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Biology 1002 Part 3 - final.pdf

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Western University
Biology 1202B
Tom Haffie

Cancer March-13-13 10:31 AM Lecture 1. four most commontypes of cancer in Canada - Breast cancer - Prostatecancer - Lung cancer - Colon cancer 2. likely factors contributing to cancer incidence in Canada - Heritability, but heritability of cancer is actually quite low 3. role of cyclin/CDKcomplexesin cell cycle regulation - CDK (inase) (Cyclin dependent kinase) ○ Kinase: transfers phosphate groups (phosphorylation),a powerful way to regulate proteins (posttranslational) - CDK are only active when bound to other proteins (cyclins) (another posttranslational regulation) ○ Cyclin production cycles with the cell cycle - Once bound to cyclin CDK causes phosphorylation,releasing G1/S check point, there are different cyclin and different CDK but the mechanism is the same 4. role of proto-oncogenes,tumor suppressor genes and oncomirsin caner a. Proto-Oncogenes - Before-oncogenes(inactivated oncogenes) - These genes are just us, there is no such thing as a cancer gene, these are genes are required for cell cycling - EGFR- (epidermal growth factor) a protein that stimulates growth (cell division), has a receptor that recognizes EGF, a signalling domain that conveysto the cell the presence of EGF ○ When deregulated results in unregulated growth - Transcription factors turn on cell cycling genes - The EGF pathway is RIDICULOUS b. Tumor Suppressor Genes - They are embryo suppressor genes, they shut down cell division and are perfectly normal in natural genes - P53, a DNA binding protein can: ○ Increase DNA repair ○ Cell cycle arrest by blocking cyclin/CDK ○ Apoptosis - Stops cells from getting past the G1 checkpoint if there is too much damage, if it is missing cells will get through the checkpoint with mutations and will undergo uncontrolled growth c. Oncomirs - Inappropriate expression of miRNA can promote cyclin e.g. oncomirs - miRNA play an important role in regulating cycling - The particular distribution of miRNA is very diagnostic for particular types of tumours 5. role of p53 gene - Increase DNA repair - Cell cycle arrest by blocking cyclin/CDK - Apoptosis - Apoptosis 6. explanation for why increased cancer risk can be inherited - The heritability of cancer is fairly low, there is some genetic variability but there is a very high environmentalcomponent - There are genes that supress cell division (tumor suppression) - You may suffer a mutation over time on one or both alleles but this is rare and takes a long time and can result in cancer - Families where it is more commoninheriting a defective allele, increasing the chance of mutation in that single allele is much higher 7. explanation for why cancer incidence tends to increase with age - Cancer is progressive,it is accumulative,very rare for a single mutation giving rise to a tumour - A mutation can predispose the gene to another mutation,eventually enough mutations to cause cancer 8. role of stem cells in tumor growth - All cells have renewability,stem cells are pleuripotent: desendence can becomea wide range of specialized cells - Stem cells, the progenitors and the descendents can regenerate into a cancer cell, a stem cell that is out of control - We thought that all cells in a tumour were the same - In many tumours the bad cells are the stem cells, likely that not all the cells in a tumour are the same 9. evidence that epigenetic regulation may be relevant in cancer - Mouse embryoscloned from brain tumors - A mouse born with high risk of brain cancer, within the tumour of that mouse, the cell does not have a tumour suppressor, after taking the nucleus from that cell and putting it into an enucleated egg cell, a mouseis born that is perfectly fine - The tumour nucleus can drive developmentof a perfectly normal mouse - Maybe cancer is epigenetically altered, not genetically ○ Tumour suppressors are turned off by methylation?Being bullied increases the risk of cancer? - Epigenetic changes occur and then mutations occur Molecular Homology March-24-13 1:25 PM Lecture 1. strategies for determining if features are homologous - Comparativegenomics - Comparing the genomesof different organisms 2. sequences detected by annotation programs to detect open reading frames (ORF) Genome annotation: ascribing biological function to the genome - Regulatory elements - Intron/exonboundaries have a specific sequence - Translating DNA and removingintrons leaves a sequence with 6 possible reading frames, the longest open reading frame (largest number of amino acids from start to stop) is most likely the protein coding sequence 3. characteristics that are, and are not, commonbetween homologousgenes - Structurally or functionally similar, evolutionarilyrelated, determined by sequence similarity - In global alignment you are trying to align sequences from the front to end (Clustal) - In a local alignment you are trying to align local regions of similarity (Blast) - Genes may be homologous,not having the same but a similar nucleotide or protein sequence 4. usefulness of BLAST analysis of sequences in Genebank at NCBI - Shows that two different gene sequences do not have the same nucleotide sequence, howeverthey are similar - Taking the predicted proteins and looking for a similar sequence in a genebank - Using sequence alignments one of the genes in (ex. Volvox)may be similar - From this informationwe can infer structurally or functionally similar, as well as evolutionaryrelatedness 5. reasons why amino acid sequence comparisonsare more informativethan nucleotide sequence comparisons 1. There is moreinformation in amino acids sequence - How many bits of informationin each letter of a four letter alphabet? - 2 - I is the total information in a message with G symbolswritten in an “alphabet” of n letters - I = (G ln n)/ln2 - Bits of information in a single amino acid? G= 1 n=20 (n is the total number of possible amino acids) 2. The genetic code is redundant - There are 64 different combinations, but only coding for 20 amino acids, there are more than one codon that can specify the same amino acid - The amino acid sequence is highly conserved, selectionwill allow the nucleotide sequence to change but the amino acid sequence will not change as much 3. DNA databases are much larger - There is a lot of junk DNA 6. mathematicalrelationship among total information,# of symbols, # letters in the alphabet - I is the total information in a message with G symbolswritten in an “alphabet” of n letters - I = (G ln n)/ln2 - I = (G ln n)/ln2 7. relative number of bits of information in a single nucleotide vs. single amino acid - Bits of information in a single amino acid? G= 1 n=20 (n is the total number of possible amino acids) = 4.3 - Bits of inforamtion in a single nucleotide? G = 1 n=4 = 2 8. relationship between E-value and likelihood of homology The E-value describes the random background noise that exists for matches between sequences. For example, an E-value of 1 assigned to a hit can be interpreted as meaning that in a database of the current size, one might expect to see one match with a similar score simply by chance. - The lower the E-value, or the closer it is to “0”, the higher is the “significance” of the match, the greater the chances that they are homologous MentalFloss: 1. What is the informationcontent in a sequence that consists of a single amino acid. - A nucleotide has the possibility of being any one of four possible nucleotides that represent two bits of information (A 00 G 01 C 10 T 11) - An amino acid is comprised of 3 different amino acids - I = (G ln n)/ln2 I = (1 ln 20)/ln2 I = 4.3 2. Think about underlying reasons to explain that two proteins have weak global aligment (CLUSTAL) but strong local alignment (BLAST). 1) The local similarities suggests similar funcions Maybe those similarities are highly conserved,and used by many different proteins. For example, ATP-domaincassette is highly conserved and are parts of many different transporter protein complexes. Or it may be that specific sequence of amino acids are required for certain enzymes to work, namely kinases 2) Maybe they happen to be transposons. Molecular Convergence March-24-13 1:35 PM Lecture 1. synonymous vs nonsynonymousmutations Synonymousmutation: changein the nucleotide sequencebut there is no effect on the amino acid sequence Non-synonymousmutations: changein the nucleotidesequencecausing a changein the amino acid sequence 2. characteristics of the neutral theory of molecular evolution - We see that lots of mutationsexist that don’t effect the protein at all 3. relationship between frequencyof amino acid substitutions in given proteinsvs. time since common ancestor - The number of differences between protein sequences of different species are proportional to the time since those species diverged 4. relative rates of accumulation of synonymous vs. non-synonymousmutations - Synonymous rate is higher,mutations that don’t effect the aminoacid sequenceoccur faster (silent and replacementsubstitutions) 5. variables that affect the rate of evolution of a particular protein - Some proteins are more sensitive to changes, you cannotchange it too much withoutchanging the protein making it deleterious - Other proteinscan be changed much more - The is a stronger constraintof proteins that cannot be changed too much 6. deducetime of divergence given number of amino acid changes in particularprotein - If the rate of amino acid substitutionsis the same than you can use the number of differencesto deducethe time of divergence 7. characteristics of the "molecular"clock - A rate at which mutationsoccur, usuallyneutralin effect over time since divergenceof the species - The rate is relatively constant,can be used to determine time since divergence - Changesfor different proteins 8. regions of two unrelated proteins that would be expected to be similar if they were the productsof convergent evolution - Expect local areas of similarity but not the entire sequence  Location of cysteins (disulfideS-S bonding)  Amino acids necessaryfor catalysis  DNA binding domains,receptor binding sites… - These are not represented by the entire sequence 9. function of lysozyme - Attacks peptidoglycan - Has evolved as a stomach enzyme in ruminants 10. characteristics of ruminant organisms that enablethem to extract energy from cellulose - Enzyme has been recruited as a stomach enzyme in ruminantsto break down bacteria 11. role of lysozyme in digestive physiology of ruminants,langur monkeys and hoisan birds - Lysozyme is used to break down the bacteriato keep the nutrients - Must be functional in low pH and able to protect from proteases such as pepsin - There is only a limited amountof ways that this convergencecan happen 12. characteristics that distinguish "digestive" lysozyme from "conventional" lysozyme 12. characteristics that distinguish "digestive" lysozyme from "conventional" lysozyme - Comparison of lysozyme aminoacid sequencesof different species - Baboonand langur monkey are closely related but their lysozyme are more divergentthan you predict based on the overall phylogeny - The cow and the langur monkey are not related at all but share 5 amino acids in common - Lysozymes do not share a common ancestor - Althoughthere are two different types of lysozyme they all have specific areas or nucleotidesthat are the same through convergence - Can be distinguished through:  The time it takes for one lysozyme to be digestedby pepsin in comparison to the other  The amountof urea it takes for one lysozyme to denaturecompared to the other  In general the digestive enzyme has a more stable structural conformation  Lacks the aspartic acid-proline bond, which is susceptible to cleavage at a low pH  There is an overall negativecharge, this repels pepsin (pepsinalso has a negative charge) Experimental Evolution March-27-13 8:09 PM Pre-Lecture 1. what is meant by Potentiation,Actualization and Refinement a. Potentiation:the developmentof an unknown mutation that allows for the potential to develop another mutation causing actualization b. Actualization: the developmentof a mutation that allows for some (poor) growth or developmentof a new phenotype c. Refinement:duplication or rearrangement of DNA that allows for robust growth or development 2. Look back at your respiration notes...where does glucose and citrate comein to cellular respiration? a. Glucose: enters into glycolysis in the cytosol b. Citrate: used in the citric acid cycle in the mitochondrion Lecture 1. characteristics of model systemsthat can be used for experimentalevolution - They don’t all contain mitochondria, they don’t all contain chloroplast - They reproduce very quickly, a short generation time - Systems that divides quickly, you can look at evolution in real time 2. origins of genetic novelty (variation) - Spontaneous mutation, there is a selective advantage, most of the time its neutral or deleterious, but can give rise to genetic novelty a. Gene duplication: duplication of a promoteror a region around the gene can result in genetic novelty i. One of the copies is usually deleted, resulting in no effect ii. Neofunctionalization:the second copy may be retained, the rate of mutation can be higher and can lead to new structure/function,it can evolve faster than the first iii. Sub-functionalization: if only the promoteror regulatory region is changed there may be a new activator of the gene, different tissue specificity (new regulatory element) b. Gene Rearrangement:two genes separated by a distance in the genome, they can be rearranged so that a positional dependant promotermovesto be linked to a new gene giving rise to genetic novelty, the new gene is under new distinct control 3. relative impact of selectionon duplicated genes - Since there are two genes doing the same thing, only one is essential, the selective pressure on the second copy is reduced, if mutations occur they are not lethal because there is a second copy, the rate of mutation can be higher and can lead to new structure/function,it can evolve faster than the first 4. design of Lenski's long term evolutionaryexperiment(LEE) with E. coli - Taking one colony of E. coli, grown from a single cell - Started 12 identical population and allows them to evolve - Every day 0.1 ml of a culture are put into fresh media so the cells can continue to grow - 5x10^8 cells transferred every day - Every 500 generations (75 days) freeze - Can comparecultures at various generations by unfreezes older generations - Can comparecultures at various generations by unfreezes older generations 5. value of cryopreservationto LEE - Because E. coli can be frozen and unfrozen different generations can be compared over time - When a mutation occurs and the culture evolvespast generations can be compared to determine when the mutation arose 6. where citrate enters metabolism - Used in the citric acid cycle in the mitochondrion 7. role of glucose limitation in LEE experiment - 25 ug/ml glucose became depleted after about 8 hours remainder of the time in stationary phase - If you can acquire the ability to take up citrate under normal oxygen conditions there is a large selective advantage, you can acquire the citrate and use it for growth for longer than 8hrs 8. how to determineif Cit+ phenotype arose from one single mutation or was dependent on previous mutations? - Testing the early lineages on a citrate agar gel you can test if the cells can take up citrate - Later in the lineage there is a slight citrate positive phenotype (actualization) - After this there was a mutation that gave rise to a considerable increase in the amount of citrate that can be taken up (refinement) - Looking at multiple culture lineages there is no capacity to generate Cit+ before 20,000 generations - This tells you that it wasn’t a single rare mutation but the age of the generations was important - The Cit+ mutation is contingent on other mutations 9. genetic changes giving rise to potentiation,actualization and refinementof Cit+ phenotype - Potentiation:a mutation that gives the ability to have the potential to give rise to a Cit+ mutation (unknown mutation) - Actualization: a part of the genomewas amplified and put back in slightly downstream  The citT gene is now downstream of the rnk promoter,the rnk promoteris a constitutivelyexpressed promoterthat is always on especially in the presence of oxygen  Promotersare position dependant, if you move genes around they are expressed in a totallydifferent way, under control of a different promoter - Refinement:the duplication of the rnk-citG modules 10. result of "replaying" evolutionof Cit+ phenotype - There are different variations in the range of where the mutations took place in then genome that gave rise to the Cit+ phenotype but they impinge on the same general area of the genome - Because there is no ability to utilize glucose up to a certain number of generations we can determinethat there was no single mutation that gave rise to the Cit+ phenotype, it was contingent on other mutations before it 11. why Cit+ lines do not drive Cit- lines to extinction - Cit- are moreefficient at glucose utilization The Elysia/Vaucheria system March-27-13 8:09 PM Lecture 1. location of PsbO gene in photoautotrophicorganisms - Gene resides in the nucleus of all photoautotrophs 2. location and role of PsbO gene product in photosyntheticelectrontransport - The PsbO gene produces a part of the oxygen evolving complex which sits on the luminal side of PSII - PSII would not work without psbO being expressed and the protein being made - Required for electron donation to from H2O leading to oxygen release 3. purpose of molecular size markers in electrophoresis - Shows the presence of the mRNA in vaucheria and elysia, synthesis of mRNA corresponding to psbO - Can infer the protein sequence given the mRNA sequence - Shows the synthesis of the mRNA rather than just a gene present that codes for a gene 4. interpretation of agarose gel data - Elysia feeds once a juvenile, 5 months later you can see the presence of a transcript - When consuming the chloroplast it is doubtful that mRNA would stay around for 5 months, more likely that Elysia has psbO 5. mechanism of polymerasechain reaction - Using heat stable DNA and gene specific primers - Amplification of the DNA allows for manipulation 6. role of thermal cycling in polymerasechain reaction 1. Increase the temperatureto denature the DNA into single strands 2. Decreasethe temperatureto allow specific primers to bind to the DNA 3. Increase the temperatureagain to allow for polymeraseto replicate the sequence of DNA from the primer 7. role of primers in polymerasechain reaction - PCR is based on extremespecificity of the primers 8. role of Taq polymerasein polymerasechain reaction - A thermal stable polymerase,takes from a thermophile - Because temperatures are changed in order to denature the DNA there must be a polymerasethat will not be effected by the changes 9. implications of lack of proofreading in Taq polymerase - The purpose of polymerasechain reaction is to amplify DNA, if there is an improper nucleotide inserted into the new strand of DNA and it goes undetected it will amplify a copy of the DNA that is not the same as the original 10. why primers are seldom less than 16 bases or longer than 30 bases - In any DNA sequence: ○ 1 in 4 chance of finding an A, G, C or T ○ 1 in 16 chance of finding any dinucleotide sequence ○ 1 in 256 chance of finding a given 4-base sequence. ○ 1 in 4^16 chance of finding a given 16-base sequence. - There is a very low chance that this sequence would occur more than once, there - There is a very low chance that this s
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