strikinghelix

I just came up with the topic for my grant proposal project in Fungal Genetics.

Behold its magnificence.

The filamentous fungus Neurospora crassa exhibits a phenomenon known as RIP, or repeat-induced point mutation. This mean that the more copies there are of a certain nucleotide sequence within its genome, the more they tend to accumulate point mutations, or changes in the identity of a single nucleotide.
Recently, the complete sequence of the N. crassa genome was published, and one of the main conclusions offered was that RIP has had a profound effect on the evolution of this organism and has resulted in a genome with an unusually low proportion of duplicated/closely related genes.
The mechanism behind RIP remains unclear. This project aims to identify genes within Neurospora that are responsible for this unique phenomenon.
My approach will be to mutate a population of fungus cells with a mutagen known as T-DNA. T-DNA is derived from Agrobacterium tumefaciens, a plant pathogen whose mode of survival in nature is to insert bits of its own DNA into plant genomes, forcing them to create food that it cannot make for itself. This mutagen is a small cassette of DNA whose most unique feature is that it inserts preferentially into coding sequences (genes) rather than non-coding sequences (so-called junk DNA). Most traditional mutagens insert themselves in a much more random fashion.
Another unusual thing about Neurospora is that it has a very high proportion of coding sequences (roughly 44% of the genome) compared to most organisms (humans have less than 5% coding DNA), which is probably another evolutionary result of RIP given that most junk DNA is repetitive. Therefore, T-DNA is an excellent choice for mutating genes in this organism. It is also possible to engineer T-DNA cassettes with selectable markers so that one can cause all cells lacking T-DNA will die.

Experimental Design:
1) Allow T-DNA to insert into a population of Neurospora, and select for only those cells which have uptaken T-DNA.
2) Cause these cells to then uptake a plasmid, or small circular DNA sequence, and again select, this time for cells which have uptaken the plasmid. Plasmids can be engineered with relative ease to contain several genes, and when introduced, the hosts molecular machinery will activate these genes. The trick is to include several copies of a reporter gene (one whose product is easily assayed) on the plasmid. GFP, or green fluorescent protein, is a popular reporter geneany cell activating GFP will glow green.
3) Allow the cells to replicate for several generations. Any cells who still express the reporter gene when present at several copies will have failed to engage RIP. Normal Neurospora cells would have mutated the reporter and silenced its expression. Cells such as this would be isolated and maintained for further study, since they likely have had a T-DNA insertion occur in the middle of a gene responsible for RIP.
4) Now, a very nifty trick known as inverse PCR can be used here to obtain the DNA sequence of the region flanking the insertion. Recall that T-DNA cassettes are somewhat customizable. It is easy enough to include short sequences called PCR primers at the ends of the cassette. These primers, when combined with the right ingredients, will cause many copies to be made of the sequences next to themhowever, the primers must face each other (they must be designed to copy towards each other) on opposite strands of the double helix. However, if you put primers at the ends of the cassette facing towards each other, then the sequence that gets copied is the cassette itself, whereas we are interested in the flanking sequences. So, we cause the primers to face away from each other instead, and randomly chop the DNA into bits with various enzymes. Eventually, situations will occur where the cassette is left intact, with some flanking sequence, yet separated from the great majority of the genome. These are the pieces that inverse PCR will work on, for the next step is to circularize the DNA, causing all pieces to fold around and attach to themselves like a snake eating its own tail. Now the primers that were facing away from each other are now facing towards each other, and PCR will do its work, making many copies of the sequence between them.
5) These flanking sequences may now be decoded, and the resulting data may be searched for in the Neurospora genome database online (via BLAST algorithm) to confirm gene identity and to order clones so that more in-depth functional studies can be peformed.

So there were some controls and stuff too, but whatever.
If you understood that, youre just a fucking pimp. Dont let anyone tell you otherwise.
I tried to address this version to a more general audience without being patronizing; the real proposal will sound way smarter.

I have my FIRST GIG EVER tomorrow, playing drums with this guy who sings and plays guitar. Im gonna try real hard not to suck balls.