1. Genetic Frontiers
Chapter 15

2. LEARNING OBJECTIVE 1 Define genetic engineering
Outline the primary techniques used in recombinant DNA technology, including genetic probes and DNA cloning

3. KEY TERMS GENETIC ENGINEERING RECOMBINANT DNA TECHNOLOGY
Manipulation of genes, often through recombinant DNA technology
RECOMBINANT DNA TECHNOLOGY
Techniques used to make DNA molecules by combining genes from different organisms

4. Genetic Engineering

5. Recombinant DNA Technology 1
DNA molecules are cleaved at specific base sequences
to break them into smaller fragments
Segments of DNA from different sources are joined, forming recombinant DNA molecule
these molecules are taken into a host cell

6. Recombinant DNA Technology 2
Cells that take up the gene are identified with a genetic probe
The gene may be transcribed and translated within the host cell
leading to production of a protein not previously produced by the host organism

7. KEY TERMS GENETIC PROBE
A single-stranded nucleic acid used to identify a complementary sequence by base pairing with it

8. Genetic Probe

9. Filter with bacteria from colonies; cells are broken to
Bacterial
colonies
Filter paper 1 1
Bacterial cells are spread on solid
nutrient medium so that only one cell
is found in each location. Each cell
multiplies to give rise to genetically
identical descendants that form a
colony on the medium. A few cells
from each colony are transferred to
special filters.
Filter with bacteria from colonies; cells are broken to
expose the DNA
Radioactively
labeled nucleic acid probe is added 2 2
Radioactively labeled probe nucleic
acid is added to the filter; the probe
nucleic acid is single stranded and
contains a sequence of nucleotides
complementary to the gene of interest. 3
Some radioactive
nucleic acid probe
molecules form base pairs with the DNA of some colonies 3
Some of the probe nucleic acid forms
base pairs with the DNA of some of
the colonies.
Figure 15.6: Genetic probe. 4
Exposed X-ray film; dark spots identify colonies with desired DNA 4
DNA from bacterial cells that contain
the DNA sequence complementary to
the radioactive probe can be detected
by X-ray film.
Fig. 15-6, p. 299

10. KEY TERMS
DNA CLONING
Process of selectively amplifying DNA sequences so their structure and function can be studied

11. LEARNING OBJECTIVE 2
Explain the actions and importance of restriction enzymes and ligase

12. KEY TERMS RESTRICTION ENZYME
An enzyme used in recombinant DNA technology to cleave DNA at specific base sequences
Breaks the DNA molecule into more manageable fragments

13. Ligase
Segments of DNA from different sources are joined by the enzyme ligase

14. Restriction Enzymes

15. Site of cleavage A A G C T T T T C G A A Site of cleavage Sticky end A
Figure 15.2: Restriction enzymes.
(a) A portion of a DNA molecule (unwound for simplification) contains the recognition site for a restriction enzyme that recognizes the DNA base sequence —AAGCTT— and its complementary base pairs. Note that —AAGCTT— and its complement, —TTCGAA—, have the same base sequence in opposite directions. (b) The cleavage of DNA by this restriction enzyme results in two DNA fragments that have short single-stranded stubs—that is, sticky ends. A A G C T T T T C G A A
Sticky end
Fig. 15-2, p. 295

16. LEARNING OBJECTIVE 3
Identify the role of biological vectors, such as plasmids, in recombinant DNA technology
Describe a biological vector and a nonbiological method used to introduce genes into plant cells

17. KEY TERMS VECTOR PLASMID
An agent, such as a plasmid or virus, that transfers DNA from one organism to another
PLASMID
A small, circular DNA molecule that carries genes separate from the main DNA of a bacterial cell

18. Plasmid

19. Main bacteria DNA Bacterium Plasmid Fig. 15-3, p. 296
Figure 15.3: Plasmid.
A plasmid is a small, circular DNA molecule that contains only a few genes. Plasmids are often used in genetic engineering to transfer desired genes into bacteria or other organisms. (a) The relative sizes of a plasmid and the main DNA of a bacterium. (b) An electron micrograph of a plasmid from the common intestinal bacterium Escherichia coli.
Fig. 15-3, p. 296

20. A Biological Vector
The plasmid of Agrobacterium is an effective vector for introducing genes into many plant cells

21. Constructing Recombinant DNA

22. DNA from another Sticky Sticky organism end end . . . treated with a
Plasmid from a
bacterium . . .
. . . treated with a
restriction enzyme
Treated
with the same
restriction enzyme
Figure 15.4: Constructing a recombinant DNA molecule.
DNA molecules from two sources are cut with the same restriction enzyme, which results in complementary sticky ends. In this example, one of the DNA molecules is a bacterial plasmid, and the other is a segment of DNA from a petunia. When mixed, their sticky ends form base pairs, and ligase splices the two together.
A plasmid and
plant DNA are
spliced together
with DNA ligase
Fig. 15-4, p. 297

23. DNA from another Sticky organism end . . . treated with a
restriction enzyme
Plasmid from a
bacterium . . .
Sticky
end
Treated
with the same
restriction enzyme
Figure 15.4: Constructing a recombinant DNA molecule.
DNA molecules from two sources are cut with the same restriction enzyme, which results in complementary sticky ends. In this example, one of the DNA molecules is a bacterial plasmid, and the other is a segment of DNA from a petunia. When mixed, their sticky ends form base pairs, and ligase splices the two together.
A plasmid and
plant DNA are
spliced together
with DNA ligase
Stepped Art
Fig. 15-4, p. 297

24. Identifying Bacteria With Altered Plasmids

25. Fragment 1 Fragment 2 Fragment 3 Fragment 4
Sites of cleavage
Fragment 1
Fragment 2
Fragment 3
Fragment 4
Plant DNA
Cut with a restriction enzyme 1 1
DNA from plant cells is cut into multiple fragments with a restriction enzyme. (Only a small part of one chromosome is shown.)
Produce recombinant
DNA 2 2 2 2 2
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.
Gene for
resistance
to antibiotic 3 3
Because the recombinant plasmids retain a gene for resistance to an antibiotic (R), bacterial cells that contain the plasmids are resistant to that antibiotic.
Figure 15.5: Identifying bacteria that have taken up genetically altered plasmids.
Plate with antibiotic-containing
medium 4
Bacteria without
plasmid fail to grow 4
The bacteria are then grown on an antibiotic-containing nutrient medium, and only those that contain the recombinant plasmid survive.
Bacteria with plasmid
live and multiply
Fig. 15-5, p. 298

26. A Nonbiological Method
A nonbiological approach to introduce DNA into plant cells is a genetic “shotgun”
Researchers coat microscopic gold or tungsten fragments with DNA and then shoot them into plant cells

27. Genetic Engineering

28. Antibiotic-resistant gene
Plasmid
Foreign gene
Antibiotic-resistant gene
Genetically
engineered
plant cells
Cultured
plant cells 1
Pieces of plant tissue are placed in a suitable medium, and the cells grow to form a clump of undifferentiated cells.
Figure 15.8: Genetic engineering. 2
Foreign DNA is spliced into the crown gall plasmid. The recombinant plasmid is
inserted into Agrobacterium
tumefaciens, which infects plant cells in culture. 3
The plant cells divide
in tissue culture. Each cultured plant cell contains the foreign gene. 4
Genetically engineered plants are produced from the cultured plant
cells through the use of plant tissue culture techniques.
Fig. 15-8, p. 301

29. LEARNING OBJECTIVE 4
Define DNA sequencing

30. KEY TERMS DNA SEQUENCING
Procedure by which the sequence of nucleotides in DNA is determined

31. DNA Sequencing
Automated DNA-sequencing machines connected to powerful computers let scientists sequence huge amounts of DNA quickly and reliably

32. LEARNING OBJECTIVE 5 Define genome
Briefly describe the emerging field of genomics

33. KEY TERMS
GENOME
All the genetic material contained in an individual

34. KEY TERMS
GENOMICS
Field of biology that studies the genomes of various organisms
Tries to identify all the genes, determine their RNA or protein products, and ascertain how the genes are regulated

35. LEARNING OBJECTIVE 6
Explain how RNA interference is used to study gene function

36. KEY TERMS RNA INTERFERENCE (RNAi)
Makes use of certain small RNA molecules that interfere with the expression of genes or their RNA transcripts

37. RNA Interference (RNAi)
After a protein-coding gene is identified, the function of that gene is studied using RNAi to shut the gene off
After the gene is silenced, biologists observe changes in phenotype to determine function of missing protein

38. LEARNING OBJECTIVE 7
Describe at least one application of recombinant DNA technology in each of the following: medicine and pharmacology, DNA fingerprinting, and transgenic organisms, specifically genetically modified crops

39. Medicine and Pharmacology
Escherichia coli have been genetically engineered to produce human insulin
Significant medical benefits to insulin-dependent diabetics

40. DNA Fingerprinting Analysis of DNA from an individual Applications
Investigating crime (forensic analysis)
Studying endangered species in conservation biology
Clarifying disputed parentage

41. KEY TERMS TRANSGENIC ORGANISM GENETICALLY MODIFIED (GM) CROP
A plant or other organism that has foreign DNA incorporated into its genome
GENETICALLY MODIFIED (GM) CROP
A crop plant that has had its genes intentionally manipulated (transgenic crop plant)

42. Genetically Modified (GM) Crops
Agricultural geneticists developing GM plants that are resistant to insect pests, viral diseases, drought, heat, cold, herbicides, and salty or acidic soil

43. GM Crops

44. LEARNING OBJECTIVE 8
Discuss safety issues associated with recombinant DNA technology
Explain how these issues are being addressed

45. Safety Issues 1
Concerns: Genetically engineered organisms might be dangerous if they escaped into the environment
Scientists carried out risky experiments in facilities designed to hold pathogenic organisms

46. Safety Issues 2
So far, there is no evidence that researchers have accidentally cloned hazardous genes or released dangerous organisms into the environment
Scientists have relaxed many of restrictive guidelines for using recombinant DNA

47. Safety Issues 3
Stringent restrictions still exist where questions about possible effects on the environment are unanswered
(Example: in research that proposes to introduce transgenic organisms into the wild)

48. Animation: Restriction Enzymes
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49. Animation: Formation of Recombinant DNA
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50. Animation: Use of a Radioactive Probe
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51. Animation: Transferring Genes Into Plants
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