13 Biotechnology.

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 Come from Various Sources and Can Be Manipulated 13.4 Biotechnology Has Wide Applications

Chapter 13 Opening Question
How is biotechnology used to alleviate environmental problems?

Concept 13.1 Recombinant DNA Can Be Made in the Laboratory
Biotechnology began with the use of organisms to produce products such as alcohol and acetone. Now it is possible to modify organisms with genes from other, distantly related organisms. Recombinant DNA: a DNA molecule made in the laboratory that is derived from two or more sources

Key tools for manipulating DNA: Restriction enzymes for cutting DNA into fragments Gel electrophoresis for analysis and purification of DNA fragments DNA ligase for joining DNA fragments in new combinations

Bacteria fight bacteriophage infections with restriction enzymes, which cut DNA molecules into noninfectious fragments.

1. Restrictive enzyme cleaves phage
Figure Bacteria Fight Invading Viruses by Making Restriction Enzymes 1. Restrictive enzyme cleaves phage 2. Other enzymes degrade the DNA into Smaller DNA Figure Bacteria Fight Invading Viruses by Making Restriction Enzymes 3. Methyl groups at the restriction sites block The restriction enzyme and protect the bacteria’

Restriction enzymes cut DNA at specific sequences called restriction sites.

Restriction enzymes have two identical active sites on two subunits, which cut both DNA strands simultaneously: EcoR1

Some restriction enzymes make staggered cuts, leaving a short piece of single-stranded DNA at each end, called “sticky ends.” Sticky ends can bind complementary sequences on other DNA molecules. Other restriction enzymes make straight cuts, resulting in “blunt ends.”

To prevent the bacterial DNA from being cut into fragments, methyl groups can be added to restriction sites by methyltransferases. Restriction enzymes do not recognize the methylated sites, but unmethylated viral DNA is recognized and cleaved.

Hundreds of restriction enzymes have been purified and are used to cut DNA in the laboratory. The DNA fragments can be separated by size using gel electrophoresis. Individual fragments can be identified and purified for further analysis or experimentation.

A mixture of DNA fragments is placed in a well in a semisolid gel, and an electric field is applied across the gel, through a buffer. The negatively charged DNA fragments move towards the positive end. Smaller fragments move faster and farther than larger ones. The position of the fragments is detected with a dye.

Figure 13.2 Separating Fragments of DNA by Gel Electrophoresis
Figure Separating Fragments of DNA by Gel Electrophoresis A mixture of DNA fragments is placed in a gel, and an electric field is applied across the gel. The negatively charged DNA moves toward the positive end of the field, with smaller molecules moving faster (and farther) than larger ones. After minutes to hours for separation, the electric power is shut off and the separated fragments can be analyzed.

Electrophoresis provides three types of information about the DNA fragments: Number—depends on number of times the restriction site occurs Sizes—fragments of known sizes are included for reference By comparing fragment sizes resulting from two or more restriction enzymes, locations of the restriction sites can be mapped.

Relative abundance—indicated by intensity of bands in the gel After separation, gel regions can be cut out and the DNA purified and sequenced or used to make recombinant DNA.

DNA ligase catalyzes the joining of DNA fragments, such as Okazaki fragments, during replication. With restriction enzymes to cut fragments and DNA ligase to combine them, recombinant DNA can be made.

Figure 13.3 Cutting and Joining DNA
Figure Cutting and Joining DNA Many restriction enzymes (EcoRI is shown here) make staggered cuts in DNA. EcoRI can be used to cut two different DNA molecules (blue and orange). The exposed bases can hydrogen-bond with complementary exposed bases on other DNA fragments, forming recombinant DNA. DNA ligase stabilizes the recombinant molecule by forming covalent phosphodiester bonds in the DNA backbone.

In the 1970s, 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 antibiotics.

Figure 13.4 Recombinant DNA (Part 1)
Figure Recombinant DNA With the discovery of restriction enzymes and DNA ligase, it became possible to combine DNA fragments from different sources in the laboratory. But would such “recombinant DNA” be functional when inserted into a living cell? The results of this experiment completely changed the scope of genetic research, increasing our knowledge of gene structure and function, and ushering in the new field of biotechnology.a [a S. N. Cohen et al Proceedings of the National Academy of Sciences USA 70: 3240–3244.]

Concept 13.2 DNA Can Genetically Transform Cells and Organisms
Recombinant DNA technology can be used to clone genes (make identical copies). In transformation, genes are inserted into host bacterial or other cells. When the cell reproduces, it produces millions of cells, all with the cloned gene. Called transfection if the host cells are from animals The altered host cell is called transgenic.

Usually only a few cells exposed to the recombinant DNA are actually transformed. To determine which cells are transgenic, the recombinant DNA includes selectable marker genes, such as genes for antibiotic resistance.

Research using model organisms: Bacteria, especially E. coli; plasmids are easily manipulated to carry recombinant DNA into cells Yeasts (Saccharomyces), commonly used as eukaryotic hosts Plant cells can be induced to dedifferentiate into unspecialized stem cells that can be transformed and then grown into plants with the recombinant DNA in all their cells.

Cultured animal cells are used to study expression of human or animal genes Whole transgenic animals can also be created.

Methods for inserting recombinant DNA into a cell: Cells are treated with chemicals to make plasma membranes more permeable so that DNA can diffuse in. Electroporation—a short electric shock creates temporary pores in membranes, and DNA can enter.

Viruses can be altered to carry recombinant DNA into cells. For plants, a specific bacterium inserts DNA into some cells. Transgenic animals can be produced by injecting recombinant DNA into the nuclei of fertilized eggs. “Gene guns” can “shoot” the host cells with tiny particles of DNA.

The new DNA must also replicate as the host cell divides. The new DNA must become part of a segment with an origin of replication—a replicon or replication unit.

New DNA can become part of a replicon in two ways: It can be inserted near an origin of replication in host chromosome. It can enter the host cell as part of a carrier sequence, or vector, that already has an origin of replication.

Plasmids make good vectors: Small and easy to manipulate Have one or more restriction sites that each occur only once Many have genes for antibiotic resistance that can be selectable markers Can replicate independently of the host chromosome

The power of bacterial transformation to amplify a gene is extraordinary. Bacterial cells can contain hundreds of copies of a recombinant plasmid. Plasmids have been made to include 20 or more unique restriction sites, origins of replication for a variety of host cells, and various reporter genes.

The plamid pBR322 is used to transform E. coli:

An important vector for plants is the Ti (tumor- inducing) plasmid from the soil bacterium Agrobacterium tumefaciens, which causes tumors called crown gall. The plasmid has a region called T DNA, which is inserted into a chromosome of an infected plant cell.

The Ti plasmid carries the genes needed for transfer and incorporation of the T DNA:

T DNA genes are expressed by the host cell that cause growth of the tumor and production of sugars for the bacterium. These genes can be removed and replaced with recombinant DNA. Altered Ti plasmids are used to transform Agrobacterium cells, then the bacteria infect plant cells.

Most eukaryotic genes are too large to be inserted into a plasmid. Viruses such as bacteriophage can be used as vectors. The viral 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.

Usually only a few 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—only cells carrying the resistance gene can grow in the presence of that antibiotic.

Selectable markers are a type of reporter gene—a gene whose expression is easily observed. [Vibrio harvei] Two other common reporter genes: β-galactosidase (lacZ) gene codes for an enzyme that converts a white substrate into bright blue. Cloning plasmids may contain antibiotic resistance genes and the lacZ gene.

Figure 13.5 Selection for Recombinant DNA
Figure Selection for Recombinant DNA Selectable marker (reporter) genes are used by scientists to select for bacteria that have taken up a plasmid. In a typical experiment, most of the bacteria will not take up any DNA. Of those that do, only a small fraction will take up recombinant DNA.

Green fluorescent protein (GFP) from a jellyfish emits green light when exposed to UV light. The gene for this protein is widely used as a reporter gene.

Figure 13.6 Green Fluorescent Protein as a Reporter
Figure Green Fluorescent Protein as a Reporter The presence of a plasmid with the gene for green fluorescent protein is readily apparent in transgenic cells because they glow under ultraviolet light.

Concept 13.3 Genes Come from Various Sources and Can Be Manipulated
DNA fragments used for cloning come from a variety of sources. The first step often involves creating a genomic library—a collection of DNA fragments that comprise the genome of an organism. DNA fragments are inserted into host cells; transformed cells produce colonies on selective media, which can be probed using complementary DNA probes.

Smaller DNA libraries can be made from complementary DNA (cDNA). mRNA is extracted from cells, then cDNA is produced by complementary base pairing, catalyzed by reverse transcriptase. A cDNA library is a “snapshot” of the transcription pattern of the cell. cDNA libraries are used to compare gene expression in different tissues at different stages of development.

Figure 13.7 Constructing Libraries
Figure Constructing Libraries Intact genomic DNA is too large to be introduced into host cells. (A) A genomic library can be made by breaking the DNA into small fragments, incorporating the fragments into a vector, and then transforming host cells with the recombinant vectors. Each colony of cells contains many copies of a small part of the genome. (B) Similarly, there are many mRNAs in a cell. These can be copied into cDNAs and a library made from them. The DNA in these colonies can then be isolated for analysis.

RT-PCR uses reverse transcriptase and PCR to create and amplify a specific cDNA sequence without making a library. mRNA is isolated and cDNA is made, then a specific sequence is amplified by PCR.

Synthetic DNA: Artificial synthesis of DNA is now fully automated. Synthetic oligonucleotides used as primers in PCR can be customized. Longer synthetic sequences can be pieced together to construct completely artificial genes.

Mutations in nature have been used to demonstrate cause-and-effect relationships. Synthetic DNA can be manipulated to create specific mutations in order to study consequences of the mutation. Example: The auxin response element in promoters of plant genes that are switched on in the presence of the hormone auxin

An artificial promoter containing many copies of the element was made and ligated to a reporter gene. The recombinant DNA was used to transform Arabidopsis plants. When the plants were treated with auxin, the reporter gene was switched on at very high levels.

Another way to determine a gene’s function is to inactivate it (“knockout” experiments): In animals, these experiments often involve homologous recombination. The normal allele is inserted into a plasmid and restriction enzymes are used to insert a reporter gene into the normal gene. The extra DNA prevents functional mRNA from being made.

The recombinant plasmid is used to transfect mouse embryonic stem cells. Stem cells—unspecialized cells that divide and differentiate into specialized cells The gene sequence in the plasmid lines up with the homologous sequence on the mouse chromosome, and may be swapped by recombination.

The transfected stem cell is then transplanted into an early mouse embryo. The resulting mouse is examined for consequences of carrying an inactivated gene. Knockout mouse models have been developed for many diseases.

Figure 13.8 Making a Knockout Mouse
Figure Making a Knockout Mouse Animals carrying mutations are rare. Homologous recombination is used to replace a normal mouse gene with an inactivated copy of that gene, thus “knocking out” the gene. Discovering what happens to a mouse with an inactive gene tells us much about the normal role of that gene.

Another way to study gene function is to block translation. RNA interference (RNAi): Translation of mRNA can be blocked by microRNAs and small interfering RNAs (siRNAs) MicroRNAs and siRNAs are antisense RNA. Antisense RNA can be synthesized and added to cells to prevent translation—the effects of the missing protein can then be determined.

Figure 13.9 Using siRNA to Block the Translation of mRNA
Figure Using siRNA to Block the Translation of mRNA (A) Normally an mRNA is translated to produce a protein. (B) Translation of a target mRNA can be prevented with a small interfering RNA (siRNA) that is complementary to part of the target mRNA.

DNA microarrays (“gene chips”) have a series of DNA sequences attached to a solid surface in an array of microscopic spots, each containing thousands of copies of a particular sequence. Each oligonucleotide can hybridize with only one DNA or RNA sequence, and thus is a unique identifier of a gene.

Microarrays can be used to examine patterns of gene expression in different tissues under different conditions and to identify individual organisms with particular mutations. Example: Women with a propensity for recurring breast cancer tumors have a gene expression signature that can be identified.

Figure 13.10 Using DNA Microarrays for Clinical Decision-Making
Figure Using DNA Microarrays for Clinical Decision-Making The pattern of expression of 70 genes in tumor tissues (the pattern of colored spots) indicates whether breast cancer is likely to recur. Actual arrays have more dots than shown here.

Concept 13.4 Biotechnology Has Wide Applications
Bacteria and yeast cells can be transformed with almost any gene and induced to express that gene at high levels. Expression vectors are designed with extra sequences required for the transgene to be expressed in the host cell (e.g., promoters and poly A tails).

Figure 13.11 A Transgenic Cell Can Produce Large Amounts of the Transgene’s Protein Product
Figure A Transgenic Cell Can Produce Large Amounts of the Transgene’s Protein Product To be expressed in E. coli, a gene derived from a eukaryote requires bacterial sequences for transcription initiation (promoter), transcription termination, and ribosome binding. Expression vectors contain these additional sequences, enabling the eukaryotic protein to be synthesized in the prokaryotic cell.

Expression vectors may include: Inducible promoters that respond to specific signals such as hormones Tissue-specific promoters, expressed only in certain tissues at certain times Signal sequences to direct the gene product to the appropriate place (e.g., secreted to the environment)

PCR https://www. youtube. com/watch. v=IaKXv1JqB tY https://www
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Many medically useful products are being made using biotechnology. Insulin was the first to be made by recombinant DNA technology in E. coli.

Table 13.1

The two insulin polypeptides were synthesized separately using a vector with the lacZ (β- galactosidase) gene. The insulin genes were transcribed and translated along with the lacZ genes. After synthesis, the polypeptides were cleaved and the two insulin peptides were combined to make a functional human insulin molecule.

Figure 13.12 Human Insulin: From Gene to Drug
Figure Human Insulin: From Gene to Drug Human insulin chains are made by recombinant DNA technology and then combined to produce the widely used drug.

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

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.

Figure Pharming Figure Pharming An expression vector carrying a desired gene can be put into an animal egg, which is implanted into a surrogate mother. The transgenic offspring produce the new protein in their milk. The milk is easily harvested and the protein isolated, purified, and made clinically available for patients.

Human growth hormone (hGH) can now be produced by transgenic cows. With only 15 such cows, we could supply hGH for all the children in the world that suffer from pituitary dwarfism. An enzyme to treat Gaucher’s disease (inherited disorder that affects lipid breakdown in lysosomes) has been developed using transgenic carrot cells.

Through selective breeding, humans have been altering the traits of plants and animals for thousands of years. Limitations include: Many desirable traits are controlled by multiple genes Combinations of desirable genes can be separated during sexual reproduction Time required for organisms to reach maturity and reproduce

Recombinant DNA technology has several advantages: Specific genes can be targeted Any gene from any organism can be introduced into any other organism New organisms can be generated quickly Recombinant DNA technology has many applications in agriculture.

Figure 13.14 Genetic Modification of Plants versus Conventional Plant Breeding
Figure Genetic Modification of Plants versus Conventional Plant Breeding Plant biotechnology offers many potential advantages over conventional breeding. In the hypo-thetical example here, the objective is to transfer gene(s) for short, strong stems into a wheat plant that has high grain production but a tall, weak stem.

Table 13.2

Crop plants that make their own insecticides: The bacterium Bacillus thuringiensis produces a protein (Bt) that kills insect larvae.

Bt is highly toxic but breaks down rapidly in the environment. (Bacillus thuringiensis: gram (+) endotoxin in soil: commonly used in pesticides). Genes for the toxin have been isolated, cloned, modified, and inserted into plant cells using the Ti plasmid vector. Transgenic corn, cotton, soybeans, tomatoes, and other crops are being grown, and pesticide use is reduced.

Crops with improved nutritional characteristics: Golden rice contains genes from daffodils or corn and a bacterium, for enzymes involved in β-carotene production. β-carotene is converted to vitamin A in humans and is critical for good health. Many people suffer from vitamin A deficiencies because rice is a major part of their diet and the wild-type rice does not have β-carotene.

Figure 13.15 Transgenic Rice Rich in β-Carotene
Figure Transgenic Rice Rich in -Carotene Right and middle: The grains from these transgenic rice strains are colored because they make the pigment -carotene, which is converted to vitamin A in the human body. Left: Wild-type rice grains do not contain -carotene.

Crops that are tolerant of environmental conditions: Example: Plants that are salt-tolerant Genes from a protein that moves Na+ from the cytoplasm to the vacuole were isolated from Arabidopsis thaliana and inserted into tomato plants, wheat, and rapeseed. This may allow cultivation of currently unproductive soils.

Figure 13.16 Salt-tolerant Tomato Plants
Figure Salt-tolerant Tomato Plants Transgenic plants containing a gene for salt tolerance thrive in salty water (A), whereas plants without the transgene die (B). This technology may allow crops to be grown on salty soils.

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.

Concerns about genetically modified crops center on these claims: 1. Genetic manipulation is an unnatural interference in nature. 2. Genetically altered foods are unsafe to eat. 3. Genetically altered crop plants are dangerous to the environment.

1. Advocates of biotechnology point out that all crop plants have been manipulated by humans. 2. Since only single genes for plant function are inserted into crop plants, advocates contend they are safe for human consumption. Genes that affect human nutrition may raise more concerns.

3. Concern over environmental effects centers on escape of transgenes into wild populations. Example: If a gene for herbicide resistance were transferred to a closely related weed

Widespread use of glyphosate on glyphosate- resistant crops has resulted in selection of rare mutations in weeds that make them resistant to glyphosate. More than 10 resistant weed species have appeared in the United States.

Answer to Opening Question
Bioremediation: using organisms to remove contaminants from the environment Composting and wastewater treatment use bacteria to break down large molecules, human wastes, paper, and household chemicals.

Oil spills can be remediated by encouraging growth of microorganisms that digest components of crude oil. Nitrogen fertilizer was used to encourage these microbes in the Exxon Valdez oil spill and in Kuwait after the Gulf War.

Figure 13.17 The Spoils of War
Figure The Spoils of War Massive oil spills occurred in Kuwait during the 1990–1991 Gulf War.

Genes that encode the oil-degrading enzymes from such bacteria have been cloned and used to transform bacterial species in oil-spill regions. But they have not been released due to concerns regarding environmental impacts.

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