Like the  TOC translocon , the TIC translocon has a large core  complex  surrounded by some loosely associated peripheral proteins like  Tic110 ,  Tic40 , and  Tic21 . [ 54 ]
The core complex weighs about one million  daltons  and contains  Tic214 ,  Tic100 ,  Tic56 , and  Tic20 I , possibly three of each. [ 54 ]


Tic20  is an  integral  protein thought to have four  transmembrane   α-helices . [ 43 ]  It is found in the 1 million  dalton  TIC complex. [ 54 ]  Because it is similar to  bacterial   amino acid  transporters and the  mitochondrial  import protein  Tim17 [ 43 ]  ( t ranslocase  on the  i nner  m itochondrial  m embrane ), [ 55 ]  it has been proposed to be part of the TIC import channel. [ 43 ]  There is no  in vitro  evidence for this though. [ 43 ]  In  Arabidopsis thaliana , it is known that for about every five  Toc75  proteins in the outer chloroplast membrane, there are two  Tic20 I  proteins (the main  form  of Tic20 in  Arabidopsis ) in the inner chloroplast membrane. [ 54 ]


Unlike  Tic214 ,  Tic100 , or  Tic56 , Tic20 has  homologous  relatives in  cyanobacteria  and nearly all chloroplast lineages, suggesting it evolved before the first chloroplast endosymbiosis.  Tic214 ,  Tic100 , and  Tic56  are unique to  chloroplastidan  chloroplasts, suggesting that they evolved later. [ 54 ]


Tic214  is another TIC core complex protein, named because it weighs just under 214  kilodaltons . It is 1786  amino acids  long and is thought to have six  transmembrane domains  on its  N-terminal  end. Tic214 is notable for being coded for by chloroplast DNA, more specifically the first  open reading frame   ycf1 . Tic214 and  Tic20  together probably make up the part of the one million  dalton  TIC complex that spans the  entire membrane . Tic20 is buried inside the complex while Tic214 is exposed on both sides of the  inner chloroplast membrane . [ 54 ]


Tic100  is a  nuclear encoded  protein that's 871  amino acids  long. The 871 amino acids collectively weigh slightly less than 100 thousand  daltons , and since the mature protein probably doesn't lose any amino acids when itself imported into the chloroplast (it has no  cleavable transit peptide ), it was named Tic100. Tic100 is found at the edges of the 1 million dalton complex on the side that faces the  chloroplast intermembrane space . [ 54 ]


Tic56  is also a  nuclear encoded  protein. The  preprotein  its gene encodes is 527 amino acids long, weighing close to 62 thousand  daltons ; the mature form probably undergoes processing that trims it down to something that weighs 56 thousand daltons when it gets imported into the chloroplast. Tic56 is largely embedded inside the 1 million dalton complex. [ 54 ]


Tic56 and  Tic100  are highly  conserved  among land plants, but they don't resemble any protein whose function is known. Neither has any  transmembrane domains . [ 54 ]


A  chromatid  (Greek  khrōmat-  'color' +  -id ) is one half of a duplicated  chromosome . Before replication, one chromosome is composed of one  DNA  molecule. In replication, the DNA molecule is copied, and the two molecules are known as chromatids. [ 1 ]  During the later stages of cell division these chromatids separate longitudinally to become individual chromosomes. [ 2 ]


Chromatid pairs are normally genetically identical, and said to be  homozygous . However, if mutations occur, they will present slight differences, in which case they are  heterozygous . The pairing of chromatids should not be confused with the  ploidy  of an organism, which is the number of  homologous versions  of a chromosome.


Chromatids may be sister or non-sister chromatids. A  sister chromatid  is either one of the two chromatids of the same  chromosome  joined together by a common  centromere . A pair of sister chromatids is called a dyad. Once sister chromatids have separated (during the  anaphase  of mitosis or the anaphase II of  meiosis  during  sexual reproduction ), they are again called chromosomes, each having the same genetic mass as one of the individual chromatids that made up its parent. The DNA sequence of two sister chromatids is completely identical (apart from very rare DNA copying errors).


Sister chromatid exchange  (SCE) is the exchange of genetic information between two  sister chromatids .  SCEs can occur during  mitosis  or  meiosis .  SCEs appear to primarily reflect DNA recombinational  repair  processes responding to  DNA damage  (see article  Sister chromatid exchange ).


Non-sister chromatids , on the other hand, refers to either of the two chromatids of paired  homologous chromosomes , that is, the pairing of a paternal chromosome and a maternal chromosome. In  chromosomal crossovers , non-sister (homologous) chromatids form  chiasmata  to exchange genetic material during the  prophase I  of  meiosis  (See  Homologous chromosome pair ).


Chromatin bridge  is a mitotic occurrence that forms when  telomeres  of sister chromatids fuse together and fail to completely segregate into their respective daughter cells. Because this event is most prevalent during  anaphase , the term  anaphase bridge  is often used as a substitute. After the formation of individual daughter cells, the DNA bridge connecting  homologous chromosomes  remains fixed. As the daughter cells exit  mitosis  and re-enter  interphase , the chromatin bridge becomes known as an interphase bridge. These phenomena are usually visualized using the laboratory techniques of  staining  and  fluorescence microscopy . [ 1 ] [ 2 ]


The faithful inheritance of genetic information from one cellular generation to the next heavily relies on the duplication of  deoxyribonucleic acid  (DNA), as well as the formation of two identical daughter cells. This complicated cellular process, known as mitosis, depends on a multitude of cellular checkpoints, signals, interactions and signal cascades for accurate and faithful functioning.  Cancer , characterized by uncontrollable cell growth mechanisms and high tendencies for proliferation and  metastasis , is highly prone to mitotic mistakes. As a result, several forms of chromosomal aberrations occur, including, but not limited to,  binucleated cells ,  multipolar spindles  and  micronuclei . [ 3 ]  Chromatin bridges may serve as a marker of cancer activity. [ citation needed ]


Chromatin bridges may form by any number of processes wherein chromosomes remain topologically entangled during mitosis. One way in which this may occur is the failure to resolve joint molecules formed during homologous recombination mediated DNA repair, a process that ensures that replicated chromosomes are intact before chromosomes are segregated during cell division. In particular, genetic studies have demonstrated that the loss of the enzymes BLM (Bloom's Syndrome Helicase) or FANCM each result in a dramatic increase in the number of chromatin bridges. This occurs because loss of these genes causes an increase in chromosome fusions, either in an end-to-end manner or through topological entrapment (e.g., catenation or unresolved DNA cross-links), have also been associated with chromatin bridge formation. When viewed under a fluorescence microscope and immunostained for cytological markers, these chromatin bridges appear to emanate from either centromeres, telomeres or DNA crosslinks (as marked by FANCD2). [ 4 ]


Chromatin bridges can be viewed utilizing a laboratory technique known as  fluorescence microscopy . Fluorescence is the process that involves excitation of a  fluorophore  (a molecule with the ability to emit fluorescent light in the visible light spectrum) using  ultraviolet light . After the fluorophore becomes chemically excited by the presence of UV light, it emits visible light at a specific wavelength, producing different colors. Fluorophores may be added as a molecular tag to different portions of a cell.  DAPI  is a fluorophore that specifically binds to DNA and fluoresces blue. In addition,  immunofluorescence  may be used as a laboratory technique to tag cells with specific fluorophores using  antibodies , immune proteins created by  B lymphocytes . Antibodies are utilized by the immune system in the identification and binding of foreign substances.  Tubulin  is a  monomer  of  microtubules  that compose the cellular  cytoskeleton . The antibody anti-tubulin specifically binds to these tubulin monomeric subunits. A fluorophore can be chemically attached to the anti-tubulin antibody, which then fluoresces green. Numerous antibodies may bind to microtubules in order to amplify the fluorescent signal. Fluorescence microscopy allows for the observation of different components of the cell against a dark background for high intensity and specificity. [ citation needed ]


Chromatin bridges are easiest and most readily visible when observing chromosomes stained with DAPI. DNA bridges appear to be a blue, "string-like" connection between two separated daughter cells. This effect is created when sticky ends of chromosomes remain connected to one another, even after mitosis. A chromatin bridge may also be observed using indirect immunofluorescence, in which anti-tubulin emits a green coloration when bound to microtubules in the presence of UV light. Because microtubules maintain the positions of the chromosomes during mitosis, they appear to be densely pinched between the two dividing, daughter cells. Chromatin bridges can be difficult to locate utilizing fluorescence microscopy, as this phenomenon is not incredibly abundant and tend to appear faint against the dark background. [ citation needed ]


Recently, chromatin bridges have been implied as a diagnostic marker for cancer, while having been linked to  tumorigenesis  in humans. [ 5 ]  This premise is based on the fact that as the mitotic cell divides and the daughter cells move further apart, stress on the DNA bridge leads to breakages in the chromosome at random points. As previously stated, the disruptions in the chromosome may lead to single chromosome  mutations , including  deletion ,  duplication  and  inversion , among others. This instability, defined as frequent changes in chromosomal structure and number, may be the basis of the development of cancer. While the frequency of chromatin bridges may be greater in tumor cells relative to normal cells, it may not be practical to utilize this phenomenon as a diagnostic tool. The process of staining and mounting sample cells using indirect immunofluorescence is time-consuming. Even though DAPI staining is quick, neither laboratory technique can guarantee the presence of the bridges under the fluorescence microscope. The rarity of chromatin bridges, even in cancerous cells, makes this phenomenon difficult to be widely accepted diagnostic marker for cancer. [ citation needed ]


A  chromomere , also known as an  idiomere , is one of the serially aligned beads or granules of a  eukaryotic   chromosome , resulting from local coiling of a continuous  DNA  thread. [ 1 ]  Chromomeres are regions of chromatin that have been compacted through  localized contraction.  In areas of  chromatin  with the absence of transcription, condensing of DNA and protein complexes will result in the formation of chromomeres. [ 2 ]  It is visible on a chromosome during the  prophase  of  meiosis  and  mitosis . Giant banded ( Polytene ) chromosomes resulting from the replication of the chromosomes and the synapsis of homologs without cell division is a process called endomitosis. These chromosomes consist of more than 1000 copies of the same chromatid that are aligned and produce alternating dark and light bands when stained. The dark bands are the chromomere.


It is unknown when chromomeres first appear on the  chromosome . Chromomeres can be observed best when chromosomes are highly condensed. [ 2 ]  The chromomeres are present during leptotene phase of prophase I during meiosis. During zygotene phase of prophase I, the chromomeres of homologs align with each other to form homologous rough pairing (homology searching). These chromomeres helps provide a unique identity for each homologous pairs. They appear as dense granules during  leptotene stage


There are more than 2000 chromomeres on 20 chromosomes of maize.


Chromomeres are organized in a discontinuous linear pattern along the condensed chromosomes (pachytene chromosomes) during the prophase stage of   meiosis . [ 2 ]  The linear pattern of chromomeres is linked to the arrangement of genes along the chromosome. [ 2 ] [ 3 ]  Chromomeres contain genes and sometimes clusters of genes within their structure. [ 3 ]  Aggregates of chromomeres are known as chromonemata. [ 4 ]


Cohesive proteins  SMC3  and hRAD21(plays a role in  sister chromatid   cohesion ) are found within chromomeres at high concentrations, and maintain the proper structure of chromomeres. The protein XCAP-D2 is also present at high concentrations within the chromomere, and acts as a  condensin  component. High concentrations of  tandem repeats  of the  heterochromatin  protein HP1β builds up within the chromomere. In regions where loops attach to chromomeres, there is hyper acetylation  of  histone  4. The extra acetylation loosens chromatin from a condensed form, making it more accessible to proteins involved in  transcription . [ 2 ]


Chromomeres are known as the structural subunit of a chromosome. The arrangement of chromomere structure can aid in control of gene expression. [ 3 ]


Maps of chromomeres can be made for use in genetic and evolutionary studies. Chromomeric maps can be used to locate the exact position of genes on a chromosome. Chromomeric maps can be used to analyze chromosome aberrations, and find correlations between the aberrations and their effects on genes near breakpoints. [ 3 ]


Chromomeres display different properties and behaviours when associated with  lampbrush  chromosomes. Although found all across the lampbrush chromosome, they are not organized in a clear pattern along as they are in normal pachytene chromosomes of meiosis. [ 2 ] [ 5 ]  The two sister chromatids of a lampbrush chromosome separate fully, forming lateral loops that extend from chromomeres, and act as transcription complexes. The lateral loops are areas where the chromosomes are transcriptionally active. [ 2 ] [ 5 ]  The loops define where the chromomere will be positioned. The chromomere is an organization of regions of non-transcribed chromatin. These regions of chromatin that have not been transcribed are located at the ends of the loops that were formed by the sister chromatids of a lampbrush chromosome. [ 2 ]  Each chromomere can have up to several pairs of loops from lampbrush chromosomes originating from it, as well as micro-loops that cannot be detected with a  light microscope . [ 6 ]


Chromomeres of lampbrush chromosomes act as structural units, and are not considered to be genetic units participating in transcription. The arrangement of a chromosome into chromomeric units happens almost simultaneously with the start of transcription. [ 2 ]  The loop structures extending from chromomeres are maintained by a high level of transcriptional activity and structural proteins of the chromosome. Chromatin within the chromomere are held in position by a variety of histone modifications and  epigenetic markers . [ 5 ]


Methyl-CpG-binding protein 2  (MeCP2) is a protein that binds to methylated DNA. MeCP2 has been found to associate most strongly with transcriptionally inactive chromomere domains. MeCP2 binding patterns to chromomere domains are proportional to the density of chromomeric DNA found on a chromosome. The binding of MeCP2 to chromomeric regions represents regions of chromatin that are highly dynamic on the lampbrush chromosome. [ 6 ]


Chromoplexy  refers to a class of complex DNA rearrangement observed in the genomes of cancer cells. This phenomenon was first identified in  prostate cancer  by  whole genome sequencing  of prostate tumors. [ 1 ] [ 2 ] [ 3 ]  Chromoplexy causes genetic material from one or more chromosomes to become scrambled as multiple strands of DNA are broken and ligated to each other in a new configuration. In prostate cancer, chromoplexy may cause multiple oncogenic events within a single cell cycle, providing a proliferative advantage to a (pre-)cancerous cell. Several oncogenic mutations in prostate cancer occur through chromoplexy, such as disruption of the tumor suppressor gene  PTEN  or creation of the  TMPRSS2-ERG   fusion gene . [ citation needed ]


Chromplexy was originally inferred by statistically analyzing the location of DNA breaks across the genome. [ 1 ]  Its prevalence across cancers is not known, because only a few types of tumors have been analyzed for chromoplexy in the published literature. However, it was detected in the majority of 57 prostate tumors analyzed, and has been reported in non-small cell lung cancers, melanoma and  head and neck cancers .  [ 1 ]  It has also been reported to generate the canonical gene fusion,  EWSR1-FLI1  and  EWSR1-ERG , in  Ewing sarcoma . [ 4 ]


Along with  chromothripsis , and break-fusion-bridge cycles, chromoplexy is an example of chromoanagenesis, [ 5 ]  a catch-all term for events that generate complex structural chromosomal abnormalities. [ 6 ]


The mechanism underlying complex rearrangements in chromoplexy has not been identified. A proposed model is that DNA is brought together in nuclear transcription hubs where genes across multiple chromosomes are co-regulated by transcription factors such as the  Androgen receptor . This DNA may then sustain multiple transient breaks during transcription [ 7 ]  and sets of broken DNA ends may be ligated to one another in an incorrect configuration. [ citation needed ]


This model has not been demonstrated experimentally. Its merit is that it may account for the fact that chromoplexy appears to cause DNA breaks in regions of the nucleus that are actively transcribed and correspond to open chromatin. It also may explain how DNA from multiple chromosomes may be involved in a single complex rearrangement due to the nuclear co-localization of genes from multiple chromosomes at transcription hubs. [ citation needed ]


Chromoplexy is similar to, but distinct from chromothripsis, a phenomenon whereby a single catastrophic event causes “shattering” of a chromosome. [ 8 ] [ 9 ]  The precise delineation between chromothripsis and chromoplexy is unclear, however general distinctions are [ 1 ] [ 3 ] [ 10 ]


Chromoplexy has been proposed as a means by which cancer genomes may undergo bursts of evolution by altering multiple cancer genes across the genome in a single “hit”. [ 1 ] [ 3 ]   For example, in at least one prostate tumor, a single chromoplectic event generated the TMPRSS2-ERG fusion while inactivating other tumor suppressor genes such as  SMAD4 . [ citation needed ]


An  inversion  is a  chromosome rearrangement  in which a segment of a chromosome becomes inverted within its original position. An inversion occurs when a  chromosome  undergoes a two breaks within the chromosomal arm, and the segment between the two breaks inserts itself in the opposite direction in the same chromosome arm. The breakpoints of inversions often happen in regions of repetitive nucleotides, and the regions may be reused in other inversions. [ 1 ]  Chromosomal segments in inversions can be as small as 1 kilobases or as large as 100 megabases. [ 2 ]  The number of genes captured by an inversion can range from a handful of genes to hundreds of genes. [ 3 ]  Inversions can happen either through  ectopic recombination  between  repetitive sequences , or through chromosomal breakage followed by  non-homologous end joining . [ 4 ]


Inversions are of two types:  paracentric  and  pericentric . Paracentric inversions do not include the  centromere , and both breakpoints occur in one arm of the chromosome. Pericentric inversions span the centromere, and there is a breakpoint in each arm [ 5 ] .


Inversions usually do not cause any abnormalities in carriers, as long as the rearrangement is balanced, with no extra or missing DNA. However, in individuals which are  heterozygous  for an inversion, there is an increased production of abnormal  chromatids  (this occurs when crossing-over occurs within the span of the inversion). This leads to lowered fertility, due to production of unbalanced gametes. Inversions do not involve either loss or gain of genetic information; they simply rearrange the linear DNA sequence.


Cytogenetic  techniques may be able to detect inversions, or inversions may be inferred from  genetic analysis . Nevertheless, in most species, small inversions go undetected. More recently, comparative genomics has been used to detect chromosomal inversions, by mapping the genome. [ 6 ] [ 7 ]  Population genomics may also be used to detect inversions, using areas of high  linkage disequilibrium  as indicators for possible inversion sites. Human families that may be carriers of inversions may be offered  genetic counseling  and  genetic testing . [ 8 ]


The first evidence of a chromosomal inversion was found in 1921 by  Alfred Sturtevant  in  Drosophila melanogaster . [ 9 ]  Since then, inversions have been found in a all  eukaryotes . [ 3 ]  When discovered by Sturtevant, inversions were regarded as areas of recombination suppression.


Originally, these inversions were noted in  polytene chromosomes  within the salivary glands of heterozygous  Drosophila melanogaster  larvae. [ 3 ]  In 1970,  Theodosius Dobzhansky  noted that genes within an inversion had higher fitness than those that are found outside of the inversions, although this is an area that needs further study. [ 10 ]


One of the more recent models of inversions is the Kirkpatrick and Barton Model (2006), which states that inversions are selectively advantageous by linking together adaptive alleles. By physically linking co-adapted variants at multiple genes into distinct versions (haplotypes) of an inversion, selection should be more efficient in driving these variants to high frequency in a population. This is in contrast to non-inverted regions, which may allow adaptive and maladaptive alleles to be carried. [ 11 ]


When an inversion carrying chromosome is paired with a non-inverted homologous chromosome (Inversion heterozygotes) during  meiosis , they fail to synapse properly and inversion loops are formed. A  crossing-over  within the loop can produce unbalanced  gametes . In a paracentric inversion,  recombination  results in one dicentric  chromatid  and one acentric chromatid. During  Anaphase , both recombinants are faced with problems. The acentric chromatid is pulled to one pole or the other, and the dicentric recombinant generates dicentric bridges as it is pulled in two directions. [ 12 ]


In a pericentric inversion, similar imbalanced chromosomes are produced. The recombinant chromosomes resulting from these crosses include  deletions  and  duplications . The offspring produced by such gametes are mostly inviable, and therefore, recombination is indirectly suppressed within inverted regions. [ 12 ]


The suppressed recombination between inversion heterozygotes provides an opportunity for the independent evolution of the ancestral and inverted arrangements. At the beginning, the inverted arrangement lacks variation, while the ancestral one does not. If the inverted  haplotype  is not lost (e.g. due to  drift ), the variation in the inverted arrangement can increase over time, and recombination rate in the inverted region is somewhat restored as more homozygotes are introduced. [ 13 ]


Chromosomal inversions have gained a lot of attention in evolutionary research due to their potential role in local adaptation and speciation. Because non-recombining inversion haplotypes may harbor multiple co-adapted gene variants, inversions are thought to facilitate local adaptation to different environments because natural selection is more efficient in driving such linked adaptive variants to high frequency within a population. [ 14 ]  However, empirically demonstrating the presence of linked, co-adapted gene variants within inversions is difficult because inversion haplotypes do not recombine. Moreover, this possible positive effect of chromosomal inversions for adaptation to different environments rests on the assumption that adaptive gene variants linked into distinct inversion haplotypes are indeed co-adapted. This idea is, however, likely violated in situations where populations experience spatially or temporally varying selection. Because of fluctuating selection on inversion-linked variants, the absence of recombination between inversion haplotypes harboring distinct gene variants may then constrain rather than help adaptation to distinct environments. [ 15 ]  The importance of chromosomal inversions in adaptation to different environments therefore remains an open empirical problem in evolutionary genetics.


Inversion polymorphism can be established in two ways. Genetic drift or selection can result in  fixation  of an inversion in a local population. Inversion polymorphism can result from  gene flow  between this population and a population without the inversion.  Balancing selection  can also result in inversion polymorphism by frequency dependence or  overdominance . [ 13 ]  The  fitness  differences between the inverted and the ancestral chromosome can either produce a stable polymorphism or can result in the fixation of one or the other chromosome. [ 16 ]


Inversions have been essential to  sex chromosome  evolution. In mammals, the Y chromosome is unable to recombine with the X chromosome, almost along its entire length. This non-recombining portion results from a series of inversions that overlap. Decreased recombination rate between sex determining  loci  and sex-anatagonistic genes is favored by selection. This causes linkage disequilibrium between the male determining locus and an  allele  at another locus that is beneficial to males. This can happen through inversions resulting in a non-recombining block including both loci, as is the case in the mammalian Y chromosome. [ 16 ]


Inversions can also be essential in the origination of new sex chromosomes. They can cause linkage disequilibrium between a sex-determining mutation and sex-antagonistic loci and create a new sex chromosome from an autosome. [ 17 ]


Inversions can be involved in  speciation  in multiple ways. Since heterozygote inversions can be  underdominant , they can cause hybrid fitness loss, resulting in  post-zygotic isolation . They can also accumulate selected differences between species, causing both pre- and post-zygotic isolation. [ 16 ]


Inversions often form geographical clines in frequency which can hint to their role in local adaptation. A prominent instance of such a cline is inversion 3RP in  Drosophila melanogaster  that can be observed in three different continents. [ 16 ]  When an inversion contains two or more locally adaptive alleles, it can be selected and spread. For example; in the butterfly  Heliconius numata ,  18 genes controlling colors are linked together by inversions as together they confer higher fitness. [ 18 ]


The  International System for Human Cytogenomic Nomenclature  (ISCN) is an international standard for  human chromosome   nomenclature , which includes band names, symbols, and abbreviated terms used in the description of human chromosome and chromosome abnormalities. Abbreviations include  inv  for inversions. [ 20 ]


In  genetics ,  chromosomal polymorphism  is a condition where one  species  contains members with varying  chromosome  counts or shapes.  Polymorphism  is a general concept in  biology  where more than one version of a trait is present in a population.


In some cases of differing counts, the difference in chromosome counts is the result of a single chromosome undergoing  fission , where it splits into two smaller chromosomes, or two undergoing fusion, where two chromosomes join to form one.


This condition has been detected in many species.  Trichomycterus  davisi , for example, is an extreme case where the polymorphism was present within a single  chimeric  individual. [ 1 ]


It has also been studied in  alfalfa , [ 2 ]   shrews , [ 3 ]  Brazilian rodents, [ 4 ]  and an enormous variety of other animals and plants. [ 5 ]  In one instance it has been found in a human. [ 6 ]


Another process resulting in differing chromosomal counts is  polyploidy . This results in cells which contain multiple copies of complete chromosome sets.


Possessing chromosomes of varying shapes is generally the result of a  chromosomal translocation  or  chromosomal inversion .


In a translocation, genetic material is transferred from one chromosome to another, either symmetrically or asymmetrically (a  Robertsonian translocation ).


In an inversion, a segment of a chromosome is flipped end-for-end.


All forms of chromosomal polymorphism can be viewed as a step towards  speciation . Polymorphisms will generally result in a level of reduced fertility, because some  gametes  from one parent cannot successfully combine with all gametes of the other parent. However, when both parents contain matching chromosomal patterns, this obstacle does not occur. Further mutations in one group will not flow as rapidly into the other group as they do within the group in which it originally occurred.


Further mutations can also cause absolute infertility. If an interbreeding population contains one group in which (for example) chromosomes A and B have fused, and another population in which chromosomes B and C have fused, both populations will be able to interbreed with the parent population. However, the two subpopulations will not be able to breed successfully with each other if the doubling of chromosome B is fatal. Similar difficulties will occur for incompatible translocations of material.


Chromosome conformation capture  techniques (often abbreviated to 3C technologies or 3C-based methods [ 1 ] ) are a set of molecular biology methods used to analyze the spatial  organization  of  chromatin  in a cell. These methods quantify the number of interactions between genomic  loci  that are nearby in 3-D space, but may be separated by many  nucleotides  in the linear genome. [ 2 ]   Such interactions may result from biological functions, such as  promoter - enhancer  interactions, or from random polymer looping, where undirected physical motion of chromatin causes loci to collide. [ 3 ]  Interaction frequencies may be analyzed directly, [ 4 ]  or they may be converted to distances and used to reconstruct 3-D structures. [ 5 ]


The chief difference between 3C-based methods is their scope. For example, when using  PCR  to detect interaction in a 3C experiment, the interactions between two specific fragments are quantified. In contrast,  Hi-C  quantifies interactions between all possible pairs of fragments simultaneously. Deep sequencing of material produced by 3C also produces genome-wide interactions maps.


Historically,  microscopy  was the primary method of investigating  nuclear organization , [ 6 ]  which can be dated back to 1590. [ 7 ]


All 3C methods start with a similar set of steps, performed on a sample of cells.


First, the cell genomes are  cross-linked  with  formaldehyde , [ 28 ]  which introduces bonds that "freeze" interactions between genomic loci. Treatment of cells with 1-3% formaldehyde, for 10-30min at room temperature is most common, however, standardization for preventing high protein-DNA cross linking is necessary, as this may negatively affect the efficiency of restriction digestion in the subsequent step. [ 29 ]   The genome is then cut into fragments with a  restriction endonuclease . The size of restriction fragments determines the resolution of interaction mapping. Restriction enzymes (REs)  that make cuts on 6bp recognition sequences, such as  EcoR1  or  HindIII , are used for this purpose, as they cut the genome once every 4000bp, giving ~ 1 million fragments in the human genome. [ 29 ] [ 30 ]  For more precise interaction mapping, a 4bp recognizing RE may also be used. The next step is, proximity based  ligation . This takes place at low DNA concentrations or within intact, permeabilized nuclei [ 26 ]  in the presence of  T4 DNA ligase , [ 31 ]  such that ligation between cross-linked interacting fragments is favored over ligation between fragments that are not cross-linked. Subsequently, interacting loci are quantified by amplifying ligated junctions by PCR methods. [ 29 ] [ 31 ]


The chromosome conformation capture (3C) experiment quantifies interactions between a single pair of genomic loci. For example, 3C can be used to test a candidate promoter-enhancer interaction. Ligated fragments are detected using  PCR  with known  primers . [ 2 ] [ 17 ]  That is why this technique requires the prior knowledge of the interacting regions.


Chromosome conformation capture-on-chip (4C) (also known as circular chromosome conformation capture) captures interactions between one locus and all other genomic loci. It involves a second ligation step, to create self-circularized DNA fragments, which are used to perform  inverse PCR . Inverse PCR allows the known sequence to be used to amplify the unknown sequence ligated to it. [ 32 ] [ 2 ] [ 19 ]  In contrast to 3C and 5C, the 4C technique does not require the prior knowledge of both interacting chromosomal regions. Results obtained using 4C are highly reproducible with most of the interactions that are detected between regions proximal to one another. On a single microarray, approximately a million interactions can be analyzed.  [ citation needed ]


Chromosome conformation capture carbon copy (5C) detects interactions between all restriction fragments within a given region, with this region's size typically no greater than a megabase. [ 2 ] [ 20 ]  This is done by ligating universal primers to all fragments. However, 5C has relatively low coverage. The 5C technique overcomes the junctional problems at the intramolecular ligation step and is useful for constructing complex interactions of specific loci of interest. This approach is unsuitable for conducting genome-wide complex interactions since that will require millions of 5C primers to be used. [ citation needed ]


Hi-C uses  high-throughput sequencing  to find the nucleotide sequence of fragments [ 2 ] [ 22 ]  and uses  paired end sequencing , which retrieves a short sequence from each end of each ligated fragment. As such, for a given ligated fragment, the two sequences obtained should represent two different restriction fragments that were ligated together in the proximity based ligation step. The pair of sequences are individually aligned to the genome, thus determining the fragments involved in that ligation event. Hence, all possible pairwise interactions between fragments are tested.


A number of methods use  oligonucleotide  capture to enrich 3C and Hi-C libraries for specific loci of interest. [ 33 ] [ 34 ]  These methods include Capture-C, [ 35 ]  NG Capture-C, [ 36 ]  Capture-3C, [ 35 ]  HiCap, [ 33 ] [ 37 ]  Capture Hi-C. [ 38 ]  and  Micro Capture-C . [ 39 ]  These methods are able to produce higher resolution and sensitivity than 4C based methods, [ 40 ]  Micro Capture-C provides the highest resolution of the available 3C techniques and it is possible to generate base pair resolution data. [ 39 ]


Single-cell adaptations of these methods, such as ChIP-seq and Hi-C can be used to investigate the interactions occurring in individual cells. [ 41 ] [ 42 ]


A number of methods sequence multiple ligation junctions simultaneously to detect higher-order structures where multiple regions of chromatin may be interacting. These methods include Tri-C, [ 43 ]  3way 4C/C-walks, [ 44 ]  and multi-contact 4C (MC-4C). [ 45 ]


ChIP-loop combines 3C with  ChIP-seq  to detect interactions between two loci of interest mediated by a protein of interest. [ 2 ] [ 46 ]  The ChIP-loop may be useful in identifying long-range  cis -interactions and  trans  interaction mediated through proteins since frequent DNA collisions will not occur.  [ citation needed ]


ChIA-PET  combines Hi-C with ChIP-seq to detect all interactions mediated by a protein of interest. [ 2 ] [ 23 ]  HiChIP was designed to allow similar analysis as ChIA-PET with less input material. [ 47 ]


3C methods have led to a number of biological insights, including the discovery of new structural features of chromosomes, the cataloguing of chromatin loops, and increased understanding of  transcriptional regulation  mechanisms (the disruption of which can lead to disease). [ 6 ]


3C methods have demonstrated the importance of spatial proximity of regulatory elements to the genes that they regulate. For example, in tissues that express  globin  genes, the β-globin locus control region forms a loop with these genes. This loop is not found in tissues where the gene is not expressed. [ 48 ]  This technology has further aided the genetic and  epigenetic  study of chromosomes both in model organisms and in humans. [ not verified in body ]


These methods have revealed large-scale organization of the genome into  topologically associating domains  (TADs), which correlate with epigenetic markers. Some TADs are transcriptionally active, while others are repressed. [ 49 ]  Many TADs have been found in D. melanogaster, mouse and human. [ 50 ]  Moreover,  CTCF  and  cohesin  play important roles in determining TADs and enhancer-promoter interactions. The result shows that the orientation of CTCF binding motifs in an enhancer-promoter loop should be facing to each other in order for the enhancer to find its correct target. [ 51 ]


There are several diseases caused by defects in promoter-enhancer interactions, which are reviewed in this paper. [ 52 ]


Beta thalassemia  is a certain type of blood disorder caused by a deletion of LCR enhancer element. [ 53 ] [ 54 ]


Holoprosencephaly  is cephalic disorder caused by a mutation in the SBE2 enhancer element, which in turn weakened the production of SHH gene. [ 55 ]


PPD2 (polydactyly of a triphalangeal thumb) is caused by a mutation of ZRS enhancer, which in turn strengthened the production of SHH gene. [ 56 ] [ 57 ]


Adenocarcinoma of the lung  can be caused by a duplication of enhancer element for MYC gene. [ 58 ]


T-cell acute lymphoblastic leukemia  is caused by an introduction of a new enhancer. [ 59 ]


The different 3C-style experiments produce data with very different structures and statistical properties. As such, specific analysis packages exist for each experiment type. [ 34 ]


Hi-C data is often used to analyze genome-wide chromatin organization, such as  topologically associating domains  (TADs), linearly contiguous regions of the genome that are associated in 3-D space. [ 49 ]  Several algorithms have been developed to identify TADs from Hi-C data. [ 4 ] [ 64 ]


Hi-C and its subsequent analyses are evolving. Fit-Hi-C  [ 3 ]  is a method based on a discrete binning approach with modifications of adding distance of interaction (initial spline fitting, aka spline-1) and refining the null model (spline-2). The result of Fit-Hi-C is a list of pairwise intra-chromosomal interactions with their p-values and q-values. [ 63 ]


The 3-D organization of the genome can also be analyzed via  eigendecomposition  of the contact matrix. Each eigenvector corresponds to a set of loci, which are not necessarily linearly contiguous, that share structural features. [ 65 ]


A significant confounding factor in 3C technologies is the frequent non-specific interactions between genomic loci that occur due to random  polymer  behavior. An interaction between two loci must be confirmed as specific through statistical significance testing. [ 3 ]


There are two major ways of normalizing raw Hi-C contact heat maps. The first way is to assume equal visibility, meaning there is an equal chance for each chromosomal position to have an interaction. Therefore, the true signal of a Hi-C contact map should be a balanced matrix (Balanced matrix has constant row sums and column sums). An example of algorithms that assumes equal visibility is  Sinkhorn-Knopp algorithm , which scales the raw Hi-C contact map into a balanced matrix.


The other way is to assume there is a bias associated with each chromosomal position. The contact map value at each coordinate will be the true signal at that position times bias associated with the two contact positions. An example of algorithms that aim to solve this model of bias is iterative correction, which iteratively regressed out row and column bias from the raw Hi-C contact map.  There are a number of software tools available for analysis of Hi-C data. [ 66 ]


DNA motifs  are specific short DNA sequences, often 8-20 nucleotides in length [ 67 ]  which are statistically overrepresented in a set of sequences with a common biological function. Currently, regulatory motifs on the long-range chromatin interactions have not been studied extensively. Several studies have focused on elucidating the impact of DNA motifs in promoter-enhancer interactions.


Bailey et al. has identified that ZNF143 motif in the promoter regions provides sequence specificity for promoter-enhancer interactions. [ 68 ]  Mutation of ZNF143 motif decreased the frequency of promoter-enhancer interactions suggesting that ZNF143 is a novel chromatin-looping factor.


For genome-scale motif analysis, in 2016, Wong et al. reported a list of 19,491 DNA motif pairs for K562 cell line on the promoter-enhancer interactions. [ 69 ]  As a result, they proposed that motif pairing multiplicity (number of motifs that are paired with a given motif) is linked to interaction distance and regulatory region type. In the next year, Wong published another article reporting 18,879 motif pairs in 6 human cell lines. [ 70 ]  A novel contribution of this work is MotifHyades, a  motif discovery  tool that can be directly applied to paired sequences.


The 3C-based techniques can provide insights into the chromosomal rearrangements in the cancer genomes. [ 71 ]  Moreover, they can show changes of spatial proximity for regulatory elements and their target genes, which bring deeper understanding of the structural and functional basis of the genome. [ 72 ]


Chromosome engineering  is "the controlled generation of chromosomal deletions, inversions, or translocations with defined endpoints." [ 1 ]  By combining  chromosomal translocation ,  chromosomal inversion , and  chromosomal deletion , chromosome engineering has been shown to identify the underlying genes that cause certain diseases in mice. In coming years, it is very likely that chromosomal engineering will be able to do the same identification for diseases in humans, as well as all other organisms. [ 2 ]


In an experiment pertaining to chromosome engineering that was conducted in 2006, it was found that chromosome engineering can be effectively used as a method of identifying the causes of genetic disorders such as the continuous gene and aneuploidy syndromes. The experiment was conducted by infecting mice with the human disease, ES, to see the effectiveness of chromosomal engineering in the gene identification of those diseases. After much experimenting, it was found that manipulating chromosomes, or chromosome engineering, is an excellent and efficient method of determining underlying genes in genetic orders and diseases. [ citation needed ]


In the future, chromosome engineering will experiment in removing more common disorders such as asthma, diabetes, and cancer. If it can be recognized by the medical community as effective and safe, it should be able to be used regularly in the near future. [ citation needed ]


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Chromosomal instability  ( CIN ) is a type of  genomic instability  in which  chromosomes  are unstable, such that either whole chromosomes or parts of chromosomes are duplicated or deleted. More specifically, CIN refers to the increase in rate of addition or loss of entire chromosomes or sections of them. [ 1 ]  The unequal distribution of DNA to daughter cells upon  mitosis  results in a failure to maintain euploidy (the correct number of  chromosomes ) leading to  aneuploidy  (incorrect number of chromosomes).  In other words, the daughter cells do not have the same number of chromosomes as the cell they originated from. Chromosomal instability is the most common form of genetic instability and cause of aneuploidy. [ 2 ]


These changes have been studied in solid tumors (a tumor that usually doesn't contain liquid, pus, or air, compared to liquid tumor), [ 3 ]  which may or may not be cancerous. CIN is a common occurrence in  solid  and  haematological cancers , especially  colorectal cancer . [ 4 ]  Although many tumours show chromosomal abnormalities, CIN is characterised by an increased rate of these errors. [ 5 ]


Numerical CIN is a high rate of either gain or loss of whole chromosomes; causing  aneuploidy . Normal cells make errors in chromosome segregation in 1% of cell divisions, whereas cells with CIN make these errors approximately 20% of cell divisions. Because aneuploidy is a common feature in tumour cells, the presence of aneuploidy in cells does not necessarily mean CIN is present; a high rate of errors is definitive of CIN. [ 6 ]  One way of differentiating aneuploidy without CIN and CIN-induced aneuploidy is that CIN causes widely variable (heterogeneous) chromosomal aberrations; whereas when CIN is not the causal factor, chromosomal alterations are often more clonal. [ 7 ]


Structural CIN is different in that rather than whole chromosomes, fragments of chromosomes may be duplicated or deleted. The rearrangement of parts of chromosomes ( translocations ) and amplifications or deletions within a chromosome may also occur in structural CIN. [ 6 ]


A loss in the repair systems for DNA double-stranded breaks and eroded telomeres can allow chromosomal rearrangements that generate loss, amplification and/or exchange of chromosome segments. [ 2 ]


Some inherited genetic predispositions to cancer are the result of mutations in machinery that responds to and repairs DNA double-stranded breaks. Examples include ataxia telangiectasia – which is a mutation in the damage response kinase ATM – and BRCA1 or MRN complex mutations that play a role in responding to DNA damage. When the above components are not functional, the cell can also lose the ability to induce cell-cycle arrest or apoptosis. Therefore, the cell can replicate or segregate incorrect chromosomes. [ 8 ]


Faulty rearrangements can occur when homologous recombination fails to accurately repair double-stranded breaks. Since human chromosomes contain repetitive DNA sections, broken DNA segments from one chromosome can combine with similar sequences on a non-homologous chromosome. If repair enzymes do not catch this recombination event, the cell may contain non-reciprocal translocation where parts of non-homologous chromosomes are joined together. Non-homologous end joining can also join two different chromosomes together that had broken ends. The reason non-reciprocal translocations are dangerous is the possibility of producing a dicentric chromosome – a chromosome with two centromeres. When dicentric chromosomes form, a series of events can occur called a  breakage-fusion-bridge cycle : Spindle fibers attach onto both centromeres in different locations on the chromosome, thereby tearing the chromatid into two pieces during anaphase. The result is a pair of DNAs with broken ends that can attach to other broken-ended DNA segments creating additional translocation and continue the cycle of chromosome breakage and fusion. As the cycle continues, more chromosome translocations result, leading to the amplification or loss of large DNA fragments. Some of these changes will kill the cell, however, in a few rare cases, the rearrangements can lead to a viable cell without  tumor suppressor  genes and increased expression of  proto-oncogenes  that may become a tumor cell. [ 9 ]


Telomeres  – which are a protective ‘cap’ at the end of DNA molecules – normally shorten in each replication cycle. In certain cell types, the  telomerase  enzyme can re-synthesize the telomere sequences, however, it is not present in all somatic cells. Once 25-50 divisions pass, the telomeres can be completely lost, inducing  p53  to either permanently arrest the cell or induce apoptosis. Telomere shortening and p53 expression is a key mechanism to prevent uncontrolled replication and tumor development because even cells that excessively proliferate will eventually be inhibited. [ 10 ] [ 11 ]


However, telomere degeneration can also induce tumorigenesis in other cells. The key difference is the presence of a functional p53 damage response. When tumor cells have a mutation in p53 that results in a non-functional protein, telomeres can continue to shorten and proliferate, and the eroded segments are susceptible to chromosomal rearrangements through recombination and breakage-fusion-bridge cycles. Telomere loss can be lethal for many cells, but in the few that are able to restore the expression of telomerase can bring about a “stable” yet tumorigenic chromosome structure. Telomere degeneration thereby explains the transient period of extreme chromosomal instability observed in many emerging tumors. [ 11 ]


In experiments on mice where both telomerase and p53 were knocked out, they developed carcinomas with significant chromosomal instability similar to tumors seen in humans. [ 2 ]


Spindle assembly checkpoint  (SAC) abnormalities: The SAC normally delays cell division until all of the chromosomes are accurately attached to the spindle fibers at the  kinetochore . Merotelic attachments – when a single kinetochore is connected to microtubules from both spindle poles. Merotelic attachments are not recognized by the SAC, so the cell can attempt to proceed through  anaphase . Consequently, the chromatids may lag on the mitotic spindle and not segregate, leading to aneuploidy and chromosome instability. [ 12 ]


CIN often results in  aneuploidy . There are three ways that aneuploidy can occur. It can occur due to loss of a whole chromosome, gain of a whole chromosome or rearrangement of partial chromosomes known as gross  chromosomal rearrangements  (GCR). All of these are hallmarks of some  cancers . [ 13 ]   Most cancer cells are aneuploid, meaning that they have an abnormal number of chromosomes which often have significant structural abnormalities such as chromosomal translocations, where sections of one chromosome are exchanged or attached onto another. Changes in ploidy can alter expression of proto-oncogenes or tumor suppressor genes. [ 1 ] [ 2 ]


Segmental aneuploidy can occur due to deletions, amplifications or translocations, which arise from breaks in DNA, [ 5 ]  while loss and gain of whole chromosomes is often due to errors during mitosis.


Chromosomes consist of the DNA sequence, and the proteins (such as  histones ) that are responsible for its packaging into chromosomes. Therefore, when referring to chromosome instability,  epigenetic  changes can also come into play. Genes on the other hand, refer only to the DNA sequence (hereditary unit) and it is not necessary that they will be expressed once epigenetic factors are taken into account. Disorders such as chromosome instability can be inherited via genes, or acquired later in life due to environmental exposure. One way that Chromosome Instability can be acquired is by exposure to ionizing radiation. [ 14 ]   Radiation is known to cause DNA damage, which can cause errors in cell replication, which may result in chromosomal instability. Chromosomal instability can in turn cause cancer.
However, chromosomal instability syndromes such as  Bloom syndrome ,  ataxia telangiectasia  and  Fanconi anaemia  are inherited  [ 14 ]  and are considered to be genetic diseases. These disorders are associated with tumor genesis, but often have a phenotype on the individuals as well. The genes that control chromosome instability are known as chromosome instability genes and they control pathways such as mitosis, DNA replication, repair and modification. [ 15 ]  They also control transcription, and process nuclear transport. [ 15 ]


Cancer cells often exhibit chromosomal abnormalities, including chromosomal rearrangements (such as translocations), deletions, and duplications. These abnormalities can disrupt the normal function of genes involved in  cell cycle regulation , leading to uncontrolled cell growth and tumor formation. [ 16 ]  The  chromosome theory of cancer  is a long-standing idea originated from the work of  Theodor Boveri , a German biologist, in the early 20th century. Boveri's studies on  sea urchin  eggs provided early evidence that abnormal chromosome numbers could lead to developmental defects, leading him to propose a connection between  chromosomal abnormalities  and cancer. [ 17 ]  Further research by scientists such as  David Hungerford  and  Peter Nowell  in the 1960s identified specific chromosomal abnormalities in  cancer cells , such as the  Philadelphia chromosome  in  chronic myeloid leukemia , providing more support for the chromosomal theory of cancer. [ 18 ]  The  chromosomal  theory of  cancer  is a fundamental concept in  cancer biology  that suggests cancer is caused by  genetic  changes, particularly alterations in the structure or number of  chromosomes  in  cells . These changes can lead to uncontrolled  cell growth , a hallmark of cancer. [ 19 ]


CIN is a more pervasive mechanism in cancer genetic instability than simple accumulation of point mutations. The degree of instability varies between cancer types. For example, in cancers where mismatch repair mechanisms are defective – like some colon and breast cancers – their chromosomes are relatively stable. [ 2 ]


Cancers can go through periods of extreme instability where chromosome number can vary within the population. Rapid chromosomal instability is thought to be caused by telomere erosion. However, the period of rapid change is transient as tumor cells generally reach an equilibrium abnormal chromosome content and number. [ 20 ]


The research associated with chromosomal instability is associated with solid tumors, which are tumors that refer to a solid mass of cancer cells that grow in organ systems and can occur anywhere in the body. These tumors are opposed to liquid tumors, which occur in the blood, bone marrow, and lymph nodes. [ 21 ]


Although chromosome instability has long been proposed to promote tumor progression, [ 22 ] [ 23 ]  recent studies suggest that chromosome instability can either promote or suppress tumor progression. [ 13 ]  The difference between the two are related to the amount of chromosomal instability taking place, as a small rate of chromosomal instability leads to tumor progression, or in other words cancer, while a large rate of chromosomal instability is often lethal to cancer. [ 24 ]  This is due to the fact that a large rate of chromosomal instability is detrimental to the survival mechanisms of the cell, [ 24 ]  and the cancer cell cannot replicate and dies (apoptosis). [ 25 ]   Therefore, the relationship between chromosomal instability and cancer can also be used to assist with diagnosis of malignant vs. benign tumors. [ 24 ]


The level of chromosome instability is influenced both by  DNA damage  during the  cell cycle  and the effectiveness of the DNA damage response in  repairing  damage. The DNA damage response during  interphase  of the  cell cycle  (G1, S and G2 phases) helps protect the  genome  against structural and numerical cancer chromosome instability.  However untimely activation of the DNA damage response once cells have committed to the  mitosis  stage of the cell cycle appears to undermine genome integrity and induce chromosome segregation errors. [ 26 ]


A majority of human solid malignant tumors is characterized by chromosomal instability, and have gain or loss of whole chromosomes or fractions of chromosomes. [ 5 ]  For example, the majority of colorectal and other solid cancers have chromosomal instability (CIN). [ 27 ]   This shows that chromosomal instability can be responsible for the development of solid cancers. However, genetic alterations in a tumor do not necessarily indicate that the tumor is genetically unstable, as ‘genomic instability’ refers to various instability phenotypes, including the chromosome instability phenotype  [ 5 ]


The role of CIN in carcinogenesis has been heavily debated. [ 28 ]  While some argue the canonical theory of  oncogene  activation and  tumor suppressor gene  inactivation, such as  Robert Weinberg , some have argued that CIN may play a major role in the origin of cancer cells, since CIN confers a mutator phenotype [ 29 ]  that enables a cell to accumulate large number of mutations at the same time. Scientists active in this debate include Christoph Lengauer, Kenneth W. Kinzler, Keith R. Loeb, Lawrence A. Loeb,  Bert Vogelstein  and  Peter Duesberg .


Current research in cancer genetics is focused on further understanding the role of  chromosomal abnormalities  in cancer development and progression. Advances in technology, such as next-generation  sequencing , are enabling researchers to study chromosomal abnormalities in cancer cells with greater detail and precision. [ 30 ]


Hypothetically, the heterogeneous gene expression that can occur in a cell with CIN, the rapid genomic changes can drive the emergence of drug-resistant tumor cells. While some studies show that CIN is associated with poor patient outcomes and drug resistance, conversely, others studies actually find that people respond better with high CIN tumors. [ 31 ]


Some researchers believe that CIN can be stimulated and exploited to generate lethal interactions in tumor cells. ER negative breast cancer patients with the most extreme CIN have the best prognosis, with similar results for ovarian, gastric and non-small cell lung cancers. A potential therapeutic strategy therefore could be to exacerbate CIN specifically in tumor cells to induce cell death. [ 32 ]  For example,  BRCA1 ,  BRCA2  and BC-deficient cells have a sensitivity to  poly(ADP-ribose) polymerase  (PARP) which helps repair single-stranded breaks. When PARP is inhibited, the replication fork can collapse. Therefore, PARP tumor suppressing drugs could selectively inhibit  BRCA  tumors and cause catastrophic effects to breast cancer cells. Clinical trials of PARP inhibition are ongoing. [ 33 ]


There is still a worry that targeting CIN in therapy could trigger genome chaos that actually increases CIN that leads to selection of proliferative advantages. [ 31 ]


Targeted therapies, such as  imatinib  for chronic myeloid leukemia [ 34 ]  and  trastuzumab  for  HER2-positive breast cancer , [ 35 ]  have been developed based on the specific chromosomal abnormalities associated with these cancers.


Chromosomal instability has been identified as a genomic driver of metastasis. [ 36 ]  Chromosome segregation errors during mitosis lead to the formation of structures called micronuclei. These micronuclei, which reside outside of the main nucleus have defective envelopes and often rupture exposing their genomic DNA content to the cytoplasm. [ 37 ]  Exposure of double-stranded DNA to the cytosol activates anti-viral pathways, such as the cGAS-STING cytosolic DNA-sensing pathway. This pathway is normally involved in cellular immune defenses against viral infections. Tumor cells hijack chronic activation of innate immune pathways to spread to distant organs, suggesting that CIN drives metastasis through chronic inflammation stemming in a cancer cell-intrinsic manner. [ 36 ]


Chromosomal instability can be diagnosed using analytical techniques at the cellular level. Often used to diagnose CIN is cytogenetics  flow cytometry ,  Comparative genomic hybridization  and  Polymerase Chain Reaction . [ 5 ]   Karyotyping , and  fluorescence in situ hybridization  (FISH) are other techniques that can be used. [ 38 ] [ 39 ] [ 40 ]   In Comparative genomic hybridization, since the DNA is extracted from large cell populations it is likely that several gains and losses will be identified. [ 5 ]
Karyotyping is used for Fanconi Anemia, based on 73-hour whole-blood cultures, which are then stained with Giemsa. Following staining they are observed for microscopically visible chromatid-type aberrations  [ 41 ]


Several  chromosome regions  have been defined by convenience and convention in order to talk about gene  loci . The largest regions on each chromosome are the short arm  p  and the long arm  q , separated by a narrow region near the center called the  centromere . [ 1 ]  Other specific regions have also been defined, some of which are similarly found on every chromosome, while others are only present in certain chromosomes.


Named regions include:


During  cell division , the molecules that compose chromosomes ( DNA  and  proteins ) undergo a condensation process (called the  chromatin  reticulum condensation) that forms a compact and small complex called a chromatid. The complexes containing the duplicated DNA molecules, the sister  chromatids , [ 2 ]  are attached to each other by the  centromere (where the  Kinetochore  assembles).


If the chromosome is a submetacentric chromosome (One arm big and the other arm small) then the centromere divides each chromosome into two regions: the smaller one, which is the  p  region, and the bigger one, the  q  region. The sister chromatids will be distributed to each daughter cell at the end of the cell division. Whereas if the chromosome is isobrachial (centromere at  centre and arms of equal length), the p and q system is meaningless.


At either end of a chromosome is a  telomere , a cap of  DNA  that protects the rest of the chromosome from damage. The telomere has repetitive  junk DNA  and hence any enzymatic damage will not affect the coded regions. The areas of the  p  and  q  regions close to the telomeres are the  subtelomeres , or subtelomeric regions. The areas closer to the centromere are the pericentronomic regions. Finally, the interstitial regions are the parts of the  p  and  q  regions that are close to neither the centromere nor the telomeres, but are roughly in the middle of  p  or  q .


In  biology , the  chromosome scaffold  is the backbone that supports the structure of the  chromosomes . It is composed of a group of  non-histone   proteins  that are essential in the structure and maintenance of  eukaryotic  chromosomes throughout the  cell cycle . These  scaffold proteins  are responsible for the  condensation  of  chromatin  during  mitosis . [ 1 ]


In the late 1970s,  Ulrich K. Laemmli  and colleagues discovered a backbone structure in  eukaryotic   chromosomes  after they depleted the  histone  proteins. This backbone was localized along the chromosome axis, and was termed the ‘chromosome scaffold’. [ 2 ] [ 1 ]


In  eukaryotic organisms , the  DNA  of each  cell  is organized into separated  chromosomes , which are composed of  chromatin , a mixture of DNA and many different groups of  proteins . Among them, the  structural proteins  (that are not  histones ) bind the chromatin fiber around themselves forming a long, continuous axis or backbone that gives the chromosomes their shape. For this reason they are known as the ‘scaffold’ of chromosomes. [ 1 ]


Three protein groups have been identified as the main components of the scaffold:  DNA topoisomerase IIα ,  condensins , and the  KIF4A  kinesin. When these proteins are removed, the chromosome shape does not appear and the chromatin fibers spread out. [ 1 ]


The  enzyme   DNA topoisomerase IIα  prominently appears along the chromosome axis as part of the scaffold. [ 3 ]  In mitosis, it is concentrated at the  centromeres  and the axis along the chromosome arms. It is thought that the protein has a role in untangling the DNA as the loops become more concentrated along the axis during the condensation of the chromosomes. [ 4 ]  The removal of this protein causes a dramatic loss of the chromosome structure in mitosis, and the cell cycle comes to a stop. [ 5 ]


Condensin  complexes, formed from the union of  SMC2  and  SMC4  (among other proteins), are responsible for the condensation of chromosomes. [ 6 ]  Condensin I regulates the timing of chromosome condensation and is essential for changing the chromatin organization at the beginning of mitosis, from  TADs  to an array of loops around the chromosome axis. Condensin II drives the compaction of the chromosome loops along the axis. [ 4 ]


In particular, SMC2 (present in condensin I and II) is detected in the interior of the chromosome as part of the scaffold. [ 4 ]  When SMC2 is inhibited, the structure of the mitotic chromosome suffers grave defects. [ 7 ]


KIF4A , a chromo kinesin , is implicated in the shaping of chromosomes during  mitosis . It binds to  condensin I  through the  CAP-G  subunit. It is known that KIF4A regulates the behavior of condensin I, because in absence of KIF4A the chromosome axis does not become enriched with condensin I. [ 8 ]


Chromosome segregation  is the process in  eukaryotes  by which two sister  chromatids  formed as a consequence of  DNA replication , or paired  homologous chromosomes , separate from each other and migrate to opposite poles of the  nucleus . This segregation process occurs during both  mitosis  and  meiosis . Chromosome segregation also occurs in  prokaryotes . However, in contrast to eukaryotic chromosome segregation, replication and segregation are not temporally separated. Instead segregation occurs progressively following replication. [ 1 ]


During  mitosis  chromosome segregation occurs routinely as a step in cell division (see mitosis diagram).  As indicated in the mitosis diagram, mitosis is preceded by a round of DNA replication, so that each chromosome forms two copies called  chromatids .  These chromatids separate to opposite poles, a process facilitated by a protein complex referred to as  cohesin .  Upon proper segregation, a complete set of chromatids ends up in each of two nuclei, and when cell division is completed, each DNA copy previously referred to as a chromatid is now called a chromosome.


Chromosome segregation occurs at two separate stages during  meiosis  called  anaphase I  and  anaphase II  (see meiosis diagram).  In a diploid cell there are two sets of  homologous chromosomes  of different parental origin (e.g. a paternal and a maternal set).  During the phase of meiosis labeled “interphase s” in the meiosis diagram there is a round of DNA replication, so that each of the chromosomes initially present is now composed of two copies called  chromatids .  These chromosomes (paired chromatids) then pair with the homologous chromosome (also paired chromatids) present in the same nucleus (see prophase I in the meiosis diagram).  The process of alignment of paired homologous chromosomes is called synapsis (see  Synapsis ).  During synapsis, genetic recombination usually occurs.  Some of the recombination events occur by  crossing over  (involving physical exchange between two chromatids), but most recombination events involve information exchange but not physical exchange between two chromatids (see  Synthesis-dependent strand annealing (SDSA) ).  Following recombination, chromosome segregation occurs as indicated by the stages metaphase I and anaphase I in the meiosis diagram.


Different pairs of chromosomes segregate independently of each other, a process termed  “independent assortment of non-homologous chromosomes” . This process results in each gamete usually containing a mixture of chromosomes from both original parents.


Improper chromosome segregation (see  non-disjunction ,  disomy ) can result in  aneuploid  gametes having either too few or too many chromosomes.


The second stage at which segregation occurs during meiosis is  prophase II  (see meiosis diagram).  During this stage, segregation occurs by a process similar to that during mitosis, except that in this case prophase II is not preceded by a round of DNA replication.  Thus the two chromatids comprising each chromosome separate into different  nuclei , so that each nucleus gets a single set of chromatids (now called chromosomes) and each nucleus becomes included in a haploid  gamete  (see stages following prophase II in the meiosis diagram).  This segregation process is also facilitated by  cohesin .  Failure of proper segregation during prophase II can also lead to aneuploid gametes.  Aneuploid gametes can undergo fertilization to form aneuploid zygotes and hence to serious adverse consequences for progeny.


Meiotic   chromosomal crossover  (CO) recombination facilitates the proper segregation of  homologous chromosomes .  This is because, at the end of meiotic  prophase I , CO recombination provides a physical link that holds homologous chromosome pairs together.  These linkages are established by  chiasmata , which are the cytological manifestations of CO recombination.  Together with  cohesion  linkage between sister  chromatids , CO recombination may help ensure the orderly segregation of the paired homologous chromosomes to opposite poles.  In support of this, a study of aneuploidy in single spermatozoa by whole genome sequencing found that, on average, human sperm cells with aneuploid autosomes exhibit significantly fewer crossovers than normal cells. [ 2 ]   After the first chromosome segregation in  meiosis I  is complete, there is further chromosome segregation during the second equational division of  meiosis II .  Both proper initial segregation of chromosomes in prophase I and the next chromosome segregation during equational division in meiosis II are required to generate gametes with the correct number of chromosomes.


CO recombinants are produced by a process involving the formation and resolution of  Holliday junction  intermediates.  As indicated in the figure titled "A current model of meiotic recombination", the formation of meiotic crossovers can be initiated by a  double-strand break  (DSB). The introduction of DSBs in DNA often employs the  topoisomerase -like protein SPO11. [ 3 ]   CO recombination may also be initiated by external sources of DNA damage such as X-irradiation, [ 4 ]  or internal sources. [ 5 ] [ 6 ]


There is evidence that CO recombination facilitates meiotic chromosome segregation. [ 2 ]   Other studies, however, indicate that  chiasma , while supportive, are not essential to meiotic chromosome segregation.  The budding yeast  Saccharomyces cerevisiae  is a model organism used for studying meiotic recombination.  Mutants of  S. cerevisiae  defective in CO recombination at the level of  Holliday junction  resolution were found to efficiently undergo proper chromosome segregation.  The pathway that produces the majority of COs in  S. cerevisiae , and possibly in mammals, involves a complex of proteins including the  MLH1 - MLH3   heterodimer  (called MutL gamma). [ 7 ]   MLH1-MLH3 binds preferentially to Holliday junctions. [ 8 ]   It is an  endonuclease  that makes single-strand breaks in  supercoiled  double-stranded DNA, [ 8 ] [ 9 ]  and promotes the formation of CO recombinants. [ 10 ]   Double mutants deleted for both MLH3 (major pathway) and MMS4 (which is necessary for a minor Holliday junction resolution pathway) showed dramatically reduced crossing over compared to wild-type (6- to 17-fold reduction); however  spore  viability was reasonably high (62%) and chromosomal  disjunction  appeared mostly functional. [ 10 ]


The  MSH4  and  MSH5  proteins form a hetero-oligomeric structure ( heterodimer ) in  S. cerevisiae  and humans. [ 11 ] [ 12 ] [ 13 ]   In  S. cerevisiae , MSH4 and MSH5 act specifically to facilitate crossovers between  homologous chromosomes  during meiosis. [ 11 ]   The MSH4/MSH5 complex binds and stabilizes double  Holliday junctions  and promotes their resolution into crossover products.  An MSH4 hypomorphic (partially functional) mutant of  S. cerevisiae  showed a 30% genome-wide reduction in crossover numbers, and a large number of meioses with non-exchange chromosomes. [ 14 ]   Nevertheless, this mutant gave rise to spore viability patterns suggesting that segregation of non-exchange chromosomes occurred efficiently. [ 14 ]    Thus it appears that CO recombination facilitates proper chromosome segregation during meiosis in  S. cerevisiae , but it is not essential.


The fission yeast  Schizosaccharomyces pombe  has the ability to segregate homologous chromosomes in the absence of meiotic recombination (achiasmate segregation). [ 15 ]   This ability depends on the microtubule motor  dynein  that regulates the movement of chromosomes to the poles of the  meiotic spindle .


Chromothripsis  is a mutational process by which up to thousands of clustered  chromosomal rearrangements  occur in a single event in localised and confined genomic regions in one or a few  chromosomes , and is known to be involved in both cancer and congenital diseases. It occurs through one massive genomic rearrangement during a single catastrophic event in the cell's history. It is believed that for the cell to be able to withstand such a destructive event, the occurrence of such an event must be the upper limit of what a cell can tolerate and survive. [ 1 ]  The chromothripsis phenomenon opposes the conventional theory that  cancer  is the gradual acquisition of genomic rearrangements and somatic mutations over time. [ 2 ]


The simplest model as to how these rearrangements occur is through the simultaneous fragmentation of distinct chromosomal regions (breakpoints show a non-random distribution) and then subsequent imperfect reassembly by  DNA repair pathways  or aberrant  DNA replication  mechanisms. Chromothripsis occurs early in tumour development and leads to cellular transformation by loss of  tumour suppressors  and  oncogene  amplifications. [ 3 ]  In 2015, it was found that chromothripsis can also be curative: a woman who had  WHIM  (warts, hypogammaglobulinemia, infections, and myelokathexis) syndrome, an extremely rare autosomal dominant combined immunodeficiency disease, found her symptoms disappeared during her 30s after chromothripsis of chromosome 2 deleted the disease allele. [ 4 ]


The term  chromothripsis  is a neologism coined by scientists at the  Wellcome Trust Sanger Institute [ 2 ]  that comes from the Greek words χρῶμα,  khrôma  'color' (representing  chromosomes  because they are strongly stained by particular dyes), and θρίψις,  thrípsis  'shattering into pieces'. [ 2 ]


Chromothripsis was first observed in sequencing the genome of a  chronic lymphocytic leukaemia . Through  paired end sequencing , 55 chromosomal rearrangements were found in the long arm of chromosome 8 and a significant number of rearrangements were found in regions of chromosomes 7, 12, and 15. [ 2 ]  Subsequent investigations using genome-wide paired-end sequencing and   SNP array analysis [ 5 ]  have found similar patterns of chromothripsis in various human cancers,  e.g. ,  melanomas ,  sarcomas  and  colorectal ,  lung  and  thyroid cancers . In subsequent investigations, about 25% of studied  bone cancers  displayed evidence of chromothripsis. Chromothripsis has been linked to the generation of oncogenic fusions in supratentorial  ependymoma ,  chondromyxoid fibroma , and  Ewing sarcoma , the latter two being bone tumours. [ 6 ] [ 7 ]  Chromothripsis has been seen in 2–3% of cancers across all subtypes. [ 3 ]


The most widely accepted and straightforward model for chromothripsis is that within a single chromosome, distinct  chromosomal regions  become fragmented/shattered almost simultaneously and subsequently rejoined in an incorrect orientation.  Deletion  of certain fragments, including deletions that are a few hundred  base pairs long , and hence gene segments is possible and consequently the production of  double minute chromosomes . [ 2 ]  When multiple  chromosomes  are involved in chromothripsis, fragments of both chromosomes are joined together by paired end joining and the exchange of fragments between the original chromosomes. [ 3 ]


Rejoining of fragments require very minimal or even no sequence  homology  and consequently suggesting that nonhomologous or microhomologous repair mechanisms  such as  non-homologous end joining  (NHEJ) and  microhomology-mediated break induced repair  (MMBIR) dominate  double stranded break  repair and are involved in modelling the chromothriptic landscape, opposed to  homologous recombination  which requires  sequence homology . Joining of fragments and rearrangements have also been shown to take place on paternal chromosomes. [ 5 ]


As well as in  cancer cells , chromothripsis has also been reported in patients with developmental and  congenital defects , i.e.  germ line cells . Using multiple molecular techniques of these germ line cells that have appeared to have undergone a chromothripsis like process, as well as  inversions  and  translocations ,  duplications  and triplications were also seen and hence increases in  copy number . This can be attributed to replicative processes that involve the restoration of collapsed  replication forks  such as fork stalling and template switching model (FoSTeS) or  microhomology mediated break induced replication  (MMBIR). [ 9 ]  This makes it seem that it would be more appropriate to name the phenomenon 'chromoanasynthesis' which means chromosome reconstitution rather than chromothripsis. [ 9 ]  However most samples displaying chromothripsis that are analysed have low copy states and hence have paired end joining predominating repair mechanisms. [ 2 ]


Further study of chromothripsis events and chromothriptic samples is required in order to understand the relative importance of paired end joining and replicative repair in chromothripsis. [ 3 ]


One of the main characteristic features of chromothripsis is large numbers of complex rearrangements occurring in localised regions of single chromosomes. The ability to cause such confined damage suggests that chromosomes need to be condensed e.g. in  mitosis , for chromothripsis and  chromosome rearrangements  to be initiated. [ 1 ] [ 3 ]


The mechanisms of chromothripsis are not well understood. There are multiple ideas of how chromothripsis occurs. [ citation needed ]


The Micronuclei model is the most accepted model as to how and when the breakage and repair in chromothripsis occurs.
In cancer cells, fragmentation of chromosomes has been correlated with the presence of  micronuclei . [ 3 ]  Micronuclei are structures  formed by mitotic errors in the transition from metaphase to anaphase. Cells with defective  chromosome segregation  will form micronuclei which contain whole chromosomes or fragments of chromosomes. The segregation of single chromosomes into individual micronuclei explains why  DNA fragmentation  is isolated to single chromosomes in chromothripsis. [ 10 ]


These micronuclei undergo defective  DNA replication , which is slower than DNA replication in the main nucleus and causes a proximal DNA damage response (DDR) to be initiated. However,  DNA repair  and  cell cycle checkpoint  activation fail to follow. [ 11 ]  Consequently, chromosomes that are not correctly replicated in micronuclei become fragmented. [ 10 ]  The mechanism by which the pulverization of these chromosomes occurs is not fully understood, but it is thought to be caused either by aberrant  DNA replication  or by  premature chromosome condensation , which entails semi-replicated chromosomes being compacted by  cyclin-dependent kinase  activity. [ 12 ]  The resulting fragmented chromosome segments can be joined together to give rise to a rearranged chromosome, which can subsequently be reincorporated into the main nucleus of a  daughter cell . The new chromosome can persist for several generations of  cell cycle  divisions and contribute to the development of a  cancer cell . [ 3 ] [ 10 ]


Although the  micronucleus  model is appropriate, other factors are likely to contribute towards chromothripsis for various cancer genomes. [ 3 ]


Chromosome shattering is triggered and reassembly of chromosome fragments in close proximity is caused by environmental stimuli such as high energy  ionising radiation  encountered during  mitosis . [ 1 ]


Stress stimuli such as radiation, nutrient deprivation or oxygen deprivation which causes  apoptosis  will lead to fragmentation of  chromatin  and cause most cells to apoptose. However a small subset of cells will survive  apoptosis . This cleaved DNA will require repair, and when this is done incorrectly, rearrangements will be introduced into the chromosome. There is currently speculation that chromothripsis might be driven by viruses such as  γ-herpes viruses  which cause  cancer , possibly by the inhibition of  apoptosis . However this speculation requires further investigation. [ 13 ]


Telomeric double stranded breaks or telomeric dysfunction is generated by exogenous agents or replicative stress. Telomeric dysfunctions are known to promote  chromosomal abnormalities  associated with cancer cells. For example, Telomeric double stranded breaks/ telomeric dysfunctions can cause  sister chromatid / end to end fusion and the formation of  anaphase bridges  resulting in  dicentric chromosomes  that can result in further rearrangements. This is a more plausible explanation as chromothripsis has been seen to mostly involve telomeric regions. [ 13 ]


Mutations in the TP53 gene can predispose a cell to chromothripsis. [ citation needed ]


Through genome sequencing of a  Sonic hedgehog  medulloblastoma (SHH-MB) brain tumour, a significant link between TP53 mutations and chromothripsis in SHH-MBs has been found. Further studies on the association between TP53 and chromothripsis has signified a role for  p53 , a  tumour suppressor  protein, in the massive genomic rearrangement which take place which takes place in chromothripsis. Hence there is a strong association between p53 status and chromothripsis, giving an insight into why some cancers are more aggressive. [ 14 ] [ 15 ]


It has also been shown that TP53 mutation-containing cells show a preference for low-fidelity repair mechanisms such as  non-homologous end joining .  TP53 mutations have also been expressed in cells that exhibit shorter [ clarification needed ]  and are more end-end fusion prone. It is also hypothesized that TP53 mutations may be implicated in  premature chromosome condensation . TP53 may also contribute to the ability of cells to survive the catastrophic event that normally would be considered to be too destructive to withstand. [ 1 ]


Chromothripsis has been seen to cause  oncogene  amplification, amplification of oncogene containing regions and the loss of  tumour suppressors . [ 3 ]


Chromosome segregation errors can lead to DNA  damage and  chromosomal aberrations  such as  aneuploidy   which is linked to tumour development. [ 3 ] [ 16 ]  The formation of  micronuclei  generally occurs concurrently with  aneuploidy  and aneuploidy cells are controlled by mechanisms involving  p53 . In order for micronuclei to progress through the  cell cycle  and induce chromosome damage, diminished levels of p53 have been seen to be needed. Through further investigation, chromothriptic tumours have been seen to occur in patients with p53 mutations. [ 17 ] [ 18 ]


Defects in DNA damage response can cause increased frequency of micronucleus formation and hence the occurrence of  chromothripsis. There are numerous examples of how DDR pathways affect chromothripsis and hence cause tumour development and cancers. [ 3 ]


As well as cells encompassing DDR defects, they are likely to have repressed apoptotic mechanisms which will further enhance the occurrence of mutations and  aneuploidy . [ 3 ]


Research in patients with chromothripsis-associated cancers may provide some information about prognosis.  TP53  mutations and chromothripsis have been linked in SHH medulloblastoma patients. [ 1 ]  Poor clinical outcomes in  neuroblastomas  (such as those caused by deletion of the  FANC  gene in Fanconi anaemia) have been linked to frequent chromothripsis occurrence. [ 22 ]   Screening  biopsy  materials for chromothripsis can lead to good  prognosis  estimates and better outcomes for patients. [ citation needed ]


It was pointed out that initial computational simulations underpinning the single-event nature of chromothripsis, which are central to the theory, did not necessarily prove the existence of a single event, and that known models of progressive cancer development do not contradict the occurrence of complex rearrangements. [ 23 ]  It has also been suggested that there is no single traumatic event, but that repeated  breakage-fusion-bridge cycles  might cause the complex genetic patterns. [ 24 ]


In 2015, several research groups presented experimental evidence that chromothripsis is indeed caused by a single catastrophic event. Using a combination of live cell imaging and single-cell genome sequencing of manually isolated cells, it was shown that micronucleus formation can generate a spectrum of genomic rearrangements, some of which recapitulate all known features of chromothripsis. [ 25 ]  Additionally, research studies inducing  telomere  crises followed by sequencing the resultant clones demonstrated complex DNA rearrangements that directly recapitulated the one-off chromosomal catastrophe model of chromothripsis. [ 26 ] [ 27 ]


A  circular chromosome  is a chromosome in  bacteria ,  archaea ,  mitochondria , and  chloroplasts , in the form of a molecule of circular DNA, unlike the  linear chromosome  of most  eukaryotes .


Most prokaryote chromosomes contain a circular DNA molecule. This has the major advantage of having no free ends ( telomeres ) to the  DNA . By contrast, most eukaryotes have linear DNA requiring elaborate mechanisms to maintain the stability of the telomeres and  replicate the DNA . However, a circular chromosome has the disadvantage that after replication, the two progeny circular chromosomes can remain interlinked or tangled, and they must be extricated so that each cell inherits one complete copy of the chromosome during  cell division .


The circular bacteria chromosome replication is best understood in the well-studied bacteria  Escherichia coli  and  Bacillus subtilis . Chromosome replication proceeds in three major stages: initiation, elongation and termination. The initiation stage starts with the ordered assembly of "initiator" proteins at the origin region of the chromosome, called  oriC . These assembly stages are regulated to ensure that chromosome replication occurs only once in each cell cycle. During the elongation phase of replication, the  enzymes  that were assembled at oriC during initiation proceed along each arm ( replichore ) of the chromosome, in opposite directions away from the oriC, replicating the DNA to create two identical copies. This process is known as bidirectional replication. The entire assembly of molecules involved in DNA replication on each arm is called a  replisome . At the forefront of the replisome is a  DNA helicase  that unwinds the two strands of DNA, creating a moving  replication fork . The two unwound single strands of DNA serve as templates for  DNA polymerase , which moves with the helicase (together with other proteins) to synthesise a complementary copy of each strand. In this way, two identical copies of the original DNA are created. Eventually, the two replication forks moving around the circular chromosome meet in a specific zone of the chromosome, approximately opposite oriC, called the terminus region. The elongation enzymes then disassemble, and the two "daughter" chromosomes are resolved before cell division is completed.


The  E. coli   origin of replication , called  oriC  consists of  DNA sequences  that are recognised by the  DnaA  protein, which is highly  conserved  amongst different  bacterial  species. DnaA binding to the origin initiates the regulated recruitment of other enzymes and  proteins  that will eventually lead to the establishment of two complete replisomes for bidirectional replication. [ 1 ]


DNA sequence elements within  oriC  that are important for its function include DnaA boxes, a 9-mer repeat with a highly conserved consensus sequence 5' – TTATCCACA – 3', [ 2 ]  that are recognized by the DnaA protein. DnaA protein plays a crucial role in the initiation of chromosomal DNA replication. [ 3 ]  Bound to ATP, and with the assistance of bacterial  histone -like proteins [HU] DnaA then unwinds an AT-rich region near the left boundary of  oriC , which carries three 13-mer motifs, [ 4 ]  and opens up the  double-stranded DNA  for entrance of other replication proteins. [ 5 ]


This region also contains four “GATC”  DNA unwinding element  sequences that are recognized by  DNA adenine methylase  (Dam), an enzyme that modifies the adenine base when this sequence is unmethylated or hemimethylated. The  methylation  of  adenines  is important as it alters the conformation of DNA to promote strand separation, [ 6 ]  and it appears that this region of  ori C has a natural tendency to unwind. [ 7 ]


DnaA then recruits the replicative  helicase ,  DnaB , from the DnaB-DnaC complex to the unwound region to form the pre-priming complex. [ 8 ]  After DnaB translocates to the apex of each replication fork, the helicase both unwinds the parental DNA and interacts momentarily with  primase . [ 9 ]


In order for DNA replication to continue, single stranded  binding proteins  are needed to prevent the single strands of DNA from forming secondary structures and to prevent them from  re-annealing . In addition,  DNA gyrase  is needed to relieve the topological stress created by the action of DnaB helicase.


When the replication fork moves around the circle, a structure shaped like the Greek letter  theta  Ө is formed.  John Cairns  demonstrated the  theta structure  of E. coli chromosomal replication in 1963, using an innovative method to visualize DNA replication. In his experiment, he  radioactively labeled  the chromosome by growing his cultures in a medium containing 3H- thymidine . The  nucleoside  base was incorporated uniformly into the bacterial chromosome. He then isolated the chromosomes by lysing the cells gently and placed them on an  electron micrograph  (EM) grid which he exposed to  X-ray  film for two months. This Experiment clearly demonstrates the theta replication model of circular bacterial chromosomes. [ 10 ]


As described above, bacterial chromosomal replication occurs in a bidirectional manner. This was first demonstrated by specifically labelling replicating bacterial chromosomes with  radioactive isotopes . The regions of DNA undergoing replication during the experiment were then visualized by using  autoradiography  and examining the developed film microscopically. This allowed the researchers to see where replication was taking place. The first conclusive observations of bidirectional replication was from studies of B. subtilis. [ 11 ]  Shortly after, the E. coli chromosome was also shown to replicate bidirectionally. [ 12 ]


The  E. coli   DNA polymerase III holoenzyme  is a 900 kD complex, possessing an essentially a  dimeric  structure. Each  monomeric  unit has a catalytic core, a  dimerization  subunit, and a  processivity  component . [ 13 ]  DNA Pol III uses one set of its core subunits to synthesize the  leading strand  continuously, while the other set of core subunits cycles from one  Okazaki fragment  to the next on the looped  lagging strand . Leading strand synthesis begins with the synthesis of a short  RNA primer  at the replication origin by the enzyme Primase ( DnaG  protein).


Deoxynucleotides  are then added to this primer by a single DNA polymerase III dimer, in an integrated complex with DnaB helicase. Leading strand synthesis then proceeds continuously, while the DNA is concurrently unwound at the replication fork. In contrast, lagging strand synthesis is accomplished in short Okazaki fragments. First, an RNA primer is synthesized by primase, and, like that in leading strand synthesis, DNA Pol III binds to the RNA primer and adds  deoxyribonucleotides .


When the synthesis of an Okazaki fragment has been completed, replication halts and the core subunits of DNA Pol III dissociates from the β sliding clamp [B sliding clap is the processivity subunit of DNA Pol III]. [ 14 ]  The RNA primer is removed and replaced with DNA by  DNA polymerase I  [which also possesses proofreading  exonuclease  activity] and the remaining nick is sealed by  DNA ligase , which then ligates these fragments to form the lagging strand.


A substantial proportion (10-15%) of the replication forks originating at oriC encounter a  DNA damage  or strand break when cells are grown under normal laboratory conditions (without an exogenous DNA damaging treatment). [ 15 ]   The encountered DNA damages are ordinarily processed by  recombinational  repair enzymes to allow continued replication fork progression. [ 15 ]


Termination is the process of fusion of replication forks and disassembly of the replisomes to yield two separate and complete  DNA molecules . It occurs in the terminus region, approximately opposite oriC on the chromosome (Fig 5). The terminus region contains several DNA replication terminator sites, or "Ter" sites. A special "replication terminator" protein must be bound at the Ter site for it to pause replication. Each Ter site has polarity of action, that is, it will arrest a replication fork approaching the Ter site from one direction, but will allow unimpeded fork movement through the Ter site from the other direction. The arrangement of the Ter sites forms two opposed groups that forces the two forks to meet each other within the region they span. This arrangement is called the "replication fork trap." [ 16 ]


The Ter sites specifically interact with the replication terminator protein called  Tus  in  E. coli . [ 17 ]  The Tus-Ter complex impedes the  DNA unwinding activity  of DnaB in an orientation-dependent manner. [ 18 ]


Replication of the DNA separating the opposing replication forks leaves the completed chromosomes joined as ‘ catenanes ’ or topologically interlinked circles. The circles are not covalently but mechanically linked, because they are interwound and each is covalently closed. The catenated circles require the action of  topoisomerases  to separate the circles (decatenation). In  E. coli , DNA topoisomerase IV plays the major role in the separation of the catenated chromosomes, transiently breaking both DNA strands of one chromosome and allowing the other chromosome to pass through the break.


There has been some confusion about the role  DNA gyrase  plays in decatenation. To define the nomenclature, there are two types of topoisomerases: type I produces transient single-strand breaks in DNA and types II produces transient double-strand breaks. As a result, the type I enzyme removes  supercoils  from DNA one at a time, whereas the type II enzyme removes supercoils two at a time. The topo I of both  prokaryotes  and eukaryotes are the type I topoisomerase. The eukaryotic topo II, bacterial gyrase, and bacterial topo IV belong to the type II.


DNA gyrase also has topoisomerase type II activity; thus, with it being a homologue of topoisomerase IV (also having topoisomerase II activity) we expect similarity in the two proteins' functions. DNA gyrase's preliminary role is to introduce negative super coils into DNA, thereby relaxing positive supercoils that form during DNA replication. Topoisomerase IV also relaxes positive supercoils, therefore, DNA Gyrase and topoisomerase IV play an almost identical role in removing the positive supercoils ahead of a translocating DNA polymerase, allowing DNA replication to continue unhindered by topological strain. [ 19 ]


DNA gyrase is not the sole enzyme responsible for decatenation. In an experiment by  Zechiedrich , Khodursky and Cozzarelli in 1997, it was found that topoisomerase IV is the only important decatenase of DNA replication intermediates in bacteria. [ 20 ]  When DNA gyrase alone was inhibited, most of the catenanes were unlinked. However, when Topoisomerase IV alone was inhibited, decatenation was almost completely blocked. This suggests that Topoisomerase IV is the primary protein for decatenation of interlinked chromosomes  in vivo , with DNA gyrase playing a minor role.


Several groups of bacteria, including  Brucella ,  Paracoccus denitrificans , and  Vibrio  have multiple circular chromosomes.


This is based on an article by Imalda Devaparanam and David Tribe made available under CC by SA licensing conditions from a university course activity at the Department of Microbiology and Immunology, University of Melbourne, 2007. [ citation needed ]
 This article incorporates material from the  Citizendium  article " Replication of a circular bacterial chromosome ", which is licensed under the  Creative Commons Attribution-ShareAlike 3.0 Unported License  but not under the  GFDL .


The  Chromosomal Rearrangements In Diseases  ( dbCRID ) is a database of human  chromosome rearrangements  events and diseases related to them. [ 1 ]


A  derivative chromosome  (der) is a structurally rearranged  chromosome  generated either by a  chromosome rearrangement  involving two or more chromosomes or by multiple  chromosome aberrations  within a single chromosome (e.g. an inversion and a deletion of the same chromosome, or deletions in both arms of a single chromosome). [1]  The term always refers to the chromosome that has an intact centromere.
Derivative chromosomes are designated by the abbreviation  der  when used to describe a  Karyotype . The derivative chromosome must be specified in parentheses followed by all aberrations involved in this derivative chromosome. The aberrations must be listed from  pter  to  qter  and not be separated by a comma.


For example,  46,XY,der(4)t(4;8)(p16;q22)t(4;9)(q31;q31)  would refer to a derivative chromosome 4 which is the result of a translocation between the short arm of chromosome 4 at region 1, band 6 and the long arm of chromosome 8 at region 2, band 2, and a translocation between the long arm of chromosome 4 at region 3, band 1 and the long arm of chromosome 9 at region 3, band 1. As for the initial string "46,XY", it only signifies that this translocation is occurring in an organism, which has this set of chromosomes, i.e. a human being.


An International System for Human Cytogenetic Nomenclature, Shaffer, L.G., Tommerup N. (eds); S. Karger, Basel 2005