* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Chemical Structure of Deoxyribonucleic Acid. Evidences, DNA is
Epigenetics wikipedia , lookup
Epigenetics of human development wikipedia , lookup
Holliday junction wikipedia , lookup
DNA profiling wikipedia , lookup
Genetic engineering wikipedia , lookup
Neocentromere wikipedia , lookup
Designer baby wikipedia , lookup
Comparative genomic hybridization wikipedia , lookup
SNP genotyping wikipedia , lookup
Mitochondrial DNA wikipedia , lookup
History of RNA biology wikipedia , lookup
Polycomb Group Proteins and Cancer wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Human genome wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
DNA polymerase wikipedia , lookup
Bisulfite sequencing wikipedia , lookup
Cancer epigenetics wikipedia , lookup
Genomic library wikipedia , lookup
Nucleic acid tertiary structure wikipedia , lookup
DNA damage theory of aging wikipedia , lookup
United Kingdom National DNA Database wikipedia , lookup
Microevolution wikipedia , lookup
Genealogical DNA test wikipedia , lookup
Molecular cloning wikipedia , lookup
DNA vaccination wikipedia , lookup
Point mutation wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
Cell-free fetal DNA wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Primary transcript wikipedia , lookup
Epigenomics wikipedia , lookup
Helitron (biology) wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Non-coding DNA wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
DNA supercoil wikipedia , lookup
DNA nanotechnology wikipedia , lookup
Extrachromosomal DNA wikipedia , lookup
History of genetic engineering wikipedia , lookup
Nucleic acid double helix wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
1 Chemical Structure of Deoxyribonucleic Acid. Evidences, DNA is genetic material. Chromosome and Chromatin. Anatomy of Chromosome Dr. Kamal Omer Abdalla, Associate Prof. of Biochemistry and Molecular Biology, Director of Scientific Research, University of Gadarif. Introduction The nucleic acids are the molecular repositories for genetic information and referred to as the ‘Molecules of Heredity’. Although the name nucleic acid suggests their location in the nuclei of cells, yet some of them are, however, also present in the cytoplasm. The nucleic acids are the hereditary determinants of living organisms. They are the macromolecules present in most living cells either in the free state or bound to proteins as nucleoproteins. There are two types of nucleic acids, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Both are present in all plants and animals. Viruses also contain nucleic acids, however, unlike a plant or animal has either RNA or DNA, but not both. DNA is found mainly as a component of chromatin material (a combination of DNA with proteins) of the cell nucleus whereas most of the RNA (90%) is present in the cell cytoplasm and the remaining (10%) in the nucleolus. Extra-nuclear DNA also exists, for e.g., in mitochondria and chloroplasts. Composition of nucleic acids Nucleic acids are biopolymers of high molecular weight with mononucleotide as their repeating units. Each mononucleotide consists of the following: 1. Nitrogenous bases 2. Phosphoric acid and 3. Pentose sugars Nitrogenous bases Two types of major nitrogenous bases, which account for the base composition of DNA or RNA, are found in all nucleic acids. These are: a) Purine bases and b) Pyrimidine bases. The purine and pyrimidine bases found in nucleic acids are shown in Fig. 1. 2 Structure of purine bases found in DNA and RNA structure Purine and pyrimidine bases are hydrophobic and relatively insoluble in water at the near neutral pH of cell. Purines can exist in syn or anti forms; pyrimidines can exist in anti form because of steric interference between the sugar and carbonyl oxygen at C-2 of pyrimidine. Besides, the major nitrogenous bases, some minor bases also called modified nitrogenous bases (purines and pyrimidines) also occur in polynucleotide structures. Some naturally occurring forms of modified purines are hypoxanthine, xanthine, uric acid, 6-methyladenine (6-Me),6-dimethyladenine (6-DiMe), 6-N isopentenyladenine (6-IPA), 1-3methylguanine (1-MeG), 2-dimethylguanine(2-DiMeG). Among the modified purines, some are found in tRNA. Methylation is the most common form of purine modification in microorganisms. The presence of such methylated purines is also suggested in plant genomes. Some naturally occurring forms of modified pyrimidines (e.g.5, 6-dihydrouracil, pseudouracil, 4-Thiouracil etc.) are common in tRNA. Other examples include 5-methylcytosine (5-MeC) and 5-hydroxy methyl cytosine. The 5-methylcytosine is a common component of higher plant and animal DNA. In fact up to 25% of the cytosine residues of plant genome are methylated. The DNA of plants is richer in 5-MeC than the DNA of animals. The DNA of the T-even bacteriophages (T2, T4) of E. coli has no cytosine but instead has 5-hydroxymethylcytosine and its glucoside derivatives. Phosphorus Phosphorus, present in the backbone of nucleic acids, is a constituent of phosphor-diester bond that links the two sugar moieties. The molecular formula of phosphoric acid is H3PO4. It contains three mono-valent hydroxyl groups and a divalent oxygen atom, all linked to the pentavalent phosphorus atom. Sugar Both DNA and RNA contain five-carbon aldose sugar, i.e. a pentose sugar. The essential difference between DNA and RNA is the type of sugar they contain. RNA contains the sugar D-ribose (hence called ribonucleic acid, RNA) whereas DNA contains its derivatives 2’-deoxy-D-ribose, where the 2’-hydroxyl group of ribose is replaced by hydrogen (hence called deoxyribonucleic acid, DNA). Sugars are always in closed ring βfuranose form in nucleic acids and hence are called furanose sugars because of their similarity to the heterocyclic compound furan. The structure of pentose sugars present in DNA and RNA are shown in Fig. 2. 3 Why is 2'-Deoxyribose the Sugar Moiety in DNA? Common perhydroxylated sugars, such as glucose and ribose, are formed in nature as products of the reductive condensation of carbon dioxide we call photosynthesis. The formation of deoxysugars requires additional biological reduction steps, so it is reasonable to speculate why DNA makes use of the less common 2'-deoxyribose, when ribose itself serves well for RNA. At least two problems associated with the extra hydroxyl group in ribose may be noted. First, the additional bulk and hydrogen bonding character of the 2'-OH interfere with a uniform double helix structure, preventing the efficient packing of such a molecule in the chromosome. Second, RNA undergoes spontaneous hydrolytic cleavage about one hundred times faster than DNA. This is believed due to intramolecular attack of the 2'-hydroxyl function on the neighboring phosphate diester, yielding a 2',3'-cyclic phosphate. If stability over the lifetime of an organism is an essential characteristic of a gene, then nature's selection of 2'-deoxyribose for DNA makes sense. The following diagram illustrates the intramolecular cleavage reaction in a strand of RNA. Structural stability is not a serious challenge for RNA. The transcripted information carried by mRNA must be secure for only a few hours, as it is transported to a ribosome. Once in the ribosome it is surrounded by structural and enzymatic segments that immediately incorporate its codons for protein synthesis. The tRNA molecules that carry amino acids to the ribosome are similarly short lived, and are in fact continuously recycled by the cellular chemistry. 4 The structural difference in the sugars of DNA and RNA, though minor, confers very different chemical and physical properties upon DNA than RNA. RNA is much stiffer due to steric hindrance and more susceptible to hydrolysis in alkaline conditions, perhaps explaining in part why DNA has emerged as the primary genetic material. Sugar, along with phosphate performs the structural role in nucleic acids. Scheme 1: Generalized structural units of nucleic acid Nucleosides The nucleosides are compounds in which nitrogenous bases (purines and pyrimidines) are conjugated to the pentose sugars (ribose or deoxyribose) by a β-N-glycosidic linkage. These consist of a base joined to a pentose sugar at positionC1′. The sugar C1′carbon atom is joined to the N1 atom of pyrimidine and the N9 atom of purine. This represents a β-Nglycosidic bond. Thus, the purine nucleosides are N-9 glycosides and the pyrimidine nucleosides are N-1 glycosides. These are stable in alkali. The purine nucleosides are readily hydrolyzed by acid whereas pyrimidine nucleosides are hydrolyzed only after prolonged treatment with concentrated acid. The nucleosides are generally named for the particular purine and pyrimidine present. Nucleosides possessing ribose are called ribonucleosides (riboside) and those containing deoxyribose are called deoxy-ribo-nucleosides (deoxyriboside). The nomenclature of nucleosides differs from that of the bases. In case of pseudouridine, which is otherwise identical to uracil, differs in the point of attachment to the ribose to the base. In case of pseudo-uridine, base is attached to sugar through C5 of base as opposed to that in case of uridine, where the attachment of base to sugar is through N1. Two nucleoside analogues, 3′-azidodeoxythymidine (AZT) and 2′, 3′-dideoxycytidine (DDC), have found therapeutic use for the treatment of acquired immune deficiency syndrome (AIDS) patients. Nucleotides or Nucleoside 5’-triphosphates These are phosphate esters of nucleosides i.e. nucleosides form nucleotides by joining with phosphoric acid. Esterification can occur at any free hydroxyl group, but is most common at the 5′and3′positionsin sugars. The phosphate residues are joined to the sugar ring by a phosphor monoester bond and several phosphate groups can be joined in series by phosphor anhydride bonds. These occur either in the free form or as subunits in nucleic acids. The phosphate is always esterified to the sugar moiety. The trivial names of purine nucleosides end with the suffix –sine, and those of pyrimidine nucleosides end with suffix –dine. 5 In addition to their role as structural components of nucleic acids, nucleotides also participate in a number of other functions as described below: Energy carriers: Nucleotides represent energy rich compounds that drive metabolic process, especially biosynthetic, in all cells. Hydrolysis of nucleoside triphosphate provides the chemical energy to drive a wide variety of cellular reactions. ATP is the most widely used for this purpose. UTP, GTP, CTP are also used. Nucleoside triphosphate also serves as the activated precursors of DNA and RNA synthesis. The hydrolysis of ester linkage (between ribose and α-phosphate) yields about 14 kJ / mol under standard conditions, whereas hydrolysis of each anhydride bond (between α-β and β-γ phosphates) yields about 30 kJ / mol. ATP hydrolysis often plays an important thermodynamic role in biosynthesis. Enzyme cofactors: Many enzyme cofactors include adenosine in their structure, e.g., NAD, NADP, FAD. Chemical messengers: Some nucleotides act as regulatory molecules and serve as chemical signals or secondary messengers, key links in cellular systems that respond to hormones and other extracellular stimuli and lead to adaptive changes in cells interior. Two hydroxyl groups can be esterified by the same phosphate moiety to generate a cyclic AMP (cAMP, adenosine 3’-5’ cyclic phosphate) or cyclic GMP (cGMP, guanosine 3’-5’ cyclic phosphate). Oligonucleotides Oligonucleotides are polymers containing < 100nucleotides. These nucleotides are linked by phosphodiester bond as shown in Fig. 3. The oligonucleotides occur naturally and are used as primers during DNA replication and for various other purposes in the cell. Synthetic oligonucleotides can be made by chemical synthesis and are essential for many lab techniques, e.g., DNA sequencing, PCR, in situ hybridization, nucleic acid probe, nucleic acid hybridization, gene therapy. The polymers containing >100 ribonucleotides or deoxyribonucleotides are called RNA and DNA (nucleic acids), respectively. Nomenclature of nucleic acids 6 Direction: By convention, single strand of nucleic acid is always written with the 5’ end at the left and 3’ end at the right i.e.in 5’ →3’ direction. Sugar: In the chemical nomenclature, the carbon atoms of sugars are designated by primed numbers i.e.C1’, C-2’, C-3’ etc. to avoid confusion with the base numbering system. Base: The various atoms in the bases lack the prime (Ꞌ) sign and are designated by the cardinal numbers, i.e.1, 2, 3 etc. By convention, the N,C,O atom attached directly C Base 5'3'2' CH2OCHHCCHHCO Base 3'2'POCH25'OO_O1' 4'1' 4' Phosphodiester bond 7 to ring is numbered 2, 3, 7 etc., but the exo-cyclic atom (not within the ring structure) is denoted as the atom with ring position as superscript to which it is attached e.g. Amino N attached to C-6 in adenine is N6. Bases are represented by single letters, such as adenine is represented as A, guanine as G, cytosine as C, thymine as T and uracil as U.” Nucleosides and nucleotides: While names of nucleosides and nucleotides are generally derived from the corresponding bases, there is one exception to this rule: the base corresponding to the nucleoside called inosine (and the derived nucleotides) is called hypoxanthine. Short hand notation: In short hand notation of nucleotides, phosphate group is symbolized by P, deoxyribose a vertical line from C1’ at top to C5’ at bottom. The connecting lines between nucleotides, which pass through P, are drawn diagonally from the middle (C3’) of deoxyribose of one nucleotide to bottom (C5’) of next (Fig. 4). The nucleoside and nucleotide derivatives of deoxyribose are distinguished by prefix ‘d’. Where clarity is especially important, ribonucleosides and ribonucleotides can similarly be identified with the prefix’r’, e.g. ATP = rATP. A second short hand notation is used to discriminate between 5’ and 3’ phosphates, with 5’-phosphate placed before the base (e.g. pA is adenosine 5’-monophosphate) and 3’-phosphates placed after the base (e.g. Ap is adenosine 3’-monophosphate). The deoxy prefix can be omitted from the names of thymidine derivatives because, as a predominantly DNA specific base, it is usually evident that sugar is deoxyribose. However, the full nomenclature is preferred for the sake of convention and because thymine is a minor base in RNA (thymine exists as modified base at various places, most notably in the TΨC loop of every tRNA; note that thymine is 5-methyl uracil). Where context is obvious, both DNA and RNA sequences are represented as a 7 single series of bases. The ambiguous bases are represented by the single letter representations as shown in Table 2. Structural levels of nucleic acids (a) Primary structure The nature, properties and function of the two nucleic acids (DNA and RNA) depend on the exact order of the purine and pyrimidine bases in the molecule. This sequence of specific bases is termed as the primary structure. Thus, primary structure of nucleic acid is its covalent structure and nucleotide sequence. (b) Secondary structure Secondary structure is the set of interactions between bases, i.e., parts of which is strands are bound to each other. In DNA double helix, the two strands of DNA are held together by hydrogen bonds. The nucleotides on one strand base pairs with the nucleotide on the other strand. The secondary structure is responsible for the shape that the nucleic acid assumes. 8 Watson and Crick base pairs form the basis of secondary structure interactions in nucleic acids as well as explaining Chargaff’s rule. Secondary structures in RNA, which exist primarily in single stranded form, generally reflect intra-molecular base interactions. Thus, the secondary structures arise due to following interactions: Complementary base pairing: It involves stable and specific configurations of H-bonds between bases in DNA. It is the predominant force causing nucleic acid strands to associate. The molecular basis of Chargaff’s rule is complementary base pairing between A-T and between G-C in double stranded DNA. Chargaff’s rule was later explained by double helical structure described by Watson and Crick. G:C with three H-bonds are more stable than A:T (or A:U). Base stacking: The structures are stabilized by hydrophobic interactions between adjacent bases. Alternative forms of base pairing: Watson-Crick base pairs (A: T and G:C) are predominant in the structure and function of nucleic acids. However, there are 28 possible arrangements of at least two H-bonds between bases, which provide the basis for a diverse set of interactions. The most significant to these alternative configurations are the Hoogsteen base pairs, which contribute to tRNA structure and allow the formation of triple helices. A modification to Watson-Crick base pairs is the Wobble pairs, which allow bases in the 5’anticodon position of tRNA to pair ambiguously with the mRNA. The Wobble base pairs are formed because bases are offset from their normal Watson-Crick positions and one of the H-bonds is lost. Intra-molecular base pairing: In RNA and single stranded regions of DNA (nonduplex DNA), secondary structure is determined by intramolecular base pairing. Since cellular DNA is usually present as a duplex, the bases are available for intramolecular interactions only rarely. Conversely intramolecular secondary structures are abundant in cellular RNA and underlie their functional specialization. The major classes of intramolecular nucleic acid secondary structures are bulges, bulge loops, bubbles, hairpins, stem loops, panhandle, cruciform. Lariats are often classified as secondary structures, but because they are formed by the covalent bonds joining nucleotides, they are strictly primary structures. (C) Tertiary structure 9 Tertiary structures of nucleic acid reflect interactions, which contribute to overall 3D shape. Tertiary structure is the locations of the atoms in three-dimensional space, taking into consideration geometrical and steric constraints. A higher order than the secondary structure in which large-scale folding in a linear polymer occurs and the entire chain is folded into a specific 3-dimensional shape. There are 4 areas in which the structural forms of DNA can differ. 1. 2. 3. 4. Handedness - right or left Length of the helix turn Number of base pairs per turn Difference in size between the major and minor grooves. The tertiary arrangement of DNA's double helix in space includes B-DNA, A-DNA and Z-DNA. A-B-Z-DNA Side View (D) Quaternary structure The quaternary structure of nucleic acids is similar to that of protein quaternary structure. Although some of the concepts are not exactly the same, the quaternary structure refers to a higher-level of organization of nucleic acids. Moreover, it refers to interactions of the nucleic acids with other molecules. The most commonly seen form of higher-level organization of nucleic acids is seen in the form of chromatin which leads to its interactions with the small proteins histones. Also, the quaternary structure refers to the interactions between separate RNA units in the ribosome or spliceosome. 10 DNA to Chromatin Deoxyribonucleic acid (DNA) is the genetic material in all organisms, except few viruses where RNA acts as the genetic material e.g. retroviruses. In prokaryotic cells, DNA occurs in the cytoplasm and is the only component of the chromosome. In eukaryotic cells, DNA is largely confined to the nucleus and is the main component in chromosome. It is combined with simple proteins to form deoxy-ribo-nucleoproteins (DNP). A small quantity of DNA also occurs in some cytoplasmic organelle such as mitochondria and chloroplast. This extra-nuclear DNA is naked as in prokaryotic DNA. The DNA content is fairly constant in all the cells of a given species. Just before cell division, however, the amount of DNA is doubled. The gametes have half the amount of DNA as they contain half the number of chromosomes. The amount of DNA per nucleus is constant in all body cells of a given species. Mirsky and Vendrely estimated that there is some 6x10-9 mg of DNA per nucleus in diploid somatic cells of mammals and 3 x 10-9 mg of DNA per nucleus in haploid gametes (eggs and sperms). EVIDENCES THAT DNA IS GENETIC MATERIAL DNA was first extracted from nuclei in 1870 named ‘nuclein’ after their source. Chemical analysis determined that DNA was a weak acid rich in phosphorous. Its name provides a lot of information about DNA: deoxyribose nucleic acid: it contains a sugar moiety (deoxyribose), it is weakly acidic, and is found in 11 the nucleus. Because of its: nuclear localization subsequent identification as a component of chromosomes it was implicated as a carrier of genetic information. Are genes composed of DNA or protein?. Chromosomes are also known to contain protein so early on it was a challenge to demonstrate that DNA was indeed the molecule that contained the genetic information. DNA: only four different subunits (nucleoside mono-phosphates) make up DNA. Chromosomes contain less DNA than protein by weight. Protein, 20 different subunits, greater potential variety of combinations. Chromosomes contain more protein than DNA by weight Classical experimental data confirmed DNA as the genetic material. Bacterial transformation implicates DNA as the substance of genes. 1928 Frederick Griffith experiments with smooth (S), virulent strain Streptococcus pneumoniae, and rough (R), non-virulent strain. Bacterial transformation demonstrates transfer of genetic material. In 1944 – Oswald Avery, Colin MacLeod, and MacIyn McCarty determined that DNA is the transformation Material. Griffith experiment: transformation of bacteria 12 Griffith’s Experiment Griffith observed that live S bacteria could kill mice injected with them.When he heat killed the S variants and mixed them with live R variants, and then injected the mixture in the mice, they died. Griffith was able to isolate the bacteria from the dead mice, and found them to be of the S variety. Thus the bacteria had been Transformed from the rough to the smooth version. The ability of a substance to change the genetic characteristics of an organism is known as transformation. Scientists set out to isolate this ‘transforming principle’ since they were convinced it was the carrier of the genetic information. Avery, MacLeod, McCarty Experiment: Identity of the Transforming Principle 13 Hershey and Chase experiment In 1952 Alfred Hershey and Martha Chase provide convincing evidence that DNA is genetic material. Waring blender experiment using T2 bacteriophage and bacteria. Radioactive labels 32P for DNA and 35S for protein. Hersey-Chase Experiment 14 Hershey and Chase experiment Performed in 1952, using bacteriophage, a type of virus that have a very simple structure: an outer core and an inner component. The phage is made up of equal parts of protein and DNA. It was known that the phage infect by anchoring the outer shell to the cell surface and then deposit the inner components to the cell, infecting it. Scientists were interested in finding out whether it was the protein component or the DNA component that got deposited inside the infected cell. By incorporating radiolabel either in the protein or the DNA of the infecting phage, they determined that the DNA was indeed introduced into the infected bacteria, causing proliferation of new phage. Chromosomes The cell nucleus contains the majority of the cell's genetic material, in the form of multiple linear DNA molecules organized into structures called chromosomes (complex very compact structures of DNA in association with various simple proteins). 15 During most of the cell cycle, DNAs are organized in a DNA-protein complex known as chromatin, and during cell division the chromatin can be seen to form the well defined chromosomes familiar from a karyotype. A small fraction of the cell's genes are located instead in the mitochondria. A chromosome is an organized structure of DNA and protein that is found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. In eukaryotes, nuclear chromosomes are packaged by proteins into a condensed structure called chromatin. This allows the very long DNA molecules to fit into the cell nucleus. Chromosomes may exist as either duplicated or unduplicated. Unduplicated chromosomes are single linear strands, whereas duplicated chromosomes (copied during synthesis phase) contain two copies joined by a centromere. Compaction of the duplicated chromosomes during mitosis and meiosis results in the classic four-arm structure as shown in the figure below: Chromatin Structure Chromatin is the chromosomal material found in nuclei of cells of eukaryotic organisms. Each chromatin consists of a single double-stranded DNA in combination with a nearly equal amount of small basic proteins called histones and smaller amounts of non-histone proteins (most of which are acidic and larger in size than histones) and a small quantity of RNA molecules. The double stranded helix of DNA has a length that is thousands of times larger than the diameter of the cell nucleus. Histones facilitate condensation (compactness) of DNA, so that all chromosomes can fit into the nucleus. The chromatin is composed of repeating units of dense spherical particles called nucleosomes (about 10 nm in diameter) connected by DNA filaments. There are two types of chromatin. Euchromatin is the less compact DNA form, and contains genes that are frequently expressed by the cell. The other type, heterochromatin, is the more compact form, and contains DNA that are infrequently transcribed. This structure is further categorized into facultative heterochromatin, consisting of genes that are organized as heterochromatin only in certain cell types or at certain stages of development and constitutive heterochromatin that consists of chromosome structural components such as telomeres and centromeres. 16 During interphase the chromatin organizes itself into discrete individual patches, called chromosome territories. Active genes, which are generally found in the euchromatic region of the chromosome, tend to be located towards the chromosome's territory boundary. Histones organize DNA, condense it and prepare it for further condensation by nonhistone proteins. This compaction is necessary to fit large amounts of DNA (2m/6.5ft in humans) into the nucleus of a cell. Non-histone is a general name for other proteins associated with DNA. This is a big group, with some structural proteins, and some that bind only transiently. Non-histone proteins vary widely, even in different cells from the same organism. Most have a net (-) charge, and bind by attaching to histones. Both histones and nonhistones are involved in physical structure of the chromosome. Histones are abundant, small proteins with a net (+) charge. The five main types are H1, H2A, H2B, H3, and H4. By weight, chromosomes have equal amounts of DNA and histones. Histones are highly conserved between species H1 less than the others. A possible nucleosome structure Chromatin formation involves histones and DNA condensation so it will fit into the cell, making a 10-nm fiber. Chromatin formation has two components: two molecules each of histones H2A, H2B, H3, and H4 associate to form a nucleosome core and DNA wraps around it 1-3 or 4 times for a 7-fold condensation factor. Nucleosomes form the fundamental repeating units of eukaryotic chromatin which is used to pack the large eukaryotic genomes into the nucleus. In mammalian cells approximately 2 m of linear DNA have to be packed into a nucleus of roughly 10 µm diameter. Nucleosomes are folded through a series of successively higher order structures to eventually form a chromosome. These compacts DNA and creates a regulatory control which ensures correct gene expression. 17 Nucleosomes carry epigenetically inherited information in the form of covalent modifications of their core histones. The nucleosome core particle consists of approximately 147 base pairs of DNA wrapped in lefthanded super-helical turns around a histone octamer consisting of 2 copies each of the core histones H2A, H2B, H3, and H4. Linker histones such as H1 and its isoforms are involved in chromatin compaction and sit at the base of the nucleosome near the DNA entry and exit binding to the linker region of the DNA. The nucleosome core particle at high resolution. ( a) A combined space-filling and surface representation. DNA is white; histones are colored: H2A-yellow, H2B-red-, H3-blue and H4-green. Nucleosome cores are about 11 nm in diameter. H1 further condenses the DNA by connecting nucleosomes to create chromatin with a diameter of 30nm, for an additional 6-fold condensation. The solenoid model proposes that the nucleosomes form a spiral with 6 nucleosomes per turn. Nucleosomes connected together by linker DNA and H1 histone to produce the “beads-on-a-string” extended form of chromatin: Packaging of nucleosomes into the 30-nm chromatin fiber: Beyond the 30-nm filament stage, electron microscopy shows 30–90 loops of DNA attached to a protein scaffold. SARs (scaffold-associated regions) bind non-histone proteins to form loops that radiate out in spiral fashion. Fully condensed chromosome is 10.000-fold shorter and 400-fold thicker than DNA alone. Model for the organization of 30-nm chromatin fiber into looped domains that are anchored to a nonhistone protein chromosome scaffold: 18 The many different orders of chromatin packing that give rise to the highly condensed metaphase chromosome: 19 Euchromatin & Heterochromatin: Staining of chromatin reveals two forms: euchromatin: condenses and decondenses with the cell cycle. It is actively transcribed, and lacks repetitive sequences. Euchromatin accounts for most of the genome in active cells. Heterochromatin remains condensed throughout the cell cycle. It replicates later than euchromatin is transcriptionally inactive. There are two types of heterochromatin: Constitutive heterochromatin: regions those are always heterochromatic. Permanently inactive with regard to transcription. Occurs at the same sites in both homologous chromosomes. Consists mostly of repetitive DNA (e.g., centromeres). Facultative heterochromatin: regions that can interconvert between euchromatin and heterochromatin. Varies between cell types or developmental stages, or even between homologous chromosomes. Example: Barr body. 20 Centromeric and Telomeric DNA These are eukaryotic chromosomal regions with special functions. Centromeres are the site of the kinetochore, where spindle fibers attach during mitosis and meiosis. They are required for accurate segregation of chromatids. Directions of kinetochores: in the first meiosis, the kinetochores of the two chromatids face the same direction (left), but in somatic cell division they face opposite directions (right). Kinetochores are regions found in chromosomes. They contain highly repetitive DNA sequences, and are bound to by many proteins. During cell division, microtubules are attached to these regions for chromosome segregation (kinetochore). Kinetochores are equivalent to the primary constriction sites of chromosomes in higher eukaryote. Telomeres are located at the ends of chromosomes, and needed for chromosomal replication and stability. Generally composed of heterochromatin, they interact with both the nuclear envelope and each other. All telomeres in a species have the same sequence. Centromeres and telomeres are both Heterochromatin. Unique-Sequence (non-repetitive) and Repetitive Sequence DNA Prokaryotes have mostly unique-sequence DNA. Eukaryotes have a mix of unique and repetitive sequences. Unique-sequence DNA includes most of the genes that encode proteins as well as other chromosomal regions. Human DNA contains about 65% unique sequences. Dispersed Repetitive Sequences There are two types of interspersion patterns found in all eukaryotic organisms: LINEs (long interspersed repeated sequences) with sequences of 5 kb or more. The common example in mammals is LINE-1, with sequences up to 7 kb in length that can act as transposons. SINEs (short interspersed repeated sequences) with sequences of 100–500 bp. An example is the Alu repeats found in some primates, including humans, where these repeats of 200–300 bp make up 9% of the genome. Anatomy of Chromosome Diagram of a duplicated and condensed metaphase eukaryotic chromosome. 21 1. 2. 3. 4. Chromatid, one of the two identical parts of the chromosome after S-Phase. Centromere, the point where the two chromatids touch, and where the microtubules attach. Short arm (is known as p). Long arm (is known as q). Functions of DNA DNA is the very basis of life and has five-fold roles: 1. 2. 3. 4. It carries hereditary characters from parents to offspring. It enables the cell to maintain grow and divide by directing the synthesis of structural proteins. It controls metabolism in the cell by directing the formation of necessary enzymatic proteins. It contributes to the evolution of the organism by undergoing gene mutations (changes in the sequence of base pairs). 5. It brings about differentiation of cells during development. Only certain genes remain functional in particular cell. This enables the cells having similar genes to assume different structure and function. Human Genome Human genome is the totality of DNA characteristic of all the 23 pairs of chromosomes. The human genome has about 3x109 bps in length. 97% of the human genome is non-coding regions called introns. 3% is responsible for controlling the human genetic behavior. The coding region is called extron. There are totally about 40.000 genes, over 5.000 have been identified. There are much more left. Human Genome Project is to identify the DNA sequence (every bp) of human genome (only a few individuals). For human being, most of the place in human genome are the same. Only a very small part is different among different individuals. Genes or DNA sequences them self are not control the phenotypes, they control the phenotypes through protein. Protein: like the DNA molecule that is a chain of base pair, each protein molecule is a linear chain of subunits called amino acids