Recall that the flow of information within a cell involves the transcription of DNA to mRNA and the translation of mRNA to protein. Recall also, that the flow of information between bacterial generations involves DNA replication and distribution to two equal daughter cells.Therefore, we would exect a change in microbial DNA to be replicated and passed on to future generations and to effect protein structure and function if the change occurred in the gene that coded for that protein. In this chapter we take a closer look at the cause and effect of changes in DNA. These changes are called mutations.
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- Point mutations are the most common type of mutation.
- A single point mutation, also called a base substitution, occurs when a single nucleotide is replaced with a different nucleotide.
- A point mutation results in a base pair substitution after replication and possibly a mutant protein after transcription and translation.
- There are three types of point mutations:
- Silent Mutation:
- A silent mutation causes no change in the activity of the protein.
- A silent mutation is usually the result of a substitution occurring in the third location of the mRNA codon
- Because the gentic code is degenerate (most amino acids are coded for by several alternative codons), the resulting new codon may still code for the same amino acid.
- Missence Mutation:
- A missence mutation is a nucleotide substitution that changes a codon so that it codes for a different amino acid in the protein.
- This usually results in a change of the activity of the protein. The change may be harmful or beneficial to the protein.
- Nonsense Mutation:
- A nonsense mutation is the same as a missense mutation except the resulting codon codes for a STOP signal.
- The result is a premature termination of translation.
- The protein is shorter than usual and does not contain all the amino acids that it should. Therefore, this protein is most likely nonfunctional.
- Another type of mutation is a frameshift mutation which is caused by the insertion or a deletion of a base pair.
- An inserted or deleted nucleotide alters the triplet grouping of nucleotides into codons and shifts the reading frame so that all nucleotides downstream from the mutation will be improperly grouped.
- The result is a protein with extensive missense ending sooner or later in nonsense.
- A mutation can be the result of different events.
- Errors made during replication, repair, or recombination can all lead to point or frameshift mutations. Mutations resulting from such errors are spontaneous mutations.
- A mutation can also result from the action of physical and chemical agents known as mutagens. We will now explore three mutagens: nitrous acid, base analogs, and UV light.
- Nitrous Acid:
- Nitrous Acid affects DNA complementation.
- The acid randomly modifies the base adenine so that it will pair with cytosine instead of thymine.
- This change is made evident during DNA replication when a new base pair appears in daughter cells in a later generation.
- A Base Analog:
- A base analog is a compound sufficiently similar to one of the four DNA bases but have different pairing properties.
- For example, 5-bromouracil is the analog of thymine but sometimes pairs with guanine and 2-aminopurine is the analog of adanine but sometimes pairs with cytosine.
- The incorporation of a base analog will to a base pair substitution in that appears in daughter cells in a later generation.
- UV Light:
- Exposure to direct UV light induces covalent linking between adjacent thymine nucleotides on a DNA strand forming a thymine dimer.
- These dimers cause the strand to buckle, disrupting normal base pairing. This prevents proper replication and transcription.
- Bacteria have enzymes to fix the damage created by UV light.
- An enzyme cuts the DNA at two point and removes the damaged portion.
- DNA polymerase synthesizes a new DNA segment using the healthly strand as a template.
- DNA ligase joins the new fragment to the old strand.
- Mutations are random events and there is no way of knowing when a mutation will occur.
- Genes do, however mutate spontaneously at a characteristic rate, making it possible to assign probabilities to certain mutation events.
- The probability that a gene will mutate when a cell divides is called the mutation rate.
- Spontanoeus mutation rate for the average gene is 0.000000001.
- This means a mutation event is estimated to occur once in every million genes replicated.
- The presence of a mutagen increases the rate of mutation to 0.00001 to 0.001.
- This means that a mutation event is estimated to occur once in every hundred thousand to one hundred thousand genes in the presence of a mutagen.
- How can you tell if there are any mutant colonies in a culture?
By either positive (direct) selection or by negative (indirect) selection.
- Positive Selection:
- Positive selection entails the growing the culture on a medium that will alow for the growth of only the mutant colonies.
- If, for example, we want to find mutants that resistant to penicillin, we grow the culture on a medium that contains pencillin. Only those colonies that are resistant to penicillin will grow and we ca identify them directly.
- Negative Selection:
- Negative selection is used to identify mutants that have lost the ability to perform a certain function that their parents had.
- Auxotrophic mutants, for example, are bacteria that have lost the ability to synthesize an essential nutrient.
- The replica-plating technique is used to identify mutants by negative selection.
- the replica-plating technique can be used, for example, to identify mutants that have lost the ability to synthesize the amino acid histidine.
Therefore, mutants are His- and require histidine in order to survive.
- Inoculate a histidine enriched medium with bateria. Incubate so that cells can form colonies. This is the master plate.
- Press a sterile velvet surface into the colonies of the master plate. Some cells from each of the colonies adhere to the velvet.
- Prepare two mediums, one with histidine, the other without histidine.
- Transfer cells from the velvet to each plate.
- Compare growth on the two plates after incubation. Colonies that grow on the histindine enriched medium but not on the medium lacking histidine are His- mutants.
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