Silent mutation program

It starts from a DNA sequence with user-specified reading frame. WatCut then scans the sequence for restriction sites that can be introduced without changing the encoded protein sequence.

It will find sites created by any number of mutations, with both non-degenerate and degenerate recognition sequences. Restriction analysis works very much as usual. WatCut can display the results in a graphical format, as a plain table, or in a complete textual format along with the DNA and translated protein sequences.

All of these displays can also be formatted for printing. Several other human diseases are also caused by expansions of polyglutamine codons Table Some diseases associated with mental retardation result from trinucleotide expansions in the leader region of a gene, giving a fragile site , a position where the chromosome is likely to break Sutherland et al.

Expansions involving intron and trailer regions are also known. How triplet expansions are generated is not precisely understood. The size of the insertion is much greater than occurs with normal replication slippage, such as that seen with microsatellite sequences, and once the expansion reaches a certain length it appears to become susceptible to further expansion in subsequent rounds of replication, so that the disease becomes increasingly severe in succeeding generations.

The possibility that expansion involves formation of hairpin loops in the DNA has been raised, based on the observation that only a limited number of trinucleotide sequences are known to undergo expansion, and all of these sequences are GC-rich and so might form stable secondary structures.

There is also evidence that at least one triplet expansion region - for Friedreich's ataxia - can form a triple helix structure Gacy et al.

Studies of similar triplet expansions in yeast have shown that these are more prevalent when the RAD27 gene is inactivated Freudenreich et al. This might indicate that a trinucleotide repeat expansion is caused by an aberration in lagging-strand synthesis. Many chemicals that occur naturally in the environment have mutagenic properties and these have been supplemented in recent years with other chemical mutagens that result from human industrial activity.

Physical agents such as radiation are also mutagenic. Most organisms are exposed to greater or lesser amounts of these various mutagens, their genomes suffering damage as a result. This definition is important because it distinguishes mutagens from other types of environmental agent that cause damage to cells in ways other than by causing mutations Table There are overlaps between these categories for example, some mutagens are also carcinogens but each type of agent has a distinct biological effect.

This type of damage may block replication and cause the cell to die, but it is not a mutation in the strict sense of the term and the causative agents are therefore not mutagens.

Categories of environmental agent that cause damage to living cells. Mutagens cause mutations in three different ways: Some act as base analogs and are mistakenly used as substrates when new DNA is synthesized at the replication fork. Some react directly with DNA , causing structural changes that lead to miscopying of the template strand when the DNA is replicated.

These structural changes are diverse, as we will see when we look at individual mutagens. Some mutagens act indirectly on DNA. They do not themselves affect DNA structure, but instead cause the cell to synthesize chemicals such as peroxides that have a direct mutagenic effect. The range of mutagens is so vast that it is difficult to devise an all-embracing classification. We will therefore restrict our study to the most common types. For chemical mutagens these are as follows: Base analogs are purine and pyrimidine bases that are similar enough to the standard bases to be incorporated into nucleotides when these are synthesized by the cell.

The resulting unusual nucleotides can then be used as substrates for DNA synthesis during genome replication. For example, 5-bromouracil 5-bU ; Figure The mutagenic effect arises because the equilibrium between the two tautomers of 5-bU is shifted more towards the rarer enol form than is the case with thymine. This means that during the next round of replication there is a relatively high chance of the polymerase encountering enol -5bU, which like enol -thymine pairs with G rather than A Figure This results in a point mutation Figure Deaminating agents also cause point mutations.

A certain amount of base deamination removal of an amino group occurs spontaneously in genomic DNA molecules, with the rate being increased by chemicals such as nitrous acid, which deaminates adenine, cytosine and guanine thymine has no amino group and so cannot be deaminated , and sodium bisulfite, which acts only on cytosine.

Deamination of guanine is not mutagenic because the resulting base, xanthine, blocks replication when it appears in the template polynucleotide. Deamination of adenine gives hypoxanthine Figure Deamination of these two bases therefore results in point mutations when the template strand is copied.

Alkylating agents are a third type of mutagen that can give rise to point mutations. Chemicals such as ethylmethane sulfonate EMS and dimethylnitrosamine add alkyl groups to nucleotides in DNA molecules, as do methylating agents such as methyl halides which are present in the atmosphere, and the products of nitrite metabolism. The effect of alkylation depends on the position at which the nucleotide is modified and the type of alkyl group that is added.

Methylations, for example, often result in modified nucleotides with altered base-pairing properties and so lead to point mutations. Other alkylations block replication by forming crosslinks between the two strands of a DNA molecule, or by adding large alkyl groups that prevent progress of the replication complex. Intercalating agents are usually associated with insertion mutations.

The best known mutagen of this type is ethidium bromide , which fluoresces when exposed to UV radiation and so is used to reveal the positions of DNA bands after agarose gel electrophoresis see Technical Note 2.

Ethidium bromide and other intercalating agents are flat molecules that can slip between base pairs in the double helix, slightly unwinding the helix and hence increasing the distance between adjacent base pairs Figure See the text for details.

Hypoxanthine is a deaminated version of adenine. The nucleoside that contains hypoxanthine is called inosine see Table The mutagenic effect of ethidium bromide. A Ethidium bromide is a flat plate-like molecule that is able to slot in between the base pairs of the double helix.

B Ethidium bromide molecules are shown intercalated into the helix: the molecules are viewed more The most important types of physical mutagen are as follows: UV radiation of nm induces dimerization of adjacent pyrimidine bases, especially if these are both thymines Figure Purine dimers are much less common. UV-induced dimerization usually results in a deletion mutation when the modified strand is copied.

Another type of UV-induced photoproduct is the lesion in which carbons number 4 and 6 of adjacent pyrimidines become covalently linked Figure Ionizing radiation has various effects on DNA depending on the type of radiation and its intensity. Some types of ionizing radiation act directly on DNA, others act indirectly by stimulating the formation of reactive molecules such as peroxides in the cell.

The sugar-phosphate that is left is unstable and rapidly degrades, leaving a gap if the DNA molecule is double stranded Figure This reaction is not normally mutagenic because cells have effective systems for repairing nicks Section Gaps do, however, lead to mutations under certain circumstances, for example in E. Photoproducts induced by UV irradiation. A segment of a polynucleotide containing two adjacent thymine bases is shown.

A A thymine dimer contains two UV-induced covalent bonds, one linking the carbons at position 6 and the other linking the carbons more The mutagenic effect of heat. B Schematic representation of the effect of heat-induced hydrolysis on a double-stranded DNA molecule.

The baseless more When considering the effects of mutations we must make a distinction between the direct effect that a mutation has on the functioning of a genome and its indirect effect on the phenotype of the organism in which it occurs.

The direct effect is relatively easy to assess because we can use our understanding of gene structure and expression to predict the impact that a mutation will have on genome function. The indirect effects are more complex because these relate to the phenotype of the mutated organism which, as described in Section 7. Many mutations result in nucleotide sequence changes that have no effect on the functioning of the genome. These silent mutations include virtually all of those that occur in intergenic DNA and in the non-coding components of genes and gene-related sequences.

In other words, some Mutations in the coding regions of genes are much more important. First, we will look at point mutations that change the sequence of a triplet codon. A mutation of this type will have one of four effects Figure A synonymous change is therefore a silent mutation because it has no effect on the coding function of the genome: the mutated gene codes for exactly the same protein as the unmutated gene.

It may result in a non-synonymous change, the mutation altering the codon so that it specifies a different amino acid. The protein coded by the mutated gene therefore has a single amino acid change.

This often has no significant effect on the biological activity of the protein because most proteins can tolerate at least a few amino acid changes without noticeable effect on their ability to function in the cell, but changes to some amino acids, such as those at the active site of an enzyme, have a greater impact. A non-synonymous change is also called a missense mutation. The mutation may convert a codon that specifies an amino acid into a termination codon.

This is a nonsense mutation and it results in a shortened protein because translation of the mRNA stops at this new termination codon rather than proceeding to the correct termination codon further downstream.

The effect of this on protein activity depends on how much of the polypeptide is lost: usually the effect is drastic and the protein is non-functional. The mutation could convert a termination codon into one specifying an amino acid, resulting in readthrough of the stop signal so the protein is extended by an additional series of amino acids at its C terminus. Most proteins can tolerate short extensions without an effect on function, but longer extensions might interfere with folding of the protein and so result in reduced activity.

Effects of point mutations on the coding region of a gene. Four different effects of point mutations are shown, as described in the text. The readthrough mutation results in the gene being extended beyond the end of the sequence shown here, the leucine more Deletion and insertion mutations also have distinct effects on the coding capabilities of genes Figure If the number of deleted or inserted nucleotides is three or a multiple of three then one or more codons are removed or added, the resulting loss or gain of amino acids having varying effects on the function of the encoded protein.

Deletions or insertions of this type are often inconsequential but will have an impact if, for example, amino acids involved in an enzyme's active site are lost, or if an insertion disrupts an important secondary structure in the protein. On the other hand, if the number of deleted or inserted nucleotides is not three or a multiple of three then a frameshift results, all of the codons downstream of the mutation being taken from a different reading frame from that used in the unmutated gene.

This usually has a significant effect on the protein function, because a greater or lesser part of the mutated polypeptide has a completely different sequence to the normal polypeptide. It is less easy to make generalizations about the effects of mutations that occur outside of the coding regions of the genome. Any protein binding site is susceptible to point, insertion or deletion mutations that change the identity or relative positioning of nucleotides involved in the DNA -protein interaction.

These mutations therefore have the potential to inactivate promoters or regulatory sequences, with predictable consequences for gene expression Figure Origins of replication could conceivably be made non-functional by mutations that change, delete or disrupt sequences recognized by the relevant binding proteins Section There is also little information about the potential impact on gene expression of mutations that affect nucleosome positioning Section 8.

Two possible effects of deletion mutations in the region upstream of a gene. One area that has been better researched concerns mutations that occur in introns or at intron-exon boundaries. In these regions, single point mutations will be important if they change nucleotides involved in the RNA -protein and RNA-RNA interactions that occur during splicing of different types of intron Sections This may mean that the intron is not removed from the pre- mRNA , but it is more likely that a cryptic splice site see page will be used as an alternative.

It is also possible for a mutation within an intron or an exon to create a new cryptic site that is preferred over a genuine splice site that is not itself mutated.

Both types of event have the same result: relocation of the active splice site, leading to aberrant splicing. This might delete part of the resulting protein, add a new stretch of amino acids, or lead to a frameshift. Mutation detection. Rapid procedures for detecting mutations in DNA molecules. Many genetic diseases are caused by point mutations that result in modification or inactivation of a gene product.

Methods for detecting these mutations are important in two more Now we turn to the indirect effects that mutations have on organisms, beginning with multicellular diploid eukaryotes such as humans. The first issue to consider is the relative importance of the same mutation in a somatic cell compared with a germ cell. Because somatic cells do not pass copies of their genomes to the next generation, a somatic cell mutation is important only for the organism in which it occurs: it has no potential evolutionary impact.

In fact, most somatic cell mutations have no significant effect, even if they result in cell death, because there are many other identical cells in the same tissue and the loss of one cell is immaterial. An exception is when a mutation causes a somatic cell to malfunction in a way that is harmful to the organism, for instance by inducing tumor formation or other cancerous activity.

Mutations in germ cells are more important because they can be transmitted to members of the next generation and will then be present in all the cells of any individual who inherits the mutation. Most mutations, including all silent ones and many in coding regions, will still not change the phenotype of the organism in any significant way. Those that do have an effect can be divided into two categories: Loss-of-function is the normal result of a mutation that reduces or abolishes a protein activity.

Most loss-of-function mutations are recessive Section 5. This is the explanation for a few genetic diseases in humans, including Marfan syndrome which results from a mutation in the gene for the connective tissue protein called fibrillin. Gain-of-function mutations are much less common. The mutation must be one that confers an abnormal activity on a protein. Many gain-of-function mutations are in regulatory sequences rather than in coding regions, and can therefore have a number of consequences.

For example, a mutation might lead to one or more genes being expressed in the wrong tissues, these tissues gaining functions that they normally lack. Alternatively the mutation could lead to overexpression of one or more genes involved in control of the cell cycle, thus leading to uncontrolled cell division and hence to cancer.

Because of their nature, gain-of-function mutations are usually dominant. A loss-of-function mutation is usually recessive because a functional version of the gene is present on the second chromosome copy.

Assessing the effects of mutations on the phenotypes of multicellular organisms can be difficult. Not all mutations have an immediate impact: some are delayed onset and only confer an altered phenotype later in the individual's life. Others display non-penetrance in some individuals, never being expressed even though the individual has a dominant mutation or is a homozygous recessive.

With humans, these factors complicate attempts to map disease-causing mutations by pedigree analysis Section 5. Mutations in microbes such as bacteria and yeast can also be described as loss-of-function or gain-of-function, but with microorganisms this is neither the normal nor the most useful classification scheme. Instead, a more detailed description of the phenotype is usually attempted on the basis of the growth properties of mutated cells in various culture media.

This enables most mutations to be assigned to one of four categories: Auxotrophs are cells that will only grow when provided with a nutrient not required by the unmutated organism. For example, E. If one of these genes is mutated in such a way that its protein product is inactivated, then the cell is no longer able to make tryptophan and so becomes a tryptophan auxotroph. It cannot survive on a medium that lacks tryptophan, and can grow only when this amino acid is provided as a nutrient Figure Unmutated bacteria, which do not require extra supplements in their growth media, are called prototrophs.

Conditional-lethal mutants are unable to withstand certain growth conditions: under permissive conditions they appear to be entirely normal but when transferred to restrictive conditions the mutant phenotype is seen. Temperature-sensitive mutants are typical examples of conditional-lethal mutants. Temperature-sensitive mutants behave like wild-type cells at low temperatures but exhibit their mutant phenotype when the temperature is raised above a certain threshold, which is different for each mutant.

Usually this is because the mutation reduces the stability of a protein, so the protein becomes unfolded and hence inactive when the temperature is raised. Inhibitor-resistant mutants are able to resist the toxic effects of an antibiotic or another type of inhibitor. There are various molecular explanations for this type of mutant. In some cases the mutation changes the structure of the protein that is targeted by the inhibitor, so the latter can no longer bind to the protein and interfere with its function.

This is the basis of streptomycin resistance in E. Another possibility is that the mutation changes the properties of a protein responsible for transporting the inhibitor into the cell, this often being the way in which resistance to toxic metals is acquired. Regulatory mutants have defects in promoters and other regulatory sequences. This category includes constitutive mutants, which continuously express genes that are normally switched on and off under different conditions.

For example, a mutation in the operator sequence of the lactose operon Section 9. A tryptophan auxotrophic mutant. Two Petri-dish cultures are shown.

Both contain minimal medium, which provides just the basic nutritional requirements for bacterial growth nitrogen, carbon and energy sources, plus some salts. The medium on the left more The effect of a constitutive mutation in the lactose operator. The operator sequence has been altered by a mutation and the lactose repressor can no longer bind to it. The result is that the lactose operon is transcribed all the time, even when lactose more In addition to these four categories, many mutations are lethal and so result in death of the mutant cell, whereas others have no effect.

The latter are less common in microorganisms than in higher eukaryotes, because most microbial genomes are relatively compact, with little non-coding DNA.

Mutations can also be leaky , meaning that a less extreme form of the mutant phenotype is expressed. For example, a leaky version of the tryptophan auxotroph illustrated in Figure Is it possible for cells to utilize mutations in a positive fashion, either by increasing the rate at which mutations appear in their genomes, or by directing mutations towards specific genes?

Both types of event might appear, at first glance, to go against the accepted wisdom that mutations occur randomly but, as we shall see, hypermutation and programmed mutations are possible without contravening this dogma. Hypermutation occurs when a cell allows the rate at which mutations occur in its genome to increase.

Several examples of hypermutation are known, one of these forming part of the mechanism used by vertebrates, including humans, to generate a diverse array of immunoglobulin proteins. We have already touched on this phenomenon in Section Additional diversity is produced by hypermutation of the V-gene segments after assembly of the intact immunoglobulin gene Figure This enhanced mutation rate appears to result from the unusual behavior of the mismatch repair system which normally corrects replication errors.

At all other positions within the genome, the mismatch repair system corrects errors of replication by searching for mismatches and replacing the nucleotide in the daughter strand, this being the strand that has just been synthesized and so contains the error see Section At V-gene segments, the repair system changes the nucleotide in the parent strand, and so stabilizes the mutation rather than correcting it Cascalho et al.

The mechanism by which this is achieved has not yet been described. Hypermutation of the V-gene segment of an intact immunoglobulin gene. See Figure An apparent increase in mutation rate arising from modifications to the normal DNA repair process does not contradict the dogma regarding the randomness of mutations.

However, problems have arisen with reports, dating back to Cairns et al. The original experiments involved a strain of E. The bacteria were spread on an agar medium in which the only carbon source was lactose.

This meant that a cell could grow and divide only if a second mutation occurred in the lactose operon, reversing the effects of the nonsense mutation and therefore allowing the lactose enzymes to be synthesized.

Mutations with this effect appeared to occur significantly more frequently than expected, and at a rate that was greater than mutations in other parts of the genomes of these E. Programmed mutations? In startling results were published suggesting that under some circumstances Escherichia coli bacteria are able to mutate in a directed way that enables cells to adapt to an environmental stress. The randomness of mutations more These experiments suggested that bacteria can program mutations according to the selective pressures that they are placed under.

In other words, the environment can directly affect the phenotype of the organism, as suggested by Lamarck, rather than operating through the random processes postulated by Darwin. With such radical implications, it is not surprising that the experiments have been debated at length, with numerous attempts to discover flaws in their design or alternative explanations for the results. Variations of the original experimental system have suggested that the results are authentic, and similar events in other bacteria have been described.

Models based on gene amplification rather than selective mutation are being tested Andersson et al. In view of the thousands of damage events that genomes suffer every day, coupled with the errors that occur when the genome replicates, it is essential that cells possess efficient repair systems. Without these repair systems a genome would not be able to maintain its essential cellular functions for more than a few hours before key genes became inactivated by DNA damage. Similarly, cell lineages would accumulate replication errors at such a rate that their genomes would become dysfunctional after a few cell divisions.

Most cells possess four different categories of DNA repair system Figure Excision repair Section Mismatch repair Section Recombination repair Section Four categories of DNA repair system. Most if not all organisms also possess systems that enable them to replicate damaged regions of their genome without prior repair. We will examine these systems in Section Most of the types of DNA damage that are caused by chemical or physical mutagens Section This is often the case with nicks resulting from the effects of ionizing radiation.

Some forms of alkylation damage are directly reversible by enzymes that transfer the alkyl group from the nucleotide to their own polypeptide chains. Enzymes capable of doing this are known in many different organisms and include the Ada enzyme of E.

Ada removes alkyl groups attached to the oxygen groups at positions 4 and 6 of thymine and guanine, respectively, and can also repair phosphodiester bonds that have become methylated.

Other alkylation repair enzymes have more restricted specificities, an example being human MGMT O 6 -methylguanine- DNA methyltransferase which, as its name suggests, only removes alkyl groups from position 6 of guanine. Cyclobutyl dimers are repaired by a light-dependent direct system called photoreactivation.

When stimulated by light with a wavelength between and nm the enzyme binds to cyclobutyl dimers and converts them back to the original monomeric nucleotides. Photoreactivation is a widespread but not universal type of repair: it is known in many but not all bacteria and also in quite a few eukaryotes, including some vertebrates, but is absent in humans and other placental mammals.

A similar type of photoreactivation involves the photoproduct photolyase and results in repair of lesions. Neither E. The direct types of damage reversal described above are important, but they form a very minor component of the DNA repair mechanisms of most organisms. This point is illustrated by the draft human genome sequences, which appear to contain just a single gene coding for a protein involved in direct repair the MGMT gene , but which have at least 40 genes for components of the excision repair pathways Wood et al.

These pathways fall into two categories: Base excision repair involves removal of a damaged nucleotide base, excision of a short piece of the polynucleotide around the AP site thus created, and resynthesis with a DNA polymerase. Nucleotide excision repair is similar to base excision repair but is not preceded by removal of a damaged base and can act on more substantially damaged areas of DNA. Base excision is the least complex of the various repair systems that involve removal of one or more damaged nucleotides followed by resynthesis of DNA to span the resulting gap.

It is used to repair many modified nucleotides whose bases have suffered relatively minor damage resulting from, for example, exposure to alkylating agents or ionizing radiation Section Each DNA glycosylase has a limited specificity Table Most organisms are able to deal with deaminated bases such as uracil deaminated cytosine and hypoxanthine deaminated adenine , oxidation products such as 5-hydroxycytosine and thymine glycol, and methylated bases such as 3-methyladenine, 7-methylguanine and 2-methylcytosine Seeberg et al.

Other DNA glycosylases remove normal bases as part of the mismatch repair system Section Most of the DNA glycosylases involved in base excision repair are thought to diffuse along the minor groove of the DNA double helix in search of damaged nucleotides, but some may be associated with the replication enzymes. Base excision repair. A Excision of a damaged nucleotide by a DNA glycosylase. B Schematic representation of the base excision repair pathway.

Alternative versions of the pathway are described in the text. This creates an AP or baseless site see Figure This step can be carried out in a variety of ways. Some AP endonucleases can also remove the sugar from the AP site, this being all that remains of the damaged nucleotide, but others lack this ability and so work in conjunction with a separate phosphodiesterase.

The single nucleotide gap is filled by a DNA polymerase , using base-paring with the undamaged base in the other strand of the DNA molecule to ensure that the correct nucleotide is inserted. Like all proteins, P-gp is comprised of amino acid building blocks. DNA consists of a sequence of chemical bases, and the code for individual amino acids is represented by specific sets of three adjacent DNA bases called codons.

Since several different codons can contain the code for the same amino acid, this SNP only altered the gene by converting one common codon to a rare one, but did not change the amino acid for which it coded. Since silent SNPs are frequently found in nature, their biological role has largely been overlooked. For more information on Dr.

Department of Health and Human Services. NIH is the primary federal agency conducting and supporting basic, clinical, and translational medical research, and is investigating the causes, treatments, and cures for both common and rare diseases. For more information about NIH and its programs, visit www. A "silent" polymorphism in the MDR1 gene changes substrate specificity. Science Express , December 21,