Archives for category: centromeres

Mitosis – fun with modelling clay

April 20, 2014 //
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Mitosis has to be one of the more beautiful things in nature. It’s a choreographed dance of the chromosomes. It’s so small that we can’t see it without a microscope, but it goes on in our bodies billions of times a day.

DNA is a very long molecule made up of the genetic alphabet (which has four letters: A, C, G, T). A gene is made of a certain sequence of DNA letters (or bases) and spells out an instruction for a step in the complex workings of our bodies (such as the structure of insulin). The genes are strung together along the chromosome, and each cell has a set of chromosomes. For our bodies to grow, these cells need to make copies of themselves. The problem of how to distribute the copied chromosomes evenly to the two “daughter cells” is handled very elegantly.

 

Chromosomes arrested at mitosis and stained with Giemsa (unbanded).

Human metaphase chromosomes stained with Giemsa (unbanded). The two halves of each chromosome are copies of each other.

 

Mitosis is the solution. Mitosis is broken up into a series of phases: interphase, prophase, metaphase, anaphase, telophase. You could break prophase up further by adding prometaphase: the part of prophase between the nuclear membrane breaking down and metaphase (where the chromosomes line up at the metaphase plate).

Now follow the captions under the pictures.

The interphase nucleus - the DNA from all the chromosomes intertwined with each other is represented by grey modelling clay. (Actually it seems that the chromosomes stay in relatively distinct domains - but under the microscope they appear as one entity.)

The interphase nucleus.   The DNA from all the chromosomes, intermingled with each other, is represented by grey modelling clay. (Actually it seems that the chromosomes stay in relatively distinct domains – but under the microscope they appear as one entity.) The DNA in the interphase nucleus copies itself as the cell grows.

 

The DNA in the nucleus starts to package and take shape as prophase chromosomes.

The DNA in the nucleus starts to coil up in a pre-determined order and take shape as prophase chromosomes.

 

The DNA folds up in a pre-determined order to make recognisable chromosomes. When the cell is ready to divide each chromosome has two chromatids or identical halves, joined at the centromere.

The DNA folds up further to make recognisable chromosomes. When the cell is ready to divide each chromosome has two chromatids or identical halves, joined at the centromere.

 

At metaphase the chromosomes meet in the middle of the cell at the metaphase plate. Then as the cell divides to become two daughter cells, the two halves of the centromere split and travel along the microtubules in opposite directions, pulling the two halves of the chromosome behind them.

Metaphase - the chromosomes line up in the centre of the cell at the metaphase plate. They are attached by their centromeres to microtubules which stretch across the cell.

Metaphase – the chromosomes line up in the centre of the cell at the metaphase plate. They are attached by their centromeres to microtubules which stretch across the cell.

 

At anaphase the two chromatids (half chromosomes) become the new chromosomes as they separate and move in opposite directions along the microtubules.

At anaphase the two chromatids (half chromosomes) become the new chromosomes as they separate and move in opposite directions along the microtubules.

 

The chromosomes start to unravel to form the two new daughter interphase nuclei. The cell membrane (the outer covering) pinches at the centre and the one cell finally becomes two (cytokinesis).

The chromosomes start to uncoil to form the two new daughter nuclei – telophase. The cell membrane (the outer covering) pinches at the centre (cytokinesis).

 

The cell membrane pinches at the centre (cytokinesis) so the cell finally becomes two cells.

Cytokinesis finishes and we have two new cells in interphase.

 

If a chemical that destroys the microtubules is added to a laboratory culture, the chromosomes are stopped at metaphase. Cytogeneticists (chromosome scientists) use this technique to get enough metaphase chromosomes for analysis. Chromosome banding helps us recognise the chromosomes and identify any changes when an abnormality is suspected. Of course, the cell is also full of other organelles that have to be shared between the new cells.

The modelling clay images above are from my claymation showing mitosis. Modelling clay is a great medium for demonstrating and thinking about how things work, move and change. For the claymation I used a phone camera resting face down on a glass coffee table over the models.

(Cross-posted from Fireside Science at SciFund Challenge.)

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Categories cell biology, centromeres, chromosomes, genetics

Chromosomes that cause cancer Part 3: Real-life examples of the breakage-fusion-bridge cycle

January 6, 2014 //
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Cancer has been described as the most common genetic disease. This doesn’t necessarily mean it’s hereditary – usually the genetic mistakes that cause cancer arise in the body’s organs or tissues and can’t be inherited. We’re continually learning of new cancer-causing genetic mistakes.

If we think of genes as words spelling out the instructions for our bodies to function, there are different types of mistakes or “typos” that can cause cancer. Some of these are like spelling mistakes – an incorrect letter or two. The mistakes I’ll be talking about here involve whole words (or lots of them). For example one or more copies of a word are added – ” very big” becomes “very very big” – extra copies of a cancer gene (we call them oncogenes) can cause or accelerate cancer growth. Or if a word is lost – “don’t grow” becomes “grow” – this illustrates loss of a tumour suppressor gene.

I described the breakage-fusion-bridge cycle a few weeks back. The BFB cycle was a theory developed from studies with maize, but it also applies to some cancers. This is an example of basic research,  inspired by curiosity but eventually being useful in ways we never imagined. If you look back at that post it shows how the BFB cycle can cause gain or loss of genes. If a cancer gene is  multiplied, or if a tumour suppressor gene is in the part that’s lost, the cell can gain a growth advantage over other cells, which is part of the process causing cancer.

Here’s an example.

Cancer cell lines are cancer cells that can be grown indefinitely in the laboratory. I’ve just published a paper on HEL, which is a leukaemia cell line. It’s popular for studying how cells make globin (molecules in red blood cells that help us process the air we breathe).

The chromosomes of HEL are very abnormal. I’ve used a combination of techniques to show how the chromosomes are reorganised as well as which parts have been lost, gained and amplified.  It’s very complicated.

One of the gene abnormalities in HEL is amplification of the JAK2 gene. JAK2 is a well-known cancer gene that is often abnormal in blood cancers. The normal gene can be mutated to become a cancer gene, for example by a “spelling mistake” in the DNA. By adding extra copies of this abnormal gene the effects can be magnified. This is known as gene amplification. There are a few cancer genes that are commonly amplified in cancers.

To cut a long story short, JAK2 is amplified in the HEL cell line. And a nearby tumour suppressor gene (CDKN2A) has been lost. But only by looking at the chromosomes does the reason become clear. Some detective work tells us that there were some breakage-fusion-bridge events. I won’t go into the detail – if you’re interested it’s in the paper. But we have chromosomes whose ancestors had two centromeres, and if we use a DNA tag for the region between the centromeres we can see “stripes”.

Here’s an example from HEL that shows DNA amplified by the BFB cycle – we can show where a gene is on the chromosomes by labelling it with a fluorescence tagged DNA “probe”. The striped pattern reminds us of the yellow dots in the modelling clay demonstration:

The red is DNA that's normally at one end of some of the chromosomes. The stripes tell us that the end of a chromosome (22) is in the middle of these chromosomes and there are extra copies. This helps us work out how these chromosomes were made. It's a strong clue that BFB cycles were involved and the ancestral chromosome had two centromeres.

The red is DNA that’s normally at one end of some of the chromosomes. The stripes tell us that the end of a chromosome (22) is in the middle of these chromosomes and there are extra copies. It’s a strong clue that BFB cycles were involved and the ancestral chromosome had two centromeres.

The new chromosome with four copies of the yellow gene courtesy of the breakage-fusion-bridge cycle.

The new chromosome with four copies of the yellow gene courtesy of the breakage-fusion-bridge cycle.

Recently JAK2 amplification was also reported in triple-negative breast cancer. Triple negative means that three well-known genetic causes of breast cancer are not present. So finding JAK2 amplification would help explain the cause of some triple-negative breast cancers, and could help work out an effective treatment. Perhaps this JAK2 amplification is sometimes caused by BFB cycles. Without looking at the layout of the abnormal chromosomes we may never know.

To end this story, here’s Bruce Mercer’s cartoon diagram showing the BFB cycle in HEL: http://emph.oxfordjournals.org/content/2013/1/225/F6.expansion.html

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Categories cancer, centromeres, chromosomes

Chromosomes that cause cancer: Chromosomes with two centromeres and the breakage-fusion-bridge cycle

November 3, 2013 //
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Barbara McClintock published a paper describing the breakage-fusion-bridge (BFB) cycle in 1939. Many of her ideas were well before their time. Like many such profound leaps in thinking, the BFB cycle took a long time to catch on. She wrote in 1973, “I stopped publishing detailed reports long ago when I realized, and acutely, the extent of disinterest and lack of confidence in the conclusions I was drawing …One must await the right time for conceptual change.” Her work was appreciated much later and she was awarded a Nobel Prize in 1983 for her discovery of “jumping genes“.

A few weeks back I introduced this post by describing normal chromosome division. This time we’ll look at the breakage-fusion-bridge cycle. This is one way chromosome division can go wrong. Very wrong, in the sense that it can cause the chromosomes to keep changing, and this can cause cancer.

A human chromosome with two centromeres is abnormal. Chromosomes with two centromeres are not unusual in cancer cells. In fact they’re probably a lot more common than we think, because in both research and diagnostic labs the centromeres are usually not looked at.

To recap, a normal chromosome has one centromere. Before the chromosome divides, the two identical halves (chromatids) are held together at the centromere. When the chromosome divides the centromere splits into two halves, the chromatids become the new chromosomes, and the centromeres take the two new chromosomes in different directions into the two new daughter cells.

So what happens if there are two centromeres? If they’re both aligned so that they head in the same direction it’s not a problem – together they take a complete new chromosome with them. The closer the centromeres are together the more likely this is.

Now follow the pictures and their captions. These describe chromosome division in an abnormal chromosome with two centromeres. Especially follow the yellow dots.

A twist between the two centromeres when the chromosomes align ready for chromosome division.

If the two centromeres on a chromosome go in the same direction there’s no problem. But if there’s a twist between the two centromeres when the chromosomes align ready for chromosome division….

....then, when the two halves of each centromere separate they head off in different directions.

….then, when the two halves of each centromere separate they go in opposite directions. We have a “bridge” spanning the gap between the two centromeres.

The bit of chromosome between them gets stretched and can break.

The bridge is stretched and can break.

The broken chromosomes in the new cell join together - the top daughter cell gets an extra copy of the yellow gene. The bottom cell loses this copy of yellow gene.

The broken chromosomes in the new cell join together – the top daughter cell gets an extra copy of the yellow gene. The bottom cell loses this copy of the yellow gene.

The new chromosome copies itself to make two equal halves.

The new chromosome copies itself to make two equal halves.

If this process repeats..

If this process repeats..

…

Fusion of the broken bits of chromosome in the top cell.

Fusion of the broken pieces creates a chromosome with four copies of the yellow gene.

After replication - the new chromosome with four copies of the yellow gene courtesy of the breakage-fusion-bridge cycle.

After replication.

If the yellow gene in the pictures is a cancer gene (“oncogene”) the cell with extra copies might grow and multiply faster than its neighbours. We call this natural selection – the cells that can grow faster than their neighbours become more common which means the genetic change causing that is undergoing “positive selection”. Yes, the cells in our body can evolve and we know this best as cancer.

All this change happens between the two centromeres where the bridge forms. So if we find a chromosome with this type of change on one side of the centromere only it’s a clue that this might have been caused by the breakage-fusion-bridge cycle.

These are modelling clay images from my breakage-fusion-bridge claymation. They’re a bit rough but I hope it helps you understand what happens. Many, perhaps most, images demonstrating the BFB cycle show a different version – where the abnormal chromosome is created by two chromatids of one chromosome breaking and joining together. Most examples don’t show the version I’ve presented – where two different chromosomes have joined together. Check this out on Google Images (search for breakage-fusion-bridge).

Here’s the answer to the quiz from the telomere post. The arrows point to the ring chromosomes. Being rings they have no ends, so no telomeres.

answer to ring chr

Further Reading:

B. McClintock 1939. The Behavior in Successive Nuclear Divisions of a Chromosome Broken at Meiosis. Proc Natl Acad Sci U S A. 1939 August; 25(8): 405–416.

M. Kinsella and V. Bafna 2012. Combinatorics of the Breakage-Fusion-Bridge Mechanism. J Comput Biol. 2012 June; 19(6): 662–678.

R. MacKinnon and L. Campbell 2011. The Role of Dicentric Chromosome Formation and Secondary Centromere Deletion in the Evolution of Myeloid Malignancy. Genetics Research InternationalVolume 2011 (2011), Article ID 643628.

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Categories cancer, centromeres, chromosomes, Uncategorized