# Biotechnology

### Introduction

Biotechnology refers to technology used to manipulate DNA. The procedures are often referred to as genetic engineering.

DNA is the genetic material of all living organisms and all organisms use the same genetic code. Genes from one kind of organism can be transcribed and translated when put into another kind of organism.

For example, human and other genes are routinely put into bacteria in order to synthesize products for medical treatment and commercial use. Human insulin, human growth hormone, and vaccines are produced by bacteria.

Recombinant DNA refers to DNA from two different sources.  Individuals that receive genes from other species are transgenic.

### Viruses

Viruses contain genetic material but are not living. Host cells are required for their reproduction.

#### Structure

Viruses are composed of an inner nucleic acid core (genetic material) and an outer protein coat (capsid).

Viruses that infect animals have an outer envelope (membrane) that is derived from the cell membrane of the host cell may surround the capsid.

The genetic material in some viruses is DNA; in others it is RNA.

#### Reproduction

When viral genetic material enters a cell, it is replicated, transcribed (mRNA is produced) and translated (proteins are produced from the mRNA) by the host cell. By this process, the host cell uses the genetic instructions in the virus to make more viruses.

viral DNA $$\rightarrow$$ mRNA $$\rightarrow$$ protein

If the viral genetic material is RNA, a DNA copy must first be made before transcription and translation can occur. The DNA copy of the viral RNA is called cDNA.

viral RNA $$\rightarrow$$ cDNA $$\rightarrow$$ mRNA ? protein

#### Bacteriophages

Bacteriophages are viruses that infect bacteria. They are not surrounded by a membrane as the animal-infecting viruses discussed above.

The virus attaches to the bacteria cell, a viral enzyme digests away a part of the wall and its DNA enters the host cell.

Inside the host cell, the viral DNA is transcribed, translated, and replicated. Translation produces protein coats and the enzymes needed in the construction of new virus particles.

The viral DNA is replicated.

The protein coats and DNA are assembled into new viral particles.

The host cell wall to ruptures releasing the newly formed viruses.

Upon entering the cell, the viral DNA may instead, become integrated into the bacterial DNA. It is replicated along with the host DNA when the host reproduces. Eventually, it will become transcribed and translated as discussed above.

#### DNA Animal Viruses

Animal viruses usually do not kill the cell.

The viruses have a membranous outer envelope with spikes.

The virus enters the cell by endocytosis.

Nucleic acid is released inside the cell. It is transcribed and translated to produce more viral DNA, protein coats and spikes.

The virus acquires its membrane when it is released from the cell by budding (exocytosis).

The host cell is not necessarily killed.

#### Retroviruses

The genetic material of retroviruses is RNA. The retrovirus carries an enzyme called reverse transcriptase, which is capable of creating a DNA copy of the viral RNA.

The new DNA produced from the RNA template is called cDNA.

DNA synthesis follows the production of cDNA to produce a double-helix.

The DNA then becomes incorporated into the host DNA.

The new viruses escape the host cell by budding.

The AIDS virus (HIV) is an example of a retrovirus.

### Recombinant DNA Technology

#### Vectors

Vectors are DNA used to transfer genes into a host cell.

A vector must be capable of self-replicating inside a cell.

Marker genes can be used to determine if the gene has been taken up.

Marker genes must have some distinguishable characteristic. For example if you put a gene that enables an ampicillin resistance on the same vector as the same vector as the gene for human insulin production, then any bacteria that grow on an ampicillin plate will be able to produce insulin.

##### Plasmids

The host bacterium takes up the plasmid, which includes the foreign gene.

When the bacteria reproduces, the plasmids are also reproduced. The gene is cloned.

Shuttle vectors are plasmids that are capable of existing in several different species. They are useful when transferring genes to multicellular organisms.

##### Viruses

Viruses are the vectors of choice for animal cells.

They can accept larger amounts of DNA than plasmids.

When the virus reproduces within the animal cell, it also reproduces the foreign gene that it carries. The gene is therefore cloned.

The DNA of some retroviruses becomes integrated into the host chromosome.

#### Restriction enzymes

Restriction enzymes were discovered in bacteria. Bacteria use them as a defense mechanism to cut up the DNA of viruses or other bacteria.

Hundreds of different restriction enzymes have been isolated. Each one cuts DNA at a specific base sequence. For example, EcoRI always cuts DNA at GAATTC as indicated below.

The sequence GAATTC appears three times in the DNA strand below. As a result, the strand is cut into four pieces.

Other restriction enzymes cut at different sites, some examples are listed below.

 Enzyme Cutting Site Bam HI GGATCC Hae III GGCC Pst I CTGCAG Hinf I GANTC
##### Sticky Ends

Fragments of DNA that has been cut with restriction enzymes have unpaired nucleotides at the ends called sticky ends. All of the fragments will have the same sticky ends. The sticky ends have complimentary bases, so they could rejoin.

If the vector and the gene to be cloned are both cut with the same restriction enzyme, they will both have complimentary sticky ends.

#### Making Recombinant DNA

To make recombinant DNA, restriction enzymes are used to cut DNA from two sources such as the that of a vector and a gene to be cloned. If the vector and the gene to be cloned are both cut with the same restriction enzyme, they will both have complimentary sticky ends (see above).

After cutting, the two samples of DNA are mixed. Some of the fragments from one species will stick to those of the other because they both have the same sticky ends.

DNA ligase is used to seal the fragments.

Bacteria are capable of taking up DNA from their environment. This process is called transformation. CaCl2 and a procedure called heat shock are used to make E. coli cells more permeable so that they take up the modified plasmids more readily.

### Genomic Libraries

A genome is all of the genes in a particular organism. Bacteria or virus vectors can be used to store fragments of the DNA from a species.

The DNA is cut up into fragments and the different fragments are inserted into bacteria or viruses. The collection of bacteria or viruses is called a genomic library.

#### Finding Genes in a Gene Library

##### Blue-White Screening Method

The plasmid used contains a gene for ampicillin resistance. The transformed bacteria are grown on a medium that contains ampicillin. Any bacteria that grow have been transformed because the bacteria cannot grow unless they are ampicillin-resistant.

The plasmid also contains a gene for the production of b-galactosidase. The b-galactosidase gene contains a region that can be cut with a number of different restriction enzymes. Genes inserted into this site will inactivate the b-galactosidase gene because it has been cut and the new gene has been inserted within it. A recombinant plasmid therefore will not produce b-galactosidase.

The transformed bacteria are grown on a medium that contains X-gal, a substrate for b-galactosidase. Colonies that use X-gal as a food source produce a blue compound. Colonies that have received a gene cannot use X-gal and appear white.

Bacteria that have foreign genes, therefore, will grow (resistant to ampicillin) and appear white (unable to produce b-galactosidase).

The blue-white screening method described above selects for bacteria that have any gene. Radioactive probes can be used to find colonies that have specific genes.

Probes are short, single-stranded segments of DNA whose base sequence matches part of the gene in question. It is not necessary to match the entire gene, just a small fragment.

The cells are lysed and the DNA is denatured by treating it with an alkaline solution. When the probe is mixed with the denatured DNA, it will form a double-helix in the area where the gene has complimentary bases.

If the probe is radioactive or fluorescent, it can be visualized. The gene can then be isolated or cloned as needed.

It may be possible to see the chromosome and the location on the chromosome while viewing under a microscope.

Autoradiography is a process in which film is used to show the area of the vector where the probe has attached. This area is the gene in question.

##### Obtaining Probes

Probes can be synthesized using a DNA synthesis machine. This requires a knowledge of at least a part of the sequence of the nucleotides in the gene to be probed so that a complimentary fragment can be created. If this is unknown, it may be deduced by knowing the sequence of amino acids in the protein that the gene codes. Amino acids often have more than one spelling,so it may be necessary to create several different alternative probes.

In some cases, a similar gene which has already been isolated from another organism can serve as a probe.

### Inserting DNA Into Cells

#### Eukaryotic Genes

Eukaryotic genes contain introns but bacteria do not contain the necessary enzymes to remove introns, so eukaryotic genes that are inserted into bacteria must be inserted without introns.

#### Making Intron-Free DNA:

The DNA of eukaryotes is extremely long, containing many thousands of genes. It is often not possible to find specific genes in the DNA. Artificial genes can be made, however, using mRNA as a template.

In order to synthesize a gene, one must first obtain some mRNA produced by the gene in question. Recall that the introns of mature mRNA have already been removed. Reverse transcriptase (from retroviruses, see discussion above) is used to produce a DNA copy of the mRNA. This copy is called cDNA.

#### Expression Vectors

The promoter and ribosome binding site codes in Eukaryotic DNA are likely to be different than those used by the host organism. Plasmids called expression vectors have been created that have a promoter and ribosome binding site that can be recognized by E. coli. These sites are adjacent to a restriction cutting site so that any gene inserted into the plasmid will be transcribed and translated by E. coli.

#### Shuttle Vectors

Shuttle vectors are plasmids that can be propagated in two different species because they have two origins of replication.

This is done because it is easier to clone genes in some species such as E. coli. After the cloning procedures are done, the plasmid can be transferred to a different species for expression of the cloned DNA.

#### Bacterial Artificial Chromosomes

Artificial vectors have been developed that can be used for inserting large segments of DNA. These vectors are called bacterial artificial chromosomes.

#### Yeast Artificial Chromosomes

Yeast artificial chromosomes can hold 150-1000 kb pairs. The chromosomes can grow in bacteria and yeast.

The has a ring form that is used in bacteria. It can be cleaved to produce a linear form with a centromere that replicates in yeast.

#### Transgenic Plants

The only plasmid that plant cells take up is the Ti (tumor-inducing) plasmid from the bacterium Agrobacterium. The plasmid transferred by this bacterium causes plants to form a gall. A wide variety of plant cells will take up the plasmid and move it into a chromosome. Scientists have been able to remove the gall-forming genes and insert other genes into the plasmid. As a result, this plasmid has enabled genetic engineering of a large number of plants.

#### Transgenic Animals

Animal cells generally will not take up plasmids. Other methods such as microinjection must be used.

One method has been developed where animal eggs are placed in a mixer with needle-like fragments of silicon carbide. The needles make holes in the cells, allowing DNA to enter. Using this procedure, eggs from fish and several agricultural species have been given the gene for bovine growth hormone, producing larger individuals.

#### Viruses

Viruses may serve as vectors for transferring DNA into eukaryotic cells. After the DNA enters the cell, it must become inserted into the host chromosome.

#### Other Methods of Delivering DNA

Electroporation involves using an electric current to create pores in the cell wall and plasma membrane for DNA to enter.

It is difficult to create transgenic plants because the cell wall prevents entry of DNA. One solution is to remove the cell wall. These cells (called protoplasts) are then placed in a liquid with foreign DNA. Electroporation is used to make small, temporary holes in the membrane so DNA can pass in.

A gene gun propels small gold pellets coated with DNA. The pellets penetrate the cell wall and plasma membrane and enter the cell to deliver their DNA.

### Polymerase Chain Reaction (PCR)

The polymerase chain reaction can be used to make many copies of small pieces of DNA. Because techniques in biotechnology usually require large amounts of DNA (many copies), PCR has allowed much of the biotechnology development that we have seen in recent years.

#### Materials and Procedure

##### Materials needed

The procedure requires primers, DNA polymerase, and nucleotides.

Primers are short chains of about 20 nucleotides that are complimentary to a region in the DNA to be amplified. They are needed because the enzyme that copies the DNA (DNA polymerase) cannot start the process unless it has already been started.

DNA polymerase from the thermophile Thermus aquaticus is used because this species thrives at temperatures that are near boiling. It's DNA polymerase (called Taq polymerase) is stable at relatively high temperatures and functions optimally at 70 degrees C. This is important because high temperatures will be used to separate the strands of the double helix.

Nucleotides are needed because DNA is composed of nucleotide "building blocks".

##### Procedure

The DNA in question is heated to approximately 95 degrees C to separate the two strands of the double helix.

After the strands are separated, the DNA is cooled to about 50 degrees and the primers attach.

The temperature is raised to approximately 70 degrees C. This is the optimal temperature for Taq polymerase. The polymerase attaches to and copies the strand.

The solution is then heated and cooled again as described above at regular intervals. Each time it is cycled through this heating and cooling procedure, the DNA replication process repeats itself and the amount of DNA produced is doubled.

### DNA Fingerprinting (RFLP Analysis)

In RFLP analysis, the DNA of an organism is cut up into fragments using restriction enzymes. A large number of short fragments of DNA will be produced.

Restriction enzymes always cut at the same base sequence. Because no two individuals have identical DNA, no two individuals will have the same length fragments. For example, the enzyme EcoRI always cuts DNA at the sequence GAATTC. Different people are going to have different numbers of this particular sequence and will therefore have different fragment lengths. In addition, some of them will be at different locations on the chromosome.

Electrophoresis is a technique used to separate the DNA fragments according to their size. They are placed on a sheet of gelatin and an electric current is applied to the sheet. DNA is charged and will move in an electric field toward the positive pole.

In the diagram below, holes (wells) in the gelatin can be seen. DNA samples placed in these wells will migrate through the gelatin toward the + side after an electric current is applied.

The smallest fragments will move the fastest because they are able to move through the pores in the gelatin faster. Bands will be produced on the gelatin where the fragments accumulate. The shortest fragments will accumulate near one end of the gelatin and the longer, slower-moving ones will remain near the other end.

In the diagram below, four samples of DNA were placed on the gelatin. After an electric current was applied for a period of time, the fragments separated. Notice that sample D on the right does not match the other three samples.

The DNA bands must be stained to make them visible. Ethidium bromide-stained DNA will fluoresce when illuminated with UV light.

PCR techniques are used to produce sufficient quantities of DNA for this technique.

##### Southern Blotting

Electrophoresis separates DNA by size but does not provide any information about the nucleotide sequence in the DNA. A technique called Southern blotting is used to search for specific base sequences in DNA that has been separated by electrophoresis. It is useful in identifying pathogens and genetic diseases.

This technique uses a probe that has a complimentary base sequence to a portion of the DNA of interest. The probe is labeled with radioactivity, a fluorescent dye, or a colored dye so that it can be visualized.

First, the DNA to be searched is digested using restriction enzymes and separated using gel electrophoresis as described above.

The fragments on the gelatin are transferred to a filter by blotting.

The filter is treated with an alkaline solution to separate the strands of DNA.

The probe is added to the filter paper and then rinsed off. Because the probe has a base sequence that is complimentary to the DNA of interest, it will only attach to the DNA of interest, the rest will rinse off.

The DNA fragment containing a sequence that is complimentary to the probe can be seen because the probe has been labeled.

##### Uses for Electrophoresis

Electrophoresis requires a large amount of DNA so it is often used in conjunction with PCR discussed above. Some uses are identification of diseased genes including oncogenes, identification of viral infections, determining family relationships among individuals, and identifying tissue found at a crime scene.

For example, suppose that this procedure is used to identify cells found at a crime scene. Samples A and B (above) came from the scene of the crime and samples C and D came from two different suspects. What can you conclude?

Some genetic diseases that can be identified using this procedure are Sickle Cell disease, Huntington’s disease, Duchenne muscular dystrophy.

Taxonomists can use this technique to explore evolutionary relationships. Individuals of the same species, while not identical, will be more similar than individuals of different species.

The procedure for sequencing and mapping DNA requires RFLP analysis.

### Gene Therapy

Gene therapy uses technology to change the genetic composition of a cell.

#### Ex vivo

Ex vivo methods are done outside the organism. Cells are removed, treated and returned to the individual.

Retroviruses are often used as the vector. The retroviruses contain recombinant RNA which includes the gene to be added. Once in the cell, the enzyme reverse transcriptase makes a DNA copy of the RNA.

Currently, there are more than 100 clinical trials of this technique.

##### Example of ex vivo gene therapy

This procedure has been used to treat severe combined immunodeficiency syndrome (SCID). People with this disease are susceptible to infections because their white blood cells do not produce an enzyme needed by their immune systems. This disease has been treated in two different ways. In a short-term solution, the white blood cells were removed and infected with a retrovirus that carried the needed gene. After the cells were replaced, many of the cells contained the gene. White blood cells, however, are short-lived and a long-term solution is to apply this technique to the cells that produce the white blood cells (called stem cells).

#### In vivo

In vivo gene therapy treats cells in the individual without removing them.

Retroviruses can be used to introduce genes directly into the body.

Synthetic carriers like liposomes can also be used to carry genes. Liposomes are microscopic lipid vesicles that are readily taken up by cells. If they are coated with DNA, the DNA is also taken up.

#### Transgenic Bacteria

##### Protein products

Genes that code for the desired protein can be inserted into plasmids and the plasmids are used to transform cells. Many useful human proteins are now synthesized by transgenic bacteria. Some of these are listed below.

Human growth hormone is used to treat dwarfism. It previously took the pituitary glands from over 50 cadavers to make one dose.

Human Insulin is used to treat diabetes.  Insulin was previously obtained from the pancreas of slaughtered cattle and pigs. It sometimes caused allergic reactions.

Tissue plasminogen activator dissolves blood clots in heart attack victims.

Clotting factor VIII is one of several factors needed for blood to clot. Most cases of hemophilia are due to the absence of this factor.

Human lung surfactant is used in premature infants with respiratory distress syndrome.

Atrial natriuretic hormone can be used to treat hypertension.

Bovine growth hormone (bGH) increases milk production in cows by about 10%.

##### Vaccines

Vaccines were previously made by killing or weakening a virus or bacteria and then injecting it. Its surface proteins caused an immune reaction. Occasionally, these vaccines would make people ill. Using biotechnology, some of the proteins of the pathogen can be made by cloning the gene that codes for them. Vaccines containing the protein component of pathogens are called subunit vaccines. They are sufficient to stimulate the immune system but are incapable of causing an infection.

Vaccines for hepatitis B and human papilloma virus (Gardasil) are now produced using biotechnology.

Hundreds of vaccines are currently are being developed.

Vaccines for hoof-and-mouth disease and scours (a form of dysentery) have been developed for farm animals.

##### Other Uses of Recombinant Bacteria

Bacteria have been produced that inhibit the formation of ice crystals. These bacteria have been released onto crop plants to protect them from frost damage.

A bacteria species that normally colonize corn roots have been given a gene that enables it to produce an insect-killing toxin (pesticide).

Bacteria are being developed that do a better job at breaking down oil. These may be useful to help clean up oil spills.

Bacteria have been developed that are capable of removing some kinds of toxins from the air and water.

Bacteria have been engineered to extract metals from low-grade ore (bioleaching). This technique is currently being tested.

#### Transgenic plants

Bt-toxin is an insecticide naturally produced by the bacterium Bacillus thuringiensis. This toxin is toxic only to insects because it must bind to receptors in their gut to function. Several different kinds of crop plants have been genetically engineered to produce this toxin.

Agricultural plants have been developed that are resistant to the effects of the herbicide RoundupTM. This herbicide kills non-resistant plants and is biodegradable.

Presently, there are approximately 50 types of genetically engineered plants that resist insects, viruses, and herbicides.

Rice has been engineered to produce B-carotene and increased levels of iron.

A weed called mouse-eared cress has been designed to produce a biodegradable plastic called polyhydroxubutrate (PHB).

##### Possibilities for the Future

In the future, biotechnology may be able to improve crop yields and produce plants that contain all of the amino acids required for human consumption.

If plants could be produced that can fix atmospheric nitrogen, they would require considerably less fertilizer.

Plants engineered to grow under harsh environmental conditions could allow wastelands to be more productive.

#### Transgenic Animals

Pharmaceutical companies are developing techniques to produce chemicals using animals. The drug is produced in the milk of females. For example, goats have been developed to produce antithrombin III, used to prevent blood clots. Clinical trials of this drug will begin soon.

Lactoferrin is added to infant formula to transport iron and to prevent bacterial infections in the gastrointestinal tract. A transgenic bull has been produced that carries a gene for the production of human lactoferrin. Females will produce milk with lactoferrin.

A sheep has been bioengineered to produce tPA (tissue plasminogen activator) in her milk. Tisue plasminogen activator is a "clot-busting" drug that is often administered to people who have suffered a heart attack or stroke.

A pig has been produced that can produce human hemoglobin. Artificial blood may soon be a reality.

#### Cloning Mammals

Cloning animals refers to producing offspring that are genetically identical to the animal being cloned. This process has been done by removing the nucleus of an egg and replacing it with a diploid nucleus from the organism to be cloned. The egg is then treated so that it begins dividing. It is placed in the uterus of a host animal where it continues to grow.

### The Human Genome Project

The Human Genome Project is a massive, government-funded project whose goal is to determine the base sequence of all of the human chromosomes.

It would take 200 volumes of 1,000 pages each just to list the letters of the bases.

Knowledge of the sequences of all genes making it easier to study, diagnose, and treat many kinds of human genetic diseases. Information on gene sequences is important in the development of gene therapy techniques.

The information is expected to lead to a better understanding of genetic systems, and ultimately answers to mysteries surrounding such topics as gene regulation and cancer.

### Safety and Ethical Issues

Harmful organisms may be accidentally produced. For example a genetically-engineered organism may be pathogenic or capable of causing ecological destruction.

To minimize this risk, most laboratories work only with microorganisms that have been modified so that they cannot survive in nature in case they are accidentally released. Organisms that are intended to be released into the environment may be engineered with genes that will eventually kill them.

Ethical questions have been raised over whether we should modify the genes of humans. Treating a person with a severe disease may seem to be appropriate use but it must be decided what a "severe disease" is. Treating a person's somatic (body) cells alters only that person but treating the person's germ (sex) cells alters future generations.

There is little legislation on the use of genetic screening and information produced by screening. There are presently few guidelines in place that protect an individual's privacy and limit access to screening information. For example, potential employers or insurance companies might find screening information useful and require employees or clients to undergo screening.

The technology is increasing the ability to diagnose genetic diseases prenatally, adding new complexity to the abortion controversy.

Genetic screening and gene therapy are expensive and may be unavailable to the poor.

Biological weapons could be created using biotechnology.