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Undergraduate Biomedical Training Program
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A Progress Report by Mary Ramsey
Human ER Evolution ExperimentExperimental design – Using a yeast 2-plasmid expression system, artificial selection will be used to evolve ligand discrimination in human ER protein.
The yeast model system requires 2 plasmids containing particular sequences –
CUP1-hER: yeast promoter fused to hER. The latest hER sequence was given to us directly from Greene. The sequence contains the same mutation as the original mutation present on the plasmid we received from McLachlan The mutation is located in the hormone binding domain, and alters the protein sequence (ala to gly).
On the original hER plasmid, ubiquitin was fused to hER sequence to enhance stability and expression of hER in yeast. Ubiquitin has been removed from the system to prevent selection for changes in ubiquitin transcription or expression that could alter hER activity with E2 or DES. Without ubiquitin, it is possible that hER expression may drop significantly – I cannot measure it until I get a functional ER plasmid into yeast.
ERE-LACZ: makes lacZ in the presence of activated ER. This construct is used to quantify ER transcription activity.
ERE-URA3: makes ura3 gene product in presence of ligand-bound ER. It is used for negative selection in the presence of 5-FOA.
What We Have Done
The majority of the time spent on the project has involved plasmid reconstruction. In the original system, a multi-copy hER plasmid (with ER fused to ubiquitin) was designed for high ER expression and used to determine estrogenic properties of various compounds. The ERE reporter plasmid did not provide a method for negative selection. With this original system, I tested many candidate environmental estrogens before settling on DES. Also, I have determined dose-response curves for E2 and DES.
However, problems with the original plasmids made it necessary to move the original ER and ERE sequences into new vectors (obtained from Clarence Chan).
For hER -
Ubiquitin had to be removed so that it would not interfere with evolution in the ER sequence.
Multiple ER plasmids within one yeast cell could mask mutant ER with the desired phenotype.
The original plasmid appears to have undergone some recombination or manipulations before we obtained it – for example, there was a 42 nt insertion between ubiquitin and ER.
For hERE-
Because no negative selection mechanism existed on the original plasmid, an ERE-URA3 fusion had to be created and inserted into the reporter plasmids.
The original ERE reporter plasmid also appears to have undergone both recombination and manipulations before we got it. The plasmid’s restriction sites did not correspond with the map we received from the O’Malley lab, and was quite a bit larger than it should have been.
The old reporter plasmid was a multi-copy plasmid, and Clarence Chan suggested that an integrating plasmid might work even better, so the new ERE-lacZ and ERE-ura3 sequences needed to go into both multi-copy and integrating plasmid types.
Plasmid Reconstruction --- hERMuch of this work has been completed. For hER, I removed ubiquitin and attached hER sequence directly to a yeast promoter. Sequence data from hER revealed a mutation in the hormone-binding domain that changed the amino acid sequence of the hER protein. Two new mutations in the hormone-binding domain arose in the course of amplifying sequence fragments to create the recombinant CUP1-hER sequence (despite using a proofreading DNA polymerase). Design and execution of site-directed mutagenesis experiments using PCR were successful in correcting pre-existing mutations. Unfortunately, two novel mutations arose (within the same 500 bp region of the HBD as the original 3 mutations.) It seems probable that the ER sequence within the HBD may have secondary structure or some other unknown problem that makes it particularly mutation-prone in PCR reactions. As a consequence, the original hER plasmid (McLaclan) was abandoned as a source of hER sequence.
Detour into Rattus norvegicus
Original hER as sequence source abandoned, enter the rat. Rat ER was sequenced and several mutations were discovered. Two of them were actually mistakes in the original rat ER published sequence but there appear to be other sequence problems.
Greene’s hER
At this point, we decided to ask Greene for hER clone. It is here, and I have transformed it into bacteria and sequenced it (and found the mutation mentioned earlier).
Plasmid Reconstruction --- hERE
All of the proposed work on the ERE reporter plasmids has been completed. The original hERE/LACZ construct has been moved into the new reporter plasmids. hERE and the URA3 coding sequence were fused through a beautiful piece of recombinant PCR, and then inserted into the new reporter plasmid. I’ve sent sequencing reactions across the street to verify insertion of hERE/URA3 and hERE/LACZ. I have inserted the negative/positive selection sequences into both integrating and multi-copy plasmids.
What We Will Do and How Long It Will Take
hER Plasmid
Now that we have Greene’s hER in hand, the next step is to attach it to a yeast promoter and insert it into a single-copy plasmid. To avoid creating any new mutations, my plan is the ligate the promoter and ER fragments together, then digest the recombinant piece and ligate it directly into the vector without amplifying it. To create the new CUP1/hER, I will need to re-amplify the yeast promoter piece with the appropriate restriction site engineered into it. The primers arrived 8.28, and the yeast promoter piece has been amplified. It should take 2 weeks to create the appropriate yeast promoter sequence, ligate it to ER, ligate it into the vector, transform into bacteria, and screen transformants. After that, I will need to send it across the street for sequencing, but the sequencing primers are all on-hand. It generally takes 3-5 working days to get sequence data back. Finally, all that will be left to do is a maxi-prep on the new hER plasmid, and then transform (both the hER and hERE plasmids) into yeast. The yeast transformation itself only takes 1 day, but it will be about 4 days before colonies are visible. These colonies will then be tested for function in the yeast estrogen assay. Each assay takes 3 days.
Total estimated time for ER plasmid construction (all the way into yeast) – 4 weeks.
hERE Plasmid
The yeast transformations are double transformations—both the ER and ERE plasmids are taken up at the same time, so no extra time is budgeted for ERE plasmid work.
Behold the Yeast
Yeast expression assays will tell us if all of these manipulations have been successful. It is highly probable that I will need to do some tinkering with the system to make the experimental set-up work. I will also need to experiment with 5-FOA and determine the correct concentrations for negative selection. I will start out with low concentrations of 5-FOA and then gradually increase negative selection pressure as the experiment progresses.
Total estimated time for establishing the new plasmid system in yeast and determining the correct 5-FOA concentrations – 2 months.
