I need help from all of you

[kevinsuper]

Here is the question.I don't even know how to approach~~~Thanks for detailed help!Please be Specific!

Three lacZ-E.Coli strains which have nonsense mutations each one.i.e 5'TAA,TAG and TGA.You wish to convert each of nonsense one to sense and choose UV irradiation to revert them.Using the knowledge of lesion bypass by DNA ploymerase V and the "A-rule" show the expected results for each nonsense mustions.Do the results suggest if UV is effective for converting nonsense to sense?Be specific!Thanks a lot!

[scicop]

I think there are missing components to the question. If i'm correct those sequences are stop codons. Thus one way to assess mutagenecity would be to place those codons in frame with a reporter gene (i.e. gfp, lucierase or antibiotic resistance). You would have to design primers that flank both 5' and 3' sides of your stop codons and use sequencing analysis in your transformed mutants (if any) to assess what base changes were made.

that's my take given the information you gave, but how that would related to DNA Pol "V" activity, i'm not sure, that's a whole different question.

 

UV tends to make pyrimadine dimers (between Ts), and inorder to assess DNA Polymerase activity past such lesions you would have to conduct kinetic assays, which have been done my many others in the past (see work by Kornberg, Grollman, Teebor, Sietz).

 

Good luck.

[CharonY]

Actually I think you are thinking a little bit too complicated here, sciop.

This is probably only a exam question.

Since it is stated that it is a lacZ negative E. coli, one can assume that the mutation is in the lacZ gene (otherwise this info had no meaning). Now the mutation that is sought will be a combination of the UV (dimer formation) and polV (sloppy replication across the resultion lesions).

PolV is known to be able to bypass stalled replication forks but it has a low fidelity. Furthermore it is known that PolV not only bypasses for instance TT-dimers with high efficiency but also tends to incorporate a guanine opposite the 3'-thymine of a TT-dimer. I think the rest should be obvious.

[Immunologist]

I think you should find this answer by yourself, but take the hints of people:

I am rephrasing your question as follow:

 

You have 3 non-sense mutations in a LacZ gene in 3 strains of E.coli.

You will use UV on these strains to try and revert these non-sense mutations to coding codons. For each of the 3 stated codons (TAA,TAG and TGA), what is the expected codon after UV irradiation when referring to the "A rule" (hint: what UV do to nucleotides??? How these modified nucleotides will be interpreted by polymerases?).

The post-UV irradiation codons will result in what? 1. which amino acid (or stop)

2. functional or not functional gene.

 

Everything revolves around the effect of UVs on nucleotides and the mutation induced by UVs...

 

Good luck!

[kevinsuper]

After I read the valuable replies from all of you,I've still got some confusions~~~