DNA Structure and Analysis: Foundations of Genetic Material

DNA Structure and Analysis

Learning Objectives

• Understand the four essential characteristics of genetic material.

• Review key experiments that established DNA, not protein, as the genetic material.

• Recognize that RNA serves as genetic material in some viruses.

• Describe nucleic acid chemistry and the structure of DNA and RNA.

• Identify analytical techniques for investigating DNA and RNA.

The Four Characteristics of Genetic Material

Essential Properties

Genetic material must fulfill four criteria to serve its biological role:

1. Replication: Ability to copy itself accurately for transmission to the next generation.

2. Information Storage: Capacity to store all necessary genetic information.

3. Expression of Information: Enable the flow of genetic information (central dogma: DNA → RNA → protein).

4. Variation by Mutation: Allow for genetic diversity through mutation.

Information Flow: The Central Dogma

Pathways of Genetic Information

• Transcription: DNA is transcribed into RNA.

• Translation: RNA is translated into protein via ribosomes.

The central dogma is summarized as:

$\text{DNA} \xrightarrow{\text{transcription}} \text{RNA} \xrightarrow{\text{translation}} \text{Protein}$

Historical Perspective: Protein or DNA as Genetic Material?

Early Views and Key Experiments

• In the 1940s, proteins were favored as genetic material due to their diversity and abundance.

• Chromosomes consist of both DNA and proteins.

Griffith's Experiment (1927)

• Showed that avirulent strains of Streptococcus pneumoniae could be transformed into virulent strains.

• Transformation depended on a polysaccharide capsule.

Strain	Capsule	Virulence
IIIS	Present	Virulent
IIR	Absent	Avirulent

Avery, MacLeod, and McCarty (1944)

• Demonstrated that DNA, not protein, is the transforming principle.

• DNase destroyed transforming activity, but RNase and protease did not.

Hershey and Chase (1952)

• Used Escherichia coli and bacteriophage T2.

• Radioisotopes $^{32}P$ (labels DNA) and $^{35}S$ (labels protein) showed that DNA enters the bacterial cell and directs viral reproduction.

Evidence for DNA as Genetic Material in Eukaryotes

Indirect Evidence

• Distribution of DNA: DNA is found in the nucleus, mitochondria, and chloroplasts; proteins are distributed throughout the cell.

• Correlation: Amount of DNA correlates with chromosome number in gametes and diploids; no such correlation for proteins.

Organism	DNA Content (pg)
Human	3.5
Mouse	2.7
Trout	2.8

Mutagenesis by UV Light

• UV light induces mutations most effectively at 260 nm, the absorption maximum for DNA/RNA.

Direct Evidence

• Recombinant DNA technology: Eukaryotic genes introduced into bacteria are replicated and expressed.

• Genomics: Comparison of genomes reveals mutations responsible for heritable disorders.

RNA as Genetic Material in Some Viruses

Examples

• Tobacco mosaic virus (TMV)

• Phage Qβ

• SARS-CoV-2

• Pathogenic fish viruses (IPNV, ISAV, SAV)

RNA Replicase and Reverse Transcriptase

• RNA-dependent RNA polymerase (RdRp): Replicates viral RNA.

• Reverse transcriptase: Synthesizes complementary DNA (cDNA) from an RNA template (e.g., retroviruses like HIV).

Nucleic Acid Chemistry

Nucleotides: Building Blocks of Nucleic Acids

• Composed of a nitrogenous base (purine or pyrimidine), pentose sugar (deoxyribose or ribose), and phosphate group (mono-, di-, or tri-).

Nucleoside vs. Nucleotide

• Nucleoside: Nitrogenous base + pentose sugar

• Nucleotide: Nucleoside + phosphate group

Nitrogenous Bases

• Pyrimidines: Cytosine (C), Thymine (T), Uracil (U)

• Purines: Adenine (A), Guanine (G)

Base	DNA	RNA
Adenine (A)	✔	✔
Guanine (G)	✔	✔
Cytosine (C)	✔	✔
Thymine (T)	✔
Uracil (U)		✔

Pentose Sugars

• DNA: Deoxyribose

• RNA: Ribose

Mono-, Di-, and Triphosphates

• NMP: Nucleoside monophosphate (one phosphate)

• NDP: Nucleoside diphosphate (two phosphates)

• NTP: Nucleoside triphosphate (three phosphates)

Phosphodiester Bonds

• Nucleotides are linked by phosphodiester bonds between the phosphate group at the C-5' position and the OH group on the C-3' position.

Structure of DNA

Watson and Crick Model (1953)

• DNA is a double helix with two antiparallel strands connected by base pairing.

• Base pairs: Adenine-Thymine (A-T), Guanine-Cytosine (G-C)

• Hydrogen bonds: A-T (2 bonds), G-C (3 bonds)

• Helix diameter: 20 Å; one complete turn: 34 Å (10 bp)

• Major and minor grooves present.

Chargaff's Rules

• Amount of A = T; amount of G = C

• Percentage of purines (A+G) equals pyrimidines (C+T)

Alternative Forms of DNA

• B-DNA: Standard Watson-Crick model

• A-DNA: More compact, forms under high salt/dehydration

• Z-DNA: Left-handed helix

• C-, D-, E-, P-DNA: Other right-handed forms, less common

Structure of RNA

Chemical Similarity and Differences

• Single-stranded (ss) in most cases

• Ribose sugar instead of deoxyribose

• Uracil replaces thymine

Three Major Classes of Cellular RNAs

• Messenger RNA (mRNA): Template for protein synthesis

• Ribosomal RNA (rRNA): Structural component of ribosomes

• Transfer RNA (tRNA): Carries amino acids for protein synthesis

Analytical Techniques for DNA and RNA Investigation

Absorption of UV Light

• Nucleic acids absorb UV light at 254–260 nm.

• Denaturation increases absorbance (hyperchromic shift).

• Melting temperature ($T_m$): Temperature at which 50% of DNA strands are unwound.

Molecular Hybridization

• Denaturation and renaturation of nucleic acids.

• Base complementarity between strands; probes can be radioactive or fluorescently labeled.

Fluorescent In Situ Hybridization (FISH)

• Uses fluorescent probes to detect specific DNA sequences in chromosomes.

Nucleic Acid Electrophoresis

• Separates DNA/RNA fragments by size using agarose gel.

• Smaller fragments migrate faster than larger ones.

Bioinformatics: BLAST

• BLAST (Basic Local Alignment Search Tool) is used to compare nucleotide or protein sequences to sequence databases.

Summary Table: Key Experiments Establishing DNA as Genetic Material

Experiment	Organism	Key Finding
Griffith (1927)	Streptococcus pneumoniae	Transformation of avirulent to virulent strains
Avery, MacLeod, McCarty (1944)	Streptococcus pneumoniae	DNA is the transforming principle
Hershey & Chase (1952)	Escherichia coli, phage T2	DNA, not protein, enters cell and directs viral reproduction

Review Questions

1. What is genetic material and which properties recognize a genetic material?

2. Which experiments contributed to proving that DNA, not protein, is the genetic material?

3. Describe and draw the building blocks in DNA and RNA.

4. Mention some analytic techniques that can be used to investigate DNA and RNA.

Additional info:

• Some context and terminology were expanded for clarity and completeness.

• Tables were reconstructed and summarized from slide images and text.