Biotechnology and Recombinant DNA

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1 Chapter 9 Biotechnology and Recombinant DN Introduction to Biotechnology Recall from Chapter 8 that recombination, the reshuffling of genes between two DN molecules, forming recombinant DN, occurs naturally in microorganisms. It is also possible to manipulate DN artificially to combine genes from two different sources, even from vertebrates to bacteria. rtificial gene manipulation is known as genetic engineering, and the term biotechnology usually means the industrial use of genetically engineered microorganisms. Overview of Recombinant DN Procedures he gene of interest is first inserted into vector DN (or cloning vector). his DN molecule used as a carrier must be self-replicating, such as a plasmid or viral genome. his recombinant vector DN must enter a cell where it can multiply, forming a clone of genetically identical cells. he gene itself may be the desired product, or it may be a protein product expressed by the gene. ools of Biotechnology Restriction Enzymes special class of DN-cutting enzymes, restriction enzymes, are the technical basis of genetic engineering. Restriction enzymes have the natural function of protecting the bacteria from attack by phages by hydrolyzing their DN. he DN of the bacteria is protected from the enzyme by addition of a methyl group to some cytosines, methylates. he enzyme recognizes and cuts only one particular sequence of nucleotide bases. Many restriction enzymes make staggered cuts in the two DN strands cuts that are not directly opposite each other (Figure 9.2 in the text). he stretches of single-stranded DN at the ends of the DN fragments are called sticky ends. hey stick to complementary stretches of single-stranded DN by base pairing. If two fragments of DN from different sources have been produced by the same restriction enzymes, the sets of sticky ends can be spliced (recombined) easily. DN ligase then links the DN pieces. Vectors Figure 9.1 diagrams the cloning of a DN fragment by using a plasmid for a vector. he host cell can be induced to take up the plasmid vector by chemical treatment. Plasmids that can exist in several species are shuttle vectors. Polymerase Chain Reaction recent development in DN analysis is the polymerase chain reaction (PCR) (Figure 9.4 in the text). Starting with just one gene-sized piece of DN, PCR can make billions of copies in only a few hours. he target piece of DN is heated to separate the DN strands, which serve as templates for DN 103

2 104 Chapter 9 Bacterium 1 Vector such as a plasmid is isolated 2 DN is cleaved by an enzyme into fragments DN containing gene of interest Bacterial chromosome Plasmid 3 ene is inserted into plasmid Recombinant DN (plasmid) ene of interest 4 Plasmid is taken up by a cell such as a bacterium Recombinant bacterium 5 Cells with gene of interest are cloned oal may be to make copies of gene OR oal may be to make protein product of gene Plasmid RN 6B Cells make a protein product Protein product 6 Copies of gene are harvested 7 Copies of protein are harvested ene for pest resistance is inserted into plants ene alters bacteria for cleaning up toxic waste myase, cellulase, and other enzymes prepare fabrics for clothing manufacture Human growth hormone treats stunted growth FIURE 9.1 ypical genetic engineering procedures with examples of applications.

3 Biotechnology and Recombinant DN 105 synthesis. DN polymerase enzyme, which forms DN by linking the nucleotides, is supplied with DN s four nucleotides and short pieces of primer nucleic acid. Each newly synthesized piece of DN serves in turn as a template for more new DN. echniques of enetic Engineering Inserting Foreign DN into Cells Plasmid vectors can be inserted into many cells by chemical treatments that make transformation possible. Such cells are then called competent, or able to take up external DN. he walls of cells can be enzymatically removed (forming a protoplast) and exchange DN by protoplast fusion. Polyethylene glycol may be used to improve efficiency. ransfer of DN in this manner can also be enhanced by using an electric field to form minute pores in the protoplast membranes electroporation. Foreign DN can be introduced into plant cells by coating microscopic particles of tungsten with DN and firing it through the plant cell wall using a gene gun. DN can be introduced into a cell through a minute glass micropipette by microinjection. Obtaining DN enes from a particular organism are isolated by cutting up the entire genome with restriction enzymes, splicing as many as possible into vectors, and then introducing the genes into bacterial cells. collection of bacterial clones containing different DN fragments is called a gene library. Cloning genes from a eukaryotic organism generally requires removal of introns, stretches of DN that do not code for protein. his can be done by splicing; introns are removed when an RN transcript of a gene is converted to mrn. What remains are exons, stretches of DN that code for protein. n artificial gene that contains only exons can be made with the enzyme reverse transcriptase. his will synthesize complementary DN (cdn) from an mrn template. his is the most common method of obtaining eukaryotic genes. enes of synthetic DN can also be made with the help of DN-synthesizing machines. he smaller chains of about 40 nucleotides synthesized in this way can be linked together to make an entire gene. Selecting a Clone One of the most common methods of selecting a desired gene is blue/white screening from the color of the bacterial colonies formed at the end of the process. Briefly, only white colonies are of interest, because they contain foreign DN incorporated into a plasmid. Further work is then needed to identify this foreign DN. For example, colony hybridization is a common method. Short segments of single-stranded DN, consisting of a sequence of nucleotides unique to the gene sought, can be synthesized. hese molecules, called DN probes, are radioactively labeled so they can be located later. he clone-carrying bacteria from the library are grown into colonies on a plate of nutrient medium treated to break open the cells and separate the DN into single strands. he labeled probe, added to the plate, will react with DN in any bacterial colony that base-pairs with the probe (colony hybridization). he radioactive tag allows the colonies containing the desired gene to be identified. similar probe based on labeled antibodies against protein products of the cells is also used. Making a ene Product Escherichia coli is often used as the genetically engineered organism to produce a desired gene product. It has disadvantages; it produces endotoxins that cause fever and shock in animals. lso, it does not secrete the product; the cells must be harvested, ruptured, and the product recovered.

4 106 Chapter 9 Organisms such as the gram-positive bacterium Bacillus subtilis and yeasts are more likely to secrete their products. nimal viruses, such as the vaccinia virus, have been genetically engineered to produce vaccines. Mammalian cells and plant cells are often engineered to produce useful products. pplications of enetic Engineering Medically important products made by genetic engineering are insulin, somatotropin growth hormone, tissue-plasminogen activator (t-p), and subunit vaccines. herapeutic, Scientific, and Medical pplications Recombinant DN technology is also the basis of DN analysis of genetic abnormalities responsible for various diseases, and it contributes to advances in gene therapy, in which abnormal genes might be replaced with normal genes in a living individual. o isolate a fragment of DN containing a gene, the method of Southern blotting is commonly used. DN fragments from each candidate clone are treated with the same restriction enzyme, and the resulting fragments separated by gel electrophoresis. he fragments are called restriction fragment length polymorphisms (RFLPs). he bands representing the fragments are then blotted onto a special filter, which is bathed with radioactively tagged probes of DN complementary to the gene desired. he desired bands can be cut out of the gel and recovered by soaking in solvent. he genes may be studied by DN sequencing to determine the sequence of nucleotides. In random shotgun sequencing small pieces of the genome are sequenced and then assembled by computer analysis. hese techniques are vital to the Human enome Project discussed in the next paragraph. DN sequencing, which is often highly automated, can be combined with PCR. his permits recovery of detectable, identifiable DN from extremely small samples. hese procedures are used for so-called DN fingerprinting. his is used for analysis of crime-scene samples, tissue identification, and paternity testing. he Human enome Project he goal of the Human enome Project is to map the 35,000 70,000 genes of the human genome, approximately 3 billion nucleotide pairs. his is now nearly complete. he next goal is the Human Proteome Project. his will map all the proteins expressed in human cells. gricultural pplications he most elegant method of introducing recombinant DN into a plant cell is by the i plasmid. bacterium that infects plants normally carries this plasmid. he infection causes a tumorlike growth called a crown gall (i means tumor inducing). he plasmid also serves as a vehicle for insertion of genetically engineered DN into a plant. Other applications are to make crop plants resistant to herbicides that then selectively kill weeds, and to improve the ability to fix nitrogen in certain symbiotic bacteria. bacterium, Bacillus thuringiensis (Bt), has been engineered into plants to produce a toxin that kills certain plant pathogens that feed on the plant. genetically engineered product, bovine growth hormone, increases milk production in dairy herds. Safety Issues and the Ethics of enetic Engineering Laboratories engaged in recombinant DN research must meet rigorous safety standards to avoid accidentally releasing genetically engineered microbes. he microbes may also be engineered to contain suicide genes that prevent them from surviving outside the laboratory environment. enetic screening for hereditary diseases and birth defects in the fetus introduces ethical questions not yet resolved.

5 Biotechnology and Recombinant DN 107 Self-ests In the matching section, there is only one answer to each question; however, the lettered options (a, b, c, etc.) may be used more than once or not at all. I. Matching 1. DN-cutting enzymes that often form sticky ends. 2. self-replicating DN molecule used as a carrier to transmit a gene from one organism to another. 3. n enzyme that links short pieces of DN into longer pieces. a. Restriction enzymes b. DN ligase c. DN polymerase d. Vector 4. n enzyme that links nucleotides to form DN. II. Matching 1. Probably the most common cloning vector used in genetic engineering. 2. he reshuffling of genes between two DN molecules forms DN of this type. 3. he kind of DN synthesized by using mrn as a template. a. Recombinant b. Complementary c. Probe d. Protoplast fusion e. Plasmid 4. DN exchange between cells in this process uses polyethylene glycol to improve efficiency. 5. Short segments of single-stranded DN used to recognize a DN sequence in a gene. III. Matching 1. In the blue/white screening procedure, the foreign DN is in these colonies. 2. hese stretches of DN of eukaryotes do not code for proteins. a. Blue b. White c. Exon d. Intron

6 108 Chapter 9 Fill in the Blanks 1. Sticky ends stick to each other by complementary stretches of single-stranded DN by pairing. 2. collection of bacterial clones each containing a different DN fragment is called a. 3. o isolate a fragment of DN containing a gene, DN fragments of clones are separated by gel electrophoresis. his is an early step in the technique for DN analysis. 4. he procedure by which billions of copies of a sequence of DN can be made in a few hours is called the reaction. 5. he most elegant way of introducing recombinant DN into a plant cell is by means of the plasmid, carried naturally by grobacterium tumefaciens. 6. he Human Proteome Project has the goal of identifying all the produced by human cells.

7 Biotechnology and Recombinant DN 109 Label the rt Recognition sites 1 a. cuts (arrows) double-stranded DN at its particular b., which are shown in dark. DN Cut C C Cut Cut C C Cut 2 hese cuts produce a DN fragment with two c.. C C C C C Sticky end C DN from another source, perhaps a plasmid. 3 When two such fragments of DN cut by the same restriction enzyme come together, they can join by d.. C C C C C C 4 he joined fragments will usually form either a linear molecule or a circular one, as shown here for a plasmid. Other combinations of fragments can also occur. C C 5 he enzyme e. is used to unite the backbones of the two DN fragments, producing a molecule of f.. C C C C

8 110 Chapter 9 Critical hinking 1. During the investigation of a robbery, police discover a small quantity of blood and skin on a piece of broken glass from the window through which the perpetrator gained entry. here was insufficient blood on the glass to type conventionally. No usable fingerprints were obtained. How might the police tie their prime suspect to the crime? 2. What are some of the advantages of using genetic engineering to produce human hormones such as insulin and somatotropin? 3. What method would be most appropriate for inserting foreign DN in each of the following examples? a. Inserting DN into an animal cell. b. Inserting DN into a plant cell. c. Inserting DN into a yeast. 4. You are asked to develop a protocol for the industrial production of a protein from a genetically engineered bacterium. bacterium of what ram designation would most likely be the best choice? Why? 5. What are subunit vaccines? re they safer than avirulent vaccines? Why?

9 Biotechnology and Recombinant DN 111 nswers Matching I. 1. a 2. d 3. b 4. c II. 1. e 2. a 3. b 4. d 5. c III. 1. b 2. d Fill in the Blanks 1. base 2. gene library 3. Southern blot 4. polymerase chain 5. i 6. proteins Label the rt a. Restriction enzyme b. Recognition sites c. Sticky ends d. Base pairing e. DN ligase f. Recombinant DN Critical hinking 1. Police could use recombinant DN techniques to amplify the DN from the skin sample or from the white cells in the blood sample. his material would be compared to the suspect s DN using the Southern blot method to do a DN fingerprint. If the samples match, then the police have positive identification tying the suspect to the crime that is, unless the suspect has an identical twin! 2. enetic engineering produces human hormones that are less expensive, less allergenic, available in large quantities, and pose no risk of transmitting disease. 3. a. nimal cells may be made competent to take up external DN by soaking them in a solution of calcium chloride. fter this treatment many of the animal cells will take up the DN; recombination will occur in some of the cells. Direct introduction of foreign DN into animal cells can be achieved by microinjection using a glass micropipette. b. Plant cells may have foreign DN introduced by first enzymatically removing the cell wall to create protoplasts. Protoplasts are then fused by adding polyethylene glycol to form a hybrid cell. he DN in the hybrid cell may then undergo recombination naturally. nother method utilizes a gene gun that shoots DN-coated tungsten or gold particles through the cell wall. Some cells will incorporate and express the new DN. c. Yeast cells may also be made competent by soaking them in a calcium chloride solution or through the process of electroporation. his involves using an electrical current to form microscopic pores in the cell membrane. DN is able to enter through these pores. 4. gram-positive bacterium would be the best choice for two reasons. First, gram-positive bacteria lack a cell-wall component called endotoxin that is found in gram-negative bacteria. Endotoxin causes fever and shock in animals and would pose a serious problem if present in gene products. Second, gram-negative cells such as E. coli don t usually secrete protein products, and the process used to harvest them is too expensive for industrial applications. 5. Subunit vaccines consist of a protein portion of a pathogen produced by a genetically engineered organism for example, a yeast. Because the protein is made by a yeast rather than a treated pathogen or a portion of the actual pathogen, there is no chance that the disease will be transmitted.

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