Life 102, Week 14 Notes
Life 102, Week 14 Notes Life 102
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This 8 page Class Notes was uploaded by Sydney Dingman on Thursday April 21, 2016. The Class Notes belongs to Life 102 at Colorado State University taught by Erik N Arthun in Winter 2016. Since its upload, it has received 17 views. For similar materials see Attributes of Living Systems in Biology at Colorado State University.
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Date Created: 04/21/16
LIFE 102, Week 14 Notes 4/18/16, Chapter 19 cont. Lysogenic Cycle o The lysogenic cycle replicates the phage genome without destroying the host o The viral DNA molecule is incorporated into the host cell’s chromosome o This integrated viral DNA is known as a prophage o Every time the host divides, it copies the phage DNA and passes the copies to daughter cells o An environmental signal can trigger the virus genome to exit the bacterial chromosome and switch to the lytic mode RNA as Viral Genetic Material o The broadest variety of RNA genomes is found in viruses that infect animals o Retroviruses use reverse transcriptase to copy their RNA genome into DNA Reverse transcription: RNA template DNA o HIV (human immunodeficiency virus) is the RNA retrovirus that causes AIDS: Acquired Immunodeficiency Syndrome HIV Reproductive Cycle o The viral DNA is integrated into the host genome is called a provirus o Unlike a prophage, a provirus remains a permanent resident of the host cell o The host’s TNA polymerase transcribes the proviral DNA into RNA molecules o The RNA molecules function both as mRNA for synthesis of viral proteins and as genomes for new virus particles released from the cell HIV: an RNA retrovirus o RNADNAinserts in host genomeNew viral RNA, proteins virus assembly, cell lysis Transcription, translation Protection from Viral Infections o Viral infections cannot be treated by antibiotics o Vaccines are harmless derivative of pathogenic microbes that stimulate the immune system to mount defenses against the harmful pathogen o Vaccines can prevent certain viral illnesses o Antiviral drugs can help to treat, though not cure, viral infections Emerging Viruses o Emerging viruses are those that suddenly become apparent Human Immunodeficiency Virus Ebola virus West Nile virus o Viral diseases in a small isolated population can emerge and become global o New viral diseases can emerge when viruses spread from animals to humans o Viral strains that hump species can exchange genetic information with other viruses to which humans have no immunity Epidemics o Recently, a general outbreak (epidemic) of a flu-like illness appeared in Mexico and the United States, caused by an influenza virus names H1N1 o Flu epidemics are caused by new strains of influenza virus to which people have little immunity Prions: The simplest of infectious agents o Prions are slow-acting, virtually indestructible infectious proteins that cause brain diseases in mammals o Prions propagate by converting normal proteins into the defectively-folded prion version o Scrapie in sheep, mad cow disease, chronic wasting disease, and Creutzfeldt-Jakob disease in humans are all caused by prions o Prions propagate by converting normal proteins into the defectively-folded prion version 2 4/18/16, Chapter 20: Biotechnology The manipulation of organisms or their genetic components to make useful products Genetic engineering: the artificial manipulation of genetic information o Changing the properties of organisms o Tailor them for our benefit o Other examples: sequencing DNA from a crime scene can identify a criminal by genotype, not phenotype o Diseases normally treated by protein-replacement therapy might be treated by actually fixing the underlying mutation Applications of Genetic Engineering o Agriculture, Medicine, Genetic Screening, Forensics, Pollution Cleanup DNA Technology o Recombinant DNA Techniques Isolate DNA molecules Cut & Paste recombine them Transfer to organism o Genetically engineered organism Transgenic organism GMO Tools used in DNA technology o Enzymes that can modify DNA o Vectors that can carry foreign DNA into a host cell and replicate there o Hosts to replicate the DNA of interest Enzymes: Cut & paste DNA from different sources o Cloning Vector: Bacterial plasmids Small circular DNA molecules that replicate separately from the bacterial chromosome o Cloning Host: bacterium itself Enzymes used in DNA technology 3 o Restriction enzymes Bacterial enzymes that cut DNA molecules at specific DNA sequences Cut DNA in a staggered way, producing fragments with “sticky ends” Sticky ends can be used to join DNA from different organisms by cutting both organisms’ DNA with the same restriction enzyme All fragments will have complementary ends o DNA Ligase Enzyme that seals the bonds between DNA fragments Enzymes o Cut vector and gene of interest: restriction enzymes o To paste vector and gene of interest together: ligase o DNA polymerase Used for polymerase chain reaction 1 DNA copy billions of copies Making copies of DNA sequences: Polymerase Chain Reaction (PCR) o Small amounts of DNA or RNA are limiting factor in some tests aimed at analyzing the genetic information o PCR rapidly-increases the amount of DNA in a sample o Can amplify a target DNA from a few copies to billions in a few hours! o Can detect a single cancer cell or a single gene from a pathogen! o Cycle: Denaturation Heat to 94 degrees Celsius to separate DNA into 2 strands Cool to 50-65 degrees Celsius Priming Primers added, bind to complementary strand of test DNA Prepares the 2 DNA strands for synthesis 4 Extension 72 degrees Celsius DNA polymerase and nucleotides are added Polymerases extended the molecule Repeat the steps 25-40 times 4/20/16, Chapter 20 cont. PCR o Target DNA sequence is amplified o Automated PCR machines can perform 20 cycles on ~100 samples in 1-2 hours Enzymes (1) o mRNA Reverse transcriptase complementary DNA (cDNA) no introns cDNA from Eukaryotes o Need to get rid of intron sequences o Isolate mRNA o Reverse transcription o mRNAcDNA (free of introns) Cloning Vectors (2) o Isolated genes are typically spliced into “cloning vectors” Thousands are available commercially o Examples of Vectors: Plasmids (transferred by host by transformation) Bacteriophages (naturally inject DNA into host via transduction) Cloning Hosts (3) o Bacteria or Yeast o A problem with expressing Eukaryotic genes in prokaryotic cells: prokaryotic cells (such as E. coli) cannot splice mRNA because their genes do not have introns How are genes cloned? 5 o Isolate and propagate o Make a gene library o Collection of all the genes from a cell o Pick out the gene of interest Genomic Library o DNA library from the entire genome (100,000) cDNA library o DNA library from the expressed genes o ~5000 5000 mRNAs Reverse Transcriptase 5000 cDNAs How is the Gene of Interest Identified? o Use a molecule that recognizes gene/gene product (protein) Nucleic Acid Probe” Complementary DNA sequence that binds to gene of interest Antibody Protein that binds specifically to another protein Nucleic Acid Hybridization and Probes o Two different nucleic acids can “hybridize” by uniting at their complementary regions o Nucleic acid probes: Short stretches of ssDNA of a known sequence Will base-pair with ssDNA containing a complementary sequence Can detect specific nucleotide sequences in unknown samples Probes carry reporter molecules so they can be visualized “labeled” via fluorescence, radiation, luminescence, or enzymes o Example of Hybridization Technique: Southern Blot Gel Electrophoresis 6 o Used for DNA analysis o DNA samples are placed in wells in an agarose gen Subjected to an electrical current o (-) charged DNA passes through gel toward (+) charge Large fragments move slowest Small fragments move fastest o DNA fragments are stained and observed for a pattern o Produces a readable pattern of DNA fragments for characterization and comparison “Southern Blot” o Locates specific DNA sequences o DNA is run on a gel o Transfer to membrane o Add labeled probe and let it hybridize o Wash off any unbound probes o Detect bound probes using label Using a cloned gene to transform an organism o Add regulatory sequences (promoter, etc.) o Add selection marker (resistance gene) o Transform gene construct to cell o Regenerate transformed cell into organism Biotechnology applications o Diagnostics: diseases, pregnancy o Genetic screening o Medicine production: insulin, growth factors o Forensics: rape/murder suspects o Biofuel production: oils, plastics o Bioremediation: pollution cleanup o Crop improvement: resistance, yield Genetic Treatments: Gene Therapy o Some diseases are due to the lack of a specific protein 7 Type I Diabetes is caused by a lack of insulin o Goal of Gene Therapy: to permanently cure a physiological dysfunction by correcting or repairing a faulty gene o Two strategies: In vivo: naked normal DNA or cloned virus vector is introduced directly into patient tissues Ex vivo: Clone normal gene in virus vector Incubate virus with tissues removed from patient Reintroduced transfected cells into patient o Examples of uses: immunodeficiency diseases, hemophilia, cancers… o Some diseases are due to the lack of protein o Introduce good protein by introducing normal gene o Phenotype is corrected 8
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