Themes: 1

The light microscope structure. Morphology of the cell.

Life cycle of cell. Mitosis. Reproduction.

Morphology of chromosomes. Human karyotype. Organization of information flow in the cell

 

I. The light microscope structure. Morphology of the cell.

1.     Light microscope structure: mechanical, optical, illuminative parts.

2.     The main principles of working with light microscope.

3.     The Cell Theory in its modern form. Importance for medicine.

4.     Cellular level of life organization: prokaryotic and eukaryotic cells.

5.     The common functions and structures of cells: cytoplasm, nucleus and cell membrane.

6.     Structural compounds of cytoplasm: cytosceleton, organelles and inclusions.

7. Fluid-mosaic model of the plasma membrane.

Microscoping — the basic method of study of preparation—utilized in biology already has been more than 300 years. There are two main kinds of modern microscopes: 1) light microscope and 2) electron microscope. In the light microscope light rays passing through a specimen are brought to focus by a set of glass lenses, and the resulting image is then viewed by the human eye. In the transmission electron microscope, electrons passing through a specimen are brought to a focus by a set of magnetic lenses, and the resulting image is projected onto a flujrescent screen or photographic film.

Cell theory, in its modern form, includes the following principles:

1.   Cell is elementary structural and functional unit of living things, within which the life processes of metabolism and heredity occur.

2.   Cell is the unit of development of all organisms.

3.   Cells are like in all organisms.

4.   Cell arise only by division of previously existing cell.

5.   Multicellular organism is composed of many cells, which form tissues, tissues form organs, organs form  systems of organs, which neuroendocrine system regulates.

Cells are of two fundamental types according to presence or absence of a nucleus: prokaryotic and eukaryotic.

Some more differences between Prokaryotic and Eukaryotyc cells

 

The main components of eukaryotic cell are:

1) Cell membrane (is also known as plasma membrane, plasmalemma);

2) Nucleus;

3) Cytoplasm.

1.                       Plasma membrane envelops the cell and aids in maintaining its structural and functional integrity. It is composed of a lipid bilayer and assosiated proteins. Lipid bilayer is composed by phospholipids (hydrophilic and hydriphobic), glycolipids and cholesterol. Membrane proteins may be integral, which dissolved in the lipid bilayer and peripheral proteins, which don‘t extend into the lipid bilayer. They may be divided into three groups: channel-forming proteins, receptors and markers. On an outside surface of plasma membrane (in animal cells) is glycocalyx. It is consists of glycoproteins and glycolipids.

 

The functions of plasma membrane (semipermeable membrane between the cytoplasm and the extracellular environment) are: passage of water, passage of bulb material (phagocytosis and pinocytosis), selective transport of molecules, reception of information, expression of cell identity, physical connection with other cells, enzyme activity.

2. Nucleus consists of nuclear envelope, nucleolus, ucleoplasm and chromatin                  (chromosomes). Nuclear envelope surrounds the nuclear material, consists of two parallel membranes separated from each other by a narrow perinuclear cisternae. Nuclear envelope is perforated at intervals by openings called nuclear pores.

Nucleolus is a well-defined nuclear inclusion (sometime more than one). It is present in the cells that are actively synthesizing proteins. Become detectably only when the cell is in interphase. It is involved in the synthesis of rRNA and its assembly into precursors of ribosomes.

Nucleoplasm is the portion of the protoplasm that is surrounded by the nuclear envelope. It is consists of a matrix and various types of particles.

Chromatin is double-stranded DNA complexed with histones and acidic proteins. It is responsible for RNA – synthesis, resides within the nucleus in two forms: heterochromatin and euchromatin.

 

The main functions of the nucleus are: 1) direct protein synthesis in the cytoplasm via ribosomal ribonucleic acid (rRNA), messenger RNA (mRNA) and transfer RNA (tRNA), which are synthesized in the nucleus; 2) nucleus contains the genetic apparatus encoded in the deoxyribonucleic acid (DNA) of chromosomes.

3. Structural components of the cytoplasm are: 1) cytoskeleton; 2) organelles; 3)  inclusions. The fluid component is called cytosol.

The cytoplasm is dynamic functional interactions among certain organelles that result in the uptake and release of material by the cell, protein synthesis and intracellular digestion.

Cytosceleton is the structural framework within the cytosol. Cytosceleton functions in maintaining cell shape, stabilizing cell attachments, facilitating endocytosis and exocytosis, promoting cell motility. Cytosceleton includes such components as microtubules, microfilaments, intermediate filaments, microtrabecular lattice.

Organelles are the complex of the cells contain specialized structures, which are metabolically active units of living matter and usually are limited by a membrane. They divide into two groups: 1) for general purpose – ribosomes, Rough Endoplasmic reticulum (RER), Smooth Endoplasmic reticulum (SER), annulate lamella, Golgi apparatus, lisosomes, mitochondria, centrosome, vacuoles; 2) for special purpose – myofibrils, cilia and flagella.

 

Structure and functions of organelles for general purpose

 

ENDOPLASMIC RETICULUM                                  CENTRIOLES

 

 

                                             

 

GOLGI APPARATUS                                    LYSOSOMES

 

 

                                         

 

 

MITOCHONDRIA                                          RIBOSOMES

 

                        

Organelles for special purpose characterized for cells that perform special function. They are:

1) myofibrils in muscles tissue;

2)  cilia – thousands of shorter hair like structures all the cell surface for movement. Cilia can have different functions: to sweep fluid and particles across the stationary cell. In cells that line the human lungs, for example, cilia sweep dusts particles out toward the air passages to eventually be expelled in mucus or swallowed.

3)  flagella – tail like single appendage,  which enable the cell to move, f.e. sperm.

Inclusions are the “lifeless” accumulations of material that are not metabolically active and usually are present in the cytosol only temporarily. They divide into three groups: 1) trophic  (lipid droplets, glycogen, protein); 2) secretary (pancreas secret); 3) special – in high developed cells (hemoglobin in erythrocyte).

 

 

II. Life cycle of cell. Mitosis. Reproduction.

1. The main forms of cell division.

2. Cell (mitotic) cycle, its stages: G₁-, S-, G₂- phases, mitosis.

3. Mitosis, its stages. Biological significance of mitosis. Renewal, expanding and static cell population.

4. Cytological and cytogenetical characteristics of meiosis, mechanisms of the gametes genetic diversity formation. Biological significance of meiosis.

5. Gametogenesis in human.

Cell division is a process by which a cell, called the parent cell, divides into two or more cells, called daughter cells. Cell division is usually a small segment of a larger cell cycle. This type of cell division in eukaryotes is known as mitosis, and leaves the daughter cell capable of dividing again. The corresponding sort of cell division in prokaryotes is known as binary fission. In another type of cell division present only in eukaryotes, called meiosis, a cell is permanently transformed into a gamete and cannot divide again until fertilization.

Three types of cell division

The cell cycle, or cell-division cycle, is the series of events that take place in a cell leading to its replication. In prokaryotes, the cell cycle occurs via a process termed binary fission. In eukaryotes, the cell cycle can be divided in two brief periods: interphase — during which the cell grows, accumulating nutrients needed for mitosis and duplicating its DNA — and the mitotis (M) phase, during which the cell splits itself into two distinct cells, often called "daughter cells". The cell-division cycle is a vital process by which hair, skin, blood cells, and some internal organs are renewed.

Mitosis (Gr. Mitos, “thread”) – is the form of cell division by which a somatic (nonsex) cell duplicates. One of the basic characteristic of mitotic cell division is that one maternal cell divides into two identical ''daughter'' cells, each with its own set of the genetic material. After mitosis, the chromosome number in each daughter nucleus is the same as it was in the original dividing cell – it is the biological significance of mitosis.

Before the initiation of mitosis, the following events have occurred in the preceding interphase, which is divided into phases of gap (designed “G”) and synthesis “S”, that are of great importance for successful completion of the mitotic process. Cells that have temporarily or reversibly stopped dividing are said to have entered a state of quiescence called G₀ phase.

G₀ phase is a resting phase where the cell has left the cycle and has stopped dividing. Nonproliferative cells in multicellular eukaryotes generally enter the quiescent G₀ state from G₁ and may remain quiescent for long periods of time, possibly indefinitely (as is often the case for neurons). This is very common for cells that are fully differentiated.

G₁ phase (lasts from a few hours to several days):

1) is the gap phase just after mitosis during which cell growth in size and the cellular organelles increase in number;

2) RNA and protein synthesis occur.

S phase (lasts 8-12 hours in most cells):

1) is the synthetic  phase during which DNA replication occur, resulting in duplication of the chromosomes: one replicated chromosome consisting of two chromatids;

2) is the period when centrioles are self-duplicated;

G₂ phase (lasts 2 - 4 hours):

1) the gap phase, which follows the S-phase and extends to mitosis;

2) when the cell prepares to divide; the centrioles grow to maturity; energy required for the completion of mitosis is stored; RNA and proteins necessary for mitosis are synthesized.

During interphase a nucleus is limited from the cytoplasm by nuclear envelope. There are one or two nucleoli in it. Chromosomes are not condensed, that’s why they are not visible. The start of chromosome condensation at the end of G₂ also signals impending mitosis.

Mitosis, or M phase (lasts 1-3 hours):

1) involves division of the nucleus (karyokinesis) and division of the cytoplasm (cytokinesis), resulting in the production of two identical daughter cells;

2) includes 4 major phases: : prophase, metaphase, anaphase and telophase;

Prophase (lasts 30-60 minutes) begins when

1) the chromosomes condense and become rodlike and distinct chromosomes suddenly appear under the light microscope;

2) nucleoli and nuclear envelope disappear;

3) a mitotic spindle forms from microtubules: a centrosome contains centrioles and is the principal microtubule – organizing center of the cell; centrioles migrates to opposite poles of the cell and give rise to the spindle fibers and astral rays of the mitotic spindle; kinetochores begin to develop at the centromere region.

Metaphase:

1) chromosomes are aligned in a plane on the metaphase plate at equator.  

2) each chromosome consists of two identical chromatids, held together at a single point, the centromere;

3) spindle microtubules attach to chromosomes by kinetochores, special sites located at the centromere of each chromosome.

Anaphase begins as

1) kinetochores separate pulling sister chromatids apart (at this time they called chromosomes);

2) diploid set of daughter chromosomes move toward each opposite poles;

3) is associated with elongation of the spindle;

4) is also characterized ( in its later stages) by a cleavage furrow that begins to form the cell due to contraction of a band of actin filaments called the contractile ring (cytokinesis begins).

Telophase (lasts 30-60 minutes) is opposite to prophase and characterized:

1) by a deeping of the cleavage furrow, which leaves the midbody (containing overlapping polar microtubules) between the newly forming two identical daughter cells;

2) reformation of the nuclear envelope around the condensed chromosomes in the daughter cells;

3) reappearance of nucleoli, which arise from specific nucleolar organizer regions (called secondary constriction sites) on the chromosomes;

4)    mitotic spindle dissolves;

5) is completed as the daughter nuclei gradually enlarge and the dense chromosomes disperse to form the typical interphase nucleus with hetero- and euchromatin.

 Human chromosomes and chromatids during mitosis

	Begin Interphase	After Interphase	After Prophase	After Metaphase	After Anaphase	After Telophase
№ of Chromosomes	46	46	46	46	92	46
№ of Chromatids	46	92	92	92	92	46

 

Regulation of the cell cycle involves processes crucial to the survival of a cell, including the detection and repair of genetic damage as well as the prevention of uncontrolled cell division. The molecular events that control the cell cycle are ordered and directional; that is, each process occurs in a sequential fashion and it is impossible to "reverse" the cycle. Two key classes of regulatory molecules, cyclins and cyclin-dependent kinases, determine a cell's progress through the cell cycle.

Two families of genes, the cip/kip family and the INK4a/ARF (Inhibitor of Kinase 4/Alternative Reading Frame) prevent the progression of the cell cycle. Because these genes are instrumental in prevention of tumor formation, they are known as tumor suppressors.

Clinical considerations.

1) Transformed cells have lost their ability to respond to regulatory signals controlling the cell cycle. A disregulation of the cell cycle components may lead to tumor formation. Some genes like the cell cycle inhibitors, RB, p53 etc., when they mutate, may cause the cell to multiply uncontrollably, forming a tumor. Although the duration of cell cycle in tumor cells is equal to or longer than that of normal cell cycle, the proportion of cells that are in active cell division (versus quiescent cells in G0 phase) in tumors is much higher than that in normal tissue. They may undergo cell division indefinitely, thus becoming cancerous. The cells which are actively undergoing cell cycle are targeted in cancer therapy as the DNA is relatively exposed during cell division and hence susceptible to damage by drugs or radiation.

2) Vinca alkaloids may arrest these cells in mitosis. Treatment with colchicine and treatment with Nocodazole halt the cell in M.

3) Oncogenes represent mutations of certain regulatory genes, called proto-oncogenes, which normally stimulate or inhibit cell proliferation and development. Bladder cancer and acute myelogenous leukemia are caused by oncogenes.

Amitosis (direct cell division) is a type of cell division in which splitting of nucleus is followed by cytoplasm constriction. It is the mean of asexual reproduction in unicellular organisms like bacteria and protozoans and also a method of multiplication or growth in fetal membranes of some vertebrates.

Endomitosis is such kind of cell division in which chromosome reproduction doesn‘t lead to to nucleus division. Whole diploid sets of chromosomes may be multiplied; an individual tissue, or cell that has three or more sets of chromosomes is said o be polyploid (it‘s leads to polyploidy). Endomitosis is present in liver cells.

Polythenia is a chromonemas reproduction in the chromosomes. The chromosome number doesn‘t change (remains still). But the chromosomes increase in sizes considerably. It is observes in some special cells.

Cell populations are certain proportions of cells in a particular stage of the cell cycle in a tissue. In a renewal cell population the cells are actively dividing (epidermis of skin and epithelium GI tract). In the human body, renewal cell populations’ replace many trillions of damaged cells each day. In the expanding cell population, up to 3% of the cells are dividing. The remaining cells of the expanding population are not actively dividing, but they can enter mitosis when a tissue is injured and new cells are required to repair it. Fast-growing tissues of young organisms, as well as kidney, pancreas, and bone marrow tissues, consist of expanding cell populations.

Static cell populations are inactive and don‘t contain dividing cells. Nerve cells form this population. These cells grow by enlarging rather than dividing. A single nerve cell may grow to a meter in length, but it cannot divide.

Meiosis is a special form of cell division on which the chromosome number is reduced from diploid (2n) to haploid (n). It is occurs in developing germs (spermatozoa and oocytes) in preparation for sexual reproduction.

Meiosis is divided in the following stages:

Reductional division (meiosis I) – occurs following interphase during which the 46 chromosomes are duplicated.

Prophase I is divided into the following five stages:

Leptotene, during which the chromatin condenses into the visible chromosomes, each of which contain two chromatids joined at the centromere;

Zygotene, during which homologous maternal and paternal chromosomes pair and make physical contact (synapsis), forming a tetrad;

Pachytene, during which the chiasmata are formed and crossing over (random exchanging of genes between segments of homologous chromosomes) occurs – an event that is crucial for increasing generic diversity;

Diplotene, during which the chromosomes continue to condense and chiasmata can be observed, indicating where crossing over has taken place;

Diakinesis, during which the nucleolus disappears, chromosomes are condensed maximally, and the nuclear envelope disappears.

Metaphase I:

1) includes alignment of homologous chromosomes on the equatorial platter of the meiotic spindle in a random arrangement, thus facilitating genetic mixing;

2) includes attachment of spindle fibers from either pole to the kinetochore of any one of the chromosome pairs, thus assuring that genetic mixing takes place.

Anaphase I:

1) is similar to anaphase in mitosis except that each chromosome consists of two chromatids that remain held together;

2) involves migration of chromosomes to the poles.

Telophase I:

1) is similar to telophase in mitosis;

2) includes reinformation of the nucleus and cytokinesis, forming two daughter cells;

3) each daughter cell now contains 23 chromosomes (the haploid number – n), but has the diploid number – 2n of DNA;

4) each chromosome is composed of two sister chromatids, which are similar but not genetically identical.

 Human chromosomes and chromatids during Meiosis I

	Begin Interphase	After Interphase	After Prophase I	After Metaphase I	After Anaphase I	After Telophase I
№ of Chromosomes	46	46	46	46	46	23
№ of Chromatids	46	92	92	92	92	46

 

Equatorial division (meiosis II) - begins soon after the completion of meiosis I, following a very brief interphase without DNA replication.

1) Involves separation of sister chromatids in the two daughter cells formed in meiosis I and their distribution as chromosomes into two daughter cells, each containing its own unique recombined generic material (the haploid number – n of DNA);

2) Involves events similar to those in mitosis; thus the stages are named similarly (prophase II, metaphase II, anaphase II, and telophase II);

3) occurs more rapidly than mitosis.

Human chromosomes and chromatids during Meiosis II

	After Prophase II	After Metaphase II	After Anaphase II	After Telophase II
№ of Chromosomes	23	23	46	23
№ of Chromatids	46	46	46	23

 

The biological significance of meiosis:

Meiosis enables a species’ chromosome number to remain constant over generation. Meiosis produces novel combination of genes. Genes that are on the same chromosome recombine by crossing over that occurs between them during prophase I. Meiosis produces novel combination of non homologies chromosomes.

Gametogenesis refers to formation of mature ova and sperm, through a process involving meiosis. In human, the cells which will ultimately differentiate into eggs and sperm arise from primordial germ cells set aside from the somatic cells (oogonia and spermatogonia). The final products of gametogenesis are the large, sedentary egg cells, and the smaller, motile sperm cells. Each type of gamete is haploid. After fertilization and the formation of the polar bodies, the haploid sperm and egg nuclei (pronuclei) fuse, thus restoring the normal diploid complement of chromosomes.

Spermatogenesis. In the mature male functional sperm cells are produced within the seminiferous tubules of the testes. Around the periphery of the seminiferous tubules are located specialized cells known as spermatogonia. Spermatogonia are diploid cells set aside early in embryonic development. They may divide by mitosis to generate more spermatogonia. Spermatogonia destined to undergo meiosis first differentiate into primary spermatocytes which undergo the two divisions of meiosis. After the first division the cells are referred to as secondary spermatocytes which in turn undergo the second division to become spermatids.

Schematic drawimg of spermatogenesis

1. Meiosis in males is a continual process from puberty until death.

2. When spermatogonia reproduce by mitosis and  becomes a spermatocyte, ready to undergo meiosis.

3. At the completion of meiosis, there are four haploid spermatids, which resemble other cells in having cytoplasm and nucleus.

4. By the process of spermiogenesis, the spermatids change their physical structure to become mature spermatozoa (sperm). This involves getting rid of cytoplasm and developing an acrosomal body and tail. The time required is about 50 days from primary spermatocyte to spermatozoon.

5. All four haploid products become functional sperm.

6. 200-500 million sperm/ejaculate several times per week; 2-5 trillion per lifetime.

Oogenesis. Oogenesis is the process when haploid ova are formed and it occurs within the ovary. The cytoplasm of the primary oocyte increases greatly during the meiotic prophase and often contains large quantities of yolk accumulated from the blood. A diploid primary oocyte divides by meiosis producing, after the first division, a secondary oocyte and a polar body. In the second division the secondary oocyte divides giving rise to an ootid and another polar body. This second division does not occur unless fertilization of the secondary oocyte by a sperm occurs.

Schematic drawimg of oogenesis

1. Meiosis begins simultaneously in all primary oocytes in late embryonic/early fetal period, proceeding to late prophase I.

2. All oocytes then go into a resting phase until an ovarian follicle develops further.

3. At that point, meiosis resumes. The mature ovum that is released is a secondary oocyte.

4. In telophase I, one of the meiotic products becomes a polar body, with very little cytoplasm; the other product receives virtually all the cytoplasm.

5. If fertilization occurs, MII (meiosis II) is completed, with formation of a second polar body.

6. Only ovum of the four haploid cells is functional.

7. The polar bodies play no role in the formation of the embryo.

8. In female embryos, there are several million ovarian follicles; at birth, only 2 million; only 400 mature during lifetime.

 

III. Morphology of chromosomes. Human karyotype.

1. Structural and functional states of the chromosomes. Euchromatin and heterochromatin.

2. The levels of organization of eukariotic chromosomes.

3. Normal human karyotype characteristics. Сhromosomes types: metacentric, submetacentric, acrocentric, telocentric chromosomes.

4. Haploid and diploid chromosome number (set up). Autosomes and sex chromosomes.

5. Human chromosomes ideogram.

Hereditary information lies in chromosomes. The eukaryotic chromosomes are located within the nucleus of the cell.

Chromosomes serve important functions:

1. They carry hereditary characters from parents to offspring.

2. They help the cell to grow, to divide and to maintain itself by directing the synthesis of structural proteins.

3. They control metabolism by directing the formation of necessary enzymatic proteins.

4.      They undergo mutation and thus contribute to the evolution of the animal.

5.     They guide cell differentiation during development.

6.                     They form nucleoli in daughter cells at nucleolar organizing sites.

7.                     They bring about continuity of life by replication.

8.                     They play a role in sex determination.

Each species has a characteristic number of chromosomes: 1) Haploid number (n) is the number of chromosomes in germ cells (23 in humans). It forms during meiosis. 2) Diploid number (2n) is the number of chromosomes in somatic cells (46 in humans). It forms during mitosis.

Karyotype is a diploid number of chromosomes and it is characteristics of the number and morphology of chromosomes that is peculiarities of each species.

Karyotype is represented in humans by the 22 pairs of autosomes and the 1 pair of sex chromosomes (either XX or XY) totaling 46 chromosomes. Pair of chromosomes, with the same gene loci in the same order, is known as the homologous chromosomes. The chromosomes of each pair have characteristic size and shape. Thus, the chromosomes have individuality and are recognizable.

Schematic drawimg of chromosome (a), euchromatin and heterochromatin (b), human karyotype (c)

An ideogram is a karyotype, which displays chromosomes arranged in pairs in descending size order. Except Denver‘s classification, in which chromosomes are distinguish by their size and the position of centromere on 7 groups, there are such methods of chromosome identification as using of different methods of chromosome staining, among them fluorescent stains and laser using.

 Human chromosome classification (Denver‘s)

Groups	Number	Size, mcm	Characteristics
A	1, 2, 3	11 – 8, 3	1, 3 metacentric, large 2 – submetacentric, large
B	4, 5	7, 7	Large submetacentric
C	6 – 12, Х	7, 2 – 5, 7	Middle submetacentric
D	13 - 15	4, 2	Middle acrocentric
E	16 - 18	3,6 – 3, 2	Small submetacentric (18 – acrocentric)
F	19 - 20	2, 3 – 2, 8	Small metacentric
G	21 – 22, Y	2, 3	Small acrocentric

 

Metaphase chromosome structure. At first appearance, the chromosomes have already doubled, and each now consists of two identical rods, called sister chromatids. The chromatid is composed of a very fine filament, called as chromonema. The chromonema is a single double-stranded DNA molecule with a protein coat. The two chromatids remain attached to each other at a point of primary constriction, the centromere.  The centromere is a specific DNA sequence of about 220 nucleotides, to which is bound a disk of protein called a kinetochore. It is a place, where the spindle fibers attach during cell division. Regions on either sides of centromere are called arms. The long arm of a chromosome is designated “q” and the short arm – “p”. Some chromosomes (13, 14, 15, 21, 22) have secondary constriction site, which is a specific nucleolar organizer region, where rRNA genes located. Secondary constriction site divides arm of chromosome on satellites (the rest of the chromosome). The ends of the chromosome arms called telomeres, or tips. Telomeres keep chromosomes individuality. Satellites promote chromosomes sticking together.

Types of chromosomes.

a) 1 – centromere (primary constriction); 2 - short arm of a chromosome (p); 3 - long arm of a chromosome (q); 4 - secondary constriction site; 5- satellite.

б) 1 – centromere; 2 – chromonema.

b) Types of chromosomes: 1 – acrocentric; 2 – submetacentric; 3 – metacentric. A – arm; Б – centromere.

Chromosomes vary in the location of a centromere, and that‘s why they may be such types as:

1. Metacentric – if the centromere divides it into two equal arms.

2. Submetacentric – if the centromere is slightly displace from the center of chromosome. 

3. Acrocentric – if the centromere establishes one long arm and one short arm.

4. Telocentric – if the centromere is in the end of the chromosome and only one arm presents.

Eukariotic chromosomes have several levels of organization:

1. The DNA is associated with basic proteins called histones to form nucleosomes, each of which consists of 8 histones bead with DNA wrapped around it, plus an adjacent linker DNA with a histone attached.

2. The nucleosomes are organized into large coiled loops held together by nonhistone scaffolding proteins.

3. DNA molecules are much longer than the nuclei or the cells that contain them. The organization of DNA into chromosomes allows the DNA to be accurately replicated and segregated into daughter cells without tangling.

Chromosome functional states:

1.     Extended chromatin – chromosomes are not visible under the light microscope during interphase, when chromatin is scattered throughout the nucleoplasm.

2.     Condensed chromatin – chromosomes are visible under the light microscope during mitosis and meiosis.

Chromatin is the complex of DNA and protein found in the eukaryotic nucleus which packages chromosomes. The structure of chromatin varies significantly between different stages of the cell cycle, according to the requirements of the DNA.

The major structures in DNA compaction; DNA, the nucleosome, the 10nm "beads-on-a-string" fibre, the 30nm fibre and the metaphase chromosome.

Interphase chromatin. During interphase (the period of the cell cycle where the cell is not dividing), two types of chromatin can be distinguished:

1)    Euchromatin, which consists of DNA that is active, e.g., expressed as protein.

2)    Heterochromatin, which consists of mostly inactive DNA. It seems to serve structural purposes during the chromosomal stages. Heterochromatin can be further distinguished into two types:

·        Constitutive heterochromatin, which is never expressed. It is located around the centromere and usually contains repetitive sequences.

·        Facultative heterochromatin, which is sometimes expressed.

Heterochromatin is a highly condensed portion of chromatin, which remains permanently condensed during interphase. Its major characteristic is that transcription is limited. As such, it is a means to control gene expression, through regulation of the transcription initiation. Heterochromatin is not transcribed into RNA and appears in the light microscope as basophilic clumps of nucleoprotein. Heterochromatin is usually localized to the periphery of the nucleus.

Heterochromatin mainly consists of genetically inactive satellite sequences. Heterochromatin also replicates later in S phase of the cell cycle than euchromatin, and is found only in eukaryotes. Both centromeres and telomeres are heterochromatic.

Heterochromatin may be constitutive and facultative. Constitutiv – maintains structural integrity. Human chromosomes 1, 9, 16, and the Y chromosome contain large regions of constitutive heterochromatin. Facultative – corresponding to one of two X- chromosomes, is present in somatic cells of female mammals. X-chromosome is visible as dark-staining evagination protruding from the nucleus. This structure as called the Barr body, or sex chromatin of the second inactivated X chromosome in a female. It is situated in the nucleoplasm, near the nuclear envelope from internal side.

Euchromatin is a lightly packed (isn’t condensed) form of chromatin during interphase that is rich in gene concentration, and is often under active transcription. It is found in both eukaryotes and prokaryotes. Euchromatin comprises the most active portion of the genome within the cell nucleus. 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. Euchromatin that is “always turned on” is housekeeping genes, which codes for the proteins needed for basic functions of cell survival.

Euchromatin generally appears as light-colored bands and observed under an optical microscope; in contrast to heterochromatin, which stains darkly. This lighter staining is due to the less compact structure of euchromatin.

Clinical considerations.

Antibodies to certain types of chromatin organization, particularly nucleosomes, have been associated with a number of autoimmune diseases, such as systemic lupus erythematosus. These are known as anti-nuclear antibodies (ANA) and have also been observed in concert with multiple sclerosis as part of general immune system dysfunction.

Polytene chromosomes – giant bundles of unsepapated chromonemata occuring especially in the salivary glands of some insects. They are many (100-200) times longer than the chromosomes found in other somatic cells or germinal cells. Being unusually large, they are visiable under a light microscope.Polytene chromosomes of fruit fly have numerous (512-1024) chromonemata. They result from repeated replication of DNA without separation into daughter chromosomes.

Organization of information flow in the cell. Regulation of gene expression. Molecular mechanisms of human variation

1. Gene Expression in prokaryotes: transcription, translation.

2. Gene Expression in eukaryotic cells: transcription, processing, translation.

3. Translation stages: initiation, elongation and termination.

4. Central dogma of molecular biology (DNA® DNA® mRNA® protein).

Gene Expression. The process by which a gene produces a product, usually a protein, is called gene expression. DNA not only serves as a template for its own replication, it is also a template for RNA formation. Most often it is mRNA that is produced. The process by which a mRNA copy is made of a portion of DN A is called transcription. Follow­ing transcription, mRNA will have a sequence of bases that is complementary to that of DNA. Then, mRNA moves into the cytoplasm. Photographic data shows radioactively labeled RNA moving from the nucleus to the cytoplasm, where protein synthesis occurs. The central dogma of molecular biology also says that mRNA directs the synthesis of a polypeptide. During translation, the sequence of bases in mRNA dictates the sequence of amino acids in a protein.Gene expression requires both transcription and transla­tion. These terms are apt. Transcribing a document means making a copy of it, and translating a document means putting it in a different language.Gene expression includes the processes of transcription and translation. During transcription, DNA serves as a template for the formation of complementary RNA. During translation, the sequence of bases in RNA determines the sequence of amino acids in a protein.

Transcription. Transcription is the first step required for gene expression, the process by which a gene product is made. Most often this gene product is a protein, but we should note that the molecules tRNA and rRNA are also transcribed off DNA templates. These mol­ecules are also gene products. Just now we are interested in the formation of mRNA, which carries genetic information to the ribosomes, where protein synthesis occurs.

Messenger RNA. During transcription, a mRNA molecule is formed that has a sequence of bases complementary to a portion of one DNA strand; wherever A, T, G, or С is present in the DNA template, U, A, C, or G is incorporated into the mRNA molecule. A segment of the DNA helix unwinds and unzips, and complementary RNA nucleotides pair with DNA nucleotides of the strand that is to be transcribed. When these RNA nucleotides are joined together by an RNA polymerase, an mRNA molecule results. This molecule now carries a sequence of codons that will be used to order the sequence of amino acids in a protein. Transcription begins at a region of DNA called a pro­moter. A promoter is a special sequence of DNA bases where RNA polymerase attaches and the transcribing process begins. A promoter is at the start end of the gene to be transcribed. Some genes are on one of the DNA strands, and some are on the other strand.

Elongation of the mRNA molecule occurs as long as transcription proceeds. Only the newest portion of a RNA mol­ecule is bound to the DNA, and the rest dangles off to the side. Finally, RNA polymerase comes to a terminator sequence at the other end of the gene being transcribed. The terminator causes RNA polymerase to stop transcribing the DNA and to release the mRNA molecule, now called a RNA transcript. Many RNA polymerase molecules can be working to produce a RNA transcript at the same time. This allows the cell to produce many thousands of copies of the same mRNA molecule and eventually many copies of the same protein within a shorter period of time than otherwise.

RNA Processing. Since the advent of modern molecular techniques, investigators can compare the structure of various eukaryotic RNA transcripts and their corresponding genes. They do this by first isolating the mRNA and its corresponding gene. Then, they separate the DNA molecule into single strands and allow the mRNA to bind to its complementary strand. If the 2 molecules are indeed colinear, then the mRNA should bind along the entire length of its template DNA. Much to their surprise, researchers have found that much of human template DNA does not bind to its mRNA. The segments of a gene that do not bind to mRN A and therefore do not code for protein are called intervening sequences, or introns. The segments of a gene that do bind to mRN A and therefore do code for protein are called exons because they are expressed. By comparing the mRNA molecule present in the nuclei with that in the cytoplasm, it can be shown that both exons an introns are present in the primary mRNA transcript, but only exons are present in the mature mRNA transcript that leaves the nuclei and enters the cytoplasm. The introns are removed from the primary mRNA transcript by a process called RNA processing or RNA splicing.

Since the discovery of split (interrupted) genes in eukaryotes, 2 essential questions have been asked: How is processing carried out? and What is the function of introns in the first place? It was discovered that splicing of RNA may be done by spliceosomes, a complex that contains several kinds of ribonucleoproteins. A spliceosome cuts the primary mRNA and then rejoins the adjacent exons.

There has been much speculation about the possible role of introns in the eukaryotic genome. It's possible that introns allow crossing-over within a gene during meiosis. It's also possible that introns divide a gene up into regions that can be joined in different combinations to give novel genes and protein products, a process that perhaps facilitates evolution.

Some researchers are trying to determine whether introns exist in more primitive eukaryotes and in prokaryotes. They have found that the more primitive the eukaryote, the less likely a gene is to be interrupted by introns and the introns that do exist are shorter. At first it was thought that introns do not exist at all in prokaryotes, but an intron has been discovered in the gene for a tRNA molecule in Anabaena, a cyanobacterium. This particular intron belongs to a class of introns called "self-splicing." Self-splicing introns, which have the enzymatic capability of splicing themselves out of an RNA transcript, were discovered in the early 1980s. The finding of these so-called ribozymes did away with the belief that only proteins can function as enzymes. Ribozymes, however, are restricted in their function since each one cleaves only RNA at specific locations.

This discovery of ribozymes in prokaryotes is being used to substantiate the belief that RNA could have been the first genetic material and the first enzyme in the history of life. For many years, scientists have puzzled over which came first-DNA, which is the genetic material, or proteins, which are enzymes. Now it appears that this is an unnecessary dilemma. Possibly RNA could have fulfilled both functions in the first cell or cells. RNA molecules (mRNA, rRNA, and tRNA) are transcribed off of DNA templates. mRNA carries a copy of the genetic informa­tion needed for protein synthesis. Particularly in eukaryotes, the primary mRNA transcript is processed before it becomes a mature mRNA transcript.

Translation.Translation is the second step by which gene expression leads to protein synthesis. During translation, the sequence of codons in mRNA directs the sequence of amino acids in a protein. Two other types of RNA are needed for protein synthesis. rRNA is contained in the ribosomes, where the codons of mRNA are read, and tRNA carries amino acids to the ribosomes so that protein synthesis сan occur.

The process of translation must be extremely orderly so that the amino acids of a polypeptide are sequenced correctly. Protein synthesis involves 3 steps: initiation, elongation, and termination.

1.    Initiation of translation: A small ribosomal subunit attaches to the mRNA in the vicinity of the start codon (AUG). The first or initiator tRNA pairs with this codon. Then a large ribosomal subunit joins to the small subunit, and translation begins.

2.    Chain elongation: Each ribosome contains 2 sites, the P (for polypeptide) site and the A (for amino acid) site.During elongation, a tRNA with attached polypeptide chain is at the P site and an tRNA-amino acid complex is just arriving at the A site. The polypeptide chain is transferred and attached by a peptide bond to the newly arrived amino acid. An enzyme (peptidyl transferase), which is a part of the larger ribosomal subunit, and energy are needed to bring about this transfer. Now the tRNA molecule at the P site leaves.

Then translocation occurs: the mRNA along with the peptide-bearing tRNA moves from the A site to the empty P site. This makes it seem as if the ribosome has moved forward 3 nucleotides, especially since there is a new codon now located at the empty A site. The complete cycle-pairing of new tRNA-amino acid complex, transfer of peptide chain, translocation-is repeated at a rapid rate (about 15 times each second in E. coli).

3.  Chain termination: Termination of polypeptide chain synthesis occurs at a stop codon, codons that do not code for an amino acid. The polypeptide chain is enzymatically cleaved from the last tRNA, and it leaves the ribosome, which dissociates into its 2 subunits.

 During translation, the codons of mRNA base pair with the anticodons of tRNA molecules carrying specific amino acids.  The order of the codons determines the order of the tRNA molecules and the sequence of amino acids in a polypeptide.

Gene Expression in Review

1. DNA contains genetic information. The sequence of its bases determines the sequence of amino acids in a protein.

2. During transcription, one strand of DNA serves as a template for the formation of messenger RNA (mRNA). The bases in mRNA are complementary to those in DNA; every 3 bases is a codon that codes for an amino acid.

3. Messenger RNA is processed before it leaves the nucleus, during which time the introns are removed.

4. Messenger RNA carries a sequence of codons to the ribosomes, which are composed of ribosomal RNA (rRNA) and proteins.

5. Transfer RNA (tRNA) molecules, each of which is bonded to a particular amino acid, have anticodons that pair complementarily to the codons in mRNA.

6. During translation, tRNA molecules and their attached amino acids arrive at the ribosomes and the linear sequence of codons of the mRNA determines the order in which the amino acids become incorporated into a protein.