Eukaryotic Chromosome Organization

1. Eukaryotic and prokaryotic cells differ in a number of aspects. In Greek, Eu means true, Pro means before and karyon means nucleus. Main difference is in their nucleus. Presence of a well-defined nucleus is the main feature that distinguishes the eukaryotic cell from a prokaryotic one. Nucleus serves as the cell’s control center and genetic information is packed up there. DNA replication, transcription, RNA processing etc take place in the nucleus, and only final stage of translation occurs in cytoplasm.      

Eukaryotic nucleus has nuclear membrane, chromatin and nucleolus which is the most prominent substructure in nucleus. Nucleolus are cell’s ribosome factories.  Chromatin, RNA and nuclear proteins move freely in aqueous solution in nucleus.  

·     In prokaryotes, usually a single chromosome is present per cell in the form of covalently closed circular DNA. They also contain extra chromosomal DNA called plasmids. But in eukaryotes, several chromosomes are present per cell (2 to many). Here also extra chromosomal DNA is found in mitochondria and chloroplast. Amount of chromosome in eukaryotic cells is much more than a prokaryotic cell. A human cell contains DNA more than a 1000 times than E coli. In E coli 4x10⁶ base pairs (bp) are seen in single cell whereas in human cell 5.5x10⁹ bp of DNA is present in its diploid nucleus. Each eukaryotic species has a characteristic number of chromosomes.

·     3. C Value- For eg: In humans 46 chromosomes (diploid set) are seen, ie., 23 pairs of chromosomes. Half or haploid set is derived from the egg and half from sperm cell. Eukaryotic chromosomes exist as a single linear unbroken double stranded DNA (DS DNA) molecule running throughout the length of chromosome, coupled with double amount of protein. Chromosomes are not always distinct in a cell. They are well defined structures at the time of cell division. At other times, chromosomes are not well organized. Total amount of DNA in a haploid genome of a species is called the “Species C value”. In other words, C value is DNA contained within a haploid nucleus (e.g. a gamete) or half the amount of DNA in a diploid somatic cell of a eukaryotic organism. C value of human beings is 3.4 x 10⁶ kb. 

·                        ·     5. Chromosome organization in Eukaryotic cells- In eukaryotes, cell cycle has 4 phases viz. G₁, S, G₂ and M phase. G₁ phase is also called the first gap phase. In this phase cells grow in size, copies organelles and makes the molecular building blocks needed in later steps. During S phase, the cell synthesizes a complete copy of the DNA in its nucleus. During the second gap phase G₂, the cell grows more, makes proteins and organelles, and begins to reorganize its contents in preparation for mitosis. G₁, S and G₂ phases together are known as interphase.  In other words, interphase is the time between two mitotic divisions. As mitosis begins, G₂ phase stops. Mitosis has 4 phases called prophase, metaphase, anaphase and telophase. Mitosis is followed by Cytokinesis, the process of the parent cell division into 2 daughter cells. In G₁ phase chromosome is single. During S phase, chromosome duplicate to produce two sister chromatids held together by centromere. Chromosome continues in the same structure in G₂ phase. Centromeres separate and sister chromatids become daughter chromosomes in M phase. 

·                   6. In G₁ phase chromosome exist as one linear DS DNA molecule. This DNA is usually complexed with double amount of proteins. In S phase, sister chromatids have one linear DS DNA running the length of each chromatid. DNA is least organized in G₁ phase and most organized just before cell division. DNA is usually coupled with proteins. DNA and proteins together form the structure called chromatins.  Chromatins are stainable material in cell nucleus and amount of protein is twice the amount of DNA. Basic structure of chromatin in all eukaryotes is identical.

·     7. Chromatin structure- In chromatin, two types of proteins are associated with the DNA called histones and non-histones. Both proteins play a crucial role in the physical structure of the chromosome. Histones are the most abundant proteins. They are small basic proteins with overall positive charge. Large amount of arginine and lysine residues are responsible for the net positive charge and this property helps histones to bind with the negatively charged DNA.  5 types of histones are found in eukaryotes namely H1, H2A, H2B, H3 and H4. In chromatin amount of histone is equal to amount of DNA. Remaining protein part of chromatin is made up of non-histones. Amount and proportion of histone relative to DNA is constant from cell to cell in eukaryotes.

·     8. Among these 5 types of histones, amino acid sequences of H2A, H2B, H3 & H4 are very similar & highly conserved between even distantly related species. H3 & H4 are among the most conserved proteins.  H2A and H2B show slight species specific variation in sequence. This evolutionary conservation shows that histones play some basic role in eukaryotes in the organization of chromosomes. DNA of chromosomes in a single human cell measures >2m (6.5 feet). Human DNA is 700 times greater than E coli. Several levels of packing is necessary to reduce it into cm or even mm to nm and to pack into the nucleus of the cell. Histones play a very crucial role in this chromatin packing.

·     9. Non- histones- These are less abundant than histones. All other proteins associated with DNA apart from histones are called non- histone proteins. These include the proteins that bind during DNA replication, repair, transcription, gene regulation, recombination etc. They are mainly negatively charged. So will bind usually with the positively charged histones. Many different non-histones are present in each eukaryotic cell. Amount and types of histones vary from organism to organism. They also differ markedly in number & type from cell to cell in same organism. Even they differ at times in the same cell.

·     10. Levels of packing in Chromatin - Chromatin structure differs in different stages of the life cycle. It is least organised in the G1 phase and highly condensed at the metaphase, the phase just before cell division. The least compact structure is called 10 nm chromatin fibre. It is morphologically similar to beads on a string. Diameter of bead region is 10 nm and it forms the core region of nucleosomes which are the basic structural units of chromatin. 

·     11. Bead like region is wound around by the string of DNA. This structure is called nucleosome and its diameter is 11nm. Bead like core region consists of 8 histone proteins – 2 each of H2A, H2B, H3 & H4. Also called core histones. This core region is surrounded by DNA of 147bp length wound around by 1.65 times. This arrangement makes the linear DNA compact by a factor of about 6. Thus nucleosome formation is considered as the 1st level of chromatin packing. In other words, 10nm chromatin fibre formation is the first level of chromatin packing. Strands of DNA between nucleosomes are called “linker DNA”. Means two nucleosomes are connected by linker DNA strands. Amount of linker DNA not constant within & among organisms. In humans, linker DNA range between 38-53 bp, thus total amount of DNA per nucleosome is between 185-200 bp. 

·     12. Second level of chromatin condensation is by the formation of “30 nm chromatin fibre” or “Solenoid fibre” which is brought about by H1 Histones.  H1 are larger than other histones. H1 binds to linker DNA at one end of nucleosome and to middle of DNA segment around histone core, making the 10 nm chromatin fibre more compact.   A nucleosome core plus H1 is called Chromatosome. This condensed DNA spiral helically & becomes regular pattern with 6 nucleosome per turn. Now the diameter of DNA is  30 nm and is called “30 nm chromatin fibre” or “Solenoid fibre”.  Solenoid fibre is seen during interphase of cell cycle. This structure brings the packing ratio of DNA to ~40.

 · 13. Next level of chromosome condensation is by the formation of Chromosome Scaffold. However, the exact mechanism behind it is not clearly understood. Non-histone proteins are mainly involved in Scaffold formation. Non histone proteins bind to 30 nm chromatin fibre to form chromatin loops of 30-90 kb long DNA.  180-300 nucleosome will be usually present per loop. In humans around 2000 such looped domains are present. Stretches of DNA that bind to non-histone proteins to form the loop are called Scaffold associated Regions (SAR).  These chromatin loops attach spirally around the Chromosome Scaffold which is again made up of non-histone proteins and form “Rosettes of chromatin loops”. Around 15 loops are present per turn. This makes the chromosomes now 10,000 times shorter & 400 times thicker than naked DNA. 

·     14. Naked DS DNA is 2nm in its diameter but around 2m long. 10-20 µm is the size of a typical eukaryotic cell and its nucleus is even smaller, having a size ranging between 2-10 µm. So, in order to accommodate the very long DNA into this very small nucleus of eukaryotic cell, it has to undergo different levels of packing.  When it undergoes first level of folding into nucleosome or Bead-on-string pattern, the diameter is 11 nm. Means DNA is getting thicker but even shorter. When chromatin undergoes second level of folding into solenoid fibre, its diameter becomes 30 nm. Further folding in to scaffold loop, makes it to a dimension of   300nm in diameter. Further spiralling of loop around chromosome scaffold makes DNA 700nm in diameter. Entire condensed chromosome is 1400 nm in diameter as consists of two sister chromatids. Such 46 chromosomes are packed in the nucleus of each eukaryotic cell (somatic cells).

·     15. To Summarise, in first level of chromatin packing, DS DNA is wound around histones to form nucleosomes and 10 nm chromatin fibre is produced. H1 histones cause second level of condensation of chromatin into 30 nm chromatin fibre or Solenoid fibre. Third level of packing is brought about by non-histone proteins and Scaffold loops are formed which helically get arranged around the chromosome scaffold.  Thus DNA in chromosome becomes 10,000 times shorter and 400 times thicker and accommodated in the nucleus of eukaryotic cell.

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