Chapter 2 - DNA: The Genetic Material Search for genetic material---is it composed of 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 Organization of DNA/RNA in chromosomes Prokaryotes Eukaryotes Genome size

Search for the genetic material: Stable source of information Ability to replicate accurately Capable of change Homunculus Pangenesis

Timeline of events: 1868 Darwin proposed a pangenesis hypothesis based on inheritance of tiny diffusive particles called gemmules. 1885 Walther Flemming - founder of cytogenetics, identifies structure known as chromatin. 1890 August Weismann - cell nuclei controls development; confirmed germ cells (sperm & egg) function in heredity. 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 DNA structure 1956 First demonstration that RNA is viral genetic material.

Some of Walther Flemming’s Illustrations 1882 & 1885 polytene chromosomes

Fig. 2.2: Frederick Griffith’s Transformation Experiment - 1928 “transforming principle” demonstrated with Streptococcus pneumoniae Griffith hypothesized that the transforming agent was a “IIIS” protein. But this was only a guess, and Griffith turned out to be wrong.

Oswald T. Avery’s Transformation Experiment - 1944 Fig. 2.3: 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. Fig. 2.4 Structure of T2 phage Bacteriophages are composed of DNA & protein

https://www.ucsf.edu/news/2015/10/131906/3-ways-viruses-have-changed-science-better

Fig. 2.5: 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. Fig. 2.6: Hershey-Chase Bacteriophage Experiment - 1953 Alfred Hershey

Gierer & Schramm Tobacco Mosaic Virus (TMV) Experiment - 1956 Fig. 2.7 (2nd edition) Gierer & Schramm Tobacco Mosaic Virus (TMV) Experiment - 1956 Fraenkel-Conrat & Singer - 1957 Demonstrated that RNA (not protein) 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.

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 Hershey Nobel Prize in Physiology or Medicine 1969

Nucleotide = monomers that make up DNA and RNA (Figs. 2.8) Three components 1. Pentose (5-carbon) sugar DNA = deoxyribose RNA = ribose (compare 2’ carbons) 2. Nitrogenous base Purines (2 rings) Adenine Guanine Pyrimidines (1 ring) 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.

Some people at the University of Chicago did this experiment Some people at the University of Chicago did this experiment. The DNA was still good---they could still isolate 20,000 base pairs.   

5’ end 3’ end Fig. 2.9

Structure of DNA James 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 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

Structure of DNA James 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 Wilkins Conclusion-DNA is a helical structure with distinctive regularities, 0.34 nm & 3.4 nm. Fig. 2.11

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.

Fig. 2.13

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.

Type A, B, and Z conformations of DNA Fig. 2.14

https://en.wikipedia.org/wiki/Nucleic_acid_double_helix

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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

RNA possesses uracil (U) not thymine (T) A pairs with U and C pairs with G Examples: mRNA messenger RNA tRNA transfer RNA rRNA ribosomal RNA snRNA small nuclear RNA miRNA micro RNA siRNA small interfering RNA RNA secondary structure: single-stranded Function in transcription (RNA processing) and translation Yeast Alanine tRNA

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. Fig. 2.15 Chromosome released from lysed E. coli cell.

Stop and Play the Video Prokaryotic chromosome structure---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) Stop and Play the Video Fig. 2.17

Looped domains Fig. 2.18

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 5 main types: H1, H2A, H2B, H3, H4 ~equal in mass to DNA evolutionarily conserved Non-histones all the other proteins associated with DNA differ markedly in type and structure amounts vary widely >> 100% DNA mass << 50% DNA mass

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. See Fig. 2.20

Fig. 2.21

Fig. 2.23

Fig. 2.21 - Metaphase chromosome depleted of histones maintains its shape with a nonhistone protein scaffold.

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

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 <---TTATTATTATTATTATTATTA--->

More about genome size: C value = total amount of DNA in the haploid (1N) genome Varies widely from species to species and shows no simple relationship to structural or organizational complexity, but there is similarity among closely related species. 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

Anybody like birds? Guess who has the smallest bird genome?