The length of DNA in the nucleus is much greater than the size of the compartment in which it is found. In order to fit into this compartment, the DNA has to be compressed in some way. The degree of DNA concentration is expressed by its packing ratio.
fill rate- DNA length divided by its packaging length.
For example, the shortest human chromosome contains 4.6 x 107bp DNA (approximately 10 times the size of the genome)Escherichia coli). This corresponds to 14,000 µm of extended DNA. In their most condensed state during mitosis, chromosomes are approximately 2 µm long. This gives a fill ratio of 7,000 (14,000/2).
To achieve the overall packing ratio, the DNA is not packaged directly into the final chromatin structure. Instead, it contains multiple organizational hierarchies. Primary packaging is accomplished by wrapping DNA around a protein core to create "bead-like" structures called "beads."nucleosoma. This gives a packing ratio of approximately 6. This structure is invariant in both euchromatin and heterochromatin of all chromosomes. The second level of packing consists of winding the pearls in a helical structure, the so-called30 nm phasesIt is present on interphase chromatin and on mitotic chromosomes. This structure increases the volume ratio to approximately 40. Final packing occurs when fibers organize into rings, scaffolds, and domains, resulting in a final packing ratio of approximately 1,000 for interphase chromosomes and approximately 10,000 for interphase chromosomes. the mitotic chromosomes.
Eukaryotic chromosomes are made up of complexes of DNA and proteins that are compactly organized and allow for the storage of large amounts of DNA in the nucleus. The name of the chromosome subunit is chromatin. The basic unit of chromatin is the nucleosome.
chromatin- chromosome analysis unit; Chromatin reflects the general structure of chromosomes but is not unique to any particular chromosome.
nucleosoma- The simplest DNA packaging structure found in all eukaryotic chromosomes; DNA is wrapped around small octamers of basic proteins called histones. 146 bp wrap around the core, with the remaining bases attached to the next nucleosome; This structure leads to negative supercoiling.
Nucleosomes consist of approximately 200 bp wrapped around a histone octamer containing two copies of histones H2A, H2B, H3, and H4. These are called core histones. Histones are basic proteins with affinity for DNA and are the proteins most commonly associated with DNA. The amino acid sequences of these four histones are conserved, suggesting that all histones have similar functions.
The length of DNA associated with a nucleosomal unit varies by species, but regardless of size, two components of DNA are involved.DNA of Kernis the actual DNA associated with the histone octamer. The value is constant at 146 base pairs. Nuclear DNA forms two loops around the octamer, allowing two regions that are 80 bp apart to be close together. Thus, two distant sequences can interact with the same regulatory protein to control gene expression. The DNA between each histone is called an octamer.linker DNAThe length can vary between 8 and 114 base pairs. This variation is species-specific, but variations in adapter DNA length are also related to the developmental stage of the organism or specific regions of the genome.
The next organizational layer of chromatin consists of 30 nm fibers. It appears to be a magnetic structure with approximately 6 nucleosomes per turn. This gives a packing ratio of 40, ie 40 µm of DNA per 1 µm along the axis. The stability of this structure requires the presence of histone H1, the last member of the histone gene family. Since the experiments in which H1 was removed from chromatin preserved the nucleosomes but not the 30 nm structure, it was concluded that H1 is important in stabilizing the 30 nm structure.
The final packaging stage is characterized by 700 nm structures visible on metaphase chromosomes. Condensed chromatin fragments have a characteristic structure that can be detected in metaphase chromosomes. This appears to be the result of extensive cycling of the DNA in the chromosomes.
The final definition to be proposed is:euchromatinYheterocromatina. When stained with a dye, the chromosomes appear to have alternating light and dark areas. The light regions are euchromatin and contain a single copy of genetically active DNA. Dark stained regions are heterochromatin and contain genetically inactive repeats.
centromeres and telomeres
Centromeres and telomeres are two fundamental features of all eukaryotic chromosomes. Each offers a unique function that is absolutely essential for chromosome stability. Centromeres are required for centromere segregation during meiosis and mitosis, and telomeres provide maximum chromosome stability and ensure their survival.
centromereThey are the condensed regions within chromosomes that are responsible for the precise separation of replicated chromosomes during mitosis and meiosis. When chromosomes are stained, they usually show a dark-stained area called the centromere. During mitosis, the centromere shared by the sister chromatids must split to allow the chromatids to migrate to the poles of the cell. On the other hand, during the first meiosis the centromeres of the sister chromatids must remain intact, while during meiosis II they must function in the same way as during mitosis. Therefore, centromeres are an integral part of chromosome structure and segregation.
Within the centromeric region, most species have multiple sites where spindle fibers composed of DNA and protein attach. The actual place where the appendix occurs is known ascinetocoroComposed of DNA and proteins. The DNA sequences within these regions are calledcendecalcified nucleosaccharide nucleic acid. GOODcenDNA can pass from one chromosome to another and still give the chromosomes the ability to segregate; these sequences must not fulfill any other function.
Normally, CEN DNA is approximately 120 base pairs long and consists of several subdomains.CDE-I,CDE-IIYCDE-III. Mutations in the first two subdomains did not affect segregation, but point mutations in the CDE III subdomain completely abrogated the ability of the centromere to function during chromosome segregation. Therefore, CDE-III must actively participate in the association of the spindle fibers with the centromeres.
Only now are the protein components of the centromeres being characterized. A complex of three proteins calledCBF-IIIIt binds to normal CDE III domains but not to CDE III domains with point mutations that prevent mitotic segregation. Furthermore, mutants of the gene encoding the Cbf-III protein have also abolished the ability of chromosomes to separate during mitosis. To fully understand the mechanism of chromosome segregation, further analyzes of the DNA and protein components of the centromere are required.
telomeresIt is a region of DNA at the ends of linear eukaryotic chromosomes necessary for chromosome replication and stability. McClintock became aware of his special function when he discovered that when two chromosomes break in a cell, the end of one chromosome can join the other chromosome. others and vice versa. He has never observed the joining of broken ends to intact chromosome ends. Therefore, the ends of the broken chromosomes are sticky while the normal ends are not, suggesting that the ends of the chromosomes have unique properties. Often, but not always, telomeric DNA is heterochromatic and contains direct tandem repeats. The following table shows repeat sequences for various species. They are usually of the form (T/A)XGRAMSjwhere x is between 1 and 4 and y is greater than 1.
|slime mold||let's go|
Note that the number of TG sequences and the number of cytokines are different in yeast sequences. At least in the case of yeast, it has been shown that different strains contain telomeres of different lengths and that this length is genetically controlled.
The main difficulty with telomeres is lagging strand replication. Since DNA synthesis requires an RNA template (providing a free 3'-OH group) to initiate DNA replication, which is eventually degraded, resulting in a short single-stranded region. at the end of the chromosome. This region is vulnerable to enzymes that degrade single-stranded DNA. The result is that the chromosomes shorten after each division. But you can't see that.
actiontelomeraseThe enzyme ensures that the end of the lagging strand is properly replicated. A well-studied system includestetrahymenaProtozoan organisms. The telomeres of this organism end with the sequence 5'-TTGGGG-3'. Telomerase adds a series of 5'-TTGGGG-3' repeats to the end of the lagging strand. Hairpin structures occur when unusual base pairs occur between guanine residues in the repeat. The RNA primer is then removed and the 5' end of the lagging strand becomes available for DNA synthesis. The connection is made between the finished braided sheath and the yoke. Finally, remove the hairpin at the 5'-TTGGGG-3' repeat. This faithfully replicates the ends of the chromosomes. The following image shows these steps.
Eukaryotic genomic DNA sequence analysis.
Techniques for determining the complexity of a genomic sequence includedenaturation and renaturationDNA. DNA is denatured by heating, which melts the hydrogen bonds and makes the DNA single-stranded. If the DNA is cooled quickly, the DNA remains single-stranded. However, as DNA slowly cools, complementary sequences meet and eventually base-pair again. The speed at which the DNA is recovered (another term for renaturation) depends on the species from which the DNA is separated. The curve obtained from the single genome is shown below.
The y-axis is the percentage of DNA that remains single-stranded. This is expressed as a ratio of the ssDNA concentration (C) to the total initial DNA concentration (Cobalt). The x-axis is a logarithmic scale of the product of the initial concentration of DNA (in mol/L) times the duration of the reaction (in seconds). The given value iscradleKnown as the "Cradle" value. The curve itself is called the "Cot" curve. As can be seen, the curves are quite smooth, suggesting that the reannealing is slow but gradual over a period of time. A useful specific value iscrib ½, HecradleValue when half of the DNA is rehybridized.
Steps in DNA denaturation and renaturation experiments.
1. Shear the DNA to a size of approximately 400 bp.
2. Heat to 100°C to denature DNA.OhC.
3. Allow to cool slowly and take samples at different intervals.
4. Determine the percentage of ssDNA at each time point.
The shape of the "Cot" curve for a given species is a function of two factors:
- the size or complexity of the genome; and
- The amount of repetitive DNA in the genome.
If we draw the "Cot" curves for the genomes of three species such as the lambda phage,Escherichia coliand yeast we will see that they have the same shape butcrib ½The amount of yeast will be maximum,Escherichia colinext and lambda are minimal. Physically, the larger the genome, the longer it takes for a sequence to find its complement in solution. This is because two complementary sequences must meet in order to mate. The more complex the genome, i. h. The more unique sequences that are available, the longer it will take for two complementary sequences to find and mate. The more complex species take longer to reach Cot½ when the concentrations in the solution are similar.
repetitive DNA sequence, a DNA sequence that occurs more than once in the genome of a species, has a unique effect on the "cot" curve. If a given sequence occurs twice in the genome, it can be paired with two complementary sequences such that its Cot value is half that of a sequence that occurs only once in the genome.
In fact, eukaryotic genomes have extensive sequences represented at different levels of repetition.Single copy sequences occur one or more times in the genome.Many sequences that encode functional genes fall into this category.There are 10 to 1000 intermediate repeats of DNA in the genome.Examples include rRNA and tRNA genes and storage proteins in plants such as maize. The length of the intermediate repeat DNA can vary between 100-300 bp and 5000 bp and can be scattered throughout the genome. The most common sequences are inrepetitive and marked DNAClass. These sequences occur between 100,000 and 1 million times in the genome and vary in length from a few bases to a few hundred bases. These sequences are found in regions of chromosomes such as heterochromatin, centromeres, and telomeres and are often arranged as tandem repeats. The following are examples of tandem repeats:
atta atta atta//atta
Genomes containing these different classes of sequences anneal differently than genomes containing only single-copy sequences. Instead of a single smooth "cradle" curve, you'll see three different curves, each representing a different repeat category. The first sequences to anneal are highly repetitive sequences because they occur in many copies in the genome and because of their low sequence complexity. The second part of the genome to be rejoined is the intermediate repeat DNA and the last part to be rejoined is the single copy of the DNA. The figure below shows the "Cot" curve for a "typical" eukaryotic genome.
The following table shows the sequential distribution of selected species.
|bacteria||99.7% single copy|
|Maus||60% serving of butter|
10% strong repetitive
|humanity||70% serving of butter|
8% strong repetitive
|Cotton||61% serving of butter|
Repeated in 27%
8% strong repetitive
|Further||30% serving of butter|
20% strong repetitive
|Wheat||10% portion of butter|
4% strong repetitive
|Arabidopsis||55% serving of butter|
Repeated in 27%
10% strong repetitive
Although the genomes of higher organisms contain monocopy, moderately repetitive, and highly repetitive DNA sequences, these sequences are not arranged in the same way in all species. called prominent permutationsshort term spread. This arrangement is characterized by 100-200 bp long repeats interspersed with 1000-2000 bp long single copy sequences. This arrangement occurs in animals, fungi, and plants.
The second arrangement islong time between. It is characterized by 5,000 bp repeats distributed within a 35,000 bp single copy region of DNA.Drosophilait is an example of a species with this unusual sequence arrangement. In both cases, the repeat sequence was normally of an intermediate repeat class. We discussed the discovery of highly repetitive sequences above.
Karyotype of the eukaryotic chromosome.
Bacteria have only one chromosome while eukaryotic species have at least one pair of chromosomes. Most have more than one pair. Another related point is that detection of eukaryotic chromosomes occurs only during cell division and not in all phases of the cell cycle. When sister chromatids unite, they are in their most condensed form at metaphase. This is the initial stage of performing a cytogenetic analysis.
Each species has a characteristickaryotype. The karyotype is a description of the number of chromosomes and their size distribution in normal diploid cells. For example, human chromosomes are made up of 23 pairs of chromosomes, 22 pairs of somatic cells, and one pair of sex chromosomes. An important aspect of genetic research is to correlate changes in the karyotype with changes in the phenotype of individuals.
An important aspect of genetics is the linkage of changes in the karyotype with changes in the phenotype. For example, people with an extra chromosome 21 have Down syndrome. Experienced cytogeneticists can recognize insertions, deletions, and changes in chromosome number, but it is difficult to correlate them with specific phenotypes.
The first distinguishing parameters in the development of a karyotype are the size and number of chromosomes. While this is useful, it does not provide enough detail to develop correlations between structure and function (phenotype). For further differentiation of the chromosomes, the DNA is reproducibly stained with dyes. After dyeing, some areas are slightly stained and others heavily. As mentioned above, lightly blotchy areas are calledeuchromatinis called the dark colored areaheterocromatina. The currently selected tint isGiemsa stain, which is called the resulting patternG band type。
The genome of an organism is not only described by the number of chromosomes, but also by the amount of DNA in a haploid cell. This is usually expressed as the amount of DNA per haploid cell (often expressed in picograms) or kilobases per haploid cell, known as the C-value. An immediate feature of eukaryotes highlights a specific abnormality previously identified in molecular studies. Although eukaryotes appear to have two to ten times as many genes as prokaryotes, they have many orders of magnitude more DNA in their cells. Furthermore, the amount of DNA per genome is independent of the assumed evolutionary complexity of a species, which is expressed asThe C-value paradox: the amount of DNA in the haploid cells of an organism is independent of its evolutionary complexity.(Another important point is that there is no correlation between the number of chromosomes and the assumed evolutionary complexity of an organism.)
C values for organisms used in genetic studies.
|species||The Kilobase/Haploidy Genome|
|Escherichia coli|| |
A spectacular example of the range of C values can be seen in the plant kingdom.ArabidopsisLily (1.0 x 10^8 kb/haploid genome), which represents the lower end of complexity, represents the upper end of complexity. By weight, each haploid weighs 0.07 pg.Arabidopsisgenome and 100 pg per haploid lily genome.
By- A complete set of chromosomes inherited from a single parent; the complete composition of an individual's DNA; this definition generally excludes organelles
Copyright © 1997. Philip McLean