Review Search for genetic material---nucleic acid or protein/DNA or RNA? Griffith’s Transformation Experiment Avery’s Transformation Experiment Hershey-Chase Bacteriophage Experiment Tobacco Mosaic Virus (TMV) Experiment Nucleotides - composition and structure Double-helix model of DNA - Watson & Crick Today’s Lesson Organization of DNA/RNA in chromosomes

Search for the genetic material: Stable source of information Ability to replicate accurately Capable of change 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 Griffith’s Transformation Experiment 1944 Avery’s 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 Griffith’s Transformation Experiment - 1928 “transforming principle” demonstrated with Streptococcus pneumoniae Griffith hypothesized that the transforming agent was a “IIIS” protein.

Oswald T. Avery’s Transformation Experiment - 1944 Determined that “IIIS” DNA was the genetic material responsible for Griffith’s results (not RNA). Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Hershey-Chase Bacteriophage Experiment - 1953 Bacteriophage = Virus that attacks bacteria and replicates by invading a living cell and using the cell’s molecular machinery. Structure of T2 phage DNA & protein

Life cycle of virulent T2 phage:

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. 32P is discovered within the bacteria and progeny phages, whereas 35S is not found within the bacteria but released with phage ghosts. Hershey-Chase Bacteriophage Experiment - 1953 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: 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.

Nucleotide = monomers that make up DNA and RNA 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. 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: 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 (Chargraff’s rules) %GC content varies from organism to organism Examples: %A %T %G %C %GC Homo sapiens 31.0 31.5 19.1 18.4 37.5 Zea mays 25.6 25.3 24.5 24.6 49.1 Drosophila 27.3 27.6 22.5 22.5 45.0 Aythya americana 25.8 25.8 24.2 24.2 48.4

James D. Watson & Francis H. Crick - 1953 Double Helix Model of DNA Two sources of information: 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 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). e.g., 5’-TATTCCGA-3’ 3’-ATAAGGCT-3’ 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.

James D. Watson Francis H. Crick Maurice H. F. Wilkins What about? 1962: Nobel Prize in Physiology and Medicine James D. Watson Francis H. Crick Maurice H. F. Wilkins What about? Rosalind Franklin

Organization of DNA/RNA in chromosomes Genome = chromosome or set of chromosomes that contains all the DNA an organism (or organelle) possesses Viral chromosomes 1. single or double-stranded DNA or RNA 2. circular or linear 3. surrounded by proteins TMV T2 bacteriophage  bacteriophage Prokaryotic chromosomes 1. most contain one double-stranded circular DNA chromosome 2. others consist of one or more chromosomes and are either circular or linear 3. typically arranged in arranged in a dense clump in a region called the nucleoid

Problem: Measured linearly, the Escherichia coli genome (4.6 Mb) would be 1,000 times longer than the E. coli cell. The human genome (3.4 Gb) would be 2.3 m long if stretched linearly. Solutions: Supercoiling DNA double helix is twisted in space about its own axis, a process is controlled by topoisomerases (enzymes). (occurs in circular and linear DNA molecules) 2. Looped domains

More about genome size: C value = total amount of DNA in the haploid (1N) genome Varies widely from species to species and shows no relationship to structural or organizational complexity. Examples C value (bp)  48,502 T4 168,900 HIV-1 9,750 E. Coli 4,639,221 Lilium formosanum 36,000,000,000 Zea mays 5,000,000,000 Amoeba proteus 290,000,000,000 Drosophila melanogaster 180,000,000 Mus musculus 3,454,200,000 Canis familiaris 3,355,500,000 Equus caballus 3,311,000,000 Homo sapiens 3,400,000,000

Eukaryotic chromosome structure Chromatin complex of DNA and chomosomal proteins ~ twice as much protein as DNA Two major types of proteins: Histones abundant, basic proteins with a positive charge that bind to DNA (which is negatively charged) 5 main types: H1, H2A, H2B, H3, H4 ~equal in mass to DNA evolutionarily conserved Histones leave the DNA only transiently during DNA replication. They stay with the DNA during transcription. By changing shape and position, nucleosomes allow RNA-synthesizing polymerases to move along the DNA. Non-histones all the other proteins associated with DNA differ markedly in type and structure amounts vary widely

Packing of DNA into chromosomes: Level 1 Winding of DNA around histones to create a nucleosome structure. Level 2 Nucleosomes connected by strands of linker DNA like beads on a string. Level 3 Packaging of nucleosomes into 30-nm chromatin fiber. Level 4 Formation of looped domains.

More about different types 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.

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 (100-500 bp) LINEs long interspersed repeated sequences (>5,000 bp) Microsatellites short tandem repeats (e.g., TTA|TTA|TTA)