mutations

We know that the structure of all organisms originates in their genetic information, and that genes code for particular phenotypes. Mendel first studied characters like round or wrinkled seeds in peas. He found that these characters were passed between generations according to certain rules. Mendel had no idea what physical mechanism was causing these differences, but these differences in phenotype are due to heritable changes in genetic information, calledmutations. These heritable changes are due to changes in DNA.

Mutations can occur in both the germline and in somatic cells. The germline mutations occur in the cells that produce gametes, and so are inherited. Somatic mutations occur in body cells and are only passed on to the cells that are formed from the cell in which the mutation occurred. For example, a mutated skin cell may divide into cells that may produce a blemish or growth. These mutations are not inherited.

Do mutations always code for an altered gene product? If the mutations are in a gene, the answer is sometimes yes. But mutations may take place in both coding and non-coding DNA. As much as 97% of the human DNA is non-coding, and as a result of genome sequencing we know that most mutations are in non-coding regions. However, mutations in these regions can have an effect, by possibly altering the expression of a gene.

Mutations vary in size from tiny point mutations where a single base pair is altered, to giant chromosomal mutations where millions of base pairs are rearranged or lost. Chromosomal mutations may involve wholesale deletions of large parts of a chromosome, or translocation of parts of chromosomes between each other. A point mutation that substitutes either a purine for a purine (A for G, or G for A) or a pyrimidine for a pyrimidine (C for T or T for C) is calledtransition. If the substitution is between a pyrimidine and a purine (for example, A for C), the substitution is called atransversion.

Point mutations within a gene are easily understood by reference to the genetic code. The gene that codes for the beta chain of hemoglobin A contains the following sequence of nucleotides

...ACT CCT GAG GAG...

Coding for ...

... Thr Pro Glu Glu...

What could happen if a single one of the nucleotides were changed, that is, if a mutation occurred?

A possibility would be

...ACT CCT GTG GAG...

Coding for ...

... Thr Pro Val Glu...

This is actually a well-known mutation. Instead of the beta chain of hemoglobin A, the mutation now codes for hemoglobin S, a form of hemoglobin that is the cause of sickle cell disease. The small biochemical change causes the disease symptoms. HbS molecules clump together when deoxygenated, and as a result, red cells acquire the “sickle” shape from which the disease gets its name (Figure 1). In the US, around 50,000 people have sickle cell anemia.

The nucleotide substitution seen in sickle cell anemia is an example of a missense mutation: the new nucleotide alters the codon so as to produce an altered amino acid in the protein product. A nonsense mutation on the other hand is one that changes a codon so that the chain termination occurs, for example a glutamine codon CAG can be converted to a stop codon TAG by a single base transition. The disease Duchenne muscular dystrophy is caused by a mutation in the dystrophin gene on the X-chromosome. Dystrophin is an important structural component in muscle tissue. A nonsense mutation in this gene prevents production of protein from the defective dystrophin mRNA.

Besides base substitution mutations, other mutations insert or delete base pairs into the DNA. Mutations that arise as a result of inserting or deleting base pairs in coding DNA that are not multiples of three are called frame shift mutations. Unlike the missense mutations, these mutations can have more far-reaching consequences on the gene because the whole reading frame of each codon is shifted. In essence, every amino acid coded for after the frameshift will be “wrong.” Frameshifts often create new stop codons, but in any case any protein produced would very likely be of no use to the cell at all. There are many cases of insertions that are involved in disease states. For instance frameshifts occur in the globin genes that causes thallasemia.

Some genes have mutations in the number of copies of triplets. Huntington disease is a degenerative condition resulting from a repeated sequence within a gene on the human chromosome 4. The normal gene has a short repeating sequence of CAG;

CAGCAGCAG

and the mutated gene has the same sequence repeated many more times. The number of repeats determines the age at which the disease will manifest itself.

Fragile X syndrome is a mutation in the FMR1 gene on the X chromosome, in which the triplet CGG is repeated many times. This causes a constriction of the X chromosome making it fragile, and males with the disorder show a number of effects including mental retardation. The FMR protein is believed to play an important role in learning and memory, and this protein isn’t produced by affected individuals.

The genetic code contains redundancy, that is, several different codons encode the same amino acid. TCT for instance, codes for serine, but so does TCA or TCG. If a mutation occurs in the third base of this codon, the same amino acid is encoded. This is an example of a silent mutation, a mutation that doesn’t change the amino acid sequence.

Unlike point mutations that can be described in terms of the substitution, addition or subtraction of base pairs, DNA molecules can break and rejoin, and this can have serious effects on the function of the genetic material.

Causes of Mutations

Mutations may be spontaneous or induced, depending on whether or not any external influences are causal factors in creating the mutation.

Spontaneous mutations result from internal cellular processes.

We’ve already discussed how errors during meiosis may produce mutated chromosomes by abnormal crossing over patterns or by breakage and subsequent rejoining. What other cellular processes lead to mutation? At the DNA level, there are several mechanisms that are subject to error. The first of these is an inherent instability in the four nucleotide bases.

Each base can exist in two different forms, called tautomers, and these two forms affect the covalent bonding affinities of the base (Figure 3). Thymine for example can exist in either a keto or enol form. Usually, the keto form is present. By switching to the enol form (by shifting a proton and some electrons), the pairing properties are changed: the enol form of thymine pairs with guanine instead of the expected adenine.

Figure 1: Sickle cell and normal red cells from human blood.

This sample is a false-color SEM micrograph of a blood specimen of an 18 year old female. The sickle cell shape is produced by a point mutation which changes the characteristics of the hemoglobin produced.

Test Yourself

A DNA sequence is as follows: CCT ACT GAG GTG Coding for: Pro Thr Glu Val If a tautomeric change occurs in the first thymine base in this sequence, what would be the new base sequence on the strand produced after the DNA strand undergoes replication? Also, how would the sequence produced when the daughter strand itself replicates differ from the sequence shown above? If you have access to a codon table, what effect do you think this mutation will have on the gene product?

Besides tautomeric changes, DNA polymerase errors can result in mutations during DNA replication. Perhaps a T base is inserted opposite a G base for example. To prevent this from happening too often, most of these errors are repaired by the presence of a “proofreading” enzyme, which removes incorrectly paired bases. However, some errors do beat the proofreading system and become permanent.

There are other chemical processes that give rise to spontaneous mutation in DNA. These include base degradation. The deamination of cytosine to uracil is one type of degradation that happens at a significant rate in cells. Damage to bases by free radicals of oxygen can also cause mispairings. Bases can also be broken down or mispaired due to alkylation, in which methyl or ethyl groups are added to bases.

Induced mutations result from external mutagens.

Every time we switch on a TV news broadcast or look at a web news site there seems to be some new study that links an environmental factor to disease. Many of these factors are associated with mutations in some way. Externalmutagens are factors which cause a permanent change in DNA. The link between ionizing radiation and mutagenesis was established in the 1920s by Hermann Muller. Muller discovered that X-rays cause mutation in fruit flies. Ionizing radiation like X-rays and gamma radiation can have devastating effects on DNA (Figure 4). They produce large numbers of free radicals: oxygen molecules with a missing electron. Free radicals are extremely unstable, and will “steal” electrons from other molecules in an attempt to replace the missing electron. This results in chemical bonds being broken: new chemical bonds and cross-linkage between macromolecules can occur.

Ultraviolet radiation can damage DNA in several different ways. In one mechanism, adjacent thymine bases on the same DNA strand bind to each other instead of with the complementary base on the other DNA strand. This “thymine dimer” creates a bulge and the distorted DNA cannot be replicated correctly (Figure 5). UV radiation can also cause pairing anomalies and DNA strand breaks.

Chemical damage to chromosomes includes substances that alter the nucleotide bases: some chemicals cause the kinds of alterations that may happen spontaneously, for example tautomeric shifts or deamination. Other substances add groups to the bases. The first discovered chemical mutagen fits into this category. Charlotte Auerbach and J. M. Robson of the University of Edinburgh showed in 1942 that nitrogen mustard, a poison gas used in World War I, shared many of the characteristics of burns produced by high doses of X-rays. They tested to see if nitrogen mustard was also mutagenic, and produced mutations in Drosophila and mice using this chemical. It was later found that nitrogen mustard is an alkylating agent.

Various chemical mutagens cause disease in humans. Aromatic amines are a class of potent carcinogens that have been found in a variety of sources from dyes to smoke from tobacco products linked to a number of cancers including bladder and breast cancers. Aromatic amines form adducts with DNA bases. A DNA adduct is a piece of DNA covalently bonded to a chemical. When an adduct is formed, the damaged DNA cannot replicate correctly, and the damaged cells may either die or replicate in a mutated form.

Figure 4: Mouse blastocyst cells before and after gamma radiation.

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