I. GENETIC APPARATUS OF HUMAN CELL – SUPPORT OF HEREDITY AND VARIABILITY OF HUMAN ORGANISM Introduction: Genetics is a branch of biology that deals with the heredity and variation of organisms; it studies the laws of storage, transmission and realization of genetic information that assure the development and functioning of organisms. Heredity is characterized through the transmission (inheritance) of genetic material from parents to offspring, determining the similarities between the individuals of the same species. Variability represents the differences between the parents and offspring determined by molecular, biochemical, physiological and environmental factors. The modern science of genetics traces its roots to Gregor Johann Mendel, who studied the nature of inheritance in plants. In 1865 Mendel described the inheritance patterns of certain traits in pea plants and expressed them mathematically. The importance of Mendel's work did not gain wide understanding until after his death, when other scientists working on similar problems re-discovered his research. Rediscovery was credited to three scientists - de Vries, Correns, Tschermak, who simultaneously obtained the results confirming the laws of heredity, discovered by Mendel. William Bateson, a supporter of Mendel's work, suggested the word ''genetics'' in 1905 (the adjective ''genetic'', derived from the Greek word ''genesis'' - "origin" and that from the word ''genno'' - "to give birth", and was first used in a biological sense in 1860). In 1909 Iogansen introduced such terms as gene, genotype, phenotype. After the rediscovery of Mendel's work, scientists tried to determine the cell molecules responsible for inheritance. In 1910, Thomas Hunt Morgan argued that genes are on chromosomes (based on observations of a sex-linked white eye mutation in fruit flies). In 1913, his student Alfred Sturtevant used the phenomenon of genetic linkage to show that genes are arranged linearly on the chromosome. The new era of genetics began with the discovery of DNA structure (Watson and Crick, 1953). With the molecular understanding of inheritance, an explosion of research became possible. One important development was DNA sequencing in 1977 by Frederick Sanger, allowing scientists to read the nucleotide sequence of a DNA molecule. In 1983, Kary Banks Mullis developed the polymerase chain reaction (PCR), providing a quick way to isolate and amplify a specific fragment of a DNA from a mixture. The new techniques and discoveries gave rise to molecular genetics. Genetics and Medicine: Human genetics is a part of genetics and is both a fundamental and an applied science that deals with the most interesting organism - the human being. The Human Genetics course is intended for medical students in order to facilitate their understanding of the main pathways of transmission of genetic information from parents to offspring, laws of variation and those of inheritance of normal and pathologic traits, role of genetic predisposition and environmental factors in appearance of diseases. Genetics has become an indispensable component of almost all research in modern biology and medicine. Great advances have been made since the discovery of the structure of DNA (1953), till the map of the complete human sequence (2003). Medicine has already assimilated much of the new information. Improved techniques for prenatal diagnosis have led to a decrease in the numbers

GENETIC APPARATUS OF HUMAN CELL – SUPPORT OF HEREDITY AND VARIABILITY OF HUMAN ORGANISM VARIABILITY

of children born with particular illnesses. Reproductive technology, too, helps to ensure that only healthy children are brought into the world. Genetics has also helped in treatment. Some diseases can be treated with a missing-gene product––sometimes, as with hemophilia, with a protein made by genetically engineered animals. New tests permit early identification and treatment. All infants are screened for phenylketonuria (PKU), a condition that results from a genetic inability to break down an amino acid found in many foods. Those found to be at risk are put on a special diet that reduces the disease's severity. Other genetic tests help prevent damage. Those carrying the gene for familial polyposis coli are offered a treatment––excision of the colon––that is drastic but effective, if done early. Their relatives, too, can be screened and offered the same treatment. Methods used in human genetics: The following methods are applied to study human genetics: * genealogical method; * twins method; * dermatoglyphic method; * cytogenetic method; * biochemical method; * population statistics method; * methods of molecular biology and genetic engineering. The development of different techniques and approaches has led to the development of many fields of specialization: Formal genetics studies segregation and linkage relationship of genes. Human cytogenetics deals with the study of human chromosomes in health and disease. Human biochemical genetics deals with the study of metabolic genetic diseases, proteins and enzymes in normal and mutant individuals. Clinical genetics deals with diagnosis, prognosis and to some extent treatment of various genetic diseases. Population genetics deals with the distribution of genes in large groups. Behavior genetics is a science that studies factors underlying behavior in health and diseases. Developmental genetics studies genetic mechanisms of normal and abnormal development. Human molecular genetics has its emphasis in the identification and analysis of genes at the DNA level. Thus, the field of human genetics is large, and its borders are indistinct. The rapid development of human genetics during recent decades has created many interactions with other fields of science and medicine. Apart from general and molecular genetics, these interactions are especially close with cell biology, biochemistry, and immunology and with many clinical specialties. Genetic Apparatus of Human Cell The support of heredity and variability of human organism is assured by the genetic apparatus of human cell, which is represented by DNA molecules stored in cell nucleus and mitochondria and by structures assuring the realization of genetic material (transcription apparatus, translation apparatus, ribosomes) and its exact distribution to daughter cells (replication apparatus, centrosomes). Genetic apparatus is represented by:

2

GENETIC APPARATUS OF HUMAN CELL – SUPPORT OF HEREDITY AND VARIABILITY OF HUMAN ORGANISM VARIABILITY

Gene, which is a discrete unit of DNA (or RNA in some viruses) that encodes RNA or protein product that contributes to or influences the phenotype of the cell. Genes may be quite short or may extend over hundreds of kilobases (kb). Individual regions of genes are defined by specific sequence features. One of the most prominent features of human genes is the presence of distinct segments, some of them responsible for protein-coding information (exons) and others separating such coding sequences (introns). Chromosomes are complex arrays of both the linear DNA molecule and its associated proteins (histones and non-histones). Each chromosome contains a single molecule of DNA organized into several levels of packaging to construct a metaphase chromosome. During interphase DNA is associated (bound) with proteins to form a structure termed as chromatin. The entire chromosome complement of an individual or cell, as seen during mitotic metaphase is called karyotype. All species have a characteristic number of chromosomes in their cells: the diploid number (2n) in somatic cells and the haploid number (n) in gametes. A diploid human cell includes 46 chromosomes or 23 pairs, out of which 44 (22 pairs) are autosomic; the remaining two (X and Y) are known as sex or gonosomal chromosomes. Genome - the total complement of cellular DNA. In the cell could be identified nuclear genome (46 molecules of linear DNA) and mitochondrial genome (several identical molecules of circular DNA) Genotype - the total complement of genes contained in a cell or virus; commonly used in eukaryotes to refer to all genes present in one complete haploid set of chromosomes. There are approximately 30.000 – 40.000 genes in human genome. the genetic constitution of an organism as distinguished from its appearance of phenotype, often used to refer to the allelic composition of one or a few genes of interest. If both alleles are the same the genotype is homozygous, if the alleles are different the genotype is heterozygous. Gene pool is described as a complete set of the genes of a population of organisms. The frequency of alleles in a gene pool of human population tends to remain stable. But there are some factors (mutation, migration, gene flow, genetic drift, nonrandom matting) that alters the frequency of the alleles in the gene pool and respectively the frequency of the genotypes and phenotypes. Organization of genetic material depending on cell cycle period: Chromosomes, representing the IVth level of DNA condensation, are best seen during the metaphase. As mitosis begins, the double-stranded chromosomes condense into short structures, which can be stained and easily observed under the light microscope. During intherphase, DNA is presented in I, II and III levels of condensation, forming chromatin. Chromatin can be observed under a light microscope in two forms: euchromatin and heterochromatin: Euchromatin forms the main body of the chromosome and has a relatively high density of coding regions of genes. It is most abundant in active, transcribing cells. Heterochromatin is the condensed form of chromatin organization. It is considered transcriptionally inactive and is devided into:. - Constitutive heterochromatin is located around the centromeres, telomeres of all chromosomes, in the long arm of the Y chromosome, and in the satellites highly repetitive elements of DNA, used as chromosomal markers. 3

GENETIC APPARATUS OF HUMAN CELL – SUPPORT OF HEREDITY AND VARIABILITY OF HUMAN ORGANISM VARIABILITY

-

Facultative heterochromatin is euchromatin in transcriptionally inactive state (in humans, a good example is inactivation of one X chromosome in females, as a means of compensation for the double dosage of X chromosome genes, compared with the single copy of males, i.e. dosage compensation). The inactive X chromosome remains highly condensed and strains darkly during interphase, forming a Barr body.

The particularities of nuclear genome There are approximately three billion (3 x 109) base pairs in human DNA. The sequences comprising a human genome can be classified in three groups: 1) nonrepetitive sequences (65%) : are unique and represented in a single copy; most structural genes are located in nonrepetitive DNA; consist of distinct segments, some of them responsible for protein-coding information (exons) and other separating such coding sequences (introns). they can make up so-called gene families, which represented groups of genes of often similar structure and function. For example, actins - 5-30 members, keratins - more than 20, tubulins 15. By far the best-studied gene families are those for the globin genes. The -globin genes of human chromosome 11 and the -globin genes in human chromosome 16 have been studied in great detail. They represent a group of related genes and include globin genes that are transcribed at different periods of ontogenesis. pseudogenes - are no longer functional and cannot make proteins. Presumably, they have arisen as evolutional derivates of their parent functional genes losing the ability of being transcribed or successfully translated. Pseudogenes are examples of historical genetic remodeling and recombination events but appear to have no functional significance in themselves; 2) moderately repetitive sequences (20%): are dispersed and repeated a small number of times in the form of related but not identical copies; make up the families of genes for histones and tandem gene families for rRNA and tRNA (cells need large amount of products of some genes); participate in regulation of gene expression and DNA replication; can be represented by transposons that may cause mutations and increase (or decrease) the amount of DNA in the genome. 3) highly repetitive sequences (15%): are short sequences (2-200 nt.) usually repeated as a tandem array; are found in areas such as telomeres at the end of chromosomes; telomeres have tandem arrays of simple DNA sequences (TTAGGG in humans) that do not code for proteins, but are required for correct replication of a linear DNA molecule and centromeres that are essential for cell division; are likely to play important roles in DNA structure, in the packing of DNA in chromosomes, and in recombination and replication; in form of mini- and microsatellites may be used as molecular markers for the gene map and genetic markers for personal identification. Examples of repetitive sequences: LINEs (Long interspersed elements). 4

GENETIC APPARATUS OF HUMAN CELL – SUPPORT OF HEREDITY AND VARIABILITY OF HUMAN ORGANISM VARIABILITY

The human genome contains over 500,000 LINEs (representing 16% of the genome). LINEs are long DNA sequences that represent reverse-transcribed RNA molecules originally transcribed by RNA polymerase I; that is, messenger RNAs. Lacking introns as well as the necessary control elements like promoters, these genes are not expressed. However, some LINEs do encode a functional reverse transcriptase and/or integrase. These enable them to mobilize not only themselves but also other, otherwise nonfunctional, LINEs and Alu sequences. Because transposition is done by copy-paste, the number of LINEs can increase in the genome. The diversity LINEs between individual human genomes make them useful markers for DNA fingerprinting. SINEs (Short interspersed elements). SINEs are short DNA sequences that represent reverse-transcribed RNA molecules originally transcribed by RNA polymerase III; that is, molecules of tRNA, 5s rRNA, and some other small nuclear RNAs. The most abundant SINEs are the Alu elements. There are about million copies in the human genome (representing about 11% of the total DNA). Alu elements consist of a sequence of 300 base pairs containing a site that is recognized by the restriction enzyme Alu I. They appear to be reverse transcripts of 75 RNA, part of the signal recognition particle. The mitochondrial genome Mitochondria contain their own genetic system consisting of mtDNA and the apparatus to transcribe and translate it. MtDNA is a double-stranded circular molecule, comprising only 16,569 bp; its entire sequence is known. Most cells contain hundreds or thousands of mitochondria, each containing on average 5 (2-10) identical mtDNA molecules. In a normal case all mtDNA molecules of an individual are identical - homoplasmy. The presence of more than one type of mtDNA is termed heteroplasmy. mtDNA determines the plasmotype. Mitochondrion is also a place of all main molecular processes, such as DNA replication, RNA transcription and protein translation. Replication starts from two discrete origins, making the process asynchronous both spatially and temporally. Transcription. The mitochondrial genome is remarkably compact. The total gene content consists of 13 protein genes, 2 rRNA genes and 22 tRNA genes. The compactness is so extreme that a translational stop codon (UAA) is introduced by post-transcriptional processing in most cases, and two pairs of protein-coding genes share overlapping reading frames. MtDNA is transcribed in three polycistronic units. Translation is carried out on mitochondrial ribosomes (55S) that are composed of RNA encoded by mtDNA and proteins encoded by nuclear DNA. The number of protein products from mitochondrial transcription is limited. Five known gene products are produced from the mammalian mitochondrial genome. These include subunits I, II and III for cytochrome oxidase, the apoprotein for citochrome b and subunit 6 of the mitochondrial ATPase. The remainder of subunits of this seven-subunit protein is encoded in the nucleus. Thus, the transfer of genetic information during the evolution of eukaryotic cells has also required the development of cooperative gene expression systems between the organelle and the nucleus. Transmission of mtDNA and inheritance of mitochondrial genetic traits is fully maternal. The portion of the sperm that penetrates and fertilizes the egg generally contains no mitochondria. Thus, the mitochondria of fertilized egg derives exclusively from the ovum. Mutations in mtDNA do cause diseases. The features of mitochondrial disorders can be summarized as follows:

5

GENETIC APPARATUS OF HUMAN CELL – SUPPORT OF HEREDITY AND VARIABILITY OF HUMAN ORGANISM VARIABILITY

Since each cell contains hundreds of mitochondria and thousands of copies of its genome, the effect of the mutated mitochondria may be diluted out. Mutation in mtDNA occur more frequently than in nuclear genes involved in oxidative phosphorylation, because of the short size and gene-rich sequences of mitochondrial genome. The physiologic effect of defective mitochondrial function depends on the energy requirements of the cell. Thus, nerves and muscle often show clinical changes first. Mitochondria in somatic cells can acquire and accumulate mutations with age. Although these are not transmissible, they affect the rate and severity of clinical changes. Aging patient may show a more severe disease phenotype.

6

GENETIC APPARATUS OF HUMAN CELL – SUPPORT OF HEREDITY AND VARIABILITY OF HUMAN ORGANISM VARIABILITY

VARIABILITY AND ITS FORMS Variability represents the propriety of an organism to gain new traits, different from those of their parents. Variability is determined by different genetic, biochemical, physiological or morphological changes. Variability is a common propriety of organisms and ensures: - Survival in different environmental conditions; - Individual polymorphism; - Natural selection; - Evolution; - Appearance of different pathological states. Different variations may be induced by internal factors (hormones, metabolites, physiological states) and external factors (physical, chemical and biological). The most important physical factors are radiations: UV (non-ionizing), X-rays, and cosmic irradiations (ionizing). The chemical substances, which may affect genetic material, are oxides, compounds with chlorine, salts of heavy metals, vitamin C, and nitrates. The main biological factors are viruses, bacterial and fungal toxins.

Classification of variability There are two types of variability: hereditary (genotypic) and non-hereditary (phenotypic). Non-hereditary variability is also called modification. Hereditary variability can be a result of recombination (crossing-over during meiosis) or can be caused by mutations. Nonhereditary (phenotypic) variability Each phenotype develops under specific environmental conditions. Different factors (like sunlight, radiations, drugs) may change the phenotype, without modifying the DNA. So, the nonhereditary variations in phenotype don’t affect the genotype. The phenotypic variations are also called modifications. Usually phenotypic modifications are determined by genotype. Each character may vary in determined limits – this property is called norm of reaction. (E.g. a white man exposed to sunlight (UV irradiation) becomes darker, but not black). Usually the modifications persist as long as the modification factor acts. Characteristics of modification are: - represents nonhereditary variations of phenotype; 7

GENETIC APPARATUS OF HUMAN CELL – SUPPORT OF HEREDITY AND VARIABILITY OF HUMAN ORGANISM VARIABILITY

-

represent adaptations to environmental conditions; are reversible (if they appear in adults); different organisms present the same variations in identical conditions (in all people UV light induces skin tanning); - phenotype variation is determined by genotype – norm of reaction (under the same condition some people become darker, others – not]. Some environmental factors may have a destructive action on organism. In this case the phenotype develops beyond the limits of the norm of reaction and an abnormal modification is produced. The factors, which produce such abnormal modifications, are called teratogenes - agents, which can cause a birth defect. The degree of abnormality depends on the stage of development: earlier damages are more sever. Frequently the phenotype resembles a genetic disease (state caused by mutations), but has arisen by a completely different (environmentally determined) mechanism. Such phenotype is called phenocopy. Hereditary (genotypic) variability The genotypic variability is determined by changes at the level of DNA. All genotypic variations have the next proprieties: -

-

Are determined by changes in genetic apparatus; Are induced by physical, chemical or biological conditions; May be inherited through generations; Represent the basis of population polymorphism.

There are two types of hereditary variations: mutational and combinative.

Combinative variability The recombination of genetic material takes place during sexual reproduction. There are 3 types of genetic recombination: -

-

Genomic recombination – random mating of gametes during fecundation. Inter-chromosomal recombination – random segregation of maternal and paternal chromosomes during anaphase I. Meiosis may yield 223 = 83886068 different chromosome combinations in gametes. Intra-chromosomal recombination – represents the exchange of fragments between homologous chromosomes during prophase I – crossing-over.

8

GENETIC APPARATUS OF HUMAN CELL – SUPPORT OF HEREDITY AND VARIABILITY OF HUMAN ORGANISM VARIABILITY

Mutational variability Mutation is the process whereby changes occur in the quantity or structure of the genetic material of an organism. Mutations are permanent alterations in the genetic material, which may lead to changes in phenotype. An organism, gene, DNA sequence, etc. in which a mutation has occurred is called a mutant. Classification of mutations: 1) Depending on the cells in which mutations occur. Mutations may occur either in germ cells or in somatic cells. Germ cell mutations can be transmitted to individuals of the next generation and are usually found in all cells of the affected offspring. Somatic mutations are found only in the descendents of the mutant cell, making the individual a "mosaic". Phenotypic consequences are observable only if the mutations happen to interfere with the specific function of the affected cells. 2) Depending on the origin of the mutation. Mutations can be spontaneous (which occur naturally) and induced (they are produced when an organism is exposed to a mutagen; such mutations typically occur at much higher frequencies than spontaneous mutations do). 3) Depending on the localization of a mutation in the cell. Mutations can be nuclear (chromosome), in case the mutated gene is localized in a chromosome, or cytoplasmic, if the mutated gene is localized in mitochondrial or chloroplast DNA. 4) Depending on the influence on viability of the organism. Mutations can be lethal (they cause death of the embryo carrying the mutation), semi-lethal (they decrease drastically the viability of the organism carrying the mutation), neutral (they do not affect the viability of the individual), and enhancing (they increase viability of the individual). 5) Depending on the character of changes to the genetic material. In experimental genetics, the following types of mutations can be distinguished: Genome mutations involve alterations in the number of chromosomes. Whole sets of chromosomes may be multiplied (polyploidy), or the number of copies of a particular chromosome may be changed (aneuploidy): increased (trisomy) or decreased (monosomy). Chromosome mutations affect the structure of chromosome, allowing microscopic detection. The total number of chromosomes is not altered. Gene mutations involve different changes on gene level: substitution, deletion, duplication, insertion, reversion, unequal crossing-over. Mutations that involve one nucleotide are called point mutations.

9