Recombinant DNA and Biotechnology

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Recombinant DNA and Biotechnology
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16 Recombinant DNA and Biotechnology

• 16.1 How Are Large DNA Molecules Analyzed?
• 16.2 What Is Recombinant DNA?
• 16.3 How Are New Genes Inserted into Cells?
• 16.4 What Are the Sources of DNA Used in Cloning?
• 16.5 What Other Tools Are Used to Manipulate DNA?
• 16.6 What Is Biotechnology?

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16.1 How Are Large DNA Molecules Analyzed?

• Naturally occurring enzymes that cleave and
  repair DNA are used in the laboratory to
  manipulate and recombine DNA.

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16.1 How Are Large DNA Molecules Analyzed?

• Restriction enzymes (restriction endonucleases)
  cut double-stranded DNA into smaller pieces.
• Bacteria use these as defense against DNA from
  bacteriophage.
• DNA is cut between the 3' hydroxyl group of one
  nucleotide and the 5' phosphate group of the
  nextrestriction digestion.

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Figure 16.1 Bacteria Fight Invading Viruses with
Restriction Enzymes
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16.1 How Are Large DNA Molecules Analyzed?

• There are many restriction enzymes that cut DNA
  at specific base sequencesthe recognition
  sequence, or restriction site.

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16.1 How Are Large DNA Molecules Analyzed?

• Restriction enzymes do not cut bacterias own DNA
  because the recognition sequences are modified.
• Methylases add methyl groups after replication
  makes sequence unrecognizable by restriction
  enzyme.

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16.1 How Are Large DNA Molecules Analyzed?

• Bacterial restriction enzymes can be isolated
  from cells.
• DNA from any organism will be cut wherever the
  recognition site occurs.
• EcoRI (from E. coli) cuts DNA at this sequence

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16.1 How Are Large DNA Molecules Analyzed?

• The sequence is palindromicit reads the same in
  both directions from the 5' end.
• EcoRI occurs about once every four genes in
  prokaryotes. DNA can be chopped into small pieces
  containing a few genes.

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16.1 How Are Large DNA Molecules Analyzed?

• The EcoRI sequence does not occur anywhere in the
  genome of the phage T7. Thus it can survive in
  its host, E. coli.

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16.1 How Are Large DNA Molecules Analyzed?

• After DNA is cut, fragments of different sizes
  can be separated by gel electrophoresis.
• Mixture of fragments is place on a well in a
  porous gel. An electric field is applied across
  the gel. Negatively charged DNA fragments move
  towards positive end.
• Smaller fragments move faster than larger ones.

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Figure 16.2 Separating Fragments of DNA by Gel
Electrophoresis (Part 1)
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Figure 16.2 Separating Fragments of DNA by Gel
Electrophoresis (Part 2)
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Figure 16.2 Separating Fragments of DNA by Gel
Electrophoresis (Part 3)
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16.1 How Are Large DNA Molecules Analyzed?

• Electrophoresis provides information on
• Size of fragments. Fragments of known size
  provide comparison.
• Presence of specific sequences. These can be
  determined using probes.
• DNA is denatured while in the gel, then
  transferred to a nylon filter to make a blot.

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Figure 16.3 Analyzing DNA Fragments by Southern
Blotting
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16.1 How Are Large DNA Molecules Analyzed?

• DNA fingerprinting uses restriction analysis and
  electrophoresis to identify individuals.
• Works best with genes that are polymorphichave
  multiple alleles.

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16.1 How Are Large DNA Molecules Analyzed?

• Two types of polymorphisms
• Single nucleotide polymorphisms (SNPs) inherited
  variation involving a single base
• Short tandem repeats (STRs) moderately
  repetitive sequences side by side

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16.1 How Are Large DNA Molecules Analyzed?

• STRs are recognizable if they lie between two
  restriction sites.
• Several different STRs can be used to determine
  the unique pattern for an individual.

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Figure 16.4 DNA Fingerprinting with Short Tandem
Repeats
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16.1 How Are Large DNA Molecules Analyzed?

• DNA fingerprinting requires at least 1 µg of DNA
  (amount in about 100,000 human cells).
• This is not always available, so amplification by
  PCR is used.

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16.1 How Are Large DNA Molecules Analyzed?

• DNA fingerprinting is used in forensics.
• It is more often used to prove innocence than
  guilt.
• Only a small portion of the genome is examined
  there is the possibility that two people could
  have the same sequence.

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16.1 How Are Large DNA Molecules Analyzed?

• DNA fingerprinting has been used to analyze
  historical events.
• The skeletal remains of Russian Tsar Nicholas II
  and his family were identified from DNA in bone
  fragments.
• DNA also showed relationships with living
  descendents of the Tsar.

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Figure 16.5 DNA Fingerprinting the Russian Royal
Family
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16.1 How Are Large DNA Molecules Analyzed?

• DNA technology can be used to identify species.
• A proposal to identify all known species and look
  for unknowns has been put forth by the Consortium
  for the Barcode of Life (CBOL)
• Use a short sequence from a gene (cytochrome
  oxidase) as a barcode for each species.

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Figure 16.6 A DNA Barcode
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16.1 How Are Large DNA Molecules Analyzed?

• The barcode project could contribute to
• Evolution research
• Species diversity issues
• Identification of new species
• Identification of undesirable microbes or
  bioterrorism agents

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16.2 What Is Recombinant DNA?

• DNA fragments can be rejoined by DNA ligase.
• Any two DNA sequences can be spliced.
• First done in 1973 with two E. coli plasmids
  Recombinant DNA was born

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Figure 16.7 Making Recombinant DNA (Part 1)
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Figure 16.7 Making Recombinant DNA (Part 2)
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16.2 What Is Recombinant DNA?

• Some restriction enzymes cut both DNA strands
  exactly opposite each other.
• Others (such as EcoRI) make a staggered cut.
  Results in single-stranded tails at the ends of
  fragments.
• Tails are called sticky endscan bind by base
  pairing to other sticky ends.

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Figure 16.8 Cutting and Splicing DNA
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16.2 What Is Recombinant DNA?

• Sticky ends of fragments that were cut by the
  same restriction enzyme are all the samethus
  fragments from different species can be joined.
• When temperature is lowered, the fragments
  annealjoin by hydrogen bonding. Must be
  permanently spliced by DNA ligase.

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16.3 How Are New Genes Inserted into Cells?

• Recombinant DNA technology can be used to clone,
  or make exact copies of genes.
• The gene can be used to make a proteinbut it
  must first be inserted, or transfected, into host
  cells.
• The altered host cell is called transgenic.

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16.3 How Are New Genes Inserted into Cells?

• To determine which of the host cells contain the
  new sequence, the recombinant DNA is often tagged
  with reporter genes.
• Reporter genes have easily observed phenotypes or
  genetic markers.

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16.3 How Are New Genes Inserted into Cells?

• The first host cells used were bacteria,
  especially E. coli.
• Yeasts (Saccharomyces) are commonly used as
  eukaryotic hosts.
• Plant cells are also usedthey have totipotency,
  the ability of any differentiated cell to develop
  into a new plant.

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16.3 How Are New Genes Inserted into Cells?

• The new DNA must also replicate as the host cell
  divides. It must become a segment with an origin
  of replicationa replicon or replication unit.

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16.3 How Are New Genes Inserted into Cells?

• New DNA can become part of a replicon in two
  ways
• Inserted near an origin of replication in host
  chromosome.
• It can be part of a carrier sequence or vector
  that already has an origin of replication.

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16.3 How Are New Genes Inserted into Cells?

• A vector should have four characteristics
• Ability to replicate independently of the host
  cell
• A recognition sequence for a restriction enzyme
• A reporter gene
• Small size in comparison with hosts chromosomes

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16.3 How Are New Genes Inserted into Cells?

• Plasmids have all these characteristics.
• Plasmids are small, many have only one
  restriction site.
• Genes for antibiotic resistance can be used as
  reporter genes.
• And they have an origin of replication and can
  replicate independently.

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Figure 16.9 Vectors for Carrying DNA into Cells
(A)
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16.3 How Are New Genes Inserted into Cells?

• Plasmids can be used for genes of 10,000 bp or
  less. Most eukaryote genes are larger than this.
• Viruses can be used as vectorse.g.,
  bacteriophage. The genes that cause host cell to
  lyse can be cut out and replaced with other DNA.

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16.3 How Are New Genes Inserted into Cells?

• Bacterial plasmids dont work for yeasts because
  the origins of replication use different
  sequences.
• A yeast artificial chromosome (YAC) has been
  created contains yeast origin of replication,
  plus yeast centromere and telomere sequences.
• Also contains artificial restriction sites and
  reporter genes

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Figure 16.9 Vectors for Carrying DNA into Cells
(B)
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16.3 How Are New Genes Inserted into Cells?

• A plasmid from the soil bacterium Agrobacterium
  tumefaciens is used as a vector for plant cells.
• Plasmid Ti (tumor inducing) causes crown gall.
• Plasmid has a region called T DNA, which inserts
  copies of itself into chromosomes of infected
  plants.

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16.3 How Are New Genes Inserted into Cells?

• T DNA has several restriction sites, where new
  DNA can be inserted.
• With altered T DNA, plasmid no longer causes
  tumors, but can still insert itself into host
  chromosomes.

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Figure 16.9 Vectors for Carrying DNA into Cells
(C)
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16.3 How Are New Genes Inserted into Cells?

• Usually only a small proportion of host cells
  take up the vector, and they may not have the
  appropriate sequence.
• Host cells with the desired sequence must be
  identifiable.

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16.3 How Are New Genes Inserted into Cells?

• One method
• E. coli is host pBR322 plasmid is the vector.
• Plasmid has genes for resistance to ampicillin
  and tetracycline.
• Plasmid has only one restriction site for enzyme
  BamHI, within the gene for tetracycline
  resistance.

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16.3 How Are New Genes Inserted into Cells?

• If new DNA is inserted at that restriction site,
  it inactivates the gene for tetracycline
  resistance.
• Plasmid then has gene for ampicillin resistance,
  but not for tetracycline. This can be used to
  select for host cells with new DNA.

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Figure 16.10 Marking Recombinant DNA by
Inactivating a Gene
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16.3 How Are New Genes Inserted into Cells?

• Other reporter genes
• Artificial vectors with restriction sites within
  the lac operon. If new DNA is inserted there,
  vector no longer carries its original function
  into the host cell.
• Green fluorescent protein, which normally occurs
  in the jellyfish Aequopora victoriana.

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16.4 What Are the Sources of DNA Used in Cloning?

• DNA fragments used for cloning come from three
  sources
• Gene libraries
• Reverse transcription from mRNA
• Artificial synthesis or mutation of DNA

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16.4 What Are the Sources of DNA Used in Cloning?

• Human chromosomes contain an average of 80
  million bp each.
• The DNA is cut into fragments by restriction
  enzymes, the fragments are stored as a gene
  library.
• Each fragment is inserted into a vector, which
  goes into a host cell.

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Figure 16.11 Constructing a Gene Library
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16.4 What Are the Sources of DNA Used in Cloning?

• If phage ? is used as a vector, about 50,000
  volumes are required to store the library.
• One petri plate can hold 80,000 phage colonies,
  or plaques.
• DNA in the plaques is screened using specific
  probes.

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16.4 What Are the Sources of DNA Used in Cloning?

• Smaller DNA libraries can be made from
  complementary DNA (cDNA).
• mRNA is extracted from a tissue and the poly A
  tails allowed to hybridize with oligo dTa string
  of thymine bases.
• Oligo dT serves as a primer for reverse
  transcriptase to synthesize a complementary DNA
  strand.

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Figure 16.12 Synthesizing Complementary DNA
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16.4 What Are the Sources of DNA Used in Cloning?

• cDNA libraries are made from particular tissues
  at particular times and represent a snapshot of
  the mRNA present at that time.
• Used to compare gene expression in different
  tissues at different stages of development.
• cDNA is also used to clone eukaryotic genes.

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16.4 What Are the Sources of DNA Used in Cloning?

• DNA can be synthesized if the amino acid sequence
  of a protein is known.
• This process is now automated, and labs can make
  custom DNA sequences overnight.
• Flanking sequences for transcription initiation,
  termination, and regulation and start and stop
  codons are also added.

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16.4 What Are the Sources of DNA Used in Cloning?

• Synthetic DNA can be used to create specific
  mutations in order to study the consequences of
  the mutation.
• Called mutagenesis techniques.
• These techniques have revealed many
  cause-and-effect relationships, e.g., determining
  signal sequences.

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16.5 What Other Tools Are Used to Manipulate DNA?

• Three additional ways of manipulating DNA
• Knockout experiments
• Gene silencing
• DNA chips

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16.5 What Other Tools Are Used to Manipulate DNA?

• A knockout experiment involves homologous
  replication to replace a gene with an inactive
  gene, and determine results in a living organism.
• The normal allele of a gene is inserted into a
  plasmid restriction enzymes are used to insert a
  reporter gene in the middle of the normal gene.

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16.5 What Other Tools Are Used to Manipulate DNA?

• The gene is thus inactivated.
• The plasmid is then transfected into a stem cell
  of a mouse embryo.
• Stem cell undifferentiated cell that divides and
  differentiates to form different tissues.

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16.5 What Other Tools Are Used to Manipulate DNA?

• Much of the normal gene is still present, so
  homologous recognition takes place between the
  normal allele and the inactive allele on the
  plasmid.
• Recombination can occur, and inactive allele is
  swapped for the normal allele.
• The transfected stem cell is then inserted into
  an early mouse embryo.

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Figure 16.13 Making a Knockout Mouse (Part 1)
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Figure 16.13 Making a Knockout Mouse (Part 2)
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16.5 What Other Tools Are Used to Manipulate DNA?

• Translation of mRNA can be blocked by
  complementary micro RNAsantisense RNA.
• Antisense RNA can be synthesized, and added to
  cells to prevent translationthe effects of the
  missing protein can then be determined.

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16.5 What Other Tools Are Used to Manipulate DNA?

• Interference RNA (RNAi) is a rare natural
  mechanism that blocks translation.
• Short, double stranded RNA is unwound and binds
  to complementary mRNA by a protein complex, which
  also catalyzes the breakdown of the mRNA.
• Small interfering RNA (siRNA) can be synthesized
  in the laboratory.

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Figure 16.14 Using Antisense RNA and RNAi to
Block Translation of mRNA
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16.5 What Other Tools Are Used to Manipulate DNA?

• Antisense RNA and RNAi are also used to study
  cause-and-effect relationships.
• Example Antisense RNA is used to block
  translation of proteins essential for growth of
  cancer cellsthe cells revert to normal phenotype.

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16.5 What Other Tools Are Used to Manipulate DNA?

• DNA chip technology provides a large array of
  sequences for hybridization experiments.
• A series of DNA sequences are attached to a glass
  slide in a precise order.
• The slide has microscopic wells which each
  contain thousands of copies of sequences up to 20
  nucleotides long.

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Figure 16.15 DNA on a Chip
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16.5 What Other Tools Are Used to Manipulate DNA?

• To analyze mRNA, it is incubated with reverse
  transcriptase to make cDNA.
• The cDNA is amplified using PCR.
• Technique is called RT-PCR.
• Amplified cDNA is tagged with a fluorescent dye
  and used as a probe of the DNA on the chip.

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16.5 What Other Tools Are Used to Manipulate DNA?

• DNA chip technology has been developed to
  identify gene expression patterns in women with a
  propensity for breast cancer tumors to recura
  gene expression signature.

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16.6 What Is Biotechnology?

• Biotechnology is the use of living cells to
  produce materials useful to people.
• Examples use of yeasts to brew beer and wine,
  use of bacteria to produce cheese, yogurt, etc.
• Use of microbes to produce antibiotics such as
  penicillin, alcohol, and other products.

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16.6 What Is Biotechnology?

• Gene cloning is now used to produce proteins in
  large amounts.
• Almost any gene can be inserted into bacteria or
  yeasts, and the resulting cells induced to make
  large quantities of the product.
• Requires specialized vectors.

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16.6 What Is Biotechnology?

• Expression vectors are synthesized that include
  sequences needed for expression of the transgene
  in the host cell.

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Figure 16.16 An Expression Vector Allows a
Transgene to Be Expressed in a Host Cell
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16.6 What Is Biotechnology?

• Expression vectors can be modified by
• Inducible promoters enhancers can also be added
  so that protein synthesis takes place at high
  rates.
• Tissue-specific promoters
• Signal sequencese.g., a signal to secrete the
  product to the extracellular medium.

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Table 16.1
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16.6 What Is Biotechnology?

• Example of a medical application
• After wounds heal, blood clots are dissolved by
  plasmin. Plasmin is stored as an inactive form
  called plasminogen.
• Conversion of plasminogen is activated by tissue
  plasminogen activator (TPA).
• TPA can be used to treat strokes and heart
  attacks, but large quantities are neededcan be
  made using recombinant DNA technology.

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Figure 16.17 Tissue Plasminogen Activator From
Protein to Gene to Drug (Part 1)
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Figure 16.17 Tissue Plasminogen Activator From
Protein to Gene to Drug (Part 2)
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16.6 What Is Biotechnology?

• Pharming production of medically useful proteins
  in milk.
• Transgenes for a protein are inserted into the
  egg of a domestic animal, next to the promoter
  for lactoglobulina protein in milk. The
  transgenic animal then produces large quantities
  of the protein in its milk.

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Figure 16.18 Pharming
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16.6 What Is Biotechnology?

• Through cultivation and selective breeding,
  humans have been altering the traits of plants
  and animals for thousands of years.
• Recombinant DNA technology has several
  advantages
• Specific genes can be targeted.
• Any gene can be introduced into any other
  organism.
• New organisms are generated quickly.

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Table 16.2
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16.6 What Is Biotechnology?

• Crop plants have been modified to produce their
  own insecticides
• The bacterium Bacillus thuringiensis produces a
  protein that kills insect larvae.
• Dried preparation of B. thuringiensis are sold as
  a safe alternative to synthetic insecticides. The
  toxin is easily biodegradable.

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16.6 What Is Biotechnology?

• Genes for the toxin have been isolated, cloned,
  and modified, and inserted into plant cells using
  the Ti plasmid vector.
• Transgenic corn, cotton, soybeans, tomatoes, and
  other crops are being grown. Pesticide use is
  reduced.

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16.6 What Is Biotechnology?

• Some transgenic crops are resistant to
  herbicides.
• Glyphosate (Roundup) is widely used to kill
  weeds.
• Expression vectors have been used to make plants
  that synthesize so much of the target enzyme of
  glyphosate that they are unaffected by the
  herbicide.

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16.6 What Is Biotechnology?

• The gene has been inserted into corn, soybeans,
  and cotton.
• About half of U.S. crops of these plants contain
  this gene.

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16.6 What Is Biotechnology?

• Crops with improved nutritional characteristics
• Rice does not have ß-carotene, but does have a
  precursor molecule.
• Genes for enzymes that synthesize ß-carotene from
  the precursor are taken from daffodils and
  inserted into rice by the Ti plasmid.

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16.6 What Is Biotechnology?

• The transgenic rice is yellow, and can supply
  ß-carotene to improve the diets of many people.
• ß-carotene is converted to vitamin A in the body.

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Figure 16.19 Transgenic Rice Is Rich in ß-Carotene
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16.6 What Is Biotechnology?

• Recombinant DNA is also used to adapt a crop
  plant to an environment.
• Example plants that are salt-tolerant
• Genes from a protein that moves sodium ions into
  the central vacuole were isolated from
  Arabidopsis and inserted into tomato plants.

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Figure 16.20 Salt-Tolerant Tomato Plants
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16.6 What Is Biotechnology?

• Concerns over biotechnology
• Genetic manipulation is an unnatural interference
  in nature.
• Genetically altered foods are unsafe to eat.
• Genetically altered crop plants are dangerous to
  the environment.

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16.6 What Is Biotechnology?

• Advocates of biotechnology point out that all
  crop plants have been manipulated by humans.
• Advocates say that since only single genes for
  plant function are inserted into crop plants,
  they are still safe for human consumption.
• Genes that affect human nutrition may raise more
  concerns.

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16.6 What Is Biotechnology?

• Concern over environmental effects centers on
  escape of transgenes into wild populations
• For example, if the gene for herbicide resistance
  made its way into the weed plants.
• Beneficial insects can also be killed from eating
  plants with B. thuringiensis genes.