arrangement facilitates the sequencing of the model organism's genome,for example, by being very compact or having a low proportion of junk DNA(e.g.yeast, Arabidopsis,or pufferfish) When researchers look for an organism to use in their studies,they look for several traits.Among these are size.generation time.accessibility.manipulation. genetics.conservation of mechanisms.and potential economic benefit.As comparative molecular biology has become more common,some researchers have ught model orga isms fro a wider as tof lineages on the tree of life. 2.5.3 Several important model organisms 2.5.3.1 Bacteriophages.Bacteriophages(bacterial viruses,phages)are infectious agents that replicate as obligate intracellular parasites in bacteria.Extracellular phage particles are metabolically inert and consist principally of proteins plus nucleic acid (DNAor RNA,but not both).The proteins of the article form a protective shell (capsid)surrounding the tightly packag Phage genomes vary in size from approximately 2 to 200 kilobases per strand of nucleic acid and consist of double-stranded DNA,single-stranded DNA,or RNA.Phage genomes, like plasmids,encode functions required for replication in bacteria,but unlike plasmids they alsoencode capsid proteins and nonstructural proteins required for phage assembly.Complex phages have po lyhedral heads to which tai s and A single cycle of phage growth is shown in Figure 2.7.Infection is initiated by adsorption of phageto specific receptors on the surface of susceptible host bacteria. The capsids remain at the cell surface and the dna or rna genomes enter the tcells(penet ation)Bec infectivity ic DNA or RNA is much less ture virus, ere is a tim e immedia eclipse period during which intracellular infectious phage cannot be detected.The infecting phage RNA or DNA is replicated to produce many new copies of the phage genome,and phage-specific proteins are produced.For most phages assembly of in the release of the progeny urs by cell lysis In hages are form cell e nvelope and sed withou killing the host cells.The eclipse period ends when intracellular infe ctious progeny appear.The latent period is the interval from infection until extracellular progeny appear,and the rise period is the interval from the end of the latent period until all phage are extracellular.The average number of phage particles produced by each nfected cell called the bu is cha racteristic ranges between 50 and several hundred Phages are classified into two major groups:virulent and temperate.Growth of virulent phages in susceptible bacteria destroys the host cells.Infection of susceptible bacteria by temperate phages can have either of two outcomes:lytic growth of tempe rate and virulent bacteriophag es is similar ction of phage progeny and death of the host bacteria.Lysogeny isa specific type of latent viral infection in which the phage genome replicates as a prophage in the bacterial cell.In most lysogenic bacteria the genes required for lytic phage development are not expressed,and production of infectiousphage does not 26
26 arrangement facilitates the sequencing of the model organism's genome, for example, by being very compact or having a low proportion of junk DNA (e.g. yeast, Arabidopsis, or pufferfish). When researchers look for an organism to use in their studies, they look for several traits. Among these are size, generation time, accessibility, manipulation, genetics, conservation of mechanisms, and potential economic benefit. As comparative molecular biology has become more common, some researchers have sought model organisms from a wider assortment of lineages on the tree of life. 2.5.3 Several important model organisms 2.5.3.1 Bacteriophages. Bacteriophages (bacterial viruses, phages) are infectious agents that replicate as obligate intracellular parasites in bacteria. Extracellular phage particles are metabolically inert and consist principally of proteins plus nucleic acid (DNA or RNA, but not both). The proteins of the phage particle form a protective shell (capsid) surrounding the tightly packaged nucleic acid genome. Phage genomes vary in size from approximately 2 to 200 kilobases per strand of nucleic acid and consist of double-stranded DNA, single-stranded DNA, or RNA. Phage genomes, like plasmids, encode functions required for replication in bacteria, but unlike plasmids they also encode capsid proteins and nonstructural proteins required for phage assembly. Complex phages have polyhedral heads to which tails and sometimes other appendages (tail plates, tail fibers, etc.) are attached. A single cycle of phage growth is shown in Figure 2.7. Infection is initiated by adsorption of phageto specific receptors on the surface of susceptible host bacteria. The capsids remain at the cell surface, and the DNA or RNA genomes enter the target cells (penetration). Because infectivity of genomic DNA or RNA is much less than that of mature virus, there is a time immediately after infection called the eclipse period during which intracellular infectious phage cannot be detected. The infecting phage RNA or DNA is replicated to produce many new copies of the phage genome, and phage-specific proteins are produced. For most phages assembly of progeny occurs in the cytoplasm, and release of the progeny occurs by cell lysis. In contrast, filamentous phages are formed at the cell envelope and released without killing the host cells. The eclipse period ends when intracellular infectious progeny appear. The latent period is the interval from infection until extracellular progeny appear, and the rise period is the interval from the end of the latent period until all phage are extracellular. The average number of phage particles produced by each infected cell, called the burst size, is characteristic for each virus and often ranges between 50 and several hundred. Phages are classified into two major groups: virulent and temperate. Growth of virulent phages in susceptible bacteria destroys the host cells. Infection of susceptible bacteria by temperate phages can have either of two outcomes: lytic growth or lysogeny. Lytic growth of temperate and virulent bacteriophages is similar, leading to production of phage progeny and death of the host bacteria. Lysogeny is a specific type of latent viral infection in which the phage genome replicates as a prophage in the bacterial cell. In most lysogenic bacteria the genes required for lytic phage development are not expressed, and production of infectiousphage does not
occur.Furthermore.the lysogenic cells are immune to superinfection by the virus which they harbor as a prophage.The physical state for all temperate viruses.For example,the prophage of integrated into the bacterial chromosome at a specific site and replicates as part of the bacterial chromosome,whereas the prophage of bacteriophage PI in E coli replicates as an extrachromosomal plasmid. 30 Time (minutes 01 .Latent period 4 Rise periad Figure 2.7 One-step gr owth of bacteriophage is synchronously infected with bacteriophage added at time at low multiplicityo infection.Unabsorbed phage is inactivated shortly thereafter by addition of anti-phage antiserum,and the culture is then diluted to prevent further activity of the antiserum.Samples are taken at intervals for phage assays.Total phage(intracellular ined by testing supernatant after removal of bacteria by centrifugation or ultrafiltration.Phage titers are as the ratio of phage per infected bacterial cell. Lytic phage growth occurs spontaneously in a small fraction of lysog nic cells and a few extracellular phages are present in cultures of lysogenic bacteria.For so ysogenic bacteria,synchronous induction of lytic phagedevelopment occurs in the entire population of lysogenic bacteria when they are treated with agents that damage DNA,such as ultraviolet light or mitomycin C.The loss of prophage from a lysogenic bacterium,converting it to the nonlysogenic state and restoring by the phage that was originally present as prophage,is Some temperate phages contain genes for bacterial characteristics that are unrelated to lytic phage development or the lysogenic state,and expression of such genes is called phage conversion(or lysogenic conversion).Phage typing is the 27
27 occur. Furthermore, the lysogenic cells are immune to superinfection by the virus which they harbor as a prophage. The physical state of the prophage is not identical for all temperate viruses. For example, the prophage of bacteriophage λ in E coli is integrated into the bacterial chromosome at a specific site and replicates as part of the bacterial chromosome, whereas the prophage of bacteriophage P1 in E coli replicates as an extrachromosomal plasmid. Figure 2.7 One-step growth of bacteriophage. A culture of susceptible bacteria is synchronously infected with bacteriophage added at time 0 at low multiplicity of infection. Unabsorbed phage is inactivated shortly thereafter by addition of anti-phage antiserum, and the culture is then diluted to prevent further activity of the antiserum. Samples are taken at intervals for phage assays. Total phage (intracellular plus extracellular) is determined by testing the sample after treating it to disrupt infected bacteria, and extracellular phage is determined by testing supernatant after removal of bacteria by centrifugation or ultrafiltration. Phage titers are as the ratio of phage per infected bacterial cell. Lytic phage growth occurs spontaneously in a small fraction of lysogenic cells, and a few extracellular phages are present in cultures of lysogenic bacteria. For some lysogenic bacteria, synchronous induction of lytic phagedevelopment occurs in the entire population of lysogenic bacteria when they are treated with agents that damage DNA, such as ultraviolet light or mitomycin C. The loss of prophage from a lysogenic bacterium, converting it to the nonlysogenic state and restoring susceptibility to infection by the phage that was originally present as prophage, is called curing. Some temperate phages contain genes for bacterial characteristics that are unrelated to lytic phage development or the lysogenic state, and expression of such genes is called phage conversion (or lysogenic conversion). Phage typing is the
testing of strains of a narticular bacterial species for suscentibility to specific bacteriophages.The patterns of susceptibility to the set of typing phages provide information about th possible relatedness of individual clinica solates.Such information is particularly useful for epidemiologica investigation Bacteriophage ),which infects E.coli,typifies the temperate phages(Figure 2.8) This phage has one of the most studied genomes and is used extensively in DNA cloning Q ·的 Lysogen &Prophage 9 ral DNA 9用 /mw2 Figure 2.8).bacteriophage ergoes either lytic replication or lysogen col The linear doubl stranded DNA is converted to a circular form immediately after infection.Left)If the nutritional state of the host cell is favorable,most infected cells undergo lytic replication,similar to lytic replication of cells by bacteriophage T4.(Right) If the nutritionl state of the host cell cannot support production of large numbers of progeny phages,lysogeny is established.In this case,viral genes required for the lytic cvcle are repressed,and host-cell enzymes synthesize viral proteins that integrate the viral DNA into a specific sequence in the host-cell chromosome where no host-cell genes are disrupted.The prophage DNA then is replicated along with the host-cell chro e as the lysog cell (called a h and divides. Repression of the viral genes required for lytic replication is maintained in progeny cells.At infrequent intervals,the prophage in a lysogen is induced,or activated, leading to expression of viral proteins that precisely remove the prophage DNA from the host-cell chromosome and to derepression of the genes required for the lytic sues Transduction In transduction,bacteriophages function as vectors to introduce DNA from donor bacteria into recipient bacteria by infection.For some phages,called generalized transducing phages,a small fraction of the virions produced during lytic growth are
28 testing of strains of a particular bacterial species for susceptibility to specific bacteriophages. The patterns of susceptibility to the set of typing phages provide information about the possible relatedness of individual clinical isolates. Such information is particularly useful for epidemiological investigations Bacteriophage λ, which infects E. coli, typifies the temperate phages (Figure 2.8). This phage has one of the most studied genomes and is used extensively in DNA cloning. Figure 2.8 λ bacteriophage undergoes either lytic replication or lysogeny following infection of E. coli. The linear double-stranded DNA is converted to a circular form immediately after infection. (Left) If the nutritional state of the host cell is favorable, most infected cells undergo lytic replication, similar to lytic replication of cells by bacteriophage T4. (Right) If the nutritional state of the host cell cannot support production of large numbers of progeny phages, lysogeny is established. In this case, viral genes required for the lytic cycle are repressed, and host-cell enzymes synthesize viral proteins that integrate the viral DNA into a specific sequence in the host-cell chromosome where no host-cell genes are disrupted. The prophage DNA then is replicated along with the host-cell chromosome as the lysogenized cell (called a lysogen) grows and divides. Repression of the viral genes required for lytic replication is maintained in progeny cells. At infrequent intervals, the prophage in a lysogen is induced, or activated, leading to expression of viral proteins that precisely remove the prophage DNA from the host-cell chromosome and to derepression of the genes required for the lytic cycle. As a result, a normal cycle of lytic replication ensues. Transduction In transduction, bacteriophages function as vectors to introduce DNA from donor bacteria into recipient bacteria by infection. For some phages, called generalized transducing phages, a small fraction of the virions produced during lytic growth are
aberrant and contain a random fragment of the bacterial genome instead of phage genes,segment of the bacterial genome .Transduction mediate by populations of such phages is called generalized transduction,because each part of the bacterial genome has approximately the same probability of being transferred from donor to recipient bacteria.When a generalized transducing phage infects a recipient cell,expression of the transferred donor genesoccurs.Abortive transduction refers to the genes withou formation of recombinant progeny,whereas complete transduction is characterized by production of stable recombinants that inherit donor genes and retain the ability to express them.In abortive transduction the donor DNA fragment does not replicate and among the progeny of the original transductant only one bacterium contains the donor DNA F In all othe er progeny the dond gene p progressively dilut d after each generation of bacterial growth until the dono phenotype can no longer be expressed.On selective medium upon which only bacteria with the donor phenotype can grow,abortive transductants produce minute colonies that can be distinguished easily from colonies of stable transductants.The of abortive tr duction is e to tu reter thn the trsaueney ofduction inncm nitude ung tha at most cells infected by generalized transducing phages do not produce recombinant progeny. Specialized transduction differs from generalized transduction in several ways.It is mediated only by specific temperate phages.and only a few specific donor genes can be transferred to rec ipient bacteria nsducing nhages are formed nter the lytic and idarre rombnnt which lac phage progeny part of th normal phage genome and contain part of the bacterial chromosome located adjacent to the prophage attachment site.Many specialized transducing phages are defective and cannot complete the lytic cycle of phage growth in infected cells unless helper are p nt to provi ide r issing hage functions.Specialized transduction romysogenio te recipient bacterium by the speciad tranduins phage and expression of the donor genes.Phage conversion and specialized transduction have many similarities,but the origin of the converting genes in temperate converting phages is unknown. 2 5 32 Escherichia oli Escherichia coli (e coli is a bacterium that is commonly found in the ower intestine of warm ded animals.Most E strains are harmless,but some,such as serotype 0157:H7,can cause serious food poisoning in humans,and are occasionally responsible for costly product recalls.The harmless strains are part of the normal flora of the gut,and can benefit their hosts by producing vitamin k3.or by preventing the establishment of pathogenic bacteria intestine E.not always confined to the intestine,and their ability to survive for brief periods outside the body makes them an ideal indicator organism to test environmental samples for fecal contamination.The bacteria can also be grown easily and its genetics are comparatively simple and easily-manipulated,making it
29 aberrant and contain a random fragment of the bacterial genome instead of phage DNA. Each individual transducing phage carries a different set of closely linked genes, representing a small segment of the bacterial genome. Transduction mediated by populations of such phages is called generalized transduction, because each part of the bacterial genome has approximately the same probability of being transferred from donor to recipient bacteria. When a generalized transducing phage infects a recipient cell, expression of the transferred donor genes occurs. Abortive transduction refers to the transient expression of one or more donor genes without formation of recombinant progeny, whereas complete transduction is characterized by production of stable recombinants that inherit donor genes and retain the ability to express them. In abortive transduction the donor DNA fragment does not replicate, and among the progeny of the original transductant only one bacterium contains the donor DNA fragment. In all other progeny the donor gene products become progressively diluted after each generation of bacterial growth until the donor phenotype can no longer be expressed. On selective medium upon which only bacteria with the donor phenotype can grow, abortive transductants produce minute colonies that can be distinguished easily from colonies of stable transductants. The frequency of abortive transduction is typically one to two orders of magnitude greater than the frequency of generalized transduction, indicating that most cells infected by generalized transducing phages do not produce recombinant progeny. Specialized transduction differs from generalized transduction in several ways. It is mediated only by specific temperate phages, and only a few specific donor genes can be transferred to recipient bacteria. Specialized transducing phages are formed only when lysogenic donor bacteria enter the lytic cycle and release phage progeny. The specialized transducing phages are rare recombinants which lack part of the normal phage genome and contain part of the bacterial chromosome located adjacent to the prophage attachment site. Many specialized transducing phages are defective and cannot complete the lytic cycle of phage growth in infected cells unless helper phages are present to provide missing phage functions. Specialized transduction results from lysogenization of the recipient bacterium by the specialized transducing phage and expression of the donor genes. Phage conversion and specialized transduction have many similarities, but the origin of the converting genes in temperate converting phages is unknown. 2.5.3.2 Escherichia coli. Escherichia coli (E. coli) is a bacterium that is commonly found in the lower intestine of warm-blooded animals. Most E. coli strains are harmless, but some, such as serotype O157:H7, can cause serious food poisoning in humans, and are occasionally responsible for costly product recalls. The harmless strains are part of the normal flora of the gut, and can benefit their hosts by producing vitamin K2, or by preventing the establishment of pathogenic bacteria within the intestine. E. coli are not always confined to the intestine, and their ability to survive for brief periods outside the body makes them an ideal indicator organism to test environmental samples for fecal contamination. The bacteria can also be grown easily and its genetics are comparatively simple and easily-manipulated, making it
one of the best-studied prokaryotic model organisms,and an important species in biotechnology. Becaus bacteria are proka otes,they do not undergo meiosis.However,there are several other ways in which bacteria genomes can unite and recombine. Therefore the approach to the genetic analysis of recombination in these organisms is remarkably similar to that for eukaryotes-namely,combine two different genomes in the same cell to afford an opportunity for recombination,and then look that afford pp nities for recombination,we shall first examine conjugation,a natural union cells during which one cell transfers DNA segments to another.Second,a bacteria cell can pick up DNA from the environment and incorporate this DNA into its own chromosome;this process,called transformation,permanently changes the genotype Third,certain phag occasionally pick up a pi ce of DNA from one bacteria cell and inject it into anothe where it ca be incorporated into the chron osome,in a process known as transduction.In all these processes.the gene transfer is partial and unidirectional.In other words,only a part of the genome of one organism is transferred and becomes incorporated into the complete genome of another.The strain that contributes the partial genomic fragment iscalled the donor,and the one hat contributes h e genome is the The dond r fragmen is called the exogenote.and the recipient genome is the endogenote.A cell containing an exogenote is a partial diploid.or merozygote.This unidirectional and partial system of genetic union in bacteria contrasts with the zygotes of eukaryotes,in which(with the exception of organelle genes)both parents contribute equally and both contribute complet The ote pro ortunity for recombin tion the genes thatare in the partially diploid segmen Thus a merozygote provides the same type of recombination opportunities found in the zygotes of eukaryotes,although on a more limited scale.The merozygote also provides opportunities for testing various types of gene interaction. E.coli and related bacteria possess the ability to transfer DNA via bacterial conjuga ransdu ction or transformation,which allows genetic material to spread horizontally through an existing population. Because of its long history of laboratory culture and ease of manipulation.E.coli also plays an important role in modern biological engineering and industrial microbiology e coli is used plasmids and restriction enzymes to create recombinant DNA,be e a foundation of bi hnology.E.coli is fr ently used as a expression vector.Genetic systems have also been developed which all ow the production of recombinant proteins using E.coli.One of the first useful applications of recombinant DNA technology was the manipulation of E.coli to produce human insulin 2 5 3 3 Sacchar revisiae.Sacchar myces cerevisiae is a species of budding yeast.It is perhaps the most useful yeast owing to its use since ancient tm in baking and brewing.It is believed that it was originally isolated from the skins of grapes(one can see the yeast as a component of the thin white film on the skins of some dark-colored fruits such as plums;it exists among the waxes of the cuticle).It 30
30 one of the best-studied prokaryotic model organisms, and an important species in biotechnology. Because bacteria are prokaryotes, they do not undergo meiosis. However, there are several other ways in which bacteria genomes can unite and recombine. Therefore the approach to the genetic analysis of recombination in these organisms is remarkably similar to that for eukaryotes—namely, combine two different genomes in the same cell to afford an opportunity for recombination, and then look for recombinants in the descendant cells. Of the processes that afford opportunities for recombination, we shall first examine conjugation, a natural union of bacteria cells during which one cell transfers DNA segments to another. Second, a bacteria cell can pick up DNA from the environment and incorporate this DNA into its own chromosome; this process, called transformation, permanently changes the genotype. Third, certain phages can occasionally pick up a piece of DNA from one bacteria cell and inject it into another, where it can be incorporated into the chromosome, in a process known as transduction. In all these processes, the gene transfer is partial and unidirectional. In other words, only a part of the genome of one organism is transferred and becomes incorporated into the complete genome of another. The strain that contributes the partial genomic fragment is called the donor, and the one that contributes the complete genome is the recipient. The donor fragment is called the exogenote, and the recipient genome is the endogenote. A cell containing an exogenote is a partial diploid, or merozygote. This unidirectional and partial system of genetic union in bacteria contrasts with the zygotes of eukaryotes, in which (with the exception of organelle genes) both parents contribute equally and both contribute complete genomes. The merozygote provides an opportunity for genetic recombination to occur between the genes that are in the partially diploid segment. Thus a merozygote provides the same type of recombination opportunities found in the zygotes of eukaryotes, although on a more limited scale. The merozygote also provides opportunities for testing various types of gene interaction. E. coli and related bacteria possess the ability to transfer DNA via bacterial conjugation, transduction or transformation, which allows genetic material to spread horizontally through an existing population. Because of its long history of laboratory culture and ease of manipulation, E. coli also plays an important role in modern biological engineering and industrial microbiology. E. coli, is used plasmids and restriction enzymes to create recombinant DNA, became a foundation of biotechnology. E. coli is frequently used as a expression vector. Genetic systems have also been developed which allow the production of recombinant proteins using E. coli. One of the first useful applications of recombinant DNA technology was the manipulation of E. coli to produce human insulin. 2.5.3.3 Saccharomyces cerevisiae. Saccharomyces cerevisiae is a species of budding yeast. It is perhaps the most useful yeast owing to its use since ancient times in baking and brewing. It is believed that it was originally isolated from the skins of grapes (one can see the yeast as a component of the thin white film on the skins of some dark-colored fruits such as plums; it exists among the waxes of the cuticle). It