What's a replicate line?

What's a replicate line?

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The methods section inside a paper I'm reading make mention of replicate lines.
Example: "We founded 10 replicate lines from a single clone". This is in the context of experimental evolution and artificial selection of Chlamydomonas in a laboratory.

Can you please explain to me what a replicate line is?

Here's a reference to the paper:
Collins, S., Sültemeyer, D., & Bell, G. (2006). Rewinding the tape: selection of algae adapted to high $CO_2$ at current and Pleistocene levels of $CO_2$. Evolution 60, 7, 1392-401. DOI: 10.1111/j.0014-3820.2006.tb01218.x

In the context of experimental evolution, replicate lines are simply separate experimental (or control) lines the are established from the founder population at the beginning of the experiment. In this paper, the authors must have established 10 separate experimental lines that then evolved in different carbon dioxide levels. For example, 10 separate aliquots of algae are drawn from the same source population and then separately subjected to the agent of selection. These replicate lines should remain separate and not be mixed or crossed.

Replicate lines are useful in experimental evolution for a few reasons. Because you are studying a population that is experiencing a selection pressure, you expect that genetic changes are in response to that pressure. However, changes can also occur due to drift. Replicate lines (and a consistent response to selection among them) is important to ensure that changes are a response to selection rather than to some other force (drift, mutation). Some drift is bound to happen when the lines are founded, because you are selecting a subset of the population, which is actually a good thing. Each line gets a random subset of genes which then evolve together. This potentially allows discovery of different genetic pathways to the same adaptive response.

The background and lots of examples are in Garland and Rose, Experimental Evolution (UC Press, 2009).

Cell Cycle

The cell cycle is the process by which a cell grows, duplicates its DNA, and divides into identical daughter cells. Cell cycle duration varies according to cell type and organism. In mammals, cell division occurs over a period of approximately twenty-four hours.

In multicellular organisms, only a subset of cells go through the cycle continuously. Those cells include the stem cells of the hematopoietic system, the basal cells of the skin, and the cells in the bottom of the colon crypts . Other cells, such as those that make up the endocrine glands, as well as liver cells, certain renal (kidney) tubular cells, and cells that belong to connective tissue, exist in a nonreplicating state but can enter the cell cycle after receiving signals from external stimuli. Finally, postmitotic cells are incapable of cell division even after maximal stimulation, and include most neurons, striated muscle cells, and heart muscle cells.

The cell cycle is functionally divided into discrete phases. During the DNA synthesis (S) phase, the cell replicates its chromosomes. During the mitosis (M) phase, the duplicated chromosomes are segregated, migrating to opposite poles of the cell. The cell then divides into two daughter cells, each having the same genetic components as the parental cell. Mammalian cells undergo two gap, or growth, phases (G1 and G2). G1 occurs prior to the S phase, and G2 occurs before the M phase.

Starting DNA replication

The process of DNA replication begins at a specific site along a strand of DNA called the ‘origins of replication’. The origins of replication are short sections on a DNA molecule that contain a specific set of nucleotides.

Prokaryotic cells will often have only one origin of replication for their ring of DNA. Eukaryotic cells on the other hand can have hundreds to thousands of origins.

The process is started by a set of proteins that recognise the set of nucleotides at the origins of replication. These proteins are able to separate the two strands of the DNA double helix and create a ‘bubble’ between the two strands.

DNA replication moves in both directions along the two strands of DNA. The bubble increases in size as several other proteins continue to unwind, straighten and separate the two strands of DNA.

As the two strands are separated, binding proteins latch on to the single strands of DNA and prevent them from bonding back together. Both strands are then able to be used as templates for building two new strands of DNA.

The new strand of DNA begins with a short segment of a molecule called RNA. The short segment is known as an RNA primer and it is usually around 5-10 nucleotides long. The new DNA strand begins by attaching a DNA nucleotide to the RNA primer.

Positive Regulation of the Cell Cycle

Two groups of proteins, called cyclins and cyclin-dependent kinases (Cdks), are responsible for the progress of the cell through the various checkpoints. The levels of the four cyclin proteins fluctuate throughout the cell cycle in a predictable pattern (Figure 2). Increases in the concentration of cyclin proteins are triggered by both external and internal signals. After the cell moves to the next stage of the cell cycle, the cyclins that were active in the previous stage are degraded.

Figure 2 The concentrations of cyclin proteins change throughout the cell cycle. There is a direct correlation between cyclin accumulation and the three major cell cycle checkpoints. Also note the sharp decline of cyclin levels following each checkpoint (the transition between phases of the cell cycle), as cyclin is degraded by cytoplasmic enzymes. (credit: modification of work by “WikiMiMa”/Wikimedia Commons)

Cyclins regulate the cell cycle only when they are tightly bound to Cdks. To be fully active, the Cdk/cyclin complex must also be phosphorylated in specific locations. Like all kinases, Cdks are enzymes (kinases) that phosphorylate other proteins. Phosphorylation activates the protein by changing its shape. The proteins phosphorylated by Cdks are involved in advancing the cell to the next phase (Figure 3). The levels of Cdk proteins are relatively stable throughout the cell cycle however, the concentrations of cyclin fluctuate and determine when Cdk/cyclin complexes form. The different cyclins and Cdks bind at specific points in the cell cycle and thus regulate different checkpoints.

Figure 3 Cyclin-dependent kinases (Cdks) are protein kinases that, when fully activated, can phosphorylate and thus activate other proteins that advance the cell cycle past a checkpoint. To become fully activated, a Cdk must bind to a cyclin protein and then be phosphorylated by another kinase.

Since the cyclic fluctuations of cyclin levels are based on the timing of the cell cycle and not on specific events, regulation of the cell cycle usually occurs by either the Cdk molecules alone or the Cdk/cyclin complexes. Without a specific concentration of fully activated cyclin/Cdk complexes, the cell cycle cannot proceed through the checkpoints.

Separation of Sister Chromatids during Anaphase

During the congression of chromosomes at the metaphase plate, when some kinetochores are unattached to the spindle, an active signal inhibits the onset of anaphase. This involves the Mitotic Checkpoint Complex or the MCC. The MCC contains proteins that primarily inhibit the activity of the Anaphase Promoting Complex (APC).

Unattached kinetochore → Activates Mitotic Checkpoint Complex –| Inhibits Anaphase Promoting Complex

The primary role of the APC is to attach a small regulatory polypeptide called ubiquitin to its target protein. When a protein is tagged with a chain of ubiquitin molecules, it is seen as a signal for the protein to be degraded by the proteasome.

During the metaphase to anaphase transition, APC targets securin and tags it for degradation by the proteasome. The absence of securin allows another enzyme called separase to act on cohesin molecules holding the two chromatids together. When cohesins are no longer resisting the pull of microtubules in the spindle, sister chromatids separate and move towards opposite poles. Cell membrane invagination then leads to the formation of two distinct daughter cells, having one chromatid of each chromosome, therefore becoming genetic copies of the parent cell. Thus, a cascade of reactions leads to the dramatic events of anaphase, and contribute towards making it one of the shortest phases in the cell cycle.

APC → Degradation of securin → Activation of separase → Sister chromatids pulled by spindle

Any deficiency in the cellular levels of cohesin lead to improper segregation and difficulties in the alignment of chromosomes on the metaphase plate. This results in aneuploidy, where daughter cells have an irregular number of chromosomes.

Basics of DNA Replication

Figure 1. The three suggested models of DNA replication. Grey indicates the original DNA strands, and blue indicates newly synthesized DNA.

The elucidation of the structure of the double helix provided a hint as to how DNA divides and makes copies of itself. This model suggests that the two strands of the double helix separate during replication, and each strand serves as a template from which the new complementary strand is copied. What was not clear was how the replication took place. There were three models suggested: conservative, semi-conservative, and dispersive (see Figure 1).

In conservative replication, the parental DNA remains together, and the newly formed daughter strands are together. The semi-conservative method suggests that each of the two parental DNA strands act as a template for new DNA to be synthesized after replication, each double-stranded DNA includes one parental or “old” strand and one “new” strand. In the dispersive model, both copies of DNA have double-stranded segments of parental DNA and newly synthesized DNA interspersed.

Meselson and Stahl were interested in understanding how DNA replicates. They grew E. coli for several generations in a medium containing a “heavy” isotope of nitrogen ( 15 N) that gets incorporated into nitrogenous bases, and eventually into the DNA (Figure 2).

Figure 2. Meselson and Stahl experimented with E. coli grown first in heavy nitrogen ( 15 N) then in 14 N. DNA grown in 15 N (red band) is heavier than DNA grown in 14 N (orange band), and sediments to a lower level in cesium chloride solution in an ultracentrifuge. When DNA grown in 15 N is switched to media containing 14 N, after one round of cell division the DNA sediments halfway between the 15 N and 14 N levels, indicating that it now contains fifty percent 14 N. In subsequent cell divisions, an increasing amount of DNA contains 14 N only. This data supports the semi-conservative replication model. (credit: modification of work by Mariana Ruiz Villareal)

The E. coli culture was then shifted into medium containing 14 N and allowed to grow for one generation. The cells were harvested and the DNA was isolated. The DNA was centrifuged at high speeds in an ultracentrifuge. Some cells were allowed to grow for one more life cycle in 14 N and spun again. During the density gradient centrifugation, the DNA is loaded into a gradient (typically a salt such as cesium chloride or sucrose) and spun at high speeds of 50,000 to 60,000 rpm. Under these circumstances, the DNA will form a band according to its density in the gradient. DNA grown in 15 N will band at a higher density position than that grown in 14 N. Meselson and Stahl noted that after one generation of growth in 14 N after they had been shifted from 15 N, the single band observed was intermediate in position in between DNA of cells grown exclusively in 15 N and 14 N. This suggested either a semi-conservative or dispersive mode of replication. The DNA harvested from cells grown for two generations in 14 N formed two bands: one DNA band was at the intermediate position between 15 N and 14 N, and the other corresponded to the band of 14 N DNA. These results could only be explained if DNA replicates in a semi-conservative manner. Therefore, the other two modes were ruled out.

During DNA replication, each of the two strands that make up the double helix serves as a template from which new strands are copied. The new strand will be complementary to the parental or “old” strand. When two daughter DNA copies are formed, they have the same sequence and are divided equally into the two daughter cells.

In Summary: Basics of DNA Replication

The model for DNA replication suggests that the two strands of the double helix separate during replication, and each strand serves as a template from which the new complementary strand is copied. In conservative replication, the parental DNA is conserved, and the daughter DNA is newly synthesized. The semi-conservative method suggests that each of the two parental DNA strands acts as template for new DNA to be synthesized after replication, each double-stranded DNA includes one parental or “old” strand and one “new” strand. The dispersive mode suggested that the two copies of the DNA would have segments of parental DNA and newly synthesized DNA. Experimental evidence showed DNA replication is semi-conservative.


Animal Coronaviruses

Coronaviruses cause a large variety of diseases in animals, and their ability to cause severe disease in livestock and companion animals such as pigs, cows, chickens, dogs, and cats led to significant research on these viruses in the last half of the twentieth century. For instance, Transmissible Gastroenteritis Virus (TGEV) and Porcine Epidemic Diarrhea Virus (PEDV) cause severe gastroenteritis in young piglets, leading to significant morbidity, mortality, and ultimately economic losses. PEDV recently emerged in North America for the first time, causing significant losses of young piglets. Porcine hemagglutinating encephalomyelitis virus (PHEV) mostly leads to enteric infection but has the ability to infect the nervous system, causing encephalitis, vomiting, and wasting in pigs. Feline enteric coronavirus (FCoV) causes a mild or asymptomatic infection in domestic cats, but during persistent infection, mutation transforms the virus into a highly virulent strain of FCoV, Feline Infectious Peritonitis Virus (FIPV), that leads to development of a lethal disease called feline infectious peritonitis (FIP). FIP has wet and dry forms, with similarities to the human disease, sarcoidosis. FIPV is macrophage tropic and it is believed that it causes aberrant cytokine and/or chemokine expression and lymphocyte depletion, resulting in lethal disease [63]. However, additional research is needed to confirm this hypothesis. Bovine CoV, Rat CoV, and Infectious Bronchitis Virus (IBV) cause mild to severe respiratory tract infections in cattle, rats, and chickens, respectively. Bovine CoV causes significant losses in the cattle industry and also has spread to infect a variety of ruminants, including elk, deer, and camels. In addition to severe respiratory disease, the virus causes diarrhea (“winter dysentery” and “shipping fever”), all leading to weight loss, dehydration, decreased milk production, and depression [63]. Some strains of IBV, a γ-coronavirus, also affect the urogenital tract of chickens causing renal disease. Infection of the reproductive tract with IBV significantly diminishes egg production, causing substantial losses in the egg-production industry each year [63]. More recently, a novel coronavirus named SW1 has been identified in a deceased Beluga whale [64]. Large numbers of virus particles were identified in the liver of the deceased whale with respiratory disease and acute liver failure. Although, electron microscopic images were not sufficient to identify the virus as a coronavirus, sequencing of the liver tissue clearly identified the virus as a coronavirus. It was subsequently determined to be a γ-coronavirus based on phylogenetic analysis but it has not yet been verified experimentally that this virus is actually a causative agent of disease in whales. In addition, there has been intense interest in identifying novel bat CoVs, since these are the likely ancestors for SARS-CoV and MERS-CoV, and hundreds of novel bat coronaviruses have been identified over the past decade [65]. Finally, another novel family of nidoviruses, Mesoniviridae, has been recently identified as the first nidoviruses to exclusively infect insect hosts [66, 67]. These viruses are highly divergent from other nidoviruses but are most closely related to the roniviruses. In size, they are

20 kb, falling in between large and small nidoviruses. Interestingly, these viruses do not encode for an endoribonuclease, which is present in all other nidoviruses. These attributes suggest these viruses are the prototype of a new nidovirus family and may be a missing link in the transition from small to large nidoviruses.

The most heavily studied animal coronavirus is murine hepatitis virus (MHV), which causes a variety of outcomes in mice, including respiratory, enteric, hepatic, and neurologic infections. These infections often serve as highly useful models of disease. For instance, MHV-1 causes severe respiratory disease in susceptible A/J and C3H/HeJ mice, A59 and MHV-3 induce severe hepatitis, while JHMV causes severe encephalitis. Interestingly, MHV-3 induces cellular injury through the activation of the coagulation cascade [68]. Most notably, A59 and attenuated versions of JHMV cause a chronic demyelinating disease that bears similarities to multiple sclerosis (MS), making MHV infection one of the best models for this debilitating human disease. Early studies suggested that demyelination was dependent on viral replication in oligodendrocytes in the brain and spinal cord [69, 70] however, more recent reports clearly demonstrate that the disease is immune-mediated. Irradiated mice or immunodeficient (lacking T and B cells) mice do not develop demyelination, but addition of virus-specific T cells restores the development of demyelination [71, 72]. Additionally, demyelination is accompanied by a large influx of macrophages and microglia that can phagocytose infected myelin [73], although it is unknown what the signals are that direct immune cells to destroy myelin. Finally, MHV can be studied under BSL2 laboratory conditions, unlike SARS-CoV or MERS-CoV, which require a BSL3 laboratory, and provides a large number of suitable animal models. These factors make MHV an ideal model for studying the basics of viral replication in tissue culture cells as well as for studying the pathogenesis and immune response to coronaviruses.

Human Coronaviruses

Prior to the SARS-CoV outbreak, coronaviruses were only thought to cause mild, self-limiting respiratory infections in humans. Two of these human coronaviruses are α-coronaviruses, HCoV-229E and HCoV-NL63, while the other two are β-coronaviruses, HCoV-OC43 and HCoV-HKU1. HCoV-229E and HCoV-OC43 were isolated nearly 50 years ago [74�], while HCoV-NL63 and HCoV-HKU1 have only recently been identified following the SARS-CoV outbreak [77, 78]. These viruses are endemic in the human populations, causing 15� % of respiratory tract infections each year. They cause more severe disease in neonates, the elderly, and in individuals with underlying illnesses, with a greater incidence of lower respiratory tract infection in these populations. HCoV-NL63 is also associated with acute laryngotracheitis (croup) [79]. One interesting aspect of these viruses is their differences in tolerance to genetic variability. HCoV-229E isolates from around the world have only minimal sequence divergence [80], while HCoV-OC43 isolates from the same location but isolated in different years show significant genetic variability [81]. This likely explains the inability of HCoV-229E to cross the species barrier to infect mice while HCoV-OC43 and the closely related bovine coronavirus, BCoV, are capable of infecting mice and several ruminant species. Based on the ability of MHV to cause demyelinating disease, it has been suggested that human CoVs may be involved in the development of multiple sclerosis (MS). However, no evidence to date suggests that human CoVs play a significant role in MS.

SARS-CoV, a group 2b β-coronavirus, was identified as the causative agent of the Severe Acute Respiratory Syndrome (SARS) outbreak that occurred in 2002� in the Guangdong Province of China. It is the most severe human disease caused by any coronavirus. During the 2002� outbreak approximately 8,098 cases occurred with 774 deaths, resulting in a mortality rate of 9 %. This rate was much higher in elderly individuals, with mortality rates approaching 50 % in individuals over 60 years of age. Furthermore, the outbreak resulted in the loss of nearly $40 billion dollars in economic activity, as the virus nearly shut down many activities in Southeast Asia and Toronto, Canada for several months. The outbreak began in a hotel in Hong Kong and ultimately spread to more than two dozen countries. During the epidemic, closely related viruses were isolated from several exotic animals including Himalayan palm civets and raccoon dogs [82]. However, it is widely accepted that SARS-CoV originated in bats as a large number of Chinese horseshoe bats contain sequences of SARS-related CoVs and contain serologic evidence for a prior infection with a related CoV [83, 84]. In fact, two novel bat SARS-related CoVs have been recently identified that are more similar to SARS-CoV than any other virus identified to date [85]. They were also found to use the same receptor as the human virus, angiotensin converting enzyme 2 (ACE2), providing further evidence that SARS-CoV originated in bats. Although some human individuals within wet animal markets had serologic evidence of SARS-CoV infection prior to the outbreak, these individuals had no apparent symptoms [82]. Thus, it is likely that a closely related virus circulated in the wet animal markets for several years before a series of factors facilitated its spread into the larger population.

Transmission of SARS-CoV was relatively inefficient, as it only spread through direct contact with infected individuals after the onset of illness. Thus, the outbreak was largely contained within households and healthcare settings [86], except in a few cases of superspreading events where one individual was able to infect multiple contacts due to an enhanced development of high viral burdens or ability to aerosolize virus. As a result of the relatively inefficient transmission of SARS-CoV, the outbreak was controllable through the use of quarantining. Only a small number of SARS cases occurred after the outbreak was controlled in June 2003.

SARS-CoV primarily infects epithelial cells within the lung. The virus is capable of entering macrophages and dendritic cells but only leads to an abortive infection [87, 88]. Despite this, infection of these cell types may be important in inducing pro-inflammatory cytokines that may contribute to disease [89]. In fact, many cytokines and chemokines are produced by these cell types and are elevated in the serum of SARS-CoV infected patients [90]. The exact mechanism of lung injury and cause of severe disease in humans remains undetermined. Viral titers seem to diminish when severe disease develops in both humans and in several animal models of the disease. Furthermore, animals infected with rodent-adapted SARS-CoV strains show similar clinical features to the human disease, including an age-dependent increase in disease severity [91]. These animals also show increased levels of proinflammatory cytokines and reduced T-cell responses, suggesting a possible immunopathological mechanism of disease [92, 93].

While the SARS-CoV epidemic was controlled in 2003 and the virus has not since returned, a novel human CoV emerged in the Middle East in 2012. This virus, named Middle East Respiratory Syndrome-CoV (MERS-CoV), was found to be the causative agent in a series of highly pathogenic respiratory tract infections in Saudi Arabia and other countries in the Middle East [94]. Based on the high mortality rate of

50 % in the early stages of the outbreak, it was feared the virus would lead to a very serious outbreak. However, the outbreak did not accelerate in 2013, although sporadic cases continued throughout the rest of the year. In April 2014, a spike of over 200 cases and almost 40 deaths occurred, prompting fears that the virus had mutated and was more capable of human-to-human transmission. More likely, the increased number of cases resulted from improved detection and reporting methods combined with a seasonal increase in birthing camels. As of August 27th, 2014 there have been a total of 855 cases of MERS-CoV, with 333 deaths and a case fatality rate of nearly 40 %, according to the European Center for Disease Prevention and Control.

MERS-CoV is a group 2c β-coronavirus highly related to two previously identified bat coronaviruses, HKU4 and HKU5 [95]. It is believed that the virus originated from bats, but likely had an intermediate host as humans rarely come in contact with bat secreta. Serological studies have identified MERS-CoV antibodies in dromedary camels in the Middle East [96], and cell lines from camels have been found to be permissive for MERS-CoV replication [97] providing evidence that dromedary camels may be the natural host. More convincing evidence for this comes from recent studies identifying nearly identical MERS-CoVs in both camels and human cases in nearby proximities in Saudi Arabia [98, 99]. In one of these studies the human case had direct contact with an infected camel and the virus isolated from this patient was identical to the virus isolated from the camel [99]. At the present time it remains to be determined how many MERS-CoV cases can be attributed to an intermediate host as opposed to human-to-human transmission. It has also been postulated that human-to-camel spread contributed to the outbreak.

MERS-CoV utilizes Dipeptidyl peptidase 4 (DPP4) as its receptor [100]. The virus is only able to use the receptor from certain species such as bats, humans, camels, rabbits, and horses to establish infection. Unfortunately for researchers, the virus is unable to infect mouse cells due to differences in the structure of DPP4, making it difficult to evaluate potential vaccines or antivirals. Recently, a small animal model for MERS-CoV has been developed using an Adenoviral vector to introduce the human DPP4 gene into mouse lungs [101]. This unique system makes it possible to test therapeutic interventions and novel vaccines for MERS-CoV in any animal sensitive to adenoviral transductions.

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The researchers will apply the existing theory to new situations in order to determine generalizability to different subjects, age groups, races, locations, cultures or any such variables.

The main determinants of this study include:

To assure that results are reliable and valid

To determine the role of extraneous variables

To apply the previous results to new situations

To inspire new research combing previous findings from related studies

Suppose you are part of a healthcare team facing a problem, for instance, regarding use and efficacy of certain "pain killer medicine" in patients before surgery. You search the literature for same problem and indentify an article exactly addressing "this" problem.

Now question arise that how can you be sure that the results of this study in hand are applicable and transferable into "your" clinical setting? Therefore you decide to focus on preparation and implementation of a replication study. You will perform the deliberate repetition of previous research procedures in your clinical setting and thus will be able to strengthen the evidence of previous research finding, and correct limitations, and thus overall results may be in favor of the results of previous study or you may find completely different results.

A question may arise that how to decide if a replication study can be carried out or not? Following are the guidelines or criteria proposed to replicate an original study:

A replication study is possible and should be carried out, when

The original research question is important and can contribute to the body of information supporting the discipline

The existing literature and policies relating to the topic are supporting the topic for its relevance

The replication study if carried out carries the potential to empirically support the results of the original study, either by clarifying issues raised by the original study or extending its generalizability.

The team of researchers has all expertise in the subject area and also has the access to adequate information related to original study to be able to design and execute a replication.

Any extension or modifications of the original study can be based on current knowledge in the same field.

Lastly, the replication of the same rigor as was in original study is possible.

In field conditions, more opportunities are available to researchers that are not open to investigations in laboratory settings.

Also, laboratory investigators commonly have only small number of potential participants in their research trials. However in applied settings such as schools, classrooms, patients at hospitals or other settings with large proportion of participants are often generously available in field settings.

It is therefore possible in field settings to repeat or replicate a research on large scale and more than once too.

Mechanism of Replication (Basic)

Knowing the structure of DNA, scientists speculated and then proved that DNA is the template for copying the genetic code. See how information in DNA is copied to make new DNA molecules.

Duration: 1 minutes, 5 seconds

Using computer animation based on molecular research, we are now able to see how DNA is actually Using computer animation based on molecular research, we are now able to see how DNA is actually copied in living cells. You are looking at an assembly line of amazing miniature biochemical machines that are pulling apart the DNA double helix and cranking out a copy of each strand. The DNA to be copied enters the production line from bottom left. The whirling blue molecular machine is called helicase. It spins the DNA as fast as a jet engine as it unwinds the double helix into two strands. One strand is copied continuously and can be seen spooling off to the right. Things are not so simple for the other strand because it must be copied backwards. It is drawn out repeatedly in loops, and copied one section at a time. The end result is two new DNA molecules.

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Overview of Autophagy

Selective Degradation of Retrotransposon (Transposon via DNA Intermediates)

Retrotransposons (RTPs) are ubiquitous components of the DNA of many eukaryotic organisms, especially that of plants. Approximately, 45–48% of the human genome is composed of RTPs or their remnants, and

42% of such genome is made up of RTPs DNA transposons account for only

2–3% ( Lander et al., 2001 ). The DNA sequences are first transcribed into RNA, then converted back into identical DNA sequences via reverse transcription. Finally, these sequences are inserted into the genome at target sites.

RTPs are a major source of genetic variation among species, individuals, and cells in a single individual ( Guo et al., 2014 ). As stated above, RTPs reinsert themselves in the human genome, which results not only in genetic changes but also involvement in a large number of diseases ( Hancks and Kazazian, 2012 ). Considering the important role played by RTPs in health and disease (genetic variation), the importance of elucidating the molecular mechanism underlying targeting P bodies and eliminating stress granules is apparent.

The replicative mode of RTPs via an RNA intermediate results in a rapid increase in the copy numbers of these elements, which increases the genome size. Thus RTPs can induce mutations by inserting themselves near or within genes. Such mutations are relatively stable because the sequence at the insertion site is retained as they transpose via the replication mechanism.

A recent study indicates that autophagy degrades LINE-1 RNA and tempers the rate of insertion of LINE-1 and Alu RTPs by using autophagy receptors NDP52 and p62 ( Guo et al., 2014 ). Autophagy also selectively degrades ribonucleic acids (RNAs) using autophagy receptors such as NDP52 (CALCOO2 gene symbol) and p62 (SQSTM1 gene symbol). This can be accomplished because autophagosome-lysosome also delivers catabolic enzymes, including RNAses for the degradation of RNAs.


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