Lecture 1 Introduction. Human genetics, its goals and role in modern medicine. Nuclear and mitochondrial genomes. 1. Introduction into human genetics. 2. Methods of studying in human genetics. 3. Levels of organization of genetic material. 4. Organization of genetic material depending on cell cycle period. 5. Genetic apparatus of human cell. 6. Nuclear genome: the particularities of organization. 7. Transposons. 8. Mitochondrial genome: the particularities of organization. 1. Introduction into human genetics. Genetics is a brunch of biological science that examines the laws of storage, transmission and realization of information for development and functions of living organisms. The year of birth of genetics as a science is considered to be 1900. In fact, the main laws of genetics were discovered by G.Mendel in 1865, but during following 35 years they remained unknown to the most biologists. Rediscovery of Mendel s laws is credited to three scientists - de Vries, Correns, Tschermak. They simultaneously obtained the results confirming the laws of heredity, discovered by Mendel. The name of the new science - genetics - was suggested by Batson in 1906. In 1909 Iogansen introduced such notions as gene, genotype, phenotype. The work of T.Morgan and his school (1910-1925) gave rise to the chromosome theory of heredity. The new era of genetics began with the decoding of DNA structure (Watson and Crick, 1953). This discovery gave rise to molecular genetics. The course named Human genetics is intended for medical students, which need to learn it in order to understand how genetic information is passed to offspring from their parents and of what determines the transfer of genetic information. Human genetics studies phenomena of inheritance and variability in human populations, laws of transmission of normal and pathological 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. A large proportion of human ill health has a genetic basis. For example, it has been estimated that at least 30% of pediatric hospital admissions have a genetic component. However, current research is revealing more and more genetic predispositions to different serious common diseases, so this figure is almost certainly an underestimate. Human genetics is both a fundamental and an applied science. As a fundamental science, it is part of genetics. It concerns itself with the most interesting organism - the human being. This concern with our own species makes us scrutinize scientific results in human genetic not only for theoretical, but also for their practical value for human welfare. Thus, human genetics is also an applied science. 2. Methods of studying 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 methods 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. 3. Levels of organization of genetic material. Gene 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. Of particular importance in considering genetic contributions to medicine is an appreciation of the structure of individual genes. Genes represent regions of DNA; they may be quite short or may extend over hundreds of kilobases. 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. Each chromosome contains a single molecule of DNA organized into several orders of packaging to construct a metaphase chromosome. DNA in chromosomes is associated (bound) with proteins known as histones and nonhistones 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. These 44 form 22 pairs and are autosomic. The remaining two are known as sex or gonosomal chromosomes. 2
Genome - 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 (~26.000 according to last data) genes in human genome. Genotype - 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. 4. Organization of genetic material depending on cell cycle period. With exception of mitochondrial DNA, all cellular DNA is found in chromosomes in the nucleus. Chromosomes are complex arrays of both the linear DNA polymer and its associated proteins. Progressive compaction of DNA into chromosomes moves through several identifiable levels of organization. The nucleosomes are the first level. Each nucleosome consists of a tightly bound package of 8 histones (two each of H2A, H2B, H3 and H4) with DNA helix wound twice around the surface. Nucleosomes are packed tinghtly together with the aid of histone H1 to form a basic chromatin fiber seen in interphase nuclei when using electron microscopy. This fiber is further packed into a system of looped domains. The loops, which contains from 20 000 to 100 000 base pairs of DNA, are formed by nonhistone proteins binding specific sites along the basic fiber. Evidence suggests that loops house individual units of DNA transcription and replication. Therefore they have both a structural and a functional significance. The process of condensation continues during prophase of mitosis. In the condensed state, nearby loops are held together by protein interactions that form a network or scaffold. The chromatin network is arranged in a helical fashion along the axis of the chromosome. The width of an individual chromatid of a metaphase chromosome is about 700 nm, reflecting a high degree of compression of the initial fiber. Chromosomes are best seen during the metaphase. Before a cell gets ready to divide by mitosis, each chromosome is duplicated (during S phase of the cell cycle). As mitosis begins, the duplicated chromosomes condense into short structures, which can be stained and easily observed under the light microscope. Chromatin is seen 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 chromatin that is either devoid of genes or has inactive genes. Heterochromatin segments of the genome remain more condensed in interphase than euchromatin. Heterochromatin is considered transcriptionally inactive. Constitutive heterochromatin is located around the centromeres of all chromosomes, in the long arm of the Y chromosome, and in the satellites of highly repetitive elements of DNA with no known function. 3
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. 5. Genetic apparatus of human cell. Genetic apparatus of human cell consists of the DNA (nuclear and mitochondrial) and the system of realization and inheritance of genetic material (ribosomes, cell centre). Nucleus is the most important component of the genetic apparatus, which contains 98% of cell DNA. The main functions of cell nucleus are: to store, transmit and realize hereditary information, to regulate most of the cell functions. DNA is the master molecule of cells since they play such vital roles in the continuation of all processes. They have two main functions: regulation of all metabolic activities within the cell; maintenance of genetic continuity between generations. In human cells, the DNA is situated in nucleus (98%) in the form of compacted chromosomes and in mitochondria (2%). Ribosomes are cell organelles which consist of proteins and rrna. The main function of ribosomes is: to serve as the site of mrna translation (=protein synthesis, the assembly of amino acids into proteins). During this process the two subunits of ribosome are joined by the mrna from the nucleus, the ribosome translate the mrna into a specific sequence of amino acids, or a polypeptid chain. Centrioles are found in pairs adjacent to the nucleus of a cell. Each is formed from nine groups of microtubules attached to each other by a triple bond and arranged in a circle. Centrioles are involved in the formation of spindle fibres during mitotic prophase. Before mitotic cell division they replicate and in late prophase each pair moving to opposite poles of the cell. Their function is: to maintain the spindle fibres, position the chromosomes at the equatorial plate and then to move them to the poles. Any defect in the centrioles effectively prevents a cell from dividing. 6. The particularities of nuclear genome There are approximately three billion (3 x 10 9 ) 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, 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 4
are transcribed and synthesized at different times in development. In general, adults synthesize only adult globin genes but fetal globin genes remain present, although unexpressed, in adults; pseudogenes - are no longer functional and cannot make proteins. Presumably, they have arisen as evolutional derivates of there parent functional genes but cannot themselves be 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; form transposons and in this process, they may cause mutations and increase (or decrease) the amount of DNA in the genome. 3) higly repetitive sequences (15): are short and usually repeated as a tandem array; these include 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 needed for correct replication of a linear DNA molecule; they repeats in centromeric heterochromatin and are essential for cell division; are likely have important roles in DNA structure, in the packing of DNA in chromosomes, and in recombination and replication; may be used as a molecular markers for the gene map and genetic markers for personal indetification. 7. Transposons Transposons - are segments of DNA that can move around to different positions in the genome. In this process, they may cause mutations and increase (or decrease) the amount of DNA in the genome. These mobile segments of DNA are sometimes called «jumping genes». There are two distinct types: 1) transposons consisting only of DNA that moves directly from place to place 2) retrotransposons first step is transcription of DNA into RNA and then use reverse transcriptase to make a DNA copy of the RNA to insert in a new location. Transposons move by a cut and paste process: the transposon is cut out of its location and inserted into a new location. This process requires an enzyme - a transposase- that is encoded within the transposon itself. Transposase binds to: both ends of the transposon, which consist of inverted repeats: that is, identical sequences reading in opposite directions, and a sequence of DNA that makes up the target site. Some transposases require a specific sequence as there target site; other can insert the transposon anywhere in the genome. Retrotransposons move by a copy and paste mechanism but in contrast to the transposons described above, the copy is made of RNA, not DNA. The RNA copies are then transcribed back into DNA - using a reverse transcriptase - and these are inserted into new locations in the genome. Many retrotransposons have long terminal repeats (LTRs) at their ends that may contain over 1000 base pairs in each. Like DNA transposons, retrotransposons generate direct repeats at their new sites of insertion. In fact, it is the presence of these direct repeats that often is the clue that the intervening 5
stretch of DNA arrived there by retrotransposition. About 40% of the entire human genome consists of retrotransposons. LINEs (Long interspersed elements). The human genom 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. Transposons and Disease. Transposons are mutagens. They have been found to be cause of the mutations responsible for some cases of human genetic disease, including: X-linked severe combined immunodeficiency (SCID); predisposition to colon polyps and cancer; Duchenne muscular dystrophy. 8. 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. This normal case, in which each mtdna molecule in an individual is identical, is called homoplasmy. The presence of more than one type of mtdna is termed heteroplasmy. It is important to note that all the basic functions of the Central Dogma of Molecular Genetics are found in mitochonria. This function include DNA replication, RNA transcription and protein translation. Replication takes place from two discrete origins such that replication of the two strands is asynchronous both spatially and temporally. The regulatory region contains the origin of heavy (H) strand replication, and the single promoters for both H and L (light) strand transcription. 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 (TAA) is introduced by post-transcriptional polyadenylation in most cases, and two pairs of protein-coding genes share overlapping reading frames. MtDNA is transcribed in three polycistronic units. 6
Translation is carried out on mitochondrial ribosomes 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 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 wholly maternal. While in meiosis and mitosis have similar final effects in males and females and transmit an identical number of chromosomes from both parents to their progeny, the genetical transmission of mitochondria and mitochondrial DNA genetic information is different. The portion of the sperm that penetrates and fertilizes the egg generally contains no mitochondria. Thus, the mitochondria of fertilized egg are usually derived exclusively from the ovum. Mutations in mtdna do cause diseases. The features of mitochondrial disorders can be summarized as follows: Since each cell contains hundreds of mitochondria and thousand of copies of the genome, the effect of the mutated mitochondria may be diluted out. Mutation in mtdna occur more frequently than those in nuclear genes involved in oxidative phosphorylation, because there is such a shirt sequence and very heavy information content in sequence. 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. 7