B.Sc. Botany Hons. Part III Paper- VI CYTOGENETICS MOLECULAR BIOLOGY AND PLANT BREEDING GROUP- A: Cytogenetics
Dr. Akanksha Priya Assistant Professor RLSY College, Bakhtiyarpur, Patliputra University
1. Structure and Physico-chemical organization of Eukaryotic chromosome with Nucleosome- Solenoid Concept: Polytene chromosome and Lampbrush chromosome a) Eukaryotic chromosome: structure and physico-chemical organization: ➢ Eukaryotic chromosome structure refers to the levels of packaging from the raw DNA molecules to the chromosomal structures seen during metaphase in mitosis or meiosis. ➢ Chromosomes contain long strands of DNA containing genetic information. ➢ A eukaryotic chromosome is linear and contains a single DNA molecule of enormous length. For example, the largest chromosome in the D. melanogaster. Drosophila genome has a DNA content of about 65,000 kb (6.5 × 10 7 nucleotide pairs), which is equivalent to a continuous linear duplex about 22 mm long. ➢ Eukaryotic chromosomes are stored in the nucleus of the cell. ➢ In conventional electrophoresis, the electric field is maintained in a constant state, usually at constant voltage. The fragments move in response to the field according to their size, smaller fragments moving faster. However, conventional electrophoresis can separate only molecules smaller than about 20 kb. All molecules larger than about 20 kb have the same electrophoretic mobility under these conditions and so form a single band in the gel. ➢ Electrophoretic separation yields a visual demonstration that each of the chromosomes contains a single DNA molecule that runs continuously throughout its length.
The Nucleosome ➢ It is the basic structural unit of chromatin. ➢ The DNA of all eukaryotic chromosomes is associated with numerous protein molecules in a stable, ordered aggregate called ‘chromatin’. ➢ Some of the proteins present in chromatin determine chromosome structure and the changes in structure that occur during the division cycle of the cell. Other chromatin proteins appear to play important roles in regulating chromosome functions.
Nucleosome Core Particles ➢ The simplest form of chromatin is present in nondividing eukaryotic cells, when chromosomes are not sufficiently condensed to be visible by light microscopy. ➢ Chromatin isolated from such cells is a complex aggregate of DNA and proteins. ➢ The major class of proteins comprises the histone structure of chromatin. Five major types — proteins. Histones (are small proteins) are largely responsible for the H1, H2A, H2B, H3, and H4 — are present in the chromatin of nearly all eukaryotes in amounts about equal in mass to that of the DNA. ➢ Histones also bind tightly to each other; both DNA- histone and histone-histone binding are important for chromatin structure.
Solenoid ➢ The solenoid structure of chromatin is a model for the structure of the 30 nm fibre. It is a secondary chromatin structure which helps to package eukaryotic DNA into the nucleus. ➢ The solenoid model was first proposed by John Finch and Aaron Klug in 1976. They used electron microscopy images and X-ray diffraction patterns to determine their model of the structure. This was the first model to be proposed for the structure of the 30 nm fibre. ➢ DNA in the nucleus is wrapped around nucleosomes, which are histone octamers formed of core histone proteins; two histone H2A-H2B dimers, two histone H3 proteins, and two histone H4 proteins. The primary chromatin structure, the least packed form, is the 11 nm, or “beads on a string” form, where DNA is wrapped around nucleosomes at relatively regular intervals, as Roger Kornberg proposed. ➢ Histone H1 protein binds to the site where DNA enters and exits the nucleosome, wrapping 147 base pairs around the histone core and stabilising the nucleosome, this structure is a chromatosome. ➢ In the solenoid structure, the nucleosomes fold up and are stacked, forming a helix. They are connected by bent linker DNA which positions sequential nucleosomes adjacent to one another in the helix. ➢ The nucleosomes are positioned with the histone H1 proteins facing toward the centre where they form a polymer. ➢ The solenoid structure's most obvious function is to help package the DNA so that it is small enough to fit into the nucleus. ➢ The "beads on a string" structure can compact DNA to 7 times smaller. The solenoid structure can increase this to be 40 times smaller. When DNA is compacted into the solenoid structure can still be transcriptionally active in certain areas. It is the secondary chromatin structure that is important for this transcriptional repression as in vivo active genes are assembled in large tertiary chromatin structures.
Fig.: Eukaryotic chromosome Fig.: Packaging of DNA
b) POLYTENE CHROMOSOMES
➢ A typical eukaryotic chromosome contains only a single DNA molecule. However, in the nuclei of cells of the salivary glands and certain other tissues of the larvae of Drosophila and other two winged (dipteran) flies, there are giant chromosomes, called polytene chromosomes. ➢ Each of these chromosomes contain about 1000 DNA molecules laterally aligned and have a sectional diameter many times greater than those of the corresponding chromosome at mitotic metaphase in ordinary somatic cells, as well as a constant and distinctive pattern of transverse banding. ➢ The polytene structures are formed by repeated replication of the DNA in a closely synapsed pair of homologous chromosomes without separation of the replicated chromatin strands or of the two chromosomes. ➢ Polytene chromosomes are atypical chromosomes and are formed in "terminal" cells; that is, the larval cells containing them do not divide and are eliminated in the formation of the pupa. Although they do not contribute to the tissues in the adult fly. ➢ The darkly staining transverse bands in polytene chromosomes have about a tenfold range in width. These bands result from the side-side alignment of tightly folded regions of the individual chromatin strands that are often visible in mitotic and meiotic prophase chromosomes as chromomeres. ➢ More DNA is present within the bands than in the interband (lightly stained) regions. About 5000 bands have been identified in the polytene chromosomes. ➢ This linear array of bands, which has a pattern that is constant and characteristic for each species, provides a finely detailed cytological map of the chromosomes. The banding pattern can be understood by observing them. ➢ Due to their large size and finely detailed morphology, polytene chromosomes are exceedingly useful for a process called nucleic acid hybridization.
Fig.: Polytene chromosome
c) LAMPBRUSH CHROMOSOMES ➢ Lampbrush chromosomes are a special form of chromosome found in the growing oocytes (immature eggs) of most animals, except mammals. ➢ They were first described by Walther Flemming in 1882. Lampbrush chromosomes of tailed and tailless amphibians, birds and insects are described best of all. ➢ Chromosomes transform into the lampbrush form during the diplotene stage of meiotic prophase I due to an active transcription of many genes. ➢ They are highly extended meiotic half-bivalents, each consisting of two sister chromatids. ➢ Lampbrush chromosomes are clearly visible even in the light microscope, and appear as organized into a series of chromomeres with large chromatin loops extended laterally. Amphibian and avian lampbrush chromosomes can be microsurgically isolated from oocyte nucleus (germinal vesicle) with either forceps or needles. ➢ Giant chromosomes in the lampbrush form are useful model for studying chromosome organization, genome function and gene expression during meiotic prophase, since they allow the individual transcription units to be visualized. ➢ Moreover, lampbrush chromosomes are widely used for high-resolution mapping of DNA sequences and construction of detail cytological maps of individual chromosomes.
Fig.: Lampbrush chromosome