBI and SIB together produced the Swiss-Prot and TrEMBL databases and these databases coexisted with differing protein sequence coverage and annotation priorities. Swiss-Prot aimed to provide reliable protein sequences associated with a level of annotation. Recognizing that sequence data were being generated at a pace exceeding Swiss-Prots ability to keep up, meanwhile, PIR maintained the PIR-PSD and related databases, including iProClass, a database of protein sequences and curated families. The consortium members pooled their resources and expertise, and launched UniProt in December 2003. UniProt provides four core databases, UniProtKB, UniParc, UniRef, UniProt Knowledgebase is a protein database partially curated by experts, consisting of two sections, UniProtKB/Swiss-Prot and UniProtKB/TrEMBL. As of 19 March 2014, release 2014_03 of UniProtKB/Swiss-Prot contains 542,782 sequence entries, UniProtKB/Swiss-Prot is a manually annotated, non-redundant protein sequence database. It combines information extracted from literature and biocurator-evaluated computational analysis. The aim of UniProtKB/Swiss-Prot is to all known relevant information about a particular protein. Annotation is regularly reviewed to keep up with current scientific findings, the manual annotation of an entry involves detailed analysis of the protein sequence and of the scientific literature. Sequences from the gene and the same species are merged into the same database entry. Differences between sequences are identified, and their cause documented, a range of sequence analysis tools is used in the annotation of UniProtKB/Swiss-Prot entries. Computer-predictions are manually evaluated, and relevant results selected for inclusion in the entry and these predictions include post-translational modifications, transmembrane domains and topology, signal peptides, domain identification, and protein family classification. Relevant publications are identified by searching databases such as PubMed, the full text of each paper is read, and information is extracted and added to the entry
7.
Chromosome
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A chromosome is a DNA molecule with part or all of the genetic material of an organism. Prokaryotes usually have one single circular chromosome, whereas most eukaryotes are diploid, chromosomes in eukaryotes are composed of chromatin fiber. Chromatin fiber is made of nucleosomes, a nucleosome is a histone octamer with part of a longer DNA strand attached to and wrapped around it. Chromatin fiber, together with associated proteins is known as chromatin, chromatin is present in most cells, with a few exceptions, for example, red blood cells. Occurring only in the nucleus of cells, chromatin contains the vast majority of DNA, except for a small amount inherited maternally. Chromosomes are normally visible under a microscope only when the cell is undergoing the metaphase of cell division. Before this happens every chromosome is copied once, and the copy is joined to the original by a centromere resulting in an X-shaped structure, the original chromosome and the copy are now called sister chromatids. During metaphase, when a chromosome is in its most condensed state, in this highly condensed form chromosomes are easiest to distinguish and study. In prokaryotic cells, chromatin occurs free-floating in cytoplasm, as these cells lack organelles, the main information-carrying macromolecule is a single piece of coiled double-helix DNA, containing many genes, regulatory elements and other noncoding DNA. The DNA-bound macromolecules are proteins that serve to package the DNA, chromosomes vary widely between different organisms. Some species such as certain bacteria also contain plasmids or other extrachromosomal DNA and these are circular structures in the cytoplasm that contain cellular DNA and play a role in horizontal gene transfer. Chromosomal recombination during meiosis and subsequent sexual reproduction plays a significant role in genetic diversity. In prokaryotes and viruses, the DNA is often densely packed and organized, in the case of archaea, by homologs to eukaryotic histones, small circular genomes called plasmids are often found in bacteria and also in mitochondria and chloroplasts, reflecting their bacterial origins. Some use the term chromosome in a sense, to refer to the individualized portions of chromatin in cells. However, others use the concept in a sense, to refer to the individualized portions of chromatin during cell division. The word chromosome comes from the Greek χρῶμα and σῶμα, describing their strong staining by particular dyes, schleiden, Virchow and Bütschli were among the first scientists who recognized the structures now so familiar to everyone as chromosomes. The term was coined by von Waldeyer-Hartz, referring to the term chromatin, in a series of experiments beginning in the mid-1880s, Theodor Boveri gave the definitive demonstration that chromosomes are the vectors of heredity. His two principles were the continuity of chromosomes and the individuality of chromosomes and it is the second of these principles that was so original
8.
Prokaryote
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A prokaryote is a unicellular organism that lacks a membrane-bound nucleus, mitochondria, or any other membrane-bound organelle. The word prokaryote comes from the Greek πρό before and καρυόν nut or kernel, prokaryotes can be divided into two domains, Archaea and bacteria. In contrast, species with nuclei and organelles are placed in the domain Eukaryota, in the prokaryotes, all the intracellular water-soluble components are located together in the cytoplasm enclosed by the cell membrane, rather than in separate cellular compartments. Bacteria, however, do possess protein-based bacterial microcompartments, which are thought to act as primitive organelles enclosed in protein shells, some prokaryotes, such as cyanobacteria may form large colonies. Others, such as myxobacteria, have multicellular stages in their life cycles, molecular studies have provided insight into the evolution and interrelationships of the three domains of biological species. Eukaryotes are organisms, including humans, whose cells have a well defined membrane-bound nucleus, the division between prokaryotes and eukaryotes reflects the existence of two very different levels of cellular organization. Distinctive types of prokaryotes include extremophiles and methanogens, these are common in extreme environments. Prokaryotes have a cytoskeleton, albeit more primitive than that of the eukaryotes. At least some also contain intracellular structures that can be seen as primitive organelles. Membranous organelles are known in some groups of prokaryotes, such as vacuoles or membrane systems devoted to special metabolic properties, in addition, some species also contain carbohydrate-enclosed microcompartments, which have distinct physiological roles. Most prokaryotes are between 1 µm and 10 µm, but they can vary in size from 0.2 µm to 750 µm, Bacteria and archaea reproduce through asexual reproduction, usually by binary fission. DNA transfer between prokaryotic cells occurs in bacteria and archaea, although it has mainly studied in bacteria. In bacteria, gene transfer occurs by three processes and these are bacterial virus -mediated transduction, plasmid-mediated conjugation, and natural transformation. Transduction of bacterial genes by bacteriophage appears to reflect an occasional error during intracellular assembly of virus particles, the transfer of bacterial DNA is under the control of the bacteriophage’s genes rather than bacterial genes. Conjugation in the well-studied E. coli system is controlled by plasmid genes, infrequently during this process, a plasmid may integrate into the host bacterial chromosome, and subsequently transfer part of the host bacterial DNA to another bacterium. Plasmid mediated transfer of host bacterial DNA also appears to be a process rather than a bacterial adaptation. Natural bacterial transformation involves the transfer of DNA from one bacterium to another through the intervening medium, for a bacterium to bind, take up and recombine donor DNA into its own chromosome, it must first enter a special physiological state called competence. About 40 genes are required in Bacillus subtilis for the development of competence, the length of DNA transferred during B. subtilis transformation can be as much as a third to the whole chromosome
9.
DNA polymerase
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In molecular biology, DNA polymerases are enzymes that synthesize DNA molecules from deoxyribonucleotides, the building blocks of DNA. These enzymes are essential to DNA replication and usually work in pairs to two identical DNA strands from a single original DNA molecule. During this process, DNA polymerase reads the existing DNA strands to create two new strands that match the existing ones. Every time a cell divides, DNA polymerases are required to duplicate the cells DNA. In this way, genetic information is passed down generation to generation. Before replication can take place, an enzyme called helicase unwinds the DNA molecule from its tightly woven form and this opens up or unzips the double-stranded DNA to give two single strands of DNA that can be used as templates for replication. In 1956, Arthur Kornberg and colleagues discovered DNA polymerase I and they described the DNA replication process by which DNA polymerase copies the base sequence of a template DNA strand. Kornberg was later awarded the Nobel Prize in Physiology or Medicine in 1959 for this work, DNA polymerase II was also discovered by Thomas Kornberg and Malcolm E. Gefter in 1970 while further elucidating the role of Pol I in E. coli DNA replication. The main function of DNA polymerase is to synthesize DNA from deoxyribonucleotides, the DNA copies are created by the pairing of nucleotides to bases present on each strand of the original DNA molecule. This pairing always occurs in specific combinations, with cytosine along with guanine, by contrast, RNA polymerases synthesize RNA from ribonucleotides from either RNA or DNA. When synthesizing new DNA, DNA polymerase can add free nucleotides only to the 3 end of the newly forming strand and this results in elongation of the newly forming strand in a 5-3 direction. No known DNA polymerase is able to begin a new chain, it can add a nucleotide onto a pre-existing 3-OH group. Primers consist of RNA or DNA bases, in DNA replication, the first two bases are always RNA, and are synthesized by another enzyme called primase. It is important to note that the directionality of the newly forming strand is opposite to the direction in which DNA polymerase moves along the template strand. Since DNA polymerase requires a free 3 OH group for initiation of synthesis, hence, DNA polymerase moves along the template strand in a 3-5 direction, and the daughter strand is formed in a 5-3 direction. This difference enables the resultant double-strand DNA formed to be composed of two DNA strands that are antiparallel to each other, the function of DNA polymerase is not quite perfect, with the enzyme making about one mistake for every billion base pairs copied. Error correction is a property of some, but not all DNA polymerases and this process corrects mistakes in newly synthesized DNA. When an incorrect base pair is recognized, DNA polymerase moves backwards by one pair of DNA
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. PMID 4927672.
