Chapter 16 Recombinant DNA and Genetic Engineering
I. Life and Death on the Threshold of a New Technology
A. Humans are developing technologies that will correct gene mistakes and deficiencies.
B. A four-year-old girl with a deficiency of the gene for adenosine deaminase was the first human to be allowed to participate in experimental gene therapy.
II. Introduction
A. For at least 3 billion years, mutation, crossing over, random gene mixing at fertilization, and hybridizations between species have contributed to the diversity of life on earth.
B. By domesticating animals and plants, humans have been manipulating the genetic character of many species for thousands of years.
C. Today, we can “engineer” genetic changes through recombinant DNA technology.
1. DNA from different species can be cut, spliced together, and inserted into bacteria which then multiply the DNA necessary for protein production.
2. Genetic engineering has great promise for agriculture, medicine, and industry, but it has also raised ecological, social, and ethical questions.

III. Recombinations in Nature: Some Examples
A. Transfer of Plasmid Genes
1. Plasmids are circular DNA, or RNA, molecules in bacteria that carry only a few genes and can replicate independently of the single “main” chromosome.
2. The F plasmid is responsible for bacterial conjugation—a transfer of DNA from one cell to another.
a. The F+ (donor) has genes for construction of a pilus that attaches to the F– (recipient).
b. Then the F+ plasmid DNA unwinds and is copied, and the replicate is transferred to the F– cell through the pilus.
3. Occasionally, the donated plasmid becomes integrated into the recipient’s chromosome.
B. Transfer of Viral Genes
1. Lambda bacteriophage can inject its viral DNA into a bacterium where it may become integrated into the bacterial chromosome.
2. The modified bacterial chromosome is replicated and passed on to offspring.
3. Later, the viral DNA may move out of the chromosome and initiate a cycle of bacteriophage production.

IV. Recombinant DNA Technology
A. By the early 1970s, scientists were able to use enzymes to cut DNA and “package” the fragments into plasmids for insertion into cells; they also began to identify DNA nucleotide sequences and map their positions.
B. Producing Restriction Fragments
1. Bacteria possess restriction enzymes whose usual function is to cut apart foreign DNA molecules.
2. Each enzyme cuts only at sites that possess a specific base sequence, allowing researchers to produce DNA fragments with specific genes.
a. Many times the “sticky ends” that result from the cut can be used to pair up with another DNA fragment cut by the same enzyme.
b. DNA ligase can be used to splice together cut plasmids and chromosome fragments.
3. The collection of DNA fragments that have been incorporated into plasmids is a DNA library.
C. DNA Amplification
1. By cloning: bacteria and yeasts are hosts for DNA library replications, which yield cloned DNA.
2. By reverse transcription: reverse transcriptase can be used to produce a single strand of DNA from an mRNA template; other enzymes then convert it to a double-stranded form known as cDNA (copied DNA).
3. By PCR: the polymerase chain reaction uses several steps to split DNA into two strands, portions of which are then copied and reassembled into millions of double-stranded forms that can be separated from each other by gel electrophoresis.
D. Identifying Modified Host Cells
1. A plasmid with antibiotic-resistant genes is selected as the cloning vector so that only the bacterial colonies with the desired genes will survive in the antibiotic-laced growth medium.
2. The colonies are then identified by nucleic acid hybridization techniques using a radioactive cDNA probe.
3. A replica plate of the colonies is made and the transformed colonies identified and allowed to grow.
E. Expressing the Gene of Interest
1. Even after a desired gene has been isolated and amplified, it may not be translated into functional protein by the bacteria because introns (noncoding regions) are still present.
2. Researchers minimize this problem by using cDNA, which is made from “mature” transcripts.

V. Risks and Prospects of the New Technology
A. Uses in Basic Biological Research
1. The microscope allowed us to see into the “micro” world of cells and organelles.
2. Mendel speculated about hereditary factors; we now can use DNA probes to identify gene sequences.
3. Darwin puzzled over life’s diversity; we now can compare species at the DNA level.
4. Researchers have embarked on the human genome project to map all of the human chromosomes by sequencing the approximately 3 billion nucleotides.
B. Applications of RFLPs
1. When DNA fragments are cut by restriction enzymes and separated on gel electrophoresis, distinct banding patterns reveal the slight variations of DNA that make each organism unique.
2. Such differences in banding patterns are called “restriction fragment length polymorphisms” (RFLPs).
a. RFLPs have increased the sites available for mapping the human genome.
b. RFLP analysis can identify a mutant allele in a genetic disorder.
c. RFLP analysis reveals a unique genetic fingerprint useful in solving cases of disputed parenthood, rape, and murder.
C. Genetic Modification of Organisms: Some Examples
1. Genetic Engineering of Bacteria
a. The bacteria used are harmless and have been modified to not survive outside the laboratory.
b. Even after a harmful gene (called “ice-forming”) was removed from a bacterium that lives on the leaves of strawberries, the public objected to experiments; years of litigation were needed before the experiments proceeded.
2. Genetic Modification of Plants
a. Whole plants can be grown from cultured cells.
1) Mutations result in new varieties.
2) Disease resistance can be identified in a plant and passed on to others by hybridization.
b. An early experiment showed that a plasmid from a bacterium that normally causes tumors in plants could be modified by replacing the tumor genes with desirable genes.
c. Genetically modified crop plants could increase production by naturally resisting insect pests.
3. Genetic Modification of Animals
a. In 1982, the rat gene for somatotropin production was introduced into mouse eggs; the mice subsequently expressed the rat gene by growing larger than normal.
b. In large mammals, a variety of undesirable disorders—such as arthritis—have developed after similar genetic engineering experiments.
c. Gene incorporation could activate oncogenes, cause mutations, or alter gene expression.