CONNECTION: Many viruses cause disease in animals and plants

Transcription

1 Outline: Gene Technology Central Dogma: RNA Protein Viruses & bacteria Mutation Cancer Gene Splicing and Cloning PCR Gel Electrophoresis Biotechnology Applications Genomics CONNECTION: Many viruses cause disease in animals and plants Both viruses and RNA viruses cause disease in animals & plants Reproductive cycle of an RNA virus Entry Glyco spikes contact host cell receptors Viral envelope fuses with host plasma membrane Uncoating of viral particle to release the RNA genome mrna synthesis using a viral enzyme Protein synthesis RNA synthesis of new viral genome Assembly of viral particles Human Immunodeficiency Virus HIV, the AIDS virus A retrovirus Envelope Glyco Human Immunodeficiency Virus Viral RNA (genome) VIRUS Plasma membrane of host cell 1 Entry Glyco spike Protein coat Membranous envelope Protein coat RNA (two identical strands) Reverse transcriptase Viral RNA (genome) Uncoating RNA synthesis by viral enzyme 1

2 Viral RNA strand Doublestranded Viral RNA and s Human Immunodeficiency Virus CYTOPLASM NUCLEUS Chromosomal Provirus 4 RNA Emerging viruses threaten human health How do emerging viruses cause human diseases? Mutation RNA viruses mutate rapidly Contact between species Viruses from other animals spread to humans Spread from isolated populations HIV replication animation 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. Bacteria can transfer in three ways Three mechanisms allow transfer of bacterial Transformation is the uptake of from the surrounding environment Transduction is gene transfer through bacteriophages Conjugation is the transfer of from a donor to a recipient bacterial cell through a cytoplasmic bridge (pilus) Fate of new entering a bacterium (1) Recombination of the transferred with the host bacterial chromosome () Uptake of a plasmid (small circular loop of )

3 Plasmids transfer genes for antibiotic resistance by conjugation History of Staphylococcus antibiotic resistance Penicillin 1947 Methicillin 1961 Plasmids Tetracycline 1990s Erythromycin 1990s Vancomycin late 1990s Linezolid 00 Superbugs: Staphylococcus necrotizing fasciitis Escherichia coli hamburger disease Streptococcus pneumonia, meningitis Pseudomonas lung, blood infections Enterococcus diverticulitis, meningitis Mutation Mutation 1. Definition: Change in. Frequency: 1 in 50 million base pairs 1 in a million gametes Albino rainbow trout Mutation Mutation = change in the nucleotide sequence of Why mutation? 1. Spontaneous errors in replication errors in recombination. Induced to form by mutagens High-energy radiation Chemicals Blue Trout White grapes Seedless navel orange

4 Mutation gene alteration Point mutations Changes in 1-few nucleotides Normal gene AUGAAGU U U mrna G G C GCA Protein Met Lys Phe Gly Ala Mutation: Altered Genes Point mutations alter one or a few bases. What happens when a point mutation occurs? Silent no change in mrna codon Nonsense create stop codon Frameshift shifts reading of mrna codons Base substitution Base deletion mrna Protein mrna Protein AUGAA G U U U A G C GCA Met Lys Phe Ser Ala U Missing AUG AA G U U GGCGCAU Met Lys Leu Ala His mrna Normal Silent Nonsense Frameshift Mutation ATG ATA ATT TTAGGCC TTAGCGCC UAC UAU UAA UCG Base insertion Amino acid Tyrosine Tyrosine Stop Serine 14 Examples of Mutation Sickle Cell Anemia Examples of Mutation Cystic Fibrosis Normal hemoglobin Mutant hemoglobin 50,000 base pairs 7 Exons + Introns 61,000 base pair mrna mrna mrna 1,480 Amino Acid Sequence of CTFR Normal hemoglobin Glu Sickle-cell hemoglobin Val 4

5 Mutation: Altered Genes Chromosomal mutations change chromosome structure. deletion part of chromosome is lost duplication part of chromosome is copied inversion part of chromosome in reverse order translocation part of chromosome moves to a new location Chromosomal mutations Deletion part of chromosome is lost Duplication part of chromosome is copied Inversion part of chromosome is reversed in order Translocation 17 chromosome segments move/swap places Chromosomal Mutations Transposition = Jumping genes Chromosomal mutations Transposons Chromosome A Transposon Chromosome B Consequences of transposition (48% Human genome = transposons) 1. Cause mutations. May disable functional genes. 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

6 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 5,000-50,000 Detected by amniocentesis Mental retardation May live normal life span but usually die in early childhood Chromosome Philadelphia chromosome Activated cancer-causing gene Reciprocal translocation Damage and Repair Damage and Repair Xeroderma pigmentosa 6

7 THE GENETIC BASIS OF CANCER Cancer results from mutations in genes that control cell division 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 Cancer & Proto-Oncogenes Cancer & Proto-Oncogenes Proto-oncogene 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 locus, under new controls Converting a proto-oncogene to an oncogene can occur by Mutation causing increased activity Increased number of gene copies causing more to be produced Change in location putting the gene under control of new promoter for increased transcription Hyperactive growthstimulating in normal amount Oncogene Normal growthstimulating in excess New promoter Normal growthstimulating in excess 7

8 Cancer & Tumor-suppressor genes Tumor-suppressor gene Normal growthinhibiting Cell division under control Mutated tumor-suppressor gene Promote cancer when both copies are mutated Defective, nonfunctioning Cell division not under control Signal Transduction Pathways & Proto-Oncogenes Signaling cell 1 Target cell Transcription factor (activated) Signaling molecule Receptor 4 Relay s Plasma membrane Nucleus 5 mrna Transcription New 6 Translation Cell Division Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Proteins Regulate Cell Cycle Ras Cytoplasm Src kinase Rb Nucleus p5 Cell cycle checkpoints PROTO-ONCOGENES Growth factor receptor: more per cell in many breast cancers. Ras : activated by mutations in 0 0% of all cancers. Src kinase: activated by mutations in 5% of all cancers. TUMOR-SUPPRESSOR GENES Rb : mutated in 40% of all cancers. p5 : mutated in 50% of all cancers. Activity of Abnormal p5 gene ABNORMAL p5 Abnormal p5 Step 1 Step p5 fails to damage stop cell division caused by and repair. heat, Cell division radiation, continues without chemicals. repair. Step Benzopyrene Cancer cell Damaged cells may turn cancerous if other mutations appear. 8

9 Cancer in the United States Cancer Carcinogens Prostate Testosterone; dietary fat Breast Estrogen; possibly dietary fat Lung Cigarette smoke Colon & Rectum High dietary fat; low dietary fiber Bladder Cigarette smoke Skin Ultraviolet light Kidney Cigarette smoke Mouth and Throat Tobacco & alcohol Pancreas Cigarette smoke Stomach Table salt; cigarette smoke Cervix Viruses; cigarette smoke Cases in ,00 176,00 171,600 19,400 54,00 44,00 0,000 9,800 8,600 1,900 1,800 Chromosomes 1 mutation Normal cell Multiple mutations lead to cancer Mutation of Tumor Suppressor Gene APC Increased Cell Division Mutation of Proto- Oncogene K-ras mutations Benign polyp Mutation of Tumor Suppressor Gene DCC mutations Benign polyp Mutation of Tumor Suppressor Gene p5 4 mutations Malignant Cell & metastasis Gene Cloning & Gene Technology END 9

10 Gene Technology Manipulating Cleaving, Splicing & Cloning Transferring & Storing Genetic engineering Procedures related to gene technology PCR Fingerprinting Applications of gene technology Cleaving, Splicing & Cloning Restriction enzyme cuts the into fragments Restriction enzyme recognition sequence Sticky end Cleaving, Splicing & Cloning Restriction enzyme recognition sequence Cleaving, 1 Splicing & Cloning Restriction enzyme cuts into fragments from species A Addition of a fragment from another source Restriction enzyme cuts into fragments Fragments stick together by 4 base-pairing from species B Addition of a fragment from Species B 10

11 Restriction enzyme recognition sequence 1 Restriction enzyme cuts the into fragments Sticky end Cleaving, Splicing & Cloning Cloning a Gene in a Bacterial Plasmid E.coli Plasmid Human cell Isolate from 1 two sources Cut both s with same restriction enzyme Sticky ends Addition of a fragment from another source Mix s; Join by base-pairing Fragments stick together by base-pairing Recombinant plasmid Add ligase to bond the covalently Gene of interest ligase pastes strands 4 5 Recombinant molecule Recombinant bacterium Bacterial clone carrying many copies of the human gene Insert plasmid into bacterium Clone bacterium Cloned genes can be stored in genomic libraries Genomic library = fragments containing all of an organism s genes. Constructed & stored in cloned bacterial plasmids or phages. Recombinant plasmid or Genome cut up with restriction enzyme Recombinant phage Cell nucleus Exon Intron Exon Intron Exon Eukaryote RNA primary transcript mrna 1 Transcription RNA splicing Reverse transcriptase Isolation of mrna and addition of reverse transcriptase; synthesis of strand Bacterial clone Phage clone c strand being synthesized 4 Breakdown of RNA Plasmid library Phage library c of gene (no introns) 5 Synthesis of second strand 11

12 Recombinant technology Can be used to produce new genetic varieties of plants and animals, genetically modified (GM) organisms Agrobacterium tumefaciens containing gene for desired trait Plant cell Plant with new trait T Ti plasmid Restriction site 1 Recombinant Ti plasmid Insert plant gene into plasmid using restriction enzyme and ligase Introduction Into plant cells Regeneration of plant T carrying new gene within plant chromosome Fig Examples of genetically modified (GM) crops Glyphosate Resistance 1. Cotton. Corn. Soybeans 4. Canola 5. Wheat Bt Crops Cotton Corn Other engineered crops 1. Papaya virus resistance Enhancement of Nutritional Value/Longevity. Carnation longevity 1. Rice. Flax herbicide resistance. 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 1.11 profiles and Genetic Marker Analysis profiling is the analysis of fragments to determine whether they come from a particular individual PCR amplification of markers Gel Electrophoresis Sizes of fragments are compared Compares genetic markers from noncoding regions that show variation between individuals 1

13 Polymerase Chain Reaction (PCR) copies Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Cycle 1 Target sequence 1 copy 1. Heat Denature Cool 1 & add primers. Add polymerase & Nucleotides. New synthesized Mixture of molecules Of different sizes Gel Electrophoresis Longer molecules 4 copies Cycle Repeat 1, & Power source Gel 8 copies Cycle Repeat 1, & + + Completed gel Shorter molecules How Restriction Fragments Reflect Sequence Restriction fragment length polymorphisms (RFLPs) Reflect differences in the sequences of samples Short tandem repeats (STRs) are genetic markers STRs are short sequences repeated many times in a row at the same location. Number of STR units differs between individuals. STR site 1 STR site Crime scene Suspect Crime scene w x Cut C G GC z AT CG CC G Suspect s Number of short tandem repeats match Number of short tandem repeats do not match y Cut C G GC y Cut G C GC G C Figure 1.11A from chromosomes 1

14 Short tandem repeats (STRs) are genetic markers Fingerprinting Crime scene Suspect s Crime scene Suspect s fragments separated by Gel Electrophoresis 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

15 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Genetically Engineered Golden Rice Beans Aspergillus fungus Wild rice Daffodil Golden Rice Ferritin gene is transferred into rice from beans. Phytase gene is transferred into rice from a fungus. Metallothionin gene is transferred into rice from wild rice. β-carotene enzyme Synthesis genes are transferred into rice from daffodils. Rice Fe Pt chromosome S A 1 A A A 4 Ferritin increases iron content of rice. Phytate, which inhibits iron reabsorption, is destroyed by the phytase enzyme. Metallothionin supplies extra sulfur to increase iron uptake. β-carotene, a precursor to vitamin A, is synthesized. 1.8 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 Improved qualities Production of s or therapeutics 1.7 CONNECTION: technology has changed the pharmaceutical industry and medicine Products of 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 15

16 1.7 CONNECTION: technology has changed the pharmaceutical industry and medicine Products of technology Vaccines Stimulate an immune response by injecting Protein from the surface of an infectious agent A harmless version of the infectious agent A harmless version of the smallpox virus containing genes from other infectious agents 1.7 CONNECTION: technology has changed the pharmaceutical industry and medicine Advantages of recombinant products Identical to human Purity Quantity Adenosine deaminase deficiency patient Table

17 1.17 Genomics is the scientific study of whole genomes 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 Genomic studies showed a 96% similarity in sequences between chimpanzees and humans Functions of human disease-causing genes have been determined by comparisons to similar genes in yeast Exons (regions of genes coding for or giving rise to rrna or trna) (1.5%) Repetitive that includes transposable elements and related sequences (44%) Introns and regulatory sequences (4%) Repetitive unrelated to transposable elements (15%) Unique noncoding (15%) 1.18 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 1,000 genes in. billion nucleotide pairs Only 1.5% of the codes for s, trnas, or rrnas The remaining 88.5% of the contains Control regions such as promoters and enhancers Unique noncoding Repetitive 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 END GENE TECHNOLOGY 17