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March Term Test Outcomes.docx

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Western University
Biology 1002B
Tom Haffie

March Term Test Tuesday, March 12, 2013 12:44 AM Lecture 10: Evolution of Eukaryotes  meaning of endosymbiosis, cyanobacteria, lateral gene transfer  The uptake of aerobic bacterium occurred before that of cyanobacterium as suggested by the fact that all cells have mitochondrion but not all have chloroplasts  origin of endomembrane system, nuclear membrane, ER etc.  Develops the endomembrane system derived from the infolding of the plasma mm'n forming the nuclear envelope and the ER  The ability to separately and tightly regulate transcription and translation allowing for specialized control; something that is not possible in bacteria  origin of mitochondria and chloroplasts  Theory: mitochondria and chloroplasts were derived bya from free-living cells = endosymbiosis  Modern day mitochondrion used to be an ancestral aerobic bacterium  Ancestral anaerobic would be at an advantage to take in mitochondrion (aerobic bacterium) and chloroplasts (cyanobacterium) through phagocytosis  evidence supporting theory of endosymbiosis  Morphology  Mitochondria look like bacteria, look like E.coli  Formation/division  Mitochondria/chloroplasts divide using very similar processes as bacteria within the cell  No mitochondria/chloroplast gene in the nucleus  Electron transport chains  Free living cells need this so mitochondria/chloroplasts should have this  Other organelles don't have ETC  Genomes  They have their own genomes  Have their own genetic information  Transcription and translation machinery  No dependent on any other cell for transcriptional and translational machinery so can be free-living  factors driving development of early eukaryotic cells  Oxygen is the key, the ability to use oxygen was the drive  Earliest bacteria were anaerobic  Uses glycolysis and fermentation to make ATP (this does not make very much ATP, so they didn't have enough ATP for major functions…)  2.2 bya cyanobacteria developed and they were able to use oxygenic photosynthesis to split water into oxygen and protons  Produced oxygen and evolved oxygen and released it into the atmosphere  This eventually increased the concentration of oxygen in the atmosphere  Bacteria that undergoes aerobic respiration  Oxphos  Lots more ATP was produced and this allowed for more functions on the part of the cell  why eukaryotic cells can be larger and more complex than prokaryotic cells  Eukaryotes are much bigger so they have a low plasma mm'n surface area/volume ratio  But the plasma mm'n is not the site of oxphos for eukarya, they have mitochondria each with many oxphos centers  The internal mm'n structure is much larger than in bacteria  Lots of energy is produced  This allows the cell to support a larger genome and thereby lots of other functions that are dictated by the genome  DNA replication = 2% cell energy, protein synthesis = 75% cell energy (prokarya don't have enough ATP to synthesize novel pns  Bacteria = 13000 ribosomes, Eukarya = 13000 x 10000 ribosomes  Greater diversity of genome size as well  evidence for lateral gene transfer from organelles to the nucleus  The proto-mitochondria was free-living with its own DNA info, but after being engulfed, its info conflicted with that of the cell's own nucleus  The mitochondria has be totally coordinated on the cellular level with the rest of the cell (glycolysis….other functions…)  One of the strategies to cope with this  Over millions of years, the organellar genes from the mitochondria have relocated to the nucleus = lateral/horizontal gene transfer  Genes which had originally been in the mitochondria/chloroplast moves into nucleus and the control was obtained by nucleus to develop the more integrated system  general idea about how lateral gene transfer is detected (Southern blot)  Southern blot  Single stranded Genomic DNA is run on a gel and hybridized with single-stranded probe is used to identify the presence of a particular gene  Isolate mtDNA and nDNA and run it on the gel  Note: hgt is still did not stop  Role of cpn60 in tracing endosymbiotic and lateral gene transfer event in eukaryotes  Cpn60 is an essential mitochondrial pn whose genes are in the nucleus (hgt)  Giardia doesn't have mito but has cpn60  The mitochondrial gene is in the nucleus shows that the cell has a mito at one point but then lost it and the spn60 no longer has any function in the cell even though its still there  Ancestor has mito and then this split into two groups  Giardia like orgs that lost their mito  Other eukaryotes that kept their mito Lecture 11: Intro to Prokaryotic Gene Structure  relative sizes of typical mitochondrial, chloroplast and nuclear genomes  Nucleus > mitochondrion > chloroplast  rubisco structure and assembly from components coded by different genomes   possible reasons why modern organelle genomes have become dramatically smaller over evolutionary time  Most of the genes have been laterally transferred to the nucleus of the cell so they are no longer present in the actual organelle  possible reasons why genes have moved to the nucleus from organelles over evolutionary time  Nucleus could take over control  The mitoc doesn't need it anymore to function  An enzyme for glycolysis  What can genes do in nucleus that they can't do in mitoc: sex recomb  So that the nucleus can have coordinated control  Genes can get transcribed in the mitoc/nuc  Safer in the nuc (away from ROS) as mitoc/chloro are sites of ROS production  The organelles have their own gene expression machinery. Organelle genomes are transcribed/translated inside the organelle itself (you have ribosomes inside mitoc/chloro)  possible reasons why certain genes have not moved to the nucleus from organelles  Genes you need a lot of product of, it makes sense to have those genes localized  Maybe it takes too much time or are too hard to transport  Maybe the genes are already optimal where they are  Maybe its just chance/genetic drift and there hadn't been enough time yet for it to move  Maybe it just doesn't work in the nucleus  There are ribosomes inside mitoc  Genes are probably still in the mitoc b/c they haven't gotten the chance to get out (too big) --- (the general environ in your cells is prok but the environ inside you nucleus is euk)  basic mechanism of transcription and translation in prokaryotic organelles vs. eukaryotic nuclear environments  Prok transcription  Transcription is the process by which information codded in DNA is transferred to a complementary RNA copy  Transcription beings when an RNA pol binds to a promoter sequence in the DNA and starts synthesizing mRNA  Pol then adds RNA nucs in sequence according to the DNA template  At the end, the terminator sequence causes a hairpin look destabilizing the base pairing b/w mRNA and DNA and causes the mRNA to fall off  Euk transcription  Transcription is the process by which information codded in DNA is transferred to a complementary RNA copy  Transcription beings when an RNA pol binds to a promoter sequence in the DNA and starts synthesizing mRNA  Pol then adds RNA nucs in sequence according to the DNA template  At the end, a poly(A) tail signals the end of the gene and the mRNA falls off and transcriptions ends  Prok/Euk translation  Translation is the assembly of aa into polypeptides  Translation occurs on ribosomes  The P, A, E sites of the ribosomes are used for the stepwise addition of aa to the polypeptide as directed by the mRNA  Occurs simultaneously as the mRNA is being made only in prok  Aa are brought to the ribosomes attached to tRNA  A ribosomes assembles with an mRNA molecules (the SD Box in the UTR base pairs with the rRNA to stabilize initiation)  Aa are added one at a time  A protein release factor terminates translation  There is no tRNA that binds to stop codon, so when the codon appears, it provides an opportunity for the release factor to get in there  The release factor is always present and always trying to get in there, but it is outcompeted by the tRNA  The release factor though is a protein, and so it DOES NOT base pair with the stop codon at the A site  basic structure and function of RNA polymerase and ribosome  RNA polymerase  Knows how to make copies of itself  DNA pol is like a pn in the sense that the DNA pol gene has figured out how to replicate itself  The gene codes for the pn that makes more of the gene  Seeks out promoters and is responsible for synthesizing mRNA from the DNA template strand in transcription  Ribosomes  Has a 3D structure and also pairs with itself to give them their needed structure and to be ribozymes  Ribosomal RNA base pairs with itself and is catalytic  Ribosomes are primarily RNA machines  The RNA is catalytic  The protein give it the structure it needs to do the work (the pn is structural)  examples of complementary base pairing in gene expression  DNA/DNA  mRNA/DNA  mRNA/rRNA  mRNA/tRNA  mRNA/mRNA  tRNA/tRNA  DNA/tRNA Lecture 12: Prokaryotic Gene Function  mechanism by which each signal is interpreted, or understood, by the cell  Promoters attract the attention of RNA pol which interacts with the DNA  -10 TATA, -35, these sequences from the start point: the location where reading of the gene begins  The startpoint is just downstream of the bubble  relationship between DNA sequence of signals and their function (ie. how would low efficiency promoters be different than high efficiency promoters?  Maybe the high efficiency terminator is longer and makes the hairpin more stable  Maybe the high efficiency terminator codes for G and C bases which make 3 H-bonds which is stronger than just 2 and this would make the loop more stable  characteristics of promoters that require a particular position and direction  The promoter has special structures at -10 and -35, these structures give the promoter direction  change in amino acid coded, given a change in the DNA sequence (and Genetic Code table)  Some aa have multiple codons  Some aa only have one codon  base sequence of start and stop codons as mRNA and DNA  Start DNA  3` TAC 5`  Start RNA  5` AUG 3`  Stop DNA  3` ATT 5`  3` ATC 5`  3` ACT 5`  Stop RNA  5` UAA 3`  5` UAG 3`  5` UGA 3` Lecture 13: Prokaryotic Gene Regulation  identify the main features of bacterial operons  The operon is a unit of transcription  A cluster of prok genes and the DNA sequences involved in their regulation  It is a polycistronic message  Contains most of the regulatory DNA sequences  Bring the many genes under the control of a single operator  Operator  A short segment that is a binding sequence for a regulatory pn  identify the function of repressor proteins  A regulatory pn  When bound to DNA, it reduces the likelihood that genes will be transcribed  identify location of various components of the lac operon  Just outside of the lac operon to the left:  Promoter (1)  Lacl gene (regulatory gene that produces the lac repressor)  Lac operon left to right:  Sequences that control the expression of the operon  Promoter (binds RNA pol)  Operator (binds lac repressor)  Transcription initiation site  Transcription unit of the three lac structural genes β  lacZ ( -galactosidase)  lacY (permease)  lacA (transacetylase)  Transcription termination site  DNA signals in RNA-coding genes  Promoter  Terminator  Coding sequence  DNA sequence of anticodon in tRNA gene, given the codon  mRNA codon: 5` UGG 3`  tRNA codon: 3` ACC 5`  DNA codon: 5` TGG 3`  likely effect of base sequence substitutions in various DNA signals  What if there is a mutation in the promoter  The efficiency of the promoter could be positive or negative  The promoter could become more/less attractive to RNA pol  What if there is a mutation in the SD Box?  Unless the mutation creates a start codon, it might make the SD Box more or less attractive to rRNA  What if there is a mutation in the start codon?  Pretty much all you can do to a start codon is break it,  Kills the gene  No transcription  What if there is a mutation in the coding region  There are redundancies  Trp: UGG  Because of redundancies, when a mutation happens, the effects of mutation depends on where and how the mutation affects the codon  Silent mutation: sub one codon for another but code for the same aa  Missense codons: sub one codon for another and another aa  Nonsense codons: creating stop codons  Indel mutation: cause a shift in reading frame  What if there is a stop codon as a mutation,  then translation won’t stop, the translational machinery will just continue  Often times there are redundant stop codons to avoid such issues  What if there is a mutations in the terminator?  Depends on if we strengthen or weaken the terminator resulting in a stronger or weaker look and therefore a more or less efficient termination  mechanism of action of lac repressor  Binds to operator so that RNA pol cannot bind the DNA  functioning as a dimer blocks the operator so that RNA pol cannot bind to the DNA  It stabilized the blue loop (loop of double stranded DNA) and there is no way that the RNA pol can access it  function of lac operon in the presence, and absence, of lactose  Presence:  the lactose is converted to allolactose which binds to the lac repressor and keeps it from blocking the operator  RNA pol is able to access the gene and transcribe lacZ, lacY and lacA  Absence:  the lac repressor pn, functioning as a dimer blocks the operator so that RNA pol cannot bind to the DNA  possible location of mutations in lac operon that give rise to a given phenotype  Mutation in repressor genes  Mutation in operator  phenotype that would arise from a given mutation in lac operon under given conditions  Mutation in repressor genes  No repressor produced - continuous transcription of lacz,y,a  Mutated repressor - cannot bind to operator - continuous transcription of lacz,y,a  Mutated repressor - does not bind to allolactose - no transcription of lacz,y,a at all  Mutation in the operator  Repressor cannot bind to it - continuous transcription of lacz,y,a  RNA pol cannot bind - no transcription of lacz,y,a Lecture 14: Eukaryotic Genes  basic structure of eukaryotic vs prokaryotic cell with respect to gene expression
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