PCB3063 Exam 3 Chapter 9 9 1 Genetic Material Must Exhibit Four Characteristics Replication Storage of Information Expression of Information Variation by Mutation Expression of the information is the basis of information flow through the cell Transcription mRNA tRNA and rRNA are synthesized mRNA is translated to proteins Translation mRNA directs construction of polypeptide chain of amino acids which then folds into a protein Central dogma of genetics DNA makes RNA makes Proteins Genetic material is source of variation through mutation 9 2 Until 1944 Proteins Were Favored as Genetic Material Many geneticists favored proteins as role of genetic material because more was known about them and they were diverse 1868 DNA first studied by Friedrich Miescher isolated nuclei and derived what he called nuclein contained DNA DNA seemed to lack necessary chemical diversity Tetranucleotide hypothesis Levene observed incorrectly that DNA contained four repeating molecules in equal proportions called nucleotides In late 1940s Chargaff showed that Levene was incorrect when he demonstrated that organisms to not contain equal proportions of the four nucleotides 9 3 Evidence Favoring DNA as Genetic Material Avery MacLeod and McCarty published the chemical nature of a transforming principle in bacteria that led to DNA being accepted Transformation Studies Virulent strains cause disease have polysaccharide capsule o Form smooth colonies S Avirulent Strains Do not cause pneumonia do not have polysaccharide heads are destroyed by phagocytic cells in immune system o Form rough colonies R Serotypes the types of each strain of Diplococcus pneumonia Griffith used serotypes IIR avirulent and IIIS virulent Only living virulent cells produce pneumonia in mice Injected living mice with living IIR and heat killed IIIS strains neither cell type killed the mice alone but when injected together the mice died after 5 days o Blood analysis showed living IIIS bacteria Control mice injected with IIR bacteria did not develop pneumonia proved that IIR did not mutate but interacted with IIIS PCB3063 Exam 3 Transformation Griffith called transformation from IIR to IIIS transformation and suggested that the transforming principle may be some part of polysaccharide capsule 1931 Henry Dawson showed that transformation could occur in vitro injection into mice was not necessary for change to occur 1933 Alloway filtrate from heat killed cells was as effective in inducing transformation as were the intact cells 1944 Avery MacLeod and McCarty published their work classic paper in field of genetics and declared they had obtained transforming principle in highly purified state and that there was no doubt that DNA was responsible o IIIS were centrifuged collected and heat killed o Derived soluble filtrate that was able to induce transformation in IIR cells o Filtrate treated with protein digesting enzyme protease and RNA digesting enzyme ribonuclease o This destroyed activity of proteins and RNA proved neither were o Then they used deoxyribonuclease transformation did not occur proved it responsible was DNA The evidence presented supports the belief that a nucleic acid of the deoxyribose type is the fundamental unit of the transforming principle oof Pneumococcus type III The Hershey Chase Experiment E coli and bacteriophage T2 Phage virus consists of a protein coat surrounding a core of DNA hexagonal head and tail Published results to clarify events leading to phage reproduction Hershey and Chase knew that o T2 were 50 protein and 50 DNA o Infection initated by adsorption of phage by its tail into the bacterial cell o Production of new viruses happens in bacterial cell Used radioisotopes to track molecular components of phage to see which part directed viral reproduction 32P labels DNA not protein because protein doesn t have sulfur and 35S labels protein because proteins do not have phosphorus this is important E coli grown in either 32P or 35S phage will have either labeled DNA or labeled protein coat Most of 32P labeled DNA had transferred to bacterial cell following adsorption almost all of 35S remained outside cell Progeny contained 32P and NO 35S Showed that phage DNA enters cell and is responsible for phage reproduction Transfection Experiments destroying the bacterium 1957 if E coli is treated with lysozyme the outer wall can be removed without PCB3063 Exam 3 Protoplasts enzymatically treated cells that contain only membrane as outer boundary Spizizen and Fraser used protoplasts and were able to initiate phage reproduction with disrupted T2 particles virus did not have to be intact for infection to occur Transfection infection by only viral nuclei acid proves that phage DNA alone contains necessary information to produce mature viruses 9 4 Indirect and Direct Evidence Supports Concept that DNA is Genetic Material in Eukaryotes Indirect Evidence distribution Genetic material should be found where it functions Protein and DNA fit this criteria DNA not present in cytoplasm protein is DNA present in mitochondria and chloroplasts protein is not Comparisons of genetic material can be made for somatic cells and gametes Indirect Evidence Mutagenesis UV Light capable of inducing mutations When data are plotted an action spectrum is obtained and can be compared to absorption spectrum to be mutagenic Molecule serving as genetic material is expected to absorb at wavelength shown UV light most mutagenic at 260nm DNA and RNA absorb a lot at 260 protein absorbs a lot at 280nm Direct Evidence Recombinant DNA Studies Recombinant DNA technology segments of eukaryotic DNA corresponding to specific genes isolated and spliced into bacterial DNA Complex inserted into bacterial cell and gene expression is monitored Products of human genes specifying insulin and interferon are produced by bacteria that have had these genes spliced into them Transgenic animals Mintz demonstrated that DNA that codes for B globin protein is injected into mice and passed on to their offspring 9 5 RNA serves as the Genetic Material in Some Viruses Some viruses have RNA core instead of DNA core Tobacco Mosaic Virus TMV 1956 when purified RNA from TMV was spread on tobacco leaves the lesions associated with TMV appeared on the leaves showing that TMV RNA was genetic material 1955 1956 Pace and Spiegelman RNA from QB can be isolated in vitro RNA Replicase enzyme isolated from E coli cells following normal infection When RNA replicated in vitro is added to E coli
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