1. You have purified a protein fro that causes cells in tissue culturetcteriu ti
ID: 256618 • Letter: 1
Question
1. You have purified a protein fro that causes cells in tissue culturetcteriu tissue culture to increase their thal D levels. You subsequently cloned a fragment of NA from bacterium Y that contained two ORFs, one of which you identified as the gene encoding the pro- tein you isolated, which you called yctA (for Y bac- terium cytotoxin A). You then determined that ex- pression of the YctA protein occurs only under iron-restrictive conditions (i.e., when iron is absent from the medium). A. Describe how you would determine if the ex- pression of YctA is regulated by iron at the tran- scriptional level. B. Describe how you would determine whether the oth codes a possible transcriptional regulator. er ORF, which you have named yctR, en- ctk is a transcriptional regulator, how could ermine if it is an activator or repressor pro- tein?Explanation / Answer
Ans. A).The regulation of the expressio of YctA at transcription level by iron can be shown by growing the bacteria in iron containing medium, such as EMB and medium lacking iron at different test tubes. The expression of the gene an be checked by run the smaple in PCR with marker for ORF YctA. In iron containing medium, sequence for YctA will not found, while, in iron-defficient medium, the sequene for YctA will be observed. Hence, we could prroved that the iron regulates expression of YctA.
B). By growing the bacteria in an iron-lacking medium, we can keep the expression of YctA gene. If yctR is a transcription regulator for YctA, then cutting the ORF for yctR will affect the transcription of gene for YctA, which can be seen by run the sample into PCR by using marker for YctA.
C). If yctR encodes for a transcriptional regulator for YctA, it may an activator or a repressor. If it is an activator, removal of ORF for yctR will leads to stop of trancription of YctA gene, and if it is a repressor, removal of ORF for yctR will results in transcription of gene for YctA. Both the outcomes can be checked by using PCR.
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