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47-438: Xenotropic viruses replicate or reproduce in cells other than those of the host species. Murine refers to the rodent family Muridae , which includes common household rats and mice. Subsequent research established that XMRV was in fact a laboratory contaminant, rather than a novel pathogen, and had been generated unintentionally in the laboratory through genetic recombination between two mouse retroviruses during propagation of

94-449: A T m (melting temperature) too much higher than the reaction's annealing temperature may mishybridize and extend at an incorrect location along the DNA sequence. A T m significantly lower than the annealing temperature may fail to anneal and extend at all. Additionally, primer sequences need to be chosen to uniquely select for a region of DNA, avoiding the possibility of hybridization to

141-586: A diastema is present. Murids lack canines and premolars. Generally, three molars (though sometimes only one or two) are found, and the nature of the molars varies by genus and feeding habit. Some murids are highly social, while others are solitary. Females commonly produce several litters annually. In warm regions, breeding may occur year-round. Though the lifespans of most genera are generally less than two years, murids have high reproductive potential and their populations tend to increase rapidly and then drastically decline when food resources have been exhausted. This

188-483: A glutamine instead of an arginine at position 462 of the RNase L enzyme, reducing its catalytic activity. A man with two copies of this mutation has twice the risk of prostate cancer; one copy raises the risk by 50%. Klein and Silverman hypothesized that "the putative linkage of RNase L alterations to HPC might reflect enhanced susceptibility to a viral agent" and conducted a viral screen of prostate cancer samples, leading to

235-495: A complementary strand. DNA polymerase adds nucleotides after binding to the RNA primer and synthesizes the whole strand. Later, the RNA strands must be removed accurately and replace them with DNA nucleotides forming a gap region known as a nick that is filled in using an enzyme called ligase. The removal process of the RNA primer requires several enzymes, such as Fen1, Lig1, and others that work in coordination with DNA polymerase, to ensure

282-548: A group of researchers to conclude that XMRV "might not be a genuine human pathogen". Xenotropic viruses (xenos Gr. foreign; tropos Gr. turning) were initially discovered in the New Zealand Black (NZB) mouse and later found to be present in many other mouse strains including wild mice. XMRV was discovered in the laboratories of Joseph DeRisi at the University of California, San Francisco , and Robert Silverman and Eric Klein of

329-439: A hopping motion, while others have broad feet and prehensile tails to improve their climbing ability, and yet others have neither adaptation. They are most commonly some shade of brown in color, although many have black, grey, or white markings. Murids generally have excellent senses of hearing and smell . They live in a wide range of habitats from forest to grassland , and mountain ranges. A number of species, especially

376-406: A prostate-cancer cell line in the mid-1990s. These findings raised serious questions concerning the findings of XMRV-related studies which purported to find connections between XMRV and human diseases. As of 2022, there is no evidence that XMRV infects humans, nor that XMRV is associated with or causes any human disease. XMRV is a murine leukemia virus (MLV) that formed through the recombination of

423-596: A result, many of the key publications which did claim an association were voluntarily retracted. This included the initial study which had linked XMRV to CFS, which was retracted at Silverman's request; one of the co-authors, Judy Mikovits , was also accused of scientific misconduct . Muridae The Muridae , or murids , are either the largest or second-largest family of rodents and of mammals , containing approximately 870 species, including many species of mice, rats, and gerbils found naturally throughout Eurasia, Africa, and Australia. The name Muridae comes from

470-744: A similar sequence nearby. A commonly used method for selecting a primer site is BLAST search, whereby all the possible regions to which a primer may bind can be seen. Both the nucleotide sequence as well as the primer itself can be BLAST searched. The free NCBI tool Primer-BLAST integrates primer design and BLAST search into one application, as do commercial software products such as ePrime and Beacon Designer . Computer simulations of theoretical PCR results ( Electronic PCR ) may be performed to assist in primer design by giving melting and annealing temperatures, etc. As of 2014, many online tools are freely available for primer design, some of which focus on specific applications of PCR. Primers with high specificity for

517-450: A specific site on the template DNA. In solution, the primer spontaneously hybridizes with the template through Watson-Crick base pairing before being extended by DNA polymerase. The ability to create and customize synthetic primers has proven an invaluable tool necessary to a variety of molecular biological approaches involving the analysis of DNA. Both the Sanger chain termination method and

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564-474: A strand of DNA . A class of enzymes called primases add a complementary RNA primer to the reading template de novo on both the leading and lagging strands . Starting from the free 3’-OH of the primer, known as the primer terminus, a DNA polymerase can extend a newly synthesized strand. The leading strand in DNA replication is synthesized in one continuous piece moving with the replication fork , requiring only an initial RNA primer to begin synthesis. In

611-479: A subset of DNA templates in the presence of many similar variants can be designed using by some software (e.g. DECIPHER ) or be developed independently for a specific group of animals. Selecting a specific region of DNA for primer binding requires some additional considerations. Regions high in mononucleotide and dinucleotide repeats should be avoided, as loop formation can occur and contribute to mishybridization. Primers should not easily anneal with other primers in

658-419: Is a short, single-stranded nucleic acid used by all living organisms in the initiation of DNA synthesis . A synthetic primer may also be referred to as an oligo , short for oligonucleotide. DNA polymerase (responsible for DNA replication) enzymes are only capable of adding nucleotides to the 3’-end of an existing nucleic acid, requiring a primer be bound to the template before DNA polymerase can begin

705-403: Is approximately spherical and 80 to 100 nm in diameter. Several XMRV genomic sequences have been published to date. These sequences are almost identical, an unusual finding as retroviruses replicate their genomes with relatively low fidelity, leading to divergent viral sequences in a single host organism. In 2010 the results of phylogenetic analyses of XMRV and related murine retroviruses led

752-441: Is known as the long flap pathway. In this pathway several enzymes are recruited to elongate the RNA primer and then cleave it off. The flaps are elongated by a 5’ to 3’ helicase , known as Pif1 . After the addition of nucleotides to the flap by Pif1, the long flap is stabilized by the replication protein A (RPA). The RPA-bound DNA inhibits the activity or recruitment of FEN1, as a result another nuclease must be recruited to cleave

799-584: Is often seen in a three- to four-year cycle. The murids are small mammals, typically around 10 cm (3.9 in) long excluding the tail , but ranging from 4.5 to 8 cm (1.8 to 3.1 in) in the African pygmy mouse to 50 cm (20 in) in the northern Luzon giant cloud rat . They typically have slender bodies with scaled tails longer than the body, and pointed snouts with prominent whiskers , but with wide variation in these broad traits. Some murids have elongated legs and feet to allow them to move with

846-634: The Pied Piper of Hamelin , retold in many versions since the 14th century, including one by the Brothers Grimm , a rat-catcher lures the town's rats into the river, but the mayor refuses to pay him. In revenge, the rat-catcher lures away all the children of the town, never to return. Mice feature in some of Beatrix Potter 's small books, including The Tale of Two Bad Mice (1904), The Tale of Mrs Tittlemouse (1910), The Tale of Johnny Town-Mouse (1918), and The Tailor of Gloucester (1903), which last

893-501: The Cleveland Clinic . Silverman had previously cloned and investigated the enzyme ribonuclease L (RNase L), part of the cell's natural defense against viruses. When activated, RNase L degrades cellular and viral RNA to halt viral replication. In 2002, the "hereditary prostate cancer 1" locus (HPC1) was mapped to the RNase L gene, implicating it in the development of prostate cancer. The cancer-associated "R462Q" mutation results in

940-552: The Latin mus (genitive muris ), meaning "mouse", since all true mice belong to the family, with the more typical mice belonging to the genus Mus . Murids are found nearly everywhere in the world, though many subfamilies have narrower ranges. Murids are not found in Antarctica or many oceanic islands. Although none of them are native to the Americas, a few species, notably

987-591: The evolution of the murids is not well known, as few fossils survive. They probably evolved from hamster -like animals in tropical Asia some time in the early Miocene , and have only subsequently produced species capable of surviving in cooler climates. They have become especially common worldwide during the current geological epoch , as a result of hitching a ride commensally with human migrations. The murids are classified in five subfamilies , around 150 genera , and about 834 species . Murids feature in literature, including folk tales and fairy stories. In

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1034-659: The gerbils , are adapted to desert conditions and can survive for a long time with minimal water . They consume a wide range of foods depending on the species, with the aid of powerful jaw muscles and gnawing incisors that grow throughout life. The dental formula of murids is 1.0.0.1-3 1.0.0.1-3 . Murids breed frequently, often producing large litters several times per year. They typically give birth between twenty and forty days after mating, although this varies greatly between species. The young are typically born blind, hairless, and helpless, although exceptions occur, such as in spiny mice . As with many other small mammals,

1081-525: The 5’ overhanging flap. This method is known as the short flap pathway of RNA primer removal. The second way to cleave a RNA primer is by degrading the RNA strand using a RNase , in eukaryotes it’s known as the RNase H2. This enzyme degrades most of the annealed RNA primer, except the nucleotides close to the 5’ end of the primer. Thus, the remaining nucleotides are displayed into a flap that is cleaved off using FEN-1. The last possible method of removing RNA primer

1128-432: The DNA polymerase reaches to the 5’ end of the RNA primer from the previous Okazaki fragment, it displaces the 5′ end of the primer into a single-stranded RNA flap which is removed by nuclease cleavage. Cleavage of the RNA flaps involves three methods of primer removal. The first possibility of primer removal is by creating a short flap that is directly removed by flap structure-specific endonuclease 1 (FEN-1), which cleaves

1175-443: The DNA template, primase intersperses RNA primers that DNA polymerase uses to synthesize DNA from in the 5′→3′ direction. Another example of primers being used to enable DNA synthesis is reverse transcription . Reverse transcriptase is an enzyme that uses a template strand of RNA to synthesize a complementary strand of DNA. The DNA polymerase component of reverse transcriptase requires an existing 3' end to begin synthesis. After

1222-417: The DNA will amplify them all, eliminating the purpose of PCR. A few criteria must be brought into consideration when designing a pair of PCR primers. Pairs of primers should have similar melting temperatures since annealing during PCR occurs for both strands simultaneously, and this shared melting temperature must not be either too much higher or lower than the reaction's annealing temperature . A primer with

1269-473: The amplified region. One application for this practice is for use in TA cloning , a special subcloning technique similar to PCR, where efficiency can be increased by adding AG tails to the 5′ and the 3′ ends. Some situations may call for the use of degenerate primers. These are mixtures of primers that are similar, but not identical. These may be convenient when amplifying the same gene from different organisms , as

1316-422: The blood of patients with CFS, prostate cancer, and other illnesses. Separate from these concerns, alarms were raised over the possibility that XMRV might be transmissible by blood transfusion since the virus was recovered from lymphocytes (white blood cells). XMRV is closely related to several known xenotropic mouse viruses which can recognize and enter cells of non-rodent species (including humans) by means of

1363-835: The cell surface xenotropic and polytropic retrovirus receptor 1 (XPR1). As a result, the AABB (formerly the American Association of Blood Banks) established a task force to determine the prevalence of XMRV in the United States' blood donation supply and the suitability of different detection methods. In September 2011, the Scientific Research Working Group (SRWG) arm of the AABB task force released its findings that current assays could not reliably identify XMRV in human blood samples which had previously tested as XMRV/MLV positive;

1410-415: The discovery of XMRV. Detection of XMRV was reported in several articles. However, subsequent studies and retractions cast doubt on these findings. In one study, XMRV was detected in a small percentage of patients with weakened immune systems, but other studies found no evidence of XMRV in immunosuppression. Concerns arose as multiple subsequent studies failed to replicate the positive findings of XMRV in

1457-626: The flap. This second nuclease is DNA2 nuclease , which has a helicase-nuclease activity, that cleaves the long flap of RNA primer, which then leaves behind a couple of nucleotides that are cleaved by FEN1. At the end, when all the RNA primers have been removed, nicks form between the Okazaki fragments that are filled-in with deoxyribonucleotides using an enzyme known as ligase1 , through a process called ligation . Synthetic primers, sometimes known as oligos, are chemically synthesized oligonucleotides , usually of DNA, which can be customized to anneal to

Xenotropic murine leukemia virus–related virus - Misplaced Pages Continue

1504-417: The genomes of two parent MLVs known as preXMRV-1 and preXMRV-2. MLVs belong to the virus family Retroviridae and the genus gammaretrovirus and have a single-stranded RNA genome that replicates through a DNA intermediate. The name XMRV was given because the discoverers of the virus initially thought that it was a novel potential human pathogen that was related to but distinct from MLVs. The XMRV particle

1551-728: The house mouse and black rat, have been introduced worldwide. Murids occupy a broad range of ecosystems from tropical forests to tundras. Fossorial , arboreal , and semiaquatic murid species occur, though most are terrestrial animals . The extensive list of niches filled by murids helps to explain their relative abundance. A broad range of feeding habits is found in murids, ranging from herbivorous and omnivorous species to specialists that consume strictly earthworms, certain species of fungi, or aquatic insects. Most genera consume plant matter and small invertebrates, often storing seeds and other plant matter for winter consumption. Murids have sciurognathous jaws (an ancestral character in rodents) and

1598-479: The insertion of Okazaki fragments , the RNA primers are removed (the mechanism of removal differs between prokaryotes and eukaryotes ) and replaced with new deoxyribonucleotides that fill the gaps where the RNA primer was present. DNA ligase then joins the fragmented strands together, completing the synthesis of the lagging strand. In prokaryotes, DNA polymerase I synthesizes the Okazaki fragment until it reaches

1645-430: The lagging strand, the template DNA runs in the 5′→3′ direction . Since DNA polymerase cannot add bases in the 3′→5′ direction complementary to the template strand, DNA is synthesized ‘backward’ in short fragments moving away from the replication fork, known as Okazaki fragments . Unlike in the leading strand, this method results in the repeated starting and stopping of DNA synthesis, requiring multiple RNA primers. Along

1692-420: The melting temperature of the primers and the annealing temperature of the reaction itself. Moreover, the DNA binding sequence of the primer in vitro has to be specifically chosen, which is done using a method called basic local alignment search tool (BLAST) that scans the DNA and finds specific and unique regions for the primer to bind. RNA primers are used by living organisms in the initiation of synthesizing

1739-404: The mixture; this phenomenon can lead to the production of 'primer dimer' products contaminating the end solution. Primers should also not anneal strongly to themselves, as internal hairpins and loops could hinder the annealing with the template DNA. When designing primers, additional nucleotide bases can be added to the back ends of each primer, resulting in a customized cap sequence on each end of

1786-455: The only two labs which reported positive findings of XMRV in samples which were previously reported as positive (the WPI and NCI/Ruscetti labs) also reported positive findings in samples which were known XMRV negative. Multiple contemporary studies concluded that XMRV was most likely a result of incidental recombination of mouse viruses during prostate cancer research in the 1990s. Positive findings of

1833-400: The previous RNA primer. Then the enzyme simultaneously acts as a 5′→3′ exonuclease , removing primer ribonucleotides in front and adding deoxyribonucleotides behind. Both the activities of polymerization and excision of the RNA primer occur in the 5′→3′ direction,  and polymerase I can do these activities simultaneously; this is known as “Nick Translation”. Nick translation refers to

1880-546: The removal of the RNA nucleotides and the addition of DNA nucleotides. Living organisms use solely RNA primers, while laboratory techniques in biochemistry and molecular biology that require in vitro DNA synthesis (such as DNA sequencing and polymerase chain reaction ) usually use DNA primers, since they are more temperature stable. Primers can be designed in laboratory for specific reactions such as polymerase chain reaction (PCR). When designing PCR primers, there are specific measures that must be taken into consideration, like

1927-427: The sequences are probably similar but not identical. This technique is useful because the genetic code itself is degenerate , meaning several different codons can code for the same amino acid . This allows different organisms to have a significantly different genetic sequence that code for a highly similar protein. For this reason, degenerate primers are also used when primer design is based on protein sequence , as

Xenotropic murine leukemia virus–related virus - Misplaced Pages Continue

1974-472: The specific sequence of codons are not known. Therefore, primer sequence corresponding to the amino acid isoleucine might be "ATH", where A stands for adenine , T for thymine , and H for adenine , thymine , or cytosine , according to the genetic code for each codon , using the IUPAC symbols for degenerate bases . Degenerate primers may not perfectly hybridize with a target sequence, which can greatly reduce

2021-630: The specificity of the PCR amplification. Degenerate primers are widely used and extremely useful in the field of microbial ecology . They allow for the amplification of genes from thus far uncultivated microorganisms or allow the recovery of genes from organisms where genomic information is not available. Usually, degenerate primers are designed by aligning gene sequencing found in GenBank . Differences among sequences are accounted for by using IUPAC degeneracies for individual bases. PCR primers are then synthesized as

2068-487: The synchronized activity of polymerase I in removing the RNA primer and adding deoxyribonucleotides . Later, a gap between the strands is formed called a nick, which is sealed using a DNA ligase . In eukaryotes the removal of RNA primers in the lagging strand is essential for the completion of replication. Thus, as the lagging strand being synthesized by DNA polymerase δ in 5′→3′ direction, Okazaki fragments are formed, which are discontinuous strands of DNA. Then, when

2115-513: The virus were likely due to contamination rather than true presence of the virus in humans. A subsequent analysis also found that the primers used to detect and replicate traces of XMRV in PCR testing are, in fact, neither selective nor specific to XMRV and will actually react to various non-XMRV sequences naturally found in mammalian genomes. In the meantime, multiple other studies also failed to find any link between XMRV and CFS or prostate cancer. As

2162-457: The “ Next-Gen ” method of DNA sequencing require primers to initiate the reaction. The polymerase chain reaction (PCR) uses a pair of custom primers to direct DNA elongation toward each other at opposite ends of the sequence being amplified. These primers are typically between 18 and 24 bases in length and must code for only the specific upstream and downstream sites of the sequence being amplified. A primer that can bind to multiple regions along

2209-496: Was described by J. R. R. Tolkien as perhaps the nearest to his idea of a fairy story , the rest being "beast- fables ". Among Aesop's Fables are The Cat and the Mice and The Frog and the Mouse . In James Herbert 's first novel, The Rats , (1974), a vagrant is attacked and eaten alive by a pack of giant rats; further attacks follow. Primer (molecular biology) A primer

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