Chapter 26: Biotechnology

Cloning of a Gene Cloning is the production of identical copies through asexual means. Cloning occurs naturally in new plant shoots, bacterial colonies, and identical human twins. Gene cloning is the production of many identical copies of a gene.

Recombinant DNA Technology Recombinant DNA (rDNA) contains DNA from two or more different sources. To make rDNA, a technician needs a vector by which rDNA will be introduced into the host cell. A plasmid (a small accessory ring of DNA in bacteria) or virus can be used as a vector to insert foreign DNA into a host cell. Restriction enzymes are so-called because they restrict the growth of viruses, but they also act as molecular scissors to cleave any piece of DNA at a spoecific site.

Cloning a human gene Human DNA and plasmid DNA are cleaved by a specific type of restriction enzyme and spliced together by the enzyme DNA ligase. Gene cloning is achieved when a host cell takes up the recombinant plasmid, and as the cell reproduces, the plasmid is replicated. Multiple copies of the gene are now available to an investigator. If the insulin gene functions normally as expected, insulin may be retrieved.

Restriction enzymes cleave the vector DNA and the source DNA at a specific sequence, leaving “sticky” ends, that allow a portion of source DNA to be inserted into the vector DNA. DNA ligase then seals the openings and recombinant DNA is formed. Bacterial cells take up recombinant plasmids and clone the new DNA.

The restriction enzyme EcoRI always cuts double-stranded DNA as shown when it has this sequence of bases. Notice that there is now a gap into which a piece of foreign DNA can be placed if it ends in bases complementary to those exposed by the restriction enzyme. The single-stranded, but complementary, ends of the two DNA molecules are called “sticky ends” because they can bind a piece of foreign DNA by complementary base pairing.

To express a human gene in a bacterium it must not have introns. When a viral vector is used the cloned DNA is inside newly formed bacteriophages. To express a human gene in a bacterium it must not have introns. Reverse transcriptase can be used to make a DNA copy of mRNA; this complementary DNA does not contain introns. Alternatively, it is possible to manufacture small pieces of DNA in the laboratory. A machine called a DNA synthesizer joins together the correct sequence of nucleotides, and this resulting DNA also lacks introns.

The Polymerase Chain Reaction The polymerase chain reaction (PCR) produces many copies of a single gene or piece of DNA. PCR requires DNA polymerase and a supply of nucleotides for the new DNA strands. PCR is a chain reaction because the targeted DNA is repeatedly replicated as long as the process continues.

Polymerase chain reaction (PCR) PCR allows the production of many identical copies of DNA in a laboratory test tube.

Analyzing DNA Segments The entire genome of an individual can be cut by restriction enzymes to yield variable fragment lengths. Gel electrophoresis separates fragment lengths and the use of probes results in a pattern unique to the individual, sometimes called a DNA fingerprint. A DNA fingerprint resembles that of one’s parents because it is inherited.

It is possible identify a person who has committed a crime, identify a cancer gene, or tell who is related to whom by performing a DNA fingerprint. Since PCR can amplify the smallest amount of DNA, a single sperm, or one cell on a toothbrush, provides enough DNA to be identified by comparison with sample DNA.

Biotechnology Products Today, bacteria, plants, and animals are genetically engineered to make biotechnology products. Organisms that have had a foreign gene inserted into them are called transgenic organisms. There are now transgenic bacteria, transgenic plants, and transgenic animals.

Transgenic Bacteria Recombinant DNA technology is used to produced transgenic bacteria, which are grown in huge vats called bioreactors. Transgenic bacteria are used to produce: insulin, human growth hormone, tissue plasminogen activator, and hepatitis B vaccine. They also add insecticidal toxins to plants, reduce frost damage on plants, degrade wastes, produce chemicals, and help mine metals.

Biotechnology products Products such as clotting factor VIII, which is administered to hemophiliacs, can be made by transgenic bacteria, plants, or animals. After being processed and packaged, it is sold as a commercial product.

Transgenic Plants Foreign genes are added to protoplasts using an electric current. Foreign genes in cotton, corn, and potatoes have given them pest resistance; soybeans are made resistant to herbicide for no-till farming. Transgenic plants produce human hormones, clotting factors, and antibodies in their seeds; one weed has even been engineered to produce plastic granules. Foreign genes are inserted into protoplasts or immature plant embryos.

Transgenic Animals Foreign genes can be inserted into animal eggs by hand or by vortex mixing. Gene pharming uses transgenic farm animals to produce pharmaceuticals in their milk. Animals may produce drugs for the treatment of cystic fibrosis, cancer, and blood diseases.

Transgenic animals DNA containing the gene of interest is injected into donor eggs. Following in vitro fertilization, the genetically engineered egg develops in a host to produce a transgenic goat that gives a biotechnology product in its milk. Nuclei from the transgenic goat are transferred into donor eggs, which develop into transgenic goats.

Cloning Transgenic Animals Cloning of mammals was once considered impossible, but has now been accomplished with sheep, calves and goats. A 2n nucleus from a bioengineered animal is inserted into enucleated eggs from a donor. A surrogate mother gives birth to the cloned animals. Cloning of an adult vertebrate requires that all the genes of an adult cell be turned on again if development is to proceed. Cloned animals are another way to obtain biotechnology products.

Animal Organs as Biotechnology Products Scientists are genetically engineering pigs to serve as organ donors for humans who need transplants. Transplants of organs across species is called xenotransplantation. Researchers are trying to make organs less antigenic to humans.

One concern is whether pig organs might carry animal viruses into humans; HIV is a virus that jumped from monkeys into humans. Tissue engineering is an alternative method of securing bioartificial organs – transplant material from culturing human tissue from a mixture of cells and synthetic polymers.

The Human Genome Project The Human Genome Project has two goals: to construct a map that shows the sequence of bases on all the human chromosomes and to construct a map that shows the sequence of genes along all the human chromosomes.

The Base Sequence Map The first goal has been completed and researchers know the sequence of three billion base pairs after 15 years of research. The two agencies that completed the task are The International Human Genome Sequencing Consortium and Celera Genomics, a private company.

DNA sequences are similar among organisms and the differences may be due to regulation of genes. It has also been determined that humans share a large number of genes with very simple organisms such as bacteria.

The Genetic Map Much research must be done to locate the genes on each human chromosome. The number of protein-encoding genes appears to be very low, about 30,000. Some believe each gene could code for three different proteins by using different combinations of exons.

Genetic map of chromosome 17 This map shows the sequence of mutant genes that cause the diseases noted.

A gene map could help discover mutant genes and lead to the development of medicines to treat these disorders. Germline therapy, that is done before a child is born, may eventually be possible. Many ethical questions surround how human genome maps should be used.

Gene Therapy Gene therapy is the insertion of genetic material into human cells for the treatment of a disorder. A patient would be given healthy genes to make up for any faulty genes. Gene therapy includes ex vivo (outside the body) and in vivo (inside the body) methods.

Ex Vivo Gene Therapy Using the ex vivo method, bone marrow stem cells are withdrawn from the body, a retrovirus is used to insert a normal gene into them, and the stem cells are returned to the body. This method of gene therapy works for severe combined immunodeficiency (SCID) and may work for familial hypercholesterolemia where liver cells lack a receptor for removing blood cholesterol.

Ex vivo gene therapy in humans Bone marrow stem cells are withdrawn from the body, an RNA retrovirus is used to insert a normal gene into them, and the cells are returned to the body.

In Vivo Gene Therapy Cystic fibrosis patients lack a gene that codes for a membrane carrier of chloride ions; researchers try to deliver the gene by nose sprays containing adenoviruses or liposomes. A gene that codes for new blood vessel growth is being injected to stimulate new coronary blood vessels. Liposomes are microscopic vesicles that spontaneously form when lipoproteins are put into a solution.

Many researchers are trying to cure cancer by inserting genes to make healthy cells tolerant of chemotherapy or use gene p53 to bring about apoptosis of cancer cells.

Chapter Summary Recombinant DNA technology utilizes genetically altered bacteria and viruses to clone a gene. The polymerase chain reaction (PCR) rapidly makes copies of DNA segments; this allows detailed analysis of the DNA for a wide range of uses.

Transgenic bacteria, agricultural plants, and farm animals have been genetically engineered to produce commercially valuable products and applications. Agricultural plants and farm animals have been genetically engineered to improve their yield. Farm animals have been genetically engineered to serve as a source of organs for human transplant patients.

It is now possible to clone animals; cloning is used to produce multiple copies of farm animals that have been genetically engineered. The Human Genome Project aims to sequence the DNA bases of each chromosome and to map the genes on each chromosome; the first goal is completed. Gene therapy is now being used to replace defective genes with healthy genes and to help cure various human ills.