Antibody  allotypes came back to spotlight due to development and use of therapies based on  monoclonal antibodies . These  recombinant  human  glycoproteins  and  proteins  are now well established in clinical practise, but sometimes leads to adverse effects such as generation of antitherapeutic  antibodies  that negates therapy or even cause severe reactions to the therapy. This reaction may be attributed to differences between therapeutics itself or may arise between same therapeutics produced by different companies or even between different lots produced by the same company. To prevent production of such antitherapeutic  antibodies , ideally, all clinical used  proteins  and  glycoproteins  should poses same allotype as natural patient’s product, this way the presence of ‘altered self‘ which poses a potential target for  immune system , is limited. Whilst many parameters connected to developing and manufacturing process that might predispose  monoclonal antibodies  to cause  immune response  are well known and appropriate steps are taken to monitor and control these unwanted effects, complications linked with administration of  monoclonal antibodies  to genetically diverse human population are less well described. Humans exhibit abundance of  genotypes  and  phenotypes , however all currently licensed IgG therapeutic  immunoglobulins  are developed as single allotypic/  polymorphic  form. Patients that are homozygous for alternative  phenotype  are therefore at higher risk of developing potential  immune response  to the therapy. [ 4 ]


Amelogenins  are a group of  protein isoforms  produced by  alternative splicing  or  proteolysis  from the  AMELX  gene, on the  X chromosome , and also the  AMELY  gene in males, on the  Y chromosome . [ 1 ]  They are involved in  amelogenesis , the development of  enamel . [ 2 ]  Amelogenins are type of  extracellular matrix  protein, which, together with  ameloblastins ,  enamelins  and  tuftelins , direct the mineralization of enamel to form a highly organized matrix of  rods ,  interrod crystal  and proteins.


Although the precise role of amelogenin(s) in regulating the  mineralization  process is unknown, it is known that amelogenins are abundant during amelogenesis. Developing human enamel contains about 70% protein, 90% of which are amelogenins.


Amelogenins are believed to be involved in the organizing of  enamel rods  during  tooth development . The latest research indicates that these proteins regulate the initiation and growth of  hydroxyapatite  crystals during the mineralization of enamel. In addition, amelogenins appear to aid in the development of  cementum  by directing cementoblasts to the tooth's root surface.


The amelogenin  gene  has been most widely studied in humans, where it is a single copy gene, located on the  X  and  Y chromosomes  at Xp22.1–Xp22.3 and Yp 11.2 [5]. [ 3 ]  The amelogenin gene's location on sex chromosomes has implications for variability both between the X chromosome form ( AMELX ) and the Y chromosome form ( AMELY ), and between alleles of AMELY among different populations. This is because AMELY exists in the  non-recombining  region of chromosome Y, effectively isolating it from normal  selection pressures . Other sources of amelogenin variation arise from the various  isoforms  of AMELX obtained from alternative splicing of mRNA transcripts. Specific roles for isoforms have yet to be established. Among other organisms, amelogenin is well conserved among  eutherians , and has homologs in  monotremes , reptiles and amphibians.


Differences between the  X chromosome  and  Y chromosome  versions of the amelogenin gene (AMELX and AMELY respectively) enable it to be used in sex determination of unknown human samples.  AMELX ’s intron 1 contains a 6-base-pair deletion relative to  intron  1 of  AMELY . This can be detected at low cost using  polymerase chain reaction  (PCR) of intron 1, followed by  gel electrophoresis . Two  bands  of DNA, at 555 bps and 371 bps, are resolved if both the AMELX and AMELY versions of the gene are present (i.e. the sample is from a male) or one band of DNA, at 555 bps, if the AMELX version only is present (i.e. the sample is from a female). [ 4 ]


However, because of AMELY variation among individuals and populations, this method of sex determination is not 100% accurate. Mutation in regions of AMELY intron 1 commonly used as  primer annealing sites  may disable PCR amplification. A 6bp  insertion  to AMELY intron 1 results in an amplicon identical in length to that of AMELX. In some males AMELY may be deleted entirely. In any of these cases only one band is visualized during gel electrophoresis of PCR products, causing misidentification of the sample as female. [ 4 ]  The misidentification rate may vary among populations, but in general appears to be low. In one study in Spain, the amelogenin sex determination test using AMELX (977bps) and AMELY (790bps) bands was performed for 1224 individuals of known gender with a 99.84% (1222/1224) accuracy rate. [ 5 ]  Another study in India, however, found 5 of its 270 men studied (1.85%) possessed an AMELY deletion, terming them "deleted-amelogenin males" (DAMs). In response the authors suggested that while the amelogenin sex test may be accurate in general, other Y chromosome markers such as  SRY , STR, or 50f2 can be used for less ambiguous gender identification. [ 6 ]


In archaeology where DNA is too broken down to be analyzed by PCR,  Liquid chromatography–tandem mass spectrometry  (LC-MS/MS) is used to directly detect the presence of the peptides corresponding to either version from tooth enamel samples. [ 7 ]  This method has been used on samples as old as the  Gravettian . [ 8 ]


Mutations in AMELX can cause  amelogenesis imperfecta , a disorder of tooth enamel development. [ 9 ]


The  Anaerococcus and Onthovivens genetic code  (tentative code number 36) translates CGG to  tryptophan , as determined by the codon assignment software Codetta; [ 1 ]  it was further shown that this recoding is associated with a special tRNA with the appropriate anticodon and tRNA identity elements appropriate for such decoding. As currently known, this code is limited to two distinct clades, the genus  Anaerococcus  in the class  Clostridia  and the genus  Onthovivens  in the class  Bacilli , as defined by the GTDB taxonomy system [ 2 ]  release 220. Codetta originally called the Anaerococcus and Onthovivens code for the following genome assemblies: GCA_000024105.1, GCA_900445285.1, GCA_902500265.1, GCA_900258475.1, GCA_002399785.1, GCA_004558005.1, GCA_900540365.1, GCA_900540395.1, GCA_900545015.1.


Genetic analysis  is the overall process of studying and  researching  in fields of science that involve  genetics  and  molecular biology . There are a number of applications that are developed from this research, and these are also considered parts of the process. The base system of analysis revolves around general genetics. Basic studies include identification of genes and  inherited disorders . This research has been conducted for centuries on both a large-scale physical observation basis and on a more microscopic scale.
Genetic analysis can be used generally to describe methods both used in and resulting from the sciences of genetics and molecular biology, or to  applications  resulting from this research.


Genetic analysis may be done to identify genetic/inherited disorders and also to make a  differential diagnosis  in certain somatic diseases such as  cancer . Genetic analyses of cancer include detection of  mutations ,  fusion genes , and DNA copy number changes.


Much of the research that set the foundation of genetic analysis began in prehistoric times. Early humans found that they could practice  selective breeding  to improve crops and animals. They also identified inherited traits in humans that were eliminated over the years. The many genetic analyses gradually evolved over time.


Modern genetic analysis began in the mid-1800s with research conducted by  Gregor Mendel . Mendel, who is known as the "father of modern genetics", was inspired to study variation in plants. Between 1856 and 1863, Mendel cultivated and tested some 29,000 pea plants (i.e., Pisum sativum). This study showed that one in four pea plants had purebred recessive alleles, two out of four were hybrid and one out of four were purebred dominant. His experiments led him to make two generalizations, the  Law of Segregation  and the  Law of Independent Assortment , which later became known as Mendel's Laws of Inheritance. Lacking the basic understanding of heredity, Mendel observed various organisms and first utilized genetic analysis to find that traits were inherited from parents and those traits could vary between children. Later, it was found that units within each cell are responsible for these traits. These units are called genes. Each gene is defined by a series of amino acids that create proteins responsible for genetic traits.


Genetic analyses include molecular technologies such as  PCR ,  RT-PCR ,  DNA sequencing , and  DNA microarrays , and  cytogenetic  methods such as  karyotyping  and  fluorescence in situ hybridisation .


DNA sequencing  is essential to the applications of genetic analysis. This process is used to determine the order of  nucleotide bases . Each molecule of DNA is made from  adenine ,  guanine ,  cytosine  and  thymine , which determine what function the genes will possess. This was first discovered during the 1970s. DNA sequencing encompasses biochemical methods for determining the order of the nucleotide bases, adenine, guanine, cytosine, and thymine, in a DNA oligonucleotide. By generating a DNA sequence for a particular organism, you are determining the patterns that make up genetic traits and in some cases behaviors.


Sequencing methods have evolved from relatively laborious gel-based procedures to modern automated protocols based on dye labelling and detection in capillary  electrophoresis  that permit rapid large-scale sequencing of genomes and transcriptomes. [ 1 ]  Knowledge of DNA sequences of genes and other parts of the genome of organisms has become indispensable for basic research studying biological processes, as well as in applied fields such as diagnostic or forensic research. The advent of DNA sequencing has significantly accelerated biological research and discovery.


Cytogenetics  is a branch of genetics that is concerned with the study of the structure and function of the cell, especially the chromosomes.  Polymerase chain reaction  studies the amplification of DNA. Because of the close analysis of chromosomes in cytogenetics, abnormalities are more readily seen and diagnosed.


A  karyotype  is the number and appearance of chromosomes in the nucleus of a eukaryotic cell. The term is also used for the complete set of chromosomes in a species, or an individual organism.


Karyotypes describe the number of chromosomes, and what they look like under a light microscope. Attention is paid to their length, the position of the  centromeres , banding pattern, any differences between the sex chromosomes, and any other physical characteristics.
 Karyotyping  uses a system of studying chromosomes to identify genetic abnormalities and evolutionary changes in the past.


A  DNA microarray  is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome. When a gene is expressed in a cell, it generates  messenger RNA  (mRNA). Overexpressed genes generate more mRNA than underexpressed genes. This can be detected on the microarray.
Since an array can contain tens of thousands of probes, a microarray experiment can accomplish many genetic tests in parallel. Therefore, arrays have dramatically accelerated many types of investigations.


The polymerase chain reaction (PCR) is a biochemical technology in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.
PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. These include  DNA cloning  for sequencing, DNA-based  phylogeny , or functional analysis of genes; the diagnosis of  hereditary diseases ; the identification of genetic fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases.


Numerous practical advancements have been made in the field of genetics and molecular biology through the processes of genetic analysis. One of the most prevalent advancements during the late 20th and early 21st centuries is a greater understanding of cancer's link to genetics. By identifying which genes in the cancer cells are working abnormally, doctors can better diagnose and treat cancers.


Research has been able to identify the concepts of genetic mutations, fusion genes and changes in DNA copy numbers, and advances are made in the field every day. Much of these applications have led to new types of sciences that use the foundations of genetic analysis. Reverse genetics uses the methods to determine what is missing in a genetic code or what can be added to change that code. Genetic linkage studies analyze the spatial arrangements of genes and chromosomes. There have also been studies to determine the legal and social and moral effects of the increase of genetic analysis. Genetic analysis may be done to identify genetic/inherited disorders and also to make a differential diagnosis in certain somatic diseases such as cancer. Genetic analyses of cancer include detection of  mutations ,  fusion genes , and DNA copy number changes.


The  antagonistic pleiotropy hypothesis  (APT) is a theory in  evolutionary biology  that suggests certain genes may confer beneficial effects early in an organism's life, enhancing reproductive success, while also causing detrimental effects later in life, contributing to the aging process.


APT was first proposed in a 1952 paper on the evolutionary theory of ageing by  Peter Medawar  and developed further in a paper by  George C. Williams  in 1957 [ 1 ]  as an explanation for  senescence . [ 2 ]   Pleiotropy  is the phenomenon where a single  gene  influences more than one phenotypic trait in an organism. [ 3 ] [ 4 ]  It is one of the most commonly observed attributes of genes. [ 5 ]  A gene is considered to exhibit antagonistic pleiotropy if it controls more than one  phenotypic trait , where at least one of these traits is beneficial to the organism's  fitness  and at least one is detrimental to fitness.


This line of genetic research began as an attempt to answer the following question: if survival and reproduction should always be favoured by natural selection, why should ageing – which in evolutionary terms can be described as the age-related decline in survival rate and reproduction – be nearly ubiquitous in the natural world?" [ 2 ]  The antagonistic pleiotropy hypothesis provides a partial answer to this question. As an evolutionary explanation for ageing, the hypothesis relies on the fact that reproductive capacity declines with age in many species and, therefore, the strength of natural selection also declines with age (because there can be no natural selection without reproduction). [ 6 ] [ 7 ]  Since the strength of selection declines over the life cycles of human and most other organisms, natural selection in these species tends to favor " alleles  that have early beneficial effects, but later deleterious effects". [ 8 ]


Antagonistic pleiotropy also provides a framework for understanding why many  genetic disorders , even those causing life threatening health impacts (e.g.  sickle cell anaemia ), are found to be relatively prevalent in populations. Seen through the lens of simple evolutionary processes, these genetic disorders should be observed at very low frequencies due to the force of natural selection. Genetic models of populations show that antagonistic pleiotropy allows genetic disorders to be maintained at reasonably high frequencies "even if the fitness benefits are subtle". [ 9 ]  In this sense, antagonistic pleiotropy forms the basis of a "genetic trade-off between different fitness components." [ 10 ]


In the theory of evolution, the concept of fitness has two components: mortality and reproduction. Antagonistic pleiotropy gets fixed in genomes by creating viable trade-offs between or within these two components.  The existence of these trade-offs has been clearly demonstrated in human, botanical and insect species. For example, an analysis of global gene expression in the fruit fly,  Drosophila melanogaster , revealed 34 genes whose expression coincided with the genetic trade-off between larval survival and adult size. The joint expression of these candidate 'trade-off' genes explained 86.3% of the trade-off. These tradeoffs can result from selection at the level of the organism or, more subtly, via mechanisms for the allocation of scarce resources in cellular metabolism. [ 11 ]


Another example is found in a study of the yellow monkey flower, an  annual plant . The study documents a trade-off between days-to-flower and reproductive capacity. This genetic balancing act determines how many individuals survive to flower in a short growing season (viability) while also influencing the seed set of survivors (fecundity). The authors find that tradeoffs between plant viability and fecundity can engender a stable polymorphism under surprisingly general conditions. Thus, for this annual flower, they reveal a tradeoff between mortality and fecundity and, according to the authors, this tradeoff is also relevant for other annual, flowering plants. [ 10 ]


Senescence refers to the process of physiological change in individual members of a species as they age. [ 9 ] [ 12 ]  An antagonistically pleiotropic gene can be selected for if it has beneficial effects in early life while manifesting its negative effects in later life because genes tend to have larger impacts on fitness in an organism's prime than in their old age. [ 13 ]   Williams's 1957 article has motivated many follow-up studies on the evolutionary causes of ageing. [ 14 ]  These studies show clear trade-offs involving early increases in fecundity and later increases in mortality. For example, two experiments with  Drosophila melanogaster  have shown that increased fertility is associated with reduced longevity. Likewise, for humans, infertile women live longer on average than fertile women. [ 15 ] [ 16 ]


One such study tests the hypothesis that death due to cardiovascular disease in women is linked to an antagonistic pleiotropy operating through inflammation and linked to fertility. Because the human immune system evolved in an ancestral environment characterized by abundant pathogens, protective, pro-inflammatory responses (which helped individuals to avoid and survive infections) were undoubtedly selected for in these environments. However, in terms of cardiovascular risk, these same inflammatory responses have turned out to be harmful as the material conditions of human existence improved – in affluent countries, where life expectancy is much longer than in the ancestral environment, strong inflammatory responses carry greater risks of cardiovascular disease as individuals age. The study looks at mortality, over a period of 3 to 5 years, in a group of 311, 85-year old Dutch women. Information on their reproductive history as well the results of blood tests, genetic tests and physical examinations was recorded. The study found that individuals with a higher pro-inflammatory ratio TNFα/IL-10 had a significantly higher incidence of death due to cardiovascular disease in old age. It also linked specific  alleles  to a combination of higher fertility, stronger inflammatory response and greater cardiovascular problems in old age. This finding supports the hypothesis that this gene was prevalent because it helped women in the ancestral environment to more effectively combat infection during their reproductive years. However, the pleiotropic costs of the gene in terms of cardiovascular risks are now clear because people live long enough to die of cardiovascular disease.  [ 15 ]


The survival of many serious genetic disorders in human evolutionary history has led researchers to explore the role of antagonistic  pleiotropy  in disease. If genetic disorders are caused by mutations to a single deleterious allele, then  natural selection  should eliminate carriers of this allele over evolutionary time, thereby lowering the frequency of mutations. Yet, research shows that the incidence of such alleles in studied populations is often stable and relatively high. [ 9 ]  In a 2011 review article, Carter and Nguyen discuss several genetic disorders, arguing that, far from being a rare phenomenon, antagonistic pleiotropy might be a fundamental mechanism by which "alleles with severe deleterious health effects can be maintained at medically relevant frequencies with only minor beneficial pleiotropic effects." [ 9 ]


An example of this is  sickle cell anaemia , which results in an abnormality in the oxygen-carrying protein  haemoglobin  found in  red blood cells . [ 17 ]  Possessors of the deleterious allele have much lower life expectancies, with  homozygotes  rarely reaching 50 years of age. However, this allele also enhances resistance to  malaria . Thus, in regions where malaria exerts or has in the past exerted a strong selective pressure, sickle cell anaemia has been selected for its conferred partial resistance to the disease. While homozygotes will have either no protection from malaria or a dramatic propensity to sickle cell anemia,  heterozygotes  have fewer physiological effects and a partial resistance to malaria. Thus, the gene that is responsible for sickle cell disease has fixed itself with relatively high frequencies in populations threatened by malaria by engendering a viable tradeoff between death from this non-communicable disease and death from malaria. [ 18 ]


In another study of genetic diseases, 99 individuals with  Laron syndrome  (a rare form of dwarfism) were monitored alongside their non-dwarf kin for a period of ten years. Patients with Laron syndrome possess one of three genotypes for the  growth hormone receptor  gene (GHR). Most patients have an A->G  splice site mutation  in position 180 in exon 6. Some others possess a  nonsense mutation  (R43X), while the rest are heterozygous for the two mutations. Laron syndrome patients experienced a lower incidence of cancer mortality and diabetes compared to their non-dwarf kin. [ 19 ]  This suggests a role for antagonistic pleiotropy, whereby a deleterious mutation is preserved in a population because it still confers some survival benefit. [ 9 ]


Another instance of antagonistic pleiotropy is manifested in  Huntington's disease , a rare  neurodegenerative  disorder characterized by a high number of CAG repeats within the  Huntingtin  gene. The onset of Huntington's is usually observed post-reproductive age and generally involves involuntary muscle spasms, cognitive difficulties and psychiatric problems. The high number of CAG repeats is associated with increased activity of  p53 , a tumor suppressing protein that participates in  apoptosis . It has been hypothesized that this explains the lower rates of cancer among Huntington's patients. Huntington's disease is also correlated with high  fecundity . [ 9 ]


Other pleiotropic diseases include:  beta-thalassemia  (also protects against malaria in the heterozygous state);  cystic fibrosis  (increased fertility); and osteoporosis in old age (reduced risk of osteoporosis in youth). [ 9 ]


Sexual selection is a process of  natural selection  in which individuals of one sex choose to mate with individuals of the other sex based on certain observable characteristics (intersexual selection, the exaggerated tail in  peacocks , for example) or they compete with other individuals of the same sex for access to mating opportunities with individuals of the opposite sex (intrasexual selection, as when males of certain mammalian species fight for the right to mate). [ 20 ]  It is generally accepted that the evolution of secondary sexual characteristics persists until the relative costs of survival outweigh the benefits of reproductive success. [ 21 ]


At the level of  genes , this means a trade-off between variation and expression of selected traits. Strong, persistent sexual selection should result in decreased genetic variation for these traits. However, higher levels of variation have been reported in sexually-selected traits compared to non-sexually selected traits. [ 22 ]  This phenomenon is especially clear in  lek  species, where males' courtship behavior confers no immediate advantage to the female. Female choice presumably depends on correlating male displays (secondary sexual characteristics) with overall genetic quality. If such directional sexual selection depletes variation in males, why would female choice continue to exist? Rowe and Houle answer this question (the  lek paradox ) using the notion of  genetic capture , which couples the sexually-selected traits with the overall condition of the organism. They posit that the genes for secondary sexual characteristics must be pleiotropically linked to condition, a measure of the organism's fitness. In other words, the genetic variation in secondary sexual characteristics is maintained due to variation in the organism's condition. [ 23 ]


DNA repair  capability in hamsters is relatively high during development in early  mitotic  brain cells, but is present at a reduced level in post-mitotic brain cells throughout adulthood. [ 24 ]     In humans DNA repair capability declines in older individuals. [ 25 ]   These findings suggest that DNA repair activity is regulated at a level that facilitates vigorous function during youth, but at an insufficient level during maturity that results in aging.  Thus proteins that regulate the level of DNA repair activity appear to have the antagonistic pleiotrophic effect of being beneficial during youthful development, but insufficient to prevent aging during maturity. [ citation needed ]


Advances in genome mappings have greatly facilitated research into antagonistic pleiotropy.  Such research is now often carried out in laboratories, but also in wild populations. The latter context for testing has the advantage of introducing the full complexity of the selection experience – competitors, predators, and parasites – though it has the disadvantage of introducing idiosyncratic factors that are specific to given locations. In order to be able to assert with confidence that a given pleiotropy is, indeed, an antagonistic pleiotropy and not due to some other competing cause (e.g. the  mutation accumulation  hypothesis), one must have knowledge of the precise gene that is pleiotropic. This is now increasingly possible with organisms that have detailed genomic mappings (e.g. mice, fruit flies and humans). A 2018 review of this research finds that "antagonistic pleiotropy is somewhere between very common or ubiquitous in the animal world .... and potentially all living domains... ". [ 2 ]


In  biology , an  atavism  is a modification of a biological structure whereby an ancestral genetic  trait  reappears after having been lost through  evolutionary  change in previous generations. [ 3 ]  Atavisms can occur in several ways, [ 4 ]  one of which is when  genes  for previously existing  phenotypic  features are preserved in  DNA , and these become expressed through a  mutation  that either knocks out the dominant genes for the new traits or makes the old traits dominate the new one. [ 3 ]  A number of traits can vary as a result of shortening of the  fetal development  of a trait ( neoteny ) or by prolongation of the same. In such a case, a shift in the time a trait is allowed to develop before it is fixed can bring forth an ancestral phenotype. [ 5 ]  Atavisms are often seen as  evidence of evolution . [ 6 ]


In  social sciences , atavism is the tendency of reversion: for example, people in the modern era reverting to the ways of thinking and acting of a former time.


The word  atavism  is derived from the  Latin   atavus —a great-great-great-grandfather or, more generally, an ancestor.


Evolutionarily traits that have disappeared phenotypically do not necessarily disappear from an organism's DNA. The gene sequence often remains, but is inactive. Such an unused gene may remain in the genome for many generations. [ 4 ] [ 7 ]  As long as the gene remains intact, a fault in the genetic control suppressing the gene can lead to it being expressed again. Sometimes, the expression of dormant genes can be induced by artificial stimulation.


Atavisms have been observed in humans, such as with infants born with  vestigial tails  (called a "coccygeal process", "coccygeal projection", or "caudal appendage"). [ 8 ]  Atavism can also be seen in humans who possess large teeth, like those of other primates. [ 9 ]  In addition, a case of "snake heart", the presence of "coronary circulation and myocardial architecture [that closely] resemble those of the reptilian heart", has also been reported in medical literature. [ 10 ]  Atavism has also recently been induced in avian dinosaur (bird) fetuses to express dormant ancestral non-avian dinosaur (non-bird) features, including teeth. [ 11 ]


Other examples of observed atavisms include:


Atavism is a term in  Joseph Schumpeter 's explanation of  World War I  in twentieth-century  liberal  Europe. He defends the  liberal international relations theory  that an international society built on commerce will avoid war because of war's destructiveness and comparative cost. His reason for World War I is termed "atavism", in which he asserts that senescent governments in Europe (those of the  German Empire ,  Russian Empire ,  Ottoman Empire , and  Austro-Hungarian Empire ) pulled the liberal Europe into war, and that the liberal regimes of the other continental powers did not cause it. He used this idea to say that liberalism and commerce would continue to have a soothing effect in international relations, and that war would not arise between nations which are connected by commercial ties. [ 29 ]  This latter idea is very similar to the later  Golden Arches theory .


University of London  professor  Guy Standing  has identified three distinct sub-groups of the  precariat , one of which he refers to as "atavists", who long for what they see as a lost past. [ 30 ]


During the interval between the acceptance of  evolution  in the mid-1800s and the rise of the modern understanding of  genetics  in the early 1900s, atavism was used to account for the reappearance in an individual of a trait after several generations of absence—often called a "throw-back". [ citation needed ]  The idea that atavisms could be made to accumulate by  selective breeding , or  breeding back , led to breeds such as  Heck cattle . [ citation needed ]  This had been bred from ancient  landraces  with selected primitive traits, in an attempt of "reviving" the  aurochs , an  extinct  species of wild cattle. [ citation needed ]  The same notions of atavisms were used by  social Darwinists , who claimed that "inferior" races displayed atavistic traits, and represented more primitive traits than other races. [ citation needed ]  Both atavism's and  Ernst Haeckel 's  recapitulation theory  are related to  evolutionary progress , as development towards a greater complexity and a superior ability. [ citation needed ]


In addition, the concept of atavism as part of an individualistic explanation of the causes of criminal deviance was popularised by the Italian criminologist  Cesare Lombroso  in the 1870s. [ 31 ]  He attempted to identify physical characteristics common to criminals and labeled those he found as atavistic, 'throw-back' traits that determined 'primitive' criminal behavior. His statistical evidence and the closely related idea of  eugenics  have long since been abandoned by the scientific community, but the concept that physical traits may affect the likelihood of criminal or unethical behavior in a person still has some scientific support. [ 32 ]


Autogamy depression  can be defined as the "lowered viability of  autogamous  progeny relative to  geitonogamous  progeny". [ 1 ]  Viability has also been evaluated in terms of percent fruit set or  seed set rather than reproductive  fitness  of the progeny. [ 2 ]  The experimental design for observing the occurrence of autogamy depression is called an "autogamy depression test" which has been described by researchers as analogous to a test for  inbreeding depression . [ 1 ]  The ability for fitness of autogamous progeny to differ from geitonogamous progeny comes from the understanding that plants can accumulate heritable mutational variation through both  mitotic division  and  meiotic division . [ 3 ]  Because plants have  indeterminate growth , the  apical meristems  that contribute to the development of the reproductive structures of a plant have the potential to undergo continual mitosis resulting in the accumulation of  somatic mutations  (acquired mutations). [ 1 ]  It has been demonstrated through research that long lived plants can have higher per generation mutation rate (based on occurrences of more mitotic cell divisions compared to short lived plants). [ 2 ]  Any deleterious mutations that appear during mitotic growth are filtered out through cell lineage selection, in which deleterious mutations that are subject to  developmental selection  during mitotic growth are replaced by vigorous cell lineages, however, somatic mutations that are not expressed will not be subject to selection during growth of the plant and will accumulate in the apical meristem. [ 4 ]


There is evidence of the  phenotypic  effects of somatic mutations in increased  chlorophyll  mutants of some long-lived plants. Chlorophyll mutants are inherently easy to observe because of the phenotypic effects of the chlorophyll mutations. Because of the highly conserved nature of  photosynthetic  processes, these chlorophyll mutation rates can be generalized to most  angiosperms . [ 5 ]  Somatic mutations accumulating during vegetative growth have also been found to affect the fitness of seedlings in the next generation. [ 3 ]


Individual crowns are treated as "independent  mitotic  mutation-accumulation lines" [ 1 ]  and so the appearance of deleterious somatic mutations in the autogamous crosses will be  heterozygous  or  homozygous  at the same locus (~25% homozygous) [ 4 ]  and the appearance of deleterious somatic mutations in the geitonogamous crosses will be heterozygous. [ 1 ]  The autogamy depression can be calculated through the simple equation AD = 1 − (w a /w g ), where AD is the autogamy depression, w a  is the fitness of the autogamous progeny and w g  is the fitness of the geitonogamous progeny. When the fitnesses are equal the AD is 0. The difference can be calculated by the equation D = w g  − w a. [ 2 ]


Backcrossing  is a crossing of a  hybrid  with one of its  parents  or an individual genetically similar to its parent, to achieve offspring with a genetic identity closer to that of the parent. It is used in horticulture, animal breeding, and  production of  gene knockout  organisms.


Backcrossed hybrids are sometimes described with  acronym  "BC"; for example, an  F1 hybrid  crossed with one of its parents (or a genetically similar individual) can be termed a BC1 hybrid, and a further cross of the BC1 hybrid to the same parent (or a genetically similar individual) produces a BC2 hybrid. [ 1 ]


York radiate groundsel ( Senecio eboracensis ) is a naturally occurring hybrid species of Oxford ragwort ( Senecio squalidus ) and common groundsel ( Senecio vulgaris ). It is thought to have arisen from a backcrossing of the F1 hybrid with  S. vulgaris . [ 2 ]


Again, the pure tall (TT) and pure dwarf (tt) pea plants when crossed in the parental generation, produce all heterozygote (Tt) tall pea plants in the first filial generation. The cross between first filial heterozygote tall (Tt) pea plant and pure tall (TT) or pure dwarf (tt) pea plant of the parental generation is also an example for the back-crossing between two plants. In this case, the filial generation formed after the back cross may have a phenotype ratio of 1:1 if the cross is made with recessive parent or else all offspring may be having phenotype of dominant trait if the backcross is with a parent having the dominant trait. The former of these traits is also called a test cross.


In plants, the term inbred backcross line (IBL) refers to a  line  (i.e.  population ) of plants derived from the repeated backcrossing of a line with artificially  recombinant DNA  with the  wild type , operating some kind of  selection  that can be  phenotypical  or through a  molecular marker  (for the production of an  introgression line ).


Backcrossing may be deliberately employed in animals to transfer a desirable trait in an animal of inferior genetic background to an animal of preferable genetic background. In gene-knockout experiments in particular, where the knockout is performed on easily  cultured   stem cell  lines, but is required in an animal with a different genetic background, the knockout animal is backcrossed against the animal of the required genetic background. As the figure shows, each time that the mouse with the desired trait (in this case the lack of a gene (i.e. a knockout), indicated by the presence of a  positive selectable marker ) is crossed with a mouse of a constant genetic background, the average percentage of the genetic material of the offspring that is derived from that constant background increases. The result, after sufficient reiterations, is an animal with the desired trait in the desired genetic background, with the percentage of genetic material from the original stem cells reduced to a minimum (on the order of 0.01%). [ 3 ]


Due to the nature of meiosis, in which chromosomes derived from each parent are randomly shuffled and assigned to each nascent gamete, the percentage of genetic material deriving from either cell line varies between offspring of a single crossing, but will have an  expected value . The genotype of each member of offspring may be assessed to choose not only an individual that carries the desired genetic trait, but also the minimum percentage of genetic material from the original stem cell line. [ 4 ]


A consomic strain is an inbred strain with one of its chromosomes replaced by the homologous chromosome of another inbred strain via a series of marker-assisted backcrosses. [ 5 ]


A  Barr body  (named after discoverer  Murray Barr ) [ 1 ]  or  X-chromatin  is an inactive  X chromosome .  In species with   XY sex-determination  (including humans), females typically have two X chromosomes, [ 2 ]  and one is rendered inactive in a process called  lyonization . Errors in chromosome separation can also result in male and female individuals with extra X chromosomes.  The  Lyon hypothesis  states that in cells with multiple  X chromosomes , all but one are inactivated early in  embryonic  development  in  mammals . [ 3 ] [ 4 ]  The  X chromosomes that become inactivated are chosen randomly, except in  marsupials  and in some extra-embryonic tissues of some placental mammals, in which the X chromosome from the sperm is always deactivated. [ 5 ]


In humans with  euploidy , a  genotypical  female (46, XX  karyotype ) has one Barr body per somatic  cell nucleus , while a genotypical male (46, XY) has none. The Barr body can be seen in the  interphase  nucleus as a darkly staining small mass in contact with the nucleus membrane. Barr bodies can be seen in  neutrophils  at the rim of the nucleus.


In humans with more than one X chromosome, the number of Barr bodies visible at interphase is always one fewer than the total number of X chromosomes. For example, people with  Klinefelter syndrome  (47, XXY) have a single Barr body, and people with a  47, XXX  karyotype have two Barr bodies.


Someone with two X chromosomes (such as the majority of  human  females) has only one Barr body per  somatic cell , while someone with one X chromosome (such as most human males) has none.


Mammalian  X-chromosome inactivation  is initiated from the X inactivation centre or  Xic , usually found near the  centromere . [ 6 ]  The center contains twelve  genes , seven of which code for  proteins , five for untranslated  RNAs , of which only two are known to play an active role in the X inactivation process,  Xist  and  Tsix . [ 6 ]  The centre also appears to be important in chromosome counting: ensuring that random inactivation only takes place when two or more X-chromosomes are present. The provision of an extra artificial  Xic  in early  embryogenesis  can induce inactivation of the single X found in male cells. [ 6 ]


The roles of  Xist  and  Tsix  appear to be antagonistic. The loss of  Tsix  expression on the future inactive X chromosome results in an increase in levels of  Xist  around the  Xic . Meanwhile, on the future active X  Tsix  levels are maintained; thus the levels of  Xist  remain low. [ 7 ]  This shift allows  Xist  to begin coating the future inactive chromosome, spreading out from the  Xic . [ 2 ]  In non-random inactivation this choice appears to be fixed and current evidence suggests that the maternally inherited  gene  may be  imprinted . [ 3 ]  Variations in Xi frequency have been reported with age, pregnancy, the use of oral contraceptives, fluctuations in menstrual cycle and neoplasia. [ 8 ]


It is thought that this constitutes the mechanism of choice, and allows downstream processes to establish the compact state of the Barr body. These changes include  histone  modifications, such as histone H3  methylation  (i.e.  H3K27me3  by  PRC2 which is recruited by Xist ) [ 9 ]  and histone H2A  ubiquitination , [ 10 ]  as well as direct modification of the  DNA  itself, via the methylation of  CpG sites . [ 11 ]  These changes help inactivate  gene  expression on the inactive X-chromosome and to bring about its compaction to form the Barr body.


Reactivation  of a Barr body is also possible, and has been seen in breast cancer patients. [ 12 ]  One study showed that the frequency of Barr bodies in breast carcinoma were significantly lower than in healthy controls, indicating reactivation of these once inactivated X chromosomes. [ 12 ]


Barr bodies  are condensed, inactive X chromosomes found in the somatic cells of female mammals. Their detection in ancient samples provides a powerful tool for gender identification in extinct species, offering insights into population dynamics, biology, and evolution.


Recent advancements in histological and genomic techniques have made it possible to observe Barr bodies in ancient remains, including fossilized bones and tissues:


In a notable example, Barr bodies were detected in osteocytes from ancient mammalian remains, demonstrating the potential of this approach for studying gender in extinct populations [ 13 ] .


Further research into the detection of Barr bodies may enhance our ability to:


Links to full text articles are provided where access is free, in other cases only the abstract has been linked.


The biosynthesis of  benzoxazinone , a cyclic hydroxamate and a natural  insecticide , has been well-characterized in  maize  and related grass species. [ 1 ]  In maize, genes in the pathway are named using the symbol  bx . Maize Bx-genes are tightly linked, a feature that has been considered uncommon for plant genes of a biosynthetic pathways. Especially notable are genes encoding the different enzymatic functions BX1, BX2 and BX8 and which are found within about 50 kilobases. [ 1 ] [ 2 ]  Results from  wheat  and  rye  indicate that the cluster is an ancient feature. [ 3 ]  In wheat the cluster is split into two parts. The wheat genes Bx1 and Bx2 are located in close proximity on chromosome 4 and wheat Bx3, Bx4 and Bx5 map to the short arm of chromosome 5; an additional Bx3 copy was detected on the long arm of chromosome 5B. [ 3 ]  Recently, additional biosynthetic clusters have been detected in other plants for other biosynthetic pathways and this organization might be common in plants. [ 4 ]


The  bx1  gene encodes a protein, BX1, that forms  indol  from indol-3-glycerol phosphate in the  plastid . It is the first step in the pathway and determines much of the natural variation in levels of  DIMBOA  in maize. [ 5 ]  The next steps in the pathway occur in the  endoplasmic reticulum , also referred to as the  microsomes  in  cell fractionation  experiments, and are carried by proteins encoded by genes bx2, bx3, bx4, and bx5.


The  bioecological model of development  is the mature and final revision of Urie Bronfenbrenner's  ecological system theory . The primary focus of ecological systems theory is on the systemic examination of contextual variability in development processes. It focuses on the world outside the developing person and how they were affected by it. After publication of The Ecology of Human Development, [ 1 ]  Bronfenbrenner's first comprehensive statement of ecological systems theory, additional refinements were added to the theory. [ 2 ]  Whereas earlier statements of ecological systems theory focused on characteristics of the environment, the goal of the bioecological model was to explicate how characteristics of the developing person influenced the environments to which the person was exposed and how they were affected by the environment. [ 3 ]  The bioecological model is strongly influenced by Bronfenbrenner's collaborations with  Stephen Ceci . [ 4 ]  Whereas much of Bronfenbrenner's work had focused on social development and the influence of social environments on development, Ceci's work focuses on memory and intelligence. [ 5 ]  The bioecological model reflects Ceci's work on contextual variability in intelligence and cognition and Bronfenbrenner's interest in developmentally instigative characteristics - how people help to create their own environments. [ 3 ]


Bronfenbrenner's initial investigations into contextual variability in developmental processes can be seen in the 1950s in the analysis of differences in methods of parental discipline as a function of historical time and social class. [ 6 ]  It was further developed in his work on the differential effects of parental discipline on boys and girls in the 1960s and of convergence of socialization processes in the US and USSR in the 1970s. [ 3 ]  These works were expressed in the experimental variations built in the development and implementation of the  HeadStart  program. [ 7 ]  informally discussed new ideas concerning  Ecological Systems Theory  throughout the late 1970s and early 1980s during lectures and presentations to the psychological community. [ 8 ]  Bronfenbrenner published a major statement of ecological systems theory in  American Psychologist , [ 9 ]  articulated it in a series of propositions and hypotheses in his most cited book,  The Ecology of Human Development  and further developing it in  The Bioecological Model of Human Development  and later writings. [ 10 ] [ 11 ] [ 12 ]


Bronfenbrenner's early thinking was strongly influenced by other developmentalists and social psychologists who studied developmental processes as contextually bound and dependent on the meaning of experience as defined by the developing person. [ 13 ]  One strong influence was  Lev Vygotsky , a Russian  psychologist who emphasized recognized that learning always occurs and cannot be separated from a social context. A second influence was  Kurt Lewin , a German forerunner of ecological systems models who focused on a person's psychological activities that occur within a kind of psychological field, [ 14 ]  including all the events in the past, present, and future that shape and affect an individual. The centrality of the person's interpretation of their environment and phenomenological nature was built on the work of Thomas & Thomas: “(i)f men define situations as real they are real in their consequences”. [ 15 ]


Bronfenbrenner was also influenced by his colleague,  Stephen J. Ceci , with whom he co-authored the article “Nature-nurture reconceptualized in developmental perspective: A bioecological theory” in 1994. [ 5 ]  Ceci is a developmental psychologist who redefined modern developmental psychology's approach to intellectual development. He focused on predicting a pattern of associations among ecological, genetic, and cognitive variables as a function of proximal processes. Together, Bronfenbrenner and Ceci published the beginnings of the bioecological model and made it an accessible framework to use in understanding developmental processes. [ 5 ]


The history of bioecological systems theory is divided into two periods. The first period resulted in the publication of Bronfenbrenner's theory of ecological systems theory, titled  The Ecology of Human Development , in 1979. [ 16 ]  Bronfenbrenner described the second period as a time of criticism and evaluation of his original work. [ 17 ]


The development of ecological systems theory arose because Bronfenbrenner noted a lack of focus on the role of context in terms of development. He argued the environment in which children operate is important because development may be shaped by their interactions with the specific environment. He urged his colleagues to study development in terms of ecological contexts, that is the normal environments of children (schools, homes, daycares). Researchers heeded his advice and a great deal of research flourished in the early 1980s that focused on context.


However, where prior research was ignoring context, Bronfenbrenner felt current research focused too much on context and ignored development. In his justification for a new theory, Bronfenbrenner wrote he was not pleased with the direction of research in the mid 1980s and that he felt there were other realms of development that were overlooked. [ 8 ]


In comparison to the original theory, bioecological systems theory adds more emphasis to the person in the context of development. Additionally, Bronfenbrenner chose to leave out key features of the ecological systems theory (e.g.,  ecological validity  and ecological experiments) during his development of bioecological systems theory.  As a whole, Bronfenbrenner's new theory continued to go through a series of transformations as he continuously analyzed different factors in human development. Critical components of bioecological systems theory did not emerge all at once. Instead, his ideas evolved and adapted to the research and ideas of the times.  For example, the role of proximal processes, which is now recognized as a key feature of bioecological systems theory, did not emerge until the 1990s. [ 17 ]  This theory went through a series of transformations and elaborations until 2005 when Bronfenbrenner died. [ 18 ]


Bronfenbrenner further developed the model by adding the chronosystem, which refers to how the person and environments change over time. He also placed a greater emphasis on processes and the role of the biological person. The Process–Person–Context–Time Model (PPCT) has since become the bedrock of the bioecological model. PPCT includes four concepts. The interactions between the concepts form the basis for the theory. [ 8 ]


Thus, the bioecological model highlights the importance of understanding a person's development within environmental systems. It further explains that both the person and the environment affect one another bidirectionally. Although even Bronfenbrenner himself critiqued the falsifiability of the model, [ 19 ]  the bioecological model has real world applications for developmental research, practice, and policies (as demonstrated below).


In addition to adding to the theoretical understanding of human development, the bioecological model lends itself to changes in the conceptualization of the research endeavor. In some of his earliest comments on the state of developmental research, Bronfenbrenner lamented that developmental research concerned itself with studying “strange behavior of children in strange situations for the briefest possible period of time”. [ 20 ]  He proposed, rather, that developmental science should take as its goal a study of children in context in order to best determine which processes are naturally “developmentally generative” (promote development) and which are naturally “developmentally disruptive” (prevent development).


Bronfenbrenner set up a contrast to the traditional “confirmatory” approach to hypothesis testing (in which research is done to “confirm” that a hypothesis is correct or incorrect) when specifying the types of research needed to support the bioecological model of development. [ 21 ]   In Bronfenbrenner's view, the dynamic nature of the model calls for “primarily generative” research designs that explore interactions between proximal processes (see Proposition 1) and the developing person, environment, time, and developmental outcome (Proposition 2).  Bronfenbrenner called this type of research the “discovery mode” of developmental science.


To best capture such dynamic processes, developmental research designs would ideally be  longitudinal  (over time), rather than  cross-sectional  (a single point in time), and conducted in children's natural environments, rather than a laboratory. Such designs would thus occur in schools, homes, day-care centers, and other environments in which proximal processes are most likely to occur. The bioecological model also proposes that the most scientifically rich studies would include more than one distinct but theoretically related proximal process in the same design. [ 8 ]  Indeed, studies that claim to be based upon bioecological theory should include elements of process, person, context, and time, and should include explicit explanation and acknowledgement if one of the elements is lacking. [ 17 ]  Based on the interactions of proposed elements of the PPCT model, appropriate statistical analyses of PPCT data would likely include explorations of mediation and moderation effects, as well as multilevel modeling of data to account for the nesting of different components of the model. Moreover, research that includes both genetic and environmental components would capture even more of the bioecological model's elements.


The ecological systems theory emerged before the advent of Internet revolution and the developmental influence of then available technology (e.g., television) was conceptually situated in the child's microsystem. Johnson and Puplampu, for instance, proposed in 2008 the ecological techno-subsystem, a dimension of the microsystem. [ 22 ]  This microsystem comprises both child interaction with living (e.g., peers, parents, teachers) and non-living (e.g., hardware, gadgets) elements of communication, information, and recreation technologies in immediate or direct environments. Johnson published a validation study in 2010. [ 23 ]


Whereas the theory of the techno-subsystem merely highlights the influence that digital technologies have on the development of an individual within the microsystem, Navarro and Tudge argue that the virtual world be given its own consideration throughout the Bioecological model. [ 24 ]  They suggest two key modifications as a way to incorporate Bonfenbrenner's theory into our technologized world:


A  blood type  (also known as a  blood group ) is  a classification of  blood , based on the presence and absence of  antibodies  and  inherited   antigenic  substances on the surface of  red blood cells  (RBCs). These antigens may be  proteins ,  carbohydrates ,  glycoproteins , or  glycolipids , depending on the blood group system. Some of these antigens are also present on the surface of other types of  cells  of various  tissues . Several of these red blood cell surface antigens can stem from one  allele  (or an alternative version of a gene) and collectively form a blood group system. [ 1 ]


Blood types are inherited and represent contributions from both parents of an individual. As of 31 December 2023, [update]  a total of 45 [ 2 ]   human blood group systems  are recognized by the  International Society of Blood Transfusion  (ISBT). [ 3 ]  The two most important blood group systems are  ABO  and  Rh ; they determine someone's blood type (A, B, AB, and O, with + or − denoting  RhD  status) for suitability in  blood transfusion .


A complete blood type would describe each of the 45 blood groups, and an individual's blood type is one of many possible combinations of blood-group antigens. [ 3 ]  Almost always, an individual has the same blood group for life, but very rarely an individual's blood type changes through addition or suppression of an antigen in  infection ,  malignancy , or  autoimmune disease . [ 4 ] [ 5 ] [ 6 ] [ 7 ]  Another more common cause of blood type change is a  bone marrow transplant . Bone-marrow transplants are performed for many  leukemias  and  lymphomas , among other diseases. If a person receives bone marrow from someone of a different ABO type (e.g., a type O patient receives a type A bone marrow), the patient's blood type should eventually become the donor's type, as the patient's  hematopoietic stem cells  (HSCs) are destroyed, either by ablation of the bone marrow or by the donor's T-cells. Once all the patient's original red blood cells have died, they will have been fully replaced by new cells derived from the donor HSCs. Provided the donor had a different ABO type, the new cells' surface antigens will be different from those on the surface of the patient's original red blood cells. [ 8 ]


Some blood types are associated with inheritance of other diseases; for example, the  Kell antigen  is sometimes associated with  McLeod syndrome . [ 9 ] For another example,  Von Willebrand disease  may be more severe or apparent in people with blood type O.  Certain blood types may affect susceptibility to infections.  For example, people with blood type O may be less susceptible to pro-thrombotic events induced by  COVID-19  or  long covid . [ 10 ] [ 11 ]  Another example being the resistance to specific  malaria  species seen in individuals lacking the  Duffy antigen . [ 12 ]  The Duffy antigen, presumably as a result of  natural selection , is less common in population groups from areas having a high incidence of malaria. [ 13 ]


The ABO blood group system involves two antigens and two antibodies found in human blood. The two antigens are antigen A and antigen B. The two antibodies are antibody A and antibody B. The antigens are present on the red blood cells and the antibodies in the  serum . Regarding the antigen property of the blood all human beings can be classified into four groups, those with antigen A (group A), those with antigen B (group B), those with both antigen A and B (group AB) and those with neither antigen (group O). The antibodies present together with the antigens are found as follows: [ citation needed ]


There is an  agglutination  reaction between similar antigen and antibody (for example, antigen A agglutinates the antibody A and antigen B agglutinates the antibody B). Thus, transfusion can be considered safe as long as the serum of the recipient does not contain antibodies for the blood cell antigens of the donor. [ citation needed ]


The ABO system is the most important blood-group system in human-blood transfusion. The associated anti-A and anti-B antibodies are usually  immunoglobulin M , abbreviated  IgM , antibodies. It has been hypothesized that ABO IgM  antibodies  are produced in the first years of life by sensitization to environmental substances such as food,  bacteria , and  viruses , although blood group compatibility rules are applied to newborn and infants as a matter of practice. [ 14 ]  The original terminology used by  Karl Landsteiner  in 1901 for the classification was A/B/C; in later publications "C" became "O". [ 15 ]  Type O is often called  0  ( zero , or  null ) in other languages. [ 15 ] [ 16 ]


The Rh system (Rh meaning  Rhesus ) is the second most significant blood-group system in human-blood transfusion with currently 50 antigens. The most significant Rh antigen is the D antigen, because it is the most likely to provoke an immune system response of the five main Rh antigens. It is common for D-negative individuals not to have any anti-D IgG or IgM antibodies, because anti-D antibodies are not usually produced by sensitization against environmental substances. However, D-negative individuals can produce  IgG  anti-D antibodies following a sensitizing event: possibly a fetomaternal transfusion of blood from a fetus in pregnancy or occasionally a blood transfusion with D positive  RBCs . [ 17 ]   Rh disease  can develop in these cases. [ 18 ]  Rh negative blood types are much less common in Asian populations (0.3%) than they are in European populations (15%). [ 19 ]


The presence or absence of the Rh(D) antigen is signified by the + or − sign, so that, for example, the A− group is ABO type A and does not have the Rh (D) antigen. [ 20 ]


As with many other genetic traits, the distribution of ABO and Rh blood groups varies significantly between populations. [ citation needed ] [ 21 ]  While theories are still debated in the scientific community as to why blood types vary geographically and why they emerged in the first place, evidence suggests that the evolution of blood types may be driven by genetic selection for those types whose antigens confer resistance to particular diseases in certain regions – such as the prevalence of blood type O in malaria-endemic countries where individuals of blood type O exhibit the highest rates of survival. [ 22 ]


As of 31 December 2022, [update]  42 blood-group systems have been identified by the International Society for Blood Transfusion in addition to the ABO and Rh systems. [ 3 ]  Thus, in addition to the ABO antigens and Rh antigens, many other antigens are expressed on the RBC surface membrane. For example, an individual can be AB, D positive, and at the same time M and N positive ( MNS system ), K positive ( Kell system ), Le a  or Le b  negative ( Lewis system ), and so on, being positive or negative for each blood group system antigen. Many of the blood group systems were named after the patients in whom the corresponding antibodies were initially encountered. Blood group systems other than ABO and Rh pose a potential, yet relatively low, risk of complications upon mixing of blood from different people. [ 23 ]


Following is a comparison of clinically relevant characteristics of antibodies against the main human blood group systems: [ 24 ]


Transfusion medicine is a specialized branch of  hematology  that is concerned with the study of blood groups, along with the work of a  blood bank  to provide a  transfusion  service for blood and other blood products. Across the world, blood products must be prescribed by a medical doctor (licensed  physician  or  surgeon ) in a similar way as medicines. [ citation needed ]


Much of the routine work of a  blood bank  involves testing blood from both donors and recipients to ensure that every individual recipient is given blood that is compatible and as safe as possible. If a unit of incompatible blood is  transfused  between a  donor  and recipient, a severe  acute hemolytic reaction  with  hemolysis  (RBC destruction),  kidney failure  and  shock  is likely to occur, and death is a possibility. [ 27 ]  Antibodies can be highly active and can attack RBCs and bind components of the  complement system  to cause massive hemolysis of the transfused blood. [ 28 ]


Patients should ideally receive their own blood or type-specific blood products to minimize the chance of a  transfusion reaction . It is also possible to use the patient's own blood for transfusion. This is called  autotransfusion , which is always compatible with the patient. The procedure of washing a patient's own red blood cells goes as follows: The patient's lost blood is collected and washed with a saline solution. The washing procedure yields concentrated washed red blood cells. The last step is reinfusing the packed red blood cells into the patient. There are multiple ways to wash red blood cells. The two main ways are centrifugation and filtration methods. This procedure can be performed with microfiltration devices like the Hemoclear filter. Risks can be further reduced by  cross-matching  blood, but this may be skipped when blood is required for an emergency. Cross-matching involves mixing a sample of the recipient's serum with a sample of the donor's red blood cells and checking if the mixture  agglutinates , or forms clumps. If agglutination is not obvious by direct vision, blood bank technologist usually check for  agglutination  with a  microscope . If agglutination occurs, that particular donor's blood cannot be transfused to that particular recipient.  In a blood bank it is vital that all blood specimens are correctly identified, so labelling has been standardized using a  barcode  system known as  ISBT 128 .


The blood group may be included on  identification tags  or on  tattoos  worn by military personnel, in case they should need an emergency blood transfusion. Frontline German  Waffen-SS had blood group tattoos  during  World War II .


Rare blood types can cause supply problems for  blood banks  and hospitals. For example,  Duffy -negative blood occurs much more frequently in people of African origin, [ 29 ]  and the rarity of this blood type in the rest of the population can result in a shortage of Duffy-negative blood for these patients.  Similarly, for RhD negative people there is a risk associated with travelling to parts of the world where supplies of RhD negative blood are rare, particularly  East Asia , where blood services may endeavor to encourage Westerners to donate blood. [ 30 ]


A  pregnant  woman may carry a  fetus  with a blood type which is different from her own. Typically, this is an issue if a Rh- mother has a child with a Rh+ father, and the fetus ends up being Rh+ like the father. [ 31 ]  In those cases, the mother can make  IgG  blood group antibodies. This can happen if some of the fetus' blood cells pass into the mother's blood circulation (e.g. a small fetomaternal  hemorrhage  at the time of childbirth or obstetric intervention), or sometimes after a therapeutic  blood transfusion .  This can cause  Rh disease  or other forms of  hemolytic disease of the newborn  (HDN) in the current pregnancy and/or subsequent pregnancies.  Sometimes this is lethal for the fetus; in these cases it is called  hydrops fetalis . [ 32 ]  If a pregnant woman is known to have anti-D antibodies, the Rh blood type of a  fetus  can be tested by analysis of fetal DNA in maternal plasma to assess the risk to the fetus of Rh disease. [ 33 ]  One of the major advances of twentieth century medicine was to prevent this disease by stopping the formation of Anti-D antibodies by D negative mothers with an injectable medication called  Rho(D) immune globulin . [ 34 ] [ 35 ]  Antibodies associated with some blood groups can cause severe HDN, others can only cause mild HDN and others are not known to cause HDN. [ 32 ]


To provide maximum benefit from each blood donation and to extend shelf-life,  blood banks   fractionate  some whole blood into several products. The most common of these products are packed RBCs,  plasma ,  platelets ,  cryoprecipitate , and  fresh frozen plasma  (FFP).  FFP is quick-frozen to retain the labile  clotting factors   V  and  VIII , which are usually administered to patients who have a potentially fatal clotting problem caused by a condition such as advanced  liver  disease, overdose of  anticoagulant , or  disseminated intravascular coagulation  (DIC). [ citation needed ]


Units of packed red cells are made by removing as much of the plasma as possible from whole blood units.


Clotting factors  synthesized by modern  recombinant  methods are now in routine clinical use for  hemophilia , as the risks of infection transmission that occur with pooled blood products are avoided.


Table note
1. Assumes absence of atypical antibodies that would cause an incompatibility between donor and recipient blood, as is usual for blood selected by cross matching.


An Rh D-negative patient who does not have any anti-D antibodies (never being previously sensitized to D-positive RBCs) can receive a transfusion of D-positive blood once, but this would cause sensitization to the D antigen, and a female patient would become at risk for  hemolytic disease of the newborn .  If a D-negative patient has developed anti-D antibodies, a subsequent exposure to D-positive blood would lead to a potentially dangerous transfusion reaction. Rh D-positive blood should never be given to D-negative women of child-bearing age or to patients with D antibodies, so blood banks must conserve Rh-negative blood for these patients. In extreme circumstances, such as for a major bleed when stocks of D-negative blood units are very low at the blood bank, D-positive blood might be given to D-negative females above child-bearing age or to Rh-negative males, providing that they did not have anti-D antibodies, to conserve D-negative blood stock in the blood bank. The converse is not true; Rh D-positive patients do not react to D negative blood.


This same matching is done for other antigens of the Rh system as C, c, E and e and for other blood group systems with a known risk for immunization such as the Kell system in particular for females of child-bearing age or patients with known need for many transfusions.


Blood plasma  compatibility is the inverse of red blood cell compatibility. [ 39 ]  Type AB plasma carries neither anti-A nor anti-B antibodies and can be transfused to individuals of any blood group; but type AB patients can only receive type AB plasma. Type O carries both antibodies, so individuals of blood group O can receive plasma from any blood group, but type O plasma can be used only by type O recipients.


Table note
1. Assuming absence of strong atypical antibodies in donor plasma


Rh D antibodies are uncommon, so generally neither D negative nor D positive blood contain anti-D antibodies.  If a potential donor is found to have anti-D antibodies or any strong atypical blood group antibody by antibody screening in the blood bank, they would not be accepted as a donor (or in some blood banks the blood would be drawn but the product would need to be appropriately labeled); therefore, donor blood plasma issued by a blood bank can be selected to be free of D antibodies and free of other atypical antibodies, and such donor plasma issued from a blood bank would be suitable for a recipient who may be D positive or D negative, as long as blood plasma and the recipient are ABO compatible. [ citation needed ]


In transfusions of packed red blood cells, individuals with type O Rh D negative blood are often called universal donors. Those with type AB Rh D positive blood are called universal recipients. However, these terms are only generally true with respect to possible reactions of the recipient's anti-A and anti-B antibodies to transfused red blood cells, and also possible sensitization to Rh D antigens. One exception is individuals with  hh antigen system  (also known as the Bombay phenotype) who can only receive blood safely from other hh donors, because they form antibodies against the H antigen present on all red blood cells. [ 41 ] [ 42 ]


Blood donors with exceptionally strong anti-A, anti-B or any atypical blood group antibody may be excluded from blood donation. In general, while the plasma fraction of a blood transfusion may carry donor antibodies not found in the recipient, a significant reaction is unlikely because of dilution.


Additionally, red blood cell surface antigens other than A, B and Rh D, might cause adverse reactions and sensitization, if they can bind to the corresponding antibodies to generate an immune response.  Transfusions are further complicated because  platelets  and  white blood cells  (WBCs) have their own systems of surface antigens, and sensitization to platelet or WBC antigens can occur as a result of transfusion.


For transfusions of  plasma , this situation is reversed.  Type O plasma, containing both anti-A and anti-B antibodies, can only be given to O recipients.  The antibodies will attack the antigens on any other blood type.  Conversely, AB plasma can be given to patients of any ABO blood group, because it does not contain any anti-A or anti-B antibodies.


Typically, blood type tests are performed through addition of a blood sample to a solution containing antibodies corresponding to each antigen. The presence of an antigen on the surface of the blood cells is indicated by  agglutination .


In addition to the current practice of serologic testing of blood types, the progress in molecular diagnostics allows the increasing use of blood group genotyping. In contrast to serologic tests reporting a direct blood type phenotype, genotyping allows the prediction of a phenotype based on the knowledge of the molecular basis of the currently known antigens. This allows a more detailed determination of the blood type and therefore a better match for transfusion, which can be crucial in particular for patients with needs for many transfusions to prevent allo-immunization. [ 43 ] [ 44 ]


Blood types were first discovered by an Austrian physician,  Karl Landsteiner , working at the Pathological-Anatomical Institute of the University of Vienna (now  Medical University of Vienna ). In 1900, he found that blood sera from different persons would clump together (agglutinate) when mixed in test tubes, and not only that, some human blood also agglutinated with animal blood. [ 45 ]  He wrote a two-sentence footnote:


The serum of healthy human beings not only agglutinates animal red cells, but also often those of human origin, from other individuals. It remains to be seen whether this appearance is related to inborn differences between individuals or it is the result of some damage of bacterial kind. [ 46 ]


This was the first evidence that blood variation exists in humans. The next year, in 1901, he made a definitive observation that blood serum of an individual would agglutinate with only those of certain individuals. Based on this he classified human bloods into three groups, namely group A, group B, and group C. He defined that group A blood agglutinates with group B, but never with its own type. Similarly, group B blood agglutinates with group A. Group C blood is different in that it agglutinates with both A and B. [ 47 ]  This was the discovery of blood groups for which Landsteiner was awarded the Nobel Prize in Physiology or Medicine in 1930. (C was later renamed to O after the German  Ohne , meaning without, or zero, or null. [ 48 ] ) Another group (later named AB) was discovered a year later by Landsteiner's students Adriano Sturli and Alfred von Decastello without designating the name (simply referring it to as "no particular type"). [ 49 ] [ 50 ]  Thus, after Landsteiner, three blood types were initially recognised, namely A, B, and C. [ 50 ]


Czech serologist  Jan Janský  was the first to recognise and designate four blood types in 1907 that he published in a local journal, [ 51 ]  using the Roman numerical I, II, III, and IV (corresponding to modern O, A, B, and AB respectively). [ 52 ]  Unknown to Janský, American physician  William L. Moss  introduced an almost identical classification in 1910, [ 53 ]  but with Moss's I and IV corresponding to Janský's IV and I. [ 54 ]  Thus the existence of two systems immediately created confusion and potential danger in medical practice. Moss's system was adopted in Britain, France, and the US, while Janský's was preferred in most other European countries and some parts of the US. It was reported that "The practically universal use of the Moss classification at that time was completely and purposely cast aside. Therefore in place of bringing order out of chaos, chaos was increased in the larger cities." [ 55 ]  To resolve the confusion, the  American Association of Immunologists , the  Society of American Bacteriologists , and the Association of Pathologists and Bacteriologists made a joint recommendation in 1921 that the Jansky classification be adopted based on priority. [ 56 ]  But it was not followed particularly where Moss's system had been used. [ 57 ]


In 1927, Landsteiner, who had moved to the  Rockefeller Institute for Medical Research  in New York, and as a member of a committee of the  National Research Council  concerned with blood grouping suggested to substitute Janský's and Moss's systems with the letters O, A, B, and AB, first instroduced by Polish physician  Ludwik Hirszfeld  and German physician  Emil von Dungern . [ 58 ]  There was another confusion on the use of O which was introduced in 1910. [ 59 ]  It was never clear whether it was meant for the figure 0, German  null  for zero or the upper case letter O for  ohne , meaning without; Landsteiner chose the latter. [ 60 ]


In 1928 the Permanent Commission on Biological Standardization adopted Landsteiner's proposal and stated:


The Commission learns with satisfaction that, on the initiative of the Health Organization of the League of Nations, the nomenclature proposed by von Dungern and Hirszfeld for the classification of blood groups has been generally accepted, and recommends that this nomenclature shall be adopted for international use as follows: 0 A B AB. To facilitate the change from the nomenclature hitherto employed the following is suggested:


This classification became widely accepted and after the early 1950s it was universally followed. [ 62 ]


Hirszfeld and Dungern discovered the inheritance of blood types as  Mendelian genetics  in 1910 and the existence of sub-types of A in 1911. [ 59 ] [ 63 ]  In 1927, Landsteiner, with  Philip Levine , discovered the  MN blood group system , [ 64 ]  and the  P system . [ 65 ]  Development of the  Coombs test  in 1945, [ 66 ]  the advent of  transfusion medicine , and the understanding of  ABO hemolytic disease of the newborn  led to discovery of more blood groups. As of October 2024 [update] , the International Society of Blood Transfusion (ISBT) recognizes 47 blood groups. [ 3 ]


A popular  pseudoscientific  belief in Eastern Asian countries (especially in  Japan  and  South Korea [ 67 ] ) known as 血液型  ketsuekigata / hyeoraekhyeong  is that a person's ABO blood type is predictive of their  personality ,  character , and  compatibility with others . [ 68 ]  Researchers have established no scientific basis exists for blood type personality categorization, and studies have found no "significant relationship between personality and blood type, rendering the theory 'obsolete' and concluding that no basis exists to assume that personality is anything more than randomly associated with blood type." [ 67 ]


In  bioinformatics , the  BLOSUM  ( BLO cks  SU bstitution  M atrix) matrix is a  substitution matrix  used for  sequence alignment  of  proteins . BLOSUM matrices are used to score alignments between  evolutionarily divergent  protein sequences.  They are based on local alignments. BLOSUM matrices were first introduced in a paper by  Steven Henikoff  and Jorja Henikoff. [ 1 ]  They scanned the  BLOCKS database  for very  conserved regions  of protein families (that do not have  gaps  in the sequence alignment) and then counted the relative frequencies of  amino acids  and their substitution probabilities. Then, they calculated a  log-odds  score for each of the 210 possible substitution pairs of the 20 standard amino acids. All BLOSUM matrices are based on observed alignments; they are not extrapolated from comparisons of closely related proteins like the  PAM Matrices .


The genetic instructions of every replicating  cell  in a living organism are contained within its DNA. [ 2 ]  Throughout the cell's lifetime, this information is transcribed and replicated by cellular mechanisms to produce proteins or to provide instructions for daughter cells during  cell division , and the possibility exists that the DNA may be altered during these processes. [ 2 ] [ 3 ]  This is known as a  mutation . At the molecular level, there are regulatory systems that correct most — but not all — of these changes to the DNA before it is replicated. [ 3 ] [ 4 ]


The functionality of a protein is highly dependent on its structure. [ 5 ]  Changing a single amino acid in a protein may reduce its ability to carry out this function, or the mutation may even change the function that the protein carries out. [ 3 ]  Changes like these may severely impact a crucial function in a cell, potentially causing the cell — and in extreme cases, the organism — to die. [ 6 ]  Conversely, the change may allow the cell to continue functioning albeit differently, and the mutation can be passed on to the organism's offspring. If this change does not result in any significant physical disadvantage to the offspring, the possibility exists that this mutation will persist within the population. The possibility also exists that the change in function becomes advantageous.


The 20 amino acids translated by the  genetic code  vary greatly by the physical and chemical properties of their side chains. [ 5 ]  However, these amino acids can be categorised into groups with similar physicochemical properties. [ 5 ]  Substituting an amino acid with another from the same category is more likely to have a smaller impact on the structure and function of a protein than replacement with an amino acid from a different category.


Sequence alignment is a fundamental research method for modern biology. The most common sequence alignment for protein is to look for similarity between different sequences in order to infer function or establish evolutionary relationships. This helps researchers better understand the origin and function of genes through the nature of  homology  and  conservation . Substitution matrices are utilized in algorithms to calculate the similarity of different sequences of proteins; however, the utility of  Dayhoff   PAM  Matrix has decreased over time due to the requirement of sequences with a similarity more than 85%. In order to fill in this gap,  Henikoff  and Henikoff introduced BLOSUM (BLOcks SUbstitution Matrix) matrix which led to marked improvements in alignments and in searches using queries from each of the groups of related proteins. [ 1 ]


Several sets of BLOSUM matrices exist using different alignment databases, named with numbers. BLOSUM matrices with high numbers are designed for comparing closely related sequences, while those with low numbers are designed for comparing distant related sequences. For example, BLOSUM80 is used for closely related alignments, and BLOSUM45 is used for more distantly related alignments. The matrices were created by merging (clustering) all sequences that were more similar than a given percentage into one single sequence and then comparing those sequences (that were all more divergent than the given percentage value) only; thus reducing the contribution of closely related sequences. The percentage used was appended to the name, giving BLOSUM80 for example where sequences that were more than 80% identical were clustered.


BLOSUM matrices are obtained by using blocks of similar amino acid sequences as data, then applying statistical methods to the data to obtain the similarity scores.
Statistical Methods Steps :
 [ 8 ]


Eliminate the sequences that are more than r% identical. There are two ways to eliminate the sequences. It can be done either by removing sequences from the block or just by finding similar sequences and replace them by new sequences which could represent the cluster. Elimination is done to remove protein sequences that are more similar than the specified threshold.


A database storing the sequence alignments of the most conserved regions of protein families. These alignments are used to derive the BLOSUM matrices. Only the sequences with a percentage of identity lower than the threshold are used.
By using the block, counting the pairs of amino acids in each column of the multiple alignment.


It gives the ratio of the occurrence each amino acid combination in the observed data to the expected value of occurrence of the pair.
It is rounded off and used in the substitution matrix.


L
 o
 g
 O
 d
 d
 R
 a
 t
 i
 o
 =
 2

 log

 2

 (

 P

 (
 O
 )

 P

 (
 E
 )

 )

 {\displaystyle LogOddRatio=2\log _{2}{\left({\frac {P\left(O\right)}{P\left(E\right)}}\right)}}


where

 P

 (
 O
 )

 {\displaystyle P\left(O\right)}

  is the probability of observing the pair and

 P

 (
 E
 )

 {\displaystyle P\left(E\right)}

  is the expected probability of such a pair occurring, given the background probabilities of each amino acid.


The odds for relatedness are calculated from log odd ratio, which are then rounded off to get the substitution matrices BLOSUM matrices.


A scoring matrix or a table of values is required for evaluating the significance of a sequence alignment, such as describing the probability of a biologically meaningful amino-acid or nucleotide residue-pair occurring in an alignment. Typically, when two nucleotide sequences are being compared, all that is being scored is whether or not two bases are the same at one position. All matches and mismatches are respectively given the same score (typically +1 or +5 for matches, and -1 or -4 for mismatches). [ 9 ]  But it is different for proteins. Substitution matrices for amino acids are more complicated and implicitly take into account everything that might affect the frequency with which any amino acid is substituted for another. The objective is to provide a relatively heavy penalty for aligning two residues together if they have a low probability of being homologous (correctly aligned by evolutionary descent). Two major forces drive the amino-acid substitution rates away from uniformity:  substitutions occur with the different frequencies, and lessen functionally tolerated than others. Thus, substitutions are selected against. [ 7 ]


Commonly used substitution matrices include the blocks substitution (BLOSUM)  [ 1 ]   and point accepted mutation (PAM)  [ 10 ] [ 11 ]   matrices. Both are based on taking sets of high-confidence alignments of many homologous proteins and assessing the frequencies of all substitutions, but they are computed using different methods. [ 7 ]


Scores within a BLOSUM are log-odds scores that measure, in an alignment, the logarithm for the ratio of the likelihood of two amino acids appearing with a biological sense and the likelihood of the same amino acids appearing by chance. The matrices are based on the minimum percentage identity of the aligned protein sequence used in calculating them. [ 12 ]  Every possible identity or substitution is assigned a score based on its observed frequencies in the alignment of related proteins. [ 13 ]  A positive score is given to the more likely substitutions while a negative score is given to the less likely substitutions.


To calculate a BLOSUM matrix, the following equation is used:


Here,

 p

 i
 j

 {\displaystyle p_{ij}}

  is the probability of two amino acids

 i

 {\displaystyle i}

  and

 j

 {\displaystyle j}

  replacing each other in a homologous sequence, and

 q

 i

 {\displaystyle q_{i}}

  and

 q

 j

 {\displaystyle q_{j}}

  are the background probabilities of finding the amino acids

 i

 {\displaystyle i}

  and

 j

 {\displaystyle j}

  in any protein sequence. The factor

 λ

 {\displaystyle \lambda }

  is a scaling factor, set such that the matrix contains easily computable integer values.


BLOSUM80: more related proteins


BLOSUM62: midrange


BLOSUM45: distantly related proteins


An article in  Nature Biotechnology [ 14 ]  revealed that the BLOSUM62 used for so many years as a standard is not exactly accurate according to the algorithm described by Henikoff and Henikoff. [ 1 ]  Surprisingly, the miscalculated BLOSUM62 improves search performance. [ 14 ]


The BLOSUM62 matrix with the amino acids in the table grouped according to the chemistry of the side chain, as in (a). Each value in the matrix is calculated by dividing the frequency of occurrence of the amino acid pair in the BLOCKS database, clustered at the 62% level, divided by the probability that the same two amino acids might align by chance. The ratio is then converted to a logarithm and expressed as a log odds score, as for PAM. BLOSUM matrices are usually scaled in half-bit units. A score of zero indicates that the frequency with which a given two amino acids were found aligned in the database was as expected by chance, while a positive score indicates that the alignment was found more often than by chance, and negative score indicates that the alignment was found less often than by chance.


BLOSUM scores was used to predict and understand the surface gene variants among hepatitis B virus carriers [ 15 ]  and T-cell epitopes. [ 16 ]


DNA sequences of HBsAg were obtained from 180 patients, in which 51 were chronic HBV carrier and 129 newly diagnosed patients, and compared with consensus sequences built with 168 HBV sequences imported from GenBank. Literature review and BLOSUM scores were used to define potentially altered antigenicity. [ 15 ]


A novel input representation has been developed consisting of a combination of sparse encoding, Blosum encoding, and input derived from hidden Markov models. this method predicts T-cell epitopes for the genome of hepatitis C virus and discuss possible applications of the prediction method to guide the process of rational vaccine design. [ 16 ]


BLOSUM matrices are also used as a scoring matrix when comparing DNA sequences or protein sequences to judge the quality of the alignment. This form of scoring system is utilized by a wide range of alignment software including  BLAST . [ 17 ]


In addition to BLOSUM matrices, a previously developed scoring matrix can be used. This is known as a  PAM . The two result in the same scoring outcome, but use differing methodologies. BLOSUM looks directly at mutations in motifs of related sequences while PAM's  extrapolate  evolutionary information based on closely related sequences. [ 1 ]


Since both PAM and BLOSUM are different methods for showing the same scoring information, the two can be compared but due to the very different method of obtaining this score, a PAM100 does not equal a BLOSUM100. [ 18 ]


There are several software packages in different programming languages that allow easy use of Blosum matrices.


Examples are the  blosum  module for  Python , or the  BioJava  library for  Java .


Bulked segregant analysis  ( BSA ) is a technique used to identify  genetic markers  associated with a mutant  phenotype . This allows geneticists to discover genes conferring certain traits of interest, such as disease resistance or susceptibility.


This technique involves forming two groups that display opposing phenotypes for a trait of interest. For example, the individuals in one group are resistant to a disease, whereas those in the second group are not. Two bulked DNA samples are then created by pooling the DNA of all individuals in each group.


These two bulked samples can then be analysed using techniques such as  Restriction fragment length polymorphism  or  RAPD  to detect similarities and differences in the various  loci  of the genome. The two groups will have a random distribution of  alleles  in all loci of the genome except for loci that are associated with the mutation. [ 1 ]  A consistent difference on a locus between the two bulked samples likely means that the locus is  associated  with the mutation of interest.


In animals, the individuals making up the two testing groups are usually produced by a cross between two siblings  heterozygous  for the mutation of interest. The use of siblings is necessary to ensure that the alleles contributing to the mutation are the same among the individuals.


There must be a minimum amount of heterozygosity in the various loci of the groups to allow the genes that are associated with the trait of interest to be identified. Since most laboratory strains are inbred,  outcrossing  of the homozygous mutated individual with a polymorphic strain is essential to generate effective testing groups. The offspring are crossed with each other to generate testing groups. [ 2 ]


Bulked DNA samples can be analysed using  Southern blotting . Use of restriction enzymes or PCR amplification on the DNA is required for  RFLP  or RAPD analysis respectively. In these techniques, the loci that are analysed are the restriction digest sites and the sequences on which PCR primers attach to. These sites are usually located throughout the genome. Once linked loci are detected, they can be  mapped  and the linkage distances between them determined. [ 3 ]


The  bump-and-hole method  is a tool in  chemical genetics  for studying a specific  isoform  in a protein family without perturbing the other members of the family. The unattainability of isoform-selective inhibition due to structural homology in protein families is a major challenge of chemical genetics. With the bump-and-hole approach, a  protein–ligand  interface is engineered to achieve selectivity through  steric  complementarity while maintaining biochemical competence and orthogonality to the wild type pair. Typically, a "bumped" ligand/inhibitor analog is designed to bind a corresponding "hole-modified" protein. Bumped ligands are commonly bulkier derivatives of a  cofactor  of the target protein. Hole-modified proteins are  recombinantly expressed  with an amino acid substitution from a larger to smaller residue, e.g. glycine or alanine, at the cofactor binding site. The designed ligand/inhibitor has specificity for the engineered protein due to steric complementarity, but not the native counterpart due to steric interference. [ 1 ]


Inspiration for the bump-and-hole method was drawn from mutant  E. coli  strains which carried an A294S mutant version of phenylalanine  tRNA synthetase  and survived exposure to  p -FluoroPhe, a slightly bumped phenylalanine analog which is cytotoxic when incorporated in  translation . The A294S mutant strain was able to incorporate Phe, but not the bumped  p -FluoroPhe due to steric crowding from the hydroxymethylene of S294. [ 2 ]  Later work in the labs of  Peter G. Schultz  and  David A. Tirrell  showed that a hole-modified A294G phenylalanine tRNA synthetase mutant was able to incorporate the bumped  p -FluoroPhe in translation, demonstrating that steric manipulation can successfully broaden substrate scope, even for the highly specific aminoacyl synthetase. [ 3 ]


The first bump-and-hole pair, developed by  Stuart Schreiber  and colleagues, was a bumped  cyclosporin A  small-molecule with an Ile replacing Val at position 11, and a hole-modified (S99T/F113A)  cyclophilin  mutant. [ 5 ]  Cyclosporin A is a  chemical inducer of dimerization (CID)   of cyclophilin. This first bump-and-hole pair was engineered to improve the binding efficiency between wild type cyclosporin A and cyclophilin, thereby giving more efficient CID. The bumped cyclosporin A was found to interact efficiently with the hole-modified cyclophilin mutant, but not endogenous cyclophilin. The orthogonal CID pair was used to inhibit  calcineurin -mediated  dephosphorylation  of nuclear factor of activated  T cells  in a cell- and tissue-specific manner. [ 6 ]  More recently, this first bump-and-hole pair was used to induce the assembly of  ten-eleven translocation 2  dioxygenase in cells for temporally controlled  DNA demethylation . [ 7 ]


As structural information about protein-ligand interfaces have become available, bump-and-hole pairs have been used to elucidate the substrates of specific proteins from various protein classes, as well as develop orthogonal neoenzyme-neosubstrate therapeutics.


Human protein  kinases  use  ATP  as a cofactor to  phosphorylate  substrate proteins. Kinases play critical roles in complex cell signaling networks. Conserved ATP binding sites and similar catalytic mechanisms pose a challenge to selectively inhibiting a particular kinase to determine its function.  Kevan Shokat's  lab has developed bump-and-hole pairs using kinase mutants with bulky "gatekeeper" residues in the ATP-binding pocket replaced  by Gly or Ala, and bulky ATP analogs. In early work, v-Src kinase I338A/G mutants were shown to accept [γ- 32 P]-labeled bumped N 6 -cyclopentyl and N 6 -benzyl ATP analogs as alternative cofactors to radiolabel its substrates. [ 8 ]  Only the mutant kinase was able to bind the bumped ATP analogs, allowing labeling of substrates specific to the engineered v-Src kinase. Purification and  MS-based proteomics  yielded the substrates of v-Src kinase. Hole-modified kinase and bumped ATP analog pairs enabled substrate profiling of several other kinases, including  CDK1 , Pho85,  ERK2 , and JNK. [ 9 ]


While bumped ATM analogs can help deconvolute kinase substrate profiles, one drawback of this strategy is the cell impermeability of the bumped analogs. To get around this, the Shokat group demonstrated that a bumped ATP analog, kinetin ATP or KTP, could be synthesized endogenously in cells cultured with  kinetin . Once synthesized, it can activate a  PINK1  kinase mutant, which is otherwise inactive in the absence of the bumped analog. Inactive PINK1 is implicated in  Parkinson's disease  (PD). In the context of PD, the mutant PINK1-KTP pair represents an orthogonal neoenzyme-neosubstrate therapeutic. [ 10 ]


The Shokat group also applied the bump-and-hole approach to develop selective, cell-permeable bumped inhibitors of mutant kinases. For the I338G v-Src kinase, a 4-amino-l- tert -butyl-3-( p -methylphenyl)pyrazolo[3,4-d]pyrimidine (PP1) derivative called  p - t ButPhe-PP1 was developed for selective inhibition; steric bulk precluded binding to the wild type v-Src kinase. In mammalian cell lines, active v-Src kinase is required for transformation by  Rous sarcoma virus . In cell lines expressing I338G v-Src kinase and transfected with RSV, treatment with  p - t ButPhe-PP1 caused the reversal of transformation, suggesting inhibition of the kinase mutant. [ 11 ]  Later, the group developed bumped inhibitors 1-naphthyl PP1 (NA-PP1) and 1-methylnaphthyl PP1 (MN-PP1), which inhibited hole-modified yeast kinases with  IC50  values in low nanomolar concentrations. [ 12 ]