In organisms that use this method of dosage compensation, the presence of one or more X chromosomes must be detected early on in development, as failure to initiate the appropriate dosage compensation mechanisms is lethal. [ 17 ]  Male specific lethal proteins (MSLs) are a family of four proteins that bind to the X chromosome exclusively in males. The name "MSL" is used because mutations in these genes cause inability to effectively upregulate X-linked genes appropriately, and are thus lethal to males only and not their female counterparts. [ 17 ]  SXL regulates pre-messenger RNA in males to differentially splice MSLs and result in the appropriate increase in X chromosome transcription observed in male  Drosophila . The immediate target of SXL is male specific lethal-2 (MSL-2). [ 22 ]  Current dogma suggests that the binding of MSL-2 at multiple sites along the SXL gene in females prevents proper MSL-2 translation, and thus, as previously stated, represses the possibility for X-linked genetic upregulation in females. However, all other transcription factors in the MSL family—maleless, MSL-1, and MSL-3—are able to act when SXL is not expressed, as in the case in males. These factors act to increase male X chromosome transcriptional activity. Histone acetylation and the consequent upregulation of X-linked genes in males is dictated by the MSL complex. [ 23 ]  Specifically, special roX non-coding RNAs on the MSL complexes facilitate binding to the single male X chromosome, and dictate acetylation of specific loci along the X chromosome as well as the formation of euchromatin. [ 24 ]  Though these RNAs bind at specific sites along the male X chromosome, their effects spread along the length of the  chromosome  and have the ability to influence large-scale chromatin modifications. The implications of this spreading epigenetic regulation along the male X chromosome is thought to have implications for understanding the transfer of epigenetic activity along long genomic stretches. [ 14 ]


Other species that do not follow the previously discussed conventions of XX females and XY males must find alternative ways to equalize X-linked gene expression among differing sexes. For example, in  Caenorhabditis elegans  (or  C. elegans ), sex is determined by the  ratio  of X chromosomes relative to autosomes; [ 25 ]  worms with two X chromosomes (XX worms) develop as  hermaphrodites , whereas those with only one X chromosome (XO worms) develop as males. [ 26 ]  This system of sex determination is unique, because there is no male specific chromosome, as is the case in XX/XY sex determination systems. However, as is the case with the previously discussed mechanisms of dosage compensation, failure to express X-linked genes appropriately can still be lethal. [ 27 ]


In this XX/XO sex determination system, gene expression on the X chromosome is equalized by downregulating expression of genes on both X chromosomes of hermaphroditic XX organisms by half. [ 26 ]  In these XX organisms, the dosage compensation complex (DCC) is assembled on both X chromosomes to allow for this tightly regulated change in transcription levels. The DCC is often compared to the condensin complex, [ 28 ]  which is conserved across the mitotic and meiotic processes of many species. This complex is crucial to the condensation and segregation of chromosomes during both meiosis and mitosis. Because data substantiates the theory that dosage compensation in other species is caused by chromatin-wide modifications, many theorize that the DCC in particular functions similar to the condensin complex in its ability to condense or remodel the chromatin of the X chromosome. [ 29 ]


The role of the DCC in this form of dosage compensation was postulated by  Barbara J. Meyer  in the 1980s, and its individual components and their cooperative function were later parsed out by her lab. Notably, in 1999, data from Meyer's lab showed that SDC-2 is a particularly important transcriptional factor for targeting the DCC to the X chromosome and for assembling DCC components onto the X chromosomes in XX embryos. [ 30 ]  More recently, Meyer's lab has shown that proteins known as X-linked signal elements (XSEs) operate in concert with SDC-2 to differentially repress and activate other genes in the dosage compensation pathway. [ 31 ]  By selectively mutating a panel of genes hypothesized to contribute to dosage compensation in worms, Meyer's group demonstrated which XSEs specifically play a role in determining normal dosage compensation. They found that during embryonic development, several X-linked genes—including sex-1, sex-2, fox-1, and ceh-39—act in a combinatorial fashion to selectively repress transcriptional activity of the xol-1 gene in hermaphrodites. [ 32 ] [ 33 ]  Xol-1 expression is tightly regulated during early development, and is considered the most upstream gene in sex determination of C. elegans. In fact, xol-1 is often referred to in the literature as the master sex regulatory gene of C. elegans. XX C. elegans embryos have much lower xol-1 expression than their XO counterparts, resulting from overall increases in the amount of SEX-1, SEX-2, CEH-39, and FOX-1 transcription produced in the female embryos. This consequent decrease in xol-1 expression then allows higher SDC-2 expression levels, which aids in the formation and function of the DCC complex in the XX hermaphroditic worms, and in turn results in equalized expression of X-linked genes in the hermaphrodite. [ citation needed ]


Though all of the above-mentioned XSEs act to reduce xol-1 expression, experimentally reducing expression levels of these individual XSEs has been shown to have a minimal effect on sex determination and successful dosage compensation. [ 31 ]  This could be in part because these genes encode different proteins that act cooperatively rather than in an isolated fashion; for example, SEX-1 is a nuclear hormone receptor, while FOX-1 is an RNA-binding protein with properties capable of inducing post-transcriptional modifications in the xol-1 target. [ 31 ] [ 33 ] [ 34 ]  However, reducing the level of more than one XSE in different combinational permutations seems to have an additive effect on ensuring proper sex determination and resultant dosage compensation mechanics. [ 31 ]  This supports the hypothesis that these XSEs act together to achieve the desired sex determination and dosage compensation fate. Thus, in this model organism, the achieved level of X-chromosome expression is directly correlated to the activation of multiple XSEs that ultimately function to repress xol-1 expression in a developing worm embryo. A summary of this  C. elegans  mechanism of dosage compensation is illustrated below.


The  ZZ/ZW sex system  is used by most birds, as well as some reptiles and insects. In this system the Z is the larger chromosome so the males (ZZ) must silence some genetic material to compensate for the female's (ZW) smaller W chromosome. Instead of silencing the entire chromosome as humans do, male chickens (the model ZZ organism) seem to engage in selective Z silencing, in which they silence only certain genes on the extra Z chromosome. [ 35 ] [ 36 ]  Thus, male chickens express an average of 1.4-1.6 of the Z chromosome DNA expressed by female chickens. [ 37 ]  The Z chromosome expression of male zebra finches and chickens is higher than the autosomal expression rates, whereas X chromosome expression in female humans is equal to autosomal expression rates, [ 38 ]  illustrating clearly that both male chickens and male zebra finches practice incomplete silencing. Few other ZZ/ZW Systems have been analyzed as thoroughly as the chicken; however a recent study on silkworms  [ 39 ]  revealed similar levels of unequal compensation across male Z chromosomes. Z-specific genes were over-expressed in males when compared to females, and a few genes had equal expression in both male and female Z chromosomes. [ citation needed ]


In chickens, most of the dosage compensated genes exist on the Zp, or short, arm of the chromosome while the non-compensated genes are on the Zq, or long, arm of the chromosome. The compensated (silenced) genes on Zp resemble a region on the primitive platypus sex chromosome, suggesting an ancestor to the XX/XY system. [ 40 ]


The sex chromosomes of birds evolved separately from those of mammals and share very little sequence  homology  with the XY chromosomes. [ 41 ]  As such, scientists refer to bird sex chromosomes as a ZW sex-determining system, with males possessing two Z chromosomes, and females possessing one Z chromosome and one W. Thus, dosage compensation in birds could be hypothesized to follow a pattern similar to the random X-inactivation observed in most mammals. Alternatively, birds might show decreased transcription of the two Z chromosomes present in the male heterogametic sex, similar to the system observed in the two hermaphrodite X chromosomes of  C. elegans . However, bird mechanisms of dosage compensation differ significantly from these precedents. Instead, male birds appear to selectively silence only a few genes along one of their Z chromosomes, rather than randomly silencing one entire Z chromosome. [ 42 ]  This type of selective silencing has led some people to label birds as "less effective" at dosage compensation than mammals. [ 38 ]  However, more recent studies have shown that those genes on the Z chromosome which are not inactivated in birds may play an important role in recruiting dosage compensation machinery to the Z chromosome in ZZ organisms. [ 43 ]  In particular, one of these genes, ScII has been demonstrated to be an ortholog of xol-1, the master sex regulator gene in C. elegans. [ 43 ] [ 44 ]  Thus, the function of the selective silencing may be to spare dosage compensation of genes crucial for sex determination of homologous pairing. [ citation needed ]


Recent studies are focusing on how  epigenetic  mechanisms could contribute to dosage compensation in birds, with a particular emphasis on  methylation . It is already known that some regions on the Z chromosome of birds are heavily methylated, called MHM regions. So far, only two such regions have been well studied: one located at around 27.3 Mb and the other at 73.16–73.17 Mb (designated MHM2). The first MHM region discovered consists of tandem repeats of a BamHI 2.2-kb sequence and has a high degree of methylation on the cytosine of  CpG islands  (segments of cytosine-phosphate-guanine that are more readily methylated and silenced than other DNA segments) in both copies of the Z chromosome in males, and less so in the Z chromosome of females. This region is transcribed only in females and produces a long non-coding RNA, which gathers at the transcription site next to the DMRT1 gene. [ 4 ]  The second MHM region, at 73.16 Mb, is not as extensively studied due to its recent discovery. It appears to be smaller in size and contains three long non-coding RNA sequences with higher expression in females. [ 9 ]


Findings also suggest that the mechanism is more gene-specific, as certain genetic variants, called methylation quantitative trait loci (meQTLs), can affect methylation. These meQTLs are hypothesized to impact a larger part of the Z chromosome in males and are mostly located on autosomes, affecting the Z chromosome in a trans manner. [ 3 ]


Monotremes  are a class of basal mammals that also lay eggs. [ 45 ]  They are an order of mammals that includes platypuses and four species of echidna, all of which are egg-laying mammals. While monotremes use an XX/XY system, unlike other mammals, monotremes have more than two sex chromosomes. The male short-beaked echidna, for example, has nine sex chromosomes—5 Xs and 4 Ys, and the male platypus has 5 Xs and 5 Ys. [ citation needed ]


Platypuses are a monotreme species whose mechanism of sex determination has been extensively studied. There is some contention in academia about the evolutionary origin and the proper  taxonomy  of platypuses. A recent study [ 46 ]  revealed that four platypus X chromosomes, as well as a Y chromosome, are homologous to some regions on the avian Z chromosome. Specifically, platypus X1 shares homology with the chicken Z chromosome, and both share homology with the human chromosome 9. This homology is important when considering the mechanism of dosage compensation in monotremes. In 50% of female platypus cells, only one of the alleles on these X chromosomes is expressed while in the remaining 50% multiple alleles are expressed. This, combined with the portions that are homologous to chicken Z and human 9 chromosomes imply that this level of incomplete silencing may be the ancestral form of dosage compensation.


Regardless of their ambiguous evolutionary history, platypuses have been empirically determined to follow an  XY sex-determination system , with females possessing five pairs of X chromosomes as the homogametic sex, and males possessing five X and five Y chromosomes as the heterogametic sex. [ 47 ]  Because the entire genome of the platypus has yet to be completely sequenced (including one of the X chromosomes), [ 46 ]  there is still continued investigation as to the definitive mechanism of dosage compensation that Platypuses follow. Research from the laboratory of Jennifer Graves used qPCR and SNP analysis of BACs containing various genes from X chromosomes in order to find whether multiple alleles for particular X-linked genes were being expressed at once, or were otherwise being dosage compensated. [ 46 ]  Her group found that in female platypuses, some X-linked genes only expressed an allele from one X chromosomes, while other genes expressed multiple alleles. [ 46 ]  This appears to be a system similar to the selective silencing method of dosage compensation observed in birds. However, about half of all X-linked genes also seemed to stochastically express only one active copy of said gene, [ 46 ]  alluding to the system of random X-inactivation observed in humans. These findings suggest that platypuses may employ a hybrid form of dosage compensation that combines feature from mammals as well as birds. Understanding the evolution of such a system may have implications for solidifying the true ancestral lineage of monotremes.


In addition to humans and flies, some plants also make use of the XX/XY dosage compensation systems.  Silene latifolia  plants are also either male (XY) or female (XX), with the Y chromosome being smaller, with fewer genes expressed, than the X chromosome. Two separate studies  [ 48 ]  have shown male  S. latifolia  expression of X-linked genes to be about 70% of the expression in females. If the  S. latifolia  did not practice dosage compensation, the expected level of X-linked gene expression in males would be 50% that of females, thus the plant practices some degree of dosage compensation but, because male expression is not 100% that of females, it has been suggested that  S. latiforia  and its dosage compensation system is still evolving. Additionally, in plant species that lack dimorphic sex chromosomes, dosage compensation can occur when aberrant meiotic events or mutations result in either  aneuploidy  or  polyploidy . Genes on the affected chromosome may be upregulated or down-regulated to compensate for the change in the normal number of chromosomes present.


Research into dosage compensation has been carried out in six species of toxicoferan reptiles and in one species of softshell turtle. Two species of caenophidian snake (one which belongs to the family Viperidae and the other to the family Colubridae) have been investigated and both of these exhibit female heterogametic sex determination systems (ZZ\ZW) and have incomplete compensation without balance. [ 49 ]  The Komodo dragon exhibits incomplete compensation without dosage balance in their independently evolved ZZ/ZW system. [ 50 ]  In the XX/XY system of  Basiliscus vittatus  and multiple neo-sex chromosomes with male heterogamety in the pygopodid gecko  Lialis burtonis  incomplete compensation without dosage balance were also seen. [ 51 ] [ 52 ]  The Green anole ( Anolis carolinensis ; Dactyloidea), has XX/XY sex determination and unlike the other squamates studied to date has complete dosage compensation with dosage balance. [ 53 ]  In the Florida softshell turtle ( Apalone ferox)  with ZZ/ZW sex chromosomes, the lack of dosage balance in the expression of Z-linked genes was also found. [ 54 ]


XCI is initiated very early during female embryonic development or upon differentiation of female embryonic stem (ES) cells and results in inactivation of one X chromosome in every female somatic cell. This process is initiated very early during development, around the two- to eight-cell stage and is maintained in the developing extra-embryonic tissues of the embryo, including the fetal placenta. [ 55 ]  Xist RNA induces heterochromatinization of the X chromosome by attracting chromatin modifiers, involved in gene silencing. Xist RNA is tightly associated with the Xi and it is required for X Chromosome Inactivation to occur in cis. Knockout studies in female ES cells and mice have shown that X chromosomes bearing a deletion of the Xist gene are unable to inactivate the mutated X. Most of the human female ES cell lines display an inactivated X chromosome already in the undifferentiated state characterized by XIST expression, XIST coating and accumulated markers of heterochromatin on the Xi. [ 55 ]


It is widely thought that human embryos do not employ XCI prior to implantation. [ 56 ]  Female embryos have an accumulation of Xist RNA on one of the two X chromosomes, beginning around the 8-cell stage. Xist RNA accumulates at the morula and blastocyst stages and is shown to be associated with transcriptional silencing of the Xist-coated chromosomal region, therefore indicating dosage compensation has occurred. [ 56 ]  Recently, however, it has become increasingly apparent that XCI of the paternal X chromosome is already present from the 4-cell stage onward in all cells of preimplantation mouse embryos, not the 8-cell stages. [ citation needed ]


Xite and Xist, are both long non-coding RNAs that regulate and facilitate the process of X-inactivation and are important in the silencing of genes within the X chromosome that is being inactivated. [ 57 ]  These work in combination with Tsix, which is non-coding RNA that is an antisense which downregulates the effects of Xist on the X chromosome in which it is expressed on the maternal X chromosome upon the start regulation of X-inactivation. [ 58 ]  These three RNAs regulate the X-X pair in a  cis orientation in order to be able to have both chromosomes available for inhibitory actions. Tsix and Xite have basic lncRNAs functions in addition to X-inactivation and regulate the X-X pair in the  trans orientation. This ensures exclusive silencing for both X chromosomes. Xite and Tsix are both essential within the orientational directional processes in  cis  and  trans  as it is seen that without Tsix and Xite in trans it perturbs pairing and counting of genes. [ 57 ] [ 58 ]


Once the Xist is turned off and no longer regulates the process, the Tsix will slowly decrease in expression as well until both RNAs are no longer being changed by Xic. [ 58 ]  Xite is the locus that harbors intergenic transcription start sites from hypersensitive sites of allelic crossovers/differences. [ 57 ]  When X-inactivation begins, the transcription of Xite increases and signals for the downregulation of Tsix in  cis orientation, which is on the silent X chromosome, all while promoting the Tsix persistence on the active X chromosome. [ 59 ]  Xite also has major roles to play in the asymmetry of Tsix expression and generates X chromosome inequality through moving and helping orient the chromosomes to be acted upon by the correct subsequent lncRNA, either Tsix or Xist. [ 58 ]


The monarch butterfly  Danaus plexippus  belongs to the order Lepidoptera and has 30 chromosomes one of which is a neo-sex chromosome which is the result of a fusion between one of the sex chromosomes and an autosome. A study using a combination of methods (Hi-C assembly, coverage analysis and ChIp-seq) found that the neo-Z segment exhibits complete dosage compensation which is achieved by increased transcription in ZW females. Interestingly, the ancestral Z segment exhibits dosage balance with transcription levels being equal between both genders but less than the expected ancestral level, and this is achieved by decreased transcription in ZZ males. [ 60 ]


Sex-linked barring  is a plumage pattern on individual feathers in chickens, which is characterized by alternating pigmented and apigmented bars. [ 1 ]  The pigmented bar can either contain red pigment ( phaeomelanin ) or black pigment ( eumelanin ) whereas the apigmented bar is always white. The  locus  is therefore often referred to as an ‘eumelanin diluter’ or ‘melanin disruptor’. [ 2 ]  Typical sex-linked barred breeds include the  Barred Plymouth Rock , Delaware, Old English Crele Games as well as  Coucou de Renne . [ 3 ]


The presence of a white bar on a dark background is distinguishing sex-linked barring from Autosomal barring, another plumage pattern in chickens which is created by a black bar on a light color background (white/ beige or brown) as exemplified by the breed  Egyptian Fayoumi . [ 1 ]  The absence of pigment in the white bar has been attributed to a lack of pigment producing cells ( melanocytes ) in the feather follicle during feather growth. [ 4 ] [ 5 ]  Initially it was proposed that this lack was the result of cell death as a consequence of the  B   locus  mutation but later research demonstrated that the lack is the result of premature  cell differentiation  rather than  apoptosis . [ 6 ]


Male chicken of traditional sex-linked barred breeds like the  Barred Plymouth Rock  usually show much wider and clearer white bands than females of the same breed. [ 3 ]  Further characteristics of sex-linked barred chickens are the dilution of skin pigment in the legs as well as a white dot at the top of the head of freshly hatched chicks which can be used for  autosexing : homozygous males have a much bigger spot than hemizygous females. [ 3 ]


Sex-linked barring has been established as the  dominant  locus  B  by traditional  mendelian  genetics in the beginning of the 20th century. [ 3 ]  The responsible gene was predicted to be located on the  Z chromosome [ 7 ] [ 8 ]  and since male birds are  homogametic  (ZZ), they can be either  hetero - or  homozygous  for sex-linked barring. Females are always  hemizygous  at this locus (ZW).
In 2010 Swedish scientist have identified four  mutations  located in or around the  tumor suppressor   locus   CDKN2A , which appear to be associated with sex-linked barring. [ 1 ]  The four mutations are organized in 3 different  alleles  named  B0 ,  B1  and  B2 . All alleles carry two  non-coding   mutation  located in regulatory regions of the gene (the  promoter  and  intron ) but only  B1  and  B2  carry two additional  missense mutations  in a functional important domain of the  protein . The  B1   allele  is causing the typical sex-linked barring  phenotype / appearance and is present in most modern sex-linked barred chicken breeds. Females or male chickens carrying the  B2   allele  in the  heterozygous  condition show a defined barring pattern but in the  homozygous  condition, males are essentially white with very little  pigmentation . [ 9 ]  This  phenotype  has been initially described as a distinct but closely related  mutation , [ 10 ]  however, it was later assigned to the same  gene  and termed ‘Sex-linked Dilution'. [ 9 ]


The  B0  allele only carries the two  non-coding   mutations  and its contribution to the barring pattern remained unknown as it only occurred in breeds that also carry the Dominant white mutation which is masking the effect at the  B   locus . [ 1 ] ). Recently scientists have removed the Dominant white  mutation  from chickens of those lines and were able to show that those chickens show a very light barring pattern. They named the phenotype ‘Sex-linked Extreme Dilution’. [ 11 ]  As chickens with the  B0  allele show the weakest barring pattern compared to those that have the coding mutation in addition, the scientists propose an evolutionary scenario in which the  non-coding  mutations occurred first and the two  missense mutations  later in time and independently. As only the combination of both  non-coding  and  missense  mutations give the desired and pronounced barring pattern,  B0  alone is not present in modern Sex-linked barred breeds anymore. [ 11 ]


Scientists were able to show that either both or one of the  non-coding   mutations  present in all  B  alleles , cause an up-regulation of the activity of  CDKN2A . [ 11 ]  ). With more of the gene product, which is called  ARF  (Alternate Reading frame Protein) in the cell, more of  p53  is protected from degradation.  p53  is a  transcription factor  which in turn activates more  genes  involved in  cell cycle  regulation and  apoptosis . As a consequence the cell stops dividing and starts to prematurely produce pigment.
The missense mutations in the  B1  and  B2   allele , however, have an opposite effect. [ 11 ]  Both  missense mutations  lead to a malfunctioning  ARF  protein, which is counteracting the effect of the higher activity of the  gene  to some degree. The premature production of pigment is still obvious but less strong as observed in the  B0  allele. It is intriguing that the  missense mutation  in the  B1   allele  is much more disruptive for the protein function than the one in the  B2   allele  and the scientists believe that this is the reason for the observed  phenotypic  differences between those two  alleles .


When the  melanocyte   progenitor  cells start to migrate up from the bottom of the follicle into the  barbs  where they will make pigment, they further divide until a sufficient number of  pigment cells  is achieved. As a consequence of the up-regulation of  CDKN2A , most cells will stop dividing and make new cells but instead start producing pigment- a black bar is emerging from the feather. But eventually, there will not be enough  pigment cells  anymore. As they are recruited from the bottom of the feather follicle, the feather keeps on growing, creating the white bar. With the new set of pigment cells, the cyclic behavior starts again, creating alternating pigmented and apigmented bars.


Mutations in  CDKN2A  have been associated with the occurrence of familial  melanoma  in humans. [ 12 ] [ 13 ] [ 14 ]  Changes in its gene product  ARF  often cause the cell to lose their ability for  self-induced cell death  or  cell cycle arrest , which are mechanisms of cells to manage uncontrolled cell divisions and therefore the occurrence of cancer. It is intriguing that chickens carrying the  B1  or  B2  allele with a malfunctioning  ARF  do not show any higher prevalence to any type of cancer and are usually considered very sturdy and easy to keep breeds. It is also astonishing that the majority of the egg and meat production industry is relying on chickens, which have a defect in a  tumor suppressor gene . [ 11 ]


The  shadow effect [ 1 ]  is a phenomenon seen in genetic studies that use noninvasive  genetic  data collection methods. It occurs when there are not enough loci and/or loci that have low variance of alleles within the population. As a result, researchers can capture two separate individuals and mistakenly label them as the same individual. This can create a negative bias in the data and portray a population as smaller and less  genetically diverse  than it is. This is most commonly seen in collection methods that rely on  environmental DNA  (eDNA) which is collected directly from the environment (such as feces or hair removed from the ground). [ 2 ]  The accuracy of non-invasive collection data can be increased by increasing the amount of loci being examined during the study. [ 1 ]


There are several types of  rarefaction methods  that can be used to estimate the size of a hard monitor species. The study of population size and density falls under  demography , the study of populations of any kind of organism. [ citation needed ]


Mark and recapture  is a common form of  data collection  involving species with large populations. Being able to capture and mark a species in a noninvasive way allows for accurate readings of the population's size, both total and effective over several rounds of recapture. However, for species that are difficult to capture or view directly such as  endangered species , it can be near impossible to use the mark-recapture method to obtain genetic samples. [ citation needed ]


Another method for population size estimation is a  real-time polymerase chain reaction  (qPCR). qPCR is a molecular approach that measures the amplification of DNA over time rather than just at the end of the reaction. [ 3 ]  This method is useful because it can rely on eDNA to give an estimate of how abundant a species is in a given habitat. [ citation needed ]


Noninvasive forms of data collection can be achieved through the collection of fur, feces or other fragments of DNA-rich material left behind (eDNA). [ 1 ]  Once considered costly, modern advancements have allowed for non-invasive data collection to become easier and provide more genetic information about a population. [ 4 ]   DNA-Barcoding  is a method of species identification that uses eDNA to determine the unique genetic makeup of individuals and their species. This method is that it requires a balancing act: Maintaining enough loci markers in the genetic data, and remaining cost-effective. [ citation needed ]


Probability of Identity (PI AV ) is the probability of a randomly selected sample from a population producing the same genotype twice if not enough loci are used during the study. Should loci with little genetic variation or few loci are selected, it is likely that multiple individuals will be identified as too genetically similar, with individuals being excluded from the data set. This creates a  negative statistical bias , pushing the results towards a smaller, less genetically diverse mean than is accurate to the population. This is known as the shadow effect. [ 1 ]


The  Cabrera vole ( Microtus cabrerae )  is a small, endangered rodent that belongs to the  Microtus  genus. Existing primarily in Portugal, populations can be difficult to estimate using typical mark-recapture methods due to their small size and ability to quickly disperse over large swaths of prairie land. [ 4 ]  With the introduction and reduced cost of using environmental DNA (in this case feces) were able to be used in a relatively low cost experiment to estimate the population size of the Cabrera vole in southern Portugal in return for sacrificing demographic (age, sex, health) information. [ 4 ]


Snijders Blok–Campeau syndrome  is a  genetic disorder  caused by mutations in the  CHD3 gene . [ 1 ]  It is characterized by  impaired intellectual development ,  macrocephaly ,  dysarthria  and  apraxia of speech , and certain  distinctive facial features . [ 2 ]


Snijders Blok–Campeau syndrome is typically a  de novo mutation [ 3 ] [ 4 ]  which generally occurs during the  early embryonic stages  of development or during the formation of the parent's  reproductive cells . [ 2 ]  This allows for  prenatal diagnosis . [ 5 ]


Snijders Blok–Campeau syndrome almost always comes with both physical and intellectual disabilities. Those with the condition will typically have trouble in the development of speech and language. Around one half typically have some form of macrocephaly, while around one third show signs of  autism  or similar conditions. [ 4 ]


The CHD3 gene is required for  chromatin remodeling , a process that regulates gene expression. [ 7 ]  By allowing for the creation of  chromatin , the CHD3 gene affects how tightly  DNA  is packed into  chromosomes . A mutation of the CHD3 gene changes the amount of chromatin produced, causing over or underexpression of other genes. [ 7 ] [ 8 ]


Due to the rarity of the condition, with only approximately 60 cases documented in scientific literature, [ 7 ]  Snijders Blok–Campeau syndrome was only discovered in 2018 by  clinical geneticist   Lot Snijders Blok  and  clinician-scientist   Philippe M Campeau . The mutation was first documented in the paper " CHD3 helicase domain mutations cause a neurodevelopmental syndrome with macrocephaly and impaired speech and language ". [ 3 ]


A  SNPSTR  is a compound  genetic marker  composed of one or more  SNPs  and one  microsatellite  (STR).  Autosomal  SNPSTRs, which contain an SNP and a microsatellite within 500 base pairs of one another, were discovered in 2002. [ 1 ]  More recently a  database  that contains all SNPSTRs in five model genomes, including human, has been created. [ 2 ]


There has been widespread and growing interest in genetic markers suitable for drawing population genetic inferences about past demographic events and to detect the effects of selection. Single nucleotide polymorphisms ( SNPs ) and  microsatellites  (or short tandem repeats, STRs) have received great attention in the analysis of human population history, even though they have both disadvantages. It was thus suggested that the combination of these two markers could give rise to better conclusions.


The  SOS chromotest  is a biological assay to assess the  genotoxic  potential of chemical compounds. The test is a colorimetric assay which measures the expression of genes induced by genotoxic agents in  Escherichia coli , by means of a fusion with the structural gene for  β-galactosidase . The test is performed over a few hours in columns of a 96-well microplate with increasing concentrations of test samples. This test was developed as a practical complement or alternative to the traditional  Ames test  assay for  genotoxicity , which involves growing bacteria on agar plates and comparing natural mutation rates to mutation rates of bacteria exposed to potentially mutagenic compounds or samples. The SOS chromotest is comparable in accuracy and sensitivity to established methods such as the Ames test and is a useful tool to screen genotoxic compounds, which could prove carcinogenic in humans, in order to single out chemicals for further in-depth analysis. [ 1 ] [ 2 ]


As with other bacterial gentoxicity and mutagenicity assays, compounds requiring metabolic activation for activity can be investigated with the addition of S9 microsomal rat liver extract.


The  SOS response  plays a central role in the response of  E. coli  to genotoxic compounds because it responds to a wide array of chemical agents. Triggering of this system can and has been used as an early sign of DNA damage. Two genes play a key role in the SOS response: lexA encodes a repressor for all the genes in the system, and recA encodes a protein able to cleave the LexA repressor upon activation by an SOS inducing signal (caused in this case by the presence of a genotoxic compound). Although the exact mechanism of the SOS response is still unknown, it is induced when DNA lesions perturb or stop DNA replication. . [ 3 ]


Various end-points are possible indicators of the triggering of the SOS system; activation of the RecA protein, cleavage of the LexA repressor, expression of any of the SOS genes, etc. One of the simplest assays consists of monitoring the expression of an SOS gene by means of fusion with lacZ, the structural gene for  E. coli  β-galactosidase.


The SOS chromotest consists of incubating the  E. coli  with increasing concentrations of the chemical to be tested. After allowing time for protein synthesis, β-galactosidase activity is assayed using a simple colorimetric assay. By including a lactose analog which yields a colored compound upon degradation, an easily observable or quantifiable change in colour is used as a metric. Since the chemical tested may inhibit protein synthesis at higher concentrations, which would lead to an underestimation of B-galactosidase induction, alkaline phosphatase is assayed simultaneously with β-galactosidase in order to scale the data to survivability of the cells.


The assay can easily be completed in a number of hours. If using a micro-plate reader, the test is quantitative and dose-response curves have an initial linear region. The slope of this linear region allows unequivocal association of each compound with a single parameter, the SOS-inducing potency (SOSIP), which reflects the inducing activity of the compound. [ 4 ]


This assay provides both a qualitative (visible observation of colour gradient) for screening purposes, or quantitative measurement (spectrophotometry) for calculation of commonly accepted metrics. In the quantitative assay, the dose-response (colour production linked to beta-galactosidase production) of a compound is plotted, with the slope of the initial linear region used as a universal parameter, the SOS-inducing potency (SOSIP), which reflects the ability of a compound to induce the SOS response (measured indirectly through production of beta-galactosidase and the breakdown of a lactose analog). Typically, the lactose analog is  X-Gal , which produces a blue colour when cleaved by beta-galactosidase. The dose response is also scaled by the survival of cells, measured through the breakdown of alkaline phosphatase (which produces a yellow colour), allowing for the calculation of the SOSIP.


Although the SOSIP is a concentration-based metric, the same method can be used for complex environmental mixtures where the concentration or even compounds of interest are unknown. Through the calculation of the intermediate SOS-induction factor (SOSIF), which can be plotted against dilution in the same manner in order to give an illustration of the dose-response without analytical measurements of samples beforehand.


THE SOS chromotest is considered to be the most simple and rapid short-term test for genotoxicity. It serves as a useful and cost effective complement to the traditional Ames test for a number of reasons. Firstly, because of its simplicity and rapidity, the SOS chromotest may be used as a screening test for a large number of potentially genotoxic compounds. Secondly, it may allow the detection of genotoxic chemicals which yield false negatives in the Ames test (such as estradiol, a compound of growing concern). Thirdly, it can prove an effective method to discriminate false positive results in the Ames test.


SoxC group  is group C of  Sry -related  HMG  box proteins  transcription factors . SoxC genes play an important role in determining the cell fate of neuronal  mesenchymal progenitor cells  in many developmental processes. [ 1 ]


In  Drosophila melanogaster  (fly),  Caenorhabditis elegans  (worm), and other lower animals SoxC is made up of only one member, but humans, mice and most other vertebrates have three members of the SoxC group. [ 2 ]  The three are Sox4, Sox11, and Sox12. These three are extremely similar to one another, more so than other proteins, but they are all highly distinct in the way that they bind DNA and active  transcription   in vitro  with different affinity. [ 3 ]  The three found in humans and other vertebrates are single- exon  genes. [ 2 ]  The SoxC proteins have 2  domains , the first an Sry-related HMG box DNA binding domain that is located near the  N-terminal  and the second a trans-activation domain, known as TAD, located near the  C-terminal . [ 3 ]   Although these are transcription factors, to this date there is no evidence of  post-translational modifications  on SoxC members, but they can work cooperatively with other proteins though, such as transcription factors Brn2 and Brn1. [ 3 ]


All of the SoxC proteins share 67% identity and 94% similarity in the 33 residues of the C-terminal domain. [ 3 ]   All SoxC genes show 84% identity and 95% similarity to one another in the HMG box, which is just slightly more highly conserved than the C-terminal domain. [ 3 ]  The SoxC proteins only have between 45% and 67% identity to Sry and the other Sox proteins. [ 3 ]  The SoxC genes found in the  Drosophila melanogaster  (fly),  Caenorhabditis elegans  (worm), and other lower animals are closely linked to those found in humans and other vertebrates. [ 3 ]  The SoxC genes are highly  conserved  through vertebrate evolution and are similar enough to those in invertebrates and other lower animal species to speculate that these are based on an evolutionary necessity from before vertebrates and invertebrates separated through evolution. [ 3 ]


If these SoxC proteins were missing during developmental stages it would cause widespread problems through the body of mice, and in many cases death. [ 3 ]   These have not been studied in humans though, because no occurrences have been linked to congential malformations of any of these proteins. [ 2 ]  In mice however, mice embryos without Sox4 die of heart defects, but mice newborns without Sox11 do live but have widespread defects. [ 3 ]   Mice without Sox12 are viable and show no outward signs of malformation. [ 3 ]   Some of the common human  malformations  are also seen in mice with mutated SoxC, such as  cleft palate  or heart outflow tract malformation. [ 3 ]   There is no evidence of the correlation between SoxC mutations in humans and these malformations, but there is speculation.


Sox4 facilitates differentiation of  lymphocytes ,  osteoblasts , pancreatic  beta cells  and along with Sox11 promotes neural  differentiation . In recent years, the belief has raised that SoxC genes may lead to tumor prognosis at elevated levels. [ 2 ]  Increased expression of Sox11 and Sox4 are seen in numerous tumors and cancers and it is possible that the tumors differ depending on the circumstance and primary transformation mechanism. [ 2 ]


The  staggered extension process  (also referred to as StEP) is a common technique used in  biotechnology  and  molecular biology  to create new, mutated genes with qualities of one or more initial genes.


The technique itself is a modified  polymerase chain reaction  with very short (approximately 10 seconds) cycles.
In these cycles the elongation of DNA is very quick (only a few hundred base pairs) and synthesized fragments anneal with complementary fragments of other strands. In this way, mutations of the initial genes are shuffled and in the end genes with new combinations of mutations are amplified. [ 1 ] [ 2 ]


The StEP protocol has been found to be useful as a method of  directed evolution  for the discovery of enzymes useful to industry. [ 3 ]


State switching  (a.k.a.  phenotypic switching ) is a fundamental physiological process in which a cell/organism undergoes spontaneous, and potentially reversible, transitions between different  phenotypes .  Thus, the ability to switch states/phenotypes ( phenotypic plasticity ) is a key feature of development and normal function of cells within most multicellular organisms that enables the cell to respond to various intrinsic and extrinsic cues and stimuli in a concerted fashion enabling them to ‘make’ appropriate cellular decisions. [ 1 ]  Although state switching is essential for normal functioning, the repertoire of phenotypes in a normal cell is limited.


In contrast to normal cells, a striking characteristic of cancer cells is the remarkable degree of phenotypic plasticity they exhibit. For example, cancer cells undergo  epithelial to mesenchymal transition  (EMT) that plays important roles in their survival, proliferation, and development of resistance to therapeutic treatments, [ 2 ] [ 3 ]  or switch to a phenotype that mimics stem cell-like features – the so-called  Cancer Stem Cells  (CSCs) or Tumour-initiating Cells. Unlike in the case of normal cells, state switching in cancer cells is widely believed to arise due to  somatic mutations . [ 4 ]  However, there is growing concern that such a deterministic view of a phenomenon that is reversible is not entirely consistent with multiple lines of evidence which indicate that stochasticity may also play an important role in driving phenotypic plasticity. [ 5 ]


A hallmark of the factors implicated in phenotypic switching whether in cancer or in normal cells is that they are  Intrinsically disordered proteins  (IDPs). That is, they lack a rigid 3D-structure under physiological conditions at least in vitro and exist as conformational ensembles instead. [ 6 ] [ 7 ]  However, many IDPs can transition from disorder to order upon interacting with a target [ 8 ]  or in response to post-translational modifications such as phosphorylation. [ 9 ]  IDPs are particularly enriched in transcriptional regulation, signaling and splicing, and are overexpressed in many pathological states including cancer. [ 10 ]  Thus, the products of most  oncogenes  such as Jun, Fos, Myc, [ 11 ] [ 12 ]  the  Yamanaka factors  namely,  OCT3/4 ,  SOX2 ,  MYC ,  NANOG , and  KLF4  that induce  reprogramming  of pluripotent stem (iPS) cells, [ 13 ]  and >90% of the  Cancer/Testis Antigens [ 14 ]  several of which are implicated in EMT [ 15 ] [ 16 ]  are predicted, and in many cases experimentally verified, to be IDPs. [ 17 ] [ 18 ] [ 19 ] [ 20 ]  Consistent with these observations, the key factors implicated in EMT/MET namely, OVOL1/2, ZEB1 and SNAI1 are also strongly predicted to be IDPs. [ 21 ]


Until the 1990s, it was tacitly assumed that most networks adopt a random architecture wherein an edge (connection) between each pair of nodes has equal probability, independent of the other edges ( Erdős–Rényi model ). However, pioneering work by Barabási and colleagues ( Barabási–Albert model ) [ 22 ]  indicated that biological networks, like many other networks they interrogated, adopt an architecture wherein the degree distribution P(k) exhibits a power-law behavior as a function of the degree k. In particular, P(k) ~ k-γ, with only a few nodes (hub nodes) has numerous edges while the majority of the nodes have very few edges. As such, these networks are robust to failure of random nodes but vulnerable to failure of hubs. [ 23 ]  In light of the scale-free topology of the PIN, Mahmoudabadi et al. [ 24 ]  set out to elucidate whether perturbation to IDP hub proteins could account for such dramatic changes as state switching in the absence of DNA mutations (changes to the DNA sequence) and whether these changes could be passed onto the progeny.


The authors proposed a theoretical model (hereby named the ‘MRK model’ after the main proponents- Gita  M ahmoudabadi,  Govindan  R angarajan , and Prakash  K ulkarni) which envisaged that because IDPs have multiple conformational states and rapid conformational dynamics, they are prone to engage in ‘promiscuous’ interactions. These stochastic interactions between the IDPs and their partners result in  ‘noise’ , defined as conformational noise. [ 24 ]  Indeed, many biological processes are driven by probabilistic events underscoring the importance of ‘noise’ in biological systems. [ 25 ]  However, while research on biological noise focused on low gene copy numbers as the predominant source of noise, noise arising from stochastic IDP interactions due to the conformational dynamics of IDPs had not been considered. Thus, the central tenet of the MRK model is that, just as transcriptional noise plays an important role in probabilistic differentiation and adaptation, noise inherent in protein interactions could underlie the activation of latent pathways and cellular transformation. Although the authors chose to highlight the role of IDPs in propagation of transcriptional noise because this type of noise has systematically been studied and is a common feature of cellular processes in both normal and diseased conditions, they also point out that the role of IDPs is not limited to the propagation of transcriptional noise. Rather, the model emphasizes that IDPs could likely relay, and perhaps, even amplify, other intrinsic and extrinsic types of noise and perturbations in the system. Consistent with this argument, there are now numerous examples of remodeling of the IDP conformational ensemble in response to binding and/or post-translational modifications [ 9 ] [ 26 ]  to populate a different conformation with huge functional consequences.


Together, these observations tend to suggest that contrary to the prevailing wisdom that phenotype specification is highly deterministic, stochasticity may be a confounding factor in specifying cell fate. This thinking may also help explain how a given cell can reversibly switch phenotypes as seen in EMT and MET or for that matter, a drug-sensitive cell from developing resistance and switching back to drug sensitivity, [ 27 ]  or the transformation of a normal cell to a malignant one and its reversal to normalcy. [ 28 ]  Indeed, such stochasticity in phenotypic switching is also thought to underlie cellular differentiation, [ 25 ]  generation of induced pluripotent stem cells (iPS cells), [ 29 ]  tumor heterogeneity [ 30 ] [ 31 ]  and emergence of cancer stem cells from non-stem cancer cells. [ 31 ]  Implicit in the MRK model, the PIN configuration contains information that can specify the cell's phenotype.


It seems quite reasonable to assume that organisms acquire useful adaptations during their lifetime. Such adaptations are the result of an exploratory search which samples various iterations of potential outputs in order to discern and select the most appropriate ones. Thus, it is plausible that ‘learning’, which can be described as an elaborate and iterative form of phenotypic modification that allows an organism to adjust its response to the same inputs over time based on the outcomes of previous outputs, could have a significant influence on evolution of a new species in the long run. Therefore, it would be quite wasteful to forego the advantage of the exploration performed by the organism to facilitate the evolutionary search for increased fitness if information about the acquired (learned) characteristics (new phenotypes) was not transferred to the genotype or at least retained in some (non-genomic) fashion to facilitate transgenerational inheritance. Indeed, this type of interaction between learning and evolution was independently proposed in the late 1800s by Baldwin, [ 32 ]  Osborn [ 33 ]  and Morgan [ 11 ]  and is often referred to as the ‘ Baldwin effect ’.  However, the Baldwin effect remained under-appreciated because of its Lamarckian connotation and consequently it was inferred by many that learning  cannot  guide evolution.


In 1987, Hinton & Nowlan [ 12 ]  using a computer simulation demonstrated that indeed, learning (they actually meant phenotypic plasticity) can be very effective in guiding the evolutionary search. In fact, the authors observed that learning alters (smoothens) the shape of the search space in which evolution operates and predicted that in difficult evolutionary searches that may require many possibilities to be tested, each learning trial can be almost as helpful to the evolutionary search as the production and evaluation of a whole new organism. Thus, logically speaking, the ‘efficiency’ of evolution is greatly enhanced since a learning trial is much faster and far less energy-intensive than that required for the production of a whole organism by random mutations. [ 12 ]  Subsequent studies by Behara and  Nanjundiah [ 34 ] [ 35 ] [ 36 ]  demonstrated that although the relationship may not be as straightforward as was assumed by Hinton & Nowlan, phenotypic plasticity can potentiate evolution even when more realistic fitness schemes are simulated.


Although these computational studies are tantalizing, the real question is, can cancer cells (or other  protists , for that matter) really ‘learn’ or ‘make’ decisions? To describe the cell's physiological response to a stimulus as learning/decision making is perhaps a matter of semantics. However, several observations made in protists that lack even the rudiments of a nervous system, much less a brain, suggest that they possess sophisticated mechanisms through which they respond to, ‘anticipate’, and even ‘learn’ from fluctuations and challenges in their environment. [ 37 ] [ 38 ] [ 39 ]


While cancer cells are not protists per se, they exhibit several characteristics that are typical of these simple forms of life. In fact, cancer has been postulated to represents some sort of reversion to a more primitive phenotype – an  atavism . [ 40 ]  In the atavistic model of cancer progression, tumor cell dedifferentiation is interpreted as a reversion to phylogenetically earlier capabilities. [ 41 ]  For example, cancer cells, develop drug resistance, exhibit traits of the  persister phenotype  (an extremely slow-growing physiological state which makes them insensitive to drug treatment) and  quorum sensing  (a system of stimulus and response correlated to population density), and display many other collective behaviour capabilities and cooperative strategies necessary for survival under extreme stress. [ 42 ]  These characteristics present cancer cells in a different light – smart communicating cells – and tend to portray tumours as societies of cells capable of making decisions. [ 42 ]   Thus, the MRK model posits that the stochasticity in interactions of IDPs that are overexpressed in cancer cells could facilitate learning by exploring the network search space and rewiring the network.


But how is the organization of the networks specified? What determines the network dynamics? How does this affect learning? Kulkarni [ 43 ]  hypothesized that analogous to the computational models developed by Hinton & Nowlan, and Behera & Nanjundiah, the basic design of the PINs is specified by the genome inasmuch as the expression of the critical nodes in space, time and amplitude are concerned. However, the ultimate organization of the PIN and its ground state threshold would be determined by learning and adapting to the environment in which the organism finds itself. [ 43 ]


For adaptive learning to be inherited, one would anticipate that changes in the genome, whether genetic or  epigenetic , would be necessary implying a reversal of information flow from phenotype to genotype. In response to dynamic environmental fluctuations, an organism's PINs constantly process information and organize and reorganize themselves. However, it is postulated [ 43 ]  that in response to ‘unanticipated’ environmental changes, several IDPs are overexpressed and the organism explores numerous iterations of network connections many of which are due to the promiscuous nature of these interactions. [ 44 ]  This results in a specific output that the organism benefits from, and in resetting the network to a new set-point (threshold). Further, it is postulated that information derived from PIN rewiring can operate across diverse timescales. [ 43 ]  Thus, while some of the information particularly that which operates over relatively short timescales maybe retained within the PINS, information that operates over long periods such as cellular transformation, development and evolution, is directly transferred to the genome to effect heritable genetic/epigenetic changes, or via a mechanism similar to genetic assimilation of the acquired character proposed by  Waddington . [ 45 ]  Several proteins that are involved in epigenetically sculpturing the chromatin are IDPs. [ 46 ]


Insofar as genetic changes are concerned, emerging evidence suggests that a nexus between  transcription factors  and  chromatin remodelers , [ 47 ]  and between transcription factors and  DNA repair  proteins [ 48 ]  that are part of large PINs, can facilitate such changes. With regard to  genetic assimilation , Waddington proposed that it is the process in which an environmental stimulus that affects the phenotype has been superseded by an internal genetic factor during the course of evolution. While such mechanisms could potentially account for permanent changes in the diploid genome of the cancer cell or other unicellular organisms, how information to activate such an internal genetic switch is transmitted to the germline for stable inheritance in metazoans reproducing sexually remains an important and intriguing question.


Notwithstanding the molecular mechanisms however, an equally important question that needs to be considered here is the evolutionary timescale. A key point in  Darwinian evolution  is that it works very slowly, over millions of years of geological time, through the gradual, incremental acquisition of small differences. Then how can a cancer cell evolve in such a short time? Perhaps, as has been suggested, [ 49 ]  under certain conditions evolution could occur more rapidly than previously envisioned. For example, in the extreme case, in a population of just a few individuals, all sorts of unusual mutations could become fixed simply because the number of individuals was so small and each mutation has a much higher likelihood of survival because competition among mutant forms is lower. Through this process a new species can arise in a few generations. However, in either case, mutations that hold the key arise by chance and without foresight for the potential advantage or disadvantage of the mutation. Furthermore, the underlying implication would be a unidirectional flow of information from genotype to phenotype.


On the other hand, in the scenario resulting from the MRK model wherein learning can guide evolution, changes to the genome arise due to necessity after trial and error and not just by chance, and in a few generations, are fixed. Episodes of rapid change - network rewiring to uncover latent pathway interactions in response to environmental perturbations - could lead to genotypic changes in a relatively short order. In other words, a species need not originate in a series of gradual steps, each resulting from a mutation with a small effect, slowly changing ancestor into descendant. Rather, the genetic changes that lead to the formation of new species have large effects and happen over relatively few generations. Thus, the MRK model implies that informational flow would be bidirectional and has parallels to ideas enunciated by  Lamarck  although empirical evidence for informational flow from phenotype to genotype is lacking at present. In fact, the inheritance of characteristics induced by the environment has often been opposed to the theory of evolution by natural selection. However, it is important to note that the emergence of non-conventional modes and the diversity of mechanisms for generating and transmitting variations such as the transmission of small interfering RNAs, the transmission of conformational states of IDPs such as prions, or, at the cellular level, the transmission of self-sustaining states of gene regulation, and, at the organismal level, so-called parental effects [ 50 ]  could provide additional support for the MRK theory.


Indeed, a recently proposed mathematical model to compare the adaptive value of different schemes of inheritance by  Rivoire  and  Leibler [ 50 ]  does appear to lend credence to the MRK model. The authors considered three biological phenomena that are often considered to be either irrelevant to evolution, or absent because “forbidden” namely, i) role of phenotypic plasticity and developmental canalization, ii) reverse flow of information from phenotype to genotype (or its absence), and iii) conditions under which direct integration of information into the transmitted genotype is logically excluded as a consequence of natural selection. Their model allows for variations to be inherited, randomly produced, or environmentally induced, and, irrespectively, to be either transmitted or not during reproduction. The adaptation of the different schemes for processing variations is quantified for a range of fluctuating environments, following an approach that links quantitative genetics with stochastic control theory.  When the authors conducted a  Gedankenexperiment  (German for a thought-experiment) that allowed them to compare the Darwinian and Lamarckian “modalities,” and test the conjecture that each of them is tuned to a different type of selective pressure, they found that the main controlling parameter in the model appears to be the correlation a of the environmental fluctuations, with the Lamarckian modality systematically becoming more favorable when this correlation is large, in line with the intuition that transmitting acquired information is beneficial when the selective pressure experienced by the offspring is sufficiently similar to that experienced by the parents.


In  genetics , a  subclade  is a subgroup of a  haplogroup . [ 1 ]


Although  human mitochondrial DNA  (mtDNA) and  Y chromosome DNA  (Y-DNA) haplogroups and subclades are named in a similar manner, their names belong to completely separate systems. [ 2 ]


mtDNA haplogroups are defined by the presence of a series of  single-nucleotide polymorphism  (SNP)  markers  in the  hypervariable regions  and the  coding region  of  mitochondrial DNA . They are named with the capital letters A through Z, with further subclades named using numbers and lower case letters. [ 2 ] [ 3 ] [ 4 ]


Y-DNA haplogroups are defined by the presence of a series of SNP markers on the  Y chromosome . Subclades are defined by a  terminal SNP , the SNP furthest down in the Y chromosome phylogenetic tree. [ 5 ]


The  Y Chromosome Consortium  (YCC) developed a system of naming major human Y-DNA haplogroups with the capital letters A through T, with further subclades named using numbers and lower case letters (YCC longhand  nomenclature ). YCC shorthand nomenclature names Y-DNA haplogroups and their subclades with the first letter of the major Y-DNA haplogroup followed by a dash and the name of the defining terminal SNP. [ 6 ]  Y-DNA haplogroup nomenclature is changing over time to accommodate the increasing number of SNPs being discovered and tested, and the resulting expansion of the Y chromosome phylogenetic tree. This change in nomenclature has resulted in inconsistent nomenclature being used in different sources. [ 7 ]  This inconsistency, and increasingly cumbersome longhand nomenclature, has prompted a move towards using the simpler shorthand nomenclature.


In  molecular biology , the  protein domain   SWAP  is derived from the term Suppressor-of-White-APricot, a splicing regulator from the  model organism   Drosophila  melanogaster . The  protein domain  is found in regulators that control  splicing . It is found in  splicing   regulatory   proteins . [ 1 ]
When a gene is  expressed  the  DNA  must be  transcribed  into  messenger RNA  (mRNA). However, it  sometimes contains intervening or interrupting sequences named  introns .   mRNA splicing  helps to remove these sequences, leaving a more favourable sequence. mRNA splicing is an essential event in the  post-transcriptional modification  process of  gene expression . [ 2 ]  SWAP helps to control this process in all cells except  gametes .


The role of the protein domain SWAP is to control sex-independent pre- mRNA  processing in   somatic  cells, that is, in every cell except the  sex cells  This includes autoregulation, whereby it regulates the splicing of its own pre-mRNA. [ 3 ]  The mammalian homologue of SWAP acts as a thyroid hormone regulated gene. This mean it is controlled by the thyroid. [ 4 ] [ 5 ]


SWAP  proteins  share a colinearly arrayed series of novel  sequence motifs . [ 3 ]  This means that they have been conserved over time. The SWAP protein in different organisms share some similarity in terms of sequence and may have been related at some point in evolutionary history.


Synaptic tagging , or the synaptic tagging hypothesis, has been proposed to explain how neural signaling at a particular  synapse  creates a target for subsequent plasticity-related product (PRP) trafficking essential for sustained  LTP  and  LTD .  Although the molecular identity of the tags remains unknown, it has been established that they form as a result of high or low frequency  stimulation , interact with incoming PRPs, and have a limited lifespan. [ 1 ]


Further investigations have suggested that plasticity-related products include  mRNA  and  proteins  from both the  soma  and dendritic shaft that must be captured by  molecules  within the  dendritic spine  to achieve persistent LTP and LTD.  This idea was articulated in the synaptic tag-and-capture hypothesis.  Overall, synaptic tagging elaborates on the molecular underpinnings of how L-LTP is generated and leads to  memory  formation.


Frey, a researcher at the  Leibniz Institute for Neurobiology  (later at the Medical College of Georgia and the Lund University), and Morris, a researcher at the  University of Edinburgh , [ 2 ]  laid the groundwork for the synaptic tagging hypothesis, stating:


"We propose that LTP initiates the creation of a short-lasting protein-synthesis-independent 'synaptic tag' at the potentiated synapse which sequesters the relevant protein(s) to establish late LTP. In support of this idea, we now show that weak tetanic stimulation, which ordinarily leads only to early LTP, or repeated tetanization in the presence of protein-synthesis inhibitors, each results in protein-synthesis-dependent late LTP, provided repeated tetanization has already been applied at another input to the same population of neurons. The synaptic tag decays in less than three hours. These findings indicate that the persistence of LTP depends not only on local events during its induction, but also on the prior activity of the neuron." [ 2 ]


L-LTP inducing  stimulus  induces two independent processes including a dendritic biological tag that identifies the synapse as having been stimulated, and a genomic cascade that produces new mRNAs and proteins (plasticity products). [ 3 ]  While weak stimulation also tags synapses, it does not produce the cascade.  Proteins produced in the cascade are characteristically promiscuous, in that they will attach to any recently tagged synapse. However, as Frey and Morris discovered, the tag is temporary and will disappear if no protein presents itself for capture. Therefore, the tag and protein production must overlap if L-LTP is to be induced by the  high-frequency stimulation .


The experiment performed by Frey and Morris involved the stimulation of two different sets of  Schaffer collateral  fibers that synapsed on same population of  CA1  cells. [ 3 ]  They then recorded field  EPSP  associated with each stimulus on either S1 or S2 pathways to produce E-LTP and L-LTP on different synapses within the same  neuron , based on the intensity of the stimulus. Results showed 1) that E-LTP produced by weak stimulation could be turned into L-LTP if a strong S2 stimulus was delivered before or after and 2)that the ability to convert E-LTP to L-LTP decreased as the interval between the two stimulations increased, creating temporal dependence. When they blocked protein synthesis prior to the delivery of strong S2 stimulation, the conversion to L-LTP was prevented, showing importance of translating the mRNAs produced by the genomic cascade.


Subsequent research has identified an additional property of synaptic tagging that involves associations between late LTP and LTD. This phenomenon was first identified by Sajikumar and Frey in 2004 and is now referred to as "cross-tagging". [ 4 ]  It involves late-associative interactions between LTP and LTD induced in sets of independent  synaptic inputs : late-LTP induced in one set of synaptic inputs can transform early-LTD into late-LTD in another set of inputs. The opposite effect also occurs: early LTP induced in the first synapse can be transformed into late  LTP if followed by a late LTD-inducing stimulus in an independent synapse. This phenomenon is seen because the synthesis of nonspecific plasticity related proteins (PRPs) by late-LTP or -LTD in the first synapse is sufficient to transform early-LTD/LTP to late-LTD/LTP in the second synapse after synaptic tags have been set.


Blitzer and his research team proposed a modification to the theory in 2005, stating that the proteins captured by the synaptic tag are actually local proteins that are translated from mRNAs located in the dendrites. [ 3 ]  This means that mRNAs are not a product of genomic cascade initiated by strong stimulus, but rather, is delivered as a result of continual  basal transcription . They proposed that even weakly stimulated synapses that were tagged can accept proteins produced from a strong stimulation nearby despite lacking the genomic cascade.


Synaptic tagging/ tag-and-capture theory potentially addresses the significant problem of explaining how mRNA, proteins, and other molecules may be specifically trafficked to certain dendritic spines during late phase LTP. It has long been known that the late phase of LTP depends on protein synthesis within the particular dendritic spine, as proven by injecting  anisomycin  into a dendritic spine and observing the resulting absence of late LTP. [ 5 ]  To achieve translation within the dendritic spine, neurons must synthesize the mRNA in the nucleus, package it within a  ribonucleoprotein  complex, initiate transport, prevent translation during transport, and ultimately deliver the RNP complex to the appropriate dendritic spine. [ 6 ]  These processes span a number of disciplines and synaptic tagging/tag-and-capture cannot explain them all; nevertheless, synaptic tagging likely plays an important role in directing mRNA trafficking to the appropriate dendritic spine and signaling the mRNA-RNP complex to dissociate and enter the dendritic spine.


A cell's identity and the identities of  subcellular  structures are largely determined by  RNA  transcripts. Considering this premise, it follows that cellular transcription, trafficking, and translation of mRNA undergo modification at a number of different junctures. [ 7 ]  Beginning with transcription, mRNA molecules are potentially modified via alternate splicing of  exons  and  introns . The alternate splicing mechanisms allow cells to produce a diverse set of proteins from a single gene within the genome. Recent developments in next-generation sequencing have allowed for greater understanding of the diversity  eukaryotic cells  achieve through splice variants. [ 8 ]


Transcribed mRNA must reach the intended  dendritic spine  for the spine to express L-LTP.  Neurons may transport mRNA to specific dendritic spines in a package along with a transport ribonucleoprotein (RNP) complex; the transport RNP complex is a subtype of an RNA granule. Granules containing two proteins of known importance to synaptic plasticity, CaMKII (Calmodulin-dependent Kinase II) and the immediate early gene Arc, have been identified to associate with a type of the motor protein kinesin, KIF5. [ 9 ]  Furthermore, there is evidence that polyadenylated mRNA associates with microtubules in mammalian neurons, at least in vitro. [ 10 ]  Since mRNA transcripts undergo polyadenylation prior to export from the nucleus, this suggests that the mRNA essential for late-phase LTP may travel along the microtubules within the dendritic shaft prior to reaching the dendritic spine.


Once the RNA/RNP complex arrives via motor protein to an area within the vicinity of the specific dendritic spine, it must somehow get “captured” by a process within the dendritic spine. This process likely involves the synaptic tag created by synaptic stimulation of sufficient strength. Synaptic tagging may result in capture of the RNA/RNP complex via any number of possible mechanisms such as:


Since the 1980s, it has become more and more clear that the  dendrites  contain the  ribosomes , proteins, and RNA components to achieve local and autonomous protein translation. Many mRNAs shown to be localized in the dendrites encode proteins known to be involved in LTP, including AMPA receptor and CaMKII subunits, and  cytoskeleton -related proteins  MAP2  and  Arc . [ 12 ]


Researchers [ 13 ]  provided evidence of local synthesis, by examining the distribution of Arc mRNA after selective stimulation of certain synapses of a hippocampal cell. They found that Arc mRNA was localized at the activated synapses, and Arc protein appeared there simultaneously. This suggests that the mRNA was translated locally.


These mRNA transcripts are translated in a cap-dependent manner, meaning they use a "cap" anchoring point to facilitate ribosome attachment to the 5' untranslated region. Eukaryotic initiation factor 4 group (eIF4) members recruit ribosomal subunits to the mRNA terminus, and assembly of the eIF4F initiation complex is a target of translational control: phosphorylation of eIF4F exposes the cap for rapid reloading, quickening the rate-limiting step of translation. It is suggested that eIF4F complex formation is regulated during LTP to increase local translation. [ 12 ]  In addition, excessive eIF4F complex destabilizes LTP.


Researchers have identified sequences within the mRNA that determine its final destination - called localization elements (LEs), zipcodes, and targeting elements (TEs). These are recognized by RNA binding proteins, of which some potential candidates are MARTA and ZBP1. [ 14 ] [ 15 ]  They recognize the TEs, and this interaction results in formation of ribonucleotide protein (RNP) complexes, which travel along cytoskeleton filaments to the spine with the help of motor proteins. Dendritic TEs have been identified in the untranslated region of several mRNAs, like MAP2 and alphaCaMKII. [ 16 ] [ 17 ]


Synaptic tagging is likely to involve the acquisition of molecular maintenance mechanisms by a synapse that would then allow for the conservation of synaptic changes. [ 18 ]  There are several proposed processes through which synaptic tagging functions. [ 19 ]  One model suggests that the tag allows for local protein synthesis at the specified synapse that then leads to modifications in synaptic strength. One example of this suggested mechanism involves the anchoring of  PKMzeta  mRNA to the tagged synapse. This anchor would then restrict the activity of translated PKMzeta, an important plasticity related protein, to this location. A different model proposes that short-term synaptic changes induced by the stimulus are themselves the tag; subsequently delivered or translated protein products act to strengthen this change. For example, the removal of AMPA receptors due to low-frequency stimulation leading to LTD is stabilized by a new protein product that would be inactive at synapses where internalization had not occurred. The tag could also be a latent  memory trace , as another model suggests. The activity of proteins would then be required for the memory trace to lead to sustained changes in synaptic strength. According to this model, changes induced by the latent memory trace, such as the growth of new  filipodia , are themselves the tag. These tags require protein products for stabilization, synapse formation, and synapse stabilization. Finally, another model proposes that the required molecular products get directed into the appropriate dendritic branches and then find the specific synapses under efficacy modification, by following Ca++ microconcentration gradients through voltage-gated Ca++ channels. [ 20 ]


While the concept of the synaptic tagging hypothesis mainly resulted from experiments applying stimulation to synapses, a similar model can be established considering the process of learning in a broader - behavioral - sense. [ 21 ]  Fabricio Ballarini and colleagues developed this behavioral tagging model by testing spatial object recognition, contextual conditioning, and conditioned taste aversion in rats with weak training. The applied training normally only results in alterations of short-term memory. However, they paired this weak training with a separate, arbitrary behavioral event that is assumed to induce protein synthesis. When the two behavioral events were coupled within a certain time frame, the weak training was sufficient to elicit task-related changes in long-term memory. The researchers believed that the weak training lead to a "learning tag". During the subsequent task, the cleavage of proteins resulted in the formation of long-term memory for this tag. The behavioral tagging model corresponds to the synaptic tagging model. A weak stimulation establishes E-LTP that may serve as the tag used in converting the weak potentiation to the stronger, more persistent L-LTP, once the high-intensity stimulation is applied.


Syntelog :  a special case of  gene   homology  where sets of genes are derived from the same  ancestral  genomic region.  This may arise from  speciation  events, or through whole or partial  genome duplication  events (e.g.  polyploidy ).  This term is distinct from  ortholog ,  paralog , in-paralog, out-paralog, and  xenolog  because it refers only to genes' evolutionary history evidenced by sequence similarity and relative genomic position. [ 1 ]


Comparison between two genomic regions of  Arabidopsis thaliana  derived from its most recent genome duplication event. Syntelogs are indicated by  red lines connecting regions of sequence similarly (red boxes):


Sequence analysis and visualization of syntelogs performed by GEvo. [ 2 ]   Regerate this analysis in CoGe's GEvo using  this link . GEvo Sequences were compared using the  BlastZ  algorithm.


Synthesis-dependent strand  annealing  ( SDSA ) is a major mechanism of homology-directed repair of DNA  double-strand breaks  (DSBs). Although many of the features of SDSA were first suggested in 1976, [ 1 ]  the  double-Holliday junction model  proposed in 1983 [ 2 ]  was favored by many researchers. In 1994, studies of double-strand gap repair in  Drosophila  were found to be incompatible with the double-Holliday junction model, leading researchers to propose a model they called synthesis-dependent strand annealing. [ 3 ]  Subsequent studies of meiotic recombination in  S. cerevisiae  found that non-crossover products appear earlier than double-Holliday junctions or crossover products, challenging the previous notion that both crossover and non-crossover products are produced by double-Holliday junctions and leading the authors to propose that non-crossover products are generated through SDSA. [ 4 ]


In the accompanying Figure, the first step labeled “5’ to 3’ resection” shows the formation of a 3’ ended single DNA strand that in the next step invades a homologous DNA duplex.  RNA polymerase III is reported to catalyze formation of a transient RNA-DNA hybrid at double-strand breaks as an essential intermediate step in the repair of the breaks by homologous recombination. [ 5 ]  Formation of the RNA-DNA hybrid would protect the invading single-stranded DNA from degradation.  After the transient RNA-DNA hybrid intermediate is formed the RNA strand is replaced by the  Rad51  protein which catalyzes the subsequent stage of strand invasion.


In the SDSA model, repair of double-stranded breaks occurs without the formation of a double Holliday junction, so that the two processes of homologous recombination are identical until just after D-loop formation. [ 6 ]  In yeast, the D-loop is formed by strand invasion with the help of proteins  Rad51  and  Rad52 , [ 7 ]  and is then acted on by DNA  helicase  Srs2 to prevent formation of the double Holliday junction in order for the SDSA pathway to occur. [ 8 ]  The invading 3' strand is thus extended along the recipient homologous DNA duplex by  DNA polymerase  in the 5' to 3' direction, so that the D-loop physically translocates – a process referred to as bubble migration DNA synthesis. [ 9 ]  The resulting single Holliday junction then slides down the DNA duplex in the same direction in a process called  branch migration , displacing the extended strand from the template strand. This displaced strand pops up to form a 3' overhang in the original double-stranded break duplex, which can then anneal to the opposite end of the original break through  complementary base  pairing. Thus DNA synthesis fills in gaps left over from annealing, and extends both ends of the still present single stranded DNA break,  ligating  all remaining gaps to produce recombinant non-crossover DNA. [ 10 ]


SDSA is unique in that D-loop translocation results in conservative rather than  semiconservative replication , as the first extended strand is displaced from its  template strand , leaving the homologous duplex intact. Therefore, although SDSA produces non-crossover products because flanking markers of heteroduplex DNA are not exchanged,  gene conversion  may occur, wherein nonreciprocal genetic transfer takes place between two homologous sequences. [ 11 ]


Assembly of a nucleoprotein filament comprising single-stranded DNA (ssDNA) and the  RecA  homolog,  Rad51 , is a key step necessary for  homology  search during  recombination . In the budding yeast  Saccharomyces cerevisiae , Srs2 translocase dismantles Rad51 filaments during  meiosis . [ 12 ]   By directly interacting with Rad51, Srs2 dislodges Rad51 from nucleoprotein filaments thereby inhibiting Rad51-dependent formation of joint molecules and  D-loop  structures.  This dismantling activity is specific for Rad51 since Srs2 does not dismantle  DMC1  (a meiosis-specific Rad51 homolog),  Rad52  (a Rad 51 mediator) or replication protein A ( RPA , a single-stranded DNA binding protein).  Srs2 promotes the non-crossover SDSA pathway, apparently by regulating RAD51 binding during strand exchange. [ 13 ]


Divergence between SDSA and double-Holliday junction occurs when the D-loop is disassembled allow the nascent strand to anneal to other resected end of the DSB (in the double-Holliday junction model the strand displaced by D-loop extension anneals to the other end of the DSB in "2nd end capture"). Research in  Drosophila melanogaster  identified the  Bloom syndrome helicase (Blm)  as the enzyme promoting dissassembly of the D-loop. [ 14 ] [ 15 ] [ 16 ]  Similarly,  S. cerevisiae  Sgs1, an  ortholog  of BLM, appears to be a central regulator of most of the  recombination  events that occur during  S. cerevisiae   meiosis . [ 17 ]   Sgs1(BLM) may disassemble  D-loop  structures analogous to early strand invasion intermediates and thus promote NCO formation by SDSA. [ 17 ]   The Sgs1 helicase forms a conserved complex with the topoisomerase III ( Top3 )- RMI1  heterodimer (that catalyzes DNA single strand passage). This complex, called STR (for its three components), promotes early formation of NCO recombinants by SDSA during meiosis. [ 18 ]


As reviewed by Uringa et al. [ 19 ]  the RTEL1 helicase is proposed to regulate recombination during meiosis in the worm  Caenorhabditis elegans . RTEL1 is a key protein in repair of DSBs. It disrupts  D-loops  and is thought to promote NCO outcomes through SDSA.


The number of DSBs created during meiosis can substantially exceed the number of final CO events.  In the plant  Arabidopsis thaliana , only about 4% of DSBs are repaired by CO recombination, [ 20 ]  suggesting that most DSBs are repaired by NCO recombination.  Data based on tetrad analysis from several species of fungi show that only a minority (on average about 34%) of recombination events during meiosis are COs (see Whitehouse, [ 21 ]  Tables 19 and 38 for summaries of data from  S. cerevisiae ,  Podospora anserina ,  Sordaria fimicola  and  Sordaria brevicollis ).  In the fruit fly  D. melanogaster  during meiosis in females there is at least a 3:1 ratio of NCOs to COs. [ 22 ]    These observations indicate that the majority of recombination events during meiosis are NCOs, and suggest that SDSA is the principal pathway for recombination during meiosis.


Synthetic genetic array analysis  ( SGA ) is a  high-throughput  technique for exploring  synthetic lethal  and synthetic sick  genetic interactions  ( SSL ). [ 1 ]  SGA allows for the systematic construction of double mutants using a combination of  recombinant genetic techniques , mating and selection steps. Using SGA methodology a query gene deletion mutant can be crossed to an entire genome deletion set to identify any  SSL  interactions, yielding functional information of the query gene and the genes it interacts with. A large-scale application of SGA in which ~130 query genes were crossed to the set of ~5000 viable deletion mutants in yeast revealed a genetic network containing ~1000 genes and ~4000 SSL interactions. [ 2 ]  The results of this study showed that genes with similar function tend to interact with one another and genes with similar patterns of genetic interactions often encode products that tend to work in the same pathway or complex. Synthetic Genetic Array analysis was initially developed using the model organism  S. cerevisiae .  This method has since been extended to cover 30% of the  S. cerevisiae  genome. [ 3 ]  Methodology has since been developed to allow SGA analysis in  S.pombe [ 4 ] [ 5 ]  and   E. coli . [ 6 ] [ 7 ]


Synthetic genetic array analysis was initially developed by Tong et al. [ 1 ]  in 2001 and has since been used by many groups working in a wide range of biomedical fields. SGA utilizes the entire genome yeast knock-out set created by the yeast genome deletion project. [ 8 ]


Synthetic genetic array analysis is generally conducted using colony arrays on petriplates at standard densities (96, 384, 768, 1536). To perform a SGA analysis in  S.cerevisiae , the query gene deletion is crossed systematically with a deletion mutant array (DMA) containing every viable knockout  ORF  of the yeast genome (currently 4786 strains). [ 9 ]   The resulting  diploids  are then sporulated by transferring to a media containing reduced nitrogen. The  haploid  progeny are then put through a series of selection platings and incubations to select for double mutants. The double mutants are screened for SSL interactions visually or using imaging software by assessing the size of the resulting colonies.


Due to the large number of precise replication steps in SGA analysis, robots are widely used to perform the colony manipulations. There are a few systems specifically designed for SGA analysis, which greatly decrease the time to analyse a query gene. Generally these have a series of pins which are used to transfer cells to and from plates, with one system utilizing disposable pads of pins to eliminate washing cycles. Computer programs can be used to analyze the colony sizes from images of the plates thus automating the SGA scoring and chemical-genetics profiling.


There are six major components:


Synthetic lethality  is defined as a type of genetic interaction where the combination of two genetic events results in cell death or death of an organism. [ 1 ]  Although the foregoing explanation is wider than this, it is common when referring to synthetic lethality to mean the situation arising by virtue of a combination of deficiencies of two or more genes leading to cell death (whether by means of apoptosis or otherwise), whereas a deficiency of only one of these genes does not. In a synthetic lethal  genetic screen , it is necessary to begin with a mutation that does not result in cell death, although the effect of that mutation could result in a differing  phenotype  (slow growth for example), and then systematically test other mutations at additional loci to determine which, in combination with the first mutation, causes cell death arising by way of deficiency or abolition of expression.


Synthetic lethality has utility for purposes of molecular targeted cancer therapy. The first example of a molecular targeted therapeutic agent, which exploited a synthetic lethal approach, arose by means of an inactivated  tumor suppressor gene  ( BRCA1  and 2), a treatment which received FDA approval in 2016 ( PARP inhibitor ). [ 2 ]  A sub-case of synthetic lethality, where vulnerabilities are exposed by the deletion of passenger genes rather than tumor suppressor is the so-called "collateral lethality". [ 3 ]


The phenomenon of synthetic lethality was first described by  Calvin Bridges  in 1922, who noticed that some combinations of mutations in the model organism  Drosophila melanogaster  (the common fruit fly) confer lethality. [ 1 ]   Theodore Dobzhansky  coined the term "synthetic lethality" in 1946 to describe the same type of genetic interaction in wildtype populations of  Drosophila . [ 4 ]  If the combination of genetic events results in a non-lethal reduction in fitness, the interaction is called synthetic sickness. Although in classical genetics the term synthetic lethality refers to the interaction between two genetic perturbations, synthetic lethality can also apply to cases in which the combination of a mutation and the action of a chemical compound causes lethality, whereas the mutation or compound alone are non-lethal. [ 5 ]


Synthetic lethality is a consequence of the tendency of organisms to maintain buffering schemes (i.e. backup plans) which engender phenotypic stability notwithstanding underlying  genetic variations, environmental changes or other random events, such as mutations. This  genetic robustness  is the result of parallel redundant pathways and  "capacitor" proteins  that camouflage the effects of mutations so that important cellular processes do not depend on any individual component. [ 6 ]  Synthetic lethality can help identify these buffering relationships, and what type of disease or malfunction that may occur when these relationships break down, through the identification of gene interactions that function in either the same biochemical process or pathways that appear to be unrelated. [ 7 ]


High-throughput synthetic lethal screens may help illuminate questions about how cellular processes work without previous knowledge of gene function or interaction. Screening strategy must take into account the organism used for screening, the mode of genetic perturbation, and whether the screen is  forward  or  reverse . Many of the first synthetic lethal screens were performed in  Saccharomyces cerevisiae . Budding yeast has many experimental advantages in screens, including a small genome, fast doubling time, both haploid and diploid states, and ease of genetic manipulation. [ 8 ]  Gene ablation can be performed using a  PCR -based strategy and complete libraries of knockout collections for all annotated yeast genes are publicly available.  Synthetic genetic array  (SGA), synthetic lethality by microarray (SLAM), and genetic interaction mapping (GIM) are three high-throughput methods for analyzing synthetic lethality in yeast. A genome scale genetic interaction map was created by SGA analysis in  S. cerevisiae  that comprises about 75% of all yeast genes. [ 9 ]


Collateral lethality is a sub-case of synthetic lethality in personalized cancer therapy, where vulnerabilities are exposed by the deletion of passenger genes rather than tumor suppressor genes, which are deleted by virtue of chromosomal proximity to major deleted tumor suppressor loci. [ 3 ]


Mutations in genes employed in  DNA mismatch repair  (MMR) cause a high mutation rate. [ 10 ] [ 11 ]   In tumors, such frequent subsequent mutations often generate "non-self" immunogenic antigens.  A human Phase II clinical trial, with 41 patients, evaluated one synthetic lethal approach for tumors with or without MMR defects. [ 12 ]   In the case of sporadic tumors evaluated, the majority would be deficient in MMR due to epigenetic repression of an MMR gene (see  DNA mismatch repair ).  The product of gene  PD-1  ordinarily represses cytotoxic immune responses.  Inhibition of this gene allows a  greater immune response.  In this Phase II clinical trial with 47 patients, when cancer patients with a defect in MMR in their tumors were exposed to an inhibitor of PD-1, 67% - 78% of patients experienced immune-related progression-free survival.  In contrast, for patients without defective MMR, addition of PD-1 inhibitor generated only 11% of patients with immune-related progression-free survival.  Thus inhibition of PD-1 is primarily synthetically lethal with MMR defects.


The analysis of 630 human primary tumors in 11 tissues shows that  WRN   promoter   hypermethylation  (with loss of expression of WRN protein) is a common event in tumorigenesis. [ 13 ]   The  WRN  gene promoter is hypermethylated in about 38% of  colorectal cancers  and  non-small-cell lung carcinomas  and in about 20% or so of  stomach cancers ,  prostate cancers ,  breast cancers ,  non-Hodgkin lymphomas  and  chondrosarcomas , plus at significant levels in the other cancers evaluated.  The  WRN  helicase protein is important in  homologous recombinational  DNA repair and also has roles in  non-homologous end joining  DNA repair and  base excision DNA repair . [ 14 ]


Topoisomerase inhibitors  are frequently used as chemotherapy for different cancers, though they cause bone marrow suppression, are cardiotoxic and have variable effectiveness. [ 15 ]   A 2006 retrospective study, with long clinical follow-up, was made of colon cancer patients treated with the topoisomerase inhibitor  irinotecan .  In this study, 45 patients had  hypermethylated   WRN  gene  promoters  and 43 patients had unmethylated  WRN  gene promoters. [ 13 ]   Irinitecan was more strongly beneficial for patients with hypermethylated  WRN  promoters (39.4 months survival) than for those with unmethylated  WRN  promoters (20.7 months survival).  Thus, a topoisomerase inhibitor appeared to be synthetically lethal with deficient expression of  WRN .  Further evaluations have also indicated synthetic lethality of deficient expression of  WRN  and topoisomerase inhibitors. [ 16 ] [ 17 ] [ 18 ] [ 19 ] [ 20 ]


As reviewed by Murata et al., [ 21 ]  five different PARP1 inhibitors are now undergoing  Phase I, II and III  clinical trials, to determine if particular PARP1 inhibitors are synthetically lethal in a large variety of cancers, including those in the prostate, pancreas, non-small-cell lung tumors, lymphoma, multiple myeloma, and Ewing sarcoma.  In addition, in preclinical studies using cells in culture or within mice, PARP1 inhibitors are being tested for synthetic lethality against epigenetic and mutational deficiencies in about 20 DNA repair defects beyond BRCA1/2 deficiencies.  These include deficiencies in  PALB2 ,  FANCD2 ,  RAD51 ,  ATM ,  MRE11 ,  p53 ,  XRCC1  and  LSD1 .


ARID1A , a chromatin modifier, is required for  non-homologous end joining , a major pathway that repairs double-strand breaks in DNA, [ 22 ]  and also has transcription regulatory roles. [ 23 ]   ARID1A  mutations are one of the 12 most common carcinogenic mutations. [ 24 ]   Mutation or epigenetically decreased expression [ 25 ]  of  ARID1A  has been found in 17 types of cancer. [ 26 ]   Pre-clinical studies in cells and in mice show that synthetic lethality for deficient  ARID1A  expression occurs by either inhibition of the methyltransferase activity of EZH2, [ 27 ] [ 28 ]  by inhibition of the DNA repair kinase ATR, [ 29 ]  or by exposure to the kinase inhibitor dasatinib. [ 30 ]


There are two pathways for  homologous recombinational  repair of double-strand breaks.  The major pathway depends on  BRCA1 ,  PALB2  and  BRCA2  while an alternative pathway depends on RAD52. [ 31 ]   Pre-clinical studies, involving epigenetically reduced or mutated  BRCA -deficient cells (in culture or injected into mice), show that inhibition of RAD52 is synthetically lethal with  BRCA -deficiency. [ 32 ]


Although treatments using synthetic lethality can stop or slow progression of cancers and prolong survival, each of the synthetic lethal treatments has some adverse side effects.  For example, more than 20% of patients treated with an inhibitor of PD-1 encounter fatigue, rash,  pruritus , cough, diarrhea, decreased appetite, constipation or  arthralgia . [ 33 ]   Thus, it is important to determine which DDR deficiency is present, so that only an effective synthetic lethal treatment can be applied, and not unnecessarily subject patients to adverse side effects without a direct benefit.


Synthetic rescue  (or  synthetic recovery  or  synthetic viability  when a lethal  phenotype  is rescued  [ 1 ] [ 2 ] ) refers to a genetic interaction in which a cell that is nonviable, sensitive to a specific drug, or otherwise impaired due to the presence of a  genetic mutation  becomes viable when the original mutation is combined with a second mutation in a different gene. [ 1 ]  The second mutation can either be a  loss-of-function mutation  (equivalent to a knockout) or a  gain-of-function mutation . [ 2 ]


Synthetic rescue could potentially be exploited for  gene therapy , but it also provides information on the function of the genes involved in the interaction.


Dosage-mediated suppression occurs when the suppression of the mutant phenotype is mediated by the over expression of a second suppressor gene. This can occur when the initial mutations destabilize a protein-protein interaction and over expression of the interacting protein bypass the negative effect of the initial mutation.


Interaction-mediated suppression occurs when a deleterious mutation in a component of a protein complex destabilizes the complex. A compensatory mutation in another component of the protein complex can then suppress the deleterious phenotype by re-establishing the interaction between the two proteins. It usually means that the deleterious mutation and the suppressive mutation occurs in two residues that are closely located in the tridimensional structure of the multi-protein complex. As thus, this kind of suppression provides indirect information on the molecular structure of the proteins involved.


The strongest form of synthetic rescues, in which the deleterious impact of a gene knockout is mitigated by an additional genetic perturbation that is also deleterious when considered in isolation, was modeled and predicted theoretically for gene interactions mediated by the metabolic network. [ 1 ]
This strong form of synthetic rescue has been recently observed in experiments in both  Saccharomyces cerevisiae . [ 3 ]
and  Escherichia coli . [ 4 ]
Patient survival analysis was also shown to predict synthetic rescues and other types of interactions. [ 5 ]


Genetic suppression can be mediated by  tRNA  genes when a mutation alters their  anticodon  sequence. For example, a tRNA designated for the recognition of the codon TCA and the corresponding insertion of  serine  in the growing polypeptide chain can mutate so that it recognize a TAA stop codon and promote the insertion of serine instead of the termination of the polypeptide chain. This could be particularly useful when a nonsense mutation (TCA >TAA) prevents the expression of a gene by either leading to a partially completed polypeptide or degradation of the  mRNA  by  nonsense-mediated decay . The redundancy of tRNA genes makes sure that such mutation would not prevent the normal insertion of serines when the TCA codon specifies them.


The  Systems Biology Ontology  (SBO) is a set of controlled, relational vocabularies of terms commonly used in  systems biology , and in particular in computational modeling.