A hybrid may have normal viability but is typically deficient in terms of reproduction or is sterile. This is demonstrated by the  mule  and in many other well known hybrids. In all of these cases sterility is due to the interaction between the genes of the two species involved; to chromosomal imbalances due to the different number of chromosomes in the parent species; or to nucleus-cytoplasmic interactions such as in the case of  Culex  described above. [ 3 ]


Hinnies  and mules are  hybrids  resulting from a cross between a  horse  and a  donkey  or between a mare and a donkey, respectively. These animals are nearly always sterile due to the difference in the number of chromosomes between the two parent species. Both horses and donkeys belong to the genus  Equus , but  Equus caballus  has 64 chromosomes, while  Equus asinus  only has 62. A cross will produce offspring (mule or hinny) with 63 chromosomes, that will not form pairs, which means that they do not divide in a balanced manner during  meiosis . In the wild, the horses and donkeys ignore each other and do not cross. In order to obtain mules or hinnies it is necessary to train the progenitors to accept copulation between the species or create them through  artificial insemination .


The sterility of many interspecific hybrids in  angiosperms  has been widely recognised and studied. [ 33 ]
Interspecific sterility of hybrids in plants has multiple possible causes. These may be genetic, related to the genomes, or the interaction between nuclear and cytoplasmic factors, as will be discussed in the corresponding section. Nevertheless, in plants, hybridization is a stimulus for the creation of new species – the contrary to the situation in animals. [ 34 ]
Although the hybrid may be sterile, it can continue to multiply in the wild by  asexual reproduction , whether  vegetative propagation  or  apomixis  or the production of seeds. [ 35 ] [ 36 ] [ clarification needed ]
Indeed, interspecific hybridization can be associated with  polyploidia  and, in this way, the origin of new species that are called  allopolyploids .  Rosa canina , for example, is the result of multiple hybridizations. [ 37 ]  The  common wheat  ( Triticum aestivum ) is an allohexaploid (allopolyploid with six chromosome sets) that contains the genomes of three different species. [ 38 ] [ 39 ]


In general, the barriers that separate species do not consist of just one mechanism.  The twin species of   Drosophila ,  D. pseudoobscura  and  D. persimilis , are isolated from each other by habitat ( persimilis  generally lives in colder regions at higher altitudes), by the timing of the mating season ( persimilis  is generally more active in the morning and  pseudoobscura  at night) and by behavior during mating (the females of both species prefer the males of their respective species). In this way, although the distribution of these species overlaps in wide areas of the west of the United States of America, these isolation mechanisms are sufficient to keep the species separated. Such that, only a few fertile females have been found amongst the other species among the thousands that have been analyzed. However, when hybrids are produced between both species, the  gene flow  between the two will continue to be impeded as the hybrid males are sterile. Also, and in contrast with the great vigor shown by the sterile males, the descendants of the backcrosses of the hybrid females with the parent species are weak and notoriously non-viable. This last mechanism restricts even more the genetic interchange between the two species of fly in the wild. [ 3 ]


Haldane's rule  states that when one of the two sexes is absent in interspecific hybrids between two specific species, then the sex that is not produced, is rare or is sterile is the  heterozygous  (or heterogametic) sex. [ 40 ]  In mammals, at least, there is growing evidence to suggest that this is due to high rates of mutation of the genes determining masculinity in the  Y chromosome . [ 40 ] [ 41 ] [ 42 ]


It has been suggested that Haldane's rule simply reflects the fact that the male sex is more sensitive than the female when the sex-determining genes are included in a  hybrid genome . But there are also organisms in which the heterozygous sex is the female:  birds  and  butterflies  and the law is followed in these organisms. Therefore, it is not a problem related to sexual development, nor with the sex chromosomes. Haldane proposed that the stability of hybrid individual development requires the full gene complement of each parent species, so that the hybrid of the heterozygous sex is unbalanced (i.e. missing at least one chromosome from each of the parental species). For example, the hybrid male obtained by crossing   D. melanogaster  females with    D. simulans  males, which is non-viable, lacks the X chromosome of   D. simulans . [ 12 ]


The genetics of  ethological  isolation barriers will be discussed first. Pre-copulatory isolation occurs when the genes necessary for the sexual reproduction of one species differ from the equivalent genes of another species, such that if a male of species A and a female of species B are placed together they are unable to copulate. Study of the genetics involved in this reproductive barrier tries to identify the genes that govern distinct sexual behaviors in the two species. The males of   Drosophila melanogaster  and those of   D. simulans  conduct an elaborate courtship with their respective females, which are different for each species, but the differences between the species are more quantitative than qualitative.  In fact the  simulans  males are able to hybridize with the   melanogaster  females. Although there are lines of the latter species that can easily cross there are others that are hardly able to. Using this difference, it is possible to assess the minimum number of genes involved in pre-copulatory isolation between the  melanogaster  and  simulans  species and their chromosomal location. [ 12 ]


In experiments, flies of the  D. melanogaster  line, which hybridizes readily with  simulans ,  were crossed with another line that it does not hybridize with, or rarely.  The females of the segregated populations obtained by this cross were placed next to  simulans  males and the percentage of hybridization was recorded, which is a measure of the degree of reproductive isolation. It was concluded from this experiment that 3 of the 8 chromosomes of the  haploid  complement of   D. melanogaster   carry at least one gene that affects isolation, such that substituting one chromosome from a line of low isolation with another of high isolation reduces the hybridization frequency. In addition, interactions between chromosomes are detected so that certain combinations of the chromosomes have a multiplying effect. [ 12 ]
Cross incompatibility or incongruence in plants is also determined by major genes that are not associated at the  self-incompatibility   S locus . [ 43 ] [ 44 ] [ 45 ]


Reproductive isolation between species appears, in certain cases, a long time after fertilization and the formation of the zygote, as happens – for example – in the twin species  Drosophila pavani  and  D. gaucha . The hybrids between both species are not sterile, in the sense that they produce viable gametes, ovules and spermatozoa. However, they cannot produce offspring as the sperm of the hybrid male do not survive in the semen receptors of the females, be they hybrids or from the parent lines. In the same way, the sperm of the males of the two parent species do not survive in the reproductive tract of the hybrid female. [ 12 ]  This type of post-copulatory isolation appears as the most efficient system for maintaining reproductive isolation in many species. [ 46 ]


The development of a zygote into an adult is a complex and delicate process of interactions between genes and the environment that must be carried out precisely, and if there is any alteration in the usual process, caused by the absence of a necessary gene or the presence of a different one, it can arrest the normal development causing the non-viability of the hybrid or its sterility. It should be borne in mind that half of the chromosomes and genes of a hybrid are from one species and the other half come from the other. If the two species are genetically different, there is little possibility that the genes from both will act harmoniously in the hybrid. From this perspective, only a few genes would be required in order to bring about post copulatory isolation, as opposed to the situation described previously for pre-copulatory isolation. [ 12 ] [ 47 ]


In many species where pre-copulatory reproductive isolation does not exist, hybrids are produced but they are of only one sex. This is the case for the hybridization between females of  Drosophila simulans  and  Drosophila melanogaster  males: the hybridized females die early in their development so that only males are seen among the offspring. However, populations of  D. simulans  have been recorded with genes that permit the development of adult hybrid females, that is, the viability of the females is "rescued". It is assumed that the normal activity of these  speciation  genes is to "inhibit" the expression of the genes that allow the growth of the hybrid. There will also be regulator genes. [ 12 ]


A number of these genes have been found in the  melanogaster  species group. The first to be discovered was "Lhr" (Lethal hybrid rescue) located in Chromosome II of  D. simulans . This  dominant allele  allows the development of hybrid females from the cross between  simulans  females and  melanogaster  males. [ 48 ]  A different gene, also located on Chromosome II of  D. simulans  is "Shfr" that also allows the development of female hybrids, its activity being dependent on the temperature at which  development  occurs. [ 49 ]  Other similar genes have been located in distinct populations of species of this group. In short, only a few genes are needed for an effective post copulatory isolation barrier mediated through the non-viability of the hybrids.


As important as identifying an isolation gene is knowing its function. The  Hmr  gene, linked to the X chromosome and implicated in the viability of male hybrids between  D. melanogaster  and  D. simulans , is a gene from the  proto-oncogene  family  myb , that codes for a transcriptional regulator. Two variants of this gene function perfectly well in each separate species, but in the hybrid they do not function correctly, possibly due to the different genetic background of each species. Examination of the allele sequence of the two species shows that change of direction substitutions are more abundant than  synonymous substitutions , suggesting that this gene has been subject to intense natural selection. [ 50 ]


The  Dobzhansky –Muller model proposes that reproductive incompatibilities between species are caused by the interaction of the genes of the respective species. It has been demonstrated recently that  Lhr  has functionally diverged in  D. simulans  and will interact with  Hmr  which, in turn, has functionally diverged in  D. melanogaster  to cause the lethality of the male hybrids.  Lhr  is located in a heterochromatic region of the genome and its sequence has diverged between these two species in a manner consistent with the mechanisms of positive selection. [ 51 ]  An important unanswered question is whether the genes detected correspond to old genes that initiated the speciation favoring hybrid non-viability, or are modern genes that have appeared post-speciation by mutation, that are not shared by the different populations and that suppress the effect of the primitive non-viability genes. The  OdsH  (abbreviation of  Odysseus ) gene causes partial sterility in the hybrid between  Drosophila simulans  and a related species,  D. mauritiana , which is only encountered on  Mauritius , and is of recent origin. This gene shows  monophyly  in both species and also has been subject to natural selection. It is thought that it is a gene that intervenes in the initial stages of speciation, while other genes that differentiate the two species show  polyphyly .  Odsh  originated by duplication in the genome of  Drosophila  and has evolved at very high rates in  D. mauritania , while its  paralogue ,  unc-4 , is nearly identical between the species of the group  melanogaster . [ 52 ] [ 53 ] [ 54 ] [ 55 ]  Seemingly, all these cases illustrate the manner in which speciation mechanisms originated in nature, therefore they are collectively known as "speciation genes", or possibly, gene sequences with a normal function within the populations of a species that diverge rapidly in response to positive selection thereby forming reproductive isolation barriers with other species. In general, all these genes have functions in the transcriptional  regulation  of other genes. [ 56 ]


The  Nup96  gene is another example of the evolution of the genes implicated in post-copulatory isolation. It regulates the production of one of the approximately 30 proteins required to form a  nuclear pore . In each of the  simulans  groups of  Drosophila  the protein from this gene interacts with the protein from another, as yet undiscovered, gene on the X chromosome in order to form a functioning pore. However, in a hybrid the pore that is formed is defective and causes sterility. The differences in the sequences of  Nup96  have been subject to adaptive selection, similar to the other examples of   speciation genes  described above. [ 57 ] [ 58 ]


Post-copulatory isolation can also arise between chromosomally differentiated populations due to  chromosomal translocations  and  inversions . [ 59 ]  If, for example, a reciprocal translocation is fixed in a population, the hybrid produced between this population and one that does not carry the translocation will not have a complete  meiosis . This will result in the production of unequal gametes containing unequal numbers of chromosomes with a reduced fertility. In certain cases, complete translocations exist that involve more than two chromosomes, so that the meiosis of the hybrids is irregular and their fertility is zero or nearly zero. [ 60 ]  Inversions can also give rise to abnormal gametes in heterozygous individuals but this effect has little importance compared to translocations. [ 59 ]  An example of chromosomal changes causing sterility in hybrids comes from the study of  Drosophila nasuta  and  D. albomicans  which are twin species from the Indo-Pacific region. There is no sexual isolation between them and the  F1 hybrid  is fertile. However, the F2 hybrids are relatively infertile and leave few descendants which have a skewed ratio of the sexes. The reason is that the X chromosome of  albomicans  is translocated and linked to an  autosome  which causes abnormal meiosis in hybrids.  Robertsonian translocations  are variations in the numbers of chromosomes that arise from either: the fusion of two  acrocentric  chromosomes into a single chromosome with two arms, causing a reduction in the haploid number, or conversely; or the fission of one chromosome into two acrocentric chromosomes, in this case increasing the haploid number. The hybrids of two populations with differing numbers of chromosomes can experience a certain loss of fertility, and therefore a poor adaptation, because of irregular meiosis.


A large variety of mechanisms have been demonstrated to reinforce reproductive isolation between closely related plant species that either historically lived or currently live in  sympatry . This phenomenon is driven by strong selection against hybrids, typically resulting from instances in which hybrids suffer reduced fitness. Such negative fitness consequences have been proposed to be the result of negative  epistasis  in hybrid genomes and can also result from the effects of  hybrid sterility . [ 61 ]  In such cases, selection gives rise to population-specific isolating mechanisms to prevent either fertilization by interspecific gametes or the development of hybrid embryos.


Because many sexually reproducing species of plants are exposed to a variety of interspecific  gametes , natural selection has given rise to a variety of mechanisms to prevent the production of hybrids. [ 62 ]  These mechanisms can act at different stages in the developmental process and are typically divided into two categories, pre-fertilization and post-fertilization, indicating at which point the barrier acts to prevent either zygote formation or development. In the case of  angiosperms  and other pollinated species, pre-fertilization mechanisms can be further subdivided into two more categories, pre-pollination and post-pollination, the difference between the two being whether or not a pollen tube is formed. (Typically when pollen encounters a receptive stigma, a series of changes occur which ultimately lead to the growth of a pollen tube down the style, allowing for the formation of the zygote.) Empirical investigation has demonstrated that these barriers act at many different developmental stages and species can have none, one, or many barriers to hybridization with interspecifics.


A well-documented example of a pre-fertilization isolating mechanism comes from study of Louisiana iris species.  These iris species were fertilized with interspecific and conspecific pollen loads and it was demonstrated by measure of hybrid progeny success that differences in pollen-tube growth between interspecific and conspecific pollen led to a lower fertilization rate by interspecific pollen. [ 63 ]  This demonstrates how a specific point in the reproductive process is manipulated by a particular isolating mechanism to prevent hybrids.


Another well-documented example of a pre-fertilization isolating mechanism in plants comes from study of the 2 wind-pollinated birch species. Study of these species led to the discovery that mixed conspecific and interspecific pollen loads still result in 98% conspecific fertilization rates, highlighting the effectiveness of such barriers. [ 64 ]  In this example, pollen tube incompatibility and slower generative mitosis have been implicated in the post-pollination isolation mechanism.


Crosses between diploid and tetraploid species of Paspalum provide evidence of a post-fertilization mechanism preventing hybrid formation when pollen from tetraploid species was used to fertilize a female of a diploid species. [ 65 ]  There were signs of fertilization and even endosperm formation but subsequently this endosperm collapsed. This demonstrates evidence of an early post-fertilization isolating mechanism, in which the hybrid early embryo is detected and selectively aborted. [ 66 ]  This process can also occur later during development in which developed, hybrid seeds are selectively aborted. [ 67 ]


Plant hybrids often suffer from an autoimmune syndrome known as hybrid necrosis. In the hybrids, specific gene products contributed by one of the parents may be inappropriately recognized as foreign and pathogenic, and thus trigger pervasive cell death throughout the plant. [ 68 ]  In at least one case, a pathogen receptor, encoded by the most variable gene family in plants, was identified as being responsible for hybrid necrosis. [ 69 ]


In brewers' yeast  Saccharomyces cerevisiae ,  chromosomal rearrangements  are a major mechanism to reproductively isolate different strains. Hou et al. [ 70 ]  showed that reproductive isolation acts postzygotically and could be attributed to chromosomal rearrangements. These authors crossed 60 natural isolates sampled from diverse niches with the reference strain S288c and identified 16 cases of reproductive isolation with reduced offspring viabilities, and identified reciprocal chromosomal translocations in a large fraction of isolates. [ 70 ]


In addition to the genetic causes of reproductive isolation between species there is another factor that can cause post zygotic isolation: the presence of  microorganisms  in the cytoplasm of certain species. The presence of these organisms in a species and their absence in another causes the non-viability of the corresponding hybrid. For example, in the  semi-species  of the group  D. paulistorum  the hybrid females are fertile but the males are sterile, this is due to the presence of a  Wolbachia [ 71 ]  in the cytoplasm which alters  spermatogenesis  leading to sterility. It is interesting that incompatibility or isolation can also arise at an intraspecific level. Populations of  D. simulans  have been studied that show hybrid sterility according to the direction of the cross. The factor determining sterility has been found to be the presence or absence of a microorganism  Wolbachia  and the populations tolerance or susceptibility to these organisms. This inter population incompatibility can be eliminated in the laboratory through the administration of a specific  antibiotic  to kill the microorganism. Similar situations are known in a number of insects, as around 15% of species show infections caused by this  symbiont . It has been suggested that, in some cases, the speciation process has taken place because of the incompatibility caused by this bacteria. Two  wasp  species  Nasonia giraulti  and  N. longicornis  carry two different strains of  Wolbachia . Crosses between an infected population and one free from infection produces a nearly total reproductive isolation between the semi-species. However, if both species are free from the bacteria or both are treated with antibiotics there is no reproductive barrier. [ 72 ] [ 73 ]   Wolbachia  also induces incompatibility due to the weakness of the hybrids in populations of spider mites ( Tetranychus urticae ), [ 74 ]  between  Drosophila recens  and  D. subquinaria [ 75 ]  and between species of  Diabrotica  (beetle) and  Gryllus  (cricket). [ 76 ]


In 1950  K. F. Koopman  reported results from experiments designed to examine the hypothesis that  selection  can increase reproductive isolation between populations. He used  D. pseudoobscura  and  D. persimilis  in these experiments. When the flies of these species are kept at 16 °C approximately a third of the matings are interspecific. In the experiment equal numbers of males and females of both species were placed in containers suitable for their survival and reproduction. The progeny of each generation were examined in order to determine if there were any interspecific hybrids. These hybrids were then eliminated. An equal number of males and females of the resulting progeny were then chosen to act as progenitors of the next generation. As the hybrids were destroyed in each generation the flies that solely mated with members of their own species produced more surviving descendants than the flies that mated solely with individuals of the other species. In the adjacent table it can be seen that for each generation the number of hybrids continuously decreased up to the tenth generation when hardly any interspecific hybrids were produced. [ 77 ]  It is evident that selection against the hybrids was very effective in increasing reproductive isolation between these species. From the third generation, the proportions of the hybrids were less than 5%. This confirmed that selection acts to reinforce the reproductive isolation of two genetically divergent populations if the hybrids formed by these species are less well adapted than their parents.


These discoveries allowed certain assumptions to be made regarding the origin of reproductive isolation mechanisms in nature. Namely, if selection reinforces the degree of reproductive isolation that exists between two species due to the poor adaptive value of the hybrids, it is expected that the populations of two species located in the same area will show a greater reproductive isolation than populations that are geographically separated (see  reinforcement ). This mechanism for "reinforcing" hybridization barriers in sympatric populations is also known as the " Wallace effect ", as it was first proposed by  Alfred Russel Wallace  at the end of the 19th century, and it has been experimentally demonstrated in both plants and animals. [ 78 ] [ 79 ] [ 80 ] [ 81 ] [ 82 ] [ 83 ]


The sexual isolation between  Drosophila miranda  and  D. pseudoobscura , for example, is more or less pronounced according to the geographic origin of the flies being studied. Flies from regions where the distribution of the species is superimposed show a greater sexual isolation than exists between populations originating in distant regions.


On the other hand, interspecific hybridization barriers can also arise as a result of the adaptive divergence that accompanies  allopatric speciation . This mechanism has been experimentally proved by an experiment carried out by  Diane Dodd  on  D. pseudoobscura . A single population of flies was divided into two, with one of the populations fed with  starch -based food and the other with  maltose -based food.  This meant that each sub population was adapted to each food type over a number of generations. After the populations had diverged over many generations, the groups were again mixed; it was observed that the flies would mate only with others from their adapted population. This indicates that the mechanisms of reproductive isolation can arise even though the interspecific hybrids are not selected against. [ 84 ]


a.   ^   The DNA of the mitochondria and chloroplasts is inherited from the maternal line, i.e. all the progeny derived from a particular cross possess the same cytoplasm (and genetic factors located in it) as the female progenitor. This is because the zygote possesses the same cytoplasm as the ovule, although its nucleus comes equally from the father and the mother. [ 3 ]


A  restriction map  is a map of known  restriction sites  within a sequence of  DNA . Restriction mapping requires the use of  restriction enzymes . In  molecular biology , restriction maps are used as a reference to engineer plasmids or other relatively short pieces of DNA, and sometimes for longer genomic DNA. There are other ways of mapping features on DNA for longer length DNA molecules, such as mapping by  transduction . [ 1 ]


One approach in constructing a restriction map of a DNA molecule is to sequence the whole molecule and to run the sequence through a computer program that will find the recognition sites that are present for every restriction enzyme known.


Before sequencing was automated, it would have been prohibitively expensive to  sequence  an entire DNA strand. To find the relative positions of restriction sites on a plasmid, a technique involving single and double restriction digests is used. Based on the sizes of the resultant DNA fragments the positions of the sites can be inferred. Restriction mapping is a very useful technique when used for determining the orientation of an insert in a cloning vector, by mapping the position of an off-center restriction site in the insert. [ 2 ]


The experimental procedure first requires a sample of purified plasmid DNA for each digest to be run. Digestion is then performed with each enzyme(s) chosen. The resulting samples are subsequently run on an  electrophoresis  gel, typically on  agarose  gel.


The first step following the completion of electrophoresis is to add up the sizes of the fragments in each lane. The sum of the individual fragments should equal the size of the original fragment, and each digest's fragments should also sum up to be the same size as each other. If fragment sizes do not properly add up, there are two likely problems. In one case, some of the smaller fragments may have run off the end of the gel. This frequently occurs if the gel is run too long.   A second possible source of error is that the gel was not dense enough and therefore was unable to resolve fragments close in size.  This leads to a lack of separation of fragments which were close in size. If all of the digests produce fragments that add up one may infer the position of the REN (restriction endonuclease) sites by placing them in spots on the original DNA fragment that would satisfy the fragment sizes produced by all three digests


In this technique the cells are lysed in alkaline conditions. The DNA in the mixture is denatured (strands separated) by disrupting the hydrogen bonds between the two strands. The large genomic DNA is subject to tangling and staying denatured when the pH is lowered during the neutralization. In other words, the strands come back together in a disordered fashion, basepairing randomly. The circular  supercoiled plasmids'  strands will stay relatively closely aligned and will renature correctly. Therefore, the genomic DNA will form an insoluble aggregate and the  supercoiled  plasmids will be left in solution. This can be followed by  phenol extraction  to remove proteins and other molecules. Then the DNA can be subjected to  ethanol precipitation  to concentrate the sample.


A  reverse transcriptase  ( RT ) is an  enzyme  used to convert  RNA genome  to  DNA , a process termed  reverse transcription . Reverse transcriptases are used by  viruses  such as  HIV ,  COVID-19 , and  hepatitis B  to replicate their genomes, by   retrotransposon  mobile genetic elements to proliferate within the host genome, and by  eukaryotic  cells to extend the  telomeres  at the ends of their  linear chromosomes . Contrary to a widely held belief, the process does not violate the flows of genetic information as described by the classical  central dogma , as transfers of information from RNA to DNA are explicitly held possible. [ 2 ] [ 3 ] [ 4 ]


Retroviral RT has three sequential biochemical activities: RNA-dependent  DNA polymerase  activity,  ribonuclease H  (RNase H), and DNA-dependent DNA polymerase activity. Collectively, these activities enable the enzyme to convert single-stranded RNA into double-stranded cDNA. In retroviruses and retrotransposons, this cDNA can then integrate into the host genome, from which new RNA copies can be made via host-cell  transcription . The same sequence of reactions is widely used in the laboratory to convert RNA to DNA for use in  molecular cloning ,  RNA sequencing ,  polymerase chain reaction  (PCR), or  genome analysis .


Reverse transcriptases were discovered by  Howard Temin  at the  University of Wisconsin–Madison  in  Rous sarcoma  virions [ 5 ]  and independently isolated by  David Baltimore  in 1970 at  MIT  from two RNA tumour viruses:  murine leukemia virus  and again  Rous sarcoma virus . [ 6 ]  For their achievements, they shared the 1975  Nobel Prize in Physiology or Medicine  (with  Renato Dulbecco ).


Well-studied reverse transcriptases include:


The enzymes are encoded and used by viruses that use reverse transcription as a step in the process of replication. Reverse-transcribing  RNA viruses , such as  retroviruses , use the enzyme to reverse-transcribe their RNA  genomes  into DNA, which is then integrated into the host genome and replicated along with it. Reverse-transcribing  DNA viruses , such as the  hepadnaviruses , can allow RNA to serve as a template in assembling and making DNA strands. HIV infects humans with the use of this enzyme. Without reverse transcriptase, the viral genome would not be able to incorporate into the host cell, resulting in failure to replicate. [ citation needed ]


Reverse transcriptase creates double-stranded DNA from an RNA template.


In virus species with reverse transcriptase lacking DNA-dependent DNA polymerase activity, creation of double-stranded DNA can possibly be done by host-encoded  DNA polymerase δ , mistaking the viral DNA-RNA for a primer and synthesizing a double-stranded DNA by a similar mechanism as in  primer removal , where the newly synthesized DNA displaces the original RNA template. [ citation needed ]


The process of reverse transcription, also called retrotranscription or retrotras, is extremely error-prone, and it is during this step that mutations may occur. Such mutations may cause  drug resistance . [ citation needed ]


Retroviruses , also referred to as class VI  ssRNA-RT  viruses, are RNA reverse-transcribing viruses with a DNA intermediate. Their genomes consist of two molecules of  positive-sense  single-stranded RNA with a  5' cap  and  3' polyadenylated tail . Examples of retroviruses include the human immunodeficiency virus ( HIV ) and the human T-lymphotropic virus ( HTLV ). Creation of double-stranded DNA occurs in the  cytosol [ 10 ]  as a series of these steps:


Creation of double-stranded DNA also involves  strand transfer , in which there is a translocation of short DNA product from initial RNA-dependent DNA synthesis to acceptor template regions at the other end of the genome, which are later reached and processed by the reverse transcriptase for its DNA-dependent DNA activity. [ 11 ]


Retroviral RNA is arranged in 5' terminus to 3' terminus. The site where the  primer  is annealed to viral RNA is called the primer-binding site (PBS). The RNA 5'end to the PBS site is called U5, and the RNA 3' end to the PBS is called the leader. The tRNA primer is unwound between 14 and 22  nucleotides  and forms a base-paired duplex with the viral RNA at PBS. The fact that the PBS is located near the 5' terminus of viral RNA is unusual because reverse transcriptase synthesize DNA from 3' end of the primer in the 5' to 3'  direction (with respect to the newly synthesized DNA strand). Therefore, the primer and reverse transcriptase must be relocated to 3' end of viral RNA. In order to accomplish this reposition, multiple steps and various enzymes including  DNA polymerase , ribonuclease H(RNase H) and polynucleotide unwinding are needed. [ 12 ] [ 13 ]


The HIV reverse transcriptase also has  ribonuclease  activity that degrades the viral RNA during the synthesis of cDNA, as well as  DNA-dependent DNA polymerase  activity that copies the  sense  cDNA strand into an  antisense  DNA to form a double-stranded viral DNA intermediate (vDNA). [ 14 ]  The HIV viral RNA structural elements regulate the progression of reverse transcription. [ 15 ]


Self-replicating stretches of  eukaryotic  genomes known as  retrotransposons  utilize reverse transcriptase to move from one position in the genome to another via an RNA intermediate. They are found abundantly in the genomes of plants and animals.  Telomerase  is another reverse transcriptase found in many eukaryotes, including humans, which carries its own  RNA  template; this RNA is used as a template for  DNA replication . [ 16 ]


Initial reports of reverse transcriptase in prokaryotes came as far back as 1971 in France ( Beljanski  et al., 1971a, 1972) and a few years later in the USSR (Romashchenko 1977 [ 17 ] ).  These have since been broadly described as part of bacterial  Retrons , distinct sequences that code for reverse transcriptase, and are used in the synthesis of  msDNA . In order to initiate synthesis of DNA, a primer is needed. In bacteria, the primer is synthesized during replication. [ 18 ]


Valerian Dolja of Oregon State argues that viruses, due to their diversity, have played an evolutionary role in the development of cellular life, with reverse transcriptase playing a central role. [ 19 ]


The reverse transcriptase employs a "right hand" structure similar to that found in other  viral nucleic acid polymerases . [ 20 ] [ 21 ]  In addition to the transcription function, retroviral reverse transcriptases have a domain belonging to the  RNase H  family, which is vital to their replication. By degrading the RNA template, it allows the other strand of DNA to be synthesized. [ 22 ]  Some fragments from the digestion also serve as the primer for the  DNA polymerase  (either the same enzyme or a host protein), responsible for making the other (plus) strand. [ 20 ]


There are three different replication systems during the life cycle of a retrovirus. The first process is the reverse transcriptase synthesis of viral DNA from viral RNA, which then forms newly made complementary DNA strands. The second replication process occurs when host cellular DNA polymerase replicates the integrated viral DNA. Lastly, RNA polymerase II transcribes the proviral DNA into RNA, which will be packed into virions. Mutation can occur during one or all of these replication steps. [ 23 ]


Reverse transcriptase has a high error rate when transcribing RNA into DNA since, unlike most other  DNA polymerases , it has no  proofreading  ability. This high error rate allows  mutations  to accumulate at an accelerated rate relative to proofread forms of replication. The commercially available reverse transcriptases produced by  Promega  are quoted by their manuals as having error rates in the range of 1 in 17,000 bases for AMV and 1 in 30,000 bases for M-MLV. [ 24 ]


Other than creating  single-nucleotide polymorphisms , reverse transcriptases have also been shown to be involved in processes such as  transcript fusions ,  exon shuffling  and creating artificial  antisense  transcripts. [ 25 ] [ 26 ]  It has been speculated that this  template switching  activity of reverse transcriptase, which can be demonstrated completely  in vivo , may have been one of the causes for finding several thousand unannotated transcripts in the genomes of model organisms. [ 27 ]


Two  RNA   genomes  are packaged into each retrovirus particle, but, after an infection, each virus generates only one  provirus . [ 28 ]   After infection, reverse transcription is accompanied by template switching between the two genome copies (copy choice recombination). [ 28 ]   There are two models that suggest why RNA transcriptase switches templates. The first, the forced copy-choice model, proposes that reverse transcriptase changes the RNA template when it encounters a nick, implying that recombination is obligatory to maintaining virus genome integrity. The second, the dynamic choice model, suggests that reverse transcriptase changes templates when the RNAse function and the polymerase function are not in sync rate-wise, implying that recombination occurs at random and is not in response to genomic damage. A study by Rawson et al. supported both models of recombination. [ 28 ]  From 5 to 14 recombination events per genome occur at each replication cycle. [ 29 ]   Template switching (recombination) appears to be necessary for maintaining genome integrity and as a repair mechanism for salvaging damaged genomes. [ 30 ] [ 28 ]


As  HIV  uses reverse transcriptase to copy its genetic material and generate new viruses (part of a retrovirus proliferation circle), specific drugs have been designed to disrupt the process and thereby suppress its growth. Collectively, these drugs are known as  reverse-transcriptase inhibitors  and include the nucleoside and nucleotide analogues  zidovudine  (trade name Retrovir),  lamivudine  (Epivir) and  tenofovir  (Viread), as well as non-nucleoside inhibitors, such as  nevirapine  (Viramune). [ citation needed ]


Reverse transcriptase is commonly used in research to apply the  polymerase chain reaction  technique to  RNA  in a technique called  reverse transcription polymerase chain reaction  (RT-PCR).  The classical  PCR  technique can be applied only to  DNA  strands, but, with the help of reverse transcriptase, RNA can be transcribed into DNA, thus making  PCR  analysis of RNA molecules possible. Reverse transcriptase is used also to create  cDNA libraries  from  mRNA . The commercial availability of reverse transcriptase greatly improved knowledge in the area of molecular biology, as, along with other  enzymes , it allowed scientists to clone, sequence, and characterise RNA. [ citation needed ]


Revive & Restore  is a nonprofit wildlife conservation organization focused on use of  biotechnology  in  conservation . Headquartered in  Sausalito , California, the organization's mission is to enhance  biodiversity  through the genetic rescue of  endangered  and  extinct  species. [ 2 ] [ 3 ]  The organization was founded by  Stewart Brand  and his wife, Ryan Phelan.


Revive & Restore has created a "Genetic Rescue Toolkit" for wildlife conservation – a suite of biotechnology tools adapted from human medicine and commercial agriculture that can improve wildlife conservation outcomes. [ 4 ] [ 5 ] [ 6 ]  The toolkit includes  biobanking  and cell culturing,  genetic sequencing , and advanced reproductive technologies, such as  cloning . The toolkit complements traditional conservation practices, such as  captive breeding  and  habitat restoration . [ citation needed ]


Revive & Restore has caused controversy. In particular, Brand's work in de-extinction has been characterized as "playing god" and criticized for taking time and money away from traditional conservation efforts. [ 7 ] [ 8 ] [ 9 ]  In addition, many are concerned by the concept of  cloning , even in the context of conservation. [ 10 ] [ 11 ] [ 12 ]


Revive & Restore was co-founded in 2012 by  Stewart Brand  and Ryan Phelan with the idea of bringing biotechnology solutions to conservation. [ 13 ]  The group was incubated by the  Long Now Foundation  until 2017, when it became an independent  501(c)(3) organization .


In 2013 Revive & Restore organized the first public meeting on de-extinction. [ 14 ]  Their founding projects include the de-extinction of the  passenger pigeon , [ 15 ]   heath hen , [ 16 ]  and  woolly mammoth . [ 17 ]  Since then, Revive & Restore has established partnerships with research institutions, governmental agencies, and conservation organizations on a broad range of genetic rescue programs. [ citation needed ]


Revive & Restore is a member of the  International Union for Conservation of Nature  (IUCN) and has long-standing partnerships with the  US Fish & Wildlife Service ,  The San Diego Zoo Wildlife Alliance ,  Morris Animal Foundation , and  ViaGen Pets & Equine , among others. [ citation needed ]


The Advanced Coral Toolkit supports research teams in the development and field testing of biotechnologies that benefit coral reef management and restoration efforts. Projects include coral cryopreservation methods for large scale  biobanking [ 18 ]  and fieldable devices for measuring genetic information or molecular signals associated with coral stress. [ 19 ]  Launched in 2019, the program has funded 10 research teams. [ 20 ]


Wild Genomes is a funding program to provide genomic tools to field scientists, wildlife managers, and citizens working to protect their local biodiversity. As of 2023, Wild Genomes has funded 30 individual projects. Program categories include Terrestrial Species, Marine Species, Amphibians, [ 21 ]  and Kelp Ecosystems. [ 22 ]


To help mitigate  inbreeding depression  for two endangered species, the  black-footed ferret   (Mustela nigripes)  and  Przewalski's horse   (Equus ferus przewalskii) , Revive & Restore facilitates on-going efforts to clone individuals from historic cell lines stored at the San Diego Zoo Wildlife Alliance Frozen Zoo.


On December 10, 2020, the world's first cloned  black-footed ferret  was born. This ferret, named Elizabeth Ann, marked the first time a U.S. endangered species was successfully cloned. [ 23 ]


On August 6, 2020, the world's first cloned  Przewalski’s horse  was born. [ 24 ] [ 25 ] [ 26 ]  Since the  oocyte  used was from a domestic horse, this was an example of interspecies  somatic cell nuclear transfer  (SCNT). [ 27 ]  In 2022, the horse, named Kurt, was paired with a female Przewalski's horse at the San Diego Zoo Wildlife Safari Park to learn the behaviors of his species. [ 28 ]  On February 17, 2023, a second cloned Przewalski's horse was born from the same historic cell line. [ 29 ] [ 30 ]  Kurt and the new foal are genetic twins that may become the first cloned animals to restore lost genetic variation to their species. [ 31 ]


In 2020, Revive & Restore developed a campaign around the concept of "Intended Consequences" – focusing on the benefits of conservation interventions, as opposed to focusing on the fears of unintended consequences. [ 32 ]  That year, Revive & Restore hosted a virtual workshop that resulted in the publication of a special issue in the journal  Conservation Science and Practice . [ 33 ]


Ribosomal  frameshifting , also known as  translational frameshifting  or  translational recoding , is a biological phenomenon that occurs during  translation  that results in the production of multiple, unique  proteins  from a single  mRNA . [ 1 ]  The process can be programmed by the  nucleotide  sequence of the mRNA and is sometimes affected by the  secondary, 3-dimensional mRNA structure . [ 2 ]  It has been described mainly in  viruses  (especially  retroviruses ),  retrotransposons  and  bacterial  insertion elements, and also in some cellular  genes . [ 3 ]


Small molecules, proteins, and nucleic acids have also been found to stimulate levels of frameshifting. In December 2023, it was reported that  in vitro -transcribed (IVT)  mRNAs  in response to  BNT162b2  (Pfizer–BioNTech) anti-COVID-19 vaccine caused ribosomal frameshifting. [ 4 ]


Proteins are translated by reading tri-nucleotides on the mRNA strand, also known as  codons , from one end of the  mRNA  to the other (from the  5'  to the  3' end ) starting with the amino acid  methionine  as the start (initiation) codon AUG.  Each codon is translated into a single  amino acid . The code itself is considered  degenerate , meaning that a particular amino acid can be specified by more than one codon. However, a shift of any number of nucleotides that is not divisible by 3 in the reading frame will cause subsequent codons to be read differently. [ 5 ]  This effectively changes the ribosomal  reading frame .


In this example, the following sentence of three-letter words makes sense when read from the beginning:


However, if the reading frame is shifted by one letter to between the  T  and H of the first word (effectively a +1 frameshift when considering the 0 position to be the initial position of  T ),


then the sentence reads differently, making no sense.


In this example, the following sequence is a region of the  human mitochondrial genome  with the two  overlapping genes   MT-ATP8  and  MT-ATP6 .
When read from the beginning, these codons make sense to a ribosome and can be translated into amino acids (AA) under the  vertebrate mitochondrial code :


However, let's change the reading frame by starting one nucleotide downstream (effectively a "+1 frameshift" when considering the 0 position to be the initial position of  A ):


Because of this +1 frameshifting, the DNA sequence is read differently. The different codon reading frame therefore yields different amino acids.


In the case of a translating ribosome, a frameshift can either result in  nonsense mutation , a premature  stop codon  after the frameshift, or the creation of a completely new protein after the frameshift. In the case where a frameshift results in nonsense, the  nonsense-mediated mRNA decay  (NMD) pathway may destroy the mRNA transcript, so frameshifting would serve as a method of  regulating  the  expression  level of the associated gene. [ 6 ]


If a novel or off-target protein is produced, it can trigger other unknown consequences. [ 4 ]


In viruses this phenomenon may be programmed to occur at particular sites and allows the virus to encode multiple types of proteins from the same mRNA. Notable examples include  HIV-1  (human immunodeficiency virus), [ 7 ]  RSV ( Rous sarcoma virus ) [ 8 ]  and the  influenza  virus (flu), [ 9 ]  which all rely on frameshifting to create a proper ratio of 0-frame (normal translation) and "trans-frame" (encoded by frameshifted sequence) proteins. Its use in viruses is primarily for compacting more genetic information into a shorter amount of genetic material.


In eukaryotes it appears to play a role in regulating gene expression levels by generating premature stops and producing nonfunctional transcripts. [ 3 ] [ 10 ]


The most common type of frameshifting is  −1 frameshifting  or  programmed −1 ribosomal frameshifting (−1 PRF) . Other, rarer types of frameshifting include +1 and −2 frameshifting. [ 2 ]  −1 and +1 frameshifting are believed to be controlled by different mechanisms, which are discussed below. Both mechanisms are  kinetically driven .


In −1 frameshifting, the ribosome slips back one nucleotide and continues translation in the −1 frame. There are typically three elements that comprise a −1 frameshift signal: a  slippery sequence , a spacer region, and an  RNA secondary structure.  The slippery sequence fits a X_XXY_YYH motif, where XXX is any three identical nucleotides (though some exceptions occur), YYY typically represents UUU or AAA, and H is A, C or U. Because the structure of this motif contains 2 adjacent 3-nucleotide repeats it is believed that −1 frameshifting is described by a tandem slippage model, in which the ribosomal P-site tRNA anticodon re-pairs from XXY to XXX and the A-site anticodon re-pairs from YYH to YYY simultaneously. These new pairings are identical to the 0-frame pairings except at their third positions. This difference does not significantly disfavor anticodon binding because the third nucleotide in a codon, known as the  wobble position , has weaker tRNA anticodon binding specificity than the first and second nucleotides. [ 2 ] [ 11 ]  In this model, the motif structure is explained by the fact that the first and second positions of the anticodons must be able to pair perfectly in both the 0 and −1 frames. Therefore, nucleotides 2 and 1 must be identical, and nucleotides 3 and 2 must also be identical, leading to a required sequence of 3 identical nucleotides for each tRNA that slips. [ 12 ]


The slippery sequence for a +1 frameshift signal does not have the same motif, and instead appears to function by pausing the ribosome at a sequence encoding a rare amino acid. [ 13 ]  Ribosomes do not translate proteins at a steady rate, regardless of the sequence. Certain codons take longer to translate, because there are not equal amounts of  tRNA  of that particular codon in the  cytosol . [ 14 ]  Due to this lag, there exist in small sections of codons sequences that control the rate of ribosomal frameshifting. Specifically, the ribosome must pause to wait for the arrival of a rare tRNA, and this increases the kinetic favorability of the ribosome and its associated tRNA slipping into the new frame. [ 13 ] [ 15 ]  In this model, the change in reading frame is caused by a single tRNA slip rather than two.


Ribosomal frameshifting may be controlled by mechanisms found in the mRNA sequence (cis-acting). This generally refers to a slippery sequence, an RNA secondary structure, or both. A −1 frameshift signal consists of both elements separated by a spacer region typically 5–9 nucleotides long. [ 2 ]  Frameshifting may also be induced by other molecules which interact with the ribosome or the mRNA (trans-acting).


Slippery sequences  can potentially make the reading ribosome "slip" and skip a number of  nucleotides  (usually only 1) and read a completely different frame thereafter. In programmed −1 ribosomal frameshifting, the slippery sequence fits a X_XXY_YYH motif, where XXX is any three identical nucleotides (though some exceptions occur), YYY typically represents UUU or AAA, and H is A, C or U. In the case of +1 frameshifting, the slippery sequence contains codons for which the corresponding tRNA is more rare, and the frameshift is favored because the codon in the new frame has a more common associated tRNA. [ 13 ]  One example of a slippery sequence is the  polyA  on mRNA, which is known to induce ribosome slippage even in the absence of any other elements. [ 16 ]


Efficient ribosomal frameshifting generally requires the presence of an RNA secondary structure to enhance the effects of the slippery sequence. [ 12 ]  The RNA structure (which can be a  stem-loop  or  pseudoknot ) is thought to pause the ribosome on the slippery site during translation, forcing it to relocate and continue replication from the −1 position. It is believed that this occurs because the structure physically blocks movement of the ribosome by becoming stuck in the ribosome mRNA tunnel. [ 2 ]  This model is supported by the fact that strength of the pseudoknot has been positively correlated with the level of frameshifting for associated mRNA. [ 3 ] [ 17 ]


Below are examples of predicted secondary structures for frameshift elements shown to stimulate frameshifting in a variety of organisms. The majority of the structures shown are stem-loops, with the exception of the ALIL (apical loop-internal loop) pseudoknot structure. In these images, the larger and incomplete circles of mRNA represent linear regions. The secondary "stem-loop" structures, where "stems" are formed by a region of mRNA base pairing with another region on the same strand, are shown protruding from the linear DNA. The linear region of the HIV ribosomal frameshift signal contains a highly conserved UUU UUU A slippery sequence; many of the other predicted structures contain candidates for slippery sequences as well.


The mRNA sequences in the images can be read according to a set of guidelines. While A, T, C, and G represent a particular nucleotide at a position, there are also letters that represent ambiguity which are used when more than one kind of nucleotide could occur at that position. The rules of the International Union of Pure and Applied Chemistry ( IUPAC ) are as follows: [ 18 ]


These symbols are also valid for RNA, except with U (uracil) replacing T (thymine). [ 18 ]


Small molecules, proteins, and nucleic acids have been found to stimulate levels of frameshifting. For example, the mechanism of a  negative feedback  loop in the  polyamine  synthesis pathway is based on polyamine levels stimulating an increase in +1 frameshifts, which results in production of an inhibitory  enzyme . Certain proteins which are needed for codon recognition or which bind directly to the mRNA sequence have also been shown to modulate frameshifting levels.  MicroRNA  (miRNA) molecules may hybridize to an RNA secondary structure and affect its strength. [ 6 ]


Right To Know  is a  non profit  support project for those who discover via  genealogical   genetic testing  that their lineage is not what they had supposed it to be due to family secrets and misattributed parentage,  thus raising existential issues of  adoption ,  race ,  ethnicity ,  culture ,  rape , etc. [ 1 ] [ 2 ] [ 3 ] [ 4 ] [ 5 ]


In  evolutionary biology ,  robustness  of a  biological system  (also called biological or genetic robustness [ 1 ] ) is the persistence of a certain characteristic or trait in a system under perturbations or conditions of uncertainty. [ 2 ] [ 3 ]   Robustness in development is known as  canalization . [ 4 ] [ 5 ]   According to the kind of perturbation involved, robustness can be classified as  mutational ,  environmental ,  recombinational , or  behavioral  robustness  etc . [ 6 ] [ 7 ] [ 8 ]  Robustness is achieved through the combination of many  genetic  and  molecular mechanisms  and can  evolve  by either direct or indirect  selection . Several  model systems  have been developed to experimentally study robustness and its evolutionary consequences.


Mutational robustness (also called mutation tolerance) describes the extent to which an organism's phenotype remains constant in spite of  mutation . [ 9 ]  Robustness can be empirically measured for several  genomes [ 10 ] [ 11 ]  and individual  genes [ 12 ]  by inducing mutations and measuring what proportion of mutants retain the same  phenotype , function or  fitness . More generally robustness corresponds to the neutral band in the  distribution of fitness effects  of mutation (i.e. the frequencies of different fitnesses of mutants). Proteins so far investigated have shown a tolerance to mutations of roughly 66% (i.e. two thirds of mutations are neutral). [ 13 ]


Conversely, measured mutational robustnesses of organisms vary widely. For example, >95% of point mutations in  C. elegans  have no detectable effect [ 14 ]  and even 90% of single gene knockouts in  E. coli  are non-lethal. [ 15 ]  Viruses, however, only tolerate 20-40% of mutations and hence are much more sensitive to mutation. [ 10 ]


Biological processes at the molecular scale are inherently stochastic. [ 16 ]  They emerge from a combination of stochastic events that happen given the physico-chemical properties of molecules. For instance, gene expression is intrinsically noisy. This means that two cells in exactly identical  regulatory states  will exhibit different  mRNA  contents. [ 17 ] [ 18 ]  The cell population level log-normal distribution of mRNA content [ 19 ]  follows directly from the application of the  Central Limit Theorem  to the multi-step nature of  gene expression regulation . [ 20 ]


In varying  environments , perfect  adaptation  to one condition may come at the expense of adaptation to another. Consequently, the total selection pressure on an organism is the average selection across all environments weighted by the percentage time spent in that environment. Variable environment can therefore select for environmental robustness where organisms can function across a wide range of conditions with little change in  phenotype  or  fitness (biology) . Some organisms show adaptations to tolerate large changes in temperature, water availability, salinity or food availability. Plants, in particular, are unable to move when the environment changes and so show a range of mechanisms for achieving environmental robustness. Similarly, this can be seen in proteins as tolerance to a wide range of  solvents ,  ion concentrations  or  temperatures .


Genomes mutate by environmental damage and imperfect replication, yet they display remarkable tolerance. This comes from robustness both at many different levels.


There are many mechanisms that provide genome robustness. For example,  genetic redundancy  reduces the effect of mutations in any one copy of a multi-copy gene. [ 21 ]  Additionally the  flux  through a  metabolic pathway  is typically limited by only a few of the steps, meaning that changes in function of many of the enzymes have little effect on fitness. [ 22 ] [ 23 ]  Similarly  metabolic networks  have multiple alternate pathways to produce many key  metabolites . [ 24 ]


Protein mutation tolerance is the product of two main features: the structure of the  genetic code  and  protein structural  robustness. [ 25 ] [ 26 ]  Proteins are resistant to mutations because many sequences can fold into highly similar  structural folds . [ 27 ]  A protein adopts a limited ensemble of native conformations because those conformers have lower energy than unfolded and mis-folded states (ΔΔG of folding). [ 28 ] [ 29 ]  This is achieved by a distributed, internal network of cooperative interactions ( hydrophobic ,  polar  and  covalent ). [ 30 ]  Protein structural robustness results from few single mutations being sufficiently disruptive to compromise function. Proteins have also evolved to avoid  aggregation [ 31 ]  as partially folded proteins can combine to form large, repeating, insoluble  protein fibrils  and masses. [ 32 ]  There is evidence that proteins show negative design features to reduce the exposure of aggregation-prone  beta-sheet  motifs in their structures. [ 33 ]
Additionally, there is some evidence that the  genetic code  itself may be optimised such that most point mutations lead to similar amino acids ( conservative ). [ 34 ] [ 35 ]  Together these factors create a  distribution of fitness effects  of mutations that contains a high proportion of neutral and nearly-neutral mutations. [ 12 ]


During  embryonic development , gene expression must be tightly controlled in time and space in order to give rise to fully functional organs. Developing organisms must therefore deal with the random perturbations resulting from gene expression stochasticity. [ 36 ]  In  bilaterians , robustness of gene expression can be achieved via  enhancer  redundancy. This happens when the expression of a gene under the control of several enhancers encoding the same regulatory logic (ie. displaying binding sites for the same set of  transcription factors ). In  Drosophila melanogaster  such redundant enhancers are often called  shadow enhancers . [ 37 ]


Furthermore, in developmental contexts were timing of gene expression in important for the phenotypic outcome, diverse mechanisms exist to ensure proper gene expression in a timely manner. [ 36 ]   Poised promoters  are transcriptionally inactive  promoters  that display  RNA polymerase II  binding, ready for rapid induction. [ 38 ]  In addition, because not all transcription factors can bind their target site in compacted  heterochromatin ,  pioneer transcription factors  (such as  Zld  or  FoxA ) are required to open chromatin and allow the binding of other transcription factors that can rapidly induce gene expression. Open inactive enhancers are call  poised enhancers . [ 39 ]


Cell competition  is a phenomenon first described in  Drosophila [ 40 ]  where mosaic  Minute  mutant cells (affecting  ribosomal proteins ) in a wild-type background would be eliminated. This phenomenon also happens in the early mouse embryo where cells expressing high levels of  Myc  actively kill their neighbors displaying low levels of  Myc  expression. This results in homogeneously high levels of  Myc . [ 41 ] [ 42 ]


Patterning mechanisms such as those described by the  French flag model  can be perturbed at many levels (production and stochasticity of the diffusion of the morphogen, production of the receptor, stochastic of the  signaling cascade , etc). Patterning is therefore inherently noisy. Robustness against this noise and genetic perturbation is therefore necessary to ensure proper that cells measure accurately positional information. Studies of the  zebrafish   neural tube  and antero-posterior patternings has shown that noisy signaling leads to imperfect cell differentiation that is later corrected by transdifferentiation, migration or cell death of the misplaced cells. [ 43 ] [ 44 ] [ 45 ]


Additionally, the structure (or topology) of  signaling pathways  has been demonstrated to play an important role in robustness to genetic perturbations. [ 46 ]  Self-enhanced degradation has long been an example of robustness in  System biology . [ 47 ]  Similarly, robustness of dorsoventral patterning in many species emerges from the balanced shuttling-degradation mechanisms involved in  BMP signaling . [ 48 ] [ 49 ] [ 50 ]


Since organisms are constantly exposed to genetic and non-genetic perturbations, robustness is important to ensure the stability of  phenotypes .  Also, under mutation-selection balance, mutational robustness can allow  cryptic genetic variation  to accumulate in a population.  While phenotypically neutral in a stable environment, these genetic differences can be revealed as trait differences in an environment-dependent manner (see  evolutionary capacitance ), thereby allowing for the expression of a greater number of heritable phenotypes in populations exposed to a variable environment. [ 51 ]


Being robust may even be a favoured at the expense of total fitness as an  evolutionarily stable strategy  (also called survival of the flattest). [ 52 ]  A high but narrow peak of a  fitness landscape  confers high fitness but low robustness as most mutations lead to massive loss of fitness. High mutation rates may favour population of lower, but broader fitness peaks. More critical biological systems may also have greater selection for robustness as reductions in function are more damaging to  fitness . [ 53 ]  Mutational robustness is thought to be one driver for theoretical  viral quasispecies  formation.


Natural selection  can select directly or indirectly for robustness. When  mutation rates  are high and  population sizes  are large, populations are predicted to move to more densely connected regions of  neutral network  as less robust variants have fewer surviving mutant descendants. [ 54 ]  The conditions under which selection could act to directly increase mutational robustness in this way are restrictive, and therefore such selection is thought to be limited to only a few  viruses [ 55 ]  and  microbes [ 56 ]  having large population sizes and high mutation rates. Such emergent robustness has been observed in experimental evolution of  cytochrome P450s [ 57 ]  and  B-lactamase . [ 58 ]  Conversely, mutational robustness may evolve as a byproduct of natural selection for robustness to environmental perturbations. [ 59 ] [ 60 ] [ 61 ] [ 62 ] [ 63 ]


Mutational robustness has been thought to have a negative impact on  evolvability  because it reduces the mutational accessibility of distinct heritable phenotypes for a single genotype and reduces selective differences within a genetically diverse population. [ citation needed ]  Counter-intuitively however, it has been hypothesized that phenotypic robustness towards mutations may actually increase the pace of heritable phenotypic adaptation when viewed over longer periods of time. [ 64 ] [ 65 ] [ 66 ] [ 67 ]


One hypothesis for how robustness promotes evolvability in asexual populations is that connected networks of fitness-neutral genotypes result in mutational robustness which, while reducing accessibility of new heritable phenotypes over short timescales, over longer time periods, neutral mutation and  genetic drift  cause the population to spread out over a larger  neutral network  in genotype space. [ 68 ]  This genetic diversity gives the population mutational access to a greater number of distinct heritable phenotypes that can be reached from different points of the neutral network. [ 64 ] [ 65 ] [ 67 ] [ 69 ] [ 70 ] [ 71 ] [ 72 ]  However, this mechanism may be limited to phenotypes dependent on a single genetic locus; for polygenic traits, genetic diversity in asexual populations does not significantly increase evolvability. [ 73 ]


In the case of proteins, robustness promotes evolvability in the form of an excess free energy of  folding . [ 74 ]  Since most mutations reduce stability, an excess folding free energy allows toleration of mutations that are beneficial to activity but would otherwise destabilise the protein.


In sexual populations, robustness leads to the accumulation of cryptic genetic variation with high evolutionary potential. [ 75 ] [ 76 ]


Evolvability may be high when robustness is reversible, with  evolutionary capacitance  allowing a switch between high robustness in most circumstances and low robustness at times of stress. [ 77 ]


There are many systems that have been used to study robustness.  In silico  models have been used to model   promoters , [ 78 ] [ 79 ]   RNA secondary structure ,  protein lattice models , or  gene networks . Experimental systems for individual genes include enzyme activity of  cytochrome P450 , [ 57 ]   B-lactamase , [ 58 ]   RNA polymerase , [ 13 ]  and  LacI [ 13 ]  have all been used. Whole organism robustness has been investigated in  RNA virus  fitness, [ 10 ]  bacterial  chemotaxis ,  Drosophila  fitness, [ 15 ]  segment polarity network, neurogenic network and  bone morphogenetic protein  gradient,  C. elegans  fitness [ 14 ]  and  vulval  development, and mammalian  circadian clock . [ 9 ]


The  rule of six  is a feature of some  paramyxovirus   genomes . These  RNA viruses  have genes made from  RNA  and not  DNA , and their whole genome – that is the number of  nucleotides  – is always a multiple of six. This is because during their replication, these viruses are dependent on  nucleoprotein   molecules  that each bind to six nucleotides. [ 1 ]


In molecular biology,  S4 domain  refers to a small RNA-binding  protein domain  found in a  ribosomal protein  named uS4 (called  S9  in eukaryotes). The S4 domain is approximately 60-65  amino acid  residues long, occurs in a single copy at various positions in different proteins and was originally found in  pseudouridine  syntheses, a  bacterial  ribosome-associated protein. [ 1 ]


The S4 protein helps to initiate assembly of the 16S rRNA. In this way proteins serve to organise and stabilise the rRNA  tertiary structure . [ 2 ] [ 3 ]


The function of the S4 domain is to be an RNA-binding protein. S4 is a multifunctional protein, and it must bind to the  16S ribosomal RNA . In addition, the S4 domain binds a complex  pseudoknot  and represses translation. More specifically, this protein domain delivers nucleotide-modifying enzymes to RNA and to regulates translation through structure specific RNA binding. [ 1 ]


The S4 protein domain is composed of three  alpha helices  and five  beta strands . It is organized as an  antiparallel  sheet in a Greek key motif. [ 4 ]


The  scleraxis   protein  is a member of the  basic helix-loop-helix  (bHLH) superfamily of  transcription factors . [ 1 ]   Currently two genes ( SCXA  and  SCXB  respectively) have been identified to code for identical scleraxis proteins.


It is thought that early scleraxis-expressing  progenitor cells  lead to the eventual formation of  tendon  tissue and other muscle attachments. [ 1 ]   Scleraxis is involved in  mesoderm  formation and is expressed in the syndetome (a collection of embryonic tissue that develops into tendon and blood vessels) of developing  somites  (primitive segments or compartments of embryos). [ 2 ]


The syndetome location within the somite is determined by  FGF  secreted from the center of the myotome (a collection of embryonic tissue that develops into  skeletal muscle )- the FGF then induces the adjacent  anterior  and  posterior  sclerotome (a collection of embryonic tissue that develops into the  axial skeleton ) to adopt a tendon cell fate. This ultimately places future scleraxis-expressing cells between the two tissue types they will ultimately join.  [ 3 ]


Scleraxis expression will be seen throughout the entire sclerotome (rather than just the sclerotome directly anterior and posterior to the myotome) with an overexpression of  FGF8 , demonstrating that all sclerotome cells are capable of expressing scleraxis in response to FGF signaling. While the FGF interaction has been shown to be necessary for scleraxis expression, it is still unclear as to whether the FGF signaling pathway directly induces the syndetome to secrete scleraxis, or indirectly through a secondary signaling pathway. Most likely, the syndetomal cells, through careful reading of the FGF  concentration  (coming from the myotome), can precisely determine their location and begin expressing scleraxis. [ 3 ]  Much of embryonic development follows this model of inducing specific cell fates through the reading of surrounding signaling molecule concentration gradients.


bHLH transcription factors have been shown to have a wide array of functions in  developmental  processes. [ 4 ]  More precisely, they have critical roles in the control of  cellular differentiation ,  proliferation  and regulation of  oncogenesis . [ 4 ] [ 5 ] [ 6 ]  To date, 242  eukaryotic  proteins belonging to the HLH superfamily have been reported. They have varied expression patterns in all eukaryotes from yeast to humans. [ 7 ]


Structurally, bHLH proteins are characterised by a “highly conserved domain containing a stretch of basic  amino acids  adjacent to two  amphipathic   α-helices  separated by a loop”. [ 8 ] [ 9 ]


These helices have important functional properties, forming part of the DNA binding and transcription activating domains. With respect to scleraxis, the bHLH region spans amino acid residues 78 to 131.  A proline rich region is also predicted to lie between residues 161–170. A stretch of basic residues, which aids in DNA binding, is found closer to the N terminal end of scleraxis. [ 1 ] [ 10 ]


HLH proteins that lack this basic domain have been shown to negatively regulate the activities of bHLH proteins and are called inhibitors of differentiation (Id). [ 11 ]  Basic HLH proteins function normally as dimers and bind to a specific hexanucleotide DNA sequence (CAANTG) known as an  E-box  thus switching on the expression of various genes involved in cellular development and survival.


A  selection limit  is a term from  animal breeding  and  quantitative genetics  that refers to a cessation of progress even when continued directional selection is being applied to a trait, such as body  size .  In other words, a breeder or scientist is using  selective breeding  (artificial selection) and choosing individuals as breeders within a population based on some  phenotypic trait  or traits.  If this is done, then the  average  value of the population typically evolves across generations in the direction being favored by selection (i.e., for higher or lower values of the trait), but then at some point the population stops evolving.  The trait under selection is then said to have reached a limit or plateau at that value.


The existence of limits in artificial selection experiments was discussed in the  scientific literature  in the 1940s or earlier. [ 1 ]   The most obvious possible cause of reaching a limit (or plateau) when a population is under continued  directional selection  is that all of the additive-genetic variation (see  additive genetic effects ) related to that trait gets "used up" or fixed. [ 2 ]   For example, if a trait, such as body mass, is under selection to increase, then, over time (i.e., across generations), the  alleles  (genetic variants) at all  loci  (most simply, positions on chromosomes) that tend to make individuals larger than average will increase in frequency, while those that tend to make an individual smaller than average will decrease in frequency.  Eventually, in principle, the favored alleles at all relevant loci will become the only ones remaining at those loci.  In reality,  mutation , random  genetic drift  (especially in small populations), and  gene flow  from immigrants may stop some loci from becoming  fixed  for the "good" alleles.


However, other factors may interfere with the realization of genetic gains before loss of genetic variation causes a selection limit.  As noted by Lerner and Dempster, [ 1 ]  these factors are generally one of two types: 1) negative relations with Darwinian fitness; 2) non-additive gene action and/or  genotype-environment interaction  (although others are possible [ 3 ]
 [ 4 ]
 [ 2 ]
 [ 5 ] ).


A negative relation with  Darwinian fitness  is a situation in which an allele that is "good" for the trait under directional selection is "bad" with respect to lifetime reproductive success.  For example, an allele that tends to confer larger body size might also lead to  infertility , thus reducing the ability of individuals with that allele to produce  offspring , limiting further response to selection, and sometimes even leading to extinction of the selected line. [ 6 ]


Non-additive gene action refers to such situations as  heterozygote advantage , where heterozygous individuals have higher (or lower) values for a trait (such as body size) than do either of the two homozygotes.  In such a case, selection will tend to maintain more than one allele in the population, and a selection limit may be reached while additive-genetic variation (narrow-sense  heritability ) remains for the trait under directional selection.


Genotype-environment interaction  occurs when the  phenotype  produced by a particular set of alleles (at one or more loci) confers relatively higher or lower values of a trait depending on the environmental circumstances in which an individual is born or raised, or under which the trait is measured.  For instance, somewhat different  genes  (a term that can refer to alleles or loci) tend to give the highest value of a trait depending on the  season .  If this occurs, then directional selection will act to favor some genes in winter and others in summer, for example. Again, the result may be that a selection plateau is attained while the population retains some additive-genetic variance for the trait under directional selection. [ citation needed ]


Some traits have a natural physical limit beyond which a trait cannot possibly go. [ 2 ]   For example, replicated selection for the building of small thermoregulatory  nests  in mice reached a limit at or near zero (i.e., none of the provided cotton was being used to make nests). [ 7 ]   Similarly, lines of  maize  selected for low oil or protein content in the  kernels  reached limits near to zero percent. [ 4 ]


Aside from absolute physical limits, and whatever their cause, limits or plateaus have often been observed in artificial selection experiments with animals, including: bristle number in fruit flies ( Drosophila ); [ 8 ]  avoidance behavior in  laboratory rats ; [ 9 ]  and large body size, [ 4 ]  large  litter size , [ 10 ]  large nest size, [ 7 ]  and high voluntary  wheel -running behavior in laboratory house mice. [ 11 ]


Experimental approaches to probe the causes of selection are of two general types, quantitative genetic and functional.  The former asks general questions about the  genetic architecture  of the trait when a limit has been attained (e.g., has narrow-sense heritability gone to zero?), whereas the latter attempts to determine what aspect of physiological or other function might have reached a limit or constraint.  Experimental studies may involve attempts to break an apparent selection limit.  As an example of a genetic approach, two replicate lines of mice at a limit for large nest size were crossed and selection was continued on this new population, resulting in further increase in nest size. [ 12 ]   From a functional perspective, in lines of mice at a selection limit for high wheel running, administration of an  erythropoietin  analog increased the  maximal rate of oxygen consumption during forced exercise , but did not increase wheel running, a result suggesting that  motivation  for  exercise  may be limiting the behavior, rather than inherent ability to run on wheels. [ 13 ]


In  evolutionary biology ,  sequence space  is a way of representing all possible sequences (for a  protein ,  gene  or  genome ). [ 1 ] [ 2 ]  The sequence space has one dimension per  amino acid  or  nucleotide  in the sequence leading to  highly dimensional spaces . [ 3 ] [ 4 ]


Most sequences in sequence space have no function, leaving relatively small regions that are populated by naturally occurring genes. [ 5 ]  Each protein sequence is adjacent to all other sequences that can be reached through a single  mutation . [ 6 ]  It has been estimated that the whole functional protein sequence space has been explored by life on the Earth. [ 7 ]  Evolution by natural selection can be visualised as the process of sampling nearby sequences in sequence space and moving to any with improved  fitness  over the current one.


A sequence space is usually laid out as a grid. For  protein  sequence spaces, each  residue  in the protein is represented by a  dimension  with 20 possible positions along that axis corresponding to the possible amino acids. [ 3 ] [ 4 ]  Hence there are 400 possible  dipeptides  arranged in a 20x20 space but that expands to 10 130  for even a small protein of 100 amino acids arranged in a space with 100 dimensions. Although such overwhelming multidimensionality cannot be visualised or represented diagrammatically, it provides a useful abstract model to think about the range of proteins and  evolution  from one sequence to another.


These highly multidimensional spaces can be compressed to 2 or 3 dimensions using  principal component analysis . A fitness landscape is simply a sequence space with an extra vertical axis of fitness added for each sequence. [ 8 ]


Despite the diversity of protein superfamilies, sequence space is extremely sparsely populated by functional proteins. Most random protein sequences have no fold or function. [ 9 ]   Enzyme superfamilies , therefore, exist as tiny clusters of active proteins in a vast empty space of non-functional sequence. [ 10 ] [ 11 ]


The density of functional proteins in sequence space, and the proximity of different functions to one another is a key determinant in understanding  evolvability . [ 12 ]  The degree of interpenetration of two  neutral networks  of different  activities  in sequence space will determine how easy it is to evolve from one activity to another. The more overlap between different activities in sequence space, the more  cryptic variation  for  promiscuous activity  will be. [ 13 ]


Protein sequence space has been compared to the  Library of Babel , a theoretical library containing all possible books that are 410 pages long. [ 14 ] [ 15 ]  In the  Library of Babel , finding any book that made sense was impossible due to the sheer number and lack of order. The same would be true of protein sequences if it were not for natural selection, which has selected out only protein sequences that make sense. Additionally, each protein sequences is surrounded by a set of neighbours (point mutants) that are likely to have at least some function.


On the other hand, the effective "alphabet" of the sequence space may in fact be quite small, reducing the useful number of amino acids from 20 to a much lower number. For example, in an extremely simplified view, all amino acids can be sorted into two classes (hydrophobic/polar) by  hydrophobicity  and still allow many common structures to show up. Early life on Earth may have only four or five types of amino acids to work with, [ 16 ]  and researches have shown that functional proteins can be created from wild-type ones by a similar alphabet-reduction process. [ 17 ] [ 18 ]  Reduced alphabets are also useful in  bioinformatics , as they provide an easy way of analyzing protein similarity. [ 19 ] [ 20 ]


A major focus in the field of  protein engineering  is on creating  DNA libraries  that  sample  regions of sequence space, often with the goal of finding mutants of proteins with enhanced functions compared to the  wild type . These libraries are created either by using a wild type sequence as a template and applying one or more  mutagenesis  techniques to make different variants of it, or by creating proteins from scratch using  artificial gene synthesis . These libraries are then  screened or selected , and ones with improved  phenotypes  are used for the next round of mutagenesis.


Sex linked  describes the sex-specific reading patterns of  inheritance  and  presentation  when a  gene   mutation  ( allele ) is present on a  sex chromosome  (allosome) rather than a non-sex chromosome ( autosome ). In humans, these are termed  X-linked recessive ,  X-linked dominant  and  Y-linked . The inheritance and presentation of all three differ depending on the sex of both the parent and the child. This makes them characteristically different from autosomal  dominance and recessiveness .


There are many more X-linked conditions than Y-linked conditions, since humans have several times as many genes on the  X chromosome  than the  Y chromosome . Only females are able to be carriers for X-linked conditions; males will always be affected by any X-linked condition, since they have no second X chromosome with a healthy copy of the gene. As such, X-linked recessive conditions affect males much more commonly than females.


In X-linked recessive inheritance, a son born to a carrier mother and an unaffected father has a 50% chance of being affected, while a daughter has a 50% chance of being a carrier, however a fraction of carriers may display a milder (or even full) form of the condition due to a phenomenon known as  skewed X-inactivation , in which the normal process of  inactivating half of the female body's X chromosomes  preferably targets a certain parent's X chromosome (the father's in this case). If the father is affected, the son will not be affected, as he does not inherit the father's X chromosome, but the daughter will always be a carrier (and may occasionally present with symptoms due to aforementioned skewed X-inactivation).


In X-linked dominant inheritance, a son or daughter born to an affected mother and an unaffected father both have a 50% chance of being affected (though a few X-linked dominant conditions are embryonic lethal for the son, making them appear to only occur in females). If the father is affected, the son will always be unaffected, but the daughter will always be affected. A Y-linked condition will only be inherited from father to son and will always affect every generation.


The inheritance patterns are different in animals that use  sex-determination systems  other than  XY . In the  ZW sex-determination system  used by birds, the mammalian pattern is reversed, since the male is the homogametic sex (ZZ) and the female is  heterogametic  (ZW).


In  classical genetics , a mating experiment called a  reciprocal cross  is performed to test if an animal's trait is sex-linked.


Each child of a mother affected with an X-linked dominant trait has a 50% chance of inheriting the mutation and thus being affected with the disorder. If only the father is affected, 100% of the daughters will be affected, since they inherit their father's X chromosome, and 0% of the sons will be affected, since they inherit their father's Y chromosome. [ citation needed ]


There are fewer X-linked dominant conditions than X-linked recessive, because dominance in X-linkage requires the condition to present in females with only a fraction of the reduction in gene expression of autosomal dominance, since roughly half (or  as many as 90% in some cases ) of a particular parent's X chromosomes are  inactivated  in females. [ citation needed ]


Females possessing one X-linked recessive mutation are considered carriers and will generally not manifest clinical symptoms of the disorder, although differences in  X chromosome inactivation  can lead to varying degrees of clinical expression in carrier females since some cells will express one X  allele  and some will express the other. All males possessing an X-linked recessive mutation will be affected, since males have only a single X chromosome and therefore have only one copy of X-linked genes. All offspring of a carrier female have a 50% chance of inheriting the mutation if the father does not carry the recessive allele. All female children of an affected father will be carriers (assuming the mother is not affected or a carrier), as daughters possess their father's X chromosome. If the mother is not a carrier, no male children of an affected father will be affected, as males only inherit their father's Y chromosome. [ citation needed ]


The incidence of X-linked recessive conditions in females is the square of that in males: for example, if 1 in 20 males in a human population are  red–green color blind , then 1 in 400 females in the population are expected to be color-blind ( 1 / 20 )*( 1 / 20 ).


It is important to distinguish between sex-linked characters, which are controlled by genes on sex chromosomes, and two other categories. [ 5 ]


Sex-influenced or sex-conditioned traits are phenotypes affected by whether they appear in a male or female body. [ 6 ]  Even in a homozygous dominant or recessive female the condition may not be expressed fully. Example:  baldness  in humans.


These are characters only expressed in one sex. They may be caused by genes on either autosomal or sex chromosomes. [ 6 ]  Examples: female sterility in  Drosophila ; and many  polymorphic  characters in insects, especially in relation to  mimicry . Closely linked genes on autosomes called " supergenes " are often responsible for the latter. [ 7 ] [ 8 ] [ 9 ]


Dosage compensation  is the process by which organisms equalize the expression of genes between members of different biological sexes. Across species, different  sexes  are often characterized by different types and numbers of  sex chromosomes . In order to neutralize the large difference in  gene dosage  produced by differing numbers of sex chromosomes among the sexes, various  evolutionary branches  have acquired various methods to equalize  gene expression  among the sexes. Because sex chromosomes    contain different numbers of  genes , different species of organisms have developed different mechanisms to cope with this inequality. Replicating the actual  gene  is impossible; thus organisms instead equalize the  expression  from each gene. For example, in  humans , female (XX) cells randomly silence the transcription of one  X chromosome , and transcribe all information from the other, expressed X chromosome. Thus, human females have the same number of expressed X-linked genes per cell as do human males (XY), both sexes having essentially one X chromosome per cell, from which to transcribe and express genes. [ 1 ]


Different lineages have evolved different mechanisms to cope with the differences in gene copy numbers between the sexes that are observed on sex chromosomes. Some lineages have evolved dosage compensation, an epigenetic mechanism which restores expression of X or Z specific genes in the heterogametic sex to the same levels observed in the ancestor prior to the evolution of the sex chromosome. [ 2 ] [ 3 ]  Other lineages equalize the expression of the X- or Z- specific genes between the sexes, but not to the ancestral levels, i.e. they possess incomplete compensation with "dosage balance". One example of this is X-inactivation which occurs in humans. The third documented type of gene dose regulatory mechanism is incomplete compensation without balance (sometimes referred to as incomplete or partial dosage compensation). In this system gene expression of sex-specific loci is reduced in the heterogametic sex i.e. the females in ZZ/ZW systems and males in XX/XY systems. [ 4 ]


There are three main mechanisms of achieving dosage compensation which are widely documented in the literature and which are common to most species. These include random inactivation of one female X chromosome (as observed in humans and  Mus musculus ; this is called  X-inactivation ), a two-fold increase in the transcription of a single male X chromosome (as observed in  Drosophila melanogaster ), and decreased transcription by half in both of the X chromosomes of a hermaphroditic organism (as observed in  Caenorhabditis elegans ). These mechanisms have been widely studied and manipulated in model organisms commonly used in the laboratory research setting. A summary of these forms of dosage compensation is illustrated below. However, there are also other less common forms of dosage compensation, which are not as widely researched and are sometimes specific to only one species (as observed in certain bird and  monotreme  species).


One logical way to equalize gene expression amongst males and females that follow a XX/XY sex differentiation scheme would be to decrease or altogether eliminate the expression of one of the X chromosomes in an XX, or female,  homogametic  individual, such that both males and females then express only one X chromosome. This is the case in many mammalian organisms, including humans and mice. [ 1 ]


The evidence for this mechanism of dosage compensation was discovered prior to scientists' understanding of what its implications were. In 1949,  Murray Barr  and Ewert Bertram published data describing the presence of "nucleolar satellites, [ 5 ]  which they observed were present in the mature somatic tissue of different female species. Further characterization of these satellites revealed that they were actually packages of condensed  heterochromatin , but a decade would pass before scientists grasped the significance of this specialized DNA. [ citation needed ]


Then, in 1959  Susumu Ohno  proved that these satellite-like structures found exclusively in female cells were actually derived from female X chromosomes. [ 6 ]  He called these structures  Barr bodies  after one of the investigators who originally documented their existence. Ohno's studies of Barr bodies in female mammals with multiple X chromosomes revealed that such females used Barr bodies to inactivate all but one of their X chromosomes. Thus, Ohno described the "n-1" rule to predict the number of Barr bodies in a female with n number of X chromosomes in her karyotype. [ 6 ]


Simultaneously,  Mary F. Lyon  began investigating manipulations of X-linked traits that had phenotypically visible consequences, particularly in mice, whose fur color is a trait intimately linked to the X chromosome. Building on work done by Ohno and his colleagues, Lyon eventually proved that either the maternal or paternal X chromosome is randomly inactivated in every cell of the female body in the species she was studying, [ 7 ]  which explained the  heterogeneous  fur patterns she observed in her mosaic mice. This process is known as  X-inactivation , and is sometimes referred to as "lyonization". [ 1 ]  This discovery can be easily extrapolated to explain the mixed color patterns observed in the coats of  tortoiseshell cats . The fur patterns characteristic of tortoiseshell cats are found almost exclusively in females, because only they randomly inactivate one X chromosome in every somatic hair cell. [ 8 ]  Thus, presuming that hair color determining genes are X-linked, it makes sense that whether the maternal or paternal X chromosome is inactivated in a particular hair cell can result in differential fur color expression.


Compounding on Lyon's discoveries, in 1962  Ernest Beutler  used female  fibroblast  cell lineages grown in culture to demonstrate the heritability of lyonization or random X-inactivation. [ 9 ]  By analyzing the differential expression of two existing, viable alleles for the X-linked enzyme  glucose-6-phosphate dehydrogenase  (G6PD) gene, Beutler observed that the inactivation of the gene was heritable across passaged generations of the cells. [ 10 ]


This pattern of dosage compensation, caused by random X-inactivation, is regulated across development in female mammals, following concerted patterns throughout development; for example, at the beginning of most female mammal development, both X chromosomes are initially expressed, but gradually undergo epigenetic processes to eventually achieve random inactivation of one X. [ 10 ]  In  germ cells , inactivated X chromosomes are then once again activated to ensure their expression in  gametes  produced by female mammals. [ 1 ]


Thus, dosage compensation in mammals is largely achieved through the silencing of one of two female X chromosomes via X-inactivation. This process involves  histone  tail modifications, DNA  methylation  patterns, and reorganization of large-scale  chromatin  structure encoded by the X-ist gene. [ 1 ]  In spite of these extensive modifications, not all genes along the X chromosome are subject to X-inactivation; active expression at some loci is required for  homologous recombination  with the pseudo-autosomal region ( PAR ) of the  Y chromosome  during meiosis. [ 11 ]  Additionally, 10-25% of human X chromosome genes, [ 12 ]  and 3-7% of mouse X chromosome genes  [ 13 ]  outside of the PARs show weak expression from the inactive X chromosome.


Random X-inactivation demands that the cell can determine if it contains more than one active X-chromosome before acting to silence any extraneous X-chromosome(s). This process is known as "counting". [ 1 ]  The exact molecular mechanism of counting is still unknown, but a popular model posits that  autosomes  produce factors that repress X-inactivation, while X-chromosome products that promote X-inactivation. These two conflicting forces are balanced such that if there is more than one X-chromosome X-inactivation will occur, but if there is only one, the autosomal products will successfully prevent the process. [ 1 ]


Not all random X-inactivation is entirely random. Some alleles, generally mutations in the X-inactivation center on the X-chromosome have been demonstrated to confer a bias towards inactivation for the chromosome on which they sit. [ 1 ]  Truly random X-inactivation may also appear to be non-random if one X-chromosome carries a deleterious mutation. This can result in fewer cells which express the lower-fitness X-chromosome to be present in the body as these cells are selected against. [ 1 ]


Another mechanism common for achieving equal X-related genetic expression between males and females involves two-fold increased transcription of a single male X chromosome. Thus, heterogametic male organisms with one X chromosome may match the level of expression achieved in homogametic females with two active X chromosomes. This mechanism is observed in  Drosophila . [ 14 ]


The concept of dosage compensation actually originated from an understanding of organisms in which males upregulated X-linked genes two-fold, and was much later extended to account for the observation of the once mysterious Barr bodies. As early as 1932,  H.J. Muller  carried out a set of experiments which allowed him to track the expression of eye color in flies, which is an X-linked gene. Muller introduced a  mutant  gene that caused loss of pigmentation in fly eyes, and subsequently noted that males with only one copy of the mutant gene had similar pigmentation to females with two copies of the mutant gene. This led Muller to coin the phrase "dosage compensation" to describe the observed phenomenon of gene expression equalization. [ 15 ]


Despite these advances, it was not until Ardhendu Mukherjee and W. Beermann performed more advanced  autoradiography  experiments in 1965 that scientists could confirm that transcription of genes in the single male X chromosome was double that observed in the two female X chromosomes. [ 16 ]  Mukherjee and Beermann confirmed this by designing a cellular autoradiography experiment that allowed them to visualize incorporation of [3H]uridine into ribonucleic acid of the X chromosomes. Their studies showed equal levels of [3H]uridine incorporation in the single male X chromosome and the two female X chromosomes. Thus, the investigators concluded that the two-fold increase in the rate of  RNA synthesis  in the X chromosome of the male relative to those of the female could account for Muller's hypothesized dosage compensation.


In the case of two-fold increased transcription of a single male X chromosome, there is no use for a Barr body, and the male organism must use different genetic machinery to increase the transcriptional output of their single X chromosome. It is common in such organisms for the Y chromosome to be necessary for male  fertility , but not for it to play an explicit role in  sex determination . [ 17 ] [ 18 ]  In  Drosophila , for example, the sex lethal (SXL) gene acts as a key regulator of sexual differentiation and maturation in  somatic tissue ; in XX animals,  SXL  is activated to repress increased transcription, while in XY animals SXL is inactive and allows male development to proceed via increased transcription of the single X. [ 18 ]  Several binding sites exist on the  Drosophila  X chromosome for the dosage compensation complex (DCC), a ribonucleoprotein complex; these binding sites have varying levels of affinity, presumably for varying expression of specific genes. [ 19 ]  The Male Specific Lethal complex, composed of protein and RNA binds and selectively modifies hundreds of X-linked genes, [ 20 ] [ 21 ]  increasing their transcription to levels comparable to female  D. melanogaster .