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PART III

What Genes Are and What They Do

CHAPTER

Anatomy and Function of a Gene: Dissection Through Mutation

OUTLINE
3.1 Mutations: Primary Tools of Genetic Analysis 3.2 What Mutations Tell Us About Gene Function 3.4 Examples found in eukaryotic gene regulation lecture

Types of mutations in the coding sequence of genes

Fig. 8.28a
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Mutations in the coding sequence of a gene can alter the gene product

Missense mutations replace one amino acid with another Conservative chemical properties of mutant amino acid are similar to the original amino acid
e.g. aspartic acid [(-)charged] glutamic acid [(-)charged]

Nonconservative chemical properties of mutant amino acid are different from original amino acid
e.g. aspartic acid [(-)charged] alanine (uncharged)

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Mutations in the coding sequence of a gene can alter the gene product (cont)
Nonsense mutations change codon that encodes an amino acid to a stop codon (UGA, UAG, or UAA)

Frameshift mutations result from insertion or deletion of nucleotides with the coding region
No frameshift if multiples of three are inserted or deleted

Silent mutations do not alter the amino acid sequence


Degenerate genetic code most amino acids have >1 codon

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A nonsense mutation in a protein-coding gene creates a truncated, nonfunctional protein

Fig 8.32a

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Nonsense suppression
A second, nonsense suppressing mutation in the anticodon of a tRNA gene allows production of a (mutant) full-length polypeptide

Fig 8.32b
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Mutations classified by their effect on DNA

Fig. 7.2a - c
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Mutations classified by their effect on DNA (cont)

Fig. 7.2d

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Mutations classified by their effect on DNA (cont)

Fig. 7.2e

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How natural processes can change the information stored in DNA


Depurination
1000/hr in every cell

Deamination of C C changed to U Normal C-G A-T after replication


Fig. 7.6a, b
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How natural processes can change the information stored in DNA (cont)
X-rays break the sugar phosphate backbone of DNA

Ultraviolet (UV) light causes adjacent thymines to form abnormal covalent bonds (thymine dimers)
Fig. 7.6c, d
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How natural processes can change the information stored in DNA (cont)
Irradiation causes formation of free radicals (e.g. reactive oxygen) that can alter individual bases 8-oxodG mispairs with A Normal G-C mutant T-A after replication

Fig. 7.6e
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Mistakes during DNA replication

Incorporation of incorrect bases by DNA polymerase is exceedingly rare (< 10-9 in bacteria and humans)
Two ways that replication machinery minimizes mistakes Proofreading function of DNA polymerase (Fig 7.7)
3'-to-5' exonuclease recognizes and excises mismatches

Methyl-directed mismatch repair (later in this chapter)


Corrects errors in newly replicated DNA

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DNA polymerases proofreading function

Mispaired base is recognized and excised by 3'-to-5' exonuclease of DNA polymerase

Improves fidelity of replication 100-fold

Fig. 7.7
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Rates of spontaneous mutation


Rates of recessive forward mutations at five coat color genes in mice
11 mutations per gene every 106 gametes

Mutation rates in other organisms


2 12 mutations per gene every 106 gametes

Fig. 7.3b
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Different genes, different mutation rates


Mutation rates are <10-9 to >10-3 per gene per gamete Differences in gene size Susceptibility of particular genes to various mutagenic mechanisms Average mutation rate in gamete-producing eukaryotes is higher than that of prokaryotes Many cell divisions take place between zygote formation and meiosis in germ cells
More chance to accumulate mutations

Can diploid organisms tolerate more mutations than haploid organisms?


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Unequal crossing-over can occur between homologous chromosomes


Pairing between homologs during meiosis can be out of register
Unequal crossing-over results in a deletion on one homolog and a duplication on the other homolog

Fig. 7.8a

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Transposable elements (TEs) move around the genome


TEs can "jump" into a gene and disrupt its function Two mechanisms of TE movement (transposition)

Fig. 7.8b

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How mutagens alter DNA: Chemical action of mutagen


Replace a base: Base analogs - chemical structure almost identical to normal base

Fig. 7.10a

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How mutagens alter DNA: Chemical action of mutagen


Alter base structure and properties: Hydroxylating agents add an OH group

Fig. 7.10b
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How mutagens alter DNA: Chemical action of mutagen (cont)


Alter base structure and properties (cont): Alkylating agents add ethyl or methyl groups

Fig. 7.10b

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How mutagens alter DNA: Chemical action of mutagen (cont)


Alter base structure and properties (cont): Deaminating agents remove amine (-NH2) groups

Fig. 7.10b
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How mutagens alter DNA: Chemical action of mutagen (cont.)


Insert between bases: Intercalating agents

Fig. 7.10c
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Mutations outside the coding sequence can disrupt gene expression

Fig. 8.28b

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Mutations classified by their effects on protein function

Table 8.2

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DNA repair mechanisms that are very accurate


Reversal of DNA base alterations Alkyltransferase removes alkyl groups

Photolyase splits covalent bond of thymine dimers


Homology-dependent repair of damaged bases or nucleotides

Base excision repair (Fig 7.11)


Nucleotide excision repair (Fig 7.12) Correction of DNA replication errors Methyl-directed mismatch repair (Fig 7.13)
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Base excision repair removes damaged bases


Different glycosylases cleave specific damaged bases Particularly important for removing uracil (created by cytosine deamination) from DNA

Fig. 7.11
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Nucleotide excision repair corrects damaged nucleotides


UvrA UvrB complex scans for distortions to double helix (e.g. thymine dimers) UvrB UvrC complex nicks the damaged DNA
4 nt to one side of damage 7 nt to the other side of damage
Fig. 7.12
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