Genetic Frontiers Chapter 15. LEARNING OBJECTIVE 1 Define genetic engineering Define genetic engineering Outline the primary techniques used in recombinant.

Genetic FrontiersGenetic Frontiers

Chapter 15 Chapter 15

LEARNING OBJECTIVE 1LEARNING OBJECTIVE 1

• Define Define genetic engineeringgenetic engineering

• Outline the primary techniques used in Outline the primary techniques used in recombinant DNA technologyrecombinant DNA technology, , including including genetic probesgenetic probes and and DNA DNA cloning cloning

KEY TERMSKEY TERMS

• GENETIC ENGINEERINGGENETIC ENGINEERING • Manipulation of genes, often through Manipulation of genes, often through

recombinant DNA technologyrecombinant DNA technology

• RECOMBINANT DNA TECHNOLOGYRECOMBINANT DNA TECHNOLOGY• Techniques used to make DNA molecules by Techniques used to make DNA molecules by

combining genes from different organismscombining genes from different organisms

Genetic EngineeringGenetic Engineering

Recombinant DNA Technology 1Recombinant DNA Technology 1

• DNA molecules are cleaved at specific DNA molecules are cleaved at specific base sequencesbase sequences• to break them into smaller fragments to break them into smaller fragments

• Segments of DNA from different sources Segments of DNA from different sources are joined, forming recombinant DNA are joined, forming recombinant DNA moleculemolecule• these molecules are taken into a host cell these molecules are taken into a host cell

Recombinant DNA Technology 2Recombinant DNA Technology 2

• Cells that take up the gene are identified Cells that take up the gene are identified with a with a genetic probegenetic probe

• The gene may be transcribed and The gene may be transcribed and translated within the host celltranslated within the host cell• leading to production of a protein not leading to production of a protein not

previously produced by the host organism previously produced by the host organism

KEY TERMSKEY TERMS

• GENETIC PROBEGENETIC PROBE • A single-stranded nucleic acid used to identify A single-stranded nucleic acid used to identify

a complementary sequence by base pairing a complementary sequence by base pairing with itwith it

Genetic ProbeGenetic Probe

Bacterialcolonies

Filter paper

Radioactivelylabeled nucleic acid probe is added

Filter with bacteria from colonies; cells are broken toexpose the DNA

Some radioactivenucleic acid probemolecules form base pairs with the DNA of some colonies

Exposed X-ray film; dark spots identify colonies with desired DNA

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Bacterial cells are spread on solidnutrient medium so that only one cellis found in each location. Each cellmultiplies to give rise to geneticallyidentical descendants that form acolony on the medium. A few cellsfrom each colony are transferred tospecial filters.

Radioactively labeled probe nucleicacid is added to the filter; the probenucleic acid is single stranded andcontains a sequence of nucleotidescomplementary to the gene of interest.

Some of the probe nucleic acid formsbase pairs with the DNA of some ofthe colonies.

DNA from bacterial cells that containthe DNA sequence complementary tothe radioactive probe can be detectedby X-ray film.

Fig. 15-6, p. 299

KEY TERMSKEY TERMS

• DNA CLONINGDNA CLONING • Process of selectively amplifying DNA Process of selectively amplifying DNA

sequences so their structure and function can sequences so their structure and function can be studiedbe studied

LEARNING OBJECTIVE 2LEARNING OBJECTIVE 2

• Explain the actions and importance of Explain the actions and importance of restriction enzymesrestriction enzymes and and ligaseligase

KEY TERMSKEY TERMS

• RESTRICTION ENZYMERESTRICTION ENZYME • An enzyme used in recombinant DNA An enzyme used in recombinant DNA

technology to cleave DNA at specific base technology to cleave DNA at specific base sequencessequences

• Breaks the DNA molecule into more Breaks the DNA molecule into more manageable fragments manageable fragments

LigaseLigase

• Segments of DNA from different sources Segments of DNA from different sources are joined by the enzyme are joined by the enzyme ligaseligase

Restriction EnzymesRestriction Enzymes

Site of cleavage

Site of cleavage

Sticky end

Sticky end

A A

A A

A

A

A

A

G

G

G

G

T T

T T

T T

T T

C

C

C

C

Fig. 15-2, p. 295

LEARNING OBJECTIVE 3LEARNING OBJECTIVE 3

• Identify the role of biological Identify the role of biological vectorsvectors, , such as such as plasmidsplasmids, in recombinant DNA , in recombinant DNA technology technology

• Describe a biological vector and a Describe a biological vector and a nonbiological method used to nonbiological method used to introduce genes into plant cellsintroduce genes into plant cells

KEY TERMSKEY TERMS

• VECTOR VECTOR • An agent, such as a plasmid or virus, that An agent, such as a plasmid or virus, that

transfers DNA from one organism to anothertransfers DNA from one organism to another

• PLASMIDPLASMID • A small, circular DNA molecule that carries A small, circular DNA molecule that carries

genes separate from the main DNA of a genes separate from the main DNA of a bacterial cellbacterial cell

PlasmidPlasmid

Plasmid

BacteriumMain bacteria DNA

Fig. 15-3, p. 296

A Biological VectorA Biological Vector

• The plasmid of The plasmid of Agrobacterium Agrobacterium is an is an effective vector for introducing genes into effective vector for introducing genes into many plant cells many plant cells

Constructing Recombinant DNAConstructing Recombinant DNA

A plasmid andplant DNA are

spliced togetherwith DNA ligase

DNA fromanother

organismStickyend

Stickyend

Treatedwith the samerestriction enzyme

. . . treated with arestriction enzyme

Plasmid from abacterium . . .

Fig. 15-4, p. 297

A plasmid andplant DNA are

spliced togetherwith DNA ligase

. . . treated with arestriction enzyme

Plasmid from abacterium . . .

Stickyend

Stickyend

Fig. 15-4, p. 297

DNA fromanother

organism

Treatedwith the samerestriction enzyme

Stepped Art

Identifying Bacteria With Identifying Bacteria With Altered PlasmidsAltered Plasmids

Fragment2

Fragment1

Fragment3

Fragment4

Plant DNA Cut with a restriction enzyme

Produce recombinantDNA

Gene forresistanceto antibiotic

Plate with antibiotic-containingmedium

Bacteria with plasmidlive and multiply

Bacteria withoutplasmid fail to grow

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Sites of cleavage

DNA from plant cells is cut into multiple fragments with a restriction enzyme. (Only a small part of one chromosome is shown.)Recombinant plasmids are formed by cutting plasmids with the same restriction enzyme, mixing the plasmids with the segments of plant DNA, and treating with ligase.

Because the recombinant plasmids retain a gene for resistance to an antibiotic (R), bacterial cells that contain the plasmids are resistant to that antibiotic.

The bacteria are then grown on an antibiotic-containing nutrient medium, and only those that contain the recombinant plasmid survive.

Fig. 15-5, p. 298

A Nonbiological MethodA Nonbiological Method

• A nonbiological approach to introduce A nonbiological approach to introduce DNA into plant cells is a genetic “shotgun” DNA into plant cells is a genetic “shotgun”

• Researchers coat microscopic gold or Researchers coat microscopic gold or tungsten fragments with DNA and then tungsten fragments with DNA and then shoot them into plant cellsshoot them into plant cells

Genetic EngineeringGenetic Engineering

Culturedplant cells

Geneticallyengineeredplant cells

Plasmid

Foreign geneAntibiotic-resistant gene

Pieces of plant tissue are placed in a suitable medium, and the cells grow to form a clump of undifferentiated cells.

Foreign DNA is spliced into the crown gall plasmid. The recombinant plasmid isinserted into Agrobacteriumtumefaciens, which infects plant cells in culture.

The plant cells dividein tissue culture. Each cultured plant cell contains the foreign gene.

Genetically engineered plants are produced from the cultured plantcells through the use of plant tissue culture techniques.

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Fig. 15-8, p. 301

LEARNING OBJECTIVE 4LEARNING OBJECTIVE 4

• Define Define DNA sequencingDNA sequencing

KEY TERMSKEY TERMS

• DNA SEQUENCINGDNA SEQUENCING • Procedure by which the sequence of Procedure by which the sequence of

nucleotides in DNA is determinednucleotides in DNA is determined

DNA SequencingDNA Sequencing

• Automated DNA-sequencing machines Automated DNA-sequencing machines connected to powerful computers let connected to powerful computers let scientists sequence huge amounts of DNA scientists sequence huge amounts of DNA quickly and reliablyquickly and reliably

LEARNING OBJECTIVE 5LEARNING OBJECTIVE 5

• Define Define genomegenome

• Briefly describe the emerging field of Briefly describe the emerging field of genomicsgenomics

KEY TERMSKEY TERMS

• GENOMEGENOME • All the genetic material contained in an All the genetic material contained in an

individual individual

KEY TERMSKEY TERMS

• GENOMICSGENOMICS • Field of biology that studies the genomes of Field of biology that studies the genomes of

various organisms various organisms • Tries to identify all the genes, determine their Tries to identify all the genes, determine their

RNA or protein products, and ascertain how RNA or protein products, and ascertain how the genes are regulatedthe genes are regulated

LEARNING OBJECTIVE 6LEARNING OBJECTIVE 6

• Explain how Explain how RNA interferenceRNA interference is used is used to study gene function to study gene function

KEY TERMSKEY TERMS

• RNA INTERFERENCERNA INTERFERENCE (RNAi)(RNAi) • Makes use of certain small RNA molecules Makes use of certain small RNA molecules

that interfere with the expression of genes or that interfere with the expression of genes or their RNA transcriptstheir RNA transcripts

RNA Interference (RNAi)RNA Interference (RNAi)

• After a protein-coding gene is identified, After a protein-coding gene is identified, the function of that gene is studied using the function of that gene is studied using RNAi to shut the gene off RNAi to shut the gene off

• After the gene is silenced, biologists After the gene is silenced, biologists observe changes in phenotype to observe changes in phenotype to determine function of missing proteindetermine function of missing protein

LEARNING OBJECTIVE 7LEARNING OBJECTIVE 7

• Describe at least one application of Describe at least one application of recombinant DNA technology in each recombinant DNA technology in each of the following: medicine and of the following: medicine and pharmacology, DNA fingerprinting, and pharmacology, DNA fingerprinting, and transgenic organisms, specifically transgenic organisms, specifically genetically modified cropsgenetically modified crops

Medicine and PharmacologyMedicine and Pharmacology

• Escherichia coli Escherichia coli have been genetically have been genetically engineered to produce human insulinengineered to produce human insulin• Significant medical benefits to insulin-Significant medical benefits to insulin-

dependent diabetics dependent diabetics

DNA FingerprintingDNA Fingerprinting

• Analysis of DNA from an individualAnalysis of DNA from an individual

• ApplicationsApplications• Investigating crime (forensic analysis)Investigating crime (forensic analysis)• Studying endangered species in conservation Studying endangered species in conservation

biology biology • Clarifying disputed parentage Clarifying disputed parentage

KEY TERMSKEY TERMS

• TRANSGENIC ORGANISMTRANSGENIC ORGANISM • A plant or other organism that has foreign A plant or other organism that has foreign

DNA incorporated into its genomeDNA incorporated into its genome

• GENETICALLY MODIFIED (GM) CROPGENETICALLY MODIFIED (GM) CROP • A crop plant that has had its genes A crop plant that has had its genes

intentionally manipulated (transgenic crop intentionally manipulated (transgenic crop plant)plant)

Genetically Modified (GM) CropsGenetically Modified (GM) Crops

• Agricultural geneticists developing GM Agricultural geneticists developing GM plants that are resistant to insect pests, plants that are resistant to insect pests, viral diseases, drought, heat, cold, viral diseases, drought, heat, cold, herbicides, and salty or acidic soilherbicides, and salty or acidic soil

GM CropsGM Crops

LEARNING OBJECTIVE 8LEARNING OBJECTIVE 8

• Discuss safety issues associated with Discuss safety issues associated with recombinant DNA technologyrecombinant DNA technology

• Explain how these issues are being Explain how these issues are being addressed addressed

Safety Issues 1Safety Issues 1

• Concerns: Genetically engineered Concerns: Genetically engineered organisms might be dangerous if they organisms might be dangerous if they escaped into the environment escaped into the environment • Scientists carried out risky experiments in Scientists carried out risky experiments in

facilities designed to hold pathogenic facilities designed to hold pathogenic organisms organisms

Safety Issues 2Safety Issues 2

• So far, there is no evidence that So far, there is no evidence that researchers have accidentally cloned researchers have accidentally cloned hazardous genes or released dangerous hazardous genes or released dangerous organisms into the environment organisms into the environment • Scientists have relaxed many of restrictive Scientists have relaxed many of restrictive

guidelines for using recombinant DNA guidelines for using recombinant DNA

Safety Issues 3Safety Issues 3

• Stringent restrictions still exist where Stringent restrictions still exist where questions about possible effects on the questions about possible effects on the environment are unanswered environment are unanswered • ((Example:Example: in research that proposes to in research that proposes to

introduce transgenic organisms into the wild)introduce transgenic organisms into the wild)

Animation: Restriction EnzymesAnimation: Restriction Enzymes

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Animation: Formation of Animation: Formation of Recombinant DNARecombinant DNA

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Animation: Use of a Radioactive Animation: Use of a Radioactive ProbeProbe

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Animation: Transferring Genes Into Animation: Transferring Genes Into PlantsPlants

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