last  page PLNT3140 Introductory Cytogenetics
Lecture 11, part 2 of 2
next page

2. Higher-level coiling

A summary of chromosome coiling can be found at

http://www.ncbi.nlm.nih.gov/books/NBK21576/figure/A2272/

a.  SEM Studies of Human Chromosomes

Allen TD, Jack EM, Harrison CJ (1988) The three dimensional structure of human metaphase chromosomes determined by scanning electron microscopy. In Adolph KW (1988) Chromosomes and Chromatin Volume II, CRC Press. Boca Raton FL. Chapter 10, pp. 51-72.

We finally have all the pieces in place to complete our model of the eukaryotic chromosome by defining its high-order structure
.
At 51000X, it is possible to identify rounded projections of about 1.5-2x the diameter of chromatin. These have been interpreted as the loop domains described above. Although the resolution of SEM is good enough, sample preparation technology has not yet made it possible to observe finer structure in chromatin loops. It is also worth pointing out that in the figure we are actually seeing two sister chromatids, which will separate during anaphase. This is evidenced by the gap between the chromatids. Note the interchromatid fibers, which are about the right size for individual chromatin solenoids.

(Allen et al.,Fig. 4 p57) Detail of a single chromosome viewed at high resolution in the SEM. The majority of the chromosome surface displays a twisted loop configuration with approximately twice the diameter of the individual fibers which are seen mainly as interchromatid fibers (arrowed). (Magnification x 51000).
Undisplayed Graphic

Now let's zoom out by a factor of 5 to look at complete chromosomes.

(Allen et al. Fig. 3, p56) FIG. 3. Part of a metaphase spread preparation prepared without banding techniques, illustrating small circumferential grooves on the surface of the chromatids. (Magnification x 9500).

Some segmentation and indentations of chromosomes apparent, but now compare with scanning EM of G-banded chromosomes.

Undisplayed Graphic
Here, we can see 'circumferential grooves' that run as a helix down each chromatid. Again, this indicates helical organization at the highest level of chromosome structure. It's important to realize that when we do any kind of banding technique, we are altering the chromosome in some way,such that they take up dye more readily. Usually, a partial hydrolysis of protein with the protease trypsin is used. So you have to remember that what we're seeing is not the way the untreated chromosome would actually look, if we could see it with light, but rather an altered structure whose purpose is to bring out structural features.
Undisplayed Graphic


(Allen et al., Fig. 5, p58) Low-power micrograph of a G-banded metaphase spread. The chromatid arms are segmented by circumferential grooves which run as a helix down each chromatid. (Magnification x 2800).



b.  Evidence for at least 2 levels of coiling above chromatin attachment to the scaffold (or, what Chinese hamsters can teach us about chromosomes)

Yuri G. Strukov 2 Yan Wang 1 , Andrew S. Belmont 1 , 2 (2003)  Engineered chromosome regions with altered sequence composition demonstrate hierarchical large-scale folding within metaphase chromosomes  J. Cell Biol. vol. 162 no. 1 23-35 The Rockefeller University Press, doi: 10.1083/jcb.200303098  http://jcb.rupress.org/content/162/1/23.full

Two alternative models of high-level chromatin folding during prophase are presented in A.
  • The top model postulates that as chromatin condenses, the nuclear matrix joins a uniform number of chromatin domains into a disk-shaped floret of chromatin domains. This would mean the chromosome could be visualized as a stack of discs, each containing a floret.
  • The second model postulates that chromatin condenses into a less-regular coil on indeterminate configuration. The thickness of each unit would be larger than the a chromatin domain, as in the first model. These coils, in turn, coil into a helix, to give the final mitotic chromosome.

Figure 9. Licensed under Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/ and http://creativecommons.org/licenses/by-nc-sa/3.0/legalcode.


Note on 30 nm label in A.
At the time this paper was written, the 30 nm fiber was considered to the primary organizational structure for chromatin. The authors have labeled the width of a floret as being 30 nm, consistent with that model. However, the alternative structure, that chromatin largely consists of disorganized bundles of 10-nm fibers, is still consistent with the models postulated in this paper.




To test these hypotheses, constructs were made containing 8 tandem repeats of the E. coli lac operator sequence, flanked by SAR sequences. This construct was transfected into Chinese Hamster Ovary (CHO) cells in culture. Transformed cells, which had incorporated the construct into chromosomes, were screened by flow cytometry for cells that contained large numbers of copies of the construct. One cell line, dSAR-d11, which had approx. 1000 tandem copies of the construct inserted at a single locus.


To enable detection of the lac operator sequences in a chromosome, cells were co-transfected with another construct that expressed the lac repressor. The lac repressor protein binds to the lac operator repeats in-vivo.

The images in Fig. 9B are from chromosomes incubated with gold-tagged antibodies to the lac repressor protein. This immuno-gold staining method detects lac repressor binding to the chromosomes as bright gold particles. Fig. 9B a-c are examples of slices visualized by electron microscopy, in X, Y and Z axes. A stack or serial images, spanning the thickness of each chromosome visualized, can be deconvoluted in software to create a 3D-image of the chromosome, showing the location of the lac operator sequence in the chromosome.


Fig. 9C is a 3D-reconstruction of one chromosome.
  • If the first hypothesis was true, we would see the lac operator sequence as occupying a thin strip along the entire width of each chromosome.
  • The observations of numerous chromosomes from this cell line are consistent with the second hypothesis. Signal for the lac operator only spans a localized region about 250 - 300 nm wide, on each chromosome. In other words, the band of lac operator sequences only takes up part of the width of the chromosome. If the first hypothesis was true, then the operator sequences would always span the entire width of the chromosome. This indicates that the entire 1000 unit repeat is included in a region which folds first, and then coils as part of the higher-level coiling of the chromosome.


Studies of numerous chromosomes support the hypothesis that at least two higher-order levels of chromatin folding occur during condensation of chromatin in prophase.

c.  The highest level of coiling appears to be a helix

Click here for an MPEG movie (589 k) for a 3D tomographic movie of a human chromosome, from
Electron Microscopy Tomography (EMT) of DNA-Depleted Human (HeLa) Cell-Line Chromosome

Peter Engelhardt, Department of Virology, University of Helsinki, Helsinki, Finland
Juha Ruokolainen,  CSC, Espoo, Finland
http://www.csc.fi/jpr/emt/movies/chromosome.html

Human chromosomes were treated with DNAseI to remove most of the DNA, and slides were scanned in layers by electron microscopy. Layers were reconstructed into a 3-dimensional image.

H. Summary


1.The fundamental unit of chromosomal structure is a single DNA molecule
2.DNA is complexed with histone core particles into nucleosomes.
3.Nucleosomes coil in a left-handed solenoid with histone H1 at the core.
4.Loops of chromatin are joined to the nuclear matrix at intervals of ~50 Kb, forming discrete domains which condense when inactive and are expanded to facilitate gene expression.
5. Chromatin domains undergo an additional level of coiling, which is visualized as a thick cylinder. The nature of this level of coiling requires further study.
6.The highest level of chromosome organization appears to be a high-order helical coil of chromatin to form a cylindrical chromosome.


The highest levels of coiling are probably mostly absent in the interphase nucleus. However, at any given time, different regions of chromosomes may be coiled or uncoiled, to one degree or another. Highly-coiled chromosomal regions will not be genetically active.

Unless otherwise cited or referenced, all content on this page is licensed under the Creative Commons License Attribution Share-Alike 2.5 Canada

 
last  page PLNT3140 Introductory Cytogenetics
Lecture 11, part 2 of 2
next page