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Genetic Engineering: Recombinant DNA Technology The discovery of enzymes that cut DNA at specific locations, along with enzymes that piece DNA segments back together, gave biologists the ability to move genes from one location to another. Biologists can determine a gene’s base sequence once they have obtained many copies of the gene—by inserting it into loops of DNA called plasmids in bacterial cells and then allowing the cells to grow or by performing a polymerase chain reaction. • Pituitary dwarfism results from the abnormal production of growth hormone, encoded by the GH1 gene. •Humans affected by pituitary dwarfism grow slowly, reaching a maximum adult height of about 4 feet. •Early trials showed that people with pituitary dwarfism could be treated successfully with growth hormone therapy, but only if the protein came from humans. Growth hormones isolated from pigs, cows, or other animals were ineffective. •Growth hormone purified from the pituitary glands of human cadavers is expensive and has been banned from human treatment due to contamination with prions—protein particles that have been implicated as the cause of various neurodegenerative disorders. • The recombinant DNA strategy for producing human growth hormone involved cloning the human gene, introducing the gene into bacteria (or yeast), and having these microbes synthesize the hormone. •Reverse transcriptase was used to make complementary DNA (cDNA) from mRNA isolated from pituitary cells How Are Plasmids Used in Cloning? • Plasmids are small, circular DNA molecules that replicate independently of the chromosome and can be used to carry recombinant genes in bacteria. Using Restriction Endonucleases to Cut DNA • Restriction endonucleases are enzymes that cut DNA at specific base sequences called recognition sites. Most recognition sites are palindromic sequences (Figure 19.3). • Plasmids can be introduced into bacteria by transformation—the process of taking up DNA from the environment and incorporating it into the genome. • Cells transformed with a plasmid carrying an antibiotic resistance gene will be able to grow in the presence of the antibiotic. • The resulting transformed cells make up a cDNA library—a collection of bacterial cells, each containing a vector with one cDNA (Figure 19.1, step 6). Using Nucleic Acid Hybridization to Find a Target Gene • A probe is a single-stranded fragment of a labeled, known gene that binds to a complementary sequence in the sample being analyzed. • A DNA probe was constructed based on the approximate growth hormone amino acid sequence. The probe was labeled and hybridized to screen for bacterial colonies containing a plasmid with the growth hormone gene (Figure 19.4). • The U.S Food and Drug Administration has approved use of the hormone only for children projected to reach adult heights of less than 5'3" for males and less than 4'11" for females. •Growth hormone has also become a popular performanceenhancing drug for athletes. Should athletes be able to enhance their physical skills by taking hormones or other types of drugs? Is the drug safe at the dosages being used by athletes? These kinds of questions form the basis for ethical concerns about genetic engineering. Di-deoxy sequencing of DNA – Sanger Technique: How Was the Huntington’s Disease Gene Found? • Huntington's disease is a rare but devastating genetic illness. •An analysis of pedigrees from families affected by the disease suggested that the trait results from a single, autosomal dominant allele. Researchers set out to identify the gene or genes involved and to document that one or more genes are altered in affected individuals. •A genetic map (or linkage map) was used to localize the Huntington's gene relative to other genetic markers (a gene that has been mapped previously). • One gene within the isolated chromosomal region that was abnormal in people with Huntington's disease had an unusual number of CAG codons at the 5' end of the coding region. Healthy individuals have about 11–25 of these repeats, whereas affected individuals have 40 or more. Genetic Testing • A genetic test for Huntington's disease uses PCR to determine the number of CAG repeats. Ethical Concerns over Genetic Testing • Genetic testing raises many ethical issues, including whether a pregnancy should be terminated if a debilitating disease is found in the fetus and whether health insurance companies can deny coverage for individuals with a genetic disease. Can Gene Therapy Cure Inherited Diseases in Humans? Research on Severe Immune Disorders Using Gene Therapy to Treat XLinked Immune Deficiency • Gene therapy has been used to treat a type of severe combined immunodeficiency (SCID), a fatal genetic disease whose sufferers have a profoundly weakened immune system. •The gene responsible for SCID-X1 encodes gc polypeptide, a key component of several plasma membrane proteins. When these proteins do not function correctly, T cells do not mature properly and infants are helpless to ward off bacterial and viral infections. •The gene responsible for SCID-X1 encodes gc polypeptide, a key component of several plasma membrane proteins. When these proteins do not function correctly, T cells do not mature properly and infants are helpless to ward off bacterial and viral infections. •Within four months after treatment, nine of the ten boys had normal levels of functioning T cells; but 30 months later, two had developed a type of cancer characterized by unchecked growth of T cells. 19.5 Biotechnology in Agriculture: The Development of Golden Rice • Most strategies for genetic engineering in agriculture focus on reducing herbivore damage, making the crop more resistant to herbicides, and improving the quality of the food product. Rice as a Target Crop • Although half the world's population depends on rice as a staple food, it contains no vitamin A. Lack of vitamin A in the diet leads to blindness, diarrhea, respiratory infections, and childhood diseases such as measles. Synthesizing b-Carotene in Rice • b-carotene is a precursor of vitamin A. Scientists set out to develop rice enriched in b-carotene. • The synthetic pathway for bcarotene has three enzymes (Figure 19.15). The Agrobacterium Transformation System • Agrobacterium tumefaciens is often used for genetic transformation of plants through transfer of its Ti (tumor-inducing) plasmid (Figure 19.16). Golden Rice • Agrobacterium was used to transfer the b-carotene synthetic genes into cells from a rice plant, eventually producing a transgenic plant now called golden rice because it is yellow from the high concentration of bcarotene (Figure 19.17). • Enthusiasm about the potential benefits of golden rice and other genetically modified foods is tempered by concern about the risks of releasing large numbers and types of recombinant crop plants into the environment.