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Biology 2581B Midterm: Genetics Testable Readings

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Western University
Biology 2581B
David R Smith

Reading lecture 2 Comparative genomics - Analysis of nucleotide sequences of genomes - Two divisions cell and viral - Cellular empire o Bacteria o Archaea o Eukarya - Creates netlike routes of evolution to depict history of life - Eukaryotes = mitochondria endosymbiosis Cells, Viruses and the classification of Organisms - Cells are o compartments that contain chromosomes (DNA) o DNA is machinery for genome replication and expression o Cell division § Segregation of genome into daughter cells o Eukaryotes contain cytoskeleton and intracellular membrane partitions - Viruses o Intracellular parasites o Encode some proteins essential for viral replication but not the RNAs required for translation or metabolism. They use the cell to produce components - Taxonomy o Linnaeus’ system § Similarities between species o Darwin Tree of Life o Post Darwin § Monophyletic taxa · Groups of organisms that share a common ancestry and form a branch in the TOL · Based on phenotypic similarities · Unicellular organisms like bacteria were hard to compare o Carl Woese § Compared nucleotide sequences of a molecule (rRNA) § 16S rRNA because it is in all cells § Derived a global phylogeny of cellular organisms § Found three domains of life · Bacteria · Archaea · Eukarya Cellular Domains: Archaea, Bacteria and Eukarya - Three breakthrough by Woese o Molecules undergo evolutionary change o Detection of 16S rRNA sequence conservation in all cellular life supported Darwin’s common ancestry theory o LUCA really existed - 1908s they placed bacteria on one side and archaea and eukarya on other side of LUCA o Cellular similarity and common ancestry are different Networks of Genome Evolution Replace Tree of Life - Scientists create phylogenetic trees for individual genes - Genes have distinct evolutionary history - Bacteria and Archaea become mixed in these trees o Horizontal gene transfer Symbiosis of Two Prokaryotic Cells at the Origin of Eukaryotes - Horizontal Gene Transfer o Less common in eukaryotes o Contain a small amount archaeal (dogma proteins) and bacterial (metabolic enzymes and transporters) gene homologue o Eukaryotes are archaebacterial genetic chimeras § Genes from two different organisms § Endosymbiosis · Invasion of host cell by another organelle like mitochondria · Transfer of genes from mitochondrion to nucleus The world of Viruses - Dominant biological entities on earth - Greatest number of unique genes without homologs - Genetic cycles o DNA or RNA genomes (ss and ds) - Small genomes 1000 to 1000000 nucleotides - Hallmark genes in virus are missing in cellular life forms o Essential for reproduction of virus o Evolutionary unity of the viral empire o Mimivirus has a bigger genome than some bacteria and archaea The Two Empires and Three Domains of Life in the Postgenomic Age ● Comparative genomics - analysis of the nucleotide sequences of genomes ○ There are two empires, cellular and viral ○ The cellular empire consists of Bacteria, Archaea, and Eukarya Viruses: ● Electron microscopy allows the visualization of tiny particles that are much smaller than cells; viruses ● Viruses are obligate intracellular parasites that contain genetic elements that encode essential proteins ○ They exploit cells to produce their components ● Viruses are the dominant biological entity on earth, both in terms of number and variety ○ Eg. variations in their genome type ● A small core of viral hallmark genes exist that are missing in cellular life ○ These are essential for reproduction ● They also invade others and are major agents of gene transfer Classification: ● Used the nucleotide sequence of the 16S rRNA since it was well conserved ○ Derived a phylogeny of cellular organisms for the first time ○ This conservation throughout all forms of cellular life is the strongest possible support for Darwin's theory ○ Provided evidence that a LUCA existed ○ Archaea and eukaryotes share a common ancestor to the exclusion of bacteria ■ The replication system of archaea is unrelated to bacteria, but homologous to eukaryotes ■ Archaeal membrane and proteins involved in its formation are unique, while bacteria and eukaryotes share homologous membranes ● But when scientists build trees for the numerous genes encoding metabolic enzymes, the separation of archaea and bacteria is almost never precisely reproduced ○ The archaeal and bacterial branches are mixed ○ This means that genome evolution in proks is not a treelike process but is rather filled with many horizontal connections Horizontal Gene Transfer: ● The exchange of genes between different species and it is the principle mechanism of evolutionary innovation in prokaryotes ○ Eg. antibiotic resistance among pathogenic bacteria ● HGT is less common in eukaryotes but also the majority are closely related to bacterial homologs, whereas a minority appear to be of archaeal origin ○ The archaeal genes encode proteins involved in information processing ○ The bacterial genes encode mostly operation proteins such as metabolic enzymes and membrane transporters ● Eukaryotes - archaebacterial genetic chimeras (combinations of genes from two very different organisms) ○ This is explained by endosymbiosis ■ An invasion of one (host) cell by another, followed by the degradation of the invader (endosymbiont), which became an organelle ■ One hypothesis says that the host was a primitive euk cell and the other says that endosymbiosis triggered the evolution of euk Reading Lecture 3 Lecture 3 readings 1. Mongrel Microbe Tests Story of Complex Life - Helps solve how simple microbes transformed into the complex cells that produced animals plants and fungi - Christa Schleper went to Slovenia o Searching for Loki § Archaea but share features with complex life forms - Eukaryotes o Mitochondria, nucleus with DNA, molecular architecture (cytoskeleton) - Endosymbiosis o One cell swallowed another and began to work together as one o Two theories § Eukaryotes emerged in a rapid burst driven by mitochondria. § Step wise process, mitochondria couldn’t have developed in simple cells, some level of complexity must have evolved before mitochondria came onboard. o Studying modern organism’s DNA and piece history together o Loki § Placed as archaea but possesses genes like eukaryotes § Engulf other cells capability Mitochondrial Merger - Eukaryotes vs archaea o Nucleus which has lots of DNA o Archaeon or primitive eukaryote engulfed a bacterium § Symbiotic relationship and bacteria became more and more dependent on host - Mitochondria early theory o Primitive archaeon engulfed bacterium and drove development of eukaryotes o Bacteria in bacteria - Mitochondria late theory o Protoeukaryotes had already developed complex features when mitochondria came on board o Ancient eukaryotes should lack mitochondria - Loki o Middle ground o Cytoskeleton to move and engulf - Have never seen loki it is just DNA right now - Loki could lack power to engulf other microbes without mitochondrion 2. Steps on the road to eukaryotes - Eukaryotic lineage originated within the archaeal domain o Related to archaea called TACK superphylum - Reconstructed genomes from ocean sediment and found closely related members of archaeal group which is Loki - Loki is the closest prokaryotic relative of eukaryotes yet discovered - Loki contains more eukaryotic signature genes than do other prokaryotes including o Actin proteins, ubiquitin, etc - More closely related archaeal relatives of eukaryotes will be discovered 3. Mitochondria in the second act - Host cell from which eukaryotes evolved was already chimeric before mitochondrial symbiosis suggesting that mitochondria evolved later in eukaryotic evolution that was previously presumed - Mitochondria do oxphos and were previously alpha proteobacteria - Mitochondria entered Loki and archaea but now thinking that eukaryotes was already largely established (mito late) o Eukaryotes emerged before symbiosis o Popularity decreasing of this model § Eukaryotes that do not have mitochondria are thought to have diverged before mit evolved to organelles - Mito-early states that primitive host cell and the endosymbiont was main driving force of eukaryogenesis - Syntrophic interaction o One lives of the other o Reallocation of energy from host cells to mitochondria o Host provided with surplus of energy to trigger more complex cellular features - Bacterial genes in eukaryotes cannot be traced back to alleged ancestor of mitochondria o Originate from unrelated bacteria - LECA o Tracing back to LECA identified protein according to timing of appearance in eukaryotes o Oldest LECA proteins are dominated by archaea related proteins that envolved in cellular functions such as replication o LECA proteins dominated by bacterial proteins mostly alphaproteobacteria o Bacterial ancient genes are probably due to horizontal gene transfer Mongrel Microbe Tests Story of Complex Life ● Loki, newly discovered group of organisms ○ Belong to archaea but seem to share some features with more complex life forms, like us ○ Its genes have potential for providing a starter kit for the emergence of eukaryotes (eg. endosymbiosis) ■ Has genes linked to the dynamic, shape shifting cytoskeleton, allowing the cell membrane to change shape, so it is able to engulf prey ○ What is odd about them is that we can't grow them in a lab: you can just isolate their bacteria and infer what it does ● Some parasites, such as giardia, are missing mitochondria ○ It appears that they lost it over the course of their evolution Evolution: Steps on the road to eukaryotes ● Loki are the closest prokaryotic relatives of eukaryotes yet discovered ● They both have: ○ Cytoskeleton ○ Membrane remodelling ○ Ubiquitin modification ○ Endocytosis and/or phagocytosis Evolution: Mitochondria in the second act ● The host cell from which eukaryotes evolved was already genetically chimeric before the mitochondrial symbiosis, suggesting that mitochondria evolved later in eukaryotic evolution than previously presumed ● Mito Late Model - eukaryotes emerged before the mitochondrial endosymbiont was acquired ● Mito Early Model - the interaction between a primitive host cell and the mitochondrial endosymbiont was the main driving force for eukaryogenesis ● The oldest LECA proteins are dominated by Archaea related proteins that are involved in cellular functions such as replication, translation and transcription, in addition to proteins that are located in the ER and the golgi apparatus ○ Maybe these came from previous endosymbiotic interactions or even waves of HGT Reading lecture 4 What do human parasites do with a chloroplast? Apicomplexans - Pathogens that include agents that cause malaria, toxoplasmosis, cryptosporidiosis - Single celled eukaryotic parasites evolved from photosynthetic algae - Remnant chloroplast called apicoplast o Essential for parasite growth and development o Target for drug therapy, human cells lack these so they can be attacked Apicomplexan parasites, the dark side of the algal world - Apicomplexans are protists that live as intracellular parasites in animals - Cause serious disease o Malaria - Evolved from photosynthetic ancestor - Branch called chromalveolates o Includes kelp, diatoms, dinoflagellates, etc… o Primary carbon fixation in the ocean - Merger of two eukaryotes o Protist host and red algal endosymbiont o Alga was transformed into a chloroplast like organelle - Apicomplexans are kept alive by their plastid even though they have lost photosynthetic capability Why did parasites maintain pointless organelle? - Cant afford to lose it - Chloroplasts have functions other than photosynthesis that are crucial - Non photosynthetic functions are conserved in the apicoplast - Biosynthetic pathways for fatty acids, heme pathway, etc… - Has to be fed energy due to loss of energy producing capability: photosynthesis - All maintenance of apicoplast including gene expression is up to host cell To IPP or not to be that is the function - Two main biosynthetic functions for apicoplast o Fatty acid synthesis o Isoprenoid precursors - In the RBC the parasite can salvage fatty acids from its host - Isoprenoid is a large class of biological compounds including rubbers, cholesterol, ubiquinone, dolichol o IPP is a 5 carbon precursor feeds the DOXP pathway o ​DOXP pathway found in plants and bacteria not animals - Parasite can grow in presence of IPP and antibiotic and plastid is lost The Apicoplast and its potential for drugs and vaccines - DOXP pathway is prime target for antiparasitic drugs - Fosmidomycin is effective in treatment What Do Human Parasites Do with a Chloroplast Anyway? ● Apicomplexans are an important group of pathogens that include the causative agents of malaria, toxoplasmosis and cryptosporidiosis ● These single celled euk parasites evolved from the photosynthetic age and they have apicoplasts (a remnant chloroplast) ○ Use the apicoplasts to target these parasites since humans and animals don’t have it ● The apicoplast is a home to synthetic pathways for fatty acids, isoprenoids, iron sulfur cluster assembly, and a segment heme pathway ● Two important biosynthetic pathways known for their importance in the biology of the plant chloroplast emerged as candidates for the most critical apicoplast functions: → The synthesis of fatty acids and isoprenoid precursors ● Both pathways are of cyanobacterial origin and are different than the mammalian pathways ● The DOXP pathway is a prime target for the development of new ant parasitic drugs Reading Lecture 7 -Nik Testable Reading - Chapter 8 237-252 Mutations: Primary Tools of Genetic Analysis - Three main themes from studies of genes - Mutations are heritable changes in base sequence that can affect phenotype - Physically, a gene is usually a specific protein encoding segment of DNA in a discrete region of a chromosome - Some genes encode for RNA which is not translated - A gene is divisible and each nucleotide can mutate independently and can recombine with each other - Polymorphic: genes with several common alleles - Wild Type allele: allele gound in the large majority of chromosomes in the population - Must be 1% or greater to be wild type, otherwise called common variant Mutations are Heritable changes in DNA base sequences - Forward mutation: mutation that changes a wild type allele to a different allele - Can be recessive or dominant to the WT - A+ → a recessive - b+ → B when dominant - Reverse mutation/reversion: mutation that causes mutant allele to revert back to WT - WT is always + - Capital means recessive or dominant Mutations may be classified by how they change DNA - Substitution: when a base at a certain position in one strand of the DNA molecule is replaced by one of the other 3 bases - Transitions: one purine replaces another purine (A or G), or pyrimidine to another pyrimidine (C or T) - Transversions: one purine changes to pyrimidine or vice versa - Deletion: block of one or more nucleotides is lost from DNA - Insertion: block of one or more nucleotides is gained - Inversions: 180 rotation and reverse of a segment of the DNA molecule - Reciprocal translocations: parts of two nonhomologous chromosomes change places Spontaneous mutations occur at a very low rate - Must examine a large number of individuals, average in coat colours of mice was 11 of every 1 million gametes - Multiply the rate of mutations per gene times 20 000 (# genes in the human genome) - In humans 2 to 12 x10^-6 average mutation rate per gene - Mutations affecting phenotype occur VERY rarely Different genes, Different mutation rates - Average mutation rate in eukaryotes higher than bacteria as there are more chances for mutations to accumulate between formation of zygote and meiosis. Two copies of human chromosomes allows for toleration of mutation. Bacteria will have a single mutation on their only copy of their genome. - Different genes mutate at different rates Gene function: Easy to disrupt, hard to restore - Reversion rate can be found and is usually significantly lower than the rate of forward mutation - Makes sense easy to mutate but harder to restore - Deletions are impossible to revert Spontaneous mutations arise from many kinds of random events - Bacteria - Easy to grow thus easier to find a mutated individual - Form colonies, some show resistance to antibiotics as a mutation when exposed to antibiotics on a sugar agar plate - Luria Delbruck​ experiment to examine bacterial resistance - Hypothesis 1 - Resistance is a physiological response to bacteriophage - Equal colonies in fluctuation test - Hypothesis 2 - resistance arises from random mutation - Differing colonies in fluctuation test - Fluctuation test results - Hypothesis 2: bacterial resistance arises from mutations that exist before exposure to bacteriophage - Replica plating - Press master plate on velvet then replicate on new plate with penicillin - Only penicillin resistance colonies will grow - Repeat multiple times will still see that the same colonies in the same place will go - Resistance is a previous random spontaneous mutation NOT a response Natural processes that alter DNA - Depurination: hydrolysis of a purine base A or G resulting in an apurinic site that cannot specify a complementary base - Introduces random base opposite to apurinic site - Deamination: removal of an amino group - Changes C to U and U pairs with A not G - CG to TA (transition) - Xrays break the DNA backbone - UV light produces thymine dimers - Oxidation creating active oxygen species - GC to TA Mistakes during DNA replication - Mistakes occur every 10^9 bp - Polymerase makes a mistake every 10^6 bp - 3’ to 5’ nuclease recognizes mispaired base and excises it allowing polymerase to correct the mistake - Without nuclease mistake every 10^4 bp Unequal crossing-over and transposable elements - Unequal crossing-over - Two closely related DNA sequences that are located in different places on two chromosomes can pair with each other during meiosis - Unequal results in deletion in one and duplication in other - Transposable elements - Jump from place to place in the genome - Replicative transposition: jump but copy remains - Conservative transposition: jump and no copy remains Unstable trinucleotide repeats - CGG or CAG, CTG, GAA etc - Number of repeats differs between cells - Expand and contract - Causes delay in DNA synthesis, polymerase falls off every few repeats - Fragile X syndrome caused by CGG repeat expansion - Causes mental retardation - Usually mother is a carrier Mutagens Induce Mutations - Exposing to Xrays cause higher rate of mutation - Mutagen: increases spontaneous mutation rate - Base analogues: mutagens similar in structure to normal base - Look above Figure 8.10 for all these mutations - Intercalators: flat planar molecules that sandwich themselves between successive base pairs and disrupt replication, repair. Causes insertion or deletion of a bp. DNA repair mechanisms minimize mutation Reversal of DNA base alterations - Alkyltransferase enzymes: remove methylation and ethylation added to guanine - Photolyase: thymine dimer reversal, works only in presence of light Removal of damaged bases or nucleotides - Types of homology dependent repair - Remove small region that contains altered nucleotide - Use other strand as template - Base excision repair - DNA glycosylases remove the base and leave apurinic/apyrimidinic (AP) site in the chain - Usually removes Uracil from deamination - Endonuclease creates a nick in the DNA backbone at the AP site, exonuclease removes nucleotides and DNA polymerase fills, then ligase seals. - Nucleotide excision repair - Protein machines (UvrB and C endonucleases) detect irregularities in double helix - Excises entire region on one strand to be replaced by polymerase and sealed by ligase - Thymine dimers Correction of DNA replication errors - Methyl directed mismatch repair: repairs DNA polymerase mistakes - In bacteria!: Attaches methyl to adenine every GATC - Old template will have methyl marks, new strand won’t contain this - MutS and MutL detects weirdness, contacts MutH near GATC creating loop, MutH creates nick on new strand, exonuclease removes, and DNa polymerase replaces Error Prone repair systems: A last resort - SOS system - Sloppy DNA polymerase, add random nucleotides to the damaged bases - Double strand breaks fixed by nonhomologous end joining - Both strands broken caused by Xrays or reactive oxygen molecules - Randomly join ends together Mutations in genes encoding DNA repair proteins can allow mutations to accumulate throughout the genome leading to cancer Reading Lecture 8 - Parisa Testable Reading - Chapter 7 215-219 and 17 555-561 Chapter 7 215-219 The Effect of Mutations on Gene Expression and Gene Function Mutations in a gene’s coding sequences may alter the gene product - Silent mutations: change codon into a mutant codon that specifies the same amino acid - Missense mutations: change codon into a mutant codon that specifies a different amino acid. Conservative means similar property amino acid. Non conservative means different amino acid property - Nonsense mutations: change codon to stop codon. Causes truncated protein. Worse if closer to the start. - Frameshift mutation: insertion or deletion of nucleotides within the coding sequence. If not divisible by 3 then will cause reading frame change. Destroys polypeptide if occurs early on. Mutations outside the coding sequence can also alter gene expression - Promoters and termination signals - Changes in promoter make it hard for RNA polymerase to recognize the site and prevent or diminish transcription - Splice acceptor sites that allow splicing to join exons together - Changes in this can obstruct splicing and result in bad protein - Ribosome binding sites and in frame stop codons can be mutated - Inefficient translation and longer proteins result Most Mutations that affect gene expression reduce gene function - Loss of function mutation: mutation inside or outside coding region that reduces or abolishes protein activity Recessive loss-of function alleles - Null/amorphic: completely block the function of a protein - Prevent synthesis or make it incapable of function - If occurs in one allele and is recessive, the other allele will create functional protein (A+/a) - Usually (not always) half of the protein made by A+/A+ cell - If the single A+ amount is above the biochemical threshold of the cell, the phenotype will be WT - Hypomorphic mutation: loss-of-function that produces less of a protein or same amount but lower activity - B+/b heterozygote where b is hypomorphic, amount of protein will be somewhat greater than half the amo
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