Unit 4 Molecular Genetics

Search for the genetic material:

1. 2. 3. Stable source of information Ability to replicate accurately Capable of change

Timeline of events:
1890 1900 1928 Weismann - substance in the cell nuclei controls development. Chromosomes shown to contain hereditary information, later shown to be composed of protein & nucleic acids. Griffiths Transformation Experiment

1944
1953 1953

Averys Transformation Experiment

Hershey-Chase Bacteriophage Experiment Watson & Crick propose double-helix model of DNA

1956

Gierer & Schramm/Fraenkel-Conrat & Singer Demonstrate RNA is viral genetic material.

Fig. 2.2: Frederick Griffiths Transformation Experiment - 1928 transforming principle demonstrated with Streptococcus pneumoniae

Griffith hypothesized that the transforming agent was a IIIS protein.

Fig. 2.3: Oswald T. Averys Transformation Experiment - 1944 Determined that IIIS DNA was the genetic material responsible for Griffiths 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 cells molecular machinery.

Fig. 2.4 Structure of T2 phage

DNA & protein

Fig. 2.5: Life cycle of virulent T2 phage:

Fig. 2-6: Hershey-Chase Bacteriophage Experiment - 1953 1. 2. T2 bacteriophage is composed of DNA and proteins: Set-up two replicates:

3.

Label DNA with 32P Label Protein with 35S

Infected E. coli bacteria with two types of labeled T2

32P

4.

is discovered within the bacteria and progeny phages, whereas 35S is not found within the bacteria but released with phage ghosts.

1969: Alfred Hershey

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.

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.

Fig. 2.11

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 %A %T %G %C %GC

Examples:

Homo sapiens Zea mays Drosophila Aythya americana

31.0 25.6 27.3 25.8

31.5 25.3 27.6 25.8

19.1 24.5 22.5 24.2

18.4 24.6 22.5 24.2

37.5 49.1 45.0 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. Fig. 2.13

Double Helix Model of DNA: Six main features

1. 2. 3. 4. 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

5. 6.

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. 2.15

Fig. 2.14 B-DNA

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
T2 bacteriophage

TMV

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: 1. 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

Fig. 2.24

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 T4 HIV-1 E. Coli Lilium formosanum Zea mays Amoeba proteus Drosophila melanogaster Mus musculus Canis familiaris Equus caballus Homo sapiens C value (bp) 48,502 168,900 9,750 4,639,221 36,000,000,000 5,000,000,000 290,000,000,000 180,000,000 3,454,200,000 3,355,500,000 3,311,000,000 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:

1.

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

2.

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:

1. 2. Level 1 Level 2 Winding of DNA around histones to create a nucleosome structure. Nucleosomes connected by strands of linker DNA like beads on a string. Packaging of nucleosomes into 30-nm chromatin fiber.

3.

Level 3

4.

Level 4

Formation of looped domains.

Figs. 2.25-29

Fig. 2.31

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. At extreme ends of the chromosome, maintain stability, and consist of tandem repeats. Play a role in DNA replication and stability of DNA.

Telomeric DNA

Unique-sequence DNA
Repetitive-sequence DNA

Often referred to as single-copy and usually code for genes.

May be interspersed or clustered and vary in size.

SINEs
LINEs

short interspersed repeated sequences (100-500 bp)

long interspersed repeated sequences (>5,000 bp) short tandem repeats (e.g., TTA|TTA|TTA)

Microsatellites