A  dicentric chromosome  is an abnormal  chromosome  with two  centromeres . It is formed through the fusion of two chromosome segments, each with a centromere, resulting in the loss of acentric fragments (lacking a centromere) and the formation of dicentric fragments. [ 1 ]  The formation of dicentric chromosomes has been attributed to genetic processes, such as  Robertsonian translocation [ 1 ]  and  paracentric inversion. [ 2 ]  Dicentric chromosomes have important roles in the mitotic stability of chromosomes and the formation of pseudodicentric chromosomes. [ 1 ] [ 3 ]  Their existence has been linked to certain natural phenomena such as  irradiation  and have been documented to underlie certain clinical syndromes, notably  Kabuki syndrome . [ 3 ] [ 4 ]  The formation of dicentric chromosomes and their implications on centromere function are studied in certain clinical  cytogenetics  laboratories. [ 5 ]


Most dicentric chromosomes are known to form through  chromosomal inversions , which are rotations in regions of a chromosome due to chromosomal breakages or intra-chromosomal recombinations. [ 2 ]  Inversions that exclude the centromere are known as paracentric inversions, which result in unbalanced  gametes  after meiosis. [ 2 ]  During prophase of  meiosis I , homologous chromosomes form an  inversion loop  and crossover occurs. If a paracentric inversion has occurred, one of the products will be acentric, while the other product will be dicentric. [ 1 ] [ 2 ]  The dicentric chromatid is pulled apart during anaphase of meiosis I with such force that the chromosome breaks at random positions. [ 2 ]  These broken fragments result in deletions of genes that lead to genetically unbalanced gametes. This can have severe consequences, contributing to the development of genetic disorders such as Kabuki syndrome and Edwards syndrome. [ 2 ]


Irradiation


Radiation  is known to induce abnormalities in the nuclei of cells. [ 5 ]  Dicentric chromosomes were first detected in lymphocytes from blood smears of civil and military personnel who were assigned to deal with the aftermath of the 1986  Chernobyl nuclear disaster  ( liquidators ). [ 4 ]  Radiation increases the probability that dicentric chromosomes form after every mitotic event, creating physical bridges between them in anaphase and telophase. [ 5 ]  As these chromosomes are pulled apart, the chromosome bridges break, resulting in the formation of "tailed" nuclei, protrusions of the nuclei into the cytoplasm. [ 4 ]


Telomeres


When  telomeres  of chromosomes shorten with continued cell divisions, the chromosome ends may also fuse, forming dicentric chromosomes. [ 6 ]  This is considered "crisis", a type of  cell cycle arrest , and most cells in this state suffer  apoptosis . [ 6 ]  The resulting dicentric chromosomes are highly unstable, giving rise to  chromosomal translocations ,  deletions  and amplifications, such as the  Robertsonian translocation . This results in broken, deleted gene products of dicentric fragments. [ 1 ]


Dicentric chromosomes are studied in  model organisms  such as yeast ( Saccharomyces cerevisiae ). [ 7 ]  The use of  S. cerevisiae  as a classical genetic system dates back to the 1950s [ 7 ]  due to its feasibility in  transformation  by  recombinant  DNA. [ 7 ]  Broken chromosomes that lead to acentric and dicentric fragments can be studied in  S. cerevisiae  due to its known ability to tolerate  aneuploidy , an abnormal number of chromosomes. [ 8 ]  Strains of  S. cerevisiae  that tolerate aneuploidy can stabilize products of broken chromosomes during proliferation, which can be recovered and studied in a laboratory setting. [ 7 ] [ 8 ]


Cytogenetics


Centromere function has been the focus of many laboratory analyses, involving techniques like fluorescence  in situ  hybridization ( FISH ) and  chromosomal banding  (C-banding). [ 9 ]  FISH involves the use of fluorescent probes to detect and localize specific DNA sequences on chromosomes, and centromere-specific probes can be used to measure dicentric chromosome  frequencies . [ 10 ]  This technique allows for the testing of human blood samples and tissue samples, which have been historically adopted by radiation laboratories. [ 4 ] [ 5 ] [ 10 ]  C-banding is a staining technique that can be used to visualize dicentric chromosomes. It specifically stains  constitutive heterochromatin , which are regions of the chromosome at or near the centromere. [ 9 ]


The existence of dicentric chromosomes has clinically relevant consequences for individuals, which may live with intellectual, neurological and physical disabilities. Tailed nuclei are signatures of radiation exposure in human tissue,  microbiota , and aquatic invertebrates from the wake of recent nuclear disasters. [ 5 ]  Additionally, they are used as potential  biomarkers  to screen for certain genetic syndromes present in human populations. [ 3 ]


Pseudodicentric chromosomes


Dicentric chromosomes may lead to pseudodicentric mutations, in which one of the centromeres on the chromosome becomes inactivated. [ 1 ]  This can have severe clinical consequences for patients, as observed in severe progressive neurological and intellectual disorders [ 11 ]  such as  Kabuki syndrome , a pediatric  congenital  disorder. [ 3 ]  Kabuki syndrome is one of the most widely studied syndromes that involve the pseudodicentric chromosome. [ 3 ]  Other characterized syndromes have also been linked to pseudodicentric chromosomes, such as  Edwards syndrome , [ 12 ]  a  trisomy  of chromosome 18, and  Turner syndrome , [ 13 ]  a loss (or partial loss) of the X chromosome. Pseudodicentric chromosomes alone do not define these syndromes, because the contribution of other chromosomal abnormalities are also considered, such as the presence of  isochromosomes  in Turner patients. [ 13 ]


Eukaryotic chromosome fine structure  refers to the structure of sequences for  eukaryotic   chromosomes . Some fine sequences are included in more than one class, so the classification listed is not intended to be completely separate.


Some sequences are required for a properly functioning chromosome:


Throughout the eukaryotic kingdom, the overall structure of chromosome ends is conserved and is characterized by the  telomeric tract  - a series of short G-rich repeats. This is succeeded by an extensive subtelomeric region consisting of various types and lengths of repeats - the telomere associated sequences (TAS). [ 1 ]  These regions are generally low in gene density, low in transcription, low in recombination, late replicating, are involved in protecting the end from degradation and end-to-end fusions and in completing replication. The subtelomeric repeats can rescue chromosome ends when telomerase fails, buffer subtelomerically located genes against transcriptional silencing and protect the genome from deleterious rearrangements due to ectopic recombination. They may also be involved in fillers for increasing chromosome size to some minimum threshold level necessary for chromosome stability; act as barriers against transcriptional silencing; provide a location for the adaptive amplification of genes; and be involved in secondary mechanism of telomere maintenance via recombination when telomerase activity is absent.


Other sequences are used in replication or during interphase with the physical structure of the chromosome.


Regions of the genome with protein-coding genes include several elements:


Many regions of the DNA are transcribed with RNA as the functional form:


Other RNAs are transcribed and not translated, but have undiscovered functions.


Repeated sequences  are of two basic types: unique sequences that are repeated in one area; and repeated sequences that are interspersed throughout the genome.


Satellites  are unique sequences that are repeated in tandem in one area.  Depending on the length of the repeat, they are classified as either:


Interspersed sequences  are nonadjacent repeats, with sequences that are found dispersed across the genome. They can be classified based on the length of the repeat as:


Both of these types are classified as  retrotransposons .


Retrotransposons  are sequences in the DNA that are the result of retrotransposition of RNA. LINEs and SINEs are examples where the sequences are repeats, but there are non-repeated sequences that can also be retrotransposons.


Typical eukaryotic chromosomes contain much more DNA than is classified in the categories above.  The DNA may be used as spacing, or have other as-yet-unknown function.  Or, they may simply be random sequences of no consequence.


G-banding ,  G banding  or  Giemsa banding  is a technique used in  cytogenetics  to produce a visible  karyotype  by staining condensed  chromosomes . It is the most common chromosome banding method. [ 1 ]  It is useful for identifying  genetic diseases  (mainly  chromosomal abnormalities ) through the photographic representation of the entire chromosome complement. [ 2 ]


The  metaphase  chromosomes are treated with  trypsin  (to partially digest the chromosome) and  stained  with  Giemsa stain .  Heterochromatic  regions, which tend to be rich with  adenine  and  thymine  (AT-rich) DNA and relatively gene-poor, stain more darkly in G-banding. In contrast, less condensed chromatin ( Euchromatin )—which tends to be rich with  guanine  and  cytosine  ( GC-rich ) and more  transcriptionally  active—incorporates less  Giemsa stain , and these regions appear as light bands in G-banding. [ 3 ]  The pattern of bands are numbered on each arm of the  chromosome  from the  centromere  to the  telomere . This numbering system allows any  band  on the  chromosome  to be identified and described precisely. [ 4 ]  The reverse of G‑bands is obtained in  R‑banding . Staining with Giemsa confers a purple color to chromosomes, but micrographs are often converted to  grayscale  to facilitate data presentation and make comparisons of results from different laboratories. [ 5 ]


The less condensed the chromosomes are, the more bands appear when G-banding. This means that the different chromosomes are more distinct in  prophase  than they are in metaphase. [ 6 ]


It is difficult to identify and group chromosomes based on simple  staining  because the uniform colour of the structures makes it difficult to differentiate between the different chromosomes. Therefore, techniques like G‑banding were developed that made "bands" appear on the chromosomes. These bands were the same in appearance on the  homologous chromosomes , thus, identification became easier and more accurate.


Other types of cytogenic banding are listed below:


A  Holliday junction  is a branched  nucleic acid  structure that contains four double-stranded arms joined. These arms may adopt one of several  conformations  depending on  buffer  salt concentrations and the  sequence  of  nucleobases  closest to the junction. The structure is named after  Robin Holliday , the  molecular biologist  who proposed its existence in 1964.


In biology, Holliday junctions are a key intermediate in many types of  genetic recombination , as well as in  double-strand break repair . These junctions usually have a symmetrical sequence and are thus mobile, meaning that the four individual arms may  slide  through the junction in a specific pattern that largely preserves  base pairing . Additionally, four-arm junctions similar to Holliday junctions appear in some  functional RNA  molecules.


Immobile Holliday junctions, with asymmetrical sequences that lock the strands in a specific position, were artificially created by scientists to study their structure as a model for natural Holliday junctions. These junctions also later found use as basic structural building blocks in  DNA nanotechnology , where multiple Holliday junctions can be combined into specific designed geometries that provide molecules with a high degree of  structural rigidity .


Holliday junctions may exist in a variety of  conformational isomers  with different patterns of  coaxial stacking  between the four double-helical arms. Coaxial stacking is the tendency of nucleic acid  blunt ends  to bind to each other, by interactions between the exposed bases. There are three possible conformers: an unstacked (or open-X) form and two stacked forms. The unstacked form dominates in the absence of  divalent   cations  such as  Mg 2+ , because of electrostatic repulsion between the negatively charged backbones of the strands. In the presence of at least about 0.1 m M  Mg 2+ , the electrostatic repulsion is counteracted and the stacked structures predominate. As of 2000, it was not known with certainty whether the electrostatic shielding was the result of site-specific binding of cations to the junction, or the presence of a diffuse collection of the ions in solution. [ 1 ]


The unstacked form is a nearly square planar, extended conformation. On the other hand, the stacked conformers have two continuous double-helical domains separated by an angle of about 60° in a  right-handed  direction. Two of the four strands stay roughly helical, remaining within each of the two double-helical domains, while the other two cross between the two domains in an  antiparallel  fashion. [ 1 ]


The two possible stacked forms differ in which pairs of the arms are stacked with each other; which of the two dominates is highly dependent on the base sequences nearest to the junction. Some sequences result in an equilibrium between the two conformers, while others strongly prefer a single conformer. In particular, junctions containing the sequence A-CC bridging the junction point appear to strongly prefer the conformer that allows a hydrogen bond to form between the second cytosine and one of the phosphates at the junction point. While most studies have focused on the identities of the four bases nearest to the junction on each arm, it is evident that bases farther out can also affect the observed stacking conformations. [ 1 ]


In junctions with symmetrical sequences, the branchpoint is mobile and can migrate in a  random walk  process. The rate of branch migration varies dramatically with ion concentration, with single-step times increasing from 0.3 to 0.4 ms with no ions to 270−300 ms with 10 mM Mg 2+ . The change in rate is correlated with the formation of the stacked versus the unstacked structures. [ 1 ]


Holliday junctions with a  nick , or break in one of the strands, at the junction point adopt a perpendicular orientation, and always prefer the stacking conformer that places the nick on a crossover strand rather than a helical strand. [ 1 ]


RNA Holliday junctions assume an antiparallel stacked conformation at high magnesium concentrations, a perpendicular stacked conformation at moderate concentrations, and rotate into a parallel stacked conformation at low concentrations, while even small calcium ion concentrations favor the antiparallel conformer. [ 1 ]


The Holliday junction is a key intermediate in  homologous recombination , a biological process that increases genetic diversity by shifting genes between two  chromosomes , as well as  site-specific recombination  events involving  integrases . They are additionally involved in  repair of double-strand breaks . [ 1 ]  In addition,  cruciform structures  involving Holliday junctions can arise to relieve helical strain in symmetrical sequences in  DNA supercoils . [ 2 ]  While four-arm junctions also appear in  functional RNA  molecules, such as  U1 spliceosomal RNA  and the  hairpin ribozyme  of the  tobacco ringspot virus , these usually contain unpaired nucleotides in between the paired double-helical domains, and thus do not strictly adopt the Holliday structure. [ 1 ]


The Holliday junctions in homologous recombination are between identical or nearly identical sequences, leading to a symmetric arrangement of sequences around the central junction. This allows a  branch migration  process to occur where the strands move through the junction point. [ 1 ]  Cleavage, or resolution, of the Holliday junction can occur in two ways. Cleavage of the original set of strands leads to two molecules that may show  gene conversion  but not  chromosomal crossover , while cleavage of the other set of two strands causes the resulting recombinant molecules to show crossover. All products, regardless of cleavage, are  heteroduplexes  in the region of Holliday junction migration. [ 3 ]


Many proteins are able to recognize or distort the Holliday junction structure. One such class contains  junction-resolving enzymes  that cleave the junctions, sometimes in a sequence-specific fashion. Such proteins distort the structure of the junction in various ways, often pulling the junction into an unstacked conformation, breaking the central base pairs, and/or changing the angles between the four arms. Other classes are branch migration proteins that increase the exchange rate by orders of magnitude, and  site-specific recombinases . [ 1 ]  In prokaryotes, Holliday junction resolvases fall into two families, integrases and nucleases, that are each structurally similar although their sequences are not conserved. [ 3 ]


In eukaryotes, two primary models for how homologous recombination repairs double-strand breaks in DNA are the double-strand break repair (DSBR) pathway (sometimes called the  double Holliday junction model ) and the synthesis-dependent strand  annealing  (SDSA) pathway. [ 4 ]  In the case of double strand breakage, the 3' end is degraded and the longer 5' end invades the contiguous sister chromatid, forming a replication bubble. As this bubble nears the broken DNA, the longer 5' antisense strand again invades the sense strand of this portion of DNA, transcribing a second copy. When replication ends, both tails are reconnected to form two Holliday Junctions, which are then cleaved in a variety of patterns by proteins. [ 5 ]  An animation of this process can be seen  here . [ 6 ]


Double-strand DNA breaks in bacteria are repaired by the  RecBCD  pathway of homologous recombination. Breaks that occur on only one of the two DNA strands, known as single-strand gaps, are thought to be repaired by the  RecF pathway . Both the RecBCD and RecF pathways include a series of reactions known as branch migration, in which single DNA strands are exchanged between two intercrossed molecules of duplex DNA, and  resolution , in which those two intercrossed molecules of DNA are cut apart and restored to their normal double-stranded state. [ 7 ]  Homologous recombination occurs in several  groups  of viruses. In  DNA viruses  such as  herpesvirus , recombination occurs through a break-and-rejoin mechanism like in bacteria and eukaryotes. [ 8 ]  In bacteria, branch migration is facilitated by the  RuvABC  complex or  RecG  protein, molecular motors that use the energy of  ATP  hydrolysis to move the junction. The junction must then be resolved into two separate duplexes, restoring either the parental configuration or a crossed-over configuration. Resolution can occur in either a horizontal or vertical fashion during homologous recombination, giving patch products (if in same orientation during double strand break repair) or splice products (if in different orientations during double strand break repair). [ 9 ] [ 10 ]  RuvA and RuvB are branch migration proteins, while RuvC is a junction-resolving enzyme. [ 1 ]


There is evidence for recombination in some  RNA viruses , specifically  positive-sense ssRNA viruses  like  retroviruses ,  picornaviruses , and  coronaviruses . There is controversy over whether homologous recombination occurs in  negative-sense ssRNA viruses  like  influenza . [ 11 ]


In budding yeast  Saccharomyces cerevisiae , Holliday junctions can be resolved by four different pathways that account for essentially all Holliday junction resolution  in vivo . [ 12 ]   The pathway that produces the majority of  crossovers  in  S. cerevisiae  budding yeast, and possibly in mammals, involves proteins  EXO1 ,  MLH1 - MLH3  heterodimer (called MutL gamma) and  SGS1  (ortholog of  Bloom syndrome helicase ). [ 12 ]    The MLH1-MLH3 heterodimer binds preferentially to Holliday junctions. [ 13 ]   It is an endonuclease that makes single-strand breaks in supercoiled double-stranded DNA. [ 13 ] [ 14 ]   The MLH1-MLH3 heterodimer promotes the formation of  crossover recombinants . [ 15 ]   While the other three pathways, involving proteins  MUS81 -MMS4, SLX1 and YEN1, respectively, can promote Holliday junction resolution in vivo, absence of all three nucleases has only a modest impact on formation of crossover products.


Double mutants deleted for both MLH3 (major pathway) and MMS4 (minor pathway) showed dramatically reduced crossing over compared to wild-type (6- to 17-fold); however spore viability was reasonably high (62%) and chromosomal disjunction appeared mostly functional. [ 15 ]


Although MUS81 is a component of a minor crossover pathway in the meiosis of budding yeast, plants and vertebrates, [ 16 ]  in the protozoan  Tetrahymena thermophila , MUS81 appears to be part of an essential, if not the predominant crossover pathway. [ 16 ]  The MUS81 pathway also appears to be the predominant crossover pathway in the fission yeast  Schizosaccharomyces pombe . [ 16 ]


The  MSH4  and  MSH5  proteins form a hetero-oligomeric structure (heterodimer) in yeast and humans. [ 17 ] [ 18 ] [ 19 ]   In the yeast  Saccharomyces cerevisiae  MSH4 and MSH5 act specifically to facilitate crossovers between homologous chromosomes during meiosis. [ 17 ]   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. [ 20 ]   Nevertheless, this mutant gave rise to spore viability patterns suggesting that segregation of non-exchange chromosomes occurred efficiently.   Thus in  S. cerevisiae  proper segregation apparently does not entirely depend on crossovers between homologous pairs.


DNA nanotechnology is the design and manufacture of artificial nucleic acid structures as engineering materials for  nanotechnology  rather than as the carriers of genetic information in living cells. The field uses branched DNA structures as fundamental components to create more complex, rationally designed structures. Holliday junctions are thus components of many such DNA structures. As isolated Holliday junction complexes are too flexible to assemble into large ordered arrays,  structural motifs  with multiple Holliday junctions are used to create rigid " tiles " that can then assemble into larger "arrays". [ 22 ] [ 23 ]


The most common such motif is the double crossover (DX) complex, which contains two Holliday junctions in close proximity to each other, resulting in a rigid structure that can self-assemble into larger arrays. The structure of the DX molecule forces the Holliday junctions to adopt a conformation with the double-helical domains directly side by side, in contrast to their preferred angle of about 60°. The complex can be designed to force the junctions into either a parallel or antiparallel orientation, but in practice the antiparallel variety are more well-behaved, and the parallel version is rarely used. [ 22 ] [ 23 ]


The DX structural motif is the fundamental building block of the  DNA origami  method, which is used to make larger two- and three-dimensional structures of arbitrary shape. Instead of using individual DX tiles, a single long scaffold strand is folded into the desired shape by a number of short staple strands. When assembled, the scaffold strand is continuous through the double-helical domains, while the staple strands participate in the Holliday junctions as crossover strands. [ 25 ]


Some tile types that retain the Holliday junction's native 60° angle have been demonstrated. One such array uses tiles containing four Holliday junctions in a parallelogram arrangement. This structure had the benefit of allowing the junction angle to be directly visualized via  atomic force microscopy . Tiles of three Holliday junctions in a triangular fashion have been used to make periodic three-dimensional arrays for use in  X-ray crystallography  of biomolecules. These structures are named for their similarity to structural units based on the principle of  tensegrity , which utilizes members both in tension and compression. [ 22 ] [ 23 ]


Robin Holliday  proposed the junction structure that now bears his name as part of his model of homologous recombination in 1964, based on his research on the organisms  Ustilago maydis  and  Saccharomyces cerevisiae .  The model provided a molecular mechanism that explained both  gene conversion  and  chromosomal crossover . Holliday realized that the proposed pathway would create   heteroduplex DNA segments  with base mismatches between different versions of a single gene. He predicted that the cell would have a mechanism for mismatch repair, which was later discovered. [ 3 ]  Prior to Holliday's model, the accepted model involved a  copy-choice mechanism [ 26 ]  where the new strand is synthesized directly from parts of the different parent strands. [ 27 ]


In the original Holliday model for homologous recombination, single-strand breaks occur at the same point on one strand of each parental DNA. Free ends of each broken strand then migrate across to the other DNA helix. There, the invading strands are joined to the free ends they encounter, resulting in the Holliday junction. As each crossover strand reanneals to its original partner strand, it displaces the original complementary strand ahead of it. This causes the Holliday junction to migrate, creating the heteroduplex segments. Depending on which strand was used as a template to repair the other, the four cells resulting from  meiosis  might end up with three copies of one allele and only one of the other, instead of the normal two of each, a property known as gene conversion. [ 3 ]


Holliday's original model assumed that heteroduplex DNA would be present on both chromosomes, but experimental data on yeast refuted this. An updated model by  Matt Meselson  and  Charley Radding  in 1975 introduced the idea of branch migration. [ 26 ]  Further observations in the 1980s led to the proposal of alternate mechanisms for recombination such as the double-strand break model (by  Jack Szostak ,  Frank Stahl , and others) and the single-strand annealing model. A third, the synthesis-dependent strand annealing model, did not involve Holliday junctions. [ 3 ]


The first experimental evidence for the structure of the Holliday junction came from  electron microscopy  studies in the late 1970s, where the four-arm structure was clearly visible in images of  plasmid  and  bacteriophage  DNA. Later in the 1980s, enzymes responsible for initiating the formation of, and binding to, Holliday junctions were identified, although as of 2004 the identification of mammalian Holliday junction resolvases remained elusive (however, see section "Resolution of Holliday junctions," above for more recent information). In 1983, artificial Holliday junction molecules were first constructed from synthetic  oligonucleotides  by  Nadrian Seeman , allowing for more direct study of their physical properties. Much of the early analysis of Holliday junction structure was inferred from  gel electrophoresis ,  FRET , and  hydroxyl radical  and  nuclease  footprinting studies. In the 1990s,  crystallography  and  nucleic acid NMR  methods became available, as well as computational  molecular modelling  tools. [ 1 ] [ 3 ] [ 28 ]


Initially, geneticists assumed that the junction would adopt a parallel rather than  antiparallel  conformation, because that would place the homologous duplexes in closer alignment to each other. [ 1 ]  Chemical analysis in the 1980s showed that the junction actually preferred the antiparallel conformation, a finding that was considered controversial, and Robin Holliday himself initially doubted the findings. [ 1 ] [ 3 ]  The antiparallel structure later became widely accepted due to X-ray crystallography data on  in vitro  molecules, although as of 2004 the implications for the  in vivo  structure remained unclear, especially the structure of the junctions is often altered by proteins bound to it. [ 3 ]


The conceptual foundation for DNA nanotechnology was first laid out by  Nadrian Seeman  in the early 1980s. [ 29 ]   A number of natural branched DNA structures were known at the time, including the DNA  replication fork  and the mobile Holliday junction, but Seeman's insight was that immobile nucleic acid junctions could be created by properly designing the strand sequences to remove symmetry in the assembled molecule, and that these immobile junctions could in principle be combined into rigid crystalline lattices.  The first theoretical paper proposing this scheme was published in 1982, and the first experimental demonstration of an immobile DNA junction was published the following year. [ 23 ] [ 30 ]  Seeman developed the more rigid double-crossover (DX)  motif , suitable for forming two-dimensional lattices, demonstrated in 1998 by him and  Erik Winfree . [ 22 ]  In 2006,  Paul Rothemund  first demonstrated the  DNA origami  technique for easily and robustly creating folded DNA structures of arbitrary shape. This method allowed the creation of much larger structures than were previously possible, and which are less technically demanding to design and synthesize. [ 31 ]  The synthesis of a three-dimensional lattice was finally published by Seeman in 2009, nearly thirty years after he had set out to achieve it. [ 32 ]


Homogeneously staining regions  ( HSRs ) are chromosomal segments with various lengths and uniform staining intensity after  G banding . This type of  aberration  is also known as Copy Number Gains or Amplification.


An HSR is one type of change in a  chromosome 's structure which is frequently observed in the  nucleus  of human  cancer   cells . In the region of a chromosome where an HSR occurs, a segment of the chromosome, which presumably contains a  gene  or genes that give  selective advantage  to the progression of the cancer, is amplified or duplicated many times.  As a result of the duplication this chromosomal segment is greatly lengthened and expanded such that when it is stained with a  fluorescent probe  specific to the region ( Fluorescent in situ hybridization ), rather than causing a focal fluorescent signal as in a normal chromosome, the probe "paints" a broad fluorescent signal over the whole of the amplified region.  It is because of the appearance of this broadly staining region that this chromosomal abnormality was named a homogeneously staining region. The homogenously staining region was first observed in chromosome 2 by  June Biedler   and Barbara Spengler in cells that had been made resistant to methotrexate.  The HSR was found to due to the amplification of the  DHFR  gene.


A pair of  homologous chromosomes , or  homologs , is a set of one maternal and one paternal  chromosome  that pair up with each other inside a cell during  fertilization . Homologs have the same  genes  in the same  loci , where they provide points along each chromosome that enable a pair of chromosomes to align correctly with each other before separating during  meiosis . [ 1 ]  This is the basis for  Mendelian inheritance , which characterizes inheritance patterns of genetic material from an  organism  to its offspring parent developmental cell at the given time and area. [ 2 ]


Chromosomes are linear arrangements of condensed  deoxyribonucleic acid  (DNA) and  histone  proteins, which form a complex called  chromatin . [ 2 ]  Homologous chromosomes are made up of chromosome pairs of approximately the same length,  centromere  position, and staining pattern, for genes with the same corresponding  loci . One homologous chromosome is inherited from the organism's mother; the other is inherited from the organism's father. After  mitosis  occurs within the daughter cells, they have the correct number of genes which are a mix of the two parents' genes. In  diploid  (2n) organisms, the genome is composed of one set of each homologous chromosome pair, as compared to tetraploid organisms which may have two sets of each homologous chromosome pair. The  alleles  on the homologous chromosomes may be different, resulting in different phenotypes of the same genes. This mixing of maternal and paternal traits is enhanced by crossing over during  meiosis , wherein lengths of chromosomal arms and the DNA they contain within a homologous chromosome pair are exchanged with one another. [ 3 ]


Early in the 1900s,  William Bateson  and  Reginald Punnett  were studying genetic  inheritance  and they noted that some combinations of alleles appeared more frequently than others. That data and information was further explored by  Thomas Morgan . Using  test cross  experiments, he revealed that, for a single parent, the alleles of genes near to one another along the length of the chromosome move together. Using this logic he concluded that the two genes he was studying were located on homologous chromosomes.
Later on during the 1930s,  Harriet Creighton  and  Barbara McClintock  were studying meiosis in corn cells and examining gene loci on corn chromosomes. [ 2 ]  Creighton and McClintock discovered that the new allele combinations present in the offspring and the event of crossing over were directly related. [ 2 ]  This proved interchromosomal genetic recombination. [ 2 ]


Homologous chromosomes are pairs of chromosomes in a diploid organism that have similar genes, although not necessarily identical. [ 4 ]  There are two main properties of homologous chromosomes: 1) the length of chromosomal arms and 2) the placement of the centromere. [ 5 ]


The actual length of the arm, in accordance with the gene locations, is critically important for proper alignment. Centromere placement on the chromosome can be characterized by four main arrangements, either  metacentric ,  submetacentric ,  acrocentric , or  telocentric . Both of these properties (i.e., the length of chromosomal arms, and the placement of the chromosomal centromere) are the main factors for creating structural homology between chromosomes. Therefore, when two chromosomes containing the relatively same structure exist (e.g., maternal chromosome 15 and paternal chromosome 15), they are able to pair together via the process of synapsis to form homologous chromosomes. [ 6 ]


Since homologous chromosomes are not identical and do not originate from the same organism, they are different from  sister chromatids . Sister chromatids result after  DNA replication  has occurred, and thus are identical, side-by-side duplicates of each other. [ 7 ]


Humans  have a total of 46 chromosomes, but there are only 22 pairs of homologous  autosomal  chromosomes. The additional 23rd pair is the sex chromosomes,  X  and  Y .


Note that the pair of  sex chromosomes  may or may not be homologous, depending on the sex of the individual. For instance, females contain XX, thus have a homologous pair of sex chromosomes. This means that females have 23 pairs of homologous chromosomes in total (i.e., 22 pairs of non-sex chromosomes (autosomes), 1 pair of sex chromosomes). Conversely, males contain XY, which means that they have a non-homologous pair of sex chromosomes as their 23rd pair of chromosomes.


In humans, the 22 pairs of homologous autosomal chromosomes contain the same genes but code for different traits in their allelic forms, as one was inherited from the mother and one from the father. [ 8 ]


So, humans have two sets of 23 chromosomes in each cell that contains a nucleus. One set of 23 chromosomes (n) is from the mother (22 autosomes, 1 sex chromosome (X only)) and one set of 23 chromosomes (n) is from the father (22 autosomes, 1 sex chromosome (X or Y)). Ultimately, this means that humans are  diploid  (2n) organisms.  [ 2 ]


Homologous chromosomes are important in the processes of meiosis and mitosis. They allow for the recombination and random segregation of genetic material from the mother and father into new cells. [ 9 ]


Meiosis is a round of two cell divisions that results in four haploid daughter cells that each contain half the number of chromosomes as the parent cell. [ 10 ]  It reduces the chromosome number in a  germ cell  by half by first separating the homologous chromosomes in  meiosis I  and then the sister chromatids in  meiosis II . The process of meiosis I is generally longer than meiosis II because it takes more time for the chromatin to replicate and for the homologous chromosomes to be properly oriented and segregated by the processes of pairing and  synapsis  in meiosis I. [ 7 ]
During meiosis, genetic recombination (by random segregation) and crossing over produces daughter cells that each contain different combinations of maternally and paternally coded genes. [ 10 ]  This recombination of genes allows for the introduction of new allele pairings and genetic variation. [ 2 ]   Genetic variation  among organisms helps make a population more stable by providing a wider range of genetic traits for  natural selection  to act on. [ 2 ]


In  prophase I  of meiosis I, each chromosome is aligned with its homologous partner and pairs completely. In prophase I, the DNA has already undergone replication so each chromosome consists of two identical chromatids connected by a common centromere. [ 10 ]  During the zygotene stage of prophase I, the homologous chromosomes pair up with each other. [ 10 ]  This pairing occurs by a synapsis process where the  synaptonemal complex  – a protein scaffold – is assembled and joins the homologous chromosomes along their lengths. [ 7 ]   Cohesin  crosslinking occurs between the homologous chromosomes and helps them resist being pulled apart until  anaphase . [ 8 ]  Genetic  crossing-over , a type of recombination, occurs during the pachytene stage of prophase I. [ 10 ]   In addition, another type of recombination referred to as  synthesis-dependent strand annealing  (SDSA) frequently occurs.  SDSA recombination involves information exchange between paired homologous  chromatids , but not physical exchange.  SDSA recombination does not cause crossing-over.


In the process of crossing-over, genes are exchanged by the breaking and union of homologous portions of the chromosomes' lengths. [ 7 ]  Structures called  chiasmata  are the site of the exchange. Chiasmata physically link the homologous chromosomes once crossing over occurs and throughout the process of chromosomal segregation during meiosis. [ 7 ]  Both the non-crossover and crossover types of  recombination  function as processes for repairing  DNA damage , particularly double-strand breaks.  At the diplotene stage of prophase I the synaptonemal complex disassembles before which will allow the homologous chromosomes to separate, while the sister chromatids stay associated by their centromeres. [ 7 ]


In  metaphase I  of meiosis I, the pairs of homologous chromosomes, also known as  bivalents  or  tetrads , line up in a random order along the  metaphase plate . [ 10 ]  The random orientation is another way for cells to introduce genetic variation. Meiotic spindles emanating from opposite spindle poles attach to each of the homologs (each pair of sister chromatids) at the  kinetochore . [ 8 ]


In anaphase I of meiosis I the homologous chromosomes are pulled apart from each other. The homologs are cleaved by the enzyme  separase  to release the cohesin that held the homologous chromosome arms together. [ 8 ]  This allows the chiasmata to release and the homologs to move to opposite poles of the cell. [ 8 ]  The homologous chromosomes are now randomly segregated into two daughter cells that will undergo meiosis II to produce four haploid daughter  germ cells . [ 2 ]


After the tetrads of homologous chromosomes are separated in meiosis I, the sister chromatids from each pair are separated. The two haploid daughter cells (the number of chromosomes has been reduced to half: earlier two sets of chromosomes were present, but now each set exists in two different daughter cells that have arisen from the single diploid parent cell by meiosis I) resulting from meiosis I undergo another cell division in meiosis II but without another round of chromosomal replication. The sister chromatids in the two daughter cells are pulled apart during anaphase II by nuclear spindle fibers, resulting in four haploid daughter cells. [ 2 ]


Homologous chromosomes do not function the same in mitosis as they do in meiosis. Prior to every single mitotic division a cell undergoes, the chromosomes in the parent cell replicate themselves. The homologous chromosomes within the cell will ordinarily not pair up and undergo genetic recombination with each other. [ 10 ]  Instead, the replicants, or sister chromatids, will line up along the metaphase plate and then separate in the same way as meiosis II – by being pulled apart at their centromeres by nuclear mitotic spindles. [ 11 ]  If any crossing over does occur between sister chromatids during mitosis, it does not produce any new recombinant genotypes. [ 2 ]


Homologous pairing in most contexts will refer to germline cells, however also takes place in somatic cells. For example, in humans, somatic cells have very tightly regulated homologous pairing (separated into chromosomal territories, and pairing at specific loci under control of developmental signalling). Other species however (notably  Drosophila ) exhibit homologous pairing much more frequently. In  Drosophila  the homologous pairing supports a gene regulatory phenomenon called  transvection  in which an allele on one chromosome affects the expression of the homologous allele on the homologous chromosome. [ 12 ]  One notable function of this is the  sexually dimorphic  regulation of  X-linked  genes. [ 13 ]


There are severe repercussions when chromosomes do not segregate properly. Faulty segregation can lead to  fertility  problems,  embryo death ,  birth defects , and  cancer . [ 14 ]  Though the mechanisms for pairing and adhering homologous chromosomes vary among organisms, proper functioning of those mechanisms is imperative in order for the final  genetic material  to be sorted correctly. [ 14 ]


Proper homologous chromosome separation in meiosis I is crucial for sister chromatid separation in meiosis II. [ 14 ]  A failure to separate properly is known as nondisjunction. There are two main types of nondisjunction that occur:  trisomy  and  monosomy . Trisomy is caused by the presence of one additional chromosome in the zygote as compared to the normal number, and monosomy is characterized by the presence of one fewer chromosome in the zygote as compared to the normal number. If this uneven division occurs in meiosis I, then none of the daughter cells will have proper chromosomal distribution and non-typical effects can ensue, including Down's syndrome. [ 15 ]  Unequal division can also occur during the second meiotic division. Nondisjunction which occurs at this stage can result in normal daughter cells and deformed cells. [ 5 ]


While the main function of homologous chromosomes is their use in nuclear division, they are also used in repairing  double-strand breaks  of  DNA . [ 16 ]  These double-stranded breaks may occur in  replicating DNA  and are most often the result of interaction of DNA with naturally occurring damaging molecules such as  reactive oxygen species . Homologous chromosomes can  repair  this damage by aligning themselves with chromosomes of the same genetic sequence. [ 16 ]  Once the base pairs have been matched and oriented correctly between the two strands, the homologous chromosomes perform a process that is very similar to recombination, or crossing over as seen in meiosis. Part of the intact DNA sequence overlaps with that of the  damaged chromosome's  sequence.  Replication proteins  and complexes are then recruited to the site of damage, allowing for repair and proper replication to occur. Through this functioning, double-strand breaks can be repaired and DNA can function normally. [ 16 ]


Current and future research on the subject of homologous chromosome is heavily focused on the roles of various proteins during recombination or during DNA repair. In a recently published article by Pezza et al. [ which? ]  the protein known as HOP2 is responsible for both homologous chromosome synapsis as well as double-strand break repair via homologous recombination. The deletion of HOP2 in mice has large repercussions in meiosis. [ 17 ]  Other current studies focus on specific proteins involved in homologous recombination as well.


There is ongoing research concerning the ability of homologous chromosomes to repair double-strand DNA breaks. Researchers are investigating the possibility of exploiting this capability for regenerative medicine. [ 18 ]  This medicine could be very prevalent in relation to cancer, as DNA damage is thought to be contributor to carcinogenesis. Manipulating the repair function of homologous chromosomes might allow for bettering a cell's damage response system. While research has not yet confirmed the effectiveness of such treatment, it may become a useful therapy for cancer. [ 19 ]


Somatic  pairing  of  homologous chromosomes  is similar to pre- and early  meiotic  pairing (see article:  Homologous chromosome#In meiosis ), and has been observed in  Diptera [ 1 ]  ( Drosophila ), and  budding yeast , [ 2 ]  for example (whether it evolved multiple times in metazoans is unclear [ 3 ] ). Mammals show little pairing apart from in germline cells, taking place at specific loci, and under the control of developmental signalling (understood as a subset of other long-range interchromosomal interactions such as looping, and organisation into  chromosomal territories ). [ 4 ]


While meiotic pairing has been extensively studied, the role of somatic pairing has remained less well understood, and even whether it is mechanistically related to meiotic pairing is unknown. [ 5 ]


The first review of somatic pairing was made by Metz in 1916, [ 1 ]  citing the first descriptions of pairing made in 1907 [ 6 ]  and 1908 by  N. M. Stevens  in germline cells, who noted: [ 7 ]


“it may therefore be true that pairing of homologous chromosomes occurs in connection with each mitosis throughout the life history of these insects” (p.215)


Stevens noted the potential for communication and a role in heredity. [ 7 ]


While meiotic homologous pairing subsequently became well studied, somatic pairing remained neglected due to what has been described as " limitations in cytological tools for measuring pairing and genetic tools for perturbing pairing dynamics ". [ 5 ]


In 1998 it was determined that homologous pairing in  Drosophila  occurs through independent initiations (as opposed to a directed, 'processive zippering' motion). [ 4 ] [ 8 ]


The first  RNAi  screen (based on  DNA FISH [ 9 ] ) was carried out to identify genes regulating  D. melanogaster  somatic pairing in 2012, [ 10 ]  described at the time as providing "an extensive “parts list” of mostly novel factors". These comprised 40 pairing promoting genes and 65 'anti-pairing' genes (of which 2 and 1 were already known, respectively), many of which have human  orthologs . [ 5 ]


An earlier RNAi screen in 2007 showed the disruption of  Topoisomerase  II activity impairs somatic pairing within  Drosophila  tissue culture, [ 11 ]  indicating a role for topoisomerase-mediated organisation (or the direct interactions of topoisomerase enzymes) in pairing. [ 4 ]  Condensin (despite dependent interactions with Topoisomerase II) is antagonistic to  Drosophila  homologous pairing. [ 12 ]


The  human β-globin  locus is composed of five  genes  located on a short region of  chromosome 11 , responsible for the creation of the beta parts (roughly half) of the oxygen transport protein  Haemoglobin . This locus contains not only the  beta globin gene  but also  delta ,  gamma-A ,  gamma-G , and  epsilon  globin.  Expression of all of these genes is controlled by single  locus control region  (LCR), and the genes are differentially expressed throughout development. [ 1 ]


The order of the genes in the beta-globin cluster is: 5' -  epsilon  –  gamma-G  –  gamma-A  –  delta  –  beta  - 3'. [ citation needed ]


The arrangement of the genes directly reflects the temporal differentiation of their expression during development, with the early-embryonic stage version of the gene located closest to the LCR. If the genes are rearranged, the gene products are expressed at improper stages of development. [ citation needed ]


Expression of these genes is regulated in embryonic  erythropoiesis  by many  transcription factors , including  KLF1 , [ 2 ]  which is associated with the upregulation of  adult hemoglobin  in adult definitive  erythrocytes , and  KLF2 , [ 3 ]  which is vital to the expression of  embryonic hemoglobin .


Many  CRMs  have been mapped within the cluster of genes encoding β-like globins expressed in embryonic (HBE1), fetal (HBG1 and HBG2), and adult (HBB and HBD) erythroid cells. All are marked by  DNase I hypersensitive sites  and  footprints , and many are bound by  GATA1  in  peripheral blood derived erythroblasts  (PBDEs). A DNA segment located between the HBG1 and HBD genes is one of the DNA segments bound by  BCL11A  and several other proteins to negatively regulate HBG1 and HBG2. It is sensitive to  DNase I  but is not conserved across  mammals . An  enhancer  located 3′ of the HBG1 gene is bound by several proteins in PBDEs and  K562 cells  and is sensitive to DNase I, but shows almost no signal for mammalian constraint. [ 4 ]


An  isochromosome  is an unbalanced  structural abnormality  in which the arms of the  chromosome  are mirror images of each other. [ 1 ]  The chromosome consists of two copies of either the  long (q) arm  or the  short (p) arm  because isochromosome formation is equivalent to a simultaneous  duplication  and  deletion  of  genetic material . Consequently, there is  partial trisomy  of the genes present in the isochromosome and  partial monosomy  of the genes in the lost arm. [ 2 ]


An isochromosome can be abbreviated as i(chromosome number)(centromeric breakpoint). For example, an isochromosome of chromosome 17 containing two q arms can be identified as i(17)(q10).( Medulloblastoma )


Isochromosomes can be created during  mitosis  and  meiosis  through a misdivision of the  centromere  or U-type strand exchange. [ 1 ]


Under normal separation of  sister chromatids  in  anaphase , the centromere will divide longitudinally, or parallel to the long axis of the chromosome. [ 3 ]  An isochromosome is created when the centromere is divided transversely, or perpendicular to the long axis of the chromosome. The division is usually not occurring in the centromere itself, but in an area surrounding the centromere, also known as a pericentric region. [ 2 ]  It is proposed that these sites of exchange contain homologous sequences between sister chromatids. [ 4 ]  Although the resulting chromosome may appear monocentric with only one centromere, it is  isodicentric  with two centromeres very close to each other; resulting in a potential loss of genetic material found on the other arms. [ 2 ] [ 4 ]  Misdivision of the centromere can also produce monocentric isochromosomes, but they are not as common as dicentric isochromosomes. [ 1 ]


A more common mechanism in the formation of isochromosomes is through the breakage and fusion of sister chromatids, most likely occurring in early  anaphase  of mitosis or meiosis. [ 3 ]  A  double-stranded break  in the pericentric region of the chromosome is repaired when the sister chromatids, each containing a centromere, are fused together. [ 2 ]   This U-type exchange of genetic material creates an isodicentric chromosome. [ 5 ]  Misdivision of the centromere and U-type exchange can occur in sister chromatids, thus creating an isochromosome with genetically identical arms. However, U-type exchange can also occur for  homologous chromosomes  which creates an isochromosome with  homologous arms . This exchange between homologues is most likely due to homologous sequences containing  low copy repeats . Regardless of the chromosome involved in U-type exchange, the  acentric fragment  of the chromosome is lost, thus creating a partial monosomy of genes located in that portion of the acentric chromosome. [ 2 ]


The most common isochromosome is the  X sex chromosome . [ 4 ]  Acrocentric  autosomal chromosomes   13 ,  14 ,  15 ,  21 , and  22  are also common candidates for isochromosome formation. [ 1 ]  Chromosomes containing smaller arms are more likely to become isochromosomes because the loss of genetic material in those arms can be tolerated. [ 2 ]


Turner syndrome  is a condition in females in which there is  partial or complete loss  of one X chromosome. This causes symptoms such as  growth  and  sexual development  problems. In 15% of Turner syndrome patients, the structural abnormality is isochromosome X, which is composed of two copies of the q arm (i(Xq)). [ 1 ] [ 2 ]  A majority of i(Xq) are created by U-type strand exchange. A breakage and reunion in the pericentric region of the p arm results in a dicentric isochromosome. [ 4 ]  Some of the p arm can be found in this formation of i(Xq), but a majority of the genetic material on the p arm is lost so it is considered absent. Since the p-arm of the X chromosome contains genes that are necessary for normal sexual development, Turner's syndrome patients experience phenotypic effects. [ 3 ]  Alternatively, the increase in dosage of genes on the q arm may be involved in a 10-fold increase in risk of i(Xq) Turner's patients developing  autoimmune thyroiditis , a disease in which the body creates antibodies to target and destroy thyroid cells. [ 6 ]


Neoplasia  is uncontrolled cell growth, resulting in the creation of a tumour. In many different forms of neoplasia, isochromosome 17q is the most frequent neoplasia associated isochromosome and corresponds with poor patient survival. [ 7 ] [ 8 ]  Unique DNA sequences, known as low copy repeats, occur in the pericentric region of the p arm, so a  crossover event  in that area can create a dicentric isochromosome through U-type strand exchange. [ 9 ]  The neoplasia created from i(17q) is caused by a decrease and increase in gene dosage from the monosomy of the p arm and trisomy of the q arm, respectively. Many candidate  tumour suppressor genes  are found on the lost p arm, allowing the tumour cell population to be maintained. [ 8 ]  It is debated whether the loss of tumour suppressor gene  p53 , located on 17p, is involved in the central  pathogenesis  of some neoplasia. The presence of one p53 gene can be functionally active, but its relation to other  oncogenes  can alter its expression levels when present only in one copy. [ 7 ] [ 8 ] [ 9 ]  Since the genetic sequences involved in i(17q) neoplasia are large, it is difficult to determine which genes, or combination of genes, are involved in tumour growth.


Lampbrush chromosome  are a special form of chromosome found in the growing  oocytes  (immature eggs) of most animals, except mammals. They were first described by  Walther Flemming  and Ruckert in 1882. [ 1 ]  Lampbrush chromosomes of tailed and tailless amphibians, birds and insects are described best of all. [ 2 ] [ 3 ] [ 4 ]   Chromosomes  transform into the lampbrush form during the diplotene stage of meiotic  prophase I  due to an active  transcription  of many genes. They are highly extended meiotic half-bivalents, each consisting of 2 sister chromatids. Lampbrush chromosomes are clearly visible even in the  light microscope , where they are seen to be organized into a series of chromomeres with large chromatin loops extended laterally. Continuous RNA transcription is required to maintain typical chromomere-loop structure of lampbrush chromosomes. Inhibition of transcription leads to retraction of lateral loops into chromomeres and chromosome condensation.


Lampbrush chromosomes produce a large number of mRNAs and non-coding RNAs, which are synthesized on the lateral loops. These transcripts are used during oogenesis and at the early stages of embryogenesis. Each lateral loop contains one or several transcription units with polarized  RNP -matrix coating the DNA axis of the loop. [ 5 ] [ 6 ] [ 7 ]


Amphibian and avian lampbrush chromosomes can be microsurgically isolated from oocyte  nucleus  (germinal vesicle) with either forceps or needles. [ 8 ] [ 9 ]  Giant chromosomes in the lampbrush form are useful model for studying  chromosome  organization,  genome  function and  gene  expression during meiotic prophase, since they allow the individual transcription units to be visualized. [ 10 ]  Moreover, lampbrush chromosomes are widely used for high-resolution mapping of DNA sequences and construction of detail cytological maps of individual chromosomes. [ 11 ]


General


Specific


A  linear chromosome  is a  chromosome  which is linear in shape, and contains terminal ends. In most  eukaryotic cells , DNA is arranged in multiple linear chromosomes. In contrast, most  prokaryotic cells  generally contain a singular  circular chromosome .


In general, the factors which led to the evolution of linear chromosomes in eukaryotes are not well understood. One potential  selective pressure  in favor of linear chromosomes relates to the size of an organism's  genome : linear chromosomes may make  transcription  and replication of large genomes easier. In an organism with a very large genome, circular chromosomes could potentially cause problems relating to  torsional strain . [ citation needed ]


Linear chromosomes are also in some ways disadvantageous or problematic, one of the biggest potential issues being the  end replication problem . This is a phenomenon which occurs due to the directionality of DNA replication enzymes, resulting in the gradual loss of genetic material at the ends of linear chromosomes after each subsequent cycle of cell and DNA replication. In order to mitigate the negative effects of this gradual loss of genetic material, eukaryotes have evolved repetitive,  non-coding  terminal DNA sequences known as  telomeres  on the ends of chromosomes. These repetitive, non-coding sequences are lost instead of important coding DNA, and they are replenished using enzymes known as  telomerases . [ 1 ]  However, telomeres do not fully prevent the loss of coding DNA at the terminal ends of linear chromosomes. In fact, the eventual loss of coding DNA in cellular lines within an organism is thought to play a role in  senescence . [ citation needed ]  Furthermore, evidence suggests that telomeres can be unstable and can be prone to mutations which lead to tumor development. [ 2 ]  Mutations which lead to the constitutive activity of telomerase can result in the loss of  cellular mortality  in tumor cell lines, which is associated with the development of  cancer . [ citation needed ]


Linear chromosomes are not limited to eukaryotic organisms; some prokaryotic organisms have linear chromosomes as well.  Borrelia burgdorferi  was the first bacterium to be found to have a linear chromosome, but new examples, such as various  Streptomyces  and  Coxiella burnetii , have been found. Some bacteria, such as  Agrobacterium tumefaciens , even have one linear chromosome  and  one circular chromosome. These bacteria have also independently invented their own versions of telomeres to protect DNA, though they are not foolproof:  Streptomyces  has telomeres capped by proteins, yet is known to represent "one of the most spectacular examples of genetic instability among prokaryotes". [ 3 ]


Experiments in which the circular chromosomes of prokaryotic organisms have been linearized have demonstrated that some prokaryotes can maintain viability even with linear chromosomes. [ 4 ]


The genomes of most eukaryotic  mitochondria  and  plastids  are in a single circular chromosome, in line with their bacterial ancestor. However, a good number of eukaryotic species do harbor linear  Mitochondrial DNA  (mtDNA), some even broken into multiple molecules, across a wide variety of taxa: animals (mammals, medusozoans, sponges), fungi (especially yeast), plants, and  Alveolatas . In yeast and plants, the shape of mtDNA depends on life-cycle: during some points they may be circular, but during others a linear branched shape is found, consisting of concatenated copies of the original genome. [ 5 ]  In these genomes,  gene conversion  help expand the repeats of their telomeres. [ 6 ]


Similar variations can be found in plastid genomes.  Maize  seedlings have  Chloroplast DNA  (cpDNA) mostly in a branched linear form. [ 7 ]   Acetabularia  have a true linear cpDNA. [ 8 ]


In  genetics , a  locus  ( pl. :  loci ) is a specific, fixed position on a  chromosome  where a particular  gene  or  genetic marker  is located. [ 1 ]  Each chromosome carries many genes, with each gene occupying a different position or locus; in humans, the total number of  protein-coding genes  in a complete  haploid  set of 23 chromosomes is estimated at 19,000–20,000. [ 2 ]


Genes may possess multiple variants known as  alleles , and an allele may also be said to reside at a particular locus.  Diploid  and  polyploid  cells whose chromosomes have the same allele at a given locus are called  homozygous  with respect to that locus, while those that have different alleles at a given locus are called  heterozygous . [ 3 ]  The ordered list of loci known for a particular  genome  is called a  gene map .  Gene mapping  is the process of determining the specific locus or loci responsible for producing a particular  phenotype  or  biological trait .  Association mapping , also known as "linkage disequilibrium mapping", is a method of mapping quantitative trait loci (QTLs) that takes advantage of historic linkage disequilibrium to link phenotypes (observable characteristics) to genotypes (the genetic constitution of organisms), uncovering genetic associations.


The shorter arm of a chromosome is termed the  p arm  or  p-arm , while the longer arm is the  q arm  or  q-arm . The chromosomal locus of a typical gene, for example, might be written  3p22.1 , where: [ citation needed ]


Thus the entire locus of the example above would be read as "three P two two point one". The  cytogenetic bands  are areas of the chromosome either rich in actively-transcribed DNA ( euchromatin ) or packaged DNA ( heterochromatin ). They appear differently upon staining (for example, euchromatin appears white and heterochromatin appears black on  Giemsa staining ). They are counted from the  centromere  out toward the  telomeres . [ citation needed ]


A range of loci is specified in a similar way. For example, the locus of gene  OCA1  may be written "11q1.4-q2.1", meaning it is on the long arm of chromosome 11, somewhere in the range from sub-band 4 of region 1 to sub-band 1 of region 2. [ citation needed ]


The ends of a chromosome are labeled  "pter"  and  "qter" , and so  "2qter"  refers to the terminus of the long arm of chromosome 2. [ citation needed ]


The  Escherichia coli   chromosome  shows two main levels of global organization: macrodomains and  replichores . Macrodomains were discovered by cytological studies. They contain loci showing the same intracellular positioning and choreography during the cell cycle. [ 1 ]


A  mega-telomere  (also known as an  ultra-long telomere  or a  class III telomere ), is an extremely long  telomere  sequence that sits on the end of  chromosomes  and prevents the loss of genetic information during  cell replication . Like regular telomeres, mega-telomeres are made of a repetitive sequence of  DNA  and associated proteins, and are located on the ends of chromosomes. However, mega-telomeres are substantially longer than regular telomeres, ranging in size from 50 kilobases to several megabases (for comparison, the normal length of vertebrate telomeres is usually between 10 and 20 kilobases). [ 1 ]


Telomeres act like protective caps for the chromosome. During cell division, a cell will make copies of its DNA. The  enzymes  in the cell that are responsible for copying the DNA cannot copy the very ends of the chromosomes. This is sometimes called the "end replication problem". If a cell did not contain telomeres, genetic information from the DNA on the ends of chromosomes would be lost with each division. However, because chromosomes have telomeres or mega-telomeres on their ends, repetitive non-essential sequences of DNA are lost instead (See:  Telomere shortening ). [ 2 ] [ 3 ]  While the chromosomes in most  eukaryotic  organisms are capped with telomeres, mega-telomeres are only found in a few species, such as mice [ 4 ]  and some birds. [ 5 ]  The specific function of mega-telomeres in vertebrate cells is still unclear.


Telomeric regions of DNA were first identified in the late 1970s (See:  Discovery of Telomeric DNA ). However, extremely long regions of telomere sequence were not recognized in vertebrates until over a decade later. These sequences, which ranged from 30 to 150 kilobases in size, were first identified in laboratory mice by David Kipling and Howard Cooke in 1990. [ 4 ]


In 1994, extremely long telomeric regions were identified in chickens. [ 6 ]  Telomeric sequences ranging from 20 kilobases to several megabases have also been identified in several species of birds. [ 5 ]  These large regions were termed "ultra-long" telomeres in the literature when they were identified using  southern blotting [ 5 ]  and "mega-telomeres" when identified by  cytogenetic  methods. [ 7 ]  The currently accepted terminology for these sequences is "mega-telomeres" [ 1 ]


Mega-telomeres in vertebrates consist of repeats of a six base-pair sequence, TTAGGG, of DNA. Mega-telomeric DNA also binds to various proteins to form complex structures on the ends of chromosomes. [ 8 ]
Telomeres are identified by  telomere arrays . A telomere array is a unique arrangement of telomeres within a sample (cell, individual, etc.) that is defined by the number of sequence repeats, the pattern of fragments given by restriction digest, the chromosome on which it is found, and the specific location of the sequence on that chromosome. In the literature, mega-telomeres are referred to as Class III telomeres based on the characteristics of their arrays. [ 5 ]


Many studies in model organisms have established the significance of telomere structure and function in regulating genome stability,  cellular aging , and  oncogenesis . [ 9 ]  It has been suggested that mega-teleomeres may serve as protective mechanism against  senescence  in long-lived organisms. [ 9 ]  However, there is some debate on the topic, since telomeric length does not seem to affect lifespan in mice [ 4 ]  and birds with both long and short life-spans have been shown to have mega-telomeres. [ 5 ]


The presence of mega-telomeres varies between species. For example, human chromosomes do not have mega-telomeres while mice and many species of birds do. There is also variation in their structure and location within the same species. In mice and birds, mega-telomeres regions are observed to be hypervariable, meaning that there is a high degree of  polymorphism  in the size and position of mega-telomeres between individuals, including those of highly inbred lines. [ 5 ]  Analysis of siblings from highly inbred chicken-lines have suggested that these ultra-long telomeric sequences are extremely  heterogenous . [ 5 ] [ 9 ]  Similar observations of heterogeneity have also been made in mice. [ 10 ]


In birds, whose cells contain  microchromosomes , it has been suggested that there was a correlation between the presence of mega-telomeres and the number of microchromosomes present in a species, such that bird genomes with large numbers of microchromosomes also possessed larger amounts of telomeric DNA sequence. It was thought that these telomeric sequences might protect genes on these tiny chromosomes from erosion during cell division. [ 5 ]  However, subsequent studies showed that mega-telomeres are not necessarily present in all species with microchromosomes, nor are they found on all microchromosomes within a cell. [ 11 ]  Mega-telomeres are also thought contribute to the high recombination rate of chicken microchromosomes. The longest mega-telomere in chickens is associated with the W (female) chromosome, suggesting that mega-telomeres may also affect sex chromosome organization and the generation of genetic variation. [ 7 ]


The current research exploring mega-telomeres has indicated unexpected heterogeneity and non-Mendelian segregation of mega-telomere profiles between subsequent generations of inbred chicken ( Gallus gallus ) lines. This heterogeneity or inconsistency from generation to generation, despite nearly identical genomic sequences, is evidence that mega-telomeres promote recombination during meiosis. [ 7 ] [ 11 ]  Furthermore, the preferential location on microchromsomes and the discovery of an extremely large mega-telomere on the female-specific W chromosome of avian species also signify the role of mega-telomeres. [ 7 ] [ 8 ]


Microchromosomes  are known to be gene-dense [ 12 ]  and particularly susceptible to damage, thus mega-telomeres may act specifically to protect these gene-rich but fragile chromosomes from erosion [ 7 ] [ 8 ]  or other forms of chromosomal damage. The nearly 3MB telomeric array on the W chromosome suggests that mega-telomeres also play a role in sex-chromosome organization or distribution during, meiosis, however a mechanism is yet to be identified. It does not appear that the presence of mega-telomeres in a genome can alter the "telomere clock" or extend an organism's lifespan. [ citation needed ]


Mega-telomeres have been best described in vertebrate species, specifically inbred mice and chicken lines. In fact, some of the largest mega-telomere arrays were reported in highly inbred and nearly homozygous chicken lines, including UCD 003 [ 13 ]  and ADOL Line 0. [ 1 ]  Normal vertebrate telomere array sizes range from 10 to 20 Kb, [ 14 ]  however, many genetic lines of mouse and chicken possess extreme 50kb or more telomere size arrays. A few other avian species, including Japanese quail ( Coturnix japonica ), ostrich ( Struthio camelus ), and emu ( Dromaius novaehollandiae ). Although most avian genomes are three times smaller than mammalian genomes, their genomes are enriched with telomeric sequence and class III (mega-telomere) arrays, perhaps due to the relatively large number of microchromosomes. [ citation needed ]


The presence of mega-telomeres may be enhanced by the process of domestication or development of highly inbred vertebrate lines. The largest chicken arrays were discovered in the most inbred genetic lines. Studies of full siblings and their progeny from the UCD 003 line, [ 13 ]  established in 1956 and maintained by full-sibling matings, established a consistent profile with 200 Kb or larger telomeres. [ 7 ]  However, less inbred Red jungle fowl families (the hypothesized ancestor of chickens) have slightly shorter Class II arrays and other avian species, such as the American bald eagle ( Haliaeetus leucocephalus ), the northern goshawk ( Accipter gentilis ), possess fewer mega-telomeres and have a considerably smaller telomere size range. Furthermore, laboratory inbred mouse strains ( Mus musculus ) exhibit extremely long telomeres of 30–150 Kb in length, however the wild mouse species ( Mus spretus ) has significantly shorter telomeres ranging from 5–15 Kb. [ 4 ]


A variety of  cytogenetic  and  molecular  methods have been utilized to identify and study mega-telomeres in  vertebrate  species. Many of these techniques allow researchers to both discover the presence of a mega-telomere in a genome but also to characterize telomere arrays. [ citation needed ]


Cytogenetic studies employ  fluorescence in situ hybridization  (FISH) with telomeric probes [ 1 ] [ 8 ]  to label telomeres on chemically-treated cells fixed to glass slides. More specifically, telomere-peptide nucleic acid fluorescein probes are frequently used to identify telomeric sequence repeats on mitotic metaphase and interphase or meiotic pachytene-stage chromosomes. FISH images allow both the identification of mega-telomeric chromosomes and the visualization of chromosome structure, GC-rich DNA regions, and, depending on the experiment, co-localization with genetic regions or genes. [ citation needed ]


Molecular techniques for quantifying telomeric sequences include pulse-field gel electrophoresis ( PFGE ),  slot blot , horizontal gel electrophoresis, and Contour-clamped homogeneous electric field pulse field gel electrophoresis (CHEF-PFGE). In these techniques, purified genomic DNA is isolated and digested with  restriction enzymes , such as  HaeIII , HinfI, AluI, Sau3AI,  EcoRI , EcoRV, PstI, SstI, BamHI, HindIII or BglII, and quantified by fluorometry. [ 8 ] [ 15 ]


The digestion of DNA into smaller fragments by  restriction enzymes , separation of variable-sized DNA fragments via electrophoresis, and labeling of fragments containing telomeric DNA using a specific radio- or fluorescently-labeled probe are the essential steps completed within many molecular techniques. In many cases, the DNA fragments are transferred to distinctive membranes before labeling via blotting techniques (i.e. Southern blot). Specialized protocols have demonstrated the ability to isolate high molecular weight Class III telomeric DNA from Class I and II fragments as well as characterize the size ranges found within each class. The pattern of the telomeric fragments on the stained or labeled membrane is typically unique to the DNA sample (i.e. telomere arrays are rarely identical). Molecular weight markers are usually separated via electrophoresis through agarose gel along with genomic DNA fragments to aid in sizing telomeric arrays and identifying array inter- and intra- individual variability. Slot blot, however, is conducted without DNA fragmentation or separation, rather whole genomic DNA is used to quantify the total concentration of telomeric DNA. The flaw of this technique is that the size of the labelled DNA molecules cannot be identified. In  slot blot  (or  dot blot ), total genomic DNA is attached to a membrane and labeled with a telomere-probe that produces a sample-specific  chemiluminescence  signal, which is captured and quantified by fluorometer equipment and software. A known concentration standard must be labeled and quantified simultaneously in order to accurately determine the telomeric sequence concentration in the DNA samples. [ 15 ]


The  monocentric chromosome  is a chromosome that has only one  centromere  in a  chromosome  and forms a narrow constriction.


Monocentric centromeres are the most common structure on highly repetitive DNA in plants and animals. [ 1 ]


Monocentric chromosomes  as compared to  holocentric chromosomes  where the entire length of the chromosome acts as the centromere. In monocentric chromosomes there is one primary constriction and the centromere its CenH3 loci at this location. [ 2 ]


Holocentric chromosomes are found throughout the plant and animal kingdoms such as the nematode  Caenorhabditis elegans . [ 3 ]  Holocentric chromosomes do have an evolutionary advantage by preventing the loss of chromosome after a DNA double-strand break. [ 4 ]


The centromere is the point of attachment for the  mitotic apparatus [ 5 ]


Deletions, duplications and translocations can produce a  polycentric chromosome .  This is troublesome for cells that divide often since at the time of  anaphase  the polycentric chromosome does not move to opposite poles of spindle fiber and the cell dies. [ 5 ]


Muton  is a term in  genetics  that means the smallest unit in a  chromosome  that can be changed by  mutations . [ 1 ]


The term Muton was created by  Seymour Benzer  in 1955 after his work about the mapping of  bacteriophages   T4 . [ 2 ]


Neocentromeres  are new  centromeres  that form at a place on the chromosome that is usually not centromeric. They typically arise due to disruption of the normal centromere. [ 1 ]  These neocentromeres should not be confused with “knobs”, which were also described as “neocentromeres” in maize in the 1950s. [ 2 ]  Unlike most normal centromeres, neocentromeres do not contain  satellite sequences  that are highly repetitive but instead consist of unique sequences. Despite this, most neocentromeres are still able to carry out the functions of normal centromeres [ 1 ]  in regulating chromosome segregation and inheritance. This raises many questions on what is  necessary versus what is sufficient  for constituting a centromere.


As neocentromeres are still a relatively new phenomenon in cell biology and genetics, it may be useful to keep in mind that neocentromeres may be somewhat related to point centromeres, holocentromeres, and regional centromeres. Whereas point centromeres are defined by sequence, regional and holocentromeres are epigenetically defined by where a specific type of  nucleosome  (the one containing the centromeric  histone H3 ) is located. [ 3 ]


It may also be analytically helpful to take into account that the centromere is generally defined in relation to the  kinetochore , specifically as the “part of the chromosome that links two sister chromatids together via the kinetochore”. However, the emergence of research in neocentromeres troubles this conventional definition and questions the function of a centromere beyond being a “landing pad” for kinetochore formation. [ 4 ]  This expands the scope of the centromere's function to include regulating the function of the kinetochore and the  mitotic spindle .


Neocentromeres were discovered relatively recently. They were first observed by Andy Choo in a human  karyotype  clinic case in 1997, [ 5 ]  using  fluorescent in situ hybridization (FISH)  and  cytogenetic  analysis. The neocentromeres were observed on chromosome 10 of a patient, who was a child with developmental delays.


Cytogenetic and FISH analyses of his chromosomes found three  marker chromosomes : one was a bisatellited chromosome, and two were derived from chromosome 10. Out of the two derived from chromosome 10, one formed a ring and the other a “deleted” version of chromosome 10, labelled as mar del(10). [ 5 ]  The two derivations were confirmed to be from chromosome 10 using characterization of YACs and BACs; [ 6 ]  this confirmation serves as a form of  endogenous control  for the neocentromere-instigating experiment. The mar del (10) and bisatellited chromosomes were present in every cell examined, but the ring chromosome was only present in 4–8% of the cells. [ 5 ]  Some have attributed this statistic to the possible mitotic instability of ring chromosomes. [ 7 ]


Centromeres are conventionally defined by dark-staining  heterochromatin  which consists of the primary constriction site [ 7 ] —this convention exists because heterochromatin is usually found flanking the centromere. With this in mind, centromeric heterochromatin was found on the bisatellited and ring markers, but not on the mar del(10) marker. However, mar del(10) was still able to stably segregate in vivo and in vitro which implied the presence of a functional centromere and kinetochore. There has been an assumption that centromeric heterochromatin is just as important as the kinetochore in segregating and stabilizing chromosomes, because heterochromatin is associated with protein recruitment and has the defining ability to silence gene expression. [ 8 ]  However, this notion is challenged by the observation in mar del (10).


A decade after the initial observation of neocentromeres, 60 more cases of human neocentromeres from across the genome, not just chromosome 10, were documented by 2002. [ 7 ]  These cases were also typically observed in children with developmental delay or  congenital  abnormalities. [ 7 ]  By 2012, more than 90 cases of human neocentromeres across 20 different chromosomes have been described. [ 9 ]


Studies suggest that neocentromeres are ultimately formed from  epigenetic  processes, rather than from changes in DNA sequence. [ 7 ]  There is a general consensus that neocentromeres result from an attempt to fix chromosomes that lack a conventional centromere, through chromosome rearrangements.


The most common type of rearrangement leading to a neocentromere is an inverted duplication, categorized as Class I. The resulting marker chromosome consists of two copies of the chromosome segment. Each of the two copies is a mirror image to the chromosome segment. The neocentromere forms at an interstitial site, between the break point and one of the  telomeres . [ 7 ]  Despite having identical sequences in two halves of the chromosome, the neocentromere is only formed once. In some cases, segregation of these chromosomes result in partial  trisomy , and at other times, a partial  tetrasomy . In partial tetrasomy, the karyotype appears normal except for the marker chromosome.


Mechanisms of formation of neocentromeres are still unclear, but a few have been proposed. It is strongly speculated that neocentromeres form during  mitosis  or  meiosis . [ 10 ]


For Class I, the proposed mechanism is that chromatid breakage occurs during mitosis, resulting in a chromosome fragment. That acentric chromosome fragment may segregate with the intact chromatid and result in partial tetrasomy; or, it may segregate with the complementary broken chromatid and result in partial trisomy because the broken chromatid may be saved by telomere restitution. [ 10 ]  In both cases, the inverted duplicated marker forms only after cell division and replication by rejoining the broken, replicated ends of the fragment. [ 10 ]  It is also suggested that  U-type  exchange during meiosis I may lead to partial tetrasomy.


On the other hand, Class II marker chromosomes result from the second most common type of rearrangement: interstitial deletions. A chromosome is rearranged to give a ring chromosome, and a linear chromosome. Hence, in hindsight, the first observation of neocentromeres made by Andy Choo in 1997 was most likely an example of a Class II pericentric interstitial deletion, followed by a complex rearrangement. The neocentromere can either appear on the linear chromosome or on the ring chromosome, depending on which ever one lacks a centromere. [ 10 ]


For Class II, it is unclear when the rearrangement occurs. General postulations include the chromosome breaking twice, with the ends rejoining. The alternative argument is that during meiosis I, looping and  homologous recombination  within a sister chromatid can cause this rearrangement. [ 10 ]


A rough estimation approximates that neocentromere formation on inverted duplicated chromosomes happens every 70,000–200,000 live births. [ 10 ]  However, this statistic does not include Class II rearrangements.


Human centromeres usually consist of 2000–4000 kilobases of a 171 base pair repeat unit. This alpha satellite is not present at all in human neocentromeres. [ 10 ]  By 2008, more than 90 reported cases of human neocentromeres were detected on marker chromosomes that experienced a loss of centromere, and a subsequent rearrangement. [ 10 ]  Common traits among these human neocentromeres include being  analphoid , staining C-band negative, containing a primary constriction site, and binding to essential centromere proteins that signal the presence of a kinetochore. [ 10 ]


Neocentromeres have been detected in human cancers, [ 10 ]  even though the observed frequency of neocentromeres in the population is relatively low. There is a strong correlation between neocentromeres and a specific class (ALP-WDLPS) of  lipomatous tumors . [ 11 ]  It has been observed that lipomatous tumors with alphoid sequences in the centromere are more aggressive and metastatic. Hence, one suggestion is that neocentromeres may quickly evolve into a centromere with alpha satellite but the correlation between repetitive sequence and aggressiveness of tumors remains unclear. [ 10 ]


Neocentromeres have also been reported in  lung carcinoma  and acute  myeloid leukemia . [ 10 ]  It may be worth noting that neocentromeres in cancer may occur more frequently than currently observed, because many cancer screenings involving karyotyping do not use assays that detect neocentromere formations.


Although not discussed explicitly in relation to neocentromeres, it is worth noting that the  breakage-fusion-bridge cycle  involves ring chromosomes too. [ 12 ]  Particularly, these ring chromosomes involved in breakage-fusion-bridge cycles have a relatively high percentage of prevalence in lipomatous tumors and a lower but still notable prevalence in lung and acute myelogenous leukemia, according to the Mitelman Database. Perhaps further studying of ring chromosomes and diseases, from the perspective of neocentromere formation, may shed light on mechanisms of tumor formation due to chromosome abnormalities. For example, one may question whether the ring chromosomes present in tumors contain a neocentromere following a Class II deletion, because the neocentromere may or may not be present on the ring.


As well as autosomes, human neocentromeres have also been observed in sex chromosomes, and correlate with some sex-linked diseases. In 1999, analysis of a neocentromere in the Y chromosome with a constriction site in the amniocytes of a 38-year-old woman, and in her husband and brother-in-law served as experimental evidence that neocentromeres can be stably transmitted through meiosis into the next generation. [ 13 ]  Not only is the neocentromere viable on a chromosome, it is also sufficient for allowing proper male sex determination. [ 14 ]  These meiotically transmitted neocentric markers show  mosaicism , which many think are due to mitotic instability. [ 7 ]  However, in contrary, others also think that mosaicism could be developed post-zygotically and hence may not be a result of mitotic instability. [ 5 ]  Post-zygotic formation happens when the function of the neocentromere is not established at the time of meiotic rearrangement.


Another case of mosaicism has been observed in a 15-year-old girl with a neocentric X chromosome. [ 9 ]  Her clinical features, along with cytogenetic and FISH analyses confirm that this is the first case of a mosaic  Turner syndrome  involving a neocentromere. During the time of this experiment, only two other cases of neocentric X chromosomes had been observed thus far, making this experiment the third. One of the two previous cases was also confirmed to be Turner syndrome but was not mosaic. An interaction of interest here would be between neocentromeres and the  XIST gene , which is responsible for  X-inactivation . It has been suggested that the abnormality caused by neocentromeres may account for the selective inactivation of the abnormal X chromosome in this patient. Taking into account that only fewer than 5% of Turner syndrome cases are mosaic, [ 9 ]  one may consider, likewise with neocentromere assays in cancer, [ 10 ]  that neocentromeres may occur at a higher frequency in mosaic Turner syndrome than observed.


As aforementioned, the formation of the centromere is well known to be regulated epigenetically. However, those epigenetic mechanisms are still debated contentiously; fortunately, neocentromeres provide a model system for studying different proposed mechanisms. [ 7 ] [ 15 ]


Centromeres are well-associated with specific proteins that are involved in the formation of the kinetochore and the mitotic spindle. Because neocentromeres do not contain repetitive sequences, they are good candidates for studying the epigenetic regulation of distributing centromeric proteins, using  chromatin immunoprecipitation (ChIP)  methods. [ 1 ]


All  centromeres are associated with  centromere protein A  (CENPA). [ 8 ]  CENPA also been widely studied as an important player in centromere regulation as it binds and specifies centromere location for both normal centromeres and neocentromeres, regardless of DNA sequence. There is a general consensus that CENPA assembles into octameric nucleosomes, with two copies of CENPA replacing two copies of histone H3 at the target locus [ 16 ] —this is the simplest model and much is still unknown about the composition of CENPA nucleosomes.


Neocentromeres have been useful tools in investigating the effects of CENPA in the absence of satellite sequences. Those studies conclude that the ability of CENPA to wrap DNA seem to be independent of DNA sequence. [ 16 ]  This leads to several questions: how does CENPA, then, decide where to wrap? What is the purpose of satellite sequences (that are present at most eukaryotic centromeres) if they are not needed for CENPA wrapping? To complicate the matter even further, introducing alpha satellite DNA into cells can cause  de novo  formation of centromeres. [ 7 ]  So far, this suggests that the repetitive sequences may play a fundamental but not necessary role in centromere formation. Moreover, non-repetitive centromeres have also been recently observed in horses, orangutans and chickens. [ 1 ] [ 17 ]


Overexpression of CENPA and  CENPH  (centromere protein H) is also associated with colorectal cancer. It may be worth noticing that the overexpression of these centromeric proteins are also related to neocentromerization. Hence, this may serve as the start of the explanation to how neocentromeres may lead to cancer. Overexpression may be due to a loss of function in regulation of CENPA by proteolysis at inappropriate times of the cell cycle. [ 18 ]  However, this link needs to be further investigated. [ 10 ]


That neocentromeres and conventional centromeres do not share consistent chromatin environment should also be taken into account in questioning the epigenetic regulation of centromere formation. [ 19 ]  The N-terminal tails of  histones  can be modified in several ways, including  phosphorylation ,  acetylation ,  methylation  and  ubiquitination . Although certain histone modifications at centromeres seem to serve a purpose—for example, contributing to a higher order organization of chromatins in mouse centromeres [ 18 ] —conventional and neocentromeres share very few modifications but arguably still maintain the same function of a centromere.


Replenishment of CENPA every cycle, which is important for reestablishing centromere identity, is carried out by  HJURP  (Holliday Junction Recognition Protein), or Scm3 in fungi and CAL1 in  Drosophila . [ 19 ]  Tethering HJURP to a non-centromeric locus can give rise to a neocentromere, even after the disassociation of HJURP. There seems to be a co-evolutionary relationship between the  Drosophila  chaperone CAL1 and CENPA which accounts for species incompatibility—this is discussed more below. [ 20 ]


Whereas deposition of CENPA happens during S phase for  S. cerevisiae , two pathways of CENPA deposition in  S. pombe  determine when CENPA is deposited, namely S phase and G2. In  Arabidopsis thaliana ,  experiments suggest that CENPA deposition via a replication-independent mechanism in G2. For humans, the time seems to be during early G1. [ 18 ]


This temporal regulation is important as it reveals the composition of centromeric chromatin during the time of kinetochore assembly in mitosis. Perhaps reimaging the formation of neocentromeres from the perspective of the cell-cycle might reveal more about what type of regulation is necessary for centromere formation.


One may expect, since the centromere plays such a significant role in chromosome segregation and general inheritance, that the centromere would be highly conserved, in sequence or in epigenetic regulation. However, even though the CENPA histone variant is in fact conserved, there is a surprisingly large amount of diversity in the organization of centromeric chromatin, across different lineages. [ 21 ]  Another striking thing in centromere evolution is that while centromere function is conserved among all eukaryotes the underlying DNA is rapidly evolving, this phenomenon has been coined as the "centromere paradox". [ 22 ]


Yeast,  Drosophila  and mammals all have heterochromatin flanking their centromeres. [ 8 ]  Although the vertebrate chaperone HJURP and the yeast chaperone Scm3 have diverged, their N-terminal domains show striking conservation. [ 23 ]  On the other hand, frogs and chickens have domains in their chaperones that are not at all shared with that of yeast. Hence, further research into the mechanistic properties of these chaperones may potentially reveal how they help determine where and what type of centromere and neocentromere form.


Another element to consider from the evolutionary standpoint is that because neocentromeres are viable and can be transmitted meiotically from one generation to the next, they may play a role in species evolution. Recently, it was shown in  Drosophila  that co-evolution of CENPA and its chaperone CAL1 may explain species incompatibility. [ 20 ]  This incompatibility exists between centromeric histones. This observation encourages neocentromeres to be studied along with their chaperones, to see if neocentromeres may also perhaps have “neo-chaperones” accompanying them.


Over time neocentromeres may also spread in a population and "mature" into full centromeres by accumulating repetitive elements, this can then lead to something known as evolutionary new centromeres. [ 24 ] [ 25 ] [ 26 ]  Centromere repositioning and evolutionary new centromeres are also thought to be involved in  speciation  as incompatibility between centromeres may give rise to  reproductive barriers . [ 27 ] [ 28 ]


Other phenomena or studies, that may or may not be explicitly related to neocentromeres, may pertain to connections that have not been made in scientific literature yet.


Holocentromeres, which are point centromeres distributed throughout the chromosome, have been most extensively studied in worms,  C. elegans . [ 8 ]  Holocentromeres serve as model comparisons to neocentromeres because holocentromeres have seemingly arbitrary “seeds” of CENPA distributed throughout the chromosome, which work together to make a functional kinetochore. [ 1 ]  It is important to note that these CENPA seeds are excluded from genes or loci that are transcribed in the germline or early embryo. This leads to the thought that the seemingly random scattering of these seeds is not inherited, and that each generation or meiosis has its own distinct scatter.


Findings in model organisms, namely inducing neocentromerization in chicken and fungal systems, have brought up a few more correlations for thought. [ 19 ]  Specifically, in chicken DT40 cells, it was found that neither histone modifications nor early replication timing is associated with neocentromere formation. [ 29 ]  Moreover, it was also found that neocentromeres form on both transcriptionally active and inactive loci, [ 29 ]  challenging the widely accepted notion that centromeres do not appear on coding regions of the chromosome. This leads to questions of how neocentromeres may disrupt transcription and expression of those genes.


Finally, a recent discovery is having double-stranded RNA specify centromere location. [ 8 ]  It seems that the arrays of repetitive sequence flanking the centromere are transcribed into RNA that may in turn become  RNAi  machinery that aids the formation of heterochromatin. This may possibly relate to regulating the levels of centromeric proteins, similar to how CENPA levels are regulated by the cell cycle and proteolysis.


Neuroacanthocytosis  is a label applied to several genetic neurological conditions in which the blood contains misshapen, spiculated  red blood cells  called  acanthocytes .


The 'core' neuroacanthocytosis syndromes, in which acanthocytes are a typical feature, are  chorea acanthocytosis  and  McLeod syndrome . Acanthocytes are seen less frequently in other conditions including  Huntington's disease-like syndrome 2 (HDL2)  and  pantothenate kinase-associated neurodegeneration  (PKAN).


The neuroacanthocytosis syndromes are caused by a range of genetic mutations and produce a variety of clinical features but primarily produce  neurodegeneration  of the brain, specifically the  basal ganglia .


The diseases are hereditary but rare.


The hallmark of the neuroacanthocytosis syndromes is the presence of  acanthocytes  in peripheral blood.  Acanthocytosis  originated from the Greek word  acantha , meaning thorn.  Acanthocytes are spiculated red blood cells and can be caused by altered distribution of membrane  lipids  or membrane protein/skeleton abnormalities.  In neuroacanthocytosis, acanthocytes are caused by protein but not lipid membrane abnormalities [ 1 ]


The 'core' neuroacanthocytosis syndromes are  chorea acanthocytosis  and  McLeod syndrome . Acanthocytes are nearly always present in these conditions and they share common clinical features. Some of these features are also seen in the other neurological syndromes associated with neuroacanthocytosis. [ 2 ]


A common feature of the core syndromes is  chorea : involuntary dance-like movements. In neuroacanthocytosis, this is particularly prominent in the face and mouth which can cause difficulties with speech and eating. These movements are usually abrupt and irregular and present during both rest and sleep. [ 3 ]


Individuals with neuroacanthocytosis also often have  parkinsonism , the uncontrolled slowness of movements, and  dystonia , abnormal body postures. Many affected individuals also have cognitive (intellectual) impairment and psychiatric symptoms such as  anxiety ,  paranoia ,  depression , obsessive behavior, and pronounced emotional instability. [ 4 ]  Seizures may also be a symptom of neuroacanthocytosis. [ 5 ]


Onset differs between individual neuroacanthocytosis syndromes but is usually between ages 20 and 40. [ 6 ]  Affected individuals usually live for 10–20 years after onset. [ 7 ]


Chorea acanthocytosis is an  autosomal recessive  disorder caused by mutations in the  VPS13A , also called  CHAC , on chromosome 9q21. The gene encodes the protein  Vacuolar protein sorting-associated protein 13A , also known as chorein. The protein's function is unknown. [ 8 ] [ 9 ]


Chorea acanthocytosis is characterised by  dystonia , chorea and progressive cognitive, behavioural changes and  seizures . Strikingly, many people with chorea acanthocytosis uncontrollably bite their tongue, lips, and the inside of the mouth. Eye movement abnormalities are also seen. [ 9 ]


There are about 500–1,000 cases of chorea acanthocytosis worldwide and it is not specific to any particular ethnic group. [ 9 ]


McLeod syndrome is an  X-linked recessive  disorder caused by mutations in the  XK  gene encoding the  Kx   blood type   antigen , one of the  Kell antigens . [ 10 ]


Like the other neuroacanthocytosis syndromes, McLeod syndrome causes movement disorder, cognitive impairment and psychiatric symptoms. The particular features of McLeod syndrome are heart problems such as  arrhythmia  and  dilated cardiomyopathy  (enlarged heart). [ 10 ]