2 Mutation and Recombination, Mutations and Mutants
3 Mutation is a heritable change in DNA sequence that can lead to a change in phenotype. By definition, a mutant differs from its parental strain in genotype, the nucleotide sequence of the genome.hisC (1,2,3,…) mutants of E. coliAuxotroph – nutritional mutant rewiring a growth factors, amino acids (e.g. His-).Prototroph – wild type
4 Selectable mutations are those that give the mutant a growth advantage under certain environmental conditions and are especially useful in genetic research.If selection is not possible, mutants must be identified by screening.
5 Although screening is always more tedious than selection, methods are available for screening large numbers of colonies in certain types of mutations.Penicillin selection – pen kills growing but not mutant (non growing) cells in minimal mediumNutritionally defective mutants can also be detected by the technique of replica plating (Figure 10.2).
7 Molecular Basis of Mutation Mutations, which can be either spontaneous or induced, arise because of changes in the base sequence of the nucleic acid of an organism's genome.
8 A point mutation, which results from a change in a single base pair, can lead to a single amino acid change in a polypeptide or to no change at all, depending on the particular codon involved (Figure 10.3).
10 In a nonsense mutation, the codon becomes a stop codon and an incomplete polypeptide is made. In a missense mutation, the sequence of amino acids in the ensuing polypeptide is changed, resulting in an inactive protein or one with reduced activity.Temperature-sensitive mutations/conditionally lethal mutations
11 Deletions and insertions cause more dramatic changes in the DNA, including frameshift mutations, and often result in complete loss of gene function (Figure 10.4).
14 Mutation RatesDifferent types of mutations can occur at different frequencies. For a typical bacterium, mutation rates of 10–7 to 10–11 per base pair are generally seen.
15 Although RNA and DNA polymerases make errors at about the same rate, RNA genomes typically accumulate mutations at much higher frequencies than DNA genomes.
16 MutagenesisMutagens are chemical, physical, or biological agents that increase the mutation rate. Mutagens can alter DNA in many different ways, but such alterations are not mutations unless they can be inherited.
17 Table 10.2 gives an overview of some of the major chemical and physical mutagens and their modes of action.
18 There are several classes of chemical mutagens, one being the nucleotide base analogs (Figure 10.5).
22 Some DNA damage can lead to cell death if not repaired Some DNA damage can lead to cell death if not repaired. A complex cellular mechanism called the SOS regulatory system is activated as a result of some types of DNA damage and initiates a number of DNA repair processes, both error-prone and high-fidelity (Figure 10.7).
34 Certain prokaryotes exhibit competence, a state in which cells are able to take up free DNA released by other bacteria.
35 Incorporation of donor DNA into a recipient cell requires the activity of single-stranded binding protein, RecA protein, and several other enzymes. Only competent cells are transformable (Figure 10.14).
40 In specialized transduction (Figure 10 In specialized transduction (Figure 10.16), the DNA of a temperate virus excises incorrectly and takes adjacent host genes along with it; transducing efficiency in this case may be very high.
42 Plasmids: General Principles Plasmids are small circular or linear DNA molecules that carry any of a variety of unessential genes. Although a cell can contain more than one plasmid, they cannot be closely related genetically.
43 Figure shows a genetic map of the F (fertility) plasmid, a very well characterized plasmid of Escherichia coli.
47 Types of Plasmids and Their Biological Significance
48 The genetic information that plasmids carry is not essential for cell function under all conditions but may confer a selective growth advantage under certain conditions.
49 Examples include antibiotic resistance (Figure 10 Examples include antibiotic resistance (Figure 10.20), enzymes for degradation of unusual organic compounds, and special metabolic pathways. Virulence factors of many pathogenic bacteria are often plasmid-encoded.
53 Conjugation: Essential Features Conjugation is a mechanism of DNA transfer in prokaryotes that requires cell-to-cell contact.
54 Genes carried by certain plasmids (such as the F plasmid) control conjugation, and the process involves transfer of the plasmid from a donor cell to a recipient cell (Figure 10.22). Plasmid DNA transfer involves replication via the rolling circle mechanism.
57 10.12 The Formation of Hfr Strains and Chromosome Mobilization, p. 279
58 The donor cell chromosome can be mobilized for transfer to a recipient cell. This requires that the F plasmid integrate into the chromosome to form the Hfr phenotype. Transfer of the host chromosome is rarely complete but can be used to map the order of the genes on the chromosome.
59 F' plasmids are previously integrated F plasmids that have deintegrated and excised some chromosomal genes.
60 Integration of the F plasmid into the host chromosome can occur at several specific sites, called IS (for insertion sequence) sites (Figure 10.23). These sites are regions of DNA sequence homology between chromosomal and F plasmid DNA.
64 This is often necessary because mutations in different genes in the same pathway may give the same phenotype. Complementation tests do not involve recombination.
65 Transposons and Insertion Sequences Transposons and insertion sequences are genetic elements that can move from one location on a chromosome to another by a process called transposition, a type of site-specific recombination (Figure 10.30).
75 An in vitro recombination procedure uses restriction enzymes and DNA ligase to produce the hybrid DNA molecule. Once introduced into a suitable host, the cloning vector can control production of large amounts of the target DNA.
76 Making a gene library by cloning random fragments of a genome is called shotgun cloning, and it is a widely practiced technique in gene cloning and genomic analyses.
77 Plasmids as Cloning Vectors Plasmids are useful cloning vectors (Figure 10.36) because they are easy to isolate and purify and can multiply to high copy numbers in bacterial cells.
84 Larger amounts of foreign DNA can be cloned with lambda than with many other plasmids. In addition, the recombinant DNA can be packaged in vitro for efficient transfer to a host cell. Plasmid vectors containing the lambda cos sites are called cosmids, and they can carry a large fragment of foreign DNA.
85 In Vitro and Site-Directed Mutagenesis Site-directed mutagenesis allows synthetic DNA molecules of desired sequence to be made in vitro and used to construct a mutated gene directly or to change specific base pairs within a gene (Figure 10.40).
87 Inserting DNA fragments, called cassettes, into genes can also cause gene disruption (Figure 10.41). The inserted cassette eliminates the function of the wild-type gene while conferring a new, and usually selectable, phenotype on the cell.