How can circles within chromosomes be hiding in plain site?

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Above is a mouse L1210 cell as it appears stained with acridine orange under a fluorescent microscope. The green color is due to the high DNA content of the cell which has very little cytoplasm. The cells have been subjected to what is called “serum arrest” which means they are dividing very slowly, if at all.

degrading cell83

As a cell begins to die under the scope, remarkable things begin to happen. In some cases, circular vesicles of various sizes begin to pop out of the cell. Some of these structures migrate away from the cell on thin tethers.

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In other cases, vesicles of very similar sizes line up in singular arrays, resembling primitive fungal elements.

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In still other cases, vesicles of various sizes are seen passing through tubules with many of them reaching the ends. The obvious initial reaction to these photographs should be dumbfounded amazement. What in the heck is going on here? At least, that was my initial reaction when I first saw them. The drama goes even further when these dying cells are treated briefly with 0.1N hydrochloric acid:

slide 7

They transform into extremely complex circular structures from which smaller beaded circles are ejected. Some of these smaller circles have “hubs” associated with them. Others are empty.

Circles

As time passes, more and more smaller circles are released until they become predominant.

91b

As more time progresses and the stain begins to fade, even more remarkable transformations take place. Larger, amorphous structures begin to appear that look like beads strung on a necklace floating in water.

8

Eventually, these structures are replaced by even larger “beaded necklaces”.

When you look at these transformation products under visible light phase contrast  you see a remarkable variety of structures.

69slide-3 crop

There appears to be some kind of whitish scaffolding to which is attached small black circular objects. Upon closer inspection, some of these black circles contain hubs within them and others do not. These circular objects can be ejected from the scaffolding as if they were grapes on a vine.

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Furthermore, they come in a variety of shapes, and can break up into linear elements that strongly resemble mitotic chromosomes.

slide-3 crop

It is obvious these structures are somehow related even though their internal compositions can vary widely. Other related structures can also be found that are either concentric rings of chromatin-like structures (or left-over scaffolding) or structures that appear similar to the radiating tubules shown with internal acridine orange stained substructures.

slide-1 crop

How does one account for such a diversified array of structures? The most logical explanation would be that the cells were in various stages of the cell cycle during serum arrest. If true, that still leaves a lot to be explained about them.

Since these circular elements obviously come in a large variety of sizes, the question of what constitutes the lower size limit should be addressed. This requires going to the level of the transmission electron microscope. The photographs below are of purified mouse nuclear DNA.

39DNA rosette

black bars (center bottom) = 0.5 microns and 0.2 microns

Note the “hub” and connecting circular elements on the figure to the right.

If cells are labeled with tritiated thymidine, lysed quiescently in gel inserts, placed on microscope slides, subjected to DC electrical current (electrophoresis), and coated with photographic emulsion for a few months, this is what is seen after the slides are developed.

Autoradiography

The grains of silver shown in this slide illustrate three circular structures connected together by a thread. Thymidine is taken up specifically by DNA. Compare this with a structure stained with acridine orange and photographed using UV microscopy.

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So let’s summarize what we have up to now:

  1. We have circles with size ranges much larger than the original cell from which they came, all the way down to those that can only be seen with the electron microscope.

2. Some of these circles have hubs, others do not.

3. There are a variety of circular elements with varying kinds of internal structure that suggest they are in varying stages of the cell cycle. Other kinds of more amorphous structures can also be found. In some of the circles, these beads appear as circular elements which may or may not have internal hubs. In others, the beads seem to join tightly together and break off into linear elements resembling mitotic chromosomes. In more amorphous forms, the “beads” travel through tubules or are tethered to tendrils.

4. Both circular and amorphous structures seem to be composed of a scaffolding that anchors compact black beads together. In some structures, the scaffolding is seen as completely devoid of these beads.

5. When circles are first released from cells, they are within certain size limits that begin to increase as time progresses.

So what in the world can be concluded from all of this, if anything?

Well, that’s what this blog had been all about from the very beginning: Explaining these structures the best way that I can. I will use highly schematic models as explanations for what may be going on here.

FigX

This model is of two different kinds of cellular nuclei. The one on the top would be similar to mammalian nuclei. The one on the bottom would be that of a more primitive animal like an insect or nematode worm. The model on the bottom gave rise to the one on top through evolution. The little cross shaped, beaded structures that are connected together within these nuclei are similar to the one shown in the election micrograph. You can learn more about them by reading through the blog.

Fig35 Fig34

The model on the left is composed of circles with hubs and the one on the right contains no hubs. In reality, both of these structure would coexist together. Note the variety of ways these structures can break down over time. In some cases, circular elements are simply released from one another. In other cases, circles fuse together to form larger circles. In the photomicrographs the “beads” on the circular necklaces may be compact chromatin particles composed of smaller circular DNA’s which may themselves be made of even smaller chromatin beads of circular DNA’s. I call this hierarchical DNA superstructure.

How can circular fusions and separations occur at the same time? One explanation would be the presence of 2’3’5′ phosphodiester bonds as occurs in some kinds of RNA. How does RNA fit into DNA? It does so in the form of Okasaki fragments. You can learn more about this by browsing through the blog.

How would such complex circular arrays fit inside a chromosome? We can start by dissecting a hypothetical chromosome to show exactly what be inside it in terms of hierarchical DNA superstructure:

Fig44

Fig A shows a eukaryotic chromosome composed of 12 fused vesicles. Vesicles 2, 4, 6, and 10 contain the active genetic components. The remaining vesicles are structural. Fig B shows how this chromosome is composed of seven subchromatids with fragile sites, sensitive to radiation and  capable of massive DNA rearrangments. Fig C shows four units of recombination and how masses of circular elements can undergo crossing over during recombination.

Fig45aeFig45fg

Now we strip away everything until only the DNA remains (A,B, and C). The chromosomal circular elements begin to fuse together to generate bigger and bigger circles of DNA. Of course, they may also be breaking off at the same time.

At this point, I believe it is time to end this particular post. Obviously, there is a lot here that is not being shown or explained. You can learn more about this by delving into the blog or contacting me via e mail at fabernathy@sbcglobal.net.

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About frankabernathy

I am a retired cell biologist and alumnus of Ohio State University. I became interested in chromosomes as far back as the 1960's when I wrote a term paper on the effects of radiomimetic drugs on chromosomes. I was fascinated at how they could break apart and reform new structures so easily. I became further involved in the early 1970's after taking a cytogenetics course at the University of Arkansas. I took that knowledge with me to Ohio State in 1980 where I eventually worked on my research and completed my Ph.D. dissertation, "Studies on Eukaryotic DNA Superstructure". My studies and later research suggested that the DNA within the eukaryotic chromosome is not the simple, linear molecular thread so widely suggested in all the classic textbooks published today. Instead, it may be the culmination of a geologically rapid set of endosymbiotic events where microorganisms plug into each other to create something greater than themselves. Feel free to contact me at fabernathy@sbcglobal.net.
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