Packaging of eukaryotic chromosomes

Eukaryotic species consist of a specific number of chromosomes. For example, humans have a total of 46 chromosomes. An average human chromosome consists of less than 5 cm of DNA. The DNA gets highly organized and packed into the chromosomes. Hence, the chromosomes are the most efficient genome packets.  The chromosomes have a different shape during every phase of the cell cycle.

However, the shape and the structure of the chromosomes get confusing in some species. The interphase and mitosis phase include the two phases of the cell cycle. The interphase consists of G1, S, and G2 phases. The chromosomes contain a thin and extended structure in the G1 phase. Hence, they do not appear clearly in this phase. The S phase is known as the phase of DNA replication. Hence, the chromosome gets duplicated by forming sister chromatids. The chromosome begins to condense in the G2 phase. The mitosis phase accompanies cell division. Hence, light microscopy helps to visualize the chromosomes in M phase. The metaphase chromosomes are thick and rod-shaped. However, the chromosomes in anaphase look like rods, J or V-shaped. Metaphase chromosomes form after DNA replication. The linking between the two chromatids involves a centromere. The chromosome arms are known as chromatids. A telomere is an extreme tip or the end of the chromosome. Metaphase chromosomes are present in a highly condensed form. 

Image: Packaging of eukaryotic chromosomes

Nucleosomes:

DNA packaging becomes difficult to study without understanding the nucleosome.  It is visible under an electron microscope. Each bead is known as a nucleosome. The string depicts the DNA. Each nucleosome consists of eight proteins known as histones. Five types of histones include H1, H2A, H2B, H3, and H4. Hence, each nucleosome consists of four main histones, each two in number. These proteins form a barrel-shaped core octamer with a DNA string wound twice around the barrel. The DNA linking the two nucleosomes is known as a linker DNA. The chromatin or a DNA histone complex gets stained with a suitable dye. The histones also contain large amounts of arginine and lysine. The charge of the amino acids is positive. Hence, it facilitates an easy binding with a negatively charged DNA. Like linker DNA, vertebrates consist of linker histones such as H1 a-e, H10, H1t, and H5. Each nucleosome gets attached to a linker histone acting like a clamp and preventing the coiled DNA from getting detached. Hence, the nucleosomes associate to form a 30nm fiber as revealed by the cell-breakage techniques. It involves interphase chromosomes in a highly condensed state or metaphase.

Non- histones are equally essential:

Non-histones prevail less abundantly as compared to the histones.  However, they play an equally important role. DNA binding proteins are known as histone proteins. They play an important role in DNA replication, repair, transcription, and recombination. They bind with the histones due to their negative charge. The proteins remaining in the chromatin after the removal of the histones are known as non-histones. Hence, the construction of the scaffold structure involves non-histone proteins. A scaffold structure is also known as the central framework of the chromosome. To this, a DNA solenoid gets attached as a loop(s). It also includes a class of enzymes known as topoisomerases. 

Two important models of a chromatin fiber:

1.   Solenoid model: A solenoid structure is known as a supercoiled arrangement of the DNA in the nuclear chromosome. This arrangement gets produced due to nucleosome string coiling. The process of coiling takes place continuously. It consists of 7 nucleosomes per turn.

2.     Helical ribbon model: It reveals the higher order structure of the chromatin.

Looped domains:

Selection micrographs show 30-90 kb loops of DNA. These loops get attached to a scaffold with a proteinaceous property. The X shape of the scaffold occurs due to the pairing of the sister chromatids. A human chromosome consists of 2000 looped domains. The location of the non-histone protein is at the base of the scaffold. Special regions get associated with the loops. They are known as scaffold-associated regions or SARs.

They involve a stretch of DNA binding to the non-histone proteins. The SARs determine the loops arranged spirally. The protrusion of the loops arises from the centromere.  They resemble petals of a flower with 15 loops per turn. Thus, the complex scaffolding helps in understanding the chromosomes in a better way.

The study of human chromosome:

Study of human chromosome involves cytogenetic technique such as karyotyping. Different banding techniques help in identifying the chromosomes. A typical metaphase chromosome in a human consists of a satellite, a centromere, a secondary constriction, and chromatids. The study of the sex chromatin and Barr body involves a separate study.

CEN sequences (DNA-protein interactions) in the yeast:

The centromere of the yeast has a single sequence with an approximately 125 base pairs length. This sequence consists of short regions known as CDE I and CDE III. A region known as CDEII lies between CDEI and CDEIII. CDEII is a longer element and is variable and AT-rich. The sequence of CDE I and CDE III are highly conserved. A histone H3 analog is known as Cse 4 protein. It is present in the yeast. This protein combines with another protein MIf2 and forms a core around which the CDEII sequence gets wrapped. Two more proteins get involved in the process. Cbf1 recognizes and attaches to the CDEI sequence. Cbf3 protein attaches to CDEIII. They altogether form the kinetochore. Unlike humans, yeast lacks nucleosomes. 

References:

[1] Cells: Molecules and Mechanisms, E.V. Wong

[2] Molecular Biology of the Gene, Watson

[3] Epigenetics, Lyle Armstrong

[4] Human Molecular Genetics, Tom Strachan

© Copyright, 2018 All Rights Reserved.

Packaging of prokaryotic chromosomes

Most of the prokaryotic chromosomes have single-stranded or a double-stranded DNA. They have a linear or a circular structure. A nucleoid is a dense clump in the bacterial and the archaeal cells. The viruses have a double-stranded or a single-stranded DNA or RNA. Most of the viruses possess a single chromosome with a segmented genome. Bacteria consist of one or few chromosomes. The DNA in the E. coli bacteria is in a supercoiled form. Its length is a thousand times that of its cell. If we try turning the one end of an E. coli DNA segment to the left in the opposite direction of the twists, the strands unwind a bit. A negative supercoil gets produced as a result of the force. Hence, think the DNA double helix to be a spiral staircase. It turns in a clockwise direction. A positive supercoil gets produced when you try turning the DNA by one or more complete turn. A class of enzymes known as topoisomerases controls the process of DNA supercoiling.

The enzyme creates or removes supercoiling in the duplex DNA by creating transitory breaks in the DNA strands. The supercoiling is a fundamental property of the DNA and the chromatin structures. The DNA gets organized in loop domains in the E. coli bacteria. The bacterial chromosomal DNA gets a thousandfold compact structure forming looped domains. In E. coli, each domain consists of a 40Kb loop coiled negatively. An E. coli genome consists of approximately a hundred domains. The nucleoid isolation studies revealed that the E. coli DNA does not freely rotate once a break gets introduced into the DNA. Introduction of breaks affects the rotation of the double helix. Hence, it also affects the supercoiling property of the DNA. There arises a loss of DNA supercoiling due to the breaks. It happens mainly because the DNA is not alone, but attached to proteins. The structure of an E. coli nucleoid seems to be very interesting. The supercoiled DNA loops radiate from the central protein core. The protein component consists of DNA gyrase and DNA topoisomerase-I. They help in maintaining the supercoiled state of the DNA. The E. coli consists of total 40 to 50 supercoiled DNA loops. Each loop consists of 100 Kb supercoiled DNA. It is the amount of DNA that unwinds after introducing a break.

Image: Packaging of prokaryotic chromosomes (Looped domains in E. coli)

The packaging proteins of the bacterial DNA:

The packaging of the DNA involves four main proteins. HU is the most abundant packaging protein. It is a histone-like protein. An E. coli cell consists of 60,000 HU proteins. The wrapping of the DNA among HU proteins determines the level of supercoiling.

Comparison studies distinguishing the relaxed DNA from the supercoiled DNA:

These studies compare the relationship between the binding of the trimethylpsoralen and the dosage of the radiation. Trimethylpsoralen is also known as Trioxysalen. It is a furanocoumarin and a psoralen derivative obtained from a plant source. It creates inter-strand crosslinks. The experiment involves photoactivation of DNA. A pulse of light with a wavelength of 360 nm participates in the photoactivation. Trimethylpsoralen binds to double-stranded DNA due to photoactivation. The rate of binding is directly proportional to the degree of torsional stress possessed by the molecule. Next, the assay involves measuring the degree of supercoiling. The E. coli cells get irradiated for introducing breaks in the DNA. The binding of the trimethylpsoralen depends on the radiation dose. The relation between the dose of radiation involves a direct proportion of the binding.

An advantage of an E. coli nucleoid organized in the domain is that it protects the nucleoid from complete damage occurring due to breaks. The presence of the domains is very important for the presence of the nucleoid. If there were no domains, the radiations would have destroyed the nucleoid completely.

Borrelia and Agrobacterium chromosomes:

Borrelia burgdorferi consists of an increased number of linear chromosomes. It is an organism causing Lyme disease. The linear molecules have free ends. These structures require terminal ends similar to telomeres. They protect the DNA from breaks. Borrelia and Agrobacterium DNA have real chromosome ends. The Borrelia also contains few circular molecules. Agrobacterium studies reveal the presence of one linear and one circular chromosome.

The core genome and the mobile elements Salmonella are good to study. All the Salmonella strains have a single chromosome with core genes.

Multipartite genome in bacteria:

The multipartite genomes are those genomes divided into two or more DNA molecules. For example, Vibrio cholera consists of two circular molecules with the main chromosome and a megaplasmid. In such cases of the multipartite genomes, the difference between the genuine part of the genome and the plasmid becomes difficult to identify. Certain bacteria consist of more than one chromosome with a larger primary chromosome and conserved housekeeping genes. Plasmids are known as small circular DNA capable of replicating autonomously. They coexist with the main chromosome. Multipartite genomes in the bacteria arise due to the origin of additional chromosomes. They arise due to single chromosome splitting, duplication or other factors.

References:

[1] Genes And Genomes, Maxine Singer, Paul Berg
[2] Genomes, T.A. Brown

 © Copyright, 2018 All Rights Reserved.