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19.1- A virus Consists of a Nucleic Acid with a protein Coat Adam Maurer, D.J. Fezler, Julia Ceh-Synnott The Discovery of viruses: Scientific inquiry • In 1883, Adolf Mayer, discovered that he could transmit the tobacco mosaic disease. • Searched for the cause of TMV • Concluded that the cause of disease was smaller than a bacteria cell • In 1935, Wendell Stanley crystallized the infectious particle • Concluded that the cause was not living because you cannot crystallize a cell Structure of viruses • Virus • A particle containing a nucleic acid • surrounded by a protein coat or a Capsid • Some viruses surrounded by a membranous envelope Viral Genomes • Virus Genomes • Can be a double-stranded DNA, single-stranded DNA, double-stranded RNA, or singlestranded RNA • Smallest virus = 4 genes • Largest virus = several hundred to a thousand genes Capsids and Envelopes • Capsid • –The protein shell that surrounds the viral genome • Shapes: rod-shapes, polyhedral, or more complex structures • Made up of capsomers (protein subunits) • TMV – rod shaped • Helical viruses – rod shaped (Continued) • Some viruses have structures to help them infect the host • Bacteriophages (phages) • Complex capsids that infect bacteria • Elongated icosahedral heads enclosing DNA • Protein tail with fibers that attach to a bacterium 19.2 Viruses replicate only in host cells Steven Norlock, Brandon Geiringer, and Eric Lakomek Viruses lack metabolic enzymes and equipment for making proteins; ribosomes ■ They are obligate intracellular parasites – Can only replicate in a host cell ■ Each virus has a host range – Limited number of host cells that it can infect It is fair to say that viruses in isolation are merely packaged sets of genes in transit from one host cell to another. Viral Reproduction ■ Once a viral genome has entered the cell – It begins to manufacture viral proteins – By using the host cells materials ■ Enzymes ■ Ribosomes ■ tRNA ■ Amino acids ■ ATP Phages ■ Best understood of all viruses ■ They have two reproductive cycles – Lytic – Lysogenic Even though phages are the most understood they are the most complex. Lytic Cycle ■ Culminates the death of the host cell – Produces new phages – Digests cell wall – Releasing the progeny virus ■ Phage that produces only by lytic cycle is a virulent phage ■ Defense against phages – Restriction enzymes ■ Recognize and cut up certain phages of DNA Lysogenic Cycle ■ Replicates the phage genome without destroying it ■ DNA molecule is incorporated into the host cells chromosome – Called a prophage ■ Every time host divides it copies phage DNA and passes it to daughter cells ■ Viruses that can be lysogenic or lytic are called – Temperate phages Replicative Cycles of Animal Viruses ■ Viral Envelopes – Uses outer membrane to enter the host cell Some viruses have envelopes not derived in plasma membrane -- Herpesviruses (nuclear envelope) -- reoccur throughout lifetime RNA as Viral Genetic Material ■ RNA viruses – Single stranded RNA genomes – If class IV, can become mRNA and translate immediately – after infection Retroviruses(Class VI) -- most complicated repetitive cycles -- enzyme called reverse transcription --HIV --AIDS Evolution of Viruses ■ Viruses don’t fit definition of living organisms ■ Evolved as bits of cellular nucleic acid ■ Source of viral genomes are plasmids, circular DNA in bacteria and yeasts, and transposons, and small mobile DNA segments ■ANY Questions I’m the new virus in town 19.3-SIMPLEST FORMS OF VIRAL INFECTIONS Jordan Smith Cammie Voss Kayla Tate Jake Gilliam Background Info Diseases that stem from viral infections affect a variety of organisms (humans, crops, livestock) Two categories of viral infections are viroids and prions Viroids affect plants while prions affect animals How viruses work Viruses inflict damage by removing hydrolytic enzymes from lysosomes Enzymes break down liquids and cell is harmed when liquid becomes too abundant Many symptoms then arise from the body’s effort to defend itself Vaccinations vaccine- harmless derivative of a pathogen that stimulates the immune system to defend itself against the harmful pathogen Increases the awareness of the immune system Example: The World Health Organization (WHO) was able to remove the smallpox virus with a vaccination program Since antibiotics are powerless against viruses, vaccinations and antiviral drugs have been responsible for interfering with the growth of pathogens Antiviral Drugs Antiviral drugs interfere with the enzymes required for the assembly and replication of viruses Example: acyclovir and azido thymidine both impede the replication of STDs Emerging viruses- viruses that spontaneously arise AIDS virus and Ebola virus Emerging viruses can be stopped early if antiviral drugs prohibit their replication Spreading of disease Epidemic- general outbreak of a virus Example: The flu virus (H1N1) infected over 600,000 people and killed 8,000 The event was declared a pandemic – when a disease has spread between continents – was declared by the WHO 3 ways viral diseases emerge 1) The mutation of existing viruses. RNA viruses tend to have an unusually high rate of mutation because the process with replicates their genomes does not have a proofreading enzyme. 2) The dissemination of a viral disease from an isolated human population. International travel, blood transfusions, sexual promiscuity, and drug abuse lead to this. 3) The spread of viruses from other animals. Animals most likely to transmit a disease are unaffected while living with it. 3 types of influenza Type A infects a wide range of animals, occasionally humans Not likely to cause an epidemic Types B and C infect only humans Prime causes of epidemics Flu examples: 1918 pandemic , H1N1 virus (2009), H5N1/Avian Flu (1997) Viral diseases in plants Viral infections spread between plants in 2 ways: 1) horizontal transmission- the plant is infected by an external source of the virus Physical damage to a plant allows an invading virus to break through the epidermis 2) vertical transmission- plant inherits a viral infection through either sexual or asexual reproduction Plasmodesmata allows a virus to replicate and therefore allows the spread of viral proteins throughout the cells in a plant Simple viruses Viroids- circular RNA molecules (a few hundred nucleotides long) that infect plants Cause errors in regulatory systems that control plant growth Prions- infectious proteins which evidently cause a number of degenerative brain diseases in various animal species most likely transmitted through foods Mad cow disease Mad Cow Disease Plagued European beef industry Consumers became concerned whether or not the infected beef would affect them The severity of prions Prions have shown scientists two very alarming traits: 1) They act slowly, with an incubation period of 10 years before symptoms show 2) Virtually indestructible, not destroyed by being heated to normal cooking temperatures A prion is essentially a misfolded form of a protein that is normally found in brain cells. It has the ability to convert normal proteins to misfolded ones within body tissue. 20.1 – DNA CLONING A LY S S A G I L L , V I C T O R I A R I C O , J O S E C A P E T I L L O , A N D A N D R E W L A M PA R S K I DNA CLONING YIELDS MULTIPLE COPIES OF A GENE • DNA molecules are very long -a single molecule carries many genes • Genes only occupy small proportions of chromosomal DNA -majority is noncoding nucleotide sequences -a single human gene only counts for 1/100,000 of a chromosomal DNA • To work directly with specific genes scientists have developed a process called DNA cloning BACTERIA IN DNA CLONING • One common method for cloning DNA uses bacteria (E. coli) • circular DNA • plasmids : small circular DNA molecules that can replicate separately from the bacterial chromosome • Contain small amounts of genes • Useful but not necessary for survival CLONING DNA 1) Obtain a bacterial plasmid 2) Insert “foreign” DNA into it -resulting plasmid is a recombinant DNA molecule 3) Plasmid is returned to the bacteria -produces a recombinant bacterium • Replicated daughter cells will then carry this foreign DNA/ and genes it carries -this is called gene cloning : production of multiply copies of a gene • Is useful for the purpose of: -making copies of a certain gene -producing a protein product ASSISTANCE FROM ENZYMES IN DNA CLONING • Gene cloning and genetic engineering rely on enzymes that cut DNA molecules at specific locations -called restriction enzymes • These enzymes protect the bacterial cell -cuts up foreign DNA from other organisms -very specific in recognizing a certain DNA sequence *Called a restriction site • Most restriction sites are symmetrical -sequence of nucleotides are same on both sides when read in 5’ 3’ direction -recognizes sequences containing 4 to 8 nucleotides ASSISTANCE FROM ENZYMES IN DNA CLONING • Restriction enzymes will make many cuts in a long DNA molecule -results in restriction fragments • Most useful restriction enzymes cleave the sugar-phosphate backbones in the 2 DNA strands in a staggered manner -creates a “sticky end” *when at least one end is single-stranded *forms hydrogen-bonded pairs with complementary “sticky ends” • This bonding is temporary -can be permanent with help from DNA ligase -forms covalent bonds CLONING A EUKARYOTIC GENE IN A BACTERIAL PLASMID • Bacterial plasmids are used for many reasons – Easy to obtain for commercial suppliers – Can be manipulated to form recombinant plasmids by insertion of foreign DNA, and then introduced into bacterial cells CLONING CELLS: HUMMINGBIRDS 1. Isolate genomic DNA from the hummingbird and cloning vector (e. coli) that has been engineered 2. Cut with the same restriction enzyme 3. Mix together, add DNA ligaments to seal them 4. Mix with bacteria cells that have mutations 5. Place the bacteria on the agar and incubate • Diagram on pg. 339 of textbook STORING CLONED GENES IN DNA LIBRARIES • Shotgun approach – no single gene is targeted for cloning • Genomic library – the complete set of plasmid-containing cell clones • May be stored in multi-welled plastic plates SCREENING A LIBRARY FOR CLONES • We can detect a gene's DNA by its ability to base-pair with a complementary sequence on another nucleic acid -to do this they use nucleic acid hybridization -nucleic acid hybridization: process of base pairing between a gene and a complementary sequence on another nucleic acid molecule • The nucleic acid probe is the complementary molecule -nucleic acid probe- single-stranded nucleic acid that can be DNA or RNA CLONING A PARTICULAR GENE • Once they're cloned in a host cell its protein product can be produced in large amounts -for research purposes -valuable practical applications • Cloned genes can be expressed as protein -bacterial and eukaryotic cells GETTING GENES TO FUNCTION • -In order to get a cloned eukaryotic gene to function, scientists employ an expression vector •expression vector: a cloning vector that contains a highly active bacterial promoter -bacterial sites will recognize the promoter and begin to express the foreign gene INTRONS -introns prevent correct expression of the gene by bacterial cells •this is another issue with expressing cloned eukaryotic genes in bacteria •this can be avoided by using a cDNA form of the gene -only includes exons EUKARYOTIC CLONING AND EXPRESSION SYSTEMS • Molecular biologists use eukaryotic cells (such as yeasts) as hosts for cloning or expressing eukaryotic genes • Several benefits to using yeasts – As easy to grow as bacteria – Have plasmids • Eukaryotic proteins will not function unless they are modified after translation • Electroporation – a brief electrical pulse applied to a solution containing cells creates temporary holes in their plasma membranes, through which DNA can enter CROSS-SPECIES GENE EXPRESSION & EVOLUTIONARY ANCESTRY • Genes are able to be taken from one species and still function when put into a very different species – EXAMPLE: Pax-6 gene (found in several mammals and fruit flies) • Leads to creation of single-lens eye in vertebrates, compound eye in flies • When taken from one species to another, still functions properly – May have a common ancestor (evidence of evolution) POLYMERASE REACTION CHAIN (PCR) • The PCR is a quick, selective way to prepare large quantities of a gene sequence. – Can make billions of copies of a DNA segment in a few hours • Three Step Cycle 1. Reaction mixture is heated, resulting in the DNA strands being denatured 2. The mixture is then cooled to allow hydrogen bonding of DNA primers to sequences on opposite strands at each end of the target sequence 3. DNA polymerase extends the primers in the 5’ to 3’ direction • ONLY WORKS FOR SHORT DNA SEGMENTS POLYMERASE REACTION CHAIN (PCR) • Has allowed for faster biological research april.russell26@gmail.com DNA Technology • Allows us to study the sequence, expression, and function of a gene. Gel electrophoreis in southern blotting • Many approaches for studying DNA molecules involve electrophoreis. • This technique uses a gel made of a polymer, it acts as a molecular seeve,10 separate nueleoic acids or protein on the bases of size, electrical charge and other properties. • Nucleic acid molecules travel to the positive pole because their phosphate groups have negative charges. • Gel electrophoreis separate a mixture of linear DNA molecules into bands. • Cach band consists of many thousands of DNA molecules. Gel electrophoreis in southern blotting • Southern blotting combines gel electrophoreis and nucleic acid. hybridization. ( developed by british biochemist Edwin Southern). • This allows us to detect those bands include parts of the B~ globin gene. • The probe is usually radio active or otherwise labeled as a single- stranded DNA molecule that is complementary to the gene of interest. DNA Sequencing • Once a gene is cloned, its nucleotide sequence can be determined. • Today sequencing is carried out by sequencing machines (automated). • The first automated procedure was called the “dideeoxyribonucleotide Chain termination method.” • Developed by British Biochemist Fredrick Sanger. • In the last 10 years “next- generation sequencing” techniques have been developed that don’t rely on chain termination. • Knowing the sequence of a gene allows researchers to compare it directly with genes and other species. Analyzing Gene Expression • Having cloned a given gene , researchers can mate labeled nucleic acid probes that can provide information about when or where in a organism the gene is transcribed. Studying the expression of single genes • Two techniques; northern blotting and reverse transcriptase/ polymerase chain reaction (RT- PCR). • NORTHERN BLOTTING • Gel electrophoresas are carried on samples of mRNA from hummingbird embryos at different stages of development. • Transfer the samples to a nitrocellulose membrane. • Then the mRNA’s on the membrane hybridize with a labeled prob to recognize B~ globin mRNA. • Transcriptase- polymerase chain reflection. The isolation of mRNA’s from different developmental stages are used. • Reverse transcriptase is added next to make cDNA. • When the products are run on a gel, copes of the amplified region will be observed as bonds. Determining Gene Function • Scientists determined the function of the gene by disabling the gene, the observing the consequences in the cell or organism. • In the Vitvo Mutagenesis; specific mutations are introduced into a cloned gene, and then the mutated gene is returned to a cell in a way it disables the normal cellular copies of the same gene. CLONING OF MULTICELLULAR ORGANISMS ANGIE HASKELL, COURTNEY GRAY, MICAELA KNOX, BRIANNA DINGEY, KAREN LEIN. INTRO • Cloning methods have evolved to duplicate multicellular organisms • Organismal cloning • Main focus of organismal cloning is to create stem cells KEY VOCABULARY • Totipotent- able to give rise to all specialized cell types of the organism • Stem Cell- an unspecialized cell that can reproduce indefinitely and differentiate into specialized cells • Pluripotent- capable of differentiating into many different cell types • Organismal Cloning- cloning an entire organism instead of a single gene • Blastocyst Stage- the stage where stem cells start to specialize CLONING PLANTS • The first successful cloning of plants was: in the 1950’s Done by F.C. Steward at Cornell University Conducted on carrot plants • Scientists found that differentiated cells taken from the root and incubated could grow into normal adult plants • Genetically identical to parent plant • Results showed that mature plant cells can “dedifferentiate” and give rise to all specialized cell types • Totipotent • Also proves that differentiation does not always involve irreversible DNA changes CLONING PLANTS • Used extensively in agriculture • In some cases, cloning is the only practical way to reproduce • Orchid plants • Cloning can be used to reproduce plants with valuable characteristics • Ability to resist pathogens REPRODUCTIVE CLONING OF MAMMALS • Nuclear Transplantation- Cloning achieved by removing the nucleus of an unfertilized or fertilized cell and replacing it with the nucleus of a differentiated cell • Dolly the sheep * A successful example of nuclear transportation * Dolly died early due to complications found normally in older sheep (lung problems and arthritis) • Reproductive Cloning- the deliberate production of genetically identical individuals *Clones are not always the same *For example: color pattern, behaviors, etc. *External influences play key roles during development COMPLICATIONS WITH ANIMAL CLONING • Only a small percentage of cloned embryos develop normally to birth • Many cloned animals exhibit defects • Obesity, pneumonia, liver failure, premature death, etc. • Even seemingly normal clones are likely to have defects WHY DOES CLONING RESULT IN SO MANY ABNORMALITIES? • DNA in calls from cloned embryos have more methyl groups than DNA from normal embryos • This means that that chromatin from the donating nuclei must be restructured • This process occurs incompletely during cloning procedures • DNA methylation helps regulate gene expression • Misplaced methyl groups interfere with the pattern of gene expression normal for embryonic development • So, successful cloning may depend on whether or not the donor nucleus’ chromatin can be artificially modified to resemble a fertilized egg STEM CELLS • Embryonic stem (ES) cells have special characteristics • Can reproduce indefinitely • Can differentiate into a wide variety of specialized cells • Can be isolated from early embryos at blastocyst stage • If scientists could clone human embryos to the blastocyst stage, then the clones could be used as a renewable source of ES cells • May also create specialized treatment for disease CHAPTER 20.4 The Practical Applications of DNA Technology Affect Our Lives In Many Ways MEDICAL APPLICATIONS • Important uses of DNA technology • Identification of human genes whose mutation plays a role in genetic disease • Leads to diagnoses, treatments, and prevention • Contribution to the understanding of nongenetic diseases • Person’s genes influence susceptibility to some diseases • Comparison of gene expression in healthy and diseased tissue • Helps figure out what “turn on” certain genes DIAGNOSES AND TREATMENT OF DISEASES • Polymerase Chain Reaction (PCR) and labeled nucleic probes • Track down pathogens • PCR with primers • Target genes associated with disorders • Sequence to reveal the presence/absence of disease-causing mutations • Can reveal carriers of a disease • Single Nucleotide Polymorphisms (SNPs) • Linked to disease-causing alleles • What is traced by PCR HUMAN GENE THERAPY • Gene therapy- introducing genes into an affected individual for therapeutic purposes • Could potentially treat disorders traceable to a gene • Figure 20.23 on page 418 • Trails have been done using bone marrow cells that weren’t producing a special protein • One trial showed success in improving 9 out of 10 children; however, three developed leukemia and one died • Gene therapy has technical challenges and provokes ethical questions PHARMACEUTICAL PRODUCTS Has significant benefits from advances in DNA technology and genetic research Applied to development of useful drugs that help treat diseases Synthesized by use of either organic chemistry or biotechnological methods SYNTHESIS OF SMALL MOLECULES FOR THE USE OF DRUGS Trying to determine the sequence and structure of proteins that are crucial for tumor cell survival is essential Helped identify small molecules that combat certain cancers by blocking protein cancers Imatinib (Gleevec) Small molecule Inhabits specific receptor tyrosine kinase Overexpression of this receptor causes chronic myelogenous leukemia Cancer patients, when given imatinib, have nearly complete remission of the cancer This type of advance is only useful in which cancer molecules are fairly well understood PROTEIN PRODUCTION IN CELL CULTURES Host cells can secrete a protein as large quantities Simplify task of purifying it by traditional biochemical methods Insulin, human growth hormone (HGH) First products “manufactured” this way Insulin: diabetes HGH: used with kids with dwarfism Tissue plasminogen activator (TPA) Dissolves blood clots if distributed shortly after a heart attack PROTEIN PRODUCTION BY “PHARM” ANIMALS Transgenic animal: when a gene from an animal of one genotype is introduced into the genome of another First remove female eggs of recipient and then fertile them in vitro while they have cloned the desired gene from another organism Inject cloned DNA into nuclei of fertilized eggs Some cell are able to express foreign gene Engineered embryos implanted in surrogate mother If embryo develops successfully then the animal is non transgenic Goats and chickens are main “pharm” animals FORENSIC EVIDENCE AND GENETIC PROFILES If a lot of blood or tissue is available forensic laboratories can determine blood or tissue type by using antibodies to detect cell-surface proteins Can only exclude a suspect, not provide strong evidence DNA testing can identify the guilty since the DNA sequence of every person is unique Genetic profile: an individual’s unique set of genetic markers FBI started using DNA technology in 1988 They used rep analysis by blotting to detect similarities and differences This method requires small blood or tissue samples FORENSIC EVIDENCE AND GENETIC PROFILE Today they use a more sensitive method, short tandom repeats (stks) These are tandemly repeated of two-five base sequences in specific regions of a genome For one individual, 2 alleles of an STR can differ from each other Can then be determined by electrophories Quicker than RFLP analysis PCR used to amplify STRs by using primers labeled with different color tags; length of regions + number of repeats PCR allows use of DNA when in poor conditions or in very little quantities (20 cells enough) In murders it can be used to compose DNA samples FORENSIC EVIDENCE AND GENETIC PROFILES Scientists would use 13 STR markers and it would provide a useful genetic profile The probability that 2 people would have identical markers is very small The innocence project (organization dedicated to overturning wrong convictions) uses STR analysis and has released 250 innocent people from prison Another useful purpose is to settle questioned paternity Can also identify victims of mass casualty FORENSIC EVIDENCE AND GENETIC PROFILES How reliable is a genetic profile? The greater number of markers in a sample, the more likely that the profile is unique to one individual Exact probability depends on the frequency of those markers in the general populations Forensic scientists now can make extremely accurate calculations ENVIRONMENTAL CLEANUP Ability of microorganisms to transform chemicals is exploited for environmental cleanup if growth needs of microbes make them unsuitable for direct use, scientists can now transfer the genes for their valuable metabolic capabilities into other organisms Ex. Some bacteria extract heavy metals (copper) and form compounds (copper sulfite) Genetic engineering microbes important for mining minerals and cleaning up highly toxic mining wastes ENVIRONMENTAL CLEANUP • Biotechnologists also trying to engineer microbes that can degrade chlorinated hydrocarbons and other harmful compounds • These can be used in wastewater treatment plants or by manufacturers before compounds are released into the environment AGRICULTURAL APPLICATIONS Using DNA technology to improve agricultural productivity Selective breeding of livestock and crops exploited naturally occurring mutations and genetic recombination DNA tech enables scientists to produce transgenic animals, speeds up selective breeding Better animals Transfer DNA to one another Low fertility or increased susceptibility to disease not uncommon Health and welfare important when developing transgenic animals Crops made with preferable traits- delayed ripening, resistance to spoilage and disease AGRICULTURAL APPLICATIONS • Genetic manipulations can be performed on an ordinary somatic cell and used to generate organism with new traits • Ti plasmid used to introduce new genes to cells • Comes from soil bacteria (Agrobacterium tumefaciens) • Integrates segments of DNA called T DNA into chromosomal DNA of its host plant cells • Scientists use non- disease causing plasmid that have been engineered to carry genes of interest to TDNA • Genetic engineering is replacing traditional plant-breeding programs, especially for helpful traits • Herbicide and pest resistance • Determined by one or few genes • Nervous that hazardous new pathogens might be created • To guard against the rogue microbes scientists developed a set of guidelines • Adopted in the U.S. and a few other countries • Few cautions taken are genetically crippling bacteria • Bacteria cannot survive outside of the lab for a long time • Some dangerous experiments banned • Most public concern has to do with genetically modified (GM) organisms • Majority of GM products are plants • GM products not required to be labeled • Products in Europe are carnivorous • Many Europeans are concerned about the safety of these products and the possible environmental consequences CONCERNS • Bio Safety protocol • Early 2000s, negotiators from 130 countries agreed to it • Requires exporters to identify GM organisms present in bulk food shipments • Tensions with transgenic plants • People fear that transgenic plants might pass on their genes to close relatives in wild areas • E.x. lawn grass commonly exchange genes with wild relatives with pollen transfer • Fear that due to gene sharing. super weeds could be produced or that protein products od transgenes could cause serious allergic reactions • Advocates of GM’s claim that this can be tested ahead of time and it is unlikely that such a thing can happen ETHICAL QUESTIONS • Today Governments are trying to facilitate how to deal with use of biotechnology and ethical questions about it • Biotech agencies are constantly evaluated in the U.S. by multiple regulatory agencies • Should people be allowed access to someone else’s genome? • Should a genome be considered when determining eligibility for a job or insurance? • All these factors can only be approached with caution and trial and error processes • A lot of testing still needed