mutually exclusive rad scenarios?

Claudia Wiese, Eloϊse Dray, Torsten Groesser, Joseph San Filippo, Idina Shi, David W. Collins, Miaw-Sheue Tsai, Gareth Williams, Bjorn Rydberg, Patrick Sung, and David Schild. 2007. Promotion of homologous recombination and genomic stability by RAD51AP1 via RAD51 recombinase enhancement. Molecular Cell

As an alternative to David Goodsell's triple helix model (see below) Rad51 may instead encircle the bound template and recipient DNA strands, as gene conversion takes place, leaving the donor's complementary strand (displacement loop) out..

A similar, earlier view on rad51 and rad54 nucleoprotein filament and D-loops:

The development and regulation of gene repair

Li Liu, Hetal Parekh-Olmedo & Eric B. Kmiec

Nature Reviews Genetics 4, 679-689

photosynth and protein complexity

(A tangent:)

Also, I've been wondering about what my level of complexity should be in animating Ig gene conversion. Since my audience will be at the researcher level, (and possibly also aimed at upper year undergrads), I would like to be as molecularly 'accurate'  as possible. 

(David Goodsell. 2005. The Molecular Perspective: Rad51 and BRCA2. The Oncologist. 7: 555-556.) Showing Rad51 binding to 2 DNA molecules. One ssDNA (pink) exchanges partners with a strand of the yellow double helix, and inside the Rad51 enclosure, a triple DNA helix exists.

I really like the concept of protein-DNA interactions, and I think many visualizations of DNA miss out on a lot of the story. i.e. They show two lone strands of DNA that, of their own volition, bend and twist around, bind onto template strands, without any kind of chaperone, scaffolding, or conformation-changing proteins to effect those forces.. 

And yet mutants with knockouts for these proteins show serious handicaps (AID, compromised immune system), increased cancer susceptibility (Brca1, breast cancer), or outright mortality (Rad51). These examples are all involved in chicken Ig gene conversion, and also are implicated in human disease and DNA regulation..

In short, I would like to explore a more complex protein-DNA story of gene conversion; with proteins as the protagonists, and DNA as their construction site...

visual references continued

YouTube searches for gene conversion animations have revealed:

Gene Conversion by Dr. Nguyen Thank Cong

A homologous recombination repair pathway 

(could be a good reference model for strand resolution / DNA repair, after templated gene conversion)

A Neat Overview of Transcription and Translation 

(shown in 'real time', with jerky camera/claymation/David Goodsell color pallette elements)

Histone Deacetylation Animation

(histone deacetylation inhibitors increase gene conversion rates, so it's probable that DNA access and its association with histones is an important aspect of which gene segments undergo gene conversion, and why this occurs only in IG DNA)

(this animation may lack some important details, such as the histone tails which are modified in acetylation/deacetylation reactions)

More Recent Histone Acetylation / Deacetylation Animation

More histone and chromosome visualizing

DNA Packaging

aesthetically pleasing

Courtesy of Albert and Wensi, the epitome of awesome intracellular and interstitial rendering:

Amgen

Brought to you by: Random42

Wow

1st research methods class + feedback

After presenting my visual question, and current state of research in class today, a few very interesting suggestions were made:

- the possibility of using a random (or nearly, depending on how random the selection of pseudogene sequences is; I think there may be evidence that pseudogenes nearer to the VJ segment are preferentially incorporated) formula / generating code (in maya?) to select pseudogene base pairs for gene conversion

- then I could show many b cells undergoing gene conversion simultaneously, but producing different converted genes depending on what pseudogene elements were selected by the formula / generator

- I could extend this further by then showing the differences in protein structure between b cells after gene conversion (i.e. after transcription and translation), and colouring the mutated/converted amino  acids so that they stand out?

- I wonder if it would be possible to have a 'program / code' that could incorporate the base pairs, and another that could take base pairs and 'translate' these into amino acid sequences (i.e. I would have many long strings of dna and want to 'magically' convert these into different amino acids at the level of the 3D animation, not at the pre-rendering stage when I could use a table or amino acid generator from the internet)

- the idea of periodically using flattened, 2D animations, like the Engelward Lab's, to clarify otherwise complex DNA nicking, inserting, winding and unwinding processes

- this is along the same lines as using sliding stills

- and other non-photorealistic / more schematic visual approaches

- further research direction: justifying my cinematic decisions (i.e. why is a 3D animation beneficial in depicting gene conversion)

Note on the idea of the formula/generator:  its primary purpose would be to simplify the rendering process

visual: heavy chain loci

Creating an annotated bibliography is a great motivator / propeller for finding helpful visuals:

Taken from: Weill, Jean-Claude, and Claude-Agnes Reynaud. 1996. Rearrangement/hypermutation/gene conversion: when, where and why? Immunology Today. 17: 92-97.

This image compares, schematically, Ig heavy chain DNA of sharks, chickens, rabbits and mice. The paper explores the mechanisms used by different species to develop diverse B cell repertoires; 

- sharks have many (cassette-type) clusters of partially fused VDJ sequences, primary B cell repertoire is then generated mostly by hypermutation, intracluster V(D)J rearrangement also takes place

- chickens have upstream pseudogenes that modify the one functional V sequence in both light and heavy chains

- rabbits have ~100 functional and ~100 pseudogene V elements, these all serve as gene conversion donor templates for the V sequence most proximal to the DJ region

- mice have ~9 V regions.

Evolution of B cell diversification: the paper postulates that so long as the requirement for one antibody -> one B cell is satisfied, the means of generating a diverse array of B cell antibodies could vary from species to species.

Interesting fact: a pathogenic microorganism, Borrelia hermsii, employs gene conversion techniques very similar to chicken to generate diverse outer membrane proteins (to avoid detection / neutralization by its host/victim's immune system)

GC Visual (& B Cell Repertoire Diversification)

Taken from Jean-Claude Weill, and Claude-Agnes Reynaud. 2008. DNA polymerases in adaptive immunity. Nature Reviews Immunology. 8: 302-312.

After gene rearrangement (V(D)J recombination) antibody DNA can be further modified and diversified by three mechanisms:

 - somatic hypermutation (initiated by AID, occurs during B cell proliferation in germinal centers, happens in humans, largely a random process, associated with class switching, affinity maturation [where Ab acquires better binding ability to antigen over time], happens in CDRs [complementariy-determining region] more frequently than framework and J segments, mutation rate is 100 000x higher than in other genes)

- gene conversion (occurs after gene rearrangement, only one functional  V  and J region in chickens, pseudogene elements largely incorporated into V region)

- class switch recombination (enables production of different classes of antibodies: IgG1-4, IgA, IgM, IgD, IgE, in order of decreasing quantity found in blood in humans. In chickens, homologs of IgG, IgM and IgA are present, in order of decreasing serum concentrations in blood)

Visual Question(s) v2

1. How to make animation as lecture-friendly as possible?

    -> 2 different scripts

    -> scene selection capabilities

   -> increased length to accommodate a longer script:

        -> sliding stills (less rendering)

         -> frozen scenes 

2. How to translate many theories on mechanism of gene conversion into an animation?

  -> 3 different scenarios

Documentary Sites

An interesting site with archives of short, independent documentaries:

FourDocs

Animations, continued

I've started scouring the internet for animations, and have found a neat one called 'Exploring Our Molecular Selves' that has an interactive scene-selection component:

Exploring Our Molecular Selves  (The animation is at the bottom of the page ... the other applications look informative too)

And a textbook site animation:

Homologous Recombination

Animations: DSB repairs

Here is a site, from the Engelward lab at MIT, that has some flash animations depicting repair of double strand breaks, a broken replication fork, and subsequent DNA mutations:

http://web.mit.edu/engelward-lab/animations.htm