PowerLecture: Chapter 16 Studying and Manipulating Genomes

Golden Rice, or Frankenfood? Ordinary daffodil Fig. 16-1a, p.242

Impacts, Issues: Golden Rice, or Frankenfood? 124 million children around the world have vitamin A deficiencies Golden rice –Rice plants engineered with genes from daffodils allowing it to produce beta-carotine in its seeds (rice) –Beta carotine is the precursor to Vitamin A Rice is the main food for 3 billion people

Golden Rice, or Frankenfood? 2nd generation 1st generation Regular rice Fig. 16-1b, p.242

Impacts, Issues: Golden Rice, or Frankenfood? Many crops plants have been modified, including corn, beets, potatoes, and cotton Potentially less harmful to the environment than current agricultural practices

Golden Rice, or Frankenfood? Today's corn Ancestral corn p.243

Discovery of Restriction Enzymes Hamilton Smith was studying how Haemophilus influenzae defend themselves from bacteriophage attack Discovered bacteria have an enzyme that chops up viral DNA

Specificity of Cuts Restriction enzymes cut DNA at a specific sequence Number of cuts made in DNA will depend on number of times the “target” sequence occurs

G another DNA fragment 5’ 3’ one DNA fragment C T A 3’ 5’ G C T A nick DNA ligase action G C T A 3’ 5’ Fig. 16-2, p.244

Terms DNA ligase – seals cuts in DNA Recombinant DNA – any molecule consisting of base sequences from 2 or more organisms of the same or different species Cloning vector – a plasmid that has accepted foreign DNA and can be slipped into host

Using Plasmids Plasmid is small circle of bacterial DNA Foreign DNA can be inserted into plasmid Forms recombinant plasmids Plasmid is a cloning vector Can deliver DNA into another cell

Plasmids Fig. 16-3a, p.244

Using Plasmids Fig. 16-4, p.245 e The DNA fragments and the plasmid DNA are mixed with DNA ligase. a A restriction enzyme cuts a specific base sequence everywhere it occurs in DNA. b The DNA fragments have sticky ends. f The result? A collection of recombinant plasmids that incorporate foreign DNA fragments. c The same enzyme cuts the same sequnece in plasmid DNA. d The plasmid DNA also has sticky ends g Host cells that can divide rapidly take up the recombinant plasmids. Fig. 16-4, p.245

Making cDNA Use reverse transcriptase onto one strand of complementary DNA (cDNA)‏ DNA polymerase strips RNA bases Copies cDNA into a second strand Fig. 16-5, p.245

Gene Libraries Bacteria that contain different cloned DNA fragments Genomic library cDNA library

Use of a Probe Colonies on plate You want to find which bacteria in a library contain a specific gene Need a probe for that gene A radioisotope- labeled piece of DNA Will base-pair with gene of interest Cells adhere to filter Cells are lysed; DNA sticks to filter Probe is added Location where probe binds forms dark spot on film, indicates colony with gene

Making a Probe Make a primer if the sequence is known. If not usually DNA from closely related species

Amplifying DNA Fragments can be inserted into fast-growing microorganisms Polymerase chain reaction (PCR)

Polymerase Chain Reaction Sequence to be copied is heated Primers are added and bind to ends of single strands DNA polymerase uses free nucleotides to create complementary strands Doubles number of copies of DNA Animation

Polymerase Chain Reaction Double-stranded DNA to copy DNA heated to 90°– 94°C Primers added to base-pair with ends Mixture cooled; base-pairing of primers and ends of DNA strands DNA polymerases assemble new DNA strands Stepped Art Fig. 16-6, p. 256

Polymerase Chain Reaction Mixture heated again; makes all DNA fragments unwind Mixture cooled; base-pairing between primers and ends of single DNA strands DNA polymerase action again doubles number of identical DNA fragments Stepped Art Fig. 16-6, p. 256

Recording the Sequence T C C A T G G A C C T C C A T G G A C T C C A T G G A T C C A T G G T C C A T G T C C A T T C C A electrophoresis gel T C C DNA is placed on gel Fragments move off gel in size order; pass through laser beam Color each fragment fluoresces is recorded on printout T C one of the many fragments of DNA migrating through the gel T one of the DNA fragments passing through a laser beam after moving through the gel T C C A T G G A C C A

Recording the Sequence http://www.dnalc.org/ddnalc/resources/cycseq.html Fig. 16-8b, p.248

Genome Sequencing 1995 - Sequence of bacterium Haemophilus influenzae determined Automated DNA sequencing now main method Draft sequence of entire human genome determined in this way

Genome Sequencing Fig. 16-10a, p.250

Nucleotides for Sequencing Standard nucleotides (A, T, C, G)‏ Modified versions of these nucleotides Labeled so they fluoresce Structurally different so that they stop DNA synthesis when they are added to a strand

Reaction Mixture Copies of DNA to be sequenced Primer DNA polymerase Standard nucleotides Modified nucleotides

DNA Fingerprints Unique array of DNA fragments Inherited from parents in Mendelian fashion Even full siblings can be distinguished from one another by this technique

Tandem Repeats Short regions of DNA that differ substantially among people Many sites in genome where tandem repeats occur Each person carries a unique combination of repeat numbers

RFLPs Restriction fragment length polymorphisms DNA from areas with tandem repeats is cut with restriction enzymes Because of the variation in the amount of repeated DNA, the restriction fragments vary in size Variation is detected by gel electrophoresis

Gel Electrophoresis DNA is placed at one end of a gel A current is applied to the gel DNA molecules are negatively charged and move toward positive end of gel Smaller molecules move faster than larger ones http://www.dnalc.org/ddnalc/resources/elec trophoresis.html

Gel Electrophoresis Fig. 16-9b, p.249

Analyzing DNA Fingerprints DNA is stained or made visible by use of a radioactive probe Pattern of bands is used to: Identify or rule out criminal suspects Identify bodies Determine paternity

Genomics Structural genomics: actual mapping and sequencing of genomes of individuals Comparative genomics: concerned with possible evolutionary relationships of groups of organisms

Reactions Proceed Nucleotides are assembled to create complementary strands When a modified nucleotide is included, synthesis stops Result is millions of tagged copies of varying length

DNA Chips Microarrays of thousands of gene sequences representing a large subset of an entire genome (p251)‏ Stamped onto a glass plate the size of a small business card (p251)‏

Genetic Engineering Genes are isolated, modified, and inserted into an organism Made possible by recombinant technology Cut DNA up and recombine pieces Amplify modified pieces

Engineered Proteins Bacteria can be used to grow medically valuable proteins Insulin, interferon, blood-clotting factors Vaccines

Cleaning Up the Environment Microorganisms normally break down organic wastes and cycle materials Some can be engineered to break down pollutants or to take up larger amounts of harmful materials

Can Genetically Engineered Bacteria “Escape”? Genetically engineered bacteria are designed so that they cannot survive outside lab Genes are included that will be turned on in outside environment, triggering death

p.252

Engineered Plants Cotton plants that display resistance to herbicide Aspen plants that produce less lignin and more cellulose Tobacco plants that produce human proteins Mustard plant cells that produce biodegradable plastic

Engineered Plants Fig. 16-12a, p.253

Engineered Plants Fig. 16-12b, p.253

The Ti plasmid Researchers replace tumor- causing genes with beneficial genes Plasmid transfers these genes to cultured plant cells plant cell foreign gene in plasmid

The Ti plasmid b The bacterium infects a plant and transfers the Ti plasmid into it. a A bacterial cell contains a Ti plasmid (purple) that has a foreign gene (blue). e Young plants with a fluorescent gene product. c The plant cell divides. d Transgenic plants. Fig. 16-13, p.253

Genetic Changes Humans have been changing the genetics of other species for thousands of years Artificial selection of plants and animals Natural processes also at work Mutation, crossing over

First Engineered Mammals Experimenters used mice with hormone deficiency that leads to dwarfism Fertilized mouse eggs were injected with gene for rat growth hormone Gene was integrated into mouse DNA Engineered mice were 1-1/2 times larger than unmodified littermates

Transgenic Mice Fig. 16-15, p.254

Designer Cattle Genetically identical cattle embryos can be grown in culture Embryos can be genetically modified create resistance to mad cow disease engineer cattle to produce human serum albumin for medical use

Genetically Modified Animals Fig. 16-14a, p.254

Genetically Modified Animals Fig. 16-14b, p.254

Genetically Modified Animals Fig. 16-14c, p.254

Xenotransplantation Researchers knockout the Ggta1genes in transgenic piglets Ggta1 gene produces proteins that human antibodies recognize Pig’s organs are less prone to rejection by a human

Safety Superpathogens DNA from pathogenic or toxic organisms used in recombination experiments Hok genes NIH guidelines for DNA research

The Human Genome Initiative Goal - Map the entire human genome Initially thought by many to be a waste of resources Process accelerated when Craig Ventner used bits of cDNAs as hooks to find genes Sequencing was completed ahead of schedule in early 2001

Results of Gene Therapy Modified cells alive in woman’s liver Blood levels of LDLs down 20 percent No evidence of atherosclerosis Cholesterol levels remain high Remains to be seen whether procedure will prolong her life

Using Human Genes Even with gene in hand it is difficult to manipulate it to advantage Viruses usually used to insert genes into cultured human cells but procedure has problems Very difficult to get modified genes to work where they should

Ethical Issues Who decides what should be “corrected” through genetic engineering? Should animals be modified to provide organs for human transplants? Should humans be cloned?