Regulation of metabolic pathways

Bacterial control of gene expression Operon: cluster of related genes with on/off switch Three Parts: 1. Promoter where RNA polymerase attaches 2. Operator on/off, controls access of RNA poly 3. Genes code for related enzymes in a pathway

Regulatory gene: produces repressor protein that binds to operator to block RNA poly

Repressible Operon (ON OFF)

Normally ON Repressible Operon Anabolic (build organic molecules) Organic molecule product acts as corepressor binds to repressor to activate it Operon is turned OFF Eg. trp operon

trp operon

Normally OFF Inducible Operon Catabolic (break down food for energy) Repressor is active inducer binds to and inactivates repressor Operon is turned ON Eg. lac operon

lac operon

Typical human cell: only 20% of genes expressed at any given time Different cell types (with identical genomes) turn on different genes to carry out specific functions Differences between cell types is due to differential gene expression

Eukaryotic gene expression regulated at different stages

Chromatin Structure: Tightly bound DNA less accessible for transcription DNA methylation: methyl groups added to DNA; tightly packed; transcription Histone acetylation: acetyl groups added to histones; loosened; transcription

Transcription Initiation: Control elements bind transcription factors Enhances gene expression

Transcription Initiation Complex Enhancer regions bound to promoter region by activators

Regulation of mrna: micro RNAs (mirnas) and small interfering RNAs (sirnas) can bind to mrna and degrade it or block translation

Control of Cell Cycle: 1. Proto-oncogene = stimulates cell division 2. Tumor-suppressor gene = inhibits cell division Mutations in these genes can lead to cancer

Proto-oncogene Oncogene

Genes involved in cancer: Ras gene: stimulates cell cycle (protooncogene) Mutations of ras occurs in 30% of cancers p53 gene: tumor-suppresor gene Functions: halt cell cycle for DNA repair, turn on DNA repair, activate apoptosis (cell death) Mutations of p53 in 50+% of cancers

Cancer results when mutations accumulate (5-7 changes in DNA) Active oncogenes + loss of tumorsuppressor genes The longer we live, the more likely that cancer might develop

Bacteria vs. Viruses Bacteria Prokaryotic cell Most are free-living (some parasitic) Relatively large size Antibiotics used to kill bacteria Virus Not a living cell (genes packaged in protein shell) Intracellular parasite 1/1000 size of bacteria Vaccines used to prevent viral infection Antiviral treatment

Viruses Very small (<ribosomes) Components = nucleic acid + capsid Nucleic acid: DNA or RNA (double or single-stranded) Capsid: protein shell Some viruses also have viral envelopes that surround capsid Limited host range (eg. human cold virus infects upper respiratory tract) Reproduce within host cells

Simplified viral replicative cycle

Bacteriophage Virus that infects bacterial cells

Lytic Cycle of T4 Phage

Bacteriophage Reproduction Lytic Cycle: Use host machinery to make copies of virus Death of host cell by rupturing it (lysis) Virulent phages replicate by this method Lysogenic Cycle: Phage DNA incorporated into host DNA and replicated along with it Phage DNA = prophage Temperate Phage: uses both methods of replication

Lytic Cycle vs. Lysogenic Cycle

Animal viruses have a membranous envelope Host membrane forms around exiting virus Difficult for host immune system to detect virus

Retrovirus RNA virus that uses reverse transcriptase (RNA DNA) Newly made viral DNA inserted into chromosome of host Host transcribes viral DNA (= provirus) to make new virus parts Example: HIV (Human Immunodeficiency Virus)

HIV = Retrovirus

HIV Infects white blood cells HIV+: provirus (DNA inserted) AIDS: active viral reproduction

Vaccines Weakened virus or part of pathogen that triggers immune system response

Emerging viruses = mutation of existing viruses

Viroids Small, circular RNA molecules that infect plants Cause errors in regulatory systems that control plant growth Eg. coconut palms in Philippines

Prions Misfolded, infectious proteins that cause misfolding of normal proteins Eg. mad cow disease (BSE),Creutzfeldt-Jakob disease (humans), scrapie (sheep)

Diseases caused by prions Prions act slowly incubation period of at least 10 years before symptoms develop Prions are virtually indestructible (cannot be denatured by heating) No known cure for prion diseases

Tools of Genetic Engineering Restriction enzymes (restriction endonucleases): used to cut strands of DNA at specific locations (restriction sites) Restriction Fragments: have at least 1 sticky end (singlestranded end) DNA ligase: joins DNA fragments Cloning vector: carries the DNA sequence to be cloned (eg. bacterial plasmid)

Using a restriction enzyme (RE) and DNA ligase to make recombinant DNA

Gene Cloning

Applications of Gene Cloning

Techniques of Genetic Engineering Transformation: bacteria takes up plasmid (w/gene of interest) Nucleic acid hybridization: used to track gene of interest PCR (Polymerase Chain Reaction): amplify (copy) piece of DNA without use of cells Gel electrophoresis: used to separate DNA molecules on basis of size and charge using an electrical current (DNA + pole) Southern blotting: used to find a specific human gene DNA microarray assays: study many genes at same time

PCR (Polymerase Chain Reaction): amplify (copy) piece of DNA without use of cells

Gel Electrophoresis: used to separate DNA molecules on basis of size and charge using an electrical current (DNA + pole)

Cloning Organisms Nuclear transplantation: nucleus of egg is removed and replaced with nucleus of body cell

Nuclear Transplantation

Problems with Reproductive Cloning Cloned embryos exhibited various defects DNA of fully differentiated cell have epigenetic changes

Stem Cells Stem cells: can reproduce itself indefinitely and produce other specialized cells Zygote = totipotent (any type of cell) Embryonic stem cells = pluripotent (many cell types) Adult stem cells = multipotent (a few cell types) or induced pluripotent, ips (forced to be pluripotent)

Applications of DNA Technology 1. Diagnosis of disease identify alleles, viral DNA 2. Gene therapy alter afflicted genes 3. Production of pharmaceuticals 4. Forensic applications DNA profiling 5. Environmental cleanup use microorganisms 6. Agricultural applications - GMOs

DNA Fingerprinting

RFLPs ( rif-lips ) Restriction Fragment Length Polymorphism Cut DNA with different restriction enzymes Each person has different #s of DNA fragments created Analyze DNA samples on a gel for disease diagnosis Outdated method of DNA profiling (required a quarter-sized sample of blood)

RFLPs Disease Diagnosis

STR Analysis STR = Short Tandem Repeats Non-coding DNA has regions with sequences (2-5 base length) that are repeated Each person has different # of repeats at different locations (loci) Current method of DNA fingerprinting used only need 20 cells for analysis

Human DNA 3 billion base pairs ~20,000 genes Only 1.5% codes for proteins (or RNA) Repetitive DNA: sequences present in multiple copies

Evolutionary Development (Evo-devo) Compares developmental processes to understand how changes can lead to evolution of organisms

Genetic Diversity in Prokaryotes Factors: 1.Rapid reproduction (binary fission) 2.Mutations errors in replication 3.Genetic recombination

Genetic Recombination in Bacteria 1. Transformation: uptake of foreign DNA from surroundings 2. Transduction: viruses transfer genes between prokaryotes 3. Conjugation: DNA transferred from one to another

Transformation Uptake of foreign DNA from surroundings Observed by Griffith (bacteria & mice)

Plasmids Small ring of DNA that carries a few genes Replicates separately from bacterial chromosome Can carry genes for antibiotic resistance Used frequently in genetic engineering for gene cloning

AP Bio Lab 6A - Transformation

Using plasmids and bacteria in genetic engineering

Transduction Viruses (bacteriophages) carry bacterial genes from one host cell to another Recombine DNA of donor and recipient cell

Conjugation One cell donates DNA to another Donors cell extends a sex pilus ( mating bridge ) through which DNA is transferred Requires the presence of a piece of DNA called the F factor to produce the pilus