DNA Structure and Analysis Required Characteristics of Genetic Material replication Information storage Expression of information Variation by mutation Central dogma of molecular genetics Information is stored in DNA is transcribed into RNA tRNA mRNA and rRNA which are used to translate that information into proteins DNA makes RNA makes Proteins Friedrich Miescher 1844 1895 Isolated nuclein from nucleii of pus cells later na DNA but The many asymmetric carbon atoms of protein allow such a colossal number of stereoisomers that the richness and variety of hereditary transmission may find their expression in it Evidence favoring DNA as Genetic Material 1927 Frederick Griffith found that heat killed virulent strains of Diplococcus pneumoniae could transform living avirulent strains and restore pathogenicity Strains of Diplococcus pneumoniae used by Griffith in his Original Transformation Experiments Serotype IIR IIIS Colony Morphology Rough Smooth Capsule Absent Present Virulence Avirulent Virulent Figure 9 2 dies lives Smooth colony IIIS living IIIS virulent were injected into the mouse the mouse Rough colony IIR living IIR avirulent were injected into the mouse the mouse heat killed IIIS is injected into the mouse mouse lives living IIR and heat killed IIIS are injected into the mouse mouse dies and the analization reveals that living IIIS is recovered Evidence favoring DNA as genetic material 1944 Avery McCarthy and McLeod performed genetic transformation studies in Diplococcus pneumoniae An extension of Griffiths experiments Virulent strains from bacterial colonies that are smooth S in appearance whereas avirulent strains appear rough Found that DNA alone from S strains could change R strains to smooth figure Concluded that A DNA is the fundamental unit of the transforming principle of 9 2 Pneumococcus type III 1952 Hershey and Chase found that DNA of bacteriophage is responsible for directing reproduction of the virus during replication in E coli Figure 9 3a Avery MacLeod McCarty DNA transforming principle IIIS cells in liquid culture medium put in centrifuge IIIS cells spun to bottom of test tube heat kill homogenize cells recover IIIS filtrate extract carbohydrates lipids and proteins treat with protease treat with ribonuclease threat with deoxyribonuclease results IIR IIIS filtrate transformation occurs IIR cells protease treated IIIS filtrate transformation occurs IIR RNase treated IIIS filtrate transformation occurs IIR cells DNase treated IIIS filtrate NO transformation occurs Figure 9 3b IIR cells DNase treated IIIS filtrate no transformation Only IIR cells Conclusion Active factor is DNA IIR RNase treated IIIS filtrate transformation occurs IIR cells IIIS cells Conclusion Active factor is NOT RNA IIR cells protease treated IIIS filtrate transformation occurs IIR cells IIIS cells Conclusion Active factor is NOT protein IIR IIIS filtrate transformation occurs IIR cells IIIS cells Control IIIS contains active factor Bacteriophage attach to surface of a bacterial cell but do not enter what does attachment of phage tail fibers to bacteria wall phage genetic materil is injected into the bacterium phage reproductive cycle begins components accumulate assembly of mature phages occurs cell lysis occurs and new phages released Figure 9 5a Set up of Hershey Chase experiment Phated are about 50 DNA and 50 protein The differential labeling of DNA with P32 and protein with S35 was the key to the Only 32P labeled material was injected into the host so DNA not protein was the design of this experiment genetic material 9 4 Indirect evidence that DNA is the genetic material 1 DNA is found in the nucleus where genetic material needs to function also is protein But protein is also in the cytoplasm whereas DNA is NOT 2 The DNA content of haploid cells is half that of diploid cells 3 The action spectrum of UV light in damaging genetic material correlates with the absorption spectrum of DNA 4 Recombinant DNA technology proves traits can be transferred from organism to organism by DNA alone Some viruses use RNA as the genetic material Although the use of DNA is near universal there are some viruses that use RNA as the genetic material ex the QB virus that infects bacteria it uses an RNA replicase to make new copies of RNA inside the host cell Retroviruses have RNA as the genetic storage material but go through a DNA stage when replicating this is carried out by an enzyme known as reverse transcriptase Structural analysis of DNA 1953 proposal of double helix structure of DNA by Jim Watson and Francis Crick revealed a structure with required properties of genetic material Know basic chemical structures of RNA and DNA Do not need to draw but be able to recognize bases and what 5 and 3 ends are and phosphodiester bonds Erwin Chargaff base composition data 1949 1953 amount of A residues is proportional to about of T residues and amount of G residues is proportional to C Sum of purines A G sum of pyrimidines C T of G C does not necessarily equal of A T 9 7 difference between ribose and deoxyribose ribose has OH deoxyribose has H Watson Crick Model of DNA two long polynucleotide chains coiled around a central axis forming a right handed double helix two chains are antiparallel C 5 to C 3 orientations run in opposite directions bases of both chains are flat perpendicular to axis stacked 0 34 nm Nitrogenous bases paired via hydrogen bonds with only A T and G C pairs permitted complementarity One complete turn of the helix every 3 4 nm 10 bases turn Major and minor grooves apparent along DNA axis Helix is 2 nm in diameter RNA is similar to DNA but single stranded RNA has ribose instead of deoxyribose and uracil instead of thymine Although most RNA is single stranded some RNA folds back on itself to make small regions of double stranded RNA antiparallel There are three major classes of RNA 1 ribosomal RNA rRNA 2 messenger RNA mRNA 3 transfer RNA tRNA Several other classes of RNA small nuclear RNA snRNA microRNA miRNA and short interfering RNA siRNA Separation of DNA molecules by Electrophoresis DNA is negatively charged so when it is placed in an electrical field is will move Small DNA molecules will move faster through a gel than large one This is an important technique known as DNA gel electrophoresis DNA samples are loaded into depressions wells in the gel and immersed in a buffer An electric field is applied and the negatively charged DNA moves towards the positively charged DNA moves toward the