of DNA polymerase along the strand. To prevent this, single-strand binding proteins bind to the DNA until the second strand is synthesized, preventing secondary structure formation.
Termination of DNA replication requires that the progress of the DNA replication fork be stopped. Termination involves the interaction between two components: a specific “termination site sequence” in the DNA, and a protein which binds to this sequence to physically stop DNA replication. In various bacterial species, this protein is named the DNA replication terminus site-binding protein, or Ter protein.
Because bacteria have circular chromosomes, termination of replication occurs when the two replication forks that started out from the origin meet each other on the opposite side of the chromosome.
5.6.6 Plasmids
Organisms carry DNA in forms other than their primary chromosomal DNA. A plasmid is a small DNA molecule that is separate and can replicate independently of the chromosomal DNA in a cell, although it is considered to be part of the total genome of an organism (Figure 5.15). Plasmids are most commonly found as small circular, double-stranded DNA molecules in bacteria, archaea, and eukaryotic organisms. They carry genes that can benefit the survival of the organism in the natural environment. Examples include genes for antibiotic resistance (which makes them of central significance in the transfer of antibiotic resistance between bacteria in hospitals) and resistance to heavy metals (which allows organisms to live in diverse environments with heavy metals, from volcanic hot springs to human-polluted industrial sites). Plasmids can also provide bacteria with the ability to fix nitrogen gas from the atmosphere into useful nitrogen compounds or to degrade certain organic compounds that provide an advantage when nutrients are scarce. Plasmids vary in size from about a thousand to over a million base pairs, and the number of identical plasmids in a single cell can range from one to several thousand depending upon the environment and the species.
Figure 5.15 Plasmids are small circular pieces of DNA. They can be introduced into microorganisms by genetic engineers to produce industrially important products. The plasmid shown here has an origin of replication, which is the sequence that allows it to be copied. It contains a “coding sequence,” which could be a DNA sequence producing an important drug. The “promoter” is a DNA sequence that allows the coding region to be read. The transcription termination sequence tells the genetic machinery when to stop transcribing. It also contains a gene for antibiotic resistance which allows for bacteria successfully carrying the plasmid to be selected by researchers if they want to make sure that only the drug-producing bacteria are growing. The other sites marked on this diagram (e.g. SpeI, kpnI) are locations where specific enzymes (restriction enzymes) cut the DNA and allow the researcher to insert or remove bits of DNA.
Artificial plasmids are widely used in molecular cloning, where they serve as “carriers” of foreign genes that the researcher might wish to incorporate and have read inside a particular organism. For this reason, they are one of the most important tools in genetic engineering (Figure 5.15).
Plasmids are sometimes also called replicons, capable of replicating autonomously within a suitable host. However, plasmids, like viruses, are not considered to be a form of life. You might like to consider whether you agree with this view, and why, in the light of discussions in Chapter 2.
Discussion Point: What Is the Minimum Size of a Cell?
The minimum size of a cell is an important measure, since it defines the size below which we would question whether a putative fossil or living cell was a self-replicating living organism. For a cell to grow and reproduce, it must have some minimal machinery to produce proteins needed for division. It must have other molecules and proteins to gather energy and carry out basic functions such as replicating genetic information. Consider the following statement from a United States National Research Council report on the “Size Limits of Very Small Microorganisms”: “Free-living organisms require a minimum of 250–450 proteins along with the genes and ribosomes necessary for their synthesis. A sphere capable of holding this minimal molecular complement would be 250–300 nm in diameter, including its bounding membrane. Given the uncertainties inherent in this estimate, the panel agreed that 250 ± 50 nm constitutes a reasonable lower size limit for life as we know it.” Discuss this statement and whether you agree with the assessment of the minimum number of proteins required for life. How would the minimum size of a cell be different if it required 10 times this number of proteins or 50 times this number?
United States National Research Council (1999). Size Limits of Very Small Microorganisms: Proceedings of a Workshop, 1999. Washington, DC: National Academies Press (available online: http://www.nap.edu/books/0309066344/html).
5.6.7 eDNA
One of the more enigmatic occurrences of DNA is extracellular deoxyribonucleic acid (eDNA), which is found outside the cell. Once thought to be DNA that had simply been released from dead cells, it is now understood to be involved in a variety of processes, including the formation of biofilms in bacteria. It may also provide a rich source of genetic information that can be sequestered by other organisms in the process of transformation, whereby naked DNA in the environment is taken up and incorporated into the genome of organisms. This process is discussed in more detail in Chapter 8.
5.7 Eukaryotic Cells
Eukaryotic cells have a number of crucial differences from prokaryotic cells (Figure 5.16), the most notable is the presence of a cell nucleus. In the prokaryotic cell, the DNA is free floating in the fluid, or cytoplasm, of the cell and is sometimes referred to as the nucleoid. It is not surrounded by a nuclear membrane. In contrast, in eukaryotic cells, the DNA is found in the nucleus. Transcription occurs in the nucleus before the mRNA is moved into the cytoplasm for processing. The nucleus is constructed with a nuclear membrane containing pores allowing for the movement of material, such as the transcribed mRNA, in and out. Eukaryotic DNA also has important differences to prokaryotic DNA. The DNA contains sequences called introns. These are removed once the mRNA has been synthesized in the nucleus to leave only the exons (the parts of the mature mRNA that will be used to translate the genes). Eukaryotic cells contain histones. These are proteins around which the chromosomal DNA is packaged. Without them, the DNA would be long and unwieldy. For example, a typical diploid human cell contains about 1.8 m of DNA. Wound around the histones, the DNA can be effectively packaged in the nucleus.
Figure 5.16 A typical plant eukaryotic cell with some of its components. The cell shown is about 10 microns across. A typical size of a prokaryotic cell is shown for comparison.
A variety of other adaptations are found in specific eukaryotic cells (Figure 4.16). For example, a cell wall made of the polysaccharide, cellulose, which is constructed from repeating glucose molecules (Chapter 4), is found in plants. In fungi, the cell wall contains chitin, a polysaccharide made from N-acetylglucosamine, a derivative of glucose. Keratins are another type of tough filamentous protein. These are excreted in mammalian skin cells and account for the tough exterior of skin. When excreted outside the cell, keratins make hair and nails.
Many eukaryotic cells have a cytoskeleton, made up of a variety of structures including tubes and filaments of the proteins tubulin (microtubules) and actin (microfilaments). These molecules provide a rigid structure