SELF INSTRUCTIONAL MATERIAL
M.Sc. PREVIOUS (BOTANY)
PAPER –I CELL & MOLECULAR
BIOLOGY OF PLANTS
BLOCK -2
UNIT III – Nucleus, Ribosomes
MADHYA PRADESH BHOJ ( OPEN) UNIVERSITY, BHOPAL
Editor: Dr. (Smt.) Renu Mishra
HOD, Botany & Microbiology
Sri Sathya Sai College for Women, Bhopal
Writer: Smt. Shikha Mandloi
Asst. Prof. Microbiology
Sri Sathya Sai College for Women, Bhopal
Paper-I
M.Sc. Prev.
Cell and Molecular Biology of Plants
UNIT-IIINUCLEUS
STRUCTURE
3.1INTRODUCTION
3.2OBJECTIVE
3.3NUCLEUS
3.4NUCLEOLUS & NUCLEAR PORE
3.5NUCLEOSOME ORGANISATION
3.6DNA STRUCTURE
3.7FORMS OF DNA
3.8REPLICATION
3.9DAMAGE AND REPAIR
3.10TRANSCRIPTION
3.11RNA SPLICING
3.12RIBOSOME STRUCTURE, r-RNA & BIOSYNTHESIS
3.13MECHANISM OF TRANSLATION
3.14SUM UP
3.15CHECK YOUR PROGRESS KEY
3.16ASSIGNMENT
3.17REFERENCES
3.1 Introduction
Since plant cell is a eukaryotic cell, the DNA of the cell remains organized in the form of nucleosome. It remains surrounded by double membrane structure called as nuclear membrane.
This organizational complex form nucleus in plants
The various functions of nucleus are
1. A site of DNA replication, transcription, translation etc.
2. Participates in ribosomal biosynthesis
3. Participates in biosynthesis of nucleotides
4. Exchange of materials with cell through nuclear pore.
During the process of growth of cell, replication of DNA occurs before cell division. All the machinery required for "duplication" of DNA is located in nucleus. Further, the flow of genetic information follows the pathway: -
DNA RNAProtein
transcriptiontranslation
The Information coded in DNA cannot act directly as a template for protein synthesis but must be first transcribed into messenger RNA. This RNA is synthesized by RNA Polymerase Enzyme. The translation of the four base codes into a 20-amino acid protein sequence involves many cellular components like nRNA, 4 RNAs, 70 protein molecules assembled together in the nucleolus.
Apart from this, other process like ribosomal biosynthesis, nucleotide synthesis is associated with nucleus. Thus in this unit, you will be learning in sequence a detailed study of eukaryotic nucleus.
3.2 Objective
After studying this unit you are expected to
1. Understand the structure and functioning of nucleus.
2. Know the structural and conformational details of DNA and RNAs.
3. Understand the process of Protein synthesis.
4. Fulfill the basic requirement to move a step towards molecular and genetic studies.
Nucleus : In Greek language the term nucleus means Karyon.
Robert Brown for the first time in 1833 discovered and coined the term Nucleus.
On the basis of the absence of well-defined nucleus, living organisms were earlier classified into two groups by molecular biologist viz.
Prokaryotes: Cells not having well defined nucleus as in bacteria and blue green algae.
Eukaryotes: Included remaining type of organisms having well-organized nucleus.
A nucleus may be described as having three important parts, viz
Nuclear membrane or nuclear envelope
Nucleolus
Chromosomes
The fluid in which nucleolus and chromosomes are present and which is enclosed in nuclear membrane is called nucleoplasm.
3.3 NUCLEUS
Nucleus was first discovered by Robert Brown (1831) in orchid cells. It is the most important part of the cell which directs and controls all the cellular functions. That’s why nucleus is very often regarded as ‘director of the cell’. Presence of true nucleus with nuclear membrane and linear chromosomes is the characteristic of all the eukaryotic cells. However, there are some exceptions viz., mature mammalian RBCs, sieve tubes of phloem, tracheids and vessels of xylem.
As far the number of nucleus in a cell is concerned, most of eukaryotic cells have single nucleus within them. However, the number may vary in some cells.
Depending on the number of nuclei cells may be of following types :
Anucleate (without nucleus):Mammalian RBCs.
Uninuclate:Most of Eukaryotic Cells.
Binucleate:Basidiomycetes, Paramoecium
Multinucleate:Phycomycetes like Mucor, Rhizopus etc., Red
Algae.
The true Nucleus may be defined as: ‘The cellular structure limited externally by a nuclear membrane surrounded by cytoplasm which contains linear nucleoproteinous chromosomes and carry genetic information’s from generation to generation’. The carrier of genetic information nature of nuclei was established by Hammerling (1953) who worked on the macroscopic unicellular alga, Acetabularia and concluded that the morphology of the plant is solely determined by the type of nucleus contained in the plant body.
STRUCTURE OF NUCLEUS
Study of the cell cycle has revealed that each cell has two phases in its cycle:
Interphase and
Phase of cell division.
In fact, interphase is the phase between two cell divisions. This is much longer than the phase of cell division, structure of nucleus is studied in this interphase only.
The electron microscopic studies of interphase nucleus have revealed that the nucleus may consists of following four parts:
1. Nuclear Membrane: It limits the nucleus externally and also known as karyothica. It is bilayedred, lipoproteineous and trilaminar in nature. Outer membrane is called ectokaryotheca and the inner is endokaryotheca. The outer membrane is studded with ribosomes while the inner is free of that. The two membranes have a thickness of 75-90 Å
And are apart from each other by a distance of 100-300Å. This space is called perinuclear space.
The nuclear membrane has many pores. Its number may vary from 1000-10000 in a nucleus. Each pore is about 400-1000 Å in diameter. The number and size of pores may depend on the needs of the cell. Each nuclear pore is fitted with a cylindrical structure called annulus. The pore and the annulus both collectively form the pore complex or pore basket.
Figure: nucleus
2. Nucleoplasm: It is transparent semi fluid, homogenous, colloidal ground substance inside the nuclear membrane. It is also called nuclear sap, karyolymph or karyoplasm. Nuclear chromatin and nucleolus are embedded within nucleoplasm, chemically, it is formed of water, sugars, minerals (Mn2+, Mg2+, etc.), Nucleotides, ribosomes, enzymes, DNA and RNA polymerases, mRNA, tRNA molecules etc. It is alkaline in nature (pH = 7.4 ).
Functions :
Nucleoplasm forms the skeleton of nuclei and helps in maintaining their shape.
The process of transcription takes place in the nucleoplasm in which different molecules of RNA are formed.
It supports nuclear chromatin and the nucleolus.
Ribosomal subunits are synthesized in the nucleoplasm.
3. Chromatin Net or thread : Electron microscopic studies of well stained eukaryotic nuclei have revealed that presence of darkly stained network of long, fine and interwoven threads which is called chromatin net or thread. It is also known as nuclear reticulatum. It was first reported by Fleming in 1882. During the phase of cell division, the chromatin net is transformed into chromosomes due to high condensation of DNA molecules. These chromosomes are rod like and have definite shape and size chracteristic of an organism.
The chromatin is chemically nucleoprotein and formed of nucleic acid (DNA) and base proteins i.e., histones . It may be classified in to two categories:
- Heterochromatin : It is made of comparatively thick regions which is darkly stained. DNA strands in this chromatin are more condensed. Transcriptionally, it is inactive and late replicative. It does not contain active genes.
Euchromatin : It is true chromatin and is formed of thick and less darkly stained areas. It has loose, less condensed DNA which is trancriptionally, inactive and early replicating.
4. Nucleolus : Within each nucleus, there is a darkly stained, granular, naked and large organelle without limiting membrane. It was discovered by Fontana in 1781. The term nucleolus was coined by Bowman (1840). The size of nucleolus is comparatively larger in those cells which have rapid rates of protein biosynthesis.
The position of nucleolus is generally definite within nucleus. It is associated with nucleolar organizer region (NOR) of nuclear chromosome. It is absent in muscle fibres , RBC, Yeast, sperm and prokaryotes. In general, each nucleus has one or two nucleoli. Its number depends on the number of chromosomes in the species. For each haploid set of chromosomes in the nucleus, there is a single nucleolus. However, a pair of nucleoli may be found in haploid nuclei. In human beings, two pairs of nucleoli are found in each diploid nuclei. In human beings, two pairs of nucleoli are found in each diploid nucleus Xenopus oocytes may contain upto 1000 nucleoli in the nucleoli in the nucleus.
Ultrastructure : The ultrastructure of nucleolus was studied by Borysko and Bang in 1951 and again by Berhard in 1952. On the basis of electron microscopic studies of the structure of nucleolus, de Robertis et al., (1971) described it to be made up of four parts:
- Fibrillar regions: This part is made up of ribonucleoprotein fibres. It is also called nucleolemma. Each fibre has a length of around 50-80 Å.
- Granular regions : This part has many granules each having the diameter of 150-200 Å. These are derived from nucleolar fibres, chemically, these granules are also ribonucleoproteins.
- Protein region: This proteinous part is also called parsamorpha. This is the fluid part of nucleolus in which other parts are found.
- Chromatin part: It is made up of chromatin fibres containing DNA. These DNA molecules function as template for RNA synthesis. The chromatin part may be differentiated into two parts
a)Perinucleolar Chromatin: it forms a covering or envelope around nucleolus. It may have ingrowths at certain places inside the nucleolus, which are called trabeculae.
b)Intranucleolar chromatin: These chromatin fibres are found in internal protein region. These form many septa like structures.
Each nucleolus has dense fibrillar region due to presence of which it is associated with nuclear organizer region of chromosomes. These region have been reported to contain many copies of DNA responsible for synthesis of ribosomal RNA. These rRNA molecules are rapidly synthesized in this region. The protein of ribosomes are synthesized in the cytoplasm which is transported to nucleus and finally to nucleolus. The rRNA and protein molecules combine to form complete ribosome molecules. These newly synthesized ribosomes are associated with thin fibrils of RNA and look like beaded string. This structure is called nucleonema. On the basis of the presence and structure of nucleonema, following three types of nucleoli may be recognized:
Nucleolus with nucleonema which is more common is all types of cells.
Nucleolus without nucleonema which is commonly found in salivary gland cells.
Ring shaped nucleolus containing ribonucleoprotein granules and RNA fibrils.
This is common in endothelial cells and muscle cells.
Functions:
Nucleoli are the site of rRNA biosysthesis.
It stores rRNA.
It helps in the biogenesis of ribosomes.
It helps in the formation of spindle fibres.
It plays important role in mitosis.
Functions of Nucleus
It controls all the cellular functions.
It controls the synthesis of all the structural and enzymatic proteins.
Synthesis of all the 3 types of RNA (mRNA, tRNA and rRNA) takes place in the nucleus.
It plays important role in cell division.
Cell growth is controlled by nucleus
Nucleus controls cellular differentiation by regulating differential gene expression
It induces genetic variation and thus helps in organic evolution.
Sexual reproduction happens due to fusion of two nuclei gametes of opposite sex.
Due to presence of all these organelles and other structures, a cell functions as self-regulatory systems and provides a definite set of characteristics to different organisms.
3.4 NUCLEOLUS & Nuclear Pore
Nucleolus can be seen as a very conspicuous structure in the interphase nucleus. It disapperar during mitosis and reappears at the next interphase. The process by which the nucleolus is formed, is described as nucleologenesis. During prometaphase to early telophase, when the nucleolus remains disappeared, a number of non-ribosomal nucleolar proteins as well as U3 s- RNA are found in (i) the peripheral regions of chromosomes and in the (ii) nucleolus derived foci (NDF) found as cytoplasmic particles 1-2 in diameter;
Fig.UltraStructure of Nucleolus
CHROMOSOMES
Chromosomes are rod like or filamentous bodies, which are typically, present in nucleus and become visible during the stage of cell division. Presence of true chromosomes is the characteristic of eukaryotic cells.
Literally, the term chromosomes have been derived from two Greek words; Chroma and soma meaning by ‘colored body’. This is named because they appear as darkly stained bodies during cell division when stained with a suitable dye and viewed under compound microscope.
Chromosomes can well be defined in following way “Chromosomes are individual protoplasmic entities found in the nuclei of almost all eukaryotic cells which multiply themselves through sequential cell divisions and provide physiological and morphological stability to protoplasm and so to a particular individual”
Some important points to remember about chromosomes.
- Chromosomes were first observed by Straburger (1875) in mitotically dividing cells and the name ‘chromosome’ was proposed by Waldeyer in 1888.
- Each species has a definite and constant number of chromosomes in their cells. The chromosome number found in somatic cells of the species is called somatic chromosome number and is usually represented by ‘2n’. This is because, ordinarily, somatic cells contain two copies of each chromosome which are morphologically identical and also have same gene content and gene location. They are known as homologous chromosomes.
- Chromosomes can darkly be stained by treating the dividing cells by acetocarmine, acetoarcine, feulgan and some other basic dyes.
- In plant kingdom, lowest number of chromosome is found in Haplopappus gracilis and highest in Ophioglossum reticulatum. In animal kingdom, Ascaris mega-locaphala has been found to have lowest number of chromosome.
- Chromosomes are chemically nucleio protein consisting of DNA and proteins. The bear genes therefore, regarded as ‘bearer of hearedity.’
- Each chromosome is made up of two longitudinally held chromatids which are visible during mitotic metaphase.
- The two chromatids of a chromosome are joined at centromere the main function of which is the formation of spindle fibres during cell division.
- Nucleolus within nucleus is associated with secondary constriction of chromosomes. Therefore, the later is called ‘nucleolar organiser.
3.5 NUCLEOSOME ORGANIZATION
This model was proposed by Kornberg and Thomas in 1974 to explain the structure of chromatin fibres. This has been widely accepted all over the world. According to this model, chromatin is composed of a repeating unit called nucleosome.
Important points of this model are as follows:
Chromatin fibres of a chromosome are made up of DNA and histone proteins.
The repeating unit of chromatin is called nucleosome. It is a disc like structure 11nm in diameter and 6nm in height. The core of a nucleosome is made up of an octamer of proteins having two molecules each of H2A, H2B, H3 and H4 histones.
Around this octamer, a DNA segment having the length of 200 base pairs is wound round making one 3/4 turns. This segment of DNA in chromatin fibre is nuclease resistant. The structure of nucleosome is invariable in all the eukaryotes.
P. Oudet et al. (1975) worked extensively on the structure of nucleosome and proposed that the length of DNA segment in the core of nucleosome is 146 base pairs. Two nucleosome units are joined with a segment of DNA, which is called linker. It consists of 50-70 base pairs. H1 histone is associated with this linker DNA which makes a connection between two adjacent nucleosomes.
The nucleosome, model explains the ‘string of beads’ concept of chromatin. This is just opposite, to the concept of ‘beads on string’ explaining the interrelationship of genes and chromosomes. Aron Clug (1977-80) made further electron microscopic studies of chromosomes and chromatin and proposed ‘Solenoid model of nucleosome’. This model describes the dense compaction of DNA in chromosomal chromatids. It further illustrates that chromatin fibres tightly coil in a chromosome and form lump like structure. The average diameter of this chromatin lump is 300 Å in which several nucleosomes of 100 Å diameter are found. As has been mentioned earlier each nucleosome is made up of protein octamer around which DNA segment of 200 base pairs was found forming one3/4 turn. Through the process of super coiling, such nucleosomes with the help of linker DNA easily form the solenoid like structure.
SOLENOID MODEL:
It was also shown that 11nm wide fibre of nucleosomes gets coiled upon itself to form – 30nm wide helix with five or six nucleosomes per helix. In this helix successive nucleosome units came close together, so that their centre to centre distance was about 10 nm. This 30nm structure was called a solenoid. Formation of solenoid from nucleosomes can be compared with winding of a cable on a spool and then folding of wrapped spools.
It was also proved that H1 protein helped in folding of 110 A wide fibre in to 300 armstrong wide solenoid, It has been shown that H1 molecules aggregate by cross linking to form polymers and may thus control the formation of solenoid. The above account gives patterns of coiling and packing of DNA. Since 60 nm along DNA is coiled in a nucleosome, only 6nm long, and then nucleosomes are coiled in 30nm wide solenoid fibres, it gives DNA a packing ratio of 1:50. However, in highly condensed chromosomes, the packing ratio is actually 1:5000, which is 100 times greater than provided by solenoid, would take place by further coiling and folding of solenoid.
Ubiquitination, acetylation, methylation and phosphorylation of histones in the nucleosome.
The histone proteins, which are integral parts of nucleosome undergo a variety of modifications to bring about decondensation of chromatin, to allow access of DNA replication or transcription machinery to naked DNA. These modifications include ubiquitination, acetylation, methylation and phosphorylation of some specific amino acid residues of histones.
Acetylation and methylation occur on the free amino groups of lysines residues. Methylation also occurs on arginine and histidine. Similarly, phosphorylation occurs on the hydroxyl group of serine and histidine. Methylation and acetylation remove the positive charge on NH3+, while phosphorylation introduces a negative charge in the form of phosphate group.
3.6 DNA STRUCTURE