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Microorganisms: Applications in Molecular Biology

  1. Jill B Keeney

Published Online: 21 DEC 2007

DOI: 10.1002/9780470015902.a0000971.pub2

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Keeney, J. B. 2007. Microorganisms: Applications in Molecular Biology. eLS. .

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  1. Juniata College, Huntingdon, Pennsylvania, USA

Publication History

  1. Published Online: 21 DEC 2007

Growth of Microorganisms

  1. Top of page
  2. Growth of Microorganisms
  3. Bacteria: Prokaryotic Unicellular Organisms
  4. Bacterial Genes and Genome
  5. Bacterial Genetics
  6. Plasmids as Vectors for Amplifying Foreign DNA
  7. Bacteriophages as Vectors for Amplifying Foreign DNA
  8. Yeast: Eukaryotic Unicellular Organisms
  9. Yeast Genes and Genome
  10. Yeast Genetics
  11. Tissue Culture Cells
  12. Shuttle Vectors: Plasmids that can Replicate in Two Different Hosts
  13. Summary
  14. Further Reading
  15. Web Links

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, effective 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 sufficient numbers of cells for isolating deoxyribonucleic acid (DNA) and many proteins. A yeast cell takes ∼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. See also Yeasts

Microorganisms are grown in the laboratory on solid media in Petri dishes or in liquid media in flasks. 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 defined components that allow for selective growth of cells, especially cells containing recombinant DNA (Table 1). 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. See also Cell Culture: Basic Procedures, and Cell Culture Media

Table 1. Applications of media types
Media typePurpose
Solid mediaUsed to isolate colonies originating from a single cell (or clone)
Liquid mediaUsed to grow cells to logarithmic growth phase, for transformation or for isolating nucleic acid and protein
Rich mediaCells grow more quickly as compared to minimal media. No selection for added recombinant DNA
Minimal mediaCells tend to grow slower than in rich media. Allows selection for recombinant DNA containing metabolic enzymes that are mutated in the host cell
Antibiotic-containing mediaAllows selection for recombinant DNA containing an antibiotic resistance gene

Bacteria: Prokaryotic Unicellular Organisms

  1. Top of page
  2. Growth of Microorganisms
  3. Bacteria: Prokaryotic Unicellular Organisms
  4. Bacterial Genes and Genome
  5. Bacterial Genetics
  6. Plasmids as Vectors for Amplifying Foreign DNA
  7. Bacteriophages as Vectors for Amplifying Foreign DNA
  8. Yeast: Eukaryotic Unicellular Organisms
  9. Yeast Genes and Genome
  10. Yeast Genetics
  11. Tissue Culture Cells
  12. Shuttle Vectors: Plasmids that can Replicate in Two Different Hosts
  13. Summary
  14. Further Reading
  15. Web Links

Organisms are generally classified as prokaryotic (no nucleus or other organelles) or eukaryotic (containing organelles). Bacteria are unicellular prokaryotes: the organism is one cell. Some bacteria have very simple growth requirements. This, in combination with their rapid 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 later) from many different organisms. Since the genetic code is conserved among living organisms, DNA from any source can be replicated in E. coli. See also Escherichia coli as an Experimental Organism, and Gene Expression in Escherichia 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 difficult 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 which have been cloned and are now essential to molecular biology research. Probably the most widely used are the DNA polymerases isolated from thermophilic (heat-loving) bacteria, used in high-throughput sequencing and amplification of DNA by a technique called polymerase chain reaction (PCR). Molecular biologists also use restriction enzymes to clone and analyse DNA. Restriction enzymes, isolated from many different species of bacteria, act as a natural host defence mechanism to prevent bacteriophage infection. These enzymes cut DNA molecules at specific sequences and many are routinely used in research and forensic DNA analysis. See also Extreme Thermophiles, Polymerase Chain Reaction (PCR), and Restriction Enzymes

Bacterial Genes and Genome

  1. Top of page
  2. Growth of Microorganisms
  3. Bacteria: Prokaryotic Unicellular Organisms
  4. Bacterial Genes and Genome
  5. Bacterial Genetics
  6. Plasmids as Vectors for Amplifying Foreign DNA
  7. Bacteriophages as Vectors for Amplifying Foreign DNA
  8. Yeast: Eukaryotic Unicellular Organisms
  9. Yeast Genes and Genome
  10. Yeast Genetics
  11. Tissue Culture Cells
  12. Shuttle Vectors: Plasmids that can Replicate in Two Different Hosts
  13. Summary
  14. Further Reading
  15. Web Links

The bacterial genome consists of one circular chromosome, attached to the bacterial cell wall. This chromosome contains all the genes the bacterium needs to replicate DNA and make proteins needed for cell growth. Each time a bacterium divides, the entire chromosome is replicated once and a copy goes with the new cell. This is regarded as asexual reproduction, and the new cell is an exact copy, or clone, of the original cell. See also Bacterial Cell Division, and Bacterial Cells

Bacterial genes and promoters

Bacterial cells regulate when genes are expressed at the level of transcription and translation. This regulation assures that the cell does not waste energy expressing a protein that is not needed. One of the best understood examples of gene regulation is the control of the lac operon of E. coli, which contains the genes needed to break down the sugar lactose (Figure 1). These genes are regulated together so that they are only expressed when the bacterium has lactose available to use as an energy source. Expression of the operon is regulated by DNA sequences located near the site on the DNA where transcription begins. Ribonucleic acid (RNA) polymerase is able to specifically bind to a specific sequence, called the promoter, and initiate transcription. Operons also contain operator sequences bound by proteins which help RNA polymerase bind better (activators) or which prevent binding of RNA polymerase (repressors). For example, the operator of the lac operon normally has a repressor bound to it, so RNA polymerase 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. See also Bacterial Transcription Regulation, and Escherichia coli Lactose Operon

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Figure 1. The E. coli lac operon. The lac operon contains three genes (lacZYA) whose products are needed for E. coli to utilize lactose as an energy source. These genes are regulated by operator (O) and promoter (P) sequences immediately adjacent to the genes. (a) When no lactose is present in the bacterial cell environment, repressor protein bound at the operator (O) prevents RNA polymerase from binding at the promoter (P). (b) When lactose is present, inducer (a lactose byproduct, represented as •) is produced, which can bind to the repressor. The repressor then no longer binds the operator, and the promoter is accessible to RNA polymerase.

Bacterial genome

Most bacterial genomes are between 1 million and 10 million base pairs (Mbp) in size. High throughput sequencing techniques allow these relatively small genomes to be sequenced in a very short time and have revolutionized the study of bacterial genomes. For example, scientists can now rapidly obtain the sequence of new pathogenic strains of bacteria and compare the entire genomic sequence to nonpathogenic strains to determine key genetic differences that may be involved in pathogenicity. In a new area of study, termed metagenomics, ecologists are now able to by-pass the very difficult task of isolating each bacterial species in an ecosystem and directly obtain the genetic sequences present.

These types of projects, combined with other government and private sequencing initiatives, have provided the complete sequence of hundreds of completed genomes with hundreds more in progress. Many of the genomic sequences are available publicly through on-line genome project sites, e.g. NCBI, the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/Genomes/). This vast amount of sequence information has greatly expanded the fields of genome biology (the study of the evolution and structure of genomes) and computational biology (the development of computer algorithms to efficiently compare vast amounts of sequence information.) These developments are rapidly changing the procedures and tools that scientists use to study bacterial genomes, and have greatly increased the demand for scientists trained in both molecular biology and computer science.

Bacterial Genetics

  1. Top of page
  2. Growth of Microorganisms
  3. Bacteria: Prokaryotic Unicellular Organisms
  4. Bacterial Genes and Genome
  5. Bacterial Genetics
  6. Plasmids as Vectors for Amplifying Foreign DNA
  7. Bacteriophages as Vectors for Amplifying Foreign DNA
  8. Yeast: Eukaryotic Unicellular Organisms
  9. Yeast Genes and Genome
  10. Yeast Genetics
  11. Tissue Culture Cells
  12. Shuttle Vectors: Plasmids that can Replicate in Two Different Hosts
  13. Summary
  14. Further Reading
  15. Web Links

As mentioned earlier, 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 specific 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 (see Bacterial Genetic Exchange for illustrations of all 3 mechanisms):

  • 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 later).

  • Transduction is the exchange of genetic information mediated by bacteriophages (discussed later). 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 later. See also Bacterial Reproduction and Growth

Plasmids as Vectors for Amplifying Foreign DNA

  1. Top of page
  2. Growth of Microorganisms
  3. Bacteria: Prokaryotic Unicellular Organisms
  4. Bacterial Genes and Genome
  5. Bacterial Genetics
  6. Plasmids as Vectors for Amplifying Foreign DNA
  7. Bacteriophages as Vectors for Amplifying Foreign DNA
  8. Yeast: Eukaryotic Unicellular Organisms
  9. Yeast Genes and Genome
  10. Yeast Genetics
  11. Tissue Culture Cells
  12. Shuttle Vectors: Plasmids that can Replicate in Two Different Hosts
  13. Summary
  14. Further Reading
  15. Web Links

In addition to the circular chromosome, bacteria may carry smaller extrachromosomal circular DNA, called plasmids, which are replicated in the cell but not necessarily carried along in cell division. Often these plasmids carry nonessential genes that allow bacteria to grow in special environmental conditions, such as genes to degrade unique nutrients in the environment (e.g. oil) or genes to break down toxins in the environment (e.g. antibiotics). Over the past few decades, bacterial geneticists have isolated and altered these plasmids, and now hundreds of different specialized plasmids exist. Plasmids are used by molecular biologists in all fields of research as vectors for cloning and amplifying DNA from many different organisms. The structure and composition of DNA is universal among all living organisms. Thus, DNA from any other ‘foreign’ organism, if inserted or cloned into a bacterial vector, can be amplified and then isolated from a bacterial cell. This process allows scientists to produce large quantities of a specific DNA sequence for experimental study. See also Antibiotic Resistance Plasmids in Bacteria, Bacterial Plasmids, and Bioremediation

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 β 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 an enzyme (ligase or topoisomerase) joins the ends together, forming a circular plasmid containing foreign DNA (Figure 2), and restriction enzymes.

thumbnail image

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).

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. See also Electroporation

The transformation process is inefficient, 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 β-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 purified plasmid for further study and analysis.

Bacterial vectors can efficiently replicate inserts of DNA of about 10 000 bp in length. While this is adequate for some genes and sequences, often scientists need to work with longer fragments of DNA. Specialized vectors called BACs, for bacterial artificial chromosomes, have been developed which can allow cloning of inserts 100–300 000 bp in length. The vectors are essential for cloning very large genes and transforming these genes into other cell types, such as insect, plant or mammalian cells. BACs are essential to the sequencing of large genomes, as they allow for sequencing of much larger continuous pieces of DNA, significantly reducing the time-consuming task of piecing together many small segments of DNA. See also Artificial Chromosomes

Expressing genes in bacteria

Sometimes further analysis of a gene requires expressing the protein product. Expressing a gene from another species in E. coli requires more specialized conditions than simply replicating the DNA. The promoters of genes from other organisms are not likely to be recognized by the bacterial RNA polymerase. Thus, the gene to be expressed must be cloned in such a way that the transcription start site is spaced properly with a bacterial promoter. The use of an inducible promoter, such as the promoter from the lac operon, allows for tight control of gene expression. The cloned gene is not expressed until the cells are cultured with inducer, a chemical substance resembling lactose. Transcription is then activated and the bacterial cell produces large quantities of the protein. If the cloned DNA is from a eukaryote, protein expression in E. coli may also require removal of introns from the gene prior to cloning, as bacteria lack the cellular machinery to splice RNA. This is accomplished by isolating messenger RNA (mRNA) from the eukaryotic cell and reverse transcribing it into DNA, thus creating complementary DNA (cDNA). The cDNA, if cloned in proper alignment with a bacterial promoter, can then be transcribed and translated, producing large quantities of the protein for purification. This process is used to isolate large quantities of proteins for use in biotechnology and medicine. See also Protein Production for Biotechnology

Bacteriophages as Vectors for Amplifying Foreign DNA

  1. Top of page
  2. Growth of Microorganisms
  3. Bacteria: Prokaryotic Unicellular Organisms
  4. Bacterial Genes and Genome
  5. Bacterial Genetics
  6. Plasmids as Vectors for Amplifying Foreign DNA
  7. Bacteriophages as Vectors for Amplifying Foreign DNA
  8. Yeast: Eukaryotic Unicellular Organisms
  9. Yeast Genes and Genome
  10. Yeast Genetics
  11. Tissue Culture Cells
  12. Shuttle Vectors: Plasmids that can Replicate in Two Different Hosts
  13. Summary
  14. Further Reading
  15. Web Links

Bacteriophages are viruses of the bacterial world. Their entire life cycle, shown in Figure 3, exists within the confines of a bacterial cell. During this replication process, bacterial DNA may be packaged into the phage particles with the phage DNA. This DNA is then ultimately transferred to another bacterial cell in a process called transduction. Foreign DNA can be inserted into the phage genome so that it is packaged into the phage particles. The bacteriophage genome is much larger than plasmids, and can be used as a tool to clone fragments of DNA up to 24 000 bp. When bacteria are infected with the recombinant phage, the foreign DNA is then amplified along with the phage DNA. As the phages replicate and lyse the bacterial cells, phage particles are released into the culture supernatant. These phage particles can then be isolated and the cloned DNA recovered in large quantities for use in other experiments. See also Bacteriophages

thumbnail image

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.

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. See also Bacteriophages in Industry

Yeast: Eukaryotic Unicellular Organisms

  1. Top of page
  2. Growth of Microorganisms
  3. Bacteria: Prokaryotic Unicellular Organisms
  4. Bacterial Genes and Genome
  5. Bacterial Genetics
  6. Plasmids as Vectors for Amplifying Foreign DNA
  7. Bacteriophages as Vectors for Amplifying Foreign DNA
  8. Yeast: Eukaryotic Unicellular Organisms
  9. Yeast Genes and Genome
  10. Yeast Genetics
  11. Tissue Culture Cells
  12. Shuttle Vectors: Plasmids that can Replicate in Two Different Hosts
  13. Summary
  14. Further Reading
  15. Web Links

Yeast, like bacteria, is a unicellular organism; however, yeast is characterized as eukaryotic because cellular functions are compartmentalized into distinct organelles, such as the nucleus, the endoplasmic reticulum and the mitochondria. As with bacteria, scientists study a number of different yeast species, many of them pathogenic. Some yeast species are easy to grow, replicate quickly and are easy to manipulate genetically. Yet, because yeasts are eukaryotic, they can also be used as a tool to study complex cellular functions such as the cell cycle, chromosome segregation, transcription, intracellular signalling and protein modification. Many processes that occur in larger eukaryotic cells, such as human cells, can be more readily studied in yeast. The most commonly used species are the budding yeast Saccharomyces cerevisiae (also known as brewer's or baker's yeast) and the fission yeast Schizosaccharomyces pombe. This discussion deals with S. cerevisiae, the most commonly used yeast in molecular biology research. See also Yeasts

Yeast Genes and Genome

  1. Top of page
  2. Growth of Microorganisms
  3. Bacteria: Prokaryotic Unicellular Organisms
  4. Bacterial Genes and Genome
  5. Bacterial Genetics
  6. Plasmids as Vectors for Amplifying Foreign DNA
  7. Bacteriophages as Vectors for Amplifying Foreign DNA
  8. Yeast: Eukaryotic Unicellular Organisms
  9. Yeast Genes and Genome
  10. Yeast Genetics
  11. Tissue Culture Cells
  12. Shuttle Vectors: Plasmids that can Replicate in Two Different Hosts
  13. Summary
  14. Further Reading
  15. Web Links

S. cerevisiae contains 16 chromosomes. During mitotic cell division each of these chromosomes must be replicated and then segregated. S. cerevisiae is called a budding yeast because, as the cell divides, the parent cell puts out a small bud. One set of the duplicated chromosomes is segregated to this bud which then ‘pinches’ off and becomes a separate daughter cell.

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 upstream 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. Like bacteria, yeast regulate when each gene is expressed in the cell, so specific yeast promoters can be used to regulate the expression of other genes for genetic studies. For example, a natural gene promoter can be replaced with the promoter of a gene that is turned on in the presence of copper; the presence or absence of copper in the media allows the researcher to control expression of that gene. In yeast and other eukaryotes, cellular DNA is organized by extensive wrapping of the DNA around proteins called histones, which collectively form a structure called chromatin. The study of the yeast genome (see later) has revealed an extensive network of enzymes that chemically modify histones located near promoter sequences to regulate gene expression. See also Transcription Activation in Eukaryotic Cells

Yeast genome

In 1996, S. cerevisiae earned the distinction of being the first eukaryote to have its genome entirely sequenced, a total of 12 million bases. Table 2 compares the genome properties for some common research organisms. The sequence data of the yeast genome has been analysed to determine putative gene coding regions. It contains ∼6000 putative genes, and a significant number of these probable gene sequences are of unknown function. A major component of the yeast genome project is developing strategies to determine the cellular function of each putative gene. The Saccharomyces Genome Database (www.yeastgenome.org) is a comprehensive, public database that lists each gene with extensive annotations to sequence data, protein data, cellular localization studies, known functions, published references, gene expression data (i.e. from DNA microarrays) and interactions with other genes. The Saccharomyces Genome Deletion Project (http://sequence-www.stanford.edu/group/yeast_deletion_project/deletions3.html) is an innovative resource created by scientists in the yeast community working together to construct a collection of yeast strains, each containing a different gene deletion. This collection is used by many scientists to study the phenotypes of strains harbouring gene deletions and to decipher genetic interactions between genes. Essential genes, which are lethal if deleted, can also be studied by deleting these genes in diploids and combining them with other gene deletions to recover viability.

Table 2. Comparison of genomes of several commonly studied organisms
OrganismApproximate size of genome (in Mbp or millions of base pairs)Number of chromosomesApproximate number of proteinsYear when first sequence completed
  1. Source: Information compiled from http://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj

Bacterium: E. coli5142001997
Yeast: S. cerevisiae121658001996
Fruitfly: Drosophila melanogaster180419 7002000
Mouse: Mus musculus30002027 2952002
Human: Homo sapiens30402328 7001999
Flowering plant: Arabidopsis thaliana119532 8002000

The comparative ease of working with yeast has also allowed scientists to use high-throughput techniques to map the physical interactions between the yeast proteins. Currently, a project is underway to construct a collection of strains containing each of the possible double gene deletion combinations. Using this information, yeast geneticists are collaborating with computational biologists to work on constructing a map of the network of protein interactions in the cell, with the eventual goal of a comprehensive understanding of cellular function.

Yeast Genetics

  1. Top of page
  2. Growth of Microorganisms
  3. Bacteria: Prokaryotic Unicellular Organisms
  4. Bacterial Genes and Genome
  5. Bacterial Genetics
  6. Plasmids as Vectors for Amplifying Foreign DNA
  7. Bacteriophages as Vectors for Amplifying Foreign DNA
  8. Yeast: Eukaryotic Unicellular Organisms
  9. Yeast Genes and Genome
  10. Yeast Genetics
  11. Tissue Culture Cells
  12. Shuttle Vectors: Plasmids that can Replicate in Two Different Hosts
  13. Summary
  14. Further Reading
  15. Web Links

Yeasts generally exist as haploid organisms of two different mating types, termed ‘a’ and ‘α’ 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 shuffled and sorted so that each spore produced contains 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. See also Yeast Mating Type

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 effective 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 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 different 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 shuffling.

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 create a gene deletion. 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 affect gene function. The ability to introduce and select for specific mutations in yeast genes and to move plasmids easily in and out of yeast for genetic studies has resulted in significant advances in our understanding of eukaryotic cellular processes. See also Yeast as a Model Genetic Organism

Cloning large pieces of DNA

Sometimes it is desired to have a very large piece of DNA cloned into a yeast cell. This can be accomplished by constructing a yeast artificial chromosome, or YAC. A YAC contains an auxotrophic marker and a centromeric and a telomeric sequence found on a yeast chromosome; the rest of the DNA can be from another organism. Whereas plasmids are generally not much bigger than 10 000 bp, YACs can be millions of base pairs, allowing very large pieces of DNA to be cloned into yeast. Since many yeast and human genes are functionally related, YACs can be used to study large human genes in yeast. See also Yeast Artificial Chromosomes

Expressing genes in yeast

Yeast plasmids are also used to express large quantities of a specific protein for research and the biotechnology industry. For example, the hepatitis B virus vaccine is commercially produced in yeast. A foreign gene to be 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 modifications specific to eukaryotic cells, is more likely to be folded correctly, and will be free of potentially toxic bacterial cell components. See also Saccharomyces cerevisiae: Applications

Tissue Culture Cells

  1. Top of page
  2. Growth of Microorganisms
  3. Bacteria: Prokaryotic Unicellular Organisms
  4. Bacterial Genes and Genome
  5. Bacterial Genetics
  6. Plasmids as Vectors for Amplifying Foreign DNA
  7. Bacteriophages as Vectors for Amplifying Foreign DNA
  8. Yeast: Eukaryotic Unicellular Organisms
  9. Yeast Genes and Genome
  10. Yeast Genetics
  11. Tissue Culture Cells
  12. Shuttle Vectors: Plasmids that can Replicate in Two Different Hosts
  13. Summary
  14. Further Reading
  15. Web Links

Tissue culture allows scientists to study the individual cell types of larger multicellular organisms (both plants and animals) by growing cells individually in a flask. As with microorganisms, the cells will divide if given the appropriate environment and nutrients. The growth requirements of cells of larger eukaryotes are not as simple as those of microorganisms, so special media and environments are required. For example, in growing mammalian cells, the environment that a cell normally experiences in the whole organism must be duplicated in the laboratory. In our bodies, our cells are kept at a very constant temperature, supplied a very precise amount of oxygen and bathed in blood or plasma that is kept at a very specific pH. Special incubators and media that mimic these conditions are required for growing mammalian cells. The media, in addition to carbon and nitrogen sources, must also contain other growth factors and serum components. These are generally supplied by bovine (cow) serum that is added to the media. In our bodies, our skin and immune system keep bacteria and yeast out of our blood and tissues. In tissue culture, the media and cells are in direct contact with the environment of the laboratory. Work with these cultures must be done in sterile cabinets, called laminar flow hoods. The persons culturing the cells must be sure their hands are scrubbed of all microorganisms before working with the cultures. Without these precautions, the faster replicating microorganisms will overrun the culture. See also Safety Considerations in the Tissue Culture Laboratory

Primary lines and transformed lines

Cells that are normally part of a multicellular organism are programmed to stop dividing at a certain cell density and to die at a specified time through the cellular process of apoptosis. Thus, when cells are moved from an organism into tissue culture, they normally will grow to a certain cell density and then die. In order to maintain cells in culture over long periods of time, cells must be immortalized or transformed. Tumour or cancer cells have already acquired this characteristic. If placed into culture and given a fresh supply of media every few days they can be grown indefinitely. Scientists often use chemicals or viruses to immortalize certain cell types in the laboratory so that they can be studied over long periods of time. As with microorganisms, cultured cells can be transformed with cloned DNA to study the effects of gene expression on cellular processes. See also Primary Cell Cultures and Immortal Cell Lines

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. See also Monoclonal Antibodies

Shuttle Vectors: Plasmids that can Replicate in Two Different Hosts

  1. Top of page
  2. Growth of Microorganisms
  3. Bacteria: Prokaryotic Unicellular Organisms
  4. Bacterial Genes and Genome
  5. Bacterial Genetics
  6. Plasmids as Vectors for Amplifying Foreign DNA
  7. Bacteriophages as Vectors for Amplifying Foreign DNA
  8. Yeast: Eukaryotic Unicellular Organisms
  9. Yeast Genes and Genome
  10. Yeast Genetics
  11. Tissue Culture Cells
  12. Shuttle Vectors: Plasmids that can Replicate in Two Different Hosts
  13. Summary
  14. Further Reading
  15. Web Links

Regardless of the organism being studied, plasmid DNA to be transformed into any cell type is primarily prepared in E. coli. These plasmids, called ‘shuttle vectors’, must have the ability to be selected and to replicate in two different organisms, E. coli and the recipient host cell. A shuttle vector has the basic components of an E. coli plasmid, but in addition must have a selectable marker appropriate to the host cell and, if applicable, an origin of replication recognized by the host cell. For example, a yeast shuttle vector has the major components of a bacterial vector (replication of origin, selectable marker and multiple cloning site), and also contains a yeast replication origin and a yeast selectable marker (such as HIS3, discussed earlier). Scientists working with a given organism must make themselves familiar with the appropriate selections and shuttle vectors available for that organism.

Summary

  1. Top of page
  2. Growth of Microorganisms
  3. Bacteria: Prokaryotic Unicellular Organisms
  4. Bacterial Genes and Genome
  5. Bacterial Genetics
  6. Plasmids as Vectors for Amplifying Foreign DNA
  7. Bacteriophages as Vectors for Amplifying Foreign DNA
  8. Yeast: Eukaryotic Unicellular Organisms
  9. Yeast Genes and Genome
  10. Yeast Genetics
  11. Tissue Culture Cells
  12. Shuttle Vectors: Plasmids that can Replicate in Two Different Hosts
  13. Summary
  14. Further Reading
  15. Web Links

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 S. pombe, are also commonly used for selecting specific 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 purified proteins. Shuttle vectors allow cloned DNA to be easily replicated in bacteria and then transformed into another host cell.

Glossary
Conjugation

The process by which bacteria exchange DNA through specialized protein structures.

Eukaryote

Organisms with organelles and a nucleus surrounding the genome.

Logarithmic growth phase

The growth phase of cells in liquid media when the cells are dividing at the most rapid rate.

Prokaryote

Organisms with no nucleus or other organelles.

Plasmid

Small, circular, extra-chromosomal DNA replicated by the cell but not necessarily carried along in cell division.

Transformation

The process by which cells pick up DNA from their surrounding environment. This can happen naturally, and is also a very common procedure in the laboratory.

Transduction

Bacteriophage-mediated exchange of DNA between bacteria.

Further Reading

  1. Top of page
  2. Growth of Microorganisms
  3. Bacteria: Prokaryotic Unicellular Organisms
  4. Bacterial Genes and Genome
  5. Bacterial Genetics
  6. Plasmids as Vectors for Amplifying Foreign DNA
  7. Bacteriophages as Vectors for Amplifying Foreign DNA
  8. Yeast: Eukaryotic Unicellular Organisms
  9. Yeast Genes and Genome
  10. Yeast Genetics
  11. Tissue Culture Cells
  12. Shuttle Vectors: Plasmids that can Replicate in Two Different Hosts
  13. Summary
  14. Further Reading
  15. Web Links

Web Links

  1. Top of page
  2. Growth of Microorganisms
  3. Bacteria: Prokaryotic Unicellular Organisms
  4. Bacterial Genes and Genome
  5. Bacterial Genetics
  6. Plasmids as Vectors for Amplifying Foreign DNA
  7. Bacteriophages as Vectors for Amplifying Foreign DNA
  8. Yeast: Eukaryotic Unicellular Organisms
  9. Yeast Genes and Genome
  10. Yeast Genetics
  11. Tissue Culture Cells
  12. Shuttle Vectors: Plasmids that can Replicate in Two Different Hosts
  13. Summary
  14. Further Reading
  15. Web Links
  • http://www.ncbi.nlm.nih.gov/ NCBI (National Center for Biotechnology Information) provides public assess to vast amounts of scientific information and tools, including scientific references, genomic and gene sequences, human genetic diseases, and nucleic acid and protein structures.
  • www.yeastgenome.org The Saccharomyces Genome Database is a comprehensive, public database that lists each yeast gene with extensive annotations to sequence data, protein data, cellular localization studies, known functions, published references, gene expression data (i.e. from DNA microarrays), and interactions with other genes.
  • http://sequence-www.stanford.edu/group/yeast_deletion_project/deletions3.html The Saccharomyces Genome Deletion Project is a collaborative effort among many researchers studying yeast to create a library of yeast strains, each with a different gene deleted.