Molecular Biology Exam Study Notes
Model Organisms
Aspects of a Model Organism:
Represent many other organisms and processes.
Readily available and easy to maintain.
Quick reproduction rate.
Extensive existing information about them.
Genome is sequenced or easily sequenced.
Cellular Aspects Not Required for Modeling Sub-Cellular Processes
Multicellularity is not required.
Key Model Organisms and Their Limitations
1. Modeling Sub-cellular Processes
Escherichia coli
Model organism for manipulating transcription/translation.
Most widely used prokaryotic model organism.
Notes: Easily obtained, cultured, and manipulated.
Limitations: Lacks endoplasmic reticulum, Golgi apparatus, and mRNA splicing; cell wall structure means fundamental processes may differ from multicellular organisms.
Saccharomyces cerevisiae (yeast)
Model eukaryotic cell.
Limitations: As with E. coli, differences exist due to unicellular nature.
Mycoplasma genitalium
Known for the smallest genome.
First artificial organism, modeled in-silico.
2. Modeling Cellular Interactions
Chlamydomonas reinhardtii
Unicellular and multicellular life stages; capable of photosynthesis and problem-solving.
Notes: Requires a pyrenoid for carbon fixation in photosynthesis.
Limitations: Similar issues as unicellular models regarding fundamental processes.
Dictyostelium discoideum
Slime mold with life stages allowing both unicellularity and multicellularity.
Model Organisms in Plants
Physcomitrella patens
Moss that grows readily in vitro; can form single cells or whole plants.
Arabidopsis thaliana
Widely recognized as the model plant.
Small, rapid generation time, easy to grow.
Limitations: Small genome means lower gene copy and sequence complexity; fewer specialized mechanisms compared to perennials.
Medicago truncatula
Small plant, known for fast generation time and association with nitrogen-fixing bacteria.
Oryza sativa
Model cereal plant and monocot.
Limitations: Primarily diploid, although most cereals are polyploidy.
Model Organisms in Animals
Caenorhabditis elegans
Small number of cells, allowing tracking of individual cells during development.
Drosophila melanogaster (fruit fly)
Genetic model organism; known for inheritance studies.
Limitations: Not suitable for modeling immune responses due to short lifespan.
Danio rerio (zebrafish)
Model vertebrate; known for its transparent body/egg during early development.
Mus musculus (mouse)
Model mammal for a wide range of studies.
Macropus eugenii (wallaby)
Model marsupial, studying embryonic development.
Macaca mulatta (Rhesus Monkey)
Model primate, closely related to humans.
Disadvantages in Model Organisms
Short generation times can miss strategies for longer lifespans.
Immune responses in shorter-lived organisms (e.g., fruit flies) are less developed.
Molecular Biology Review
DNA Structure and Stability
Polar vs. Apolar Amino Acids:
Affect the stabilization of structures if polarized or non-polar configurations change.
DNA Characteristics:
Double-stranded, reverse complementary structure.
Each strand has a 3’ end (sugar –OH group) and a 5’ end (phosphate group).
Base pairs consist of adenine-thymine (A-T), cytosine-guanine (C-G), or adenine-uracil (A-U in RNA).
Stability of DNA:
Maintains stability millions of years due to:
Double helical structure that shields hydroxyl groups in base pairs.
Strong phosphate backbone that prevents degradation.
Disadvantage of High Stability:
Requires unwinding by DNA helicase for replication; DNA polymerase synthesizes new strands in a 5’ to 3’ direction.
Limitations of DNA
DNA is present as a single copy in cells; consequently, there are duplicates of genes that may not be needed.
RNA serves to transport necessary information outside the nucleus, as DNA cannot pass through nuclear pores.
Advantages of RNA:
More flexible, less stable due to ribose's extra –OH group; capable of moving through nuclear pores.
RNA Challenges
Being single-stranded means there is no backup if a mutation occurs.
Possible structures formed by RNA include:
Complementary structures with proteins (like ribosomes) or other RNA molecules.
Transcription and Translation
Transcription:
RNA polymerase utilizes DNA template to build RNA from the 5’ to 3’ end.
Starts at promoter regions with assistance from transcription factors; terminates at terminator regions.
Translation:
Involves converting mRNA to proteins through ribosomes, using tRNA to bring amino acids.
Location of Translation:
Occurs in free ribosomes in the cytoplasm or on ribosomes bound to the rough endoplasmic reticulum.
Importance of Separation in Signals:
Allows for faster protein synthesis to respond to environmental changes.
Types of Mutations
Silent Mutation: No amino acid change; sequence alteration does not affect the protein.
Missense Mutation: Results in a change to a different amino acid.
Nonsense Mutation: Introduces a premature stop codon that truncates the protein.
Insertion/Deletion Mutations:
Frame Shift Mutation: Changes in nucleotide number alter the reading frame, impacting protein length and structure.
Molecular Regulation (Chapter 18.1)
Basic Measures of Regulation
Tightly wrapped DNA around histone proteins prevents transcription by hindering RNA polymerase access.
Levels of Regulation
Packaging: Histone modifications can either condense or relax DNA structure.
Transcription Regulation: Control occurs at the promoter level.
RNA Processing: Involves removing non-coding regions (introns) before translation.
Translation Regulation: Signals for ribosome attachment can be modified to control translation initiation.
Protein Targeting: Processes within the Golgi apparatus ensure proteins reach their designated locations.
Protein Activation: Phosphorylation can activate proteins by changing their shape to expose active sites.
Special Notes on Transcriptional Regulation
Most studied because it is universally active in all cells.
Single transcriptional units may encode multiple genes and proteins, known as polycistronic mRNA.
Types of Transcriptional Control:
Negative Control: Blockage preventing transcription.
Positive Control: Activation promoting transcription.
Example of Lac Operon
Positive Control: Upon lactose presence, it induces enzyme synthesis (needed for lactose breakdown).
Negative Control: The lacI repressor can bind to the operator to inhibit transcription in the absence of lactose.
Mutations in Lac Operon:
Constitutive mutants express the lac operon continuously.
Non-inducible mutants never express the lac operon when they should.
Tryptophan Operon
Negative Control: Trp is synthesized only if absent from the environment, facilitated by trp repressor.
Repressible System: Operates when trp is deficient, otherwise transcription is repressed.
Catabolite Repression in E. coli
When glucose is available, E. coli preferentially uses it over lactose; mediated by the CAP (catabolite activator protein) which is responsive to cAMP levels.
cAMP levels inversely relate to glucose levels.
Positive Control in Lac Operon: CAP enhances RNA polymerase binding in the absence of glucose, increasing transcription.
Summary of Conditions for Lac Operon Transcription
Glucose + Lactose: no transcription due to absence of CAP activator.
Only Glucose: repressor binds at the operator, inhibiting transcription.
No Lactose: repressor binds to operator.
Lactose only: initiates fast transcription when no repressor is present due to CAP binding.
Fate of Cells in Animal Development (Chapter 11.5, 47)
Differences Between Animals and Plants
Global Insight: Animals cannot generate new tissues and organs throughout life.
Both developed multicellularity independently.
Stages of Embryonic Development
Cleavage:
From fertilization to blastula formation.
No growth between divisions; gradient of proteins in egg cell sets animal and vegetal poles.
Gastrulation:
Formation of ectoderm (animal pole) and endoderm (vegetal pole); mesoderm formed through inward migration.
Organogenesis:
In vertebrates, mesoderm organizes new structures like the notochord, somites, and organizer regions for limb formation.
Development of Germ Layers
Ectoderm: Develops into epidermis, nervous systems, hair, and glands.
Endoderm: Forms digestive and respiratory systems, liver, pancreas, and various glands.
Mesoderm: Constitutes skeletal, muscular, excretory, reproductive systems, and dermis.
Protein Gradients and Differentiation Signals
Cells inherit intercellular protein gradients that initiate specific processes through gene expression based on gradients.
Regulating Development (Chapters 11.5, 18.4, 35.5)
Protein Gradients and Translation
Protein gradients are produced through translation on free ribosomes, enabling localized distributions in cytoplasm.
The first differentiation point occurs at the zygote level due to uneven protein distributions influenced by maternal genotype.
Homeotic Genes
Responsible for signaling cascades that direct determination and differentiation.
Example: myoD gene activates differentiation of mesoderm cells into myoblasts.
Spatial Signaling in Plants
GLABRA-2: Gene expression varies based on interactions with adjacent cells, affecting root hair production.
KNOTTED-1 Gene: A receptor involved in determining leaf morphology in response to hormonal gradients.
Requirements for Tissue Formation
Cells must:
Adhere: Requires cell adhesion molecules to maintain connections.
Migrate: Ensure proper movement through the extracellular matrix.
Shape Change: Microtubules and actin facilitate elongation and contraction.
Undergo Apoptosis: Programmed cell death is essential for sculpting tissues (e.g., separation of digits, xylem development).
Working with Genes (Chapter 20)
Viewing and Analyzing DNA
Techniques:
Spooling, Staining: Various dyes for visibility in separation processes.
Gel Electrophoresis: Separates DNA based on size/charge.
Restriction Endonucleases
Serve bacterial defense by cutting foreign DNA at specific sequences.
Recognition Sites: Often palindromic, allowing for precise cuts.
Sticky Ends Formation
Result from uneven cuts by restriction enzymes, enabling facilitated joining of DNA fragments.
DNA Synthesis Functions
Important for fragment isolation, genome management, genetic fingerprinting, and cloning tasks.
DNA Ligase
Critical for joining DNA strands; only functions if sequences have matching sticky ends.
T4 DNA Ligase: Functions in the absence of complementary strands, useful in laboratory applications.
Polymerase Chain Reaction (PCR)
Steps in PCR
Melting: Heating DNA to 94°C to separate strands.
Annealing: Cooling to allow primers to bind around 60°C.
Extension: Increasing back to 72°C for DNA synthesis by Taq polymerase.
Repetition: Each cycle doubles the target DNA, enabling amplification.
Amplification Results
Shorter molecules increase slowly; longer ones increase exponentially, leading to targeted amplification success.
Reverse Transcriptase and Sanger Sequencing
Reverse Transcriptase Functionality
Converts mRNA to DNA using a poly-T primer that binds to adenines in the tail.
Key for retroviruses that need to integrate into host DNA.
Sanger Sequencing Process
Melting DNA.
Annealing: Introduces a single primer to sequence one strand.
Utilization of ddNTPs: Incorporates terminators halting sequence elongation at various lengths.
Electrophoresis: Fragment sizes reveal nucleotide sequences through charge differences.
Molecular Cloning Overview
Involves recombinant DNA creation for expression in living organisms.
Plasmids and various vectors facilitate the introduction and transformation of DNA.
Genetically Modified Organisms (GMOs)
Types of GMOs
Cisgenic: Introduce genes from the same species.
Transgenic: Transfer genes from different species to manipulate traits.
Cloning Plants and Animals
Techniques such as cuttings, mericlones, and somatic embryogenesis used in plants.
Animal transformation is more complex; stable inheritance requires incorporation into germ lines.
Examples of GMOs
BT Corn: Contains bacteria-derived toxin for pest resistance.
Golden Rice: Enriched with Vitamin A through genetic modifications.
Malaria-resistant Mosquitoes: Aim to reduce malaria transmission.
Enviropig: Genetically modified to produce less phosphate in waste.
Summary Points to Know
Model Organisms: E. coli, Mouse, Rhesus Monkey, their advantages and limitations.
Molecular Techniques: Staining, Gel Electrophoresis, Restriction Enzymes, PCR, and Sanger Sequencing.
Genetic Engineering: Use of vectors and transformation techniques, including plasmids and artificial chromosomes.