Introduction to DNA Chapter 4

4.1 DNA Structure and Function Manipulations of DNA and RNA are center for biotech research “Central Dogma” Small sections of DNA (genes) mRNA proteins Transcription Translations at ribosome

4.1 DNA Structure and Function Purines Pyrimidines

4.1 DNA Structure and Function Made of 4 nucleotide monomers that contain 4 nitrogenous bases Form double helix (2 sides) of repeating nucleotides, several million pb in length ie…same DNA structure A from one strand always with T from another and C from one strand always with G from another and ….so equal amounts of each A,T, C, G are stacked 0.34 nm apart with 10 nitrogen bases per complete turn of the helix Number of DNA strands in the cells of an organism (i.e. # of chromosomes) Length in bp of DNA strand Number and types of genes Shape of DNA strand (circular or linear chromosomes) Similarities in DNA Molec. Among Organisms Variations in DNA Molec.

4.2 Sources of DNA Prokaryotic Bacterial cell culture Eukaryotic Mammalian cell culture Viral

4.2 Prokaryotic DNA Bacteria cells ex E.Coli Do not contain nucleus or membrane bound organelle DNA is floating in cytoplasm Typical bacterium contain only one circular long DNA (a chromosome) Some bacteria have extra small rings of DNA (plasmids)

4.2 Prokaryotic DNA Most familiar plasmids are R plasmids Contain antibiotic resistance genes Bacteria with these genes can survive antibiotics that would normally kill them Bacteria can transfer plasmids ie genetic info between themselves So, genes for antibiotic resistance can be transferred btw bacteria and lead to deadly antibiotic resistant disease causing bacteria Gives bacteria a way of evolving

4.2 Prokaryotic DNA Different bacteria have different plasmid Some bacteria have more than one kind Some bacteria have none Plasmids are small and easy to extract from cells Often used as rDNA vectors to transform cells

4.2 Prokaryotic DNA DNA fragments (genes) can be cut and pasted into plasmid vector Recombinant plasmid can then get introduced to cell Cell will read DNA code on the r-plasmid Then start synthesizing proteins coded for the gene Same procedures Genentech, Inc used to produce human insulin from E-Coli

Gene Expression Process Prokaryotic DNA’s gene expression (genes turned on or off ) is simple with only few controls An operon (1 or more genes and their controlling elements) RNA polymerase (enzyme that synthesize mRNA molec.) attach to DNA segment at a promoter region of operon this “turns on” gene RNA polymerase works its way down DNA strand to structural gene to built mRNA mRNA is decoded into a peptide at a ribosome Operator can then “turn off” the gene

Operon Section that codes for mRNA which later get translated to proteins

Gene Expression Process In case of regulatory molec. Attach at operon Operon get “turned off” b/c RNA polymerase is blocked from continuing down the strand to the gene No protein is produced Blocking and unblocking is how bacteria make certain proteins certain times Example: Lactose

Bacterial Cell Culture To manipulate bacteria DNA, cells are needed Some bacteria prefer Liquid medium (Broth) Solid medium (agar) Grow well in either

Agar Mixture of water and protein molec. Preparation: Powdered agar and water are mixed, heated until agar completely suspended Agar is sterilized at high temps (121C or higher) and high pressure (15 psi or higher) cooled to 65C and poured under sterile condition in Petri dishes Agar cools and solidifies in min Plates can be used after 24 hr.

Broth Also water and protein molec. Cultures grow as suspension of million of floating cells

4.2 Eukaryotic DNA From protist, fungi, plants, and animal cells Same as prokaryotic Same nucleotides (A, C, G, T) Same double helix of repeating nucleotides Each antiparallel strand bound to the other by H-bonds Different from prokaryotic Packed into chromosomes (regulated and expressed different from bacteria) Several chromosomes/cell vs. only 1/cell Each chromosome is single, linear, very long molec of DNA Lack of operators Chromosomes range or more Human have 46 chromosomes, fruit flies 8, ferns more than 1000 Amount of DNA/cell is not directly related to organism’s complexity

Gene Expression Controlled differently than prokaryotes Eukaryotic gene expressed at very low level Increase in expression happen when enhancer (section of DNA) molec. interact with RNA polymerase or with enhancer DNA regions Molecules attach to DNA and increase gene transcription

Gene Expression Gene contains promoter region where RNA molec. recognize the gene RNA polymerase attach and move down DNA molec. To get to structural genes At structural genes RNA polymerase builds a complementary mRNA transcript from one side of DNA strand The enzyme transcribes the entire gene until reaches a termination sequence

Gene Expression Prokaryotic mRNA transcript is immediately translated into polypeptide at a ribosome No introns and exons at structural genes Operator to regulate gene expression Eukaryotic mRNA is often modified before translation Structural genes are made of intron and exon sections Exons: DNA sections that contain the protein code Introns: spacer DNA No operator so produce molec. called transcription factors to turn genes on Also regulated by way chromosome coiled (around histones proteins) Coiling makes genes buried and RNA polymerase can not get to them DNA has to uncoil

Mammalian Cell Culture Growing them more challenging that bacteria Normally grown in broth culture

4.2 Eukaryotic DNA Viral DNA Viruses Do not have cellular structure Collection of protein and nucleic acid molec. that become active once they are within a suitable cell Very small, measure from nm Based on type of cell they attach classified as: Bacterial, plant, or animals Have thick protein coat surrounding nucleic acid core of either DNA or RNA Animal virus

Viral DNA Within a cell: Nucleic acid of a virus is released Viral genes are read by host cell enzyme Decoded into viral mRNA Translated into viral proteins New virus molec. are assembled and released and may infect other cells

Viral DNA Viral DNA and RNA molec are short, easy to manipulate Called vectors

4.3 Isolating and Manipulating DNA Genetic engineering (G.E.): All modifications of DNA code of an organism Process: ID of molec produced by living things that could be produced more economically or easily through G.E. (ex. insulin) Isolation of gene for production of molec (insulin gene) Manipulation of DNA instruction Harvesting of molecule or product

Recombinant DNA Technology Methods used to create new DNA molec by piecing together different DNA molec When cells accept rDNA and start expressing the new genes by making new proteins….they are considered genetically engineered Many items on market with rDNA technology Ex recombinant rennin, chymosin

Site-Specific Mutagenesis Process of inducing changes in certain sections of a particular DNA code Accomplished through use of chemicals, radiation, viruses Sometimes “directed”(on purpose by scientist to make better changes) Ex: subtilisin marketed by Genecor international (tide) An enzyme (protease) that degrades proteins Added to laundry detergent to stains like blood, gravy

Gene Therapy Process of correcting faulty DNA codes that causes genetic diseases and disorders Common way to use virus to carry a normal gene into cells containing defective ones (gene replacement) Correct diseases like Parkinson's, diabetes, cystic fibrosis (CF), and some cancers CF: One of every 3000 babies born with it Causes build up of thick mucus that clogs the respiratory and digestive systems

Gene Therapy In 2002: Modified cold virus used to transfer normal copy of the gene cystic fibrosis transmembrane conductance regulator (CFTR) To cells lining the nose CFTR gene is defective in CF patients Regulates flow of Cl- ions into epithelial cells lining respiratory and digestive systems This method called adeno-assosiated virus (AAV) delivery system b/c uses modified adenovirus (cold virus) Worked only in small # still in clinical trials

4.4 Gel Electrophoresis Uses electricity to separate charged molec. on a gel slab Separation based on size, shape and charge Ex: DNA and RNA fragments, and proteins Gel: Powdered agarose (carb. derived from seaweed) Dissolve in boiling buffer soln. Most common agarose is polyacrylamide (PAGE) Gel solidify and placed in get box and covered with buffer soln.

Gel Electrophoresis

Agarose used when separating DNA pieces no smaller than 500bp and no larger than 25,000bp Made at a specific concentrations ranging (0.6- 3%) figure 4.32 High concentration for smaller molec

Gel Electrophoresis Gel stains: Nucleic acids are colorless Must be stained DNA stains: Ethidium bromide (EtBr)…orange when mixed with DNA under UV light Methylene blue…dark blue…not as sensitive as EtBr viewed with white light

Gel Electrophoresis Note: Only negative charge would run on this one Only one DNA type most common Sizing standard Plasmid restriction digestion DNA sample from bacterial chromosome RNA Smears (thousands of different size molec in small concentration) No nucleic acids DNA so large will not load Ex: eukaryotic genome

Homework Sec 4.3 Review question 1, 2, 3, 4 Sec 4.2 Review questions 1, 2, 3, 4 Sec 4.4 Review questions 2, 3, 4 Think like Biotech 2, 3, 6, 7, 8

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