Discussion paper 1

Question 1

What is the underlying hypothesis of the paper?

Answer 1

An isomerase exists which converts atRE to 11cisROL

Question 2

What findings from previous studies suggest that Rpe65 could be the retinoid isomerase? (2)

Answer 2

1. Rpe65 is abundant in RPE cells and is capable of binding to retinyl esters
2. Rpe65 knockout mice contain only the apoform of the opsin and accumulate all-trans-retinyl-esters

Question 3

What findings suggest that Rpe65 is not the isomerase? (3)

Answer 3

1. Partially purified Rpe65 did not have isomerase activity in vitro
2. If you take away Rpe65 through saline extraction, isomerase activity persisted
3. There is a high abundance of Rpe65 in Rpe65 positive cells (enzymes are not usually that abundant)

Question 4

What is the rationale behind the creation of the LRAT, Rpe65, and CRALBP cell lines?

Answer 4

To generate cell lines which stably express the three proteins to observe what effect differential expression has on 11cROL production

Question 5

To go from RNA to cDNA, this process must occur using this enzyme

Answer 5

Process: reverse transcription
Enzyme: reverse transcriptase

Question 6

What is cDNA useful for?

Answer 6

Using plasmid vectors to express different proteins in cells which do not normally express that protein

Question 7

In this study, what was the source of cDNA and why was it selected?

Answer 7

Bovine RPE

Selected because: cow retinas are large, work well in explant culture, useful because it is a mammal model

Question 8

The authors of the paper made their cDNA library and pooled the cDNA to transfect 293T-LRC cells. What are the concerns with this process?

Answer 8

When you pool all that cDNA together into these cells it is easy to lose cDNA - a lot can go wrong

Question 9

When assaying cells for presence of 11cROL, why was this done in the dark?

Answer 9

Because the light converts 11cROL into atROL

Question 10

What is pRK5 and why was it used in the study?

Answer 10

It is a mammalian expression plasmid
In one of the figures it is the control for the study: transfected cells with the plasmid in the absence of RNA for the gene of interest and assessed 11cROL activity

Question 11

When subpools were divided up and they found peaks in subpool 7:22, why were the researchers disappointed to find LRAT and Rpe65?

Answer 11

Those were the elements which they had transfected into the cell which they knew were a part of the visual cycle somehow but did not suspect either one to be the isomerase during the trial phase of the study

Question 12

What is LRAT’s role in the visual cycle?

Answer 12

It catalyses the reaction which creates the substrate for the isomerase

Question 13

Prior to the study, what did the researchers suspect Rpe65’s role was in the visual cycle?

Answer 13

Thought it was some kind of “helper” in the reaction for the “true” isomerase

Question 14

When LRAT and Rpe65 were expressed together in 293T cells, what happened to 11cROL production?

Answer 14

It was significantly higher when both were combined

Question 15

Does LRAT have endogenous isomerase activity?

Answer 15

No

Question 16

Based on their experiments on only the 293T cells, what were the two possibilities for the role of Rpe65?

Answer 16

Either it is the isomerase or it is required for the endogenous isomerase which may have been in 293T cells

Question 17

How did the authors attempt to prove that Rpe65 was the isomerase and not a “helper” for the isomerase?

Answer 17

By repeating their transfection experiment in Sf9 (insect) cells

Question 18

Do insects have a retinoid isomerase?

Answer 18

No, they photoregenerate 11cROL