The following points highlight the two main adaptational forms of chromosomes. The forms are: 1. Polytene Giant Chromosomes 2. Lampbrush Chromosomes.
Form # 1. Polytene Giant Chromosomes:
Polytene giant chromosomes are normal interphase chromosomes where homologues become paired and undergo several replications; all the copies so produced remain associated together. Polytene giant chromosomes are typically found in salivary gland cells of Dipteran larvae.
They have also been described in several other organisms and in a variety of cell types. They have been found in intestinal cells of mosquitoes, malpighian tubules, ovarian nurse cells and gut epithelia etc. In plants, polytene chromosomes have been reported in the nutritive tissues such as suspensor cells of kidney bean (Phaseolus vulgaris L.).
Salivary gland giant chromosomes were first discovered by Balbiani in 1881. But their cytogenetic importance was realized several years later. Similarities between their banded structure and the linearly arranged genes were pointed out during early 1930s by Kostoff and Calvin Bridges.
In 1933. Painter, and Heitz and Bauer established that each visible chromosome consisted of homologous chromosomes synapsed intimately.
In the salivary gland anlage of Drosophila melanogaster, mitotic activity ceases after 18 hr of embryo development; further development takes place only by cell growth. Their chromosome complement replicates several times.
This growth process produces “giant cells” whose volumes may be thousands of times greater than that of normal cells. Cytophotometric measurements of DNA on pre-pupal salivary glands of D. melanogaster revealed that the chromosome complement has undergone 8 to 9 successive replications during the larval development.
The reduplicated chromosomes do not separate, and all the sister chromatids remain together; there are up to 1024 to 2048 strands in a polytene chromosome. The process of this type of chromosome replication is called endomitosis. When the sister chromatids separate after one endomitosis, the chromosome number is doubled; this condition is called endopolyploidy.
Polyteny and endopolyploidy may coexist in the same cell. For instance, a diploid cell becomes a polyploid cell through endomitosis; further endo-mitotic division may occur in which the chromatids may not separate and produce polyteny.
Such cases have been reported in salivary gland nuclei of various Cecidomyiidae. Endopolyploidy is a normal process in certain tissues of plants and animals. The tapetal cells of anthers in some species become polyploid due to endomitosis.
Endopolyploidy has been reported in roots of spinach, root nodule cells of legumes etc. In salivary glands of water strider, Gerri’s lateralis (20 + XO = ♂; 20 + XX =♀), the cells possess giant nuclei and the ploidy livel is as high as 512x, 1024x or even 2048x.
Morphological Characteristics:
The salivary gland chromosomes of Drosophila are the largest chromosomes known and have been the most thoroughly studied. In the late larval stage (3rd instar) of D. melanogaster, these chromosomes are approximately 100 times the length of somatic metaphase chromosomes.
On stretching, these chromosomes become 2000 µm long. In the salivary gland cells of the midge Chironomus, a pair of the giant chromosomes is 20 µm in diameter and 270 µm in length. Polytene chromosomes show distinctive patterns of darkly staining regions, called bands, separated from one another by lighter staining areas known as inter-bands (Fig. 7.11).
As compared to bands, the inter-band regions are much lower in DNA content.
Each band represents a tightly packed chromomere. Mutations involving deletions of specific genes are accompanied with the disappearance of particular bands. Based on these observations, Calvin Bridges in early 1903s proposed that each band corresponds to a single gene.
Recent experiments tend to support this basic view. But essential gene functions occasionally map in the inter-band region as well. The D. melanogaster genome contains a total of about 5000 bands as estimated from electron-micrographs. Therefore, it has been suggested that there are roughly 5000 genes in Drosophila genome.
The bands represent chromomeres which represent the tightly folded and coiled chromatin. Electron micrographs reveal fibres of 30 nm diameter both in the band and inter-band regions; these fibres resemble the chromatin fibres of typical eukaryotic cells. The fibres are densely packed within the bands, while in the inter-band regions they are arranged rather loosely.
In his simplified model of polytene chromosomes, DuPraw in 1970 showed the polytene chromosomes to be constructed from numerous chromatin fibres that are lined up in register and pass continuously from one end of the chromosome to the other. A tight folding of the chromatin fibres gives rise to the chromomeres and the bands (Fig. 7.12).
In D. melanogaster, the centromeric regions of all the salivary gland chromosomes fuse to form a chromo Centre (Fig. 7.11). The chromo Centre includes all the segments immediately adjacent to the centromeres, heterochromatic arm of the X chromosome, and the complete Y chromosome in males.
Mean DNA content of a single salivary gland nucleus at pre-pupal stage at this stage, the nuclei have undergone 8-9 replication cycles (1024 C). In 1965, Rudkin showed that polytene nuclei in the salivary glands of D. melanogaster do not contain exact geometric multiples of the DNA content of mitotic interphase nuclei.
This may be, at least in part, due to the fact that the DNA of chromo Centre stops replication just after the nuclei begin to become polytene.
One minute and five long strands are observed radiating from the chromo Centre. These are:
(i) Single very short strand representing chromosome 4,
(ii) One long strand formed by the X chromosome,
(iii) Two long strands consisting of the long short arms of chromosomes 2, and
(iv) Two long strands arising the two arms of the chromosome 3.
Puff and Balbiani Ring:
When a gene is activated, a specific band of the salivary gland chromosome enlarges dramatically and assumes a puffed out configuration; hence it is known as puff. Puffs were first observed by Bridges in 1935. The size of puffs varies from very small to more than double the usual diameter of the concerned band.
When a puff becomes exceptionally large, it is called a Balbiani ring (Fig. 7.11). These are restricted to the family Chiromidae. In 1961, Beerman reported that in Chironomus the expression of specific genetic traits was correlated with the development of specific Balbiani ring. In Ch. pallidivitattus, the salivary gland secretion is a granular fluid, while in the related species Ch. tentans it is a clear fluid.
A comparison of their salivary gland chromosomes revealed that a large Balbiani ring present in Ch. pallidivitattus is missing in Ch. tentans. Salivary secretions of the hybrids between the two species were half as granular as that of Ch. pallidivitattus and the size of the particular Balbiani ring was also half of that in Ch. pallidivitattus.
These observations led to the conclusion that the formation of this Balbiani ring represents activation of the gene responsible for the granularity of the salivary secretion. Puffing involves unfolding of the DNA present in the band regions (Fig. 7.12). All the genes are not required to function in the organ where polytene chromosomes occur and most of the genes would, therefore, remain repressed.
Understandably, all the bands are not involved in puffing. There are two types of puffs: (1) DNA puffs in which additional DNA is synthesized during the period of puffing and (2) RNA puffs which support massive RNA synthesis. However, both types of puff are active in RNA synthesis. DNA puffs have been observed in the family Sciaridae. They are supposed to be the sites of specific gene amplification.
Puffing of certain bands can be induced artificially by the application of the steroid hormone “ecdysone”, which is involved in natural molting. When this hormone is injected to the non- molting larvae of Ch. tentans, puffs characteristics of normal molting appear prematurely.
A similar puffing pattern occurs when the isolated salivary glands are incubated with ecdysone. Ecdysone is known to bind with a specific protein receptor; the resulting complex activates transcription by binding to specific sites of the chromosomal DNA.
Transcription occurs in the puffs and each puff synthesizes a specific type of RNA unique to that particular DNA segment. The newly synthesized RNA is complexed with protein to form a ribonucleoprotein granule. These granules are transported into the cytoplasm through the nuclear pores. The RNA component of the ribonucleoprotein complex is processed (splicing) ; it subsequently becomes associated with the ribosomes and functions as mRNA.
Form # 2. Lampbrush Chromosomes:
In the primary oocytes of most vertebrates and some lower animals, meiosis is arrested in the diplotene stage which lasts for a very long time which may be several years. During this period, large amounts of RNA and proteins are synthesized resulting in an extensive increase in the cellular mass.
The chromosomes de-condense and take a unique appearance of brushes used for cleaning chimneys of the old fashion and oil-burning lamps. The term lampbrush chromosomes was derived from this specific appearance of chromosomes.
Lampbrush chromosomes were first discovered in shark in 1892 by Ruckert. They have subsequently been studied in several animal species such as, in Triturus (newt) and Xenopus (an African frog). Similar chromosomes may also occur in spermatocytes of some vertebrates. The Y chromosomes in Drosophila hydei and D. neohydei spermatocytes also generate lampbrush like structure.
General Structure:
When the diplotene is blocked and bivalents de-condense, the stage is called Dictyotene. In human, this stage is reached before the birth of the females and lasts for a period of 12 to 50 years. The homologues of the bivalents remain attached through chiasmata.
Each chromosome of the bivalent contains a long central fibre and thousands of thin chromatin loops which extend laterally from the central axis (Fig. 7.13). In urodele amphibia, the length of a lampbrush chromosome reaches up to one millimeter (1000 pm).
In Triturus viridescens (2n = 22), the bivalents range from 350 to 800 pm in length and the total length of the 11 bivalents becomes 5900 µm during the period of their maximum extension. Lampbrush chromosomes can be stretched using micro needles; stretched chromosomes are many times their original length. Interestingly, they assume their normal size when the stretching is relieved.
The loops occur in pairs and extend in the opposite sides of the chromosome; they originate from the dark stained swellings called chromosomes; 1 to 9 loops may arise. The size of the loops varies in an individual and also among different organisms.
The average length of loops in frog is 9.5 µm, whereas in the newt some loops measure 200 µm. In each pair of loops, one loop is derived from each of the two chromatids forming the chromosome. Chiasma is always located between loops but never within a loop. Each loop contains a single DNA double helix.
According to Gall and Callan, about 5% of the chromosomal DNA is present in the loops at any one time. The loop axis is surrounded by granules and fuzzy mass of fibrils some of which may reach 20 pm in length, giving each loop a characteristic appearance.
These fibrils and granules consist of ribonucleoproteins. Callan in 1967 proposed that genetic units in chromosomes of higher organisms consist of serially repeated coding sequences. Each series consists of a master sequence, followed by a slave sequence.
Thus the DNA present in a chromomere is the main sequence (master), while the DNA present in the loops is composed of the multiple copies (slaves) of the master sequence. During ovulation, the meiotic process is resumed. Prior to metaphase I, the lateral loops are withdrawn. Chromosome contraction then occurs leading to short and compact chromosomes with smooth outlines.
RNA and Protein Synthesis:
The loops have a core of DNA in highly de-condensed state. Auto-radiographic studies of lampbrush chromosomes present in cells incubated in the presence of 3H -uridine have revealed that the loops are engaged in active RNA synthesis. Different loops have different rates of transcription activity. However, different portions of a single loop show equal rates of accumulation of labeled RNA.
The end of the loop at which transcription is just being initiated is thinner because the RNA transcripts are small. As one proceeds from this end towards the opposite end of the loop, the amount of RNA becomes greater and the region becomes thicker.
The RNA is quickly associated with non-histone proteins to form ribonucleoprotein granules. There is considerable compaction so that the granules are only 1/10 as long as the RNA chain. The RNA-protein complexes stick out from the loops to form the loop matrix.
Large amounts of RNA are produced during the diplotene phase. Some of the RNA is utilized for the extensive protein synthesis which occurs during the growth of the egg cell. A major amount of RNA synthesized during this period is stored in the egg for use after fertilization.
Yolky amphibian oocytes have large multiple nucleoli which are active in RNA synthesis. The overall rates of RNA synthesis in these free nucleoli was estimated to be approximately 50% higher than chromosomal RNA synthesis. In non-yolky oocytes, the RNA synthesis is mostly chromosomal.
In the spermatocytes of Drosophila hydei and D. neohydei, the Y chromosome produces morphologically distinguishable lampbrush loops. Labelling with 3H-thymidine and fragmentation of the loops with DNAase have indicated that these structures contain a DNA axis.
The loops appear in prophase I and disappear before metaphase I. Intensive RNA synthesis occurs on these loops. The number, position and order of loops along the Y chromosome is well known. An absence of any loop from the Y chromosome in the spermatocyte of D. hydei males results in sterility. In such a condition, spermatogenesis is blocked at a specific stage depending on which of the loops is absent.