Contrast: mutagenesis, mutation, mutant

Mutagenesis is the occurrence of a change in DNA.  A mutation is a stably maintained change; replication is necessary for this. A mutant is an organism that expresses this mutation.

List possible outcomes for mutations in regulatory genes such as lacP or lacO.

Anything could happen.

1. Expression could be impaired,
2. Expression could be enhanced,
3. Expression could be unaffected,
4. Expression could be destroyed.

How might mutations in structural genes affect protein structure and function?

Again, anything could happen.

1. Protein function could be impaired (including conditional lethals),
2. Protein function could be enhanced,
3. Protein function could be unaffected,
4. Protein function could be destroyed.

How do these suppressor mutations (supE, supF) act to suppress the effects of point mutations in structural genes?

The suppressor mutations are mutant tRNA’s that code for stop codons.  What then occurs is that a point mutation that produces a stop codon, which would normally produce a nonfunctional prematurely terminated protein, now would produce a protein with some random amino acid at the point of mutation.  The amino acid may drastically impair the protein, or it might not even work at all, but there is a higher probability that it will work due to the fact that the protein is now allowed to be translated to completion.

Point mutations of coding regions for structural genes may generate missense, nonsense, or silent mutations. Describe each of these types of mutations and their possible effects on protein function.

Silent mutation: There is no change in translation, therefore there is no change in protein function.
Missense mutation: The codon is changed, so there is a different amino acid at that position.  This may have no effect on protein function, it might impair it, enhance it, or destroy it.
Nonsense mutation: The codon is changed to a stop codon.  This type of mutation almost always destroys protein function, because the protein cannot be completed.

Describe frameshift mutations and their possible effects on protein structure and function.

A frameshift mutation is normally a deletion or insertion that disrupts the reading frame of the message. This causes a profound shift in the amino acid translation; the protein products are virtually always nonfunctional. Luckily, there are stop codons hidden out-of-frame, so that frameshifts cause nonsense mutations to crop up.

Class I revertants generate wild-type amino acid sequences.  Class II revertants generate silent mutations in protein sequences - What is meant by thins? Class III revertants can be suppressed by second site mutations - what is meant by this?

Silent mutation, in this context, means that the protein is still functional, so there is no expression of the mutation on the phenotype. Class III second site suppression indicates that there is a second mutation in another place on the chromosome, that activates transcription of the mutant sup tRNA’s. This allows suppression of stop codons; it is important to recognize that all such stop codons are suppressed, not just the one created by the initial mutation.

How are each of these procedures used to isolate either spontaneous or induced mutants when studying biosynthetic pathways: screening and enrichment (eg, suicide selection?)

Both these procedures can be used to isolate nutritional mutants, aka auxotrophs.  By selecting for “losers”, we eliminate prototroph cells.

Describe a positive selection for mutations in genes for enzymes such as DNA gyrase or RNA polymerase.

DNA gyrase: nalidixic acid interferes with DNA gyrase activity, so it would be reasonable to hypothesize that naladixic acid resistant bacteria have mutant genes for DNA gyrase.  Therefore, adding nalidixic acid to growth medium, then search out for surviving cells, should yield cells with mutations in DNA gyrase.

Contrast a scoring procedure (such as beta galactosidase blue/white) with a positive selection procedure.

Selection, any type of selection, involves preferential growth of one group of cells.  Scoring, on the other hand, merely labels the cells with something easy to observe, such as color change, or fluorescence.

Mutations in essential genes are often lethal and cannot be studied. What is meant by conditional lethal mutations and in what ways could bacteria possessing these types of mutations be isolated.

Conditional lethal mutations only are lethal under certain conditions (duh, right?), such as increased temperature, pH, etc, that the organism normally would be able to withstand. One way to isolate (say, a temperature sensitive mutant that can’t grow past 35 degrees), would be to do replica plating, then growing one plate at 25 degrees, the other at 37.

Describe how these chemical changes to DNA bases can each contribute to a mutagenic event: deamination of cytosine/tautomeric forms of bases during replication/alkylating agents/ incorporation of base analogs/ mistakes in the error prone repair system.

The DNA repair section of the website describes these in detail.

Compare Transition and transversion.

Transition is a purine -> purine, or pyrimidine -> pyrimidine mutation.  A transversion is a purine -> pyrimidine, or vice versa, mutation. It is held that transversions are more “dangerous” mutations than transitions.