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Chapter 21 Model Organism. 2003 级生科 2 班 苏亮 200331060158. Model Organism.
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Chapter 21 Model Organism 2003级生科2班 苏亮 200331060158
Model Organism • Two important feature of all model systems: first, the availability of powerful tools of and study the organism genetically. Second, ideas, methods, tools, and strains could be shared among scientists investigating the same organism, facilitating rapid progress.
The choice of a model organism depends on what question is being asked. • In this chapter we will describe some of the most commonly studied experimental organisms and advantages of each as a model system.
Bacteriophage • Bacteriophage (and viruses in general) offer the simplest system to examine the basic processes of life. Phage typically consist of a genome (DNA and RNA, most commonly the former) packaged in a coat of protein subunits, some of which form a head structure (in which the genome is stored) and some a tail stricture.
Each phage attaches to a specific cell surface molecule (usually a protein) and so only cells bearing that “receptor” can be infected by a given phage. • Phage come in two basic types-lytic and temperate. The former, examples of which include the T phage, grow only lytically.
Temperate phage can also replicate lytically. But they can adopt an alternative developmental pathway called lysogeny. (figure 21-2) • In this integrated, repressed state the phage is called a prophage. • The lysogenic state can be maintained in this way for many generations but is also poised to switch to lytic growth at any time. This switch from the lysogenic to lytic pathway, called induction.
Assays of phage growth • For bacteriophage to be use ful as an experimental system, methods are needed to propagate and quantify phage. To quantify the numbers of phage particles in a solution, a plaque assay is used.
Figure 21-3 Plaques firmed by phage infection of a lawn of bacterial cells.
Plaque is the result of multiple round of infection, a circular clearing in the otherwise opaque lawn of densely grown uninfected bacterial cells. Knowing the number of plaques on a given plate, and the extent to which the original stock was diluted before plating, makes it trivial to calculate the number of phage in that original stock.
The single-step growth curve • This classic experiment revealed the life cycle of a typical lytic phage and paved the way for many subsequent experiments that examined that life cycle in detail. The essential feature of this procedure is the synchronous infection of a population of bacteria and the elimination of any re-infection by the progeny.
The time lapse between infection and release of progeny is called the latent period, and the number of phage released is called the burst size.
Phage crosses ancomplementation tests • Differences in host range and plaque morphologies of the phage were very often the result of genetic differences between otherwise identical phage. • The ability to perform mixed infection-in which a single cell is infected with two phage particles at once-makes genetic analysis possible in two ways.
First, it allow one to perform phage crosses. • Second, co-infection also allow one to assign mutations to complementation groups; that is, one can identify when two or more mutations are in the sane or in different genes.
Transduction and recombinant DNA • The process involves a site-specific recombination event, and if that event occurs at slightly the wrong position, phage DNA is lost and bacterial DNA included is as known as specialized transduction.
Because of the ability to promote specialized transduction, it was natural that phage λ was chosen as one of the original cloning vectors. • Many different λ vectors were developed, all differing in the restriction sites used and in how recombinant phage could be identified.
Bacteria • The attraction of bacteria such as E. coli or B. subtilis as experimental systems is that they are relatively simple cells and can be grown and manipulated with comparative ease. • Molecular biology owes its origin to experiments with bacterial and phage model systems.
Assays of bacterial growth • Bacterial cells are large enough（about 2µm in length）to scatter light, allowing the growth of a bacterial culture to be monitored conveniently in liquid culture by the increase in optical density.
The number of bacteria can be determined by diluting the culture and plating the cells o solid (agar) medium in a petri dish. Knowing how many colonies are on the plate and how much the culture was diluted makes it possible to calculate the concentration of cells in the original culture.
Bacteria exchange DAN by sexual conjugation, phage-mediated transduction, and DAN-mediated transformation • Bacterial often harbor autonomously replicating DNA elements known as plasmids.
The F-factor can undergo conjugation only with other E .coli strains, promiscuous conjugative plasmids provide a convenient means for introducing DNA into bacterial strains that are otherwise lacking in their own systems of genetic exchange.
Yet another powerful tool for genetic exchange is phage-mediated transduction. • Generalized transduction is mediated by phage that occasionally package fragment of chromosomal DNA during maturation of the virus rather than viral DNA.
Another kind of phage-mediated transduction is called specialized transduction. It involves a lysogenic phage such as λ that has incorporated a segment of chromosomal DNA in place of a segment of phage DNA.
DNA-mediated transformation: certain experimentally important bacterial species possess a natural system of genetic exchange that enables them to take up and incorporate linear, naked DNA into their own chromosome by recombination. • Often the cells must be in a specialized state known as “genetic competence” to take up and incorporate DNA from their environment.
Bacterial plasmids can be used as cloning vectors • Circular DNA elements in bacterial known as plasmids can serve as convenient vectors for bacterial DNA as well as foreign DNA.
Transposons can be used to generate insertional mutations and gene and operon fusion • Transposons are enormously useful tools for carrying out molecular genetic manipulations in bacterial.
It have two important advantages over traditional mutations induced by chemical mutagenesis. • One is that the insertion of a transposon into a gene is more lifely to result in complete inactivation of the gene than a simple nucleotide switch created by a mutagen. • The second is that ,having inactivated the gene, the presence of the inserted DNA makes it easy to isolate and clone that gene.
Transposons can also be used to create gene and operon fusions on a genome-wide basis. such a fusion is know as an operon or transcriptional fusion.
And a fusion in which the reporter is joined both transcriptionally and translationally to the target gene is known as a gene fusion
Studies on the molecular biology of bacteria have been enhanced by recombinant DNA technology, whole-genome sequencing, and transcriptional profiling
With the advent of recombinant DNA technologies revolutionized molecular biological studies of higher cells. But they have had an impact on the study of bacterial model systems as well. • The availability of microarrays representing all of the genes in a bacterium has made it possible to study gene expression on a genome basis.
And the availability of whole-genome sequences and promiscuous conjugative plasmids has created opportunities for carrying out molecular genetic manipulations in bacterial species that otherwise lack sophisticated, traditional tools of genetics.
Biochemical analysis is especially powerful in simple cells with well-developed tools of traditional and molecular genetics • There are three reasons for this:
first, large quantities of bacterial cells can be grown in a defined and homogenous physiological state. • Second , the tools of traditional and molecular genetics. • Third, the machinery for carrying out DNA replication, gene transcription, protein synthesis, and so forth is much simpler (having far fewer components) in bacterial than in higher cells.
Bacterial are accessible to cytological analysis • Despite their small size, bacteria are accessible to the tools of cytology, such as immunofluoresence microscopy for localizing proteins in fixed cells with specific antibodies, fluorescence microscopy with the Green Fluorescent Protein for localizing proteins in living cells, and fluorescence in situ hybridization (FISH) for localizing chromosomal regions and plasmids within cells.
Cytological methods are an important part of the arsenal for molecular studies on the bacterial cell.
Phage and bacteria told us most of the fundamental things about the gene • Molecular biology owes its origin to experiments with bacterial and phage model systems. Indeed, groundbreaking work with a pneumococcus bacterium led to the discovery that the genetic material is DNA. Since then, experiments with E .coli and its phage have the way.
There are countless examples where, by choosing these simplest of systems, fundamental processes of life were understood. An important example comes from the classic work of Seymor Benzer, who examined intensely a single genetic locus in phage T4, called rⅡ.
BAKER’S YEAST, Saccharomyces cerevisiae • Unicellular eukaryotes offer many advantages as experimental model systems. And the best studied unicellular eukaryote is the budding yeast S. cerevisiae.
The existence of haploid and diploid cells facilitate genetic analysis of S. cerevisiae • S. cerevisiae exists in three forms. Two haploid cell types, a and α, and the diploid product of mating between these two.
Figure 21-10 The lifecycle of the budding yeast S. cerevisiae
These cell types can be manipulate to perform a variety of genetic assays. • Genetic complementation can be performed the two mutations whose complementation is being tested. • If the mutations complement each other, the diploid will be a wild type for mntations can be made in haploid cells in which there is only a single copy of that gene.
Generating precise mutations in yeast is easy • The genetic analysis of S. cerevisiae is further enhanced by the availability of techniques used to precisely and rapidly modify individual genes.
