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Yeast Artificial Chromosome
Yeast artificial chromosomes (YACs) are genetically engineered chromosomes derived from the DNA of the yeast, ''Saccharomyces cerevisiae'', which is then ligated into a bacterial plasmid. By inserting large fragments of DNA, from 100–1000 kb, the inserted sequences can be cloned and physically mapped using a process called chromosome walking. This is the process that was initially used for the Human Genome Project, however due to stability issues, YACs were abandoned for the use of Bacterial artificial chromosomes (BAC). Beginning with the initial research of the Rankin et al., Strul et al., and Hsaio et al., the inherently fragile chromosome was stabilized by discovering the necessary autonomously replicating sequence (ARS); a refined YAC utilizing this data was described in 1983 by Murray et al. The primary components of a YAC are the ARS, centromere, and telomeres from ''S. cerevisiae''. Additionally, selectable marker genes, such as antibiotic resistance and a visib ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Saccharomyces Cerevisiae
''Saccharomyces cerevisiae'' () (brewer's yeast or baker's yeast) is a species of yeast (single-celled fungus microorganisms). The species has been instrumental in winemaking, baking, and brewing since ancient times. It is believed to have been originally isolated from the skin of grapes. It is one of the most intensively studied eukaryotic model organisms in molecular and cell biology, much like '' Escherichia coli'' as the model bacterium. It is the microorganism behind the most common type of fermentation. ''S. cerevisiae'' cells are round to ovoid, 5–10 μm in diameter. It reproduces by budding. Many proteins important in human biology were first discovered by studying their homologs in yeast; these proteins include cell cycle proteins, signaling proteins, and protein-processing enzymes. ''S. cerevisiae'' is currently the only yeast cell known to have Berkeley bodies present, which are involved in particular secretory pathways. Antibodies against ''S. ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Plasmid
A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; however, plasmids are sometimes present in archaea and eukaryotic organisms. In nature, plasmids often carry genes that benefit the survival of the organism and confer selective advantage such as antibiotic resistance. While chromosomes are large and contain all the essential genetic information for living under normal conditions, plasmids are usually very small and contain only additional genes that may be useful in certain situations or conditions. Artificial plasmids are widely used as vectors in molecular cloning, serving to drive the replication of recombinant DNA sequences within host organisms. In the laboratory, plasmids may be introduced into a cell via transformation. Synthetic plasmids are available for procurement over the int ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Autonomously Replicating Sequence
An autonomously replicating sequence (ARS) contains the origin of replication in the yeast genome. It contains four regions (A, B1, B2, and B3), named in order of their effect on plasmid stability. The A-Domain is highly conserved, any mutation abolishes origin function. Mutations on B1, B2, and B3 will diminish, but not prevent functioning of the origin. Element A is highly conserved, consisting of the consensus sequence: (where ''Y'' is either pyrimidine and ''R'' is either purine). When this element is mutated, the ARS loses all activity. As seen above the ARS are considerably A-T rich which makes it easy for replicative proteins to disrupt the H-bonding in that area. ORC protein complex (origin recognition complex) is bound at the ARS throughout the cell cycle, allowing replicative proteins access to the ARS. Mutational analysis for the yeast ARS elements have shown that any mutation in the B1, B2 and B3 regions result in a reduction of function of the ARS element. A muta ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Human Artificial Chromosome
A human artificial chromosome (HAC) is a microchromosome that can act as a new chromosome in a population of human cells. That is, instead of 46 chromosomes, the cell could have 47 with the 47th being very small, roughly 6–10 megabases (Mb) in size instead of 50–250Mb for natural chromosomes, and able to carry new genes introduced by human researchers. Ideally, researchers could integrate different genes that perform a variety of functions, includindisease defense Alternative methods of creating transgenes, such as utilizing yeast artificial chromosomes and bacterial artificial chromosomes, lead to unpredictable problems. The genetic material introduced by these vectors not only leads to different expression levels, but the inserts also disrupt the original genome. HACs differ in this regard, as they are entirely separate chromosomes. This separation from existing genetic material assumes that no insertional mutants would arise. This stability and accuracy makes HACs preferabl ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Genetic Engineering
Genetic engineering, also called genetic modification or genetic manipulation, is the modification and manipulation of an organism's genes using technology. It is a set of technologies used to change the genetic makeup of cells, including the transfer of genes within and across species boundaries to produce improved or novel organisms. New DNA is obtained by either isolating and copying the genetic material of interest using recombinant DNA methods or by artificially synthesising the DNA. A construct is usually created and used to insert this DNA into the host organism. The first recombinant DNA molecule was made by Paul Berg in 1972 by combining DNA from the monkey virus SV40 with the lambda virus. As well as inserting genes, the process can be used to remove, or " knock out", genes. The new DNA can be inserted randomly, or targeted to a specific part of the genome. An organism that is generated through genetic engineering is considered to be genetically modified (G ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Fosmid
Fosmids are similar to cosmids but are based on the bacterial F-plasmid. The cloning vector is limited, as a host (usually '' E. coli'') can only contain one fosmid molecule. Fosmids can hold DNA inserts of up to 40 kb in size; often the source of the insert is random genomic DNA. A fosmid library is prepared by extracting the genomic DNA from the target organism and cloning it into the fosmid vector. The ligation mix is then packaged into phage particles and the DNA is transfected into the bacterial host. Bacterial clones propagate the fosmid library. The low copy number offers higher stability than vectors with relatively higher copy numbers, including cosmids. Fosmids may be useful for constructing stable libraries from complex genomes. Fosmids have high structural stability and have been found to maintain human DNA effectively even after 100 generations of bacterial growth. Fosmid clones were used to help assess the accuracy of the Public Human Genome Sequence. Discovery The fe ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Cosmid
A cosmid is a type of hybrid plasmid that contains a Lambda phage ''cos'' sequence. They are often used as a cloning vector in genetic engineering. Cosmids can be used to build genomic libraries. They were first described by Collins and Hohn in 1978. Cosmids can contain 37 to 52 (normally 45) kb of DNA, limits based on the normal bacteriophage packaging size. They can replicate as plasmids if they have a suitable origin of replication (ori): for example SV40 ori in mammalian cells, ColE1 ori for double-stranded DNA replication, or f1 ori for single-stranded DNA replication in prokaryotes. They frequently also contain a gene for selection such as antibiotic resistance, so that the transformed cells can be identified by plating on a medium containing the antibiotic. Those cells which did not take up the cosmid would be unable to grow. Unlike plasmids, they can also be packaged in vitro into phage capsids, a step which requires ''cohesive ends'', also known as ''cos'' sites al ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Sequence-tagged Site
A sequence-tagged site (or STS) is a short (200 to 500 base pair) DNA sequence that has a single occurrence in the genome and whose location and base sequence are known. Usage STSs can be easily detected by the polymerase chain reaction (PCR) using specific primers. For this reason they are useful for constructing genetic and physical maps from sequence data reported from many different laboratories. They serve as landmarks on the developing physical map of a genome. When STS loci contain genetic polymorphisms (e.g. simple sequence length polymorphisms, SSLPs, single nucleotide polymorphisms), they become valuable genetic markers, i.e. loci which can be used to distinguish individuals. They are used in shotgun sequencing, specifically to aid sequence assembly. STSs are very helpful for detecting microdeletions in some genes. For example, some STSs can be used in screening by PCR to detect microdeletions in Azoospermia (AZF) genes in infertile Infertility is the inability ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
PBR322
pBR322 is a plasmid and was one of the first widely used '' E. coli'' cloning vectors. Created in 1977 in the laboratory of Herbert Boyer at the University of California, San Francisco, it was named after Francisco Bolivar Zapata, the postdoctoral researcher and Raymond L. Rodriguez. The p stands for "plasmid," and BR for "Bolivar" and "Rodriguez." pBR322 is 4361 base pairs in length and has two antibiotic resistance genes – the gene '' bla'' encoding the ampicillin resistance (AmpR) protein, and the gene ''tetA'' encoding the tetracycline resistance (TetR) protein. It contains the origin of replication of pMB1, and the '' rop'' gene, which encodes a restrictor of plasmid copy number. The plasmid has unique restriction sites for more than forty restriction enzymes. Eleven of these forty sites lie within the TetR gene. There are two sites for restriction enzymes HindIII and ClaI within the promoter of the TetR gene. There are six key restriction sites inside the AmpR ge ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Primer Walking
Primer walking is a technique used to clone a gene (e.g., disease gene) from its known closest markers (e.g., known gene). As a result, it is employed in cloning and sequencing efforts in plants, fungi, and mammals with minor alterations. This technique, also known as "directed sequencing," employs a series of Sanger sequencing reactions to either confirm the reference sequence of a known plasmid or PCR product based on the reference sequence (sequence confirmation service) or to discover the unknown sequence of a full plasmid or PCR product by designing primers to sequence overlapping sections (sequence discovery service). Primer walking: a DNA sequencing method Primer walking is a method to determine the sequence of DNA up to the 1.3–7.0 kb range whereas chromosome walking is used to produce the clones of already known sequences of the gene. Too long fragments cannot be sequenced in a single sequence read using the chain termination method. This method works by dividing t ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Gene Mapping
Gene mapping describes the methods used to identify the locus of a gene and the distances between genes. Gene mapping can also describe the distances between different sites within a gene. The essence of all genome mapping is to place a collection of molecular markers onto their respective positions on the genome. Molecular markers come in all forms. Genes can be viewed as one special type of genetic markers in the construction of genome maps, and mapped the same way as any other markers. In some areas of study, gene mapping contributes to the creation of new recombinants within an organism. Genetic vs physical There are two distinctive types of "maps" used in the field of genome mapping: genetic maps and physical maps. While both maps are a collection of genetic markers and gene loci, genetic maps' distances are based on the genetic linkage information, while physical maps use actual physical distances usually measured in number of base pairs. While the physical map ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Posttranslational Modification
Post-translational modification (PTM) is the covalent and generally enzymatic modification of proteins following protein biosynthesis. This process occurs in the endoplasmic reticulum and the golgi apparatus. Proteins are synthesized by ribosomes translating mRNA into polypeptide chains, which may then undergo PTM to form the mature protein product. PTMs are important components in cell signaling, as for example when prohormones are converted to hormones. Post-translational modifications can occur on the amino acid side chains or at the protein's C- or N- termini. They can extend the chemical repertoire of the 20 standard amino acids by modifying an existing functional group or introducing a new one such as phosphate. Phosphorylation is a highly effective mechanism for regulating the activity of enzymes and is the most common post-translational modification. Many eukaryotic and prokaryotic proteins also have carbohydrate molecules attached to them in a process called gl ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
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