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. Growth of Microorganisms . Bacteria: Prokaryotic Unicellular Organisms . Bacterial Genes and Genome . Bacterial Genetics . Plasmids as Vectors for Amplifying Foreign DNA . Bacteriophages as Vectors for Amplifying Foreign DNA . Yeast: Eukaryotic Unicellular Organisms . Yeast Genes and Genome . Yeast Genetics
Growth of Microorganisms
A variety of microorganisms are used by molecular biologists to study gene structure and function. Some scientists study microorganisms because they are pathogenic to plants, humans or other animals, and through learning more about these pathogenic organisms, eective drugs and strategies for infection control can be developed. Microorganisms, particularly bacteria and yeasts, are also used by many scientists as a tool for molecular biology research. Bacteria cultures grow very quickly, and most strains used in molecular research divide in less than 45 min. Thus, a single bacterial cell can, in 16 h, produce sucient numbers of cells for isolating deoxyribonucleic acid (DNA) and many proteins. A yeast cell takes approximately 1.5 h to divide, and thus also requires fairly short culture times. By contrast, a mammalian cell requires 18 h to complete cell division, and several days of growth are needed to produce comparable cell numbers. Microorganisms are grown in the laboratory on solid media in Petri dishes or in liquid media in asks. Media contain essential components needed for cell growth, including a carbon source, a nitrogen source, and essential vitamins and cofactors. Additionally, media often contain antibiotics or dened components that allow for selective growth of cells, especially cells containing recombinant DNA. An essential skill for culturing microorganisms is aseptic technique. Media prepared in the laboratory must be kept sterile, and cultures must be free of contamination by other microorganisms present in the laboratory.
. Tissue Culture Cells . Shuttle Vectors: Plasmids that can Replicate in Two Different Hosts . Summary
division time, makes them ideal research organisms. By far the most frequently used bacterial species in molecular biology is Escherichia coli. E. coli is a Gram-negative bacteria found in the intestines of many mammals and can often be pathogenic. However, the commonly used laboratory strains do not carry toxins and therefore are not considered pathogenic. E. coli is regularly used for cloning genes and growing plasmid DNA (discussed below) from many dierent organisms. Since the genetic code is conserved among living organisms, DNA from any source can be replicated in E. coli. Not all bacteria are, however, easy to culture. For example, the bacterium that causes tuberculosis grows very slowly in the laboratory, and thus is dicult to culture. Other bacteria are naturally found in extreme or unique environments and thus require specialized conditions for culture in the laboratory. Some examples include bacteria that grow in deep oceanic thermal vents, in anaerobic environments, or in acid lakes. Although these bacteria are not routinely cultured in the laboratory, they contain enzymes that are essential to molecular biology research. Probably the most widely used are the DNA polymerases isolated from thermophilic (heat-loving) bacteria, used in a technique called polymerase chain reaction (PCR). Molecular biologists also use restriction enzymes to clone and analyse DNA. Restriction enzymes, isolated from many dierent species of bacteria, act as a natural host defence mechanism to prevent bacteriophage infection. These enzymes cut DNA molecules at specic sequences and many are routinely used in research and forensic DNA analysis.
ENCYCLOPEDIA OF LIFE SCIENCES 2001, John Wiley & Sons, Ltd. www.els.net
cannot bind the promoter and transcribe the genes. The presence of lactose in the cell causes the removal of the repressor protein, and the genes are then expressed. The study of transcriptional regulation in E. coli has led to major advances in understanding how transcription is regulated in all organisms.
Bacterial genome
Most bacterial genomes are between 1 million and 10 million base pairs in size. Although the function and regulation of some bacterial genes have been studied extensively, the function of many bacterial genes remains unknown. Recent sequencing initiatives have produced completed genomic sequences of several bacterial species, which are available to scientists on the Internet. The sequences of these dierent species can then be compared to each other, yielding valuable clues about gene function, mechanisms of pathogenicity and evolutionary relationships.
Bacterial Genetics
As mentioned above, bacterial cells reproduce asexually, always producing an exact clone. For evolutionary survival, bacteria also have several mechanisms by which they trade or share their DNA with other individual bacterial cells, resulting in variability in genomic content. Genes that are critical for survival in specic situations, such as genes for antibiotic resistance, are exchanged between individuals, including cross-species exchange. There are three main mechanisms for exchanging DNA between individual bacterial cells: . Transformation is the process by which bacteria pick up DNA from their immediate surroundings. In the natural environment, the source can be DNA fragments released from dead bacterial cells. In the laboratory, this is the most common method used in molecular biology to clone and replicate genes in bacteria (see below). . Transduction is the exchange of genetic information mediated by bacteriophages (discussed below). Bacteriophages are also commonly used tools for cloning and manipulating DNA. . Conjugation is the process by which bacteria exchange DNA through specialized protein structures called sex pili. Each of these mechanisms of DNA transfer is employed in a variety of molecular biology techniques to manipulate DNA. Some applications of transformation and phage infection are discussed below.
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Foreign DNA Multiple cloning site Vector Origin of replication Selectable marker (ampicillin resistance)
Ligation
(a)
(b)
Figure 2 Ligating, transforming and selecting plasmid DNA. (a) Linearized vector is ligated to a fragment of foreign DNA, forming a circular plasmid. When this plasmid is transformed into bacteria, the ampicillin resistance gene is expressed, producing an enzyme that degrades ampicillin. (b) When the bacteria are plated on to media containing ampicillin, cells containing the plasmid (and thus ampicillin-degrading enzyme) grow and form colonies (large, dark circles). Many of the cells do not contain the plasmid and fail to divide (represented as small, faint circles, although they would not be at all visible).
Most standard bacterial vectors have three essential components: 1. A multiple cloning site containing several unique restriction enzyme sites, allowing foreign DNA to be easily inserted into the plasmid. 2. A selectable marker to select the bacteria which received the vector DNA during a bacterial transformation. The selectable marker is often an antibiotic resistance gene such as b-lactamase, an enzyme that degrades ampicillins. 3. An origin of replication so that the bacterial DNA polymerase will replicate the plasmid, including the inserted foreign DNA. Origins of replication vary so that some vectors will be present in only a few copies, while other vectors may have many copies within an individual cell. Circular vector DNA can be cut, or linearized, at one or two of the restriction sites in the multiple cloning site. Foreign DNA is then combined with the linearized vector and the enzyme ligase joins the ends together, forming a circular plasmid containing foreign DNA (Figure 2).
A plasmid can be put into a bacterial cell by transformation, as outlined in Figure 2. In the laboratory this is easily accomplished by placing freshly grown cells into calcium chloride solution, adding the plasmid DNA, and allowing the DNA to adsorb to the bacterial membrane. Following a brief heat shock, some of the bacterial cells will take up the DNA. The cells are then plated and allowed to grow overnight to form bacterial colonies. Electroporation is another common transformation process, in which a quick electrical pulse is delivered to cells. During the temporary disruption of the cellular membrane, DNA enters the cell. The transformation process is inecient, so that only a small fraction of the bacterial cells actually take up the plasmid DNA. The selectable marker on the plasmid allows for selection of these cells by plating the cells on media containing antibiotic. For example, after transformation of vector DNA carrying the b-lactamase gene, the bacterial cells are plated on to media containing ampicillin. Bacterial cells that have obtained the plasmid begin producing the enzyme that degrades ampicillin and are able to begin cell division, forming colonies after about 16 h. The ampicillin inhibits growth of all bacterial cells lacking the plasmid. A single colony, or clone, containing the plasmid can then be transferred into a large volume of liquid media, and allowed to grow overnight. This culture will yield large quantities of the puried plasmid for further study and analysis.
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Figure 3 Bacteriophage life cycle. The bacteriophage, a capsule of proteins (called a phage particle) surrounding the phage DNA, anchors on to the surface of the bacterial cell (1) and injects its DNA into the cell (2). Once inside the bacterial cell, the bacteriophage DNA is transcribed into RNA (3), and also replicated to produce many copies of the phage DNA (4). The RNA is translated by the bacterial ribosomes (5), so that numerous phage particles, containing phage DNA, can be assembled (6). The phages then signal lysis of the bacterial cells, releasing a multitude of new phages (7), which can then infect neighbouring bacteria.
sequencing. Some phages, most notably M13, package only one DNA strand into the phage particle, and thus are commonly used to isolate single-stranded DNA. Phagemids are common vectors that contain the required sequences both for replicating in bacteria and for packaging DNA into phage particles. When transformed into bacterial cells, the bacterial replicating sequence allows for selection of clones. When a clone is infected with an M13 helper phage, the M13 phage sequences direct replication and packaging of the DNA into phage particles, allowing isolation of single-stranded DNA.
stream regulatory sequences and are targets of nuclear proteins (transcriptional regulators) that regulate the activity of RNA polymerase at the promoter. The function of these sequences is often studied by cloning the promoter (including the regulatory sequences) next to a reporter gene, an enzyme for which activity can be easily assayed. The cloned promoter is mutated to determine what sequences are important for activation and suppression of gene expression. Much of our knowledge of eukaryote gene regulation is a result of such yeast studies.
Yeast genome
In 1996, S. cerevisiae earned the distinction of being the rst eukaryote to have its genome entirely sequenced, a total of 12 million bases. The sequence and much supporting data are available on the Internet (see Further Reading), providing an extremely valuable resource to yeast geneticists and to genetic studies. The sequence data has been analysed to determine where putative gene coding regions and repeated regions of DNA are located. A large number of probable gene sequences are of unknown function. A major component of the yeast genome project is developing strategies to determine the cellular function of these genes.
Yeast Genetics
Yeasts generally exist as haploid organisms of two dierent mating types, termed a and a in S. cerevisiae. Two strains of opposite mating type can be cultured together to form a diploid cell. The diploid cell normally undergoes rapid meiosis and sporulation, producing four haploid spores. During this process, genetic information is randomly shued so that each spore produced contains some genetic information from each parent. Yeast strains commonly used in research have been altered so that both haploids and diploids can be cultured for genetic analysis.
Yeast plasmids
Yeast plasmids, like bacterial plasmids, also have a multiple cloning site, a selectable marker, and often, but not always, an origin of replication. The selectable marker may be a drug eective against yeast cells, but is most commonly an auxotrophic marker. Yeast does not have any essential amino acids and can synthesize all the amino acids from carbon and nitrogen supplied in growth medium. Mutations in enzymes needed to synthesize certain amino acids, called auxotrophic markers, are important selection tools for yeast geneticists. For example, a yeast strain carrying a mutation in a gene for synthesizing histidine (his3) cannot grow on media lacking histidine. However, if a plasmid containing the missing
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Yeast promoters
The promoters of yeast and other eukaryotes are more complex than prokaryotic promoters. Eukaryotic promoters often contain a TATA box sequence near the site where transcription initiates, but must also have other, more distant, sequences to direct RNA polymerase to begin transcription at the proper location and to control how often transcription initiates. These are called up-
gene (HIS3) is transformed into the yeast cell, the individual cells that obtain the plasmid can now grow and form a colony on media lacking histidine. Several dierent common auxotrophic markers exist and thus a given yeast strain can harbour several plasmids at once. Since the auxotrophic marker is not essential to cell growth in rich medium, these plasmids can also easily be removed from the cell, in a process called plasmid shuing. Using various auxotrophic markers, plasmids have been constructed as tools for studying the function of yeast genes and cellular processes. As with E. coli, plasmids can be easily transformed into yeast. Yeast plasmids can exist as extrachromosomal DNA, replicating as an independent unit, if they contain an origin of replication. Yeast molecular biologists also use integrating plasmids or simply transform a fragment of DNA containing a selectable marker. The DNA being transformed will insert into the chromosome that has DNA sequences matching those on the transformed DNA, in a process called homologous recombination. This characteristic of yeast has proven to be a very powerful tool, as researchers can easily replace a wild-type yeast gene with any mutation, or remove it completely, to see the eects on the cell. Yeast plasmids also allow for selection of important mutations in studying gene function. Many mutations are introduced into a cloned gene, which is then transformed into yeast, and the resulting colonies selected for a particular phenotype. The plasmid from the selected colonies can be isolated and transformed into E. coli for DNA sequencing to determine which mutations aect gene function. The ability to introduce and select for specic mutations in yeast genes and to move plasmids easily in and out of yeast for genetic studies has resulted in huge advances in our understanding of eukaryotic cellular processes.
expressed can be cloned next to an inducible yeast promoter, like the process used for expressing proteins in bacteria. Some advantages of using yeast cells for protein production are that the expressed protein will contain carbohydrate modications specic to eukaryotic cells, is more likely to be folded correctly, and will be free of potentially toxic bacterial cell components.
cloned DNA to study the eects of gene expression on cellular processes. Tissue culture is a routine procedure, and a central part of biomedical research, biotechnology and pharmaceutical production. Many drugs, vaccines, monoclonal antibodies and other substances are produced from cell cultures.
Summary
Researchers studying a particular aspect of molecular biology in any organism use the bacterium E. coli for cloning, replicating and manipulating DNA. Yeasts, namely S. cerevisiae and Schizosaccharomyces pombe, are also commonly used for selecting specic genetic mutants useful in studying eukaryotic cellular process. The knowledge gained from studying cloned genes in yeast leads to a greater understanding of cellular processes in more complex eukaryotic organisms. Specialized yeast and bacterial expression vectors are used in the biotechnology industry to produce large quantities of puried proteins. Shuttle vectors allow cloned DNA to be easily replicated in bacteria and then transformed into another host cell.
Further Reading
Atlas RM (1997) Methods for studying microorgansims, pp. 6376; Genetic mutation, recombination and mapping, pp. 319356; Industrial microbiology and biotechnology, pp. 820-843. In: Principles of Microbiology. Dubuque, IA: WC Brown. Glazier AN and Nikaido H (1995) Microbial Biotechnology. New York: Freeman. Micklos DA and Freyer GA (1990) DNA Science. Cold Spring Harbor: Cold Spring Harbor Laboratory Press and Carolina Biological Supply Company. Primrose SB (1991) Molecular Biotechnology. Cambridge, MA: Blackwell Scientic. Stanford University Saccharomyces Genome Database. [http://genomewww.stanford.edu/Saccharomyces]. Watson JD, Hopkins NH, Roberts JW, Steitz JA and Weiner AM (1987) Yeasts as the E. coli of eukaryotic cells; Recombinant DNA at work. In: Molecular Biology of the Gene, chaps 1819, pp. 550618. Menlo Park, CA: Benjamin/Cummings.