13 Biotechnology Chapter 13 Biotechnology Key Concepts 13

13 Biotechnology

Chapter 13 Biotechnology Key Concepts • 13. 1 Recombinant DNA Can Be Made in the Laboratory • 13. 2 DNA Can Genetically Transform Cells and Organisms • 13. 3 Genes and Gene Expression Can Be Manipulated • 13. 4 Biotechnology Has Wide Applications

Concept 13. 1 Recombinant DNA Can Be Made in the Laboratory It is possible to modify organisms with genes from other, distantly related organisms. Recombinant DNA is a DNA molecule made in the laboratory that is derived from at least two genetic sources.

Concept 13. 1 Recombinant DNA Can Be Made in the Laboratory Three key tools: • Restriction enzymes for cutting DNA into fragments • Gel electrophoresis for analysis and purification of DNA fragments • DNA ligase for joining DNA fragments together in new combinations

Concept 13. 1 Recombinant DNA Can Be Made in the Laboratory Restriction enzymes recognize a specific DNA sequence called a recognition sequence or restriction site. 5′……. GAATTC…… 3′ 3′……. CTTAAG…… 5′ Each sequence forms a palindrome: the opposite strands have the same sequence when read from the 5′ end.

Figure 13. 1 Bacteria Fight Invading Viruses by Making Restriction Enzymes

Concept 13. 1 Recombinant DNA Can Be Made in the Laboratory Some restriction enzymes cut DNA leaving a short sequence of single-stranded DNA at each end. Staggered cuts result in overhangs, or “sticky ends; ” straight cuts result in “blunt ends. ” Sticky ends can bind complementary sequences on other DNA molecules. Methylases add methyl groups to restriction sites and protect the bacterial cell from its own restriction enzymes.

Concept 13. 1 Recombinant DNA Can Be Made in the Laboratory Many restriction enzymes with unique recognition sequences have been purified. In the lab they can be used to cut DNA samples from the same source. A restriction digest combines different enzymes to cut DNA at specific places. Gel electrophoresis analysis can create a map of the intact DNA molecule from the formed fragments.

Concept 13. 1 Recombinant DNA Can Be Made in the Laboratory DNA fragments cut by enzymes can be separated by gel electrophoresis. A mixture of fragments is placed in a well in a semisolid gel, and an electric field is applied across the gel. Negatively charged DNA fragments move towards the positive end. Smaller fragments move faster than larger ones.

Concept 13. 1 Recombinant DNA Can Be Made in the Laboratory DNA fragments separate and give three types of information: • The number of fragments • The sizes of the fragments • The relative abundance of the fragments, indicated by the intensity of the band

Figure 13. 2 Separating Fragments of DNA by Gel Electrophoresis (Part 1)

Figure 13. 2 Separating Fragments of DNA by Gel Electrophoresis (Part 2)

Concept 13. 1 Recombinant DNA Can Be Made in the Laboratory After separation on a gel, a specific DNA sequence can be found with a singlestranded probe. The gel region can be cut out and the DNA fragment removed. The purified DNA can be analyzed by sequence or used to make recombinant DNA.

Concept 13. 1 Recombinant DNA Can Be Made in the Laboratory DNA ligase is an enzyme that catalyzes the joining of DNA fragments, such as Okazaki fragments during replication. With restriction enzymes to cut fragments and DNA ligase to combine them, new recombinant DNA can be made.

Figure 13. 3 Cutting, Splicing, and Joining DNA

Concept 13. 1 Recombinant DNA Can Be Made in the Laboratory Recombinant DNA was shown to be a functional carrier of genetic information. Sequences from two E. coli plasmids, each with different antibiotic resistance genes, were recombined. The resulting plasmid, when inserted into new cells, gave resistance to both of the antibiotics.

Concept 13. 2 DNA Can Genetically Transform Cells and Organisms Recombinant DNA technology can be used to clone (make identical copies) genes. Transformation: Recombinant DNA is cloned by inserting it into host cells (transfection if host cells are from an animal). The altered host cell is called transgenic.

Concept 13. 2 DNA Can Genetically Transform Cells and Organisms Usually only a few cells exposed to recombinant DNA are actually transformed. To determine which of the host cells are transgenic, the recombinant DNA includes selectable marker genes, such as genes that confer resistance to antibiotics.

Concept 13. 2 DNA Can Genetically Transform Cells and Organisms Most research has been done using model organisms: • Bacteria, especially E. coli • Yeasts (Saccharomyces), commonly used as eukaryotic hosts • Plant cells, able to make stem cells— unspecialized, totipotent cells • Cultured animal cells, used for expression of human or animal genes—whole transgenic animals can be created

Concept 13. 2 DNA Can Genetically Transform Cells and Organisms Methods for inserting the recombinant DNA into a cell: • Cells may be treated with chemicals to make plasma membranes more permeable —DNA diffuses in. • Electroporation—a short electric shock creates temporary pores in membranes, and DNA can enter.

Concept 13. 2 DNA Can Genetically Transform Cells and Organisms • Viruses and bacteria can be altered to carry recombinant DNA into cells. • Transgenic animals can be produced by injecting recombinant DNA into the nuclei of fertilized eggs. • “Gene guns” can “shoot” the host cells with particles of DNA.

Concept 13. 2 DNA Can Genetically Transform Cells and Organisms The new DNA must also replicate as the host cell divides. DNA polymerase does not bind to just any sequence. The new DNA must become part of a segment with an origin of replication—a replicon or replication unit.

Concept 13. 2 DNA Can Genetically Transform Cells and Organisms New DNA can become part of a replicon in two ways: • Inserted near an origin of replication in host chromosome • It can be part of a carrier sequence, or vector, that already has an origin of replication

Concept 13. 2 DNA Can Genetically Transform Cells and Organisms Plasmids make good vectors: • Small and easy to manipulate • Have one or more restriction enzyme recognition sequences that each occur only once • Many have genes for antibiotic resistance which can be selectable markers

Concept 13. 2 DNA Can Genetically Transform Cells and Organisms • Have a bacterial origin of replication (ori) and can replicate independently of the host chromosome Bacterial cells can contain hundreds of copies of a recombinant plasmid. The power of bacterial transformation to amplify a gene is extraordinary.

In-Text Art, Ch. 13, p. 249

Concept 13. 2 DNA Can Genetically Transform Cells and Organisms Most eukaryotic genes are too large to be inserted into a plasmid. Viruses can be used as vectors—e. g. , bacteriophage. The genes that cause host cells to lyse can be cut out and replaced with other DNA. Because viruses infect cells naturally they offer an advantage over plasmids.

Concept 13. 2 DNA Can Genetically Transform Cells and Organisms Usually only a small proportion of host cells take up the vector (1 cell in 10, 000) and they may not have the appropriate sequence. Host cells with the desired sequence must be identifiable. Selectable markers such as antibiotic resistance genes can be used.

Figure 13. 6 Green Fluorescent Protein as a Reporter

Concept 13. 3 Genes and Gene Expression Can Be Manipulated DNA fragments used for cloning come from three sources: • Gene libraries • Reverse transcription from m. RNA • Products of PCR • Artificial synthesis or mutation of DNA

Concept 13. 3 Genes and Gene Expression Can Be Manipulated A genomic library is a collection of DNA fragments that comprise the genome of an organism. The DNA is cut into fragments by restriction enzymes, and each fragment is inserted into a vector. A vector is taken up by host cells which produce a colony of recombinant cells.

Concept 13. 3 Genes and Gene Expression Can Be Manipulated Smaller DNA libraries can be made from complementary DNA (c. DNA). m. RNA is extracted from cells, then c. DNA is produced by complementary base pairing, catalyzed by reverse transcriptase. A c. DNA library is a “snapshot” of the transcription pattern of the cell. c. DNA libraries are used to compare gene expression in different tissues at different stages of development.

Figure 13. 7 Constructing Libraries

Concept 13. 3 Genes and Gene Expression Can Be Manipulated DNA can be synthesized by PCR if appropriate primers are available. The amplified DNA can then be inserted into plasmids to create recombinant DNA and cloned in host cells. Artificial synthesis of DNA is now fully automated.

Concept 13. 3 Genes and Gene Expression Can Be Manipulated RNA interference (RNAi) is a rare natural mechanism that blocks translation. RNAi occurs via the action of small interfering RNAs (si. RNAs). An s. RNA is a short, double stranded RNA that is unwound to single strands by a protein complex, which also catalyzes the breakdown of the m. RNA. Small interfering RNA (si. RNA) can be synthesized in the laboratory.

Concept 13. 3 Genes and Gene Expression Can Be Manipulated DNA microarray technology provides a large array of sequences for hybridization experiments. A series of DNA sequences are attached to a glass slide in a precise order. The slide has microscopic wells, each containing thousands of copies of sequences up to 20 nucleotides long.

Concept 13. 3 Genes and Gene Expression Can Be Manipulated DNA microarrays can be used to identify specific single nucleotide polymorphisms or other mutations. Microarrays can be used to examine gene expression patterns in different tissues in different conditions. Example: Women with a propensity for breast cancer tumors to recur have a gene expression signature.

Figure 13. 10 Using DNA Microarrays for Clinical Decision-Making

Concept 13. 4 Biotechnology Has Wide Applications Expression vectors may also have: • Inducible promoters that respond to a specific signal • Tissue-specific promoters, expressed only in certain tissues at certain times • Signal sequences—e. g. , a signal to secrete the product to the extracellular medium

Concept 13. 4 Biotechnology Has Wide Applications Many medically useful products are being made using biotechnology. The two insulin polypeptides are synthesized separately along with the βgalactosidase gene. After synthesis the polypeptides are cleaved, and the two insulin peptides combined to make a functional human insulin molecule.

Concept 13. 4 Biotechnology Has Wide Applications Before giving it to humans, scientists had to be sure of its effectiveness: • Same size as human insulin • Same amino acid sequence • Same shape • Binds to the insulin receptor on cells and stimulates glucose uptake

Concept 13. 4 Biotechnology Has Wide Applications Pharming: Production of pharmaceuticals in farm animals or plants. Example: Transgenes are inserted next to the promoter for lactoglobulin—a protein in milk. The transgenic animal then produces large quantities of the protein in its milk.

Concept 13. 4 Biotechnology Has Wide Applications Human growth hormone (for children suffering deficiencies) can now be produced by transgenic cows. Only 15 such cows are needed to supply all the children in the world suffering from this type of dwarfism.

Concept 13. 4 Biotechnology Has Wide Applications Through cultivation and selective breeding, humans have been altering the traits of plants and animals for thousands of years. Recombinant DNA technology has several advantages: • Specific genes can be targeted • Any gene can be introduced into any other organism • New organisms can be generated quickly

Table 13. 2 Potential Agricultural Applications of Biotechnology

Concept 13. 4 Biotechnology Has Wide Applications Crop plants have been modified to produce their own insecticides: • The bacterium Bacillus thuringiensis produces a protein that kills insect larvae • Dried preparations of B. thuringiensis are sold as a safe alternative to synthetic insecticides. The toxin is easily biodegradable.

Concept 13. 4 Biotechnology Has Wide Applications • Genes for the toxin have been isolated, cloned, and modified, and inserted into plant cells using the Ti plasmid vector • Transgenic corn, cotton, soybeans, tomatoes, and other crops are being grown. Pesticide use is reduced.

Concept 13. 4 Biotechnology Has Wide Applications Recombinant DNA is also used to adapt a crop plant to an environment. Example: Plants that are salt-tolerant. Genes from a protein that moves sodium ions into the central vacuole were isolated from Arabidopsis thaliana and inserted into tomato plants.

Figure 13. 16 Salt-tolerant Tomato Plants (Part 1)

Figure 13. 16 Salt-tolerant Tomato Plants (Part 2)

Concept 13. 4 Biotechnology Has Wide Applications Instead of manipulating the environment to suit the plant, biotechnology may allow us to adapt the plant to the environment. Some of the negative effects of agriculture, such as water pollution, could be reduced.

Concept 13. 4 Biotechnology Has Wide Applications Concerns over biotechnology: • Genetic manipulation is an unnatural interference in nature • Genetically altered foods are unsafe to eat • Genetically altered crop plants are dangerous to the environment

Concept 13. 4 Biotechnology Has Wide Applications Advocates of biotechnology point out that all crop plants have been manipulated by humans. Advocates say that since only single genes for plant function are inserted into crop plants, they are still safe for human consumption. Genes that affect human nutrition may raise more concerns.

Concept 13. 4 Biotechnology Has Wide Applications Concern over environmental effects centers on escape of transgenes into wild populations: • For example, if the gene for herbicide resistance made its way into the weed plants • Beneficial insects can also be killed from eating plants with B. thuringiensis genes