Mendel's Genetics Experiments

Mendel’s Experiments in Genetics (Mid-1800s)

• Gregor Mendel, an Austrian monk, used pea plants (Pisum sativum) in experiments
• The goal was to understand how traits pass from parents to offspring
• This work became the basis for classical genetics
• Pea plants had clear, contrasting traits
• They can self-pollinate or cross-pollinate for controlled breeding
• They have a short generation time and produce many offspring
• P Generation (Parental) refers to true-breeding plants (always purple or white)
• F1 Generation (First Filial) refers to offspring from a cross between two P plants
• F2 Generation refers to offspring from a cross between F1 individuals
• Mendel crossed true-breeding purple-flowered and white-flowered plants
• All F1 offspring had purple flowers, showing purple is dominant
• When F1 plants were crossed, the F2 generation had a 3:1 ratio of purple to white
• Traits are controlled by factors, now called genes, with each individual carrying two factors (alleles)
• These factors don’t blend but remain separate
• A dominant allele can mask a recessive one
• The blending inheritance theory was disproved
• The Law of Segregation states each individual has two alleles for a trait, but only passes one to offspring
• The Law of Independent Assortment states genes for different traits are inherited independently unless linked on the same chromosome

Hammerling’s Alga Experiments (1930s)

• Joachim Hammerling used Acetabularia to show genetic information resides in the nucleus
• Acetabularia has a foot (containing the nucleus), stalk, and cap
• Two species used include A. mediterranea (disk-shaped cap) and A. crenulata (branched cap)
• Caps were cut off and stalks swapped between two species
• The regenerated cap always matched the species of the foot, which had the nucleus
• The nucleus contains the instructions for cell regeneration and morphology
• Genetic information is not stored in the cytoplasm, but in the nucleus
• This confirmed the nucleus is the genetic control center, years before the identification of DNA

Griffith’s Transformation Experiment (1928)

• Frederick Griffith studied two strains of Streptococcus pneumoniae
• S strain (Smooth) has a capsule and causes disease (virulent)
• R strain (Rough) has no capsule and is harmless (non-virulent)
• Mice were injected with live R strain and lived
• Mice were injected with live S strain and died
• Mice were injected with heat-killed S strain and lived
• Mice were injected with heat-killed S + live R and died
• Live S strain was recovered from the dead mouse
• Something from the dead S strain transformed the live R strain into a deadly S form
• This was called the “transforming principle"
• The identity of the "something" was unknown at the time

Avery, MacLeod, and McCarty (1944)

• Griffith’s work was continued to identify the transforming molecule
• Heat-killed S strain was taken and different molecules were systematically destroyed
• Transformation still happened after removing proteins
• Transformation still happened after removing RNA
• Transformation stopped after removing DNA
• DNA is the transforming principle
• DNA must carry the genetic instructions
• This was the first strong evidence that DNA (not protein) is the genetic material

Hershey and Chase Experiment (1952)

• Alfred Hershey and Martha Chase worked with bacteriophages
• Bacteriophages are viruses that infect bacteria
• The protein coat is on the outside and DNA is inside
• DNA was labeled with radioactive phosphorus (P-32)
• Protein was labeled with radioactive sulfur (S-35)
• Phages were allowed to infect E. coli bacteria
• A blender separated the phage coats from the bacteria
• Only P-32 was found inside the bacterial cells, not S-35
• Only DNA entered the bacteria, and it directed the production of new viruses
• DNA, not protein, is the genetic material

Central Dogma of Molecular Biology

• This is the core concept for understanding how cells use genetic information
• DNA is transcribed into RNA
• RNA is translated into protein
• This process is universal among living organisms

DNA Replication

• The purpose is to make an exact copy of DNA before cell division
• This occurs in the cytoplasm in prokaryotes
• This occurs in the nucleus in eukaryotes
• Each new DNA molecule contains one original and one new strand (semiconservative)
• DNA polymerase builds the new strand by matching complementary bases (A-T, C-G)
• Topoisomerase unwinds supercoils to relieve tension ahead of the replication fork
• Helicase unzips the double helix by breaking hydrogen bonds between bases
• The replication fork is the Y-shaped region where the DNA splits into two strands for copying
• The leading strand is made continuously toward the replication fork
• The lagging strand is made in short segments (Okazaki fragments) away from the fork because DNA polymerase only works 5’ to 3’
• DNA ligase seals the gaps between Okazaki fragments

Transcription: Making RNA from DNA

• This occurs in the cytoplasm in prokaryotes
• This occurs in the nucleus in eukaryotes
• RNA polymerase binds to a promoter (start site on DNA)
• It unwinds the DNA and reads one strand (the template strand)
• It creates a complementary RNA strand, replacing T with U
• RNA synthesis stops at a termination signal
• The resulting RNA is called a transcript
• Eukaryotes have post-transcriptional modifications
• A 5’ Cap helps ribosomes recognize mRNA
• A Poly-A tail protects mRNA from degradation
• Splicing removes introns (noncoding regions), and exons (coding parts) are joined

Translation: RNA → Protein

• This occurs in the cytoplasm of both prokaryotes and eukaryotes
• mRNA carries the genetic code
• tRNA brings the correct amino acid to the ribosome
• rRNA is part of the ribosome structure and catalyzes the reaction
• The A site (Arrival) is where tRNA carrying an amino acid first binds
• The P site (Peptide) is where the growing protein chain is held
• The E site (Exit) is where empty tRNA leaves
• Each set of 3 RNA bases is 1 codon and equals 1 amino acid
• mRNA with AUG codes for Methionine (Start codon)
• Anticodon (on tRNA) is UAC
• Polycistronic messages in prokaryotes mean one mRNA can code for multiple proteins at once
• Each mRNA usually codes for one protein in eukaryotes

Mutations and Their Effects

• Mutation refers to a change in the DNA sequence
• Silent mutations: Changes a base but doesn’t change the amino acid (no effect)
• Missense mutations: Changes a base, changes the amino acid (possible effect on protein function)
• Nonsense mutations: Changes a base, resulting in a STOP codon, and a short/nonfunctional protein
• Frameshift mutations: Caused by insertion or deletion, changing the reading frame and often is harmful
• A mutagen is anything that causes mutations
• Examples include radiation, chemicals, and viruses

RNA Viruses and Retroviruses

• Viruses use RNA but cells use DNA for long-term storage
• RNA less stable and more prone to mutations
• Some viruses use RNA because it's quicker to replicate
• Retroviruses like HIV use RNA as their genetic material
• Use reverse transcriptase to convert RNA back to DNA within a host cell
• That DNA integrates into the host genome
• Humans don’t have reverse transcriptase, viruses bring their own

Genotype vs. Phenotype

• Genotype refers to the genetic makeup (e.g., gene for brown eyes)
• Phenotype is the physical expression of that gene (e.g., you have brown eyes)
• A mutation in genotype may or may not lead to a change in phenotype

Recombination: Mixing Genes

• This increases genetic diversity
• Vertical gene transfer occurs from parent to offspring
• Horizontal gene transfer occurs between individuals of the same generation
• Three types of horizontal gene transfer in bacteria exist
• Transformation: Bacteria take in naked DNA from the environment
• Griffith’s experiment is the classic example
• Transduction: Bacteriophages accidentally transfer bacterial genes between bacteria
• Conjugation: Requires direct contact
• One bacterium transfers DNA through a pilus
• This often involves plasmids, circular DNA
• It can spread drug resistance genes

Gene Regulation in Prokaryotes

• An operon is a group of genes regulated together

Definitions You Must Know (with Examples)

• A fomite is a nonliving object that can harbor pathogens and transmit infection
• Examples: doorknobs, hospital bed rails, stethoscopes, used bandages
• Disinfection eliminates/reduces harmful microorganisms (except spores) from inanimate objects
• Example: using bleach to wipe a countertop
• Sanitization reduces microbial populations to safe levels on inanimate surfaces
• Example: commercial dishwasher or cleaning restaurant utensils

Sterilization, Antisepsis, and Degerming

• Sterilization is the complete destruction/removal of all microbial life, including spores
• Example: autoclaving surgical tools
• Antisepsis reduces/kills microbes on living tissue
• Example: Using iodine before surgery
• Degerming is the mechanical removal of microbes from a limited area
• Example: Scrubbing skin with alcohol before drawing blood

Categories of Control Agents

• Control agents can be physical and chemical
• Moist heat (boiling, autoclaving, pasteurization)
• Autoclave: 121°C, 15 psi, 15–20 min → sterilizes equipment
• Boiling disinfects but doesn't kill all spores including prions
• Pasteurization reduces spoilage organisms/pathogens in food
• Dry heat includes incineration and hot-air ovens
• Incineration burns everything and is used for loops in the lab
• The dry oven is 160-170°C for 2-3 hours and is used for glassware
• Refrigeration slows microbial growth (bacteriostatic)
• Freezing slows growth further but doesn’t reliably kill all microbes
• Filtration removes microbes from air or liquids
• This is used for heat-sensitive solutions and HEPA filters filter air in hospital rooms
• Ionizing radiation (X-rays, gamma rays) breaks DNA and sterilizes medical supplies
• Non-ionizing radiation (UV light) damages DNA and disinfects air and surfaces

Chemical Methods

• Alcohols denature proteins and dissolve membranes
• They are effective against bacteria and enveloped viruses
• Ethanol (70%) and isopropanol (rubbing alcohol) serve as examples
• Phenolics disrupt membranes and proteins such as Lysol and triclosan
• Halogens oxidize and damage macromolecules
• Examples include iodine (antiseptic) and chlorine (disinfects water)
• Heavy metals bind to and inactivate proteins
• Examples include silver nitrate and copper sulfate
• Aldehydes inactivate proteins and nucleic acids
• An example is glutaraldehyde
• Peroxygens are strong oxidizers that damage proteins and membranes
• Examples include hydrogen peroxide and peracetic acid
• Surfactants (soaps and detergents) lower surface tension and help mechanically remove microbes (degerming)
• Quats kill some bacteria and fungi and are used in mouthwash and surface disinfectants
• Gaseous agents are used for sterilization of heat-sensitive materials like ethylene oxide

Levels of Resistance to Microbial Control

• Prions are the most resistant to microbial control
• Bacterial endospores
• Mycobacteria
• Protozoan cysts
• Gram-negative bacteria
• Fungi
• Gram-positive bacteria
• Enveloped viruses are the easiest to kill