Polytene Chromosome:

Polytene chromosomes provided the first evidence that eukaryotic gene activity is regulated at the level of RNA synthesis. When dipteran chromosomes become polytenic, the DNA replicates by endomitosis, and the resulting daughter chromatids remain aligned side by side.

There chromatids are visible during interphase and have a characteristic morphology of dark bands and alternating interbands. Within these chromosomes it is possible to observe the genetic activity of specific loci at local enlargements called puffs, which represent DNA undergoining intense gene transcription. Puff distribution varies from one tissue to another and can be induced experimentally, indicating the cell specialization results from variable gene transcription.

Polytene chromosomes constitute a valuable material for the study of gene regulation because their gene transcription can be visualized directly in the microscope.

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Some cells of dipteran (flies, mosquitoes, midges) larvae become very large and have a high DNA content. The most prominent ones are located in the salivary gland, but other cells from the gut, fat body, and malpighian tubules of the larva also become ‘polytenic’. (Polyteny differs from polyploidy, in which there is also excess DNA per nucleus, but in which the new chromosomes are separate from each other).

A polytene chromosome of Drosophila salivary glands has about 1000 DNA molecules arranged side by side which arise from 10 rounds of DNA replication (210 = 1024). Other dipteran species have even more DNA molecules per polytene chromosome, for example, chironomus has 16,000.

In polytene cells, the chromosomes are visible during interphase, and the chromomeres (regions in which the chromatin is more tightly coiled) alternate with regions where the DNA fibres are folded more loosely. The alignment of many chromosomes give polytene chromosomes their characteristic morphology, in which a series of dark bands alternate with clear zones called interbands. There are about 5000 bands in the Drosophilla genome. They have characteristic morphology and positions, which permit detailed chromosome mapping.

An additional characteristic of polytene chromosomes is that the maternal and paternal homologue remain associated side by side, in what is called ‘somatic pairing’. This permits the identification of deletions, inversions, duplication as regions looped out of the chromosomes. The pericentromeric heterochromatin of all the Drosophila chromosomes coalesces in a chromocenter, where the chromosomes are joined together. The satellite DNAs of the chromocenter are underreplicated with respect to the rest of the chromosome, (i.e. they undergo fewer rounds of replication). Polytene cells are unable to undergo mitosis and are destined to die. Not all the cells in a dipteran larva have polytene chromosomes. Those destined to produce the adult structures after metamorphosis (imaginal discs) remains diploid.

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Polytene chromosomes have become even more important with the advent of recombinant DNA techniques because they make it possible to map any DNA segment to specific chromosomal loci by in sit hybridization. Polytene chromosomes are very suitable for in situ hybridization because their 1000 DNA molecules are aligned side by side, there by greatly facilitating the detection of single copy genes.

The bands have greatly helped in the mapping of chromosomes in cytogenetics studies. The bands occasionally form reversible “puffs” known as “chromosome puffs” or “Balbiani rings” which are associated with differential gene activation. A puff can be considered a band in which the DNA unfolds into open loops as a consequence of intense gene transcription, i.e. “puffs are sites of intense gene transcription”.

In salivary glands, the appearance of some puffs has been correlated with the production of specific proteins which are secreted in large amounts in the larval saliva, e.g. Chironomous has at the base of the salivary glands four specialized cells that contain cytoplasmic granules of a special secretary protein. The gene for this protein is located in a distinct puff that appears only in the four specialized cells. These results show that cell specialization results from variable gene transcription.

Puffing is a cyclic and reversible phenomenon. The steroid hormone ‘ecdysone’, which induces molting in insects, will induce the formation of specific puffs when injected into larvae or when added to salivary glands in culture by temperature shock. When Drosophila larvae normally grown at 25°C, are exposed to a temperature of 37°C, a series of specific genes is activated while most other genes are repressed.

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Five minutes after the heat shock, nine new puffs are seen on the giant chromosome of the salivary glands. There puffs are active in RNA synthesis as can be demonstrated by H-uridine labeling and radio autography. These new RNAs are accumulated in cytoplasm after being released from the puffs and give rise to eight specific heat shock proteins, so the induction of specific proteins is due to an increased transcription of individual genes RNA polymerase II accumulates in the heat shock puffs, while it disappears from other regions of chromosomes.

In D. melanogaster the giant chromosomes are found in the form of five long and one short strands radiating from a single more or less amorphous mass known as chromocentre. One long strand corresponds to the X-chromosome and the remaining four long strands are the arms of IInd and IIIrd chromosomes. The short strand which is small dot like is IVth chromosome. The centromeres of all there chromosomes fuse to form chromocentre. In the male files, the Y chromosome is also found fused within the chromocentre and is therefore not seen as a separate strand.

Lampbrush Chromosomes:

Special kind of chromosomes known as lampbrush chromosomes are found during the prolonged diplotene stage of first meiotic division and in spermatocyte nuclei of Drosophila. They are characterized by a remarkable change in structure. The change in structure includes an enormous increase in length. These chromosomes may sometimes become even larger than polytenic giant salivary gland chromosomes.

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The largest chromosome having a length up to 1 mm has been observed in urodele amphibian. The chromosomes seem to have a chromomeric pattern with loops projecting in pairs from a single chromomere. The size of loops varies from an average of 9.5 µ in frog to upto 200 µ in newt.

The chromomeres are connected by inter-chromomeric fibres. These pair of loops in these chromosomes give them the characteristic lampbrush appearance. Frequently there loops exhibit a thin axis (which probably consists of one DNA double helix) from which fibres project which are covered with a loop matrix consisting of RNA and protein. The number of pairs of loops gradually increases in meiosis till it reaches maximum in diplotene.

As meiosis proceeds further, number of loops gradually decreases and the loops ultimately disappear due to disintegration rather then reabsorption back into the chromomere. (H. Ris., had though that the loops were integral parts of chromonemata which are extended in the form of major coils.) It is also believed that the loops represent the modified chromosome structures at the loci of active genes.

It has been observed that, if the activity of their genes is checked by actinomycin D, the loops will collapse. Numerous small nucleoli are commonly formed from the lampbrush chromosomes due to the rings detached from the loops at specific loci.

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Lampbrush chromosomes occur at the diplotene stage of meiotic prophase of all animal species. Highly condensed chromomeres form the chromosome axis, from which loops of DNA extend laterally as a result of intense RNA synthesis. Each loop has an axis formed by a single DNA molecule which is covered by a matrix of nascent RNA with hn RNA-binding proteins attached to it. Transcriptional units in oocyte may be extremely long-upto 100 n. Some experi­ments suggest that the long transcriptional units of lampbrush chromosomes may be due to a failure of transcriptional termination.

Lampbrush chromosomes were first observed by Flemming in 1882 and were described in detail in shark oocyte by Ruckert in 1892. He coined the name because the chromosomes look like the brushes used in those times to clean the chimneys of oil lamps.

Lampbrush chromosomes occur at the diplotene stage of meiotic prophase in oocytes of all animal species, in spermatocytes of several species, and even in the giant nucleus of the unicellular alga ‘Acetabularia’. They are visualized best in Salamander oocytes because they have high DNA content and therefore very large chromosomes.

Because Lampbrush chromosomes are found in meiotic prophase, they are present in the form of ‘bivalents’ in which the maternal and paternal chromosomes are held together by chiasmata at those sites where crossing over has previously occurred. Each bivalent has four chromatids, two in each nomologue.

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The axis of each homologue consists of a row of granules or chromomeres from which lateral loops extend. The loops are always symmetrical, each chromosome having two of them, one for each chromatid (two additional symmetrical loops will be found on the homologue because cells in prophase of meiosis have a 4C DNA content-equivalent to a tetraploid cell.

The loops can be distinguished by size, thickness, and other morphologic characteristics. Each loop appears at a constant position in the chromosome, and detailed chromosome maps can be drawn. There are about 10,000 loops per chromosome set.

Each loop has an axis formed by a single DNA molecule that is unfolded from the chromosome as a result of intense RNA synthesis. About 5 to 10% of the DNA is in the lateral loops; the rest being tightly condensed in the chromomeres of the chromosome axis, which are transcriptionally inactive.

Lampbrush chromosomes are very good material for in situ hybridization of cloned DNA to RNA because the thousands of nascent RNA molecules aligned side by side along the loop greatly amplify the hybridization signal. Studies with there techniques have shown that in some loops simple sequence satellite DNAs are transcribed; which is unusual because satellite DNA are normally not expressed.

The hybrization results are due to initiation of transcription at a histone gene promoter that then foils to terminate normally. Enormous transcripts are produced which result from read through into the satellite DNA. Eventually transcription stops when the next transcriptional unit is reached. This failure of termination may be generalized throughout the lamp- brush chromosome and may explain the long loops.

Bacterial Chromosome:

Bacterial and blue green algae are designated as the akaryobionta as against the karyobionta having better differentiated nuclei. However, feulgen positive bodies are seen in bacteria and blue-green algae, a reaction meant for the nucleus of chromosomes.

Their bodies can be clearly observed with the help of electron microscope. It has also been demonstrated that their bodies consist of a network of fine thread. Further work by a number of workers demonstrated that the network of threads consists of a single chromosome in the form of a ring.

However, the exact three dimensional arrangement by which 1100 µ -1400 µ long DNA chain is packed in a nucleoid is not very clear. The cross sections showed 500-900 strands indicating that a single DNA strand is folded back and forth several hundred times. It is also known that large part of the DNA in bacterial nucleoid is not combined with proteins.

It has also been shown that the nucleoid is not combined with proteins. It has also been shown that the nucleoid of Escherichia coli, one of the most extensively studied bacterium often has a point of contact with the plasma membrane. This is perhaps the point at which the replication of DNA strand is initiated.