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Download AP Bio Ch.18 “Genetics of Viruses and Bacteria” The Genetics of Viruses
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AP Bio Ch.18 “Genetics of Viruses and Bacteria” The Genetics of Viruses Objectives: 1. Explain why viruses are obligate intracellular parasites. They can reproduce only within a host cell Contain no: • Metabolic enzymes • ribosomes • Other equipment for making proteins 5. Explain how a virus identifies its host cell. • Recognition systems evolved in the virus • Lock-and-Key” fit between proteins on the outside of the virus and specific receptor molecules on the surface of cells. • Some viruses have broad host range o West Nile virus: mosquitoes, birds and humans • Some have only one species Poliovirus, measles infect humans only 6. Describe bacterial defenses against phages. • Natural selection favors bacterial mutants with receptor sites that are no longer recognized by a particular type of phage • Restriction enzymes (restriction endonucleases) chop up foreign DNA (bacterial DNA is chemically modified) • Lysogenic cycle: coexist with host 7. Distinguish between the lytic and lysogenic reproductive cycles, using phage lambda as an example. Lytic: (T4) fig. 18.6 • attachment • phage DNA injected • Phage gene makes enzyme to degrade host DNA • Viral genome transcribed using host enzymes • Self-Assembly of viral proteins & DNA • Last phage gene makes enzyme to damage bacterial cell wall, fluid enters, cell bursts, releasing 100-200 new viruses. Lysogenic: (Phage λ - lambda) fig. 18.7 • Attachment • Phage DNA injected • λ DNA circularizes • Phage DNA integrates into host DNA (prophage) • Phage gene makes protein, keeps other phage genes silent. • Bacterium reproduces by binary fission (prophage copied) • Prophage might exit bacterial chromosome, initiating lytic cycle. Temperate Viruses: Phages using both cycles Mrs. Loyd Page 1 of 5 3/26/12 8. Describe the reproductive cycle of an enveloped virus. Fig. 18.8 Nearly all animal viruses with RNA genomes have an envelope, few bacteriophages do. Glycoproteins on the viral envelope bind to specific receptor molecules on the host cell, promoting viral entry into the cell. • The capsid and viral genome enter the cell. Digestion of the capsid by cellular enzymes releases the viral genome. • The viral genome functions as a template for synthesis of complementary RNA strands by a viral enzyme. • New copies of viral genome RNA are made using complementary RNA strands as templates. • Complementary RNA strands also function as mRNA, which is translated into both capsid proteins (cytosol) and glycoproteins (ER) for the viral envelope. • Vesicles transport envelope glycoproteins to the plasma membrane • A capsid assembles around each viral genome molecule. • Each new virus buds from the cell, its envelope studded with viral glycoproteins embedded in membrane derived from the ER. Explain the reproductive cycle of the herpesvirus. Fig. 18.9, 10 • Envelope derived from the nuclear membrane of the host • Have double-stranded DNA genome • Reproduce within the host cell nucleus using combo of viral and cellular enzymes to replicate and transcribe their DNA. • Minichromosomes in nuclei of nerve cells are copies of herpesvirus DNA • Remain latent until physical or emotional stress triggers new round of active viruses (blisters). • 9. Describe the reproductive cycle of retroviruses. Fig. 18.10 Come equipped with enzyme reverse transcriptase transcribes RNA into DNA. Opposite of central paradigm (DNA→RNA→ Protein) Retroviruses: HIV (human immunodeficiency virus causes AIDS: acquired immunodeficiency syndrome) • All retroviruses contain two identical single-stranded RNA and two molecules of reverse transcriptase. • After entry, reverse transcriptase synthesizes two strands of complementary DNA. • Viral DNA enters cell’s nucleus and integrates into the DNA of a chromosome. • Now called a provirus, permanent. • Viral proteins made: capsid, rev. transc.(cytoplasm), envelope glycoproteins (ER). • Vesicles transport the glycoproteins from the ER to the cell’s plasma membrane. • Capsids are assembled around viral genomes and rev. trnsc. • New viruses bud off from host cell. 10. List some characteristics that viruses share with living organisms and explain why viruses do not fit our usual definition of life. Mrs. Loyd Page 2 of 5 3/26/12 • • • • Biologically inert unable to replicate its genes (no ribosomes, enzymes) unable to regenerate its own supply of ATP unable to reproduce independently 11. Describe the evidence that viruses probably evolved from fragments of cellular nucleic acids. • Depend on cells so evolved after cells • May have evolved several times • Viral genomes have more in common with host genome than other viruses • Some viral genes are essentially identical to genes of the host 12. Define and describe mobile genetic elements. Possible Original sources of viral genomes Plasmids • Small circular DNA molecules • Found in bacteria and yeasts • Exist apart from cell’s genome • Can replicate independently of genome • Occasionally transferred between cells Transposons • DNA segments that can move from one location to another within a cell’s genome Mobile genetic elements • Plasmids • Transposons • Viruses 13. Describe the best current medical defenses against viruses. • Antibiotics kill bacteria by inhibiting enzyme-catalyzed processes specific to the pathogen; viruses don’t have their own enzymes. • Vaccines are harmless variants of pathogenic microbes that stimulate the immune system to mount defenses against the actual pathogen or virus. (Viral flu vaccine, smallpox, chicken pox.) • Although vaccines can prevent certain viral illnesses, medical technology can do little, at present, to cure most viral infections once they occur. • Antiviral drugs resemble nucleosides (pentose sugar plus nitrogen base, like adenosine in ATP) and as a result, interfere with viral nucleic acid synthesis. • Currently, multidrug treatments, or cocktails, have been found to be most effective. These are combinations of two nucleoside mimics and a protease inhibitor, which interferes with an enzyme required for assembly of virus particles. Explain how AZT helps to fight HIV infections. • AZT (azidothymidine) curbs HIV reproduction by interfering with the synthesis of DNA by reverse transcriptase. ❏ Acyclovir impedes herpesvirus reproduction by inhibiting viral polymerase that synthesizes viral DNA The Genetics of Bacteria 24. Describe the significance of R plasmids. • R plasmids confer resistance to antibiotics for the bacteria that carry them. They can be transmitted thru sex pili to other bacteria. Mrs. Loyd Page 3 of 5 3/26/12 24. Explain how the widespread use of antibiotics contributes to R plasmid-related disease. • Exposure of bacteria to antibiotics will kill all susceptible bacteria but not those that happen to have R plasmids with genes that counter the antibiotic. • Natural selection predicts that the small fraction of resistant bacteria that survive will go on to repopulate the organism or environment. • Many R plasmids have genes that encode sex pili and enable plasmid transfer from one bacterial cell to another by conjugation. • Some R plasmids carry as many as ten genes for resistance to that many antibiotics. 30. Using the trp operon as an example, explain the concept of an operon and the function of the operator, repressor, and corepressor. Fig. 18.21 Operon: A unit of genetic function common in bacteria and phages, consisting of coordinately regulated clusters of genes with related functions. Operator: a master switch that controls a cluster of functionally related genes. Repressor: A protein that suppresses the transcription of a gene. Binds the operator to turn off the operon. Corepressor: A small molecule that cooperates with a repressor protein to switch an operon off. • The trp operon contains 5 genes needed to synthesize (anabolic) tryptophan. • The trp operon: regulated synthesis of repressible enzymes. • In the presence of tryptophan, it acts as a corepressor, binding the repressor and turning the operon off. • In the absence of tryptophan (the corepressor), the repressor is inactive, the operon turns on and the enzymes to synthesize tryptophan are made. 32. Describe how the lac operon functions and explain the role of the inducer, allolactose. Fig. 18.22 • The lac operon contains 3 genes needed to metabolize (catabolic) lactose. • The lac operon: regulated synthesis of inducible enzymes. • Lactose absent, repressor active, binds operator, turns operon off. • The lac repressor is innately active, and in the absence of lactose it switches off the operon by binding to the operator. • Lactose present, repressor inactive, operon on. • Allolactose, an isomer of lactose, derepresses the operon by inactivating the repressor, The enzymes for lactose utilization are induced. Allolactose is the inducer. In summation: Trp (anabolic): repressor + corepressor (tryptophan) = active repressor Lac (catabolic): repressor + inducer (allolactose) = inactive repressor Mrs. Loyd Page 4 of 5 3/26/12 34. Distinguish between positive control by CAP (fig. 18.23) and negative control by the lac repressor (fig. 18.22a) and give examples of each from the lac operon. • • • • Bacteria preferentially use glucose for glycolysis instead of lactose. How can they detect glucose concentration? Mechanism depends of the interaction of an allosteric regulatory protein with a small organic molecule Small organic molecule: cyclic AMP (cAMP) which accumulates when glucose is scarce. The regulatory protein, called catabolite activator protein (CAP), is an activator of transcription. Mrs. Loyd Page 5 of 5 3/26/12 Ch. 20 DNA Technology 1. Explain how advances in recombinant DNA technology have helped scientists study the eukaryotic genome. Genetic Engineering: The direct manipulation of genes for practical purposes. Examples: • Manufacture of hundreds of protein products (hormones and blood-clotting factors) • Recombinant DNA can be made then introduced into cultured cells that replicate the DNA and express its genes, yielding a desired protein. Biotechnology: the manipulation of organisms or their components to make useful products. Examples: • Using microbes to make wine and cheese • Selective breeding of livestock, crops, flowers. • Both use naturally occurring mutations and genetic recombinations. • In vitro manipulation is modern and enables modification of specific genes and to move them between organisms as distinct as bacteria, plants and animals. 2. Describe the natural function of restriction enzymes and explain how they are used in recombinant DNA technology. Restriction Endonucleases: In nature, bacteria protect themselves from invasion by viral bacteriophages (“bacteria eaters”) or other bacteria by cutting up foreign DNA, a process called “restriction.” These enzymes cut DNA molecules at a limited number of locations. Gene cloning and genetic engineering were made possible by discovery of restriction enzymes. They gave scientists a pair of microscopic scissors that cut at specific sites consistently. 3. Explain how the creation of sticky ends by restriction enzymes is useful in producing a recombinant DNA molecule. • Example: EcoR1, cuts DNA at a specific 6-base sequence. • Creates “sticky ends” that will anneal easily with complementary sticky ends. • Use same restriction enzyme with different DNA. • Mix together. • Any complementary bases across sticky ends will join • Forms recombinant DNA. Mrs. Loyd Page 6 of 5 3/26/12 4. Outline the procedures for cloning a eukaryotic gene in a bacterial plasmid. Cloned genes are useful for two main purposes: • to make many copies of a particular gene • to produce a protein product Examples of uses: • sequencing the gene • producing the protein • gene therapy • basic research 5. Describe techniques that allow identification of recombinant cells that have taken up a gene of interest. • ampR • restriction enzyme cuts lacZ gene, if gene is taken in there, (recombined) the X-gal will not be hydrolyzed, white colonies. • Non-recombined plasmids, colonies will be blue; they hydrolyze X-gal. 6. Define and distinguish between genomic libraries using plasmids, phages, and cDNA. Genomic Libraries: A set of thousands of DNA segments from a genome, each carried by a plasmid, or other cloning vectors. (Each plasmid or virus is analogous to a book in the library.) “Other vectors” include • viral DNA • cDNA Mrs. Loyd Page 7 of 5 3/26/12 cDNA: contains only a subset of genes that were transcribed into mRNA in the original cell. One advantage of a plasmid or viral vector over cDNA is that they contain regulatory sequences and introns which mRNA and therefore, cDNA, would not. cDNA or complementary DNA Procedure: • extract mRNA from cell of interest • reverse transcriptase used in vitro to make singlestranded DNA (transcripts of mRNA). • Get rid of mRNA by enzyme degradation • Add DNA polymerase to add the second strand to single-stranded DNA. • Restriction Enzyme recognition sequence at each end • Insert cDNA into vector DNA to create library Creates “stripped-down” versions of genes. Good for: • When you don’t want whole genome, just mRNA or protein. • studying genes from specific cell types • following changes in pattern of gene expression thru development. 7. Describe the role of an expression vector. • To overcome differences between prok/euk gene expression (DNA control sequences like promoters) expression vectors are used. • Contains a highly active prokaryotic promoter just upstream of a restriction site where the eukaryotic gene can be inserted in the correct reading frame. • Euk have introns, prok don’t have introns and do not have RNA-splicing machinery. Use cDNA, includes only exons. Just add bacterial promoter and other control elements. 8. Describe two advantages of using yeast cells instead of bacteria as hosts for cloning or expressing eukaryotic genes. 1. They are just as easy to grow as bacteria • Have plasmids (rarity in euk). Will accept recombinant plasmids and express the genes. • YAC: Yeast Artificial Chromosomes will behave normally during mitosis, replicating foreign DNA. • Can carry a much longer DNA segment than plasmids • More likely to contain an entire gene. 2. Eukaryotic proteins will not function unless they are modified after translation like adding carbs and lipids 9. Describe two techniques to introduce recombinant DNA into eukaryotic cells. • Electroporation: brief electrical pulse creates temporary holes in plasma membrane • Injection • Plants: Agrobacterium (soil bacterium) 10. Describe the polymerase chain reaction (PCR) and explain the advantages and limitations of this procedure. • ADVANTAGES: A small quantity of DNA can be amplified, in vitro, to a useable amount. (To be sequenced or introduced into a vector: yeast, plasmid, bacterium) • LIMITATIONS: Occasional errors during PCR replication imposes limits of the # of good copies that can be made. Mrs. Loyd Page 8 of 5 3/26/12 Procedure for PCR: Starting materials: • double-stranded DNA containing the target nucleotide sequence to be copied • Heat resistant DNA polymerase • All four nucleotides • Two short, single-stranded DNA molecules (primers) One primer is complementary to one end, the other primer is complementary to the other end. During each PCR cycle the samples are warmed, then cooled. • Each cycle will double the target DNA sequence. • After about 20 cycles, target sequence DNA increased a billion-fold. 11. Explain how gel electrophoresis is used to analyze nucleic acids and to distinguish between two alleles of a gene. P.393 12. Describe the process of nucleic acid hybridization. Once you have made a genomic library with bacteria, how can you tell which are for the gene of interest? The key is the radiolabeled probe DNA that will anneal with its complementary DNA (a segment of the target gene). Mrs. Loyd Page 9 of 5 3/26/12 13. Describe the Southern blotting procedure and explain how it can be used to detect and analyze instances of restriction fragment length polymorphism (RFLP). The Southern blot was named after the inventor’s name of Southern. Subsequent procedures for similar blotting techniques were named: Northern, Western, Eastern. Fig. 20.10 Shows how to detect specific nucleotide sequences within different samples of genomic DNA. The example given compares three individual with different genotypes: • homozygous normal hemoglobin • homozygous sickle-cell hemoglobin • heterozygous (one normal allele, one sickle-cell allele.) Mrs. Loyd Page 10 of 5 3/26/12 14. Explain how RFLP analysis facilitated the process of genomic mapping. Cytogenetic mapping: created starting point for genomic mapping. Karyotyping determines # of chromosomes and banding pattern FISH: Fluorescence in situ hybridization of whole chromosomes Linkage Mapping: Ordering of genetic markers such as RFLPs, simple sequence DNA, and other polymorphisms (about 200 per chromosome. Based on recombination frequencies. Resulted in map with 5,000 markers. Physical Mapping: Made by cutting the DNA of each chromosome into a number of restriction fragments and then determining the original order of the fragments. • The key is to make fragments that overlap and then use probes to find the overlaps. First round uses: • YAC (yeast artificial chromosome) and • BAC (bacterial artificial chromosome) • because they can be so large. After these long fragments are ordered, • each fragment is cut into smaller pieces • which are cloned in plasmids or phages • ordered in turn, and finally • sequenced. DNA Analysis and Genomics 15. Explain the goals of the Human Genome Project. 1990-2003 • Read out and make freely available the complete DNA sequence of the human being. • To gain a comprehensive picture of how the genes are turned on and off in different tissues and at different times, so we can see how genes collaborate in modules or circuits. • It’s an ensemble picture that we’ve never had before. Genomes Sequenced: E. coli: Saccharomyces cerevisiae: Caenorhabditis elegans: Drosophila melanogaster: Mus musculus: Hemophilus influenzae: Homo sapiens: enteric bacteria yeast nematode fruit fly mouse flu humans 16. Explain how linkage mapping, physical mapping, and DNA sequencing each contributed to the genome mapping project. These were the three successive stages of the HGP. Linkage Mapping: Ordering of genetic markers such as RFLPs, simple sequence DNA, and other polymorphisms (about 200 per chromosome. Based on recombination frequencies. Resulted in map with 5,000 markers. Physical Mapping: Made by cutting the DNA of each chromosome into a number of restriction fragments and then determining the original order of the fragments. • The key is to make fragments that overlap and then use probes to find the overlaps. First round uses: • YAC (yeast artificial chromosome) and • BAC (bacterial artificial chromosome) Mrs. Loyd Page 11 of 5 3/26/12 • because they can be so large. After these long fragments are ordered, • each fragment is cut into smaller pieces • which are cloned in plasmids or phages • ordered in turn, and finally • sequenced. 17. Describe the alternate approach to whole-genome sequencing pursued by J. Craig Venter and the Celera Genomics Company. Skip genetic mapping and physical mapping stages and start directly with the sequencing of random DNA fragments. Powerful computer programs would then assemble the resulting very large number of overlapping short sequences into a single continuous sequence. 18. Explain how researchers recognize protein-coding genes within DNA sequences. • DNA sequences are collected in computer banks available on the internet. Software scans sequences for: • transcriptional and translational start and stop signals • RNA-splicing sites, • Other telltale signs of protein-coding genes • Short coding sequences similar to those present in known genes. (ESTs: expressed sequence tags) are cataloged in computer databases. • Identifies gene candidates (putative genes) 19. Describe the surprising results of the Human Genome Project. • Genome size generally increases from prok to euk, it does always correlate with biological complexity among eukaryotes. • The number of genes an organism has is often lower than expected from the size of its genome. • Human genome is approx. 25,000 genes, much lower than the 50,000 – 100,000 expected • Human genome is only 1.5x the number found in the fruit fly and the nematode worm. • Relative to other organisms studied, human genes account for a much smaller fraction of the human genome. 20. Explain how the vertebrate genome, including that of humans, generates greater diversity than the genomes of invertebrate organisms. • Gene expression is regulated in more subtle and complicated ways in vertebrates than in other organism. • Non-coding DNA may function in these regulatory mechanisms. • Alternative splicing of RNA transcripts • Nearly all human genes contain multiple exons. o About 75% of these multi-exon genes are alternatively spliced. o Assume each alternatively spliced gene specifies 3 different polypeptides Mrs. Loyd Page 12 of 5 3/26/12 o • • Total # of different polypeptides = 75,000 added to post-transcriptional cleavage or addition of carb groups to increase variation of polypeptides. Much larger number of possible interactions between gene products that result from greater polypeptide diversity 23. Define and compare the fields of proteomics and genomics. Genomics: The systematic study of full sets of genes and their interactions. Proteomics: the systematic study of full protein sets (proteomes) encoded by genomes. • Because proteins, not genes, actually carry out the activities of the cell, we must study when and where they are produced in an organism, and also how they interact, if we are to understand the functioning of cells and organisms. • A first step in systems-biology approach is defining gene circuits and protein interaction networks. • With the use of computer science and math to process and integrate vast amounts of biological data, researchers can detect and quantify the many combinations of interactions. 24. Explain the significance of single nucleotide polymorphisms in the study of human evolution. • Much of human diversity is in single nucleotide polymorphisms (SNP’s or snips). • These occur about once in 1,000 bp. • Comparing your personal gene sequence with someone of same gender you would be 99.9% identical. • SNP’s make useful genetic markers in studying human evolution • Such as migratory routes • ID disease genes and genes that affect our health in more subtle ways. • Likely to change practice of medicine in 21st century. PRACTICAL APPLICATIONS OF DNA TECHNOLOGY 25. Describe how DNA technology can have medical applications in such areas as the diagnosis of genetic disease, the development of gene therapy, vaccine production, and the development of pharmaceutical products. diagnosis of genetic disease: • Use of PCR and labeled nucleic acid probes to detect RNA of disease in blood using RT-PCR (take RNA + reverse transcriptase→cDNA then PCR and probe for HIV sequence. Examples of diseases that can be diagnosed this way: o Sickle-cell disease o Hemophilia o Cystic fibrosis o Huntington’s disease o Duchennne muscular dystrophy o o RFLP analysis: to find genetic markers with diseasecausing alleles that are different than normal allele. gene therapy: Mrs. Loyd Page 13 of 5 3/26/12 The alteration of an afflicted individual’s genes with normal genes. • Cells receiving normal allele would need to reproduce throughout lifetime of individual. Bone marrow cells (include stem cells for blood and immune system) are prime candidate. Fig. 20.16 p. 403 • Challenge: DNA receiving vector DNA may insert near gene involved in proliferation and development of blood cells, causing leukemia. • Worry: eugenics, some researchers see no difference between gene therapy and organ transplant. vaccine production: • Two types: Inactivated, Attenuated • Both present viral surface proteins to immune system to prime it for the next time it comes in contact. • The protein alone could be created using DNA techniques. pharmaceutical products: • Human insulin: for diabetics • Human growth hormone treats people with too little HGH caused by a form of dwarfism. 26. Explain how DNA technology is used in the forensic sciences. • DNA Fingerprinting: DNA testing reveals unique DNA sequence from body fluids of tissue left at crime scene. • Presented in court as autoradiograph. 27. Describe how gene manipulation has practical applications for environmental and agricultural work. Environmental Cleanup: • Certain microorganisms’ ability to transform chemicals are being engineered into other microorganisms • Then used to treat environmental problems • Example: bacteria can extract heavy metals from environment turn into compounds that are easily recoverable. May become important in mining minerals and cleaning toxic mining wastes. Agricultural: • Transgenic animals and plants to create better products. • Pharm animals: producers of large amounts of an otherwise rare biological substance for medical use, like, human hormone or blood clotting factor. o Cows secrete chemical in milk o Chickens secrete chemical in eggs o Product can be purified more easily than from a cell culture. 28. Describe how plant genes can be manipulated using Ti plasmid carried by Agrobacterium as a vector. See fig. 20.19 29. Explain how DNA technology can be used to improve the nutritional value of crops and to develop plants that can produce pharmaceutical products. Increased Nutritional Value: Mrs. Loyd Page 14 of 5 3/26/12 • • • • • Transgenic rice plants that produce yellow rice grains containing beta-carotene, which our body uses to make vitamin A. “Golden Rice” could help prevent vit A deficiency in the half of the world’s population that depends on rice as a staple food. Currently, large numbers of young children in SE Asia suffer from vit A deficiency, which leads to vision impairment and increases susceptibility to disease. Pharm Crops: • Human proteins for medical use • Viral proteins for use as vaccines • Vaccines for hepatitis B • antibody produced by transgenic tobacco plants that interferes with bacteria that cause tooth decay. 30. Discuss the safety and ethical questions related to recombinant DNA studies and the biotechnology industry. Hazardous new pathogens might be created Cancer cell genes transferred into bacteria or viruses • Strict laboratory procedures designed to: • protect researchers from infection by engineered microbes and • to prevent the microbes from accidentally leaving the lab. • Strains of microorganisms to be used in recombinant DNA experiments are genetically crippled to ensure that they cannot survive outside the lab. • Obviously dangerous experiments have been banned. GM organisms: A Genetically Modified organism is one that has acquired by artificial means one or more genes from the same or another species. Most are crops Fears: • Transgeneic plants might pass their new genes to close relatives in nearby wild areas. • Crop plants that carry resistance to herbicide, disease, or insect pests cross-pollinated with wild relatives, it may create “super weeds.” • Protein products of transgenes might lead to allergic reactions Mrs. Loyd Page 15 of 5 3/26/12