18 Recombinant DNA and Biotechnology. 18 Recombinant DNA and Biotechnology 18.1 What Is Recombinant...

18Recombinant DNA and Biotechnology

18 Recombinant DNA and Biotechnology

18.1 What Is Recombinant DNA?

18.2 How Are New Genes Inserted into Cells?

18.3 What Sources of DNA Are Used in Cloning?

18.4 What Other Tools Are Used to Study DNA Function?

18.5 What Is Biotechnology?

18.6 How Is Biotechnology Changing Medicine and Agriculture?

18 Recombinant DNA and Biotechnology

Opening Question:Are there other uses for microorganisms in environmental cleanup?

Bioremediation is the use of microorganisms to remove pollutants.

Some microbes can digest some components of crude oil, but researchers are developing genetically modified organisms that can clean up oil more rapidly and effectively.


Recombinant DNA is a DNA molecule made in the laboratory using at least two different sources of DNA.

Restriction enzymes and DNA ligase are used to cut DNA into fragments and then splice them together in new combinations.


The first recombinant DNA was made in 1973 using plasmids from E. coli.

This research was the start of recombinant DNA technology.

Figure 18.1 Recombinant DNA


Some restriction enzymes recognize palindromic DNA sequences:

5′…….GAATTC……3′

3′…….CTTAAG……5′

Some make straight cuts, others make staggered cuts, resulting in overhangs, or sticky ends.


Sticky ends can bind by base pairing to other sticky ends.

Fragments from different sources can be joined.

Then ligase catalyzes formation of covalent bonds between adjacent nucleotides at fragment ends, joining them to form a single, larger molecule.

Working with Data 18.1: Recombinant DNA

In 1973, the first recombinant plasmid was made using the restriction enzyme EcoRI and two plasmids with resistance to different antibiotics:

• pSC101 had a gene for tetracycline resistance.

• pSC102 had a gene for kanamycin resistance.


Question 1:

In one experiment, some pSC101 was cut with EcoRI but not sealed with DNA ligase.

Cut or intact pSC101 were used to transform E. coli cells, which were grown on media containing tetracycline or kanamycin.

What can you conclude from this experiment?


Question 2:

In another experiment, pSC101 and pSC102 were mixed and treated in three ways:


Did treatment with DNA ligase improve the efficiency of genetic transformation by the cut plasmids?

What is the quantitative evidence for your statement?


Question 3:

How did the antibiotic-resistant bacteria arise in the “None” DNA treatment?


Question 4:

Did the EcoRI + DNA ligase treatment result in an increase in doubly-resistant bacteria over controls?

What data provide evidence for your statement?


Question 5:

For the EcoRI + DNA ligase treatment, compare the number of transformants that were resistant to either tetracycline or kanamycin alone to the number that were doubly resistant.

What accounts for the large difference?

Figure 18.2 Cutting, Splicing, and Joining DNA


Recombinant DNA technology can be used to clone, or make identical copies, of 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.


Usually only a few cells are transformed.

To determine which of the host cells contain the new sequence, the recombinant DNA includes selectable marker genes, such as genes that confer resistance to antibiotics.


The first host cells used were bacteria, especially E. coli.

Yeasts (Saccharomyces) are commonly used as eukaryotic hosts.

Plant cells are also used—they have the ability to make stem cells (unspecialized, totipotent cells).


Cultured animal cells can be used to study expression of human or animal genes.

Whole transgenic animals can also be created.


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.


• Viruses can be altered to carry recombinant DNA into cells.

• Plants are often transformed using a bacterium that inserts DNA into plant cells.

• Transgenic animals can be produced by injecting recombinant DNA into the nuclei of fertilized eggs.


The new DNA must also replicate as the host cell divides.

It 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:

• Inserted near an origin of replication in host chromosome

• 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 enzyme recognition sequences that each occur only once

• Many have genes for antibiotic resistance that can be used as selectable markers


• 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. 18, p. 377 (1)


A plasmid from the soil bacterium Agrobacterium tumefaciens is used as a vector for plant cells.

Plasmid Ti (tumor inducing) causes crown gall.

The plasmid has a region called T DNA, which inserts copies of itself into chromosomes of infected plants.

In-Text Art, Ch. 18, p. 377 (2)


T DNA genes are removed and replaced with foreign DNA.

Altered Ti plasmids transform Agrobacterium cells, then the bacterium cells infect plant cells.

Whole plants can be regenerated from transgenic cells, or germ line cells can be infected.


Most eukaryotic genes are too large to be inserted into a plasmid.

Viruses can be used as vectors (e.g., bacteriophage).

Because viruses infect cells naturally, they offer a great advantage over plasmids.


Usually only a small proportion of host cells take up the vector, 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.


Selectable markers or reporter genes: genes whose expression is easily observed.

There are several types:

• Antibiotic resistance in a plasmid or other vector. A transformed host cell will grow on medium containing the antibiotic.


• The lacZ gene codes for an enzyme that can convert the substrate X-Gal into a bright blue product.

If foreign DNA is inserted within the lacZ gene, and the plasmid transforms bacterial cells, they will not be able to convert X-Gal, and will produce white colonies. Untransformed cells produce blue colonies.

Figure 18.3 Selection for Recombinant DNA


• Green fluorescent protein (GFP), which normally occurs in a jellyfish, emits visible light when exposed to UV light.

The gene for this protein has been isolated and incorporated into vectors as a reporter gene.

It has also been modified to produce other colors.

Figure 18.4 Green Fluorescent Protein as a Reporter


DNA fragments used for cloning come from four sources:

• Gene libraries

• Reverse transcription from mRNA

• Products of PCR

• Artificial synthesis or mutation of DNA


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, which is used to produce a colony of recombinant cells.

Figure 18.5 Constructing Libraries


If bacteriophage λ is used as a vector, about 160,000 “volumes” are required to store the library.

One petri plate can hold thousands of phage colonies, or plaques.

DNA in the plaques is screened using specific 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.


mRNAs do not last long in the cytoplasm and are often present in small amounts, so 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.


RT-PCR: reverse transcriptase and PCR are used to create and amplify a specific cDNA sequence.

This is used to study expression of particular genes in cells and organisms.


Artificial DNA with specific sequences can be synthesized by PCR.

The process is now fully automated and is used to create PCR primers and DNA with specific characteristics, such as restriction sites or specific mutations.

Fragments can be pieced together to form artificial genes.


A way to study a gene and its protein: express it in cells that do not normally express the gene or in a different organism.

The gene must have a promoter and regulatory sequences for the host cell.


Another way to study a gene: overexpress it so that more product is made.

A copy of the coding region is inserted downstream of a different, stronger promoter, and cells are transformed with the recombinant DNA.


Mutations can be created in the laboratory in synthetic DNA.

Consequences of the mutation can be observed when the mutant DNA is expressed in host cells.


Genes can also be studied by inactivating them (e.g., transposon mutagenesis) to define the minimal genome.

In animals, this is called a knockout experiment.


Homologous recombination can knock out a specific gene.

Homologous recombination occurs during meiosis or as part of the DNA repair process.


The normal allele of a gene is inserted into a plasmid, with a reporter gene in the middle of the normal allele.

The recombinant plasmid transfects mouse embryonic stem cells.

The sequences line up with homologous sequences, and if recombination occurs, the normal allele is lost because the plasmid cannot replicate in mouse cells.

Figure 18.6 Making a Knockout Mouse


The transfected stem cell is transplanted into an early mouse embryo.

The mouse and its progeny will have the inactive allele in all cells. The mice are inbred to produce a homozygous line.

Phenotypic changes provide clues to the normal allele function.


Complementary RNA:

Translation of mRNA can be blocked by complementary micro RNAs—antisense RNA.

Antisense RNA can be synthesized and added to cells to prevent translation—the effects of the missing protein can then be determined.


Interference RNA (RNAi) is a natural mechanism that blocks translation.

Short, double stranded RNA is unwound and binds to complementary mRNA by a protein complex, which also catalyzes the breakdown of the mRNA.

Small interfering RNA (siRNA) can be synthesized in the laboratory to inhibit gene expression.

Figure 18.7 Using Antisense RNA and siRNA to Block Translation of mRNA


DNA microarray technology provides a large array of sequences for hybridization experiments.

DNA sequences are attached to a glass slide in a precise order.

The slide has microscopic wells which each contain thousands of copies of sequences up to 20 nucleotides long.

Figure 18.8 DNA Microarray for Medical Diagnosis


DNA microarrays have been developed to identify gene expression patterns in women with a propensity for breast cancer tumors to recur—a gene expression signature.


Biotechnology is the use of living cells or organisms to produce materials useful to people.

Examples:

• Using yeasts to brew beer and wine

• Using bacteria to make cheese, yogurt, etc.

• Using microbes to produce antibiotics, alcohol, and other products


Gene cloning is now used to produce proteins in large amounts.

Almost any gene can be inserted into bacteria or yeasts, and the resulting cells are induced to make large quantities of the product.

Requires specialized vectors.


Expression vectors include all the sequences needed for expression of a transgene in a host cell, including promoters, termination signals, poly A–addition sequences, etc.

Figure 18.9 Expression of a Transgene in a Host Cell Produces Large Amounts of Its Protein Product


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)


Many medically useful products are being made using biotechnology.

Example: The manufacture of tissue plasminogen activator (TPA).

Table 18.1


After wounds heal, blood clots are dissolved by plasmin. Plasmin is stored as an inactive form called plasminogen.

Conversion of plasminogen is activated by TPA.

TPA can be used to treat strokes and heart attacks. The large quantities needed can be made using recombinant DNA technology.

Figure 18.10 Tissue Plasminogen Activator


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 18.11 Pharming


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.


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

Figure 18.12 Genetic Modification of Plants versus Conventional Plant Breeding

Table 18.2


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 an alternative to insecticides. The toxin is easily biodegradable.

Genes for the toxin have been isolated, cloned, 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.


Some transgenic crops are resistant to herbicides.

• Glyphosate is widely used to kill weeds.

• Expression vectors have been used to make plants that synthesize so much of the target enzyme of glyphosate that they are unaffected by the herbicide.


• The gene has been inserted into corn, soybeans, and cotton.

• The crops can be sprayed with glyphosate, and only the weeds will be killed.


Crops with improved nutritional characteristics:

• Rice does not have -carotene, but does have a precursor molecule.

• Genes for enzymes that synthesize β-carotene from the precursor are taken from daffodils or corn and inserted into rice by the Ti plasmid.


• The transgenic rice is yellow, and can supply -carotene to improve the diets of many people.

• -carotene is converted to vitamin A in the body.

Figure 18.13 Transgenic Rice Rich in -Carotene


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 18.14 Salt-Tolerant Tomato Plants


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 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.


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.


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.


Widespread use of glyphosate on fields of glyphosate-resistant crops has resulted in the selection of weeds that are resistant to glyphosate.

More than ten resistant weed species have appeared in the United States.


Microorganisms developed to break down components of crude oil have not been released into the environment because of the unknown effects they might have on natural ecosystems.

Because of the potential benefits of biotechnology, scientists believe that it is wise to proceed with caution.

18 Answer to Opening Question

We use microorganisms to decompose compost and treat wastewater.

The radiation-resistant bacterium Deinococcus radiodurans has been engineered to precipitate heavy metals and break down crude oil components.

It may be useful for bioremediation at radioactively contaminated sites.

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