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mRNA transcripts, cis/trans-acting questions

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mRNA transcripts, cis/trans-acting questions

Postby animus31 » Fri Nov 09, 2012 11:58 am

I need some help with these two homework questions. I have no idea where/how to start to figure these out.

You have isolated several yeast mutants unable to metabolize galactose. All of the mutants fail to transcribe the GAL2 gene, which encodes the enzyme for the first step in metabolizing galactose. For mutants a. – e. below, use the description to determine whether it is: a cis-acting mutation of a regulatory sequence for GAL2 itself (“cis”); a trans-acting mutation in GAL4, which activates transcription of GAL2 (“GAL4”); or a trans-acting mutation in GAL3, which interacts with GAL80 to de-repress GAL4 when galactose is present (“GAL3”). NOTE: Yeast are normally haploid, but the diploid cells resulting from mating can persist as diploid colonies until induced to undergo meiosis.

______ a. When this mutant is crossed to a known GAL4 recessive mutant strain, the resulting diploid yeast colonies are all unable to metabolize galactose.
______ b. You do a cross and find this mutation maps to the vicinity of GAL3, and not to GAL4 or GAL2.
______ c. A double mutant haploid with both this mutation and a GAL80 mutation restores galactose metabolism, and GAL2 is transcribed even when galactose is absent.
______ d. All other enzyme-encoding genes (other than GAL2) in the galactose metabolism pathway are transcribed normally in this mutant.
______ e. The DNA sequence region containing the UAS sites upstream of GAL2 is deleted in this mutant.

You are studying the genetics of a negatively regulated operon in E. coli, in which a single mRNA transcript encodes two genes (A and B) that are separately translated into two proteins A and B. You have isolated mutations in both genes (A - and B -) that result in non-functional proteins. You have also isolated two constitutive regulatory mutations that you are calling m1 and m2, respectively. (I.e. both mutants synthesize proteins A and B even without induction.) When you test different genotypes and different partial diploids using F’ strains, you obtain the following results. (The mutations are present only on copies labeled as m1 or m2.)
Protein A produced? Protein B produced?
Genotype no induction with induction no induction with induction
A+B+ (wild-type) no yes no yes
A -B+ no no no yes
m1A+B+ yes yes yes yes
m2A+B+ yes yes yes yes
A+B+ / F’: m1A+B+ yes yes yes yes
A+B+ / F’: m2A+B+ no yes no yes
A - B+ / F’: m1A+B - yes yes no yes
A - B+ / F’: m2A+B - no yes no yes

Which of the mutations (m1 or m2) is:
a. cis-acting? b. trans-acting?

c. probably an operator sequence mutation?

d. probably a loss-of-function mutation in the gene encoding a repressor to the operon?
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