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Biology 1002B
Tom Haffie

Biology 1002 Natty Liu Final Exam Outcomes Lecture 18: Cancer Four most common types of cancer in Canada • Breast, prostate, lung, colorectal • Leading cause of premature death in Canada, 25% of Canadians can expect to die of cancer Likely factors contributing to cancer incidence in Canada • Men are at higher risk for cancer than women • Rates among youth are rising Role of cyclin/CDK complexes in cell cycle regulation  Cyclin / CDKinase complexes regulate cycling  G1S checkpoint prevents cell from replicating it‟s DNA if it‟s damaged until everything is repaired which is monitored by CDK (cyclin-dependent kinase)  Cyclin production cycles are with cell cycle – produced early on and binds & activates CDK  CDK complex then phosphorylates whole bunch of targets downstream, releasing the G1S checkpoint  Example of post-translational regulation of protein function Role of proto-oncogenes, tumor suppressor genes and oncomirs in cancer • Oncogenes – “cancer genes” aren‟t actually cancer genes but really embryo genes • Normal genes become an oncogene due to mutations or deregulation • Expression of proto-oncogenes promotes cell cycling: Epidermal growth factor receptor (needed for rapid cell division, if they are over expressed they can promote tumor growth) • Expression of tumor suppressor genes slow cell cycling: p53 • “TP53” is a master tumor suppressor gene, coding a transcription factor whose activity can result in: increased DNA repair, cell cycle arrest by blocking cyclin / CDK, apoptosis • Did not evolve to shut down tumor, evolved to shut down cell division • Prevent cell division, if they become mutated then we get more out of control division • Inappropriate expression of miRNA can promote cycling: oncomirs 1 Biology 1002 Natty Liu • Micro RNAs turn out to be very integral in cell division / regulation • HeatMap shows miRNA overexpressed (red) / underexpressed (blue) • In tumors have lots of blue, each tumor has its own fingerprint of miRNA expression Role of p53 gene  Tumor suppressor gene, transcription factor (regulates activity of other genes)  Normal functions to shut down cell division when a cell is stressed  When DNA is damaged, p53 activates genes that stop cell growth / trigger death  Mutation to this gene can cause tumors Explanation for why increased cancer risk can be inherited • Cancer can run in families, related people are at higher risk sometimes • Sporadic cancer requires new loss of function mutations in both alleles • BRCA1 puts breaks on cell division, but mutation in both those alleles and now you have lost the tumor suppressor activity and the cells divide out of control • Familial cancer requires loss of function mutations in one allele • Much higher risk if you inherited one mutation from parent (need 1 instead of 2 in order to develop a tumor) Explanation for why cancer incidence tends to increase with age  Cancer is progressive, accumulated more mutations when you‟re older  Most cancers require several mutations to occur before you get a tumor  Accumulated deregulation gives rise to a tumor  Death rates are high because there‟s more old people (problem in aging populations) Role of stem cells in tumor growth  Cancer may begin as alterations to gene expression in stem cells  Pluri potent stem cells are able to differentiate into a wide range of tissue types (know how to be different types of cells)  One is the stem cell, one becomes progenitor that differentiate into what is needed  Stem cells, progenitors and differentiated can all suffer mutations and become cancer stem cells  Tumors may be driven by cancer stem cells, cells are not the same in tumors Evidence that epigenetic regulation may be relevant in cancer  Some tumor cell nuclei can be re-programmed  Tumors have mutations but maybe they don‟t start with mutations but epigenetic changes that are potentially reversible  Experiment: mice at high risk for brain cancer inheriting one defective tumor suppressor and suffer another mutation that can give risk to tumor (dividing out of control due to loss of tumor suppressor gene) 2 Biology 1002 Natty Liu  Mouse egg cell – take nucleus out of the cell and replaced with nucleus from the tumor cell and “fertilize”…tumor makes a mouse not a tumor  When you put the tumor in the environment of an egg, it reprograms and is normal How does HPV cause cervical cancer? • HPV is a DNA virus – infects the cells on the cervix Lecture 19: Molecular Homology Strategies for determining if features are homologous • Homology means common ancestry, does not mean similar • Comparative Genomics: genome sequences of different species are compared • Sequence genomes  genome annotation  protein prediction  align sequences  determine homology Sequences detected by annotation programs to detect open reading frames (ORF)  Use of computer algorithms to detect: promoter elements, intron / exon boundaries, other conserved DNA motifs  Need to determine the longest open reading frame from start to stop (largest number of amino acids)  Six possible reading frames from the predicted protein-coding sequence Characteristics that are, and are not, common between homologous genes  Look for similar sequences in a database – NCBI Genbank  Arrange sequences to show regions of similarity  From sequence similarity one can infer structural and functional similarity as well as evolutionary relatedness Usefulness of BLAST analysis of sequences in Genbank at NCBI  BLAST – Basic local alignment search tool, much faster just looks for regions of very high similarity, doesn‟t try to force two sequences together  CLUSTAL – Global algorithm that starts at the beginning of the protein coding sequence and tries to arrange similar bases (introduce gap if they don‟t) Reasons why amino acid sequence comparisons are more informative than nucleotide sequence comparisons  More information in an amino acid sequence of same length  The genetic code is redundant, amino acids are much more reserved  DNA databases are much larger, protein database only have translated sequence that codes for a protein predicted, much more curated (specific & refined) Mathematical relationship among total information, # of symbols, # letters in the alphabet  How many bits of information in each letter of a four-letter alphabet?  A 00 G 01 C 10 T 11  I is the total information in a message with G symbols written in an “alphabet” of n letters  I = G lnn / ln2 Relative number of bits of information in a single nucleotide vs. single amino acid  A string of 20 amino acid has much more information 20 nucleotides 3 Biology 1002 Natty Liu  One piece of information (bit) – every base conveys two bits (using binary) Relationship between E-value and likelihood of homology  Two or more genes are homologous is a conjecture, proposition that is unproven  We don‟t know what the common ancestor was, no sequence thus homology determination is based on probability  The E-value describes the random background noise that exists for matches between sequences – lower the E-value, or the closer it is to “0”, the higher is the “significance” of the match (less likely they aren‟t homologous)  Higher the similarity between two sequences the lower the probability that they originated independently of each other and became similar just by chance, probably homologous What‟s the information content in a sequence that consists of a single amino acid?  Amino acid sequence based on larger "alphabet" of 20 characters, therefore has more information  I = ln(20)/ln(2) = 4.32  Information conveyed by a single amino acid is more than double that of the info conveyed by a single nucleotide Think about underlying reasons to explain that two proteins have weak global alignment (CLUSTAL) but strong local alignment (BLAST)  Local similarities suggests similar functions, could be highly conserved and used by many different proteins  Specific sequence of amino acids are required for certain enzymes to work, namely kinases Lecture 20: Molecular Convergence Synonymous vs nonsynonymous mutations  Synonymous: change in nucleotide but has no effect on amino acid sequence  Nonsynonymous: nucleotide mutation that alters the amino acid sequence of a protein Characteristics of the neutral theory of molecular evolution  Many mutations have no effect – they are neutral  Most evolutionary change at the molecular level is driven by random drift rather than natural selection  Neutral theory does not suggest that random drift explains all evolutionary change, natural selection is still needed to explain adaptation  Evolution at the level of the DNA and proteins is dominated by random processes, most evolution at the molecular level would then be non-adaptive Relationship between frequency of amino acid substitutions in given proteins vs. time since common ancestor  If they diverged a long time ago, you expect more mutations than if they diverged more recently  Fewer amino acid differences if the organisms diverged more recently  The further out you get, the more differences 4 Biology 1002 Natty Liu Relative rates of accumulation of synonymous vs. non-synonymous mutations  Rate of synonymous mutations would be higher than non-synonymous mutations  Synonymous mutations do not change the amino acid sequence leading to the protein to still be functional and have weak constraint whereas non-synonymous mutations change the amino acid sequence which affects the protein's functionality  To prevent more mutations that change the functionality, the rate is placed under strong constraint lowering the rate  Many non-synonymous mutations would be deleterious to the organism so the rate at which those remain in a population would be much lower Variables that affect the rate of evolution of a particular protein  Degree of constraint dictates rate of evolution, change very fast = lots of mutations  One is more sensitive to changes (constrained), can‟t be changed without being deleterious / changing function  Weak constraint – you can change a lot Deduce time of divergence given number of amino acid changes in particular protein  Straight line of change as a function of time (rate), change is not occurring at the same rate in the lines  Within all of them, there seems to be a constant rate – mutations occur at a constant rate because they‟re pretty neutral Characteristics of the "molecular" clock  Mutations may build up in any given stretch of DNA at a reliable rate (molecular clock)  Powerful tool for estimating the dates of lineage-splitting events  Measure of evolutionary change over time at the molecular level based on specific DNA sequences / proteins spontaneously mutate at constant rates used for estimating how long ago two related organisms diverged from a common ancestor Regions of two unrelated proteins that would be expected to be similar if they were the products of convergent evolution  Proteins tend to be modular – active sites / binding sites  You would expect localized areas of similarity: location of cysteins (give rise to specific disulfide S-S bonding), amino acids necessary for catalysis, DNA binding domains, receptor binding sites  Proteins converge but doesn‟t have to be over the entire sequence Function of lysozyme  Small (130 aa) enzyme, antibacterial activity that attacks peptidoglycan and found in blood, tears, mucus, egg white  Enzyme has been recruited as a stomach enzyme in ruminants Characteristics of ruminant organisms that enable them to extract energy from cellulose  Cows, who eat a lot of grass, can eat cellulose and there are bacteria in that mixture (microbes in the rumen) and the bacteria break down the cellulose  Bacteria + temperature = cellulose break down  Cows don‟t have the enzyme; the bacteria do  Enzyme must acquire new properties – catalytic active at low pH to protect itself from pepsin (proteases) 5 Biology 1002 Natty Liu Role of lysozyme in digestive physiology of ruminants, langur monkeys and hoisan birds  Independent evolution of this lysozyme that can function in low pH in three distinct different lines  Cow, langur, bird have restrictive enzyme through convergent evolution  Two very different forms of lysozyme, not homologous – lysozymes are independent Characteristics that distinguish "digestive" lysozyme from "non-digestive" lysozyme  Digestive enzyme is much more resistant to pepsin treatment than the non-digestive one  Digestive enzyme – 3D shape doesn‟t have that much difference, greater structural stability prevents pepsin access, doesn‟t flex very much Lecture 21: Experimental Evolution Characteristics of model systems that can be used for experimental evolution  Model systems: viruses, bacteria, chlamy, drosophila, yeast  All model systems have very fast life cycles, generation time is short  You can actually look at evolution over real time, look at selection / changes in the genome Origins of genetic novelty (variation)  Random spontaneous mutation to DNA can give rise to genetic novelty  Most of the time, mutation is bad or neutral (doesn‟t convey any huge advantage)  Gene duplication = gene amplification, make more copies of the gene  Gene rearrangement – one promoter (positional-dependent) moves to the other gene under very different control compared to when it had its original promoter Possible fates of duplicated genes  Most of the time, one of the two copies gets lost (deletion, degeneration) but sometimes, it gets retained (power of duplication)  Now you have two genes doing the exact same thing, but you only need one copy  Neo-Functionalization – protein does something different, change in the structural gene  Sub-functionalization – not affecting the structural gene, just promoter Relative impact of selection on duplicated genes 6 Biology 1002 Natty Liu  If you have a second copy of the same gene, then the selective pressure on that copy is lower than on the original gene  If mutations occur, they‟re not lethal to the organism in which it resides  More freedom for this second copy to mutate and change Design of Lenski's long term evolutionary experiment (LEE) with E. coli  Lenski group MSU used E. coli (grows very fast) to see how evolution would lead to adaptation  Generation time = very short, taking one colony (single cell that divides), no genetic recombination, totally asexual  Any mutation that arises is spontaneous  One cell / one flask  started 12 identical populations – genetically identical, let populations go (evolve)  Every day, take 0.1 ml cells to inoculate a new flask Value of cryopreservation to LEE  Every 500 generations (75 days) freeze (easy to freeze E.Coli – compare the evolved strains to the ancestral strains)  Looking at how does 10,000 generation compare to ancestral? Subject populations to adaptations to low temperature…etc and compare that to the ancestor Where citrate enters metabolism  Citric acid cycle starts with the reaction between the acetyl group of Acetyl CoA and oxaloacetate to form citrate  Two pyruvates are converted into two acetyl-CoA, two carbon dioxide molecules, and two NADH  During the series of eight reactions that make up the citric acid cycle, acetyl-coA molecules are oxidized, yielding two more carbon dioxide and 2 ATP Role of glucose limitation in LEE experiment  Glucose became depleted after about 8 hours remainder of the time in stationary phase  Cells stop growing, unable to metabolize anything else How to determine if Cit+ phenotype arose from one single mutation or was dependent on previous mutations  Take cells from different generations and grow for 3,770 generations more  If it‟s a rare mutations, some of those along the line will acquire the Cit+ phenotype Genetic changes giving rise to potentiation, actualization and refinement of Cit+ phenotype  Citrate transporter is not expressed with normal oxygen conditions  In actualization, gene duplication (part of the genome was amplified) produced a new construct, now downstream of the rnk promoter  Rnk is always on especially with oxygen, now Cit T is expressed when oxygen is around  Refinement – icrease in number of rnk-citT modules = increased cit transport (duplicating number of modules accounts for growth) Result of "replaying" evolution of Cit+ phenotype  Mutations in replay experiments are similar but unique  No capacity to generate Cit+ before 20,000 7 Biology 1002 Natty Liu  Wasn‟t one single mutation, but the age of the culture was important  Contingent on other mutations  This suggests a rare mutation, you need some sort of background mutation upon which you build this Cit+ phenotype Why Cit+ lines do not drive Cit- lines to extinction  Cit- more efficient at glucose utilization  Cit+ may be dominant but it didn‟t drive Cit- to extinction  Cit+ can use glucose and citrate, cit- have greater ability to bring in glucose, represent small percentage of the population but still exists! Lecture 22: The Elysia / Vaucheria system Location of PsbO gene in photoautotrophic organisms • Gene resides in the nucleus of all photoautotrophs • Elysia doesn‟t have any genes in the nucleus that code for chloroplast protein • But through LGT, photosynthetic gene psbO from nucleus of vaucheria to nucleus of elysia which can explain why it can support the chloroplast for so long Location and role of PsbO gene product in photosynthetic electron transport  Encodes part of the oxygen evolving complex  Sits on the luminal side of the thylakoid membrane  Required for PSII to work Purpose of molecular size markers in electrophoresis  To identify the approximate size of a molecule run on a gel (approx. bp length) Interpretation of agarose gel data  Assess the quality and quantity of DNA present in a sample  Darker bands have more DNA, no bands are present when the concentration is either very low or DNA is not present  Amplified portion of psbO transcript shows expression of psbO in elysia, evidence that elysia has the gene and making the transcript Mechanism of polymerase chain reaction  After 30 cycles…10 x amplification (billion-fold)  Break hydrogen bonds through heat, cool down which enables gene specific primers to anneal hydrogen bond to complementary sequences on either strand, heat it up for Taq polymerase to elongate after primer 8 Biology 1002 Natty Liu Role of thermal cycling in polymerase chai
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