Microchromosomes are characteristically very small and often  cytogenetically  indistinguishable in a  karyotype , which makes ordering and identifying chromosomes into a coherent  karyotype  particularly difficult. While originally thought to be insignificant fragments of chromosomes, in species where they have been studied they have been found to be rich in  genes  and high in  GC content . In  chickens , microchromosomes have been estimated to contain between 50 and 75% of all genes. [ 5 ] [ 6 ]   During  metaphase , they appear merely as 0.5-1.5  μm  long specks. Their small size and poor condensation into  heterochromatin  means they generally lack the diagnostic  banding  patterns and distinct  centromere  locations used for chromosome identification. [ 7 ]


Microchromosomes are found in many vertebrates, but not in most mammals. [ 1 ]  Important comparisons were made using the genomic organization of the  Florida lancelet  – part of a  sister group  to all  vertebrates  – suggests that the ancestral  amniote  (and vertebrates in general) genome consisted entirely of microchromosomes. Comparison between lancelet and modern vertebrate chromosomes shows that the macrochromosomes were the result of fusion between ancestral microchromosomes. In addition, retention of microchromosomes is shown to be the  norm ; the complete loss of them in mammals is the outlier instead. [ 3 ]


Lancelet


Garfish


Lungfish


Amphibians


Mammals


Squamata  (snakes, lizards)


Turtles


Crocodilians


Birds


Chickens have a  diploid  number of 78 (2 n  = 78) chromosomes, and as is usual in birds, the majority are microchromosomes. Classification of chicken chromosomes varies by author. Some classify them as 6 pairs of macrochromosomes, one pair of sex chromosomes, with the remaining 32 pairs being intermediate or microchromosomes. [ 5 ]  Other arrangements such as that used by the International Chicken Genome Sequencing Consortium include five pairs of macrochromosomes, five pairs of intermediate chromosomes, and twenty-eight pairs of microchromosomes. [ 4 ] [ 8 ]  Microchromosomes represent approximately one third of the total genome size, and have been found to have a much higher gene density than macrochromosomes. Because of this, it is estimated that the majority of genes are located on microchromosomes, [ 6 ]  though due to the difficulty in physically identifying microchromosomes and the lack of  microsatellite markers , it has been difficult to place genes on specific microchromosomes. [ 8 ]


Birds (except  Falconidae ) usually have karyotypes of approximately 80 chromosomes ( 2n = 80 ), with only a few being distinguishable macrochromosomes and an average of 60 being microchromosomes. [ 7 ]   They are more abundant in birds than any other group of animals. Chickens ( Gallus gallus ) are an important model organism for studying microchromosomes. [ 7 ]  Examination of microchromosomes in birds has led to the hypotheses that they may have originated as conserved fragments of ancestral macrochromosomes, and conversely that macrochromosomes could have arisen as aggregates of microchromosomes. [ 7 ]  Comparative  genomic  analysis shows that microchromosomes contain genetic information which has been conserved across multiple classes of chromosomes. This indicates that at least ten chicken microchromosomes arose from fission of larger chromosomes and that the typical bird karyotype arose 100–250  mya . [ 6 ]


Replication timing  and recombination rates have been found to differ between micro- and macrochromosomes in chickens. Microchromosomes replicate earlier in the  S phase  of  interphase  than macrochromosomes. [ 5 ]  Recombination rates have also been found to be higher on microchromosomes. [ 9 ]  Possibly due to the high recombination rates, chicken chromosome 16 (a microchromosome) has been found to contain the most genetic diversity of any chromosome in certain  chicken breeds . [ 9 ]  This is likely due to the presence on this chromosome of the major histocompatibility complex (MHC).


For the many small linkage groups in the chicken genome which have not been placed on chromosomes, the assumption has been made that they are located on the microchromosomes. Groups of these correspond almost exactly with large sections of certain human chromosomes. For example, linkage groups E29C09W09, E21E31C25W12, E48C28W13W27, E41W17, E54 and E49C20W21 correspond with  chromosome 7 . [ 8 ]


The  turkey  has a  diploid  number of 80 (2 n  = 80) chromosomes.  The karyotype contains an additional chromosomal pair relative to the chicken due to the presence of at least two fission/fusion differences (GGA2 = MGA3 and MGA6 and GGA4 = MGA4 and MGA9).  Given these differences involving the macrochromosomes, an additional fission/fusion must also exist between the species involving the microchromosomes if the diploid numbers are valid.   Other rearrangements have been identified through comparative genetic maps, [ 10 ]  physical maps and whole genome sequencing. [ 11 ]


Microchromosomes play a key role in  sex determination  in  soft-shelled turtles . [ 12 ]


Microchromosomes are absent from the karyotypes of  mammals [ 3 ]  and some  amphibians . [ 13 ]  (The  monotreme   platypus  has an intermediate karyotype with smaller chromosomes that are not quite "micro".) [ 3 ]


In rare cases, microchromosomes have been observed in the karotypes of individual humans. A link has been suggested between microchromosome presence and certain genetic disorders like  Down syndrome [ 14 ]   and  fragile X syndrome . [ 15 ]  The smallest chromosome in humans is normally  chromosome 21 , which is 47 Mb.


17q12 microdeletion syndrome , also known as  17q12 deletion syndrome , is a rare  chromosomal anomaly  caused by the  deletion  of a small amount of material from a region in the long arm of  chromosome 17 . It is typified by deletion of the  HNF1B  gene, resulting in kidney abnormalities and  renal cysts and diabetes syndrome . It also has neurocognitive effects, and has been implicated as a genetic factor for  autism  and  schizophrenia .


17q12 microdeletion syndrome is not to be confused with 17q12 microduplication syndrome, caused by the addition of genetic material in the same region from which it is removed in the microdeletion, or 17q21.31 microdeletion syndrome, another name for  Koolen–De Vries syndrome .


17q12 microdeletions have a variable phenotype, ranging from few or no symptoms to severe disability. The condition is thought to be underdiagnosed, and cases with milder phenotypes may not reach clinical attention unless they have an affected child themselves. [ 1 ] [ 2 ]  The most characteristic symptom is  renal cysts and diabetes syndrome  (RCAD), also known as "type 5 diabetes", which is caused by deletion of the associated HNF1B gene in the region. [ 3 ]  RCAD is associated with kidney abnormalities and a characteristic form of diabetes that causes atrophy of the  pancreas . However, some people with 17q12 microdeletions have normal renal function. [ 2 ]  RCAD is diagnosed in approximately 40% of people with 17q12 microdeletions, usually prior to age 25, while kidney abnormalities more broadly occur in approximately 85-90%. [ 4 ]


People with 17q12 microdeletions have a characteristic facial phenotype, albeit a subtle one not usually obvious in daily life.  Macrocephaly  is common, along with high arched eyebrows, flattening of the  malar region , and  epicanthic folds . [ 4 ]  Pathological  short stature  is possible, and a characteristic "short and stocky" body shape occurs in many cases. [ 1 ]


17q12 microdeletions are associated with neurocognitive and developmental involvement of variable severity. Some have mild to moderate  intellectual disability ; however, such impairment is not universal. [ 5 ] [ 6 ]  Average intelligence is in the average to low average range. [ 7 ]   Speech delay  is common, regardless of intellectual functioning. The most striking association between 17q12 microdeletions and neurodevelopment is the raised prevalence of  autism spectrum disorder , with significant increases in both diagnosis and subclinical autistic traits. [ 7 ] [ 8 ]  17q12 microdeletions have been implicated as one of the major genetic causes of high-functioning autistic spectrum disorders. [ 1 ]   Schizophrenia  is also a significant psychiatric complication of 17q12 microdeletion syndrome. 17q12 microdeletions are estimated to occur in approximately 1 in 1,600 people with schizophrenia, compared to an estimation of below 1 in 50,000 in the general population. [ 8 ]   Epilepsy , usually mild, occurs in approximately one-third of cases. [ 6 ]


Reproductive system anomalies are associated with 17q12 microdeletions, particularly in females. 17q12 microdeletions have been linked to uterine malformations, most frequently  Müllerian agenesis , where the  uterus  and part of the  vaginal canal  are absent. [ 4 ] [ 9 ]


17q12 microdeletion syndrome is an  autosomal dominant  disorder, where one copy of the relevant mutation is enough to cause the condition. Most cases are  de novo , or spontaneous mutations that do not occur in the  proband 's parents; [ 10 ]  approximately 75% are de novo, while 25% are inherited. [ 4 ]  People with 17q12 microdeletions who have normal fertility have a 50% chance of passing the deletion down to their offspring. [ 1 ]  Environmental factors have not been implicated in the syndrome. [ 1 ]


Like other chromosomal microdeletions, 17q12 microdeletion syndrome is diagnosed via  fluorescence in situ hybridization . [ 11 ]  Traditional  karyotyping , used to diagnose major chromosomal disorders such as  aneuploidy , is rarely sensitive enough to detect microdeletions. [ 11 ]


As the underlying 17q12 microdeletion is an innate genetic disorder, it cannot by itself be treated. Rather, treatment is symptomatic and supportive. The high prevalence of kidney disease indicates routine monitoring of renal function, particularly in people taking potentially nephrotoxic medications such as  lithium . The comorbities involved in 17q12 microdeletion syndrome require caution in medical treatment; for instance, the increased risk of diabetes requires strict monitoring for post-transplantation diabetes mellitus in kidney transplant patients, as does the risk of weight gain and diabetes from  neuroleptic  drugs in those with a mental health diagnosis. [ 4 ]


The prevalence of 17q12 microdeletion syndrome is unknown, and it is likely to be underdiagnosed. 17q12 microdeletions are estimated to occur in approximately 1 in 600 people on the autism spectrum and 1 in 1,600 with schizophrenia, but are far rarer in the general population. [ 8 ]  General prevalence is estimated to be between 1 in 14,000 [ 4 ]  and 1 in 62,500. [ 12 ]


In addition to the increased prevalence in autism and schizophrenia, some other clinical populations have increased prevalence of 17q12 microdeletion syndrome. The condition occurs in approximately 2% of those with congenital kidney abnormalities and 3-6% of women with Müllerian agenesis. [ 4 ]  It is one of the ten most common microdeletions amongst children with idiopathic  developmental delay . [ 1 ]


17q12 microduplication syndrome is far rarer than the corresponding microdeletion, estimated to occur roughly one-fifth as frequently as 17q12 microdeletion syndrome. Due to its rarity and the overlap between their phenotypes, 17q12 microduplications are usually discussed as an adjunct to microdeletions. [ 3 ]  Like the microdeletion syndrome, the microduplication syndrome has a broad phenotypic range, ranging from asymptomatic to profound disability; intellectual disability is frequently but not always more severe than the microdeletion, while physical health is often better. [ 13 ] [ 14 ]  Epilepsy is a frequent finding. A case of  sex reversal  has been reported. [ 13 ]  While autism comorbid with 17q12 microduplication has been reported, it appears far rarer than in the microdeletion. [ 15 ]  Physical anomalies associated with 17q12 microduplication syndrome include  syndactyly , microcephaly, epicanthic folds, and thick eyebrows or a  unibrow . [ 13 ] [ 14 ]  The 17q12 microduplication appears to have a low  penetrance , as many cases are inherited from asymptomatic parents. [ 13 ]


An  autosome  is any  chromosome  that is not a  sex chromosome . [ 1 ]  The members of an autosome pair in a  diploid  cell have the same  morphology , unlike those in  allosomal  ( sex chromosome ) pairs, which may have different structures. The  DNA  in autosomes is collectively known as  atDNA  or  auDNA . [ 2 ]


For example,  humans  have  a diploid genome  that usually contains 22 pairs of autosomes and one  allosome  pair (46 chromosomes total). The autosome pairs are labeled with numbers (1–22 in humans) roughly in order of their sizes in base pairs, while allosomes are labelled with their letters. [ 3 ]  By contrast, the allosome pair consists of two  X chromosomes  in females or one X and one  Y chromosome  in males. Unusual combinations  XYY ,  XXY ,  XXX ,  XXXX ,  XXXXX  or  XXYY , among other irregular combinations, are known to occur and usually cause developmental abnormalities.


Autosomes still contain sexual determination  genes  even though they are not sex chromosomes. For example, the  SRY  gene on the Y chromosome encodes the transcription factor  TDF  and is vital for male sex determination during development. TDF functions by activating the  SOX9  gene on  chromosome 17 , so mutations of the  SOX9  gene can cause humans with an ordinary Y chromosome to develop as females. [ 4 ]


All human autosomes have been identified and mapped by extracting the chromosomes from a cell arrested in  metaphase  or  prometaphase  and then staining them with a type of dye (most commonly,  Giemsa ). [ 5 ]  These chromosomes are typically viewed as  karyograms  for easy comparison. Clinical geneticists can compare the karyogram of an individual to a reference karyogram to discover the cytogenetic basis of certain  phenotypes . For example, the karyogram of someone with  Patau Syndrome  would show that they possess three copies of  chromosome 13 . Karyograms and staining techniques can only detect large-scale disruptions to chromosomes—chromosomal aberrations smaller than a few million base pairs generally cannot be seen on a karyogram. [ 6 ]


Autosomal genetic disorders can arise due to a number of causes, some of the most common being  nondisjunction  in parental germ cells or  Mendelian inheritance  of deleterious alleles from parents. Autosomal genetic disorders which exhibit Mendelian inheritance can be inherited either in an  autosomal dominant  or recessive fashion. [ 7 ]  These disorders manifest in and are passed on by either sex with equal frequency. [ 7 ] [ 8 ]  Autosomal dominant disorders are often present in both parent and child, as the child needs to inherit only one copy of the deleterious  allele  to manifest the disease. Autosomal recessive diseases, however, require two copies of the deleterious allele for the disease to manifest. Because it is possible to possess one copy of a deleterious allele without presenting a disease phenotype, two phenotypically normal parents can have a child with the disease if both parents are carriers (also known as  heterozygotes ) for the condition.


Autosomal  aneuploidy  can also result in disease conditions. Aneuploidy of autosomes is not well tolerated and usually results in miscarriage of the developing fetus. Fetuses with aneuploidy of gene-rich chromosomes—such as  chromosome 1 —never survive to term, [ 9 ]  and fetuses with aneuploidy of gene-poor chromosomes—such as  chromosome 21 — are still miscarried over 23% of the time. [ 10 ]  Possessing a single copy of an autosome (known as a monosomy) is nearly always incompatible with life, though very rarely some monosomies can survive past birth. Having three copies of an autosome (known as a trisomy) is far more compatible with life, however. A common example is  Down syndrome , which is caused by possessing three copies of  chromosome 21  instead of the usual two. [ 9 ]


Partial aneuploidy can also occur as a result of  unbalanced translocations  during meiosis. [ 11 ]  Deletions of part of a chromosome cause partial monosomies, while duplications can cause partial trisomies. If the duplication or deletion is large enough, it can be discovered by analyzing a karyogram of the individual. Autosomal translocations can be responsible for a number of diseases, ranging from  cancer  to  schizophrenia . [ 12 ] [ 13 ]  Unlike single gene disorders, diseases caused by aneuploidy are the result of improper  gene dosage , not nonfunctional gene product. [ 14 ]


Azoospermia factor  ( AZF ) is one of several proteins or their genes, which are coded from the  AZF region  on the human male  Y chromosome . [ 1 ]  Deletions in this region are associated with inability to produce  sperm . [ 2 ]  Subregions within the AZF region are AZFa (sometimes AZF1), AZFb and AZFc (together referred to as AZF2). AZF microdeletions are one of the major causes of  male infertility  for azoospermia (complete absence of sperm in the ejaculate [ 3 ] [ 4 ] ) and severe oligozoospermia (less than 5 million spermatozoa in the ejaculate [ 4 ] ) males. [ 5 ] [ 6 ]   AZF  is the term used by the HUGO Gene Nomenclature Committee.


Of the 15% of couples who are affected by  infertility , 50% of those cases are due to the male partner. [ 3 ]  15-30% of male factor infertility cases can be correlated with genetic abnormalities. [ 5 ]  One of the most commonly identified genetic abnormalities in male factor infertility are microdeletions on the long arm of the Y chromosome (Yq), specifically at a region known as the azoospermic factor (AZF) region. [ 3 ]


In certain circumstances, men with AZF mutations can turn to assisted reproductive technologies (ART), such as intracytoplasmic sperm injection (ICSI), to help them overcome their suboptimal sperm quality. However, it may be more important for clinicians to screen for Yq microdeletions, due to a growing body of evidence that AZF microdeletions have the capability to be vertically transmitted to male offspring. [ 3 ] [ 4 ] [ 6 ]  Minor et al. demonstrated that an AZFc mutation was vertically transmitted over three generations via fathers receiving reproductive assistance through ICSI. [ 7 ]


The AZF1 ( Azoospermia Factor 1 ) gene is likely located in the  euchromatic  part of the  long arm  in Yq11.23. AZF1 is 792kb long and just  distal  to the  centromere  of the  Y chromosome . [ 8 ]  AZF1 genes are involved in  spermatogenesis  in the  testes .


Common phenotypic manifestations of deletions in this region are azoospermia and Sertoli cell-only syndrome. [ 3 ]  Men with a complete deletion in the AZFa region are unable to produce testicular spermatozoa for ICSI. [ 4 ]  There are several candidate genes in the AZFa region that have been shown to cause infertility in males: Ubiquitin Specific Peptidase 9, Y-Linked (USP9Y), DEAD Box RNA helices, Box3, Y-linked (DBY), Ubiquitously Transcribed Tetratricopeptide Repeat Containing, Y-linked (UTY), and Thymosin Beta 4, Y-Linked (TB4Y). [ 3 ] [ 4 ] [ 6 ]


Originally, the AZFb and AZFc genes were identified and thought to be separate regions.  They were later found to be overlapping and are sometimes referred to as AZF2.


The AZFb subregion is located in the middle region of Yq11. [ 6 ]  Genes in this region have been found to support the growth and maturity of sperm and are critical for efficient progression of spermatogenesis. [ 4 ]  Common phenotypic manifestations of deletions in this region are spermatogenic arrest and azoospermia. [ 3 ] [ 4 ]  There are multiple candidate genes in the AZFb region that have been shown to cause infertility in males: RNA Binding Motif Protein, Y-linked (RBMY), PTPN13-like, Y-linked (PRY), Chromosome Y Open Reading Frame 15 (CYorf15), Ribosomal Protein S4, Y-linked (RPS4Y1), Eukaryotic Translation Initiation Factor 1A, Y-linked (EIF1AY), Lysine Demethylase 5D (KDM5D), X Linked Kell Blood Group Precursor, Y-linked (XKRY), and Heat Shock Transcription Factor, Y-linked (HSFY). [ 3 ] [ 4 ] [ 6 ]


The AZFc subregion is located in the distal part of Yq11. [ 6 ]  Genes in this region have a diverse role, but overall, they are essential to complete spermatogenesis. [ 3 ] [ 4 ]  AZFc deletions have been associated with drastic reduction in sperm count, and there are subsets of men with AZFc microdeletions that experience progressive declines in their sperm count. There are multiple candidate genes in the AZFc region that have been shown to cause infertility in males: Deleted in Azoospermia (DAZ), Chromodomain Protein, Y-linked (CDY), and Basic Protein, Y-linked, 2 (BPY2). [ 3 ] [ 4 ] [ 6 ]


AZFc is one of the most genetically dynamic regions in the human genome, possibly serving as counter against the  genetic degeneracy  associated with the lack of a partner chromosome during  meiosis . [ 9 ]  However, such strategy comes has the adverse effects that some rearrangements represent a risk factor or a de facto causative agent of spermatogenic disruption. [ 9 ]


A specific partial deletion of AZFc called  gr/gr deletion  is significantly associated with  male infertility  among Caucasians in Europe and the Western Pacific region. [ 10 ]


Mutations or deletions in the AZF genes are associated with inability or lessened ability to create sperm. It may cause  azoospermia  (not having any measurable level of sperm in semen). Deletions in the  USP9Y  gene, which is located within AZF1, are usually associated with inability to form sperm.


In addition to the normal  karyotype , wild populations of many animal, plant, and fungi  species  contain  B chromosomes  (also known as  supernumerary,   accessory ,  (conditionally-)dispensable,  or  lineage-specific chromosomes ). [ 1 ]  By definition, these  chromosomes  are not essential for the life of a species, and are lacking in some (usually most) of the individuals. Thus a population would consist of individuals with 0, 1, 2, 3 (etc.) B chromosomes. [ 1 ]  B chromosomes are distinct from  marker chromosomes  or additional copies of normal chromosomes as they occur in  trisomies .


The evolutionary origin of supernumerary chromosomes is obscure, but presumably, they must have been derived from heterochromatic segments of normal chromosomes in the remote past. In general "we may regard supernumeraries as a very special category of  genetic polymorphism  which, because of manifold types of accumulation mechanisms, does not obey the ordinary Mendelian laws of inheritance." [ 1 ]


Next generation sequencing has shown that the B chromosomes from  rye  are amalgamations of the rye A chromosomes. [ 2 ]  Similarly, B chromosomes of the cichlid fish  Haplochromis latifasciatus  also have been shown to arise from rearrangements of normal A chromosomes. [ 3 ]


Most B chromosomes are mainly or entirely  heterochromatic  (i.e. largely  non-coding ), but some contain sizeable  euchromatic  segments [ 4 ]  (e.g. such as the B chromosomes of maize). In some cases, B chromosomes act as  selfish genetic elements . In other cases, B chromosomes provide some positive adaptive advantage. For instance, the British  grasshopper   Myrmeleotettix maculatus  has two structural types of B chromosomes: metacentrics and submetacentric. The supernumeraries, which have a  satellite DNA , occur in warm, dry environments, and are scarce or absent in humid, cooler localities.


There is evidence of deleterious effects of supernumeraries on pollen fertility, and favourable effects or associations with particular habitats are also known in a number of species. [ citation needed ]


B chromosomes have a tendency to accumulate in meiotic cell products resulting in an increase of B number over generations, thereby acting as  selfish genetic elements . However, this effect is counterbalanced for selection against infertility.


Chromosome polymorphisms are very common among  fungi . Different isolates of the same species often have a different chromosome number, with some of these additional chromosomes being unnecessary for normal growth in culture. The extra chromosomes are known as conditionally dispensable, or supernumerary, because they are dispensable for certain situations, but may confer a selective advantage under different environments. [ 5 ]


Supernumerary chromosomes do not carry genes that are necessary for basic fungal growth but may have some functional significance. For example, it has been discovered that the supernumerary chromosome of the pea pathogen  Haematonectria haematococca  carries genes that are important to the disease-causing capacity of the fungus. This supernumerary DNA was found to code for a group of enzymes that metabolize toxins, known as  phytoalexins , that are secreted by the plant's immune system. [ 5 ]  It is possible that these supernumerary elements originated in  horizontal gene transfer  events because sequence analysis often indicates that they have a different evolutionary history from essential chromosomal DNA. [ 5 ]


The wheat-infecting fungal pathogen  Zymoseptoria tritici  contains 8 dispensable B-chromosomes, which is the largest number of dispensable chromosomes observed in fungi. [ 6 ]


B-chromosomes are a significant reflection of genetic diversity between varying plant species. [ 7 ]  These supernumerary chromosomes are commonly observed in  angiosperms , specifically the flowering plants bred through outcrossing. [ 7 ]


The obscure development of B-chromosomes is supported by the irregularity of their appearances in specific species populations. The number of B-chromosomes copied between cells within individual members of a plant population fluctuates. [ 8 ]  For example, the sister species  Aegilops speltoides  and  Aegilops mutica  possess copies of B-chromosomes within their aerial tissues, while their roots exhibit an absence of these supernumerary chromosomes. [ 8 ]


The morphological structure and size of B-chromosomes is different from normally-occurring  chromosomes  in both plants and mammals. [ 8 ]  Most often in plants, B-chromosomes are notably “non-homologous and smaller than the smallest A-chromosome”. [ 8 ]


Balancer chromosomes  (or simply  balancers ) are a type of  genetically engineered   chromosome  used in laboratory biology for the maintenance of  recessive lethal  (or sterile) mutations within living organisms without interference from  natural selection . Since such mutations are viable only in  heterozygotes , they cannot be stably maintained through successive generations and therefore continually lead to production of  wild-type  organisms, which can be prevented by replacing the  homologous  wild-type chromosome with a balancer. In this capacity, balancers are crucial for genetics research on model organisms such as  Drosophila melanogaster , the common fruit fly, for which stocks cannot be archived (e.g. frozen). They can also be used in  forward genetics   screens  to specifically identify recessive lethal (or sterile) mutations. For that reason, balancers are also used in other model organisms, most notably the nematode worm  Caenorhabditis elegans  and the mouse. [ 1 ]


Typical balancer chromosomes are designed to (1) carry recessive lethal mutations themselves, eliminating homozygotes which do not carry the desired mutation; (2) suppress  meiotic recombination  with their homologs, which prevents  de novo  creation of wild-type chromosomes; and (3) carry  dominant   genetic markers , which can help identify rare recombinants and are useful for screening purposes.


Balancer chromosomes were first used in the fruit fly by  Hermann Muller , who pioneered the use of radiation for organismal  mutagenesis . [ 2 ]


In the modern usage of balancer chromosomes, random mutations are first induced by exposing living organisms with otherwise normal chromosomes to substances which cause  DNA  damage; in flies and nematodes, this usually occurs by feeding larvae  ethyl methanesulfonate  (EMS). The DNA-damaged larvae (or the adults into which they develop) are then screened for mutations. When a  phenotype  of interest is observed, the line expressing the mutation is  crossed  with another line containing balancer chromosomes in order to maintain their lineage. [ 3 ]  In one instance, balancers were used to genetically screen a population of  Caenorhabditis elegans . By this point in time, scientists had already realized the benefits of being able to genetically screen populations of organisms for genetic study. Equally as important, they also realized that they could limit  crossing over  in these populations as well as give them very consistent genetic compositions. [ 4 ]


The use of balancer chromosomes has since evolved into a well known and widely used method for  genetic screening  of model organisms. They are even being used to investigate the role of  heterochromatin  packing and the effect it has on genes, [ 5 ]  as well as studies of the effects that  telomeres  have on  gene silencing . [ 6 ]


In  diploid  organisms, mutations without recessive lethal (or sterile) phenotypes can simply be bred to  homozygosity  and maintained stably and indefinitely by crossing homozygotes. However, homozygotes for recessive lethal mutations are by definition non-viable, because the presence of the recessive lethal allele on both chromosomal homologs causes the organism to die early in development; an organism that is homozygous for a recessive mutation that causes sterility yields essentially the same result (i.e. its genetic material cannot be passed on to progeny, even if the sterile individual itself survives to maturity). This problem forces geneticists wanting to study recessive lethal/sterile mutations to maintain the mutation in  heterozygous  organisms instead (in which a chromosome containing a recessive lethal/sterile mutation is complemented by a homolog that functions as wild-type at the same locus, allowing the organism to survive and reproduce more or less normally).


Crosses between heterozygotes yield wild-type organisms in addition to heterozygotes and the non-viable homozygotes. To maintain a purely heterozygous line, wild-type offspring must be identified and prevented from mating. This can be prohibitively resource-intensive, especially if long-term maintenance of the recessive mutation is the goal.


Substituting a balancer chromosome for the wild-type homolog of the chromosome carrying the recessive mutation prevents the establishment of wild-type organisms in various ways. First, a balancer carries its own independent recessive lethal mutation, which makes the organism non-viable if two copies of balancer are inherited (i.e. no copy of the desired mutation). However,  recombination  between the balancer and the homolog containing the mutated allele may also result in the  de novo  creation of a wild-type chromosome. To suppress recombination, balancers usually harbor multiple, nested  chromosomal inversions  so that  synapsis  between the homologous chromosomes is disrupted. [ 7 ]  If crossing over does occur, it is often unbalanced, with each resulting  chromatid  lacking some genes and carrying two copies of others. The process can also lead to dicentric or acentric chromosomes (chromosomes with two  centromeres  or no centromere), which are inherently unstable and usually end up breaking up and mutating or being lost during subsequent mitosis. All of these outcomes are very likely to be lethal.


Finally, flies with balancer chromosomes are easily identified by genetic marker mutations. For example, curly wings or stubbled hair. These phenotypes allow researchers to easily recognize flies that carry the balancer. [ 8 ]  In the unlikely case of viable recombination, the marker may be lost, thus alerting researchers to the event.


Importantly, suppression of recombination by nested inversions only occurs at the inverted intervals, while other regions (usually peri-centromeric and sub-telomeric regions) are free to recombine. Likewise, if the desired mutation is in the same locus as the balancer's recessive lethal mutation (i.e. is in strong  linkage disequilibrium  with it), recombination resulting in a wild-type chromosome is very unlikely, regardless of recombination suppressive inversions.


In addition to simply maintaining an isolated recessive lethal (or sterile) mutation, balancer chromosomes are also useful in  forward genetic screens  to identify such mutations. In such screens randomly mutagenized organisms carrying a balancer are crossed with each other. Offspring that carry the balancer, identified by the dominant marker, can be crossed with littermates. Any such cross that does not produce marker-negative animals is likely the result of a recessive lethal mutation in the non-balancer chromosome. Of course, only the genomic interval covered by the inversions in the balancer can be screened in this way, with recessive lethal mutations in other intervals and on other chromosomes being lost.


Balancer chromosomes are named for the chromosome they serve to stabilize and for the phenotypic or  genetic marker  the balancer carries. [ 9 ]  The naming of balancer chromosomes in  D. melanogaster  has been standardized as follows: the first letter of the chromosome's name represents the number of the chromosome it stabilizes.  F  stands for the first chromosome,  S  for second, and  T  for third. The small fourth chromosome does not undergo recombination and therefore does not require balancing. This letter is then followed by an  M  for "multiply inverted". The  M  is followed by a number to distinguish balancers of the same chromosome. Additionally, the genetic marker or markers within the balancer are listed after the name and separated by a comma. Generally, mutations with easily observable  dominant  phenotypic traits that are often homozygous lethal are used to ensure that all progeny are heterozygous. For example, the commonly used  TM3, Sb  balancer stabilizes the third chromosome and carries a mutant  Sb  ("stubble") gene as a dominant marker. All flies containing the  TM3, Sb  balancer will have shortened or stubbly hairs on the back of their abdomens, which are easily seen when viewed through a microscope. The  3  distinguishes this balancer from other third-chromosome balancers such as  TM1  and  TM2 .


A line is said to be "double-balanced" if it is heterozygous for two different balancer chromosomes (for example,  TM6, Tb/TM3, Ser ) on one chromosome and a homozygous-lethal, heterozygous-visible mutant on the other,  wild-type  chromosome (for example,  D/TM3, Ser ). Most balancer chromosomes also carry a recessive allele such as the "ebony" mutation that is only manifest in these stocks with two balancer chromosomes. Such stocks are often used to provide sources of easily traceable traits when breeding two different lines together, so that the correct progeny of each cross can be selected. Stocks double-balanced at both the second and third chromosomes in  Drosophila  are widely available from fly stock repositories.


Balancer chromosomes give geneticists a reliable method for genetically screening organisms for a specific mutation and maintaining that mutation consistently in subsequent generations. A new technique using balancer chromosomes is explored in the paper  "The Autosomal Flp-Dfs Technique for Generating Germline Mosaics in Drosophila Melanogaster" , which showed for the first time that it is possible to screen for a recessive mutation that only shows a phenotype when homozygous. Using old balancer chromosome methods, genetic screening only allowed for the selection of heterozygous dominant mutations. This experiment uses clonal screening to detect homozygous individuals and keep them in a constant line. [ 12 ]  They achieved this by using the  FLP recombinase  gene, isolated from yeast, which causes large  chromosomal inversions . Through trial and error they found that the chromosomes could be recombined such that each had the recessive mutation while the other half contained half of a balancer chromosome with a physical marker and a lethal recessive. The other homolog did not contain the lethal recessive in the lines that survived. Figure one in the paper illustrates the screen. This new technique allowed recessive screening in 95% of the  Drosophila  genome. It also greatly improved yields in germ-line mutations. [ 12 ]


Another published paper that employed the use of balancer chromosomes is  "Inhibition of RNA Interference and Modulation of Transposable Element Expression by Cell Death in Drosophila" . This paper demonstrates the power of balancer chromosomes and what can be accomplished with genetically stable lines. A line was established that exhibited low levels of cell death and was named EGFPir hs-hid. When the  RNAi  levels were analyzed, the authors found interesting results in the cells undergoing low levels of cell death and the surrounding cells in the tissue. They found that these cells would shut down their RNAi mechanism via maintaining RNA in a double-stranded state; i.e. if RNA remains in a double-stranded state, then the RNAi mechanism of gene silencing is effectively disabled.


The authors speculated that this response was an evolutionary trend toward a redundant immune response against RNA viruses. If one cell is already undergoing cell death to attempt to stop the spread of a virus, then the RNAi immune response has been ineffective. This causes another immune response that attempts to stop the virus, which is binding double-stranded RNA and keeping it double-stranded so that it cannot be transcribed into viral proteins. The precise mechanism by which double-stranded RNA is maintained is not known. [ 13 ]


In  cellular biology , a  bivalent  is one pair of  chromosomes  (homologous chromosomes) in a  tetrad . A tetrad is the association of a pair of   homologous chromosomes  (4 sister  chromatids ) physically held together by at least one  DNA crossover . This physical attachment allows for alignment and segregation of the homologous chromosomes in the first  meiotic  division. In most organisms, each replicated chromosome (composed of two identical sisters chromatid) elicits formation of DNA double-strand breaks during the leptotene phase. These breaks are repaired by  homologous recombination , that uses the homologous chromosome as a template for repair. The search for the homologous target, helped by numerous proteins collectively referred as the  synaptonemal complex , cause the two homologs to pair, between the leptotene and the pachytene phases of meiosis I.


The formation of a bivalent occurs during the first division of  meiosis  (in the  zygotene  stage of meiotic prophase 1). In most organisms, each replicated chromosome (composed of two identical sister  chromatids [ 1 ] [ 2 ] ) elicits formation of DNA double-strand breaks during the leptotene phase. [ 3 ]  These breaks are repaired by  homologous recombination , that uses the homologous chromosome as a template for repair. The search for the homologous target, helped by numerous proteins collectively referred as the  synaptonemal complex , cause the two homologs to pair, between the leptotene and the pachytene phases of meiosis I. [ 4 ]  Resolution of the DNA recombination intermediate into a  crossover  exchanges DNA segments between the two homologous chromosomes at a site called a  chiasma (plural: chiasmata) . This physical strand exchange and the cohesion between the sister chromatids along each chromosome ensure robust pairing of the homologs in diplotene phase. The structure, visible by microscopy, is called a bivalent. [ 5 ]  Resolution of the DNA recombination intermediate into a  crossover  exchanges DNA segments between the two homologous chromosomes at a site called a  chiasma (plural: chiasmata) . This physical strand exchange and the cohesion between the sister chromatids along each chromosome ensure robust pairing of the homologs in diplotene phase. The structure, visible by microscopy, is called a bivalent. An intricate molecular machinery is at the core of gene expression regulation in every cell. During the initial stages of organismal development, the coordinated activation of diverse transcriptional programs is crucial and must be carefully executed to shape every organ and tissue. Bivalent which promoters and poised enhancers are regulatory regions decorated with histone marks that are associated with both positive and negative transcriptional outcomes. Finally, we highlight the potential link between bivalency and cancer which could drive biomedical research in disease etiology and treatment.


The information of a one gene should be the different in executive way in the cell types to achieve main program in this diversity. Chromatin is carrier of the instructions and also the DNA surrounded by the histones shows impact of the nucleosome which we can see this is the basic unit. The packed gives information for regulation nucleosome of physical barrier they show impact on the chromatin remodelers parts N- terminal parts of histone particle, histone tails, covalent post-translational modifies and also creates an epigenetics of [PCG] and [TRXG] plays an initial role these mutations caused in groups from transformation in Drosophila shows a clearcut information


A bivalent is the association of two replicated homologous chromosomes having exchanged DNA strand in at least one site called chiasmata. Each bivalent contains a minimum of one chiasma and rarely more than three. This limited number (much lower than the number of initiated DNA breaks) is due to  crossover interference , a poorly understood phenomenon that limits the number of resolution of repair events into crossover in the vicinity of another pre-existing crossover outcome, thereby limiting the total number of crossovers per homologs pair. [ 4 ]  Bivalent gene is a gene marked with both H3K4me3 and H3K27me3 epigenetic modification in the same area of this kind and is proposed to play a pivotal role related to pluripotency in embryonic stem (ES) cells. Bivalent promoters marked with both H3K27me3 and H3K4me3 histone modifications are characteristic of poised promoters in embryonic stem (ES) cells. The model of poised promoters postulates that bivalent chromatin in ES cells is resolved to Mono valency upon differentiation. With the availability of single-cell RNA sequencing (scRNA-seq) data, subsequent switches in transcriptional state at bivalent promoters can be studied more closely.


At the meiotic metaphase I, the  cytoskeleton  puts the bivalents under tension by pulling each homolog in opposite direction (contrary to mitotic division where the forces are exerted on each chromatid). The anchorage of the cytoskeleton to the chromosomes takes place at the  centromere  thanks to a protein complex called  kinetochore . This tension results in the alignment of the bivalent at the center of the cell, the chiasmata and the distal cohesion of the sister chromatids being the anchor point sustaining the force exerted on the whole structure. Impressively, human female primary  oocytes  remains in this tension state for decades (from the establishment of the oocyte in metaphase I during embryonic development, to the  ovulation  event in adulthood that resume the meiotic division), highlighting the robustness of the chiasma and the cohesion that hold the bivalents together. The cell transcription regulates of developmental genes We develop an approach for capturing genes undergoing transcriptional switching by detecting 'bimodal' gene expression patterns from scRNA-seq data. We integrate the identification of bimodal genes in ES cell differentiation with analysis of chromatin state and for kind of then identify clear cell-state dependent patterns of bimodal, bivalent genes. We show that binarization of bimodal genes can be used to identify differentially expressed genes from fractional ON/OFF proportions. In time series data from differentiating cells, we build a pseudo time approximation and use a hidden Markov model to infer gene activity switching pseudo times, which we use to infer a regulatory network. We identify pathways of switching during differentiation, novel details of those pathway, and transcription factor coordination with downstream targets.


Conclusions: Genes with expression levels too low to be informative in conventional scRNA analysis can be used to infer transcriptional switching networks that connect transcriptional activity to chromatin state.  in with analysis of chromatin state and for kind of then identify clear cell-state dependent patterns of bimodal, bivalent genes. We show that binarization of bimodal genes can be used to identify differentially expressed genes from fractional ON/OFF proportions. In time series data from differentiating cells, we build a pseudo time approximation and use a hidden Markov model to infer gene activity switching pseudo times, which we use to infer a regulatory network. We identify pathways of switching during differentiation, novel details of those pathway, and transcription factor coordination with downstream targets. This offers a novel and productive means of inferring regulatory networks from scRNA-seq data.


Keywords: Bimodality; Bivalency; Chromatin state; Embryonic stem cells; Genome regulatory network; Hidden Markov model; Pseudo time; scRNA-seq.


Blanco E., Gonzalez Ramirez M., Alcaine-colet , A., Aranda the bivalent genome ; characterization structure trends in genetics


. Thomson JA, itskovitz-Eldor J,Shapiro SS, et al.


. Santos-Rosa H, Schneider R, Beernstein BE, et al. Methylation of histone H3 K4


. Ringrose L, ehret H, paro R. District contribution of histones H3 lysine 9 and 27


c7orf26 (Chromosome 7, Open Reading Frame 26)  is a  gene  in humans that encodes a  protein  known as c7orf26 (uncharacterized protein c7orf26). Based on properties of c7orf26 and its conservation over a long period of time, its suggested function is targeted for the  cytoplasm  and it is predicted to play a role in regulating  transcription .


Chromosome 7  is one of the 23 pairs of chromosomes in the human body, and spans about 159 million base pairs and represents about 5-5.5% of the total DNA in cells. [ 1 ]  Changes to the structure of chromosome 7 can result in a number of genetic abnormalities, including  Williams Syndrome  which causes structural and cosmetic changes to the human body, ultimately resulting in a shorter lifespan. [ 2 ]  There are hundreds of known  open reading frames  (ORF) along the domain of chromosome 7, however there is not much known about the 26th reading frame, which is of considerable interest.


Currently, two  isoforms  of c7orf26 are known in  Homo Sapiens  and are referred to as isoforms 1 and 2, respectively. [ 3 ]


c7orf26 (accession: NM_024067 / NP_076972; alias: MGC-2178) is located on the long arm of  chromosome 7  (7p22.1), starting at 6590021 and ending at 6608726. The c7orf26 gene spans 2178  base pairs  and is orientated on the + strand. The coding region is made up of a protein sequence measuring 449  amino acids  long. It is divided into 6 transcripts containing a total of 24  exons  on the forward strand and has 5952 unique  Single Nucleotide Polymorphisms  (SNPs). [ 4 ]


Genes  ZDHHC4 , ZNF853 and ZNF316 neighbor c7orf26 on chromosome 7. [ 5 ]  Gene ZDHHC4 is a zinc-finger protein involved with  cytochrome-c oxidase  activity and protein-cysteine S-palmitoyltransferase activity and has overlapping regions with c7orf26. [ 6 ]  Gene GRID2IP lies upstream by >2000 bp of c7orf26, and is heavily involved with in  synaptogenesis  and synaptic plasticity. [ 7 ]


c7orf26 is highly expressed in lymphatic, reproductive, and nervous tissue. These include the brain (frontal and occipital cortex), thymus glands, salivary glands, endometrium, cervix, and prostate. It is intermediately expressed in the lungs. [ 8 ]


No  paralogs  of c7orf26 have been found in the human genome, however, six unique  isoforms  have been identified. They are c7orf26 isoform (X1, X2, X3, X4) and isoform 2 (two sub-isoforms identified). [ 9 ]


Below is a table of a variety of   orthologs  of the human c7orf26. The table include closely, intermediately and distantly related orthologs. [ 10 ]  Orthologs of the human protein c7orf26 are listed above in descending order of the date of divergence. c7orf26 is highly conserved throughout all orthologs, this is demonstrated with a 65% identity in the least similar ortholog. c7orf26 has evolved slowly and evenly over time.


Below is a phylogenetic tree showing the evolutionary history of c7orf26 and its nearest orthologs.


The  molecular weight  of c7orf26 is 50  kilodaltons . The  isoelectric point  is 7.61. The protein sequence is uniquely rich for  leucine  at 15.8% of its composition, this may indicate a  leucine-zipper . Further analysis from PSORT indicates that a leucine-zipper region is found at amino acid 318 and lasts until position 340 (22 amino acids long). There are no extremes with regards to acidity and alkalinity. c7orf26 has a positive charge cluster from amino acid 245 – 275 and does not have any negative, or mixed charge clusters. [ 11 ]


An even distribution of amino acids compose c7orf26. The percent composition of each amino acid is fairly consistent throughout the orthologs of the protein. The most distant ortholog displays the most variance in amino acid composition. There is a higher percent composition of  tyrosine ,  histidine  and  leucine  and a lower composition of  valine  and  alanine .


c7orf26 is highly  phosphorylated   post modified . There are 66 predicted phosphorylated sites according to the NetPhos predictor of phosphorylation sites. [ 12 ]  There are 4 unique  sumoylation  sites according to SUMOplot/SUMOsp programs. [ 13 ]  Sumoylation sites are involved in a number of cellular processes, including nuclear-cytosolic transport, transcriptional regulation and protein stability.


According DAS-TMFilter Server, [ 14 ]  c7orf26 has zero predicted transmembrane sites or transmembrane protein coding regions, therefore, it can be inferred with certainty that c7orf26 is  not  a transmembrane protein.


Using the GOR ( G arnier- O sguthorpe- R obson) [ 15 ]  method, it can be inferred that c7orf26 has unique  secondary structure  composed of alpha helices, random coil regions and extended strands. Random coil regions are most found in c7orf26, as they constitute 53.23% of the protein, while alpha helices constitute 34.30% and extended strands 12.47%.


According to  PSORT , c7orf26 is predicted to be localized in the cytoplasm with 70.6% confidence. [ 16 ]


c7orf26 interacts uniquely with 11 different proteins, according to the Mentha  interactome  browser. [ 17 ]  In particular, c7orf26 interacts with the entire family of ' INTS ' (Integrator Complex Subunit 1–7). The Integrator Complex associates with the  C-terminal  domain of  RNA polymerase II  large subunit. It is involved in the transcription and processing of their transcripts. INTS mediates recruitment of cytoplasmic  dynein  to the  nuclear envelope .


Outside of the INTS gene family, c7orf26 interacts with AK5, [ 18 ]   HDGF , and ASUN. [ 19 ]


According to Guirato et al. (2018), there may be some evidence that regions on  chromosome 7  may be directly linked to a nuclear estrogen receptor (ESR2) that modulates cancer cell proliferation and tumor growth. [ 20 ]  In another journal article by Fu et al. (2014), there is further indication that regions along chromosome 7, located between  open reading frames  20-30, directly correlate to  cellular functions of a hepatoma-derived growth factor (HDGF), another way of expressing normal function in tumorigenesis. [ 21 ]


NM_001031748 NM_001319247


NP_001026918 NP_001306176


C12orf40 , also known as  Chromosome 12 Open Reading Frame 40 ,  HEL-206 , and  Epididymis Luminal Protein 206  is a  protein  that in  humans  is encoded by the  C12orf40   gene . [ 3 ]


In humans, the gene for  C12orf40  is located on  chromosome 12 . There are 13 exons in the canonical isoform that is transcribed into an  mRNA  of 2797 base pairs. [ 4 ]  Three other isoforms have been isolated. [ 3 ]


Homologs exist as distant as the  green sea turtle  and  chickens  at approximately 60% sequence identity, suggesting that the gene may have arisen in the  amniotes  after their divergence from other  tetrapods ; [ 5 ]  the first 4 exons are conserved with 36% identity as distantly as the  anemone .


The human  C12orf40  protein is 652 amino acids in length. [ 6 ]  Its molecular weight is predicted to be 74.52 kDa, [ 7 ]  and its  isoelectric point  7.822. [ 8 ]  Amino acids 229-652 contain a domain of unknown function ( DUF4552 ) which is conserved in  vertebrates . [ 6 ]   C12orf40  is predicted to be a soluble protein with no transmembrane segments. [ 9 ]  Its secondary and tertiary structures are not currently known.


Experimental evidence shows that C12orf40 has a physical interaction with dynein light chain 2 ( DYNLL2 ). [ 10 ]  This protein is part of a complex that regulates the function of the motor protein dynein.


Within the cell,  C12orf40  is predicted to be present in the nucleus based on signals within its sequence. [ 11 ]  An analysis of normal human tissues shows that  C12orf40  expression occurs primarily in the  testis , [ 12 ]  suggesting importance to the male reproductive system.


The function of  C12orf40  is not yet well understood. However, the  three prime untranslated region  (3' UTR) of  C12orf40  is highly similar to the 3' UTR of the  cystic fibrosis transmembrane conductance regulator  (CFTR), which may mean that the two genes share certain expression patterns. [ 13 ]  In the  fibroblasts  of hypertrophic scars, exposure to the immunosuppressant  Tacrolimus  causes  C12orf40  up-regulation. [ 14 ]   In pigs, a region homologous to human  C12orf40  plays a role in  arthrogryposis , a disease characterized by congenital fibrosis. [ 15 ]  The common thread of these studies suggests that  C12orf40  may have a connection to the formation of healthy connective tissue.


Chromosome 18  open reading frame  63  is a  protein  which in humans is encoded by the C18orf63  gene . [ 5 ]  This protein is not yet well understood by the scientific community. Research has been conducted suggesting that C18orf63 could be a potential  biomarker  for early stage  pancreatic cancer  and  breast cancer . [ 6 ] [ 7 ]


This gene is located at band 22, sub-band 3, on the long arm of  chromosome 18 .  It is composed of 5065  base pairs  spanning from 74,315,875 to 74,359,187 bp on chromosome 18. [ 5 ]  The gene has a total of 14  exons . [ 5 ]   C18orf63 is also known by the alias DKFZP78G0119. [ 8 ]  No isoforms exist for this gene. [ 5 ]


C18orf63 has high expression in the  testis . [ 5 ]  The gene shows low expression in the kidneys, liver, lung, and pelvis. [ 9 ]  There is no  phenotype  associated with this gene. [ 5 ] [ 10 ]


The  promoter region  for C18orf63 is 1163 bp long starting at 74,314,813 bp and ending at 74,315,975 bp. [ 11 ]  The promoter ID is GXP_4417391. The presence of multiple y-box binding transcription factors and SRY transcription factor binding sites suggest that C18orf63 is involved in male sex determination. [ 12 ]


The C18orf63 protein is composed up of 685  amino acids  and has a molecular weight of 77230.50 Da, with a predicted  isoelectric point  of 9.83. [ 5 ] [ 13 ]   No  isoforms  exist for this protein. [ 14 ]  This protein is rich in  glutamine ,  isoleucine ,  lysine , and  serine  when compared to the average protein, but lacks in  aspartic acid  and  glycine . [ 15 ] [ 16 ]


In the predicted secondary structure for this protein there are a number of  beta turns ,  beta strands  and  alpha helices . For C18orf63 48.6% of the protein is expected to form alpha helices and 28.6% of the structure is expected to be composed of beta strands. [ 17 ] [ 18 ]


The protein contains one  domain of unknown function , DUF 4709, spanning from the 7th amino acid to the 280th amino acid. [ 19 ]   Motifs  that are predicted to exist include an N-terminal motif, RxxL motif, and KEN conserving motif, which all signal for  protein degradation . [ 20 ]  Another motif that is predicted to exist is a Wxxx motif, which facilitates entrance of PTS1 cargo proteins into the organellar lumen, and a RVxPx motif which allows protein transport from the  trans-Golgi network  to the  plasma membrane  of the  cilia . [ 21 ] [ 22 ]  There is also a bipartite  nuclear localization signal  at the end of the protein sequence. [ 23 ]  There is no trans-membrane domain present, indicating that C18orf63 is not a trans-membrane protein. [ 24 ]


Post-translational modifications  the protein is predicted to undergo include  SUMOylation ,  PKC  and  CK2   phosphorylation ,  N -glycosylation , amiditation, and cleavage. [ 25 ] [ 26 ] [ 27 ] [ 28 ]  There are six total PKC phosphorylation sites and 2 CK2 phosphorylation sites, 2 SUMOylation sites, and 2  N -glycosylation sites. There are no signal peptides present in this sequence. [ 28 ]


Due to the nuclear localization signal at the end of the protein sequence, C18orf63 is predicted to be  nuclear . C18orf63 has also been predicted to be targeted to the  mitochondria  in addition to the nucleus. [ 29 ] [ 30 ] [ 31 ]


Orthologs  have been found in most  eukaryotes , with the exception of the class  Amphibia . [ 14 ]  No human  paralogs  exist for C18orf63. [ 14 ] [ 32 ]  The most distant homolog detectable is  Mizuhopecten yessoensis , sharing a 37% identity with the human protein sequence. The domain of unknown function was the only homologous domain present in the protein sequence, it was found to be highly conserved in all orthologs. The table below shows some examples of various orthologs for this protein.


C18orf63 is a mildly slow evolving protein. The protein evolves faster than  Cytochorme C  but slower than  Betaglobin . [ 14 ]


Transcription factors of interest predicted to bind to the regulatory sequence include  p53 tumor suppressors ,  SRY testis determining factors ,  Y-box binding transcription factors , and  glucocorticoid responsive elements . [ 11 ]  The JUN protein was found to interact with C18orf63 through anti-bait  co-immunoprecipitation . [ 33 ]  The JUN protein binds to the USP28 promoter in  colorectal cancer cells  and is involved in the activation of these cancer cells. [ 34 ] [ 35 ]


A variety of  missense mutations  occur in the human population for this protein. In the regulatory sequence missense mutations occur at two transcription factor binding sites. [ 32 ]  Transcription factors affected are  glucocorticoid responsive elements  and  E2F-myc cell cycle regulars . There are eleven common mutations that occur that affect the protein sequence itself. [ 32 ]  None of these mutations affect predicted post-translational modifications the protein sequence undergoes.


C18orf63 has been associated with  personality disorders ,  obesity , and  type two diabetes  through a  genome-wide association study . [ 36 ] [ 37 ] [ 38 ]  Currently research has not shown if C18orf63 plays a direct role in any of these diseases.


The  centromere  links a pair of sister  chromatids  together during cell division. This constricted region of chromosome connects the sister chromatids, creating a short arm (p) and a long arm (q) on the chromatids. During  mitosis ,  spindle fibers  attach to the centromere via the  kinetochore .


The physical role of the centromere is to act as the site of assembly of the  kinetochores  – a highly complex multiprotein structure that is responsible for the actual events of  chromosome segregation  – i.e. binding  microtubules  and signaling to the cell cycle machinery when all chromosomes have adopted correct attachments to the  spindle , so that it is safe for  cell division  to proceed to completion and for cells to enter  anaphase .


There are, broadly speaking, two types of centromeres. "Point centromeres" bind to specific  proteins  that recognize particular  DNA   sequences  with high efficiency. Any piece of DNA with the point centromere DNA sequence on it will typically form a centromere if present in the appropriate species. The best characterized point centromeres are those of the budding yeast,  Saccharomyces cerevisiae . "Regional centromeres" is the term coined to describe most centromeres, which typically form on regions of preferred DNA sequence, but which can form on other DNA sequences as well. The signal for formation of a regional centromere appears to be  epigenetic . Most organisms, ranging from the fission yeast  Schizosaccharomyces pombe  to humans, have regional centromeres.


Regarding mitotic chromosome structure, centromeres represent a constricted region of the chromosome (often referred to as the primary constriction) where two identical  sister chromatids  are most closely in contact. When cells enter mitosis, the sister chromatids (the two copies of each chromosomal DNA molecule resulting from  DNA replication  in chromatin form) are linked along their length by the action of the  cohesin  complex. It is now believed that this complex is mostly released from chromosome arms during prophase, so that by the time the chromosomes line up at the mid-plane of the mitotic spindle (also known as the metaphase plate), the last place where they are linked with one another is in the chromatin in and around the centromere.


In humans, centromere positions define the chromosomal  karyotype , in which each chromosome has two arms,  p  (the shorter of the two) and  q  (the longer). The short arm 'p' is reportedly named for the French word "petit" meaning 'small'. [ 1 ]  The position of the centromere relative to any particular linear chromosome is used to classify chromosomes as  metacentric, submetacentric, acrocentric, telocentric, or holocentric. [ 2 ] [ 3 ]


Metacentric means that the centromere is positioned midway between the chromosome ends, resulting in the arms being approximately equal in length. When the centromeres are metacentric, the chromosomes appear to be "x-shaped."


Submetacentric means that the centromere is positioned below the middle, with one chromosome arm shorter than the other, often resulting in an L shape.


An acrocentric chromosome's centromere is situated so that one of the chromosome arms is much shorter than the other. The "acro-" in acrocentric refers to the Greek word for "peak." The  human genome  has six acrocentric chromosomes, including five autosomal chromosomes ( 13 ,  14 ,  15 ,  21 ,  22 ) and the  Y chromosome .


Short acrocentric p-arms contain little genetic material and can be translocated without significant harm, as in a balanced  Robertsonian translocation . In addition to some protein coding genes, human acrocentric p-arms also contain  Nucleolus organizer regions  (NORs), from which  ribosomal RNA  is transcribed. However, a proportion of acrocentric p-arms in cell lines and tissues from normal human donors do not contain detectable NORs. [ 4 ]  The  domestic horse  genome includes one metacentric chromosome that is  homologous  to two acrocentric chromosomes in the  conspecific  but undomesticated  Przewalski's horse . This may reflect either fixation of a balanced Robertsonian translocation in domestic horses or, conversely, fixation of the fission of one metacentric chromosome into two acrocentric chromosomes in Przewalski's horses. A similar situation exists between the human and great ape genomes, with a reduction of two acrocentric chromosomes in the great apes to one metacentric chromosome in humans (see  aneuploidy  and the  human chromosome 2 ).


Many diseases from the result of unbalanced translocations more frequently involve acrocentric chromosomes than other non-acrocentric chromosomes. Acrocentric chromosomes are usually located in and around the  nucleolus . As a result, these chromosomes tend to be less densely packed than chromosomes in the nuclear periphery. Consistently, chromosomal regions that are less densely packed are also more prone to chromosomal translocations in cancers.


Telocentric chromosomes have a centromere at one end of the chromosome and therefore exhibit only one arm at the cytological (microscopic) level. They are not present in humans but can form through cellular chromosomal errors. Telocentric chromosomes occur naturally in many species, such as the  house mouse , in which all chromosomes except the Y are telocentric.


Subtelocentric chromosomes' centromeres are located between the middle and the end of the chromosomes, but reside closer to the end of the chromosomes.


An acentric chromosome is fragment of a chromosome that lacks a centromere. Since centromeres are the attachment point for spindle fibers in cell division, acentric fragments are not evenly distributed to daughter cells during cell division. As a result, a daughter cell will lack the acentric fragment and deleterious consequences could occur.


Chromosome-breaking events can also generate acentric chromosomes or acentric fragments.


A  dicentric chromosome  is an abnormal chromosome with two centromeres, which can be unstable through cell divisions. It can form through translocation between or fusion of two chromosome segments, each with a centromere. Some rearrangements produce both dicentric chromosomes and acentric fragments which can not attach to spindles at mitosis. [ 5 ]  The formation of dicentric chromosomes has been attributed to genetic processes, such as  Robertsonian translocation [ 6 ]  and  paracentric inversion. [ 7 ]  Dicentric chromosomes can have a variety of fates, including mitotic stability. [ 8 ]  In some cases, their stability comes from inactivation of one of the two centromeres to make a functionally monocentric chromosome capable of normal transmission to daughter cells during cell division. [ 9 ]


For example, human  chromosome 2 , which is believed to be the result of a Robertsonian translocation at some point in the evolution between the great apes and  Homo , has a second, vestigial, centromere near the middle of its long arm. [ 10 ]


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. [ 11 ]


Unlike monocentric chromosomes, holocentric chromosomes have no distinct primary constriction when viewed at mitosis. Instead, spindle fibers attach along almost the entire (Greek: holo-) length of the chromosome. In holocentric chromosomes centromeric proteins, such as  CENPA  (CenH3) are spread over the whole chromosome. [ 12 ]  The nematode,  Caenorhabditis elegans , is a well-known example of an organism with holocentric chromosomes, [ 13 ]  but this type of centromere can be found in various species, plants, and animals, across eukaryotes. Holocentromeres are actually composed of multiple distributed centromere units that form a line-like structure along the chromosomes during mitosis. [ 14 ]  Alternative or nonconventional strategies are deployed at meiosis to achieve the homologous chromosome pairing and segregation needed to produce viable gametes or gametophytes for sexual reproduction.


Different types of holocentromeres exist in different species, namely with or without centromeric repetitive DNA sequences and with or without  CenH3 . Holocentricity has evolved at least 13 times independently in various green algae, protozoans, invertebrates, and different plant families. [ 15 ]  Contrary to monocentric species where acentric fragments usually become lost during cell division, the breakage of holocentric chromosomes creates fragments with normal spindle fiber attachment sites. [ 16 ]  Because of this, organisms with holocentric chromosomes can more rapidly evolve karyotype variation, able to heal fragmented chromosomes through subsequent addition of telomere caps at the sites of breakage. [ 17 ]


Polycentric chromosomes have several kinetochore clusters, i.e. centromes. The term overlaps partially with "holocentric", but "polycentric" is clearly preferred when discussing defectively formed monocentric chromosomes. There is some actual ambiguity as well, as there is no clear line dividing up the transition from kinetochores covering the whole chromosome to distinct clusters. In other words, the difference between "the whole chromosome is a centrome" and "the chromosome has no centrome" is hazy and usage varies. Beyond "polycentricity" being used more about defects, there is no clear preference in other topics such as evolutionary origin or kinetochore distribution and detailed structure (e.g. as seen in  tagging  or  genome assembly  analysis). [ 18 ] [ 19 ] [ 20 ] [ 21 ]


Even clearly distinct clusters of kinetochore proteins do not necessarily produce more than one constriction: "Metapolycentric" chromosomes feature one elongated constriction of the chromosome, joining a longer segment which is still visibly shorter than the chromatids. [ 22 ]  Metapolycentric chromosomes may be a step in the emergence and suppression of centromere drive, a type of  meiotic drive  that disrupts parity by monocentric centromeres growing additional kinetochore proteins to gain an advantage during meiosis. [ 23 ]


Based on the micrographic characteristics of size, position of the centromere and sometimes the presence of a  chromosomal satellite , the human chromosomes are classified into the following groups: [ 24 ]


There are two types of centromeres. [ 25 ]  In regional centromeres,  DNA  sequences contribute to but do not define function. Regional centromeres contain large amounts of DNA and are often packaged into  heterochromatin . In most  eukaryotes , the centromere's DNA sequence consists of large arrays of repetitive DNA (e.g.  satellite DNA ) where the sequence within individual repeat elements is similar but not identical. In humans, the primary centromeric repeat unit is called α-satellite (or alphoid), although a number of other sequence types are found in this region. [ 26 ]  Centromere satellites are hypothesized to evolve by a process called layered expansion. They evolve rapidly between species, and analyses in wild mice show that satellite copy number and heterogeneity relates to population origins and subspecies. [ 27 ]  Additionally, satellite sequences may be affected by inbreeding. [ 27 ]


Point centromeres are smaller and more compact. DNA sequences are both necessary and sufficient to specify centromere identity and function in organisms with point centromeres. In budding yeasts, the centromere region is relatively small (about 125 bp DNA) and contains two highly conserved DNA sequences that serve as binding sites for essential  kinetochore  proteins. [ 26 ]


Since centromeric DNA sequence is not the key determinant of centromeric identity in  metazoans , it is thought that  epigenetic inheritance  plays a major role in specifying the centromere. [ 28 ]  The daughter chromosomes will assemble centromeres in the same place as the parent chromosome, independent of sequence. It has been proposed that histone H3 variant  CENP-A  (Centromere Protein A) is the epigenetic mark of the centromere. [ 29 ]  The question arises whether there must be still some original way in which the centromere is specified, even if it is subsequently propagated epigenetically. If the centromere is inherited epigenetically from one generation to the next, the problem is pushed back to the origin of the first metazoans.


On the other hand, thanks to comparisons of the centromeres in the X chromosomes, epigenetic and structural variations have been seen in these regions. In addition, a recent assembly of the human genome has detected a possible mechanism of how pericentromeric and centromeric structures evolve, through a layered expansion model for αSat sequences. This model proposes that different αSat sequence repeats emerge periodically and expand within an active vector, displacing old sequences, and becoming the site of kinetochore assembly. The αSat can originate from the same, or from different vectors. As this process is repeated over time, the layers that flank the active centromere shrink and deteriorate. This process raises questions about the relationship between this dynamic evolutionary process and the position of the centromere. [ 30 ]


The centromeric DNA is normally in a  heterochromatin  state, which is essential for the recruitment of the  cohesin  complex that mediates sister chromatid cohesion after DNA replication as well as coordinating sister chromatid separation during anaphase. In this chromatin, the normal  histone  H3 is replaced with a centromere-specific variant, CENP-A in humans. [ 31 ]  The presence of CENP-A is believed to be important for the assembly of the kinetochore on the centromere. CENP-C has been shown to localise almost exclusively to these regions of CENP-A associated chromatin. In human cells, the histones are found to be most enriched for  H4K20me 3 and  H3K9me3 [ 32 ]  which are known heterochromatic modifications. In  Drosophila, Islands of retroelements are major components of the centromeres. [ 33 ]


In the yeast  Schizosaccharomyces pombe  (and probably in other eukaryotes), the formation of centromeric heterochromatin is connected to  RNAi . [ 34 ]  In nematodes such as  Caenorhabditis elegans , some plants, and the insect orders Lepidoptera and Hemiptera, chromosomes are "holocentric", indicating that there is not a primary site of microtubule attachments or a primary constriction, and a "diffuse" kinetochore assembles along the entire length of the chromosome.


In rare cases,  neocentromeres  can form at new sites on a chromosome as a result of a repositioning of the centromere. This phenomenon is most well known from human clinical studies and there are currently over 90 known human neocentromeres identified on 20 different chromosomes. [ 35 ] [ 36 ]  The formation of a neocentromere must be coupled with the inactivation of the previous centromere, since chromosomes with two functional centromeres ( Dicentric chromosome ) will result in chromosome breakage during mitosis. In some unusual cases human neocentromeres have been observed to form spontaneously on fragmented chromosomes. Some of these new positions were originally euchromatic and lack alpha  satellite DNA  altogether.  Neocentromeres  lack the repetitive structure seen in normal centromeres which suggest that centromere formation is mainly controlled  epigenetically . [ 37 ] [ 38 ]  Over time a neocentromere can accumulate repetitive elements and mature into what is known as an evolutionary new centromere. There are several well known examples in primate chromosomes where the centromere position is different from the human centromere of the same chromosome and is thought to be evolutionary new centromeres. [ 37 ]  Centromere repositioning and the formation of evolutionary new centromeres has been suggested to be a mechanism of  speciation . [ 39 ]


Centromere proteins are also the autoantigenic target for some  anti-nuclear antibodies , such as  anti-centromere antibodies .


It has been known that centromere misregulation contributes to mis-segregation of chromosomes, which is strongly related to cancer and miscarriage. Notably, overexpression of many centromere genes have been linked to cancer malignant phenotypes. Overexpression of these centromere genes can increase genomic instability in cancers. Elevated genomic instability on one hand relates to malignant phenotypes; on the other hand, it makes the tumor cells more vulnerable to specific adjuvant therapies such as certain chemotherapies and radiotherapy. [ 40 ]  Instability of centromere repetitive DNA was recently shown in cancer and aging. [ 41 ]


When  DNA breaks  occur at centromeres in the  G1 phase  of the  cell cycle , the cells are able to recruit the  homologous recombinational  repair machinery to the damaged site, even in the absence of a  sister chromatid . [ 42 ]  It appears that homologous recombinational repair can occur at centromeric breaks throughout the cell cycle in order to prevent the activation of inaccurate mutagenic DNA repair pathways and to preserve centromeric integrity. [ 42 ]


The word  centromere  ( / ˈ s ɛ n t r ə ˌ m ɪər / [ 43 ] [ 44 ] ) uses  combining forms  of  centro-  and  -mere , yielding "central part", describing the centromere's location at the center of the chromosome.


Chloroplast DNA  ( cpDNA ), also known as  plastid DNA  ( ptDNA ) is the DNA located in chloroplasts, which are photosynthetic organelles located within the cells of some eukaryotic organisms. Chloroplasts, like other types of  plastid , contain a  genome  separate from that in the cell  nucleus . The existence of chloroplast DNA was identified biochemically in 1959, [ 1 ]  and confirmed by electron microscopy in 1962. [ 2 ]  The discoveries that the chloroplast contains ribosomes [ 3 ]  and performs protein synthesis [ 4 ]  revealed that the chloroplast is genetically semi-autonomous. The first complete chloroplast genome sequences were published in 1986,  Nicotiana tabacum  (tobacco) by Sugiura and colleagues and  Marchantia polymorpha  (liverwort) by Ozeki et al. [ 5 ] [ 6 ]  Since then,  tens of thousands of chloroplast genomes  from various species have been  sequenced .


Chloroplast  DNAs are circular, and are typically 120,000–170,000  base pairs  long. [ 7 ] [ 8 ] [ 9 ]  They can have a contour length of around 30–60 micrometers, and have a mass of about 80–130 million  daltons . [ 10 ]


Most chloroplasts have their entire chloroplast genome combined into a single large ring, though those of  dinophyte algae  are a notable exception—their genome is broken up into about forty small  plasmids , each 2,000–10,000  base pairs  long. [ 11 ]  Each minicircle contains one to three genes, [ 11 ]  but blank plasmids, with no  coding DNA , have also been found.


Chloroplast DNA has long been thought to have a circular structure, but some evidence suggests that chloroplast DNA more commonly takes a linear shape. [ 12 ]  Over 95% of the chloroplast DNA in  corn  chloroplasts has been observed to be in branched linear form rather than individual circles. [ 11 ]


Many chloroplast DNAs contain two  inverted repeats , which separate a long single copy section (LSC) from a short single copy section (SSC). [ 9 ]


The inverted repeats vary wildly in length, ranging from 4,000 to 25,000  base pairs  long each. [ 11 ]  Inverted repeats in plants tend to be at the upper end of this range, each being 20,000–25,000 base pairs long. [ 9 ] [ 13 ]
The inverted repeat regions usually contain three  ribosomal RNA  and two  tRNA  genes, but they can be expanded or  reduced  to contain as few as four or as many as over 150 genes. [ 11 ]
While a given pair of inverted repeats are rarely completely identical, they are always very similar to each other, apparently resulting from  concerted evolution . [ 11 ]


The inverted repeat regions are highly  conserved  among land plants, and accumulate few mutations. [ 9 ] [ 13 ]  Similar inverted repeats exist in the genomes of cyanobacteria and the other two chloroplast lineages ( glaucophyta  and  rhodophyceæ ), suggesting that they predate the chloroplast, [ 11 ]  though some chloroplast DNAs like those of  peas  and a few  red algae [ 11 ]  have since lost the inverted repeats. [ 13 ] [ 14 ]  Others, like the red alga  Porphyra  flipped one of its inverted repeats (making them direct repeats). [ 11 ]  It is possible that the inverted repeats help stabilize the rest of the chloroplast genome, as chloroplast DNAs which have lost some of the inverted repeat segments tend to get rearranged more. [ 14 ]


Each chloroplast contains around 100 copies of its DNA in young leaves, declining to 15–20 copies in older leaves. [ 15 ]  They are usually packed into  nucleoids  which can contain several identical chloroplast DNA rings. Many nucleoids can be found in each chloroplast. [ 10 ]


Though chloroplast DNA is not associated with true  histones , [ 16 ]  in  red algae , a histone-like chloroplast protein (HC) coded by the chloroplast DNA that tightly packs each chloroplast DNA ring into a  nucleoid  has been found. [ 17 ]


In primitive  red algae , the chloroplast DNA nucleoids are clustered in the center of a chloroplast, while in green plants and  green algae , the nucleoids are dispersed throughout the  stroma . [ 17 ]


More than 5000 chloroplast genomes have been  sequenced  and are accessible via the NCBI organelle genome database. [ 18 ]  The first chloroplast genomes were sequenced in 1986, from tobacco ( Nicotiana tabacum ) [ 19 ]  and liverwort ( Marchantia polymorpha ). [ 20 ]  Comparison of the gene sequences of the cyanobacteria  Synechocystis  to those of the chloroplast genome of  Arabidopsis  provided confirmation of the  endosymbiotic  origin of the chloroplast. [ 21 ] [ 22 ]  It also demonstrated the significant extent of  gene transfer  from the cyanobacterial ancestor to the nuclear genome.


In most plant species, the chloroplast genome encodes approximately 120 genes. [ 23 ] [ 24 ]  The genes primarily encode core components of the photosynthetic machinery and factors involved in their expression and assembly. [ 25 ]  Across species of land plants, the set of genes encoded by the chloroplast genome is fairly conserved. This includes four  ribosomal RNAs , approximately 30  tRNAs , 21  ribosomal proteins , and 4 subunits of the plastid-encoded  RNA polymerase  complex that are involved in plastid gene expression. [ 25 ]  The large  Rubisco  subunit and 28 photosynthetic  thylakoid  proteins are encoded within the chloroplast genome. [ 25 ]


Over time, many parts of the chloroplast genome were transferred to the  nuclear genome  of the host, [ 7 ] [ 8 ] [ 26 ]  a process called  endosymbiotic gene transfer .
As a result, the chloroplast genome is heavily  reduced  compared to that of free-living cyanobacteria. Chloroplasts may contain 60–100 genes whereas cyanobacteria often have more than 1500 genes in their genome. [ 27 ]  The parasitic  Pilostyles  have even lost their plastid genes for  tRNA . [ 28 ]  Contrarily, there are only a few known instances where genes have been transferred to the chloroplast from various donors, including bacteria. [ 29 ] [ 30 ] [ 31 ]


Endosymbiotic gene transfer is how we know about the  lost chloroplasts  in many  chromalveolate  lineages. Even if a chloroplast is eventually lost, the genes it donated to the former host's nucleus persist, providing evidence for the lost chloroplast's existence. For example, while  diatoms  (a  heterokontophyte ) now have a  red algal derived chloroplast , the presence of many  green algal  genes in the diatom nucleus provide evidence that the diatom ancestor (probably the ancestor of all chromalveolates too) had a  green algal derived chloroplast  at some point, which was subsequently replaced by the red chloroplast. [ 32 ]


In land plants, some 11–14% of the DNA in their nuclei can be traced back to the chloroplast, [ 33 ]  up to 18% in  Arabidopsis , corresponding to about 4,500 protein-coding genes. [ 34 ]  There have been a few recent transfers of genes from the chloroplast DNA to the nuclear genome in land plants. [ 8 ]


Of the approximately three-thousand proteins found in chloroplasts, some 95% of them are encoded by nuclear genes. Many of the chloroplast's protein complexes consist of subunits from both the chloroplast genome and the host's nuclear genome. As a result,  protein synthesis  must be coordinated between the chloroplast and the nucleus. The chloroplast is mostly under nuclear control, though chloroplasts can also give out signals regulating  gene expression  in the nucleus, called  retrograde signaling . [ 35 ]


Protein synthesis within chloroplasts relies on an  RNA polymerase  coded by the chloroplast's own genome, which is related to RNA polymerases found in bacteria. Chloroplasts also contain a mysterious second RNA polymerase that is encoded by the plant's nuclear genome. The two RNA polymerases may recognize and bind to different kinds of  promoters  within the chloroplast genome. [ 36 ]  The  ribosomes  in chloroplasts are similar to bacterial ribosomes. [ 37 ]


RNA editing  is the insertion, deletion, and substitution of nucleotides in a mRNA transcript prior to translation to protein. The highly oxidative environment inside chloroplasts increases the rate of mutation so post-transcription repairs are needed to conserve functional sequences. The chloroplast editosome substitutes C -> U and U -> C at very specific locations on the transcript. This can change the codon for an amino acid or restore a non-functional pseudogene by adding an AUG start codon or removing a premature UAA stop codon. [ 38 ]


The editosome recognizes and binds to cis sequence upstream of the editing site. The distance between the binding site and editing site  varies by gene and proteins involved in the editosome.  Hundreds of different  PPR proteins  from the nuclear genome are involved in the RNA editing process. These proteins consist of 35-mer repeated amino acids, the sequence of which determines the cis binding site for the edited transcript. [ 38 ]


Basal land plants such as liverworts, mosses and ferns have hundreds of different editing sites while flowering plants typically have between thirty and forty. Parasitic plants such as  Epifagus virginiana  show a loss of RNA editing resulting in a loss of function for photosynthesis genes. [ 39 ]


The mechanism for chloroplast DNA (cpDNA) replication has not been conclusively determined, but two main models have been proposed. Scientists have attempted to observe chloroplast replication via  electron microscopy  since the 1970s. [ 40 ] [ 41 ]  The results of the microscopy experiments led to the idea that chloroplast DNA replicates using a double displacement loop (D-loop). As the  D-loop  moves through the circular DNA, it adopts a theta intermediary form, also known as a Cairns replication intermediate, and completes replication with a rolling circle mechanism. [ 40 ] [ 12 ]  Replication starts at specific points of origin. Multiple  replication forks  open up, allowing replication machinery to replicate the DNA. As replication continues, the forks grow and eventually converge. The new cpDNA structures separate, creating daughter cpDNA chromosomes.


In addition to the early microscopy experiments, this model is also supported by the amounts of  deamination  seen in cpDNA. [ 40 ]  Deamination occurs when an  amino group  is lost and is a  mutation  that often results in base changes. When adenine is deaminated, it becomes  hypoxanthine  (H). Hypoxanthine can bind to  cytosine , and when the HC base pair is replicated, it becomes a GC (thus, an A → G base change). [ 42 ]


In cpDNA, there are several A → G deamination gradients. DNA becomes susceptible to deamination events when it is single stranded. When replication forks form, the strand not being copied is single stranded, and thus at risk for A → G deamination. Therefore, gradients in deamination indicate that replication forks were most likely present and the direction that they initially opened (the highest gradient is most likely nearest the start site because it was single stranded for the longest amount of time). [ 40 ]  This mechanism is still the leading theory today; however, a second theory suggests that most cpDNA is actually linear and replicates through homologous recombination. It further contends that only a minority of the genetic material is kept in circular chromosomes while the rest is in branched, linear, or other complex structures. [ 40 ] [ 12 ]


One of the main competing models for cpDNA asserts that most cpDNA is linear and participates in  homologous recombination  and replication structures similar to  bacteriophage T4 . [ 12 ]  It has been established that some plants have linear cpDNA, such as maize, and that more still contain complex structures that scientists do not yet understand; [ 12 ]  however, the predominant view today is that most cpDNA is circular. When the original experiments on cpDNA were performed, scientists did notice linear structures; however, they attributed these linear forms to broken circles. [ 12 ]  If the branched and complex structures seen in cpDNA experiments are real and not artifacts of concatenated circular DNA or broken circles, then a D-loop mechanism of replication is insufficient to explain how those structures would replicate. [ 12 ]  At the same time, homologous recombination does not explain the multiple A → G gradients seen in plastomes. [ 40 ]  This shortcoming is one of the biggest for the linear structure theory.


The movement of so many chloroplast genes to the nucleus means that many chloroplast  proteins  that were supposed to be  translated  in the chloroplast are now synthesized in the cytoplasm. This means that these proteins must be directed back to the chloroplast, and imported through at least two chloroplast membranes. [ 43 ]


Curiously, around half of the protein products of transferred genes aren't even targeted back to the chloroplast. Many became  exaptations , taking on new functions like participating in  cell division ,  protein routing , and even  disease resistance . A few chloroplast genes found new homes in the  mitochondrial genome —most became nonfunctional  pseudogenes , though a few  tRNA  genes still work in the  mitochondrion . [ 27 ]  Some transferred chloroplast DNA protein products get directed to the  secretory pathway [ 27 ]  (though many  secondary plastids  are bounded by an outermost membrane derived from the host's  cell membrane , and therefore  topologically  outside of the cell, because to reach the chloroplast from the  cytosol , you have to cross the  cell membrane , just like if you were headed for the  extracellular space . In those cases, chloroplast-targeted proteins do initially travel along the secretory pathway). [ 44 ]


Because the cell acquiring a chloroplast  already  had  mitochondria  (and  peroxisomes , and a  cell membrane  for secretion), the new chloroplast host had to develop a unique  protein targeting system  to avoid having chloroplast proteins being sent to the wrong  organelle . [ 43 ]


Polypeptides , the precursors of  proteins , are chains of  amino acids . The two ends of a polypeptide are called the  N-terminus , or  amino end , and the  C-terminus , or  carboxyl end . [ 45 ]  For many (but not all) [ 46 ]  chloroplast proteins encoded by  nuclear  genes,  cleavable transit peptides  are added to the N-termini of the polypeptides, which are used to help direct the polypeptide to the chloroplast for import [ 43 ] [ 47 ]  (N-terminal transit peptides are also used to direct polypeptides to plant  mitochondria ). [ 48 ]
N-terminal transit sequences are also called  presequences [ 43 ]  because they are located at the "front" end of a polypeptide— ribosomes  synthesize polypeptides from the N-terminus to the C-terminus. [ 45 ]


Chloroplast transit peptides exhibit huge variation in length and  amino acid sequence . [ 47 ]  They can be from 20 to 150 amino acids long [ 43 ] —an unusually long length, suggesting that transit peptides are actually collections of  domains  with different functions. [ 47 ]  Transit peptides tend to be  positively charged , [ 43 ]  rich in  hydroxylated  amino acids such as  serine ,  threonine , and  proline , and poor in  acidic  amino acids like  aspartic acid  and  glutamic acid . [ 47 ]  In an  aqueous solution , the transit sequence forms a random coil. [ 43 ]


Not all chloroplast proteins include a N-terminal cleavable transit peptide though. [ 43 ]  Some include the transit sequence within the  functional part  of the protein itself. [ 43 ]  A few have their transit sequence appended to their  C-terminus  instead. [ 49 ]  Most of the polypeptides that lack N-terminal targeting sequences are the ones that are sent to the  outer chloroplast membrane , plus at least one sent to the  inner chloroplast membrane . [ 43 ]


After a chloroplast  polypeptide  is synthesized on a  ribosome  in the  cytosol ,  ATP  energy can be used to  phosphorylate , or add a  phosphate group  to many (but not all) of them in their transit sequences. [ 43 ]   Serine  and  threonine  (both very common in chloroplast transit sequences—making up 20–30% of the sequence) [ 50 ]  are often the  amino acids  that accept the  phosphate group . [ 48 ] [ 50 ]  The  enzyme  that carries out the phosphorylation is  specific  for chloroplast polypeptides, and ignores ones meant for  mitochondria  or  peroxisomes . [ 50 ]


Phosphorylation changes the polypeptide's shape, [ 50 ]  making it easier for  14-3-3 proteins  to attach to the polypeptide. [ 43 ] [ 51 ]  In plants,  14-3-3 proteins  only bind to chloroplast preproteins. [ 48 ]  It is also bound by the  h eat  s hock  p rotein   Hsp70  that keeps the polypeptide from  folding  prematurely. [ 43 ]  This is important because it prevents chloroplast proteins from assuming their active form and carrying out their chloroplast functions in the wrong place—the  cytosol . [ 48 ] [ 51 ]  At the same time, they have to keep just enough shape so that they can be recognized and imported into the chloroplast. [ 48 ]


The heat shock protein and the 14-3-3 proteins together form a cytosolic guidance complex that makes it easier for the chloroplast polypeptide to get imported into the chloroplast. [ 43 ]


Alternatively, if a chloroplast preprotein's transit peptide is not phosphorylated, a chloroplast preprotein can still attach to a heat shock protein or  Toc159 . These complexes can bind to the  TOC complex  on the outer chloroplast membrane using  GTP  energy. [ 43 ]


The  TOC complex , or  t ranslocon  on the  o uter  c hloroplast membrane , is a collection of proteins that imports preproteins across the  outer chloroplast envelope . Five  subunits  of the TOC complex have been identified—two  GTP -binding proteins  Toc34  and  Toc159 , the protein import tunnel  Toc75 , plus the proteins  Toc64 [ 43 ]  and  Toc12 . [ 46 ]


The first three proteins form a core complex that consists of one Toc159, four to five Toc34s, and four Toc75s that form four holes in a disk 13  nanometers  across. The whole core complex weighs about 500  kilodaltons . The other two proteins, Toc64 and Toc12, are associated with the core complex but are not part of it. [ 46 ]


Toc34  is an  integral protein  in the outer chloroplast membrane that's anchored into it by its  hydrophobic [ 53 ]   C-terminal  tail. [ 43 ] [ 51 ]  Most of the protein, however, including its large  guanosine triphosphate  (GTP)-binding  domain  projects out into the stroma. [ 51 ]


Toc34's job is to catch some chloroplast  preproteins  in the  cytosol  and hand them off to the rest of the TOC complex. [ 43 ]  When  GTP , an energy molecule similar to  ATP  attaches to Toc34, the protein becomes much more able to bind to many chloroplast preproteins in the  cytosol . [ 43 ]  The chloroplast preprotein's presence causes Toc34 to break GTP into  guanosine diphosphate  (GDP) and  inorganic phosphate . This loss of GTP makes the Toc34 protein release the chloroplast preprotein, handing it off to the next TOC protein. [ 43 ]  Toc34 then releases the depleted GDP molecule, probably with the help of an unknown  GDP exchange factor . A  domain  of  Toc159  might be the exchange factor that carry out the GDP removal. The Toc34 protein can then take up another molecule of GTP and begin the cycle again. [ 43 ]


Toc34 can be turned off through  phosphorylation . A  protein kinase  drifting around on the outer chloroplast membrane can use  ATP  to add a  phosphate group  to the Toc34 protein, preventing it from being able to receive another  GTP  molecule, inhibiting the protein's activity. This might provide a way to regulate protein import into chloroplasts. [ 43 ] [ 51 ]


Arabidopsis thaliana  has two  homologous  proteins,  AtToc33  and  AtToc34  (The  At  stands for  A rabidopsis  t haliana ), [ 43 ] [ 51 ]  which are each about 60% identical in  amino acid sequence  to Toc34 in  peas  (called  ps Toc34). [ 51 ]  AtToc33 is the most common in  Arabidopsis , [ 51 ]  and it is the functional  analogue  of Toc34 because it can be turned off by phosphorylation. AtToc34 on the other hand cannot be phosphorylated. [ 43 ] [ 51 ]


Toc159  is another  GTP  binding TOC  subunit , like  Toc34 . Toc159 has three  domains . At the  N-terminal  end is the A-domain, which is rich in  acidic amino acids  and takes up about half the protein length. [ 43 ] [ 53 ]  The A-domain is often  cleaved  off, leaving an 86  kilodalton  fragment called  Toc86 . [ 53 ]  In the middle is its  GTP  binding domain, which is very similar to the  homologous  GTP-binding domain in Toc34. [ 43 ] [ 53 ]  At the  C-terminal  end is the  hydrophilic  M-domain, [ 43 ]  which anchors the protein to the outer chloroplast membrane. [ 53 ]


Toc159 probably works a lot like Toc34, recognizing proteins in the cytosol using  GTP . It can be regulated through  phosphorylation , but by a different  protein kinase  than the one that phosphorylates Toc34. [ 46 ]  Its M-domain forms part of the tunnel that chloroplast preproteins travel through, and seems to provide the force that pushes preproteins through, using the energy from  GTP . [ 43 ]


Toc159 is not always found as part of the TOC complex—it has also been found dissolved in the  cytosol . This suggests that it might act as a shuttle that finds chloroplast preproteins in the cytosol and carries them back to the TOC complex. There isn't a lot of direct evidence for this behavior though. [ 43 ]


A family of Toc159 proteins,  Toc159 ,  Toc132 ,  Toc120 , and  Toc90  have been found in  Arabidopsis thaliana . They vary in the length of their A-domains, which is completely gone in Toc90. Toc132, Toc120, and Toc90 seem to have specialized functions in importing stuff like nonphotosynthetic preproteins, and can't replace Toc159. [ 43 ]


Toc75  is the most abundant protein on the outer chloroplast envelope. It is a  transmembrane  tube that forms most of the TOC pore itself. Toc75 is a  β-barrel  channel lined by 16  β-pleated sheets . [ 43 ]  The hole it forms is about 2.5  nanometers  wide at the ends, and shrinks to about 1.4–1.6 nanometers in diameter at its narrowest point—wide enough to allow partially folded chloroplast preproteins to pass through. [ 43 ]


Toc75 can also bind to chloroplast preproteins, but is a lot worse at this than Toc34 or Toc159. [ 43 ]


Arabidopsis thaliana  has multiple  isoforms  of  Toc75  that are named by the  chromosomal  positions of the  genes  that code for them.  AtToc75 III  is the most abundant of these. [ 43 ]


The  TIC translocon , or  t ranslocon on the  i nner  c hloroplast membrane  translocon [ 43 ]  is another protein complex that imports proteins across the  inner chloroplast envelope . Chloroplast polypeptide chains probably often travel through the two complexes at the same time, but the TIC complex can also retrieve preproteins lost in the  intermembrane space . [ 43 ]