Number of base pairs in the human genome 3 * 10^9 Gene therapy is proposed to help cure this disease Cystic Fibrosis Cystic Fibrosis has this type of hereditary influence autosomal recessive Cystic Fibrosis affects these tissues Respiratory System - multiple infections; Pancreas - must replace enzymes; Gut - poor digestion due to lack of enzymes; Male genital duct - sterility; Sweat Gland - excessive salt How Cystic Fibrosis was originally diagnosed By tasting the excessive salt present on skin Gene related to Cystic Fibrosis CFTR - Cystic Fibrosis Transmembrane Regulator Where Cystic Fibrosis gene is located Chromosome 7 (7q31) Responsible for 70% of Cystic Fibrosis disease ĘPhe508 This results in Cystic Fibrosis causing the mucus to be dry The chloride ion channel is blocked, so it cannot move out of the cell and since no ionic gradient is present, no water moves out from the cell into the mucus. Gene Therapy requires knowledge of these 5 things Cell function; gene identity and function; gene expression; function of gene product; mechanisms of gene delivery Two forms of Gene Therapy Somatic GT and Germline GT Three methods of somatic gene delivery ex vivo, in vivo, in situ Advantages of DNA vaccines vs. traditional vaccines simple to develop; inexpensive; effective: protein produced in native form; easy to transport and store; safe: no risk of infection Barriers to gene delivery in the body Elimination by cells of RES; Binding of serum proteins affects biodistribution; Target separated by a biological barrier (BBB or intestinal barrier); Low permeability of target tissues; Degradation of nucleic acids Barriers to gene delivery in the cell Need endocytosis; Must avoid lysosomes which would degrade the gene DNA; Must escape endosomes or lysosomes; Must enter the nucleus; Must avoid DNA purging and replication The 7 requirements for a successful gene delivery system Be efficient in introducing the genes to recipient cells; Capable of short-term or long-term expression as directed; Flexible w.r.t. the target tissues; Harmless to patient; Able to handle a wide range of therapeutic genes; Able to demonstrate a dose-response relationship; Supplied in a form familiar to medical professionals The Central Dogma DNA --> RNA --> Protein RNA can be enyzmatic with these Ribozymes Proposed Cell Theory Mathias Schleiden in 1838 for plants, Theodor Schwann in 1839 for animals Cell Theory Cells make up all living matter; All cells arise from other cells; Genetic material passes from generation to generation; The chemical reactions in an organism (metabolism) occurs within cells Examples of unicellular eukaryotes Fungi, protists Size of a typical prokaryote 1 - 10 µM Two types of prokaryotes Eubacteria and Archaebacteria Three types of archaebacteria Methanogens, halophiles, thermophiles Membrane phospholipids have a hydrophilic end and a hydrophobic end. Such an arrangement is said to be amphipathic Membrane functions permeability barrier; generate & store energy; provide surface for enzymes; responsible for cell-cell communication Components of cell-cell communication cell attachment molecules; receptors for signaling molecules Components of Eubacterial cell surface plasma membrane; cell wall; periplasmic space; outer membrane; locomotion organelles; mesosome Plasma membrane functions primary permeability barrier; only small molecules can diffuse (O2, CO2); amino acid and sugar transport Cell wall in prokaryotes is composed of this peptidoglycan Cell wall in prokaryotes provides this rigidity; capacity for high osmotic pressures (> 20 atm) Periplasmic space function contains enzymes secreted by the cell Outer membrane function more porous proteins (porins) create channels for large molecules Locomotion organelles flagella, cilia Mesosome An infolding of the membrane Mesosome function photosynthesis, chromosome attachment The contents of the cell excluding organelles and membranes cytosol Eubacteria contents are dominated by this ribosomes The prokaryotic genome a single circular chromosome Location of prokaryotic chromosome Attached to membrane (mesosomes) Number of base pairs in E. coli genome 4.7 * 10^6 Prokaryotic strategy Simplicity, fast growth; miniaturization; advantage in harsh changing environments Eukaryotic strategy Specialization, complexity, slow growth; Advantage in stable environment with limited resources Number of proteins per human cell 10,000 Number of genes expressed per human cell 40,000 Approximate size of eukaryotic cell 10 - 100 µM Contains 95% of a eukaryotic cell's DNA nucleus Contains 5% of a eukaryotic cell's DNA mitochondria Chromatin is composed of this DNA and histones Heterochromatin Densely packed; unexpressed DNA Euchromatin Dispersed; expressed DNA; condenses during mitosis Nucleolus A densely staining region of the nucleus Nuceolus function Site of ribosomal RNA synthesis and ribosome assembly Nuclear Matrix is composed of this Cytoskeleton fibers; Lamins Nuclear Matrix function Compartmentalize and control movement within the nucleus Lamins Line inner membrane of the envelope; intermediate filaments of the cytoskeleton; give shape to envelope, bind DNA; phosphorylated during mitosis Nuclear Envelope Double membrane; outer membrane is continuous with the endoplasmic reticulum; outer and inner membranes fuse to create nuclear pores The largest membrane in the cell endoplasmic reticulum Lumen The interior of the ER's series of membrane enclosed ducts Two types of endoplasmic reticulum Smooth (no ribosomes), Rough (with ribosomes) Steps of Cellular Protein Transport Synthesis in rough ER lumen; travel through ER (modification); enter transfer vesicle; Bud from ER into transfer vesicle; Fuse with Golgi Complex The Golgi Complex Membrane system of flattened, fluid filled sacs; routes proteins to final destination; proteins may be modified with carbohydrates, lipids, or phosphates Three regions of Golgi Cis, Medial, Trans Cis Golgi function Fusion of transfer vesicles from ER Medial Golgi function Modification, indicates final destination Trans Golgi Budding of proteins into secretory vesicles Lysosomes Degrade non-functional or damaged molecules or organelles, as well as molecules transported into the cell via endocytosis Lysosomal Enzymes Are acidic hydrolases; phosphatases, nucleases, proteases, polysaccharide hydrolases, lipases Peroxisomes Degrade long chain fatty acids, using oxidases to produce H2O2 which must be removed with catalase Defects in the ability to degrade cause these two diseases Tay Sachs, Adrenoleukodystrophy (ALD) Defects in the ability to transport proteins can cause this disease Cystic Fibrosis Tay Sachs has this type of hereditary influence autosomal recessive Lack of this enzyme causes Tay Sachs Hexosaminidase A (HEX A) How Tay Sachs can be detected Fluorescence assay in utero Adrenoleukodystrophy (ALD) has this type of hereditary influence X-linked Mutation of this enzyme causes Adrenoleukodystrophy (ALD) Acyl CoA synthetase Vacuoles are most common in Plants Vacuole function Storage of water, ions, N-compounds, sugars, degradative enzymes; Creates osmotic pressure (turgor) Mitochondria account for this percentage of cell volume 25% Mitochondria function Oxidize glucose and fat to ATP Cytoskeleton is composed of this Microtubules (tubulin), Microfilaments (actin), Intermediate filaments (lamins)