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Sunday, 9 May 2010

Meet the chickens - sister chromatid labelling

Yo all,

welcome again in Meet the chickens section. Today a specific labelling of chromosomes (actually sister chromatids) will be presented.

As you probably remember from previous post (Meet the chickens - chook chromosomes at hand), there is a way to examine karyotype (set of chromosomes) of chicken DT40 cell line (or any other cell line:). In this post we will combine this technique with a labelling procedure. This protocol will give us oportunity to see differential staining of sister chromatids.

Ok lets go.

In this protocol a nucleotide derivative, called BrdU (5-bromo deoxyuridine) is used to labell DNA for two cell cycles (see the picture below).


Simply saying, cells are treated with BrdU, which is incorporated to DNA during S-phase (DNA synthesis phase of cell cycle). It is important that labelling is performed for exactly two cell cycles. This is because after two cell cycles (two subsequent S-phases) one of sister chromatids is complately substituted with BrdU where other one is half substituted half not. This feature of labelling can be now used to distinguish between sister chromatids (they simply have different physical and chemical properties).

First cells are stopped in metaphase (cell division phase) using a specific drug (usually colcemid a microtubule depolymerysing agent) that prevents mitotic spindle formation. After that, cells are swollen (to make them fragile) and fixed (to fix their state:). Further, cells are droped onto slide to open them and release chromosome spreads. Next few steps leads to differential staining of sister chromatids (see picture below).

In the first step, cells are treated with a DNA intercalating dye called Hoechst 33258 (this is a fluorophore that adsorbs UVC light at 258nm). Intercalating means that it gets in between the DNA base pairs stacks (see picture below)

Red bars represent an intercalating agent bound to DNA/
Picture taken from http://en.wikipedia.org/wiki/File:DNA_intercalation.jpeg

As you remember one of the chromatids is complately BrdU substituted. BrdU contains bromide and occupy more space than a regular nucleotide (remember that bromide atom is way more bigger than carbon, hydrogen or nitrogen). Additionally BrdU nucleotide is light sensitive. Hoeachst intercalation occurs only in monosubstituted chromatid but not in disubstituted one (there is not enough space for Hoechst intercalation, because of two bromide atoms).
Further, slides are exposed to UVC light. Hoechst 33258 adsorbs the UVC light protecitng monosubstituted chromatid from UV light degradation. Subsequently DNA is stained with another dye called Giemsa stain. Degraded DNA do not stain with Giemsa dye revealing a differential effect as on picture below. Additionally to UVC light effect it is believed that different set of proteins are bound to BrdU disubstituted chromatid comparing to monosubstituted (what leads to differential staining). 

Picture taken by Kliszczak M.

As you can see on the picture above mono- and disubstituted chromatid are indicated with blue and red arrowheads, respectively. Monosubstituted chromatid appears darker (as a result of Giemsa staining) than disubstituted (pale colour, no staining).
This technique is being used to assay chromosomal stability. Any DNA damage that occurs in the cell and is repaired by recombination (exchange of DNA information between the sister chromatids), will be visualized by this technique. You can see such events on the picture above. Two of them are indicated with black arrowheads. Some gene mutations (for example Rad54 or Rad51 - these genes code for proteins involved in DNA recombination pathways) leads to decreased number of Sister Chromatid Exchanges (SCE). Usually a specific drug (in this case Mitomycin C) is used to induced SCE's. After induction SCE's frequencies between Wild Type and mutant strains are comapred.

I hope you enjoy it.

Maciek GGSTEAM

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