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Cell and Molecular Biology for Teachers

by: Marcella Lebsack

Cell and Molecular Biology for Teachers BIOL 5312

Marketplace > Texas Tech University > Biology > BIOL 5312 > Cell and Molecular Biology for Teachers
Marcella Lebsack
GPA 3.81


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This 35 page Class Notes was uploaded by Marcella Lebsack on Thursday October 22, 2015. The Class Notes belongs to BIOL 5312 at Texas Tech University taught by Densmore in Fall. Since its upload, it has received 23 views. For similar materials see /class/226495/biol-5312-texas-tech-university in Biology at Texas Tech University.


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Date Created: 10/22/15
quotmu are here Finally at least two critters that know how you folks feel being in this course Chapter 6 DNA as the genetic material Today it is taken for granted that DNA is genetic material but the development of the field of molecular biology was greatly impacted by the search for the source of heredity Although the Austrian surgeon Fredrich Miescher had discovered nuclein ie DNA in 1869 without the chromosome theory of inheritance to took almost 50 years before people ever even began to Seriously consider DNA as a critical component In 1910 Levene recognized that nucleoproteins as contained bases but was way farr off base with his tetranucleotide structure mamas Thymi i quotLi f Su r E39 4r Vulgar quot 3 1S ugar u cytosine Adenine The Transformation experiments Griffith et al the real beginning of understanding the what and wherefore of DNA Several sets of experiments Griffith 1 2 strains of bacteria 3 S strain kills mouse Encapsulated 8 strain DIS virulent Live mouse b R strain does not kill mouse 0 0 39 o Nonencapsulated R strain illFt nonviruient Live mouse rt WW inject mouse Encapsulated S strain EllS virulent recovered Dead mouse No bacteria recovered Griffith experiments 2 If the 8 strain is heat denatured c Heatkilled 5 strain does not kill mouse 39 V man I quot No bacteria We recovered w gt mouse w gt Heat Live encapsulated 8 strain gt Heatkilled Live mouse DI S virulent S strain Live mouse d Fl strain plus heatkilled S strain both of which are separately nonlethal kill mouse 69 695 gt 39 0 0 9 H o Live encapsulated gt Heatkilled Encapsulated 8 strain DIS 8 strain 8 strain DIS virulent nonvirulent virulent recovered However if 8 strain is heat denatured and then mixed with live R strain Live Fl strain llFl nonvirulent Somehow there was a factor or principle in the S strain that even when heat killed could transform the R non leth strain into a virulent strain Thus Griffith began to call the unknown substance the transforming principle Preparation of transforming principle from S strain 5 LySiS CelHree Filtration gt extract gt Trfntform39m Centrifugation Erlncglet I precipitation 0m sraln Encapsulated S strain Addition of transforming principle to Ft strain 0 0 0 Growth 39 39 o o 39 o O o O o S transforming R strain Culture containing principle both 8 and Ft cells It took 15 years to fully resolve the problem Avery Macleod and McCarty partially purified the transforming principle showing that the principle at least included some DNA Analyzed using chromatography 3 Heal kill Lipids H olysacc arides Protein NA DNA w W W W w Transformation I Their evidence supported that for at least one type of organism DNA was the fundamental unit of the transforming principle A very important paper was Chargaff s recognition that DNA isolated from different species shared similar properties such as that the amount of the base A was always equal to the amount of T as was the amount of the base C equal to the amount of G Thus DNA could be the genetic material For most people proof came with the blendor experiments of Hershey and Chase DNA can be labeled with 3P Protein can be labeled with 358 Head Tail with fibers a Fig 65 Why Hershey and Chase could use a bacteriophage to distinguish whether DNA or a protein was really the genetic material Phage Empty phage B acterium N gt gt lnjects DNA DNA replicates Phages Adsorbs assembled to living bacterium Lysis I t lt U I Broken bacterium Fig 66 The life cycle of a bacteriophage fsss protein r Bl m e briefly Radioactivity 35S associated with phage ghosts Phage ghosts b a 32p DNA Blend 5 briefly C 3 gt gt Radioactivity 32P entered bacterial host and was transmitted J to progeny phage Fig 67 The famous Hershey Chase experiment that showed that DNA was really the inherited genetic material Fig 67 FrankelConradt and Singer 1957 show that RNA is the genetic material in some viruses Characteristics that any genetic material DNA is obviously the major one must have 1 DNA has the ability to store genetic information which can be expressed in the cell as needed 2 This information can be transmitted to daughter cells with minimal error This process requires complex enzymes and repair mechanisms 3 DNA possesses both physical and chemical stability so information present in duplicate is not lost over long periods of time years 4 DNA has the potential for heritable change without major loss of parental information In order to be valuable as a genetic storage information molecule genomes with the more highly reactive less stable RNA must compensate in some ways In most RNA viruses the nucleic acid is completely protected by a protein coat Often the RNA is converted to DNA so that it can insert into the host s genome ie the retroviruses RNA is generally considered to be representative of the first genetic information molecule Chapter 7 DNA replication in virtually all organisms this is one of the most complex processes that is performed and in most cases is dependent upon more polypeptides that must interact together in humans gt23 in a complex unit than any other major pathway Replica Why DNA replication is so complex Must unwind helix and energy is normally required SsDNA tends to form intrastrand base pairs that can potentially halt the process ie cruciform structures A single enzyme can realistically only catalyze a limited number of different types of reactions replication complexes are typically one of the most complex enzymatic associations that exist in any organism Safeguards have evolved to recognize and prevent errors and to repair most of those that do occur Circularity in prokaryotes and largely linear molecules in eukaryotes along with great ranges in size of organisms genomes 50000 gt10 x 109 bp add great physical constraints on replication systems No single mode of replication is absolutely shared by all organisms Replication is always a doublestranded event Some important proteins that are required in prokaryotes Single stranded binding protein when strands separate this comes in SSB DNA Pol l major repair enzyme in prokaryotes DNA ligase ligates sews together DNA pieces into one large piece DnaB actually the helicase activity that unwinds the double stranded parental DNA also important in initiation Primase synthesizes the RNA primer that is needed for replication to Start DNA Pol III the prokaryotic replicase at most replication fork it is present in two copies Topoisomerases Top helps relax supercoiled DNA to help start replication DNA gyrase Top ll reintroduces supercoiling to compensate for helicase unwinding Topoisomerases as we learned enzymes that relax or introduce supercoils into DNA Enzyme has phophotyrosine residue that recognizes site and cleaves DNA k Fig 75 Mechanism of action for Topoisomerase type enzyme type II enzymes make cuts in both strands New strand A Primer De novo Covalent extension initiation initiation Fig 76 Two types of initiation of DNA replication in prokaryotes complex Initial complex AT rich region 13 bp repeats Replication V Also needed SS binding protein Replicase RNA primer etc Supercoiled template Fig 77 De novo initiation at oriC in E coli the paradigm 3 OH on leading strand 3 OH 5 P l Concatamer lt consists of serially repeated units 39 5 P 3 OH Fig 79 Initiation by covalent extension leading to sigma 039 replication COMPARISON OF Pill I AND POL lll Activity pol l pol Ill 5 P gt 3 OH polymerization Replication 3 OH gt 5 P polymerization 5 P gt 3 OH exonuclease Repair function 3 OH a 539P exonuclease Repair function Reaction rates 3000 basesmin 50000 basesmin There are two major prokaryotic enzymatic replication activities Pol a repair enzyme of one subunit and Pol III the primary replicase having multiple subunits Notice the differences in their activities Demonstration of how antiparallel strands caused problems in understanding what at the replication fork occurred at the replication fork If overall growth is in both the 5 gt 3 direction and the 3 gt 5 direction then there must be 2 polymerases Replication fork Parental DNA 5 N 3 Overall growth of daughter writs 3 Fig 715 Only a single type of synthesis could ever be isolated however always in the 5 gt 3 direction 3 5 nunus O n 3 OH Lagging strand wding strand 5 P Fig 716 Growing replication fork showing both leading and lagging strand synthesis Newly synthesized daughter strands W Pol III Fig 710 General events at the replication fork in a replication what most bacteria have see below Total DNA Figure 826 a The type of data obtained by alkaline sedimentation of pulselabeled DNA black and pulsechased DNA red Total DNA is the sum of the nonradioactive and radioactive DNA as might be indicated by the optical absorbence The s value of the sedimenting material increases from right to left b The location of radioactive DNA red at the time of pulse labeling and after a chase The radioactive molecules present in alkali are shown The fragmentation resulting from removal of uracil see later in text accounts for the fact that all pulse Iabeled DNA has a low 3 before the chase Radioactivity Tube F a bottom Direction of sedimentation How we know about what happens in Fig 710 5 3 J denatures 3 Pulse 3 39 and 5 5 Alkali Remove U 5 3 PUISe 3 J V 5 chase 5 3 and Alkali 5 b um How Okazaki made his landmark discovery of lagging strand synthesis Lagging strand What we think a replication fork actually is like RNA primer Earlier Okazaki ediled out by fragment pol I and gap sealed by ligase synthesized RNA primer Previously synthesized primase FlNA primer Primosome helicase I 5v DNA gyrase RNA primer edited oul 1 Leading ws slrand 3 Figure 722 Summary of events al the DNA replication fO k Dna A protein Primase binding ori Helicase Off g l W l m39 I Helicase I I Primase Fig 724 Many replication forks are really bidirectional this allows for even faster replication this is why most bacterial forks are called 6 Replication in Eukaryotes It is much more complex part of the problem is the length of the chromatin structure of the DNA requiring more sites of initiation part of the problem is the number of subunits le different proteins involved and part of the problem is that we do not believe that all of the replication occurs at a fork that moves some replication forks appear to be stationary permanently attached to certain regions of the inside of the nucleus this effectively means that some replication is done at factories in eukaryotes Some additional slides that might allow you to better understand and therefore explain replication to your biology classes The best current model for replication leading strand DNA polymerase T VDNA nelicase u i 4 39 39 lagging strand DNA polymerase Cnpyiigni 2004 Pearson Education inc publishing as Benjamin Cummings b H ow it begins lagging strand Pol completing previous Okazaki fragment gt wmonm n 1 H 39xs39 39 DNAprimase isreleased completion 0 Okazaki fragmem Copyright 2004 Pearson Eduumon Inn publishing as Benjamin Cummings DNA pmymerase synthesizes new Okazaki agmen 3 5 Copyright 2004 Pearson Educanon Inc publishing as Benjamin Cummings The slide involving arabinose should say that the levels of arabinose influence the pathway that synthesizes the proteins that are involved in its utilization And by the way I apologize I got confused on the time and actually thought that it was 850 pm not 750 pm so I let you guys about an hour early I am sorry to have cheated you tonight but I guess it is ok We will just finish chapter 11 the week after the exam November 12 Good luck studying and I will see you next week Lou


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