Field of Science

A new mutation causing hypercompetence

I described last month how we were revisiting on old hypercompetent mutant whose causative gene was unknown.  I rechecked its phenotype and prepped DNA to sequence, from both the original EMS-induced mutant strain (strain RR735) and from a 'backcross' strain where the unknown mutation had been transferred to an unmutagenized genetic background by transformation (strain R753). 

Here's the phenotype again.  The lower graph shows that it grows slightly slower than wildtype, and the upper graph that it has a 10-200 times higher transformation frequency in the rich medium sBHI.

The postdoc just emailed me the sequencing results.  Both the original and backcross strains have the same single mutation, an amino acid substitution in the rpoD gene, which encodes the sigma-70 transcription factor.

This is a surprisingly clear result.  We had expected to find many EMS-induced mutations in the original strain, and probably several mutations in the segments of DNA transferred in the backcross transformation, and were planning another series of analyses to sort out which mutation causes the phenotype.  But both strains have only the one rpoD mutation, suggesting that our EMS mutagenesis wasn't nearly as heavy as we had thought.  As controls we had sequenced the original and backcross strains of another hypercompetence mutant (RR749, known to have a mutation in I, and both these strains also had only the single known mutation.

A hypercompetence mutation in rpoD fits very nicely into my thinking about how competence is regulated.  I'll write a separate post about this.

Cell preps for RNAseq are all done

I'm pretty sure that I've now done all of the cell preps for our big planned RNAseq analysis, more or less as diagrammed in the previous post. 

Instead of a cya knockout mutant as a negative control (pink in the diagram I used a crp knockout.  cya encodes the enzyme that synthesizes cyclic AMP (cAMP), and crp encodes the transcriptional activator CRP, whose ability to induce transcription is entirely dependent on cAMP, so the two mutants have the same phenotype - inability to induce both the competence genes and the energy-balance genes in the CRP regulon.  I decided to use the crp mutant partly because that strain grew up first and partly just in case there are traces of cAMP in our sBHI medium.  I did one MIV-competence time course with this mutant (4 samples) and one sBHI time course (3 samples rather than the 2 in the diagram).

I also did 3 sBHI-timecourse samples of the sxy knockout as another negative control (also pink in the diagram).  I think I now have two more samples than will fit in 3 lanes of sequencing (24 samples multiplexed per lane), so I'll probably omit the OD=0.6 samples of the negative control sBHI timecourses.  But I'll process the RNA from them just in case something goes wrong with another sample.

I found my missing DNase.  I hadn't lost it, just ordered the wrong kind.  So now I have $250 worth of a high-quality DNase I don't really need, and will need to order the right kind.

I'll get to the RNA preps once I get some teaching responsibilities under control - partly the grading for my face-to-face Human Ecology course but mainly the need to rerecord ~150 lecture videos for Useful Genetics/Genetics for Life.

RNA-seq progress, problems and plans

I've been growing the cell preps for the RNA-seq analysis, as shown in the planning figure below.

I did Day C's cultures, freezing 1 ml of cells for later transformation testing if needed, and 2 ml of cells for RNA purification.  The first snag was the invisibility of the cells!  Following instructions from the former RA, I mixed 2 ml cells from each of the first samples with 4 ml of the magic 'RNAprotect' reagent, let the mixture sit for 5 min at room temperature, and spun down the cells (2 ml of mixture in each of three 2 ml tubes) in our mini-spin microcentrifuge.  The plan was to discard the liquid and freeze the cell pellets at -80 °C, for later RNA preps.  But there was no visible cell pellet!  This amount of cells typically forms a small but easily visible pellet when centrifuged, but the bottoms of the plastic tubes looked perfectly clean.

I spun the tubes again - still no pellet.  So I pretended a pellet was present, discarded the liquid, and froze the apparently empty tubes anyway.  I checked the RNAprotect instruction booklet which reassuringly said that sometimes the pellet might be invisible, but I also contacted the RA, who said that her preps had given clearly visible pellets.  Later I thawed out one tube and did a wilful-suspension-of-disbelief RNA prep using the RNeasy kit, which produced the same concentration of (high-quality) RNA as the RA's original preps.  So I'm now assuming that there are invisible cell pellets in all the tubes, and I've done Day D's preps.

The RNAeasy kit also had some surprises, a solution that was supposed to be clear (or with a bit of particulates that could be removed by centrifugation), instead was very cloudy, and centrifuging it raised the cloudy material to the top (as a diffuse scum) rather than pelletting it.  I couldn't get rid of the scum (it just redispersed when I tried to pipette it), so I went on to the next step (adding 100% ethanol), which eliminated the cloudiness completely.  (Maybe this cloudiness came from the presence of too much residual RNAprotect in my tubes - because I couldn't see any pellet I didn't thoroughly drain the tubes before freezing them.)

I would have also tested the DNA-elimination step, which uses Turbo Dnase and a DNase-inactivator chemical.  But my brand new box of TurboDNase ($250) has gone missing.  I've searched the freezer a couple of times, and racked my brain in case I put it somewhere special for 'safekeeping', with no success. My next step is to go down to Stores and have them show me exactly what the TurboDNase box looks like, so I know I have the right search image.

I've also revised my plans for the cells I'll test.  As I wrote earlier, I'm only going to do one replicate of the ∆hfq mutant in MIV, since we really should use special precautions to avoid losing small RNAs from the prep (and maybe to do strand-specific sequencing).  Since we planned on three full lanes of Illumina sequencing, each 24-fold multiplexed, this change opens up space for 8 additional samples. Four of these will come from a MIV time-course using a crp or cya knockout strain (in Day E, which will be tomorrow).  This is an excellent control since it lets us identify all of transcripts dependent on the transcription factor CRP.  I'll also include both crp (or cya) and sxy knockout strains in the rich medium cultures on Day H; taking two time points for each will give the four additional cultures to complete the first two lanes of sequencing. 

Big prep of MAP7 DNA

We're almost out of the standard DNA that we use in our transformation assays.  It's chromosomal DNA of a strain called MAP7 (because it contains point mutations conferring resistance to seven different antibiotics).

So I grew up a liter of cells and prepped DNS from them.  Now I have 25 ml of nicely viscous DNA solution.  It's transparent and colourless but I suspect it's not really pure yet, so I'm doing a second purification on 0.5 ml just to check if the apparent concentration or purity changes when examined with the Nanodrop spectrophotometer.  I'll also do test transformations, with the last of the old DNA preps as a control.

Later:  The Nanodrop spec found that the repurified DNA had half the concentration of the big stock. So I ran a gel and found that the big stock still contains a lot of RNA.  My original RNase step must not have worked very well, probably because of the high concentration of SDS and of proteinase K.  So I';ve now incubated the prep with more RNase overnight.

Getting ready for RNA-seq cell/RNA preps

The RA's missing notebook hasn't turned up, so I don't have her notes of how she prepared the samples for the RNA-seq analysis. Luckily the main procedures are ones she used in many experiments and are described in her earlier notebooks and in an email she sent me.

The basic procedures:

Collecting samples:
  1. Grow cells to desired state in rich medium (sBHI) or competence medium (MIV).
  2. Mix 2 ml with 4 ml RNAprotect reagent (Qiagen); leave 5 min at RT.  We have 100 ml and can get more quickly through LSC Stores.
  3. Mix 2 x 1 ml with 0.25 ml 80% glycerol and freeze at -80°C. (for later competence assays).
  4. Pellet RNAprotect cells and freeze at -80°C.
Preparing RNAs:
  1. Thaw cell pellets
  2. Use Qiagen RNA prep kit.  We have lots and can get more quickly through LSC Stores.
  3. Don't use the DNase step.
  4. Elute in 40 µl H20.
  5. Measure concentration of 1 µl with Nanodrop.
  6. Run 4 µl in a 1% agarose TAE gel at 60V.
Treat to remove DNA:
  1. Use volume containing 1 µg RNA (using RNA concentration from Nanodrop)
  2. Use Ambion Turbo DNase 
  3. Use protocol in RA's notebook #1 (not missing); 2 x 30 min incubations
Check RNA quality (and concentration?):
  1. Use the 'Bioanalyzer' (high-tech equivalent of gel electrophoresis) to check the size distribution of the RNAs in each prep.  Expect to see 2 strong rRNA peaks.
Treat to remove rRNA:
  1. Use Ribo-Zero kit to remove the rRNA from each sample.
Check RNA quality (and concentration?) again:
  1. Use the 'Bioanalyzer' (high-tech equivalent of gel electrophoresis) to check the size distribution of the RNAs in each prep.  Expect to see no rRNA peaks.

Here's the chart showing the MIV-competence samples I had planned.  I'm only going to do one set of the ∆hfq strain now, because our procedures aren't optimized for small RNAs (poor recovery and no strand information).  One of our summer-Honours students will be working on this mutant, and he can take my preliminary data and use it to help design an optimized RNAseq procedure.  So I think on Day C I'll only do the three strains (sxy-, ∆659 and ∆6759/660), and if this goes smoothly scale up to four strains on Day D.  Maybe on Day E I'll replace the ∆hfq strain with something else I want preliminary data about.

She lost me at the Central Limit Theorem

I've been saying for ages that I need to learn the statistical programing language R, so that I can work with all the bioinformatic data we're generating.  So yesterday I looked through the Coursera offerings and found an introductory statistics course that taught R (Data Analysis and Statistical Inference, taught by Mine Cetinkaya-Rundel of Duke University.  It started a few weeks ago, and I've spent yesterday watching the Week 1 videos and doing the Week 1 R lab. 

The labs are excellent.  They use a web-based R learning platform called DataCamp - each lab is a long series of guided exercises: with each exercise you're given a bit of instruction and asked to use it to display something or graph something or calculate something.  Integrated multiple-choice questions check your understanding - DataCamp automatically sends your results back to Coursera.

It's also very good that they're part of a basic statistics course, since I've always been disgracefully ignorant of this.  The video lectures are good -short but many, and aimed at the complete beginner.  I was initially quite pleased to be learning what 'marginal' means, and the differences between variance and standard deviation and standard error.  The course materials are very well designed.

But I started getting frustrated when I tried to think more deeply about quantifying variation.  I can sort-of understand why we want to know how variable the members of the population are, but this was never really explained, and I have no idea why we measure it the way we do.  To me it seems simplest to measure variation by calculating how different each individual is from the mean (the deviations), summing the absolute values of these and dividing by the number of individuals.  But that's not what's done.  Instead we square each of the deviations and sum that, to get the 'variance'.  But we don't use the variance (at least not yet), instead we take its square root, which we call the 'standard deviation' and use as our measure of variation.  Why is this cumbersome measurement better that just taking the mean of the deviations?  The instructor doesn't explain; the textbook doesn't explain.

In the first lecture of Week 3 we come to something called The Central Limit Theorem'.  This is apparently a big deal, but I don't know what the point is.  We've moved from considering the properties of a single sample from a population to considering the properties of many independent samples (size n) from the same population - I have no idea why.  The Central Limit Theorem tells us that, if we take many samples and calculate the mean of each one, the mean of these means will be the same as the population mean (is this not expected?), and that the shape of the distribution of means will be 'nearly normal', and that the standard deviation of the means will be smaller than that of the population, by a factor of 1/√n.  So what?  What do we gain by repeating our sampling many times.  Seems like a lot of work, to what end?

Then we're supposed to learn a list of conditions under which the Central Limit Theorem applies.  But without understanding the point, this was too much like rote memorization to me, and why should I bother?

Does fructose inhibit development of competence?

I gave a seminar at Michigan State yesterday, invited by the Microbiology graduate students (Thanks guys!).  While I was there I met with a research group that works on Actinobacillus succinogenes, a relative of H. influenze.  They were interested in improving the competence levels of this species, and I explained that induction of the H. influenzae competence regulon was controlled by CRP and cAMP, with cAMP levels determined by the availability of external fructose to the phosphotransferase system’ (PTS) sugar-uptake system.

The PI then asked me whether we’d tested the obvious prediction that adding frucose to the culture medium should inhibited the development of competence, and I was shocked to realize that we hadn’t.  At least I don’t think we have.  The PTS and fructose studies were done by a very competent PhD student about 15 years ago, and I’m pretty sure I would have remembered the result of this experiment, since it would either have nicely confirmed our hypothesis or disproved it.  I’ll check her thesis when I get back (I ‘m writing this in the East Lansing airport).

Assuming she didn’t do this, I’ll do it right away.  Two experiments really, a time course of competence development in rich medium and induction by transfer to MIV starvation medium.  I’ll grow wildtype cells in regular sBHI, and at OD =0 0.2 (log phase) I’ll collect them and resuspend them in either MIV or fresh sBHI containing either fructose or glucose (as control).  What sugar concentration should I use - is 0.5% standard?

Hmm, I just remembered some old experiments I did (20 years ago?) testing the effect of adding glucose to MIV  I vaguely remember that the cells became very unhappy  - did they die from unbalanced nutrients???  I know that glycerol added to MIV doesn’t have any effect on competence (very old publication confirmed by me).  I could use a different sugar (ribose? xylose?  one that we know H. influenzae can use).

Woohoo! RRResearch made a top-ten list!

If you've come from the list of The 10 Must Read #womeninscience Blogs, you might be a bit disappointed to see that the most recent posts are descriptions of day-to day work in my lab.  That's typical for RRResearch, but here are a few posts you might find more interesting:

Checking the hypercompetence mutants

Here's the results of the transformation time courses of the control strain 'K' (= KW20) and the hypercompetence mutants (RR735 and its backcross descendant RR735).

The lower graph is just culture growth (CFU/ml).  You can see that the two mutants (red and green lines) grew slightly slower than the control (blue line).  The upper graph is the transformation frequencies.  You can see that the two mutants behave identically, and that their transformation frequencies are 100-fold higher than wildtype in log-phase growth and 10-fold higher as growth slows. The dotted red circle indicates two samples that gave no transformants, so the values plotted are upper-limit estimates of the true transformation frequencies.

I also made the chromosomal DNA preps from these two mutants and from a parallel pair of mutants whose mutation has been identified (200 µl of each, at about 150 ng DNA/µl).  I set 50 µl aside for the postdoc's sequencing.

More hypercompetent mutants Part 1

Well, I did submit a CIHR proposal after all.  Not on the regulation of competence as I had originally planned, but a resubmission of our last proposal (from 18 months ago) to develop the knowledge base to predict transformation in vivo.  Now that's done I'm planning some research to identify new mutations that cause hypercompetence.

One project will start right away, to identify the mutation causing hypercompetence in a strain we isolated about 10 years ago.  I wrote about this a few months ago, but we didn't do anything then.  We're now going to do sequence both the original EMS-mutagenized strain (RR735) and its backcrossed derivative RR753.  As controls we'll sequence the original and backcrossed versions of a parallel isolate whose mutation is well-characterized (RR727 and RR749).

My job is to prepare the DNA preps for sequencing.  I've streaked out the strains from the freezer stocks so I can grow up the cells - making DNA is easy.  If I inoculate the cultures this morning I can make DNA later today.  (Later: inoculated them in the afternoon; will make DNA tomorrow.)

But I'll also repeat the transformation time courses for RR735 and RR753, with wildtype cells as a control. I should try to get that done tomorrow (in between appointments) so I can get the data analyzed over the weekend, since I'll be out of town Monday to Wednesday (giving seminar at Michigan State!). (Later: too many appointments tomorrow so will set things up for Saturday.  If the colonies aren't ready to count on Sunday I'll have the grad student put them in the fridge on Monday so I can count them when I get back.)

Fortunately all the strains are NovS, so I can select for NovR (that's the easiest marker to work with because it gives a low background and doesn't need expression time).

There's a chance that this sequencing will discover something interesting about EMS mutagenesis.  As far as I know nobody has used genome sequencing to examine the effects of EMS in bacteria (I surveyed the community via Twitter but nothing was known), though the post-doc thinks this may have been done in Drosophila.

Part 2 of this post will be on strategies to isolate large numbers of new hypercompetence mutations.

Who really discovered trisomy 21? (righting an old wrong)

A few days ago a French student in my Useful Genetics Coursera course posted a link to an article in Le Monde (sorry, it's both in French and behind a paywall, but this link might get you a translation).  It reported that a Jan. 31 award ceremony for the discovery of the cause of Down syndrome, part of the 7th Human and Medical Genetics Congress  in Bordeaux, had been blocked by a Down syndrome support organization (Fondation Jerome-Lejeune).  The back story is very depressing, an egregious example of a woman scientist being denied credit for her discovery.

Photo source: Le Monde
The woman is Dr. Marthe Gautier, now 88 years old.  In 1956 she was a young physician, returning to Paris from a year's study of pediatric cardiology at Harvard.  She was given a clinical/teaching position at a local hospital, with no funds for research.  The Head of the Pediatric Unit, Raymond Turpin, was interested in mongolism (as Down syndrome was then called); years earlier he had proposed that it might be caused by a chromosome abnormality.  Human cytogenetics was not well understood, but a big breakthrough came this same year, when the true chromosome number was finally established as 46 (not 48).  When Turpin complained that nobody was investigating his hypothesis, Gautier proposed that she take this problem on, since her Harvard training had introduced her to both cell culture and histology.  Turpin agreed to provide a tissue sample from a patient.

For this work she was given a disused laboratory with a fridge, a centrifuge, and a poor quality microscope, but no funding.  And of course she still had her other responsibilities.  But she was keen and resourceful, so she took out a personal loan to buy glassware, kept a live cockerel as a source of serum, and used her own blood when she needed human serum.

By the end of 1957 she had everything working with normal human cells, and could clearly distinguish the 46 chromosomes.  So she asked Prof. Turpin for the patient sample.  After 6 months wait it arrived, and she quickly was able to prepare slides showing that it had not 46 but 47 chromosomes, with three copies of a small chromosome.  But her microscope was very poor, and she could not identify the chromosome or take the photographs of her slides that a publication would need.

All this time Prof. Turpin had never visited her lab, but she'd had frequent visits from a protege of his, Jerome Lejeune.  When she showed Lejeune her discovery, he offered to take the slides to another laboratory where they could be photographed.  She never saw the slides again, but the photographs appeared in Montreal two months later (August 1958), where Lejeune announced to the International Conference of Human Genetics in Montreal that he had discovered the cause of Down syndrome!  Lejeune and Turpin quickly wrote up 'their' discovery, with Gautier as middle author, but Gautier only learned about this publication the day before it appeared in print.

These were tough times for a woman scientist in France, and Gautier decided not to fight for the credit for her discovery, instead returning to her clinical and teaching work on congenital heart diseases.

Lejeune became not just a renowned researcher but the darling of the French Catholic right-to-life movement.  You can read long flattering Wikipedia biographies in both French and English. He was showered with awards and given a prestigious Chair of Human Genetics at the Paris School of Medicine, bypassing the usual competition.

When prenatal diagnosis became available Lejeune campaigned against it on religious grounds. He became a friend of Pope John Paul II and was appointed President of the Pontifical Academy for Life (Wikipedia), the Catholic think-tank for medical ethics.  He died in 1994.  The Fondation Jerome-Lejeune was established in his honour; there's an American branch too.  This foundation provides funds for research into Down syndrome and support for families and patients, but only in the context of very strong opposition to abortion.  They're also campaigning to have Lejeune beatified by the Vatican.

But Gautier's role in the discovery of trisomy 21 was not totally forgotten.  It has been very well described in a 2009 article in the journal Human Genetics by Gautier and Peter Harper, the author of a major history of cytogenetics (paywalled but try this link to a pdf), and in a 2013 interview.  There's also a French Wikipedia page about her.  But few people in the field know about this injustice, and cytogenetics textbooks and courses still credit Lejeune for the discovery.  Gautier has no English Wikipedia page, and the Wikipedia pages on Lejeune describe her contribution as follows:
"Using a new tissue culture technique brought back from the United States by his colleague Marthe Gautier, Lejeune began working with her to count the number of chromosomes in children with Down syndrome. The laboratory notebook begun by Dr. Lejeune on July 10, 1957 indicates that on May 22, 1958, he succeeded in showing, for the first time, the presence of 47 chromosomes in a child with Down syndrome."
Not surprisingly, the Fondation Jerome-Lejeune strongly opposes any correction of the scientific record, since this would reveal the intellectual theft at the base of their hero's reputation.  That's why they sent in the bailiffs to record her award ceremony.

I'm of course outraged to learn about this situation, and this post is one attempt to set the record straight.  My other venue is Wikipedia, which I've been learning to edit.  So far I've added sentences crediting Gautier with the discovery to the Wikipedia entries on Down Syndrome and on Jerome Lejeune.  Someone else had added a mention of the dispute to his French entry.  I'm going to expand these each into a paragraph.  I've also created an empty page for Marthe Gautier and requested that the Wikipedia translation people fill it from her French entry.

So please spread the word.  Marthe Gautier discovered that trisomy 21 is the cause of Down syndrome, and Jerome Lejeune's saintly reputation is based on scientific fraud.

Later:  I've corrected an error: Gautier could not identify the trisomic chromosome with her poor microscope.  And here are links to news articles about this controversy, in Nature and Science.

Plans for RNA-seq analyses

I should have posted this after last week's lab meeting but am only now getting to it.  I sensibly took snapshots of the whiteboard at the end of that lab meeting, so I could check what we'd decided.

The issues:  We have several Haemophilus influenzae mutants whose gene-expression profiles we want to examine, either during competence development in the MIV starvation medium or during normal growth in the rich culture medium sBHI.  For most of these (i) we want samples from several timepoints over a few hours, (ii)we want wildtype controls done in the same experiment, and (iii) we want three replicate samples from experiments done on different days.  And it would be nice to have multiples of 24 samples, since the kits and sequencing are most efficient with that.

That's a lot of constraints , but we came up with a plan that meets them all:  The first two days are the samples that have already been prepared and sequenced; the other 6 days are for me to generate the samples.

The samples will consist of viable cells frozen in glycerol (one or two 1.5 ml tubes), and duplicate pellets of cells that have been briefly incubated with a RNA-protection reagent to stabilize their RNA.  Later the frozen viable cells will be thawed and transformed to check that they have the expected level of competence - I can probably do all of them in a few days.

After all the samples have been collected, the RNA-prep pellets will be thawed and the RNA isolated using a kit.  After a quick check of RNA concentration and size, the contaminating DNA is removed by treatment with Turbo-DNase.

All the samples are then checked for concentration and quality using a special something (the post-doc recommends, using equipment in another lab), then treated to remove the bulk of the ribosomal RNA (this kit costs a lot, I think $200 per sample), then rechecked for concentration and quality using the special something.

Finally the samples are ready to be made into multiplexed sequencing libraries (expensive service on campus) and then sequenced (another service on campus).

To order (first check what we have on hand):
  •  RNA protection reagent for 72 samples
  • RNA prep kits for 72 samples
  • Turbo-DNase for 72 samples
  • Does the 'special something' require special reagents?
  • rRNA-removing kits for 72 samples
The reasons for doing these experiments are described briefly in this post.  

Because my hydroxyurea experiments found that cell doubling is substantially faster when cultures are at very low cell density, for the first samples in experiments F. G and H I'll use large volumes of cells at a lower cell density than the usual OD600=0.2.  Getting the right density will be a bit tricky since we can't use OD600 to check densities of very dilute cultures.  In the table above I said I'd use an OD600 of 0.02, but I'll start by growing cells from a lower density to about this OD and then diluting them 16-fold so they'll have another four doublings before I sample them.  The hydroxyurea-experiment cultures were much more dilute than this, but I need to balance culture density and culture volume so I'll have enough cells for the RNA extractions.  With this plan I'll be collecting cells from about 2 x 250 ml of culture for the first samples, and combining the remaining volumes in the two flasks for the later samples.

Other experiments to do:  We need to sequence the genome of strain RR753, to identify candidate mutations that cause its hypercompetence.  Before doing this I should recheck it and the backcross strain I made - if they both are hypercompetent we'll want to sequence both.

But it's MY figure!

The postdoc and I have a minireview coming out in an ASM journal, and we're at the 'permissions' stage.

One of the figures is an explanatory diagram I drew for this article (figure on the left below).  It's similar to an explanatory diagram I drew for an article we published in Genetics a few years ago (on the right below).  Because one of the reviewers asked if our minireview figure was adapted from the published figure, in the Figure Legend we wrote '(adapted from (91)' even though it wasn't. 

Now the Production Editor is asking us for the formal permission from Genetics to republish this figure. I explained that it wasn't really adapted from the published one, but because the figures are similar she recommends that we request permission.


So Genetics has a 'Get Permissions' link beside each article, and that sends me to the Copyright Clearance Center, where I specify that I want permission to republish their content in a journal.  I click on Price & Order (not a good sign). 

That takes me to another form, asking me questions I don't understand ('Duration of use'?  'Lifetime unit quantity'?).  Whatever.

I click on 'Get Price'.

I click on 'Continue'.  This takes me to a 'Login' page.

 Of course I don't have an account.  I create an account.

Now I log in with my account, but I don't see any record that they remember what I was asking for.  So I do it all again.

Several more pages.

 I click 'Add to Cart'.

My order is not complete until I click 'Checkout'.

My order is not complete until I accept the Terms and Conditions.

OK, my order is complete.  They're sending me an email confirmation.  They say it could take 15 days....

On the other hand, I  just also requested permission to use a figure from our Nucleic Acids Research paper.  Very quick and easy - permission granted immediately.

List of RNAseq analyses to do

In case we have money for lots of RNAseq runs, here's a list of every sample that might be useful:

24 samples = 1 run:
Replicates of the samples we've already done:  MIV competence induction at t=0, t=10, t=30 and t=100:
  • 1 x KW20, 
  • 1 x sxy-, 
  • 2 x ∆HI0659, 
  • 2 x ∆HI0660.
Miscellaneous samples (3 replicates of each?):
  • KW20 in late-log (time of max 'spontaneous' competence)
  • KW20 in log phase + hydroxyurea: t=0, t=30? t=60? 
  • HI0659/0660 double mutant, in MIV at t=0 (= log phase) and t=???
  • murE749 mutant in log phase growth and stationary phase
  • Other hypercompetence mutants:  murE750, 751, 752?  in log phase growth
  • mystery hypercompetence mutant RR753 or backcross, in log phase and late-log?
  • KW20 in log phase growth as control (duplicating earlier MIV t=0?)
  • gcvA mutant in MIV?  or in murE749 background in log phase?
  • hfq mutant in MIV?  in log phase too.
  • purR? purH, crp? cya?
  • Cells in MIV + AMP.

What I've done lately

('Lately' being the 20 months since I last updated the Table of Contents of my lab notebook.)

I've been keeping a Table of Contents of my lab notebooks since I was a grad student, initially on paper but for the past 20+ years as a Word file. As can be seen from the screenshot above, each experiment has a number, and I record the date and a few words about what I was trying to do and what I found. Before I kept this blog, searching it was the easy way to find experiments on a particular problem, and it's still a very valuable resource.

One good thing about keeping a Table of Contents is that updating it forces me to go back over every experiment I've done lately.  So I've just done that for everything since May 2012, and it was very informative.  Here I'm going to write a summary of the experiments that I'd like to now follow up on, especially noting where RNAseq would be appropriate.

Experiments with the HI0659/0660 antitoxin/toxin genes:  We had found that a HI05659 knockout mutation completely prevents competence, that a HI0660 mutation doesn't affect competence, and that both genes were homologous to a known antitoxin/toxin pair.  This suggested that HI0660 encodes a toxin that blocks competence and HI0659 encodes an antitoxin that blocks this.  Now I've shown: Competence is not restored by the sxy1 or murE749 hypercompetence mutations, which act by increasing sxy expression. The HI0659 mutation does not cause a dramatic change in expression of lacZ fusions to the competence genes comA and rec2.  A HI0659/0660 double mutant transforms normally, confirming the hypothesis that HI0659's job is to block the activity of HI0660.  The HI0659 mutant grows just like wildtype in a BioScreen culture, suggesting that the toxin is either not expressed in noncompetent cells or has no activity that affects growth.  Gene expression in these mutants has been examined by RNAseq.  I'll describe these results in another post, but replication is needed.

Experiments with the hypercompetence mutation murE749 and peptidoglygan recycling mutations:  I first remade all the strains and rechecked the MIV-induced competence phenotypes of our set of four peptidoglygan recycling mutants, in a wildtype background with and without added cyclic AMP and in sxy1 and murE749 backgrounds.  There was quite a bit of variation, but all were approximately normal on replication.  The occasional dramatic differences may just be noise, or could result from some sensitivity to the details of the experiments.  The only consistent difference is that the gcvA mutation (a putative regulator) was about 3-5-fold less competent than the others.  We should definitely do RNAseq of the murE749 mutant, and maybe of the gcvA mutant too.

Phenotype of a hfq mutant:  hfq encodes an RNA-binding protein that interacts with many regulatory small RNAs, so we and others have hypothesized that it plays a role in regulating the translatability of sxy mRNA.  The RA made a knockout, which I found has normal growth but a consistent 10-fold reduction in transformation in late-log, in MIV and in a sxy1 hypercompetent background.  The drop was more extreme in an overnight culture - this should be retested. This mutant is a good candidate for RNAseq analysis

Effect of hydroxyurea:  The small molecule hydroxyurea (HU) specifically inhibits the enzyme ribonucleotide reductase, which converts NTPs (RNA precursors) to dNTPs (DNA precursors).  Thus HU depletes dNTP pools and stalls replication forks (demonstrated in E. coli).  If H. influenzae competence is a response to blockage of DNA replication fork progression, HU should induce competence.  But it doesn't.  If competence protects H. influenzae cells from the harmful effects of stalled replication forks, competent cells should be less sensitive to HU.  But they aren't: cells that become transformed show the same sensitivity to HU as non-competent cells, and mutations that cause hypercompetence do not reduce sensitivity to growth arrest or killing by HU.

BUT. these is a connection between competence and HU.  One competence-induced gene turns out to provide substantial protection against the harmful effects of HU.  dprA is in all characterized competence regulons, where it coats incoming DNA and promotes homologous recombination.  it's also present in many more species not know to ever become competent, although no non-competence function is known.  A dprA knockout has about the same effect on HU sensitivity as a mutation in recBC, which is well established as serving mainly to help DNA replication recover from stalling.  But an E. coli dprA mutant isn't more sensitive to HU.

Isolating more hypercompetent mutants:  Highly desirable, but a badly executed series of experiments.  It did produce lots of EMS-mutagenized cells stored at -80°C.  I should first test these for induced novR mutations, and if seen then redo the selection for hypercompetent mutants.   These mutants, if obtained, would be good candidates for RNAseq analysis.

Planning more RNAseq experiments

The postdoc and former RA generated some great RNAseq data, which I'll write about in another post.  But we have some money that needs to be spent on sequencing in the next couple of months, so we need to decide which additional RNA seq runs we should do.  And then I'm going to grow the cultures and prep the RNAs.

We have data sets showing how RNA levels change after transfer to competence-inducing MIV medium for several Haemophilus influenzae strains: wildtype (2 expts), sxy- (2 expts), HI0659- (1 (antitoxin?, 1 expt) and HI0660- (toxin?, 1 expt).  For each we have samples at t=0, t=10, t=30 and t=100 minutes.  (The figure shows a comparison between wildtype and sxy- at t=0 and t=10; the red circles are CRP-regulated genes and the blue ones are competence genes.)  We need to do at least one replicate of the HI0659 and HI0660 cultures.  If we also did another replicate of everything, that would be a full 24 sample run (one lane?) for the sequencer, and enough data that we could do proper statistical analyses.

But I also want to get RNAseq data for strains with other mutations, especially the hypercompetence-causing mutation murE749 in exponential growth.  This would be a single condition, replicated once or twice, so 2 or 3 samples total.  I might be able to squeeze this in with the run described above; depending on what other experiments we plan to do with these strains, two replicates of some might be enough.  Or it might be better to do a second run since we have the funds, doing a more comprehensive analysis of other conditions and other mutants too.

One condition I'd like to examine is 'late-log' growth, where wildtype cells develop moderate levels of competence.  I want to see if these levels are comparable to those in the HI0659 antitoxin mutant, which shows no competence at all although it appears to have (compared to wildtype cells) only a slight decrease in competence gene expression at t=10, no decrease at t=30, and a moderate decrease at t=100.  It's possible that the toxin acts only by decreasing mRNA levels of other competence genes, but the disproportion between its absolute competence defect and modest RNA defect makes me wonder if it also does something else.

We have three other hypercompetence mutants with mutations in murE - I don't know if it would be worth doing one sample of each of these in exponential growth.  We also have a hypercompetent mutant of unknown genotype - RNAseq might find the mutation as well as show us the RNA changes.  Might it be worth testing a crp mutant or cya mutant, to confirm our understanding of cAMP/CRP regulation?  Or the purR or purH mutant, or cells whose MIV-induced competence development has been blocked by adding AMP?  Or cells whose DNA replication has been blocked by hydroxyurea (depletes dNTP pools).  Or the hfq mutant, which has 10-fold lower competence.