Exam One Notes
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825 Notes LECTURE ONE INTRODUCTION TO MOLECULAR BIOLOGY L211 What is Molecular Biology 0 The study of biology at the molecular level 0 quotThe branch of biology that deals with the nature of biological phenomena at the molecular level through the study of DNA and RNA proteins and other macromolecules involved in genetic information and cell function characteristically making use of advanced tools and techniques of separation manipulation imaging and analysis 0 In other words the study of the structure and function of macromolecules o Polysaccharides Lipids Nucleic acids Proteins 000 Understanding how these macromolecules function in the cell their cellular processes and how scientists study them Molecular Biology of the Gene 0 Watson and Crick with some help discovered the structure of DNA is a double helix 0 quotIt has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material Watson and Crick Nature 1953 I Determining the structure of DNA 9 questions that still haven t been answered 0 Structure suggested how DNA was replicated 0 DNA was a double helix with a bases of double bonding 0 Proposed easy for hydrogen bonds to be broken and unzipped for each strand to serve as the template 0 Also suggested DNA might contain a genetic code 0 Bases themselves might code for protein 0 which led to further questions 0 How are genes passed on to new generations molecular level 0 How do genes encode characteristics 0 How are genes expressed and regulated The Central Dogma 0 Proposed by Francis Crick 1956 o In simpler terms the central dogma explains the directional flow of genetic information in a cell 0 DNA 9 RNA 9 Protein 0 Arrows show the transfer of information 0 DNA is transcribed to RNA and mRNA a type of RNA is translated to protein Molecular Biology in the News New Life of Ancient DNA Scientific American Aug 2012 o Extinct creatures understanding comes from fossils or preserved carcasses 0 Physical specimen of the carcass tell us about physical characteristics 0 But not about cellular processes I For example How did wooly mammoths adapt to cold 0 Hemoglobin a protein binds oxygen in the lungs and delivers it to tissues 0 Oxygen from blood hemoglobin 9 oxygen released to tissue cells At low temperatures hemoglobin has difficulty releasing oxygen 0 A potential problem in the extremities of arctic animals Woolly mammoths Arctic are evolutionarily related to Asian and African elephants O Hypothesis Wooly mammoths migrated from warmer climate Mammoth hemoglobin evolved to improve oxygen release in cold temperatures What the researchers did 0 1 Sequence mammoth hemoglobin gene from fragments I DNA is fragile and tends to be fragmented because the carcass is old I Make many copies of the fragments stich them together gene 0 Use evolutionarily related elephants hemoglobin gene as a template 2 Compared sequences I Differed at 3 nucleotides 3 Make an intact mammothlike gene I Can t use DNA fragments 9 mutate elephant hemoglobin gene corresponding 3 nucleotides 4 Introduce gene into e coli I Use as a proteinmaking factory 5 Expose the purified hemoglobin to a chemical environment similar to that inside blood cells I Measured ability of mammoth and elephant hemoglobin to release oxygen at various temps 6 Observe how readily mammoth hemoglobin releases oxygen at various physiologically relevant temperatures I Three nucleotide mutations allowed for hemoglobin release at cold temperatures The Ebola Crisis in West Africa Largest outbreak ever At least approximately 1800 cases in West Africa No licensed drugs or vaccines for ebola 825 Notes LECTURE TWO MENDELIAN GENETICS Eukaryotic cells have a nucleus 0 The nucleus houses the genome which is the entire set of chromosomes comprised of DNA and proteins 0 A human somatic cell has 23 pairs of chromosomes 0 The nucleus also compartmentalizes cellular functions 0 Replication and transcription take part in the nucleus Understanding Genetics 0 Family members often look alike because they share genetic information 0 Though we now understanding how inheritance works it took us a long time to get there Early attempts to understand genetics The Theory of Pangenesis 0 Ancient Greeks all parts of the body exhibit hereditary differences 9 theory of pangenesis The Theory of Pangenesis 0 6th century BC developed by the ancient Greeks o Remained current until the 19th century 0 Darwin was unable to provide genetic information because he was thinking about his theory in the terms of pangenesis o Pangenes in every organ move throughout the body via the blood are delivered to the reproductive organs and are passed on to offspring 0 Ex Pangenes from your parents are in their genitals 9 pangenes are passed on to offspring Aristotle s problems with pangenesis o Aristotle debated the theory 0 What about a soldier who loses an arm in battle but produces a child with two arms 0 What about traits that develop following procreation I Example premature graying o What about traits that skip a generation 0 Suggested that hereditary information provides the potential for a characteristic not the characteristic itself Lamarckism o Named after JeanBaptiste Lamarck o Organisms can pass on characteristics acquired throughout a lifetime to their offspring 0 Ex Giraffes reach higher to reach taller leaves and pass on elongated necks to their offspring The Germ Plasm Theory Disproving Pangenesis and Lamarckism 0 August Weismann tested if an organism could pass on acquired characteristics to its offspring 0 Experiment cut off tails of mice 9 mate them together 9 check offspring for tails I Offspring had tails continued for 22 consecutive generations 0 Weismann developed the Germ Plasm theory 0 Hereditary material does not come from each organ or tissue ONLY germ sperm and egg cells contain hereditary information which is passed on to offspring o Germ cells are not affected by acquired characteristics 0 Genetic information are not passed on by somatic cells The Blending Theory of Inheritance 19th Century 0 Any single inherited trait can only be within the upper and lower values of its two parents o The offspring of a red flower and a white flower will be pink 0 Major problem would result in convergence of all variations into one 0 Example flower color 0 Color would be diluted through the crosses 9 color is diluted into one o If true color variation would be lost in a few generations Mendelian Genetics 0 Gregor Mendel tested blending theory by mating true breeding pea plants where self pollination of seeds always produces the same trait TrLle breeding parents Parental generation Mated pnenotypically different pea plants round seed x wrinkled seed gametes hybrid F1 generation Remained the same F1 generation phenotype as one parent NOT a blend of Hr parental traits 0 Observed the same patterns using seven other pea plant characteristics 0 Disproved blending because the pea plant was not a blending of the parents 0 The statistical laws of heredity derived by these experiments comprise modern genetics Language of Genetics 0 Gene a segment of DNA that controls an individual trait o Locus the physical location of a gene on a chromosome 0 Allele a version of a gene the gene controlling pea shape is either R or r o Allele is round vs wrinlltled Allele for round seed coat One allelefrorn 39 Pair ofhomoloous each parent chromosomes Allele for wrinkled seed coat 0 Phenotype the appearance or manifestation of a feature wrinlltled or round 0 Genotype Set of alleles that an individual possess genetic instructions RR Rr or rr 0 Homozygous possessing two of the same alleles of a gene 0 Heterozygous possessing two different alleles of the same gene 0 Dominant an allele expressed in both homozygotes and heterozygotes o Denoted as R or r 0 only dominant allele is expressed in heterozygotes o Recessive an allele whose phenotype is only seen when homozygous o Denoted as r or r39 First Law of Inheritance The Principle of Independent Segregation Observations leading to the principle eNDtE that the recessive allele was not lost in the F1 generation parental 1 generation Ft rr39 lv 39lr gametes quotrill I l ly39ljlrid r I i F1 generation FL generation All rolund seeds Fir l f f 1I 1I female l malle gametes metes 1 F2 generation F1 x Fl 31 ratio of round to wrinlkled Mlendei s first law independent segregation F2 39generation Mendel s first law independent segregation Alleles of the same gene segregate independently from each other during gamete formation to end up in different gametes o Came from observation of F2 generation The Testcross An exercise in determining genotypes The green alleles Y is dominant to the yellow allele y What is the phenotype of the green plant boxed in red Testcross cross with a homozygous recessive individual 0 Draw Punnett squares for predictions Two possibilities homozygous heterozygous W W W W O C O 0 V V V V Y W W Y W W lithe plant was W5 Ethel 5531 waS YY F1 50 green 1 a green Y W W V W W 50 yellow The Principle of Independent Assortment 0 Do the alleles for one trait seed shape assort dependently or independently of the alleles for another trait color 0 Traits alleles located on different genes and different chromosomes panamaquot O Dihybrid cross round and yellow wrinkled and green rryy at gametes lP generation Only two possible gamete types 0 F1 generation er y t i l i i er a gametes m or F1 generation l l l 7 l l Fggimramn g v F2 Observed a 9331 ratio with all possible gametes 5y aw gametes combinations of seed coats observed W am Halyo W 2W HrWoaaygb armies l art yfl arm27 am w o a Two new phenotypes not previously seen I lard o Mendel s second law independent assortment alleles of different genes sort independently of each other during gamete formation 0 Only applies to genes located on different chromosomes Chromosomal Theory of Heredity 0 Walter S Sutton 0 Worked with grasshopper chromosomes 0 Observed that chromosomes occur in pairs which segregate during meiosis o Devised the Chromosomal Theory of Heredity proposes that chromosomes contain hereditary information 0 Also hypothesized that traits are controlled by genes located on chromosomes Molecular Biology in the news 0 Women preferred scents of Tshirts worn by men whose MHC genes were different from their own 0 MHC genes play roles in immune recognition and fighting infection o If a male and a female have different MHC genes their children will have a more diverse immune system 829 Notes LECTURE THREE DROSOPHILA AS A MODEL SYSTEM EXCEPTIONS TO MENDELIAN GENETICS Model Organisms A widely studied nonhuman species used to understand a specific disease trait or phenomenon A few examples at IU O O O O Saccharomyces cerevisiae yeast Drosophila melanogaster fruit fly Arabidopsis thaliana flowering plant Escherichia coli bacterium Typical characteristics 0 O 0 Easy to grow or maintain in labs I Do not have to wait long for the next generation I Don t take much space and they can be quite cheap Sequenced genome I How genes are regulated I How proteins are modified Similarity to humans I Genes or pathways that are related to those pathways in humans Drosophila Melanogaster as a Model System Drosophila have been used in research for 100 years 0 Used to study genetics development neuroscience eye formation etc What makes drosophila a good model organism 0 Easy to maintain in lab I Both females and males are easy to distinguish 0 Females are larger than the males I Millions of flies can live in vials Rapid life cycle I 10 days at room temp life cycle Only four chromosomes I 3 autosomes and 1 sex chromosome Easy to genetically manipulate Sequenced genome I Published in journals for the scientific community uploaded to databases online Examples of different phenotypes O 0 Wild type typical phenotype observed in the wild in their normal environment Mutants locus form mutations have been mapped I Short winged can t fly but can hop around on the bench 0 Used to study wing formation I Antennapedia prevents antenna formation 0 Legs form where the antenna are I White eyed 0 Usually have red eyes I Eyeless I Proboscipedia turns mouthparts into legs 0 It is viable 9 the organism will survive 0 Maintenance 0 Kept in vials with media 0 Change food every 1014 days 0 Shift temperature to adjust life cycle 0 Working with them 0 Must be anesthetized because fruit flies fly 0 Cooled or put to sleep with C02 Drosophila A Very Brief Early History 0 1 William Ernst Castle Publishes the first scientific article using Drosophila o Geneticist that established Drosophila as a model organism o 2 Thomas Hunt Morgan Wins Noble Prize for his contributions to understand the role of chromosomes in heredity o Inspired by Castle to use Drosophila o 3 Herman Muller Wins Nobel Prize for showing that exposing flies to Xrays induces mutations 0 First way of showing mutagenesis 0 Important because US had just dropped two bombs on Japan Drosophila at IU 0 Home to the Bloomington Drosophila Stock Center 0 Maintains gt 50000 mutant strains of flies o Distributes flies around the world 0 Located in Jordan Hall 0 Maintains the database FlyBase 0 Access to all drosophila papers 0 Information regarding 15000 identified genes 0 Information on mutant flies o 9 faculty members and counting use fruit flies in their research Molecular Biology in the News Ill Leads the Way in Study of Genetic Workhorse The Humble Fruit Fly 0 Prior to their study Drosophila genome had already been sequenced o The scientists studied fruit files at different stages of development and exposed flies to insults stressors 9 flies experience changes in the result of stresses 0 New genes and old genes are turned on I No one knows what they do and if there are human counterparts o Discovered 1468 new genes 0 Promise for research 0 Drosophila as a human disease model 0 Why It has 75 of the disease genes that humans have 0 Example I A man s cancer was replicated in a fruit fly and then the fly was exposed to drugs 0 Once the fly s condition improved those drugs were used in the human whose condition also improved 0 A process that only took a couple of weeks Discovery of SexLinked Traits 0 Thomas Hunt Morgan and his students screen thousands of flies for spontaneous mutants 0 Trying to find a random mutation o The first mutant found was a male with white eyes instead of red eyes 0 Normal phenotype wild type Red eyes 0 Gene W or w o Mutant phenotype white type 0 Gene W or W39 o In Drosophila the gene is named after the MUTATION Exceptions to Mendel s Laws of Heredity Linked Genes 0 Law of independent assortment Each pair of alleles segregates independently of each other pair of alleles during gamete formation 0 Only applies to genes on di erent chromosomes like the genes Mendel studied 0 Genes on the same chromosome do not assort independently they are linked 0 Cross wild type red female x mutant white male 0 P generation Female XWXW Male wa There is no corresponding locus on the 0 F1 generation Female XWXw Male wa eye color It is only on X the chromosor I All offspring had red eyes I Conclusion White eyes must be recessive o This conclusion came about because the white eye phenotype was not seen in F1 generation 0 Cross F1 red female x F1 red Male 0 F1 generation Female xWxW Male ny 0 F2 generation Female xwa or xWxW Male ny or ny I 100 of females had red eyes I 50 of males have red eyes and 50 of males had white eyes I All white eyed flies are males 0 Conclusion Eye color is located on the X chromosome it is sexlinked I More specifically it is Xlinked o Morgan s work provided the strongest evidence for the chromosomal theory of inheritance 0 Sutton used grasshopper chromosomes to make the theory 0 Morgan provided experimental evidence a parentalgeneration red phenotype white 3 Viv5 xquot I x W or w is located on the X chromosome WW genotype wY l l There Is no corresponding locus on the Y W 39gimi ii i l chromosome F1 generation 1 red red 3 K 7 FL generation All progeny had red eyes gtlt Note White eve allele recessive w w 1 1 F 2 generation red 9 red red 6 while 7 r All females had red eyes males had red eyes BoleFiglla w Wm M W Vamales had whiteeyes o If there is only one X chromosome present recessive Xlinked gene will be expressed 100 of the time in males Recombination and Genetic Linkage Morgan observed Some combinations of alleles were inherited together at a greater frequency than other combinations Alfred Sturtevant Genes more closely positioned on a chromosome are more tightly linked together than genes that are farther apart 0 Bands on microscopy correspond to a gene proven by Kaufman Recombination Exchange of genetic material between multiple chromosomes or between different regions of the same chromosomes Recombination frequency correlates directly with the distance between two genes 0 An idea proposed by Alfred Sturtevant proven by Barbara McClintock Crossing Over and Meiosis Sister chromatids i lHD mologo LIIE Eli romosom ES During interphase of meiosis 1 individual chromosomes duplicate After DNA replication each chromosome will have two sister chromatids Crossing over a form of recombination which occurs during prophase I of meiosis Genes that are close together have a higher probability of remaining together during meiosis o 1 Synapsis 9 sister chromatids to form tetrads o 2 Nonsister chromatids of homologous chromosomes cross over 0 3 Each chromatid will break at a point of contact and fuse with a portion of the other nonsister homologous chromosome I This is how genes transfer from momgene to dadgene to form babygene Occurs between nonsister chromatids of homologous chromosomes Does NOT occur during mitosis syoisiosis oi dopiiosteo i chromosomes to 39 Beotromere i iorm tetrads iN onsisier39 chromatids two ohzrome tios bend Nonhomologoos chromosomes across esoh other il l Ii Hiomoiogioos chromosomes r7 i eeoh chromatid breaks ii at point of contact and iuses wiiih a portion of the other Creating a Genetic Map Recombination frequency can be used to create a genetic map Example Determine location of 3 genes A B and C on the same chromosome Experiment Perform three different twofactor crosses Results Parental AB x ab AC x ac BC x bc genotype A a A a B b B AB aB C AC aC C BC bC b Ab ab c Ac Ac c Bc bc F1 AB ab Ab aB AC ac Ac aC BC bc Bc bC Recombination frequency recombinants 30 10 25 recombinance progeny x 100 Each cross yields 4 progeny types two parental genotypes and two new types recombinants Each cross produces a ratio of parental to recombinant progeny recombination frequency Creating a Genetic Map Recombination frequency measures the frequency with which a single chromosomal crossover will take place between two genes during meiosis Genes closer together on a chromosome have a lower combination frequency more closely linked o If recombination frequency is less than 50 then the genes are linked Using the provided recombination frequencies what is the gene order of A B and C o ACB Barbara McClintock Chromosomes and Crossing Over Recipient of Noble Prize 1983 for the discovery of transposons mobile genetic elements Also showed that crossing over involves the physical exchange of genetic information Late 19205 Studying how genes move during breeding of maize corn plants 1931 Publishes article with Harriet Creighton on crossing over 19505 Her work on transposons is considered too radical and is largely ignored 19705805 Her work on transposons is replicated and McClintock is validated Cr055ing Over Involves a Physical Exchange of Material Harriet Creighton and Barbara McClintock noticed changing patterns of coloration in maize kernels between generations One member of homologous chromosome pair had markers Along with observing phenotypes could also follow movement of physical markers Knob Extrachlromosomal material C W C 2 color aquot l l c colorless il iz Wx waxy C WX wx n0nwaxy Maize chromosomes just after prolphase l in meiosis parental genotypes extraohromoso meal 6 Wx material a Wit 2quoty l C wx c not knob l nonorossover progeny a W r 3 l c W3 0 Wx la c wx C W I l C W wx IfI I li 7 if C WK 1 crossover progeny C color c colorless W waxy wx nonwaxy C WK L C ilf C W Example Cl l 8 wt Genetically Hlomozygousc wx c w progeny couldl only arise by crossing over between C and c W wx IOCI Wx i C l l Cytologically Cauld SEE adldlition of extralcl iromosomal material using a microscope Plumohr39ut of F1 Hume olon39rdm39sl Commit551 Colomdlelarchy Ioi39ml quot lullcrm lmrllijquot Modems mail The date Eilmber oiquot Kernels quotquottm39 39l39F fy llulugiinl JHlIPEHI l EE offhwmowm 39539 liiul F 39sylrimf Kll hlhrll ll l ill 3 i d 25 i 74 fquot In Kllnllilll mannl39mnl r mr Kllnln lirs trnnslnrn om iij a 41quot III Kuuhllleexl39li39vumlumlioin Iquot J Euoljllbedmmms w 7 i r39 1 r39Ii lhssljbli hue tilmmm on Ellielath wars ashamed MIMI PL mhmthmsmw menus 39om pmlen Dal Emu Harrie1 11 C DELSJIMII and Bauhai ilt lmmdi 193 ll 1 Etwlebamu M uhmtal and EHLEIIJJEII ETMHIIE Int 1 PIE1353 ikzll l SELL511 H 193 49 lllicl a E Tmmm er ieeur During inlete iFnI39Innrlnn iIIl FHIIE39FIII39II A Follulu Nutmil Tee i Tr 11quot Tee W Exceptions to Mendel s Laws of Heredity Incomplete Dominance o In Mendel s crosses one allele was clearly dominant o This is not always true in nature 0 F1 generation seems to support blending theory 0 F2 generation disproves it because the parental phenotypes of red and White return Parental Generation AA x A A Aa Aa 1 Aa Aa F1 generation Aa Aa Aa Aa Gametes A and a F2 generation A a A AA Aa a Aa 0 Able distinguish 121 ratio phenotypically in F2 generation Exceptions to Mendel s Laws of Heredity Codominance 0 Sometimes neither allele of the same gene is dominant or recessive to the other 0 Progeny that are heterozyogous for codnominant alleles appear mosaic for both parental phenotypes Parental Generation RR x quot quot R R Rquot quot R 1 Rquot quot R 1 F1 generation Rquot fquot Rquot quot Rfquot Rquot quot 0 Another example Blood type Parent A0 39Cordl mirlant Parent BO l 1 I o A is dominant to 0 Blood Type A 1 H Blood Type B 0 Neither the A nor the B is dominant to each other In H g l A allele B allele ll 0 allele V V Ema Type BWTrPEAB Bland was aleoars o Ced omlnanll UEQ llal hnral Lilillargr of l1nlejl1iru3 If an individual is AB neither allele is dominant or recessive to the other the alleles are codorninant 93 Notes REVIEW FROM LAST LECTURE Crossing Over A form of recombination that occurs during prophase I of meiosis Genes that are close together have a higher probability of remaining together during meiosis Occurs between nonsister chromatids of homologous chromosomes Barbara McClintock Crossing over involves the physical exchange of genetic information Crossing Over Involves A Physical Exchange Of Material Harriet Creighton and Barbara McClintock 1931 Noticed changing patterns of coloration in maize kernels between generation One member of homologous chromosome pair had Imarkers a knoblike structure and extrachromosomal material Along with observing phenotypes could also follow movement of physical markers 0 O Genotypes I C color I c colors I Wx waxy I wx not waxy non crossover progeny c Wx or C wx Genetically homozygous c wx progeny could only arise by crossing over between C and w loci Cytologically McClintock could SEE addition of extrachromasomal material and the knob using a microscope Observe c wx kernels not present in P generation First time a physical exchange between chromosomes was shown Exceptions to Mendel s Laws of Heredity Incomplete Dominance In Mendel s crosses one allele was clearly dominant 0 Not always true in nature AA red x aa white 9 Aa pink snapdragon O 0 Intermediate phenotype between parental generation At F1 level seems to support blending theory I At F2 level red and white are back in the game therefore not support blending LECTURE FOUR BACTERIA 8 BACTERIOPHAGE AS MODEL SYSTEMS THE TRANSFORMING PRINCIPLE EXPERIMENTS PROPERTIES OF DNA Why do we care about bacteria in L211 0 Bacteria o Sustain life on earth I Provide oxygen we breath fix nitrogen decompose carbon both in and on our bodies they help keep us healthy 0 Help us understand the fundamental processes of life I Model organism to understand replication identify proteins the central dogma o Enabled the development of many commonly used molecular biology techniques Bacteria as Model Organisms 0 Just because they are small doesn t mean they re not complex 0 Shape 0 Habitat growth conditions 0 Development 0 Metabolism Etc 0 What mallte bacteria good model organisms 0 Easy to work with in a lab I They are small so you can grow millions in a limited space 0 They can be grown on different media I Pea tree plates have media jelly lillte substances that provided different nutrients growing conditions Easy to genetically manipulate Single chromosome Grow fast usuallysometimes I Fastest Double amount of bacteria in 20 minutes I Slowest Double in a week or longer 0 Many have sequenced genomes Tree Of Life Evolutionary relatedness of organisms o Phylogenetic trees show evolutionary relationships between organisms o Nodes indicate common evolutionary ancestors 0 Everything except animals fungi and plants Bacteria Archaea Eucawa We39re in here are microbes f 0 Prolltaryotes bacteria Green MDIMSLMHI I Mr warr malaria J and archaea lacllt nucleus 7 in W y V 7395quot Memanosarcma o 99 of the time refer Smmclwtes mommy Halobaclerma mm bacteria MeiI m 7 pFDIE UDH flema Themiococcus magnum In gnawa to T managing The nomast b hemmprormis CUECVUS r CY n na mlm Pyrodichum aCte Filla39uohactc rna E Impmw 0 EUkaIYOtes have a nucleus The rm I m 93 AWHHEI MM mil 3min What are Bacteria o Nucleoid single circular chromosome 0 Not membrane bound 0 No organelles 0 Have a unique cell wall peptidoglycan 0 Not similar plant cell wall of cellulose o Medically antibiotics attack peptidoglycan we don t have it but bacteria does 0 May have plasmids circular piece of DNA that carries nonessential information o Gives extra phenotype 0 May move via a flagellum or flagella through liquid or across a surface 0 May be surrounded by a capsule sticky substance on the outside 0 Capsules can mask the bacteria from the immune system 0 Can cause clumping of bacteria which can make it more difficult to be gulfed by macrophages Eukaryotes Bacteria Large Small Nucleus Nucleoid Membranebound organelles No organelles 808 ribosomes antibiotic target with different size 708 ribosomes antibiotic target with different size No wall animalscellulose wall plantsfungi Peptidoglycan Fairly limited metabolism Diverse metabolism essentially grow everywhere Fairly limited tolerance Many are extremophiles essentially grow everywhere Linear chromosomes Circular chromsomes Single genes Operons of genes Transcription translation are spatially separate Transcriptiontranslation NOT spatially separate Due to organelles No organelles therefore in the same space What do bacteria do 0 Cycle carbon 0 Convert C02 to sugars 0 Important in the carbon cycle 0 Make oxygen 0 Cyanobacteria in the ocean provide much of the oxygen that we breath 0 Fix nitrogen o Symbiotic relationship with bacteria and plant root module fix nitrogen I Change nitrogen into a form that can be used by plants 0 Most keep us healthy 0 Some cause disease 0 Some decompose garbage 0 They are single celled 0 but they also build communities cooperate communicate and prey on each other I Communicate via chemicals Molecular Biology in the News 0 Human body 0 23000 genes 10 trillion cells 0 Microbiome the bacteria that live on us and in our gut o 3 million genes 100 trillion bacteria o Microbiome 0 Composition differs between individuals I Can even differ between twins 0 Contributes to digestion vitamin production defense against pathogens etc I Break down milk I Synthesize vitamins I Outcompete pathogens from entering our body on skin and within 0 Disruption associated with numerous diseases 0 How can you fix a brollten microbiome o Probiotics bacteria in yogurt I Hasn t been tested in healthy individuals I Might be useful in actual IBS o Fecal transplants used to teach clostridium difficile infection I Clostridium difficile is a bacteria that is resistant to many antibiotics I Feces from healthy individual and enema in another individual 9 replaces microbiome in the sick individual The Transforming Principle 0 Frederick Gri ith 1928 Used a pathogenic strain of pneumococcal bacterial S strain and a non pathogenic strain R strain and performed the following experiment o Capsule is caps inject neat killed 5 strain reSPOHSible for capsule gene cnromosornie HHSZH r v 39 g V I h H H 3153 0 Heat lltilled S strain r i eat 0 l 9 v a i I 1 gh i capSiragment 2 f l39 and R strain mouse dies 9 up a Yr released 1quot I39Mquot 3 39 r naequot cap The R strain was a in A at pathogenic if transformed by a substance SCsmootti cell firt p 1 capS39ftz apR femmbmation aps with f h t r quotin and cell division f rom e ea 1 Ir i Ly 1by Jeri killedfragment S strain r quot 39 Ii39 quot g of 0 Release of ca 5 391 nJECt Stra I n nonpathogenic entryr of Chromosome p miouse Enough cell fragment bearing 39 allOWEd for It to be inject R strain capsinm mpg 6 3 mouse lives transformed into the nonpathogenic cell 9 translation of capsule 9 pathogenic 9 mouse dies Inject mixture of heat killed 5 strain and R strain nrlouse dies Why Foundation for later experiments that proved the transforming substance was DINA The Transforming Agent is DNA The Avery Experiment 0 Oswald Avery 1944 Repeated Griffith s experiments but included a set of controls that showed that nucleic acids and 39 not protein or i Eal iiillian lipids are the hereditary material a Heat killed and fragmented S strain Condusmn The f transforming agent 1 I Separated nucleic acid protein and lipid fractions 15 nude aud c3353l Mixed fraction with the R strain injected into mice quot R 39 Mixtures containingthe R strain and either the protein or tar Fl roughi cell lipid fraction mouse lived recombination and cell division I r I Bacteriophage Viruses that Infect Bacteria Can be Viewed on an electron micrograph Probably the most abundant specie on earth Phages vary greatly but typically consist of o A genome DNA or RNA Mixture containingthe R strain and nucleic acid mouse died 0 A capsid or head protein coat that surrounds the genome o Tails sometimes Two types of phages o Lytic undergo the lytic cycle 0 Temperate lysogenic Undergo the lysogenic cycle and when induced the lytic cycle Lytic Phages Undergo the Lytic Cycle 7 1 Phage infects bacterium injects its genome 5 magi b I I usually DNA 2 DNA is replicated we Phage particles are produced 3 4 5 Phage is released Bacterium bursts 3M Temperate Phages Undergo the Lysogenic Cycle 4 gierial 1 o 1 Phage infects a bacterium injects its genome usually DNA W m 0 2 Phage DNA integrates into the bacterial chromosome now ggm called a prophage considered recombination o 3 Indefinite replication of prophage a part of the bacterial i 2 genome o 4 Can switch to lytic cycle when induced usually by DNA i V damage prophage excises lytic cycle begins The Transforming Agent is DNA The HersheyChase Experiment 0 Alfred Hershey and Martha Chase 1952 Used phage to confirm that DNA is the heredity material GE slabeled eeei pretein 3239 I b H d Labeled phage coat proteins w1th radioactive sulfur 39 tEILE E if DNA 7 r Labeled genome with radioactive phosphorous mixing at virus with heel eelle as 32quot Welles Phage infected bacteria injected genome into bacterial egreviein cell rlpmtem must Removed phage particles with gentle agitation labeled with 35 New phage particles isolated as 35513139663 0 Some contained radiolabeled genomes 0 Others contained nonradioactive genomes 0 Both genomes packaged within nonradioactive particles multipli a m d o No radioactive sulfur identified virei ehremeeeme end predueiien of new phage o The DNA was 1nected not the protein 0 Also showed that bacteriophage had DNA Conclusion The transforming agent is nucleic acid DNA is a Double Helix periieiee 0 Early 19503 Knew DNA contained nucleotide bases sugar molecules and phosphate did not know structure 0 Franklin and Maurice Wilkins 1953 Take the Xray diffraction image 0 Watson and Crick 1953 Shown the picture by Wilkins deduce the structure of DNA 0 1962 Watson Crick and Francis receive the Nobel Prize The Helical Structure of DNA 0 DNA two polynucleotide chains twisted in a double helix Twisting creates two grooves a major groove and a minor groove DNA binding proteins interact with DNA at the major groove and histone proteins interact at the minor groove 0 Histone proteins help to localize the group and compact DNA 9 make chromosomes smaller DNA Is Comprised of Four Nucleotides Nucleotides consist of a nitrogenous purinepyrmidine base a sugar deoxyribose and a phosphate group The four bases adenine A guanine g cytosine C and T The bases are divided into two structural classes 0 Pyrimidine cytosine and thymine o Purine adenine and guanine Chargaff s Rules A pairs with T and G pairs with C Purine bases form hydrogen bonds with pyrmidine AT hydrogen bondGC Organisms differ in GC content 0 The amount of GC pairing or AT pairs differs I An organism can have more GC pairs than AT pairs But pyrinepyrmidine ration will always be 11 Base Pairing and Phosphodiester Bonds in DNA Nucleotides are joined by phosphodiester bonds 0 5 phosphate 3 hydroxyl Hydrogen bonds between purines and pyrimidines connect the two strands o AT forms 2 H bonds 0 GC forms 3 H bonds DNA strands are antiparallel run in opposite 5 9 3 directions and complementary o Antiparallel due to phosphodiester bonds in DNA 0 Complementary knowing the sequence base pairs of left strand leads you to conclude the answer for right strand GC Content and Denaturation High temperatures near 100C or conditions of high pH cause DNA to denature separate Denaturation is reversible Melting temperature Tm is determined by GC content and by ionic strength of the solution The higher GC content the higher the temperature must be to denature the DNA strand Why 0 GC forms 3H bonds rather than 2 therefore there needs to be more energy to rip apart the Hbonds and denature the DNA Hypothesis higher GC content 9 better adaptability in hotter temperatures 95 Notes GENOME ORGANIZATION A Preview of the Next Few Lectures Chromatin complex of DNA and protein Histones proteins that compact eukaryotic chromosomes absent in prokaryotes Nucleosome unit of DNA wrapped around histones Why is DNA so BIG Every Cell Maintains a Certain Number of Chromosomes apparatus chromosomes endoplasmic h haploid bacteria moleold lchzrmosome Iasmid diploid cell nucleus reticulum 39 mgitooharidria 7 haploid tell 0 Bacterial cell usually have one chromosome 0 Somatic cell two copies of each chromosome in the nucleus diploid o Gamete one copy of each chromosome haploid Genome Size and Organism Complexity 0 Trends 0 Genome size varies between organisms I Prokaryotes tend to have a smaller genome size than eukaryotes I Mega base pairs 1 million base pairs 0 How genome size is calculated Genome size is roughly correlated with apparent complexity BUT gene number correlates more closely to complexity More complex organisms tend to have decreased gene density I Gene density average number of genes per MB 0 How many genes that fit within a defined region Gene Density 0 What accounts for discrepancy in gene density between organisms o junk DNA not really junk 0 Example in 65000 base pairs 0 E coli 57 genes 0 Yeast 31 genes 0 Drosophila 9 genes 0 Human 2 genes 0 Bacteria don t have introns 0 Therefore their gene density is much higher Introns Exons and Intergenic Regions Introns Exons o Nonprotein coding regions DNA that o Coding regions interrupts a gene 0 Separate coding exons 0 Will be transcribed into mRNA and translated 0 Removed by RNA splicing into protein 0 Example Avg Transcribed region of a human gene 27 kb 27000 base pairs Avg Proteincoding region of a human gene 13 kb 1300 base pairs 0 Over 20000 base pairs are introns that must be removed by RNA splicing o Intergenic regions 0 DNA sequences found between genes 0 Do not contain coding sequences or structural RNAs Repetitive Sequences 0 Two general classes 0 Microsatellites another form of junk DNA I Short 13 base pairs or less tandem repeats I Commonly dinucleotide repeats I Due to errors in copying DNA I Example CACACACACACA 0 Hard to accurately copy long stretches of DNA o Genomewide re eats Segment P of DNA I F I I Large over 100 base pa1r but many are over x l K v base pairs Dispersed repeats a quot a Segment I Can be tandem or dlsperesed m Dim innIi I Can be transposable elements aka transposons WEWEPEE DNA Transposon Structure and Function 0 Transposon mobile genetic element ljumping gene 0 Transposase encodes by transposon o Recognizes inverted terminal repeats o Excises the element form original location host DNA 0 Inserts it in a new location target DNA which may cause mutation or duplication I Mutation inserting of a transposase in a gene can cause mutation if it disrupts a coding sequence ofr a gene and causing a frame shift Composite Transposons o 0 Very common in bacteria 0 Often the extra gene is an antibiotic resistance cassette 0 Source of new genetic information for the organism CutandPaste Transposition 0 Cutandpaste transposition excision of transposon from old site and insertion into new site element in old DNA iocatim39i anking host terminal inverted repeats DNA iEu g binding to imiLllliiimer of transposase Multimer of transposase Transposase recognizes inverted repeats cieaves transposon DNA DNA sleevege of both strands DNA sissvsgs sf hath strands excised transpsssn Elfgait DNA tg39 I DN t strsn transfer DNArepair synthssis ts ll gaps slismsnt tn nsw DNA tssatisn Itlgstlsn sat mudts new DNA t 5139 v 39 i7 739 l llzl tsrgs tstts tsrgs tstts duptllisattsn duplication 0 Result in target site duplication Transpsssn at initial sits ligase fill in gaps Trsnspsssn st Insw sits DNA polymerase and Jr fluvial13d mineral Target sits duplication DNA Strand Transfer 0 3 OH ends of transposon DNA attack the phosphodiester bonds of target DNA transposon DNA and target DNA strands join 0 Note the sticky ends of the target DNA 0 The cut is staggered so there is a single stranded staggered fragment that has no nucleotides sticky ends 0 Ligase fills in the single starred fragment A Better View of Sticky Ends and Target Site Duplication o recognition sequence for transposase is the target site 0 There is a staggered cut which ligase fills in using the target site DNA Pseudogenes 0 Reverse transcriptase polymerase and nuclease subunits 0 Use RNA a template to produce dsDNA called complementary DNA or cDNA 0 They do transcription in reverse in which they follow the central dogma backwards 0 Only expressed by some viruses 0 Upon infection 339 0 Using reverse transcriptase 9 RNA is J transcribed to DNA 9 reintegrated in genome o Pseudogenes are not expressed lack regulatory sequeces also lacllt introns o It won t have upstream regulatory reverse sequences transcr39pt39on 0 Because the template is RNA RNA is spliced without introns re uegra on l Elli l J l fune onalgene i pseudogene o Pseudogenes Molecular Biology in the News Gene Patents 0 The Gene Hunt Should Finders be Keepers 0 Mutation in BRCAl or BRCAZ genes 4085 chance of developing breast cancer though only 5 10 of breast cancer patients have a mutation o Myriad Genetics Inc I Isolated sequenced and identified mutaitons in BRCA1 and BRCA2 genes I Patented BRCAli and BRCA2 I Must use their cancer risk test 0 Gene Patent Issues I Is it ethical I Will it cost patients more money I Does it impede research 0 US Supreme Court Srtrikes Down Gene Patents 0 Supreme Court unamously ruled human genes can t be patented I Ruled that gene occur in nature and are not a human invention 0 However ruled that cDNAs can be patented I Ruled that cDNA is different than DNA is manmade 0 Supreme Court cDNA is distinct from the DNA from which it was derived As aresult cDNA is not a product of nature and is patent eligble I But cDNA CAN occur naturally some viruses can copy mRNA to cDNA infact this is how scientists learned the technique 98 Notes CELL CYCLE Cell Division 0 Cell division involves o Replication of DNA in the parent cell 0 Distribution of DNA to the daughter cells 0 Occurs in I Prokaryotes binary fission o Asexual reproduction I Somatic cells example epithelial mitosis I Gametes meiosis o Occurs as part of a larger cell cycle Cell Division in Bacteria o Binary Fission asexual reproduction o 1 Cell Replicates its DNA 0 2 The cytoplasmic membrane elongates separating DNA molecules 0 3 Cross wall forms peptidoglycan membrane invaginates o 4 Cross wall forms peptidoglycan completely 0 5 Daughter cells Cell Division in Eukaryotes 0 Somatic cell any cell in an organism besides the gametes o of Chromosomes 46 Zn diploid made up of 2 sets of 23 chromosomes 0 Gametes Egg and sperm 0 of Chromosomes 23 1n haploid 1 set of 23 chromosomes 0 Mitosis occurs in somatic cells 2n to Zn produces two identical daughter cells 0 Meiosis occurs in gametes 2n to 1n produces foar genetically di erent daughter cells 0 Mitosis and meiosis are part of a larger cell cycle The Somatic Cell Cycle 0 lnterphase consists of G1 S and G2 phases 0 G1 prepares for chromosome segregation checkpoints that there are no mutations duplication of organelles nutrient accumulation mreaPJIASE DNEA sfmhesis S replication 0 G2 Prepare for cell division o Mitosis M phase Chromosome segregation 9 final product two new daughter cells 0 Division of nucleus 0 Cytokinesis physical separation of two daughter cells 0 Division of cytoplasm 0 Throughout the cell cycle the structure of DNA changes lnterphase S phase 0 Chromosomes decondense o Unwrapped from histones o Allows replication enzymes to have access to the strands of DNA 0 DNA replication occurs 0 Sister chromatid cohesion is established 0 Cohesion holds sister chromatids together I Occurs immediately following DNA replication 0 Cohesin ringshaped protein complex that encircles two copies of recently replicated DNA Mitosis Prophase o Chromosomes condense Xshaped 0 Nuclear envelope breaks down 0 Each duplicated chromosome contains two identical sister chromatids joined at a centromere region at which the sister chromatids are attached Metaphase o Chromosomes line up along metaphase plate imaginary plane 0 Bivalent attachment sister chromatid pairs attach to microtubules from opposite pole 0 One sister chromatid is attached to S pole the other sister chromatid on the other side of the centromere is attached to N Pole 0 Monovalent attachment is improper and can result in aneuploidy tumor formation and cancer Anaphase o Cohesion proteloyzed o The rings that held the sister chromatids together is digested 0 Sister chromatids pulled apart and migrate to opposite poles due to bivalent attachment Telophase 0 Each chromosome at the spindle pole 0 Nuclear envelope reforms 0 DNA decondenses as cell prepares to reenter interphase o Mitosis is complete Cytokinesis 0 Final separation of the two daughter cells membranes and cytoplasm 0 Through invagination of the cytoplasm Meiosis 1 Separation of Homologous Chromosomes Interphase 0 DNA replication Prophase I o Chromosomes condense 0 Pairs of homologous chromosomes align 0 Crossing over between nonsister chromatids within the homologous pair occurs 0 A homologous pair with one coming from the mother and the one coming from the father Metaphase I 0 Pairs of homologous chromosomes arrange on the metaphase plate 0 Homologs attach to microtubules from opposite poles Anaphase I o Breakdown of cohesion along arms of paired homolgous cohesion remains at centromeres sister chromatids remain paired 0 Sister chromatids pair move together toward the same pole Telophase I 6 Cytokinesis 0 Each half of the cell ahs a complete haploid set of replicated chromosomes 0 Two daughter cells are formed 0 Sister chromatids are not identical due to crossing over Meiosis II Separation of Sister Chromatids Prophase II o Chromosomes still composed of two sister chromatid associated at the centromere Metaphase II o Chromosomes align on metaphase plate 0 Sister chromatids attach to microtubules from opposite poles bivalently Anaphase II o Cohesion rings break down 0 Sister chromatids separate and move toward opposite poles Telophase II and Cytokinesis o Nuclei form chromosomes begin to decondense and cytolltinesis occurs 0 During meiosis 11 DNA is not replicated 9 four haploid genetically distinct daughter cells 0 Mitosis 2 diploid Mitosis Meiosis daughter cells genetically V lnlwp hnsc Ilnl erplwse 7 Chromosomes 39W it Chmmommcs Identlcal rel 2quot l1 ll ll f P if P o Me1os1s 4 hap101d l 7 H Ml lu lll l W V V V 39 Pmphase Pmphml daughter cells genet1cally r l quota HamMUSHUE 1m chromosomes U 39ld39 g different synapsis unclemsle V 1 mar occurs in Metaphase faquot Metaphase l Chmmmmcs line up am Chmmmmcs line up individually at metaphase quot u by homologous palm plate Iquot I all nrclaphas plat an n Anaphase I ii Ad quot f llnmt rlugs mpamlui Anaphase f q chromatids Daugm H 1513mm j cells of in nnaphase II slums WWW quot Sistcrchmmmids r 39 E f separate D aughllercellls l a 2 I r I V l I I gt 21 In Iaquot 7 thl r 391 0 mulmls I I l I 1 v 2n k I 39 39 j n n 311 xx n 2n Daughter cells of meiosis ll 910 Notes THE NUCLEOSOME Molecular Biology in the News 0 Hello Mothers Hello Father 0 All mitochondria is inherited maternally I During fertilization sperm mitochondria enter the egg but are chemically marked for destruction 0 Mitochondria Transplantation I 1 Collect eggs from mother with mutated mtDNA and from donor with healthy mitochondria I 2 Remove nuclei from both eggs I 3 Discard nuclei from healthy egg I 4 Transplant nuclei from diseased mother egg to healthy nucleusfree egg 0 Hybrid egg has nuclear DNA from mother and mtDNA from donor I 5 Fertilize with sperm I 6 Embryo has genetic information from three people Eukaryotic Chromosomes are compacted by Histones 0 DNA must be compacted to fit in the nucleus 0 This is mediated by proteins called histones 0 Chromatin complex of DNA and protein 0 Nucleosome a single unit of DNA wrapped around an octamer of histones o Prokaryotes do not have histones or nucleosomes o Is DNA negatively or positively charged negatively charged 0 Why phosphate groups on backbone I Histones are charged I The charge helps determine if chromosomes are condensed or decondensed I If a histone is positively charged will it interact more or less tightly with DNA more tightly o The charge is important since the structure of DNA varies during the cell cycle Nucleosomes o Nucleosome a single DNAhistone octamer 8 histone subunits core complex 0 Core DNA DNA most tightly associated with the nucleosome like thread on a spool 0 Always 147 base pairs invariant never change 0 Linker DNA DNA between each nucleosome 0 Average linker DNA length varies by organism 0 DNA not packaged in nucleosomes typically involved in gene expression recombination or replication usually associated with nonhistone proteins Histones N lelr milnal tail histone folld domain 5 Core histones comprised the octamer HZA HZB H3 H4 Gterminus Linker histone H1 Histonefold domain Nterminus rE Helps the core complex form an I xla ilx 39 H n 3 alpha Memes 2 unstructured luopsl turns l 1ntermed1ate structure 1n the absence of DNA 0 Histone fold domain consists of a helixturn helixturn helix HTHTH motif Assembly of a Nucleosome 1 H3 and H4 form a tetramer H2A and H213 form a dimer intermediate structures that must form prior to binding DNA A lt w iquot quotI H I 1 x l 3 I 39 rh 39 i a 4quot I va iquot N H3H4 tetra Inner H2A H 28 d imer 2 H3H4 tetramer binds dsDNA first complex to associate with DNA 3 Two molecules of the HZAHZB dimer join the complex The nucleosome is formed Modification of Nterminal histone tails lls3 N 0 N terminal tails of each histone are outside the DNA loop Cterminal heads are in the inside 0 Can be modified to change the charge of the core proteins this will affect the condensation or decondensation of DNA 0 Occasionally the histone fold domain can be modified too 0 Histone modifications 0 Are reversible 0 Do not alter the DNA sequence 0 There are several posttranslational the protein was already translated before the modification took place modifications to histones we ll discuss two Methylation of Histones 0 Adding a methyl group has a neutral effect on the overall charge of histones 0 Because the electronegativity of carbon and hydrogen are relatively neutral 0 Makes histones more hydrophobic 9 package more tightly in the nucleus condense 0 Because the environment of the nucleus is hydrophilic o Histone Methyl Transferases HMTs 0 Add methyl groups Me to histones I Is it always to the N terminal tail 7 7 7 o Differ in substrate specificity and in number of methyl groups 1 2 or 3 can add o Histone Demethylases HDMs 0 Remove methyl groups 0 Makes histones less hydrophobic Acetylation of histones 0 Makes the overall charge of histones more negative 0 Reasoning polar double bond CO 0 May help decondense chromosomes in late telophase and late interphase 0 So that the DNA is more open during replication of DNA 0 Removal of acetyl groups from histones may promote condensation in late Gz and prophase o Histone acetyltransferases HATS Add acetyl groups Ac to histones o Histone deactylases HDACs remove acetyl groups Histone Modification Affects Transcriptional Regulation DNA compaction affects accessibility of DNA binding proteins to regulatory regions Histone acetylation shown below is also true for methylation phosphorylation etc FEETDIME E y aii l ETFE E Ewpl i l ll39r llia m In In as lling i gh1 laigli rJl Ewen thrma m Histme EIE39EEEE39EFI align an silencing Genes can be turned off if transcription factors can t get to these sites and vice versa Epigenetics regulation of gene expression by chemical modification of histones or of DNA Molecular Biology in the News Grandma s Curse Remember Lamarckism Organisms can pass on characteristics acquired throughout a lifetime to their offspring 0 Example giraffes stretched to reach high branches 9 acquired long necks 9 acquired trait long neck was passed to offspring Epigenetics chemical modifications may be passed on to new generations In this article Smoking effects may be passed on from grandparent to grandchild The experiment 0 Pregnant rats injected with nicotine day 6 through day 21 birth of offspring I Nicotine acquired characteristic 0 Among other symptoms F1 pups had asthmatic lungs 0 F1 pups also used in breeding where NOT exposed to nicotine 0 F2 pups progeny of F1 generation had similar smokingrelated effects as F1 asthmatic lungs Results 0 Saw a nicotineinduced increase in histone H3 acetylation in the lung 0 A molecule called RGZ protects lungs against the effects of nicotine 0 Treatment with RGZ blocked histone H3 acetylation o Hypothesized that H3 acetylation caused asthmatic lungs Conclusion hypothesized that H3 acetylation passed on to offspring 9 asthmatic lungs HigherOrder Remodeling of Chromatin Once nucleosomes formed 0 Histones H1 binds to linker DNA 0 Brings neighboring nucleosomes closer Regulated by phosphorylation Phosphorylated histone H1 peaks in early mitosis 0 When chromosomes begin to condense Next level of compaction 30 nm fiber 0 Stabilized by histone aminoterminal tails I Nterminal tails that stick out of the loops Several additional levels of chromosome packaging occur but do not involve histones HistoneDNA Interactions Many DNAbinding proteins prefer binding to histonefree DNA 0 How do these proteins gain access to histonefree DNA I HistoneDNA association is not permanent I Any region of DNA may be transiently released from interaction with histone octamer 0 Proposed Model I DNA unwraps from the nucleosome rather than briefly coming off the nucleosome o What about the H1 t0 linkage DNA complex Or the 30 nm bers I Protein binding sites become accessible Nucleosomeremodeling complexes Multiprotein complexes that promote changes in nucleosome location or DNA interaction Use ATP hydrolysis Facilitate three types of changes 0 1 Sliding Nucleosome slides along DNA 0 2 Ejection Nucleosome ejected from DNA creating larger nucleosomefree region 0 3 Dimer exchange H2AHZB dimer exchanged with unmodified or variant dimers I Happens at double stranded breaks I Requires energy Nucleosomes preferentially form on DNA that bends easily AzT rich region in the minor groove facing the histone octamer However such sequences are NOT required for nucleosome assembly HistoneDNA interaction is determined by charge not sequence Allows nucleosomes to form along the entire length of the chromosomes 912 Notes REPLICATION I How is DNA Replicated During S phase of eukaryotic cell cycle chromosomes decondense o Replication enzymes have direct access to entire genome Watson and Crick determined that DNA is a double helix 0 Two polynucleotide strands held together by hydrogen bonds twisted into a helical structure Proposed that during DNA replication the chains of double helix would separate to provide templates for making a complementary strand of DNA Three Possible Mechanisms for DNA Replication Dispersive model favored model parental strands broken into double stranded fragments and used as templates Semiconservative model each parental strand serves as a template Conservative model parental strands remain together The MeselsonStahl Experiment Goal determine mechanism for DNA replication Overview grow bacteria containing one of two different isotopes of nitrogen heavy or light 0 Why nitrogen each of the four bases contains nitrogen 0 Allow bacteria to grow and replicate 0 Separate DNA by density 1 Grow bacteria in media containing nucleotide precursors labeled with 15N heavy isotope 0 Take sample 2 Transfer media containing nucleotide precursors labeled with 14N 0 Take sample one generation after transfer to 14N 3 Allow media to grow with 14N 0 Take sample gen2 after transfer to 14N 4 Place the three samples in an ultracentrifuge and pellet through a cesium chloride CsCl gradient that separates DNA by density 5 Observe location of DNA molecules Conclusion DNA replication 9 semi conservative Why Walk through predictions A 14Ni14N light DNA mm Less dense 15 14 hybrid DNA 1 r r a h5N 15EBNA mm leavy before transf r one cell two generations More dense to N generation after after transfer transfer to quotMN to 1 4N V Predictions C m frFliW Wife Semimm m f39ie Semiconservative model 9 hybrid light and dark band ParentalDMA because there is parental new strands in a DNA in first I I in generation Second generation 9 one light band because Fi t em tmquot NA there is a newnew strand 1 l l Ex EE Ledba ds E B a Dispersive model 9 hybrid light and dark band because there is parental new strands in a DNA in first Second generation generation Second generation 9 hybrid light and dark l i band because there is parental new strands in a DNA in Expmedbandg first generation Conservative model 9 light band and dark band because parental DNA is always conserved to replicate itself and stay together and never mix with new DNA vice versa DNA Synthesis 0 Origins of replication sequence specific positions where replication is initiated 0 Hundreds to thousands in eukaryotes one in bacterial chromosome PreReplication Complex preRC complex of several proteins that locally melt the double helix create replication bubbles origwrepncm parentlama Replication bubbles have two replication forks a o In future diagrams we will only consider one replication fork 0 DNA synthesis occurs in both directions 9 0 During S phase replication bubbles will grow phase and come into contact a Two daughter DNA molecules The Replication Fork 0 Numerous events occur at or near the replication for including but not limted to 0 DNA unwound by DNA helicases 0 Primers synthesized by primases o Singlestranded DNA bound by DNA polymerase o Replication occurs in the 5 to 3 direction 0 Leading strand strand that is being synthesized in one long continous stretch I Continous because it adds DNA at the 3 end 0 Lagging strand strand that is synthesized in several short bursts I As DNA is unwound the 5 region is available but there needs to be a long enough stretch available for DNA polymerase to replicate DNA in the 5 to 3 direction I Okazaki fragment short segments made on the lagging strand Hexameric ringshaped protein 3r I l r Enci rclesssDNA 5r 1 v 39 ti ATP hydrolysis 339E Jf j39ng hwgi j MWWI ulj 3 KW NEE pyrocessivewunwinds DNA 0 Processively more than one strand at a time is niL calmquot unwound 539lile 11mmimi fl 13 suCCH 39 Topoisomerase Relieves Structural Tension 39 39 rree Elication machiiner P y Unwinding of dsDNA double stranded DNA puts strains on unwound DNA replication 93936 Positive supercoils accumulate in front of the replication fork g V 7 l Can stall replication V l Coiling the DNA in a right handed helix that makes it l quot tighter topoisomerase II break DNA Topoisomerase protein that acts ahead of the replication fork and breaks one or both strands of DNA Topoisomerase Holds on to DNA Passes strands through the break p355 DNA Puts ends back together through the break negative supercoill Result negatlve superco s natural conformat1on of DNA L mmmmot oowwmu Left handed hehx tw1st 18 normal SingleStranded Binding Proteins Prevent Premature Annealing o Helicase unwinds dsDNA double stranded DNA creating ssDNA single stranded DNA 0 The two DNA strands could prematurely pair undoing helicase activity 0 ssDNAbinding ssBs o Bind newly separated strands as soon as helicase unzips the DNA o Keeps ssDNA from becoming dsDNA again PrimerTemplate Junction 0 Primase binds ssDNA adds short 10 bp primer 0 Primer can be DNA or RNA will be removed at end of replication complementary to but shorter than the template 0 PrimerTemplate Junction composed of template strand and primer 0 Template strand Provides template for DNA synthesis b annealed primer I W V t n I V g g I fgrowing end of DNA 39 1 template strand cJEGTEGNELM EDNA ssDNA 0 Why is a primer necessary 0 DNA polymerase can t simply bind the template strand and begin replication 0 It requires a starting point for replication primerztemplate junction DNA Polymerase o Resembles hand 3 domains 0 Palm 0 Fingers 0 Thumb 0 Palm catalyzes DNA synthesis and monitors base pairing 0 Where nucleotides are added 0 Ensures that the correct nucleotide is added at the end of the template strand 0 Fingers bends template strand positions next base to be paired 0 Points the bases away from the catalytic side of the palm so a nucleotide isn t added where it should be 0 Positions the next base that needs to be paired so that the incoming nucleotide can be placed in the next position 0 Thumb maintains association between DNA polymerase and DNA DNA Synthesis 0 Deoxynucleoside triphosphate dNTP dATP dCTP dCTP dTTP 0 Individual dNTPs are added to the growing dNA strand 0 Template strand determines which dNTP is added 0 Notice there are three phosphate groups alpha or beta and gamma y o Phosphodiester bond formation requires removal of p and y 0 Template strand determines which dNTP is added 7 3quot 0H 0f Primer 0 Phosphodiester bond between 3 OH and attacks rotphosphate of imcommngTP alpha Of the dNTP pr H118 r 539 3 and vphosphates removed p 7cm Phos hodiesterbond forms e e 3 P 915 Notes LECTURE NINE REPLICATION 11 DNA Polymerase is Processive Processive adds many dNTPs before falling off template strand 1 Dwipmg sem l dNTP is a nucleotide quotquot 3FL W 39 39 0 Though still adds dNTPs one at a time can add 1000 dNTPssec quot 539 o Replication is very fast H quot1H u 9391 l I iii quot Il I g 5quot I i 1 fast l 39 5quot quot Lmf39l quota 5 Jud 339Q39 V39L I 4w rlql 1 mmz I quotFu39 ll lil39 lll In 14 rmc T J 1 DNA polymerase releases I quot quott i f quot I V a 7 u it 7 li lllii 17L mm 5 LIL I quot 1 quotr quot i 39 3 J HHHHHIHHIJIW NIH lithls Iquot DNA Synthesis Overview 0 Template strand determines which dNTP is added 3 OH primer attacks or phosphate of incoming dNTP o B and yphosphates removed 0 Phosphodiester bond forms DNA Polymerase Uses an Exonuclease to Proofread 0 Errors do occur proofreading exonuclease removes improperly basepaired dNTPs 0 Slow or No DNA Synthesis 0 If an incorrect nucleotide is added I Alters positioning of the 3 OH I Reduces the rate of DNA synthesis 0 Because the end is no longer in the proper orientation in the active site 0 Removal of mismatched nucleotide o Primerztemplate junction destabilized I Becomes singlestranded gt no high affinity to the palm o Singlestranded is a high affinity for exonuclease active site 0 Moves to exonuclease active site incorrect nucleotide removed 0 Only removes the most recent errors have to be at the end of the growing strand 0 Resume DNA synthesis 0 Primerztemplate junction reforms I Once the incorrect nucleotide has been removed 0 Returns to polymerase active site 0 DNA synthesis resumes The Sliding Clamp Increases DNA Polymerase Processivity o Sliding clamp protein 0 Assembly of multiple subunits o Surrounds DNA like a donut I Encircles DNA Binds DNA polymerase Keeps DNA polymerase from diffusing away from DNA I Interaction between sliding clamp and DNA polymerase is stronger than the interaction between DNA polymerase and DNA 0 Keeps both sliding clamp and DNA polymerase together and DNA polymerase and DNA together 0 Without a sliding clamp DNA polymerase adds 1015 bases before falling off the template strand 0 With a sliding clamp Can add thousands of bases at a time o How Sliding Clamp increases DNA Polymerase ProcessiVity o EBRPRBR o 1 Sliding clamp encircles DNA binds DNA polymerase o 2 DNA polymerase releases from 3 end of primertemplate junction 0 3 Sliding clamp prevents DNA polymerase from diffusing away I Due to its affinity to the sliding clamp I Ensures that the polymerase stays at the same nucleotide o 4 DNA polymerase rebinds continues DNA synthesis primer to primer 0 5 In the absence of a primertemplate junction DNA polymerase releases I When it gets doublestranded DNA in its active site 9 lose affinity for sliding clamp Primer Synthesis in Eukaryotes o Primase o Synthesizes primer using the template strand as a primer then falls off 0 DOES NOT interact with sliding clamp 9 low processiVity o Primase does not require the primertemplate junction 0 Note that DNA polymerase will extend DNA from one primer to the next The Sliding Clamp Loader Loads Sliding Clamps BOPRC binds opens places releases Closes a g Sliding clamp loader ATP bound clamp loader binds and opens sliding clamp Places sliding clamp at primertemplate junction ATP hydrolysis releases clamp loader from DNA and sliding clamp DNA polymerase can come in and start synthesizing DNA Sliding Clamp spontaneously closes around DNA Synthesizing Leading and Lagging Strands Leading and lagging strands synthesized simultaneously at the replication fork DNA polymerase reads template 3 to 5 synthesizes new strands 5 to 3 0 Difference in reading is due to antiparallel of DNA Leading Strand Lagging Strand Made in the same direction that the replication fork moves Made in short discontinuous fragments Made in long continuous stretches Also known as Okazaki fragments Few primer sequences Many primer sequences How does DNA Polymerase know to jump ahead to start the next Okazaki fragments The E Coli Holoenzyme and Replisome Holoenzyme o Physically links 4 enzymes I 3 DNA polymerases 1 sliding clamp loader 0 DNA polymerases are oriented in opposite directions I 1 replicates leading strand 2 replicate lagging strand see Fig 923 de I Allows 5 3 synthesis to occur on antiparallel strands All of the proteins acting together at the replication fork form the replisome Replisome is similar in eukaryotes but less understood Finishing Replication Most of RNA primer removed by RNase H an endonuclease o Endonuclease removes nucleotides internal to DNA 0 RNase cleaves nucleotides within the primer but can t cleave the last nucleotide I Because the last nucleotide is RNA and the second nucleotide is DNA and RNase H can only go between two RNA molecules Last nucleotide ribonucleotide removed by 5 exonuclease o Exonuclease removes nucleotides from the end of the DNA Gap filled by DNA polymerase because it recognizes the primertemplate junction DNA ligase fills in the nick 0 Forms the final phosphodiester bond in the strand I Connects the nucleotides The End Replication Problem of Lagging Linear Chromosomes Telomeres ends of linear chromosomes 0 Standard repeated T and A rich sequences that is repeated at the end of chromosome RNase H removes primers Lagging strand DNA polymerase can t fill in the end of the chromosome needs a primer that was just removed 0 Shorter chromosome will result in the next round of replication I The short chromosome will be template which will only be shorter next time because DNA polymerase will still not be able to fill in the end of the chromosome on the lagging strand 0 The problem is repeated in new lagging strands 0 Problem is solved by telomerase Telomerase replicates the ends of linear chromosomes 0 Composed of protein and DNA Telomerase binds to lagging strand RNA component extends over the end and serves as a template Adds bases that are complementary to own RNA Repeats process Decreased telomerase activity has been implicated in the aging process Replication Enzymes and Disease Werner Syndrome Incidence 1 in 100000 Inheritance Pattern Autosomal Recessive Symptoms Early graying of hair Wrinkling and sagging of skin Decrease in muscle mass Premature loss of stature OOOO Cataracts Shorter life span death by 50 Most have a mutation in WRN a gene encoding a helicaselike protein WRN unwinds DNA but also involved in DNA repair 0 Loss of WRN results in accumulation of replication errors 0 Loss of WRN also implicated in accelerated telomere shortening 0 Loss of WRN is implicate in decreased telomerase 915 Notes LECTURE TEN POLYMERASE CHAIN RXN CHAIN SEQUENCING RESTRICTION ENDONUCLEASES Polymerase Chain Reaction PCR In vitro process modeled after cellular DNA replication 0 In vitro outside the cell Method for amplifying small quantities of DNA Three Basic Steps 0 1 Denaturing separating the two DNA stands I Breaking of hydrogen bonds between base pairs I Done through heating I No replication forks form 0 2 Annealing attaching primers to the DNA strand I At a cool temp for hydrogen bonds to form amongst base pair I 30 seconds 0 Only the primers to anneal not longer stretches of sstA o 3 Extension DNA polymerase extends the primer sequence in 5 9 3 direction I DNA synthesis occurs in continuous stretches o No leading or lagging strand 0 Due to no replication fork Cycle completion of all three basic steps 0 After each cycle the amount of DNA doubles Final amount of DNA 2X 0 X number of cycles Ingredients for PCR DNA template 0 Genomic or plasmid A set of oligonucleotide more than just one nucleotide primers 0 Synthetic I Do not require use of primase 0 Designed to amplify specific region DNA polymerase o Modified to I Withstand high reaction temperatures I Have a high degree of fidelity I Be processive in the absence of sliding clamp A solution containing dNTPs and a physiologically relevant salt mixture 0 Mimic conditions required when synthesizing DNA in the body PCR Replication Both Separation Use heat Use helicase Primers DNA primers RNA primers Processivity Clamp loader sliding No clamp clamp loaderclamp loader Primase No primase Yes primase Exonuclease No Yes to remove primase RNaseH No Yes primers stay on and are simply extend DNA is made 5 9 3 Both use DNA polymerase it is modified in PCR Enzymes No to topoisomerase single stranded binding proteins ssBPs Strand formation Both strands are made Leading and lagging continuously in strand stretches Agarose Gel Electrophoresis Method to analyze DNA fragments from PCR to restriction digests 0 Uses an electrical charge to spate fragm ents based on relative size 1 Agarose gel made contains ethidium bromide o Ethidium bromide inserts in between stacllting bases 2 Gel placed into a chamber filled with salt buffer 3 DNA mixed with heavy dye loaded into well 0 Dye helps dNA sink into the wells 4 Electric current applied to the gel DNA fragments migrate according to size smaller pieces migrate further 0 Why does DNA move toward the anode because DNA is negatively charged 5 Viewed by placing gel on an ultraviolet light emitting box 0 Large fragments at the top they get stuck in the agarose o Smaller fragments near the bottom 1 Power Source 2 Chamber filled with buffer 3 Wells loaded with DNA samples 4 Negative electrode cathode 5 Agarose gel 6 Positive electrode anode DNA Sequencing ChainTerminating Method This technique utilizes chain terminating nucleotides 0 Normal nucleotides 3 OH 0 Chain terminating nucleotides 3 H I AGTC can be made chainterminating Remember DNA synthesis occurs by joining the 3 OH of one nucleotide to one 5 phosphate of another Chainterminating reactions can t attack the 5 of another nucleotide 9 DNA synthesis stops Reaction ingredients do a separate reaction for each chainterminating phosphate 0 Single stranded DNA to be sequenced template strand 0 Primer anneals to template like in PCR 0 DNA polymerase o All four normal nucleotides I With 3 OH 0 One chainterminating nucleotide per reaction either A G T or C I With 3 H time Example Reaction with Chainterminating G 0 Sometimes normal G is added 0 Elongating chain will stop if chainterminating nucleotide is added 0 Generates fragments of various lengths which can be separated on gel Then repeat for all reactions using same template Each lane corresponds to a separate reaction containing the indicated chainterminating nucleotide Fragments separate on gel according to size Read fro top of the gel to the bottom of the gel to reduce sequence PCR and Sequencing Real World Applications Forensics 0 Identify human remains o Convict or exonerate individuals accused of committing crimes Study Extinct animals Medical Diagnostic Tests 0 Use PCR to amplify early stages of cancer or other diseases Restriction Endonucleases Enzymes that act like molecular scissors for DNA analysis Found in Bacteria and Archaea Sold commercially Example ECORI 0 First restriction enzyme discovered in e coli 0 Recognizes and cuts 5 GAATTC3 Used in research labs to cut DNA into smaller pieces Can separate pieces on an agarose gel Example Linear piece of DNA with 6 copies of GAATTC sequence 0 EcoRl cuts DNA into seven fragments o Agarose gel electrophoresis separates fragments based on size Uses for Restriction Endonucleases 0 Can be used for diagnostic tests 0 Example large pieces of DNA cut with restriction enzymes 0 Run on agarose gel 0 0 Compare restriction digest fragments banding pattern on gel Make rough judgments on degree of similarity between pieces of DNA 0 Used frequently in DNA cloning O O O Amplify region of DNA using PCR Digest with restriction endonucleases Glue products of restriction digests back together with DNA ligase I But sticky ends must be compatible digested by the same enzyme to be ligated 0 So that sticky ends can complimentary base pair 919 Notes LECTURE TEN POLYMERASE CHAIN RXN CHAIN SEQUENCING RESTRICTION ENDONUCLEASES Initiation of DNA Replication in E Coli Driven by proteinDNA and proteinprotein interactions 0 O O O 1 DnaAATP binds 9mer 9bp repeats at ori 2 DNA strands at 13mer repeats separate 3 DNA helicase DnaB and DNA helicase loader DnaC associate with the DnaAbound origin I Analogous to sliding clamp and sliding clamp loader 4 Helicase protein ring opens and is placed around the origin 5 Helicase recruits primase helicase loader and DnaA are released helicase is activated 6 Sliding clamp assemble on each primer DNA polymerase initiates synthesis on leading strand I Remember DNA is made 5 9 3 7 Sliding clamps load on lagging strands helicase must have opened up a significant amount of DNA for this to occur DNA polymerase initiates synthesis on lagging strand 8 Two replication forks have been assembled I Initiation of replication is complete Do they have single stranded binding proteins Growth and Replication Paradox in Bacteria For rapidly growing cells it takes as little as 20 minutes to divide But it takes at least 40 minutes to replicate the chromosome If a cell must have a chromosome how is it possible to divide faster than replication 0 Use multiple replication forks Multiple Replication Forks New rounds of replication begin before old round ends 0 Each black dot represents a replication fork PICTURE Replication is bi directional Many rounds of replication can occur at one time Each daughter cell receives an actively replicating chromosome after binary fission O Binary fission cell elongates 9 chromosome replicates 9 peptidoglycan invaginates 9 daughter cells Ensures entire genome is replicated before cell division Chromosome is actively replicated as it s segregated unlike eukaryotes O Eukaryotic chromosomes replicate only once and cells will not divide until chromosome is replicated
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