In this article we will discuss about the chemical composition of chromatin.
Chromatin is composed of DNA (30-40%), RNA (1-10%) and proteins (50-60%). These constituents vary in different organisms and even in the different tissues of the same species (Table 8.2). Even in the same call, the proportions of DNA, RNA and proteins vary with the stage of cell cycle.
For example, the proportion of DNA in metaphase chromosomes is lower than that in interphase chromosomes. As opposed to this, the proportion of proteins is higher in the metaphase than in the interphase chromosome.
The part of DNA active in transcription is also variable in different organisms and in different tissues of a single organism. About 6% of the total DNA is active in transcription in vegetative buds of pea, while 32% of it is involved in transcription in its growing cotyledons (Table 8.2).
The DNA content varies among the cells of different organisms (Table 8.3). The gametes (eggs and sperms) of a species have only one-half amount of the DNA present in its somatic tissues. In human, DNA content of egg or sperm is 2.8 picogram (pg) per cell, while in cow it is 3.3 pg per cell.
The DNA content per cell also varies during the different stages of cell cycle. The haploid DNA content of a cell is denoted by 2C. DNA content of a somatic cell is 2C is G1 it doubles during the S-phase and becomes 4C in G2; it remains at 4C during prophase and metaphase stages of mitosis, and prophase I and metaphase I of meiosis (Fig. 8.1).
The 4C DNA of meiotic cells is reduced to half (2C) in the two daughter cells forming the dyad (due to the first meiotic division). Finally, each of the four component cells of the tetrad produced after the completion of meiosis contains 1C DNA which replicates to become 2C DNA of the gametes (Fig. 8.1).
Denaturation and Renaturation of DNA:
One of the procedures for understanding the organisation of DNA is the technique of denaturation and renaturation. (re-association). Separation of double stranded DNA or RNA into single strands by heating or by exposure to high pH is called denaturation.
When denatured DNA (or RNA) is slowly cooled (in case of heat denaturation) or is subjected to low pH (in case of high pH denaturation), the single strands re-associate by complementary base pairing ; this is known as renaturation.
During renaturation, the complementary strands collide randomly and undergo re-association. Using this technique, DNA-DNA and DNA-RNA hybrids can be produced; it is then called nucleic acid hybridization. DNA/RNA re-association depends chiefly on two parameters; concentration of DNA (moles per litre) and time (seconds).
The re-association rate of a DNA segment is proportional to the number of copies of that segment in the DNA solution.
The reaction can be written as:
where, Co = initial DNA concentration (nucleotide moles) at time “to”
C = DNA concentration which remains single-stranded after time “t”
t = time of incubation under conditions favouring renaturation, and K = re-association rate constant.
Generally “Cot” is represented as “Cot ½” where ‘t½” is the time at which the re-association reaction is half complete, The “Cot” is equal to the concentration of DNA in ‘moles x seconds/litre’. The fraction of DNA that has re-associated (1-C/C0)is plotted against the log of ‘Cot’ and the ‘Cot curve’ is drawn. A direct relationship is observed between the “Cot ½” and the amount of DNA in the genome. Therefore, the complexity of genomes can be estimated by studying the “Cot½” values. E. coli genome (4.2 x 106 bp) is a unique genome and it is used as a standard to measure the complexity of any genome as follows:
For re-association studies, the eukaryotic genome is cut into small pieces of ~ 400 bp, denatured and then allowed to re-associate. Such studies have shown three components of DNA.
1. The component that re-natures fast; it contains highly repetitive DNA.
2. The component showing intermediate re-association rate between the fast and the late re-associating components; it contains moderately repetitive DNA.
3. The third component re-associates very slowly; it contains non-repetitive or unique DNA.
Thus denaturation-renaturation studies have revealed that the eukaryotic genome is composed of the following two types of base sequences:
(i) Unique or non-repetitive DNA and
(ii) Repetitive DNA.
These are the sequences of DNA which are present in a single copy, in each genome. The base sequences of unique DNA are not repeated in the genome. The proportion of unique DNA varies in different eukaryotic organisms (Table 8.4).
It constitutes 8% of the rye genome. 25% of pea, 40% of snail and 70% of human genome. A large number of genes, e.g., most of the structural genes, are present in single copy in the genome. Bacterial genome is considered to be composed r unique DNA; it contains only 0.3% repetitive DNA which, in fact, is rDNA and codes for isomers.
DNA sequences present in more than one copy per genome are called repetitive DNA. They consist of families of sequences that are not exactly similar but are related. However, the members of each family consist of a set of base sequences which are sufficiently similar to re-associate with one another.
Differences among individual sequences occur due to deletion, insertion and substitution. Unequal crossing over plays a role in changing the size of these sequences.
Repetitive DNA sequences are classified into two main groups:
(i) Moderately repetitive sequences, and
(ii) Highly repetitive sequences.
Moderately repetitive sequences:
In the case of moderately repetitive sequences, the number varies from 2 to less than 105 copies per genome. The proportion of these sequences is variable in different species (Table 8.5). Drosophila melanogaster has 12% of its DNA in the form of moderately repetitive sequences, while in man, 13% of the DNA is of this type.
In Nicotiana tabacum, these sequences constitute 65% of the genome. Several genes are present in the form of moderately repetitive DNA sequences, e.g., genes for ribosomal RNA, genes for ribosomal proteins, genes for histones and several others. Several families of transposable elements are also grouped under this class of DNA.
Highly repetitive DNA:
Highly repetitive DNA constitutes a smaller proportion of the genome (Table 8.5). Generally, it consists of very short sequences which are repeated tandemly in large clusters. Highly repetitive DNA is also called simple sequence DNA due to its short repeating units. They may be present in more than 105 copies, even up to millions of copies per genome.
These sequences are located in the constitutive heterochromatin, present mostly in the centromeric and telomeric religions of chromosomes (section 8.4, 8.5). In mammalian genomes, the proportion of highly repetitive DNA is generally below 10%, while in Drosophila melanogaster, it is 17% (Table 8.5). Another Drosophila species, D. virilis, contains more than 40% highly repetitive DNA sequences per genome.
The short tandemly repeated DNA sequences are identical in some cases hut are related in the others. There exists a great variation among different individuals of a single species regarding the size of the tandem clusters; therefore, they can be used in “DNA finger printing”, for characterization of individual genomes.
Highly repetitive DNA sequences may differ from other classes of DNA regarding the buoyant density so that they usually form a separate minor band under CsCl density gradient ultracentrifugation. This minor band of DNA is called a satellite DNA or sat DNA. The buoyant density depends on G : C content of the DNA and it can be expressed by the following formula.
Buoyant density (p) = 1.660 + 0.00098 (G = C%) g/cm3 …(8.3)
When mouse DNA is analysed, it gives one main and one minor band (satellite band) under the density gradient centrifugation. The main band DNA has the buoyant density of 1.701 g/cm3, while the satellite DNA has the buoyant density of 1.690 g/cm3.
This indicates that the main band DNA has a higher average content of G s C as compared to the satellite band DNA. The main band DNA in mouse constitutes 92% of the genomic DNA, while the minor band DNA makes up 8% of the genome. Further, the main band DNA consists of 42% G = C, while the satellite band DNA has much lower viz., 30%, G = C content.
Sometimes the repetitive DNA does not form a minor band but it lies within the main band; it is then called cryptic satellite. The same species may show several satellite bands. For example, there occur three satellite bands I, II and III in D. virilis ; in addition, it also contains a cryptic satellite band.
The DNA of satellite band I constitutes 25% of the genome, while the DNAs of e satellite bands II and III constitute 8% each, making a total of 41% highly repetitive DNA sequences per genome. Predominant base sequences of the satellite bands I, II and III are ACAAACT 3′, 5′ ATAAACT3′ and 5′ ACAAATT 3′, respectively.
Most of the satellite DNA consists of the tandem repeats of these sequences. The total length of satellite DNA sequence of d I is 1.1 x 107 bp, whereas the satellite DNA of each of the bands II and III is 3.6 x 106 along, per genome of D. virilis. Analyses of satellite band DNAs in different Drosophila species have shown that the band I has given rise to the bands II and III during evolution.
Mammalian satellite DNAs are composed of hierarchies of repeating units. Mouse satellite can be cleaved by the enzyme Eco RII into a predominant fragment of 234 bp. This sequence is repeated in about 60% of the satellite DNA; it is composed of two repeating units 117 base pairs.
Further analysis shows that two repeating subunits of 58 bp have constituted the 117 bp unit. Thus the large 234 bp repeated sequence is composed of 4 repeats of 58 bp each.
Lastly it was found that a 9 bp sequences has given rise to larger sequences by saltatory replication, mutations and insertion. A sudden type of DNA replication that produces families of similar base sequences is called saltatory replication. In the organization of satellite DNA, unequal crossing over has played a major role.
Certain tandem repeats of short DNA sequences occur less frequently. They may range from 5 to 50 repeats in mammalian genomes. Such repetitive sequences are called minisatellite or variable number tandem repeats (VNTR) as the size of these repeats are variable. Genetic recombination causes the variation in the number of the repeats. Minisatellites are useful in “DNA finger printing” in human.
RNA constitutes a very small proportion of chromatin (Table 8.2); it is 3.5% in human, 0.3% in cow and 10% in pea. Most of the RNAs are ribosomal RNA, mRNA and tRNA. But apart from these, a special class of RNAs called “chromosomal RNA” is associated with the chromosomes.
Chromosomal RNA constitutes about 5% of the total chromosome weight. These RNAs are small molecules containing 40 to 60 nucleotides. They may be involved in the structural organization of chromatin fibres and gene regulation.
Proteins constitute more than half of the total mass of chromosomes. They belong to two classes:
(i) Histones or basic proteins, and
(ii) Non-histone proteins. Another class of proteins called “protamines” are found associated with chromosomes in sperms of certain animals. Protamines are acidic proteins with molecular weights ranging from 1000 to 5000; they replace histones from the chromatin of the sperms.
Histones are basic proteins or acid soluble proteins and have a net positive charge. These proteins are of 5 types namely, HI, H2A, H2B H3 and H4. These proteins have low molecular mass (Table 8.6). The amino acid compositions of different histone types in pea bud histone are given in Table 8.7. The histones are divided into three classes.
(i) Very rich in lysine:
They are the largest histone molecules. They possess little or no a-helix and are relatively weakly bound to DNA e.g., HI histone. The HI histone accounts for 20-25% of calf thymus whole histones and 15% of pea bud histones. The proportion of lysine is 25.5 per 100 moles of amino acids in pea bud HI histone (Table 8.7).
(ii) Moderately rich in lysine:
H24 and H2B are the histones of this type. H2B has lysine/ arginine ratio of about 2.5, while H2A has a lysine/arginine ratio of about 1.2. Therefore, H2A histone is also called “lysine-arginine rich” histone. In pea, moderately lysine, rich histones constitute about 50% of total pea bud histones.
(iii) Arginine rich histone:
The two types of arginine rich histones are designated as H3 and H4. The H3 histone is larger than H4. Molecular weight H4 histone is 11000 and it is the smallest histone molecule. The proportion of arginine as moles per 100 moles of amino acids is 13.1 in H3 histone, while 15.6 in H4 histone of pea bud (Table 8.7).
Non-histone proteins occur in much lower proportion than histones, and their proportion in the total chromosome mass varies considerably in the different organisms. These proteins make up about 4% in pea vegetative bud, 16% in growing pea cotyledon, while 25% in human HeLa cells (Table 8.2).
Non-histone proteins consist of various enzymes involved in different metabolic functions, e.g., DNA polymerase, RNA polymerase, nucleases, polynucleotide ligase, DNA methylase, proteases, histone methylases proteases, histone methylases, histone actylases, histone deacetylases, histone kinases etc.
Apart from these enzymes, certain non-histone proteins are found that have high electrophoretic mobility; they are called HMG (high mobility group) proteins. Some of the HMG proteins form association with chromatin fibres during transcription.