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Main Ideas for Bio 1002B Part III.docx

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Western University
Biology 1002B
Denis Maxwell

Main Ideas for Bio 1002B Part III Cancer  4 most common: Prostate, breast, lung, colorectal  Predisposition of inheriting one defective allele increases risk  More environmental – progressive and accumulative mutations Cyclic dependent Kinase (CDK)  Cell cycle regulation  G1 checkpoint – G1 cyclin E bind to CDK2 to activate protein phosphorylation to allow DNA replication o Mutations cause no DNA replication ∴ sterile or allow damage DNA to replicate  S phase – S cyclin A bind to CDK2 to initiate DNA replication  G2 – accumulation of M cyclin B bind to CDK1 to activate protein phosphorylation allow G2 checkpoint  Each cyclin is degraded in the next stage  EGF binds to EGFR and stimulates growth by signalling gene transcription that promote cell cycling, mitosis o Mutation can cause rapid/excessive cell division and can form a tumour Ways that cells can turn to be cancerous  Proto-oncogene (normal cell growth and differentiation gene for rapid embryonic cell division)  oncogene o Mutation in promoter or genome rearrangement could ↑ expression o ↑ mRNA stability or ↓ mRNA decay or gene duplication ↑ protein o Any mutation or deregulation in EGF pathway  Oncomir (usually over expression of miRNA but each tumour has a specific expression of miRNA) o Bind to mRNA to block translation and causes mRNA destabilization/decay of tumour suppressor genes o Underexpression of miRNA that causes overproduction of unnecessary proteins  TP53 tumor suppressor gene (slow cell cycling in embryology) helps prevent cancer by preventing mutation o Prevent G1 checkpoint by initiating apoptosis to get rid of damaged DNA and repairing it  Stem cells  stem cell or specialized progenitor  differentiation cell (asymmetrical fate) o Mutated or deregulated self-renewal gene can make them go back to be an out of control stem cell  Epigenetic (cancer = deregulation and regulation = epigenetic) o Changed expression/reprogrammed of tumour nuclei when placed inside another enucleated egg o Maybe suppressor genes turned off by methylation Molecular Homology and Comparative Genomics  Sequence genome – easy, cheap and no math involved  Genome annotation – look at regulatory elements, biological functions of similar to predict what gene codes for  Protein prediction – most probable reading frame out of the 6 possible is the longest open frame  Blast/local  Clustal/global o High similarity regions w/out force o Forced to align o Faster and stronger o Slower and weaker  Interpretation o Weak global and strong local = similarity shows conserved protein functioning but diff structure as whole o Strong global: overall similarity, when suspect high in homology o Homology means common ancestor and not similarity  E.g. query: predicted protein coding for chlamy, subject: volvox glsA gene o Can’t have everything identical but similar (protein, amino acids, length, function) if homologous o a.a. codes > proteins – redundant code ∴ evolution and selection act on phenotype and not genotype o Blast/local used and shows local similarity and homology was found (orthologue)  Information theory o More info in amino acid sequence than nucleotide of same length due to larger alphabet o = total info (nucleotide = 2; amino acid = 20, more conserved) o G = # of symbols, n = # of alphabet characters o ↑ similarity = ↑ homology = ↓E value = ↓ convergence Molecular Convergence  Selection theory – mutations are deleterious advantageous or not (most are not)  Neutral hypothesis – neutral synonymous/silent mutation rate (change genotype not phenotype)>nonsynonymous  # of differences in proteins vs time since diverged o Molecular clock – straight constant rate of molecular change (based on neutral theory) o Genetic equidistance = equally diff e.g. cytochrome of 2 species – 20 a.a. diff, diverge 400 mya o Slow mutation – can’t alter protein too much without changing structure, function e.g. histone h4 o Fast mutation – function less depend on 3D shape e.g. fibrinopeptide (clot blood), α-globin Functional convergence  Convergent evolution – local similarities (modular protein function) and not similarity through entire sequence o Location of cysteines and disulphide bonding for tertiary structure o Amino acids for catalysts, DNA binding domain, receptor binding Lysozyme  Both types of gene duplication in ruminants – digest peptidoglycan wall of bacteria that digests cellulose o Low pH – stronger hydrogen bonds and tertiary structure o Protect from pepsin  Structural stability since less flexible to induced fit  Lack aspartic acid (intermediate in making denaturer urea) more resistant to denaturation  Lack proline bond cleavage which is acid labile (easily changed and broken down in acid)  Negative charge repels pepsin  Convergent since langur and baboon more related but more similarities in langur, cow and hoatzin bird o Lysozyme is different than humans – aspartic acid (75) and asparagine (87) o Human lysozyme break down bacteria in blood and tears and not in stomach due to denature in low pH Experimental Evolution  Model systems have short generation time and reproduce asexually e.g. Lenki’s experiment with E. Coli o Transfer 0.1 mL (5 million cells) of 12 isogenetic identical culture into 9.9 mL media daily o Cryopreservation (freeze) every 500 generations (75 days) to compare with ancestor o Ara – 3 change turbidity more cells/mL – use citrate in oxic conditions (only enough glucose for 8hrs/day)  Citric acid cycle in mitochondrial matrix makes citrate as the first product  Citrate originally used to keep iron in solution to be taken up by cells and hemoglobin  Citrate mutation dependent/contingent on previous mutation o Potentiation (first 20 000 generations) mutation that set up genome for cit + phenotype o Actualization – faint positive citrate phenotype since 2 copies of rnk-citT expressed  Genome rearrangement – oxic promoter for rnk now closer/upstream of cit T & G (diff regulation) o Refinement – 9 copies of rnk-citT through amplification o Cit – is not the dominant form but is not extinct since it is more efficient at glucose utilization  Gene duplication – only one copy is kept o Subfunctionalization gene duplication (diff promoter = diff regulation; ↑ expression) o Neofunctionalization – whichever mutates faster, change structure (keep promoter)  Rare mutation – replay evolution and get mutation at various times  Spontaneous mutation – rare but usually deleterious or neutral Elysia/Vaucheria System  Hypothesis: isolated chloroplast will decrease in function but doesn’t in elysia because of lateral gene transfer o Isolated chloroplast cannot repair parts coded in the nucleus genome  pbsO in nuclear genome to split water in chloroplast in photosystem II  Secondary endosymbiosis – eukaryote engulf primary endosymbiosis (chloroplast only – 4 membranes/heterokont) o Has mitochondria, nucleus, kept only the chloroplast from primitive eukaryote o Vaucheria tripartite targeting sequence of psbO gene for chloroplast ER (outer two), next two, lumen o Elysia’s chloroplast only has 2 membranes because of digestion o Protein cannot be imported – inner and outer membrane does not recognize signal for chloroplast ER Polymerase Chain Reaction  Replicate a portion of DNA only  denaturation (94°C) to separate strands  annealing (45 - 65°C) of hybridization/complementary base pairing of primers  extension (72°C) DNA synthesis from primers  Anneal: 2 unique primers 16 16 – 30 bp (1 in 4 chance similar; not too specific) pair to each end o To design a primer, find similar sequence from various organisms for vaucheria o Make variations of degenerate primers keeping conserved (*) sequences  Extension/amplification by thermostable taq DNA polymerase (from thermophile) add monomers for DNA (dATP, dGTP, dCTP, dTTP) to 3’ end (read 3’ to 5’) o No 3’ to 5’ proofreading and doesn’t stop until it reaches until the end of the sequence (no signal to stop) o # of DNA molecules after cycle n = 2ycle(exclude original 2 copies) o N cycles, # of target sequence pairs: 3, 2; 4,8; 5, 22  Size of amplified DNA same band (molecular size marker in electrophoresis) as target DNA then PCR successful  RT – PCR to amplify mRNA of elysia since DNA polymerase amplifies DNA only o First primer used consists of T’s (complementary to 3’ poly A tail) o Reverse transcriptase amplify strand to make cDNA o PCR begins when another primer makes the other cDNA and the rest is the same o Put in Northern Blot to look at the abundance of transcript (darkest = most abundant)  Slight expression of psbO in elysia after 5 months (not possible) because mRNA will have decayed  Therefore gene was there before also proven by that elysia egg has it not by feeding Oxygen and Aging
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