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Biology 1002 Exam Notes on Lecture 18-24.pdf

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Biology
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Biology 1002B
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Tom Haffie

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• Bio lecture 18+ Lecture 18: Cancer The success of multi-cellular life depends on most individual cells agreeing to limit their reproductive capacity;  however, sometimes some cells break this reproductively repressive agreement and go their own way.  We call this cancer.  We don't like it. learning and memory is highly connected to epigenetic regulation (methylation) in canada, men at higher risk than women • • women have a 40% possibility of getting cancer • about 27% of dying from it • men have a 45% of dying from cancer • and a 30%+ possibility of dying from it • top four: prostate, breast, lung, and colon cancer • 25% of Canadians can expect to die of cancer • rates among youth are rising • heritability of cancer: depends on the kind of cancer, but on low end (0.27-0.42) Embryogenesis rapidly dividing cells • all involve rapid cell division from single cell to multicellular • not out of control but very rapid • fig 8.18 pg. 175 • G/S phase checkpoint stops unprepared (mutated or problematic cells) from duplicating • CDK (cyclin dependent kinase) • kinase phosphorylates targets • CDK are only active when bound to cyclins • cyclins are called that because their production cycles with the cell cycle • produced early in the cell cycle and then binds to CDK which then phosphorylates things, which releases the G1 checkpoint (posttranslational regulation) Expression of proto-oncogenes promotes cell cycling:e.g. EGFR • cells must be able to respond to the environment (when they should divide) • EGFR: epidermal growth factor receptor • factor means protein • when there is EGF, it binds onto the receptor • embryonic gene • Bio lecture 18+ • EGFR can cause tumours to grow because its on all the time which causes the cells to continue to divide • call them cancer genes or oncogenes...stupid...they are genes for embryonic genes • • 2nd year cell bio....sooo dont take it Expression of Tumour suppressor genes slow cell cycling •Bio lecture 18+ •“TP53” is a master tumour suppressor gene, coding a transcription factor whose activity can result in... • increased DNA repair • cell cycle arrest by blocking cyclin/CDK (in charge of first G1 checkpoint) • apoptosis •these genes are embryotic suppressors not cancer specific cell repressors Sporadic cancer requires new lose of function mutations in both alleles Breast cell at first mutation second mutation birth it can run in families • •now lost tumour seppressor activity •rare Familial Cancer requires loss of function mutations in one allele if your father already has that mutation u only need one more mutation to give you a • tumour Inappropriate Expression of miRNA can promote cycling e.g. oncomirs •miRNA is diagonstic normal tissue it is overexpressed • •in tumour it is underexpressed Cancer is DeRegulation •uncontrolled growth can arise from upsetting the balance between the activity of gene products that promote cell cycling vs. those products that suppress cell cycling • Bio lecture 18+ • • haffie is 54..... damn • cancers require several mutations (express enco genes, suppress miRNA...) more of a problem is aging populations • • Bio lecture 18+ Cancer is Progressive Cancer may Begin as alterations to gene expression in stem cells • • pleuripotent stem cells can turn into a wide range of cells ex all skin cells • • when they divide to make two daughter cells...one stays a stem cell and the other differentiates into something • Progenitors: those that differentiate • stem and progentiors and differentiated cells all suffer mutation and become cancer stem cells all tumour cells are different! • • cancer maybe driven by cancer stem cells • Bio lecture 18+ Some tumour cell nuclei can be re-programmed! • mouse family that is at high risk of brain cancer because they are inheriting a mutated suprressor • over life span they have another mutated repressor • cells dividing out of control because of loss of tumour suppressing gene • take mouse egg cell and take out its nucleus and replace it with the tumour nucleus • tumour nucleus in mouse egg, little electric shock and it things its been fertilized • starts to divide under the control of a tumour nucleus, that tumour makes a mouse not a tumour!!!!!!! • SO ITS GETS REPROGRAMMED!!!! • maybe cancer stars with epigenetics????????? IS cancer contagious? Human Papilloma Virus(HPV) (not on midterm) • DNA virus • Many different strains cause increased growth (warts) in different tissues in both men and women • some strains are oncogenic, particularly strong association of strains 16 and 18 with cervical cancer • total HPV prevalence, 27% women 14- 59 • 45% in women aged 20-24 • total incidence of “high risk” type 16 and 18 was 2.3% In multiple choice questions, identify the  four most common types of cancer in Canada likely factors contributing to cancer incidence in Canada role of cyclin/CDK complexes in cell cycle regulation role of proto-oncogenes, tumor suppressor genes and oncomirs in caner role of p53 gene explanation for why increased cancer risk can be inherited explanation for why cancer incidence tends to increase with age role of stem cells in tumor growth evidence that epigenetic regulation may be relevant in cancer Lecture 19: Molecular Homology • Molecular evolution • Gene evolution: • mutation • duplication • rearrangement • loss • Bio lecture 18+ • all those can change phenotype which causes evolution • ex. how does a change/mutation in an enzyme increase its affinity to substrate? • this evolution is then subject to selection • evolution and selection effect phenotype not genotype Homology • means common ancestry • gene in chlammy is homologous to gene in volvox If GLsA in Volvox and GLsA in Chlammy are homologous-what do they have in common? A. identical nucleotide sequence B. Identical amino acid sequence C. identical length of polypeptide D. same function • none of these are the right answer LOOKING FOR SIMILARITY Comparative Genomics Sequence Genomes---> Genome annotation---> Protein prediction---> Align sequences---> determine homology • genome sequencing is easy...just all the base pairs (atcgatggct..) • genome annotation is linking those sequences to their function • attach biological meaning to sequence • gene prediction • regulatory elements • biological function through similarity searches • automated genome annotation • protein coding genes( has computer algorithms) • program detects: promoter elements, intron/exon boundaries, other conserved DNA motifs • can splice out exons and then they have a deduced gene section called a predicted protein coding sequence (just know their is a protein coding gene somewhere but not sure where, 6 reading frames...) • which of those 6 are the opening reading frame for that protein? Protein prediction: find longest ORF (computer does this) • translate all 6 • whats the longest/largest open reading frame from start to stop • Bio lecture 18+ National Center for Biotechnology Information • Chlamydomonas: 15000 predicted proteins • now what? • look for similar genes in the database • NCBI: • genbank • sequence database • 23500 total (whole) genomes • massive amounts of genome sequences Sequence Alignment( DNA or protein) • arrange sequences to show regions of similarity (also computerized) • search database for similarities • sequence similarity infers structural and functional similarity, and evolutionary relatedness • 2 algorigthms to use • BLAST: basic local alignment search tool • local • CLUSTAl • global • • Bio lecture 18+ • Clustal looks at the beginning of the gene and align the similar bases and work to the end • Blast doesn’t do that, it looks for regions for very high similarity • Global not strong, local very strong---> means ...check forum? • 155 BLAST hits against Chlammy gene (GLSA) • 155 sequences at NCBI that are similar to GLSA • very high similarity with Volvox gene Volvox vs. Chlamy: Nucleotide Alignment • asymmetric division in volvox the function in Chlammy’s gene isn’t really know • • homologous doesn’t mean they have identical nucleotides...above picture shows this...BLAST shows this • can look at DNA or translate to protein • fair amount of similarity • Bio lecture 18+ Volvox vs. Chlammy:Amino Acid alignment homologous doesn’t mean the amino acid sequence is the same either • Amino acid sequence comparisons are more informative than nucleotide 1.More information in an amino acid sequence of same length • How many bits of information in each letter of a four-letter alphabet/base pair!? • every base then is 2 bits A:00 • • G:01 • C:01 • T:11 • I is the total information in a message with G symbols written in an “alphabet” of n letters. I= GIn n / In2▯ ▯ ▯ ▯ ▯ • • Bits of information in a single amino acid? • on forum • 20 different amino acids so 20 characters not just four • Bio lecture 18+ 2. The genetic code is redundant. • meaning it has 64 possible triplets that codes for only 20 amino acids • one codon can code for the same amino acid • more noise in nucleotides • amino acid sequence more highly conserved • why? what is selection acting upon? 3. DNA databases are much larger • non coding junk DNA is also in the database, which makes it hard to search Amino Acid Sequence vs. Nucleotide • 90 nucleotides in length 30 amino acids here • • ten nucleotide differences (mutations between chlammy and volvox) • but when you translate them they code for the same thing • differences don’t show up in proteins Homology determination is based on probability you can never that two sequences are homologous with 100% certainty • • a conclusion that 2 or more genes are homologous is a conjecture • unproven and not based on any experimental data • don’t have the GLSA from common ancestor • then an experiment could be done • decisions about homology are made based on similarity-numercial and correlated with probability • The higher the similarity between two sequences the lower the probability that they originated independently of each other and became similar just by chance. The E-value • 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. • Bio lecture 18+ • The lower the E-value, or the closer it is to “0”, the higher is the “significance” of the match. Lecture 20: Molecular Convergence   Proteins do not have to be homologous to have similarities in structure/ function. • synonymous vs nonsynonymous mutations • synonymous a change or mutation in the nucleotide that does not change the amino acid • nonsynonmous=amino acid change • a lot of variation in the nucleotide sequence (lots of mutation) because its been millions of years since the common ancestor of volvox and chlammy but certain mutations do not effect the amino acid • selection is on the amino acids (phenotype) • selection theory: most mutations will be harmful (deleterious) and be selected out of population • and have advantageous ones that are selected for • neutral theory:lots mutations that don’t effect the protein at all, therefore there are neutral mutations , and a very few are advantageous • Bio lecture 18+ • we can how sequences change from one species to another because of the new databases • what is found is that the number of differences in protein sequences of different species is proportional to the time since those species diverged • • more differences in sequences the longer ago it was when they diverged • all straight lines of change as a function of time= A RATE • not at the same rate • hard to explain this theory with selection theory • relates more with neutral theory • straight rate= “molecular clock”: rate of mutation is constant • ex. 2 unknown organisms and sequences hemoglobin and found a certain amount of amino acid substitutions. Plot them on graph and can identify when they diverged from their common ancestor • human-chimpanzee= 5 million years • Bio lecture 18+ • • Sequence Conservation • synonymous is higher!!! (the ones that don’t effect the amino acid sequence) • silent substitutions =synonymous • replacement substitutions=nonsynonmous • Bio lecture 18+ Degree of Constraint Dictates Rate of Evolution hmm why? • • histones are more sensitive, if you change them a lot it becomes very delterious to their function • CONSTRAINT: the amount a sequence can change b4 its function is compromised • weak= can change a lot, strong= cant change a lot Molecular Convergence • Why is similarity between sequences considered to be evidence of homology? • why can’t sequences converge to be similar? • phenotype can be similar due to homologue and convergence? Functional Convergence There is no reason for two proteins to share high identity across their entire • sequences...is there? • volvox and chlammy are very similar at the nucleotide level • you would not expect that if two proteins were distantly related and converged to a similar phenotype • you would expect localized areas of similarity wouldn’t expect entire sequence to be similar • • Location of cysteins (disulfide (S-S) bonding) helps in protein folding • amino acids necessary for catalysis • DNA bind domains, receptor binding sites • all of these are in specific parts of the sequence • PROTEINS CAN CONVERGE BUT IN LOCALIZATION NOT NECESSARILY THE ENTIRE SEQUENCE •Bio lecture 18+ •cytochrome C is very constrained/conserved does not mutate much..very similar from human to bacterium •its entire sequence is similar which means it due to homology •sequences similar in its entirety means its homologous Lysozyme: A case study of convergent Evolution •small antibacterial activity •attacks peptidoglycan •found in blood, tears, mucus, egg whites •enzyme has been recruited as a stomach enzyme in ruminants (e.g. Cows) •What’s a ruminant? •animals that can eat cellulose (grass) and break it down via bacterium that breaks it down •now the cow wants to break down the bacteria and it does this via lysozymes in the stomach •bacteria breaks down cellulose--->lysozymes breakdown bacteria •we cant •enzyme in a stomach must be catalytically active at a very low pH (acidic) •pepsin in stomach...lysoyme must protect itself from proteoases (pepsin) Picture of A cow and a langore monkey •both evolved the digestive lysozyme •but they aren’t related at all •they have acquired this ability independently Lysosyme in different species •comparison f lysozyme amino acid sequences of different species SPECIE Lang Baboo Huma Rat Cow Horse S ur n n Langur 14 18 38 32 65 Baboon 0 14 33 39 65 Human 0 1 37 41 64 Rat 0 1 0 55 64 •Bio lecture 18+ SPECIE Lang Baboo Huma Rat Cow Horse S ur n n Cow 5 0 0 0 71 Horse 0 0 0 0 1 •above line is the number of differences •human and langur are closer related but their are 14 differences •the sequence of the lyzsome from the langur monkey and the baboon are more divergent than predicted •below the line is how many amino acids do they uniquely share •cow and monkey aren’t related at all by share 5 common amino acids •lyzoymes do not share a common ancestor Convergence to same amino acids •Hoatzin bird has the enzyme too •3 unrelated organisms...there is a problem? •need an enzyme that can function at low pH and resist pepsin •they are all similar • Bio lecture 18+ • • special stomach enzyme are very similar!!!!!!! showing convergence of the molecular scale Amino Acid Substitutions at specific sites •Bio lecture 18+ 14 21 41 75 76 83 87 126 R R R N A A D Q Human K K E D A A N K Langur R R Q N A A D Q Baboon R Y Q N A A D R Rat K K K D G E N E Cow Chicken R R Q N L A S R R V T N A K D R Pigeon E E E D G E N K Hoatzin Horse A G R N A K E A •D-aspartic acidigestive enzymes •N-asparagine Biochemical Consequences Non digestive▯ ▯ ▯ digestive • Bio lecture 18+ Unfolding ▯ ▯ ▯ Urea • digestive enzyme is much more resistant to pepsin then non-digestive • shows denaturation in graph • ND=non digestive=triangle • D=digestive=circle Digestive enzyme • • greater structural stability prevents pepsin access • lack of aspartic acid- proline bond (acid labile) this bond is very susceptible to cleavage at low pH • very popular in lysozymes except the digestive ones • negative charged surface repels pepsin (pepsin has a negative charge as well) Lecture 21: Experimental Evolution • Charles Darwin remarked in 1859 that “in looking for the gradations by which an organ in any species has been perfected, we ought to look exclusively to its lineal ancestors; but this is scarcely ever possible, and we are forced in each case to look to species of the same group, that is to the collateral descendants from the same original parent- form”. • Volvox and Chlammy are collateral descendants • Model systems for EE • Viruses • Bacteria Chlamydomonas • • Drosophila • Bio lecture 18+ • Yeast • Testing hypothesis about evolution using controlled experiments • what do all the models have in common? • they reproduce very quickly/ a short generation time • Chlammy nine hours • drosophilia 7 days • can look at evolution over real time of these short generation time species • can look at mutations and adaptation The Origins of Genetic Novelty • spontaneous mutation can give rise to genetic novelty ( pretty rare) • besides this there are two other thing that cause genetic novelty 1.Gene duplication • not duplicating specific gene but duplicating part of it (not very precise) • when this occurs usually one of the copies is deleted • could have that copy survive though! • when you have two copies the selective pressure on the second copy is REDUCED • if mutations occur they are not lethal to the organism in which they reside cuz of the other copy • more freedom for the second copy to mutate and change then the original • the rate of mutation and a possible change in function is higher (evolution) • structural gene is now different, evolves faster • this is called NEO-FUNCTIONALIZATION BECAUSE IT CAN EVOLVE FASTER AND IT HAS A NEW FUNCTION • what if we don’t effect the structural element at all? but the promoter? • maybe now its switched on in low O2? Low temp? Normal temp? expressed in new tissue? • same gene... but change the regulatory element called SUB- FUNCTIONALIZATION • Gene duplication is also called gene amplification 2. Gene rearrangement • genes have quite a distance from each other • the promoter which is position dependent, can be paired with another gene when two genes are switched • now the gene is regulated completely differently Long Term Evolution Experiment (LEE) • Can evolution produce adaptation if it depends on random mutations (most of which are harmful/neutral and rare)? • Lenski group MSU • E. coli • Asexual (no recombination) • Spontaneous mutation, gene duplication, gene rearrangement • Bio lecture 18+ • Population size is huge (new alleles come about and can sustain themselves in the population) • What Lenski did? • started with 12 identical populations • populations are genetically identical and lets them evolve • overtime watches them evolve • everyday take 0.1 ml cells and puts it into a fresh flash for each 12 populations • 5x10 e8 • every 500 generations (75 days) freeze • E.coli can be frozen then reanimated back to life • you can compare a culture of a generation of 500 to a generation of 2000 • 50000 generation of E.coli • 6.6 generations/day= Generation time of 3.63 hours • 22 years of evolution Mental Floss • Given human generation time of around 20 years • how many years is 50000 generations? • 1 million years of human evolution!! • humans have only been homo sapiens sapiens for 150000 Evolution of Citrate Utilization • after more than 30000 generations one of the 12 populations (Ara-3) changed... • it is more turbid (more cell concentration) • those cells acquired the ability to metabolize citrate • can use citrate as a carbon source! • where does citrate enter cellular respiration? • number of mutations over 30000 generations? • billions of mutations have occurred • genome is only 4.6 million bp • every mutation was “tried” “changed” many times • RARE Citrate=Ecological Opportunity • Medium contains citrate • but citrate is not used for the carbon source • the citrate is in the medium to give Iron • Fe-citrate • these cells have the inability to grown on citrate under oxic conditions (lots O2) • citrate transporter is not expressed • normal cells use Glucose • 25ug/ml glucose became depleted after about 8 hours remainder of the time is stationary phase • they grow and divide and use the little bit of glucose then stop because of no glucose • Bio lecture 18+ • if you could acquire the ability to use citrate you could divide and grow for much more than 8 hours...would be very advantageous • uses citrate for growth! A CARBON SOURCE • how was the citrate positive phenotype acquired? • can go back in time and grow its ancestors and see when it began • can sequence genomes and find when the phenotype occurred Rare Mutation or Contingency? • Was the Cit+ phenotype the result of a unusually rare mutation or contingent upon prior mutations? • contiginent? a mutation that occurs on what happened b4? e r t u C • • thaw out frozen Ara-3 cells from previous generations • grow them with citrate and see anything happening? • 30 000 and 31000=no! they cannot use any citrate at all • 32000 and 33000 there is a slight positive citrate phenotype • this is called ACTUALIZATION • another mutation occurred that allowed for use of citrate so strongly Replaying Evolution in Ara-3 line • Bio lecture 18+ • grow it for 300700 generations...? • no capacity to generate Cit+ before 20000 years • it wasn’t one single rare mutation • it suggests its contingent , not just a rare event Potentiation, Actualization, Refinement • Bio lecture 18+ • before 20000 there was a mutation • this mutation allowed for the potential of the citrate positive phenotype • around 30000 almost there!!! • refinement they can use it a lot Actualization of Cit+ • all has to do with the expression of the citrate transporter • not expressed when O2 is around • what happened in the actualization step? • gene duplication • a part of the genome was amplified • see diagram in lecture slides • the ciT gene (transport gene) is now downstream of the rnk promoter • the rnK promoter is the strong constituently expressed promoter • it is always on! • especially when there is lots of O2 around • now ciT is expressed when O2 is around • need to know ciT is downstream of rnK promoter! • “promoters are position dependent • Bio lecture 18+ Refinement of Cit+ • increase in number of rnk-ciT molecules (increase cit transport) • many copies of the modules • gene duplication • huge capacity to use citrate which increases culture density! • Mutations in replay experiments are similar but unique • Red box – boundary of amplification of original Cit+ line • Blue boxes boundary of amplifcation of 14 replay expts. • they are all around this region all require gene duplication and rearrangement • • all slightly different in length that they amiplfy • but they all still give rise to cit+ phenotype • Bio lecture 18+ Evolution of A New Species • now have a line of E.coli that can grow on citrate • now have a genome that is reorganized • Defining characteristic of E.coli is... • they don’t grow on citrate • Question is: Does this Ara-3 line deserve to be a new species??? • The rise of Cit+ did not drive Cit- to extinction • Cit- more efficient at glucose utilization. • cit+ were 99% of the culture • both have different ecological niches! Lecture 22: The Elysia/Vaucheria System • Horizontal gene transfer of the algal nuclear gene psbO to the photosynthetic sea slug Elysia chlorotica Photosynthetic Competency • kleptoplasty steal the chloroplasts from vaucheria those chloroplasts still have high rate of photosynthesis • • spinach (very good model system to isolate chloroplasts), after 6-12 hours the chloroplasts stop working • what potentially can account for the high photosynthesis in the elysia gut? • Mental Floss question: why does photosynthesis decrease when isolated? • Photosynthesis does go down over time in Elysia as well. why? Horizontal (Lateral( Gene Transfer) • vaucheria has both mitochondria and chloroplast • most genes are in nucleus Vaucheria Nucleus • • Bio lecture 18+ Elysia Nucleus • then steals chloroplasts • the hypothesis that they can retain photosynthetic competency for so long is that there is horizontal gene transfer from vaucheria to elysia, of a photosynthetic gene • gene thought to be transfered is psbO • not the only gene transferred sequence genome of elysia has photosynthetic gene • • elysia would get exposed to vaucheria DNA and it makes sense there was a transfer PsbO encodes a component of PS2 • gene resi
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