Cytogenetics: DNA Replication & Protein Synthesis PDF

CYTOGENETICS polymerase to bind complementary bases, A with T and G with C DNA Replication & 4. Sugar-phosphate backbones of...

CYTOGENETICS polymerase to bind complementary bases, A with T and G with C DNA Replication & 4. Sugar-phosphate backbones of daughter strands close Protein Synthesis Enzymes in DNA Replication DNA Replication Is Semiconservative Helicase - unwinds parental double helix Binding proteins - stabilize separate strands Primase - adds short primer to template Every double helix in a generation of an strand organism is consist of one complete old strand DNA polymerase - binds nucleotides to and one complete new strand wrap around form new strands each other. Ligase - joins Okazaki fragments and seals other nicks in sugar-phosphate backbone. DNA Replication - left out all spaces that has no DNA fragment Matthew Maselson & Franklin Stahl, 1957 Activities at the Replication Fork Demonstrated the semiconservative mechanism of DNA replication with a series of density shift experiments Labeled replicating DNA from bacteria with a heavy form of nitrogen and traced its pattern of distribution 1. Helicase binds to origin and Higher-density nitrogen was separates strands incorporated into one strand of each daughter 2. Binding proteins keep strands apart double helix 3. Primase make a short stretch of RNA on the DNA template Overview of DNA Replication 1. Parent DNA molecule 2. Parental strands unwind and separate at several points and hydrogen bonds 4. DNA polymerase adds DNA break nucleotides to the RNA primer 3. Each parental 5. DNA polymerase proofreading strand provides a activity checks and replaces template for DNA incorrect bases.  PCR Thermal cycler - used to produce large amount required for research purposes USES Pharmaceutical research labs - analyze and duplicate RNA and DNA samples Clinical healthcare - diagnose infected patients Forensic - amplify DNA samples 6. Continuous strand synthesis continues in a 5’ to 3’ direction 4 components in PCR 7. Discontinuous synthesis produces DNA or RNA samples - skin, saliva, hair Okazaki fragments on the 5’ to 3’ DNA primers - short single stranded DNA template that promote synthesis of complementary strand of nucleotides DNA polymerase - enzymes that aid in the synthesis of complementary strand of DNA Nucleotide solution mix - containing adenine, thymidine, cytosine and guanine 8. Enzymes remove RNA primers. Ligase seals sugar-phosphate Steps in Amplifying DNA Using PCR backbone Replication Bubbles The sites of replication resemble bubbles that coalesce as the daughter double helices form. 1. Select target sequence in virus genome 2. Primers Polymerase Chain Reaction (PCR) Preparation 3. Free-nucleotides DNA amplification technique 4. Heat-resistant polymerase Temperature shift Uses DNA polymerase to rapidly replicate a 5. Target sequence (Up) specific DNA sequence in a test tube 6. Heat separates strands (Down) - Yields more than 10 billion copies of the Hybridization target DNA sequence 7. Primers hybridize due to base - Useful in forensic investigations to amplify complementarity small DNA samples 8. DNA fills in  9. Repeat process many times Each amino acid has a central carbon atom Amplification that bonds to an amino group (NH2) and an acidic group (COOH), a hydrogen atom (H), and a variable “R” group Amino Acid Structure Gene Expression DNA of the human genome which encodes protein is the exome 1. Denaturation However, this represents only a small part of Heated to 94˚C or 201.2 F using the genome thermal cycler heat breaks the hydrogen bonds of Much of the human genome controls protein the original DNA samples and separates the synthesis DNA into a single strand including the time, speed and location Genes encoded 20,325 types of proteins 2. Annealing cold— 120 - 140 F or 50 - 60˚C, Production of protein from instruction on the which will allow DNA primers and DNA DNA polymerase to bind to the individual strands of Gene expression requires several steps: DNA resulted in heating process Transcription - Synthesizes an RNA molecule 3. Extension Translation - Uses the information in the once joined together it form new RRNA to manufacture a protein by aligning complementary strands of DNA and joining specified amino acids Folding of the protein into specific 3-D form Proteins Exome - protein coding gene Central Proteins serve many vital functions: Dogma -contractile -structural directional -regulatory -transport flow of genetic -enzymatic -clotting information Proteins are comprised of one or more long chains of amino acids called polypeptides There 20 naturally occurring amino acids Transcription Setting the Stage for Transcription to Begin Factors Interact and form an apparatus that binds DNA at certain sequences Initiates transcription at specific sites on chromosomes Respond to signals from outside the cell Link the genome to the environment Mutations in transcription factors may cause a wide range of effects Transcription of RNA from DNA Steps Transcription is described in three steps: Initiation Elongation Termination In transcription initiation, transcription factors and RNA polymerase are attracted to a promoter RNA polymerase joins the complex, binding in front of the start of the gene sequence In transcription elongation, enzymes unwind the DNA double helix Simultaneous Transcription of mRNAs free RNA nucleotides bond with exposed complementary bases on the DNA template Several mRNAs may be transcribed from the strand same template DNA strand at a time RNA polymerase adds the RNA nucleotide, in the sequence the DNA specifies A terminator sequence in the DNA indicates where the gene’s RNA-encoding region ends. RNA Processing Messenger RNA Processing — the Maturing of the Message In eukaryotes, mRNA must exit the nucleus to enter the cytoplasm Several steps process pre-mRNA into mature mRNA 1. A methylated cap is added to the 5’ end Recognition site for protein synthesis 2. A poly A tail is added to the 3’ end Necessary for protein synthesis to begin and stabilized the mRNA 3. Splicing occurs Introns (“Intervening sequences”) are removed Ends of the remaining molecule Translation are spliced together Exons are parts of mRNA that Assembles a protein using the information in remain, translated into amino acid sequences the mRNA sequence Note that introns may outnumber particular mRNA codons correspond to and outsize exons particular amino acids 4. mRNA is proofread and the mature Occurs on the ribosome mRNA is sent out of the nucleus Alternate Splicing Mechanism of combining exons of a gene in different ways: Cell types can use versions of the same protein in slightly different ways in different tissues The Genetic Code the correspondence between the chemical languages of mRNA and proteins  In the 1960s, researchers used logic and clever experiments on simple genetic systems to decipher the genetic code consists of mRNA triplets and the amino acids that they specify It is universal Evidence that all life evolved from a common ancestor Different codons that specify the same amino acid are termed synonymous codons Nonsynonymous codons encode different amino acids Three at a Time It is a triplet code Three successive mRNA bases form a codon There are 64 codons Altering the DNA sequence by one or two bases produced a different amino acid sequence due to disruption in the reading frame Adding a base at one point and deleting a base at another point disrupted the reading frame between the sites. It is nonoverlapping Reading Frame In an overlapping DNA sequence, certain amino acids would follow others, constraining protein structure It includes controls Includes directions for starting and stopping translation An open reading frame does not include a stop codon  Position of the sites on the ribosome remain the same, covering different parts of the Translation—Building a Protein mRNA as the ribosome moves. The P site bears growing amino acid chain Requires mRNA, tRNAs with amino acids, The A site holds the next amino acid to be ribosomes, energy molecules (ATP, GTP) and added to the chain protein factores Amino acids link by a peptide bond, with the Divided into three steps: help of rRNA that functions as a ribozyme Initiation Elongation The polypeptide builds one amino acid at a Termination time Each piece is brought in by a tRNA whose Translation Initiation anticodon corresponds to a consecutive mRNA codon as the ribosome moves down The leader sequence of the mRNA forms H the mRNA bonds with the small ribosomal subunit The start codon (AUG) attracts an initiator tRNA that carries methionine Building a Polypeptide This completes the initiation complex Translation Begins as the Initiation Complex Forms Translation Elongation The large ribosomal subunit joins GGA bonds to its complementary anticodon, which is part of a free tRNA that carries the amino acid glycine Two amino acids attached to their tRNAs align Translation Termination Protein Structure Occurs when a stop codon enters the A site of the ribosome Protein fold into one or more 3-D shapes or A protein release factor frees the polypeptide conformations The ribosomal subunits separate and are Based on attraction and repulsion between recycled New polypeptide is released atoms of proteins, and interactions of proteins with chemicals in the environment There are four levels for protein structure: Primary (1˚) structure Secondary (2˚) structure Tertiary (3˚) structure Quaternary (4˚) structure Four Levels of Protein Structure Multiple copies of a protein can be made simultaneously Primary Structure - sequence of amino acids in a polypeptide chain Secondary Structure - loops, coils, sheets, or other shapes formed by hydrogen bonds between neighboring carboxyl and amino groups Tertiary Structure - three-dimensional forms shaped by bonds between R groups, interaction between R groups and water  Quaternary Structure - protein complexes As the protein moves through the tunnel, it is formed by bonds between separate straightened and dismantled polypeptides Proteasomes also destroy properly folded proteins that are in excess or no longer needed Protein Folding Proteins misfold from a mutation, or by Protein begin to fold after the amino acid having more than one conformation chain winds away from the ribosome A mutation alters the attractions First few amino acids in a protein secreted in and repulsions between parts of the protein a membrane form a “signal sequence” Prion protein can fold into any of Leads it and the ribosome into a several conformations pore in the ER membrane Moreover, it can be passed on to Not found on proteins synthesized other proteins upon contact, propagating like on free ribosomes an infectious agent. Chaperone Proteins In several disorders that affect the brain, Stabilize partially folded regions in their the misfolded proteins aggregate correct form The protein masses that form clog Prevent a protein from getting stuck in an the proteasomes and inhibit their function intermediate form Developed into drugs to treat diseases that result from misfolded proteins Protein Misfolding Prion Diseases They have been found in 85 species Scrapie in sheep In humans Kuru Misfolded proteins are tagged with ubiquitin Creutzfedt-Jakob disease Protein with more than one tag is taken to a proteasome, a tunnel-like multiprotein Prion Diseases of Humans structure Creutzfeldt-Jakob disease Fatal familial insomnia  Gerstmann-Straüssler-Scheiner Disease Prions  1790 U.S. patent act enacted. A patented invention must be new, useful, and not obvious. 1873 Louis Pasteur is awarded first patent on a life form, for yeast used in industrial processes. DNA TECHNOLOGIES 1930 New plant variants can be patented. Biotechnology is the use or alteration of cells 1980 First patent awarded on a genetically modified organism, a bacterium given or biological molecules for specific four DNA rings that enable it to applications such as: metabolize components of crude oil. Providing breakthrough products and technologies to combat debilitating and rare 1988 First patent awarded for a transgenic organism, a mouse that manufactures diseases human protein in its milk. Harvard Reduce environmental foot print University granted a patent for Feed the hungry "OncoMouse," transgenic for human cancer. Use less and cleaner energy Have safer, cleaner, and more efficient 1992 Biotechnology company awarded industrial manufacturing processes patent for all forms of transgenic cotton. Groups concerned that this will limit the rights of subsistence farmers Transgenic organism contest the patent several times. has DNA from different species introduced using biotechnology. 1996- Companies patent partial gene Recombinant 1999 sequences and certain disease-causing genes for developing specific medical DNA comes from more than one type of tests. organism. 2000 With gene and genome discoveries Patentable pouring into the Patent and Trademark To qualify for patent protection, a transgenic Office, requirements for showing utility of a DNA sequence are organism must be new, useful, and non- tightened. obvious. Patent law has had to evolve to keep up with 2003 Attempts to enforce patents on modern biotechnology. non-protein-encoding parts of the human genome anger researchers who A DNA sequence alone does not warrant support open access to the patent protection: It must be useful as a tool information. for research or as a novel or improved product, such as a diagnostic test or drug. 2007 Patent requirements must embrace a new, more complex definition of a DNApatentingisevolvingtoembrace gene genome-wide applications. 2009 Patents on breast cancer genes Technology Timeline challenged. Patenting Life and Genes 2010 Direct-to-consumer genetic testing  The cutting action of many of these enzymes companies struggle to license DNA patents for multigene and SNP generates single-stranded extensions called association tests. “sticky ends.” Patents on breast cancer genes Cloning vector invalidated. Carries DNA from cellsofonespeciesintothe 2011 U.S. government considers changes to cells of another gene patent laws. provide a basic backbone for the DNA insert to be reproduced and generally have the 2013 U.S. Supreme Court declares genes common features just described, but these unpatentable. vectors are useful only for cloning and not for expressing a protein product. Because DNA testing companies are forming faster than the Patent and Trademark Office Commonly used vectors include: can evaluate patent applications, Plasmids the US government is exploring ways around Bacteriophages the patent thicket: Disabled retroviruses Ban the patenting of associations between DNA sequence variants and disease. Recombining DNA Exempt the use of DNA in a diagnostic or Please take note: EcoR1 risk assessment test from patent infringement. Exempt physicians and researchers from litigation if they use patented DNA sequences Patent Thicket The effect of multiple patents on a gene. Means that a company or researcher developing a tool or test based on a particular gene or its encoded protein might infringe upon several patents that are based on essentially the same information. Recombinant DNA technology Also known as gene cloning adds genes from one type of organism to the genome of another. First gene modification biotechnology Was initially done in bacteria to produce peptides and proteins useful as drugs. creating Recombinant DNA Molecules Restriction enzymes Cut donor and recipient DNA at the same sequence/ palindromic.  Factor VIII Promotes blood clotting in Three types of recipient cells can result from treatment of hemophilia A attempt to introduce a DNA molecule into a bacterial cell. Glucocerebrosida Corrects enzyme deficiency Cells that lack plasmids se in Gaucher disease Cells with plasmids that do not contain Human growth Promotes growth of muscle foreign hormone and bone in people with genes very short stature due to Cells that contain plasmids with foreign hormone deficiency genes Insulin Allows cells to take up Applications of Recombinant DNA glucose in treatment of type Recombinant DNA is used to: 1 diabetes Study the biochemical properties or genetic pathways of that protein Interferons Treat genital warts; hairy cell leukemia; hepatitis B Mass-produce proteins (e.g., insulin) and C; Kaposi sarcoma; Sometimes conventional methods are still multiple sclerosis the better choice because of economics Textile industry can produce indigo dye in Interleukin-2 Treats kidney cancer recurrence E. coli by genetically modifying genes of the glucose pathway and introducing genes from Lung surfactant Helps lung alveoli to inflate another bacterial species protein in infants with respiratory distress syndrome Drugs Produced Using Recombinant DNA Renin inhibitor Lowers blood pressure Technology Somatostatin Decreases growth in muscle and bone in pituitary gigantism Drug Use Superoxide Prevents further damage to Atrial natriuretic Dilates blood vessels, dismutase heart muscle after heart peptide promotes urination attack Colony- Help restore bone marrow Thrombin Stops postsurgical bleeding stimulating after marrow transplant; factors restore blood cells following Tissue Dissolves blood clots in cancer chemotherapy plasminogen treatment of heart attack, activator stroke, and pulmonary Deoxyribonucleas Thins secretions in lungs of embolism e (DNase) people with cystic fibrosis Epidermal growth Accelerates healing of Transgenic Organisms factor wounds and burns; treats An even more efficient way to express some gastric ulcers recombinant genes is in the body fluid of a Erythropoietin Stimulates production of red transgenic animal. (EPO) blood cells in cancer Transgenic sheep, cows, and goats have all patients expressed human genes in their milk. Clotting factors  Clot busters Collagen Genetically Modified Foods Antibodies Forms Of Genetic Modification Based On phenotype, such as taste or appearance Several techniques are used to insert DNA Genetically Modified Organisms into cells to create transgenic animals: - Organisms altered to have genes from other Chemicals that open transient holes in species or to over- or under express their own plasma membrane genes Liposomes that carry DNA into cells Objections: Field tests may not adequately predict the Transgenic Animals effects on ecosystems Finally,an organism must be regenerated Lead to genetic uniformity from the altered cell. If the trait is dominant, the transgenic animal Some Genetically Modified Organisms must express it in the appropriate tissue at the right time in development. If the trait is recessive, crosses between Organism Altered Trait heterozygotes may be necessary to yield homozygotes that express the trait. Grapes Less sugar Fluorescent Transgenic Mice Cassava, papaya, Resist viral Mice containing the jellyfish gene for green plum infection fluorescent protein (GFP) Cattle Resist mad cow Animal Models disease Transgenic animals are far more useful as Sugar beets, corn, Tolerate an models of human diseases. soybeans herbicide Example: Inserting the mutant human beta globin gene that causes sickle cell anemia into Bananas, rice More iron and mice vitamin A Drug candidates can be tested on these animal models before testing on humans. Canola Altered fatty acid Will be abandoned if they cause significant composition side effects Salmon Faster growth Transgenic animal models have limitations: Researchers cannot control where a Bioremediation transgene inserts, and how many copies do so. The use of bacteria or plants to detoxify The level of gene expression necessary for a environmental pollutants phenotype may differ in the model and Examples: humans. Nickel-contaminated soils Animal models may not mimic the human Mercury-tainted soils condition exactly because of differences in Trinitrotoluene (TNT) in land mines development or symptoms. Monitoring Gene Function Microarrays (gene chips) are devices that detect and display the mRNAs in a cell. Piece of glass or plastic that is about 1.5 centimeters square The color and intensity pattern of the microarray provides a glimpse of gene expression following spinal cord injury. DNA Microarray DNA microarrays enable researchers to track gene expression. In a DNA microarray experiment: DNA pieces of known sequence (mRNAs) are extracted from the samples, and cDNAs are made. Time After Injury Type of Increased These are differentially labeled and then (rats) Gene Expression applied to the microarray. The patterns and color intensities of spots Day 1 Protective genes to indicate which genes are expressed. preserve remaining A laser scanner detects and a computer tissue algorithm interprets the results. Day 3 Growth, repair, cell division DNA MICROARRAY EXPERIMENT: Gene Expression in Response to Spinal Cord Injury Day 10 Repair of connective tissues Angiogenesis Day 30-90 Blood vessel mature New type of connective tissue associated with healing Gene Silencing and Genome Editing Gene Silencing Techniques Block Synthesis of, or degrade, mRNA. Genome Editing Techniques Create Double - stranded breaks in the DNA double helix, enabling insertion of a desired DNA sequence or removal of a sequence. Gene Silencing by Use of Ribozyme Ribozyme is a RNA-based enzyme in the ribosome. ItfitstheshapesofcertainRNAmolecules, and can cut the RNA molecule. Because it can cut the RNA molecule, it could be used to destroy RNA from pathogens, such as HIV. Gene Silencing by Use of RNA interference (RNAi) Gene Silencing by Antisense Technology Block gene expression by introducing RNA that is complementary to the gene’s mRNA transcript. TheintroducedRNA,calledantisenseRNA, binds to the mRNA, preventing its translation into a protein. Gene Silencing by Use of Morpholinos A morpholino consists of 25 DNA bases bonded to each other by organic groups that are not the sugar-phosphate ones in DNA. The morpholinos can block splice-site mutations that would delete exon sites in a gene. One use is in Duchenne muscular dystrophy. The morpholino blocks an exon site necessary to produce effective dystrophin.