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Polytene Chromosomes

Polytene cells, which are seen in flies such as the fruit fly Drosophila, are an additional distinctive sort of cell that has contributed to a greater understanding. Certain types of cells in many different kinds of animals go through many cycles of DNA synthesis without ever dividing into new cells, which leads these cells to grow in an unnatural way.


These cells are considered to be polyploid due to the fact that they have an abnormally high number of standard chromosomes. In the salivary glands of fly larvae, this process is carried out to its logical conclusion, leading to the formation of huge cells that contain hundreds or thousands of copies of the genome. In addition, in order to create huge polytene chromosomes, all of the copies of each chromosome are lined up next to one another in precise register, much like drinking straws are when they are arranged in a box. These make it feasible to see characteristics that are generally difficult to distinguish but that are thought to be shared with ordinary interphase chromosomes.

On polytene chromosomes taken from the salivary glands of flies, visible under a light microscope are obvious alternating black bands and light interbands comprised of 1,000 identical DNA sequences ordered side by side in register. These bands and interbands make up polytene chromosomes. In polytene chromosomes, bands account for around 95% of the DNA, while interbands account for only 5% of the DNA. A very narrow band may only have 3,000 nucleotide pairs in each of its chromatin strands, whereas a thick band may have 200,000 nucleotide pairs in each of its chromatin strands. The chromatin in each band appears darker because the DNA in each band is more tightly packed than the DNA in the interbands. There may also be more proteins present in the chromatin in each band. The banding pattern is very similar to the arrangement that can be seen in the chromosomes of frog lampbrushes, as was mentioned earlier.


There are approximately 3700 bands and 3700 interbands across the entirety of the set of Drosophila polytene chromosomes. A "map" of the fly's chromosomes, which is an index to the fly's entire genome sequence, has been created by giving each band a unique number. The bands can be distinguished from one another based on the varying widths and distances between them.

The polytene chromosomes of Drosophila are a good resource to check at if you are interested in learning more about the large-scale organization of chromatin. In the last part of this chapter, we discovered that there are many distinct types of chromatin, and that each of these types has nucleosomes that are composed of modified histones in a particular order. On these nucleosomes, certain protein complexes other than histones can assemble in a variety of different ways, each of which can have an effect on the activity of the cell. A number of these non-histone proteins are able to be recruited along extensive stretches of DNA, which provides substantial parts of the genome with chromatin structures that are analogous to one another. These regions are separated by barrier proteins from neighboring domains of the chromatin that have a more consistent structure. At low resolution, the interphase chromosome can be viewed as a mosaic of different chromatin structures. Each of these structures has a unique set of nucleosome modifications that are related to a distinct group of proteins that are not histones. In addition to enabling us to examine some of the changes that are associated with gene expression, polytene chromosomes make it possible for us to see specifics of this mosaic of domains when viewed under a light microscope.

ChIP is a more modern technique that can be used in place of Drosophila polytene chromosomes to map the sites of histone and non-histone proteins in chromatin over the whole DNA sequence of an organism's genome. This mapping process can also be done using Drosophila polytene chromosomes (chromatin immunoprecipitation analysis).

Using this technology, researchers have been able to pinpoint the location of over fifty unique chromatin proteins and histone modifications in Drosophila. Based on the data, it may be deduced that the organism in question possesses two primary forms of chromatin on actively transcribed genes and three primary forms of repressive chromatin, and that each kind of chromatin is linked to a different complex of non-histone proteins. Therefore, the so-called Polycomb type of heterochromatin is composed of a comparable number of proteins from a distinct set, but classical heterochromatin has more than six of these proteins, one of which is known as heterochromatin protein 1 (HP1) (PcG proteins). In addition to the five major chromatin types, it appears that there are also other, smaller chromatin types, each of which may be regulated in a distinct manner and serve a variety of roles within the cell.


A separate collection of proteins will be bound as a component of the chromatin at a particular locus, and this will vary according to the type of cell and the stage of development the cell is in. These alterations alter the degree to which specific genes are accessible in the various tissues, which contributes to the cell diversification that takes place during embryonic development.


The development of an insect is accompanied by the activation of new genes and the inactivation of old genes, which results in the appearance of discrete chromosomal puffs and the disappearance of old puffs in the insect's polytene chromosomes. It would appear that the majority of puffs come from the decondensation of a single chromosomal band when a puff is inspected closely and the banding pattern is still discernible.

Under the scrutiny of an electron microscope, one can make out the individual chromatin strands that compose a puff. When the conditions are just perfect, loops that are analogous to those discovered in the chromosomes of amphibian lampbrushes can be observed. When the genes included in the loop are not being expressed, the structure of the loop may be similar to that of a folded 30-nm fiber. However, when gene expression is taking place, the structure of the loop grows. In electron micrographs, the chromatin that can be seen on either side of the decondensed loop appears to be noticeably more compact. This suggests that a loop is a distinct functional zone of chromatin structure.

In human cells, observations suggest that when a gene within those cells is expressed, highly folded chromatin loops extend to fill a greater volume. This is implied by the fact that the loops fill a larger volume. For example, fluorescence microscopy can be used to observe dormant chromosomal regions in an interphase nucleus as compact dots. These regions can range in size from 0.4 million to 2 million nucleotide pairs. When a gene is expressed, the initial dot is replaced by longer, punctate structures, and it is noted that the same DNA occupies a bigger region. This happens when a gene is activated.


According to new methods of seeing individual chromosomes, the 46 interphase chromosomes that are found in a human cell tend to inhabit their own distinct area within the nucleus. This suggests that the chromosomes are not very closely intertwined with one another. However, these pictures can only provide a general idea of what each chromosome's DNA looks like. Experiments that explicitly locate the heterochromatic sections of a chromosome demonstrate that they are typically strongly related to the nuclear lamina. This finding holds true regardless of the chromosome that is being investigated. DNA probes that selectively stain gene-rich regions of human chromosomes give a striking image of the interphase nucleus. It is likely that this is a reflection of the varying average positions for active and dormant genes.


A robust extension of the chromosome conformation capture method that was previously described can be used to map the connections between the various one-megabase (Mb) segments of the human genome in human interphase chromosomes. This method takes advantage of a high throughput DNA sequencing technology known as massive parallel sequencing. According to the findings, the vast majority of human chromosomes are folded into a structure that is referred to as a fractal globule. This is a knot-free arrangement that permits optimum dense packing while still preserving the flexibility of the chromatin fiber.


I

Polytene Chromosomes

Polytene cells, which are seen in flies such as the fruit fly Drosophila, are an additional distinctive sort of cell that has contributed to a greater understanding. Certain types of cells in many different kinds of animals go through many cycles of DNA synthesis without ever dividing into new cells, which leads these cells to grow in an unnatural way.


These cells are considered to be polyploid due to the fact that they have an abnormally high number of standard chromosomes. In the salivary glands of fly larvae, this process is carried out to its logical conclusion, leading to the formation of huge cells that contain hundreds or thousands of copies of the genome. In addition, in order to create huge polytene chromosomes, all of the copies of each chromosome are lined up next to one another in precise register, much like drinking straws are when they are arranged in a box. These make it feasible to see characteristics that are generally difficult to distinguish but that are thought to be shared with ordinary interphase chromosomes.

On polytene chromosomes taken from the salivary glands of flies, visible under a light microscope are obvious alternating black bands and light interbands comprised of 1,000 identical DNA sequences ordered side by side in register. These bands and interbands make up polytene chromosomes. In polytene chromosomes, bands account for around 95% of the DNA, while interbands account for only 5% of the DNA. A very narrow band may only have 3,000 nucleotide pairs in each of its chromatin strands, whereas a thick band may have 200,000 nucleotide pairs in each of its chromatin strands. The chromatin in each band appears darker because the DNA in each band is more tightly packed than the DNA in the interbands. There may also be more proteins present in the chromatin in each band. The banding pattern is very similar to the arrangement that can be seen in the chromosomes of frog lampbrushes, as was mentioned earlier.


There are approximately 3700 bands and 3700 interbands across the entirety of the set of Drosophila polytene chromosomes. A "map" of the fly's chromosomes, which is an index to the fly's entire genome sequence, has been created by giving each band a unique number. The bands can be distinguished from one another based on the varying widths and distances between them.

The polytene chromosomes of Drosophila are a good resource to check at if you are interested in learning more about the large-scale organization of chromatin. In the last part of this chapter, we discovered that there are many distinct types of chromatin, and that each of these types has nucleosomes that are composed of modified histones in a particular order. On these nucleosomes, certain protein complexes other than histones can assemble in a variety of different ways, each of which can have an effect on the activity of the cell. A number of these non-histone proteins are able to be recruited along extensive stretches of DNA, which provides substantial parts of the genome with chromatin structures that are analogous to one another. These regions are separated by barrier proteins from neighboring domains of the chromatin that have a more consistent structure. At low resolution, the interphase chromosome can be viewed as a mosaic of different chromatin structures. Each of these structures has a unique set of nucleosome modifications that are related to a distinct group of proteins that are not histones. In addition to enabling us to examine some of the changes that are associated with gene expression, polytene chromosomes make it possible for us to see specifics of this mosaic of domains when viewed under a light microscope.

ChIP is a more modern technique that can be used in place of Drosophila polytene chromosomes to map the sites of histone and non-histone proteins in chromatin over the whole DNA sequence of an organism's genome. This mapping process can also be done using Drosophila polytene chromosomes (chromatin immunoprecipitation analysis).

Using this technology, researchers have been able to pinpoint the location of over fifty unique chromatin proteins and histone modifications in Drosophila. Based on the data, it may be deduced that the organism in question possesses two primary forms of chromatin on actively transcribed genes and three primary forms of repressive chromatin, and that each kind of chromatin is linked to a different complex of non-histone proteins. Therefore, the so-called Polycomb type of heterochromatin is composed of a comparable number of proteins from a distinct set, but classical heterochromatin has more than six of these proteins, one of which is known as heterochromatin protein 1 (HP1) (PcG proteins). In addition to the five major chromatin types, it appears that there are also other, smaller chromatin types, each of which may be regulated in a distinct manner and serve a variety of roles within the cell.


A separate collection of proteins will be bound as a component of the chromatin at a particular locus, and this will vary according to the type of cell and the stage of development the cell is in. These alterations alter the degree to which specific genes are accessible in the various tissues, which contributes to the cell diversification that takes place during embryonic development.


The development of an insect is accompanied by the activation of new genes and the inactivation of old genes, which results in the appearance of discrete chromosomal puffs and the disappearance of old puffs in the insect's polytene chromosomes. It would appear that the majority of puffs come from the decondensation of a single chromosomal band when a puff is inspected closely and the banding pattern is still discernible.

Under the scrutiny of an electron microscope, one can make out the individual chromatin strands that compose a puff. When the conditions are just perfect, loops that are analogous to those discovered in the chromosomes of amphibian lampbrushes can be observed. When the genes included in the loop are not being expressed, the structure of the loop may be similar to that of a folded 30-nm fiber. However, when gene expression is taking place, the structure of the loop grows. In electron micrographs, the chromatin that can be seen on either side of the decondensed loop appears to be noticeably more compact. This suggests that a loop is a distinct functional zone of chromatin structure.

In human cells, observations suggest that when a gene within those cells is expressed, highly folded chromatin loops extend to fill a greater volume. This is implied by the fact that the loops fill a larger volume. For example, fluorescence microscopy can be used to observe dormant chromosomal regions in an interphase nucleus as compact dots. These regions can range in size from 0.4 million to 2 million nucleotide pairs. When a gene is expressed, the initial dot is replaced by longer, punctate structures, and it is noted that the same DNA occupies a bigger region. This happens when a gene is activated.


According to new methods of seeing individual chromosomes, the 46 interphase chromosomes that are found in a human cell tend to inhabit their own distinct area within the nucleus. This suggests that the chromosomes are not very closely intertwined with one another. However, these pictures can only provide a general idea of what each chromosome's DNA looks like. Experiments that explicitly locate the heterochromatic sections of a chromosome demonstrate that they are typically strongly related to the nuclear lamina. This finding holds true regardless of the chromosome that is being investigated. DNA probes that selectively stain gene-rich regions of human chromosomes give a striking image of the interphase nucleus. It is likely that this is a reflection of the varying average positions for active and dormant genes.


A robust extension of the chromosome conformation capture method that was previously described can be used to map the connections between the various one-megabase (Mb) segments of the human genome in human interphase chromosomes. This method takes advantage of a high throughput DNA sequencing technology known as massive parallel sequencing. According to the findings, the vast majority of human chromosomes are folded into a structure that is referred to as a fractal globule. This is a knot-free arrangement that permits optimum dense packing while still preserving the flexibility of the chromatin fiber.


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