The goal of this series of experiments is to find out how important the individual positions within the H. influenzae uptake signal sequence (USS) are for DNA uptake. The experiments were originally done by a technician several years ago, but the data had a lot of variability (big error bars) because the results were not very reproducible from one experiment to the next. So they were never published.
Now a post-doc is repeating them so we can get better (more reproducible and therefore publishable) data. But there are still problems with sensitivity and reproducibility. In this post I want to consider several questions: 1. How can we improve these specific experiments? 2. What steps should we take now towards getting the paper written? 3. Do we have other data that should be included in the paper? 4. Should we do additional new experiments?
The USS is a 29bp segment of DNA whose presence in DNA fragments strongly influences whether cells will take them up. It was originally studied by DNA uptake experiments using cloned and/or synthetic DNAs. The sequence is strongly over-represented in H. influenzae DNA and most of our recent inferences have come from comparisons of the many (~1500) copies in the chromosome The bases at 9 positions in the USS are usually considered to be the 'core' (this is what there are ~1500 copies of), possibly only because this specificity is the easiest to recognize. Two other 5-6bp flanking segments on one side also show a strong consensus, but we have little or no direct evidence about their importance in DNA uptake.
The goal of the original series of experiments was to directly test the importance of individual positions within the USS for DNA uptake, to find out whether the base frequencies at each position of the many genomic copies are really good predictors of their importance for DNA uptake.
1. How can we improve these specific experiments?
The experiments use a series of cloned restriction fragments, each differing from the standard USS at a single (or two) positions. The changes replace the most-common base with the least common base for that position. The original experiments used short fragments (~200bp I think) synthesized and 32P-labeled as PCR products. For the new series these short fragments have been cloned in an E. coli vector (total length ~4000bp?) which is linearized and end-labeled using kinase. The labeled fragments are then incubated with competent H. influenzae cells, and the cell-associated 32P is measured after extensive washing of the cells by centrifugation.
The labeling experiments appear to work fine, but the cells never take up as much DNA as we think they should. Hmm, I don't think we have directly calculated how much DNA the cells should be taking up. This is important because the new fragments are about 20x longer than those used previously. Would we get better results with shorter fragments?
2. What steps should we take now towards getting the paper written?
We (the post-doc and I) should start writing it now. I think bits of it (the old Methods, probably not very applicable now as the methods have changed) were written up years ago. Writing now will help us clarify what else we need to do.
3. Do we have other data that should be included in the paper?
Should we include our analysis of Sol Goodgal's old taken-up sequences? I did this analysis 6 or more years ago, and it was going to be included in another paper, by a former post-doc. Oops, or maybe the present uptake data was going to be included in the former post-doc's paper - I need to sort this out NOW.
Any other data we already have?
4. Should we do other new experiments for the paper?
One of the early papers investigating uptake specificity (from Ham Smith's lab) used DNA ethylation (?) to randomly modify positions in an end-labeled fragment containing a USS, and then measured how modifications at different positions interfered with binding (and uptake?) by competent cells, by examining the modifications in the fragments that were (or were not?) taken up. We should check whether this experiment could be improved now that we know more about the USS and (maybe) have more specific questions to address.
Can we find out anything about how such modifications are expected to change the structure of the DNA? Maybe its ability to bend or kink?
Real tough guys - lichens
3 hours ago in The Phytophactor