Until 2015 all known mammalian prion diseases were considered to be caused by the prion protein,  PrP ; in 2015  multiple system atrophy  was found to be transmissible and was hypothesized to be caused by a new prion, the misfolded form of a protein called  alpha-synuclein . [ 10 ]  The endogenous, properly folded form of the prion protein is denoted PrP C  (for  C ommon  or  C ellular ), whereas the disease-linked, misfolded form is denoted PrP Sc  (for  Sc rapie ), after one of the diseases first linked to prions and neurodegeneration. [ 26 ] [ 11 ]  The precise structure of the prion is not known, though they can be formed spontaneously by combining PrP C , homopolymeric polyadenylic acid, and lipids in a  protein misfolding cyclic amplification  (PMCA) reaction even in the absence of pre-existing infectious prions. [ 69 ]  This result is further evidence that prion replication does not require genetic information. [ 85 ]


It has been recognized that prion diseases can arise in three different ways: acquired, familial, or sporadic. [ 86 ]  It is often assumed that the diseased form directly interacts with the normal form to make it rearrange its structure. One idea, the "Protein X" hypothesis, is that an as-yet unidentified cellular protein (Protein X) enables the conversion of PrP C  to PrP Sc  by bringing a molecule of each of the two together into a complex. [ 87 ]


The primary method of infection in animals is through ingestion. It is thought that prions may be deposited in the environment through the remains of dead animals and via urine, saliva, and other body fluids. They may then linger in the soil by binding to clay and other minerals. [ 88 ]


A  University of California  research team has provided evidence for the theory that infection can occur from prions in manure. [ 89 ]  And, since manure is present in many areas surrounding water reservoirs, as well as used on many crop fields, it raises the possibility of widespread transmission. Although it was initially reported in January 2011 that researchers had discovered prions spreading through airborne transmission on  aerosol  particles in an  animal testing  experiment focusing on  scrapie  infection in  laboratory mice , [ 90 ]  this report was retracted in 2024. [ 90 ]  Preliminary evidence supporting the notion that prions can be transmitted through use of urine-derived  human menopausal gonadotropin , administered for the treatment of  infertility , was published in 2011. [ 91 ]


The majority of human prion diseases are classified as sporadic Creutzfeldt–Jakob disease (sCJD). Genetic research has identified an association between susceptibility to sCJD and a polymorphism at codon 129 in the PRNP gene, which encodes the prion protein (PrP). A homozygous methionine/methionine (MM) genotype at this position has been shown to significantly increase the risk of developing sCJD when compared to a heterozygous methionine/valine (MV) genotype. Analysis of multiple studies has shown that individuals with the MM genotype are approximately five times more likely to develop sCJD than those with the MV genotype. [ 92 ]


In 2015, researchers at  The University of Texas Health Science Center at Houston  found that plants can be a vector for prions. When researchers fed hamsters grass that grew on ground where a deer that died with  chronic wasting disease  (CWD) was buried, the hamsters became ill with CWD, suggesting that prions can bind to plants, which then take them up into the leaf and stem structure, where they can be eaten by herbivores, thus completing the cycle. It is thus possible that there is a progressively accumulating number of prions in the environment. [ 93 ] [ 94 ]


Infectious particles possessing  nucleic acid  are dependent upon it to direct their continued replication. Prions, however, are infectious by their effect on normal versions of the protein. Sterilizing prions, therefore, requires the  denaturation  of the protein to a state in which the molecule is no longer able to induce the abnormal folding of normal proteins. In general, prions are quite resistant to  proteases , heat,  ionizing radiation , and  formaldehyde  treatments, [ 95 ]  although their infectivity can be reduced by such treatments. Effective prion decontamination relies upon protein  hydrolysis  or reduction or destruction of  protein tertiary structure . Examples include  sodium hypochlorite ,  sodium hydroxide , and strongly acidic  detergents  such as LpH. [ 96 ]


The  World Health Organization  recommends any of the following three procedures for the sterilization of all heat-resistant surgical instruments to ensure that they are not contaminated with prions:


134 °C (273 °F) for 18 minutes in a pressurized steam  autoclave  has been found to be somewhat effective in deactivating the agent of disease. [ 98 ] [ 99 ]   Ozone  sterilization has been studied as a potential method for prion denaturation and deactivation. [ 100 ]  Other approaches being developed include  thiourea - urea  treatment,  guanidinium chloride  treatment, [ 101 ]  and special heat-resistant  subtilisin  combined with heat and detergent. [ 102 ]  A method sufficient for sterilizing prions on one material may fail on another. [ 103 ]


Renaturation of a completely denatured prion to infectious status has not yet been achieved; however, partially denatured prions can be renatured to an infective status under certain artificial conditions. [ 104 ]


Overwhelming evidence shows that prions resist degradation and persist in the environment for years, and  proteases  do not degrade them. Experimental evidence shows that  unbound  prions degrade over time, while soil-bound prions remain at stable or increasing levels, suggesting that prions likely accumulate in the environment. [ 105 ] [ 106 ]  One 2015 study by US scientists found that repeated drying and wetting may render soil bound prions less infectious, although this was dependent on the soil type they were bound to. [ 107 ]


More recent studies suggest scrapie prions can be degraded by diverse cellular machinery. Inhibition of autophagy accelerates prion accumulation whereas encouragement of autophagy promotes prion clearance. [ 108 ]  The  ubiquitin proteasome system  appears to be able to degrade small enough aggregates. [ 108 ]  In addition,  keratinase  from  B. licheniformis , [ 109 ] [ 110 ]  alkaline  serine protease  from  Streptomyces sp , [ 111 ]   subtilisin -like pernisine from  Aeropyrum pernix , [ 112 ]  alkaline protease from  Nocardiopsis  sp , [ 113 ]   nattokinase  from  B. subtilis , [ 114 ]  engineered subtilisins from  B. lentus [ 115 ] [ 116 ]  and serine protease from three lichen species [ 117 ]  have been found to degrade PrP Sc .


Proteins showing prion-type behavior are also found in some  fungi , which has been useful in helping to understand mammalian prions.  Fungal prions  do not always cause disease in their hosts. [ 118 ]  In yeast, protein refolding to the prion configuration is assisted by  chaperone proteins  such as  Hsp104 . [ 119 ]  All known prions induce the formation of an  amyloid  fold, in which the protein polymerises into an aggregate consisting of tightly packed  beta sheets . Amyloid aggregates are fibrils, growing at their ends, and replicate when breakage causes two growing ends to become four growing ends. The  incubation period  of prion diseases is determined by the  exponential growth  rate associated with prion replication, which is a balance between the linear growth and the breakage of aggregates. [ 66 ]


Fungal proteins exhibiting templated conformational change [ further explanation needed ]  were discovered in the yeast  Saccharomyces cerevisiae  by  Reed Wickner  in the early 1990s. For their mechanistic similarity to mammalian prions, they were termed  yeast prions . Subsequent to this, a prion has also been found in the fungus  Podospora anserina . These prions behave similarly to PrP, but, in general, are nontoxic to their hosts.  Susan Lindquist 's group at the  Whitehead Institute  has argued some of the fungal prions are not associated with any disease state, but may have a useful role; however, researchers at the NIH have also provided arguments suggesting that fungal prions could be considered a diseased state. [ 120 ]  There is evidence that fungal proteins have evolved specific functions that are beneficial to the microorganism that enhance their ability to adapt to their diverse environments. [ 121 ]  Further, within yeasts, prions can act as vectors of  epigenetic  inheritance, transferring traits to offspring without any  genomic  change. [ 122 ] [ 123 ]


Research into  fungal prions  has given strong support to the protein-only concept, since purified protein extracted from cells with a prion state has been demonstrated to convert the normal form of the protein into a misfolded form  in vitro , and in the process, preserve the information corresponding to different strains of the prion state. It has also shed some light on prion domains, which are regions in a protein that promote the conversion into a prion. Fungal prions have helped to suggest mechanisms of conversion that may apply to all prions, though fungal prions appear distinct from infectious mammalian prions in the lack of cofactor required for propagation. The characteristic prion domains may vary between species – e.g., characteristic fungal prion domains are not found in mammalian prions. [ citation needed ]


There are no effective treatments for prion diseases. [ 125 ]  Clinical trials in humans have not met with success and have been hampered by the rarity of prion diseases. [ 125 ]  Although some potential treatments have shown promise in the laboratory, none have been effective once the disease has commenced. [ 126 ]


Prion-like domains have been found in a variety of other mammalian proteins. Some of these proteins have been implicated in the ontogeny of age-related neurodegenerative disorders such as  amyotrophic lateral sclerosis  (ALS),  frontotemporal lobar degeneration with ubiquitin-positive inclusions  (FTLD-U),  Alzheimer's disease ,  Parkinson's disease , and  Huntington's disease . [ 127 ] [ 128 ] [ 12 ]  They are also implicated in some forms of systemic  amyloidosis  including  AA amyloidosis  that develops in humans and animals with inflammatory and infectious diseases such as  tuberculosis ,  Crohn's disease ,  rheumatoid arthritis , and  HIV/AIDS . AA amyloidosis, like prion disease, may be transmissible. [ 129 ]  This has given rise to the 'prion paradigm', where otherwise harmless proteins can be converted to a pathogenic form by a small number of misfolded, nucleating proteins. [ 130 ]


The definition of a prion-like domain arises from the study of fungal prions. In yeast, prionogenic proteins have a portable prion domain that is both necessary and sufficient for self-templating and protein aggregation. This has been shown by attaching the prion domain to a reporter protein, which then aggregates like a known prion. Similarly, removing the prion domain from a fungal prion protein inhibits prionogenesis. This modular view of prion behaviour has led to the hypothesis that similar prion domains are present in animal proteins, in addition to PrP. [ 127 ]  These fungal prion domains have several characteristic sequence features. They are typically enriched in asparagine, glutamine, tyrosine and glycine residues, with an asparagine bias being particularly conducive to the aggregative property of prions. Historically, prionogenesis has been seen as independent of sequence and only dependent on relative residue content. However, this has been shown to be false, with the spacing of prolines and charged residues having been shown to be critical in amyloid formation. [ 131 ]


Bioinformatic screens have predicted that over 250 human proteins contain prion-like domains (PrLD). These domains are hypothesized to have the same transmissible, amyloidogenic properties of PrP and known fungal proteins. As in yeast, proteins involved in gene expression and RNA binding seem to be particularly enriched in PrLD's, compared to other classes of protein. In particular, 29 of the known 210 proteins with an RNA recognition motif also have a putative prion domain. Meanwhile, several of these RNA-binding proteins have been independently identified as pathogenic in cases of ALS, FTLD-U, Alzheimer's disease, and Huntington's disease. [ 132 ]


The pathogenicity of prions and proteins with prion-like domains is hypothesized to arise from their self-templating ability and the resulting exponential growth of amyloid fibrils. The presence of  amyloid  fibrils in patients with degenerative diseases has been well documented. These amyloid fibrils are seen as the result of pathogenic proteins that self-propagate and form highly stable, non-functional aggregates. [ 132 ]  While this does not necessarily imply a causal relationship between amyloid and degenerative diseases, the toxicity of certain amyloid forms and the overproduction of amyloid in familial cases of degenerative disorders supports the idea that amyloid formation is generally toxic. [ 133 ]


Specifically, aggregation of  TDP-43 , an RNA-binding protein, has been found in ALS/MND patients, and mutations in the genes coding for these proteins have been identified in familial cases of ALS/MND. These mutations promote the misfolding of the proteins into a prion-like conformation. The misfolded form of TDP-43 forms cytoplasmic inclusions in affected neurons, and is found depleted in the nucleus. In addition to ALS/MND and FTLD-U, TDP-43 pathology is a feature of many cases of Alzheimer's disease, Parkinson's disease and Huntington's disease. The misfolding of TDP-43 is largely directed by its prion-like domain. This domain is inherently prone to misfolding, while pathological mutations in TDP-43 have been found to increase this propensity to misfold, explaining the presence of these mutations in familial cases of ALS/MND. As in yeast, the prion-like domain of TDP-43 has been shown to be both necessary and sufficient for protein misfolding and aggregation. [ 127 ]


Similarly, pathogenic mutations have been identified in the prion-like domains of heterogeneous nuclear riboproteins hnRNPA2B1 and hnRNPA1 in familial cases of muscle, brain, bone and motor neuron degeneration. The wild-type form of all of these proteins show a tendency to self-assemble into amyloid fibrils, while the pathogenic mutations exacerbate this behaviour and lead to excess accumulation. [ 134 ]


Prions could theoretically be employed as a  weaponized agent . [ 135 ] [ 136 ]  With potential fatality rates of 100%, prions could be an effective bioweapon, sometimes called a "biochemical weapon", because a prion is a biochemical. An unfavorable aspect is prions' very long incubation periods. Persistent heavy exposure of prions to the  intestine  might shorten the overall onset. [ 137 ]  Another aspect of using prions in warfare is the difficulty of detection and  decontamination . [ 138 ]


In the 18th and 19th centuries, exportation of sheep from Spain was observed to coincide with a disease called  scrapie . This disease caused the affected animals to  "lie down, bite at their feet and legs, rub their backs against posts, fail to thrive, stop feeding and finally become lame" . [ 139 ]  The disease was also observed to have the long incubation period that is a key characteristic of  transmissible spongiform encephalopathies (TSEs) . Although the cause of scrapie was not known back then, it is probably the first transmissible spongiform encephalopathy to be recorded. [ 140 ]


In the 1950s,  Carleton Gajdusek  began research which eventually showed that  kuru  could be transmitted to chimpanzees by what was possibly a new infectious agent, work for which he eventually won the 1976  Nobel prize . During the 1960s, two London-based researchers, radiation biologist  Tikvah Alper  and biophysicist  John Stanley Griffith , developed the hypothesis that the  transmissible spongiform encephalopathies  are caused by an infectious agent consisting solely of proteins. [ 141 ] [ 142 ]  Earlier investigations by  E.J. Field  into  scrapie  and kuru had found evidence for the transfer of pathologically inert polysaccharides that only become infectious post-transfer, in the new host. [ 143 ] [ 144 ]  Alper and Griffith wanted to account for the discovery that the mysterious infectious agent causing the diseases scrapie and  Creutzfeldt–Jakob disease  resisted  ionizing radiation . [ 145 ]  Griffith proposed three ways in which a protein could be a  pathogen . [ 142 ]


In the first  hypothesis , he suggested that if the protein is the product of a normally suppressed  gene , and introducing the protein could induce the gene's expression, that is, wake the dormant gene up, then the result would be a process indistinguishable from replication, as the gene's expression would produce the protein, which would then wake the gene in other  cells . [ citation needed ]


His second hypothesis forms the basis of the modern prion theory, and proposed that an abnormal form of a cellular protein can convert normal proteins of the same type into its abnormal form, thus leading to replication. [ citation needed ]


His third hypothesis proposed that the agent could be an  antibody  if the antibody was its own target  antigen , as such an antibody would result in more and more antibody being produced against itself. However, Griffith acknowledged that this third hypothesis was unlikely to be true due to the lack of a detectable  immune response . [ 146 ]


Francis Crick  recognized the potential significance of the Griffith protein-only hypothesis for scrapie propagation in the second edition of his " Central dogma of molecular biology " (1970): While asserting that the flow of sequence information from protein to protein, or from protein to RNA and DNA was "precluded", he noted that Griffith's hypothesis was a potential contradiction (although it was not so promoted by Griffith). [ 147 ]  The revised hypothesis was later formulated, in part, to accommodate  reverse transcription  (which both  Howard Temin  and  David Baltimore  discovered in 1970). [ 148 ]


In 1982,  Stanley B. Prusiner  of the  University of California, San Francisco , announced that his team had purified the hypothetical infectious protein, which did not appear to be present in healthy hosts, though they did not manage to isolate the protein until two years after Prusiner's announcement. [ 149 ] [ 14 ]  The protein was named a prion, for "proteinacious infectious particle", derived from the words protein and infection. When the prion was discovered, Griffith's first hypothesis, that the protein was the product of a normally silent gene was favored by many. It was subsequently discovered, however, that the same protein exists in normal hosts but in different form. [ 150 ]


Following the discovery of the same protein in different form in uninfected individuals, the specific protein that the prion was composed of was named the prion protein (PrP), and Griffith's second hypothesis that an abnormal form of a host protein can convert other proteins of the same type into its abnormal form, became the dominant theory. [ 146 ]  Prusiner was awarded the  Nobel Prize in Physiology or Medicine  in 1997 for his research into prions. [ 151 ] [ 152 ]


A  pronucleus  ( pl. :  pronuclei ) denotes the  nucleus  found in either a  sperm  or  egg cell  during the process of  fertilization . The  sperm cell  undergoes a transformation into a pronucleus after entering the egg cell but prior to the fusion of the genetic material of both the sperm and egg. In contrast, the egg cell possesses a pronucleus once it becomes  haploid , not upon the arrival of the sperm cell. Haploid cells, such as sperm and egg cells in humans, carry half the number of  chromosomes  present in  somatic cells , with 23 chromosomes compared to the 46 found in somatic cells. It is noteworthy that the male and female pronuclei do not physically merge, although their genetic material does. Instead, their membranes dissolve, eliminating any barriers between the male and female chromosomes, facilitating the combination of their chromosomes into a single  diploid  nucleus in the resulting  embryo , which contains a complete set of 46 chromosomes.


The presence of two pronuclei serves as the initial indication of successful fertilization, often observed around 18 hours after  insemination , or  intracytoplasmic sperm injection  (ICSI) during  in vitro fertilization . At this stage, the  zygote  is termed a  two-pronuclear zygote  (2PN). Two-pronuclear zygotes transitioning through 1PN or 3PN states tend to yield poorer-quality embryos compared to those maintaining 2PN status throughout development, [ 1 ]  and this distinction may hold significance in the  selection of embryos  during in vitro fertilization (IVF) procedures.


The pronucleus was discovered the 1870s  microscopically  using  staining  techniques combined with  microscopes  with improved magnification levels. The pronucleus was originally found during the first studies on  meiosis .  Edouard Van Beneden  published a paper in 1875 in which he first mentions the pronucleus by studying the eggs of rabbits and bats. He stated that the two pronuclei form together in the center of the cell to form the embryonic nucleus. Van Beneden also found that the sperm enters into the cell through the membrane in order to form the male pronucleus. In 1876,  Oscar Hertwig  did a study on sea urchin eggs because the eggs of sea urchins are transparent, so it allowed for much better magnification of the egg. Hertwig confirmed Van Beneden's finding of the pronucleus, and also found the formation of the female pronucleus involves the formation of  polar bodies . [ 2 ]


The female pronucleus is the female egg cell once it has become a  haploid cell , and the male pronucleus forms when the sperm enters into the female egg. While the sperm develops inside of the male  testes , the sperm does not become a pronucleus until it decondenses quickly inside of the female egg. [ 3 ]  When the sperm reaches the female egg, the sperm loses its outside membrane as well as its tail. The sperm does this because the membrane and the tail are no longer needed by the female ovum. The purpose of the cell membrane was to protect the  DNA  from the acidic  vaginal fluid , and the purpose of the tail of the sperm was to help move the sperm cell to the egg cell. The formation of the female egg is asymmetrical, while the formation of the male sperm is symmetrical. Typically in a female mammal, meiosis starts with one diploid cell and becomes one haploid ovum and typically two  polar bodies , however one may later divide to form a third polar body. [ 4 ]  In a male, meiosis starts with one diploid cell and ends with four sperm. [ 5 ]  In mammals, the female pronucleus starts in the center of the egg before fertilization. When the male pronucleus is formed, after the sperm cell reaches the egg, the two pronuclei migrate towards each other. However, in brown alga  Pelvetia , the egg pronucleus starts in the center of the egg before fertilization and remain in the center after fertilization. This is because the egg cells of brown alga  Pelvetia , the egg pronucleus is anchored down by  microtubules  so only the male pronucleus migrates towards the female pronucleus. [ 6 ]


The calcium concentration within the egg cell  cytoplasm  has a very important role in the formation of an activated female egg. If there is no calcium influx, the female diploid cell will produce three pronuclei, rather than only one. This is due to the failure of release of the second polar body. [ 7 ]


In sea urchins, the formation of the  zygote  starts with the fusion of both the inner and outer nuclei of the male and female pronuclei. It is unknown if one of the pronuclei start the combination of the two, or if the microtubules that help the dissolution of membranes commence the action. [ 8 ]  The microtubules that make the two pronuclei combine come from the sperm's  centrosome . There is a study that strongly supports that microtubules are an important part of the fusion of the pronuclei.  Vinblastine  is a chemotherapy drug that affects both the plus and minus ends of microtubules. [ 9 ]  When vinblastine is added to the ovum, there is a high rate of pronuclear fusion failure. This high rate of pronuclear fusion failure highly suggests that microtubules play a major role in the fusion of the pronucleus. [ 10 ]  In mammals, the pronuclei only last in the cell for about twelve hours, due to the fusion of the genetic material of the two pronuclei within the egg cell. Many studies of pronuclei have been in the egg cells of sea urchins, where the pronuclei are in the egg cell for less than an hour. The main difference between the process of fusion of genetic materials in mammals versus sea urchins is that in sea urchins, the pronuclei go directly into forming a zygote nucleus. In mammalian egg cells, the  chromatin  from the pronuclei form chromosomes that merge onto the same  mitotic spindle . The diploid nucleus in mammals is first seen at the two-cell stage, whereas in sea urchins it is first found at the zygote stage. [ 3 ]


The term  proofreading  is used in genetics to refer to the error-correcting processes, first proposed by  John Hopfield  and  Jacques Ninio , involved in  DNA replication ,  immune system  specificity, and enzyme-substrate recognition among many other processes that require enhanced specificity. The proofreading mechanisms of Hopfield and Ninio are non-equilibrium active processes that consume ATP to enhance specificity of various biochemical reactions.


In  bacteria , all three  DNA polymerases  (I, II and III) have the ability to proofread, using 3’ → 5’  exonuclease  activity. When an incorrect base pair is recognized, DNA polymerase reverses its direction by one base pair of DNA and excises the mismatched base.  Following base excision, the polymerase can re-insert the correct base and replication can continue.


In  eukaryotes , only the polymerases that deal with the elongation (delta and epsilon) have proofreading ability (3’ → 5’ exonuclease activity). [ 1 ]


Proofreading also occurs in  mRNA translation  for  protein  synthesis. [ 2 ]  In this case, one mechanism is the release of any incorrect  aminoacyl-tRNA  before  peptide bond  formation. [ 3 ]


The extent of proofreading in DNA replication determines the  mutation rate , and is different in different species. [ 4 ]
For example, loss of proofreading due to mutations in the  DNA polymerase epsilon  gene results in a hyper-mutated genotype with >100 mutations per Mbase of DNA in human colorectal cancers. [ 5 ]


The extent of proofreading in other molecular processes can depend on the  effective population size  of the species and the number of genes affected by the same proofreading mechanism. [ 6 ]


Bacteriophage (phage) T4  gene 43 encodes the phage's  DNA polymerase  replicative enzyme.   Temperature-sensitive ( ts ) gene 43 mutants  have been identified that have an antimutator  phenotype , that is a lower rate of spontaneous  mutation  than wild type. [ 7 ]   Studies of one of these mutants,  tsB120 , showed that the DNA polymerase specified by this mutant copies DNA templates at a slower rate than the wild-type polymerase. [ 8 ]   However, the 3’ to 5’  exonuclease  activity was no higher than wild-type. During  DNA replication  the ratio of  nucleotides  turned over to those stably incorporated into newly formed DNA is 10 to 100 times higher in the case of the  tsB120  mutant than in wild-type. [ 8 ]   It was proposed that the antimutator effect may be explained by both greater accuracy in nucleotide selection and an increased efficiency of removal of noncomplementary nucleotides (proofreading) by the  tsB120  polymerase.


When phage T4 virions with a  wild-type  gene 43 DNA polymerase are exposed to either  ultraviolet  light, which introduces cyclobutane  pyrimidine dimer  damages in DNA, or  psoralen -plus-light, which introduces pyrimidine adducts, the rate of mutation increases. However, these mutagenic effects are inhibited when the phage's DNA synthesis is catalyzed by the  tsCB120  antimutator polymerase, or another antimutator polymerase,  tsCB87 . [ 9 ]   These findings indicate that the level of induction of mutations by DNA damage can be strongly influenced by the gene 43 DNA polymerase proofreading function.


Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 pandemic.  The SARS-CoV-2 RNA virus genome encodes a replication-and transcription complex, a multisubunit protein machine that carries out viral genome replication and transcription, processes essential to the virus life cycle.  One of the proteins specified by the coronavirus genome is a non-structural protein, nsp14, that is a 3’-to-5’ exoribonuclease (ExoN).  This protein resides in the protein complex nsp10-nsp14 that enhances replication fidelity by proofreading RNA synthesis, an activity critical for the virus life cycle. [ 10 ]   Furthermore, the coronavirus proofreading exoribonuclease nsp14-ExoN is required for maintaining genetic recombination generated during infection. [ 11 ]


Prophase  (from  Ancient Greek     προ-  ( pro- )  'before' and    φάσις  (phásis)  'appearance') is the first stage of  cell division  in both  mitosis  and  meiosis . Beginning after  interphase ,  DNA  has already been replicated when the  cell  enters prophase. The main occurrences in prophase are the condensation of the  chromatin  reticulum and the disappearance of the  nucleolus . [ 3 ]


Microscopy  can be used to visualize condensed  chromosomes  as they move through  meiosis  and  mitosis . [ 4 ]


Various DNA  stains  are used to treat cells such that condensing  chromosomes  can be visualized as the move through prophase. [ 4 ]


The  giemsa   G-banding  technique is commonly used to identify  mammalian   chromosomes , but utilizing the technology on  plant cells  was originally difficult due to the high degree of chromosome compaction in plant cells. [ 5 ] [ 4 ]   G-banding  was fully realized for plant chromosomes in 1990. [ 6 ]  During both  meiotic  and  mitotic  prophase,  giemsa staining  can be applied to cells to elicit  G-banding  in  chromosomes . [ 2 ]  Silver staining, a more modern technology, in conjunction with  giemsa staining  can be used to image the  synaptonemal complex  throughout the various stages of  meiotic  prophase. [ 7 ]  To perform  G-banding ,  chromosomes  must be fixed, and thus it is not possible to perform on living cells. [ 8 ]


Fluorescent stains  such as  DAPI  can be used in both live  plant  and  animal cells . These stains do not band  chromosomes , but instead allow for DNA probing of specific regions and  genes . Use of  fluorescent microscopy  has vastly improved  spatial resolution . [ 9 ]


Prophase is the first stage of  mitosis  in  animal cells , and the second stage of  mitosis  in  plant cells . [ 10 ]  At the start of prophase there are two identical copies of each  chromosome  in the cell due to replication in  interphase . These copies are referred to as  sister chromatids  and are attached by  DNA  element called the  centromere . [ 11 ]  The main events of prophase are: the condensation of  chromosomes , the movement of the  centrosomes , the formation of the  mitotic spindle , and the beginning of  nucleoli  break down. [ 3 ]


DNA  that was  replicated  in  interphase  is condensed from DNA strands with lengths reaching 0.7 μm down to
0.2-0.3 μm. [ 3 ]  This process employs the  condensin  complex. [ 11 ]  Condensed chromosomes consist of two  sister chromatids  joined at the  centromere . [ 12 ]


During prophase in  animal cells ,  centrosomes  move far enough apart to be resolved using a  light microscope . [ 3 ]   Microtubule  activity in each  centrosome  is increased due to recruitment of  γ-tubulin . Replicated  centrosomes  from  interphase  move apart towards opposite poles of the cell, powered by  centrosome associated motor proteins . [ 13 ]  Interdigitated interpolar  microtubules  from each  centrosome  interact with each other, helping to move the  centrosomes  to opposite poles. [ 13 ] [ 3 ]


Microtubules  involved in the  interphase  scaffolding break down as the replicated  centrosomes  separate. [ 3 ]  The movement of  centrosomes  to opposite poles is accompanied in  animal cells  by the organization of individual radial  microtubule  arrays (asters) by each centriole. [ 13 ]  Interpolar  microtubules  from both  centrosomes  interact, joining the sets of  microtubules  and forming the basic structure of the  mitotic spindle . [ 13 ]  Plant cells do not have centrosomes and the  chromosomes  can  nucleate   microtubule  assembly into the  mitotic apparatus . [ 13 ]  In  plant cells ,  microtubules  gather at opposite poles and begin to form the  spindle apparatus  at locations called foci. [ 10 ]  The  mitotic spindle  is of great importance in the process of  mitosis  and will eventually segregate the  sister chromatids  in  metaphase . [ 3 ]


The  nucleoli  begin to break down in prophase, resulting in the discontinuation of ribosome production. [ 3 ]  This indicates a redirection of cellular energy from general cellular metabolism to  cellular division . [ 3 ]  The  nuclear envelope  stays intact during this process. [ 10 ]


Meiosis  involves two rounds of  chromosome segregation  and thus undergoes prophase twice, resulting in prophase I and prophase II. [ 12 ]  Prophase I is the most complex phase in all of meiosis because  homologous chromosomes  must pair and exchange  genetic information . [ 3 ] : 98   Prophase II is very similar to  mitotic  prophase. [ 12 ]


Prophase I is divided into five phases: leptotene, zygotene, pachytene, diplotene, and diakinesis. In addition to the events that occur in  mitotic  prophase, several crucial events occur within these phases such as pairing of  homologous chromosomes  and the reciprocal  exchange of genetic material  between these  homologous chromosomes . Prophase I occurs at different speeds dependent on  species  and  sex . Many species arrest  meiosis  in diplotene of prophase I until  ovulation . [ 3 ] : 98   In humans, decades can pass as  oocytes  remain arrested in prophase I only to quickly complete meiosis I prior to  ovulation . [ 12 ]


In the first stage of prophase I, leptotene (from the Greek for "delicate"),  chromosomes  begin to condense. Each chromosome is in a  diploid  state and consists of two  sister chromatids ; however, the  chromatin  of the  sister chromatids  is not yet condensed enough to be resolvable in  microscopy . [ 3 ] : 98    Homologous  regions within  homologous chromosome  pairs begin to associate with each other. [ 2 ]


In the second phase of prophase I, zygotene (from the Greek for "conjugation"), all maternally and paternally derived  chromosomes  have found their  homologous  partner. [ 3 ] : 98   The homologous pairs then undergo synapsis, a process by which the  synaptonemal complex  (a proteinaceous structure) aligns corresponding regions of  genetic information  on maternally and paternally derived non-sister  chromatids  of  homologous chromosome  pairs. [ 3 ] : 98  [ 12 ]   The paired homologous chromosome bound by the  synaptonemal complex  are referred to as  bivalents  or tetrads. [ 10 ] [ 3 ] : 98    Sex (X and Y) chromosomes  do not fully synapse because only a small region of the chromosomes are homologous. [ 3 ] : 98


The  nucleolus  moves from a central to a peripheral position in the  nucleus . [ 14 ]


The third phase of prophase I, pachytene (from the Greek for "thick"), begins at the completion of synapsis. [ 3 ] : 98    Chromatin  has condensed enough that  chromosomes  can now be resolved in  microscopy . [ 10 ]  Structures called recombination nodules form on the  synaptonemal complex  of  bivalents . These recombination nodules facilitate  genetic exchange  between the non-sister chromatids of the  synaptonemal complex  in an event known as  crossing-over  or genetic recombination. [ 3 ] : 98   Multiple recombination events can occur on each bivalent. In humans, an average of 2-3 events occur on each chromosome. [ 13 ] : 681


In the fourth phase of prophase I, diplotene (from the Greek for "twofold"),  crossing-over  is completed. [ 3 ] : 99  [ 10 ]   Homologous chromosomes  retain a full set of genetic information; however, the  homologous chromosomes  are now of mixed maternal and paternal descent. [ 3 ] : 99   Visible junctions called chiasmata hold the  homologous chromosomes  together at locations where recombination occurred as the  synaptonemal complex  dissolves. [ 12 ] [ 3 ] : 99   It is at this stage where meiotic arrest occurs in many  species . [ 3 ] : 99


In the fifth and final phase of prophase I, diakinesis (from the Greek for "double movement"), full chromatin condensation has occurred and all four  sister chromatids  can be seen in  bivalents  with  microscopy . The rest of the phase resemble the early stages of mitotic  prometaphase , as the meiotic prophase ends with the  spindle apparatus  beginning to form, and the  nuclear membrane  beginning to break down. [ 10 ] [ 3 ] : 99


Prophase II of  meiosis  is very similar to prophase of  mitosis . The most noticeable difference is that prophase II occurs with a  haploid  number of  chromosomes  as opposed to the  diploid  number in mitotic prophase. [ 12 ] [ 10 ]  In both  animal  and  plant cells  chromosomes may de-condense during  telophase  I requiring them to re-condense in prophase II. [ 3 ] : 100  [ 10 ]  If chromosomes do not need to re-condense, prophase II often proceeds very quickly as is seen in the  model organism   Arabidopsis . [ 10 ]


Female mammals and birds are born possessing all the oocytes needed for future ovulations, and these  oocytes  are arrested at the prophase I stage of  meiosis . [ 15 ]   In humans, as an example, oocytes are formed between three and four months of  gestation  within the fetus and are therefore present at birth.  During this prophase I arrested stage ( dictyate ), which may last for decades, four copies of the  genome  are present in the oocytes.  The adaptive significance of prophase I arrest is still not fully understood.  However, it has been proposed that the arrest of oocytes at the four genome copy stage may provide the informational redundancy needed to  repair damage in the DNA  of the  germline . [ 15 ]   The repair process used appears to be  homologous recombinational  repair [ 15 ] [ 16 ]  Prophase arrested oocytes have a high capability for efficient repair of  DNA damages . [ 16 ]   DNA repair capability appears to be a key quality control mechanism in the female germ line and a critical determinant of  fertility . [ 16 ]


The most notable difference between prophase in  plant cells  and  animal cells  occurs because plant cells lack  centrioles . The organization of the  spindle apparatus  is associated instead with foci at opposite poles of the cell or is mediated by chromosomes. Another notable difference is  preprophase , an additional step in plant  mitosis  that results in formation of the  preprophase band , a structure composed of  microtubules . In  mitotic  prophase I of plants, this band disappears. [ 10 ]


Prophase I in  meiosis  is the most complex iteration of prophase that occurs in both  plant cells  and  animal cells . [ 3 ]  To ensure pairing of  homologous chromosomes  and  recombination of genetic material  occurs properly, there are  cellular checkpoints  in place. The meiotic checkpoint network is a  DNA damage  response system that controls  double strand break  repair,  chromatin  structure, and the movement and pairing of  chromosomes . [ 17 ]  The system consists of multiple pathways (including the  meiotic recombination checkpoint ) that prevent the cell from entering  metaphase I  with errors due to recombination. [ 18 ]


Proton-coupled amino acid transporters  belong to the  SLC26A5  family; they are protein receptors whose main function is the  transmembrane  movement of amino acids and their derivatives. This family of receptors is most commonly found within the luminal surface of the small intestine as well as in some lysosomes. The  solute carrier family  (SLC) of genes includes roughly 400 membrane proteins that are characterized by 66 families in total. The SLC36 family of genes maps to chromosome 11. The diversity of these receptors is vast, with the ability to transport both charged and uncharged amino acids along with their derivatives. In research and practice, SLC36A1/2 are both targets for drug-based delivery systems for a wide range of disorders.


The human protein acid transporter (hPAT1) is 5585 base pairs long and codes for a protein 476 amino acids long. The transporter has nine transmembrane regions where the amino terminus faces the cytoplasm. The rat protein acid transporter (rPAT1) has been widely studied and an 85% amino acid sequence match was found between hPAT1 and rPAT1. The hPAT1 gene is located on chromosome 5q31-33 and has 11 exons that are coding regions. Its translation site begins in exon 2 and exon 11 contains the termination site. [ 1 ]


The molecular weight of  Proton-coupled amino acid transporter 1  is 53.28 kDA; the molecular weight of  Proton-coupled amino acid transporter 2  is 53.22 kDA. PAT1 has been found in lysosomes in brain neurons but also in the apical membrane of intestinal epithelial cells where it is associated with the brush border. Proton-coupled amino acid transporter 1 has a higher affinity for proline than it does for glycine and alanine Proton-coupled amino acid transporter 2 is found subcellularly in the kidneys, lungs, spinal cord, and brain and likely has a role in myelinating neurons. [ 2 ]  It has an overall higher affinity for glycine, alanine, and proline than PAT1 but is more specific for what can inhibit it. [ 3 ] [ 4 ]


Unlike most amino acid transporters in the exchange of Na+ with amino acid symporters, proton-coupled amino acid transporters function as H+ with amino acid symporters. They are located within the luminal surface of the small intestine and within lysosomes, so their action functions in absorption in the intestine and in the efflux pathway after intralysosomal digestion. Unlike typical mammalian amino acid transporters which function in exchanging Na+/amino acid symporters, these- transporters function in exchanging H+/amino acid symporters. The activity of transporters, such as  Proton-coupled amino acid transporter 1  and Proton-coupled amino acid transporter 2 can be measured at the  apical membrane  of the human epithelial layer of cells which are loaded with pH sensitive dyes. The change in membrane potential can be measured by the absorption of pH sensitive dyes and the associated influx of H+ ions. The proteins involved in these transporters are consider anion exchangers


The function of proton-coupled amino acid transporters is the transmembrane movement of amino acids and their derivatives for absorption by the luminal surface of the small intestine or digestion by intralysosomal proteins. In  Drosophila  models, the expression of SLC family genes that code for proton-coupled amino acid transporters is directly linked to the nutrient-dependent growth. In humans, similar expression patterns are observed and their function correlates to their location anatomically. Being located within the lamina of the small intestine allows for functional absorption of transported amino acids and derivatives. The majority of nutrient absorption takes place within this region of the intestines, and makes sense that these transporters are located throughout this tissue.


In hereditary disease iminoglycinuria, there is a defect in the human proton-coupled amino acid transporter 1 and 2 genes which results in a defect in the absorption of proline and glycine. Iminoglycinuria is an autosomal recessive disorder of the renal tubular. Lack of glycine and proline absorption leads to excess urinary excretions containing amino acids. If the transporters are not working properly, a drug that they usually help gain entry in to the cell might not be absorbed [ 3 ]  Their function can also be inhibited by tryptophan derivatives and allow for exploration into the function of hPAT1 and hPAT2. Additionally, mutations that lead to structural changes in amino acid binding sites play a role in their functional transport. [ 5 ]


The DNA sequence of these transporters is transcribed in the nucleus of the cell by RNA polymerase and undergoes splicing and capping before it travels to the cytoplasm. In the cytoplasm, translation begins via a sequence in exon 2 of the mRNA. Subsequently, protein folding and packaging insert the transporter into the membrane. The protein has a signal recognition particle that is recognized as it leaves the ribosome. N-glycosylation at various sites on hPAT1 is necessary for its transport function. Three of its extracellular residues are glycosylated and determine transport efficacy. [ 6 ]


PAT1 mRNA is expressed in the GI tract between the stomach and descending colon, but is generally absent in the esophagus, caecum, and rectum. This allows for different treatments that affect the affinity of the carrier protein for its substrates, giving the potential to treat various amino-acid related diseases. HPAT1 and HPAT2 are important in the absorption of certain drugs, especially pharmaceutically active amino acids derivatives. [ 3 ]  They have also been targeted with medications used as anticonvulsants, for prostate cancer, and for bladder cancer. [ 7 ]  HPAT1 and 2 are integral to the central nervous system because they transport GABA and its analogues which can induce and inhibitory and excitatory effect in the brain. [ 1 ]


Pseudoallelism  is a state in which two genes with similar functions are located so close to one another on a chromosome that they are genetically linked. [ 1 ] [ 2 ]  This means that the two genes (pseudoalleles) are nearly always inherited together. Since the two genes have related functions, they may appear to act as a single gene. In rare cases, the two linked pseudoalleles can be separated, or recombined. One hypothesis is that pseudoalleles are formed as a result of gene duplication events, and the duplicated genes can undergo gene evolution to develop new functions.


Characteristic of pseudoalleles:


Example:


Red eye colour of Drosophila has different mutants like white and apricot. They affect pigmentation i.e., affect the same character. So, they are allelic. They can undergo recombination, i.e., they are nonallelic.


In  genetics ,  pseudolinkage  is a characteristic of a  heterozygote  for a  reciprocal translocation , in which genes located near the  translocation  breakpoint behave as if they are  linked  even though they originated on  nonhomologous chromosomes .


Linkage  is the proximity of two or more  markers  on a  chromosome ; the closer together the markers are, the lower the probability that they will be separated by  recombination .  Genes  are said to be linked when the frequency of  parental  type progeny exceeds that of recombinant progeny.


During  meiosis  in a translocation homozygote, chromosomes segregate normally according to  Mendelian  principles. Even though the genes have been rearranged during  crossover , both  haploid  sets of chromosomes in the individual have the same  rearrangement . As a result, all chromosomes will find a single partner with which to pair at meiosis, and there will be no deleterious consequences for the  progeny .


In translocation heterozygote, however, certain patterns of chromosome segregation during meiosis produce genetically unbalanced  gametes  that at  fertilization  become deleterious to the  zygote . In a translocation heterozygote, the two haploid sets of chromosomes do not carry the same arrangement of genetic information. As a result, during  prophase  of the first  meiotic  division, the translocated chromosomes and their normal  homologs  assume a crosslike configuration in which four chromosomes, rather than the normal two, pair to achieve a maximum of  synapsis  between similar regions. We denote the chromosomes carrying translocated material with a T and the chromosomes with a normal order of genes with an N. Chromosomes N1 and T1 have homologous  centromeres  found in wild type on chromosome 1; N2 and T2 have centromeres found in wild type on chromosome 2.
During  anaphase  of meiosis I, the mechanisms that attach the  spindle  to the chromosomes in this crosslike configuration still usually ensure the disjunction of homologous centromeres, bringing homologous chromosomes to opposite spindle poles. Depending on the arrangement of the four chromosomes on the  metaphase plate , this normal disjunction of homologous produces one of two equally likely patterns of segregation.


In the alternate segregation pattern, the two translocation chromosomes (T1 and T2) go to one pole, while the two normal chromosomes (N1 and N2) move to the opposite pole. Both kinds of gametes resulting from this segregation (T1, T2, and N1, N2) carry the correct haploid number of genes; and the zygotes formed by union of these gametes with normal gamete will be viable.


In the adjacent-1 segregation pattern, homologous centromeres disjoin so that T1 and N2 go to one pole, while the N1 and T2 go to the opposite pole. Consequently, each gamete contains a large  duplication  (of the region found in both the normal and the translocated chromosome in that gamete) and a correspondingly large  deletion  (of  the region found in neither  of the chromosomes in that gametes), which make them genetically unbalanced. Zygotes formed by union of these gametes with a normal gametes are usually not  viable .


Because of the unusual cruciform pairing configuration in translocation heterozygotes,  nondisjunction  of homologous centromeres occurs at a measurable but low rate. This nondisjunction produces an adjacent-2 segregation pattern in which the homologous centromeres N1 and T1 go to the same spindle pole while the homologous centromeres N2 and T2 go to the other spindle pole. The resulting genetic imbalances are  lethal  after fertilization to the zygotes containing them.


Thus, in a translocation heterozygote, only the  alternate segregation  pattern yields viable  progeny  in outcrosses, the equally likely adjacent-1 pattern and the rare adjacent-2 pattern do not.
Because of this, genes near the translocation breakpoints on the nonhomologous chromosomes participating in a reciprocal translocation exhibit pseudolinkage: They behave as if they are linked.


Q-system  is a  genetic  tool that allows to  express   transgenes  in a living  organism . [ 1 ]  Originally the Q-system was developed  [ 2 ] [ 3 ]  for use in the  vinegar fly   Drosophila melanogaster , and was rapidly adapted for use in cultured  mammalian cells , [ 2 ]   zebrafish , [ 4 ]   worms [ 5 ]  and  mosquitoes . [ 6 ]  The Q-system utilizes  genes  from the  qa  cluster [ 7 ]  of the  bread fungus   Neurospora crassa , and consists of four components: the  transcriptional activator  (QF/QF2/QF2 w ), the  enhancer  QUAS, the  repressor  QS, and the chemical de-repressor  quinic acid . Similarly to  GAL4/UAS [ 8 ]  and LexA/LexAop, [ 9 ]  the Q-system is a binary expression system that allows to express  reporters  or effectors (e.g.  fluorescent proteins ,  ion channels ,  toxins  and other genes) in a defined subpopulation of  cells  with the purpose of visualising these cells or altering their function. In addition, GAL4/UAS, LexA/LexAop and the Q-system function independently of each other and can be used simultaneously to achieve a desired pattern of reporter expression, or to express several reporters in different subsets of cells.


The Q-system is based on two out of the seven  genes  of the  qa  gene cluster of the  bread fungus   Neurospora crassa . [ 7 ]  The genes of the  qa  cluster are responsible for the catabolism of quinic acid, which is used by the fungus as a carbon source in conditions of low glucose. [ 7 ]  The cluster contains a transcriptional activator  qa-1F , a transcriptional repressor  qa-1S , and five structural genes. The  qa-1F  binds to a specific DNA sequence, found upstream of the  qa  genes. The presence of quinic acid disrupts interaction between  qa-1F  and  qa-1S , thus disinhibiting the transcriptional activity of  qa-1F .
Genes  qa-1F ,  qa-1S  and the DNA binding sequence of  qa-1F  form the basis of the Q-system. The genes were renamed to simplify their use as follows: transcriptional activator  qa-1F  as QF, repressor  qa-1S  as QS, and the DNA binding sequence as QUAS. [ 2 ]  The quinic acid represents the fourth component of the Q-system.
The original transactivator QF appeared to be toxic when expressed broadly in  Drosophila . To overcome this problem, two new transactivators were developed: QF2 and QF2 w. [ 3 ]


The Q-system functions similarly to, and independently of, the GAL4/UAS [ 8 ]  and the LexA/LexAop  [ 9 ]  systems. QF, QF2 and QF2 w  are analogous to GAL4 and LexA, and their expression is usually under the control of cell-type specific promoter, such as  nsyb  (to target neurons) or  tubulin  (to target all cells). QUAS is analogous to UAS and LexAop, and is placed upstream of an effector gene, such as  GFP . QS is analogous to GAL80, and may be driven by any promoter (e.g.  tubulin-QS ). Quinic acid is a unique feature of the Q-system, and it must be fed to the flies or maggots in order to alleviate the QS-induced repression. In some ways, quinic acid is analogous to temperature in the case of GAL80 ts .
In its basic form, two transgenic fly lines, one containing a QF transgene and the other one containing a QUAS transgene, are crossed together. Their progeny that had both a QF transgene and a QUAS transgene will be expressing a reporter gene in a subset of cells (e.g.  nsyb-QF2, QUAS-GFP  flies express GFP in all neurons). If a fly also expresses QS in some of the cells, the activity of QF will be repressed in these cells, but it may be restored of a fly is fed quinic acid (e.g. a  nsyb-QF2, QUAS-GFP, tub-QS  fly expresses no GFP when its diet doesn't contain quinic acid, and expresses GFP in its neurons when fed quinic acid). [ 2 ] [ 3 ]  The use of QS repressor and quinic acid allows to fine-tune the temporal control of transgene expression.


Chimeric transactivators GAL4QF [ 3 ]  and LexAQF [ 3 ]  allow to combine the use of all three binary expression systems. GAL4QF binds to UAS, and may be repressed by QS while being unaffected by GAL80. Similarly, LexAQF binds to LexAop, and may be repressed by QS. LexAQF represents a useful extension of the LexA/LexAop system that doesn't have its own repressor.


A variety of expression patterns may be achieved by combination of the three binary expression systems and the FLP/FRT or other recombinases. [ 10 ]  Expression patterns may be constructed as AND, OR, NOR etc. logic gates  [ 1 ] [ 2 ]  to e.g. narrow down expression patterns of available GAL4 lines. The resulting expression pattern somewhat depends on the developmental timing of activation of the transcription factors (discussed in  [ 1 ] ).


Q-system appeared to be working successfully in a variety of organisms. It has been used to drive expression of luciferase, as a proof of principle, in cultured  mammalian cells . [ 2 ]  In  zebrafish [ 4 ]  the Q-system has been successfully used with several tissue-specific promoters, and was shown to work independently of the GAL4/UAS system when expressed in the same cell. In  C. elegans [ 5 ]  the Q-system has been shown to work in muscles and in neuronal tissue. In 2016, the Q-system was used to target, for the first time, the olfactory neurons of malaria mosquitoes  Anopheles gambiae . [ 6 ]  In 2019, the Q-system in  Anopheles  mosquitoes was used to examine the functional responses of olfactory neurons to odors. [ 11 ]  In 2019, the Q-system was introduced into the  Aedes aegypti  mosquito to capture tissue specific expression patterns. [ 12 ]  These successes make the Q-system the system of choice when developing genetic tools for other organisms. Currently the main shortcoming of the Q-system is the low number of available transgenic lines, but it will be overcome as the scientific community creates and shares these resources, such as by the use of the GAL4>QF2 HACK system to convert existing GAL4 transgenic insertions to QF2. [ 13 ]  DNA binding domain of QF2 fused with VP16 transcriptional activator domain was successfully applied in  Penicillium  to gain control over the penicillin producing secondary metabolite gene cluster in a scalable manner.  [ 14 ]


Radiation reduced hybrid  is procedure in discovering the location of genetic markers relative to one another.  The relative location of these markers can be combined into a  physical map  or a  genetic map .  The radiation hybrid technique begins as another way to amplify and purify  DNA , the first step in any sequencing project; radiation is used to break the DNA into pieces and these pieces are incorporated into the hybrid cell; the hybrid can be grown in large quantities.  One can then check for the presence of various genetic markers using  PCR  and linkage analysis to resolve the distance between the markers.  That is, if two  genetic markers  are near each other, they are less likely to be separated by the DNA-breaking radiation.  The technique is similar to traditional linkage analysis, where we depend on  genetic recombination  to calculate the distance between two genetic markers.


The procedure utilizes two cell lines, neither of which can survive in toxic media,
but contain genes that can resist the toxin when contained by the same cell.  The cell line under study is irradiated, causing breaks in the DNA.  These cells are fused with the other cell line, producing a hybrid.  If the hybrid incorporates genes from both cells, it will be able to survive in the toxic media.


The cells that survive can be grown in large quantities, thus amplifying the DNA that was incorporated from the irradiated cell line.  One can prepare a sample of DNA from the hybrid cell line and use PCR to amplify two specific genetic markers.  By running the PCR products on a gel, one can determine if both markers are in the cell line.  If both markers are usually in the hybrid, we can conclude that they are near each other.


RCCX  is a complex, multiallelic, and tandem  copy number variation  (CNV) human  DNA   locus  on  chromosome  6p21.3, a cluster located in the  major histocompatibility complex (MHC)   class III  region. [ 1 ] [ 2 ]  CNVs are segments of  DNA  that vary in copy number compared to a  reference genome  and play a significant role in human  phenotypic variation  and disease development. The RCCX cluster consists of one or more modules each having a series of genes close to each other:  serine/threonine kinase  19 ( STK19 ),  complement 4  ( C4 ),  steroid 21-hydroxylase  ( CYP21 ), and  tenascin-X  ( TNX ). [ 3 ]


The RCCX abbreviation is composed of the names of the genes  R P (a former name for  STK19  serine/threonine kinase 19), [ 2 ] [ 3 ]   C 4 ,  C YP21  and  TN X ). [ 4 ]  The RCCX abbreviation was first mentioned in a 1994 article published in  Immunogenetics , an academic journal, for a study by Dangel et al. [ 5 ]


The number of RCCX segments varies between one and four in a  chromosome , [ 2 ]  with the prevalence of approximately 15% for monomodular, 75% for bimodular (STK19-C4A-CYP21A1P-TNXA-STK19B-C4B-CYP21A2-TNXB), [ 3 ] [ 6 ]  and 10% for trimodular in Europeans. [ 7 ]  The quadrimodular structure of the RCCX unit is very rare. [ 8 ] [ 2 ] [ 7 ]


In a monomodular structure, all of the genes are functional i.e.  protein-coding , but if a module count is two or more, there is only one copy of each functional gene rest being non-coding  pseudogenes  with the exception of the  C4  gene which always has active copies. [ 2 ] [ 7 ]  Each copy of the C4 gene, due to five adjacent  nucleotide substitutions  cause four amino acid changes and immunological subfunctionalization (different functions related to the immune system), [ 7 ]  can be of one of two types:  C4A  and  C4B . [ 9 ]  Each C4 gene contains 41  exons  and has a dichotomous size variation (existence of two distinct sizes) between approximately 22 kb and 16 kb, with the longer variant being the result of the integration of the endogenous retrovirus  HERV-K (C4) into intron 9. [ 10 ] [ 3 ]


The RCCX module is the most complex gene cluster in the human genome. [ 3 ] [ 9 ] [ 11 ]  It is part of the major histocompatibility complex (MHC) class III ( MHC class III ), [ 12 ] [ 13 ]  which is the most gene-dense region of the human genome, containing many genes that yet have unknown function or structure. [ 14 ] [ 15 ] [ 16 ] [ 17 ]  RCCX modules exhibit a high degree of  linkage disequilibrium , meaning that genes are inherited together more frequently than would be expected by chance. It indicates that there is a non-random association or correlation between the alleles of different genes within the RCCX modules. The high degree of  linkage disequilibrium  observed in the RCCX modules suggests that the genes within this module are inherited as a group, rather than independently. This makes the RCCX module well-suited for genetic association studies, especially in the context of  autoimmune diseases . [ 10 ] [ 2 ]


The RCCX module is involved in the synthesis of the  steroid   hormones   cortisol ,  aldosterone , and  androgen  precursors, in extracellular matrix glycoprotein synthesis, and in  innate immune system . [ 7 ]


The RP gene (a former name for the  STK19  gene) is involved in cell growth and  differentiation , but its exact functions remain unclear. [ 18 ]  Current knowledge suggests that the STK19 gene encodes the protein called nuclear  serine/threonine kinase  19. This protein probably plays a role in regulating the activity of  neuroblastoma RAS viral oncogene homolog  (NRAS), a protein involved in cellular signaling. STK19  phosphorylates  NRAS, which means it adds a  phosphate   functional group  to NRAS. This  phosphorylation  event facilitates interactions between NRAS and its downstream  effectors , which are molecules that carry out specific cellular functions. By increasing the activation of the  mitogen-activated protein kinase  (MAPK) cascade, STK19 ultimately influences cellular processes such as cell growth, proliferation, and differentiation. [ 19 ] [ 20 ] [ 21 ]


The C4 gene encodes the  complement component 4 , which is involved in the  complement system  and is an important part of the  innate immune system . The gene has two forms:  C4A  and  C4B , encoding form A and B of the complement component 4 protein, respectively. [ 22 ]


The  CYP21A2  gene encodes the  enzyme   21-hydroxylase  involved in synthesizing  cortisol  and  aldosterone . [ 23 ]


The  TNXB  gene encodes the  Tenascin X , an extracellular matrix glycoprotein. Tenascin X is involved in the formation and maintenance of the extracellular matrix, which provides structural support and regulates cell behavior. It is also involved in tissue repair and regeneration and musculoskeletal development. Tenascin X interacts with other extracellular matrix proteins such as  fibrillin-1  and  collagen  and is thought to play a role in regulating their organization and function. [ 24 ]


The RCCX module is related to personality traits such as novelty seeking and impulsivity [ 25 ]  as major histocompatibility complex (MHC), where the RCCX module is located, may affect these traits through its role in immune function and  neurodevelopment , still, the exact mechanisms are not fully understood. [ 3 ]


Variations in complement component  C4  genes within the RCCX module have been associated with  psychiatric disorders  such as  schizophrenia  and  neurodegenerative diseases  such as  Alzheimer's disease . [ 3 ]


The RCCX module may be involved in developing  autoimmune diseases  such as  rheumatoid arthritis ,  systemic lupus erythematosus , and  multiple sclerosis : the  C4A  gene may be associated with an increased risk of  systemic lupus erythematosus , while the C4B gene may be associated with an increased risk of  rheumatoid arthritis . [ 26 ] [ 27 ] [ 28 ]  The  HERV-K  retrovirus within the C4 gene has also been associated with  autoimmune diseases  such as  systemic lupus erythematosus  and  rheumatoid arthritis , probably because retrovirus may activate the C4 gene, leading to increased production of  C4  proteins, which can contribute to autoimmune responses, and can probably lead to  neuroinflammation , and increased risk of developing diseases such as  schizophrenia  and  bipolar disorder . [ 29 ] [ 30 ] [ 31 ]


The presence of multiple RCCX modules is also associated with an increased risk of  autoimmune diseases . [ 3 ]


Genetic variations in the RCCX module have been linked to many other disorders, including  autism spectrum disorder , and  drug addiction . [ 32 ]


The  CYP21  gene is associated with developing  congenital adrenal hyperplasia due to 21-hydroxylase deficiency  (CAH), [ 33 ] [ 34 ] [ 35 ]  a genetic disorder that affects the  adrenal glands  and causes  cortisol  deficiencies and excessive androgen biosynthesis (that may lead to  virilization  of female infants) and in severe cases also  aldosterone  deficiencies (that may lead to salt wasting - large amounts of sodium in urine that causes such life-threatening consequences as  hypotension ,  hyponatremia , and  hyperkalemic   metabolic acidosis ). [ 36 ]


The  TNXB  gene, also known as tenascin-X, is associated with such disorders of connective tissue, such as   Ehlers-Danlos syndrome  (EDS), characterized by  joint hypermobility , skin hyperextensibility, and tissue fragility. Another disorder, when recombination events occur between a pseudogene  TNXA [ 37 ]  and the functional gene  TNXB [ 38 ]  within the RCCX module, resulting in  CYP21A2  deletion along with impaired  TNXB  function, is called CAH-X Syndrome [ 39 ] [ 6 ]  and leads to both  congenital adrenal hyperplasia  (CAH) symptoms and features consistent with EDS. [ 3 ]  This impaired function of the  TNXB  gene refers to the decreased production or abnormal structure of the tenascin-X protein due to genetic changes within the  TNXB  gene. The exact molecular mechanisms through which alterations or deficiencies in the  TNXB  gene or its impaired function lead to these conditions (the EDS and the CAH-X syndrome) are not fully understood yet but are believed to be related to defects in  extracellular matrix  organization and  cell adhesion processes  mediated by tenascin-X protein. [ 3 ]


An "RCCX theory", a hypothesis developed by Sharon Meglathery, a US psychiatrist, [ 40 ]  an author of a few publications on psychiatry, [ 41 ] [ 42 ]  and oncology, [ 43 ] [ 44 ]  highlights the links between certain autoimmune and psychiatric disorders due to variations in the RCCX cluster. According to the hypothesis, these variations contribute to the development of autoimmune disorders, such as lupus and rheumatoid arthritis, as well as psychiatric conditions, such as anxiety and depression. The hypothesis provides insights into the genetic basis of these disorders. It highlights the importance of considering both immunological and psychological factors in their diagnosis and treatment, suggesting shared genetic underpinnings of these disorders and aiming to bridge the gap between immunology and psychiatry, ultimately paving the way for more comprehensive approaches to diagnosis and treatment strategies for patients suffering from these conditions. [ 45 ] [ 46 ]  Meglathery encountered obstacles in initiating bench research for her hypothesis such as skepticism from the scientific community. [ 47 ]


In  molecular biology , a  reading frame  is a way of dividing the  sequence of nucleotides  in a  nucleic acid  ( DNA  or  RNA ) molecule into a set of consecutive, non-overlapping triplets. Where these triplets equate to  amino acids  or stop signals during  translation , they are called  codons .


A single strand of a  nucleic acid  molecule has a  phosphoryl  end, called the  5′-end , and a  hydroxyl  or  3′-end . These define the  5′→3′ direction . There are three reading frames that can be read in this 5′→3′ direction, each beginning from a different nucleotide in a triplet. In a double stranded nucleic acid, an additional three reading frames may be read from the other,  complementary  strand in the 5′→3′ direction along this strand. As the two strands of a double-stranded nucleic acid molecule are antiparallel, the 5′→3′ direction on the second strand corresponds to the 3′→5′ direction along the first strand. [ 1 ] [ 2 ]


In general, at the most, one reading frame in a given section of a nucleic acid, is biologically relevant ( open reading frame ). Some viral transcripts can be translated using multiple, overlapping reading frames. There is one known example of overlapping reading frames in mammalian  mitochondrial DNA : coding portions of genes for 2 subunits of ATPase overlap.


DNA encodes protein sequence by a series of three-nucleotide  codons . Any given sequence of DNA can therefore be read in six different ways: Three reading frames in one direction (starting at different nucleotides) and three in the opposite direction. During  transcription , the RNA polymerase read the template DNA strand in the 3′→5′ direction, but the mRNA is formed in the 5′ to 3′ direction. [ 3 ]  The mRNA is single-stranded and therefore only contains three possible reading frames, of which only one is  translated . The codons of the mRNA reading frame are translated in the 5′→3′ direction into  amino acids  by a  ribosome  to produce a  polypeptide chain .


An  open reading frame  (ORF) is a reading frame that has the potential to be  transcribed  into RNA and  translated  into protein. It requires a continuous sequence of DNA which may include a  start codon , through a subsequent region which has a length that is a multiple of 3 nucleotides, to a  stop codon  in the same reading frame. [ 4 ]


When a putative amino acid sequence resulting from the translation of an ORF remained unknown in mitochondrial and chloroplast genomes, the corresponding open reading frame was called an unidentified reading frame (URF). For example, the  MT-ATP8  gene was first described as URF A6L when the complete human  mitochondrial genome  was sequenced. [ 5 ]


The usage of multiple reading frames leads to the possibility of  overlapping genes ; there may be many of these in viral, prokaryote, and mitochondrial  genomes . [ 6 ]  Some viruses, e.g.  hepatitis B virus  and  BYDV , use several overlapping genes in different reading frames.


In rare cases, a ribosome may shift from one frame to another during translation of an mRNA ( translational frameshift ). This causes the first part of the mRNA to be translated in one reading frame, and the latter part to be translated in a different reading frame. This is distinct from a  frameshift mutation , as the nucleotide sequence (DNA or RNA) is not altered—only the frame in which it is read.


Reassortment  is the mixing of the  genetic material  of a species into new combinations in different individuals. The product of reassortment is called a  reassortant . It is particularly used when two similar  viruses  that are infecting the same cell exchange genetic material. More specifically, it refers to the swapping of entire segments of the genome, which only occurs between viruses with segmented genomes. [ 1 ]  (All known viruses with segmented genomes are RNA viruses.)


The classical example of reassortment is seen in the  influenza viruses , whose genomes consist of eight distinct segments of RNA.  These segments act like mini-chromosomes, and each time a flu virus is assembled, it requires one copy of each segment.


If a single host (a human, a chicken, or other animal) is infected by two different strains of the influenza virus, then it is possible that new assembled viral particles will be created from segments whose origin is mixed, some coming from one strain and some coming from another. The new reassortant strain will share properties of both of its parental lineages.


Reassortment is responsible for some of the major  antigenic shifts  in the history of the influenza virus. In the 1957 " Asian flu " and 1968 " Hong Kong flu "  pandemics , flu strains were caused by reassortment between an avian virus and a human virus. [ 2 ] [ 3 ]  In addition, the  H1N1  virus responsible for the  2009 swine flu pandemic  has an unusual mix of swine, avian and human influenza genetic sequences. [ 4 ]


When  influenza viruses  are inactivated by  UV  irradiation or  ionizing radiation , they remain capable of multiplicity reactivation in infected host cells. [ 5 ] [ 6 ] [ 7 ]   If any of a virus's  genome  segments is damaged in such a way as to prevent replication or expression of an essential  gene , the virus is inviable when it, alone, infects a host cell (single infection).  However, when two or more damaged viruses infect the same cell (multiple infection), the infection can often succeed (multiplicity reactivation) due to reassortment of segments, provided that each of the eight genome segments is present in at least one undamaged copy. [ 8 ]


The  reptarenavirus  family, responsible for  inclusion body disease  in snakes, shows a very high degree of genetic diversity due to reassortment of genetic material from multiple strains in the same infected animal.


The  RecF pathway , also called the  RecFOR pathway , is a  pathway  of  homologous recombination  that  repairs DNA  in  bacteria .  It repairs breaks that occur on only one of DNA's two strands, known as single-strand gaps.  The RecF pathway can also repair double-strand breaks in DNA when the  RecBCD pathway , another pathway of homologous recombination in bacteria, is inactivated by mutations. [ 1 ]   Like the RecBCD pathway, the RecF pathway requires  RecA  for strand invasion.  The two pathways are also similar in their phases of  branch migration , in which the  Holliday junction  slides in one direction, and resolution, in which the Holliday junctions are cleaved apart by enzymes. [ 2 ] [ 3 ]


The RecF pathway begins when RecJ, an  exonuclease  that cleaves single-stranded DNA in the  5 → 3′  direction, binds to the 5' end of a single-strand gap in DNA and starts moving upstream while cleaving the 5' strand.  Although RecJ can function without them,  single-strand binding protein  (SSBP) and the  RecQ  helicase greatly increase how much the 5' is cut back.  When present, SSBP binds to the 3' overhang remaining after RecJ finishes cutting back the 5' strand. [ 3 ]   By binding to the single-stranded DNA, SSBP ensures that the 3' DNA overhang does not stick to itself through self- complementation . [ 4 ]


The RecA protein can be loaded onto the SSBP-coated 3' overhang in one of two distinct pathways, one that requires the RecFOR enzyme or one that requires the RecOR enzyme. [ 5 ]   In the RecFOR pathway, the RecFR complex binds where the single-strand DNA of the 3' meets the double-strand DNA.  RecO then displaces SSBP from the ssDNA, although SSBP remains attached to RecO. RecFOR then loads RecA onto a recessed 5' end of this ssDNA-dsDNA junction.  The RecR subunit in RecFR then interacts with RecO to form the RecFOR complex.  In doing so, the RecR subunit helps to both detach the SSBP molecules from RecO and load molecules of the RecA protein onto the 3' overhang. [ 6 ]


The RecOR pathway of RecA loading differs from the RecFOR pathway in several respects, most notably its molecular interaction requirements and its ideal DNA substrate. [ 5 ]   Unlike the RecFOR pathway, the RecOR pathway requires an interaction between RecO and the  C-terminus  of SSBP.  The RecOR pathway also does not need a ssDNA-dsDNA junction to begin loading RecA onto the 3' overhang, whereas the RecFOR pathway typically does to work efficiently.  Thus, the RecOR pathway in most conditions is more efficient than the RecFOR pathway in loading RecA. [ 5 ]


Reciprocal silencing , a  genetic  phenomenon that primarily occurs in plants, refers to the pattern of redundant  genes  being  silenced  following a  polyploid  event. Polyploidy (wholesale  genome  duplication) is common in  plants  and constitutes an important method of speciation. [ 1 ]  When a  polyploid  species arises, its  genome  contains  homoeologs , duplicated  chromosomes  with equivalent genetic information. However silencing of redundant genes occurs rapidly in new polyploids through genetic and  epigenetic  means. This primarily occurs because redundancy allows one of the two genes present for each locus to be silenced without affecting the  phenotype  of the organism, and thus  mutations  that eliminate  gene expression  are much less likely to be deleterious or lethal. [ 1 ] [ 2 ]  This allows mutations that would be lethal in diploid populations to accumulate in polyploids. Reciprocal silencing refers to the specific pattern of silencing where equivalent loci in are both silenced and expressed in a reciprocal manner. This phenomenon is observed on two distinct scales.


Allopolyploids  are species whose increased complement of genetic material is the result of hybridization of two closely related  species . Thus homeologous chromosomes in allopolyploids are equivalent, but not identical. These differences mean that the precise pattern of  silencing  and expression can have important  phenotypic  effects. Reciprocal silencing on the  population  level refers to the case where two populations are each descended from the same  allopolyploid . In one population, one of the two equivalent loci (A) is expressed while the other (B) has been silenced, while in the other population the reciprocal pattern occurs, with B being expressed and A silenced. It is important to note that this refers to equivalent loci, specific locations within the genome, rather than the entire homeologous  chromosome .


Reciprocal silencing on the population level has been proposed as a means of  allopatric speciation  following a  polyploid  event. [ 1 ]  Allopatric speciation occurs when two populations of the same species become spatially separated and accumulate enough genetic differences to lose the ability to interbreed. As redundant genes are silenced in allopolyploids there is the potential for rapid genetic differences to accumulate through reciprocal silencing. These differences can lead to the loss of ability to interbreed between separated populations at a faster rate than other methods of  speciation , given the relative speed with which genes are silenced following a polyploid event. Faster still, redundant genes can be silenced through  epigenetic  means, although the importance of this phenomenon is not fully understood. [ 2 ]


Reciprocal silencing on the  tissue  level refers to the same pattern of  silencing  and expression of homeologous loci. However, in this case, the differences in silencing and expression occur between two types of tissue within the same individual, rather than in individuals of different populations. This is an example of  neofunctionalization , a process where duplicated genes that were once at equivalent loci evolve to carry out two separate functions. [ 3 ]  Since different tissues require different genes to be expressed, reciprocal silencing can occur between  tissues . Importantly, while the pattern of gene expression is the same as in the population case, the genetic means by which this pattern is achieved are very different. While  silencing mutations  are thought to be the main source of reciprocal silencing
at the population level, at the  tissue  level only epigenetic factors are in play, since expressible copies of both homeologous loci must exist in all cells in an individual if different tissues express different homeologs.


RecLOH  is a term in  genetics  that is an abbreviation for " Recombinant  Loss of  Heterozygosity ".


This is a type of  mutation  which occurs with  DNA  by  recombination . From a pair of equivalent ("homologous"), but slightly different ( heterozygous ) genes, a pair of identical genes results. In this case there is a non-reciprocal exchange of genetic code between the chromosomes, in contrast to  chromosomal crossover , because genetic information is lost.


In  genetic genealogy , the term is used particularly concerning similar seeming events in  Y chromosome  DNA. This type of mutation happens within one chromosome, and does not involve a reciprocal transfer. Rather, one homologous segment "writes over" the other. The mechanism is presumed to be different from RecLOH events in  autosomal chromosomes , since the target is the very same chromosome instead of the homologous one.


During the mutation one of these copies overwrites the other. Thus the differences between the two are lost. Because differences are lost,  heterozygosity  is lost.


Recombination on the  Y-chromosome  does not only take place during  meiosis , but virtually at every  mitosis  when the Y chromosome condenses, because it doesn't require pairing between chromosomes. Recombination frequency even exceeds the frame shift mutation frequency ( slipped strand mispairing ) of (average fast)  Y-STRs , however many recombination products may lead to infertile germ cells and "daughter out".


Recombination events (RecLOH)  can be observed if YSTR databases are searched for twin alleles at 3 or more duplicated markers on the same palindrome ( hairpin ).
E.g.  DYS459, DYS464 and DYS724  (CDY) are located on the same palindrome P1. A high proportion of 9-9, 15-15-17-17, 36-36 combinations and similar twin allelic patterns will be found. PCR typing technologies have been developed (e.g.  DYS464X ) that are able to verify that there are most frequently really two alleles of each, so we can be sure that there is no  gene deletion . Family genealogies have proven many times, that parallel changes on all markers located on the same palindrome are frequently observed and the result of those changes are always twin alleles. So a 9–10, 15-16-17-17, 36-38 haplotype can change in one recombination event to the one mentioned above, because all three markers ( DYS459, DYS464 and DYS724 ) are affected by one and the same recLOH event.


The  red-suffusion rose-faced lovebird  ( Agapornis roseicollis ), also known as the  red-pied lovebird , is not a true colour mutation of lovebird species. Many breeders believe it is due to a health issue, most likely dealing with the bird's liver. Some think the red-pied has some genetic relations with the  Lutino rosy-faced lovebird mutation , as many cases of red spots appear in Lutino lovebirds. Although many breeders of parrots have claimed that this is a  genetic mutation , no one has been able to successfully reproduce it through a series of generations. [ 1 ]


Replication protein A  ( RPA ) is the major  protein  that binds to single-stranded  DNA  (ssDNA) in  eukaryotic  cells. [ 1 ] [ 2 ]   In vitro , RPA shows a much higher affinity for ssDNA than RNA or double-stranded DNA. [ 3 ]  RPA is required in  replication ,  recombination  and repair processes such as  nucleotide excision repair  and  homologous recombination . [ 2 ] [ 4 ]   It also plays roles in responding to damaged DNA. [ 4 ]


RPA is a  heterotrimer , composed of the subunits RPA1 (RPA70) (70kDa subunit), RPA2 (RPA32) (32kDa subunit) and RPA3 (RPA14) (14kDa subunit). The three RPA subunits contain six  OB-folds (oligonucleotide/oligosaccharide binding) , with  DNA-binding domains  (DBD) designated DBDs A-F, that bind RPA to  single-stranded DNA . [ 2 ] [ 3 ]


DBDs A, B, C and F are located on RPA1, DBD D is located on RPA2, and DBD E is located on RPA3. [ 4 ]   DBDs C, D, and E make up the trimerization core of the protein with  flexible linker  regions connecting them all together. [ 4 ]   Due to these flexible linker regions RPA is considered highly flexible and this supports the dynamic binding that RPA is able to achieve.  Because of this dynamic binding, RPA is also capable of different conformations that leads to varied numbers of nucleotides that it can engage. [ 4 ]


DBDs A, B, C and D are the sites that are involved in ssDNA binding. [ 5 ]   Protein-protein interactions between RPA and other proteins happen at the  N-terminal  of RPA1, specifically DBD F, along with the  C-terminal  of RPA2. [ 5 ]   Phosphorylation  of RPA takes place at the N-terminus of RPA2. [ 5 ]


RPA shares many features with the  CST complex  heterotrimer, although RPA has a more uniform 1:1:1  stoichiometry . [ 6 ]


During DNA replication, RPA prevents single-stranded DNA (ssDNA) from winding back on itself or from forming secondary structures. It also helps protect the ssDNA from being attacked by  endonucleases . [ 2 ]  This keeps DNA unwound for the  polymerase  to replicate it.  RPA also binds to ssDNA during the initial phase of homologous recombination, an important process in  DNA repair  and  prophase I  of  meiosis .


RPA has a key role in the maintenance of the  recombination  checkpoint during  meiosis  of the yeast  Saccharomyces cerevisiae . [ 7 ]   RPA appears to act as a sensor of single-strand DNA for the activation of the meiotic DNA damage response. [ 7 ]


Hypersensitivity to DNA damaging agents can be caused by  mutations  in the RPA gene. [ 8 ]   Like its role in DNA replication, this keeps ssDNA from binding to itself (self-complementizing) so that the resulting  nucleoprotein  filament can then be bound by  Rad51  and its cofactors. [ 8 ]


RPA also binds to DNA during the nucleotide excision repair process. This binding stabilizes the repair complex during the repair process. A bacterial homolog is called  single-strand binding protein  (SSB).


The mechanisms of  reproductive isolation  are a collection of  evolutionary  mechanisms,  behaviors  and  physiological processes  critical for  speciation . They prevent members of different  species  from producing  offspring , or ensure that any offspring are sterile. These barriers maintain the integrity of a species by reducing  gene flow  between related species. [ 1 ] [ 2 ] [ 3 ] [ 4 ]


The mechanisms of reproductive isolation have been classified in a number of ways. Zoologist  Ernst Mayr  classified the mechanisms of reproductive isolation in two broad categories: pre-zygotic for those that act before  fertilization  (or before  mating  in the case of  animals ) and post-zygotic for those that act after it. [ 5 ]  The mechanisms are  genetically  controlled and can appear in species whose geographic distributions overlap ( sympatric speciation ) or are separate ( allopatric speciation ).


Pre-zygotic isolation mechanisms are the most economic in terms of the  natural selection  of a population, as resources are not wasted on the production of a descendant that is weak, non-viable or sterile. These mechanisms include physiological or systemic barriers to fertilization.


Any of the factors that prevent potentially fertile individuals from meeting will reproductively isolate the members of distinct species.  The types of barriers that can cause this isolation include: different  habitats , physical barriers, and a difference in the time of  sexual maturity  or flowering. [ 6 ] [ 7 ]


An example of the ecological or habitat differences that impede the meeting of potential pairs occurs in two fish species of the family  Gasterosteidae  (sticklebacks). One species lives all year round in fresh  water , mainly in small streams.  The other species lives in the sea during winter, but in spring and summer individuals migrate to river estuaries to reproduce. The members of the two populations are reproductively isolated due to their adaptations to distinct salt concentrations. [ 6 ]
An example of reproductive isolation due to differences in the mating season are found in the toad species  Bufo americanus  and  Bufo fowleri . The members of these species can be successfully crossed in the laboratory producing healthy, fertile hybrids. However, mating does not occur in the wild even though the geographical distribution of the two species overlaps. The reason for the absence of  inter-species mating  is that  B. americanus  mates in early summer and  B. fowleri  in late summer. [ 6 ]
Certain plant species, such as  Tradescantia canaliculata  and  T. subaspera , are sympatric throughout their geographic distribution, yet they are reproductively isolated as they flower at different times of the year. In addition, one species grows in sunny areas and the other in deeply shaded areas. [ 3 ] [ 7 ]


The different  mating rituals  of animal species creates extremely powerful reproductive barriers, termed sexual or behavior isolation, that isolate apparently similar species in the majority of the groups of the animal kingdom. In  dioecious  species, males and females have to search for a partner, be in proximity to each other, carry out the complex mating rituals and finally copulate or release their gametes into the environment in order to breed. [ 8 ] [ 9 ] [ 10 ]


Mating dances, the songs of males to attract females or the mutual grooming of pairs, are all examples of typical courtship behavior that allows both recognition and reproductive isolation. This is because each of the stages of courtship depend on the behavior of the partner.  The male will only move onto the second stage of the exhibition if the female shows certain responses in her behavior.  He will only pass onto the third stage when she displays a second key behavior. The behaviors of both interlink, are synchronized in time and lead finally to copulation or the liberation of gametes into the environment.  No animal that is not physiologically suitable for fertilization can complete this demanding chain of behavior.  In fact, the smallest difference in the courting patterns of two species is enough to prevent mating (for example, a specific song pattern acts as an isolation mechanism in distinct species of grasshopper of the genus  Chorthippus [ 11 ] ).
Even where there are minimal morphological differences between species, differences in behavior can be enough to prevent mating. For example,  Drosophila melanogaster  and  D. simulans  which are considered twin species due to their morphological similarity, do not mate even if they are kept together in a laboratory. [ 3 ] [ 12 ]   Drosophila ananassae  and  D. pallidosa  are twin species from  Melanesia . In the wild they rarely produce hybrids, although in the laboratory it is possible to produce fertile offspring. Studies of their sexual behavior show that the males court the females of both species but the females show a marked preference for mating with males of their own species. A different regulator region has been found on Chromosome II of both species that affects the selection behavior of the females. [ 12 ]


Pheromones  play an important role in the sexual isolation of insect species. [ 13 ]  These compounds serve to identify individuals of the same species and of the same or different sex. Evaporated molecules of  volatile  pheromones can serve as a wide-reaching chemical signal. In other cases, pheromones may be detected only at a short distance or by contact.


In species of the  melanogaster  group of  Drosophila , the pheromones of the females are mixtures of different compounds, there is a clear dimorphism in the type and/or quantity of compounds present for each sex. In addition, there are differences in the quantity and quality of constituent compounds between related species, it is assumed that the pheromones serve to distinguish between individuals of each species. An example of the role of pheromones in sexual isolation is found in 'corn borers' in the genus  Ostrinia . There are two twin species in Europe that occasionally cross. The females of both species produce pheromones that contain a volatile compound which has two  isomers , E and Z; 99% of the compound produced by the females of one species is in the E isomer form, while the females of the other produce 99% isomer Z. The production of the compound is controlled by just one  locus  and the  interspecific hybrid  produces an equal mix of the two isomers.  The males, for their part, almost exclusively detect the isomer emitted by the females of their species, such that the hybridization although possible is scarce.  The perception of the males is controlled by one  gene , distinct from the one for the production of isomers, the  heterozygous  males show a moderate response to the odour of either type.  In this case, just 2 'loci' produce the effect of ethological isolation between species that are genetically very similar. [ 12 ]


Sexual isolation between two species can be asymmetrical.  This can happen when the mating that produces descendants only allows one of the two species to function as the female progenitor and the other as the male, while the reciprocal cross does not occur.  For instance, half of the  wolves  tested in the  Great Lakes  area of  America  show  mitochondrial DNA  sequences of  coyotes , while mitochondrial DNA from wolves is never found in coyote populations. This probably reflects an asymmetry in inter-species mating due to the difference in size of the two species as male wolves take advantage of their greater size in order to mate with female coyotes, while female wolves and male coyotes do not mate. [ 14 ] [a]


Mating pairs may not be able to couple successfully if their genitals are not compatible. The relationship between the reproductive isolation of species and the form of their genital organs was signaled for the first time in 1844 by the French  entomologist   Léon Dufour . [ 15 ]  Insects' rigid carapaces act in a manner analogous to a lock and key, as they will only allow mating between individuals with complementary structures, that is, males and females of the same species (termed  co-specifics ).


Evolution has led to the development of genital organs with increasingly complex and divergent characteristics, which will cause mechanical isolation between species.  Certain characteristics of the genital organs will often have converted them into mechanisms of isolation.  However, numerous studies show that organs that are anatomically very different can be functionally compatible, indicating that other factors also determine the form of these complicated structures. [ 16 ]


Mechanical isolation also occurs in plants and this is related to the adaptation and  coevolution  of each species in the attraction of a certain type of pollinator (where  pollination  is  zoophilic ) through a collection of  morphophysiological  characteristics of the flowers (called  pollination syndrome ), in such a way that the transport of  pollen  to other species does not occur. [ 17 ]


The synchronous  spawning  of many species of coral in marine  reefs  means that inter-species hybridization can take place as the gametes of hundreds of individuals of tens of species are liberated into the same water at the same time. Approximately a third of all the possible crosses between species are compatible, in the sense that the gametes will fuse and lead to individual hybrids. This hybridization apparently plays a fundamental role in the evolution of coral species. [ 18 ]  However, the other two-thirds of possible crosses are incompatible.  It has been observed that in  sea urchins  of the genus  Strongylocentrotus  the concentration of  spermatocytes  that allow 100% fertilization of the ovules of the same species is only able to fertilize 1.5% of the ovules of other species. This inability to produce hybrid offspring, despite the fact that the gametes are found at the same time and in the same place, is due to a phenomenon known as  gamete incompatibility , which is often found between marine invertebrates, and whose physiological causes are not fully understood. [ 19 ] [ 20 ]


In some  Drosophila  crosses, the swelling of the female's  vagina  has been noted following insemination.  This has the effect of consequently preventing the fertilization of the ovule by sperm of a different species. [ 21 ] [ clarification needed ]


In plants the pollen grains of a species can germinate in the  stigma  and grow in the  style  of other species.  However, the growth of the pollen tubes may be detained at some point between the stigma and the ovules, in such a way that fertilization does not take place.  This mechanism of reproductive isolation is common in the  angiosperms  and is called  cross-incompatibility  or  incongruence . [ 22 ] [ 23 ]   A relationship exists between self-incompatibility and the phenomenon of cross-incompatibility. In general crosses between individuals of a self-compatible species (SC) with individuals of a self-incompatible (SI) species give hybrid offspring.  On the other hand, a reciprocal cross (SI x SC) will not produce offspring, because the pollen tubes will not reach the ovules. This is known as  unilateral incompatibility , which also occurs when two SC or two SI species are crossed. [ 24 ]


A number of mechanisms which act after fertilization preventing successful inter-population crossing are discussed below.


A type of incompatibility that is found as often in plants as in animals occurs when the egg or  ovule  is fertilized but the  zygote  does not develop, or it develops and the resulting individual has a reduced viability. [ 3 ]  This is the case for crosses between species of the  frog  order, where widely differing results are observed depending upon the species involved. In some crosses there is no segmentation of the zygote (or it may be that the hybrid is extremely non-viable and changes occur from the first  mitosis ). In others, normal segmentation occurs in the  blastula  but  gastrulation  fails. Finally, in other crosses, the initial stages are normal but errors occur in the final phases of  embryo development . This indicates differentiation of the embryo development genes (or gene complexes) in these species and these differences determine the non-viability of the hybrids. [ 25 ]


Similar results are observed in mosquitoes of the genus  Culex , but the differences are seen between  reciprocal crosses , from which it is concluded that the same effect occurs in the interaction between the genes of the  cell nucleus  (inherited from both parents) as occurs in the genes of the  cytoplasmic organelles  which are inherited solely from the female progenitor through the cytoplasm of the ovule. [ 3 ]


In Angiosperms, the successful development of the embryo depends on the normal functioning of its  endosperm . [ 26 ]


The failure of endosperm development and its subsequent abortion has been observed in many  interploidal crosses  (that is, those between populations with a particular degree of intra or interspecific  ploidy ), [ 26 ] [ 27 ] [ 28 ] [ 29 ] [ 30 ]  and in certain crosses in species with the same level of ploidy. [ 30 ] [ 31 ] [ 32 ]  The collapse of the  endosperm , and the subsequent abortion of the hybrid embryo is one of the most common post-fertilization reproductive isolation mechanism found in  angiosperms .