Northern blot analysis is performed on cellular mRNA isolated from E. coli. The probe used in the northern blot analysis hybridizes to a portion of the lacY sequence. Below is an example of the gel from northern blot analysis for a wild-type lac⁺ bacterial strain. In this gel, lane 1 is from bacteria grown in a medium containing only glucose (minimal medium). Lane 2 is from bacteria in a medium containing only lactose. Following the style of this diagram, draw the gel appearance for northern blots of the bacteria listed below. In each case, lane 1 is for mRNA isolated after growth in a glucose-containing (minimal) medium, and lane 2 is for mRNA isolated after growth in a lactose-only medium.
lac⁻ bacteria with the genotype I⁺ P⁺ O⁺ Z⁺ Y⁺ and a mutation that prevents CAP–cAMP binding to the CAP site

Question

Northern blot analysis is performed on cellular mRNA isolated from E. coli. The probe used in the northern blot analysis hybridizes to a portion of the lacY sequence. Below is an example of the gel from northern blot analysis for a wild-type lac⁺ bacterial strain. In this gel, lane 1 is from bacteria grown in a medium containing only glucose (minimal medium). Lane 2 is from bacteria in a medium containing only lactose. Following the style of this diagram, draw the gel appearance for northern blots of the bacteria listed below. In each case, lane 1 is for mRNA isolated after growth in a glucose-containing (minimal) medium, and lane 2 is for mRNA isolated after growth in a lactose-only medium.
Northern blot showing mRNA bands for lanes 1 and 2 from E. coli grown in glucose and lactose media, respectively.

lac⁻ bacteria with the genotype I⁺ P⁺ O⁺ Z⁺ Y⁺ and a mutation that prevents CAP–cAMP binding to the CAP site

Northern blot showing mRNA bands for lanes 1 and 2 from E. coli grown in glucose and lactose media, respectively.

lac⁻ bacteria with the genotype I⁺ P⁺ O⁺ Z⁺ Y⁺ and a mutation that prevents CAP–cAMP binding to the CAP site

Accepted Answer

Hi, everyone. Let's look at our next problem. Suppose a researcher is studying the regulation of the LAC operon and E coli and performs a northern block analysis. They isolate M R N A from two bacterial type strains. One with a wild type black operon genotype I plus P plus O plus Z plus Y plus and another with a deletion mutation in the lac eye gene. The researcher hybridizes the M R N A with a probe specific to the lac Z gene. Suppose the researcher includes a sample from an E coli strain with a deletion mutation in a lax Z gene in the northern blood analysis. How would this affect the results of the hybridization? Choice? A, the hybridization will show strong visible bands. Choice B. The hybridization will create overlapping bands, choice C, the hybrid hybridization will not show any bands or choice D, none of the options is correct. So let's think through what's going on here in our northern blot and in our samples, recalling that we do have that option of choice D that there's no correct answers here. So we recall that in northern blots, our gel has M R N a samples. And that means that the only bands that would be present on the Joe would be from jeans that were transcribed. So we need to keep that in mind. And then the labeled probe is composed of C DNA that's complementary to a specific gene of interest. So that allows us to see which genes are actually being transcribed. So we have initially two different samples. We have one that's wild type and one that has a mutation in the lack I gene. So our wild type sample will act as this OPERON usually does, which is that we will see expression or we'll see bands in cases where the OPERON is induced. And of course, what are those situations that would be in the presence of lactose and the absence of glucose just acting as it does in nature. And we could look at, you know that there's uh low levels of transcription if glucose is present, but lactose is also present. And we can say there will be high levels of expression if lactose is present and glucose is absent and no transcription of lactose is absent. So the usual conditions and we could look at how those conditions affect the expression of the gene. If there's a mutation to lack I gene, we will always see a band visible. And that's because mutation in the lack igene will mean that the lac repressor is not transcribed. So no repressor. So we'll always have some transcription of those genes, it will now be constitutive transcription, always going on at some level. So now, finally, our question says, how would it affect the hybridization if we have a mutation in the lac Z gene causing it not to operate? And our probe is for the lax Z gene? Well, in this case, we're going to see no visible bands because if we've interfered with the gene that causes the lax Z to be expressed or to be produced, then we won't see any M R N A for the lay gene. So our answer here is going to be choice. C the hybridization will not show any bands. I like that. Since due to the mutation and the lax E gene, we have no M R N A being transcribed from that gene. See you in the next video.

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Key Concepts

Northern Blotting

Lac Operon

CAP-cAMP Complex