Genetic Engineering

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Genetic Engineering

Genetic engineering is the altering of an organism's deoxyribonucleic acid (DNA) to create a desired effect. Genetic engineers follow a set of techniques that allows them to remove genetic material from two or more species, recombine the genetic material (to create recombinant DNA), and integrate it into a host's genome, or genetic material. Genetic engineering is used for a variety of scientific, agricultural, and medical purposes.

Restriction Enzymes

All recombinant DNA technology requires the use of restriction enzymes. These enzymes are naturally occurring in bacteria that fight phage, or virus, DNA, but geneticists can use restriction enzymes as tools to cut DNA into manageable fragments.

Restriction enzymes recognize specific sequences along the DNA where it can be cut. These sequences contain four or more bases and occur randomly, and each enzyme cuts at a different sequence. Most restriction enzymes cut double-stranded DNA in a staggered fashion, so that a single strand extends from each end. These complementary "sticky" ends tend to bond to each other in solution.

Geneticists often need to isolate specific DNA molecules from a mixture. Restriction enzymes cut DNA into many small fragments. These fragments can be run through an electrophoretic gel, which distributes them according to size. In a technique known as Southern blotting, an absorbent membrane is then placed on the gel, transferring the ordered DNA fragments. Finally, a radioactive DNA probe is applied to the membrane, binding to any complementary DNA sequences and thereby labeling them.

Restriction enzymes can also be used to map genes. Restriction sites often vary by one or more nucleotides within a species. Genetic variation at a restriction site can produce a change in the length of a DNA sequence, also known as restriction fragment length polymorphism (RFLP). RFLP can then be measured, what is often called "DNA fingerprinting." Any genetic variation that is correlated with the RFLP is likely to be in the same part of the genetic material because correlations between distant genetic locations (loci) tend to break down over time as a result of recombination between chromosomes.

Recombinant DNA

Making recombinant DNA requires isolating the DNA that will be cut, donor DNA, and the DNA into which the donor DNA will be inserted, vector DNA. The combination of donor DNA and vector DNA is recombinant DNA.

A vector is a small piece of DNA that carries the donor DNA into host cells. A common vector is a plasmid, a small ring of bacterial DNA that replicates independently of the main chromosome. Plasmids are useful vectors because they easily enter bacterial cells. A plasmid vector must first be removed from the rest of the bacterial genome. The DNA is removed and centrifuged so that the smaller plasmids sink farther than the larger chromosomes. Alternatively, an alkaline pH degrades genomic DNA but not plasmids, and the genomic DNA can be precipitated out of the solution.

Donor DNA and vector DNA must be digested with a restriction enzyme so that they can be spliced together. Some sticky ends will anneal (bind) vector DNA to donor DNA. Although their initial bonding is temporary, adding DNA ligase creates stronger bonds between the joined ends to make a continuous molecule.

To improve the efficiency of this procedure, annealing between two vector molecules or two donor molecules can be prevented by adding complementary nucleotides to one strand of each molecule, for example, As to the donor molecules and Ts to the vector molecules. Furthermore, two different restriction enzymes may be used for donors and vectors, allowing greater flexibility in choosing splice sites.

Cloning

Plasmid vectors are introduced into bacteria by transformation. The plasmid replicates within each bacterium, and the bacteria divide many times. Consequently, a single DNA donor molecule can be amplified into billions of copies. This set of copies is referred to as a clone.

Most plasmids used to make recombinant DNA carry genes for drug resistance. This feature allows geneticists to apply an antibiotic to select bacteria that have been transformed by the recombinant plasmid. The antibiotics destroy bacteria that do not carry the plasmid. Plasmids also have particular restriction target sites that tailor them to the use of certain restriction enzymes. Vectors can have two different drug resistance genes, one of which contains the restriction site. If an insertion of donor DNA occurs, drug resistance is lost, and application of the drug will destroy all bacteria carrying the insert. In this way bacterial colonies carrying the desired gene can be identified and allowed to grow.

Lambda phage, a bacterial virus, can also be used as a cloning vector. Phage heads selectively package chromosomes about fifty kilobases in length. If a piece of phage DNA is removed and replaced by a donor DNA fragment of approximately the same size, the recombinant DNA can still be packaged. However, if the insertion is not successful, the phage head will not form and the DNA cannot be injected into bacteria. Successful transfection of donor DNA into a bacterial colony can be detected as a plaque indicative of infection.

There are several other types of vectors. Cosmids are hybrids of lambda phages and plasmids. Because they are larger than either phages or plasmids, they are capable of inserting larger DNA fragments. Expression vectors allow foreign genes produce proteins in a bacterium. Yeast artificial chromosomes (YAC) can carry very large inserts into yeast cells for replication. Bacterial artificial chromosomes (BAC) are capable of carrying large inserts into bacteria.

DNA Libraries

One goal of cloning genetic material is to develop a DNA library. A library is a collection of clones covering part or all of the genome of interest. It may consist of phage, bacteria, or yeast, depending on the vector used. A library may be genomic or cDNA. A cDNA library contains only coding DNA because it is synthesized from messenger RNA, which is the template from which proteins are made. A cDNA library is useful to researchers interested in genes that are expressed in particular tissues, from which they can obtain mRNA. Because the donor cDNA encodes protein, what is known as a cDNA expression library can also transcribe and translate genes into proteins.

Once a library is created, it can be screened with a probe to find a gene of interest. A probe is a piece of single-stranded DNA or an antibody that is radioactively labeled. A DNA probe will bind to a complementary strand of DNA in the library, whereas an antibody probe will bind to a protein. cDNA probes can be synthesized as an oligonucleotide, a short piece of synthetic DNA, from the amino acid sequence of the protein of interest. Because of the redundancy of the genetic code, many different cDNA's can code for the same protein. Therefore, a cocktail is often made of many different cDNA oligonucleotides.

Positional cloning is a process of using information about the location of a gene so as to clone it more efficiently. Clones can be ordered by their positions along a chromosome by doing a chromosome walk. A chromo-some walk uses the ends of each clone to probe the rest of the library for overlapping clones. If another gene is closely linked to the gene of interest and its position is known, only the clones near the first gene need be probed to find the gene of interest.

Tagging is a process in which a known DNA sequence (tag) is allowed to insert at random places in a genome. If by chance an insertion occurs in the gene of interest, it will likely cause a mutation that limits the functioning of the gene's product. Mutant lines are selected and used to construct a genomic library, which is then tested with the tag as a probe to identify the gene of interest.

DNA Sequencing

Once a gene is cloned, its DNA sequence can be determined. First, the gene of interest is copied with a polymerase enzyme, a primer to direct the polymerase to the gene, and nucleotides. The nucleotides are not all the same, however. Some are missing an oxygen atom, which prevents them from adding another nucleotide to the end. Thus, the sequencing reaction results in an array of partial copies of the gene. If the reaction is performed with some deoxygenated thymine, then all of the copies will end in thymine. A different sequencing reaction can be performed for each deoxygenated base to generate four different sets of partial copies. The next step is to separate the partial copies according to size. Larger fragments travel more slowly through a gel, and mobility through an acrylamide gel is extremely sensitive to size. If one were to run two sets of fragments differing by just one base in size, the difference would be evident as two distinct bands on an acrylamide gel. Thus the products of a sequencing reaction will show up as an array of bands on an acrylamide gel, with each band representing the position of the deoxygenated nucleotide used in the sequencing reaction. Alternatively, just one sequencing reaction may be done with some of each nucleotide deoxygenated if each is labeled with a unique fluorescent dye, resulting in color-coded bands.

If the precise location of a gene is not known, sequencing can reveal its location. A functional gene usually appears as an open reading frame (ORF), which is a sequence of DNA uninterrupted by a stop signal. A sequence can be analyzed by a computer program, which examines all three possible reading frames on each strand. Unusually long ORFs indicate the presence of a gene, because otherwise stop codons are expected to appear at random.

If a gene has been cloned and sequenced, it can be copied from any individual in a population by polymerase chain reaction (PCR). Primers, short oligonucleotides that attach to complementary DNA and act as targets for the polymerase enzyme, are designed from the gene sequence. The strands of DNA are separated at a high temperature. The temperature is lowered again, one primer attaches to each strand of DNA, and the polymerase extends each one in opposite directions. This process is repeated many times by a machine, providing billions of copies of the gene in a few hours. PCR is useful for quickly amplifying DNA from many individuals in a population.

Gene Alteration and Expression

All of the above recombinant techniques allow geneticists to identify and describe genes. Once a gene has been sufficiently characterized, it can be manipulated to produce a desired outcome. In vitro mutagenesis can change a single target nucleotide to another nucleotide. First the gene is cloned into a single-stranded phage vector. An oligonucleotide primer is constructed from the gene's DNA sequence. The primer is complementary to the gene with the exception of the target site, which is changed to the desired nucleotide. (Although not completely complementary to the template DNA, hybridization occurs under certain conditions.) The phage is allowed to replicate so that its copy incorporates the new nucleotide. Continued copying will produce mostly mutant genes, which can be identified with the mutant primer as a probe under conditions that prevent hybridization with nonmutants.

Synthetic oligonucleotides can also be used to construct entire genes up to sixty bases in length. The automated reaction adds bases one at a time to the growing oligonucleotide, which is embedded in a resin. Overlapping oligonucleotides can be pieced together to synthesize longer sequences. Herbert Boyer's lab synthesized the gene for somatostatin, a human growth hormone, in this manner. The scientists added a restriction site to each end of the gene and a methionine codon to one end. Restriction enzymes were used to insert the gene into a bacterial plasmid that carried the same restriction sites. The insertion occurred in the middle of a bacterial beta-galactosidase gene, which the bacteria transcribed and translated. The resulting protein was a chimera (of genetically diverse tissue) of somatostatin and beta-galactosidase, separated by a methionine residue. Cyanogen bromide was used to cleave the protein at the methionine residue and recover the somatostatin.

There are other ways to produce eukaryotic (non-bacterial) gene products in bacteria. For example, phage T7 contains promoters that generate a large amount of protein during a late stage of infection. First, a gene is inserted next to a T7 promoter. The promoter interacts with T7 RNA polymerase, which is synthesized in the presence of lactose. Therefore, the addition of lactose to bacteria carrying the recombinant T7 will produce large quantities of the gene product. Insulin, somatostatin, and many pharmaceutical drugs are now produced with engineered bacteria and fungi.

Genetic engineering is not restricted to placing eukaryotic genes in bacteria. Eukaryotic cells can be altered by inserting foreign DNA into their genomes. An organism derived from such a cell is referred to as transgenic. There are several ways to produce transgenic organisms. For example, the gene of interest (the transgene) can be paired with a eukaryotic promoter in a vector and injected into the nucleus of the organism's gamete. The promoter will cause the gene to be expressed whenever the promoter's natural gene is expressed. This may be useful in determining the expression pattern of the natural gene if the product is not as easily detectable as that of the transgene. Or, the transgene's product alone may be valuable. For example, crops can be endowed with genes for pesticides or metabolizing nitrogen. Unfortunately, the ecological consequences of introducing transgenic organisms into nature are unknown.

Gene Therapy and Screening

Gene therapy is the insertion of a normal gene into a chromosome carrying a defective copy of the gene. The first case of gene therapy in a mammal was the cure of a growth hormone deficiency in mice. A gene was inserted into the ova of mice carrying two defective promoters for the gene. The gene was paired with a promoter-regulator for the metallothionein gene, which activates in the presence of heavy metals. Offspring carrying the transgene showed higher growth rates in the presence of heavy metals than did those without the transgene, indicating that a functional metal-lothionein promoter had taken control of the growth hormone gene.

Human gene therapy can be either germinal or somatic. Germinal gene therapy introduces transgenes into both somatic cells and germ line cells of the early embryo. This therapy has been performed on mice but not humans. Somatic gene therapy inserts transgenes only into affected tissues using a viral vector. This form of therapy has proven successful in treating severe combined immunodeficiency disease and atherosclerosis. Because most transgene vectors insert randomly throughout the genome, one potential side effect of gene therapy is a mutation caused by insertion into a healthy gene.

Although gene therapy for humans remains highly experimental, genetic screening for diseases is already being applied. Embryonic cells are collected from the amniotic fluid or the placenta. Some genetic abnormalities can be detected by their effects on restriction sites. A probe constructed from the cDNA of the gene of interest is used to identify fragments of DNA carrying the gene. These fragments are digested by a restriction enzyme whose target site contains the deleterious mutation. Mutant genes will not be cleaved by the restriction enzyme, whereas normal genes will. This can be detected as two different banding patterns on an electrophoretic gel. A related technique takes advantage of the often tight linkage between mutant genes and restriction fragment length polymorphisms (RFLP), which can serve as indicators for the presence of mutant genes.

PCR can also be used to amplify DNA for sequencing or another form of testing.

see also Genes; Genetically Engineered Foods; Genetics.

Brian R. West