Entry into  S-phase  during endoreduplication (and mitosis) is regulated through the formation of a  prereplicative complex  (pre-RC) at  replication origins , followed by recruitment and activation of the  DNA replication  machinery. In the context of endoreduplication these events are facilitated by an oscillation in  cyclin E - Cdk2  activity. Cyclin E-Cdk2 activity drives the recruitment and activation of the replication machinery, [ 33 ]  but it also inhibits pre-RC formation, [ 34 ]  presumably to ensure that only one round of replication occurs per cycle. Failure to maintain control over pre-RC formation at replication origins results in a phenomenon known as " rereplication " which is common in cancer cells. [ 2 ]  The mechanism by which cyclin E-Cdk2 inhibits pre-RC formation involves downregulation of  APC - Cdh1 -mediated proteolysis and accumulation of the protein  Geminin , which is responsible for sequestration of the pre-RC component  Cdt1 . [ 35 ] [ 36 ]


Oscillations in  Cyclin E - Cdk2  activity are modulated via  transcriptional  and post-transcriptional mechanisms. Expression of cyclin E is activated by  E2F  transcription factors that were shown to be required for endoreduplication. [ 37 ] [ 38 ] [ 39 ]  Recent work suggests that observed oscillations in E2F and cyclin E protein levels result from a  negative-feedback loop  involving  Cul4 -dependent  ubiquitination  and degradation of E2F. [ 40 ]  Post-transcriptional regulation of cyclin E-Cdk2 activity involves  Ago/Fbw7 -mediated proteolytic degradation of cyclin E  [ 41 ] [ 42 ]  and direct inhibition by factors such as Dacapo and  p57 . [ 43 ] [ 44 ]


The unisexual salamanders (genus  Ambystoma ) are the oldest known unisexual vertebrate lineage, having arisen about 5 million years ago. [ 45 ]  In these polyploid unisexual females, an extra premeiotic endomitotic replication of the genome, doubles the number of chromosomes. [ 46 ]  As a result, the mature eggs that are produced subsequent to the two meiotic divisions have the same ploidy as the somatic cells of the adult female salamander. Synapsis and recombination during meiotic prophase I in these unisexual females is thought to ordinarily occur between identical sister chromosomes and occasionally between homologous chromosomes. Thus little, if any, genetic variation is produced. Recombination between homeologous chromosomes occurs rarely, if at all. [ 46 ]


An enhanceosome is a protein complex that assembles at an  enhancer region  on DNA and helps to regulate the expression of a target gene. [ 1 ]


Enhancers are bound by  transcription activator proteins  and transcriptional regulation is typically controlled by more than one activator. Enhanceosomes are formed in special cases when these activators cooperatively bind together along the enhancer sequence to create a distinct three-dimensional structure. Each enhanceosome is unique towards its specific enhancer. This assembly is facilitated by energetically favorable  protein: protein  and protein: DNA interactions. Therefore, all the necessary activators need to be present for the enhanceosome to be formed and able to function. [ 1 ]


Once the enhanceosome has been formed, it recruits  coactivators  and  general transcription factors  to the  promoter region  of the target gene to begin transcription. [ 2 ]  The effectiveness of this is dependent on DNA conformation. As a result, the enhanceosome also recruits non histone architectural transcription factors, called  high-mobility group (HMG) proteins , which are responsible for regulating  chromatin  structure. [ 3 ]  These factors do not bind to the enhancer, but instead are used to restructure the DNA to ensure that the genes can be accessed by the transcription factors.


Most enhanceosomes have been discovered pertaining to genes requiring tight regulation, like those associated with the cells defense system. [ 4 ]   Using more than one kind of transcriptional activator protein could help to ensure that a gene is not transcribed prematurely.  Furthermore, the use of multiple factors enables gene regulation through a combination of cellular stimuli that function through multiple  signaling cascades.


The best known example of the enhanceosome acts on the  human   interferon-beta  gene, which is upregulated in cells that are infected by  viruses . [ 5 ]  Three activator proteins— NF-κB , an interferon activator protein such as  IRF-3 , and the  ATF -2/c-Jun complex—cooperatively bind to the upstream enhancer region upon viral infection. The interaction is mediated by a fourth protein  HMG-I , which assists in stabilizing the complex by promoting inter-protein interactions. The assembled enhanceosome recruits transcriptional machinery such as  RNA polymerase  to the promoter region to initiate  gene expression . [ 1 ] [ 5 ] [ 6 ]


An  enhancer trap  is a method in  molecular biology . The enhancer trap construct contains a  transposable element  and a  reporter gene . The first is necessary for (random) insertion in the genome, the latter is necessary for identification of the spatial regulation by the enhancer. On top of this, the construct usually includes a  genetic marker , e.g., the  white gene  producing red-colored eyes in  Drosophila , or ampicillin resistance in  E. coli .


The most common and basic enhancer traps are: P[ lacZ ] from the  bacterium   E. coli  and P[ GAL4 ] from  yeast . There exists a large number of fly stocks containing GAL4 insertions and an equally large number of fly stocks containing an UAS DNA sequence followed by a gene of interest, which permits the expression of a large number of genes with different GAL4 "drivers".  Rather than generating transgenic flies with the enhancer linked directly to the gene of interest (which takes about a year when starting without the appropriate DNA construct), one transgenic fly is simply mated (crossed) with another transgenic fly. [ 1 ] [ 2 ] [ 3 ]


Effector cell peptidase receptor 1 , also known as  EPR1 , is a withdrawn database record. [ 1 ]


This locus represents an antisense transcript of the  survivin  locus. This record was withdrawn in collaboration with HGNC. It was defined by L26245.1, which appears to be a cloning artifact ( Zaman GJ, Conway EM (July 2000). "The elusive factor Xa receptor: failure to detect transcripts that correspond to the published sequence of EPR-1".  Blood .  96  (1):  145– 8.  doi : 10.1182/blood.v96.1.145 .  PMID   10891443 . ).(This information come from  https://www.ncbi.nlm.nih.gov/gene?term=L26245.1 ) [ 1 ]


The  evo-devo gene toolkit  is the small subset of  genes  in an organism's  genome  whose products control the organism's  embryonic development .  Toolkit genes  are central to the synthesis of  molecular genetics ,  palaeontology ,  evolution  and developmental biology in the science of  evolutionary developmental biology  (evo-devo). Many of them are ancient and highly  conserved  among animal  phyla .


Toolkit genes are highly  conserved  among  phyla , meaning that they are ancient, dating back to the  last common ancestor of bilaterian animals . For example, that ancestor had at least 7  Pax genes  for  transcription factors . [ 1 ]


Differences in deployment of toolkit genes affect the body plan and the number, identity, and pattern of body parts. The majority of toolkit genes are components of signaling pathways and encode for the production of transcription factors,  cell adhesion  proteins, cell surface  receptor  proteins (and signalling  ligands  that bind to them), and secreted  morphogens ; all of these participate in defining the fate of undifferentiated cells, generating spatial and temporal patterns that, in turn, form the  body plan  of the organism. Among the most important of the toolkit genes are those of the  Hox gene  cluster, or complex. Hox genes, transcription factors containing the more broadly distributed  homeobox  protein-binding DNA motif, function in patterning the body axis. Thus, by combinatorially specifying the identity of particular body regions, Hox genes determine where  limbs  and other  body  segments will grow in a developing  embryo  or  larva . A  paradigmatic  toolkit gene is  Pax6 /eyeless , which controls  eye  formation in all animals. It has been found to produce eyes in mice and  Drosophila , even if mouse  Pax6/eyeless  was expressed in  Drosophila . [ 2 ]


This means that a big part of the morphological evolution undergone by organisms is a product of variation in the genetic toolkit, either by the genes changing their expression pattern or acquiring new functions. A good example of the first is the enlargement of the beak in Darwin's large ground-finch ( Geospiza magnirostris ), in which the gene  BMP  is responsible for the larger beak of this bird, relative to the other finches. [ 3 ]


The loss of legs in  snakes  and other  squamates  is another good example of genes changing their expression pattern. In this case the gene  Distal-less  is very under-expressed, or not expressed at all, in the regions where limbs would form in other  tetrapods . [ 4 ]
In 1994,  Sean B. Carroll 's team made the groundbreaking discovery that this same gene determines the  eyespot pattern  in  butterfly   wings , showing that toolkit genes can change their function. [ 5 ] [ 6 ] [ 7 ]


Toolkit genes, as well as being highly conserved, also tend to evolve the same function  convergently  or  in parallel . Classic examples of this are the already mentioned  Distal-less  gene, which is responsible for appendage formation in both tetrapods and insects, or, at a finer scale, the generation of wing patterns in the butterflies  Heliconius erato  and  Heliconius melpomene . These butterflies are  Müllerian mimics  whose coloration pattern arose in different evolutionary events, but is controlled by the same genes. [ 8 ]
This supports  Marc Kirschner  and  John C. Gerhart 's theory of  Facilitated Variation , which states that morphological evolutionary novelty is generated by regulatory changes in various members of a large set of conserved mechanisms of development and physiology. [ 9 ]


The  exome  is composed of all of the  exons  within the  genome , the sequences which, when transcribed, remain within the mature  RNA  after  introns  are removed by  RNA splicing . This includes  untranslated regions  of  messenger RNA  (mRNA), and  coding regions .  Exome sequencing  has proven to be an efficient method of determining the genetic basis of more than two dozen  Mendelian  or  single gene disorders . [ 1 ]


The human exome consists of roughly 233,785  exons , about 80% of which are less than 200  base pairs  in length, constituting a total of about 1.1% of the total  genome , or about 30 megabases of  DNA . [ 2 ] [ 3 ] [ 4 ]  Though composing a very small fraction of the  genome ,  mutations  in the exome are thought to harbor 85% of  mutations  that have a large effect on disease. [ 5 ]


It is important to note that the exome is distinct from the  transcriptome , which is all of the transcribed RNA within a cell type. While the exome is constant from cell-type to cell-type, the  transcriptome  changes based on the structure and function of the cells. As a result, the entirety of the exome is not  translated  into protein in every cell. Different cell types only  transcribe  portions of the exome, and only the  coding regions  of the exons are eventually translated into proteins.


Next-generation sequencing  (NGS) allows for the rapid sequencing of large amounts of DNA, significantly advancing the study of genetics, and replacing older methods such as  Sanger sequencing . This technology is starting to become more common in healthcare and research not only because it is a reliable method of determining genetic variations, but also because it is cost effective and allows researchers to sequence entire genomes in anywhere between days to weeks. This compares to former methods which may have taken months. Next-gen sequencing includes both  whole-exome sequencing  and  whole-genome sequencing . [ 6 ]


Sequencing an individual's exome instead of their entire genome has been proposed to be a more cost-effective and efficient way to diagnose rare  genetic disorders. [ 7 ] [ 8 ]  It has also been found to be more effective than other methods such as  karyotyping  and  microarrays . [ 9 ]  This distinction is largely due to the fact that phenotypes of genetic disorders are a result of mutated exons. In addition, since the exome only comprises 1.5% of the total genome, this process is more cost efficient and fast as it involves sequencing around 40 million bases rather than the 3 billion base pairs that make up the genome. [ 10 ]


On the other hand,  whole genome sequencing  has been found to capture a more comprehensive view of variants in the DNA compared to  whole-exome sequencing . Especially for  single nucleotide variants , whole genome sequencing is more powerful and more sensitive than whole-exome sequencing in detecting potentially disease-causing mutations within the exome. [ 11 ]  One must also keep in mind that  non-coding regions  can be involved in the regulation of the exons that make up the exome, and so whole-exome sequencing may not be complete in showing all the sequences at play in forming the exome.


With either form of  sequencing , whole-exome sequencing or whole genome sequencing, some have argued that such practices should be done under the consideration of medical ethics. While physicians strive to preserve patient autonomy, sequencing deliberately asks laboratories to look at  genetic variants  that may be completely unrelated to the patient's condition at hand and have the potential of revealing findings that were not intentionally sought. In addition, such testing have been suggested to have imply forms of discrimination against particular groups for having certain genes, creating the potential for stigmas or negative attitudes towards that group as a result. [ 12 ]


Rare mutations that affect the function of essential proteins constitute the majority of  Mendelian diseases . In addition, the overwhelming majority of disease-causing mutations in  Mendelian loci  can be found within the coding region. [ 5 ]  With the goal of finding methods to best detect harmful mutations and successfully diagnose patients, researchers are looking to the exome for clues to aid in this process.


Whole-exome sequencing  is a recent technology that has led to the discovery of various genetic disorders and increased the rate of diagnoses of patients with rare genetic disorders. Overall, whole-exome sequencing has allowed healthcare providers to diagnose 30–50% of patients who were thought to have rare Mendelian disorders. [ citation needed ]  It has been suggested that whole-exome sequencing in clinical settings has many unexplored advantages. Not only can the exome increase our understanding of genetic patterns, but under clinical settings, it has the potential to the change in management of patients with rare and previously unknown disorders, allowing physicians to develop more targeted and personalized interventions. [ 13 ]


For example,  Bartter Syndrome , also known as salt-wasting nephropathy, is a hereditary disease of the kidney characterized by  hypotension  (low blood pressure),  hypokalemia  (low potassium), and  alkalosis  (high blood pH) leading to muscle fatigue and varying levels of fatality. [ 14 ]  It is an example of a rare disease, affecting fewer than one per million people, whose patients have been positively impacted by whole-exome sequencing. Thanks to this method, patients who formerly did not exhibit the classical mutations associated with Bartter Syndrome were formally diagnosed with it after the discovery that the disease has mutations outside of the loci of interest. [ 5 ]  They were thus able to gain more targeted and productive treatment for the disease.


Much of the focus of exome sequencing in the context of disease diagnosis has been on protein coding "loss of function" alleles. Research has shown, however, that future advances that allow the study of non-coding regions, within and without the exome, may lead to additional abilities in the diagnoses of rare Mendelian disorders. [ 15 ]  The  exome  is the part of the  genome  composed of  exons , the sequences which, when transcribed, remain within the mature  RNA  after  introns  are removed by  RNA splicing  and contribute to the final protein product encoded by that gene.  It consists of all DNA that is transcribed into mature RNA in cells of any type, as distinct from the  transcriptome , which is the RNA that has been transcribed only in a specific cell population. The exome of the  human genome  consists of roughly 180,000  exons  constituting about 1% of the total  genome , or about 30 megabases of  DNA . [ 16 ]  Though composing a very small fraction of the  genome ,  mutations  in the exome are thought to harbor 85% of  mutations  that have a large effect on disease. [ 17 ] [ 18 ]   Exome sequencing  has proved to be an efficient strategy to determine the genetic basis of more than two dozen  Mendelian  or  single gene disorders . [ 19 ]


In  molecular biology ,  exon skipping  is a form of  RNA splicing  used to cause cells to “skip” over faulty or misaligned sections ( exons ) of genetic code, leading to a truncated but still functional protein despite the genetic mutation.


Exon skipping is used to restore the  reading frame  within a gene.  Genes  are the genetic instructions for creating a protein, and are composed of  introns  and  exons . Exons are the sections of DNA that contain the instruction set for generating a protein; they are interspersed with non-coding regions called introns. The introns are later removed before the protein is made, leaving only the coding exon regions.


Splicing naturally occurs in  pre-mRNA  when introns are being removed to form mature-mRNA that consists solely of exons. Starting in the late 1990s, scientists realized they could take advantage of this naturally occurring cellular splicing to downplay genetic  mutations  into less harmful ones. [ 1 ] [ 2 ]


The mechanism behind exon skipping is a mutation specific  antisense oligonucleotide  (AON). An antisense oligonucleotide is a synthesized short nucleic acid polymer, typically fifty or fewer base pairs in length that will bind to the mutation site in the pre-messenger RNA, to induce exon skipping. [ 3 ]  The AON binds to the mutated exon, so that when the gene is then translated from the mature mRNA, it is “skipped” over, thus restoring the disrupted reading frame. [ 3 ]  This allows for the generation of an internally deleted, but largely functional protein.


Some mutations require exon skipping at multiple sites, sometimes adjacent to one another, in order to restore the reading frame. Multiple exon skipping has successfully been carried out using a combination of AONs that target multiple exons. [ 4 ]


Exon skipping is being heavily researched for the treatment of  Duchenne muscular dystrophy  (DMD), where the muscular protein  dystrophin  is prematurely truncated, which leads to a non-functioning protein. Successful treatment by way of exon skipping could lead to a mostly functional dystrophin protein, and create a phenotype similar to the less severe  Becker muscular dystrophy  (BMD). [ 1 ] [ 5 ]


In the case of Duchenne muscular dystrophy, the protein that becomes compromised is dystrophin. [ 5 ]  The dystrophin protein has two essential functional domains that flank a central rod domain consisting of repetitive and partially dispensable segments. [ 6 ]  Dystrophin’s function is to maintain muscle fiber stability during contraction by linking the extra cellular matrix to the cytoskeleton. Mutations that disrupt the  open reading frame  within dystrophin create prematurely truncated proteins that are unable to perform their job. Such mutations lead to muscle fiber damage, replacement of muscle tissue by fat and fibrotic tissue, and premature death typically occurring in the early twenties of DMD patients. [ 6 ]  Comparatively, mutations that do not upset the open reading frame, lead to a dystrophin protein that is internally deleted and shorter than normal, but still partially functional. Such mutations are associated with the much milder Becker muscular dystrophy. Mildly affected BMD patients carrying deletions that involve over two thirds of the central rod domain have been described, suggesting that this domain is largely dispensable.


Dystrophin can maintain a large degree of functionality so long as the essential terminal domains are unaffected, and exon skipping only occurs within the central rod domain. Given these parameters, exon skipping can be used to restore an open reading frame by inducing a deletion of one or several exons within the central rod domain, and thus converting a DMD phenotype into a BMD phenotype.


The genetic mutation that leads to Becker muscular dystrophy is an  in-frame deletion . This means that, out of the 79 exons that code for dystrophin, one or several in the middle may be removed, without affecting the exons that follow the deletion. This allows for a shorter-than-normal dystrophin protein that maintains a degree of functionality. In Duchenne muscular dystrophy, the genetic mutation is out-of-frame. Out-of-frame mutations cause a premature stop in protein generation - the ribosome is unable to “read” the RNA past the point of initial error - leading to a severely shortened and completely non-functional dystrophin protein. [ 6 ]


The goal of exon skipping is to manipulate the splicing pattern so that an out-of-frame mutation becomes an in-frame mutation, thus changing a severe DMD mutation into a less harmful in-frame BMD mutation.


One exon-skipping drug was approved in 2016, by the US FDA:  eteplirsen  (ExonDys51), a  Morpholino  oligo from  Sarepta Therapeutics  targeting exon 51 of human dystrophin. Another exon-skipping Morpholino,  golodirsen  (Vyondys 53) (targeting dystrophin exon 53), was approved in the United States in December 2019. [ 7 ]  A third  antisense oligonucleotide ,  viltolarsen  (Viltepso), targeting dystrophin exon 53 was approved for medical use in the United States in August 2020. [ 8 ]


Genetic testing , usually from blood samples, can be used to determine the precise nature and location of the DMD mutation in the dystrophin gene. It is known that these mutations cluster in areas known as the 'hot spot' regions — primarily in exons 45–53 and to a lesser extent exons 2–20. [ 4 ]  As the majority of DMD mutations occur in these 'hot spot' regions, a treatment which causes these exons to be skipped could be used to treat up to 50% of DMD patients. [ 4 ] [ 5 ] [ 9 ]


In  molecular biology , an  exonic splicing enhancer  (ESE) is a DNA  sequence motif  consisting of 6 bases within an  exon  that directs, or enhances, accurate  splicing  of heterogeneous nuclear RNA ( hnRNA ) or  pre-mRNA  into messenger RNA ( mRNA ).


Short sequences of DNA are transcribed to  RNA ; then this RNA is translated to a  protein .  A gene located in DNA will contain  introns  and  exons .  Part of the process of preparing the RNA includes  splicing  out the introns, sections of RNA that do not code for the protein. The presence of exonic splicing enhancers is essential for proper identification of splice sites by the cellular machinery.


SR proteins  bind to and promote exon splicing in regions with ESEs, while  heterogeneous ribonucleoprotein particles  (hnRNPs) bind to and block exon splicing in regions with  exonic splicing silencers .  Both types of proteins are involved in the assembly and proper functioning of  spliceosomes . [ 1 ]


During  RNA splicing ,  U2 small nuclear RNA auxiliary factor 1  (U2AF35) and  U2AF2  (U2AF65) interact with the branch site and the 3' splice site of the intron to form the lariat.  It is thought that SR proteins that bind to ESEs promote exon splicing by increasing interactions with U2AF35 and U2AF65. [ 2 ]


Mutation of exonic splicing enhancer motifs is a significant contributor to genetic disorders and some cancers. Simple point mutations in ESEs can inhibit affinity for splicing factors and alter  alternative splicing , leading to altered mRNA sequence and protein translation. A field of genetic research is dedicated to determining the location and significance of ESE motifs  in vivo . [ 3 ]


Computational methods were used to identify 238 candidate ESEs. [ 4 ]  ESEs are clinically significant because synonymous point mutations previously thought to be  silent mutations  located in an ESEs can lead to exon skipping and the production of a non functioning protein.


Disruption of an exon splicing enhancer in exon 3 of  MLH1  gene is the cause of  HNPCC  (hereditary nonpolyposis colorectal cancer) in a Quebec family. [ 5 ]


There is evidence that these 236 hexamers that signal splicing are evolutionarily conserved. [ 6 ]


An  exonic splicing silencer  (ESS) is a short region (usually 4-18 nucleotides) [ 1 ]  of an  exon  and is a  cis-regulatory element . A set of 103 hexanucleotides known as FAS-hex3 has been shown to be abundant in ESS regions. [ 2 ]  ESSs inhibit or silence splicing of the  pre-mRNA  and contribute to constitutive and  alternate splicing . To elicit the silencing effect, ESSs recruit proteins that will negatively affect the core splicing machinery. [ 1 ]


Exonic splicing silencers work by inhibiting the splicing of pre-mRNA strands or promoting exon skipping. The single stranded pre-mRNA molecules need to have their  intronic  and exonic regions spliced in order to be translated. ESSs silence splice sites adjacent to them by interfering with the components of the core splicing complex, such as the  snRNP's ,  U1  and  U2 . [ 3 ]  This causes proteins that negatively influence splicing to be recruited to the splicing machinery.


ESSs have four general roles: [ 2 ]


Myotonic dystrophy  (MD) is most noticeably caused by inheriting an unstable  CTG triplet expansion  in the  DMPK  gene . In healthy genotypes two isoforms of an insulin receptor mRNA transcript exist. The isoform  IR-A  lacks exon 11 and is expressed ubiquitously in cells. Isoform  IR-B  contains exon 11 and is expressed in cells of the liver, muscles, kidney, and adipocytes. In individuals with MD,  IR-A  is  upregulated  in high amounts in skeletal muscle leading to the disease phenotype. [ 4 ]


The ESS nucleotide sequence exists within intron 10 and is thought to be dependent on the CUG triplet repeat in order to silence the splicing of exon 11. Silencing exon 11 splicing leads to the increased transcription of the  IR-A  isoform. [ 4 ]


Mutations in the  CFTR  gene are responsible for causing  cystic fibrosis . A particular mutation occurs in the  CFTR  pre-mRNA and leads to the exclusion of exon 9, mRNA lacking this exon folds a truncated protein (a protein shortened by a mutation). [ 4 ]


Exclusion of exon 9 is mediated by a  polymorphic locus  with variable TG repeats and stretches of T nucleotides – this is abbreviated as (TG)mT(n). This locus is an exonic splicing silencer and is located upstream of the exon 9 splice site (site 3c). The silencing is related to the high number of TG repeats and decreased stretches of T repeats (T tracts). A combination of both these factors is shown to increase levels of exon skipping. [ 4 ]


The  TDP-43  protein is responsible for physically silencing the exon splicing site once it is recruited by the exonic splicing silencer (TG)mT(n). TDP-43 is a DNA binding protein and repressor, it binds to the TG repeat to cause exon 9 skipping. The role of the T tracts is not well understood. [ 4 ]


Spinal muscular atrophy  is caused by the  homozygous  loss of the  SMN1  gene. Humans have two isoforms of the SMN (survival motor neuron) gene,  SMN1  and  SMN2 . The  SMN1  gene produces a complete transcript, while  SMN2  produces a transcript without exon 7 which results in a truncated protein. [ 4 ]


The ESS that contributes to the disease phenotype is the UAGACA nucleotide sequence. This sequence arises when a C-to-T mutation occurs at position +6 in exon 7 of the  SMN2  gene. This  transition point mutation  leads to the exclusion of exon 7 from the mRNA transcript, it is also the only difference between the  SMN2  and  SMN1  gene. [ 4 ]


The UAGACA ESS is thought to work by disrupting an  exonic splicing enhancer  and attracting proteins that inhibit splicing by binding sequences on exon 7. [ 4 ]


Mutations in the  ATM  gene are responsible for  ataxia telangiectasia . These mutations are generally single base pair  substitutions ,  deletions , or micro-insertions. A 4-nucleotide deletion within intron 20 of the  ATM  gene disrupts an exonic splicing silencer and causes the inclusion of a 65-nucleotide cryptic exon in the mature transcript. The inclusion of the cryptic exon results in protein truncation and atypical splicing patterns. [ 4 ]


Extranuclear inheritance  or  cytoplasmic inheritance  is the transmission of genes that occur outside the  nucleus . It is found in most  eukaryotes  and is commonly known to occur in cytoplasmic  organelles  such as  mitochondria  and  chloroplasts  or from cellular parasites like  viruses  or  bacteria . [ 1 ] [ 2 ] [ 3 ]


Mitochondria are organelles which function to transform energy as a result of  cellular respiration . Chloroplasts are organelles which function to produce sugars via  photosynthesis  in plants and algae. The  genes  located in  mitochondria  and chloroplasts are very important for proper cellular function. The  mitochondrial DNA  and other extranuclear types of DNA replicate independently of the DNA located in the nucleus, which is typically arranged in chromosomes that only replicate one time preceding cellular division. The extranuclear genomes of mitochondria and chloroplasts however replicate independently of cell division. They replicate in response to a cell's increasing energy needs which adjust during that cell's lifespan. Since they replicate independently, genomic recombination of these genomes is rarely found in offspring, contrary to nuclear genomes in which recombination is common.


Mitochondrial diseases are inherited from the mother, not from the father. Mitochondria with their mitochondrial DNA are already present in the egg cell before it gets fertilized by a sperm. In many cases of fertilization, the head of the sperm enters the egg cell; leaving its middle part, with its mitochondria, behind. The mitochondrial DNA of the sperm often remains outside the  zygote  and gets excluded from inheritance.


Extranuclear transmission of viral genomes and symbiotic bacteria is also possible. An example of viral genome transmission is  perinatal  transmission. This occurs from mother to fetus during the perinatal period, which begins before birth and ends about 1 month after birth. During this time viral material may be passed from mother to child in the bloodstream or breastmilk. This is of particular concern with mothers carrying  HIV  or  hepatitis C  viruses. [ 2 ] [ 3 ]  Symbiotic cytoplasmic  bacteria are also inherited in organisms such as insects and protists. [ 4 ]


Three general types of extranuclear inheritance exist.


Poky  is a mutant of the fungus  Neurospora crassa  that has extranuclear inheritance.  Poky  is characterized by slow growth, a defect in  mitochondrial ribosome  assembly and deficiencies in several  cytochromes . [ 10 ]   The studies of  poky  mutants were among the first to establish an extranuclear mitochondrial basis for inheritance of a particular genotype.  It was initially found, using genetic crosses, that  poky  is maternally inherited. [ 11 ]  Subsequently, the primary defect in the  poky  mutants was determined to be a  deletion  in the  mitochondrial  DNA sequence encoding the small subunit of mitochondrial  ribosomal RNA . [ 12 ]


The  fim  switch in  Escherichia coli  is the mechanism by which the  fim  gene cluster, encoding  Type I Pili , [ 1 ]  is transcriptionally controlled.


These pili are virulence factors involved in adhesion, especially important in  uropathogenic  Escherichia coli . The gene undergoes  phase variation  mediated via two  recombinases  and is a model example of site specific inversion.


The operon consists of the promoter region  fim  S, the main constituent  fim  A, its gene product forming a rod like structure and  fim  H, coding for an adhesin at the tip, to name just a few important elements. The  fim  S region is flanked by 9bp repeats that are mirror images of each other. [ 2 ]  These mirror images serve as substrates for two  ATP -dependent recombinases,  fim  B and  fim  E. These recombinases can invert the orientation of the   fim  S region and only one orientation allows for 3' to 5' transcription. [ citation needed ]


fim  B "flips" the promoter region both ways, from the "on" position to the "off" position and  vice versa , whereas  fim  E can only facilitate recombination from "on" to "off". This equilibrium, shifted towards maintaining the "off" position, due to higher  fim  E activity, [ 3 ]  serves as a mode of expressing pili only when adhesion is needed. Another level of transcriptional control in  E. coli  is mediated by the sensitivity of the recombinases to pH and osmolarity, [ 4 ]  further ensuring appropriate expression levels of type-I pili, given the stark differences in osmolarity inside and outside an animal's body. Type-I pili are expressed by many species of  Enterobacteriaceae . The transcriptional control can differ widely between species, [ 5 ]  in  Salmonella typhimurium , for example much influence is exerted by a  leucine-responsive regulatory protein  and there is no  fim  S element. [ 5 ]


Fisher's geometric model  ( FGM ) is an  evolutionary  model of the  effect sizes  and effect on  fitness  of spontaneous  mutations [ 1 ]  proposed by  Ronald Fisher  to explain the distribution of effects of mutations that could contribute to  adaptative  evolution. [ 2 ]


Sometimes referred to as the Fisher–Orr model, Fisher's model addresses the problem of adaptation (and, to some extent,  complexity ), and continues to be a point of reference in contemporary research on the genetic and evolutionary consequences of  pleiotropy . [ 3 ]


The model has two forms, a geometric formalism, and a  microscope  analogy.  A microscope which has many knobs to adjust the lenses to obtain a sharp image has little chance of obtaining an optimally functioning image by randomly turning the knobs. The chances of a clear image are not so bad if the number of knobs is low, but the chances will decrease dramatically if the number of adjustable  parameters  (knobs) is larger than two or three. Fisher introduced a geometric metaphor, which eventually became known as Fisher's geometric model. [ 3 ] [ 1 ]


In his model, Fisher argues that the functioning of the microscope is analogous to the fitness of an organism. The performance of the microscope depends on the state of various knobs that can be manipulated, corresponding to distances and orientations of various lenses, whereas the fitness of an organism depends on the state of various  phenotypic  character such as body size and  beak  length and depth. The increase in the fitness of an organism by random changes is then analogous to the attempt to improve the performance of a microscope through randomly changing the positions of the knobs on the microscope.


The analogy between the microscope and an evolving organism can be formalized by representing the phenotype of an organism as a point in a  high-dimensional data space , where the dimensions of that space correspond to the  traits  of the organism. The more independent dimensions of variation the phenotype has, the more difficult is improvement resulting from random changes. If there are many different ways to change a phenotype it becomes very unlikely that a random change affects the right combination of traits in the right way to improve fitness. Fisher noted that the smaller the effect, the higher the chance that a change is beneficial. At one extreme, changes with infinitesimally small effect have a 50% chance of improving fitness. This argument led to the widely held position that evolution proceeds by small mutations.


Furthermore, Orr discovered that both the fixation probability of a beneficial mutation and the fitness gain that is conferred by the fixation of the beneficial mutation decrease with organismal complexity. [ 4 ]  Thus, the predicted rate of adaptation decreases quickly with the rise in organismal complexity, a theoretical finding known as the ‘cost of complexity’.


Forward genetics  is a  molecular genetics  approach of determining the genetic basis responsible for a phenotype. Forward genetics provides an unbiased approach because it relies heavily on identifying the genes or genetic factors that cause a particular phenotype or trait of interest. [ 1 ]


This was initially done by using naturally occurring mutations or inducing mutants with radiation, chemicals, or  insertional mutagenesis  (e.g.  transposable elements ). Subsequent breeding takes place, mutant individuals are isolated, and then the gene is  mapped .  Forward genetics can be thought of as a counter to  reverse genetics , which determines the function of a gene by analyzing the phenotypic effects of altered DNA sequences. [ 2 ]  Mutant phenotypes are often observed long before having any idea which gene is responsible, which can lead to genes being named after their mutant phenotype (e.g.  Drosophila   rosy  gene which is named after the eye colour in mutants). [ 3 ]


Forward genetics provides researchers with the ability to identify genetic changes caused by mutations that are responsible for individual phenotypes in organisms. [ 1 ]  There are three major steps involved with the process of forward genetics which includes: making random mutations, selecting the phenotype or trait of interest, and identifying the gene and its function. [ 4 ]  Forward genetics involves the use of several mutagenesis processes to induce DNA mutations at random which may include:


Chemical mutagenesis is an easy tool that is used to generate a broad spectrum of mutant alleles. Chemicals like ethyl methanesulfonate (EMS) cause random  point mutations  particularly in G/C to A/T transitions due to guanine alkylation. [ 3 ]  These point mutations are typically loss-of-function or null alleles because they generate stop codons in the DNA sequence. [ 5 ]  These types of mutagens can be useful because they are easily applied to any organism but they were traditionally very difficult to  map , although the advent of next-generation sequencing has made this process considerably easier.


Another chemical such as ENU, also known as N-ethyl-N-nitrosourea works similarly to EMS. ENU also induces random point mutations where all codons are equally liable to change. These point mutations modify gene function by inducing different alleles, including gain or loss of function mutations in protein-coding or noncoding regions in the genome. [ 6 ]


Other methods such as using radiation to cause large deletions and  chromosomal rearrangements  can be used to generate mutants as well. [ 3 ]  Ionizing radiation can be used to induce genome-wide mutations as well as chromosomal duplications, inversions, and translocations.


Similarly, short wave UV light works in the same way as ionizing radiation which can also induce mutations generating chromosomal rearrangements. When DNA absorbs short wave UV light, dimerizing and oxidative mutations can occur which can cause severe damage to the DNA sequence of an organism.


Mutations can also be generated by  insertional mutagenesis . Most often, insertional mutagenesis involves the use of transposons, which introduces dramatic changes in the genome of an organism. Transposon movements can create random mutations in the DNA sequence by changing its position within a genome, therefore modifying gene function, and altering the organism’s genetic information. For example,  transposable elements  containing a  marker  are mobilized into the genome at random. These transposons are often modified to transpose only once, and once inserted into the genome a selectable marker can be used to identify the mutagenized individuals. Since a known fragment of DNA was inserted this can make mapping and cloning the gene much easier. [ 3 ] [ 7 ]


Once mutagenized and  screened , typically a  complementation test  is done to ensure that mutant  phenotypes  arise from the same genes if the mutations are recessive. [ 3 ] [ 8 ]  If the progeny after a cross between two recessive mutants have a wild-type phenotype, then it can be inferred that the phenotype is determined by more than one gene. Typically, the allele exhibiting the strongest phenotype is further analyzed. A genetic map can then be created using  linkage  and genetic markers, and then the gene of interest can be cloned and sequenced. If many alleles of the same genes are found, the screen is said to be saturated and it is likely that all of the genes involved producing the phenotype were found. [ 8 ]


Human diseases and disorders can be the result of mutations. [ 9 ]  Forward genetics methods are employed in studying heritable diseases to determine the genes that are accountable. [ 10 ]  With single-gene or mendelian disorders a  missense mutation  can be significant;  single nucleotide polymorphisms  (SNPs) can be analyzed to identify gene mutations that are associated with the disorder phenotype. Before 1980 very few human genes had been identified as disease loci until advances in DNA technology gave rise to  positional cloning  and reverse genetics. In the 1980s and 1990s, positional cloning consisted of genetic mapping, physical mapping, and discerning the gene mutation. [ 11 ]  Discovering disease loci using old forward genetic techniques was a very long and difficult process and much of the work went into mapping and cloning the gene through  association studies  and chromosome walking. [ 3 ] [ 12 ]  Despite being laborious and costly, forward genetics provides a way to obtain objective information regarding a mutation's connection to a disease. [ 13 ]  Another advantage of forward genetics is that it requires no prior knowledge about the gene being studied. [ 10 ]   Cystic fibrosis  however demonstrates how the process of forward genetics can elucidate a human genetic disorder. Genetic-linkage studies were able to map the disease loci in cystic fibrosis to chromosome 7 by using protein markers. Afterward,  chromosome walking  and  jumping  techniques were used to identify the gene and sequence it. [ 14 ]  Forward genetics can work for single-gene-single phenotype situations but in more complicated diseases like cancer, reverse genetics is often used instead. [ 12 ]  This is usually because complex diseases tend to have multiple genes, mutations, or other factors that cause or may influence it. [ 9 ]  Forward and  reverse genetics  operate with opposite approaches, but both are useful for genetics research. [ 10 ]  They can be coupled together to see if similar results are found. [ 10 ]


By the  classical genetics  approach, a researcher would locate (map) the gene on its chromosome by  crossbreeding  with individuals that carry other unusual traits and collecting statistics on how frequently the two traits are inherited together. Classical geneticists would have used phenotypic traits to map the new mutant alleles. Eventually the hope is that such screens would reach a large enough scale that most or all newly generated mutations would represent a second hit of a locus, essentially saturating the genome with mutations. This type of saturation mutagenesis within classical experiments was used to define sets of genes that were a bare minimum for the appearance of specific phenotypes. [ 15 ]  However, such initial screens were either incomplete as they were missing redundant loci and epigenetic effects, and such screens were difficult to undertake for certain phenotypes that lack directly measurable phenotypes. Additionally, a classical genetics approach takes significantly longer.


Gregor Mendel experimented with pea plant phenotypes and published his conclusions about genes and inheritance in 1865. [ 10 ]  Around the early 1900s  Thomas Hunt Morgan  was mutating  Drosophila  using radium and attempting to find heritable mutations. [ 16 ]  Alfred Sturtevant later began mapping genes of  Drosophila  with mutations they had been following. [ 17 ]  In the 1990s forward genetics methods were utilized to better understand  Drosophila  genes significant to development from embryo to adult fly. [ 18 ]  In 1995 the Nobel Prize went to Christiane Nüsslein, Edward Lewis, and Eris Wieschaus for their work in developmental genetics. [ 18 ]  The human genome was mapped and the sequence was published in  2003 . [ 19 ]  The ability to identify genes that contribute to Mendelian disorders has improved since 1990 as a result of advances in genetics and technology. [ 9 ]


Functional divergence  is the process by which genes, after  gene duplication , shift in function from an ancestral function. Functional divergence can result in either  subfunctionalization , where a paralog specializes one of several ancestral functions, or  neofunctionalization , where a totally new functional capability evolves. It is thought that this process of gene duplication and functional divergence is a major originator of molecular novelty and has produced the many large  protein families  that exist today. [ 1 ] [ 2 ]


Functional divergence is just one possible outcome of  gene duplication  events. Other fates include  nonfunctionalization  where one of the  paralogs  acquires  deleterious   mutations  and becomes a  pseudogene  and  superfunctionalization  (reinforcement), [ 3 ]  where both paralogs maintain original function. While gene, chromosome, or whole genome duplication events are considered the canonical sources of functional divergence of  paralogs ,  orthologs  (genes descended from speciation events) can also undergo functional divergence  [ 4 ] [ 5 ] [ 6 ] [ 7 ]  and  horizontal gene transfer  can also result in multiple copies of a gene in a genome, providing the opportunity for functional divergence.


Many well known protein families are the result of this process, such as the ancient gene duplication event that led to the divergence of  hemoglobin  and  myoglobin , the more recent duplication events that led to the various subunit expansions (alpha and beta) of vertebrate  hemoglobins , [ 8 ]  or the expansion of  G-protein alpha subunits  [ 9 ]


The GAL4-UAS system  is a  biochemical  method used to study  gene expression  and function in organisms such as the  fruit fly . It is based on the finding by Hitoshi Kakidani and Mark Ptashne, [ 1 ]  and Nicholas Webster and Pierre Chambon [ 2 ]  in 1988 that Gal4 binding to UAS sequences activates gene expression. The method was introduced into flies by  Andrea Brand  and  Norbert Perrimon  in 1993 [ 3 ]  and is considered a powerful technique for studying the  expression of genes . [ 4 ]  The system has two parts: the  Gal4  gene, encoding the  yeast   transcription activator   protein   Gal4 , and the UAS ( Upstream Activation Sequence ), an enhancer to which GAL4 specifically binds to activate gene  transcription .


The  Gal4  system allows separation of the problems of defining which cells express a gene or protein and what the experimenter wants to do with this knowledge. Geneticists have created genetic variants of model organisms (typically fruit flies), called  GAL4 lines , each of which expresses GAL4 in some subset of the animal's tissues. For example, some lines might express GAL4 only in muscle cells, or only in nerves, or only in the antennae, and so on. For fruit flies in particular, there are tens of thousands of such lines, with the most useful expressing GAL4 in only a very specific subset of the animal—perhaps, for example, only those  neurons  that connect two specific compartments of the fly's brain. The presence of GAL4, by itself, in these cells has little or no effect, since GAL4's main effect is to bind to a UAS region, and most cells have no (or innocuous) UAS regions.


Since  Gal4  by itself is not visible, and has little effect on cells, the other necessary part of this system are the "reporter lines". These are strains of flies with the special UAS region next to a desired gene. These genetic instructions occur in every cell of the animal, but in most cells nothing happens since that cell is not producing GAL4. In the cells that  are  producing GAL4, however, the UAS is activated, the gene next to it is turned on, and it starts producing its resulting protein. This may report to the investigator which cells are expressing GAL4, hence the term "reporter line", but genes intended to manipulate the cell behavior are often used as well.


Typical  reporter genes  include:


For example, scientists can first visualize a class of neurons by choosing a fly from a GAL4 line that expresses GAL4 in the desired set of neurons, and crossing it with a reporter line that express GFP.  In the offspring, the desired subset of cells will make GAL4, and in these cells the GAL4 will bind to the UAS, and enable the production of GFP.  So the desired subset of cells will now fluoresce green and can be followed with a  fluorescence microscope . Next, to figure out what these cells might do, the experimenter might express  channelrhodopsin  in each of these cells, by crossing the same GAL4 line with a channelrhodopsin reporter line. In the offspring the selected cells, and only those cells, will contain channelrhodopsin and can be triggered by a bright light. Now the scientist can trigger these particular cells at will, and examine the resulting behavior to see what these cells might do.


Gal4  is a  modular protein  consisting broadly of a  DNA-binding domain  and an activation domain. The UAS to which GAL4 binds is CGG-N 11 -CCG, where N can be any  base . [ 6 ]  Although GAL4 is a yeast protein not normally present in other organisms it has been shown to work as a  transcription activator  in a variety of organisms such as  Drosophila , [ 7 ]  and human cells, highlighting that the same mechanisms for gene expression have been conserved over the course of evolution. [ 2 ]


For study in  Drosophila , the GAL4 gene is placed under the control of a native gene  promoter , or driver gene, while the UAS controls expression of a target gene. GAL4 is then only expressed in cells where the driver gene is usually active. In turn, GAL4 should only activate gene transcription where a UAS has been introduced. For example, by fusing a gene encoding a visible marker like GFP ( Green Fluorescent Protein ) the expression pattern of the driver genes can be determined. GAL4 and the UAS are very useful for studying gene expression in  Drosophila  as they are not normally present and their expression does not interfere with other processes in the cell. For example, GAL4/UAS-regulated transgenes in  Drosophila  have been used to alter glial expression to produce arrhythmic behavior in a known  rhythmic circadian  output called  pigment dispersing factor  (PDF). [ 8 ]  However, some research has indicated that over-expression of GAL4 in  Drosophila  can have side-effects, probably relating to immune and stress responses to what is essentially an alien protein. [ 9 ]


The GAL4-UAS system has also been employed to study gene expression in organisms besides  Drosophila  such as the African clawed frog  Xenopus [ 10 ]  and  zebrafish . [ 11 ]


The GAL4/UAS system is also utilized in  Two-Hybrid Screening , a method of identifying interactions between two proteins or a protein with DNA.


Gal4  expression can be made even more specific by means of "intersectional strategies". These can combine two different GAL4 lines—say, A and B—in a way that GAL4 is only expressed in the cells that are in line A but not line B, or those that are in both lines A and B. When combined with intrinsically sparse GAL4 lines, this offers very specific selection, often limited to a single cell type. The disadvantage is that at least three independent insertion sites are required, so the lines must use different and independent insertion sites, and creating the desired final organisms needs more than a single cross. This is a very active field of research, and there are many such intersectional strategies, of which two are discussed below.


One way to create GAL4 expression in the cells that are in line A but not line B, requires line A to be made to express GAL4, and line B made to express  Gal80 , which is a GAL4 inhibitor.  Therefore, only the cells that are in A but not B will have active GAL4, which can then drive the  reporter gene . [ 12 ] [ 13 ]


To express GAL4 in only the cells contained in both A and B, a technique called "split-GAL4" can be used. Line A is made to express half of the GAL4 protein, which is inactive by itself.  Similarly, line B is made to express the other half of GAL4, also inactive by itself.  Only the cells that are in both lines make both halves, which self-assemble by  leucine zipper  into GAL4 and activate the reporter gene. [ 14 ]


In biology, a  gene cassette  is a type of  mobile genetic element  that contains a gene and a  recombination  site. Each cassette usually contains a single gene and tends to be very small; on the order of 500–1,000  base pairs . They may exist incorporated into an  integron  or freely as circular DNA. [ 1 ]  Gene cassettes can move around within an organism's genome or be transferred to another organism in the environment via  horizontal gene transfer . These cassettes often carry  antibiotic resistance   genes . An example would be the  kanMX  cassette which confers  kanamycin  (an  antibiotic ) resistance upon  bacteria .


Integrons are genetic structures in  bacteria  which express and are capable of acquiring and exchanging gene cassettes. The integron consists of a  promoter , an attachment site, and an  integrase  gene that encodes a  site-specific   recombinase [ 2 ]  There are three classes of integrons described. [ 1 ]  The mobile units that insert into integrons are gene cassettes. [ 2 ]  For cassettes that carry a single gene without a promoter, the entire series of cassettes is  transcribed  from an adjacent promoter within the integron. [ 3 ]  The gene cassettes are speculated to be inserted and excised via a circular intermediate. [ 4 ]  This would involve recombination between short sequences found at their termini and known as 59 base elements (59-be)—which may not be 59 bases long. The 59-be are a diverse family of sequences that function as recognition sites for the site-specific integrase (enzyme responsible for integrating the gene cassette into an integron) that occur downstream from the gene coding sequence. [ 5 ]


The ability of genetic elements like gene cassettes to excise and insert into genomes results in highly similar gene regions appearing in distantly related organisms. The three classes of integrons are similar in structure and are identified by where the insertions occur and what systems they coincide with. Class 1 integrons are seen in a diverse group of bacterial genomes and likely are all descendant from one common ancestor. The prevalence of the integron has shaped bacterial evolution by allowing rapid transfer of genes that are novel to an organism, such as antibiotic resistance genes. [ 6 ]


In  genetic engineering , a gene cassette is a manipulable fragment of DNA carrying, and capable of expressing, one or more genes of interest between one or more sets of  restriction sites . It can be transferred from one DNA sequence (usually on a  vector ) to another by 'cutting' the fragment out using  restriction enzymes  and 'pasting' it back into the new context. The vectors containing the gene of interest typically also carry an antibiotic resistance gene called a  selectable marker  to easily identify cells that have successfully integrated the vector into their genome.


To introduce a vector into a target cell, a state of  competence  must be inferred on the cell. This state is induced in the lab by incubating cells with calcium chloride before a brief heat shock, or by  electroporation . This makes the cells more susceptible to the plasmid that is being inserted. Once the plasmid has been added, the cells are grown in the presence of an antibiotic to confirm the uptake and expression of the new genetic elements.


The usage of CRISPR/Cas9 systems has shown success in inserting genes into eukaryotic genomes. [ 7 ]  While CRISPR modification is still in its infancy, there is significant evidence for usage in combination with other techniques to produce high throughput (HTP) genome editing systems. [ 8 ]  Genetic engineering of bacteria for production of a variety of industrial products, including biofuels and specialty chemicals/nutraceuticals is a major area of research. [ 9 ]


Horizontal gene transfer (HGT) is the transfer of genetic elements between cells other than parental inheritance. HGT is responsible for much of the spread of antibiotic resistance among bacteria. [ 10 ]  Gene cassettes containing antibiotic resistance genes, or other virulence factors such as exotoxins, can be transferred from cell to cell via phage,  transduction , taken up from the environment,  transformation , [ 11 ]  or by bacterial conjugation. [ 12 ]  The ability to transfer gene cassettes between organisms has played a large role in the evolution of prokaryotes. Many commensal organisms, such as  E. coli , regularly harbor one or more gene cassettes that convey antibiotic resistance. [ 13 ]  Horizontal transfer of genetic elements from non-pathogenic commensals to unrelated species results in highly virulent pathogens that can carry  multiple antibiotic resistance  genes. The increasing prevalence of resistance creates challenging questions for researchers and physicians.


Gene mapping  or  genome mapping  describes the methods used to identify the  location  of a  gene  on a  chromosome  and the distances between genes. [ 2 ] [ 3 ]   Gene mapping can also describe the distances between different sites within a gene.


The essence of all  genome  mapping is to place a collection of  molecular markers  onto their respective positions on the genome. Molecular markers come in all forms. Genes can be viewed as one special type of genetic markers in the construction of genome maps, and mapped the same way as any other markers. In some areas of study, gene mapping contributes to the creation of new recombinants within an organism. [ 4 ]


Gene maps  help describe the spatial arrangement of genes on a  chromosome . Genes are designated to a specific location on a chromosome known as the  locus  and can be used as  molecular markers  to find the distance between other genes on a chromosome. Maps provide researchers with the opportunity to predict the inheritance patterns of specific traits, which can eventually lead to a better understanding of disease-linked traits. [ 5 ]


The genetic basis to gene maps is to provide an outline that can potentially help researchers carry out  DNA sequencing . A gene map helps point out the relative positions of genes and allows researchers to locate regions of interest in the  genome . Genes can then be identified quickly and  sequenced  quickly. [ 6 ]


Two approaches to generating gene maps ( gene mapping ) include physical mapping and genetic mapping. Physical mapping utilizes molecular biology techniques to inspect chromosomes. These techniques consequently allow researchers to observe chromosomes directly so that a map may be constructed with relative gene positions. Genetic mapping on the other hand uses genetic techniques to indirectly find association between genes. Techniques can include cross-breeding ( hybrid ) experiments and examining  pedigrees . These technique allow for maps to be constructed so that relative positions of genes and other important sequences can be analyzed. [ 6 ]


There are two distinctive mapping approaches used in the field of genome mapping: genetic maps (also known as linkage maps) [ 7 ]  and physical maps. [ 3 ]  While both maps are a collection of  genetic markers  and gene  loci , [ 8 ]  genetic maps' distances are based on the  genetic linkage  information, while physical maps use actual physical distances usually measured in number of  base pairs . While the physical map could be a more accurate representation of the genome, genetic maps often offer insights into the nature of different regions of the chromosome, for example the genetic distance to physical distance ratio varies greatly at different genomic regions which reflects different recombination rates, and such rate is often indicative of euchromatic (usually gene-rich) vs heterochromatic (usually gene-poor) regions of the genome. [ citation needed ]


Researchers begin a genetic map by collecting samples of blood, saliva, or tissue from family members that carry a prominent disease or trait and family members that do not. The most common sample used in gene mapping, especially in personal genomic tests is saliva. Scientists then isolate DNA from the samples and closely examine it, looking for unique patterns in the DNA of the family members who do carry the disease and the DNA of those who do not carry the disease do not have. These unique molecular patterns in the DNA are referred to as polymorphisms, or markers. [ 9 ]


The first steps of building a genetic map are the development of  genetic markers  and a mapping population. The closer two markers are on the chromosome, the more likely they are to be passed on to the next generation together. Therefore, the "co-segregation" patterns of all markers can be used to reconstruct their order. With this in mind, the genotypes of each genetic marker are recorded for both parents and each individual in the following generations. The quality of the genetic maps is largely dependent upon these factors: the number of genetic markers on the map and the size of the mapping population. The two factors are interlinked, as a larger mapping population could increase the "resolution" of the map and prevent the map from being "saturated". [ citation needed ]


In gene mapping, any sequence feature that can be faithfully distinguished from the two parents can be used as a genetic marker. Genes, in this regard, are represented by "traits" that can be faithfully distinguished between two parents. Their linkage with other genetic markers is calculated in the same way as if they are common markers and the actual gene loci are then bracketed in a region between the two nearest neighboring markers. The entire process is then repeated by looking at more markers that target that region to map the gene neighborhood to a higher resolution until a specific causative locus can be identified. This process is often referred to as " positional cloning ", and it is used extensively in the study of plant species. One plant species, in particular in which positional cloning is utilized is in  maize . [ 10 ]  The great advantage of genetic mapping is that it can identify the relative position of genes based solely on their phenotypic effect. [ citation needed ]


Genetic mapping is a way to identify exactly which chromosome has which gene and exactly pinpointing where that gene lies on that particular chromosome. Mapping also acts as a method in determining which gene is most likely to recombine based on the distance between two genes. The distance between two genes is measured in units known as centimorgan or map units, these terms are interchangeable. A centimorgan is a distance between genes for which one product of meiosis in one hundred is recombinant. [ 11 ] [ 4 ]  The farther two genes are from each other, the more likely they are going to recombine. If it were closer, the opposite would occur. [ 12 ]


The basis to  linkage analysis  is understanding chromosomal location and identifying disease genes. Certain genes that are  genetically linked  or associated with each other reside close to each other on the same chromosome. During  meiosis , these genes are capable of being inherited together and can be used as a  genetic marker  to help identify the  phenotype  of diseases. Because linkage analysis can identify inheritance patterns, these studies are usually family based. [ 13 ]


The earliest gene maps were done by linkage analysis of fruitflies, in the research group around  Thomas Hunt Morgan . The first was published in 1913. [ 15 ]


Gene association analysis is population based; it is not focused on inheritance patterns, but rather is based on the entire history of a population. Gene association analysis looks at a particular population and tries to identify whether the frequency of an  allele  in affected individuals is different from that of a control set of unaffected individuals of the same population. This method is particularly useful to identify complex diseases that do not have a  Mendelian inheritance  pattern. [ 16 ]


Since actual base-pair distances are generally hard or not possible to directly measure, physical maps are actually constructed by first shattering the genome into hierarchically smaller pieces. By characterizing each single piece and assembling back together, the overlapping path or "tiling path" of these small fragments would allow researchers to infer physical distances between genomic features. [ citation needed ]


Restriction mapping  is a method in which structural information regarding a segment of  DNA  is obtained using  restriction enzymes . Restriction enzymes are  enzymes  that help cut segments of DNA at specific recognition sequences. The basis to restriction mapping involves digesting (or cutting) DNA with restriction enzymes. The digested DNA fragments are then run on an agarose gel using  electrophoresis , which provides one with information regarding the size of these digested fragments. The sizes of these fragments help indicate the distance between restriction enzyme sites on the DNA analyzed, and provides researchers with information regarding the structure of DNA analyzed. [ 16 ]  The resulting pattern of DNA migration – its  genetic fingerprint  is used to identify what stretch of DNA is in the  clone . By analyzing the fingerprints,  contigs  are assembled by automated (FPC) or manual means (pathfinders) into overlapping DNA stretches. Now a good choice of clones can be made to efficiently sequence the clones to determine the  DNA sequence  of the organism under study. [ citation needed ]


In physical mapping, there are no direct ways of marking up a specific gene since the mapping does not include any information that concerns traits and functions. Genetic markers can be linked to a physical map by processes like  in situ hybridization . By this approach, physical map contigs can be "anchored" onto a genetic map. The clones used in the physical map contigs can then be sequenced on a local scale to help new genetic marker design and identification of the causative loci. [ citation needed ]


Macrorestriction is a type of physical mapping wherein the high molecular weight DNA is digested with a restriction enzyme having a low number of restriction sites. [ citation needed ]


There are alternative ways to determine how  DNA  in a group of clones overlaps without completely sequencing the clones. Once the map is determined, the clones can be used as a resource to efficiently contain large stretches of the genome. This type of mapping is more accurate than genetic maps. [ citation needed ]


Restriction mapping  is a method in which structural information regarding a segment of  DNA  is obtained using  restriction enzymes . Restriction enzymes are  enzymes  that help cut segments of DNA at specific recognition sequences. The basis to restriction mapping involves digesting (or cutting) DNA with restriction enzymes. The digested DNA fragments are then run on an agarose gel using  electrophoresis , which provides one with information regarding the size of these digested fragments. The sizes of these fragments help indicate the distance between restriction enzyme sites on the DNA analyzed, and provides researchers with information regarding the structure of DNA analyzed. [ 16 ]


Fluorescence in situ hybridization  (FISH) is a method used to detect the presence (or absence) of a DNA sequence within a cell. [ 17 ]  DNA probes that are specific for chromosomal regions or genes of interest are labeled with  fluorochromes . By attaching fluorochromes to probes, researchers are able to visualize multiple DNA sequences simultaneously. When a probe comes into contact with DNA on a specific chromosome, hybridization will occur. Consequently, information regarding the location of that sequence of DNA will be attained. FISH analyzes single stranded DNA ( ssDNA ). Once the DNA is in its single stranded state, the DNA can bind to its specific probe. [ 6 ]


A  sequence-tagged site  (STS) is a short sequence of DNA (about 100 - 500  base pairs  in length) that is seen to appear multiple times within an individual's genome. These sites are easily recognizable, usually appearing at least once in the DNA being analyzed. These sites usually contain genetic  polymorphisms  making them sources of viable genetic markers (as they differ from other sequences). Sequenced tagged sites can be mapped within our genome and require a group of overlapping DNA fragments.  PCR  is generally used to produce the collection of DNA fragments. After overlapping fragments are created, the  map distance  between STSs can be analyzed. In order to calculate the map distance between STSs, researchers determine the frequency at which breaks between the two markers occur (see  shotgun sequencing ) [ 16 ]


In the early 1950s the prevailing view was that the genes in a  chromosome  are discrete entities, indivisible by  genetic recombination  and arranged like beads on a string.  During 1955 to 1959, Benzer performed  genetic recombination  experiments using  rII  mutants of  bacteriophage T4 .  He found that, on the basis of recombination tests, the sites of  mutation  could be mapped in a linear order. [ 18 ] [ 19 ]  This result provided evidence for the key idea that the gene has a linear structure equivalent to a length of  DNA  with many sites that can independently mutate. [ citation needed ]


In 1961, Francis Crick, Leslie Barnett, Sydney Brenner and Richard Watts-Tobin performed genetic experiments that demonstrated the basic nature of the  genetic code  for proteins. [ 20 ]  These experiments, involving mapping of mutational sites within the rIIB gene of bacteriophage T4, demonstrated that three sequential  nucleobases  of the gene's DNA specify each successive amino acid of its encoded protein. Thus the genetic code was shown to be a triplet code, where each triplet (called a codon) specifies a particular amino acid. They also obtained evidence that the codons do not overlap with each other in the DNA sequence encoding a protein, and that such a sequence is read from a fixed starting point. [ citation needed ]


Edgar et al. [ 21 ]  performed mapping experiments with r mutants of bacteriophage T4 showing that recombination frequencies between rII mutants are not strictly additive. The recombination frequency from a cross of two rII mutants (a x d) is usually less than the sum of recombination frequencies for adjacent internal sub-intervals (a x b) + (b x c) + (c x d). Although not strictly additive, a systematic relationship was demonstrated [ 22 ]  that likely reflects the underlying molecular mechanism of  genetic recombination .


Genome sequencing  is sometimes mistakenly referred to as "genome mapping" by non-biologists. The process of  shotgun sequencing [ 23 ]  resembles the process of physical mapping: it shatters the genome into small fragments, characterizes each fragment, then puts them back together (more recent sequencing technologies are drastically different). While the scope, purpose and process are totally different, a genome assembly can be viewed as the "ultimate" form of physical map, in that it provides in a much better way all the information that a traditional physical map can offer. [ citation needed ]


Identification of genes is usually the first step in understanding a genome of a species; mapping of the gene is usually the first step of identification of the gene. Gene mapping is usually the starting point of many important downstream studies.


The process to identify a genetic element that is responsible for a  disease  is also referred to as "mapping". If the locus in which the search is performed is already considerably constrained, the search is called the  fine mapping  of a gene. This information is derived from the investigation of disease manifestations in large families ( genetic linkage ) or from populations-based  genetic association  studies.


Using the methods mentioned above, researchers are capable of mapping disease genes. Generating a gene map is the critical first step towards identifying disease genes. Gene maps allow for variant alleles to be identified and allow for researchers to make predictions about the genes they think are causing the  mutant  phenotype. An example of a disorder that was identified by Linkage analysis is  Cystic Fibrosis . For example, with Cystic Fibrosis (CF), DNA samples from fifty families affected by CF were analyzed using linkage analysis. Hundreds of markers pertaining to CF were analyzed throughout the genome until CF was identified on the  long arm  of chromosome 7. Researchers then had completed linkage analysis on additional DNA markers within chromosome 7 to identify an even more precise location of the CF gene. They found that the CF gene resides around 7q31-q32 (see  chromosomal nomenclature ). [ 16 ]


Genetic regulatory circuits  (also referred to as  transcriptional regulatory circuits ) is a concept that evolved from the  Operon  Model discovered by  François Jacob  and  Jacques Monod . [ 1 ]   [ 2 ] [ 3 ]  They are functional clusters of  genes  that impact each other's expression through inducible  transcription factors  and  cis-regulatory elements . [ 4 ] [ 5 ]


Genetic regulatory circuits are analogous in many ways to  electronic circuits  in how they use signal inputs and outputs to determine  gene regulation . [ 4 ] [ 5 ]  Like electronic circuits, their organization determines their efficiency, and this has been demonstrated in circuits working in series to have a greater sensitivity of gene regulation. [ 4 ] [ 6 ]  They also use inputs such as trans and cis sequence regulators of genes, and outputs such as  gene expression  level. [ 4 ] [ 5 ]  Depending on the type of circuit, they respond constantly to outside signals, such as sugars and hormone levels, that determine how the circuit will return to its fixed point or periodic equilibrium state. [ 7 ]  Genetic regulatory circuits also have an ability to be evolutionarily rewired without the loss of the original transcriptional output level. [ 8 ] [ 9 ]  This rewiring is defined by the change in regulatory-target gene interactions, while there is still conservation of regulatory factors and target genes. [ 8 ] [ 10 ]


These circuits can be modelled  in silico  to predict the dynamics of a genetic system. [ 8 ] [ 11 ]  Having constructed a  computational model  of the natural circuit of interest, one can use the model to make testable predictions about circuit performance. [ 12 ] [ 13 ]  When designing a synthetic circuit for a specific engineering task, a model is useful for identifying necessary connections and parameter operating regimes that give rise to a desired functional output. Similarly, when studying a natural circuit, one can use the model to identify the parts or parameter values necessary for a desired biological outcome. [ 12 ] [ 14 ]  In other words, computational modelling and experimental synthetic perturbations can be used to probe biological circuits. [ 12 ] [ 14 ]  However, the structure of the circuits have shown to not be a reliable indicator of the function that the regulatory circuit provides for the larger cellular regulatory network. [ 7 ]


Understanding of genetic regulatory circuits are key in the field of  synthetic biology , where disparate genetic elements are combined to produce novel  biological functions . [ 1 ] [ 12 ]  These biological gene circuits can be used synthetically to act as physical models for studying regulatory function. [ 15 ] [ 16 ]


By engineering genetic regulatory circuits, cells can be modified to take information from their environment, such as nutrient availability and developmental signals, and react in accordance to changes in their surroundings [ 17 ] [ 18 ]  . [ 19 ] [ 20 ]  In plant synthetic biology, genetic regulatory circuits can be used to program traits to increase crop plant efficiency by increasing their robustness to environmental stressors. [ 18 ] [ 21 ]  Additionally, they are used to produce  biopharmaceuticals  for medical intervention. [ 18 ] [ 21 ]


A  gene signature  or  gene expression signature  is a single or combined group of genes in a cell with a uniquely characteristic pattern of gene expression [ 1 ]  that occurs as a result of an altered or unaltered biological process or pathogenic medical condition. [ 2 ]  This is not to be confused with the concept of  gene expression profiling . Activating pathways in a regular physiological process or a physiological response to a  stimulus  results in a cascade of  signal transduction  and interactions that elicit altered levels of gene expression, which is classified as the gene signature of that physiological process or response. [ 3 ]  The clinical applications of gene signatures breakdown into prognostic, diagnostic [ 4 ] [ 5 ]  and predictive signatures. The phenotypes that may theoretically be defined by a gene expression signature range from those that predict the survival or prognosis of an individual with a disease, those that are used to differentiate between different subtypes of a disease, to those that predict activation of a particular  pathway . Ideally, gene signatures can be used to select a group of patients [ 6 ]  for whom a particular treatment will be effective. [ 7 ] [ 8 ]


In 1995, 2 studies conducted identified unique approaches to analyzing global gene expression of a genome which collectively promoted the value of identifying and analyzing gene signatures for physiological relevance. The first study reports a technique that improves  expressed sequence tag (EST)  analysis, known as  Serial Analysis of Gene Expression  (SAGE) that hinged on sequencing and quantifying mRNA samples which acquired levels of gene expression that eventually revealed characteristic gene expression patterns. [ 9 ]


The second study identified a technique that is now widely known as the  microarray  which quantifies complementary DNA (cDNA) hybridization on a glass slide to analyze the expression of many genes in parallel. [ 10 ]  These studies drew greater attention to the wealth of information that analysis of gene signatures bear that may or may not be physiologically relevant.


Pressing forward, the latter technique has revolutionized research in genetics and DNA chip technology [ 11 ]  as it is a widely adopted technique to profile gene expression signatures such that these physiological responses can be cataloged [ 12 ]  in repositories such as  NCBI Gene Expression Omnibus . This catalogue of prognostic, diagnostic and predictive gene expression signatures allow for predictions of onset of pathogenic diseases in patients, [ 13 ]  tumour and cancer classification, [ 14 ]  and enhanced therapeutic strategies that predict the optimal target candidates subjects and genes. [ 15 ]


Today, microarrays and other quantitative methods such as  RNA-seq  that encompass  gene expression profiling , are moving towards promotion of re-analysis and integration of the large, publicly available database of gene expression signatures and profiles to uncover the full threshold of information these expression signatures hold. [ 16 ]


Prognostic refers to predicting the likely outcome or course of a disease. Classifying a biological phenotype or medical condition based on a specific gene signature or multiple gene signatures, can serve as a prognostic biomarker for the associated phenotype or condition. This concept termed prognostic gene signature ,  serves to offer insight into the overall outcome of the condition regardless of therapeutic intervention. [ 17 ]  Several studies have been conducted with focus on identifying prognostic gene signatures with the hopes of improving the diagnostic methods and therapeutic courses adopted in a clinical settings. It is important to note that prognostic gene signatures are not a target of therapy; they offer additional information to consider when discussing details such as duration or dosage or drug sensitivity etc. in therapeutic intervention. The criteria a gene signature must meet to be deemed a prognostic marker include demonstration of its association with the outcomes of the condition, reproducibility and validation of its association in an independent group of patients and lastly, the prognostic value must demonstrate independence from other standard factors in a multivariate analysis. [ 3 ]  The applications of these prognostic signatures include prognostic assays for  breast cancer , [ 18 ] [ 19 ]   hepatocellular carcinoma , [ 20 ]   leukaemia [ 21 ]  and are continually being developed for other types of cancers and disorders as well.


A diagnostic gene signature serves as a  biomarker  that distinguishes phenotypically similar medical conditions that have a threshold of severity consisting of mild, moderate or severe phenotypes. [ 5 ]  Establishing verified methods of diagnosing clinically indolent and significant cases allows practitioners to provide more accurate care and therapeutic options that range from no therapy, preventative care to symptomatic relief. These diagnostic signatures also allow for a more accurate representation of test samples used in research. [ 6 ]  Similar to the procedure of validation of prognostic gene signature, a criterion exists for classifying a gene signature as a biomarker for a disorder or diseases outlined by Chau et al. [ 22 ] [ 23 ]


A predictive gene signature is similar to a predictive biomarker, where it predicts the effect of treatment in patients or study participants that exhibit a particular disease phenotype. A predictive gene signature unlike a prognostic gene signature can be a target for therapy. [ 17 ]   The information predictive signatures provide are more rigorous than that of prognostic signatures as they are based on treatment groups with therapeutic intervention on the likely benefit from treatment, completely independent of prognosis. [ 24 ]  Predictive gene signatures addresses the paramount need for ways to personalize and tailor therapeutic intervention in diseases. These signatures have implications in facilitating  personalized medicine  through identification of more novel therapeutic targets and identifying the most qualified subjects for optimal benefit of specific treatments. [ 3 ] [ 25 ] [ 26 ]


In  bioethics  and  law ,  gene theft  or  DNA theft  is the act of acquiring the  genetic material  of another individual, usually from  public places , without his or her permission. The  DNA  may be harvested from a wide variety of common objects such as discarded cigarettes, used condoms, coffee cups, and hairbrushes. In addition, a variety of people can be interested on collecting someone's genetic material. This includes the police, political parties, historians, professional sports teams, personal enemies, etc. [ 1 ]  DNA contains adequate amount of information about someone and it can be used for many purposes such as establishing  paternity , proving  genealogical connections  or even unmasking  private medical  conditions. [ 2 ]


Currently, there are not many laws pertaining to the punishment that one may receive from obtaining the genetic material of others without their consent. However, due to the  Health Insurance Portability and Accountability Act (HIPAA) , one's genetic material cannot be given to his or her school or employer as the genome is a part of one's personal health data, but, law enforcement can have access to it without consent. This only occurs when a person is either a victim or a suspect of a criminal investigation. [ 3 ]


Great Britain  criminalized the acquisition of DNA without  consent  in 2006 at the urging of the  Human Genetics Commission . [ 4 ] [ 5 ]   Australia's  legislature debated a two-year jail sentence for such theft in 2008. [ 6 ] [ 7 ]  In the United States, eight states currently have criminal or civil prohibitions on such  non-consensual  appropriation of genetic materials. [ 8 ]  In  Alaska ,  Florida ,  New Jersey ,  New York  and  Oregon , individuals caught swiping DNA face fines or short jail sentences. [ 8 ]   Lawsuits against "gene snatchers" are permitted in  Minnesota ,  New Hampshire  and  New Mexico . [ 8 ]   In jurisdictions where such non-consensual taking of DNA is illegal, exceptions are generally made for law enforcement.


Many  bioethicists  believe that such conduct is an unethical invasion of  human privacy . [ 8 ]  Professor  Jacob Appel  has warned that criminals may acquire the capability to copy DNA of innocent people and deposit it at crimes scenes, endangering the blameless and undermining a key tool of forensic investigation." [ 8 ]  In addition, there have been ethical concerns on law enforcement using the DNA of the family members of criminals to catch them. This concept was used for the  Golden State Killer  case in  California , who was connected to at least 50 rapes and 12 murders between 1976 and 1986. After the case went cold, investigators used a website that compared the genetic information of those who had uploaded their information and found a relative of the killer. [ 9 ]


However, others defend the appropriation of genetic material on the grounds that doing so may further human knowledge in productive ways. [ 2 ]  One particularly controversial case which received widespread attention in the media was that of Derrell Teat, a wastewater coordinator, who sought to acquire without consent the DNA of a man who was allegedly the last male descendant of her great-great-great-grandfather's brother. [ 2 ] [ 10 ]  Another prominent case was a United States paternity suit involving film producer  Steve Bing  and billionaire investor  Kirk Kerkorian . [ 11 ]


Gene transfer agent s ( GTAs ) are DNA-containing  virus -like particles that are produced by some  bacteria  and  archaea  and mediate  horizontal gene transfer . Different GTA types have originated independently from viruses in several bacterial and archaeal lineages. These cells produce GTA particles containing short segments of the  DNA  present in the cell. After the particles are released from the producer cell, they can attach to related cells and inject their DNA into the cytoplasm.  The DNA can then become part of the recipient cells' genome. [ 1 ] [ 2 ] [ 3 ] [ 4 ]


GTAs are classified as  viriforms  in the  ICTV taxonomy . Among the GTAs mentioned by the article, RcGTA and DsGTA are now in the family  Rhodogtaviriformidae , BaGTA in  Bartogtaviriformidae , and VSH-1 in  Brachygtaviriformidae . [ 5 ]  Dd1 and VTA do not yet have a classification.


The first GTA system was discovered in 1974, when mixed cultures of  Rhodobacter capsulatus  strains produced a high frequency of cells with new combinations of genes. [ 6 ]  The factor responsible was distinct from known gene-transfer mechanisms in being independent of cell contact, insensitive to deoxyribonuclease, and not associated with phage production.  Because of its presumed function it was named gene transfer agent (GTA, now RcGTA)   More recently other gene transfer agent systems have been discovered by incubating filtered (cell-free) culture medium with a genetically distinct strain. [ 3 ]


The genes specifying GTAs are derived from  bacteriophage  (phage) DNA that has integrated into a host chromosome. Such  prophages  often acquire mutations that make them defective and unable to produce phage particles.  Many bacterial genomes contain one or more defective prophages that have undergone more-or less-extensive mutation and deletion. Gene transfer agents, like defective prophages, arise by mutation of prophages, but they retain functional genes for the head and tail components of the phage particle (structural genes) and the genes for DNA packaging. The phage genes specifying its regulation and DNA replication have typically been deleted, and expression of the cluster of structural genes is under the control of cellular regulatory systems. Additional genes that contribute to GTA production or uptake are usually present at other chromosome locations.  Some of these have regulatory functions, and others contribute directly to GTA production ( e.g.  the phage-derived lysis genes) or uptake and recombination ( e.g.  production of cell-surface capsule and DNA transport proteins)  These GTA-associated genes are often under coordinated regulation with the main GTA gene cluster. [ 7 ]   Phage-derived cell-lysis proteins (holin and endolysin) then weaken the cell wall and membrane, allowing the cell to burst and release the GTA particles. The number of GTA particles produced by each cell is not known.


Some GTA systems appear to be recent additions to their host genomes, but others have been maintained for many millions of years.  Where studies of sequence divergence have been done (dN/dS analysis), they indicate that the genes are being maintained by natural selection for protein function (i.e. defective versions are being eliminated). [ 8 ] [ 9 ]


However, the nature of this selection is not clear. Although the discoverers of GTA assumed that gene transfer was the function of the particles, the presumed benefits of gene transfer come at a substantial cost to the population. Most of this cost arises because GTA-producing cells must lyse (burst open) to release their GTA particles, but there are also genetic costs associated with making new combinations of genes because most new combinations will usually be less fit than the original combination. [ 10 ]    One alternative explanation is that GTA genes persist because GTAs are genetic parasites that spread infectiously to new cells. However this is ruled out because GTA particles are typically too small to contain the genes that encode them. For example, the main RcGTA cluster (see below) is 14 kb long, but RcGTA particles can contain only 4–5 kb of DNA.


Most bacteria have not been screened for the presence of GTAs, and many more GTA systems may await discovery. Although DNA-based surveys for GTA-related genes have found homologs in many genomes, but interpretation is hindered by the difficulty of distinguishing genes that encode GTAs from ordinary prophage genes. [ 8 ]   [ 9 ]


In laboratory cultures, production of GTAs is typically maximized by particular growth conditions that induce transcription of the GTA genes; most GTAs are not induced by the DNA-damaging treatments that induce many prophages.  Even under maximally inducing conditions only a small fraction of the culture produces GTAs, typically less than 1%. [ 11 ] [ 12 ]


The steps in GTA production are derived from those of phage infection. The structural genes are first transcribed and translated, and the proteins assembled into empty heads and unattached tails.  The DNA packaging machinery then packs DNA into each head, cutting the DNA when the head is full, attaching a tail to the head, and then moving the newly-created DNA end on to a new empty head. Unlike prophage genes, the genes encoding GTAs are not excised from the genome and replicated for packaging in GTA particles.  The two best studied GTAs (RcGTA and BaGTA) randomly package all of the DNA in the cell, with no overrepresentation of GTA-encoding genes. [ 11 ] [ 13 ]  The number of GTA particles produced by each cell is not known.


Whether release of GTA particles leads to transfer of DNA to new genomes depends on several factors.   First, the particles must survive in the environment – little is known about this, although particles are reported to be quite unstable under laboratory conditions. [ 14 ]  Second, particles must encounter and attach to suitable recipient cells, usually members of the same or a closely related species.  Like phages, GTAs attach to specific protein or carbohydrate structures on the recipient cell surface before injecting their DNA.  Unlike phage, the well-studied GTAs appear to inject their DNA only across the first of the two membranes surrounding the recipient cytoplasm, and they use a different system,  competence -derived rather than phage-derived, to transport one strand of the double-stranded DNA across the inner membrane into the cytoplasm. [ 15 ] [ 16 ]


If the cell's recombinational repair machinery finds a chromosomal sequence very similar to the incoming DNA, it replaces the former with the latter by homologous recombination, mediated by the cell's  RecA  protein. If the sequences are not identical this will produce a cell with a new genetic combination.  However, if the incoming DNA is not closely related to DNA sequences in the cell it will be degraded, and the cell will reuse its nucleotides for DNA replication.


The GTA produced by the  alphaproteobacterium   Rhodobacter capsulatus , named  R. capsulatus  GTA (RcGTA), is currently the best studied GTA. When laboratory cultures of  R. capsulatus  enter stationary phase, a subset of the bacterial population induces production of RcGTA, and the particles are subsequently released from the cells through  cell lysis . [ 12 ]   Most of the RcGTA structural genes are encoded in a ~ 15 kb genetic cluster on the bacterial chromosome. However, other genes required for RcGTA function, such as the genes required for cell lysis, are located separately. [ 2 ] [ 17 ]   RcGTA particles contain 4.5 kb DNA fragments, with even representation of the whole chromosome except for a 2-fold dip at the site of the RcGTA gene cluster.


Regulation of GTA production and transduction has been best studied in  R. capsulatus , where a quorum-sensing system and a CtrA-phosphorelay control expression of not only the main RcGTA gene cluster, but also a holin/endolysin cell lysis system, particle head spikes, an attachment protein (possibly tail fibers), and the capsule and DNA processing genes needed for RcGTA recipient function. An uncharacterized stochastic process further limits expression of the gene cluster is  to only 0.1-3% of the cells.


RcGTA-like clusters are found in a large subclade of the alphaproteobacteria, although the genes also appear to be frequently lost by deletion.  Recently, several members of the order  Rhodobacterales  have been demonstrated to produce functional RcGTA-like particles. Groups of genes with  homology  to the RcGTA are present in the chromosomes of various types of alphaproteobacteria. [ 8 ]


D. shibae , like  R. capsulatus , is a member of the Order Rhodobacterales, and its GTA shares a common ancestor and many features with RcGTA, including gene organization, packaging of short DNA fragments (4.2 kb) and regulation by quorum sensing and a CtrA phosphorelay. [ 18 ]   However, its DNA packaging machinery has much more specificity, with sharp peaks and valleys of coverage suggestion that it may preferentially initiate packaging at specific sites in the genome.  The DNA of the major DsGTA gene cluster is packaged very poorly.


Bartonella  species are members of the Alphaproteobacteria like  R. capsulatus  and  D. shibae , but  BaGTA  is not related to RcGTA and DsGTA. [ 19 ]   BaGTA particles are larger than RcGTA and contain 14 kb DNA fragments.  Although this capacity could in principle allow BaGTA to package and transmit its 14 kb GTA cluster, measurements of DNA coverage show reduced coverage of the cluster. An adjacent region of high coverage is thought to be due to local DNA replication. [ 13 ]


Brachyspira  is a genus of spirochete; several species have been shown to carry homologous GTA gene clusters.  Particles contain 7.5 kb DNA fragments.  Production of VSH-1 is stimulated by the DNA-damaging agent mitomycin C and by some antibiotics.  It is also associated with detectable cell lysis, indicating that a substantial fraction of the culture may be producing VSH-1. [ 20 ]


D. desulfuricans  is a soil bacterium in the deltaproteobacteria; Dd1 packages 13.6 kb of DNA fragments. It is unclear which genes encode for this GTA: there is one 17.8 kb area with phage-like structural genes in the bacterial genome, but their link to GTA production is not yet experimentally proven. [ 21 ]


M. voltae  is an archaean; its GTA is known to transfer 4.4 kb DNA fragments but has not been otherwise characterized, [ 22 ]  although a defective provirus related to  Methanococcus  head-tailed viruses ( Caudoviricetes ) in  M. voltae  A3 genome has been suggested to represent the GTA locus. [ 23 ]  A possible  terL  terminase ( D7DSG2 ) was again identified in 2019. [ 24 ]


Gene trapping  is a  high-throughput  approach that is used to introduce insertional  mutations  across an organism's genome.


Trapping is performed with gene trap vectors whose principal element is a gene trapping cassette consisting of a promoterless reporter gene and/or selectable  genetic marker , flanked by an upstream 3' splice site (splice acceptor; SA) and a downstream  transcriptional  termination sequence ( polyadenylation  sequence; polyA).


When inserted into an  intron  of an expressed gene, the gene trap cassette is transcribed from the endogenous promoter of that gene in the form of a fusion transcript in which the exon(s) upstream of the insertion site is spliced in frame to the reporter/selectable marker gene. Since transcription is terminated prematurely at the inserted polyadenylation site, the processed fusion transcript encodes a truncated and nonfunctional version of the cellular  protein  and the reporter/selectable marker. Thus, gene traps simultaneously inactivate and report the expression of the trapped gene at the insertion site, and provide a  DNA  tag (gene trap sequence tag, GTST) for the rapid identification of the disrupted  gene . [ 1 ] [ 2 ]


The International Gene Trap Consortium is centralizing the data and supplies modified cell lines. [ 3 ]


The  Generation of Animals  (or  On the Generation of Animals ;  Greek :  Περὶ ζῴων γενέσεως  ( Peri Zoion Geneseos );  Latin :  De Generatione Animalium ) is one of the biological works of the  Corpus Aristotelicum , the collection of texts traditionally attributed to  Aristotle  (384–322 BC). The work provides an account of animal  reproduction ,  gestation  and  heredity .