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[交流] gene 8 word 文档形式 chapter1 1.1-1.4

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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gene 8 word 文档形式 chapter1 1.5-1.9

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.
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gene 8 word 文档形式 chapter1 1.9

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.
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gene 8 word 文档形式 chapter1 1.10-1.13

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.
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gene 8 word 文档形式 chapter1 1.14-1.16

1.14 Many hotspots result from modified bases
Key Concepts
• A common cause of hotspots is the modified base  5-methylcytosine, which is spontaneously deaminated to thymine.  Amajor cause of spontaneous mutation results from the presence  of an unusual base in the DNA. In addition to the four bases that  are inserted into DNA when it is synthesized, modified bases are sometimes  found. The name reflects their origin; they are produced by chemically  modifying one of the four bases already present in DNA. The  most common modified base is 5-methylcytosine, generated by a  methylase enzyme that adds a methyl group to certain cytosine residues  at specific sites in the DNA.  Sites containing 5-methylcytosine provide hotspots for spontaneous  point mutation in E. coli. In each case, the mutation takes the form of a  GC to AT transition. The hotspots are not found in strains of E. coli  that cannot methylate cytosine.  The reason for the existence of the hotspots is that cytosine bases  suffer spontaneous deamination at an appreciable frequency. In this reaction,  the amino group is replaced by a keto group. Recall that deamination  of cytosine generates uracil (see Figure 1.20). Figure 1.24  compares this reaction with the deamination of 5-methylcytosine where  deamination generates thymine. The effect in DNA is to generate the  base pairs GU and GT, respectively, where there is a mismatch between  the partners.  All organisms have repair systems that correct mismatched base  pairs by removing and replacing one of the bases. The operation of  these systems determines whether mismatched pairs such as GU and  GT result in mutations.  Figure 1.25 shows that the consequences of deamination are different  for 5-methylcytosine and cytosine. Deaminating the (rare) 5-methylcytosine  causes a mutation, whereas deamination of the more common cytosine  does not have this effect. This happens because the repair systems  are much more effective in recognizing GU than G-T.  E. coli contains an enzyme, uracil-DNA-glycosidase, that removes  uracil residues from DNA (see 15.22 Base flipping is used by methylases  and glycosylases). This action leaves an unpaired G residue, and a "repair  system" then inserts a C base to partner it. The net result of these reactions  is to restore the original sequence of the DNA. This system  protects DNA against the consequences of spontaneous deamination of  cytosine (although it is not active enough to prevent the effects of the increased  level of deamination caused by nitrous acid; see Figure 1.20).  But the deamination of 5-methylcytosine leaves thymine. This creates"  a mismatched base pair, G-T. If the mismatch is not corrected before the  next replication cycle, a mutation results. At the next replication, the  bases in the mispaired G-T partnership separate, and then they pair with  new partners to produce one wild-type G-C pair and one mutant AT pair.  Deamination of 5-methylcytosine is the most common cause of production  of G-T mismatched pairs in DNA. Repair systems that act on  G-T mismatches have a bias toward replacing the T with a C (rather than  the alternative of replacing the G with an A), which helps to reduce the  rate of mutation (see 15.24 Controlling the direction of mismatch repair).  However, these systems are not as effective as the removal of U  from GU mismatches. As a result, deamination of 5-methylcytosine  leads to mutation much more often than does deamination of cytosine.  5-methylcytosine also creates hotspots in eukaryotic DNA. It is  common at CpG dinucleotides that are concentrated in regions called  CpG islands (see 21.19 CpG islands are regulatory targets). Although  5-methylcytosine accounts for - 1 % of the bases in human DNA, sites  containing the modified base account for -30% of all point mutations.  This makes the state of 5-methylcytosine a particularly important determinant  of mutation in animal cells.  The importance of repair systems in reducing the rate of mutation is  emphasized by the effects of eliminating the mouse enzyme MBD4, a  glycosylase that can remove T (or U) from mismatches with G. The result  is to increase the mutation rate at CpG sites by a factor of 3x. (The  reason the effect is not greater is that MBD4 is only one of several systems  that act on G-T mismatches; we can imagine that elimination of all  the systems would increase the mutation rate much more.)  The operation of these systems casts an interesting light on the use of T  in DNA compared with U in RNA. Perhaps it relates to the need of DNA  for stability of sequence; the use of T means that any deaminations of C  are immediately recognized, because they generate a base (U) not usually  present in the DNA. This greatly increases the efficiency with which repair  systems can function (compared with the situation when they have to  recognize G-T mismatches, which can be produced also by situations  where removing the T would not be the appropriate response). Also, the  phosphodiester bond of the backbone is more labile when the base is U.
1.15 A gene codes for a single polypeptide
Key Concepts
• The one gene: one enzyme hypothesis summarizes the basis of modern genetics: that a gene is a stretch of DNA coding for a single polypeptide chain.
• Most mutations damage gene function.
The first systematic attempt to associate genes with enzymes  showed that each stage in a metabolic pathway is catalyzed by a  single enzyme and can be blocked by mutation in a different gene. This  led to the one gene: one enzyme hypothesis. Each metabolic step is catalyzed  by a particular enzyme, whose production is the responsibility of  a single gene. A mutation in the gene alters the activity of the protein  for which it is responsible.  A modification in the hypothesis is needed to accommodate proteins  that consist of more than one subunit. If the subunits are all the same,  the protein is a homomultimer, represented by a single gene. If the subunits  are different, the protein is a heteromultimer. Stated as a more  general rule applicable to any heteromultimeric protein, the one gene:  one enzyme hypothesis becomes more precisely expressed as one gene:  one polypeptide chain.  Identifying which protein represents a particular gene can be a protracted  task. The mutation responsible for creating Mendel's wrinkledpea  mutant was identified only in 1990 as an alteration that inactivates  the gene for a starch branching enzyme!  It is important to remember.that a gene does not directly generate a  protein. As shown previously in Figure 1.2, a gene codes for an RNA,  which may in turn code for a protein. Most genes code for proteins, but  some genes code for RNAs that do not give rise to proteins. These  RNAs may be structural components of the apparatus responsible for  synthesizing proteins or may have roles in regulating gene expression.  The basic principle is that the gene is a sequence of DNA that specifies  the sequence of an independent product. The process of gene expression  may terminate in a product that is either RNA or protein.  A mutation is a random event with regard to the structure of the  gene, so the greatest probability is that it will damage or even abolish  gene function. Most mutations that affect gene function are recessive:  they represent an absence of function, because the mutant gene has  been prevented from producing its usual protein. Figure 1.26 illustrates  the relationship between recessive and wild-type alleles. When a  heterozygote contains one wild-type allele and one mutant allele, the  wild-type allele is able to direct production of the enzyme. The wildtype  allele is therefore dominant. (This assumes that an adequate  amount of protein is made by the single wild-type allele. When this is  not true, the smaller amount made by one allele as compared to two  alleles results in the intermediate phenotype of a partially dominant  allele in a heterozygote.)  
1.16 Mutations in the same gene cannot
complement
Key Concepts
• A mutation in a gene affects only the protein coded by the mutant copy of the gene, and does not affect the protein coded by any other allele.
* Failure of two mutations to complement (produce wild-phenotype)
when they are present in trans configuration in a heterozygote  means that they are part of the same gene.  How do we determine whether two mutations that cause a similar  phenotype lie in the same gene? If they map close together, they  may be alleles. However, they could also represent mutations in two dif-  ferent genes whose proteins are involved in the same function. The  complementation test is used to determine whether two mutations lie in  the same gene or in different genes. The test consists of making a heterozygote  for the two mutations (by mating parents homozygous for  each mutation).  If the mutations lie in the same gene, the parental genotypes can be  represented as  The first parent provides an ml mutant allele and the second parent  provides an m-i allele, so that the heterozygote has the constitution  No wild-type gene is present, so the heterozygote has mutant phenotype.  If the mutations lie in different genes, the parental genotypes can be  represented as  Each chromosome has a wild-type copy of one gene (represented by  the plus sign) and a mutant copy of the other. Then the heterozygote has  the constitution  in which the two parents between them have provided a wild-type  copy of each gene. The heterozygote has wild phenotype; the two  genes are said to complement.  The complementation test is shown in more detail in Figure  1.27. The basic test consists of the comparison shown in the top  part of the figure. If two mutations lie in the same gene, we see a  difference in the phenotypes of the trans configuration and the  cis configuration. The trans configuration is mutant, because  each allele has a (different) mutation. But the cis configuration is  wild-type, because one allele has two mutations but the other  allele has no mutations. The lower part of the figure shows that  if the two mutations lie in different genes, we always see a wild  phenotype. There is always one wild-type and one mutant allele  of each gene, and the configuration is irrelevant.  Failure to complement means that two mutations are part of the  same genetic unit. Mutations that do not complement one another  are said to comprise part of the same complementation group. Another  term that is used to describe the unit defined by the complementation  test is the cistron. This is the same as the gene. Basically  these three terms all describe a stretch of DNA that functions as a  unit to give rise to an RNA or protein product. The properties of the gene  with regards to complementation are explained by the fact that this product  is a single molecule that behaves as a functional unit.  double mutant  Mutations in
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gene 8 word 文档形式 chapter1 1.17-1.20

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)
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gene 8 word 文档形式 chapter1 1.22-1.24

. 1.21 The genetic code is triplet Key Concepts •
The genetic code is read in triplet nucleotides called codons. • The triplets are nonoverlapping and are read from a fixed starting point. • Mutations that insert or delete individual bases cause a shift in the triplet sets after the site of mutation. • Combinations of mutations that together insert or delete 3 bases (or multiples of three) insert or delete amino acids but do not change the reading of the triplets beyond the last site of mutation.  Each gene represents a particular protein chain. The concept that  each protein consists of a particular series of amino acids dates  from Sanger's characterization of insulin in the 1950s. The discovery  that a gene consists of DNA faces us with the issue of how a sequence  of nucleotides in DNA represents a sequence of amino acids in  protein.  A crucial feature of the general structure of DNA is that it is independent  of the particular sequence of its component nucleotides. The  sequence of nucleotides in DNA is important not because of its structure  per se, but because it codes for the sequence of amino acids that  constitutes the corresponding polypeptide. The relationship between a  sequence of DNA and the sequence of the corresponding protein is  called the genetic code.  The structure and/or enzymatic activity of each protein follows from  its primary sequence of amino acids. By determining the sequence of  amino acids in each protein, the gene is able to carry all the information  needed to specify an active polypeptide chain. In this way, a single type of  structure—the gene—is able to represent itself in innumerable polypeptide  forms.  Together the various protein products of a cell undertake the catalytic  and structural activities that are responsible for establishing its phenotype.  Of course, in addition to sequences that code for proteins, DNA also  contains certain sequences whose function is to be recognized by regulator  molecules, usually proteins. Here the function of the DNA is determined  by its sequence directly, not via any intermediary code. Both types  of regions, genes expressed as proteins and sequences recognized as  such, constitute genetic information.  The genetic code is deciphered by a complex apparatus that interprets  the nucleic acid sequence. This apparatus is essential if the information  carried in DNA is to have meaning. In any given region, only  one of the two strands of DNA codes for protein, so we write the genetic  code as a sequence of bases (rather than base pairs).  The genetic code is read in groups of three nucleotides, each group  representing one amino acid. Each trinucleotide sequence is called a  codon. A gene includes a series of codons that is read sequentially from a  starting point at one end to a termination point at the other end. Written in  the conventional 5'—>3' direction, the nucleotide sequence of the DNA  strand that codes for protein corresponds to the amino acid sequence of  the protein written in the direction from N-terminus to C-terminus.  The genetic code is read in nonoverlapping triplets from a fixed  starting point:  ' Nonoverlapping implies that each codon consists of three nucleotides  and that successive codons are represented by successive trinucleotides.  * The use of a fixed starting point means that assembly of a protein  must start at one end and work to the other, so that different parts of  the coding sequence cannot be read independently.  The nature of the code predicts that two types of mutations will have  different effects. If a particular sequence is read sequentially, such as  UUU AAA GGG CCC (codons)  aal aa2 aa3 aa4 (amino acids)  then a point mutation will affect only one amino acid. For example, because  only the second codon has been changed, the substitution of an A  by some other base (X) causes aa2 to be replaced by aa5:  UUU AAX GGG CCC  aal aa5 aa3 aa4  But a mutation that inserts or deletes a single base will change the  triplet sets for the entire subsequent sequence. A change of this sort is  called a frameshift. An insertion might take the following form:  UUU AAX AGG GCC C  aal aa5 aa6 aa7  Because the new sequence of triplets is completely different from  the old one, the entire amino acid sequence of the protein is altered beyond  the site of mutation. So the function of the protein is likely to be  lost completely.  Frameshift mutations are induced by the acridines, compounds that  bind to DNA and distort the structure of the double helix, causing additional  bases to be incorporated or omitted during replication. Each mutagenic  event sponsored by an acridine results in the addition or  removal of a single base pair.  If an acridine mutant is produced by, say, addition of a nucleotide, it  should revert to wild type by deletion of the nucleotide. But reversion  can also be caused by deletion of a different base, at a site close to the  first. Combinations of such mutations provided revealing evidence  about the nature of the genetic code.  Figure 1.33 illustrates the properties of frameshift mutations. An  insertion or a deletion changes the entire protein sequence following  the site of mutation. But the combination of an insertion and a deletion  causes the code to be read incorrectly only between the two sites of mutation;  correct reading resumes after the second site.  Genetic analysis of acridine mutations in the rll region of the phage  T6 in 1961 showed that all the mutations could be classified into one  of two sets, described as (+) and (-). Either type of mutation by itself  causes a frameshift, the (+) type by virtue of a base addition, the (-) .  type by virtue of a base deletion. Double mutant combinations of the  types (+ +) and (—) continue to show mutant behavior. But combinations  of the types (+ -) or (- +) suppress one another, giving rise to a  description in which one mutation is described as a suppressor of the  other. (In the context of this work, "suppressor" is used in an unusual  sense, because the second mutation is in the same gene as the first.)  These results show that the genetic code must be read as a sequence  that is fixed by the starting point, so additions or deletions compensate  for each other, whereas double additions or double deletions remain mutant.  But this does not reveal how many nucleotides make up each codon.  When triple mutants are constructed, only (+ + +) and ( ) combinations  show the wild phenotype, while other combinations remain  mutant. If we take three additions or three deletions to correspond respectively  to the addition or omission overall of a single amino acid,  this implies that the code is read in triplets. An incorrect amino acid sequence  is found between the two outside sites of mutation, and the sequence  on either side remains wild type, as indicated in Figure 1.33.

1.22 Every sequence has three possible reading frames
Key Concepts
• Usually only one reading frame is translated and the other two are blocked by frequent termination signals. If the genetic code is read in nonoverlapping triplets, there are three possible ways of translating any nucleotide sequence into protein, depending on the starting point. These called reading frames. For the sequence ACGACGACGACGACGACG the three possible reading frames are
ACG ACG ACG ACG ACG ACG ACG
CGA CGA CGA CGA CGA CGA CGA
GAC GAC GAC GAC GAC GAC GAC
A reading frame that consists exclusively of triplets representing amino acids is called an open reading frame or ORF. A sequence that istranslated into protein has a reading frame that starts with a special initiation codon (AUG) and that extends through a series of triplets representing amino acids until it ends at one of three types of termination codon (see 5 Messenger RNA).  By comparing the nucleotide sequence of a gene with the amino  acid sequence of a protein, we can determine directly whether the  gene and the protein are colinear: whether the sequence of nucleotides in  the gene corresponds exactly with the sequence of amino acids in the  protein. In bacteria and their viruses, there is an exact equivalence. Each  gene contains a continuous stretch of DNA whose length is directly  related to the number of amino acids in the protein that it represents. A  gene of SA'bp is required to code for a protein of N amino acids, according  to the genetic code.  The equivalence of the bacterial gene and its product means that a  physical map of DNA will exactly match an amino acid map of the protein.  How well do these maps fit with the recombination map?  The colinearity of gene and protein was originally investigated in  the tryptophan synthetase gene of E. coli. Genetic distance was measured  by the percent recombination between mutations; protein distance  was measured by the number of amino acids separating sites of replacement.  Figure 1.35 compares the two maps. The order of seven sites of  mutation is the same as the order of the corresponding sites of amino  acid replacement. And the recombination distances are relatively similar  to the actual distances in the protein. The recombination map expands  the distances between some mutations, but otherwise there is  little distortion of the recombination map relative to the physical map.  The recombination map makes two further general points about the  organization of the gene. Different mutations may cause a wild-type  amino acid to be replaced with different substituents. If two such mutations  cannot recombine, they must involve different point mutations at  the same position in DNA. If the mutations can be separated on the genetic  map, but affect the same amino acid on the upper map (the con-  A reading frame that cannot be read into protein because terition  J mination codons occur frequently is said to be blocked. If a sei  : quence is blocked in all three reading frames, it cannot have the  function of coding for protein.  :kedS When the sequence of a DNA region of unknown function is  issEi obtained, each possible reading frame is analyzed to determine  whether it is open or blocked. Usually no more than one of the  three possible frames of reading is open in any single stretch of  DNA. Figure 1.34 shows an example of a sequence that can be read in  only one reading frame, because the alternative reading frames are  blocked by frequent termination codons. A long open reading frame is  unlikely to exist by chance; if it were not translated into protein, there  would have been no selective pressure to prevent the accumulation of  termination codon-s. So the identification of a lengthy open reading  frame is taken to be prima facie evidence that the sequence is translated  into protein in that frame. An open reading frame (ORF) for which no  protein product has been identified is sometimes called an unidentified  reading frame (URF).  necting lines converge in the figure), they must involve point mutations  at different positions that affect the same amino acid. This happens because  the unit of genetic recombination (actually 1 bp) is smaller than  the unit coding for the amino acid (actually 3 bp).
1.24 Several processes are required to express the protein product of a gene
In comparing gene and protein, we are restricted to dealing with the  sequence of DNA stretching between the points corresponding to  the ends of the protein. However, a gene is not directly translated into  protein, but is expressed via the production of a messenger RNA (abbreviated  to mRNA), a nucleic acid intermediate actually used to synthesize  a protein (as we see in detail in 5 Messenger RNA).  Messenger RNA is synthesized by the same process of complementary  base pairing used to replicate DNA, with the important difference  that it corresponds to only one strand of the DNA double helix. Figure  1.36 shows that the sequence of messenger RNA is complementary  with the sequence of one strand of DNA and is identical (apart from the  replacement of T with U) with the other strand of DNA. The convention  for writing DNA sequences is that the top strand runs 5'—>3', with the  sequence that is the same as RNA.  The process by which a gene gives rise to a protein is called gene expression.  In bacteria, it consists of two stages. The first stage is transcription,  when an mRNA copy of one strand of the  DNA is produced. The second stage is translation of the  mRNA into protein. This is the process by which the sequence  of an mRNA is read in triplets to give the series  of amino acids that make the corresponding protein.  A messenger RNA includes a sequence of nucleotides  that corresponds with the sequence of amino acids  in the protein. This part of the nucleic acid is called the  coding region. But the messenger RNA includes additional  sequences on either end; these sequences do not  directly represent protein. The 5' nontranslated region is  called the leader, and the 3' nontranslated region is  called the trailer.  The gene includes the entire sequence represented in  messenger RNA. Sometimes mutations impeding gene  function are found in the additional, noncoding regions,  confirming the view that these comprise a legitimate  part of the genetic unit.  Figure 1.37 illustrates this situation, in which the  gene is considered to comprise a continuous stretch of DNA, needed to  produce a particular protein. It includes the sequence coding for that  protein, but also includes sequences on either side of the coding region.  A bacterium consists of only a single compartment,  so transcription and translation occur in the same place,  as illustrated in Figure 1.38.  In eukaryotes transcription occurs in the nucleus, but  the RNA product must be transported to the cytoplasm in  order to be translated, as shown in Figure 1.39. For the  simplest eukaryotic genes (just like in bacteria) the transcript  RNA is in fact the mRNA. But for more complex  genes, the immediate transcript of the gene is a premRNA  that requires processing to generate the mature  mRNA. The basic stages of gene expression in a eukaryote  are outlined in Figure 1.40.  The most important stage in processing is RNA splicing.  Many genes in eukaryotes (and a majority in higher  eukaryotes) contain internal regions that do not code for  protein. The process of splicing removes these regions  from the pre-mRNA to generate an RNA that has a continuous  open reading frame (see Figure 2.1). Other processing  events that occur at this stage involve the  modification of the "5' and 3' ends of the pre-mRNA (see  Figure 5.16).  Translation is accomplished by a complex apparatus  that includes both protein and RNA components. The actual  "machine" that undertakes the process is the ribosorne,  a large complex that includes some large RNAs  (ribosomalRNAs, abbreviated to rRNAs) and many small  proteins. The process of
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gene 8 word 文档形式 chapter1 1.23-1.28

1.26 Genetic information can be provided by DNA or RNA
The central dogma defines the paradigm of molecular biology.  Genes are perpetuated as sequences of nucleic acid, but function  by being expressed in the form of proteins. Replication is responsible  for the inheritance of genetic information. Transcription and translation  are responsible for its conversion from one form to another.  Figure 1.44 illustrates the roles of replication, transcription, and  translation, viewed from the perspective of the central dogma:  • The perpetuation of nucleic acid may involve either DNA or RNA as  the genetic material. Cells use only DNA. Some viruses use RNA,  and replication of viral RNA occurs in the infected cell.  • The expression of cellular genetic information usually is unidirectional.  Transcription of DNA generates RNA molecules that can be  used further only to generate protein sequences; generally they cannot  be retrieved for use as genetic information. Translation of RNA  into protein is always irreversible.  These mechanisms are equally effective for the cellular genetic information  of prokaryotes or eukaryotes, and for the information carried  by viruses. The genomes of all living organisms consist of duplex DNA.  Viruses have genomes that consist of DNA or RNA; and there are examples  of each type that are double-stranded (ds) or single-stranded  (ss). Details of the mechanism used to replicate the nucleic acid vary  among the viral systems, but the principle of replication via synthesis  of complementary strands remains the same, as illustrated in Figure  1.45.  Cellular genomes reproduce DNA by the mechanism of semi-conservative  replication. Double-stranded virus genomes, whether DNA or  RNA, also replicate by using the individual strands of the duplex as  templates to synthesize partner strands.  Viruses with single-stranded genomes use the single strand as a template  to synthesize a complementary strand; and this complementary  strand in turn is used to synthesize its complement, which is, of course,  identical with the original starting strand. Replication may involve  the formation of stable double-stranded intermediates or may use doublestranded  nucleic acid only as a transient stage.  The restriction to unidirectional transfer from DNA to RNA is not  absolute. It is overcome by the retroviruses, whose genomes consist of  single-stranded RNA molecules. During the infective cycle, the RNA is  converted by the process of reverse transcription into a single-stranded  DNA, which in turn is converted into a double-stranded DNA. This duplex  DNA becomes part of the genome of the cell, and is inherited like  any other gene. So reverse transcription allows a sequence of RNA to be  retrieved and used as genetic information.  The existence of RNA replication and reverse transcription establishes  the general principle that information in the form of either type of  nucleic acid sequence can be converted into the other type. In the usual  course of events, however, the cell relies on the processes of DNA replication,  transcription, and translation. But on rare occasions (possibly  mediated by an RNA virus), information from a cellular RNA is converted  into DNA and inserted into the genome. Although reverse transcription  plays no role in the regular operations of the cell, it becomes a  mechanism of potential importance when we consider the evolution of  the genome.  The same principles are followed to perpetuate genetic information  from the massive genomes of plants or amphibians to the tiny genomes  of mycoplasma and the yet smaller genetic information of DNA or  RNA viruses. Figure 1.46 summarizes some examples that illustrate  the range of genome types and sizes.  Throughout the range of organisms, with genomes varying in total  content over a 100,000 fold range, a common principle prevails. The DNA  codes for all the proteins that the cell(s) of the organism must synthesize;  and the proteins in turn (directly or indirectly) provide the functions  needed for survival. A similar principle describes the function of the  genetic information of viruses, whether DNA or RNA. The nucleic acid  codes for the protein(s) needed to package the genome and also for any  functions additional to those provided by the host cell that are needed to  reproduce the virus during its infective cycle. (The smallest virus, the  satellite tobacco necrosis virus [STNV], cannot replicate independently,  but requires the simultaneous presence of a "helper" virus [tobacco  necrosis virus, TNV], which is itself a normally infectious virus.)
1.27 Some hereditary agents are extremely small
Viroids are infectious agents that cause diseases in higher plants.  They are very small circular molecules of RNA. Unlike viruses,  where the infectious agent consists of a virion, a genome encapsulated in a_  protein coat, the viroid RNA is itself the infectious agent. The viroid consists  solely of the RNA, which is extensively but imperfectly base paired,  forming a characteristic rod like the example shown in Figure 1.47. Mutations  that interfere with the structure of the rod reduce infectivity.  A viroid RNA consists of a single molecular species that is replicated  autonomously in infected cells. Its sequence is faithfully perpetuated in  its descendants. Viroids fall into several groups. A given viroid is identified  with a group by its similarity of sequence with other members of the  group. For example, four viroids related to PSTV (potato spindle tuber  viroid) have 70-83% similarity of sequence with it. Different isolates of a  particular viroid strain vary from one another, and the change may affect  the phenotype of infected cells. For example, the mild and severe strains  of PSTV differ by three nucleotide substitutions.  Viroids resemble viruses in having heritable nucleic acid genomes.  They fulfill the criteria for genetic information. Yet viroids differ from  viruses in both structure and function. They are sometimes called subviral  pathogens. Viroid RNA does not appear to be translated into protein.  So it cannot itself code for the functions needed for its survival.  This situation poses two questions. How does viroid RNA replicate?  And how does it affect the phenotype of the infected plant cell?  Replication must be carried out by enzymes of the host cell, subverted  from their normal function. The heritability of the viroid sequence  indicates that viroid RNA provides the template.  Viroids are presumably pathogenic because they interfere with normal  cellular processes. They might do this in a relatively random way,  for example, by sequestering an essential enzyme for their own replication  or by interfering with the production of necessary cellular RNAs.  Alternatively, they might behave as abnormal regulatory molecules,  with particular effects upon the expression of individual genes.  An even more unusual agent is scrapie, the cause of a degenerative  neurological disease of sheep and goats. The disease is related to the  human diseases of kuru and Creutzfeldt-Jakob syndrome, which affect  brain function.  The infectious agent of scrapie does not contain nucleic acid. This  extraordinary agent is called a prion (proteinaceous infectious agent). It is  a 28 kD hydrophobic glycoprotein, PrP. PrP is coded by a cellular gene  (conserved among the mammals) that is expressed in normal brain. The  protein exists in two forms. The product found in normal brain is called  PrPc. It is entirely degraded by proteases. The protein found in infected  brains is called PrPsc. It is extremely resistant to degradation by proteases.  PrPc is converted to PrPsc by a modification or conformational change that  confers protease-resistance, and which has yet to be fully defined.  As the infectious agent of scrapie, PrPsc must in some way modify  the synthesis of its normal cellular counterpart so that it becomes  infectious instead of harmless (see 23.24 Prions cause diseases in  mammals). Mice that lack a PrP gene cannot be infected to develop  scrapie, which demonstrates that PrP is essential for development of the  disease.
1.28 Summary
Two classic experiments proved that DNA is the genetic material.  DNA isolated from one strain of Pneumococcus bacteria  can confer properties of that strain upon another strain. And DNA is  the only component that is inherited by progeny phages from the  parental phages. DNA can be used to transfect new properties into  eukaryotic cells.  DNA is a double helix consisting of antiparallel strands in which  the nucleotide units are linked by 5'-3' phosphodiester bonds. The  backbone provides the exterior; purine and pyrimidine bases are  stacked in the interior in pairs in which A is complementary to T  while G is complementary to C. The strands separate and use complementary  base pairing to assemble daughter strands in semiconservative  replication. Complementary base pairing is also used to  transcribe an RNA representing one strand of a DNA duplex.  A stretch of DNA may code for protein. The genetic code describes  the relationship between the sequence of DNA and the sequence  of the protein. Only one of the two strands of DNA codes for  protein. A codon consists of three nucleotides that represent a single  amino acid. A coding sequence of DNA consists of a series of  codons, read from a fixed starting point. Usually only one of the  three possible reading frames can be translated into protein.  A chromosome consists of an uninterrupted length of duplex  DNA that contains many genes. Each gene (or cistron) is transcribed  into an RNA product, which in turn is translated into a polypeptide  sequence if the gene codes for protein. An RNA or protein product of  a gene is said to be frans-acting. A gene is defined as a unit on a single  stretch of DNA by the complementation test. A site on DNA that  regulates the activity of an adjacent gene is said to be c/s-acting.  A gene may have multiple alleles. Recessive alleles are caused by  a loss-of-function. A null allele has total loss-of-function. Dominant  alleles are caused by gain-of-function.  A mutation consists of a change in the sequence of AT and GC  base pairs in DNA. A mutation in a coding sequence may change the  sequence of amino acids in the corresponding protein. A frameshift  mutation alters the subsequent reading frame by inserting or deleting  a base; this causes an entirely new series of amino acids to be  coded after the site of mutation. A point mutation changes only the  amino acid represented by the codon in which the mutation occurs.  Point mutations may be reverted by back mutation of the original  mutation. Insertions may revert by loss of the inserted material, but  deletions cannot revert. Mutations may also be suppressed indirectly  when a mutation in a different gene counters the original defect.  The natural incidence of mutations is increased by mutagens.  Mutations may be concentrated at hotspots. A type of hotspot responsible  for some point mutations is caused by deamination of the  modified base 5-methylcytosine.  Forward mutations occur at a rate of ~10 6 per locus per generation;  back mutations are rarer. Not all mutations have an effect on  the phenotype.  Although all genetic information in cells is carried by DNA, viruses  have genomes of double-stranded or single-stranded DNA or RNA.  Viroids are subviral pathogens that consist solely of small circular  molecules of RNA, with no protective packaging. The RNA does not  code for protein and its mode of perpetuation and of pathogenesis is  unknown. Scrapie consists of a proteinaceous infectious agent.
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不知道怎么发附件，先发第一章，如果你觉得有用，回个帖，谢谢！
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