Life of a Lab Rat

When I get to the bottom
I go back to the top of the slide

Where I stop and turn

and I go for a ride

A bit of a frustrating week.

The thermal cycler broke down just before it had completed a crucial experiment (but I think it went far enough to get some useful data), someone put agar plates with no antibiotic into the ampicillin plate bag (screwing up my cloning), a Western blot is tantalizingly inconclusive and a beautiful hypothesis appears to have been brutally slain.

On the other hand EMBOSS 4 seems to have installed (via Fink) quite nicely, and I'm toying with the idea of advertising it to the group. I did try, a couple of months ago, to get Jemboss up and running but no one else seems to have done it on Intel Macs yet and it was turning into a full-time job, so I gave up. But I have opened a guest account on this machine so maybe I'll let the clued have SSH access and get at the EMBOSSy goodness.

Okay, background. My protein-de-jour, which I shall call 'Z', is a potential splicing factor. There are reports that it changes the pattern of alternative splicing of certain gene transcripts, but the evidence is not strong and it has been difficult to duplicate it. Z itself exists in at least two splice forms, and there was a very bad paper in an obscure Chinese publication that suggested that Z affects its own splicing. This would be quite exciting. Additionally, we know which part of the protein binds to messenger RNA and I'm trying to characterize the activity of the whole protein more fully.

I have antisera (developed before I got to the lab) that does not work very well in Western blots using cultured cell extracts. So I cleaned up the antisera but it still did not work — it did not detect a specific band. I hypothesized that the epitope (the bit of protein that the antibody recognizes) was altered in cells. Briefly, the epitope contains a protein kinase A recognition motif, which means that potentially the cells are sticking a phosphate group onto it. This might prevent my antibody from recognizing the epitope. I treated the cell extracts with an enzyme that removes phosphate groups, and a faint band appeared on the Western. This is merely 'tantalizing' and not 'significant' because there are still background bands around. To add to my intrigue, the band could actually be a doublet — as one might expect if there were two splice variants.

As for the beautiful hypothesis, my latest RT-PCR appears to show that Z does not affect its own splicing. To turn this around, I've failed to disprove the null hypothesis, which is a shame because now I have to go fishing and do another set of experiments in order to find out which messenger RNA(s) it actually is affecting. Oh hum.

But there is an upside, which got the honours student ('P') excited: She made some stable transfectants of our model cell line; one expressing Z and the other a control. And when I ran the RT-PCR that looked at RNA isolated from P's two cell populations, it appeared that there is less Z-encoding messenger RNAin the cells that express Z. Now, this is not real-time so it's not strictly quantitative, but the non-specific (background) band is the same intensity in both samples. To me it looks as if Z is inhibiting its own expression rather than splicing, which may or may not be significant. Exciting stuff, eh?