Presentation on theme: "Chapter 2 - DNA: The Genetic Material"— Presentation transcript:
1 Chapter 2 - DNA: The Genetic Material Search for genetic material---is it composed of nucleic acid or protein/DNA or RNA?Griffith’s Transformation ExperimentAvery’s Transformation ExperimentHershey-Chase Bacteriophage ExperimentTobacco Mosaic Virus (TMV) ExperimentNucleotides - composition and structureDouble-helix model of DNA - Watson & CrickOrganization of DNA/RNA in chromosomesProkaryotesEukaryotes
2 Search for the genetic material: Stable source of informationAbility to replicate accuratelyCapable of changeTimeline of events:1890 Weismann - substance in the cell nuclei controls development.1900 Chromosomes shown to contain hereditary information, later shown to be composed of protein & nucleic acids.1928 Griffith’s Transformation Experiment (incorrectly guessed protein!)1944 Avery’s Transformation Experiment (DNA not RNA)1953 Hershey-Chase Bacteriophage Experiment (DNA not protein)1953 Watson & Crick propose double-helix model of DNA1956 First demonstration that RNA is viral genetic material.
3 Fig. 2.2: Frederick Griffith’s Transformation Experiment - 1928 “transforming principle” demonstrated with Streptococcus pneumoniaeGriffith hypothesized that the transforming agent was a “IIIS” protein. But this was only a guess, and Griffith turned out to be wrong.
5 Hershey-Chase Bacteriophage Experiment - 1953 Bacteriophage = Virus that attacks bacteria and replicates by invading a living cell and using the cell’s molecular machinery.Fig. 2.4Structure of T2 phageBacteriophagesare composed ofDNA & protein
7 T2 bacteriophage is composed of DNA and proteins: Set-up two replicates:Label DNA with 32PLabel Protein with 35S3. Infected E. coli bacteria with two types of labeled T24. 32P is discovered within the bacteria and progeny phages, whereas 35S is not found within the bacteria but released with phage ghosts.Fig. 2.6: Hershey-Chase Bacteriophage ExperimentAlfred Hershey
8 Gierer & Schramm Tobacco Mosaic Virus (TMV) Experiment - 1956 Fig. 2.7 (2nd edition)Gierer & Schramm Tobacco Mosaic Virus (TMV) ExperimentFraenkel-Conrat & SingerDemonstrated that RNA is the genetic material of TMV.Numerous viruses uses RNA as the genetic material. But no known prokaryotes or eukaryotes have RNA as their genetic material.
9 Conclusions about these early experiments: Griffith 1928 & Avery 1944: DNA (not RNA) is transforming agent.Hershey-Chase 1953:DNA (not protein) is the genetic material.Gierer & Schramm 1956/Fraenkel-Conrat & Singer 1957:RNA (not protein) is genetic material of some viruses, but no known prokaryotes or eukaryotes use RNA as their genetic material.Alfred HersheyNobel Prize in Physiology or Medicine1969
10 Nucleotide = monomers that make up DNA and RNA (Figs. 2.8) Three components1. Pentose (5-carbon) sugarDNA = deoxyriboseRNA = ribose(compare 2’ carbons)2. Nitrogenous basePurines (2 rings)AdenineGuaninePyrimidines (1 ring)CytosineThymine (DNA)Uracil (RNA)3. Phosphate group attached to 5’ carbon
11 Nucleotides are linked by phosphodiester bonds to form polynucleotides. Covalent bond between the phosphate group (attached to 5’ carbon) of one nucleotide and the 3’ carbon of the sugar of another nucleotide.This bond is very strong, and for this reason DNA is remarkably stable. DNA can be boiled and even autoclaved without degrading!No kidding, you can autoclave a mouse and get good PCR!5’ and 3’The ends of the DNA or RNA chain are not the same. One end of the chain has a 5’ carbon and the other end has a 3’ carbon.
13 Structure of DNAJames D. Watson/Francis H. Crick 1953 proposed the Double Helix Model based on two sources of information:Base composition studies of Erwin Chargaff (Chargaff’s Rules)indicated double-stranded DNA consists of ~50% purines (A,G) and ~50% pyrimidines (T, C)amount of A = amount of T and amount of G = amount of C%GC content varies from organism to organismExamples: %A %T %G %C %GCHomo sapiensZea maysDrosophilaAythya americana
14 Structure of DNAJames D. Watson/Francis H. Crick 1953 proposed the Double Helix Model based on two sources of information:X-ray diffraction studies by Rosalind Franklin & Maurice WilkinsConclusion-DNA is a helical structure withdistinctive regularities, 0.34 nm & 3.4 nm.Fig. 2.11
15 Double Helix Model of DNA: Six main features Two polynucleotide chains wound in a right-handed (clockwise) double-helix.Nucleotide chains are anti-parallel: 5’ 3’3’ 5’Sugar-phosphate backbones are on the outside of the double helix, and the bases are oriented towards the central axis.Complementary base pairs from opposite strands are bound together by weak hydrogen bonds.A pairs with T (2 H-bonds), and G pairs with C (3 H-bonds).5’-TATTCCGA-3’3’-ATAAGGCT-5’Base pairs are 0.34 nm apart. One complete turn of the helix requires 3.4 nm (10 bases/turn).Sugar-phosphate backbones are not equally-spaced, resulting in major and minor grooves.
17 Fig. 2.12 Type B-DNA Other DNA forms include: A-DNA: Right-handed double helix with 11 bases per turn; shorter and wider at 2.2 nm diameter. Exists in some DNA-protein complexes.Z-DNA:Left-handed double helix with 12 bases per turn; longer and thinner at 1.8 nm diameter.
18 Type A, B, and Z conformations of DNA Fig. 2.14
20 James D. Watson Francis H. Crick Maurice H. F. Wilkins What about? 1962: Nobel Prize in Physiology and MedicineJames D.WatsonFrancis H.CrickMaurice H. F.WilkinsWhat about?Rosalind Franklin
21 RNA possesses uracil (U) not thymine (T) A pairs with U and C pairs with GExamples:mRNA messenger RNAtRNA transfer RNArRNA ribosomal RNAsnRNA small nuclear RNAmiRNA micro RNAsiRNA small interfering RNARNA secondary structure:single-strandedFunction in transcription(RNA processing) and translationYeast Alanine tRNA
22 Organization of DNA/RNA in chromosomes Genome = chromosome or set of chromosomes that contains all theDNA an organism (or organelle) possessesViral chromosomes 1. single or double-stranded DNA or RNA2. circular or linear3. surrounded by proteinsTMV T2 bacteriophage bacteriophageProkaryotic chromosomes1. most contain one double-stranded circularDNA chromosome2. others consist of one or more chromosomesand are either circular or linear3. typically arranged in arranged in a denseclump in a region called the nucleoid
23 Problem:Measured linearly, the Escherichia coli genome (4.6 Mb) would be 1,000times longer than the E. coli cell.The human genome (3.4 Gb) would be 2.3 m long if stretched linearly.Fig. 2.15Chromosome released from lysed E. coli cell.
24 Stop and Play the Video Prokaryotic chromosome structure---solutions: Supercoiling DNA double helix is twisted in space about itsown axis, a process is controlled bytopoisomerases (enzymes).(occurs in circular and linear DNA molecules)Stop and Play the VideoFig. 2.17
26 Eukaryotic chromosome structure Chromatin complex of DNA and chomosomal proteins~ twice as much protein as DNATwo major types of proteins:Histones abundant, basic proteins with a positive chargethat bind to DNA5 main types: H1, H2A, H2B, H3, H4~equal in mass to DNAevolutionarily conservedNon-histones all the other proteins associated with DNAdiffer markedly in type and structureamounts vary widely>> 100% DNA mass<< 50% DNA mass
27 Packing of DNA into chromosomes: Level 1 Winding of DNA around histones to create a nucleosome structure.Level 2 Nucleosomes connected bystrands of linker DNA likebeads on a string.Level 3 Packaging of nucleosomes into30-nm chromatin fiber.Level 4 Formation of looped domains.See Fig. 2.20
31 Fig Metaphase chromosome depleted of histones maintains its shape with a nonhistone protein scaffold.
32 Two other regions of DNA you should know about: Centromeric DNA (CEN) Center of chromosome, specialized sequences function with the microtubles and spindle apparatus during mitosis/meiosis.Telomeric DNA At extreme ends of the chromosome, maintain stability, and consist of tandem repeats. Play a role in DNA replication and stability of DNA.Fig. 2.25
33 Repeated DNA:Unique-sequence DNA Often referred to as single-copy and usually code for genes.Repetitive-sequence DNA May be interspersed or clustered and vary in size.SINEs short interspersed repeated sequences ( bp)LINEs long interspersed repeated sequences (>5,000 bp)Microsatellites short tandem repeats<---TTATTATTATTATTATTATTA--->
34 More about genome size: C value = total amount of DNA in the haploid (1N) genomeVaries widely from species to species and shows no simple relationship to structural or organizational complexity.Examples C value (bp) ,502T ,900HIV ,750E. Coli ,639,221Lilium formosanum 36,000,000,000Zea mays 5,000,000,000Amoeba proteus 290,000,000,000Drosophila melanogaster ,000,000Mus musculus 3,454,200,000Canis familiaris 3,355,500,000Equus caballus 3,311,000,000Homo sapiens 3,400,000,000
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