Mutation PDF

Mutation Genetic code most likely consisted of a series of blocks of information called codons, each corresponding to an amino acid in the encoded protein. The information within one codon was probably a sequence of three nucleotides. The codons lie immediately adjacent to each other, forming a con...

Mutation Genetic code most likely consisted of a series of blocks of information called codons, each corresponding to an amino acid in the encoded protein. The information within one codon was probably a sequence of three nucleotides. The codons lie immediately adjacent to each other, forming a continuous sequence of nucleotides. Thus, any addition or elimination of nucleotides will alter the entire sentence. Between transcription in the nucleus and export of a mature mRNA to the cytoplasm, a number of modifications occur to the initial transcripts.  At the 5' end of mRNAs, the first base in the transcript is usually an adenine (A) or a guanine (G), and this is further modified by the addition of GTP to the 5' PO4 group, forming what is known as a 5' cap.  A series of adenine (A) residues, called the 3' poly-A tail (100-200 A), is added. The poly-A tail appears to play a role in the stability of mRNAs by protecting them from degradation  Many eukaryotic genes appeared to contain sequences that were not represented in the mRNA that exported to ribosomes. Consequently, they have no corresponding sequences of amino acids in the protein. Sequences that are not represented in the mRNA and the protein (noncoding DNA) that interrupts the sequence of the gene are called ―intervening sequences,‖ or introns, and we call the coding sequences exons because they are expressed. The primary transcript is cut and put back together to produce the mature mRNA. The latter process is referred to as pre- mRNA splicing, and it occurs in the nucleus prior to the export of the mRNA to the cytoplasm. 1 Mutation is change in DNA molecule ranging from one base pair to the disappearance of an entire chromosome. In the following part we will focus on alterations of single base pair of DNA or of small number of base pairs (point mutations). Mutations are both the sustainer of life and the cause of great suffering. On the one hand, mutation is the source of all genetic variation, the raw material of evolution. The ability of organisms to adapt to environmental change depends critically on the presence of genetic variation in natural populations, and genetic variation is produced—at least partly—by mutation. On the other hand, many mutations have detrimental effects, and mutation is the source of many diseases and disorders. 2 Mutations are also useful for probing fundamental biological processes. Finding or creating mutations that affect different components of a biological system and studying their effects can often lead to an understanding of the system. This method, referred to as genetic dissection. In unicellular organisms, mutations may give rise to descendants carrying modified phenotypes. Accumulation of mutations may ultimately produce new species. In multicellular organisms, we can distinguish between two broad categories of mutations: somatic mutations and germ-line mutations. Somatic mutations arise in somatic tissues, which do not produce gametes. When a somatic cell with a mutation divides (mitosis), the mutation is passed on to the daughter cells, leading to a population of genetically identical cells (a clone). The earlier in development that a somatic mutation occurs, the larger the clone of cells within that individual organism that will contain the mutation. Because of the huge number of cells present in a typical eukaryotic organism, somatic mutations are numerous. Typically, a mutation arises once in every million cell divisions, and so hundreds of millions of somatic mutations must arise in each person. Many somatic mutations have no obvious effect on the phenotype of the organism, because the function of the mutant cell (even the cell itself) is replaced by that of normal cells. However, cells with a somatic mutation that stimulates cell division can increase in number and spread; this type of mutation can give rise to cells with a selective advantage and is the basis for cancers. Germ-line mutations arise in cells that ultimately produce gametes. A germ-line mutation can be passed to future generations, producing individual organisms that carry the mutation in all their somatic and germ-line cells. Historically, mutations have been partitioned into those that affect a single gene, called gene mutations, and those that affect the number or structure of chromosomes, called chromosome mutations. Chromosome mutations can be observed directly, by looking at chromosomes with a microscope. Gene mutations could be detected only by observing their phenotypic effects. Now, DNA based techniques allow the detection of gene mutations. The term chromosome mutation is used for a large-scale genetic alteration that affects chromosome structure or the number of chromosomes. The term gene mutation is used for a relatively small DNA lesion that affects a single gene. 3  I. Spontaneous (naturally occurring) 1. Errors in DNA replication a. Tautomeric shifts Purine and pyrimidine bases exist in different chemical forms called tautomers. The two tautomeric forms of each base are in dynamic equilibrium, although one form is more 4 common than the other. The standard Watson-and-Crick base pairings—adenine with thymine, and cytosine with guanine—are between the common forms of the bases, but, if the bases are in their rare tautomeric forms, other base pairings are possible. When a mismatched base has been incorporated into a newly synthesized nucleotide chain, an incorporated error is said to have occurred. Suppose that, in replication, guanine (which normally pairs with cytosine) mispairs with thymine. In the next round of replication, the two mismatched bases separate, and each serves as template for the synthesis of a new nucleotide strand. This time, guanine pairs with cytosine, producing another copy of the original DNA sequence. On the other strand, however, the incorrectly incorporated thiamine serves as the template and pairs with adenine, producing a new DNA molecule that has an error—a T · A pair in place of the original G · C pair (a G · C→T · A base substitution). The original incorporated error leads to a replication error, which creates a permanent mutation, because all the base pairings are correct and there is no mechanism for repair systems to detect the error. 5  b. Strand slippage Mutations due to small insertions and deletions also arise spontaneously in replication and crossing over. Strand slippage can occur when one nucleotide strand forms a small loop. If the looped-out nucleotides are on the newly synthesized strand, an insertion results. At the next round of replication, the insertion will be replicated and both strands will contain the insertion. If the looped-out nucleotides are on the template strand, then the newly replicated strand has a deletion, and this deletion will be perpetuated in subsequent rounds of replication. 6  c. Unequal crossing over Another process that produces insertions and deletions is unequal crossing over. In normal crossing over, the homologous sequences of the two DNA molecules align, and crossing over produces no net change in the number of nucleotides in either molecule. Misaligned pairing can cause unequal crossing over, which results in one DNA molecule with an insertion and the other with a deletion. 2. Spontaneous lesions In addition to spontaneous mutations that arise in replication, mutations also result from spontaneous chemical changes in DNA. One such change is a. Depurination, The loss of a purine base from a nucleotide. It results when the covalent bond connecting the purine to the 1′-carbon atom of the deoxyribose sugar breaks, producing an apurinic site, a nucleotide that lacks its purine base. An apurinic site cannot act as a template for a complementary base in replication. In the absence of base-pairing constraints, an incorrect nucleotide (most often adenine) is incorporated into the newly synthesized DNA strand opposite the apurinic site, frequently leading to an incorporated error. The incorporated error is then transformed into a replication error at the next round of replication. 7  b. Deamination Another spontaneously occurring chemical change that takes place in DNA is deamination, the loss of an amino group (NH2) from a base. Deamination can be spontaneous or be induced by mutagenic chemicals. Deamination can alter the pairing properties of a base: the deamination of cytosine, for example, produces uracil, which pairs with adenine in replication. After another round of replication, the adenine will pair with thymine, creating a T · A pair in place of the original C · G pair (C · G→U · A→T · A); this chemical change is a transition mutation. This type of mutation is usually repaired by enzymes that remove uracil whenever it is found in DNA. The ability to recognize the product of cytosine deamination may explain why thymine, not uracil, is found in DNA. 8  II. Induced (through the action of certain agents called mutagens) A number of environmental agents are capable of damaging DNA, including certain chemicals and radiation. Any environmental agent that significantly increases the rate of mutation above the spontaneous rate is called a mutagen. 1. Chemical Mutagens a. Base analogs One class of chemical mutagens consists of base analogs, chemicals with structures similar to that of any of the four standard bases of DNA. DNA polymerases cannot distinguish these analogs from the standard bases; so, if base analogs are present during replication, they may be incorporated into newly synthesized DNA molecules. For example, 5-bromouracil (5BU) is an analog of thymine; it has the same structure as that of thymine except that it has a bromine (Br) atom on the 5-carbon atom instead of a methyl group. Normally, 5-bromouracil pairs with adenine just as thymine does, but it occasionally mispairs with guanine, leading to a transition (T · A→5BU · A→5BU · G→C · G). Through mispairing, 5-bromouracil can also be incorporated into a newly synthesized DNA strand opposite guanine. In the next round of replication 5-bromouracil pairs with adenine, leading to another transition (G · C→G · 5BU→A · 5BU→A · T). b. Alkylating agents Alkylating agents are chemicals that donate alkyl groups, such as methyl (CH3) and ethyl (CH3–CH2) groups, to nucleotide bases. For example, ethylmethylsulfonate (EMS) adds an ethyl group to guanine, producing O6-ethylguanine, which pairs with thymine. Thus, EMS produces C · G→T · A transitions. 9 EMS is also capable of adding an ethyl group to thymine, producing 4-ethylthymine, which then pairs with guanine, leading to a T · A→C · G transition. Because EMS produces both C · G→T · A and T · A→C · G transitions, mutations produced by EMS can be reversed by additional treatment with EMS. Mustard gas is another alkylating agent. c. Nitrous acid Deamination In addition to its spontaneous occurrence, deamination can be induced by some chemicals. For instance, nitrous acid deaminates cytosine, creating uracil, which in the next round of replication pairs with adenine, producing a C · G→T · A transition mutation. Nitrous acid changes adenine into hypoxanthine, which pairs with cytosine, leading to a T · A→C · G transition. Nitrous acid also deaminates guanine, producing xanthine, which pairs with cytosine just as guanine does; however, xanthine can also pair with thymine, leading to a C · G→T · A transition. 10 Nitrous acid produces exclusively transition mutations and, because both C · G→T · A and T · A→C · G transitions are produced, these mutations can be reversed with nitrous acid. d. Hydroxylamine Hydroxylamine is a very specific base modifying mutagen that adds a hydroxyl group to cytosine, converting it into hydroxylaminocytosine. This conversion increases the frequency of a rare tautomer that pairs with adenine instead of guanine and leads to C · G→T · A transitions. Because hydroxylamine acts only on cytosine, it will not generate T · A→C · G transitions; thus, hydroxylamine will not reverse the mutations that it produces. e. Intercalating agents Proflavin, acridine orange, ethidium bromide, and dioxin are intercalating agents, which produce mutations by sandwiching themselves (intercalating) between adjacent bases in DNA, distorting the three-dimensional structure of the helix and causing single- nucleotide insertions and deletions in replication. These insertions and deletions frequently produce frameshift mutations, and so the mutagenic effects of intercalating agents are often severe. Because intercalating agents generate both additions and deletions, they can reverse the effects of their own mutations. 11  2. Physical Mutagens a. Ionizing radiation Because of their high energies, X-rays, gamma rays, and cosmic rays are all capable of penetrating tissues and damaging DNA. These forms of radiation, called ionizing radiation, dislodge electrons from the atoms that they encounter, changing stable molecules into free radicals and reactive ions, which then alter the structures of bases and break phosphodiester bonds in DNA. Ionizing radiation also frequently results in double-strand breaks in DNA. Attempts to repair these breaks can produce chromosome mutations b. Ultraviolet (UV) Ultraviolet (UV) light has less energy than that of ionizing radiation and does not eject electrons but is nevertheless highly mutagenic. Purine and pyrimidine bases readily absorb UV light, resulting in the formation of chemical bonds between adjacent pyrimidine molecules on the same strand of DNA and in the creation of pyrimidine dimers. Pyrimidine dimers consisting of two thymine bases (called thymine dimers) are most frequent, but cytosine dimers and thymine–cytosine dimers also can form. Dimers distort the configuration of DNA and often block replication. Most pyrimidine dimers are immediately repaired but some escape repair and inhibit replication and transcription. When pyrimidine dimers block replication, cell division is inhibited and the cell usually dies; for this reason, UV light kills bacteria and is an effective sterilizing agent. 12  Molecular Consequences of Gene Mutations I. Molecular consequences of point mutations in regulatory sequences Molecular consequences of point mutations in non coding regions (regulatory sequences). This mutation may disrupt (or creates) a binding site: II. Molecular consequences of point mutations in coding sequences 13 Not all silent mutations, however, are truly silent For example, silent mutations may have phenotypic effects when different isoaccepting tRNAs (Different tRNAs that accept the same amino acid but have different anticodons) are used for different synonymous codons. Because some isoaccepting tRNAs are more abundant than others, which synonymous codon is used may affect the rate of protein synthesis. The rate of protein synthesis can influence the phenotype by affecting the amount of protein present in the cell and, in a few cases, the folding of the protein. Other silent mutations may alter sequences near the exon–intron junctions that affect splicing. Still other silent mutations may influence the folding of the mRNA, affecting its stability. 14 Frameshift Mutation Not all insertions and deletions lead to frameshifts, however; insertions and deletions consisting of any multiple of three nucleotides will leave the reading frame intact, although the addition or removal of one or more amino acids may still affect the 15 phenotype. Mutations not affecting the reading frame are called in-frame insertions and deletions, respectively. Important Definitions Forward and reverse mutations A mutation that alters the wild-type phenotype is called a forward mutation, whereas a reverse mutation (a reversion) changes a mutant phenotype back into the wild type. Neutral Mutation It is a missense mutation that alters the amino acid sequence of the protein but does not change its function. It occurs when one amino acid is replaced by another that is chemically similar or when the affected amino acid has little influence on protein function. For example, neutral mutations occur in the genes that encode hemoglobin; although these mutations alter the amino acid sequence of hemoglobin, they do not affect its ability to transport oxygen. Loss-of-function mutations The mutations that cause the complete or partial absence of normal protein function. A loss-of-function mutation so alters the structure of the protein that the protein no longer works correctly or the mutation can occur in regulatory regions that affect the transcription or translation. Gain-of-function mutations They produce an entirely new trait or it causes a trait to appear in an inappropriate tissue or at an inappropriate time in development. For example, a mutation in a gene that encodes a receptor for a growth factor might cause the mutated receptor to stimulate growth all the time, even in the absence of the growth factor. Suppressor Mutations A suppressor mutation is a genetic change that hides or suppresses the effect of another mutation. This type of mutation is distinct from a reverse mutation, in which the mutated site changes back into the original wild-type sequence. A suppressor mutation occurs at a site that is distinct from the site of the original mutation. Thus, an individual with a suppressor mutation is a double mutant, possessing both the original mutation and the suppressor mutation but exhibiting the phenotype of an 16 unmutated wild type. Like other mutations, suppressors arise randomly. Geneticists distinguish between two classes of suppressor mutations: intragenic and intergenic. Intragenic Suppressor Mutation An intragenic suppressor mutation is in the same gene as that containing the mutation being suppressed and may work in several ways. For example, the suppressor may change a second nucleotide in the same codon altered by the original mutation, producing a codon that specifies the same amino acid as that specified by the original, unmutated codon. Intergenic Suppressors Mutations An intergenic suppressor mutation, in contrast, occurs in a gene other than the one bearing the original mutation. For example, the original DNA sequence is AAC (UUG in the mRNA) and specifies leucine. This sequence mutates to ATC (UAG in mRNA), a termination codon. The ATC nonsense mutation could be suppressed by a second mutation in a different gene that encodes tyrosine tRNA (anticodon AUA mutated to AUC that pairs with the UAG stop codon). Thus, instead of translation terminating at the UAG codon, tyrosine would be inserted into the protein and a full-length protein would be produced. Although tyrosine substituted leucine, the effect of this change would depend on the role of this amino acid in the overall structure of the protein, but the effect of the suppressor mutation is likely to be less detrimental than the effect of the nonsense mutation, which would halt translation prematurely. 17 18  The Ames Test: A correlation between carcenogenecity and mutagenicity of compounds. Bacteria would be a model for evaluating the mutagenicity of compounds as a first level of detection of carcinogenic potential. Salmonella typhimurium had one of several mutant alleles of gene responsible for histidine synthesis that reverts (return to wild-type phenotype) only by certain additional mutations. But not all carcinogens were themselves mutagenic; rather some carcinogens metabolites produced in liver and converted by enzymes (that does not occur in bacteria) into bioactive (mutagenic) metabolites. Thus liver enzymes should be addad to reaction mixture 19