Genetic Frontiers Chapter 15

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

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

Genetic Engineering

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

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

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

Genetic Probe

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

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

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

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

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

Restriction Enzymes

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

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

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

Plasmid

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

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

Constructing Recombinant DNA

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

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

Identifying Bacteria With Altered Plasmids

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

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

Genetic Engineering

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

LEARNING OBJECTIVE 4 Define DNA sequencing

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

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

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

KEY TERMS GENOME All the genetic material contained in an individual

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

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

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

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

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

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

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

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)

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

GM Crops

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

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

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

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)

Animation: Restriction Enzymes CLICK TO PLAY

Animation: Formation of Recombinant DNA CLICK TO PLAY

Animation: Use of a Radioactive Probe CLICK TO PLAY

Animation: Transferring Genes Into Plants CLICK TO PLAY