9 Геном, плазмиды, вирусы (Лекции)

Лекция 9.Геном, плазмиды, вирусыC H A P T E RGenes and ChromosomesEvery cell of a multicellular organism generally contains the same genetic material. One has only to look at a human being to marvel at thewealth of information contained in each human cell. It should come asno surprise that the DNA molecules containing the cellular genes areby far the largest macromolecules in cells.

They are commonly packaged into structures called chromosomes. Most bacteria and viruseshave a single chromosome; eukaryotes usually have many. A singlechromosome typically contains thousands of individual genes. The sumof all the genes and intergenic DNA on all the different chromosomes ofa cell is referred to as the cellular genome.Measurements carried out in the 1950s indicated that the largestDNAs had molecular weights of 106 or less, equivalent to about 15,000base pairs. But with improved methods for isolation of native DNAs,their molecular weights were found to be much higher. Today we knowthat native DNA molecules, such as those from E. coli cells, are so largethat they are easily broken by mechanical shear forces, and thereforeare not readily isolated in intact form.The size of DNA molecules represents an interesting biologicalproblem in itself.

Chromosomal DNAs are often many orders of magnitude longer than the biological packages (cells or viruses) that containthem (Fig. 23-1). In this chapter we move from the secondary structure of DNA considered in Chapter 12 to the extraordinary degree oforganization required for the tertiary packaging of DNA into chromosomes. First we examine the size of viral DNAs and cellular chromosomes and the organization of genes and other sequences within them.We then turn to the discipline of DNA topology to give formal definitionto the twisting and coiling of DNA molecules.

Finally, we consider theprotein-DNA interactions that organize chromosomes into compactstructures.Figure 23-1 Electron micrograph of bacteriophageT2 surrounded by its single, linear molecule ofDNA. The DNA was released by lysing the bacteriophage in distilled water and allowing the DNA tospread on the water surface.The Size and Sequence Structure ofDNA MoleculesWe begin with a survey of the DNA molecules of viruses and of cells,both prokaryotic and eukaryotic. Chromosomes contain, in addition togenes, special-function sequences that aid in the packaging and segregation of chromosomes to daughter cells at cell division.

The structureof chromosomes will be examined, with a focus on the various types ofDNA sequences found within them.791792Part IV Information PathwaysViral DNA Molecules Are SmallViruses generally require considerably less genetic information thancells, because they rely on many functions of a host cell to reproducethemselves. Viral genomes can be made up of either RNA or DNA.Almost all plant viruses and some bacterial and animal viruses containRNA. RNA viruses tend to have particularly small genomes. The genomes of DNA viruses, in contrast, span a wide range of sizes (Table23-1). From the molecular weight of a double-stranded (duplex) viralDNA it is possible to calculate its contour length (its helix length),given that each nucleotide pair has an average molecular weight ofabout 650 and there is one nucleotide pair for every 0.36 nm of theduplex (see Fig.

12-15). Note that the DNA found in some viruses issingle-stranded rather than double-stranded.Table 23-1 The DNA and particle sizes of some bacterial virusesVirus0X174 (duplex form)T7A (lambda)T2, T4kViralparticleweight (xlO6)Longdimensionof particle (nm)Number ofbase pairs6385022025781902105,38639,93648,502182,000*The complete base sequence of T2 and T4 DNA is not known; this is an approximationMany viral DNAs have covalently linked ends and are thereforecircular (in the sense of an endless belt, rather than a perfect round)during at least part of their life cycle.

During viral replication within ahost cell, specific types of viral DNA called replicative forms mayappear; for example, linear DNAs often become circular and all singlestranded DNAs become double-stranded.A typical medium-sized DNA virus is bacteriophage A (lambda) ofE. coli.

In its replicative form inside cells, its DNA is a circular doublehelix. Double-stranded A DNA contains 48,502 base pairs and has acontour length of 17.5 fim. Bacteriophage 0X174 is a much smallerDNA virus; the DNA in a c/>X174 viral particle is a single-strandedcircle. Its double-stranded replicative form contains 5,386 base pairs.Another important point about viral DNAs will be echoed in sections tofollow: their contour lengths are much greater than the long dimensions of the viral particles in which they are found. The DNA of bacteriophage T2, for example, is about 3,500 times longer than the viralparticle itself (Fig.

23-1).Bacteria Contain Chromosomes and Extrachromosomal DNABacteria contain much more DNA than the DNA viruses. For example,a single E. coli cell contains almost 200 times as much DNA as a bacteriophage A particle. The DNA in an E. coli cell is a single, covalentlyclosed double-stranded circular molecule. It contains about 4.7 x 106base pairs and has a contour length of about 1.7 mm, some 850 timesthe length of an E. coli cell (Fig.

23-2). Again, the DNA molecule musthave a tightly compacted tertiary structure.In addition to the very large, circular DNA chromosome found inthe nucleoid, many species of bacteria contain one or more small, circular DNA molecules that are free in the cytosol. These extrachromosomal elements are called plasmids (Fig.

23-3). Many plasmids areonly a few thousand base pairs long, but some contain over 105 basepairs. Plasmids carry genetic information and undergo replication toyield daughter plasmids, which pass into the daughter cells at celldivision. Ordinarily, plasmids exist separately, detached from thechromosomal DNA.

A few classes of plasmid DNAs are sometimes inserted into the chromosomal DNA and later excised in a precise manner by means of specialized recombination processes.Figure 23-2 The length ofthe E. coli chromosome(1.7 mm) is depicted relativeto the length of a typicalE. coli cell (2 /xm).Figure 23-3 Electron micrograph of DNA from alysed E. coli cell. Several small, circular plasmidDNAs are indicated by arrows. The black spots andwhite specks are artifacts of the preparation.793794Part IV Information PathwaysPlasmids have been found in yeast and other fungi as well as inbacteria. In many cases plasmids confer no obvious advantage on theirhost, and their sole function appears to be self-propagation. However,some plasmids carry genes that make a host bacterium resistant toantibacterial agents.

For example, plasmids carrying the gene for theenzyme /3-lactamase confer resistance to /3-lactam antibiotics such aspenicillin and amoxicillin. Plasmids also may pass from an antibioticresistant cell to an antibiotic-sensitive cell of the same or another bacterial species, thus rendering the latter resistant. The extensive use ofantibiotics has served as a strong selective force for the spread of theseplasmids in disease-causing bacteria, creating multiply resistant bacterial strains, particularly in hospital settings.

Physicians are becoming reluctant to prescribe antibiotics unless a bacterial infection is confirmed. For similar reasons, the widespread use of antibiotics inanimal feeds is being curbed.Plasmids are useful models for the study of many processes inDNA metabolism.

They are relatively small DNA molecules and hencecan quite easily be isolated intact from bacterial and yeast cells. Plasmids have also become a central component of the modern technologiesassociated with the isolation and cloning of genes. Genes from a variety of species can be inserted into isolated plasmids, and the modifiedplasmid can then be reintroduced into its normal host cell. Such aplasmid will be replicated and transcribed, and may also cause thehost cell to make the proteins coded by the foreign gene, even though itis not part of the normal genome of the cell. Chapter 28 describes howsuch recombinant DNAs are made.Eukaryotic Cells Contain More DNA than ProkaryotesTable 23-2 Normalchromosome numberin different organisms*BacteriaFruit flyRed cloverGarden peaYeastHoneybeeCornFrogHydraFoxCatMouseRatRabbitHumanChicken181414161620263034384042444678* For all eukaryotic organismslisted, the diploid chromosomenumber is shownAn individual cell of a yeast, one of the simplest eukaryotes, has fourtimes more DNA than an E.

coli cell. Cells of Drosophila, the fruit flyused in classical genetic studies, have more than 25 times as muchDNA as E. coli cells. Each cell of human beings and many other mammals has about 600 times as much DNA as E. coli, and the cells ofmany plants and amphibians have an even greater amount. Note thatthe nuclear DNA molecules of eukaryotic cells are linear, not circular.The total contour length of all the DNA in a single human cell isabout 2 m, compared with 1.7 mm for E. coli DNA.

In the approximately 1014 cells of the adult human body, the total length of all theDNA would be about 2 x 1014 m or 2 x 1011 km. Compare this with thecircumference of the earth (4 x 104 km) or the distance between theearth and the sun (1.5 x 108 km). Once again it becomes clear thatDNA packaging in cells must involve an extraordinary degree of organization and compaction.Microscopic observation of nuclei in dividing eukaryotic cells hasshown that the genetic material is subdivided into chromosomes, theirdiploid number depending upon the species of organism (Table 23-2).Human cells, for example, have 46 chromosomes.