Chromosome: Structure, Gene Sequence and Types | Genetics

In this article we will discuss about:- 1. Gross Structure of Chromosomes 2. Specific Gene Sequences in Chromosomes 3. Chromosome Banding 4. Synaptonemal Complex 5. Strandedness 6. Types of Specialised.

Gross Structure of Chromosomes:

The chromosome consists of two rod-like structures called chromatids joined with each other at the primary constriction or centromere. The portions of the chromatid on either side of the centromere are the arms of the chromosome.

The ends of the chromosome are called telomeres. If the centromere lies midway along the length, the chromosome is said to be metacentric; if the centromere is slightly off-centre, then sub-metacentric; if the centromere is very near one end so that one arm is exceedingly short, the other equally long, the chromosome is acrocentric.

There has been some dispute about the existence of telocentric (terminal centromere) chromosomes. Studies on the fine structure of the centromere indicate that telocentric chromosomes do exist.

Sometimes a secondary constriction is present (in addition to primary constriction) at specific places in specific chromosomes. In human beings chromosome numbers 1,9 and 16 have it. The nucleolus organisers of many species are located at the secondary constriction. Some secondary constrictions have large amounts of satellite DNA.

In chromosomes fixed in OsO₄ the centromeric region of each chromatid appears to have a small intensely staining granule, called the spindle spherule. The spindle fibers become attached to this spherule rather than to the whole region of the primary constriction. The term kinetochore is applied to this granule.

The terms centromere and kinetochore are often used synonymously which is incorrect. The term centromere includes the entire area of the primary constriction at which the chromatids are held together; it is visible at metaphase stage in the light microscope.

The kinetochore is a specialised structure within the primary constriction to which the spindle fibers become attached; it is seen only in the electron microscope. Whole mount preparations of chromosomes in EM show the centromere to be a region with lesser number of fibres, and is also the region where fibres of the two chromatids intermingle.

Kinetochore:

Its structure is not the same in all organisms. There are two main types:

(1) The trilaminar or stratified structure as in many animals and lower plants;

(2) Ball and cup structure as in higher plants.

The trilaminar type consists of an outer dense layer 30-40 nm thick, a middle layer of low density 15-60 nm thick, and an inner dense layer, 15-40 nm thick, which is granular like the chromatin, and is dense and compact (Fig. 19.1).

The middle layer is structure less, and has a clear area called the corona. The kinetochore usually takes after the shape of the centromere in which it lies. In some elongated centromeres the kinetochore may be 1.4 nm long and only 0.4 nm wide. Its size is increased by spindle poisons.

The ball and cup type of kinetochore has a depression (the cup) about 1.5 µm across, in the surface of the chromosome on the side facing the spindle pole. In the middle of the depression is an amorphous mass, the ball, about 0.8 µm across. The ball is attached to the bottom of the cup, and the spindle microtubules appear to be attached all-round the sides of the ball.

Diffuse centromere:

Sometimes there is no localised centromere in a chromosome. Such holocentric chromosomes are present in certain plants like Luzula, some members of Cyperaceae, algae, protozoa and insects like Steatococcus and Tamalia. The kinetochore is said to be diffuse throughout the length of the chromosome; the spindle fibres also attach along the entire length. When such a chromosome is broken into small fragments by radiation, all the fragments move independently to the poles.

Heterochromatin and Euchromatin:

Depending upon the degree of condensation, Heitz (1928, 1933) distinguished two types of chromosome material—heterochromatin which remains highly condensed throughout interphase, and euchromatin which unravels at the end of mitosis and stains weakly in interphase nucleus. Brown (1966) suggested two distinct types of heterochromatin, facultative and constitutive.

Facultative heterochromatin is present in one or the other of a pair of homologous chromosomes, not both. For example, the inactivation of one chromosome in mammalian females during early stages of development. In adults it is visible as the Barr Body in cells of the buccal mucosa (Fig. 19.2).

It is absent from male cells. Genetic experiments have indicated that although the DNA of facultative heterochromatin contains structural gene sequences, these are permanently repressed. The sequences on the homologous chromosome are euchromatic and expressed.

The mealy bug (Planococcus citri) is an interesting example of facultative heterochromatin. In males the entire set of chromosomes inherited from the male parent becomes heterochromatic, genetically inactive, and eventually eliminated during spermatogenesis. These same chromosomes were euchromatic in the father.

The set of chromosomes from the maternal parent remains euchromatic and contains active genes. The maternal set is passed on to the next generation. In males of the subsequent generation, this paternal set becomes inactivated, but in female offspring’s it remains euchromatic.

Constitutive heterochromatin is permanently condensed and is found in the same locations in both homologous chromosomes. It is often present in specific regions of chromosomes such as the centromeres (Fig. 19.2c), telomere, nucleolus organising regions and other secondary constrictions. Biochemical experiments suggest that coding sequences in constitutive heterochromatin are inactive.

Centromere Function:

The cellular DNA is replicated during S (synthetic) phase of interphase resulting in formation of two copies of DNA prior to mitosis. During cell division metaphase chromosomes reveal two identical sister chromatids held together at the centromere. Spindle fibres consisting of bundles of microtubules bind to the centromere, and pull the sister chromatids apart to the two poles.

Centromeres consist of specific DNA sequences to which centromere-associated proteins bind. The DNA-protein complex is the kinetochore. These proteins act as molecular motors that drive the movement of chromatids to the two poles. Centromeric DNA sequences have been studied in yeast (Saccharomyces cerevisiae) by following the segregation of plasmids at mitosis.

Plasmids that contain functional centromeres segregate like chromosomes and are equally distributed to the daughter cells formed after mitosis. In the absence of a functional centromere, the plasmid is not able to segregate properly, with the result that many daughter cells fail to receive plasmid DNA. These studies led to the determination of sequences required for centromere function.

In yeast, the centromere sequences are contained in approximately 125 base pairs consisting of three sequence elements, that is, two short sequences of 8 and 25 base pairs separated by 78 to 86 base pairs of every AT-rich DNA.

The centromere sequences of another species of yeast, S. pombe are much larger, spanning 40 to 100 kb of DNA, making them about 1000 times larger than those of S. cerevisiae. Moreover, the centromere in S. pombe consists of a central core of 4 to 7 kb of single copy DNA flanked by repetitive sequences, making it more complex than that of S. cerevisiae.

The centromere in Drosophila spans 420 kb, out of which about 85% consists of two highly repeated satellite DNAs having sequences AATAT and AAGAG. The remainder of the centromere consists of interspersed transposable elements, that may also be present at other sites in the genome, and a non-repetitive region of AT-rich DNA.

Deletion of the satellite sequences, transposable elements and the non-repetitive DNA reduced functional activity of the centromere. This implies that both repetitive and non-repetitive sequences are required for kinetochore formation and centromere function.

Centromeres in humans and mammals have been studied through their centromere- associated proteins. Mammalian centromeres include extensive heterochromatin regions that contain highly repeated satellite DNA sequences. The precise function of centromere components in mammalian cells is not known.

Telomeres:

The telomere is a specialised structure at the extremity of a linear chromosome that is essential for the maintenance of chromosome stability. Classical studies recognised telomeres as structures whose absence produced sticky ends and unstable chromosomes. Telomeres contain sequences that play important roles in chromosome replication.

Experiments in which telomeres from the protozoan Tetrahymena were added to the ends of linear molecules of yeast plasmid DNA allowed plasmids to replicate as linear chromosome-like molecules in yeast. It became clear that telomeres are required for replication of linear DNA molecules.

DNA sequences in telomeres are similar across a wide range of lower and higher eukaryotes, the same type of sequence is present in plants and humans. Each telomere consists of tandem arrays (arranged end-to-end or head to tail) of highly repeated sequences of DNA containing clusters of G-C residues on one strand. Thus, the sequence of telomere repeats in humans and various mammals is 5′-TTAGGG-3′ and in Tetrahymena it is 5′-GGGGTT-3′.

These sequences span up to several kilo-bases, being repeated hundreds or thousands of times. One unusual feature of the telomere sequence is extension of the G-C rich strand, by about 14 to 16 bases, as an overhanging tail of single-stranded DNA. DNA replication cannot begin precisely at the 3′ end of a template strand, therefore, the 3′ end of the replicated duplex DNA must terminate in a short stretch in which the DNA is single-stranded.

The single-stranded overhanging DNA is subject to degradation by the action of nucleases during replication. If there was no mechanism to restore the end digested by nucleases, the DNA molecule in a chromosome would become slightly shorter with each round of replication. Such a mechanism exists, and studies of mutant cells in which this mechanism is defective indicate that each chromosome end does become shorter in each replication due to degradation.

The mechanism for restoring ends of a DNA molecule in a chromosome involves the enzyme telomerase. The enzyme first discovered in the ciliated protozoan Tetrahymena, functions by adding tandem repeats of a simple sequence—TTGGGG-3′ to the 3′ end of a RNA strand; in humans this enzyme adds the sequence -TTAGGG-3′. The tandem repeats of these sequences constitute the telomere. As the repeating telomere sequence is being elongated, DNA replication takes place resulting in synthesis of a partner strand.

A few copies of the telomere repeat seem to be necessary to prime the telomerase to add additional copies and form a telomere. The telomerase enzyme is remarkable because it incorporates an essential RNA molecule referred to as a guide RNA, that contains sequences complementary to the telomere repeat.

The guide RNA serves as a template for telomere synthesis and elongation. The guide RNA undergoes base-pairing with the telomere repeat and serves as a template for telomere elongation by the addition of more repeat units.

Notably, the complementary DNA strand of the telomere is synthesised by the cellular DNA synthesizing enzymes. Recent studies provide evidence that the repeated sequences of telomeric DNA form loops at the ends of the chromosomes, perhaps to protect the chromosome terminus from degradation (Figure below).

The telomeric DNA is anchored to the nuclear matrix by proteins called Ku proteins. Ku is an abundant nuclear protein. It exists as a heterodimer of two subunits of 70 kDa and 80 kDa. Ku functions in capping the telomeres, preventing chromosome end fusions, and in telomere length control. Ku binds DNA ends in a sequence-independent manner.

Structure of Human Y Chromosome:

Out of the 46 human chromosomes, the sex chromosomes X and Y stand apart from the 44 autosomal chromosomes because of their distinctive role in mammalian sex determination. A human with XY in chromosome complement is male, while XX is female. Evidence suggests that X and Y originated a few hundred million years ago from a single ancestral autosome.

The two then diverged in sequence in such a manner that today we find that only relatively short regions at either end of the Y chromosome are homologous to the corresponding region of the X chromosome. The remaining 95% of the present day Y chromosome is male specific referred to as MSY (male- specific region of Y).

Recent studies of Skaletsky (2003) describe the MSY region as a mosaic of discrete sequence classes, namely heterochromatic (genetically inert) sequences as well as three classes of euchromatic (transcriptionally active) sequences designated X-transposed, X- degenerated and ampliconic. About 15% of MSY consists of X-transposed sequences, and as expected, they are even today 99% identical to their corresponding sequences in X chromosome.

These sequences consist predominantly of a high proportion of dispersed repetitive sequences and contain only two genes. About 20% of the MSY is comprised of X-degenerate sequences that are most distantly related to the X chromosome and have a higher gene content.

The remaining portion of MSY consists of a web of Y-specific repetitive sequences called amplicons, that constitute a series of palindromes. These palindromes display a range of sizes, up to 3 Mb in length. The ampliconic DNA has the highest gene content and also a very high pseudo-gene content compared with the rest of the MSY.

Chromatin Organisation in Nucleus:

Components within the nucleus are organised in relation to their functions. The nucleolus, chromatin, chromosomes, RNAs and nuclear proteins are localised to discrete sites. Consequently, their functions such as DNA replication, transcription of rRNA genes and assembly of ribosomal subunits in nucleolus, and processing of pre-mRNA become localised to distinct nuclear domains.

DNA in the non-dividing nucleus is organised as chromatin fibres about 30 nm in thickness. The 30 nm fibre is organised into higher order structures. Folding of the chromatin fibres produces chromatin loops which have a DNA content of about 100 kb each. The loop-domain organisation is most clearly visualised in the highly transcribed chromosomes (lampbrush chromosomes) of amphibian oocytes.

The fibres and loops are further organised into chromatin domains with DNA content of about 1 Mega base each. When a cell becomes committed to divide, the chromatin undergoes condensation and organises distinct linear chromosomes that can be visualised during the stages of cell division.

That chromosomes are distributed in a nonrandom manner was first suggested by C. Rabl, way back in 1885, and proved correct a hundred years later in 1984 by studies on polytene chromosomes in salivary glands of Drosophila. Each chromosome occupies a discrete region of the nucleus with centromeres and telomeres clustered at opposite poles.

Further studies have shown that individual chromosomes occupy discrete territories within the nucleus. Chromosome territories may differ in position in different cell types at different times in development. Chromosome territories have been found to correlate with gene densities and gene expression.

The territories of chromatin domains containing relatively few genes tend to be located near the periphery of the nucleus or near the nucleolus, whereas the territories of domains that are relatively gene rich tend to be located toward the interior of the nucleus.

For example, human chromosome 18 that is about 85 Mega base in size, is relatively gene poor while chromosome 19, about 67 Mega base in size is relatively gene rich. In the nucleus, chromosome 18 territories tend to be at the nuclear periphery, whereas those of chromosome 19 tend to be in the interior.

Experiments reveal that the position of a gene in chromosomal DNA affects the level at which the gene is expressed. For example, in the case of genes introduced into transgenic mice, the transcriptional activity of these genes depends on their sites of integration in the mouse genome. This effect of chromosomal position on gene expression may be overcome by sequences referred to as locus control regions, which result in a high level of expression of the introduced genes, regardless of their site of integration.

Locus control regions are found to stimulate only transfected genes that have been integrated into chromosomal DNA; they do not influence the expression of un-integrated plasmid DNAs. The mechanism of action of locus control regions suggests that they are not involved with individual promoters, but seem to activate large chromosomal domains, perhaps by inducing long-range alterations in chromatin structure.

The chromosomal domains appear to be separated from each other by boundary sequences called insulator elements, which prevent the chromatin structure of one domain from spreading to its neighbours. Insulators also prevent enhancers in one domain from acting on promoters located in an adjacent domain.

Further, the spaces between chromatin domains are seen to form a network of channels like the holes in a sponge. The channels are large enough to permit passage of molecules for the nuclear processes such as replication, transcription and RNA processing. Evidence suggests that these molecules move to their site of action in chromatin by passive diffusion.

DNA replication in mammalian cells appears to take place in discrete clustered sites resulting in multiple DNA molecules. Experiments by labeling cells with bromodeoxyuridine, an analogue of thymidine, have indicated that DNA replication appears to take place in large structures that contain multiple replication complexes organised into distinct functional domains.

Transcriptionally active genes seem to be distributed throughout the nucleus. But components of the splicing process are located in discrete structural domains within the nucleus.

Fluorescence microscopy of nuclei stained with immuno-fluorescent techniques using antibodies against small nuclear RNPs and splicing factors have demonstrated that components of the splicing apparatus are localised in 20 to 50 discrete structures referred to as nuclear speckles. Thus, splicing components seem to be recruited from their storage site, speckles, to the transcribed genes for pre-mRNA processing.

Specific Gene Sequences in Chromosomes:

The way genes for ribosomal RNA, 5S RNA and histones are organised in chromosomes has been analysed.

These are described below:

1. Ribosomal RNA Genes:

A cluster of genes associated with the nucleolus represent ribosomal RNA genes. They have been studied most in the toad Xenopus laevis. The cluster consists of 450 tandemly repeated (arranged one behind the other) units.

Each unit is made up of two regions, one which is transcribed to produce the rRNA molecule, and a spacer region which is not transcribed. The transcribed region is richer in A + Tnucleotides. The spacer region is heterogenous in nucleotide composition and also varies in length within the same species.

2. Genes for 5S RNA:

These have been sequenced completely in a number of organisms. They have been however, analysed best in two species of Xenopus, X. laevis and X mulleri. The genes are repeated 10,000 to 25,000 times in X. laevis and are located in the telomere regions of 15 out of the 18 chromosomes. There is a transcribed region of 120 nucleotide pairs, and a non-transcribed spacer region about 600 nucleotide pairs long in X. laevis.

3. Genes for Histones:

The development stages of sea urchin embryos have been extensively analysed for histones genes. This is a favourable material because it provides histone mRNAs in large quantities; the mRNAs are used as probes for locating their complementary sequences (histone genes) in chromosomal DNA.

The histone genes are repeated 300-1,000 times in Xenopus, but only 10-20 times in man. By use of restriction endonucleases, the genes for the 5 types of histones have been determined.

The genes are clustered together and the repeat unit is about 6,000 to 7,000 nucleotides long (i.e., 6-7 kilo-base). Out of the 6-7 kilo-base repeat unit only 2 kb are required to code for the five histones. It was on the basis of this finding that spacers were discovered in histone genes. The transcribed region is rich in G + C nucleotides and the spacer is A + T rich.

Inverted Sequences:

A small proportion of DNA in higher organisms is present as inverted repeated sequences, also known as palindromes. If a nucleotide sequence is repeated and reversed on the same linear molecule, each of the repeated single strands will contain two sequences complementary to each other. Each single strand folds back on itself to form a hairpin duplex structure.

Such structures are visible in the electron microscope. They are also known as fold-back or snapback DNA. The inverted repeats may be adjacent to each other, or separated by a number of nucleotides (Fig. 19.3). Inverted repeats are widely distributed throughout the genome. Their function is not known clearly, although some of them appear to be transcribed.

Chromosome Banding:

Until about the 70s the only visible differentiation in mitotic chromosomes was the centromere and arm length. Around 1971 Caspersson and his colleagues in Sweden initiated staining of chromosomes with quinacrine mustard.

A new substructure in the form of horizontal bands was revealed in the chromosomes. Later on a number of dyes were found to produce bands which were arranged in patterns characteristic for specific chromosomes (Fig. 19.4).

A classification has been proposed for the various banding techniques at the Paris Conference (1971) as follows:

1. Q-Banding:

Chromosomes are stained with quinacrine mustard, quinacrine or Hoechst 33258, or some other dyes and observed in fluorescence microscope. The A + T rich regions of chromosomes show intensely fluorescent bands, the G + C rich regions do not (Fig. 19.4).

2. G-Bands:

Chromosome spreads are first incubated in saline then stained with Giemsa. The slides are examined in the light microscope. Treatment with urea and detergents has the same effect. G-bands (Fig. 19.4A) appear to be related in some way to differences in the state of protein sulfur along the chromosome.

3. R-Bands:

Chromosome preparations are incubated in a buffer at high temperature, and stained with Giemsa. The banding pattern is reverse of the one observed with G-bands.

4. C-Bands:

Chromosome preparations are treated with a moderately strong alkali followed by warm saline and staining with Giemsa. The satellite DNA present around the centromeres becomes deeply stained to form C-bands.

Importance of Banding:

The chromosomes of some materials like mouse and ox are mostly acrocentric and form a continuous gradation in size. Banding pattern is useful for identifying individual autosomes with certainty. The bands can identify each chromosome of a normal complement. They can also identify small parts of chromosomes involved in structural rearrangements such as deletions and translocations.

The banding technique has established the Philadelphia (Ph1) chromosome which is associated with chronic myeloid lukemia in humans. This chromosome was earlier believed to be a deleted chromosome 21, but banding pattern showed that it was in fact chromosome 22.

Moreover, the positions of bands also indicated that in many cases the deleted portion of chromosome 22 was translocated to the end of the long arm of chromosome 9. The banding technique is also useful for assigning different linkage groups to specific chromosomes and for accurate gene mapping.

Synaptonemal Complex of Chromosome:

During their studies of paired meiotic chromosomes, Moses (1956) and Fawcett (1956) independently observed ribbon-like structures in the electron microscope. These were called synaptonemal complex (SC). The complexes were considered to represent the state of chromosome pairing.

The complex consists of two dense parallel lines called lateral elements (LE) and a less dense filament in the centre called the central element (CE). Across the space from the CE run transverse filaments towards each lateral element. This tripartite structure has chromatin material of paired homologous chromosomes lying on either side of the complex, next to each lateral element (Fig. 19.6).

Thus each LE with its associated chromatin comprises one homologue. However, sister chromatids belonging to each homologue are not distinguishable in the complex. The dimensions of the complex are known; the LE is about 40 nm thick, and the CE 60-80 nm thick.

Usually SCs are attached at both ends to the nuclear envelope. Digestion studies with trypsin and some other proteolytic enzymes have established that both LE and CE are made up of protein; they are resistant to DNAse digestion.

There may be one or more dense bodies in the central element called nodes, first observed in Neurospora and yeast, later in other fungi. Nodes are a common feature in SCs of Drosophila oocytes where they are about 100 nm in diameter. They are also called recombination nodules although their involvement in genetic exchange is not clear.

In spermatocytes of the Chinese hamster, Dresser and Moses (1980) found that at early pachytene the LEs are single elements, at mid pachytene they start appearing double; by late pachytene all the LEs are double along their lengths.

Besides the typical tripartite form, SCs with different configurations have been observed. In some insects the complex consists of multiple elements. In the triploid anthers of lily, each of the 3 homologues has its own dense axis at leptotene.

Each axis then joins first with one and then with another of the remaining two homologues to form a double synaptonemal complex. The haploid spermatids of Gryllus contain multiple core complexes resembling stacked SCs somewhat separated from the chromosomes.

The synaptonemal complex first becomes visible at leptotene when unpaired chromosomes develop single dense axes, each of which will become a lateral element of a future SC. At zygotene the dense axes form parallel pairs similar to SC although CE is not yet clearly visible. As pairing advances, the complete tripartite structure of SC is seen. At pachytene full length SCs are visible, often twisted around their long axes.

At diplotene SCs disintegrate and the paired homologues separate. However, the dense single axes remain visible resembling the leptotene configurations. The relationship between SCs and crossing over is not fully known.

Strandedness of Chromosomes:

The eukaryotic cell contains an enormous amount of DNA. A human diploid cell has DNA which could be stretched to a length of 174 cm. This much DNA is distributed into 46 distinct chromosomes. The largest chromosome contains more than 7.3 cm, and the smallest 1.4 cm. But the highly condensed metaphase chromosomes are many thousand times shorter than the length of the DNA they contain.

Obviously the DNA is greatly compressed and packaged inside the chromosome. This raises two questions: first, how many linear, duplex molecules of DNA are present in a chromosome? In other words, is there one DNA duplex in a chromatid (unineme or single strandedness), or more than one (multineme or multistrandedness)?

The second question concerns the organisation of the DNA molecule within a chromosome and will be discussed under nucleosome. The question regarding strandedness of chromosomes has been much debated. It is now generally accepted that the chromosome is unineme, containing a single DNA duplex per chromatid.

The following evidences point to the unineme structure of chromosomes:

1. Taylor’s experiments with autoradiography of root tip chromosomes in Vicia faba have shown segregation of newly synthesised DNA to only one chromatid in two generations. This finding is difficult to explain if two or more molecules of DNA are present in a chromatid.

2. Enzyme digestion studies with the giant lampbrush chromosomes in amphibian oocytes have shown that whole chromosomes are broken by the treatment with DNAse, but not by proteolytic enzymes or RNAse. This suggests the absence of non-DNA linkers made of protein or RNA. Gall (1963) studied kinetics of DNAase digestion of lampbrush chromosomes and concluded that each chromatid consists of a single DNA duplex.

It was assumed that the number of breaks required at a particular site to produce a visible break could indicate the number of subunits present at the site of the break. It was possible to show that four hits or fractures are required to cause a break in a pair of sister chromatids. Since DNAase I produces single strand breaks, it suggests that a single chromatid consists of one DNA duplex.

3. Petes and Fangman (1972) applied the technique of sedimentation velocity analysis to chromosomal DNA in yeast (Saccharomyces cerevisiae). The haploid nucleus of yeast contains about 8.4 – 12.0 x 10⁹ Daltons (1 Dalton = mass of 1 hydrogen atom = 3.32 x 10^-21 gm.). Yeast has 17 chromosomes, therefore the average DNA content per chromosome is from 4.9 to 7.1 x 10⁸ Daltons. Petes et al (1973) observed linear DNA molecules of yeast in the electron microscope and calculated that the above mentioned range in the amount of DNA is expected if there is a single DNA molecule in each chromosome.

4. Kavenoff and Zimm (1973) applied the viscoelastometric method which is suitable for measurement of long DNA molecules to yeast. In this method a cylinder is rotated inside another fixed cylinder which contains lysed cells. The long molecules which are stretched by rotation return to the relaxed state by forming coils; during this process they rotate the inner cylinder back to its initial position.

The rate of the recoil movement is proportional to the molecular weight and the number of the largest DNA molecules in solution. Although the technique is not free from error, the data support the unineme model.

Types of Specialised Chromosomes:

Two types of specialised chromosomes, the polytenic and lampbrush chromosomes exhibit some unique features and provide a system by which gene function mainly transcription can be visualised and studied.

1. Polytene Chromosomes:

When repeated duplications of chromosomal material occur without anaphasic segregation, it results in an interphase nucleus having a number of parallel chromatids organised into a giant- sized chromosome. Called polytenic or salivary gland chromosomes, they were first described in salivary glands of Chironomus tentans by Balbiani (1881). Thereafter many species of Dipteran insects including Drosophila were found to have these chromosomes in the salivary glands and some other tissues.

Besides the enormous length which could reach up to 2,000 µm, a distinctive feature of the giant polytene chromosomes is the presence of horizontal bands ranging in number between 2,000 and 5,000 per chromosome. The cross-bands stain deeply and alternate with lightly stained inter-bands.

The thickness of individual bands in the larvae of various species of Chironomus, Drosophila and Sciara ranges between 0.05 µm and 0.5 µm; the narrowest bands being visible only under the electron microscope. The inter bands show longitudinal striations, or fibrils which are interpreted as indicating polyteny (meaning multistrandedness).

It has been calculated that the thinnest fibre represents an individual chromatid. The number and arrangement of bands along the length of a chromosome is constant for the species. The banding pattern is also identical in all cells of the same tissue and have been used for chromosome mapping.

Deletions and inversions in chromosomes are also readily identified from the position of the bands. Bridges (1935) was able to determine that the linear order of bands would correspond with the linear arrangement of genes on the chromosome.

Some of the bands are visible in an expanded form known as puff. When the compact DNA in a band becomes active, it unravels to form a swollen puff. When a puff becomes very much enlarged it is called a Balbiani ring. There is a characteristic pattern of puffing in different tissues and at different times during larval development.

It was demonstrated that the presence of a specific puff is related with the appearance of a cellular phenotype, that is, a specific protein. For example, the salivary proteins were shown to be associated with a particular puff.

That puffs represent sites of RNA synthesis (gene transcription) was shown by Pelling (1964) by the auto-radiographic technique. Transcription occurs also in the bands but to a very small extent. The accumulation of ribonucleoprotein has been demonstrated cytochemically in the region of a puff.

Inhibitors of transcription such as actinomycin- D and α-amanitin prevent puff formation and lead to some amount of regression of existing puffs. There is an increase in puffing activity during those stages of larval development at which the moulting hormone ecdysone is released from the prothoracic gland. This has also been shown experimentally by injection of ecdysone into fourth instar larvae which respond by increased formation of puffs.

2. Lampbrush Chromosomes:

These are giant chromosomes present during meiotic prophase in oocytes of amphibians, birds and mammals including man. First described by Ruckert (1882), these chromosomes were so designated because of their resemblance to brushes used for cleaning the chimneys of Victorian lamps.

At the diplotene stage of meiosis the oocytes of amphibians contain chromosomes which become modified into extremely long bivalents with lateral loops projecting from the chromomeres. This is considered to be an adaptation to the intense metabolic and synthetic activities of primary oocytes.

This modified diplotene stage has been called the dictyate stage. In mammalian females, dictyotene is an arrested state of meiosis in which the oocytes remain for a very long time from birth until shortly before ovulation.

It must be noted however, that the giant size of chromosome and clear lateral loops are visible only in amphibian oocytes. In most other species the chromosomes are not enlarged, and the loops are few and indistinct, in some there are hair-like lateral appendages or only fuzziness may be visible.

Lampbrush chromosomes have rarely been recorded at male meiotic prophase from spermatocytes of insects, birds, vertebrates and also in man (Fig. 15.7). Among plants, they have been reported in tomato, wild onions, and the fungus Neurospora. In spermatocytes of two species of Drosophila large prominent lateral loops are present on the Y chromosome.

Due to their large size, lampbrush chromosomes can be dissected out of the oocytes and analysed chemically, enzymatically, or by auto-radiographic techniques. Whole mounts of these chromosomes can be examined in an inverted phase contrast microscope. The lateral loops arise from the chromomeres, and consist of an axis of DNA from which numerous fine fibres project outwards (Fig. 15.6).

That fibres consist of ribonucleoprotein (RNP; nascent RNA molecules coated with protein) was shown by Gall and Callan (1962). The loops are said to be asymmetrical as they have very long RNP fibres at one end, gradually shorter ones midway, and smallest fibres near the other end. There are usually no loops in the region of the centromere nor in the nucleolus organising region of specific chromosomes.

Enzyme digestion studies have shown that DNA is continuous throughout a lampbrush chromosome, being present in the inter-chromomeric regions, the chromomeres and the loop axis. Maximum growth of lampbrush chromosomes occurs at the dictyotene stage; as the cell enters metaphase the loops regress and finally disappear. Interestingly, each pair of loops has its own characteristic morphology.

It has been possible to construct cytological maps on the basis of loop morphology. The specific features of a particular loop are transmitted as in Mendelian inheritance. An organism could be homozygous or heterozygous with respect to the morphology of a specific loop pair.

The pattern of loops can be altered by mutations and can be observed in the heterozygotes. That loops are the sites of gene transcription has been shown by Gall and Callan (1962) and others. Soon after formation, the nascent RNA molecules become associated with newly synthesised proteins. When RNA synthesis is blocked by actinomycin-D, it leads to complete or partial collapse of loops.

Miller and Beatty (1969) pioneered a new spreading technique for lampbrush chromosomes. It is possible to visualize how transcriptionally active chromatin is organised in lampbrush chromosomes, at the level of the electron microscope. The method has been extensively applied to transcribing chromatin in diverse materials such as embryos of rabbit and sea urchins. HeLa cells, hen erythrocytes, rat liver, the green alga Acetabularia and some others.

The spreading technique has revealed that the chromomeres are either compact or composed of numerous extended chromatin fibrils. The loop axes are densely covered with lateral RNP fibrils of increasing length, all these together constituting a transcription unit. Each lateral RNP fibril shows a basal granule identified to be an RNA polymerase molecule, at the point where the RNP fibril emerges from the DNA axis.

In some cases the loop has a single transcription unit. In others termination and reinitiation of transcription occur within a loop and produce several transcription units.

The loops of lampbrush chromosomes in amphibian oocytes containing repeated genes for ribosomal RNA produce tandemly arranged transcription units separated by silent regions or spacers (christmas trees; Fig. 15.6, 8). One of the highly transcribed genes, the silk fibroin gene of Bombyx mori has been identified by the spreading technique.

Molecular hybridisation techniques have been used to identify specific loops on lampbrush chromosomes. In this method DNA of known sequence labelled with radioactive isotopes is hybridised to nascent lampbrush loop RNA. Pukilla (1975) utilized purified DNA coding for 5S ribosomal RNA for hybridising with nascent RNA transcribed on the loops. The genes for 5S rRNA were thus located near the centromere of chromosomes 1, 2 and 6.

The study of lampbrush and polytenic chromosomes has shown that gene activity occurs on loops formed from chromomeres in the case of lampbrush chromosomes and in regions of bands in the case of polytenic chromosomes.

Both chromomeres and bands consist of compact chromatin which appears deeply stained in the light microscope. Compact chromatin has been generally considered to be genetically inert. On the contrary however, neither the inter-chromomeric regions of lampbrush chromosomes nor inter-bands of polytene chromosomes have been found to show transcriptional activity.