BIMM 100 Lecture 13

BIMM 100 Lecture 13 - Lecture 13: BIMM100 ­...

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Unformatted text preview: Lecture 13: BIMM100 ­ Centromeres and teleomeres Reading: pages 257 ­266, 243 ­247 and 269 ­281 Remember: email me your clicker # if you are not geIng points registered on Ted! Get your test back to me by Thursday if you want a regrade! The point of syteny analysis? To idenPfy unknown genes! CSF3 synteny conserved among humans, mice, opossums, chicken, finches, and lizards. Looks like they’re two copies in fish (a and b) that evolved millions of years before these other organisms! Bartunek Chromosomes and genomes •  Experiments & techniques –  defining funcPonal elements of chromosomes –  determining how the RNA component of telomerase provides template informaPon •  Genomics –  Gene structures and expression on a genome ­ wide scale –  BioinformaPcs Chromosomes and your genome •  Things to know: –  Chromosome •  structure •  Required parts •  A special case: telomere replicaPon –  Sequence informaPon •  Comparisons and predicPons with genomics and bioinformaPcs –  RegulaPon of gene expression: transcripPon Similar species have different karyotypes (genomic informaPon can be organized in a mulPtude of different ways) Reeves muntjac: female: 2n=6 chromosomes Male 2n+1=7 chromosomes Indian muntjac: Female and male: 2n=46 chromosomes The number, size, and structure (like the posiPon and consPtuency of telomeres) is species specific! Similar species have different karyotypes: how the internet can lead you astray… What elements do chromosomes require for their funcPon? 1.  An origin of DNA replicaPon 2.  Centromere (as a means of segregaPon during mitosis) 3.  Telomeres (for stabilizaPon) What elements are required for chromosome funcPon? Experiments done in yeast… Origin of DNA replicaPon: ARS element. Stands for “autonomous replicaPon sequence” What elements are required for chromosome funcPon? Experiments done in yeast… CEN (centromere) sequences are required for segregaPon of the chromosomes ­ In yeast, it is a simple sequence about 110bp long. In more “complex” metazoans, it is 2 ­4 megabases of simple sequence repeats What elements are required for chromosome funcPon? Experiments done in yeast… TEL (telomere) sequences are required when the DNA is linear in the cells (like it is in chromosomes!). These data led to the development of yeast arPficial chromosomes (YACs) ­ important for molecular biology assays Centromeres: more than just repePPve DNA •  Three regions of yeast centromeres: –  I: short, well conserved sequences –  II: rich in A and T, no consensus sequence –  III: short, well conserved sequences •  I and III bind to 30 different proteins that bind to microtubules •  II is bound to a nucleosome with a variant of histone H3 (called CENP ­A in humans). Kinetochores •  In “higher” eukaryotes •  Complex protein structure that assembles at the centromere and directs binding of microtubule spindle fibers during mitosis Blue: DAPI (DNA dye) Green: microtubules (spindle) Pink: kinetochore ReplicaPon problem: what to do at the end of the lagging strand? •  Leading strand? No problem! •  Lagging strand: would get shorter every replicaPon Eventually the ends of chromosomes would erode! The soluPon? Telomerase! Telomerase •  Protein ­RNA complex called telomere terminal transferase (telomerase) –  Telomerase ­associated RNA serves as a template for addiPon of dNTPs to the ends of telomeres –  The protein performs the enzymaPc acPvity •  Basically, a specialized form of reverse transcriptase that carries its own internal RNA template! •  No telomerase? –  Ends of chromosomes shorten every generaPon Telomeres: how they work Controlled “slippage” Old DNA New DNA Telomerase associated RNA •  AcPon of the telomerase of Oxytricha (a protozoan) is depicted, but it is similar to other telomerases. •  Remember that each type of telomerase will add different types of sequences to the ends of DNA. Telomeres: how they work Telomerase keeps making copies by “slippage,” lengthening the DNA ends Old DNA New DNA Telomerase associated RNA Telomerase animaPon How do researchers know that RNA in telomerase provides the template for replicaPon? •  The experiment: a mutant telomerase RNA gene was introduced into cells. •  The observa1on: the telomeric DNA sequences had the mutant sequence incorporated •  The conclusion: the telomerase complex uses it’s own template! This template is RNA… Telomerase is involved in aging •  Germ cells and ES cells express telomerase; they need to be able to divide regularly. •  Most other cells in the body do not express a lot of it, unless they need to divide regularly (like progenitors in the immune system). •  Aging is associated with a loss of telomerase acPvity –  January 2011 (Nature): Telomerase reac5va5on reverses 5ssue degenera5on in aged telomerase ­deficient mice (see hpp:// nature09603.html) •  Many diseases are associated with telomere deficiencies –  Ex: Werner syndrome ­ premature aging, autosomal recessive. Rapid decay of telomeres Ashkenazi Jews: a populaPon well studied by genePcists •  Why? –  Jewish populaPons, and parPcularly the large Ashkenazi populaPon, are ideal for such research studies because they exhibit a high degree of endogamy (marrying within a specific ethnic, class, or social group), yet they are sizable (unlike groups like the Amish). –  Importantly, Jewish communiPes are also comparaPvely well informed about genePcs research, and have been supporPve of efforts to study and prevent genePc diseases (again, unlike populaPons like the Amish). •  Link to telomeres? –  Studied traits important for increased lifespan in this populaPon. The result? Discovery that the longest living have more acPve telomerase! This hyperacPve telomerase leads to increased longevity! PNAS, 2010. So, we should encourage more telomerase acPvity, right? •  Wrong! –  Telomerase is inappropriately expressed by many types of cancer cells to promote replicaPon and division! –  Great example: HeLa cells (an immortal cancer cell line derived from Henriepa Lacks that has been used in the laboratory since 1951). HeLa cells •  Why is it immortal? AcPve form of telomerase (these cells do not normally have telomerase acPvity). Basically, cells have gone through so many forms of mutaPons that this has been selected for over Pme… Telomerase acPvity varies in different cell types Changing gears… A look at bioinformaPcs •  Sequencing: genomes of hundreds of organisms now reside in two locaPons –  Genbank (NIH, Bethesda, Maryland) –  EMBL Sequence Database (European Molecular Biology Laboratory, Heidelberg, Germany) •  Use of computers to analyze these sequences is called bioinforma1cs •  The comparison of enPre genomes is called genomics Where are the genes? •  Proteins with similar funcPons osen contain similar aa sequences that correspond to important structures and funcPons •  Usually across species, protein similariPes are somewhat conserved (more than the DNA that codes for them). Why is this? –  Use BLAST (basic local alignment search tool) ­ algorithm for comparing your protein sequence to the database. It will even give you a probability that these correlaPons are due to chance (a p value) •  The lower the p value the beper! Making connecPons… Linking human diseases and gene mechanisms with studies performed in other organisms. Genome Transcriptome Proteome Great example: comparaPve analysis of neurofibromatosis type I Human gene: NF1 is associated with neurofibromatosis type I (mulPple tumors develop in the CNS). Has homology to yeast gene called Ira, a GTPase acPvaPng protein that modulates Ras (which is essenPal for cell replicaPon and differenPaPon control). The homology suggested the funcPon, which could then be verified by other means! Comparisons of proteins also inform us of evoluPonary changes Protein families can be recognized as being shared among many related species. It is likely, based on their sequences and funcPons, that these genes all derived from a common ancestral gene early in evoluPon. Comparisons of proteins also inform us of evoluPonary changes Homologous: common ancestor Orthologous: genes arose by speciaPon Paralogous: genes arose because of duplicaPon Of these three, orthologous sequences are most likely to share the same funcPon! Comparisons of the genome can disPnguish differences between individuals "I've always said that at the end of the world there will be roaches, Ozzy and Keith Richards," said Sharon, Osbourne's wife. See: hpp:// id=ozzy ­osbourne ­genome "Ozzy carries several hundred thousand variants that have never been seen by scienPsts," Nathaniel Pearson, Knome's director of research, told ScienPfic American. "Many of the variants in his genome are about how the brain processes dopamine," he conPnued. Osbourne is 2.6 Pmes more likely to experience hallucinaPons while on marijuana, has an increased risk of cocaine addicPon and "an increased predisposiPon for alcohol dependence of something like six Pmes higher", Pearson said. Osbourne's genes also suggest that he is slow to metabolise coffee. "Ozzy's kryptonite is caffeine," a Knome rep explained. A funcPoning change to the singer's TTN gene, which has been linked to the nervous system, may be connected to Osbourne's hearing or to his tremors. How to find genes with bioinformaPcs •  Bacteria and yeast –  Scan genomic sequences for ORFs •  Defined as a stretch of DNA containing at least 100 codons (starts with “ATG”, ends with a stop codon) •  Can idenPfy over 90% genes this way •  More complex eukaryotes –  More complicated ­ most genes are short exons interspersed between long intronic sequences –  Algorithms have been created that factor in •  HybridizaPon of query sequence to full length cDNA •  Alignment to parPal cDNA sequence (usually 200 ­400bp in length known as an expressed sequence tag [EST]) •  FiIng to models of exon, intron, and splice sequences •  Sequence similarity to other organisms! InteresPng finding: protein ­coding gene number is not directly related to complexity of the organism Key: the number is not necessarily the most important thing! Remember: 1.  AlternaPve splicing can make mulPple funcPonal mRNAs from one transcript 2.  Proteins are post ­ translaPonally modified, which can differenPally affect their funcPons 3.  Increased biological complexity results from increased numbers of cells based on the same kinds of proteins (larger #s of cells can interact in more complex combinaPons) Changing gearsagain: moving on to gene expression •  Overview •  Bacterial principles •  The polymerases •  Regulatory sequences •  Finding them •  Building complexity •  Regulators •  Finding them RNA polymerase acts in concert with σ (sigma) protein factors Sigma factors: help “find” the promoter and bring RNA pol closer to it… Core polymerase: α, β, β , ω = apoenzyme (inacPve) The apoenzyme + σ = holoenzyme (the acPve form) RNA pol σ factor  ­50  ­35  ­10 +1 +20 σ Factor also separates DNA strands and “feeds” the coding strand into the acPve site of RNA pol RNA σ factors •  Most common factor? –  σ 70 in eubacterial cells •  Not the only one, though! •  Specific sequence recogniPon sites:  ­35 region TTGACAT 15 ­17bp  ­10 region TATAAT σ factor is an ini#a#on factor: something required for transcripPon iniPaPon, but not for RNA strand elongaPon Many other “flavors” of σ factors Different consensus binding sites! Important for the expression of different types of genes: heat shock and nutrient deprivaPon responses, moPlity and sporulaPon IniPaPon and repression of transcripPon: a look at the lac operon •  Lac operon: responsible for responding to different metabolites •  When E. coli is in an environment that lacks lactose, the synthesis of this mRNA is repressed –  Why? The cell doesn’t want to waste energy making enzymes to process something that isn’t there! Operator: lac repressor binds here when there is no lactose Glucose is low? cAMP increases. Then, it binds to CAP, which changes shape and apaches to special site in the promoter Lac operon is even more complex than that! •  Lac repressor actually binds two sites at the same Pme (O1 is the primary site, and O2 & O3 are possible secondary ones) •  Property of DNA makes this possible? It’s flexible! Lac operon has a weak promoter Key behind this repression? The lac operon is a weak promoter. What controls that property? The σ Factor! RNA pol σ factor  ­50  ­35  ­10 +1 +20 How? The σ Factor of the lac operon does not have an “opPmal” σ factor binding site!  ­35 region Strong promoter: TTGACAT 15 ­17bp  ­10 region TATAAT Not all σ factors bind at the same •  σ54 is different sites… from the other factors •  Bind at enhancer elements 80 ­160bp upstream from the start site. •  Best characterized? NtrC ­ it sPmulates transcripPon from the glnA promoter (important for the synthesis of glutamine) Not all σ factors bind at the same sites… Forms a loop in the DNA! •  σ54 and RNApol bind to the promoter, but transcripPon doesn’t happen unPl it is acPvated by NtrC •  Not the only level of regulaPon! –  NtrC is regulated by NtrB ATP hydrolysis is required for this acPvaPon of σ54 and RNApol by phosphorylated NtrC ­ σ70 doesn’t require ATP input! Bacterial responses are controlled by two ­ component regulatory systems •  Just like NtrC and NtrB, most bacterial regulatory systems have two components •  PhoR and PhoB: responsible for responding to levels of free phosphorous ­ when it’s present, the system is inacPve. •  Takes ATP! •  Turn on transcripPon that helps cell deal with low phosphate PhoR: “sensor” & PhoB: “response regulator” Tomorrow: more transcripPon! •  EukaryoPc gene control •  RNA polymerases (there are more than one!) •  Regulatory elements and their importance ...
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