THE ORGANIZATION OF DNA IN CHROMOSOMES

• Prokaryotes have a single type of chromosome while most eukaryotes have diploid number of chromosomes in somatic cells and each cell has many chromosomes.

• In bacteria the chromosome is a circular, double-stranded DNA molecule.

• Each eukaryotic chromosome consists of one linear, unbroken double-stranded DNA molecule running throughout its length.

• The chromosome consists of a complex of DNA and proteins. This is called chromatin

• There are two types of chromatin: euchromatin and heterochromatin. When chromosomes are stained with dyes, they appear to have alternating lightly and darkly stained regions. The lightly-stained regions are euchromatin and the darkly-stained regions are heterochromatin.

• Euchromatin is uncoiled during interphase, but becomes condensed during mitosis. Most of the genome consists of euchromatin.

• Heterochromatin is the chromatin region that is more highly condensed than the euchromatin . Heterochromatin is found near centromeres, at telomeres and else where in repetitive sequences.

• Functionally euchromatin is genetically active (contains genes that are being expressed), whereas heterochromatin is genetically inactive, either it contains no genes or genes that cannot be expressed.

• Two classes of heterochromatin can be distinguished: constitutive heterochromatin and facultative heterochromatin.

• Constitutive heterochromatin is always condensed and genetically inactive. Centromeric and telomeric heterochromatin are examples of constitutive heterochromatin.

• Facultative heterochromatin is sometimes, but not always condensed. It has the potential to become condensed to heterochromatin state. It may contain genes that are made inactive when the chromatin becomes condensed. Barr bodies (inactivated X chromosomes in female mammals) are examples of constitutive heterochromatin.

• There are two major types of protein associated with DNA in chromatin: histones and nonhistones.

• Histones and nonhistones play an important role in determining the physical structure of the chromomosome. The DNA is wrapped around a core of histone molecules, and the nonhistones are somehow associated with that complex.

• Weight for weight there is about an equal amount of histone proteins and of DNA in chromatin. The amount of nonhistone may be as much as the amount of DNA and histone proteins combined.

• The histones are relatively small basic proteins, at normal pH of a cell they have a net positive charge. This facilitates their binding to the negatively charged DNA.

• There are five main types of histones associated with DNA; H1, H2A, H2B, H3 and H4. The amount and proportion of these histones relative to DNA amount are relatively constant from cell to cell in eukaryotic organisms.

• Nonhistones are usually acidic proteins, they have a net negative charge at normal pH, and are likely to bind to positively charged histones in the chromatin.

• Some nonhistones play a structural role and others are enzymes and proteins involved in replication, transcription and regulation of gene expression.

• Nonhistone proteins differ markedly in number and type from cell type to cell type within an organism, at different times in the same cell type and from organism to organism.

Packing of chromosomes into nucleus

• The length of DNA in the nucleus is far greater than the size of the compartment in which it is contained. To fit into this compartment the DNA has to be condensed in some manner. The degree to which DNA is condensed is expressed as its packing ratio.

• Packing ratio - the length of DNA divided by the length into which it is packaged. For example, the shortest human chromosome contains 4.6 x 107 bp of DNA. This is equivalent to 14,000 µm of extended DNA. In its most condensed state during mitosis, the chromosome is about 2 µm long. This gives a packing ratio of 7000 (14,000/2).

• To achieve the overall packing ratio, DNA has several hierarchies of organization. The first level of packing is achieved by the winding of DNA around histones in a structure called nucleosome. This gives a packing ratio of about 6.

• The nucleosome consists of about 200 bp of DNA wrapped around a histone octamer that contains two copies of histone proteins H2A, H2B, H3 and H4. These are known as the core histones. In addition there is a single molecule of the H1(a linker histone).

• Histones are basic proteins that have an affinity for DNA and are the most abundant proteins associated with DNA.

• The length of DNA that is associated with the nucleosome unit varies between species.

• Two DNA components are involved: Core DNA is the DNA that is actually associated with the histone octamer. This value is invariant and is 146 base pairs. The core DNA forms two loops around the octamer, and this permits two regions that are 80 bp apart to be brought into close proximity.

• The DNA that is between each histone octamer is called the linker DNA and can vary in length from 8 to 180 base pairs.

• The winding of DNA around the histone core makes the chromatin fiber to appear like beads on a string.

Coiling of DNA around histones

• The second level of packing is the coiling of beads in a helical structure called the 30 nm chromatin fiber that is found in both interphase chromatin and mitotic chromosomes. This appears to be a solenoid structure with about 6 nucleosomes per turn. This gives a packing ratio of 40. The stability of this structure requires the presence of the last member of the histone gene family, histone H1. Hence, H1 is important for the stabilization of the 30 nm structure.

• The final packaging occurs when the chromatin fiber is organized in loops, scaffolds and domains that give a final packing ratio of about 1000 in interphase chromosomes and about 10,000 in mitotic chromosomes.

• An interphase chromosome may have a diameter of 300 nm while a metaphase chromosome may have a diameter of 700 nm.

• The condensed piece of chromatin has a characteristic scaffolding structure that can be detected in metaphase chromosomes. This appears to be the result of extensive looping of the DNA in the chromosome.

The orders of chromatin packing into mitotic chromosome

Centromeres and Telomeres

• The behaviour of chromosomes in mitosis and meiosis is a function of the centromeres, the site at which chromosomes attach to mitotic and meiotic spindles, and the telomeres, the ends of the chromosomes.

• The centromere region of each chromosome is responsible for the accurate segregation of the replicated chromosomes to the daughter cells during during mitosis and meiosis.

• In many eukaryotes the centromere is seen as a constricted region at one point along the chromosome. The region contains kinetochore to which spindle fibers attach during cell division.

• The DNA sequence within these regions is called CEN DNA. CEN DNA provide the chromosome with the ability to segregate.

• Telomeres are the region of DNA at the end of the linear eukaryotic chromosome that are required for the replication and stability of the chromosome.

• Teleomeres provide terminal stability to the chromosome and ensure its survival.

• Telomeric DNA is usually, but not always, heterochromatic and contains direct tandemly repeated sequences.

• Telomeric sequences may be divided into two types: Simple telomeric sequences –are at the extreme ends of the chromosomal DNA molecules.

• Simple telomeric sequences are the essential functional components of telomeric regions in that they provide a chromosomal end with stability.

• Telomere-associated sequences are regions near but not at the ends of chromosomes. These sequences often contain repeated DNA sequences extending many thousands of base pairs in from the chromosome end.

The Karyotype

• A complete set of all the metaphase chromosomes in a cell is called its karyotype.

• Usually all the chromosomes are arranged in order of size and position of centromeres. They are number for easy of identification.

• For most organisms, all cells have the same karyotype.

• Each species has a characteristic karyotype, and within any species, each sex has a characteristic karyotype.

• Karyotypes of different species differ in the shape, size and number of their chromosomes.

• Within any species, all chromosomes occur in pairs. Somatic cells contain pairs of homologous chromosomes, hence, are diploid. Sex cells (sperm and egg) have half the number of chromosomes of somatic cells, hence are described as haploid.

• In females (for mammals) both members of each chromosome pair have the same size and shape. In males all, but two chromosomes occur in pairs. The two chromosomes are the sex chromosomes. These have different size and shape.

• Chromosomes are often classified according to whether the centromere is at one end (acrocentric), closer to one end than the other (sub-metacentric) or in the middle (metacentric).

Chromosomal banding patterns

• A variety of treatments involving denaturation or enzymatic digestion of chromatin, followed by incorporation of a DNA-specific binding dye can stain certain regions of the chromosomes more intensely than other regions and cause them to appear as a series of alternating light and dark bands.

• Banding patterns are specific for each chromosome, enabling each chromosome in the karyotype to be distinguished clearly.

• One of the staining techniques is called G banding. In this procedure, metaphase chromosomes are first treated with mild heat or proteolytic enzymes (enzymes that digest protein) to partially digest the chromosomal protein, and then stained with Giemsa stain to produce dark bands called G bands. G bands reflect regions of DNA that are rich in adenine and thymine.

• Another staining technique is called Q banding, in which chromosomes are stained with a quinacrine dye which binds preferentially to AT-rich regions of DNA. This procedure produce what are known as Q bands.

The nucleus contains chromosomes.Chromosomes can be arranged to

form a KaryotypeHuman male karyotype