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Genetic mapping. Gene mapping methods. Sevostyanova Natalia Vladimirovna Doctor of Medical Sciences, Professor of the Department of Biology and Genetics

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Lecture plan Gene mapping. Chromosome maps. Cytological maps. Gene mapping methods. Testing mutations for allelism. Chromosomal mutations.

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Genetic mapping is the determination of the position of the mapped gene relative to other genes on a given chromosome. The more genes known in a given species, the more accurate the results.

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The genetic map of a chromosome is a diagram of the relative arrangement of genes located in the same linkage group. As is known, D. melanogaster has four pairs of chromosomes in its diploid set.

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Such a map can be compiled only for objects in which a large number of mutant genes have been studied. For example, in Drosophila, over 500 genes localized in 4 linkage groups have been identified. Group I - sex chromosomes (XX - females, XY - males), II, III, IV - autosomes.

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Corn has over 400 genes distributed in 10 linkage groups

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To compile genetic maps of chromosomes, it is necessary to identify many mutant genes and conduct numerous crosses.

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Genetic chromosome maps are compiled for each pair of homologous chromosomes. Clutch groups are numbered sequentially as they are discovered. The full or abbreviated names of mutant genes and their distances in morganids are also indicated. Indicates the location of the centromere.

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In less studied objects, the number of detected linkage groups is less than the haploid number of chromosomes. Bacteria, which are haploid organisms, have one, most often continuous, circular chromosome and all genes form one linkage group. Genetic map of the E. coli chromosome.

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Methods of gene mapping Physical determination using restriction maps of electron microscopy of electrophoresis variants of intergenic distances - in nucleotides Genetic determination of recombination frequencies between genes, in particular, in family analysis, etc. Cytogenetic in situ hybridizations, production of monochromosomal cell hybrids, deletion method, etc.

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Mutation allelism testing A functional allelism test that allows you to determine whether mutant alleles belong to the same locus or to different ones. Hybrids (heterokaryons) are obtained in which the two mutations under study are located on different homologous chromosomes - Trans position.

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If both mutations act on different independent functions (affect two different genes), then such a hybrid has a wild phenotype, since a diheterozygote is formed in which normal alleles dominate over the mutant ones. If the mutations under study affect the same function (damage the same gene), then the hybrid should have a mutant phenotype.

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Cis test - hybrids are obtained in which both of the studied mutations were introduced by one of the parents, while the chromosomes of the others contain normal alleles. Hybrids with cis mutations should have a wild-type phenotype, regardless of whether the mutations being tested are in the same or different genes. This is the reason why the cis test is rarely used.

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Meiotic and mitotic crossing over are used to construct a genetic map of eukaryotic chromosomes. Comparison of genetic maps of chromosomes constructed by different methods in the same species reveals the same order of gene arrangement, although the distance between specific genes on meiotic and mitotic genetic maps of chromosomes may vary. Some of these errors can be observed using cytogenetic methods

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Meiotic crossing over is difficult process, during which errors are possible. Crossover exchange is carried out according to the break-reunion type. A cytological illustration of this mechanism is meiotic crossing over between differently colored sister chromatids. Sometimes chromatid reunification does not occur correctly, and this can lead to the formation of dicentric chromosomes and acentric fragments.

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Normally, the genetic maps of chromosomes in eukaryotes are linear. When constructing genetic maps of chromosomes in heterozygotes for translocation, a genetic map of chromosomes is obtained in the form of a cross. This indicates that the shape of the maps reflects the nature of chromosome conjugation.

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Crossover exchanges involving errors in chromatid reunification are called U exchanges. U exchanges are found in many plant and animal species. They have been studied in most detail in rye (Jones, Brumpton, 1971). The frequency of U-configurations in rye can reach 30-40% per cell, or 4-5% per bivalent. The frequency of nonsister U exchanges is significantly higher than that of sister exchanges. Improper chromatid reunification may be one of the factors leading to the formation of unbalanced gametes.

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A cytological map is compiled based on the study of polytene chromosomes, which makes it possible to compare the structure of the synthesized protein with a certain region of the chromosome (genome), since the transcribed region is determined under a microscope in the form of a puff. This makes it possible to determine the location of the gene.

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A cytological map of a chromosome is a photograph or precise drawing of a chromosome that shows the sequence of genes. It is built on the basis of a comparison of the results of analyzing crossings and chromosomal rearrangements. For example, if a chromosome with dominant genes consistently loses individual loci (when exposed to mutagens), then recessive traits will begin to appear in the heterozygote. The order in which traits appear will indicate the sequence of genes.

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The cytological map method is based on the use of chromosomal rearrangements. When irradiated and exposed to mutagens, losses (deletions) or insertions (duplications) of small fragments comparable in size to one or more loci are often observed in chromosomes. For example, you can use heterozygotes on chromosomes, one of which will carry a group of consecutive dominant alleles, and the homologous one will carry a group of recessive alleles of the same ABCDE / abcde genes. If a chromosome with dominant genes has lost individual genes, for example DE, then the ABC/abcde heterozygote will exhibit recessive traits de. The method of overlapping deletions, used in the construction of cytological maps, is based on this principle!!!

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Cytogenetic maps of chromosomes are compiled based on differential coloring (dark and light bands) and mapping genes at individual chromosomal loci.

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Cytogenetic maps provide information about the location of a gene on a chromosome relative to its regions identified by differential staining methods. Thanks to this staining, the chromosome appears “cross-striped” in the field of view of the microscope.

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The location of colored areas (bands) is specific to each chromosome.

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The use of the FISH method makes it possible to construct cytogenetic maps with a resolution of 2-5 Mb, and its modifications for interphase chromosomes - 0.1 Mb. Thus, the localization of a gene mapped using the FISH method can be established with an accuracy of subsegment and locus band.

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Mapping genes using chromosomal mutations Intrachromosomal mutations are the transformation of genetic material within one chromosome. Interchromosomal - rearrangements, as a result of which two non-homologous chromosomes exchange their sections. Chromosomal mutations are changes in the structure of chromosomes

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Inversions Inversions - chromosomal rearrangements associated with the rotation of individual chromosome sections by 180°, were discovered by A. Sturtevant in 1926.

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Paracentric inversion - two chromosome breaks occur, both on the same side of the centromere. In the area between the break points, a 180 turn occurs. Pericentric inversion - the break points are located on both sides of the centromere.

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In individuals heterozygous for inversion, a loop is formed in the chromosomes. In individuals homozygous for inversions, crossing over occurs without changes.

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In heterozygous individuals by paracentric inversion, crossing over is “locked” as follows: in the case of crossover between genes C and D, two products are formed: acentric chromosomes and dicentric chromosomes, i.e. without a centromere and with two centromeres, respectively. Both combinations are lethal.

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The dicentric forms a "chromosomal bridge" in anaphase 1 of meiosis, which is visible under a microscope. Both combinations are lethal. Thus, as a result of crossing over, non-viable gametes are formed, and there are no offspring.

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With pericentric inversion, in the case of crossover between genes C and D, two products are also obtained. Duplication A and deletion F. Each of the resulting chromosomes carries a duplication of one non-inverted chromosome region and a deletion of another. As a result, such gametes are not viable and crossovers are not detected. Just like paracentric inversions, pericentric inversions “lock” crossing over. Since the crossing over in the inverted region of the chromosome is “locked”, blocks of mutations can be formed in it, different from those that are localized in the homologous fragment of the chromosome, but not the inverted one. This phenomenon is called inversion polymorphism of populations.

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Chromosomes with multiple inversions are used to create balancers, i.e., lines that allow the maintenance of lethal mutations and mutations in fertility. One example is line C L B. More reliable balancers, i.e. containing several inversions, are lines Base, Binsn. The construction of balancer chromosomes essentially represents the first example of genetic engineering. Another example of balancers is the Su line (curved wings, lethality), in which a dominant mutation is associated with a long inversion that covers almost the entire second chromosome. In the offspring of crossing heterozygotes for Cy, only flies of the parental classes survive, i.e. the line is balanced, and the lethal f under study is constantly maintained in a heterozygous state.

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The use of deletions to localize genes has been called deletion mapping. Deletions A deletion is the loss of a section of a chromosome. Deletions were discovered in 1917 by K. Bridges using genetic methods. In a normal chromosome, genes are located in a certain order: tABCDEF When a fragment of a chromosome is lost, two fundamental options are possible: ABEF or ABC, i.e. the middle or end part of the chromosome can be lost.

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Translocations Chromosomal rearrangements, as a result of which part of a chromosome is transferred to another place on the same chromosome or to another chromosome. But the total number of genes does not change!!! Translocations were discovered by K. Bridges in 1923 in Drosophila.

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Intrachromosomal translocations occur as a result of the formation of three breaks and the transfer of a chromosomal segment to another region of the same chromosome. Interchromosomal reciprocal translocations arise as a result of the formation of two breaks and the exchange of sections of non-homologous chromosomes.

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Two chromosomes, as a result of reciprocal exchange of fragments, form a heterozygous translocation. If three breaks are formed and a chromosome fragment is removed from one chromosome and inserted into another, this is an insertional translocation.

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The most striking example where a gene has been mapped using translocation is Duchenne myopathy. The Duchenne myopathy gene is located on the X chromosome and usually manifests as severe myopathy in boys. However, several cases of a typical clinical picture of myopathy in women were found. They turned out to be associated with translocations between chromosome X and autosomes, and in chromosome X the break was always localized in the Xp21 region.

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Gene mapping can sometimes be achieved by exploiting the gene dosage effect. In the case of a deletion, a 50% decrease in the gene product (this may primarily be an enzyme) should be expected. It was in this way that the erythrocyte acid phosphatase gene was mapped on chromosome 2.

Gene mapping gene mapping, mapping- gene mapping.

Determining the position of a given gene on any 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 recombination frequencies between genes, in particular, in family analysis, etc.) and cytogenetic (in situ hybridization<in situ hybridization>, obtaining monochromosomal cell hybrids<monochromosomal cell hybrid>, deletion method<deletion mapping> etc.); in human genetics, 4 degrees of reliability of the localization of a given 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 definitively specified data from one laboratory); Appendix 5 provides a summary (as of 1992-93) of structural genes, oncogenes and pseudogenes in the human and - including some mutations - mouse genomes.

(Source: “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 any chromosome relative to other genes; three main groups of K.g. methods are used. physical (determination using restriction maps, electron microscopy and some variants of electrophoresis... ...

Gene mapping- determination of the position of a given gene on any chromosome relative to other genes. Genetic mapping involves determining distances based on recombination frequencies 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 analyzing the splitting of variant alleles that are polymorphic in restriction fragment lengths; This method is most common in mapping genes in... ... Technical Translator's Guide

Backcross mapping [genes] using backcrossing. A genetic mapping method based on obtaining backcross hybrids of related forms and analyzing the splitting of allele variants that are polymorphic in restriction lengths... ...

Comparative gene mapping of mammals- * mapping of mammalian genes * comparative mapping of mammalian genes (informative comparison of genetic maps of humans and any other species of mammals). They must be both well studied and far apart...

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

Mapping using irradiated hybrids [cells]- * mapping of dapamogay apramenennyh hybrids [cells] * radiated hybrid mapping modification of the gene mapping method using somatic cell hybridization. Cells of the hybrid clone “rodent-human” containing only chromosome 1... ... Genetics. encyclopedic Dictionary

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

Establishing the order of location of genes and the relative distance between them in a linkage group... Large medical dictionary

Alfred Sturtevant (Morgan's collaborator) suggested that the frequency of crossing over in a region between genes localized on the same chromosome could serve as a measure of the distance between genes. In other words, the frequency of crossing over, expressed as the ratio of the number of crossover individuals to the total number of individuals, is directly proportional to the distance between genes. Crossing over frequency can then be used to determine the relative positions 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; This unit is called in honor of Morgan Morganida(M), or centimorganide (sM).

At the first stage of mapping, it is necessary to determine whether a gene belongs to a linkage group. The more genes known for 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, at D. melanogaster 4 linkage groups, in corn - 10, in mouse - 20, in humans - 23 linkage groups. If sex chromosomes are present, 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. Thus, the fruit fly has one (IV) point chromosome (when analyzed under a light microscope). Accordingly, the number of genes in it is many times less than in the others, which are significantly longer than it. It should also be noted that in heterochromatic regions of chromosomes there are no or almost no genes, therefore extended areas of constitutive heterochromatin can somewhat change the proportionality of the number of genes and chromosome length.

Based on genetic mapping, genetic maps are compiled. On genetic maps, the outermost gene (i.e., the one furthest from the centromere) corresponds to the zero (starting) point. The distance of a gene from the zero point is indicated in morganids.

If the chromosomes are long enough, then the distance of the gene 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. localization of genes on 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.

After a brief review of the main methods most often used in molecular genetics to study the structure and mechanisms of gene functioning, it seems appropriate, using the example of the human genome, to take a closer look at the practical application of these methods and their modifications for the study of large genomes. In order to comprehensively study the human genome, this colossal repository of genetic information, a special international program “Human Genome Project” has recently been developed and is being implemented. The main goal of the program is to construct comprehensive, high-resolution genetic maps of each of the 24 human chromosomes, which should ultimately culminate in determining the complete primary DNA structure of these chromosomes. Currently, work on the project is in full swing. If it is successfully completed (and this is planned to happen in 2003), humanity 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 control human biology, and to establish the causes of most pathological conditions of its body .

1. Basic approaches to mapping the human genome

Solving the main task of the Human Genome program includes three main stages. At the first stage, it is necessary to specifically divide each individual chromosome into smaller parts, allowing their further analysis using 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 actually determine the primary DNA structure of each of the characterized chromosome fragments and compile 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. The passage of the three above stages is required not only to obtain comprehensive characteristics of the human genome, but also of any other large genome.

1. Genetic linkage maps

Genetic linkage maps represent one-dimensional diagrams of the relative position genetic markers on individual chromosomes. Genetic markers are understood as any heritable phenotypic characteristics that differ among individual individuals. The phenotypic traits that meet the requirements of genetic markers are very diverse. They include both behavioral characteristics or predisposition to certain diseases, as well as morphological characteristics of whole organisms or their macromolecules that differ in structure. With the development of simple and effective methods for studying biological macromolecules, such features, known as molecular markers, have become most often used in the construction of genetic linkage maps. Before moving on to considering methods for constructing such maps and their significance for genome research, it is necessary to recall that the term " clutch" is used in genetics to indicate the probability of joint transmission of two traits from one parent to the offspring.

When sex cells (gametes) are formed in animals and plants at the stage of meiosis, synapsis (conjugation) of homologous chromosomes usually occurs. Sister chromatids of homologous chromosomes are connected along the entire length to each other, and as a result crossing over(genetic recombination between chromatids) their parts are exchanged. The farther two genetic markers are located from each other on the chromatid, the more likely it is that the chromatid break necessary for crossing over will occur between them, and the two markers on the new chromosome belonging to the new gamete will be separated from each other, i.e. their adhesion will be broken. The linkage unit of genetic markers is Morganida(Morgan unit, M), which contains 100 centimorganide(cm). 1 cM corresponds to the physical distance on the genetic map between two markers, recombination between which occurs at a frequency of 1%. Expressed in base pairs, 1 cM corresponds to 1 million bp. (m.b.o.) DNA.

Genetic linkage maps correctly reflect the order of genetic markers on chromosomes, but the resulting distances between them do not correspond to real physical distances. This fact is usually associated with the fact that the efficiency of recombination between chromatids on individual chromosome sections can vary greatly. In particular, it is suppressed in heterochromatic regions of chromosomes. On the other hand, recombination hotspots often occur in chromosomes. Using recombination frequencies to construct physical genetic maps without taking these factors into account will lead to distortions (underestimation or overestimation, respectively) of the actual distances between genetic markers. Thus, genetic linkage maps are the least accurate of all the types of genetic maps available, and can only be considered as a first approximation to actual physical maps. However, in practice, it is they and only they that make it possible to localize complex genetic markers (for example, those associated with symptoms of a 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 located on an individual chromosome would be passed from parents to offspring together, since they are physically linked to each other. Therefore, individual chromosomes form gene linkage groups, and one of the first tasks in constructing genetic linkage maps is to assign the gene or nucleotide sequence under study to a specific linkage group. In table II.4 are listed modern methods, which, according to V.A. McKusick were most often used to construct genetic linkage maps until the end of 1990.