|last page||PLNT3140 Introductory Cytogenetics
Lecture 11, part 2 of 2
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).
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.
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.
|Two alternative models of
high-level chromatin folding during prophase are presented
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|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.
|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.
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
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.
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.
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|last page||PLNT3140 Introductory
Lecture 11, part 2 of 2