1. Copyright © 2010 Pearson Education Inc.
Lecture 05 - DNA Structure Based on Chapter 2 - DNA: The Genetic Material
Copyright © 2010 Pearson Education Inc.

2. The Search for the Genetic Material
1.1. From the early 1900’s until the publication of a structure for DNA by Watson & Crick in 1956, geneticists (and genomic biologists) were focused on the basic tools of gene mapping in intensively studied genetic organisms like Drosophila (fruit fly) and others. Chromosomes were shown to be the carriers of hereditary information, and in eukaryotes chromosomes are composed of both DNA and protein. Most scientists initially believed that protein must be the genetic material.
1.2. Some substance must be responsible for passage of traits from parents to offspring. What are the properties a substance must have?
Stable enough to store information for long periods.
Able to replicate accurately.
Capable of change to allow evolution.

3. 2. Griffith’s Transformation Experiment
2.1. Frederick Griffith’s 1928 experiment with Streptococcus pneumoniae bacteria in mice showed that something passed from dead bacteria into nearby living ones, allowing them to change their cell surface (Figures 2.1 and 2.2).
Experiment explanation –
Type IIR – rough and avirulent (doesn’t kill mouse)
Type IIIS – smooth and virulent (kills mouse)
Heat kill type IIIS – smooth (but dead), and does not kill mouse
Mix IIR and Heat killed IIIS – kills mouse, and only live type IIIS can be isolated from the dead mouse
Conclusion: Something transferred from the dead IIIS to the live avirulent IIR that make them phenotypically IIIS (virulent and smooth).
2.2. Griffith called this agent the transforming principle, but did not know what it was or how it worked.

4. 3. Avery’s Transformation Experiment
Avery Experiment
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3.1. In 1944, Avery, MacLeod, and McCarty published results of a study that identified the transforming principle from S. pneumoniae. Their approach was to break open dead cells, chemically separate the components (e.g., protein, nucleic acids), and determine which was capable of transforming live S. pneumoniae cells (Figure 2.3).
3.2. Only the nucleic acid fraction was capable of transforming the bacteria.
3.3. Critics noted that the nucleic acid fraction was contaminated with proteins. The researchers treated this fraction with either RNase or protease and still found transforming activity, but when it was treated with DNase, no transformation occurred, indicating that the transforming principle was DNA.

5. 4. Hershey and Chase’s Bacteriophage Experiment
Hershey Chase Experiment
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4.1. In 1953, more evidence for DNA as the genetic material resulted from Alfred Hershey and Martha Chase’s work on E. coli infected with bacteriophage T2 (Figure 2.4). T2 phage replicates by a lytic life cycle (Figure 2.5).
4.2. In one part of the experiment, T2 proteins were labeled with 35S, and in the other part, T2 DNA was labeled with 32P. Then each group of labeled viruses was mixed separately with the E. coli host. After a short time, phage attachment was disrupted with a kitchen blender, and the location of the label determined (Figure 2.6).
4.3. The 35S-labeled protein was found outside the infected cells, while the 32P-labeled DNA was inside the E. coli, indicating that DNA carried the information needed for viral infection. This provided additional support for the idea that genetic inheritance occurs via DNA.

6. 5. RNA as Viral Genetic Material
All known cellular organisms and many viruses have DNA as their genetic material. Some viruses, however, use RNA instead.
Examples of RNA viruses include:
Bacteriophages such as MS2 and Qb.
Animal viruses such as poliovirus and human immunodeficiency virus (HIV).
Plant viruses such as tobacco mosaic virus (TMV) and barley yellow dwarf virus.
5.1. All known cellular organisms and many viruses have DNA as their genetic material. Some viruses, however, use RNA instead.
5.2. Examples of RNA viruses include:
Bacteriophages such as MS2 and Qb.
Animal viruses such as poliovirus and human immunodeficiency virus (HIV).
Plant viruses such as tobacco mosaic virus (TMV) and barley yellow dwarf virus.

7. 6a. The Composition and Structure of DNA and RNA
6.1. DNA and RNA are polymers composed of monomers called nucleotides.
6.2. Each nucleotide has three parts:
A pentose (5-carbon) sugar.
A nitrogenous base.
A phosphate group.
6.3. The pentose sugar in RNA is ribose, and in DNA it is deoxyribose. The only difference is at the 2’ position, where RNA has a hydroxyl (OH) group, while DNA has only a hydrogen (Figure 2.7).

8. 6b. The Composition and Structure of DNA and RNA
6.4. There are two classes of nitrogenous bases (Figure 2.8):
Purines (double-ring, nine-membered structures) include adenine (A) and guanine (G).
Pyrimidines (one-ring, six-membered structures) include cytosine (C), thymine (T) in DNA, and uracil (U) in RNA.

9. 6c. The Composition and Structure of DNA and RNA
6.5. The structure of nucleotides has these features:
The base is always attached by a covalent bond between the 1’ carbon of the pentose sugar and a nitrogen in the base (specifically, the 9 nitrogen in purines and the 1 nitrogen in pyrimidines).
The sugar–base combination is a nucleoside. When a phosphate is added (always to the 5’ carbon of the pentose sugar), it becomes a nucleoside phosphate, or simply nucleotide.

10. 6d. The Composition and Structure of DNA and RNA
Nucleotide naming conventions are given in Table 2.1.

11. 6e. The Composition and Structure of DNA and RNA
6.6. Polynucleotides of both DNA and RNA are formed by stable covalent bonds (phosphodiester linkages) between the phosphate group on the 5’ carbon of one nucleotide and the 3’ hydroxyl on another nucleotide (Figure 2.9). This creates the “backbone” of a nucleic acid molecule.
6.7. The asymmetry of phosphodiester bonds creates 3’ to 5’ polarity within the nucleic acid chain.

12. 7a. The DNA Double Helix
7.1. James Watson and Francis Crick published the famous double-helix structure in 1953 (Figure 2.10). When they began their work, it was known that DNA is composed of nucleotides, but how the nucleotides are assembled into nucleic acid was unknown. Two additional sources of data assisted Watson and Crick with their model:

13. 7b. The DNA Double Helix Chargaff’s Rules –
From inspection of data such as that shown in Table 2.2. above Erwin Chargaff concluded that:
the amount of purine always equals the amount of pyrimidine
the amount of G equals C, and the amount of A equals T
7.2. Base Composition Studies - Erwin Chargaff’s ratios obtained for DNA derived from a variety of sources showed that the amount of purine always equals the amount of pyrimidine, and further, that the amount of G equals C, and the amount of A equals T (Table 2.2).

14. 7c. The DNA Double Helix
7.3. X-Ray Diffraction Studies - Rosalind Franklin’s X-ray diffraction images of DNA showed a helical structure with regularities at 0.34 nm and 3.4 nm along the axis of the molecule (Figure 2.11).

15. 7d. The DNA Double Helix
7.4. Watson and Crick’s three-dimensional model (Figure 2.12) has these main features:
It is two polynucleotide chains wound around each other in a right-handed helix.
The two chains are antiparallel.
The sugar–phosphate backbones are on the outside of the helix, and the bases are on the inside, stacked perpendicularly to the long axis like the steps of a spiral staircase.

16. 7e. The DNA Double Helix
The bases of the two strands are held together by hydrogen bonds between complementary bases (two for A-T pairs and three for G-C pairs). Individual H-bonds are relatively weak, and so the strands can be separated (by heating, for example). Complementary base pairing means that the sequence of one strand dictates the sequence of the other strand (Figure 2.13).
The base pairs are 0.34 nm apart, and one full turn of the DNA helix takes 3.4 nm, so there are 10 bp in a complete turn. The diameter of a dsDNA helix is 2 nm.
Because of the way the bases H-bond with each other, the opposite sugar–phosphate backbones are not equally spaced, resulting in a major and minor groove. This feature of DNA structure is important for protein binding.
7.5. The 1962 Nobel Prize in Physiology or Medicine was awarded to Francis Crick, James Watson, and Maurice Wilkins (the head of the lab in which Franklin worked). Franklin had died and was not eligible posthumously.

17. 8. RNA Structure
Similarities and differences between DNA and RNA structure:
RNA structure is very similar to that of DNA.
It is a polymer of ribonucleotides (the sugar is ribose rather than deoxyribose).
Three of its bases are the same (A, G, and C) while it contains U rather than T.
Functional RNA in a cell is single-stranded, but internal base pairing can produce secondary structure in the molecule.
Some viruses use either dsRNA or ssRNA for their genomes. Double-stranded RNA is structurally very similar to dsDNA.
8.1. RNA structure is very similar to that of DNA.
It is a polymer of ribonucleotides (the sugar is ribose rather than deoxyribose).
Three of its bases are the same (A, G, and C) while it contains U rather than T.
8.2. Functional RNA in a cell is single-stranded, but internal base pairing can produce secondary structure in the molecule.
8.3. Some viruses use either dsRNA or ssRNA for their genomes. Double-stranded RNA is structurally very similar to dsDNA.

18. 9. The Organization of DNA in Chromosomes
Cellular DNA is organized into chromosomes.
A genome is the chromosome or set of chromosomes that contains all the DNA of an organism.
In prokaryotes the genome is usually a single circular chromosome.
In eukaryotes, the genome is one complete haploid set of nuclear chromosomes. Mitochondrial and sometimes chloroplast DNA are also present.
9.1. Cellular DNA is organized into chromosomes. A genome is the chromosome or set of chromosomes that contains all the DNA of an organism.
9.2. In prokaryotes the genome is usually a single circular chromosome.
9.3. In eukaryotes, the genome is one complete haploid set of nuclear chromosomes. Mitochondrial and sometimes chloroplast DNA are also present.

19. 10a. Prokaryotic Chromosomes
10.1. The typical prokaryotic genome is one circular dsDNA chromosome, but some prokaryotes are more exotic, with a main chromosome and one or more smaller ones. When a minor chromosome is dispensable to the life of the cell, it is called a plasmid. Some examples:
Borrelia burgdorferi (Lyme disease in humans) has a 0.91-Mb linear chromosome, plus an additional 0.53 Mb of DNA in 17 different linear and circular molecules.
Agrobacterium tumefaciens (crown gall disease of plants) has a 3.0-Mb circular chromosome and a 2.1-Mb linear chromosome.
10.2. Archaebacteria also vary in chromosomal organization, but only circular forms have been found. Examples:
Methanococcus jannaschii has three chromosomes of 1.66 Mb, 58 kb, and 16 kb.
Archaeoglobus fulgidus has one 2.2-Mb circular chromosome.
10.3. Both Eubacteria and Archaebacteria lack a membrane-bounded nucleus, hence their classification as prokaryotes. Their DNA is densely arranged in a cytoplasmic region called the nucleoid.
10.4. In an experiment where E. coli is gently lysed, it releases one 4.6-Mb circular chromosome, highly supercoiled (Figures 2.15 and 2.16). A 4.6-Mb double helix is about 1 mm in length, about 103 times longer than an E. coli cell. DNA supercoiling helps it fit into the cell.

20. 10b. Prokaryotic Chromosomes
DNA Supercoiling
10.5. A molecule of B-DNA, with 10 bp/turn of the helix, is in relaxed conformation. If turns of the helix are removed and the molecule circularized, the DNA will form superhelical turns to compensate for the added tension.
10.6. A nick in supercoiled DNA will allow it to return to a relaxed DNA circle (Figure 2.17).
10.7. Either overwinding or underwinding DNA will create a structure where 10 bp/turn of the helix is not the most energetically favored conformation, and supercoils will be induced. Both positive and negative supercoils will condense the DNA.
10.8. All organisms contain topoisomerase enzymes to supercoil their DNA.

21. 10c. Prokaryotic Chromosomes
10.9. Prokaryotes also organize their DNA into looped domains, with the ends of the domains held so that each is supercoiled independently (Figure 2.18). In E. coli there are about 400 domains of varying lengths.

22. 11. Eukaryotic Chromosomes
11.1. The genome of most prokaryotes consists of one chromosome, while most eukaryotes have a diploid number of chromosomes.
11.2. A genome is the information in one complete haploid chromosome set. The total amount of DNA in the haploid genome of a species is its C-value (Table 2.3). The structural complexity and the C-value of an organism are not related, creating the C-value paradox.
11.3. The form of eukaryotic chromosomes changes through the cell cycle:
In G1, each chromosome is a single structure.
In S, chromosomes duplicate into sister chromatids but remain joined at centromeres through G2.
At M phase, sister chromatids separate into daughter chromosomes.
11.4. In G1, eukaryotic chromosomes are linear dsDNA and contain about twice as much protein as DNA by weight. The DNA–protein complex is called chromatin, and it is highly conserved in all eukaryotes.

23. 12a. The Structure of Chromatin
12.1. Chromatin is a stainable DNA and protein complex in the nucleus. Its structure is the same in all eukaryotes.
12.2. Both histones and nonhistones are involved in physical structure of the chromosome.
12.3. Histones are abundant, small proteins with a net (+) charge. The five main types are H1, H2A, H2B, H3, and H4. By weight, chromosomes have equal amounts of DNA and histones.
12.4. Histones are highly conserved between species (H1 less than the others).
12.5. Histones organize DNA, condensing it and preparing it for further condensation by nonhistone proteins. This compaction is necessary to fit large amounts of DNA (2 m/6.5 ft in humans) into the nucleus of a cell.
12.6. Nonhistone is a general name for other proteins associated with DNA. This is a big group, with some structural proteins and some that bind only transiently. Nonhistone proteins vary widely, even in different cells from the same organism. Most have a net (-) charge and bind by attaching to histones.

24. 12b. The Structure of Chromatin
12.7. Chromatin formation involves histones and condenses the DNA so it will fit into the cell, making a 10-nm fiber (Figure 2.19). Chromatin formation has two components:
Two molecules each of histones H2A, H2B, H3, and H4 associate to form a nucleosome core, and DNA wraps around it 1.65 times for a six-fold condensation factor (Figure 2.20). Nucleosome cores are about 11 nm in diameter.
H1 further condenses the DNA by connecting nucleosomes to create chromatin with a diameter of 30 nm, for an additional six-fold condensation. The solenoid model proposes that the nucleosomes form a spiral with 6 nucleosomes per turn (Figure 2.21).

25. 12c. The Structure of Chromatin
12.8. Beyond the 30-nm filament stage, electron microscopy shows 30–90 loops of DNA attached to a protein scaffold (Figure 2.22). Each loop is 180–300 nucleosomes of the 30-nm fiber. SARs (scaffold-associated regions) bind nonhistone proteins to form loops that radiate out in spiral fashion (Figure 2.23).
12.9. Fully condensed chromosome is 10,000-fold shorter and 400-fold thicker than DNA alone.

26. 13. Euchromatin and Heterochromatin
The cell cycle affects DNA packing, with DNA condensing for mitosis and meiosis and then decondensing during interphase, being most dispersed at S phase.
Staining of chromatin reveals two forms:
Euchromatin condenses and decondenses with the cell cycle. Euchromatin accounts for most of the genome in active cells.
Heterochromatin remains condensed throughout the cell cycle. There are two types based on activity:
Constitutive heterochromatin
Facultative heterochromatin varies between cell types or developmental stages
13.1. The cell cycle affects DNA packing, with DNA condensing for mitosis and meiosis and then decondensing during interphase, being most dispersed at S phase.
13.2. Staining of chromatin reveals two forms:
Euchromatin condenses and decondenses with the cell cycle. It is actively transcribed and lacks repetitive sequences. Euchromatin accounts for most of the genome in active cells.
Heterochromatin remains condensed throughout the cell cycle. It replicates later than euchromatin and is transcriptionally inactive. There are two types based on activity:
Constitutive heterochromatin occurs at the same sites in both homologous chromosomes of a pair and consists mostly of repetitive DNA (e.g., centromeres).
Facultative heterochromatin varies between cell types or developmental stages, or even between homologous chromosomes. It contains condensed, and thus inactive, euchromatin (e.g., Barr bodies).

27. 14. Unique- and Repetitive-Sequence DNA
Sequences vary widely in how often they occur within a genome. The categories are:
Unique-sequence DNA, present in one or a few copies.
Moderately repetitive DNA, present in a few to 105 copies.
Highly repetitive DNA, present in about 105–107 copies.
Prokaryotes - mostly unique-sequence DNA
Eukaryotes have a mix of unique and repetitive sequences.
Unique-sequence DNA includes most of the genes that encode proteins.
Human DNA contains about 65% unique sequences.
Repetitive-sequence DNA includes the moderately and highly repeated sequences. They may be dispersed throughout the genome or clustered in tandem repeats.
14.1. Sequences vary widely in how often they occur within a genome. The categories are:
Unique-sequence DNA, present in one or a few copies.
Moderately repetitive DNA, present in a few to 105 copies.
Highly repetitive DNA, present in about 105–107 copies.
14.2. Prokaryotes have mostly unique-sequence DNA, with repeats only of sequences like rRNAs and tRNAs. Eukaryotes have a mix of unique and repetitive sequences.
14.3. Unique-sequence DNA includes most of the genes that encode proteins, as well as other chromosomal regions. Human DNA contains about 65% unique sequences.
14.4. Repetitive-sequence DNA includes the moderately and highly repeated sequences. They may be dispersed throughout the genome or clustered in tandem repeats.
14.5. Dispersed repetitive sequences occur in families that have a characteristic sequence. Often the same few sequences are highly repeated and comprise most of the dispersed repeats in the genome. There are two types of interspersion patterns found in all eukaryotic organisms:
LINEs (long interspersed repeated sequences) with sequences of 1,000–7,000 bp or more. The common example in mammals is LINE-1, with sequences up to 7 kb in length, that can act as transposons.
SINEs (short interspersed repeated sequences) with sequences of 100–500 bp. An example is the Alu repeats found in some primates, including humans, where these repeats of 200–300 bp make up 9% of the genome. SINEs are also transposons but are dependent on LINES for transposase genes.
14.6. Tandemly repetitive sequences are common in eukaryotic genomes, ranging from very short sequences (1–10 bp) to genes and even longer sequences. This group includes centromere and telomere sequences as well as rRNA and tRNA genes.