Biotechnology Chapter 20

Gene technology

Biotechnology Manipulation of organisms to make useful products

Genetic engineering Manipulation of genes Gene cloning: Multiple copies of a single gene Produce a specific product

Fig. 20-2 Cell containing gene of interest Bacterium 1 Gene inserted into plasmid Bacterial chromosome Plasmid Gene of interest Recombinant DNA (plasmid) DNA of chromosome 2 Plasmid put into bacterial cell Recombinant bacterium 3 Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Gene of Interest Protein expressed by gene of interest Copies of gene Protein harvested Figure 20.2 A preview of gene cloning and some uses of cloned genes 4 Basic research and various applications Basic research on gene Basic research on protein Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hor- mone treats stunted growth

Recombinant DNA 1970’s Combining genes from different sources Even different species Combined into single DNA Example: Bacteria & mammal

Recombinant DNA Genetically modified bacteria Mass produce beneficial chemicals Insulin Growth hormone Cancer drugs Pesticides

Plasmid

Plasmid Small separate circular DNA Replicated same as main DNA Foreign DNA added to plasmid Replicated along with plasmid

Recombinant DNA Nucleases: Enzymes that degrade DNA Restriction endonulceases: Restriction enzymes Cut DNA into fragments Specific points

Recombinant DNA Restriction sites: Places where DNA is cut Short DNA sequence

Recombinant DNA Restriction enzyme Recognizes short sequences in DNA Cuts at these sequences Staggered cut Leaves single-stranded ends Called “sticky ends”

Recombinant DNA D:\Chapter_20\A_PowerPoint_Lectures\20_Lecture_Presentation\2003RestrictionEnzymesA.html

                                                        

Recombinant DNA Sticky ends Enables insertion of other DNA DNA fragments from other sources Match ends by base pairs (complementary sequences) DNA ligase: Enzyme combines ends Forms a phosphodiester bond

                                                        

                                                        

Recombinant DNA (Process) 1. Isolate gene of interest & bacterial plasmid 2. Cut DNA & plasmid into fragments 3. Mix DNA fragments with cut plasmid. Fragment with gene of interest is inserted into the plasmid 4. Recombinant plasmid is mixed with bacteria

Recombinant DNA (Process) 5. Bacteria with recombinant DNA reproduce 6. Isolate bacterial clones that contain gene of interest Producing protein of interest 7. Grow large quantities of bacteria that produce the protein

Recombinant DNA (Process) D:\Chapter_20\A_PowerPoint_Lectures\20_Lecture_Presentation\2004CloningAGeneA.html

Fig. 20-4-4 TECHNIQUE Hummingbird cell Bacterial cell lacZ gene Restriction site Sticky ends Gene of interest ampR gene Bacterial plasmid Hummingbird DNA fragments Nonrecombinant plasmid Recombinant plasmids Bacteria carrying plasmids RESULTS Colony carrying non- recombinant plasmid with intact lacZ gene Colony carrying recombinant plasmid with disrupted lacZ gene One of many bacterial clones

Recombinant DNA Vector: DNA molecule that carry foreign DNA Enters & replicates in the host Plasmids & phages are common vectors Phages are larger than plasmid Can handle inserts up to 40 kilobases

PCR Polymerase chain reaction Amplify DNA Makes large quantities of DNA 1985

PCR Heated Denatured DNA primer Heat stable DNA polymerase Makes DNA

molecules; 2 molecules (in white boxes) match target sequence Fig. 20-8 TECHNIQUE 5 3 Target sequence Genomic DNA 3 5 1 Denaturation 5 3 3 5 2 Annealing Cycle 1 yields 2 molecules Primers 3 Extension New nucleo- tides Figure 20.8 The polymerase chain reaction (PCR) Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence

Gel electrophoresis Study DNA Polymer (gel) Restriction fragments Separates DNA based on charge & size Nucleic acids negative charge (Phosphates) Migrate towards + end (red)

Fig. 20-9 Figure 20.9 Gel electrophoresis TECHNIQUE Mixture of DNA mol- ecules of different sizes Power source – Cathode Anode + Gel 1 Power source – + Longer molecules 2 Shorter molecules RESULTS Figure 20.9 Gel electrophoresis

Fig. 20-10 Normal -globin allele Normal allele Sickle-cell allele 175 bp 201 bp Large fragment DdeI DdeI DdeI DdeI Large fragment Sickle-cell mutant -globin allele 376 bp 201 bp 175 bp 376 bp Large fragment DdeI Figure 20.10 Using restriction fragment analysis to distinguish the normal and sickle-cell alleles of the β-globin gene DdeI DdeI (a) DdeI restriction sites in normal and sickle-cell alleles of -globin gene (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles

Cloning Multicellular organisms come from a single cell. Offspring are identical

Cloning 1950 Carrots Totipotent: Mature cells that undifferentiated Give rise to any type of cells Common in plants

Cloning Nuclear transplantation Nucleus of an unfertilized/fertilized egg is removed Replaced with nucleus of differentiated cell Direct development of cell into tissues etc.

Cloning Removed nuclei from an egg Mammary cells Fused with egg cells Dolly, 1997, identical to mammary cell donor Died prematurely age 6 Arthritis & lung disease

TECHNIQUE RESULTS Fig. 20-18 Mammary cell donor Egg cell donor Egg cell from ovary Nucleus removed Cultured mammary cells 3 Cells fused 3 Nucleus from mammary cell 4 Grown in culture Early embryo 5 Implanted in uterus of a third sheep Surrogate mother 6 Embryonic development Lamb (“Dolly”) genetically identical to mammary cell donor RESULTS

Fig. 20-19

Cloning Few develop normally Abnormalities Epigenetic changes to the chromatin More methylation of chromatin Reprogram chromatin of differentiated cell

Stem cells Started 1998 at UW Early embryonic cells Potential to become any type of cell Master cell generates specialized cells Such as muscle cells, bone cells, or blood cells

Stem cells Embryos Bone marrow Umbilical cord blood Blood stem cells ?? Turn skin cells into embryonic stem cells Therapeutic cloning

From bone marrow in this example Fig. 20-20 Embryonic stem cells Adult stem cells Early human embryo at blastocyst stage (mammalian equiva- lent of blastula) From bone marrow in this example Cells generating all embryonic cell types Cells generating some cell types Cultured stem cells Different culture conditions Different types of differentiated cells Liver cells Nerve cells Blood cells

Medical applications Genetic markers Detect abnormal disease SNP Single nucleotide polymorphisms Single base pair site where variation is found RFLP Restriction fragment length polymorphisms

Disease-causing allele Fig. 20-21 DNA T Normal allele SNP C Figure 20.21 Single nucleotide polymorphisms (SNPs) as genetic markers for disease-causing alleles Disease-causing allele

Medical applications Gene therapy Treat genetic defects Alters person’s genes 2 girls with rare blood disease CF (vectors are viruses) SCID (immune disorder) Injected viral DNA with normal gene

Insert RNA version of normal allele into retrovirus. Fig. 20-22 Cloned gene 1 Insert RNA version of normal allele into retrovirus. Viral RNA 2 Let retrovirus infect bone marrow cells that have been removed from the patient and cultured. Retrovirus capsid 3 Viral DNA carrying the normal allele inserts into chromosome. Bone marrow cell from patient Bone marrow 4 Inject engineered cells into patient.

Medical applications Transgenic animal Gene from one animal is inserted into another Goat milk protein anti-thrombin Isolated from milk “pharm” animals

Animals Transgenic animals engineered for specific traits Genetically create a racehorse Not have to breed Sheep with better wool??

Agricultural applications Manipulate tomatoes Do not ripen as fast “Flavr-Savr” Slows down ethylene production

Agricultural applications Introduce genes to plants Enable them to “fix” nitrogen Convert N2 to NH3 Help eliminate use fertilizers Cut $$

Agricultural applications Herbicide resistance Plant genetically resists the herbicide Insect resistance

Agricultural applications Transgenic rice “golden rice” Rice with genes that code for better absorption of iron and beta carotene First of many genetically engineered foods Helps dietary deficiencies

Forensics Genetic profile: Individual genetic markers “DNA fingerprint” RFLP STR Short tandem repeats Occur in specific regions in genome Unique

Fig. 20-24 (a) This photo shows Earl Washington just before his release in 2001, after 17 years in prison. Source of sample STR marker 1 STR marker 2 STR marker 3 Figure 20.24 STR analysis used to release an innocent man from prison For the Discovery Video DNA Forensics, go to Animation and Video Files. Semen on victim 17, 19 13, 16 12, 12 Earl Washington 16, 18 14, 15 11, 12 Kenneth Tinsley 17, 19 13, 16 12, 12 (b) These and other STR data exonerated Washington and led Tinsley to plead guilty to the murder.

Concerns over genetic engineering Genetically modified foods Harmful? Genetically engineered gametes Blonde and blue eyes??