Microbial Genetics

All of a cell's hereditary information is encoded by the genome. In prokaryotes, the genome consists of the bacterial chromosome(s) and plasmids; in eukaryotes, the genome is encoded by the chromosomes. The genome contains the genes that direct the formation of all the proteins the cell needs to survive. Genes are stretches of deoxyribonucleic acid (DNA) that code for polypeptides and ribonucleic acid (RNA). DNA is a double-stranded molecule (think of it as a twisted ladder) constructed from repeating units of a phosphate backbone, a sugar (deoxyribose), and one of four possible nitrogenous bases - adenine, thymine, cytosine, or guanine. Repeating units of these structures are called nucleotides. DNA strands are held together by hydrogen bonds between complementary nucleotides: adenine to thymine (A-T) and cytosine to guanine (C-G). In order for the genes to be expressed as proteins, the DNA is first transcribed into ribonucleic acid (RNA) then the RNA is translated into a protein. The genome constitutes the genotype, or potential genetic properties, of an organism, while the actual expressed proteins determine an organism's phenotype. Before bacteria can divide into two daughter cells, the DNA must be replicated. Replication creates two identical daughter chromosomes. These identical chromosomes are then segregated to different sides of the bacterial cell so that each daughter cell receives a chromosome. The daughter chromosomes are usually identical to the parent chromosome due to the DNA polymerase's proofreading accuracy. The polymerase scans the growing DNA chain to ensure that the correct nucleotide has been added in the proper position (i.e., if there is an A nucleotide on the separated DNA strand, a T must be added to the growing DNA strand. If there is a G nucleotide on the separated strand, a C must be added to the new DNA strand). If DNA polymerase detects an incorrect nucleotide, it cuts that nucleotide out and replaces it with the correct one. The efficiency of DNA polymerase is 1 incorrect nucleotide for every 10⁵ nucleotide added. However, the range for polymerase fidelity is 10^-4 to 10^-8 depending on specific polymerase types and organisms. Together, with other repair mechanisms, the overall error rate is approximately 10^-8 o 10^-9 (as an average of various data research).

Figure 1: Schematic of DNA replication. The top half of the figure represents the parent DNA that is unwound by the enzyme helicase. The exposed nucleotides are then “read” and the complementary nucleotides are added to the free strands by DNA polymerase in a 5' to 3' (based on the carbon number of the sugar molecule) direction. This results in two new daughter DNAs that are divided between the two halves of a dividing bacteria.

Figure 2: Nucleotides. Notice how the G and C bases bind together, and the A and T bases bind together.	Figure 3: DNA double helix structure.

Transcription

DNA is transcribed into mRNA with the help of RNA polymerase. RNA polymerase binds to special sequences in the DNA called promoter sequences. The promoter region is always 5�Œ to the gene encoded in the DNA. RNA polymerase incorporates ribose-containing nucleotides into a growing strand off of the DNA. Then, RNA polymerase falls off of the DNA template and stops transcription when it reaches a stop region encoded the DNA. mRNA is the intermediary between the genome and proteins, the molecules coded for by DNA that perform specific functions within the organism

Figure 4: Example of a point mutation in DNA. In this example, two DNA sequences are shown (only the 5' strand is shown for simplicity). The bottom sequence shows an A (arrow; green) where a G (arrow; black) should be (top sequence). This point mutation may result in a silent, nonsense, or missense mutation in the resulting protein.

Translation

mRNA takes the message out of the nucleus and into the cytoplasm, where it encounters a ribosome. With the coordination of a few additional proteins, new proteins can now be translated from the RNA template on the ribosome. Specialized RNA molecules called transfer RNA (or tRNA) read the genetic code by binding to the mRNA. The code is read in groups of three nucleotides that code and direct the incorporation of the necessary amino acids to form proteins. Calculating the number of possible combinations that could arise from four nucleotides that can be grouped by three yields 64 codons. However, there are only 20 amino acids that constitute proteins. Therefore, some amino acids are encoded by multiple codons; this is called degeneracy or redundancy of the genetic code. Additionally, three codons do not code for any amino acid. These codons are called stop codons (non-sense codons) and signal the ribosome to fall off of the RNA and release the new protein.

Mutations

Mutations, or changes in the nucleotide sequence of DNA, sometimes occur due to proofreading errors by DNA polymerase or environmental mutagens (DNA interacting chemicals and various forms of radiation: xrays, gamma rays and ultraviolet). If the mutation alters a resulting protein's function, it is called a missense mutation. Mutations that do not alter protein function are called silent mutations. Mutations that create a stop codon in the middle of an RNA, thereby prematurely ending translation and resulting in a shortened (and likely non-functional) protein are called nonsense mutations.

Mutations are commonly classified as point mutations. A point mutation occurs when a nucleotide is replaced with an incorrect nucleotide (for example: A for G, G for C, etc.). Point mutations are also called base substitutions. Frameshift mutations occur when one or more nucleotide pairs are inserted or deleted from the DNA. This can cause the genetic code to be misread during translation and result in an dysfunctional or nonfunctional protein. Mutations that occur in bacterial genomes are particularly significant because they can lead to the development of antibiotic-resistant bacteria or bacteria with increased ability to cause disease. Fortunately for humans, cells have a system called nucleotide excision repair (NER) that can repair DNA mutations and help prevent bacteria from becoming resistant. During this process, DNA is scanned for certain mutations such as thymine dimers, caused by UV radiation. If damaged DNA is discovered, it is cut out (excised) by repair enzymes. Then, DNA polymerase can then add the correct nucleotide(s) using the complementary strand as a guide.

The Ames Test

The Ames test uses bacteria to determine whether a chemical has mutagenic properties. Most substances that cause mutations in bacterial DNA can also cause mutations in humans. The Ames test uses a strain of Salmonella that lacks the ability to produce the amino acid histidine (his^-), which is required for survival. These bacteria are called histidine auxotrophs, since histidine must be supplied to them in order for them to survive. The histidine auxotrophs are grown on minimal agar plates and the test substance is applied to them. The auxotrophs will only be able to grow on the agar if they revert to being able to produce histidine (these are called revertants). Reversion only happens if the auxotroph DNA is mutated by the test substance. The Ames test greatly facilitated mutagen identification as the test was previously performed in animals, which is time consuming and costly.

Recombinant DNA

Recombination is the mixing of genes between two disparate DNA molecules. For instance, a gene from a human can be integrated into bacterial DNA and that newly formed DNA can be inserted into bacteria and used to make the human protein. Two discoveries greatly facilitated recombinant DNA (or rDNA) technology: bacterial plasmids and restriction enzymes. Plasmids are small (2,000-10,000 base pairs) extrachromosomal, circular DNAs that replicate independently from the bacterial chromosome. Plasmids can be easily transferred from one bacteria to another and they can be manipulated to contain a foreign gene. Restriction enzymes are DNA cutting enzymes derived from certain bacteria that recognize and cut (or digest) one specific DNA sequence. Each restriction enzyme has a unique recognition sequence and cuts any DNA containing that sequence the same way every time. Restriction enzymes are used to digest DNA and plasmids into specific pieces that can recombine. They can cut DNA symmetrically on both strands, resulting in “blunt” ends or they can cut DNA asymmetrically on both strands, resulting in “sticky” ends that have complementary overhanging nucleotides.

Figure 5: Sticky ends are generated by restriction enzymes. In this example, the DNA sequence GAATTC is recognized and cut asymmetrically by the restriction enzyme EcoRI. Notice in the figure that the restriction enzyme recognition sequence is the same on both strands, but they run in opposite directions. Most restriction enzyme sites have this arrangement and are called palindromic. Overhanging DNA end fragments that are not base paired are provided in this example. If two separate DNA sequences have the EcoRI recognition site, both will have un-base paired, overhanging DNA ends that can stick together by hydrogen bonding. Combining these two DNA fragments with DNA ligase (an enzyme that links DNA strands together with covalent bonds between the phosphate backbone) creates a new DNA sequence.

Figure 5 demonstrates the steps used to clone a “foreign” gene into a plasmid by using a restriction enzyme. After the foreign gene is inserted into the plasmid DNA, the plasmid must be introduced into bacteria in order for many copies of the foreign gene to be produced. Bacteria are transformed with the plasmid DNA by either heat shock or by applying an electric field to the bacteria. Both processes cause holes to temporarily form in the bacteria cell membrane and allow the bacteria to take up the DNA. The bacteria are then grown in culture and many identical copies of the foreign gene are produced. This process is called cloning the gene and plasmids used are often called cloning plasmids (also called vectors). The plasmid DNA usually contains a selection marker, which is a gene that confers resistance to an antibiotic such as ampicillin or kanamycin. The transformed bacteria are grown in media containing the antibiotic to select for the growth of only those bacteria that have taken up the plasmid containing the foreign DNA. The DNA cloned can be studied itself or the protein product can be harvested and used in many different ways. Cloning foreign genes in bacterial plasmids has wide ranging application and is used in numerous fields.

Polymerase Chain Reaction (PCR)

Another method that is widely used to make many copies of a DNA sequence is the Polymerase Chain Reaction (PCR). PCR uses extremely small amounts of starting DNA to generate huge amounts for analysis or cloning purposes. Under ideal conditions, PCR follows an exponential growth pattern, similar to bacterial growth. Therefore, the amount of DNA doubles with every reaction cycle. A PCR setup includes the template DNA that you want to amplify, primers that are complementary to the 5' ends of each DNA strand, free nucleotides, and a thermostable DNA polymerase (derived from extremophile bacteria living in thermal springs). The mixture is heated to just below boiling temperature to separate (denature) the DNA strands, then cooled to a temperature that facilitates hydrogen bonds to form between the primers and the newly separated DNA strands. Next, the temperature is raised slightly to allow DNA polymerase to use the primer- DNA hybrids as templates to synthesize two new DNA molecules. The mixture is then heated again to denature the strands and the process starts over. This is repeated for 30-40 cycles, producing millions of copies of the original DNA target. This procedure has revolutionized molecular biology, genomics, forensics, and diagnostic testing. The cause of many viral and bacterial infections can now be identified based on PCR screening of samples.

Medical Applications of Recombinant DNA

Recombinant DNA technology is used to produce a number of therapeutics. Diabetics rely on insulin to control their blood sugar. Insulin was originally obtained from pigs and cows, which is expensive. Additionally, animal insulin does not work as well as human insulin in human patients. DNA for the human insulin gene was cloned into plasmid vectors then the plasmids were inserted into bacteria (a process called transformation). The bacteria are grown in large cultures and they produce the insulin protein, which is purified and sold commercially. Other medicines that are produced by recombinant DNA technology include human growth hormone (to treat abnormal growth), clotting factor VIII (to treat hemophilia), epidermal growth factor (wound and burn treatment), taxol (anti-cancer agent), as well as many others

Vaccines targeting HIV, influenza, hepatitis, and other infectious viruses also rely on recombinant DNA technology. The vaccines are made by cloning a portion of the viral genome into a plasmid where the expression of the viral gene is under control of a human protein. The plasmids are transformed into bacteria and the bacteria are used to produce the DNA vaccine. The DNA vaccine is injected into a patient and the patient's cells then express the viral proteins, which are recognized as foreign by the patient's immune system and elicits an immune response. These types of vaccines are still in development but hold great promise as they can trigger a broader immune response than can traditional vaccines.

Gene therapy is being studied as a way to replace defective genes with normally functioning genes. Adenosine deaminase deficiency (ADA) is a rare genetic disorder that causes severe combined immunodeficiency in children. Patients with ADA deficiency suffer from opportunistic infections that can be lifethreatening. Gene therapy for ADA deficiency involves cloning the normal ADA gene into a vector and inserting it into white blood cells (part of the immune system) from the patient. These cells with the “corrected” gene are infused back to the patient and the ADA deficiency is relieved. Gene therapy is being explored for many other human diseases.

Industrial Applications

Recombinant DNA technology is used extensively in industry to increase process efficiency, reduce costs, reduce energy demand, and minimize environmental impact. Most detergents contain proteases that degrade proteins on clothes during washing. The proteases are recombinant DNA products and replace harsh chemicals in the washing process. Polyester is produced by a genetically modified bacteria that makes trimethylene glycol from sugar; the trimethylene glycol is then converted into polyester for fabric production. Modified bacteria are used to produce large quantities of enzymes that are then used in chemical production, reducing the negative environmental consequences of chemical synthesis.

Agricultural Applications

The bacteria Agrobacterium tumefaciens contains a plasmid called Ti (Tumor inducing) that integrates into the genome of a plant infected with the bacteria. The Ti plasmid has been used to engineer plants that 1) are resistant to the herbicide glyphosate and 2) express the Bt toxin (a bacterially derived chemical that kills insects that damage crops). These modifications increase yield and decrease costs associated with weed and pest irradication. Genes for drought and viral resistance have also been inserted into agriculturally important crops.

Various health conditions have also been addressed through genetic engineering of agricultural products. Recombinant DNA has been used to create tomatoes with increased levels of vitamins C and E in an effort to reduce the risk developing cancer and heart disease. Genetically modifi ed rice that has a much higher iron content than normal rice is being used to treat anemia world-wide. Transgenic animals, which carry a portion of human DNA in their genome, are used as “drug factories” to produce a number of therapeutic proteins in their milk. This reduces the cost and infection risk that is inherent when these proteins are isolated from human donors.