9:00a – 11:45p
- project 2 team meeting
- project 2 design new experiments
- start analysis
- wordpress site / notebook running very slow today.
- imaged beads
- imaging E07 on prism2
- ~4/11 cells (RNase treated), G06-p1 + G08-p3
- ~4/11 cells (RNase treated), F07-p1 + F06-p3
- ~4/11 cells (RNase treated), F06-p1 + F07-p3
- 4/9 cells, newly RNase treated L3E08
- 4/9 cells, newly RNase treated L3E09
- 4/9 cells, newly RNase treated L3E10
Thicker glass, lower temperature (coverglass vs. microscope slide)
MNase digestion — nucleosome phasing
26 different histone modifications mapped genome wide. in 20kb of yeast chrIII
chromosome conformation capture
mapping 300 nm
the 30 nm fiber mode
- solenoid model, regular 30 nm model
- zigzag, 3 nucleosomes / turn, less regular
- non-existent in vivo, only in dilute in vitro preps
- using MNase to map nucleosome-nucleosome contact maps.
- different fiber folding predict different peaks
- challenge, need to repair ends of MNase digested DNA while crosslinked to allow ligation
- claim: Nuclesome resolution HiC yeast map. semi-blocky structure on scale of 100 nucleosomes.
- clear relation between local gene structure
- in yeast squares on scale 2kb
- is this really contact or is it co-accessabililty?
- difference between triangle and mountain
- triangle structure are highly expressed.
- tandem orientation genes more likely in same block.
- divergent genes, clean break at promoter.
- (I suspect this is all transcription driven not topology driven)
- no evidence for regular 30 nm fiber
- ‘Gene loops?’
- +1 touches N-2 nucleosome etc maybe just as well or better than the N.
- ‘gene crumples’ rather than ‘gene loops’
- access to data about DNA as a chromatin polymer
Pt II: inheritance of acquired characteristics
- compare high protein vs. low protein fed males
- hundreds of (liver genes) can distinguish the difference between dads
- upregulated genes enriched in cholesterol and lipid biosynthesis
- downregulated are nothing in particular
- see phenotype differences as well.
- other work
- starve males in utero, up-regulate glucose and colesterol
- in utero during dutch hunger winter — increased diabetes etc. bodies hoarde calories
- connection between metabolic phenotype and later generations
- hypothesis ‘sperm epigenome?’
- also data showing molecules in the sperm fluids
- artificial insemination doesn’t work in mice. Need to do IVF.
- phenotype less penetrant than natural mating but still passable. Can sequence the rest of the sperm sample or the blastocyst. Look for cytosine methylation patterns.
- these methylation patterns and histone modifiation patterns don’t seem to carry the info.
- small RNAs?
- small RNAs (under 40bp).
- types: 70% are tRNA fragments. Also 19bp RNAs and microRNAs.
- most abundant form is 25-30% of all small RNA in sperm
- see differences in these guys in males with different parental effects
- tRNAs directly linked to metabolism, so this is a reasonable messanger. The particular ones here are logical choices.
- tRNAs are used in retro-element replication. (e.g. HIV packaging)
- tRNAs prime reverse transcriptase for example. Also true for endogenous retro-elements
- can out-compete other
- are these refuse from earlier sperm metabolism or functional?
- charged testes tRNAs don’t correlate with deitary effects of sperm on tRFs.
- testes small RNAs vs sperm — testes don’t make lots of tRNA fragments. more abundant in sperm (50 fold).
- tRNA fragments accumulate during sperm maturation in epididymis.
- deep sequence somatic cells from epididymis, find lots of these. (transfered from soma to germline?)
- Sperm maturation
- change lipid composition
- gain 200 proteins (despite being translationally inactive) shipped in vesicles from epididymis.
- isolate and sequence epididymesomes, similar tRNA profile as mature sperm.
- mostly come from distal epididymis, not proximal.
- do immature caput sperm have tRNA fragments?
- yes but less than mature ones.
- increasingly find other examples of soma-germline communication
- mostly mediated by small RNAs
- evidence against this hypothesis? Find high levels of intact tRNA in caput sperm.
- follow up
- genetically express modified label tRNAs in somatic cells, look for these in germline.
- make library of tRNAs express in virus, look for trasnfection into germline cells.
- Epididymisis as a sensory organ for gamete RNA engineering
- try to squirt in epididymesomes into oocytes.
- knockdown GlyC tRNA, see strong overexpresseion of ~30 genes.
- genes that are de-repressed in knockdown are repressed in low protein sperm.
- All 25 genes are regulated by MERVL, an endogenous retro-element
- MERVL uses tRNA leucine (not Gly) for its replication. odd.
- turned on in 2 cell totipotent stage. Turns off in 4 cell stage and back on in 8 cell stage.
- in an ES cell colony, 1:100 cells is oct4 negative and is MERVL positive, cells cycle in and out of this state.
- FACS these cells and implent. MERVL negative cells are pluripotent (can’t make extra-embryonic tissues)
- MERVL cells are totipotent, also make placenta.
- phenotypes seen are similar to uterine implantation phenotypes. Possible model:
- tRNAs affect implantation and placenta vs embryo growth?
9:30a – 10:00p
- Need to finish review
- Need to make slides for symposium at Penn
- send revised
- Troubleshooting sudden failure in staining
Background of problem:
Stained new cells on Saturday, went to image these on Monday — no staining. Suspected extended hybe time could have been problematic, or something wrong with new multi-color probes. One idea I had about the extended hybe time was that the rubber cement seal is not very good and there’s a fair amount of condensation onto the slide, which may have leached out the probe concentration. I tried staining another three coverslips of cells using a probes that had previously worked and using a dry box. I still observed condensation and I still observed no staining. Now I thought I might have screwed up the cell prep, so I repeated the cell prep with a fresh batch of cells on Wed and stained these with E3 probes for which I very recently got beautiful staining. Yet again, no staining. To really see if this was hybe buffer / staining conditions or cell-prep, I took an old coverslip (previously stained for C8, a very small weak staining region) and stained again with the large / strong C1 probe. I used this same probe mix and hybe buffer to stain some of the cells prepped Wed, one with and one without RNase treatment. The C1 on the old cells stains great, the new prepped cells both no staining.
The cell prep is reasonably straightforward and I had been using all the same buffers and stuff for a while, so I’ve no explanation of why this should suddenly fail. I did notice a little growth in my PBS glycerol, which I promptly replaced. Yesterdays cells were briefly exposed to this before I noticed the tiny cloudy clusters in the glycerol. I have a hard time believing though that this brief exposure to some extra bacteria or mold eating the glycerol would completely and utterly destroy the ability to hybridize FISH probes to the genome…
All New Buffers
- made completely new PBS
- made new 1/2x PBST and 5% formaldehyde
- made new 0.5% Triton-X in PBS
- have new 20% glycerol in PBS (using old stock of 10x PBS though…)
- rinsed PBS squirt bottle. Maybe we should not use it for this round just to be sure.
- made new 0.1 M HCl from 37% stock
- bleached and washed bench
- bleached and washed pipettes
- new pipette tips
- set up new clean-zone at desk with bench guard.
New Cell Culture
- started new cell culture from frozen stock.
- Probably enough cells here to plate 1 well and try staining? They may be a little pissed just coming out of LN2 today, but it’s a comparison.
New cell staining controls
- Resuspend new cells and plate in 4 wells
- Resuspend old cells and plate in 8 wells
- allow cells 1 hour to attach
- follow my protocol
- stain new cells with C1 probe (no RNase)
- stain old cells with C1 probe (no RNase)
- stain new cells (with RNase 32 min at 37 treatment) with E4 probe. This sample kept in separate dish for this step.
More control ideas:
- RNase treat C7 and then stain it with C1. (see if RNase treatment is a problem / contaminate). It is a new batch of RNase dilution (same original enzyme lot).
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