7.2.2.

3 Nuclear chromatin

Chromatin is substance of which chromosomes are made. It consists of DNA filaments associated with proteins named histones. The chromatin in interphase is the relaxed form of chromosomes; during cell division, the chromatin condenses to form the chromosomes. With electron microscope the chromatin can be observed as a network within nucleus and which attaches to the nuclear lamina. The chromatin can be also observed in light microscopy. Because of its DNA content, the chromatin is intensely stained with basic dyes like hematoxylin and Tripan blue. According to the differentiated staining two types of chromatin were described. The first one is euchromatin, observed as a network of fine filaments that are weak stained. The second one is heterochromatin, a condensed form and more intense stained, with the aspect of granules of different sizes. Therefore the euchromatin and heterochromatin contain the same substances, but with different wrapping degrees. The euchromatin is a metabolic active form the genetic information contained in DNA is transcribed into RNA , and the heterochromatin is an inactive form has genetic information that is not transcribed. A cell with a higher ratio of euchromatin has a more intense metabolic activity. This is the case of young cells and since the malignant cells are very young and very active cells, the content in euchromatin represents another criterion in the diagnosis of cancer. There are two types of heterochromatin: constitutive and facultative. The constitutive heterochromatin is permanently condensed in all cells of a tissue; the facultative heterochromatin is condensed only in certain cell types and in certain periods of development. Sexual chromatin described by Barr and Bertram in 1949 is a component of the facultative heterochromatin; it is named the Barr body and is bigger than other heterochromatin granules. It has usually a triangle shape, with the base on the nuclear membrane. The sexual chromatin is an inactive X chromosome that remains condensed in interphase. It is normally present only in women which have two X-chromosomes (46, XX). In men, the unique X chromosome is active and doesnt form a Barr body. Any extra Barr body, (47, XXX) the absence of Barr body in women (45, X Turner syndrome), or its presence in men (47, XXY Klinefelter syndrome) represent chromosomal abnormalities. The detection of Barr body is done by the Barr test which is a specific technique of cytogenetics. Y chromosome may be observed in the nucleus of quinacrine stained cells, as an intense fluorescent body, named the F body. By this method some abnormalities like the presence of two F bodies may be detected.
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7.2.2.4 Nucleolus

Nucleolus is present in all eukaryotic cells that accomplish protein synthesis; it is absent in embryonic cells that are fed by vitellus. The nucleolus was discovered by Valentin in 1836. The essential function of nucleolus is biogenesis of ribosomes. This function is achieved through synthesis of ribosomal RNA molecules according to the genetic information located in certain regions on several pairs of chromosomes. These regions are known as nucleolar organizers. In humans there are 5 nucleolar organizers, represented by 5 pairs of chromosomes on which genes for different types of rRNA molecules are repeatedly replicated. On each one of these genes rRNA molecules are intensely transcribed. During the transcription process the rRNA molecules associate with ribosomal proteins arrived from cytoplasm. As the transcription ends, the nucleo-proteic complexes result in ribosomal precursors; they pass form nucleus to cytoplasm through the nuclear pores, and eventually associate becoming functional ribosomes. In the light microscopy the nucleolus is observed as refringent corpuscle since it contains 85% dry substance, from which 3% is DNA (the nucleolar organizers), 7% rRNA and 90% nucleolar and ribosomal proteins. The nucleolus may be stained with pyronin, by the double staining method Brachet. By the Feulgen method, an intense stained heterochromatin ring is observed, that surrounds the nucleolus. In the electron microscopy, three different regions can be observed within the nucleolus: a fibrillar, a granular and an amorphous one. In the fibrillar zone there are thin filaments of DNA from nucleolar organizers and the rRNA formed on these DNA filaments. A specific protein named nucleolin was identified in this region. The granular zone consists of ribosomal precursors and may be disposed as islets between the other components. The amorphous zone fills the spaces between the other components and some authors consider it as a part of karyoplasm. The nucleolus has 1-2 m, a round or ovoid shape and may be situated anywhere in nucleus. The shape changes according to the age and activity of cell. In the malignant cell the nucleoli are multiple (around 20), big, and with irregular and monstrous shapes. The nucleolus/nucleus ratio is normally 1/3, but in the malignant cells is bigger. The criteria of malignancy are: big hypochromic nucleus (weak stained), multiple nucleoli, and low abundant basophilic cytoplasm. The old cell has a small hyperchromic nucleus, and abundant eosinophilic cytoplasm, because of the smooth ER.
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7.2.2.5 Karyoplasm

Karyoplasm is also known as the nuclear matrix. It is the fourth component of the nucleus that occupies the spaces outside the nucleolus and chromatin. It is part of nucleoplasm the whole content of nucleus, surrounded by the nuclear envelope. As the cytoplasmic matrix, the nuclear matrix has a well defined molecular structure. It has two parts: the true matrix and the matrix labile fraction. The true matrix is a network of fibrous, stable proteins, with high molecular weight that represents the nuclear equivalent of cytoskeleton. A specific protein nucleoplasmin was identified at this level. The labile fraction interacts with the true matrix and with nucleolus and is weakly linked to the true matrix. It consists of soluble proteins with small molecular weight and very heterogeneous with regard to their structures and functions. In the karyoplasm there are proteins with structural role and with roles in gene expression, DNA and RNA polymerases, nucleases that cut the nucleic acids and protein kinases with various functions. The karyoplasm also contains nucleotides, ATP, water and ions that interact with the chromatin. In both components there are non-histone proteins, with different electric charges, acidic or basic, with MW of 104-105 Da. The nuclear matrix controls nuclear shape through its fibrous network, it participates in DNA and RNA synthesis, intermediates the effects of some steroid hormones and participates to other metabolic processes. The nuclear matrix displays contractility depending on the bivalent cations Ca2+ and Mg2+ and not on ATP like other contractile structures. The monovalent cations like Na+ and K+ control the volume of nucleus. The concentrations of these ions in nucleus are different as compared to cytoplasm. The water content of nuclear matrix is also very high.

Chapter 8. Eukaryotic chromosomes

Chromosomes are the main cellular structures of heredity property of living organisms to have alike offspring in certain environmental conditions. All hereditary features of organisms such as the morphological, physiological or biochemical ones, and the most complex ones, such as behavior, are genetically determined. Genetic information is given by nucleotide sequence in genes (100-1,000 nucleotides/gene).

8.1 Functions of chromosomes

Chromosomes have the following functions: 1. The first important function of chromosomes is storage of genetic information. The chromosomes contain most of the genetic information in a eukaryotic cell; apart from the genetic information stored in nucleus (in chromosomes) the animal and human cells also have genes in mitochondria, and the plant cells, in mitochondria and chloroplasts. 2. Transmission of genetic information. In interphase chromosomes replicate (auto-replication) each chromosome is made of an individual molecule of DNA that will be doubled , and in division the doubled amount of DNA is equally distributed to the daughter cells. 3. Expression of genetic information is performed in two distinct steps. In the first one the genetic information stored into a gene (in DNA) is transcribed (copied) into a molecule of messenger RNA (mRNA) that leaves the nucleus and arrives in cytoplasm, in order to carry the genetic information to ribosomes. The second step in the expression of the genetic information is translation, in which the information of mRNA is transferred into amino acid sequence of a protein. The auto-replication of DNA and the next two consecutive steps represent the flux of genetic information within cells that is also known as the central dogma of molecular biology (the Cricks central dogma):

Auto-replication

DNA

Transcription

RNA

Translation

Proteins

4. Chromosomes have a role in evolution of genetic information. De novo (new) random mutations (changes in the nucleotide sequence of genes) result in individuals with different
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features within the same species (variability). The mutations are crucial for the evolution of species since the individuals most adapted to the environment will survive (natural selection as was described by Charles Darwin).

8.2 General morphological features of chromosomes

Morphological features of chromosomes are studied in their condensed form during cellular division, in anaphase, when they have aspect of sticks intensely stained with basic dyes. The general morphological features of chromosomes are useful in cytogenetics. The chromosomes have three important general morphological features. 1. Chromosomes have a narrower region named centromere (or primary constriction), that appears weak stained. The centromere (one centromere in human chromosomes monocentric chromosomes) is the place where the arms of chromosomes are attached to each other and where fibers of the mitotic spindle also attach to the chromosome. There are 4 types of chromosomes depending on the position of centromere: - metacentric chromosomes, with the centromere placed at the middle of chromosome, have two equal arms and consequently a V shape in anaphase; - submetacentric chromosomes, with the centromere situated close to the middle of chromosome, have two unequal arms and a L shape. Most of the human chromosomes are submetacentric chromosomes; - acrocentric chromosomes, with the centromere near the extremity of one arm of the chromosome, have an I shape; - telocentric chromosomes, with the centromere placed at the extremity of the chromosome, have an I shape. 2. Certain chromosomes (5 pairs in humans) have another narrower zone on one of their arms where the arm doesnt change the direction. This region is known as secondary constriction and represent is equivalent with a nucleolar organizer. 3. Some chromosomes have at one of the extremities a small fragment as a sphere linked by a pedicle to the arm. This sphere is satellite and the chromosomes are named SATchromosomes.

8.3 Human karyotype and notions about chromosomal anomalies

Karyotype represents the number and the morphology of chromosomes in metaphase. A metaphasic chromosome consists of two sister chromatids connected at the level of centromere. At the end of metaphase the chromosome splits in length into the two chromatids. The karyotype is a constant of species, and displays identical characters for all individuals of the same species. There are two types of cells in pluricellular organisms according to the number of chromosomes: - Diploid cells (somatic cells) have two sets of chromosomes (2n where n = number of chromosomes/set). The somatic cells are diploid (2n) because they have two sets of chromosomes, one set from either parent. - Haploid cells have one set of chromosomes (n); they are gametes and the precursor cells of gametes. After the fusion of 2 gametes with n chromosomes the egg cell will have 2n chromosomes and will be diploid. In humans, the number of chromosomes/set is n=23 (also the number of chromosomes in haploid cells), and the total number of chromosomes in diploid cells is 2n=46. A complete chromosomal formula includes 46 chromosomes. There are two categories of chromosomes: somatic chromosomes named autosomes (in humans there are 22 pairs of autosomes), and sex chromosomes named also gonosomes or heterosomes (one pair in humans). The sex chromosomes are represented by 2 X chromosomes in women, wile in men they are different: one X and one Y. Chromosomal formula is in women 46, XX and in men !46, XY. It includes

the total number of chromosomes, and the gonosomes are also indicated. In each pair of chromosomes of a diploid cell, one has maternal and the other has paternal origin. The two chromosomes of the same pair are also known as homologous chromosomes; they have the same size and morphology, and genes for the same characters (allele genes), arranged in the same sequence. As compared to autosomes, the gonosomes are not homologous: in men X and Y have different genetic information (but they may have certain areas of homology), and in women one of the two X is condensed during the interphase as a Barr body. For the human karyotype the chromosomes are arranged in pairs numbered from 1 to 22 according to the decreasing order of their size, and the pairs are also classified into groups labeled from A to G. The first group (A) contains the biggest chromosomes (in pairs 1, 2, 3); group B has the chromosomes in pairs 4, and 5; group C, the chromosomes in pairs 6-12 and the X chromosome(s); group D, the chromosomes in pairs 13-15; group E, the chromosomes in pairs
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16-18; group F, the chromosomes in pairs 19-20, and group G, the chromosomes in pairs 21-22. Separate is the Y chromosome. In some cases, the X and Y chromosomes are separately arranged, after the group G. The human karyotype is one of the most important methods in cytogenetics. In most of the cases the karyotype is obtained from lymphocytes separated from the peripheral blood collected on an anticoagulant (heparin). The lymphocytes are cultured for three days on special culture media that stimulate cell division and proliferation, in order to increase the number of cells. Cell division is next inhibited with colchicine and the lymphocytes in division are arrested in metaphase. The cells are broken and the chromosomes are spread on a microscopic slide. The chromosomes are next fixed and stained. Uniform staining of chromosomes allows identification of numeric anomalies but not structural anomalies. For accurate identification of pairs banding techniques are used and in this way successive bands are obtained on the chromosomal arms. G bands are obtained by a partial protein digestion of chromosomes with trypsin and then they are stained according to the Giemsa method. On the chromosomes dark and light bands appear after an alternative pattern. Using another banding technique, R bands are obtained, with a reversed pattern, as compared to the G bands. Using quinacrine in the so-called Q banding technique, green fluorescent bands can be observed on the chromosomes arms under a UV microscope. Finally, the chromosomes are photographed and then they are cut from photos and are stuck on a card in the order of chromosomal groups. The human karyotype is useful in order to identify numeric and structural chromosomal anomalies. There are numeric chromosomal anomalies involving gonosomes or autosomes. Among the numeric anomalies concerning gonosomes, Klinefelter syndrome (47, XXY), triple X syndrome (X-trisomy 47, XXX), and Turner syndrome (45, X) were previously mentioned. Among the numeric chromosomal anomalies concerning autosomes, presence of 3 chromosomes in pair 21 gives the most frequent autosomal abnormality, trisomy 21 (47, XY + 21), also known as Down syndrome. Patients with this syndrome display multiple malformations, and mental retardation; the frequency increases with the mothers age (35-40 years). The numeric chromosomal anomalies appear in 1/250 births but the real number of these anomalies is bigger since some of them are not compatible with life and drive to death of embryo and spontaneous abortion. The structural chromosomal anomalies are identified taking into consideration the position of bands on the chromosomal arms. Among the structural anomalies can be mentioned: breaks, intrachromosomal rearrangements (a chromosomal fragment is inserted in another position),
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deletions (absence of parts of chromosomes), inversions (a fragment is broken and is stuck in inversed position), ring chromosomes, interchromosomal rearrangements such as translocations (change of fragments between two different chromosomes). Some chromosomal anomalies can be detected in utero, on karyotypes obtained from the cells of chorion villi, or from the amniotic cells collected by amniocentesis after the 14th week of pregnancy.

8.4 Chemical composition, ultrastructure and molecular organization of chromatin

From a chemical point of view, chromatin in eukaryotes consists of DNA, which is the genetic material and the main component, a low amount of RNA transcribed on DNA, and proteins. Two categories of proteins are found in chromatin: non-histone proteins (described for the karyoplasm), and histones, which are specific chromatin proteins in eukaryotes. The histones are low molecular weight (10,000-20,000 Da), basic proteins. They are classified into 5 classes: H1, H2A, H2B, H3 and H4. The histones of different classes have specific amino acid composition and different amino acid sequences. H1, H2A and H2B histones have lysine in high amounts, while H3 and H4 histones contain arginine as the most important amino-acid. Taking into account the amino-acid sequence, it is important to mention that H1 histones have variable sequences at different species, while H2A and H2B histones have a much more constant sequence, and H3 and H4 have a very constant amino-acid sequence regardless the species. Ultrastructure of chromatin. Examined on electron micrographs the eukaryotic chromosomes in interphase can be seen as fibers or granules made of chromatin fibers. Each chromatid has a single DNA molecule. Levels of molecular organization of chromatin Nucleosome is the fundamental unit of chromatin organization. It consists of a protein core surrounded by DNA and a linker DNA between two adjacent nucleosomes. The core is an octamer, made of 8 histones (two of H2A, H2B, H3 and H4), and resembles to a flat cylinder, of 10-11 nm diameter and 5.5 nm thickness. The protein octamer is surrounded by two spires of DNA. The DNA surrounding the core has the same length in all species 146 base pairs. The linker DNA has variable length according to species, between 0 to 100 base pairs. Therefore, the nucleosome has a total of 146-246 base pairs. The H1 histones link to the linker DNA. Thin chromatin filament of 10-11 nm is made of the lined nucleosomes suchlike the pearls in a necklace. Its diameter it corresponds to the diameter of nucleosomes.
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Thick chromatin fiber of 30 nm diameter is formed by a three dimensional arrangement of nucleosomes, in most cases by coiling of the thin chromatin filament. Chromatin loop or domain is made of the thick chromatin fiber folded in loops of different sizes. In order to form a loop specific regions on the thick fiber adhere to a protein network of chromosome, known as the chromosomal scaffold (with a spiral shape in the condensed chromosome). These regions are the scaffold attachment regions (SAR). The chromatin loop represents a functional unit of chromatin, because all the genes from a loop are transcribed together or are not transcribed at all; each loop also has an origin point for replication. Chromosomal bands result by spatial wrapping of loops. In regions with more compact spires dark bands appear on the chromosomal arms (when stained with the G banding method). In the other regions with less packed loops light bands can be observed.