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gene 8 word 文档形式 chapter1 1.1-1.4
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GENE EIGHT GENES is continuously updated on the web site, www.ergito.com with revisions posted weekly. This allows readers to check for revised sections and relate them to the printed book. The web site can be viewed as either sections from the book or as a slide show of the figures from the book. Some of the figures shown are animated and there are references hyperlinked to the original sources. Other features of the web site include a glossary, sophisticated searches, and ancillary material such as the essays in the Great Experiments and Structures Series. To subscribe to this site, please visit www.ergito.com. Genes are DNA 1.1 Introduction The hereditary nature of every living organism is defined by its genome, which consists of a long sequence of nucleic acid that provides the information needed to construct the organism. We use the term "information" because the genome does not itself perform any active role in building the organism; rather it is the sequence of the individual subunits (bases) of the nucleic acid that determines hereditary features. By a complex series of interactions, this sequence is used to produce all the proteins of the organism in the appropriate time and place. The proteins either form part of the structure of the organism, or have the capacity to build the structures or to perform the metabolic reactions necessary for life. The genome contains the complete set of hereditary information for any organism. Physically the genome may be divided into a number of different nucleic acid molecules. Functionally it may be divided into genes. Each gene is a sequence within the nucleic acid that represents a single protein. Each of the discrete nucleic acid molecules comprising the genome may contain a large number of genes. Genomes for living organisms may contain as few as <500 genes (for a mycoplasma, a type of bacterium) to as many as >40,000 for Man. In this chapter, we analyze the properties of the gene in terms of its basic molecular construction. Figure 1.1 summarizes the stages in the transition from the historical concept of the gene to the modern definition of the genome. The basic behavior of the gene was defined by Mendel more than a century ago. Summarized in his two laws, the gene was recognized as a "particulate factor" that passes unchanged from parent to progeny. A gene may exist in alternative forms. These forms are called alleles. In diploid organisms, which have two sets of chromosomes, onecopy of each chromosome is inherited from each parent. This is the same behavior that is displayed by genes. One of the two copies of each gene is the paternal allele (inherited from the father), the other is the maternal allele (inherited from the mother). The equivalence led to the discovery that chromosomes in fact carry the genes. Each chromosome consists of a linear array of genes. Each gene resides at a particular location on the chromosome. This is more formally called a genetic locus. We can then define the alleles of this gene as the different forms that are found at this locus. The key to understanding the organization of genes into chromosomes was the discovery of genetic linkage. This describes the observation that alleles on the same chromosome tend to remain together in the progeny instead of assorting independently as predicted by Mendel's laws. Once the unit of recombination (reassortment) was introduced as the measure of linkage, the construction of genetic maps became possible. On the genetic maps of higher organisms established during the first half of this century, the genes are arranged like beads on a string. They occur in a fixed order, and genetic recombination involves transfer of corresponding portions of the string between homologous chromosomes. The gene is to all intents and purposes a mysterious object (the bead), whose relationship to its surroundings (the string) is unclear. The resolution of the recombination map of a higher eukaryote is restricted by the small number of progeny that can be obtained from each mating. Recombination occurs so infrequently between nearby points that it is rarely observed between different mutations in the same gene. By moving to a microbial system in which a very large number of progeny can be obtained from each genetic cross, it became possible to demonstrate that recombination occurs within genes. It follows the same rules that were previously deduced for recombination between genes. Mutations within a gene can be arranged into a linear order, showing that the gene itself has the same linear construction as the array of genes on a chromosome. So the genetic map is linear within as well as between loci: it consists of an unbroken sequence within which the genes reside. This conclusion leads naturally into the modern view that the genetic material of a chromosome consists of an uninterrupted length of DNA representing many genes. A genome consists of the entire set of chromosomes for any particular organism. It therefore comprises a series of DNA molecules (one for each chromosome), each of which contains many genes. The ultimate definition of a genome is to determine the sequence of the DNA of each chromosome. The first definition of the gene as a functional unit followed from the discovery that individual genes are responsible for the production of specific proteins. The difference in chemical nature between the DNA of the gene and its protein product led to the concept that a gene codes for a protein. This in turn led to the discovery of the complex apparatus that allows the DNA sequence of gene to generate the amino acid sequence of a protein. Understanding the process by which a gene is expressed allows us to make a more rigorous definition of its nature. Figure 1.2 shows the basic theme of this book. A gene is a sequence of DNA that produces another nucleic acid, RNA. The DNA has two strands of nucleic acid, and the RNA has only one strand. The sequence of the RNA is determined by the sequence of the DNA (in fact, it is identical to one of the DNA strands). In many, but not in all cases, the RNA is in turn used to direct production of a protein. Thus a gene is a sequence of DNA that codes for an RNA; in protein-coding genes, the RNA in turn codes for a protein. From the demonstration that a gene consists of DNA, and that a chromosome consists of a long stretch of DNA representing many genes, we move to the overall organization of the genome in terms of its DNA sequence. In 2 The interrupted gene we take up in more detail the organization of the gene and its representation in proteins. In 3 The content of the genome we consider the total number of genes, and in 4 Clusters and repeats we discuss other components of the genome and the maintenance of its organization. 1.2 DNA is the genetic material of bacteria The idea that genetic material is nucleic acid had its roots in the discovery of transformation in 1928. The bacterium Pneumococcus kills mice by causing pneumonia. The virulence of the bacterium is determined by its capsular polysaccharide. This is a component of the surface that allows the bacterium to escape destruction by the host. Several types (I, II, III) of Pneumococcus have different capsular polysaccharides. They have a smooth (S) appearance. Each of the smooth Pneumococcal types can give rise to variants that fail to produce the capsular polysaccharide. These bacteria have a rough (R) surface (consisting of the material that was beneath the capsular polysaccharide). They are avirulent. They do not kill the mice, because the absence of the polysaccharide allows the animal to destroy the bacteria. When smooth bacteria are killed by heat treatment, they losetheir ability to harm the animal. But inactive heat-killed S bacteria and the ineffectual variant R bacteria together have a quite different effect from either bacterium by itself. Figure 1.3shows that when they are jointly injected into an animal, the mouse dies as the result of a Pneumococcal infection. Virulent S bacteria can be recovered from the mouse postmortem. In this experiment, the dead S bacteria were of type III. The live R bacteria had been derived from type II. The virulent bacteria recovered from the mixed infection had the smooth coat of type III. So some property of the dead type III S bacteria can transform the live R bacteria so that they make the type III capsular polysaccharide, and as a result become virulent. Figure 1.4 shows the identification of the component of the dead bacteria responsible for transformation. This was called the transforming principle. It was purified by developing a cell-free system, in which extracts of the dead S bacteria could be added to the live R bacteria before injection into the animal. Purification of the transforming principle in 1944 showed that it is deoxyribonucleic acid (DNA). 1.3 DNA is the genetic material of viruses Having shown that DNA is the genetic material of bacteria, the next step was to demonstrate that DNA provides the genetic material in a quite different system. Phage T2 is a virus that infects the DNA is the genetic material of bacteria SECTION 1.2 Key Concepts ; * Phage infection proved that DNA is the genetic material of : • viruses. When the DNA and protein components of bacteriophages i ; are labeled with different radioactive isotopes, only the DNA is : transmitted to the progeny phages produced by infecting bacteria. • Key Concepts I * Bacterial transformation provided the first proof that DNA is the : : genetic material. Genetic properties can be transferred from one • : bacterial strain to another by extracting DNA from the first strain : : and adding it to the second strain. • bacterium E. coli. When phage particles are added to bacteria, they adsorb to the outside surface, some material enters the bacterium, and then -20 minutes later each bacterium bursts open (lyses) to release a large number of progeny phage. Figure 1.5 illustrates the results of an experiment in 1952 in which bacteria were infected with T2 phages that had been radioactively labeled either in their DNA component (with 32P) or in their protein component (with 35S). The infected bacteria were agitated in a blender, and two fractions were separated by centrifugation. One contained the empty phage coats that were released from the surface of the bacteria. The other fraction consisted of the infected bacteria themselves. Most of the 32P label was present in the infected bacteria. The progeny phage particles produced by the infection contained ~30% ofthe original 32P label. The progeny received very little—less than 1%—of the protein contained in the original phage population. The phage coats consist of protein and therefore carried the 35S radioactive label. This experiment therefore showed directly that only the DNA of the parent phages enters the bacteria and then becomes part of the progeny phages, exactly the pattern of inheritance expected of genetic material. A phage (virus) reproduces by commandeering the machinery of an infected host cell to manufacture more copies of itself. The phage possesses genetic material whose behavior is analogous to that of cellular genomes: its traits are faithfully reproduced, and they are subject to the conclusion that the genetic material is BNA, wriemeir part of me genome of a cell or virus. 1.4 DNA is the genetic material of animal cells When DNA is added to populations of single eukaryotic cells growing in culture, the nucleic acid enters the cells, and in some of them results in the production of new proteins. When a purified DNA is used, its incorporation leads to the production of a particular protein. Figure 1.6 depicts one of the standard systems. Although for historical reasons these experiments are described as transfection when performed with eukaryotic cells, they are a direct counterpart to bacterial transformation. The DNA that is introduced into the recipient cell becomes part of its genetic material, and is inherited in the same way as any other part. Its expression confers a new trait upon the cells (synthesis of thymidine kinase in the example of the figure). At first, these experiments were successful only with individual cells adapted to grow in a culture medium. Since then, however, DNA has been introduced into mouse eggs by microinjection; and it may become a stable part of the genetic material of the mouse (see 18.18 Genes can be injected into animal eggs). Such experiments show directly not only that DNA is the genetic material in eukaryotes, but also that it can be transferred between different species and yet remain functional. The genetic material of all known organisms and many viruses is DNA. However, some viruses use an alternative type of nucleic acid, ribonucleic acid (RNA), as the genetic material. The general principle of the nature of the genetic material, then, is that it is always nucleic acid; in fact, it is DNA except in the RNA viruses. |
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wangzhuoyjs
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gene 8 word 文档形式 chapter1 1.17-1.20
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1.17 Mutations may cause loss-of-function or gain-of-function Key Concepts • Recessive mutations are due to loss-of-function by the protein product. • Dominant mutations result from a gain-of-function. • Testing whether a gene is essential requires a null mutation (one that completely eliminates its function). • Silent mutations have no effect, either because the base change does not change the sequence or amount of protein, or because the change in protein sequence has no effect. • Leaky mutations do affect the function of the gene product, but are not revealed in the phenotype because sufficient activity remains. Figure 1.28 Mutations that do not affect protein sequence or function are silent. Mutations that abolish all protein activity are null. Point mutations that cause loss-of-function are recessive; those that cause gain-of-function are dominant. The various possible effects of mutation in a gene are summarized in Figure 1.28. When a gene has been identified, insight into its function in principle can be gained by generating a mutant organism that entirely lacks the gene. A mutation that completely eliminates gene function, usually because the gene has been deleted, is called a null mutation. If a gene is essential, a null mutation is lethal. To determine what effect a gene has upon the phenotype, it is essential to characterize a null mutant. When a mutation fails to affect the phenotype, it is always possible that this is because it is a leaky mutation—enough active product is made to fulfill its function, even though the activity is quantitatively reduced or qualitatively different from the wild type. But if a null mutant fails to affect a phenotype, we may safely conclude that the gene function is not necessary. Null mutations, or other mutations that impede gene function (but do not necessarily abolish it entirely) are called loss-of-function mutations. A loss-of-function mutation is recessive (as in the example of Figure 1.26). Sometimes a mutation has the opposite effect and causes a protein to acquire a new function; such a change is called a gain-of-function mutation. A gain-of-function mutation is dominant. Not all mutations in DNA lead to a detectable change in the phenotype. Mutations without apparent effect are called silent mutations. They fall into two types. Some involve base changes in DNA that do not cause any change in the amino acid present in the corresponding protein. Others change the amino acid, but the replacement in the protein does not affect its activity; these are called neutral substitutions. If a recessive mutation is produced by every change in a gene that prevents the production of an active protein, there should be a large number of such mutations in any one gene. Many amino acid replacements may change the structure of the protein sufficiently to impede its function. Different variants of the same gene are called multiple alleles, and their existence makes it possible to create a heterozygote between mutant alleles. The relationship between these multiple alleles takes various forms. In the simplest case, a wild-type gene codes for a protein product that is functional. Mutant allele(s) code for proteins that are nonfunctional. But there are often cases in which a series of mutant alleles have different phenotypes. For example, wild-type function of the white locus of D. melanogaster is required for development of the normal red color of the eye. The locus is named for the effect of extreme (null) mutations, which cause the fly to have a white eye in mutant homozygotes. To describe wild-type and mutant alleles, wild genotype is indicated by a plus superscript after the name of the locus (w+ is the wild-type allele for [red] eye color in D. melanogaster). Sometimes + is used by itself to describe the wild-type allele, and only the mutant alleles are indicated by the name of the locus. An entirely defective form of the gene (or absence of phenotype) may be indicated by a minus superscript. To distinguish among a variety of mutant alleles with different effects, other superscripts may be introduced, such as w' or wa. The w+ allele is dominant over any other allele in heterozygotes. There are many different mutant alleles. Figure 1.29 shows a (small) sample. Although some alleles have no eye color, many alleles produce some color. Each of these mutant alleles must therefore represent a different mutation of the gene, which does not eliminate its function entirely, but leaves a residual activity that produces a characteristic phenotype. These alleles are named for the color of the eye in a homozygote. (Most w alleles affect the quantity of pigment in the eye, and the examples in the figure are arranged in [roughly] declining amount of color, but others, such as wsp, affect the pattern in which it is deposited.) When multiple alleles exist, an animal may be a heterozygote that carries two different mutant alleles. The phenotype of such a heterozygote depends on the nature of the residual activity of each allele. The relationship between two mutant alleles is in principle no different from that between wild-type and mutant alleles: one allele may be dominant, there may be partial dominance, or there may be codominance. 1.19 A locus may have more than one wild-type allele Key Concepts • A locus may have a polymorphic distribution of alleles, with no individual allele that can be considered to be the sole wild-type. There is not necessarily a unique wild-type allele at any particular locus. Control of the human blood group system provides an example. Lack of function is represented by the null type, O group. But the functional alleles A and B provide activities that are codominant with one another and dominant over O group. The basis for this relationship is illustrated in Figure 1.30. The O (or H) antigen is generated in all individuals, and consists of a particular carbohydrate group that is added to proteins. The ABO locus codes for a galactosyltransferase enzyme that adds a further sugar group to the O antigen. The specificity of this enzyme determines the blood group. The A allele produces an enzyme that uses the cofactor UDP-N-acetylgalactose, creating the A antigen. The B allele produces an enzyme that uses the cofactor UDP-galactose, creating the B antigen. The A and B versions of the transferase protein differ in 4 amino acids that presumably affect its recognition of the type of cofactor. The O allele has a mutation (a small deletion) that eliminates activity, so no modification of the O antigen occurs. This explains why A and B alleles are dominant in the AO and BO heterozygotes: the corresponding transferase activity creates the A or B antigen. The A an"d B alleles are codominant in AB heterozygotes, because both transferase activities are expressed. The OO homozygote is a null that has neither activity, and therefore lacks both antigens. Neither A nor B can be regarded as uniquely wild type, since they represent alternative activities rather than loss or gain of function. A situation such as this, in which there are multiple functional alleles in a population, is described as a polymorphism (see 3.5 Individual genomes show extensive variation). 1.20 Recombination occurs by physical exchange of DNA Key Concepts • Recombination is the result of crossing-over that occurs at chiasmata and involves two of the four chromatids. • Recombination occurs by a breakage and reunion that proceeds via an intermediate of hybrid DNA. Genetic recombination describes the generation of new combinations of alleles that occurs at each generation in diploid organisms. The two copies of each chromosome may have different alleles at some loci. By exchanging corresponding parts between the chromosomes, recombinant chromosomes can be generated that are different from the parental chromosomes. Recombination results from a physical exchange of chromosomal material. This is visible in the form of the crossing-over that occurs during meiosis (the specialized division that produces haploid germ cells). Meiosis starts with a cell that has duplicated its chromosomes, so that it has four copies of each chromosome. Early in meiosis, all four copies are closely associated (synapsed) in a structure called a bivalent. Each individual chromosomal unit is called a chromatid at this stage. Pairwise exchanges of material occur between the chromatids. The visible result of a crossing-over event is called a chiasma, and is illustrated diagrammatically in Figure 1.31. A chiasma represents a site at which two of the chromatids in a bivalent have been broken at corresponding points. The broken ends have been rejoined crosswise, generating new chromatids. Each new chromatid consists of material derived from one chromatid on one side of the junction point, with material from the other chromatid on the opposite side. The two recombinant chromatids have reciprocal structures. The event is described as a breakage and reunion. Its nature explains why a single recombination event can produce only 50% recombinants: each individual recombination event involves only two of the four associated chromatids. The complementarity of the two strands of DNA is essential for the recombination process. Each of the chromatids shown in Figure 1.31 consists of a very long duplex of DNA. For them to be broken and reconnected without any loss of material requires a mechanism to recognize exactly corresponding positions. This is provided by complementary base pairing. Recombination involves a process in which the single strands in the region of the crossover exchange their partners. Figure 1.32 shows that this creates a stretch of hybrid DNA in which the single strand of one duplex is paired with its complement from the other duplex. The mechanism of course involves other stages (strands must be broken and resealed), and we discuss this in more detail in 75 Recombination and repair, but the crucial feature that makes precise recombination possible is the complementarity of DNA strands. The figure shows only some stages of the reaction, but we see that a stretch of hybrid DNA forms in the recombination intermediate when a single strand crosses over from one duplex to the other. Each recombinant consists of one parental duplex DNA at the left, connected by a stretch of hybrid DNA to the other parental duplex at the right. Each duplex DNA corresponds to one of the chromatids involved in recombination in Figure 1.31. The formation of hybrid DNA requires the sequences of the two recombining duplexes to be close enough to allow pairing between the complementary strands. If there are no differences between the two parental genomes in this region, formation of hybrid DNA will be perfect. But the reaction can be tolerated even when there are small differences. In this case, the hybrid DNA has points of mismatch, at which a base in one strand faces a base in the other strand that is not complementary to it. The correction of such mismatches is another feature of genetic recombination (see 15 Recombination and repair) |
6楼2006-05-05 22:34:01
wangzhuoyjs
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gene 8 word 文档形式 chapter1 1.5-1.9
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1.5 Polynucleotide chains have nitrogenous bases linked to a sugar-phosphate backbone The basic building block of nucleic acids is the nucleotide. This has three components: • a nitrogenous base; • a sugar; • and a phosphate. The nitrogenous base is a purine or pyrimidine ring. The base is linked to position 1 on a pentose sugar by a glycosidic bond from Ni of pyrimidines or N9 of purines. To avoid ambiguity between the numbering systems of the heterocyclic rings and the sugar, positions on the pentose are given a prime (')• Nucleic acids are named for the type of sugar; DNA has 2'-deoxyribose, whereas RNA has ribose. The difference is that the sugar in RNA has an OH group at the 2' position of the pentose ring. The sugar can be linked by its 5' or 3' position to a phosphate group. A nucleic acid consists of a long chain of nucleotides. Figure 1.7 shows that the backbone of the polynucleotide chain consists of an alternating series of pentose (sugar) and phosphate residues. This is constructed by linking the 5' position of one pentose ring to the 3' position of the next pentose ring via a phosphate group. So the sugar-phosphate backbone is said to consist of 5'-3' phosphodiester linkages. The nitrogenous bases "stick out" from the backbone. Each nucleic acid contains 4 types of base. The same two purines, adenine and guanine, are present in both DNA and RNA. The two pyrimidines in DNA are cytosine and thymine; in RNA uracil is found instead of thymine. The only difference between uracil and thymine is the presence of a methyl substituent at position C5. The bases are usually referred to by their initial letters. DNA contains A, G, C, T, while RNA contains A, G, C, U. The terminal nucleotide at one end of the chain has a free 5' group; the terminal nucleotide at the other end has a free 3' group. It is conventional to write nucleic acid sequences in the 5'—>3' direction—that is, from the 5' terminus at the left to the 3' terminus at the right. 1.6 DNA is a double helix The observation that the bases are present in different amounts in the DNAs of different species led to the concept that the sequence of bases is the form in which genetic information is carried. By the 1950s, the concept of genetic information was common: the twin problems it posed were working out the structure of the nucleic acid, and explaining how a sequence of bases in DNA could represent the sequence of amino acids in a protein. Three notions converged in the construction of the double helix model for DNA by Watson and Crick in 1953: • X-ray diffraction data showed that DNA has the form of a regular helix, making a complete turn every 34 A (3.4 nm), with a diameter of ~20 A (2 nm). Since the distance between adjacent nucleotides is 3.4 A, there must be 10 nucleotides per turn. • The density of DNA suggests that the helix must contain two polynucleotide chains. The constant diameter of the helix can be explained if the bases in each chain face inward and are restricted so that a purine is always opposite a pyrimidine, avoiding partnerships of purine-purine (too wide) or pyrimidine-pyrimidine (too narrow). • Irrespective of the absolute amounts of each base, the proportion of G is always the same as the proportion of C in DNA, and the proportion of A is always the same as that of T. So the composition of any DNA can be described by the proportion of its bases that is G + C. This ranges from 26% to 74% for different species. Watson and Crick proposed that the two polynucleotide chains in the double helix associate by hydrogen bonding between the nitrogenous bases. G can hydrogen bond specifically only with C, while A can bond specifically only with T. These reactions are described as base pairing, and the paired bases (G with C, or A with T) are said to be complementary. The model proposed that the two polynucleotide chains run in opposite directions (antiparallel), as illustrated in Figure 1.8. Looking along the helix, one strand runs in the 5'—>3' direction, while its partner runs 3'—»5'. The sugar-phosphate backbone is on the outside and carries negative charges on the phosphate groups. When DNA is in solution in vitro, the charges are neutralized by the binding of metal ions, typically by Na+. In the cell, positively charged proteins provide some of the neutralizing force. These proteins play an important role in determining the organization of DNA in the cell. The bases lie on the inside. They are flat structures, lying in pairs perpendicular to the axis of the helix. Consider the double helix in terms of a spiral staircase: the base pairs form the treads, as illustrated schematically in Figure 1.9. Proceeding along the helix, bases are stacked above one another, in a sense like a pile of plates. Each base pair is rotated ~36° around the axis of the helix relative to the next base pair. So ~10 base pairs make a complete turn of 360°. The twisting of the two strands around one another forms a double helix with a minor groove (~12 A across) and a major groove (~22 A across), as can be seen from the scale model of Figure 1.10. The double helix is right-handed; the turns run clockwise looking along the helical axis. These features represent the accepted model for what is known as the B-formofDNA. It is important to realize that the B-form represents an average, not a precisely specified structure. DNA structure can change locally. If it has more base pairs per turn it is said to be overwound; if it has fewer base pairs per turn it is underwound. Local winding can be affected by the overall conformation of the DNA double helix in space or by the binding of proteins to specific sites. 1.7 DNA replication is semiconservative It is crucial that the genetic material is reproduced accurately. Because the two polynucleotide strands are joined only by hydrogen bonds, they are able to separate without requiring breakage of covalent bonds. The specificity of base pairing suggests that each of the separated parental strands could act as a template strand for the synthesis of a complementary daughter strand. Figure 1.11 shows the principle that a new daughter strand is assembled on each parental strand. The sequence of the daughter strand is dictated by the parental strand; an A in the parental strand causes a T to be placed in the daughter strand, a parental G directs incorporation of a daughter C, and so on. The top part of the figure shows a parental (unreplicated) duplex that consists of the original two parental strands. The lower part shows the two daughter duplexes that are being produced by complementary base pairing. Each of the daughter duplexes is identical in sequence with the original parent, and contains one parental strand and one newly synthesized strand. The structure of DNA carries the information needed to perpetuate its sequence. The consequences of this mode of replication are illustrated in Figure 1.12. The parental duplex is replicated to form two daughter duplexes, each of which consists of one parental strand and one (newly synthesized) daughter strand. The unit conserved from one generation to the next is one of the two individual strands comprising the parental duplex. This behavior is called semiconservative replication. The figure illustrates a prediction of this model. If the parental DNA "heavy,, density label because the organism has been grown in medium containing a suitable isotope (such as 15N), its strands can be distinguished from those that are synthesized when the organism is transferred to a medium containing normal "light" isotopes. The parental DNA consists of a duplex of two heavy strands (red). After one generation of growth in light medium, the duplex DNA is "hybrid" in density—it consists of one heavy parental strand (red) and one light daughter strand (blue). After a second generation, the two strands of each hybrid duplex have separated; each gains a light partner, so that now half of the duplex DNA remains hybrid while half is entirely light (both strands are blue). The individual strands of these duplexes are entirely heavy or entirely light. This pattern was confirmed experimentally in the Meselson- Stahl experiment of 1958, which followed the semiconservative replication of DNA through three generations of growth of E. coll. When DNA was. extracted from bacteria and its density measured by centrifugation, the DNA formed bands corresponding to its density— heavy for parental, hybrid for the first generation, and half hybrid and half light in the second generation. 1.8 DNA strands separate at the replication fork Key Concepts • Replication of DNA is undertaken by a complex of enzymes that separate the parental strands and synthesize the daughter strands. • The replication fork is the point at which the parental strands are separated. • The enzymes that synthesize DNA are called DNA polymerases; the enzymes that synthesize RNA are RNA polymerases. • Nucleases are enzymes that degrade nucleic acids; they include DNAases and RNAases, and can be divided into endonucleases and exonucleases. Replication requires the two strands of the parental duplex to separate. However, the disruption of structure is only transient and is reversed as the daughter duplex is formed. Only a small stretch of the duplex DNA is separated into single strands at any moment. The helical structure of a molecule of DNA engaged in replication is illustrated in Figure 1.13. The nonreplicated region consists of the parental duplex, opening into the replicated region where the two daughter duplexes have formed. The double helical structure is disrupted at the junction between the two regions, which is called the replication fork. Replication involves movement of the replication fork along the parental DNA, so there is a continuous unwinding of the parental strands and rewinding into daughter duplexes.The synthesis of nucleic acids is catalyzed by specific enzymes, which recognize the template and undertake the task of catalyzing the addition of subunits to the polynucleotide chain that is being synthesized. The enzymes are named according to the type of chain that is synthesized: DNA polymerases synthesize DNA, and RNA polymerases synthesize RNA. Degradation of nucleic acids also requires specific enzymes: deoxyribonucleases (DNAases) degrade DNA, and ribonucleases (RNAases) degrade RNA. The nucleases fall into the general classes of exonucleases and endonucleases: Endonucleases cut individual bonds within RNA or DNA molecules, generating discrete fragments. Some DNAases cleave both strands of a duplex DNA at the target site, while others cleave only one of the two strands. Endonucleases are involved in cutting reactions, as shown in Figure 1.14. Exonucleases remove residues one at a time from the end of the molecule, generating mononucleotides. They always function on a single nucleic acid strand, and each exonuclease proceeds in a specific direction, that is, starting at either a 5' or at a 3' end and proceeding toward the other end. They are involved in trimming reactions, as shown in Figure 1.15. |
2楼2006-05-05 22:27:27
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gene 8 word 文档形式 chapter1 1.9
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1.9 Nucleic acids hybridize by base pairing Key Concepts • Heating causes the two strands of a DNA duplex to separate. • The Tm is the midpoint of the temperature range for denaturation. • Complementary single strands can renature when the temperature is reduced. • Denaturation and renaturation/hybridization can occur with DNA-DNA, DNA-RNA, or RNA-RNA combinations, and can be intermolecular or intramolecular. • The ability of two single-stranded nucleic acid preparations to hybridize is a measure of their complementarity. Acrucial property of the double helix is the ability to separate the two strands without disrupting covalent bonds. This makes it possible for the strands to separate and reform under physiological conditions at the (very rapid) rates needed to sustain genetic functions. The specificity of the process is determined by complementary base pairing. The concept of base pairing is central to all processes involving nucleic acids. Disruption of the base pairs is a crucial aspect of the function of a double-stranded molecule, while the ability to form base pairs is essential for the activity of a single-stranded nucleic acid. Figure 1.16 shows that base pairing enables complementary single-stranded nucleic acids to form a duplex structure. • An intramolecular duplex region can form by base pairing between two complementary sequences that are part of a single-stranded molecule. • A single-stranded molecule may base pair with an independent, complementary single-stranded molecule to form an intermolecular duplex. Formation of duplex regions from single-stranded nucleic acids is most important for RNA, but single-stranded DNA also exists (in the form of viral genomes). Base pairing between independent complementary single strands is not restricted to DNA-DNA or RNA-RNA, but can also occur between a DNA molecule and an RNA molecule. The lack of covalent links between complementary strands makes it possible to manipulate DNA in vitro. The noncovalent forces that stabilize the double helix are disrupted by heating or by exposure to low salt concentration. The two strands of a double helix separate entirely when all the hydrogen bonds between them are broken. The process of strand separation is called denaturation or (more colloquially) melting. ("Denaturation" is also used to describe loss of authentic protein structure; it is a general term implying that the natural conformation of a macromolecule has been converted to some other form.) Denaturation of DNA occurs over a narrow temperature range and results in striking changes in many of its physical properties. The midpoint of the temperature range over which the strands of DNA separate is called the melting temperature (Tm). It depends on the proportion of GC base pairs. Because each G-C base pair has three hydrogen bonds, it is more stable than an A-T base pair, which has only two hydrogen bonds. The more G-C base pairs are contained in a DNA, the greater the energy that is needed to separate the two strands. In solution under physiological conditions, a DNA that is 40% G-C—a value typical of mammalian genomes—denatures with a Tm of about 87°C. So duplex DNA is stable at the temperature prevailing in the cell. The denaturation of DNA is reversible under appropriate conditions. The ability of the two separated complementary strands to reform into a double helix is called renaturation. Renaturation depends on specific base pairing between the complementary strands. Figure 1.17 shows that the reaction takes place in two stages. First, single strands of DNA in the solution encounter one another by chance; if their sequences are complementary, the two strands base pair to generate a short doublehelical region. Then the region of base pairing extends along the molecule by a zipper-like effect to form a lengthy duplex molecule. Renaturation of the double helix restores the original properties that were lost when the DNA was denatured. Renaturation describes the reaction between two complementary sequences that were separated by denaturation. However, the technique can be extended to allow any two complementary nucleic acid sequences to react with each other to form a duplex structure. This is sometimes called annealing, but the reaction is more generally described as hybridization whenever nucleic acids of different sources are involved, as in the case when one preparation consists of DNA and the other consists of RNA. The ability of two nucleic acid preparations to hybridize constitutes a precise test for their complementarity since only complementary sequences can form a duplex structure. The principle of the hybridization reaction is to expose two singlestranded nucleic acid preparations to each other and then to measure the amount of double-stranded material that forms. Figure 1.18 illustrates a procedure in which a DNA preparation is denatured and the single strands are adsorbed to a filter. Then a second denatured DNA (or RNA) preparation is added. The filter is treated so that the second preparation can adsorb to it only if it is able to base pair with the DNA that was originally adsorbed. Usually the second preparation is radioactively labeled, so that the reaction can be measured as the amount of radioactive label retained by the filter. The extent of hybridization between two single-stranded nucleic acids is determined by their complementarity. Two sequences need not be perfectly complementary to hybridize. If they are closely related but not identical, an imperfect duplex is formed in which base pairing is interrupted at positions where the two single strands do not correspond. |
3楼2006-05-05 22:27:51
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gene 8 word 文档形式 chapter1 1.10-1.13
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1.10 Mutations change the sequence of DNA Key Concepts * All mutations consist of changes in the sequence of DNA. • Mutations may occur spontaneously or may be induced by mutagens. Mutations provide decisive evidence that DNA is the genetic material. When a change in the sequence of DNA causes an alteration in the sequence of a protein, we may conclude that the DNA codes for that protein. Furthermore, a change in the phenotype of the organism may allow us to identify the function of the protein. The existence of many mutations in a gene may allow many variant forms of a protein to be compared, and a detailed analysis can be used to identify regions of the protein responsible for individual enzymatic or other functions. All organisms suffer a certain number of mutations as the result of normal cellular operations or random interactions with the environment. These are called spontaneous mutations; the rate at which they occur is characteristic for any particular organism and is sometimes called the background level. Mutations are rare events, and of course those that damage a gene are selected against during evolution. It is therefore difficult to obtain large numbers of spontaneous mutants to study from natural populations. The occurrence of mutations can be increased by treatment with certain compounds. These are called mutagens, and the changes they cause are referred to as induced mutations. Most mutagens act directly by virtue of an ability either to modify a particular base of DNA or to become incorporated into the nucleic acid. The effectiveness of a mutagen is judged by how much it increases the rate of mutation above background. By using mutagens, it becomes possible to induce many changes in any gene. Spontaneous mutations that inactivate gene function occur in bacteriophages and bacteria at a relatively constant rate of 3-4 x 1(T3 per genome per generation. Given the large variation in genome sizes between bacteriophages and bacteria, this corresponds to wide differences in the mutation rate per base pair. This suggests that the overall rate of mutation has been subject to selective forces that have balanced the deleterious effects of most mutations against the advantageous effects of some mutations. This conclusion is strengthened by the observation that an archaeal microbe that lives under harsh conditions of high temperature and acidity (which are expected to damage DNA) does not show an elevated mutation rate, but in fact has an overall mutation rate just below the average range. Figure 1.19 shows that in bacteria, the mutation rate corresponds to ~1(T6 events per locus per generation or to an average rate of change per base pair of 10~9-10~10 per generation. The rate at individual base pairs varies very widely, over a 10,000 fold range. We have no accurate measurement of the rate of mutation in eukaryotes, although usually it is thought to be somewhat similar to that of bacteria on a per-locus pergeneration basis. We do not know what proportion of the spontaneous events results from point mutations. 1.11 Mutations may affect single base pairs or longer sequences Key Concepts • A point mutation changes a single base pair. • Point mutations can be caused by the chemical conversion of one base into another or by mistakes that occur during replication. • A transition replaces a G-C base pair with an A-T base pair or vice-versa. • A transversion replaces a purine with a pyrimidine, such as changing A-T to T-A. • Insertions are the most common type of mutation, and result from the movement of transposable elements. • Chemical modification of DNA directly changes one base into a different base. • A malfunction during the replication of DNA causes the wrong base to be inserted into a polynucleotide chain during DNA synthesis. Point mutations can be divided into two types, depending on the nature of the change when one base is substituted for another: • The most common class is the transition, comprising the substitution of one pyrimidine by the other, or of one purine by the other. This replaces a GC pai* with an AT pair or vice versa. • The less common class is the transversion, in which a purine is replaced by a pyrimidine or vice versa, so that an AT pair becomes a T A or C G pair. The effects of nitrous acid provide a classic example of a transition caused by the chemical conversion of one base into another. Figure 1.20 shows that nitrous acid performs an oxidative deamination that converts cytosine into uracil. In the replication cycle following the transition, the U pairs with an A, instead of with the G with which the original C would have paired. So the CG pair is replaced by a TA pair when the A pairs with the T in the next replication cycle. (Nitrous acid also deaminates adenine, causing the reverse transition from AT to GC.) Transitions are also caused by base mispairing, when unusual partners pair in defiance of the usual restriction to Watson-Crick pairs. Base mispairing usually occurs as an aberration resulting from the incorporation into DNA of an abnormal base that has ambiguous pairing properties. Figure 1.21 shows the example of bromouracil (BrdU), an analog of thymine that contains a bromine atom in place of the methyl group of thymine. BrdU is incorporated into DNA in place of thymine. But it has ambiguous pairing properties, because the presence of the bromine atom allows a shift to occur in which the base changes structure from a keto ( O) form to an enol (-OH) form. The enol form can base pair with guanine, which leads to substitution of the original AT pair by a GC pair. The mistaken pairing can occur either during the original incorporation of the base or in a subsequent replication cycle. The transition is induced with a certain probability in each replication cycle, so the incorporation of BrdU has continuing effects on the sequence of DNA. Point mutations were thought for a long time to be the principal means of change in individual genes. However, we now know that insertions of stretches of additional material are quite frequent. The source of the inserted material lies with transposable elements, sequences of DNA with the ability to move from one site to another (see 16 Transposons and 17 Retroviruses and retroposons). An insertion usually abolishes the activity of a gene. Where such insertions have occurred, deletions of part or all of the inserted material, and sometimes of the adjacent regions, may subsequently occur. A significant difference between point mutations and the insertions/ deletions is that the frequency of point mutation can be increased by mutagens, whereas the occurrence of changes caused by transposable elements is not affected. However, insertions and deletions can also occur by other mechanisms—for example, involving mistakes made during replication or recombination—although probably these are less common. And a class of mutagens called the acridines introduce (very small) insertions and deletions. 1.12 The effects of mutations can be reversed Key Concepts • Forward mutations inactivate a gene, and back mutations (or revertants) reverse their effects. • Insertions can revert by deletion of the inserted material, but deletions cannot revert. • Suppression occurs when a mutation in a second gene bypasses the effect of mutation in the first gene. Figure 1.22 shows that the isolation of revertants is an important" characteristic that distinguishes point mutations and insertions from deletions: • A point mutation can revert by restoring the original sequence or by gaining a compensatory mutation elsewhere in the gene. • An insertion of additional material can revert by deletion of the inserted material. • A deletion of part of a gene cannot revert. Mutations that inactivate a gene are called forward mutations. Their effects are reversed by back mutations, which are of two types. An exact reversal of the original mutation is called true reversion. So if an AT pair has been replaced by a GC pair, another mutation to restore the AT pair will exactly regenerate the wild-type sequence. Alternatively, another mutation may occur elsewhere in the gene, and its effects compensate for the first mutation. This is called secondsite reversion. For example, one amino acid change in a protein may abolish gene function, but a second alteration may compensate for the first and restore protein activity. A forward mutation results from any change that inactivates a gene, whereas a back mutation must restore function to a protein damaged by a particular forward mutation. So the demands for back mutation are much more specific than those for forward mutation. The rate of back mutation is correspondingly lower than that of forward mutation, typically by a factor of ~\ 0. Mutations can also occur in other genes to circumvent the effects of mutation in the original gene. This effect is called suppression. A locus in which a mutation suppresses the effect of a mutation in another locus is called a suppressor. 1.13 Mutations are concentrated at hotspots Key Concepts ' The frequency of mutation at any particular base pair is determined by statistical fluctuation, except for hotspots, where the frequency is increased by at least an order of magnitude. So far we have dealt with mutations in terms of individual changes in the sequence of DNA that influence the activity of the genetic unit in which they occur. When we consider mutations in terms of the inactivation of the gene, most genes within a species show more or less similar rates of mutation relative to their size. This suggests that the gene can be regarded as a target for mutation, and that damage to any part of it can abolish its function. As a result, susceptibility to mutation is roughly proportional to the size of the gene. But consider the sites of mutation within the sequence of DNA; are all base pairs in a gene equally susceptible or are some more likely to be mutated than others? What happens when we isolate a large number of independent mutations in the same gene? Many mutants are obtained. Each is the result of an individual mutational event. Then the site of each mutation is determined. Most mutations will lie at different sites, but some will lie at the same position. Two independently isolated mutations at the same site may constitute exactly the same change in DNA (in which case the same mutational event has happened on more than one occasion), or they may constitute different changes (three different point mutations are possible at each base pair). The histogram of Figure 1.23 shows the frequency with which mutations are found at each base pair in the lad gene of E. coli. The statistical probability that more than one mutation occurs at a particular site is given by random-hit kinetics (as seen in the Poisson distribution). So some sites will gain one, two, or three mutations, while others will not gain any. But some sites gain far more than the number of mutations expected from a random distribution; they may have 10x or even 100x more mutations than predicted by random hits. These sites are called hotspots. Spontaneous mutations may occur at hotspots; and different mutagens may have different hotspots. |
4楼2006-05-05 22:30:44
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