Mutation and Variation

1. Mutation and Variation In genetics, a mutation is a change of the nucleotide sequence of the genome of an organism, virus, or extrachromosomal genetic element. Mutations result from unrepaired damage to DNA or to RNA genomes (typically caused by radiation or chemical mutagens), errors in the process of replication, or from the insertion or deletion of segments of DNA by mobile genetic elements. Mutations may or may not produce discernible changes in the observable characteristics (phenotype) of an organism. Mutations play a part in both normal and abnormal biological processes including: evolution, cancer, and the development of the immune system.

2. Directed mutation in bacteria? • Despite the apparently conclusive results of the fluctuation test, there have been suggestions that something akin to directed mutation can be observed in bacteria under certain circumstances. • This originated with work by Cairns and others which showed that a strain of E. coli that was unable to ferment lactose (Lac) reverted to Lac‏ at a higher frequency when supplied with lactose as the sole carbon source (i.e. conditions under which the ability to ferment lactose was advantageous) than when supplied with glucose (the ability to ferment lactose therefore being of no advantage)

3. Types of mutations • Point mutations • base substitution : replacing one nucleotide by another • Silent mutations(synonymous codons) • nonsense mutation : are referred to as stop or termination codons (UAG : amber , UAA : ochre , UGA opal) • frameshift mutation : deletion or addition of a single nucleotide (or of any number other than a multiple of three) • Polarity : If a point mutation results in premature termination of translation, it may also affect the expression of other genes downstream in the same operon

4. Frameshift mutation and suppression. (a) Initial (mRNA) sequence and translated product. (b) Deletion of a single base alters the subsequent reading frame producing a different amino acid sequence and encountering a stop codon. (c) Addition of a base at a different position restores the original reading frame and may suppress the mutation

5. Types of mutations • Conditional mutants There are many genes that do not affect resistance to antibiotics or bacteriophages, biosynthesis of essential metabolites or utilization of carbon sources. Some of these genes are indispensable and any mutants defective in those activities would die (or fail to grow).

6. Variation due to larger scale DNA alterations • Deletions • Insertion mutations • Inversion (salmonella typhimurium) • Extra chromosomal agents and horizontal gene transfer • Plasmids • bacteriophages

7. Phenotypes • There are strains that require an additional growth supplement, such as a specific amino acid auxotrophs and prototroph • Mutants that are defective in their ability to use a certain substrate (Lac־) • The third type of commonly used mutation confers antibiotic resistance (mutation in the gene coding for one of the ribosomal proteins can make the cell resistant to streptomycin) • Unfortunately, observation of the phenotype does not always tell us much about the genotype (Trp־)

8. Genetic nomenclature • Ampʳ means the strain is resistant to ampicillin • His denotes a histidine auxotroph • Lac indicates inability to ferment lactose • hisA::Tn10., indicating that the mutation in the hisA gene is due to insertion of the transposon Tn10 • hisA/FʹhisA‏ has a mutation in the chromosomal • hisA gene, but contains an Fʹ plasmid which carries a functional hisA gene • There are other genes with a trp designation that are not part of the tryptophan operon and where mutation does not lead to auxotrophy (trpT: tryptophan-specific tRNA , trpS : tryptophanyl-tRNA synthetase , TrpR protein is a repressor protein involved in the regulation of the trp operon)

9. Restoration of phenotype • A mutant strain with the UUU codon (phenylalanine) may undergo a further mutation which restores the UUA codon (a true back mutation) • The effect of a mutation can also be negated by a second, unrelated mutation; this effect is known as suppression. There are two types of suppression that are of more general importance. • The first occurs with frameshift mutations • Another important type of suppression occurs with ‘nonsense’ mutations, where a stop codon has been created within the coding sequence • Permissive host : Phagescarrying an amber mutation will show a mutant phenotype (or fail to grow) on a normal bacterial host, but will show a wild-type phenotype when an appropriate amber suppressor host is used

10. Suppression of a nonsense mutation. A base substitution changes CAG to the stop codon UAG, causing premature termination of translation. This can be suppressed by a separate mutation in a tRNA gene, giving rise to a tRNA that can recognize the UAG codon

11. Complementation • Another way in which a mutant phenotype can be converted back to the wild type is by acquisition of a plasmid that carries a functional version of the affected gene Recombination general or homologous recombination site-specific recombinational mechanisms

13. Mechanisms of mutation Spontaneous mutation Spontaneous mutation occurs through errors in the replication of DNA • The normal pairing of adenine and thymine : adenine in the amino form and the keto form of thymine • The imino form of adenine, will base pair with cytosine rather than thymine, while the enol form of thymine will hydrogen bond to guanine

14. Mechanisms of mutation Chemical mutagens Nitrous acid causes oxidative deamination of bases Nitrous acid causes an oxidative deamination in which amino groups are converted to keto groups and thus cytosine residues for example will be converted to uracil

15. Mechanisms of mutationUltraviolet irradiation Structure of thymine dimers • The principal effect of UV irradiation with which we are concerned is the production of pyrimidine dimers (commonly referred to as thymine dimers) • These pyrimidine dimers cannot be replicated and are therefore lethal to the cell unless it is able to repair the damage

16. Photoreactivation The best defence that is mounted against damage by UV irradiation is known as photoreactivation. This is catalysed by an enzyme (photolyase) within the cells that in the presence of visible light can break the covalent bonds linking the two pyrimidine residues, thus re-establishing the original nature of the base sequence at that point

17. T^T dimers may be repaired by two mechanisms. In photoreactivation repair, the PRE enzyme activated by blue light breaks the dimer, restoring the normal base pairing. Note that blue light is at the same end of the spectrum as UV radiation. (b) In excision repair, the uvr system excises the dimer, and the gap is filled in by the proof-reading activity of DNAPol

18. Non-SOS repair is basically error-free, but SOS repair is error-prone. This is why UV is a mutagen. May be due to RecA binding ssDNA in lesions, which could then bind to DNA Pol III complex passing through this area of the DNA and inhibit 3'>5' exonuclease (proofreading) ability. This makes replication faster but also results in more mutations. This affect on proofreading seems to involve UmuD'-UmuC complex as well. RecA facilitates proteolytic cleavage of UmuD to form UmuD'. The UmuD'-UmuC complex may bind to the RecA-Pol III complex and promote error-prone replication