The Search for the Genetic Material of Life What is a gene? Stable source of information Ability to replicate accurately Capable of change The Search for the Molecular Basis of Heredity Search for genetic material---nucleic acid or protein/DNA or RNA?
Griffiths Transformation Experiment Averys Transformation Experiment Hershey-Chase Bacteriophage Experiment Tobacco Mosaic Virus (TMV) Experiment Nucleotides - composition and structure Double-helix model of DNA - Watson & Crick
Original Source for portions of slide content: http://mercury.bio.uaf.edu/~kevin_mccracken/genetics/lectures/chapter_02.ppt by Kevin McCracken University of Alaska Fairbanks. Timeline 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 Griffiths Transformation Experiment 1944 Averys Transformation Experiment 1953
Hershey-Chase Bacteriophage Experiment 1953 Watson & Crick propose double-helix model of DNA 1956 Gierer & Schramm/Fraenkel-Conrat & Singer Demonstrate RNA is viral genetic material. Frederick Griffiths Transformation Experiment - 1928 transforming principle demonstrated with Streptococcus pneumoniae
Griffith hypothesized that the transforming agent was a IIIS protein. Oswald T. Averys Transformation Experiment 1944 Determined that IIIS DNA was the genetic material responsible for Griffiths results (not RNA). Hershey-Chase Bacteriophage Experiment - 1953 Bacteriophage = Virus that attacks bacteria and replicates by invading a living cell and using the cells molecular machinery.
Structure of T2 phage DNA & protein Life cycle of virulent T2 phage: Hershey-Chase Bacteriophage Experiment - 1953 1. 2. T2 bacteriophage is composed of DNA and proteins: Set-up two replicates: Label DNA with 32P
Label Protein with 35S 3. Infected E. coli bacteria with two types of labeled T2 4. 32 P is discovered within the bacteria and progeny phages, whereas 35S is not found within the bacteria but released with phage ghosts.
1969: Alfred Hershey Gierer & Schramm Tobacco Mosaic Virus (TMV) Experiment 1956 & Fraenkel-Conrat & Singer - 1957 Used 2 viral strains to demonstrate RNA is the genetic material of TMV Conclusions about these early experiments: Griffith 1928 & Avery 1944: Hershey-Chase 1953:
DNA (not RNA) is transforming agent. DNA (not protein) is the genetic material. Gierer & Schramm 1956/Fraenkel-Conrat & Singer 1957: RNA (not protein) is genetic material of some viruses. Nucleotide = monomers that make up DNA and RNA (Figs. 2.9-10) Three components 1. Pentose (5-carbon) sugar DNA = deoxyribose RNA = ribose
(compare 2 carbons) 2. Nitrogenous base Purines Adenine Guanine Pyrimidines Cytosine Thymine (DNA) Uracil (RNA) 3. Phosphate group attached to 5 carbon Nucleotides are linked by phosphodiester bonds to form polynucleotides. Phosphodiester bond 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! 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. 5 end 3 end James D. Watson & Francis H. Crick - 1953 Double Helix Model of DNA Two sources of information: 1. Base composition studies of Erwin Chargaff
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 (Chargraffs rules) %GC content varies from organism to organism Examples: %A
%T %G %C %GC Homo sapiens Zea mays Drosophila Aythya americana 31.0 25.6 27.3 25.8
48.4 James D. Watson & Francis H. Crick - 1953 Double Helix Model of DNA Two sources of information: 2. X-ray diffraction studies - Rosalind Franklin & Maurice Wilkins Conclusion-DNA is a helical structure with distinctive regularities, 0.34 nm & 3.4 nm. Double Helix Model of DNA: Six main features 1. Two polynucleotide chains wound in a right-handed (clockwise) double-helix.
2. Nucleotide chains are anti-parallel: 3. Sugar-phosphate backbones are on the outside of the double helix, and the bases are oriented towards the central axis. 4. Complementary base pairs from opposite strands are bound together by weak hydrogen bonds. 5 3 3 5 A pairs with T (2 H-bonds), and G pairs with C (3 H-bonds).
e.g., 5-TATTCCGA-3 3-ATAAGGCT-3 5. Base pairs are 0.34 nm apart. One complete turn of the helix requires 3.4 nm (10 bases/turn). 6. Sugar-phosphate backbones are not equally-spaced, resulting in major and minor grooves. 1962: Nobel Prize in Physiology and Medicine James D.
Watson Francis H.Maurice H. F. Crick Wilkins What about? Rosalind Franklin RNA (A pairs with U and C pairs with G) Examples: mRNA tRNA rRNA snRNA messenger RNA transfer RNA
ribosomal RNA small nuclear RNA RNA secondary structure: Yeast Alanine tRNA single-stranded Function in transcription (RNA processing) and translation Organization of DNA/RNA in chromosomes Genome = chromosome or set of chromosomes that contains all the DNA an organism (or organelle) possesses Prokaryotic chromosomes 1. most contain one double-stranded circular
DNA molecule 2. typically arranged in arranged in a dense clump in a region called the nucleoid Eukaryotic chromosomes 1. Eukaryotic chromosome structure Chromatin - complex of DNA and chomosomal proteins ~ twice as much or more protein as DNA. 2. Eukaryotic chromosomes or chromatin found in the nucleus of the cell. 3. Cells from different species contain varying numbers of chromosome of different sizes and morphologies -the karyotype (e.g., pea, 2N = 14; human, 2N = 46, fruit fly, 2N= 8).
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