Protists and their plastids

A quick skim through this blog reveals fairly quickly that I have a slight fixation on bacteria. I like to research them, read about them, and then blog about them, most specifically about their cell walls. However life contains more than just bacteria, and occasionally, strange though it might seem, people write papers about such non-bacterial things, and they end up on my desk with a small post-it attached reminding me that I have a presentation for my supervision group coming up.

So for the sake of my supervision, and to prevent myself becoming too scientifically blinkered, I took a quick foray this weekend into the murky world of protists, the strange and wonderful organisms that occupy the taxonomic equivalent of the 'misc.' draw in a filing cabinet. The creatures that are neither plant, nor animal, nor demonstrably bacteria. Many of them are single celled, some of them photosynthesise, and they all seem to occupy little evolved niches of their own, producing proteins with no noticeable homologues in any other branch of life.

The paper has the rather terrifying title of : "Rampant polyuridylylation of plastid gene transcripts in the dinoflagellate Lingulodinium". And I am not ashamed to admit that I had to go double-check the meaning of several of those words.

Dinoflagellates are little organisms that live in water, and mostly look a little like the picture on the right. Many of them are marine organisms, making up a large amount of the photosynthesising biomass in the ocean, and occasionally blooming to form 'red tides', leading to whole sweeps of water turning bright red (possibly occasionally on biblical command). The photosynthetic ones contain chloroplasts, which are wrapped up in three membranes, rather than the usual two. These, like all chloroplasts, contain their own genetic material (known as plastid genes), although unlike plant plastids, they don't seem to contain very many, and those that they do posess are found on little minicircles.

What the paper is interested in is whether there are any other genes in the chloroplast which aren't in minicircle form. There are, afterall, only 12 genes encoded on the minicircles, which is a small amount for a plastid. In order to explore this, it uses a characteristic property of the dinflagellate species it's working with. All organisms, when making proteins, make them from an mRNA copy of the genetic code. This mRNA copy tends to have a long string of adenosine residues added to the end, in order to prevent the mRNA getting degraded. This happens in our dinoflagellate species as well, but it doesn't happen to the plastid genes.

However instead of getting multiple adenosine repeats the plastid genes get multiple uracil repeats. It's just a different base, but it allows the mRNA made in the nucleus, and the mRNA made by the chloroplast to be separated. You can probe for adenosine enriched and adenosine depleted mRNA as shown on the gel below (A and B show different species). The psbA mRNA is clearly strongly present A+ (adenosine enriched) and therefore codes for a nuclear encoded protein. Conversely, the 23S RNA is A- (adenosine depleted) and is coded for in the chloroplast, from a plastid gene.

(Image taken from reference below)

The paper selected 300 random poly-uridine mRNAs (A-) and sequenced them to see if they corresponded to genes found in minicircles, or whether they might be plastid genes held in some different architecture. All the A- mRNA corresponded to the 12 genes discovered in the minicircle. They carried out rarefaction analysis to see if their sample size was large enough, apparently it was, in fact 300 clones was way in excess of the amount needed to find a further, non-minicircled-gene.

This suggests that minicircles are the only architecture for plastid genes and, importantly, that there really are only 12 genes contained in the chloroplast of the dinoflagellate Lingulodinium. This is a very small number of genes, all the rest have somehow migrated to the nucleus, leaving these 12 behind. And it's still very much an open question about why these have been left behind. The paper, in its discussion section puts forward the possibility of size. The genes that have been left behind all code for some of the longer proteins usually found in chloroplasts, although the paper does have the good grace to admit that that's not the most convincing of arguments.

It's worlds away from my little bacteria. But still just as fascinating.
---
Wang, Y. (2006). Rampant polyuridylylation of plastid gene transcripts in the dinoflagellate Lingulodinium Nucleic Acids Research, 34 (2), 613-619 DOI: 10.1093/nar/gkj438

Yes but what does it do...

I am currently trying to get myself to finished writing an essay (rather terrifyingly my first essay of term) on the different approaches to gene annotation in vertebrates. As I've just woken up (afternoon naps seem like such a good idea until you wake up with a mouth that feels like a hamster died in it) I thought I'd give a quick summary of gene annotation methods:

Gene annotation is the 'interesting' bit of genomics. Quite a lot of gene sequencing work has been done, some of it (especially the human bits) very highly publicised. And while genome sequencing is probably useful (more on that maybe in a more ethically-inclined post) on it's own it's not terribly exciting. You're left with a big database full of mindless streams of nucleotides and one bit embarrassing question:

What does it all do?

Gene annotation attempts to answer that; trying to work out which proteins each gene codes for, essentially what the end function of the genome is, what each piece of DNA is used for. There are two main methods: just using DNA, and using data from protein/cDNA sources. Both of these methods can be either comparative or non-comparative:

1) Just using DNA: Non-Comparative
This relies on getting a program such as GENSCAN to, quite literally, scan along the DNA looking for the beginning and end of genes based on sequence patterns it had been told to recognise. Not so good for function, but useful enough for finding the damn genes in the first place. Also relatively cheap and you can go run it overnight.

2)Just using DNA: Comparative
Like it says, this compares your DNA with other previously annotated pieces of DNA to see if there are any very similar bits it can ascribe function to. It's a good starting point, especially now the pool of annotated genomes is increasing, but it's really bad at finding gene start point, especially when there are 'introns', or bits of DNA that are not actually turned into protein. Which is around 95% of the human genome incidentally. (an e.g of this, if anyones interested, is TWINSCAN)

3) cDNA/Protein data: Non-comparitive
cDNA, just to clarify, is DNA that has been reverse transcribed from RNA templates; i.e itt's all the DNA that will get turned into protein, and without any of the introns. A good way to use this is to make cDNA 'libraries' i.e all the cDNA within the cell stored on plasmids, choose one at random, see what it makes and, at the same time, find where it is in the genome. Simple and useful.

4) cDNA/Protein data: Comparative
This compares your genome with bits of cDNA from other genomes, where the cDNA has known function. Protein comparison is even more useful as seeing what protein your protein most resembles provides structural information, as well as functional and allows you to build up homologous families of proteins with similar function (if you have enough genomes). Also if you have enough protein data you can say you're doing 'proteomics' and the more 'omics' words in your project, the more funding you're likely to get :)

By the way, all of these comparative methods are based on homologous evolutionary relationships between the genomes, so anyone who says that scientists never use evolution is WRONG. (and probably pissing off the evodevo people as well)

As always, any questions are welcomed, leave them in the comments and I'll get back to you.

Disclaimer: This post was written while half asleep. Any spelling/grammer mistakes are therefore completely the fault of the writers Brain On Sleep.

Presenting Histones

Bravery is an interesting word. It's one of those words that has many different subtle shades of definition; ranging from altruism to stupidity. The big question is, of course, was it brave or stupid of me to volunteer to do a presentation at the first supervision of term?

To put this more in context, I haven't actually done a scientific presentation since, well, at all. We did some mini ones in our supervision group last year, but they did not go so well. I haven't had to speak in front of a large crowd of people for about three years, not since the upper sixth performance of 'Dracula' where I stumbled on stage for a few minutes to play Translyvanian Peasant With Godawful Accent.

Here is the title and the link (for those who can get it) for what I have to present:
"Rb targets histone H3 methylation and HP1 to promoters"

I'm going to go through the paper now and try to provide a quick summary of what it is about. I have no idea how I'm meant to present it (hopefully there will be a brief meeting at some point to discuss this) but I can't help but feel things will go slightly better if I actually know what the paper is talking about.

okay... a look at the abstract and one brief scribbled diagram later this is what I've got:

Pretty pictures if you follow the links!

There is lots of DNA in the cell, so in order for it to fit into the nucleus it has to be coiled. One method for coiling involves wrapping the DNA around histone proteins (beads on a string) to keep them coiled. As well as keeping the DNA wound up, histones can also signal to transcription factors (proteins that start the complex process of turning DNA into protein) which bits of the DNA they need to read by displaying chemical signals.

One such signal is the methyl group, -CH3. Sticking a methyl group onto the end of a histone signals to the cells that this DNA is in Do Not Disturb mode, and should not be turned into protein. The study the paper was doing focused on a protein that goes around putting up all the nuclear Do Not Disturb signs; SUV39H1 (which shall henceforth be known as SUVy). This methylated the histones at a certain point (lysine 9 of histone H3 for anyone interested) and keeps the DNA associated with them from being expressed. It does this by recruting HP1 which binds to the DNA and, as far as I can work out from this, just sits there and stops it being expressed.

There are two forms that DNA in the nucleus can take: heterochromatin, which is all coiled up and not doing anything, and enchromatin, which is being actively expressed. This paper was getting fairly excited because while it was known that SUVy and HP1 were good at keeping heterochromatin quiet, they found them interacting with euchromatin! What's more they were consorting with Rb, a very well known protein that is involved in all sorts of processes that supress the expression of DNA, particularly in different parts of the cell cycle.

By doing various assays involving pulling out the Rb bound to DNA and then finding what bit of DNA it was bound to, they discovered that it methylated the same H3 on the lycine that HP1 did. Furthermore, Rb can interact with SUVy, due to a 'pocket domain' which SUVy fits into quite well. The end conclusion of all this is that Rb and SUVy interact together, methylate a part of the DNA which people hadn't really known SUVy was methylating, and then HP1 comes and sits on it.

The exciting thing here (alright not that exciting, but fairly interesting at the least) is that they put forward at the end that there may be other euchromatin repressor proteins out there that bind to SUVy and mobilise the DNA repression in euchromatin. Also, as Rb is involved in cell cycle control, it helps to build a bigger picture of just what is going on in the cell cycle (which cancer reseachers tend to like).

And woohoo I get to do a presentation on it. :)