PBG 212 Classical and Molecular Cytogenetics Notes PDF

PBG 212 Classical and Molecular Cytogenetics S.No. Title 1. Definition- Brief history of developments of genetics and cytogenetics 2. Cell division- mitosis and meiosis- significance 3. Chromosome structure- chromatid, chromomeres, centromere, telomere, secondary constriction, nucleolar organizer region, satellite; 4. Chromatin chemical composition; Chromosome landmarks-Euchromatin and heterochromatin; centromere, bands, chromosome ends, knobs 5. Types of chromosomes classified based on- position of centromere, number of centromeres, shape at anaphase, structure and appearance, essentiality, role in sex determination, structure and function 6. Special chromosomes –polyteneandlampbrushchromosomes,other types of chromosomes - B, ring and isochromosomes 7. Differential staining of chromosomes- Q banding, G banding, R banding and C banding 8. FISH- Steps in FISH- applications and limitations 9. Mid semester Examination 1 1. Definition- Brief history of developments of genetics and cytogenetics GENETICS 1. Genetics is the science of heredity and variation. 2. Genetics is a study of the mechanism of transmission of characters from parents to their offspring, origin of variation and gene action. The word genetics was derived from the Greek root gen which means to become or to grow into and it was coined by Bateson in 1906. HEREDITY & VARIATION Heredity may be defined as the potential of an individual to transmit its characters to the offspring while variation are the differences that the individual acquires due to the interaction with the environment. The main aim of genetics is the study of Heredity and variation, while heredity tries to maintain uniformity, variation brings in plasticity. INHERITANCE Inheritance is the transmission of genetic information from parents and ancestors to offspring. CYTOLOGY Cytology (Greek words, Kytos = hollow vessel or cell; logous = to discourse) orcell biology is the biological science which deals with the study of structure, function, molecular organization, growth, reproduction and genetics of the cells. A Cell may be defined as the structural and functional unit of a living being.The theory, that the cell is the basic unit of life, and all plants and animals are composed of one or more cells, was enunciated in 1833 by two German scientists, Schleiden and Schwann. The cells arise only from pre-existing cells is an equally important generalization made by another German scientist, Virchow in his ‘Theory of Cell Lineage’, proposed in 1858. Every plant or animal starts its life only as a single cell. This gives rise to two new cells by division. Each of these again divides into two and the process is repeated. In unicellular organisms, the division of cells is a process of asexual reproduction. The 2 first cell of a new individual arising from sexual reproduction is formed by the union of the egg nucleus from the female and the sperm nucleus from the male. The physical links between the parents and the offspring are thus the nuclei of the egg and the sperm, and the hereditary material passed on from one generation to another be contained in the nuclei. Thus, the nuclei are the carriers of heredity. History of genetics and cytogenetics The history of genetics started with the work of the Augustinian friar Gregor Johann Mendel. His work on pea plants, published in 1866, described what came to be known as Mendelian Inheritance. In the centuries before—and for several decades after—Mendel's work, a wide variety of theories of heredity proliferated. 1900 marked the "rediscovery of Mendel" by Hugo de Vries, Carl Correns and Erich von Tschermak, and by 1915 the basic principles of Mendelian genetics had been applied to a wide variety of organisms—most notably the fruit fly Drosophila melanogaster. Led by Thomas Hunt Morgan and his fellow "drosophilists", geneticists developed the Mendelian model, which was widely accepted by 1925. Alongside experimental work, mathematicians developed the statistical framework of population genetics, bringing genetic explanations into the study of evolution. With the basic patterns of genetic inheritance established, many biologists turned to investigations of the physical nature of the gene. In the 1940s and early 1950s, experiments pointed to DNA as the portion of chromosomes (and perhaps other nucleoproteins) that held genes. A focus on new model organisms such as viruses and bacteria, along with the discovery of the double helical structure of DNA in 1953, marked the transition to the era of molecular genetics. In the following years, chemists developed techniques for sequencing both nucleic acids and proteins, while others worked out the relationship between the two forms of biological molecules: the genetic code. The regulation of gene expression became a central issue in the 1960s; by the 1970s gene expression could be controlled and manipulated through genetic engineering. In the last decades of the 20th century, many biologists focused on large-scale genetics projects, sequencing entire genomes. 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 2. Cell division- mitosis and meiosis- significance All living cells increase in size by physical growth, the very basis of life. This growth occurs by increase in volume and an enlargement of the outer membrane. The surface area of the spherical call increases by the square of its radius 9r) and its volume proportionately increases by the cube of the radius. therefore an increase in size of a cell produces relatively smaller increase in surface area (r2) than in cell volume (r3). Hence the inner constituents have lesser surface area to obtain food, oxygen and various metabolites, as well as more metabolite wastes to be excreted. In the absence of cell division, death would quickly ensure. hence, some form of cell division have been a necessity for the very existence as well as for maintenance of life. The process of reproduction or formation of new cells from pre –existing cell is called cell division. The cell which undergoes division is called the mother cell and the new cells which are formed by the process of cell division are termed daughter cells. In prokaryotes such as bacteria, cell division takes place by binary fission (cleavage) of pre existing cell. But in case of eukaryotes (higher organism), the cell division occurs in a specialized manner. In eukaryotes, there are two types of cell division viz., mitosis and meiosis. A cell has to maintain continuity from one generation to another and the hereditary material has to copy itself most faithfully. the cell has to divide in growing tissues in such a manner that the two daughter cells are similar to each other and resemble the parent cell from which there were produced. This is accomplished by mitotic mechanism. that reduced an even cellular division of essential hereditary components. In the life cycle of plants and animals, the only link between parents and offspring are the sex cells or gametes. Therefore the structures responsible for inheritance namely the chromosomes (the physical basis of inheritance_ should pass through the gametes. Hence the behaviour of chromosomes during cell division should govern the pattern of inheritance. This is accomplished by meiotic mechanism in which the chromosome number is reduced to half, which is restored to normal diploid number at the time of zygote formation (fertilization). Mitosis 29 The term mitosis was coined by W.Flemming in 1882. All somatic cells of a multicellular organism are descendents of one original cell, the fertilized egg or zygote through a divisional process called Mitosis. Mitosis occurs in somatic ells. The two basic function of mitosis if 1. To conserve an exact copy of each chromosome by nuclear division (Karyokinesis) 2. To distribute each set (original set and a copy) to the two progeny (daughter) cells through the division of the original (mother) cell by cytoplasmic division(Cytokinesis). Through mitosis, two daughter cells which resemble each other and also the parent cell quantitatively and qualitatively. The manipulation of chromosomes occur through microtubules that is organized into spindle shaped bodies generated by the centrioles. The basic outline of mitosis is the same in all kinds of living forms. It consists of flowing stages forming a cell cycle. Mitosis is a continuous process and its division into stages is done only for convenience of description. The cell cycle The period in which one cycle of cell division is completed is called a cell cycle. A cell cycle consists of two major phases viz., Interphase and Mitotic phase. The interpahse is generally known as DNA synthesis phase and the mitotic phase is known as nuclear division. The time required to complete one cell cycle differs from species to species. For example in Vicia faba root tip mitosis takes approximately 31 hours to complete a mitotic cell cycle. The details of duration of various stages of mitotic cell cycle are given in the following table. Different phases of mitotic cell cycle ( in root tip mitosis of Vicia faba) Parts of cell cycle Phase Description of phase Duration (hrs) Interphase G1 Pre – DNA synthesis phase 12 S DNA synthesis phase 6 G2 post DNA Synthesis phase 12 Mitosis M Mitotic phase 1 30 Interphase Interphase is the period between two mitotic phase. Interphase consists of G 1, S and G2 sub phases. G1 phase: It is the resting phase. It is called pre – DNA synthesis phase. It takes place between mitotic telophase and S phase of interphase. It is the longest phase in cell cycle and it takes 12 hours of duration in Vicia faba. The synthesis of protein and RNA takes place in this phase. S phase: This comes after G1 Phase. The chromosomal and DNA replication takes place during this phase. This phase takes six hours in Vicia faba. G2 phase: This is resting phase after DNA synthesis phase. It is called post DNA synthesis phase. This is last sub stage of interphase. This phase also takes 12 hours in Vicia faba. Here also protein and RNA synthesis takes place. Mitotic phase The mitotic division has four phases viz., prophase, Metaphase, Anaphase and Telophase. Prophase 1. The chromosomes condense and become visible in the light microscope first as a thin thread and then progressively shorter and thicker. (Early prophase). 2. The chromosome condenses further and each chromosome is visible with two chromatids held together by a common centromere. 3. The centrioles of animal cell migrate to opposite ends of the cell and establish the polar region from which the spindle apparatus begins to develop. 4. The spindle fibers of the apparatus consist of microtubules formed by the polymerization of the protein tubulin. 5. The nuclear membrane begins to degenerate and by the end of late prophase it completely disappears. 31 Metaphase 1. The spindle tubules start appear and get attached to individual chromosomes at centromeres (also called kinetochore). The spindle fibers are called centromeric fibers. 2. The spindle fibers also get attached with certain points on the sister chromatid arms. These fibers are called continuous fibers. 3. The replicated chromosomes align along the equator with their centromeres lying on the equator 4. A double pled spindle shaped structure formed by the centromeric fibers and continuous fibers with the chromosomes at the equatorial plate form characteristic spindle typical to the metaphase stage. Anaphase 1. This the shortest of mitotic phase 32 2. The centromeres divides (by replication of the DNA in that region) allowing the sister chromatids to be pulled to opposite poles by the centromeric fibers of the spindle apparatus. 3. Once the sister chromatids are no longer connected by a common centromere and thus become new chromosomes. 4. The new chromosome move to opposite poles by the motive force created by cross- bridging between the continuous fibres. The two kinds of fiber slide relative to one another similar to the mechanism analogues to the interaction of actin and myosin fibers during muscle contraction. This is referred as Sliding filament hypothesis. 5. During anaphase the arms of each chromosome drag behind their centromere. giving them characteristic shapes upon the location of centromeres. The meta- centric chromosome appears V shaped, sub-metacentric chromosome appears J shaped and the telocentric chromosome appear rod shaped. Telophase 1. Identical set of chromosomes assembles at each pole of the cell 2. the chromosomes begin to uncoil and returns to an interphase condition 3. the spindle degenerate 4. The nuclear membrane reforms on each set of nuclei ad now two ne daughter nuclei exist. 33 Cytokinesis At the end of telophase, of the cytoplasm divides by a process called cytokinesis. In animals cytokinesis is accomplished by the formation of a cleavage furrow that deepens and eventually pinches the cell in two. But in plants cytokinesis involves the construction of a cell plate of pectin originating in the center of the cell and spreading laterally to the cell wall. In this cell plate formation the golgi body derived vesicles migrate to the equator where their content fuse to form the new wall and their membrane make up the plasma membrane of the new cells. Significance of mitosis Mitosis is responsible for the development of zygote into an adult organism Mitosis is responsible for the normal growth and development of living organisms the pattern of mitotic division gives shape to a specific organism 34 In Plants mitosis leads to formation of new parts viz., roots, stem, branches and leaves Mitosis help to repair the damaged parts In case of vegetatively propagated crops, mitosis help in asexual propagation leading to production of identical progeny Mitosis is useful in maintaining the purity of types, because it leads to production of identical daughter cells without recombination and segregation of genetic materials In animals it helps in continuous replacement of old tissues with new ones, such as blood cells and gut epithelium. Cell division - Meiosis and their significance MEIOSIS The term meiosis was coined by J.B. Farmer in 1905. This type of division is found in 35 organisms in which there is sexual reproduction. The term has been derived from Greek word; Meioum = diminish or reduce. The cells that undergo meiosis are called meiocytes. Three important processes that occur duringmeiosis are: 1. Pairing of homologous chromosomes (synapsis) 2. Formation of chiasmata and crossing over 3. Segregation of homologous chromosomes The first division of meiosis results in reduction of chromosome number to half and is called reduction division. The first meiotic division is also called heterotypic division. Two haploid cells are produced at the end of first meiotic division and in the second meiotic division, the haploid cells divide mitotically and results in the production of four daughter cells (tetrad), each with haploid number ofchromosomes. In a tetrad, two daughter cells will be of parental types and the remaining two will be recombinant types. The second meiotic division is also known as homotypic division. Both the meiotic divisions occur continuously and each includes the usual stages viz., prophase, metaphase, anaphase andtelophase. Interphase Meiosis starts after an interphase. During the premeiotic interphase DNA duplication occurs during the S phase. But approximately 0.3% of the total DNA present in the nucleus does not replicate during the 'S' phase this DNA replicates during the zygotine sub stage of prophase I. I. Meiosis-I (1) Prophase-I It is of a very long duration and is also very complex. It has been divided into the following sub-stages: Leptotene Zygotene Pachytene Diplotene Diakinesis a) Leptotene or Leptonema 36 Chromosomes at this stage appear as longthread like structures that are loosely interwoven. In some species, on these chromosomes, bead-like structures called chromomeres are found all along the length of the chromosomes.RNA and protein synthesis takes place during leptotene. b)Zygotene or Zygonema It is characterized by pairing of homologouschromosomes (synapsis), which form bivalents. The paired homologouschromosomes are joined by a protein containing frame work known as synaptonemal complex. The bivalents have four strands. c) Pachytene or Pachynema The chromosomes appear as thickenedthread-like structures. At this stage, exchange of segments between non-sister chromatids of homologous chromosomes known as crossing overoccurs. During crossing over, only one chromatid from each of the two homologous chromosomes takes part. The nucleolus still persists. d) Diplotene or Diplonema At this stage further thickening and shortening of chromosomes takes place. Homologous chromosomes startseparating from one another. Separation starts at the centromere and travels towards the ends (terminalization). Homologouschromosomes are held together only at certain points along thelength. Such points of contact are known as chiasmata and represent the places of crossing over. The process of terminalisation iscompleted at this stage. e) Diakinesis Chromosomes continue to undergo further contraction.The bivalents appear as round darkly stained bodies and they are evenly distributed throughout the cell. The nuclear membrane andnucleolus disappear. 2) Metaphase-I: The chromosomes are most condensed and have smooth outlines. The centromeres of a bivalent are connected to the poles through the spindle fibres. The bivalents will migrate to the equatorbefore they disperse to the poles. The centromeres of the bivalents are arranged on either side of the equator and this type of orientation is called co-orientation. 3) AnaphaseI: 37 The chromosomes in a bivalent move to opposite poles(disjunction). Each chromosome possesses two chromatids. Thecentromere is the first to move to the pole. Each pole has a haploidnumber of chromosomes. Reduction in chromosome number from diploid to haploid sets in as haploid chromosomes groups at the poles. 4) TelophaseI: Nuclear membranes are formed around the groups ofchromosomes at the two poles. The nucleus and nucleolus are re-organized. Interkinesis This is a short interphase before the second meiotic division. The meiotic interphase chromosomes are not physically extended as in mitosis. Mechanical division of cell (cytokinesis) may occur during this stage(as in corn) or may be postponed until simultaneous formation of four daughter cells at the end of second meiotic division. This interphase does not involve S phase as the chromosomes already have two chromatids. II. Meiosis-II: The second meiotic division is similar to mitosis, but here division is with haploid set of chromosomes as against diploid set in mitosis. The second meiotic division has four phases viz., Prophase II, Metaphase II, Anaphase II and Telophase II. 1) Prophase-II: The chromosomes condense again. The nucleolus andnuclear membrane disappear. The chromosomes with two chromatids each become short and thick. 2) Metaphase -II: Spindle fibres appear and the chromosomes get arranged on the equatorial plane(auto-orientation). This plane is at right angle to the equatorial plane of the first meiotic division. 3) Anaphase-II: Each centromere divides and separates the two chromatids,whichmove towards the opposite poles. 4) Telophase-II The chromatids move to the opposite poles. The nuclearenvelope and the nucleolus reappears. Thus at each pole, there is re-organization of haploid nucleus. 38 Cytokinesis The division of cytoplasm takes place by cell plate method in plants and by furrow method in animals. The cytokinesis may take place after meiosis I and meiosis II separately or sometimes may take place at the end of meiosis II only. Synaptonemal Complex Moses first discovered synaptonemal complex in 1956. This is a triparti protein framework found between paired chromosomes at Prophase I of meiosis I. It consists of one central and two lateral elements. The transverse fibres are electron dense filaments that interconnect the central element with the lateral elements. These lateral elements are attached to the homologous chromosomes. The lateral elements are composed of fibres wider than 10 nm and are called synaptomeres. The central element is a ladder like configuration in the center of synaptonemal complex. The SC is associated with homologous chromosome pairing and recombination. Significance of Meiosis 1. It helps in maintaining a definite and constant number of chromosomes in a species. 2. Meiosis results in production of gametes with haploid (half) chromosome number. Union of male and female gametes leads to formation of zygote which receives half chromosome number from male gamete and half from the female gamete and thus the original somatic chromosome number is restored. 3. Meiosis facilitates segregation and independent assortmentofchromosomes and genes. 4. It provides an opportunity for the exchange of genes through the process of crossing over. Recombination of genes results in generation of variability in a biological population which is important from evolution points of view. 5. In sexually reproducing species, meiosis is essential for the continuity of generation. Because meiosis results in the formation of male and female gametes and union of such gametes leads to the development of zygotes and thereby new individual. 39 40 41 Differences between mitosis and meiosis S. Mitosis Meiosis No. 1 Consists of one nuclear Consists of two nuclear divisions divisions. 2 One cell cycle results in One cell cycle results in production production of two daughtercells. of four daughter cells 3 The chromosome number of Daughter cells contain half the daughter cells is the same as that chromosome number of mother cell of mother cell (2n). (n) 4 Daughter cells are identical with Daughter cells are different from mother cell in structure and mother cell in chromosome number chromosome composition. and composition 5 It occurs in somatic cells. It occurs in reproductive cells 6 Total DNA of nucleus replicates About 0.3% of the DNA is not during S phase. replicated during S phase and it occurs during the zygotene stage 7 The prophase is not divided into The prophase I is divided into five sub stages. sub stages 8 There is no pairing between Homologous chromosomes pair homologous chromosomes. during Pachytene 9 Segregation and recombination Crossing over takes place during do not occur. pachytene 10 Chromosomes are in the form of Chromosomes are in the form of dyad at metaphase. tetrad at metaphase 42 11 The centromeres of all the The centromeres of all the chromosomes lie on the chromosomes lie on either side of equatorial plate (auto orientation) the equatorial plate (co-orientation) during metaphase. during metaphase I 12 At metaphase, centromere of The centromere does not divide at each bivalent divides metaphase I longitudinally. 13 One member of One member of sisterchromatids moves to homologouschromosomes moves opposite pole during to opposite poles during the Anaphase. anaphase I 14 Maintains purity due to lack of Generates variability due to segregation and recombination. segregation and recombination. 3. Chromosome structure- chromatid, chromomeres, centromere, telomere, secondary constriction, nucleolar organizer region, satellite; Chromosomes are rod shaped, dark stained bodies seen during metaphase. Chromosomes were first described by Strausberger in 1875. The term ‘chromosome’ was first used by Waldeyer in 1888. (Chroma- colour, soma =body), Deeply stained, while cytoplasm remained unstained. Each species has a definite chromosome number, represented by 2n. Somatic cells contains two copies of each chromosomes, which are identical in morphology, gene content and gene order and they are known as homologous chromosomes. Gametic chromosome number is precisely one half of the somatic number, represented by ‘n’. Zygote is produced by fusion of one male and one female gamete (n + n=2n). The chromosome have been considered as the physical bases of heredity because they have a special organization, individuality, functions and are capable of 43 self-reproduction. Their main chemical constituent is DNA, universally accepted genetic or hereditary materials found to carry genetic information from one generation to next generation. Through the electron microscope , chromosome structure of eukaryote can be visualized at various levels of organization. “ The chromatic fibers are the basic unit of chromosome structure. The organization of this chromatin fiber into a chromosome was proposed by different models. the most familiar models were 1. The folded fiber model put forth by Dupraw in 1965 2. Nuclesome- Solenoid model given by Korenberg and Thomas in 1974 Among the two of the nucleosome Solenoid model was the most accepted model. 44 Structure of a typical chromosome The chromosome are usually studied in the cells of root tip during mitotic metaphase under light microscope. A tyical chromosome consists of the following eleven parts as detailed below 1.Centromere The region of chromosome with which the spindle fibers are attached during metaphase is known as centromere or primary constriction or kinetochore. Centromere has four important functions viz., (i). Orientation of metaphase chromosomes at the equatorial plane. (ii.) Movement of chromosomes during anaphase, (iii). Division of centromere for chromatid segregation during mitotic anaphase and during meiotic anaphase II (iv.) Deciding chromosome shape. Since centromere is associated with movement of chromosomes at anaphase, it is also called as kinetochore. The shape of the chromosome varies based on the position centromere. Generally each chromosome has one centromere, but in some cases, the number of centromeres may vary from nil to many. 2.Chromatid One of the two distinct longitudinal sub units of a chromosome is called chromatid. These sub units get separated at mitotic anaphase and at second meiotic anaphase II. The chromatids arises due to replication during S phase of mitosis or Meiosis. The chromatids that arise from same chromosome that is still attached to a common centromere is called sister chromatids and the chromatid of non homologous chromosomes that involve in crossing over during prophase I of meiosis is called non sister chromatids. After centromere division each chromatid become a chromosomes. 3.Secondary constriction 45 The constricted or narrow region other than centromere is called Secondary constriction. It has constant position and there for can be used as a marker. It is generally found in the short arm of a chromosome, away from the centromere. 4.Satellite A chromosome segment separated from the main body of chromosome by one secondary constriction is known as satellite. A chromosome with secondary constriction is referred as satellite chromosome or sat chromosome. The sat chrosomosmes are associated with nucleolar organiser. 5.Chromomeres The linearly arranged bead like structures found on the chromosomes are known as chromomeres. These are clearly visible in polytene chromosomes. Aailable evidences indicate that chromomeres represent a unit DNA replication, chromosome coiling, RNA synthesis and RNA processing 6.Chromonema Under light microscope, thread like coiled structures are found in chromosomes and chromatids which are called chromonema (plural chromonemata). Chromonema is associated with size of the chromosome, duplication of chromosome and is the gene bearing portion of chromosome. 46 7.Telomere The terminal region of a chromosome on either end of a chromosome is called a telomere. the telomere if one chromosome cannot unite with the telomere of another chromosome due to polarity effect. The telomere of one chromosome cannot unite with the telomere of another chromosome due to polarity effect. The telomere effect avoids translocation between non homologous chromosomes by preventing their pairing. 8.Euchromatin region The region of chromosome that shows relatively uncoiled chromonema (the chromosome strand)that is transcriptionally active and shows staining behaviour characteristics of majority of the chromosomal complement, uncoiled during interphase and condensed during mitosis reaching a maximum density at metaphase. 9.Heterochromatin region Highly condensed region appearing during interphase stage having maximal condensation, composed of highly repetitive DNA. Heterochromatin is late replicate and is transcriptionally inactive. 10.Matrix A mass of achromatic region of chromosome is which the chromonema are embedded is called the matrix. Matrix of a chromosome is a non genetic material. 47 11.Pellicle The matrix of a chromosome is enclosed in a sheath called Pellicle. This is also a non genetic material of chromosome. The most ideal stage to study the structure of chromosomes is the metaphase of mitosis. A metaphase chromosome consists of two identical components called chromatids, which are held together at a specific region called primary constriction. It is usually found in the centre and hence it is commonly described as centromere. It shows a plate-like proteinaceous structure called kinetochore where the microtubules of the spindle become attached during cell division. The portions of a chromatid found on either side of a centromere, are called arms which may be equal (isobrachial) or unequal (heterobrachial), based on the position of centromere. Chemically, the eukaryotic chromosomes are composed of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), histone and non-histone proteins and certain metallic ions. The most important enzymatic proteins of chromosomes are phosphoproteins, DNA polymerase, RNA-polymerase, DPN-pyro phosphorylase, and nucleoside triphosphatase. The metal ions as Ca+ and Mg+ are supposed to maintain the organization of chromosomes intact. NUCLEOSOMES 48 A nucleosome is a section of DNA that is wrapped around a core of proteins. Inside the nucleus, DNA forms a complex with proteins called chromatin, which allows the DNA to be condensed into a smaller volume. Each nucleosome is composed of a little less than two turns of DNA wrapped around a set of eight proteins called histones, which are known as a histone octamer. Each histone octamer is composed of two copies each of the histone proteins H2A, H2B, H3, and H4. The chain of nucleosomes is then compacted further and forms a highly organized complex of DNA and protein called a chromosome. Sometimes, a chromosome may have an additional constriction apart from the centromere, called secondary constriction. Portion of the chromatid situated beyond the secondary constriction, is called as a satellite body. Such a chromosome with a satellite body is known as a sat-chromosome. Secondary constrictions most often take part in the formation of nucleolus. Hence, such secondary constrictions are called as nucleolar organizers. 49 The terminal end of a chromosome is called as telomere. It is functionally different from the rest of the chromosome. A chromatid may breakup into pieces and the pieces may rejoin, but no segment becomes connected to the telomere. Thus, the telomere shows a sort of polarity. The chromatid, when observed under the electron microscope appears to be composed of a very fine filamentous structure called chromonema. It represents a molecule of double stranded DNA extending from one end of the chromosome to the other. The chromonema occurs in a highly coiled state. The chromonema is in turn composed of a chain of tiny, bead-like structures called nucleosomes. A nucleosome consists of a core particle surrounded by a DNA strand. The core particle is formed by a group of proteins called histones. In a nucleosome there is an octomer of 8 histone molecules, two each of H2A, H2B, H3 and H4. In between the nucleosomes is the DNA portion not associated with histones and is called Linker DNA. A linker DNA with the nucleosome is described as a chromatosome. Chemically, the chromosomes are described as nucleo-proteins, since they contain DNA and proteins. The chromosomal proteins have nothing to do with the genetic potency of the organism. These proteins, called histones, regulate the gene action. Autosomes Autosomes are structures that contain the hereditary information. They do not contain information related to reproduction and sex determination. They are identical in 50 both sexes, i.e., male and female species of humans. There are 46 (2n) chromosomes in humans. Of these 46 chromosomes, there are 44 pairs of autosomes and contain information related to the phenotypic characters. Allosomes / Heterosomes The allosomes are sex chromosomes that are different from autosomes in form, behaviour and size. There are a pair of allosomes in humans. The X chromosomes are present in the ovum and either the X or Y chromosome can be present in the sperm. These chromosomes help in determination of sex of the progeny. If the offspring receives X chromosome from the mother as well as father, it results in a female child (XX). If the offspring receives one X and one Y chromosome from the parents, it results in a male child (XY). In simple words, it is the donation of X or Y chromosome by the father that helps in determination of the sex of the child. Apart from these two categories, chromosomes can further be divided according to the location of the centromere and number of centromeres. A chromatid is one of the two identical halves of a chromosome that has been replicated in preparation for cell division. The two “sister” chromatids are joined at a constricted region of the chromosome called the centromere A chromomere, also known as an idiomere, is one of the serially aligned beads or granules of a eukaryotic chromosome, resulting from local coiling of a continuous DNA thread. Chromates are regions of chromatin that have been compacted through localized reduction. 51 The centromere appears as a constricted region of a chromosome and plays a key role in helping the cell divide up its DNA during division (mitosis and meiosis). Specifically, it is the region where the cell's spindle fibers attach. Telomeres, the specific DNA–protein structures found at both ends of each chromosome, protect genome from nucleolytic degradation, unnecessary recombination, repair, and interchromosomal fusion. Telomeres therefore play a vital role in preserving the information in our genome. Secondary constrictions are the constricted or the narrow region found at any point of the chromosome other than that of centromere (primary constriction). The difference between the two constrictions can be noticed during anaphase, as chromosomes can only bend at the site of primary constriction. The specific region at which the nucleolus is attached to the chromatin fiber is called as nucleolar organizer region. In humans, the NORs are located on the short arms of the acrocentric chromosomes 13, 14, 15, 21 and 22, the genes RNR1, RNR2, RNR3, RNR4, and RNR5 respectively... 52 Satellite or SAT chromosomes are chromosomes th at contain secondary constructs that serve as identification. They are observed in Acrocentric chromosomes. It is commonly associated with the short arm of an acrocentric chromosome in humans. Satellite chromosomes are chromosomes 13,14,15,16,21,22 in human. These are often referred to as (sat) chromosomes or marker chromosome numbers. CENTROMERE The region where two sister chromatids of a chromosome appeared to held together is known as ‘centromere’. When chromosomes are stained they typically show a dark-stained region that is the centromere, also termed as Primary constriction. Under light microscope, centromere generally appears as a constriction in the chromosome, here it is also termed as ‘primary constriction’. Centromeres are the first part moving towards the opposite poles during anaphase. 53 The remaining regions lag behind and appear as if they were being pulled by the centromere. Therefore, chromosome movement is due to the centromeres of chromosomes. Hence they are also known as ‘Kinetochores’. In most species, each chromosome has a single centromere in a fixed position which does not change except due to structural chromosome aberrations. Therefore, the position of centromere serves as an important land mark in the identification of different chromosomes of a species. Each chromosomes is divided into two transverse parts by its centromere. These parts are called 'arms'. Functions Centromere is an important component of 1) Chromosome structure and segregation 2) Orientation of chromosomes at metaphase 3) Movement of chromosomes during anaphase 4) Formation of chromatids TELOMERE The two ends of a chromosomes are known as ‘Telomeres’. They are highly stable and do not fuse with other chromosomes. It is generally accepted that, the structural integrity and individuality of chromosomes is maintained due to the telomeres and that all stable chromosome ends are composed of telomeres. CHROMATIN Chromatin describes, DNA that is complexed with proteins. The primary protein components of chromatin are histones, which are highly basic proteins that associate readily with DNA. Histones combined with DNA form nucleosomes, which are the subunit of chromatin. Chromatin can exist as either euchromatin or heterochromatin. 54 CHROMOMERE In some species like maize, rye etc. chromosomes in pachytene stage of meiosis show small bead like structures called chromomeres. Chromomeres are visible during meiotic prophase (pachytene) and invisible in mitotic metaphase chromosomes. The distribution of chromosome show considerable variation in size. They may differ in size as in the case of maize or they may be of uniform size as in the case of rye. CHROMONEMA A chromosome consists of two chromatids and each chromatid consists of thread like coiled structures called chromonema (plural chromonemata). The term chromonema was coined by Vejdovsky in 1912. The chromonemata form the gene bearing portion of chromosomes. Nucleolus Organizer Regions (NORs) Nucleolus organizer region or NOR is a region in the chromosome around which the nucleolus forms. It is a region which is active in nucleolus formation and which functions in the synthesis of ribosomal RNA. Nucleolar organizer regions (NORs) are chromosomal landmarks that consist of tandemly repeated sequences of ribosomal 55 genes. Treating chromosomes with silver nitrate solution selectively stain the NORs. Since the NORs are located on the satellite stalks of the acrocentric chromosomes, silver staining will yield dark regions in these areas. SATELLITE CHROMOSOME In some chromosomes a second constriction, in addition to that due to centromere (primary constriction) is also present. It is known as “Secondary constriction. It is present in short arm near one end, or in many chromosomes they are located in the long arm nearer to the centromere. The region between the secondary constriction and the nearest telomere is known as satellite. Therefore, chromosomes having secondary constriction are called “Satellite Chromosome” or “Sat -Chromosomes''. The position of secondary constriction in Sat-Chromosome is fixed and remains constant. Nucleolus is always associated with the secondary constriction of Sat-Chromosomes. Therefore, secondary constrictions are also called as ‘Nucleolus organizerRegion” (NOR) and Sat- Chromosomes are often referred as Nucleolus organizer chromosome (NOC).NOR contains several hundred copies of the gene coding for ribosomal RNA (rRNA). 56 4. Chromatin chemical composition; Chromosome landmarks-Euchromatin and heterochromatin; centromere, bands, chromosome ends, knobs Chemical composition of Chromosome The chromosomes are composed of DNA and protein. Generally DNA is about 40% and protein 60%. The DNA consists of nucleotides while the protein is histone which consists of amino acids argine and lysine in abundance besides other amino acids. The presence argine and lysine amino acids make the histone protein, a positively charged particle. The DNA show negative charge due to phosphate. A polynucleotide part consisting of 200 nucleotides base pairs of the DNA takes two turn on the core of eight histone proteins forming a complex known as nucleosome. The negatively charged coil of polynucleotide is strongly attracted by the positively charged histone protein. The nucleosome appears as a beaded structure on the string under electron microscope. Two nucleosomes are joined by linker DNA consisting of up to 200 nucleotides. Each nucleosome units further wrap up into higher order coil called super coil. Highly condensed portions of chromatin are called heterochromatin. If these remain permanently condensed, they do not transcribe. The chromatin which is condensed only during cell division but remains in open configuration in an undividing cell is transcribed and thus genes are expressed. It is euchromatin. CHROMATIN CHEMICAL COMPOSITION Chromatin is a complex of DNA and protein found in eukaryotic cells. The primary function is to package long DNA molecules into more compact, denser structures. This prevents the strands from becoming tangled and also plays important roles in reinforcing the DNA during cell division, preventing DNA damage, and regulating gene expression and DNA replication. During mitosis and meiosis, chromatin facilitates proper segregation of the chromosomes in anaphase; the characteristic shapes of chromosomes visible during this stage are the result of DNA being coiled into highly condensed chromatin. 57 The primary protein components of chromatin are histones. An octamer of two sets of four histone cores (Histone H2A, Histone H2B, Histone H3, and Histone H4) bind to DNA and function as "anchors" around which the strands are wound. In general, there are three levels of chromatin organization: 1. DNA wraps around histone proteins, forming nucleosomes and the so- called beads on a string structure (euchromatin). 2. Multiple histones wrap into a 30-nanometer fiber consisting of nucleosome arrays in their most compact form (heterochromatin).[a] 3. Higher-level DNA supercoiling of the 30 nm fiber produces the metaphase chromosome (during mitosis and meiosis). Chromatin is composed of DNA (30-40%), RNA (1-10%) and proteins (50-60%). These constituents vary in different organisms and even in the different tissues of the same species. Even in the same call, the proportions of DNA, RNA and proteins vary with the stage of cell cycle. DNA content The DNA content varies among the cells of different organisms. The gametes (eggs and sperms) of a species have only one-half amount of the DNA present in its somatic tissues. In human, DNA content of egg or sperm is 2.8 picogram (pg) per cell, while in cow it is 3.3 pg per cell. The DNA content per cell also varies during the different stages of cell cycle. The haploid DNA content of a cell is denoted by 2C. DNA content of a somatic cell is 2C is G1 it doubles during the S-phase and becomes 4C in G2; it remains at 4C during prophase and metaphase stages of mitosis, and prophase I and metaphase I of meiosis. The 4C DNA of meiotic cells is reduced to half (2C) in the two daughter cells forming the dyad (due to the first meiotic division). Finally, each of the four component cells of the tetrad produced after the completion of meiosis contains 1C DNA which replicates to become 2C DNA of the gametes. Chemical Compounds in Chromatin Class 1. Nucleic Acids: The nucleic acids are of two types: i) Deoxyribonucleic acid (DNA) 58 ii) Ribonucleic acid (RNA) i) Deoxyribonucleic acid (DNA) DNA is present in the cells of all plants, animals, prokaryotes and in a number of viruses. DNA molecule appears to be the most stable substance that is present in the chromosome. It has also been reported in some cytoplasmic organelles like chloroplast, mitochondia and centrioles. The amount of DNA per set of chromosomes is constant for a particular species. The DNA content of a diploid somatic cell (2n) is exactly double that of haploid cells (n). There is a close relationship between DNA content and chromosome size. It has been found that about 31cm of double helix DNA equals a pikogram (Ipg = 10 -12g). In man a diploid cell contains about 5.6 pg of DNA which may correspond to about 174 cm of DNA. Denaturation and Renaturation of DNA: One of the procedures for understanding the organisation of DNA is the technique of denaturation and renaturation. (re-association). Separation of double stranded DNA or RNA into single strands by heating or by exposure to high pH is called denaturation. When denatured DNA (or RNA) is slowly cooled (in case of heat denaturation) or is subjected to low pH (in case of high pH denaturation), the single strands re-associate by complementary base pairing ; this is known as renaturation. During renaturation, the complementary strands collide randomly and undergo re- association. Using this technique, DNA-DNA and DNA-RNA hybrids can be produced; it is then called nucleic acid hybridization. DNA/RNA re-association depends chiefly on two parameters; concentration of DNA (moles per litre) and time(seconds). 1. The component that re-natures fast; it contains highly repetitive DNA. 2. The component showing intermediate re-association rate between the fast and the late re-associating components; it contains moderately repetitive DNA. 3. The third component re-associates very slowly; it contains non-repetitive or unique DNA. Thus denaturation-renaturation studies have revealed that the eukaryotic genome is composed of the following two types of base sequences: i) Unique or non-repetitive DNA 59 ii) Repetitive DNA. (i).Unique DNA: These are the sequences of DNA which are present in a single copy, in each genome. The base sequences of unique DNA are not repeated in the genome. The proportion of unique DNA varies in different eukaryotic organisms. It constitutes 8% of the rye genome. 25% of pea, 40% of snail and 70% of human genome. A large number of genes, e.g., most of the structural genes, are present in single copy in the genome. Bacterial genome is considered to be composed r unique DNA; it contains only 0.3% repetitive DNA which, in fact, is rDNA and codes for isomers. (ii.)Repetitive DNA: DNA sequences present in more than one copy per genome are called repetitive DNA. They consist of families of sequences that are not exactly similar but are related. However, the members of each family consist of a set of base sequences which are sufficiently similar to re-associate with one another. Differences among individual sequences occur due to deletion, insertion and substitution. Unequal crossing over plays a role in changing the size of these sequences. Repetitive DNA sequences are classified into two main groups: i) Moderately repetitive sequences ii) Highly repetitive sequences. (i).Moderately repetitive sequences: In the case of moderately repetitive sequences, the number varies from 2 to less than 105 copies per genome. The proportion of these sequences is variable in different species. Drosophila melanogaster has 12% of its DNA in the form of moderately repetitive sequences, while in man, 13% of the DNA is of this type. In Nicotiana tabacum, these sequences constitute 65% of the genome. Several genes are present in the form of moderately repetitive DNA sequences, e.g., genes for ribosomal RNA, genes for ribosomal proteins, genes for histones and several others. Several families of transposable elements are also grouped under this class of DNA. (ii). Highly repetitive DNA: 60 Highly repetitive DNA constitutes a smaller proportion of the genome. Generally, it consists of very short sequences which are repeated tandemly in large clusters. Highly repetitive DNA is also called simple sequence DNA due to its short repeating units. They may be present in more than 105 copies, even up to millions of copies per genome.These sequences are located in the constitutive heterochromatin, present mostly in the centromeric and telomeric religions of chromosomes. In mammalian genomes, the proportion of highly repetitive DNA is generally below 10%, while in Drosophila melanogaster, it is 17%. Another Drosophila species, D. virilis, contains more than 40% highly repetitive DNA sequences per genome.The short tandemly repeated DNA sequences are identical in some cases hut are related in the others. There exists a great variation among different individuals of a single species regarding the size of the tandem clusters; therefore, they can be used in “DNA finger printing”, for characterization of individual genomes. (2) Ribonucleic acid (RNA) RNA constitutes a very small proportion of chromatin ; it is 3.5% in human, 0.3% in cow and 10% in pea. Most of the RNAs are ribosomal RNA, mRNA and tRNA. But apart from these, a special class of RNAs called “chromosomal RNA” is associated with the chromosomes. Chromosomal RNA constitutes about 5% of the total chromosome weight. These RNAs are small molecules containing 40 to 60 nucleotides. They may be involved in the structural organization of chromatin fibres and gene regulation. According to function, RNAs are of two kinds: (a) Genetic RNA: In some viruses which contain RNA, RNA is the sole genetic material which carries all hereditary responsibilities. (b) Non-genetic RNA: In cellular organisms DNA is genetic material and RNA molecules do not carry out genetic functions. In these organisms different genetically controlled cellular functions are performed by different kinds of RNA called non-genetic RNAs. 61 Class 2. Histones or Basic Proteins: Chromatin of all eukaryotic cells is a nucleoprotein complex in which DNA strand is associated with proteins. The associated proteins are of two kinds; basic proteins (histones) and acidic proteins (non-histones). Histones are small proteins that contain between 100 and 200 aminoacids of which 20 to 30% are lysine and arginine. The histones bear positive charges which enable them to bind to DNA primarily by electrostatic attraction to the negatively charged phosphate groups in sugar-phosphate backbone of DNA. Histones also bind tightly to each other and both DNA-histone and histone-histone binding are important for chromatin structure. The histones found in all eukaryotic chromosomes are five distinct types: (i) H1 which is very rich in the basic amino acid lysine (mol. weight-approximately 21,500) (ii) H2a and H2b which are lysine-rich (mol. wts. 14000 and 13775 respectively). (iii) H3 and H4 which are rich in basic amino acid arginine (mol. wts. 15,320 and 11,280 respectively). The five histones are present in molar ratios of approximately IH1: 2H2a : 2H2b : 2H3 : 2H4. They are complexed with DNA to produce the basic structural subunits of chromatin, the nucleosome. The histones have been highly conserved during evolution and four of the five types of histones are similar in all higher eukaryotes. The sperms of a few eukaryotes are exceptions where the histones are replaced another class of small basic proteins called protamines.The chromatin shows about equal amounts of histones and DNA. Class 3. Non-Histone Proteins Non-histone proteins occur in much lower proportion than histones, and their proportion in the total chromosome mass varies considerably in the different organisms. These proteins make up about 4% in pea vegetative bud, 16% in growing pea cotyledon, while 25% in human HeLa cells. Non-histone proteins consist of various enzymes involved in different metabolic functions, e.g., DNA polymerase, RNA polymerase, nucleases, polynucleotide ligase, DNA methylase, proteases, histone methylases proteases, histone methylases, histone actylases, histone deacetylases, histone kinases etc. 62 Apart from these enzymes, certain non-histone proteins are found that have high electrophoretic mobility; they are called HMG (high mobility group) proteins. Some of the HMG proteins form association with chromatin fibres during transcription.The non- histones or acidic proteins are found more or less firmly associated with DNA-histone complexes. In contrast to the histones, the molecules of non-histone proteins are numerous and heterogenous. Their number in most of the organisms may be more than 100 and they may include the enzymes involved in the synthesis of DNA and RNA, i.e., DNA polymerase, RNA polymerase and protein molecules involved in the control of DNA and RNA synthesis. The content of non-histones varies throughout the cell cycle and from one differentiated cell to another but the histones remain constant in both these instances. The amount of non-histone proteins generally equals the DNA-histones combined. CENTROMERE : The centromere links a pair of sister chromatids together during cell division. This constricted region of chromosome connects the sister chromatids, creating a short arm (p) and a long arm (q) on the chromatids. During mitosis, spindle fibers attach to the centromere via the kinetochore. The physical role of the centromere is to act as the site of assembly of the kinetochores – a highly complex multiprotein structure that is responsible for the actual events of chromosome segregation – i.e. binding microtubules and signaling to the cell cycle machinery when all chromosomes have adopted correct attachments to the spindle, so that it is safe for cell division to proceed to completion and for cells to enter anaphase. Regarding mitotic chromosome structure, centromeres represent a constricted region of the chromosome (often referred to as the primary constriction) where two identical sister chromatids are most closely in contact. When cells enter mitosis, the sister chromatids (the two copies of each chromosomal DNA molecule resulting from DNA replication in chromatin form) are linked along their length by the action of the cohesin complex. It is now believed that this complex is mostly released from chromosome arms during prophase, so that by the time the chromosomes line up at 63 the mid-plane of the mitotic spindle (also known as the metaphase plate), the last place where they are linked with one another is in the chromatin in and around the centromere. CENTOMERE POSITION : Metacentric : Metacentric means that the centromere is positioned midway between the chromosome ends, resulting in the arms being approximately equal in length. When the centromeres are metacentric, the chromosomes appear to be "x-shaped." Submetacentric : Submetacentric means that the centromere is positioned below the middle, with one chromosome arm shorter than the other, often resulting in an L shape. Acrocentric : An acrocentric chromosome's centromere is situated so that one of the chromosome arms is much shorter than the other. The "acro-" in acrocentric refers to the Greek word for "peak." The human genome has six acrocentric chromosomes, including five autosomal chromosomes (13, 14, 15, 21, 22) and the Y chromosome. Telocentric : Telocentric chromosomes have a centromere at one end of the chromosome and therefore exhibit only one arm at the cytological (microscopic) level. They are not present in human but can form through cellular chromosomal errors. Telocentric chromosomes occur naturally in many species, such as the house mouse, in which all chromosomes except the Y are telocentric. Subtelocentric : Subtelocentric chromosomes' centromeres are located between the middle and the end of the chromosomes, but reside closer to the end of the chromosomes. CENTROMERE TYPES : Acentric : An acentric chromosome is fragment of a chromosome that lacks a centromere. Since centromeres are the attachment point for spindle fibers in cell division, acentric fragments are not evenly distributed to daughter cells during cell division. As a result, a daughter cell will lack the acentric fragment and deleterious consequences could 64 occur.Chromosome-breaking events can also generate acentric chromosomes or acentric fragments. Dicentric : A dicentric chromosome is an abnormal chromosome with two centromeres, which can be unstable through cell divisions. It can form through translocation between or fusion of two chromosome segments, each with a centromere. Some rearrangements produce both dicentric chromosomes and acentric fragments which can not attach to spindles at mitosis. The formation of dicentric chromosomes has been attributed to genetic processes, such as Robertsonian translocation and paracentric inversion. Dicentric chromosomes can have a variety of fates, including mitotic stability. In some cases, their stability comes from inactivation of one of the two centromeres to make a functionally monocentric chromosome capable of normal transmission to daughter cells during cell division. For example, human chromosome 2, which is believed to be the result of a Robertsonian translocation at some point in the evolution between the great apes and Homo, has a second, vestigial, centromere near the middle of its long arm. Monocentric : The monocentric chromosome is a chromosome that has only one centromere in a chromosome and forms a narrow constriction.Monocentric centromeres are the most common structure on highly repetitive DNA in plants and animals. Holocentric : Unlike monocentric chromosomes, holocentric chromosomes have no distinct primary constriction when viewed at mitosis. Instead, spindle fibers attach along almost the entire (Greek: holo-) length of the chromosome. In holocentric chromosomes centromeric proteins, such as CENPA (CenH3) are spread over the whole chromosome.The nematode, Caenorhabditis elegans, is a well-known example of an organism with holocentric chromosomes,but this type of centromere can be found in various species, plants, and animals, across eukaryotes. Holocentromeres are actually composed of multiple distributed centromere units that form a line-like structure along the chromosomes during mitosis.Alternative or nonconventional strategies are deployed at meiosis to achieve the homologous 65 chromosome pairing and segregation needed to produce viable gametes or gametophytes for sexual reproduction. Different types of holocentromeres exist in different species, namely with or without centromeric repetitive DNA sequences and with or without CenH3. Holocentricity has evolved at least 13 times independently in various green algae, protozoans, invertebrates, and different plant families.] Contrary to monocentric species where acentric fragments usually become lost during cell division, the breakage of holocentric chromosomes creates fragments with normal spindle fiber attachment sites. Because of this, organisms with holocentric chromosomes can more rapidly evolve karyotype variation, able to heal fragmented chromosomes through subsequent addition of telomere caps at the sites of breakage. Karyotype and idiogram ✓ The number, size and morphology of the chromosome set of a cell, individual or species is referred as karyotype. This term is often used for photomicrographs of chromosome preparations. ✓ Karotype refers to the characteristics features of chromosome of a species. In other words, karotype is a phenotypic appearance of chromosome of a particular species. ✓ Karotype is genetically identical for a particular species, but differ from species to species. In the study of karyotype the chromosome number, position of centromere, size of chromosome, position of satellite, and the degree and distribution of heterochromatic are taken into consideration. ✓ Karotype is represented by the gametic chromosome number. ✓ Karyotype is defined as the study of chromosome morphology of a chromosome complement in the form of size, shape, position of primary constriction or centromere, secondary constriction, satellite, definite individuality of the somatic chromosomes and any other additional features. ✓ Karyotype highlights closely or distantly related species based on the similarity or dissimilarity of the karyotypes. 66 Asymmetric karyotype Asymmetric karyotype is defined as the huge difference between the largest and smallest chromosome as well as less number of metacentric chromosomes in a chromosome complement. Symmetric karyotype Symmetric karyotype is defined as the small difference between the largest and smallest chromosome as well as more number of metacentric chromosomes in a chromosome complement. Karyotypes showing a deviation from this state are called asymmetrical. It is believed that, perfectly symmetrical karyotypes represent a primitive state from which more advanced asymmetrical karyotypes have evolved through structural changes in chromosomes. Idiogram A diagrammatic representation of the karotype of an organism such as G banding is referred as Idiogram. The ideogram is generally depicted in descending order of chromosome length. The study of karyotype and ideogram helps in understansing the evolutionary process. 67 When the karyotype of a species is represented by the diagram then such diagrams are called Idiogram. In an Idiogram the chromosomes of a haploid set of an organism are ordered in a series of decreasing size. It is represented by arranging the chromosomes in a descending order of size keeping their centromeres in a straight line. Each chromosome in the karyotype is designated by a serial number according to its position. A perfectly symmetrical karyotype has all metacentric chromosomes of the same size. Ideograms are a schematic representation of chromosomes. They show the relative size of the chromosomes and their banding patterns. A banding pattern appears when a tightly coiled chromosome is stained with specific chemical solutions and then viewed under a microscope. Some parts of the chromosome are stained (G-bands) while others refuse to adopt the dye (R-bands). The resulting alternating stained parts form a characteristic banding pattern which can be used to identify a chromosome. The bands can also be used to describe the location of genes or interspersed elements on a chromosome. Chromosome some banding techniques Chromosome banding is the process of staining chromosomes to help researchers better understand and identify their structural composition. Chromosome banding can be compared to chromosome tie-dying. The term “chromosome banding” refers to the tagging and identifying of chromosomes by giving the appearance of various coloured bands on stained chromosomes. 68 Chromosome banding technique is the staining of different regions of the chromosomes using specific dyes for staining AT rich/ GC rich or other type of regions of the chromosome with necessary pre-treatment to visualize unique type of alternating light and dark stripes or bands. The horizontal bands appear along the length of the chromosomes Banding patterns are chromosomal patterns of bright and dark transverse bands. These bands identify where genes are located on a chromosome. The bright and dark bands are visible when the chromosome is stained with a chemical solution and examined under a microscope. Because stains create patterns of bands down the length of the chromosome, staining of chromosomes is also known as the “banding technique.” According to one or more banding techniques, a band is the region of a chromosome that may be easily distinguished from its neighbouring sections by appearing lighter or darker. Basic principle of chromosome banding Chromosome banding is developed based on the presence of heterochromatin and euchromatin. Heterochromatin is darkly stained whereas euchromatin is lightly stained during chromosome staining 69 Band of a chromosome is defined as that part of a chromosome which is clearly distinguishable from its adjacent segments by appearing darker or brighter with one or more banding techniques. The pattern of chromosome banding is highly specific in each chromosome of species. Classification of Banding of Chromosomes Banding techniques fall into the following two groups I.Bands distributed along the length of the whole chromosome 1. Giemsa banding (G-banding) 2. Quinacrine banding (Q-banding) 3. Reverse banding (R-banding) Staining of chromosomes in such a way those light and dark banded areas occurs along the length of the chromosomes for lateral comparison and identification of Pairs of a homologue. Bands are defined as parts of chromosomes that appear lighter of darker than adjacent regions, when treated with particular staining method. 1.G banding: The chromosomes are treated with trypsin and then stained with Giemsa. This banding technique is useful to distinguish between euchromatin and heterochromatic regions of a chromosome. The euchromatic region stains lightly and most of the heterochromatic region stain darkly by this staining. The dark regions tend to be heterochromatic, late replicating AT rich and inactive. The light regions tend to be euchromatic, early replicating and GC rich. i).Positive G-bands The darkly stained bands are positive G-bands. These areas are hydrophobic and facilitate the precipitation of the thiazine-eosin compound. The hydrophobic proteins are responsible for hydrophobicity. These proteins maintain the condensed areas. They are primarily AT-rich regions and constitute the late replicating heterochromatin. (ii).Negative G-bands 70 Negative G-bands are light-stained bands. These are less condensed early replicating euchromatin. Base pairs from GC are abundant in these areas. These areas are less hydrophobic and less conducive to the precipitation of thiazine-eosin. 2.Q banding: The chromosomes are stained with a flourochrome dye, usually quinacrine mustard, or quinacrine dihydrochloride and are viewed under ultra violet light, the banding techniques is useful in identifying the Y chromosomes and polymorphism that are not easily demonstrated by the G banding techniques. Pattern of bands is very similar to that seen in G-banding 3.R banding This produced by treating chromosomes with heat in phosphate buffer and then stained with Giemsa to produce pattern that is the reverse of G banding , thereby allowing the evaluation of terminal bands that are lighter after G banding. II.Bands that stain specific chromosome structures 1.Centromeric heterochromatin staining (C-banding) 2. Nucleolar-organizer-region staining (NOR staining) 3. T-banding 1.C- banding This is produced by treating chromosomes with alkali and controlling the hydrolysis with buffer salt solutions. This emphasizes the centromeric region. Usually, during staining process, a suitable dye is applied after cells have been arrested during cell division by a solution of colchicine. For humans, white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture. Sometimes observations may be made on non-dividing (interphase) cells. Most (but not all) species have a standard karyotype. The normal human karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes. Normal karyotypes for females contain two X chromosomes and are denoted 46, XX; males have both an X and a Y chromosome denoted 46,XY. Any variation from the standard karyotype may lead to developmental abnormalities. 71 Six different characteristics of karyotypes are usually observed and compared: 1. differences in absolute sizes of chromosomes. Chromosomes can vary in absolute size by as much as twenty-fold between genera of the same family: Lotus tenuis and Vicia faba(legumes), both have six pairs of chromosomes (n=6) yet V. fabachromosomes are many times larger. This feature probably reflects different amounts of DNA duplication. 2. differences in the position of centromeres. This is brought about by translocations. 3. differences in relative size of chromosomes can only be caused by segmental interchange of unequal lengths. 4. differences in basic number of chromosomes may occur due to successive unequal translocations which finally remove all the essential genetic material from a chromosome, permitting its loss without penalty to the organism (the dislocation hypothesis). Humans have one pair fewer chromosomes than the great apes, but the genes have been mostly translocated (added) to other chromosomes. 5. differences in number and position of satellites, which (when they occur) are small bodies attached to a chromosome by a thin thread. 6. differences in degree and distribution of heterochromatic regions. Heterochromatin stains darker than euchromatin, indicating tighter packing, and mainly consists of genetically inactive repetitive DNA sequences. A full account of a karyotype may therefore include the number, type, shape and banding of the chromosomes, as well as other cytogenetic information. Variation is often found: 1. Between the sexes 2. Between the germ-line and soma (between gametes and the rest of the body) 3. Between members of a population (chromosome polymorphism) 4. Geographical variation between races 5. Mosaics or otherwise abnormal individuals 2. N (Nucleolar organizing regions) banding 72 ✓ Nucleolar-organizer-region staining (NOR staining) is a technique that stains NOR regions that contain genes for ribosomal RNA. NOR are located in the satellite stalks of acrocentric chromosomes 13, 14, 15, 21, and 22. ✓ Acrocentric chromosomes have long and short arms with stalks and satellite regions without euchromatic regions. ✓ This stain uses a silver nitrate solution and is viewed with a brightfield microscope. ✓ The NOR regions could be selectively stained by techniques involving either giemsa or silver staining. ✓ NOR banding is useful to study some chromosome polymorphisms and to identify satellite stalks in nonacrocentric chromosomes. 3.T (Telomeric) banding ✓ T bands are, in fact, the segments of the R bands that are most resistant to the heat treatment ✓ The clear marking of telomeric regions of chromosome with T banding enables the detailed analyses of the structural rearrangements at the ends of chromosomes. ✓ It also allows the detection of human chromosome 22 and its involvement in translocation. The usefulness of this method is for the detection of dicentric rings that were undetectable by other procedures. Applications of banding techniques Useful for i. the identification of individual chromosomes of a species ii. the Identification of structural chromosomal changes viz., deletion, duplication, inversion and translocation iii. assigning linkage groups to specific chromosome and for gene mapping studies iv. Establishing evolutionary relationships between different species 73 Chromosome ends A telomere is a region of repetitive nucleotide sequences associated with specialized proteins at the ends of linear chromosomes (see Sequences). Telomeres are a widespread genetic feature most commonly found in eukaryotes. In most, if not all species possessing them, they protect the terminal regions of chromosomal DNA from progressive degradation and ensure the integrity of linear chromosomes by preventing DNA repair systems from mistaking the very ends of the DNA strand for a double-strand break. Telomeres are the repetitive nucleotide sequences that are present on the chromosomal endings. It is present in eukaryotic chromosomes. These are non-coding regions and do not code for any protein.Muller coined the term ‘telomere’. Barbara McClintock showed that broken chromosomes have sticky ends as compared to natural chromosomal ends, which are stable and do not show the tendency to fuse. Telomeres ensure that the chromosomes do not stick together and protect from deterioration. Telomeres contain non-coding repetitive sequences, which are rich in Guanine nucleotides. In humans, the repetitive sequence is 5’-TTAGGG-3’, which is repeated multiple times. Telomere Function – Role of Telomeres Telomeres are produced as a result of incomplete replication at the end of the chromosomes. In each replication cycle, a part of the DNA is lost. These protective end caps ensure that genetic information is preserved and not lost in the process. They play a vital role in ageing.They are essential for attracting telomerase replication machinery towards the terminus of the chromosomes and in regulation their function there. Additionally, telomeres are needed to stabilize the eukaryotic chromosomes in several ways. Telomeres protect the chromosomal termini from identification by the cell’s DNA damage response system. It caps the ends of the chromosomes thus preventing its degradation or their fusion. There are chances of fused chromosomes getting 74 missegregated in meiosis or mitosis. Often, telomeres are situated beneath the nuclear envelope and its particular association with the spindle pole body in the fission yeast is essential for the normal conduction of meiotic recombination. Telomere Replication Telomeres are the terminals of the linear chromosomes. They are the repetitive sequences coding for not a specific gene. The telomeres are involved in protecting the vital genes from deletion during cell division and shortening of DNA strands at the time of replication.Some of the telomeric sequences after every round of replication tend to get lost at the 5′ terminal of the synthesized strand on each of the daughter DNA. Since, these are noncoding sequences; its loss has no serious impact on the cell. These sequences however are not limitless. After adequate rounds of replication, the telomeric repeats get lost. There is a risk of DNA losing the coding sequences in the following rounds.Thus, telomeres play an important role in preserving genes on DNA and cellular ageing. It protects the genome from degradation, unnecessary repair and recombination and fusion between two chromosomes. Knobs In cytogenetics, a heavily staining enlarged chromomere that may serve as a landmark, allowing certain chromosomes to be identified readily in the nucleus. In maize, knobbed chromatids preferentially enter the outer cells of a linear set of four megaspores during megasporogenesis and are therefore more likely to be included in the egg nucleus (see meiotic drive); genetic markers close to a knob tend to appear more frequently in gametes than those far from a knob. Chromatin is a relative term used to describe the association of DNA and histone proteins in the formation of chromosomes. It is observed that chromatin is made of proteins (50-60%), DNA (30-40%), and RNA (1-10%). The chromatin structure is referred to as ‘beads on a string’ because the DNA threads are wrapped around histone proteins and observed during the replication process (mitosis/meiosis). 75 Chromatin fibers are stainable; hence, it is easy to find the cell replication stages based on chromatin bands. Chromatin functions include packaging genetic material to fit inside the nucleus and regulation of gene expression via replication of DNA, transcription, chromosome segregation, and recombination. Based on the structure, compaction, and location in a chromosome, chromatin fibers are classified as Heterochromatin, Euchromatin and Centromeric chromatin. 1. Euchromatin constitutes the chromatin structure associated with the active transcription of genes. 2. Heterochromatin makes up less accessible chromatin fibers thatare associated with silencing. 3. Centromeric chromatin are fibers recruited for spindle binding during chromosomal segregation. EUCHROMATIN & HETEROCHROMATIN Emil Heitzin the year 1928, coined the termHeterochromatin and Euchromatin. Euchromatin is chromatin fibers with transcriptionally active genes, with wider spaces. Between the nucleosomes (DNA + histone protein = repeating units of chromatin structure) hence they are also called Open Chromatin. Euchromatin fibers facilitate active transcription by allowing histone modifications and higher accessibility for transcription.Machinery to act, It is observed that euchromatin consists of more nucleosome beads with specific nucleusomalpositioning.This exact position of nucleosome aids in the Transcription regulation associated with precise. Recognition by histone proteins of DNA sequence motifs.Theeuchromatic sequence of the human genome 92% comprises the euchromatin structure FUNCTIONS Transcription 76 Euchromatin participates in the active transcription of DNA to mRNA products. The unfolded structure allows gene regulatory proteins and RNA polymerase complexes to bind to the DNA sequence, which can then initiate the transcription process. While not all euchromatin is necessarily transcribed, as the euchromatin is divided into transcriptionally active and inactive domains, euchromatin is still generally associated with active gene transcription. Epigenetics Epigenetics involves changes in the phenotype that can be inherited without changing the DNA sequence.This can occur through many types of environmental interactions. Regarding euchromatin, post-translational modifications of the histonescan alter the structure of chromatin, resulting in altered gene expression without changing the DNA. Additionally, a loss of heterochromatin and increase in euchromatin has been shown to correlate with an accelerated aging process, especially in diseases known to resemble premature aging. Euchromatin has loose chromatin structure and active for transcription. Heterochromatin has condensed chromatin structure and is inactive for transcription. When the non-dividing cells of the nucleus were observed under the light microscope, it exhibited the two regions, on the ground of concentration or intensity of staining. The dark stained areas are said as heterochromatin and light stained areas are said as euchromatin. Heterochromatin is further divided into two subcategories: constitutive and facultative heterochromatin. Constitutive heterochromatin 77 It is the stable form of heterochromatin, i.e. it does not loosen up to form euchromatin, and contains repeated sequences of DNA called satellite DNA. It can be found in centromeres and telomeres, and is usually involved in structural functions. Facultative heterochromatin Facultative heterochromatin, on the other hand, is reversible, i.e. its structure can change depending on the cell cycle, and is characterized by another kind of repeated DNA sequences known as LINE sequences. Eg: X chromosomes in mammalian female, one of which is active and remains euchromatic whereas other is inactive and forms at interphase. Difference between Heterochromatin and Euchromatin Euchromatin Heterochromatin Heterochromatin is a tightly Euchromatin is more lightly packed packed or condensed DNA DNA that is characterized by less That is characterized by intense staining and DNA Definition intense stains when stained sequences. That are with nuclear stains and Transcriptionally active or might transcriptionally inactive become transcriptionally- active at sequences some point during growth In heterochromatin, is the DNA is tightly bound in In Euchromatin is the DNA is lightly condensed DNA bound or compressed The DNA in heterochromatin conformation The DNA in euchromatin is is folded with the histone unfolded to form a beaded structure proteins The genes present in The genes present in euchromatin Staining heterochromatin are usually are either already active or will be inactive active during growth. Heterochromatin is darkly Euchromatin is lightly stained under Genes stained under nuclear genes nuclear stains. 78 Heterochromatin is Euchromatin is Transcriptionally- Transcription transcriptionally inactive active Heterochromatin has more Euchromatin less amount of DNA amount of DNA tightly lightly compressed with The histone DNA content compressed with the histone proteins proteins Heterochromatin Forms a Euchromatin a mere significant part Content in smaller part of the genome. In of the genome in humans it makes genome humans, it makes about 3- about 90- 92% The genome. 10% of the genome. Heterochromatin is found only Euchromatin is found in both Found in in eukaryotes prokaryotes and eukaryotes Heterochromatin exist two Euchromatin exists in a single form Types forms ;facultative and constitutive euchromatin constitutive heterochromatin Heterochromatin is present Euchromatin is present in the inner Location with in towards the periphery of the body of the nucleus the nucleus nucleus Heterochromatin exhibits Euchromatin doesn't exhibits Heteropycnosis heteropycnosis heteropycnosis Heterochromatin is a late Euchromatin early replicative that Replicative replicative that replicate later replicate earlier than Than euchromatin heterochromatin. Heterochromatin is not Euchromatin is affected by various Genetics affected by genetic processes. genetic processes that result in process Where the alleles are net variation within nucleus varied Heterochromatin maintains Euchromatin allows the genes To the structural integrity of the Function be transcribed and variation occur genome and allows the with in nucleus regulate gene expression 79 MATRIX The mass of acromatic material which surrounds the chromonemata is called matrix. The matrix is enclosed in a sheath which is known as pellicle. Both matrix and pellicle are non genetic materials and appear only at metaphase, when the nucleolus disappears. Genetic Significance of Chromosomes The chromosomes are considered as the organs of heredity because of following reasons: (i).They form the only link between two generations. (ii). A diploid chromosome set consists of two morphologically similar (except the X and Y sex chromosomes) sets, one is derived from the mother and another from the father at fertilization. (iii) The genetic material, DNA or RNA is localized in the chromosome and its contents are relatively constant from one generation to the next. (iv) The chromosomes maintain and replicate the genetic informations contained in their DNA molecule and this information is transcribed at the right time in proper sequence into the specific types of RNA molecules which directs the synthesis of different types of proteins to form a body form like the parents. 80 5. Types of chromosomes classified based on- position of centromere, number of centromeres, shape at anaphase, structure and appearance, essentiality, role in sex determination, structure and function TYPES OF CHROMOSOMES I.Depending on position of the centromeres, chromosomes can be grouped as a) Metacentric: Centromere is located exactly at the centerofchromosome, i.e. both arms are equal in size. Suchchromosomesassume ‘V’shape at anaphase. Human chromosome 1 and 3 are metacentric. b) Sub metacentric: Centromere is located on one side of the centerpoint such that one arm is longer than the other. These chromosomes become ‘J’ or ‘L’ shaped at anaphase. Human chromosomes 4, 12 are submetacentric. c) Acrocentric: Centromere is located close to one end of thechromosome and thus giving a very short arm and a very long arm. These chromosomes acquire ‘ J’ shape or rod shape during anaphase. Human chromosomes 13,15, 21, and 22 are acrocentric. d) Telocentric: Centromere is located at one end of the chromosome so that the chromosome has only one arm. These chromosomes are ‘I”Shaped or rod shaped. Humans do not possess telocentric chromosomes but they are found in other species such as mice. 81 II. Depending on the number of centromerepresent , chromosomes can be classified as 1.Monocentric having one centromere ; All types of plants and animals. 2.Acentric (without centromere) ; Chromosomes without centromere are called Acentric Chromosomes. 3.Dicentric (with two centromeres): Chromosomes having two centromereare 82 called Dicentric Chromosomes. Translocation and paracentric inversion are known to form Dicentric chrom. It is used as biomarkers to study the genetic syndromes. 4.Holocentric chromosomes: In this type of Chromosomes Diffuse the centromere is non-localized. 5.Polycentric chromosomes: Chromosomes having more than two centromeres are called Polycentric Chromosomes. Formed as a result of translocation, deletion and duplication. No such significance because the cells tend to die. Acentricand dicentric chromosomes are produced due to chromosomal aberrationsand cannot orient properly on the equatorial plate and lag behind other chromosomes during anaphase movements. 83 Some species have diffuse centromeres with microtubules attached along the length of the chromosomes and are called holocentric chromosomes. III. Depending upon their role in sex determination, chromosomes can be classified as Autosomes Differ in morphology and number in male and female sex and contain sex determining genes. Allosomes Do not differ in morphology and number in male and female sex and rarely contain sex determining genes. IV. Depending upon the structure and appearance, chromosome can be grouped as a) Linear Linear structure or having both endsfree. It is found in eukaryotes. b) Circular Circular shape and structure.It is found in bacteria and virus. V.Depending upon the structure and function, chromosome can be grouped as a) Normal chromosomes Normal members;Essential for normal growth and development b) Special chromosomes 84 Significantly differ in structure and function; Ex. Lamp brush, polytene and Bchromosomes Sex determination The most important structures in a cell during division are the chromosomes, which contain DNA. This is because they are responsible for the transmission of the hereditary information from one generation to the next. There are two types of chromosomes. Those are autosomes and sex chromosomes. Sex chromosomes are important in sex determination. Autosomes Any chromosome, which is not a sex chromosome, is an autosome. There are 22 pairs of autosomes in humans. Each autosome contains a large number of genes arranges in a definite sequence. In these homologous pairs, the 2 chromosomes are of the same length. The position of the centromere is the same. Mitosis is the process by which all these chromosomes duplicate and give one copy of each chromosome to each of the daughter cells. This ensures that all somatic cells of an organism’s body carry an identical set of genes. Allosomes H.Henking (1891) first identified the chromosome involved in sex-determination. Allosomes are otherwise called as sex-chromosomes. These are the chromosomes responsible for the determination of sex. The allosomes are of two types viz., X and Y. Modern geneticists have reported many different mechanisms of determination of sex in living organisms. Some important and common mechanisms of ‘sex’ determination are the following. Humans have 23 pairs of chromosomes. One of them is known as sex chromosomes. Other 22 are autosomes. Sex chromosomes are of 2 types; X and Y. Y is smaller, and X and Y are partially homologous. However, they pair and segregate into daughter cells during meiosis. Males have X and Y combinations. Females have X and X combination. All female gametes carry only one X chromosome. Male gametes may carry X or Y. The number carrying X is equal to the number 85 carrying Y. If an ovum is fertilized by a sperm carrying X, the result is a female XX. If an ovum is fertilized by a sperm carrying Y, the result is a male XY. Sex linked characters show deviations from Mendel’s laws. All the genes carried by X chromosomes do not determine sexuality. Many of the genes have other functions similar to genes carried in autosomes. Sex linked inheritance shown by genes carried with X chromosomes is not represented in Y chromosomes. These genes normally have recessive alleles and are shown by recessive mutations. These defective alleles are very rare in human population. Therefore, they are not expressed in females as they carry 2X chromosomes. Heterozygous females are carriers, and they may pass the gene on to their sons. They are expressed in males because they have only one X chromosome. What is the difference between Autosomes and Sex Chromosomes? Autosomes Sex Chromosomes 1. homologous chromosomal partially homologous chromosomal pairs pairs 2. sex determination do not involve in sex determination 3. autosomal pairs both the In sex chromosomes, Y chromosomes are of the chromosome is shorter same height 4. the position of the position of the centromere may not centromere is the same. be the same Bisexual/ hermaphroditic flower Bisexual flowers are complete flowers, containing both androecium and gynoecium in one flower. Therefore, bisexual flowers contain both stamens and pistils in the same flower. Hence, bisexual flowers are called hermaphrodite or androgynous flowers as well. In bisexual plants, both self pollination and cross pollination can occur due to the presence of both reproductive organs in the same flower itself. During self pollination, the stigma of a plant is pollinated by the pollen 86 grains of a genetically identical flower. Hence, self pollination produces genetically identical offspring to the parent. It occurs in three ways: autogamy, geitonogamy, and cleistogamy. The pollination within the same flower is called autogamy. Geitonogamy is the pollination between different flowers on the same plant. Cleistogamy is the pollination of the flower before its opening. Ex: Rose, rice, mustard, sweet pea, hibiscus Unisexual/incomplete flower Unisexual flowers are incomplete flowers, containing either male or female reproductive organs in the flower. That means, androecium, which is the male reproductive structure and gynoecium, which is the female reproductive structure, are found in separate flowers. The flowers containing the androecium are called male flowers and the flowers containing gynoecium are called female flowers. In some plants, both male and female flowers occur in the same plant. These plants are called monoecious plants. Ex.: Corn is the most common monoeci

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