10.1-10.2 book
10.1 Using Microbiology to Discover the Secrets of Life & 10.2 Structure and Function of DNA
Overview of Heredity and DNA Discovery
Early 20th Century Beliefs: DNA was not recognized as the genetic material.
Prevailing incorrect belief: Inheritance involved a blending of parental traits, producing intermediate offspring.
This appeared correct due to continuous variation, which arises from multiple genes determining characteristics (e.g., human height).
The blending theory stated parental traits were lost or absorbed, which is incorrect.
Foundations of Genetics (Mid-Late 1800s): Two separate research lines began to converge in the 1920s.
Microbial systems played a significant role in understanding the molecular basis of genetics.
Discovery and Characterization of Nucleic Acid
Friedrich Miescher (1860s):
First to isolate phosphorus-rich chemicals from leukocytes (white blood cells) in pus from bandages.
Named these chemicals, which would later be known as RNA and DNA, "nuclein" because they were isolated from cell nuclei.
Richard Altmann (20 years later):
Termed it "nucleic acid" after discovering its acidic nature.
Albrecht Kossel (Last two decades of 19th century):
German biochemist who isolated and characterized the five different nucleotide bases composing nucleic acid:
Adenine (A)
Guanine (G)
Cytosine (C)
Thymine (T – in DNA)
Uracil (U – in RNA)
Received the Nobel Prize in Physiology or Medicine in 1910 for his work on nucleic acids and proteins (including histidine discovery).
Foundations of Genetics: Mendel's Pea Plants
Despite DNA discovery, its link to heredity wasn't made for decades.
Johann Gregor Mendel (1856-1860s): Austrian monk and botanist.
Experimented with garden peas (Pisum sativum), a diploid organism, because it self-fertilizes and is highly inbred, producing "true-breeding" lines.
Demonstrated basic patterns of inheritance, now known as Mendel's laws.
Hybridizations: Mated two true-breeding individuals (P generation) with different traits.
Examined offspring: first filial generation ($ ext{F}_{1}$).
Examined self-fertilized $ ext{F}{1}$ offspring: second filial generation ($ ext{F}{2}$).
Example (Figure 10.2): Violet flowers (true-breeding) crossed with white flowers (true-breeding).
$ ext{F}_{1}$ generation: all violet flowers.
$ ext{F}_{2}$ generation: approx. three-quarters violet, one-quarter white flowers.
Presentation and Publication (1865-1866): Presented results from ~ pea plants, showing traits are transmitted faithfully and independently.
Initial Reception: Work was virtually unnoticed; the scientific community incorrectly upheld the blending theory.
Rediscovery: Mendel's work was rediscovered, reproduced, and revitalized in 1900, on the brink of discovering the chromosomal basis of heredity.
The Chromosomal Theory of Inheritance
Late 1800s: Improved microscopic techniques allowed cell biologists to visualize subcellular structures like chromosomes during meiosis.
Observations: Chromosomes replicating, condensing into X-shaped bodies, and migrating to separate cellular poles.
Theodor Boveri (1902): Observed in sea urchins that nuclear components (chromosomes) determined proper embryonic development.
Walter Sutton (1902): Observed chromosomes separating into daughter cells during meiosis.
Development of Chromosomal Theory: Boveri and Sutton's observations led to the theory, identifying chromosomes as the genetic material for Mendelian inheritance.
Thomas Hunt Morgan (Early 1900s):
Provided first experimental evidence to support the theory using fruit flies (Drosophila melanogaster).
Carried out crosses, meticulous microscopic observations of fly chromosomes, correlating them with resulting characteristics.
Publication (1915): The Mechanism of Mendelian Heredity identified chromosomes as cellular structures responsible for heredity.
Received Nobel Prize in Physiology or Medicine in 1933.
Barbara McClintock (Late 1920s - 1950s):
Developed chromosomal staining techniques for maize (corn).
Identified a breakage event on chromosome , named the dissociation locus (Ds), which could change position.
Identified an activator locus (Ac), an element (transposase enzyme) that could activate Ds chromosome breakage.
These "jumping genes" (now called transposons) were initially not accepted by the scientific community.
Later acceptance: Discovered in bacteriophages, bacteria, and Drosophila in the 1960s and later.
Significance of Transposons: Mobile DNA segments that can move within a genome, regulating gene expression, protein expression, and virulence.
Microbes and Viruses in Genetic Research
Advantages of Microbes/Viruses as Model Systems:
Easily propagated in the laboratory.
Grow to high population densities in small spaces and short times.
Structurally simpler, making genetic manipulation easier.
Biochemical Unity of Life: Despite differences, all organisms share the same underlying molecules for heredity (Jacques Monod: "What is true for E. coli is also true for the elephant").
Joachim Hämmerling (1930s-1940s): Used the single-celled alga Acetabularia as a microbial model.
Discovery: Established that genetic information in a eukaryotic cell is housed within the nucleus.
Acetabularia spp. are unusually large (2-6 cm), asymmetric algal cells with a foot (containing nucleus, for attachment), a stalk, and an umbrella-like cap (Figure 10.3).
Experiment 1: Removed either the cap or the foot.
Removed foot: new feet did not grow.
Removed caps: new caps regenerated.
Conclusion: Hereditary information was in the nucleus-containing foot.
Experiment 2 (Figure 10.4): Used two species with different cap morphologies (A. crenulata and A. mediterranea).
Grafted stalk from A. crenulata onto A. mediterranea foot, and vice versa.
Observed that cap morphology of the regenerated cap was dictated by the species of the nucleus-containing foot.
Conclusion: Nucleus is the location of genetic material dictating cell properties.
George Beadle and Edward Tatum (1941): Used red bread mold Neurospora crassa.
Model organism choice: Simpler than fruit flies, grows on minimal medium by synthesizing its own vitamins and amino acids.
Experiment (Figure 10.5): Irradiated mold with X-rays to induce mutations.
Mated irradiated spores and grew them on complete medium (with supplements) and minimal medium (lacking supplements).
Looked for mutants that grew on complete medium but not minimal medium.
Systematically tested mutants to determine which vitamin or amino acid they couldn't produce.
Subsequent Work (Figure 10.6): Isolated different classes of arginine mutants.
By supplementing minimal medium with intermediates (citrulline or ornithine) in the arginine biosynthesis pathway, they identified three classes.
Each mutant had a defect in a different gene in the pathway.
One Gene-One Enzyme Hypothesis (1945): Proposed that each gene encodes one enzyme.
Revised later to "one gene-one polypeptide" hypothesis due to transcription/translation knowledge.
Remains largely true, especially in microbes, although some genes encode tRNAs or rRNAs.
Significance: Nobel Prize in Physiology and Medicine in 1958; basis for modern molecular genetics.
DNA as the Molecule Responsible for Heredity
Early 20th Century: Despite extensive research, DNA was considered too simple (four nucleotides) to encode complex genetic information.
Proteins: Believed to be the genetic material due to their complexity (20 different amino acids).
Frederick Griffith (1928): Griffith's Transformation Experiments (Figure 10.7)
British bacteriologist who showed horizontal gene transfer (between same generation) rather than vertical (parent to offspring).
Bacterial Transformation: External DNA is taken up by a cell, changing its characteristics.
Organism: Streptococcus pneumoniae (causes pneumonia).
Rough (R) strain: Nonpathogenic, lacks capsule, colonies appear rough.
Smooth (S) strain: Pathogenic, has capsule (escapes phagocytosis), colonies appear smooth.
Experiments with mice:
Live S strain $\rightarrow$ mice died.
Live R strain $\rightarrow$ mice survived.
Heat-killed S strain $\rightarrow$ mice survived.
Mixture of live R strain + heat-killed S strain $\rightarrow$ mice died.
Key Finding: Only S strain bacteria were recovered from dead mice given the mixture.
Conclusion: Something passed from the heat-killed S strain to the live R strain, "transforming" it into the pathogenic S strain. He called this the "transforming principle."
Oswald Avery, Colin MacLeod, and Maclyn McCarty (1944): (Figure 10.8)
Followed up on Griffith's work to identify the transforming principle.
Isolated S strain extract from infected mice, heat-killed it, and systematically inactivated components using enzymes:
Enzymes degrading proteins.
Enzymes degrading RNA.
Enzymes degrading DNA.
Mixed each treated S extract with R strain bacteria and observed for transformation (diffuse S strain growth in culture).
Result: Only when DNA was degraded was the mixture unable to transform the R strain.
Conclusion: DNA was the transforming principle.
Scientific Community Reaction: Many scientists initially did not accept this, believing protein contaminants were responsible.
Alfred Hershey and Martha Chase (1952): Confirmatory Evidence (Figure 10.9)
Studied bacteriophage T2 (a virus infecting E. coli).
Bacteriophage Structure: Protein coat (capsid) and a nucleic acid core (DNA or RNA).
Phage infection: T2 attaches to E. coli, injects nucleic acids, host machinery makes copies, host cell bursts, releasing new phages.
Labeling Strategy:
Protein coat: Labeled with radioactive sulfur, (sulfur in methionine/cysteine, not nucleic acids).
DNA: Labeled with radioactive phosphorus, (phosphorus in DNA/RNA, not typically protein).
Experiment: Each batch of labeled phage infected E. coli separately.
Blender: Detached phage coats from host cells.
Centrifuge: Separated heavier bacterial cells (pellet) from lighter phage particles (supernatant).
Results:
Protein-labeled tube (): Radioactivity remained in the supernatant (phage coats).
DNA-labeled tube (): Radioactivity detected only in the bacterial cells (pellet).
Conclusion: Phage DNA was injected into the cell and carried the genetic information to produce new phages, proving DNA, not protein, was the genetic material.
Impact: Led to broader acceptance of DNA as the molecule responsible for heredity.
Culmination: By the early 1950s, over years of research converged, leading to the general agreement that DNA was the genetic material (Figure 10.10), setting the stage for molecular biology and biotechnology.
Structure and Function of DNA
Nucleic Acids: Fourth class of macromolecules, composed of monomers called nucleotides.
Base Sequence: The specific order of nucleotides in a strand, carrying and retaining hereditary information.
DNA Nucleotides (Deoxyribonucleotides): Building blocks of DNA.
Three Components (Figure 10.11a):
Five-carbon sugar: Deoxyribose (carbons numbered , , , , ).
Phosphate group.
Nitrogenous base: Nitrogen-containing ring structure responsible for complementary base pairing.
Nucleoside: Comprises the five-carbon sugar and nitrogenous base.
Nitrogenous Bases (Figure 10.12):
Purines: Double-ring structure (six-carbon fused to five-carbon ring)
Adenine (A)
Guanine (G)
Pyrimidines: Single six-carbon ring structure
Cytosine (C)
Thymine (T) – unique to DNA
Uracil (U) replaces Thymine (T) in RNA.
Polymerization of Nucleotides (Figure 10.13):
Individual nucleoside triphosphates (dNTPs) combine via phosphodiester bonds (linkages).
Phosphate group at carbon of one sugar bonds to hydroxyl group of carbon of the next sugar.
Forms the sugar-phosphate backbone, the alternating sugar-phosphate framework.
During polymerization, two terminal phosphates are released from dNTP as pyrophosphate, driving the reaction.
Resulting strand has a free phosphate group at the carbon end and a free hydroxyl group at the carbon end.
Discovering the Double Helix
Erwin Chargaff (Early 1950s): Chargaff's Rules.
Examined DNA content across species: A, T, G, C not in equal quantities, varied by species but not within individuals of the same species.
Discovered: Amount of adenine () was equal to amount of thymine (); amount of cytosine () was equal to amount of guanine ().
Formula: and . Also, .
Linus Pauling (1952): Proposed an incorrect triple-stranded model of DNA based on X-ray diffraction.
Rosalind Franklin and R.G. Gosling: Used X-ray diffraction to understand DNA structure.
Franklin's expertise produced well-defined X-ray diffraction images (Figure 10.14) that clearly showed the overall double-helix structure.
James Watson and Francis Crick (1950s): (Figure 10.15)
Used Chargaff's rules and Franklin and Wilkins' X-ray diffraction images.
Published their double helix model in Nature in April 1953.
Nobel Prize (1962): Awarded to James Watson, Francis Crick, and Maurice Wilkins.
Franklin had died and Nobel Prizes were not awarded posthumously.
Alexander Rich (1973): Analyzed DNA crystals to further confirm and elucidate DNA structure.
DNA Structure
Double Helix Model (Watson and Crick): (Figure 10.16)
Two strands twisted around each other to form a right-handed helix.
Antiparallel: The end of one strand faces the end of the other.
end: free hydroxyl group.
end: free phosphate group.
Sugar-phosphate backbones: On the outside of the helix.
Nitrogenous bases: Stacked inside, forming the "rungs" of the ladder.
Turns: Approximately bases per turn.
Grooves: Asymmetrical spacing of backbones creates:
Major grooves: Backbone far apart.
Minor grooves: Backbone close together.
These grooves are binding sites for proteins, which can alter DNA structure, regulate replication, or regulate transcription.
Base Pairing (Figure 10.17): Occurs between a purine and a pyrimidine, explaining Chargaff's rules.
Adenine (A) and Thymine (T): Complementary, form two hydrogen bonds.
Cytosine (C) and Guanine (G): Complementary, form three hydrogen bonds.
DNA Denaturation and Reannealing (Figure 10.18):
Denaturation: Exposing DNA to high temperatures or certain chemicals breaks hydrogen bonds, separating strands into single-stranded DNA (ssDNA).
Reannealing/Renaturing: Cooling or removing denaturants allows hydrogen bonds to reform, returning to double-stranded DNA (dsDNA).
GC Content: DNA with high GC content is more difficult to denature due to the three hydrogen bonds between C and G, compared to two between A and T.
This artificial manipulation is crucial for biotechnology techniques.
DNA Function
Information Storage: DNA stores the information needed to build and control the cell.
Vertical Gene Transfer: Transmission of information from mother to daughter cells occurs through DNA replication.
A cell makes a duplicate copy of its DNA, then divides, ensuring correct distribution of one DNA copy to each resulting cell.
Nutrient Source: DNA can be enzymatically degraded and used as a source of nucleosides and nucleotides for the cell.
No Structural Role: Unlike other macromolecules, DNA does not primarily serve a structural role in cells.
Eye on Ethics: Women in Science and Health Professions
Historical Underrepresentation: Women have been underrepresented in science and medicine, with pioneering contributions often unnoticed.
Rosalind Franklin: Her X-ray diffraction studies were crucial for demonstrating the double helical structure of DNA, yet Watson and Crick received more initial credit and the Nobel Prize. Controversy exists regarding data acquisition and gender bias.
Barbara McClintock: Discovered transposons ("jumping genes") in maize (corn) from the 1930s-1950s, but her work wasn't recognized until much later, receiving a Nobel Prize in 1983 (Figure 10.19a).
Current Disparities: Women remain underrepresented, particularly in advanced scientific and medical careers.
More than half of undergraduate science degrees go to women, but only 46 ext{%} of doctoral degrees in science.
In academia, women hold less than one-third of Ph.D.-level tenure-track positions and less than one-quarter of full professorships.
In health professions, women are often underrepresented in many careers and earn significantly less than male counterparts (2013 JAMA study).
Contributing Factors: Complex, likely involve societal conditioning, lack of support, and gender bias (e.g., Tim Hunt's 2015 controversial comments).
Suggested Solutions (Figure 10.19b):
Greater support for girls in science and math from a young age (e.g., STEM programs by AAUW and NASA).
Increased public awareness of women's contributions in science.
Marketing targeting young girls should include more images of successful female scientists and medical professionals to encourage careers in STEM.
Clinical Focus Part 1 & 2: Traveler's Diarrhea
Alex's Case: 22-year-old college student experienced abdominal cramping and extensive watery diarrhea after vacationing in Puerta Vallarta, Mexico.
Initial Suspect Conditions: Bacterial (e.g., enterotoxigenic E. coli, Vibrio cholerae, Campylobacter jejuni, Salmonella), viral (rotavirus, norovirus), or protozoan (Giardia lamblia, Cryptosporidium parvum, Entamoeba histolytica) infections.
Diagnosis Steps:
Physician ordered a stool sample to look for causative agents (bacteria, cysts) and blood (indicative of some agents).
Stool sample showed neither blood nor cysts.
Based on symptoms and travel history, physician suspected traveler's diarrhea caused by enterotoxigenic E. coli (ETEC).
Confirmation: Physician ordered a diagnostic lab test of the stool sample to look for DNA sequences encoding specific virulence factors of ETEC.
Treatment: Alex instructed to drink lots of fluids.
Pathogenicity of ETEC: Produces plasmid-encoded virulence factors:
Heat-labile enterotoxin (LT) and Heat-stabile enterotoxin (ST): Cause excretion of chloride ions from intestinal cells to the intestinal lumen, leading to water loss and diarrhea.
Colonization factor (CF): Bacterial protein aiding adherence to the lining of the small intestine.
Why Genetic Analysis? Alex's physician used genetic analysis instead of bacterial isolation or direct Gram stain alone because:
ETEC is a strain of E. coli, which is a common inhabitant of the gut; isolation alone wouldn't distinguish pathogenic ETEC from normal flora.
Gram staining would identify E. coli as a Gram-negative rod, but not its specific virulence factors.
Genetic analysis allowed specific identification of DNA sequences for virulence factors (LT, ST, CF), confirming the presence of pathogenic ETEC. This is a more precise and accurate method for confirming the specific pathogenic strain rather than just the presence of a common bacterium.