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Transcript
Outline: Gene Technology Central Dogma: DNA Æ RNA Æ Protein Viruses & bacteria CONNECTION: Many viruses cause disease in animals and plants Both DNA viruses and RNA viruses cause disease in animals & plants Reproductive cycle of an RNA virus Mutation – Entry Cancer Gene Splicing and Cloning – Glycoprotein spikes contact host cell receptors – Viral envelope fuses with host plasma membrane PCR Gel Electrophoresis – Uncoating of viral particle to release the RNA genome Biotechnology Applications – mRNA synthesis using a viral enzyme Genomics – Protein synthesis – RNA synthesis of new viral genome – Assembly of viral particles Copyright © 2009 Pearson Education, Inc. Human Immunodeficiency Virus • HIV, the AIDS virus – A retrovirus Human Immunodeficiency Virus Glycoprotein spike Protein coat Membranous envelope VIRUS Viral RNA (genome) Envelope Glycoprotein Plasma membrane of host cell 1 Entry 2 Uncoating 3 RNA synthesis by viral enzyme Protein coat RNA (two identical strands) Reverse transcriptase Viral RNA (genome) Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 1 Human Immunodeficiency Virus Viral RNA CYTOPLASM 1 DNA strand NUCLEUS Chromosomal DNA 2 Doublestranded DNA 3 Viral RNA and proteins 5 Provirus DNA 4 Emerging viruses threaten human health How do emerging viruses cause human diseases? ¾Mutation RNA viruses mutate rapidly ¾Contact between species RNA Viruses from other animals spread to humans ¾Spread from isolated populations 6 HIV replication animation Copyright © 2009 Pearson Education, Inc. Emerging viruses threaten human health Examples of emerging viruses – HIV – Ebola virus – West Nile virus – RNA coronavirus - severe acute respiratory syndrome (SARS) – Avian flu virus 10.22 Bacteria can transfer DNA in three ways Three mechanisms allow transfer of bacterial DNA – Transformation is the uptake of DNA from the surrounding environment – Transduction is gene transfer through bacteriophages – Conjugation is the transfer of DNA from a donor to a recipient bacterial cell through a cytoplasmic bridge (pilus) Fate of “new” DNA entering a bacterium (1) Recombination of the transferred DNA with the host bacterial chromosome (2) Uptake of a plasmid (small circular loop of DNA) Copyright © 2009 Pearson Education, Inc. Copyright © 2009 Pearson Education, Inc. 2 Plasmids transfer genes for antibiotic resistance by conjugation History of Staphylococcus antibiotic resistance Penicillin Æ 1947 Methicillin Æ 1961 Plasmids Tetracycline Æ ∼1990s Erythromycin Æ ∼1990s Mu ta t io n Vancomycin Æ late 1990s Linezolid Æ 2003 Superbugs: Staphylococcus Æ necrotizing fasciitis Escherichia coli Æ “hamburger disease” Streptococcus Æ pneumonia, meningitis Pseudomonas Æ lung, blood infections Enterococcus Æ diverticulitis, meningitis Mutation Mutation 1. Definition: Change in DNA 2. Frequency: Mutation = change in the nucleotide sequence of DNA 1 in 50 million base pairs 1 in a million gametes Why mutation? 1. Spontaneous errors in DNA replication errors in DNA recombination Albino rainbow trout 2. Induced to form by mutagens High-energy radiation Chemicals Blue Trout White grapes Seedless navel orange Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 3 Mutation – gene alteration Mutation: Altered Genes Point mutations Æ Changes in 1-few nucleotides Normal gene Base substitution mRNA Protein mRNA Protein A U G A AG U U U G G C G C A Met Lys mRNA Protein Ala Normal A U G A A G U U U A G C G C A Met Lys Ser Phe U Base deletion Gly Phe Point mutations Æ alter one or a few DNA bases. What happens when a point mutation occurs? Silent Æ no change in mRNA codon Nonsense Æ create stop codon Frameshift Æ shifts reading of mRNA codons Ala Lys Nonsense Frameshift TTAGGCC DNA ATG ATA ATT mRNA UAC UAU UAA Missing TTAGCGCC AU G AA G U U GG C G C A U Met Silent Mutation Leu Ala UCG His Amino Tyrosine acid Base insertion Tyrosine Stop Serine 14 Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Examples of Mutation – Sickle Cell Anemia Examples of Mutation – Cystic Fibrosis 250,000 base pairs Normal hemoglobin DNA Mutant hemoglobin DNA 27 Exons + Introns 61,000 base pair mRNA mRNA 1,480 Amino Acid Sequence of CTFR protein mRNA Normal hemoglobin Sickle-cell hemoglobin Glu Val 4 Chromosomal mutations Mutation: Altered Genes Deletion Chromosomal mutations change chromosome structure. deletion Æ part of chromosome is lost duplication Æ part of chromosome is copied inversion Æ part of chromosome in reverse order translocation Æ part of chromosome moves to a new location part of chromosome is lost Duplication part of chromosome is copied Inversion part of chromosome is reversed in order Translocation 17 Chromosomal Mutations Transposition = Jumping genes chromosome segments move/swap places Chromosomal mutations – Transposons Chromosome A Transposon Chromosome B Consequences of transposition (48% Human genome = transposons) 1. Cause mutations 2. May disable functional genes 3. May cause cancer by insertion of transposon promoter near cancer-causing gene 4. Transposon diseases: Hemophilia, SCID, Muscular Distrophy 5. Viruses like HIV behave like transposons 5 Alterations of chromosome structure - Deletion Reciprocal translocation associated with chronic myelogenous leukemia (CML) Chromosome 9 Deletion in Chromosome #5 Cri du chat Syndrome Chromosome 5 deletion 1 in 25,000-50,000 Detected by amniocentesis Mental retardation May live normal life span but usually die in early childhood Chromosome 22 Reciprocal translocation “Philadelphia chromosome” Activated cancer-causing gene Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings DNA Damage and Repair DNA Damage and Repair Xeroderma pigmentosa 6 11.18 Cancer results from mutations in genes that control cell division THE GENETIC BASIS OF CANCER Mutations in two types of genes can cause cancer – Oncogenes – Proto-oncogenes normally promote cell division – Mutations to oncogenes enhance activity – Tumor-suppressor genes – Normally inhibit cell division – Mutations inactivate the genes and allow uncontrolled division to occur Copyright © 2009 Pearson Education, Inc. 11.18 Cancer & Proto-Oncogenes Cancer & Proto-Oncogenes Proto-oncogene DNA Promote cancer when present in a single copy Can be viral genes inserted into host chromosomes Can be mutated versions of proto-oncogenes, normal genes that promote cell division and differentiation Mutation within the gene Multiple copies of the gene Gene moved to new DNA locus, under new controls Converting a proto-oncogene to an oncogene can occur by – Mutation causing increased protein activity – Increased number of gene copies causing more protein to be produced – Change in location putting the gene under control of new promoter for increased transcription New promoter Oncogene Hyperactive growthstimulating protein in normal amount Normal growthstimulating protein in excess Normal growthstimulating protein in excess Copyright © 2009 Pearson Education, Inc. 7 Cancer & Tumor-suppressor genes Tumor-suppressor gene Mutated tumor-suppressor gene Normal growthinhibiting protein Defective, nonfunctioning protein Cell division under control Cell division not under control Signal Transduction Pathways & Proto-Oncogenes Signaling cell 1 2 Signaling molecule Receptor protein Plasma membrane 3 Target cell Relay proteins Transcription factor (activated) 4 Cell Division Nucleus DNA 5 mRNATranscription New protein 6 Promote cancer when both copies are mutated Translation Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Proteins Regulate Cell Cycle Ras protein Cytoplasm Src kinase Rb protein Nucleus p53 protein Cell cycle checkpoints PROTO-ONCOGENES Growth factor receptor: more per cell in many breast cancers. Ras protein: activated by mutations in 20–30% of all cancers. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Activity of Abnormal p53 gene Benzopyrene ABNORMAL p53 Abnormal p53 protein Src kinase: activated by mutations in 2–5% of all cancers. TUMOR-SUPPRESSOR GENES Rb protein: mutated in 40% of all cancers. p53 protein: mutated in 50% of all cancers. Cancer cell Step 1 Step 2 DNA damage caused by heat, radiation, chemicals. p53 protein fails to stop cell division and DNA repair. Cell division continues without repair. Step 3 Damaged cells may turn cancerous if other mutations appear. 8 Multiple mutations lead to cancer Cancer in the United States Cancer Carcinogens Cases in 1999 Prostate Testosterone; dietary fat 179,300 Breast Estrogen; possibly dietary fat 176,300 Lung Cigarette smoke 171,600 Colon & Rectum High dietary fat; low dietary fiber 129,400 Bladder Cigarette smoke 54,200 Skin Ultraviolet light 44,200 Kidney Cigarette smoke 30,000 Mouth and Throat Tobacco & alcohol 29,800 Pancreas Cigarette smoke 28,600 Stomach Table salt; cigarette smoke 21,900 Cervix Viruses; cigarette smoke 12,800 Mutation of Tumor Suppressor Gene APC Chromosomes Normal cell 1 mutation Increased Cell Division Mutation of ProtoOncogene K-ras Mutation of Tumor Suppressor Gene DCC Mutation of Tumor Suppressor Gene p53 2 mutations 3 mutations 4 mutations Benign polyp Benign polyp Malignant Cell & metastasis Gene Cloning & Gene Technology D EN 9 Gene Technology Cleaving, Splicing & Cloning DNA Manipulating DNA – Cleaving, Splicing & Cloning Restriction enzyme recognition sequence Transferring & Storing DNA DNA Genetic engineering Procedures related to gene technology PCR DNA Fingerprinting Restriction enzyme cuts the DNA into fragments Applications of gene technology Sticky end Cleaving, Splicing & Cloning DNA Restriction enzyme recognition sequence DNA Cleaving, Splicing & Cloning DNA 1 Restriction enzyme cuts DNA into fragments DNA from species A 2 Restriction enzyme cuts DNA into fragments 3 3 Addition of a DNA fragment from another source Fragments stick together4 by base-pairing DNA from species B Addition of a DNA fragment from Species B 10 Restriction enzyme recognition sequence 1 DNA Restriction enzyme cuts the DNA into fragments Cleaving, Splicing & Cloning DNA Cloning a Gene in a Bacterial Plasmid E.coli Human cell DNA Isolate DNA 1 two sources from Cut both DNAs with same restriction enzyme Plasmid 2 Sticky ends Sticky end Addition of a DNA fragment from another source Mix DNAs; Join by base-pairing 3 Add DNA ligase to bond the DNA covalently Fragments stick together by base-pairing 4 Recombinant DNA plasmid Recombinant bacterium DNA ligase pastes DNA strands Recombinant DNA molecule 5 Bacterial clone carrying many copies of the human gene Gene of interest Insert plasmid into bacterium Clone bacterium Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Cloned genes can be stored in genomic libraries Cell nucleus Exon Intron Exon Intron Exon Genomic library = DNA fragments containing all of an organism’s genes. Eukaryote DNA 1 Transcription Constructed & stored in cloned bacterial plasmids or phages. Genome cut up with restriction enzyme Recombinant plasmid or Recombinant phage DNA RNA primary transcript 2 RNA splicing mRNA 3 Isolation of mRNA Reverse transcriptase Bacterial clone Plasmid library Phage clone Phage library cDNA strand being synthesized and addition of reverse transcriptase; synthesis of DNA strand 4 Breakdown of RNA 5 Synthesis of second cDNA of gene (no introns) DNA strand Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 11 • Recombinant DNA technology – Can be used to produce new genetic varieties of plants and animals, genetically modified (GM) organisms Agrobacterium tumefaciens DNA containing gene for desired trait Ti plasmid 1 Recombinant Plant cell 2 Plant with new trait 3 Insert plant geneTi plasmid Introduction Regeneration into plasmid using of plant Into restriction enzyme plant cells T DNA and DNA ligase Restriction T DNA carrying new site gene within plant chromosome Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Fig. 16.20 Examples of genetically modified (GM) crops Glyphosate Resistance 1. Cotton 2. Corn 3. Soybeans 4. Canola 5. Wheat Bt Crops • Cotton • Corn Other engineered crops 1. Papaya virus resistance Enhancement of Nutritional Value/Longevity 2. Carnation longevity 1. Rice 3. Flax herbicide resistance 2. “Flavr Savr” Tomato 4. Lentil herbicide resistance 5. Potato insect resistance 6. Squash virus resistance 7. Sugar beet herbicide resistance 8. Cucumber virus resistance 9. Watermelon virus resistance 12.11 DNA profiles and Genetic Marker Analysis DNA profiling is the analysis of DNA fragments to determine whether they come from a particular individual – PCRÆ PCR amplification of DNA markers – Gel Electrophoresis Æ Sizes of fragments are compared Compares genetic markers from noncoding regions that show variation between individuals Copyright © 2009 Pearson Education, Inc. 12 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Polymerase Chain Reaction (PCR) Gel Electrophoresis Target sequence 1 copy 1. Heat ÆDenature DNA Cool & add primers 1 Cycle 1 Mixture of DNA molecules Of different sizes 2.2 Add DNA polymerase & Nucleotides 3 New DNA synthesized 3. Repeat 1, 2 & 3 4 copies Longer molecules – – 2 copies Cycle 2 Repeat 1, 2 & 3 Power source Gel + + Completed gel 8 copies Shorter molecules Cycle 3 Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings How Restriction Fragments Reflect DNA Sequence • Restriction fragment length polymorphisms (RFLPs) Short tandem repeats (STRs) are genetic markers STRs are short DNA sequences repeated many times in a row at the same location. Number of STR units differs between individuals. • Reflect differences in the sequences of DNA samples STR site 1 STR site 2 Crime scene DNA Crime scene Suspect w G Cut C CG GC GC z x y Number of short tandem Number of short tandem repeats match repeats do not match AT CG GC GC Suspect’s DNA G Cut C CG GC GC y CG Cut C G GC GC DNA from chromosomes Figure 12.11A Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 13 Short tandem repeats (STRs) are genetic markers DNA Fingerprinting Crime scene DNA Suspect’s DNA Crime scene DNA Suspect’s DNA DNA fragments separated by Gel Electrophoresis Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Nutritional Deficiencies Iron Main component of hemoglobin Supports energy production enzymes May have anti-cancer properties Powerful immune-system booster Symptoms of Iron Deficiency Anemia Tiredness Sleep problems Impaired mental / intellectual function Learning, growth and behavioural disturbances Frequent infections Some types of deafness Nutritional Deficiencies Vitamin A Roles •Vision •Immune defense •Reducing morbidity of measles •Reducing respiratory infections •Cell differentiation and morphogenesis 14 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Genetically Engineered Golden Rice Beans Aspergillus fungus Ferritin gene is transferred into rice from beans. Fe Phytase gene is transferred into rice from a fungus. Pt Wild rice Metallothionin gene is transferred into rice from wild rice. Rice chromosome Ferritin protein Phytate, which increases iron inhibits iron content of rice. reabsorption, is destroyed by the phytase enzyme. Daffodil β-carotene enzyme Synthesis genes are transferred into rice from daffodils. S Metallothionin protein supplies extra sulfur to increase iron uptake. A1 A2 A3 A4 β-carotene, a precursor to vitamin A, is synthesized. 12.8 CONNECTION: Genetically modified organisms are transforming agriculture Genetically modified (GM) organisms contain one or more genes introduced by artificial means Transgenic organisms contain at least one gene from another species GM plants – Resistance to herbicides – Resistance to pests – Improved nutritional profile GM animals Golden Rice 12.7 CONNECTION: DNA technology has changed the pharmaceutical industry and medicine Products of DNA technology – Therapeutic hormones – Insulin to treat diabetes – Human growth hormone to treat dwarfism – Diagnosis and treatment of disease – Testing for inherited diseases – Detecting infectious agents such as HIV – Improved qualities – Production of proteins or therapeutics Copyright © 2009 Pearson Education, Inc. Copyright © 2009 Pearson Education, Inc. 15 12.7 CONNECTION: DNA technology has changed the pharmaceutical industry and medicine Products of DNA technology – Vaccines – Stimulate an immune response by injecting – Protein from the surface of an infectious agent – A harmless version of the infectious agent 12.7 CONNECTION: DNA technology has changed the pharmaceutical industry and medicine Advantages of recombinant DNA products – Identical to human protein – Purity – Quantity – A harmless version of the smallpox virus containing genes from other infectious agents Copyright © 2009 Pearson Education, Inc. Copyright © 2009 Pearson Education, Inc. Table 09.02 Adenosine deaminase deficiency patient 16 12.17 Genomics is the scientific study of whole genomes Exons (regions of genes coding for protein or giving rise to rRNA or tRNA) (1.5%) Genomics is the study of an organism’s complete set of genes and their interactions – Initial studies focused on prokaryotic genomes – Many eukaryotic genomes have since been investigated Evolutionary relationships can be elucidated Repetitive DNA that includes transposable elements and related sequences (44%) – Genomic studies showed a 96% similarity in DNA sequences between chimpanzees and humans Introns and regulatory sequences (24%) Unique noncoding DNA (15%) Repetitive DNA unrelated to transposable elements (15%) – Functions of human disease-causing genes have been determined by comparisons to similar genes in yeast Copyright © 2009 Pearson Education, Inc. 12.18 CONNECTION: The Human Genome Project revealed that most of the human genome does not consist of genes Results of the Human Genome Project – Humans have 21,000 genes in 3.2 billion nucleotide pairs – Only 1.5% of the DNA codes for proteins, tRNAs, or rRNAs – The remaining 88.5% of the DNA contains END GENE TECHNOLOGY – Control regions such as promoters and enhancers – Unique noncoding DNA – Repetitive DNA – Found in centromeres and telomeres – Found dispersed throughout the genome, related to transposable elements that can move or be copied from one location to another Copyright © 2009 Pearson Education, Inc. 17
