Chapter 16 Studying and Manipulating Genomes

Objectives   1. Know how DNA can be cleaved, spliced, cloned, and sequenced.   2.Understand what plasmids are and how they may be used to insert new genes into recombinant DNA molecules.   3.Explain the types of genetic experiments that nature has been performing for billions of years.

Objectives – cont.   4.Understand how one organism can produce the products of another.   5.Be aware of several limits and possibilities for future research in genetic engineering.   6.Explain how a new field of research called genomics has come about from the Human Genome Project.

 124 million children around the world have vitamin A deficiencies, they do not grow or develop normally, and may become blind.  Three genes (beta carotene –yellow pigment) from daffodils have been transferred into rice plants.  Rice plants engineered with genes produce beta-carotene in its seeds ( Golden rice)  Beta carotene is the precursor to Vitamin A  Rice is the main food for 3 billion people 16.0 Golden Rice, or Frankenfood?

 Humans have been experimenting with new breeds of organisms for thousands of years.  Many crops plants have been modified, including corn, beets, potatoes, and cotton  Potentially less harmful to the environment than current agricultural practices – less pesticide is needed. Golden Rice, or Frankenfood?

16.1 A Molecular Toolkit  In 1970, Hamilton Smith was studying how Haemophilus influenzae (bacteria) defend themselves from bacteriophage attack  He discovered bacteria have an enzyme that chops up viral DNA (foreign molecules) before it can get inserted into the bacterial chromosome.

Restriction Enzymes  Restriction enzymes (scissors) cut DNA at a specific base sequence (4 to 8 base pairs in length).  Number of cuts made in DNA will depend on number of times the “target” sequence occurs, in the 5’  3’ direction on both strands of DNA.

Restriction Enzymes  Many times the “sticky ends” that results from the cut can be used to pair up with another DNA fragment cut by the same enzyme.  DNA fragments produced by restriction enzymes are treated with DNA ligase to splice the DNA fragments together to form a recombinant DNA molecule.

Fig. 16-2, p.244 Stepped Art G CTTAA AATTCG 3’ 5’ 3’ 5’ CTTAA AATTC G cut fragments G DNA ligase action nick G CTTAA 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 G CTTAA AATTCG 3’ 5’

Cloning Vectors  Plasmids are circular DNA molecules in bacteria that carry only a few genes and can replicate independently of the single “main” chromosome.  Modified plasmids that are capable of accepting, replicating, and delivering DNA to another host cell are called cloning vectors.

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

cDNA cloning  Researches use DNA made from “mature” mRNA transcripts ( i.e. introns have been removed from original DNA)  Restriction enzymes will not cut single- stranded molecules.  Reverse transcriptase catalyzes the assembly of a single DNA strand on an mRNA template, forming a hybrid.

cDNA (cont.)  DNA polymerase replaces the mRNA with another DNA strand.  The result is a double stranded DNA, that may be inserted into a plasmid for amplification or cloning.

cDNA Cloning Fig. 16-5, p.245

16.2 Gene Libraries  A gene library is a collection of host cells (bacteria) that house different cloned fragments of DNA.  A genomic library are the cloned fragments of an entire genome.  A cDNA library is derived from mRNA.

Using a Probe to Find a Gene  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 that will base-pair with gene of interest A radioisotope-labeled piece of DNA that 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

Amplifying DNA  Fragments can be inserted into fast-growing microorganisms  Polymerase chain reaction (PCR) is a method of rapidly and exponentially amplifying the number of particular DNA fragments, used to make millions of copies of cDNA.

Polymerase Chain Reaction  Sequence to be copied is heated  Primers (short nucleotide sequences) are added and bind to ends of single strands. They are start tags.  DNA polymerase (heat resistant) uses free nucleotides to create complementary strands  Doubles number of copies of DNA

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 Fig. 16-6, p. 256 Stepped Art

Polymerase Chain Reaction 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

16.3 Automated DNA sequencing  This techniques used DNA polymerase to partially replicate a DNA template.  Laboratories use automated DNA sequencing to determine the unknown sequence of bases in any DNA sample (cloned or PCR-amplified DNA) in just a few hours.

Automated DNA sequencing  The machine builds DNA molecules but uses eight kinds of bases: four normal (A, T, C, G) and four that are modified to fluoresce in different colors in laser light, Structurally different so that they stop DNA synthesis when they are added to a strand.  Researchers mix these eight bases with a single stranded DNA template, a primer, and DNA polymerase.

Automated DNA sequencing  The automated DNA sequencer separates the set of fragments by gel electrophoresis.  The “tag” base at the end of each fragment in the set is identified by the laser beam.  The computer program assembles the information to reveal the entire DNA sequence.

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 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

16.4 DNA Fingerprints  They are unique array of DNA fragments  Inherited from parents in Mendelian fashion  More than 99% of DNA is the same in all humans – only 1% is different  Even full siblings can be distinguished from one another by this technique

Tandem Repeats  The technique focuses on tandem repeats – copies of the same short regions of DNA that differ substantially among people  There are 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 (vary by individual)  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  DNA may be amplified by PCR if the sample is small.

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

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 or rule out criminal suspects Identify bodies – 9/11 victims Identify bodies – 9/11 victims Determine paternity Determine paternity

16.5 Genome Sequencing  Sequence of bacterium Haemophilus influenzae determined  Automated DNA sequencing now main method  Draft sequence of entire human genome determined in this way  Human Genome Project began in 1988

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 2003

Genome sequencing  About 99% of the coding regions of human DNA have been deciphered  About 20,000 confirmed genes  Protein encoding genes make up less than 2 percent of our genome.

Genomics  Genomics is a new research field.  Structural genomics: actual mapping and sequencing of genomes of individuals  Comparative genomics: concerned with possible evolutionary relationships of groups of organisms

Genomics  Genomics has potential for human gene therapy – the transfer of one or more normal or modified genes into a person’s body cells to correct a genetic defect or boost resistance to disease.  Some gene therapies use viruses as vectors, deliver modified cells into a patients tissue.

DNA Chips  Researchers can pinpoint which genes are silent and which are being expressed with the use of DNA chips.  DNA Chips are microarrays of thousands of gene sequences representing a large subset of an entire genome  Stamped onto a glass plate the size of a small business card (p251)

DNA Chips  DNA chips are being used to compare different genes expression patterns between cells. Examples are yeasts grown in the presence or absence of oxygen. See fig  Green – genes active during fermentation  Red – genes used in aerobic respiration  Yellow – genes active in both

16.6 Genetic Engineering  Genetic Engineering is the deliberate modification of an individual’s genome.  Genes from another species may be transferred to an individual.  The individual may have its own genes isolated, modified and copied, and then receive copies of the modified genes.

16.6 Genetic Engineering  Genetic engineering started with bacterial species.  The kinds that take up plasmids are now widely used in basic research, agriculture, medicine and industry.  Made possible by recombinant technology

Engineered Proteins  Bacteria can be used to grow medically valuable proteins Insulin, somatotropin (growth hormone), hemoglobin, interferon, blood-clotting factors Insulin, somatotropin (growth hormone), hemoglobin, interferon, blood-clotting factors Vaccines Vaccines

Cleaning Up the Environment  Microorganisms normally break down organic wastes and cycle materials  Some bacteria can be engineered to break down pollutants or to take up larger amounts of harmful materials; oil, heavy metals, and radioactive wastes.

16.7 Designer Plants  Pressured to produce more food at lower cost and with less damage to the environment, farmers are turning to genetically engineered crop plants.  This leads to a decrease in pesticide use that can harm humans, animals, and beneficial insects.

Engineered Plants  Cotton plants that display resistance to herbicide that will kill weeds.  Aspen plants that produce less lignin and more cellulose for paper making  Tobacco plants that produce human proteins  Mustard plant cells that produce biodegradable plastic

The Ti 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

16.8 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

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

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

Transgenic Animals  Transgenic Animals are used routinely for medical research.  They can be the source of medically valued proteins.

Xenotransplantation  This is the transfer of an organ from one species to another.  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

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

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

16.10 Modified Human?  The goal of human gene therapy is to transfer normal or modified genes into body cells to correct genetic defects.  There are risks associated with these procedures, as noted with “Bubble” children suffering from a severe immune deficiency.  Eugenic engineering – selecting the most desired human traits.

Using Human Genes  Even with gene in hand it is difficult to manipulate it to advantage  Viruses are usually used to insert genes into cultured human cells, but the procedure has problems  It is 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?