Genetic mapping. Genetic mapping strategy and its role in the identification of new genes of hereditary diseases Genetic mapping of human disease genes examples

Alfred Sturtevant (Morgan's collaborator) suggested that the frequency of crossing over between genes located on the same chromosome can serve as a measure of the distance between genes. In other words, the crossover frequency, expressed as the ratio of the number of crossover individuals to the total number of individuals, is directly proportional to the distance between genes. The crossover frequency can then be used to determine the relative position of genes and the distance between genes.

Genetic mapping is the determination of the position of a gene in relation to (at least) two other genes. The constancy of the percentage of crossing over between certain genes allows them to be localized. The unit of distance between genes is 1% crossing over; in honor of Morgan, this unit is called morganida (M), or santimorganide (CM).

The number of linkage groups corresponds to the haploid set of chromosomes. For example, in D. melanogaster 4 clutch groups, maize 10, mice 20, humans 23 clutch groups. If there are sex chromosomes, they are indicated additionally (for example, a person has 23 linkage groups plus a Y chromosome).

As a rule, the number of genes in linkage groups depends on the linear dimensions of the corresponding chromosomes. So, a fruit fly has one (IV) point (when analyzed under a light microscope) chromosome. Accordingly, the number of genes in it is many times less than in the others, significantly exceeding it in length. It should also be noted that in heterochromatic regions of chromosomes there are no genes or almost none; therefore, extended regions of constitutive heterochromatin can somewhat change the proportionality of the number of genes and the length of the chromosome.

Based on genetic mapping, genetic maps are drawn up. On genetic maps, the extreme gene (i.e., the most distant from the centromere) corresponds to the zero (initial) point. The remoteness of a gene from the zero point is indicated in morganids.

If the chromosomes are long enough, then the gene removal from the zero point can exceed 50 M - then a contradiction arises between the distances marked on the map, exceeding 50%, and the position postulated above, according to which 50% of the crossovers obtained in the experiment, in fact, should mean the absence of linkage. i.e. e. localization of genes in different chromosomes. This contradiction is explained by the fact that when compiling genetic maps, the distances between the two closest genes are summed up, which exceeds the experimentally observed percentage of crossing over.

KAZAKH NATIONAL UNIVERSITY NAMED AFTER AL-FARABI

Faculty: biology and biotechnology

Department: biotechnology

"ESSAY"

On the topic of: GENETIC CLUTCH AND MAPPING OF HUMAN GENES.

Completed : 3-year students (medical bt.)

Nuralibekov S.Sh.

Davronova M.A.

Checked : ph.D. , associate professor of the departmentmolecular

biology and genetics Omirbekova N.Zh.

ALMATY 2018

Genetic linkage maps ……………………………………………………… ..3

Modern methods for constructing genetic linkage maps …… .......... …… ...… .5

PCR in studies of the human genome ……………………………… .... …………. …… 8

Low resolution physical maps ………………………………………… ..….… .9

High resolution physical maps …………… .. ……………………… .. ……… 11

List of sources used ……………… ... …………… .. ………………… .13

Mapping and determination of the primary structure of the human genome

After a brief consideration of the main methods most often used in molecular genetics to study the structure and mechanisms of gene functioning, it seems appropriate to take a closer look at the practical application of these methods and their modifications to study large genomes using the example of the human genome. In order to comprehensively study the human genome, this colossal storage of its genetic information, a special international program "Human Genome Project" was recently developed and is being implemented. The main task of the program is the construction of comprehensive genetic maps of high resolution for each of the 24 human chromosomes, which, ultimately, should be completed with the determination of the complete primary structure of the DNA of these chromosomes. Currently, work on the project is in full swing. In the event of its successful completion (and this is planned to happen in 2003), mankind will have prospects for a thorough study of the functional significance and mechanisms of functioning of each of its genes, as well as the genetic mechanisms that govern human biology, and establishing the causes of most of the pathological conditions of its body ...

Basic approaches to mapping the human genome

The solution to the main task of the Human Genome program includes three main stages. At the first stage, it is necessary to divide each individual chromosome in a specific way into smaller parts, allowing their further analysis by known methods. The second stage of research involves determining the relative position of these individual DNA fragments relative to each other and their localization in the chromosomes themselves. At the final stage, it is necessary to make the actual determination of the primary structure of DNA for each of the characterized chromosome fragments and draw up a complete continuous sequence of their nucleotides. The solution to the problem will not be complete if it is not possible to localize all the genes of the organism in the found nucleotide sequences and determine their functional significance. Passage of the above three stages is required not only to obtain comprehensive characteristics of the human genome, but also any other large genome.

Genetic linkage maps

Genetic linkage maps are one-dimensional patterns of the mutual arrangement of genetic markers on individual chromosomes. Genetic markers are understood as any inherited phenotypic traits that differ in individual individuals. Phenotypic traits that meet the requirements of genetic markers are very diverse. They include both behavioral features or predisposition to certain diseases, and morphological signs of whole organisms or their macromolecules, which differ in structure. With the development of simple and effective methods for studying biological macromolecules, such traits, known as molecular markers, have become most often used in the construction of genetic linkage maps. Before proceeding to the consideration of the methods for constructing such maps and their implications for the study of the genome, it is necessary to recall that the term "linkage" is used in genetics to denote the probability of joint transmission of two traits from one parent to the offspring.

During the formation of germ cells (gametes) in animals and plants at the stage of meiosis, as a rule, synapsis (conjugation) of homologous chromosomes occurs. Sister chromatids of homologous chromosomes are connected along their entire length with each other, and as a result of crossing over (genetic recombination between chromatids), their parts are exchanged. The further the two genetic markers are located from each other on the chromatid, the more likely it is that the chromatid rupture required for crossing over will occur between them, and the two markers in the new chromosome belonging to the new gamete will be separated from each other, i.e. their cohesion will be broken. The unit of linkage of genetic markers is morganida (Morgan's unit, M), which contains 100 centimeters (cM). 1 cM corresponds to the physical distance on the genetic map between two markers, the recombination between which occurs with a frequency of 1%. Expressed in base pairs, 1 cM corresponds to 1 million bp. (m.p.) DNA.

Genetic linkage maps correctly reflect the order in which genetic markers are located on chromosomes; however, the obtained values \u200b\u200bof the distances between them do not correspond to the actual physical distances. Usually, this fact is associated with the fact that the efficiency of recombination between chromatids in individual regions of chromosomes can vary greatly. In particular, it is suppressed in the heterochromatic regions of chromosomes. On the other hand, recombination hot spots are common in chromosomes. The use of recombination frequencies for constructing physical genetic maps without taking into account these factors will lead to distortions (respectively, underestimation or overestimation) of real distances between genetic markers. Thus, genetic linkage maps are the least accurate of all the available types of genetic maps, and can only be considered as a first approximation to real physical maps. Nevertheless, in practice, it is they and only they that make it possible to localize complex genetic markers (for example, associated with symptoms of the disease) at the first stages of the study and make it possible to further study them. It must be remembered that in the absence of crossing over, all genes on an individual chromosome would be passed from parents to offspring together, since they are physically linked to each other. Therefore, individual chromosomes form linkage groups of genes, and one of the first tasks of constructing genetic linkage maps is to assign the studied gene or nucleotide sequence to a specific linkage group. In the next. The table lists modern methods that, according to V.A. McCusick were most often used to construct genetic linkage maps until the end of 1990.

Modern methods for constructing genetic linkage maps

Method	Number of mapped loci
Somatic cell hybridization	1148
In situ hybridization	687
Family	466
Determination of dose effect	159
Restriction mapping	176
Use of chromosomal aberrations	123
Using synthenia	110
Radiation-induced gene segregation	18
Other methods	143
Total	3030

Hybridization of somatic cells. One of the most popular methods for assigning a genetic marker (functionally active gene) to a specific linkage group is hybridization (fusion with each other) of somatic cells of different biological species of organisms, one of which is the studied one. In interspecific hybrids of somatic cells in the process of cultivation, the loss of chromosomes occurs, mainly of one of the biological species. The loss of chromosomes is, as a rule, random, and the resulting clones of cells contain the remaining chromosomes in different combinations. Analysis of clones containing different sets of chromosomes of the studied species makes it possible to determine with which of these remaining chromosomes the expression of the studied marker is associated, and, consequently, to localize the gene on a particular chromosome.

In situ hybridization. The in situ hybridization technique is also widely used to map nucleotide sequences on chromosomes. For this purpose, preparations of fixed chromosomes are hybridized (incubated at an elevated temperature with subsequent cooling) with the nucleotide sequences under investigation labeled with a radioactive, fluorescent or other label. After washing off the unbound label, the remaining labeled nucleic acid molecules are associated with chromosomal regions containing sequences complementary to the studied labeled nucleotide sequences. The resulting hybrids are analyzed with a microscope either directly or after autoradiography. This group of methods is characterized by a higher resolution than hybridization of somatic cells, since they allow localizing the studied nucleotide sequences on chromosomes. As the Human Genome Program progresses, researchers have more and more isolated nucleotide sequences that can be used as probes for in situ hybridization. In this regard, these methods in terms of frequency of use have recently firmly come out on top. The most popular is a group of methods called fluorescence in situ hybridization (FISH), which uses polynucleotide probes containing a fluorescent label. In particular, in 1996, more than 600 papers were published describing the use of this method.

Family genetic linkage analysis. This group of methods is often used in medical genetics to identify the link (linkage) between the symptoms of a disease caused by a mutation in an unknown gene and other genetic markers. In this case, the symptoms of the disease themselves act as one of the genetic markers. A large number of polymorphisms, including RFLP, have been found in the human genome. RFLPs are distributed more or less evenly in the human genome at a distance of 5–10 cm from each other. The closer the individual polymorphic loci are located to the gene responsible for the disease, the less likely they are to separate during recombination in meiosis and the more often they will occur together in a sick individual and together are transmitted from parents to offspring. Having cloned an extended genome region, including the corresponding polymorphic marker (its selection from the genomic DNA clone library is carried out using a probe), it is possible to simultaneously isolate the gene that causes a hereditary disease with it. Such approaches have, in particular, been successfully applied for family analysis and isolation of the corresponding genes in Duchenne muscular dystrophy, cystic fibrosis of the kidneys (cystic fibrosis) and myotonic dystrophy. The information content of individual RFLPs of the human genome depends on the level of their heterozygosity in the studied population. The measure of the informativeness of RFLP as a genetic marker, as suggested by D. Botstein et al. (1980), is considered to be the value of the polymorphism information content (PIC), which is the ratio of the number of crosses in which at least one of the parents has the studied polymorphic marker in a heterozygous state, to all crosses.

Gene dose effect determination and use of chromosomal aberrations ... These methods reveal correlations between the level of expression of the studied gene and the number of specific chromosomes in aneuploid cell lines or structural rearrangements of chromosomes (chromosomal mutations - aberrations). Aneuploidy is the presence of a number of chromosomes in a cell, tissue or whole organism that is not equal to that typical for a given biological species. Chromosomal aberrations in the form of translocations of chromosome regions into heterochromatic regions of the same or other chromosomes are often accompanied by suppression of transcription of genes located in translocated regions or in the acceptor chromosome (mosaic effect of position).

Using synthenia. Synthenia is the structural similarity of gene linkage groups in organisms of different biological species. In particular, several dozen synthenic groups of genes are known in the human and mouse genomes. The presence of the phenomenon of synthenia makes it possible to narrow the search for the site of localization of the studied gene on the chromosomes, limiting it to the region of known genes belonging to a specific synthenic group.

Gene segregation induced by ionizing radiation. Using this method, the distance between the genes under study is determined by assessing the probability of their separation (segregation) after the cells are irradiated with a certain standard dose of ionizing radiation. The irradiated cells are saved from death by hybridization with somatic cells of rodents, and the presence of the studied markers of irradiated cells is determined in somatic hybrids in culture. As a result, it is possible to conclude about the presence or absence of linkage (physical distance) between these genes.

Among other methods Mention should be made of methods based on the use of large DNA fragments produced by large-cleavage restriction enzymes for mapping genes. After cleavage of genomic DNA, the resulting fragments are separated by electrophoresis in a pulsed electric field and then they are hybridized according to Southern with probes corresponding to the mapped genes. If, after hybridization, the signals of both probes are localized on the same large DNA fragment, this indicates a close linkage of such genes.

PCR in studies of the human genome

The polymerase chain reaction is central to the development of approaches to the practical implementation of the Human Genome Program. As discussed above, using PCR, it is possible to quickly and efficiently amplify almost any short region of the human genome, and the resulting PCR products can then be used as probes for mapping the corresponding regions on chromosomes by Southern or in situ hybridization.

STS concept. One of the key concepts underlying the mapping of human genes in the framework of the discussed program is the concept of sequence-tagged sites (STS). In accordance with this concept, all DNA fragments used to construct genetic or physical maps can be uniquely identified using a nucleotide sequence of 200-500 bp that will be unique for a given fragment. Each of these sites must be sequenced, which will make it possible to further amplify them using PCR and use as probes. The use of STS would make it possible to use their sequences in the form of PCR products as probes for the targeted isolation of any DNA fragment of a particular genome region from the collection of genomic sequences. As a result, databases can be created that include the localization and structure of all STSs, as well as the primers required for their amplification. This would eliminate the need for laboratories to store numerous clones and send them to other laboratories for research. In addition, STS provide the basis for the development of a single language in which different laboratories could describe their clones. Thus, the end result of the development of the STS concept would be a comprehensive map of the STS of the human genome. Theoretically, to construct a genetic map of 1 cm in size, 3000 fully informative, polymorphic DNA markers are required. However, since polymorphic markers are unevenly distributed in the genome and only a few of them are fully informative, the actual number of markers required to build a map of this size is estimated at 30–50 thousand. To obtain markers corresponding to the studied regions of chromosomes, primers corresponding to dispersed repetitive sequences are often used, among which Alu sequences were the first to be used.

Alu-PCR.Dispersed repeating Alu sequences are characteristic of the human genome. Primers specific for Alu sequences are used to amplify DNA regions of the human genome enclosed between Alu repeats, which are located on average at a distance of 4–10 kbp. apart. Another option for Alu-PCR is the directed synthesis of DNA probes with its help to chromosome regions obtained after laser fragmentation, individual chromosomes isolated using flow cytometry, or DNA of hybrid cells containing a certain part of the human genome. In addition, Alu-PCR is used to obtain unique fingerprints characterizing cell hybrids in terms of their genome stability, as well as to characterize human DNA fragments cloned into YAC vectors, cosmids, or vectors based on bacteriophage DNA. The uniqueness of Alu sequences for the human genome makes it possible to use them for "walking along chromosomes", as well as for expanding existing contigs. Since\u003e 90% of moderately repetitive sequences in the human genome are represented by the Alu and KpnI families, it is not surprising that the latter are also used in PCR for the same purposes as Alu. However, here the profiles of PCR products are less complex, since the KpnI sequences are less frequently repeated in the genome and have a characteristic localization in chromosomes.

PCR is actively used to identify polymorphic molecular markers when constructing genetic linkage maps, the basic principles of which were discussed above. This method is also useful in DNA sequencing, as well as in the construction of high-resolution physical maps for the human genome. The last two areas of application of PCR will be discussed in more detail below.

Low resolution physical maps

In contrast to the genetic linkage maps discussed above, physical maps of the genome reflect the real distance between markers, expressed in base pairs. Physical maps differ in their degree of resolution, i.e. on the details of the genome structure that are presented on them. A comprehensive physical map of the human genome of maximum resolution will contain the complete nucleotide sequence of all his chromosomes. At the other extreme of physical maps with minimal resolution are chromosomal (cytogenetic) maps of the genome.

Four types of genetic maps of genomic DNA and their relationship

1 - genetic linkage map, 2 - physical restriction map, spaces indicate the sites of DNA cleavage by restriction enzymes, 3 - physical map of contigs, showing overlapping DNA clones obtained using YAC vectors, 4 - comprehensive physical map in the form of DNA nucleotide sequence. All maps show the same chromosome region

Chromosome maps. Chromosomal maps of the human genome are obtained by localizing genetic markers on individual chromosomes using cytogenetic methods, including autoradiography and FISH. In the last two cases, radioactive or fluorescent labels associated with the studied genetic loci of intact chromosomes are detected using light microscopy. Not so long ago, chromosome maps made it possible to localize the studied DNA fragment on a chromosome 10 mp in length. Modern in situ hybridization methods using metaphase chromosomes, mainly the FISH method, localize polynucleotide markers within 2–5 bp. Moreover, during in situ hybridization with interphase chromosomes, in which the genetic material is in a less compact form, the resolution of chromosome maps approaches 100 kbp.

The accuracy of chromosome maps is also improved with the use of modern genetic methods. For example, the ability of PCR to amplify DNA segments of a single spermatozoon makes it possible to study a large number of meiosis, as it were, conserved in individual sperm samples. As a result, it becomes possible to check the relative position of genetic markers localized on chromosome maps by more crude methods.

CDNA maps... CDNA maps reflect the position of the expressed DNA regions (exons) relative to known cytogenetic markers (bands) on metaphase chromosomes. Since such maps provide an idea of \u200b\u200bthe localization of transcribed regions of the genome, including genes with unknown functions, they can be used to search for new genes. This approach is especially useful when searching for genes whose damage causes human diseases, if the approximate localization of such chromosome regions has already been previously carried out on genetic linkage maps as a result of family genetic analysis.

High resolution physical maps

Two Strategies for Building Physical DNA Maps

a - “top-down” strategy: the DNA of the whole chromosome is cleaved by large-cleavage restriction enzymes, a restriction map is constructed for each of the individual DNA fragments; b - bottom-up strategy, individual YAC clones are combined into contigs after identification

In attempts to build high-resolution human genome maps, two alternative approaches have been experimentally implemented, called top-down mapping and bottom-up mapping. When mapping from top to bottom, the initial analysis is a DNA preparation of an individual human chromosome. DNA is cut with large-cleavage restriction enzymes (for example, NotI) into long fragments, which, after separation by electrophoresis in a pulsed electric field, are subjected to further restriction analysis with other restriction enzymes. As a result, a macrorestriction map is obtained, on which all the sequences of the studied chromosome or its part are sufficiently fully represented, but its resolution is low. It is very difficult to localize individual genes on such a map. In addition, each individual map rarely covers extended DNA segments (as a rule, no more than 1–10 mp).

When mapping the human genome from bottom to top, based on the preparation of the total DNA of the genome or individual chromosome, a series of random clones of extended DNA sequences (10–1000 kb) are obtained, some of which overlap with each other. In this case, artificial mini-chromosomes of bacteria (BAC) or yeast (YAC) are often used as a vector for cloning, described in detail in section 7.2.4. A series of partially overlapping and complementary clones form a contiguous DNA nucleotide sequence called a contig. The correctness of the obtained contigs is confirmed by in situ hybridization (FISH) with their simultaneous binding to certain regions of the studied chromosomes. Contig-based maps provide complete information about the structure of individual chromosome segments and allow you to localize individual genes. However, such maps are difficult to use for the reconstruction of entire chromosomes or their extended sections due to the absence of the corresponding clones in the existing clone libraries of genes.

The main problem that has to be solved when using both approaches to the construction of high-resolution physical maps is the unification of scattered DNA fragments into contiguous nucleotide sequences. Most often, special cloned DNA fragments are used for this, called linking clones. DNA fragments from binding clones contain nucleotide sequences of large-cleavage restriction endonucleases in their inner parts and, therefore, represent the junctions of DNA fragments used in the first stages of physical mapping. By Southern hybridization, during which DNA fragments of binding clones are used as probes, DNA fragments of physical maps containing nucleotide sequences in the vicinity of the restriction sites of large-cleavage restriction endonucleases are determined. If two such fragments are found, then the corresponding linking clone overlaps both of these fragments and is a part of them. Binding clones, in turn, are selected from gene banks using probes, which are nucleotide sequences of large-cleavage restriction enzyme restriction sites.

LIST USED SOURCES

1) Clark M.S. Comparative genomics: The key to understanding the Human Genome Project // BioEssays. 1999. Vol. 21. P. 21-30.

2) Billings P.R., Smith C.L., Cantor C.L. New techniques for physical mapping of the human genome // FASEB J. 1991. Vol. 5. P. 28–34.

3) Georgiev G.P. Genes of higher organisms and their expression. Moscow: Nauka, 1989.254 p.

4) http://referatwork.ru/refs/source/ref-8543.html

Soon after the rediscovery of Mendel's laws, the German cytologist Theodor Boveri (1902) presented evidence in favor of the participation of chromosomes in the processes of hereditary transmission, showing that the normal development of the sea urchin is possible only if all chromosomes are present. At the same time (1903), the American cytologist William Setton drew attention to the parallelism in the behavior of chromosomes in meiosis and hypothetical factors of heredity, the existence of which had already been predicted by Mendel himself.

William Setton suggested that several genes can be found on one chromosome. In this case, there should be linked inheritance of traits, i.e. several different traits can be inherited as if they were controlled by a single gene. In 1906, W. Batson and R. Pennett discovered linked inheritance in sweet peas. They studied joint inheritance: flower colors (purple or red) and pollen grain shapes (elongated or round). When crossing diheterozygotes in their offspring, a splitting of 11.1: 0.9: 0.9: 3.1 was observed instead of the expected 9: 3: 3: 1. It seemed that the pollen color and shape factors tend to stay together when the inclinations recombine. The authors called this phenomenon "mutual attraction of factors", but they failed to find out its nature.

Further study of chromosomes as carriers of information took place in the first decades of the twentieth century in the laboratory of Thomas Hunt Morgan (USA) and his collaborators (A. Sturtevant, C. Bridges, G. Möller). Morgan used the fruit fly Drosophila melanogaster as his main object of research, which turned out to be a very convenient model object:

- Firstly, this fly is easily cultivated in laboratory conditions.

- Secondly, it is characterized by a small number of chromosomes (2 n \u003d 8).

- Thirdly, in the salivary glands of Drosophila larvae there are giant (polytene) chromosomes that are convenient for direct observation.

- And, finally, Drosophila is distinguished by high variability of morphological characters.

Based on experiments with the fruit fly Drosophila Morgan and his students, the chromosomal theory of heredity was developed.

The main provisions of the chromosomal theory of heredity:

1. Gene - it is an elementary hereditary factor (the term "elementary" means "indivisible without loss of quality"). A gene is a section of a chromosome that is responsible for the development of a specific trait. In other words, genes are located on chromosomes.

2. One chromosome can contain thousands of genes arranged in a linear fashion (like beads on a string). These genes form linkage groups. The number of linkage groups is equal to the number of chromosomes in the haploid set. A collection of alleles on one chromosome is called a haplotype. Examples of haplotypes: ABCD (only dominant alleles), abcd (only recessive alleles), AbCd (various combinations of dominant and recessive alleles).

3. If genes are linked to each other, then there is an effect of linked inheritance of traits, i.e. several traits are inherited as if they were controlled by a single gene. With linked inheritance, the original combinations of traits are preserved in a succession of generations.

4. The linkage of genes is not absolute: in most cases, homologous chromosomes exchange alleles as a result of crossing (crossing over) in the prophase of the first meiotic division. As a result of crossing over, crossover chromosomes are formed (new haplotypes appear, i.e. new combinations of alleles.). With the participation of crossover chromosomes in subsequent generations, new combinations of traits should appear in crossover individuals.

5. The likelihood of new combinations of traits due to crossing over is directly proportional to the physical distance between genes. This allows you to determine the relative distance between genes and build genetic (crossover) maps of different types of organisms.

CROSSINGOVER

Crossover (from the English crossing-over - crossing) is a process of exchange of homologous regions of homologous chromosomes (chromatids).

Crossover usually occurs in meiosis I.

When crossing over, there is an exchange of genetic material (alleles) between chromosomes, and then recombination occurs - the appearance of new combinations of alleles, for example, AB + ab → Ab + aB.

Break-Reunion Crossover Mechanism

According to the Janssens – Darlington theory, crossing over occurs in the prophase of meiosis. Homologous chromosomes with AB and ab chromatids form bivalents. In one of the chromatids in the first chromosome, a rupture occurs in the A – B region, then in the adjacent chromatid of the second chromosome, a rupture occurs in the a – b region. The cell seeks to repair the damage with the help of repair-recombination enzymes and attach fragments of chromatids. However, in this case, it is possible to join crosswise (crossing over), and recombinant chromatids Ab and aB are formed. In the anaphase of the first division of meiosis, there is a divergence of two-chromatid chromosomes, and in the second division, a divergence of chromatids (one-chromatid chromosomes). Chromatids that did not participate in crossing over retain the original combinations of alleles. Such chromatids (one-chromatid chromosomes) are called non-crossover; with their participation, non-crossover gametes, zygotes, and individuals will develop. Recombinant chromatids that are formed during crossing over carry new combinations of alleles. Such chromatids (one-chromatid chromosomes) are called crossover; with their participation, crossover gametes, zygotes and individuals will develop. Thus, due to crossing over, recombination occurs - the appearance of new combinations of hereditary inclinations in chromosomes.

According to other theories, crossing over is associated with DNA replication: either in pachytene of meiosis, or in interphase. In particular, it is possible to change the matrix in the replication fork.

Genetic (crossover) maps

Alfred Sturtevant (Morgan's collaborator) suggested that the frequency of crossing over between genes located on the same chromosome may serve as a measure of the distance between genes. In other words, the crossover frequency, expressed as the ratio of the number of crossover individuals to the total number of individuals, is directly proportional to the distance between genes. The crossover frequency can then be used to determine the relative position of genes and the distance between genes. The unit of distance between genes is 1% crossing over; in honor of Morgan, this unit is called morganida (M).

Based on genetic mapping, genetic maps - diagrams reflecting the position of genes in chromosomes relative to other genes. On genetic maps, the extreme gene (i.e., the most distant from the centromere) corresponds to the zero (initial) point. The remoteness of a gene from the zero point is indicated in morganids.

The construction of genetic maps of various organisms is of great importance in health care, breeding and ecology. When studying human traits (and in particular, genetic diseases), it is important to know which gene determines the trait in question. This knowledge makes it possible to make predictions in medical and genetic counseling, in the development of methods for treating genetic diseases, incl. and for genome correction. Knowledge of the genetic maps of cultivated plants and domestic animals allows planning the breeding process, which contributes to obtaining reliable results in a short time. The construction of genetic maps of wild plants and wild animals is also important from the point of view of ecology. In particular, the researcher gets the opportunity to study not just phenotypic traits of organisms, but specific, genetically determined traits.

Double and multiple crossing over

Morgan suggested that crossing over between two genes can occur not only at one, but also at two or even more points. An even number of crosses between two genes, ultimately, does not lead to their transfer from one homologous chromosome to another; therefore, the number of crossing over and, consequently, the distance between these genes, determined in the experiment, decreases. This usually refers to genes located far enough from each other. Naturally, the probability of a double cross is always less than the probability of a single cross. In principle, it will be equal to the product of the probability of two single acts of recombination. For example, if a single cross will occur with a frequency of 0.2, then a double cross - with a frequency of 0.2 × 0.2 \u003d 0.04. Later, along with double crossing over, the phenomenon of multiple crossing over was also discovered: homologous chromatids can exchange regions at three, four, or more points.

Interference - This is the suppression of crossing over in the areas immediately adjacent to the point of the exchange that took place.

Consider the example described in one of Morgan's early works. He investigated the frequency of crossing over between genes w (white - white eyes), y (yellow - yellow body) and m (miniature - small wings), localized on the X chromosome of D. melanogaster. The distance between genes w and y in percentage of crossing over was 1.3, and between genes y and m - 32.6. If two crossover events are observed by chance, then the expected double crossing over frequency should be equal to the product of the crossing over frequencies between the y and w genes and the w and m genes. In other words, the double crossover rate will be 0.43%. In fact, only one double crossing over was found in the experiment per 2205 flies, that is, 0.045%. Morgan's student G. Möller proposed to determine the intensity of interference quantitatively by dividing the actually observed double crossing over frequency by the theoretically expected (in the absence of interference) frequency. He called this indicator the coefficient of coinci- dence, that is, coincidence. Möller showed that in the X chromosome of Drosophila the interference is especially great at short distances; with an increase in the interval between genes, its intensity decreases and at a distance of about 40 morganids and more, the coefficient of co-incidence reaches 1 (its maximum value).

Cytological evidence of crossing over

Direct cytological evidence of the exchange of parts of chromosomes during crossing-over was obtained in the early 1930s in Drosophila and maize.

Consider Stern's experiment on D. melanogaster. Usually, two homologous chromosomes are morphologically indistinguishable. Stern examined X chromosomes, which had morphological differences and, therefore, were not completely homologous. However, the homology between these chromosomes was preserved for most of their length, which allowed them to mate normally and segregate in meiosis (that is, to be normally distributed among daughter cells). One of the X-chromosomes of the female as a result of translocation, i.e., the movement of a fragment of the Y-chromosome, acquired an L-shaped form. The second X chromosome was shorter than the normal one, since part of it was transferred to chromosome IV. Females were obtained that were heterozygous for the indicated two, morphologically different, X-chromosomes, as well as heterozygous for two genes localized on the X-chromosome: Bar (B) and carnation (cr). Gene Bar Is a semi-dominant gene that affects the number of facets and, therefore, the shape of the eye (mutants with the B allele have stripe eyes). The cr gene controls eye coloration (the cr + allele causes normal eye coloration, and the cr allele determines the color of the red carnation eyes). The L-shaped X chromosome carried the wild-type B + and cr + alleles, the truncated chromosome carried the B and cr mutant alleles. Females of the indicated genotype were crossed with males with a morphologically normal X chromosome with the cr and B + alleles. In the offspring of females, there were two classes of flies with non-crossover chromosomes (crB / crB + and cr + B + / crB +) and two classes of flies whose phenotype corresponded to crossovers (crB + / crB + and cr + B / crB +). A cytological study showed that the crossover individuals exchanged sections of the X chromosomes, and, accordingly, their shape changed. All four classes of females had one normal, i.e., rod-shaped, chromosome received from the father. Crossover females contained in their karyotype X chromosomes transformed as a result of crossing over - a long rod-shaped or two-armed with short shoulders. These experiments, as well as simultaneously obtained similar results on corn, confirmed the hypothesis of Morgan and his co-workers that crossing over is an exchange of regions of homologous chromosomes and that genes are indeed localized on chromosomes.

Somatic (mitotic) crossing over.

In somatic cells, exchanges sometimes occur between chromatids of homologous chromosomes, as a result of which a combinative variability is observed, similar to that which is regularly generated by meiosis. Often, especially in Drosophila and lower eukaryotes, homologous chromosomes are synapsed in mitosis. One of the autosomal recessive mutations in humans, in a homozygous state, leading to a serious disease known as Blum's syndrome, is accompanied by a cytological picture that resembles the synapse of homologues and even the formation of chiasmata.

Evidence for mitotic crossing over was obtained on Drosophila when analyzing the variability of traits determined by genes in (yellow - yellow body) and sn (singed - singed bristles), which are located on the X chromosome. A female with genotype y sn + / y + sn is heterozygous for genes y and sn, and therefore, in the absence of mitotic crossing over, her phenotype will be normal. However, if crossing over occurs at the stage of four chromatids between chromatids of different homologues (but not between sister chromatids), and the exchange site is between the sn gene and the centromere, then cells with genotypes y sn + / y + sn + and y + sn / y + sn are formed. In this case, on the gray body of a fly with normal bristles, twin mosaic spots will appear, one of which will be yellow with normal bristles, and the other gray with scorched bristles. For this, it is necessary that after crossing over, both chromosomes (former chromatids of each of the homologues) y + sn moved to one pole of the cell, and chromosomes y sn + to the other. Descendants of daughter cells, multiplying at the pupal stage, and will lead to the appearance of mosaic spots. Thus, mosaic spots are formed when two groups (more precisely, two clones) of cells are located next to each other, phenotypically differing from each other and from the cells of other tissues of a given individual.

Unequal crossing over

This phenomenon was studied in detail using the example of the Bar gene (B - stripe eyes), localized on the X chromosome of D. melanogaster. Unequal crossing over is associated with the duplication of a site in one of the homologues and its loss in another homolog. It was found that gene B can be present in the form of tandem, that is, following one after another, repeats consisting of two or even three copies. Cytological analysis confirmed the assumption that unequal crossing over can lead to tandem duplications. In the region corresponding to the localization of the B gene, an increase in the number of disks proportional to the gene dose was noted on the polytene chromosome preparations. It is assumed that in evolution, unequal crossing over stimulates the creation of tandem duplications of different sequences and their use as raw genetic material for the formation of new genes and new regulatory systems.

Crossover regulation

Crossover Is a complex physiological and biochemical process that is under the genetic control of a cell and is influenced by environmental factors. Therefore, in a real experiment, we can talk about the crossing-over frequency, bearing in mind all the conditions in which it was determined. There is practically no crossover between heteromorphic X and Y chromosomes. If it happened, then the chromosomal mechanism of sex determination would be constantly destroyed. Blocking of crossing over between these chromosomes is associated not only with the difference in their size (it is not always observed), but also due to Y-specific nucleotide sequences. A prerequisite for the synapse of chromosomes (or their sections) is the homology of nucleotide sequences.

The vast majority of higher eukaryotes are characterized by approximately the same crossing over frequency in both homogametic and heterogametic sexes. However, there are species in which Crossover is absent in individuals of the heterogametic sex, while in individuals of the homogametic sex it proceeds normally. This situation is observed in heterogametic Drosophila males and silkworm females. It is significant that the frequency of mitotic crossing over in these species in males and females is practically the same, which indicates different control elements for individual stages of genetic recombination in germ and somatic cells. In heterochromatic regions, in particular pericentromeric regions, the frequency of crossing over is reduced, and therefore the true distance between genes in these regions can be changed.

Genes have been found that function as crossover blockers, but there are also genes that increase its frequency. They can sometimes induce a noticeable number of crossovers in male Drosophila. Chromosomal rearrangements, in particular inversions, can also act as crossover inhibitors. They disrupt the normal conjugation of chromosomes in the zygotene.

It was found that the frequency of crossing over is influenced by the age of the organism, as well as by exogenous factors: temperature, radiation, salt concentration, chemical mutagens, drugs, hormones. With most of these influences, the frequency of crossing over increases.

In general, crossing over is one of the regular genetic processes controlled by many genes, both directly and through the physiological state of meiotic or mitotic cells. The frequency of various types of recombinations (meiotic, mitotic crossing over, and sister chromatid exchanges) can serve as a measure of the action of mutagens, carcinogens, antibiotics, etc.

The biological significance of crossing over

Thanks to linked inheritance, successful combinations of alleles are relatively stable. As a result, groups of genes are formed, each of which is like a single supergene that controls several traits. At the same time, during crossing over, recombinations occur - i.e. new combinations of alleles. Thus, crossing over increases the combinative variability of organisms.

The evolutionary meaning of linked inheritance. As a result of linkage, one chromosome can contain both favorable alleles (for example, A) and neutral or relatively unfavorable ones (for example, N). If a certain haplotype (for example, AN) increases the fitness of its carriers due to the presence of favorable A alleles, then both favorable alleles and neutral or relatively unfavorable N linked to them will accumulate in the population.

Example. The AN haplotype has an advantage over the “wild type” (++) haplotype due to the presence of a favorable allele A, and then the N allele will accumulate in the population if it is selectively neutral or even relatively unfavorable (but its negative effect on fitness is compensated by the positive effect of allele A ).

The evolutionary significance of crossing over. As a result of crossing over, unfavorable alleles, initially linked to favorable ones, can pass to another chromosome. Then new haplotypes arise that do not contain unfavorable alleles, and these unfavorable alleles are eliminated from the population.

Example. The Al haplotype turns out to be unfavorable in comparison with the "wild type" (++) haplotype due to the presence of the lethal allele l. Therefore, allele A (favorable, neutral ooze somewhat reducing fitness) cannot manifest itself in the phenotype, since this haplotype (Al) contains the lethal allele l. As a result of crossing over, the recombinant haplotypes A + and + l appear. Haplotype + l is eliminated from the population, and haplotype A + is fixed (even if allele A somewhat reduces the fitness of its carriers).

ADDITIONS

Principles of genetic mapping

At the first stage of mapping, it is necessary to determine the belonging of a gene to a linkage group. The more genes are known in a given species, the more accurate the mapping results. All genes are divided into linkage groups. The number of linkage groups corresponds to the haploid set of chromosomes. For example, D. melanogaster has 4 linkage groups, corn has 10, mice have 20, and humans have 23 linkage groups. As a rule, the number of genes in linkage groups depends on the linear dimensions of the corresponding chromosomes. So, a fruit fly has one (IV) point (when analyzed under a light microscope) chromosome. Accordingly, the number of genes in it is many times less than in the others, significantly exceeding it in length. It should also be noted that in the heterochromatic regions of chromosomes there are no genes or almost none, therefore, extended regions of constitutive heterochromatin can somewhat change the proportionality of the number of genes and the length of the chromosome.

If the chromosomes are long enough, then the gene removal from the zero point can exceed 50 M - then a contradiction arises between the distances marked on the map, exceeding 50%, and the position postulated above, according to which 50% of the crossovers obtained in the experiment should actually mean the absence of linkage. i.e. e. localization of genes in different chromosomes. This contradiction is explained by the fact that when compiling genetic maps, the distances between the two closest genes are summed up, which exceeds the experimentally observed percentage of crossing over.

Cytogenetic mapping

This method is based on the use of chromosomal rearrangements. In the case of giant polytene chromosomes, it allows direct comparison of the results of genetic analysis of the distances between the studied loci and their mutual arrangement with data on the physical sizes of certain chromosomal regions. Irradiation and the action of other mutagens in chromosomes often result in deletions (deletions) or insertions of small fragments comparable in size to one or more loci. For example, you can use heterozygotes for chromosomes, one of which will carry a group of successive dominant alleles, while a homologous to it will carry a group of recessive forms of the same genes. If a chromosome with dominant genes consistently loses individual loci, then recessive traits will appear in the heterozygote. The order in which recessive traits appear indicates the sequence in which genes are located.

With the order of the AbC genes, in the case of a deletion capturing gene C, in flies with a truncated chromosome that has lost a fragment equal to gene C, alleles c, b, and A will appear in the phenotype.

In general, a comparison of genetic (crossing over) and cytological maps shows their correspondence: the greater the percentage of crossing over separates a pair of genes, the greater the physical distance between them. However, the discrepancy between the distances determined by these two methods can be influenced by two factors. First, these are areas where crossing over is difficult or absent (for example, in heterochromatic areas); secondly, the physical distance will be greater than the genetic one if the genes are separated by a zone of "silent" DNA. Bridges' calculations showed that each crossover unit on the map of polytene chromosomes of the salivary glands of D. melanogaster corresponds to 4.2 μm in length of polytene chromosomes. This length is at least equal to two to three average genes.

Features of constructing genetic maps in prokaryotes

To build genetic maps in prokaryotes, the phenomenon of conjugation is used - the transfer of genetic material from one cell to another with the help of special circular DNA molecules (plasmids, in particular, with the help of an F-plasmid).

The probability of transfer of a particular gene into a recipient cell depends on its removal from the F – plasmid DNA, or rather, from the point O, at which replication of the F – plasmid DNA begins. The longer the conjugation time, the higher the likelihood of the transfer of a given gene. This makes it possible to create a genetic map of bacteria in minutes of conjugation. For example, in E. coli, the thr gene (an operon of three genes that control threonine biosynthesis) is located at the zero point (that is, directly next to the F-plasmid DNA), the lac gene is transferred after 8 minutes, the recE gene - after 30 minutes, the argR gene - after 70 minutes, etc.

This issue will be considered in more detail when studying the genetics of prokaryotes.

Human chromosome mapping

Gene mapping is based on linkage grouping. The more mutations are known and the fewer the number of chromosomes, the easier it is to map. In this regard, a person (in addition to the fact that he cannot have classical hybridological analysis) as an object is doubly unfavorable for mapping: he has relatively few known genes (at least it was so until the end of the 70s), and the haploid number of chromosomes is quite large - 22 (excluding sex). This means that the probability that two newly discovered genes will be linked is 1/22. For these reasons, pedigree analysis, which to some extent replaces hybridological analysis, provides rather limited information on the nature of the linkage.

Methods of somatic cell genetics turned out to be more promising for mapping human genes. The essence of one of them is as follows. Cellular engineering techniques allow different types of cells to be combined. The fusion of cells belonging to different biological species is called somatic hybridization. The essence of somatic hybridization is to obtain synthetic cultures by fusion of protoplasts of various types of organisms. Various physicochemical and biological methods are used for cell fusion. After fusion of protoplasts, multinucleated heterokaryotic cells are formed. Subsequently, during the fusion of nuclei, synkaryotic cells are formed, containing chromosome sets of different organisms in the nuclei. When such cells divide in vitro, hybrid cell cultures are formed. Currently obtained and cultivated cell hybrids "human × mouse", "human × rat" and many others.

In hybrid cells derived from different strains of different species, one of the parental chromosome sets, as a rule, replicates faster than the other. Therefore, the latter gradually loses chromosomes. These processes are intensively occurring, for example, in cell hybrids between mice and humans - species that differ in many biochemical markers. If at the same time follow any biochemical marker, for example the enzyme thymidine kinase, and simultaneously carry out cytogenetic control, identifying chromosomes in clones formed after their partial loss, then, in the end, the disappearance of a chromosome can be associated simultaneously with a biochemical trait. This means that the gene encoding this trait is localized on this chromosome. So, the thymidine kinase gene in humans is located on chromosome 17.

Some information about the localization of genes can be obtained by analyzing the numerical and structural mutations of chromosomes, by the occurrence in families of chromosomes with morphological variations, and by taking into account hereditary traits. Partial monosomies resulting from deletions are also used for the same purpose. However, in these cases, it should be borne in mind that sometimes the gene under study remains in the centric fragment, but its manifestation can be drastically weakened as a result of the position effect or some other regulatory mechanisms (change in the order of replication, detachment of the promoter region, etc.) ... In the late 60s, an in situ hybridization method was developed, which is based on the specificity of complementary interactions between a gene and its copy (mRNA, as well as complementary DNA obtained by reverse transcription). The resolution of this method is much higher on polytene chromosomes than on human mitotic chromosomes, but it is constantly being improved.

Gene mapping gene mapping, mapping - gene mapping.

Determination of the position of a given gene on a chromosome relative to other genes; use three main groups of methods K.g. - physical (determination using restriction maps, electron microscopy and some variants of electrophoresis of intergenic distances - in nucleotides), genetic (determination of the frequencies of recombinations between genes, in particular, in family analysis, etc.) and cytogenetic (hybridization in situ<in situ hybridization\u003e, obtaining monosomal cell hybrids<monochromosomal cell hybrid\u003e, deletion method<deletion mapping\u003e etc.); in human genetics, 4 degrees of reliability of the localization of this gene are accepted - confirmed (established in two or more independent laboratories or on the material of two or more independent test objects), preliminary (1 laboratory or 1 analyzed family), contradictory (discrepancy between data from different researchers), doubtful (not finalized data from one laboratory); Appendix 5 provides a summary (as of 1992-93) of structural genes, oncogenes, and pseudogenes in human genomes and - including some mutations - in mice.

(Source: "The English-Russian Explanatory Dictionary of Genetic Terms." Arefiev V.A., Lisovenko L.A., Moscow: VNIRO Publishing House, 1995)

See what "gene mapping" is in other dictionaries:

gene mapping - Determination of the position of a given gene on a chromosome relative to other genes; use three main groups of methods K.g. physical (determination using restriction maps, electron microscopy and some variants of electrophoresis ... ...

Gene mapping - determination of the position of a given gene on a chromosome relative to other genes. Genetic mapping involves determining the distances by the frequency of recombinations between genes. Physical mapping uses some techniques ... ... Dictionary of Psychogenetics

mapping [genes] using backcrossing - Genetic mapping method based on obtaining backcross hybrids of related forms and analysis of cleavage of allele variants polymorphic in length of restriction fragments; this method is most widespread in gene mapping in ... ... Technical translator's guide

Backcross mapping mapping [genes] using backcrossing. Genetic mapping method based on obtaining backcross hybrids of related forms and analysis of splitting of variants of alleles polymorphic in length restriction ... ...

Mapping comparative genes in mammals - * cartovanne paranal genes of mammals * comparative mapping of mammalian genes informative comparison of genetic maps of humans and any of other mammalian species). They must be both well studied and far from each other ...

Mapping - * cartovanne * mapping establishing the positions of genes or some specific sites (see) along the DNA strand (. Map) ... Genetics. encyclopedic Dictionary

Mapping with irradiated hybrids [cells] - * a map of the dapamogay of the applied hybrydў [cell] * radiated hybrid mapping modification of the gene mapping method using hybridization of somatic cells. Cells of the hybrid clone "rodent H human" containing only chromosome 1 ... ... Genetics. encyclopedic Dictionary

Radiation hybrid mapping using irradiated hybrids [cells]. Modification of the gene mapping method using hybridization of somatic cells cells of the hybrid clone “rodent ˟ human” containing only 1 chromosome ... ... Molecular biology and genetics. Explanatory dictionary.

Establishing the order of the genes and the relative distance between them in the linkage group ... Big Medical Dictionary

Mapping the human genome

We have no need to disturb the gods in vain -

There are the insides of the victims to guess about the war,

Slaves to be silent and stones to build!

Osip Mandelstam, "Nature is the same Rome ..."

Genetics is a young science. The evolution of species was really discovered only in the late 50s of the XIX century. In 1866, the Austrian monk Gregor Mendel published the results of his experiments on pollination of peas. Until the end of the century, no one paid attention to its discovery. And Galton, for example, never found out about them. Even the mechanism of fertilization - the fusion of the nuclei of male and female germ cells - was discovered only in 1875. In 1888, little bodies called chromosomes were found in the nuclei of cells, and in 1909 Mendelian factors of inheritance were named genes. The first artificial insemination (in a rabbit and then in monkeys) was carried out in 1934; and finally, in 1953, a fundamental discovery was made - the double helical structure of DNA was established. As you can see, all this happened quite recently, so that early eugenicists, in general, were very little aware of the technique of their craft.

The mapping of the human genome is still in its early stages. What we know is a tiny fraction of what we do not know. There are three billion nucleotide sequences that form twenty-six to thirty-eight thousand genes that directly code for proteins. But how genes and the proteins they produce interact is still poorly understood.

However, the role of genes in human society is quickly recognized. In 1998, Diana Paul (University of Massachusetts) recalled what fourteen years ago she called

The “biologically deterministic” view, according to which genes influence differences in intelligence and temperament — using these terms as if their meaning had been specified. Today, their use would be controversial, as these labels seem to call this point of view into question, while it is widely accepted by both scientists and the public. ".

Be that as it may, our knowledge is replenished literally every day, and in the very near future we will be able to analyze with great accuracy genetic load,which we impose on future generations.

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PART II. HUMAN GENOME FUNCTION THE QUEEN IS DIED - HAIL THE QUEEN! What we know is limited, and what we don’t know is infinite. P. Laplace Science is always wrong. She will never solve an issue without raising a dozen new ones. B. Shaw So,

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PART III. ORIGIN AND EVOLUTION OF THE HUMAN GENOME

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How different is the human genome from the chimpanzee? A genome is a collection of genes contained in a haploid (single) set of chromosomes of a given organism. The genome is not a characteristic of an individual, but of a species of organisms. In February 2001 in American

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Chapter 11 Deciphering the Human Genome What will you say when, climbing with the last of your strength to the top of a mountain that no one has ever visited, you suddenly see a person climbing up a parallel path? In science, cooperation is always much more fruitful,