FUNDAMENTALS OF MICROBIOLOGY

Lesson 23. GENETIC MUTATION

Module 6. Bacterial genetics

Lesson 23

GENETIC MUTATION

23.1 Mutations

A mutation is any change in the sequence of the DNA encoding a gene. Most of these mutations are recognized because the phenotype of the organism has changed Originally genes were thought to be beads-on-string, where each bead was a single entity responsible for a phenotype. This theory lead to the concept that only single mutation was possible for a specific gene. Detailed genetic experiments proved that the gene actually consists of many individual units, and specific changes in these units can lead to several mutant phenotypes. We now know those units are nucleotides. Therefore understanding the nature of mutations is important to our understanding of a gene.

23.2 Germinal and Somatic Mutations

Eukaryotic organisms have two primary cell types: germ and somatic. Mutations can occur in either cell type. If a gene is altered in a germ cell, the mutation is termed a germinal mutation. Because germ cells give rise to gametes, some gamete s will carry the mutation and it will be passed on to the next generation when the individual successfully mates. Typically germinal mutations are not expressed in the individual containing the mutation. The only instance in which it would be expressed is if it negatively (or positively) affected gamete production.

Somatic cells give rise to all non-germline tissues. Mutations in somatic cells are called somatic mutations. Because they do not occur in cells that give rise to gametes, the mutation is not passed along to the next generation by sexual means. To maintain this mutation, the individual containing the mutation must be cloned. Two examples of somatic clones are navel oranges and red delicious apples. Horticulturists first observed the mutants. They then grafted mutant branches onto the stocks of "normal" trees. After the graft was established, cuttings from that original graft were grafted onto tree stocks. In this way the mutation was maintained and proliferated.

Most tissues are derived from a cell or a few progenitor cells. If a mutation occurs in one of the progenitor cells, all of its daughter cells will also express the mutation. For this reason, somatic mutations generally appear as a sector on the mutated individual.

Cancer tumors are a unique class of somatic mutations. The tumor arises when a gene involved in cell division, a protooncogene, is mutated. All of the daughter cells contain this mutation. The phenotype of all cells containing the mutation is uncontrolled cell division. This result in a tumor that is a collection of undifferentiated cells called tumor cells.

23.3 Causes

Two classes of mutations are spontaneous mutations (molecular decay) and induced mutations caused by mutagens.

23.3.1 Spontaneous mutation

In general, the appearance of a new mutation is a rare event. Most mutations that were originally studied occurred spontaneously. This class of mutation is termed spontaneous mutations. The spontaneous mutation rate varies. Large gene provide a large target and tend to mutate more frequently. A study of the five coat color loci in mice showed that the rate of mutation ranged from 2 x 10^-6 to 40 x 10^-6 mutations per gamete per gene. Data from several studies on eukaryotic organisms shows that in general the spontaneous mutation rate is 2-12 x 10^-6 mutations per gamete per gene. Given that the human genome contains 100,000 genes, we can conclude that we would predict that 1-5 human gametes would contain a mutation in some gene. Spontaneous mutations on the molecular level can be caused by:

23.3.1.1 Tautomerism

A base is changed by the repositioning of a hydrogen atom, altering the hydrogen bonding pattern of that base resulting in incorrect base pairing during replication.

23.3.1.2 Depurination

Loss of a purine base (A or G) to form an apurinic site (AP site).

23.3.1.3 Deamination

Hydrolysis changes a normal base to an atypical base containing a keto group in place of the original amine group. Examples include C → U and A → HX (hypoxanthine), which can be corrected by DNA repair mechanisms; and 5MeC (5-methylcytosine) → T, which is less likely to be detected as a mutation because thymine is a normal DNA base.

23.3.1.4 Slipped strand mispairing

It is denaturation of the new strand from the template during replication, followed by renaturation in a different spot (slipping). This can lead to insertions or deletions.

23.3.2 Induced mutation

Spontaneous mutations clearly represent only a small number of all possible mutations. To genetically dissect a biological system further, new mutations were created by scientists by treating an organism with a mutagenizing agent. These mutations are called induced mutations. Induced mutations on the molecular level can be caused by:

• Chemicals
• Radiations
• Transposable elements

23.3.2.1 Chemicals

• Hydroxylamine NH₂OH
• Base analogs (e.g. BrdU)
• Alkylating agents (e.g. N-ethyl-N-nitrosourea). These agents can mutate both replicating and non-replicating DNA. In contrast, a base analog can only mutate the DNA when the analog is incorporated in replicating the DNA. Each of these classes of chemical mutagens has certain effects that then lead to transitions, transversions, or deletions.
• Agents that form DNA adducts (e.g. ochratoxin A metabolites)
• DNA intercalating agents (e.g. ethidium bromide)
• DNA crosslinkers
• Oxidative damage
• Nitrous acid converts amine groups on A and C to diazo groups, altering their hydrogen bonding patterns which leads to incorrect base pairing during replication.

23.3.2.2 Radiation

• Ultraviolet radiation (nonionizing radiation). Two nucleotide bases in DNA – cytosine and thymine – are most vulnerable to radiation that can change their properties. UV light can induce adjacent pyrimidine bases in a DNA strand to become covalently joined as apyrimidine dimer. UV radiation, particularly longer-wave UVA, can also cause oxidative damage to DNA.
• Ionizing radiation
• Radioactive decay, such as 14C in DNA

23.3.2.3 Transposable elements

Scientists are now using the power of transposable elements to create new mutations. Transposable elements are mobile pieces of DNA that can move from one location in a genome to another. Often when they move to a new location, the result is a new mutant. The mutant arises because the presence of a piece of DNA in a wild type gene disrupts the normal function of that gene. As more and more is being learned about genes and genomes, it is becoming apparent that transposable elements are a power source f or creating insertional mutants.

The detailed knowledge of the structure and function of transposable elements is now being applied in the pursuit of new mutations. Stocks are created in which a specific type of element is present. This stock is then crossed to a genetic stock that doe s not contain the element. Once that element enters the virgin stock, it can begin to move around that genome. By carefully observing the offspring, new mutants can be discovered. This approach to developing mutants is termed insertional mutagenesis.

23.4 Classification of Mutations

23.4.1 By effect on structure

The sequence of a gene can be altered in a number of ways. Gene mutations have varying effects on health depending on where they occur and whether they alter the function of essential proteins. Mutations in the structure of genes can be classified as:

23.4.1.1 Point mutations

Often caused by chemicals or malfunction of DNA replication, exchange a single nucleotide for another. These changes are classified as transitions or transversions. Most common is the transition that exchanges a purine for a purine (A ↔ G) or a pyrimidine for a pyrimidine, (C ↔ T). A transition can be caused by nitrous acid, base mis-pairing, or mutagenic base analogs such as 5-bromo-2-deoxyuridine (BrdU). Less common is atransversion, which exchanges a purine for a pyrimidine or a pyrimidine for a purine (C/T ↔ A/G). An example of a transversion is adenine (A) being converted into a cytosine (C). A point mutation can be reversed by another point mutation, in which the nucleotide is changed back to its original state (true reversion) or by second-site reversion (a complementary mutation elsewhere that results in regained gene functionality). Point mutations that occur within the protein coding region of a gene may be classified into three kinds, depending upon what the erroneous codon codes for:

23.4.1.2 Silent mutations

Silent mutations are those that cause no change in the final protein product and can only be detected by sequencing the gene. Most amino acids that make up a protein are encoded by several different codons (see genetic code). So, if for example, the third base in the 'cag' codon is changed to an 'a' to give 'caa', a glutamine (Q) would still be incorporated into the protein product, because the mutated codon still codes for the same amino acid. These types of mutations are 'silent' and have no detrimental effect. (Fig.23.1)

Fig. 23.1 Silent mutations

23.4.1.3 Missense mutations

In a missense mutation, the new base alters a codon resulting in a different amino acid being incorporated into the protein chain. This is what happens in sickle cell anaemia. The 17th nucleotide of the gene for the beta chain of haemoglobin is changed from an 'a' to a 't'. This changes the codon from 'gag' to 'ggt' resulting in the 6th amino acid of the chain being changed from glutamic acid to valine. This apparently trivial alteration to the beta globin gene alters the quaternary structure of haemoglobin, which has a profound influence on the physiology and wellbeing of the individual (Fig. 23.2).

Fig. 23.2 Missense mutations

The picture shows an example of a missence mutation, a substitution of 'a' in the second codon to 'g' leads to an amino acid substitution of glutamine (Q) to arginine (R).

23.4.1.4 Nonsense mutations

In a nonsense mutation, the new base changes a codon that specified an amino acid into one of the stop codons (taa, tag, tga). This will cause translation of the mRNA to stop prematurely and a truncated protein to be produced. This truncated protein will be unlikely to function correctly. Nonsense mutations occur in between 15% to 30% of all inherited diseases including cystic fibrosis, haemophilia, retinitis pigmentosa and duchenne muscular dystrophy (Fig. 23.3).

The picture shows an example of a nonsense mutation, where 'c' in the second codon is changed to 't'. In the normal nucleotide sequence the second codon 'cag' codes for glutamine (Q) whilst in the mutated second codon 'tag' codes for Stop leading to premature termination of the protein.

Fig. 23.3 Nonsense mutations

Insertions add one or more extra nucleotides into the DNA. They are usually caused by transposable elements, or errors during replication of repeating elements (e.g. AT repeats). Insertions in the coding region of a gene may altersplicing of the mRNA (splice site mutation), or cause a shift in the reading frame (frameshift), both of which can significantly alter the gene product. Insertions can be reverted by excision of the transposable element (Fig. 23.4).

Fig. 23.4 Frameshift mutation

23.4.1.5 Deletions

Deletion removes one or more nucleotides from the DNA. Like insertions, these mutations can alter the reading frame of the gene. They are generally irreversible: though exactly the same sequence might theoretically be restored by an insertion, transposable elements able to revert a very short deletion (say 1–2 bases) in any location are either highly unlikely to exist or do not exist at all. Note that a deletion is not the exact opposite of an insertion: the former is quite random while the latter consists of a specific sequence inserting at locations that are not entirely random or even quite narrowly defined (Fig. 23.5).

Fig. 23.5 Deletions

23.4.1.6 Large-scale mutations in chromosomal structure, including

• Amplifications (or gene duplications) leading to multiple copies of all chromosomal regions, increasing the dosage of the genes located within them.
• Deletions of large chromosomal regions, leading to loss of the genes within those regions.
• Mutations whose effect is to juxtapose previously separate pieces of DNA, potentially bringing together separate genes to form functionally distinct fusion genes (e.g. bcr-abl). These include
• Chromosomal translocations
• Interchange of genetic parts from non-homologous chromosomes
• Interstitial deletions
• An intra-chromosomal deletion that removes a segment of DNA from a single chromosome, thereby apposing previously distant genes. For example, cells isolated from a human astrocytoma, a type of brain tumor, were found to have a chromosomal deletion removing sequences between the "fused in glioblastoma" (fig) gene and the receptor tyrosine kinase "ros", producing a fusion protein (FIG-ROS). The abnormal FIG-ROS fusion protein has constitutively active kinase activity that causes oncogenic transformation (a transformation from normal cells to cancer cells).
• Chromosomal inversions
• Reversing the orientation of a chromosomal segment.
• Loss of heterozygosity
• Loss of one allele, either by a deletion or recombination event, in an organism that previously had two different alleles.