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Chapter 19: Viruses Structure of Viruses Virus = DNA/RNA + capsid Capsid – protein coat Capsomeres- small units that make up the capsid Viral envelope – - Are derived from membranes of host cells: as a virus is brought into a cell, it brings part of the host cell membrane in through endocytosis - May cloak the capsids of viruses found in animals Viral genomes may be single or double stranded DNA or single or double stranded RNA. - Viral genes are usually contained on a single linear or circular nucleic acid molecule May be rod-shaped (helical viruses), polyhedral (icosahedral viruses), or more complex in shape Some viruses also contain a few viral enzymes. Viruses are specific to organisms and tissues - Colds/flus = different strains and mutations Ways of entering the cell = Inject genome, use endocytosis or budding (=passes through membrane without breaking it) Bacteriophage – viruses that infect bacteria only - Complex capsids are found among phages - Inject viral DNA into host bacteria cell - Use bacteria cell to produce viral DNA proteins (capsid and capsomeres) - Cell gets packed with self assembled virus particles - The cell lyses and virus particles are released to infect other cells *Same process in all living organisms evolution, continuity and change - Use bacteria for DNA replication and protein synthesis with transcription and translation Frederick Griffith – tested mice – mixed nonpathogenic and pathogenic bacteria killed mice - Nonpathogenic bacteria were transformed into pathogenic bacteria Transforming factor = DNA Caused inactive DNA of dead bacterial cell leaking from one cell into another live bacterial and becoming active Zone of inhibition: penicillin (protein) zone of inhibition: -chemicals diffuse out of the penicillin and prevents bacteria from growing their cell walls 1) Transformation: nonliving cell’s DNA is picked up by a living cell - Avery, McCarty, and McCleod – discovered it was DNA - Alexander Flemming – discovered penicillin (antibiotic) 2) Transduction: virus injects DNA into host cell and takes over its machinery to produce viral DNA and proteins - Plasmids act as resistance factors - Example = E.coli (Eschercia (genus) coli (species) ) 3) Conjugation: living bacteria cells transmit DNA between each other; called bacteria sex - A conjugation bridge - Contributes to variation Reproduction of Virus Viruses are obligate intracellular parasites that lack metabolic enzymes and other equipment needed to reproduce Each virus type has a limited host range due to proteins on the outside of the virus that recognize only specific receptor molecules on the host cell surface General Features of Viral Reproductive Cells Once the viral genome enters the host cell, the cell’s enzymes, nucleotides, amino acids, ribosomes, ATP, and other resources are used to replicate the viral genome and produce capside proteins - The virus basically takes over the cell and turns it into a virus factory - DNA viruses use host DNA polymerases to copy their genome, whereas RNA viruses use virus-encoded polymerases for replicating their RNA genome After replication, viral nucleic acid and capside proteins spontaneously assemble to form new viruses within the host cell, a process called “self-assembly” Hundreds or thousands of newly formed virus particles are released, often destroying the host cell in the process Reproductive Cycles of Phages 1. There are 2 different types of viral reproductive cycles: Lytic cycle Lytic cycle = a reproductive cycle of a phage that culminates in lysis of the host cell and release of newly produced phages - The virus attaches to the cell by sticking to a receptor site on the surface of a cell - The virus injects its DNA into the host cell, leaving behind the empty capside - The cell begins to transcribe and translate phage genes, one of which codes for an enzyme that chops up host cell DNA. - Virus parts assemble: Capsid proteins are made and assembled into phage tails, tail fibers, heads - Virus particles are released from the cell, damaging its cell wall and often lysing the cell Virulent = very aggressive infection 2. - - - - Lowering viral infection in bacteria: 1. Mutations that change their receptor sides 2. Restriction enzymes that chop up foreign viral DNA once it enters the cell Lysogenic cycle Lysogenic cycle = a virus reproduces its genome without killings its host by incorporating itself into the DNA of the host cell but doesn’t express itself Temperate phages can reproduce by the lytic and lysogenic cycles. Temperate phage – a virus that stays in your body and can go from active to inactive to active When the phage injects its DNA into a cell, it can begin a lytic cycle, or its DNA may be incorporated into the host cell’s chromosomes and begin a lysogenic cycle as prophage. Prophage/ provirus – unexpressed bacteria / unexpressed virus Most of the genes of the inserted phage genome are repressed by a protein coded for by a prophage gene. Reproduction of the host cell replicates the phage DNA along with the bacterial DNA. the prophage may exit the bacterial chromosome, usually in response to environmental stimuli and start the lytic cycle. Expression of prophage genes may cause a change in the bacterial phenotype. Several diseasecausing bacteria would be harmless except for the expression of prophage genes that code for toxins. Retroviruses Retrovirus – contains RNA (not DNA) Retroviruses convert viral RNA into DNA with the viral enzyme reverse transcriptase The viral DNA is then integrated into a chromosome, where it is transcribed by the host cell into viral RNA which acts both as a new viral genome and as mRNA for viral proteins HIV (human immunodeficiency virus) is a retrovirus that causes AIDS (acquired immunodeficiency syndrome) The integrated viral DNA remains as a provirus (=unexpressed virus) within the host cell DNA New viruses, assembled with 2 copies of both the RNA genome and reverse transcriptase, bud off covered in host cell plasma membrane studded with viral glycoproteins Viral Diseases in Animals The symptoms of a viral infection may be caused by viral-programmed toxins produced by infected cells, cells killed or damaged by the virus, or the body’s defense mechanisms fighting the infection. Vaccines are variants or derivatives of pathogens that induce the immune system to react against the actual disease agent. - Vaccines – stimulates body’s immune system and shortens the lag time for antibodies to find antigens - Can be made from a protein coat or an attenuated virus (=dead/inactive form of the virus) Unlike bacteria, viruses use the host’s cellular machinery to replicate, and few drugs have been found to treat or cure viral infections. Some antiviral drugs resemble nucleosides and interfere with viral nucleic acid synthesis. Emerging Viruses Emerging viruses: new viruses that appear and disappear The emergence of “new” viral diseases may be linked to the mutation of an existing virus (as in influenza viruses), the dissemination of an existing virus to a more widespread population (as in HIV), or the spread from one host species to another (as in SARs). Many new human diseases are estimated to originate by the third mechanism. A general or local outbreak of a disease is called an epidemic. Flue epidemics may be caused by new strains of viruses to which people have little immunity. Pandemics are global outbreaks. A recombinant virus may be highly pathogenic Viroids and Prions: The Simplest Infectious Agents - Viroids are very small infectious molecules of circular RNA that can replicate in host plant cells, causing abnormal development and stunted growth. Prions are protein infectious agents that may be linked to several degenerative brain diseases such as mad cow disease and Creutzfeldt-Jacob disease in humans. Prions apparently spread disease by converting normal cellular proteins into the defective form of the protein. Bacterial Diseases vs. Bacteria shapes: rod, spherical, spiral Treat with antibiotics Salmonella Cholera Legionnaire’s disease Stomach ulcers Syphilis Gonorrhea Lyme disease Botulism Strep/staph infection Black death (bubonic plague) E.coli Viral Diseases Viruses: variety of shapes (icosahedrons, rod) Treat with vaccines or antiviral drugs Warts Cold sores Chicken pox Small pox Polio Rhinovirus Hepatitis SARS Influenza Measles Mumps Rabies HIV Ebola Defenses: Antigens: foreign agents - Take time to infect the body’s cells B-CELLS Antibodies: Y-shaped proteins that tag and secure foreign particles for the immune system to fend against and get rid of them - Look for and recognize matching virus spikes - Virus spikes: can mutate so the virus particles are not recognized by the immune system T-CELLS Natural killer cells: - HIV – kills T cells - Ebola – high virulence and causes “bleeding out” in a few days mRNA splicing: exons joined, introns out - Gives you specific proteins to recognize antigens Chapter 27: Bacteria and Archaea Structure and Function 3 shapes: spherical (cocci), rod-shaped (bacilli), spiral Cell-Surface Structure Structure: - Nucleoid= single loop of DNA - Plasmids= extra loops of DNA that resist antibiotics - Cell wall= made of peptidoglycan; can have an outer membrane that makes it more - resistant maintain cell shape and prevent the cell from bursting in a hypotonic surrounding Gram positive = bacteria with walls containing just peptidoglycan Gram negative = bacteria with more complex walls like an outer membrane Plasma membrane= phospholipid bilayer imbedded with proteins - Capsule= sticky polysaccharide coating that makes bacteria more resistant to attack by a host’s immune system and serves as a glue for adhering to a subrate or other prokaryotes - Fimbriae/sex pili = allow bacteria to stick to their substrate or to other individuals/ allow bacteria to exchange DNA Motility Many prokaryotes are equipped with flagella, either scattered over the cell surface or concentrated at one or both ends of the cell. These flagella differ from eukaryotic flagella in their lack of a plasma membrane cover, structure, and function. Many motile prokaryotes exhibit taxis, an oriented movement in response to chemical, light, or other stimuli. Reproduction and Adaptation Prokaryotes have a huge reproductive potential. The growth of colonies usually stops due to the exhaustion of nutrients or the toxic accumulation of wastes Some bacteria produce endospores, which are tough-walled dormant cells formed in response to a a lack of nutrients Due to short generation times, favorable mutations are rapidly expanded, increasing adaptive evolution to environmental changes Prokaryotes vs. Bacteria Eukaryotes Protists, fungi, plants, animals Archea Eubacteria or “extremophiles” Genetic variation 1) Rapid reproduction - Prokaryotes reproduce very rapidly and have large population sizes 2) Mutation - Mutations, although statistically rare, produce a great deal of genetic diversity because the mutated cells reproduce so fast 3) Genetic recombination 1. Transformation= nonliving cell’s DNA from the environment or plasmid is picked up by a living one - The foreign DNA is integrated into the bacterial chromosome by an exchange of homologous DNA segments. - Many bacteria have surface proteins specialized for the uptake of DNA from closely related species. 2. Transduction = exchange between bacteria and bacteriophage/virus - A random piece of host DNA may be accidently packaged within a phage, introduced into a new bacterium, and replace the homologous region of a bacterial chromosome 3. Conjugation = transfer of genetic material (often plasmids) between living bacteria cells; called bacteria sex - Sex pili form the conjugation bridge for plasmid exchange - F- factor is required to produce sex pili and donate DNA. Bacterial cells containing the F factor on the F plasmid are called F+ cells - During conjugation, a single strand of the plasmid DNA is transferred through a mating bridge to the recipient cell Growth Curve for Bacteria Number of bacteria Bacteria start dying because they’ve used up their resources Best for transformation experiment Time (days) The Harmful and Beneficial Impacts of Prokaryotes Pathogenic Prokaryotes - - About half of all human diseases are caused by pathogenic prokaryotes Pathogens most commonly cause disease by producing toxins. Exotoxins are proteins secreted by prokaryotes that cause such diseases as botulism and cholera. Endotoxins are lipopolysaccharides released from the outer membrane of gram negative bacteria, and they cause diseases like typhoid fever Improved sanitation and development of antibiotics have decreased the incidence of bacterial disease in developed countries. The evolution of antibiotic-resistant strains of pathogenic bacteria, however, poses a serious health threat. R plasmids carry genes that code for various mechanisms of resistance to antibiotics. R plasmids also have genes coding for sex pili and are transferred to nonresistant cells during conjugation, creating the medical problem of antibiotic-resistant pathogens Horizontal gene transfer also spreads genes connected with virulence Some pathogenic prokaryotes are potential bioterrorism weapons Prokaryotes in Research and Technology Bioremediation is the use of organisms to remove environmental pollutants. Prokaryotes are used to treat sewage and clean up oil spills They are used in the mining industry to recover metals from ores. They have been engineered to make vitamins (vitamin K), antibiotics, and other products Chapter 18: Regulating Gene Expression Feedback inhibition Bacteria can respond to short-term environmental fluctuations by regulating enzyme activity through feedback inhibition Feedback inhibition is when the product inhibits an enzyme earlier in the process 1) Prevents overproduction of a product 2) Energy conservation: turns the process on or off to prevent waste Eukaryotic chromosomes are more complex than prokaryotes - It’s easier to understand prokaryotic gene expression because their DNA is in a single loop, not strands like eukaryotes Prokaryotic Gene Expression Genes can be under coordinated control by a single on-off switch An operator is a segment of DNA within or near the promoter that controls the access of RNA polymerase to the genes An operon is the entire stretch of DNA that includes the group of structural genes that will make a protein, the operator, and the promoter The operon can be switched off by a protein repressor Repressor: protein that binds to a specific operator, blocking attachment of RNA polymerase and thus turning an operon “off” Prevents gene transcription Repressors are produced by a separate regulatory gene Regulatory genes code for repressor proteins. These allosteric proteins, which may assume active or inactive shapes, are usually produced at a slow but continuous rate. The activity of the repressor protein may be determined by the presence or absence of a corepressor. 2 types of operons: 1) Lac operon - RNA polymerase: makes a complementary transcript of DNA and always binds to the promoter Operator: where the repressor will bind If the repressor binds off switch If the repressor doesn’t bind on switch 2) Trp operon - Tryptophan absent repressor inactive gene is transcribed Tryptophan is the corepressor that binds to the repressor protein, changing it into its active shape, which has a high affinity for the operator and switches off the trp operon If Tryptophan is absent: repressor proteins aren’t bound with try the operator is no longer repressed RNA polymerase attaches to the promoter mRNA for the enzymes needed to tryptophan synthesis is produced Differential Gene Expression Almost all the cells in an organism are genetically identical - Differences between cell types result from differential gene expression: the expression of different genes by cells with the same genome Basically: not every cell of every organism expresses every gene at the same time Only a small portion of a cell’s DNA codes for proteins; some codes for RNA; and most is noncoding DNA Errors in gene expression can lead to diseases like cancer Gene expression is regulated at many stages Example: embryonic development It is most commonly regulated at transcription Regulation of Chromatin Structure DNA packing and the location of a gene’s promoter relative to nucleosomes may help control which genes are available for transcription Genes within highly packed heterochromatin are not usually expressed Heterochromatin is tightly wound up while euchromatin is long and skinny Chemical modifications to histones and DNA of chromatin influence both chromatin structure and gene expression These modifications can either allow or stop transcription: 1) Acetylation – CH2CH3 promotes initiation of transcription - Loosens attachment of DNA to proteins 2) Methylation – CH3 stops transcription - Condenses and tightens DNA around proteins - It “silences” gene expression According to the histone code hypothesis, the specific patterns of modifications determine chromatin shape and influence transcription DNA methylation - In a process called DNA methylation, methyl groups are added to DNA bases (usually cytosine) Genes are more heavily methylated in cells in which they are not expressed. Some proteins that bind to methylated DNA also interact with histone deacetylation enzymes, reinforcing the transcription repression. DNA methylation may reinforce gene regulatory decisions of early development as enzymes act during DNA replication to methylate daughter DNA strands and pass on methylation records. Such methylation patterns account for genomic imprinting in mammals in which either maternal or paternal allele of certain genes is permanently regulated. Epigenetics - Epigenetics means “above the genome” It is a second “code” that “sits on top” of our regular DNA and controls how are genes are expressed by turning them on and off with altered consequences This 2nd code consists of gene regulators that turn genes on or off. (e.g. methylation turns genes off) Epigenic inheritance is the transmission of traits by mechanisms that do not involve changes in DNA sequences, such as chromatin modifications that affect gene expression in future generations of cells. Regulation of Transcription Initiation A typical gene consists of a promoter sequence, where a transcription initiation complex that includes RNA polymerase II attaches, and a sequence of introns interspersed among the coding exons. After transcription, RNA processing of the pre-mRNA removes the introns and adds a 5’ cap and a poly-A tail at the 3’ end. Control elements are noncoding sequences to which transcription factors bind that help regulate transcription. mRNA processing Alternative mRNA splicing may produce different mRNA molecules when regulatory proteins control which segments of the primary transcript are chosen as introns and which are chosen as exons. Introns are taken out, exons are joined together by ligase Provides diversity and variation in terms of proteins mRNA Degradation - In contrast to prokaryotic mRNA that is degraded after a few minutes, eukaryotic mRNA can last hours or even weeks. The hydrolysis of mRNA by nucleases is usually preceded by the enzymatic shortening of the poly-A tail and removal of the 5’ cap. Poly A tail and methyl-guanine cap help to prevent mRNA degradation Initiation of Translation Translation may begin when enzymes add more A nucleotides to poly-A tails or when translation initiation factors are activated following fertilization The translation of eukaryotic mRNA can be delayed by the binding of regulatory proteins to the 5’ UTR (untranslated region) that prevent ribosome binding A great deal of mRNA is synthesized and stored in egg cells Protein Processing and Degradation Following translation, polypeptides are often cleaved or chemical groups added to yield an active protein. Some proteins must be transported to target locations. Selective degredation of proteins may serve as a control mechanism in the cell. Molecules of the protein ubiquitin are added to mark proteins for destruction Proteasomes recognize and degrade the marked proteins Cancer results from genetic changes that affect cell control Types of Genes Associated with Cancer Chemical carcinogens, X-rays, or certain viruses most often cause the changes in the genes that regulate cell growth and division that lead to cancer. Viruses that can cause cancer in animals are called tumor viruses. Mutations in tumor-suppressor genes can contribute to the onset of cancer when they result in a decrease in the activity of proteins that prevent uncontrolled cell growth Chapter 20: Biotechnology The DNA Toolbox DNA technology began with techniques for making recombinant DNA, which combines nucleotide sequences from different organisms or species into the same DNA molecule. Genetic engineering, the direct manipulation of genetic material for practical purposes, has begun a revolution in biotechnology. Biotechnology is the use of living organisms or their components to manufacture desirable produces, and new advances have resulted in hundreds of new products and applications. Stanley Cohen and Herbert Boyer (1980s)- demonstrated that the gene for frog ribosomal RNA could be transferred into bacterial cells and expressed by them DNA Cloning An Overview of DNA Cloning The “gene of interest” is the gene being placed into the bacterial/eukaryotic cell Plasmids of bacterial cells can be used to clone DNA Recombinant DNA can be made by inserting foreign DNA into plasmids. These plasmids are put back into bacterial cells where they will replicate as the recombinant bacteria reproduce to form clones of identical cells. 2 purposes of gene cloning: 1) Provides multiple copies of a particular gene 2) Produces a protein product Using Restriction Enzymes to Make Recombinant DNA Restriction enzymes “cut” DNA at specific restriction sites, short nucleotide sequences. - Common restriction enzymes: EcoRI, HindIII, BamHI - In bacteria, restriction enzymes defend against viruses by cutting up the foreign DNA - The cell protects its own DNA from restriction by methylating nucleotide bases within its own restriction sequences. Restriction enzymes cut the sugar-phosphate backbone of DNA in specific staggered patterns, forming sticky ends on both sides of the resulting restriction fragment DNA from different sources can be combined in the laboratory when the DNA is cut by the same restriction enzyme it needs to be cut by the same restriction enzyme so that sticky ends are complementary and can form base pairs DNA ligase is used to seal the strands together. Cloning a Eukaryotic Gene in a Bacterial Plasmid Cloning vectors are DNA molecules that can move foreign DNA into a cell and replicate there. Recombinant plasmids returned to bacterial cells will replicate the foreign DNA as the bacteria reproduce. Producing Clones of Cells Carrying Recombinant Plasmids The plasmid method of gene cloning involves treating plasmids containing an antibiotic-resistant gene with a restriction enzyme that cuts the DNA ring at a single restriction site and disrupts a gene The clipped plasmids are mixed with the DNA containing the gene of interest, which has been treated with the same restriction enzyme - The sticky ends of the cut plasmids are complementary to the sticky ends of the DNA with the gene of interest - The sticky ends form hydrogen bonds with each other, and DNA ligase seals the recombinant molecules The challenge is getting recombinant plasmid back into the bacteria. There are different processes for inserting the recombinant plasmid: 1. Electrophoration – an electric pulse briefly opens holes in the plasma membrane through which DNA can enter 2. Gene gun – injection of plasmids into cells using microscopically thin needles 3. Bacterial transformation – the bacteria cell’s DNA picks up the DNA of the recombinant plasmid *Competent cells are likely to take up a plasmid (18–20% chance) For example: E.coli (control-not transformed) vs. E.coli with plasmid (hopefully transformed) -The plasmid has an ampicillin resistance factor -If placed in ampicillin (an antibiotic): *E.coli cells will not grow the ampicillin kills them *E.coli cells that have been transformed with recombinant plasmids will GROW they will not be killed because the plasmid has an amp resistance factor After it is proved that E.coli cells have been transformed, scientists take the transformed cells and grow them in a culture flask. - Fermentation tanks are usually glass, metal or plastic tanks used in the pharmaceutical industry for the growth of specialized pure cultures of bacteria, fungi and yeast, and the production of enzymes and drugs Storing Cloned Genes in DNA Libraries An organism’s DNA can be cut into thousands of pieces with restriction enzymes and inserted into plasmids. A genomic library is the collection of the thousands of clones of bacteria that contain recombinant plasmids. Bacteriophages are also used as vectors for creating genomic libraries. - DNA fragments are spliced into phage DNA, which is packaged into capsids and used to infect bacteria. The production of new phage clones the foreign DNA. Cloning vectors also include bacterial artificial chromosomes (BACs) which are large plasmids that carry longer inserts that standard plasmid or phage vectors. BAC clones are often stored in multiwelled plastic plates. A partial genomic library can be produced using the mRNA molecules isolated from a cell. Using reverse transcriptase from retroviruses, mRNA is used as a template to produce complementary DNA (cDNA) Restriction sites are added to the ends and the cDNA is inserted into vector DNA, which is used to create a cDNA library. Screening a Library for Clones Carrying a Gene of Interest A genomic library can consist of thousands of bacterial colonies, so how do you find the specific gene? The colonies with recombinant plasmids may be tested to find the ones that contain the gene of interest by nucleic acid hybridization, using a nucleic acid probe that has complementary sequences to segments of the gene. Nucleic acid probes identify a specific gene. - They have complementary sequences to segments of the gene, and this complementary molecule is labeled with a radioactive isotope or fluorescent dye. - It’s called a probe because it can be used to find the gene. To find the bacterial clone that holds the gene: - DNA is obtained from each colony of bacteria and treated to separate the DNA strands. - The probe is then mixed with the DNA strands and it only bonds with the recombinant DNA with a complementary base sequence. Once you have figured out which bacterial colony in the library contains the gene of interest, you can grow these bacteria in larger amounts. Polymerase Chain Reaction (PCR) The polymerase chain reaction (PCR) amplifies DNA by taking a small segment and copying it, increasing the amount of DNA - It can produce billions of copies of a section of DNA in only a few hours. PCR is often used prior to cloning a gene in cells. - By amplifying the gene prior to cloning, the later task of identifying clones carrying the desired gene is simplified. Even though PCR produces so many copies so fast, gene cloning is a necessary process. - There is a limit to the number of accurate copies that can be made due to the accumulation of relatively rare copying errors. - Large quantities of a gene are better prepared by DNA cloning in cells. Expressing Cloned Eukaryotic Genes Bacterial Expression Systems Differences between prokaryotic and eukaryotic mechanisms for gene expression can be overcome by using an expression vector An expression vector is a cloning vector that has an active prokaryotic promoter just upstream from the eukaryotic gene insertion site. Using a cDNA gene removes the problem of long introns, especially since bacteria don’t have RNA-splicing machinery Eukaryotic Cloning and Expression Systems Yeast cells are eukaryotic hosts used to clone and express eukaryotic genes - They are easy to grow and have plasmids that serve as vectors. Yeast artificial chromosomes (YACs) carry foreign DNA and contain an origin for DNA replication, a centromere, and 2 telomeres - These vectors behave normally in mitosis and are able to clone large pieces of DNA Plant and animal cells in culture can serve as hosts and may be necessary when a protein must be modified following translation DNA Technology Gel electrophoresis - Many of the methods for analyzing and comparing DNA involve gel electrophoresis, a technique that separates nucleic acids and proteins on the basis of their SIZE and electrical CHARGE Gel electrophoresis is used to separate fragments of DNA according to their size Due to the negative charge of their phosphate groups, DNA molecules are negative. As a result, when it is placed in the electric field of the gel, the DNA will migrate toward the positive pole. The process: - The gel used in gel electrophoresis is called agarose. This is very similar to the agar used in microbiology and is like Jello. When the agarose is poured into the tray, a device called a “comb” is in place. When the agarose gels, or solidifies, the comb is removed, leaving small holes or “wells” in the gel. Different sized fragments of DNA are loaded into wells with capillary tubes. The electric current is turned on and the DNA begins to move to the positive end. The rate depends on SIZE: -The smaller the DNA the faster and further it will move -The longer the DNA the slower and less far it will move - Restriction Analysis Restriction fragment analysis is the process used to produce DNA fingerprints. Another name for this is RFLP (=Restriction Fragment Length Polymorphisms) In the process of DNA fingerprinting: 1. A small sample of human DNA is cut with a particular restriction enzyme 2. The resulting restriction fragments are separated by gel electrophoresis This produces a series of characteristic patterns of bands (called a DNA FINGERRPINT) that show the lengths of the fragments. - A unique pattern of bands (=DNA FINGERPRINT) can be established for each individual in the world. 20.3 – Cloning organisms may lead to production of stem cells I don’t know how much detail we have to know, so just read over it. 20.4 – The practical applications of DNA technology Medical Applications Diagnosis of Diseases DNA technology is identifying genes responsible for genetic diseases; it is hoped this will lead to new ways to diagnose, treat, and even prevent those disorders. DNA microarray assays allow comparisons of gene expression in healthy and diseased tissues. Even if a gene hasn’t yet been cloned, a disease allele may be diagnosed when closely linked with a genetic marker, a sequence of variation, or polymorphism, found among individuals - Single nucleotide polymorphisms (SNPs) are variations that occur in at least 1% of humans. - When such variations change a restriction site, they result in restriction fragment length polymorphisms (RFLPs). Human Gene Therapy Gene therapy may provide the means for correcting genetic disorders in individuals by replacing or supplementing defective genes. New genes would be introduced into somatic cells of the affected tissue. For the correction to be permanent, the cells must be actively reproducing within the body, like bone marrow cells. Pharmaceutical Products Many pharmaceutical proteins are produced using biotechnology. Cancer: New approaches to cancer treatments include genetically engineered proteins that block a protein necessary for a tumor cell’s survival. Vaccines: Recombinant DNA techniques can be used to make vaccines that consist of surface molecules on pathogens, or to modify the genome of a pathogen as to attenuate it (make it nonpathogenic) so it can be used as a vaccine Forensic Evidence and Genetic Profiles Criminal cases: RFLP analysis has been used in criminal cases to compare the genetic profile of a suspect to that of crime DNA samples. Environmental Cleanup Mining: Genetically engineered microorganisms that are able to extract heavy metals, such as copper, may be important in mining and cleaning up mining waste. Fuel: Biofuels use microorganisms to ferment plant sugars to “bioethanol” or plant oils to “biodiesel” Agricultural Applications Animal husbandry: DNA technology allows scientists to produce transgenic animals which speed up the selective breeding process. Transgenic animals (“Pharm” animals) are produced by injecting a foreign gene into egg cells fertilized in vitro (=in a test tube). The eggs are then transplanted into surrogate mothers.