Chapter 4 - DNA and Chromosomes PDF

Genes contain the instructions that determine the characteristics of a species. Hereditary information is carried on chromosomes. Chromosomes are made of DNA and protein of equal amounts. The 2 DNA strands run antiparallel to each other (5' phosphate end, 3' hydroxyl end). A and T have 2 H-bonds...

Genes contain the instructions that determine the characteristics of a species. Hereditary information is carried on chromosomes. Chromosomes are made of DNA and protein of equal amounts. The 2 DNA strands run antiparallel to each other (5' phosphate end, 3' hydroxyl end). A and T have 2 H-bonds. C and G have 3 H-bonds. A and G are purines (larger, 2 rings), C and T are pyrimidines (smaller, 1 ring) because of which each base pair is of similar width. Phosphodiester bond connects one sugar to the next, it is the bond between the phosphate group of one sugar and the hydroxyl group of another sugar. Chains of nucleotides in DNA are directional and linear. Information to specify an organism is carried in a chemical form because DNA is a linear polymer formed from 4 different kinds of monomers strung out in a long sequence. Information is duplicated and copied from generation to generation because the 2 strands of a DNA helix are complementary and serve as templates to synthesize copies. Genes contain the instructions from producing proteins and since genes are made of DNA, DNA encodes proteins. The linear sequence of nucleotides in a gene spell out the linear sequence of amino acids in a protein. This correspondence is called the genetic code. Genome: store of information that specifies all the RNA molecules and proteins an organism will ever make. Nuclear Envelope: two concentric lips bilayer membranes punctured by nuclear pores, outer nuclear membrane is continuous with the ER, nuclear envelope is supported by nuclear lamina. Each chromosome contains one long DNA molecule along with proteins that fold the DNA thread into a more compact structure and some RNA molecules that are required for gene expression, DNA duplication, and DNA repair. Chromatin: Complex of DNA and tightly bound protein. Gametes, cells that cannot multiply, lack DNA or multiply without completing the cell cycle, do not have homologous chromosomes. Human cell (other than the ones mentioned) have two copies of each chromosome called homologous chromosomes. Sex chromosomes are non-homologous. DNA painting is based on DNA hybridization where short stand of nucleic acid sequences are attached to probes that color them and these then bind to their complementary sequences on the DNA molecule. Chromosomes can also be distinguished by staining them with dyes to produce a banded appearance that is unique for every chromosome. The bands represent variations in chromatin structure and base composition. Karyotype: display of 46 human chromosomes at mitosis. In addition to genes, genome contains larger quantities of interspersed DNA some which is used for control of gene expression. Differences in the non-coding DNA account for variation in the genome size. Differences in the amount of noncoding DNA cause variations of a hundredfold in genomes of closely related organisms even though they contain roughly the same number of genes. How the genome is divided into chromosomes also differs from one eukaryotic species to the next. Half of the chromosomal DNA is made up of mobile pieces of DNA that have inserted themselves in the chromosomes over time. These are called transposable elements. The noncoding region inside a gene is intron and the coding is exon. Most of the gene consists of introns. Each gene has regulatory DNA sequences that are responsible for ensuring that the gene is turned on and off at the right time. The regulatory sequences are spread out over hundreds of thousands of nucleotide pairs. During interphase, the cell is actively expressing its genes and synthesizing proteins, and DNA is replicated to form sister chromatids. M phase occurs one DNA replication is complete. During M phase, the nucleus is divide into 2 daughter nuclei, chromosomes condense, nuclear envelop breaks, mitotic spindle forms, one complete set of chromosomes is pulled by the mitotic spindle, nuclear envelop reforms around each set. During interphase, chromatin is in long threads. 3 specialized sites on the DNA that control replication and separation of sister chromatids: 1. Replication origin: location at which bi-directional duplication begins. Eukaryotic chromosomes have many to ensure rapid replication. 2. Centromere: allows sister chromatids to be pulled into each daughter cell. Kinetochore form at centromeres and allow mitotic spindle to pull the sister chromatids apart at the centromere. 3. Telomeres: ends of chromosomes that enable ends of chromosomes to be replicated and they form structures that protect the end of the chromosome from being mistaken as broken DNA. In budding yeast cells, the 3 types of sites are short. Telomere sequences for eukaryotes are short, Centromeres and replication origins are long and complex. The sequence that codes for centromeres isn't a well-defined sequence. (it can be any sequence). Interphase chromosomes decondense and recondense in order to allow gene expression, DNA repair, and replication. Proteins that bind to DNA are histone and non-histone chromosomal proteins. Histones and DNA make up nucleosomes. Nucleases cut DNA and allow the nucleosome core particles to be isolated. Histone octamer is made up of 2 dimers of H2A and H2B and 2 dimers of H3 and H4. Linker DNA separates each nucleosome and can very in length. Each histone protein is made up of a histone fold formed from 3 α helices connected by 2 loops. Half of the 142 hydrogen bonds that form between DNA and histone are formed between sugar phosphate backbone of DNA and amino acid backbone of histone. Hydrophobic interactions and salt linkages are also present. Lysine and Arginine and present in the core histones whose positive charges neutralize negatively charged DNA. Bending of DNA compresses the minor grove of DNA helix. Certain dinucleotides bind more tightly to histones. Each histone core has unstructured N-terminal amino acid tail that are sites for covalent modifications. Histones are the most highly conserved eukaryotic protein. Histones H2A and H2B have changed more than H3 and H4 over the course of evolution. This means that histones involve all their amino acids so that a change in any position is deleterious to cell. Eukaryotic organisms produce specialized variant core histones. ATP-dependent chromatin-remodelling complexes have a subunit that hydrolyses ATP (ATPase) which binds to the histone core of nucleosome and to the DNA and moves the DNA to change the structure of the nucleosome temporarily resulting in nucleosome sliding. Other types of remodelling complexes remove all or part of the nucleosome core by catalysing either an exchange of its H2A-H2B histones or complete removal of octameric core. Nucleosome positioning is majorly influenced by the presence of other tightly bound proteins on the DNA. Nucleosomes are packed on top of one another forming arrays in a zigzag model. Nucleosome to nucleosome attractions between the histone tails especially H4 tails lead to nucleosome stacking. Histone h1 (linker histone) is larger than core histones and has been less conserved during evolution. Nucleosome binds to H1 and then the H1 changes the path of the DAN that exits from the nucleosome. This helps compact nucleosomal DNA. Epigenetic inheritance: inheritance that is not based on a change in the DNA sequence. Two types of chromatins in interphase; heterochromatin (highly condensed) and euchromatin (less condensed). Open and active chromatin (20% of genome): euchromatin Closed and inactive (80% of genome): heterochromatin and quiescent euchromatin. Heterochromatin prevents gene expression and is commonly found at centromere and telomeres as well as other regions depending on the type of cell. Constitutive heterochromatin is permanently condensed. Facultative heterochromatin can be regulated to control gene expression. Euchromatic chromosome can accidently translocate to region of heterochromatin, through errors in DNA repair, this silences the normally active gene. This is called position effect. The zone of silencing spreads for different distances and is inherited by daughter cells. This is position effect variegation. Genes that enhance or suppress position effect variegation can be determined by genetic screens. Genetic screens involve the production of mutant genes then the one that causes an abnormality is picked. These genes code of non histone chromosome proteins (reader-writer-remodeling protein complexes). Core histones are covalently modified at many different sites by acetylation and mono, di, tri methylation of lysine and phosphorylation of serine. Most modifications occur on the 8 tails. However, there are more than 20 specific side chain modifications on the nucleosome's globular core. Enzymes that perform these modifications are recruited by transcription regulatory proteins (transcription factor). These factors recognize and bind to specific DNA sequences. Covalent modifications of heterochromatin remain long after the transcription factors are removed and are inherited. Acylation of lysine removes positive charge and loosens chromatin structure. Trimethylation of one lysine on H3 tail attracts protein HP1 and promotes the spread of a type pf heterochromatin. All histones except H4 have variants. Major histones are synthesized during the S phase. Histone variants are assembled during interphase. Histone variants replace histones in a nucleosome in a histone exchange process by ATP-dependent chromatin-remodelling complex. Histone modifications occur in coordinated sets, and these have specific meanings like chromatin has been newly replicated, chromatin has been damaged or how gene expression should take place. Domains of regulatory protein are linked together as modules in a large protein complex and these bind to specific marks and recognize them. These are called reader protein complexes. Modification order: Name of histone is written followed by amino acid side chain followed by the distance from that histone's amino terminus followed by the type of modification. Ex: H3K9ac Reader protein and writer enzyme form a protein complex. Writer enzyme marks histone (covalent modification). Reader protein from same protein complex binds allosterically to mark and activates writer enzymes that marks adjacent nucleosome. 2 major classes of heterochromatin: 1. H3K9me3: forms centromere, silences a variety of selfish DNA elements, blocks the frequent genetic recombination. 2. H3K27me3: generated by polycomb repressive complex (PRC), highly regulated, facultative heterochromatin Erasers are also part of reader writer protein complex. Barrier DNA sequences mark the boundaries of chromatin domains. They contain a cluster of binding sites for histone acetylase enzymes as methylation is required for the spread of heterochromatin and acetylation of a lysine side chain is incompatible with methylation of the same side chain. This is why the barrier DNA has acetylase enzymes. Centromeres have centromeric chromatin that persists through interphase. This chromatin contains variant of H3 histone (CENP-A) and additional proteins that form kinetochores. However, centromeres do not contain a centromere-specific DNA sequence. They instead consist of short, repeated DNA sequences (alpha satellite DNA). But these satellites are again not specific to centromeres only as they are found in other places on the genome too. Neocentromeres form spontaneously on fragmented chromosomes at positions that were originally euchromatic **and do not have alpha satellite DNA**. Hence, centromeres are defined by assembly of proteins rather than DNA sequences. De novo centromere formation starts with a seeding event on alpha satellite DNA sequence followed by the formation of a specialized DNA-protein structure that contains nucleosomes formed with CENP-A variant of H3. This then spreads to create a centromere and it passed down to daughter cells. The spreading of a particular chromatin is the action of reader-writer complexes. In regard to the inheritance of a particular chromatin, the H3-H4 tetramers from each nucleosome of the parent DNA helix is passed down to the daughter cells. Then 2 H2A-H2B dimers complete the half-old nucleosome. Cancer occurs because of sequential changes(that are passed on to daughter cells and accumulate ) of the sequence of DNA for regulatory proteins like protein kinase. Tumour progression is driven by changes to the chromatin structure (changes affecting readers, writers) A change in a single amino acid in a histone can cause cancer. Usually, mutations are not that dangerous because each histone is coded by multiple copies of its histone genes so when a mutation occurs it only cause a 10% change. However, when the mutation is large enough to override the normal histone, an oncohistone gets produced with is a predominant cancer driver. H3K27me3 changes to H3K27M, this causes DIPG in babies and acute myeloid leukaemia and melanomas in adults. Each chromosome is folded into a series of large loops by ring-shaped SMC. Lampbrush chromosomes: stiff and enormously extended amphibian oocytes chromosomes that have a series of large chromatin loops emanating from a linear chromosomal axis. Most of the DNA is located near the junction of 2 chromatids and it is highly condensed. The loops are less condensed and are highly transcribed regions. Lampbrush chromosomes are found in oocytes of all vertebrates except for mammals. However human sperm can form lampbrush chromosomes if incubated with amphibian oocyte cytoplasm. Polytene chromosome: multiples copies of each chromosome aligned side by side in polyploid cells. These chromosomes allow features that are hard to see to be detected. 95% of the DNA in polytene chromosomes is in bands and 5% in interbands. Polytene chromosomes can be stained with antibodies then using chromatin immunoprecipitation analysis (ChIP), the locations of specific histone modifications and non-histone proteins can de mapped out. Drosophila DNA sequence has 3 types of repressive chromatin and 2 types of chromatin on actively transcribed genes, and other minor forms. Chromosome puffs from when genes are expressed and arise from the decondensation of a single chromosome band. Heterochromatic region of a chromosome is closely related with the nuclear envelope. Euchromatic (high density of active genes) extend into the nucleoplasm. The interior of the nucleus is heterogenous, and it made of biomolecular condensates that speed different biochemical processes. Ex: nucleolus for producing ribosome and nuclear speckles for RNA production. Hi-C: chromosome conformation captures that asses the frequency with which any two genes are held together. (lets us know the location of genes, which genes are close to each other, and which are not). Hi-C has revealed that chromosomes are folded in long series of topologically associated domains (TADS) Chromosomal DNA in each TAD is organised into loops by large protein rings called SMC. SMC protein complex: large protein ring that binds and encircles DNA helix, subunits are pair of coiled-coil SMC proteins, each protein has a globular ATPase domain. The ATPase domain of SMC protein complex is associated with additional proteins. SMC protein complex: 1. Role in bacteria: Multiple SMC protein complexes are loaded on to the parent DNA near the replication origin and then these create a lop on each new DNA helix that comes out of the replication origin when the DNA divides and then the protein complexes proceed to move continuously along the DNA separating the two daughter chromosomes. 2. Role in eukaryotes: During interphase, cohesion (SMC complex) is loaded at multiple sites along the chromosome and folds it into a series of loops. Cohesin stops at specific sites where CTCF protein is present, and a loop is formed there. CTCF stops cohesin rings, holds ends of loops together, is an insulator protein that helps to maintain discrete domains of chromatin function. The chromatin in each domain that is separated by CTCF differs in both its non-histone proteins and its covalent histone modifications. Most of the loops of eukaryote mammal's chromosomes are unstable. H3K9me3 is located near the nucleus periphery, H3K27me3 is next to it on the interior, open chromatin (euchromatin) is in the inner regions. Reason why heterochromatin is at the nuclear periphery is unclear. Exception is inverted nuclei of rod cells in nocturnal mammals where the euchromatin and heterochromatin are swapped. Sister chromatids are covered with large amounts of RNA-protein complexes. Chromatids have loops of chromatin emanating from a central scaffolding. Condensation of chromosomes beings in M phase where gene expression stops and histones are modified to reorganize chromatin. SMC condensin and topoisomerase II drive the compaction and form a linear chromosome axis. Topoisomerase and condensin II also play a role in separating the chromosome into 2 separate chromatids. Mitotic chromosome condensation is the final level of chromosome packaging. Condensation allows easy separation of sister chromatids and protects DNA molecules from being broken when pulled apart. HOW GENOMES EVOLVE Homologous genes are genes that are similar in sequence and function. Sequence of genes are more conserved that the overall genome. Genome size, number of chromosomes, order of genes, size of introns, amount of repetitive DNA differs but the sequence of genes doesn't. Exons are short segments that code of proteins. These segments are in a sea of noncoding DNA, and this makes it difficult to identify all the exons and determine where a gene begins and ends. Conserved DNA regions: regions that encode functionally important exons and RNA molecules. 4.5% of human genome consists of multispecies conserved sequences. One fourth of these are protein coding, remaining code for clusters of protein binding sites and RNA molecules. 5% of human genome has reduced variation which means that this 5% is very important. 10% of the human genome contains sequences that truly matter. ENCODE scientists wrongfully stated that 76% of our genome is functional and produces RNA molecules and that previously undetected genes code for RNA molecules. However, most of the genome is junk and this junk sometimes produces RNA (which might make it look like its functionally important when in fact it's not) due to errors in gene expression (background noise). Transposable DNA elements play a role in evolution. Transpons are mobile DNA elements that act as parasitic DNA sequences that spread within genome. This leads to disruption or alteration of existing genes or they create novel genes. Point mutation: local change ex. Substitution of one base pair for another. Large scale genome rearrangements: deletions, duplications, translocations of DNA from one chromosome to another. The close similarity of humans and chimpanzees is due to the short time available for mutation rather than the fact that mutations didn't happen because of function. This is because some sequences that can mutate freely (mutation doesn't affect their function) like synonymous codons are identical in chimpanzees and humans. Purifying selection: selection based on function. Fossil record: source of absolute dates based on radioisotope decay in the rock formation however this has many gaps and precise divergence times are difficult to establish so they are used with phylogenetic trees. Changes in the sequences of genes occur at a nearly constant rate. This provides us with a molecular clock. Clocks run rapidly in introns that lack splicing or regulatory signals, in synonymous codons, and in irreversibly inactivated genes. Clocks are slow in histones and RNA subunits. Rapid change can be seen in previously highly conserved e=sequence due a selective advantage introduced by a mutation. Mitochondrial DNA sequences have clocks that run much faster than those of nuclear sequences. Mitochondrial DNA clock is used to show the divergence of neanderthal lineage from homo sapiens. Molecular clocks are more reliable and accurate than fossil records. Human, chimpanzees, and mouse genomes contain the same size and nearly identical sets of genes. Rodent lineages have unusually fast molecular clocks that resulted them to diverge from the human linage more rapidly than expected. Chromosome breakage and rejoining events moved large blocks of DNA sequences between humans and mice. So, although the number of chromosomes are similar, their structures are different. Even after these breakage and rejoining events, some genes have the same order in mice and humans and are called regions of synteny. In human and mice, small blocks of DNA have been added and removed. Sequence gains from many small chromosome duplications and from the multiplication of transposons have compensated for these deletions. Genome size can change without a drastic effect on the organism or its number of genes. The small size of Fugu genome is due to small size of its introns and intergenic regions however their positions are the same.