Gene mutations are changes in the structure of one gene. This is a change in the nucleotide sequence: deletion, insertion, substitution, etc. For example, replacing a with t. Causes - violations during DNA doubling (replication)
Gene mutations are molecular changes in DNA structure that are not visible in a light microscope. Gene mutations include any changes in the molecular structure of DNA, regardless of their location and effect on viability. Some mutations have no effect on the structure or function of the corresponding protein. Another (large) part of gene mutations leads to the synthesis of a defective protein that is unable to perform its inherent function. It is gene mutations that determine the development of most hereditary forms of pathology.
The most common monogenic diseases in humans are: cystic fibrosis, hemochromatosis, adrenogenital syndrome, phenylketonuria, neurofibromatosis, Duchenne-Becker myopathies and a number of other diseases. Clinically, they manifest themselves as signs of metabolic disorders (metabolism) in the body. The mutation may be:
1) in replacing a base in a codon, this is the so-called missense mutation(from English, mis - false, incorrect + lat. sensus - meaning) - replacement of a nucleotide in the coding part of a gene, leading to replacement of an amino acid in a polypeptide;
2) in such a change in codons that will lead to a stop in reading information, this is the so-called nonsense mutation(from Latin non - no + sensus - meaning) - replacement of a nucleotide in the coding part of a gene leads to the formation of a terminator codon (stop codon) and cessation of translation;
3) a violation of information reading, a shift in the reading frame, called frameshift(from the English frame - frame + shift: - shift, movement), when molecular changes in DNA lead to changes in triplets during translation of the polypeptide chain.
Other types of gene mutations are also known. Based on the type of molecular changes, there are:
division(from Latin deletio - destruction), when a DNA segment ranging in size from one nucleotide to a gene is lost;
duplications(from Latin duplicatio - doubling), i.e. duplication or reduplication of a DNA segment from one nucleotide to entire genes;
inversions(from Latin inversio - turning over), i.e. a 180° rotation of a DNA segment ranging in size from two nucleotides to a fragment including several genes;
insertions(from Latin insertio - attachment), i.e. insertion of DNA fragments ranging in size from one nucleotide to an entire gene.
Molecular changes affecting one to several nucleotides are considered a point mutation.
The fundamental and distinctive feature of a gene mutation is that it 1) leads to a change in genetic information, 2) can be transmitted from generation to generation.
A certain part of gene mutations can be classified as neutral mutations, since they do not lead to any changes in the phenotype. For example, due to the degeneracy of the genetic code, the same amino acid can be encoded by two triplets that differ in only one base. On the other hand, the same gene can change (mutate) into several different states.
For example, the gene that controls the blood group of the AB0 system. has three alleles: 0, A and B, the combinations of which determine 4 blood groups. The ABO blood group is a classic example of genetic variation in normal human characteristics.
It is gene mutations that determine the development of most hereditary forms of pathology. Diseases caused by such mutations are called genetic, or monogenic, diseases, i.e., diseases whose development is determined by a mutation of one gene.
Genomic and chromosomal mutations are the causes of chromosomal diseases. Genomic mutations include aneuploidies and changes in the ploidy of structurally unchanged chromosomes. Detected by cytogenetic methods.
Aneuploidy- a change (decrease - monosomy, increase - trisomy) in the number of chromosomes in a diploid set, not a multiple of the haploid set (2n + 1, 2n - 1, etc.).
Polyploidy- an increase in the number of sets of chromosomes, a multiple of the haploid one (3n, 4n, 5n, etc.).
In humans, polyploidy, as well as most aneuploidy, are lethal mutations.
The most common genomic mutations include:
trisomy- the presence of three homologous chromosomes in the karyotype (for example, on the 21st pair in Down syndrome, on the 18th pair in Edwards syndrome, on the 13th pair in Patau syndrome; on sex chromosomes: XXX, XXY, XYY);
monosomy- the presence of only one of two homologous chromosomes. With monosomy for any of the autosomes, normal development of the embryo is impossible. The only monosomy in humans that is compatible with life, monosomy on the X chromosome, leads to Shereshevsky-Turner syndrome (45, X0).
The reason leading to aneuploidy is the nondisjunction of chromosomes during cell division during the formation of germ cells or the loss of chromosomes as a result of anaphase lag, when during movement to the pole one of the homologous chromosomes may lag behind all other nonhomologous chromosomes. The term "nondisjunction" means the absence of separation of chromosomes or chromatids in meiosis or mitosis. Loss of chromosomes can lead to mosaicism, in which there is one uploid(normal) cell line, and the other monosomic.
Chromosome nondisjunction most often occurs during meiosis. Chromosomes that would normally divide during meiosis remain joined together and move to one pole of the cell during anaphase. Thus, two gametes arise, one of which has an additional chromosome, and the other does not have this chromosome. When a gamete with a normal set of chromosomes is fertilized by a gamete with an extra chromosome, trisomy occurs (i.e., there are three homologous chromosomes in the cell); when a gamete without one chromosome is fertilized, a zygote with monosomy occurs. If a monosomal zygote is formed on any autosomal (non-sex) chromosome, then the development of the organism stops at the earliest stages of development.
Chromosomal mutations- These are structural changes in individual chromosomes, usually visible under a light microscope. A chromosomal mutation involves a large number (from tens to several hundreds) of genes, which leads to a change in the normal diploid set. Although chromosomal aberrations generally do not change the DNA sequence of specific genes, changes in the copy number of genes in the genome lead to genetic imbalance due to a lack or excess of genetic material. There are two large groups of chromosomal mutations: intrachromosomal and interchromosomal.
Intrachromosomal mutations are aberrations within one chromosome. These include:
—deletions(from Latin deletio - destruction) - loss of one of the sections of the chromosome, internal or terminal. This can cause disruption of embryogenesis and the formation of multiple developmental anomalies (for example, division in the region of the short arm of the 5th chromosome, designated as 5p-, leads to underdevelopment of the larynx, heart defects, and mental retardation). This symptom complex is known as the “cry of the cat” syndrome, since in sick children, due to an abnormality of the larynx, the crying resembles a cat’s meow;
—inversions(from Latin inversio - inversion). As a result of two chromosome break points, the resulting fragment is inserted into its original place after a 180° rotation. As a result, only the order of the genes is disrupted;
— duplications(from Latin duplicatio - doubling) - doubling (or multiplication) of any part of a chromosome (for example, trisomy on one of the short arms of the 9th chromosome causes multiple defects, including microcephaly, delayed physical, mental and intellectual development).
Patterns of the most common chromosomal aberrations:
Division: 1 - terminal; 2 - interstitial. Inversions: 1 - pericentric (with capture of the centromere); 2 - paracentric (within one chromosome arm)
Interchromosomal mutations, or rearrangement mutations- exchange of fragments between non-homologous chromosomes. Such mutations are called translocations (from the Latin tgans - for, through + locus - place). This:
Reciprocal translocation, when two chromosomes exchange their fragments;
Non-reciprocal translocation, when a fragment of one chromosome is transported to another;
- “centric” fusion (Robertsonian translocation) - the connection of two acrocentric chromosomes in the region of their centromeres with the loss of short arms.
When chromatids break transversely through centromeres, “sister” chromatids become “mirror” arms of two different chromosomes containing the same sets of genes. Such chromosomes are called isochromosomes. Both intrachromosomal (deletions, inversions and duplications) and interchromosomal (translocations) aberrations and isochromosomes are associated with physical changes in chromosome structure, including mechanical breaks.
The presence of common species characteristics allows us to unite all people on earth into a single species, Homo sapiens. Nevertheless, we easily, with one glance, single out the face of a person we know in a crowd of strangers. The extreme diversity of people - both within groups (for example, diversity within an ethnic group) and between groups - is due to their genetic differences. It is currently believed that all intraspecific variation is due to different genotypes arising and maintained by natural selection.
It is known that the haploid human genome contains 3.3x10 9 pairs of nucleotide residues, which theoretically allows for up to 6-10 million genes. At the same time, modern research data indicate that the human genome contains approximately 30-40 thousand genes. About a third of all genes have more than one allele, that is, they are polymorphic.
The concept of hereditary polymorphism was formulated by E. Ford in 1940 to explain the existence in a population of two or more distinct forms when the frequency of the rarest of them cannot be explained by mutational events alone. Since gene mutation is a rare event (1x10 6), the frequency of the mutant allele, which is more than 1%, can only be explained by its gradual accumulation in the population due to the selective advantages of carriers of this mutation.
The multiplicity of segregating loci, the multiplicity of alleles in each of them, along with the phenomenon of recombination, creates inexhaustible human genetic diversity. Calculations show that in the entire history of mankind there has not been, is not, and will not occur in the foreseeable future, genetic repetition, i.e. Every born person is a unique phenomenon in the Universe. The uniqueness of the genetic constitution largely determines the characteristics of the development of the disease in each individual person.
Humanity has evolved as groups of isolated populations living for a long time under the same environmental conditions, including climatic and geographic characteristics, dietary patterns, pathogens, cultural traditions, etc. This led to the consolidation in the population of combinations of normal alleles specific for each of them, most adequate to environmental conditions. Due to the gradual expansion of the habitat, intensive migrations, and resettlement of peoples, situations arise when combinations of specific normal genes that are useful in certain conditions do not ensure the optimal functioning of certain body systems in other conditions. This leads to the fact that part of the hereditary variability, caused by an unfavorable combination of non-pathological human genes, becomes the basis for the development of so-called diseases with a hereditary predisposition.
In addition, in humans as a social being, natural selection proceeded over time in increasingly specific forms, which also expanded hereditary diversity. What could be discarded by the animals was preserved, or, conversely, what the animals retained was lost. Thus, fully meeting the needs for vitamin C led in the process of evolution to the loss of the L-gulonodactone oxidase gene, which catalyzes the synthesis of ascorbic acid. In the process of evolution, humanity also acquired undesirable characteristics that are directly related to pathology. For example, in the process of evolution, humans have acquired genes that determine sensitivity to diphtheria toxin or to the polio virus.
Thus, in humans, like in any other biological species, there is no sharp line between hereditary variability leading to normal variations in characteristics and hereditary variability causing the occurrence of hereditary diseases. Man, having become the biological species Homo sapiens, seemed to pay for the “reasonableness” of his species by accumulating pathological mutations. This position underlies one of the main concepts of medical genetics about the evolutionary accumulation of pathological mutations in human populations.
Hereditary variability of human populations, both maintained and reduced by natural selection, forms the so-called genetic load.
Some pathological mutations can persist and spread in populations for a historically long time, causing the so-called segregation genetic load; other pathological mutations arise in each generation as a result of new changes in the hereditary structure, creating a mutational load.
The negative effect of genetic load is manifested by increased mortality (death of gametes, zygotes, embryos and children), decreased fertility (reduced reproduction of offspring), decreased life expectancy, social disadaptation and disability, and also causes an increased need for medical care.
The English geneticist J. Hoddane was the first to draw the attention of researchers to the existence of genetic load, although the term itself was proposed by G. Meller back in the late 40s. The meaning of the concept of “genetic load” is associated with the high degree of genetic variability necessary for a biological species in order to be able to adapt to changing environmental conditions.
Variability is the result of the reaction of the genotype in the process of individual development of the organism (ontogenesis) to environmental conditions.
Variation is one of the main factors of evolution. It serves as a source of natural and artificial selection.
Distinguish hereditary And non-hereditary variability. Hereditary variability includes such changes in characteristics that are determined by the genotype and persist over a number of generations. Hereditary variability arises as a result mutations(mutational variability) or as a result recombination genetic material of two individuals, for example, parents (combinative variability).
Combinative variability is the result of recombination of genes and recombination of chromosomes carrying different alleles, and is expressed in the emergence of a diversity of organisms - descendants that have received new combinations of genes, already existing in parent forms.
In eukaryotic organisms, combinative variation occurs due to recombination of the genetic material of the parents during sexual reproduction. Gene recombination occurs in various ways. This process may be associated with the redistribution of entire chromosomes. This mechanism, in accordance with Mendel’s third law, ensures the independent inheritance of unlinked genes and traits. Most often, recombination in the narrow sense of the word is associated with crossing over, that is, with the recombination of genes localized on homologous chromosomes.
Three mechanisms for combining and recombining genetic material have been found in bacteria: transformation, conjugation and transduction.
Non-hereditary variability includes changes in the characteristics of an organism that are not preserved during sexual reproduction. This is the so-called modification variability is the property of organisms to change their phenotype depending on environmental conditions while maintaining the stability of the genotype. Modification changes are of a massive adaptive nature and disappear when conditions change. They are not of interest to evolution because they are not inherited. The limits within which an organism is able to respond to environmental conditions are called reaction norm. A wide reaction rate ensures good adaptive ability of the body. The reaction rate is determined by the genotype of the individual.
Epigenetic variation associated with changes in gene expression without changing their structure. The set of working genes changes during the process of individual development and in response to external influences. These changes can be either non-heritable or persist over several generations.
Mutational variability.
The term “mutation” was proposed at the beginning of the 20th century by G. De Vries. As a result of many years of research on the evening primrose plant, he discovered a number of forms that differed from the main mass, and these differences persisted from year to year. Summarizing his observations, De Vries formulated the mutation theory: “A mutation is a phenomenon of spasmodic, intermittent changes in a hereditary characteristic.”
Basic provisions of mutation theory.
Subsequently, all provisions of this theory, except point 3, were confirmed.
In the modern sense Mutations are inherited changes in genetic material.
There are several types of mutation classification
TO genomic mutations include changes in the number of chromosomes. The minimum set of chromosomes, when each chromosome is represented by one copy, is called haploid. Gametes are haploid. The haploid set of chromosomes is designated by the letter n. Usually present in somatic cells diploid a set of chromosomes containing a double set of chromosomes compared to the haploid one (2 n). In the life cycles of eukaryotes, cases of supernormal multiplication of the number of chromosomes occur. If such changes are proportional (multiple) to the haploid set, then they speak of polyploidization. If the number of copies of only one or several chromosomes of a set changes, then we speak of aneuploidy.
Polyploidy is widely and unevenly distributed in nature. Polyploid fungi and algae are known, and polyploids are often found among flowering plants. Macronuclei of ciliates are highly polyploid (more than 100 n).
Autopolyploidy- repetition of the same chromosome set in a cell. One of the ways of occurrence of polyploids is the formation of unreduced gametes. Doubling the number of chromosomes may be the result of endoreduplication of genetic material: cells that were in the G2 phase in the original plant re-enter the S phase instead of mitosis. Then such cells with double the number of chromosomes divide and give rise to polyploid clones. Another reason for the appearance of polyploid cells is endomitosis - the process of chromosome nondisjunction in anaphase due to dysfunction of the spindle. To artificially produce polyploids, agents are used that block the divergence of duplicated chromosomes, for example, colchicine, produced by the crocus plant, vinblastine, obtained from another plant - periwinkle, camphor.
Allopolyploids– organisms containing sets of chromosomes of two or more species obtained as a result of hybridization and polyploidization. Some plant species are natural allopolyploids; for example, the genome of common wheat includes two genomes of related diploid wheats and the genome of Aegilops. An example of an artificial allopolyploid is a hybrid of radish and cabbage, obtained in 1927 by G.D. Karpechenko.
Polyploidy often leads to more powerful and productive organisms. However, the fertility of polyploids is reduced due to incorrect conjugation of chromosomes in meiosis and uneven divergence of chromosomes among gametes; triploids do not produce offspring
Chromosomal mutations associated with chromosome rearrangements - aberrations. Aberrations are distinguished as intrachromosomal (parts of one chromosome are involved) and interchromosomal (parts of different non-homologous chromosomes are involved).
Intrachromosomal rearrangements:
Deficiency - terminal shortages;
Deletions are loss of parts of a chromosome that do not affect the telomere;
Duplications – doubling (multiplication) of a part of a chromosome;
Inversions are changes in the alternation of genes in a chromosome as a result of rotating a section of the chromosome by 180 degrees.
Interchromosomal rearrangements- translocation - movement of part of one chromosome to another, not homologous to it.
A special position is occupied by transpositions, or insertions - changes in the localization of small sections of genetic material, including one or more genes. Transpositions can occur both within one chromosome and between chromosomes. Therefore, transpositions occupy an intermediate position between intrachromosomal and interchromosomal rearrangements.
Gene (point) mutations These are changes in the sequence of nucleotides in DNA. Point mutations are divided into the following groups:
a) transitions - replacement of purine with purine; pyrimidine to pyrimidine;
b) transversions - replacement of pyridine with purine and vice versa;
c) insertion of an extra pair of nucleotides;
d) loss of a pair of nucleotides.
The main reason for the occurrence of mutations is “errors of the three Ps”: replication, repair and recombination. Such errors occur when these three processes are dysregulated. A positive correlation has been shown between the frequency of mutations and defects in DNA polymerases and other replication and repair enzymes.
DNA bases can exist in several tautomeric forms. If adenine is in its normal amine form, it pairs with thymine. Being in a rare imine form, adenine forms pairs with cytosine. This tautomeric transition of adenine during subsequent replication can lead to AT-GC transition. The rare enol tautomer of thymine is capable of pairing with guanine, which will also result in a nucleotide pair substitution. All transitions and transversions can be explained by some ambiguity in the correspondence between nucleotides in complementary DNA strands.
The frequency of spontaneous, that is, mutations that arose without the influence of external factors, varies from 10 -4 to 10 -10. For example, streptomycin resistance mutations in Escherichia coli are observed with a frequency of 4 . 10 -10, and the appearance of white eyes in Drosophila is 4. 10 -5. In various microorganisms - bacteria, bacteriophages, fungi - the overall frequency of spontaneous mutation in terms of genome replication is approximately the same - about 1%. Several (many) genes can mutate at the same time.
In 1925-1927 The mutagenic effect of X-rays was discovered. In the 30s of the twentieth century, the mutagenic effect of a number of chemicals was discovered. Physical mutagens include, in addition to X-rays, ultraviolet and gamma radiation, fast neutrons. Chemical mutagens are very diverse in their chemical structure and mechanism of action. For example, nitrous acid causes deamination of nucleic acid bases, and alkylating supermutagens cause the addition of methyl or ethyl groups to them. This results in incorrect mating. Acridine compounds promote the appearance of nucleotide insertions.
Particularly mobile migrating genetic elements have been found in the genomes of many organisms. They were first discovered by the American researcher B. McClintock in 1940. While studying the mutation of grain color in corn, she found an unstable mutation that reverted to the wild type with increased frequency. Unstable mutations were often accompanied by chromosomal abnormalities. Genes that cause chromosome breaks have been named mobile elements, because they could move from one part of the chromosome to another. These elements are characterized by the following properties:
The maize genome contains several families of transposable elements. The members of each family can be divided into two classes:
Autonomous elements that can be cut and transposed. Their introduction leads to the appearance of unstable alleles.
Non-autonomous elements that can only be activated to tan positions by certain autonomous elements (members of the same family).
In maize, the best studied families are Ac-Ds (activator-dissociator), Spm (suppressor-mutator), and Dt. The Ac element is 4563 bp long and has inverted repeats at the ends. It encodes the enzyme transposase, which ensures the movement of Ac and Ds. Ds elements arise from deletions of internal regions of the Ac gene.
Currently, mobile elements have been discovered in many species of plants, animals and microorganisms. IS elements (insertion sequences) were found in E.coli. They are characterized by the following characteristic features:
1) at the ends, IS elements carry inverted (rotated 180 degrees relative to each other) repeats from several pairs to several tens of nucleotide pairs.
2) most IS elements contain a transposase gene, which controls the synthesis of the enzyme responsible for their movement.
3) at the point of insertion of each IS element, a duplication in direct orientation of 4-9 nucleotide pairs in length is always detected on its flanks.
Typically, the E. coli chromosome contains several IS elements.
Subsequently, more complex MEs were discovered in bacteria - transposons, which differ from IS elements in that they include some genes that are not related to the transposition process itself, for example, the gene for resistance to antibiotics, heavy metals and other inhibitors. Transposons are usually flanked by long direct or inverted repeats, which are often IS elements.
The ME of eukaryotes, for example, Ty 1 of yeast, and multiple dispersed genes of Drosophila, are also structured similarly.
Based on the mechanisms of transposition, MEs are divided into two classes. Elements of the first class move using reverse transcriptase, that is, DNA is synthesized on the RNA matrix of the mobile element. Reverse transcriptase (revertase) not only synthesizes the DNA strand into RNA, but also synthesizes the second complementary strand of DNA, and the LNA matrix disintegrates and is removed. Double-stranded DNA is synthesized in the cytoplasm and then moves into the nucleus and can be integrated into the genome. Such mobile elements are called retrotransposons. Retrotransposons make up more than 2% of the genome in Drosophila and up to 40% in plants. Elements of the second class move directly as DNA elements and are called transposons. They all have short inverted repeats at the ends.
Functional significance of mobile elements.
1. Movement and introduction of ME into genes can cause mutations. About 80% of spontaneous mutations in different Drosophila loci are caused by ME insertions. By introducing itself into a gene, ME can damage an exon, breaking it. In this case, the gene will no longer code for the protein. Once in the region of protomores or enhancers, a mobile element can damage the regulatory zone of a gene and change its expression. An insertion into the intron region may be harmless.
2. The state of gene activity may change. Long terminal repeats of retrotransposons and the retrotransposons themselves contain nucleotide sequences that act as transcription enhancers. Therefore, the movement of these signals in the genome can change the regulation of gene activity.
3. As a result of crossing over between similarly oriented elements, duplication and deletion of the material located between the insertions occurs. If MEs are oriented in opposite directions, inversion occurs.
In recent decades there has been enormous progress in the study epigenetic variability, which is understood as a variety of heritable, although possibly reversible, changes in gene expression that are not associated with a violation of the structure of the genetic material. It is now clear that epigenetic factors play a significant role in developmental differentiation, and disruption of this system is associated with many pathological conditions. The regulation of many genes is carried out through DNA-protein interactions. This applies, in particular, to the control of gene expression by transcription factors, the reverse regulation of a gene by its product or the products of other genes when they reach certain concentrations. If, under the influence of some external influences, changes occur in such regulatory proteins, their consequences will be expressed in the form of disruption of the expression of certain genes.
Epigenetic changes can be inherited not only at the cellular level, but also at the level of the whole organism. Gene expression is influenced by the nature of chromosome heterochromatinization, which depends not only on endogenous, but also on exogenous factors. This phenomenon was first studied by A. A. Prokofieva-Belgovskaya, who in the materials of her doctoral dissertation convincingly showed that “the development of a trait in the body is not determined only by the presence of a certain gene on a section of a chromosome, but is also controlled by the state of this section, detectable at the microscopic level, that is, is this region of the chromosome in interphase in a decondensed state or is it condensed.” The activity of many proteins is determined by their post-translational modifications - phosphorylation, acetylation, methylation. In particular, such modifications affecting histone proteins or proteins involved in the regulation of gene function can significantly affect their transcription. An important role in the regulation of gene expression is played by the spatial relationships between genes and the corresponding regulatory complexes. All these features of gene work determine a phenomenon well known to geneticists, called “ position effect" - that is, the different nature of the phenotypic manifestation of a gene depending on its localization in specific regions of the genome. The list of phenomena that can be explained in terms of epigenetic variability can be continued.
One of the most well-studied epigenetic mechanisms is DNA methylation, passing, most often, at the 5th carbon of cytosine. This DNA modification plays a significant role in regulating eukaryotic gene expression. The 5' untranslated regions of genes contain sequences enriched in CpG pairs, the so-called CpG islands. In many cases, gene inactivation is achieved through methylation of these sequences, and this state can be stably maintained over many generations of cells. Methyl groups disrupt interactions between DNA and proteins, thereby preventing the binding of transcription factors. In addition, methylated regions of DNA can interact with transcriptional repressors.
Changes in structural genes.
1) "Reading frame shift"- insertion or deletion of a pair or several pairs of nucleotides. For example, the original order of nucleotides is: AGGACTTCGA..., and after inserting a nucleotide: AAGGACTCGA...; Depending on the place of insertion or deletion of nucleotides, fewer or more codons change.
2) Transition- replacement of bases: purine to purine, or pyrimidine to pyrimidine; for example: A ↔ G, C ↔ T; in this case, the codon in which the transition occurred changes.
3) Transversion- replacement of a purine base with a pyrimidine base or a pyrimidine base with a purine base. For example: A ↔ C, G ↔ T; the codon in which the transversion occurred changes.
Changes in structural genes lead to: a) miscension mutations- replacement of a nucleotide in the coding part of a gene, leading to replacement of an amino acid in a polypeptide; b) nonsense mutations- the formation of “senseless” codons (UAA, UAG, UGA) that do not code for amino acids (terminators that determine the end of reading), and the cessation of translation.
1) The repressor protein “does not fit” the operator gene (“the key does not fit into the keyhole”) - structural genes work constantly (proteins are synthesized all the time).
2) The repressor protein is tightly “attached” to the operator gene and is not removed by the inducer (“the key does not come out of the keyhole”) - structural genes constantly do not work, and proteins encoded in this transcripton are not synthesized.
3) Violation of the alternation of repression and induction - in the absence of an inducer, a specific protein is synthesized, and in its presence, it is not synthesized. The above-mentioned disruptions in the functioning of transcriptons are associated with mutations in the regulator gene or operator gene.
Some mutations do not affect the function of the corresponding polypeptide. A significant portion of gene mutations disrupt the functioning of the gene and lead to the development of gene diseases. Phenotypically, gene mutations manifest themselves as signs of metabolic disorders, the frequency of which in human populations is 1-2% (phenylketonuria, hemophilia, neurofibromatosis, cystic fibrosis, Duchenne-Becker muscular dystrophy , hemoglobinopathy S). They are detected by biochemical methods and recombinant DNA methods.
According to the consequences gene mutations are classified into neutral, regulatory And dynamic.
Neutral(silent) mutation- does not have a phenotypic expression (for example, a nucleotide substitution that does not lead to an amino acid substitution due to the degeneracy of the genetic code).
Regulatory mutation- mutation in 5"- or 3" - untranslated regions of the gene; disrupts gene expression.
Dynamic Mutations- are caused by an increase in the number of trinucleotide repeats in functionally significant parts of the gene. Such mutations can lead to inhibition or blockade of transcription, and the acquisition of properties by protein molecules that disrupt their normal metabolism.
CHROMOSOMAL MUTATIONS
On chromosomal level of organization hereditary material has all the characteristics of the substrate of heredity and variability, including the ability to acquire changes that can be passed on to a new generation. Under the influence of various influences, the physicochemical and morphological structure of chromosomes can change. Chromosome breaks occur naturally during crossing over when homologous chromosomes exchange corresponding sections. Crossing-over disorder, in which chromosomes exchange unequal genetic material, leads to the appearance new clutch groups with a change in the number of genes. At chromosomal mutations The sequence of nucleotides in genes usually does not change, but changes in the number or position of genes can lead to a genetic imbalance, which has a detrimental effect on the normal development of the body.
Chromosomal aberrations can be intrachromosomal, interchromosomal and isochromosomal.
TO intrachromosomal These include rearrangements within one chromosome.
1) Deletions(deficiencies) - loss of a part of a chromosome (loss of a DNA segment ranging in size from one nucleotide to entire genes) - can cause disruption of embryogenesis and the formation of multiple developmental anomalies. For example, deletion of a section of the short arm of the 5th (5p-) chromosome in humans leads to the development cry the cat syndrome(underdevelopment of the larynx, congenital heart defects, mental retardation; in sick children, due to an anomaly of the larynx, crying resembles a cat’s meow). When the telomeres of both arms of a chromosome are deleted, a closure of the remaining structure into a ring is often observed - ring chromosomes. When the centromeric region falls out, decentral chromosomes.
2) Duplication- duplication of a chromosome region. The result of duplication in the second chromosome of the Drosophila fly can be the appearance of strip-shaped eyes. For example, trisomy on the short arm of chromosome 9 leads to the appearance of multiple congenital defects, including microcephaly, delayed physical, mental and intellectual development.
3) Inversion- tearing off a chromosome fragment, rotating it 180° and attaching it to the tear site. In this case, a violation of the order of gene arrangement is observed.
4) Insertion- insertion of DNA fragments ranging in size from one nucleotide to an entire gene.
5) Transposition- attachment of a fragment to its own chromosome, but in a different place.
Deletions And duplications always manifest themselves phenotypically, since the set of genes changes and partial monosomies (monosomies of a part of a chromosome) are observed in case of deficiencies and partial trisomies in case of duplications. Inversions And translocations do not always appear phenotypically; they can be balanced when there is no increase or decrease in genetic material and the overall balance of genes in the genome is maintained. With inversions and translocations, the conjugation of homologous chromosomes becomes difficult, which can cause a disturbance in the distribution of genetic material between daughter cells.
Interchromosomal rearrangements occur between non-homologous chromosomes.
Translocation- This is the exchange of segments between non-homologous chromosomes. There are translocations:
- reciprocal when two chromosomes exchange segments;
- non-reciprocal when segments of one chromosome are transferred to another;
- Robertsonian, when two acrocentric chromosomes are connected at their centromeric regions with the loss of short arms; As a result, one metacentric chromosome is formed.
Isochromosomal aberrations- the formation of identical but mirror fragments of two different chromosomes containing the same sets of genes. This occurs as a result of the transverse breaking of chromatids through the centromeres (hence the other name - centric connection).
Chromosomal aberrations are detected by cytogenetic methods.
GENOMIC MUTATIONS
Genomic mutations characterized by changes in the number of chromosomes. The cause of genomic mutations may be disruption of processes occurring in meiosis. Violation of the divergence of bivalents in anaphase leads to the appearance of gametes with a different number of chromosomes (for every gamete with an extra chromosome there is another - without one chromosome). Fertilization of such gametes by normal germ cells leads to a change in the total number of chromosomes in the karyotype due to a decrease ( monosomy) or increase ( trisomy) number of individual chromosomes.
Aneuploidy- multiple haploid decrease or increase in the number of chromosomes (2n±1, 2n±2, etc.). Types of aneuploidy: a) trisomy- three homologous chromosomes in the karyotype, for example, with Down syndrome(trisomy on chromosome 21), Edwards syndrome(trisomy on the 18th pair of chromosomes), Patau syndrome(trisomy 13); b) monosomy- the set contains only one of a pair of homologous chromosomes. The only monosomy compatible with life in humans - on the X chromosome - leads to the development Shereshevsky-Turner syndrome(45,X0). Monosomies on the first large pairs of chromosomes are lethal mutations for humans. Nulisomy- absence of a pair of chromosomes (lethal mutation).
If the mechanism of distribution of homologous chromosomes is damaged, the cell remains undivided, and diploid gametes are formed. Fertilization of such gametes leads to increase in the number of sets of chromosomes.
Polyploidy- an increase in the number of sets of chromosomes, a multiple of the haploid one (Зn, 4n, 5n, ...). Reasons: double fertilization and absence of the first meiotic division. Polyploidy is typically used in plant breeding and leads to increased yield. In mammals and humans, polyploidy, as well as most aneuploidies, lead to the formation of lethals.
Haploidy(1n) - a single set of chromosomes - for example, in bee drones. The viability of haploids is reduced, since in this case all recessive genes contained in the singular appear. For mammals and humans - a lethal mutation.
Genomic mutations are detected by cytogenetic methods. They always manifest themselves phenotypically.
Pathological effects of chromosomal and genomic mutations manifest themselves at all stages of ontogenesis, as they cause disturbances:
Overall genetic balance;
Coordination in the work of genes;
Systematic regulation.
They appear in two interrelated variants:
Mortality;
Congenital malformations.
Lethal outcome of chromosomal mutations- one of the main factors of intrauterine death, which is quite common in humans. Numerous cytogenetic studies of material from spontaneous abortions, miscarriages and stillbirths make it possible to objectively judge the effects of different types of chromosomal abnormalities in the prenatal period of individual development. The total contribution of chromosomal mutations to the number of cases of intrauterine death in humans is 45%. Among perinatally dead fetuses, the frequency of chromosomal abnormalities is 6%. In these cases, the lethal effects are combined with developmental defects, or rather, are realized through the defects.
Almost all chromosomal abnormalities lead to congenital malformations. Their more severe forms lead to earlier termination of pregnancy. The role of chromosomal and genomic mutations is not limited to their influence on the development of pathological processes in the early periods of ontogenesis. Their effects can be seen throughout life.
In the postnatal period, chromosomal mutations occur in somatic cells constantly with a low frequency (about 2%). Such cells contain proteins specific to them and are normally eliminated by the immune system if they manifest themselves as foreign. However, in some cases, chromosomal abnormalities are the cause of malignant growth.
Irradiation and chemical mutagens that induce chromosomal aberrations cause cell death and, thereby, contribute to the development of radiation sickness and bone marrow aplasia. There is experimental evidence of the accumulation of cells with chromosomal aberrations during aging.
GENETIC CONCEPTS OF CARCINOGENESIS
It has now been established that when carcinogenesis changes occur at the molecular genetic level and affect the mechanisms responsible for cell reproduction, growth and differentiation. Chronologically, several theories (concepts) of carcinogenesis can be distinguished.
1. Mutation concept. First G. de Vries (1901) suggested that tumors are the result of mutations in somatic cells. T. Boveri (1914) believed that carcinogenesis is based on genomic or chromosomal mutations. Subsequently, it was shown that the process of carcinogenesis can occur without structural changes in the genome, and chromosomal and genomic mutations found in tumor cells are a consequence of cell degeneration.
2. Virus-genetic concept. Back in 1911 F. Routh first showed that viruses are the cause of sarcoma in chickens. Then the viral nature of some other tumors was established (leukemia in chickens, mice and rats, warts in humans and rabbits). L.A. Zilber(1944-1968) considered viruses to be the universal cause of malignant growth. Mutagens and carcinogens stimulate the activity of viruses; their genome is incorporated into the cell's DNA and changes its properties.
3. Epigenomic concept.Yu.M. Olenov (1967) And A.Yu. Bronovitsky (1972) believed that the transformation of a normal cell into a tumor cell is based on persistent dysregulation of gene activity, i.e. functional genes are damaged.
4. Oncogene concept. R. Huebner (1969) And G.I. Abelev (1975) combined the second and third concepts. The DNA of the cells of any organism contains certain sections - proto-oncogenes. The body receives proto-oncogenes from its ancestors, or they are introduced by integrative viruses. They can remain in an inactive (repressed) state for a long time. Activation of proto-oncogenes can be caused by their mutation, the introduction of a viral promoter into the cell, etc. They are converted into oncogenes, which determine the synthesis of transforming proteins that transform a normal cell into a tumor cell.
LEVELS OF PATHOGENESIS OF HEREDITARY DISEASES
The main link in the pathogenesis of many hereditary diseases is at the cellular level, where pathological processes characteristic of this particular nosological form occur. The point of application of the primary action of the mutant gene is individual cell structures, different for different diseases: lysosomes, peroxisomes, membranes, mitochondria.
At the lysosomal level Pathogenetic processes unfold in storage diseases due to impaired activity of lysosomal enzymes. Thus, the accumulation of glycosaminoglycans (mucopolysaccharides) in the cells, and then in the main intercellular substance, leads to the development of serious diseases - mucopolysaccharidoses. The reason for the excess content of these polymers is the lack of their degradation in lysosomes, which is associated with defects in the group of specific enzymes that catalyze the entire degradation cycle.
If the point of application of the action of the mutant gene becomes peroxisomes, then develop peroxisomal diseases. Clinically, these diseases manifest themselves as multiple congenital malformations, generally similar in different nosological forms (multiple craniofacial dysmorphia, cataracts, renal cysts and other manifestations).
Cell membranes may also be key elements in the pathogenesis of genetic diseases. Thus, the absence of specific protein receptor molecules on the cell surface that bind low-density lipoproteins leads to familial hypercholesterolemia
The cellular level of the pathogenesis of gene diseases can manifest itself not only in specific organelles, but also in the form disturbances in the coordination of cell activity. For example, mutations affecting oncogene regions lead to to the removal of control of cell reproduction and, accordingly, to malignant growth.
At the organ level In different diseases, the target of the pathological process is different organs. Their defeat may be primary or secondary:
Copper deposition in the liver and extrapyramidal system of the brain during hepatolenticular degeneration(Wilson-Konovalov disease) - primary process;
Hemosiderosis of parenchymal organs with primary hemochromatosis or thalassemia develops secondary due to increased breakdown of red blood cells.
CLASSIFICATION OF HEREDITARY DISEASES
1. According to the type of cells in which the mutations occurred: a) gametic; b) somatic.
2. According to clinical manifestations: a) predisposition to the disease; b) the disease itself; c) special forms of the disease - deformities.
3. By type of heredity and abnormal gene: a) dominant; b) recessive; c) semi-dominant.
4. The most practical is the classification depending on level of organization of hereditary material: a) genetic; b) chromosomal.
Depending on the type of primary affected cells The following groups of diseases have been identified:
diseases due to mutations in germ cells- “gametic”, i.e. actual hereditary diseases (for example, phenylketonuria, hemophilia);
diseases due to mutations in somatic cells- “somatic” (for example, tumors, some diseases of immune autoaggression); these diseases are not inherited;
diseases due to a combination of mutations in germ and somatic cells(eg, familial retinoblastoma).
Consider separately lethal, sublethal And hypogenital diseases:
lethal diseases– lead to death during intrauterine development (for example, autosomal monosomy, haploidy, most polyploidy);
sublethal diseases– lead to the death of an individual before puberty (for example, hereditary immunodeficiencies such as Swiss type agammaglobulinemia, Louis-Bar syndrome, some hemophilia);
hypogenital diseases– combined with infertility (for example, Shereshevsky-Turner, Klinefelter syndromes).
Division of hereditary diseases into genetic And chromosomal informal: gene mutations are passed on from generation to generation in accordance with Mendel's laws, while chromosomal diseases caused by aneuploidy are not inherited at all (lethal effect from a genetic point of view), and structural adjustments(inversions, translocations) are transmitted with additional recombinations.
The basis genetic classification of hereditary pathology the etiological principle is laid down - type of mutation And the nature of the interaction of hereditary factors with the environment.
Genetic diseases of somatic cells isolated into a separate group after the discovery of specific chromosomal rearrangements in cells of malignant neoplasms that cause activation of oncogenes. These changes in the genetic material of cells are etiopathogenetic for malignant growth and can therefore be classified as genetic pathology.
Diseases that occur when mother and fetus are incompatible with antigens, develop as a result of the mother’s immune reaction to fetal antigens. In general, this group makes up a significant part of the pathology and is quite common in medical practice. The most typical and well-studied disease of this group is hemolytic disease of the newborn, resulting from incompatibility of mother and fetus with the Rh antigen. The disease occurs when the mother has an Rh-negative blood type and the fetus has inherited an Rh-positive allele from the father.
Clinical classification of hereditary diseases no different from the classification of non-hereditary diseases according to organ, systemic characteristics or by type of metabolic damage.
Since hereditary diseases are the same etiological principle (mutations), the basis for their classification is, first of all, systemic and organ principle: nervous; neuromuscular; skin; ophthalmic; musculoskeletal system; endocrine; blood; lungs; of cardio-vascular system; genitourinary system; gastrointestinal tract. This approach is controversial. Very few inherited diseases can be found in which one system is selectively affected. Most gene mutations, and especially chromosomal and genomic ones, cause generalized damage to any tissue or affect several organs. Therefore, many hereditary diseases manifest themselves in the form syndromes or a complex of pathological signs.
Despite the obvious convention, the clinical classification helps the doctor of the relevant profile to concentrate on hereditary diseases encountered in the practice of this specialty.
Pathogenetic classification based on identifying the main pathogenetic link.
Biochemical classification of hereditary diseases , manifested in metabolic disorders, unites genetically th and clinical approach and is carried out by type of damage to the primary metabolic link.
Depending on the nature of the metabolic disorder, there are:
· Hereditary defects in carbohydrate metabolism.
Galactosemia, impaired lactose metabolism, mucopolysaccharidosis, impaired breakdown of polysaccharides.
· Defects in lipid and lipoprotein metabolism.
Sphingolipidoses-disorders of the breakdown of structural lipids and other forms of lipid metabolism disorders.
· Defects in amino acid metabolism.
Phenylketonuria, albinism-disruption of the synthesis of melanin pigment from tyrosine, etc.
· Defects in vitamin metabolism.
Homocystinuria-develops as a result of a genetic defect in the coenzyme of B vitamins.
· Defects in the metabolism of purine and pyrimidine bases.
· Defects in hormone biosynthesis.
Adrenogenital syndrome, testicular feminization.
· Hereditary defects of red blood cell enzymes.
· Collagen diseases are defects in the biosynthesis and breakdown of collagen, a structural component of connective tissue.
Ellers-Danlos disease, Marfan disease and a number of other diseases.
Diseases caused by defects in genes encoding enzymes are inherited autosomal recessive type, and those caused by genes encoding modulatory protein functions or receptors - by autosomal dominant or autosomal recessive. Diseases caused by transcription factor genes belong to the group autosomal dominant.In the human genome, this rule is sometimes violated, which is due to various mutations in the same gene. This must be taken into account during medical genetic counseling.
The basis of diseases, the pathogenesis of which leads to anomalies of morphogenesis, is a violation of cell differentiation, leading to congenital malformations (polydactyly, Hall-Oram, Crouzon syndromes, etc.).
However, the vast majority of monogenic hereditary diseases belong to third group- conditional a combination of metabolic disorders and morphogenesis anomalies (cystic fibrosis, achondroplasia, muscular dystrophy, etc.).
The formation of hereditary diseases over time also has patterns associated with the function of the primary gene product:
transcription factor diseases develop in utero;
enzyme pathology- during the first year of life;
receptors- from 1 year to puberty;
modular protein function- for a period up to 50 years.
TYPES AND REGULARITIES OF THE COURSE OF GENE DISEASES
Widely known to the scientific and medical community and updated daily Catalog of hereditary human diseases(OMIM, Online Mendelian Inheritance in Man
Gene diseases- a group of hereditary diseases, diverse in clinical manifestations, caused by mutations at the gene level. The basis for combining them into one group is etiological genetic characteristics And patterns of inheritance in families and populations. According to numerous studies of various hereditary diseases and the human genome as a whole, we can talk about a variety of types of mutations in the same gene. Any of these types of mutations can lead to hereditary diseases. Even the same gene disease can be caused by different mutations.
When studying the protein products of mutant genes, two groups of mutations are distinguished. First group associated with qualitative changes in protein molecules, i.e. the presence of abnormal proteins in patients (for example, abnormal hemoglobins), which is caused by mutations of structural genes. Another group diseases are characterized by quantitative changes in the content of normal protein in the cell (increase or decrease), which is most often caused by mutations of functional genes, i.e. associated with dysregulation of genes.
Mutations that cause hereditary diseases can affect structural, transport, embryonic proteins, and enzymes. Hereditary abnormalities may occur at all levels of regulation of protein synthesis, caused by the corresponding enzymatic reactions:
pretranscriptional(carried out by increasing or decreasing the number of gene copies);
transcriptional(genetic defects in spacers, introns, transposons, regulatory proteins can lead to disruption of the transcription of the entire gene, causing a change in the volume of synthesis of the corresponding protein);
processing And splicing pro-i-RNA (impairments at the level of destruction of non-informative sections of pro-i-RNA and fusion of informative sections);
broadcast(disturbances at the level of direct assembly of the protein molecule in the ribosome);
post-translational(violations at the level of formation of the secondary, tertiary and quaternary structure of the protein molecule).
Since mutations in individual genes are the etiological factor of gene diseases, then the patterns of their inheritance correspond to the Mendelian rules of segregation in offspring.
By revising gene diseases as mendelian characteristics of the body it is believed that we are talking about the so-called full forms, that is, forms caused by gametic mutations. These may be new or inherited mutations from previous generations. Consequently, in these cases, pathological genes are present in all cells of the body.
However, theoretically it is possible to imagine the appearance and mosaic forms. Any mutations, including gene mutations, can occur in the early stages of division of the zygote in one of the cells, and then the individual will be mosaic for this gene. In some cells it will have a normal allele, in others it will be a mutant or pathological one.
In the case of gene diseases, the clinical picture of the disease can be formed due to the pathogenetic effects of mutations of different genes, that is A similar phenotypic manifestation of the disease can be caused by several different mutations. Consequently, cases that are different from a genetic point of view (mutations in different loci or different mutations in one locus) will fall into one group - gene copies. At the same time, although rare, there may be phenocopies gene diseases - cases in which damaging external factors, usually acting in utero, cause a disease whose clinical picture is generally similar to hereditary. The opposite condition, when with a mutant genotype as a result of environmental influences (medicines, diet, etc.) the disease does not develop, is called standard copying.
The concepts of geno- and phenocopies give the doctor the following opportunities: to make the correct diagnosis; more accurately determine the prognosis of the patient’s health or the likelihood of having a sick child; in a particular case, prevent the development of the disease in a child who has inherited a pathological gene.
In accordance with genetic classification principle gene diseases are divided into groups by type of inheritance
· autosomal dominant;
· autosomal recessive;
· X-linked dominant;
· X-linked recessive;
· Y-linked (holandric) and mitochondrial.
Assigning a disease to one group or another allows the doctor to orient himself regarding the situation in the family and determine the type of medical and genetic assistance.
Two years ago, the CRISPR/Cas9 genome modification technology was invented. In 2015, she made a real revolution in genetic engineering. The technology is based on the molecular defense mechanism of microorganisms, thanks to which DNA fragments can be edited and cut out with increased precision. Moreover, this can be done directly in living cells of any organism!
Of course, today manipulation of genes will not surprise anyone, but work with them was previously carried out in specially equipped laboratories at major institutes. But CRISPR/Cas9 technology can become available to everyone. NASA molecular biologist Josiah Zayner intends to develop a kit that would allow experiments with gene modification at home. He will allow him to change the genome of yeast and microorganisms in his kitchen.
The abbreviation CRISPR can be literally translated into Russian as “clustered regularly interspaced short palindromic repeats”; they were first found in the genes of archaea and bacteria. Then it was discovered that microorganisms that managed to survive the attack of the virus inscribe a portion of the enemy’s gene into their own DNA. Thanks to this, the cells formed by the body will be able to recognize such a strain. If the “database” of genes contains information about an enemy, then when they encounter him, microorganisms use a special molecular mechanism. It attaches to the viral DNA in the place that corresponds to the preserved region. Next, Cas group proteins are used to cut it and destroy the virus. Scientists have determined that similar scissors for cutting molecules can be used for any part of the genetic code of mammals, and humans are no exception. With their help, you can replace or edit various genes.
According to Mr. Zayner, CRISPR/Cas9 should become publicly available, and even novice researchers and amateurs should be able to conduct experiments with this method. For this purpose, the online store The ODIN was developed. Its goal is to help conduct home experiments with artificially created bacteria. Today, Zayner’s company is raising funds on the Indiegogo crowdfunding platform, offering complete kits and reagents for gene editing as a “reward.”
Available sets
The products sold are similar to educational kits for conducting chemical experiments by schoolchildren and students. For $75, you can buy a kit here that allows you to add a fluorescent protein to the genome of bacteria, causing them to glow in the dark. To create a genetically modified strain of bacteria that can survive in extreme conditions, you need to buy a kit for $130. But a kit for 160 US dollars will allow you to make changes to the gene code of yeast, adding red pigment to them.
The company also offers more expensive sets. For example, for $200 you can get a kit that gives bacteria the ability to fertilize soil and break down plastic. For $500 you can buy a classroom kit - the client can specify the type of kits that will be sent in quantities of 20 for group use. The tools in this set can give bacteria the ability to glow in the dark or change color.
A $3,000 kit will allow you to create a real home laboratory for conducting experiments in molecular and synthetic biology. It includes: centrifuges, pipettes, reagents, electrophoresis gels, chemicals and much more. The included kit allows you to use the CRISPR system for various studies.
The most incredible is the offer for $5,000: the authors of the project promise the opportunity to create a new, unique living organism. With its help, you can isolate the desired characteristic of yeast or bacteria and change it. The owner of such a kit can independently breed genetically modified organisms. The company helps you determine the parameters that will help you achieve your goals! Detailed instructions included with each kit will help you carry out experiments without outside help, although the authors readily promise to provide consultation if necessary.
Future plans
CRISPR technology is capable of making changes to human genes. However, Zayner does not plan to sell kits that would help fight baldness or grow an additional kidney.
To achieve his goal, Zayner launched a crowdfunding campaign on the Indiegogo website. You can view the company. Thanks to growing interest in the CRISPR method, the company's authors managed to obtain the $10,000 needed to create portable kits ahead of schedule. According to Investtok.ru experts, by the end of the campaign, the project’s authors can raise ten times more funds than originally planned, since the audience’s interest in the new technology is enormous.
In the fifties of the 20th century, scientists encountered a strange phenomenon. They noticed that some viruses infect different strains of the same bacterium differently. Some strains - for example, E. coli - became easily infected and quickly spread the infection throughout the colony. Others became infected very slowly or were completely resistant to the viruses. But once it had adapted to one strain or another, the virus subsequently infected it without difficulty.
It took biologists two decades to understand this selective resistance of bacteria. As it turned out, the ability of certain strains of bacteria to resist viruses - it was called restriction (that is, “limitation”) - is explained by the presence of special enzymes that physically cut viral DNA.
The peculiarity of these proteins - restriction enzymes - is that they recognize a small and strictly defined DNA sequence. Bacteria “target” restriction enzymes to rare sequences that they themselves avoid in their genes - but which may be present in viral DNA. Different restriction enzymes recognize different sequences.
Each strain of bacteria has a specific arsenal of such enzymes and, thus, reacts to a specific set of “words” in the genome of the virus. If we imagine that the genome of the virus is the phrase “mom washed the frame,” then the virus will not be able to infect a bacterium that recognizes the word “mom,” but a bacterium that targets the word “uncle” will be defenseless. If the virus manages to mutate and turn into, say, a “woman washed the frame,” then the first bacterium will lose its protection.
Why did the discovery of “bacterial immunity” appear at the very top of the list of the most important achievements in molecular biology? It's not the bacteria themselves or even the viruses.
The scientists who described this mechanism almost immediately drew attention to the most important detail of this process. Restriction enzymes (more precisely, one of the types of these enzymes) are capable of cutting DNA at a clearly defined point. Returning to our analogy, an enzyme that targets the word "mom" in the DNA binds to that word and cuts it, for example, between the third and fourth letter.
Thus, for the first time, researchers were able to “cut” the DNA fragments they needed from genomes. With the help of special “gluing” enzymes, the resulting fragments could be stitched together - also in a certain order. With the discovery of restriction enzymes, scientists had all the necessary tools for “assembling” DNA in their hands. Over time, a slightly different metaphor took root to denote this process - genetic engineering.
Although other methods of working with DNA exist today, the vast majority of biological research of the last twenty to thirty years would not have been possible without restriction enzymes. From transgenic plants to gene therapy, from recombinant insulin to induced stem cells, any work involving genetic manipulation uses this “bacterial weapon.”
The immune system of mammals - including humans - has both innate and acquired defense mechanisms. The innate components of the immune system usually react to something common that unites many enemies of the body at once. For example, the innate immune system can recognize components of the bacterial cell wall that are common to thousands of different microbes.
Acquired immunity relies on the phenomenon of immunological memory. It recognizes specific components of specific pathogens, “remembering” them for the future. Vaccination is based on this: the immune system “trains” on a killed virus or bacteria, and later, when a live pathogen enters the body, it “recognizes” it and destroys it on the spot.
Innate immunity is a border inspection point. It protects against everything at once and nothing in particular. Acquired immunity is a sniper who knows the enemy by sight. As it turned out in 2012, bacteria also have something similar.
If restriction is a bacterial analogue of innate immunity, then the role of acquired immunity in bacteria is performed by a system with the rather cumbersome name CRISPR/Cas9, or “Crisper”.
The essence of Crisper's work is as follows. When a bacterium comes under viral attack, it copies part of the virus’s DNA to a special place in its own genome (this “repository” of information about viruses is called CRISPR). Based on these stored “photo images” of the virus, the bacterium then makes an RNA probe that can recognize the viral genes and bind to them if the virus tries to infect the bacterium again.
The RNA probe itself is harmless to the virus, but this is where another player comes into play: the Cas9 protein. It is a “scissors” responsible for the destruction of viral genes - like a restriction enzyme. Cas9 grabs onto the RNA probe and, as if on a leash, is delivered to the viral DNA, after which it is given a signal: cut here!
In total, the entire system consists of three bacterial components:
1) DNA storage of “photo identikit” of old viruses;
2) an RNA probe made on the basis of these “photo images” and capable of identifying the virus from them;
3) protein “scissors” attached to an RNA probe and cutting the viral DNA exactly at the point from which the “photo sketch” was taken last time.
Almost instantly after the discovery of this “bacterial immunity,” everyone forgot about bacteria and their viruses. The scientific literature has exploded with enthusiastic articles about the potential of the CRISPR/Cas9 system as a tool for genetic engineering and future medicine.
As with restriction enzymes, the Crisper system is capable of cutting DNA at a strictly defined point. But compared to the “scissors” discovered in the seventies, it has enormous advantages.
Restriction enzymes are used by biologists to “assemble” DNA exclusively in a test tube: you must first produce the desired fragment (for example, a modified gene), and only then introduce it into a cell or organism. “Crisper” can cut DNA on the spot, right in a living cell. This makes it possible not only to produce artificially introduced genes, but also to “edit” entire genomes: for example, to remove some genes and insert new ones in their place. Just recently one could only dream of this.
As has become clear over the past year, the CRISPR system is unpretentious and can work in any cell: not only bacterial, but also mouse or human. “Installing” it in the desired cell is quite simple. In principle, this can be done even at the level of entire tissues and organisms. In the future, this will make it possible to completely remove defective genes, such as those that cause cancer, from the genome of an adult.
Let’s say that the phrase “mom washed the frame” that is present in your genome causes in you a painful craving for gender stereotypes. To get rid of this problem, you need a Cas9 protein - always the same one - and a pair of RNA probes targeting the words "mama" and "rama". These probes can be anything - modern methods make it possible to synthesize them in a few hours. There are no restrictions on quantity at all: you can “cut” the genome at least at a thousand points simultaneously.
But the value of the Crisper is not limited to the scissor function. As many authors note, this system is the first tool known to us with which it is possible to organize a “meeting” of a certain protein, a certain RNA and a certain DNA at the same time. This in itself opens up enormous opportunities for science and medicine.
For example, you can turn off the scissor function of the Cas9 protein, and instead bind another protein to it - say, a gene activator. With the help of a suitable RNA probe, the resulting pair can be sent to the desired point in the genome: for example, to a poorly functioning insulin gene in some diabetics. By organizing the meeting of an activating protein and a switched-off gene in this way, it is possible to precisely and finely tune the functioning of the body.
You can bind not only activators, but anything in general - say, a protein that can replace a defective gene with its “backup copy” from another chromosome. In this way, in the future it will be possible to cure, for example, Huntington's disease. The main advantage of the CRISPR system in this case is precisely its ability to “send expeditions” to any point in the DNA that we can program without much difficulty. What the task of each specific expedition is is determined only by the imagination of the researchers.
Today it is difficult to say exactly what problems the CRISPR/Cas9 system will be able to solve in a few decades. The global community of geneticists now resembles a child who was allowed into a huge hall filled to capacity with toys. The leading scientific journal Science recently released a review of the latest advances in the field called "The CRISPR Craze." And yet, it is already obvious: bacteria and fundamental science have once again given us a technology that will change the world.
In January, reports emerged of the birth of the first primates whose genome was successfully modified by the CRISPR/Cas9 system. As a test experiment, monkeys were given mutations in two genes: one associated with the functioning of the immune system, and the other responsible for fat deposition, which opaquely hints at the possible application of the method to homo sapiens. Perhaps solving the problem of obesity using genetic engineering is not such a distant future.
