C HAPTER 16 Studying and Manipulating Genomes

I MPACTS, I SSUES : G OLDEN R ICE, OR F RANKENFOOD ? 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

G ENETIC C HANGES 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

D ISCOVERY OF R ESTRICTION E NZYMES Hamilton Smith was studying how Haemophilus influenzae defend themselves from bacteriophage attack Discovered bacteria have an enzyme that chops up viral DNA

S PECIFICITY OF C UTS Restriction enzymes cut DNA at a specific sequence Number of cuts made in DNA will depend on number of times the “target” sequence occurs

M AKING R ECOMBINANT DNA 5’ 3’ G C T T A A A A T T C G G C T T A AG 3’ 5’ one DNA fragment another DNA fragment 3’ 5’

M AKING R ECOMBINANT DNA nick 5’ 3’ 5’ GA A T T C C T T A AG nick GA A T T C C T T A AG DNA ligase action

Fig. 16-2, p.244 Stepped Art GCTTAA AATTCG 3’ 5’ 3’ 5’ CTTAA AATTCG cut fragments G DNA ligase action nick GCTTAA AATTCG 3’ 5’ 3’ 5’ another DNA fragment AATTC 3’ 5’3’ 5’ G one DNA fragment 3’ 5’ G CTTAA 3’ 5’ enzyme recognition site GCTTAA AATTCG 3’ 5’

U SING P LASMIDS 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

Fig. 16-3b, p.244 Plasmids

Restriction enzyme cuts molecule of chromosomal DNA or cDNA recombinan t plasmids containing foreign DNA host cells containing recombinant plasmids Same enzyme cuts same sequence in plasmid DNA Foreign DNA, plasmid DNA, and modification enzymes are mixed DNA or cDNA fragments with sticky ends plasmid DNA with sticky ends Stepped Art Fig. 16-4, p.245

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

M AKING C DNA Fig. 16-5, p.245

G ENE L IBRARIES Bacteria that contain different cloned DNA fragments Genomic library cDNA library

U SING A P ROBE TO F IND A G ENE 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

a Bacterial colonies, each derived from a single cell, grow on a culture plate. Each colony is about 1 millimeter across. b A nitrocellulose or nylon filter is placed on the plate. Some cells of each colony adhere to it. The filter mirrors how the colonies are distributed on the culture plate. c The filter is lifted off and put into a solution. Cells stuck to it rupture; the cellular DNA sticks to the filter. d The DNA is denatured to single strands at each site. A radioactively labeled probe is added to the filter. The probe binds to DNA with a complementary base sequence. e The probe’s location is identified by exposing the filter to x-ray film. The image that forms on the film reveals the colony that has the gene of interest. Fig. 16-6, p.246 Use of a Probe

F AMILIAL H YPERCHOLESTEROLEMIA Gene encodes protein that serves as cell’s LDL receptor Two normal alleles for the gene keep blood level of LDLs low Two mutated alleles lead to abnormally high cholesterol levels & heart disease

E XAMPLE OF G ENE T HERAPY Woman with familial hypercholesterolemia Part of her liver was removed Virus used to insert normal gene for LDL receptor into cultured liver cells Modified liver cells placed back in patient

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

P OLYMERASE C HAIN R EACTION 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

P OLYMERASE C HAIN R EACTION 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 Fig. 16-6, p. 256 Stepped Art

P OLYMERASE C HAIN R EACTION Stepped Art 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 Fig. 16-6, p. 256

R ECORDING THE S EQUENCE 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 T C C T C T electrophoresis gel one of the many fragments of DNA migrating through the gel 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 DNA is placed on gel Fragments move off gel in size order; pass through laser beam Color each fragment fluoresces is recorded on printout

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

T ANDEM R EPEATS 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

RFLP S 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

G EL E LECTROPHORESIS 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

Fig. 16-9b, p.249 G EL E LECTROPHORE SIS

A NALYZING DNA F INGERPRINTS 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

B OZEMAN VIDEO — DNA FINGERPRINTING

G ENOME S EQUENCING Sequence of bacterium Haemophilus influenzae determined Automated DNA sequencing now main method Draft sequence of entire human genome determined in this way

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

G ENETIC E NGINEERING Genes are isolated, modified, and inserted into an organism Made possible by recombinant technology Cut DNA up and recombine pieces Amplify modified pieces

E NGINEERED P ROTEINS Bacteria can be used to grow medically valuable proteins Insulin, interferon, blood-clotting factors Vaccines

C LEANING U P THE E NVIRONMENT 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

C AN G ENETICALLY E NGINEERED B ACTERIA “E SCAPE ”? 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

E NGINEERED P LANTS 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

T HE T I PLASMID Researchers replace tumor- causing genes with beneficial genes Plasmid transfers these genes to cultured plant cells foreign gene in plasmid plant cell

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

F IRST E NGINEERED M AMMALS 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

T RANSGENIC M ICE Fig , p.254

C LONING D OLLY A sheep cloned from an adult cell Nucleus from mammary gland cell was inserted into enucleated egg Embryo implanted into surrogate mother Sheep is genetic replica of animal from which mammary cell was taken

D ESIGNER C ATTLE 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

X ENOTRANSPLANTATION 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

Fig c, p.254 Genetically Modified Animals

T HE H UMAN G ENOME I NITIATIVE 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

U SING H UMAN G ENES 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

E THICAL I SSUES Who decides what should be “corrected” through genetic engineering? Should animals be modified to provide organs for human transplants? Should humans be cloned?