The field of ultra-structure of the chromatin is still the area where electron microscope had failed to provide us a clear picture of the organization of DNA in the chromatin. For the study of chromosomes with the help of electron microscope, whole chromosome mounts as well as sections of chromosomes were studied. Such studies had demonstrated that chromosomes have very fine fibrils having thickness of 2nm-4 nm. Since DNA is 2nm wide, there is a possibility that a single fibril corresponds to a single DNA molecule.
It is well established that the chromosome contains DNA and protein. But the main concerns have been on how the DNA are arranged in the chromosome. Several models have been proposed to explain the association of DNA and protein in the chromosome.
When chromosomes are compared in related species which differ widely in DNA content, such differences may be attributed to one of two causes:
(1) lateral multiplication of chromonemata leading to multiple or multi-strandedness, or (2) tandem duplication of DNA or chromonemata where lengthwise duplication is responsible for chromatin differences.
Folded-fibre Model and Nucleosome Concept
If we presume that a single chromatid has a single long DNA molecule, we have no choices but to believe that DNA should be present in a coiled or folded manner. The manner of coiling and folding of DNA was a matter of debate and dozens of models were available for this purpose; of them only two stand out and are important.
A popular model was the folded-fibre model, proposed by E.J.Dupraw in 1965.
He studied whole mounts of human leucocytes under the electron microscope and found that sister chromatids consist of irregularly folded fibres 200 to 500A in diameter. Since very few or no free fibre ends were found, it was concluded that each chromatid consist of a single fibre (Type B fibre) folded both longitudinally and transversely.
The details of chromosome structure according to the DuPraw are as follows:
The 2oA double helix, 56 microns long, is spirally packed in protein to form a fibril. This fibril is coiled would form a fibre 10 to 100A in diameter and 7-8 microns long. This fibre is called the Type A fibre. The DNA is packed inside the Type A fibre in packing ratio of at least 6:1. The type A fibre is then in turn coiled in a packing ratio of 10:1 to form a type B fibre 200-250A in diameter. The total packing ratio of DNA in the type B fibre averages 56:1. The 200-250A type B fibre is extensively folded to form the chromatid. This theory has been supported by McDermott (1968), Abuelo and Moore (1969), Lampert (1969).
DuPraw’s model of DNA-histone association is now considered unlikely by the discovery that DNA itself is looped around histone beads to form nucleosomes.
Nucleosomes are the simplest packaging structure of all eukaryotic chromatin having fundamental repeating sub-units. They pack DNA into chromosomes inside the cell nucleus and control gene expression. They are made up of DNA and four pairs of proteins called histones, and resemble ‘beads on a string of DNA’. Woodcock (1973) showed that under the electron microscope chromatin appears to have a “tring of beads” structure and is made up of a number of repeating units. These units (the ‘beads’) have been called nucleosomes. The nucleosome concept represents the latest chromosome model of DNA-protein association. The nucleosome concept was proposed by Roger Romberg in 1974.
The structure of nucleosome consists of 200 bp DNA wrapped around an octamer of small basic proteins called histones H2A, H2B, H3 and H4, all of which exist in two copies known as core histones. 146 bp of DNA is wrapped around the core and the remaining bases link to the next nucleosome called linker DNA. This structure causes negative supercoiling. Available data suggest that the DNA is located on the outside the nucleosome, and that it is in the B structure. It is assumed that the DNA double helix is wound around the histone core in a superhelix. It is estimated that there are 75-82 base pairs per turn of the superhelix. Thus for the 146 bp there are about 1¾ turns of the superhelix around the histone core. The DNA double helices are approximately in phase on the two turns, bringing the phosphate groups periodically close together
According to the crystal structure, the histone octamer likely interacts with the DNA around it roughly every 10 bp. Each of the four histone dimers contains three regions of interaction with the DNA. The central interaction site for each dimer is formed by an alpha helix from each histone in the pair pointing at a single phosphate group on the DNA to which they all hydrogen bond.
Finch and Clug (1976) have proposed an arrangement of chromatin in the chromosome model of nucleosomes. The coiled DNA according to them is in the string of nucleosomes. The nuleosome string is then coiled into 300A wide solenoid, which in turn coils to form a supersolenoid with a diametre of 4000A. This super solenoid structure is the unit fibre (chromonema seen in light microscope) whose coils constitute the chromosomes.
In the condensed form, nucleosomes are packed into a 30 nm fibre with about 6 nucleosomes per turn. A string of nucleosomes is coiled into a solenoid configuration by the fifth histone, called HI. One molecule of HI binds to the site at which DNA enters and leaves each nudeosome, and a chain of HI molecules coils the string of nucleosomes into the solenoid structure of the chromatin fibre.
Nucleosomes not only neutralize the charges of DNA, but they have other consequences as well. First, they are an efficient means of packaging. DNA becomes compacted by a factor of six when wound into nucleosomes and by a factor of about 40 when the nucleosomes are coiled into a solenoid chromatin fibre. The winding into nucleosomes also allows some inactive DNA to be folded away in inaccessible conformations, a process that probably contributes to the selectivity of gene expression.
The final level of packaging is characterized by the 700 nm structure seen in the metaphase chromosome. The condensed piece of chromatin has a characteristic structure that can be detected in metaphase chromosomes. This appears to be the result of extensive looping of the DNA in the chromosome.
Arrangement of chromatin in chromosomes:
Mitotic chromosomes are made up by the coiling of a large cylindrical fibre, the unit fibre. This fibre is presumed to be formed by three levels of coiling.
- The first level of coiling of DNA is in the string of nucleosomes.
- The string of nucleosomes is then coiled into a 300A diameter solenoid.
- The solenoid is further coiled into a super solenoid structure with a diameter of 4000A, and a wall 300 A thick. This super solenoid structure is the unit fibre. The unit fibre corresponds to the “chromonema” of light microscopy. It is further coiled in the chromosome.